Design, synthesis and biological evaluation of 6-(nitroimidazole-1 H -alkyloxyl)-4-anilinoquinazolines as efficient EGFR inhibitors exerting cytotoxic effects both under normoxia and hypoxia

Article abstract of DOI:10.1016/j.ejmech.2014.11.010

A series of novel 6-(nitroimidazole-1H-alkyloxyl)-4-anilinoquinazoline derivatives (15a-15r) were designed, synthesized and evaluated as efficient EGFR inhibitors through introduction of hypoxia activated nitroimidazole moiety into the quinazoline scaffold of EGFR inhibitors. The majority of these newly synthesized compounds exhibited comparable EGFR inhibitory activities to gefitinib and moderate to excellent anti-proliferative activities against HT-29 cells under normoxia and hypoxia. The most promising compound 15c displayed the IC₅₀ value of 0.47 nM against EGFR kinase and excellent cytotoxic effect against HT-29 cells under normoxia and hypoxia with the IC₅₀ values of 2.21 μM and 1.62 μM, respectively. The mimic reductive activation study revealed that compound 15c exerted reductive activation properties under hypoxia, which were consistent with the in vitro metabolic study, wherein 15c was easily reductive activated under hypoxia and much more stable under normoxia. All these results suggested that 15c was a potential cancer therapeutic agent both under normoxia and hypoxia and was worth of further development.

Full text of DOI:10.1016/j.ejmech.2014.11.010

European Journal of Medicinal Chemistry 89 (2015) 826e834
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of 6-(nitroimidazole-1H-
alkyloxyl)-4-anilinoquinazolines as efficient EGFR inhibitors exerting
cytotoxic effects both under normoxia and hypoxia
Weiyan Cheng, Shijun Zhu, Xiaodong Ma, Ni Qiu, Peng Peng, Rong Sheng*, Yongzhou Hu*
Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 6-(nitroimidazole-1H-alkyloxyl)-4-anilinoquinazoline derivatives (15ae15r) were
designed, synthesized and evaluated as efficient EGFR inhibitors through introduction of hypoxia acti-
vated nitroimidazole moiety into the quinazoline scaffold of EGFR inhibitors. The majority of these newly
synthesized compounds exhibited comparable EGFR inhibitory activities to gefitinib and moderate to
excellent anti-proliferative activities against HT-29 cells under normoxia and hypoxia. The most prom-
ising compound 15c displayed the IC50 value of 0.47 nM against EGFR kinase and excellent cytotoxic
Received 5 August 2014
Received in revised form
27 October 2014
Accepted 5 November 2014
Available online 6 November 2014
effect against HT-29 cells under normoxia and hypoxia with the IC50 values of 2.21 mM and 1.62 mM,
Keywords:
respectively. The mimic reductive activation study revealed that compound 15c exerted reductive acti-
vation properties under hypoxia, which were consistent with the in vitro metabolic study, wherein 15c
was easily reductive activated under hypoxia and much more stable under normoxia. All these results
suggested that 15c was a potential cancer therapeutic agent both under normoxia and hypoxia and was
worth of further development.
EGFR inhibitor
4-Anilinoquinazoline
Nitroimidazole
Normoxia
Hypoxia
© 2014 Published by Elsevier Masson SAS.
1. Introduction
potency to overcome EGFR up-regulation under hypoxia may exert
better effects for cancer therapy.
As a member of the HER family, epidermal growth factor re-
ceptor (EGFR) plays a vital role in the cellular signaling transduction
processes [1]. The over-expression of EGFR has been observed in
many solid tumors, such as colon [2], ovarian [3], and non-small cell
lung cancer (NSCLC) [4]. Therefore, EGFR inhibition has been
developed as one of the most efficient strategies for cancer therapy.
In the past few years, more than 20 EGFR inhibitors have been
advanced into clinical trials, and four 4-anilinoquinazoline de-
rivatives, termed as gefitinib (1), erlotinib (2), lapatinib (3), and
afatinib (4), have been approved by FDA for the treatment of NSCLC,
pancreatic cancer and advanced breast cancer (Fig. 1) [5].
As an inevitable circumstance in most solid tumors, hypoxia is
responsible for the resistance to radiotherapy and chemotherapy
and presents a tremendous challenge to cancer therapy [6]. Espe-
cially, the expression of EGFR is up-regulated when tumors are
under hypoxia, which is closely related to tumor survival [7,8].
Therefore, design and development of EGFR inhibitors with
The specific hypoxic micro-environment of tumor cells also
makes it as an attractive and exploitable therapeutic target. 2-
Nitroimidazole derivatives have been developed as hypoxia acti-
vated radio-sensitization and chemotherapy agents since 1960s [9].
Under hypoxia, the 2-nitroimidazole moiety was reduced by
nitroreductase to yield reactive radicals, which could deplete tumor
endogenous protective agents such as glutathione (GSH) to make
tumors be sensitive to radiotherapy [10]. Moreover, the irreversibly
binding of reactive radicals to the cellular protein and nucleic acids
makes them accumulate therein and exert cytotoxic effects [11]. Up
to now, several radioactive 2-nitroimidazole derivatives have been
advanced into clinical trials for the noninvasive assessment of
hypoxia in cancer, including [18F]FMISO (5), [18F]FAZA (6) and [18F]
HX4 (7) (Fig. 1) [12,13]. Based on the above reports, we envisioned
that EGFR inhibitors containing hypoxia activated pharmacophore
may exert efficient antitumor activities both under normoxia and
hypoxia.
The crystal structures of 4-anilinoquinazolineeEGFR complexes
reveal that the quinazoline scaffold confers an H-bond interaction
to the hinge domain of the kinase and the aniline moiety inserts
into the hydrophobic pocket, which are crucial for EGFR inhibitory
* Corresponding authors.
E-mail addresses: shengr@zju.edu.cn (R. Sheng), huyz@zju.edu.cn (Y. Hu).
http://dx.doi.org/10.1016/j.ejmech.2014.11.010
0223-5234/© 2014 Published by Elsevier Masson SAS.

W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
827
Fig. 1. Structures of selected EGFR inhibitors and 2-nitroimidazole derivatives.
activity. The side chains at the C-6 and C-7 positions are projected
to the solvent portion which exerts good compatibility for long
ether chains or other moieties [14e17] (Fig. 2). Therefore, a series of
novel EGFR inhibitors were designed and synthesized through
incorporating 2-nitroimidazole and its analogue 2-methyl-5-
nitroimidazole into the C-6 position of the 4-anilinoquinazoline
scaffold. Different kinds of anilines from gefitinib, erlotinib, lapa-
tinib and other advanced EGFR inhibitor were employed to the C-4
position of the quinazoline template, as well as the length of linker
between the nitroimidazole and quinazoline was probed for the
SAR study (Fig. 2).
1.18 nM, which were comparable to that of gefitinib
(IC50 ¼ 0.45 nM). The introduction of the bulky substituents on
aniline resulted in a dramatic decrease in activity. For instance,
compounds 15ke15n only demonstrated EGFR inhibition with the
IC50 values ranging from 15.1 to 51.2 nM, which were more than 30
fold less potent than gefitinib. Other compounds with small sub-
stituents on aniline demonstrated favorable inhibitory activity with
the IC50 values ranging from 0.32 to 4.90 nM, indicating that bulky
moiety on aniline may prevent the aniline from adapting the hy-
drophobic pocket of EGFR kinase. The length of linker between the
imidazole and quinazoline also significantly affected the activities.
The compromised EGFR inhibitory activities of compound 15a, 15b,
15o and 15p compared to 15c and 15q indicated that longer linkers
(n ¼ 3e5) were more favorable than shorter ones (n ¼ 1e2).
2. Results and discussion
2.1. Chemistry
2.3. In vitro anti-proliferative activity
The synthetic route to compounds 15ae15r was displayed in
Scheme 1. Compounds 9ae9e were prepared via alkylation of 2-
The human colorectal adenocarcinoma HT-29 cells were widely
used to evaluate the biological activities of the hypoxia-activated
drugs, and this kind of cells also expressed high levels of EGFR
[20e23]. Therefore, the anti-proliferative activities of compounds
15ae15r were evaluated against HT-29 cells under normoxia and
hypoxia. As shown in Table 1, most of these compounds demon-
strated moderate to excellent anti-proliferative activities. Espe-
cially, five compounds (15c, 15i, 15j, 15l, and 15m) were more
potent than gefitinib both under normoxia (IC50 values:
nitroimidazole (8) with
a, u-dibromoalkane in the presence of
potassium carbonate, and compounds 11ae11c were obtained in a
similar way by using 2-methyl-5-nitroimidazole (10) as the start
material. Condensation of compound 12 with substituted anilines
afforded 4-anilinoquinazoline derivatives 13ae13d [18], followed
by de-acetylation with aqueous ammonia provided 14ae14d as the
key intermediates. Finally, reaction of 14ae14d with 9ae9e or
11ae11c in the presence of potassium carbonate furnished title
compounds 15ae15r.
1.87e3.55
exhibited less potent anti-proliferative activity under hypoxia
M), probably because
mM) and hypoxia (IC50 values: 1.36e3.46 mM). Gefitinib
2.2. In vitro EGFR inhibitory activity
(IC50 ¼ 5.21
mM) than normoxia (IC50 ¼ 3.63
m
the over-expression of EGFR made tumors be more resistant under
hypoxia. For the newly synthesized compounds, most of them
(15ae15i and 15me15p) exhibited comparable or more potent
anti-proliferative activities under hypoxia than under normoxia.
We deduced that the cytotoxicities contributed by the reductive
activation of these compounds were partly offset by the increased
The EGFR inhibitory activities of compounds 15ae15r were
evaluated using a well established Z0-Lyte assay and gefitinib was
employed as the positive control [19]. As shown in Table 1, most of
the tested compounds (15be15j, 15q, and 15r) exhibited potent
EGFR inhibitory activities with the IC50 values ranging from 0.32 to
Fig. 2. Design of new EGFR inhibitors containing hypoxia activated pharmacophores.

828
W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
Scheme 1. Synthesis of 15ae15r. Reagents and conditions: (i)
aniline, isopropanol, reflux, 1 h; (iv) ammonia/methanol, rt, 3 h; (v) 9ae9e, 11ae11c, K2CO3, DMF, 80 ꢀC, 5 h.
a, u a-bromo-u
-dibromoalkane, K2CO3, DMF, 60 ꢀC, 4 h. (ii) -chloro-alkane, K2CO3, DMF, 60 ꢀC, 4 h. (iii) substituted
vitalities of the HT-29 cells under hypoxia, and this led to the
relatively less obvious difference in the IC50 values between nor-
moxia and hypoxia.
vs 15r), indicating that 2-nitroimidazole was a more favorable
hypoxia-activated moiety [24]. In addition, although compound 15l
and 15m displayed compromised EGFR inhibitory activities
(IC50 ¼ 28.5, 51.2 nM, respectively) compared to gefitinib, they
demonstrated excellent anti-proliferative activities with the IC50
Compounds with 2-nitroimidazole moieties exhibited more
potent
anti-proliferative
activities
than
2-methyl-5-
nitroimidazole-containing ones (15b vs 15p, 15c vs 15q, and 15f
values of 1.87 and 3.09 mM under normoxia, 2.07 and 1.36 mM under
Table 1
In vitro EGFR inhibitory and anti-proliferative activities of compounds 15ae15r.
Compd.
R1
R2
n
R3
R4
EGFRa (IC50, nM)
HT-29 (IC50,
Normoxia
m
M)a
Hypoxia
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
15q
15r
Cl
Cl
Cl
Cl
Cl
Br
Ethynyl
Ethynyl
F
F
F
F
F
H
H
H
1
2
3
4
5
2
2
3
4
5
2
3
4
5
1
2
3
2
e
e
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
CH3
CH3
CH3
CH3
e
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
NO2
NO2
NO2
e
3.05 0.52
0.89 0.31
0.47 0.10
0.51 0.14
0.32 0.11
0.66 0.13
1.08 0.34
0.56 0.17
0.49 0.23
0.50 0.12
38.3 4.83
28.5 6.65
51.2 12.43
15.1 4.18
4.90 0.76
3.43 0.54
0.53 0.21
1.18 0.13
0.45 0.12
e
>50
10.02 2.56
7.17 1.87
1.62 0.39
5.59 1.08
9.81 1.34
4.01 0.57
5.53 0.89
6.90 1.02
3.05 0.63
3.46 0.91
20.00 3.31
2.07 1.02
1.36 0.45
24.31 3.34
>50
8.39 2.32
2.21 0.54
17.58 4.12
12.89 2.49
4.48 0.68
5.55 1.06
10.08 2.34
3.55 0.78
2.93 0.82
15.48 3.23
1.87 0.73
3.09 0.93
>50
Ethynyl
Ethynyl
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
e
3-Fluorobenzyloxy
3-Fluorobenzyloxy
3-Fluorobenzyloxy
3-Fluorobenzyloxy
F
F
F
>50
23.24 4.23
8.21 3.25
15.72 4.51
3.63 0.58
>50
11.52 2.31
9.98 2.03
5.41 1.32
5.21 0.93
9.45 2.81
H
e
e
Gefitinib
TPZb
e
e
e
a
The data were means from at least three independent experiments.
TPZ: Tirapazamine.
b

W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
829
respectively. The peak D (m/z ¼ 391.1349) was deduced as the
degeneration product of the reactive radical.
According to the LCeMS results of mimic reduction reaction of
15c, we deduced that compound 15c experienced the following
reductive process (Fig. 4).
2.5. Metabolic stability of compound 15c under normoxia and
hypoxia
To further evaluate the metabolic stability of compound 15c
under normoxia and hypoxia, it was incubated with mouse liver
microsomes using NADPH as the activator [29] and gefitinib as the
reference. As shown in Fig. 5, gefitinib exerted similar metabolic
stability profile under normoxia and hypoxia, with more than 90%
remained after 30 min incubation. While for compound 15c, about
70% remained under normoxia and less than 10% remained under
hypoxia. These results indicated that compound 15c was relatively
stable under normoxia and could be easily metabolic activation
under hypoxia.
Fig. 3. The HPLC chromatograms of products in the mimic reductive activation of
compound 15c.
hypoxia, respectively. The most promising compound 15c displayed
extraordinary cytotoxic activities against HT-29 cells under both
normoxia and hypoxia with the IC50 values of 2.21
respectively, and it was selected for further study.
mM and 1.62 mM,
2.4. Mimic the reductive activation of compound 15c
2.6. Molecular docking study
As reported, the 2-nitroimidazole derivatives may experience
reductive activation under tumor hypoxia and zinc dust was used to
mimic this process [25,26]. To confirm this speculation, compound
15c was chosen to react with zinc dust and cysteine was used to
catch the produced reactive radicals [27,28]. The reaction was car-
ried out in the phosphate buffer saline at 37 ꢀC under hypoxia, and
the resulting mixture was detected by HPLC-Q-ToF-MS. As shown in
Fig. 3, several peaks were appeared within the retention time of
36 min. Among them, peak A (m/z ¼ 457.1569) was the main
product of the reaction and was confirmed as the 2-
To predict the possible binding mode of this series of com-
pounds with EGFR, the docking study of compound 15c with EGFR
kinase (PDB ID: 2ITY) was performed using gefitinib as a compar-
ison. As illustrated in Fig. 6, compound 15c favorably fitted into
EGFR in a similar way to gefitinib, the 3-chloro-4-fluoro-aniline
moiety inserted into the hydrophobic pocket formed by Met766,
Glu762, Lys745 and Leu788, and the fluorine atom interacted with
Lys745 through a hydrogen bond with a distance of 2.92 Å. Another
hydrogen bond was formed between Met793 and N-1 of the qui-
aminoimidazole derivative. The appearance of peak
B
(m/
nazoline with a distance of 2.99 Å. As expected, the 2-
z ¼ 695.1663) and peak C (m/z ¼ 576.1618) indicated that two or
nitroimidazole side chain was positioned at the solvent portion of
the kinase which was similar to the morpholine moiety of gefitinib.
one molecular cysteines were bounded to one reactive radical,
Fig. 4. The proposed reductive activation process of compound 15c.

830
W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
potential cancer therapeutic agent under normoxia as well as
hypoxia and was worth of further development.
4. Experimental
4.1. Chemistry
4.1.1. General methods/instruments
Melting points were recorded on a B-540 Büchi melting-point
apparatus and are uncorrected, 1H NMR were recorded on a
BRUKER AVIII 500 MHz or AVII 400 MHz and 13C NMR were
recorded on a BRUKER AVII 100 MHz spectrometer with TMS as the
internal standard. Proton chemical shifts are expressed in parts per
million (ppm) and coupling constants in Hz. Mass spectra (ESI-MS)
were performed on a Finnigan LCQ Deca XP ion trap mass spec-
trometry. High resolution mass spectra were measured on an Agi-
lent 1290 HPLC-6224 Time of Fight Mass Spectrometer.
Fig. 5. The percentage of intact compound 15c and gefitinib after 30 min of in vitro
metabolism reaction.
4.1.2. General synthetic procedure of nitroimidazole derivatives
(9ae9e, 11ae11c)
3. Conclusion
Dibromo-alkane (0.04 mol) was added to a stirred mixture of
nitroimidazole derivative (8 or 10, 0.01 mol) and potassium car-
bonate (1.38 g, 0.02 mol) in DMF (5 mL). The mixture was heated to
60 ꢀC and stirred for about 4 h. After cooling to room temperature,
the mixture was filtered and the filtrate was concentrated to dry-
ness. The residue was purified by silica gel column chromatography
to get pure 9ae9e and 11ae11c.
To overcome the EGFR up-regulation under tumor hypoxia, a
novel series of EGFR inhibitors were synthesized by incorporating
the nitroimidazole moieties into the C-6 position of the 4-
anilinoquinazolines with proper linkers. Most of the compounds
exhibited excellent EGFR inhibitory activities in vitro and the SARs
revealed that bulky substituents on aniline and short linkers be-
tween nitroimidazole and quinazoline were detrimental to the
enzymatic activity. The molecular docking study of 15c with EGFR
has revealed that it could fit well into EGFR kinase in a similar way
to gefitinib. Most of these compounds demonstrated moderate to
excellent anti-proliferative activities against HT-29 cells under
normoxia and hypoxia. The most promising compound 15c
exhibited excellent anti-proliferative activities with the IC50 values
4.1.2.1. 1-(2-Bromoethyl)-2-nitro-1H-imidazole (9a). Yellow solid,
yield: 87%, mp: 62e64 ꢀC.
4.1.2.2. 1-(3-Bromopropyl)-2-nitro-1H-imidazole (9b). Yellow oil,
yield: 82%. 1H NMR (500 MHz, CDCl3)
d 7.21 (s, 1H), 7.17 (s, 1H), 4.62
of 2.21 and 1.62
mM under normoxia and hypoxia, respectively,
(t, J ¼ 6.5 Hz, 2H), 3.38 (t, J ¼ 6.0 Hz, 2H), 2.45e2.38 (m, 2H). ESI-MS
which were more potent than that of gefitinib. The mimic reductive
activation study revealed that compound 15c could be reduced to
generate reactive radicals and covalently bound to nucleophiles
under hypoxia. Moreover, the metabolic stability study indicated
that compound 15c could be easily reductive activated under
hypoxia. All these results demonstrated that compound 15c was a
m/z: 234 [MþH]þ.
4.1.2.3. 1-(4-Bromobutyl)-2-nitro-1H-imidazole (9c). Yellow oil,
yield: 86%. 1H NMR (500 MHz, CDCl3)
d 7.15 (s, 1H), 7.11 (s, 1H), 4.46
(t, J ¼ 7.5 Hz, 2H), 3.42 (t, J ¼ 6.5 Hz, 2H), 2.08e2.00 (m, 2H),
1.95e1.88 (m, 2H). ESI-MS m/z: 248 [MþH]þ.
4.1.2.4. 1-(5-Bromopentyl)-2-nitro-1H-imidazole (9d). Yellow oil,
yield: 82%. 1H NMR (500 MHz, CDCl3)
d 7.14 (s, 1H), 7.10 (s, 1H), 4.42
(t, J ¼ 7.5 Hz, 2H), 3.40 (t, J ¼ 6.5 Hz, 2H), 1.93e1.84 (m, 4H),
1.55e1.49 (m, 2H). ESI-MS m/z: 262 [MþH]þ.
4.1.2.5. 1-(6-Bromohexyl)-2-nitro-1H-imidazole (9e). Yellow oil,
yield: 89%. 1H NMR (500 MHz, CDCl3)
d 7.14 (s, 1H), 7.08 (s, 1H), 4.41
(t, J ¼ 7.5 Hz, 2H), 3.40 (t, J ¼ 7.0 Hz, 2H), 1.91e1.81 (m, 4H),
1.53e1.47 (m, 2H), 1.41e1.35 (m, 2H). ESI-MS m/z: 276 [MþH]þ.
4.1.2.6. 1-(2-Chloroethyl)-2-methyl-5-nitro-1H-imidazole
(11a).
(11b).
(11c).
White solid, yield: 74%, mp: 73e75 ꢀC (lit. 78e79 ꢀC) [30].
4.1.2.7. 1-(3-Chloropropyl)-2-methyl-5-nitro-1H-imidazole
White solid, yield: 78%, mp: 61e63 ꢀC (lit. 65e67 ꢀC) [31].
4.1.2.8. 1-(4-Chlorobutyl)-2-methyl-5-nitro-1H-imidazole
White solid, yield: 79%, mp: 67e69 ꢀC. 1H NMR (500 MHz, CDCl3)
Fig. 6. The docked poses of compound 15c (C: yellow; N: blue; O: red) or gefitinib (C:
green; N: blue; O: red) at the ATP binding cleft of EGFR kinase. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
d
7.69 (s, 1H), 3.97 (t, J ¼ 7.5 Hz, 2H), 3.58 (t, J ¼ 6.5 Hz, 2H), 2.43 (s,
3H), 2.03e1.93 (m, 2H), 1.87e1.79 (m, 2H). ESI-MS m/z: 218
[MþH]þ.

W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
831
4.1.3. General synthetic procedure of 4-anilinoquinazoline
derivatives (14ae14d)
yield: 68%, mp: 180e182 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
9.51 (s,
1H), 8.48 (s, 1H), 8.09e8.11 (m, 1H), 7.81e7.74 (m, 2H), 7.71 (d,
J ¼ 0.5 Hz, 1H), 7.43 (t, J ¼ 9.0 Hz, 1H), 7.20e7.17 (m, 2H), 4.42 (t,
J ¼ 7.0 Hz, 2H), 4.12 (t, J ¼ 6.5 Hz, 2H), 3.91 (s, 3H), 1.92e1.81 (m,
Compound 12 [18] (10.0 mmol) and substituted aniline
(15.0 mmol) were mixed in isopropanol (10.0 mL) and refluxed for
1 h. After cooling to room temperature, the white solid was filtered
to get compounds 13ae13d without further purification. Com-
pounds 13ae13d was added to the mixture of methanol (15.0 mL)
and 25% ammonia (2.0 mL) and stirred for 3e4 h at room tem-
perature. The solid was filtered and washed with chilled methanol
and dried in vacuum to obtain 14ae14d.
4H),1.50e1.44 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 156.2,154.7,
152.8, 153.4 (d, JCeF ¼ 242.5 Hz), 152.8, 148.5, 147.2, 144.8 (d,
JCeF ¼ 2.7 Hz), 137.0 (d, JCeF ¼ 3.0 Hz), 128.1 (2), 123.7, 122.5 (d,
JCeF ¼ 6.9 Hz), 119.0 (d, JCeF ¼ 18.5 Hz), 116.7 (d, JCeF ¼ 21.5 Hz),
109.0, 107.5, 102.7, 68.8, 56.1, 49.5, 29.8, 28.3, 22.8. ESI-MS m/z: 501
[MþH]þ. HRMS (ESI): m/z calcd for (C23H22ClFN6O4
þ
H)þ:
501.1453; found: 501.1459.
4.1.4. General synthetic procedure of final compounds (15ae15r)
Nitroimidazole derivatives (9ae9e and 11ae11c, 0.18 mmol)
were added to a mixture of 4-anilinoquinazoline derivatives
(14ae14d, 0.15 mmol) and potassium carbonate (0.3 mmol) in DMF
(3.0 mL). The reaction mixture was heated to 80 ꢀC and stirred for
about 5 h. After cooling to room temperature, the mixture was
filtered and the filtrate was concentrated to dryness. H2O (15 mL)
was added to the residue and extracted with ethyl acetate
(15 mL ꢁ 3). The combined organic layer was washed with brine,
dried over anhydrous Na2SO4. The solvent was evaporated to dry-
ness and the residue was purified with silica gel chromatography to
give title compounds 15ae15r.
4.1.4.5. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((6-(2-nitro-1H-
imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (15e). Yellow solid,
yield: 66%, mp: 160e162 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.51 (s,
1H), 8.48 (s, 1H), 8.09e8.11 (m, 1H), 7.81e7.74 (m, 2H), 7.69 (d,
J ¼ 0.5 Hz, 1H), 7.43 (t, J ¼ 9.0 Hz, 1H), 7.18 (s, 1H), 7.16 (d, J ¼ 0.5 Hz,
1H), 4.38 (t, J ¼ 7.5 Hz, 2H), 4.11 (t, J ¼ 6.5 Hz, 2H), 3.92 (s, 3H),
1.87e1.77 (m, 4H), 1.53e1.47 (m, 2H), 1.41e1.34 (m, 2H). 13C NMR
(100 MHz, DMSO-d6)
d
156.0,153.2 (d, JCeF ¼ 242.7 Hz), 152.5,148.4,
146.8, 144.5, 136.8 (d, JCeF ¼ 3.1 Hz), 127.8, 127.8, 123.5, 122.4 (d,
JCeF ¼ 6.7 Hz), 118.8 (d, JCeF ¼ 18.3 Hz), 116.5 (d, JCeF ¼ 21.8 Hz),
108.7, 107.2, 102.4, 68.6, 55.9, 49.3, 29.7, 28.5, 25.6, 25.1. ESI-MS m/z:
515 [MþH]þ. HRMS (ESI): m/z calcd for (C24H24ClFN6O4 þ H)þ:
515.1610; found: 515.1612.
4.1.4.1. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(2-(2-nitro-1H-
imidazol-1-yl)ethoxy)quinazolin-4-amine (15a). White solid, yield:
43%, mp: 229e231 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
9.52 (s, 1H),
4.1.4.6. N-(3-bromophenyl)-7-methoxy-6-(3-(2-nitro-1H-imidazol-
8.50 (s, 1H), 8.08e8.13 (m, 1H), 7.82 (s, 1H), 7.80e7.73 (m, 1H), 7.71
(s, 1H), 7.44 (t, J ¼ 9.2 Hz, 1H), 7.22 (s, 1H), 7.18 (s, 1H), 4.95 (t,
J ¼ 5.2 Hz, 2H), 4.51 (t, J ¼ 5.2 Hz, 2H), 3.91 (s, 3H). 13C NMR
1-yl)propoxy)quinazolin-4-amine (15f). White solid, yield: 48%, mp:
111e113 ꢀC. 1H NMR (500 MHz, MeOD)
d 8.73 (s, 1H), 8.01 (t,
J ¼ 2.0 Hz, 1H), 7.99 (s, 1H), 7.72e7.69 (m, 1H), 7.53 (d, J ¼ 1.0 Hz,
1H), 7.50e7.48 (m, 1H), 7.40 (t, J ¼ 8.0 Hz, 1H), 7.22 (s, 1H), 7.13 (d,
J ¼ 1.0 Hz, 1H), 4.79 (t, J ¼ 6.5 Hz, 2H), 4.38e4.28 (m, 2H), 4.06 (s,
(100 MHz, DMSO-d6)
d
156.3, 154.6, 153.4 (d, JCeF ¼ 242.7 Hz), 152.1,
147.6, 147.5, 145.2, 136.9 (d, JCeF ¼ 2.7 Hz), 128.5, 127.9, 123.7, 122.6
(d, JCeF ¼ 6.8 Hz), 119.0 (d, JCeF ¼ 18.3 Hz), 116.8 (d, JCeF ¼ 21.5 Hz),
108.8, 107.7, 103.4, 67.6, 56.2, 48.6. ESI-MS m/z: 459 [MþH]þ. HRMS
(ESI): m/z calcd for (C20H16ClFN6O4 þ H)þ: 459.0984; found:
459.0986.
3H), 2.59e2.46 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 158.5,
156.8, 149.4, 148.8, 145.0, 138.8, 135.7, 130.8, 129.1, 128.1 (2), 127.5,
123.9, 121.3, 107.5, 105.4, 99.8, 67.3, 56.7, 47.1, 29.2. ESI-MS m/z: 501
[MþH]þ. HRMS (ESI): m/z calcd for (C21H19BrN6O4 þ H)þ: 501.0709;
found: 501.0715.
4.1.4.2. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(2-nitro-1H-
imidazol-1-yl)propoxy)quinazolin-4-amine
(15b). White
solid,
11.55
4.1.4.7. N-(3-ethynylphenyl)-7-methoxy-6-(3-(2-nitro-1H-imidazol-
yield: 62%, mp: 199e201 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
1-yl)propoxy)quinazolin-4-amine (15g). White solid, yield: 63%,
(s, 1H), 8.89 (s, 1H), 8.36 (s, 1H), 8.00e8.03 (m, 1H), 7.75e7.70 (m,
2H), 7.55 (t, J ¼ 9.0 Hz, 1H), 7.31 (s, 1H), 7.17 (d, J ¼ 1.0 Hz, 1H), 4.62
(t, J ¼ 6.5 Hz, 2H), 4.30 (t, J ¼ 6.0 Hz, 2H), 3.98 (s, 3H), 2.43e2.37 (m,
mp: 209e211 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.48 (s, 1H), 8.51
(s,1H), 7.98 (t, J ¼ 1.5 Hz,1H), 7.90e7.86 (m, 1H), 7.84 (s, 1H), 7.69 (d,
J ¼ 1.0 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 7.23e7.20 (m, 2H), 7.19 (d,
J ¼ 1.0 Hz, 1H), 4.63 (t, J ¼ 7.0 Hz, 2H), 4.29e4.15 (m, 3H), 3.94 (s,
2H). 13C NMR (100 MHz, DMSO-d6)
d 156.1, 154.6, 153.2 (d,
JCeF ¼ 242.8 Hz), 152.7, 147.9, 146.9, 144.7, 136.7 (d, JCeF ¼ 2.7 Hz),
127.9, 127.8, 123.5, 122.3 (d, JCeF ¼ 6.9 Hz), 118.8 (d, JCeF ¼ 18.3 Hz),
116.5 (d, JCeF ¼ 21.5 Hz), 108.6, 107.2, 102.9, 66.1, 55.9, 47.0, 29.1. ESI-
MS m/z: 473 [MþH]þ. HRMS (ESI): m/z calcd for
(C21H18ClFN6O4 þ H)þ: 473.1140; found: 473.1147.
3H), 2.45e2.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 158.2,
156.5, 149.1, 148.5, 144.7, 137.2, 135.3, 129.3, 129.1, 127.9, 127.8 (2),
125.5, 121.9, 107.3, 105.3, 99.4, 82.9, 81.3, 67.1, 56.4, 46.9, 29.0. ESI-
MS m/z: 445 [MþH]þ. HRMS (ESI): m/z calcd for
(C23H20N6O4 þ H)þ: 445.1624; found: 445.1633.
4.1.4.3. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(2-nitro-1H-
imidazol-1-yl)butoxy)quinazolin-4-amine (15c). White solid, yield:
51%, mp: 188e190 ꢀC. 1H NMR (500 MHz, DMSO-d6) 1H NMR
4.1.4.8. N-(3-ethynylphenyl)-7-methoxy-6-(4-(2-nitro-1H-imidazol-
1-yl)butoxy)quinazolin-4-amine (15h). White solid, yield: 62%, mp:
207e209 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 11.51 (s, 1H), 8.85 (s,
(500 MHz, DMSO-d6)
d
9.53 (s, 1H), 8.50 (s, 1H), 8.11e8.13 (m, 1H),
1H), 8.39 (s, 1H), 7.86 (s, 1H), 7.80e7.74 (m, 2H), 7.48 (t, J ¼ 8.0 Hz,
1H), 7.42e7.38 (m, 1H), 7.33 (s, 1H), 7.18 (d, J ¼ 0.5 Hz, 1H), 4.50 (t,
J ¼ 7.0 Hz, 2H), 4.32e4.23 (m, 3H), 3.97 (s, 3H), 2.03e1.94 (m, 2H),
7.85e7.73 (m, 3H), 7.45 (t, J ¼ 9.5 Hz, 1H), 7.21 (s, 2H), 4.52 (t,
J ¼ 7.0 Hz, 2H), 4.19 (t, J ¼ 6.0 Hz, 2H), 3.94 (s, 3H), 2.07e1.98 (m,
2H), 1.89e1.80 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d
158.2,
1.86e1.78 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 158.2, 156.5,
156.5, 155.0 (d, JCeF ¼ 243.3 Hz), 149.4, 148.44, 144.6, 135.1, 134.2 (d,
JCeF ¼ 2.8 Hz), 127.9, 127.8, 126.7, 125.4 (d, JCeF ¼ 7.3 Hz), 119.0 (d,
JCeF ¼ 18.6 Hz),116.7 (d, JCeF ¼ 22.0 Hz),107.3,105.2, 99.5, 69.3, 56.5,
49.1, 26.5, 25.1. ESI-MS m/z: 487 [MþH]þ. HRMS (ESI): m/z calcd for
(C22H20ClFN6O4 þ H)þ: 487.1297; found: 487.1297.
149.4, 148.6, 144.58, 137.3, 135.4, 129.3, 129.1, 128.0, 127.9, 127.7,
125.4, 122.0, 107.2, 105.0, 100.0, 82.9, 81.3, 69.2, 56.5, 49.1, 26.5,
25.2. ESI-MS m/z: 459 [MþH]þ. HRMS (ESI): m/z calcd for
(C24H22N6O4 þ H)þ: 459.1781; found: 459.1784.
4.1.4.9. N-(3-ethynylphenyl)-7-methoxy-6-((5-(2-nitro-1H-imida-
4.1.4.4. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-((5-(2-nitro-1H-
imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (15d). Yellow solid,
zol-1-yl)pentyl)oxy)quinazolin-4-amine (15i). White solid, yield:
64%, mp: 197e199 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 11.29 (s, 1H),

832
W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
8.84 (s, 1H), 8.26 (s, 1H), 7.84 (t, J ¼ 2.0 Hz, 1H), 7.77e7.72 (m, 1H),
7.72 (d, J ¼ 1.0 Hz,1H), 7.49 (t, J ¼ 8.0 Hz,1H), 7.42e7.38 (m,1H), 7.31
(s, 1H), 7.17 (d, J ¼ 1.0 Hz, 1H), 4.42 (t, J ¼ 7.5 Hz, 2H), 4.28 (s, 1H),
4.21 (t, J ¼ 6.5 Hz, 2H), 3.98 (s, 3H), 1.91e1.82 (m, 4H), 1.51e1.41 (m,
JCeF ¼ 21.1 Hz), 114.3, 114.0 (d, JCeF ¼ 21.8 Hz), 108.7, 107.2, 102.5,
69.4 (d, JCeF ¼ 1.1 Hz), 68.5, 55.8, 49.3, 29.5, 28.1, 22.5. ESI-MS m/z:
607 [MþH]þ. HRMS (ESI): m/z calcd for (C30H28ClFN6O5 þ H)þ:
607.1872; found: 607.1879.
2H). 13C NMR (100 MHz, DMSO-d6)
d 157.8, 156.1, 152.2, 149.3, 144.5,
138.9,137.7, 129.1, 128.8, 127.9, 127.8,127.2, 124.9,122.0,107.6,104.3,
101.1, 83.0, 81.2, 69.2, 56.4, 49.3, 29.4, 27.9, 22.4. ESI-MS m/z: 473
[MþH]þ. HRMS (ESI): m/z calcd for (C25H24N6O4 þ H)þ: 473.1937;
found: 473.1947.
4.1.4.14. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-methoxy-6-
((6-(2-nitro-1H-imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (15n).
White solid, yield: 51%, mp: 123e125 ꢀC. 1H NMR (400 MHz, DMSO-
d6)
d
9.41 (s, 1H), 8.45 (s, 1H), 7.94 (d, J ¼ 2.8 Hz, 1H), 7.78 (s, 1H),
7.73e7.66 (m, 2H), 7.50e7.45 (m, 1H), 7.36e7.29 (m, 2H), 7.27 (d,
J ¼ 9.2 Hz, 1H), 7.22e7.14 (m, 3H), 5.25 (s, 2H), 4.40 (t, J ¼ 7.2 Hz,
2H), 4.12 (t, J ¼ 6.4 Hz, 2H), 3.93 (s, 3H),1.90e1.78 (m, 4H),1.56e1.48
4.1.4.10. N-(3-ethynylphenyl)-7-methoxy-6-((6-(2-nitro-1H-imida-
zol-1-yl)hexyl)oxy)quinazolin-4-amine (15j). White solid, yield:
57%, mp: 174e176 ꢀC. 1H NMR (400 MHz, DMSO-d6)
d
9.46 (s, 1H),
(m, 2H), 1.44e1.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 162.2
8.50 (s, 1H), 7.99 (t, J ¼ 1.6 Hz, 1H), 7.92e7.87 (m, 1H), 7.82 (s, 1H),
7.70 (d, J ¼ 0.8 Hz,1H), 7.40 (t, J ¼ 8.0 Hz,1H), 7.23e7.19 (m, 2H), 7.18
(d, J ¼ 0.8 Hz, 1H), 4.40 (t, J ¼ 7.2 Hz, 2H), 4.20 (s, 1H), 4.14 (t,
J ¼ 6.4 Hz, 2H), 3.94 (s, 3H), 1.89e1.79 (m, 4H), 1.58e1.47 (m, 2H),
(d, JCeF ¼ 243.6 Hz), 156.2, 154.3, 152.8, 149.4, 148.2, 146.7, 144.5,
139.7 (d, JCeF ¼ 7.4 Hz), 133.5, 130.5 (d, JCeF ¼ 8.1 Hz), 127.8 (2),
124.0, 123.3 (d, JCeF ¼ 2.7 Hz), 122.2, 121.0, 114.7 (d, JCeF ¼ 20.9 Hz),
114.3, 114.0 (d, JCeF ¼ 21.8 Hz), 108.7, 107.2, 102.4, 69.4, 68.6, 55.8,
49.3, 29.7, 28.5, 25.6, 25.1. ESI-MS m/z: 621 [MþH]þ. HRMS (ESI): m/
z calcd for (C31H30ClFN6O5 þ H)þ: 621.2028; found: 621.2029.
1.45e1.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 156.3, 154.6,
152.9, 148.5, 147.1, 144.7, 140.0, 129.1, 128.0 (2), 126.5, 125.0, 122.8,
121.9, 109.1, 107.5, 102.6, 83.7, 80.8, 68.8, 56.0, 49.5, 29.9, 28.7, 25.8,
25.3. ESI-MS m/z: 487 [MþH]þ. HRMS (ESI): m/z calcd for
(C26H26N6O4 þ H)þ: 487.2094; found: 487.2103.
4.1.4.15. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(2-(2-methyl-5-
nitro-1H-imidazol-1-yl)ethoxy)quinazolin-4-amine
White solid, yield: 41%, mp: 244e246 ꢀC. 1H NMR (500 MHz,
DMSO-d6) 9.54 (s, 1H), 8.49 (s, 1H), 8.40 (s, 1H), 8.12e8.05 (m, 1H),
(15o).
4.1.4.11. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-methoxy-6-
(3-(2-nitro-1H-imidazol-1-yl)propoxy) quinazolin-4-amine (15k).
White solid, yield: 43%, mp: 168e170 ꢀC. 1H NMR (500 MHz, DMSO-
d
7.82 (s, 1H), 7.75 (d, J ¼ 6.5 Hz, 1H), 7.44 (t, J ¼ 9.0 Hz, 1H), 7.20 (s,
1H), 4.53 (t, J ¼ 4.5 Hz, 2H), 4.44 (t, J ¼ 4.5 Hz, 2H), 3.92 (s, 3H). 13
C
d6)
d
9.40 (s, 1H), 8.46 (s, 1H), 7.94 (d, J ¼ 2.5 Hz, 1H), 7.80 (s, 1H),
NMR (100 MHz, DMSO-d6)
d
156.1, 154.2, 153.2 (d, JCeF ¼ 242.8 Hz),
7.72e7.64 (m, 2H), 7.52e7.43 (m, 1H), 7.36e7.30 (m, 2H), 7.27 (d,
J ¼ 9.0 Hz, 1H), 7.22e7.15 (m, 3H), 5.25 (s, 2H), 4.63 (t, J ¼ 7.0 Hz,
2H), 4.19 (t, J ¼ 5.5 Hz, 2H), 3.93 (s, 3H), 2.44e2.32 (m, 2H). 13C NMR
152.9, 152.0, 147.3 (2), 146.0, 145.4, 136.7 (d, JCeF ¼ 3.0 Hz), 123.4,
122.5, 122.2 (d, JCeF ¼ 6.7 Hz), 118.8 (d, JCeF ¼ 18.3 Hz), 116.6 (d,
JCeF ¼ 21.7 Hz), 108.6, 107.5, 103.1, 67.8, 56.0, 45.8, 12.6. ESI-MS m/z:
473 [MþH]þ. HRMS (ESI): m/z calcd for (C21H18ClFN6O4 þ H)þ:
473.1140; found: 473.1144.
(100 MHz, DMSO-d6)
d
162.18 (d, JCeF ¼ 243.6 Hz), 156.2, 154.4,
152.9, 149.4, 147.8, 147.0, 144.7, 139.7 (d, JCeF ¼ 7.4 Hz), 133.5, 130.5
(d, JCeF ¼ 8.2 Hz), 127.9, 127.8, 123.9, 123.3 (d, JCeF ¼ 2.8 Hz), 122.1,
121.0, 114.7 (d, JCeF ¼ 21.0 Hz), 114.3, 114.0 (d, JCeF ¼ 21.9 Hz), 108.6,
107.3, 102.9, 69.4 (d, JCeF ¼ 1.0 Hz), 66.0, 55.8, 47.0, 29.1. ESI-MS m/z:
579 [MþH]þ. HRMS (ESI): m/z calcd for (C28H24ClFN6O5 þ H)þ:
579.1559; found: 579.1565.
4.1.4.16. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-(2-methyl-5-
nitro-1H-imidazol-1-yl)propoxy)-quinazolin-4-amine
White solid, yield: 44%, mp: 207e209 ꢀC. 1H NMR (500 MHz,
DMSO-d6) 9.56 (s, 1H), 8.50 (s, 1H), 8.41 (s, 1H), 8.12e8.08 (m, 1H),
(15p).
d
7.80 (s, 1H), 7.79e7.71 (m, 1H), 7.44 (t, J ¼ 9.0 Hz, 1H), 7.21 (s, 1H),
4.1.4.12. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-methoxy-6-
4.21 (t, J ¼ 7.0 Hz, 2H), 4.14 (t, J ¼ 5.5 Hz, 2H), 3.92 (s, 3H), 2.38e2.31
(4-(2-nitro-1H-imidazol-1-yl)butoxy)quinazolin-4-amine
White solid, yield: 45%, mp: 146e148 ꢀC. 1H NMR (400 MHz, DMSO-
d6)
(15l).
(m, 5H). 13C NMR (100 MHz, DMSO-d6)
d 156.1, 154.5, 153.2 (d,
JCeF
¼
242.9 Hz), 152.7, 147.8, 146.8, 145.5, 145.1, 136.7 (d,
d
9.41 (s, 1H), 8.45 (s, 1H), 7.94 (d, J ¼ 2.8 Hz, 1H), 7.80 (s, 1H),
JCeF ¼ 2.5 Hz), 123.5, 122.3 (d, JCeF ¼ 6.8 Hz), 122.0, 118.8 (d,
JCeF ¼ 18.4 Hz), 116.5 (d, JCeF ¼ 21.6 Hz), 108.6, 107.2, 102.9, 65.6,
55.9, 43.6, 28.9, 12.4. ESI-MS m/z: 487 [MþH]þ. HRMS (ESI): m/z
calcd for (C22H20ClFN6O4 þ H)þ: 487.1297; found: 487.1303.
7.77 (d, J ¼ 1.2 Hz, 1H), 7.72e7.66 (m, 1H), 7.51e7.44 (m, 1H),
7.36e7.29 (m, 2H), 7.27 (d, J ¼ 9.2 Hz, 1H), 7.21 (d, J ¼ 1.2 Hz, 1H),
7.20e7.15 (m, 2H), 5.25 (s, 2H), 4.52 (t, J ¼ 6.8 Hz, 2H), 4.18 (t,
J ¼ 6.0 Hz, 2H), 3.93 (s, 3H), 2.08e1.95 (m, 2H), 1.89e1.78 (m, 2H).
13C NMR (100 MHz, DMSO-d6)
d
162.2 (d, JCeF ¼ 243.6 Hz), 156.2,
4.1.4.17. N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(4-(2-methyl-5-
154.4, 152.8, 149.4, 148.0, 146.8, 144.6, 139.7 (d, JCeF ¼ 7.7 Hz), 133.5,
130.5 (d, JCeF ¼ 8.2 Hz), 127.9, 127.8 (d, JCeF ¼ 4.1 Hz), 124.0, 123.3 (d,
JCeF ¼ 2.6 Hz), 122.1, 121.0, 114.7 (d, JCeF ¼ 20.7 Hz), 114.3, 114.0 (d,
JCeF ¼ 21.9 Hz), 108.7, 107.3, 102.7, 69.4, 68.5, 55.8, 49.2, 26.7, 25.4.
ESI-MS m/z: 593 [MþH]þ. HRMS (ESI): m/z calcd for
(C29H26ClFN6O5 þ H)þ: 593.1715; found: 593.1716.
nitro-1H-imidazol-1-yl)-butoxy)-quinazolin-4-amine
White solid, yield: 48%, mp: 158e160 ꢀC. 1H NMR (500 MHz,
DMSO-d6) 9.66 (s, 1H), 8.51 (s, 1H), 8.40 (s, 1H), 8.12e8.08 (m, 1H),
(15q).
d
7.84 (s, 1H), 7.80e7.76 (m, 1H), 7.44 (t, J ¼ 9.0 Hz, 1H), 7.20 (s, 1H),
4.19 (t, J ¼ 6.0 Hz, 2H), 4.12 (t, J ¼ 7.0 Hz, 2H), 3.94 (s, 3H), 2.38 (s,
3H), 2.01e1.91 (m, 2H), 1.87e1.75 (m, 2H). 13C NMR (100 MHz,
DMSO-d6)
145.3, 145.2, 144.9, 136.4 (d, JCeF ¼ 2.8 Hz), 123.9, 122.7 (d,
JCeF 6.8 Hz), 122.1, 118.8 (d, JCeF 18.4 Hz), 116.5 (d,
d
156.3, 154.8, 153.4 (d, JCeF ¼ 243.3 Hz), 152.0, 148.3,
4.1.4.13. N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-methoxy-6-
((5-(2-nitro-1H-imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (15m).
White solid, yield: 48%, mp: 134e136 ꢀC. 1H NMR (400 MHz, DMSO-
¼
¼
JCeF ¼ 21.6 Hz), 108.5, 106.1, 103.0, 68.6, 56.0, 46.1, 26.4, 25.2, 12.6.
ESI-MS m/z: 501 [MþH]þ. HRMS (ESI): m/z calcd for
(C23H22ClFN6O4 þ H)þ: 501.1453; found: 501.1456.
d6)
d
9.41 (s, 1H), 8.44 (s, 1H), 7.94 (d, J ¼ 2.8 Hz, 1H), 7.78 (s, 1H),
7.72 (d, J ¼ 0.8 Hz, 1H), 7.70e7.67 (m, 1H), 7.51e7.44 (m, 1H),
7.36e7.30 (m, 2H), 7.27 (d, J ¼ 9.2 Hz,1H), 7.23e7.14 (m, 2H), 5.25 (s,
2H), 4.44 (t, J ¼ 7.2 Hz, 2H), 4.13 (t, J ¼ 6.4 Hz, 2H), 3.92 (s, 3H),
1.95e1.83 (m, 4H),1.55e1.44 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
4.1.4.18. N-(3-bromophenyl)-7-methoxy-6-(3-(2-methyl-5-nitro-1H-
imidazol-1-yl)propoxy)quinazolin-4-amine (15r). White solid, yield:
d
162.2 (d, JCeF ¼ 243.6 Hz), 156.2, 154.3, 152.8, 149.4, 148.2, 146.8,
49%, mp: 94e96 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d 9.47 (s, 1H),
144.5, 139.7 (d, JCeF ¼ 7.4 Hz), 133.5, 130.5 (d, JCeF ¼ 8.2 Hz), 127.9,
127.8, 124.0, 123.3 (d, JCeF ¼ 2.7 Hz, 1H), 122.2, 121.0, 114.7 (d,
8.51 (s, 1H), 8.40 (s, 1H), 8.12 (s, 1H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.80 (s,
1H), 7.34 (t, J ¼ 8.0 Hz, 1H), 7.26 (d, J ¼ 8.0 Hz, 1H), 7.21 (s, 1H), 4.21

W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
833
(t, J ¼ 6.5 Hz, 2H), 4.15 (t, J ¼ 5.5 Hz, 2H), 3.92 (s, 3H), 2.37e2.32 (m,
5H). 13C NMR (100 MHz, DMSO-d6)
155.9, 154.5, 152.8, 147.8, 147.2,
145.5, 145.1, 141.2, 130.4, 125.6, 124.0, 122.0, 121.2, 120.5, 108.8,
107.4, 102.9, 65.6, 55.9, 43.6, 28.9, 12.4. ESI-MS m/z: 515 [MþH]þ.
HRMS (ESI): m/z calcd for (C22H21BrN6O4 þ H)þ: 515.0865; found:
515.0871.
metabolism reaction was initiated by the addition of 5
m
M test
d
compound to the incubation mixtures. At 0 and 30 min, the reac-
tion mixture was quenched with equal volume of acetonitrile
containing 0.1% formic acid. The samples were centrifuged for
10 min at 10,000 g. The supernatant was transferred to a vial for
HPLC analysis.
4.2. Pharmacology
4.5. Molecular modeling
Human colorectal adenocarcinoma HT-29 cells were purchased
from Cell Bank of China Science Academy (Shanghai, China). The
above cells were cultured in RPMI-1640 (Invitrogen Corp., Carlsbad,
CA) medium with heat-inactivated 10% fetal bovine serum, peni-
cillin (100 units/mL) and streptomycin (100 mg/mL) and incubated
in hypoxic atmosphere with 2% O2, 5% CO2 and in normoxic at-
mosphere with 20% O2, 5% CO2 at 37 ꢀC.
The EGFR-gefitinib proteineligand complex crystal structure
(PDB ID: 2ITY) was chosen as the template to study the docking
mode of compound 15c with EGFR. The molecular docking proce-
dure was performed by using C-DOCKER protocol within Discovery
Studio 2.1. For ligand preparation, the 3D structure of compound
15c was generated and minimized. For enzyme preparation, the
hydrogen atoms were added, and the CHARMm force field was
employed. The whole EGFR enzyme was defined as a receptor and
the site sphere was selected based on the ligand binding location of
gefitinib, then gefitinib was removed and compound 15c was
placed during the molecular docking procedure. Types of in-
teractions of the docked enzyme with ligand were analyzed after
end of molecular docking. Ten docking poses were saved for each
ligand and the final docked conformation was scored and selected
based on calculated C-DOCKER-energy.
4.2.1. In vitro enzymatic activity assay
Wild type EGFR and the Z0-Lyte Kinase Kit were purchased from
Invitrogen. Ten concentration gradients were set for all the tested
compounds. The experiments were performed according to the
instructions of the manufacturer.
4.2.2. Cytotoxicity assay
The cytotoxic activity in vitro was measured using sulfo-
rhodamin B (SRB) assay. All the compounds were dissolved in
DMSO at the concentrations 10.0 mg/mL and were then diluted to
the appropriate concentrations. Cells were plated in 96-well plates
(5 ꢁ 103 per well) for 24 h and subsequently treated with different
concentrations of all tested compounds under normoxia or hypoxia
for 72 h, respectively. Cells were then washed with PBS and fixed
with 10% (w/v) trichloroacetic acid at 4 ꢀC for one hour. After
washing, the cells were stained for 30 min with 0.4% SRB dissolved
in 1% acetic acid. Then the cells were washed by 1% acetic acid for 5
times, and protein-bound dye was extracted with 10 mmol un-
buffered Tris base. The absorbance was measured at 515 nm using a
multiscan spectrum (Thermo Electron Co., Vantaa, Finland). The
inhibition rate on cell proliferation of each well was calculated as
(A515 control cells A515 treated cells)/A515 control cells ꢁ 100%,
and the IC50 values were determined by Logit method.
Acknowledgments
We thank the support of the National Natural Science Founda-
tion of China (81072575). We thank Dr. Ke Ding (Guangzhou In-
stitutes of Biomedicine and Health, Chinese Academy of Sciences)
for performing the EGFR activity evaluation. We also thank Mrs
Jianyang Pan (Pharmaceutical Informatics Institute, Zhejiang Uni-
versity) for performing the NMR and MS spectrometry tests and
Mrs Xiaodan Wu (Analysis Center of Agrobiology and Environ-
mental Sciences, Zhejiang University) for performing the HPLC-Q-
ToF-MS test.
References
[1] N.E. Hynes, H.A. Lane, ERBB receptors and cancer: the complexity of targeted
inhibitors, Nat. Rev. Cancer 5 (2005) 341e354.
[2] R.L. Rego, N.R. Foster, T.C. Smyrk, M. Le, M.J. O'Connell, D.J. Sargent,
H. Windschitl, F.A. Sinicrope, Prognostic effect of activated EGFR expression in
human colon carcinomas: comparison with EGFR status, Br. J. Cancer 102
(2010) 165e172.
[3] C. Cao, S. Lu, A. Sowa, R. Kivlin, A. Amaral, W.M. Chu, H. Yang, W. Di, Y.S. Wan,
Priming with EGFR tyrosine kinase inhibitor and EGF sensitizes ovarian cancer
cells to respond to chemotherapeutical drugs, Cancer Lett. 266 (2008)
249e262.
[4] T. Mitsudomi, Y. Yatabe, Epidermal growth factor receptor in relation to tumor
development: EGFR gene and cancer, Febs J. 277 (2010) 301e308.
[5] R. Patel, H.Y. Leung, Targeting the EGFR-family for therapy: biological chal-
lenges and clinical perspective, Curr. Pharm. Des. 18 (2012) 2672e2679.
[6] W.R. Wilson, M.P. Hay, Targeting hypoxia in cancer therapy, Nat. Rev. Cancer
11 (2011) 393e410.
[7] T. Wang, T. Niki, A. Goto, S. Ota, T. Morikawa, Y. Nakamura, E. Ohara,
S. Ishikawa, H. Aburatani, J. Nakajima, M. Fukayama, Hypoxia increases the
motility of lung adenocarcinoma cell line A549 via activation of the epidermal
growth factor receptor pathway, Cancer Sci. 98 (2007) 506e511.
[8] H. Nishi, K.H. Nishi, A.C. Johnson, Early growth response-1 gene mediates up-
regulation of epidermal growth factor receptor expression during hypoxia,
Cancer Res. 62 (2002) 827e834.
[9] G.C. Lancini, E. Lazzari, V. Arioli, P. Bellani, Synthesis relationship between
structure and activity of 2-nitroimidazole derivatives, J. Med. Chem. 12 (1969)
775e780.
[10] M.B. Noss, R. Panicucci, R.A. McClelland, A.M. Rauth, 1-Methyl-2-
nitrosoimidazole e cytotoxic and glutathione depleting capabilities, Int. J.
Radiat. Oncol. 16 (1989) 1015e1019.
[11] L. Hoigebazar, J.M. Jeong, J.Y. Lee, D. Shetty, B.Y. Yang, Y.S. Lee, D.S. Lee,
J.K. Chung, M.C. Lee, Syntheses of 2-nitroimidazole derivatives conjugated
with 1,4,7-triazacyclononane-N,N0-diacetic acid labeled with F-18 using an
aluminum complex method for hypoxia imaging, J. Med. Chem. 55 (2012)
3155e3162.
4.3. Mimic the reductive activation of compound 15c
Compound 15c (2.0 mg, 4.1
41.1 mol) was firstly dissolved in 0.5 mL dimethyl sulfoxide and
then added to 3 mL phosphate buffer saline (pH ¼ 7.4, 50 M), then
Zn dust (6.7 mg, 102.7 mol) and ammonium chloride (11.0 mg,
205.4 mol) were added to the reaction mixture. The reaction flask
mmol) and cysteine (5.0 mg,
m
m
m
m
was evacuated and backfilled with nitrogen three times. The reac-
tion mixture was stirred at 37 ꢀC under a nitrogen balloon for 12 h
and then filtered through 0.45
the filtrate was analyzed through HPLC-Q-ToF-MS (AB Sciex Triple
TOF 5600þ). The column was Eclipse Plus C18 (5 m,
4.6 250 mm), the mobile phase was MeOH:H2O (0.1%
mm microfiltration membrane and
m
ꢁ
HCOOH þ 5 mM AcONH4) ¼ 44:56 at the flow rate of 1.0 mL/min,
and the detected wavelength was 330 nm, with column tempera-
ture at 30 ꢀC.
4.4. Metabolic stability assay [21,32]
The assay mixtures contained the following substance with the
indicated final concentrations: 50 mM potassium phosphate buffer
(pH 7.4), 5 mM MgCl2, microsomes (1 mg/mL) with or without
2 mM NADPH. The mixtures were preincubated at 37 ꢀC both under
normoxia (20% O2, 5% CO2) and hypoxia (2% O2, 5% CO2). The

834
W. Cheng et al. / European Journal of Medicinal Chemistry 89 (2015) 826e834
[12] P. Kumar, G. Shustov, H. Liang, V. Khlebnikov, W.Z. Zheng, X.H. Yang,
C. Cheeseman, L.I. Wiebe, Design, synthesis, and preliminary biological eval-
uation of 6-O-glucose-azomycin adducts for diagnosis and therapy of hypoxic
tumors, J. Med. Chem. 55 (2012) 6033e6046.
[13] J. van Loon, M.H.M. Janssen, M. Ollers, H. Aerts, L. Dubois, M. Hochstenbag,
A.M.C. Dingemans, R. Lalisang, B. Brans, B. Windhorst, G.A. van Dongen,
H. Kolb, J. Zhang, D. De Ruysscher, P. Lambin, PET imaging of hypoxia using F-
18 HX4: a phase I trial, Eur. J. Nucl. Med. Mol. Imaging 37 (2010) 1663e1668.
[14] C.H. Yun, T.J. Boggon, Y.Q. Li, M.S. Woo, H. Greulich, M. Meyerson, M.J. Eck,
Structures of lung cancer-derived EGFR mutants and inhibitor complexes:
mechanism of activation and insights into differential inhibitor sensitivity,
Cancer Cell 11 (2007) 217e227.
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) 2412e2420.
[22] M.P. Hay, F.B. Pruijn, S.A. Gamage, H.D.S. Liyanage, M.S. Kovacs, A.V. Patterson,
W.R. Wilson, J.M. Brown, W.A. Denny, DNA-targeted 1,2,4-benzotriazine 1,4-
dioxides: potent analogues of the hypoxia-selective cytotoxin tirapazamine,
J. Med. Chem. 47 (2004) 475e488.
[23] S. Maya, B. Sarmento, V.K. Lakshmanan, D. Menon, R. Jayakumar, Actively
targeted cetuximab conjugated gamma-poly(glutamic acid)-docetaxel nano-
medicines for epidermal growth factor receptor over expressing colon cancer
cells, J. Biomed. Nanotechnol. 10 (2014) 1416e1428.
[24] M.V. Papadopoulou, W.D. Bloomer, Exploiting hypoxia in solid tumors with
DNA-targeted bioreductive drugs, Drug. Future 29 (2004) 807e819.
[25] A.J. Varghese, G.F. Whitmore, Misonidazole-glutathione conjugates in CHO
cells, Int. J. Radiat. Oncol. 10 (1984) 1341e1345.
[15] J. Stamos, M.X. Sliwkowski, C. Eigenbrot, Structure of the epidermal growth
factor receptor kinase domain alone and in complex with
a 4-
anilinoquinazoline inhibitor, J. Biol. Chem. 277 (2002) 46265e46272.
[16] E.R. Wood, A.T. Truesdale, O.B. McDonald, D. Yuan, A. Hassell, S.H. Dickerson,
B. Ellis, C. Pennisi, E. Horne, K. Lackey, K.J. Alligood, D.W. Rusnak, T.M. Gilmer,
L. Shewchuk, A unique structure for epidermal growth factor receptor bound
to GW572016 (Lapatinib): relationships among protein conformation, inhib-
itor off-rate, and receptor activity in tumor cells, Cancer Res. 64 (2004)
6652e6659.
[17] F. Solca, G. Dahl, A. Zoephel, G. Bader, M. Sanderson, C. Klein, O. Kraemer,
F. Himmelsbach, E. Haaksma, G.R. Adolf, Target binding properties and cellular
activity of afatinib (BIBW 2992), an irreversible erbb family blocker,
J. Pharmacol. Exp. Ther. 343 (2012) 342e350.
[18] V. Chandregowda, A.K. Kush, G.C. Reddy, Synthesis and in vitro antitumor
activities of novel 4-anilinoquinazoline derivatives, Eur. J. Med. Chem. 44
(2009) 3046e3055.
[19] S.H. Chang, L.W. Zhang, S.L. Xu, J.F. Luo, X.Y. Lu, Z. Zhang, T.F. Xu, Y.X. Liu,
Z.C. Tu, Y. Xu, X.M. Ren, M.Y. Geng, J. Ding, D.Q. Pei, K. Ding, Design, synthesis,
and biological evaluation of novel conformationally constrained inhibitors
targeting epidermal growth factor receptor threonine(790)/methio-
nine(790) mutant, J. Med. Chem. 55 (2012) 2711e2723.
[26] H. Nagasawa, Y. Uto, K.L. Kirk, H. Hori, Design of hypoxia-targeting drugs as
new cancer chemotherapeutics, Biol. Pharm. Bull. 29 (2006) 2335e2342.
[27] A.J. Varghese, G.F. Whitmore, Properties of 2-hydroxylaminoimidazoles and
their implications for the biological effects of 2-nitroimidazoles, Chem. Biol.
Interact. 56 (1985) 269e287.
[28] C. Carmi, E. Galvani, F. Vacondio, S. Rivara, A. Lodola, S. Russo, S. Aiello,
F. Bordi, G. Costantino, A. Cavazzoni, R.R. Alfieri, A. Ardizzoni, P.G. Petronini,
M. Mor, Irreversible inhibition of epidermal growth factor receptor activity by
3-aminopropanamides, J. Med. Chem. 55 (2012) 2251e2264.
[29] V.S. Gopinath, J. Pinjari, R.T. Dere, A. Verma, P. Vishwakarma, R. Shivahare,
M. Moger, P.S.K. Goud, V. Ramanathan, P. Bose, M.V.S. Rao, S. Gupta, S.K. Puri,
D. Launay, D. Martin, Design, synthesis and biological evaluation of 2-
substituted quinolines as potential antileishmanial agents, Eur. J. Med. Chem.
69 (2013) 527e536.
[30] L. Kumar, A. Sarswat, N. Lal, V.L. Sharma, A. Jain, R. Kumar, V. Verma,
J.P. Maikhuri, A. Kumar, P.K. Shukla, G. Gupta, Imidazole derivatives as
possible microbicides with dual protection, Eur. J. Med. Chem. 45 (2010)
817e824.
[20] R.F. Borch, J.W. Liu, J.P. Schmidt, J.T. Marakovits, C. Joswig, J.J. Gipp,
R.T. Mulcahy, Synthesis and evaluation of nitroheterocyclic phosphoramidates
as hypoxia-selective alkylating agents, J. Med. Chem. 43 (2000) 2258e2265.
[21] J.X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J.W. Evans, L. Lan, G. Lorente,
M. Banica, D. Jung, J.W. Wang, H.Y. Ma, X.M. Li, Z.J. Yang, R.M. Hoffman,
[31] P. Kowalski, K. Nowak, B. Szpakiewicz, Synthesis and prediction of bioactivity
of new nitroimidazole derivatives, J. Heterocycl. Chem. 42 (2005) 883e888.
[32] M.V. Papadopoulou, M. Ji, M.K. Rao, W.D. Bloomer, Reductive metabolism of
the nitroimidazole-based hypoxia-selective cytotoxin NLCQ-1 (NSC 709257),
Oncol. Res. 14 (2003) 21e29.