Synthesis of hydrazine containing piperazine or benzimidazole derivatives and their potential as α-amylase inhibitors by molecular docking, inhibition kinetics and in vitro cytotoxicity activity studies

Article abstract of DOI:10.1007/s00044-021-02785-8

The α-amylase is the main product of pancreas and is necessarily involved in the hydrolysis of carbohydrates into glucose so that it has been known to be a pioneer target for type 2 Diabetes mellitus (DM). Type 2 DM has no certain cure and the global increase in the cases of DM requires effective and extensive number of drug candidates. Drug discovery studies using organic biochemistry approaches are of important to describe novel compounds. This study aimed to reveal inhibitory potential of 13 novel compounds containing piperazine or benzimidazole moieties on α-amylase. The novel compounds were synthesized, structurally corroborated by various spectral analysis (FTIR, UV-Vis, ¹H NMR and ¹³C NMR) and screened for anti α-amylase activity. Among the synthesized derivatives, compound 14 was found to be the most potent inhibitor of α-amylase having IC₅₀ 64.8 ± 1.8 μM. Inhibition types and K_i values of the most effective molecules (14 and 10a with different moieties) were further investigated. Molecular docking studies were conducted to correlate the outcome of in vitro biochemical kinetic assays and therefore rationalize the binding interactions. In vitro cytotoxicity studies on pancreatic cancer (AR42J) cells were then performed for compound14, and the compound was found to be more effective compared to the positive control, acarbose. Prediction of in silico ADME properties of all tested molecules were determined.

Full text of DOI:10.1007/s00044-021-02785-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-021-02785-8
ORIGINAL RESEARCH
Synthesis of hydrazine containing piperazine or benzimidazole
derivatives and their potential as α-amylase inhibitors by molecular
docking, inhibition kinetics and in vitro cytotoxicity activity studies
1 ●
1 ●
1 ●
2 ●
3 ●
Ummuhan Cakmak
Ahmet Colak
Fulya Oz-Tuncay
Serap Basoglu-Ozdemir
Elif Ayazoglu-Demir
İlke Demir
1 ●
4 ●
3 ●
1
Selcen Celik-Uzuner
Safiye Sag Erdem
Nuri Yildirim
Received: 6 May 2021 / Accepted: 10 August 2021
The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
©
Abstract
The α-amylase is the main product of pancreas and is necessarily involved in the hydrolysis of carbohydrates into
glucose so that it has been known to be a pioneer target for type 2 Diabetes mellitus (DM). Type 2 DM has no certain
cure and the global increase in the cases of DM requires effective and extensive number of drug candidates. Drug
discovery studies using organic biochemistry approaches are of important to describe novel compounds. This study
aimed to reveal inhibitory potential of 13 novel compounds containing piperazine or benzimidazole moieties on α-
amylase. The novel compounds were synthesized, structurally corroborated by various spectral analysis (FTIR, UV-Vis,
1
13
H NMR and C NMR) and screened for anti α-amylase activity. Among the synthesized derivatives, compound 14 was
found to be the most potent inhibitor of α-amylase having IC50 64.8 ± 1.8 μM. Inhibition types and K values of the most
i
effective molecules (14 and 10a with different moieties) were further investigated. Molecular docking studies were
conducted to correlate the outcome of in vitro biochemical kinetic assays and therefore rationalize the binding
interactions. In vitro cytotoxicity studies on pancreatic cancer (AR42J) cells were then performed for compound14, and
the compound was found to be more effective compared to the positive control, acarbose. Prediction of in silico ADME
properties of all tested molecules were determined.
●
●
●
●
Keywords Piperazine Benzimidazole α-amylase inhibition Molecular docking Cell culture
Introduction
α-Amylase (EC 3.2.1.1) is the key digestive enzyme that
catalyzes the hydrolysis of α-(1,4) glycosidic linkages in
starch to produce maltose, maltotriose, and limit dextrins
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
[
1]. Rapid digestion of carbohydrate by α-amylase enzyme
is associated with sudden increase in blood glucose level
2], resulting in type 2 diabetes, and there are also other
0
21-02785-8.
[
*
Ahmet Colak
acolak@ktu.edu.tr
disorders such as cardiovascular diseases, neuropathy,
angiopathy, nephropathy, related to carbohydrate intake
[3, 4]. α-Amylase inhibitors are used to reduce the glucose
levels by slowing down the mechanism for conversion of
starch into glucose. Acarbose, miglitol and metformin are
currently used as α-amylase inhibitors but they lead to some
side effects including bloating abdominal discomfort,
flatulence, diarrhea, abdominal and liver disorders [2, 5]. In
addition, over the past years, the overuse of common anti-
microbial drugs has resulted in the emergence of resistant
bacteria and a decrease in the effectiveness of them [6–10].
1
2
Department of Chemistry, Faculty of Science, Karadeniz
Technical University, 61080 Trabzon, Turkey
Department of Chemistry and Chemistry Processing Technologies,
Macka Vocational High School, Karadeniz Technical University,
6
1750 Trabzon, Turkey
3
4
Department of Chemistry, Faculty of Arts and Sciences, Marmara
University, 34722 Istanbul, Turkey
Department of Molecular Biology and Genetics, Faculty of
Science, Karadeniz Technical University, 61080 Trabzon, Turkey

Medicinal Chemistry Research
These limitations have emphasized the need of new drug
discovery strategies focusing on the designs to increase
affinity and specificityin concordance with minimizing
current side effects.
Results and discussion
Chemistry
A number of N-containing heterocyclic compounds
are an important part of organic and medical chemistry.
Among them piperazines are advantageous to have
flexible core structure for the design and synthesis of
new bioactive compounds. These flexible heterogeneous
compounds display several biological roles as, anti-oxi-
dant, anti-cancer, anti-microbial, anti-fungal, anti-bac-
terial, anti-viral (anti-HIV), anti-inflammatory, anti-
diabetic, anti-depressant, anti-malarial, anti-convulsant,
and anti-anxiety [11, 12]. Imidazole ring is the compo-
nent of many natural products such as purine, histamine,
histidine, and nucleic acids. Due to its polar and ioniz-
able ability, it proves to be a characteristic pharmaco-
kinetic for lead molecules by enhancing their solubility
The treatment of the starting compound, 1-(2-fluorophenyl)
piperazine with ethyl bromoacetate to give Ethyl [4-(2-
fluorophenyl)piperazin-1-yl]acetate (2). The substitution of
ester group by hydrazine hydrazide generated 2-[4-(2-
Fluorophenyl)piperazine-1-yl]acetohydrazide (3), which
were confirmed by the appearance of broad signal for
-NHNH group in FT IR spectrum. This group was recorded
2
1
at 4.25 (NH ) and 8.94 (NH) ppm in the H NMR spectrum.
2
The nucleophilic addition of compound 3 to alkyl(aryl)iso
(thio) cyanates afforded the corresponding carbo(thio)
amides (4a, 4b). FT IR spectra of compounds 4a and 4b
−
1
revealed the presence of C=S group 1235 cm while C=O
−
1
group 1666 cm . Another evidence for the formation of
carbo(thio)amides was the presence of three NH- signals at
1
[
13]. Benzimidazole derivatives are highly effective
7.85–9.65 ppm in the H NMR spectrum. The FT IR spectra
compounds with both inhibitory activities and appro-
priate selectivity ratios, and they are of great interest
with their biological activities and clinical applications
of compound 5 has C=O and -NH stretching bands at 1701
−1
1
and 3387 cm , respectively. In the H NMR spectra,
resonance assigned to the -NH proton on 1,2,4-triazole
ring was recorded at 11.77 ppm (NH) in the spectrum
(Scheme 1).
[
14, 15].
Structure-based and ligand-based computer-aided drug
design (CADD) methods are frequently used in rational
drug design since discovering new drug lead compounds
is a challenging and costly procedure [16]. Molecular
docking is one of the most common structure-based
CADD methods that calculates binding affinities via
noncovalent protein-ligand interactions such as electro-
static, van der waals, coulomb, hydrogen bonds and pre-
dicts ligand conformations in the binding site of the
macromolecule. Predicted binding poses are ranked as
docking scores. Autodock Vina calculates Gibbs free
energy of binding (ΔG) as the docking score, which can
The syntheses of triazoles 10a, 10b were carried out by the
treatment of NaOH in water under reflux condition to intro-
duce the 1,2,4-triazole nucleus to piperazine skeleton, because
it is well known that more efficacious bioactive compounds
can be designed by joining two or more biologically active
heterocyclic systems together in a single molecular frame-
work [21–23] (Scheme 2). It is also well known that 1,2,4-
triazol-3-(thi)ol derivatives can exist as mercapto-thioxo (or
enol-keto) tautomeric forms. The -SH proton due to mercapto
form resonates at about 13–14 ppm, while NH signal origi-
nated from thioxo tautomer appears at 9–12 ppm as D O
2
also be converted to inhibition constant (K ) [17–20].
exchangeable signals [22, 24]. These compounds exhibited
i
1
13
In the present study, molecular docking calculations
were performed to understand the mechanism of
enzyme-ligand binding interactions by predicting the
preferred orientation of newly synthesized ligands in α-
amylase and calculating their binding energies. In addi-
tion, absorption, distribution, metabolism and excretion
FT-IR, H NMR and C NMR spectra consistent with the
assigned structures. In compound 10a the -SH signal was
recorded at 13.65 ppm, for the NH signal 10b 11.58 ppm was
13
recorded. C NMR spectra of these compounds have reso-
nances of triazole C-3 and C-5 at 149.3–167.3 ppm and
144.5–155.6 ppm, respectively.
(
ADME) properties of the synthesized compounds
For the synthesis of compound number 14, starting from 2-
aminobenzimidazole, it was treated with ethyl bromoacetate,
hydrazine hydrate and phenyl isocyanate, respectively
(Scheme 3). NH signals of the compound 14 were recorded in
were predicted to examine the bioavailability of these
compounds.
Herein we report for the first time the synthesis of 13
novel piperazine and benzimidazole derivatives. Designed
compounds were synthesized, characterized and screened
for in vitro α-amylase inhibitory activity. Besides, inhi-
bition kinetics and molecular docking of synthesized
compounds were also performed. Once and for all, the
most effective compound was evaluated by the MTT
assay against pancreatic cancer cell line AR42J.
1
the range of 8.28–12.10 ppm in the H NMR spectrum.
α-Amylase inhibition activity
Before performing the inhibition studies to reveal the
inhibition potential of the synthesized organic molecules on
α-amylase activity, the commercially available pancreatic

Medicinal Chemistry Research
Scheme 1 Synthesis of compounds starting from 2-fluorophenyl piperazine
Scheme 2 Synthesis of compounds starting from 4-nitrophenyl piperazine
Scheme 3 Synthesis of compounds starting from 2-aminobenzimidazole
α-amylase was optimized and the values found are given in
Table 1.
Moderate inhibition was observed by the compounds 4a, 4b
and 10a with values of 896.1 ± 7.7 μM, 598.4 ± 3.3 μM and
372.1 ± 4.0 μM, respectively. The compound 9a showed the
minimum α-amylase inhibition as 2796.7 ± 4.9 μM. It can
be extracted from this study that compound with benzimi-
dazole core (compound 14) was found to be more effective
in terms of amylase inhibition potential than the most potent
compound with piperazine core (10a). It is also observed
that aromatic rings and large hydrophobic groups that added
to the molecular structure increased anti-amylase activity
performance of molecules containing each of the core. In
addition, 1,2,4-triazole ring added to the structure enhanced
the inhibitory effectiveness of the piperazine derivative
For newly synthesized organic molecules, relative
activity (%) was plotted against the different concentrations
of inhibitor concentrations and the inhibitor concentration at
which caused a 50% decrease of α-amylase activity speci-
fied as the IC50 value. Acarbose was used as the reference
standard inhibitor and IC50 value was found as 6.9 ±
0
.1 µM. The results of the α-amylase inhibition study are
summarized in Table 1. The synthesized compounds 4a, 4b,
a, 10a and 14 show varying effects on glucose utilization
9
at the tested concentrations. Amongst these, 14 showed
maximum inhibition with the IC50 value of 64.8 ± 1.8 µM.

Medicinal Chemistry Research
Table 1 Reaction conditions for α-amylase activity and α-amylase
8
7
6
0
0
0
a
inhibition potentials of tested compounds
Compound no
IC50, µM
Max inhibition
%
[I], µM
50
4
3
0
0
2
3
>30,000
>40,400
896.1 ± 7.7
598.4 ± 3.3
>3600
7.3 ± 0.5
12.2 ± 0.9
76.5 ± 4.0
90.1 ± 0.5
18.7 ± 1.1
28.6 ± 0.4
4.4 ± 0.1
30,000
40,400
1200
1000
3600
12,840
5805
7800
11,940
1000
1815
10,080
150
- [I]
2
0 μM
4
4
5
7
8
9
9
1
1
1
1
a
20
10
0
8
5 μM
b
>12,840
>5805
-2
0
2
4
6
8
10
-
1
1/[S] (μM)
8
7
0
0
b
a
2796.7 ± 4.9
>11,940
372.1 ± 4.0
>1815
80.2 ± 0.4
7.4 ± 0.2
b
0a
0b
3
85.1 ± 0.8
24.8 ± 0.2
7.5 ± 0.4
60
50
>10,080
64.8 ± 1.8
6.9 ± 0.1
4
3
2
0
0
0
-
[I]
4
77.7 ± 0.2
94.9 ± 0.7
Acarbose
50
200 μM
600 μM
Reaction Conditions: pH = 7.00, Temperature = 35 °C, Starch con-
centration = 0.32%, Effective enzyme concentration = 34 μg/mL
10
0
-
5
0
5
10
1
/[S] (μM)-1
compound. Finally, this study supported the ability of
synthesized compound 14 to serve as an effective anti-
diabetic agent.
Fig. 1 Lineweaver–Burk plots for compounds 14 (a) and 10a (b)
against α-amylase
Kinetic study of α-amylase inhibition
be ≤5 (or MLogP ≤ 4.15), the number of hydrogen bond
acceptors should be ≤10 and the number of hydrogen bond
donors should be ≤5 [25]. All compounds pass Lipinski’s
drug-likeness filter as well as most of the other filters. In
addition, all compounds have fewer than 10 rotatable bonds
According to the IC50 results, compounds 10a and 14
representing each group that derive from the same core
displayed the most potent inhibition effects on pancreatic α-
amylase. Hereby, the inhibition kinetic constants and the
inhibition types of these two tested compounds against
pancreatic α-amylase were evaluated, using varying sub-
strate concentrations and various inhibitor concentrations
2
and less than 140 A topological polar surface areas
(TPSAs), confirming their good oral bioavailability [26].
Compounds show considerable percent absorption range
from 64.44 to 97.69% as well.
(
Fig. 1, Table 2). Figure 1a revealed that Vmax decreases,
and the Km value is not correlated with increasing con-
centration of compound 14 suggesting the inhibition of
pancreatic α-amylase by compound 14 in a noncompetitive
manner. A series of approximate parallel lines were seen in
Molecular docking calculation
According to the docking results, all 13 compounds bind to
the active site of the α-amylase enzyme. The binding ener-
gies for the compounds calculated were at range from −7.3
and −10.6 kcal/mol. The binding energies and inhibition
constants of the conformations that bind to the active site of
the enzyme with the greatest affinity are given in Table 5.
The compounds 4a, 4b, 9a, 10a and 14 showed high
inhibition activities according to the results of in vitro
biochemical kinetic studies. Thus, possible enzyme-
inhibitor interactions of these compounds responsible for
high inhibition activity were determined. Amino acid resi-
dues and interaction types that play important roles in
binding are presented in Fig. 2A, B and Table 6.
Fig. 1b. K and V
were both decreased with increasing
max
m
concentration of compound 10a, indicating that it is un-
competitive inhibition against pancreatic α-amylase.
ADME properties
ADME properties predicted from Molinspration for 13
compounds are given in Table 3. Additional ADME pre-
dictions from SwissADME are given in Table 4. Drug-
likeness of drug lead compounds are generally evaluated
based on Lipinski’s Rule of Five. According to this rule,
molecular weight (MW) should be ≤500 Da, LogP should

Medicinal Chemistry Research
Table 2 Comparison of the
kinetic parameters related to the
α-amylase enzyme in the
presence and absence of
inhibitors and the type of
inhibition
Compound no
Inhibitor, (µM)
0
Km (%)
Vmax (µmol/dk)
Type of inhibition
Noncompetitive
Ki (µM)
1
4
0,50
0.50
0.50
0.50
0.37
0.25
0.12
0.11
0.09
0.12
0.09
0.06
265.13
2
0
85
0
1
0a
Uncompetitive
629.63
2
6
00
00
Table 3 ADME properties of the
newly synthesized compounds
%
ABS TPSA
n-ROTB MV
MW ≤ 500 LogP ≤ 5 n-ON ≤ n-OHNH ≤ 5 n-Violation ≤ 1
2
(
A )
10
2
3
4
4
5
7
8
9
9
1
1
1
1
97.69
87.75
88.43
82.54
89.28
81.88
32.78
61.60
59.63
76.70
57.16
78.61
5
3
7
6
5
6
4
8
6
5
5
3
6
249.04 266.32
2.29
−0.30
1.48
4
0
3
3
3
1
0
3
3
3
0
1
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
230.38 252.29
305.12 339.44
351.09 385.44
332.51 367.43
267.44 293.32
248.79 279.30
323.53 366.45
314.65 350.38
305.60 348.43
296.06 332.36
180.74 205.22
387.83 443.47
5
a
6
b
1.96
7
2.31
6
2.13
7
71.94 107.42
72.62 105.45
66.73 122.52
−0.45
1.32
8
a
9
b
0.78
10
8
0a 80.36
83.02
2.01
0b 73.47 102.99
0.94
9
3
4
75.94
95.83
−0.73
3.24
6
64.44 129.17
10
As seen in Table 5, compounds 4b, 5 and 14 show the
highest binding affinities (ΔG ≤ −10.0 kcal/mol), respec-
tively, by strongly interacting with the enzyme. Compounds
with Tyr62 residue. In addition, in the active site, charge
and hydrogen bond interactions with catalytic Asp197 and
Asp300 residues, π-interactions with Trp59 and Val163
residues are mostly detected. Thus, these amino acid resi-
dues are important for the inhibition mechanism of α-
amylase enzyme and hydrogen bond, charge and π-inter-
actions are mostly responsible for their high binding affinity
in the active site.
Also, phenyl, p-nitro phenyl, piperazine, benzimidazole
and triazole rings are predicted pharmacophore groups by
examining interactions between compounds 4a, 4b, 9a, 10a
and 14 and α-amylase enzyme according to docking results.
Compound 14, which has the highest binding affinity in
in vitro and in silico studies, exhibits π-σ, π-alkyl, π-π T-
shaped, π-π stacked, π-cation and π-donor hydrogen bond
interactions with benzimidazole ring, π-π stacked and π-
alkyl interactions with phenyl rings (Table 6).
Beside these results, according to biochemical kinetic
studies, inhibition type of compounds 14 and 10a were
determined as non-competitive and un-competitive inhibi-
tion, respectively. Since such inhibitors generally prefer to
bind to a region other than the active site of the enzyme in
these inhibition types, we also explored the feasibility for
binding of compound 10a and 14 to different sites as
4
a, 9a, 9b and 10b also exhibit significant binding affinities
(
ΔG ≤ −8.5 kcal/mol). These results are in agreement with
in vitro enzyme inhibition tests where compound 14, 10a
and 4b were found to be among the most effective ones,
respectively (Table 1). Besides, the docking scores of
compounds 4a and 9a are compatible with the in vitro
studies and they bind to the enzyme with good affinities as
well. In addition, compound 10a which exhibits a high
inhibition profile in in vitro studies, binds α-amylase quite
effectively (−8.3 kcal/mol) also in silico calculations.
There are three amino acid residues in the active site of
the α-amylase enzyme important in catalytic effect. In the
active site of the X-ray structure (1OSE) of the α-amylase
enzyme used in docking studies, Asp197, Glu233 and
Asp300 residues constitute a catalytic triad [27, 28]. Similar
interactions with these important residues are observed
when enzyme-inhibitor interactions of compounds with
high inhibition activity are investigated in silico. In Table 6,
it is observed that all of the compounds 4a, 4b, 9a, 10a and
1
4 exhibit π, charge and hydrogen bond interactions with
the catalytic Glu233 residue. They also show π-interactions

Medicinal Chemistry Research
Table 4 ADME and pharmacokinetic properties of newly synthesized compounds calculated by SwissADME
Pharmacokinetics
Compound GI absorption BBB
permeant
P-gp
substrate
CYP1A2
inhibitor
CYP2C19
inhibitor
CYP2C9
inhibitor
CYP2D6
inhibitor
CYP3A4
inhibitor
2
3
4
4
5
7
8
9
9
1
1
1
1
High
High
High
High
High
High
High
High
High
High
High
High
High
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
Yes
No
No
Yes
No
Yes
No
No
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
Yes
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
a
b
No
a
Yes
Yes
Yes
Yes
No
b
0a
0b
3
4
No
Compound
Log S (ESOL)
Solubility class
Drug-likeness
Lipinski
Ghose
Veber
Egan
Muegge
2
3
−2.81
−1.74
−2.62
−3.29
−3.91
−2.72
−1.64
−2.53
−2.06
−2.95
−2.68
−1.75
−4.74
Soluble
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Very soluble
Soluble
4
4
5
7
8
9
9
1
1
1
1
a
Yes
Yes
Yes
Yes
No
b
Soluble
Soluble
Soluble
Very soluble
Soluble
a
Yes
Yes
Yes
Yes
Yes
Yes
b
Soluble
Yes
Yes
Yes
Yes
Yes
0a
0b
3
Soluble
Soluble
Very soluble
Moderately soluble
4
possible allosteric sites by employing blind docking similar
to the in silico calculations on amylase in the literature [29].
We identified four different binding sites based on the top
docking scores from blind docking (Fig. 3). Three of them
are consistent with the previous study by Wang et al. using
the same crystal structure [29]. They confirmed Site 2 and
Site 3 as the possible allosteric sites while Site 1 is the active
site [29]. Moreover, we identified an additional pocket, Site 4
in Fig. 3, which has not been reported by Wang et al. [29].
The noncompetitive inhibitor compound 14 binds to Site 2
with the higest score in blind docking. Therefore, we per-
formed a subsequent flexible docking calculation at Site 2
where compound 14 binds with quite good score (ΔG =
noncompetitive behavior which is in line with in vitro mea-
surements (Fig. 2C). As seen in Table 7, compound 14
interacts with Arg267, Trp316, Ala310, Gly309, Arg346, and
Asp317 residues in this site by exhibiting hydrogen bond and
π interactions. In addition, to mimic the un-competitive
inhibition by compound 10a, it was also docked into the
enzyme-substrate complex after blind docking. Flexible
docking results reveal that it can bind to this complex with
good affinity (−7.3 kcal/mol) (Fig. 2C) at Site 2. Compound
10a exhibits hydrogen bond, π interactions and amide- π
stacked interaction with Arg267, Ala310, Glu352, Asp317,
Phe348, and Arg303-Gly304 residues at this site (Table 7).
The fact that these compounds can bind to different binding
pockets in the enzyme other than the active site with good
−8.7 kcal/mol
and
Ki = 0.42 μM)
supporting
its

Medicinal Chemistry Research
Table 5 Binding energies (ΔG) and inhibition constants (Ki) obtained
from flexible docking to the α-amylase
chemicals in drug design for cancer therapy is
fluoroquinolone-derived compounds so that a large scale of
drug development studies currently focuses on the design
and synthesize these compounds due to their clinical
importance [31, 32]. There are some studies investigating
the cytotoxic effects of various compounds containing
fluoroquinolone on pancreatic, leukemia, prostate, lung,
breast, ovary and colon and melanoma cancer cells [33–
Compound
ΔG Kcal/mol
Ki (µM)
2
−7.3
−8.1
4.452
3
1.154
4
4
5
7
8
9
9
1
1
1
1
a
−8.5
0.587
b
−10.0
−10.4
−7.5
0.047
0.024
3
9]. Some derivatives were reported to have cytotoxic
3.176
effects on breast cancer cell lines and IC50 values varied in
the range of 2,1–621 μM [40]. On the other hand, the new
Schiff–Mannich triazole derivatives of fluoroquinolones
exhibited cytotoxic effects on leukemia cell line (HL60)
−8.2
0.974
a
−8.5
0.587
b
−8.8
−8.3 (−7.3)a
0.354
0.823 (4.452)a
0a
0b
3
[
41]. Novel N-1 alkylated fluoroquinolones were also
−8.6
0.496
shown to have a cytotoxic effect on kidney (HEK-293)
cells and the IC50 values were determined between 2.56 and
15.21 µg/mL [42]. Fluoroquinolone antibiotic, cipro-
floxacin exhibited moderate cytotoxic effect on cervix
(HeLa) cells [43]. The findings of this study confirm the
previous studies that showed fluoroquinolone compounds
can cause cytotoxicity in various cancer cells. But one of
the most important challenges of current chemotherapeutics
is to be potential mutagenic for normal cells. Enhancing
selectivity of drug candidates is one of main approaches to
deal with this limitation. Candidate compound used in this
study as an amylase inhibitor showed cytotoxic selectivity
on specifically amylase-secreting cancer cells. This study
reports that compound 14 can be considered as an anti-
metabolite chemotherapeutic interfering with the pathways
involved in amylase activation. The findings should be
further enhanced to reveal the mechanism of action by
investigating all cellular effects of the drug candidate using
gene expressions and protein-based methodologies.
−7.8
−10.6 (−8.7)b
1.914
0.016 (0.418)b
4
Ki = exp((ΔG.1000)/(R.T))
a
Data in parenthesis is obtained from mimicking uncompetitive
inhibition
bData in parenthesis is obtained from mimicking noncompetitive
inhibition
affinities and the inhibition constants of these conformations
(
Table 5) are more compatible with biochemical kinetic
studies demonstrate that the results of in silico studies largely
support in vitro studies.
In conclusion, considering in vitro and in silico results and
the predicted ADME properties, compounds 4a, 4b, 9a, 10a
and 14 have the potential to be effective inhibitors of the
α-amylase enzyme and bioavailable drug lead candidates.
Cell culture
This study reports that a novel organic molecule containing
fluoroquinolone, compound 14, was designed and synthe-
sized and also showed its cytotoxic effect on pancreatic
cancer cells. The compound 14 induced cytotoxicity on
AR42J and hGF cells in a dose-dependent manner (Figs. 4,
Conclusion
Despite of many diverse biological activities exhibited by
piperazine and benzimidazole scaffold, it is rarely reported
for the α-amylase inhibitory activity. For that purpose, a
series of novel derivatives of these compounds were syn-
thesized and evaluated for their ability to inhibit the
enzyme α-amylase and drug design profiles. The synthetic
molecules were characterized by FT IR, C NMR and H
NMR spectroscopic techniques. Kinetic analysis revealed
that the most potent compounds 10a and 14 (representing
each group that is derived from the same core) inhibited α-
amylase in noncompetitive and uncompetitive inhibition
manner, respectively. In vitro and in silico studies have
conformingly identified the binding interactions of 10a and
14 molecules with the enzyme. As a result of cell culture
studies, it was determined that compound 14 was more
effective than positive control acarbose, which is an α-
5
). The compound 14 was more effective compared to
positive control acarbose, an α-amylase inhibitor [30] (Fig.
). The IC50 values obtained for each cell are shown in
Table 8. The findings suggest the effect of novel compound
4 on the selective cytotoxicity on AR42J cells. The
6
1
3
1
1
selectivity index of the tested compound was 1.5. IC50
value could not be calculated for acarbose at 500 μM which
was the highest concentration used for the compound 14,
because the cell survival did not reduce up to 50%. There
are a range of drugs developed chemically or using bio-
technological tools. Anti-cancer drugs are generally either
cytotoxic (cellular they are targeted as virulent drugs) or
cytostatic (anti-proliferative drugs). These drugs are used to
induce decrease in tumor growth [31]. One of the group of

Medicinal Chemistry Research
Fig. 2 3-D and 2-D binding modes of compound 10a (A) and of compound 14 (B) in the active site of α-amylase enzyme. Interaction distances are
given in Å. C Surface view of the enzyme complexed with substrate analogue in the active site (yellow) and binding conformations of 10a (green,
left) and 14 (cyan, right) in binding site 2
amylase inhibitor, and induced cytotoxicity on AR42J
cells in a dose-dependent manner. Conclusively, the find-
ings suggest a novel drug candidate, compound 14, in
consideration for the treatment of type 2 diabetes. The
anti-diabetic effect of compound needs to be evaluated by
in vivo mice studies in the future.

Medicinal Chemistry Research
Table 6 Interactions between the compounds having IC50 values and α-amylase active site
Compound
ΔG (kcal/
mol)
Residue
Interaction type
Interaction 0
distance (A )
4
a
−8.5
Glu233(O)…Ligand4a-NH(H)
Asp197(O)…Ligand4a-NH(N)
Tyr62…Ligand4a(piperazine ring)
Tyr62-C=O(O)…Ligand4a(F)
Leu165(C)…Ligand4a
H-Bond
2.43
4.94
5.50
3.33
5.33
Attractivecharge
Pi-Alkyl
Halogen (Fluorine)
Pi-Alkyl
(
phenyl ring)
Val163(C)…Ligand4a
phenyl ring)
Pi-Alkyl
5.25
(
Trp59…Ligand4a(phenyl ring)
Trp59…Ligand4a(phenyl ring)
His305…Ligand4a
Pi-Pi stacked
Pi-Pi stacked
Pi-Alkyl
4.74
4.80
4.67
(
piperazine ring)
Asp300(O)…Ligand4a-NH(H)
Asp300(O)…Ligand4a-NH(N)
Trp59…Ligand4b(phenyl ring)
Trp59…Ligand4b(phenyl ring)
Val163(C)…Ligand4b
H-Bond
2.38
4.05
4.16
4.50
5.23
Attractivecharge
Pi-Pi stacked
Pi-Pi stacked
Pi-Alkyl
4
b
−10.0
(
phenyl ring)
Tyr62…Ligand4b-NH(N)
Asp300(O)…Ligand4b-NH(N)
Asp300(O)…Ligand4b-NH(N)
Asp300(O)…Ligand4b-NH(H)
Asp197(O)…Ligand4b(N)
Glu233(O)…Ligand4b(C)
Ala198(C)…Ligand4b
Pi-Cation
4.83
3.06
3.76
2.78
5.45
3.66
4.52
Attractivecharge
Attractivecharge
Attractivecharge
Attractivecharge
Carbon H-Bond
Pi-Alkyl
(
piperazine ring)
His201-NH(H)…Ligand4b
phenyl ring)
Pi-cation, Pi–donor
H-Bond
2.98
(
Tyr151…Ligand4b(phenyl ring)
Ile235(C)…Ligand4b(phenyl ring)
Pi-Pi stacked
Pi-Sigma
5.57
3.85
2.81
9
a
−8.5
Gln63-NH (H)…Ligand9a-NO2
H-Bond
2
(
O)
Val163(C)…Ligand9a
phenyl ring)
His305…Ligand9a
piperazine ring)
Pi-Alkyl
Pi-Alkyl
5.10
4.83
(
(
Asp300(O)…Ligand9a-NH(N)
Asp300(O)…Ligand9a(S)
Attractivecharge
H-Bond
5.22
3.57
3.02
3.82
5.33
4.58
4.50
3.80
Glu233(O)…Ligand9a-NH(N)
Asp197(O)…Ligand9a-NH(N)
Tyr62…Ligand9a(piperazine ring)
Trp59…Ligand9a(phenyl ring)
Trp59…Ligand9a(phenyl ring)
Glu233(O)…Ligand10a
Attractivecharge
Attractivecharge
Pi-Alkyl
Pi-Pi stacked
Pi-Pi stacked
Pi-Anion
1
0a
−8.3
(
triazole ring)
Glu233(O)…Ligand10a-SH(H)
Asp300(O)…Ligand10a(C)
Asp300(O)…Ligand10a(C)
H-Bond
2.67
3.50
3.55
Carbon H-Bond
Carbon H-Bond

Medicinal Chemistry Research
Table 6 (continued)
Compound
ΔG (kcal/
mol)
Residue
Interaction type
Interaction 0
distance (A )
Asp300(O)…Ligand10a(C)
Tyr62…Ligand10a(C)
Carbon H-Bond
Pi-Sigma
3.68
3.93
Trp59…Ligand10a(phenyl ring)
Trp59…Ligand10a(phenyl ring)
Gln63-NH2(H)…Ligand10a-NO2
Pi-Pi stacked
Pi-Pi stacked
H-Bond
4.80
5.00
2.92
(
O)
Leu162(C)…Ligand10a(C)
His201…Ligand10a-SH(S)
Ala198(C)…Ligand10a
Alkyl
4.23
4.76
4.34
Pi-Sulfur
Pi-Alkyl
(
triazole ring)
Ala198-NH(H)…Ligand10a(N)
Glu233(O)…Ligand14-NH(H)
Asp197(O)…Ligand14-NH(H)
Asp197(O)…Ligand14-NH(H)
His299-NH(H)…Ligand14-C=O
H-Bond
H-Bond
H-Bond
H-Bond
H-Bond
2.80
2.53
2.30
2.92
2.00
1
4
−10.6
(
O)
Tyr62…Ligand14(phenyl ring)
Pi-Pi stacked
Pi-Sigma
4.86
4.00
Ile235(C)…Ligand14
(
benzimidazole ring)
Ile235…Ligand14
benzimidazole ring)
His201…Ligand14
benzimidazole ring)
His201-NH(H)…Ligand14
benzimidazole ring)
Leu162(C)…Ligand14
benzimidazole ring)
Tyr151…Ligand14
benzimidazole ring)
Val163(C)…Ligand14
phenyl ring)
Pi-Alkyl
5.11
4.66
2.53
5.40
4.84
5.30
(
Pi-Pi T-shaped
(
Pi-cation, Pi–donor
H-Bond
(
Pi-Alkyl
(
Pi-Pi stacked
Pi-Alkyl
(
(
Fig. 3 Top-scored different
binding conformations obtained
from blind docking of
compound10a (A) and
compound 14 (B). Site 1 and the
possible allosteric sites (Site 2
and Site 3) are the same as
reported by Wang et al. [29]

Medicinal Chemistry Research
Table 7 Interactions of
compound 10a and 14 with α-
amylase enzyme in Site 2
Compound ΔG (kcal/ Residue
InteractionType
H-Bond
Interaction Distance
(A )
0
mol)
1
0a
−7.3
Arg267-NH2(H)…Ligand10a(N)
2.29
4.15
3.29
Asp317(O)…Ligand10a(triazole ring) Pi-Anion
Ala310-C=O(O)…Ligand10a Carbon H-Bond
piperazine ring)
(
Phe348…Ligand10a(phenyl ring)
Phe348…Ligand10a(C)
Pi-Pi T-shaped
Pi-Alkyl
5.12
4.58
3.34
Glu352(C)…Ligand10a-NO2(O)
Arg303(C),Gly304(N)…Ligand10a
Carbon H-Bond
Amide-Pi Satcked 4.10
(
phenyl ring)
Arg267(N)…Ligand14
benzimidazole ring)
Asp317(O)…Ligand14
benzimidazole ring)
1
4
−8.7
Pi-Cation
Pi-Anion
4.51
4.81
(
(
Ala310(C)…Ligand14(phenyl ring)
Ala310(C) …Ligand14
Pi-Alkyl
Pi-Alkyl
4.54
4.89
(
benzimidazole ring)
Ala310(C) …Ligand14
benzimidazole ring)
Pi-Alkyl
5.41
(
Gly309-C=O(O)…Ligand14-NH(H) H-Bond
Gly309-C=O(O)…Ligand14-NH(H) H-Bond
2.24
2.79
2.31
Arg346-NH2(H) …Ligand14-C=O
O)
Arg346-NH (H) …Ligand14-C=O
H-Bond
H-Bond
H-Bond
(
2.44
2.26
2
(
O)
Arg346-NH2(H) …Ligand14-C=O
O)
(
Trp316(C) …Ligand14-C=O(O)
Trp316…Ligand14(phenyl ring)
Trp316…Ligand14(phenyl ring)
H-Bond
3.35
4.45
4.64
Pi-Pi Stacked
Pi-Pi Stacked
1
1
20
00
120
1
00
80
*
8
6
4
2
0
0
0
0
0
*
*
*
6
0
*
*
40
*
2
0
0
0
2.5
5
10
25
50
100
250
500
0
2.5
5
10
25
50
125
250
500
[
14], ꢀM
[
14], ꢀM
Fig. 5 The cytotoxic effect of compound 14 on normal fibroblast
hGF) cells. Cell viabilities (%) are shown in cells after treatment with
compound 14 at various concentrations. Error bars are the repre-
sentative of standard deviation of at least three independent experi-
ments. *p < 0.05
Fig. 4 The cytotoxic effect of compound 14 on pancreatic cancer
AR42J) cells. Cell viabilities (%) are shown in cell after treatment with
compound 14 at various concentrations. Error bars are the representative
(
(
of standard deviation of at least three independent experiments. *p < 0.05
Material and methods
further purification. Melting points of the synthesized
compounds were determined in open capillaries on a
Büchi B-540 melting point apparatus and are uncor-
rected. Reactions were monitored by thin-layer chroma-
tography (TLC) on silica gel 60 F254 aluminum sheets.
FT-IR spectra were recorded using a Perkin Elmer
Chemistry
All the chemicals used in synthesis were purchased from
Merck, Fluka and Sigma-Aldrich and used without

Medicinal Chemistry Research
1
1
20
00
2
(3)
-[4-(2-Fluorophenyl)piperazine-1-yl]acetohydrazide
*
*
*
8
6
4
2
0
0
0
0
0
White solid was obtained. This crude intermediate product
was crystallized from ethyl acetate: diethyl ether (1:3).Yield:
−1
95%, m.p: 84–86 °C. FT IR (νmax, cm ): 3256 and 3225
(NH ), 3174 (NH), 3037 (Aromatic CH), 2819 (Aliphatic
2
1
CH), 1692 (C=O). H NMR (DMSO-d , δ ppm): 2.59 (t, 4H,
6
0
2.5
5
10
25
50
100
250
500
2
4
1
CH , J = 4.8 Hz), 2.98 (s, 2H, CH ), 3.03 (t, 4H, 2CH , J =
.8 Hz), 4.25 (s, 2H, NH ), 6.94–6.96 (m, 1H, ArH), 7.02 (d,
H, ArH, J = 8.0 Hz), 7.07–7.13 (m, 2H, ArH), 8.94 (s, 1H,
2 2 2
[
Acarbose], ꢀM
2
Fig. 6 The cytotoxic effect of acarbose on AR42J cells. Cell viabilities
%) are shown in cell after treatment with acarbose at various con-
centrations. Error bars are the representative of standard deviation of at
1
3
(
NH). C NMR (DMSO-d , δ ppm): 50.4 (CH ), 50.5 (CH ),
6
2
2
53.3 (2CH ), 60.3 (CH ), arC: [116.4 (d, CH, J
=
2
2
C–F
least three independent experiments. *p < 0.05
21.0 Hz), 119.6 (d, CH, JC–F = 3.0 Hz), 122.7 (d, CH,
JC–F = 8.0 Hz) 125.3 (d, CH, JC–F = 4.0 Hz), 140.3 (d, C,
JC–F = 8.0 Hz), 155.4 (d, C, JC–F = 243.0 Hz)], 168.6 (C=O).
Table 8 IC50 values (µM) selectivity index values calculated for 14 on
AR42Jand hGF cell lines (n = 3)
Test compounds
AR42J
hGF
Select
1.5
N-Ethyl-2-{[4-(2-fluorophenyl)piperazine-1-yl]acetyl}
hydrazinecarbothioamide (4a)
IC50 (µM)
205,34 ± 3.9
IC50 (µM)
304.45 ± 4.1
1
4
White solid was obtained. This crude product was crystal-
lized from acetone:diethyl ether (1:3).Yield: 95%, m.p:
156–157 °C. FT IR (νmax, cm ): 3211 (2NH), 3152 (NH),
600 series FT IR spectrometer. H NMR and 13C NMR
1
−1
1
spectra were registered in DMSO-d6 on a BRUKER
AVANCE II 400 MHz NMR spectrometer (400.13 MHz
for H and 100.62 MHz for C). The mass spectra were
obtained on a Thermo TSQ Quantum Access Max
Quattro Instrument.
All chemicals and solutions used in the cell culture stu-
dies were purchased from American Type Culture Collec-
tion (ATCC, Manassas, VA, USA), Gibco (NY, USA) and
Sigma-Aldrich (St. Louis, MO, USA).
3078 (Aromatic CH), 2955 (Aliphatic CH), 1667 (C=O),
1
1235 (C=S). H NMR (DMSO-d , δ ppm): 1.06 (t, 3H,
6
1
13
CH , J = 6.8 Hz), 2.64 (s, 4H, 2CH ), 3.03 (s, 4H, 2CH ),
3
2
2
3.08 (s, 2H, CH ), 3.44 (q, 2H, CH , J = 6.4 Hz), 6.93–7.08
2
2
(m, 1H, ArH), 7.10–7.14 (m, 1H, ArH), 7.14 (s, 2H, ArH),
7.85 (s, 1H, NH), 9.13 (s, 1H, NH), 9.65 (brs, 1H, NH). 13
NMR (DMSO-d , δ ppm): 14.9 (CH ), 38.9 (CH ), 50.3
C
6
3
2
(CH ), 50.4 (CH ), 53.3 (2CH ), 60.2 (CH ), arC: [116.4 (d,
2
2
2
2
CH, JC-F = 20.0 Hz), 119.6 (d, CH, JC–F = 3.0 Hz), 122.7
d, CH, JC–F = 8.0 Hz), 125.3 (d, CH, JC–F = 3.0 Hz), 140.3
(d, C, JC–F = 9.0 Hz), 155.40 (d, C, JC–F = 243.0 Hz)],
69.3 (C=O), 181.7 (C=S). LC MS/MS m/z (%): 362.06
(
Synthetic procedures and characterizations
1
+
+
In this study, compounds 2, 3 and 4a were synthesized
following the procedure reported by us earlier [44]. Com-
pounds 7, 8, 9a and 10a were synthesized following the
procedure reported in the literature [45].
([M+Na] , 28), 339.94 ([M] , 100).
General procedure for the synthesis of compounds
(4b, 9b and 14)
Ethyl [4-(2-fluorophenyl)piperazin-1-yl]acetate (2)
A mixture compound of 3, 8 and 13 (10 mmol) and the
corresponding isocyanate (20 mmol) in dichloromethane
was stirred at room temperature for 24 h. After evapor-
ating the solvent under reduced pressure, a solid is
obtained. The crude product was crystallized appropriate
solvent.
Colorless liquid intermediate product was obtained. FT IR
−
1
(
(
νmax, cm ): 3066 (Aromatic CH), 2982 (Aliphatic CH), 1745
1
C=O). H NMR (DMSO-d , δ ppm): 1.20 (t, 3H, CH , J =
6
3
6
.8 Hz), 2.68 (t, 4H, 2CH , J = 5.2 Hz), 3.01 (t, 4H, 2CH ,
2
2
J = 4.8 Hz), 3.40 (s, 2H, CH ), 4.10 (q, 2H, CH , J = 7.2 Hz),
2
2
13
6
.94–7.04 (m, 2H, ArH), 7.05–7.13 (m, 2H, ArH). C NMR
N-benzyl-2-{[4-(2-fluorophenyl)piperazine-1-yl]
(
DMSO-d , δ ppm): 14.6 (CH ), 50.4 (CH ), 50.5 (CH ), 52.4
acetyl}hydrazinecarboxamide (4b)
6
3
2
2
(
2CH ), 58.8 (CH ), 60.3 (CH ), arC: [116.3 d, CH, J
=
=
=
2
2
2
C–F
2
8
8
1.0 Hz), 119.7 (d, CH, JC–F = 3.0 Hz), 122.7 (d, CH, JC–F
.0 Hz), 125.2 (d, CH, JC–F = 4.0 Hz), 140.3 (d, C, JC–F
.0 Hz), 155.4 (d, C, JC–F = 243.0 Hz)], 170.3 (C=O).
White solid was obtained. This crude product was crystal-
lized from dimethyl sulfoxide: water (1:3). Yield: 77%, m.
−
1
p: 185–186 °C. FT IR (νmax, cm ): 3316 (2NH), 3191

Medicinal Chemistry Research
(
(
3
NH), 3065 (Aromatic CH), 2940 (Aliphatic CH), 1666
(s, 2H, CH ), 3.46 (t, 4H, 2CH , J = 4.8 Hz), 4.09 (q, 2H,
2 2
1
2 C=O). H NMR (DMSO-d , δ ppm): 2.68 (s, 4H, 2CH ),
CH , J = 7.2 Hz), 7.02 (d, 2H, ArH, J = 9.2 Hz), 8.05 (d,
6
2
2
1
3
.05 (s, 4H, 2CH ), 3.11 (s, 2H, CH ), 3.38 (s, 2H, H O
2H, ArH, J = 9.2 Hz). C NMR (DMSO-d , δ ppm): 14.6
2
2
2
6
+
CH ), 6.94–6.98 (m, 2H, ArH), 7.04–7.15 (m, 3H, ArH),
(CH ), 46.8 (2CH ), 51.9 (2CH ), 58.5 (CH ), 60.4 (CH ),
2
3
2
2
2
2
7
8
.26 (t, 2H, ArH, J = 8.0 Hz), 7.45 (d, 2H, ArH, J =
arC: [113.1 (2CH), 126.2 (2CH), 137.3 (C), 155.2 (C)],
170.3 (C=O).
.0 Hz), 8.04 (s, 1H, NH), 8.76 (s, 1H, NH), 9.61 (s, 1H,
1
3
NH). C NMR (DMSO-d , δ ppm): 43.1 (2CH ), 50.3
6
2
(
CH ), 53.2 (2CH ), 60.1 (CH ), arC: [116.4 (d, CH, J =
2-[4-(4-Nitrophenyl)piperazine-1-yl]acetohydrazide
(8)
2
2
2
C–F
2
1.0 Hz), 119.7 (d, CH, JC–F = 3.0 Hz), 122.7 (d, CH, JC–F
8.0 Hz), 125.3 (d, CH, JC–F = 3.0 Hz), 127.0 (CH), 127.4
CH), 127.5 (CH), 128.5 (CH), 128.6 (CH), 140.2 (C),
=
(
Yellow solid was obtained. This crude product was crystal-
1
1
40.9 (d, C, JC–F = 9.0 Hz), 155.4 (d, C, JC–F = 243.0 Hz)],
58.6 (C=O), 169.50 (C=O).
lized from ethanol. Yield: 75%, m.p: 163–164 °C. FT IR
−1
(υmax, cm ): 3328 and 3248 (NH+NH ), 3006 (Aromatic
2
1
CH), 1631 (C=O), 1474 and 1322 (NO ). H NMR (DMSO-
2
General procedure for the synthesis of compounds
and 10b
d , δ ppm): 2.54–2.65 (m, 4H, 2CH ), 2.98 (s, 2H, CH ), 3.47
6
2
2
5
(brs, 4H, 2CH ), 4.27 (brs, 2H, NH ), 7.02 (d, 2H, ArH, J =
2 2
13
9
.2 Hz), 8.04 (d, 2H, ArH, J = 8.0 Hz), 9.00 (s, 1H, NH).
C
A solution of the corresponding compound 4b and 9b
10 mmol) was refluxed in the presence of a 2% NaOH
NMR (DMSO-d , δ ppm): 46.7 (2CH ), 52.7 (2CH ), 59.9
6
2
2
(
(CH ), arC: [113.0 (CH), 113.1 (CH), 126.2 (2CH), 137.2
2
solution for (8 h for 5), (5 h for 10b). Then, the solution
(C), 155.1 (C)],168.5 (C=O).
was cooled to room temperatureand acidified to pH 7 with
3
7% HCl. The precipitate formed was filtered and washed
N-ethyl-2-{[4-(4-nitrophenyl)piperazine-1-yl]acetyl}
with water. These compounds were recrystallized from
appropriate solvent.
hydrazinecarbothioamide (9a)
Yellow solid was obtained. This crude product was crystal-
4
-Benzyl-5-{[4-(2-fluorophenyl)piperazine-1-yl]
lized from ethanol. Yield: 80%, m.p: 124–125 °C. FT IR
−1
methyl}-2,4-dihydro-3H-1,2,4 triazole-3-one (5)
(υmax, cm ): 3286 (NH), 3228 (NH), 3202 (NH), 1743
1
(
C=O), 1485 ve 1319 (NO ). H NMR (DMSO-d , δ ppm):
2
6
White solid was obtained. This crude product was crys-
tallized from dimethyl sulfoxide: water (1:3). Yield: 72%,
m.p: 249–250 °C. FT IR (νmax, cm ): 3378 (NH), 3065
1.06 (t, 3H, CH , J = 6.8 Hz), 2.60 (s, 4H, 2CH ), 3.08 (s, 2H,
3
2
CH ), 3.43–3.51 (m, 6H, 3CH ), 7.04 (d, 2H, ArH, J =
2
2
−
1
9.6 Hz), 7.87 (brs, 1H, NH), 8.05 (d, 2H, ArH, J = 9.6 Hz),
13
(
(
Aromatic CH), 2937 (Aliphatic CH), 1701 (C=O), 1498
9.13 (brs, 1H, NH), 9.68 (brs, 1H, NH). C NMR (DMSO-d ,
6
1
C=N). H NMR (DMSO-d , δ ppm): 2.45 (s, 4H, 2CH ),
δ ppm): 14.9 (CH ), 38.9 (CH ), 46.7 (2 CH ), 52.7 (2CH ),
6
2
3
2
2
2
2
6
7
.81 (s, 4H, 2CH ), 3.32 (s, 2H, CH ), 4.89 (s, 2H, CH ),
59.9 (CH ), arC: [113.1 (2CH), 126.2 (2CH), 137.2 (C), 155.2
(C)], 169.3 (C=O), (C=S) not observed.
2
2
2
2
.94 (d, 2H, ArH, J = 8.4 Hz), 7.07 (d, 2H, ArH, J =
1
3
.6 Hz), 7.24–7.35 (m, 5H, ArH), 11.77 (s, 1H, NH).
C
NMR (DMSO-d , δ ppm): 43.1 (CH ), 44.2 (CH ), 50.1
N-Ethyl-2-{[4-(4-nitrophenyl)piperazine-1-yl]acetyl}
hydrazinecarboxamide (9b)
6
2
2
(
CH ), 52.7 (2CH ), 53.4 (CH ), arC: [116.3 (d, CH,
2
2
2
JC–F = 20.0 Hz), 119.6 (d, CH, JC–F = 3.0 Hz), 122.8 (d,
CH, JC–F = 8.0 Hz), 125.3 (d, CH, JC–F = 3.0 Hz), 126.9
Yellow solid was obtained. This crude product was crys-
tallized from dimethyl formamide: water (1:3). Yield: 90%,
(
1
=
CH), 127.5 (CH), 127.7 (CH), 128.5 (CH), 128.9 (CH),
37.7 (C), 140.1 (d, C, JC–F = 8.0 Hz), 155.4 (d, C, JC–F
243.0 Hz)], 144.73 (triazole C-3), 155.9 (triazole C-5).
−
1
m.p: 225–226 °C. FT IR (υmax, cm ): 3274 (3NH), 3099
1
(Aromatic CH), 1655 (2C=O), 1497 and 1337 (NO ). H
2
+
LC MS/MS m/z (%): 390.13 ([M+Na] , 46), 368.01
NMR (DMSO-d , δ ppm): 0.99 (t, 3H, CH , J = 7.2 Hz),
6
3
+
(
[M+1] , 100).
2.60 (t, 4H, 2CH , J = 4.4 Hz), 3.00–3.05 (m, 4H, 2CH ),
2
2
3
.49 (t, 4H, 2CH , J = 4.4 Hz), 6.30 (t, 1H, NH, J =
2
Ethyl [4-(4-nitrophenyl)piperazine-1-yl]acetate (7)
5.6 Hz), 7.03 (d, 2H, ArH, J = 9.6 Hz), 7.72 (s, 1H, NH),
1
3
8.05 (d, 1H, ArH, J = 9.2 Hz), 9.42 (s, 1H, NH). C NMR
Yellow solid was obtained. This crude product was crys-
(DMSO-d , δ ppm): 15.9 (CH ), 34.7 (CH ), 46.7 (2CH ),
6
3
2
2
tallized from ethanol. Yield: 84%, m.p: 115–116 °C. FT IR
52.6 (2CH ), 59.9 (CH ), arC: [113.2 (2CH), 126.1 (2CH),
2 2
−
1
(
1
νmax, cm ): 3023 (Aromatic CH), 1742 (C=O) 1485 and
137.3 (C), 155.1 (C)], 158.5 (C=O), 169.5 (C=O).
1
+
390 (NO ). H NMR (DMSO-d , δ ppm): 1.19 (t, 3H,
LC MS/MS m/z (%): 373.08 ([M+Na] , 100), 351.10
2
6
+
CH , J = 7.2 Hz), 2.64 (t, 4H, 2CH , J = 4.8 Hz), 3.29
([M+1] , 43).
3
2

Medicinal Chemistry Research
1
4-Ethyl-5-{[4-(4-nitrophenyl)piperazine-1-yl]methyl}-
CH), 1645 (C=O), 1539 (C=N). H NMR (DMSO-d , δ
6
4
H-1,2,4-triazole-3-thiol (10a)
ppm): 4.34 (s, 2H, NH ), 4.58 (brs, 2H, CH ), 6.41 (s, 2H,
2
2
2NH), 6.86 (t, 1H, ArH, J = 8.0 Hz), 6.94 (t, 1H, ArH, J =
Yellow solid was obtained. This crude product was crys-
8.0 Hz), 7.02 (d, 1H, ArH, J = 8.0 Hz), 7.13 (d, 1H, ArH,
1
3
tallized from ethanol. Yield: 75%, m.p: 215–216 °C.FT IR
J = 8.0 Hz), 9.43 (s, 1H, NH). C NMR (DMSO-d , δ
6
−
1
(
1
υmax, cm ): 3088 (Aromatic CH), 2830 (SH), 1589 and
ppm): 43.4 (CH ), arC: [108.0 (CH), 115.4 (CH), 119.4
2
1
336 (NO ). H NMR (DMSO-d , δ ppm): 1.30 (t, 3H,
(CH), 121.4 (CH), 134.3 (C), 142.0 (C), 155.6 (C)], 166.5
2
6
+
CH , J = 8.0 Hz), 2.56 (t, 4H, 2CH , J = 4.0 Hz), 3.46 (brs,
(C=O). LC MS/MS m/z (%): 206.02 ([M+1] , 100).
3
2
4
H, 2CH ), 3.65 (s, 2H, CH ), 4.05 (q, 2H, CH , J =
2 2 2
8
.0 Hz), 7.03 (d, 2H, ArH, J = 8.0 Hz), 8.03 (d, 2H, ArH,
2-({2-[2-(anilinecarbonyl)hydrazino]-2-oxoethyl}
amino)-N-phenyl 1Hbenzimidazole-1-carboxamide
(14)
13
J = 8.0 Hz), 13.65 (brs, 1H, SH). C NMR (DMSO-d , δ
ppm): 13.8 (CH ), 39.28 (CH ),46.78 (2CH ), 52.1 (CH ),
6
3
2
2
2
5
1
2.4 (2CH ), arC: [113.2 (2CH), 126.2 (2CH), 137.4 (C),
55.1 (C)], 149.3 (triazole C-3), 167.3 (triazole C-5).
2
White solid was obtained. This crude product was crystal-
lized from ethanol. Yield: 74%, m.p: 231–232 °C. FT IR
−
1
4-Ethyl-5-{[4-(4-nitrophenyl)piperazine-1-yl]methyl}-
(νmax, cm ): 3313 (NH), 3222 (2NH), 3120 (2NH), 3064
2,4-dihydro-3H-1,2,4-triazole-3-one (10b)
(Aromatic CH), 2971 (Aliphatic CH), 1677 (2 C=O), 1646
1
(
C=O), 1515 (C=N). H NMR (DMSO-d , δ ppm): 4.85 (s,
6
Yellow solid was obtained. This crude product was crys-
2H, CH ), 6.90–6.98 (m, 2H, ArH), 7.12–7.14 (m, 2H,
2
tallized from ethanol. Yield: 55%, m.p: 251–252 °C. FT IR
ArH),7.21–7.25 (m, 5H, ArH), 7.40–7.46 (m, 3H, ArH),
7.68 (d, 2H, ArH, J = 8.0 Hz), 8.28 (s, 1H, NH), 8.78
(s, 1H, NH), 9.11 (s, 1H, NH), 10.18 (s, 1H, NH), 12.10 (s,
−
1
(
(
υmax, cm ): 3160 (NH), 3075 (Aromatic CH), 2955
1
Aliphatic CH), 1688 (C=O), 1500 and 1334 (NO ). H
2
1
3
NMR (DMSO-d , δ ppm): 1.21 (t, 3H, CH , J = 8.0 Hz),
1H, NH). C NMR (DMSO-d , δ ppm): 43.9 (CH ), arC:
6
3
6
2
2
3
8
.50–2.54 (m, 4H, 2CH +DMSO), 3.46 (s, 6H, 3CH ),
[109.3 (CH), 111.4 (CH), 115.1 (CH), 118.3 (CH), 119.0
(CH), 121.5 (CH), 122.1 (CH), 122.3 (2 CH), 122.5 (CH),
128.9 (2CH), 129.1 (2CH), 130.1 (C), 130.4 (C), 139.9 (C),
141.8 (C)],152.9 (C=N), 161.5 (2C=O), 167.1 (C=O). LC
MS/MS m/z (%): 466.18 ([M+Na] , 52), 444.27 ([M+1] ,
15), 325.20 (100).
2
2
.68 (q, 2H, CH , J = 8.0 Hz), 7.03 (d, 2H, ArH, J =
2
.0 Hz), 8.06 (d, 2H, ArH, J = 8.0 Hz), 11.58 (s, 1H, NH).
C NMR (DMSO-d , δ ppm): 14.7 (CH ), 36.1 (CH ), 46.8
1
3
6
3
2
+
+
(
(
(
2CH ), 52.4 (2CH ), 53.5 (CH ), arC: [113.2 (2CH), 126.2
2CH), 137.4 (C), 155.1 (C)], 144.5 (triazole C-3), 155.6
triazole C-5). LC MS/MS m/z (%): 355.16 ([M+Na] ,
2
2
2
+
+
1
00), 333.11 ([M+1] , 27).
α-Amylase assay
Ethyl [(1H-benzimidazol-2-yl)]aminno]acetate(12)
α-Amylase activity was determined by the adapted
method with minor modifications from the study of Taha
et al. [46]. 60 µL of starch solution (0.64%) and 60 µLof
α-amylase (porcine pancreatic) solution (68 µg/mL in
0.02 M, pH 7.00 sodium phosphate buffer containing
0.006 M sodium chloride) were incubated at 35 °C for
10 min, and the reaction was stopped by addition of
100 µL of 3,5-dinitrosalicylic acid (DNS) solution. The
mixture was incubated in boiling water for 5 min, and then
cooled to room temperature. The reaction mixture was
diluted by adding 1 mL distilled water. Finally, the
absorbance was measured at 540 nm using a spectro-
photometer. One-unit enzyme activity was determined as
the amount of 1 μmol maltose released in 1 min under
reaction conditions [47].
Ethyl bromoacetate (10 mmol) was added to the solution of
2
-aminobenzimidazole (10 mmol) in tetrahydrofuran drop
wise, and the mixture was stirred at room temperature in the
presence of triethylamine (15 mmol) for 24 h. The solid
formed was removed by filtration, and the solvent was
evaporated under reduced pressure. These crude products
were crystallized from appropriate solvent. Colorless liquid
−
1
product was obtained. FT IR (νmax, cm ): 3312 (NH), 3156
(
NH), 3025 (Aromatic CH), 2979 (Aliphatic CH), 1738
(
C=O), 1547 (C=N).
2-(1H-Benzimidazole-2-ylamino)acetohydrazide (13)
Hydrazine hydrate (25 mmol) was added to the solution of
compound 12 (10 mmol) in absolute ethanol, and the mix-
ture was refluxed 5 h. Then, the precipitated white solid was
filtered and crystallized crystallized from ethanol. Yield:
Optimization of enzyme activity
−
1
8
0%, m.p: 245–246 °C. FT IR (νmax, cm ): 3331 ve 3275
Before inhibition studies, the enzyme optimization studies
including determination of optimum pH and temperature,
(
NH ), 3159 (NH), 3045 (Aromatic CH), 2920 (Aliphatic
2

Medicinal Chemistry Research
optimum protein concentration and, Km value, were per-
formed separately.
1.5%). Their inhibition types were defined based on
Lineweaver–Burk plots and their inhibition constants (Ki)
were calculated according to two inhibition kinetics
models [Eq. (1): uncompetitive inhibition; Eq. (2): non-
competitive inhibition] [49–51].
To determine the optimum pH was used sodium acetate
(
6
20 mM, pH 5.00), sodium phosphate (20 mM, pH
.00–8.00) and Tris-HCl (20 mM, pH 9.00) buffers. Rela-
tive activity % graphs for the pH and optimum pH value
were then plotted.
v ¼ ðVmaxSÞ=ð1 þ Km=S þ I=KiÞ
v ¼ ðVmaxSÞ=ðKm þ SÞ=ð1 þ I=KiÞ
ð1Þ
ð2Þ
The optimal temperature for enzyme activities was
determined in optimum pH value with 5 °C increments at
temperatures ranging from 20 to 40 °C. The optimum
temperature was expressed as a percentage of relative
activity.
where V is the reaction rate; Vmax is the maximum rate; Km
is the Michaelis constant, K is the inhibition constant; S and
i
To determine the effect of enzyme concentration on
activity, a series of activity measurements were performed
at optimum pH and temperature, with a final enzyme con-
centration of 1–250 µg/mL. Activity graphs for enzyme
amount were plotted, and the initial velocity measurements
were performed at an appropriate enzyme concentration.
Reaction mixtures were prepared at substrate concentrations
ranging from 0.05 to 1.4% for enzyme activity and the enzyme
activity was determined. The Lineweaver–Burk plot was
drawn using the obtained data, and K and V values were
I are the substrate and inhibitor concentrations, respectively.
In silico determination of ADME properties
ADME properties of 13 hybrid compounds were calcu-
lated using Molinspiration and SwissADME web tool
[52, 53]. Percentage absorption (% ABS), TPSA, number
of rotatable bonds (n-ROTB), molecular volume (MV),
MW, logarithm of n-octanol/water partition coefficient
(miLog P), number of hydrogen bond acceptors (n-ON),
number of hydrogen bond donors (n-OHNH) and phar-
macokinetics were examined to estimate ADME proper-
ties and bioavailability of the potential drug lead
compounds. Percentage absorption (%ABS) was calcu-
lated using TPSA with the following equation: %ABS =
109-(0.345xTPSA) [54].
m
max
calculated [48].
Inhibition studies
Firstly, stock solutions of synthesized organic molecules
were prepared by dissolving in a minimum quantity of
DMSO. Inhibitor solutions of different concentrations
were prepared from these stock solutions. For inhibition
studies, 3.6 µL of inhibitor solutions and 40 µL of α-
amylase solution (102 µg/mL) were added in an eppendorf
tube and preincubated at 35 °C for 10 min. After this,
Ligand preparation and molecular docking
The 13 compounds were prepared in Spartan16 software
[55] as ligands for molecular docking calculations of α-
amylase enzyme. First, conformer distribution calculations
were performed with molecular mechanics MMFF method.
The conformations generated were ranked according to
increasing energy values. The top 50 lowest energy con-
formations were selected for each compound and optimized
using semi-empirical PM6 method [56]. Then, the most
stable conformation obtained from PM6 method was further
optimized with a much higher level of theory utilizing
density functional theory M06-2X/6-31G** method
[57, 58]. The X-ray crystal structure of porcine pancreatic
α-amylase enzyme (PDB ID: 1OSE, resolution 2.30 A°, co-
crystalized with acarbose) [28], was obtained from Protein
Data Bank. Molecular docking calculations were performed
with AutoDock Tools and AutoDock Vina software for all
compounds [17, 59]. His305, Asp197, Glu233, Asp300,
Trp58, Trp59, Tyr62, Gln63 residues in enzyme’s active
site and, ligand were selected as flexible. Water molecules,
acarbose inhibitor and other ligands were deleted from the
X-ray structure and hydrogen atoms were added. The
charges of chlorine and calcium atoms were set manually to
3
6.4 µL of sodium phosphate buffer (20 mM, pH 7.00)
and 40 µL of starch solution (0.96%) were added to each
reaction mixture and incubated at 35 °C for 10 min. After
incubation, the reactions were stopped with 100 µL of
DNS solution and boiling for 5 min. The boiled reaction
mixtures were cooled to room temperature. Finally, the
reaction mixtures were diluted with distilled water (1 mL),
the absorbance was recorded at 540 nm. Acarbose was
used as the positive control. Inhibitor concentrations were
drawn toward the relative enzyme activity (%) for each
organic molecule, and the IC50 values of organic mole-
cules were calculated.
Determination of inhibition type
The mechanism of enzyme inhibition was determined
using multiple concentrations of 10a and 14 compounds
(
with the lowest IC50 values) in terms of representing each
group that derives from the same core in the presence of
different substrate concentrations (ranging from 0.1 to

Medicinal Chemistry Research
−
1 and +2, respectively. A gridbox size of 50 × 50 × 54
Measurement of cell viability
Cell viability was evaluated with a colorimetric MTT assay
Angstroms in x, y and z directions was built in the active
site. In order to validate the docking protocol, prior to
docking studies, the acarbose removed from the X-ray
structure and re-docked into the enzyme’s active site. The
RMSD value for the co-crystallized and the re-docked
acarbose was calculated to be smaller than 2 Angstroms
3
4
[68]. Briefly, 5 × 10 AR42J cells and 1*10 hGF cells were
seeded into each well of sterile 96-well cell culture plates.
Media was refreshed after overnight incubation followed by
treatment with various concentrations of compound 14
(2.5–500 μM) or acarbose (0.05–500 μM) for 24 h. Subse-
quently, 10 μL of MTT dye (0.25 mg/mL) was added to
each well for 2 h at 37 °C. After 2 h, the dye was removed
and 200 μL of DMSO was added to each well to dissolve
the formazan crystals. Shades of purple color were observed
and absorbance was read at 570 nm using a microplate
reader (Molecular Devices Versamax, CA, USA) [69].
Optical densities were used to evaluate viabilities (%) of
treated cells compared to untreated control cells. A loga-
rithmic plot of log concentrations against cell viability was
plotted to determine the IC50 value [63, 65]. The IC50 values
of compound 14 in both cell lines were used to calculate
selectivity index with the following formula [38].
(
0.524) validating the employed docking protocol. Docking
calculations were run ten times for each compound and nine
binding poses were obtained in each run. Among the 90
conformations generated, best docking pose in the active
site with the highest affinity was chosen. Blind docking was
performed for compound 10a and 14 by targeting the whole
enzyme with SwissDock webserver [60, 61] to predict other
binding sites which can be possible allosteric regions. For
each compound, top five conformations which bind to four
different sites in the enzyme were determined (Fig. 3). In
order to mimic un-competitive inhibition by compound 10a,
firstly enzyme-substrate complex was created. Alpha mal-
tose structure was selected as substrate analogue and docked
into the active site of amylase enzyme. Then compound 10a
was docked into enzyme-substrate complex with the same
flexible docking method of AutoDock Vina described above
by targeting the top-scored blind docking site. To mimic
and examine non-competitive inhibition by compound 14,
flexible docking calculation was also performed targeting
Site 2 in Fig. 3B. Gridbox size of 40 × 40 × 40 Angstroms in
x, y and z directions was built and Asp356, Glu352,
Arg346, Ala310 and compound 14 were selected as flex-
ible. All enzyme-ligand interactions were examined using
Discovery Studio 4.5 Client software [62].
Selectivity Index ¼ hGF cells IC =AR42J cells IC
50
50
Statistical analysis
All experiments were conducted as at least three indepen-
dent repeats and the data were represented by arithmetic
means ± standard deviations. One-Way ANOVA was used
to determine inhibition significance between treatments. For
cell culture studies, statistical analyze were carried out using
SPSS (Statistics Program for Social and Science) software
(
Version 13.0.1). The suitability of the data to normal dis-
Cell culture
tribution was evaluated with the Kolmogorov–Smirnov test.
One-Way ANOVA test was used for parametric data and
then Post-hoc Tukey test for multiple comparisons between
groups. p value < 0.05 was considered as statistically
significant.
Mammalian pancreatic cancer cells (AR42J, Cat. No.
9
3100618) and human normal fibroblast cells (hGF, Cat.
No. PCS-201-018) were purchased from ECACC (UK,
England) and ATCC, respectively. AR42J cells and hGF
cells were cultured in Rosewell Park Memorial Institute-
Data availability
1
640 Medium (RPMI-1640) or, MEM (Minimum Essential
Medium), respectively, and media included 10% heat
inactivated fetal bovine serum and 1% penicillin/strepto-
mycin. Cells were incubated at 37 °C supplied with 5% CO2
The datasets generated during/or analyzed during the cur-
rent study are available from the corresponding author on
reasonable request. The datasets supporting the results of
this paper are included within the paper and its
additional files.
[
63, 64].
Drug preparation and treatment
Author contributions UC: Methodology, Formal analysis, Investiga-
tion, Writing—Original draft, Writing—Review & Editing, Visuali-
zation, Data curation. FOT: Methodology, Formal analysis,
Investigation, Writing—Original draft. SBO: Methodology, Formal
analysis, Investigation, Writing—Original draft, Visualization, Data
curation. EA-D: Formal analysis, Investigation, Writing—Original
draft. ID: Methodology, Software, Formal analysis, Investigation,
Writing—Original Draft, Visualization. AC: Conceptualization,
Acarbose and compound 14 were dissolved in DMSO.
Acarbose was used as a positive control due to is a standard
α-amylase inhibitor [65, 66] and therefore included in the
experimental design for cytotoxicity. Final solvent con-
centrations were adjusted to 0.1% in the medium during cell
culture [67].

Medicinal Chemistry Research
Resources, Supervision, Writing—Original draft, Writing—Review &
Editing Project administration, Funding acquisition. SC-U: Metho-
dology, Resources. SSE: Methodology, Software, Resources. NY:
Formal analysis, Resources.
11. Basoglu S, Ulker S, Alpay-Karaoglu S, Demirbas N.
Microwave-assisted synthesis of some hybrid molecules con-
taining penicillanic acid or cephalosporanic acid moieties and
investigation of their biological activities. Med Chem Res.
2
014;23:3128–43.
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1
2. Sharma S, Wakode F, Fayaz S, Khasimbi F, Pottoo H, Kaur A. An
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therapeutic targets. Curr Pharm Des. 2020;26:1–13.
3. Adegboye AA, Khan KM, Salar U, Aboaba SA, Kanwal, Chi-
gurupati S, et al. 2-Aryl benzimidazoles: Synthesis, in vitro α-
amylase inhibitory activity, and molecular docking study. Eur J
Med Chem. 2018;150:248–60.
Funding The authors are grateful to the Research Fund of the
TUBITAK (The Scientific and Technological Research Council of
Turkey) or this support with Project No 117Z199.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Consent to participate All authors have their consent to participate
Consent to publish The participants gave their consent for publication
14. Kazimierczuk ZJ, Upcroft A, Upcroft P, Gorska A, Starosciak B,
Laudy A. Synthesis, antiprotozoal and antibacterial activity of
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