Design, synthesis and evaluation of novel hybrids between 4-anilinoquinazolines and substituted triazoles as potent cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2018.10.016

In this research several series of novel dioxygenated ring fused 4-anilinoquinazolines (10a-d) and 4-anilinoquinazoline-substituted triazole hybrid compounds (11–14) have been designed and synthesized. Their biological significance was highlighted by evaluating in vitro for anticancer activities, wherein several compounds displayed excellent activity specifically against three human cancer cell lines (KB, epidermoid carcinoma; HepG2, hepatoma carcinoma; SK-Lu-1, non-small lung cancer). Especially, compound 13a exhibited up to 100-fold higher cytotoxicity in comparison with erlotinib. Docking the most cytotoxic compounds (11d, 13a, 13b, and 14c) into the ATP binding site of different EGFR tyrosine kinase domains was perfomed to predict the analogous binding mode of these compounds to the EGFR targets.

Full text of DOI:10.1016/j.bmcl.2018.10.016

Accepted Manuscript
Design, synthesis and evaluation of novel hybrids between 4-anilinoquinazo-
lines and substituted triazoles as potent cytotoxic agents
Giang Le Nhat Thuy, Thuy Van Dinh, Hai Pham-The, Hung Nguyen Quang,
Nga Nguyen Thi, Tuyet Anh Dang Thi, Phuong Hoang Thi, Tu Anh Le Thi, Ha
Thanh Nguyen, Phuong Nguyen Thanh, Trung Le Duc, Tuyen Van Nguyen
PII:
S0960-894X(18)30806-0
https://doi.org/10.1016/j.bmcl.2018.10.016
BMCL 26075
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
18 April 2018
9 October 2018
10 October 2018
Please cite this article as: Thuy, G.L.N., Van Dinh, T., Pham-The, H., Quang, H.N., Thi, N.N., Thi, T.A.D., Thi,
P.H., Thi, T.A.L., Nguyen, H.T., Thanh, P.N., Duc, T.L., Nguyen, T.V., Design, synthesis and evaluation of novel
hybrids between 4-anilinoquinazolines and substituted triazoles as potent cytotoxic agents, Bioorganic & Medicinal
Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.016
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DESIGN, SYNTHESIS AND EVALUATION OF NOVEL HYBRIDS BETWEEN 4-
ANILINOQUINAZOLINES AND SUBSTITUTED TRIAZOLES AS POTENT
CYTOTOXIC AGENTS
a,b
b
c
c
Giang Le Nhat Thuy , Thuy Van Dinh , Hai Pham-The , Hung Nguyen Quang , Nga Nguyen
b
a,b
a
a
a
Thi , Tuyet Anh Dang Thi , Phuong Hoang Thi , Tu Anh Le Thi , Ha Thanh Nguyen , Phuong
d
d
a,b
Nguyen Thanh , Trung Le Duc , Tuyen Van Nguyen *
a
Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
b
Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau
Giay, Hanoi, Vietnam
c
Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi, Vietnam
Chu Van An High School, 10, Thuy Khue, Hanoi, Vietnam
d
*
Corresponding author. Tel.: +84 917683979.
E-mail address: ngvtuyen@hotmail.com, ngvtuyen@ich.vast.vn (T. Van Nguyen)
ABSTRACT
In this research several series of novel dioxygenated ring fused 4-anilinoquinazolines
(
10a-d) and 4-anilinoquinazoline-substituted triazole hybrid compounds (11-14) have been
designed and synthesized. Their biological significance was highlighted by evaluating in vitro
for anticancer activities, wherein several compounds displayed excellent activity specifically
against three human cancer cell lines (KB, epidermoid carcinoma; HepG2, hepatoma
carcinoma; SK-Lu-1, non-small lung cancer). Especially, compound 13a exhibited up to 100-
fold higher cytotoxicity in comparison with erlotinib. Docking the most cytotoxic compounds
(
11d, 13a, 13b, and 14c) into the ATP binding site of different EGFR tyrosine kinase domains
was perfomed to predict the analogous binding mode of these compounds to the EGFR
targets.
Keywords: 4-anilinoquinazolines; triazole; hybrids; cytotoxic agents; EGFR inhibitors
As a class of nitrogen-containing heterocyclic compounds, quinazoline derivatives have
been widely known as powerful inhibitors of the epidermal growth factor receptors (EGFRs)
1
2,3
of tyrosine kinase, ligands for benzodiazepine and GABA receptors in the CNS system or
4
as DNA binders. Quinazoline derivatives have shown remarkable biological activities, such
as antiinflammatory, antibacterial, antiviral, and most importantly, anticancer activities
5
-7
8
9
10-
1
4
that makes them as one important pharmacophore widely used in the developing novel
anticancer drugs. Representative drugs, like gefitinib, erlotinib, and lapatinib (Fig. 1) have
been approved by the FDA and were successfully applied in clinic for the treatment of
1
5,16
16
17
multiple cancers, such as non-small cell lung cancer,
pancreatic cancer, breast cancer.
The structure-activity relationship (SAR) of EGFR inhibitors shows that the 4-
anilinoquinazoline scaffold is crucial to EGFR inhibitory activity, and the C6- and C7-
position side chains are mainly contribute to their physicochemical properties with good
1
8-21
compatibility for bulky moieties.
As a result, in recent years, numerous 4-
anilinoquinazoline derivatives have been designed and synthesized in succession. Among
which, crown ether fused anilinoquinazoline analogues were synthesized as novel EGFR
2
2-24
tyrosine kinase inhibitors.
SAR studies indicated that oxygen containing heterocycles with
1

ring size higher than 12 member are the favorable fused anilinoquinazolines, and the preferred
substituent on the 4-anilino is a halogen such as chlorine, bromine, or phenyl group at the
2
3,24
meta-position.
Some of them proved to be active in an EGFR-mediated intracellular
2
3
tyrosine phosphorylation assay in human tumor cell line A431, while another showed a very
potent antiproliferative activity by the inhibition of both receptor tyrosine kinase including
EGFR, VEGFR, PDGFR, and nonreceptor TKs including C-Src and Abl kinase with superior
inhibition activity against EGFR. In the light of the results mentioned above, and taking
erlotinib as the leading compound, we have devised and synthesized the serie of dioxygenated
ring fused quinazolines containing the ethynyl group at the meta-position of anilino ring, with
the aim of obtaining agents displaying more potent anticancer activities.
2
4
Fig 1. Structures of some EGFR inhibitors
On the other hand, 1,2,3-triazole fragment is of great importance in medicinal chemistry
and can be used for the synthesis of numerous heterocyclic compounds with different
2
5-27
28-30
biological activitives, including anticancer activities,
and antiproliferative properties.
Remarkably, this framework was widely used as a building block in the design of anticancer
3
1
agents because of its ability to form hydrogen-bonding interactions with drug targets.
3
2
33-35
Recently, several 1,2,3-triazole-dithiocarbamate hybrids, triazole-quinazolinone hybrids,
and other 1,2,3-triazole derivatives have been synthesized and demonstrated impressive
3
6,37
biological activities.
However, the combination of two bioactive moieties 1,2,3-triazole
and 4-anilinoquinazoline into single molecule has not been reported in the literature so far.
These findings have led to the hypothesis that the introduction of 1,2,3-triazolyl group into
targeted dioxygenated ring fused quinazolines at the anilino side chain could give the
promising potent biological compounds. It was expected that this triazole scaffold would
create more hydrogen interaction with the bingding pocket in the hydrophobic environment,
thus increasing the EGFR inhibitory potency and improving the anticancer activities.
Moreover, the ethynyl group in our designed erlotinib analogues easily takes part in the
"Click" reaction with various azides to form the 1,2,3-triazole ring. In this context, in view of
the above fact and to discover completely new cytotoxic agents with a novel skeleton, a
relatively small library of 4-aminoquinazoline-substituted triazole hybrids was constructed via
3
8
the copper(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction and the biological
2

significance of the novel synthesized quinazolines was highlighted by evaluating them for
their cytotoxic activities.
Despite the widespread utility of 4-anilinoquinazolines, the reported syntheses of these
compounds require multistep and low-yielding pathways. The most common synthetic
approach to these compounds involves the preparation of 4(3H)-quinazolinone intermediates,
which are mainly obtained by Niementowski reaction between anthranilic acid derivatives and
3
9-42
formamide.
The key 4(3H)-quinazolinones are then submitted to chlorination at the 4
position and finally the chlorine atom is substituted with the appropriate aniline moiety.
Anilinoquinazolines can be afforded by the reaction of anthranilonitrile with N,N-
dimethylformamide (DMF-DMA), followed by cyclization of produced formamidine
4
3,44
derivatives with different amines in acetic acid.
Scheme 1: Preparation of dioxygenated ring fused quinazolines 10a-d. Reagents and conditions: (a)
NH OH.HCl, NaOH, MeOH, H O, rt, 30-60 min, 95-98%; (b) Ac O, reflux, 8-12 h, 90-95%; (c) Na , H O,
COOH, 50 C, 4 h, 90-95%; (e) DMF-DMA, acetic acid, toluene, reflux,
2
2
2
2
S
2
O
4
2
o
o
5
4
0-65 C, 3-4 h, 80-85%; (d) HNO
-6 h; (f) 3-ethynylaniline, acetic acid, toluene, 60 C-110 C, 4-6 h, 50-63%.
3 3
, CH
o
o
In this study, the synthesis of dioxygenated ring fused quinazolines 10a-d were
achieved in 5-6 steps from various benzaldehydes as depicted in Scheme 1. The aldoximes
a,b and 5a,b, prepared by the treatment of benzaldehydes 1a,b or 4a,b with hydroxylamine,
were subjected to intramolecular dehydration with anhydride acetic to afford the benzonitriles
a,b and 6a,b, respectively (up to quantitative yields). Reduction of the nitro group of 3a,b
2
3
using sodium dithionite in acidic solution gave the corresponding amines 8a,b. In the case of
compounds containing two or three methylene groups in the dioxygenated ring fused, the
o
nitrile compounds 6a,b were nitrated with 65% nitric acid in acetic acid at 50 C and the
resulted nitroderivatives 7a,b were subsequently submitted to reduction with sodium
dithionite in acidic solution to furnish the corresponding amines 8c,d. Afterwards, the
coupling of amines 8a-d with DMF-DMA gave the formamidine intermediates 9a-d, which
3

were subjected to cyclization with 3-ethynylanilines to afford the target quinazolines 10a-d in
0-63% yields. The structures of the synthesized compounds 10a-d were determined
5
1
straightforwardly based on analysis of spectroscopic data, including IR and H NMR.
The second section of the work deals with the syntheses of 4-anilinoquinazoline–
substituted triazole hybrids, showing different quinazolines and constant triazole substitution
patterns. Most of the EGFR-tyrosine kinase inhibitors have the same 4-anilinoquinazoline
skeleton, only the substituents and the side chains are variable. Therefore, the replacement of
the acetylene moiety at the C3 position of the phenyl ring by a triazole nucleus could rigid the
resulting structure by only one single bonding between the triazole and the quinazoline
nucleus. Moreover, hydrogen bonding between the triazole ring and the peptide backbone of
the EGF receptor could afford specific conformations, improving the inhibitory activities of
the resulting derivatives. Besides, with respect to the triazolyl substituent, we consider
bioactive functionalization including nitrophenyl and cyanotrifluoromethylphenyl. Due to the
specific chemical and physical properties of nitrogen and fluorine, the introduction of a NO
CF , and CN moieties in pharmacologically active compounds is known to convey beneficial
biological effects to the resulting molecules, hence the increasing interest from organic and
2
,
3
4
5-52
medicinal chemists in polyfunctional NO
2
-, CF
3
-, and CN-substituted scaffolds.
In that
respect, copper(I) catalyzed click reaction (CuAAC) of the alkyne key intermediates 10a-d
with nitrophenyl- and cyanotrifluoromethylphenylazides generated the target 4-
anilinoquinazoline–substituted triazole hybrid compounds 11-14 in 70-90% yields (Scheme
1
13
2
). The structure of hybrid compounds 11-14 was determined by their H NMR, C NMR,
1
and MS (ESI) spectra. Notably, the H NMR spectrum showed a singlet at 9.17-9.65 ppm
corresponding to the triazolyl proton, while the C NMR spectrum showed peaks at 120-123
ppm and 147-149 ppm corresponding to CH and Cq characteristic to the triazole core unit.
1
3
Scheme 2. Synthesis of 4-anilinoquinazoline–substituted triazole hybrids. Reagents and conditions: 1 equiv 4-
anilinoquinazoline 10, 1.1 equiv azide, 12 equiv DIPEA, 0.2 equiv CuI, THF, rt, 1-2 days, 70-90%.
The synthesized compounds were evaluated for their cytotoxicity against three human
cancer cell lines, including KB (epidermoid carcinoma cancer), HepG2 (hepatoma carcinoma
cancer) and SK-Lu-1 (non-small lung cancer). Erlotinib, erlotinib hydrochloride and
ellipticine were used as positive controls. The results are presented in Table 1. As shown in
Table 1, in general, the synthesized compounds exhibited good cytotoxic inhibitory effects
with IC50 values in submicromolar range in most cases. Most of the active compounds
exhibited higher inhibitory activity than those of the reference drugs erlotinib and erlotinib
hydrochloride.
4

Table 1
Cytotoxicity of the synthesized compounds against three human cancer cell lines
1
2
Entry
R ,R
R
-
2-NO
3-NO
4-NO
Compound
IC50 (KB), µM
5.46 ± 0.14
5.50 ± 0.19
104.20 ± 3.20
5.47 ± 0.24
1.46 ± 0.07
75.60 ± 2.30
193.33 ± 6.42
6.35 ± 0.02
30.10 ± 2.04
4.59 ± 0.01
2.60 ± 0.81
0.04 ± 0.01
3.51 ± 0.12
79.09 ± 0.43
0.27 ± 0.83
54.83 ± 1.85
20.44 ± 1.21
53.17 ± 2.37
1.49 ± 0.09
13.01 ± 0.61
IC50 (HepG2), µM
4.16 ± 0.09
4.64 ± 0.12
286.75 ± 4.70
8.11 ± 0.37
1.86 ± 0.07
26.17 ± 0.69
207.01 ± 4.31
6.88 ± 0.02
11.29 ± 0.56
6.10 ± 0.03
2.84 ± 0.09
0.14 ± 0.03
0.88 ± 0.03
230.02 ± 0.85
6.09 ± 2.27
69.89 ± 2.34
43.35 ± 1.95
76.81 ± 3.94
1.61 ± 0.08
25.01 ± 1.24
IC50 (Lu), µM
4.16 ± 0.08
4.76 ± 0.12
259.74 ± 1.35
9.16 ± 0.42
4.50 ± 0.21
64.88 ± 1.93
216.84 ± 5.78
6.66 ± 0.02
44.48 ± 2.62
27.46 ± 0.15
3.36 ± 0.08
1.03 ± 0.03
5.67 ± 0.18
54.77 ± 0.32
4.44 ± 0.94
80.67 ± 2.14
14.75 ± 0.81
230.40 ± 7.91
1.81 ± 0.09
99.76 ± 4.21
1
2
3
4
5
6
7
8
9
10a
11a
11b
11c
11d
10b
12a
12b
12c
12d
10c
13a
13b
13c
13d
10d
14a
14b
14c
2
2
2
H
3-CN-4-CF
-
3
2-NO
3-NO
4-NO
2
2
2
2
-OCH O-
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
3-CN-4-CF
-
3
2-NO
3-NO
4-NO
2
2
2
-O(CH
2
)
2
O-
3-CN-4-CF
-
2-NO
3-NO
3-CN-4-CF
Erlotinib
3
2
-
O(CH
2
)
3
O-
2
3
2
2
1
2
Erlotinib.HCl
Ellipticine
49.62 ± 0.16
1.95 ± 0.05
14.17 ± 0.05
2.72 ± 0.04
31.15 ± 0.09
1.38 ± 0.04
Substitution by different oxygen substituent heterocycles on the positions 6 and 7 of
quinazolines skeleton affected the cytotoxic inhibition differently. Compound 10a and the
dioxane derivative 10c are clearly more preferred than dioxolane and dioxepine derivatives
1
0b, 10d. The effect of these unfavorable substitutions may result from steric hindrance.
Moreover, compounds 10a and 10c were found to be more potent cytotoxic inhibitors against
all three cancer cell lines than erlotinib and erlotinib hydrochloride with IC50-values ranging
from 2 to 6 µM.
The coupling of substituted 1,2,3-triazolyl groups with targeted dioxygenated ring fused
quinazolines 10a-d at the anilino side chain was found to greatly enhance the cytotoxicity of
the resulting hybrid compounds 11-14. It is important to note that these separate
pharmacophores and the reference drugs display considerably less potent cytotoxic activities
(
1
IC50-values ranging from 2 to 100 µM) as compared to the most promising conjugates
1a,c,d, 12b,d, 13a,b,d and 14a,c (IC50-values ranging from 0.04 µM to 25 µM) showing a
reasonable activity against these cancer cell lines. Preliminary investigation of the structure-
activity relationships (SARs) of these synthesized hybrid compounds 11-14 revealed that the
nature of the oxygen substituent heterocycles and the aryl group which connected to the
triazole influenced the cytotoxicity activity remarkably. For instance, the result revealed that
the cytotoxic activity of dioxane substituted analogues 13a-d are more potent than those of
the corresponding dioxolane, dioxepine counterparts, and the analogues without oxygen
substituent heterocycles (IC50: 13a > 11a > 14a > 12a, 13b > 12b > 14b > 11b, 13d > 14c ≈
1
1d > 12d). Especially, compound 13a displayed the most potent inhibitory activity against
KB, HepG2, and Lu with IC50-values of 0.04 µM, 0.14 µM, and 1.03 µM, respectively, which
was up to 100 fold higher than those of erlotinib. The introduction of an electron-withdrawing
groups such as NO
2
, CF , and CN of the aryl which connected to the triazole can remarkable
3
improve the cytotixic activities. Substitution by a trifluoromethyl group of the aryl seemed to
be better than a nitro (IC50: 11d > 11a > 11c > 11d, 12d > 12b > 12c > 12b, 14c > 14a >
5

1
4b). In addition, the effects of the substituent position seemed to be also dependent on the
substituent nature. In fact, with a nitro substituent, substitution at orto-position was better than
at meta- or para-positions in most cases.
Among the 4-anilinoquinazoline derivatives designed, compounds 11d, 13a-b, and 14c
were identified as novel hits given their excellent in vitro citotoxicity against three human
cancer cell lines. In particular, 13a exhibited up to 100-fold higher cytotoxicity in comparison
with erlotinib, a potent inhibitor that bind both active and inactive conformations of the EGFR
5
3
tyrosine kinase domain. In order to explore the structure-activity relationships at molecular
target, docking simulation of 11d, 13a-b, and 14c into ATP binding site of different
conformations of EGFR were performed. To this end, three common protein structures of
EGFR were modelled, including active (1M17), inactive (4HJO), and L858R mutant (2ITV)
conformations. The docking protocol was evaluated by using the initial conformation of
Erlotinib obtained from 1M17.
As can be visualized in Fig. 2, the catalytic domains of these EGFR kinase proteins
comprise two lobes: an NH -terminal lobe (N-lobe) formed from mostly β-trands and one α-
2
helix, and a larger COOH-terminal lobe (C-lobe) mostly consisted of α-helical. The binding
site is positioned between these two lobes and comprises two regions: the front cleft which
contains ATP-binding site, and the back cleft that contains important elements for the
5
4
regulation of kinase catalysis.
The main difference between active and inactive
conformations relies on the rotation of the α-helix by 45° away from the active site in the N-
lobe and the switch of Asp-Phe-Gly (DFG) motif in the activation loop in the C-lobe that
5
4,55
significantly alter the conformation of the active site.
However, the re-docked orientations
of erlotinib on PDB entries 1M17 and 4HJO revealed that this drug similarly bound to active
and inactive EGFR conformations with binding affinities of -7.8 and -7.1 kCal/mol,
respectively. The same hydrogen-bonding network was observed in two systems, including
quinazoline N-1 and Met769 (distance: 2.3-2.9 Å), and a water-mediated H-bond between
quinazoline N-3 and Thr766 side chain. These findings were in agreement with the
5
3
crystallographic coordinates found for erlotinib binding. On the other hand, erlotinib was
docked into the L858R mutant domains of EGFR using the crystal structures established by
5
6
Yun et al. As shown in amino acid alignment (Fig. 2), this point mutation made a leucine-to-
arginine substitution at position 858, and would lead to enhance the stabilization of active
5
7
conformation of EGFR. The docking result of erlotinib on PDB entry 2ITV revealed that
this drug stricly bound to the L858R mutant with dG of -9.9 kCal/mol, significantly higher
than affinity calculated with non-mutated active conformation. Three H-bonds were formed
between erlotinib and the “gatekeeper” residues Thr790, Met793, and Gly796. In addition, the
drug was mainly accomodated in the ATP-binding site which is a small hydrophobic pocket,
and form multiple pi-alkyl stacking interactions involving quinazoline moiety of erlotinib,
toward Leu718, Val726, Ala743 and Leu844 of L858R mutant. Given the selectivity of
5
8
erlotinib toward L858R mutant over other wild-type EGFRs in clinical trials, current
docking protocol provided acceptable accuracy of the binding mode predictions.
6

Fig 2. Docking poses of erlotinib with protein structures (top panel) and amino acid alignment
bottom panel) of different conformations of EGFR. In black, the residues are conserved
(
between all kinases. Residues in red refer to the amino acid replacements observed in L858R
mutant and inactive EGFRs.
The next steps involved in docking selected hit compounds 11d, 13a-b, and 14c into
binding sites of difference EGFR conformations. The topological docking conformation and
interactions between these compounds and three PDB entries were depicted in Fig. 3. To
better depict the role of 1,2,3-triazolyl group and dioxygenated ring fused quinazolines in
targeting EGFR proteins, table 2 was added with special focus on the interactions of these
moieties.
According to the docking results in the active conformation (1M17), compounds 13a-b
and 14c showed higher affinity (-10.0 to -10.6 kCal/mol) compared to 11d and erlotinib
(
Table 2). Visual inspection of the interactions of 14c with the binding pocket of active EGFR
revealed that two H-bonds were formed in the similar way to erlotinib between dioxygenated
ring fused quinazoline moiety and Thr766 (through a water bridge) and Met769. A H-bond
was also generated between 3-CN substituent and the Lys721 side chain. The 4-
anilinoquinazoline rings deeply inserted into the hydrophobic pocket of active EGFR and
showed multiple pi-alkyl stacking interactions with Leu694, Val702, Ala719, Lys721, and
Leu820.
To the inactive conformation of EGFR (4HJO), all the compounds are more strongly
bound than erlotinib, with 13b and 14c having the highest binding energies of -12.3 and -12.1
kCal/mol, respectively. In cases of 11d and 13a-b, the quinazoline moieties can make one or
two H-bonds with Met769, and the 1,2,3-triazole ring can form an additional water-mediated
7

H-bond with Thr766 or Lys721. For 13a-b and 14c, the dioxygenated fused 4-
anilinoquinazoline structures assembled multiple interactions with the residues in the
hydrophobic ATP-binding cleft and plays a significant role in the binding to the target.
Interestingly, the docking orientations of 13b and 14c extended to outside the hydrophobic
ATP-binding cleft and formed three H-bonds with the side chain residues Thr830, Asp831,
and Phe832. These interactions are quite different from those of erlotinib.
Fig 3. 2D diagrams of docking interactions of compounds 11d, 13a-b and 14c (atoms colored
by element type) in the active sites of three conformations of EGFR protein. Number in
windows represent the free binding energies (kCal/mol) of the compounds.
On the other hands, all four compounds displayed similar affinity to erlotinib (dG
ranges from -9.9 to -10.1 kCal/mol) when bound to L858R mutant domains, with the highest
value corresponding to 13a. The quinazoline and triazole moieties received two hydrogen
bonds from two water molecules which took part in the H-bonding network with Thr790,
Thr854, Ser720, and Phe723. In cases of compounds 13a and 13b an additional H-bond was
formed between triazole ring and Gly724. The docking results also revealed the favourable
van der Waals interactions between all four compounds and the residues in the back of the
ATP-binding cleft (Val726, Ala743, Lys745, and Leu844) of L858R mutant. In general,
compounds 13a and 13b are in very similar positions to the crystal structures of erlotinib
5
6
determined by Yun et al. These findings suggested that our hit compounds may have less
activity against L858R mutated domain compared to the other conformations analyzed herein.
However, more extensive analysis is required in order to identify the difference between
synthesized quinazolines and erlotinib, which ultimately should aid in rationalizing the design
of potent and selective kinase inhibitors.
8

Table 2.
Docking results of binding energies and interactions between quinazoline/triazole moieties
and residues in the active site of different EGFR conformations
Active EGFR (1M17)
Inactive EGFR (4HJO)
L858R Mutation EGFR (2ITV)
Interactions
Interactions
Interactions
with
Interactio
ns with
1,2,3-
Cpd.
code
Interactions
with 1,2,3-
Triazole
Interactions
with 1,2,3-
Triazole
with
3
with
3
3
dG
dG
dG
Quinazoline
2
Quinazoline
2
Quinazoline
1
1
1
2
system
system
system
Triazole
HB*
Thr766
Val702,
,
HB*
HB
Thr766
Val702,
Ala719
,
Thr790
Ala743,
Leu844
,
HB
Cys773,
Arg817(2)
Gly724
Lys745
,
1
1d
Ala719(2),
Lys721,
Leu820(2)
Arg817
-9.5
-10.0
-10.2
-10.6
-10.6
-12.3
-9.9
-10.1
-9.9
HB*$
Thr766
Lys721
,
,
HB
Thr790
Thr854
,
,
HB$
HB$
Thr766
,
HB
HB
HB
Val702(2)
Ala719,
Leu820(2)
Lys721
Phe699
,
Leu694,
Val702(2),
Ala719,
Lys721
Asp831
,
1
3a
Val726,
Ala743,
Leu844
NE
Leu820(2)
HB$
Lys745
Thr790
,
,
HB$*
HB*
Thr766
,
Thr766
Thr830
,
HB$
Leu694(3),
Ala719,
Leu820
HB*,
Val702,
Ala719(2),
Leu820(2)
13b
NE
Val726(2),
Ala743(2),
Leu844
Lys745
Val702,
Ala719,
HB*$
HB$
Thr766
Met769
,
Thr790
Thr854
,
HB$*
Thr766
,
HB
HB$
,
,
Leu694,
Leu694,
Val726(2),
Ala743(2),
Val720(2),
Ala719(2),
$
14c
Val702(2) ,
Ala719(2),
Phe699
-10.6
Ser696
-12.1
Lys745
-10.0
$
Lys745 ,
$
Lys721 ,
$
$
Lys721 ,
Leu788 ,
Leu820(2),
Leu820(2),
Leu844(2)
HB*
Thr790
Met793
,
HB*
HB*
Thr766
Met769
Leu694,
,
,
Thr766
Met769
Leu694,
Ala719,
,
,
HB
,
HB
HB
Leu718,
Erlotinib
NE
-7.8
NE
-7.1
NE
-9.9
Val726(2),
Ala743(2),
Leu844(2)
Ala719(2),
Leu820(2)
Leu820(2)
1
2
3
Residues interacting with dioxygenated fused quinazoline moieties; residues interacting with 1,2,3-triazole ring; free
HB
energy binding (kCal/mol) of the ligands and EGFR proteins computed by affinity scoring function; hydrogen bonding
interaction; Water mediated hydrogen bonds; interaction with oxygen heterocycles; NE: not exists; in parenthesis ():
number of interactions.
*
$
In summary, we have reported a serie of dioxygenated fused 4-anilinoquinazolines and
three series of 4-anilinoquinazoline–substituted triazole hybrid compounds with potent
cytotoxicity against three human cancer cell lines, including KB (epidermoid carcinoma
cancer), HepG2 (hepatoma carcinoma cancer), and SK-Lu-1 (non-small lung cancer). Several
compounds, e.g. 10a,c, 11a,c,d, 12b,d, 13a,b,d and 14a,c displayed up to 100-fold more
potent than reference drugs erlotinib and erlotinib hydrochloride in term of cytotoxicity.
Preliminary investigation of the structure-activity relationships (SARs) of these synthesized
hybrid compounds 11-14 revealed that the nature of the oxygen substituent heterocycles and
the aryl group which connected to the triazole influenced the cytotoxicity activity remarkably.
The size of the fused dioxygenated ring was crucial for the biological activity, the dioxane
derivatives being the most promissing class of these series. The different substitutents and the
9

effects of the substituent position substantially influenced cytotoxicity of the resulting
compounds. The molecular docking studies showed that 13a-b and 14c are more strongly
bound than erlotinib to the ATP binding site of the active and inactive conformations of
EGFR, meanwhile they are equally potent binders to the L858R mutated EGFR. In particular,
the H-bonding interactions of 1,2,3-triazole ring as well as the dense hydrophobic interaction
network of dioxygenated ring fused quinazolines with the residues in the ATP pocket play an
important role in EGFR binding. The results we obtained from this study suggest that the
introduction of 1,2,3-triazolyl group into dioxygenated ring fused quinazolines at the anilino
side chain increases the EGFR inhibitory potency and improves the anticancer properties.
Acknowledgement
The authors are indebted to the Vietnamese National Foundation for Science and
Technology Development (NAFOSTED, code: 104.01-2016.22) for financial support.
A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/10.1016/j.bmcl.
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1

1
2

GRAPHICAL ABSTRACT
DESIGN, SYNTHESIS AND EVALUATION OF NOVEL HYBRIDS BETWEEN 4-
ANILINOQUINAZOLINES AND SUBSTITUTED TRIAZOLES AS POTENT CYTOTOXIC AGENTS
1
3

DESIGN, SYNTHESIS AND EVALUATION OF NOVEL HYBRIDS BETWEEN 4-
ANILINOQUINAZOLINES AND SUBSTITUTED TRIAZOLES AS POTENT CYTOTOXIC AGENTS
a,b
b
c
c
b
Giang Le Nhat Thuy , Thuy Van Dinh , Hai Pham-The , Hung Nguyen Quang , Nga Nguyen Thi ,
a,b
a
a
d
Tuyet Anh Dang Thi , Phuong Hoang Thi , Ha Thanh Nguyen , Phuong Nguyen Thanh , Trung Le
Duc , Tu Anh Le Thi , Tuyen Van Nguyen *
d
a
a,b
a
Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi,
Vietnam
b
Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet,
Cau Giay, Hanoi, Vietnam
c
Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi, Vietnam
d
Chu Van An High School, 10, Thuy Khue, Hanoi, Vietnam
*
Corresponding author. Tel.: +84 917683979.
E-mail address: ngvtuyen@hotmail.com, ngvtuyen@ich.vast.vn (T. Van Nguyen)

Fifteen new 4-anilinoquinazoline-substituted triazole hybrid compounds were designed
and synthesized.



Their anticancer activities against human cancer cell lines were evaluated.
Compound 13a exhibited up to 100-fold higher cytotoxicity in comparison with erlotinib.
Molecular docking suggests greater affinity of 13a-b and 14c than erlotinib for the ATP
binding sites of the active and inactive conformations of EGFR.
1
4