Discovery of 5-(or 6)-benzoxazoles and oxazolo[4,5-b]pyridines as novel candidate antitumor agents targeting hTopo IIα

Article abstract of DOI:10.1016/j.bioorg.2021.104913

Discovery of novel anticancer drugs which have low toxicity and high activity is very significant area in anticancer drug research and development. One of the important targets for cancer treatment research is topoisomerase enzymes. In order to make a con

Full text of DOI:10.1016/j.bioorg.2021.104913

Bioorganic Chemistry 112 (2021) 104913
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of 5-(or 6)-benzoxazoles and oxazolo[4,5-b]pyridines as novel
candidate antitumor agents targeting hTopo II
α
,
,
,
,d,2
Esin Karatasa 1, Egemen Fotob 1, Tugba Ertan-Bolellia 2, Gozde Yalcin-Ozkatc
,
,
,*
Serap Yilmaze 2, Sanaz Ataeic, Fatma Zilifdarf, Ilkay Yildiza
a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
b Necmettin Erbakan University, Faculty of Science, Department of Biotechnology, Konya, Turkey
c Ankara University, Biotechnology Institute, 0fef0 Yenimahalle, Ankara, Turkey
d Recep Tayyip Erdogan University, Faculty of Engineering, Bioengineering Department, Rize, Turkey
e Trakya University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Edirne, Turkey
f Selcuk University Faculty of Science, Department of Biochemistry, Konya, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Discovery of novel anticancer drugs which have low toxicity and high activity is very significant area in anti-
cancer drug research and development. One of the important targets for cancer treatment research is topo-
isomerase enzymes. In order to make a contribution to this field, we have designed and synthesized some 5(or 6)-
nitro-2-(substitutedphenyl)benzoxazole (1a-1r) and 2-(substitutedphenyl)oxazolo[4,5-b]pyridine (2a-2i) de-
rivatives as novel candidate antitumor agents targeting human DNA topoisomerase enzymes (hTopo I and hTopo
Benzoxazoles
Oxazolo[4,5-b]pyridines
Topoisomerase I and II
α inhibition
Antitumor
Molecular docking
IIα). Biological activity results were found very promising for the future due to two compounds, 5-nitro-2-(4-
Molecular dynamic simulation
butylphenyl)benzoxazole (1i) and 2-(4-butylphenyl)oxazolo[4,5-b]pyridine (2i), that inhibited hTopo IIα with
2 µM IC50 value. These two compounds were also found to be more active than reference drug etoposide.
However, 1i and 2i did not show any satisfactory cyctotoxic activity on the HeLa, WiDR, A549, and MCF7 cancer
cell lines. Moreover, molecular docking and molecular dynamic simulations studies for the most active com-
pounds were applied in order to understand the mechanism of inhibition activity of hTopo IIα. In addition, in
silico ADME/Tox studies were performed to predict drug-likeness and pharmacokinetic properties of all the tested
compounds.
1. Introduction
concerted breakage and reunion of DNA strands during normal cell
growth [6]. They solve DNA replication, transcription, recombination,
Cancer characterized by unregulated proliferation of cells is a major
human health problem in the worldwide and the second leading cause of
death after cardiovascular disease [1,2]. Hence, on-going researches and
discovery of anticancer drugs that have low toxicity and highly potent
are still important fields in anti-cancer drug research and development
[3]. In drug discovery, “one disease–one target–one drug” approach is
more common implementation to reduce unwanted side effects [4]. The
development of synthetic topoisomerase inhibitors is an important
group of drugs in the treatment of many types of cancer [5].
repair, and chromatin assembly in the regulation of DNA topology
[7–10]. There are two types of DNA topoisomerases (Type I and Type II)
[11,12] based on the number of DNA strands cleaved (one or two, for
type I or II, respectively), the nature of the covalent phosphotyrosyl
intermediate formed (5′ or 3′ linkage), and other aspects of enzyme
structure and catalysis. Nevertheless, these enzymes all share a common
mechanism of transient breakage and reunion of DNA strand(s) [13].
Topo I, changing the DNA topology by breaking the phosphodiester
bond between DNA strands is based on the same general mechanism.
The phosphoryl group of DNAs is attacked by tyrosyl group of Topo I.
This creates a covalent bond between the tyrosyl group and one side of
DNA Topoisomerases (Topo) are enzymes which regulate the
conformational or topological changes of DNA by catalyzing the
* Corresponding author.
E-mail address: iyildiz@pharmacy.ankara.edu.tr (I. Yildiz).
1
These authors (E.K. and E.F.) contributed equally as a first name.
2
These authors (G.Y., T.E.B. and S.Y) contributed equally as a second name.
https://doi.org/10.1016/j.bioorg.2021.104913
Received 6 December 2020; Received in revised form 11 March 2021; Accepted 11 April 2021
Available online 14 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
the broken DNA. At the same time, the free hydroxylated strand is
released and rotated. The hydroxyl end of the free strand of DNA attacks
the formed phosphotyrosine bond, rebuilds the phosphodiester bond
between the two strands and releases the enzyme to the next catalytic
cycle [14,15]. Topo II changing the topology of DNA by cleaving both
strands of DNA duplex with Mg2+ and energy from ATP hydrolysis. Topo
II covalently attaches tyrosine to the 5′ end of broken DNA, release a free
3′ end and allows to passing a second DNA duplex (the transported or T-
segment) through a gap (the gate or G-segment) Topo II enzymes can
relax both positive and negative supercoils in DNA. [15,16]. Recently,
some studies have indicated that Topo II levels increase during cell
proliferation and this enzyme appears to be the isoform involved in
mitosis [17]. Therefore, inhibition of these enzymes has been considered
as targets for the development of novel anticancer agents. Some of the
topoisomerase inhibitors such as camptothecin, topotecan irinotecan,
etoposide have notable therapeutic efficacy as antitumor drugs [18–23].
All topoisomerase directed agents are able to interfere with at least
one step of the catalytic cycle of topoisomerases. Agents able to stabilize
the covalent DNA topoisomerase complex (the cleavable complex) are
traditionally called Topo II poisons, while agents acting on any of the
other steps in the catalytic cycle are called catalytic inhibitors. Catalytic
Topo II inhibitors are a heterogeneous group of compounds that might
interfere with the binding between DNA and Topo II (aclarubicin and
suramin), stabilize noncovalent DNA Topo II complexes (merbarone,
ICRF-187, and structurally related bisdioxopiperazine derivatives), or
inhibit ATP binding (novobiocin) [24]. Classical Topo II-inhibiting
agents such as epipodophyllotoxins or anthracyclines interfere with
the breakage-reunion reaction of Topo II by stabilizing this cleavable
complex. The stabilization of the cleavable complex and not the inhi-
bition of the Topo II activity is supposed to play the decisive role in the
cytotoxic effect of the classical Topo II interacting agents [25]. The
stabilized cleavable complex leads to both single- and double-strand
DNA breaks, which can trigger cellular signal transduction pathways
leading to cell death [26]. Accordingly, resistance against classical Topo
II-inhibiting agents can result from any process that leads to an altered
binding of Topo II to drugs or DNA and a reduced formation of cleavable
complexes. Indeed, it was demonstrated that decreased Topo II catalytic
activity can mediate drug resistance to cancer cells [27]. Since these
drug-resistant tumor cells showed cross resistance to other drugs, this
phenotype was designated as altered Topo II multidrug resistance (at-
MDR) [28]. The decrease in Topo II activity can be caused by diminished
expression levels of both Topo II isoforms [29] as well as by missense
mutations within the Topo II isoenzyme encoding genes [30].
an electron withdrawing substitution in 5 (or 6) position positively
affected the activity and in order to demonstrate this, 3D QSAR studies
of these compounds (Fig. 1) were performed using CoMFA (Comparative
Molecular Field Analysis) and CoMSIA (Comparative Molecular Simi-
larity indices Analysis) methods in 2005 and 2006 [32,33]. With these
CoMFA and CoMSIA analysis, the properties of the regions responsible
for the activity of the Topo II enzyme have been revealed using potent
compounds. These previoulsy published our studies demonstrated that
hydrophobic interactions have significant role for enhancing Topo II
enzyme inhibitory activity. It was also noticed that hydrophilic sub-
stituent on the 5th or 6th position of heterocyclic core was more signif-
icant than hydrophobic ones. The other significant knowledge was a
hydrophobic group had to have been at the ortho and para of the phenyl
ring of the 2nd position of heterocyclic ring. Moreover, the 3D CoMFA
analysis study showed that both an electronegative group at the 5th or
6th position of the heterocyclic structure and an electropositive group on
the ortho of the phenyl ring in the 2nd position of heterocyclic ring sys-
tem would increase Topo II enzyme inhibition effect. In addition, both
CoMFA and CoMSIA studies indicated that steric properties were found
to be important for the activity and activity was reported to be increased
by the inclusion of bulky groups at the ortho and para positions of the
phenyl ring in 2nd position of the heterocyclic ring.
Under the light of these studies, in here, an electron-withdrawing
and hydrophilic grup for 5 (or 6) position and hydrophobic alkyl
chains for 2nd position, especially on the para of the phenyl ring were
selected. Therefore, some of novel 2-(substitutedphenyl)benzoxazole
derivatives bearing the nitro group at position 5 (or 6) and 2-(sub-
stitutedphenyl)oxazolo[4,5-b]pyridines were designed, synthesized in
order to determine for their human DNA Topo I and IIα inhibitory ef-
fects. Their activity was also compared to camptothecine (CPT) (for
Topo I) and etoposide (for Topo II), which are well known clinically used
drug. Moreover, the most active compounds were analyzed the potential
antitumor activity in various cancer lines, as well. In silico studies give us
some prediction related to how the interaction between ligand and
enzyme is and about the ADME/Tox properties. Therefore, in this study
¨
we applied molecular docking (Schrodinger) and molecular dynamic
(AMBER v14) studies in order to see the interactions between hTopo II
α
(PID: 5GWK) and the most active compounds. Additionally, ADME/Tox
properties of these compounds were predicted by using Accelrys Dis-
covery Studio 3.5 in order to find the “best” drug candidate as hTopo II
α
inhibitors or antitumor agents.
2. Materials and methods
Since drug resistance is a major problem in treatment of cancer
diseases, it is important to design alternative chemotherapeutic agents.
In 2004, Pinar et al., screened some synthesized 2,5,6-substituted
benzoxazole, benzimidazole, benzothiazole and oxazolo[4,5-b]pyri-
dine derivatives for their eukaryotic DNA Topo II inhibitory activity in
cell free system [31]. Among these compounds, 2-phenoxymethylbenzo-
thiazole (IC50 = 11.4 µM), 2-(2-methoxyphenyl)-6-nitrobenzoxazole
(IC50 = 17 µM), 5-methylcarboxylate-2-(phenylthiomethyl)benzimid-
azole (IC50 = 17 µM), and 6-methyl-2-(2-nitrophenyl)benzoxazole
(IC50 = 18.8 µM), were found to be more potent than the clinically
used reference drug etoposide (IC50 = 21.8 µM). It was determined that
2.1. Chemical synthesis
All chemicals and solvents were purchased from commercial vendors
and used without purification. All reactions were monitored by thin
layer chromatography onready-made silica gel 60 GF254 aluminia plates
(Merck) with visualization of Camag UV light. The melting points were
measured with a capillary melting point apparatus (Buchi B540) and
were uncorrected. The IR spectra were directly recorded on an Agi-
lentCary 630 FTIR-Diamond ATR1 spectrophotometer. The 1H NMR
(Supporting Information) and 13C NMR spectra were recorded
R2
X
N
R1
Y
A
R3
R
Z
Z: CH, N; X: O, NH, S; Y: -, CH2, C2H4, CH2O, CH2S; A: Phenyl, Cyclohexyl, Cyclopentyl
R: H, Cl, CH3, NO2, NH2, COOCH3; R1: H, CH3, NO2; R2: F, CH3, NO2, OCH3; R3: H, Cl,
CH3, C2H5, C(CH3)3, NH2, NHCH3, OCH3, OC2H5
Fig. 1. Heterocyclic compounds used in CoMFA and CoMSIA studies [32,33].
2

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
employing a VARIAN Mercury 400 High Performance Digital FT-NMR
Spectrometer 400 MHz and 100 MHz, respectively. Chemical shifts
were reported in ppm (δ) relative to TMS and coupling constants (J)
were reported in hertz. Mass spectra were taken on a Waters2965 Alli-
ance Micromass ZQ LC/MS using the ESI method. Elemental anayles
were performed by Leco CHNS-932 CHNS-O analyzer. The result of the
elemental analyses (C, H, N, S) were within ±0.4% of the calculated
amounts.
δ ppm: 2.36 (s, 3H, CH3) 2.62 (s, 3H, CH3), 7.32 (t, 1H, H-4′), 7.45 (d,
1H, J = 7,6 Hz, H-7), 7.89 (d, 1H, J = 7,6 Hz, H-5′), 8.01 (d, 1H, J = 8,4
Hz, H-6′), 8.33 (dd, 1H, J = 9,2 Hz, J = 2,4 Hz, H-6), 8.65 (d, 1H, J = 2,4
Hz, H-4); 13C NMR (DMSO‑d6, 100 MHz); δ ppm: 16.776, 20.232,
111.557, 115.680, 121.335, 124.932, 125.926, 127.984, 133.415,
137.339, 138.378, 141.834, 153.439, 165.930; ESI(+): 269.36 (M+
+
H) (100%); Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44
Found: C, 67.23; H, 4.59; N, 10.49.
2.2.6. 2-(2,4-dimethylphenyl)-5-nitrobenzoxazole (1f)
2.2. General method of synthesis of 2-(substitutedphenyl)benzoxazole
(1a-1r) and 2-(substitutedphenyl)oxazolo[4,5-b]pyridine (2a-2i)
derivatives
Yield: 55.66%; mp: 155–158 ◦C; FT-IR (νmax): 3108, 1545–1613,
1518, 1433, 1340, 1144–1254, 816–868 cmꢀ 1; 1H NMR (DMSO‑d6, 400
MHz); δ ppm: 2.35 (s, 3H, CH3), 2.71 (s, 3H, CH3), 7.26 (t, 2H, H-3′, H-
5′), 8.00 (d, 1H, J = 9,2 Hz, H-7), 8.04 (d, 1H, J = 8,4 Hz, H-6′), 8.31 (dd,
1H, J = 8,6 Hz, J = 2,4 Hz, H-6), 8.63 (s, 1H, H-4); 13C NMR (DMSO‑d6,
100 MHz); δ ppm: 20.861, 21.600, 107.205, 119.725, 120.525, 121.592,
127.147, 129.936, 132.618, 138.928, 142.585, 144.529, 146.85,
148.773, 167.099; ESI(+): 269.28 (M+ + H) (100%); Anal. Calcd. for
The derivatives were synthesized by heating 0.01 mol appropriate
amine with 0.015 mol suitable acid in 24 g polyphosphoric acid (PPA)
and stirring for 2–3 h at 120 ◦C ꢀ 140 ◦C. At the end of reaction period,
the residue was poured into an ice-water mixture and neutralized with
an excess %10 NaOH solution. The residue was boiled with 200 mg
charcoal in ethanol and filtered. The crude product was obtained and
recrystallized from ethanol. In the present study, all the compounds are
original except compounds 1a, 1c, 1j, 1l, 2a, 2c-2h [34–38].
C
15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found: C, 66.82; H, 4.60; N,
10.34.
2.2.7. 2-(2,5-dimethylphenyl)-5-nitrobenzoxazole (1 g)
Yield: 45.58%; mp: 135–143 ◦C; FT-IR (νmax): 3106, 2924,
2.2.1. 2-(4-tert-butylphenyl)-5-nitrobenzoxazole (1a)
1602–1617, 1522, 1435, 1334, 1176–1256, 987–703 cmꢀ 1
;
1H NMR
Yield: 46,80% mp 158–161 ◦C (Ref. mp: 158 ◦C) [37]; ESI(+): 297,48
(M+ + H) (100%); Anal. Calcd. for C17H16N2O3. 0,1 H2O C, 68.48; H,
5.47; N, 9.40 Found: C, 68.14; H, 5.72; N, 9.82.
(DMSO‑d6, 400 MHz); δ ppm: 2.34 (s, 3H, CH3), 2.66 (s, 3H, CH3), 7.31
(s, 2H, H-3′, H-4′), 7.90 (s, 1H, H-6′), 7.97 (d, 1H, J = 8,8 Hz, H-7), 8.28
(dd, 1H, J = 8,8, J = 2 Hz, H-6), 8.58 (d, 1H, J = 2 Hz, H-4); 13C NMR
(DMSO‑d6, 100 MHz); δ ppm: 20.312, 21.311, 111.503, 115.587,
121.318, 124.084, 129.997, 131.971, 132.801, 135.666, 135.849,
141.778, 144.841, 153.322, 165.007; ESI(+): 269.33 (M+ + H) (100%);
Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found: C, 66.87;
H, 4.72; N, 10.34.
2.2.2. 2-(4-isopropylphenyl)-5-nitrobenzoxazole(1b)
Yield: 43.15%; mp: 149–153 ◦C; FT-IR (νmax): 3117, 2961,
1556–1617, 1518, 1490, 1343, 1179–1256, 918 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz); δ ppm: 1.24 (d, 6H, J = 6,8 Hz, CH3), 2.95–3.02
(m, 1H, CH), 7.48 (d, 2H, J = 8,8 Hz, H-3′, H-5′), 7.98 (d, 1H, J = 9,2 Hz,
H-7), 8.1 (d, 2H, J = 8,4 Hz, H-2′, H-6′), 8.28 (dd, 1H, J = 9,2 Hz, J = 2,4
Hz, H-6), 8.57 (d, 1H, J = 2,4 Hz, H-4); 13C NMR (DMSO‑d6, 100 MHz); δ
ppm: 23.300, 33.419, 111.488, 115.321, 121.097, 122.971, 127.315,
127.764, 141.877, 141.925, 153.688, 153.833, 165.324; ESI(+): 283,34
(M+ + H) (100%); Anal. Calcd. for C16H14N2O3 C, 68.07; H, 5.00; N,
9.92 Found: C, 68.16; H, 4.87; N, 9.88
2.2.8. 2-(3,5-dimethylphenyl)-5-nitrobenzoxazole (1h)
Yield: 53.87%; mp: 172–178 ◦C; FT-IR (νmax): 3104, 2916,
1604–1621, 1518, 1435, 1345, 1058–1231, 691–926 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz); δ ppm: 2.36 (s, 3H, CH3), 7.29 (s, 1H, H-4′), 7.79
(s, 2H, H-2′, H-6′), 7.98 (d, 1H, J = 9,2 Hz, H-7), 8.29 (dd, 1H, J = 8,8 Hz
, J = 2,4 Hz, H-6), 8.58 (d, 1H, J = 2 Hz, H-4); 13C NMR (DMSO‑d6, 100
MHz); δ ppm: 20.747, 111.686, 115.564, 121.401, 125.364, 134.348,
138.852, 141.900, 145.032, 153.955, 165.530; ESI(+): 269.30 (M++H)
(100%); Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found:
C, 67.02; H, 4.15; N, 10.54.
2.2.3. 2-(4-(trifluoromethyl)phenyl)-5-nitro-benzoxazole (1c)
Yield: 44.09%; mp: 141–146 ◦C; FT-IR Spektrumu (νmax): 3112,
2908, 1504–1625, 1531, 1435, 1345, 1120, 1064–1112, 691–943 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz); δ ppm: 7.98 (d, 2H, J = 8,4 Hz, H-2′, H-
6′), 8.06 (d, 1H, J = 9,2 Hz, H-7), 8.35 (dd, 1H, J = 8,8 Hz, J = 2,4 Hz, H-
6), 8.38 (d,2H, J = 8 Hz, H-3′, H-5′), 8.68 (d, 1H, J = 2,4 Hz, H-4); 13C
NMR (DMSO‑d6, 100 MHz); δ ppm: 112.044, 116.098, 121.958,
122.323, 125.036, 126.358, 128.511, 129.220, 131.963, 132.283,
141.602, 145.176, 154.016, 163.830; ESI(+): 309.09 (M+ + H) (100%);
Anal. Calcd. for C14H7F3N2O3 C, 54.56; H, 2.29; N, 9.09 Found: C, 54.45;
H, 2.09; N, 9.25
2.2.9. 2-(4-butylphenyl)-5-nitrobenzoxazole (1i)
Yield: 45.24%; mp: 108–113 ◦C; FT-IR (νmax): 3071, 2955,
1608–1522, 1340, 1235–1060, 920–732 cmꢀ 1; 1H NMR (DMSO‑d6, 400
MHz); δ ppm: 0.92 (t, 3H, CH3), 1.29–1.38 (m, 2H, CH2), 1.56–1.64 (m,
2H, CH2), 2.67 (t, 2H, CH2), 7.43 (d, 2H, J = 8 Hz, H-3′, H-5′), 7.98 (d,
1H, J = 8,8 Hz, H-7), 8.08 (d, 2H, J = 8,8 Hz, H-2′, H-6′), 8.29 (dd, 1H, J
= 9,2 Hz, J = 2,4 Hz, H-6), 8.58 (d, 1H, J = 2,4 Hz, H-4); 13C NMR
(DMSO‑d6, 100 MHz); δ ppm: 13.652, 21.669, 32.581, 34.745, 111.556,
115.389, 121.180, 122.864, 127.696, 129.296, 141.922, 144.948,
147.904, 153.860, 165.408; ESI(+): 297.4 (M++H) (100%); Anal.
Calcd. for C17H16N2O3 C, 68.91; H, 5.44; N, 9.45 Found: C, 69.19; H,
5.75; N, 9.47.
2.2.4. 2-(3-(methylthio)phenyl)-5-nitrobenzoxazole (1d)
Yield: 40.33%; mp: 130–134 ◦C; FT-IR (νmax): 3101, 1574–1619,
1522, 1430, 1340, 1079–1267, 788–926 cmꢀ 1; 1H NMR (DMSO‑d6, 400
MHz); δ ppm: 2.56 (s, 3H, CH3), 7.52–7.57 (m, 2H, H-2′, H-4′),
7.93–7.96 (m, 2H, H-5′, H-6′), 8.01 (d, 1H, J = 8,8 Hz, H-7), 8.31 (dd,
1H, J = 9,2 Hz, J = 2,4 Hz, H-6), 8.63 (d, 1H, J = 2 Hz, H-4); 13C NMR
(DMSO‑d6, 100 MHz); δ ppm: 14.341, 111.750, 115.666, 121.488,
123.888, 123.911, 126.083, 129.779, 129.870, 140.089, 141.682,
144.996, 153.889, 164.725; ESI(+): 287.29 (M+ + H) (100%); Anal.
Calcd. for C14H10N2O3S C, 58.74; H,3.49; N, 9.79; S, 11.19 Found: C,
58.94; H, 3.44; N, 9.92; S, 11.30.
2.2.10. 2-(4-tert-butylphenyl)-6-nitrobenzoxazole (1j)
Yield: 44.15%; mp 164–166 ◦C (Ref. mp: 160–165 ◦C) [34]; ESI(+):
297.40 (M++H) (100%); Anal. Calcd. for C17H16N2O3 C, 68.91; H, 5.44;
N, 9.45 Found: C, 69.11; H, 5.67; N, 9.38
2.2.11. 2-(4-isopropylphenyl)-6-nitrobenzoxazole (1k)
Yield: 39.40%; mp: 112–123 ◦C; FT-IR (νmax): 3110, 2963,
2.2.5. 2-(2,3-dimethylphenyl)-5-nitrobenzoxazole (1e)
1554–1606, 1543, 1464, 1341, 1060–1170, 922 cmꢀ 1
;
1H NMR
Yield: 55,17%; mp: 143–148 ◦C; FT-IR (νmax): 3106, 1522–1610,
1449, 1345, 1131–1248, 795–875 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz);
(DMSO‑d6, 400 MHz); δ ppm: 1.26 (d, 6H, J = 6,8 Hz, CH3), 3.01 (m, 1H,
3

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
CH), 7.52 (d, 2H, J = 8 Hz, H-3′, H-5′), 7.98 (d, 1H, J = 8,8 Hz, H-4),
8.14 (d, 2H, J = 8 Hz, H-2′, H-6′), 8,29 (dd, 1H, J = 9,2 Hz J = 2,4 Hz, H-
5), 8.68 (d, 1H, H-7); 13C NMR (DMSO‑d6, 100 MHz); δ ppm: 23.348,
33.506, 107.391, 119.697, 120.772, 122.997, 127.417, 127.996,
144.516, 146.955, 149.386, 154.034, 166.805; ESI(+): 283.32 (M++H)
(100%); Anal. Calcd. for C16H14N2O3..0.4 H2O C, 66.38; H, 5.15; N, 9.67
Found: C, 66.72; H, 5.05; N, 9.73.
2.2.17. 2-(4-butylphenyl)-6-nitrobenzoxazole (1r)
Yield: 28%; mp: 84–86 ◦C; FT-IR (νmax): 3106, 2933, 1614–1517,
1340, 1269–1125, 881–732 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz); δ
ppm: 0.91 (t, 3H, CH3), 1.28–1.37 (m, 2H, CH2), 1.55–1.62 (m, 2H,
CH2), 2.66 (t, 2H, CH2), 7.41 (d, 2H, J = 8,4 Hz, H-3′, H-5′), 7.94 (d, 1H,
J = 8,8 Hz, H-4), 8.07 (d, 2H, J = 8 Hz, H-2′, H-6′), 8.26 (dd, 1H, J = 8
Hz, J = 2,4 Hz, H-5), 8.62 (d, 1H, J = 2,4 Hz, H-7); 13C NMR (DMSO‑d6,
100 MHz); δ ppm: 13.706, 21.768, 32.604, 34.821, 107.350, 119.695,
120.77, 122.788, 127.840, 129.288, 144.460, 146.959, 148.194,
149.337, 166.818; ESI(+): 297.3 (M++H) (100%); Anal. Calcd. for
C17H16N2O3 C, 68.91; H, 5.44; N, 9.45 Found: C, 68.92; H, 5.58; N, 9.45.
2.2.12. 2-(4-(trifluoromethyl)phenyl)-6-nitro-benzoxazole (1l)
Yield: 44.46%; mp: 129–133 ◦C; FT-IR (νmax): 3121, 1556–1619,
1524, 1426, 1343, 1259, 1131, 1067–1259, 756–818 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz); δ ppm: 8.00 (d, 3H, J = 8,4 Hz, H-2′, H-6′), 8.06
(d, 1H, J = 8,4 Hz, H-4), 8.32 (dd, 1H, J = 8,8 Hz, J = 2,4 Hz, H-5), 8.41
(d, 2H, J = 8 Hz, H-3′, H-5′), 8.76 (d, 1H, J = 2 Hz, H-7); 13C NMR
(DMSO‑d6, 100 MHz); δ ppm: 107.928, 120.482, 121.059, 122.329,
125.041, 126.445, 128.721, 129.286, 132.145, 132.466, 145.194,
146.540, 149.701, 165.250; ESI(+): 309.45 (M++H) (100%); Anal.
Calcd. for C14H7F3N2O3 C, 54.56; H, 2.29; N, 9.09 Found: C, 54.37; H,
2.17; N, 9.20
2.2.18. 2-(4-tert-butylphenyl)oxazolo[4,5-b]pyridine (2a)
Yield: 43.39%; mp 140–144 ◦C (Ref. mp: 142 ◦C) [36]; ESI(+):
253.18 (M++H) (100%); Anal. Calcd. for C16H16N2O C, 76.16; H, 6.39;
N, 11.10 Found: C, 76.67; H, 6.64; N, 11.06.
2.2.19. 2-(4-isopropylphenyl)oxazolo[4,5-b]pyridine (2b)
Yield: 43.1%; mp: 101–103 ◦C; FT-IR (νmax): 3065, 2957, 1615,
1548, 1496, 1261–1179, 934–918 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz);
δ ppm: 1.26 (d, 6H, J = 6,4 Hz, CH3), 3.34 (s, 1H, CH), 7.46 (m, 1H, H-6),
7.52 (d, 2H, J = 8 Hz, H-3′, H-5′), 8.18 (d, 2H, J = 8 Hz, H-2′, H-6′), 8.23
(dd, 1H, J = 8 Hz, J = 1,6 Hz, H-7), 8.55 (dd, 1H, J = 4,8 Hz, J = 1,6 Hz,
H-5); 13C NMR (DMSO‑d6, 100 MHz); δ ppm: 23.414, 33.503, 118.856,
120.525, 123.497, 127.353, 127.772, 142.624, 146.396, 153.619,
155.578, 164.897; ESI(+): 239.04 (M++H) (100%); Anal. Calcd. for
2.2.13. 2-(2,3-dimethylphenyl)-6-nitrobenzoxazole (1m)
Yield: 50.86%; mp: 143–149 ◦C; FT-IR (νmax): 3108, 1589–1610,
1518, 1435, 1345, 1058, 726–825 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz);
δ ppm: 2.34 (s, 3H, CH3), 2.61 (s, 3H, CH3), 7.31 (d, 1H, J = 7,6 Hz, H-
4′), 7.44 (d, 1H, J = 6,4 Hz, H-4), 7.89 (d,1H, J = 6,8 Hz, H-5′), 8.,00 (d,
1H, J = 8.4 Hz, H-6′), 8.27 (d, 1H, J = 7,6 Hz, H-5), 8.66 (s, 1H, H-7); 13C
NMR (DMSO‑d6, 100 MHz); δ ppm: 16.748, 20.153, 107.380, 119.966,
120.556, 124.993, 125.923, 128.116, 133.592, 137.452, 138.401,
144.723, 146.704, 148.967, 167.478; ESI(+): 269.34 (M++H) (100%);
Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found: C, 66.77;
H, 4.10; N, 10.63.
C
15H14N2O C, 75.61; H, 5.92; N, 11.76 Found: C, 75.38; H, 6.10; N,
11.63.
2.2.20. 2-(4-(trifluoromethyl)phenyl)oxazolo[4,5-b]pyridine (2c)
Yield: 40.32%; mp 163–165 ◦C (Ref. mp: 162–164 ◦C) [38]; ESI(+):
265.12 (M++H) (100%); Anal. Calcd. for C13H7F3N2O. 0.05 H2O C,
58.89; H, 2.69; N, 10.56 Found: C, 58.67; H, 2.66; N, 10.58.
2.2.14. 2-(2,4-dimethylphenyl)-6-nitrobenzoxazole (1n)
Yield: 40%; mp: 164–170 ◦C; FT-IR (νmax): 3106, 2957, 1540–1606,
1518, 1433, 1340, 1121–1144, 816–868 cmꢀ 1; 1H NMR (DMSO‑d6, 400
MHz); δ ppm: 2.35 (s, 3H, CH3), 2.71 (s, 3H, CH3), 7.26 (t, 2H, H-3′, H-
5′), 7.97 (d, 1H, J = 8,4 Hz, H-4), 8.04 (d, 1H, J = 8,4 Hz, H-6′), 8.27 (dd,
1H, J = 8,8 Hz, J = 2,4 Hz, H-5), 8.64 (d, 1H, H J = 2 Hz, H-7);13C NMR
(DMSO‑d6, 100 MHz); δ ppm: 20.861, 21.600, 107.205, 119.725,
120.525, 121.592, 127.147, 129.936, 132.618, 138.928, 142.585,
144.529, 146.853, 148.773, 167.099; ESI(+): 297.48 (M++H) (100%);
Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found: C, 66.97;
H, 4.47; N, 10.37.
2.2.21. 2-(3-(methylthio)phenyl)oxazolo[4,5-b]pyridine (2d)
Yield: 42.99% mp 83–85 ◦C (Ref. mp: 82–84 ◦C) [35]; ESI(+): 243.3
(M++H) (100%); Anal. Calcd. for C13H10N2OS. 0,1 H2O C, 63.96; H,
4.21; N, 11.47; S, 13.13 Found: C, 63.92; H, 4.35; N, 11.30; S, 13.10.
2.2.22. 2-(2,3-dimethylphenyl)oxazolo[4,5-b]pyridine (2e)
Yield: 57.14%; mp 72–74 ◦C (Ref. mp: 72–73 ◦C) [35]; ESI(+):
225.38 (M++H) (100%); Anal. Calcd. for C14H12N2O C, 74.98; H, 5.39;
N, 12.49 Found: C, 75.13; H, 5.52; N, 12.35.
2.2.15. 2-(2,5-dimethylphenyl)-6-nitrobenzoxazole (1o)
2.2.23. 2-(2,4-dimethylphenyl)oxazolo[4,5-b]pyridine (2f)
Yield: 47.74%; mp: 94–96 ◦C (Ref. mp: 93–95 ◦C) [35]; ESI(+):
225.06 (M++H) (100%); Anal. Calcd. for C14H12N2O C, 74.98; H, 5.39;
N, 12.49 Found: C, 75.25; H, 5.53; N, 12.40.
Yield: 40.14%; mp: 133–135 ◦C; FT-IR (νmax): 3108, 2920,
1539–1620, 1513, 1435, 1349, 1062, 689–930 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz); δ ppm: 2.36 (s, 3H, CH3), 2.68 (s, 3H, CH3), 7.33
(t, 2H, H-3′, H-4′), 7.94 (s, 1H, H-6′), 7.99 (d, 1H, J = 9,2 Hz, H-4), 8.27
(dd, 1H, J = 9,2 Hz, J = 2,4 Hz, H-5), 8.66 (d, 1H, J = 2,4 Hz, H-7); 13C
NMR (DMSO‑d6, 100 MHz): δ ppm: 20.277, 21.314, 107.322, 119.888,
120.589, 124.071, 130.122, 132.004, 133.010, 135.662, 136.020,
144.608, 146.749, 148.814, 166.996; ESI(+): 269.35 (M++H) (100%);
Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44 Found: C, 67.54;
H, 4.57; N, 10.45.
2.2.24. 2-(3,5-dimethylphenyl)oxazolo[4,5-b]pyridine (2g)
Yield: 53.69%; mp 157–159 ◦C (Ref. mp: 155–157 ◦C) [35]; ESI(+):
225.25 (M++H) (100%); Anal. Calcd. for C14H12N2O.0,6 H2O C, 71.53;
H, 5.66; N, 11.91 Found: C, 71.29; H, 5.31; N, 11.75.
2.2.25. 2-(2,5-dimethylphenyl)oxazolo[4,5-b]pyridine (2h)
Yield: 44.57%, mp 87–89 ◦C (Ref. mp: 86–87 ◦C) [35]; ESI(+):
225.07 (M++H) (100%); Anal. Calcd. for C14H12N2O C, 74.98; H, 5.39;
N, 12.49 Found: C, 75.18; H, 5.54; N, 12.38.
2.2.16. 2-(3,5-dimethylphenyl)-6-nitrobenzoxazole (1p)
Yield: 42.68%; mp: 161–169 ◦C; FT-IR (νmax): 3099, 2918,
1545–1600, 1507, 1464, 1343, 1056–1265, 684–829 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz); δ ppm: 2.38 (s, 6H, CH3), 7.3 (s, 1H, H-4′), 7.79 (s,
2H, H-2′, H-6′), 7.94 (d, 1H, J = 8.4 Hz, H-4), 8.27 (dd, 1H, J = 8.6 Hz, J
= 2 Hz, H-5), 8.61 (d, 1H, J = 2 Hz, H-7); 13C NMR (DMSO‑d6, 100
MHz); δ ppm: 20.556, 107.213, 119.664, 120.647, 125.120, 125.341,
134.356, 138.653, 144.529, 146.761, 149.276, 166.795; ESI(+): 269.21
(M++H) (100%); Anal. Calcd. for C15H12N2O3 C, 67.16; H, 4.51; N,
10.44 Found: C, 66.91; H, 4.28; N, 10.48.
2.2.26. 2-(4-butylphenyl)oxazolo[4,5-b]pyridine (2i)
Yield: 47.38%; mp: 76–79 ◦C; FT-IR (νmax): 3060, 2953, 1578, 1466,
1049–1265, 795–930 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz); δ ppm:
0.916 (t, 3H, CH3), 1.31–1.38 (m, 2H, CH2), 1.57–1.65 (m, 2H, CH2),
2.69 (t, 2H, CH2), 7.44–7.48 (m, 3H, H-6, H-3′, H-5′), 8.16 (d, 2H, J =
8,4 Hz, H-2′, H-6′), 8.23 (dd, 1H, J = 8 Hz, J = 1,6 Hz, H-7), 8.54 (dd,
1H, J = 5 Hz, J = 1,2 Hz, H-5); 13C NMR (DMSO‑d6, 100 MHz); δ ppm:
4

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
13.691, 21.699, 32.649, 34.776, 118.872, 120.548, 123.352, 127.665,
129.296, 142.639, 146.403, 147.798, 155.593, 164.951; ESI(+): 253.20
(M++H) (100%); Anal. Calcd. for C16H16N2O. 0,6 H2O C, 73.03; H, 6.53;
N, 10.64 Found: C, 72.89; H, 6.41; N, 10.59.
was assumed to have no inhibitory activity (NE) on human DNA Topo I
and IIα.
2.3.2. In vitro anticancer activities
The cytotoxicity of test compounds was assessed using a cell death
assay based on detection of cells by sulforhodamine-B (SRB) according
to procedure of Vichai and Kitikira [41,42].
2.3. Biological activity studies
In this study, four different human cancer cell lines [HeLa (human
epithelial adenocarcinoma cell line of the cervix), Widr (human colo-
rectal adenocarcinoma cell line of the colon), A549 (human epithelial
carcinoma cell line of the lung), MCF7 (human epithelial adenocarci-
noma cell line of the breast)] were used. All cell lines were cultured in
DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10%
FBS, 45 IU/ml penicillin and 45 IU/ml streptomycin at 37 ◦C in a hu-
midified atmosphere of 5% CO2. The cells were inoculated into 96-well
micro titer plates at a density of 1 × 104 cells per well. After cell inoc-
ulation, the micro titer plates were incubated for 24 h to attached cells
on well surface. Test compounds prepared in DMSO were dissolved in
cell culture medium at appropriate final concentrations and added each
well. After the addition of compounds, plates were incubated for 48 h at
37 ◦C and 5% CO2. Cells were fixed by the gentle addition of 50 mL of
cold 10% (w/v) and incubated for 60 min at 4 ◦C. After incubation were
plates washed with tap water for four times and SRB solution at 0.4%
(w/v) in 1% acetic acid has been added to each well and the plates were
incubated for 30 min at room temperature. Then the plates were rinsed
quickly four times with 1% acetic acid to removed unbound excess dye.
Bound stain was subsequently eluted with 10 mM tris base solution (pH
10.5) and the absorbance was read on an Elisa plate reader at 510 nm.
Percentage growth for each concentrations of compound was calculated
on a plate-by-plate basis for test wells relative to control wells. Analyses
of cell growth percentage were performed using GraphPad Prism
(GraphPad Software Inc and calculated 50% cell growth inhibition
(IC50) value of each test compounds.
2.3.1. Topoisomerase I and II
α
enzyme inhibition
(hTopo I and hTopo IIα)
Human DNA topoisomerase I and II
α
inhibitory activities of the tested compounds were determined by
plasmid relaxation assay which could measure the conversion of
supercoiled pBR322 plasmid DNA to its relaxed form [39,40]. In all
experiments, the final concentration of DMSO was 1% and control
samples contained an equivalent amount of vehicle. Recombinant pu-
rified human Topo I and IIα were purchased from TopoGEN (PortOr-
ange, FL, USA). All other common laboratory chemicals were of the
highest grade available.
Firstly, hTopo I and II
α enzymes inhibition of derivatives were
screened at the concentration of 3 mM, and dose-dependent experiments
were carried out with various concentrations (0.5–3 mM for hTopo I and
8–1000 µM for hTopo IIα) of the most effective compounds that signif-
icantly inhibited the enzymes. Finally, optical intensity of each con-
centration of the compounds was compared with the control to calculate
inhibition percentages. With these percentages, IC50 (50% inhibition
concentrations) values of the compounds were calculated using the S
probit analysis program.
For hTopo I relaxation assay, reaction mixture included 0.2 μg of
supercoiled pBR322 plasmid DNA, 1 U of hTopo I and enzyme buffer (10
mMT ris-HCl (pH 7.5), 100 mM NaCl, 1 mM PMSF, 1 mM ß-mercap-
toethanol). Six different concentrations (0.5–3 mM) of the compounds
were added reaction mixture without plasmid, the enzyme and test
compounds were preincubated for 5 min at 37 ◦C. Enzyme reactions
◦
were started by addition of DNA and incubated 30 min at 37 C. The
All experiments were repeated for a minimum of two times with each
experiment done in three replicates. Camptothecin and Etoposide were
used as the positive control drugs.
reactions were terminated by stop solution containing 1% SDS and 6X
loading buffer.
For hTopo II
volume of 10
α relaxation assay, the reaction mixture had a total
μ
L containing 10 mM TrisHCl (pH 7.9), 50 mM NaCl, 50
2.4. Molecular docking studies
mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mL/mL bovine serum albumin
(BSA), 1 mM ATP, 0.3 mM pBR322 plasmid DNA, 1 U of human DNA
¨
Molecular docking studies were performed by using Schrodinger
Topo II
α enzyme and different concentrations of compounds. The
¨
molecular modeling software (Schrodinger Release 2018-2, LLC, New
mixture was incubated for 1 h at 37 ◦C. After incubation period, 3 mL of
loading buffer in electrophoresis buffer TAE (60 mMTris, 30 mM acetic
acid and 1.5 mM EDTA, pH 8.0) was added.
York, NY, USA) [43,44]. Ligands were prepared by using LigPrep
module, the 2D structures of the ligands converted to the full 3D
structure by assigning the OPLS-2005 force field. LigPrep can generate
the expected ionized forms at significant concentrations corresponding
to the pH 7.0 ± 3.0; generate variations, verification and optimize the
structures. It generates maximum 32 stereochemical structures per
ligand. Binding of ligands to the receptors adopts more than one
conformation and the lowest energy conformer is important for docking
After hTopo I and hTopo IIα enzyme and derivatives were incubated
and reactions were terminated, the samples were electrophoresed in a
horizontal 1% agarose gel in TAE buffer (40 mM Tris acetate, 2 mM
EDTA, pH 8.0) at 45 V for 3 h at room temperature. The gels were
stained with ethidium bromide (1 μg/ml) and photographed under UV
illumination. Band distributions were analysed by a GDS 8000 Complete
Gel Documentation and Analysis System (Gel Works 1D Intermediate,
version 2.5; UltraViolet Products).
studies. Crystal structure of human Topo IIα enzyme complexed with
known inhibitor; etoposide was extracted from the Protein Data Bank
(PDB ID: 5GWK) (Fig. 3a). Prior to docking the ligands onto the pro-
tein’s active site, the protein was prepared using protein preparation
Standard drugs, camptothecin (CPT) for hTopo I and etoposide for
hTopo IIα, were used as positive controls. All experiments were repeated
¨
wizard of Schrodinger software. During the protein preparation all
for a minimum of two times.
hetero atoms and water molecules were removed. Hydrogen atoms were
added, and the active site of the protein was defined for generating the
grid. The grid box was limited to the size of 20 Å at the active site.
Finally docking studies were carried out using GLIDE (Grid-based Ligand
2.3.1.1. Analysis of data for relaxation assays. Topoisomerase enzyme
activities were measured by the optical rate of different forms of DNA
bands on agarose gel electrophoresis. Inhibition percentages were
calculated by bands of intensity difference between control and com-
pound applied wells. We analyzed supercoiled-relaxed DNA bands for
catalytic inhibition. We assumed supercoiled DNA without enzyme well
as a one hundred percent of enzyme was inhibited. We used direct
proportion to calculate inhibition percentages of the remaining super-
coiled DNA intensity that belonged to tested compounds in each well. If
inhibition was not obtained at any concentration of a tested compound it
¨
Docking with Energetics) module of Schrodinger Software, the ligands
were docked into the prepared grid by using “Extra precision mode” and
no constraints were defined. The docking method was first validated by
docking of the known inhibitor, etoposide with 0,42 Å RMSD (root-
mean-square deviation) value. To determine the spatial fit into the
active site of receptor, favorable ligand poses were generated, and best
fitted conformations of the ligands were evaluated and minimized for
generating glide scores. To predict the binding affinities and best
5

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Fig. 2. Structure-hTopo IIα inhibiton activity relationship of the synthesized compounds.
alignment of the compounds at the active site of the enzyme, hydrogen
bonds and pi interactions formed with the surrounding amino acids and
glide scores were used. All the results were presented in Table 3.
complex species as well as its parameterization was implemented by
AMBER14SB force field available in the LEAP module of AMBER v14
[49] suite of programs. The complex molecule was solvated in LEAP by
putting together small units of TIP3 water boxes in dimensions of
18.774 Å × 18.774 Å × 18.774 Å to constitute a rectangular water box.
Solvent unit box means; size of the box of 216 water molecules (WAT-
BOX216) that will be used to overlay the solute, remove water molecules
that are too close to or inside the solute, and be trimmed to the desired
size. The distance between the outer boundary of the octahedral solvent
box and the solute surface was set to 10 Å. A space of 0.4 Å was used to
set water molecules at the solute–solvent boundary.
2.5. Molecular dynamic simulations methods
2.5.1. Structural models
The atomic coordinates of DNA Topo IIα from human have been
deposited in the Protein Data Bank (www.rcsb.org; PDB accession no.
5GWK) [45] (Fig. 3a). hTopo IIα was co-crystallized with a part of DNA
helix and an inhibitor of receptor, etoposide.
Relaxation and temperature equilibration of the solvated complex
structure were implemented by the PMEMD module of AMBER v14, and
molecular dynamics (MD) computations were carried out by the
PMEMD.CUDA module of AMBER v.14, running at the TR-Grid e-
Infrastructure of Turkey. The protein-DNA helix-ligand systems were
preheated up to 298.15 K over 100 ps (50000 iterations and 2 fs for each
of them), during which the protein and the ligand were strongly
restrained. Finally, at least 75 nsec of production MD runs were imple-
mented with no restrains for the complex systems at 1 bar and 298.15 K,
which utilized the Langevin dynamics algorithm (with a collision fre-
quency of 1 ps ꢀ 1 and a velocity limit of 10 Kelvin) for keeping the
temperature constant and SHAKE algorithm. Structural changes in MD
results of enzyme-DNA complex system had visualized and analyzed by
Chimera v1.14 [50] molecular modeling program (Figs. 7–10).
Receptor crystallized in dimer form. Subunit A was chosen was used
for docking protocol. All the other heteroatoms except the interested
part of DNA helix (i.e., nonreceptor atoms such as redundant water
molecules, ions, co-crystallized ligand, etc.,) were also removed. For the
preparation of receptor Gasteiger charges and polar hydrogens were
assigned, and the receptor input file was prepared in PDBQT format for
AutoDock Vina by using the Auto-Dock Tools package [46]. Ligands
were drawn with Discovery Studio Client 3.5 [47]. The conformations
were fixed with the “clean geometry” option of this package. Clean
Geometry tool uses a fast, DREIDING-like force field to optimize the
structure geometry. The tool improves the geometry of the molecule and
results in an approximate 3D structure. For all ligands, including O95,
the nonpolar hydrogen atoms were merged, and the Gasteiger charges
were assigned. Later, ligand input files were also arranged in PDBQT
format using the AutoDock Tools package. The binding sphere was
selected around the inhibitor EVP using the binding site tools.
2.6. In silico ADME/Tox studies
2.5.2. Small molecule docking
2.6.1. Molecular property analysis
Compounds were docked into the binding site of D2R by Auto-
dock_vina v1.5.6. The docking area was determined in the crystal
structure, by a grid box of 60 Å 50 Å 40 Å using a 0.375 Å grid point
spacing in AutoGrid. The docking grid box was defined with the x, y, and
z centers as 18.464, ꢀ 40.422, and ꢀ 61.005, respectively. Auto-Dock
Vina uses a global heuristic optimizer algorithm, Iterated Local
Search, with a local optimization algorithm called Broyed- Fletcher-
Goldfarb-Shanno (BFGS) [46]. The docking conformations of ligands
in the binding sites of the receptor were searched with this Iterated Local
Search Global Optimizer algorithm with a Monte Carlo sampling tech-
nique as a molecular mechanic method. Semiflexible algorithm was used
in docking protocol. Docked coordinates were visualized and selected by
MGLtools v1.5.6 [48].
Chemical structures of compounds were prepared in mol file using
ChemDraw 12.0 and molecular properties were calculated using
Accelrys Discovery Studio 3.5. All the ligands were screened for Lip-
inski’s Rule of 5 [51] and Veber rules [52]. All of the data was presented
in Table 4.
2.6.2. ADME/Tox prediction
ADME/Tox (ADMET) protocol in Discovery studio 3.5 [47] were
used for deploying ADMET properties of all synthesized molecules (1a-
1r, 2a-2i). This model calculates some pharmacokinetic properties like
aqueous solubility, blood brain barrier penetration (BBB), cytochrome
P450 (CYP450) 2D6 inhibition, hepatotoxicity, plasma protein binding
and human intestinal absorption (HIA) from compounds structure and
also predicts some toxicity properties such as AMES and developmental
toxicity potential (DTP).
2.5.3. Simulation procedure
Protonation, ion addition and solvation of the initial structure of the
Aqueous solubility calculations use base 10 logarithm of the molar
6

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Fig. 3. (a) Crystal structure of hTopo II
α
enzyme complexed with etoposide (PDB ID: 5GWK). (b) Superposition of 2i and etoposide: both have pi interactions with
Arg487. (c) Docked position of etoposide: compound revealed H-bond with deoxyguanosin DG13, and Asp463; pi-pi stacking with deoxyguanosin DG13; pi-cation
interactions with Arg487. (d) Docked position of 2i: compound revealed pi-pi stacking with deoxyadenosine DA12 and deoxyguanosin DG13, pi-cation interaction
with Arg487. (e) Docked position of 1i: compound revealed pi-pi stacking with deoxycytidine DC8, deoxyadenosine DA12 and deoxyguanosin DG13, pi-cation
interaction with Arg487.
solubility as predicted by linear regression to predict the solubility of
each compound in water at 25 ◦C [53].
Plasma protein binding of drug molecules can affect the efficiency of a
drug, because the bound fraction is temporarily shielded from meta-
bolism. On the other hand, only the unbound fraction exhibits phar-
macological effects. The classification whether a compound is highly
bounded (≥90% bound) to plasma proteins using the cutoff Bayesian
score of ꢀ 2.226 (obtained by minimizing the total number of false
positives and false negatives) [57,58].
BBB model predicts the blood brain barrier penetration of a molecule
after oral administration by using quantitative linear regression model,
as well as 95% and 99% confidence ellipses in the PSA_2D and AlogP98
plane [54].
The cytochrome P450 2D6 (CYP2D6) model predicts CYP2D6
enzyme inhibition using 2D chemical structure as input. The classifica-
tion whether a compound is an CYP2D6 inhibitor using the cutoff
Bayesian score of 0.162 (obtained by minimizing the total number of
false positives and false negatives) [55].
The results of ADME/Tox prediction of the benzoxazole (1) and
oxaxolo[4,5-b]pyridine (2) derivatives were shown in Table 5.
Human intestinal absorption (HIA) is defined as a percentage
absorbed after oral administration. A well-absorbed compound is one
that is absorbed at least 90% into the bloodstream in humans. The in-
testinal absorption model includes 95% and 99% confidence ellipses in
the ADMET_PSA_2D, ADMET_AlogP98 plane [54,59].
The hepatotoxicity model predicts potential organ toxicity for a wide
range of structurally diverse compounds. The classification whether a
compound is hepatotoxic using the cutoff Bayesian score of ꢀ 0.4095
(obtained by minimizing the total number of false positives and false
negatives) [56].
The ellipses define regions where well-absorbed compounds are ex-
pected to be found: 95% of well-absorbed compounds are expected to
fall within the 95% ellipse, while 99% of well-absorbed compounds
should fall within the 99% ellipse. In general, however, absorption tends
The plasma protein binding model predicts whether a compound is
likely to be highly bound (≥90% bound) to carrier proteins in the blood.
7

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
to drop off quite rapidly outside the 95% ellipse [54]. ADME plot of all
benzoxazole (1a-1r) and oxazolo[4,5-b]pyridine (2a-2i) derivatives was
given in Fig. 11.
seen in Scheme 1. In the present study, all the compounds are original
except compounds 1a, 1c, 1j, 1l, 2a, 2c-2h [34–38].
3.2. Biological evaluation
2.6.3. Ames mutagenicity
The mutagenicity QSTR model of the TOPKAT program has been
developed from compounds assayed according to the US EPA GeneTox
protocol. According to this protocol, a chemical is tested against five
strains of Salmonella typhimurium, namely: TA100, TA1535, TA1537,
TA1538, and TA98, using the Histidine Reversion Assay [60].
Tests are performed both with and without S9 activation. A chemical
is labeled a mutagen if a positive response, that is, a significant increase
in number of reversions as compared to the background reversions, is
observed against one or more strains, with or without S9 activation. A
chemical is considered a non-mutagen if a negative response, that is, no
significant increase in number of reversions as compared to the back-
ground reversions, is observed in all of these five bacterial strains with or
without S9 activation. Therefore, when a query structure is assessed by
TOPKAT to be a non-mutagen (computed probability of mutagenicity
between 0.0 and 0.3), it indicates that, there is a high probability of the
query chemical producing a negative response in the Histidine Rever-
sion Assay, with or without S9 activation, against all of the five bacterial
strains.
3.2.1. hTopo I and IIα inhibitions
Inhibition of hTopo I and hTopo Iiα from the conversion of super-
coiled pBR322 plasmid DNA by benzoxazole (1) and oxazolo[4,5-b]
pyridine (2) derivatives were monitored using a relaxation assay. IC50
values for inhibition of hTopo II
α were in the micromolar range
(Table 1). Clinically well-known Topo I and Topo II inhibitors CPT and
etoposide, respectively were used as positive controls.
Most of the compounds did not show any activity against hTopo I
enzyme. Only, 1a, 1m, 1o, (2273, 3133, 2769 µM, respectively) indi-
cated less effect. Suprisingly, the effects on hTopo IIα enzyme of these
series either benzoxazole (1) and oxazolo[4,5-b]pyridines (2) exhibited
very significant results that would allow further studies. Namely, all
compounds showed more or less some activities between 2 µM and over
3 mM with IC50 values. Among the tested compounds only 5-nitro-2-(p-
butylphenyl)benzoxazol (1i) and 2-(p-butylphenyl)oxazolo[4,5-b]pyr-
idin (2i) displayed the most potency for hTopo IIα with 2 µM IC50 value.
These two small structures were found to be more effective than stan-
dard drug, etoposide (IC50 value = 10 µM). In parallel with earlier
findings [31–33], n-butyl substitution on the para position of phenyl
displayed lower IC50 values (1i, 2i) than the other substituents. Ac-
cording to previously CoMFA [32] and CoMSIA [33] studies we reported
2.6.4. The developmental toxicity potential
The Developmental Toxicity Potential (DTP) Module of the TOPKAT
package comprises three statistically significant and cross-validated
quantitative structure–toxicity relationship (QSTR) models, and the
data from which these models are derived. Each model applies to a
specific class of chemicals. Molecular structure is the only input required
to conduct a Developmental Toxicity Potential assessment. These
discriminant models compute the probability of a submitted chemical
structure being a developmental toxicant in the rat; a probability below
0.3 indicates no potential for developmental toxicity (NEG), and prob-
ability above 0.7 signifies developmental toxicity potential (POS). The
probability range between 0.3 and 0.7 refers to the “indeterminate” zone
(IND).
–
an electron-withdrawing and a hydrophilic group such as NO2 or
–
COOCH3 at the 5 and/or 6 position of the heterocyclic ring system
(benzazoles) increased the Topo II inhibitory activity. In addition, we
also reported over there a hydrophobic and a bulky group at the ortho
and para positions of the phenyl ring played a very important role for
enhancing the Topo II inhibitory activiy. In the present study, it has been
observed that the most effective compounds were those that bearing n-
butyl group at the para position of the phenyl ring. It is noticed that the
para position is more important than others and, on this position, had to
have more hydrophobic and steric substituent in order to achieve the
best Topo IIα inhibition effect. Even though the compound 1r has n-
butyl at the para location of phenyl ring, did not show good activiy as
much as 1i. This result brings to mind a question of what the effect of the
position of nitro group on the activity is. It can be considered that the
position of 5 of benzoxazole ring is more substantial than 6 when we
3. Results and discussion
3.1. Chemistry
compare the position of nitro group. For increasing hTopo IIα inhibition
The synthetic pathways for preparation of the target compounds
listed in Table 1 are shown in Scheme 1. The synthesis of compounds
(1a-1r, 2a-2i) was performed by condensing of appropriate amine and
suitable acids in polyphoshoric acid [34,61,62] in one step procedure as
potent, 5-nitrobenzoxazole or oxazolo[4,5-b]pyridine as main pharma-
cophore cores can be used for further studies. On the other hand, the
compounds 1k (22 µM), and 2d (28 µM) demonstrated moderate activity
even if they showed lower potency than etoposide.
Scheme 1. Reaction pathway of synthesized compounds (1a-1r, 2a-2i).
8

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Table 1
R2
R3
R5
R1
R
O
N
IC50 Values of benzoxazoles and oxazolo[4,5-b]pyridines versus hTopo I, hTopo II α.
R4
X
CompNo
X
R
R1
R2
R3
R4
R5
Topo Inhibition
IC50 (µM)
hTopo I
hTopo IIα
1a
1b
1c
1d
1e
1f
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
C(CH3)3
CH(CH3)2
CF3
2273
NE
NE
NE
NE
NE
NE
NE
NT
NE
NE
NE
3133
NE
2769
NE
NT
NE
NE
NE
NE
NE
NE
NE
NE
NT
34
188
133
116
652
376
191
884
263
2
SCH3
CH3
CH3
CH3
CH3
CH3
1g
1h
1i
CH3
CH3
CH3
C4H9
1j
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
C(CH3)3
CH(CH3)2
CF3
657
22
1k
1l
698
214
1043
143
3339
483
> 3 mM
7457
2909
28
1m
1n
1o
1p
1r
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C4H9
2a
2b
2c
2d
2e
2f
C(CH3)3
CH(CH3)2
CF3
N
N
N
SCH3
CH3
N
CH3
CH3
1390
277
426
315
2
N
CH3
2g
2h
2i
N
CH3
CH3
CH3
N
CH3
N
C4H9
PC*
PC*
CPT
ETOP
NE
NE
10
*Positive control; CPT: Camptothecin, ETOP: Etoposide, NT: Not tested, NE: No effect.
3.2.1.1. SAR for hTopo II
α
. The results of structure-hTopo II
α
inhibition
etc.) routinely used as topoisomerase inhibitors on cancer cell lines are
examined, it is seen that the effect can be quite different according to the
cell line. A good example that we can give to this situation is the mer-
barone compound, which is routinely used as an hTopo II catalytic in-
hibitor. Although the IC50 values of the effects of this compound on
MCF7, Hela, DU145 cells are 83.9, 62.3 and 18.9 µM, it is known to be
much more effective in different types of cancer cell lines (MT-4 (T-cell
Leukaemia) etc.) even at lower IC50 concentrations (12 µM) [63,64].
The relaxation assay, in which we tested topoisomerase inhibition, is
a cell-free system. However, in order to observe the same effect in the
cell-dependent system, the first requirement is that the sufficient
amount of the compound can enter the cell and reach the nucleus stably.
It suggests that the reason why the high activity we detected in the cell-
free system in our experiment could not affect the cell environment in
the same way may be that the compounds could not enter the cell at
sufficient concentrations.
activity of the synthesized benzoxazoles and oxazolo[4,5-b]pyridines is
summarized in Fig. 2.
Presence of an electron withdrawing group ( NO2) in the 5th posi-
–
tion has a positive effect on activity rather than the 6th position. At the
same time oxazolo[4,5-b]pyridine core is slightly important than 6-
nitrobenzoxazole for improving hTopo IIα inhibition effect. Among the
substituents on the phenyl ring located in the 2nd position, the para
position shows higher activity, and gradually decreases towards the
meta and ortho position. The fact that the substituent on the phenyl ring
is an electron donating group increases the activity. While the straight
alkyl chain located in para position of phenyl ring also enhances the
activity, presence of a branched structure such as tert-butyl reduces ac-
tivity due to steric shield.
3.2.2. Cyctotoxic activity
In this study, the most active compounds (1i and 2i) were also tested
using some of cancer lines such as HeLa (Human Epithelial Cervix
Adenocarcinoma), WiDR (Human Epithelial Colorectal Adenocarci-
noma), A549 (Human Lung Carcinoma), MCF7 (Human Breast Adeno-
carcinoma) for evaluating of the anti-tumor cyctotoxicity (Table 2).
However, 1i did not show any good activity on these cell lines. Com-
pound 2i indicated 79,9 and 80,53 µM IC50 values for HeLa and WiDR,
respectively. Although these two compounds displayed very significant
In conclusion, in the light of these data we have obtained, the effects
of compounds should be examined on different cell lines and possible
problems with the cell pathways should be determined.
Table 2
IC50 Values of compounds 1i and 2i versus some cancer cell lines.
CompNo
In vitro Anticancer Activity
IC50 (µM)
activity for hTopo IIα, but they did not show satisfactory cyctotoxicity
HeLa
WiDR
A549
MCF7
on the tested cancer cell lines. It can be said that these compounds are
not suit for HeLa, WiDR, A549, and MCF7. It can be considered that they
have to test on different cancer cell lines.
1i
>100
79,9
5,27
4,98
100
>100
>100
0,809
22,85
>100
>100
1,959
6,023
2i
80,53
2,50
11,02
CPT
ETOP
When the cytotoxic effects of anticancer agents (e.g. CPT, etoposide,
9

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
affinities and best alignment of the compounds at the active site of the
enzyme, hydrogen bonds and pi interactions formed with the sur-
rounding amino acids and glide scores were used. All the results were
shown in Table 3.
Table 3
Docking results of the most active hTopo IIα inhibitors.
Compounds
Docking Score
Glide Score
1i
ꢀ 5.452
ꢀ 4.823
ꢀ 10.193
ꢀ 5.452
ꢀ 4.823
ꢀ 10.193
According to the docking results, compounds 2i and 1i showed
strong interactions between one of the important active site residues,
Arg487 and DNA similar to etoposide. When we look at the super-
position of 2i and etoposide, both have pi interactions with Arg487
(Fig. 3b). Compounds 2i revealed pi-pi stacking with DA12 and DG13, pi-
cation interaction with Arg487 (Fig. 3c); Structure 1i revealed pi-pi
stacking with DC8, DA12 and DG13, pi-cation interaction with Arg487
(Fig. 3d).
2i
Etoposide
3.2.3. Molecular docking studies
The 3D molecular docking of the most active compounds (1i, 2i)
molecules on crystal structure of hTopo II
α
(PDB ID: 5GWK) (Fig. 3a)
¨
was performed by using Schrodinger molecular modeling software
¨
(Schrodinger Release 2018–2, LLC, New York, NY, USA) [43,44] for the
first time to evaluate the binding energies as well as their mode of
interaction with the active site of the enzyme. To predict the binding
3.2.4. Molecular dynamics simulations
We selected compounds 1i and 2i having the best hTopo IIα inhibi-
tory activity for the Molecular Dynamics (MD) simulations using
AMBER. Human Topo II (5GWK) in complex with DNA and etoposide
from protein data bank was used for this study, as well. Molecular dy-
α
namics simulations between hTopo IIα and etoposide were implemented
Huang et al. [65] ALA505, GLU506, LYS489, ARG487, GLY462,
ASP463, MET762, MET766 residues and DA6, DG5, DG2, DC3, DG4,
bases were found in binding pocket (Fig. 4) [65].
For the analysis of binding profile for compounds 1i and 2i of MD
simulations were implemented for 75 nsec. The plots of RMSD curves for
complex are shown in Figs. 5 and 6. In there, blue line, green line, black
line, and red line represent whole system, protein structure, DNA helix,
compounds, respectively. Initial coordinates of the peptide, DNA and
compound (Supporting Information) were taken as reference in RMSD
computations. As seen in the Fig. 5, although compound 2i was con-
formationally stabilized after 30 nsec of MD (red lines), the protein
structure was stabilized after 65 nsec. This situation in the MD study
indicates that the last 10 nsec of the MD curve can be used for under-
standing of the binding profile.
While compound 1i conformationally is stabilized after 45 nsec of
MD (red lines), the protein structure is stabilized after 65 nsec as seen in
Fig. 6. It is pointed out that the last 10 nsec of the MD trajectory can be
Fig. 4. Binding pocket with etoposide [65].
Fig. 5. RMSD curve for compound 2i and protein complex (blue line refers to whole system; green line refers to protein structure; black line refers to DNA helix; red
line refers to compound). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. RMSD curve for compound 1i and protein complex.
10

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
utilized to understand of the binding profile.
Superposition of the X-ray structure of complex, PDB ID: 5GWK [45]
with the last snapshot coordinates of system that was extracted from the
75 nsec MD trajectory, as shown in Fig. 7Fig. 7A-B. This clearly indicates
that the integrity of α-helical and β-pleated sheet structures throughout
protein is mainly preserved after MD computation of compound 2i and
hTopo IIα complex. However, DNA helix twists on counterclockwise,
protein structure shows a elastic recovery by the shrinking of the helix
parts.
The integrity of α-helical and β-pleated sheet structures throughout
protein is mainly preserved after MD computation of 1i and hTopo II
α
complex as well as 2i (see Fig. 8A–B). Besides, DNA helix protects its
position, shrinking of the protein structure could be seen easily.
It was observed that 2-(p-butylphenyl)-5-nitrobenzoxazole (1i)
mainly interacted with the binding pocket of protein through the last 30
nsec of MD trajectory. Nitro group of compound 1i interacted with the
guanidinium group of ARG487 (Fig. 9). This hydrogen bond was pro-
tected for whole last 30 nsec.
2-(p-Butylphenyl)oxazolo[4,5-b]pyridine (2i) was also interacted
with the binding pocket of protein through the last 10 ns of MD trajec-
tory. Oxazole part of oxazolo[4,5-b]pyridine ring of 2i provides
hydrogen bond acceptor points by both “O” and “N” atoms. While “O” of
oxazole ring of 2i interacted with the amino group of residue GLU506,
“N” formed hydrogen bond with DG5 of purine ring (Fig. 10). However,
Fig. 9. Hydrogen bonding between ARG487 and 2-(p-butylphenyl)-5-nitro-
benzoxazole (1i).
Fig. 7. Superposition of initial structure and the complex structure after 75 nsec MD. (A) Base view of the complex. DNA helix twisting counterclockwise. Blue helix
shows initial coordinates, red helix shows the structure after MD. (B) Front view of complex. Shrinking on the helixes was shown with arrows. Blue protein shows the
initial coordinates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Superposition of initial structure and the complex structure after 75 nsec MD. (A) Base view of the complex. DNA helix protects its position. Red helix shows
initial coordinates, red helix shows the structure after MD. (B) Front view of complex. Shrinking on the helixes was shown with arrows. Light brown protein shows
the initial coordinates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
11

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Fig. 10. (a) Hydrogen bonding between ARG487, LYS489 and 2-(p-butylphenyl) oxazolo[4,5-b]pyridine (2i) at the beginning of MD simulation. (b) Hydrogen
bonding between GLU506, DG5 and 2i at last 10 nsec of MD.
at the beginning of the MD simulation, compound 2i had interacted with
ARG487. It was seen that the compound 2i formed two hydrogen bonds
with GLU506 residue and DG5 base in the last 10 nsec of MD simulation.
In here, it was noticed that GLU506 prevents the exit of 2i from the
binding groove of receptor. (Please refer to the link for the movement of
2i in the Supporting Information)
that the newly developed drugs could have better properties [66]. In
silico ADME calculations are extensively used for molecular modeling
studies to understand structure–property relationships and to predict
drug metabolism and pharmacokinetic properties [67].
In this study, the drug-likeness, molecular and ADME/Tox properties
of all synthesized and tested compounds were calculated by using Dis-
covery Studio 3.5 [47]. First of all, we screened the drug-likeness pro-
files of compounds in terms of their suitability to Lipinski [51] and Veber
[52] rules. The data was given in Table 4. All compounds except 1i and
1r whose AlogP values slightly >5, were detected to be suitable
3.2.5. In silico ADME/Tox properties
Nowadays, in silico calculations have been came into prominence in
order to minimize the unfavorable ADME/Tox properties and to ensure
Table 4
Molecular properties of benzoxazoles (1a-1r) and oxazolo[4,5-b]pyridines (2a-2i),
Comp. No
AlogP
MW
n_HBA
n_HBD
n_rot
n_ring
n_Ar
PSA_2D
n_vio.
1a
4,575
4,369
4,117
3,716
4,147
4,147
4,147
4,147
5,029
4,575
4,369
4,117
4,147
4,147
4,147
4,147
5,029
3,726
3,519
3,268
2,867
3,298
3,298
3,298
3,298
4,18
296,321
282,294
308,212
286,306
268,267
268,267
268,267
268,267
296,321
296,321
282,294
308,212
268,267
268,267
268,267
268,267
296,321
252,311
238,284
264,203
242,296
224,258
224,258
224,258
224,258
252,311
<500
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
≤5
3
3
3
3
2
2
2
2
5
3
3
3
2
2
2
2
5
2
2
2
2
1
1
1
1
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
66,638
35,076
35,076
35,076
35,076
35,076
35,076
35,076
35,076
35,076
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
≤1
0
1b
3
1c
3
1d
4
1e
3
1f
3
1g
3
1h
3
1i
3
1j
3
1k
3
1l
3
1m
3
1n
3
1o
3
1p
3
1r
3
2a
2
2b
2
2c
2
2d
3
2e
2
2f
2
2g
2
2h
2
2i
2
Lipinski’s Rule
Veber Rule
≤5
≤10
≤12
≤10
≤140
ALogP: the log value of octanol–water partition coefficient,
MW: molecular weight.
n_HBA: number of hydrogen bond acceptor.
n_HBD: number of hydrogen bond donor.
n_rot: number of rotatable bonds.
n_ring: number of rings.
n_Ar: number of aromatic rings.
PSA_2D: 2D polar surface area.
n_vio.: violations from Lipinski’s rule of five or Veber rules.
12

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Table 5
ADME/Tox properties of synthesized compounds (1a-1r, 2a-2i).
Comp.
No
Sol.
BBB
Abs.
Lev.
CYP2D6
CYP2D6
Pred.
Hepatot.
Hepatot.
Pred.
PPB
PPB
DTP
DTP
AMES
Prob.
AMES
Pred.
Pred.
Prob.
Pred.
1a
1b
1c
1d
1e
1f
ꢀ 5,981
ꢀ 5,688
ꢀ 5,621
ꢀ 5,060
ꢀ 5,591
ꢀ 5,585
ꢀ 5,59
0,206
0,142
0,064
ꢀ 0,06
0,073
0,073
0,073
0,073
0,346
0,206
0,142
0,064
0,073
0,073
0,073
0,073
0,346
0,523
0,46
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
ꢀ 4,50583
ꢀ 4,48847
ꢀ 1,37569
ꢀ 4,44524
ꢀ 4,89291
ꢀ 5,86512
ꢀ 5,66685
ꢀ 6,16644
ꢀ 5,22693
ꢀ 4,1141
false
false
false
false
false
false
false
false
false
false
false
false
false
false
false
true
6,89242
8,08332
3,9971
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
10,2943
9,51438
11,2172
8,20364
7,38375
7,84808
6,7395
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
true
0,462518
0,428538
0,429121
0,478355
0,462383
0,453826
0,45066
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
Non-
Tox.
0,204236
0,282564
0,162506
0,542507
0,594832
0,593382
0,635974
0,578182
0,33227
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
Non-
Mut.
7,51601
4,66038
4,64077
5,03583
5,92295
7,06498
5,868
1g
1h
1i
ꢀ 5,585
ꢀ 6,028
ꢀ 5,981
ꢀ 5,688
ꢀ 5,621
ꢀ 5,591
ꢀ 5,585
ꢀ 5,590
ꢀ 5,585
ꢀ 6,028
ꢀ 5,343
ꢀ 5,034
ꢀ 4,983
ꢀ 4,386
ꢀ 4,914
ꢀ 4,909
ꢀ 4,908
ꢀ 4,913
ꢀ 5,395
7,305
0,46448
11,0665
9,1346
0,515893
0,447309
0,414559
0,41512
1j
0,137759
0,202474
0,105731
0,542393
0,54077
1k
1l
ꢀ 4,09675
ꢀ 0,983972
ꢀ 4,50119
ꢀ 5,4734
7,0589
8,3547
2,97268
3,92563
3,90601
4,30108
4,89853
6,04056
2,73681
3,85068
ꢀ 0,114222
3,4907
10,0575
6,57104
7,03537
5,92679
6,14533
9,90679
3,40257
2,45855
4,47375
1,33802
2,74554
3,20987
1,97287
2,10129
5,05187
1m
1n
1o
1p
1r
2a
2b
2c
2d
2e
2f
0,447179
0,438917
0,435863
0,449206
0,499128
0,440437
0,414299
0,387302
0,445249
0,42364
ꢀ 5,27513
ꢀ 5,77472
ꢀ 4,83521
ꢀ 1,42132
ꢀ 1,49928
0,642698
ꢀ 2,23427
ꢀ 3,66074
ꢀ 4,63295
ꢀ 4,93428
ꢀ 4,43468
ꢀ 2,17439
0,588996
0,500051
0,246615
0,346366
0,431082
0,289392
0,611188
0,638715
0,651816
0,642121
0,674526
0,48933
false
false
false
true
0,382
0,258
0,391
0,391
0,391
0,391
0,664
false
false
false
false
false
false
1,54716
1,52755
2,23041
1,92261
2,31379
0,41587
2g
2h
2i
0,425548
0,413003
0,460808
Sol. (Aqueous Solubility): log(Sw) < -8.0: Extremely low (0); ꢀ 8.0 < log(Sw) < -6.0: No, very low, but possible (1); ꢀ 6.0 < log(Sw) < -4.0: Yes, low (2); ꢀ 4.0 < log(Sw)
< -2.0: Yes, good (3); ꢀ 2.0 < log(Sw) 0.0: Yes, optimal (4); 0.0 < log(Sw): No, too soluble (5).
logBB (BBB): logBB ≥ 0.7: Very high penetrants (0); 0 ≤ logBB < 0.7: High penetrants (1), ꢀ 0.52 < logBB < 0: Medium penetrants (2), logBB ≤ -0.52: Low penetrants
(3).
Abs. Lev. (Absorbtion levels): 0:Good; 1:Moderate; 2:Poor; 3:Very Poor.
CYP2D6 binding: true: inhibition of CYP2D6; false: no inhibition; CYP2D6 Pred. (CYP2D6 Prediction).
Hepatot. (Hepatotoxicity): true: hepatotoxic, false: non-hepatotoxic; Hepatot. Pred. (Hepatotoxicity prediction).
PPB: true: binding to plasma protein, false: no binding; PPB Pred. (PPB prediciton).
DTP: developmental toxicity potential. The probability below 0.3 indicates no potential for developmental toxicity (NEG), and probability above 0.7 signifies
developmental toxicity potential (POS); DTP Prob. (DTP probability); DTP Pred. (DTP prediciton); Non-Tox. (Non-Toxic).
AMES: The probability below 0.3 indicates non-mutagen (NEG), and probability above 0.7 signifies mutagen (POS).; AMES Prob. (AMES probability); AMES Pred.
(AMES prediction); Non-Mut. (Non-Mutagen).
according to Lipinski’s rule of five and Veber rules.
to the blood–brain barrier. In ADME plot (Fig. 11) of all compounds
were placed into the prediction confidence space (95% and 99%) for the
Blood Brain Barrier Penetration and Human Intestinal Absorption
models, especially oxazolo[4,5-b]pyridine derivatives (2a-2i) were
located in the middle of prediction confidence ellipses. Besides all
compounds tended to bind plasma proteins. CYP2D6 was not played a
The results of pharmacokinetic properties of the compounds were
presented in Table 5. According to these datas, it was noticed that ADME
properties of tested benzoxazoles and oxazolo[4,5-b]pyridines were
found to be very satisfactory. This study showed us that they had an
acceptable solubility, a very good absorbtion level, and highly penetrant
13

E. Karatas et al.
Bioorganic Chemistry 112 (2021) 104913
Fig. 11. Two dimensional ADME image for benzoxazoles (1a-r) and oxazolo[4,5-b]pyridines (2a-i).
role in metabolism of compounds except 1p and 2c. On the other hand,
AMES and developmental toxicity potential (DTP) tests were calculated
as toxicology tests and none of the compounds were predicted to exhibit
mutagenicity and toxicity. Considering all these results, the synthesized
compounds were thought to indicate good drug-likeness and pharma-
cokinetic properties, therefore, are estimated that they will exhibit good
bioavailability.
of hTopo IIα enzyme, as lead anticancer compounds.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4. Conclusion
Acknowledgments
In this study, we designed and synthesized a novel class 5(or 6)-nitro-
2-(substitutedphenyl)benzoxazole (1a-1r) and 2-(substitutedphenyl)
oxazolo[4,5-b]pyridine (2a-2i) compounds as candidate antitumor
agents targeting human DNA topoisomerase enzymes (hTopo I and
The Central Laboratory of the Faculty of Pharmacy, Ankara Uni-
versity supported the acquisition of the NMR, Mass spectra and
elemental analyses in this work.
hTopo IIα). None of the compounds indicated significant inhibition ac-
tivity for hTopo I enzyme. Suprisingly, two original structures, 5-nitro-2-
Appendix A. Supplementary material
(4-butylphenyl)benzoxazole (1i) and 2-(4-butylphenyl)oxazolo[4,5-b]
pyridine (2i), showed very significant effect on the hTopo IIα with 2 µM
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104913.
IC50 value. Even these two small compounds displayed more activity
than standard drug etoposide (10 µM IC50 value). These two molecules
were also tested on some cancer cell lines such as HeLa, WiDR, A549,
and MCF7. Unfortunately, they did not display any notable cytotoxicity.
At this point, we can say that the compounds should be tried on different
cancer cell lines. According to molecular dynamic simulations study,
compound 2i showed very close relationships with residue and nucle-
otide which are in the nearby of the active site of receptor, such as
GLU506 and DG5, unlike the docking study. Compound 1i formed
hydrogen bond with ARG487 in the binding pocket similar as the mo-
lecular docking results. Moreover, ADME/Tox properties of all mole-
cules were predicted using Discovery Studio 3.5 software programme. It
can be noticed that the tested compounds were thought to indicate good
drug-likeness and pharmacokinetic properties. Therefore, it can be
estimated that they would exhibit good bioavailability. Considering all
these results, especially original two small compounds 1i and 2i ob-
tained from this study can be useful in designing of new potent inhibitors
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