Design and synthesis of vandetanib derivatives containing nitroimidazole groups as tyrosine kinase inhibitors in normoxia and hypoxia

Article abstract of DOI:10.3390/molecules21121693

Sixteen novel epidermal growth factor receptor (EGFR)/vascular endothelial growth factor (VEGF)-2 inhibitors (nitroimidazole-substituted 4-anilinoquinazoline derivatives (16a-p)) were designed and prepared via the introduction of a nitroimidazole group in the piperidine side chain and modification on the aniline moiety of vandetanib. Preliminary biological tests showed that comparing with vandetanib, some target compounds exhibited excellent EGFR inhibitory activities and anti-proliferative over A549/H446 cells in hypoxia. Meanwhile, several of the above compounds demonstrated better bioactivity than vandetanib in VEGF gene expression inhibition. Owing to the excellent IC50 value (1.64 μmol/L), the inhibition ratios of 16f over A549 and H446 cells were 62.01% and 59.86% at the concentration of 0.5 μM in hypoxia, respectively. All of these results indicated that 16f was a potential cancer therapeutic agent in hypoxia and was worthy of further development.

Full text of DOI:10.3390/molecules21121693

molecules
Article
Design and Synthesis of Vandetanib Derivatives
Containing Nitroimidazole Groups as Tyrosine
Kinase Inhibitors in Normoxia and Hypoxia
1
1
3
1
1
1,
Huiqiang Wei , Deguan Li , Xiangbo Yang , Haihua Shang , Saijun Fan , Yiliang Li *
2
,
and Dan Song *
1
Tianjin 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; wakie0208@163.com (H.W.); ldguan@163.com (D.L.);
shanghaihua2005@163.com (H.S.); fansaijun@163.com (S.F.)
Chongqing Technical Center for Drug Evaluation & Certification, Chongqing 400014, China
School of Petrochemical Engineering, Changzhou University, Changzhou 213164, China;
2
3
1
5122363480@163.com
*
Correspondence: liyiliang@irm-cams.ac.cn (Y.L.); songdan1995@163.com (D.S.);
Tel.: +86-22-8568-2077 (Y.L.); +86-23-6367-6135 (D.S.)
Academic Editor: Diego Muñoz-Torrero
Received: 19 October 2016; Accepted: 6 December 2016; Published: 14 December 2016
Abstract: Sixteen novel epidermal growth factor receptor (EGFR)/vascular endothelial growth
factor (VEGF)-2 inhibitors (nitroimidazole-substituted 4-anilinoquinazoline derivatives (16a–p)) were
designed and prepared via the introduction of a nitroimidazole group in the piperidine side chain
and modification on the aniline moiety of vandetanib. Preliminary biological tests showed that
comparing with vandetanib, some target compounds exhibited excellent EGFR inhibitory activities
and anti-proliferative over A549/H446 cells in hypoxia. Meanwhile, several of the above compounds
demonstrated better bioactivity than vandetanib in VEGF gene expression inhibition. Owing to the
excellent IC50 value (1.64
and 59.86% at the concentration of 0.5
6f was a potential cancer therapeutic agent in hypoxia and was worthy of further development.
µmol/L), the inhibition ratios of 16f over A549 and H446 cells were 62.01%
µM in hypoxia, respectively. All of these results indicated that
1
Keywords: tyrosine kinase inhibitor; vandetanib; nitroimidazole; hypoxia
1
. Introduction
Hypoxia is a common phenomenon in most solid tumors [
from blood vessels and general drugs are difficult to enter internal hypoxic tissues [
cells are resistant to radiotherapy and liable to transform from the G0 phase into normal proliferative
ones after oxygen concentration increased [ ]. Moreover, the expression of hypoxia-inducible factor-1
HIF-1) can be promoted by hypoxia and enhance gene expression levels of some cell growth
factors [4–6] and tyrosine kinase receptors, such as vascular endothelial growth factor (VEGF),
platelet-derived growth factor- (PDGF- ), and epidermal growth factor receptor (EGFR) [ ]. These cell
growth factors and tyrosine kinase receptors induce a series of cellular circumstances, such as
angiogenesis, tumor cells proliferation and migration, through tyrosine kinase signaling pathways [ ].
1
,2]. Hypoxic tumor cells are distant
2
]. Hypoxic tumor
3
(
β
β
7
5
Hence, traditional radiotherapy and chemotherapy are invalid towards hypoxic tumor cells. In recent
years, multi-targeted TKIs and irreversible TKIs have been hot products on market, which are not
effective towards hypoxic tumor cells, yet. Therefore, overcoming hypoxic resistance of solid tumors
has become a tremendous challenge in cancer therapy [8].
Molecules 2016, 21, 1693; doi:10.3390/molecules21121693
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1693
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Nitroimidazole derivatives have been developed as hypoxia radiosensitizers in radiotherapy [9,10]
because of their high affinities to hypoxic tissues, specifically. As electron-affinic compounds,
radiosensitizers fix the radiation-induced damages in DNAs and enhance the effects of
radiotherapy [11,12]. Cytotoxic metabolites of nitroimidazole radiosensitizers in hypoxia are
hydroxylamines, nitroso compounds, and amines, which are liable to bind to DNAs irreversibly
and lethal to tumor cells [13]. Now nitroimidazole derivatives have been used as hypoxia radiotracers
labeled with 9 Tc, Ga and F for tumor hypoxic regions imaging [14
–17]. The DNA alkylating agent
9m
68
18
Evofosfamine (TH-302) also contains a 2-nitroimidazole group and targets to hypoxic tissues in soft
tissue sarcoma (STS) and pancreatic adenocarcinoma [18]. Based on the above studies, we conceived
novel 4-anilinoquinazoline tyrosine kinase inhibitors containing nitroimidazole and anticipated their
efficient antitumor activities both in normoxia and hypoxia.
In this study, vandetanib (Figure 1) was chosen as a small molecule tyrosine kinase inhibitor
targeted to EGFR/VEFR-2, and 2-nitroimidazole hypoxia radiosensitizer KIN-806 [19] (Figure 1) as
lead compounds. According to previous researches [20], substituted 4-anilinoquinazoline scaffold in
vandetanib is the crucial pharmacophore for EGFR/VEFR-2 inhibition, which donates key H-bonds
by N1 atom of quinazoline moiety and inserts into the hydrophobic pocket by aniline moiety
in corresponding crystal structures of EGFR and VEFR-2. The N-methyl-piperidine side chain of
vandetanib is the kinetophore of compatibilities and we merged the amid group of KIN-806 and its
analogues onto the piperidine N4 position in the side chain of vandetanib to obtain target compounds
(Figure 1). Based on this strategy, sixteen target compounds were constructed and evaluated their
molecular docking study as well as in vitro biological activities.
X
2
X
2
X
1
X
3
X
1
X
3
R1
n
N
HN
N
N
N
HN
N
O
O
N
O
R2
N
KIN-806: R
1
= NO2,
R
2
= H, n = 1
R
1
O
O
n
N
N
N
N
O
Vandetanib: X
1
= F, X
2
= H, X
3
=Br
R
2
16a-p
Figure 1. Structures of vandetanib, KIN-806, and target compounds.
2
. Results and Discussion
2
.1. Chemistry
Target compounds were synthesized from substituted 4-anilino-6-methoxyquinazoline (1a
The synthetic route displayed in Scheme 1 was started from 4-piperidine methanol ( ) and
6c). The p-toluene sulfonyl substituted 4-piperidinyl methanol mesylate (
via Boc-protection, sulfonylation [21], and deprotection [22], as reported in
(excepted for 8a) were obtained with good yields (up to 96%)
by reaction of nitroimidazoles (6a ) with -/ -/ -bromo-carboxylic esters in the presence of
base (K CO ) in refluxing acetonitrile; 8a was prepared from methyl acrylate and 6a by Michael
–g).
2
nitroimidazoles (6a
–
5)
was prepared from
2
early studies. Intermediates 7–9
–
c
α β γ
2
3
addition with N,N-diisopropylethylamine (DIPEA) as base in acetonitrile at room temperature with
a good yield of 95.6%. The 2-(substituted-nitroimidazol-1-yl)acetic acid (10a ) were prepared by
acidic hydrolysis in 30% TFA/CH Cl solution; 3-(substituted-nitroimidazol-1-yl)propionic acid (11a
–
c
)
2
2
and 4-(substituted-nitroimidazol-1-yl)butyric acid (12a
–
b
) were prepared by alkaline hydrolysis in
◦
saturated Na CO solution at 85 C for 2.5 h and then acidizing with 6 N HCl solution. Catalyzed by
2
3
carbonyldiimidazole (CDI) and triethylamine (TEA), the key intermediates 13
from and 10 12 in DMF at room temperature with acceptable yields varying from 78%–91%.
Target compounds 16a
–15 were synthesized
5
–
–
p
were obtained by intermediates 13
–
15 connecting with 1a
–
g
in the presence
◦
of K CO in DMF at 85 C with moderate yields of 52%–76%.
2
3

Molecules 2016, 21, 1693
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Scheme 1. Synthesis of target compounds. Reagents and conditions: (
i
) (Boc) O, MeOH, r.t.;
2
◦
◦
(
ii) p-TsCl, pyridine, 0 C to r.t.; (iii) methylsulphonic acid, toluene, i-PrOH, 50 C ; (iv) t-Butyl
bromoacetate, K CO , ACN, reflux, (7a
–
c
); (
v) methyl acrylate, DIPEA, ACN, r.t., 7 days, (8a); (vi) Ethyl
2
3
−
◦
4
0
-bromobutyrate, K CO , ACN, reflux, (9a
.5–3 h, then H , r.t., (11a, 12a–b); (ix) CDI, TEA, DMF, r.t.; and (x) K CO , DMF, 85 C.
–
b
); (vii) TFA, CH Cl , r.t., (10a
–
c
); (viii) OH , 85 C,
2
3
2
2
+
◦
2
3
2
.2. Molecular Docking Study
To predict the possible binding mode of target compounds with EGFR and VEFR-2, two docking
studies of compound 16f were performed using vandetanib as a comparison. As illustrated in Figure 2,
the crystal structure of the complex between 16f and EGFR (Figure 2A) reveals that N1 atom of
quinazoline moiety formed a H-bond with the backbone-NH of Met793 with a distance of 2.823 Å
in a similar way to vandetanib, and the aniline moiety inserted into the hydrophobic pocket but
did not form any additional H-bonds; In another crystal structure complex between 16f and VEFR-2
(
Figure 2B), 16f favorably fitted into VEFR-2 with the aniline moiety occupying the narrow deep
hydrophobic pocket and a critical H-bond between quinazoline N1 atom and Cys919 with a distance of
.099 Å and it was in a similar way to vandetanib. As we expected, the nitroimidazole side chain was
3
positioned at the solvent portion of the kinase. However, an extra H-bind was located at the opening
of the hydrophobic pocket in the crystal structure complex between 16f and VEFR-2, which formed
between the nitro group and the side chain amino group of Lys838 residue with an non-normal distance
of 3.491 Å. We inferred that halogen atoms on the aniline moiety contributed more hydrophobicity and
electronegativity, which caused a reinforced affinity to corresponding receptors; from another aspect,
since the nitroimidazole side chain was positioned at the solvent portion of the kinase, advisable bigger
and heavier halogen atoms on aniline would balance the “barycenter” of target compounds and fixed
it in the complex. Bioassay results have proved this.
Figure 2. The docked poses of compound 16f (C: yellow; N: blue; O: red) or vandetanib (C: green;
N: blue; O: red) at the ATP binding cleft of EGFR kinase (A), and VEGFR-2 kinase (B).
2
.3. Biological Evaluation
In vitro EGFR inhibitory activity, in vitro anti-proliferative activity in normoxia/hypoxia, and
VEGF/EGF gene expression inhibitory activity of target compounds have been determined in

Molecules 2016, 21, 1693
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this section. Human epidermoid carcinoma A431 cells and human non-small cell lung cancer
H1975 cells were used in in vitro EGFR inhibitory activity assays of target compounds by standard
(
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS))
method with vandetanib as a comparison [23]. Human non-small cell lung cancer A549 cells, human
lung cancer H446 cells and human cervical cancer Hela cells were used in in vitro normoxic/hypoxic
anti-proliferative activity tests by CCK-8 method with admixing cobalt chloride to establish a hypoxic
model. VEGF/EGF gene expression inhibitory assays in hypoxia and normoxia were referred to
a previous literature [24] by quantitative polymerase chain reaction (Q-PCR).
2
.3.1. In Vitro EGFR Inhibitory Activity
Two EGFR over-expressed cell lines of human epidermoid carcinoma A431 cells and human
non-small cell lung cancer H1975 cells (T790M mutation [25]) were used to evaluate the in vitro
biological activities of EGFR inhibition by MTS method. As shown in Table 1, more than a half of
target compounds demonstrated acceptable anti-proliferative activities towards A431 cells and 16f
showed the best IC50 value of 1.64
However, 16a exhibited undesirable inhibitory activities over T790M-mutated H1975 cells with
IC50 values all over 40 mol/L, showing much lower activities than vandetanib (IC50 = 4.81 mol/L).
-Bromo-2-fluoro and 3,4-dichloro-2-fluoro substituted compounds (16a 16n and 16p) showed
relatively better inhibitory activities than other substituted 4-anilinoquinazoline compounds; smaller
and lighter fluorine atoms substituted compounds (16j 16l, and 16m) all showed obvious lower
µmol/L, but all were inferior to vandetanib (IC50 = 0.85 µmol/L).
–
p
µ
µ
4
–f,
,
inhibitory activities. The bioassay results coincided well with the aforementioned inference in a DOCK
study. With higher single electron reduction potentials, 2-nitroimidazole derivatives showed more
potent hypoxia radiosensitizing activity than 5(4)-nitroimidazole derivatives as radiosensitizers; side
chain length would contribute to molecular flexibility efficiently, which might cause reduced activities
and unanticipated toxicities. However, the results demonstrated that nitro group’s position and side
chain length (no more than three carbon atoms) did not impact the inhibitory activity observably.
It indicated that introducing different nitroimidazole substituents and different lengths of side chains
to piperidine moiety of vandetanib did not alter its wild-type EGFR inhibitory activity drastically,
but led to detrimental binding interactions with T790M mutant EGFR kinase. Nevertheless, the
in vitro anti-tumor assays proved that target compounds remain normal EGFR inhibitory activities
and nitroimidazole side-chains did not temper with the original EGFR-selective inhibitory activity.
Table 1. In vitro EGFR inhibitory activity assay results of target compounds 16a–p.
Substituent
IC50 (µmol/L)
Compound
R1
R2
n
X1 X2
X3
A431
H1975
1
1
1
1
1
1
1
1
6a
6b
6c
6d
6e
6f
6g
6h
CH3 NO2
CH3 NO2
CH3 NO2
1
2
3
1
3
1
1
1
2
2
2
2
2
2
1
1
-
F
F
F
F
F
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
Br
F
1.82
3.42
2.03
2.10
4.81
1.64
39.76
58.46
49.52
53.42
60.12
40.15
60.01
71.80
78.25
78.82
87.90
74.84
>100
>100
46.22
58.55
37.85
4.81
NO2
NO2
H
H
H
H
H
NO2
NO2
NO2
F
Cl
H
Cl
H
H
H
H
F
Br CH3 12.34
H
F
Br CH3 17.95
F
H
Cl
H
Cl
H
1
1
6i
6j
CH3 NO2
CH3 NO2
CH3 NO2
CH3 NO2
CH3 NO2
CH3 NO2
F
F
15.14
65.42
1
6k
1
6l
6m
6n
H
F
Cl
F
Cl
Br
78.94
66.75
6.75
24.84
9.45
1
1
1
6o
NO2
NO2
-
H
H
-
Cl
F
F
1
6p
Vadetanib
0.85

Molecules 2016, 21, 1693
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2
.3.2. In Vitro Anti-Proliferative Activity Assays in Normoxia and Hypoxia
To confirm whether target compounds showed better anti-tumor activities in hypoxia than
in normoxia, compounds 16a
–
f
,
16h
,
16i
mol/L) over three cell lines (A549, H446 and Hela) through
mol/L cobalt
, 16n, and 16p were tested the inhibition ratios at
two concentrations (5 mol/L and 0.5
µ
µ
CCK-8 method in normoxia and hypoxia. Hypoxia in these assays was induced by 200
µ
chloride solutions. As shown in Table 2, A549 cells inhibition ratios of target compounds were close to
H446 cells (except for 16d) in identical conditions. In hypoxic conditions, nearly all compounds (except
for 16d) exhibited inhibition ratios approaching or exceeding 50%. 16b
anti-proliferative activities over both A549 and H446 cells at the concentration of 0.5
compounds demonstrated more potent inhibitory activities than vandetanib in hypoxic conditions
at both concentrations, especially over A549 cells at the concentration of 0.5 mol/L (except for 16d).
Inhibition ratios of target compounds in hypoxia were all significantly improved compared with
normoxia (except for 16d over A549 cells at 0.5 mol/L), especially 16b and 16f. It was also observed
,
16c, and 16f exhibited favorable
µmol/L. All target
µ
µ
in vandetanib groups, but not as obvious as mentioned above. The results demonstrated that the nitro
group’s position did not impact the inhibitory activity observably in hypoxia. We deduced that the
more potent inhibitory activities of target compounds in hypoxia were attributed to the cytotoxicities
by the reductive activation of nitroimidazole groups, which could counteract the hypoxia-activated
tumor cells to some extent. A recent research [26] has also proved it. However, all target compounds
showed low inhibition ratios over HeLa cells at both two concentrations in normoxia and hypoxia,
and only 16c exhibited the best inhibition ratio barely more than 20% at low concentration in hypoxia.
Compared with vandetanib, target compounds over HeLa cells (except for 16p) showed evidently
inferior inhibition ratios at high concentration and comparable inhibition ratios at low concentration,
notwithstanding that none of them exhibited inhibitory activities over 40%.
Table 2. In vitro anti-proliferative activity assay results of target compounds in normoxia and hypoxia a.
Compound
Vandet-anib 16a
16b
16c
16d
16e
16f
16h
16i
16n
16p
Normoxia
Hypoxia
37.30
40.82
40.43
46.64
37.32
57.95
43.78
71.04
46.74
41.33
66.09
85.70
12.02
52.95
33.17
65.81
38.76
50.29
42.23
56.11
55.84
80.33
A549
5
µM
inhibition
ratio (%)
Normoxia
Hypoxia
27.09
43.01
47.07
44.81
41.56
58.84
35.36
48.42
45.57
60.77
51.87
68.21
30.00
45.74
39.20
56.60
36.92
53.64
31.53
57.32
49.60
70.19
H446
Hela b
A549
Hypoxia
36.43
10.83
26.21
21.40
18.19
23.46
8.00
13.66
18.93
10.53
36.24
Normoxia
Hypoxia
11.22
29.95
4.02
58.08
6.81
69.30
15.24
69.93
11.20
17.62
24.43
50.97
0.52
62.01
15.42
25.70
7.04
30.87
12.75
47.96
22.67
47.13
0
.5 µM
inhibition
ratio (%)
Normoxia
Hypoxia
12.53
37.41
16.95
42.33
8.97
62.94
11.44
62.52
17.92
53.82
22.31
43.74
3.69
59.86
28.79
36.06
12.79
47.72
15.21
52.92
26.04
42.98
H446
Hela b
Hypoxia
14.78
8.09
15.91
21.01
15.48
18.47
11.59
<0
10.54
12.44
18.55
a
Control group in hypoxia was performed with 200 µmol/L cobalt chloride solution and corresponding amount of DMSO.
Anti-proliferative activity test over HeLa cells in normoxia was not determined.
b
2
.3.3. In Vitro VEGF/EGF Gene Expression Inhibitory Activity
We deemed that the cytotoxicities in nitroimidazole reductive activation were not the only reasons
for the enhanced anti-tumor activities in hypoxia and it could not explain the low inhibition ratios over
HeLa cells. Some cell growth factors such as VEGF and EGF played critical roles in tumor proliferation,
survival, invasion, and migration [4]. We conceived that target compounds could impact their genes
expression and promote tumor cell death. To validate the assumptions, we conducted a “black-box”
experiment by testing in vitro VEGF/EGF gene expression levels in hypoxia and normoxia.
Hypoxia in these tests was induced by cobalt chloride and quantitative polymerase chain reaction
(
Q-PCR) was used to evaluate the expression levels of VEGF and EGF genes over A549 cells by testing
VEGF and EGF mRNA amounts. In this assay, compounds 16a were tested at the concentration
of 5 mol/L. As shown in Table 3, The VEGF mRNA levels of target compounds groups in hypoxia
were all lower than control group and vandetanib group significantly by over 1000-fold and 400-fold,
–
f
µ

Molecules 2016, 21, 1693
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respectively. This indicated that all of the target compounds exhibited excellent VEGF gene expression
inhibitory activities and were distinctly superior to vandetanib. As a comparison, VEGF gene
expression levels of target compounds groups in normoxia were all higher than that in hypoxia
by over 100-fold; VEGF gene expression level in normoxia were also higher that in hypoxia by nearly
three-fold in the vandetanib group. However, EGF gene expression level assays exhibited nearly
opposite results. In hypoxia, EGF gene expression levels raised to different extents (except for 16f
)
and the results in vandetanib groups were augmented more obviously than that in target compounds
groups. In normoxia, EGF gene expression level all decreased excepted for 16b and 16e showed a
slightly increased expression level. In these assays, we confirmed that 16f demonstrated all evident
decreased expression levels in hypoxia and normoxia and showed the best inhibitory activities in both
VEGF and EGF gene expression assays.
Table 3. VEGF/EGF gene expression level assay results of target compounds 16a–f.
Compound
Control b,c
Vandetanib
16a
16b
16c
16d
16e
16f
VEGF gene
expression level
Normoxia
Hypoxia
1.0000
1.0000
0.6457
0.2457
0.1070
0.0009
0.1387
0.0009
0.0949
0.0007
0.0606
0.0008
0.1866
0.0006
0.0706
0.0002
a
EGF gene
expression level
Normoxia
Hypoxia
1.0000
1.0000
0.3440
5.4907
0.5260
2.5398
0.9732
2.8108
0.5471
1.7447
0.4366
1.9433
1.1758
2.4923
0.5614
0.3199
a
a
Concentration of cobalt chloride in control and experimental groups was 200 µ
mol/L. b Control group was
performed with a corresponding amount of DMSO. c VEGF/EGF gene expression levels in control groups in
hypoxia or normoxia was calculated as one, respectively and independently.
Hypoxia-inducible factor-1
with HIF-1 subunit to form the heterodimer of HIF-1, which could bind with and activate VEGF
promoter as a transcription factor in the cell nucleus, enhancing the expression level of VEGF mRNA
and inducing angiogenesis and metastatic processes in tumors [ ]. However, the VEGF gene expression
α (HIF-1α) became stabilized in hypoxic tumor tissues and combined
β
4
assays exhibited very distinct decreased expression levels in hypoxia, which were incompatible with
what we predicted above. Moreover, the EGF gene showed different expression levels in hypoxia and
normoxia. We inferred that target compounds impacted VEGF/EGF gene expression levels through
influencing the signaling pathways by some unknown mechanisms and more correlational research is
required in our further work.
3
3
3
. Experimental Section
.1. Materials and Methods
.1.1. General Methods
Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 UV254 pre-coated
silica gel plates. Detection was by means of a UV lamp. Flash column chromatography was
performed on 300–400 mesh silica gel. Melting points were determined on an X-5 melting-point
1
13
apparatus (Taike technology Co., Ltd., Beijing, China) and were not corrected. H-NMR, C-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-d solution without TMS or CFCl as internal
6
3
standards. Mass spectra (ESI-MS) were performed on Agilent 1200 HPLC-6310 liquid chromatography
mass spectrometer (Agilent Ltd., Palo Alto, CA, USA). All reagents and solvents were used as received
from commercial sources without further purification.

Molecules 2016, 21, 1693
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3
.1.2. Synthesis
General Synthetic Procedure of tert-Butyl 2-(Substituted-nitroimidazol-1-yl)acetate (7a–7c)
To a solution of 6a
◦
–
c
(1 eq) in ACN was added K CO (1.1 eq) and stirred for 0.5 h at 50 C.
2
3
Then a solution of tert-Butyl bromoacetate (1.2 eq) in ACN was added dropwise to the first solution.
The resulting mixture was refluxed until no 6a appeared on TLC. The reaction mixture was cooled to
–
c
room temperature and filtered. The filtrate was evaporated under reduced pressure, and the residue
was dissolved in a mixture of EA and water. The organic layer was separated and the aqueous layer
was extracted twice. The organic extracts were combined, dried (anhydrous Na SO ) and evaporated
2
4
to dryness to afford crude 7a–7c without further purification.
tert-Butyl 2-(2-Methyl-5-nitroimidazol-1-yl)acetate (7a). Following the general procedure. Yield: 95.6%.
◦
+ 1
Faint yellow solid. m.p. 109.6–111.2 C; ESI-MS m/z = 242.17 [M + H] ; H-NMR (400 MHz, DMSO-d )
6
δ 1.43 (s, 9H), 2.27 (s, 3H), 4.94 (s, 2H), 8.25 (s, 1H).
tert-Butyl 2-(2-Nitroimidazol-1-yl)acetate (7b). Following the general procedure. Yield: 94.6%.
◦
1
Faint yellow solid. m.p. 89.9–91.7 C; H-NMR (400 MHz, DMSO-d )
δ
1.41 (s, 9H), 5.12 (s, 2H),
6
.21 (s, 1H), 7.62 (s, 1H). Yield: 94.0%. White solid. m.p. 137.7–138.2 C; ESI-MS m/z = 228.13 [M + H]+
H-NMR (400 MHz, DMSO-d ) δ 1.44 (s, 9H), 4.97 (s, 2H), 7.81 (s, 1H), 8.34 (s, 1H).
◦
;
7
1
6
tert-Butyl 2-(5-Nitroimidazol-1-yl)acetate (7c). Following the general procedure. Yield: 94.0%; White solid.
◦
+ 1
m.p. 137.7–138.2 C; ESI-MS m/z = 228.13 [M + H] ; H-NMR (400 MHz, DMSO-d ):
δ (ppm): 1.44 (s,
6
9
H), 4.97 (s, 2H), 7.81 (s, 1H), 8.34 (s, 1H).
Methyl 3-(2-Methyl-5-nitroimidazol-1-yl)propionate (8a)
To a solution of 6a (20g, 157.40 mmol, 1.0 eq) in DMF (100 mL) were added methyl acrylate
(14.90 g, 173.14 mmol, 1.1 eq) and DIPEA (20.0 g, 157.40 mmol, 1.0 eq) under nitrogen atmosphere.
The mixture was stirred at room temperature for 7d. Then reaction mixture was evaporated under
reduced pressure, and the residue was dissolved in mixture of EA (400 mL) and water (400 mL).
The organic layer was washed by brine (200 mL
×
2), dried (anhydrous Na SO ) and evaporated
2 4
+
to dryness to afford the product 8a as brown oil (32.08 g, 95.6%). ESI-MS m/z = 214.09 [M + H] ;
1
H-NMR (400 MHz, DMSO-d6)
.29 (s, 1H).
δ 2.37 (s, 3H), 2.92 (t, J = 6.8 Hz, 2H), 3.60 (s, 3H), 4.21 (t, J = 6.8 Hz, 2H),
8
General Synthetic Procedure of Ethyl 4-(Substituted-nitroimidazol-1-yl)butyrate (9a–b)
a–b was prepared according to the synthetic procedure of 7a–c from 6a–b.
9
Ethyl 4-(2-Methyl-5-nitroimidazol-1-yl)butyrate (9a). Following the general procedure. Yield: 93.2%.
Brown oil without further purification.
Ethyl 4-(2-Nitroimidazol-1-yl)butyrate (9b). Following the general procedure. Yield: 85.7%. Yellow solid.
◦
+ 1
m.p. 69.3–70.9 C; ESI-MS m/z = 227.11 [M + H] ; H-NMR (400 MHz, DMSO-d )
3
7
δ
1.15 (t, J = 7.2 Hz,
H), 2.04 (m, 2H), 2.34 (t, J = 7.2 Hz, 2H), 4.00 (q, J = 7.2 Hz, 2H), 4.40 (t, J = 7.2 Hz, 2H), 7.16 (s, 1H),
.64 (s, 1H).
6
General Synthetic Procedure of 2-(Substituted-nitroimidazol-1-yl)acetic acid (10a–c)
To a solution of CF COOH (1 eq) in CH Cl was added 7a (1 eq). The mixture was stirred at
–
c
3
2
2
room temperature for 2.5 h, and evaporated under reduced pressure. The residue was dissolved in the
mixture solvent (EA/PE, 1:1, v/v) under vigorous stirring. The mixture was filtrated to afford the solid
product 10a–c.

Molecules 2016, 21, 1693
8 of 15
2
-(2-Methyl-5-nitroimidazol-1-yl)acetic acid (10a). Following the general procedure. Yield: 84.3%.
◦
−
H] ; H-NMR (400 MHz, DMSO-d )
6
1
White solid. m.p. 220.9–221.9 C; ESI-MS m/z = 184.04 [M
δ 2.28 (s, 3H), 4.93 (s, 2H), 8.25 (s, 1H), 13.44 (br s, 1H).
−
2
-(2-Nitroimidazol-1-yl)acetic acid (10b). Following the general procedure. Yield: 87.5%. Faint yellow
◦
−
H] ; H-NMR (400 MHz, DMSO-d ):
6
1
solid. m.p. ~138 C (decomposition); ESI-MS m/z = 170.03 [M
δ (ppm): 5.21 (s, 2H), 7.20 (s, 1H), 7.62 (s, 1H), 13.42 (br s, 1H).
−
2
-(5-Nitroimidazol-1-yl)acetic acid (10c). Following the general procedure. Yield: 90.5%. White solid.
◦
−
1
m.p. 179.3–180.6 C; ESI-MS m/z = 170.03 [M
.81 (s, 1H), 8.34 (s, 1H), 13.41 (br s, 1H).
−
H] ; H-NMR (400 MHz, DMSO-d )
δ
4.97 (s, 2H),
6
7
General Synthetic Procedure of 3-(Substituted-nitroimidazol-1-yl)propionic acid (11a) and
-(Substituted-nitroimidazol-1-yl)butyric acid (12a–b)
4
◦
To a solution of saturated Na CO solution was added 8a
(9a
–b
). The mixture was attired at 85 C
2
3
for 2.5 h and then cooled to room temperature. The pH of the reaction solution was adjusted to 4, and
the solid precipitated out. The crude product (11a, 12a–b) was obtained by filtration, washed with EA
and dried in the vacuum.
3
-(2-Methyl-5-nitroimidazol-1-yl)propionic acid (11a). Following the general procedure. Yield: 87.5%.
◦
−
H] ; H-NMR (400 MHz, DMSO-d )
6
1
White solid. m.p. 204.9–206.3 C; ESI-MS m/z = 198.06 [M
δ 2.37 (s, 3H), 2.81 (t, J = 6.8 Hz, 2H), 4.16 (t, J = 6.8 Hz, 2H), 8.28 (s, 1H), 12.54 (br s, 1H).
−
4
-(2-Methyl-5-nitroimidazol-1-yl)butyric acid (12a). Following the general procedure. Yield: 82.0%.
◦
−
1
Faint yellow solid. m.p. 126.8–129.3 C; ESI-MS m/z = 212.07 [M
DMSO-d6) 1.93–1.98 (m, 2H), 2.27 (t, J = 6.6 Hz, 2H), 2.36 (s, 3H), 4.01 (t, J = 6.6 Hz, 2H), 8.32 (s, 1H),
2.22 (br s, 1H).
−
H] ; H-NMR (600 MHz,
δ
1
4
-(2-Nitroimidazol-1-yl)butyric acid (12b). Following the general procedure. Yield: 83.4%. Yellow solid.
◦
−
H] ; H-NMR (400 MHz, DMSO-d ) δ 2.00 (m, 2H),
6
1
m.p. 111.3–113.2 C; ESI-MS m/z = 198.06 [M
2
−
.26 (t, J = 7.2 Hz, 2H), 4.40 (t, J = 7.2 Hz, 2H), 7.16 (s, 1H), 7.65 (s, 1H), 12.14 (br s, 1H).
General Synthetic Procedure of p-Toluenesulfonate Intermediates (13a–c, 14a and 15a–b)
To a solution of 10a (1.0 eq) in DMF, was added dropwise the solution of carbonyldiimidazole
CDI) (1.0 eq) in DMF. The mixture was stirred at room temperature for 1h. Then the solution of
(1.0 eq) and triethylamine (1.3 eq) in DMF was added dropwise into the first reaction mixture.
–c
(
5
The mixture was stirred under room temperature overnight. The reaction solution poured into water
under vigorous mechanically stirring and filtered. The residue was dissolved in EA and the organic
layer was separated, dried (anhydrous Na SO ) and evaporated to dryness to afford the corresponding
2
4
products (14a–b, 15a–b) without further purification.
N-[2-(2-Methyl-5-nitroimidazol-1-yl)acetyl]piperidin-4-yl-methyl p-toluenesulfonate (13a). Following the
+
1
general procedure. Yield: 89.5%. Brown jelly. ESI-MS m/z = 436.15 [M + H] ; H-NMR (400 MHz,
DMSO-d6) 1.01 (m, 1H), 1.21 (m, 1H), 1.60 (d, J = 12.0 Hz, 1H), 1.68 (d, J = 12.8 Hz, 1H), 1.92 (m, 1H),
.20 (s, 3H), 2.42 (s, 3H), 2.60 (t, J = 12.4 Hz, 1H), 3.04 (t, J = 12.4 Hz, 1H), 3.78 (d, J = 13.6 Hz, 1H),
.91 (J = 6.4 Hz, 2H), 4.26 (d, J = 13.2 Hz, 1H), 5.04 (d, J = 17.2 Hz, 1H), 5.09 (d, J = 17.2 Hz, 1H), 7.48
δ
2
3
(d, J = 8.4 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 8.14 (s, 1H).
N-[2-(2-Nitroimidazol-1-yl)acetyl]piperidin-4-yl-methyl p-toluenesulfonate (13b). Following the general
+
1
procedure. Yield: 85.2%. Brown jelly. ESI-MS m/z = 423.14 [M + H] ; H-NMR (400 MHz, DMSO-d )
6
δ
0.96 (m, 1H), 1.20 (m, 1H), 1.58 (d, J = 12.4 Hz, 1H), 1.70 (d, J = 12.0 Hz, 1H), 1.93 (m, 1H), 2.42 (s, 3H),
.60 (t, J = 12.0 Hz, 1H), 3.07 (t, J = 12.0 Hz, 1H), 3.80 (d, J = 13.6 Hz, 1H), 3.91 (J = 6.0 Hz, 2H), 4.22
2
(
2
d, J = 12.8 Hz, 1H), 5.36 (d, J = 16.4 Hz, 1H), 5.40 (d, J = 16.4 Hz, 1H), 7.16 (s, 1H), 7.48 (d, J = 8.0 Hz,
H), 7.53 (s, 1H), 7.79 (d, J = 8.0 Hz, 2H).

Molecules 2016, 21, 1693
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N-[2-(5-Nitroimidazol-1-yl)acetyl]piperidin-4-yl-methyl p-toluenesulfonate (13c). Following the general
◦
+ 1
procedure. Yield: 91.2%. White powder. m.p. 112.5–115.1 C; ESI-MS m/z = 423.14 [M + H] ; H-NMR
400 MHz, DMSO-d6) 1.00 (m, 1H), 1.20 (m, 1H), 1.59 (d, J = 12.4 Hz, 1H), 1.68 (d, J = 12.0 Hz, 1H),
.91 (m, 1H), 2.42 (s, 3H), 2.59 (t, J = 12.0 Hz, 1H), 3.02 (t, J = 12.0 Hz, 1H), 3.77 (d, J = 13.6 Hz, 1H),
(
1
3
δ
.90 (J = 6.0 Hz, 2H), 4.27 (d, J = 13.2 Hz, 1H), 5.09 (d, J = 16.4 Hz, 1H), 5.13 (d, J = 16.8 Hz, 1H), 7.48
(d, J = 8.0 Hz, 2H), 7.71 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), 8.22 (s, 1H).
N-[3-(2-Methyl-5-nitroimidazol-1-yl)propionyl]piperidin-4-yl-methyl p-toluenesulfonate (14a). Following
+
1
the general procedure. Yield: 82.2%. Brown jelly ESI-MS m/z = 451.18 [M + H] ; H-NMR (400 MHz,
DMSO-d6) 0.90 (m, 1H), 1.04 (m, 1H), 1.57 (t, J = 12.8 Hz, 2H), 1.85 (m, 1H), 2.37 (s, 3H), 2.41 (s, 3H),
.89 (t, J = 7.2 Hz, 2H), 3.79 (d, J = 13.6 Hz, 1H), 3.86 (d, J = 6.4 Hz, 2H), 4.13 (t, J = 7.2 Hz, 2H), 4.30 (t,
δ
2
J = 13.2 Hz, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.4 Hz, 2H), 8,27 (s,1H).
N-[4-(2-Methyl-5-nitroimidazol-1-yl)butyryl]piperidin-4-yl-methyl p-toluenesulfonate (15a). Following the
+
1
general procedure. Yield: 78.6%. Faint yellow jelly. ESI-MS m/z = 465.18 [M + H] ; H-NMR (600 MHz,
DMSO-d6) 0.89 (m, 1H), 1.14 (m, 1H), 1.58 (dd, J = 12.6 Hz, J = 30.0 Hz, 2H), 1.86 (m, 1H), 1.93
δ
1
2
(
(
(
t, J = 6.6 Hz, 2H), 2.32 (t, J = 6.6 Hz, 2H), 2.35 (s, 3H), 2.43 (s, 3H), 2.48 (t, J = 12.0 Hz, 1H), 2.91
t, J = 12.0 Hz, 1H), 3.79 (d, J = 13.2 Hz, 1H), 3.91 (d, J = 5.4 Hz, 2H), 3.97 (t, J = 7.2 Hz, 2H), 4.33
d, J = 12.6 Hz, 1H), 7.49(d, J = 7.8 Hz, 2H), 7.79 (d, J = 7.8 Hz, 2H), 8.31 (s, 1H).
N-[4-(2-Nitroimidazol-1-yl)butyryl]piperidin-4-yl-methyl p-toluenesulfonate (15b). Following the general
+
1
procedure. Yield: 80.6%. Brownish jelly. ESI-MS m/z = 451.17 [M + H] ; H-NMR (600 MHz, DMSO-d )
6
δ
0.90 (m, 1H), 1.03 (m, 1H), 1.56 (m, 2H), 1.84 (m, 1H), 2.00 (m, 2H), 2.30 (t, J = 7.2 Hz, 2H), 2.41
(
(
s, 3H), 2.43 (m, 1H), 2.89 (t, J = 12.0 Hz, 1H), 3.76 (d, J = 13.6 Hz, 1H), 3.87 (d, J = 6.0 Hz, 2H), 4.29
d, J = 12.8 Hz, 1H), 4.38 (t, J = 7.2 Hz, 2H), 7.14 (s, 1H), 7.47(d, J = 8.0 Hz, 2H), 7.63 (s, 1H), 7.77 (d,
J = 8.0 Hz, 2H).
General Synthetic Procedure of Target Compounds (16a–p)
To a solution of 4-anilino-6-methoxyquinazoline (1a
The mixture was stirred at 50 C for 0.5 h. Then the solution of 13
was added dropwise to the reaction mixture. The resulting mixture was stirred at 50 C until no
–
g
) (1.0 eq) in DMF was added K CO .
2
3
◦
(
14 or 15, 1 eq) in DMF
◦
4-anilino-6-methoxyquinazoline appeared (1a
–g) on TLC. The reaction mixture was poured into water
under vigorous stirring. Half an hour later, the mixture was filtrated. The residue was dissolved in
methanol, and the organic layer was dried (anhydrous Na SO ) and evaporated to dryness to afford a
2
4
crude solid, which was washed by EA to afford the product.
1
5
5
-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(2-methyl-
-nitroimidazol-1-yl)ethanone (16a). Dark green powder prepared from 13a and 1a with the yield of
◦
+
−
6.8%. m.p. 201.4–203.1 C; ESI-MS m/z = 628.2, 630.2 [M + H] , 626.3, 628.1 [M
−
H] ; ESI-HRMS
+
1
calcd for C H BrFN O m/z = 628.1319, 630.1299 [M + H] , found: 628.1310, 630.1295; H-NMR
27
28
7
5
(400 MHz, DMSO-d6)
δ
1.24 (m, 1H), 1.42 (m, 1H), 1.89 (m, 2H), 2.17 (m, 1H), 2.24 (s, 3H), 2.74 (t,
J = 12.0 Hz, 1H), 3.17 (t, J = 12.0 Hz, 1H), 3.87 (d, J = 12.8 Hz, 1H), 3.95 (s, 3H), 4.06 (d, J = 6.0 Hz, 2H),
.36 (d, J = 12.4 Hz, 1H), 5.09 (d, J = 17.2 Hz, 1H), 5.16 (d, J = 17.2 Hz, 1H), 7.22 (s, 1H), 7.46 (d, 1H), 7.53
4
13
(
t, 1H), 7.64 (d, 1H), 7.80 (s, 1H), 8.33 (s, 1H), 8.36 (s, 1H), 9.52(s, 1H); C-NMR (100 MHz, DMSO-d6)
δ
13.15, 28.72, 29.41, 31.31, 35.64, 42.19, 44.64, 56.86, 72.98, 102.72, 108.49, 109.33, 119.86, 120.09, 124.10,
26.80, 128.13, 130.18, 145.69, 146.81, 147.60, 149.77, 153.60, 154.36, 157.56, 164.50; 1 F-NMR (376 MHz,
9
1
DMSO-d ) δ −115.47.
6
1-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-
5
6
-nitroimidazol-1-yl)propyl-1-one (16b). Faint green powder prepared from 14a and 1a with the yield of
◦
4.7%. m.p. 193.6–194.4 C; ESI-MS m/z = 642.27, 644.25 [M + H] ; ESI-HRMS calcd for C H BrFN O
28 30 7 5
+
+
1
m/z = 642.1476, 644.1455 [M + H] , found: 642.1473, 644.1457; H-NMR (600 MHz, DMSO-d )
δ
1.16
6
(
m, 1H), 1.25 (m, 1H), 1.83 (m, 2H), 2.11 (m, 1H), 2.41 (s, 3H), 2.63 (t, J = 12.6 Hz, 1H), 2.95 (m, 2H), 3.04

Molecules 2016, 21, 1693
10 of 15
(t, J = 12.6 Hz, 1H), 3.90 (d, J = 12.6 Hz, 1H), 3.95 (s, 3H), 4.01 (d, J = 6.0 Hz, 2H), 4.19 (t, J = 6.6 Hz, 2H),
4
8
4
.43 (d, J = 12.6 Hz, 1H), 7.20 (s, 1H), 7.47 (d, 1H), 7.54 (t, 1H), 7.66 (d, 1H), 7.80 (s, 1H), 8.33 (s, 1H),
.37 (s, 1H), 9.54 (s, 1H); 13C-NMR (151 MHz, DMSO-d6)
δ
16.85, 28.13, 28.83, 32.59, 35.04, 40.71, 42.71,
4.36, 56.18, 72.31, 101.98, 107.74, 108.60, 117.46, 119.22, 122.15, 126.35, 127.46, 129.49, 145.26, 146.91,
1
49.04, 152.90, 155.78, 156.84, 157.44, 167.57; 19F-NMR (376 MHz, DMSO-d ) δ −115.46.
6
1-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-4-(2-methyl-
5
-nitroimidazol-1-yl)butyl-1-one (16c). Faint green powder prepared from 15a and 1a with the yield
◦
+
of 53.5%. m.p. 213.7–215.2 C; ESI-MS m/z = 656.26, 658.22 [M + H] ; ESI-HRMS calcd for
C H BrFN O m/z = 656.1632, 658.1612 [M + H] , found: 656.1629, 658.1612; H-NMR (600 MHz,
+
1
29
32
7
5
DMSO-d6)
.60 (t, J = 12.0 Hz, 1H), 3.04 (t, J = 12.0 Hz, 1H), 3.89 (d, J = 12.6 Hz, 1H), 3.95 (s, 3H), 4.02 (m, 4H), 4.43
d, J = 12.6 Hz, 1H), 7.20 (s, 1H), 7.47 (d, 1H), 7.54 (t, 1H), 7.66 (d, 1H), 7.81 (s, 1H), 8.34 (s, 1H), 8.37 (s,
H), 9.54 (s, 1H); 13C-NMR (151 MHz, DMSO-d6)
12.49, 25.22, 28.14, 28.74, 35.15, 40.76, 44.40, 45.96,
δ 1.14 (m, 1H), 1.25 (m, 1H), 1.84 (t, J = 15.0 Hz, 2H), 1.96 (m, 2H), 2.10 (m, 1H), 2.39 (m, 5H),
2
(
1
δ
5
1
6.10, 72.33, 101.97, 107.70, 108.61, 117.44, 119.20, 119.36, 122.00, 127.45, 129.48, 144.95, 145.38, 146.90,
49.05, 152.90, 153.65, 155.77, 156.85, 157.43, 169.15; 19F-NMR (376 MHz, DMSO-d ) δ −115.46.
6
1-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(2-
nitroimidazol-1-yl)ethanone (16d). Faint green powder prepared from 13b and 1a with the yield of 59.5%.
◦
+
m.p. 157.6–160.1 C; ESI-MS m/z = 614.23, 616.20 [M + H] ; ESI-HRMS calcd for C H BrFN O m/z
26
26
7
5
+
1
=
614.1163, 616.1142 [M + H] , found: 614.1157, 616.1142; H-NMR (400 MHz, DMSO-d )
δ
1.22 (m,
6
1
H), 1.42 (m, 1H), 1.98 (dd, J = 12.0 Hz, J = 32.8 Hz, 2H), 2.17 (m, 1H), 2.74 (t, J = 11.6 Hz, 1H), 3.19 (t,
1
2
J = 12.0 Hz, 1H), 3.88 (d, J = 12.0 Hz, 1H), 3.95 (s, 3H), 4.05 (d, J = 5.2 Hz, 2H), 4.32 (d, J = 12.0 Hz, 1H),
5
7
5
.41 (d, J = 16.4 Hz, 1H), 5.47 (d, J = 16.4 Hz, 1H), 7.20 (d, 2H), 7.45 (d, 1H), 7.53 (m, 2H), 7.64 (d, 1H),
.80 (s, 1H), 8.36 (s, 1H), 9.52 (s, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ
28.80, 29.48, 35.67, 42.24, 44.61,
1.73, 56.85, 72.96, 102.68, 108.49, 109.32, 118.15, 119.98, 127.10, 128.14, 129.10, 130.21, 145.92, 147.61,
1
49.78, 153.60, 154.37, 156.06, 157.56, 158.57, 164.34; 19F-NMR (376 MHz, DMSO-d ) δ −115.45.
6
1-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-4-(2-
nitroimidazol-1-yl)butyl-1-one (16e). Faint green powder prepared from 15b and 1a with the yield of
◦
2.5%. m.p. 192.1–193.8 C; ESI-MS m/z = 642.22, 644.18 [M + H] ; ESI-HRMS calcd for C H BrFN O
28 30 7 5
+
5
+
1
m/z = 642.1476, 644.1455 [M + H] , found: 642.1466, 644.1451; H-NMR (400 MHz, DMSO-d ):
δ
1.15
6
(
m, 1H), 1.26 (m, 1H), 1.81 (t, 2H), 2.05 (m, 3H), 2.38 (m, 2H), 2.57 (t, J = 11.6 Hz, 1H), 3.01 (t, J = 12.0 Hz
,
1
1
H), 3.85 (d, J = 13.2 Hz, 1H), 3.93 (s, 3H), 4.01 (d, J = 6.4 Hz, 2H), 4.41 (m, 3H), 7.18 (d, 2H), 7.43 (d,
H), 7.50 (t, 1H), 7.64 (m, 2H), 7.80 (s, 1H), 8.34 (s, 1H), 9.56 (br, 1H); C-NMR (100 MHz, DMSO-d6)
25.94, 28.78, 29.57, 29.73, 35.87, 41.45, 45.15, 49.77, 56.84, 73.04, 102.75, 108.43, 109.37, 118.43, 109.37,
13
δ
1
1
18.08, 119.84, 120.08, 128.14, 128.48, 130.16, 145.36, 147.55, 149.73, 153.56, 154.34, 156.04, 157.53, 158.54,
69.78; 19F-NMR (376 MHz, DMSO-d ) δ −115.42.
6
1-{4-[({4-[(4-Bromo-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(5-
nitroimidazol-1-yl)ethanone (16f). Dark green powder prepared from 13c and 1a with the yield of 64.5%.
◦
+
m.p. 144.6–146.1 C; ESI-MS m/z = 614.24, 616.22 [M + H] ; ESI-HRMS calcd for C H BrFN O
5
26
26
7
+
1
m/z = 614.1163, 616.1142 [M + H] , found: 614.1153, 616.1138; H-NMR (600 MHz, DMSO-d )
δ
1.27
6
(
(
m, 1H), 1.41 (m, 1H), 1.90 (dd, J = 12.0 Hz, J = 31.8 Hz, 2H), 2.17 (m, 1H), 2.74 (t, J = 12.6 Hz, 1H), 3.16
1
2
t, J = 12.6 Hz, 1H), 3.88 (d, J = 12.6 Hz, 1H), 3.97 (s, 3H), 4.06 (d, J = 6.6 Hz, 2H), 4.39 (d, J = 12.6 Hz
,
1
1
H), 5.17 (d, J = 16.2 Hz, 1H), 5.22 (d, J = 16.8 Hz, 1H), 7.23 (s, 1H), 7.47 (d, 1H), 7.55 (t, 1H), 7.64 (d,
H), 7.77 (s, 1H), 7.82 (s, 1H), 8.29 (s, 1H), 8.38 (s, 1H), 9.56 (s, 1H); C-NMR (151 MHz, DMSO-d6)
13
δ
27.97, 28.65, 34.93, 41.41, 43.81, 48.67, 56.16, 72.29, 102.01, 107.80, 108.64, 117.54, 119.37, 123.15, 127.47,
29.49, 138.50, 146.51, 146.91, 149.06, 152.92, 153.64, 155.78, 156.87, 157.45, 164.11; 1 F-NMR (376 MHz,
9
1
DMSO-d ) δ −115.46.
6
1
-{4-[({4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(5-nitro-
imidazol-1-yl)ethanone (16g). Yellow solid powder prepared from 13c and 1b with the yield of 73.4%.

Molecules 2016, 21, 1693
11 of 15
◦
+
m.p. 143.3–145.0 C; ESI-HRMS calcd for C H ClFN O m/z = 570.1668 [M + H] , found: 570.1661;
26
26
7
5
1
H-NMR (400 MHz, DMSO-d6)
J = 12.0 Hz, 1H), 3.17 (t, J = 12.0 Hz, 1H), 3.88 (d, J = 12.8 Hz, 1H), 3.94 (s, 3H), 4.05 (d, J = 6.4 Hz,
H), 4.37 (d, J = 12.8 Hz, 1H), 5.15 (d, J = 16.8 Hz, 1H), 5.19 (d, J = 16.4 Hz, 1H), 7.20 (s, 1H), 7.29 (td,
δ 1.24 (m, 1H), 1.41 (m, 1H), 1.89 (t, 2H), 2.16 (m, 1H), 2.73 (t,
2
J = 8.4 Hz, J = 2.8 Hz, 1H), 7.56 (m, 2H), 7.74 (s, 1H), 7.82 (s, 1H), 8.28 (d, J = 10.8 Hz, 2H), 9.51 (s, 1H);
1
3
2
1
C-NMR (100 MHz, DMSO-d6)
δ 27.97, 28.65, 34.92, 41.42, 43.82, 56.17, 72.30, 89.31, 102.04, 107.86,
1
1
08.43, 114.81, 116.95, 123.13, 127.55, 131.20, 132.20, 132.93, 138.49, 146.51, 146.78, 148.99, 153.05, 153.56,
57.61, 158.64, 161.09, 164.12; 19F-NMR (376 MHz, DMSO-d ) δ −114.05.
6
1
-{4-[({4-[(3-Bromo-4-methylphenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(5-nitro-
imidazol-1-yl)ethanone (16h). Green powder prepared from 13c and 1c with the yield of 66.6%. m.p.
◦
+
1
6
50.6–152.3 C; ESI-HRMS calcd for C H BrN O m/z = 610.1414, 612.1393 [M + H] , found:
2
7
29
7
5
1
10.1406, 612.1390; H-NMR (600 MHz, DMSO-d )
δ
1.24 (m, 1H), 1.39 (m, 1H), 1.92 (m, 2H), 2.15 (m,
6
1
4
(
1
H), 2.33 (s, 1H), 2.73 (t, J = 12.0 Hz, 1H), 3.15 (t, J = 12.0 Hz, 1H), 3.86 (d, J = 12.8 Hz, 1H), 3.96 (s, 3H),
.04 (d, J = 6.0 Hz, 2H), 4.36 (d, J = 12.8 Hz, 1H), 5.15 (d, J = 16.4 Hz, 1H), 5.19 (d, J = 16.4 Hz, 1H), 7.20
s, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.79 (m, 2H), 8.13 (d, J = 1.6 Hz, 1H), 8.26 (s, 1H), 8.48 (s,
H), 9.45 (s, 1H); 13C-NMR (151 MHz, DMSO-d6)
21.67, 27.97, 28.65, 34.94, 41.42, 43.82, 48.67, 56.31,
2.29, 101.93, 107.96, 108.81, 121.10, 123.12, 123.37, 124.84, 130.59, 131.49, 138.49, 138.76, 146.51, 146.91,
δ
7
1
49.06, 152.70, 153.55, 156.03, 164.12.
1
-{4-[({4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-
5
-nitro-imidazol-1-yl)propyl-1-one (16i). Faint yellow powder prepared from 14a and 1b with the yield
◦
+
of 71.1%. m.p. 128.7–130.3 C; ESI-HRMS calcd for C H ClFN O m/z = 598.1981 [M + H] , found:
5
28
30
7
5
1
98.1972; H-NMR (400 MHz, DMSO-d ) δ 1.18 (m, 2H), 1.83 (d, J = 11.6 Hz, 2H), 2.10 (m, 1H), 2.41
6
(
(
s, 3H), 2.63 (t, J = 12.0 Hz, 1H), 2.95 (m, 2H), 3.04 (t, J = 12.0 Hz, 1H), 3.89 (m, 1H), 3.94 (s, 3H), 4.01
d, J = 4.0 Hz, 2H), 4.19 (t, J = 6.8 Hz, 2H), 4.43 (d, J = 12.8 Hz, 1H), 7.19 (s, 1H), 7.31 (td, J = 8.4 Hz,
1
1
3
J2 = 2.8 Hz, 1H), 7.51–7.67 (m, 2H), 7.83 (s, 1H), 8.32 (d, J = 11.6 Hz, 2H), 9.54 (br, 1H); C-NMR
100 MHz, DMSO-d6) 13.14, 28.62, 29.34, 33.09, 35.53, 41.21, 43.22, 44.86, 56.64, 72.80, 102.48, 108.28,
15.09, 117.46, 122.68, 132.65, 134.43, 145.78, 147.27, 149.45, 153.54, 154.04, 158.10, 168.08; F-NMR
(
1
δ
19
(
376 MHz, DMSO-d ) δ −114.07.
6
1
-{4-[({4-[3,4-Difluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-5-nitro-
imidazol-1-yl)propyl-1-one (16j). Faint yellow powder prepared from 14a and 1d with the yield of 63.9%.
◦
+
m.p. 174.6–177.1 C; ESI-HRMS calcd for C H F N O m/z = 582.2776 [M + H] , found: 582.2770;
28
30
2
7
5
1
H-NMR (400 MHz, DMSO-d6)
δ
1.19 (m, 2H), 1.82 (s, 2H), 2.08 (m, 1H), 2.41 (s, 3H), 2.61 (t, J = 12.0 Hz
,
1H), 2.84–3.14 (m, 3H), 3.82–3.99 (m, 6H), 4.19 (t, J = 6.4 Hz, 2H), 4.43 (d, J = 12.0 Hz, 1H), 7.16 (s, 1H),
7
.43 (dd, J = 19.2 Hz, J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.78 (s, 1H), 8.07 (dd, J1 = 11.6 Hz
,
1
2
13
J2 = 8.0 Hz, 1H), 8.32 (s, 1H), 8.49 (s, 1H), 9.53 (br, 1H); C-NMR (100 MHz, DMSO-d6)
δ
13.08, 28.62,
2
9.32, 33.09, 35.57, 41.21, 43.19, 44.85, 56.73, 72.77, 102.27, 108.31, 109.20, 111.27, 111.48, 117.24, 117.42,
1
1
18.46, 122.62, 137.07, 137.16, 144.56, 145.73, 145.78, 146.83, 147.43, 147.94, 148.07, 149.52, 150.35, 150.48,
53.01, 154.05, 156.37, 168.05; 1 F-NMR (376 MHz, DMSO-d6) δ −137.77 (d, JFF = 23.2 Hz),
9
−
145.10 (d,
JFF = 23.2 Hz).
1-{4-[({4-[(3-Bromo-4-methylphenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-
5
6
-nitro-imidazol-1-yl)propyl-1-one (16k). Green powder prepared from 14a and 1c with the yield of
◦
6.6%. m.p. 141.2–143.4 C; ESI-HRMS calcd for C H BrN O m/z = 638.1727, 640.1706 [M + H] ,
29 33 7 5
+
1
found: 638.1716, 640.1701; H-NMR (400 MHz, DMSO-d )
δ
1.17 (m, 2H), 1.81 (s, 2H), 2.08 (m, 1H),
.32 (s, 3H), 2.41 (s, 3H), 2.60 (t, J = 12.0 Hz, 1H), 2.88–3.04 (m, 3H), 3.88–3.96 (m, 6H), 4.18 (s, 2H), 4.43
d, J = 12.0 Hz, 1H), 7.14 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.81 (d, 2H), 8.16 (s, 1H), 8.32 (s, 1H), 8.49 (s,
H), 9.45 (br, 1H); 13C-NMR (100 MHz, DMSO-d6)
13.11, 22.16, 28.62, 29.33, 33.10, 35.57, 43.19, 44.86,
6.74, 72.74, 102.32, 108.28, 109.27, 121.49, 122.64, 123.86, 125.24, 131.00, 131.85, 139.51, 145.73, 145.78,
47.37, 149.46, 153.32, 153.95, 156.44, 168.03.
6
2
(
1
5
δ
1

Molecules 2016, 21, 1693
12 of 15
1
-{4-[({4-[3-Fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-5-nitro-
imidazol-1-yl)propyl-1-one (16l). Faint yellow powder prepared from 14a and 1e with the yield of 63.3%.
◦
+
m.p. 175.8–178.3 C; ESI-HRMS calcd for C H FN O m/z = 564.2371 [M + H] , found: 564.2375;
28
31
7
5
1
H-NMR (400 MHz, DMSO-d6) δ 1.18 (m, 2H), 1.82 (s, 2H), 2.08 (m, 1H), 2.41 (s, 3H), 2.61 (t, J = 12.0 Hz,
1
H), 2.96–3.02 (m, 3H), 3.89–3.97 (m, 6H), 4.19 (s, 2H), 4.43 (d, J = 12.0 Hz, 1H), 6.91 (s, 1H), 7.17 (s, 1H),
7
(
.41 (d, J = 4.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.92 (d, J = 11.6 Hz, 1H), 8.32 (s, 1H), 8.52
13.10, 28.62, 29.32, 33.09, 35.56, 41.21, 43.19,
s, 1H), 9.56 (s, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ
4
1
4.85, 56.77, 72.77, 102.36, 108.33, 108.83, 109.09, 109.38, 109.70, 109.91, 117.82, 122.63, 130.22, 141.91,
42.02, 145.74, 145.78, 147.51, 149.54, 153.05, 154.06, 156.44, 161.31, 163.70, 168.05; 1 F-NMR (376 MHz,
9
DMSO-d ) δ −112.66.
6
1
-{4-[({4-[4-Fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-methyl-5-nitro-
imidazol-1-yl)propyl-1-one (16m). Faint yellow powder prepared from 14a and 1f with the yield of
◦
9.1%. m.p. 171.3.3–172.7 C; ESI-HRMS calcd for C H FN O m/z = 564.2371 [M + H] , found:
28 31 7 5
+
5
5
3
2
9
7
1
64.2373; H-NMR (400 MHz, DMSO-d )
δ 1.17 (m, 2H), 1.82 (d, J = 11.6 Hz, 2H), 2.08 (m, 1H), 2.41 (s,
6
H), 2.61 (t, J = 12.0 Hz, 1H), 2.90–3.06 (m, 3H), 3.90 (d, J = 12.4 Hz, 1H), 3.97 (m, 5H), 4.19 (t, J = 6.8 Hz,
H), 4.43 (d, J = 12.4 Hz, 1H), 7.17 (s, 1H), 7.23 (t, J = 8.0 Hz, 2H), 7.80 (m, 3H), 8.33 (s, 1H), 8.44 (s, 1H),
.50 (s, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ 13.11, 28.62, 29.33, 33.09, 35.57, 41.21, 43.20, 44.85, 56.73,
2.15, 102.44, 108.33, 109.17, 115.33, 115.55, 122.65, 124.76, 124.81, 136.19, 136.22, 145.75, 145.77, 147.33,
1
49.41, 153.30, 153.92, 156.80, 157.56, 159.95, 168.05; 19F-NMR (376 MHz, DMSO-d ) δ −119.21.
6
1-{4-[({4-[(3,4-Dichloro-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-3-(2-
methyl-5-nitro-imidazol-1-yl)propyl-1-one (16n). Faint yellow powder prepared from 14a and 1g with the
yield of 68.6%. m.p. 157.1–159.5 C; ESI-HRMS calcd for C H Cl FN O m/z = 632.1591 [M + H]+
◦
,
28
29
2
7
5
1
found: 632.1586; H-NMR (400 MHz, DMSO-d )
δ
1.19 (m, 2H), 1.82 (s, 2H), 2.08 (m, 1H), 2.41 (s,
6
3
H), 2.61 (t, J = 12.0 Hz, 1H), 2.84–3.14 (m, 3H), 3.82–3.99 (m, 6H), 4.19 (t, J = 6.4 Hz, 2H), 4.43 (d,
13
J = 12.0 Hz, 1H), 7.19 (m, 1H), 7.57 (m, 2H), 7.81 (s, 1H), 8.32 (s, 1H), 8.44 (s, 1H), 9.78 (s, 1H); C-NMR
100 MHz, DMSO-d6) 13.15, 22.17, 28.65, 29.33, 33.11, 35.56, 43.20, 44.86, 56.71, 72.72, 102.40, 108.19,
(
δ
1
1
09.15, 119.98, 120.19, 125.78, 127.24, 127.92, 128.92, 145.72, 149.67, 152.28, 153.27, 154.31, 154.76, 157.17,
64.18; 19F-NMR (376 MHz, DMSO-d ) δ −113.50.
6
1
-{4-[({4-[(2-Chloro-4-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(2-nitro-
imidazol-1-yl)ethanone (16o). Faint yellow powder prepared from 13b and 1b with the yield of
◦
5.7%. m.p. 158.6–159.9 C; ESI-HRMS calcd for C H ClFN O m/z = 570.1668 [M + H] , found:
26 36 7 5
+
7
5
1
70.1661; H-NMR (400 MHz, DMSO-d )
δ 1.19 (m, 1H), 1.41 (m, 1H), 1.83 (d, J = 11.6 Hz, 1H), 1.92 (d,
6
J = 11.6 Hz, 1H), 2.16 (m, 1H), 2.73 (t, J = 12.0 Hz, 1H), 3.18 (t, J = 12.0 Hz, 1H), 3.91 (d, J = 12.0 Hz
H), 3.97 (s, 3H), 4.03 (d, J = 5.6 Hz, 2H), 4.31 (d, J = 12.0 Hz, 1H), 5.46 (d, J = 16.0 Hz, 1H), 5.53 (d,
J = 16.0 Hz, 1H), 7.20 (d, 2H), 7.29 (td, J = 9.6 Hz, J = 2.8 Hz 1H), 7.54 (m, 2H), 7.66 (s, 1H), 8.09 (s,
,
1
1
2
13
1
H), 8.33 (s, 1H), 10.15 (br, 1H); C-NMR (100 MHz, DMSO-d6)
δ 28.61, 29.28, 35.43, 42.04, 44.45,
5
1
1.57, 57.01, 72.81, 103.42, 107.36, 108.88, 115.03, 115.25, 117.16, 117.42, 125.91, 127.87, 128.68, 128.97,
31.62, 132.77, 133.43, 145.70, 149.53, 152.97, 154.21, 158.30, 159.16, 161.61, 164.18; 1 F-NMR (376 MHz,
9
DMSO-d ) δ −113.92.
6
1-{4-[({4-[(3,4-Dichloro-2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]piperidin-1-yl}-2-(2-
nitro-imidazol-1-yl)ethanone (16p). Faint yellow powder prepared from 13b and 1g with the yield of
◦
0.3%. m.p. 166.6–168.7 C; ESI-HRMS calcd for C H Cl FN O m/z = 604.1278 [M + H] , found:
26 25 2 7 5
+
7
6
1
04.1272; H-NMR (400 MHz, DMSO-d )
δ 1.24 (m, 1H), 1.43 (m, 1H), 1.86 (d, J = 11.6 Hz, 1H), 1.94 (d,
6
J = 11.6 Hz, 1H), 2.18 (m, 1H), 2.75 (t, J = 12.0 Hz, 1H), 3.21 (t, J = 12.0 Hz, 1H), 3.92 (d, J = 12.0 Hz,
H), 3.97 (s, 3H), 4.07 (d, J = 5.6 Hz, 2H), 4.35 (d, J = 12.0 Hz, 1H), 5.43 (d, J = 16.0 Hz, 1H), 5.49 (d,
J = 16.0 Hz, 1H), 7.21 (m, 2H), 7.59 (m, 3H), 7.81 (s, 1H), 8.42 (s, 1H), 9.73 (s, 1H); 13C-NMR (100 MHz,
DMSO-d6) 28.63, 29.29, 35.49, 42.05, 44.43, 51.55, 56.65, 72.79, 102.42, 108.21, 109.14, 119.99, 120.18,
25.78, 127.23, 127.91, 128.92, 145.73, 149.67, 152.28, 153.27, 154.29, 154.77, 157.13, 164.15; F-NMR
1
δ
19
1
(
376 MHz, DMSO-d ) δ −113.50.
6

Molecules 2016, 21, 1693
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3
.2. Molecular Docking Study
Two docking studies of compound 16f with EGFR (PDB ID: 4I23) and VEFR-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).
For ligand preparation, the 3D structure of compound 16f was generated and minimized. For enzyme
preparation, the hydrogen atoms were added and the Chemistry at HARvard Macromolecular
Mechanics (CHARMM) force field was employed. EGFR and VEFR-2 were defined as receptors and
the site spheres were selected based on the ligand binding location of vandetanib, then vandetanib was
removed and compound 16f was placed by the MacroModel module through Embrace Minimization
procedure. Types of interactions of the docked receptors with ligands were analyzed after end of
molecular docking. Ten docking poses were saved for each ligand and the final docked conformations
were evaluated by the score of the program and also by visual inspection.
3
.3. Pharmacology
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.
MTS assay kit (Promega Corp., Madison, WI, USA) and CCK-8 assay kit (BestBio Corp., Shanghai,
China) were used in corresponding assays. Primer sequences of VEGF, EGF, 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.
3
.3.1. In Vitro EGFR Inhibitory Assay
The In Vitro EGFR Inhibitory activities of compound 16a
determined by MTS method. The cells were incubated with 10% RPMI-1640 medium at 37 C in
–
f
on A431 cells and H1975 cells were
◦
4
5
% CO and seeded in 96-well tissue culture plates at the concentration of 1
×
10 cells per well.
2
The cells adhered completely after 24 h of incubation. Removed the medium and washed with D-hanks
buffer, then exposed cells to different concentrations of test compounds (0.3, 1, 3, 10, 30, and 100 mol/L
µ
in DMSO) dissolved in non-serum medium (three wells per concentration with corresponding blank
◦
groups). 0.1% DMSO was added to the control group. After 48 h of incubation at 37 C in 5% CO ,
2
added 20 µL MTS solution to per well and incubated for another 0.5 h. Absorbance was measured
at 490 nm in plate reader (RT-6100 Rayto, Shenzhen, China) and calculated for inhibition ratios and
IC50 values.
3
.3.2. In Vitro Anti-Proliferative Activity Assays in Normoxia and Hypoxia
A549 and H446 cells were divided into hypoxic and normoxic groups (Hela cells were only
determined in hypoxia). Both groups were incubated and exposed to two concentrations of test
compounds (0.5 and 5 mol/L in DMSO) in the same way with above assay. In hypoxic groups,
µ
cobalt (II) chloride solution (100
µ
mol/L in water) was added, except for blank groups. After 48 h of
◦
incubation at 37 C in 5% CO , 20
µL CCK-8 solution was added to per well and incubated for another
2
1
h. Measured absorbance at 450 nm in plate reader and calculated for inhibition ratios.
3
.3.3. In Vitro VEGF/EGF Gene Expression Inhibition Assays
Two groups of A549 cells were seeded in 96-well tissue culture plates at the concentration of
5
1
× 10 cells per well. Cobalt (II) chloride solution (100 µmol/L) (containing 10% serum HG-DMEM)
was added to cells and setup blank and control groups. Cells were exposed to two concentrations of
test compounds (0.5 and 5 µmol/L in DMSO, three wells per concentration) and extracted RNAs by
the Trizol method after 24 h of incubation. The Q-PCR method was used for determining the amounts
of VEGF mRNA and EGF mRNA, with β-actin as internal control.

Molecules 2016, 21, 1693
14 of 15
4
. Conclusions
In summary, sixteen novel compounds were designed and synthesized by incorporating
the nitroimidazole moieties into the piperidine N-4 position of vandetanib with amide linkers.
The molecular docking study of 16f with EGFR and VEFR-2 has revealed that it could fit well into
EGFR and VEFR-2 kinase in a similar way to vandetanib and an extra H-bond formed between the
nitroimidazole group and Lys838 residue at the opening of the hydrophobic pocket in VEGFR-2,
notwithstanding that it did not seem very firm, with a distance of 3.491 Å. More than a half of the
compounds showed acceptable EGFR inhibition activities over wild-type tumor cells but obviously
weak activities over T790M mutant tumor cells. In in vitro normoxic/hypoxic anti-proliferative assays,
all compounds demonstrated superior activities to vandetanib in hypoxia and the activities were
greatly activated at low concentration comparing with normoxia. In VEGF/EGF gene expression
inhibition assays, all target compounds exhibited excellent VEGF expression inhibitory activities in
hypoxia and were distinctly superior to vandetanib; nevertheless, EGF gene expression levels are
augmented mostly in hypoxia. Among target compounds, 16f showed the most potent activities in the
above-mentioned assays and was worthy of further development as a potential cancer therapeutic
agent in hypoxia.
Acknowledgments: This work was supported by the Chinese Academy of Medical Sciences & Peking Union
Medical College Institute of Radiation Medicine Development Fund (No. 1537) and Peking Union Medical College
Youth Fund (No. 3332015102).
Author Contributions: Huiqiang Wei was responsible for the chemical experiments and analyzed the data;
Deguan Li and Xiangbo Yang were responsible for in vitro anti-proliferative activity experiments; Haihua Shang
was responsible for Q-PCR experiments; Saijun Fan was responsible for pharmacology orientations; Yiliang Li
and Dan Song were responsible for design of article, analyzed the data, Huiqiang Wei orientation and wrote the
paper. All Authors read and approved the final version of the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Van Putten, L.M.; Kallman, R.F. Oxygenation status of a transplantable tumor during fractionated radiation
therapy. J. Natl. Cancer Inst. 1968, 40, 441–451. [CrossRef] [PubMed]
Zheng, X.L.; Jin, Y.Z.; Shen, Y. Tumor Therapy Sensitizers, 1st ed.; Shanghai Science and Technical Literature
Press: Shanghai, China, 2002; pp. 12–14.
Toma-Da s¸ u, I.; Da s¸ u, A.; Karlsson, M. The relationship between temporal variation of hypoxia, polarographic
measurements and predictions of tumour response to radiation. Phys. Med. Biol. 2004, 49, 4463–4475.
[
CrossRef] [PubMed]
Pugh, C.W.; Ratcliffe, P.J. Regulation of angiogenesis by hypoxia: Role of the HIF system. Nat. Med. 2003, 9,
77–684. [CrossRef] [PubMed]
4
5
.
.
6
Maxwell, P.H.; Dach, G.U.; Gleadle, J.M. Hypoxia-inducible factor-1 modulates gene expression in solid
tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 1997, 94, 8104–8109.
[CrossRef] [PubMed]
6
.
.
Shweiki, D.; Itin, A.; Soffer, D.; Keshet, E. Vascular endothelial growth factor induced by hypoxia may
mediate hypoxia-initiated angiogenesis. Nature 1992, 359, 843–845. [CrossRef] [PubMed]
Wang, T.; Niki, T.; Goto, A.; Ota, S.; Morikawa, T.; Nakamura, Y.; Ohara, E.; Ishikawa, S.; Aburatani, H.;
Nakajima, J.; Fukayama, M. Hypoxia increases the motility of lung adenocarcinoma cell line A549 via
activation of the epidermal growth factor receptor pathway. Cancer Sci. 2007, 98, 506–511. [CrossRef]
7
[
PubMed]
Wilson, W.R.; Hay, M.P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 2011, 11, 393–410. [CrossRef]
PubMed]
Lancini, G.C.; Lazzari, E.; Arioli, V.; Bellani, P. Synthesis relationship between structure and activity of
-nitroimidazole derivatives. J. Med. Chem. 1969, 12, 775–780. [CrossRef] [PubMed]
0. Wardman, P. Chemical radiosensitizers for use in radiotherapy. Clin. Oncol. 2007, 19, 397–417. [CrossRef]
PubMed]
8
9
1
.
[
.
2
[

Molecules 2016, 21, 1693
15 of 15
1
1
1
1. Adams, G.E. Hypoxic cell sensitizers for radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 1978, 4, 135–141.
[CrossRef]
2. Adams, G.E. Accomplishments, problems and prospects: A conference summary. Int. J. Radiat. Oncol.
Biol. Phys. 1982, 8, 805–808. [CrossRef]
3. Adams, G.E.; Stratford, I.J.; Wallace, R.G.; Wardman, P.; Watts, M.E. Toxicity of nitro compounds toward
hypoxic mammalian cells in vitro: Dependence on reduction potential. J. Natl. Cancer Inst. 1980, 64, 555–560.
[PubMed]
1
4. Kumar, P.; Shustov, G.; Liang, H.; Khlebnikov, V.; Zheng, W.Z.; Yang, X.H.; Cheeseman, C.; Wiebe, L.I. Design,
synthesis, and preliminary biological evaluation of 6-O-glucose-azomycin adducts for diagnosis and therapy
of hypoxic tumors. J. Med. Chem. 2012, 55, 6033–6046. [CrossRef] [PubMed]
1
5. Ziemer, L.S.; Evans, S.M.; Kachur, A.V.; Shuman, A.L.; Cardi, C.A.; Jenkins, W.T.; Karp, J.S.; Alavi, A.;
Dolbier, W.R., Jr.; Koch, C.J. Noninvasive imaging of tumor hypoxia using the 2-nitroimidazole 18F-EF5 in
rats. Eur. J. Nucl. Med. Mol. Imaging 2003, 30, 259–266. [CrossRef] [PubMed]
1
1
6. Melo, T.; Duncan, J.; Ballinger, J.R. BRU59-21, a second-generation 99mTc-labled 2-nitroimidazole for imaging
hypoxia in tumors. J. Nucl. Med. 2000, 41, 169–176.
7. Hoigebazar, L.; Jeong, J.M.; Choi, S.Y.; Choi, J.Y.; Shetty, D.; Lee, Y.S.; Lee, D.S.; Chung, J.Y.; Lee, M.C.;
Chung, Y.K. Synthesis and characterization of citroimidazole derivatives for 68Ga-labeling and testing in
tumor xenografted mice. J. Med. Chem. 2010, 53, 6378–6385. [CrossRef] [PubMed]
1
1
8. Duan, J.; Jiao, H.; Kaizerman, J.; Stanton, T.; Evans, J.W.; Lan, L.; Lorente, G.; Banica, M.; Jung, D.; Wang, J.;
et al. Potent and highly selective hypoxia-activated achiral phosphoramidate mustards as anticancer drugs.
J. Med. Chem. 2008, 51, 2412–2420. [CrossRef] [PubMed]
9. Inomata, T.; Ogawa, Y.; Itoh, S.; Kariya, S.; Hamada, N.; Nishioka, A.; Yoshida, S.; Nagasawa, H.; Hori, H.;
Inayama, S. Lung metastasis suppression of the bifunctional new radiosensitizer KIN-806. Int. J. Mol. Med.
1
999, 4, 257–317. [CrossRef] [PubMed]
0. Zhang, W.N. Modern Drug Design, 1st ed.; China Medical Science Press: Beijing, China, 2005; pp. 240–242,
86–587.
1. Hennequin, L.F.; Stokes, E.S.E.; Thomas, A.P.; Johnstone, C.; Ple, P.A.; Ogilvie, D.J.; Dukes, M.; Wedge, S.R.;
Kendrew, J.; Curwen, J.O. 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.
2
2
5
2
002, 45, 1300–1312. [CrossRef] [PubMed]
2
2
2
2
2. Ashworth, I.W.; Cox, B.G.; Meyrick, B. Kinetics and mechanism of N-Boc cleavage: Evidence of a
second-order dependence upon acid concentration. J. Org. Chem. 2010, 75, 8117–8125. [CrossRef] [PubMed]
3. Shi, Y.; Hu, Q.Q.; Yu, B.; Li, Y.L.; Xiong, D.S. Anti-tumor activity of novel 4-aminobenzene quinazoline
tyrosine kinase inhibitors. Drugs Clin. 2014, 29, 969–973.
4. Chen, J.W.; Hu, X.J.; Lin, Q.; Mao, J.J.; Wang, L.; Bo, M.J.; Huang, M.S.; Shan, H. Effect of CoCl on expression
2
of HIF-lα and VEGF, and tumor invasion in rat RH35 hepatoma cells. J. New. Med. 2014, 45, 790–795.
5. Yun, C.; Mengwasser, K.E.; Toms, A.V.; Woo, M.S.; Greulich, H.; Wong, K.K.; Meyerson, M.; Eck, M.J.
The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc. Natl.
Acad. Sci. USA 2008, 105, 2070–2075. [CrossRef] [PubMed]
2
6. Cheng, W.Y.; Zhu, S.J.; Ma, X.D.; Qiu, N.; Peng, P.; Sheng, R.; Hu, Y.Z. Design, synthesis and biological
evaluation of 6-(nitroimidazole-1H-alkyloxyl)-4-anilinoquinazolines as efficient EGFR inhibitors exerting
cytotoxic effects both under normoxia and hypoxia. Eur. J. Med. Chem. 2015, 89, 826–834. [CrossRef]
[PubMed]
Sample Availability: Not available.
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