Identification of novel 4-anilinoquinazoline derivatives as potent EGFR inhibitors both under normoxia and hypoxia

Article abstract of DOI:10.1016/j.bmc.2014.10.038

A novel series of 4-anilinoquinazoline derivatives (19a-19t) were designed and synthesized through incorporation of the 2-nitroimidazole moiety into the 4-anilinoquinazoline scaffold of EGFR inhibitors. The most promising compound 19h displayed potent EGF

Full text of DOI:10.1016/j.bmc.2014.10.038

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of novel 4-anilinoquinazoline derivatives as potent
EGFR inhibitors both under normoxia and hypoxia
⇑
⇑
Weiyan Cheng, Youting Yuan, 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 novel series of 4-anilinoquinazoline derivatives (19a–19t) were designed and synthesized through
incorporation of the 2-nitroimidazole moiety into the 4-anilinoquinazoline scaffold of EGFR inhibitors.
The most promising compound 19h displayed potent EGFR inhibitory activity with the IC50 value of
Received 5 September 2014
Revised 27 October 2014
Accepted 27 October 2014
Available online xxxx
0.47 nM. It also strongly suppressed the proliferation of A549 and HT-29 cells with sub-micromolar
IC50 values both under normoxia and hypoxia, which were several folds more potent than gefitinib
and erlotinib. Further reductive mimic investigation revealed that 19h could be reductive activated under
hypoxia and was fully consistent with the results of cell apoptotic assay and in vitro metabolism evalu-
ation. Our results suggest that the incorporation of hypoxia-activated moiety into EGFR inhibitor scaffold
might be a tractable strategy to overcome the tumor hypoxia.
Keywords:
Hypoxia
2
-Nitroimidazole
EGFR
-Anilinoquinazoline
Ó 2014 Published by Elsevier Ltd.
4
1
. Introduction
Hypoxia is a common characteristic of solid tumors resulting
clinical trials by alone or in combination with doxorubicin,
pemetrexed, gemcitabine, and so on, for the treatment of various
tumors, including advanced renal cell carcinoma, gastrointestinal
stromal tumors, pancreatic adenocarcinoma and hepatocellular
from the insufficient blood supply which fails to meet the rapid
tumor growth. Hypoxic tumor cells confer resistance to radiother-
1
5
carcinoma.
apy and chemotherapy and present a tremendous challenge to
cancer therapy. On the other hand, this specific microenvironment
of tumor cells distinguished it from physiologically normal cells
The epidermal growth factor receptor (EGFR) plays a vital role
in mediation of growth factor signaling and EGFR overexpression
6
7
is detected in many solid tumors, including non-small cell lung,
1
8
9
10
and made it as an attractive and exploitable therapeutic target.
head and neck, ovarian, and colon tumors. Up to date, several
EGFR inhibitors (gefitinib, erlotinib, lapatinib, vandetanib, and afat-
inib) (Fig. 1) have been approved by FDA for the treatment of non-
small cell lung cancer (NSCLC), pancreatic cancer and advanced
Five classes of bioreductive agents (quinones, nitroaromatics,
aliphatic N-oxides, aromatic di-N-oxides and transition metal
complexes) have been developed as potential antitumor agents
in the past few decades.2 These compounds are hypoxia-selective
prodrugs which undergo enzymatic reduction in hypoxia and
generate toxic metabolites causing cell death.
11
breast cancer. Recent study reveals that the expression of EGFR
is up-regulated in hypoxic tumors and intimately related to poor
1
2,13
prognosis of patients.
With the inspiration from the design of
Among them, the 2-nitroimidazole derivatives have attracted
extensive attention due to their remarkable hypoxia-activated
TH-302, we envisioned that incorporation of hypoxic selective moi-
ety into the structure of EGFR inhibitor may exert better efficacy
against solid tumor due to its specific hypoxia-direct properties.
The SARs of EGFR inhibitors revealed that the 4-anilinoquinaz-
oline scaffold is crucial to EGFR inhibitory activity, and the C-6 and
3
18
properties in tumor cell. Especially, the [ F]MISO (Fig. 1) has been
advanced into phase II clinical trial as noninvasive markers of
hypoxia via PET imaging (NCT01868906). The 2-nitroimidazole
1
8
moiety of [ F]MISO can be reduced to 2-amino-imidazole radical
by reductive enzymes within hypoxic environment, followed by
covalent binding to the cellular nucleophiles and accumulating
C-7 side chains are mainly contribute to their physicochemical
properties with good compatibility for bulky moieties.1
4–17
There-
fore, a novel series of EGFR inhibitors were designed through
incorporation of 2-nitroimidazole moiety into the C-7 position of
4-anilinoquinazoline scaffold. To explore the extensive SARs of this
series of compounds, aniline moieties from gefitinib, erlotinib,
lapatinib and other EGFR inhibitor were respectively employed to
the C-4 position of the quinazoline template, as well as different
linkers between 2-nitroimidazole and quinazoline were investigated.
3
therein. Combination of 2-nitroimidazoles with cytotoxic moiety
phosphoramide mustard yielded novel antitumor drug candidate
4
TH-302 (Fig. 1). TH-302 has been evaluated in more than 15
⇑
Corresponding authors. Tel./fax: +86 571 88208460.
E-mail addresses: shengr@zju.edu.cn (R. Sheng), huyz@zju.edu.cn (Y. Hu).
http://dx.doi.org/10.1016/j.bmc.2014.10.038
968-0896/Ó 2014 Published by Elsevier Ltd.
0
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2
W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 1. Structures of selected 2-nitroimidazole derivatives and approved EGFR inhibitors.
2
2
. Results and discussion
presence of potassium carbonate. Meanwhile, coupling of methyl
vanillate (10) with benzyl bromide gave intermediate 11, subse-
quent nitration and catalytic hydrogenation provided 13, which
was subjected to cyclization to generate intermediate 14. Follow-
ing introduction of the acetyl group, the newly formed 15 was
chloridized by thionyl chloride to generate intermediate 16.18
Afterwards, condensation of 16 with substituted anilines afforded
.1. Chemistry
The synthetic route to compounds 19a–19t was shown in
Scheme 1. Firstly, intermediates 9a–9e were prepared by
alkylation of 2-nitroimidazole (8) with -dibromoalkanes in the
a,x
Scheme 1. Reagents and conditions: (i)
HCONH , HCOONH , 150 °C, 5 h; (vi) Ac
CO , DMF, 80 °C, 5 h.
a
,
x
-dibromoalkanes, K
2
CO
3
, DMF, 60 °C, 4 h; (ii) BnBr, K , Pd/C, MeOH, rt; (v)
2
CO
3
, DMF, 60 °C, 5 h; (iii) HNO
3
, AcOH, 50 °C, 5 h; (iv) H
2
.
2
4
2
O, pyridine, 90 °C, 2 h; (vii) SOCl
2
, DMF, reflux, 3 h; (viii) substituted aniline, i-PrOH, reflux, 1 h; (ix) NH H O, MeOH, rt, 3 h; (x) 9a–9e,
3 2
K
2
3
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W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
4
-anilinoquinazoline derivatives 17a–17d,19 followed by deacety-
than under normoxia, which also confirmed our conception that
the introduction of the hypoxic-directed 2-nitroimidazole moiety
was favorable for the treatment of hypoxic tumor cells.
lation using aqueous ammonia gave C-7-OH quinazoline deriva-
tives 18a–18d. Finally, alkylation of 18a–18d with intermediates
9
a–9e furnished title compounds 19a–19t.
In consistent with the potent EGFR inhibitory activity
(
IC50 = 0.47 nM), compound 19h exhibited excellent anti-proliferative
activities against A549 cells with the IC50 values of 0.77 M and
.18 M under normoxia and hypoxia, respectively, which are 9
folds and 50 folds more potent than that of erlotinib (IC50 = 7.59
under normoxia and 9.10 M under hypoxia, respectively). Similar
to that, compound 19h also strongly suppressed the proliferation
of HT-29 cells with the IC50 values of 1.31 and 0.28 M under
normoxia and hypoxia, respectively, which are several folds more
potent than that of erlotinib (IC50 = 2.98 M under normoxia and
4.37 M under hypoxia, respectively). Compound 19h was picked
2
.2. In vitro EGFR inhibitory activity
l
0
l
The EGFR inhibitory activity of compounds 19a–19t were eval-
lM
0
uated using a well established Z -Lyte assay employing gefitinib as
the positive control.
compounds displayed excellent EGFR inhibitory activities and
fourteen of them exhibited sub-nanomolar IC50 values. Compounds
l
2
0
As shown in Table 1, almost all the
l
0
1
9k–19o with 3 -fluorobenzyloxy-3-chloroanilino moiety showed
l
compromised EGFR inhibitory with the IC50 values from 11.9 to
0.5 nM, probably due to this bulky moiety cannot fit the
l
3
up for further biological evaluation to explore the hypoxia-
hydrophobic pocket of kinase very well. The length of linker
between imidazole and quinazoline also affected the EGFR inhibi-
tory activities to some extent. Compounds with 4–5 carbon linkers
displayed 1–2 fold increase in EGFR inhibitory activities in
comparison with compounds with shorter or longer linkers, such
as 19b and 19c vs 19a and 19d; 19g and 19h vs 19f; 19l and
activated mechanism.
2.4. Mimic the bioreductive activation of 19h
To mimic the bioreductive activation process of 19h under
tumor hypoxia, the zinc dust was used to replace reductive enzyme
and cysteine was employed as nucleophiles.
2
1,22
1
9m vs 19j and 19n.
The reaction was
carried out in phosphate buffer saline at 37 °C under hypoxia and
the resulting mixture was detected by HPLC-Q-ToF-MS. According
to the LC–MS results (Fig. 2 and Supporting figures), we deduced
that 19h experienced the following reductive process (Fig. 3). Com-
pound 19h was firstly reduced to the hydroxylamino-imidazole
intermediate and then to the crucial intermediate amino-imidaz-
ole radical. The radicals were transferred to the amino-imidazole
derivative (peak B) or degenerated to the guanidino derivative
(peak D) and the amino derivative (peak F). The amino-imidazole
radicals were also caught by cysteines to yield three cysteine-
containing complexes (peak A, peak C, and peak E).
2
.3. In vitro anti-proliferative activity
The anti-proliferative activities of the new compounds against
A549 cells and HT-29 cells were evaluated under normoxia and
hypoxia in vitro. As shown in Table 1, almost all the compounds
displayed good to excellent anti-proliferative activities against
the A549 and HT-29 cell lines with sub-micromolar to micromolar
IC50 values, which are similar to or more potent than that of gefiti-
nib and erlotinib. In addition, these novel EGFR inhibitors demon-
strated more potent anti-proliferative activities under hypoxia
Table 1
In vitro EGFR inhibitory and anti-proliferative activities of compounds 19a–19t
R2
HN
N
R1
O
2
N
O
O
N
N
(Linker)
N
Compd.
R
1
R
2
Linker
EGFR IC50 (nM)
A549 IC50
Normoxia
(
l
M)
HT-29 IC50
Normoxia
(lM)
Hypoxia
Hypoxia
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9a
9b
9c
9d
9e
9f
9g
9h
9i
Cl
Cl
Cl
Cl
Cl
Ethynyl
Ethynyl
Ethynyl
Ethynyl
Ethynyl
Cl
Cl
Cl
Cl
Cl
Br
Br
F
F
F
F
F
H
H
H
H
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
—
)
2 3
)
2 4
)
2 5
)
2 6
)
2 2
)
2 3
)
2 4
)
2 5
)
2 6
)
2 2
)
2 3
)
2 4
)
2 5
)
2 6
)
2 2
)
2 3
)
2 4
)
2 5
)
2 6
)
2 2
0.77
0.31
0.52
1.44
0.32
0.65
0.26
0.47
0.57
0.54
30.5
11.9
16.6
35.5
20.1
0.49
0.37
0.33
0.78
0.82
0.45
—
5.91
2.67
4.26
13.37
12.88
2.31
2.21
0.77
10.26
9.77
3.97
10.53
19.92
>50
5.06
13.54
3.09
4.31
1.41
1.74
6.52
7.64
1.17
1.08
0.18
2.70
6.43
1.52
8.69
14.38
>50
6.23
7.83
1.97
8.42
20.18
10.12
11.67
9.10
6.90
6.14
1.54
4.08
5.63
9.86
3.17
2.52
1.31
4.50
8.77
3.79
4.49
14.09
>50
7.64
10.88
4.02
5.05
10.51
10.84
3.63
2.98
>50
2.06
0.67
1.03
1.84
5.52
0.65
0.59
0.28
1.37
6.04
0.94
2.27
5.04
>50
2.59
2.82
1.43
2.54
4.30
3.44
5.21
4.37
9.45
O(CH
O(CH
O(CH
O(CH
)
2 2
)
2 2
)
2 2
)
2 2
9j
9k
9l
H
3-Fluorobenzyloxy
3-Fluorobenzyloxy
3-Fluorobenzyloxy
3-Fluorobenzyloxy
9m
9n
9o
9p
9q
9r
9s
9t
3-Fluorobenzyloxy
H
H
H
H
H
—
—
—
Br
Br
Br
—
—
—
8.78
30.23
12.97
12.17
7.59
Gefitinib
Erlotinib
TPZ
—
—
—
>50
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4
W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 2. The UV spectrum of the HPLC-MS results about the mimic reductive activation of compound 19h.
Figure 3. The proposed reductive activation process of compound 19h.
2
.5. Apoptosis assay of 19h under normoxia and hypoxia
respectively. The results further confirmed the hypoxic activation
property of compound 19h.
Propidium iodide staining for Sub-G1 content analysis was used
to characterize the apoptosis process of compound 19h. As shown
in Figure 4, after treatment with 19h (10.0 M) for 72 h under nor-
2.6. In vitro metabolism assay of compound 19h
l
moxia and hypoxia, a characteristic cleavage of DNA was observed
Compound 19h was incubated with mouse liver microsomes to
in A549 cells. The percentage of apoptotic cells induced by 19h was
evaluate its metabolic stability under normoxia and hypoxia with
2
3
2
6.15% under hypoxia, which was more than 2-fold higher than
NADPH as an activator. As shown in Figure 5, the metabolism
of 19h started in the presence of NADPH, more than 80% of 19h
was metabolized under hypoxia in comparison with less than
that under normoxia (12.46%). Spontaneous apoptosis of the con-
trols were seen in 3.94% and 4.70% under normoxia and hypoxia,
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W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 4. Cell apoptotic index analyses of A549 cells exposed to 19h under normoxia and hypoxia.
Figure 5. The in vitro metabolism of compound 19h under normoxia and hypoxia.
1
1
0% metabolism of 19h under normoxia. The easily metabolism of
9h under hypoxia also explained its more potent anti-prolifera-
tive activity against A549 and HT-29 cell lines in comparison with
under normoxia conditions.
Figure 6. The molecular docking study of compound 19h with EGFR (19h, yellow;
gefitinib, green).
2
.7. Molecular docking study
Met793 and N-1 of the quinazoline with a distance of 2.97 Å. As
expected, the 2-nitroimidazole side chain at C-7 quinazoline was
positioned at the solvent portion of the kinase.
To examine the possible binding mode of compound 19h with
EGFR kinase, a docking analysis was performed using the co-crystal
structure of gefitinib-EGFR complex (PDB ID: 2ITY). As illustrated
in Figure 6, compound 19h is favorably fitted into EGFR in a similar
3. Conclusion
0
way to gefitinib, the 3 -ethynylaniline moiety inserted into the
hydrophobic pocket formed by Asp855, Glu762, Leu788 and
Lys745, and a fluorine atomhydrogen bond was formed between
In order to design EGFR inhibitors to overcome tumor hypoxia, a
novel series of small molecules were synthesized by incorporating
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6
W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
the 2-nitroimidazole moiety into the C-7 position of the 4-anilino-
quinazolines. Most of these compounds exhibited potent EGFR
inhibitory activity and excellent anti-proliferative activities against
A549 and HT-29 cells under normoxia and hypoxia. The most
promising compound 19h displayed extraordinary EGFR inhibitory
activity (IC50 = 0.47 nM) and excellent anti-proliferative activities
4.1.3. Methyl 4-benzyloxy-3-methoxybenzoate (11)
To a solution of methyl vanillate (10) (1.82 g, 10 mmol) in DMF
2 3
(10 mL) were added K CO (1.38 g, 10 mmol) and benzyl bromide
(2.05 g, 12 mmol). The mixture was heated at 60 °C for 5 h. After
cooling to room temperature, water was added and the product
was extracted with CH
water and brine. After drying over Na
2
Cl
2
. The organic phase was washed with
SO , the solvent was
against A549 (IC50: 0.77
l
M, normoxia; 0.18
l
M, hypoxia) and
2
4
HT-29 (IC50: 1.31 M, normoxia; 0.28
l
l
M, hypoxia) cells. The
removed to yield intermediate 11 as a white solid. Yield: 97%,
mp: 77–79 °C.
mimic bioreductive activation study, cell apoptosis assay and
in vitro metabolism evaluation confirmed that compound 19h
can be reductive activated under hypoxia. Our results suggest that
the incorporation of hypoxia-activated moiety into EGFR inhibitor
scaffold might be a tractable strategy to overcome the tumor
hypoxia and compound 19h is worthy of further investigation as
a potential cancer therapeutic agent.
4
.1.4. Methyl 4-benzyloxy-5-methoxy-2-nitrobenzoate (12)
To a suspension of intermediate 11 (10.1 g, 37 mmol) in AcOH
(
3
100 mL) was slowly added 70% HNO (20 mL). Subsequently, the
mixture was heated to 50 °C for 5 h, then cooled to room temper-
ature and poured into ice water (500 mL). After stirring for 1 h, the
resulting precipitate was filtered off, washed with water and dried
to obtain the nitro compound 12 as a white solid. Yield: 86%, mp:
4
4
4
. Experimental
1
26–128 °C.
.1. Chemistry
4
.1.5. Methyl 2-amino-4-hydroxy-5-methoxybenzoate (13)
.1.1. General methods/instruments
A solution of intermediate 12 (11.7 g, 37 mmol) in MeOH
Melting points were recorded on a B-540 Büchi melting-point
(
2
250 mL) was treated with H (1.0 bar, room temperature) in the
1
apparatus and are uncorrected, H 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
presence of 10% palladium on charcoal (1.17 g, 1.1 mmol Pd). After
completion (5 h), the catalyst was removed by filtration, and the
solvent was evaporated to obtain the aniline 13 as a white solid
quantitatively. Mp: 122–124 °C.
(
ESI-MS) were performed on a Finnigan LCQ DecaXP ion trap mass
4
.1.6. 7-Hydroxy-6-methoxyquinazolin-4(3H)-one (14)
A mixture of formamide (25 mL), ammonium formate (3.59 g,
spectrometry.
5
7 mmol) and 13 (6.5 g, 33 mmol) was heated at 150 °C for 5 h.
4
(
.1.2. General synthetic procedure of nitroimidazole derivatives
9a–9e)
To a stirred solution of 2-nitroimidazole (8) (1.13 g, 0.01 mol)
The mixture was cooled to room temperature, and water
100 mL) was added in order to precipitate the quinazolinone 14.
(
After stirring for 1 h, the resulting precipitate was filtered off,
washed with water and dried to obtain intermediate 14 as a white
solid. Yield: 86%, mp: >250 °C.
and potassium carbonate (1.38 g, 0.02 mol) in DMF (5.0 mL) was
added alkyl halide (0.04 mol). The reaction mixture was heated
to 60 °C and stirred for about 4 h. After cooling to room tempera-
ture, the mixture was filtered and the filtrate was concentrated
to dryness. The residue was purified by silica gel column chroma-
tography to get pure 9a–9e.
4
.1.7. 7-Acetoxy-6-methoxyquinazolin-4(3H)-one (15)
A mixture of intermediate 14 (5.95 g, 31 mmol), Ac O (20 mL),
2
and pyridine (4.90 g, 62 mmol) was heated at 90 °C for 2 h. After
cooling to room temperature, ice water (200 mL) was added and
the mixture was stirred for 1 h. The precipitate was filtered off,
washed with water and dried to obtain intermediate 15 as a white
solid. Yield: 97%, mp: >250 °C.
4.1.2.1. 1-(3-Bromopropyl)-2-nitro-1H-imidazole (9a). Yellow oil,
1
yield: 82%. H NMR (500 MHz, CDCl
3
) d 7.21 (s, 1H), 7.17 (s, 1H), 4.62
(t, J = 6.5 Hz, 2H), 3.38 (t, J = 6.0 Hz, 2H), 2.45–2.38 (m, 2H). ESI-MS
+
m/z: 234 [M+H] .
4
.1.8. 4-Chloro-7-hydroxy-6-methoxyquinazoline (16)
A mixture of intermediate 15 (1.99 g, 8.5 mmol), SOCl (12 mL),
2
4
.1.2.2. 1-(4-Bromobutyl)-2-nitro-1H-imidazole (9b). Yellow oil,
1
yield: 86%. H NMR (500 MHz, CDCl
3
) d 7.15 (s, 1H), 7.11 (s, 1H),
and DMF (0.2 mL) was heated at reflux temperature for 3 h. The
volatiles were removed under reduced pressure. The residue was
dissolved in CH
4
1
.46 (t, J = 7.5 Hz, 2H), 3.42 (t, J = 6.5 Hz, 2H), 2.08–2.00 (m, 2H),
.95–1.88 (m, 2H). ESI-MS m/z: 248 [M+H] .
+
2
Cl
2
(50 ml) and the organic layer was washed with
4
solution and brine, and dried over Na SO ,
aqueous NaHCO
3
2
4
.1.2.3. 1-(5-Bromopentyl)-2-nitro-1H-imidazole (9c). Yellow
1
filtered and evaporated to give the crude product 16, which was
purified by silica gel column chromatography to get pure
intermediate 16 as a white solid. Yield: 57%, mp: 140–142 °C.
oil, yield: 82%. H NMR (500 MHz, CDCl
3
) d 7.14 (s, 1H), 7.10 (s,
1
H), 4.42 (t, J = 7.5 Hz, 2H), 3.40 (t, J = 6.5 Hz, 2H), 1.93–1.84 (m,
+
4
H), 1.55–1.49 (m, 2H). ESI-MS m/z: 262 [M+H] .
4
.1.2.4. 1-(6-Bromohexyl)-2-nitro-1H-imidazole (9d). Yellow oil,
4.1.9. General synthetic procedure of 4-anilinoquinazoline
derivatives (17a–17d, 18a–18d)
1
yield: 89%. H NMR (500 MHz, CDCl
3
) d 7.14 (s, 1H), 7.08 (s, 1H),
4
1
.41 (t, J = 7.5 Hz, 2H), 3.40 (t, J = 7.0 Hz, 2H), 1.91–1.81 (m, 4H),
.53–1.47 (m, 2H), 1.41–1.35 (m, 2H). ESI-MS m/z: 276 [M+H] .
To a stirred solution of compound 16 (2.53 g, 10 mmol) in iso-
propanol (10 mL) was added substituted aniline (15 mmol). The
reaction mixture was refluxed for 1 h and cooled to room temper-
ature. The white solid was filtered to get compounds 17a–17d.
Compounds 17a–17d were added to a mixture of methanol
(15.0 mL) and 25% ammonia (2.0 mL) and stirred for 3 h at room
temperature. The solid was filtered and washed with chilled meth-
anol and dried in vacuum to obtain 18a–18d.
+
4
.1.2.5. 1-(2-(2-Bromoethoxy)ethyl)-2-nitro-1H-imidazole (9e)
1
Yellow oil, yield: 68%. H NMR (400 MHz, CDCl
3
) d 7.17 (d,
J = 0.8 Hz, 1H), 7.07 (d, J = 0.8 Hz, 1H), 4.57 (t, J = 4.8 Hz, 2H), 3.80
t, J = 4.8 Hz, 2H), 3.70–3.67 (m, 2H), 3.36–3.33 (m, 2H). ESI-MS
(
+
m/z: 264 [M+H] .
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W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
4
.1.9.1. 4-((3-Chloro-4-fluorophenyl)amino)-6-methoxyquinaz-
dried over anhydrous Na
2
SO
4
. The solvent was evaporated to dry-
olin-7-yl acetate hydrochloride (17a). White solid, yield: 93%,
ness and the residue was purified with silica gel chromatography
to give title compounds 19a–19t.
1
mp: >250 °C. H NMR (500 MHz, DMSO-d
6
) d 11.83 (s, 1H), 8.92
(
(
[
s, 1H), 8.65 (s, 1H), 8.09–8.07 (m, 1H), 7.87–7.75 (m, 2H), 7.58
t, J = 9.0 Hz, 1H), 4.05 (s, 3H), 2.39 (s, 3H). ESI-MS m/z: 398
4.1.10.1. N-(3-Chloro-4-fluorophenyl)-6-methoxy-7-(3-(2-nitro-
+
M-HCl+H] .
1H-imidazol-1-yl)propoxy)quinazolin-4-amine (19a). Yellow
1
solid, yield: 61%, mp: 167–169 °C. H NMR (500 MHz, DMSO-d
6
)
1
4
.1.9.2. 4-((3-Ethynylphenyl)amino)-6-methoxyquinazolin-7-yl
6
H NMR (500 MHz, DMSO-d ) d 9.57 (s, 1H), 8.50 (s, 1H),
acetate hydrochloride (17b). White solid, yield: 92%, mp: 240–
8.14–8.12 (m, 1H), 7.83–7.77 (m, 2H), 7.68 (d, J = 1.0 Hz, 1H),
7.46 (t, J = 9.0 Hz, 1H), 7.19 (s, 1H), 7.17 (d, J = 1.0 Hz, 1H), 4.61
(t, J = 7.0 Hz, 2H), 4.20 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 2.39–2.34
1
2
8
1
4
6
42 °C. H NMR (500 MHz, DMSO-d ) d 11.51 (s, 1H), 8.89 (s, 1H),
.51 (s, 1H), 7.90 (t, J = 1.5 Hz, 1H), 7.81–7.79 (m, 1H), 7.75 (s,
1
3
H), 7.52 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 4.30 (s, 1H),
(m, 2H). C NMR (100 MHz, DMSO-d
(d, JC–F = 242.8 Hz), 152.6, 149.1, 146.8, 144.8, 136.8 (d, JC–F = 3.1 Hz),
27.8, 127.7, 123.5, 122.3 (d, JC–F = 6.7 Hz), 118.8 (d, JC–F = 18.4 Hz),
6
) d 156.0, 153.3, 153.2
+
.04 (s, 3H), 2.38 (s, 3H). ESI-MS m/z: 334 [M-HCl+H] .
1
4
.1.9.3.
4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-
Brown
solid, yield: 89%, mp: 245–247 °C. H NMR (500 MHz, DMSO-d
d 11.68 (s, 1H), 8.88 (s, 1H), 8.56 (s, 1H), 7.90 (d, J = 2.5 Hz, 1H),
116.5 (d, JC–F = 21.6 Hz), 108.9, 107.9, 101.8, 65.8, 56.2, 46.9, 28.9.
ESI-MS m/z: 473 [M+H] .
+
6-methoxyquinazolin-7-yl acetate hydrochloride (17c).
1
6
)
4.1.10.2. N-(3-Chloro-4-fluorophenyl)-6-methoxy-7-(4-(2-nitro-
7
3
.76 (s, 1H), 7.68–7.65 (m, 1H), 7.51–7.46 (m, 1H), 7.39–7.30 (m,
1H-imidazol-1-yl)butoxy)quinazolin-4-amine
(19b). White
solid, yield: 64%, mp: 126–128 °C. H NMR (500 MHz, DMSO-d
d 9.55 (s, 1H), 8.48 (s, 1H), 8.12–8.10 (m, 1H), 7.82–7.76 (m, 2H),
.75 (s, 1H), 7.43 (t, J = 9.0 Hz, 1H), 7.19 (s, 2H), 4.48 (t, J = 7.0 Hz,
2H), 4.17 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 2.00–1.94 (m, 2H), 1.83–
1
H), 7.24–7.16 (m, 1H), 5.31 (s, 2H), 4.03 (s, 3H), 2.38 (s, 3H).
6
)
+
ESI-MS m/z: 468 [M-HCl+H] .
7
4
.1.9.4. 4-((3-Bromophenyl)amino)-6-methoxyquinazolin-7-yl
1
3
acetate hydrochloride (17d). White solid, yield: 91%, mp:
1.75 (m, 2H). C NMR (100 MHz, DMSO-d
(d, JC–F = 242.7 Hz), 152.6, 149.1, 147.0, 144.6, 136.8 (d, JC–F
2.6 Hz), 127.9, 127.8, 123.4, 122.2 (d, C–F = 6.8 Hz), 118.8
6
) d 156.0, 153.6, 153.1
1
>
1
(
250 °C. H NMR (400 MHz, DMSO-d
H), 8.57 (s, 1H), 8.06 (t, J = 2.0 Hz, 1H), 7.83–7.78 (m, 1H), 7.77
s, 1H), 7.55–7.50 (m, 1H), 7.47 (t, J = 8.0 Hz, 1H), 4.04 (s, 3H),
6
) d 11.61 (s, 1H), 8.90 (s,
=
J
(d, JC–F = 18.1 Hz), 116.5 (d, JC–F = 21.5 Hz), 108.7, 107.9, 101.8,
68.0, 56.3, 49.1, 26.5, 25.2. ESI-MS m/z: 487 [M+H] .
+
+
2
.38 (s, 3H). ESI-MS m/z: 388 [M-HCl+H] .
4
.1.9.5. 4-((3-Chloro-4-fluorophenyl)amino)-6-methoxyquinaz-
4.1.10.3. N-(3-Chloro-4-fluorophenyl)-6-methoxy-7-((5-(2-nitro-
1
olin-7-ol (18a). Yellow solid, yield: 92%, mp: 202–204 °C.
NMR (500 MHz, DMSO-d
H), 8.11 (d, J = 6.5 Hz, 1H), 8.02 (s, 1H), 7.87–7.74 (m, 1H), 7.48
H
1H-imidazol-1-yl)pentyl)oxy)quinazolin-4-amine
solid, yield: 63%, mp: 166–168 °C. H NMR (500 MHz, DMSO-d
(19c). White
1
6
) d 10.88 (s, 1H), 10.26 (s, 1H), 8.58 (s,
6
)
1
d 9.54 (s, 1H), 8.48 (s, 1H), 8.12–8.10 (m, 1H), 7.86–7.74 (m, 2H),
7.71 (s, 1H), 7.44 (t, J = 9.0 Hz, 1H), 7.23–7.14 (m, 2H), 4.41 (t,
J = 7.5 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 1.92–1.79 (m,
+
(
t, J = 9.0 Hz, 1H), 7.17 (s, 1H), 3.99 (s, 3H). ESI-MS m/z: 320 [M+H] .
1
3
4
.1.9.6. 4-((3-Ethynylphenyl)amino)-6-methoxyquinazolin-7-ol
6
4H), 1.51–1.39 (m, 2H). C NMR (100 MHz, DMSO-d ) d 156.0,
1
(
(
1
7
18b). Brown solid, yield: 91%, mp: 142–144 °C.
500 MHz, DMSO-d ) d 10.84 (br s, 1H), 10.13 (s, 1H), 8.56 (s,
H), 8.05–8.00 (m, 1H), 7.97–7.96 (m, 1H), 7.88–7.87 (m, 1H),
.43 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.18 (s, 1H), 4.23
H
NMR
153.7, 153.1 (d, JC–F = 242.8 Hz), 152.6, 149.1, 147.0, 144.5, 136.8
(d, JC–F = 2.6 Hz), 127.8 (2), 123.4, 122.2 (d, JC–F = 6.8 Hz), 118.8 (d,
6
JC–F = 18.4 Hz), 116.5 (d, JC–F = 21.4 Hz), 108.7, 107.8, 101.8, 68.2,
+
56.2, 49.3, 29.4, 27.8, 22.4. ESI-MS m/z: 501 [M+H] .
+
(
s, 1H), 4.00 (s, 3H). ESI-MS m/z: 292 [M+H] .
4
.1.10.4.
N-(3-Chloro-4-fluorophenyl)-6-methoxy-7-((6-(2-nitro-
(19d). Yellow
solid, yield: 59%, mp: 219–221 °C. H NMR (400 MHz, DMSO-d
d 9.55 (s, 1H), 8.50 (s, 1H), 8.14–8.12 (m, 1H), 7.83–7.79 (m, 2H),
7.71 (d, J = 0.8 Hz, 1H), 7.45 (t, J = 9.2 Hz, 1H), 7.19 (s, 1H), 7.18
(d, J = 0.8 Hz, 1H), 4.40 (t, J = 7.2 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H),
4
.1.9.7. 4-((3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-
1H-imidazol-1-yl)hexyl)oxy)quinazolin-4-amine
1
methoxyquinazolin-7-ol (18c). Brown solid, yield: 95%, mp:
6
)
1
1
20–122 °C. H NMR (500 MHz, DMSO-d
s, 1H), 8.51 (s, 1H), 7.99 (s, 1H), 7.95 (d, J = 2.5 Hz, 1H), 7.71–
.68 (m, 1H), 7.51–7.45 (m, 1H), 7.38–7.30 (m, 2H), 7.28 (d,
J = 9.0 Hz, 1H), 7.21–7.16 (m, 1H), 7.15 (s, 1H), 5.27 (s, 2H), 3.98
6
) d 10.78 (s, 1H), 10.06
(
7
3.96 (s, 3H), 1.89–1.75 (m, 4H), 1.58–1.44 (m, 2H), 1.44–1.33 (m,
+
13
(
s, 3H). ESI-MS m/z: 426 [M+H] .
6
2H). C NMR (100 MHz, DMSO-d ) d 156.0, 153.7, 153.1 (d,
JC–F = 242.6 Hz), 152.6, 149.1, 147.0, 144.5, 136.8 (d, JC–F = 3.0 Hz),
4
.1.9.8. 4-((3-Bromophenyl)amino)-6-methoxyquinazolin-7-ol
127.8, 127.8, 123.4, 122.2 (d, JC–F = 6.8 Hz), 118.7 (d, JC–F = 18.3 Hz),
116.5 (d, JC–F = 21.6 Hz), 108.6, 107.8, 101.7, 68.2, 56.2, 49.3, 29.7,
28.2, 25.5, 25.0. ESI-MS m/z: 515 [M+H] .
1
(
18d). White solid, yield: 96%, mp: >250 °C. H NMR (400 MHz,
+
DMSO) d 10.78 (s, 1H), 10.05 (s, 1H), 8.56 (s, 1H), 8.13 (t,
J = 2.0 Hz, 1H), 7.99 (s, 1H), 7.89–7.83 (m, 1H), 7.38 (t, J = 8.0 Hz,
1
3
H), 7.35–7.32 (m, 1H), 7.16 (s, 1H), 3.99 (s, 3H). ESI-MS m/z:
46 [M+H] .
4.1.10.5. N-(3-Chloro-4-fluorophenyl)-6-methoxy-7-(2-(2-(2-nitro-
+
1H-imidazol-1-yl)ethoxy)ethoxy)quinazolin-4-amine (19e). Yellow
1
solid, yield: 64%, mp: 178–180 °C. H NMR (500 MHz, DMSO-d
6
)
4
.1.10. General synthetic procedure of final compounds (19a–19t)
d 9.57 (s, 1H), 8.49 (s, 1H), 8.11 (dd, J = 7.0, 2.5 Hz, 1H), 7.82–7.76
(m, 2H), 7.66 (d, J = 1.0 Hz, 1H), 7.44 (t, J = 9.0 Hz, 1H), 7.18 (s,
1H), 7.12 (d, J = 1.0 Hz, 1H), 4.59 (t, J = 5.0 Hz, 2H), 4.21 (t,
J = 4.0 Hz, 2H), 3.95 (s, 3H), 3.88 (t, J = 5.0 Hz, 2H), 3.82 (t,
To
18a–18d) (0.15 mmol) and potassium carbonate (0.3 mmol) in
DMF (3.0 mL) was added nitroimidazole derivatives (9a–9e)
0.18 mmol). 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. H O (15 mL)
was added to the residue and extracted with ethyl acetate
15 mL ꢀ 3). The combined organic layer was washed with brine,
a stirred solution of 4-anilinoquinazoline compound
(
1
3
(
6
J = 4.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) d 156.0, 153.4,
153.1 (d, C–F = 242.6 Hz), 152.6, 149.0, 146.8, 144.7, 136.8
J
2
(d, JC–F = 2.6 Hz), 128.2, 127.4, 123.4, 122.3 (d, JC–F = 6.8 Hz),
118.8 (d, JC–F = 18.3 Hz), 116.5 (d, JC–F = 21.6 Hz), 108.8, 107.8,
+
(
101.8, 68.8, 68.3, 67.9, 56.2, 48.9. ESI-MS m/z: 503 [M+H] .
Please cite this article in press as: Cheng, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.038

8
W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4
.1.10.6. N-(3-Ethynylphenyl)-6-methoxy-7-(3-(2-nitro-1H-
3.94 (s, 3H), 2.39–2.34 (m, 2H). 13C NMR (100 MHz, DMSO-d
)
6
imidazol-1-yl)propoxy)quinazolin-4-amine (19f). Yellow solid,
d 162.2 (d, JC–F = 243.9 Hz), 156.2, 153.2, 152.7, 149.4, 148.9,
146.6, 144.8, 139.7 (d, JC–F = 7.8 Hz), 133.5, 130.5 (d, JC–F = 8.4 -
Hz), 127.8 (2), 124.0, 123.3 (d, J = 2.9 Hz), 122.2, 121.0, 114.7
(d, JC–F = 20.6 Hz), 114.3, 114.0 (d, J = 21.9 Hz), 108.8, 107.9,
1
yield: 59%, mp: 180–182 °C. H NMR (500 MHz, DMSO-d
6
) d 9.51
(
(
(
s, 1H), 8.50 (s, 1H), 8.00–7.97 (m, 1H), 7.91–7.89 (m, 1H), 7.83
s, 1H), 7.68 (d, J = 1.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.24–7.20
+
m, 1H), 7.19 (s, 1H), 7.17 (d, J = 1.0 Hz, 1H), 4.61 (t, J = 7.0 Hz,
101.9, 69.4, 65.7, 56.2, 46.9, 28.9. ESI-MS m/z: 579 [M+H] .
3
2
H), 4.26–4.17 (m, 3H), 3.96 (s, 3H), 2.39–2.34 (m, 2H). 1 C NMR
(
6
100 MHz, DMSO-d ) d 156.1, 153.3, 152.7, 149.0, 146.8, 144.8,
4.1.10.12. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methoxy-
1
1
39.8, 128.9, 127.8 (2), 126.3, 124.8, 122.6, 121.7, 109.0, 107.9,
01.9, 83.5, 80.5, 65.7, 56.2, 46.9, 28.9. ESI-MS m/z: 445 [M+H] .
7-(4-(2-nitro-1H-imidazol-1-yl)butoxy)quinazolin-4-amine
+
1
(19l). Brown solid, yield: 53%, mp: 176–178 °C.
500 MHz, DMSO-d
H NMR
(
6
)
d
9.43 (s, 1H), 8.43 (s, 1H), 7.94 (d,
4
.1.10.7. N-(3-Ethynylphenyl)-6-methoxy-7-(4-(2-nitro-1H-
J = 2.5 Hz, 1H), 7.78 (s, 1H), 7.75 (d, J = 1.0 Hz, 1H), 7.68 (dd,
J = 9.0, 2.5 Hz, 1H), 7.51–7.41 (m, 1H), 7.35–7.28 (m, 2H), 7.25 (d,
J = 9.0 Hz, 1H), 7.21–7.13 (m, 3H), 5.23 (s, 2H), 4.48 (t, J = 7.0 Hz,
2H), 4.16 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.00–1.94 (m, 2H),
imidazol-1-yl)butoxy)quinazolin-4-amine (19g). Yellow solid,
yield: 61%, mp: 200–202 °C. H NMR (500 MHz, DMSO-d
1
6
) d 9.48
(
s, 1H), 8.48 (s, 1H), 7.98–7.97 (m, 1H), 7.90–7.88 (m, 1H), 7.82
1
3
(
(
s, 1H), 7.75 (d, J = 1.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.23–7.14
m, 3H), 4.49 (t, J = 7.0 Hz, 2H), 4.19 (s, 1H), 4.17 (t, J = 6.5 Hz,
6
1.84–1.74 (m, 2H). C NMR (100 MHz, DMSO-d ) d 162.2 (d,
JC–F = 243.7 Hz), 156.2, 153.5, 152.7, 149.4, 149.0, 146.5, 144.6,
1
3
2
H), 4.01–3.88 (m, 3H), 2.02–1.93 (m, 2H), 1.84–1.75 (m, 2H).
) d 156.1, 153.5, 152.7, 149.0, 147.0,
44.6, 139.8, 128.9, 127.9, 127.8, 126.3, 124.7, 122.5, 121.7,
08.9, 107.9, 101.9, 83.5, 80.5, 68.0, 56.3, 49.1, 26.5, 25.2. ESI-MS
C
139.7 (d, JC–F = 7.4 Hz), 133.5, 130.5 (d, JC–F = 8.4 Hz), 127.9, 127.8,
124.0, 123.3 (d, JC–F = 2.6 Hz), 122.1, 121.0, 114.7 (d, JC–F = 21.0 Hz),
114.4, 114.0 (d, JC–F = 21.9 Hz), 108.6, 107.7, 101.9, 69.4, 67.9, 56.2,
NMR (100 MHz, DMSO-d
1
1
6
+
49.1, 26.5, 25.2. ESI-MS m/z: 593 [M+H] .
+
m/z: 459 [M+H] .
4
.1.10.13. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methoxy-
4
.1.10.8. N-(3-Ethynylphenyl)-6-methoxy-7-((5-(2-nitro-1H-
imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (19h). Yellow
solid, yield: 66%, mp: 111–113 °C. H NMR (400 MHz, DMSO-d
d 9.49 (s, 1H), 8.50 (s, 1H), 8.00 (t, J = 2.0 Hz, 1H), 7.92–7.89 (m,
7-((5-(2-nitro-1H-imidazol-1-yl)pentyl)oxy)quinazolin-4-amine
1
(19m). White solid, yield: 59%, mp: 179–181 °C.
H NMR
1
6
)
(400 MHz, DMSO-d 9.44 (s, 1H), 8.44 (s, 1H), 7.96 (d,
6
) d
J = 2.4 Hz, 1H), 7.79 (s, 1H), 7.72 (d, J = 1.2 Hz, 1H), 7.70–7.67 (m,
1H), 7.50–7.45 (m, 1H), 7.32 (t, J = 8.4 Hz, 2H), 7.27 (d, J = 9.2 Hz,
1H), 7.23–7.15 (m, 3H), 5.25 (s, 2H), 4.43 (t, J = 7.2 Hz, 2H), 4.14
1H), 7.83 (s, 1H), 7.72 (d, J = 1.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H),
.24–7.16 (m, 3H), 4.43 (t, J = 7.2 Hz, 2H), 4.20 (s, 1H), 4.15 (t,
7
J = 6.4 Hz, 2H), 3.96 (s, 3H), 1.94–1.80 (m, 4H), 1.52–1.44 (m, 2H).
(t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 1.94–1.80 (m, 4H), 1.54–1.43 (m,
2H). C NMR (100 MHz, DMSO-d ) d 162.2 (d, JC–F = 243.4 Hz),
6
1
3
13
C NMR (100 MHz, DMSO-d
6
) d 156.1, 153.6, 152.7, 149.0, 147.0,
1
1
44.5, 139.8, 128.9, 127.8, 127.8, 126.3, 124.7, 122.5, 121.7,
156.2, 153.5, 152.8, 149.4, 148.9, 146.8, 144.5, 139.7 (d, JC–F =
7.4 Hz), 133.6, 130.5 (d, JC–F = 8.4 Hz), 127.9, 127.8, 123.9, 123.3
(d, JC–F = 2.5 Hz), 122.1, 121.0, 114.7 (d, JC–F = 20.7 Hz), 114.3,
08.8, 107.8, 101.8, 83.5, 80.5, 68.2, 56.2, 49.3, 29.4, 27.8, 22.4.
+
ESI-MS m/z: 473 [M+H] .
1
14.0 (d, JC–F = 21.8 Hz), 108.6, 107.8, 101.8, 69.4, 68.1, 56.2, 49.3,
+
4
.1.10.9. N-(3-Ethynylphenyl)-6-methoxy-7-((6-(2-nitro-1H-
29.4, 27.8, 22.4. ESI-MS m/z: 607 [M+H] .
imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (19i). Yellow solid,
1
yield: 63%, mp: 146–148 °C. H NMR (400 MHz, DMSO-d
6
) d 9.49
4.1.10.14. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methoxy-
(
7
7
s, 1H), 8.58–8.44 (m, 1H), 8.00–7.99 (m, 1H), 7.92–7.90 (m, 1H),
.83 (s, 1H), 7.71 (d, J = 1.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H),
.23–7.16 (m, 2H), 4.40 (t, J = 7.2 Hz, 2H), 4.20 (s, 1H), 4.14 (t,
7-((6-(2-nitro-1H-imidazol-1-yl)hexyl)oxy)quinazolin-4-amine
1
(19n). Yellow solid, yield: 54%, mp: 180–182 °C.
(400 MHz, DMSO-d
H NMR
6
) d 9.44 (s, 1H), 8.44 (s, 1H), 7.96 (d, J =
J = 6.4 Hz, 2H), 3.97 (s, 3H), 1.87–1.77 (m, 4H), 1.55–1.45 (m, 2H),
2.8 Hz, 1H), 7.79 (s, 1H), 7.71 (d, J = 0.8 Hz, 1H), 7.69 (d, J = 2.4 Hz,
1H), 7.50–7.45 (m, 1H), 7.36–7.30 (m, 2H), 7.27 (d, J = 9.2 Hz, 1H),
7.22–7.15 (m, 3H), 5.25 (s, 2H), 4.44–4.37 (m, 2H), 4.13 (t,
1
3
1
1
1
4
6
.44–1.35 (m, 2H). C NMR (100 MHz, DMSO-d ) d 156.1, 153.7,
52.7, 149.1, 147.0, 144.5, 139.8, 128.9, 127.8, 127.7, 126.3,
24.7, 122.5, 121.7, 108.8, 107.8, 101.9, 83.5, 80.5, 68.2, 56.3,
J = 6.4 Hz, 2H), 3.95 (s, 3H), 1.87–1.76 (m, 4H), 1.55–1.45 (m, 2H),
+
13
9.3, 29.7, 28.2, 25.5, 25.0. ESI-MS m/z: 487 [M+H] .
6
1.42–1.35 (m, 2H). C NMR (100 MHz, DMSO-d ) d 162.2 (d,
J = 243.8 Hz), 156.1, 153.5, 152.8, 149.3, 148.9, 146.8, 144.5, 139.7
(d, JC–F = 7.3 Hz), 133.5, 130.5 (d, JC–F = 8.6 Hz), 127.81 (2), 123.9,
123.3 (d, JC–F = 2.6 Hz), 122.1, 121.0, 114.7 (d, JC–F = 21.0 Hz),
114.3, 114.0 (d, JC–F = 22.0 Hz), 108.6, 107.7, 101.7, 69.3, 68.2,
4
.1.10.10. N-(3-Ethynylphenyl)-6-methoxy-7-(2-(2-(2-nitro-1H-
imidazol-1-yl)ethoxy)ethoxy)quinazolin-4-amine (19j). Yellow
solid, yield: 67%, mp: 86–88 °C. H NMR (500 MHz, DMSO-d
1
6
) d
+
9
7
7
4
.53 (s, 1H), 8.51 (s, 1H), 8.00–7.99 (m, 1H), 7.94–7.88 (m, 1H),
56.2, 49.3, 29.7, 28.2, 25.5, 25.0. ESI-MS m/z: 621 [M+H] .
.85 (s, 1H), 7.68 (d, J = 1.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H),
.25–7.17 (m, 2H), 7.14 (d, J = 1.0 Hz, 1H), 4.61 (t, J = 5.0 Hz, 2H),
.27–4.20 (m, 3H), 3.97 (s, 3H), 3.90 (t, J = 5.0 Hz, 2H), 3.84 (t,
4.1.10.15. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methoxy-
7-(2-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)ethoxy)quinazolin-4-
1
3
1
J = 4.0 Hz, 2H). C NMR (100 MHz, DMSO-d
6
) d 158.1, 156.1,
amine (19o). Brown solid, yield: 65%, mp: 118–120 °C. H NMR
1
1
4
53.4, 152.7, 149.0, 146.9, 139.8, 128.8, 128.1 127.4, 126.3, 124.7,
6
(500 MHz, DMSO-d ) d 9.46 (s, 1H), 8.43 (s, 1H), 7.94 (d, J = 2.5 Hz,
22.5, 121.7, 108.9, 107.9, 102.0, 83.4, 80.4, 68.8, 68.3, 67.9, 56.2,
1H), 7.79 (s, 1H), 7.72–7.62 (m, 2H), 7.49–7.42 (m, 1H), 7.35–7.28
(m, 2H), 7.26 (d, J = 9.0 Hz, 1H), 7.21–7.13 (m, 2H), 7.12 (d,
J = 1.0 Hz, 1H), 5.24 (s, 2H), 4.59 (t, J = 5.0 Hz, 2H), 4.20
(t, J = 4.0 Hz, 2H), 3.94 (s, 3H), 3.88 (t, J = 5.0 Hz, 2H), 3.82
+
8.9. ESI-MS m/z: 475 [M+H] .
4
7
.1.10.11. N-(3-Chloro-4-((3-fluorobenzyl)oxy)phenyl)-6-methoxy-
1
3
-(3-(2-nitro-1H-imidazol-1-yl)propoxy)quinazolin-4-amine
6
(t, J = 4.0 Hz, 2H). C NMR (100 MHz, DMSO-d ) d 162.2 (d, JC–F =
1
(
19k). Brown solid, yield: 51%, mp: 216–218 °C. H NMR
243.7 Hz), 156.2, 153.3, 152.8, 149.4, 148.9, 146.7, 144.7, 139.7
(d, JC–F = 7.3 Hz), 133.6, 130.5 (d, JC–F = 8.4 Hz), 128.1, 127.4,
123.9, 123.3, 122.1, 121.1, 114.6 (d, JC–F = 21.1 Hz), 114.4, 114.0
(d, JC–F = 21.9 Hz), 108.8, 107.9, 101.9, 69.4, 68.8, 68.4, 67.9, 56.2,
(
6
500 MHz, DMSO-d ) d 9.45 (s, 1H), 8.45 (s, 1H), 7.95 (d,
J = 2.5 Hz, 1H), 7.79 (s, 1H), 7.72–7.65 (m, 2H), 7.50–7.45 (m,
1
3
H), 7.37–7.30 (m, 2H), 7.27 (d, J = 9.0 Hz, 1H), 7.22–7.14 (m,
H), 5.26 (s, 2H), 4.61 (t, J = 7.0 Hz, 2H), 4.19 (t, J = 6.0 Hz, 2H),
+
48.9. ESI-MS m/z: 609 [M+H] .
Please cite this article in press as: Cheng, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.038

W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
4
.1.10.16. N-(3-Bromophenyl)-6-methoxy-7-(3-(2-nitro-1H-
4.2.1. In vitro enzymatic activity assay
0
imidazol-1-yl)propoxy)quinazolin-4-amine (19p). Yellow solid,
yield: 56%, mp: 135–137 °C. H NMR (500 MHz, DMSO-d
Wild type EGFR and the Z -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.
1
6
) d 9.53
(
s, 1H), 8.50 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.86 (d, J = 9.0 Hz,
1
H), 7.81 (s, 1H), 7.66 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.27 (d,
J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.16 (s, 1H), 4.59 (t, J = 7.0 Hz, 2H),
1
3
4
.18 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 2.37–2.32 (m, 2H). C NMR
4.2.2. Cytotoxicity assay
(
1
1
100 MHz, DMSO-d ) d 156.0, 153.4, 152.6, 149.1, 146.9, 144.8,
6
The cytotoxic activity in vitro was measured using sulforhoda-
mine 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
41.3, 130.4, 127.9, 127.8, 125.6, 124.0, 121.2, 120.6, 109.0,
+
07.9, 101.8, 65.8, 56.3, 47.0, 29.0. ESI-MS m/z: 499 [M+H] .
3
4
.1.10.17. N-(3-Bromophenyl)-6-methoxy-7-(4-(2-nitro-1H-
(5 ꢀ 10 per well) for 24 h and subsequently treated with different
imidazol-1-yl)butoxy)quinazolin-4-amine (19q). Yellow solid,
yield: 58%, mp: 189–191 °C. H NMR (500 MHz, DMSO-d
(
7
1
2
(
1
1
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 wash-
ing, 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 unbuf-
fered 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
1
6
) d 9.53
s, 1H), 8.50 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.89–7.85 (m, 1H),
.81 (s, 1H), 7.76 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.29–7.24 (m,
H), 7.22–7.17 (m, 2H), 4.48 (t, J = 7.0 Hz, 2H), 4.17 (t, J = 6.5 Hz,
1
3
H), 3.94 (s, 3H), 2.03–1.92 (m, 2H), 1.85–1.74 (m, 2H). C NMR
) d 155.9, 153.6, 152.6, 149.0, 147.0, 144.6,
100 MHz, DMSO-d
6
41.3, 130.3, 127.9, 127.8, 125.5, 123.9, 121.2, 120.5, 108.9,
+
07.8, 101.8, 67.9, 56.2, 49.1, 26.5, 25.2. ESI-MS m/z: 513 [M+H] .
(
A515 control cells—A515 treated cells)/A515 control cells ꢀ
4
.1.10.18. N-(3-Bromophenyl)-6-methoxy-7-((5-(2-nitro-1H-
100%, and the IC50 values were determined by Logit method.
imidazol-1-yl)pentyl)oxy)quinazolin-4-amine (19r).
Yellow
solid, yield: 62%, mp: 109–111 °C. H NMR (500 MHz, DMSO-d ) d
.53 (s, 1H), 8.50 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.90–7.84 (m, 1H),
1
6
4.3. Mimic the bioreductive activation of compound 19h
9
7
7
3
.81 (s, 1H), 7.71 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.29–7.24 (m, 1H),
.20–7.16 (m, 2H), 4.41 (t, J = 7.5 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H),
Compound 19h (2.0 mg, 4.2
41.1 mol) was firstly dissolved in 0.5 mL dimethyl sulfoxide and
then added to 3 mL phosphate buffer saline (pH = 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
lmol) and cysteine (5.0 mg,
l
1
3
.94 (s, 3H), 1.91–1.78 (m, 4H), 1.49–1.43 (m, 2H). C NMR
) d 156.5, 154.3, 153.1, 149.7, 147.5, 145.1,
l
(
100 MHz, DMSO-d
6
l
1
1
41.9, 130.9, 128.4 (2), 126.1, 124.5, 121.7, 121.1, 109.4, 108.3,
02.4, 68.7, 56.8, 49.8, 30.0, 28.4, 23.0. ESI-MS m/z: 527 [M+H] .
l
+
reaction flask was evacuated and backfilled with nitrogen three
times. The reaction mixture was stirred at 37 °C under a nitrogen
4
.1.10.19. N-(3-Bromophenyl)-6-methoxy-7-((6-(2-nitro-1H-
balloon for 6 h and then filtered through 0.45 lm microfiltration
imidazol-1-yl)hexyl)oxy)quinazolin-4-amine (19s).
Yellow
solid, yield: 56%, mp: 162–164 °C. H NMR (500 MHz, DMSO-d
d 9.52 (s, 1H), 8.50 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.90–7.84 (m,
H), 7.81 (s, 1H), 7.70 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.29–7.24
m, 1H), 7.18 (s, 1H), 7.16 (s, 1H), 4.43–4.35 (m, 2H), 4.12 (t,
J = 6.5 Hz, 2H), 3.95 (s, 3H), 1.85–1.73 (m, 4H), 1.50–1.44 (m, 2H),
membrane and the filtrate was analyzed through HPLC-Q-ToF-MS
(AB Sciex Triple TOF 5600+). The column was Eclipse Plus C18
1
6
)
(5
l
m, 4.6 ꢀ 250 mm), the mobile phase was MeOH/H
2
O (0.1%
1
(
HCOOH + 5 mM AcONH
4
) = 42: 58 at the flow rate of 1.0 ml/min,
and the detected wavelength was 330 nm, with column tempera-
ture at 30 °C.
1
3
1
1
1
3
.39–1.33 (m, 2H).
6
C NMR (100 MHz, DMSO-d ) d 156.45,
54.3, 153.1, 149.6, 147.6, 145.0, 141.8, 130.8, 128.3, 128.2,
26.0, 124.4, 121.6, 121.0, 109.3, 108.3, 102.4, 68.8, 56.8, 49.8,
0.2, 28.7, 26.0, 25.4. ESI-MS m/z: 541 [M+H] .
4.4. Apoptosis assay
+
To analysis apoptosis, A549 cells (5 ꢀ 104 cells/ml, 5 ml) were
2
cultured in 25 cm flasks and treated with vehicle to test com-
4
.1.10.20. N-(3-Bromophenyl)-6-methoxy-7-(2-(2-(2-nitro-1H-
pounds under normoxia and hypoxia. After 72 h of treatments,
cells were harvested and quickly washed twice with ice-cold PBS.
Detection of apoptosis by FACSCalibur flow cytometer (Becton
Dickinson, Lincoln Park, NJ) was performed using the Annexin V-
FITC/Propidium iodide (PI) apoptosis detection kit (BioVision,
Mountain View, CA).
imidazol-1-yl)ethoxy)ethoxy)quinazolin-4-amine (19t). Yellow
solid, yield: 61%, mp: 116–118 °C. H NMR (500 MHz, DMSO-d
1
6
) d
9
7
7
.54 (s, 1H), 8.51 (s, 1H), 8.13 (t, J = 2.0 Hz, 1H), 7.89–7.85 (m, 1H),
.82 (s, 1H), 7.66 (d, J = 1.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H),
.29–7.25 (m, 1H), 7.18 (s, 1H), 7.12 (d, J = 1.0 Hz, 1H), 4.59 (t,
J = 5.0 Hz, 2H), 4.22 (t, J = 4.0 Hz, 2H), 3.96 (s, 3H), 3.88 (t,
J = 5.0 Hz, 2H), 3.82 (t, J = 4.0 Hz, 2H). 13C NMR (100 MHz, DMSO-
4.5. Metabolic stability assay4,24
6
d ) d 156.0, 153.5, 152.6, 149.0, 147.0, 144.7, 141.3, 130.3, 128.2,
1
27.4, 125.6, 124.0, 121.2, 120.5, 109.0, 107.9, 101.9, 68.8, 68.3,
The incubation mixtures of compound 19h with mouse liver
microsomes contained the following at the indicated final concen-
trations: 50 mM potassium phosphate buffer (pH 7.4) and 5 mM
+
67.9, 56.2, 48.9. ESI-MS m/z: 529 [M+H] .
4
.2. Pharmacology
2
MgCl , microsomes (1 mg/mL) with or without 2 mM NADPH.
The mixtures were preincubated at 37 °C both under normoxia
Two human cancer cell lines (A549 and HT-29) were purchased
and hypoxia. The metabolism reaction was initiated by the addi-
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, penicillin (100 units/mL) and streptomycin (100 mg/mL)
tion of 5 lM test compound to the incubation mixture. After
30 min, the reaction mixture was quenched using equal volume
of a quenching solution containing 0.1% formic acid/acetonitrile.
The samples were centrifuged for 10 min at 10000g. The superna-
tant was transferred to a vial for analysis using a generic HPLC
method.
and incubated in hypoxic atmosphere with 2% O
2 2
, 5% CO and in
normoxic atmosphere with 20% O , 5% CO at 37 °C.
2
2
Please cite this article in press as: Cheng, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.038

1
0
W. Cheng et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4
.6. Molecular modeling
References and notes
1
2
3
.
.
.
Chen, Y.; Hu, L. Med. Res. Rev. 2009, 29, 29.
Wilson, W. R.; Hay, M. P. Nat. Rev. Cancer 2011, 11, 393.
Liu, K.; Zhu, H. L. Anticancer Agents Med. Chem. 2011, 11, 687.
The EGFR-gefitinib protein-ligand complex crystal structure
PDB ID: 2ITY) was chosen as the template to compare the docking
(
mode between compound 19h bound to EGFR. The molecular
docking procedure was performed by using C-DOCKER protocol
within Discovery Studio 2.1. For ligand preparation, the 3D struc-
ture of compound 19h was generated and minimized using Discov-
ery Studio 2.1. 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
the gefitinib molecule was removed and compound 19h was
placed during the molecular docking procedure. Types of interac-
tions 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. Duan, J. X.; Jiao, H.; Kaizerman, J.; Stanton, T.; Evans, J. W.; Lan, L.; Lorente, G.;
Banica, M.; Jung, D.; Wang, J. W.; Ma, H. Y.; Li, X. M.; Yang, Z. J.; Hoffman, R. M.;
Ammons, W. S.; Hart, C. P.; Matteucci, M. J. Med. Chem. 2008, 51, 2412.
5
. Available from: http://clinicaltrials.gov/.
6. Yarden, Y.; Sliwkowski, M. X. Nat. Rev. Mol. Cell Biol. 2001, 2, 127.
7. Mitsudomi, T.; Yatabe, Y. FEBS J. 2010, 277, 301.
8
9
.
.
Kalyankrishna, S.; Grandis, J. R. J. Clin. Oncol. 2006, 24, 2666.
Haldar, K.; Gaitskell, K.; Bryant, A.; Nicum, S.; Kehoe, S.; Morrison, J. Cochrane
Database Syst. Rev. 2011.
10. Rego, R. L.; Foster, N. R.; Smyrk, T. C.; Le, M.; O’Connell, M. J.; Sargent, D. J.;
Windschitl, H.; Sinicrope, F. A. Br. J. Cancer 2010, 102, 165.
11. Warnault, P.; Yasri, A.; Coisy-Quivy, M.; Cheve, G.; Bories, C.; Fauvel, B.;
Benhida, R. Curr. Med. Chem. 2013, 20, 2043.
12. Wang, T.; Niki, T.; Goto, A.; Ota, S.; Morikawa, T.; Nakamura, Y.; Ohara, E.;
Ishikawa, S.; Aburatani, H.; Nakajima, J.; Fukayama, M. Cancer Sci. 2007, 98,
506.
13. Nishi, H.; Nishi, K. H.; Johnson, A. C. Cancer Res. 2002, 62, 827.
14. Yun, C. H.; Boggon, T. J.; Li, Y. Q.; Woo, M. S.; Greulich, H.; Meyerson, M.; Eck, M.
J. Cancer Cell 2007, 11, 217.
1
5. Stamos, J.; Sliwkowski, M. X.; Eigenbrot, C. J. Biol. Chem. 2002, 277, 46265.
Acknowledgments
16. Wood, E. R.; Truesdale, A. T.; McDonald, O. B.; Yuan, D.; Hassell, A.; Dickerson,
S. H.; Ellis, B.; Pennisi, C.; Horne, E.; Lackey, K.; Alligood, K. J.; Rusnak, D. W.;
Gilmer, T. M.; Shewchuk, L. Cancer Res. 2004, 64, 6652.
We thank the support of the National Natural Science Founda-
tion of China (81072575, 21072171). We thank Dr. Ke Ding
1
7. Solca, F.; Dahl, G.; Zoephel, A.; Bader, G.; Sanderson, M.; Klein, C.; Kraemer, O.;
Himmelsbach, F.; Haaksma, E.; Adolf, G. R. J. Pharmacol. Exp. Ther. 2012, 343,
342.
(
Guangzhou Institutes of Biomedicine and Health, Chinese Acad-
1
8. Tasler, S.; Muller, O.; Wieber, T.; Herz, T.; Pegoraro, S.; Saeb, W.; Lang, M.;
Krauss, R.; Totzke, F.; Zirrgiebel, U.; Ehlert, J. E.; Kubbutat, M. H. G.; Schachtele,
C. Bioorg. Med. Chem. 2009, 17, 6728.
emy of Sciences) for performing the EGFR activity evaluation. We
also thank Mrs Jianyang Pan (Pharmaceutical Informatics Institute,
Zhejiang University) for performing the NMR and MS spectrometry
tests and Mrs Xiaodan Wu (Analysis Center of Agrobiology and
Environmental Sciences, Zhejiang University) for performing the
HPLC-Q-ToF-MS test.
19. Chandregowda, V.; Kush, A. K.; Reddy, G. C. Eur. J. Med. Chem. 2009, 44, 3046.
2
0. Chang, S. H.; Zhang, L. W.; Xu, S. L.; Luo, J. F.; Lu, X. Y.; Zhang, Z.; Xu, T. F.; Liu, Y.
X.; Tu, Z. C.; Xu, Y.; Ren, X. M.; Geng, M. Y.; Ding, J.; Pei, D. Q.; Ding, K. J. Med.
Chem. 2012, 55, 2711.
21. Varghese, A. J.; Whitmore, G. F. Chem. Biol. Interact. 1985, 56, 269.
2
2. Carmi, C.; Galvani, E.; Vacondio, F.; Rivara, S.; Lodola, A.; Russo, S.; Aiello, S.;
Bordi, F.; Costantino, G.; Cavazzoni, A.; Alfieri, R. R.; Ardizzoni, A.; Petronini, P.
G.; Mor, M. J. Med. Chem. 2012, 55, 2251.
Supplementary data
2
3. Gopinath, V. S.; Pinjari, J.; Dere, R. T.; Verma, A.; Vishwakarma, P.; Shivahare,
R.; Moger, M.; Goud, P. S. K.; Ramanathan, V.; Bose, P.; Rao, M. V. S.; Gupta, S.;
Puri, S. K.; Launay, D.; Martin, D. Eur. J. Med. Chem. 2013, 69, 527.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.038.
24. Papadopoulou, M. V.; Ji, M.; Rao, M. K.; Bloomer, W. D. Oncol. Res. 2003, 14, 21.
Please cite this article in press as: Cheng, W.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.10.038