Synthesis, anticancer evaluation and molecular docking studies of new benzimidazole- 1,3,4-oxadiazole derivatives as human topoisomerase types I poison

Article abstract of DOI:10.1080/14756366.2020.1806831

In this study, some benzimidazole-oxadiazole derivatives were synthesised and tested for their in vitro anticancer activities on five cancer cell lines, including HeLa, MCF7, A549, HepG2 and C6. Their structures were elucidated by IR, ¹H-NMR, <

Full text of DOI:10.1080/14756366.2020.1806831

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, anticancer evaluation and molecular
docking studies of new benzimidazole- 1,3,4-
oxadiazole derivatives as human topoisomerase
types I poison
Ulviye Acar Çevik , Begüm Nurpelin Sağlık , Derya Osmaniye , Serkan
Levent , Betül Kaya Çavuşoğlu , Abdullah Burak Karaduman , Özlem Atlıd ,
Özlem Atlı Eklioğlu & Zafer Asım Kaplancıklı
To cite this article: Ulviye Acar Çevik , Begüm Nurpelin Sağlık , Derya Osmaniye , Serkan
Levent , Betül Kaya Çavuşoğlu , Abdullah Burak Karaduman , Özlem Atlıd , Özlem Atlı Eklioğlu
& Zafer Asım Kaplancıklı (2020) Synthesis, anticancer evaluation and molecular docking
studies of new benzimidazole- 1,3,4-oxadiazole derivatives as human topoisomerase types
I poison, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1657-1673, DOI:
10.1080/14756366.2020.1806831
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1657–1673
https://doi.org/10.1080/14756366.2020.1806831
RESEARCH PAPER
Synthesis, anticancer evaluation and molecular docking studies of new
benzimidazole- 1,3,4-oxadiazole derivatives as human topoisomerase types
I poison
a,b
Ulviye Acar C¸evika,b , Begum Nurpelin Saglık
, Derya Osmaniyea,b , Serkan Leventa,b
,
€
ꢀ
c
d
a,b
, Abdullah Burak Karadumand , Ozlem Atlıd
, Ozlem Atlı Eklioglu
and
€
€
€
ꢀ
ꢀ
Betul Kaya C¸avus¸oglu
Zafer Asım Kaplancıklıa
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey; Doping and Narcotic Compounds
c
Analysis Laboratory, Faculty of Pharmacy, Anadolu University, Eskis¸ehir, Turkey; Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
d
€
Zonguldak Bulent Ecevit University, Zonguldak, Turkey; Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir, Turkey
ABSTRACT
ARTICLE HISTORY
Received 17 May 2020
Revised 7 July 2020
Accepted 28 July 2020
In this study, some benzimidazole-oxadiazole derivatives were synthesised and tested for their in vitro
anticancer activities on five cancer cell lines, including HeLa, MCF7, A549, HepG2 and C6. Their structures
were elucidated by IR, H-NMR, 13C-NMR, 2 D-NMR and HRMS spectroscopic methods. Among all screened
1
compounds; 5a, 5b, 5d, 5e, 5k, 5l, 5n and 5o exhibited potent selective cytotoxic activities against vari-
ous tested cancer cell lines. Especially, compounds 5l and 5n exhibited the most antiproliferative activity
than Hoechst 33342 and doxorubicin against HeLa cell line, with IC50 of 0.224 0.011 mM and
0.205 0.010 mM, respectively. Furthermore, these potent lead cytotoxic agents were evaluated in terms of
their inhibition potency against Topoisomerase I and it was determined that selected compounds inhib-
ited the Topoisomerase I. Docking studies were performed and probable interactions in the DNA-Topo I
enzyme complex was determined.
KEYWORDS
Benzimidazole; 1,3,4-
oxadiazole; anticancer; DNA
Topo I; Hoechst 33342
1. Introduction
Benzimidazoles represent a structurally unique class of Topo I
poisons that act as DNA minor groove binders such as Hoescht
33258 and Hoechst 33342 (Figure 1)18–20. These drugs are known
to hinder the breakage/reunion reaction of topoisomerase I, in
which the enzyme is reversibly trapped in a state where the DNA
is cleave6. Furthermore, benzimidazole is known to be a versatile
scaffold that possess potential anticancer, antitumor and antiproli-
ferative activities21–27. Similarly, oxadiazole has engrossed signifi-
cant attention of medicinal chemists owing to their wide range of
useful pharmacological actions particularly cytotoxic activities
DNA topoisomerases (Topo) are ubiquitous enzymes that are
required for proper chromosome structure and segregation. These
enzymes play important roles in triggering, controlling, and modi-
fying topological DNA problems during cell proliferation, differen-
tiation, and survival. Thus, topo enzymes possesses immense
importance in almost all stages of the cell cycle. As a result, the
role of mammalian DNA topoisomerases as molecular targets for
anticancer drugs has been explored, and it was found that topo-
isomerase inhibition could curb cancerous cell growth across a
against DNA topoisomerase I28–31
.
variety of cell lines1–8
.
In the light of this information, it is aimed to develop new anti-
cancer effective compounds in this research. To this aim, Hoechst
compounds have been identified as precursor compounds. In add-
ition to the hydroxy (–OH) and ethoxy (–OC2H5) substituents in
the para position of the phenyl ring in the Hoechst compounds,
the methoxy (–OCH3) substituent was also used for synthesised
compounds. Instead of bisbenzimidazole, the 1,3,4-oxadiazole ring
attached to the benzimidazole ring was synthesised. Methyl, ethyl,
pyridine, pyrimidine and phenyl substituents were used for the
fourth position of piperazine. The cytotoxicity of these synthesised
compounds to different cell lines, and Topo I inhibitory activities
were evaluated. Besides different mechanisms such as Flow cyto-
metric analysis, DNA synthesis inhibition assay was also studied in
our research. In order to determine the possible interactions of
Human Topo are divided into two classes: Topoisomerase I
(topo I), breaks only one strand of DNA and Topoisomerase II
(topo II), breaks both strands of DNA9–11. Several Topo poisons
have been approved for the chemotherapeutic treatment of differ-
ent cancer types in recent decades, including the Topo I inhibitors
camptothecin, topotecan and the Topo-II-targeting drugs etopo-
side and amsacrin12,13
.
In the field of anticancer drugs, DNA topoisomerase I has been
reported to be an important target for anticancer agents but
topoisomerase inhibitors have several barriers and limitations.
These include poor solubility, accumulation in target tissues or
organs and prolonged half-life in plasma as well as drug efflux
from the target cells and cancer cell resistance14–17
.
CONTACT Ulviye Acar C¸evik
uacar@anadolu.edu.tr,
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470, Turkey
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1658
U. A. ÇEVIK ET AL.
Figure 1. Hoechst 33258 and Hoechst 33342 compounds.
compound, showed high activity, docking studies have sulphuric acid were added into the mixture. The mixture was
been performed.
refluxed for 72 h, and the precipitate was filtered off.
2.1.1.4. Synthesis of 2-(4-Substitutedphenyl)-1H-benzo[d]imidazole-
6-carbohydrazide derivatives (3a-3c). Methyl 2–(4-substituted-
phenyl)-1H-benzo[d]imidazole-6-carboxylate (2a-2c) (0.018 mol)
and excess of hydrazine hydrate (5 ml) in EtOH (15 ml) were mixed
in a vial (30 ml) of microwave synthesis reactor (Anton-Paar
Monowave 300). The reaction mixture was heated at 240 ꢀC and
10 bar for 10 min. After cooling, the mixture was poured into iced
water, the product was filtered, washed with water, dried, and
recrystallized from EtOH.
2. Experimental
2.1. Chemistry
Whole chemicals employed in the synthetic procedure were pur-
chased from Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St.
Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt,
Germany). Melting points of the obtained compounds were deter-
mined by MP90 digital melting point apparatus (Mettler Toledo,
OH, USA) and were uncorrected. The IR spectra were obtained on
a Shimadzu, IR Prestige-21 (Shimadzu, Tokyo, Japan). 1H NMR,
HSQC, HMBC, COSY and 13C NMR spectra of the synthesised com-
pounds were registered by a Bruker 300 MHz and 75 MHz digital
FT-NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in
DMSO-d6, respectively. Splitting patterns were designated as fol-
lows: s: singlet; d: doublet; t: triplet; m: multiplet in the NMR spec-
tra. Coupling constants (J) were reported as Hertz. M þ 1 peaks
were determined by Shimadzu LC/MS ITTOF system (Shimadzu,
Tokyo, Japan). All reactions were monitored by thin-layer chroma-
tography (TLC) using Silica Gel 60 F254 TLC plates (Merck KGaA,
Darmstadt, Germany).
2.1.1.5. Synthesis of 2-((4-Substituted phenyl)-(6-(5-mercapto-1,3,4-
oxadiazol-2-yl)-1H-benzo [d]imidazole derivatives (4a-4c). 2–(4-
Substitutedphenyl)-1H-benzo[d]imidazole-6-carbohydrazide (3a-3c)
derivative (0.01 mol) was dissolved in
a solution of NaOH
(0.01 mol, 0.4 g) in ethanol and carbon disulphide (0.01 mol,
0.60 ml) was added in the mixture. The mixture was refluxed for
8 h. When the reaction was finished, the solution was cooled and
acidified to pH 4–5 with concentrated hydrochloric acid solution
to obtained target compound.
2.1.1.6. General synthesis method of target compounds (5a-5o). 2-
((4-Substitutedphenyl)-(6–(5-mercapto-1,3,4-oxadiazol-2-yl)-1H-ben-
2.1.1. General procedure for the synthesis of the compounds
2.1.1.1. Synthesis of 2-(4-substitutedphenyl)-1H-benzo[d]imidazole-
6-carboxylic acid derivatives (1a-1c). A mixture of 4-substituted
benzaldehyde (0.03 mol) and sodium disulphide (0.03 mol, 5.7 g) in
DMF were treated under microwave irradiation (Anton-Paar
Monowave 300) at 240 ꢀC and 10 bar for 5 min. After cooling the
mixture, 3,4-diamino benzoic acid (0.03 mol, 4.56 g) was added
and kept under the same reaction conditions. The progress of the
reaction was monitored by TLC. After completion of the reaction,
the mixture was poured into crushed ice. The solid obtained was
filtered, washed with water, dried and recrystallized from EtOH.
zo[d]imidazole
(4a-4c)
(0.001 mol),
potassium
carbonate
(0.001 mol, 0.138 g) and 2-chloro-1-(4-substitutedepiperazin-1-yl)
-ethane-1-on (0.0015 mol) derivative were dissolved in acetone
and refluxed for 6 h. After TLC control, the solvent was evapo-
rated, the residue was washed with water, dried and then recrys-
tallized from ethanol to afford final compounds (5a-5o).
Methyl 2-(4-hydroxyphenyl)-1H-benzo[d]imidazole-6-carboxylate (2a).
Yield: 75%, M.P. ¼ 306.1–307.5 ꢀC. FTIR (ATR, cmꢁ1): 3334 (N-H),
1687 (C¼O), 844 (1,4-disubstituted benzene). 1H-NMR (300 MHz,
DMSO-d6): d ¼ 3.88 (3H, s, –CH3), 6.95 (2H, d, J ¼ 8.70 Hz, 1,4-disub-
stituted benzene), 7.62 (1H, d, J ¼ 8.34 Hz, benzimidazole-C4), 7.82
(1H, dd, J1¼8.43 Hz, J2¼1.26 Hz, benzimidazole-C5), 8.04 (2H, d,
J ¼ 8.64 Hz, 1,4-disubstituted benzene), 8.14 (1H, s, benzimidazole-
C7), 10.05 (1H, s, N–H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 52.41,
107.48, 111.94, 116.26 (2C), 120.89, 123.40, 126.28, 129.02, 130.54
(2C), 134.46, 154.79, 160.14, 167.28. HRMS (m/z): [M þ H]þ calcd
for C15H13N2O3: 269.0910; found: 269.0921.
€
€
2.1.1.2. Synthesis of 2-chloro-1-(4-substituepiperaz-1-yl) ethan-1-
one derivatives (1d-1h). Chloroacetyl chloride (0.014 mol, 1.056 ml)
in THF (15 ml) was taken into ice bath. 4-Substituted piperazine
derivatives (0.012 mol) and TEA (0.0132 mol, 1.90 ml) in THF (50 ml)
were added dropwise to this solution. After completion of drop-
ping, the reaction mixture was stirred at room temperature for
1 h. The precipitated product was filtered washed with water
and dried.
Methyl 2-(4-methoxyphenyl)-1H-benzo[d]imidazole-6-carboxylate (2 b).
Yield: 68%, M.P.¼230.4–231.7 ꢀC. FTIR (ATR, cmꢁ1): 3250 (N–H),
1695 (C¼O), 839 (1,4-disubstituted benzene). 1H-NMR (300 MHz,
2.1.1.3. Synthesis of Methyl 2-(4-substitutedphenyl)-1H-benzo[d]imi-
dazole-6-carboxylate derivatives (2a-2c). 2–(4-Substitutedphenyl)- DMSO-d6): d ¼ 3.85 (3H, s, –OCH3), 3.87 (3H, s, –COOCH3), 7.13 (2H,
1H-benzo[d]imidazole-6-carboxylic acid (1a-1c) derivative com- d, J ¼ 8.85 Hz, 1,4-disubstituted benzene), 7.65 (1H, br.s., benzimi-
pound (0.025 mol) was dissolved in methanol and several drops of dazole-C–H), 7.83 (1H, d, J ¼ 7.95 Hz, benzimidazole C–H), 8.14 (2H,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1659
d, J ¼ 8.85 Hz, 1,4-disubstituted benzene), 8.22 (1H, br.s., benzimi- benzene), 8.13 (1H, s, benzimidazole-C7), 10.20 (1H, s, O-H). 13C-NMR
dazole, C-H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 52.43, 55.87, 111.49, (75 MHz, DMSO-d6): d ¼ 36.76, 42.47, 45.75, 52.23, 52.44, 60.63,
113.08, 115.31 (2C), 118.69, 120.49, 122.45, 123.29, 123.56, 128.88 112.96, 115.85, 116.47 (2C), 117.52, 118.66, 121.59, 129.67 (2C),
(2C), 130.43, 161.55, 167.26. HRMS (m/z): [M þ H]þ calcd for 153.79, 161.14, 163.26, 165.68, 166.19, 167.30, 169.40. HRMS (m/z):
[M þ H]þ/2 calcd for C22H23N6O3S: 226.0804; found: 226.0810.
C16H15N2O3: 283.1083; found: 283.1077.
Methyl 2-(4-ethoxyphenyl)-1H-benzo[d]imidazole-6-carboxylate (2c).
Yield: 60%, M.P.¼ 252.8–254.4 ꢀC. FTIR (ATR, cmꢁ1): 3477 (N-H),
1645 (C¼O), 844 (1,4-disubstituted benzene). 1H-NMR (300 MHz,
DMSO-d6): d ¼ 1.36 (3H, t, J ¼ 6.96 Hz, –CH3), 3.87 (3H, s, –CH3),
4.11 (2H, q, J ¼ 6.96 Hz, -CH2), 7.11 (2H, d, J ¼ 8.88 Hz, 1,4-disubsti-
tuted benzene), 7.63 (1H, d, J ¼ 8.43 Hz, benzimidazole-C4), 7.83
(1H, dd, J1¼8.40 Hz, J2¼1.59 Hz, benzimidazole-C5), 8.12 (2H, d,
J ¼ 8.88 Hz, 1,4-disubstituted benzene), 8.1 (1H, s, benzmidazole-
C7), 13.07 (1H, s, N-H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 15.04,
52.38, 63.81, 114.39, 115.33, 116.22 (2C), 118.31, 119.34, 122.31,
123.53, 128.49 (2C), 128.88, 154.40, 160.84, 167.26. HRMS (m/z):
[M þ H]þ calcd for C17H17N2O3: 297.1232; found: 297.1234.
2-((5–(2-(4-Hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1–(4-ethyl- piperazin-1-yl) -ethane-1-on (5 b). Yield: 78%,
M.P.¼ 199.9–201.2 ꢀC. FTIR (ATR, cmꢁ1): 3311 (N-H), 1645 (C¼O),
839 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
d ¼ 1.01 (3H, t, J ¼ 7.16, CH3), 2.34–2.51 (6H, m, CH2, piperazine),
3.48–3.50 (4H, m, piperazine), 4.56 (2H, s, CH2), 6.90 (2H, d,
J ¼ 8.70 Hz, 1,4-disubstituted benzene), 7.67 (1H, d, J ¼ 8.46 Hz,
benzimidazole-C4), 7.74 (1H, dd, J1¼8.39 Hz, J2:1.65 Hz, benzimida-
zole-C5), 8.06 (2H, d, J ¼ 8.70 Hz, 1,4-disubstituted benzene), 8.09
(1H, s, benzimidazole-C7). 13C-NMR (75 MHz, DMSO-d6): d ¼ 12.32,
37.04, 42.23, 45.85, 51.91, 52.31, 52.79, 113.41, 116.30, 116.45,
116.70 (2C), 120.38, 129.02 (2C), 155.20, 155.45, 161.21, 163.08,
165.06, 165.89, 166.58, 168.44. HRMS (m/z): [M þ H]þ calcd for
C23H25N6O3S: 465.1692; found: 465.1703.
2-((4-Hydroxyphenyl)-(6-(5-mercapto-1,3,4-oxadiazol-2-yl)-1H-benzo[d]
imidazole (4a). Yield: 81%, M.P.¼ 226.8–228.3 ꢀC. FTIR (ATR, cmꢁ1):
3483 (N-H), 1645 (C¼O), 844 (1,4-disubstituted benzene). 1H-NMR
(300 MHz, DMSO-d6):d ¼ 7.06 (2H, d, J ¼ 8.76 Hz, 1,4-disubstituted
benzene), 7.89 (2H, m, benzimidazole-C–H), 8.10 (1H, s, benzimida-
zole-C-H), 8.24 (2H, d, J ¼ 8.70 Hz, 1,4-disubstituted benzene), 10.75
(1H, s, O-H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 112.11, 115.27,
115.48, 116.86 (2C), 118.85, 122.86, 130.64 (2C), 134.41, 136.72,
152.40, 160.92, 162.58, 177.83. HRMS (m/z): [M þ H]þ calcd for
C15H11N4O2S: 311.0595; found: 311.0597.
2-((5–(2-(4-Hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl) thio)-1–(4-(phenyl) -piperazin-1-yl) -ethane-1-on (5c). Yield:
72%, M.P.¼ 271.1–273.4 ꢀC. FTIR (ATR, cmꢁ1): 3365 (N-H), 1645
(C¼O), 840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
d6): d ¼ 3.13–3.26 (4H, m, piperazine), 3.63–3.70 (4H, m, pipera-
zine), 4.64 (2H, s, -CH2), 6.82 (1H, t, J ¼ 7.23 Hz, phenyl C-H),
6.99–6.93 (4H, m, 1,4-disubstituted benzene, phenyl C-H), 7.24 (2H,
t, J ¼ 7.29 Hz, phenyl C-H), 7.66 (1H, br.s., benzimidazole-C4), 7.81
(1H, d, J ¼ 8.34 Hz, benzimidazole-C5), 8.01–8.06 (3H, m, 1,4-disub-
stituted benzene, benzimidazole-C7), 10.10 (1H, s, O-H), 13.09 (1H,
s, N-H).13C-NMR (75 MHz, DMSO-d6): d ¼ 37.03, 42.06, 45.66, 48.63,
48.97, 116.21 (2C), 116.28, 116.37, 116.77 (2C), 119.18, 119.85,
120.83, 121.11, 128.86, 129.03 (2C), 129.48 (2C), 151.15, 159.93,
160.18, 162.08, 163.22, 165.26, 166.46. HRMS (m/z): [M þ H]þ calcd
for C27H25N6O3S: 513.1699; found: 513.1703.
2-((4-Methoxyphenyl)-(6-(5-mercapto-1,3,4-oxadiazol-2-yl)-1H-benzo[d]
imidazole (4b). Yield: 78%, M.P.¼ 274.4–276.5 ꢀC. FTIR (ATR, cmꢁ1):
3381 (N-H), 1633 (C¼O), 837 (1,4-disubstituted benzene). 1H-NMR
(300 MHz, DMSO-d6): d ¼ 3.90 (3H, s, –OCH3), 7.24–7.28 (2H, m, 1,4-
disubstituted benzene), 7.89 (1H, s, benzimidazole C-H), 8.12 (1H,
s, benzimidazole C–H), 8.32 (2H, d, J ¼ 8.70 Hz, 1,4-disubstituted
benzene), 8.40 (1H, s, benzimidazole C-H). 13C-NMR (75 MHz,
DMSO-d6): d ¼ 56.25, 111.07, 114.23, 115.49, 115.76 (2C), 119.97,
122.60, 130.24 (2C), 130.49, 133.74, 152.25, 160.86, 162.73, 177.22.
HRMS (m/z): [M þ H]þ calcd for C16H13N4O2S: 325.0748;
found: 325.0754.
2-((5-(2-(4-Hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1-(4-(pyridin-2-yl)-piperazin-1-yl)-ethane-1-on (5d). Yield:
69%, M.P.¼ 247.6–248.9 ꢀC. FTIR (ATR, cmꢁ1): 3628 (N-H), 1645
(C¼O), 840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
d6): d ¼ 3.56–3.66 (8H, m, piperazine), 4.65 (2H, s, CH2), 6.68–6.69
(2H, m, pyridine C-H), 6.99 (2H, d, J ¼ 8.73 Hz, 1,4-disubstituted
benzene), 7.57–7.58 (2H, m, pyridine C-H), 7.72 (1H, d, J ¼ 8.37 Hz,
benzimidazole-C4), 7.82 (1H, dd, J1¼5.40 Hz, J2¼1.53 Hz, benzimi-
dazol-C5), 8.11–8.14 (2H, m, 1,4-disubstituted benzene, benzimida-
zole-C7), 10.26 (1H, s, O-H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 37.17,
41.83, 44.74, 44.99, 45.41, 107.88, 113.21, 113.79, 114.59, 116.27
(2C), 116.73, 117.11, 120.67, 120.74, 129.13 (2C), 138.24, 147.83,
154.66, 158.94, 160.34, 163.22, 165.40, 165.49, 166.47. HRMS (m/z):
[M þ H]þ calcd for C26H24N7O3S: 514.1636; found: 514.1656.
2-((4-Ethoxyphenyl)-(6–(5-mercapto-1,3,4-oxadiazol-2-yl)-1H-benzo[-
d]imidazole (4c). Yield: 84%, M.P.¼ 288.2–289.9 ꢀC. FTIR (ATR,
cmꢁ1): 3402 (N-H), 1624 (C¼O), 846 (1,4-disubstituted benzene).
1H-NMR (300 MHz, DMSO-d6): d ¼ 1.38 (3H, t, J ¼ 6.90 Hz, -CH3),
4.17 (2H, q, J ¼ 6.96 Hz, -CH2), 7.23 (2H, d, J ¼ 8.28 Hz, 1,4-disubsti-
tuted benzene), 7.91–7.93 (2H, m, benzimidazole-C-H), 8.12 (1H, s,
benzimidazole-C-H), 8.24 (2H, d, J ¼ 7.47 Hz, 1,4-disubstituted ben-
zene). 13C-NMR (75 MHz, DMSO-d6): d ¼ 14.92, 64.35, 112.17,
115.55, 115.96, 116.14 (2C), 119.30, 123.28, 130.57 (2C), 133.78,
135.98, 151.83, 155.14, 162.99, 177.88. HRMS (m/z): [M þ H]þ calcd
for C17H15N4O2S: 339.0910; found: 339.0910.
2-((5–(2-(4-Hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1-(4-(pyrimidine-2-yl)-piperazin-1-yl)-ethane-1-on (5e). Yield:
78%, M.P.¼ 181.4–183.2 ꢀC. FTIR (ATR, cmꢁ1): 3587 (N-H), 1645
(C¼O), 840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
d6): d ¼ 3.60 (2H, br.s, piperazine), 3.67 (2H, br.s., piperazine), 3.79
(2H, br.s., piperazine), 3.90 (2H, brs., piperazine), 4.64 (2H, s, CH2),
6.74 (1H, J ¼ 4.80, pyrimidine), 7.09 (2H, J ¼ 8.76 Hz, 1,4-disubsti-
2-((5–(2-(4-Hydroxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1–(4-methyl- piperazin-1-yl) -ethane-1-on (5a). Yield: 65%,
M.P.¼ 161.3–163.4 ꢀC. FTIR (ATR, cmꢁ1): 3618 (N-H), 1645 (C¼O),
840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
d ¼ 2.76 (3H, s, -CH3), 3.35 (8H, br.s., piperazine), 4.64 (2H, s, -CH2),
6.96 (2H, d, J¼ 8.64 Hz, 1,4-disubstituted benzene), 7.71 (1H, d, tuted benzene), 7.92 (1H, d, J ¼ 8.55 Hz, benzimidazole-C4), 8.03,
J¼ 8.28 Hz, benzimidazole-C4), 7.80 (1H, dd, J1¼8.40 Hz, J2¼1.15 Hz, (1H, dd, J1¼8.60 Hz, J2¼1.17 Hz, benzimidazole-C5), 8.21 (1H, s,
benzimidazole-C5), 8.09 (2H, d, J ¼ 8.61 Hz, 1,4-disubstituted benzimidazole-C7), 8.35 (2H, d, J ¼ 8.73 Hz, 1,4-disubstituted

1660
U. A. ÇEVIK ET AL.
benzene), 8.44 (2H, d, J ¼ 4.83 Hz, pyrimidine). 13C-NMR (75 MHz, (1H, s, benzimidazole-C4), 7.70 (1H, dd, J1¼8.37 Hz, J2¼1.50 Hz,
DMSO-d6): d ¼ 37.43, 41.82, 43.67, 43.95, 45.37, 110.86, 111.87, benzimidazole-C5), 8.09 (1H, s, benzimidazole-C7), 8.12–8.14 (1H,
113.24, 115.16, 117.01 (2C), 120.25, 124.09, 131.19 (2C), 132.41, m, pyridine C-H), 8.20 (2H, d, J ¼ 8.85 Hz, 1,4-disubstituted ben-
134.34, 151.27, 158.26, 159.99, 163.40, 164.30, 165.12, 165.37. zene). 13C-NMR (75 MHz, DMSO-d6): d ¼ 37.12, 41.83 44.72, 44.97,
HRMS (m/z): [M þ H]þ calcd for C25H23N8O3S: 515.1610; 45.42, 55.75, 107.77, 113.80, 114.03, 114.61, 114.82, 115.23 (2C),
found: 515.1608.
116.07, 119.53, 124.75, 128.70 (2C), 128.80, 138.12, 142.46, 148.04,
159.09, 160.87, 162.71, 165.43, 167.01. HRMS (m/z): [M þ H]þ calcd
for C27H26N7O3S: 528.1825; found: 528.1812.
2-((5-(2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl) thio)-1–(4-methyl- piperazin-1-yl)-ethane-1-on (5f). Yield: 74%,
M.P.¼ 156.6–158.7 ꢀC. FTIR (ATR, cmꢁ1): 3446 (N-H), 1645 (C¼O),
2-((5-(2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
835 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6): 2-yl)thio)-1–(4-(pyrimidin-2-yl)-piperazin-1-yl)-ethane-1-on (5j). Yield:
d ¼ 2.78 (3H, s, -CH3), 3.43 (6H, br.s., piperazine), 3.65 (2H, br.s., 80%, M.P.¼ 118.7–120.3 ꢀC. FTIR (ATR, cmꢁ1): 3406 (N-H), 1627
piperazine), 3.87 (3H, s, –OCH3), 4.64 (2H, s, -CH2), 7.17 (2H, d, (C¼O), 839 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
J ¼ 8.91 Hz, 1,4-disubstituted benzene), 7.78 (1H, d, J ¼ 8.46 Hz, d6): d ¼ 3.64 (2H, br.s, piperazine), 3.72–3.76 (6H, m, piperazine),
benzimidazole-C4), 7.87 (1H, dd, J1¼ 8.43 Hz, J2¼ 1.50 Hz benzimi- 3.82 (3H, s, -OCH3), 4.62 (2H, s, CH2), 6.68 (1H, t, J ¼ 4.71, pyrimi-
dazole-C5), 8.16 (1H, br.s. benzimidazole-C7), 8.25 (2H, d, dine), 7.12 (2H, J ¼ 8.91 Hz, 1,4-disubstituted benzene), 7.70 (1H, d,
J ¼ 8.82 Hz, 1,4-disubstituted benzene), 11.38 (1H, s, N-H).13C-NMR J ¼ 8.31 Hz, benzimidazole C4), 7.79 (1H, dd, J1¼ 8.43 Hz, J2¼
(75 MHz, DMSO-d6): d ¼ 36.82, 42.40, 42.79, 45.66, 52.19, 52.42, 1.47 Hz, benzimidazole-C5), 8.13 (1H, br.s. benzimidazole-C7), 8.20
56.00, 113.15, 113.44, 115.09 (2C), 115.98, 117.70, 120.46, 121.70, (2H, J ¼ 8.85 Hz, 1,4-disubstituted benzene), 8.40 (2H, d,
124.59, 129.56 (2C), 153.34, 162.18, 163.31, 165.67, 166.15. HRMS J ¼ 4.74 Hz, pyrimidine). 13C-NMR (75 MHz, DMSO-d6): d ¼ 37.22,
(m/z): [M þ H]þ calcd for C23H25N6O3S: 465.1703; found: 465.170.
41.94, 43.77, 45.53, 52.71, 55.71, 110.54, 110.91, 110.96, 114.09,
114.52 (2C), 116.10, 119.28, 125.13, 128.82 (2C), 158.43, 158.46,
160.74, 161.53, 161.62, 162.61, 165.49, 167.55, 168.04. HRMS (m/z):
[M þ H]þ calcd for C26H25N8O3S: 529.1755; found: 529.1765.
2-((5-(2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1–(4-ethyl-piperazin-1-yl)-ethane-1-on (5 g). Yield: 66%,
M.P.¼ 180.4–181.9 ꢀC. FTIR (ATR, cmꢁ1): 3334 (N-H), 1645 (C¼O),
840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
2-((5-(2-(4-Ethoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-2-
d ¼ 1.01 (3H, t, J ¼ 7.08 Hz, CH3), 2.34 (2H, m, CH2), 3.85 (3H, s, yl)thio)-1–(4-methyl-piperazin-1-yl)-ethane-1-on (5k). Yield: 79%,
OCH3), 3.47–3.53 (8H, m, piperazine), 4.56 (2H, s, CH2), 7.13 (2H, d, M.P.¼ 181.2–183.5 ꢀC. FTIR (ATR, cmꢁ1): 3360 (N-H), 1645 (C¼O),
J ¼ 8.79 Hz, 1,4-disubstituted benzene), 7.71 (1H, d, J ¼ 8.40 Hz, 833 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
benzimidazole-C4), 7.80 (1H, dd, J1¼8.42 Hz, J2¼1.44 Hz benzimida- d ¼ 1.37 (3H, t, J ¼ 6.81 Hz, -CH3), 2.75 (3H, s, -CH3), 3.39 (4H, br.s.,
zole-C5), 8.13 (1H, br.s., benzimidazole-C7), 8.17 (2H, d, J ¼ 8.88 Hz, piperazine), 3.60 (4H, br.s., piperazine), 4.47–4.50 (2H, m, -CH2),
1,4-disubstituted benzene). 13C-NMR (75 MHz, DMSO-d6): d ¼ 12.36, 4.66 (2H, s, -CH2), 7.21 (2H, d, J ¼ 8.67 Hz, 1,4-disubstituted ben-
37.10, 42.23, 45.85, 51.92, 52.32, 52.80, 55.85, 114.88 (2C), 119.77, zene), 7.88 (1H, d, J ¼ 8.67 Hz, benzimidazole-C4), 7.99 (1H, d,
120.02, 120.63, 122.75, 128.71, 128.93 (2C), 153.30, 154.66, 161.25, J ¼ 8.55 Hz, benzimidazole-C5), 8.22 (1H, s, benzimidazole-C7), 8.38
161.42, 163.19, 165.05, 166.48. HRMS (m/z): [M þ H]þ calcd for (2H, d, J ¼ 8.16 Hz, 1,4-disubstituted benzene), 11.55 (1H, s, N-H).
C24H27N6O3S: 479.1872; found: 479.1860.
13C-NMR (75 MHz, DMSO-d6): d ¼ 14.96, 36.87, 39.11, 42.38, 42.74,
45.66, 52.18, 64.25, 112.48, 115.79 (2C), 117.36, 119.21, 121.51,
123.22, 126.03 (2C), 128.45, 130.51, 151.88, 162.62, 163.81, 165.63,
165.80. HRMS (m/z): [M þ H]þ calcd for C24H27N6O3S: 479.1873;
found: 479.1860.
2-((5-(2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1–(4-(phenyl)-piperazin-1-yl)-ethane-1-on (5 h). Yield: 77%,
M.P.¼ 166.7–169.2 ꢀC. FTIR (ATR, cmꢁ1): 3367 (N-H), 1645 (C¼O),
840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
d ¼ 3.19 (4H, br.s., piperazine), 3.67 (4H, br.s., piperazine), 3.85 (3H,
2-((5-(2-(4-Ethoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-2-
s, -OCH3), 4.63 (2H, s, -CH2), 6.81 (1H, t, J ¼ 7.14 Hz, phenyl C-H), yl)thio)-1–(4-ethyl-piperazin-1-yl)-ethane-1-on (5 l). Yield: 77%, M.P.¼
6.97 (2H, d, J ¼ 7.92 Hz, phenyl C-H), 7.14 (2H, d, J ¼ 8.79 Hz, 1,4- 109.4–110.8 ꢀC. FTIR (ATR, cmꢁ1): 3429 (N-H), 1653 (C¼O), 827
disubstituted benzene), 7.23 (2H, d, J ¼ 7.47 Hz, phenyl C-H), 7.76 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
(1H, d, J ¼ 8.46 Hz, benzimidazole C4), 7.80 (1H, dd, J1¼ 8.37 Hz, d ¼ 1.19–1.21 (3H, m, -CH3), 1.36 (3H, t, J ¼ 6.90 Hz, -CH3),
J2¼ 1.20 Hz, benzimidazole-C5), 8.16 (1H, br.s. benzimidazole-C7), 2.93–2.98 (2H, m, -CH2), 3.80–3.90 (8H, m, piperazine), 4.10–4.13
8.29 (2H, d, J ¼ 8.70 Hz, 1,4-disubstituted benzene), 10.72 (1H, s, N- (2H, m, -CH2), 4.62 (2H, s, -CH2), 7.10 (2H, d, J ¼ 8.91 Hz, 1,4-disub-
H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 42.23, 45.85, 51.92, 52.32, stituted benzene), 7.73 (1H, d, J ¼ 8.40 Hz, benzimidazole-C4), 7.81
52.80, 55.85, 114.88 (2C), 116.12, 116.63 (2C), 117.06, 119.77, (1H, dd, J1¼8.37 Hz, J2¼1.50 Hz, benzimidazole-C5), 8.13 (1H, s,
120.02, 120.63, 122.75, 122.86 128.71 (2C), 128.93 (2C), 153.30, benzimidazole-C7), 8.22 (2H, d, J ¼ 8.82 Hz, 1,4-disubstituted ben-
154.66, 161.25, 161.42, 163.19 165.05, 166.48. HRMS (m/z): zene). 13C-NMR (75 MHz, DMSO-d6): d ¼ 15.06, 36.81, 37.88, 43.45,
[M þ H]þ calcd for C28H27N6O3S: 527.1849; found: 527.1860.
50.57, 50.98, 51.22, 60.65, 63.81, 113.08, 115.28, 116.74 (2C),
120.76, 122.30, 125.99, 127.43, 129.02 (2C), 132.31, 154.34, 160.80,
163.07, 165.51, 165.61. HRMS (m/z): [M þ H]þ calcd for
C25H29N6O3S: 493.2019; found: 493.2016.
2-((5-(2-(4-Methoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-
2-yl)thio)-1–(4-(pyridin-2-yl)-piperazin-1-yl)-ethane-1-on (5ı). Yield:
72%, M.P.¼ 168.3–170.1 ꢀC. FTIR (ATR, cmꢁ1): 3350 (N-H), 1651
(C¼O), 829 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
2-((5-(2-(4-Ethoxyphenyl)-1H-benzo [d] imidazol-6-yl)-1,3,4-oxadiazol-
d6): d ¼ 3.52–3.54 (2H, m, piperazine), 3.61–3.65 (6H, m, pipera- 2-yl) thio)-1–(4-(phenyl)-piperazin-1-yl)-ethane-1-on (5 m). Yield: 70%,
zine), 3.83 (3H, s, -OCH3), 4.62 (2H, s, CH2), 6.65–6.69 (1H, m, pyri- M.P.¼ 262.5–263.9 ꢀC. FTIR (ATR, cmꢁ1): 3367 (N-H), 1645 (C¼O),
dine C-H), 6.85–6.87 (1H, m, pyridine C-H), 7.07 (2H, d, J ¼ 8.91 Hz, 840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-d6):
1,4-disubstituted benzene), 7.53–7.58 (1H, m, pyridine C-H), 7.64 d ¼ 1.35 (3H, t, J ¼ 6.93 Hz, -CH3), 2.32 (2H, br.s., piperazine), 2.41

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1661
Table 1. Hydrogen and carbon values of compound 5e detected by 2 D NMR.
(2H, br.s., piperazine), 3.52 (4H, br.s., piperazine), 4.11 (2H, q,
J ¼ 6.96 Hz, –CH2), 4.53 (2H, s, –CH2), 7.15 (2H, d, J ¼ 8.94 Hz, 1,4-
disubstituted benzene), 7.44–7.47 (3H, m, phenyl C-H), 7.66–7.69
(2H, m, phenyl C-H), 7.76 (1H, d, J ¼ 8.46 Hz, benzimidazole-C4),
7.81–7.85 (1H, m, benzimidazole-C5), 8.15 (1H, br.s. benzimidazole-
C7), 8.28 (2H, d, J ¼ 8.85 Hz, 1,4-disubstituted benzene), 10.70 (1H,
s, N-H). 13C-NMR (75 MHz, DMSO-d6): d ¼ 36.75, 42.72, 45.68, 50.38,
50.84, 55.91, 59.07, 114.95, 115.83 (2C), 117.01, 121.02, 121.93,
128.76 (2C), 129.03, 129.09, 129.18, 129.83, 130.20, 131.01, 131.89,
154.05, 161.69, 162.99, 163.10, 165.59, 166.41, 169.44. HRMS (m/z):
[M þ H]þ calcd for C29H29N6O3S: 541.2002; found: 541.2016.
18
17
19
20
12
14
15
11
N
O
5
4
21
OH
O
S
4
9
16
6
N
N
10
8
7
18 19
H
13
N
N
N
N
2
1
5
22
3
N
5e
2
Position
1H
13C
1
2
3
6.68
8.39
–
111.0
158.5
161.4
2-((5-(2-(4-Ethoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-2-
yl) thio-1–(4-(pyridin-2-yl)-piperazin-1-yl)-ethane-1-on (5n). Yield:
71%, M.P. ¼ 168.3–170.4 ꢀC. FTIR (ATR, cmꢁ1): 3361 (N-H), 1635
(C¼O), 840 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
d6): d ¼ 1.37 (3H, t, J ¼ 6.93 Hz, –CH3), 3.52–3.54 (2H, m, piperazine),
3.59–3.65 (6H, m, piperazine), 4.13 (2H, q, J ¼ 6.99 Hz, -CH2), 4.65
(2H, s, –CH2), 6.66–6.70 (1H, m, pyridine C–H), 6.88 (1H, d,
J ¼ 8.61 Hz, pyridine-C–H), 7.11 (2H, d, J ¼ 8.85 Hz, 1,4-disubstituted
benzene), 7.54–7.60 (1H, m, benzimidazole-C4), 7.80 (2H, m, pyri-
dine C–H, benzimidazole C–H), 8.06–8.15 (2H, m, pyridine C–H, ben-
zimidazole C–H), 8.18 (2H, d, J ¼ 8.49 Hz, 1,4-disubstituted
benzene), 13.50 (1H, s, N–H). 13C-NMR (75 MHz, DMSO-d6):
d ¼ 15.07, 37.18, 41.84, 44.72, 44.98, 45.42, 63.81, 107.80, 113.82,
114.63 (2C), 115.31, 116.84, 119.76, 122.26, 123.67, 125.16, 128.96,
130.02 (2C), 138.15, 141.90, 148.04, 159.08, 160.83, 164.04, 165.40,
166.45. HRMS (m/z): [M þ H]þ calcd for C28H28N7O3S: 542.1982;
found: 542.1969.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
3.60–3.86
3.65–3.77
–
4.62
–
–
–
7.98
7.86
8.15
–
42.0–45.5
43.4–43.7
165.4
37.3
152.3
165.3
136.0
123.6
115.4
112.3
119.7
134.0
164.2
114.8
130.7
116.9
162.8
–
–
–
–
8.22
7.06
–
10.88
12.69
–
adenocarcinoma cell line), HepG2 (human liver carcinoma cell line)
and C6 (rat glioma cell line) cell lines as well as NIH3T3 (mouse
embryo fibroblast cell line). Doxorubicin and Hoechst 33342 were
used as the reference drugs in the MTT assays.
2-((5-(2-(4-Ethoxyphenyl)-1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-2-
yl)thio)-1–(4-(pyrimidine-2-yl)-piperazin-1-yl)-ethane-1-on (5o). Yield:
67%, M.P. ¼ 118.7–120.5 ꢀC. FTIR (ATR, cmꢁ1): 3334 (N-H), 1645
(C¼O), 871 (1,4-disubstituted benzene). 1H-NMR (300 MHz, DMSO-
d6): d ¼ 1.37 (3H, t, J ¼ 6.90 Hz, -CH3), 3.59 (2H, br.s., piperazine),
3.65 (2H, br.s., piperazine), 3.75–3.76 (4H, m, piperazine), 4.11 (2H,
q, J ¼ 6.99 Hz, –CH2), 4.64 (2H, s, –CH2), 6.67 (1H, t, J ¼ 4.77 Hz, pyr-
imidine), 8.09 (2H, d, J ¼ 8.73 Hz, 1,4-disubstituted benzene), 7.70
(1H, d, J ¼ 7.70 Hz, benzimidazole-C4), 7.79 (1H, dd, J1 ¼ 8.40 Hz,
J2 ¼ 1.59 Hz, benzimidazole-C5), 7.89 (1H, s, benzimidazole-C7),
8.15 (2H, d, J ¼ 8.73 Hz, 1,4-disubstituted benzene), 8.40 (2H, d,
J ¼ 4.71 Hz, pyrimidine). 13C-NMR (75 MHz, DMSO-d6): d ¼ 15.08,
37.25, 41.95, 43.41, 43.68, 45.53, 63.76, 110.99, 115.23 (2C), 116.27,
119.73, 119.96, 120.37, 122.71, 123.06, 128.72 (2C), 128.91, 153.41,
158.48, 160.54, 162.13, 163.06, 165.46, 166.66, 179.63. HRMS (m/z):
[M þ H]þ calcd for C27H27N8O3S: 543.1907; found: 543.1921.
2.2.2. DNA synthesis inhibition assay
The BrdU cell proliferation method was performed to analyse the
effects of the active compounds on proliferation of cancer cells.
Cancer cells were seeded into the 96-well plates at a density of
1 ꢂ 104 cells. Compounds were added into the each well at three
different concentrations (IC50/2, IC50 and 2xIC50) and the plates
were incubated for 24 h. At the end of the incubation period,
BrdU solution was added and cells were reincubated for 2 h at
37 ꢀC. Anti-BrdU-POD (100 ml) was added and the mixture was
incubated for 90 min. Microplates were washed with PBS for three
times. After adding substrate solution, the mixture was incubated
for 15 min. OD of the wells were determined at 492 nm.
Proliferation of control cells was assessed as 100% and growth
inhibition % of cells, treated with test compounds and cisplatin
were calculated34.
2.2. 2 D NMR
2D NMR studies including HSQC (Heteronuclear single-quantum
correlation spectroscopy), HMBC (Heteronuclear multiple-bond cor-
relation spectroscopy) and COSY (Correlation Spectroscopy) were
performed for compound 5e (Table 1 and Supporting information).
2.2.3. Flow cytometric analysis
Death pathway of the carcinogenic cell lines was detected by
Annexin V-FITC Apoptosis Detection Kit (BD, Pharmingen) as
reported previously described35. Doxorubicin and compounds,
which possess the highest cytotoxic activity, were used at their
IC50 concentration. FCS Express software was used to display the
percent of normal and apoptotic cells at different stages. In the
diagrams, Q1, Q2, Q3, and Q4 demonstrates the necrotic cells
2.3. Biological activity
2.3.1. Cytotoxicity test
The anticancer activity of compounds 5a–5o were screened
according to the MTT assays. The MTT assays were performed as
previously described32,33. Anticancer activity of final compounds
was assessed against five different cancer cell lines A549 (lung car- (positive for PI and negative for annexin/FITC), late apoptotic or
cinoma cell line), HeLa (cervical cell line), MCF-7 (human breast necrotic cells (positive for annexin and PI), live cells (negative for

1662
U. A. ÇEVIK ET AL.
Scheme 1. Synthesis way of the compounds 5a-5o.
annexin and PI), and apoptotic cells (negative for PI and positive 2.3. Molecular docking
for annexin), respectively. The experiments were carried out in
triplicates.
A structure-based in silico docking method was applied to deter-
mine the binding and interaction modes of compound 5n that
show significant anticancer activity in the series, in the active
region of the DNA-Topoisomerase I enzyme complex. A protein–
ligand interaction analysis was performed on the DNA-
Topoisomerase I enzyme complex by using X-ray crystal structure
retrieved from Protein Data Bank server (www.pdb.org) (PDB
Code: 1T8I)35.
2.2.4. DNA topoisomerase I assay
In this study, topoisomerase I assay kit (TG1018-2; TopoGen) was
used to determine if synthesised compounds showed topoisomer-
ase I inhibition. The topoisomerase I inhibition activities of final
compounds were measured as relaxation of supercoiled plasmid
DNA using agarose gel electrophoresis and camptothecin was
used as a positive control. The assay was carried out in a final vol-
ume of 20 ml reaction volume containing 2 ml of 10xTGS Buffer,
6 ml water, 2 ml supercoiled plasmid DNA, 2 ml test compound, 2 ml
of Top1, 2 ul 10% SDS, 2 ml proteinase K, 2 ml DNA loading dye.
After incubation of reaction mixtures at 37 ꢀC for 30 min, electro-
The chemical structures of ligands were drawn using the
36
€
Schrodinger Maestro interface and then were submitted to the
€
Protein Preparation Wizard protocol of the Schrodinger Suite 2016
Update 237. Then the ligands were prepared by the LigPrep 3.838
to set to protonation states at pH 7.4 1.0 and the atom types,
correctly. Bond orders were assigned, and hydrogen atoms were
added to the structures. The grid generation was formed using
phoresis was done on a 1% agarose gel at a potential 50 V for Glide 7.139. Flexible docking runs were performed with single pre-
75 min using 1xTAE buffer.
cision docking mode (SP).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1663
compound (4a-4c) was reacted with acetylated piperazine
derivatives in acetone to produce 2-((5–(2-(4-substitutedphenyl)-
Comp.
5a
R1
R2
1H-benzo[d]imidazol-6-yl)-1,3,4-oxadiazol-2-yl)
thio)-1–
(4-
-OH
-CH3
€
€
substituepiperazin- 1-yl)-ethane-1-on derivatives (5a-5o).
After the isolation and purification, the structures of newly syn-
thesised compounds (5a-5o) were characterised by using various
modern analytical techniques like IR, 1H NMR, 13C NMR, 2 D-NMR
and HRMS.
5b
5c
-OH
-OH
-C2H5
In general, for all the synthesised compounds, the stretching
bands for C¼O and N-H were observed between 1627–1653 cmꢁ1
and 3311–3628 cmꢁ1
, respectively. The stretching absorption
5d
-OH
-OH
belonging to 1,4-disubstituted benzene was determined
at 827–871 cmꢁ1
.
In the 1H-NMR spectra, protons of piperazine was seen
between 2.32–3.90 ppm. The –CH3 group protons of compounds
5a, 5f and 5k on para position of piperazine ring were observed
at 2.75–2.78 ppm as asinglet. Again, in position para of the pipera-
zine ring, methyl protons from the -C2H5 protons of compounds
5b, 5g and 5l were observed in the range of 1.01–1.19 ppm, and
-CH2 protons in the range of 2.34–2.78 ppm. The aromatic protons
of the phenyl (5d, 5l and 5n), pyridine (5d, 5l ve 5n) and pyrimi-
dine (5e, 5j and 5o) substituent attached to the piperazine ring
were observed in the range of 6.81–7.69, 6.65–8.14 and
6.67–8.44 ppm, respectively.
5e
5f
-OCH3
-OCH3
-CH3
5g
-C2H5
5h
-OCH3
-OCH3
The –CH3 group protons of compounds 5f, 5g, 5h, 5ı and 5j
on phenyl ring were observed at 3.82–3.87 ppm ppm as a singlet.
The -OC2H5 group of compounds 5k, 5l, 5m, 5n ve 5o on phenyl
ring, –OCH2 protons were observed at 4.11–4.13 ppm as a quar-
tette while for other compounds (5k, 5l,) –OCH2 protons were
appeared as multiplet. The protons of CH3 group of ethyl substitu-
ent were observed 1.35–1.37 ppm as a singlet. Methylene protons
between C¼O and -S group were recorded as a singlet peak
between 4.53–4.65 ppm.
A broad singlet due to NH proton of the benzimidazole ring
was recorded at 10.70–13.50 ppm. The aromatic protons of 4-sub-
stitutedphenyl assigned at 6.90–8.09 and 8.06–8.38 as two dou-
blets. The benzimidazole protons were visualised in the form of
doublet, doublet’s doublet and one singlet in 1H NMR spectra at
around 7.64–7.92, 7.74–8.03 and 7.89–8.22 due to H-4, H-5, and H-
7 protons.
5ı
5j
-OCH3
5k
5l
-OC2H5
-OC2H5
-CH3
-C2H5
5m
5n
-OC2H5
-OC2H5
The 13C NMR spectra showed the peaks between
165.61–169.44 ppm due to carbonyl group (C¼O). The carbons of
the piperazine ring were observed at 39.11–55.91 ppm. The
13CNMR spectra of all the derivatives showed carbon values in the
predictable regions, while the HRMS analysis confirmed the mass
with the calculated values of the target compounds. In addition
for structure elucidations using routine spectroscopic methods,
2 D NMR studies including HMBC, HSQC and COSY were per-
formed for compound 5e as seen in Table 1 and supporting
information.
5o
-OC2H5
Scheme 1. Continued.
3. Result and discussion
3.1. Chemistry
The protocol adopted for synthesis of compounds (5a-5o) was
shown in Scheme 1. In the first step, 2–(4-substitutedphenyl)-1H-
benzo[d]imidazole-6-carboxylic acid (1a-1c) derivatives were
obtained by the reaction of 4-substituted benzaldehyde with 3,4-
diamino benzoic acid using sodium bisulphite in DMF. The com-
pound (1a-1c) was converted to a methyl ester (2a-2c) by simple
esterification reaction followed by treatment with hydrazine
hydrate to obtain 2–(4-substitutedphenyl-1H-benzo[d]imidazole-6-
carbohydrazide (3a-3c). The reaction of the hydrazide derivative
(3a-3c) with carbon disulfide in boiling ethanol and KOH gave 2-
((4-substitutedphenyl)-(6–(5-mercapto-1,3,4-oxadiazol-2-yl)-1H-ben-
3.2. Biological activity
3.2.1. Cytotoxicity assay
The benzimidazole-1,3,4-oxadiazole derivatives (5a-5o) were tested
their cytotoxic effects in vitro using five cancer cell line A549 (lung
carcinoma cell line), HeLa (cervical cell line), MCF-7 (human breast
adenocarcinoma cell line), HepG2 (human liver carcinoma cell line)
and C6 (rat glioma cell line) cell lines as well as NIH3T3 (mouse
embryo fibroblast cell line). Doxorubicin and Hoechst 33342 were
used for this study as a reference drugs. The IC50 values, defined
as the half maximal inhibitory concentration, were summarised in
zo[d]imidazole derivative (4a-4c). At the last reaction step, the Table 2.

1664
U. A. ÇEVIK ET AL.
Investigations of the cytotoxic activity against A549, MCF-7, C6,
HepG-2 and HeLa indicated that the HeLa was the most sensitive
cell line to the influence of the new derivatives. It was important
to note that compounds 5l and 5n exhibited better antiprolifera-
tive activity than Hoechst 33342 and doxorubicin against HeLa
cell line, with IC50 of 0.224 0.011 mM and 0.205 0.010 mM,
respectively. The above MTT assay results confirmed our expect-
ation that compounds 5l and 5n may exhibit effective activity and
lower toxicity and thus possibly better therapeutic potency than
Hoechst 33342. Furthermore, compound 5b exhibited prominent
activity against HeLa cell line with IC50 0.698 0.032 mM and
shows poor activity against the other cell lines with IC50 ꢃ100 mM
Compound 5a emerged as potential compound against both
cell lines with IC50 5.704 0.254 mM against MCF-7 cell line and
IC50 5.695 0.283 mM against HepG2 cell line. IC50 values of com-
pounds 5d, 5e and 5o for A549 cell line were determined as
9.148 0.391, 7.318 0.352 and 6.442 0.287 mM, respectively.
Only compound 5n displayed most promising cytotoxic activity
against A549 cell line with an IC50 value of 7.388 0.325 mM.
Besides, among compounds 5a-5o, compound 5k exhibited the
most promising anticancer activity against C6 cell line with IC50
values of 3.204 0.152 mM.
According to all results, it can be concluded that 4-hydroxy-
phenyl and 4-ethoxyphenyl enhanced anticancer activity more
than 4-methoxyphenyl. The presence of methyl and ethyl group
at the 4th position of the piperazine scaffold also increased anti-
cancer activity, whereas phenyl moiety at the 4th position of the
piperazine ring led to a significant drop in anticancer activity.
However, compounds carrying pyridine and pyrimidine substitu-
ents have also been found to be active.
3.2.2. DNA synthesis inhibition assay
According to the MTT assay, compound 5n for A549 cell line;
compounds 5a, 5d, 5e, 5o for MCF-7 cell line; compound 5k for
C6 cell line; compound 5a for HepG2 cell line; compounds 5b, 5l
and 5n for HeLa cell line were selected for the DNA synthesis
inhibition assay. A549, C6, MCF-7, HepG2 and HeLa cells were
incubated with three different concentrations (IC50/2, IC50, and
IC50/2) of the compounds for 24 and 48 h time periods. The tested
compounds showed time-and dose-dependent inhibitory activity
on DNA synthesis ofthe tumour cells. Doxorubicin was used as
positive control.
Figure 2 shows the DNA % synthesis inhibitory activity of the
compound 5n and standard drug on A549 cells. Compound 5n
was found to have 78.17, 68.25 and 66.00% DNA synthesis inhib-
ition at 14.77, 7.39 and 5.39 mg/mL doses after 48 h of incubation,
It is crucial that an anticancer agent affects the cancer cell line
but having minimal or no side-effect on healthy cells. For this pur-
pose, the cytotoxic effects of the active compounds on the
NIH3T3 cell line were investigated (Table 2).
Table 2. IC50 values (mM) of the compounds 5a-5o, doxorubicin and Hoechst 33342 against A549, MCF-7, C6, HepG2, HeLa and NIH3T3.
Biles¸ik
A549
MCF-7
C6
HepG2
HeLa
NIH3T3
5a
5b
5c
5d
5e
5f
5g
5h
5ı
5j
5k
5l
5m
5n
5o
Dox.
43.256 1.785
ꢃ100
5.704 0.254
ꢃ100
15.614 0.615
ꢃ100
5.695 0.283
ꢃ100
13.960 0.65
0.698 0.032
20.285 1.101
ꢃ100
39.105 1.940
944.360 7.625
44.556 0.221
720.645 5.032
54.026 2.682
–
ꢃ100
ꢃ100
ꢃ100
21.860 0.991
ꢃ100
ꢃ100
9.148 0.391
7.318 0.352
ꢃ100
38.93 1.921
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
–
–
–
–
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
ꢃ100
22.254 1.054
31.017 1.530
ꢃ100
38.705 1.875
ꢃ100
3.204 0.152
ꢃ100
ꢃ100
11.167 0.467
0.224 0.011
ꢃ100
74.630 3.631
113.451 5.472
52.264 2.512
188.570 8.728
44.072 2.104
1110.80 8.254
7.388 0.269
ꢃ100
ꢃ100
32.167 1.552
ꢃ100
ꢃ100
7.388 0.325
ꢃ100
ꢃ100
ꢃ100
0.205 0.010
ꢃ100
6.442 0.287
10.525 0.472
2.404 0.118
24.951 1.237
28.690 1.228
1.051 0.307
59.369 2.589
16.482 0.804
27.815 1.190
12.420 0.521
0.422 0.020
14.280 0.704
0.306 0.015
Hoechst 33342
A-549 Cell Line (48 h)
A-549 Cell Line (24 h)
90
80
70
60
50
40
30
20
10
0
Dox.
5n
Dox.
5n
2xIC50
IC50
IC50/2
Figure 2. % DNA synthesis inhibition activities of compound 5n and doxorubicin against A549 cell line.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1665
MCF-7 Cell Line (24 h)
100
90
80
70
60
50
40
30
20
10
0
5d
5a
5o
2XIC50
5d
5e
5o
5e
Dox.
IC50
IC50/2
Figure 3. % DNA synthesis inhibition activities of compounds 5a, 5d, 5e, 5o and doxorubicin against MCF-7 cell line.
HepG2 Cell Line (48 h)
HepG2 Cell Line (24 h)
90
80
70
60
50
40
30
20
10
0
5a
Dox.
2xIC50
Dox.
5a
IC50
IC50/2
Figure 4. % DNA synthesis inhibition activities of compound 5a and doxorubicin against HepG2 cell line.
whereas doxorubicin was found to have 81.22, 73.43 and 62.78%
inhibition at 24.84, 12.42 and 6.21 mg/mL doses, respectively.
The DNA % synthesis inhibitory activity of the compounds 5a,
5d, 5e, 5o, and standard drug on MCF7 cells is seen in Figure 3.
Among of the compounds, compound 5e was determined as the
most active compounds. Compound 5e was observed to have an
approximate inhibitory activity with a value of 93.81% at 7.318 mg/
mL concentration whereas doxorubicin had 84.60% inhibition at
10.525 mg/mL concentration after 48-h incubation.
Figure 6 shows the DNA % synthesis inhibitory activity of the
compounds 5b, 5l, 5n and standard drug on HeLa cells. DNA %
inhibition was increased with the increasing incubation period (24
and 48 h) for all of the compounds. Compounds 5b, 5l, 5n were
found to have 49.70, 56.01 and 57.65% DNA synthesis inhibition
at 0.698, 0.224 and 0.205 mg/mL doses after 48 h of incubation
whereas doxorubicin was found to have 66.48% inhibition at
14.280 mg/mL doses, respectively.
Figure 4 shows the DNA % synthesis inhibitory activity of the
compound 5a and standard drug on HepG2 cells. Compound 5a
had 60.89 and 72.05% inhibitory activity after 24- and 48-h incu-
bation, at 5.695 mg/mL whereas doxorubicin had 42.80 and
74.22% inhibitions at 16.482 mg/mL.
The DNA % synthesis inhibitory activity of the compound 5k
and standard drug on C6 cells is seen Figure 5. DNA % inhibition
was increased with the increasing incubation period (24 and 48 h)
for the compound 5k and doxorubicin. This increase can be
clearly appreciated in particular for the compound 5k.
Measurement of the inhibition for the compound 5k at lower
3.2.3. Flow cytometric analysis
Annexin V/PI staining on tested cancer cells treated with the
active compounds was performed to investigate induction of
apoptosis. Usually, four quadrant images were generated by flow
cytometry: Q1 shows damaged cells due to any technical factor
rather than compound, Q2 area shows late apoptotic cells, Q3
denotes normal cells while Q4 illustrates early apoptotic cells.
According to the MTT assay, compound 5n for A549 cell line;
compounds 5a, 5d, 5e, 5o for MCF-7 cell line; compound 5k for
C6 cell line; compound 5a for HepG2 cell line; compounds 5b, 5l
dose (IC50/2) after 48 h incubation was eight times higher than at and 5n for HeLa cell line were selected for the flow cytometric
24 h of measurement. analysis. Furthermore, flow cytometry studies were performed for

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U. A. ÇEVIK ET AL.
C6 Cell Line (24 h)
C6 Cell Line (48 h)
100
90
80
70
60
50
40
30
20
10
0
5k
Dox.
2xIC50
Dox.
5k
IC50/2
IC50
Figure 5. % DNA synthesis inhibition activities of compound 5k and doxorubicin against C6 cell line.
HeLa Cell Line (24 h)
HeLa Cell Line (48 h)
80
70
60
50
40
30
20
10
0
5n
5b
5l
5b
Dox
5l
2xIC50
IC50
IC50/2
Figure 6. % DNA synthesis inhibition activities of compounds 5 b, 5 l, 5n and doxorubicin against HeLa cell line.
reference drug doxorubicin. Flow cytometric analysis diagrams of
tested compounds and doxorubicin at IC50/2, IC50, and 2xIC50 con-
centrations on cancer cells were presented in Figures 7–11.
Flow cytometric analysis diagram of doxorubicin with com-
Flow cytometric analysis diagram of doxorubicin and com-
pounds 5b, 5l, 5n on HeLa cell line was presented in Figure 11.
For HeLa cells, compound 5b showed the highest percentage of
17.4% whereas doxorubicin caused 17.4% apoptotic cells. The
other tested compounds 5l and 5n followed it with 8.6 and 15.2%
percentages.
pound 5n was presented in Figure
7 for A549 cell line.
Accordingly, the percentage of apoptotic cells at IC50 concentra-
tions was calculated as 19.2% for doxorubicin and 13% for com-
pound 5n.
3.2.4. DNA topoisomerase I assay
Flow cytometric analysis diagram of doxorubicin with com-
pounds 5a, 5d, 5e and 5o in MCF-7 cell line was presented in
Figure 8. Compound 5d caused the highest percentage of apop-
tosis (early þ late apoptotic cells) with 22.3% whereas doxorubicin
caused 12.7% on MCF-7 cells. The other compounds 5a, 5e and
5o possessed 15.4, 13.1 and 8.7% apoptotic cell percentages.
Flow cytometric analysis diagram of doxorubicin with com-
pound 5k was presented in Figure 9 for C6 cell line. Accordingly,
the percentage of apoptotic cells at IC50 concentrations was calcu-
lated as 13.7% for doxorubicin and 12.3% for compound 5k.
For HepG2 cell line, the percentage of apoptotic cells at IC50
concentrations was calculated as 9.4% for doxorubicin and 13.2%
for compound 5a (Figure 10). As a result, it is observed that
compound 5a induces apoptotic cell death more than doxorubi-
cin in HepG2 cell line in IC50 concentrations and 24 h incuba-
tion time.
To determine whether the DNA intercalation properties of com-
pounds 5a, 5b, 5d, 5e, 5k, 5l, 5n and 5o might result in Topo I
inhibition, agarose gel electrophores is assay was performed to
evaluate the Topo I inhibitory activity of them via agarose-gel
electrophoresis and CPT was used as the positive control. The
TopoGEN Topoisomerase I Drug Screening Kit were optimised and
implemented to accomplish this goal.
The topo I DNA relaxation data demonstrated that the super-
coiled kDNA migrates faster on the agarose gel than the relaxed
topoisomers (nicked) caused by the full activity of topo I. Therefore,
the appearance of relaxed topoisomers along with residual super-
coiled form indicates the partial activity of topoisomerase I and
thus its inhibition by the test compounds. In a previous studies, it
was said that the compounds poisoning topoisomerase caused the
formation of nick bands in such gel appearances, while the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1667
Figure 7. The flow cytometric analysis diagram of compound 5n and doxorubicin for A549 cell line.
formation of Supercoiled bands was said to be caused by catalytic Benzene and imidazole structures of benzimidazole ring forms cat-
inhibitors that bound to either DNA or enzyme40,41
.
ion-p interactions separately with amino of Arg364. Each of the
nitrogen atoms located in the same position of successive benzi-
midazole rings in the structure of Hoechst 33342 creates a hydro-
gen bond with carbonyl of thymine (DT10) in the D chain of DNA-
Topoisomerase enzyme complex. In addition, these interactions
analyse of Hoechst 33342 were found to be similar and compat-
ible with the literature data3.
The results in Figure 12 indicated that compounds 5a, 5b, 5d,
5e, 5k, 5l, 5n and 5o exhibited potent topo I inhibition activity.
By the comparison of the effective inhibited concentrations of
camptothecin with these compounds, it could be concluded that
these compounds exhibited similar topo I inhibition activity with
camptothecin.
The docking poses of compound 5n was given in Figure 14.
When the docking poses of this compound was examined, it was
seen that this compound interact with both amino acids in the
enzyme active region and certain regions of the DNA contained in
the complex crystal.
Thus, it was observed that it cause damage in DNA chain and
prevent the binding of topoisomerase enzyme to DNA (Figure
14(A)). Also, when looking at the chemical structure of the com-
pound, it has been seen that benzimidazole, oxadiazole and pyri-
dine structures show similar interactions. Benzimidazole ring
system has attracted attention in terms of polar and apolar inter-
actions. p–p interactions were observed separately between pur-
ine ring of adenine (DA113) in the DNA chain and benzene and
imidazole rings of benzimidazole in the compound 5n. Also, in
this compound, phenyl ring of benzimidazole forms p–p inter-
action with Arg364. The nitrogen atom in the benzimidazole ring
is found to be important for polar interactions.
3.3. Molecular docking
In order to determine the possible interactions of compound,
showed high activity, docking studies have been performed by
using high-resolution crystal structure of the DNA-Topoisomerase I
enzyme complex (PDB Code: 1T8I)35,42–45. Hoechst 33342 molecule
was included in the docking study with the relevant crystal struc-
ture before performing the analysis of binding modes of selected
compound which has displayed significant topoisomerase
I
enzyme activity (5n). Hence, the docking protocol was verified by
examining and comparing binding modes and interactions of
Hoechst 33342.
Two-dimensional interaction pose of Hoechst 33342 with DNA-
Topoisomerase I enzyme complex active region is given in Figure
13. The oxygen atom in the ethoxy group in the structure forms a
hydrogen bond with amino Arg488. The phenyl ring near to
ethoxy group establishes p-p interaction with imidazole ring of
Hid632. The benzimidazole ring adjacent to phenyl ring has been
The oxadiazole ring in the structure of compound 5n has been
shown to contribute to binding to DNA-Topoisomerase I enzyme
complex active site (Figure 14(B)). The oxadiazole ring forms p–p
found to be very important for both polar and apolar interactions. interaction with pyrimidine of cytosine (DC112) in DNA. It has

1668
U. A. ÇEVIK ET AL.
Figure 8. The flow cytometric analysis diagram of compounds 5d, 5e, 5o and doxorubicin for MCF-7 cell line.
been determined that carbonyl group in the chemical structures thanks to their properties. Hydrogen bond formation with amino
of selected compound is especially important in binding to the group of Ile427 is observed in the compound 5n. Van der Waals
DNA chain (Figure 14(C)). In compound 5n, carbonyl group forms and electrostatic interactions of selected compound with DNA-
a hydrogen bond with amino group in the purine ring of adenine Topoisomerase I enzyme complex active region is given in Figure
(DA113) of DNA. The oxygen atom of ethoxy group in the para 14(D). According to the docking analysis procedure followed in
position for compound 5n forms a hydrogen bond with hydroxyl Van der Waals interactions, red and pink coloured amino acids
of Thr718 in both compounds. An additional cation–p interaction
is observed between amino group of Lys532 and para-ethoxy-
pheny. It is observed that the chemical structure of selected com-
pound whose molecular docking studies are carried out include
pyridine ring adjacent to the piperazine ring. It has been observed
that nitrogen atoms in this ring systems are very effective and
important in binding to related region. As it is known, nitrogen
atom in these ring systems have the ability to make hydrogen
bonds by acting as an electron donor by keeping the unpaired
electron pairs on them outside the ring without including them in
the ring electron system. They strengthen the binding profiles of
and nucleic acids indicate strong van der Waals interactions.
Hereunder, it is seen that Asn352, Glu356, Lys425, Lys532, Asp533
and Ile535 amino acids and DT10, DC112 and DA113 nucleic acids
in DNA play an important role in terms of van der Waals interac-
tions. Strong electrostatic interactions are similarly represented by
red- and blue-coloured amino acids and nucleic acids according
to the docking analysis procedure. In terms of electrostatic inter-
actions, it is seen that Glu356, Arg364, Lys425, Arg488, Lys532,
Asp533 and Thr718 amino acids and DT10, DC112 and DA113
nucleic acids in DNA play an important role.
Table 3 presents the binding affinity and interacting residues
the compound by forming hydrogen bonds with the active region of compound 5n and Hoechst. The binding affinity was calculated

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1669
Figure 9. The flow cytometric analysis diagram of compound 5k and doxorubicin for C6 cell line.
Figure 10. The flow cytometric analysis diagram of compound 5a and doxorubicin for HepG2 cell line.

1670
U. A. ÇEVIK ET AL.
Figure 11. The flow cytometric analysis diagram of compounds 5 b, 5 l, 5n and doxorubicin for HeLa cell line.
Figure 12. Topo I activity of compounds 5a, 5 b, 5d, 5e, 5k, 5 l, 5n, 5o, Hoechst 33342 and camptothecin.
according to the parameters of docking score, Glide gscore and displayed potent antitumor activity in different cancer cell lines.
Glide emodel, and is as follow: for compound 5n ꢁ7.409, ꢁ7.889,
Especially, compounds 5l (0.224 mM) and 5n (0.205 mM) dis-
ꢁ100.082 kcal/mol and for Hoechst ꢁ4.373, ꢁ5.989, ꢁ66.563 kcal/
played potent and selective anticancer activity against HeLa cell
mol. As can be seen from this table, compound 5n showed more
line compared to doxorubicin (14.280 mM) and Hoechst 33342
(0.306 mM). Further detailed biological studies including flow
cytometric analysis, DNA synthesis inhibition assay and Topo I
inhibition studies delivered promising results. Docking studies of
compound 5n and Hoechst 33342 were performed and prob-
able interactions in the DNA-Topo I enzyme complex was deter-
mined. According to all in vitro and in silico studies, novel
benzimidazole-1,3,4-oxadiazole derivatives could be served as a
novel promising scaffold for the development of new chemo-
potent binding affinity than Hoechst.
4. Conclusion
In conclusion, 15 novel benzimidazole-1,3,4-oxadiazole deriva-
tives were synthesised and evaluated for their anticancer effects
on A549, MCF-7, C6, HepG2 and HeLa cell lines. The results of
this preliminary cytotoxicity screening using the MTT assay sug-
gested that many of them (5a, 5b, 5d, 5e, 5k, 5l, 5n and 5o) therapeutic agent.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1671
Figure 13. Three-dimensional interaction of Hoechst 33342 with the DNA-Topoisomerase I enzyme complex active site.
Figure 14. Docking poses of compound 5n. (A) Placement of the compound 5n on the DNA-Topoisomerase I enzyme complex active site. (B) The interacting mode of
compound 5n in the active region of DNA-Topoisomerase I enzyme. (C) Van der Waals interaction of DNA-Topoisomerase I enzyme complex active site of compound
5n. (D) Electrostatic interaction of DNA-Topoisomerase I enzyme complex active site of compound 5n.

1672
U. A. ÇEVIK ET AL.
Table 3. Binding affinity and interacting residues of compound 5n and Hoechst.
Binding affinity (Kcal/mol)
Interacting residues
Compounds
Docking score
Glide gscore
Glide emodel
(According to Van der Waals, coulomb, hydrogen-bonding energies)
5n
ꢁ7.409
ꢁ7.889
ꢁ100.082
Asn352, Glu356, Arg364, Lys374, Lys425, Arg488, Lys493, Gly531, Lys532,
Asp533, Ile535, Lys587, Arg590, Hid632, Gly717, Thr718, Asn722, DA7,
DC8, DT9, DT10, DC111, DC112, DA113, DA114, DG115, DT116
Asn352, Glu356, Phe361, Arg364, Lys374, Gln421, Ser423, Lys425, Arg488,
Lys493, Lys532, Asp533, Lys587, Arg590, Hid632, Gly717, Thr718, Leu721,
Asn722, DA7, DC8, DT9, DT10, DC111, DC112, DA113, DA114,
DG115, DT116
Hoechst
ꢁ4.373
ꢁ5.989
ꢁ66.563
7. Shu B, Yu Q, Hu DX, et al. Synthesis and biological evalu-
ation of novel indole-pyrazoline hybrid derivatives as poten-
tial topoisomerase 1 inhibitors. Bioorg Med Chem Lett 2020;
30:126925.
Disclosure statement
The authors declare no conflicts of interest.
Funding
8. Sharma PC, Bansal KK, Sharma A, et al. Thiazole-containing
compounds as therapeutic targets for cancer therapy. Eur J
Med Chem 2020;188:112016.
9. Lee JF, Chang TY, Liu ZF, et al. Design, synthesis, and bio-
logical evaluation of heterotetracyclic quinolinone deriva-
tives as anticancer agents targeting topoisomerases. Eur. J.
Med. Chem 2020;190:112074.
10. Joshi G, Kalra S, Yadav UP, et al. E-pharmacophore guided
discovery of pyrazolo [1,5-c] quinazolines as dual inhibitors
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2020;94:103409.
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This study was financially supported by Anadolu University
Scientific Projects Fund, Project No: 2005S036 and 1706S381.
ORCID
Ulviye Acar C¸evik
http://orcid.org/0000-0003-1879-1034
€
ꢀ
Begum Nurpelin Saglık
http://orcid.org/0000-0002-0151-6266
http://orcid.org/0000-0002-0499-436X
http://orcid.org/0000-0003-3692-163X
Derya Osmaniye
Serkan Levent
€
ꢀ
Betul Kaya C¸avus¸oglu
http://orcid.org/0000-0001-8270-4949
Abdullah Burak Karaduman
http://orcid.org/0000-0002-
0434-1334
12. Aichinger G, Lichtenberger FB, Steinhauer TN, et al. The Aza-
Analogous Benzo [c] phenanthridine P8-D6 Acts as a Dual
Topoisomerase I and II Poison, thus Exhibiting Potent
Genotoxic Properties. Molecules 2020;25:1524.
€
Ozlem Atlıd
http://orcid.org/0000-0002-6131-3399
€
ꢀ
Ozlem Atlı Eklioglu
http://orcid.org/0000-0001-8815-153X
http://orcid.org/0000-0003-2252-0923
Zafer Asım Kaplancıklı
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