Design and discovery of new 1,2,4-triazolo[4,3-c]quinazolines as potential DNA intercalators and topoisomerase II inhibitors

Article abstract of DOI:10.1002/ardp.202000237

A new series of 1,2,4-triazolo[4,3-c]quinazoline derivatives was designed and synthesized as Topo II inhibitors and DNA intercalators. The cytotoxic effect of the new members was evaluated in vitro against a group of cancer cell lines including HCT-116, HepG-2, and MCF-7. Compounds 14_c, 14_d, 14_e, 14_e, 15_b, 18_b, 18_c, and 19_b exhibited the highest activities with IC₅₀ values ranging from 5.22 to 24.24 μM. Furthermore, Topo II inhibitory activities and DNA intercalating affinities of the most promising candidates were evaluated as a possible mechanism for the antiproliferative effect. The results of the Topo II inhibition and DNA binding tests were coherent with that of in vitro cytotoxicity. Additionally, the most promising compound 18_c was analyzed in HepG-2 cells for its apoptotic effect and cell cycle arrest. It was found that 18_c can induce apoptosis and arrest the cell cycle at the G2–M phase. Finally, molecular docking studies were carried out for the designed compounds against the crystal structure of the DNA?Topo II complex as a potential target to explore their binding modes. On the basis of these studies, it was hypothesized that the DNA binding and/or Topo II inhibition would participate in the noted cytotoxicity of the synthesized compounds.

Full text of DOI:10.1002/ardp.202000237

Received: 15 July 2020
Revised: 27 October 2020
Accepted: 31 October 2020
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DOI: 10.1002/ardp.202000237
F U L L P A P E R
Design and discoveryofnew 1,2,4‐triazolo[4,3‐c]quinazolines
as potential DNA intercalators and topoisomerase II
inhibitors
Mohamed S. Alesawy1
Mohamed Alswah3 | Ahmed Belal4
| Ahmed A. Al‐Karmalawy2
| Eslam B. Elkaeed3
| Mohammed S. Taghour1 | Ibrahim H. Eissa1
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1Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University, Cairo,
Egypt
Abstract
A new series of 1,2,4‐triazolo[4,3‐c]quinazoline derivatives was designed and
synthesized as Topo II inhibitors and DNA intercalators. The cytotoxic effect of the
new members was evaluated in vitro against a group of cancer cell lines including
HCT‐116, HepG‐2, and MCF‐7. Compounds 14c, 14d, 14e, 14e, 15b, 18b, 18c, and 19b
exhibited the highest activities with IC50 values ranging from 5.22 to 24.24 µM.
Furthermore, Topo II inhibitory activities and DNA intercalating affinities of the
most promising candidates were evaluated as a possible mechanism for the anti-
proliferative effect. The results of the Topo II inhibition and DNA binding tests were
coherent with that of in vitro cytotoxicity. Additionally, the most promising com-
pound 18c was analyzed in HepG‐2 cells for its apoptotic effect and cell cycle arrest.
It was found that 18c can induce apoptosis and arrest the cell cycle at the G2–M
phase. Finally, molecular docking studies were carried out for the designed com-
pounds against the crystal structure of the DNA−Topo II complex as a potential
target to explore their binding modes. On the basis of these studies, it was
hypothesized that the DNA binding and/or Topo II inhibition would participate in
the noted cytotoxicity of the synthesized compounds.
2Department of Pharmaceutical Medicinal
Chemistry, Faculty of Pharmacy, Horus
University‐Egypt, New Damietta, Egypt
3Department of Pharmaceutical Organic
Chemistry, Faculty of Pharmacy (Boys),
Al‐Azhar University, Cairo, Egypt
4Zoology Department, Faculty of Science,
Al‐Azhar University, Cairo, Egypt
Correspondence
Ibrahim H. Eissa and Mohamed S. Alesawy,
Pharmaceutical Medicinal Chemistry and
Drug Design Department, Faculty of
Pharmacy (Boys), Al‐Azhar University, Cairo
11884, Egypt.
Email: Ibrahimeissa@azhar.edu.eg (I. H. E.)
and mohammedalesawy@azhar.edu.eg (M. S. A.)
K E Y W O R D S
anticancer, DNA intercalator, molecular docking, 1,2,4‐triazolo[4,3‐c]quinazoline,
topoisomerase II
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1
INTRODUCTION
these drugs demonstrate their antitumor activity by binding to DNA
and/or enzymes essential to normal DNA functions, causing a cellular
Cancer is a serious public health issue in all countries[1] and it marks
its beginning when cells begin to grow without control in a part of
the body.[2] According to WHO, cancer is the second leading cause of
death worldwide, which accounted for an approximated 9.6 million
deaths in 2018. Around one in six deaths worldwide are due to
cancer.[3] Millions of people are still alive with cancer.[4] There are
currently over 100 types of cancer, needing a particular diagnosis
and treatment.[3] Compounds that interfere with DNA play a sig-
nificant role in possible approaches to antitumor activity.[5] Many of
response that ultimately results in cell death.[6]
DNA intercalators are substances that bind with the helix of DNA
via an intercalation process that causes cell cycle disruption and cell
death.[7] The process of intercalation can be identified as the process by
which molecules comprising planar aromatic or heteroaromatic ring
systems, known as chromophores, are inserted into opposing DNA helix
base pairs.[8] In addition, most intercalating agents are either positively
charged or contain basic groups that under physiological conditions can
be protonated.[6] DNA intercalators in clinical use include acridine
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© 2020 Deutsche Pharmazeutische Gesellschaft
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derivatives (e.g., amsacrine 1),[8] anthracyclines (e.g., doxorubicin 2, mi-
toxantrone 3, and nogalamycin 5),[9,10] and ellipticine 4a.[11]
can be protonated at physiological pH.[7] The third feature is a
groove‐binding side chain moiety, which occupies the minor groove
of DNA (Figure 1).[28–30]
Topoisomerase II (Topo II) is a primary regulator of DNA re-
plication.[12] Topo II modifies the topology of DNA, monitors the su-
Quinazoline is a substantial scaffold and one of the most pro-
mising classes of heterocycles that is well‐tolerated in humans and
possesses anticancer activity.[31–34] It is also the basis of many
bioactive compounds that show potential activities as DNA inter-
calators and Topo inhibitors.[35,36] It has been documented that
quinazoline analogs such as EBE‐A22 6 possess a DNA intercalating
activity.[37] It has also been confirmed that several new moieties as
triazole,[38] benzylidene,[39] and thiosemicarbazide[40] have antitumor
activities.
percoiling of DNA, and controls
a variety of critical nuclear
mechanisms, such as transcription, replication, and repair.[13] It is
possible to classify compounds targeting Topo II into two groups. The
first is Topo II poisons, which increase the rate of cleavable complexes
of Topo II−DNA, resulting in a lesion in DNA strands.[14] Topo II poi-
sons include many clinically active compounds including doxorubicin
2[9] and mitoxantrone 3.[10] The second class inhibits the catalytic
activity of Topo II without increasing the rates of the cleavable com-
plexes of Topo II. The second class is known as catalytic inhibitors such
as etoposide.[15] Targeting DNA and blocking Topo II are, therefore,
effective strategies to find new anticancer agents.[16–26]
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1.1
Rationale of molecular design
There are three common essential pharmacophoric features of
DNA intercalators and Topo II poisons. The first is a polyaromatic
planar structure (chromophore) sandwiched between base pairs of
DNA.[27] The second feature is a cationic species, interacting with the
negatively charged phosphate group of DNA. The cationic center
may be an amino or nitrogen‐containing heterocyclic group, which
In continuation of our previous efforts of design and synthesis of new
anticancer agents,[41–48] especially DNA intercalators and Topo II
inhibitors,[14,49–51] ligand‐based drug design approach was con-
sidered. Accordingly, molecular hybridization of quinazoline and
other effective antitumor moieties was performed to obtain more
FIGURE 1 Some reported DNA intercalators and Topo II inhibitors exhibiting the essential pharmacophoric features

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promising anticancer agents. The new compounds were designed to
have the essential pharmacophoric features of DNA intercalators
and Topo II inhibitors.
yielded the corresponding hydrazones 18a−d, respectively. The 1
H
NMR spectra of compounds 18a−d revealed the presence of D2O‐
exchangeable singlet signals of hydrazinyl NH at a range of
δ 9.80–11.53 ppm and singlet signals for the idene moiety at a
range of δ 8.34–8.71 ppm.
The rationale of our molecular design depended on the gen-
eration of the planar aromatic system, 1,2,4‐triazolo[4,3‐c]quinazo-
line (chromophore). Different groove‐binding side chains were
incorporated into the planar system at position‐5. The side chains
may be aliphatic or aromatic amines to generate the basic center
(Supporting Information Data).
Finally, target compounds 19a−d were synthesized by the reac-
tion of 13 with appropriate isothiocyanates, namely, ethyl iso-
thiocyanate, propyl isothiocyanate, butyl isothiocyanate, and phenyl
isothiocyanate, in absolute ethanol. The IR spectra of compounds
19a−d showed sharp absorption bands of three NH groups of
thiosemicarbazide moieties in the range of 3,173−3,205 cm−1
.
The 1H NMR spectrum of compound 19b, as a representative ex-
ample, showed three exchangeable singlet broad signals at around
δ 8.24, 9.55, and 10.21 ppm, corresponding to the three NH groups.
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The procedures of the synthesis of the target compounds are illu-
strated in Scheme 1. First, commercially available 2‐aminobenzoic
acid 7 was condensed with urea 8 at 200°C for 6 h to get
quinazoline‐2,4‐dione 9 as white crystals.[52] Compound 9 was fur-
ther treated with phosphorus oxychloride in the presence of trie-
thylamine for 7 h to provide 2,4‐dichloroquinazoline 10.[53] 2‐Chloro‐
4‐hydrazinylquinazoline 11 was obtained by adding hydrazine
hydrate dropwise to the alcoholic solution of 2,4‐dichloro quinazo-
line 10.[54] Subsequently, compound 11 reacted with triethyl ortho-
formate at 100°C for 1 h to afford 5‐chloro‐[1,2,4]triazolo[4,3‐c]
quinazoline 12.[55] Compound 12 was heated with hydrazine hydrate
to afford 5‐hydrazinyl‐[1,2,4]triazolo[4,3‐c]quinazoline 13. The in-
frared (IR) spectrum of compound 13 demonstrated stretching bands
at 3,302 and 3,257 cm−1, corresponding to NH2 and NH groups, re-
spectively. Moreover, the 1H NMR (nuclear magnetic resonance)
spectrum of this compound showed exchangeable signals at δ 4.68
and 9.30 ppm, corresponding to NH2 and NH groups, respectively.
Reflux of 12 with commercially available alkyl amines, name-
ly ethylamine, n‐propylamine, n‐pentylamine, cyclopentylamine, and
diethylamine, in isopropyl alcohol (IPA) afforded the target com-
pounds 14a−e, respectively. The IR spectra of 14a−d demonstrated
stretching bands at a range of 3,212−3,279 cm−1, corresponding to
NH groups. Moreover, 1H NMR spectrum of these compounds
showed exchangeable singlet signals at a range of δ 7.82−8.09 ppm,
corresponding to NH groups. In addition, reflux of 12 with com-
mercially available aromatic amines, namely 4‐methylaniline,
4‐aminobenzoic acid, and ethyl p‐aminobenzoate, in IPA afforded
the title compounds 15a−c, respectively. The IR spectra of 15a−c
showed stretching bands at a range of 3137−3189 cm−1, corre-
sponding to the NHs. Moreover, the 1H NMR spectra of these
compounds demonstrated exchangeable singlet signals at a range of
δ 10.40–10.44 ppm, corresponding to NH groups. The reaction of
compound 12 with benzylamine and morpholine in IPA produced
compounds 16 and 17, respectively.
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2.2
Biological testing
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2.2.1
In vitro antiproliferative activities
The synthesized compounds were evaluated for their cytotoxic activ-
ities against a group of cancer cell lines: colorectal carcinoma (HCT‐
116), hepatocellular carcinoma (HepG‐2), and breast cancer (MCF‐7).
The standard 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐
2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTT) method was used in this
test,[56–58] utilizing doxorubicin as a positive control. The IC50 values are
summarized in Table 1.
The results demonstrated that some of the tested compounds
showed promising antiproliferative activities against the tested cells.
In general, compounds 14c, 14d, 14e, 15b, 18b, 18c, and 19b exhibited
good antiproliferative activities.
In particular, compound 18c was the most cytotoxic member and
exhibited superior antiproliferative activities against HCT‐116 and
HepG‐2 with IC50 values of 5.22 and 4.43 µM, respectively, com-
pared with doxorubicin (IC50 = 9.63 and 8.28 µM, respectively). Also,
compound 14d showed a higher activity than doxorubicin against
HCT‐116 (IC50 = 9.39 µM). In addition, compound 14e exhibited a
higher activity than doxorubicin against HepG‐2 with an IC50 value
of 6.92 µM.
Compounds 13, 14d, 14e, 15b, 18b, 18c, and 19b showed strong
antiproliferative activities against all tested cells with IC50 values
ranging from 4.43 to 20.53 µM.
Compounds 14c and 19c showed strong activities against only
HCT‐116 and HepG‐2 cells with IC50 values ranging from 9.79 to
14.52 µM, whereas compounds 18a and 19a showed strong activities
against only HepG‐2 cells with IC50 values of 13.16 and 16.04 µM,
respectively.
Moreover, compounds 14a, 14b, 15a, 15c, 16, 17, and 19d showed
moderate antiproliferative activities against HCT‐116 and HepG‐2
with IC50 values ranging from 20.41 to 38.78 µM, whereas com-
pounds 13, 14c, 18a, 19a, and 19c showed moderate activities against
MCF‐7 with IC50 values of 22.72, 24.24, 27.28, 29.65, and 23.27 µM,
respectively.
Condensation of compound 13 with certain aromatic alde-
hydes, namely, 4‐methylbenzaldhehyde, 4‐nitrobenzaldhehyde,
4‐hydroxybenzaldhehyde, and 2,6‐dichlorobenzaldhehyde, in
absolute ethanol with a catalytic amount of glacial acetic acid

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SCHEME 1 The general procedure for preparation of the target compounds

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TABLE 1 In vitro antiproliferative
activities against HCT‐116, HepG‐2, and
MCF‐7 cell lines, Topo II inhibitory activity,
and DNA intercalating affinity of the
synthesized compounds
Antiproliferative activities, IC50 (µM)ab
Topo II
inhibition,
IC50 (µM)a
DNA binding,
IC50 (µM)a
Compound
13
HCT‐116
HepG‐2
MCF‐7
14.23 1.1
38.78 2.2
28.02 1.9
12.49 0.4
9.39 0.4
18.03 1.3
29.87 2.1
30.88 2.3
9.79 0.5
9.00 0.4
6.92 0.3
29.45 2.1
11.66 1.1
31.40 2.4
20.41 1.8
28.04 1.9
13.16 0.4
12.63 0.5
4.43 0.1
49.01 3.2
16.04 0.9
12.44 0.5
12.93 0.3
36.97 2.1
8.28 0.5
22.72 1.6
NA
NT
NT
14a
NT
NT
14b
NA
NT
NT
14c
24.24 1.7
13.50 0.6
17.23 1.1
48.12 3.2
19.81 1.2
NA
1.93 0.1
1.05 0.1
1.78 0.1
NT
59.13 3.1
54.52 2.7
74.18 5.2
NT
14d
14e
18.56 1.2
27.34 1.7
17.32 0.9
37.46 2.0
33.23 1.9
33.96 1.7
20.73 1.1
12.84 0.7
5.22 0.3
15a
15b
3.51 0.2
NT
72.57 4.8
NT
15c
16
NA
NT
NT
17
48.33 2.8
27.28 1.5
15.42 1.2
12.22 1.0
NA
NT
NT
18a
2.78 0.2
2.39 0.2
0.86 0.1
NT
66.39 3.6
67.35 4.1
48.53 3.2
NT
18b
18c
18d
43.17 2.9
21.82 0.7
18.11 1.1
14.52 0.5
34.25 2.3
9.63 0.7
19a
29.65 1.7
20.53 1.4
23.27 1.8
43.47 3.1
7.67 0.4
4.67 0.3
3.45 0.3
NT
82.04 6.5
79.63 5.7
NT
19b
19c
19d
NT
NT
Doxorubicin
0.94 0.1
57.53 3.3
Abbreviations: NA, compounds having IC50 value > 50 µM; NT, not tested.
aIC50 values are the mean standard deviation of three separate experiments.
b0−10 µM: very strong; 10−20 µM: strong; 20−40 µM: moderate; 40−50 µM: weak.
Additionally, compounds 15a, 17, and 19d showed weak activities
The results revealed that some of the tested compounds potently
against MCF‐7 with IC50 values ranging from 43.47 to 48.33 µM,
whereas compound 18d exhibited weak activities against HCT‐116 and
HepG‐2 with IC50 values of 43.17 and 49.01 µM, respectively. Finally,
compounds 14a, 14b, 15c, 16, and 18d appeared to be inactive against
MCF‐7.
inhibit Topo II activity, such as compounds 14d and 18c with IC50 values
of 1.05 and 0.86 µM, respectively. These two compounds were 0.9 and
1.1 times as active as doxorubicin (IC50 = 0.94 µM). Additionally, some
compounds exhibited moderate Topo II inhibitory activities such as
compounds 14c (IC50 = 1.93 µM), 14e (IC50 = 1.78 µM), 18a (IC50 = 2.78
µM), and 18b (IC50 = 2.39 µM). On the contrary, compounds 15b, 19a,
and 19b showed a weak Topo II inhibitory activity.
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2.2.2
Topoisomerase II inhibitory activity
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DNA intercalation test
As mentioned in the rationale of molecular design, we aimed to ex-
amine the inhibitory activity of the synthesized compounds against
Topo II as a proposed mechanism of cytotoxic activity, such that the
most cytotoxic compounds (14c, 14d, 14e, 15b, 18a, 18b, 18c, 19a, and
19b) were further evaluated as Topo II inhibitors, based on the method
described by Patra et al.[59] In this test, doxorubicin was utilized as a
positive control. The IC50 values of the synthesized compounds against
Topo II were determined from the concentration–inhibition response
curve and summarized in Table 1.
2.2.3
The compounds that exhibited higher cytotoxic activities were further
evaluated for their DNA binding affinities. These compounds include
14c, 14d, 14e, 15b, 18a, 18b, 18c, 19a, and 19b. The DNA–methyl green
assay was applied as described by Burre et al.[60] Doxorubicin, as an
ideal DNA intercalator, was used as a positive control. The DNA
binding affinity values were reported as IC50 values and summarized in
Table 1.

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The results revealed that the two quadrant images describ-
ing damaged, necrotic/apoptotic, normal, and early apoptotic cells were
detected in quadrants Q1, Q2, Q3, and Q4, respectively, during the flow
cytometry analysis.[26] To calculate the apoptosis ratio, the values of
both quadrants Q2 and Q4 were added. As shown in Figure 2, com-
pound 18c has an apoptosis ratio of 21.71%, which is 12 times more
than the control, 0.82%. These findings revealed that compound 18c can
induce antiproliferative effect via induction of apoptosis.
TABLE 2 Apoptosis and necrosis percentage induced by
compound 18c in HepG‐2 cells
Apoptosis
Sample
Total
21.71
0.82
Early
6.75
0.54
Late
14.96
0.28
Necrosis
2.66
18c/HepG‐2
Cont. HepG‐2
1.15
The results of cell cycle analysis carried out in HepG‐2 cells are
presented in Table 3 and Figure 3. The results revealed that com-
pound 18c produced 41.82% of cell accumulation in the G2–M phase.
However, it produced only 18.24% of cell accumulation in G2–M in
control (untreated cells). These results indicate that compound 18c
induced a significant cell cycle arrest in the G2–M phase, compared
with the control cells.
The tested compounds showed versatile DNA binding affinities.
These affinities range from strong to weak. Compound 18c showed
binding affinity higher than that of doxorubicin with an IC50 value
of 48.53 µM. It was 1.16 times more active than doxorubicin.
Additionally, compounds 14c and 14d showed comparable activities
with doxorubicin with IC50 values of 59.13 and 54.52 µM, respectively.
Moreover, compounds 14e, 15b, 18a, and 18b exhibited moderate DNA
binding affinities with IC50 values ranging from 66.39 to 74.18 µM. On
the contrary, compounds 19a and 19b showed weak binding activities
with IC50 values of 82.04 and 79.63 µM, respectively.
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2.3
Computational studies
Molecular docking
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2.3.1
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2.2.4
Apoptosis and cell‐cycle analysis
Molecular docking studies were conducted for the synthesized
compounds using doxorubicin as a reference standard. The main aim
of this study was to gain further insights into the binding modes of
the synthesized compounds against the Topo II–DNA complex (ID:
3qx3). Table 4 illustrates the binding free energies (ΔG) in negative
values, reflecting the spontaneity of bindings. The reported binding
site of DNA–Topo II complex consists of Asp479, Arg503, Gln778,
Met782 Cyt8, Thy9, Cyt11, Gua13, and Ade12.[41]
Apoptosis initiation and cell cycle stages play an important role in
cancer development. In cancer management, these two factors have
a crucial role.[61,62] The most promising candidate 18c was selected
to evaluate its impact on apoptosis and cell cycle profile. As HepG‐2
cells were the most sensitive cells against the tested compounds,
they were used for testing the apoptotic effect and cell cycle profile
of compound 18c. Determination of apoptosis was carried out ac-
cording to the reported annexin V and propidum iodide (PI) double‐
staining assay protocol.[63] In this test, HepG‐2 cells were treated
with compound 18c at a concentration that equals its IC50 value on
HepG‐2 (4.43 µM) for 24 h. The flow cytometry analysis was applied
for the determination of apoptosis ratios of compound 18c in early‐
and late‐stage apoptosis. The results are reported in Table 2 and
Figure 2.
The proposed doxorubicin binding mode showed an affinity va-
lue of −58.46 kcal/mol. A hydrogen bonding interaction was formed
between the Cyt8 phosphate group and the NH2 group of the sugar
moiety. The OH group at position‐12 was involved in hydrogen
bonding interaction with Arg503. The aromatic planar system
formed aromatic stacking interactions with various key residues as
Thy9, Cyt8, Gua13, and Ade12. The side chain of sugar moiety was
positioned in a minor groove of DNA (Figure 4).
FIGURE 2 Induced apoptosis in HepG‐2 cells by compound 18c

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TABLE 3 Effect of compound 18c on cell‐cycle progression in
HepG‐2 cells
The enhancement of absorption was attributed to the polycyclic
nucleus (1,2,4‐triazolo[4,3‐c]quinazoline), which increased the lipo-
philicity. Finally, the closer the hepatotoxicity scores to one, the
more is the probability to be toxic, whereas the closer the hepato-
toxicity scores to zero, the more is the probability to be nontoxic.
Most of the designed compounds were predicted to be safer than
doxorubicin, and the others were expected to be like doxorubicin. It
is well known that numerous drug candidates have failed during
clinical tests due to problems related to their absorption proper-
ties.[64] Upon computational investigation of the synthesized com-
pounds, it was found that all the designed compounds have good
absorption behavior. Moreover, the solubility level of most com-
pounds was expected to be better than or even like that of the
reference drug (Supporting Information Data).
Cell cycle distribution (%)
Sample
G0–G1
30.25
50.47
S
G2–M
41.82
18.24
Pre‐G1
24.37
1.97
18c/HepG‐2
Cont. HepG‐2
27.93
31.29
The proposed binding mode of compound 13 showed an affinity
value of −44.52 kcal/mol with three hydrogen bonds and eight π–π
interactions. The aromatic planar framework (1,2,4‐triazolo[4,3‐c]
quinazoline) was involved in aromatic stacking interactions with
DNA base pairs, Thy9, Cyt8, and Ade12. Furthermore, in the minor
groove of DNA, the hydrazinyl group at position‐5 was oriented and
formed three hydrogen bonding interactions with the Thy9 phos-
phate group (Figure 5).
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2.4
Structure–activity relationship (SAR)
The proposed binding mode of compound 15b showed an affinity
value of −46.07 kcal/mol. It showed one hydrogen bonding interac-
tion between the C═O of COOH group and Ade12. Additionally, the
aromatic planar system (1,2,4‐triazolo[4,3‐c]quinazoline) exhibited
aromatic interactions with Thy9 and Gua13. Moreover, the terminal
benzoic acid moiety was oriented at the minor groove of DNA
(Figure 6). The binding mode of compounds 16 and 18b is illustrated
in Supporting Information Data.
As mentioned in the rationale of molecular design, we aimed at
studying the SAR of the newly synthesized quinazoline derivatives as
potential DNA intercalators and Topo II inhibitors. Observing the
results of different biological tests, we could deduce significant data
about SAR.
First, we investigated the effect of substitutions on position‐5
of 1,2,4‐triazolo[4,3‐c]quinazoline nucleus by different amines. It
was found that the substitutions with aliphatic amines (com-
pounds 14c–e) are more preferred biologically than that with aro-
matic amines (compounds 15a–c). Concerning the activity of the
different aliphatic amine derivatives, it was noted that alicyclic
aliphatic amine (compound 14d) is more active than that with open
chain with the same length (compound 14c). Moreover, the long‐
chain aliphatic amino derivative was more active than the corre-
sponding members with a short aliphatic chain (14c > 14b > 14a).
Compound 15b containing free carboxylic acid moiety was more
active than the ester form 15c.
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2.3.2
In silico ADMET analysis
Discovery Studio 2.5 was used to predict ADMET descriptors (ab-
sorption, distribution, metabolism, excretion, and toxicity) for the
designed compounds as compared with doxorubicin as a reference
drug. Predicted ADMET studies for the tested compounds are illu-
strated in Table 5. It was found that BBB penetration of the designed
compounds ranges from high to low, except compound 18b, which
was expected to be very low. This indicated that most compounds
were anticipated to be safe for the CNS. Moreover, all the designed
compounds were predicted to be absorbed better than doxorubicin.
Next, we studied the effect of substitutions at 5‐position of
1,2,4‐triazolo[4,3‐c]quinazoline nucleus by different hydrazones and
thiosemicarbazides. It was found that the hydrazones derivatives
FIGURE 3 Distribution of HepG‐2 cells upon treatment with compound 18c

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TABLE 4 The docking binding free
energies of the synthesized compounds
against DNA–Topo II complex forming
hydrogen bonding and π–π interactions
No. of H
ΔG kcal/mol bonds
No. of π–π
Compound
13
bonds
Putative interactions
−44.52
−35.62
−39.24
−45.12
−46.26
−43.15
−41.29
−46.07
−40.26
−39.28
−36.90
−44.68
−47.11
−48.07
−40.04
3
0
0
1
0
0
0
1
0
1
0
1
0
1
1
8
5
7
6
8
7
9
8
8
12
5
4
6
5
3
Ade12, Thy9, and Cyt8
14a
14b
14c
Ade12, Thy9, and Gua13
Ade12, Thy9, and Gua13
Ade12, Thy9, Gua13, and Arg503
Ade12, Thy9, and Gua13
Ade12, Thy9, and Cyt8
14d
14e
15a
15b
15c
Ade12, Thy9, and Gua13
Ade12, Thy9, and Gua13
Ade12, Thy9, and Gua13
Ade12, Thy9, and Gua13
Ade12, Thy9, and Cyt8
16
17
18a
18b
18c
Arg503, Thy9, Cyt8, and Gua13
Gua13, Thy9, and Cyt8
Asp479, Cyt8, Gua13, Ade12
18d
Arg503, Gln778, Thy9, Cyt8, and
Ade12
19a
19b
19c
19d
−39.09
−41.15
−42.67
−40.16
0
0
1
1
4
6
5
3
Gln778, Thy9, Cyt8, and Gua13
Thy9, Cyt8, and Gua13
Arg503, Cyt8, and Gua13
Gln778, Gly504, Thy9, Cyt8, and
Ade12
Doxorubicin −58.46
2
6
Ade12, Thy9, Cyt8, Gua13, and
Arg503
FIGURE 4 Doxorubicin in the active site of
Topo II–DNA complex; hydrogen bonds are
presented by green dashed lines and π–π
interactions are presented by orange lines

ALESAWY ET AL.
9 of 16
|
FIGURE 5 Compound 13 in the active site
of Topo II–DNA complex; hydrogen bonds are
presented by green dashed lines and π–π
interactions are presented by orange lines
(compounds 18a–c) are more active than thiosemicarbazides (com-
pounds 19a–d). Moreover, observing the activity of the substituted
hydrazones, it was found that the derivatives with hydrophilic sub-
stitutions (compounds 18b,c) were more active than the corre-
assay. Compounds 14c, 14d, 14e, 15b, 18b, 18c, and 19b exhibited
promising antiproliferative activities. Compound 18c was the most
cytotoxic member and exhibited superior antiproliferative activ-
ities against HCT‐116 and HepG‐2 with IC50 values of 5.22 and
4.43 µM, respectively, compared with doxorubicin (IC50 = 9.63 and
8.28 µM, respectively). Also, compound 14d showed higher activity
than doxorubicin against HCT‐116 cells (IC50 = 9.39 µM). In addi-
tion, compound 14e exhibited a higher activity than doxorubicin
against HepG‐2 with an IC50 value of 6.92 µM. The most promising
antiproliferative members were assessed for their Topo II in-
hibitory activities and DNA intercalating affinities to deduce the
proposed mechanism of antiproliferative effect. Some compounds
displayed potent inhibitory activities against Topo II such as
compounds 14d and 18c (IC50 = 1.05 and 0.86 µM, respectively).
Moreover, the DNA intercalation assay revealed that compounds
14d and 18c (IC50 = 54.52 and 48.53 µM, respectively) were more
active than doxorubicin (IC50 = 57.53 µM). Moreover, the flow
cytometry analysis demonstrated that compound 18c could
sponding members with hydrophobic ones (compounds 18a,d .
)
Regarding the activity of different thiosemicarbazide derivatives, it
was noted that aliphatic derivatives (compounds 19a–c) were more
active than aromatic one (compounds 19d). Moreover, long‐chain
aliphatic thiosemicarbazide derivative was more active than the
corresponding members with a short aliphatic chain (19c > 19b > 19a).
|
3
CONCLUSION
In conclusion, a new series of 1,2,4‐triazolo[4,3‐c]quinazoline de-
rivatives has been designed and synthesized. The synthesized
compounds were evaluated for their in vitro cytotoxicity against
three tumor cell lines (HCT‐116, HepG‐2, and MCF‐7) by the MTT
FIGURE 6 Compound 15b in the active site
of Topo II–DNA complex; hydrogen bonds are
presented by the green dashed lines and
π–πinteractions are presented by orange lines

10 of 16
ALESAWY ET AL.
|
TABLE 5 Predicted
BBB
Absorption
levelb
Hepatotoxicity
probabilityc
Solubility
Compound No. levela
CYP2D6d PPBe levelf
ADMET (absorption, distribution,
metabolism, excretion, and toxicity)
descriptors for the designed compounds
and doxorubicin
13
3
2
2
1
1
1
2
3
2
1
3
1
1
4
2
1
3
2
2
2
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0.966
0.741
0.761
0.450
0.390
0.496
0.470
0.920
0.781
0.900
0.966
0.847
0.860
0.947
0.907
0.953
0.523
0.774
0.503
0.907
0.900
0
0
0
1
1
0
1
0
1
1
0
1
0
0
0
0
1
1
1
0
0
2
2
1
2
2
1
0
2
2
2
2
2
2
2
2
2
2
0
0
2
0
3
3
2
2
2
2
3
2
2
2
3
2
2
2
2
1
2
2
2
2
2
14a
14b
14c
14d
14e
14a
15a
15b
15c
16
17
18a
18b
18c
18d
19a
19b
19c
19d
Doxorubicin
aBBB level, blood–brain barrier level, 0 = very high, 1 = high, 2 = medium, 3 = low, 4 = very low.
bAbsorption level, 0 = good, 1 = moderate, 2 = poor, 3 = very poor.
cHepatotoxicity probability, value > 0.5 means toxic, value < 0.5 means nontoxic.
dCYP2D6, cytochrome P2D6, 0 = noninhibitor, 1 = inhibitor.
ePBB, plasma protein binding, 0 means less than 90%, 1 means more than 90%, 2 means more
than 95%.
fSolubility level, 0 = extremely low, 1 = very low, 2 = low, 3 = good, 4 = optimal.
|
significantly induce apoptosis (41.82%) in HepG‐2 cells at a con-
centration of 4.43 μM and could arrest the cell cycle at the G2–M
phase. In addition, the reported results allowed to draw an in-
teresting SAR. In particular, The substitutions at 5‐position of
1,2,4‐triazolo[4,3‐c]quinazoline nucleus by different aliphatic
amines 14c–e were more preferred biologically than that with
aromatic ones 15a–c. Moreover, alicyclic aliphatic amine 14d was
more active than that with open chain with the same length 14c.
Additionally, it was found that the derivatives with hydrazones
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All melting points were determined on a Gallenkamp apparatus using
the open capillary method. The infrared spectra were reported using
the potassium bromide disc technique on a Unicam SP 1000 IR
spectrophotometer Pye. On a Jeol 400 MHz‐NMR spectrometer,
proton magnetic resonance 1H NMR spectra are reported. Tetra-
methylsilane has been used as an internal norm, and chemical shifts
have been calculated in the ppm range. The mass spectra were re-
corded on SHIMADZU GC/MS–QP5050A's Varian MAT 311‐A
(70 eV) and SHIMADZU GC/MS–QP5050A's Direct Inlet Unit
(DI‐50). All compounds in the theoretical values were within 0.4.
substitutions 18a–c are more active than thiosemicarbazides 19a–d
.
Also, docking studies were carried out to explore the binding
pattern of the designed compounds with the probable target,
DNA–Topo II complex (PDB‐code: 3qx3). Depending on the ac-
quired results, we can conclude that DNA binding and/or Topo II
inhibition may contribute to the detected cytotoxicity of the
synthesized compounds.

ALESAWY ET AL.
11 of 16
|
Thin‐layer chromatography (TLC) monitored the reactions, using TLC
sheets precoated with Merck 60 F254 UV fluorescent silica gel and
visualized as mobile phases using UV lamps and various solvents.
5‐Chloro‐[1,2,4]triazolo[4,3‐c]quinazoline 12 was obtained according
to the reported procedures.[55]
(C═N); 1H NMR (DMSO‐d6) δ ppm: 0.95–0.98 (t, 3H, CH3), 1.65–1.74
(m, 2H, CH2–CH3), 3.46–3.51 (q, 2H, NH–CH2), 7.30–7.34 (dd,
J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.50–7.52 (d, J = 7.6 Hz, 1H, H‐10
of quinazoline), 7.57–7.61 (dd, J = 7.6 Hz, 1H, H‐8 of quinazoline),
8.05–8.07 (t, 1H, NH), 8.21–8.24 (d, J = 7.6 Hz, 1H, H‐7 of quinazo-
line), and 9.45 (s, 1H, CH triazole); 13C NMR (DMSO‐d6, 100 MHz) δ
(ppm): 11.4, 21.7, 42.8, 112.1, 122.6, 123.4, 125.0, 131.5, 134.6,
141.2, 143.4, and 148.1; Anal. calcd. for C12H13N5 (227.27); C, 63.42;
H, 5.77; N, 30.82. Found: C, 63.73; H, 5.81; N, 30.78%.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
|
4.1.2
General procedure for the synthesis of
5‐hydrazinyl‐[1,2,4]triazolo[4,3‐c]quinazoline (13)
N‐Pentyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (14c)
Shiny yellow crystal (yield, 69%); m.p. = 170–172°C. IR (KBr, cm−1):
3,250 (NH), 3,087 (CH aromatic), 2,938 (CH aliphatic), and 1,632
(C═N); 1H NMR (DMSO‐d6) δ ppm: 0.87–0.91 (t, 3H, CH3), 1.33–1.41
(m, 4H, CH2–CH2–CH3), 1.65–1.72 (p, 2H, NH‐CH2‐CH2), 3.51–3.56
(q, 2H, NH–CH2), 7.32–7.36 (dd, J = 8.4 & 7.6 Hz, 1H, H‐9 of quina-
zoline), 7.52–7.53 (d, J = 7.6 Hz, 1H, H‐10 of quinazoline), 7.59–7.63
(dd, J = 8.4 Hz, 1H, H‐8 of quinazoline), 8.05–8.08 (t, 1H, NH),
8.23–8.25 (d, J = 7.6 Hz, 1H, H‐7 of quinazoline), and 9.48 (s, 1H, CH
triazole); Anal. calcd. for C14H17N5 (255.33); C, 65.86; H, 6.71; N,
27.43. Found: C, 65.98; H, 6.65; N, 27.54%.
A mixture of 5‐chloro‐[1,2,4]triazolo[4,3‐c]quinazoline 12 (0.409 g,
0.002 mol) and hydrazine hydrate (0.128 g, 0.004 mol) was heated
under reflux in isopropyl alcohol (IPA) (20 ml) for 1 h. After com-
pletion, the obtained crude solid was filtered and washed with IPA to
give yellow solid of the desired compound 13. Yellow powder (yield
85%); m.p. 227–229°C; IR (KBr, cm−1); 3,302 (NH2), 3,257 (NH),
3,051 (CH aromatic), and 1,637 (C═N); 1H NMR (dimethyl sulfoxide
[DMSO]‐d6) δ ppm: 4.68 (s, 2H, NH2), 7.38–7.42 (dd, J = 6.8 Hz, 1H,
H‐9 of quinazoline), 7.66–7.67 (d, J = 7.6 Hz, 1H, H‐10 of quinazo-
line), 7.69–7.73 (dd, J = 6.8, 8 Hz, 1H, H‐8 of quinazoline), 8.20–8.22
(d, J = 7.6 Hz, 1H, H‐7 of quinazoline), 8.59 (s, 1H, CH triazole), and
9.30 (s, 1H, NH); MS (m/z): 200 (M+, 15.40%), 167 (31.69%), 145
(59.89%), 97 (69.99%), 67 (52.69%), and 54 (100% base beak); Anal.
calcd. for C9H8N6 (200.21): C, 53.99; H, 4.03; N, 41.98; Found:
C, 54.08; H, 4.01; N, 41.92%.
N‐Cyclopentyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (14d)
Brown powder (yield, 78%); m.p. = 222–224°C. IR (KBr, cm−1): 3,238
(NH), 3,051 (CH aromatic), 2,959 (CH aliphatic), and 1,630 (C═N); 1H
NMR (DMSO‐d6) δ ppm: 1.03–1.07 (m, 1H, H‐3 of cyclopentyl),
1.54–1.68 (m, 3H, H‐3 & H‐4 of cyclopentyl), 1.74–1.76 (m, 2H, H‐2
of cyclopentyl), 2.03–2.11 (m, 1H, H‐5 of cyclopentyl), 3.42–3.46
(m, 1H, H‐5 of cyclopentyl), 4.43–4.52 (m, 1H, H‐1 of cyclopentyl),
7.31–7.35 (dd, J = 8 Hz, 1H, H‐9 of quinazoline), 7.52–7.54
(d, J = 8 Hz, 1H, H‐10 of quinazoline), 7.58–7.62 (dd, J = 8 Hz, 1H,
H‐8 of quinazoline), 7.82–7.84 (d, 1H, NH), 8.22–8.24 (d, J = 8 Hz, 1H,
H‐7 of quinazoline), and 9.55 (s, 1H, CH triazole); Anal. calcd. for
|
4.1.3
General procedure for the synthesis
of compounds 14a–e
A mixture of compound 12 (0.409 g, 0.002 mol) and appropriate
amines, namely ethylamine, n‐propylamine, n‐phenethylamine,
cyclopentylamine, and diethylamine (0.004 mol), was refluxed in
IPA (20 ml) for 2 h. After completion of the reaction, the crude solid
was filtered and washed with IPA to give the corresponding target
compounds 14a–e, respectively.
C14H15N5 (253.31); C, 66.38; H, 5.97; N, 27.65. Found: C, 66.59; H,
6.11; N, 27.78%.
N,N‐Diethyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (14e)
Yellowish brown (yield, 76%); m.p. = 180–182°C. IR (KBr, cm−1):
3,124 (NH), 3,072 (CH aromatic), 2,961 (CH aliphatic), and 1,609
N‐Ethyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (14a)
(C═N); 1H NMR (DMSO‐d6)
δ ppm: 1.23–1.27 (t, 6H, 2CH3),
Gray crystal (yield, 72%); m.p. = 240–242°C. IR (KBr, cm−1): 3,214
(NH), 3,046 (CH aromatic), 2,971 (CH aliphatic), and 1,635 (C═N); 1H
NMR (DMSO‐d6) δ ppm: 1.26–1.30 (t, 3H, CH3), 3.54–3.61 (m, 2H,
CH2), 7.33–7.38 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.52–7.54
(d, J = 7.6 Hz, 1H, H‐10 of quinazoline), 7.58–7.62 (dd, J = 7.6 Hz, 1H,
H‐8 of quinazoline), 8.07–8.09 (t, 1H, NH), 8.22–8.24 (d, J = 7.6 Hz,
1H, H‐7 of quinazoline), and 9.43 (s, 1H, CH triazole); Anal. calcd. for
3.59–3.64 (q, 4H, 2CH2), 7.44–7.48 (dd, J = 8 Hz, 1H, H‐9 of quina-
zoline), 7.60–7.62 (d, J = 8 Hz, 1H, H‐10 of quinazoline), 7.65–7.69
(dd, J = 8 & 7.6 Hz, 1H, H‐8 of quinazoline), 8.30–8.32 (d, J = 7.6 Hz,
1H, H‐7 of quinazoline), and 9.23 (s, 1H, CH triazole); Anal. calcd.
for C13H15N5 (241.30); C, 64.71; H, 6.27; N, 29.02. Found: C, 64.87;
H, 6.30; N, 29,11%.
C
11H11N5 (213.24); C, 61.96; H, 5.20; N, 32.84. Found: C, 62.14;
|
General procedure for the synthesis
H, 5.12; N, 32.99%.
4.1.4
of compounds 15a–c
N‐Propyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (14b)
Shiny yellow crystal (yield, 80%); m.p. = 211–213°C. IR (KBr, cm−1):
A
mixture of 5‐chloro‐[1,2,4]triazolo[4,3‐c]quinazoline 12 (0.409 g,
3,279 (NH), 3,057 (CH aromatic), 2,949 (CH aliphatic), and 1,630
0.002 mol) and the appropriate aromatic amines, namely 4‐methylaniline,

12 of 16
ALESAWY ET AL.
|
4‐aminobenzoic acid, and ethyl p‐aminobenzoate (0.006 mol), in IPA
(20 ml) was refluxed for 7 h. After completion, the obtained precipitate
was filtered and washed with IPA to give the corresponding title
compounds 15a–c, respectively.
7.23–7.27 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.31–7.36
(m, J = 7.6 Hz, 3H, H‐3, H‐4 and H‐5 phenyl), 7.44–7.46 (d,
J = 7.6 Hz, 2H, H‐2 and H‐6 phenyl), 7.50–7.52 (d, J = 8 Hz, 1H, H‐10
of quinazoline), 7.58–7.62 (dd, J = 8 Hz, 1H, H‐8 of quinazoline),
8.23–8.25 (d, J = 7.6 Hz, 1H, H‐7 of quinazoline), 8.68 (s, 1H, NH), and
9.49 (s, 1H, CH triazole); 13C NMR (DMSO‐d6, 100 MHz) δ (ppm):
44.2, 112.3, 122.7, 123.7, 125.1, 127.1, 127.6 (2C), 128.3 (2C), 131.5,
134.7, 138.5, 141.3, 143.3, and 148.1; Anal. calcd. for C16H13N5
(275.32); C, 69.80; H, 4.76; N, 25.44. Found: C, 69.91; H, 4.81;
N, 25.49%.
N‐(p‐Tolyl)‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (15a)
Yellowish white powder (yield, 83%); m.p. = 214–216°C; IR (KBr, cm−1):
3,137 (NH), 3,065 (CH, aromatic), and 1,629 (C═N); 1H NMR (DMSO‐
d6) δ ppm: 2.32 (s, 3H, CH3), 7.21–7.23 (d, J = 8 Hz, 2H, H‐2 and H‐6
phenyl), 7.42–7.45 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.58–7.60 (d,
J = 8 Hz, 1H, H‐10 of quinazoline), 7.64–7.68 (dd, J = 8 Hz, 1H, H‐8 of
quinazoline), 7.90–7.92 (d, J = 8 Hz, 2H, H‐3 and H‐5 phenyl), 8.29–8.31
(d, J = 8 Hz, 1H, H‐7 of quinazoline), 10.30 (s, 1H, CH triazole), and
10.44 (s, 1H, NH); Anal. calcd. for C16H13N5 (275.32); C, 69.80; H, 4.76;
N, 25.44. Found: C, 69.91; H, 4.81; N, 25.49%.
|
4.1.6
General procedure for the synthesis of
4‐([1,2,4]triazolo[4,3‐c]quinazolin‐5‐yl)morpholine (17)
A
mixture of 5‐chloro‐[1,2,4]triazolo[4,3‐c]quinazoline 12 (0.409 g,
4‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐ylamino)benzoic acid (15b)
Shiny yellow powder (yield, 78%); m.p. = 225–227°C; IR (KBr, cm−1):
3,175 (NH), 3,046 (CH, aromatic), and 1,674 (C═N); 1H NMR
(DMSO‐d6) δ ppm: 7.51–7.57 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline),
7.72–7.83 (d, J = 8 Hz, 2H, H‐2 and H‐6 phenyl), 7.98–8.02
(d, J = 8 Hz, 2H, H‐3 and H‐5 phenyl), 8.11–8.13 (d, J = 8 Hz, 1H,
H‐10 of quinazoline), 8.27–8.30 (dd, J = 8 Hz, 1H, H‐8 of quinazoline),
8.35–8.37 (dd, J = 7.6 Hz, 1H, H‐7 of quinazoline), 9.38 (s, 1H, CH
triazole), 10.42 (s, 1H, NH), and 12.35 (s, 1H, OH); Anal. calcd. for
0.002 mol) and morpholine (0.348 g, 0.004 mol) in IPA (20 ml) was re-
fluxed for 3 h. After completion, the obtained crude solid was filtered
and washed with IPA to give the desired compound 17. White powder
(yield, 88%); m.p. = 210–212°C. IR (KBr, cm−1): 3,117 (NH), 2,975 (CH
aliphatic), and 1,617 (C═N); 1H NMR (DMSO‐d6) δ ppm: 3.50–3.52
(t, 4H, 2CH2–O), 3.82–3.83 (t, 4H, 2CH2–N), 7.53–7.57 (dd, J = 8 Hz, 1H,
H‐9 of quinazoline), 7.71–7.73 (d, J = 7.6 Hz, 2H, H‐8 and H‐10 of qui-
nazoline), 8.35–8.37 (d, J = 7.6 Hz, 1H, H‐7 of quinazoline), and 9.49
(s, 1H, CH triazole); 13C NMR (DMSO‐d6, 100 MHz) δ (ppm): 49.3 (2C),
66.0 (2C), 114.4, 123.1, 126.6, 126.7, 132.1, 136.7, 142.1, 144.7, and
149.2; Anal. calcd. for C13H13N5O (255.28); C, 61.17; H, 5.13; N, 27.43.
Found: C, 61.26; H, 5.19; N, 27.52%.
C
16H11N5O2 (305.30); C, 62.95; H, 3.63; N, 22.94. Found: C, 63.08;
H, 3.71; N, 22.91%.
Ethyl 4‐([1,2,4]triazolo[4,3‐c]quinazolin‐5‐ylamino)benzoate (15c)
Shiny yellow powder (yield, 81%); m.p. = 169–171°C; IR (KBr, cm−1):
3,189 (NH), 3,055 (CH, aromatic), 1,704 (C═O), and 1,643 (C═N); 1
H
4.1.7
General procedure for the synthesis
|
NMR (DMSO‐d6) δ ppm: 1.32–136 (t, 3H, CH3), 4.29–4.34 (q, 2H,
CH2), 7.49–7.53 (dd, J = 8 Hz, 1H, H‐9 of quinazoline), 7.70–7.75
(m, 2H, H‐8 of quinazoline and H‐10 of quinazoline), 8.01–8.04
(d, J = 8 Hz, 2H, H‐2 and H‐6 phenyl), 8.13–8.15 (d, J = 8 Hz, 2H, H‐3
and H‐5 phenyl), 8.34–8.36 (d, J = 8 Hz, 1H, H‐7 of quinazoline), 9.93
(s, 1H, CH triazole), and 10.40 (s, 1H, NH); 13C NMR (DMSO‐d6,
100 MHz) δ (ppm): 14.0, 60.2, 112.8, 119.9 (2C), 122.6, 124.1, 125.2,
125.4, 129.8 (2C), 131.7, 135.4, 137.5, 141.7, 143.1, 147.8, and
165.1; Anal. calcd. for C18H15N5O2 (333.35); C, 64.86; H, 4.54;
N, 21.01. Found: C, 64.96; H, 4.58; N, 21.11%.
of compounds 18a–d
A
mixture of 5‐hydrazinyl‐[1,2,4]triazolo[4,3‐c]quinazoline 13
(0.400 g, 0.002 mol) and the appropriate aldehydes, namely 4‐
methylbenzaldhehyde, 4‐nitrobenzaldhehyde, 4‐hydroxybenzaldhe-
hyde, and 2,6‐dichlorobenzaldhehyde (0.0024 mol), was refluxed in
absolute ethanol (25 ml) in the presence of catalytic amount of glacial
acetic acid for 6 h. The mixture was cooled, filtered, and crystallized
from ethanol to afford the corresponding derivatives 18a–d
,
respectively.
(E)‐5‐[2‐(4‐Methylbenzylidene)hydrazinyl]‐[1,2,4]triazolo[4,3‐c]‐
quinazoline (18a)
|
4.1.5
General procedure for the synthesis of
N‐benzyl‐[1,2,4]triazolo[4,3‐c]quinazolin‐5‐amine (16)
Canary yellow powder (yield, 76%); m.p. = 220–222°C; IR (KBr,
cm−1): 3,131 (NH), 3,044 (CH, aromatic), and 1,627 (C═N); 1H NMR
(DMSO‐d6) δ ppm: 2.31 (s, 3H, CH3), 7.20–7.22 (d, J = 8 Hz, 2H, H‐3
and H‐5 phenyl), 7.41–7.45 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline),
7.58–7.60 (d, J = 8 Hz, 1H, H‐10 of quinazoline), 7.64–7.68 (dd,
J = 8 Hz, 1H, H‐8 of quinazoline), 7.91–7.93 (d, J = 8 Hz, 2H, H‐2 and
H‐6 phenyl), 8.29–8.31 (d, J = 8 Hz, 1H, H‐7 of quinazoline), 8.71
A mixture of 5‐chloro‐[1,2,4]triazolo[4,3‐c]quinazoline 12 (0.409 g,
0.002 mol) and benzylamine (0.428 g, 0.004 mol) in IPA (20 ml) was
refluxed for 1 h. After completion, the obtained crude solid was fil-
tered and washed with IPA to give yellow solid of the desired
compound 16. Yellow powder (yield, 89%); m.p. = 216–218°C; IR
(KBr, cm−1): 3,279 (NH), 3,066 (CH, aromatic), 2,925 (CH aliphatic),
and 1,628 (C═N); 1H NMR (DMSO‐d6) δ ppm: 4.78–4.79 (d, 2H, CH2),
(s, 1H, N═CH), 10,36 (s, 1H, CH triazole), and 10.50 (s, 1H, NH); 13
C
NMR (DMSO‐d6, 100 MHz) δ (ppm): 20.5, 113.0, 121.5 (2C), 122.7,

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124.5, 125.5 (2C), 128.9 (2C), 131.6, 132.6, 135.9, 136.0, 138.7,
142.6, 148.1; Anal. calcd. for C17H14N6 (302.34); C, 67.54; H, 4.67;
N, 27.80. Found: C, 67.61; H, 4.74; N, 27.86%.
isothiocyanate, propyl isothiocyanate, butyl isothiocyanate, and phenyl
isothiocyanate (0.003 mol), was refluxed in ethanol (25 ml) for 3 h. The
reaction mixture was cooled, and the separated solids were filtered
and crystallized from ethanol to afford the corresponding compounds
19a–d, respectively.
(E)‐5‐[2‐(4‐Nitrobenzylidene)hydrazinyl]‐[1,2,4]triazolo[4,3‐c]‐
quinazoline (18b)
Yellow powder (yield, 78%); m.p. = 230–232°C; IR (KBr, cm−1): 3,318
(NH), 3,089 (CH, aromatic), and 1,642 (C═N); 1H NMR (DMSO‐d6)
δ ppm: 7.33–7.35 (dd, 1H, H‐9 of quinazoline), 7.82–7.84 (d, J = 8 Hz,
2H, H‐2 and H‐6 phenyl), 8.01–8.03 (d, 1H, H‐10 of quinazoline),
8.11–8.13 (dd, 1H, H‐8 of quinazoline), 8.30–8.32 (d, J = 8 Hz, 2H,
H‐3 and H‐5 phenyl), 8.34 (s, 1H, N═CH), 8.53–8.55 (d, 1H, H‐7 of
quinazoline), 8.70 (s, 1H, CH triazole), and 11.53 (s, 1H, NH); Anal.
calcd. for C16H11N7O2 (333.31); C, 57.66; H, 3.33; N, 29.42. Found:
C, 57.71; H, 3.35; N, 29.38%.
2‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐yl)‐N‐ethylhydrazine‐1‐
carbothioamide (19a)
Shiny yellow powder (yield, 74%); m.p. = 243–245°C; IR (KBr, cm−1):
3,205 (NH), 2,969 (CH, aliphatic), and 1,637 (C═N); 1H NMR (DMSO‐
d6) δ ppm: 1.04–1.06 (t, 3H, CH3), 3.45–3.48 (q, 2H, CH2), 7.48–7.52
(dd, J = 7.2 Hz, 1H, H‐9 of quinazoline), 7.71–7.75 (m, 2H, H‐8 of
quinazoline and H‐10 of quinazoline), 8.24 (s, 1H, NH–CH2),
8.28–8.30 (d, J = 7.2 Hz, 1H, H‐7 of quinazoline), 8.68 (s, 1H, CH
triazole), 9.57 (s, 1H, NH‐quinazoline), and 10.20 (s, 1H, NH–C═S);
13C NMR (DMSO‐d6, 100 MHz) δ (ppm): 14.4, 38.5, 114.2, 123.2,
124.5, 125.8, 132.3, 143.1, 143.8, 151.0, 153.4, and 181.3; Anal.
calcd. for C12H13N7S (287.35); C, 50.16; H, 4.56; N, 34.12. Found: C,
50.24; H, 4.61; N, 34.18%.
(E)‐4‐{[2‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐yl)hydrazono]methyl}‐
phenol (18c)
Gray powder (yield, 71%); m.p. = 215–217°C; IR (KBr, cm−1): 3,141
(NH), 3,048 (CH, aromatic), and 1,652 (C═N); 1H NMR (DMSO‐d6) δ
ppm: 6.85–6.88 (d, J = 7.6 Hz, 2H, H‐3 and H‐5 phenyl), 7.34–7.37
(dd, J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.67–7.70 (d, J = 8 Hz, 1H,
H‐10 of quinazoline), 7.75–7.81 (m, 3H, H‐8 of quinazoline, H‐2 and
H‐6 phenyl), 8.15–8.17 (d, J = 7.6 Hz, 1H, H‐7 of quinazoline), 8.51
(s, 1H, N═CH), 8.57 (s, 1H, CH triazole), 9.25 (s, 1H, OH), and 9.80
(s, 1H, NH); 13C NMR (DMSO‐d6, 100 MHz) δ (ppm): 116.09 (2C),
116.28, 123.65, 124.04, 125.47, 126.12, 130.02, 130.58, 132,91,
151.21, 154.01, 160.11 (2C), 160.86, and 160.93; MS (m/z): 304
(M+, 2.33%), 240 (13.31%), 185 (25.25%), 147 (15.13%), 120
(22.24%), 105 (31.07%), 77 (84.29%), 65 (100% base beak), and 64
(39.69%); Anal. calcd. for C16H12N6O (304.31); C, 63.15; H, 3.97;
N, 27.62. Found: C, 63.21; H, 4.04; N, 27.54%.
2‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐yl)‐N‐propylhydrazine‐1‐
carbothioamide (19b)
Gray powder (yield, 77%); m.p. = 229–231°C; IR (KBr, cm−1): 3,173
(NH), 2,954 (CH, aliphatic), and 1,626 (C═N); 1H NMR (DMSO‐d6) δ
ppm: 0.78–0.82 (t, 3H, CH3), 1.73–1.82 (m, 2H, CH2–CH3), 4.03–4.07
(t, 2H, NH–CH2), 7.08–7.11 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline),
7.45–7.47 (d, 1H, H‐10 of quinazoline), 7.70–7.74 (dd, 1H, H‐8 of
quinazoline), 8.24–8.26 (m, 2H, NH–CH2 and H‐7 of quinazoline),
8.67 (s, 1H, CH triazole), 9.55 (s, 1H, NH‐quinazoline), and 10.21
(s, 1H, NH–C═S); Anal. calcd for C13H15N7S (301.37); C, 51.81;
H, 5.02; N, 32.53. Found: C, 51.98; H, 4.94; N, 32.61%.
2‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐yl)‐N‐butylhydrazine‐1‐
carbothioamide (19c)
(E)‐5‐[2‐(2,6‐Dichlorobenzylidene)hydrazinyl]‐[1,2,4]triazolo[4,3‐c]‐
quinazoline (18d)
White powder (yield, 79%); m.p. = 199–201°C; IR (KBr, cm−1): 3,203
(NH), 2,936 (CH, aliphatic), and 1,637 (C═N); 1H NMR (DMSO‐d6) δ
ppm: 0.80–0.83 (t, 3H, CH3), 1.22–1.24 (m, 2H, CH2–CH3), 1.44–1.47
(m, 2H, CH2–CH2–CH3), 4.42–3.44 (t, 2H, NH–CH2), 7.47–7.51 (dd,
J = 7.6 Hz, 1H, H‐9 of quinazoline), 7.68–7.70 (d, J = 7.6 Hz, 1H, H‐10
of quinazoline), 7.73–7.77 (dd, J = 7.6 Hz, 1H, H‐8 of quinazoline),
8.70 (s, 1H, NH‐CH2), 8.27‐8.29 (d, J = 7.6 Hz, 1H, H‐7 of quinazo-
line), 8.67 (s, 1H, CH triazole), 9.53 (s, 1H, NH‐quinazoline),
and 10.19 (s, 1H, NH–C═S); Anal. calcd. for C14H17N7S (315.40);
C, 53.31; H, 5.43; N, 31.09. Found: C, 53.44; H, 5.47; N, 31.21%.
Yellowish white powder (yield, 79%); m.p. = 254–256°C; IR (KBr,
cm−1): 3,148 (NH), 3,092 (CH, aromatic), and 1,645 (C═N); 1H NMR
(DMSO‐d6) δ ppm: 7.27–7.31 (dd, J = 7.6 Hz, 1H, H‐9 of quinazoline),
7.46–7.50 (dd, J = 8 Hz, 1H, H‐8 of quinazoline), 7.55–7.61
(m, J = 8 Hz, 3H, H‐3, H‐4 and H‐5 phenyl), 7.74–7.76 (d, 1H, H‐10
of quinazoline), 8.14–8.16 (d, J = 8 Hz, 1H, H‐7 of quinazoline), 8.61
(s, 1H, N═CH), 9.33 (s, 1H, CH triazole), and 11.28 (s, 1H, NH); 13C
NMR (DMSO‐d6, 100 MHz) δ (ppm): 109.4, 116.5, 123.1, 123.2,
128.7 (2C), 131.3, 131.7, 132.0, 134.2 (2C), 135.4, 137.5, 143.7,
146.5, and 151.4; Anal. calcd. for C16H10Cl2N6 (356.03); C, 53.80;
H, 2.82; N, 23.53. Found: C, 53.92; H, 2.85; N, 23.56%.
2‐([1,2,4]Triazolo[4,3‐c]quinazolin‐5‐yl)‐N‐phenylhydrazine‐1‐
carbothioamide (19d)
Pale yellow powder (yield, 70%); m.p. = 182–185°C; IR (KBr, cm−1):
3,199 (NH), 3,062 (CH, aromatic), and 1,607 (C═N); 1H NMR
|
4.1.8
General procedure for the synthesis
of compounds 19a–d
(DMSO‐d6) δ ppm: 7.12–7.16 (t, J = 7.6 Hz, 1H, H‐5 phenyl),
7.31–7.35 (dd, J = 8 Hz, 1H, H‐9 of quinazoline), 7.50–7.54 (m, 3H,
H‐3, H‐5 phenyl and H‐10 of quinazoline), 7.65–7.68 (m, 3H, H‐2, H‐6
phenyl and H‐8 of quinazoline), 8.04 (s, 1H, NH‐quinazoline),
A mixture of 5‐hydrazinyl‐[1,2,4]triazolo[4,3‐c]quinazoline 13 (0.409 g,
0.002 mol) and the appropriate isothiocyanates, namely ethyl

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8.28–8.30 (d, 1H, H‐7 of quinazoline), 9.66 (s, 1H, CH triazole), 11.07
(s, 1H, NH–C═S), and 13.41 (s, 1H, NH‐phenyl); 13C NMR (DMSO‐d6,
100 MHz) δ (ppm): 117.5, 121.2, 125.0, 127.4, 128.3, 129.5 (2C),
130.0 (2C), 130.3, 132.3, 137.4, 139.3, 143.0, 148.3, and 164.9; Anal.
calcd. for C16H13N7S (335.39); C, 57.30; H, 3.91; N, 29.23. Found: C,
57.44; H, 3.97; N, 29.36%.
reported as IC50 (50% inhibition concentration) values calculated
from the concentration–inhibition response curve.
|
4.2.3
DNA intercalation assay
Nine compounds that exhibited significant antiproliferative activities
(14c, 14d, 14e, 15b, 18a, 18b, 18c, 19a, and 19b) were further evaluated
to determine their DNA‐binding affinities. Doxorubicin as a DNA in-
tercalator was used as a positive control. In this test, methyl green dye
can bind with DNA to form a colored reversible complex of DNA/
methyl green. These complexes stay stable at neutral pH. Upon ad-
dition of intercalating agents, the methyl green is displaced from DNA
with the addition of H2O molecule to the dye, resulting in the for-
mation of the colorless carbinol, leading to a dramatic decrease in
spectrophotometric absorbance.[60] 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 GraphPad Prism 5.0 software.
The reaction was performed as follows. Calf thymus DNA (10 mg) and
methyl green (20 mg) (Sigma‐Aldrich) were added in 100 ml of 0.05 M
Tris‐HCl buffer (pH 7.5) containing 7.5 mM MgSO4. 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 con-
centrations of 10, 100, and 1,000 μM. From each well, the excess
solvent was removed under vacuum, followed by an addition of 200 μl
of the DNA/methyl green solution. The test samples were incubated in
the dark at an 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.
|
4.2
Biological testing
|
4.2.1
In vitro antiproliferative activities
The synthesized compounds were evaluated for their antiproliferative
activities against three human cancer cell lines, namely colorectal carci-
noma (HCT‐116), hepatocellular carcinoma (HepG‐2), and breast cancer
(MCF‐7). The MTT assay protocol was applied as described.[56–58]
Cell lines were cultured in the RPMI‐1640 medium with 10%
fetal bovine serum. Antibiotics, 100 units/ml penicillin and 100 µg/ml
streptomycin, were added at 37°C in a 5% CO2 incubator. The cell
lines were seeded in a 96‐well plate at a density of 1.0 × 104 cells/
well at 37°C for 48 h under 5% CO2. After incubation, the cells were
treated with different concentration of the synthesized compounds
and incubated for 24 h. Then, 20 µl of the MTT solution at a con-
centration of 5 mg/ml was added and incubated for 4 h. DMSO in
volume of 100 µl was added into each well to dissolve the purple
formazan formed. The colorimetric assay was measured and re-
corded at an absorbance of 570 nm using a plate reader (EXL 800).
The relative cell viability in percentage was calculated as (A570 of
treated samples/A570 of untreated sample) × 100. Results for IC50
values of the active compounds are summarized in Table 1.
|
4.2.2
Measurement of topoisomerase II activity
|
4.2.4
Flow cytometric analysis of the cell cycle
Nine compounds that showed high antiproliferative activities (14c,
14d, 14e, 15b, 18a, 18b, 18c, 19a, and 19b) were further assessed to
determine their inhibitory activities against Topo II. Topo II drug
screening kit (TopoGEN, Inc.) was used to determine the activity of
Topo II according to a previously reported procedure by Patra
et al.[65] Doxorubicin as a potent Topo II inhibitor was used as
positive control in this test.
According to the method described by Léonce et al.,[66] the flow
cytometric analysis was carried out. In this test, PI is used to dis-
criminate between living cells from dead ones or for cell cycle ana-
lysis. The cell cycle analysis is based on the stoichiometric binding of
PI to intracellular DNA. HepG‐2 cells were seeded in 100‐mm culture
dishes and immediately incubated with the test compound 18c. After
24 h, the cells were washed, fixed, and stained in phosphate‐buffered
saline (PBS), Triton X‐100 (0.1%), RNase A (1 mg/ml), and 0.5 ml of PI
in PBS (1 mg/ml). Then, the DNA content was determined with a flow
cytometer and the distribution of cells in pre‐G1 (apoptotic cells),
G0–G1, S, and G2–M peaks was quantified by the histogram analysis.
The obtained data represent three independent experiments.
In general, the reaction was allowed to start by incubating a
mixture of human Topo II (2 µl), substrate supercoiled pHot1 DNA
(0.25 µg), 50 µg/ml test compound (2 µl), and assay buffer (4 µl) in
37°C for 30 min. To terminate the reaction, 10% sodium dode-
cylsulphate (2 µl) and proteinase K (50 µg/ml) were added at 37°C
for 15 min, followed by incubation for 15 min at 37°C. Then, the
DNA was run on 1% agarose gel in BioRad gel electrophoresis
system for 1–2 h, followed by staining with GelRed™ stain for 2 h
and destaining for 15 min with TAE buffer (Tris base, acetic acid,
and EDTA). The gel was imaged via BioRad's Gel DocTMEZ system.
Both supercoiled and linear strands DNA were incorporated in the
gel as markers for DNA–Topo II intercalators. The results were
|
4.2.5
Apoptosis using annexin‐V‐FITC assay
Annexin V‐fluorescein isothiocyanate (FITC)/PI apoptosis detection
kit was used in staining HepG‐2 cells, which were stained with

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annexin V‐FITC and counterstained with PI. Cells were incubated
with the tested compound 18c for 24 h and 48 h. Then, they were
trypsinized and washed with cold PBS two times, and then stained
with 5 µl annexin V‐FITC in a binding buffer (10 mM HEPES, 140 mM
NaCl, and 2.5 mM CaCl2 at pH 7.4) for 15 min at room temperature
in the dark. The samples were analyzed using the flow cytometer.[63]
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How to cite this article: Alesawy MS, Al‐Karmalawy AA,
Elkaeed EB, et al. Design and discovery of new 1,2,4‐triazolo
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