[1,2,4]Triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4′,3′-c]quinazoline derived DNA intercalators: Design, synthesis, in silico ADMET profile, molecular docking and anti-proliferative evaluation studies

Article abstract of DOI:10.1016/j.bmc.2020.115958

In view of their DNA intercalation activities as anticancer agents, novel fifteen [1,2,4]triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4′,3′-c]quinazoline derivatives have been designed, synthesized and evaluated against HepG2 and HCT-116. The molecular design was performed to investigate the binding mode of the proposed compounds with DNA active site. The data obtained from biological testing highly correlated with that obtained from molecular modeling studies. HCT-116 was found to be more sensitive cell lines to the influence of the new derivatives. In particular, compounds 16, 18, 11 and 5 were found to be the most potent derivatives with IC₅₀ = 3.61, 6.72, 7.16 and 5.18 μM respectively against HepG2 cell line. Also, compounds 16, 18, 11 and 5 displayed IC₅₀ = 2.85, 3.82, 4.97 and 6.40 μM respectively against HCT-116 cell line. These derivatives displayed higher activities than doxorubicin, (IC₅₀ = 7.94 and 8.07 μM respectively) against the two HepG2 and HCT-116 cell lines. The most active anti-proliferative derivatives 5, 6, 10, 11, 13, 16, 18, 19 and 20 were further evaluated for their DNA-binding affinity which revealed the ability of these compounds to intercalate DNA. The tested compounds displayed very strong to moderate DNA-binding affinities. Compounds 16 and 18 potently intercalate DNA at IC₅₀ values of 26.03 and 28.37 μM respectively which were lower than IC₅₀ of Doxorubicin (IC₅₀ = 31.27). This finding indicated that these derivatives exhibited higher DNA binding activities than Doxorubicin. Also, compounds 11 and 5 displayed very strong DNA binding at IC₅₀ = 30.84 and 33.56 μM respectively, which were nearly equipotent to that of doxorubicin. Moreover, most of our derivatives exhibited good ADMET profile.

Full text of DOI:10.1016/j.bmc.2020.115958

Bioorg. Med. Chem. 30 (2021) 115958
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
′ ′
1,2,4]Triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]
[
quinazoline derived DNA intercalators: Design, synthesis, in silico ADMET
profile, molecular docking and anti-proliferative evaluation studies
a,
b
,
*
a
a,
*
a,*
Khaled El-Adl
, Mohamed-Kamal Ibrahim , Mohammed S.I. Alesawy , Ibrahim H. Eissa
a
Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884, Cairo, Egypt
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University for Sustainable Development, Cairo, Egypt
b
A R T I C L E I N F O
Keywords:
A B S T R A C T
In view of their DNA intercalation activities as anticancer agents, novel fifteen [1,2,4]triazolo[4,3-c]quinazoline
′
′
Bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline
Molecular docking
′
′
and bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline derivatives have been designed, synthesized and evaluated
against HepG2 and HCT-116. The molecular design was performed to investigate the binding mode of the
proposed compounds with DNA active site. The data obtained from biological testing highly correlated with that
obtained from molecular modeling studies. HCT-116 was found to be more sensitive cell lines to the influence of
the new derivatives. In particular, compounds 16, 18, 11 and 5 were found to be the most potent derivatives
with IC50 = 3.61, 6.72, 7.16 and 5.18 µM respectively against HepG2 cell line. Also, compounds 16, 18, 11 and 5
displayed IC50 = 2.85, 3.82, 4.97 and 6.40 µM respectively against HCT-116 cell line. These derivatives displayed
higher activities than doxorubicin, (IC50 = 7.94 and 8.07 µM respectively) against the two HepG2 and HCT-116
cell lines. The most active anti-proliferative derivatives 5, 6, 10, 11, 13, 16, 18, 19 and 20 were further
evaluated for their DNA-binding affinity which revealed the ability of these compounds to intercalate DNA. The
tested compounds displayed very strong to moderate DNA-binding affinities. Compounds 16 and 18 potently
intercalate DNA at IC50 values of 26.03 and 28.37 µM respectively which were lower than IC50 of Doxorubicin
DNA intercalators
Anticancer agents
(
IC50 = 31.27). This finding indicated that these derivatives exhibited higher DNA binding activities than
Doxorubicin. Also, compounds 11 and 5 displayed very strong DNA binding at IC50 = 30.84 and 33.56 µM
respectively, which were nearly equipotent to that of doxorubicin. Moreover, most of our derivatives exhibited
good ADMET profile.
7
1
. Introduction
DNA intercalators, e.g. doxorubicin I, share three common essential
diseases such as cancer. The most important group of compounds that
8
interact reversibly with the DNA double helix are intercalators. Many
anticancer drugs that interact with DNA through intercalation are in
clinical use.8 Intercalation is a process that transfers the intercalating
molecule from an aqueous environment to the hydrophobic space be-
tween two adjacent DNA base pairs.9 Extensive research over the last
four decades has focused on the small organic compounds effects that
non-covalently bind to nucleic acids.11 These interactions are known to
disrupt replication and/or transcription that culminate in cellular death.
Accordingly, DNA-binding compounds have potential applications as
anticancer agents. Small molecules may bind non-covalently to DNA
through intercalation between nucleobase pairs, major or minor groove
,9
structural features, i) a planar polyaromatic system involves fused
planar rings (chromophore), binding with DNA.1,2 ii) Cationic species
which increase the efficiency of DNA intercalators by interaction with
the negatively charged DNA sugar–phosphate backbone. The cationic
species are basic groups (ex: amino or nitrogen containing heterocyclic
groups) that can be protonated under physiological pH.3 iii) Groove
binding side chains, which can occupy the minor groove of DNA
,10
Fig. 1).4 DNA plays an essential role in a wide diversity of cellular
–6
(
processes and acts as a fundamental target for treating hallmark genetic
*
Corresponding authors at: Pharmaceutical Medicinal Chemistry and Drug Design Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City 11884,
Cairo, Egypt (K. El-Adl).
E-mail addresses: eladlkhaled74@yahoo.com, eladlkhaled74@azhar.edu.eg, khaled.eladl@hu.edu.eg (K. El-Adl), abdo_mo2007@yahoo.com (M.S.I. Alesawy),
ibrahimeissa@azhar.edu.eg (I.H. Eissa).
https://doi.org/10.1016/j.bmc.2020.115958
Received 13 August 2020; Received in revised form 10 December 2020; Accepted 11 December 2020
Available online 21 December 2020
0
968-0896/© 2020 Elsevier Ltd. All rights reserved.

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
binding and electrostatic interactions.12,13
The insertion of the planar aromatic molecule between DNA base
pairs results in a decrease in the DNA helical twist and lengthening of the
DNA. While groove binding, unlike intercalation, does not induce large
conformational changes in DNA and may be considered similar to
standard lock-and-key models for ligand-macromolecular binding. In
addition, Groove binders are usually crescent-shaped molecules that
bind to the minor groove of DNA.1
4–16
There are three types of intercalators. Classical intercalators, that
have no bulky groups, bind to DNA duplexes with essentially all of their
aromatic system inserted between base pairs from the top and bottom of
the intercalation site.7 Groove binding intercalators that have bulky
substituents that must be in one groove after the planar aromatic ring of
the drugs is bound by intercalation. To date, most evidence supports the
location of the bulky groups in the minor groove.7 The presence of two
groove binding bulky side chains as in case of nogalamycin V 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 groove (Fig. 2).17
,14
,15
Many DNA intercalators are either used already as anticancer drugs
1
9
20
Fig. 2. A representation of classical, groove binding and threading modes of
or still under clinical trials (e.g., doxorubicin I , amsacrine II
mitoxantrone III21 ellipticine IV4 nogalamycin V22) (Fig. 3).
,
drug intercalation to DNA.
Quinazoline nucleus is the backbone of many DNA intercalators.2
3–26
2
9–35
espe-
research in design and synthesis of new anticancer agents,
The discovery and development of new therapeutic DNA intercalators
cially DNA intercalators,23 we reported the design, synthesis, DNA
binding examination and docking studies of a new series of quinazoline
derivatives. These derivatives were designed according to the main
pharmacophoric features of DNA intercalators.
for the treatment of cancer is considered as one of the most important
targets in the field of medicinal chemistry.27 In recent years several
DNA-targeting agents consisting of a polycyclic planar moiety, such as
quinoline, quinazoline, or indole linked to an alkyl or aniline, have been
shown to be more active than kinase inhibitors.28 These compounds
interact with double stranded DNA and also present selective recogni-
tion of DNA sequences. The DNA interaction capacity of EBE-A22 VI
1
.1. Rationale and structure-based design
(
Fig. 3) has been documented.28
The rationale of our molecular design depended on generation of two
Based on the earlier findings, and in continuation of our previous
scaffolds of planer aromatic system containing quinazoline moiety
Fig. 1. The basic pharmacophoric features for doxorubicin as reported DNA intercalator.
2

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Fig. 3. Some reported DNA intercalators and their basic pharmacophoric features.
(
chromophore) with one or two groove binding side chains. The first one
intercalators.
The second scaffold consisted from [1,2,4]triazolo[4,3-c]quinazolin-
′
′
consisted from bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline moiety,
chromophore of four rings) containing one groove binding side chain
with different chemical substitutions at position-7, as compounds 8–12
Figs. 4 and 5). These compounds were designed as groove binding
(
5(6H)-one moiety, (chromophore of three rings) containing two groove
binding side chains with different chemical substitutions at both
positions-3 & 6, as compounds 15–21 (Figs. 4 and 5). These compounds
(
Fig. 4. The basic structural requirements and possible modifications of the new DNA-intercalators.
3

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Fig. 5. Rationale of molecular design of new DNA-intercalators.
were designed as threading DNA intercalators.
cell lines, namely, hepatocellular carcinoma (HepG2), human colorectal
carcinoma-116 (HCT-116) and breast cancer (Michigan Cancer
Foundation-7 (MCF-7)). The results prompted us to carry out further
examinations to reach a deep insight about the mechanism of action of
the synthesized compounds.
The two scaffolds were designed to comprise the main pharmaco-
phoric features of DNA intercalators; the planar polyaromatic system
(
chromophore) was represented by the quinazoline moiety and its fused
rings intercalating the base pairs of the DNA through hydrophobic
interaction. In addition, most of the designed compounds include basic
nitrogen atoms that may be protonated at physiological pH to form
cationic centers that increase the affinity and selectivity of these com-
pounds. Finally all the designed compounds contain mainly one and/or
two side chains exhibiting different antitumor activities. These side
chains bind to the minor groove and/or the major groove increasing
DNA binding affinities.
The most cytotoxic agents were further evaluated to assess their
binding affinities against DNA through DNA/methyl green assay.
Moreover, molecular docking was carried out to examine the binding
patterns with the prospective target, DNA active site (PDB-code: 4G0U).
2. Results and discussion
The choice of the different substituents was based on their relatively
high lipophilicity to pass the nuclear membranes aiming to have a strong
DNA intercalation. Moreover, the variability of substitutions enabled us
to study structure–activity relationship of the final compounds.
In general, the designed compounds were synthesized and evaluated
for their in vitro anti-proliferative activities against three human tumor
2.1. Chemistry
For synthesis of the target compounds, reactions sequence is illus-
trated in Schemes 1–3.
4

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Scheme 1. Synthetic route for preparation of the target compounds 1–7.
Scheme 2. Synthetic route for preparation of the target compounds 8–12.
5

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Scheme 3. Synthetic route for preparation of the target compounds 13–21.
2
.2. In vitro anti-proliferative activity
investigations of the cytotoxic activity indicated that HCT-116 was more
sensitive cell lines to the influence of the new derivatives. In particular,
compounds 16, 18, 11 and 5 were found to be the most potent deriva-
tive with IC50 = 3.61, 6.72, 7.16 and 5.18 µM respectively against
Anti-proliferative activities of the fifteen newly synthesized [1,2,4]
′
′
triazolo[4,3-c]quinazoline and bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]quina-
zoline derivatives 5, 6, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20 and
HepG2 cell line. Also, compounds 16, 18, 11 and 5 displayed IC50
=
2
1 were examined against two human tumor cell lines namely, hepa-
2.85, 3.82, 4.97 and 6.40 µM respectively against HCT-116 cell line.
tocellular carcinoma (HepG2) and colorectal carcinoma (HCT-116)
using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
These derivatives displayed higher activities than doxorubicin, (IC50
=
7.94 and 8.07 µM respectively) against the two cell lines.
3
6–38
(
MTT) colorimetric assay as described by Mosmann.
Doxorubicin
With respect to the HepG2 hepatocellular carcinoma cell line, com-
was included in the experiments as a reference cytotoxic drug. The re-
sults were expressed as growth inhibitory concentration (IC50) values
and summarized in Table 1. From the obtained results, it was explicated
that most of the prepared compounds displayed very good to lower
growth inhibitory activity against the tested cancer cell lines. In general,
pounds 6 and 20 displayed very strong anticancer activities with IC50
=
4.43 and 8.13 µM, respectively, while compound 13 with IC50 = 15.06
µM, displayed strong cytotoxicity. Additionally, compounds 19 and 10
with IC50 = 29.78 and 39.18 µM respectively, exhibited moderate
cytotoxicity. While compounds 21, 15 and 17 with IC50 79.45, 86.53
6

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Table 1
were obtained as IC50 values and summarized in Table 2. Doxorubicin,
as one of the most powerful DNA intercalators, was used as a positive
control.
In vitro cytotoxic activities of the newly synthesized compounds against HepG2,
HCT-116 and VERO cell lines.
a
Compound
IC50 (µM)
The tested compounds displayed very strong to moderate DNA-
binding affinities. Compounds 16 and 18 displayed very strong bind-
ing affinities. These compounds potently intercalate DNA at IC50 values
of 26.03 and 28.37 µM respectively which lower than IC50 of Doxoru-
bicin (IC50 = 31.27). This finding indicated that these derivatives
exhibited higher DNA binding activities than Doxorubicin. Also, com-
pounds 11 and 5 displayed very strong DNA binding at IC50 = 30.84 and
HepG2
HCT116
VERO
5
6
8
9
5.18 ± 0.3
4.43 ± 0.2
6.40 ± 0.4
9.28 ± 0.8
87.39 ± 5.1
85.07 ± 4.9
26.14 ± 1.7
4.97 ± 0.2
92.67 ± 5.5
17.14 ± 1.5
47.60 ± 3.6
2.85 ± 0.1
72.64 ± 4.5
3.82 ± 0.2
21.41 ± 1.6
7.34 ± 0.5
65.81 ± 4.3
8.07 ± 0.8
59.43 ± 0.16
47.24 ± 0.23
b
b
c
c
c
NA
NA
NT
NT
NT
1
1
1
1
1
1
1
1
1
2
2
0
1
2
3
5
6
7
8
9
0
1
39.18 ± 1.6
7.16 ± 0.6
55.15 ± 0.20
3
3.56 µM respectively, which was nearly equipotent to that of doxoru-
b
c
NA
NT
c
bicin. Moreover, compound 6 with IC50 = 37.12 µM displayed very
strong DNA binding which slightly lower than doxorubicin. Compounds
10 and 20 exhibited strong DNA-binding affinities with IC50 values of
15.06 ± 1.3
86.53 ± 4.6
3.61 ± 0.2
93.68 ± 5.2
6.72 ± 0.5
29.78 ± 1.8
8.13 ± 0.8
79.45 ± 4.4
7.94 ± 0.6
NT
c
NT
58.09 ± 0.21
c
NT
4
1.78 and 45.89 µM, respectively. Finally, compounds 13 and 19
55.78 ± 0.18
showed moderate DNA-binding affinities with IC50 values of 54.88 and
c
NT
6
4.63 µM respectively.
50.07 ± 0.23
c
NT
c
Doxorubicin
NT
2
.4. Structure activity relationship (SAR)
a
IC50 (µM): 1–10 (very strong); 11–20 (strong); 21–50 (moderate); 51–100
weak); > 100 (very weak).
(
a
The preliminary SAR study has focused on the effect of hydrophobic,
IC50 values are the mean ± S.D. of three separate experiments.
electronic nature, number and position of the substituents used in this
study. Also, it focused on the effect of number and length of linkers used
and the presence of one and/or two distal moieties. The data obtained
revealed that, the tested compounds displayed different levels of anti-
cancer activity and possessed a distinctive pattern of selectivity against
the HCT-116 cell lines. Generally, the [1,2,4]triazolo[4,3-c]quinazoline
b
NA: Compounds having IC50 value > 100 µM.
NT: Not Tested.
c
and 93.68 µM respectively exhibited weak activities. On the other hand,
compounds 8, 9 and 12 with IC50 > 100 µM displayed very weak
cytotoxicity.
′
′
and bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline scaffolds, ester, hy-
drazide and/or amide linkers containing (HBA-HBD), lipophilicity and
electronic nature exhibited an important role in DNA intercalations and
consequently anticancer activity. The presence of two hydrazine moi-
eties linked to the tricyclic chromophore [1,2,4]triazolo[4,3-c]quina-
zoline as in compounds 16 and 18 was found to be responsible for their
higher anticancer activity than the other compounds that containing
only one hydrazine moiety linked to the tetracyclic chromophore bis
Cytotoxicity evaluation against colorectal carcinoma (HCT-116) cell
line, discovered that compounds 20 and 6 exhibited very strong anti-
cancer activities with (IC50 = 7.34 and 9.28 µM, respectively), while
compound 13 with IC50 = 17.14 µM, displayed strong cytotoxicity.
Additionally, compounds 19, 10 and 15 with (IC50 = 21.41, 26.14 and
4
7.60 µM respectively, showed moderate cytotoxicity. While, com-
pounds 8, 9, 12, 17 and 21 with IC50 ranging from 65.81 to 92.67 µM,
exhibited weak activities.
′
′
([1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline as in compound 11 which
showed higher activities than only one hydrazine moiety attached to
tricyclic chromophore 5. The presence of two hydrazine arms enables 16
and 18 to form different hydrophilic bonding interactions and easily
sandwiched between DNA base pairs. Also, the long arms enable one of
them to be oriented into the minor groove and the other one oriented
into the major groove of DNA as shown in molecular docking studies.
From the structure of the synthesized derivatives and the data shown
in Table 1 we can divide these tested compounds into three groups. The
first group is compounds 5, 6, 11and 13. In this group SH substitutions
Finally, the most potent four derivatives 5, 6, 11, 16, 18 and 20 were
tested for their cytotoxicity against normal VERO cell lines. The results
revealed that the tested compounds have low toxicity against VERO
normal cells with IC50 values ranging from 47.24 to 59.43
μ
M. The
cytotoxicity of these compounds against the cancer cell lines was from
2
7
.85 to 9.28 M.
μ
Compounds 5, 6, 11, 16, 18 and 20 are respectively, 11.47, 10.66,
.70, 16.09, 8.30 and 6.16 fold times more toxic in hepatocellular car-
cinoma (HepG2) cell lines than in VERO normal cells.
Compounds 5, 6, 11, 16, 18 and 20 are respectively, 9.29, 5.09,
2
are present as in compounds 6 and 13 or only one short NHNH sub-
1
1.10, 20.38, 14.60 and 6.82 fold times more toxic in hepatocellular
stituent attached to the pharmacophore as in compounds 5 and 11. The
tetracyclic scaffold with only thiol group at position-7 (6) exhibited
higher activities than that attached to the tricyclic one at position-3 (13)
against both HepG2 and HCT116 cell lines respectively. The hydrazine
group attached to the tetracyclic scaffold at position-7 (11) exhibited
higher activities than that attached to the tricyclic one at position-5 (5)
against HCT116 cell lines. On the other hand, the order of activity was
reversed to be 5 > 11 against HepG2 cell lines.
carcinoma (HCT-116) cell lines than in VERO normal cells.
2
.3. In vitro DNA/Methyl green assay
The most active anti-proliferative derivatives 5, 6, 10, 11, 13, 16,
1
8, 19 and 20 were further evaluated for their DNA-binding affinity
which revealed the ability of these compounds to intercalate DNA. It was
carried out by use of methyl green dye according to the reported pro-
cedure described by Burre et al.39 The results of DNA-binding affinity
The second group is compounds 8, 9, 10 and 12 that have only one
long ester and/or amide arm attached to the tetracyclic scaffold at
position-7. Within this group, the ethyl 3-propanoate ester arm 10 dis-
played higher activities than ethyl 2-propanoate 9, ethyl acetate 8 and/
or acetamide one 12 respectively.
Table 2
DNA intercalating affinity of the tested compounds.
In the third group 15, 16, 17, 18, 19, 20 and 21 showed that; the
acetohydrazide derivative 16 exhibited higher activities than the 2-pro-
panehydrazide (18), 3-propanehydrazide (20), two ethyl 3-propaoate
ester arms at positions-3 and -6 (19), the two acetamide arms (21),
the two ethyl acetate arms (15) and ethyl 2-propaoate ester arms against
the two HCT116 and HepG2 cell lines respectively except compound 15
that showed higher activity than 21 against HepG2 cell lines.
Compound
DNA binding IC50 (µM)
Compound
DNA binding IC50 (µM)
5
6
33.56 ± 1.7
37.12 ± 1.9
41.78 ± 2.1
30.84 ± 1.6
54.88 ± 3.0
16
26.03 ± 1.3
28.37 ± 1.5
64.63 ± 3.8
45.89 ± 2.5
31.27 ± 1.8
18
1
1
1
0
1
3
19
20
Doxorubicin
7

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
The data obtained from DNA binding in Table 2 we can concluded
simulations, all the necessary grid maps were calculated prior to dock-
ing. Doxorubicin was used 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. The binding free
energies (ΔG) were presented in Table 4. All studied ligands have similar
position and orientation inside the DNA binding site.
that, the presence of one hydrazine group at position-3 and acetohy-
drazide group at position-6 linked to the tricyclic chromophore as in
compounds 16 exhibited higher DNA binding activity than that has one
hydrazine, 2-propanehydrazide 18, that has only hydrazine group at
position-7 of the tetracyclic scaffold 11, that has only hydrazine group at
position-5 of the tricyclic scaffold 5 respectively.
The most favorable pose was selected according to the minimum free
energy of the DNA–ligand complex for analyzing the interaction be-
tween the ligand and DNA.
2
.5. In silico ADMET profile
The proposed binding mode of doxorubicin revealed affinity value
of ꢀ 100.31 kcal/mol and ten H-bonds. The planar aromatic system
occupied the hydrophobic pocket formed by Arginine945, Aspara-
gine882, Lucine880, Glutamate870, Alanine869, Glycine868, Aspara-
Discovery studio 2.5 was used to predict ADMET (absorption, dis-
tribution, metabolism, excretion and toxicity) for the designed com-
pounds comparing with doxorubicin as a reference drug. Predicted
ADMET studies for the tested compounds were listed in Table 3. It was
found that BBB penetration of the designed compounds is ranging from
low to very low except three compounds (6, 9 and 10) are expected to be
medium. This indicates that, most compounds were expected to be safe
to CNS. Moreover, all of the designed compounds were predicted to be
absorbed better than doxorubicin which appeared away from our
compounds at Fig. 6 which augmented the parental use of doxorubicin
injections. The enhancement of absorption is expected to be due to the
polycyclic nucleus, triazoloquinazoline, which increases lipophilicity.
Finally, the closer the hepatotoxicity scores to one, the more probability
to be toxic, while the closer the hepatotoxicity scores to zero, the more
probability to be non-toxic. Some of the designed compounds are pre-
dicted to be safer than doxorubicin, and the others are predicted to be
nearly the same as doxorubicin. It is well known that numerous drug
candidates have failed during clinical tests because of problems related
to their absorption properties.40 Up on computational investigation of
the synthesized compound, it was found the most of them have good
absorption behavior. Moreover, the solubility level of the most com-
pounds is expected to be better than or even similar to that of reference
drug.
gine867,
Asparagine795,
Glutamine789,
Asparagine786,
Glutamine742, Lysine739, Phenyalanine738 and Glycine737. It formed
five H-bonds with the key amino acids in the active site (one H-bond
with Asparagine786, two H-bonds with Asparagine795, one H bonds
with Arginine945, one H-bond with Asparagine867). It also stacked
between the diphosphate Cytidine (Dc3, Dc4 and Dc8), diphosphate
Guanosine (Dg5 and Dg13) and Adinine monophosphate 6 (Da6). The
sugar moiety was oriented into minor groove of DNA and stabilized by
formation of five H-bonds with the key amino acids (two H-bonds with
Arginine945, one H-bonds with Leucine880 and two H-bonds with
Asparagine882) (Fig. 7).
The proposed binding mode of 16 exhibited affinity value of
ꢀ 110.55 kcal/mol and thirteen H-bonds. The planar aromatic system
occupied the hydrophobic pocket formed by Glutamate870, Alanine869,
Glycine868, Asparagine867, Phenylalanine834, Leucine799, Aspara-
gine798, Asparagine795, Glutamine789, Leucine787, Asparagine786,
Glutamine742, Lysine739, Phenylalanine738, Glycine737 and Pro-
line732. It also stacked between the diphosphate Cytidine (Dc3, Dc4,
Dc8 and Dc11) and diphosphate Guanosine (Dg2, Dg5 and Dg13). The
two hydrazine arms were oriented into minor and major grooves of DNA
and stabilized by formation of thirteen H-bonds with the key amino
acids (one H-bond with Lysine739, two H-bond with Glutamine742, five
H-bond with Asparagine795, two H-bond with Glutamate870, one H-
bond with Leucine787 and two H-bond with Asparagine786) (Fig. 8).
These interactions may explain its highest anticancer activity.
2
.6. Docking studies
All the synthesized compounds were evaluated for their DNA binding
affinity with the help of molecular docking studies using Molsoft soft-
ware. The 3D crystal structure of DNA was downloaded from the protein
databank using PDB ID of 4G0U.41 Water molecules were removed from
the downloaded DNA sequence. Polar hydrogens were added and the
protein active site was defined by placing a grid over the center of co-
The proposed binding mode of 18 exhibited affinity value of
ꢀ 106.28 kcal/mol and nine H-bonds. The planar aromatic system
occupied the hydrophobic pocket formed by Glutamate870, Alanine869,
Glycine868, Asparagine867, Phenylalanine834, Leucine799, Aspara-
gine798, Asparagine795, Glutamine789, Leucine787, Asparagine786,
crystallized ligand. Before
a protein was ready for docking
Table 3
Predicted ADMET for the designed compounds and doxorubicin.
a
b
c
d
e
f
Comp. No.
BBB level
Absorption level
Hepatotoxicity probability
CYP2D6
PPB
Solubility level
5
6
8
9
3
2
3
2
2
3
4
3
4
4
4
3
4
4
4
0
0
0
0
0
0
0
0
2
0
2
0
2
2
3
0.966
0.953
0.350
0.397
0.344
0.980
0.807
0.350
0.821
0.490
0.576
0.158
0.741
0.913
0.900
0
0
1
1
1
0
0
1
0
1
0
1
1
1
0
2
2
1
1
1
0
1
1
0
1
0
0
0
0
0
3
2
2
2
2
2
2
3
3
2
3
3
3
3
2
1
1
1
1
1
1
1
1
2
2
0
1
2
5
6
7
8
9
0
1
Doxorubicin
a
BBB level, blood brain barrier level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low.
Absorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.
b
c
d
e
f
Hepatotoxicity probability, value > 0.5 means toxic, value less than 0.5 means non-toxic.
CYP2D6, cytochrome P2D6, 0 = non inhibitor, 1 = inhibitor.
PBB, plasma protein binding, 0 means less than 90%, 1 means>90%, 2 means >95%.
solubility level, 0 = extremely low, 1 = very low, 2 = low, 3 = good, 4 = optimal.
8

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Fig. 6. The expected ADMET study of the designed compounds and the reference drugs.
formation of seven H-bonds with the key amino acids (one H-bond with
Lysine739, three H-bond with Glutamate870, two H-bond with Aspar-
agine867 and one H-bond with Leucine787) (Fig. 9). These interactions
may explain its high anticancer activity.
Table 4
The calculated free energy of binding (ΔG in Kcal/mole) for the ligands.
ΔG [kcal molꢀ
1
]
Compound
ΔG [kcal molꢀ 1
]
Compound
5
6
8
9
ꢀ 96.97
ꢀ 90.40
ꢀ 76.30
ꢀ 76.92
ꢀ 77.78
ꢀ 100.66
ꢀ 75.34
ꢀ 76.76
15
ꢀ 75.44
ꢀ 110.55
ꢀ 75.12
ꢀ 106.28
ꢀ 76.11
ꢀ 77.08
ꢀ 90.06
ꢀ 100.31
The proposed binding mode of 11 exhibited affinity value of
16
ꢀ
100.66 kcal/mol and six H-bonds. The planar aromatic system occu-
17
pied the hydrophobic pocket formed by Glutamate870, Alanine869,
Glycine868, Asparagine867, Phenylalanine834, Leucine799, Aspara-
gine798, Asparagine795, Glutamine789, Leucine787, Asparagine786,
Glutamine742, Lysine739, Phenylalanine738, Glycine737 and Pro-
line732. It formed one H-bond with Glutamate870. It also stacked be-
tween the diphosphate Cytidine (Dc3, Dc4, Dc8 and Dc11) and
diphosphate Guanosine (Dg2, Dg5 and Dg13). The hydrazine arm was
stabilized by formation of five H-bonds with the key amino acids (one H-
bond with Glutamine742, one H-bond with Lysine739 and three H-bond
with Glycine737) (Fig. 10). These interactions may explain its high
anticancer activity.
18
1
1
1
1
0
1
2
3
19
20
21
Doxorubicin
Glutamine742, Lysine739, Phenylalanine738, Glycine737 and Pro-
line732. It formed two H-bonds with Glutamine742. It also stacked be-
tween the diphosphate Cytidine (Dc3, Dc4, Dc8 and Dc11) and
diphosphate Guanosine (Dg2, Dg5 and Dg13). The two hydrazine arms
were oriented into minor and major grooves of DNA and stabilized by
Fig. 7. Binding of doxorubicin with DNA, the hydrogen bonds are represented in blue dashed lines.
9

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Fig. 8. Predicted binding mode for 16 with DNA.
Fig. 9. Predicted binding mode for 18 with DNA.
Moreover, the proposed binding mode of 5 exhibited affinity value of
96.97 kcal/mol and eight H-bonds with the key amino acids (one H-
bond with Glutamate870, two H-bonds with Asparagine795, three H-
bonds with Asparagine786, one H-bond with Glutamine742 and one H-
bond with Lysine739) (Fig. 11).
ꢀ
On the other hand, compound 21 with long tow amide arms
1
0

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
Fig. 10. Predicted binding mode for 11 with DNA.
displayed affinity value of ꢀ 90.06 kcal/mol and seven H-bonds with the
key amino acids (three H-bonds with Arginine945, one H-bond with
Serine794, one H-bond with Glutamine789 and two H-bonds with
Asparagine786). The two long amide arms were oriented into minor and
major grooves of DNA (Fig. 12).
As planned, our synthesized compounds were reported here to act
Fig. 11. Predicted binding mode for 5 with DNA.
1
1

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
through one of the three DNA intercalation mechanisms according to the
structure and substituents types. Classical intercalators, that have no
bulky groups, bind to DNA duplexes with essentially all of their aromatic
system inserted between base pairs from the top and bottom of the
intercalation site as in compounds 5, 6 and 11. Minor groove binding
intercalators that have bulky substituents that oriented in the minor
groove of DNA as in compounds 8–12. The presence of two groove
binding bulky side chains as in case of compounds 15–21 which contains
two moieties at both ends of their chromophores, lead to a special type
of interaction with DNA, called threading intercalation in which one arm
is oriented to the minor groove and the other to the major groove.
18, 19 and 20 were further evaluated for their DNA-binding affinity
which revealed the ability of these compounds to intercalate DNA. The
tested compounds displayed very strong to moderate DNA-binding af-
finities. Compounds 16 and 18 displayed very strong binding affinities.
These compounds potently intercalate DNA at decreased IC50 values of
26.03 ± 1.3 and 28.37 ± 1.5 µM respectively which lower than the
reference drug Doxorubicin (IC50 = 31.27 ± 1.8). This finding indicated
that these derivatives exhibited higher DNA binding activities than that
of Doxorubicin. Also, compounds 11 and 5 displayed very strong DNA
binding at IC50 = 30.84 ± 1.6 and 33.56 ± 1.7 µM respectively, which
nearly equipotent to that of that of doxorubicin. Moreover, compound 6
with IC50 = 37.12 ± 1.9 µM displayed very strong DNA binding which
slightly lower than doxorubicin. Compounds 10 and 20 exhibited strong
DNA-binding affinities with IC50 values of 41.78 ± 2.1 and 45.89 ± 2.5
µM, respectively. Finally, compounds 13 and 19 showed moderate DNA-
binding affinities with IC50 values of 54.88 ± 3.0 and 64.63 ± 3.8 µM
respectively.
3
. Conclusion
In summary, novel fifteen [1,2,4]triazolo[4,3-c]quinazoline and bis
′ ′
(
[1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazoline derivatives have been
designed, synthesized and evaluated against HepG2 and HCT-116. The
molecular design was performed to investigate the binding mode of the
proposed compounds with DNA active site. The data obtained from
biological testing highly correlated with that obtained from molecular
modeling studies.
Predicted ADMET studies for the tested compounds were calculated.
It was found that BBB penetration of the designed compounds is ranging
from low to very low except three compounds (6, 9 and 10) are expected
to be medium. This indicates that, most compounds were expected to be
safe to CNS. Moreover, all of the designed compounds were predicted to
be absorbed better than doxorubicin. Finally, the closer the hepatotox-
icity scores to one, the more probability to be toxic, while the closer the
hepatotoxicity scores to zero and the more probability to be non-toxic.
Some of the designed compounds are predicted to be safer than doxo-
rubicin, and the others are predicted to be nearly the same as doxoru-
bicin. Moreover, the solubility level of the most compounds is expected
to be better than or even similar to that of reference drug. Also, mo-
lecular modeling studies concluded that, our synthesized compounds
can act through one of the three DNA intercalation mechanisms ac-
cording to the structure and substituents types. Classical intercalators,
that have no bulky groups, bind to DNA duplexes with essentially all of
their aromatic system inserted between base pairs from the top and
bottom of the intercalation site as in compounds 5, 6 and 11. Minor
groove binding intercalators that have bulky substituents that oriented
in the minor groove of DNA as in compounds 8–12. The presence of two
In general, investigations of the cytotoxic activity indicated that
HCT-116 was more sensitive cell lines to the influence of the new de-
rivatives. In particular, compounds 16, 18, 11 and 5 were found to be
the most potent derivative overall the tested compounds against the two
HepG2 and HCT116 cancer cell lines with IC50 = 3.61 ± 0.2, 2.85 ± 0.1
µM, 6.72 ± 0.5, 3.82 ± 0.2 µM, 7.16 ± 0.6, 4.97 ± 0.2 µM and 5.18 ±
0
.3, 6.40 ± 0.4 µM respectively. These derivatives displayed higher
activities than doxorubicin, (IC50 = 7.94 ± 0.6 and 8.07 ± 0.8 µM
respectively) against both two cell lines. With respect to the HepG2
hepatocellular carcinoma cell line, compounds 6 and 20 displayed very
strong anticancer activities with (IC50 = 4.43 ± 0.2 and 8.13 ± 0.8 µM,
respectively). With respect to the HCT-116 colorectal carcinoma cell
lines, discovered that compounds 20 and 6 exhibited very strong anti-
cancer activities with (IC50 = 7.34 ± 0.5 and 9.28 ± 0.8 µM,
respectively).
The most active anti-proliferative derivatives 5, 6, 10, 11, 13, 16,
Fig. 12. Predicted binding mode for 21 with DNA.
1
2

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
groove binding bulky side chains as in case of compounds 15–21 which
contains two moieties at both ends of their chromophores, lead to a
special type of interaction with DNA, called threading intercalation in
which one arm is oriented to the minor groove and the other to the major
groove.
afforded compound 6.
Off white powder (yield 85%); m.p. > 300 C; IR (KBr, cm ); 3095
◦
1
–
6
(CH aromatic), 2587 (SH), 1616 (C N).; H NMR (DMSO‑d ) δ ppm:
1
–
7.71–7.74 (dd, J = 7.6 Hz, 1H, H-11 of quinazoline), 7.88–7.92 (dd, J =
8 Hz, 1H, H-10 of quinazoline), 8.37–8.39 (d, J = 8 Hz, 1H, H-12 of
quinazoline), 8.65–8.67 (d, J = 7.6 Hz, 1H, H-9 of quinazoline), 10.46 (s,
4
. Experimental
6 6
1H, CH triazole), 14.54 (s, 1H, SH).; Anal. Calcd. for C10H N S (242.26):
C, 49.58; H, 2.50; N, 34.69; Found: C, 50.01; H, 2.51; N, 34.67%.
4
4
.1. Chemistry
4
.1.1.3. General procedure for synthesis of potassium bis([1,2,4]triazolo)
′
′
′
′
.1.1. General
[4,3-a:4 ,3 -c]quinazoline-7-thiolate 7. Bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]
quinazoline-7-thiol 6 (3 g, 0.02 mol) was heated with alcoholic solution
of potassium hydroxide (0.8 g, 0.02 mol), the reaction mixture was
stirred for 30 min, the solid obtained was filtered, washed with absolute
ethanol and air dried.
All melting points were carried out by open capillary method on a
Gallen kamp Melting point apparatus at faculty of pharmacy Al-Azhar
University and were uncorrected. The infrared spectra were recorded
on pye Unicam SP 1000 IR spectrophotometer at Pharmaceutical
analytical Unit, Faculty of Pharmacy, Al-Azhar University using potas-
sium bromide disc technique. Proton magnetic resonance 1HNMR
spectra were recorded on a Bruker 400 Megahertz-nuclear magnetic
resonance (400 MHZ-NMR) spectrophotometer at Microanalytical Unit,
Faculty of pharmacy, Cairo University. Carbon-13 (C13) nuclear mag-
netic resonance (13CNMR) spectra were recorded on a Bruker 100
Megahertz-nuclear magnetic resonance (100 MHZ-NMR) spectropho-
tometer at Microanalytical Unit, Faculty of pharmacy, Cairo University.
Tetramethylsilane (TMS) was used as internal standard and chemical
shifts were measured in δ scale one part per million (ppm). The mass
spectra were carried out on Direct Probe Controller Inlet part to Single
Quadropole mass analyzer in Thermo Scientific Gas chromatogra-
phy–mass spectrometry (GCMS) model ISQ LT using Thermo X-Calibur
software at the Mycology and Biotechnology Regional Center, Al-Azhar
University. Elemental analyses (C, H, N) were performed on a carbon
hydrogen and nitrogen (CHN) analyzer at Mycology and Biotechnology
Regional Center, Al-Azhar University. All compounds were within ±0.4
of the theoretical values. The reactions were monitored by thin-layer
chromatography (TLC) using TLC sheets precoated with UV fluores-
cent silica gel Merck 60 F254 plates and were visualized using ultravi-
olet (UV) lamp and different solvents as mobile phases.
◦
+
Yellow powder (yield 85%); m.p. > 300 C); MS (m/z): 280 (M ,
16.83%), 260 (34.10%), 208 (18.31%), 146 (23.93%), 95 (67.18%), 69
(66.20%), 43 (100% base beak); Anal. Calcd. for C10H KN S (280.35): C,
5
6
42.84; H, 1.80; N, 29.98; Found: C, 42.94; H, 1.85; N, 30.05%.
4
.1.1.4. General procedure for synthesis of ethyl 2-{bis([1,2,4]triazolo)
′ ′
[
4,3-a:4 ,3 -c]quinazolin-7-yl]thio} acetate 8. Equimoler quantities of the
potassium salt 7 (2.8 g, 0.01 mol) and ethyl chloroacetate (0.01 mol) in
DMF (20 ml) were heated on water-bath for 3 hrs. The reaction mixture
was poured onto ice-water (200 ml) and stirred for 30 min. The obtained
solid was filtered and crystallized from aqueous ethanol.
◦
1
White powder (yield 69%); m.p.225–227 C; IR (KBr, cm ); 3081 (CH
–
–
–
–
1
aromatic), 2978 (CH aliphatic), 1712 (C
O), 1636 (C
N).; H NMR
(
DMSO‑d
S), 4.21–4.25 (q, 2H, CH
of quinazoline), 7.57–7.59 (d, J = 8.4 Hz, 1H, H-12 of quinazoline),
.65–7.69 (dd, J = 7.6, 8.2 Hz, 1H, H-10 of quinazoline), 8.08–8.10 (d, J
7.6 Hz, 1H, H-9 of quinazoline), 8.49 (s, 1H, CH triazole).; Anal. Calcd.
for C14 S (328.35): C, 51.21; H, 3.68; N, 25.60; Found: C, 51.25;
H, 3.70; N, 25.56%.
6
) δ ppm: 1.12–1.15 (t, J = 7.2 Hz, 3H, CH
3 2
), 4.11 (s,2H, CH -
2
-CH
3
), 7.29–7.32 (dd, J = 7.2, 7.6 Hz, 1H, H-11
7
=
12 6 2
H N O
Compounds, quinazoline-2,4-dione 1, 2,4-dichloro quinazoline 2, 2-
chloro-4-hydrazinylquinazoline 3 and 5-chloro-[1,2,4]triazolo[4,3-c]
4
.1.1.5. General procedure for synthesis of ethyl 2-{bis([1,2,4]triazolo)
′
′
[
4,3-a:4 ,3 -c]quinazolin-7-ylthio}propanoate 9. A mixture of potassium
salt of compound 7 (2.8 g, 0.01 mol) and ethyl 2-chloropropanoate
0.01 mol) in DMF (20 ml) was heated on water-bath for 3hrs. The re-
quinazoline
procedures.2
4
were obtained according to the reported
5,26,42,43
(
action mixture was poured onto ice-water (200 ml) and stirred for 30
min. The obtained solid was filtered and crystallized from aqueous
4
.1.1.1. 5-Hydrazinyl-[1,2,4]triazolo[4,3-c]quinazoline 5. A mixture of
-chloro-[1,2,4]triazolo[4,3-c]quinazoline 4 (0.409 g, 0.002 mol) and
5
ethanol.
Brown powder (yield, 62%); m.p. = 219–221 ◦C; IR (KBr, cm 1):
ꢀ
hydrazine hydrate (0.128 g, 0.004 mol) was heated under reflux in
isopropyl alcohol (IPA) (20 ml) for 1 h. After completion, the obtained
crude solid was filtered and washed with IPA to give yellow solid of the
desired compound 5.
–
–
–
–
3
066 (CH aromatic), 2944 (CH aliphatic), 1725 (C
O), 1646 (C
–CH
),4.01–4.06 (q, J = 7.2 Hz, 2H,
), 4.47–4.52 (q, J = 6.8 Hz, 1H, S-CH), 7.76–7.80 (dd, J = 7.6 Hz,
N).;
1
H NMR (DMSO‑d
6
) δ ppm: 1.07–1.11 (t, J = 7.2 Hz, 3H,CH
2
3
),
1
.59–1.61 (d, J = 6.8 Hz, 3H, CH-CH
3
◦
ꢀ 1
Yellow powder, Yield, 85%; m.p. 227–9 C; IR
ν
max (cm ): 3302
–
–
CH
2
(
NH2), 3257 (NH), 3051 (CH aromatic), 1637 (C
N); 1HNMR (400
1
H, H-11 of quinazoline), 7.97–8.01 (dd, J = 7.6 Hz, 1H, H-10 of qui-
MHz, DMSO‑d
6
): 4.68 (s, 2H, NH
2
), 7.38–7.42 (dd, J = 6.8 Hz, 1H, H-9
nazoline), 8.46–8.48 (d, J = 7.6 Hz, 1H, H-12 of quinazoline), 8.71–8.72
1
3
of quinazoline), 7.66–7.67 (d, J = 7.6 Hz, 1H, H-10 of quinazoline),
(
d,1H, H-9 of quinazoline), 8.89 (s, 1H, CH triazole).; C NMR
DMSO‑d , 100 MHz) δ (ppm):14.0 (CH , CH -CH ), 18.1 (CH , CH-
), 46.0 (CH, CH-CH ), 62.0 (CH , CH -CH ), 114.1 (C, C-12a), 116.9
7
7
.69–7.73 (dd, J = 6.8, 8 Hz, 1H, H-8 of quinazoline), 8.20–8.22 (d, J =
(
6
3
2
3
3
.6 Hz, 1H, H-7 of quinazoline), 8.59 (s, 1H, CH triazole), 9.30 (s, 1H,
CH
3
3
2
2
3
+
NH); MS (m/z):200 (M , 15.40%), 167 (31.69%), 145 (59.89%), 97
(
CH, C-12), 126.0 (CH, C-11), 128.2 (CH, C-9), 131.9 (CH, C-10), 133.2
CH, C-3), 143.5 (C, C-7), 144.8 (C, C-8a), 149.2 (C, C-12b), 154.9 (C, C-
(
69.99%), 67 (52.69%), 54 (100% base beak); Anal. Calcd. for C
9 8 6
H N
(
(
200.21): C, 53.99; H, 4.03; N, 41.98; Found: C, 54.08; H, 4.01; N,
–
4
a), 171.1 (C, C O).; Anal. Calcd for C15H N O S (342.38): C, 52.62;
–
14 6 2
4
1.92%.
H, 4.12; N, 24.55; Found: C, 52.69; H, 4.15%.
4
.1.1.2. General procedure for synthesis of bis([1,2,4]triazolo)[4,3-
4
.1.1.6. General procedure for synthesis of ethyl 3-{bis([1,2,4]triazolo)
′
′
a:4 ,3 -c]quinazoline-7-thiol 6. A mixture of compound 5 (0.5 g, 0.0024
mol), carbon disulfide (1.52 g, 1.42 ml, 0.02 mol) and potassium hy-
droxide (1.12 g, 0.02 mol) was refluxed in absolute ethanol (20 ml) for 5
h. The mixture was then allowed to reach the room temperature and
poured onto 20 ml 1 N HCl. The precipitated product was filtered,
washed with distilled water and dried. Crystallization from ethanol 80%
′
′
[
4,3-a:4 ,3 -c]quinazolin-7-yl]thio}propanoate 10. Equimoler quantities
of the potassium salt 7 (2.8 g, 0.01 mol) and ethyl 3-chloropropanoate
0.01 mol) in DMF (20 ml) were heated on water-bath for 3hrs. The
(
reaction mixture was poured onto ice-water (200 ml) and stirred for 30
min. The obtained solid was filtered and crystallized from aqueous
ethanol.
1
3

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
◦
Yellowish brown powder (yield 67%); m.p. 205–207 C; IR (KBr,
obtained was filtered, washed with absolute ethanol and air dried.
1
–
–
◦
+
cm ); 3098 (CH aromatic), 2964 (CH aliphatic), 1718 (C
O), 1656
), 2.88–2.92
), 4.06–4.10 (q,2H, CH -CH ),
.71–7.74 (dd, J = 7.6 Hz, 1H, H-11 of quinazoline), 7.88–7.92 (dd, J =
Hz, 1H, H-10 of quinazoline), 8.37–8.39 (d, J = 7.6 Hz, 1H, H-12 of
Yellow powder (yield 95%); m.p. > 300 C); MS (m/z): 294 (M ,
11.83%), 287 (44.84%), 219 (31.98%), 186 (39.64%), 156 (79.06%),
101 (59.98%), 82 (57.09%), 41 (100% base beak); Anal. Calcd. for
–
1
(
C
–
N).; H NMR (DMSO‑d
6
) δ ppm: 1.15–1.19 (t,3H, CH
3
(
t,2H, CH
2
-CO), 3.54–3.58 (t,2H, S-CH
2
2
3
7
8
9 4 2 4
C H K N OS (294.41): C, 36.72; H, 1.37; N, 19.03; Found: C, 36.81; H,
1.33; N, 19.09%.
quinazoline), 8.66–8.68 (d, J = 8 Hz, 1H, H-9 of quinazoline), 10.46 (s,
1
4
H, CH Triazole).; Anal. Calcd. for C15
H
14
N
6
O
2
S (342.38): C 52.62; H,
4.1.1.11. General procedure for synthesis of ethyl 2-{[6-(2-ethoxy-2-
oxoethyl)-5-oxo-5,6-dihydro-[1,2,4]triazolo[4,3-c] quinazolin-3-yl]thio}
acetate 15. A mixture of potassium 5-oxo-3-sulfido-5H-[1,2,4]triazolo
[4,3-c] quinazolin-6-ide 14 (2.95 g, 0.01 mol) and ethyl chloroacetate
(0.02 mol) in DMF (20 ml) was heated on water-bath for 3hrs. The re-
action mixture was poured onto ice-water (200 ml) and stirred for 30
min. The obtained solid was filtered and crystallized from aqueous
ethanol.
.12; N, 24.55; Found: C, 52.69; H, 4.15; N, 24.57%.
4
.1.1.7. General procedure for synthesis of 7-Hydrazinylbis([1,2,4]tri-
′
′
azolo)[4,3-a:4 ,3 -c]quinazoline 11. To a solution of ester derivatives 8,
and 10 (0.005 mol) in ethanol, hydrazine hydrate (0.01 mol) was
9
added. The reaction mixture was then heated under reflux for 2 h,
concentrated then cooled to precipitate the desired compound 11. The
formed precipitate was filtered and recrystallized from ethanol.
◦
–
–
1
Brown powder (yield 68%); m.p. 90–92 C; IR (KBr, cm ); 3075 (CH
◦
1
O).; 1H NMR (DMSO‑d
White powder (yield 64%); m.p. > 300 C; IR (KBr, cm ); 3327
2), 3167 (NH), 3051 (CH aromatic), 1637 (C–
aromatic), 2979 (CH aliphatic), 1734 (C
ppm: 1.20–1.25 (m, J = 7.2 Hz, 6H, 2CH ), 4.15–4.20 (q, J = 7.2 Hz, 4H,
2CH -CH ), 4.23 (s, 2H, S-CH ), 4.30 (s, 2H, N-CH
), 7.69–7.73 (dd, J =
6
) δ
1
(
NH
–
N).; H NMR
), 7.37–7.41 (dd, J = 6.8 Hz, 1H, H-
1 of quinazoline), 7.66–7.68 (d, J = 7.6 Hz, 1H, H-12 of quinazoline),
.68–7.72 (dd, J = 6.8, 8 Hz, 1H, H-10 of quinazoline), 8.19–8.21 (d, J
7.6 Hz, 1H, H-9 of quinazoline), 8.58 (s, 1H, CH triazole), 9.29 (s, 1H,
NH).; Anal. Calcd. for C10 (240.23): C, 50.00; H, 3.36; N, 46.65;
Found: C, 50.09; H, 3.35; N, 46.57%.
3
(
DMSO‑d
6
) δ ppm: 4.67 (s, 2H, NH
2
2
3
2
2
1
7
7.2, 8 Hz, 1H, H-9 of quinazoline), 7.81–7.83 (d, J = 8 Hz, 1H, H-10 of
quinazoline), 7.87–7.91 (dd, J = 7.2, 8 Hz, 1H, H-8 of quinazoline),
8.28–8.30 (d, J = 8 Hz, 1H, H-7 of quinazoline).; Anal. Calcd. for
=
H
N
8 8
17 18 4 5
C H N O S (390.41): C, 52.30; H, 4.65; N, 14.35; Found: C, 52.36; H,
4.66; N, 14.37%.
4
.1.1.8. General procedure for synthesis of N-(4-Acetylphenyl)-2-{[bis
4.1.1.12. General procedure for synthesis of 2-(3-Hydrazinyl-5-oxo-
[1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)acetohydrazide 16. A mixture
of ester 15 (3.91 g, 0.01 mol) and hydrazine hydrate (10 ml, 85%) in
′
′
(
[1,2,4]triazolo)[4,3-a:4 ,3 -c]quinazolin-7-yl]thio}acetamide
12. A
′
′
mixture of the potassium salt of bis([1,2,4]triazolo)[4,3-a:4 ,3 -c]qui-
nazoline-7-thiol 7 (2.8 g, 0.01 mol) and N-(4-acetylphenyl)-2-chlor-
oacetamide (2.11 g, 0.01 mol) in dry DMF (30 ml) in presence of
catalytic amount of KI was heated under refluxing using water bath for 5
h. After cooling to room temperature, the reaction mixture was poured
onto ice-water (200 ml) and stirred for 1 h. The obtained solid was
filtered and crystallized from aqueous ethanol.
◦
ethanol (20 ml) was stirred well and heated at 100 C for 6 h. The re-
action mixture was cooled, and the crude product was collected by
filtration, washed with water and then crystallized from ethanol.
◦
1
white powder (yield 71%); m.p. 202–204 C; IR (KBr, cm ); 3370
2
–
–
1
(NH
(DMSO‑d
exchangeable), 4.62 (s,2H, NH
), 3291 (NH), 3075 (CH aromatic), 1629 (C
δ ppm: 3.91 (s, 2H, CH ), 4.42 (s,2H, NH
), (D
O exchangeable), 7.37–7.41 (dd, J
O).; H NMR
6
)
2
2
), (D O
2
◦
1
Yellow powder (yield 75%); m.p. 240–242 C; IR (KBr, cm ); 3178
2
2
–
–
1
(
NH), 3051 (CH aromatic), 1651 (C
N).; H NMR (DMSO‑d
6
) δ ppm:
), 7.39–7.40 (d,1H, H-11 of
= 7.2, 7.6 Hz, 1H, H-9 of quinazoline), 7.64–7.66 (d, J = 8 Hz, 1H, H-10
of quinazoline), 7.69–7.73 (dd, J = 7.2, 7.6 Hz, 1H, H-8 of quinazoline),
8.13–8.15 (d, J = 7.6 Hz, 1H, H-7 of quinazoline), 9.42 (s, 2H, 2NH),
2
.51 (s,3H, CH ), 4.42 (s,2H, S-CH
3
2
quinazoline),7.59–7.69 (m,3H, H-10 of quinazoline and H-2, H-6
phenyl), 7.86–7.91 (m,3H, H-12 of quinazoline and H-3, H-5 phenyl),
8
1
2
(D
CH
(CH, C-8), 145.2 (C, C-6a), 152.3(C, C
2
O exchangeable).;13C NMR (DMSO‑d
6
2
, 100 MHz) δ (ppm):33.4 (CH ,
.21–8.22 (d,1H, H-9 of quinazoline), 8.61 (s, 1H, CH triazole), 10.84 (s,
2
), 112.8 (C, C-10a), 123.8 (CH, C-7), 125.6 (2CH, (C-9, C-10)), 132.9
–
H, NH).; Anal. Calcd. for C20
H
15
7
N O
2
S (417.45): C, 57.55; H, 3.62; N,
–
O of quinazoline), 164.0 (2C, (C-
O amidic).;Anal. Calcd. for C11
288.27): C, 45.83; H, 4.20; N, 38.87; Found: C, 45.91; H, 4.22; N,
–
3.49; Found: C, 57.51; H, 3.65; N, 23.47%.
10b, C-3)), 167.4 (C, C
–
12 8 2
H N O
(
4
.1.1.9. General procedure for synthesis of 3-mercapto-[1,2,4]triazolo
38.85%.
[
4,3-c]quinazolin-5(6H)-one 13. A mixture of compound 3 (1.95 g, 0.01
mol), carbon disulfide (2.28 g, 1.81 ml, 0.03 mol) and potassium hy-
droxide (1.12 g, 0.02 mol) was refluxed in absolute ethanol (20 ml) for 4
h. The mixture was then allowed to reach the room temperature and
poured onto 20 ml 1 N HCl. The yellow precipitated product was
filtered, washed with distilled water and dried. Crystallization from
ethanol 80% afforded compound 13.
4.1.1.13. General procedure for synthesis of ethyl 2-{[6-(1-ethoxy-1-oxo-
propan-2-yl)-5-oxo-5,6-dihydro-[1,2,4]triazolo[4,3-c]quinazolin-3-yl]
thio}propanoate 17. A mixture of potassium 5-oxo-3-sulfido-5H-[1,2,4]
triazolo[4,3-c]quinazolin-6-ide 14 (2.95 g, 0.01 mol) and ethyl 2-chlor-
opropionate (0.02 mol) in DMF (20 ml) was heated on water-bath for
3hrs. The reaction mixture was poured onto ice-water (200 ml) and
stirred for 30 min. The obtained solid was filtered and crystallized from
aqueous ethanol.
◦
1
Yellow powder (yield 86%); m.p. > 300 C; IR (KBr, cm ); 3429
–
1
(
NH), 3011 (CH aromatic), 2569 (SH), 1628 (C
DMSO‑d
) δ ppm: 7.47–7.49 (d, 1H, H-9 of quinazoline), 7.59–7.60 (d,
H, H-10 of quinazoline), 7.75–7.76 (dd, J = 6.8 Hz, 1H, H-8 of qui-
nazoline), 8.12–8.14 (d, J = 7.2 Hz, 1H, H-7 of quinazoline), 10.56 (s,
H, NH), (D O exchangeable), 14.03 (s, 1H, SH), (D O exchangeable);
Anal. Calcd. for C OS (218.23): C, 49.53; H, 2.77; N, 25.67; Found:
C, 50.01; H, 2.71; N, 25.69%.
–
N).; H NMR
◦
1
(
6
Brown powder (yield 64%); m.p. 68–70 C; IR (KBr, cm );): 3072 (CH
–
3
O).; 1H NMR (DMSO‑d
), 1.63–1.69 (q, J = 7.2 Hz,
), 4.55–4.57 (q, J =
1
aromatic), 2976 (CH aliphatic), 1735 (C
ppm: 1.17–1.21 (t, J = 6.8 Hz, 6H, 2CH -CH
6H, 2CH-CH
), 4.16–4.18 (m, J = 6.4 Hz, 4H, 2CH
–
6
) δ
2
1
2
2
3
2
9
H
N
6 4
7.2 Hz, 1H, S-CH), 4.73–4.75 (q J = 7.2 Hz, 1H, N-CH), 7.68–7.71 (dd, J
= 6.8, 7.2 Hz, 1H, H-9 of quinazoline), 7.80–7.82 (d, J = 8 Hz, 1H, H-10
of quinazoline), 7.86–7.90 (dd, J = 6.8 Hz, 1H, H-8 of quinazoline),
8.27–8.29 (d, J = 7.2 Hz, 1H, H-7 of quinazoline).; Anal. Calcd. for
4
.1.1.10. General procedure for synthesis of potassium 5-oxo-3-sulfido-5H-
[
1,2,4]triazolo[4,3-c]quinazolin-6-ide
14. 3-Mercapto-[1,2,4]triazolo
19 22 4 5
C H N O S (418.47): C, 54.53; H, 5.30; N, 13.39; Found: C, 54.59; H,
[
4,3-c]quinazolin-5(6H)-one 13 (2.19 g, 0.01 mol) was treated with
5.32; N, 13.41%.
alcoholic solution of potassium hydroxide (1.12 g, 0.02 mol), the reac-
tion mixture was heated while continuous stirring for 30 min, the solid
1
4

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
).;Anal. Calcd. for C29H N O S (568.61): C, 61.26; H, 4.25;
3 24 6 5
4
.1.1.14. General procedure for synthesis of 2-(3-Hydrazinyl-5-oxo-
2C=O-CH
[
1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)propanehydrazide 18. A solu-
N, 14.78; Found: C, 61.30; H, 4.28; N, 14.82%.
tion of compound 17 (4.19 g, 0.01 mol) in absolute ethanol (50 ml) was
treated with hydrazine hydrate (95%, 20 ml). The reaction mixture was
4.2. In vitro anti-proliferative activity
◦
stirred well and heated to 100 C for 6 h, then cooled. The crude product
was collected by filtration, washed with water and then crystallized
The cytotoxicity assays were performed at Pharmacology & Toxi-
cology Department, Faculty of pharmacy, Al-Azhar University, Cairo,
Egypt. Cancer cells from different cancer cell lines hepatocellular car-
cinoma (HepG2), colorectal carcinoma (HCT-116) and breast cancer
(MCF-7), were purchased from American type Cell Culture collection
(ATCC, Manassas, USA) and grown on the appropriate growth medium
Roswell Park Memorial Institute medium (RPMI 1640) supplemented
with 100 mg/mL of streptomycin, 100 units/mL of penicillin and 10% of
from ethanol to afford compound 18.
◦
1
White powder (yield 71%); m.p. 206–208 C; IR (KBr, cm );): 3391
), 3291 (NH), 3057 (CH aromatic), 1630 (C–
1
(
NH
DMSO‑d
.2 Hz, 1H, CH), 4.50 (s,2H,CO-NH-NH
2
–
O).; H NMR
), 4.41–4.47 (q, J =
), 4.66 (s,2H, NH
), 7.37–7.41
(
6
) δ ppm: 1.55–1.56 (d, J = 7.2 Hz, 3H, CH
3
7
2
2
(
dd, J = 6.8 Hz, 7.6 Hz, 1H, H-9 of quinazoline), 7.64–7.66 (d, J = 8.4
Hz, 1H, H-10 of quinazoline), 7.70–7.74 (dd, J = 8, 8.4 Hz, 1H, H-8 of
quinazoline), 8.14–8.16 (d, J = 7.6 Hz, 1H, H-7 of quinazoline), 9.45 (s,
heat-inactivated fetal bovine serum in a humidified, 5% (v/v) CO at-
2
◦
2
3
H, 2NH).; Anal. Calcd. for C12
H
14
N
8
O
2
(302.30): C, 47.68; H, 4.67; N,
mosphere at 37 C Cytotoxicity assay by 3-[4,5-dimethylthiazole-2-yl]-
7.07; Found: C, 47.65; H, 4.69; N, 37.10%.
2,5-diphenyltetrazolium bromide (MTT).
Exponentially growing cells from different cancer cell lines were
trypsinized, counted and seeded at the appropriate densities
(2000–1000 cells/0.33 cm2 well) into 96-well microtiter plates. Cells
then were incubated in a humidified atmosphere at 3 7˚ C for 24 h. Then,
cells were exposed to different concentrations of compounds (0.1, 10,
100 and 1000 µM) for 72 h. Then the viability of treated cells was
determined using MTT technique as follow. Cells were incubated with
4
.1.1.15. General procedure for synthesis of ethyl 3-{[6-(3-ethoxy-3-oxo-
propyl)-5-oxo-5,6-dihydro-[1,2,4]triazolo[4,3-c]quinazolin-3-yl]thio}
propanoate 19. A mixture of potassium 5-oxo-3-sulfido-5H-[1,2,4]tri-
azolo[4,3-c] quinazolin-6-ide 14 (2.95 g, 0.01 mol) and ethyl 3-chloro-
propionate (0.02 mol) in DMF (20 ml) was heated on water-bath for
3
hrs. The reaction mixture was poured onto ice-water (200 ml) and
stirred for 6 h. The obtained solid was filtered and crystallized from
aqueous ethanol.
200 l of 5% MTT solution/well (Sigma Aldrich, MO) and were allowed
μ
to metabolize the dye into colored-insoluble formazan crystals for 2 h.
The remaining MTT solution were discarded from the wells and the
formazan crystals were dissolved in 200 µl/well acidified isopropanol
for 30 min, covered with aluminum foil and with continuous shaking
using a MaxQ 2000 plate shaker (Thermo Fisher Scientific Inc, MI) at
room temperature. The colorimetric assay was measured and recorded
at absorbance of 570 nm using a Stat FaxR 4200 plate reader (Awareness
Technology, Inc., FL). The cell viability were expressed as percentage of
control and the concentration that induces 50% of maximum inhibition
of cell proliferation (IC50) were determined using Graph Pad Prism
◦
1
Yellowish brown powder(yield 61%); m.p. 63–65 C; IR (KBr, cm );
–
3
047 (CH aromatic), 2935 (CH aliphatic), 1719 (C
–
O); MS (m/z): 418
+
(
M , 13.17%), 360 (98.78%), 286 (87.48%), 195 (67.75%), 143
(
91.86%), 70 (100% base beak), 42 (95.74%).; Anal. Calcd. for
19 22 4 5
C H N O S (418.47): C, 54.53; H, 5.30; N, 13.39; Found: C, 54.49; H,
5
.33; N, 13.44%.
4
.1.1.16. General procedure for synthesis of 3-(3-Hydrazinyl-5-oxo-
[
1,2,4]triazolo[4,3-c]quinazolin-6(5H)-yl)propanehydrazide
20. A
version 5 software (Graph Pad software Inc, CA).3
.3. In vitro DNA/Methyl green assay
Nine compounds exhibited significant anti-proliferative activities (5,
6–38
mixture of ester 19 (3.03 g, 0.01 mol) and hydrazine hydrate (10 ml,
◦
8
5%) in ethanol (20 ml) was stirred well and heated at 100 C for 6 h.
4
The reaction mixture was cooled, and the crude product was collected by
filtration, washed with water and then crystallized from ethanol.
◦
1
White powder (yield 71%); m.p. 212–214 C; IR (KBr, cm ); 3361
), 3250 (NH), 3061 (CH aromatic), 1640 (C–
O); MS (m/z): 302
M , 13.27%), 265 (30.85%), 216 (48.96%), 143 (47.00%), 75
6, 10, 11, 13, 16, 18, 19 and 20) were further evaluated to determine
their DNA-binding affinities. Doxorubicin as DNA intercalator was used
as positive control. In this test, methyl green dye can bind DNA to form
colored reversible complex of DNA/methyl green. These complex still
stable at neutral pH. Upon addition of intercalating agents, the methyl
(
(
(
NH
2
–
+
14 8 2
49.71%), 55 (100% base beak).; Anal. Calcd. for C12H N O (302.30):
C, 47.68; H, 4.67; N, 37.07; Found: C, 47.61; H, 4.70; N, 37.14%.
2
green is displaced from DNA with addition of H O molecule to the dye
4
.1.1.17. General procedure for synthesis of N-(4-Acetylphenyl)-2-{[6-(2-
[4-acetylphenyl]amino}-2-oxoethyl)-5-oxo-5,6-dihydro-[1,2,4]triazolo
4,3-c]quinazolin-3-yl]sulfanyl}acetamide 21. A mixture of potassium 5-
oxo-3-sulfido-5H-[1,2,4]triazolo[4,3-c] quinazolin-6-ide 14 (2.95 g,
.01 mol) and N-(4-acetylphenyl)-2-chloroacetamide (4.22 g, 0.02 mol)
resulting in formation of the colorless carbinol, leading to a dramatic
decrease in spectrophotometric absorbance.39 ΔA value (the difference
between DNA/methyl green complex and free cabinol) provides the
simplest means for detecting the DNA-binding affinity and relative
binding strength. IC50 values were determined using the GraphPadPrism
{
[
0
in dry DMF (30 ml) in presence of catalytic amount of KI was heated
under reflux using water bath for 6 h. After cooling to room temperature,
the reaction mixture was poured onto ice-water (200 ml) and stirred for
5
.0 software. The reaction was performed as follows.
A mixture of Calf thymus DNA (10 mg) and methyl green (20 mg)
(
Sigma– Aldrich) in 100 ml of 0.05 M Tris-HCl buffer (pH 7.5) con-
3
0 min. The obtained solid was filtered and crystallized from aqueous
ethanol.
Yellowish brown powder (yield 75%); m.p. 174–176 ◦C; IR (KBr,
◦
taining 7.5 mM MgSO
4
. Then, the mixture was stirred for 24 h at 37 C.
The test samples were dissolved in ethanol and dispensed into wells of a
96-well microtiter tray at concentration of 10,100 and 1000 µM. From
each well, the excess solvent was removed under vacuum followed by
addition of 200 µl of the DNA/methyl green solution. The test samples
were incubated in a dark at ambient temperature. After 24 h, the
absorbance of each sample was determined at 642.5–645 nm. Readings
were corrected for initial absorbance and normalized as the percentage
of the untreated DNA/methyl green absorbance value.
1
cm ); 3293 (NH), 3103 (CH aromatic), 2928 (CH aliphatic), 1669
–
2
1
(
C
–
O).; H NMR (DMSO‑d
), 4.44 (s,2H, N-CH
), 7.69–7.93 (m,11H, Ar-H), 8.27–8.28 (d, J = 6
Hz, 1H, Ar-H), 10.80 (s, 1H, NH), 10.84 (s, 1H, NH).; C NMR
DMSO‑d , 100 MHz) δ (ppm):26.8 (2CH , 2CH ), 36.0 (CH , S-CH ),
, N-CH ), 115.1 (C, C-10a), 118.9 (4CH, 2C (2(C-2, C-6))),
23.8 (CH, C-7), 127.2 (CH, C-9), 128.0 (CH, C-10), 129.9 (4CH, 2C
(2C-
O of qui-
6
3
) δ ppm: 2.52 (s,6H, 2CH ), 4.40 (s,2H, S-
CH
2
1
3
(
6
3
3
2
2
3
1
6.8 (CH
2
2
6 4
H
6 4
H
(
6 4
2(C-3, C-5))), 132.4 (CH, C-8), 133.2 (C, C-6a), 143.2 (2C, 2C H
4
)), 143.5 (CH, C-3), 147.9 (2C, 2C
6
H
4
(2C-1)), 151.2 (C, C–
–
4.4. In silico ADMET analysis
nazoline), 164.9 (C, C-10b), 166.7 (2C, 2C=O amidic), 196.9 (2C,
Discovery studio 2.5 was used to predict ADMET (absorption,
1
5

K. El-Adl et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115958
distribution, metabolism, excretion and toxicity) for the designed com-
pounds comparing with doxorubicin as a reference drug.
16 Palchaudhuri R, Hergenrother P. DNA as a target for anticancer compounds: Methods
to determine the mode of binding and the mechanism of action. Curr Opin Biotechnol.
2
007;18:497–503.
1
7 Takenaka S, Takagi M. Threading intercalators as a new DNA structural probe. Bull
Chem Soc Jpn. 1999;72:327–337.
4
.5. Docking studies
19 Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem. 1989;58:
3
51–375.
2
0 Chilin A, Marzaro G, Marzano C, et al. Synthesis and antitumor activity of novel
amsacrine analogs: the critical role of the acridine moiety in determining their
biological activity. Bioorg Med Chem. 2009;17:523–529.
In the present work, all the target compounds were subjected to
docking study to explore their binding mode towards DNA. All modeling
experiments were performed using molsoft program, which provides a
unique set of tools for the modeling of protein/ligand interactions. It
predicts how small flexible molecule such as substrates or drug candi-
dates bind to a protein of known 3D structure represented by grid
interaction potentials (http://www.molsoft.com/icm_pro.html). Each
experiment used the biological target DNA downloaded from the Broo-
khaven Protein Databank (http://www.rcsb.org/pdb/explore/explore.
do?structureId=4G0U). In order to qualify the docking results in terms
of accuracy of the predicted binding conformations in comparison with
the experimental procedure, the reported DNA inhibitor drug doxoru-
bicin was used as reference ligand.
21 Shenkenberg TD, Von Hoff DD. Mitoxantrone: a new anticancer drug with significant
clinical activity. Ann Intern Med. 1986;105:67–81.
2
2
2 Liaw YC, Gao YG, Robinson H, Van der Marel GA, Van Boom JH, Wang AH.
Antitumor drug nogalamycin binds DNA in both grooves simultaneously: molecular
structure of nogalamycin-DNA complex. Biochemistry. 1989;28:9913–9918.
3 Marzaro G, Dalla Via L, Toninello A, Guiotto A, Chilin A. Benzoquinazoline
derivatives as new agents affecting DNA processing. Bioorg Med Chem. 2011;19:
1
197–1204.
24 Skibo EB, Huang X, Martinez R, Lemus RH, Craigo WA, Dorr RT.
Pyrimidoquinazoline-based antitumor agents. Design of topoisomerase II to DNA
cross-linkers with activity against protein kinases. J Med Chem. 2002;45:5543–5555.
2
2
2
2
2
5 Ma Z, Hano Y, Nomura T, Chen Y. Novel quinazoline–quinoline alkaloids with
cytotoxic and DNA topoisomerase II inhibitory activities. Bioorg Med Chem Lett. 2004;
1
4:1193–1196.
6 Park HJ, Kim Y-S, Kim JS, et al. 6-Arylamino-7-chloro-quinazoline-5, 8-diones as
novel cytotoxic and DNA topoisomerase inhibitory agents. Bioorg Med Chem Lett.
2
004;14:3385–3388.
Declaration of Competing Interest
7 Oyallon B, Brachet-Botineau M, Log ´e C, et al. Structure-based design of novel
quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors. Eur J Med Chem.
2
018;154:101–109.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
8 Garofalo A, Goossens L, Baldeyrou B, et al. Design, synthesis, and DNA-binding of N-
alkyl(anilino)quinazoline derivatives. J Med Chem. 2010;53(22):8089–8103. https://
doi.org/10.1021/jm1009605.
9 El-Adl K, Sakr H, Nasser M, Shoman FMA. 5-(4-Methoxybenzylidene)thiazolidine-
2
,4-dione-derived VEGFR-2 inhibitors: Design, synthesis, molecular docking, and
Appendix A. Supplementary material
anticancer evaluations. Arch Pharm. 2020. https://doi.org/10.1002/
ardp.202000079.
3
3
0 El-Adl K, El-Helby A-G-A, Sakr H, El-Hddad SSA. Design, synthesis, molecular
docking, and anticancer evaluations of 1-benzylquinazoline-2,4(1H,3H)-dione
bearing different moieties as VEGFR-2 inhibitors. Arch Pharm. 2020:e2000068.
https://doi.org/10.1002/ardp.202000068.
1 El-Adl K, El-Helby A-G-A, Sakr H, Eissa IH, El-Hddad SSA, Shoman FMIA. Design,
synthesis, molecular docking and anticancer evaluations of 5-benzylidenethiazoli-
dine-2,4-dione derivatives targeting VEGFR-2 enzyme. Bioorg Chem. 2020.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115958.
References
1
El-Helby AGA, et al. Design, Synthesis, Molecular Docking, and Anticancer Activity
of Phthalazine Derivatives as VEGFR-2 Inhibitors. Arch Pharm. 2017;350(12):
32 El-Helby AGA, et al. Benzoxazole/benzothiazole-derived VEGFR-2 inhibitors: Design,
synthesis, molecular docking, and anticancer evaluations. Arch Pharm. 2019;352
(12):1900178.
1
700240.
2
3
4
5
Graves DE, Velea LM. Intercalative binding of small molecules to nucleic acids. Curr
Org Chem. 2000;4:915–929.
3
3 El-Helby AGA, et al. Design, synthesis, molecular docking, and anticancer activity of
benzoxazole derivatives as VEGFR-2 inhibitors. Arch Pharm. 2019;352(10):1900113.
4 El-Helby A-G-A, et al. Design, synthesis, in vitro anti-cancer activity, ADMET profile
and molecular docking of novel Triazolo [3, 4-a] phthalazine derivatives targeting
VEGFR-2 Enzyme. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current
Medicinal Chemistry-Anti-Cancer Agents). 2018;18(8):1184–1196.
Gago F. Stacking interactions and intercalative DNA binding. Methods. 1998;14:
3
2
77–292.
Avendano C, Men ´e ndez J. DNA Intercalators and topoisomerase inhibitors. In:
Medicinal Chemistry of Anticancer Drugs. 2nd ed. Elsevier; 2008:199–228.
Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular
advances and pharmacologic developments in antitumor activity and cardiotoxicity.
Pharmacol Rev. 2004;56:185–229.
35 Bailey SA, Graves DE, Rill R. Binding of actinomycin D to the T (G) nT motif of
double-stranded DNA: determination of the guanine requirement in nonclassical,
non-GpC binding sites. Biochemistry. 1994;33:11493–11500.
6
7
Gallego J, Ortiz AR, de Pascual-Teresa B, Gago F. Structure–affinity relationships for
the binding of actinomycin D to DNA. J Comput Aided Mol Des. 1997;11:114–128.
Soni A, Khurana P, Singh T, Jayaram B. A DNA intercalation methodology for an
efficient prediction of ligand binding pose and energetics. Bioinformatics. 2017;33
36 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1–2):55–63.
37 Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium/
formazan assay for cell growth and drug sensitivity in culture using human and other
tumor cell lines. Cancer Res. 1988;48(17):4827–4833.
(
10):1488–1496. https://doi.org/10.1093/bioinformatics/btx006.
8
9
Bra n˜ a MF, Cacho M, Gradillas A, de Pascual-Teresa B, Ramos A. Intercalators as
anticancer drugs. Curr Pharm Des. 2001;7(17):1745–1780. https://doi.org/10.2174/
3
8 Freimoser FM, Jakob CA, Aebi M, Tuor U. The MTT [3-(4,5-Dimethylthiazol-2-yl)-
2,5-Diphenyltetrazolium Bromide] Assay Is a Fast and Reliable Method for
Colorimetric Determination of Fungal Cell Densities. Appl Environ Microbiol. 1999;65
1
381612013397113.
Graves DE, Velea LM. Intercalating binding of small molecules to nucleic acids. Curr
Org Chem. 2000;4:915–929.
(
8):3727–3729.
39 Burres NS, Frigo A, Rasmussen RR, McAlpine JB. A colorimetric microassay for the
detection of agents that interact with DNA. J Nat Prod. 1992;55:1582–1587.
40 Holt PA, Chaires JB, Trent JO. Molecular docking of intercalators and groove-binders
to nucleic acids using Autodock and Surflex. J Chem Inf Model. 2008;48:1602–1615.
41 Wu CC, Li TK, Farh L, et al. Structural basis of type II topoisomerase inhibition by the
anticancer drug etoposide. Science. 2011;333(6041):459–462. https://doi.org/
1
1
1
1
1
0 Goftar MK, Kor NM, Kor ZM. DNA intercalators and using them as anticancer drugs.
Int J Adv Biol Biom Res. 2014;2(3):811–822.
1 Strekowski L, Wilson B. Noncovalent Interactions With DNA: An Overview. Mutat
Res. 2007;623(1–2):3–13.
2 Paul A, Bhattacharya S. Chemistry and biology of DNA-binding small molecules. Curr
Sci. 2012;102:212–231.
1
0.1126/science.1204117.
3 Kumar S, Pandya P, Pandav K, Gupta S, Chopra A. Structural studies on ligand—DNA
system. A rubst approach in drug design. J Biosci. 2012;37:553–561.
4 Tanious FA, Yen SF, Wilson WD. Kinetic and equilibrium analysis of a threading
intercalation mode: DNA sequence and ion effects. Biochemistry. 1991;30:
42 Lipunova G, Nosova E, Mokrushina G, Sidorova L, Charushin V. Antitumor activity of
the fluorinated derivatives of condensed quinolines and quinazolines. Pharm Chem J.
2000;34:19–22.
43 Skibo EB, Huang X, Martinez R, Lemus RH, Craigo WA, Dorr RT.
Pyrimidoquinazoline-based antitumor agents. Design of topoisomerase II to DNA
cross-linkers with activity against protein kinases. J Med Chem. 2002;45:5543–5555.
1
813–1819.
5 Liu H-K, Sadler PJ. Metal complexes as DNA intercalators. Acc Chem Res. 2011;44:
49–359.
1
3
1
6