1,2,4-Triazolo[4,3-c]quinazolines: a bioisosterism-guided approach towards the development of novel PCAF inhibitors with potential anticancer activity

Article abstract of DOI:10.1039/d1nj00710f

Targeting PCAF with small inhibitor molecules has emerged as a potential therapeutic strategy for the treatment of cancer. Recently, L-45 was identified as a potent triazolophthalazine inhibitor of the PCAF bromodomain. Here, we report the bioisosteric modification of the triazolophthalazine ring system of L-45 to its bioisosteric triazoloquinazoline while maintaining other essential structural fragments for effective binding with the binding site of PCAF. Consequently, a set of sixteen triazoloquinazoline derivatives were designed, synthesized, and investigated for their anticancer activity against four human cancer cell lines. Five derivatives demonstrated comparable cytotoxic activity with that of doxorubicin as a reference anticancer drug. Among them, compound23showed the most potent activity with IC₅₀values of 6.12, 4.08, 7.17, and 6.42 μM against HePG2, MCF-7, PC3, and HCT-116, respectively. Also, compound21exhibited comparable cytotoxic effects with that of doxorubicin against the selected cancer cell lines with IC₅₀values in the range of 7.41-9.58 μM. Molecular docking and pharmacokinetic studies were additionally performed to rationalize the binding affinities of the newly designed triazoloquinazolines toward the active site of histone acetyltransferase PCAF and to evaluate the druggability of new compounds. The results of these studies suggested that PCAF binding could be the mechanism of action of these derivatives.

Full text of DOI:10.1039/d1nj00710f

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1,2,4-Triazolo[4,3-c]quinazolines: a bioisosterism-
guided approach towards the development of
novel PCAF inhibitors with potential anticancer
activity†
Cite this: New J. Chem., 2021,
45, 11136
Mohamed H. El-Shershaby,a Adel Ghiaty,a Ashraf H. Bayoumi,a Hany E. A. Ahmed,ab
de
af
Mona S. El-Zoghbi,c Khaled El-Adl
and Hamada S. Abulkhair
*
Targeting PCAF with small inhibitor molecules has emerged as a potential therapeutic strategy for the
treatment of cancer. Recently, L-45 was identified as a potent triazolophthalazine inhibitor of the PCAF
bromodomain. Here, we report the bioisosteric modification of the triazolophthalazine ring system of
L-45 to its bioisosteric triazoloquinazoline while maintaining other essential structural fragments for
effective binding with the binding site of PCAF. Consequently, a set of sixteen triazoloquinazoline
derivatives were designed, synthesized, and investigated for their anticancer activity against four human
cancer cell lines. Five derivatives demonstrated comparable cytotoxic activity with that of doxorubicin as
a reference anticancer drug. Among them, compound 23 showed the most potent activity with IC50
values of 6.12, 4.08, 7.17, and 6.42 mM against HePG2, MCF-7, PC3, and HCT-116, respectively. Also,
compound 21 exhibited comparable cytotoxic effects with that of doxorubicin against the selected
cancer cell lines with IC50 values in the range of 7.41–9.58 mM. Molecular docking and pharmacokinetic
studies were additionally performed to rationalize the binding affinities of the newly designed triazolo-
quinazolines toward the active site of histone acetyltransferase PCAF and to evaluate the druggability of
new compounds. The results of these studies suggested that PCAF binding could be the mechanism of
action of these derivatives.
Received 10th February 2021,
Accepted 17th May 2021
DOI: 10.1039/d1nj00710f
rsc.li/njc
the aberrant DNA mutations linked to carcinogenesis.1,2 Therefore,
histone acetyltransferases have emerged as chemotherapeutic
1. Introduction
Cancer is a diverse collection of diseases that involve uncontrolled targets for developing anticancer agents.1,3 Also, the unusual
cell growth and have the potential to invade all the body organs. expression of the bromodomain family of proteins has been
Over the past few decades, more than 150 anticancer agents have associated with abnormal cell proliferation.4,5 New avenues for
been approved by the FDA. Nevertheless, the majority of these developing anticancer chemotherapeutics have been emphasized
approved chemotherapeutics negatively affect the patients’ life. by the proven potent cytotoxicity of selective bromodomain inhi-
Consequently, unmet efforts for finding new effective and more bitor molecules.6 The histone acetyltransferase paralogue p300/
safe anticancer agents remain crucial to treat the global growing CBP-associated factor (PCAF) is a member of subfamily I of the
number of patients. The irregular acetylation of histone is one of phylogenetic bromodomain tree.7 Mis-regulation of the latter
bromodomain has been shown to be linked with a number of
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar
University, Nasr City 11884, Cairo, Egypt. E-mail: hamadaorganic@azhar.edu.eg
b Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College,
Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
c Pharmaceutical Chemistry Department, Faculty of Pharmacy, Menoufia University,
Gamal Abd El-Nasir Street, Shebin El-Koum, Egypt
human diseases including cancer8–10 and inflammatory dis-
orders.5 Mis-regulation of PCAF signaling is a predisposing
factor for over-expression in a variety of cancer cells including
hepatocellular,11 mammary gland,12 colorectal,10 and prostate
carcinoma.13
Quinazolines are a group of nitrogenous heterocycles that
have gained particular importance as they are embedded in the
core structures of many FDA-approved anticancer agents14 (Fig. 1).
These include gefitinib (1; Iressa), vandetanib (2; Caprelsa),
erlotinib (3; Tarceva), lapatinib (4; Tykreb), afatinib (5; Gilotrif),
and dacomitinib (6; Vizimpro). While the investigation of the
d Department of Medicinal Chemistry & Drug Design, Faculty of Pharmacy, Al-Azhar
University, Cairo, Egypt
e Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Heliopolis
University for Sustainable Development, Cairo, Egypt
f Pharmaceutical Chemistry Department, Faculty of Pharmacy, Horus University –
Egypt, International Coastal Road, 34518, New Damietta, Egypt
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1nj00710f
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Fig. 1 Structures of quinazoline-derived FDA-approved anticancer agents.
anticancer activity of triazoloquinazoline derivatives has been stacking interaction between the pyridazine ring and the Tyr1442
described in a few articles,15,16 both the quinazoline17 and residue; (ii) a hydrogen bonding interaction between N-1 of the
1,2,4-triazole18–20 derivatives have been individually recognized triazole ring and the Asn1436 residue; and (iii) an additional
as anticancer drugs.
hydrogen bond between the side chain NH functionality attached
So far, six attempts have been accomplished for the devel- to C-6 of the triazolophthalazine core and the Glu1389 residue.
opment of triazoloquinazolines with antitumor activity.21–26 The aim of the present study is to develop new 1,2,4-triazolo-
Among these, two articles considered the annelation of a triazole [4,3-c]quinazoline bioisosteric congeners of L-45 by fusion of the
ring to the quinazoline cycle to produce 1,2,4-triazolo[1,5-c]- quinazoline and triazole ring as a suggested new scaffold of
quinazolines.21,22 In the third one, a series of 1,2,4-triazolo[4,3- PCAF inhibitors with potent anticancer activity. This bio-
a]quinazolin-5-ones were designed as inhibitors of tubulin isosteric transformation is expected to furnish new derivatives
polymerization.23 The results of the latter study revealed the potent capable of interacting with the PCAF receptor site in a similar
anticancer activity of 7 and 8 against a panel of human cancer cell pattern to that of L-45.
lines. Both compounds also presented potent inhibitory effects
In medicinal chemistry, bioisosterism is a key concept that
against tubulin polymerization. In 2014, the in vitro cytotoxicity could be used for the modification of certain lead compounds
evaluation of twenty two 1,2,4-triazolo[1,5-a]quinazolines revealed to get safer and more clinically useful agents. Bioisosterism allows
the higher cytotoxic effect of 9, 10, and 11 (Fig. 2) against medicinal chemists to substitute constitutional fragments without
medulloblastoma, hepatocellular carcinoma, and melanoma cell significant perturbation in the biological activity. As initially
lines than dasatinib.24 The last pair of articles was published in the specified by Friedman, bioisosteres must include all atoms and
previous year25,26 and led to the detection of new 1,2,4-triazolo[4,3- molecules which fit the widest definition for isosteres and have a
c]quinazolines as a new class of DNA intercalators and potential similar type of biological activity.28 A more recent definition of
inhibitors of topoisomerase II. These latter studies also reported bioisosteres has been broadened to include compounds or groups
the EGFR inhibition activity of 12, 13, and 14 (Fig. 2), with IC50 that retain nearly equal molecular shapes and volumes, and
values in the range of 0.69–1.8 mM. Also, 1,2,4-triazolo[4,3-c]quina- approximately the same distribution of electrons, which exhibit
zoline derivative 15 (Fig. 2) was evaluated as a topoisomerase II similar physicochemical properties. The crucial component for
inhibitor and DNA intercalator. The obtained results verified its bioisosterism is that bioisosteres are expected to affect the same
inhibitory activity on topoisomerase II and the DNA binding biological target without considerable perturbation in the biologi-
affinity with IC50 values of 0.86 and 48.53 mM, respectively.
cal activity.29 Overall, this strategy is an excellent tool for the
A few years ago, L-45 (16) was identified as the first potent, optimization of lead compounds to obtain the desired potency
highly selective, and cell-active PCAF bromodomain inhibitor and selectivity, and improved ADMET profiles for a marketable
belonging to the class of 1,2,4-triazolo[3,4-a]phthalazines.9 Our drug.30 The in silico bioisosteric replacement mining approaches
recent couple of research articles reported the design and PCAF are divided into two types, ligand-based and structure-based. The
bromodomain inhibitory effects of certain triazolopthalazines18,27 former category uses information on ligands, whereas the latter
as congeners of L-45. The PCAF antagonistic effect of L-45 was requires knowledge of the biological target, as well as a specific
reported to be mediated through three key interactions with the understanding of the interactions between the ligand and target in
binding site of histone acetyltransferase GCN5 (Fig. 3): (i) a p–p the binding pocket.31
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Fig. 2 Structures of selected [1,2,4]triazoloquinazines with potent anticancer activity.
derivatives (Fig. 4) to examine the effect of these structural
optimizations on the anticancer and PCAF inhibitory potentials:
First, the pyrimidine ring replaced the pyridazine ring of L-45 to
keep the favorable p–p stacking interaction with the Tyr1442
residue. Second, diverse substitution patterns were introduced
to position 5 of triazoloquinazoline scaffolds to investigate the
effect on the cytotoxic activity. Last, the methyl group at C-3
of L-45 was replaced by three different substituent groups of
different electronic and spatial characteristics. With this set of
structural modifications, we decided to determine the signifi-
cance of the HBD/HBA groups at C-6 of L-45 and the new
compounds. All the new compounds were evaluated for their
in vitro anticancer activity against four cancer cell lines, namely,
hepatocellular carcinoma (HePG-2), mammary gland breast
cancer (MCF-7), human prostate cancer (PC3), and colorectal
carcinoma (HCT-116). In addition, the possible underlying
mechanisms of these compounds were investigated in silico to
rationalize their ability to bind with the active site of histone
acetyltransferase GCN5 as a proposed mode of their anticancer
activity. Also, the structure–activity relationship of the synthe-
Fig. 3 The key interactions of L-45 with the active site of histone
acetyltransferase GCN5.
1.1. Rationale and aim of the work
Considering the aforementioned facts, inspired by the versatilities sized compounds was illustrated based on the results of cyto-
of both the quinazoline and 1,2,4-triazole rings mentioned above, toxicity evaluation against four human cancer cell lines (HePG2,
and as a continuation of our recent studies32–35 on identifying MCF-7, PC3, and HCT-116). Finally, ADMET profiles of the best
new anticancer agents, synthesis of a new set of 1,2,4-triazolo- effective derivatives were evaluated to determine their potential
[4,3-c]quinazolines as L-45 bioisosteres was carried out to get to build up as good drug candidates.
new molecules with good potency. In the design of the new
compounds, we employed the ligand-based bioisosterism strategy.
Following the directions of bioisosterism, a number of con-
stitutional fragments in the structure of lead compound L-45
and FDA approved anticancer quinazolines are expected to be
2. Results and discussion
2.1. Chemistry
substituted without significant perturbation in the activity. The synthetic approach adopted for the synthesis of the starting
Accordingly, we considered keeping N-1 of the triazole ring in 3-substituted-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-ones (21–23)
L-45 as an essential fragment that maintains the interaction is presented in Scheme 1. Briefly, a solution of potassium cyanate
with the essential amino acid residue Asn1436. However, three was added drop-wise into a solution of anthranilic acid in glacial
bioisosteric modifications were adopted in the newly designed acetic acid. The produced quinazoline-2,4(1H,3H)-dione was
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Fig. 4 Rational design of new suggested PCAF inhibitors.
Scheme 1 Synthetic protocol of the starting triazoloquinazolines.
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Scheme 2 Synthetic route of new triazoloquinazoline derivatives 24–36.
treated with phosphorus oxychloride to get 2,4-dichloroquinazo- side chain. 13C NMR of 31 as a representative spectrum showed
line (19). The addition of hydrazine hydrate dropwise to the fifteen signals equivalent to fifteen carbon types, among them two in
latter at 0–5 1C gives 2-chloro-4-hydrazinylquinazoline,36,37 which the aliphatic region at 67.00 and 45.89 ppm due to (CH2OCH2) and
was finally reacted with the appropriate acid anhydride38 to (CH2NCH2), respectively. The purity of the synthesized compounds
obtain the desired starting 3-substituted-[1,2,4]triazolo[4,3-c]- was monitored by TLC and confirmed by elemental analysis.
quinazolin-5(6H)-one (21–23).
2.2. Evaluation of the biological activity
The structures of 21–23 were established based on their
elemental and spectral data. The disappearance of the biforked
2.2.1. Cytotoxicity assay. Four cancer cell lines were selected
absorption band of NH2 of the starting hydrazinyl derivative to evaluate the cytotoxic effects of the new compounds using the
together with the appearance of a new amidic CQO absorption MTT colorimetric assay.42 These cells include hepatocellular,
band in the range of 1674–1697 cmÀ1 in the IR spectra con- mammary gland, colorectal, and prostate carcinoma. The selection
firmed the construction of the tricyclic ring. Also, the amidic NH of such cancer cells depended on the over-expression of PCAF in
revealed a singlet signal in the d value range of 11.20–11.40 ppm these cell types.10–13 The cytotoxicity of the new 1,2,4-triazolo-
which is D2O exchangeable.
[4,3-c]quinazoline bioisosteres of L-45 was compared with that of
As presented in Scheme 2, our convergent synthesis approach doxorubicin as a reference anticancer agent. Results of the pre-
of final target molecules 24–36 started by using a simple and liminary antiproliferative evaluation are shown in Table 1. The
straightforward strategy. Construction of this set of compounds tabulated results showed very strong to moderate cytotoxicity for
was performed by converting the appropriate 3-substituted- six of the tested compounds against the selected four cancer cell
[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one to its 5-chloro-3-sub- lines. The concentrations of new ligands 21–26 necessary for
stituted-[1,2,4]triazolo[4,3-c]quinazoline derivative by the action 50% inhibition of tumor cell proliferation were found to be
of phosphorus oxychloride. Treating these latter with different within the range of 4.08–24.50 mM. The compounds belonging
amine derivatives yielded our target compounds in reasonable to the [1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one derivatives (21–23)
yields and acceptable purities.39–41 The selected approach and 5-chloro-[1,2,4]triazolo[4,3-c]quinazoline derivatives (24–26)
depended on the highly reliable and well-established nucleo- with no substituent group at C-5 revealed better activity than those
philic substitution reaction, exploiting the good commercial incorporating an N-alkyl/aralkyl amine substituent group at the
availability of amine derivatives. Adopting this approach, a set same position. In regard to the tested cancer cells, the breast
of ten triazoloquinazoline derivatives carrying an amine fragment cancer cells (MCF-7) and colorectal cancer cells (HCT-116) were
at position 5 were afforded in yields ranging from 73 to 86%. found to be the most sensitive to the cytotoxic effect of the new
1
All the IR, H NMR, and 13C NMR spectra of these derivatives compounds followed by the prostate cancer cells (PC3). The
were in accordance with the assumed structures. A piece of hepatocarcinoma cells (HePG2) were found to be the least sensitive
supporting evidence for the given produced structures was to the cytotoxic effect of our target compounds as indicated by the
provided by element analysis and spectral data (see the ESI†). higher IC50 values presented.
The 1H nuclear magnetic resonance (NMR) spectrum of com-
pound 27 as a representative example showed a triplet signal of
2.3. Structure–activity relationship study
four protons at 3.66 ppm equivalent to two methylene protons As outlined in the rational ligand-based drug design, studying
(CH2) at positions 3 and 5 of the morpholinyl fragment. The the SAR of the newly designed 1,2,4-triazolo[4,3-c]quinazoline
1H NMR spectrum of the same compound revealed another bioisosteres of L-45 as cytotoxic agents is a major objective of
triplet signal of another four protons at 3.60 ppm due to the this work. Doxorubicin was selected as a reference anticancer drug
two methylene protons at positions 2 and 6 of the morpholinyl to compare the cytotoxicity of the newly designed compounds as it
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Table 1 In vitro anticancer activity of the designed new triazoloquinazolinesa
b
In vitro cytotoxicity IC50 (mM)
R
R1
HePG2
MCF-7
PC3
HCT-116
DOXc
21
22
—
CF3
C6H5
4-ClC6H4
—
—
—
—
4.50 Æ 0.2
7.41 Æ 0.5
13.89 Æ 1.1
6.12 Æ 0.5
4.17 Æ 0.2
5.93 Æ 0.4
16.21 Æ 1.3
4.08 Æ 0.5
8.87 Æ 0.6
9.58 Æ 0.8
22.48 Æ 1.8
7.17 Æ 0.9
5.23 Æ 0.3
8.16 Æ 0.7
12.08 Æ 1.1
6.42 Æ 0.7
23
24
25
26
27
28
29
30
31
32
33
34
35
36
CF3
C6H5
4-ClC6H4
CF3
CF3
C6H5
C6H5
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
Cl
Cl
Cl
18.58 Æ 1.5
11.26 Æ 1.0
7.32 Æ 0.8
68.27 Æ 3.8
23.69 Æ 1.9
9.02 Æ 0.8
26.87 Æ 2.3
42.71 Æ 2.9
84.19 Æ 4.5
4100
19.57 Æ 1.7
7.84 Æ 0.7
5.38 Æ 0.6
80.54 Æ 4.4
21.04 Æ 1.9
6.38 Æ 0.6
15.81 Æ 1.3
58.39 Æ 3.2
93.48 Æ 5.3
4100
24.50 Æ 2.0
14.09 Æ 1.3
8.28 Æ 0.8
92.68 Æ 4.9
28.36 Æ 2.2
10.27 Æ 1.0
24.96 Æ 2.1
60.61 Æ 3.5
4100
4100
4100
4100
63.82 Æ 3.5
13.28 Æ 1.2
9.27 Æ 0.8
8.15 Æ 0.7
47.19 Æ 3.6
15.49 Æ 1.4
8.56 Æ 0.7
9.38 Æ 0.8
31.97 Æ 2.7
61.45 Æ 4.1
67.69 Æ 4.2
88.16 Æ 4.8
4100
Morpholin-4-yl
Piperazin-1-yl
NH(CH2)3CH3
Piperazin-1-yl
Morpholin-4-yl
NHCH2CH3
NH(CH2)2CH3
NHCH2C6H5
NHC6H4-COCH3
Piperazin-1-yl
4100
4100
40.38 Æ 2.8
4100
4100
58.16 Æ 3.4
39.17 Æ 3.1
a
Cancer cells were treated with the test compound for 72 h and the cytotoxicity is expressed as the concentration needed to inhibit 50% of tumor
b
cell proliferation (IC50). Data here are presented as the means of three independent experiments Æ SD. IC50 (mM): 1–10 (very strong), 11–20
c
(strong), 21–50 (moderate), 51–100 (weak) and above 100 (non-cytotoxic). DOX: doxorubicin.
is a potent cytotoxic agent typically used in the treatment of a C-3 (23) presented very strong cytotoxic effects with IC50 values of
variety of carcinomas including hepatocellular carcinoma, breast, 6.12, 4.08, 7.17, and 6.42 mM against the HePG2, MCF-7, PC3, and
prostate, and colorectal cancers.43,44 Based on the obtained results HCT-116 cancer cell lines, respectively, compared with 4.50, 4.17,
for the tested compounds and doxorubicin as a reference drug, the 8.87, and 5.23 mM, respectively, of doxorubicin. These findings
SAR of the anticancer activity of the synthesized compounds indicate the approximately equipotent activity of 23 and doxorubicin
revealed a number of common findings. Except for the N-butyl- on breast and colorectal cancer cells and even a better activity of 23
amine derivative 29, there is a dramatic fall in the activity upon against the prostate cancer.
attachment of N-alkyl/aralkyl amine fragments at C-5 of the 1,2,4-
On the other hand, the nature of the substituents attached
triazolo[4,3-c]quinazoline ring system. This is noticeable upon to C-3 of the triazole ring had no remarkable impact on the
comparing the in vitro cytotoxic activity of the [1,2,4]triazolo[4,3-c]- cytotoxicity. Accordingly, the compounds with substituents
quinazolin-5(6H)-one derivatives (21–23) and 5-chloro-[1,2,4]- with a ÀI effect, like CF3, and substituents with a +M effect,
triazolo[4,3-c]quinazoline derivatives (24–26) with no substituent like C6H5 and 4-ClC6H4, at this position displayed comparable
group with that of compounds 27, 28, and 30–36 incorporating an cytotoxic effects regardless of the nature of the C-3 attached
N-alkyl/aralkyl amine substituent group at the same position. This group. Similarly, among the C-5 attached amine fragments,
finding may indicate the importance of the presence of the HBD/ none of them had a positive effect on the anticancer potency.
HBA group at this position to enable the ligand to bind with the Individually, butylamine derivative 29 revealed good cytotoxic
essential amino acid Glu1389 at the target protein binding site.9 In effects against the selected four cancer cells. Exceptionally,
this context, compounds 21 and 23 presented comparable cyto- butylamine derivative 29 is approximately more than three-fold
toxic effects with that revealed by the reference drug doxorubicin more potent (IC50 = 6.38–10.27 mM) against the tested cancer cells
against all the tested cancer cells. In particular, compound 21 was than the corresponding N-alkyl/aralkyl amine derivatives (27, 28
the most potent derivative with IC50 values of 7.41, 5.93, 9.58, and and 30–36), which had moderate to weak cytotoxicity with IC50
8.16 mM compared with 4.50, 4.17, 8.87, and 5.23 mM, respectively, values Z15.49 mM. As well, the piperazinyl derivatives 28 and 30
of the reference cytotoxic agent against HePG2, MCF-7, PC3, and were found to show moderate activity (IC50 = 9.38–28.36 mM).
HCT-116, respectively. These results indicate the approximately Among the N-alkyl/aralkyl derivatives, the para-acetylphenylamino
equipotent activity of 21 and doxorubicin on breast and prostate derivative (35) revealed the least activity as an anticancer agent.
cancer cells. Also, the 1,2,4-triazolo[4,3-c]quinazoline-triazolo[4,3-c]- This lower cytotoxic effect of such a compound may be attributed
quinazolin-5(6H)-one derivative with a 4-chlorophenyl substituent at to the inability of such compounds to accommodate the target
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Table 2 Results of in silico docking scores for the new compounds and
the co-crystallized ligand (L-45) with the binding site of histone acetyl-
transferase GCN5
H-bonding
interactions
Hydrophobic
interactions
DG
RMSD
Distance
(Å)
Distance
Residue (Å)
Comp. (kcal molÀ1) (Å)
Residue
L-45
À11.52
0.81
GLU 1389 3.47
GLU 1389 2.82
ASN 1436 3.40
TYR 1442 3.63
21
22
À10.15
À10.12
0.98
1.19
ASN 1436 3.31
Fig. 5 Summary of the SAR study of the designed 1,2,4-triazolo[4,3-
c]quinazolines.
TYR 1442 3.62
TRP 1379 4.10
TRP 1379 4.34
VAL 1385 3.60
TYR 1442 3.77
protein binding site. These observations may collectively reflect
the importance of the presence of a hydrogen bond donor group
at position number six which is essential for binding with the
Glu1389 fragment in the PCAF binding site.
In the analysis of each individual cancer cell, MCF-7 was
shown to be potently inhibited by compounds 21, 23, 25, and
26, with IC50 values of 5.93, 4.08, 7.84, and 5.38 mM, respec-
23
24
À10.98
À10.26
1.00
1.20
GLU 1389 3.09
THR 1401 3.24
ASN 1436 2.84
TYR 1442 3.85
TYR 1442 3.84
PRO 1380 3.20
ASN 1436 2.98
ALA 1390 4.52
TYR 1442 3.65
tively, compared with the reference drug (doxorubicin IC50
=
4.17 mM). Whereas, compounds 21, 23, and 26 effectively
inhibited HePG2, with IC50 values of 7.41, 6.12, and 7.32 mM,
respectively, compared with the reference drug (doxorubicin
IC50 = 450 mM). The PC3 cell line was highly susceptible to the
cytotoxic effect of compounds 21, 23, and 26 with IC50 values of
9.58, 7.17, and 8.28 mM, respectively, compared with the
reference drug (doxorubicin IC50 = 8.87 mM). Similarly, the
HCT-116 cell line was also significantly inhibited by the same
latter three compounds with IC50 values of 8.16, 6.42, and
8.15 mM, respectively, compared with the reference drug
(doxorubicin IC50 = 5.23 mM).
25
26
À10.10
À10.74
0.73
1.11
ASN 1436 2.99
TYR 1442 3.94
TYR 1442 3.90
THR 1401 3.42
ASN 1436 2.84
TYR 1442 3.90
TYR 1442 3.80
27
À10.07
0.92
0.80
ASN 1436 3.45
TYR 1442 3.84
TYR 1442 3.78
28
29
30
31
À9.79
CYS 1432 3.76
ASN 1436 2.71
À11.556
À10.02
À9.53
1.162 ASN 1436 3.11
TYR 1442 3.98
TYR 1442 3.74
TRP 1379 4.15
1.20
A diagrammatic description of the SAR of the antitumor
activity for the synthesized L-54 bioisosteres is presented in
Fig. 5.
1.06
0.89
ASN 1436 2.93
TYR 1442 3.76
TYR 1442 3.75
32
À9.55
THR 1401 3.44
ASN 1436 2.85
2.4. Molecular docking study
TYR 1442 3.91
TYR 1442 3.79
A molecular docking study was performed to give guidance on
the molecular binding modes of the target compounds inside
the pocket of histone acetyltransferase. Docking was conducted
using MOE2014 to determine the free energy and binding
mode. The selection of promising molecules depends on both
the perfect binding mode and the best binding free energy. The
docking studies were validated in terms of the root mean
square deviation (RMSD). Only poses possessing RMSD values
within the acceptable range (0–1.20 Å) were considered.45 With
three main key interactions, the binding mode of L-45 in the
33
34
À10.80
À9.78
0.99
1.16
ASN 1436 2.85
TYR 1442 3.96
TYR 1442 3.75
THR 1401 3.42
ASN 1436 2.87
TYR 1442 3.95
TYR 1442 3.75
35
36
À9.89
À9.92
1.10
1.19
GLU 1389 2.97
TYR 1442 3.93
active site of histone acetyltransferase GCN5 exhibited a binding of the newly designed L-45 bioisosteres and those of the re-docked
energy of À11.52 kcal molÀ1. These interactions include p–p internal co-crystallized ligand is shown in Table 2.
stacking between Tyr1442 and the pyridazine ring of L-45. As
In all the newly designed L-45 bioisosteres, the pyrimidine
well, there is a hydrogen bond between the triazole ring and rings revealed the same p–p stacking interaction with the
Asn1436 residue. The last interaction is presented in the form of essential amino acid residue Tyr1442. Also, N-1 of the triazole
another hydrogen bond between Glu1389 residues and the ring in the majority of the designed compounds formed a hydro-
dimethylamino motif of L-45. An outline of the energy of gen bond with the second essential amino acid Asn1436 which
binding, H-bonding interactions, and hydrophobic interactions is comparable with that formed by the co-crystallized ligand.
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Unfortunately, the third interaction revealed by the co-crystallized atom attached to the terminal phenyl ring of the 26 bioisostere of
ligand with the target protein active site is missing for almost all L-45 formed a hydrogen bond with the amino acid residue Thr1401.
the new ligands. This finding may indicate the importance of the These additional desirable interactions and the relatively elevated
presence of a hydrogen bond donor fragment at position six binding free energy values (À10.10 to À10.98 kcal molÀ1) for the
whether in the triazolophthalazine or the triazoloquinazoline ring 1,2,4-triazolo[4,3-c]quinazolin-5(6H)-one and 5-chloro-3-(substituted)-
system. Individually, 23 revealed a standard interaction pattern [1,2,4]triazolo[4,3-c]quinazoline derivatives 21–26 may justify the
and exhibited the best value of the binding free energy. The same superior in vitro anticancer activity of these derivatives. With almost
compound exerted two additional favorable interactions in the much lower binding free energies, the other derivatives (27–34)
form of p–p stacking and a hydrogen bond between the triazole showed binding modes which are also similar to those presented
ring and the chlorine atom of 23 and Tyr1442 and Thr1401, by the 1,2,4-triazolo[4,3-c]quinazolin-5(6H)-one and 5-chloro-3-(sub-
respectively. In particular, the binding modes of triazoloquinazo- stituted)-[1,2,4]triazolo[4,3-c]quinazoline derivatives. Independently,
linones 21, 22, and 23 presented two essential binding interactions N-butyl derivative 29 exhibited an affinity value of À11.55 kcal molÀ1
that are similar to those of L-45 with the active site of the target and obeyed the same binding pattern with the binding site of
protein. Also, the distances of the hydrogen bonds and p–p histone acetyltransferase GCN5.
contacts are comparable for the new triazoloquinazoline bio-
Collectively, the obtained results of the molecular docking
isosteres and the co-crystallized ligand. The pyrimidine rings of studies showed that all the designed triazoloquinazoline bio-
21 and 23 showed p–p stacking interactions with Tyr1442 while N-1 isosteres of L-45 have almost similar positions and orientations
formed hydrogen bonds with Asn1436. The phenyl side chain of 22 inside the binding site of histone acetyltransferase GCN5. As
interacted with the protein binding site via p–p stacking interac- well, the distribution of binding free energies calculated from
tions with Tyr1442 and an arene-H interaction with Glu1389. Two the MOE software showed preferentially that the most active
more favorable interactions were also observed as a bidirectional derivatives are highly correlated with the biological activity.
arene-H interaction between Trp1379 and the quinazoline ring of Moreover, the absence of a hydrogen bond donor group at
22. In regards to the 5-chloro-3-(substituted)-[1,2,4]triazolo[4,3- position 6 of the triazoloquinazoline ring system prohibited the
c]quinazoline derivatives 24–26, they also presented the first two formation of a hydrogen bond between the designed compounds
essential interactions of L-45 with Tyr1442 and Asn1436 with one and the Glu1389 amino acid residue. This missing interaction may
or more additional favorable contacts. These include hydrogen explain the lower activities of the compounds with a substituent
bond and arene–H interactions between the chlorine atom and amine attached to C-5 of the triazoloquinazoline ring system. The
benzene ring of 24 and the Pro1380 and Ala1390 amino acid 2-D and 3-D interaction patterns of the co-crystallized ligand L-45
residues, respectively. The p–p stackings of 25 and 26 with Tyr1442 and compounds with the best in vitro anticancer activity with the
were found to be bidirectional to include both the triazole and active site of histone acetyltransferase PfGCN5 (homologous Brd
pyrimidine rings of the triazoloquinazoline scaffold. The chlorine with PCAF) are presented in Fig. 6 and 7.
Fig. 6 3D binding interactions of 21 (upper left), 22 (upper middle), 23 (upper right), 24 (lower left), 25 (lower middle), and 26 (lower right) with the active
site of histone acetyltransferase PfGCN5 (homologous Brd with PCAF).
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Fig. 7 3D binding interactions of 21 (upper left), 22 (upper middle), 23 (upper right), 24 (lower left), 25 (lower middle), and 26 (lower right) with the active
site of histone acetyltransferase PfGCN5 (homologous Brd with PCAF).
2.5. In silico pharmacokinetic study
skin sensitization. All parameters were studied for the best
A computational study was conducted for the compounds that
showed the best in vitro anticancer activity to evaluate their
effective new derivatives as well as to the reference anticancer
and PCAF inhibitory agents. From the obtained data shown in
physicochemical properties according to the directions of Table 3, we can suggest that the new derivatives are predicted to
Lipinski’s rule of five.46 According to the Lipinski rule, the possess superior intestinal absorption in humans over all the
intestinal absorption of a molecule is predicted to be acceptable reference drugs (92.59–97.68) compared with 62.37 and 82.25 in
if it fulfills four rules: (i) molecular weight o 500; (ii) number of
the case of doxorubicin and bromosporine. This advantage
may be attributed to the superior lipophilicity of the new
H bond donors r 5; (iii) number of H bond acceptors r 10; and
(iv) log P o 5. Also, reduced molecular flexibility (as indicated by
the count of rotatable bonds), a low polar surface area, and the
compounds, which would make them have significant good
bioavailability after oral administration.49
total count of hydrogen bond donors and acceptors are found to
In regards to the CNS permeability, our newly designed L-45
be crucial predictors of good oral bioavailability.47 In this study, bioisosteres are suggested to have a higher possibility to
the reference anticancer drug was found to violate three of penetrate the CNS (CNS permeability 2.28 to À1.39 compared
Lipinski’s rules while bromosporine and all the designed bioi- with À4.30 for doxorubicin and À3.27 for bromosporine). It is
sosteres pleasingly satisfied all Lipinski’s rules. For all the new
ligands, the numbers of hydrogen bond acceptor and donor
groups are within the acceptable range (4 and r1, respectively).
The molecular weights of and the total count of rotatable bonds
in the new ligands are much lower than those of doxorubicin
also obvious that CYP3A4, the key enzyme in the metabolism,
could be inhibited under the effect of bromosporine and five of
the new triazoloquinazolines (22, 23, 24, 25, and 26). This is
possibly due to the relatively higher lipophilicity of our new
ligands. Excretion was predicted based on the total clearance, a
and bromosporine. Additionally, the ADMET profiles of the new substantial parameter in determining dose intervals. The tabulated
triazoloquinazolines were tentatively investigated to evaluate results revealed that bromosporine and its newly designed bio-
their potential to build up as new oral drug candidates. This isosteres have comparable total clearance values (À0.03 to 0.78)
study has been performed with the aid of the pkCSM descriptors
algorithm.48
Drug absorption depends on many factors including intest-
inal absorption, membrane and skin permeabilities, and being
a P-glycoprotein substrate or inhibitor. The distribution of a
which are much lower than that of doxorubicin. Thus, doxorubicin
could be excreted faster and consequently needs shorter dosing
intervals. Dissimilar to doxorubicin, all other compounds are pre-
dicted to exhibit much slower clearance rates, which suggests the
preference of possible longer dosing intervals for bromosporine and
drug depends on the volume of distribution (VDss), the blood the new compounds.
brain barrier permeability (logBB), and the CNS permeability.
Toxicity is the last parameter analyzed in the pharmacokinetic
Metabolism is predicted depending on the CYP inhibition. profiles of our newly synthesized derivatives. As presented
Excretion is predicted based on the total clearance and the renal
OCT2 substrate. The toxicity of the drugs is predicted depended
on the AMES toxicity, hERG I & II inhibition, hepatotoxicity, and
in Table 3, all the new derivatives except 26 shared the dis-
advantage of expected AMES toxicity, which indicates the draw-
back of probable mutagenic potential of these compounds.50
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Table 3 ADMET profile of the six most active derivatives and reference anticancer and PCAF inhibitory agents
Parameter
21
22
23
24
25
26
Doxorubicin
Bromosporine
Molecular properties
Molecular weight
log P
Rotatable bonds
HB acceptors
HB donors
254.171
1.5896
0
4
262.272
2.2378
1
4
296.717
2.8912
1
4
272.617
2.9497
0
4
280.718
3.5979
1
4
315.163
4.2513
1
4
543.525
0.0013
5
12
404.452
2.34804
5
8
1
1
1
0
0
0
6
2
Surface area
96.882
113.077
123.381
103.023
119.219
129.522
222.081
161.528
Absorption
Water solubility
À4.042
0.523
92.599
À3.032
No
No
No
À3.398
0.734
96.169
À2.753
Yes
No
No
À3.509
0.829
94.648
À2.757
Yes
No
No
À3.711
1.751
93.989
À2.582
No
No
No
À3.458
1.607
97.685
À2.731
No
No
Yes
À3.611
1.458
96.101
À2.729
No
No
Yes
À2.915
0.457
62.372
À2.735
Yes
No
No
À3.035
0.703
82.254
À2.737
No
Yes
No
Caco2 permeability
Intestinal abs. (human)
Skin permeability
P-Glycoprotein substrate
P-Glycoprotein I inhibitor
P-Glycoprotein II inhibitor
Distribution
VDss (human)
Fraction unbound (human)
BBB permeability
CNS permeability
À0.378
0.321
0.449
À2.28
À0.2
À0.116
0.144
0.535
À1.98
À0.304
0.223
0.918
À1.664
0.095
0.248
1.011
À1.4
0.187
0.26
0.965
À1.396
1.647
0.215
À0.361
0.116
0.133
0.58
À1.379
À1.473
À2.097
À4.307
À3.277
Metabolism
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
No
No
Yes
No
No
No
No
No
Yes
Yes
No
No
No
Yes
No
No
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
No
Yes
No
No
Excretion
Total clearance
Renal OCT2 substrate
0.27
No
0.784
No
À0.038
0.133
No
0.215
No
À0.035
0.987
No
0.022
No
No
No
Toxicity
AMES toxicity
Yes
Yes
Yes
Yes
Yes
No
No
No
Max. tolerated dose
hERG I inhibitor
hERG II inhibitor
Oral rat acute toxicity (LD50
Oral rat chronic toxicity (LOAEL)
Hepatotoxicity
À0.2
0.407
No
Yes
2.749
1.368
Yes
0.389
No
Yes
2.753
1.308
Yes
À0.079
0.535
No
No
2.436
0.853
Yes
0.442
No
No
2.458
0.675
Yes
0.081
No
Yes
2.408
3.339
Yes
0.606
No
Yes
2.148
1.783
Yes
No
No
No
No
)
2.613
1.367
Yes
2.713
1.642
Yes
Skin sensitization
T. Pyriformis toxicity
Minnow toxicity
No
0.606
1.661
No
0.31
0.618
No
0.315
0.143
No
0.5
0.693
No
0.293
À0.048
No
0.296
À0.914
No
0.285
4.412
No
0.287
0.952
Conversely, 26 is expected to be non-mutagenic as revealed lower predicted risk of the new bioisosteres in the aquatic
from the test of AMES toxicity. However, they are expected to environment.52 Finally, the oral acute toxic doses (LD50) of the
not inhibit hERG I activity. This inability suggests their safety new compounds are expected to be almost equal to or even a
for human heart electrical activity.51 Unfortunately, two of the little higher than those of the reference anticancer and PCAF
new L-45 bioisosteres are expected to share the disadvantage of inhibitory agents.
both doxorubicin and bromosporine as they could inhibit
hERG II, which indicates the probability of cardiac arrhythmia
risk. In addition, all the triazoloquinazolines, doxorubicin, and
bromosporine shared the drawback of expected hepatotoxicity.
3. Conclusion
In regard to the maximum tolerated dose in humans, four of In conclusion, this work reports a bioisosteric guided approach
the new ligands (21, 22, 24, and 25) are suggested to have for the design of a novel series of triazoloquinazolines derivatives
intermediate tolerability between that of doxorubicin and bromos- as analogous structures of the first reported triazolophthalazine
porine, which means the advantage of wide therapeutic indices of PCAF inhibitor, L-45, with the objective of identifying new PCAF
these derivatives. Additionally, all the designed L-45 bioisosteres inhibitors with potential anticancer activity. The target bio-
are expected to show lower Minnow toxicity values than that of isosteres were designed based on keeping essential structural
doxorubicin. These lower values of Minnow toxicities disclosed the fragments that are essential for binding with the PCAF receptor
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binding site with three structural modifications of the lead hour. The formed precipitate was filtered off under a vacuum
compound. First, the pyrimidine ring replaced the pyridazine and washed with a copious amount of water (50 ml), and then
ring of L-45 to keep the favorable p–p stacking interaction with finally recrystallized from methanol to afford the corresponding
the Tyr1442 residue. Second, different substitution patterns triazoloquinazolinone derivative.
were introduced to position 5 of triazoloquinazoline scaffolds
4.2.1. 3-(Trifluoromethyl)-[1,2,4]triazolo[4,3-c]quinazolin-
to investigate the effect on the cytotoxic activity. Last, the 5(6H)-one (21).
methyl group at C-3 of L-45 was replaced by three different
substituent groups of different electronic and spatial char-
acteristics. Six of our designed bioisosteres showed moderate
to good cytotoxic effects against four human cancer cell lines
(HePG2, MCF-7, PC3, and HCT-116). Among which, compounds
21 and 23 were the most potent against all the tested cancer cell
lines, with IC50 values in the range of 5.93–9.58 mM. Addition-
Greenish white solid, yield: 95%; m.p. 259–261 1C. IR (KBr)
ally, molecular docking, SAR, and in silico pharmacokinetic
prediction studies were conducted. The results of these studies
verified the good activities of both compounds by anticipating
potential binding interactions with the active site of histone
acetyltransferase. The in silico ADMET profiling study indicated
that the designed bioisosteres presented promising pharmaco-
kinetic profiles. Overall, the most active candidates in the quest
for effective cytotoxic agents will serve as useful leads and merit
further investigation.
cmÀ1: 3163 (NH), 3059 (CH aromatic), 1697 (CQO), 1597 (CQC
1
aromatic). H NMR (DMSO-d6) d ppm: 11.19 (s, 1H, NH, D2O
exchangeable), 7.93 (d, J = 8.4 Hz, 1H, quinazoline-H10), 7.66
(dd, J = 7.6 Hz, 1H, quinazoline-H8), 7.39 (d, J = 5.2 Hz, 1H,
quinazoline-H7), 7.22 (dd, J = 8.4 Hz, 1H, quinazoline-H9). MS
(m/z): 254 (C10H5 F3N4O), 237 (C10H4 F3N4, 6.71%). Anal. calc.
for (C10H5 F3N4O) (M.W. = 254): C, 47.25; H, 1.98; N, 22.04%;
found: C, 47.49; H, 2.16; N, 22.21%.
4.2.2. 3-Phenyl-[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (22).
4. Experimental section
4.1. General
Melting points were measured using an electrothermal (Stuart
SMP30) apparatus and were uncorrected. Infrared spectra were
recorded on a Pye Unicam SP 1000 IR spectrophotometer at the
Pharmaceutical Analytical Unit, Faculty of Pharmacy, Al-Azhar
University. 1H NMR and 13C NMR spectra were recorded in
DMSO-d6 at 300 and 100 MHz, respectively, on a Varian
Mercury VXR-300 NMR spectrometer at the NMR Lab, Faculty
of Science, Cairo University. TMS was used as an internal
standard, and chemical shift and coupling constant values
are listed in ppm and Hz, respectively. Mass spectra and
elemental analyses were carried out at the Regional Center of
Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
The reaction progress was monitored with Merck silica gel
IB2-F plates (0.25 mm thickness) visualized under a UV lamp
using different solvent systems as mobile phases. The reagents
and starting compounds anthranilic acid, phosphorus oxychloride,
hydrazine, acid anhydrides, and amine derivatives were purchased
from the Aldrich chemical company and were used as received.
Compounds 25 and 26 were synthesized according to the
directions of previously reported procedures.38
Yellowish green solid, yield: 88%; m.p. 269–271 1C. IR (KBr)
cmÀ1: 3205 (NH), 3051 (CH aromatic), 1678 (CQO), 1624 (CQC
aromatic). H NMR (DMSO-d6) d ppm: 11.41 (s, 1H, NH, D2O
1
exchangeable), 7.96 (d, 2H, phenyl-H2, H6), 7.88 (d, J = 8.4 Hz,
1H, quinazoline-H10), 7.64 (dd, J = 7.6 Hz, 1H, quinazoline-H8),
7.42 (dd, J = 7.6 Hz, 3H, phenyl-H3, H4, H5), 7.20 (d, J = 8.4 Hz,
1H, quinazoline-H7), 7.19 (dd, J = 8.4 Hz, 1H, quinazoline-H9).
13C NMR (DMSO-d6) d ppm: 161.95 (triazole-C3), 135.09
(quinazoline-CQO), 135.01 (quinazoline-C4), 134.18 (quin-
azoline-C8a), 131.87 (phenyl-C1), 130.02 (phenyl-C4), 129.39
(phenyl-C3, C5), 129.03 (quinazoline-C8), 128.98 (phenyl-C2,
C6), 128.83 (quinazoline-C6), 127.92 (quinazoline-C5), 127.01
(quinazoline-C8) and 122.96 (quinazoline-C8a). MS (m/z): 262
(C15H10N4O, 11.64%), 63 (100%). Anal. calc. for (C15H10N4O)
(M.W. = 262): C, 68.69; H, 3.84; N, 21.36%; found: C, 68.92; H,
4.09; N, 21.54%.
4.2.3. 3-(4-Chlorophenyl)-[1,2,4]triazolo[4,3-c]quinazolin-
5(6H)-one (23).
4.2. General procedure for synthesis of 3-substituted-
[1,2,4]triazolo[4,3-c]quinazolin-5(6H)-one (21–23)
Into a stirred solution of 2-chloro-4-hydrazinylquinazoline 20
(0.194 g, 0.001 mol) in glacial acetic acid (20 ml), the appro-
priate acid halide (0.001 mole) was added. The reaction mixture
was heated to reflux temperature in a water bath for 6 hours.
After cooling to room temperature, the reaction mixture was Yellowish white solid, yield: 82%; m.p. 282–284 1C. IR
poured into ice-cooled water (100 ml) and stirred for one more (KBr) cmÀ1: 3225 (NH), 3045 (CH aromatic), 1674 (CQO),
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1626 (CQC aromatic). 1H NMR (DMSO-d6) d ppm: 11.43 (s, 1H,
4.3.3. 5-Chloro-3-(4-chlorophenyl)-[1,2,4]triazolo[4,3-c]-
NH, D2O exchangeable), 7.96 (d, 2H, phenyl-H2, H6), 7.88 (d, J = quinazoline (26)38
8.4 Hz, 1H, quinazoline-H10), 7.64 (dd, J = 7.60 Hz, 1H,
quinazoline-H8), 7.41 (dd, J = 7.6 Hz, 2H, phenyl-H3, H5),
7.21 (d, J = 8.4 Hz, 1H, quinazoline-H7), 7.19 (dd, J = 8.4 Hz,
1H, quinazoline-H9). 13C NMR (DMSO-d6) d ppm: 160.95
(triazole-C3), 135.08 (quinazoline-CQO), 135.00 (quinazoline-
C4), 134.17 (quinazoline-C8a), 131.86 (phenyl-C1), 130.01
.
Yellowish white solid, yield: 65%; m.p. 226–228 1C. IR
(KBr) cmÀ1: 3092 (CH aromatic), 1621 (CQN). MS (m/z): 315
(C15H8C2lN4), 315 (C15H8C2lN4, 100%), 317 (C15H8C2lN4, M + 2,
32.17%). Anal. calc. for (C15H10N4O) (M.W. = 280): C, 57.17;
H, 2.56; N, 22.50%; found: C, 57.21; H, 2.56; N, 22.59%.
(phenyl-C4), 129.36 (phenyl-C3, C5), 129.01 (quinazoline-C8),
128.97 (phenyl-C2, C6), 128.82 (quinazoline-C6), 127.91 (quin-
azoline-C5), 127.02 (quinazoline-C8) and 122.97 (quinazoline-
C8a). MS (m/z): 296 (C15H9ClN4O), 296 (C15H9N4O, 100%). Anal.
calc. for (C15H10N4O) (M.W. = 262): C, 68.69; H, 3.84; N, 21.36%;
found: C, 68.92; H, 4.09; N, 21.54%.
4.4. General procedure for synthesis of 4-(3-(substituted)-
[1,2,4]triazolo[4,3-c]quinazolin-5-yl)amine derivatives (27–36)
4.3. General procedure for synthesis of 5-chloro-3-
Into a solution of the appropriate 5-chloro-[1,2,4]triazolo-
[4,3-c]quinazoline derivative (24–26) (0.001 mol) in isopropyl
alcohol (20 ml), an equimolar amount of the appropriate amine
was added. The reaction mixture was heated to reflux at
120 1C overnight. After cooling to room temperature, the
reaction mixture was quenched in ice-cooled water (100 ml).
The obtained solid was filtered off under reduced pressure and
washed with water (50 ml), dried, and finally recrystallized from
ethanol.
(substituted)-[1,2,4]triazolo[4,3-c]quinazoline (24–26)
The appropriate 3-(4-chlorophenyl)-[1,2,4]triazolo[4,3-c]quina-
zolin-5(6H)-one (21–23) (0.001 mol) in posphorusoxy chloride
(10 ml) was heated to reflux temperature in a water bath for 7–8
hours. After cooling to room temperature, the reaction mixture
was quenched in ice-cooled water (100 ml). The obtained solid
was filtered off under a vacuum and washed with a copious
amount of water (50 ml), dried, and finally recrystallized from
ethanol to afford the corresponding 5-chloro-3-(substituted)-
[1,2,4]triazolo[4,3-c]quinazoline derivative.
4.4.1. 4-(3-(Trifluoromethyl)-[1,2,4]triazolo[4,3-c]quinazolin-
5-yl)morpholine (27).
4.3.1. 5-Chloro-3-(trifluoromethyl)-[1,2,4]triazolo[4,3-c]-
quinazoline (24).
Yellowish solid, yield: 78%; m.p. 262–264 1C. IR (KBr) cmÀ1
:
Yellowish white solid, yield: 73%; m.p. 266–268 1C. IR (KBr)
cmÀ1: 3056 (CH aromatic), 2847 (CH aliphatic), 1619 (CQC
aromatic). 1H NMR (DMSO-d6) d ppm: 7.87 (d, J = 8 Hz, 1H,
quinazoline-H10), 7.63 (dd, J = 7.6 Hz, 1H, quinazoline-H8),
7.17–7.19 (d, J = 8.4 Hz, 1H, quinazoline-H7), 7.15 (dd, J =
8.4 Hz, 1H, quinazoline-H9). MS (m/z): 274 (C10H4ClF3N4,
4.99%, M2+), 272 (C10H4ClF3N4, 64.57%), 168 (C9H4N4,
1.47%). Anal. calc. for (C10H4ClF3N4) (M.W. = 272): C, 44.06;
H, 1.48; N, 20.55%; found: C, 44.32; H, 1.72; N, 20.38%.
4.3.2. 5-Chloro-3-phenyl-[1,2,4]triazolo[4,3-c]quinazoline (25)38.
3032 (CH aromatic), 2962 (CH aliphatic), 1612 (CQN), 1566
(CQC aromatic).1H NMR (DMSO-d6) d ppm: 7.79 (d, 1H, J =
4 Hz, quinazoline-H7), 7.58 (dd, 1H, J = 12 Hz, quinazoline-H8),
7.40 (d, 1H, J = 4 Hz, quinazoline-H10), 7.13 (dd, 1H, J = 4 Hz,
quinazoline-H9), 3.66 (t, 4H, J = 4 Hz, (CH2)2N), 3.60 (t, 4H, J =
4 Hz, (CH2)2O). MS (m/z): 323 (C14H12F3N5O, 8.17%, M+), 304
(C14H12F2N5O 9.78%), 269 (C14H12F1N5, 100%). Anal. calc. for
(C14H12F3N5O) (M.W. = 323): C, 52.01; H, 3.74; N, 21.66%;
found: C, 52.27; H, 3.87; N, 21.85%.
4.4.2. 5-(Piperazin-1-yl)-3-(trifluoromethyl)-[1,2,4]triazolo-
[4,3-c]quinazoline (28).
Greenish white solid, yield: 80%; m.p. 143–145 1C. IR
(KBr) cmÀ1: 3081 (CH aromatic), 1620 (CQN). MS (m/z):
280 (C15H9ClN4), 280 (C15H9ClN4, M +, 100%), 282 (C15H9ClN4,
M + 2, 31.08%). Anal. calc. for (C15H10N4O) (M.W. = 280): Faint brown solid, yield: 76%; m.p. 258–260 1C. IR (KBr) cmÀ1
:
C, 64.18; H, 3.23; N, 19.96%; found: C, 64.20; H, 3.20; 3422 (NH), 3065 (CH aromatic), 2925 (CH aliphatic), 1609
N, 20.01%.
(CQN), 1551 (CQC aromatic). 1H NMR (DMSO-d6) d ppm:
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8.14 (d, 1H, J = 8 Hz, quinazoline-H7), 7.99 (dd, 1H, J = 7.7 Hz, White solid, yield: 69%; m.p. 269–271 1C. IR (KBr) cmÀ1: 3075
quinazoline-H8), 7.46 (d, 1H, J = 8.4 Hz, quinazoline-H10), 7.71 (CH aromatic), 2974 (CH aliphatic), 1625 (CQN), 1552 (CQC
1
(dd, 1H, J = 8.4 Hz, quinazoline-H9), 3.88 (t, 4H, (CH2)2–N), 2.81 aromatic). H NMR (DMSO-d6) d ppm: 8.42 (d, 1H, J = 7.2 Hz,
(t, 4H, (CH2)2–NH), 1.29 (s, 1H, NH). MS (m/z): 322 (C14H13F3N6, quinazoline-H7), 8.26 (d, 1H, J = 8.4 Hz, quinazoline-H10), 8.02
23.01%, M+), 278 (C12H7F3N5, 23.70%), 264 (C11H5F3N5, 9.53%), (dd, 2H, J = 8, phenyl-H2, H6), 7.65 (dd, 2H, J = 8, phenyl-H3,
57 (C3H7N, 100%). Anal. calc. for (C14H13F3N6) (M.W. = 322): C, H5), 7.42 (dd, 1H, J = 7.7 Hz, quinazoline-H8), 7.12 (dd, 1H, J =
52.17; H, 4.07; N, 26.08%; found: C, 52.40; H, 4.28; N, 25.91%. 8.4 Hz, quinazoline-H9), 3.68 (t, 4H, J = 8.4 Hz, (CH2)2N), 3.57 (t,
4.4.3. N-Butyl-3-phenyl-[1,2,4]triazolo[4,3-c]quinazolin-5-
amine (29).
4H, J = 4.5 Hz, (CH2)2O). 13C NMR (DMSO-d6) d ppm: 162.34
(quinazoline-C2), 157.80 (quinazoline-C4), 154.98 (triazole-C3),
138.82 (quinazoline-C8a),
135.62 (phenyl-C4), 134.80
(quinazoline-C7), 129.02 (phenyl-C1), 127.63 (phenyl-C2, C6),
124.63 (phenyl-C3, C5), 120.00 (quinazoline-C8), 116.70
(quinazoline-C5), 115.18 (quinazoline-C6), 110.79 (quinazoline-
C4a), 67.00, (CH2OCH2), 45.89 (CH2NCH2). MS (m/z): 367
(C19H16ClN5O, M2+), 365 (C19H16ClN5O, M+, 38.19%), 337
(C17H12ClN5O, 12.67%), 296 (C15H9ClN4O, 23.55%). Anal. calc.
for (C19H16ClN5O) (M.W. = 365): C, 62.38; H, 4.41; N, 19.14%;
found: C, 62.59; H, 4.38; N, 19.95%.
Faint greenish white solid, yield: 62%; m.p. 267–269 1C. IR (KBr)
cmÀ1: 3331 (NH), 3059 (CH aromatic), 2958 (CH aliphatic), 1627
(CQN), 1524 (CQC aromatic). 1H NMR (DMSO-d6) d ppm: 8.05 (d,
1H, J = 8 Hz, quinazoline-H7), 7.88 (d, 1H, J = 8.4 Hz, quinazoline-
H10), 7.81 (s, 1H, NH), 7.52 (dd, 2H, J = 8 Hz, phenyl-H2, H6), 7.43
(dd, 1H, J = 8.4 Hz, quinazoline-H8), 7.42 (dd, 1H, J = 8.4 Hz,
quinazoline-H9), 7.04 (dd, 3H, J = 8 Hz, phenyl-H3, H4, H5), 3.35 (t,
2H, CH2–NH), 1.51 (m, 2H, CH2), 1.35 (m, 2H, CH2), 0.93 (t, 3H, J =
8 Hz, CH3). MS (m/z): 317 (C19H19N5, 8.23%, M+), 262 (C16H14N4,
1.62%), 77 (C6H5, 100%). Anal. calc. for (C19H19N5) (M.W. = 317): C,
71.90; H, 6.03; N, 22.07%; found: C, 63.87; H, 4.95; N, 20.91%.
4.4.4. 3-Phenyl-5-(piperazin-1-yl)-[1,2,4]triazolo[4,3-c]-
quinazoline (30).
4.4.6. 3-(4-Chlorophenyl)-N-ethyl-[1,2,4]triazolo[4,3-c]qui-
nazolin-5-amine (32).
White solid, yield: 69%; m.p. 259–261 1C. IR (KBr) cmÀ1: 3232
(NH), 3078 (CH aromatic), 2931 (CH aliphatic), 1624 (CQN),
1
1558 (CQC aromatic). H NMR (DMSO-d6) d ppm: 7.83 (s, 1H,
NH, D2O exchangeable), 7.73 (d, 1H, J = 7.2 Hz, quinazoline-
H7), 7.71 (d, 1H, J = 8.4 Hz, quinazoline-H10), 7.66 (dd, 2H, J = 8
Hz, phenyl-H2, H6), 7.59 (dd, 1H, J = 7.7 Hz, quinazoline-H8),
7.46 (dd, 1H, J = 8.4 Hz, quinazoline-H9), 7.40 (dd, 2H, J = 8 Hz,
phenyl-H3, H5), 3.18 (q, 2H, CH2–NH), 1.11 (t, 3H, J = 8.4 Hz,
CH3). MS (m/z): 325 (C17H14ClN5, 12.82%, M+ + 2), 323
(C17H14ClN5, 41.8%, M+), 294 (C15H9 ClN5, 20.21%). Anal. calc.
for (C17H14ClN5) (M.W. = 323): C, 63.06; H, 4.36; N, 21.63%;
found: C, 63.28; H, 4.50; N, 21.49%.
Yellowish white solid, yield: 73%; m.p. 251–253 1C. IR (KBr) cmÀ1
:
3422 (NH), 3066 (CH aromatic), 2925 (CH aliphatic), 1610 (CQN),
1551 (CQC aromatic). 1H NMR (DMSO-d6) d ppm: 8.13 (dd, 2H, J =
8 Hz, phenyl-H2, H6), 8.07 (d, 1H, J = 8 Hz, quinazoline-H10), 7.65
(dd, 1H, J = 7.7 Hz, quinazoline-H9), 7.61 (dd, 3H, J = 8 Hz, phenyl-
H3, H4, H5), 7.56 (d, 1H, J = 8.4 Hz, quinazoline-H7), 7.19 (dd, 1H,
J = 8.4 Hz, quinazoline-H8), 3.21 (t, 4H, (CH2)2N), 2.39 (t, 4H,
(CH2)2NH), 1.21 (s, 1H, NH). MS (m/z): 330 (C19H18N6, 32.42%, M+),
77 (C6H5, 100%). Anal. calc. for (C19H18lN6) (M.W. = 330): C, 69.07;
H, 5.49; N, 25.44%; found: C, 69.15; H, 5.71; N, 25.16%.
4.4.5. 4-(3-(4-Chlorophenyl)-[1,2,4]triazolo[4,3-c]quinazolin-
5-yl)morpholine (31).
4.4.7. 3-(4-Chlorophenyl)-N-propyl-[1,2,4]triazolo[4,3-c]qui-
nazolin-5-amine (33).
Yellowish brown solid, yield: 60%; m.p. 262–264 1C. IR (KBr)
cmÀ1: 3421 (NH), 3078 (CH aromatic), 2960 (CH aliphatic), 1628
(CQN), 1544 (CQC aromatic). 1H NMR (DMSO-d6) d ppm: 8.34
(d, 1H, J = 8 Hz, quinazoline-H7), 8.18 (dd, 2H, J = 8 Hz, phenyl-
H2, H6), 8.12 (d, 1H, J = 8.4 Hz, quinazoline-H10), 7.80 (dd, 1H,
J = 7.7 Hz, quinazoline-H8), 7.74 (dd, 2H, J = 8 Hz, phenyl-H3,
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H5), 7.70 (s, 1H, NH), 7.65 (dd, 1H, J = 8.4 Hz, quinazoline-H9), 8.14 (d, 1H, J = 8 Hz, quinazoline-H7), 7.99 (dd, 1H, J = 7.7 Hz,
3.58 (t, 2H, J = 8.4, CH2–NH), 1.27 (m, 2H, CH2), 0.92 (t, 3H, J = quinazoline-H8), 7.71 (dd, 1H, J = 8.4 Hz, H9 quinazoline-H9), 7.63
8.0, CH3). MS (m/z): 339 (C18H16ClN5, 9.50%, M+ + 2), 337 (dd, 2H, J = 8 Hz, phenyl-H3, H5), 7.46 (d, 1H, J = 8.4 Hz,
(C18H16ClN5, 24.77%, M+), 322 (C17H13ClN5, 3.00%), 308 quinazoline-H10), 7.39 (dd, 2H, J = 8 Hz, p-aminoacetophenone-
(C16H11ClN5, 7.07%), 294 (C15H9ClN5, 29.36%), 279 (C15H8ClN4, H2, H6), 2.42 (s, 3H, CH3). 13C NMR (DMSO-d6) d ppm: 162.34
2.70%). Anal. calc. for (C18H16ClN5) (M.W. = 337): C, 64.00; H, (CQO), 153.89 (quinazoline-C2), 144.23 (quinazoline-C4),
4.77; N, 20.73%; found: C, 63.87; H, 4.95; N, 20.91%.
4.4.8. N-benzyl-3-(4-chlorophenyl)-[1,2,4]triazolo[4,3-c]qui-
nazolin-5-amine (34).
137.51 (triazole-C3), 135.59 (quinazoline-C8a), 133.27 (p-amino
acetophenone-C1), 132.27 (phenyl-C1), 129.53 (quinazoline-C7),
129.05 (phenyl-C3, C5), 128.95 (p-amino acetophenone-C2–C6),
124.58 (phenyl-C2, C4, C6), 124.04 (quinazoline-C5, C6), 116.54
(quinazoline-C4a), 110,68 (quinazoline-C8), and 25.92 (CH3).
MS (m/z): 415 (C23H16ClN5O, 3.68%, M+ + 2), 413 (C23H16ClN5 O,
10.78%, M+), 399 (C22H14ClN5O, 1.51%), 371 (C21H14ClN5, 1.69%),
295 (C15H10ClN5, 6.16%). Anal. calc. for (C23H16ClN5O) (M.W. = 413):
C, 66.75; H, 3.90; N, 16.92%; found: C, 66.91; H, 4.17; N, 17.05%.
4.4.10. 3-(4-Chlorophenyl)-5-(piperazin-1-yl)-[1,2,4]triazolo-
[4,3-c]quinazoline (36).
Yellowish white solid, yield: 74%; m.p. 268–270 1C. IR (KBr)
cmÀ1: 3295 (NH), 3061 (CH aromatic), 2927 (CH aliphatic), 1621
(CQN), 1548 (CQC aromatic). 1H NMR (DMSO-d6) d ppm: 8.30
(d, 1H, J = 8 Hz, quinazoline-H7), 8.22 (dd, 2H, J = 8 Hz, phenyl-
H2, H6), 8.00 (d, 1H, J = 8.4 Hz, quinazoline-H10), 7.78 (dd, 1H,
J = 4 Hz, quinazoline-H8), 7.38 (dd, 2H, J = 8 Hz, phenyl-H3,
H5), 7.26 (m, 5H, benzylic protons), 7.24 (s, 1H, NH, D2O
exchangeable), 7.09 (dd, 1H, J = 8.4 Hz, quinazoline-H9), 4.35
Yellowish white solid, yield: 81%; m.p. 254–256 1C. IR (KBr) cmÀ1
:
3451 (NH), 3050 (CH aromatic), 2924 (CH aliphatic), 1626
(CQN), 1555 (CQC aromatic). 1H NMR (DMSO-d6) d ppm:
8.33 (d, 1H, J = 8 Hz, quinazoline-H10), 8.18 (dd, 2H, J = 8 Hz,
phenyl-H2, H6), 7.85 (dd, 1H, J = 7.7 Hz, quinazoline-H9), 7.70
(d, 1H, J = 8.4 Hz, quinazoline-H7), 7.64 (dd, 2H, J = 8 Hz, phenyl-H3,
H5), 7.39 (dd, 1H, J = 8.4 Hz, quinazoline-H8), 3.36 (t, 4H, (CH2)2-N),
2.84 (t, 4H, (CH2)2-NH), 1.29 (s, 1H, NH). MS (m/z): 366 (C19H17ClN6,
3.75%, M+2), 364 (C19H17ClN6, 7.34%, M+), 76 (C6H4, 100%). Anal.
calc. for (C19H17clN6) (M.W. = 364): C, 62.55; H, 4.70; N, 23.04%;
found: C, 62.73; H, 4.79; N, 22.87%.
(s, 2H, CH2–NH). 13C NMR (DMSO-d6)
d ppm: 157.74
(quinazoline-C2), 155.69 (quinazoline-C4), 140.86 (triazole-C3),
139.02 (quinazoline-C8a), 134.52 (benzylic-C1), 130.31 (phenyl-
C4), 129.32 (quinazoline-C8), 128.72 (phenyl-C1), 128.51
(phenyl-C2, C6), 127.88 (phenyl-C3, C5), 127.54 (benzylic-C3,
C5), 127.13 (benzylic-C2, C4, C6), 121.40 (quinazoline-C6, C7),
120.57 (quinazoline-C5), 115.50 (quinazoline-C4a), and 43.39
(CH2NH). MS (m/z): 387 (C22H16ClN5, 20.90%, M+ + 2), 385
(C22H16ClN5, 46.40%, M+), 274 (C16H12N5, 29.42%), 196
(C10H6N5, 16.08%). Anal. calc. for (C22H16ClN5) (M.W. = 385):
C, 68.48; H, 4.18; N, 18.15%; found: C, 68.61; H, 4.42; N, 17.98%.
4.4.9. 1-{4-[(3-(4-Chlorophenyl)-[1,2,4]triazolo[4,3-c]quina-
zolin-5-yl)amino]phenyl}ethan-1-one (35).
4.5. Biological evaluation
4.5.1. In vitro cytotoxic activity. Four human tumor cell
lines, namely, hepatocellular carcinoma (HePG-2), mammary
gland breast cancer (MCF-7), human prostate cancer (PC3), and
colorectal carcinoma (HCT-116), were obtained from VACSERA,
Cairo, Egypt. Doxorubicin was used as a standard anticancer
drug for comparison. The inhibitory effects of our target
compounds on cell growth of the above-mentioned cell lines
were determined using the MTT assay.42 Cells were cultured in
RPMI-1640 medium with 10% fetal bovine serum. Penicillin
(100 mg mlÀ1) and streptomycin (100 units per ml) antibiotics
were added at 37 1C in a 5% CO2 incubator. The cells were then
seeded in a 96-well plate at a density of 1.0 Â 104 cells per well
at 37 1C for 48 h under 5% CO2. After incubation, the cells were
treated with different concentrations of compounds and incubated
Faint brown solid, yield: 66%; m.p. 263–265 1C. IR (KBr) cmÀ1
:
3182 (NH), 3050 (CH aromatic), 2924 (CH aliphatic), 1752 (CQO), for 24 h. After 24 h of drug treatment, 20 ml of MTT solution at
1
1625 (CQN), 1598 (CQC aromatic). H NMR (DMSO-d6) d ppm: 5 mg mlÀ1 was added and incubated for 4 h. 100 ml DMSO is
12.34 (S, 1H, NH, D2O exchanged), 8.29 (dd, 2H, J = 8 Hz, phenyl-H2, added into each well to dissolve the purple formazan formed. A
H6), 8.20 (dd, 2H, J = 8 Hz, p-aminoacetophenone-H3, H5), colorimetric assay was performed at a wavelength of 570 nm using
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a plate reader (EXL 800, USA). The relative cell viability in percentage References
was calculated. Individual IC50 values of the final target compounds
are summarized in Table 1.
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Molecular docking of the final target compounds was per-
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each case, 20 docked structures were generated using genetic
algorithm searches; Affinity dG & London dG were used for
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A. Ghiaty, A. H. Bayoumi, and H. S. Abulkhair were responsible
for the conception and rational design of the work. M. H.
El-Shershaby, A. Ghiaty, and H. S. Abulkhair were responsible
for the data collection and synthesis of the new compounds.
K. El-Adl, H. E. A. Ahmed, and H. S. Abulkhair performed the
molecular docking study. A. Ghiaty, A. H. Bayoumi, M. S.
El-Zoghbi, and H. S. Abulkhair were responsible for spectral data
analysis. M. H. El-Shershaby and M. S. El-Zoghbi conducted the
cytotoxicity assay. H E. A. Ahmed and K. El-Adl conducted the
in silico pharmacokinetic study. All authors discussed the results
and contributed to the writing and revision of the original
manuscript.
Conflicts of interest
The authors declare that they have no competing financial
interests or personal relationships that could have appeared
to influence the work reported in this article.
Acknowledgements
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H. Kusaba, K. Akashi, M. Kuwano and K. Kohno, Enhanced
Expression of PCAF Endows Apoptosis Resistance in Cisplatin-
Resistant Cells, Mol. Cancer Res., 2010, 8, 864–872, DOI:
10.1158/1541-7786.MCR-09-0458.
The authors would like to express their appreciation to Prof.
Dr Magda A. El-Sayed, Professor of Pharmaceutical Chemistry
at Horus University in Egypt, for her valuable help throughout
proofreading the article and the revision stages.
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