Synthesis of novel benzazole derivatives and evaluation of their antidepressant-like activities with possible underlying mechanisms

Article abstract of DOI:10.3390/molecules23112881

Novel benzazole derivative compounds 4a–4h were obtained by the reaction of corresponding 2-(benzazol-2-ylthio)acetohydrazide and appropriate 4-substituted benzaldehydes. The chemical structures of the synthesized compounds were elucidated by FT-IR, ¹H-NMR, ¹³C-NMR and LCMS spectroscopic methods. Antidepressant-like effects of the compounds were evaluated by tail suspension test (TST) and modified forced swimming tests (MFST). Moreover, locomotor activities of the animals were assessed by an activity cage apparatus. In the series, compounds 4a, 4b, 4e and 4f (at 50 mg/kg) significantly decreased the immobility time of mice in both of the TST and MFST. The same compounds prolonged the swimming time of animals in MFST without any change in the climbing duration. These data indicated that compounds 4a, 4b, 4e and 4f possess significant antidepressant-like activities. Moreover, pre-treatments with p-chloro-phenylalanine methyl ester (an inhibitor of serotonin synthesis), NAN-190 (a 5-HT_1A antagonist), ketanserin (a 5-HT_2A/2C antagonist), and ondansetron (a 5-HT₃ antagonist) reversed the exhibited pharmacological effects. Results of the mechanistic studies suggested the involvement of serotonergic system and contributions of 5-HT_1A, 5-HT_2A/2C and 5-HT₃ receptors to the antidepressant-like effects of compounds 4a, 4b, 4e and 4f. Furthermore, unchanged locomotor activity of mice following the administrations of these four derivatives confirmed that the presented antidepressant-like effects are specific.

Full text of DOI:10.3390/molecules23112881

molecules
Article
Synthesis of Novel Benzazole Derivatives and
Evaluation of Their Antidepressant-Like Activities
with Possible Underlying Mechanisms
Gamze Tokgöz 1, Ümide Demir Özkay 1,*, Derya Osmaniye 2,3, Nazlı Turan Yücel 1
Özgür Devrim Can 1 and Zafer Asım Kaplancıklı 2
,
1
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey;
eczgamzetokgoz@gmail.com (G.T.); nazlituran@anadolu.edu.tr (N.T.Y.); ozgurdt@anadolu.edu.tr (Ö.D.C.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470,
Turkey; dosmaniye@anadolu.edu.tr (D.O.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir 26470, Turkey
2
3
*
Correspondence: udemir@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3751)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 21 September 2018; Accepted: 1 November 2018; Published: 5 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Novel benzazole derivative compounds 4a–4h were obtained by the reaction of
corresponding 2-(benzazol-2-ylthio)acetohydrazide and appropriate 4-substituted benzaldehydes.
The chemical structures of the synthesized compounds were elucidated by FT-IR, 1H-NMR, 13C-NMR
and LCMS spectroscopic methods. Antidepressant-like effects of the compounds were evaluated
by tail suspension test (TST) and modified forced swimming tests (MFST). Moreover, locomotor
activities of the animals were assessed by an activity cage apparatus. In the series, compounds 4a, 4b,
4e and 4f (at 50 mg/kg) significantly decreased the immobility time of mice in both of the TST and
MFST. The same compounds prolonged the swimming time of animals in MFST without any change
in the climbing duration. These data indicated that compounds 4a, 4b, 4e and 4f possess significant
antidepressant-like activities. Moreover, pre-treatments with p-chloro-phenylalanine methyl ester
(an inhibitor of serotonin synthesis), NAN-190 (a 5-HT1A antagonist), ketanserin (a 5-HT2A/2C
antagonist), and ondansetron (a 5-HT3 antagonist) reversed the exhibited pharmacological effects.
Results of the mechanistic studies suggested the involvement of serotonergic system and contributions
of 5-HT1A, 5-HT2A/2C and 5-HT3 receptors to the antidepressant-like effects of compounds 4a, 4b, 4e
and 4f. Furthermore, unchanged locomotor activity of mice following the administrations of these
four derivatives confirmed that the presented antidepressant-like effects are specific.
Keywords: activity cage test; antidepressant; benzazole; modified forced swimming test;
tail suspension test
1. Introduction
Depression is a common and incapacitating mental health condition that is estimated to affect
over 300 million people worldwide. The main symptoms of depression are sad mood, loss of interest or
pleasure, low self-worth, feel of guilty, lack of concentration, anhedonia, suicidal thoughts, disrupted
sleep, appetite or energy [1,2]. Clinically used antidepressant drugs such as selective serotonin reuptake
inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors alleviate the symptoms of
major depression. However low response rates, delayed onset of therapeutic action or various side
effects associated with the use of current antidepressants are still major challenges in the management
of this disease limiting the success of treatment and causing compliance problems in patients [
3,4].
Therefore, studies for discovering and developing new antidepressant drugs are of great importance.
Molecules 2018, 23, 2881; doi:10.3390/molecules23112881
www.mdpi.com/journal/molecules

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The benzazole type compounds such as benzimidazoles and benzothiazoles are frequently
preferred rings in drug development studies because of their widespread pharmacological activity
potential. For example, various benzimidazole-based compounds have been reported for their
antibacterial [
5], anticancer [6], anti-HIV [7], anti-ulcer [8], anti-asthmatic [9], antidiabetic [10,11],
anti-inflammatory [11], antihypertensive [12], cholinesterase inhibitory, and anti-oxidant [13
]
effects. Numerous benzothiazole derivatives have also been shown to induce antibacterial,
antifungal, anticancer [14], anti-oxidant [15], antiviral [16], antituberculosis [17], antidiabetic [18],
and anti-inflammatory [19] activities, so far.
Various compounds bearing benzimidazole or benzothiazole ring systems in their chemical
structures have been demonstrated to possess potential for passing through the blood brain barrier
and triggering some central nervous system (CNS)-related pharmacological activities. Actually,
benzimidazole derivative compounds have been shown to induce antidepressant-like [20
antinociceptive [23 24] and anticonvulsant [10 11 25] activities. The same activities [19 26 32] as well as
anti-Alzheimer [33 34] and neuroprotective [35] effects have also been reported for compounds carrying
–22],
,
,
,
,
–
,
benzothiazole groups. Therefore, based on the CNS-related activity potential of benzimidazole and
benzothiazole ring systems, in the present study, we synthesized some novel compounds carrying both
of these ring systems on their chemical scaffolds and investigated their potential antidepressant-like
activities using some well-known in vivo methods.
2. Results and Discussion
2.1. Chemistry
The compounds were obtained through a four-step synthesis as presented in Scheme 1. Initially,
4-substituted benzaldehyde derivatives 1a
and appropriate secondary amines in DMF. Secondly, ethyl 2-(benzazol-2-yl-thio)acetates (2a
were gained as a result of reactions between an appropriate benzazol-2-thiol and ethyl
2-chloroacetate. Thirdly, the reaction of compounds 2a 2b and excess of hydrazine hydrate afforded
2-((5-substitutedbenzazol-2-yl)thio)acetohydrazides (3a 3b), which were reacted with the appropriate
–
1h were prepared by refluxing 4-fluorobenzaldehyde
,2b
)
,
,
4-substituted benzaldeydes 1a
–
1h to afford the target compounds 4a
–
4h. Structure elucidations of
1
the final compounds were performed by IR, H-NMR, 13C-NMR, and MS spectroscopic methods
(see Supplementary Materials).

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Comp.
X
R
4a
4b
4c
4d
4e
4f
-NH
-NH
-NH
-NH
-S
-S
-S
-S
Methyl
Ethyl
Isopropyl
Cyclopropyl
Methyl
Ethyl
Isopropyl
Cyclopropyl
4g
4h
Scheme 1. Synthesis of the target compounds 4a–4h.
2.2. Pharmacology
In this study, antidepressant-like effects of the test compounds were investigated with tail
suspension test (TST) and modified forced swimming tests (MFST), which are commonly used methods
in antidepressant-like activity screening studies [36–39].
In the TST, animals are hanged from their tails. Under this short-term and inescapable stressful
condition, animals become immobile following some escape-oriented movements. In this test,
the duration of “immobile posture” reflecting the behavioral despair is measured. It is known that
various antidepressant drugs reduce the immobility time of animals and increase the escape-related
movements in the TST [40
the immobility time of mice in the TST are illustrated in Figure 1 [F(9,60) = 10.27, p < 0.001]. Tukey HSD
multiple comparison tests following one-way ANOVA demonstrated that compounds 4a 4b 4e 4f
–43]. The effects of the tested compounds and reference drug fluoxetine on
,
,
,
and reference drug fluoxetine significantly reduced the immobility time of animals pointing out the
antidepressant-like effects of these compounds.
MFST, a modified version of Porsolt’s forced swimming test, is based on the behavioral analysis
of animals that are forced to swim in an inescapable narrow cylinder. Animals struggle to escape
from this environment and after a while, they develop an immobile posture. This immobility behavior
is considered as “behavioral despair”. Various antidepressant drugs have been reported to reduce
immobility times of rodents in MFST [42
on immobility time of mice in the MFST was shown in Figure 2 [F(9,60) = 9.95, p < 0.001]. Results of the
post-hoc analysis indicated that compounds 4a 4b 4e 4f and fluoxetine caused significant decreases
–44]. In this study, effects of the test compounds and fluoxetine
,
,
,
in the immobility time of mice in MFST. Findings of the MFST, revealing the antidepressant-like effects
of compounds 4a, 4b, 4e and 4f, supported the data obtained from the TST.

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Figure 1. The effect of 4a–4h (50 mg/kg) and fluoxetine (20 mg/kg) on immobility time of animals in
the tail suspension test. ** p < 0.01, *** p < 0.001, compared with control group; One way analysis of
variance followed by Tukey HSD multiple comparison test, n = 7.
Figure 2. The effect of 4a–4h (50 mg/kg) and fluoxetine (20 mg/kg) on immobility time of animals in
the modified forced swimming test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control group;
One way analysis of variance followed by Tukey HSD multiple comparison test, n = 7.
Different from Porsolt’s forced swimming test, in MFST, durations of active movements such
as swimming and climbing are taken into account in addition to the immobility behaviors of the
animals. Evaluation of the active behaviors is critical, since it provides some additional clues
related to the possible neurotransmitter systems playing role in the antidepressant-like effect of
a test compound [45–48]. Namely, agents affecting serotonergic neurotransmission are known to
reduce immobility and increase swimming times, in MFST. On the other hand, agents increasing
catecholaminergic neurotransmission in the synaptic cleft have been reported to enhance climbing
time while shortening the immobility durations of animals [47]. In the present study, effects of test
compounds and fluoxetine on swimming and climbing time of mice in the MFST was demonstrated
in Figure 3 [F(9,60) = 5.53, p < 0.001] and Figure 4 [F(9,60) = 0.82, p > 0.05], respectively. Results of
the post-hoc analysis indicated that compounds 4a, 4b, 4e, 4f and fluoxetine significantly increased
the swimming duration of mice while reducing the immobility time. On the other hand, none of
the test compounds altered the climbing time of the animals. Therefore, it can be suggested that
antidepressant-like effects of the compounds 4a, 4b, 4e and 4f might be related to the serotonergic
rather than catecholaminergic mechanisms in the CNS.

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Figure 3. The effect of 4a–4h (50 mg/kg) and fluoxetine (20 mg/kg) on swimming time of animals in
the modified forced swimming test. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with control group;
One way analysis of variance followed by Tukey HSD multiple comparison test, n = 7.
Figure 4. The effect of 4a–4h (50 mg/kg) and fluoxetine (20 mg/kg) on climbing time of animals in
the modified forced swimming test. One way analysis of variance followed by Tukey HSD multiple
comparison test, n = 7.
It is known that agents altering motor activity performances of animals may cause “false positive”
or “false negative” results, in TST and MFST [40,42,49]. For this reason, spontaneous locomotor
activities of animals were also evaluated, in this study. Effects of the test compounds on horizontal and
vertical locomotor activities of mice in the activity cage test were presented in Figure 5 [F(8,54) = 3.32,
p < 0.01] and Figure 6 [F(8,54) = 4.52, p < 0.001], respectively. Compounds 4a, 4b, 4e and 4f, which show
antidepressant-like activity in the TST and MFST, did not cause any significant changes in the
horizontal and vertical spontaneous locomotor activities of the animals. These findings suggest that
shortened immobility time of the animals in TST and MFST is not associated with stimulatory effects of
compounds 4a, 4b, 4e and 4f. In other words, observed antidepressant-like effects are specific. On the
other hand, compound 4d, which did not cause any changes in immobility or swimming time of mice,
triggered a significant reduction both in the horizontal and vertical locomotor activities of animals.
These decreases in the spontaneous locomotor activities may be directly related to neurosedative
activity of compound 4d or possible effect of this compound on neuromuscular junction. Further
studies are needed to clarify this issue.

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Figure 5. The effect of 4a–4h (50 mg/kg) on horizontal locomotor activities of animals in the activity
cage test. ** p < 0.01, compared with control group; One way analysis of variance followed by Tukey
HSD multiple comparison test, n = 7.
Figure 6. The effect of 4a–4h (50 mg/kg) on vertical locomotor activities of animals in the activity cage
test. * p < 0.05, compared with control group; One way analysis of variance followed by Tukey HSD
multiple comparison test, n = 7.
Following the antidepressant-like activity screening studies, we conducted some further
mechanistic experiments. Instead of MFST, we conducted our studies by TST, because of the lack of
hypothermia risk, enhanced pharmacological sensitivity, and rapid return to normal spontaneous
activity following this test [40,50].
With the aim of examining the possible involvement of the serotonergic system to the
antidepressant-like effects of the compounds 4a, 4b, 4e and 4f, we used p-chlorophenylalanine methyl
ester (PCPA). PCPA is an agent inhibiting tryptophan hydroxylase enzyme playing role in the synthesis
of serotonin. It stops the serotonin synthesis and reduces the endogenous serotonin storage by 60–90%
in the CNS of mice, when administered at 100 mg/kg daily dose for four consecutive days. PCPA does
not alter central noradrenaline or dopamine levels while reducing serotonin in brain [51
–53]. In this
study, effects of PCPA pre-treatments on anti-immobility activities of the compounds 4a
,
4b 4e and 4f
,
in the TST were exhibited in Figure 7 [F(9,60) = 11.82, p < 0.001].

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Figure 7. Effect of PCPA (100 mg/kg) pre-treatments on antidepressant-like activity induced by
compounds 4a, 4b, 4e and 4f in the tail suspension test. ** p < 0.01, *** p < 0.001, compared with
control group; b p < 0.01, compared with compound 4a administrated group; & p < 0.05, compared
with compound 4b administrated group; + p < 0.05, compared with compound 4e administrated group;
éé
p < 0.01, compared with compound 4f administrated group; One way analysis of variance followed
by Tukey HSD multiple comparison test, n = 7.
Pre-treatments with PCPA reversed the compounds 4a, 4b, 4e and 4f induced shortenings in the
immobility time of mice. This data indicated that antidepressant-like effects of these compounds are
associated with the serotonergic neurotransmission. This result is in accordance with our experimental
data obtained from the MFST tests suggesting the involvement of serotonergic system in the observed
antidepressant-like effects.
After realizing the importance of serotonergic neurotransmission in the pharmacological effects of
the active compounds, we tried to clarify possible serotonergic receptors participating the mechanism
of action. Therefore, probable contributions of 5-HT1A, 5-HT2A/2C and 5-HT3 serotonergic receptors to
the observed antidepressant-like effects were evaluated by pre-treatments with NAN-190, ketanserin
and ondansetron, respectively [54,55].
The effect of NAN-190 pre-treatment on anti-immobility activities of the compounds 4a, 4b,
4e and 4f are shown in Figure 8 [F(9,60) = 18.15, p < 0.001]. NAN-190 pre-treatments reversed the
anti-immobility effects of these compounds pointing out the participation of 5-HT1A receptors to the
antidepressant-like effects induced by compounds 4a, 4b, 4e and 4f.
Figure 9 displays the effect of ketanserin pre-treatment on anti-immobility activities of the
compounds 4a, 4b, 4e and 4f [F(9,60) = 13.66, p < 0.001].
Obtained data indicated that ketanserin pre-treatments antagonized the anti-immobility
effects of these compounds pointing out the contribution of 5-HT2A/2C receptors to the exhibited
antidepressant-like effects.

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Figure 8. Effect of NAN-190 (0.5 mg/kg) pre-treatments on antidepressant-like activity induced by
compounds 4a 4b 4e and 4f in the tail suspension test. ** p < 0.01, *** p < 0.001, compared with control
,
,
group; b p < 0.01, compared with compound 4a administrated group; &&& p < 0.001, compared with
compound 4b administrated group; +++ p < 0.001, compared with compound 4e administrated group;
ééé p < 0.001, compared with compound 4f administrated group; One way analysis of variance followed
by Tukey HSD multiple comparison test, n = 7.
200
+++
&&&
éé
b
150
100
50
**
**
***
***
0
Figure 9. Effect of ketanserin (1 mg/kg) pre-treatments on antidepressant-like activity induced by
compounds 4a, 4b, 4e and 4f in the tail suspension test. ** p < 0.01, *** p < 0.001, compared with control
group; b p < 0.01, compared with compound 4a administrated group; &&& p < 0.001, compared with
compound 4b administrated group; +++ p < 0.001, compared with compound 4e administrated group;
éé
p < 0.01, compared with compound 4f administrated group; One way analysis of variance followed
by Tukey HSD multiple comparison test, n = 7.
The effect of ondansetron pre-treatment on anti-immobility activities of the compounds 4a
and 4f are shown in Figure 10 [F(9,60) = 17.36, p < 0.001]. Ondansetron pre-treatments reversed the
anti-immobility effects induced by the compounds 4a 4b 4e and 4f suggesting the involvement of
5-HT3 receptors in the antidepressant-like effects of these compounds.
, 4b, 4e
,
,

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Figure 10. Effect of ondansetron (0.3 mg/kg) pre-treatments on antidepressant-like activity induced by
compounds 4a
,
4b
,
4e and 4f in the tail suspension test. *** p < 0.001, compared with control group;
p < 0.05, compared with compound 4a administrated group; & p < 0.05, compared with compound 4b
administrated group; +++ p < 0.001, compared with compound 4e administrated group; ééé p < 0.001,
compared with compound 4f administrated group; One way analysis of variance followed by Tukey
HSD multiple comparison test, n = 7.
a
In summary, the findings acquired from the mechanistic studies indicated that compounds
4a
5-HT3 receptors seem to contribute to these effects. These results supported the previous literature
pointing out the role of serotonergic system and 5-HT1A, 5-HT2A/2C and 5-HT3 receptors [56 59] in
, 4b, 4e and 4f presented significant antidepressant-like activities and 5-HT1A, 5-HT2A/2C and
–
the pharmacological mechanisms of various compounds possessing antidepressant efficacy. Actually,
additional radioligand binding studies will help to confirm the involvement of these three serotonergic
receptors as molecular targets of compounds 4a, 4b, 4e and 4f. On the other hand, other serotonergic
receptor subtypes might also be participated to the observed pharmacological effect. Therefore,
potential involvement of additional serotonergic receptors and even other neuromediator systems
(GABAergic, glutaminergic, and nitrergic systems etc.) to the presented antidepressant-like activities
should also be examined by further detailed studies.
Results of the in vivo studies indicated that pharmacological activities of the compounds 4a
,
4b
4a
, 4e and 4f (Figures 1–3) are independent of whether they contain benzimidazole (compounds
,4b) or benzothiazole (compounds 4e,4f) rings on the chemical scaffold. Instead, substitutions at
the 4--position of the piperazine ring seem to cause the difference in the activity. More specifically,
antidepressant-like effect was observed with the compounds bearing methyl and ethyl groups at the
piperazine 4-position (compounds 4a,4b,4e and 4f), whereas isopropyl- and cyclopropyl-substituted
compounds 4c 4d 4g and 4h were found to be ineffective. Therefore, it can be suggested that steric
,
,
effects may play a role on the pharmacological activity, which is enhanced by smaller substituents such
as methyl and ethyl, but abolished by relatively more bulky groups like isopropyl and cyclopropyl.
On the other hand, it should be noted that increasing the number of test compounds in the series might
provide a more reliable approach for the structure activity relationship related to the tested compounds.
3. Materials and Methods
3.1. Chemistry
Chemicals of the Merck (Merck KGaA, Darmstadt, Germany) or Sigma-Aldrich (Sigma-Aldrich
1
Corp., St. Louis, MO, USA) companies were used in the synthesis. The H-NMR and 13C-NMR
spectra were recorded in DMSO-d6 at 300 MHz and 75 MHz, pespectively, on a digital FT-NMR

Molecules 2018, 23, 2881
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spectrometer (Bruker Bioscience, Billerica, MA, USA). The NMR spectra signals were defined as
a singlet (s), br.s. (broad singlet), doublet (d), triplet (t), double doublet (dd), and multiplet (m).
The coupling constants (J) are reported in units of Hertz (Hz). A FT-IR spectrometer (IRAffinity-1S
Fourier transform, Shimadzu, Tokyo, Japan) was used to record the IR spectra of the compounds.
MS studies were performed on an LCMS-8040 tandem mass system (Shimadzu) and acetonitrile
containing 0.1% formic acid was used as solvent. Silica gel 60 F254 by TLC (Merck KGaA) was used to
monitor the progress of reactions.
3.1.1. Synthesis of 4-Substituted Benzaldehydes 1a–1d
A solution of 4-fluorobenzaldehyde (0.017 mol, 1.82 mL) and an appropriate secondary amine
in DMF (10 mL) was refluxed (0.017 mol) in the presence of potassium carbonate (K2CO3, 0.017 mol,
2.35 g). After completion of reaction, the mixture was cooled with ice-water (10 mL) and extracted
with ethyl acetate (3
×
20 mL). The ethyl acetate phases were combined, and the remaining product
was collected after evaporation of the solvent. The following compounds were prepar◦ed in this way:
4-(4-methylpiperazin-1-yl)benzaldehyde (1a), CAS No:27913-99-1, Yield 82%, m.p. = 55–57 C (measured),
m.p. = 58–60 ◦C (reported) [60]; 4-(4-ethylpiperazin-1-yl)benzaldehyde (1b), CAS No:197638-76-9,
Yield 79%, obtained as an oil; 4-(4-isopropylpiperazin-1-yl)benzaldehyde (1c), CAS No:197638-78-1,
Yield 84%, obtained as an oil and 4-(4-cylopropylpiperazin-1-yl)benzaldehyde (1d), CAS No:1292778-23-4,
Yield 78%, obtained as an oil.
3.1.2. Synthesis of Ethyl 2-(Benzazol-2-yl-thio)acetates 2a,2b
The corresponding benzazol-2-thiol (0.015 mol), ethyl 2-chloroacetate, (0.015 mol, 1.61 mL),
and K2CO3 (0.015 mol, 2.07 g) were refluxed in acetone (70 mL) for 8–10 h. After completion of the
reaction, the solvent was removed using reduced pressure. The precipitated product was washed with
water, dried and recrystallized from EtOH. The following compounds were preapared in this way:
ethyl 2-(benzimidazole-2-ylthio)acetate (2a), CAS No:5429-62-9, Yield 65%, m.p. = 99–101 ◦C (measured),
m.p. = 97 ◦C (reported) [61]; ethyl 2-(benzothiazol-2-ylthio)acetate (2b), CAS No:24044-88-0, Yield 72%,
m.p. = 45–47 ◦C (measured), m.p. = 42–44 ◦C (reported) [62].
3.1.3. Synthesis of 2-((5-Substitutedbenzazol-2-yl)thio)acetohydrazides 3a,3b
A solution (20 mL) of hydrazine hydrate (5 mL) in EtOH (15 mL) was added as portions
into the reaction mixture of ethyl 2-((benzazol-2-yl)thio)acetate 2a–2b (0.012 mol) in EtOH
(50 mL). The reaction mixture was stirred for 5 h. The precipitated product was filtered, washed
with cold-ethanol, dried and recrystallized from EtOH. To afford the following compounds:
◦
2-((1H-benzimidazol-2-yl)thio)acetohydrazide (3a), CAS No:30065-27-1, Yield 62%, m.p. = 164–166 C
(measured), m.p. = 165–166 ◦C (reported) [63]; 2-((1H-benzothiazole-2-yl)thio)acetohydrazide (3b),
CAS No:24044-91-5, Yield 81%, m.p. = 175–177 ◦C (measured), m.p. = 173–174 ◦C (reported) [62].
3.1.4. General Procedure for the Synthesis of the Target Compounds 4a–4h
An appropriate 2-((benzazol-2-yl)thio) acetohydrazide 3a–3b (0.002 mol), and a 4-substituted-
benzaldehyde 1a 1h (0.002 mol) and a catalytic amount of acetic acid were refluxed in EtOH (30 mL)
–
for 2 h. The mixture was cooled in an ice-bath, and the precipitated product was filtered, dried and
recrystallized from EtOH.
2-((1H-Benzimidazol-2-yl)thio)-N0-(4-(4-methylpiperazin-1-yl)benzylidene)acetohydrazide (4a). Yield: 88%,
1
FTIR (ATR, cm−1): 3572 (N-H), 2937 (C-H), 1660 (C=O), 817. H-NMR (DMSO-d6):
δ = 2.22 (3H, s,
-CH3), 2.42–2.45 (4H, m, piperazine), 3.21–3.24 (4H, m, piperazine), 3.37 (2H, s, -CH2), 6.98 (2H, d,
J = 8.85 Hz, Ar-H), 7.07 (1H, t, J = 7.80 Hz, Ar-H), 7.19 (1H, t, J = 7.44 Hz, Ar-H), 7.32–7.37 (1H, m,
Ar-H), 7.48–7.56 (3H, m, Ar-H), 8.07 (1H, s, Ar-H), 11.56 (1H, br.s, -NH).13C-NMR (DMSO-d6):
δ = 40.82,

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46.20, 47.65, 54.90, 109.68, 110.66, 115.04, 116.72, 121.49, 124.52, 128.31, 145.45, 152.24, 159.99. ESI-MS
[M + H]+: 409.00 (100%).
2-((1H-Benzimidazol-2-yl)thio)-N0-(4-(4-ethylpiperazin-1-yl)benzylidene)acetohydrazide (4b). Yield: 82%,
1
FTIR (ATR, cm−1): 3562 (N-H), 2970 (C-H), 1660 (C=O), 817. H-NMR (DMSO-d6):
δ = 1.03 (3H, t,
J = 7.17 Hz, -CH3), 2.36 (2H, q, J = 7.20 Hz, -CH2-) 2.47–2.50 (4H, m, piperazine), 3.21–3.24 (4H, m,
piperazine), 3.35 (2H, s, -CH2), 6.98 (2H, d, J = 8.85 Hz, Ar-H), 7.07 (1H, t, J = 7.74 Hz, Ar-H), 7.19 (1H, t,
J = 7.50 Hz, Ar-H), 7.35–7.37 (1H, m, Ar-H), 7.49–7.56 (3H, m, Ar-H), 8.07 (1H, s, Ar-H), 11.80 (1H, br.s,
-NH). 13C-NMR (DMSO-d6):
δ = 12.45, 40.89, 47.76, 52.08, 52.67, 109.67, 114.99, 116.71, 121.48, 124.52,
128.29, 142.94, 145.47, 148.55, 152.27, 160.00. ESI-MS [M + H]+: 423.10 (100%).
2-((1H-Benzimidazol-2-yl)thio)-N0-(4-(4-isopropylpiperazin-1-yl)benzylidene)acetohydrazide (4c). Yield: 80%,
1
FTIR (ATR, cm−1): 3562 (N-H), 2966 (C-H), 1708 (C=O), 806. H-NMR (DMSO-d6):
δ = 0.99 (6H, d,
J = 6.45 Hz, -CH3), 2.55 (4H, br.s., piperazine), 2.62–2.69 (1H, m, -CH-), 3.21 (4H, br.s., piperazine), 4.64
(2H, s, -CH2-), 6.95 (2H, d, J = 8.85 Hz, Ar-H), 7.31–7.34 (2H, m, Ar-H), 7.53 (2H, d, J = 8.76 Hz, Ar-H),
7.63–7.66 (2H, m, Ar-H), 7.93 (1H, s, Ar-H), 11.58 (1H, s, -NH). 13C-NMR (DMSO-d6):
δ = 18.67, 35.26,
48.08, 48.38, 54.12, 110.67, 114.90, 118.72, 124.04, 124.71, 125.09, 128.58, 141.73, 144.94, 148.04, 152.61,
162.74, 167.97. ESI-MS [M + H]+: 437.20 (100%).
2-((1H-Benzimidazol-2-yl)thio)-N0-(4-(4-cyclopropylpiperazin-1-yl)benzylidene)acetohydrazide (4d). Yield:
1
79%, FTIR (ATR, cm−1): 3550 (N-H), 2974 (C-H), 1718 (C=O), 810. H-NMR (DMSO-d6):
δ = 0.34–0.35
(2H, m, -CH2-), 0.42-0.44 (2H, m, -CH2-), 1.62-1.65 (1H, m, -CH-), 2.65 (4H, br.s., piperazine), 3.17–3.20
(4H, m, piperazine), 4.65 (2H, s, -CH2-), 6.93–6.98 (2H, m, Ar-H), 7.07 (1H, t, J = 7.86 Hz, Ar-H), 7.19
(1H, t, J = 6.81, Ar-H) 7.31–7.33 (1H, m), 7.51–7.57 (2H, m, Ar-H), 7.63–7.65 (1H, m, Ar-H), 11.59 (1H,
s, -NH). 13C-NMR (DMSO-d6):
δ = 6.09, 35.27, 38.52, 47.78, 52.99, 53.04, 110.66, 115.07, 118.72, 121.46,
124.71, 125.09, 128.58, 141.73, 144.92, 148.01, 152.25, 162.76, 167.97. ESI-MS [M + H]+: 435.20 (100%).
2-(Benzothiazol-2-ylthio)-N0-(4-(4-methylpiperazin-1-yl)benzylidene)acetohydrazide (4e). Yield: 81%, FTIR
1
(ATR, cm−1): 3313 (N-H), 2933 (C-H), 1660 (C=O), 819. H-NMR (DMSO-d6):
δ = 2.22 (3H, s, -CH3),
2.42–2.43 (4H, m, piperazine), 3.21–3.24 (4H, m, piperazine), 4.66 (2H, s, -CH2), 6.96 (2H, t, J = 9.63
Hz, Ar-H), 7.34–7.40 (1H, m, Ar-H), 7.44–7.55 (3H, m, Ar-H), 7.85 (1H, d, J = 8.07 Hz, Ar-H), 7.93 (1H,
s, Ar-H), 8.00–8.04 (1H, m, Ar-H), 11.54 (1H, s, -NH). 13C-NMR (DMSO-d6):
δ = 35.61, 46.20, 47.57,
54.85, 114.98, 121.58, 122.30, 124.17, 124.92, 126.84, 128.56, 135.20, 144.74, 147.99, 152.49, 162.98, 168.29.
ESI-MS [M + H]+: 426.10 (100%).
2-(Benzothiazol-2-ylthio)-N0-(4-(4-ethylpiperazin-1-yl)benzylidene)acetohydrazide (4f). Yield: 82%, FTIR
1
(ATR, cm−1): 3228 (N-H), 2966 (C-H), 1662 (C=O), 821. H-NMR (DMSO-d6):
δ = 1.02 (3H, t, J = 6.63
Hz, -CH3), 2.34 (2H, q, J = 7.32 Hz), 2.46 (4H, br.s., piperazine), 3.21 (4H, br.s., piperazine), 4.64 (2H, s,
-CH2-), 6.94 (2H, d, J = 8.73 Hz, Ar-H), 7.35 (1H, m, Ar-H), 7.43–7.51 (3H, m, Ar-H), 7.84 (1H, d, J = 7.92
Hz, Ar-H), 7.91 (1H, s, Ar-H), 8.01 (1H, d, J = 8.67 Hz, Ar-H), 11.52 (1H, s, -NH). 13C-NMR (DMSO-d6):
δ
= 17.18, 40.35, 52.43, 56.82, 57.37, 119.68, 126.33, 127.05, 128.91, 129.67, 131.60, 133.31, 139.95, 149.50,
152.75, 157.27, 167.73, 173.04. ESI-MS [M + H]+: 440.11 (100%).
0
2-(Benzothiazol-2-ylthio)-N -(4-(4-isopropylpiperazin-1-yl)benzylidene)acetohydrazide (4g). Yield: 82%, FTIR
(ATR, cm−1): 3471 (N-H), 2960 (C-H), 1668 (C=O), 833. H-NMR (DMSO-d6):
δ = 0.99 (6H, d, J = 6.51
1
Hz, -CH3), 2.50-2.51 (1H, m, -CH-), 2.54–2.57 (4H, m, piperazine), 3.19–3.22 (4H, m, piperazine), 4.66
(2H, s, -CH2-), 6.94 (2H, t, J = 9.67 Hz, Ar-H), 7.34–7.40 (1H, m, Ar-H), 7.44–7.47 (1H, m, Ar-H), 7.51
(1H, d, J = 6.30 Hz, Ar-H), 7.54 (1H, d, J = 6.12 Hz, Ar-H), 7.84–7.89 (1H, m, Ar-H), 7.93 (1H, s, Ar-H),
8.00–8.04 (1H, m, Ar-H), 11.53 (1H, s, -NH). 13C-NMR (DMSO-d6):
δ = 18.66, 35.60, 48.07, 48.37, 54.13,
114.89, 121.58, 122.30, 124.09, 124.92, 126.84, 128.55, 128.85, 135.20, 144.76, 148.02, 152.57, 162.97, 168.29.
ESI-MS [M + H]+: 454.13 (100%).

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2-(Benzothiazol-2-ylthio)-N0-(4-(4-cyclopropylpiperazin-1-yl)benzylidene)acetohydrazide (4h). Yield: 82%,
1
FTIR (ATR, cm−1): 3228 (N-H), 2821 (C-H), 1654 (C=O), 821. H-NMR (DMSO-d6):
δ = 0.34–0.36 (2H, m,
-CH2-), 0.41–0.44 (2H, m, -CH2-), 1.62–1.66 (1H, m, -CH-), 2.63–2.65 (4H, m, piperazine), 3.16–3.19 (4H,
m, piperazine), 4.65 (2H, s, -CH2-), 6.94 (2H, t, J = 9.46 Hz, Ar-H), 7.34-7.39 (1H, m, Ar-H), 7.44–7.47
(1H, m, Ar-H), 7.50 (1H, d, J = 6.24 Hz, Ar-H), 7.53 (1H, d, J = 5.94 Hz, Ar-H), 7.85 (1H, d, J = 8.10 Hz,
Ar-H), 7.92 (1H, s, Ar-H), 7.99–8.03 (1H, m, Ar-H), 11.53 (1H, s, -NH). 13C-NMR (DMSO-d6):
δ = 6.08,
35.61, 47.61, 47.70, 52.99, 115.01, 121.58, 122.29, 124.16, 124.92, 126.84, 128.56, 135.20, 144.73, 147.98,
152.51, 162.97, 168.29. ESI-MS [M + H]+: 452.12 (100%).
3.2. Pharmacology
3.2.1. Animals
Adult Balb/c mice weighing 30–35 g were used for the behavioral tests. They were group-housed
◦
and maintained in standard laboratory conditions: constant temperature (24
±
1 C) and 12-h
light/dark cycle. Animals were allowed to acclimatize to the laboratory conditions 48 h before the
experiments. The experimental protocol of this study was approved by the Local Ethical Committee
on Animal Experimentation of Anadolu University (Eskis¸ehir, Turkey; date: 13.04.2018; Project no:
2018-21).
3.2.2. Experimental Groups and Treatments
Animals were randomly placed into 10 groups with seven animals in each. Efficacies of the test
compounds (50 mg/kg, po) [21] were compared to control and fluoxetine (reference drug, 20 mg/kg,
po) groups [37]. Vehicle, fluoxetine and test compounds were administered three times to mice at 24, 5
and 1.0 h before the experiments [37,64].
3.2.3. Evaluation of Antidepressant-Like Activity
Tail Suspension Test
TST was carried out as previously described [36,41]. In this test, mice were suspended 30 cm
above the ground with an adhesive tape, which is located about one cm from tip of the tails. Animals
were evaluated as immobile when they did not perform escape-oriented movements and become
motionless. Total duration of the immobility behavior was measured by a chronometer over the last
4 min of the 6-min test period. Mice climbing their tails during the tests were eliminated from the tests.
Modified Forced Swimming Test
MFST was also performed to examine the antidepressant-like effects of test compounds. In this
test, mice were forced to swim in a Plexiglas cylinder (30 cm height, 12 cm diameter) filled with water
up to 20 cm high. The temperature of the water was set at 25
±
1 ◦C. Twenty-four hours before the
experiments, animals were let to swim individually in the cylinder for 15 min. In the experimental
phase, mice were placed again in the cylinder and durations of immobility (the movements required
to keep the head above the water), swimming (horizontal motion on the surface of the water) and
climbing (upward-directed movements of the forepaws along the side of the swim chamber) behaviors
over 5-s intervals were measured with a chronometer for 5 min [36,47,65].
The water in the cylinder was changed after each mouse. After pre-test and experimental phases,
mice were dried carefully before returning their home cages.

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3.2.4. Evaluation of Spontaneous Locomotor Activity
Activity Cage Test
Activity cage device (No. 7420, Ugo Basile, Varese, Italy) was used to assess the effects of test
compounds on motor activities of mice. In this device, numbers of movements interrupting the IR
beams are recorded, automatically. Horizontal and vertical spontaneous locomotor activities were
recorded for 4 min. After testing each animal, the apparatus was carefully cleaned with ethanol
solution [37,66].
Mechanistic Studies
Mechanistic studies were performed with PCPA (a serotonin synthesis inhibitor), NAN-190
(5-HT1A receptor antagonist), ketanserin (5-HT2A/2C receptor antagonist) and ondansetron (5-HT3
receptor antagonist) to investigate the possible involvement of the serotonergic system and serotonergic
receptors in the antidepressant-like effects of test compounds. PCPA was intraperitoneally administered
at dose of 100 mg/kg for four consecutive days. Test compounds were applied 24 h after the last
injection and 1 h later, animals were subjected to the TST [54]. NAN-190 (0.5 mg/kg i.p.), ketanserin
(1 mg/kg i.p.), ondansetron (0.3 mg/kg i.p.) were injected 15 min prior to the administration of the
test compounds. Then, TST was conducted 1 h later [36].
Statistical Analysis
Graphpad Prism ver. 6.01 (GraphPad Software, San Diego, CA, USA) was used for statistical
analyses and for the preparation of graphical presentations. Obtained data was analyzed using
one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Results were
expressed as the mean
considered as statistically significant if p < 0.05.
±
standard error of the mean (SEM). Differences between the groups were
4. Conclusions
Data obtained from this study presented that test compounds 4a, 4b, 4e and 4f possess significant
antidepressant-like efficacy mediated through serotonergic system and 5-HT1A, 5-HT2A/2C 5-HT3
serotonergic receptors. These results supported to the previous literature findings pointing out
the antidepressant capacity of benzazole derivative compounds [20–22,26,27]. On the other hand,
to clarify the exact mechanism of action possible contribution of other neurotransmitter systems
(such as opioidergic, GABAergic, glutaminergic, and nitrergic systems) should also be investigated by
further studies.
Supplementary Materials: The 13C-NMR, 1H-NMR, FTIR, and MS spectrums of compounds 4a
–4h are
available online.
Author Contributions: Conceptualization and design of the study, Ü.D.Ö. and Z.A.K.; Synthesis of the
compounds: Z.A.K. and D.O., Conducting pharmacological tests G.T. and N.T.Y.; Statistical analysis of the
data Ü.D.Ö. and Ö.D.C.; Writing of the manuscript Ü.D.Ö. and Ö.D.C.; Proofreading Z.A.K.
Acknowledgments: This research was supported from the Anadolu University Scientific Research Projects
Commission (Project No: 1805S189).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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