An efficient synthesis of novel di-heterocyclic benzazole derivatives and evaluation of their antiproliferative activities

Article abstract of DOI:10.1080/07391102.2020.1803966

A series of unsymmetrical nine di-heterocyclic compounds of benzazole derivatives were synthesized at one step via cyclization reaction. The compounds evaluated for in?vitro cytotoxic activity against A549, A498, HeLa, and HepG2 cancer cell lines. The biological evaluation results show that 23, 26 and 29 exhibit better activity against HepG2 and HeLa cancer cell lines. Compound 23 also showed good activity against A549, and A498 cancer cell lines. The analogs were further performed molecular docking studies against human cytochrome P450 2C8 monooxygenase enzyme, calculated some theoretical quantum parameters, ADMET descriptor and molecular electrostatic potential analysis. The strategy applied in this research work may act as a perspective for the rational design of potential anticancer drugs. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1803966

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
An efficient synthesis of novel di-heterocyclic
benzazole derivatives and evaluation of their
antiproliferative activities
Oztekin Algul , Ronak Haj Ersan , Mehmet Abdullah Alagoz , Nizami Duran &
Serdar Burmaoglu
To cite this article: Oztekin Algul , Ronak Haj Ersan , Mehmet Abdullah Alagoz , Nizami Duran &
Serdar Burmaoglu (2020): An efficient synthesis of novel di-heterocyclic benzazole derivatives and
evaluation of their antiproliferative activities, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1803966
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1803966
An efficient synthesis of novel di-heterocyclic benzazole derivatives
and evaluation of their antiproliferative activities
Oztekin Algula, Ronak Haj Ersana, Mehmet Abdullah Alagozb, Nizami Duranc and Serdar Burmaoglud
b
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin, Turkey; Department of Pharmaceutical
c
Chemistry, Faculty of Pharmacy, Inonu University, Malatya, Turkey; Department of Medical Microbiology, Medical Faculty, Mustafa Kemal
d
€
University, Antakya-Hatay, Turkey; Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 11 June 2020
Accepted 26 July 2020
A series of unsymmetrical nine di-heterocyclic compounds of benzazole derivatives were synthesized
at one step via cyclization reaction. The compounds evaluated for in vitro cytotoxic activity against
A549, A498, HeLa, and HepG2 cancer cell lines. The biological evaluation results show that 23, 26 and
29 exhibit better activity against HepG2 and HeLa cancer cell lines. Compound 23 also showed good
activity against A549, and A498 cancer cell lines. The analogs were further performed molecular dock-
ing studies against human cytochrome P450 2C8 monooxygenase enzyme, calculated some theoretical
quantum parameters, ADMET descriptor and molecular electrostatic potential analysis. The strategy
applied in this research work may act as a perspective for the rational design of potential anticancer
drugs.
KEYWORDS
Di-heterocyclic structure;
benzimidazole; benzothia-
zole; benzoxazole;
benzazole; polyphosphoric
acid; antiproliferative
activity; molecular
docking; ADMET
Abbreviations: dppb: 1,4-bis(diphenylphosphino)butane; DMF: N,N-dimethylformamide; DMSO:
dimethyl sulfoxide; HepG2: human hepatocellular cancer cell line; A498: human renal cancer cell line;
A549: human lung adenocarcinoma epithelial cancer cell line; HeLa: human cervical cancer cell line;
ADME: absorption, distribution, metabolism, and excretion; ADMET: absorption, distribution, metabol-
ism, excretion and toxicity; MEP: molecular electrostatic potential; MM2: molecular mechanics method;
DFT: density functional theory; HOMO: highest occupied molecular orbital; LUMO: lowest unoccupied
molecular orbitals; Egap: energy gap; EA: electron affinity energy; QSAR: quantitative structure–activity
relationship; LOO: leave-one-out; Sreg: standard deviation of regression; F: Fisher’s F value; q2: correl-
ation coefficient; ROS: reactive oxygen species; XP: extra precision; TLC: thin layer chromatography;
PPA: polyphosphoric acid; IC50: the 50% inhibitory concentration; eq: equivalent; Vero: African green
monkey kidney epithelial; MTX: methotrexate
1. Introduction
antioxidants and cancer in clinical studies. Oxidative stress
plays an important role in the pathogenesis of cancer. The
levels of reactive oxygen species (ROS) and the capacity of
oxidative defenses are well balanced in the cells of healthy
individuals. Also, many factors can disrupt this balance and
increase the risk of developing cancer (Himmetoglu et al.,
2009). Therefore, drugs possessing antioxidant and free rad-
ical scavenging properties are considered for preventing
and/or treatment of tumor diseases which are directly
related to the lack of the antioxidant capacity of
the organism.
Bis- and di-heterocyclic benzazole compounds exhibit
more potent activities, such as antitumor (Mabkhot et al.,
2013; Thurston et al., 1996) and antimicrobial activities
(Shaker, 1999) than monomeric compounds owing to their
two heterocyclic nuclei. For instance, UK-1 (1) and AJI95618
(2) are di-heterocyclic benzoxazole derivatives and Ueki et al.
The incidence and mortality of cancer are rapidly growing
worldwide. According to the World Health Organization, the
global cancer burden was estimated to have been 18.1 mil-
lion new cases and 9.6 million deaths in 2018 (Siegel et al.,
2019). In cancer drug discovery programs, researchers have
used many novel synthetic compounds as anticancer drugs.
Benzazole derivatives (e.g. benzimidazole, benzothiazole,
and benzoxazole) have a wide range of biological applica-
tions (Noel et al., 2013). The antitumor activity of benza-
zoles is well documented (Liu et al., 2019; Madia et al.,
2018) and there have been several reports in which the
benzazole nucleus is modified to improve the antitumor
activity (Aiello et al., 2008; Dubey et al., 2006; White et al.,
2004). In addition, benzazoles have been reported in the lit-
erature to increase the antioxidant level of the organism
(Can-Eke et al., 1998; Cressier et al., 2009; Han et al., 2018).
In the literature, there is
a
relationship between and Sato et al. have demonstrated the cytotoxic activity of
CONTACT Oztekin Algul
oztekinalgul@mersin.edu.tr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin,
33169, Turkey
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1803966.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
O. ALGUL ET AL.
Figure 1. Structure of the previously synthesized di-heterocyclic benzazole derivatives with antiproliferative activity.
Scheme 1. Some synthesis methods of di-heterocyclic benzazoles. Reaction conditions: (i) [A ¼ NH, Y ¼ –CH2SCH2–], 6 M HCl, reflux, 72 h (ii) NH3 (pH-9) (iii) con-
centrated HCl (to pH ¼ 7) (iv) [B ¼ O, S], 4 M HCl, refluxed 17 h, NH3 (pH ¼ 9) (v) recrystallized from ethanol/charcoal and water (vi) [A ¼ S, Y ¼ –CH2–] n-BuLi (vii)
[B ¼ NH, O], polyphosphoric acid, 180 ꢀC (viii) [A ¼ –S–, meta disubstituted –Ar–], DMSO, 180 ꢀC (ix) [B; NMe, –O–], PdCl(C3H5)dppb], Cs2CO3, DMF, 150 ꢀC (x) [A,
B ¼ NH, S, O Y ¼ without linker] Co/Cu.
these against different cancer cell lines (Figure 1) (Ueki et al., Dang et al. reported examples of di-heterocyclic compounds
1993; Sato et al., 2001). Hoechst 33258 (3) is a benzimidazole which were prepared by
a two-step procedure using
di-heterocyclic compound that has some antitumor activity PdCl(C3H5)dppb (dppb:1,4-Bis(diphenylphosphino)butane)
against murine leukemias and P388 tumors; however, a and cesium carbonate (Cs2CO3) as catalysts (Dang et al.,
phase II trial against pancreatic cancer was abandoned on 2016). Li et al. discovered that cobalt and copper can be co-
ꢀ
account of toxicity (Jenkins, 2000; Singh et al., 1992). Racane catalyzed in cross-dehydrogenative coupling reactions of
et al. illustrated that imidazole-substituted bis-benzothiazole benzazoles using air as an oxidant which leads to the forma-
derivative (4) acts as an antiproliferative agent (Figure 1) tion of symmetrical and unsymmetrical di-heterocyclic com-
ꢀ
(Racane et al., 2012).
A cyclocondensation reaction of two equivalents of 1,2- reaction conditions to obtain di-heterocyclic compounds of
phenylenediamine/2-aminophenol/2-mercaptoaniline with benzimidazole and benzothiazole using a Cu(II)-containing
pounds (Li et al., 2019). In the literature, reported different
one equivalent of bisimidate, malonic dinitrile, or dicarboxylic nano silica triazine dendrimer (Cu(II)-TD@nSiO2) and rhodium
acid is well-known for the synthesis of symmetrical bisbenza- as the catalyst (Nasr-Esfahani et al., 2013; Wu et al., 2017).
zole derivatives (Dauer et al., 2016; Elagab & Alt, 2015; Dauer et al. treated aniline derivatives with nitrile derivatives
Kretsch et al., 2019;). Although, some methods are used for in a two-step procedure for the synthesis of di-heterocyclic
the preparation of symmetrical bis-benzazole derivatives, compounds (Dauer et al., 2017). But, Matthews et al. faced
there are few methods in the literature for use in the synthe- difficulties to obtain unsymmetrical compounds using con-
sis of symmetrical di-heterocyclic derivatives.
centrated hydrochloric acid condensation reaction (Matthews
In the synthesis of di-heterocyclic compounds from benza- et al., 1996).
zole derivatives, there are difficulties in selectivity between
Nevertheless, these strategies have deficiencies because
self-binding and cross-linking products (Monguchi et al., hazardous and expensive ligands and catalysts are employed
2010). Regardless of these challenges, different strategies with low yields. The main difficulties in this area are that sim-
have been used to obtain unsymmetrical cross-coupled di- ple, economical and accessible strategies have not been
heterocyclic benzazole products, as shown in Scheme 1.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Figure 2. The molecular design strategy for the synthesis of novel di-heterocyclic benzazole derivatives bearing aliphatic linkers.
developed for the synthesis of high-yield and asymmetric determining ADMET properties before synthesizing drug can-
cross-linked di-heterocyclic products.
didate molecules is very important. In this study, ADMET
properties of the compounds were determined using
QikProp (Maestro 11.8) PRE-ADMET and DATAWARRIOR
4.07.02 softwares.
The strong to excellent antitumor potential of di-benza-
zole derivatives has fascinated our group. Thus, we synthe-
sized some di-heterocyclic compounds with different
aliphatic linkers between heterocyclic rings (Figure 2) and
evaluated their antiproliferative activity in human hepatocel-
lular (HepG2), human renal cancer (A498), human lung
adenocarcinoma epithelial (A549), and human cervical cancer
(HeLa) cell lines. Additionally, we tested the role of linker
groups between benzazole rings in regard to selectivity and
antiproliferative activity.
2.3. QSAR analysis
QSAR studies have been performed on the HepG2 cell line
where the compounds have the best activity. As a result of
DFT calculations, various quantum-chemical descriptors
(HOMO, LUMO, total dipole moment etc.) were obtained. In
addition, physicochemical parameters of the compounds
were determined by using QikProp (Maestro 11.8) software.
Correlation analysis was carried out to determine the
molecular descriptors associated with the antiproliferative
activities of the compounds. In order to develop the optimal
QSAR equation, molecular descriptors with higher correlation
to activity were determined for multiple linear regression
analysis. Then, the ’leave-one-out’ (LOO) cross-validation
method, which is one of the most frequently used methods
to edit the QSAR equation, was used. As a result of linear
regression analysis, R2 (square of correlation coefficient of
It is important that the prediction of ADME properties of
all the compounds, molecular reactivity analyses, molecular
electrostatic potential (MEP) analysis, and molecular docking
studies were also performed.
2. Material and methods
2.1. DFT/B3LYP calculations
The 3D structures of the synthesized compounds were drawn
with Chem3D and the molecular mechanics MM2 method
was used in the same program to determine the lowest
energy conformations of each molecule. Compounds are
optimized using Density Functional Theory (DFT) calculations.
Theoretical calculations of all synthesized compounds were
carried out with the Gaussian 09 W program. The calculations
were done using the (DFT)/B3LYP method, 6-311 G (d, p)
base set for H, C, N, O, S atoms, HOMO (highest occupied
molecular orbital), LUMO (lowest unoccupied molecular orbi-
tals), HOMO-LUMO energy gap (Egap). Electron affinity energy
regression), R2 (is the square of adjusted correlation coeffi-
A
cient), r (root-mean-square deviation), Sreg (standard devi-
ation of regression), F (Fisher’s F value) q2 (correlation
coefficient) and
were calculated.
p
(p value using the
F
statistics)
2.4. Molecular docking
All molecular docking studies were performed with Maestro
(EA ¼ ꢁELUMO), Ionization potential energy (IP ¼ ꢁEHOMO
)
€
11.8 (Schrodinger, LLC, NY). In these studies, HepG2 was per-
and molecular electrostatic potential (MEP) analysis of the
optimized structures were also calculated with this program
and method. Visualization of the results was done through
GaussView 5.0.8.
formed, where the compounds were the most active and the
specificity values were highest. For these cell lines, 2NNI pdb
coded crystal structures of the target proteins were obtained
from the protein data bank (www.rscb.org/). Solvent mole-
cules, ligands and segments in crystal structures have been
deleted, hydrogens have been added, charges have been
assigned, polar hydrogens have been removed using the
2.2. ADMET properties
One of the most important problems that the drug did not
develop the molecule is that the compounds do not have
good pharmacokinetic properties. Although many synthe-
sized compounds may have high activity, they cannot reach
clinical use due to some pharmacokinetic properties. In add-
ition, the compounds have various toxic effects makes it dif-
€
€
software Prime (Schrodinger, LLC, NY), Impact (Schrodinger,
€
LLC, NY), Epik (Schrodinger, LLC, NY) and Propka. Molecules
were prepared and minimized with LigPrep (Maestro 11.8)
software. Grid maps of the active regions of proteins were
created using Maestro’s receptor grid generation panel and
ficult for these compounds to be drug molecules. Therefore, the remaining ligands were docked to these maps 70 times

4
O. ALGUL ET AL.
Scheme 2. Synthesis of di-heterocyclic benzazole derivatives (12, 23-30).
€
in extra precision (XP) mode using Glide (Schrodinger, LLC,
NY) software.
3.1.3. 2-(Benzothiazol-2-ylmethyl)benzoxazole (23)
1
The above procedure was followed to yield 23. The H NMR
and 13C NMR spectra are in agreement with reported data
(Dauer et al., 2017).
3. Experimental
3.1. Chemistry
3.1.4. 2-((1H-benzimidazol-2-yl)methyl)benzothiazole (24)
1
The above procedure was followed to yield 24. The H NMR
General Commercial grade reagents and solvents were used
without further purification. The purity of all compounds was
judged by TLC analysis (single-spot/two-solvent systems)
using a UV lamp. Melting points were measured with
Gallenkamp melting point devices. 1H NMR and 13C NMR
spectra were taken on 400 and 100 MHz spectrometer with
tetramethyl silane (TMS) as an internal standard, and chem-
ical shifts were recorded in ppm values. The IR spectra were
obtained on Perkin Elmer Spectrum One FT-IR spectrometer.
Elemental analysis results were obtained on a Leco CHNS-
932 instrument.
and 13C NMR spectra are in agreement with reported data
(Chuiguk & Fedotov, 1981).
3.1.5. 2-(2-(1H-benzimidazol-2-yl)ethyl)benzoxazole (25)ꢁ1
Light brown powder, yield 73%. mp ¼ 160 ꢀC; IR (KBr, cm
)
mmax 3453, 3058, 2930, 1569, 1459, 831, 783; 1H NMR
(400 MHz, CDCl3) d 7.47 (dd, J ¼ 3.16, 5.96 Hz, 2H, Ar–H),
7.18–7.13 (m, 4H, Ar–H), 7.01 (d, J ¼ 8.39 Hz, 2H, Ar–H),
3.14–3.07 (m, 4H, –CH2); 13C NMR (100 MHz, CD3OD) d 157.6,
156.3, 133.1, 133.0, 131.0, 130.4, 130.3, 127.9, 116.6, 116.0,
114.7, 33.4, 30.2; Anal. calcd for C16H13N3O: C, 72.99; H, 4.98;
N, 15.96; O, 6.08; Found: C, 72.83; H, 4.89; N, 16.03.
3.1.1. General procedure for the synthesis of (12, 23-30)
A mixture of aniline derivatives (5, 6, 13) (1 eq) and the cor-
responding dicarboxylic acid derivatives (7, 14, 15) (1 eq)
were heated for a period of 6–8 h in PPA at 120 ꢀC then the
other aniline derivatives (5, 6, 13) (1 eq) were added to the
reaction mixture and the mixture was stirred additionally for
12–14 h at 180 ꢀC. The reaction mixture was poured onto ice
water and neutralized by mixing with 5 M NaOH till slightly
basic pH (8–9) to get the precipitate. The resulting precipi-
tate was filtered off and washed with cold water.
Recrystallized with a suitable solvent. The resulting crystalline
compounds were filtered and the vacuumed product was
dried (Scheme 2).
3.1.6. 2-(2-(Benzothiazol-2-yl)ethyl)benzoxazole (26)
Red powder, yield 71%. mp ¼ 194 ꢀC; IR (KBr, cmꢁ1) mmax
3053, 2925, 1518, 1427, 1144, 833, 732; 1H NMR (400 MHz,
DMSO-d6) d 7.99 (t, J ¼ 7.20 Hz, 1H, Ar–H), 7.83 (d, J ¼ 7.95 Hz,
1H, Ar–H), 7.69 (dd, J ¼ 3.51, 9.35 Hz, 1H, Ar–H), 7.54–7.40 (m,
2H, Ar–H), 7.33–7.28 (m, 3H, Ar–H), 3.80–3.71 (m, 2H, –CH2),
3.62–3.54 (m, 2H, –CH2); 13C NMR (100 MHz, DMSO-d6) d
162.1, 159.7, 156.8, 136.2, 135.5, 130.1, 130.0, 125.3, 115.7,
115.2, 115.0, 31.8, 29.6; Anal. calcd for C16H12N2OS: C, 68.55;
H, 4.31; N, 9.99; O, 5.71; S, 11.44; Found: C, 68.66; H, 4.08; N,
10.05; S, 11.59.
3.1.2. 2-((1H-benzimidazol-2-yl)methyl)benzoxazole (12)
Red powder, yield 72%. mp ¼ 180 ꢀC; IR (KBr, cmꢁ1) mmax
3400, 3029, 2973, 1653, 1523, 1449, 843, 762; 1H NMR
(400 MHz, CDCl3) d 11.11 (s, 1H, NH), 8.02 (d, J ¼ 8.12 Hz, 1H,
Ar–H), 7.81–7.69 (m, 2H, Ar-H), 7.52 (t, J ¼ 7.72 Hz, 1H, Ar–H),
7.39 (t, J ¼ 7.63 Hz, 2H, Ar–H), 7.26–7.22 (m, 2H, Ar–H), 4.76
(s, 2H, –CH2); 13C NMR (100 MHz, CD3OD) d 155.3, 153.4,
152.8, 139.0, 138.97, 138.92, 127.6, 117.4, 117.2, 117.0, 65.7;
Anal. calcd for C15H11N3O: C, 72.28; H, 4.45; N, 16.86; O, 6.42;
Found: C, 72.10; H, 4.37; N, 17.05.
3.1.7. 2-(2-(1H-benzimidazol-2-yl)ethyl)benzothiazole (27)
Light brown crystalline, yield 75%. mp ¼ 198 ꢀC; IR (KBr,
cmꢁ1) mmax 3300, 3016, 2904, 1514, 817, 624; 1H NMR
(400 MHz, DMSO-d6) d 7.89 (d, J ¼ 1.66 Hz, 1H, Ar–H), 7.80 (d,
J ¼ 8.77 Hz, 1H, Ar–H), 7.55 (dd, J ¼ 1.98, 8.74 Hz, 1H, Ar–H),
7.35–7.27 (m, 4H, Ar–H), 7.25–7.21 (m, 1H, Ar–H), 3.46 (d,
J ¼ 7.76 Hz, 2H, –CH2), 3.26 (d, J ¼ 7.82 Hz, 2H, –CH2); 13C
NMR (100 MHz, DMSO-d6) d 157.1, 139.7, 130.7, 130.2, 130.1,
128.3, 128.2, 123.6, 115.8, 32.2, 30.0; Anal. calcd

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
forC16H13N3S: C, 68.79; H, 4.69; N, 15.04; S, 11.48; Found: C, solution was added to each well. The plates were gently
68.65; H, 4.75; N, 14.97; S, 11.59.
agitated using mechanical plate mixer until the color reac-
tion was uniform and the OD570 was determined using
micro plate reader. The 50% inhibitory concentration (IC50)
was defined as the concentration that reduced the absorb-
ance of the untreated wells by 50% of vehicle in the
MTT assay.
The cell viability was expressed as percentage of the
viable cells in each sample with respect to the control wells.
Three independent experiments in triplicates were done for
the determination of the growth inhibition of each com-
pound. The IC50 values were calculated from concentration-
3.1.8. 2-(3-(1H-benzimidazol-2-yl)propyl)benzoxazole (28)
White powder, yield 78%. mp ¼ 201 ꢀC; IR (KBr, cmꢁ1) mmax
3407, 3245, 3063, 2954, 1523, 1414, 840, 732; 1H NMR
(400 MHz, CDCl3) d 9.96 (s, 1H, N), 7.70–7.49 (m, 2H, Ar–H),
7.29 (dd, J ¼ 3.25, 5.99 Hz, 2H, Ar–H), 7.13–6.85 (m, 4H, Ar–H),
3.11–3.03 (m, 2H, –CH2), 2.59–2.51 (m, 2H, –CH2), 2.28–2.18
(m, 2H, –CH2); 13C NMR (100 MHz, CDCl3) d 165.5, 151.1,
138.7, 135.1, 125.4, 118.9, 110.6, 27.8, 23.7, 21.7; Anal. calcd
for C17H15N3O: C, 73.63; H, 5.45; N, 15.15; O, 5.77; Found: C,
73.75; H, 5.38; N, 15.22.
response
curves
using
the
SPSS
(SPSS.
Inc.
Chicago) Software.
3.1.9. 2-(3-(Benzothiazol-2-yl)propyl)benzoxazole (29)
Beige powder; yield 74%. mp ¼ 190 ꢀC; IR (KBr, cmꢁ1) mmax
3049, 2975, 1571, 1455, 836, 747; 1H NMR (400 MHz, DMSO-
d6) d 7.99–7.28 (m, 8H, Ar–H), 3.32–3.26 (m, 2H, –CH2),
3.16–3.09 (m, 2H, –CH2), 2.55–2.47 (m, 2H, –CH2); 13C NMR
(100 MHz, DMSO-d6) d 157.5, 156.1, 156.0, 137.7, 135.0, 134.9,
128.9, 128.1, 125.6, 121.3, 32.8, 30.4, 20.6; Anal. calcd for
C17H14N2OS: C, 69.36; H, 4.79; N, 9.52; O, 5.43; S, 10.89;
Found: C, 69.25; H, 4.68; N, 9.59; S, 10.75.
4. Results and discussion
4.1. Chemistry
A synthetic strategy was developed for the formation of di-
heterocyclic compounds. The main framework was a bis-
benzazole compound in which the two aromatic ring struc-
tures are different. In the first method to optimize the syn-
thesis conditions of unsymmetrical cross-coupled di-
heterocyclic derivatives, 1,2-phenylenediamine (5) (1 eq), 2-
aminophenol (6) (1 eq) and malonic acid (7) (1 eq) in the
presence of polyphosphoric acid (PPA) were reacted at
180 ꢀC for 14 h according to previously reported method-
ology (Chhonker et al., 2009; Elagab & Alt, 2015; Peng
et al., 2016; Sun et al., 2018). A mixture of 2-(1H-benzimida-
zol-2-yl)acetic acid (8), 2-(benzoxazol-2-yl)acetic acid (9),
bis(benzoxazol-2-yl)methane (10), bis(1H-benzimidazol-2-
yl)methane (11), and 2-((1H-benzimidazol-2-yl)methyl)ben-
zoxazole (12) at 7%, 5%, 18%, 20% and 12% yields, respect-
ively was obtained (Route A). In the second method, the
reaction of 1,2-phenylenediamine (5) (1 eq) and malonic
acid (7) (1 eq) in the presence of PPA at 180 ꢀC was carried
out for 12–14 h and bis(1H-benzimidazol-2-yl)methane (11)
and 2-(1H-benzimidazol-2-yl)acetic acid (8) were obtained at
30% and 36% yields, respectively (Route B). The last reac-
tion method which was carried out at 120 ꢀC in PPA pro-
duced 2-(1H-benzimidazol-2-yl)acetic acid (8) at a good
yield of 72% (Route C) (Scheme 3).
After some preliminary experiments the best yield of the
desired di-benzazole derivatives (12, 23-30) was obtained
when the dicarboxylic acid derivatives (7, 14, 15) (1 eq)
were treated with aniline derivatives (5, 6, 13) (1 eq) at
120 ꢀC in 6–8 h without isolation. Then, the corresponding
aniline derivatives (13, 5, 6) (1 eq) were added to the reac-
tion mixture in 12–14 h at 180 ꢀC (Route C and D). This
method was an ideal strategy, thereby greatly reducing the
number of reaction steps and increasing the reaction yield
(Scheme 3).
3.1.10. 2-(3-(1H-benzimidazol-2-yl)propyl)benzothiazole (30)
Yellow powder, yield 76%. mp ¼ 189 ꢀC; IR (KBr, cmꢁ1) mmax
3383, 2981, 2918, 1603, 1511, 1232, 801, 752; 1H NMR
(400 MHz, d6-DMSO) d 8.06 (d, J ¼ 7.90 Hz, 1H, Ar–H), 7.95 (d,
J ¼ 8.10 Hz, 1H, Ar–H), 7.51–7.47 (m, 3H, Ar–H), 7.41 (t,
J ¼ 7.56 Hz, 2H, Ar–H), 7.14 (d, J ¼ 3.11 Hz, 1H, Ar–H), 3.24 (t,
J ¼ 7.47 Hz, 2H, –CH2), 2.99 (t, J ¼ 7.47 Hz, 2H, –CH2),
2.39–2.31 (m, 2H, –CH2); 13C NMR (100 MHz, DMSO-d6) d
157.4, 138.0, 135.0, 129.1, 128.9, 128.0, 126.5, 123.5, 32.6,
30.4, 20.6; Anal. calcd for C17H15N3S: C, 69.60; H, 5.15; N,
14.32; S, 10.93; Found: C, 69.51; H, 5.28; N, 14.41; S, 10.87.
3.2. Biological studies
3.2.1. Antiproliferative activity
The synthesized compounds were tested in vitro for their
cytotoxic properties against tumor cell lines panel by using
MTT assay Mosmann’s method. The MTT assay is based on
the reduction of the soluble MTT (0.5 mg mL–1.100 mL),
into a blue-purple formazan product mainly by mitochon-
drial reductase activity inside living cells (Mosdam, 1983).
The cells used in cytotoxicity assay were cultured in RPMI
1640 medium supplemented with 10% fetal calf serum,
Penicillin, and streptomycin at 37 ꢀC and humidified at 5%
CO2. Briefly cells were placed on 96-well plates at 100 mL
total volume with density of 1–2.5 ꢂ 104 cells per mL and
were allowed to adhere for 24 h before treatment with
tested drugs in DMSO solution (10-5.10-6.10-7 mol L-1 final
concentration). Triplicate wells were treated with media
and agents. Cell viability was assayed after 96 h of continu-
ous drug exposure with a tetrazolium compound. The
The optimized reaction conditions were then applied to
the synthesis of a series of unsymmetrical cross-coupled di-
supernant medium was removed and 150 mL of DMSO heterocyclic derivatives (12, 23-30) (Scheme 3).

6
O. ALGUL ET AL.
Scheme 3. Optimization of reaction conditions.
tested cancer cell lines. Compounds with benzoxazole and
benzothiazole moieties (23, 26, and 29) were the most
potent antiproliferative agents against HepG2 and HeLa can-
cer cell lines with IC50 values of 7.81 mM and 15.63 mM.
respectively. Compound 23 was two times more selective
towards HepG2 (IC50. 7.81 lM) than towards Vero. Similarly,
compound 27 was two times more selective towards the
HepG2 cancer cell line than the Vero cell line with IC50 val-
ues of 15.63 lM and 31.25 lM, respectively (Table 1).
Among the synthesized derivatives compound 23 showed
good selectivity and antiproliferative activity with IC50 values
of 7.81 and 15.63 lM against A498 and Vero cell lines,
respectively; thus compound 23 was two times more select-
4.2. Antiproliferative activity
All synthesized compounds (12, 23–30) were evaluated for
their in vitro antiproliferative activity against four human can-
cer cell lines by comparing the results with the standard
antiproliferative drug methotrexate. The in vitro antiprolifera-
tive screening of the compounds against HepG2, A498, A549,
and HeLa was performed using the MTT assay (Mosdam,
1983). The results of the in vitro antiproliferative activities are
summarized in Table 1. The selectivity of these compounds
towards cancerous cells was evaluated against Vero (African
green monkey kidney epithelial) cells to study their cyto-
toxic nature.
The synthesized compounds displayed good antiprolifera- ive towards the A498 cancer cell line than towards the Vero
tive activities against the tested human cancer cell lines. Di- line. Additionally, compound 27 showed good selectivity
heterocyclic compounds with benzoxazole and benzothiazole
rings were observed as the most potent antiproliferative
towards A498. with an IC50 value of 15.63 mM.
Compounds with a methylene linker (23 and 24) were
agents against the tested cancer cell lines. Among the syn- good antiproliferative agents against the A549 cancer cell
thesized compounds benzothiazole derivatives (23 and 27) line with an IC50 value of 15.63 mM and the selectivity was
showed promising antiproliferative activity against all the observed with benzoxazole and benzothiazole rings bearing

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Table 1. Antiproliferative activity of the synthesized benzazole derivatives.
Specificity
HeLa
Vero
HepG2
A498
IC50 (lM)
A549
Compound No
HepG2
A498
A549
HeLa
12
23
24
25
26
27
28
29
30
MTX
31.25 1.28
15.63 1.24
3.91 1.58
7.81 0.67
3.91 1.61
31.25 0.34
7.81 1.10
3.91 1.86
7.81 2.12
0.030 0.41
125 4.52
7.81 1.93
15.61 2.54
15.3 0.98
7.81 1.05
15.63 2.28
15.63 3.36
7.81 1.24
15.63 2.04
0.041 0.12
62.5 1.85
7.81 1.04
15.63 0.56
31.25 1.96
31.25 0.99
15.63 1.58
15.63 0.75
15.63 1.08
31.25 2.28
0.016 0.28
125 2.42
15.63 1.89
15.63 2.36
31.25 2.35
31.25 1.37
31.25 2.42
31.25 1.67
31.25 0.72
31.25 2.89
0.025 0.53
62.5 3.02
15.63 1.12
31.25 1.42
31.25 2.13
15.63 1.93
31.25 1.56
15.63 2.65
15.63 0.47
15.63 1.62
0.022 0.36
0.25
2.00
0.25
0.5
0.5
2.00
0.5
0.5
0.5
0.73
0.5
0.25
1.00
0.25
0.25
0.12
1.00
0.25
0.12
0.25
1.20
0.5
1.00
0.12
0.25
0.25
1.00
0.5
0.25
0.5
1.36
2.00
0.25
0.25
0.12
2.00
0.5
0.25
0.25
1.87
Table 2. Calculated quantum-chemical descriptors.
Compounds
HOMO
LUMO
Gap (DE)
IP(-EHOMO
)
EA (-ELUMO)
12
23
24
25
26
27
28
29
30
ꢁ6.07739
ꢁ6.63768
ꢁ6.10107
ꢁ6.14297
ꢁ6.59659
ꢁ6.19794
ꢁ6.07848
ꢁ6.56203
ꢁ6.15059
ꢁ1.30941
ꢁ1.35894
ꢁ1.50697
ꢁ1.09471
ꢁ1.17063
ꢁ1.28002
ꢁ1.05607
ꢁ1.10315
ꢁ1.24492
ꢁ4.76798
ꢁ5.27874
ꢁ4.5941
6.07739
6.63768
6.10107
6.14297
6.59659
6.19794
6.07848
6.56203
6.15059
1.30941
1.35894
1.50697
1.09471
1.17063
1.28002
1.05607
1.10315
1.24492
ꢁ5.04826
ꢁ5.42595
ꢁ4.91792
ꢁ5.02241
ꢁ5.45888
ꢁ4.90567
Figure 3. Molecular electrostatic potential and HOMO orbital of highest. Moderate and excessive biological activity compounds 12. 26 and 29 by DFT/B3LYP
calculations.

8
O. ALGUL ET AL.
derivative 23. Compounds with a propylene linker (28, 29,
and 30) were screened to determine their toxicity against
the normal cell line (Vero) and they were more toxic to
human cell lines than the positive control compound metho-
trexate (Table 1).
4.3. Molecular reactivity analyses
Molecular geometries of all molecular structures were fully
optimized using DFT/B3LYP. From the results of the DFT cal-
culations the quantum-chemical descriptors were obtained
for the model building as the energy levels of the molecular
orbital order HOMO (highest occupied molecular orbital) and
LUMO (lowest unoccupied molecular orbital) for molecules
give information on the possible electronic transition. The
HOMO and LUMO also indicate the electrophilic and nucleo-
philic attraction in molecule. The energy range created by
HOMO and LUMO is an indicator of the chemical stability of
the molecule and can be a critical parameter to determine
the electrical transfer properties of the compounds (Erol
et al., 2020; Reddy et al., 2016). The EHOMO-ELUMO (Gap) is the
most important parameter for the chemical reactivity.
Quantum-chemical descriptors (HOMO, LUMO, Gap, EA) of
the molecules are given in Table 2. The likelihood and select-
ivity of the reactions depend on the energies and symme-
tries of these leading orbitals.
A correlation was established between the calculated
quantum-mechanical parameters and the activities of all syn-
thesized compounds. In this correlation analysis, HOMO val-
ues were found to be in high relationship with activity.
4.4. Geometry optimization and MEP analysis
Molecular docking studies showed that benzazole groups
were effective in the antiproliferative activities and com-
pounds interact with residues in the active region of the
receptor through these groups. In QSAR studies, HOMO ener-
gies were found to be closely related to activity. Once HUMO
energy related to biological activity need to investigate the
other aspect like location of these orbitals on the molecule
(Figure 3). Such investigation of electronic densities revealed
the location of particular part in a compound influenced the
biological activity. It can be seen that HOMO orbitals settle
on molecules 12, 26 and 29, especially on benzazole groups
(Figure 3). Although HOMO orbitals are located on only one
benzazole ring in compound 12, 26 and 29 appear in both
ring systems on the bisbenzazole structure of these orbitals.
Therefore, depending on the localization of HOMO orbital
bisbenzazole-derived compounds have theoretically pro-
posed to have different biological activities.
4.5. In silico ADMET prediction
In silico ADMET studies of the compounds were conducted
to determine various physicochemical properties, inappropri-
ate pharmacokinetic properties and possible toxicities of
molecules. In silico ADMET and toxicological screening sys-
tems can provide an opportunity to predict performance

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
in vivo. Estimates of ADMET properties of all synthesized
compounds were performed with PRE ADMET and
DATAWARRIOR 4.07.02 software. The software evaluated
drug candidates’ ADMET profiles according to CMC-like, lead-
like, Lipinski’s rule of five, and WDI-like rules.
Toxicological, ADME, and Drug-like values were shown in
Table 3. Scores were observed to be within the range of
traded drugs. When the toxicological parameters (mutagenic,
carcinogenic, irritant, reproduction effects) were examined, it
was seen that all the compounds did not have an estimated
toxicity. Also, it is important points to know about these
principle predictors for the compounds were determined
using QikProp (Maestro 11.8) (details are provided in the sup-
plementary content of Table S1).
A good QSAR model; R2 must be > 0.7; q2 must be >
0.5; large F should be small r and p value < 0.001. It was
seen that compound 12 which constitutes our QSAR equa-
tion has the largest regression deviation and is accepted as
an outlier. After excluding compound 12, an optimal QSAR
model was obtained as follows and the established QSAR
equation (2) appears to be statistically successful.
-
Log 1=IC50 ¼ ꢁ5:033 ꢁ 0:626 x EHOMO
(2)
n ¼ 8; R2 ¼ 0:972; R2 ¼ 0:967; r ¼ 0:0277; q2 ¼ 0:953
A
Sreg ¼ 0:163; F ¼ 212:136; p < 0:000001
The newly created equation is very successful statistically.
While R2 value in Equation (1) is 0.437. R2 value in Equation
(2) is 0.972; the q2 value was calculated as 0.953 in Equation
(2) instead of 0.123. The p value which should be less than
0.001 has decreased considerably compared to the first
equation. According to Equation (2), EHOMO values were
found to be closely related to the antiproliferative activities
of compounds with the bisbenzazole skeleton.
In this study, a correlation was made to determine a rela-
tionship between the antiproliferative activity of the com-
pounds and the physicochemical parameters calculated.
However, the correlation coefficient (r) values of these
4.6. QSAR analyses
In QSAR studies, the relationship between the physicochemi-
cal, electronic and structural properties and biological activ-
ities of chemical compounds are examined quantitatively by
mathematical methods. In these studies, the rational design
of new lead compounds is provided and data that can con-
tribute to their development are obtained.
In this study, an optimal QSAR equation (1) containing
eight compounds was obtained by multiple linear regression parameters were found less than 0.5 and were
method with QSAR analysis.
not evaluated.
Log 1=IC50 ¼ ꢁ7:558 ꢁ 1:013 x EHOMO
(1)
n ¼ 9; R2 ¼ 0:437; R2 ¼ 0:357; r ¼ 0:29; q2 ¼ 0:123
4.7. Molecular docking
A
Sreg ¼ 0:47; F ¼ 5:444; p < 0:052
Molecular docking is a significant computer-assisted drug
design method for predicting the main binding modes of a
ligand with a protein of known three-dimensional structure
(Sepehri et al., 2017). In order to know the possible binding
studies of the di-heterocyclic compounds (12, 23-30)
molecular interaction studies of the active anti-cancer com-
pounds were performed against human P450 (CYP) 2C8
(CYP2C8) enzyme. CYP2C8 accounts for approximately 6–7%
of the total hepatic CYP content (Achour et al., 2014). The
importance of CYP2C8 causing variation in drug response via
drug-drug interactions and pharmacogenetics polymor-
phisms has been recognized only for the last two decades.
CYP2C8 participates in the metabolism of numerous drugs
and some endogenous and natural compounds. It catalyzes
Table 4. Molecular docking scores of all compounds.
Compounds
Docking Scores for protein (2NNI)
12
23
24
25
26
27
28
29
30
MTX
ꢁ3.708
ꢁ5.569
ꢁ5.216
ꢁ5.247
ꢁ5.562
ꢁ5.257
ꢁ4.842
ꢁ5.914
ꢁ5.167
ꢁ7.336
Table 5. Intermolecular interactions of the most active compounds (26 and 29) and MTX in the active region of the protein (pdb ID: 2NNI).
Compounds
Interactions Type
Protein residues
Distance in Å
4.79; 5.32
26
pi-pi stacking
Hydrophobic
Polar
PHE205
ILE106. PHE201. PHE205. LEU208. VAL233. VAL237. ALA292. ALA296. ALA297. ILE476. VAL477
THR107. ASN204. ASN209. ASN236. THR240. THR301
Charged (þ)
Charged (ꢁ)
pi-pi stacking
Hydrophobic
Polar
ARG200. ARG241
ASP293. GLU300
PHE205
29
4.92; 5.38
ILE106. LEU110. LEU208. VAL237. VAL244. VAL288. ALA292. ALA296. ALA297. ILE476. VAL477
THR107. ASN202. ASN204. ASN209. ASN236. THR240. THR301
Charged (þ)
pi-pi stacking
H-bound
ARG200. ARG241
HEM
ARG241
MTX
4.91; 4.92
1.58
Hydrophobic
ILE102. ILE103. LEU110. PHE205. VAL207. LEU208. VAL288. ALA292. ALA297. LEU361. LEU362.
VAL366. ILE476. VAL477
Polar
SER103. SER114. THR107. ASN204. ASN236. THR301. THR240. LYS108

10
O. ALGUL ET AL.
Figure 4. 2D interaction 12. 26. 29 and MTX with active site of protein (2NNI).
a variety of oxidative reactions, in particular hydroxylation, onset of a broadening scientific interest in CYP2C8, promptly
N-demethylations, and N-deethylations. Because of its large, convincing drug regulatory authorities to acknowledge
sinuous active site, CYP2C8 can accommodate substrates of CYP2C8 as one of the major drug-metabolizing
different sizes and structures. Typical substrate drugs of
CYP2C8 include anticancer, antidiabetic, antimalarial, and
CYP enzymes.
The crystal structure of the 2NNI pdb coded CYP2C8dH
lipid-lowering agents. Interestingly, some glucuronide metab- protein was used in the docking studies on the HepG2 cell
olites of drugs interact with CYP2C8. The event was the line. Although the reference molecule methotrexate has a

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
ꢁ7.336 kcal/mol docking score; the scores of the compounds studies, it was determined that the docking scores of the
23, 26 and 29 (IC50 ¼ 7.81 lM) were ꢁ5.569, ꢁ5.562 and compounds 23-30 were between ꢁ4.842 and ꢁ5.914 kcal/
ꢁ5.914 ꢁ 6.168 kcal/mol respectively; the scores of com-
mol and the compound 12 removed from the QSAR equa-
pounds 24, 25, 27, 28, and 30 (IC50 ¼ 15.3 ꢁ 15.63 lM) are
ꢁ5.216, ꢁ5.247, ꢁ5.257, ꢁ4.842 and ꢁ5.167 kcal/mol,
respectively (Table 4).
tion with the LOO method was ꢁ3.708 (Table 4). It was also
observed that compound 7 did not make significant interac-
tions with residues in the active region of the receptor.
These results explain why the predicted IC50 value (16.77 lM)
of compound 12 in the QSAR equation is different from the
experimental IC50 value (125 lM) of the compound.
The results obtained by using the QSAR equation that we
created to design new and higher antiproliferative molecules
with a bisbenzazole skeleton can be supported by molecular
docking studies. Finally, asymmetric di(benzazol-2-yl)ethanes
can be regarded as promising drug leads for the develop-
ment of effective anticancer drugs.
The lowest experimental activity result (IC50 ¼ 125 lM)
was compound 12 and the docking score was calculated as
ꢁ3.708 kcal/mol. Docking study results showed that docking
scores and activity results appear to be in harmony. It is
seen that the methotrexate structure has the highest activity.
Pi-pi stacking interaction with HEM in the active region of
the protein of the pteridine ring in the methotrexate struc-
ture were hydrogen bond with ARG241; and hydrophobic
polar and charged (þ) interactions with various residues.
Compounds 26 and 29 have the highest activity and have
been found to interact with PHE205 in the active region of
the receptor, and to interact with various residues hydropho-
bic, polar and charged (þ) (Table 5). Compound 12 with the
lowest docking score was found to do no pi-pi stacking
interaction and hydrogen bonding with residues. It is
thought that the low docking score of compound 12 is due
to the inability to fully settle in the active site and to make
strong bonds with the residues (Figure 4).
Disclosure statement
The authors have declared no conflict of interest.
Funding
We thank the Scientific and Technological Research Council of Turkey
(TUBITAK Grant Number: 115S190) and Mersin University for their finan-
cial support (BAP-SBE-2018-1-TP3-2911).
5. Conclusion
In the development of potent small and active anticancer
molecules an approach in which two bioactive heterocyclic
scaffolds are embedded in a single molecule using an easy,
simple and economical one-stage reaction strategy with
good selectivity and moderate to good isolated yields of di-
heterocyclic molecules can be used. However, the synthesis
of di-benzazoles is not as easy as that of bis-structures.
Careful examination led to the establishment of a signifi-
cantly regular structure-activity relationship which indicated
that the di-heterocyclic compounds with benzoxazole and
benzothiazole moieties are the most potent antiproliferative
agents with good selectivity against cancer cell lines.
Benzothiazole and benzoxazole rings show better anti-
cancer activity than the benzimidazole ring. Additionally, the
antiproliferative activity is higher with a methylene linker
than with a propylene linker. The flexibility of two di-hetero-
cyclic compounds should not be too high for antiproliferative
activity.
In order to explain the relationship between the activity
and structure of these compounds, QSAR study was per-
formed by calculating various physicochemical parameters
with various quantum-chemical descriptors and QikProp soft-
ware using DFT calculations. In QSAR studies, HOMO values
of the compounds were determined to be highly related to
activity and a QSAR equation was created by multiple regres-
sion analysis method (Log 1/IC50 ¼ ꢁ5.033 ꢁ 0.626EHOMO).
According to the QSAR equation predicted and experimental
IC50 values are very close to each other (Table 4). Molecular
docking studies were also conducted to control the results
of QSAR studies and to examine the interactions of mole-
cules (12, 23-30) and MTX with the receptor. In docking
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