Design, synthesis and biological activities of novel oxazolo[4,5- g ]quinazolin-2(1H)-one derivatives as EGFR inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.07.008

A series of oxazolo[4,5-g]quinazolin-2(1H)-one derivatives employing Erlotinib as lead compound were synthesized and evaluated for their EGFR inhibition activity. These compounds having variation at the 1 and 8-position, included ether and esters hydrophilic side-chain and aromatic head fragment, respectively. All these compounds were evaluated by EGFR inhibition and two anti-proliferation assays in vitro. Four compounds were found more potent than Erlotinib in EGFR-TK assay. Furthermore, compounds 18, 42 and 50 also had good to excellent anti-proliferation activity against human epidermoid cancer cell line (KB) and renal cell carcinoma cell line (A498). Finally, compound 50 presented remarkably higher inhibition efficacy towards tumor growth than Erlotinib in a mouse lewis lung cancer (LLC) xenograft model. Furthermore, compound 50 displayed the most distinguished effect on extending the survival period of the tumor-bearing mice.

Full text of DOI:10.1016/j.ejmech.2015.07.008

European Journal of Medicinal Chemistry 101 (2015) 462e475
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological activities of novel oxazolo[4,5-g]
quinazolin-2(1H)-one derivatives as EGFR inhibitors
Siyuan Yin, Liliang Zhou, Jinsheng Lin, Lingjing Xue**, Can Zhang*
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, 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 oxazolo[4,5-g]quinazolin-2(1H)-one derivatives employing Erlotinib as lead compound were
synthesized and evaluated for their EGFR inhibition activity. These compounds having variation at the 1
and 8-position, included ether and esters hydrophilic side-chain and aromatic head fragment, respec-
tively. All these compounds were evaluated by EGFR inhibition and two anti-proliferation assays in vitro.
Four compounds were found more potent than Erlotinib in EGFR-TK assay. Furthermore, compounds 18,
42 and 50 also had good to excellent anti-proliferation activity against human epidermoid cancer cell line
(KB) and renal cell carcinoma cell line (A498). Finally, compound 50 presented remarkably higher in-
hibition efficacy towards tumor growth than Erlotinib in a mouse lewis lung cancer (LLC) xenograft
model. Furthermore, compound 50 displayed the most distinguished effect on extending the survival
period of the tumor-bearing mice.
Received 16 March 2015
Received in revised form
3 July 2015
Accepted 6 July 2015
Available online 10 July 2015
Keywords:
EGFR inhibitor
Oxazolo[4,5-g]quinazolin-2(1H)-one
scaffold
© 2015 Elsevier Masson SAS. All rights reserved.
Protein tyrosine kinases (PTK)
Docking
SAR
1. Introduction
one scaffold and adopting the hydrophilic side-chain at its 1-
position for EGFR small molecule inhibitors [14]. So based on this
Epidermal growth factor receptor (EGFR) is a tyrosine kinase
receptor which plays an essential role in normal cell growth and
differentiation, and is involved in tumor proliferation and survival
[1,2]. EGFR over-expression is a common feature in many human
solid malignancies including non-small-cell lung cancer, ovarian
cancer, breast cancer, etc., which is associated with poor clinical
prognosis [3e6].
Quinazoline-containing derivatives form an important class of
synthetic products and represent an attractive scaffold for EGFR
inhibitors. Huge interest has been attracted over the past years
because of their varied biological activities, notably as kinase in-
hibitors [7e10]. The 4-anilinoquinazoline scaffold (Fig. 1) has led to
the development and the marketing of a series of anti-tumor agents
such as Gefitinib [11], Erlotinib [12], and Lapatinib [13].
hypothesis that oxazolo[4,5-g]quinazolin-2(1H)-one scaffold is
useful as a template for creation of new EGFR small molecule in-
hibitors, we synthesized a novel series of oxazolo[4,5-g]quinazolin-
2(1H)-one derivatives adopting 2-methoxyethoxyl and 4-ethyloxy-
4-oxobutyl group as side-chain at 1-position respectively and
evaluated their EGFR inhibition activity comparing with Erlotinib
(Fig. 2). Eventually, compounds 18, 42 and 50 were discovered as
promising inhibitors against EGFR Therefore, we reported com-
pounds 18, 42 and 50 as promising candidates for clinical devel-
opment as novel EGFR kinase inhibitors.
2. Results and discussion
2.1. Chemistry
Recently, we developed oxazolo[4,5-g]quinazolin-2(1H)-one
derivatives, using Gefitinib as a lead compound. In that work, we
found the potential of building oxazolo[4,5-g]quinazolin-2(1H)-
The synthetic method for the target compounds is showed in
Scheme 1 and Scheme 2. Nitration of the starting material ethyl 4-
hydroxybenzoate (4) led to ethyl 4-hydroxy-3-nitrobenzoate (5),
followed by reduction with tin (II) chloride dihydrate to give ethyl
3-amino-4-hydroxybenzoate (6), which was cyclocondensed with
di-(1H-imidazol-1-yl)-methanone (CDI) in anhydrous THF to afford
ethyl 2-oxo-2,3-dihydrobenzo-oxazole-5-carboxylate (7). Nitration
* Corresponding author.
** Corresponding author.
E-mail addresses: xuelingjing65@163.com (L. Xue), zhangcan@cpu.edu.cn
(C. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2015.07.008
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
463
Fig. 1. The structures of Gefitinib, Erlotinib and Lapatinib.
Fig. 2. The design inhibitors of EGFR.
of compound 7 with concentrated HNO3, and subsequent purifi-
Erlotinib as the positive control.
cation via column chromatography, yielded compound 8 which was
then reduced with iron power to produce compound 9.
The series I compounds were obtained by alkylation of com-
pound 9 with 1-chloro-2-methoxyethane, cyclization with for-
mamidine acetate in EtOH, chlorination with thionyl chloride using
DMF as catalyst, and nucleophilic displacement with aniline
(Scheme 1). Likewise, the series II compounds were synthesized as
described in Scheme 2.
Most compounds exhibited moderate to high inhibition activ-
ities with EGFR kinase. These results revealed that introducing the
moderate hydrophilic groups to the 1-position contributed to the
activity. As indicated by the results of compounds 49, 50, 52 on
compared to compounds 32, 38, 39, aniline moieties directly con-
juncted to the 8-position increased EGFR inhibitory activities as
compared to benzyl amino groups. Compounds containing some
functional groups at 8-position, such as halogens, hydroxyl groups,
alkoxyl groups etc., caused an increase in EGFR inhibition with the
exception of compounds 36, 44, 48 and 22. This is due in part to
their relatively large structures, which sterically hindered their
interaction with the active site of the kinase. Additionally, the
compounds which contained functional groups such as t-butyl,
sulfanilamido group, etc., also demonstrated decreased inhibition
activities like compounds 37, 52 and 20.
2.2. Biological activities
2.2.1. Kinase inhibitory activity
Kinase assay was carried out to test the inhibitory activity of the
designed compounds at the concentration of 0.04 mg/mL. As
shown in Table 1, most of compounds inhibited EGFR dramatically,
whereas compounds 21, 22, 36, and 44 had no obvious inhibitory
activity. The IC50 values of EGFR inhibition were further tested using
The IC50 values of compounds 18, 42 and 50 were 0.026
mM,
0.0073 M and 0.0087 M respectively. This is a significant
m
m

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S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
Scheme 1. Reagents and reaction condition: (a) fuming HNO3, acetic acid, DCM, ꢀ15 ꢁC, 1 h; (b) SnCl2$2H2O, conc. HCl, 45 ꢁC, 7 h; (c) CDI, anhydrous THF, 25 ꢁC, 2 h; (d) conc. HNO3,
60 ꢁC, 8 h; (e) reduced iron powder, acetic acid, MeOH, reflux, 3 h; (f) 1-chloro-2-methoxyethane, DMF, K2CO3, KI, 70 ꢁC, 12 h; (g) formamidine acetate, ethanol, reflux, 5 h; (h) SOCl2,
DMF(cat.), reflux, 16 h; (i) phenyl aniline, i-PrOH, reflux, 6e24 h.
improvement when compared to Erlotinib which has an IC50 value
of 0.035 M (Table 1).
2.3. Structureeactivity relationships
m
As shown in Fig. 4, N1, N3 atoms in pyrimidine ring are crucial for
compound binding. Introducing moderate hydrophilic side-chain,
such as 2-methoxyethoxyl and 4-ethyloxy-4-oxobutyl group can
achieve medium to good inhibition activity. The proton in Fragment
B is essential, and if phenyl amines are replaced by benzylaminos,
the inhibition activities will be reduced, such as compound 38 and
compound 39. Halogen, hydroxy, alkyl and benzyloxy groups
introduced in the phenyl amines improved the inhibition activity,
such as compound 18, 22, 40, 49, 50. However, tert-butyl and sul-
fonamide groups introduced in phenyl amines may reduce the
compound inhibition activity, such as compounds 20, 37, 52 (Fig. 4
and Table 1).
2.2.2. Anti-proliferative effects on KB and A498 cells
In our study, KB cells and A498 cells overexpressing EGFR [17,18]
were selected as cell models to evaluate the anti-proliferation of
the synthesized compounds. The cellular anti-proliferative effects
were mainly coincident with kinase inhibition activity, most com-
pounds exhibited moderate to high inhibition activities and cyto-
toxicities to KB and A498 cells. In addition, compounds with
benzoheterocycle introduced to the 8-position also showed mod-
erate cytotoxicity for KB and A498 cells. For example, compound 45
was found to be effective at concentration of 38.4
respectively, and exhibited high inhibition of EGFR with IC50 of
0.13 M. Interestingly, the compounds 26 and 54 only showed
mM and 26.8 mM,
m
moderate activity against EGFR yet exhibited promising anti-
proliferative effects for KB and A498 cells, and this result indicated
that compounds 26 and 54 may work through multiple molecular
mechanisms (Table 2).
2.4. Molecular docking study
Docking study was carried out for the target compounds into
EGFR using Glide version 7.3, Tripos Inc and Molegro virtual docker
version 2007. The crystal structure of the enzyme with Erlotinib
(ID: 1M17) was obtained from protein data bank (PDB).
2.2.3. In vivo antitumor activity
For the in vivo antitumor activity, the xenograft mouse lewis
lung cancer (LLC) tumor mice model was used. LLC cells also
overexpress EGFR [19]. Compound 50 was further evaluated for its
in vivo antitumor efficacy in the LLC xenograft mouse model. As
shown in Fig. 3, compound 50 and Erlotinib were well tolerated and
displayed significant in vivo antitumor efficacy when compared
with water as vehicle control, and compound 50 presented
remarkably higher inhibition efficacy towards tumor growth than
Erlotinib (P < 0.001) (Fig. 3a). Furthermore, compound 50 displayed
the most distinguished effect on extending the survival period of
the tumor-bearing mice (Fig. 3b), and no significant change of the
body weight during the study was seen.
It is found that Gefitinib mimic ATP and the ATP binding site is
sandwiched between the lobes where ATP forms critical hydrogen
bonding interactions and the hinge region. The binding of ATP itself
involves two important hydrogen bonding interactions with the
amino acids Gln767 and Met769 in the kinase backbone, and the
quinazoline ring of Erlotinib contains two nitrogen atoms that act
as hydrogen bond acceptors like ATP. The kinase or catalytic domain
includes an N-terminal lobe, which consists mainly of
b-strands but
contains one -helix and C helix. The C-terminal lobe is mainly a-
a
helical, and a short strand termed the hinge region connects the
two lobes [15].
The docking studies were progressed by positioning the

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
465
Scheme 2. Reagents and reaction condition: (a) ethyl 4-bromobutanoate, acetonitrile, K2CO3, KI, reflux, 4 h; (b) formamidine acetate, ethanol, reflux, 24 h; (c) SOCl2, DMF(cat.),
reflux, 7 h; (d) phenyl aniline, i-PrOH, reflux,6e24 h; (e) 1H-indazol-5-amine, i-PrOH, reflux, 13 h; (f) phenylmethanamine, i-PrOH, TEA, 50 ꢁC, 24 h.
compounds in the Erlotinib binding site in accordance with the
published crystal structures of quinazoline core derivatives binding
to EGFR [16], and the results had revealed that the oxazolo[4,5-g]
quinazolin-2(1H)-one ring bounded to a narrow hydrophobic
pocket in the N-terminal domain of EGFR active site where N5 of
the oxazolo[4,5-g]quinazolin-2(1H)-one core interacted with the
backbone NH of Met769 via a hydrogen bond. A water molecule-
mediated hydrogen bonding interaction between the N7 and the
Thr830 side chain was observed which was similar to Erlotinib.
These interactions underscored the importance of nitrogen atoms
for the binding and the subsequent inhibitory capacity. The aniline
moiety at the C4 lay in a deep hydrophobic pocket. The 30-
hydroxyphenyl group at the C4 of aniline moiety lay in the same
site of the 30-ethynyllbenzyl moiety of Erlotinib. It was glad to
found a hydrogen bonding interaction existing between the 30-OH
oxygen atom and the backbone OH of Thr766 or NH of Leu764. A
hydrogen bonding interaction between 30-OH hydrogen atom and
the backbone carbonyl oxygen of Ala719 could also be noticed in
Fig. 5.
tumor growth in a LLC xenograft mouse model and resulted in
prolonged survival time. In conclusion, the findings presented
herein showed that using the oxazolo[4,5-g]quinazolin-2(1H)-one
scaffold decorated with 2-methoxyethoxyl or 4-ethyloxy-4-
oxobutyl group in position 1 is an excellent strategy for the
development of EGFR inhibitors.
4. Experimental sections
4.1. Chemistry experiment
4.1.1. Chemistry: general procedures
All commercially available starting materials, reagents and
solvents were used without further purification unless other-
wise stated. Melting points were determined with an Electro
thermal melting point apparatus, and are uncorrected. High-
resolution mass spectra (HRMS) were recorded on QSTAR XL
Hybrid MS/MS mass spectrometer. Infrared (IR) spectra were
recorded on Thermo FTIR spectrometer (KBr disks).1H NMR and
13C NMR spectra on a Bruker AV 300 or 500 MHz spectrometer
were recorded in DMSO-d6 or CDCl3. Chemical shifts are re-
ported in d (ppm) units relative to the internal standard tetra-
methylsilane (TMS). The reaction conditions were not optimized
for reaction yields. All oxygen-sensitive or moisture-sensitive
reactions were run under nitrogen atmosphere. All the re-
actions were monitored by thin layer chromatography (TLC) on
pre-coated silica gel G plates at 254 nm under a UV laMp using
ethyl petroleum ether/acetate or DCM/MeOH as eluent. Column
chromatography separations were obtained on silica gel
(200e300 mesh).
3. Conclusion
In summary, a series of novel oxazolo[4,5-g]quinazolin-2(1H)-
one derivatives were synthesized and subjected to pharmacological
evaluation. The results showed that most of the oxazolo[4,5-g]
quinazolin-2(1H)-one derivatives possessed moderate to high
EGFR inhibition activities. Protein tyrosine kinase inhibitions assay
indicated that many of these derivatives were good inhibitors of
EGFR with IC50 of 1.0e50
mM. Furthermore, these derivatives
demonstrated high anti-proliferation potencies against KB and
A498 cells. Among them three compounds 18, 42, and 50 exhibited
more potent inhibition activities than the reference Erlotinib which
merited further evaluation. Especially, 50 substantially inhibited
4.1.2. Purity analysis
The purity of the synthesized compounds were measured by

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S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
Table 1
The inhibition and IC50 values of the designed compounds toward EGFR kinase.
Inhibitiona/%
88
Enzymatic. IC50
0.47 0.18
(mM)
b
Comp.
R1
R2
16
eCH2CH2OCH3
17
18
eCH2CH2OCH3
eCH2CH2OCH3
92
97
0.19 0.03
0.026 0.017*
19
20
eCH2CH2OCH3
eCH2CH2OCH3
91
99
5.1 1.3
>10
21
22
eCH2CH2OCH3
eCH2CH2OCH3
78
40
>10
0.34 0.08
23
24
eCH2CH2OCH3
eCH2CH2OCH3
81
78
>10
>10
25
26
eCH2CH2OCH3
eCH2CH2OCH3
92
98
0.56 0.08
1.3 0.1
27
28
eCH2CH2OCH3
eCH2CH2OCH3
94
98
5.5 0.5
0.44 0.09
29
30
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
93
96
7.8 1.4
5.7 1.3
31
eCH2(CH2)2COOC2H5
92
>10
32
33
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
93
96
>10
1.2 0.5

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
467
Table 1 (continued )
Inhibitiona/%
Enzymatic. IC50 (mM)
b
Comp.
R1
R2
34
eCH2(CH2)2COOC2H5
92
>10
35
36
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
93
97
7.5 2.1
>10
37
38
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
92
88
>10
>10
39
40
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
90
97
>10
0.78 0.23
41
42
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
96
99
>10
0.0073 0.0021*
43
44
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
99
61
>10
>10
45
46
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
99
86
0.13 0.02
2.3 0.13
47
48
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
99
78
0.14 0.01
0.053 0.019
49
eCH2(CH2)2COOC2H5
99
0.020 0.014*
50
51
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
100
99
0.0087 0.0179*
0.16 0.02
(continued on next page)

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S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
Table 1 (continued )
Inhibitiona/%
Enzymatic. IC50 (mM)
b
Comp.
R1
R2
52
eCH2(CH2)2COOC2H5
82
>10
53
eCH2(CH2)2COOC2H5
82
>10
54
55
eCH2(CH2)2COOC2H5
eCH2(CH2)2COOC2H5
85
98
0.66 0.05
1.5 0.2
Erlotinib
100
0.035 0.012
a
Inhibition of EGFR kinase of compounds under the concentration of 0.04 mg/mL.
Data represent mean SD, n ¼ 3, *P < 0.05 vs. Erlotinib.
b
Table 2
4.1.3. Ethyl 6-nitro-2-oxo-2, 3-dihydrobenzo[d]oxazole-5-
carboxylate (8)
Cellular anti-proliferative data for some of the designed compounds toward KB and
A498 cells.
Ethyl 4-hydroxybenzoate 4 (100 g, 0.602 mol) was dissolved in
the mixture of DCM (400 mL) and glacial acetic acid (150 mL) and
cooled to ꢀ15 ꢁC. Fuming nitric acid (70 mL) was slowly added and
the reaction was stirred at ꢀ15 ꢁC for 1 h. When the starting ma-
terial was completely consumed, the solvent was slowly poured to
400 mL cold water and extracted with DCM (3 ꢂ 300 mL). The
organic layer was washed with brine, dried over anhydrous Na2SO4
and evaporated. The crude product compound 5 (123 g, yield, 97%)
was obtained.
a
a
Comp.
Cell inhibition IC50
KBb A498c
1.1 0.3*
(
mM)
Comp.
Cell inhibition IC50 (mM)
KBb
A498c
18
26
37
45
47
50
54
17.6 4.2
6.2 1.7*
8.0 1.5*
26.8 4.2
36.4 5.8
7.6 1.1*
8.7 1.3*
19
28
42
46
49
51
Erlotinib
11.7 2.7
33.5 13.3
6.3 1.1*
17.1 3.7
19.5 4.1
61.6 17.6
16.6 4.3
47.0 15.6
11.1 2.3*
15.2 1.6
18.6 3.4
41.1 7.4
5.0 0.7*
25.6 5.1
6.1 1.9*
18.7 4.1
38.4 5.8
17.9 2.9
3.7 0.3*
5.5 0.6*
Compound 5 (123 g, 0.58 mol) was added to conc. HCl (370 mL),
and stannous chloride dihydrate (395 g, 1.75 mol) was portionwise
added to the mixture at 0 ꢁC, then heated to 45 ꢁC and stirred for 7 h
dramatically. The mixture was cooled to room temperature and
filtered, and the solid was washed with acetate (200 mL), then
dissolved in 500 mL water and basified with saturate NaHCO3 until
a pH of 9e10 was obtained. The resulting precipitate was filtered
and washed with water, over anhydrous Na2SO4 and evaporated to
give compound 6 as pale yellow solid (95.2 g, yield, 90%).
a
Data represent mean SD, n ¼ 3, *P < 0.05 vs. Erlotinib.
b
KB cells are a cell line derived from a human carcinoma of the nasopharynx that
overexpresses both EGFR and Src.
c
A498 cells are a renal cell carcinoma (RCC) cell line that overexpresses both
EGFR and Src.
high performance liquid chromatography (HPLC, Shimadzu LC-
2010 system, Kyoto, Japan) equipped with a Diamonsil C18 col-
umn (5
consisted of acetonitrile and water with a flow rate of 1.0 mL/min.
The detection wavelength was 340 nm and sample injected volume
was 20
had a purity of ꢃ95%.
1, 10-Carbonyl-diimidazole (CDI, 89.5 g, 0.552 mol) was added to
a solution of compound 6 (95.2 g, 0.53 mol) in anhydrous THF
(400 mL) at room temperature and stirred for 2 h at the same
temperature. Diluted aqueous HCl solution (50 mL) was added to
the mixture and the aqueous phase was extracted with EtOAc. The
mm particle size, 250 mm ꢂ 4.6 mm). The mobile phase
mL. All compounds evaluated for EGFR inhibitory potency
Fig. 3. (a) In vivo antitumor effect of compound 50 in a LLC xenograft mouse model. Eighteen mice were weighed and randomly divided into three groups (n ¼ 6): 1) Control; 2)
Erlotinib; 3) Compound 50. Mice bearing established LLC tumor xenografts were dosed with compound 50 (100 mg/kg.qd.po) over a 14 day period. Erlotinib (100 mg/kg.qd.po) was
employed as reference drug. Tumor size was measured every other day. Data are shown as the mean SD (n ¼ 6). Statistical significance (p < 0.001) for antitumor efficacy, based
upon tumor growth relative to the saline controls. (b) Survial curves of LLC tumor bearing mice received different treatments.

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
469
Fig. 4. Structureeactivity relationships identified in this study.
organic solution was washed with saturated aqueous NaCl solution,
4.1.4. Ethyl 6-amino-2-oxo-2, 3-dihydrobenzo[d]oxazole-5-
carboxylate (9)
dried over anhydrous Na2SO4, and evaporated. The resulting solid
compound 7 (103.5 g, yield, 95%) was essentially pure and could be
used to the following reaction.
A mixture of compound 8 (88.3 g, 0.35 mol), reduced iron power
(60.8 g, 1.08 mol), acetic acid (600 mL) in 2 L CH3OH was me-
chanically stirred at reflux for 3 h. To this mixture was added
aqueous ammonia until the pH was adjusted to 10. The boiling
mixture was filtered and the solid was washed with hot CH3OH
(500 mL). The solvent was removed from filtrate and the resulting
solid was extracted with boiling acetone (600 mL) and filtered. The
acetone extracts were concentrated in vacuo. The residue was
recrystallized from EtOAc (300 mL) to 71.5 g (yield, 92%) of com-
pound 9 as light brown solid. The compound was used without
Compound 7 (103.5 g, 0.500 mol) was added portionwise to
500 mL conc. HNO3 at the temperature of 0e5 ꢁC. The mixture was
heated to 60 ꢁC and stirred for 8 h at this temperature, after the
starting material was completely consumed, the solution was
poured to 2.5 L cold water, the resulting precipitate was filtered and
washed with chill water, dried, the crude product was purified by
silica gel column chromatography with petroleum ether/acetate (4/
1, v/v) as mobile phase to give compound 8 (88.3 g, yield, 70%) as a
pale yellow solid. Mp: 162e164 ꢁC, 1H NMR (300 MHz, DMSO-d6)
d:
further purification: Mp: 173e175 ꢁC, 1H NMR (300 MHz, CDCl3)
d:
12.60 (1H, bs), 8.16 (1H, s), 4.30 (2H, q, J ¼ 6.9 Hz), 1.29 (3H, t,
J ¼ 6.9 Hz); IR (film, cmꢀ1): 3289, 3062, 2990, 1788, 1712, 1626,
1540, 1477, 1372, 1292, 1247, 1104, 929, 893, 651.
8.63 (1H, bs), 7.57 (1H, s), 6.53 (1H, s), 4.35(2H, q, J ¼ 7.0 Hz), 1.39
(3H, t, J ¼ 7.0 Hz); IR (film, cmꢀ1): 3459, 3351, 3255, 2991, 1775,
1665, 1583, 1426, 1279, 1229, 1126, 1077, 929, 866, 706.
Fig. 5. Binding model of compound 50 (yellow) in complex with EGFR (PDB ID: 1M17) and Erlotinib (purple). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

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S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
4.1.5. General procedure for the synthesis of 1-(2-methoxyethyl)
oxazolo[4,5-g] quinazolin-2(1H)-one derivatives (16e28)
4.1.5.3.2. 8-(3-Ethynylphenylamino)-1-(2-methoxyethyl)oxazolo
[4,5-g]quinazolin-2(1H)-one (17). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf ¼ 0.29. White solid,
324 mg, yield, 84%; Mp: 215e217 ꢁC; HPLC purity: 97%; HRMS, ESIþ,
m/z: Calcd for C20H17N4O3 (MþH)þ, 361.1295; found, 361.1298; 1H
4.1.5.1. Ethyl 6-amino-3-(2-methoxyethyl)-2-oxo-2,3-dihydrobenzo
[d]oxazole-5-carboxylate (10). K2CO3 (24.8 g, 180 mmol), KI
(0.75 g, 4.5 mmol) and 1-chloro-2-methoxyethane (17.0 g,
180 mmol) were added successively to a solution of the compound
9 (20 g, 90.0 mmol) in DMF (120 mL) and the mixture was stirred at
70 ꢁC for 5 h. The mixture was poured to water (500 mL) and the
aqueous phase was extracted with AcOEt (3 ꢂ 400 mL). The organic
solution was washed with water follow by saturated aqueous NaCl
solution, dried over anhydrous Na2SO4, and evaporated to afford
the compound 10 (24.0 g, yield, 95%), which was used to the next
reaction without further purification. Mp: 187e189 ꢁC; 1H NMR
NMR (DMSO-d6) d: 9.64 (1H, bs), 8.53 (1H, s), 8.26 (1H, s), 7.96 (1H,
s), 7.86 (1H, d, J ¼ 8.1 Hz), 7.63 (1H, s), 7.39 (1H, dd, J ¼ 8.1 Hz,
J ¼ 7.5 Hz), 7.20 (1H, d, J ¼ 7.5 Hz), 4.16 (1H, s), 4.05 (2H, t,
J ¼ 5.3 Hz), 3.76 (2H, t, J ¼ 5.3 Hz), 3.26 (3H, s); 13C NMR (75 MHz,
DMSO-d6) d: 156.94, 153.47, 152.98, 147.19, 146.21, 139.53,130.95,
128.87, 126.65, 124.91, 123.29, 122.69, 121.76, 111.76, 106.58, 100.61,
83.40, 80.51, 58.12, 41.94.
4.1.5.3.3. 8-(3, 4-Difluorophenylamino)-1-(2-methoxyethyl)oxa-
zolo[4,5-g]quinazolin-2(1H)-one (18). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf ¼ 0.28. White solid,
313 mg, yield, 87%; Mp: 220e224 ꢁC; HPLC purity: 98%; HRMS,
ESIþ, m/z: Calcd for C18H15F2N4O3 (MþH)þ, 373.1107; found,
(500 MHz, CDCl3) d: 7.52 (1H, s), 6.51 (1H, s), 5.77 (2H, bs), 4.35 (2H,
q, J ¼ 7.1 Hz), 3.94 (2H, t, J ¼ 5.4 Hz), 3.69 (2H, t, J ¼ 5.4 Hz), 3.36 (3H,
s), 1.40 (3H, t, J ¼ 7.1 Hz); IR (film, cmꢀ1): 3468, 3358, 2987, 2891,
1780, 1691, 1584, 1489, 1432, 1370, 1328, 1280, 1226, 1111, 1064, 954,
832, 789, 746, 660.
373.1111; 1H NMR (500 MHz, DMSO-d6)
d: 9.76 (1H, bs), 8.27 (1H, s),
8.06 (1H, m), 7.71 (1H, s), 7.69 (1H, s), 7.59 (1H, m), 7.48 (1H, q,
J ¼ 9.2 Hz), 4.08 (2H, t, J ¼ 5.5 Hz), 3.79 (2H, t, J ¼ 5.5 Hz), 3.29 (3H,
4.1.5.2. 1-(2-Methoxyethyl) oxazolo[4, 5-g]quinazoline-2, 8(1H,7H)-
dione (11). A solution of compound 10 (24.0 g, 85.5 mmol) and
formamidine acetate (10.7 g, 102.6 mmol) in ethanol (120 mL) was
refluxed for 24 h under the protection of N2. The mixture was
cooled to room temperature and filtered, and the solid was washed
with EtOH (100 mL) and water (50 mL), and dried to give com-
pound 11 as white power (17.9 g, yield, 80%); Mp: 218e219 ꢁC; 1H
s); 13C NMR (75 MHz, DMSO-d6)
d: 157.76, 153.39, 152.81, 150.31 (d,
J ¼ 12.8 Hz), 147.18, 147.12, 147.01, 146.21, 143.90 (d, J ¼ 12.8 Hz),
136.18 (d, J ¼ 3.0 Hz), 136.06 (d, J ¼ 3.0 Hz), 118.38 (d, J ¼ 3.8 Hz),
118.30 (d, J ¼ 3.8 Hz), 117.03, 116.79, 111.53, 111.36, 111.08, 106.57,
100.27, 67.70, 58.10, 48.52, 41.94.
4.1.5.3.4. 1-(2-Methoxyethyl)-8-(4-methoxyphenylamino)oxazolo
[4,5-g]quinazolin-2(1H)-one (19). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf ¼ 0.22. White solid, yield,
354 mg, 90%; Mp: 206e209 ꢁC; HPLC purity: 97%; HRMS, ESIþ, m/z:
Calcd for C19H19N4O4 (MþH)þ, 367.1401; found, 367.1398; 1H NMR
NMR (500 MHz, DMSO-d6) d: 12.27 (1H, bs), 8.06 (1H, s), 7.91 (1H,
s), 7.58 (1H, s), 4.10 (2H, t, J ¼ 5.0 Hz), 3.67 (2H, t, J ¼ 5.0 Hz), 3.25
(3H, s); IR (film, cmꢀ1): 3439, 3167, 3012, 2881, 1773, 1663, 1587,
1482, 1366, 1279, 1111, 975, 909, 751, 681.
(500 MHz, DMSO-d6) d: 9.68 (1H, bs), 8.54 (1H, s), 8.28 (1H, s), 7.87
(2H, d, J ¼ 6.2 Hz), 7.66 (1H, s), 7.42 (2H, d, J ¼ 6.2 Hz), 4.07 (2H, m),
4.1.5.3. 8-Chloro-1-(2-methoxyethyl) oxazolo[4, 5-g]quinazolin-
2(1H)-one (12). Compound 11 (17.9 g, 68.4 mmol) was added to
thionyl chloride (200 mL) with magnetic stirring, then DMF
(1.0 mL) was slowly added dropwise and the reaction flask was
refluxed for 6 h. Most of the excess of thionyl chloride and DMF was
then removed under reduced pressure and the yellow residue was
purified by silica-gel column chromatography (DCM/MeOH, 40/1),
Rf ¼ 0.25. Drying give compound 12 (17.6 g, yield, 92%); Mp:
3.79 (5H, m), 3.78 (3H, s), 3.29 (3H, s); 13C NMR (75 MHz, DMSO-d6)
d: 157.38, 155.92, 153.52, 153.35, 146.97, 131.74, 130.74, 124.51,
113.73, 111.58, 106.49, 100.60, 67.67, 58.11, 55.22, 41.93.
4.1.5.3.5. 8-(4-tert-Butylphenylamino)-1-(2-methoxyethyl)oxa-
zolo[4,5-g]quinazolin-2(1H)-one (20). Purified by silica-gel column
chromatography (DCM/MeOH, 90/1), Rf ¼ 0.27. White solid, yield,
423 mg, 86%; Mp: 241e243 ꢁC; HPLC purity: 96%; HRMS, ESIþ, m/z:
Calcd for C22H25N4O3 (MþH)þ, 393.1921; found, 393.1925; 1H NMR
150e152 ꢁC; 1H NMR (500 MHz, CDCl3)
d: 8.97 (1H, s), 7.85 (1H, s),
(125 MHz, DMSO-d6) d: 9.57 (1H, bs), 8.49 (1H, s), 8.28 (1H, s), 7.69
7.80 (1H, s), 4.16 (2H, t, J ¼ 4.9 Hz), 3.79 (2H, t, J ¼ 4.9 Hz), 3.37 (3H,
s); IR (film, cmꢀ1): 3425, 3064, 2951, 2856, 2784, 1792, 1603, 1566,
1484, 1439, 1343, 1260, 1112, 951, 863, 806, 738, 685.
(2H, d, J ¼ 8.0 Hz), 7.62 (1H, s), 7.42 (1H, d, J ¼ 8.0 Hz), 4.07 (2H, m),
3.79 (2H, m), 3.29 (3H, s), 1.32 (9H, s); 13C NMR (300 MHz, DMSO-
d6) d: 157.21, 153.44, 153.18, 147.09, 146.15, 146.07, 136.31, 130.77,
4.1.5.3.1. 8-(3-Chloro-4-fluorophenylamino)-1-(2-methoxyethyl)
124.98, 122.29, 111.65, 106.47, 100.54, 67.67, 58.05, 41.88, 33.99,
31.12.
oxazolo[4,5-g]
quinazolin-2(1H)-one
(16).
3-Chloro-4-fluoroaniline (172 mg, 1.18 mmol) was added to a so-
lution of compound 12 (300 mg, 1.07 mmol) in isopropanol (10 mL)
and refluxed for 8 h. The mixture was cooled to room temperature
and filtered, the solid was washed with chill isopropanol (5 mL), the
residue was treated with aqueous NaHCO3 (10 mL) and extracted
with EtOAc/MeOH (20:1, 30 mL). The organic layer was washed
with brine, dried over Na2SO4, and concentrated. Purified by silica-
gel column chromatography (DCM/MeOH, 40/1), Rf ¼ 0.25. Drying
gave 358 mg (yield, 86%) of the title compound as white solid: Mp:
217e220 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z: Calcd for
4.1.5.3.6. 8-(4-Chlorophenylamino)-1-(2-methoxyethyl)oxazolo
[4,5-g]quinazolin-2(1H)-one (21). Purified by silica-gel column
chromatography (DCM/MeOH, 60/1), Rf ¼ 0.29. White solid, yield,
332 mg, 84%; Mp: 203e206 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z:
Calcd for C18H16 Cl N4O3 (MþH)þ, 371.0905; found, 371.0909; 1H
NMR (500 MHz, DMSO-d6) d: 9.56 (1H, bs), 8.45 (1H, s), 8.28 (1H, s),
7.65 (2H, d, J ¼ 9.0 Hz), 7.63 (1H, s), 6.99 (2H, d, J ¼ 9.0 Hz), 4.07 (2H,
m), 3.79 (2H, m), 3.29 (3H, s); 13C NMR (75 MHz, DMSO-d6)
d:
156.91, 153.39, 152.90, 147.16, 146.21, 138.03, 130.97, 128.23, 127.22,
123.76, 111.67, 106.54, 100.45, 67.66, 58.05, 41.92.
C
18H15ClFN4O3 (MþH)þ, 389.0811; found, 389.0809; 1H NMR
4.1.5.3.7. 1-(2-Methoxyethyl)-8-(4-((3-methyl-4-(2,2,2-
trifluoroethoxy)pyridin-2-yl) methoxy)phenylamino)oxazolo[4,5-g]
quinazolin-2(1H)-one (22). Purified by silica-gel column chroma-
tography (DCM/MeOH, 40/1), Rf ¼ 0.29. White solid, yield, 517 mg,
87%; Mp: 322e325 ꢁC; HPLC purity: 97%; HRMS, ESIþ, m/z: Calcd for
(DMSO-d6) d: 9.64 (1H, bs), 8.55 (1H, s), 8.19 (1H, s), 8.10 (1H, d,
J ¼ 4.8 Hz), 7.79 (1H, m), 7.63 (1H, s), 7.45 (1H, t, J ¼ 9 Hz), 4.06 (2H,
m), 3.79 (2H, m), 3.30 (3H, s); 13C NMR (125 MHz, DMSO-d6)
d:
156.78, 154.28, 153.40, 152.85, 152.35, 147.11,146.23, 136.35, 131.00,
123.61, 122.40 (d, J ¼ 6.3 Hz), 118.79 (d, J ¼ 15.0 Hz), 116.45 (d,
J ¼ 21.2 Hz), 111.55, 106.58, 100.31, 67.69, 58.11, 41.95.
Compounds 17e28 were synthesized according to the same
procedure as compound 16.
C
27H25 F3N5O5 (MþH)þ, 556.1802; found, 556.1797; 1H NMR
(500 MHz, DMSO-d6) d: 9.56 (1H, bs), 8.44 (1H, s), 8.36(1H, m), 8.27
(1H, s), 7.66 (2H, d, J ¼ 6.2 Hz), 7.63 (1H, s), 7.15 (1H, m), 7.08 (2H, d,
J ¼ 6.2 Hz), 5.20 (2H, s), 4.92 (2H, m), 4.06 (2H, m), 3.79 (2H, m),

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
471
3.28 (3H, s), 2.24 (3H, s); 13C NMR (75 MHz, DMSO-d6)
157.28, 155.39, 154.99, 153.43 (d, J ¼ 13.5 Hz), 147.63, 147.05, 146.09,
132.11, 130.73, 124.40 (q, 245.0 Hz), 121.38, 114.50 (d,
d: 161.36,
Rf ¼ 0.27. White solid, yield, 390 mg, 86%; Mp: 277e279 ꢁC; HPLC
purity: 96%; HRMS, ESIþ, m/z: Calcd for C21H21N4O6 (MþH)þ,
J
¼
425.1456; found, 425.1461; 1H NMR (500 MHz, DMSO-d6)
d: 9.40
J ¼ 16.5 Hz), 111.58 (d, J ¼ 3.0 Hz), 107.57, 106.40, 100.65, 70.45,
(1H, bs), 8.45 (1H, s), 8.28 (1H, s), 8.16 (1H, s), 7.76 (1H, d, J ¼ 6.4 Hz),
67.69, 64.90, 64.44, 58.10, 41.93, 9.93.
7.65 (1H, s), 7.06 (1H, d, J ¼ 6.4 Hz), 4.30 (2H, m), 4.08 (2H, m), 3.80
4.1.5.3.8. 8-(3-Isopropoxyphenylamino)-1-(2-methoxyethyl)oxa-
zolo[4,5-g] quinazolin-2(1H)-one (23). Purified by silica-gel column
chromatography (DCM/MeOH, 80/1), Rf ¼ 0.26. White solid, yield,
332 mg, 83%; Mp:245e247 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z:
Calcd for C21H23N4O4 (MþH)þ, 395.1714; found, 395.1715; 1H NMR
(2H, m), 3.31 (3H, s), 1.32 (3H, m); 13C NMR (125 MHz, DMSO-d6)
d:
165.38, 157.97, 156.48, 153.52, 146.93, 146.19, 130.88, 128.17, 128.01,
126.10, 120.14, 116.23, 111.69, 106.42, 100.83, 67.76, 60.14, 58.05,
41.94, 14.19.
(500 MHz, DMSO-d6)
d: 9.53 (1H, bs), 8.54 (1H, s), 8.28 (1H, s), 7.64
4.1.6. General procedure for the synthesis of 1-(4-ethoxy-4-
oxobutyl) oxazolo[4,5-g]quinazolin-2(1H)-one derivatives (29e55)
4.1.6.1. Ethyl 6-amino-3-(4-ethoxy-4-oxobutyl)-2-oxo-2,3-
(2H, d, J ¼ 6.2 Hz), 7.50 (1H, s), 7.35 (1H, d, J ¼ 7.5 Hz), 7.29 (1H, m),
6.71 (1H, d, J ¼ 7.5 Hz), 4.62 (1H, m), 4.08 (2H, m), 3.79 (2H, m), 3.29
(3H, s), 1.31 (9H, d, J ¼ 5.7 Hz); 13C NMR (125 MHz, DMSO-d6)
d
:
dihydrobenzo[d]oxazole-5-carboxylate
(13). K2CO3
(24.8
g,
157.54, 157.04, 153.41, 153.04, 147.21, 146.12, 140.23, 130.86, 129.04,
114.36, 111.74, 110.67, 110.07, 106.50, 100.48, 69.23, 67.66, 58.04,
41.88, 21.79.
180 mmol), KI (0.75 g, 4.5 mmol) and ethyl 4-bromobutanoate
(19.3 g, 99 mmol) were added successively to a solution of the
compound 9 (20 g, 90.0 mmol) in acetonitrile (220 mL) and the
mixture was refluxed for 3 h. The mixture was poured to water
(500 mL) and the aqueous phase was extracted with AcOEt
(3 ꢂ 400 mL). The organic solution was washed with brine, dried
over anhydrous Na2SO4, and the solvent was evaporated to afford
the compound 10 (29.4 g, yield, 97%), which was used to the next
reaction without further purification. Mp: 76e78 ꢁC; 1H NMR
4.1.5.3.9. 4-(1-(2-Methoxyethyl)-2-oxo-1,2-dihydrooxazolo[4,5-g]
quinazolin-8-ylamino) benzenesulfonamide (24). Purified by silica-
gel column chromatography (DCM/MeOH, 35/1), Rf ¼ 0.25. White
solid, yield, 355 mg, 80%; Mp: 285e288 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C18H18N5O5S (MþH)þ, 416.1023; found,
416.1028; 1H NMR (125 MHz, DMSO-d6)
d: 8.9e10.2 (1H, bs), 8.58
(1H, s), 8.31 (1H, s), 8.04 (2H, d, J ¼ 8.2 Hz), 7.85 (2H, d, J ¼ 8.2 Hz),
(500 MHz, CDCl3) d: 7.45 (1H, s), 6.52(1H,s), 5.0e6.0(2H, bs), 4.36
7.66 (1H, s), 6.8e7.4 (2H, bs), 4.07 (2H, m), 3.78 (2H, m), 3.27 (3H, s);
(2H, q, J ¼ 7.1 Hz), 4.14 (2H, q, J ¼ 7.0 Hz), 3.84 (2H, t, J ¼ 7.0 Hz), 2.46
(2H, t, J ¼ 7.2 Hz), 2.11 (2H, p, J ¼ 7.0 Hz), 1.41 (2H, t, J ¼ 7.1 Hz), 1.26
(2H, t, J ¼ 7.2 Hz), 2.11 (2H, t, J ¼ 7.2 Hz); IR (film, cmꢀ1): 3462, 3354,
2985,1779,1715,1680,1587,1485,1372,1285,1230,1181,1103,1061,
953, 875, 786, 749, 677.
13C NMR (500 MHz, DMSO-d6)
d: 156.88, 153.45, 152.84, 147.34,
146.38, 142.38, 138.36, 131.16, 126.28, 121.48, 111.90, 106.63, 100.62,
67.73, 58.11, 41.99.
4.1.5.3.10. 8-(3-Acetylphenylamino)-1-(2-methoxyethyl)oxazolo
[4,5-g]quinazolin-2(1H)-one (25). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf ¼ 0.30. White solid, yield,
303 mg, 75%; Mp: 259e261 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z:
Calcd for C20H19N4O4 (MþH)þ, 379.1041; found, 379.1399; 1H NMR
4.1.6.2. Ethyl4-(2,8-dioxo-7,8-dihydrooxazolo[4,5-g]quinazolin-
1(2H)-yl)butanoate (14). A solution of compound 13 (29.4 g,
87.3 mmol) and formamidine acetate (10.9 g, 104.8 mmol) in
ethanol (120 mL) was refluxed for 24 h under the protection of N2.
The mixture was cooled to room temperature and filtered, the solid
was washed with EtOH (100 mL) and water (50 mL), and dried to
give compound 14 as white power (22.7 g, yield, 82%); Mp
(125 MHz, DMSO-d6) d: 9.83 (1H, bs), 8.55 (1H, s), 8.33 (1H, s), 8.31
(1H, s), 8.22 (1H, d, J ¼ 7.4 Hz), 7.75 (1H, d, J ¼ 7.4 Hz), 7.64 (1H, s),
7.55 (1H, t, J ¼ 7.8 Hz), 4.08 (2H, m), 3.80 (2H, m), 3.29 (3H, s), 2.62
(3H, s); 13C NMR (500 MHz, DMSO-d6)
d: 197.66, 157.07, 153.46,
153.00, 147.21, 146.22, 139.82, 137.15, 130.93, 128.75, 126.79, 123.53,
121.18, 111.85, 106.54, 100.66, 67.71, 58.09, 41.94, 26.72.
187e189 ꢁC; 1H NMR (500 MHz, CDCl3)
d: 12.28 (1H, bs), 8.06 (1H,
s), 7.90 (1H,s), 7.58(1H,s), 3.97 (4H, m), 2.45 (2H, t, J ¼ 7.2 Hz), 1.98
(2H, p, J ¼ 7.0 Hz), 1.13 (2H, t, J ¼ 7.2 Hz); IR (film, cmꢀ1): 3437, 3108,
2942, 1794, 1723, 1677, 1590, 1489, 1369, 1267, 1206, 1063, 959, 914,
751.
4.1.5.3.11. 8-(4-(4-Chlorobenzyloxy)-3-chlorophenylamino)-1-(2-
methoxyethyl)
oxazolo[4,5-g]quinazolin-2(1H)-one
(26).
Purified by silica-gel column chromatography (DCM/MeOH, 45/1),
Rf ¼ 0.26. White solid, yield, 475 mg, 87%; Mp: 305e306 ꢁC; HPLC
purity: 98%; HRMS, ESIþ, m/z: Calcd for C25H21 Cl2N4O4 (MþH)þ,
4.1.6.3. Ethyl 4-(8-chloro-2-oxooxazolo[4,5-g]quinazolin-1(2H)-yl)
butanoate (15). Compound 14 (22.7 g, 71.6 mmol) was added to
thionyl chloride (150 mL) with magnetic stirring, then DMF (1.0 mL)
was slowly added dropwise and the reaction flask was heated to
reflux for 6 h. Most of the excess of thionyl chloride and DMF was
then removed under reduced pressure and the yellow residue was
purified by silica gel column chromatography with DCM/MeOH
(100/1, Rf ¼ 0.30) as mobile phase to give compound 15 (21.6 g,
511.0934; found, 511.0932; 1H NMR (500 MHz, DMSO-d6)
d: 10.37
(1H, bs), 8.67 (2H, m), 8.50 (1H, s), 7.93 (1H, m), 7.70 (2H, d,
J ¼ 9.8 Hz), 7.49 (4H, m), 7.30 (1H, d, J ¼ 7.8 Hz), 5.24 (2H, s), 4.08
(2H, m), 3.79 (2H, m), 3.29 (3H, s); 13C NMR (125 MHz, DMSO-d6)
d:
157.00, 153.59, 152.99, 149.69, 147.01, 146.35, 135.68, 133.07, 132.47,
131.09, 129.24, 128.43, 124.08, 122.26, 121.07, 114.36, 111.52, 106.46,
100.22, 67.72, 58.13, 41.98.
4.1.5.3.12. 8-(3, 4-Dimethoxyphenylamino)-1-(2-methoxyethyl)
oxazolo[4,5-g] quinazolin-2(1H)-one (27). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.31. White
solid, yield, 343 mg, 90%; Mp: 235e238 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C20H21N4O5 (MþH)þ, 397.1508; found,
yield, 90%); Mp: 187e189 ꢁC; 1H NMR (500 MHz, CDCl3)
d: 8.81 (1H,
s), 8.02 (1H,s), 7.99 (1H,s), 3.97 (4H, m), 2.45 (2H, t, J ¼ 7.2 Hz), 1.98
(2H, m), 1.12 (2H, t, J ¼ 7.0 Hz); IR (film, cmꢀ1): 3420, 2986, 2870,
1809, 1727, 1606, 1563, 1484, 1356, 1258, 1180, 1065, 961, 861, 745,
692.
397.1511; 1H NMR (500 MHz, DMSO-d6)
d: 9.50 (1H, bs), 8.46 (1H,
4.1.6.3.1. Ethyl 4-(8-(3-Chloro-4-fluorophenylamino)-2-
s), 8.37 (1H, s), 8.25 (1H, s), 7.60 (1H, s), 7.38 (1H, s), 7.30 (1H, d,
oxooxazolo[4,5-g]quinazolin-1(2H)-yl)butanoate
(29).
J ¼ 4.9 Hz), 7.01 (1H, d, J ¼ 4.9 Hz), 4.05 (2H, m), 3.77e3.79 (8H, m),
3-Chloro-4-fluoroaniline (130 mg, 0.89 mmol) was added to a so-
lution of compound 15 (250 mg, 0.74 mmol) in isopropanol (10 mL)
and stirred at reflux for 7 h. The mixture was cooled to room
temperature and filtered, the solid was washed with chill iso-
propanol (5 mL), the residue was treated with aqueous NaHCO3
(10 mL) and extracted with EtOAc/MeOH (20:1, 30 mL). The organic
layer was washed with brine, dried over Na2SO4, and concentrated.
3.29 (3H, s); 13C NMR (125 MHz, DMSO-d6)
d: 157.34, 153.50,
148.46, 147.02, 146.04, 145.58, 132.27, 130.71, 115.19, 111.84, 111.66,
108.71, 106.49, 100.57, 67.66, 58.08, 55.74, 55.60, 41.90.
4.1.5.3.13. Ethyl 4-hydroxy-3-(1-(2-methoxyethyl)-2-oxo-1, 2-
dihydrooxazolo[4,5-g]
quinazolin-8-ylamino)benzoate
(28).
Purified by silica-gel column chromatography (DCM/MeOH, 70/1),

472
S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
Purified by silica-gel column chromatography (DCM/MeOH, 50/1),
Rf ¼ 0.30, gave 286 mg (yield, 87%) of the title compound; Mp:
245e247 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z: Calcd for
ESIþ, m/z: Calcd for C22H23N4O5 (MþH)þ, 423.1663; found,
423.1670; 1H NMR (500 MHz, DMSO-d6)
d: 9.54 (1H, bs), 8.45 (1H,
s), 8.21 (1H, s), 7.64 (1H, s), 7.62 (2H, d, J ¼ 8.8 Hz), 7.00 (2H, d,
J ¼ 8.8 Hz), 3.96 (4H, m), 3.79 (3H, s), 2.47 (2H, t, J ¼ 7.3 Hz), 2.12
(2H, p, J ¼ 6.8 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR (75 MHz, DMSO-
C
21H19ClFN4O4 (MþH)þ, 445.1073; found, 445.1081; 1H NMR
(500 MHz, DMSO-d6) d: 9.72 (1H, bs), 8.58 (1H, s), 8.20 (1H, s), 8.12
(1H, s), 7.80 (1H, m), 7.68 (1H, s), 7.48 (1H, t, J ¼ 9.1 Hz), 3.97 (4H,
d6) d: 172.10, 157.40, 155.95, 153.59, 153.28, 147.04, 146.23, 131.72,
m), 2.47 (1H, t, J ¼ 7.3 Hz), 2.13 (2H, p, J ¼ 6.9 Hz), 1.10 (3H, t,
130.71, 124.64, 111.67, 111.51, 106.40, 100.17, 59.82, 55.17, 41.63,
30.68, 22.14, 13.86.
J ¼ 7.1 Hz); 13C NMR (75 MHz, DMSO-d6)
d: 172.14, 156.81, 154.91,
153.55, 152.82, 151.69, 147.17, 146.44, 136.52 (d, J ¼ 3.0 Hz), 136.43,
130.97, 123.68, 122.50 (d, J ¼ 6.8 Hz), 118.91, 118.61, 116.48 (d,
J ¼ 21.0), 111.61, 106.50, 100.07, 59.68, 41.65, 30.66, 22.18, 13.89.
4.1.6.3.2. Ethyl 4-(8-(3-ethynylphenylamino)-2-oxooxazolo[4,5-g]
quinazolin-1(2H)-yl)butanoate (30). Purified by silica-gel column
4.1.6.3.7. Ethyl 4-(8-(4-(4-methoxybenzyloxy)phenylamino)-2-
oxooxazolo[4,5-g] quinazolin-1(2H)-yl)butanoate (36). Purified by
silica-gel column chromatography (DCM/MeOH, 45/1), Rf ¼ 0.26.
White solid, 348 mg, yield, 89%; Mp: 310e312 ꢁC; HPLC purity: 98%;
HRMS, ESIþ, m/z: Calcd for C29H29N4O6 (MþH)þ, 529.2082; found,
chromatography (DCM/MeOH, 60/1), Rf
¼
0.28. White solid,
529.2092; 1H NMR (500 MHz, DMSO-d6)
d: 9.51 (1H, bs), 8.43 (1H,
256 mg, yield, 83%; Mp: 240e243 ꢁC; HPLC purity: 97%; HRMS,
ESIþ, m/z: Calcd for C23H21N4O4 (MþH)þ, 417.1557; found, 417.1566;
s), 8.20 (1H, s), 7.62 (1H, s), 7.60 (2H, d, J ¼ 9.0 Hz), 7.38 (2H, d,
J ¼ 8.8 Hz), 7.04 (2H, d, J ¼ 9.0 Hz), 6.94 (2H, d, J ¼ 8.8 Hz), 5.03 (2H,
s), 3.96 (4H, m), 3.75 (3H, s), 2.45 (2H, t, J ¼ 7.2 Hz), 2.10 (2H, p,
1H NMR (500 MHz, DMSO-d6)
d: 9.66 (1H, bs), 8.59 (1H, s), 8.24 (1H,
s), 8.00 (1H, s), 7.92 (1H, d, J ¼ 8.0 Hz), 7.45 (1H, t, J ¼ 7.8 Hz), 7.27
(1H, d, J ¼ 7.6 Hz), 4.21 (1H, s), 3.98 (4H, m), 2.49 (2H, t, J ¼ 7.2 Hz),
2.14 (2H, p, J ¼ 6.9 Hz), 1.10 (3H, t, J ¼ 7.0 Hz); 13C NMR (75 MHz,
J ¼ 6.8 Hz), 1.08 (3H, t, J ¼ 7.1 Hz); 13C NMR (75 MHz, DMSO-d6)
d:
172.11, 158.93, 157.38, 155.06, 153.60, 153.68, 147.04, 146.26, 131.82,
130.74, 129.37, 128.97, 124.60, 114.70, 113.75, 111.51, 106.42, 100.16,
69.15, 59.83, 55.03, 41.64, 30.68, 22.15, 13.88.
DMSO-d6) d: 172.14, 156.81, 154.91, 153.55, 152.82, 151.69, 147.17,
146.44, 136.54, 136.43, 130.97, 123.68, 122.54, 122.45, 118.91, 118.61,
116.62, 116.34, 111.61, 106.50, 100.07, 59.68, 41.65, 30.66, 22.18,
13.89.
4.1.6.3.8. 4-(1-(4-Ethoxy-4-oxobutyl)-2-oxo-1,2-dihydrooxazolo
[4,5-g]quinazolin-8-ylamino) benzenesulfonamide (37). Purified by
silica-gel column chromatography (DCM/MeOH, 40/1), Rf ¼ 0.24.
White solid, 289 mg, yield, 83%; Mp: 281e284 ꢁC; HPLC purity:
98%; HRMS, ESIþ, m/z: Calcd for C21H22N5O6S (MþH)þ, 472.1285;
4.1.6.3.3. Ethyl 4-(2-oxo-8-(phenylamino)oxazolo[4,5-g]quinazo-
lin-1(2H)-yl) butanoate (31). Purified by silica-gel column chro-
matography (DCM/MeOH, 50/1), Rf ¼ 0.29. White solid, 250 mg,
yield, 86%; Mp: 213e217 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z:
Calcd for C21H21N4O4 (MþH)þ, 393.1557; found, 393.1563; 1H NMR
found, 472.1290; 1H NMR (300 MHz, DMSO-d6)
d: 9.54 (1H, bs),
8.61 (1H, s), 8.29 (1H, s), 8.04 (2H, d, J ¼ 8.4 Hz), 7.86 (2H, d,
J ¼ 8.4 Hz), 7.71 (1H, s), 7.29 (2H, bs), 3.98 (4H, m), 2.27 (2H, t,
J ¼ 7.3 Hz), 2.12 (2H, p, J ¼ 6.7 Hz), 1.17 (3H, t, J ¼ 7.1 Hz); 13C NMR
(500 MHz, DMSO-d6) d: 10.20 (1H, bs), 8.61 (1H, s), 8.50 (1H, s), 7.80
(1H, d, J ¼ 7.7 Hz), 7.69 (1H, s), 7.44 (2H, t, J ¼ 7.7 Hz), 7.21 (1H, d,
(75 MHz, DMSO-d6) d: 172.17, 156.88, 153.59, 152.81, 147.40, 146.62,
J ¼ 7.4 Hz), 3.96 (4H, m), 2.49 (2H, t, J ¼ 7.4 Hz), 2.11 (2H, p,
142.39, 138.39, 131.21, 126.32, 121.53, 111.87, 106.59, 100.17, 59.87,
41.69, 30.68, 22.20, 13.90.
J ¼ 7.1 Hz), 1.10 (3H, t, J ¼ 7.2 Hz); 13C NMR (125 MHz, DMSO-d6)
d:
172.11, 157.18, 153.58, 153.07, 147.12, 146.37, 138.97, 130.91, 128.41,
123.77, 122.62, 111.64, 106.42, 100.23, 59.82, 41.65, 30.68, 22.17,
13.86.
4.1.6.3.9. Ethyl 4-(8-(4-((3-methyl-4-(2,2,2-trifluoroethoxy)pyr-
idin-2-yl)methoxy)
phenylamino)-2-oxooxazolo[4,5-g]quinazolin-
1(2H)-yl)butanoate (40). Purified by silica-gel column chromatog-
raphy (DCM/MeOH, 40/1), Rf ¼ 0.27. White solid, 398 mg, yield,
88%; Mp: 340e342 ꢁC; HPLC purity: 97%; HRMS, ESIþ, m/z: Calcd for
4.1.6.3.4. Ethyl 4-(8-(4-chlorophenylamino)-2-oxooxazolo[4,5-g]
quinazolin-1(2H)-yl) butanoate (33). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf
¼
0.30. White solid,
C
30H29 F3N5O6 (MþH)þ, 612.2064; found, 612.2074; 1H NMR
265 mg, yield, 84%; Mp: 225e227 ꢁC; HPLC purity: 98%; HRMS,
ESIþ, m/z: Calcd for C21H20ClN4O4 (MþH)þ, 427.1168; found,
(500 MHz, DMSO-d6) d: 9.55 (1H, bs), 8.44 (1H, s), 8.37 (1H, d,
J ¼ 5.2 Hz), 8.23 (1H, s), 7.63 (1H, s), 7.61 (2H, m), 7.16 (1H, d,
J ¼ 5.3 Hz), 7.09 (2H, d, J ¼ 8.4 Hz), 5.21 (2H, s), 4.92 (2H, q,
J ¼ 8.4 Hz), 3.97 (4H, m), 2.47 (2H, t, J ¼ 7.0 Hz), 2.25 (3H, s), 2.12
427.1173; 1H NMR (500 MHz, DMSO-d6)
d: 9.93 (1H, bs), 8.58 (1H,
s), 8.34 (1H, s), 7.87 (1H, d, J ¼ 8.6 Hz), 7.66 (1H, s), 7.47 (2H, t,
J ¼ 8.6 Hz), 3.96 (4H, m), 2.48 (2H, t, J ¼ 7.0 Hz), 2.11 (2H, p,
(2H, m), 1.10 (3H, t, J ¼ 7.0 Hz); 13C NMR (125 MHz, DMSO-d6)
d:
J ¼ 6.9 Hz), 1.10 (3H, t, J ¼ 7.0 Hz); 13C NMR (125 MHz, DMSO-d6)
d
:
172.12, 161.35, 157.41, 155.38, 155.03, 153.61, 153.27, 147.60, 147.06,
146.27, 132.03, 130.76, 124.62, 123.48 (q, J ¼ 240.0 Hz), 121.35,
114.62, 111.52, 107.56, 106.42, 100.22, 70.44, 64.71 (q, J ¼ 34.3 Hz),
41.65, 30.68, 22.15, 13.87, 9.91.
172.10, 157.05, 153.51, 152.66, 146.52, 146.46, 137.93, 131.10, 128.28,
127.44, 123.98, 111.55, 106.03, 100.35, 59.81, 41.67, 30.65, 22.17,
13.86.
4.1.6.3.5. Ethyl 4-(8-(4-(benzyloxy)phenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (34). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.31. White
solid, 335 mg, yield, 91%; Mp: 276e276 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C28H27ClN4O5 (MþH)þ, 499.1976; found,
4.1.6.3.10. Ethyl 4-(8-(3,4-dimethoxyphenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (41). Purified by silica-gel
column chromatography (DCM/MeOH, 45/1), Rf ¼ 0.28. White
solid, 301 mg, yield, 90%; Mp: 243e245 ꢁC; HPLC purity: 98%;
HRMS, ESIþ, m/z: Calcd for C23H25N4O6 (MþH)þ, 453.1769; found,
499.1977; 1H NMR (500 MHz, DMSO-d6)
d: 9.56 (1H, bs), 8.45 (1H,
453.1776; 1H NMR (500 MHz, DMSO-d6)
d: 9.55 (1H, bs), 8.47 (1H,
s), 8.21 (1H, s), 7.64 (1H, s), 7.61 (1H, d, J ¼ 9.2 Hz), 7.48 (2H, d,
J ¼ 8.0 Hz), 7.39 (2H, t, J ¼ 6.5 Hz), 7.34 (1H, t, J ¼ 7.2 Hz), 7.07 (2H, d,
J ¼ 9.0 Hz), 5.13 (2H, s), 3.96 (4H, m), 2.47 (2H, t, J ¼ 7.4 Hz), 2.11 (2H,
s), 8.37 (1H, s), 8.22 (1H, s), 7.63 (1H, s), 7.36 (1H, s), 7.30 (1H, d,
J ¼ 4.9 Hz), 7.01 (1H, d, J ¼ 4.9 Hz), 3.95 (4H, m), 3.79 (6H, s), 2.47
(2H, t, J ¼ 7.0 Hz), 2.12 (2H, m), 1.10 (3H, t, J ¼ 7.0 Hz); 13C NMR
p, J ¼ 6.8 Hz), 1.10 (3H, t, J ¼ 7.2 Hz); 13C NMR (75 MHz, DMSO-d6)
d:
(125 MHz, DMSO-d6) d: 172.12, 157.39, 153.27, 148.48, 147.07,
172.15, 157.15, 154.99, 153.62, 153.30, 147.06, 146.26, 137.13, 131.96,
130.74, 128.37, 127.74, 127.62, 124.62, 114.67, 111.53, 106.43, 100.21,
69.38, 59.86, 41.65, 30.69, 22.17, 13.90.
4.1.6.3.6. Ethyl 4-(8-(4-methoxyphenylamino)-2-oxooxazolo[4,5-
g]quinazolin-1(2H)-yl)butanoate (35). Purified by silica-gel column
146.26, 145.66, 132.15, 130.74, 115.38, 111.82, 111.56, 108.32, 106.44,
100.16, 59.84, 55.73, 55.60, 41.64, 30.69, 22.17, 13.88.
4.1.6.3.11. Ethyl 3-(1-(4-ethoxy-4-oxobutyl)-2-oxo-1,2-
dihydrooxazolo[4,5-g] quinazolin-8-ylamino)-4-hydroxybenzoate
(42). Purified by silica-gel column chromatography (DCM/MeOH,
50/1), Rf ¼ 0.26. White solid, 297 mg, yield, 84%; Mp: 312e313 ꢁC;
HPLC purity: 96%; HRMS, ESIþ, m/z: Calcd for C24H25N4O7 (MþH)þ,
chromatography (DCM/MeOH, 50/1), Rf
¼
0.25. White solid,
281 mg, yield, 90%; Mp: 226e229 ꢁC; HPLC purity: 98%; HRMS,

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
473
481.1718; found, 481.1721; 1H NMR (300 MHz, DMSO-d6)
d: 10.75
J ¼ 7.0 Hz); 13C NMR (125 MHz, DMSO-d6)
d
: 172.15, 157.84, 153.35,
(1H, bs), 9.41 (1H, bs), 8.68 (1H, s), 8.26 (1H, s), 8.08 (1H, s), 7.79
(1H, d, J ¼ 7.6 Hz), 7.66 (1H, s), 7.08 (1H, d, J ¼ 7.6 Hz), 4.30 (2H, m),
3.98 (4H, m), 3.26 (2H, m), 2.13 (2H, m), 1.32 (3H, t, J ¼ 7.0 Hz), 1.14
151.49, 150.53, 147.00, 135.58, 132.50, 131.91, 131.71, 129.24, 121.44,
124.98, 123.22, 121.06, 114.22, 110.89, 103.39, 101.46, 69.44, 59.82,
41.83, 30.64, 22.21, 13.90.
(3H, t, J ¼ 6.7 Hz); 13C NMR (125 MHz, DMSO-d6)
d
: 172.14, 165.32,
4.1.6.3.17. Ethyl 4-(8-(4-hydroxyphenylamino)-2-oxooxazolo[4,5-
g]quinazolin-1(2H)- yl)butanoate (49). Purified by silica-gel column
chromatography (DCM/MeOH, 40/1), Rf ¼ 0.25. White solid,
272 mg, yield, 90%; Mp: 238e241 ꢁC; HPLC purity: 97%; HRMS, ESIþ,
m/z: Calcd for C21H21N4O5 (MþH)þ, 409.1506; found, 409.1514; 1H
158.38, 153.62, 153.12, 147.01, 146.42, 130.85, 128.76, 128.22, 125.92,
120.60, 116.29, 111.60, 106.38, 100.51, 60.20, 59.84, 41.67, 30.96,
30.69, 22.14, 21.43, 14.19, 13.90.
4.1.6.3.12. Ethyl 4-(8-(p-toluidino)-2-oxooxazolo[4,5-g]quinazo-
lin-1(2H)-yl)butanoate (43). Purified by silica-gel column chroma-
tography (DCM/MeOH, 50/1), Rf ¼ 0.31. White solid, 261 mg, yield,
87%; Mp: 215e218 ꢁC; HPLC purity: 98%; HRMS, ESIþ, m/z: Calcd for
NMR (500 MHz, DMSO-d6) d: 9.45 (1H, bs), 9.32 (1H, bs), 8.42 (1H,
s), 8.20 (1H, s), 7.59 (1H, s), 7.47 (2H, d, J ¼ 8.7 Hz), 6.82 (2H, d,
J ¼ 8.7 Hz), 3.98 (2H, t, J ¼ 7.1 Hz), 3.93 (2H, t, J ¼ 6.6 Hz), 2.47 (2H, t,
J ¼ 7.2 Hz), 2.12 (2H, p, J ¼ 6.8 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR
C
22H23N4O4 (MþH)þ, 407.1714; found, 407.1718; 1H NMR (300 MHz,
DMSO-d6)
d
: 9.53 (1H, bs), 8.48 (1H, s), 8.22 (1H, s), 7.61 (2H, t,
(125 MHz, DMSO-d6) d: 172.18, 157.54, 154.27, 153.42, 146.98,
J ¼ 8.8 Hz), 7.23 (2H, t, J ¼ 8.8 Hz), 3.96 (4H, m), 2.33 (3H, s), 2.12
146.21, 130.66, 130.09, 125.08, 115.51, 111.51, 106.38, 100.21, 59.89,
41.66, 30.72, 22.18, 13.92.
(2H, m), 1.10 (3H, m); 13C NMR (125 MHz, DMSO-d6)
d: 172.10,
157.20, 153.16, 147.10, 146.26, 136.34, 132.87, 130.77, 128.83, 122.73,
111.58, 106.42, 100.15, 59.82, 41.62, 30.67, 22.16, 20.44, 13.86.
4.1.6.3.13. Ethyl 4-(8-(4-ethoxyphenylamino)-2-oxooxazolo[4,5-
g]quinazolin-1(2H)-yl)butanoate (44). Purified by silica-gel column
chromatography (DCM/MeOH, 50/1), Rf ¼ 0.30. White solid,
291 mg, yield, 90%; Mp: 230e233 ꢁC; HPLC purity: 97%; HRMS,
ESIþ, m/z: Calcd for C23H25N4O5 (MþH)þ, 437.1819; found,
4.1.6.3.18. Ethyl 4-(8-(3-hydroxyphenylamino)-2-oxooxazolo[4,5-
g]quinazolin-1(2H)-yl)butanoate (50). Purified by silica-gel column
chromatography (DCM/MeOH, 40/1), Rf ¼ 0.25. White solid,
266 mg, yield, 88%; Mp: 237e239 ꢁC; HPLC purity: 98%; HRMS,
ESIþ, m/z: Calcd for C21H21N4O5 (MþH)þ, 409.1506; found,
409.1507; 1H NMR (500 MHz, DMSO-d6)
d: 9.46 (1H, bs), 9.42 (1H,
bs), 8.52 (1H, s), 8.25 (1H, s), 7.64 (1H, s), 7.34 (1H, s), 7.18 (2H, m),
437.1829; 1H NMR (300 MHz, DMSO-d6)
d: 9.63 (1H, bs), 8.44 (1H,
6.56 (1H, m), 3.96 (4H, m), 2.47 (2H, t, J ¼ 7.2 Hz), 2.11 (2H, p,
s), 8.28 (1H, s), 7.65 (1H, s), 7.61(2H, t, J ¼ 8.3 Hz), 6.97 (2H, t,
J ¼ 8.3 Hz), 3.99 (6H, m), 2.48 (2H, t, J ¼ 6.7 Hz), 2.11 (2H, t,
J ¼ 6.9 Hz), 1.35 (3H, t, J ¼ 6.5 Hz), 1.10 (3H, t, J ¼ 6.8 Hz); 13C NMR
J ¼ 7.0 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR (75 MHz, DMSO-d6)
d:
172.18, 157.45, 157.16, 153.65, 153.14, 147.21, 146.40, 140.00, 130.98,
129.07, 113.26, 111.72, 100.97, 106.52, 100.25, 59.87, 41.68, 30.70,
22.19, 13.90.
(125 MHz, DMSO-d6)
d: 172.13, 157.39, 155.16, 153.60, 153.29,
147.04, 146.22, 131.69, 130.70, 124.57, 114.17, 111.56, 106.37, 100.36,
59.82, 41.63, 30.68, 22.17, 14.64, 13.87.
4.1.6.3.19. Ethyl 4-(8-(4-isopropoxyphenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (51). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.28. White
solid, 303 mg, yield, 91%; Mp: 233e235 ꢁC; HPLC purity: 98%;
HRMS, ESIþ, m/z: Calcd for C24H27N4O5 (MþH)þ, 451.1976; found,
4.1.6.3.14. Ethyl
amino)oxazolo[4,5-g]
4-(2-oxo-8-(4-(pyridin-2-ylmethoxy)phenyl-
quinazolin-1(2H)-yl)butanoate (46).
Purified by silica-gel column chromatography (DCM/MeOH, 40/1),
Rf ¼ 0.26. White solid, 325 mg, yield, 88%; Mp: 275e277 ꢁC; HPLC
purity: 98%; HRMS, ESIþ, m/z: Calcd for C27H26N5O5 (MþH)þ,
451.1979; 1H NMR (300 MHz, DMSO-d6)
d: 9.60 (1H, bs), 8.46 (1H,
s), 8.25 (1H, s), 7.63 (1H, s), 7.59 (2H, d, J ¼ 8.8 Hz), 6.98 (2H, d,
J ¼ 8.8 Hz), 4.61 (1H, m), 3.96 (4H, m), 2.42 (2H, t, J ¼ 7.5 Hz), 2.12
(2H, m), 1.29 (6H, d, J ¼ 6.0 Hz), 1.10 (3H, t, J ¼ 6.6 Hz); 13C NMR
500.1928; found, 500.1933; 1H NMR (300 MHz, DMSO-d6)
d: 9.56
(1H, bs), 8.60 (1H, s), 8.22 (1H, s), 8.06 (1H, m), 7.89 (2H, m),
7.57e7.66 (3H, m), 7.36 (1H, m), 7.10 (2H, m), 5.21 (2H, s), 3.96 (4H,
(125 MHz, DMSO-d6) d: 172.10, 157.39,154.14, 153.51, 153.14, 146.64,
m), 2.45 (2H, t, J ¼ 6.7 Hz), 2.09 (2H, m), 1.15 (3H, t, J ¼ 6.8 Hz); 13
C
146.28, 131.46, 130.78, 124.65, 115.55, 111.46, 106.16, 100.27, 69.37,
59.81, 41.64, 30.67, 22.14, 21.80, 13.86.
NMR (125 MHz, DMSO-d6) d: 172.20, 160.24, 157.35, 153.46, 153.25,
149.02, 146.78, 145.50, 144.11, 136.87, 130.80, 124.62, 122.85, 121.56,
114.64, 106.97, 106.42, 104.05, 67.14, 59.82, 42.96, 30.70, 21.42,
13.90.
4.1.6.3.20. Ethyl 4-(8-(4-tert-butylphenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (52). Purified by silica-gel
column chromatography (DCM/MeOH, 70/1), Rf ¼ 0.26. White
solid, 282 mg, yield, 85%; Mp: 236e239 ꢁC; HPLC purity: 98%;
HRMS, ESIþ, m/z: Calcd for C25H29N4O4 (MþH)þ, 449.2183; found,
4.1.6.3.15. Ethyl 4-(8-(3,4-difluorophenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (47). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.31. White
solid, 266 mg, yield, 84%; Mp: 255e256 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C21H19F2N4O4 (MþH)þ, 429.1369; found,
449.2188; 1H NMR (500 MHz, DMSO-d6)
d: 9.57 (1H, bs), 8.49 (1H,
s), 8.24 (1H, s), 7.68 (2H, t, J ¼ 8.7 Hz),7.64 (1H, s), 7.43 (2H, t,
J ¼ 8.7 Hz), 3.96 (4H, m), 2.47 (2H, t, J ¼ 7.2 Hz), 2.12 (2H, m), 1.32
429.1377; 1H NMR (300 MHz, DMSO-d6)
d: 9.72 (1H, bs), 8.56 (1H,
(9H, s), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR (75 MHz, DMSO-d6)
d:
s), 8.18 (1H, s), 8.04 (1H, m), 7.64 (1H, s), 7.48 (1H, m), 3.96 (4H, m),
172.17, 157.30, 153.66, 153.23, 147.18, 146.38, 136.31, 130.92, 125.51,
125.11, 122.50, 111.64, 106.51, 100.21, 59.86, 41.68, 34.09, 31.70,
22.18, 13.90.
2.48 (2H, t, J ¼ 7.2 Hz), 2.13 (2H, m), 1.10 (3H, t, J ¼ 7.0 Hz); 13C NMR
(75 MHz, DMSO-d6) d: 171.12, 158.40, 153.10, 150.28, 150.08, 148.37
(d, J ¼ 11.3 Hz), 147.38, 147.02, 146.85, 145.14, 144.97, 139.01, 134.54
(d, J ¼ 2.3 Hz), 134.41 (d, J ¼ 1.5 Hz), 132.30, 120.33 (d, J ¼ 2.3 Hz),
117.14, 116.90, 113.13, 112.85, 110.56, 102.64, 101.29, 59.74, 41.91,
30.56, 22.24, 21.39, 13.87.
4.1.6.3.21. 4-Cyanobenzyl 4-(1-(4-ethoxy-4-oxobutyl)-2-oxo-1,2-
dihydrooxazolo[4,5-g]quinazolin-8-ylamino)benzoate
(53).
Purified by silica-gel column chromatography (DCM/MeOH, 50/1),
Rf ¼ 0.24. White solid, 330 mg, yield, 81%; Mp: 340e342 ꢁC; HPLC
purity: 97%; HRMS, ESIþ, m/z: Calcd for C30H26N5O6 (MþH)þ,
4 .1. 6 . 3 .16 . E t h y l 4 - ( 8 - ( 4 - ( 4 - c h l o r o b e n z y l o x y ) - 3 -
chlorophenylamino)-2-oxooxazolo [4,5-g]quinazolin-1(2H)-yl)buta-
noate (48). Purified by silica-gel column chromatography (DCM/
MeOH, 50/1), Rf ¼ 0.31. White solid, 385 mg, yield, 92%; Mp:
322e325 ꢁC; HPLC purity: 96%; HRMS, ESIþ, m/z: Calcd for
552.1878; found, 552.1884; 1H NMR (500 MHz, DMSO-d6)
d: 9.85
(1H, bs), 8.64 (1H, s), 8.27 (1H, s), 8.08 (4H, s), 7.89 (2H, d,
J ¼ 8.2 Hz), 7.70 (1H, s), 7.69 (2H, d, J ¼ 8.2 Hz), 5.45 (2H, s),
4.00e3.95 (4H, m), 2.48 (2H, t, J ¼ 7.2 Hz), 2.14 (2H, p, J ¼ 7.0 Hz),
C
28H25Cl2N4O5 (MþH)þ, 567.1197; found, 567.1198; 1H NMR
1.10 (3H, t, J ¼ 7.2 Hz); 13C NMR (75 MHz, DMSO-d6)
d: 172.17,
(300 MHz, DMSO-d6)
d
: 10.53 (1H, bs), 8.67 (2H, m), 7.98 (1H, m),
165.06, 156.71, 153.58, 152.74, 147.48, 146.65, 144.18, 142.01, 132.43,
131.27, 130.10, 128.23, 123.35, 120.98, 118.64, 114.22, 111.93, 110.65,
106.62, 100.12, 64.97, 59.87, 41.69, 30.67, 22.20, 13.90.
7.70 (2H, d, J ¼ 9.8 Hz), 7.50 (4H, m), 7.29 (1H, d, J ¼ 7.8 Hz), 5.24
(2H, s), 3.97 (4H, m), 2.48 (2H, t, J ¼ 7.2 Hz), 2.11 (2H, m), 1.11 (3H, t,

474
S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
4.1.6.3.22. 4-Chlorobenzyl 4-(1-(4-ethoxy-4-oxobutyl)-2-oxo-1,2-
dihydrooxazolo[4,5-g]quinazolin-8-ylamino)benzoate (54).
p, J ¼ 5.7 Hz), 3.95 (3H,m), 2.44 (2H, t, J ¼ 7.2 Hz), 2.11 (2H, m), 1.63
(3H, d, J ¼ 7.0 Hz), 1.10 (3H, m); 13C NMR (125 MHz, DMSO-d6)
d:
Purified by silica-gel column chromatography (DCM/MeOH, 50/1),
Rf ¼ 0.25. White solid, 323 mg, yield, 78%; Mp: 334e337 ꢁC; HPLC
purity: 98%; HRMS, ESIþ, m/z: Calcd for C29H26ClN4O6 (MþH)þ,
171.56, 158.10, 153.61, 153.54, 146.53, 146.02, 144.47, 130.36, 128.13,
126.51, 125.94, 111.23, 106.19, 100.25, 67.09, 59.76, 49.04, 41.59,
30.92, 22.13, 21.34.
561.1535; found, 561.1546; 1H NMR (500 MHz, DMSO-d6)
d: 9.88
4.1.6.3.27. Ethyl 4-(8-(1H-indazol-5-ylamino)-2-oxooxazolo[4,5-
(1H, bs), 8.64 (1H, s), 8.29 (1H, s), 8.06 (4H, m), 7.71 (1H, s), 7.52 (2H,
g]quinazolin-1(2H)-yl)butanoate
(45). 1H-Indazol-5-amine
d, J ¼ 8.6 Hz), 7.48 (2H, d, J ¼ 8.6 Hz), 5.35 (2H, s), 3.97 (4H, m), 2.48
(118 mg, 0.89 mmol) was added to a solution of compound 15
(250 mg, 0.74 mmol) in isopropanol (10 mL) and stirred at reflux for
13 h. The mixture was cooled to room temperature and filtered, the
solid was washed with chill isopropanol (5 mL), the residue was
treated with aqueous NaHCO3 (10 mL) and extracted with EtOAc/
MeOH (20:1, 30 mL). The organic layer was washed with brine,
dried over Na2SO4, and concentrated. Purified by silica-gel column
chromatography (DCM/MeOH, 70/1), Rf ¼ 0.21. Drying gave 238 mg
(yield, 75%) of the title compound as white solid: Mp: 287e289 ꢁC;
HPLC purity: 96%; HRMS, ESIþ, m/z: Calcd for C18H15F2N4O3
(MþH)þ, 373.1107; found, 373.1111; 1H NMR (300 MHz, DMSO-d6)
(2H, t, J ¼ 7.2 Hz), 2.13 (2H, p, J ¼ 6.9 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13
C
NMR (75 MHz, DMSO-d6) d: 172.17, 165.78, 153.58, 152.70, 147.26,
146.69, 144.04,135.32,132.66,131.32,130.03, 129.81,128.48,123.64,
121.05, 111.91, 111.91, 106.49, 100.22, 65.09, 59.86, 41.69, 30.66,
22.20, 13.90.
4.1.6.3.23. Ethyl 4-(8-(3,5-dimethylphenylamino)-2-oxooxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (55). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.30. White
solid, 261 mg, yield, 84%; Mp: 224e226 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C23H25N4O4 (MþH)þ, 421.1870; found,
421.1877; 1H NMR (500 MHz, DMSO-d6)
d: 9.45 (1H, bs), 8.52 (1H,
d: 13.10 (1H, s), 9.76 (1H, bs), 8.47 (1H, s), 8.33 (1H, s), 8.15 (1H, s),
s), 8.25 (1H, s), 7.64 (1H, s), 7.44 (2H, s), 6.80 (1H, s), 3.96 (4H, m),
8.10 (1H, s), 8.06 (1H, d, J ¼ 6.7 Hz), 7.91 (1H, s), 7.64 (1H, s), 7.60
(1H, s), 7.58 (1H, d, J ¼ 6.7 Hz), 3.95 (4H, m), 2.48 (2H, t, J ¼ 7.2 Hz),
2.13 (2H, p, J ¼ 6.9 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR (125 MHz,
2.47 (2H, t, J ¼ 7.2 Hz), 2.32 (6H, s), 2.12 (2H, m), 1.32 (9H, s), 1.10
(3H, t, J ¼ 7.2 Hz); 13C NMR (75 MHz, DMSO-d6)
d: 172.16, 157.16,
153.62, 153.20, 147.15, 146.35, 138.82, 137.44, 130.92, 125.36, 120.21,
111.65, 106.51, 100.16, 59.84, 41.66, 30.69, 22.17, 21.02, 13.89.
4.1.6.3.24. Ethyl 4-(8-(benzylamino)-2-oxooxazolo[4,5-g]quina-
zolin-1(2H)-yl)butanoate (32). Phenylmethanamine (95.4 mg,
0.89 mmol) and TEA (150 mg,1.48 mmol) was added to a solution of
compound 12 (250 mg, 0.74 mmol) in isopropanol (10 mL) and
stirred at 50 ꢁC for 24 h. The mixture was cooled to room temper-
ature and concentrated in vacuo, the residue was treated with
aqueous NaHCO3 (10 mL) and extracted with EtOAc/MeOH (20:1,
30 mL). The organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated. Purified by silica-gel column
chromatography (DCM/MeOH, 90/1), Rf ¼ 0.23. Drying gave 210 mg
(yield, 70%) of the title compound as pale yellow solid: Mp:
220e223 ꢁC; HPLC purity: 96%; HRMS, ESIþ, m/z: Calcd for
DMSO-d6) d: 171.63, 157.66, 153.61, 153.32, 147.04, 146.28, 144.11,
131.60, 130.68, 123.90, 114.13, 111.57, 109.87, 106.98, 106.24, 104.05,
100.18, 67.16, 41.68, 30.29, 22.41, 22.13.
4.2. Biological evaluation
4.2.1. EGFR tyrosine kinase enzyme inhibition assay
EGFR tyrosine kinase activity was determined by an enzyme-
linked-immuno-sorbent assay (ELISA) in 96-well plates pre-
coated in a 10
compounds, 4
37 ꢁC, the reactions were stopped with 10
m
L reaction volume including 4
L substrate and 2 L ATP. After incubation for 1 h at
L 2% (v/v) H3PO4. The
mL diluted test
m
m
m
plates were then aspirated and washed twice with 200 mL 0.9% (w/
v) NaCl, and incorporation of 33Pi was determined with a micro-
plate scintillation counter. The residual kinase activities for each
concentration of compound and the compound IC50 values were
calculated using GraphPad Prism 5.0. Comparisons between all
groups were made using a two-tailed Student's t test (SPSS
version11.5 software). Data represented as means SD from three
independent experiments, and differences between groups were
considered statistically significant at P < 0.05.
C
22H23N4O4 (MþH)þ, 407.1714; found, 407.1721; 1H NMR (500 MHz,
DMSO-d6)
d
: 8.65 (1H, t, J ¼ 5.7 Hz), 8.41 (1H, s), 8.07 (1H, s), 7.55
(1H, s), 7.38 (2H, d, J ¼ 7.2 Hz), 7.33 (2H, t, J ¼ 7.7 Hz), 7.25 (2H, t,
J ¼ 7.2 Hz), 4.83 (2H, d, J ¼ 5.7 Hz), 3.94 (2H, q, J ¼ 7.1 Hz), 3.89 (2H,
t, J ¼ 7.2 Hz),2.44 (2H, t, J ¼ 7.2 Hz), 2.07 (2H, p, J ¼ 6.9 Hz), 1.08 (3H,
t, J ¼ 7.1 Hz); 13C NMR (125 MHz, DMSO-d6)
d: 172.08, 158.97,
153.59, 146.14, 145.84, 139.27, 130.47, 128.25, 127.20, 126.77, 111.24,
105.84, 100.45, 59.79, 43.58, 41.62, 30.68, 22.11, 13.86.
4.1.6.3.25. (S)-ethyl
4-(2-oxo-8-(1-phenylethylamino)oxazolo
4.2.2. Cancer cell proliferation inhibition assay
[4,5-g]quinazolin-1(2H)-yl)butanoate (38). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.33. Pale yel-
low solid, 202 mg, yield, 65%; Mp: 255e257 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C23H25N4O4 (MþH)þ, 421.1870; found,
Human epidermoid cancer cell line (KB) and renal cell carci-
noma cell line (A498) were provided by Shanghai Institute of
Biochemistry and Cell Biology, Chinese Academy of Sciences. KB cell
was cultured in DMEM medium supplemented with 10% FBS, and
Penicillin 100 U/mL and Streptomycin 100 U/mL were added. Cell
cultures were maintained in a humidified atmosphere of 5% CO2 at
37 ꢁC. Cells were seeded at respective density (2e4 ꢂ 104/mL) in
421.1869; 1H NMR (500 MHz, DMSO-d6)
d: 8.36 (1H, s), 8.24 (1H, d,
J ¼ 7.2 Hz), 8.19 (1H, s), 7.54 (1H, s), 7.44 (2H, d, J ¼ 7.6 Hz), 7.33 (2H,
t, J ¼ 7.8 Hz), 7.22 (2H, d, J ¼ 7.4 Hz), 5.65 (1H, p, J ¼ 7.2 Hz), 4.79 (1H,
p, J ¼ 5.7 Hz), 3.95 (3H,m), 2.44 (2H, t, J ¼ 7.2 Hz), 2.12 (2H, m), 1.63
(3H, d, J ¼ 7.0 Hz), 1.10 (3H, t, J ¼ 7.1 Hz); 13C NMR (125 MHz, DMSO-
96-well plates in a volume of 180 mL per well. After seeding 24 h,
the medium was removed. The test compounds were dissolved in
DMSO and diluted with culture medium to different concentrations
d6) d: 171.56, 158.10, 153.61, 153.54, 146.52, 146.03, 144.47, 130.38,
128.14, 126.51, 125.94, 111.23, 106.19, 100.25, 67.10, 59.76, 49.04,
41.59, 30.92, 22.14, 21.35.
(the final concentration of DMSO was 0.1%). 20 mL of the test
compound solution was added in duplicates, and incubation
continued for 48 h in a humidified atmosphere of 5% CO2 at 37 ꢁC.
Remove the medium, and cells were fixed with 10% trichloroacetic
4.1.6.3.26. (R)-ethyl
4-(2-oxo-8-(1-phenylethylamino)oxazolo
[4,5-g]quinazolin-1(2H)-yl)butanoate (39). Purified by silica-gel
column chromatography (DCM/MeOH, 50/1), Rf ¼ 0.33. Pale yel-
low solid, 208 mg, yield, 67%; Mp: 252e255 ꢁC; HPLC purity: 97%;
HRMS, ESIþ, m/z: Calcd for C23H25N4O4 (MþH)þ, 421.1870; found,
acid (50
and air-dried. Cells that survived were stained with 0.4% (w/v) SRB
dye solution (100 L) for 20 min at room temperature. Then,
remove the SRB dye solution, and the plate was washed with tap
water and dried. 150 L of Trizma base (10 mM) was added to each
well, and the absorbance was measured at 570 nm using a
m
L) for 1 h at 37 ꢁC, and then washed 5 times with tap water
m
421.1869; 1H NMR (500 MHz, DMSO-d6)
d: 8.36 (1H, s), 8.23 (1H, d,
J ¼ 7.2 Hz), 8.19 (1H, s), 7.53 (1H, s), 7.45 (2H, d, J ¼ 7.6 Hz), 7.33 (2H,
m
t, J ¼ 7.8 Hz), 7.23 (2H, d, J ¼ 7.4 Hz), 5.66 (1H, p, J ¼ 7.2 Hz), 4.79 (1H,

S. Yin et al. / European Journal of Medicinal Chemistry 101 (2015) 462e475
475
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (81273468, 81473153), National Basic Research
Program of China (2015CB755500), Natural Science Foundation of
Jiangsu (BK20140672), and 111 Project from the Ministry of Edu-
cation of China and the State Administration of Foreign Expert Af-
fairs of China (No. 111-2-07). We greatly appreciated Professor Tao
Lu and Ms. Yanmin Zhang at China Pharmaceutical University for
docking study.
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Appendix A. Supplementary data
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proliferative activity of 4,6-quinazolinediamines as potent EGFR-TK in-
hibitors, Eur. J. Med. Chem. 61 (2013) 132e145.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.07.008.
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T. Tamura, A. Nakayama, H. Miki, H. Kamiguchi, T. Tanaka, N. Habuka,
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human epidermal growth factor receptor 2 (HER2)/Epidermal growth factor
receptor (EGFR) dual inhibitors bearing a pyrrolo[3,2-d]pyrimidine scaffold,
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