Triazolo[4,3-a] quinoxaline and [1,2,4]triazolo[4,3- a] quinoxaline-1-thiol-derived DNA intercalators: Design, synthesis, molecular docking, in silico ADMET profiles and anti-proliferative evaluations

Article abstract of DOI:10.1039/d0nj02990d

In view of their DNA intercalation activities as anticancer agents, 17 novel [1,2,4]triazolo[4,3-a]quinoxaline derivatives have been designed, synthesized and evaluated against HepG2, HCT-116 and MCF-7 cells. Molecular docking studies were performed to investigate the binding modes of the proposed compounds with the DNA active site. The data obtained from biological testing highly correlated with those obtained from the molecular modeling studies. MCF-7 was found to be the most sensitive cell line to the influence of the new derivatives. In particular, compound 12d was found to be the most potent derivative of all the tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines, with IC50 = 22.08 ± 2.1, 27.13 ± 2.2 and 17.12 ± 1.5 μM, respectively. Although this compound displayed nearly one third of the activity of doxorubicin (IC50 = 7.94 ± 0.6, 8.07 ± 0.8 and 6.75 ± 0.4 μM, respectively), it may be useful as a template for future design, optimization, and investigation to produce more potent anticancer analogs. Compounds 12a, 10c and 10d displayed very good anticancer activities against the three HepG2, HCT116 and MCF-7 cancer cell lines, with IC50 = 31.40 ± 2.8, 28.81 ± 2.4 and 19.72 ± 1.5 μM for 12a, 33.41 ± 2.9, 29.96 ± 2.5 and 24.78 ± 1.9 μM for 10c, and 37.55 ± 3.3, 30.22 ± 2.6 and 25.53 ± 2.0 μM for 10d. The most active derivatives, 10c, 10d, 10h, 12a, 12b and 12d, were evaluated for their DNA binding activities. Compound 12d displayed the highest binding affinity. This compound potently intercalates DNA at a decreased IC50 value (35.33 ± 1.8 μM), which is nearly equipotent to that of doxorubicin (31.27 ± 1.8 μM). Compounds 12a and 10c exhibited good DNA-binding affinities, with IC50 values of 39.35 ± 3.9 and 42.35 ± 3.9 μM, respectively. Finally, compounds 10d, 10h and 12b showed moderate DNA-binding affinities, with IC50 values of 50.35 ± 3.9, 57.08 ± 3.3 and 59.35 ± 3.2 μM, respectively.

Full text of DOI:10.1039/d0nj02990d

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[1,2,4]Triazolo[4,3-a]quinoxaline and
[1,2,4]triazolo[4,3-a]quinoxaline-1-thiol-derived
DNA intercalators: design, synthesis, molecular
docking, in silico ADMET profiles and
anti-proliferative evaluations†
ab
Cite this: New J. Chem., 2021,
45, 881
Khaled El-Adl,
*
Abdel-Ghany A. El-Helby,a Helmy Sakra and Alaa Elwana
In view of their DNA intercalation activities as anticancer agents, 17 novel [1,2,4]triazolo[4,3-a]quinoxaline
derivatives have been designed, synthesized and evaluated against HepG2, HCT-116 and MCF-7 cells.
Molecular docking studies were performed to investigate the binding modes of the proposed
compounds with the DNA active site. The data obtained from biological testing highly correlated with
those obtained from the molecular modeling studies. MCF-7 was found to be the most sensitive cell line
to the influence of the new derivatives. In particular, compound 12d was found to be the most potent
derivative of all the tested compounds against the three HepG2, HCT116 and MCF-7 cancer cell lines,
with IC50 = 22.08 ꢀ 2.1, 27.13 ꢀ 2.2 and 17.12 ꢀ 1.5 mM, respectively. Although this compound displayed
nearly one third of the activity of doxorubicin (IC50 = 7.94 ꢀ 0.6, 8.07 ꢀ 0.8 and 6.75 ꢀ 0.4 mM,
respectively), it may be useful as a template for future design, optimization, and investigation to produce
more potent anticancer analogs. Compounds 12a, 10c and 10d displayed very good anticancer activities
against the three HepG2, HCT116 and MCF-7 cancer cell lines, with IC50 = 31.40 ꢀ 2.8, 28.81 ꢀ 2.4 and
19.72 ꢀ 1.5 mM for 12a, 33.41 ꢀ 2.9, 29.96 ꢀ 2.5 and 24.78 ꢀ 1.9 mM for 10c, and 37.55 ꢀ 3.3, 30.22 ꢀ 2.6
and 25.53 ꢀ 2.0 mM for 10d. The most active derivatives, 10c, 10d, 10h, 12a, 12b and 12d, were evaluated
for their DNA binding activities. Compound 12d displayed the highest binding affinity. This compound
potently intercalates DNA at a decreased IC50 value (35.33 ꢀ 1.8 mM), which is nearly equipotent to that of
doxorubicin (31.27 ꢀ 1.8 mM). Compounds 12a and 10c exhibited good DNA-binding affinities, with IC50
values of 39.35 ꢀ 3.9 and 42.35 ꢀ 3.9 mM, respectively. Finally, compounds 10d, 10h and 12b showed
moderate DNA-binding affinities, with IC50 values of 50.35 ꢀ 3.9, 57.08 ꢀ 3.3 and 59.35 ꢀ 3.2 mM,
respectively.
Received 15th June 2020,
Accepted 29th November 2020
DOI: 10.1039/d0nj02990d
rsc.li/njc
transferred from an aqueous environment to the hydrophobic
space between two adjacent DNA base pairs.3,4 Extensive
1. Introduction
DNA plays an essential role in a wide diversity of cellular research over the last four decades has focused on the effects
processes and acts as a fundamental target for treating hall- of small organic compounds that non-covalently bind to nucleic
mark genetic diseases such as cancer.1 The most important acids.5 These interactions are known to disrupt replication and/
group of compounds that interact reversibly with the DNA or transcription, which culminates in cellular death. Accordingly,
double helix are intercalators.2 Many anticancer drugs that DNA-binding compounds have potential applications as anti-
interact with DNA through intercalation are in clinical use.2,3 cancer agents. Small molecules may bind non-covalently to DNA
Intercalation is a process in which the intercalating molecule is through intercalation between nucleobase pairs, major or minor
groove binding and electrostatic interactions.6 The planar
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University,
Cairo, Egypt. E-mail: eladlkhaled74@yahoo.com, eladlkhaled74@azhar.edu.eg,
khaled.eladl@hu.edu.eg
b Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Heliopolis University for Sustainable Development, Cairo, Egypt
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj02990d
aromatic portions of molecules (known as chromophores) are
inserted between adjacent base pairs of the DNA helix perpen-
dicularly to its axis, forming strong non-covalent interactions
with DNA bases.7,8 This complex between the drug and DNA
deforms and uncoils the DNA;7 also, the structural changes
induced in DNA by intercalation lead to interference with
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recognition and function of DNA-associated proteins or
enzymes involved in replication, transcription, and DNA repair
systems, leading to failure of these processes.7
There are two types of intercalators: classic/mono-
intercalators and threading intercalators.1 The presence of
two groove-binding side chains, as in the case of nogalamycin
I, which contains two sugar moieties at both ends of its
chromophore, leads to a special type of interaction with DNA
called threading intercalation, in which one sugar unit is
oriented to the minor groove and the other to the major
groove7 (Fig. 1).
Many DNA intercalators are either being used already as
anticancer drugs or are still in clinical trials (e.g., nogalamycin
I,9 doxorubicin II,10 and amsacrine III11) (Fig. 2).
The quinoxaline nucleus is the backbone of many DNA
intercalators.12–15 The discovery and development of new
Fig. 1 Cartoon representations of the classical and threading modes of
drug intercalation with DNA.
Fig. 2 Basic pharmacophoric features of reported DNA intercalators and our derivatives.
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therapeutic DNA intercalators for the treatment of cancer is
The choice of the different substituents was based on their
considered to be one of the most important targets in the field relatively high lipophilicity, with the aim to pass through the
of medicinal chemistry.16 Quinoxaline analogues demonstrate nuclear membranes and achieve strong DNA intercalation.
excellent anticancer activities through DNA intercalation; Moreover, the variability of the substitutions enabled us
for instance, echinomycin VI, a natural DNA intercalator, to study the structure–activity relationships of the final
showed potent activities in phase I and II trials against a wide compounds.
array of cancers.17 Moreover, N3-(3-(dimethylamino)propyl)-N2-
(4-methoxybenzyl)-6-nitroquinoxaline-2,3-diamine VII is of cur- macophoric features of DNA intercalators; the planar poly-
rent interest in view of its binding to DNA.18
aromatic system (chromophore) was represented by the
All compounds were designed to comprise the main phar-
On the other hand, quinoxaline derivatives have been quinoxaline nucleus and its fused triazole ring intercalating
reported to have different cellular-based anticancer mechanisms. the base pairs of the DNA. In addition, all of the designed
These derivatives were reported to have anticancer activity compounds include basic nitrogen atoms that may be protonated
through EGFR-TK inhibition.19 Moreover, [1,2,4]triazolo[4,3-a] at physiological pH to form cationic centers that increase
quinoxaline derivatives were reported as potent anticancer agents the affinity and selectivity of these compounds; finally, all the
with dual EGFR kinase and tubulin polymerization inhibitory compounds mainly contain one or two side chains (Fig. 2),
effects.20 Furthermore, [1,2,4]triazolo[4,3-a]quinoxaline derivatives exhibiting different antitumor activities. These side chains bind
were reported as efficient adenosine A2B antagonists.21,22
to the minor groove and/or the major groove, increasing the DNA
Based on these earlier findings, and in continuation of our binding affinity.
previous research in the design and synthesis of new anticancer
agents,23–29 especially DNA intercalators,12 we report the
design, synthesis, DNA binding examination and docking studies
2. Results and discussion
2.1. Chemistry
of a new series of quinoxaline derivatives as DNA intercalators.
In general, the designed compounds were synthesized and
evaluated for their in vitro anti-proliferative activities against three For the synthesis of the target compounds, the sequence of the
human tumor cell lines, namely, hepatocellular carcinoma (HCC) reactions is illustrated in Schemes 1–3. o-Phenylenediamine 1
type (HepG2), human colorectal carcinoma-116 (HCT-116) and was reacted with oxalic acid 2 in the presence of 4 N HCl
breast cancer (Michigan Cancer Foundation-7 (MCF-7)). The to afford 2,3-(1H,4H)-quinoxalinedione 3.13,30 The latter was
results prompted us to carry out further examinations to obtain treated with thionyl chloride to afford 2,3-dichloroquinoxaline
deep insight into the mechanisms of action of the synthesized 4,13,30 which underwent reaction with hydrazine hydrate with
compounds.
continuous stirring at room temperature to produce 2-chloro-3-
The most cytotoxic agents were further evaluated to assess hydrazinylquinoxaline 5.13 Cyclization of 5 with triethylortho-
their binding affinities against DNA through DNA/methyl green formate resulted in 4-chloro[1,2,4]triazolo[4,3-a]quinoxaline
assays. Moreover, molecular docking was carried out to examine 6.13 Heating of 6 with methyl 4-aminobenzoate in acetonitrile
the binding patterns of the compounds with the prospective afforded the corresponding methyl ester 7. On the other hand,
target DNA active site (PDB-code: 4G0U).
cyclization of 2-chloro-3-hydrazinylquinoxaline 5 with CS2 in
the presence of KOH in absolute ethanol afforded the corres-
ponding 4-chloro-[1,2,4]triazolo[4,3-a]quinoxaline-1-thiol 831
1.1. Rationale and structure-based design
The rationale of our molecular design depended on the gen- (Scheme 1).
eration of a planar aromatic system containing the triazolo
The methyl ester 7 was treated with hydrazine hydrate to
[4,3-a]quinoxaline scaffold (chromophore) with one or two obtain the corresponding acid hydrazide 9, which was reacted
groove-binding side chains. The synthesis of the title com- with the appropriate benzaldehyde derivatives to afford the
pounds was carried out by two pathways. The first pathway corresponding Schiff bases 10a–j, respectively (Scheme 2).
involves molecular hybridization of the quinoxaline nucleus
Furthermore, heating of 8 with alcoholic potassium hydroxide
with 1,2,4-triazole moiety to produce [1,2,4]triazolo unfortunately afforded the dipotassium salt 11 instead of the
a
a
[4,3-a]quinoxaline scaffold (chromophore) which is substituted targeted potassium 4-chloro-[1,2,4]triazolo[4,3-a]quinoxaline-1-
at position 4 with different moieties, such as ester, acid thiolate 13, which may be due to air oxidation under the
hydrazide benzylidene, ketone, chalcone and/or pyrazole deri- reaction conditions. The obtained dipotassium salt 11 was
vatives, as a single groove-binding side chain. These com- reacted with the appropriate chloroacetanilide derivative to
pounds were designed as classical DNA intercalators.
afford the corresponding 1,5 derivatives 12a–e, respectively
1
The second pathway was performed through molecular (Scheme 3). The H NMR spectra exhibited the appearance of
hybridization of the quinoxaline nucleus with the 1,2,4- two singlet peaks at about 4.34–4.56 d ppm for the two
triazole-3-thiol moiety to produce a [1,2,4]triazolo[4,3-a]quino- introduced CH2 groups, increased the integration of the aromatic
xaline-1-thiol scaffold (chromophore) substituted at both protons and also resulted in the presence of two singlet peaks
positions 1 and 5 with different acetanilides as two groove- for the two amidic NH groups. Also, the mass spectroscopic
binding side chains. These compounds were designed as chart for compound 12a supported the presence of its molecular
threading DNA intercalators.
ion peak at 484. The reaction of 4-chloro-[1,2,4]triazolo
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Scheme 1 Synthetic route for the preparation of the target compounds 3–8.
Scheme 2 Synthetic route for the preparation of the target compounds 9–10a–j
.
[4,3-a]quinoxaline-1-thiol 6 with alcoholic potassium hydroxide chloroacetanilide derivatives to furnish the targeted corres-
was designed to produce the targeted potassium 4-chloro- ponding derivatives 14, respectively. Furthermore, the deriva-
[1,2,4]triazolo[4,3-a]quinoxaline-1-thiolate 13. This potassium tives 14a–e were designed to react with different aromatic amines
salt was designed to be reacted with the appropriate to obtain diversity of compounds 15. These reactions were
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Scheme 3 Synthetic route for the preparation of the target compounds 12a–e
.
designed to obtain different substitutions at position 1 (14a–e
)
studied ligands have similar positions and orientations inside
and also to combine the substitutions at positions 1 and 4 (13) in the DNA binding site.
order to study the effects of these substitutions at different
The proposed binding mode of doxorubicin revealed an affinity
positions. Moreover, the designed reactions afforded a diversity value of ꢁ100.31 kcal molꢁ1 and ten H-bonds. The planar
of compounds to study the effects of the combinations of the two aromatic system occupied the hydrophobic pocket formed by
substitutions at two positions in the same compound and Arginine945, Asparagine882, Lucine880, Glutamate870, Alanine869,
determine the structure activity relationships (SARs) of these Glycine868, Asparagine867, Asparagine795, Glutamine789, Aspara-
derivatives. In fact, the data proved that the obtained 12a–e gine786, Glutamine742, Lysine739, Phenyalanine738 and
derivatives were disubstituted at positions 1 and 5, and the SARs Glycine737. It formed five H-bonds with the key amino acids in
were obtained for these compounds.
the active site (one H-bond with Asparagine786, two H-bonds with
Asparagine795, one H bond with Arginine945, and one H-bond with
Asparagine867). It also stacked between the diphosphate cytidines
(Dc3, Dc4 and Dc8), diphosphate guanosines (Dg5 and Dg13) and
2.2. Docking studies
All the synthesized compounds were evaluated for their DNA adenine monophosphate 6 (Da6). The sugar moiety was oriented
binding affinity with the aid of molecular docking studies using into the minor groove of the DNA and stabilized by the formation of
Molsoft software. The 3D crystal structure of DNA was down- five H-bonds with the key amino acids (two H-bonds with Argi-
loaded from the Protein Data Bank using the PDB ID of 4G0U.32 nine945, one H-bond with Leucine880 and two H-bonds with
The binding free energies (DG) are presented in Table 1. All the Asparagine882) (Fig. 3).
The proposed binding mode of 12d exhibited an affinity
value of ꢁ105.42 kcal molꢁ1 and twelve H-bonds. The planar
aromatic system occupied the hydrophobic pocket formed
by Arginine945, Tyrosine908, Asparagine882, Glutamate870,
Alanine869, Glycine868, Asparagine867, Asparagine798, Aspara-
gine795, Glutamine789, Leucine787 and Asparagine786. It
formed eight H-bonds with the key amino acid Arginine945.
It also stacked between the diphosphate cytidines (Dc3, Dc4, Dc1
and Dc19) and diphosphate guanosines (Dg2, Dg5 and Dg13).
The two substituted arms were oriented into the minor and
major grooves of the DNA and were stabilized by the formation
of four H-bonds with the key amino acids (one H-bond with
Tyrosine908, one H-bond with Glutamine789 and two H-bond
Table 1 The calculated free energies of binding (DG in kcal molꢁ1) of the
ligands
Compound
DG [kcal molꢁ1
]
Compound
DG [kcal molꢁ1
]
7
9
ꢁ88.30
ꢁ80.92
ꢁ80.50
ꢁ80.44
ꢁ89.88
ꢁ88.50
ꢁ80.01
ꢁ79.16
ꢁ79.14
10h
10i
10j
12a
12b
12c
12d
12e
ꢁ88.33
ꢁ79.05
ꢁ80.06
ꢁ104.45
ꢁ88.30
ꢁ79.11
ꢁ105.42
ꢁ79.08
ꢁ100.31
10a
10b
10c
10d
10e
10f
10g
Doxorubicin
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Fig. 3 Binding of doxorubicin with DNA; the hydrogen bonds are represented with blue dashed lines.
with Asparagine786) (Fig. 4). These interactions may explain the Dg13). The 4-chlorophenyl moiety was oriented into the minor
highest anticancer activity of 12d. groove of the DNA. The side chain was stabilized by the
The proposed binding mode of 12a exhibited an affinity value formation of five H-bonds with the key amino acids (four
of ꢁ104.45 kcal molꢁ1 and nine H-bonds. The planar aromatic H-bonds with Arginine945 and one H-bond with Glycine868)
system occupied the hydrophobic pocket formed by Arginine945, (Fig. 6).
Tyrosine908, Asparagine882, Glutamate870, Alanine869, Gly-
As planned, the hydrophobic distal moieties and the long
cine868, Asparagine867, Lysine814, Asparagine798, Aspara- linkers and hydrogen bond acceptors present in the planar
gine795, Glutamine789, Leucine787 and Asparagine786. It aromatic scaffold and/or the long chain linkers increased the
formed five H-bonds with the key amino acid Arginine945. It also affinity towards the DNA active site. From the obtained docking
stacked between the diphosphate cytidines (Dc3 and Dc4) and results (Table 1), we concluded that the 12d and 12a derivatives,
diphosphate guanosines (Dg5 and Dg13). The two substituted which were designed as threading DNA intercalators due to
arms were oriented into the minor and major grooves of the DNA having disubstituents at positions 1 and 5, exhibited the highest
and were stabilized by the formation of four H-bonds with the key DNA intercalations. The benzylidene derivatives 10c and 10d
amino acids (one H-bond with Tyrosine908, one H-bond with showed increased hydrophobic interactions and, consequently,
Glutamine789 and two H-bonds with Lysine814) (Fig. 5). These increased affinities towards the DNA active site, which may be
interactions may explain the high anticancer activity of 12a.
due to the long chain linkers used. On the other hand, these
The proposed binding mode of 10c exhibited an affinity value modifications compensated for the hydrogen bonding and
of ꢁ89.88 kcal molꢁ1 and seven H-bonds. The planar aromatic hydrophobic interactions of the reference drug doxorubicin.
system occupied the hydrophobic pocket formed by Arginine945,
2.3. In vitro anti-proliferative activity
Tyrosine908, Asparagine882, Glutamate870, Alanine869, Gly-
cine868, Asparagine867, Leucine799, Asparagine798, Aspara- The anti-proliferative activities of the seventeen newly synthe-
gine795, Leucine787, Asparagine786, Glutamine742, Lysine739, sized [1,2,4]triazolo[4,3-a]quinoxaline derivatives 5–10a–e were
Phenylalanine738, Glycine737 and Proline732. It formed two examined against three human tumor cell lines, namely hepato-
H-bonds with the key amino acids Asparagine786 and Aspara- cellular carcinoma (HepG2), colorectal carcinoma (HCT-116) and
gine795. It also stacked between the diphosphate cytidines (Dc3, breast cancer (MCF-7), using the 3-[4,5-dimethylthiazol-2-yl]-2,5-
Dc4, Dc1 and Dc19) and diphosphate guanosines (Dg2, Dg5 and diphenyltetrazolium bromide (MTT) colorimetric assay as
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Fig. 4 Predicted binding mode of 12d with DNA.
Fig. 5 Predicted binding mode of 12a with DNA.
described by Mosmann.33–35 Doxorubicin was included in the explicated that most of the prepared compounds displayed very
experiments as a reference cytotoxic drug. The results were good to low growth inhibitory activity against the tested cancer
expressed as the growth inhibitory concentration (IC50) values cell lines. In general, the investigations of the cytotoxic activity
and are summarized in Table 2. From the obtained results, it was indicated that MCF-7 was the most sensitive cell line to the
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Fig. 6 Predicted binding mode of 10c with DNA.
Table 2 In vitro cytotoxic activities of the newly synthesized compounds against the HepG2, HCT-116, MCF-7 and VERO cell lines
a
IC50 (mM)
Compound
HepG2
HCT116
MCF-7
VERO
7
9
40.69 ꢀ 3.6
59.23 ꢀ 3.9
69.56 ꢀ 4.3
72.47 ꢀ 4.4
33.41 ꢀ 2.9
37.55 ꢀ 3.3
85.19 ꢀ 5.0
88.09 ꢀ 5.1
89.79 ꢀ 5.3
40.28 ꢀ 3.6
4100
36.47 ꢀ 3.3
47.30 ꢀ 3.9
57.95 ꢀ 4.2
61.87 ꢀ 4.3
29.96 ꢀ 2.5
30.22 ꢀ 2.6
76.88 ꢀ 4.9
66.62 ꢀ 4.5
4100
32.91 ꢀ 2.4
41.56 ꢀ 3.0
49.16 ꢀ 3.7
58.43 ꢀ 4.2
24.78 ꢀ 1.9
25.53 ꢀ 2.0
64.79 ꢀ 4.6
43.93 ꢀ 3.1
76.38 ꢀ 4.9
31.05 ꢀ 2.5
4100
59.61 ꢀ 4.3
19.72 ꢀ 1.5
32.58 ꢀ 3.6
68.65 ꢀ 5.2
17.12 ꢀ 1.5
93.89 ꢀ 5.5
6.75 ꢀ 0.4
NTb
NTb
10a
10b
10c
10d
10e
10f
10g
10h
10i
NTb
NTb
66.43 ꢀ 0.22
69.23 ꢀ 0.22
NTb
NTb
NTb
35.12 ꢀ 3.3
70.54 ꢀ 0.22
4100
NTb
10j
79.37 ꢀ 4.7
31.40 ꢀ 2.8
40.60 ꢀ 4.2
4100
71.34 ꢀ 4.8
28.81 ꢀ 2.4
36.33 ꢀ 4.1
4100
NTb
12a
12b
12c
12d
12e
Doxorubicin
65.12 ꢀ 0.17
73.12 ꢀ 0.17
NTb
22.08 ꢀ 2.1
27.13 ꢀ 2.2
74.61 ꢀ 0.19
4100
4100
NTb
7.94 ꢀ 0.6
8.07 ꢀ 0.8
b
NTb
a
IC50 values are the mean ꢀ S.D. of three separate experiments. NT: Not tested.
influence of the new derivatives. In particular, compound 12d HepG2, HCT116 and MCF-7 cancer cell lines, with the same IC50
was found to be the most potent derivative of all the tested values 4100 mM.
compounds against the three HepG2, HCT116 and MCF-7 cancer
With respect to the HepG2 hepatocellular carcinoma cell
cell lines, with IC50 = 22.08 ꢀ 2.1, 27.13 ꢀ 2.2 and 17.12 ꢀ line, compounds 12a, 10c and 10d displayed very good anti-
1.5 mM, respectively. Although this compound displayed nearly cancer activities, with IC50 values of 31.40 ꢀ 2.8, 33.41 ꢀ 2.9
one third of the activity of doxorubicin (IC50 = 7.94 ꢀ 0.6, 8.07 ꢀ and 37.55 ꢀ 3.3 mM, respectively. Compounds 10h, 12b and 7,
0.8 and 6.75 ꢀ 0.4 mM, respectively), it may be useful as a with IC50 = 40.28 ꢀ 3.6, 40.60 ꢀ 4.2 and 40.69 ꢀ 3.6 mM,
template for future design, optimization, and investigation to respectively, displayed good cytotoxicity. Compounds 9, 10a and
produce more potent anticancer analogs. On the other hand, 10b, with IC50 values ranging from 59.23 ꢀ 3.9 to 72.47 ꢀ
compound 10i exhibited the lowest activity against the three 4.4 mM, exhibited moderate cytotoxicity. Meanwhile, compounds
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10e, 10f, 10g, 10j, 12c and 12e, with IC50 values ranging from binding affinity. This compound potently intercalated DNA at
79.37 ꢀ 4.7 to 4100 mM, displayed the lowest cytotoxicity. a decreased IC50 value (35.33 ꢀ 1.8 mM) which is nearly
The cytotoxicity evaluation against the colorectal carcinoma equipotent to that of doxorubicin (31.27 ꢀ 1.8 mM). Compounds
(HCT-116) cell line revealed that compounds 12a, 10c and 10d 12a and 10c exhibited good DNA-binding affinities, with IC50
displayed very good anticancer activities, with IC50 values of values of 39.35 ꢀ 3.9 and 42.35 ꢀ 3.9 mM, respectively. Finally,
28.81 ꢀ 2.4, 29.96 ꢀ 2.5 and 30.22 ꢀ 2.6 mM, respectively. compounds 10d, 10h and 12b showed moderate DNA-binding
Compounds 10h, 12b, 7 and 9, with IC50 = 35.12 ꢀ 3.3, 36.33 ꢀ affinities, with IC50 values of 50.35 ꢀ 3.9, 57.08 ꢀ 3.3 and
4.1, 36.47 ꢀ 3.3 and 47.30 ꢀ 3.9 mM, respectively, displayed 59.35 ꢀ 3.2 mM, respectively.
good cytotoxicity. Compounds 10a, 10b, 10f and 10j, with IC50
2.5. Structure–activity relationship (SAR)
values ranging from 57.95 ꢀ 4.2 to 71.34 ꢀ 4.8 mM, exhibited
moderate cytotoxicity. Meanwhile, compounds 10e, 10g, 12c and The preliminary SAR study focused on the effects of the hydro-
12e, with IC50 values ranging from 76.88 ꢀ 4.9 to 4100 mM, phobic natures, electronic natures and positions of the
displayed the lowest cytotoxicity.
substituents used in this study. Also, it focused on the effects
The cytotoxicity evaluation against the MCF-7 cell line of the number and length of linkers used and the presence of
revealed that compounds 12a, 10c and 10d displayed very good one and/or two distal moieties. The data obtained revealed that
anticancer activities, with IC50 values of 19.72 ꢀ 1.5, 24.78 ꢀ 1.9 the tested compounds displayed different levels of anticancer
and 25.53 ꢀ 2.0 mM, respectively. Compounds 10h, 12b, 7, 9, 10f activity and possessed a distinctive pattern of selectivity against
and 10a, with IC50 = 31.05 ꢀ 2.5, 32.58 ꢀ 3.6, 32.91 ꢀ 2.4, the MCF-7 cell line. Generally, the [1,2,4]triazolo[4,3-a]quinoxa-
41.56 ꢀ 3.0, 43.93 ꢀ 3.1 and 49.16 ꢀ 3.7 mM, respectively, line scaffold, ester, acid hydrazide and/or acetamide linkers
displayed good cytotoxicity. Compounds 10b, 10e, 10j and 12c, containing (HBA-HBD), lipophilicity and electronic nature
with IC50 values ranging from 58.43 ꢀ 4.2 to 68.65 ꢀ 5.2 mM, exhibited important roles in DNA intercalation and, consequently,
exhibited moderate cytotoxicity. Meanwhile, compounds 10g in the anticancer activity. The presence of two hydrophobic distal
and 12e, with IC50 = 76.38 ꢀ 4.9 and 93.89 ꢀ 5.5 mM, respec- moieties linked to the chromophore through acetamide linkers,
tively, displayed the lowest cytotoxicity.
as in compounds 12a–e, was found to be responsible for their
Finally, the six most potent derivatives, 10c, 10d, 10h, 12a, 12b higher anticancer activity than that of the other compounds
and 12d, were tested for their cytotoxicity against normal VERO containing only one hydrophobic distal moiety, as in compounds
cells. The results revealed that the tested compounds have low 7, 8 and 10a–j. The presence of two arms enables 12a–e to form
toxicity against normal VERO cells, with IC50 values ranging different hydrophobic and hydrophilic bonding interactions; one
from 65.12 to 74.61 mM. The cytotoxicities of these compounds of the two arms is oriented into the minor groove of DNA and
against the cancer cell lines ranged from 17.12 to 40.60 mM.
the other one is oriented into the major groove, as shown in
Compounds 10c, 10d, 10h, 12a, 12b and 12d are respectively molecular docking studies.
2.68, 2.71, 2.27, 3.30, 2.24 and 4.36 fold more toxic to breast
From the structure of the synthesized derivatives and the
cancer cells (MCF-7, the most sensitive cells) than to normal data shown in Table 2, we can divide these tested compounds
VERO cells.
into three groups. The first group is compounds 7 and 9. In this
group, short chain hydrophobic ester and hydrazide linkers
were used. The ethyl ester derivative 7 showed higher activities
than the acid hydrazide 9 against the three MCF-7, HCT116 and
HepG2 cell lines, respectively. The second group is compounds
10a–j; the terminal phenyl tail was unsubstituted, as in compound
10a, or 2-substituted with a hydrophobic, electron-withdrawing
chloro group and a hydrophobic, electron-donating methoxy
group, as in compounds 10b and 10g, respectively. These
compounds were also 4-substituted with different hydrophobic
and/or hydrophilic electron-withdrawing groups, as in 10c and
10d, and/or electron-donating groups, as in 10h. Compound 10e
was 2,6-disubstituted with two chloro groups. Moreover,
compounds in this group were substituted with a hydrophobic,
electron-withdrawing nitro group at positions 3 and/or 4, as in
compounds 10i and 10j, respectively.
2.4. In vitro DNA/methyl green assay
The most active anti-proliferative derivatives, 10c, 10d, 10h, 12a,
12b and 12d, were further evaluated for their DNA-binding affi-
nities, which revealed the ability of these compounds to inter-
calate DNA. This investigation was carried out using methyl green
dye according to the reported procedure described by Burre
et al.12,13,36 The results of the DNA-binding affinity were obtained
as IC50 values and are summarized in Table 3. Doxorubicin, as one
of the most powerful DNA intercalators, was used as a positive
control.
The tested compounds displayed good to moderate DNA-
binding affinities. Compound 12d displayed the highest
Within this group, the 4-substituted phenyl tail with a
hydrophobic, electron-withdrawing chloro (+mesomeric effect
(+M effect) and ꢁ inductive effect (ꢁI effect)) group (10c)
exhibited higher anticancer activities than the less hydrophobic,
more electron-withdrawing fluoro (+M, ꢁI) group (10d) and
hydrophobic, electron-donating methoxy (+M, +I) group (10h)
against the three MCF-7, HCT116 and HepG2 cell lines,
Table 3 DNA intercalating affinities of the tested compounds
DNA binding
IC50 (mM)
DNA binding
IC50 (mM)
Compound
Compound
10c
10d
10h
12a
42.35 ꢀ 3.9
50.35 ꢀ 3.9
57.08 ꢀ 3.3
39.35 ꢀ 3.9
12b
12d
Doxorubicin
59.35 ꢀ 3.2
35.33 ꢀ 1.8
31.27 ꢀ 1.8
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respectively. The unsubstituted derivative 10a exhibited higher
In the third group, 12a–e, the 4-substituted terminal phenyl
activities than the 2-substituted derivative with the hydrophobic group with the hydrophobic electron-donating methyl (+I)
electron-withdrawing chloro (+M, ꢁI) group (10b) and that 4- group, as in 12d, exhibited higher activities than the unsubsti-
substituted with a nitro (ꢁM, ꢁI) group (10j) against the three tuted 12a and the compound substituted with the hydrophobic
MCF-7, HCT116 and HepG2 cell lines, respectively. The 2,6- electron-withdrawing chloro (+M, ꢁI) group 12b and hydrophobic
dichloro-substituted compound 10e exhibited lower activities electron withdrawing nitro (ꢁM, ꢁI) group 12e against the three
than the monosubstituted compounds against the three MCF- MCF-7, HCT116 and HepG2 cell lines, respectively. The 2,6-
7, HCT116 and HepG2 cell lines, respectively. These findings dichloro-substituted compound 12c showed lower activities than
obeyed the Hansch equation for the parabolic relationship the monosubstituted compound 12b against the three MCF-7,
between drug activity and hydrophobicity. The 4-substituted HCT116 and HepG2 cell lines, respectively. These findings obeyed
derivative with
a
hydrophilic electron-donating hydroxy the Hansch equation for a parabolic relationship between drug
(+M, ꢁI) group (10f) exhibited higher activities than that substi- activity and hydrophobicity and demonstrated that the 4-position
tuted at the 2-position with the hydrophobic, electron-donating is critical to the activity.
methoxy (+M, +I) group (10g) and that 3-substituted with the
Based on the data obtained from DNA binding in Table 3, it
hydrophobic electron-withdrawing nitro (ꢁM, ꢁI) group (10i), can be concluded that the presence of two hydrophobic distal
which indicates that the 4-position is critical to the activity.
moieties linked to the chromophore through acetamide linkers,
Table 4 ADMET profiles of the four most active compounds and doxorubicin
Parameter
10c
10d
12a
12d
Doxorubicin
Molecular properties
Molecular weight
log P
Rotatable bonds
Acceptors
441.882
4.4384
5
7
425.427
3.9241
5
7
484.541
3.4137
7
8
512.595
4.03054
7
8
543.525
0.0013
5
12
Donors
Surface area
2
2
2
2
6
187.133
180.995
203.350
216.080
222.081
Absorption
Water solubility
ꢁ3.997
1.097
92.827
ꢁ2.735
Yes
Yes
Yes
ꢁ3.684
1.25
92.132
ꢁ2.736
Yes
Yes
Yes
ꢁ3.852
0.622
90.728
ꢁ2.736
Yes
Yes
Yes
ꢁ3.897
0.424
91.679
ꢁ2.736
Yes
Yes
Yes
ꢁ2.915
0.457
62.372
12.735
Yes
No
No
Caco2 permeability
Intestinal abs. (human)
Skin permeability
P-Glycoprotein substrate
P-Glycoprotein I inhibitor
P-Glycoprotein II inhibitor
Distribution
VDss (human)
Fraction unbound (human)
BBB permeability
CNS permeability
0.108
0.062
0.089
0.097
ꢁ0.181
0.156
ꢁ0.091
0.191
1.647
0.215
ꢁ0.565
ꢁ0.597
ꢁ0.715
ꢁ0.691
ꢁ1.379
ꢁ1.947
ꢁ2.119
ꢁ2.416
ꢁ2.269
ꢁ4.307
Metabolism
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
No
No
No
No
Yes
No
Yes
Yes
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Excretion
Total clearance
Renal OCT2 substrate
ꢁ0.107
0.01
No
ꢁ0.028
ꢁ0.142
0.987
No
No
No
No
Toxicity
AMES toxicity
Yes
Yes
No
No
No
Max. tolerated dose (human)
hERG I inhibitor
hERG II inhibitor
0.495
No
Yes
2.64
1.308
Yes
0.514
No
Yes
2.628
1.287
Yes
0.886
No
Yes
2.581
1.82
Yes
0.799
No
Yes
2.585
1.59
Yes
0.081
No
Yes
2.408
3.339
Yes
Oral rat acute toxicity (LD50
)
Oral rat chronic toxicity (LOAEL)
Hepatotoxicity
Skin sensitization
T. pyriformis toxicity
Minnow toxicity
No
0.286
0.924
No
0.285
0.618
No
0.287
ꢁ1.87
No
0.287
ꢁ2.008
No
0.285
4.412
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as in compounds 12d, , exhibited higher DNA binding activity new compounds exhibited slower clearance rates, which
a
than that of a single hydrophobic distal moiety, as in compounds indicates the possibility of preferential extended dosing intervals
10c,
, h. Compound 12d 4-substituted with the hydrophobic of the novel derivatives. The last parameter examined in the
d
electron-donating (+I) methyl group displayed higher activities ADMET profiles of our newly synthesized VEGFR-2 inhibitors is
than the unsubstituted compound 12a and that substituted with toxicity. As displayed in Table 4, doxorubicin and all the new
a hydrophobic electron-withdrawing chloro (+M, ꢁI) group (12b). ligands shared the drawback of unwanted hepatotoxic effects.
Compound 10c 4-substituted with the hydrophobic, electron- In terms of the maximum tolerated dose in humans, the new
withdrawing chloro (+M, ꢁI) group exhibited higher DNA binding [1,2,4]triazolo[4,3-a]quinoxaline derivatives 12d, 12a, 10c and 10d
activity than the less hydrophobic, more electron-withdrawing showed values 9.86, 10.94, 6.11 and 6.35 fold that of the
fluoro (+M, ꢁI) group (10d) and the hydrophobic, electron- reference compound doxorubicin, respectively, which indicates
donating methoxy (+M, +I) group (10h).
the advantage of the wide therapeutic index of the new derivatives.
Finally, the oral acute toxic doses of the new compounds (LD50)
are slightly greater than that of the reference drug (2.581–2.64 for
2.6. In silico ADMET profiles
In the present study, a computational study of the four most our new derivatives compared with 2.40 for doxorubicin).
active compounds (12d, 12a, 10c and 10d) was conducted to
determine their surface areas and other physicochemical
properties37 according to the directions of Lipinski’s rule.38
Lipinski suggested that the absorption of a compound is likely
to be better if the molecule obeys at least three out of the
3. Conclusion
following four rules: (i) HB donor groups r5; (ii) HB acceptor In summary, seventeen new [1,2,4]triazolo[4,3-a]quinoxaline
groups r10; (iii) molecular weight o500; (iv) log P o 5. In this based derivatives have been designed, synthesized and evaluated
study, whereas the reference anticancer agent doxorubicin for their anticancer activities against three human tumor cell
violates three of Lipinski’s rules, compound 12d violates only lines, namely hepatocellular carcinoma (HepG2), colorectal
one (molecular weight). All the most active derivatives have a carcinoma (HCT-116) and breast cancer (MCF-7), as DNA inter-
number of hydrogen-bonding acceptor groups (7 and/or 8) and calators. The tested compounds showed variable anticancer
only 2 hydrogen-bonding donors, and these values agree with activities. Molecular docking studies were performed to investigate
Lipinski’s rules. Also, the absorption, distribution, metabolism, the binding modes of the proposed compounds with the DNA
excretion, and toxicity (ADMET) profiles of the new synthesized active site. The data obtained from the biological testing highly
derivatives were preliminarily assessed to analyze their potentials correlated with those obtained from the molecular modeling
to serve as good medication candidates. The prediction of the studies.
ADMET profiles was conducted with the aid of the pkCSM
descriptors algorithm protocol.39
In general, the investigations of the cytotoxic activity indicated
that MCF-7 was the most sensitive cell line to the influence of the
After assessing the ADMET profiles of compounds 12d, 12a, new derivatives. In particular, compound 12d was found to be
10c and 10d (Table 4), we can suggest that these derivatives have the most potent derivative of all the tested compounds against the
the advantage of better intestinal absorption in humans than three HepG2, HCT116 and MCF-7 cancer cell lines, with IC50
=
doxorubicin (90.7–92.8, compared with 62.3 in the case of 22.08 ꢀ 2.1, 27.13 ꢀ 2.2 and 17.12 ꢀ 1.5 mM, respectively.
doxorubicin). This preference can be attributed to the superior Although this compound displayed nearly one third of the activity
lipophilicity of our designed ligands, which would facilitate of doxorubicin (IC50 = 7.94 ꢀ 0.6, 8.07 ꢀ 0.8 and 6.75 ꢀ 0.4 mM,
their passage through different biological membranes.40 respectively), it may be useful as a template for future design,
Accordingly, these compounds may have significantly good optimization, and investigation to produce more potent anti-
bioavailability after oral administration. In the CNS permeability cancer analogs. Compounds 12a, 10c and 10d displayed very good
study, [1,2,4]triazolo[4,3-a]quinoxaline derivatives 12d, 12a, 10c anticancer activities against the three HepG2, HCT116 and MCF-7
and 10d demonstrated the best ability to penetrate the CNS (CNS cancer cell lines, with IC50 = 31.40 ꢀ 2.8, 28.81 ꢀ 2.4 and 19.72 ꢀ
permeability values ꢁ1.947 to ꢁ2.416), while doxorubicin is 1.5 mM for 12a, 33.41 ꢀ 2.9, 29.96 ꢀ 2.5 and 24.78 ꢀ 1.9 mM for
unable to penetrate the CNS (CNS permeability oꢁ4.0). It is 10c, and 37.55 ꢀ 3.3, 30.22 ꢀ 2.6 and 25.53 ꢀ 2.0 mM for 10b,
also clear that cytochrome P3A4, the main enzyme involved in respectively.
drug metabolism, could be inhibited under the effects of
The most active derivatives, 10c, 10d, 10h, 12a, 12b and 12d,
compounds 12d, 12a, 10c and 10d, while doxorubicin could not. were evaluated for their DNA binding activities. Compound 12d
This may also be due to the higher lipophilicity of our new displayed the highest binding affinity. This compound potently
ligands. Excretion was assessed in term of the total clearance, intercalates DNA at a decreased IC50 value (35.33 ꢀ 1.8 mM)
which is a significant parameter in deciding dose intervals. which is nearly equipotent to that of doxorubicin (31.27 ꢀ
The obtained data revealed that doxorubicin revealed the highest 1.8 mM). Compounds 12a and 10c exhibited good DNA-binding
total clearance value compared to the other ligands. On the other affinities, with IC50 values of 39.35 ꢀ 3.9 and 42.35 ꢀ 3.9 mM,
hand, the new ligands showed lower total clearance values. respectively. Finally, compounds 10d, 10h and 12b showed
Thus, doxorubicin can be excreted more quickly and accordingly moderate DNA-binding affinities, with IC50 values of 50.35 ꢀ
requires shorter dosing intervals. Dissimilar to doxorubicin, the 3.9, 57.08 ꢀ 3.3 and 59.35 ꢀ 3.2 mM respectively.
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Also, compounds 10c, 10d, 10h, 12a, 12b and 12d are respec-
NJC
Yield, 90%; m.p. 170–2 1C; IRn (cmꢁ1): 3243 (NH), 3090 (C–
max
tively 2.68, 2.71, 2.27, 3.30, 2.24 and 4.36 fold more toxic in H aromatic), 2965 (C–H aliphatic), 1725 (CO ester); 1H NMR
breast cancer cells (MCF-7, the most sensitive cells) than in (400 MHz, DMSO-d6): 3.83 (s, 3H, CH3), 7.40–7.44 (dd, H, H-7
normal VERO cells.
quinox.), 7.46–7.49 (dd, 1H, H-8 quinox.), 7.70–7.72 (d, 1H, H-9
Furthermore, ADMET profiles were calculated for the four quinox.), 7.88–7.90 (dd, 2H, H-3 and H-5 phenyl), 8.17–819
most active compounds in comparison to doxorubicin as a (d, 1H, H-6 quinox.), 8.31–8.33 (dd, 2H, H-2 and H-6 phenyl),
reference drug.
10.01 (s, 1H, CH triazole), 10.53 (s, 1H, NH-ph) (D2O exchange-
able); anal. calcd for C17H13N5O2 (319.32): C, 63.94; H, 4.10; N,
21.93. Found: C, 64.08; H, 4.23; N, 21.88.
4.1.3. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-
ylamino)benzohydrazide (9). A mixture of the ester derivative 7
(9.6 g, 0.03 mol) and hydrazine hydrate 80% (6.0 g, 0.12 mol) in
ethanol (150 ml) was stirred well and refluxed for 8 h. The
4. Experimental
4.1. Chemistry
4.1.1. General. All melting points were determined by the solvent was evaporated and the obtained solid was filtered,
open capillary method on a Gallenkamp melting point apparatus washed with water several times, dried and re-crystallized from
at the Faculty of Pharmacy of Al-Azhar University and were ethanol to afford the corresponding hydrazide derivative (9).
uncorrected. The infrared spectra were recorded on a Pye Unicam
Yield, 80%; m.p. 293–5 1C; IRn (cmꢁ1): 3299, 3229 (2NH,
max
SP 1000 IR spectrophotometer at the Pharmaceutical Analytical NH2), 3054 (C–H aromatic), and 1660 (CO amide); 1H NMR
Unit, Faculty of Pharmacy, Al-Azhar University, using the potas- (400 MHz, DMSO-d6): 4.48 (s, 2H, NH2) (D2O exchangeable),
sium bromide disc technique. Proton magnetic resonance 7.44–7.48 (dd, 1H, H-7 quinox.), 7.51–7.55 (dd, 1H, H-8 qui-
1H NMR spectra were recorded on a Bruker 400 Megahertz- nox.), 7.76–7.78 (d, 1H, H-9 quinox.), 7.86–7.88 (dd, 2H, H-3 and
nuclear magnetic resonance (400 MHz-NMR) spectrophotometer H-5 phenyl), 8.22–8.24 (d, 1H, H-6 quinox.), 8.28–8.30 (dd, 2H,
at the Microanalytical Unit, Faculty of Pharmacy, Ain Shams H-2 and H-6 phenyl), 9.71 (s, 2H, CH triazole and NH-ph (D2O
University, and on a GEMINI-400BB NMR spectrometer at the exchangeable)), 10.05 (s, 1H, NH-NH2) (D2O exchangeable); MS
Chemical Laboratories of the Ministry of Defense, Cairo. Carbon-13 (m/z): 319 (M+, C16H13N7O, 18.98%), 288 (base peak, 100%), 231
(C13) nuclear magnetic resonance (13C NMR) spectra were recorded (37.76%), 89 (10.02%), and 74 (31.33%); anal. calcd for
on a Bruker 100 megahertz-nuclear magnetic resonance (100 MHz-
C16H13N7O (319.33): C, 60.18; H, 4.10; N, 30.70. Found: C,
NMR) spectrophotometer at the Microanalytical Unit, Faculty of 60.34; H, 4.37; N, 30.97.
Pharmacy, Cairo University. Tetramethylsilane (TMS) was used as
4.1.4. General procedure for the synthesis of target com-
the internal standard, and the chemical shifts were measured on a pounds 10a–j. Equimolar amounts of acid hydrazide derivative 9
d scale in parts per million (ppm). The mass spectra were carried (0.5 g, 0.0015 mol) and the appropriate benzaldehyde deriva-
out on the Direct Probe Controller Inlet part of a single quadrupole tives, namely benzaldehyde, 2-chlorobenzaldehyde, 4-chloro-
mass analyzer (Thermo Scientific Gas chromatography-mass benzaldehyde, 4-fluorobenzaldehyde, 2,6-dichlorobenzaldehyde,
spectrometry (GCMS) model ISQ LT) using Thermo X-Calibur 4-hydroxybenzaldehyde, 2-methoxybenzaldehyde, 4-methoxy-
software at the Mycology and Biotechnology Regional Center, benzaldehyde, 3-nitrobenzaldehyde, and/or 4-nitrobenzaldehyde
Al-Azhar University. Elemental analyses (C, H, N) were performed (0.0015 mol), were refluxed in ethanol (25 ml) in the presence of
on a carbon hydrogen and nitrogen (CHN) analyzer at the Mycology few drops of glacial acetic acid for 4 h. The mixture was cooled, and
and Biotechnology Regional Center, Al-Azhar University. All com- the formed solid was filtered and re-crystallized from ethanol to
pounds were within ꢀ0.4 of the theoretical values. The reactions furnish the corresponding Schiff bases 10a–j, respectively.
were monitored by thin-layer chromatography (TLC) using TLC
sheets precoated with Merck 60 F254 UV fluorescent silica gel
plates and were visualized using an ultraviolet (UV) lamp and benzylidenebenzohydrazide (10a). Yield, 90%; m.p. 254–6 1C;
different solvents as mobile phases. IRn
(cmꢁ1): 3254, 3115 (2NH), 3060 (C–H aromatic), and
4.1.4.1. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
max
Compounds 2,3-(1H,4H)-quinoxalinedione 3, 2,3-dichloroquino- 1631 (CO); 1H NMR (400 MHz, DMSO-d6): 7.44–7.49 (m, 3H,
xaline 4, 2-chloro-3-hydrazinylquinoxaline 5, 4-chloro[1,2,4]triazolo H-3, H-4 and H-5 of C6H5), 7.52–7.56 (dd, 1H, H-7 quinox.),
[4,3-a]quinoxaline 6, 4-chloro-[1,2,4]triazolo[4,3-a]quinoxaline-1-thiol 7.73–7.75 (dd, 1H, H-8 quinox.), 7.77–7.79 (d, 1H, H-9 quinox.),
8 and potassium 4-oxo-1-sulfido-4H-[1,2,4]triazolo[4,3-a]quinoxalin-5- 7.98–8.00 (dd, 2H, H-3 and H-5 C6H4), 8.24–8.26 (dd, 2H, H-2
ide 11 were obtained according to the reported procedures.13,30
and H-6 C6H4), 8.37 (d, 1H, H-6 quinox.), 8.39 (dd, 2H, H-2 and
4.1.2. Methyl 4-([1,2,4]triazolo[4,3-a]quinoxalin-4-ylamino) H-6 of C6H5), 8.49 (s, 1H, NHNQCH-ph), 10.08 (s, 1H, CH
benzoate (7). Equimolar quantities of 4-chloro-[1,2,4]triazolo triazolo), 10.57 (s, 1H, NH-ph) (D2O exchangeable), 11.82 (s, 1H,
[4,3-a]quinoxaline 6 (2.04 g, 0.01 mol) and methyl 4-amino- NH of CONH–NQ) (D2O exchangeable); anal. calcd for
benzoate (1.51 g, 0.01 mol) in acetonitrile (CH3CN) (20 ml) in
C23H17N7O (407.44): C, 67.80; H, 4.21; N, 24.06. Found: C,
the presence of a few drops of triethylamine (TEA) were heated 68.12; H, 4.36; N, 24.13.
under reflux for 5 h. The formed precipitate was filtered,
washed with n-hexane, and air dried to obtain the corres-
ponding methyl ester derivative 7.
4.1.4.2. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
(2-chlorobenzylidene)benzohydrazide (10b). Yield, 95%; m.p. 285–
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7 1C; IRn (cmꢁ1): 3333, 3177 (2NH), 3060 (C–H aromatic), and 2H, H-30 and H-50), 7.47–7.49 (dd, 1H, H-7 of quinox.), 7.51–7.53
max
1646 (CO); 1H NMR (400 MHz, DMSO-d6): 7.42–7.44 (dd, 1H, (dd, 1H, H-8 quinox.), 7.56–7.58 (d, H, H-9 quinox.), 7.79–7.80
H-50), 7.45–7.48 (m, 2H, H-30 and H-40), 7.49–7.51 (dd, 1H, H-7 (dd, 2H, H-3 and H-5 phenyl), 7.92–7.94 (dd, 2H, H-2 and H-6
quinox.), 7.52–7.54 (dd, 1H, H-8 quinox.), 7.56–7.58 (d, 1H, H-9 phenyl), 8.25–8.27 (d, 1H, H-6 quinox.), 8.33–8.35 (dd, 2H, H-20
quinox.), 7.78–7.81 (dd, 2H, H-3 and H-5 phenyl), 7.96–8.02 (dd, and H-60), 8.36 (s, 1H, NHNQCH-ph), 9.89 (s, 1H, OH) (D2O
2H, H-2 and H-6 phenyl), 8.26–8.28 (d, 1H, H-6 quinox.), 8.36– exchangeable), 10.06 (s, 1H, CH triazolo), 10.54 (s, 1H, NH-ph)
8.38 (d, 1H, H-60), 8.87 (s, 1H, NHNQCH-ph), 10.07 (s, 1H, CH (D2O exchangeable), 11.55 (s, 1H, NH of CONH–NQ) (D2O
triazolo), 10.57 (s, 1H, NH-ph) (D2O exchangeable), 12.00 (s, 1H, exchangeable); anal. calcd for C23H17N7O2 (423.14): C, 65.24;
NH of CONH–NQ) (D2O exchangeable); anal. calcd for H, 4.05; N, 23.16. Found: C, 65.38; H, 4.23; N, 23.50.
C
23H16ClN7O (441.88): C, 62.52; H, 3.65; N, 22.19. Found: C,
62.69; H, 3.81; N, 22.37.
4.1.4.7. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
(2-methoxybenzylidene)benzohydrazide (10g). Yield, 90%; m.p.
4.1.4.3. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-(4-
257–9 1C; IRn (cmꢁ1): 3259, 3107 (2NH), 3094 (C–H aromatic),
max
2950 (C–H aliphatic), and 1676 (CO); 1H NMR (400 MHz,
DMSO-d6): 3.86 (s, 3H, CH3), 7.02 (d, 1H, H-30), 7.09–7.11 (dd,
1H, H-50), 7.40 (dd, 1H, H-7 quinox.), 7.49–7.51 (dd, 1H, H-8
quinox.), 7.54–7.56 (d, 1H, H-9 quinox.), 7.79–7.81 (dd, 2H, H-3
and H-5 phenyl), 7.95–7.97 (dd, 2H, H-2 and H-6 phenyl), 8.26–
8.28 (d, 1H, H-6 quinox.), 8.34–8.36 (dd, 2H, H-40 and H-60), 8.81
(s, 1H, NHNQCH-ph), 10.07 (s, 1H, CH triazolo), 10.55 (s, 1H,
NH-ph) (D2O exchangeable), 11.76 (s, 1H, NH of CONH–NQ)
(D2O exchangeable); MS (m/z): 437 (M+, C24H19N7O2, 11.32%),
318 (98.09%), 288 (base peak, 100%), 104 (66.46%) and 89
(17.16%); anal. calcd for C24H19N7O2 (437.46): C, 65.89; H, 4.38;
N, 22.41. Found: C, 66.11; H, 4.57; N, 22.32.
chlorobenzylidene)benzohydrazide (10c). Yield, 95%; m.p. 280–
2 1C; IRn (cmꢁ1): 3242, 3177 (2NH), 3090 (C–H aromatic), and
max
1650 (CO); 1H NMR (400 MHz, DMSO-d6): 7.48–7.50 (dd, 2H,
H-30 and H-50), 7.51–7.53 (dd, 1H, H-7 quinox.), 7.55–7.57 (dd,
1H, H-8 quinox.), 7.75–7.77 (d, 1H, H-9 quinox.), 7.79–7.81 (dd,
2H, H-3 and H-5 phenyl), 7.97–7.99 (dd, 2H, H-2 and H-6
phenyl), 8.26–8.28 (d, 1H, H-6 quinox.), 8.37–8.39 (dd, 2H,
H-20 and H-60), 8.46 (s, 1H, NHNQCH-ph), 10.08 (s, 1H, CH
triazolo), 10.58 (s, 1H, NH-ph) (D2O exchangeable), 11.86 (s, 1H,
NH of CONH–NQ) (D2O exchangeable); MS (m/z): 443 (M2+
,
1.65%), 441 (M+, 4.94%), 231 (48%), 318 (base peak, 100%), and
287 (60.69%); anal. calcd for C23H16ClN7O (441.88): C, 62.52; H,
3.65; N, 22.19. Found: C, 62.73; H, 3.79; N, 22.42.
4.1.4.8. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
4.1.4.4. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
(4-methoxybenzylidene)benzohydrazide (10h). Yield, 95%; m.p.
(4-fluorobenzylidene)benzohydrazide (10d). Yield, 85%; m.p. 273–
280–2 1C; IRn (cmꢁ1): 3258, 3134 (2NH), 3090 (C–H aromatic),
5 1C; IRn (cmꢁ1): 3331, 3194 (2NH), 3070 (C–H aromatic), and
max
2969 (CH-aliphatic), and 1676 (CO); 1H NMR (400 MHz, DMSO-
d6): 3.84 (s, 3H, CH3), 7.02–7.04 (dd, 2H, H-30 and H-50), 7.49–
7.52 (dd, 1H, H-7 quinox.), 7.55–7.59 (dd, 1H, H-8 quinox.),
7.67–7.70 (d, 1H, H-9 quinox.), 7.80–7.82 (dd, 2H, H-3 and H-5
phenyl), 7.95–7.98 (dd, 2H, H-2 and H-6 phenyl), 8.26–8.28
(d, 1H, H-6 quinox.), 8.36–8.39 (dd, 2H, H-20 and H-60), 8.42
(s, 1H, NHNQCH-ph), 10.10 (s, 1H, CH triazolo), 10.58 (s, 1H,
NH-ph) (D2O exchangeable), 11.66 (s, 1H, NH CONH–NQ) (D2O
exchangeable); 13C NMR (100 MHz, DMSO-d6): 55.76, 114.80
(2), 116.72, 120.72 (2), 123.17 (2), 125.64, 127.49, 127.54, 128.18,
128.72 (2), 129.08 (2), 136.25, 138.78, 138.83, 148.24, 143.40,
147.68, 161.22, 162.97; anal. calcd for C24H19N7O2 (437.46): C,
65.89; H, 4.38; N, 22.41. Found: C, 65.97; H, 4.62; N, 22.48.
max
1644 (CO); 1H NMR (400 MHz, DMSO-d6): 7.29–7.31 (dd, 1H,
H-30 and H-50), 7.45–7.47 (dd, 1H, H-7 quinox.), 7.50–7.52 (dd, 1H,
H-8 quinox.), 7.55–7.57 (d, 1H, H-9 quinox.), 7.77–7.79 (dd, 2H, H-3
and H-5 phenyl), 7.97–7.99 (dd, 2H, H-2 and H-6 phenyl), 8.24–8.26
(d, 1H, H-6 of quinox.), 8.37–8.39 (dd, 2H, H-20 and H-60), 8.48
(s, 1H, NHNQCH-ph), 10.08 (s, 1H, CH triazolo), 10.55 (s, 1H, NH-
ph) (D2O exchangeable), 11.81 (s, 1H, NH of CONH–NQ) (D2O
exchangeable); anal. calcd for C23H16FN7O (425.43): C, 64.94; H,
3.79; N, 23.05. Found: C, 65.21; H, 3.86; N, 23.18.
4.1.4.5. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-(2,6-
dichlorobenzylidene)benzohydrazide (10e). Yield, 90%; m.p. 292–
4 1C; IRn (cmꢁ1): 3188 (2NH), 3060 (C–H aromatic), and 1686
max
1
(CO); H NMR (400 MHz, DMSO-d6): 7.41–7.45 (t, 1H, H-40),
4.1.4.9. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
7.47–7.49 (dd, 1H, H-7 quinox.), 7.49–7.51 (dd, 1H, H-8 qui-
nox.), 7.54–7.56 (d, 1H, H-9 quinox.), 7.79–7.81 (dd, 2H, H-3 and
H-5 phenyl), 7.96–7.98 (dd, 2H, H-2 and H-6 phenyl), 8.25–8.27
(d, 1H, H-6 of quinox.), 8.36–8.38 (dd, 2H, H-30 and H-50), 8.66
(s, 1H, NHNQCH-ph), 10.07 (s, 1H, CH triazolo), 10.57 (s, 1H,
NH-ph) (D2O exchangeable), 12.05 (s, 1H, NH CONH–NQ) (D2O
exchangeable); anal. calcd for C23H15Cl2N7O (476.39): C, 58.00;
H, 3.17; N, 20.58. Found: 57.89; H, 3.40; N, 20.79.
(3-nitrobenzylidene)benzohydrazide (10i). Yield, 80%; m.p. 295–
7 1C; IRn (cmꢁ1): 3395, 3268 (2NH), 3090 (C–H aromatic), and
max
1681 (CO); 1H NMR (400 MHz, DMSO-d6): 7.50–7.52 (dd, 1H,
H-50), 7.56–7.58 (d, 1H, H-40), 7.74–7.76 (dd, 1H, H-7 quinox.),
7.79–7.81 (dd, 1H, H-8 quinox.), 7.96–7.98 (d, 1H, H-9 quinox.),
8.14–8.16 (dd, 1H, H-60), 8.23–8.25 (dd, 2H, H-3 and H-5
phenyl), 8.26–8.28 (dd, 2H, H-2 and H-6 phenyl), 8.37–8.39 (d,
1H, H-6 quinox.), 8.54 (s, 1H, H-20), 8.57 (s, 1H, NHNQCH-ph),
10.07 (s, 1H, CH triazolo), 10.58 (s, 1H, NH-ph) (D2O exchange-
4.1.4.6. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
(4-hydroxybenzylidene)benzohydrazide (10f). Yield, 80%; m.p. able), 12.03 (s, 1H, NH of CONH–NQ) (D2O exchangeable);
294–6 1C; IRn (cmꢁ1): 3336, 3221 (2NH), 3085 (C–H aromatic), anal. calcd for C23H16N8O3 (452.43): C, 61.06; H, 3.56; N, 24.77.
max
and 1658 (CO); 1H NMR (400 MHz, DMSO-d6): 6.82–6.84 (dd, Found: C, 61.30; H, 3.69; N, 24.96.
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4.1.4.10. 4-([1,2,4]Triazolo[4,3-a]quinoxalin-4-ylamino)-N0-
NJC
(17.98%), 520 (23.19%), 368 (67.37%), 77 (61.19%), 70 (base
(4-nitrobenzylidene)benzohydrazide (10j). Yield, 85%; m.p. 300– peak, 100%), and 78 (83.16%); anal. calcd for C25H16Cl4N6O3S
2 1C; IRn
(cmꢁ1): 3300, 3197 (2NH), 3060 (C–H aromatic), (622.302): C, 48.25; H, 2.59; N, 13.51. Found: C, 48.03; H, 2.87;
max
1686 and (CO); 1H NMR (400 MHz, DMSO-d6): 52–7.54 (dd, 2H, N, 13.74.
H-30 and H-50), 7.55–7.58 (dd, 1H, H-7quinox.), 7.59–7.61 (dd,
4.1.5.4. 2-{4-Oxo-1-([2-oxo-2-{p-tolylamino}ethyl]thio)-
[1,2,4]triazolo[4,3-a]quinoxalin-5(4H)-yl}-N-(p-tolyl)acetamide
(12d). Yield, 90%; m.p. 206–8 1C; IRn
(cmꢁ1): 3285 (2NH),
3047 (C–H aromatic), 2922 (C–H aliphatic), 1669 (3 CO);
1H NMR (400 MHz, DMSO-d6): 2.25 (s, 6H, 2CH3), 4.34 (s, 2H,
S-CH2), 4.43 (s, 2H, N-CH2), 7.10–8.57 (m, 12H, aromatic pro-
tons), 10.29 (s, 1H, NH) (D2O exchangeable), 10.35 (s, 1H, NH)
(D2O exchangeable); anal. calcd for C27H24N6O3S (512.59): C,
63.27; H, 4.72; N, 16.40. Found: C, 63.01; H, 4.89; N, 16.73.
1H, H-8 quinox.), 7.81–7.83 (d, 1H, H-9 quinox.), 7.85–7.87 (dd,
2H, H-3 and H-5 phenyl), 8.28–8.30 (dd, 2H, H-2 and H-6
phenyl), 8.31–8.33 (d, 1H, H-6 quinox.), 8.39–8.42 (dd, 2H,
H-20 and H-60), 8.59 (s, 1H, NHNQCH-ph), 10.09 (s, 1H, CH
triazolo), 10.61 (s, 1H, NH-ph) (D2O exchangeable), 12.11 (s, 1H,
NH of CONH–NQ) (D2O exchangeable); anal. calcd for
max
C
23H16N8O3 (452.43): C, 61.06; H, 3.56; N, 24.77. Found: C,
61.25; H, 3.74; N, 24.59.
4.1.5. General procedure for synthesis of target compounds
(12a–e). The appropriate 2-chloroacetanilides, namely 2-chloro-
acetanilide, 2-chloro-4-chloroacetanilide, 2-chloro-2,6-dichloro-
acetanilide, 2-chloro-4-methylacetanilide, and 2-chloro-4-
nitroacetanilide, (0.002 mol), were added to the dipotassium
salt (11) (0.5 g, 0.001 mol) in DMF (10 ml), and the reaction
mixture was heated under reflux for 3 h. The reaction mixture
was poured on crushed ice, and the resulting precipitate was
filtered and crystallized from ethanol to obtain the corres-
ponding di-substituted derivatives 12a–e, respectively.
4.1.5.5. N-(4-Nitrophenyl)-2-{(5-[2-{(4-nitrophenyl)amino}-2-
oxoethyl]-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-1-
yl)thio}acetamide (12e). Yield, 80%; m.p. 270–2 1C; IRn (cmꢁ1):
max
3284 (2NH), 3047 (C–H aromatic), 2899 (C–H aliphatic), 1669
(3 CO); 1H NMR (400 MHz, DMSO-d6): 4.39 (s, 2H, S-CH2), 4.49
(s, 2H, N-CH2), 7.60–8.57 (m, 12H, aromatic protons), 10.72
(s, 1H, NH) (D2O exchangeable), 10.81 (s, 1H, NH) (D2O
exchangeable); anal. calcd for C25H18N8O7S (574.53): C, 52.26;
H, 3.16; N, 19.50. Found: C, 52.39; H, 3.44; N, 19.82.
4.1.5.1. 2-(4-Oxo-1-{(2-oxo-2-[phenylamino]ethyl)thio}-
[1,2,4]triazolo[4,3-a]quinoxalin-5(4H)-yl)-N-phenylacetamide
5.1. Docking studies
(cmꢁ1): 3264 (2NH),
(12a). Yield, 90%; m.p. 183–5 1C; IRn
max
3057 (C–H aromatic), 2927 (C–H aliphatic), 1667 (3 CO);
1H NMR (400 MHz, DMSO-d6): 4.39 (s, 2H, S-CH2), 4.49
(s, 2H, N-CH2), 7.60–8.57 (m, 14H, aromatic protons), 10.72
(s, 1H, NH) (D2O exchangeable), 10.81 (s, 1H, NH) (D2O
exchangeable); 13C NMR (100 MHz, DMSO-d6): 20.91, 34.36,
116.66, 119.65 (2), 119.66 (2), 125.35 (2C), 128.26, 128.49,
129.63, 132.86 (2), 133.01 (2), 136.23, 136.95 (2), 144.04,
146.99, 152.05 (2) 165.30, 165.94; MS (m/z): 484 (M+, 8.50%),
337 (44.91%), 236 (45.20%), 102 (45.89%), 91 (base peak,
100%), and 78 (83.16%); anal. calcd for C25H20N6O3S (484.53):
C, 61.97; H, 4.16; N, 17.34. Found: C, 62.31; H, 4.28; N, 17.60.
In the present work, all the target compounds were subjected to
docking studies to explore their binding modes towards the
DNA active site. All modeling experiments were performed
using the Molsoft program, which provides a unique set of
tools for the modeling of protein/ligand interactions. It predicts
how small, flexible molecules, such as substrates or drug
candidates, will bind to a protein of known 3D structure,
represented by grid interaction potentials (http://www.mol
soft.com/icm_pro.html). Each experiment used the biological
target DNA downloaded from the Brookhaven Protein Data-
bank
(http://www.rcsb.org/pdb/explore/explore.do?structure
Id=4G0U). In order to qualify the docking results in terms of
the accuracy of the predicted binding conformations in com-
parison with the experimental procedure, the reported DNA
intercalator doxorubicin was used as a reference ligand.
4.1.5.2. N-(4-Chlorophenyl)-2-{(5-[2-{(4-chlorophenyl)amino}-
2-oxoethyl]-4-oxo-4,5-dihydro-[1,2,4]triazolo-[4,3-a]quinoxalin-1-
yl)thio}acetamide (12b). Yield, 90%; m.p. 265–7 1C; IRn (cmꢁ1):
max
3298 (2NH), 3090 (C–H aromatic), 2947 (C–H aliphatic), 1689
(3 CO); 1H NMR (400 MHz, DMSO-d6): 4.39 (s, 2H, S-CH2), 4.56
(s, 2H, N-CH2) 7.59–8.58 (m, 12H, aromatic protons), 10.71 5.2. In vitro anti-proliferative activity
(s, 1H, NH) (D2O exchangeable), 10.79 (s, 1H, NH) (D2O
The cytotoxicity assays were performed at the Pharmacology &
exchangeable); anal. calcd for C25H18Cl2N6O3S (553.42): C,
Toxicology Department, Faculty of Pharmacy, Al-Azhar Univer-
54.26; H, 3.28; N, 15.19. Found: C, 54.89; H, 3.48; N, 15.02.
sity, Cairo, Egypt. Cancer cells from different cancer cell lines,
4.1.5.3. N-(2,6-Dichlorophenyl)-2-{(5-[2-{(2,6-
dichlorophenyl)amino}-2-oxoethyl]-4-oxo-4,5-dihydro-
namely hepatocellular carcinoma (HepG2), colorectal carcinoma
(HCT-116) and breast cancer (MCF-7), were purchased from the
[1,2,4]triazolo[4,3-a]quinoxalin-1-yl)thio}acetamide (12c). Yield, American Type Cell Culture collection (ATCC, Manassas, USA)
95%; m.p. 217–9 1C; IRn
(cmꢁ1): 3289 (2NH), 3009 (C–H and grown on appropriate growth medium (Roswell Park
max
aromatic), 2925 (C–H aliphatic), 1683 (3 CO); 1H NMR Memorial Institute medium, RPMI 1640) supplemented
(400 MHz, DMSO-d6): 4.51 (s, 4H, 2CH2), 7.52–8.71 (m, 10H, with 100 mg mlꢁ1 of streptomycin, 100 units per ml of
aromatic protons), 10.35 (s, 1H, NH) (D2O exchangeable), 11.03 penicillin and 10% heat-inactivated fetal bovine serum in a
(s, 1H, NH) (D2O exchangeable); MS (m/z): 622 (M+, 6.43%), 608 humidified, 5% (v/v) CO2 atmosphere at 37 1C for the cytotoxicity
894 | New J. Chem., 2021, 45, 881--897
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Paper
assay by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium absorbance and normalized as the percentage of the untreated
bromide (MTT).
DNA/methyl green absorbance value.
Exponentially growing cells from different cancer cell lines
were trypsinized, counted and seeded at the appropriate den-
sities (2000–1000 cells per 0.33 cm2 well) into 96-well microtiter
Conflicts of interest
plates. The cells then were incubated in
atmosphere at 37 1C for 24 hours. Then, the cells were exposed
a humidified
There are no conflicts to declare.
to different concentrations of the compounds (0.1, 10, 100 and
1000 mM) for 72 hours. Then, the viability of the treated cells Acknowledgements
was determined using the MTT technique as follows. Cells were
The authors extend their appreciation and thanking to Dr
incubated with 200 ml of 5% MTT solution/well (Sigma Aldrich,
MO) and were allowed to metabolize the dye into colored-
insoluble formazan crystals for 2 hours. The remaining MTT
solution was discarded from the wells, and the formazan
crystals were dissolved in 200 ml per well acidified isopropanol
for 30 min, covered with aluminum foil and subjected to
continuous shaking using a MaxQ 2000 plate shaker (Thermo
Fisher Scientific Inc, MI) at room temperature. The colorimetric
assay was measured and recorded at an absorbance of 570 nm
using a Stat FaxR 4200 plate reader (Awareness Technology,
Inc., FL). The cell viability were expressed as the percentage of
the control, and the concentration that induces 50% of
maximum inhibition of cell proliferation (IC50) was determined
using Graph Pad Prism version 5 software (Graph Pad software
Inc, CA).33–35
Tamer Abdel-Ghany, Pharmacology & Toxicology Department,
Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt for help-
ing in the pharmacological part.
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