Design, synthesis, molecular modeling and anti-proliferative evaluation of novel quinoxaline derivatives as potential DNA intercalators and topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.06.004

New series of [1,2,4]triazolo [4,3-a]quinoxaline and bis([1,2,4]triazolo)[4,3-a:3′,4′-c]quinoxaline derivatives have been designed, synthesized and biologically evaluated for their cytotoxic activities against three tumor cell lines (HePG-2, Hep-2 and Caco-2). Compounds 16_e, 21, 25_a and 25_b exhibited the highest activities against the examined cell lines with IC₅₀ values ranging from 0.29 to 0.90 μM comparable to that of doxorubicin (IC₅₀ ranging from 0.51 to 0.73 μM). The most active members were further evaluated for their topoisomerase II (Topo II) inhibitory activities and DNA intercalating affinities as potential mechanisms for their anti-proliferative activities. Interestingly, the results of Topo II inhibition and DNA binding assays were consistent with that of the cytotoxicity data, where the most potent anti-proliferative derivatives exhibited good Topo II inhibitory activities and DNA binding affinities, comparable to that of doxorubicin. Moreover, the most active compound 25_a caused cell cycle arrest at G2/M phase and induced apoptosis in Caco-2 cells. In addition, Furthermore, molecular docking studies were performed for the novel compounds against DNA-Topo II complex to investigate their binding patterns. Based on these studies, it was concluded that DNA binding and/or Topo II inhibition may contribute to the observed cytotoxicity of the synthesized compounds.

Full text of DOI:10.1016/j.ejmech.2018.06.004

Accepted Manuscript
Design, synthesis, molecular modeling and anti-proliferative evaluation of novel
quinoxaline derivatives as potential DNA intercalators and topoisomerase II inhibitors
M.K. Ibrahim, M.S. Taghour, A.M. Metwaly, A. Belal, A.B.M. Mehany, M.A.
Elhendawy, M.M. Radwan, A.M. Yassin, N.M. El-Deeb, E.E. Hafez, M.A. ElSohly, I.H.
Eissa
PII:
S0223-5234(18)30494-X
10.1016/j.ejmech.2018.06.004
EJMECH 10474
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 April 2018
Revised Date: 31 May 2018
Accepted Date: 1 June 2018
Please cite this article as: M.K. Ibrahim, M.S. Taghour, A.M. Metwaly, A. Belal, A.B.M. Mehany, M.A.
Elhendawy, M.M. Radwan, A.M. Yassin, N.M. El-Deeb, E.E. Hafez, M.A. ElSohly, I.H. Eissa, Design,
synthesis, molecular modeling and anti-proliferative evaluation of novel quinoxaline derivatives as
potential DNA intercalators and topoisomerase II inhibitors, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.06.004.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, molecular modeling and anti-proliferative evaluation of novel
quinoxaline derivatives as potential DNA intercalators and topoisomerase II
inhibitors
a
a
b
c
d
M. K. Ibrahim , M. S. Taghour , A. M. Metwaly , A. Belal , A. B. M. Mehany , M.
e
e
f
f
g
A. Elhendawy , M. M. Radwan A. M. Yassin , N. M El-Deeb , E. E. Hafez , M. A.
e
a
ElSohly* and I. H. Eissa*
a
Medicinal Chemistry department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo11884, Egypt.
b
Pharmacognosy department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo 11884, Egypt.
c
Medicinal Chemistry department, Faculty of Pharmacy, Beni-Suef University, Beni-
Suef 62514, Egypt.
d
Zoology department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt.
e
National Center for Natural Products Research, University of Mississippi,
University, MS 38677, USA.
f
Biopharmaceutical Product Research Department, Genetic Engineering and
Biotechnology Research Institute, City of Scientific Research and Technological
Applications, Alexandria, Egypt.
g
Department of Plant Protection and Biomolecular Diagnosis, ALCRI City of
Scientific Research and Technological Applications, Alexandria, Egypt.
*
Corresponding authors:
Ibrahim H. Eissa
Medicinal Chemistry department, Faculty of Pharmacy (Boys), Al-Azhar University,
Cairo 11884, Egypt.
Email: Ibrahimeissa@azhar.edu.eg
M. A. ElSohly
National Center for Natural Products Research, University of Mississippi, University,
MS 38677, USA.
Email: mmelsohly@olemiss.edu
1

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Abstract
New series of [1,2,4]triazolo[4,3-a]quinoxaline and bis([1,2,4]triazolo)[4,3-a:3',4'-
c]quinoxaline derivatives have been designed, synthesized and biologically evaluated
for their cytotoxic activities against three tumor cell lines (HePG-2, Hep-2 and Caco-
2
). Compounds 16 , 21, 25 and 25 exhibited the highest activities against the
e a b
examined cell lines with IC values ranging from 0.29 to 0.90 µM comparable to that
5
0
of doxorubicin (IC ranging from 0.51 to 0.73 µM). The most active members were
5
0
further evaluated for their topoisomerase II (Topo II) inhibitory activities and DNA
intercalating affinities as potential mechanisms for their anti-proliferative activities.
Interestingly, the results of Topo II inhibition and DNA binding assays were
consistent with that of the cytotoxicity data, where the most potent anti-proliferative
derivatives exhibited good Topo II inhibitory activities and DNA binding affinities,
comparable to that of doxorubicin. Moreover, the most active compound 25 caused
a
cell cycle arrest at G2/M phase and induced apoptosis in Caco-2 cells. In addition,
Furthermore, molecular docking studies were performed for the novel compounds
against DNA-Topo II complex to investigate their binding patterns. Based on these
studies, it was concluded that DNA binding and/or Topo II inhibition may contribute
to the observed cytotoxicity of the synthesized compounds.
Key words: Anticancer; DNA-intercalator; Molecular docking; Quinoxaline
derivatives; Topoisomerase II;
1.1.Introduction
DNA intercalators are those compounds which interact with DNA helix through
an intercalation process causing interference with the cell cycle and cell death [1].
Intercalation process can be defined as the process by which compounds containing
planar aromatic systems are inserted between adjacent base pairs of the DNA helix
[
2]. DNA intercalators in clinical use include anthracyclines (e.g. doxorubicin 1 and
mitoxantrone 2) [3, 4], acridine derivatives (e.g. amsacrine 3) [2] and ellipticine 4a
5].
[
Topoisomerase II (Topo II ) is a pivotal regulator of DNA replication [6]. Topo II
modifies DNA topology, controls DNA supercoiling and regulates a number of vital
nuclear processes, such as transcription, replication and repair [7]. Compounds
targeting Topo II can be divided into two classes. The first one, is Topo II poisons
which increases the levels of Topo II-DNA covalent complexes, generating a lesion in
DNA strands [8]. Topo II poisons comprises many of clinically active compounds
2

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including doxorubicin 1 [3], mitoxantrone 2 [4], PD-115934 5 [9] and KW -2170 6
10]. The second class inhibits Topo II catalytic activity without increasing in levels
[
of Topo II covalent complexes. This second class is termed catalytic inhibitors as
etoposide [11]. Hence, targeting the DNA and blocking of Topo II are good
approaches for the discovery of new anticancer agents [12-22]
DNA Intercalators and Topo II poisons share three common essential structural
features. The first one is a planar polyaromatic system (chromophore) which is
sandwiched between DNA base-pairs [23]. The second feature is a cationic species,
interacting with the negatively charged phosphate group of DNA. The cationic center
may be an amino or nitrogen containing heterocyclic group, which can be protonated
at physiological pH [24] ( Fig. 1). The third feature is a groove binding side chain
moiety, occupying the minor groove of DNA [25-27].
Several quinoxaline containing compounds possess promising biological activities
such as anti-cancer [28], anti-diabetic [29, 30] and anti-convulsant [31, 32].
Quinoxaline analogues, such as echinomycin 7 [33] and 2,3-bis(2-pyridyl)-
quinoxaline (DPQ) 8 [34] were reported to have DNA intercalation effect.
Chemical synthesis of two scaffolds of quinoxaline derivatives were carried out to
reach the first and second common features of both DNA intercalators and Topo II
poisons. In attempt to obtain the third feature, a molecular hybridization of
quinoxaline moiety and other effective antitumor moieties such as triazoles [35-37],
hydrazones [31, 38], semicarbazones [39, 40] and thiosemicarbazones [36, 41] have
been proceeded.
1.2.Rationale of molecular design
In continuation of our pervious works of design and synthesis of new
anticancer agents [8, 42, 43], this study was carried out to obtain new compounds
with different chemical isosters in order to study the SAR of these compounds and the
effect of each substituent on their anti-tumor activities.
The design and synthesis of the title compounds were carried out by molecular
hybridization of quinoxaline moiety with hydrazones, semicarbazides,
thiosemicarbazides and triazoles to produce [1,2,4]triazolo[4,3-a]quinoxaline
(scaffold-1)
and bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline-3-thiol (scaffold-2)
derivatives. Then, the chemical modifications of the synthesized compounds were
carried ouy by two pathways. The first one was performed on scaffold-1 through
replacement of hydrazone, semicarbazide and thiosemicarbazide groups of the
3

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synthesized compounds by alkylamino or alkoxy groups. The second pathway was
carried out on scaffold-2 via insertion of different acetanilide, alkyl, and alkyl ester
derivatives on the thiol group. The choice of the different substituents was based on
their relatively high lipophilicity to pass the nuclear membranes aiming to have strong
DNA intercalating activity [44] (fig. 2).
Indeed, all the newly synthesized quinoxaline derivatives were evaluated for
their in vitro inhibitory activity against three different cancer cell lines; hepatocellular
carcinoma (HepG-2), human laryngeal carcinoma (Hep-2) and colorectal
adenocarcinoma (Caco-2), using neutral red assay. The excellent results drove us to
perform further examinations to understand the mechanism of action.
Firstly, the most active members were evaluated for their ability to inhibit the
catalytic activity of Topo II. Secondly, these members were tested to determine their
binding affinities against DNA through restriction enzyme cleavage inhibition and
DNA/methyl green assays. Thirdly, the most active compound (25 ) was investigated
a
for its effect on topo II relaxation activity and apoptosis induction potential in Caco-2
cell line. Fourthly, gene expression analysis was done for compound 25 in Caco-2
a
to determine the affected genes by quinoxaline derivatives. Finally, a molecular
docking of the tested compounds was carried out to investigate their binding patterns
with the prospective target, DNA-Topo II complex (PDB-code: 3qx3).
4

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Fig. 1. Basic pharmacophoric features of DNA intercalators
5

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Fig. 2: Rationale concept to new DNA-intercalators
6

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2
2
. Results and discussion
.1. Chemistry
For synthesis of the target compounds, reactions sequence is illustrated in
Schemes 1–3. At first, o-phenylenediamine was treated with oxalic acid in the
presence of 4N HCl to afford 2,3-(1H,4H)-quinoxalinedione 11 [23], and subsequent
treatment with thionyl chloride to produce 2,3- dichloroquinoxaline 12 [23]. Stirring
of the 2,3 dichloroquinoxaline with hyrazine hydrate at room temperature afforded 2-
chloro-3-hydrazinylquinoxaline 13 [45], subsequent heating with triethylorthoformate
to afford 4-chloro[1,2,4]triazolo[4,3-a]quinoxaline 14 [45] . The obtained compound
1
4 was heated with hydrazine hydrate to afford 4-hydrazinyl-[1,2,4] triazolo[4,3-
a]quinoxaline 15 [46]. Reflux of 14 with commercially available alkyl amines
namely, methylamine, 2-hydroxyethylamine, n-propylamine, n-butylamine
cyclohexylamine and benzylamine in the presence of triethylamine afforded the target
compounds 16 , respectively. The IR spectra of 16a-f demonstrated stretching bands
a-f
-1
1
at a range of 3353-3255 cm corresponding to NH groups. Moreover, H NMR of
these compounds showed exchangeable signals at a range of δ 7.91 - 8.48 ppm
corresponding to NH groups. Singlet signals appeared around δ 10.00 ppm
corresponding to aromatic protons in position-1. In addition, reflux of 14 with
commercially available primary aliphatic alcohols namely, methanol and ethanol in
1
the presence of triethylamine afforded the title compounds 17 , respectively. The H
a,b
NMR spectrum of compound 17 as representative example, showed a singlet signal
a
at δ 4.19 ppm corresponding to methyl group. The aromatic area of 17 spectra
a
showed a characteristic singlet signal at δ 10.06 ppm corresponding to the aromatic
proton in position-1. (Scheme 1).
Condensation of compound 15 with certain aromatic aldehydes namely, 2-
methoxybenzaldehyde, 2-chlorobenzaldehyde and 2,4-dichlorobenzaldehyde in
absolute ethanol with catalytic ammout of glacial acetic acid yielded the
1
corresponding hydrazones 18 , respectively . The H NMR spectra of compounds
a-c
1
8a-c revealed the presence of D O exchangeable singlet signals of hydrazinyl NHs at
2
about 12.00 ppm, and two singlet signals for the idene moiety at about 8.87, 8.94
ppm. According to the reported procedure of thiosemicarbazide and semicarbazide
synthesis [47, 48], reaction of 15 with appropriate isothiocyanates and/or isocyanates
namely,
ethylisothiocyanate,
phenylisothiocyanate,
phenylisocyanate
and
cyclohexylisocyanate in absolute ethanol afforded the corresponding
7

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thiosemicarbazides 19 , and semicarbazides 20 , respectively. The IR spectra of
a,b
a,b
compounds 19a,b and 20a,b showed sharp absorption bands of 3NH groups of
-1 1
thiosemicarbazide and semicarbazide moieties in the range of 3350 - 3233 cm . H
NMR of compound 20 as a representative example, showed three exchangeable
b
singlet broad signals at a round δ 6.25, 9.80 and 9.86 ppm corresponding to the three
NH groups. Moreover, reflux of 15 in alcoholic mixture of carbon disulphide and
potassium hydroxide afforded bis[1,2,4]triazolo[4,3-a:3',4'-c]quinoxaline-3-thiol 21.
1
The H NMR spectrum of compound 21 showed an exchangeable singlet broad signal
at δ 14.83 ppm corresponding to the SH group. Also, a singlet signal appeared at δ
1
0.45 ppm corresponding to the aromatic proton at position-10 (Scheme 2).
The treatment of compound 21 with alcoholic solution of potassium hydroxide
afforded potassium bis[1,2,4]triazolo[4,3-a]quinoxaline-4-thiolate 22. Then, heating a
mixture of compound 22 and different alkyl 2-chloroacetates, namely, methyl 2-
chloroacetate and ethyl 2-chloroacetate in dry DMF yielded the corresponding
sulfanylacetate derivatives 23 , respectively. The IR spectra of 23a,b demonstrated
a,b
-1
stretching bands at 1738 and 1733 cm corresponding to C=O groups of esters.
1
Moreover, H NMR of these compounds showed disappearance of SH signal and
appearance of aliphatic signal in the aliphatic region. On the other hand, the
potassium salt 22 was allowed to react with appropriate alkylhalides, namely,
methylbromide and benzyl bromide in dry DMF to afford the corresponding
1
sulfanylalkyl derivatives 24 , respectively. The H NMR spectra of 24 showed
a,b
a,b
appearance of different signals in the aliphatic region corresponding to the different
alkyl moieties. In addition, the reaction of 22 with chloroacetanilides namely, 2-
chloro-N-(3-methoxyphenyl) acetamide and 2-chloro-N-(4-fluorophenyl)acetamide in
dry DMF gave the corresponding acetanilide derivatives 25 , respectively. The IR
a,b
1
spectra of 25a,b demonstrated stretching bands at 1689 and 1689 cm- corresponding
1
to C=O groups. Moreover, H NMR of these compounds showed exchangeable
signals at δ10.45 ppm corresponding to NH groups. Also, singlet signals appeared at δ
1
0.02 and 10.00 ppm corresponding to the aromatic protons at position-10. Finally,
the reaction of 22 with ethyl-4-bromobutyrate in dry DMF afforded the target
-1
compound 26. The IR spectra of 26 demonstrated stretching bands at 1716 cm
1
corresponding to C=O group of ester. H NMR showed appearance of five signals in
the aliphatic region. (Scheme 3).
8

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H
NH2
NH2
O
O
N
O
N
Cl
Cl
HO
HO
ii
i
+
N
H
O
N
9
12
1
0
1
1
iii
N
N
N
N
N
v
N
N
N
NHNH2
Cl
iv
N
NHNH2
N
Cl
1
4
1
5
1
3
R-NH2
1
vi
vii
R -OH
N
N
N
N
N
N
N
a) R = CH3,
1
a) R = CH
b) R = CH CH OH
3
R1
R
2
2
1
N
b) R = C H
N
O
c) R = n. C H
2
5
3
7
H
d) R = n. C H
1
6a-f
4
9
17a,b
e) R = Cyclohexyl
f) R = Benzyl
Scheme 1. General procedure for preparation of target compounds 15, 16 , 17 ;
a-f
a,b
Reagents and conditions: (i) 4 N HCl/reflux/6h, (ii) SOCl /DCM/reflux/4h, (iii)
2
NH NH .H O/ ethanol/ room temperature, (iv) triethyl orthoformate/reflux/4h, (v)
2
2
2
NH NH .H O/ ethanol/ reflux/4h, (vi) TEA/ reflux/4h, (vii) TEA/ reflux/4h
2
2
2
9

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Scheme 2. General procedure for preparation of compounds 18 , 19 , 20 , 21;
a-c
a,b
a,b
Reagents and conditions: (i) absolute ethanol/ gl. acetic acid/ reflux, (ii) absolute
ethanol/ reflux/ 3h, (iii) absolute ethanol/ reflux/ 3h, (iv) absolute ethanol/ KOH/ CS2/
reflux/6h.
1
0

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Scheme 3. General procedure for preparation of compounds 23 , 24 , 25 , 26;
a,b
a,b
a,b
Reagents and conditions: (i) absolute ethanol/ KOH/ heating/ 10 min. (ii) & (iii) dry
DMF/KI/ heating in w.b./ 2h, (iv) & (v) dry DMF/KI/ heating in w.b./ 3h.
2
.2.Biological testing
2
.2.1. In vitro anti-proliferative activities
The current synthesized compounds have been tested for their anti-cancer activities
against three different cancer cell lines; hepatocellular carcinoma (HepG-2), human
laryngeal carcinoma (Hep-2) and colorectal adenocarcinoma (Caco-2), through
neutral red assay protocol as described by Borenfreund and Puerner [49]. The results
of cytotoxicity were reported as IC values and summarized in Table 1.
5
0
The cytotoxicity results indicated that the examined compounds showed versatile
anti-proliferative activities against the tested cell lines. In general, compounds 16 , 21,
e
2
5 and 25 were the most active members of this study, showing excellent anti-
a b
1
1

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proliferative activities against the HepG-2, Hep-2 and Caco-2 cell lines. These
compounds showed IC values near and in several cases less than the reference
5
0
compound (doxorubicin). Compound 16 showed cytotoxic activities with IC values
e
50
of 0.63, 92 and 0.46 µM, respectively, while compound 21 exhibited cytotoxic
activities with IC values of 0.56, 0.77 and 0.57 µM, respectively, also, compound
5
0
2
5 had cytotoxic activities with IC values of 0.50, 0.29 and 0.39 µM, respectively,
a 50
and compound 25 found to have cytotoxic activities with IC values of 0.76, 0.50
b
50
and 0.80 µM, respectively. Doxorubicin inhibited HepG-2, Hep-2 and Caco-2 cell
lines with IC values of 0.62, 0.51 and 0.73 µM, respectively.
5
0
In addition, compounds 16 , 18 , 19 , 20 and 23 exhibited superior anti-
c
c
b
a
b
proliferative activities against HepG-2 and Hep-2 tumor cell lines with IC values
5
0
range of 0.01 - 0.83 µM, but they showed non-significant activities against Caco-2
cell line with IC50 values of 2.25, 5.70, 1.29, 1.44 and 1.42 µM, respectively.
Meanwhile, compounds 16 , 18 and 20 exhibited good anti-proliferative activities
a
b
b
against Hep-2 and Caco-2 tumor cell lines with IC range of 0.12 - 1.48 µM, but they
5
0
showed less cytostatic activities against HepG-2 cell line with IC values of 9.89 and
5
0
2
.28 µM, respectively. The rest of compounds (16 , 17 , 17 , 18 , 19 , 23 , 24 , 24
b a b a a a a b
and 26) didn’t show promising cytotoxic activities as the mentioned compounds.
1
2

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Table 1: Anti-proliferative activities of the target compounds against HepG-2, Hep-2
and Caco-2 cell lines
a
IC (µM)
5
0
Comp.
HepG-2
Hep-2
Caco-2
1
6a
6b
0.88 ± 0.04 3.17 ± 0.16 1.48 ± 0.07
3.18 ± 0.16 0.14 ± 0.01 2.66 ± 0.14
1.08 ± 0.05 2.33 ± 0.18 3.00 ± 0.21
0.83 ± 0.07 0.01 ± 0.001 2.25 ± 0.14
0.63 ± 0.06 0.92 ± 0.07 0.46 ± 0.04
2.97 ± 0.23 40.53 ± 3.57 4.51 ± 0.32
8.05 ± 0.58 3.95 ± 0.19 8.15 ± 0.41
9.90 ± 0.67 4.21 ± 0.34 6.41 ± 0.51
2.44 ± 0.19 2.21 ± 0.17 3.76 ± 0.26
9.89 ± 0.78 0.12 ± 0.01 1.15 ± 0.09
0.06 ± 0.01 0.52 ± 0.04 5.70 ± 0.45
1.90 ± 0.15 2.37 ± 0.18 3.46 ± 0.24
0.64 ± 0.05 0.10 ± 0.01 1.29 ± 0.11
0.71 ± 0.05 0.56 ± 0.04 1.44 ± 0.12
2.28 ± 0.18 0.32 ± 0.03 0.97 ± 0.07
0.56 ± 0.04 0.77 ± 0.05 0.57 ± 0.04
9.39 ± 0.65 3.11 ± 0.15 6.33 ± 0.51
1
1
6c
6d
6e
6f
1
1
1
17a
17b
18a
18b
1
1
8c
9a
19b
20a
20b
2
1
23a
23b
24a
24b
25a
25b
0.81 ± 0.06 0.36 ± 0.03 1.42 ± 0.11
b
22.74 ± 2.04 5.79 ± 0.46
NA
b
NA
5.36 ± 0.37 10.53 ± 0.74
0.50 ± 0.04 0.29 ± 0.02 0.39 ± 0.03
0.76 ± 0.06 0.50 ± 0.03 0.80 ± 0.06
b
2
6
5.80 ± 0.46
NA
14.77 ± 1.18
Doxorubicin 0.62 ± 0.05 0.51 ± 0.04 0.73 ± 0.07
a
Three independent experiments were performed for each concentration
NA: Compounds having IC value >50 µM.
b
5
0
1
3

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2
.2.2. Topoisomerase II inhibitory activity
The most active compounds 16 , 16 , 16 , 18 , 18 , 19 , 19 , 21, 20 , 20 , 23 ,
a
c
d
b
c
a
b
a
b
b
2
5 and 25 were evaluated for their inhibitory activity against Topo II The catalytic
a
b
activity of Topo II was assessed according to reported method described by Patra et
al [50]. Doxorubicin, was used as a positive control in this procedure. The results
were recorded as a 50% inhibition- concentration values (IC ) calculated from the
5
0
concentration-inhibition response curve and summarized in Table 2.
Topo II was efficiently inhibited by most of the tested compounds which
displayed good to moderate inhibitory activities with IC values ranging from 0.68 to
5
0
4
.38 µM. These results were consistent with that of in vitro cytotoxicity activities for
a great extent. Compounds 16 , 20 , 20 , 21, 25 and 25 showed good inhibitory
e
a
b
a
b
activities with IC values of 0.68, 1.19, 1.22, 1.06, 0.85 and 0.94 µM, respectively,
5
0
comparing with that of the standard drug, doxorubicin (IC = 0.88 µM). On the other
5
0
hand, some compounds displayed moderate activities as compounds 16 , 16 , 18 , 18 ,
a
c
b
c
1
9b and 23 (IC50 = 2.09, 1.94, 1.91, 2.97 and 2.98 µM, respectively). Finally,
compound 19 displayed weak inhibitory activity with an IC value of 5.38 µM).
a
50
Table 2: IC values of the tested compounds against Topo II
5
0
a
Comp.
IC (µM)
50
1
6a
6d
2.09 ± 0.17
1.94 ± 0.15
0.68 ± 0.05
1.91 ± 0.14
2.97 ± 0.21
5.38 ± 0.38
2.98 ± 0.21
1.19 ± 0.08
1.22 ± 0.09
1.06 ± 0.07
2.12 ± 0.15
0.85 ± 0.06
0.94 ± 0.07
1
1
6e
1
8b
1
8c
9a
1
19b
20a
20b
2
1
23b
25a
25b
Doxorubicin 0.88 ± 0.06
Three independent experiments were performed for each concentration
a
1
4

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2
.2.3. DNA intercalation assay
2
.2.3.1. Restriction enzyme cleavage inhibition test
Restriction enzymes (endonucleases) cleavage inhibition is a sensitive tool for
determination of DNA binding affinities with different intercalators [51]. DNA-
intercalators inhibit the DNA cleavage caused by restriction enzymes [52]. Here, Nine
compounds that revealed strong anti-proliferative activities (16 , 16 , 16 , 18 , 18
a
d
e
b
c
1
9 , 21, 25 and 25 ) were further evaluated to determine their abilities to inhibit
b a b
DNA cleavage caused by restriction endonucleases, Sal 1 enzyme (recognition
sequence GTCGAC) and EcoR I (recognition sequence GAATTC). Doxorubicin was
used as a positive control in this assay.
Some compounds completely inhibited the cleavage activities of both Sal I
and EcoR I (16 , 18 , 19 , 21, and 25 ) while compounds 16 , 16 , 18 , and 25 ,
e
c
b
a
a
d
b
b
completely inhibited the cleavage activity of Sal I with partial inhibition of EcoR I
activity (Table 3 & Fig. 3).
2.2.3.2. DNA/methyl green assay
In order to provide additional quantitative information about binding affinity of
the synthesized compounds towards the DNA molecule, DNA/methyl green assay
was carried out for the most active anti-proliferative derivatives (16 , 16 , 16 , 18 ,
a
d
e
b
1
8 19 , 20 , 20 , 21, 23 , 25 and 25 ) according to the reported procedure described
c b a b b a b
by Burre et al [53]. DNA-binding affinities were reported as IC50 values and
summarized in Table 3. Doxorubicin was used as a positive control.
Compounds 20 , 21, 20 19 , 25 and 16 showed good binding affinities with
b
a,
b
a
e
IC values of 28.45, 29.85, 31.26. 34.30, 38.62 and 39.37 µM, respectively. On the
5
0
other hand, compounds 16 , 16 , 18 and 25 exhibited moderate IC values ranging
a
d
c
b
50
from 40.13 to 49.12. Finally, compounds 18 and 23 showed high IC values of
b
b
50
5
2.34 and 62.64 µM, respectively.
1
5

ACCEPTED MANUSCRIPT
Table 3: DNA intercalating affinity of the tested compounds.
DNA intercalation
Comp.
a
b
RECI IC ( µM )
5
0
c
1
6a
6d
PI
48.63
49.12
39.37
52.34
40.13
34.30
31.26
28.45
29.85
62.64
38.62
43.40
30.24
c
1
PI
d
1
6e
8b
8c
CI
c
1
PI
d
1
CI
d
19b
20a
20b
CI
e
e
NT
NT
d
2
1
CI
e
23b
25a
25b
NT
d
CI
c
PI
c
Doxorubicin
PI
a
b
c
Restriction enzyme cleavage inhibition
50% Inhibition concentration values of DNA/methyl green assay
d
e
Partial inhibition
Complete inhibition
Not tested
Fig. 3: Inhibition of restriction enzymes action using different synthesized
compounds. Left; (+) Lambada DNA cutting pattern using EcoRI and Sal I; (-)
Lambada DNA. Middle; L; DNA Ladder, 1: 16 , 2: 18 ; 3: 25 ; 4: 25 . Right) L;
d
b
a
b
DNA Ladder, 1: 16 ; 2: 16 ; 3: 18 ; 4: 19 ; 5: 21.
a
e
c
b
1
6

ACCEPTED MANUSCRIPT
2
.2.4. Effect on topoisomerase II relaxation activity
The effect of 25 on the relaxation of supercoiled plasmid pBS-SK (+) DNA in
a
presence and absence of Topo II was examined (Fig. 4). m-AMSA was used as a
reference drug (10 µM) [15, 18]. Different concentrations of 25 (5, 10, 25 and 50
a
µM) were used. As shown in Fig. 4, compound 25 revealed a dose-dependent effect
a
on Topo II relaxation activity. At 5 µM, no significant inhibition was observed, at 10
µM, the inhibitory effect increased slightly, while at 25 µM, it showed observable
inhibition. The inhibitory effect was detected through the increase of supercoiled
DNA amount. It was observed that the effect of compound 25 at 25 and 50 µM is
a
comparable with the effect of the reference drug.
Fig. 4: Effect of compound 25 and m-AMSA on relaxation of supercoiled pBS-SK
a
(+) DNA by human recombinant Topo II.
2
.2.5. Topo II-mediated DNA cleavage assay
Topo II-mediated DNA cleavage assay was performed for compound 25 to
a
investigate its mode of action. Based on formation of linear DNA or not, Topo II
poisoning or catalytic inhibition can be detected then [15]. Concentration of 25 and 50
µM of compound 25 and 10 µM of m-AMSA were used to study the ability of these
a
concentrations to induce linear DNA. The reference standard induced formation of
linear DNA, however, the tested concentrations of 25 showed inability to induce
a
formation of the cleavage complex (Fig. 5), this indicates that, compound 25 might
a
act as Topo II catalytic inhibitor.
1
7

ACCEPTED MANUSCRIPT
Fig. 5: Effect of compound 25a and m-AMSA on stabilization of covalent DNA-Topo
II complex.
2
.2.6. Flow cytometric analysis of the cell cycle
Flow cytometric analysis is a rapid, reliable and reproducible method allows
estimation and analysis of cell-cycle parameters of surviving cells [54].The promising
derivative 25 was selected to examine its impact on cell cycle progression and
a
induction of apoptosis in Caco-2 cell line using 0.5 µM. Cell cycle parameters in
Caco-2 cells that had been incubated for 24 h with 0.5 µM of compound 25 were
a
compared with untreated control cells. The obtained results showed accumulation of
Caco-2 cells at G2/M phase, the percent was 40.58 % (Table 4). Compound 25a
exhibited ability to induce apoptosis at priG1 phase (Fig. 6 & 7). The percentages of
cell death at Pre-G1 phase by compound 25 and in the control Caco-2 cells were
a
1
3.99 and 2.76, respectively.
Table 4: (%) of cell cycle phases and pre-G1 apoptosis upon treatment with
compounds 25 using Caco-2 cells
a
Results
Comp.
5a
Cont. Caco-2
%
G0-G1
%S
%G2-M
40.58
%Pre-G1
13.99
2
44.23
53.85
15.19
30.96
15.19
2.76
1
8

ACCEPTED MANUSCRIPT
Fig. 6: Schematic diagram for % of Caco-2 cells in each phase and apoptosis effect
upon treatment with 0.5 µM of compound 25a.
Fig. 7: Cell cycle analysis: A-control Caco-2, B- compound 25 by flow cytometery
a
using PI staining method.
1
9

ACCEPTED MANUSCRIPT
2
.2.7. Apoptosis induction
To investigate the mode of induced cell death in Caco-2 cells at pri-G1 phase,
Annexin V/propidium iodide (PI) double staining assay method was performed. Caco-
2
cells were treated with 0.5 µM of the tested compound 25 . The obtained results
a
were presented in Fig. 8 and Table 5. The percentage of apoptosis induced by
compound 25a was 11.41. So that, We can say that treatment of Caco-2 cells with
compound 25a lead to increase in the percentage of apoptotic cells from 0.48% for
control untreated cells to be 3.91% in the early stage and also lead to increase in the
apoptotic cells in the late stage to attain 7.5% in comparison to 0.69% in control
Caco-2 cells. The obtained results revealed that compound 25a induced pre G1
apoptosis and arrested cell cycle at G2/M phase.
Fig. 8: Schematic diagram for percentage of cell death induced by compound 25 on
a
Caco-2 cells
Table 5: Percent of cell death induced by compound 25 on Caco-2 cells.
a
Apoptosis %
Comp.
5a
Control
Necrosis
Total Early
Late
7.5
2
13.99
2.76
3.91
0.48
2.58
1.59
0.69
2
0

ACCEPTED MANUSCRIPT
2
.2.8. Gene expression analysis (RNA Extraction and Real-Time RT-PCR for
tested Genes)
The gene expression pattern of the most sensitive cell line (Caco-2) was treated
with the most effective member (compound 25 ). Comparing with the untreated cells
a
as shown in Fig. 9, the current results indicate that the treatment up-regulated the
expression level of cytochrome c gene and down-regulated the expression of Bax,
Beclin 1, caspase 8, cyclin B and Bcl-2 genes (Table 6). These findings provide
insights into the possible molecular mechanisms of growth inhibition by the tested
compound. In spite of the ability of compound 25 to induce the apoptotic pathway in
a
Caco-2 cells, the expression level of caspase 8 gene (apoptotic gene) was down
regulated. The mutations in caspase-8 gene in malignant cells were recorded in
various types of cancers with alteration in the stop codon [36]. In addition, Caco-2
cells are less sensitive to apoptosis induced by caspase-8 over expression. Caspase-8
mutations have also been identified in hepatocellular, gastric carcinomas and
insensitive colorectal carcinomas with defective in the activities of apoptosis in
mutants [55-57]. So we could conclude that compound 25 treatment could down-
a
regulate the mutated form of Caspase-8 gene that could be overexpressed in cancer
cells. Moreover, compound 25 decreased the expression of BCl-2, which is an
a
essential factor for apoptosis resistance (anti-apoptotic). The ability of compound 25a
to down-regulate Bcl-2 levels emphasize its potential to induce apoptosis.
Table 6: RT-PCR gene expression of Bax, Bcl1, Caspase 8, Cyclin B cytochrome c
and Bcl-2 genes of Caco-2 cells treated with compound 25a
a
Gene
Control
Regulation /25
Effect
a
BaX
Bec1
961.233
632.377
465.306
315.741
432.534
312.483
590.624
519.840
112.682
701.798
259.743
106.895
Down regulated
Down regulated
Down regulated
Up regulated
Caspase8
Cytoc2
Cyclin B
Bcl-2
Down regulated
Down regulated
a
Three independent experiments were performed.
2
1

ACCEPTED MANUSCRIPT
Fig. 9: Schematic diagram for real time PCR data for Bax, Bec 1, Casp-8, Cyto-2,
Cyclin B and Bcl-2 genes and their control
2
.3. Molecular docking
In this work, molecular docking investigational study was performed for the
synthesized compounds, and doxorubicin as a reference DNA intercalator. This study
was carried out in order to gain further insight into the binding modes of the
synthesized compounds into the DNA binding site of Topo II (ID: 3qx3). The binding
free energies (∆G) were reported Table 7. The negative values of free energies
indicates the spontaneity of bindings. The reported key binding site of DNA-Topo II
complex consists of amino acid and nucleotide residues of Asp479, Arg503, Gln778,
Met782 Cyt8, Thy9, Cyt11, Gua13 and Ade12 [58].
The proposed binding mode of doxorubicin showed affinity value equals -
5
5.51 kcal/ mol and RMSD value of 1.45. The planer aromatic system formed
aromatic stacking interactions with the different key residues of Arg503, Gln778,
Gly478, Gly776, Ala779, Asp479, Met782, Thy9, Cyt8, Gua13, and Ade12. The NH
group of sugar moiety formed a hydrogen bonding interaction with the phosphate
group of Thy9. The OH group of sugar moiety formed a hydrogen bonding interaction
with the phosphate group of Cyt8. The OH group at 12-position was involved in
hydrogen bonding interaction with Thy9. The OH group at the 8-position formed a
hydrogen bond with C=O group of Gua7 (Fig. 10).
2
2

ACCEPTED MANUSCRIPT
Fig. 10: Binding of doxorubicin with Topo II, the hydrogen bonds are represented in
green dashed lines and the pi interactions are represented in orange lines.
The proposed binding mode of compound 16 showed affinity value of - 40.41
e
kcal/mol with two hydrogen bonds and eleven pi-pi interactions. The planer aromatic
system ([1,2,4]triazolo[4,3-a]quinoxaline) was involved in aromatic stacking
interactions with base pairs of DNA; Thy9, Cyt8 and Gua13. The NH group of
butylamino side chain was involved in a hydrogen bonding interaction with the
Arg503. The butyl side chain was oriented in the minor groove of DNA (Fig. 11).
Fig.11: Binding of compound 16 with Topo II, the hydrogen bonds are represented in
e
green dashed lines and the pi interactions are represented in orange lines
2
3

ACCEPTED MANUSCRIPT
The proposed binding mode of compound 18 showed affinity value of - 41.12
b
kcal/mol. It showed one hydrogen bond interaction between NH group of hydrazinyl
moiety at 4-position and the C=O group of Arg503. In addition, the planer aromatic
system ([1,2,4]triazolo[4,3-a]quinoxaline) showed aromatic stacking interactions with
Thy9, Cyt8 and Gua13. Moreover, the terminal benzylidene moiety was oriented at
the minor groove of DNA (Fig. 12).
Fig. 12: Binding of compound 18 with Topo II, the hydrogen bonds are represented
b
in green dashed lines and the pi interactions are represented in orange lines
Compound 20 showed affinity value of - 39.65 kcal/mol. The planer
a
aromatic system demonstrated three pi-pi interaction with Cyt8. The phenyl
semicarbazide side chain was oriented in the minor groove of DNA and formed two
haydrogen bonds with Gua10. (Fig. 13).
2
4

ACCEPTED MANUSCRIPT
Fig. 13: Binding of compound 23 with Topo II, the hydrogen bonds are represented
b
in green dashed lines and the pi interactions are represented in orange lines
Compound 25 showed affinity value of - 48.20 kcal/mol. The planer
b
aromatic system of bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline moiety exhibited
nine pi-pi interaction with Thy9, Cyt8, Gua13, Ade12. In addition, the terminal 4-
fluorophenyl)acetamide moiety was oriented in the minor groove of DNA and the
carbonyl group formed one hydrogen bond with Gua10 (Fig. 14).
Fig. 14: Binding of compound 25 with Topo II, the hydrogen bonds are represented
b
in green dashed lines and the pi interactions are represented in orange lines
Table 7: The docking binding free energies of the synthesized compounds with DNA-
Topo II forming hydrogen bonding and π interactions
Comp.
∆G
No. of No. of
Putative interactions
kcal/mol H bond π bonds
16a
16b
16d
16d
- 27.97
- 35.92
- 39.10
- 34.00
1
2
1
1
8
11
5
Arg503, Gln778, Gly504, Thy9, Cyt8,
Gua13
Arg503, Gln778, Gly776, Thy9, Cyt8,
Gua13, Ade12
Arg503, Gln778, Gly504, Thy9, Cyt8,
Gua13,
7
Arg503, Gln778, Gly504, Thy9, Cyt8,
Gua13,
1
6e
- 40.41
- 24.00
- 26.04
0
0
0
5
5
Arg503, Gln778, Gly776, Thy9, Cyt8
Arg503, Thy9, Gua13, Ade12
Arg503, Thy9, Gua13, Ade12
1
6d
7a
1
11
2
5

ACCEPTED MANUSCRIPT
1
7b
8a
- 29.84
- 40.25
0
1
7
6
Arg503, Gln778, Thy9, Cyt8, Gua13
Arg503, Gln778, Gly776 Gly504, Thy9,
Cyt8, Gua13, Ade12
1
1
8b
- 41.12
- 39.80
- 36.49
- 38.91
- 39.65
1
1
0
2
2
6
1
6
5
3
Arg503, Gln778, Gly504, Thy9, Cyt8,
Gua13, Ade12
1
8c
9a
Arg503, Gln778, Gly776, Ala779
Gly504, Thy9, Gua13, Ade12, Met782
Arg503, Gln778, Gly504, Thy9, Cyt8,
Gua13
1
19b
20a
20b
Arg503, Gln778, Asp479, Thy9, Cyt8,
Gua13
Arg503, Gln778, Gly776, Ala779,
Asp479, Cyt8, Gua13, Ade12
Arg503, Gln778, Thy9, Cyt8, Ade12
Arg503, Gln778, Thy9, Cyt8, Gua13
Arg503, Gln778, Thy9, Cyt8, Gua13
Arg503, Gln778, Thy9, Cyt8, Gua13
Arg503, Gln778, Gly504, Thy9, Cyt8,
Ade12
- 39.46
- 39.97
- 38.77
- 40.52
- 31.10
2
0
0
0
0
3
7
9
8
7
2
1
2
2
2
3a
3b
4a
2
2
2
4b
5a
5b
- 43.21
- 49.70
- 48.20
- 26.51
- 55.51
0
1
1
1
3
4
5
9
5
7
Arg503, Gln778, Gly776, Ala779,
Asp479, Cyt8, Gua13
Arg503, Gln778, Gly776, Ala779,
Asp479, Met782, Thy9, Cyt8, Gua13
Arg503, Gln778, Thy9, Cyt8, Gua13,
Ade12
2
6
Arg503, Gln778, Thy9, Cyt8, Gua13,
Ade12
Doxorubicin
Arg503, Gln778, Gly776, Ala779,
Asp479, Met782, Thy9, Cyt8, Gua13,
Ade12
2
6

ACCEPTED MANUSCRIPT
2
.4. Structure-Activity Relationship (SAR)
As outlined in the rationale molecular design, we aimed at studying the SAR
of newly synthesized quinoxaline derivatives as potential Topo II inhibitors and DNA
intercalators. Observing the results of different biological tests (In vitro anti-
proliferative activity, measurement of Topo II activity and DNA intercalation assay),
we could deduce valuable data about the structure activity relationships.
Firstly, we explored the effect of substitution on 4-position of
[
1,2,4]triazolo[4,3-a]quinoxaline nucleus (scaffold-1) by different effective anti-
tumor moieties such as hydrazones, semicarbazides and thiosemicarbazides. The
decreased IC50 values of compounds 20a,b incorporating semicarbazides than the
corresponding members incorporating hydrazones 18a-c or thiosemicarbazides 19a,b,
indicated that the substitution with semicarbazides is more preferred biologically than
that with hydrazones and thiosemicarbazides. With regard to the activity of different
semicarbazide and thiosemicarbazide derivatives, it was noted that aromatic
derivatives 19 and 20 are more active than aliphatic ones 19 and 20 . On the other
b
a
a
b
side, the substitution at the phenyl ring of hydrazone derivatives decreases the activity
in the order of 2-Cl > 2,6-diCl > 2-OCH . Moreover, observing the activity of
3
substituents with alkylamino and alkoxy groups, it was found that the derivatives with
alkylamino substitutions 16a-f are more active than the corresponding members with
alkoxy derivatives 17a-b except that of benzylamine derivatives. Interestingly, the long
chain of aliphatic amino derivative 16 is more active than the corresponding
e
members with short aliphatic chain.
Next, we explored the effect of molecular hybridization between quinoxaline
and triazole moieties to produce bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline nucleus
(
scaffold-2). The decreased IC values of compounds 21, 25 and 25 against the
50 a b
three human cancer cell lines than the corresponding members incorporating
[
1,2,4]triazolo[4,3-a]quinoxaline scaffold as compounds 16 , 17 , 18 , 19 and
a-f a,b a-c a,b
2
0a,b indicated that scaffold-2 (bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline), four
aromatic ring system is more advantageous than scaffold-1([1,2,4]triazolo[4,3-
a]quinoxaline), three aromatic ring system.
We then investigated the impact of the substitution on the thiol group of
scaffold-2. Introduction of acetanilide derivatives 25a,b resulted in remarkable
increase in activity against the three tested cell lines and Topo II . With regard to the
effect of substitution in the phenyl ring of these members, it was found that 3-
methoxy acetanilide derivative 25 showed better activity than the 4-fluoro analog
a
2
7

ACCEPTED MANUSCRIPT
2
5 , indicating that grafting an electron donating substituent, as methoxy group, at the
b
phenyl ring is beneficial for the activity. On the other hand, introduction of alkyl 24a,b
or alkyl ester 23 and 26 led to dramatic decrease in the anti-proliferative activity.
a,b
3
. Conclusion
In conclusion, new series of [1,2,4]triazolo[4,3-a]quinoxaline
and
bis([1,2,4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives have been designed,
synthesized and biologically evaluated for their in vitro cytotoxicity against three
tumor cell lines (HePG-2, Hep-2 and Caco-2) by the neutral red assay. Compounds
1
6 , 21, 25 and 25 exhibited the highest activity against the three cancer cells (IC
e a b 50
ranging from 0.29 to 0.90 µM) comparable to that of doxorubicin. Additionally, the
most active members were further evaluated for their Topo II inhibitory activities and
DNA intercalating affinities as a potential mechanism for their anti-proliferative
activities. Interestingly, the results of Topo II inhibition and DNA binding assays
were consistent with that of the cytotoxicity data, where the most potent anti-
proliferative derivatives exhibited good Topo II inhibitory activities and DNA binding
affinities, comparable to that of doxorubicin. Moreover, the most active compound
2
5a was found to be able to cause cell cycle arrest in the G2/M phase and to induce
apoptosis in Caco-2 cells. Also, it was found that the effect of compound 25 on topo
a
II relaxation activity is comparable with the effect of the reference drug. Furthermore,
molecular docking studies were performed for the novel compounds to investigate
their binding pattern with the prospective target, DNA-Topo II complex (PDB-code:
3
qx3). Based on obtained results, we conclude that DNA binding and/or Topo II
inhibition may contribute to the observed cytotoxicity of the synthesized compounds.
4
. Experimental
.1. Chemistry
4
All melting points were carried out by the open capillary method on a Gallen
kamp apparatus. The infrared spectra were recorded on a pye Unicam SP 1000 IR
spectrophotometer using the potassium bromide disk technique. Proton magnetic
1
resonance H NMR spectra were recorded on a jeol 400 MHz-NMR spectrometer.
TMS was used as internal standard and chemical shifts were measured in δ scale
(ppm). The mass spectra were recorded on Varian MAT 311-A (70 e.v.) and Direct
Inlet unit (DI-50) of SHIMADZU GC/MS–QP5050A. Elemental analyses (C, H, N)
were performed on a CHN analyzer. All compounds were within ± 0.4 of the
theoretical values. The reactions were monitored by thin-layer chromatography (TLC)
2
8