Design, synthesis and biological evaluation of novel 4-anilinoquinazoline derivatives as hypoxia-selective EGFR and VEGFR-2 dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.07.055

Tyrosine kinase inhibitors (TKIs) have achieved substantial clinical effects for cancer treatment while causing a number of adverse effects. Since hypoxia is an intrinsic difference between solid tumor and healthy tissues, one strategy to overcome the adverse effects of TKIs is to enhance the specificity of anti-tumor activity by selectively targeting hypoxic region of tumors. Herein, we designed and synthesized a series of novel 4-anilinoquinazoline derivatives by introducing 3-nitro-1,2,4-triazole group to the side chain of vandetanib with modification of aniline moiety. Lead compounds, 10a and 10g, exhibited potent inhibitory activity against EGFR and VEGFR-2 kinase. Moreover, these two compounds were shown to enhance anti-proliferative activities on A549 and H446 cells under hypoxic conditions compared to vandetanib and dramatically down-regulate VEGF gene expression. In vivo studies confirmed that 10a and 10g not only inhibited tumor growth in A549 xenografts of BALB/c-nu mice but also significantly reduce toxicity associated with weight loss compared to vandetanib. These results suggest that EGFR/VEGFR-2 dual inhibitors, 10a and 10g, emerged as potential hypoxia-selective anti-tumor drugs with less toxicity for inhibiting in vitro and in vivo models of non-small cell lung cancer cells.

Full text of DOI:10.1016/j.ejmech.2019.07.055

Accepted Manuscript
Design, synthesis and biological evaluation of novel 4-anilinoquinazoline derivatives
as hypoxia-selective EGFR and VEGFR-2 dual inhibitors
Huiqiang Wei, Yuqing Duan, Wenfeng Gou, Jie Cui, Hongxin Ning, Deguan Li, Yong
Qin, Qiang Liu, Yiliang Li
PII:
S0223-5234(19)30676-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.055
EJMECH 11552
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 May 2019
Revised Date: 10 July 2019
Accepted Date: 19 July 2019
Please cite this article as: H. Wei, Y. Duan, W. Gou, J. Cui, H. Ning, D. Li, Y. Qin, Q. Liu, Y. Li, Design,
synthesis and biological evaluation of novel 4-anilinoquinazoline derivatives as hypoxia-selective
EGFR and VEGFR-2 dual inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.07.055.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Novel TKIs with enhanced anti-tumor activity under hypoxia
Hypoxia is an inherent and significant difference between most solid tumors and healthy tissues.
Herein, we designed a series of hypoxia-targeting TKIs to increase anti-tumor efficiency and
reduce the adverse effects, by introducing 3-nitro-1,2,4-triazole moiety onto the EGFR/VEGFR-2
dual inhibitor vandetanib. Two lead compounds, 10a and 10g, demonstrated enhanced
anti-tumor activities under hypoxia compared to vandetanib, which also shown enhanced
inhibitory activities on tumor growth and reduced toxicities in tumor xenograft models compared
to vandetanib and the vehicle control.

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of novel
4-anilinoquinazoline derivatives as hypoxia-selective EGFR
and VEGFR-2 dual inhibitors
Huiqiang Wei, #,1 Yuqing Duan, #,1 Wenfeng Gou,1 Jie Cui,2 Hongxin Ning,1 Deguan Li,1
,1
Yong Qin,* *
,3 Qiang Liu *,1 and Yiliang Li
1Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union
Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China
2 Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
3 Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United
States
# These authors contributed equally to the article
*Corresponding authors: Yong Qin, Qiang Liu and Yiliang Li
Tel.: +86-22-8568-3040 (Y. Li)
E-mail: yongqin@mdanderson.org (Y. Qin); liuqiang@irm-cams.ac.cn (Q. Liu); liyiliang@irm-cams.ac.cn (Y. Li)
Abstract
Tyrosine kinase inhibitors (TKIs) have achieved substantial clinical effects for cancer treatment while causing a
number of adverse effects. Since hypoxia is an intrinsic difference between solid tumor and healthy tissues, one
strategy to overcome the adverse effects of TKIs is to enhance the specificity of anti-tumor activity by selectively
targeting hypoxic region of tumors. Herein, we designed and synthesized a series of novel 4-anilinoquinazoline
derivatives by introducing 3-nitro-1,2,4-triazole group to the side chain of vandetanib with modification of aniline
moiety. Lead compounds, 10a and 10g, exhibited potent inhibitory activity against EGFR and VEGFR-2 kinase.
Moreover, these two compounds were shown to enhance anti-proliferative activities on A549 and H446 cells under
hypoxic conditions compared to vandetanib and dramatically down-regulate VEGF gene expression. In vivo
studies confirmed that 10a and 10g not only inhibited tumor growth in A549 xenografts of BALB/c-nu mice but
also significantly reduce toxicity associated with weight loss compared to vandetanib. These results suggest that
EGFR/VEGFR-2 dual inhibitors, 10a and 10g, emerged as potential hypoxia-selective anti-tumor drugs with less
toxicity for inhibiting in vitro and in vivo models of non-small cell lung cancer cells.
Keywords
1. Introduction
A number of tyrosine kinase inhibitors (TKIs) have been developed as therapeutics for cancers and shown
: Tyrosine kinase inhibitor ; 3-nitro-1,2,4-triazole ; Hypoxia; EGFR; VEGFR-2, Vandetanib
significant anti-tumor effects [1]. Among these tyrosine kinases, epidermal growth factor receptor (EGFR) and
vascular endothelial growth factor receptor (VEGFR) are among those prominent targets. However, the clinical
experience revealed that EGFR/VEGFR TKIs could induce unexpected serious toxic effects, such as cutaneous
and oral adverse reactions, hypertension, arrhythmia, and diarrhea [2]. These toxicities of EGFR/VEGFR TKIs are
mainly caused by the inhibition of EGFR/VEGFR signaling pathways in healthy tissues [3]. One feasible strategy
to decrease or avoid adverse effects is to increase the specificity of TKIs against tumor cells. Hypoxia is a known
inherent difference between solid tumors and healthy tissues, which represents a unique characteristic of a solid
tumor that 10%-50% of tumor cells exist in a hypoxic microenvironment [4]. Hypoxia plays a crucial role in solid
tumor survival and development. Previous studies have confirmed that hypoxia could promote the expression of
various transcription factors, growth factors, and tyrosine kinase receptors, including hypoxia-inducible factor-1
(HIF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-β (PDGF-β), and epidermal

ACCEPTED MANUSCRIPT
growth factor receptor (EGFR). Most of these hypoxia-driven proteins can induce angiogenesis and accelerate the
growth and migration of tumor cells through tyrosine kinase signaling pathways [5]. Moreover, hypoxia is one of
the main reasons for radio- and chemotherapy resistance of solid tumors [6]. Thus, hypoxia is potentially
exploitable in cancer treatment, and molecules in hypoxia-driven pathways, especially TKIs, represent attractive
and feasible therapeutic “targets” to distinguish tumor from healthy tissues.
For example, VEGFR inhibitors inhibit tumor angiogenesis to enhance blood vessels atrophy and tumor
hypoxia [7]. Thus, TKIs coupled with a hypoxia-targeted group, may improve the specificity of TKIs in tumor
under hypoxia and overcome some adverse effects. Several studies have been conducted to discover potent
hypoxia-targeted group for drug development. Cobalt compounds are one of these hypoxia-targeted candidates,
which can be used to induce hypoxia in cell incubation due to the Co (III)/Co (II) redox potential [8]. In the
absence of oxygen, Co (III) in drug-Co (III) complex would turn to Co (II) by a biochemical reduction. The
reduced Co (II) showed significant ligand lability, and the drug was released after dissociation, which could be
utilized as a hypoxia trigger in hypoxia-targeted prodrugs [9]. However, as a metallic element, the accumulation
and metabolism of cobalt in vivo limited its application as the anti-cancer drug. The electron-affinity compounds,
nitroimidazole derivatives, have been developed as hypoxia-activated and radiosensitizing agents in chemotherapy
since the 1960s. 2-nitroimidazole group was used in several drug designs as the hypoxia-selective trigger,
including the phosphoramidate mustard prodrug TH-302 (Evofosfamide, phase III) [10]. The 4-aminoquinazoline
pharmacophore was widely used as an EGFR/VEGFR kinase inhibitor in the past few years [11]. In our previous
research, a series of novel nitroimidazole-substituted 4-anilinoquinazoline derivatives were developed by coupling
with EGFR/VEGFR-2 dual inhibitor vandetanib (Zactima®, Figure 1). The lead compound LYL-10 (16f in ref
[12], Figure 1) from this study showed an increase of anti-tumor activity under hypoxia than normoxia [12].
However, the high electron-affinity of nitroimidazole derivatives also contributes to their potential cytotoxicity
[13] and poor pharmacokinetic properties [14] of nitroimidazole, which limit its druggability. The toxicity of
candidate compound LYL-10 was observed in previous in vivo study, and adverse reactions of vomit, diarrhea,
asthenia, weight loss, and apparent abdominal breathing were documented in more than half of the test BALB/c-nu
mice. Hence, nitroimidazole may not be an ideal candidate to construct hypoxia-selective drugs TKIs. Sanazole
(AK-2123, Figure 1), a 3-nitro-1,2,4-triazole derivative, showed similar radio-sensitizing activities and much
lower cytotoxicity than other 2-nitroimidazoles analogs, which had been tested as a radiosensitizer in Phase III
clinical trials in radiotherapy for solid tumor [15]. It indicated that 3-nitro-1,2,4-triazole had the characteristic of an
effective hypoxia-targeted group with good biosecurity and pharmacokinetic property.
Figure 1.Structures of vandetanib, LYL-10, AK-2123 and target compounds

ACCEPTED MANUSCRIPT
In this study, we developed a series of novel hypoxia-targeted EGFR/VEGFR-2 inhibitors with
3-nitro-1,2,4-triazole motif. To our knowledge, this is the first study to develop a hypoxia-selective TKI
harboring EGFR/VEGFR-2 inhibitory activity of vandetanib, which may reduce the level of active compound in
non-malignant tissue resulting in the decrease of the adverse effects while enhancing the inhibitory effect on tumor
cells under hypoxia. Moreover, two lead compounds 10a and 10g were identified as potent candidates for cancer
therapy based on our initial screening and validation by in vitro and in vivo models.
2. Results and Discussion
2.1 Design and synthesis of novel hypoxia-selective EGFR/VEGFR-2 inhibitor
Our previous research confirmed that introducing nitroimidazole groups onto the side chain of
4-anilinoquinazoline scaffold did not affect its EGFR/VEGFR-2 inhibitory activities[12]. In a similar strategy,
3-nitro-1,2,4-triazole moiety (AK-2123) was linked to the 4-anilinoquinazoline scaffold by long ether chains
(Figure 1). This long ether side chain was also found in erlotinib and gefitinib [16]. The relationship between
substitutions on the benzene ring and derivative activities has been discussed in our previous work [12], which
provided a stragtegy to modify 4-anilinoquinazoline for develeoping novel EGFR/VEGFR-2 dual inhibitor. Hence,
in this study, we selected representative substitutions to obtain several target compounds, which were further
evaluated by various in vitro and in vivo cancer models.
Fourteen target compounds were designed and synthesized by seven steps as shown in Scheme 1, which were
started from 7-benzyloxy-6-methoxy-3H-quinazolin-4-one (1) and 3-nitro-1,2,4-triazole (5). The intermediate
7-benzyloxy-4-chloro-6-methoxy-quinazoline (2) was obtained from 1 by refluxing in thionyl chloride and then
reacted with anilines in isopropanol to give crude products of 3a-4g, which were further treated by trifluoroacid to
deprotect benzyl group at 80 °C, then adjusted pH to 10 by ammonium hydroxide [17]. These steps led to the
production of white to yellow powders of intermediates 4a-4g with moderate yields of 75%–88%. The
intermediate methyl 2-(3-nitro-[1,2,4]triazol-1-yl)acetate (6) was prepared as yellow solid from 5 and methyl
chloroacetate in the presence of a base (K2CO3) in refluxing acetonitrile with a good yield of 95.1%. The amide
intermediate 7a-7c were prepared from 6 and amino polyethylene glycols by aminolysis reaction in refluxing
methanol with acceptable yields varying from 43.8%–86.7%. Sulfonylation of 7a-7c was reacted in pyridine at
0 °C with a catalytic amount of 4-dimethylaminopyridine (DMAP). 8a-8c were all faint yellow jellies with
relatively lower yields of 20.8%–65.7%. Target compounds 9a-9c, 10a-10g, and 11a-11d were obtained by
intermediates 4a-4g connecting with 8a-8c in the presence of K2CO3 in DMF at 85 °C with moderate yields of
1
52.2%–68.9%. All of the target compounds were characterized by H-NMR, 13C-NMR, 19F-NMR, and HRMS
analysis.
X2
X2
X1
X3
X1
X3
O
N
Cl
N
HN
HN
O
O
X2
ii
iii
i
O
NH
N
O
N
N
X1
X3
BnO
BnO
HO
N
BnO
N
HN
3a-3g
2
4a-4g
1
O
O
N
3a, 4a: X1 = F, X2 = H, X3 =Br;
3b, 4b: X1 = Cl, X2 = H, X3 =F;
3c, 4c: X1 = H, X2 = CH3, X3 =Br;
3d, 4d: X1, X3 = F, X2 = H;
3e, 4e: X1 , X3 = H, X2 = F;
3f, 4f: X1 , X2 = H, X3 = F;
3g, 4g: X1 = F, X2 , X3 =Cl;
H
N
N
vii
N
N
n
N
O
O2N
9a-9c, 10a-10g, 11a-11d
10a: X1 = F, X2 = H, X3 =Br, n=2;
10b: X1 = Cl, X2 = H, X3 =F, n=2;
10c: X1 = H, X2 = Br, X3 =CH3, n=2;
10d: X1 = H, X2, X3 = F, n=2;
10e: X1 , X3 = H, X2 = F, n=2;
10f: X1 , X2 = H, X3 = F, n=2;
10g: X1 = F, X2 , X3 =Cl, n=2;
9a: X1 = F, X2 = H, X3 =Br, n=1;
9b: X1 = Cl, X2 = H, X3 =F, n=1;
9c: X1 = H, X2 =Br , X3 =CH3, n=1;
11a: X1 = F, X2 = H, X3 =Br, n=3;
11b: X1 = Cl, X2 = H, X3 =F, n=3;
11c: X1 = H, X2 = Br, X3 =CH3, n=3;
11d: X1 = H, X2, X3 = F, n=3
H
H
N
N
N
O
v
iv
N
N
OTs
n
N
OH
n
vi
NH
N
N
O
N
O
N
N
N
N
O
O2N
O2N
O
O
O2N
O2N
5
6
8a-8c
7a-7c
7a, 8a: n = 0
7b, 8b: n = 1
7c, 8c: n = 2
Scheme 1. Synthesis of target compounds. Reagents and conditions: (i) SOCl2, DMF, reflux; (ii) Substituted
anilines, i-PrOH, 60°C; (iii) TFA, 80°C, then ammonium hydroxide; (iv) Methyl chloroacetate, K2CO3,

ACCEPTED MANUSCRIPT
acetonitrile, reflux; (v) amino polyethylene glycols, methanol, reflux; (vi) p-TsCl, DMAP, pyridine, 0°C to rt;
(vii) K2CO3, DMF, 85°C
2.2 In vitro biological activities
The abilities of targeted compounds to inhibit EGFR and VEGFR-2 were evaluated by kinase inhibitory assay
with LYL-10 and vandetanib as positive controls. As shown in Table 1, more than half of the tested compounds
(LYL-10, 10a, 10c, 10d, 10e, 10g, 11a, 11b, and 11c) exhibited more potent EGFR inhibitory activities with the
IC50 values ranging from 0.37 to 12.93 nM, which were superior to that of vandetanib (IC50 = 19.76 nM). Most of
targeted compounds with a long link chain between 4-anilinoquinazoline and 3-nitro-1,2,4-triazole (n= 2 or 3; 10a,
10c, 10d, 10e, 10g, 11a, 11b, and 11c; IC50 from 0.37 to 12.93 nM) showed more potent inhibitory effects on
EGFR than those compounds with a short side chain (n = 1; 9a, 9b, and 9c; IC50 from 18.54 to 43.56 nM). It
indicated that the length of the linker between trizole and quinazoline moiety could dramatically affect the EGFR
inhibitory activity of targeted compounds. Most of the target compounds showed relatively enhanced inhibitory
activities against VEGFR-2 kinase (IC50 from 36.78 to 4082.09 nM) than vandetanib. 10a showed a similar
inhibitory activity against VEGFR-2 kinase (IC50 = 36.78 nM) to vandetanib (IC50 = 33.26 nM). The IC50 values of
9a and 10g for VEGFR-2 were lower than 100 nM, but the rest of targeted compound showed a dramatically
higher IC50 for VEGFR-2, which were about 5-122 folds of increases compared to vandetanib. Moreover, LYL-10
showed a slightly more potent inhibitory activity against VEGFR-2 with a 27.13 nM IC50 than vandetanib. It
seemed that target compounds with bulky and heavy halogen atoms on aniline moiety exhibited better inhibitory
activity to VEGFR-2 kinase, and the length of the link group also significantly impact the VEGFR-2 inhibitory
activity. Several target compounds retained potent EGFR/VEGFR-2 inhibitory activities similar to vandetanib.
Among these compounds, 10a and 10g demonstrated potent inhibitory activities against EGFR and VEGFR-2 with
the IC50 values of 5.90/36.78 nM and 0.69/67.84 nM (Figure 2). 10g showed higher inhibitory activity against
EGFR than VEGFR-2, since the IC50 value of 10g against EGFR was 100-fold more potent than VEGFR-2. At the
same time, 10g still have potent inhibitory activity against VEGFR-2 with an IC50 lower than 70 nM, which was
still in the same order of magnitude as vandetanib and LYL-10. Therefore, 10g was still considered as an
EGFR/VEGFR-2 dual inhibitor.
Table 1. In vitro EGFR and VEGFR-2 kinase inhibitory activity assay results of target compounds.
Substituents
X2
IC50 (nmol/L)
Compounds
n
1
1
1
2
2
2
2
2
2
2
3
3
3
X1
F
X3
Br
F
EGFR
VEGFR-2
86.84
H
H
Br
H
H
Br
F
43.56
48.07
18.54.
5.90
9a
9b
Cl
H
F
517.33
183.56
36.78
CH3
Br
F
9c
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
Cl
H
H
H
H
F
20.83
0.37
4082.09
407.47
1104.22
1196.59
864.18
67.84
CH3
F
3.70
F
H
3.11
H
Cl
H
H
Br
F
16.43
0.69
Cl
Br
F
F
4.95
216.77
2865.05
244.76
Cl
H
12.93
6.24
CH3

ACCEPTED MANUSCRIPT
3
-
H
F
F
F
H
H
F
19.97
3.23
1883.43
27.13
11d
Br
Br
LYL-10
-
19.76
33.26
vandetanib
Figure 2. inhibition ratio curves of 10a and 10g: (A) EGFR; (B) VEGFR-2. Ten compound concentrations of
40000, 10000, 2500, 625, 156, 39, 10, 2, 1, and 0.2 nM were set in the assay.
The in vitro anti-proliferative effects of target compounds were evaluated against the A549 (adenocarcinomic
human alveolar basal epithelial cells) and H446 (human small cell lung cancer cells) cell lines by using tetrazolium
salt (WST-8) assay under normoxic or hypoxic conditions. The potential radio-sensitizing activities of targeted
compounds were also evaluated under the hypoxic condition with the exposure dose of 8 Gy. Under normoxic
conditions, 0.5 µM of target compounds, 9a, 10a, 10b, 10c, 10f, 10g, or 11a, inhibited about 5~20% of cell growth
for both cancer cell lines, which was less potent than vandetanib that inhibited 21.06% and 25.58% of cell growth
in A549 and H446 cells (Table 2 and Figure 3 A-B). Under hypoxic conditions, target compound, 10a, exhibited
stronger anti-proliferative activities (75.86% of inhibition in A549 cells and 84.37% of inhibition in H446 cells) in
both cell lines than vandetanib (67.26% of inhibition in A549 cells and 77.67% of inhibition in H446 cells) (Table
2 and Figure 3 A-B). Moreover, under hypoxic condition combining with irradiation, 10a was the only
compound that showed stronger inhibitory effects on the growth of both cancer cell lines than vandetanib (Table 2
and Figure 3 A-B).
In order to estimate the hypoxic hypoxia-enhanced anti-tumor activities of tested compounds, the inhibitory
activities caused by CoCl2 and irradiation alone need to be taken out. Herein two kinds of hypoxic sensitive
enhancement ratios, HSERhyp and HSERhyp+IR, were used to quantitatively evaluate the enhanced inhibitory
activities in normoxia/hypoxia and hypoxia/hypoxia+irradiation respectively (Table 2 and Figure 3 C-D). All
target compounds demonstrated evidently higher HSERhyp values (1.07~1.36) than vandetanib (HSERhyp = 1.05) on
A549 and H446 cells, and target compounds, 10a and 10g, exhibited the best HSERhyp values of 1.32 and 1.36 on
A549 cells; however, when combined with irradiation, tested compounds and vandetanib showed similar lower
HSERhyp+IR values (0.80~0.96) on both A549 and H446 cells (Table 2 and Figure 3 C-D). Target compounds
containing 3-nitro-1,2,4-triazole moiety, 10a and 10g, demonstrated more potent hypoxia-targeted inhibitory
activities than vandetanib on the growth of A549 and H446 cells under hypoxic conditions. Moreover, these two
target compounds only showed additive effect with irradiation. No synergistic radio-sensitizing activities were
observed in these assays.
Further quantitative polymerase chain reaction (Q-PCR) analysis showed that treatment of A549 cells with 5

ACCEPTED MANUSCRIPT
µM of vandetanib, 10a, or 10g could down-regulate VEGF expression under normoxic or hypoxic conditions
(Table 3 and Figure 4). Both targeted compounds, 10a and 10g, demonstrated more potent inhibitory effects on
VEGF expression in A549 cells compared to vandetanib.
Table 2. In vitro anti-proliferative activity assay results (inhibition ratios and HSER) in normoxia, hypoxia and
hypoxia + irradiation
Inhibition ratios（%）
c
c
Compounds Cell line
HSERhyp
HSERhyp+IR
normoxia
21.06
25.58
14.87
16.76
6.75
Hypoxia a
67.26
77.67
72.99
76.34
75.86
84.37
69.01
80.31
65.39
77.71
72.53
78.70
82.25
77.90
74.36
78.81
hypoxia + IR b
81.44
82.02
80.31
79.03
86.41
88.66
77.88
83.35
69.18
80.10
76.94
80.58
80.70
85.04
80.36
84.92
A549
vandetanib
1.05
1.04
1.19
1.07
1.32
1.17
1.17
1.14
1.15
1.08
1.25
1.13
1.36
1.07
1.20
1.08
0.94
0.88
0.90
0.85
0.96
0.93
0.89
0.89
0.80
0.86
0.86
0.86
0.87
0.91
0.90
0.91
H446
A549
9a
H446
A549
10a
H446
18.95
10.33
13.87
5.48
A549
10b
H446
A549
10c
H446
18.65
8.43
A549
10f
H446
11.08
13.76
20.52
16.04
20.38
A549
10g
H446
A549
11a
H446
a Control group in hypoxia was performed with 200 µM CoCl solution and blank group with corresponding amount of DMSO;b Irradiation was conducted at
2
c
24 h late after administration under the absorbed dose of 8 Gy, and then cultured for another 24 h before CCK-8 test; ꢀꢁꢂꢃꢂꢄ /ꢀꢁꢂꢃꢂꢄ
values in A549 and
ꢅꢆꢇꢈ
ꢅ
H446 group were 59.94%/64.13% and 65.84%/68.77%.

ACCEPTED MANUSCRIPT
Figure 3. In vitro inhibition ratios in normoxia, hypoxia and hypoxia + irradiation on A549 (A) and H446 cells (B); HSER values of
normoxia/hypoxia (C) and hypoxia/hypoxia+IR (D).
Table 3. VEGF gene expression level assay results of tested drugs in hypoxia/normoxia
Compounds
Normoxia
Hypoxia a
Control b
Vandetanib c
10a
10g
1.0000
0.6457
0.1571
0.0011
0.1833
0.0037
1.0000
0.2457
b
a hypoxia was performed with 200 µM CoCl2 solution； VEGF/EGF gene expression levels in control groups in hypoxia or normoxia was
calculated as 1.0000, respectively and independently; c VEGF gene expression levels of vandetanib were from ref [12].

ACCEPTED MANUSCRIPT
Figure 4. VEGF gene expression level assay results of tested compounds (5 µmol/L).
2.3. Molecular docking
In order to study the mechanism of target compound 10a inhibiting the activity of relevant kinases, the
molecular docking was conducted to analyze the different binding patterns of 10a or vandetanib to EGFR or
VEGFR-2 based on available crystal structures of these two receptor kinases (EGFR: PDB code 4I23 and
VEGFR-2: PDB code 2RL5). As shown in Figure 5A, the structural modeling of the drug-protein complex
showed that the binding pattern of 10a to EGFR was similar to vandetanib, which the formation of important
hydrogen bonds (H-bonds) between the backbone amino acid residue Met793 of EGFR and N1 atom of
quinazoline moiety was observed in both drugs with similar H-bond distances (10a: 2.607 Å, vandetanib: 2.685 Å).
Moreover, the aniline moieties of 10a and vandetanib were observed to insert into EGFR`s hydrophobic pocket
coplanarly, but the 3-nitro-1,2,4-triazole side chain of 10a positioned in the solvent with no any additional H-bond
with EGFR (Figure 5A). The structure model of 10a and VEGFR-2 complex showed that the aniline moieties of
10a and vandetanib occupied the narrow and deep hydrophobic pocket of VEGFR-2, and two critical H-bonds
formed between the backbone amino acid residue Cys919 of VEGFR-2 and quinazoline N1 atom of drugs with the
distances of 3.101 Å (10a) and 3.095 Å (vandetanib) (Figure 5B). Unlike the coplanar binding mode of 10a or
vandetanib to EGFR, the 3-nitro-1,2,4-triazole side chain of 10a folded as a U shape, which contributed to a π-π
stacking interaction with the Phe1047 residue of VEGFR-2 and further stabilized this folding structure. Moreover,
three additional H-bonds were formed between 10a and VEGFR-2 (Figure 5B). An extra H-bond formed between
quinazoline N2 atom of 10a and the side chain hydroxyl group of Thr916 residue of VEGFR-2 with a distance of
3.281 Å, and another two H-bonds were found between the side chain carbonyl oxygen atom of 10a and the
backbone residue Asn923 of VEGFR-2 with a distance of 2.895 Å and 3.319 Å at the gate of hydrophobic pocket
(Figure 5B).

ACCEPTED MANUSCRIPT
Figure 5. The docked poses of compound 10a (C: yellow; N: blue; O: red) or vandetanib (C: magenta; N: blue; O:
red) at the ATP binding cleft of EGFR kinase (cyan) (A), and VEGFR-2 kinase (coral) (B). H-bonds are shown in
red lines (A) or sky-blue lines (B). The pictures were generated using Chimera. Some backbone amino acid
residues were hidden.
A
schematic diagram shown in Figure 6 was given to illustrate how substituents and link group length
influenced the binding between compounds and VEGFR-2 protein. As shown in Figure 6A, bulky and heavy
halogen atoms (Br or Cl) on aniline moieties of compounds contributed more hydrophobicity and electronegativity
than fluorine atoms or methyl groups, which caused a reinforced affinity for binding to VEGFR-2, especially when
it fits into the narrow and deep hydrophobic pocket of VEGFR-2. Since the triazole side chain of compounds
situated at the solvent portion, advisable bulky and heavy halogen atoms on aniline would balance the “barycenter”
of target compounds and stabilized it in the complex. Target compounds with 4-bromo-2-fluoro- and
3,4-dichloro-2-fluoro- substituted aniline moieties showing higher VEGFR-2 kinase inhibitory activities were
attributed to that the substitutions were bulky and heavy enough to balance the barycenter as well as afforded
appropriate hydrophobicity and electronegativity to reinforce the binding complex (Figure 6A). The length of the
link group between 3-nitro-1,2,4-triazole and quinazoline also influenced the binding pattern. Triazole moiety in
10a with appropriate length (n=2) contributed to the binding between 10a and VEGFR-2, which formed a U shape
structure like a fishhook to further stabilize drug-protein interaction by the formation of key H-bonds (Figure 6A).
This special U shape structure was believed to improve the affinity of targeted compound 10a binding to
VEGFR-2. The short linking group (n=1) of 9a could prevent the formation of U shape structure resulting in the

ACCEPTED MANUSCRIPT
triazole group of the drug blocking the gate of hydrophobic pocket and reducing the formation of H-bonds to
stabilize the complex (Figure 6B). With a longer link group (n≥3), the triazole group of the drug would be
impacted by solvent effect and shift the “barycenter” to the solvent region, which was detrimental to a stable
drug-protein complex (Figure 6C); Longer link groups also contributed to the excess flexibility that might cause
unexpected pernicious influence, such as hERG potassium channel inhibition [18].
Figure 6. (A) Bulky and hydrophobic halogen atoms of aniline moiety reinforced the binding complex; the
appropriate linking group length (n=2) contributed the “fishhook” binding pattern through a π-π stacking
interaction and key H-binds formed with the residues Cys919, Asn923 and Thr916 of VEGFR-2. (B) The short
linking group caused a sterically hindered effect at the opening of the hydrophobic pocket. (C) The long linking
group shifted the “barycenter” to the solvent region, which resulted in an unstable drug-receptor complex.
Taking in vitro kinase inhibitory activities and docking study results into account, the preliminary
structure-activity relationships (SAR) were obtained: the length of linking group impacts activities dramatically,
and a proper length (n=2) was favorable to keep EGFR/VEGFR-2 inhibition; substituent on aniline was vital to
VEGFR-2 selectivity, and advisable bulky and heavy halogen atoms substituted anilines were conducive to
EGFR/VEGFR-2 inhibitory activities. More compound structure optimization would be undertaken according to
the SAR guideline in the future.
2.4 In vivo anticancer activity
The targeted compounds, 10a and 10g, were selected to further investigate their anticancer activity in mouse
modes based on their high antiproliferative activity derived from in vitro studies. The ability of 10a and 10g to
suppress tumor growth in the A549 tumor cell xenograft models were further compared to vandetanib. The male
BALB/c-nu mice were randomly divided into the vehicle control group, the experimental groups (treated with 10a
or 10g), and the positive control group (treated with vandetanib). Respectively, each group consisted of 5
tumor-bearing mice. The mice were treated with 10a, 10g, and vandetanib with oral administration of 10 mg/kg
drugs daily for 17 days, and tumor size and the body weight of the mouse were measured daily. In in vivo
dose-dependent anti-proliferation experiments, each group consisted of 3 tumor-bearing mice. The mice were
treated with 10a and 10g with oral drug administrations of 5 mg/kg, 15 mg/kg and 50 mg/kg daily for 18 days, and

ACCEPTED MANUSCRIPT
tumor size were measured at day 4, 6, 8, 11, 13, 15 and 18.
As shown in Figure 7A, 10a exhibited remarkable inhibition on A549 tumor growth with a tumor growth
inhibitions (TGI) of 63.93% at day 17 compared to controls. The TGI value of 10a at day 17 was similar to that of
vandetanib (TGI: 62.06%, Figure 7A), indicating that the in vivo antitumor activity of 10a was similar to
vandetanib. After treatment of 7 days, the tumor volume slowly increased in the 10a, 10g, and vandetanib groups,
and the 10g group showed a faster tumor growth than 10a and vandetanib, which indicated that these compounds
could not completely control tumor growth in A549 xenografts. 10g still showed a distinct inhibitory effect on
tumor growth (TGI=49.05% at day 17) compared to the vehicle control group. Thus, 10a and 10g remained potent
inhibitory activities against A549 tumor growth in mouse models. Moreover, the tumor growth inhibitions of 10a
and 10g were dose-dependent in A549 xenografts (Figure 7B). The highest TGI of 80.5% was observed in the
mice treated with 50 mg/kg of 10a at day 18. The TGI value of 10g at day 18 were found to be 28.10%, 63.90%,
76.33% for treatment of 5 mg/kg, 15 mg/kg, and 50 mg/kg respectively, while those values of 10a were 31.22%,
68.31%, and 80.55%. Evidently, the TGI values of 10g were much lower than those of 10a, indicating that the in
vivo antitumor activity of 10g was less potent than that of 10a (Figure 7B).
The in vivo toxicity of targeted compounds 10a and 10g was evaluated in BALB/c-nu mice, using vandetanib
as control. The mice were monitored for body weight change and clinical signs of toxicity. The tested mice were
well tolerated with oral administrations of 10a and 10g, and no significant weight loss was observed during the
treatment up to 17 days compared to the vehicle control group (Figure 7C). However, the mice in vandetanib
group showed a notable weight loss during the first 8 days of treatment compared to the vehicle control group,
while their weights were back on to normal gradually after day 9 (Figure 7C). The maximum tolerated dose (MTD)
assessed for 10a was 100 mg/kg (five times a week for two weeks with two days off for a weekend) on male
BALB/c-nu mice, and the treated animals exhibited no clinically observable sign of toxicity or weight loss. The
results suggested that targeted compounds, 10a and 10g, developed in this study had potent antitumor activity with
lower toxicity in A549 mouse model, which presented novel and promising EGFR/VEGFR-2 inhibitors as
anticancer reagents.
Figure 7. (A) Effect of compound 10a and 10g on tumor growth in A549 xenograft model; (B) Effect of
administration dose on tumor growth in A549 xenograft model; (C) Mice weight changes during administration;
A549 cells (1 × 107 cells) were implanted sc in the flank of BALB/c-nu mice. Seven days after implantation,

ACCEPTED MANUSCRIPT
mice were orally administrated the tested doses daily, and the body weights of mice were measured daily.
3. Conclusions
In order to overcome the toxicity of LYL-10, which is a nitro-imidazole derivative of the EGFR/VEGFR-2
dual inhibitor vandetanib, we designed and synthesized a series of 3-nitro-1,2,4-triazole derivatives based on the
structure of vandetanib as novel hypoxia-targeted antitumor compounds. The in vitro kinase inhibitory
experiments showed that most of 3-nitro-1,2,4-triazole derivatives developed in this study retained potent EGFR
inhibitory activities. Meanwhile, the lead compounds, 10a and 10g, exhibited potent VEGFR inhibitory activities
that were similar to vandetanib. The molecular docking study confirmed that 10a could bind with EGFR and
VEGFR-2 with a great affinity. Further biological studies showed that most of the targeted compounds showed
enhanced inhibitory activities in hypoxia towards A549 and H446 cells compared to vandetanib. Further gene
expression assays confirmed that 10a and 10g could strongly inhibit the VEGF gene expression in A549 cells,
which might contribute to the potent anti-proliferative activities of these two compounds in hypoxia. Based on in
vivo anticancer test, 10a and 10g showed notable inhibitory effects and safety on tumor growth and good
dose-dependence in dose-dependent anti-proliferation experiments in A549 xenograft models.
In conclusion, a series of novel 4-anilinoquinazoline TKIs were designed and synthesized. To our knowledge,
this was the first study to introducing 3-nitro-1,2,4-triazole onto vandetanib to develop a potent EGFR/VEGFR-2
dual inhibitor with low toxicity targeted hypoxic solid tumors. The lead compounds, 10a and 10g were identified
to be the most potent inhibitors by in vitro assays. 10a and 10g also showed prominent efficacy and drug safety in
tumor xenograft models. Thus, 10a and 10g provide a foundation for further structure optimization and biological
investigation to develop effective cancer therapeutic agent targeted hypoxic tumors.
4. Experimental Section
4.1 Chemical synthesis section
4.1.1 General
Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 UV254 pre-coated silica
gel plates. Detection was using a UV lamp. Flash column chromatography was performed on 300–400 mesh silica
1
gel. H-NMR, 13C-NMR, and 19F-NMR were recorded on a BRUKER AVANCE III spectrometer (Bruker Ltd.,
Faellanden, Switzerland) at 600 MHz/151 MHz/564 MHz or an AV400 spectrometer at 400 MHz/100 MHz/376
MHz. Chemical shifts were reported in ppm using DMSO-d6 solution without TMS or CFCl3 as internal standards.
Mass spectra (ESI-MS) were performed on Agilent 1200 HPLC-6310 liquid chromatography mass spectrometer
(Agilent Ltd., Palo Alto, CA, USA). High resolution mass spectrometry (HRMS) was performed on VG ZAB-HS
gas chromatography mass spectrometer (VG Instruments Ltd., England). All reagents and solvents purchased from
commercial sources without further purification.
4.1.2 Synthesis of methyl 2-(3-nitro-[1,2,4]triazol-1-yl)acetate (6)
11.41 g 5 (0.1 mol, 1 eq) and 15.29 g potassium carbonate (0.11 mol, 1.1 eq) was added to 100 mL
acetonitrile and stirred at 50 °C for 0.5 h, the mixture turned to be bright yellow. 16.05 g methyl bromoacetate
(0.105 mol, 1.05 eq) was added to the mixture and then reflux until no 5 appeared on TLC. The mixture was
cooled to room temperature and filtrated. The filtrate was evaporated under reduced pressure, and the residue was
dissolved in a mixture of ethyl acetate and water. The organic layer was separated and the aqueous layer was
extracted twice. The organic extracts were combined, dried (anhydrous Na2SO4) and evaporated to dryness to
afford 17.23 g 6 as brownish solid with the yield of 95.1%; ESI-MS m/z = 187.04 [M + H]+; 1H-NMR (400 MHz,
DMSO-d6): δ (ppm): 3.73 (s, 3H), 5.43 (s, 2H), 8.86 (s, 1H)

ACCEPTED MANUSCRIPT
4.1.3 General synthetic procedure of N-polyethyleneglycol-2-(3-nitro-[1,2,4]triazol-1-yl)acetamide (7a-7c)
Dissolved 2.00 g 6 (10.74 mmol, 1 eq) and amino polyethylene glycol (2 eq) in methanol, heated to reflux and
monitored the reaction by TLC. Evaporated solvent under reduced pressure after 6 consumed completely, the
residue was purified by silica gel column chromatography to obtain 7a-c (CH2Cl2:MeOH=30:1→15:1).
N-(2-hydroxy-ethyl)-2-(3-nitro-[1,2,4]triazol-1-yl)acetamide (7a). Faint yellow solid powder with the yield of
81.2%; ESI-MS m/z = 215.16 [M + H]+; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.17 (dd, J1 = 5.6 Hz, J2 = 11.6
Hz, 2H), 3.43 (dd, J1 = 5.6 Hz, J2 = 11.6 Hz, 2H), 4.73 (t, J = 5.6 Hz, 1H), 5.08 (s, 2H), 8.41 (t, J = 5.6 Hz, 1H),
8.82 (s, 1H)
N-[2-(2-hydroxy-ethoxy)ethyl]-2-(3-nitro-[1,2,4]triazol-1-yl)acetamide (7b). Faint yellow oil with the yield
1
88.5%; ESI-MS m/z = 259.19 [M + H]+; H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.29 (m, 2H), 3.45 (m, 4H),
3.49 (m, 2H), 4.56 (t, J = 5.2 Hz, 1H), 5.10 (s, 2H), 8.44 (t, J = 5.2 Hz, 1H), 8.82 (s, 1H)
N-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-2-(3-nitro-[1,2,4]triazol-1-yl)-acetamide (7c). Faint yellow oil with
the yield 68.5%; ESI-MS m/z = 303.21 [M + H]+; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.28 (q, J = 5.6 Hz,
2H), 3.41-3.51 (m, 6H), 3.53 (m, 4H), 4.60 (t, J = 5.2 Hz, 1H), 5.11 (s, 2H), 8.49 (t, J = 5.2 Hz, 1H), 8.83 (s, 1H)
4.1.4
General
synthetic
procedure
of
(3-nitro-[1,2,4]triazol-1-yl)acetyl
aminopolyethyleneglycol
p-toluenesulfonate (8a-8c)
7a-c (1 eq) and DMAP (0.05 eq) were dissolved in 15 mL pyridine and stirred in ice bath for 0.5 h. TsCl (1.3
eq) was dissolved in 10 mL pyridine and then added dropwise to the reaction mixture in 1 h, then removed ice bath
and stirred overnight. The mixture was extracted with ethyl acetate and 1 N HCl solution, organic layer was
washed by 1 N HCl solution, water and brine, dried with anhydrous Na2SO4, then evaporated solvent. The residue
was purified by silica gel column chromatography to obtain 8a-c (hexane : ethyl acetate =3:1→1:2).
2-[2-(3-Nitro-[1,2,4]triazol-1-yl)-acetylamino]-ethyl p-toluenesulfonate (8a). Faint yellow jelly with the yield
of 20.8%; ESI-MS m/z = 370.07 [M + H]+; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 2.29 (s, 3H), 3.14 (q, J= 5.6
Hz, 2H), 4.63 (t, J = 5.6 Hz, 2H), 5.44 (s, 2H), 7.13 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.91 (t, J = 5.6
Hz, 1H), 8.89 (s, 1H)
2-{2-[2-(3-Nitro-[1,2,4]triazol-1-yl)acetylamino]ethoxy}ethyl p-toluenesulfonate (8b). Faint yellow jelly with
the yield of 62.4%; ESI-MS m/z = 413.21 [M + H]+; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 2.41 (s, 3H), 3.21
(dd, J1 = 5.6 Hz, J2 = 1.2 Hz, 2H), 3.38 (dd, J1 = 5.6 Hz, J2 = 11.2 Hz, 2H), 3.58 (t, J = 4.4 Hz, 2H), 4.12 (t, J = 4.4
Hz, 2H), 5.09 (s, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 8.44 (t, J = 5.2 Hz, 1H), 8.82 (s, 1H)
2-(2-{2-[2-(3-nitro-[1,2,4]triazol-1-yl)-acetylamino]-ethoxy}-ethoxy)-ethyl p-toluenesulfonate (8c). Faint
1
yellow jelly with the yield of 65.7%; ESI-MS m/z = 457.23 [M + H]+; H-NMR (400 MHz, DMSO-d6): δ (ppm):
2.41 (s, 3H), 3.24-3.27 (m, 2H), 3.42 (t, J = 5.6 Hz, 2H), 3.45-3.50 (m, 2H), 3.58-3.60 (m, 2H), 3.64-3.75 (m, 2H),
4.12 (dd, J1 = 3.6 Hz, J2 = 5.2 Hz, 2H), 5.10 (s, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 2H), 8.49 (t, J =
5.2 Hz, 1H), 8.83 (s, 1H)
4.1.5 General synthetic procedure of target compounds (9a-9c, 10a-10g and 11a-11d)
The quinazolinamines 4a-4g (1 eq) was dissolved in 10 mL DMF, potassium carbonate (2 eq) was added and
stirred at 50 °C for 0.5 h. Intermediate 8a-8c (1 eq) was dissolved in DMF and then added dropwise to reaction
mixture. The reaction was monitored by TLC. The mixture was cooled to room temperature after reactant
consumed completely and then poured into water with stirring for 0.5 h. Filtrated and dissolved the precipitate with
methanol, evaporated solvent under reduced pressure after dryness with anhydrous Na2SO4. The residue was
recrystallized in methanol to obtain target compounds.
N-(2-{4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethyl-2-(3-nitro-[1,2,4]triazol-1-yl
)acetamide (9a). Faint yellow solid powder with the yield of 47.2%; ESI-MS m/z = 561.07, 563.06 [M+H]+;

ACCEPTED MANUSCRIPT
1
GC-HRMS calcd for C21H18BrFN8O5, m/z = 561.0646, 563.0625 [M+H]+, found: 561.0643, 563.0622; H-NMR
(600 MHz, DMSO-d6): δ (ppm): 3.38 (dt, J1 = 5.4 Hz, J2 = 4.2 Hz, 2H), 3.96 (s, 3H), 4.32 (t, J = 4.2 Hz, 2H), 5.13
(s, 2H), 7.24 (s, 1H), 7.48 (d, 1H), 7.54 (t, 1H), 7.67 (d, 1H), 7.82 (s, 1H), 8.37 (s, 1H), 8.54 (t, J = 5.4 Hz, 1H),
9.55 (s, 1H); 13C-NMR (151 MHz, DMSO-d6): δ (ppm): 38.85, 52.52, 56.13, 68.95, 102.00, 107.74, 108.73,
2
2
3
3
117.48 (d, JC-F = 22.2 Hz), 119.21 (d, JC-F = 24.6 Hz), 126.39 (d, JC-F = 9.2 Hz), 127.44 (d, JC-F = 11.2 Hz),
129.46, 147.94 (d, 1JC-F = 210.1 Hz), 148.10, 152.93, 155.79, 156.88, 158.06, 161.91, 164.80; 19F-NMR (376 MHz,
DMSO-d6) : δ (ppm): -115.42.
N-(2-{4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethyl-2-(3-nitro-[1,2,4]triazol-1-yl
)acetamide (9b). Yellow solid powder with the yield of 67.1%; ESI-MS m/z = 517.12 [M+H]+; GC-HRMS calcd
for C21H18ClFN8O5, m/z = 517.1151 [M+H]+, found: 517.1150; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.36 (t,
J = 5.6 Hz, 2H), 3.96 (s, 3H), 4.34 (m, 2H), 5.15 (s, 2H), 7.25 (s, 1H), 7.31 (td, J1 = 9.6 Hz, J2 = 2.8 Hz, 1H), 7.58
(m, 2H), 7.84 (s, 1H), 8.34 (s, 1H), 8.57 (t, J = 5.2 Hz, 1H), 9.57 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ
(ppm): 39.34, 53.08, 56.59, 69.46, 108.54, 108.26, 109.01, 115.22 (d, 2JC-F = 21.2 Hz), 117.34 (d, 2JC-F = 24.6 Hz),
131.63 (d, 3JC-F = 9.1 Hz), 132.67 (d, 3JC-F = 11.2 Hz), 133.44, 147.20, 148.57, 149.31, 153.71 (d, 1JC-F = 212.1 Hz),
158.07, 159.13, 161.58, 162.46, 165.32; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -114.11.
N-(2-{4-[(3-Bromo-4-methylphenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethyl-2-(3-nitro-[1,2,4]triazol-1-y
l)acetamide (9c). Faint green solid powder with the yield of 58.3%; ESI-MS m/z = 557.09, 559.09 [M+H]+;
1
GC-HRMS calcd for C22H21BrN8O5, m/z = 557.0897, 559.0876 [M+H]+, found: 557.0899, 559.0879; H-NMR
(600 MHz, DMSO-d6): δ (ppm): 2.33 (s, 3H), 3.35 (t, J = 5.6 Hz, 2H), 3.96 (s, 3H), 4.35 (m, 2H), 5.15 (s, 2H),
7.21 (s, 1H), 7.32 (d, 1H), 7.78 (m, 2H), 8.13 (s, 1H), 8.48 (s, 1H), 8.54 (t, J = 4.2 Hz, 1H), 8.83 (s, 1H), 9.45 (s,
1H); 13C-NMR (151 MHz, DMSO-d6): δ (ppm): 21.69, 38.98, 52.59, 56.24, 68.96, 101.95, 108.89, 121.11, 123.40,
124.83, 130.56, 131.49, 138.80, 146.89, 148.92, 152.68, 153.44, 156.03, 161.98, 164.80.
N-{2-[2-({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4]
triazol-1-yl)acetamide (10a). Faint yellow solid powder with the yield of 56.1%; ESI-MS m/z = 605.10, 607.09
[M+H]+; GC-HRMS calcd for C23H23BrFN8O6, m/z = 605.0908, 607.0887 [M+H]+, found: 605.0906, 607.0885;
1H-NMR (600 MHz, DMSO-d6): δ (ppm): 3.35 (t, J = 5.4 Hz, 2H), 3.58 (t, J = 5.4 Hz, 2H), 3.86 (t, J = 4.2Hz, 2H),
3.96 (s, 3H), 4.30 (t, J = 4.2 Hz, 2H), 5.13 (s, 2H), 7.24 (s, 1H), 7.48 (d, 1H), 7.54 (t, 1H), 7.67 (d, 1H), 7.82 (s,
1H), 8.37 (s, 1H), 8.55 (t, J = 5.4 Hz, 1H), 9.55 (s, 1H); 13C-NMR (151 MHz, DMSO-d6): δ (ppm): 38.96, 52.55,
56.11, 68.01, 68.46, 68.95, 102.00, 107.74, 108.72, 117.49 (d, 2JC-F = 22.2 Hz), 119.20 (d, 2JC-F = 24.6 Hz), 126.40
3
3
1
(d, JC-F = 9.2 Hz), 127.46 (d, JC-F = 11.2 Hz), 129.51, 147.90 (d, JC-F = 210.1 Hz), 148.08, 152.91, 155.79,
156.86, 157.46, 161.93, 164.75; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -115.45.
N-{2-[2-({4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4]
triazol-1-yl)acetamide (10b). Yellow solid powder with the yield of 64.1%; ESI-MS m/z = 561.13 [M+H]+;
GC-HRMS calcd for C23H22ClFN8O6 m/z = 561.1413 [M+H]+, found: 561.1411; 1H-NMR (400 MHz, DMSO-d6):
δ (ppm): 3.35 (t, J = 5.6 Hz, 2H), 3.59 (t, J = 5.6 Hz, 2H), 3.86 (m, 2H), 3.96 (s, 3H), 4.30 (m, 2H), 5.15 (s, 2H),
7.23 (s, 1H), 7.31 (td, J1 = 9.6 Hz, J2 = 2.8 Hz, 1H), 7.59 (m, 2H), 7.86 (s, 1H), 8.33 (s, 1H), 8.59 (t, J = 5.2 Hz,
1H), 9.57 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ (ppm): 39.47, 53.06, 56.57, 68.49, 68.98, 69.47, 108.54,
108.25, 109.03, 115.18 (d, 2JC-F = 22.2 Hz), 117.31 (d, 2JC-F = 25.7 Hz), 131.69 (d, 3JC-F = 9.2 Hz), 132.69 (d, 3JC-F
= 11.2 Hz), 133.43, 147.24, 148.60, 149.34, 153.73 (d, 1JC-F = 212.1 Hz), 158.11, 159.14, 161.59, 162.44, 165.28;
19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -114.06.
N-{2-[2-({4-[(3-Bromo-4-methylphenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4
]triazol-1-yl)acetamide (10c). Green solid powder with the yield of 68.9%; ESI-MS m/z = 601.11 [M+H]+;
1
GC-HRMS calcd for C24H25BrN8O6 m/z = 601.1159, 603.1138 [M+H]+, found: 601.1160, 603.1140; H-NMR
(600 MHz, DMSO-d6): δ (ppm): 2.32 (s, 3H), 3.33 (t, J = 5.6 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H), 3.84 (t, J = 4.0 Hz,

ACCEPTED MANUSCRIPT
2H), 3.96 (s, 3H), 4.30 (t, J = 4.0 Hz, 2H), 5.13 (s, 2H), 7.20 (s, 1H), 7.32 (d, 1H), 7.79 (m, 2H), 8.12 (s, 1H), 8.48
(s, 1H), 8.52 (t, J = 4.2 Hz, 1H), 8.83 (s, 1H), 9.43 (s, 1H); 13C-NMR (151 MHz, DMSO-d6): δ (ppm): 21.67, 38.99,
52.58, 56.21, 68.00, 68.48, 68.97, 101.91, 108.92, 121.09, 123.38, 124.84, 130.55, 131.47, 138.77, 146.87,
148.93, 152.68, 153.40, 156.02, 161.95, 164.76
N-{2-[2-({4-[(3,4-Difluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4]triaz
ol-1-yl)acetamide (10d). Yellow solid powder with the yield of 58.9%; ESI-MS m/z = 545.17 [M+H]+;
GC-HRMS calcd for C23H22F2N8O6 m/z = 545.1709 [M+H]+, found: 561.1711; 1H-NMR (400 MHz, DMSO-d6): δ
(ppm): 3.36 (t, J = 5.6 Hz, 2H), 3.59 (t, J = 5.6 Hz, 2H), 3.86 (m, 2H), 3.98 (s, 3H), 4.29 (m, 2H), 5.15 (s, 2H),
7.22 (s, 1H), 7.44 (dd, J1 = 19.2 Hz, J2 = 8.0 Hz, 1H), 7.60 (m, 1H), 7.84 (s, 1H), 8.08 (ddd, J1 = 13.2 Hz, J2 = 7.6
Hz, J3 = 2.0 Hz, 1H), 8.50 (s, 1H), 8.58 (t, J = 5.6 Hz, 1H), 9.60 (s, 1H); 13C-NMR (101 MHz, DMSO-d6): δ (ppm):
2
2
39.46, 53.06, 56.72, 68.48, 68.95, 69.45, 102.40, 108.36, 109.34, 111.46 (d, JC-F = 21.2 Hz), 117.36 (d, JC-F
=
17.7 Hz), 118.57, 137.13 (d, 3JC-F = 9.2 Hz), 145.73 (dd, 1JC-F = 241.4 Hz, 2JC-F = 12.7 Hz), 147.40, 148.60, 149.21
1
2
(dd, JC-F = 243.4 Hz, JC-F = 12.7 Hz), 149.43, 153.05, 153.94, 156.44, 162.43, 165.27; 19F-NMR (376 MHz,
DMSO-d6) : δ (ppm): -137.77 (d, 3JF-F = 23.2 Hz), -145.10 (d, 3JF-F = 23.2 Hz).
N-{2-[2-({4-[(3-Florophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4]triazol-1-y
l)acetamide (10e). Faint yellow solid powder with the yield of 57.1%; ESI-MS m/z = 527.18 [M+H]+; GC-HRMS
calcd for C23H23FN8O6 m/z = 527.1803 [M+H]+, found: 527.1803; 1H-NMR (400 MHz, DMSO-d6): δ (ppm):
3.35 (dd, J1 = 10.8 Hz, J1 = 5.2 Hz, 2H), 3.59 (t, J = 5.6 Hz, 2H), 3.86 (m, 2H), 3.99 (s, 3H), 4.29 (m, 2H), 5.15 (s,
2H), 6.93 (td, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.23 (s, 1H), 7.42 (dd, J1 =15.2 Hz, J2 = 8.0 Hz, 1H), 7.64 (d, 1H), 7.90
(m, 2H), 8.56 (s, 1H), 8.59 (t, J=5.2 Hz, 1H), 8.86 (s, 1H), 9.77 (brs, 1H); 13C-NMR (101 MHz, DMSO-d6): δ
(ppm): 39.45, 53.05, 56.81, 68.55, 68.92, 69.44, 102.64, 107.68, 109.17, 109.39 (d, 3JC-F = 7.8 Hz). 110.18 (d, 2JC-F
= 21.0 Hz), 118.18, 130.33 (d, 3JC-F = 9.5 Hz), 141.68 (d, 2JC-F = 11.2 Hz), 146.42, 148.60, 149.56, 152.72, 155.40
(d, 1JC-F = 254.4 Hz), 161.28, 162.43, 163.67, 165.27; 19F-NMR (376 MHz, DMSO-d6): δ (ppm): -112.68.
N-{2-[2-({4-[(4-Florophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,2,4]triazol-1-y
l)acetamide (10f). Faint yellow solid powder with the yield of 52.2%; ESI-MS m/z = 527.18 [M+H]+; GC-HRMS
calcd for C23H23FN8O6 m/z = 527.1803 [M+H]+, found: 527.1805; 1H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.35
(dd, J1 = 10.8 Hz, J2 = 5.2 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H), 3.85 (t, J = 4.0 Hz, 2H), 3.97 (s, 3H), 4.29 (m, 2H),
5.13 (s, 2H), 7.23 (m, 3H), 7.78 (dd, J1 = 8.8 Hz, J2 = 5.2 Hz, 2H), 7.83 (s, 1H), 8.43 (s, 1H), 8.56 (t, J = 5.2 Hz,
1H), 8.84 (s, 1H), 9.51 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ (ppm): 39.45, 53.05, 56.84, 68.55, 68.92, 69.43,
3
2
102.66, 107.68, 109.17, 116.87 (d, JC-F = 6.6 Hz), 119.19 (d, JC-F = 18.4 Hz), 141.65, 146.42, 148.61, 149.46,
152.72 (d, 1JC-F = 252.4 Hz), 161.29, 162.45, 163.57, 166.07; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -119.28.
N-{2-[2-({4-[(3,4-Dichloro-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)ethoxy]ethyl}-2-(3-nitro-[1,
2,4]triazol-1-yl)acetamide (10g). White solid powder with the yield of 52.2%; ESI-MS m/z=595.10 [M+H]+；
GC-HRMS calcd for C23H22Cl2FN8O6, m/z = 595.1023 [M+H]+, found: 595.1021; 1H-NMR (400 MHz, DMSO-d6):
δ (ppm): 3.33 (t, J = 5.6 Hz, 2H), 3.57 (t, J = 5.6 Hz, 2H), 3.85 (m, 2H), 3.95 (s, 3H), 4.29 (m, 2H), 5.12 (s, 2H),
7.23 (s, 1H), 7.58 (m, 2H), 7.79 (s, 1H), 8.39 (s, 1H), 8.52 (t, J = 5.4 Hz, 1H), 8.83 (s, 1H), 9.69 (br.s., 1H);
13C-NMR (400 MHz, DMSO-d6): δ (ppm): 38.96, 52.56, 56.08, 68.06, 68.44, 68.95, 101.97, 107.71, 108.73,
119.58 (d, 2JC-F = 19.2 Hz), 125.26 (d, 3JC-F = 2.0 Hz), 126.74, 127.30 (d, 2JC-F = 13.1 Hz), 128.56, 146.88, 148.07,
149.05, 152.76, 153.04 (d, 1JC-F = 251.5 Hz), 153.65, 156.64, 161.93, 164.73; 19F-NMR (376 MHz, DMSO-d6) : δ
(ppm): -113.48.
N-(2-{2-[2-({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethoxy]ethoxy}ethyl)-2-(3-
nitro-[1,2,4]triazol-1-yl)acetamide (11a). Faint yellow solid powder with the yield of 51.2%; ESI-MS m/z =
649.12, 651.12 [M+H]+; GC-HRMS calcd for C25H26BrFN8O7, m/z = 649.1170, 651.1150 [M+H]+, found:
1
649.1168, 651.1149; H-NMR (400 MHz, DMSO-d6): δ (ppm): 3.32 (t, J = 5.4 Hz, 2H), 3.58 (m , 4H), 3.86 (m,

ACCEPTED MANUSCRIPT
4H), 3.96 (s, 3H), 4.35 (t, J = 4.2 Hz, 2H), 5.11 (s, 2H), 7.25 (s, 1H), 7.45 (d, 1H), 7.54 (t, 1H), 7.65 (d, 1H), 7.80
(s, 1H), 8.35 (s, 1H), 8.56 (t, J = 5.4 Hz, 1H), 9.54 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ (ppm): 39.03, 52.57,
56.08, 68.10, 68.67, 68.86, 69.64, 69.91, 101.99, 107.78, 108.71, 117.56 (d, 3JC-F = 9.0 Hz), 119.33 (d, 2JC-F = 23.8
2
1
Hz), 126.40 (d, JC-F = 12.2 Hz), 127.52, 129.57, 147.92 (d, JC-F = 207.3 Hz), 148.13, 152.94, 153.57, 155.42,
156.89, 157.92, 161.90, 164.73; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -115.55 (t, 3JH-F = 7.5 Hz).
N-(2-{2-[2-({4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethoxy]ethoxy}ethyl)-2-(3-
nitro-[1,2,4]triazol-1-yl)acetamide (11b). Yellow solid powder with the yield of 54.9%; ESI-MS m/z = 605.17
1
[M+H]+; GC-HRMS calcd for C25H26ClFN8O7 m/z = 605.1675 [M+H]+, found: 605.1671; H-NMR (400 MHz,
DMSO-d6): δ (ppm): 3.35 (t, J = 5.6 Hz, 2H), 3.56 (m, 4H), 3.87 (m, 4H), 3.96 (s, 3H), 4.29 (t, J = 4.2 Hz, 2H),
5.14 (s, 2H), 7.23 (s, 1H), 7.30 (td, J1 = 9.6 Hz, J2 = 2.8 Hz, 1H), 7.59 (m, 2H), 7.88 (s, 1H), 8.34 (s, 1H), 8.57 (t, J
= 5.2 Hz, 1H), 9.58 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ (ppm): 39.47, 53.06, 56.57, 68.49, 68.52, 68.98,
69.11, 69.46, 108.60, 108.23, 109.11, 115.16 (d, 2JC-F = 22.2 Hz), 117.29 (d, 2JC-F = 25.7 Hz), 131.68 (d, 3JC-F = 9.2
3
1
Hz), 132.60 (d, JC-F = 11.2 Hz), 133.38, 147.25, 148.57, 149.36, 153.72 (d, JC-F = 212.1 Hz), 158.10, 159.13,
161.57, 162.45, 165.32; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -114.10.
N-(2-{2-[2-({4-[(3-Bromo-4-methylphenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethoxy]ethoxy}ethyl)-2-(3-
nitro-[1,2,4]triazol-1-yl)acetamide (11c). Faint green solid powder with the yield of 66.6%; ESI-MS m/z =
645.14, 647.13 [M+H]+; GC-HRMS calcd for C26H29BrN8O7 m/z = 645.1421, 647.1400 [M+H]+, found: 645.1423,
647.1401; 1H-NMR (600 MHz, DMSO-d6): δ (ppm): 2.30 (s, 3H), 3.35 (t, J = 5.4 Hz, 2H), 3.54 (m, 4H), 3.86 (m,
4H), 3.96 (s, 3H), 4.34 (t, J = 4.2 Hz, 2H), 5.15 (s, 2H), 7.19 (s, 1H), 7.33 (d, 1H), 7.78 (m, 2H), 8.10 (s, 1H), 8.49
(s, 1H), 8.50 (t, J = 4.2 Hz, 1H), 8.83 (s, 1H), 9.44 (s, 1H); 13C-NMR (151 MHz, DMSO-d6): δ (ppm): 21.63, 38.92,
52.59, 56.26, 68.09, 68.17, 68.42, 68.48, 68.97, 102.04, 108.94, 121.16, 123.36, 124.81, 130.56, 131.50, 138.78,
146.82, 148.87, 152.66, 153.43, 159.92, 161.52, 164.68.
N-(2-{2-[2-({4-[(3,4-Difluorophenyl)amino]-6-methoxy-quinazolin-7-yl}oxy)ethoxy]ethoxy}ethyl)-2-(3-nitro-[
1,2,4]triazol-1-yl)acetamide (11d). Yellow solid powder with the yield of 42.2%; ESI-MS m/z = 545.20 [M+H]+;
GC-HRMS calcd for C25H26F2N8O7 m/z = 589.1971 [M+H]+, found: 589.1968; 1H-NMR (400 MHz, DMSO-d6): δ
(ppm): 3.35 (t, J = 5.6 Hz, 2H), 3.57 (m, 4H), 3.86 (m, 2H), 3.98 (s, 3H), 4.28 (m, 4H), 5.15 (s, 2H), 7.22 (s, 1H),
7.42 (dd, J1 = 18.8 Hz, J2 = 7.6 Hz, 1H), 7.56 (m, 1H), 7.84 (s, 1H), 8.09 (m, 1H), 8.50 (s, 1H), 8.54 (t, J = 5.6 Hz,
1H), 9.61 (s, 1H); 13C-NMR (101 MHz, DMSO-d6): δ (ppm): 39.51, 53.09, 56.66, 68.43, 68.48, 68.89, 68.95,
2
2
3
69.42, 102.30, 108.31, 109.28, 111.36 (d, JC-F = 22.4 Hz), 117.28 (d, JC-F = 16.6 Hz), 118.48, 137.03 (d, JC-F
=
1
1
8.6 Hz), 145.47 (dd, JC-F = 237.4 Hz, 2JC-F = 11.1 Hz), 147.44, 148.10, 148.98 (dd, JC-F = 241.4 Hz, 2JC-F = 11.1
3
Hz), 149.38, 153.16, 153.92, 156.39, 162.40, 165.41; 19F-NMR (376 MHz, DMSO-d6) : δ (ppm): -137.71 (d, JF-F
= 23.2 Hz), -145.07 (d, 3JF-F = 23.2 Hz).
4.2 Molecular docking section
Docking studies of compound 10a with EGFR (PDB ID: 4I23) and VEGFR-2 (PDB ID: 2RL5) were
performed using vandetanib as a comparison. The molecular docking procedure was performed by the Glide Dock
method using Schrödinger software (Schrödinger Corp., New York, NY, USA) as previously described [12].
Docking results were visualized using Chimera software (version number: 1.13).
4.3 Biological test section
All cell lines mentioned above were purchased from Cell Bank of China Science Academy (Beijing, China).
The above cells were cultured in RPMI-1640 (Gibco Corp., USA) with 10% fetal calf serum. VEGFR-2 (Carna,
Cat.No 08-191,), EGFR (Carna, Cat.No 08-115), Peptide FAM-P22 (GL Biochem, Cat. No. 112393), ATP (Sigma,
Cat. No. A7699-1G) and staurosporine (MCE, Cat. No. HY-15141) were used for kinase inhibitory assay. CCK-8
assay kit (BestBio Corp., Shanghai, China) were applied in in vitro anti-proliferative assays. Primer sequences of

ACCEPTED MANUSCRIPT
VEGF and β-actin (Sangon Corp., Shanghai, China) were used for VEGF/EGF gene expression inhibition tests.
Cobalt chloride hexahydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the test compounds
were dissolved in DMSO for use.
4.3.1 Kinase inhibitory assay
14 target compounds, LYL-10 and vandetanib were screened in vitro using staurosporine as the reference
compound. Tested compounds and reference compound were dissolved in DMSO to obtain drug solutions with
different concentrations of 40 µM, 10 µM, 2.5 µM, 625 nM, 156 nM, 39 nM, 10 nM, 2 nM, 1 nM and 0.2 nM.
Transfer 10 µL of the compound from the source plate to a new 96-well plate as the intermediate plate. Add 90 µL
of 1x kinase buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35) to each well of the intermediate plate. Mix the
compounds in an intermediate plate for 10 min on a shaker. Transfer 5 µL of each well from the 96-well
intermediate plate to a 384-well plate in duplicates. 10 µL of 2.5 x enzyme solutions were added to each well of
the 384-well assay plate and incubated at room temperature for 10 min; then 10 µL of 2.5 x peptide solution were
added to each well of the 384-well assay plate and incubated at 28 °C for a specified period. 25 µL of stop buffer
(100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent, 50 mM EDTA) were added to stop reaction.
Collect conversion data from Caliper program; convert conversion values to inhibition values by Equation (1):
Percent inhibition (%) = (max-conversion)/(max-min) × 100.
where“max” stands for DMSO control; “min” stands for low control.
Fit the data in XLFit excel add-in version 5.4.0.8 to obtain IC50 values by Equation (2):
Y=Bottom + (Top-Bottom)/(1+(IC50/X)^HillSlope)
4.3.2 In vitro anti-proliferative assay in hypoxia and normoxia
Evaluation of the anti-proliferative activity in hypoxia and normoxia of the compounds was performed using
the CCK-8 assay as previously described [12]. In hypoxic + irradiation group, cobalt (II) chloride solution was
added (except for blank group and controlꢉꢊ ꢋꢊꢋꢌ group) and incubated for 24 h, then was irradiated at the dose of
ꢅ
8 Gy for another 24 h at 37 °C in 5% CO2, 20 µL CCK-8 solution was added to per well and incubated for another
1 h. Measured absorbance at 450 nm in a plate reader and calculated for inhibition ratios.
Hypoxic sensitive enhancement ratios (HSERhyp) were calculated using Equation (3):
ꢀꢁꢔꢕꢖ
ꢍꢎꢏꢐꢑꢒꢓ
=
ꢙ
ꢚ
× ꢀꢁ
ꢀꢁꢗꢃꢘ + 1 − ꢀꢁ
ꢗꢃꢘ
ꢂꢃꢂꢄꢅ
Where Irhyp and Irnor mean the inhibition ratios of target compounds or vandetanib in hypoxia and
normoxia; Irꢋꢊꢋꢌ means the inhibition ratios of CoCl2 group and CoCl2+irradiation group on A549 and H446
ꢅ
cells.
Irradiation-hypoxic sensitive enhancement ratios (HSERhyp+IR,) was calculated using Equation (4):
ꢀꢁꢔꢕꢖꢆꢇꢈ
ꢍꢎꢏꢐꢑꢒꢓꢆꢛꢜ
=
ꢀꢁꢑꢒꢓ + ꢝ1 − ꢀꢁꢑꢒꢓꢞ × ꢀꢁ
ꢂꢃꢂꢄꢅꢆꢛꢜ
Where Irhyp+IR means the inhibition ratios of target compounds or vandetanib in hypoxia+irradiation;
Irꢋꢊꢋꢌ ꢆꢛꢜ means the inhibition ratios of CoCl2+irradiation group on A549 and H446 cells.
ꢅ
4.3.3 In vitro VEGF gene expression inhibition assays
Evaluation of the VEGF gene expression levels in hypoxia and normoxia of the compounds was performed as
previously described[12].
4.3.4 In vivo anticancer activity assay
Mice were orthotopically implanted with in vitro cultured A549 cells (1 × 107 cells per mouse). After 6 or 7
days of cell inoculation, mice were divided into groups, and the administration of tested compounds was started.

ACCEPTED MANUSCRIPT
Tumor dimensions were measured using a caliper, and the body weight of animals was monitored daily. Tumor
volume (mm3) was calculated using the formula (a2×b)/2, where a = shortest tumor diameter in millimeters, and b
= longest tumor diameter in millimeters. Tumor growth inhibition (TGI) was calculated using Equation (5):
TGI (%) = [1-(Vt-V0)/(Vctrl-Vctrl-0)] × 100
Where Vt means tumor volume of treated mice and Vctrl that untreated control animals; V0 and Vctrl-0 mean
tumor volumes of treated mice and control animals at the day starting the administration.
Ethics statement
The study was approved by the Institutional Animal Ethical Committee of the Institute of Radiation Medicine
(Peking Union Medical College & Chinese Academy of Medical Sciences, China). Animal welfare and
experimental procedures were followed in accordance with the Guide for Care and Use of Laboratory Animals
(National Institutes of Health, US).
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31670859), CAMS
Innovation Fund for Medical Sciences (CIFMS 2016-I2M-3-022 and 2017-I2M-3-019) and Fundamental Research
Funds for the Central Universities (3332018117).
Conflict of interest
The authors declare no conflict of interest.
References:
[1]. (a) D. S. Krause, R. A. Van Etten, Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353 (2005)
172-187. (b) K. S. Bhullar, N. O. Lagarón, E. M. McGowan, I. Parmar, A. Jha, B. P. Hubbard, H. P. V.
Rupasinghe, Kinase-targeted cancer therapies: progress, challenges and future directions. Mol. Cancer 17
(2018) 48-68.
[2]. (a) C. Widakowich, G. de Castro, Jr., E. de Azambuja, P. Dinh, A. Awada, Review: side effects of approved
molecular targeted therapies in solid cancers. Oncologist 12 (2007) 1443-1455; (b) F. A. L. M. Eskens, J.
Verweij, The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial
growth factor receptor (VEGFR) targeting angiogenesis inhibitors; A review. Eur. J. Cancer. 42 (2006)
3127-3139; (c) H. Izzedine, S. Ederhy, F. Goldwasser, J. C. Soria, G. Milano, A. Cohen, D. Khayat, J. P.
Spano, Management of hypertension in angiogenesis inhibitor-treated patients. Ann. Oncol. 20 (2009)
807-815.
[3]. (a) D. R. Shah, R. R. Shah, J. Morganroth, Tyrosine kinase inhibitors: their on-target toxicities as potential
indicators of efficacy. Drug Safety. 36 (2013) 413-426; (b) T. Force, D. S. Krause, R. A. Van Etten,
Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nat. Rev. Cancer. 7 (2007) 322-344.
[4]. L. Goethals, A. Debucquoy, C. Perneel, K. Geboes, N. Ectors, H. De Schutter, F. Penninckx, W. H. McBride,
A. C. Begg, K. M. Haustermans, Hypoxia in human colorectal adenocarcinoma: comparison between
extrinsic and potential intrinsic hypoxia markers. Int. J. Radiat. Oncol. Biol. Phys. 65 (2006) 246-254.
[5]. (a) C. W. Pugh, P. J. Ratcliffe, Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9
(2003) 677-684; (b) P. H. Maxwell, G. U. Dach, J. M. Gleadle, L. G. Nicholls, A. L. Harris, I. J. Stratford, O.
Hankinson, C. W. Pugh, P. J. Ratcliffe. Hypoxia-inducible factor-1 modulates gene expression in solid tumors
and influences both angiogenesis and tumorꢀgrowth. Proc. Natl. Acad. Sci. USA. 94 (1997) 8104-8109; (c) D.
Shweiki, A. Itin, D. Soffer, E. Keshet, Vascular endothelial growth factor induced by hypoxia may mediate
hypoxia-initiated angiogenesis. Nature. 359 (1992) 843-845; (d) B. J. Moeller, Y. Cao, Z. Vujaskovic, C. Y.
Li, Z. A. Haroon, M. W. Dewhirst, The relationship between hypoxia and angiogenesis. Semin. Radiat. Oncol.

ACCEPTED MANUSCRIPT
14 (2004) 215-221.
[6]. (a) L. M. Van Putten, R. F. Kallman, Oxygenation status of a transplantable tumor during fractionated
radiation therapy. J. Natl. Cancer. I. 40 (1968) 441-451; (b) R. F. Kallman, The phenomenon of
reoxygenation and its implications for fractionated radiotherapy. Radiology 105 (1972) 135-142.
[7]. (a) K. De Bock, M. Mazzone, P. Carmeliet, Antiangiogenic therapy, hypoxia, and metastasis: risky liaisons,
or not? Nat. Rev. Clin. Oncol. 8 (2011) 393-404; (b) G. Bergers, D. Hanahan, Modes of resistance to
anti-angiogenic therapy. Nat. Rev. Cancer. 8 (2008) 592-603.
[8]. (a) I. Ott, R. Gust, Non platinum metal complexes as anti-cancer drugs. Arch. Pharm. 340 (2007) 117-126; (b)
U. Jungwirth, C. R. Kowol, B. K. Keppler, C. G. Hartinger, W. Berger, P. Heffeter, Anticancer activity of
metal complexes: involvement of redox processes. Antioxid. Redox. Sign. 15 (2011) 1085-1127.
[9]. C. Karnthaler-Benbakka, D. Groza, K. Kryeziu, V. Pichler, A. Roller, W. Berger, P. Heffeter, C. R. Kowol,
Tumor-targeting of EGFR inhibitors by hypoxia-mediated activation. Angew. Chem. Int. Ed. Engl. 53 (2014)
12930-12935.
[10]. (a) J. X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G. Lorente, M. Banica, D. Jung, J.
Wang, H. Ma, X. Li, Z. Yang, R. M. Hoffman, W. S. Ammons, C. P. Hart, M. Matteucci, Potent and highly
selective hypoxia-activated achiral phosphoramidate mustards as anticancer drugs. J. Med. Chem. 51 (2008)
2412-2420; (b) C. Karnthaler-Benbakka, D. Groza, B. Koblmüller, A. Terenzi, K. Holste, M. Haider, D. Baier,
W. Berger, P. Heffeter, C.R. Kowol, B.K. Keppler, Targeting a targeted drug: an approach toward
hypoxia-activatable tyrosine kinase inhibitor prodrugs. ChemMedChem. 11(2016) 2410-2421.
[11]. D. Das, J. Hong, Recent advancements of 4-aminoquinazoline derivatives as kinase inhibitors and their
applications in medicinal chemistry. Eur. J. Med. Chem. 170 (2019) 55-72.
[12]. H. Q. Wei, D. G. Li, X. B. Yang, H. H. Shang, S. J. Fan, Y. L. Li, D. Song, Design and synthesis of
vandetanib derivatives containing nitroimidazole groups as tyrosine kinase inhibitors in normoxia and
hypoxia. Molecules. 21 (2016) 1693-1708.
[13]. (a) B. Melgaard, H. S. Hansen, Z. Kamieniecka, O. B. Paulson, A. G. Pedersen, X. F. Tang, W. Trojaborg,
Misonidazole neuropathy: a clinical, electrophysiological and histological study. Ann. Neurol. 12 (1982)
10-17; (b) M. A. Stevenson, S. K. Calderwood, C. N. Coleman, Effects of nitroimidazoles on neuronal cells
in vitro. Int. J. Radiat. Oncol. Biol. Phys. 16 (1989) 1225-1230.
[14]. (a) T. Inomata, Y. Ogawa, A. Nishioka, N. Hamada, S. Ito, S. Kariya, S. Yoshida, H. Nagasawa,
H. Hori, S. Inayama, The immunopotentiation effects of the bifunctional radiosensitizer KIN-806 in
comparison with its analogs KIN-804 and KIN-844. Oncol. Rep. 6 (1999) 1209-1221; (b) T. Inomata, Y.
Ogawa, S. Ito, S. Kariya, N. Hamada, A. Nishioka, S. Yoshida, H. Nagasawa, H. Hori, S. Inayama, Lung
metastasis suppression of the bifunctional new radiosensitizer KIN-806. Int. J. Mol. Med. 4 (1999) 257; (c) S.
Kasai, H. Nagasawa, M. Yamashita, M. Masui, H. Kuwasaka, T. Oshidani, Y. Uto, T. Inomata, S. Oka, S.
Inayama, H. Hori, New antimetastatic hypoxic cell radiosensitizers: design, synthesis, and biological
activities of 2-nitroimidazole-acetamide, TX-1877, and its analogues. Bioorg. Med. Chem. 9 (2001) 453-464.
[15]. (a) K. Pasupathy, C. K. K. Nair, T. V. Kagiya, Effect of a hypoxic radiosensitizer, AK 2123 (Sanazole), on
yeast Saccharomyces cerevisiae. J. Radiat. Res. 42 (2001) 217-227; (b) R. Rajagopalan, T. V. Kagiya, C. K.
K. Nair, Radiosensitizer Sanazole (AK-2123) enhances γ-radiation-induced apoptosis in murine fibrosarcoma.
J. Radiat. Res. 44 (2003) 359-365; (c) C. Sugie, Y. Shibamoto, M. Ito, H. Ogino, H. Suzuki, Y. Uto, H.
Nagasawa, H. Hori, Reevaluation of the radiosensitizing effects of Sanazole and Nimorazole in vitro and in
vivo. J. Radiat. Res. 46 (2005) 453-459; (d) S. Kapoor, R. Mathew, N. G. Huilgol, T. V. Kagiya, C. K.
K. Nair, Redox reactions of Sanazole (AK-2123) in aqueous solutions: a pulse radiolysis study. J. Radiat. Res.
41 (2000) 355-366. (e) W. Dobrowskya, N. G. Huigolb, R. S. Jayatilakec, N. Kizilbashd, Sait Okkane, V. T.

ACCEPTED MANUSCRIPT
Kagiyaf, H. Tatsuzaki, AK-2123 (Sanazol) as a radiation sensitizer in the treatment of stage III cervical
cancer: Results of an IAEA multicentre randomised trial. Radiother. Oncol. 82 (2007) 24-29.
[16]. (a) R. S. Herbst, M. S. Kies. ZD1839 (Iressa) in non-small cell lung cancer, Oncologist, 2002, 7 (Suppl 4):
9215; (b) V. Grunwald, M. Hidalgo. Development of the epidermal growth factor receptor inhibitor Tarceva
(OSI2774), Adv. Exp. Med. Biol., 2003, 532: 235-246.
[17]. L. F. Hennequin, E. S. E. Stokes, A. P. Thomas, C. Johnstone, P. A. Ple, D. J. Ogilvie, M. Dukes, S. R.
Wedge, J. Kendrew, J. O. Curwen, Novel 4-anilinoquinazolines with C-7 basic side chains: design and
structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J.
Med. Chem. 45 (2002) 1300-1312.
[18]. J. C. Hancox, A. F. James, Refining insights into high-affinity drug binding to the human
ether-à-go-go-related gene potassium channel. Mol. Pharmacol, 73 (2008) 1592-1595.

ACCEPTED MANUSCRIPT
Highlights
•Fourteen novel 4-anilinoquinazoline derivatives containing 3-nitro-1,2,4-triazole moiety with
dual EGFR/VEGFR-2 kinase inhibitory activities were designed and synthesized.
•Lead compounds, 10a and 10g, showed enhanced anti-proliferative activity on cancer cells
under hypoxia compared to vandetanib.
•10a and 10g not only inhibited tumor growth in A549 xenografts but also significantly reduce
toxicity associated with weight loss compared to vandetanib.