Design, synthesis and biological evaluation of novel EGFR/HER2 dual inhibitors bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold

Article abstract of DOI:10.1016/j.ejmech.2016.04.072

For the purpose of developing novel EGFR/HER2 tyrosine kinases inhibitors with high inhibition activity and low toxicity, two novel series of oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR/HER2 dual inhibitors introducing two electrophiles 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl group as side-chain at 1-position respectively and evaluated their EGFR and HER2 inhibition activity and toxicity comparing with Lapatinib. All these compounds were evaluated by EGFR and HER2 kinase inhibition and two anti-proliferation assays in vitro. Most of the designed compounds exhibited moderate to high inhibition activity against EGFR and HER2. Especially, compounds 11o, 11p, 12e and 12f presented high inhibition against EGFR and HER2. Furthermore, compounds 11p and 12f also had well exhibition to excellent anti-proliferation activity against human lung adenocarcinoma cell line (A549) and human breast cancer cell line (SK-Br3), and 12f also exhibited the lowest toxicity against human embryonic lung fibroblast cell line (HELF) cell. Finally, compound 12f presented remarkably higher inhibition efficacy towards tumour growth than Lapatinib in a mouse lewis lung cancer (LLC) xenograft model.

Full text of DOI:10.1016/j.ejmech.2016.04.072

Accepted Manuscript
Design, synthesis and biological evaluation of novel EGFR/HER2 dual inhibitors
bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold
Siyuan Yin, Chunming Tang, Bin Wang, Ying Zhang, Liliang Zhou, Lingjing Xue, Can
Zhang
PII:
S0223-5234(16)30365-8
10.1016/j.ejmech.2016.04.072
EJMECH 8587
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2016
Revised Date: 29 April 2016
Accepted Date: 30 April 2016
Please cite this article as: S. Yin, C. Tang, B. Wang, Y. Zhang, L. Zhou, L. Xue, C. Zhang,
Design, synthesis and biological evaluation of novel EGFR/HER2 dual inhibitors bearing a
oxazolo[4,5-g]quinazolin-2(1H)-one scaffold, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.04.072.
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Design, synthesis and biological evaluation of novel EGFR/HER2
dual inhibitors bearing a oxazolo[4,5-g]quinazolin-2(1H)-one scaffold
Siyuan Yin, Chunming Tang, Bin Wang, Ying Zhang, Liliang Zhou, Lingjing Xue**,
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Can Zhang
State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for
Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials, China Pharmaceutical
University 24 Tong Jia Xiang, Nanjing, 210009, China
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Abstract
For the purpose of developing novel EGFR/HER2 tyrosine kinases inhibitors with
high inhibition activity and low toxicity, two novel series of
oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR/HER2 dual inhibitors
introducing two electrophiles 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl
group as side-chain at 1-position respectively and evaluated their EGFR and HER2
inhibition activity and toxicity comparing with Lapatinib. All these compounds were
evaluated by EGFR and HER2 kinase inhibition and two anti-proliferation assays in
vitro. Most of the designed compounds exhibited moderate to high inhibition activity
against EGFR and HER2. Especially, compounds 11o, 11p, 12e and 12f presented
high inhibition against EGFR and HER2. Furthermore, compounds 11p and 12f also
had well exhibition to excellent anti-proliferation activity against human lung
adenocarcinoma cell line (A549) and human breast cancer cell line (SK-Br3), and 12f
also exhibited the lowest toxicity against human embryonic lung fibroblast cell line
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Corresponding author.
Corresponding author.
*
E-mail: xuelingjing65@163.com (L. Xue), zhangcan@cpu.edu.cn (C. Zhang).
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(HELF) cell. Finally, compound 12f presented remarkably higher inhibition efficacy
towards tumour growth than Lapatinib in a mouse lewis lung cancer (LLC) xenograft
model.
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Keywords: EGFR/HER2 dual inhibitor, oxazolo[4,5-g]quinazolin-2(1H)-one scaffold,
antitumor proliferation, molecular docking, SAR
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1. Introduction
EGFR family contains four structurally related receptors, EGFR (ErbB1/HER1),
ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4), for which a variety of different
ligands have been characterized. EGFR has become one of the targets in anticancer
drug research because of its wide distribution and essential role in cell proliferation,
differentiation and migration [1-2]. Overexpression of the EGFR and HER2 is
frequently observed in various solid tumors, and coexpression of EGFR and HER2
has been found in different tumors such as breast, ovarian, prostate and colon cancer
[3]. Therefore, EGFR and HER2 have been the most well studied targets in current
drug research, and EGFR/HER2 dual inhibitors can be more effective than just EGFR
or HER2 inhibitors because of HER2 binding either EGFR or HER2 to form a hetero-
or homodimer for the signal transduction of EGFR signal cascade [4-5]. In recent
years, several EGFR/HER2 dual inhibitors have been approved for cancer by U.S.
Food and Drug Administration (FDA), such as the first generation EGFR/HER2
inhibitor Lapatinib (Tykerb) [6] and the secend generation EGFR/HER2 inhibitor
Afatinib (Gilotrif) [7]. As shown in Fig. 1, Lapatinib is a reversible EGFR/HER2 dual
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inhibitor while Afatinib is an irreversible EGFR/HER2 dual inhibitor. Quinoline
moiety in Lapatinib and Afatinib is an important pharmacophore for anticancer
activity and represents an attractive scaffold for EGFR inhibitors [8], and Afatinib has
higher inhibition activity than Lapatinib because Afatinib is an irreversible inhibitor
which contains a Michael addition receptor moiety which can form a covalent bind
with the conserved cysteine residue present in the lip of the EGFR ATP binding site
(Cys797) to achieve occupancy greater than the reversible inhibitor Lapatinib [9].
Unfortunately, due to toxicity-related dose-limitation, Afatinib only displayed 7%
response rate for gefitinib-resistant non-small cell lung cancer (NSCLC) patients in a
phase III clinical trial [10].
For the purpose of developing novel EGFR/HER2 tyrosine kinases inhibitors with
high inhibition activity and low toxicity, we designed two novel series of
oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as EGFR/HER2 dual inhibitors
introducing two electrophiles 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl
group as side-chain at 1-position respectively and evaluated their EGFR and HER2
inhibition activity and toxicity comparing with Lapatinib (Fig. 2). Eventually,
compounds 11o, 11p, 12e and 12f presented higher inhibition against EGFR and
HER2 than Lapatinib. Moreover, compounds 11p and 12f also had well exhibition to
excellent anti-proliferation activity against human lung adenocarcinoma cell line
(A549) and human breast cancer cell line (SK-Br3), and among which 12f showed the
lowest toxicity. Furthermore, compound 12f presented remarkably higher inhibition
efficacy towards tumour growth than Lapatinib in a mouse lewis lung cancer (LLC)
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xenograft model (P<0.05). Therefore, we reported compound 12f as a promising
candidate for clinical development as novel EGFR/HER2 dual inhibitors.
2. Results and Discussion
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2.1 Rational and design
Based on our earlier work with oxazolo[4,5-g]quinazolin-2(1H)-one inhibitors [11-12],
we planned to develop EGFR/HER2 dual inhibitors with high inhibition activity and
low toxicity by exploring the different electrophiles at 1-position.
2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl were expected to be more
promiscuous because of their (expectedly) high reactivity [13-14]. These two
side-chains in target compounds were responsible for the covalent binding to an active
site cysteine residue.
With respect to the high inhibition activity, 2-(2-bromoacetyl)ethyl group was adopted
at 1-position due to its high electrophilic reactivity and nucleophilic displacement
with aniline at 8-position to give series I compounds. Then, 2-(2-chloroacetoxy)ethyl
group which was less reactive [15] compared with 2-(2-bromoacetyl)ethyl group was
introduced at 1-position, and aniline was displaced at 8-position according to the
inhibition activity results of series I compounds to give series II compounds (Fig. 2).
2.2 Chemistry
The synthetic method for target compounds was showed in Scheme 1 and Scheme
2. Compounds 2~6 were synthesized in as described in our earlier research [12].
The series I compounds were obtained by alkylation of compound 6 with
2-bromoethan-1-ol, cyclization with formamidine acetate in EtOH, esterification with
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bromoacetyl bromide, chlorination with thionyl chloride using DMF as catalyst, and
nucleophilic displacement with aniline (Scheme 1). Likewise, the series II compounds
were synthesized in as described in Scheme 2.
2.2. Biological activities
Kinase Inhibitory Activity and Cancer Cell Proliferation Inhibitory Activity. Our
principle behind the designed compounds is to improve the inhibition activity and
reduce toxicity. Therefore, EGFR and HER2 tyrosine kinase inhibition assay and
cancer cell proliferation inhibition assay were used to assess the in vitro antitumor
activities of designed compounds. EGFR and HER2 kinase inhibition assay was
performed according to the instructions of the manufacturer, and human lung
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adenocarcinoma cell line (A549) [16] which overexpresses EGFR and human breast
cancer cell line (SK-Br3) [17] overexpressing HER2 were selected as cell models to
evaluate the anti-proliferation of the designed compounds. As shown in Table 1, most
of the designed compounds exhibited moderate to high inhibition activity against
EGFR and HER2, which revealed that introducing 2-(2-bromoacetyl)ethyl at
1-position of oxazolo[4,5-g]quinazolin-2(1H)-one scaffold contributed to the activity.
As indicated by the results that compounds containing some functional groups (such
as halogens, alkoxyl groups etc.) at 8-position caused an increase in EGFR and HER2
inhibition with the exception of compounds 11a, 11b, 11d, 11i, 11o and 11p. Among
these, the compounds which contained polar groups (such as piperazinyl, morpholino
etc.) would also demonstrate increased inhibition activities towards EGFR and HER
kinase like 11o and 11p. However, compounds containing some big functional groups
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would demonstrate decreased inhibition activities like 11e, 11f, 11g and 11h. This
may be due to their relatively large structures, which sterically hindered their
interaction with the active site on kinase. Above all, compounds 11o and 11p
exhibited promising activities with IC values of 0.008 ꢀM and 0.0010 ꢀM on EGFR,
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0.033 ꢀM and 0.021 ꢀM on HER2, repectively. This was a significant improvement
when compared to Lapatinib which had IC values of 0.026 ꢀM on EGFR and 0.017
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ꢀM on HER2 (Table 1).
The cellular anti-proliferative effects were mainly coincident with kinase inhibition
activity, most compounds exhibited moderate to high inhibition activities and
cytotoxicities to A549 and SK-Br3 cell. Compounds 11o and 11p exhibited high
activities with IC values of 2.03 ꢀM and 3.60 ꢀM against A549, 12.50 ꢀM and 2.30
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and 0.47 ꢀM against SK-Br3 (Table 1).
According the inhibition activity result of 11a-11q, 2-(2-chloroacetoxy)ethyl group
was introduced at 1-position, and aniline was displaced at 8-position to give
compounds 12a-12g. As shown in Table 1, almost all of compounds had potent
activity on EGFR and HER2 as well as A549 and SK-Br3 cells except compound 12d.
The EGFR and HER2 kinase inhibition activities of compounds 12a-12g had a slight
decrease compared with compounds 11a-11q when they had the same aniline
displaced at 8-position, such as 11d vs. 12c, 11i vs. 12b, 11p vs. 12f, etc.. The cancer
cell proliferation inhibitory activities of compounds 12a-12g were also mainly
coincident with kinase inhibition activity, most compounds exhibited moderate to
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high inhibition activities and cytotoxicities to A549 and SK-Br3 cell. We were excited
finding that compound 12f exhibited equivalent inhibition activity towards EGFR and
HER2 kinase as well as A549 and SK-Br3 cells (Table 1).
Normal Cell Cytotoxicity
As further evaluation for the selective cytotoxicity of the most promising compounds
(11p and 12f), they have been tested over normal human embryonic lung fibroblast
cell line (HELF) [18] using MTT assay. As illustrated in Table 2, compounds 11p and
12f had lower cytotoxicity to HELF cell compared with Lapatinib, and compound 12f
exhibited the lowest cytotoxicity to normal human lung cell and normal human breast
cell.
In Vivo Antitumor Activity. The xenograft mouse lewis lung cancer (LLC) tumor
mice model [12] was used to investigate the in vivo antitumor activity of designed
compound 12f. As shown in Figure 3, compound 12f and Lapatinib were well
tolerated and displayed significant in vivo antitumor efficacy when compared with
water as vehicle control, and compound 12f presented remarkably higher inhibition
efficacy towards tumour growth than Lapatinib (P<0.05) (Fig. 3A). The images of the
tumor harvested from the mice visually showed the greatest tumor shrinkage after
treatment with compound 12f (Fig. 3B), and no significant change of the body weight
during the study.
2.3 Structure-activity relationships
As illustrated in our earlier research, N , N atoms in pyrimidine ring are ATP
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competitive binding sites, which is pharmacophore of EGFR inhibitors. And also, the
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proton in aniline at 8-position is essential. As shown in Figure 4, introducing
electrophiles side-chain, such as 2-(2-bromoacetyl)ethyl and 2-(2-chloroacetoxy)ethyl
group can achieve medium to high inhibition activity, and the EGFR and HER2
kinase inhibition activities had a slight decrease when X = Cl compared with X = Br
when they had the same aniline displaced at 8-position because
2-(2-chloroacetoxy)ethyl was less reactive compared with 2-(2-bromoacetyl)ethyl
group, such as 11d vs. 12c, 11i vs. 12b, 11p vs. 12f, etc.. Halogen introduced in
phenyl amines can improve the inhibition activity (Cl> F> H), such as compounds
11a, 11i, 11k, and morpholinyl, 4-methylpiperazin-1-y introduced in phenyl amines
may increase the compound inhibition activity, such as compounds 11o, 11p, 12e, 12f.
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4-(piperidine-1-carboxamido)phenyl,
introduced in phenyl amines would decreased inhibition activities like 11c, 11e, 11f,
11g and 11h. This may be due to their relatively large structures, which sterically
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hindered their interaction with the active site on kinase. (Fig. 4 and Table 1).
2.4 Molecular Docking Study
Docking study using GOLD 4.1 was performed. GOLD is a genetic algorithm based
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docking program that allows a wide range of flexibility for the ligand and the protein
[19]. It is used to predict the binding modes and orientation of the synthesized
compounds at the active site of the ATP binding site of EGFR-TK. The crystal
structure of the enzyme with Lapatinib (ID: 1XKK) was obtained from protein data
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bank (PDB). The kinase or catalytic domain includes an N-terminal lobe, which
consists mainly of β-strands but contains one α-helix and C helix. The C-terminal lobe
is mainly α-helical, and a short strand termed the hinge region connects the two lobes
[20]. The docking studies were progressed by positioning the compound 12f in the
Lapatinib binding site, 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 Met793
via a hydrogen bond. A water molecule-mediated hydrogen bonding interaction
between the N7 and the Thr854 side chain was observed which was similar to
Lapatinib. 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 morpholinyl at the C4 of aniline moiety lay in the same
site of the 3-fluorobenzyl)oxy)phenyl moiety of Lapatinib. It was glad to found a
covalent bonding interaction existing between the 2-(2-chloroacetoxy)ethyl
side-chains and the Cys797 cysteine residue which revealed that 12f could form the
covalent binding to an active site cysteine residue of EGFR (Fig. 5).
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In summary, a series of novel oxazolo[4,5-g]quinazolin-2(1H)-one derivatives as
EGFR/HER2 dual inhibitors were synthesized and subjected to pharmacological
evaluation. The results showed that most of oxazolo[4,5-g]quinazolin-2(1H)-one
derivatives exhibited moderate to high EGFR inhibition activities. Protein tyrosine
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kinase inhibitions assay indicated that many of these derivatives exerted potent
activity on both EGFR and HER2 kinases. Moreover, these derivatives demonstrated
high anti-proliferation potencies against A549 and SK-Br3 cells. Among them two
designed compounds 11p and 12f exhibited more potent inhibition activities than the
reference compounds Lapatinib which merited further evaluation. Especially, 12f also
exhibited the lowest toxicity against HELF cell compared with Lapatinib and 11p.
Furthermore, compound 12f presented remarkably higher inhibition efficacy towards
tumour growth than Lapatinib. In conclusion, the findings presented herein showed
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scaffold is an excellent strategy for the development of EGFR/HER2 dual inhibitors.
10 4. Experimental sections
11 4.1 Chemistry experiment
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4.1.1 Chemistry: General procedures
All commercially available starting materials, reagents and solvents were used
without further purification unless otherwise 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
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spectrometer. H -NMR and C-NMR spectra on a Bruker AV 300 300 or 500 MHz
spectrometer were recorded in DMSO-d . Chemical shifts are reported in d (ppm)
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units relative to the internal standard tetramethylsilane (TMS).The reaction conditions
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were not optimized for reaction yields. All oxygen-sensitive or moisture-sensitive
reactions were run under nitrogen atmosphere. All the reactions 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 (200–300 mesh).
4.1.2 Purity analysis
The purity of the synthesized compounds were measured by high performance liquid
chromatography (HPLC, Shimadzu LC-2010 system, Kyoto, Japan) equipped with a
Diamonsil C18 column (5 ꢀm particle size, 250 mm × 4.6 mm). The mobile phase
consisted of acetonitrile and water with a flow rate of 1.0 mL/min. The detection
wavelength was 344 nm and sample injected volume was 20 ꢀL. All compounds
evaluated for EGFR/HER2 inhibitory potency had a purity of ≧ 95%.
4.1.3
Ethyl
6-amino-3-(2-hydroxyethyl)-2-oxo-2,3-dihydrobenzo[d]oxazole-5-carboxylate (7)
Compound 6 (13.2 g, 60.0 mmol) was dissolved in 75 mL DMF, and MeCN 150 mL,
2-bromoethan-1-ol (22.5 g, 180 mmol) and K CO (24.8 g, 180 mmol) were
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successively added, and the mixture was stirred at 60 °C for 4 h. After cooled to room
temperature, ice water (500 mL) was added and the aqueous phase was extracted with
AcOEt (4×200 mL). The organic layers were combined, dried (Na SO ) and
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concentrated to give the crude product. The crude product was purified by silica-gel
column chromatography (PE/EA, 4/1), R = 0.25. Drying gave compound 7 (12.8 g,
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yield, 80.1%); mp: 159-161 °C; H NMR (300 MHz, DMSO-d ) δ 10.37 (1H, s), 7.65
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(1H, s), 6.75 (2H, s), 6.68 (1H, s), 4.37 (2H, t, J = 7.2 Hz), 4.21 (2H, q, J = 6.9 Hz),
3.77 (2H, t, J = 7.2 Hz), 1.27 (3H, t, J = 6.9 Hz).
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4.1.4 1-(2- hydroxyethyl) oxazolo[4,5-g]quinazoline-2,8(1H,7H)-dione (8)
Compound 7 (12.0 g, 45.0 mmol) and formamidine acetate (5.15 g, 49.5 mmol) and
ethanol (120 mL) was successively added and the mixture was refluxed for 24 h under
the protection of N . The mixture was cooled to room temperature and filtered, and
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the solid was washed with cold EtOH (50 mL) and water (50 mL), and dried to give
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compound as white power (9.2 g, yield, 82.9%); mp: 237-239 °C; H NMR (300 MHz,
DMSO-d ) δ 12.27 (1H, br s), 8.04 (1H, s), 7.91 (1H, s), 7.57 (1H, s), 4.96 (1H, t, J =
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5.7 Hz), 3.95 (2H, t, J = 5.4 Hz), 3.70 (2H, t, J = 5.4 Hz).
4.1.5 General procedure for the synthesis of 1-(2-(2-bromoacetyl)ethyl)
oxazolo[4,5-g]quinazolin-2(1H)-one derivatives (11a-q)
4.1.5.1 1-(2-(2-bromoacetyl)ethyl)oxazolo[4,5-g]quinazoline-2,8(1H,7H)-dione (9a)
Compound 8 (3.0 g, 12.1 mmol) was dissolved in 50 mL DMF, and bromoacetyl
bromide (7.3 g, 36.4 mmol) was added dropwise at 0 °C. The reaction mixture was
stirred at room temperature for 30 min, and then the mixture was poured into ice
water (200 mL) and stirred for 15 min, followed by filtering and distilling under a
reduced pressure. The white product was purified by silica-gel column
chromatography (DCM/MeOH, 100/1), R = 0.15. Drying give compound 9a (3.8 g,
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yield, 85.4%); mp: 245-246 °C; H NMR (300 MHz, DMSO-d ) δ 12.27 (1H, br s),
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4.1.5.2
1-(2-(2-bromoacetyl)ethyl)-8-Chlorooxazolo[4,5-g]quinazolin-2(1H)-one
(10a)
Compound 9a (3.7 g, 10 mmol), thionyl chloride (40 mL) and DMF (4 drops) was
successively added and the mixture was refluxed for 24 h. After cooled to room
temperature, most of the excess of thionyl chloride and DMF was removed under
reduced pressure to give the yellow residue. The crude product was purified by
silica-gel column chromatography (DCM), R = 0.21. Drying gave compound 10a (3.3
f
1
g, yield, 85.4%); mp: 184-185 °C; H NMR (300 MHz, DMSO-d ) δ 8.43 (1H, s),
6
8.02 (1H, s), 7.65 (1H, s), 4.43 (2H, t, J = 4.4 Hz), 4.24-4.22 (4H, m).
4.1.5.2.1
1-(2-(2-bromoacetyl)ethyl)-8-(3-Chloro-4-fluorophenylamino)oxazolo[4,5-g]quinazol
in-2(1H)-one (11a)
A mixture of compound 10a (155 mg, 0.4 mmol) and 3-chloro-4-fluoroaniline (70 mg,
0.48 mmol) in isopropanol (6 mL) was stirred for 24 h. After cooled to room
temperature, the mixture was filtered and washed with chill isopropanol (3 mL) and
the residue was treated with aqueous NaHCO (10 mL) and extracted wtih
3
EtOAc/MeOH (20:1, 20 mL). The organic layer was washed with brine, dried over
Na SO , and concentrated under reduced pressure. Purified by silica-gel column
2
4
chromatography (DCM/MeOH, 100/1), R = 0.23. Drying gave 163 mg (yield, 82.3%)
f
of the title compound as white solid; mp: 229-231 °C; HPLC purity: 98%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrClFN O (M+H) , 494.9881; found, 494.9879; H
1
9
14
4
4
NMR (300 MHz, DMSO-d ) δ 11.60 (1H, br s), 9.01 (1H, s), 8.90 (1H, s), 8.08 (1H,
6
1
3

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
dd, J = 6.8, 2.5 Hz), 7.82-7.78 (2H, m), 7.54 (1H, t, J = 9.0 Hz), 4.57 (2H, t, J = 4.7
1
3
Hz), 4.29 (2H, s), 4.20 (2H, t, J = 4.7 Hz); C NMR (75 MHz, DMSO-d ) δ 167.06,
6
159.15, 153.10, 149.82, 147.69, 136.44, 134.13 (d, J = 2.7 Hz), 132.49, 126.28,
125.04 (d, J = 7.3 Hz), 116.85 (d, J = 21.8 Hz), 110.37, 103.58, 99.88, 62.05, 41.88,
25.61.
4.1.5.2.2
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)oxazolo
[4,5-g]quinazolin-2(1H)-one (11b)
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.20. Light
f
yellow solid, 200 mg, yield, 83.3%; mp: 252-254 °C; HPLC purity: 97%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrClFN O (M+H) , 601.0289; found, 601.0285; H
26
20
4
5
NMR (300 MHz, DMSO-d ) δ 11.71 (1H, br s), 9.16 (1H, s), 8.91 (1H, s), 7.75-7.65
6
(m, 1H), 7.50-7.45 (m, 1H), 7.38-7.28 (m, 2H), 7.24-7.10 (m, 1H), 5.30 (s, 2H), 4.60
1
3
(2H, t, J = 4.6 Hz), 4.33 (2H, s), 4.24 (2H, t, J = 4.6 Hz); C NMR (75 MHz,
DMSO-d ) δ 167.06, 163.80, 160.58, 158.92, 153.12, 151.54, 149.81, 147.57, 136.28,
6
132.39, 130.61 (d, J = 7.5 Hz), 126.05 (d, J = 3.2 Hz), 124.37, 123.32 (d, J = 2.6 Hz),
121.10, 114.75 (d, J = 7.7 Hz), 114.14 (d, J = 8.3 Hz), 113.91, 110.29, 103.46, 99.89,
69.38, 62.05, 41.84, 25.62.
4.1.5.2.3
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-(tert-butyl)phenyl)amino)
oxazolo[4,5-g]quinazolin-2(1H)-one (11c)
Purified by silica-gel column chromatography (DCM/MeOH, 200/1), R = 0.22. Light
f
yellow solid, 172 mg, yield, 86.1%; mp: 224-226 °C; HPLC purity: 97%; HRMS,
1
4

ACCEPTED MANUSCRIPT
+
+
1
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
ESI , m/z: Calcd for C H BrN O (M+H) , 499.0981; found, 499.0978; H NMR
2
3
24
4
4
(300 MHz, DMSO-d ) δ 11.64 (1H, br s), 9.01 (1H, s), 8.86 (1H, s), 7.86 (1H, s), 7.66
6
(2H, d, J = 8.6 Hz), 7.51 (2H, d, J = 8.6 Hz), 4.60 (2H, t, J = 4.6 Hz), 4.33 (2H, s),
1
3
4.24 (2H, t, J = 4.6 Hz), 1.32 (9H, s); C NMR (75 MHz, DMSO-d ) δ 167.04,
6
158.66, 155.04, 153.08, 149.57, 147.42, 138.90, 135.88, 132.28, 130.40, 124.60,
119.40, 110.16, 103.40, 99.70, 62.00, 41.80, 34.67, 31.35, 25.63.
4.1.5.2.4
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amino)
oxazolo[4,5-g]quinazolin-2(1H)-one (11d)
Purified by silica-gel column chromatography (DCM/MeOH, 70/1), R = 0.23. Light
f
yellow solid, 214 mg, yield, 83.9%; mp: 257-259 °C; HPLC purity: 98%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrClF N O (M+H) , 637.0001; found, 636.9997; H
2
6
18
3
4
5
NMR (300 MHz, DMSO-d ) δ 11.76 (1H, br s), 9.18 (1H, s), 8.96 (1H, s), 8.20 (1H, d,
6
J = 2.5 Hz), 7.90 (1H, dd, J = 8.8, 2.5 Hz), 7.86 (1H, s), 7.64 (1H, t, J = 7.8 Hz), 7.51
(1H, d, J = 7.8 Hz), 7.38 (1H, d, J = 8.8 Hz), 7.32-7.20 (2H, m), 4.59 (2H, t, J = 4.6
1
3
Hz), 4.31 (2H, s), 4.23 (2H, t, J = 4.6 Hz); C NMR (75 MHz, DMSO-d ) δ 167.04,
6
159.01, 157.07, 153.10, 149.88, 147.85 (d, J = 24.8 Hz), 136.69, 134.85, 132.50,
131.55, 126.58, 126.22, 124.79, 122.02, 120.76, 119.96 (d, J = 3.0 Hz), 119.50,
113.47 (d, J = 4.5 Hz), 110.47, 103.52, 100.04, 62.02, 41.86, 25.64.
4.1.5.2.5
1-(2-(2-bromoacetyl)ethyl)-8-((4-(morpholine-4-carboxamido)phenyl)amino)oxazolo[
4,5-g]quinazolin-2(1H)-one (11e)
1
5

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Purified by silica-gel column chromatography (DCM/MeOH, 50/1), R = 0.22. Light
f
yellow solid, 197 mg, yield, 86.0%; mp: 263-265 °C; HPLC purity: 98%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrN O (M+H) , 571.0940; found, 571.0933; H NMR
2
4
24
6
6
(300 MHz, DMSO-d ) δ 11.52 (1H, br s), 9.02 (1H, s), 8.88 (1H, s), 8.78 (1H, s), 7.86
6
(1H, s), 7.65-7.58 (4H, m), 4.62 (2H, t, J = 4.6 Hz), 4.34 (2H, s), 4.25 (2H, t, J = 4.6
1
3
Hz), 3.66-3.63 (4H, m), 3.49-3.46 (4H, m); C NMR (75 MHz, DMSO-d ) δ 167.06,
6
158.69, 155.06, 153.10, 149.59, 147.44, 138.91, 135.90, 132.30, 130.42, 124.61,
119.41, 110.18, 103.41, 99.68, 65.98, 62.00, 44.19, 41.80, 25.63.
4.1.5.2.6
1-(2-(2-bromoacetyl)ethyl)-8-((4-(3-cyclopentylureido)phenyl)amino)oxazolo[4,5-g]q
uinazolin-2(1H)-one (11f)
Purified by silica-gel column chromatography (DCM/MeOH, 50/1), R = 0.21. Light
f
yellow solid, 189 mg, yield, 83.2%; mp: 266-268 °C; HPLC purity: 96%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrN O (M+H) , 569.1132; found, 569.1128; H NMR
2
5
26
6
5
(300 MHz, DMSO-d ) δ 11.60 (1H, br s), 9.02 (1H, s), 8.86 (1H, s), 8.76 (1H, s), 7.86
6
(1H, s), 7.65-7.58 (4H, m), 6.24 (1H, bs), 4.60 (2H, t, J = 4.6 Hz), 4.33 (2H, s), 4.23
(2H, t, J = 4.6 Hz), 1.78-1.69 (2H, m), 1.67-1.56 (2H, m), 1.54-1.42 (1H, m),
1
3
1.30-1.03 ( 4H, m); C NMR (75 MHz, DMSO-d ) δ 167.07, 159.01, 156.89, 154.30,
6
153.12, 149.90, 149.41, 147.69, 142.44, 136.53, 133.63, 132.51, 129.97, 126.20,
124.63, 124.28, 121.09, 118.66, 112.40, 110.41, 109.70, 106.07, 103.38, 100.00,
89.40, 62.03, 47.43, 41.85, 32.86, 25.61, 22.43.
4.1.5.2.7
1
6

ACCEPTED MANUSCRIPT
1-(2-(2-bromoacetyl)ethyl)-8-((4-(piperidine-1-carboxamido)phenyl)amino)oxazolo[4
,5-g]quinazolin-2(1H)-one (11g)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
Purified by silica-gel column chromatography (DCM/MeOH, 50/1), R = 0.20. Light
f
yellow solid, 185 mg, yield, 81.1%; mp: 259-261 °C; HPLC purity: 97%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrN O (M+H) , 569.1148; found, 569.1143; H NMR
2
5
26
6
5
(300 MHz, DMSO-d ) δ 11.39 (1H, br s), 8.89 (1H, s), 8.86 (1H, s), 8.63 (1H, s), 7.84
6
(1H, s), 7.62-7.54 (4H, m), 4.61 (2H, t, J = 4.6 Hz), 4.32 (2H, s), 4.24 (2H, t, J = 4.6
1
3
Hz), 3.47-3.43 (4H, m), 1.59-1.51 (6H, m); C NMR (75 MHz, DMSO-d ) δ 167.05,
6
158.74, 154.78, 153.13, 149.66, 147.48, 139.39, 135.96, 132.35, 130.13, 124.63,
119.40, 110.20, 103.31, 99.76, 62.02, 44.68, 41.81, 25.62, 25.51, 24.08.
4.1.5.2.8
1-(2-(2-bromoacetyl)ethyl)-8-((4-(3-cyclohexylureido)phenyl)amino)oxazolo[4,5-g]qu
inazolin-2(1H)-one (11h)
Purified by silica-gel column chromatography (DCM/MeOH, 50/1), R = 0.21. Light
f
yellow solid, 192 mg, yield, 82.4%; mp: 267-268 °C; HPLC purity: 97%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrN O (M+H) , 583.1204; found, 583.1200; H NMR
2
6
28
6
5
(300 MHz, DMSO-d ) δ 11.45 (1H, br s), 8.92 (1H, s), 8.86 (1H, s), 8.80 (1H, s), 7.84
6
(1H, s), 7.59-7.48 (4H, m), 6.36 (1H, br s), 4.60 (2H, t, J = 4.6 Hz), 4.33 (2H, s), 4.22
13
(2H, t, J = 4.6 Hz), 1.88-1.46 (5H, m), 1.40-1.08 (6H, m); C NMR (75 MHz,
DMSO-d ) δ 167.02, 158.64, 154.43, 153.10, 149.60, 147.46, 139.32, 132.33, 129.40,
6
125.05, 117.20, 110.15, 103.25, 99.68, 61.99, 47.48, 41.79, 25.63, 25.25, 24.24.
4.1.5.2.9
17

ACCEPTED MANUSCRIPT
1-(2-(2-bromoacetyl)ethyl)-8-((3,4-difluorophenyl)amino)oxazolo[4,5-g]quinazolin-2(
1H)-one (11i)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.24. White
f
solid, 159 mg, yield, 82.8%; mp: 228-230 °C; HPLC purity: 98%; HRMS, ESI+, m/z:
+
1
Calcd for C H BrF N O (M+H) , 479.0166; found, 479.0164; H NMR (300 MHz,
1
9
14
2
4
4
DMSO-d ) δ 11.71 (1H, br s), 9.14 (1H,s), 8.95 (1H,s), 8.03 (1H, ddd, J = 12.5, 7.5,
6
2.5 Hz), 7.87 (1H, s), 7.70 (1H, d, J = 9.0 Hz), 7.58 (1H, dd, J = 19.4, 9.0 Hz), 4.61
1
3
(2H, t, J = 4.8 Hz), 4.32 (2H, s), 4.25 (2H, t, J = 4.8 Hz); C NMR (75 MHz,
DMSO-d ) δ 167.05, 159.11, 153.11, 149.93, 147.72, 136.74, 132.53, 121.19 (q, J =
6
3.0 Hz), 117.42 (d, J = 18.0 Hz), 114.90 (d, J = 21.0 Hz), 110.37, 103.25, 100.16,
62.02, 41.84, 25.62.
4.1.5.2.10
1-(2-(2-bromoacetyl)ethyl)-8-((4-(benzyloxy)phenyl)amino)oxazolo[4,5-g]quinazolin-
2(1H)-one (11j)
Purified by silica-gel column chromatography (DCM/MeOH, 70/1), R = 0.25. Light
f
yellow solid, 182mg, yield, 82.7%; mp: 250-251 °C; HPLC purity: 97%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrN O (M+H) , 549.0773; found, 549.0771; H NMR
2
6
22
4
5
(300 MHz, DMSO-d ) δ 11. 48 (1H, br s), 8.97 (1H, s), 8.83 (1H, s), 7.65 (1H, s),
6
7.62 (1H, s), 7.48-7.33 (5H, m), 7.14 (1H, s), 7.11 (1H, s), 5.15 (2H, s), 4.59 (2H, t, J
1
3
= 4.9 Hz), 4.34 (2H, s), 4.22 (2H, t, J = 4.9 Hz); C NMR (75 MHz, DMSO-d ) δ
6
167.02, 158.79, 156.62, 153.12, 149.82, 147.41, 136.92, 132.24, 129.72, 128.43,
127.85, 127.67, 125.96, 114.84, 110.22, 103.29, 100.02, 69.46, 62.03, 41.80, 25.61.
1
8

ACCEPTED MANUSCRIPT
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
4.1.5.2.11
1-(2-(2-bromoacetyl)ethyl)-8-((4-((4-chlorobenzyl)oxy)phenyl)amino)oxazolo[4,5-g]q
uinazolin-2(1H)-one (11k)
Purified by silica-gel column chromatography (DCM/MeOH, 70/1), R = 0.25. Light
f
yellow solid, 187mg, yield, 79.9%; mp: 251-252 °C; HPLC purity: 98%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrClN O (M+H) , 583.0333; found, 583.0331; H NMR
2
6
21
4
5
(300 MHz, DMSO-d ) δ 11.58 (1H, br s), 9.10 (1H, s), 8.97 (1H, s), 8.14-8.05 (4H,
6
m), 7.90 (1H, s), 7.57-7.48 (4H, m), 4.63 (2H, t, J = 4.9 Hz), 4.33 (2H, s), 4.28 (2H, t,
13
J = 4.9 Hz); C NMR (75 MHz, DMSO-d ) δ 167.02, 164.92, 158.98, 153.08, 149.81,
6
147.69, 141.73, 137.14, 135.17, 132.70, 132.48, 129.84, 129.76, 128.49, 126.33,
123.74, 110.70, 103.49, 100.19, 65.35, 62.04, 41.84, 25.62.
4.1.5.2.12
Ethyl-4-((1-(2-(2-bromoacetoxy)ethyl)-2-oxo-1,2-dihydrooxazolo[4,
5-g]quinazolin-8-yl)amino)benzoate (11l)
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.20. Light
f
yellow solid, 175 mg, yield, 85.0%; mp: 227-229 °C; HPLC purity: 96%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrN O (M+H) , 515.0566; found, 515.0567; H NMR
2
2
20
4
6
(300 MHz, DMSO-d ) δ 11.63 (1H, br s), 9.15 (1H, s), 8.95 (1H, s), 8.03 (4H, s), 7.88
6
(1H, s), 4.61 (2H, t, J = 4.8 Hz), 4.36-4.30 (4H, m), 4.25 (2H, t, J = 4.8 Hz), 1.34 (3H,
1
3
t, J = 7.1 Hz); C NMR (75 MHz, DMSO-d ) δ 167.05, 165.14, 158.97, 153.10,
6
149.92, 147.72, 141.44, 137.20, 132.51, 129.70, 126.85, 123.71, 110.67, 103.29,
100.28, 62.05, 60.74, 41.82, 25.63, 14.17.
1
9

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
4.1.5.2.13
1-(2-(2-bromoacetyl)ethyl)-8-((4-((4-methoxybenzyl)oxy)phenyl)amino)oxazolo[4,5-g
]quinazolin-2(1H)-one (11m)
Purified by silica-gel column chromatography (DCM/MeOH, 70/1), R = 0.23. Light
f
yellow solid, 190 mg, yield, 81.9%; mp: 256-258 °C; HPLC purity: 97%; HRMS,
+
1
ESI+, m/z: Calcd for C H BrN O (M+H) , 579.0879; found, 579.0873; H NMR
2
7
24
4
6
(300 MHz, DMSO-d ) δ 11.55 (1H, br s), 9.02 (1H, s), 8.86 (1H, s), 7.87 (1H, s), 7.66
6
(2H, d, J = 9.0 Hz), 7.43 (2H, d, J = 9.0 Hz), 7.13 (2H, d, J = 9.0 Hz), 6.97 (2H, d, J =
9.0 Hz), 5.10 (2H, s), 4.62 (2H, t, J = 5.0 Hz), 4.34 (2H, s), 4.25 (2H, t, J = 5.0 Hz),
1
3
3.79 (3H, s); C NMR (75 MHz, DMSO-d ) δ 167.08, 167.25, 159.01, 158.89,
6
156.79, 153.12, 149.73, 147.50, 135.99, 132.37, 129.52, 128.73, 126.06, 114.89,
113.82, 110.14, 103.22, 99.80, 69.21, 62.03, 61.30, 55.08, 41.81, 25.63.
4.1.5.2.14
1-(2-(2-bromoacetyl)ethyl)-8-((3-ethynylphenyl)amino)oxazolo[4,5-g]quinazolin-2(1
H)-one (11n)
Purified by silica-gel column chromatography (DCM/MeOH, 200/1), R = 0.25. Light
f
yellow solid, 158 mg, yield, 84.5%; mp: 230-231 °C; HPLC purity: 98%; HRMS,
+
+
1
ESI , m/z: Calcd for C H BrFN O (M+H) , 467.0355; found, 467.0357; H NMR
2
1
16
4
4
(300 MHz, DMSO-d ) δ 11.71 (1H, br s), 9.17 (1H, s), 8.95 (1H, s), 7.96 (1H, s), 7.88
6
(1H, s), 7.85 (1H, s), 7.52 (1H, t, J = 7.8 Hz), 7.43 (1H, d, J = 7.8 Hz), 4.61 (2H, t, J =
13
4.6 Hz), 4.33 (2H, s), 4.30 (1H, s), 4.26 (2H, t, J = 4.6 Hz); C NMR (75 MHz,
DMSO-d ) δ 167.04, 159.18, 153.10, 149.82, 147.69, 137.15, 136.42, 132.51, 129.47,
6
2
0

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
129.20, 127.41, 125.10, 122.06, 110.37, 103.40, 99.91, 82.84, 81.39, 62.05, 41.84,
25.62.
4.1.5.2.15
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-(4-methylpiperazin-1-yl)phenyl)amino)oxa
zolo[4,5-g]quinazolin-2(1H)-one (11o)
Purified by silica-gel column chromatography (DCM/MeOH, 30/1), R = 0.23. Yellow
f
solid, 184 mg, yield, 80.0%; mp: 272-275 °C; HPLC purity: 97%; HRMS, ESI+, m/z:
+
1
Calcd for C H BrClN O (M+H) , 575.0709; found, 575.0710; H NMR (300 MHz,
24
25
6
4
DMSO-d ) δ 9.88 (1H, br s), 8.57 (1H, s), 8.53 (1H, s), 8.09 (1H, d, J = 2.4 Hz), 7.86
6
(1H,dd, J = 8.8, 2.4 Hz), 7.69(1H, s), 7.28 (1H, d, J = 8.8 Hz), 4.60 (2H, t, J = 4.6 Hz),
4.34 (2H, s), 4.25 (2H, t, J = 4.6 Hz), 3.22-3.05 (4H, m), 2.96 (3H, s), 2.56-2.52 (4H,
1
3
m); C NMR (126 MHz, DMSO-d ) δ 167.04, 159.04, 153.13, 149.72, 147.67,
6
132.51, 127.04, 126.29, 124.19, 120.58, 110.36, 103.61, 99.76, 62.08, 57.04, 52.31,
46.64, 41.92, 25.62.
4.1.5.2.16
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-morpholinophenyl)amino)oxazolo[4,5-g]q
uinazolin-2(1H)-one (11p)
Purified by silica-gel column chromatography (DCM/MeOH, 40/1), R = 0.22. Yellow
f
solid, 190 mg, yield, 84.4%; mp: 275-277 °C; HPLC purity: 98%; HRMS, ESI+, m/z:
+
1
Calcd for C H BrClN O (M+H) , 562.0493; found, 562.0492; H NMR (300 MHz,
23
22
5
5
DMSO-d ) δ 11.63 (1H, br s), 9.10 (1H, s), 8.94 (1H, s), 7.95 (1H, d, J = 2.4 Hz), 7.87
6
(1H, s), 7.76 (1H, dd, J = 8.8, 2.4 Hz), 7.30 (1H, d, J = 8.8 Hz), 4.60 (2H, t, J = 4.6
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Hz), 4.34 (2H, s), 4.25 (2H, t, J = 4.6 Hz), 3.86-3.70 (4H, m), 3.10-2.98 (4H, m); C
NMR (126 MHz, DMSO-d ) δ 167.03, 159.02, 153.15, 149.74, 147.69, 132.53,
6
127.06, 126.31, 124.21, 120.60, 110.38, 103.63, 99.77, 66.35, 62.10, 54.93, 41.92,
25.61.
4.1.5.2.17
1-(2-(2-bromoacetyl)ethyl)-8-((3-chloro-4-(piperidin-1-yl)phenyl)amino)oxazolo[4,5-
g]quinazolin-2(1H)-one (11q)
Purified by silica-gel column chromatography (DCM/MeOH, 35/1), R = 0.22. Yellow
f
solid, 181 mg, yield, 80.8%; mp: 271-273 °C; HPLC purity: 97%; HRMS, ESI+, m/z:
+
1
Calcd for C H BrClN O (M+H) , 560.0600; found, 560.0599; H NMR (300 MHz,
24
24
5
4
DMSO-d ) δ 9.61 (1H, br s), 8.55 (1H, s), 8.23 (1H, s), 7.96 (1H, d, J = 2.4 Hz), 7.75
6
(1H,dd, J = 8.8, 2.4 Hz), 7.64 (1H, s), 7.20 (1H, d, J = 8.8 Hz), 4.60 (2H, t, J = 4.6
Hz), 4.33 (2H, s), 4.24 (2H, t, J = 4.6 Hz),3.01-2.85 (4H, m), 1.79-1.64 (4H, m),
1
3
1.63-1.48 (2H, m); C NMR (126 MHz, DMSO-d ) δ 167.05, 159.02, 153.15, 149.74,
6
147.69, 132.53, 127.06, 126.31, 124.21, 120.60, 110.38, 103.63, 99.77, 66.35, 62.10,
49.52, 41.92, 26.13, 25.62, 24.74.
4.1.6 General procedure for the synthesis of 1-(2-(2-chloroacetoxy)ethyl)
oxazolo[4,5-g]quinazolin-2(1H)-one derivatives (12a-g)
4.1.6.1 1-(2-(2-chloroacetoxy)ethyl) oxazolo[4,5-g]quinazoline-2, 8(1H,7H)-dione
(9b)
Compound 8 (2.0 g, 8.1 mmol) was dissolved in 50 mL DMF, and bromoacetyl
bromide (2.9 g, 24.3 mmol) was added dropwise at 0 °C. The reaction mixture was
2
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stirred at room temperature for 30 min, and then the mixture was poured into ice
water (200 mL) and stirred for 15 min, followed by filtering and distilling under a
reduced pressure. The white product was purified by silica-gel column
chromatography (DCM/MeOH, 100/1), R = 0.17. Drying give compound 9b (2.3 g,
f
1
yield, 87.8%); mp: 233-234 °C; H NMR (DMSO-d ) δ 12.27 (1H, br s), 8.05 (1H, s),
6
7.98 (1H, s), 7.57 (1H, s), 4.60 (2H, t, J = 4.6 Hz), 4.32 (2H, s), 4.24 (2H, t, J = 4.6
Hz).
4.1.6.2
1-(2-(2-chloroacetoxy)ethyl)-8-Chlorooxazolo[4,5-g]quinazolin-2(1H)-one
(10b)
Compound 9a (2.0 g, 6.1 mmol), thionyl chloride (25 mL) and DMF (4 drops) was
successively added and the mixture was refluxed for 24 h. After cooled to room
temperature, most of the excess of thionyl chloride and DMF was removed under
reduced pressure to give the yellow residue. The crude product was purified by
silica-gel column chromatography (DCM), R = 0.23. Drying gave compound 10b (1.7
f
1
g, yield, 81.5%); mp: 182-184 °C; H NMR (DMSO-d ) δ 8.43 (1H, s), 8.02 (1H, s),
6
7.65 (1H, s), 4.60 (2H, t, J = 4.6 Hz), 4.32 (2H, s), 4.24 (2H, t, J = 4.6 Hz).
4.1.6.2.1
1-(2-(2-chloroacetoxy)ethyl)-8-(3-Chloro-4-fluorophenylamino)oxazolo[4,5-g]quinaz
olin-2(1H)-one (12a)
A mixture of compound 10b (137 mg, 0.4 mmol) and 3-chloro-4-fluoroaniline (70 mg,
0.48 mmol) in isopropanol (6 mL) was stirred for 12 h. After cooled to room
temperature, the mixture was filtered and washed with chill isopropanol (3 mL) and
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the residue was treated with aqueous NaHCO (10 mL) and extracted wtih
3
EtOAc/MeOH (20:1, 20 mL). The organic layer was washed with brine, dried over
Na SO , and concentrated under reduced pressure. Purified by silica-gel column
2
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chromatography (DCM/MeOH, 100/1), R = 0.23. Drying gave 154 mg (yield, 85.6%)
f
of the title compound as white solid; mp: 228-230 °C; HPLC purity: 98%; HRMS,
+
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ESI+, m/z: Calcd for C H Cl FN O (M+H) , 451.0371; found, 451.0381; H NMR
1
9
14
2
4
4
(DMSO-d ) δ 11.90 (1H, br s), 9.26 (1H, s), 8.96 (1H, s), 8.13 (1H, dd, J = 6.8, 2.6
6
Hz), 7.90-7.84 (2H, m), 7.56 (1H, t, J = 9.1 Hz), 4.60 (2H, t, J = 4.6 Hz), 4.32 (2H, s),
13
4.24 (2H, t, J = 4.6 Hz); C NMR (75 MHz, DMSO-d ) δ 167.24, 159.18, 156.73,
6
153.10, 149.85, 147.71, 136.56, 134.14 (d, J = 2.6 Hz), 132.51, 126.32, 125.05 (d, J =
7.5 Hz), 116.86 (d, J = 21.8 Hz), 114.01, 110.39, 103.51, 99.97, 62.02, 41.87, 40.99.
4.1.6.2.2
1-(2-(2-chloroacetoxy)ethyl)-8-((3,4-difluorophenyl)amino)oxazolo[4,5-g]quinazolin-
2(1H)-one (12b)
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.24. White
f
solid, 144 mg, yield, 82.8%; mp: 227-228 °C; HPLC purity: 96%; HRMS, ESI+, m/z:
+
1
Calcd for C H ClF N O (M+H) , 435.0666; found, 435.0674; H NMR (300 MHz,
1
9
14
2
4
4
DMSO-d ) δ 11.83 (1H, br s), 9.24 (1H, s), 8.96 (1H, s), 8.08-8.01 (1H, m), 7.88 (1H,
6
s), 7.73-7.70 (1H, m), 7.63-7.53 (1H, m), 4.61 (2H, t, J = 4.6 Hz), 4.33 (2H, s), 4.25
1
3
(2H, t, J = 4.6 Hz); C NMR (75 MHz, DMSO-d ) δ 167.21, 159.07, 153.08, 149.85,
6
147.67, 136.73, 132.47, 121.11 (q, J = 3.0 Hz), 117.33 (d, J = 18.0 Hz), 113.80 (d, J =
20.0 Hz), 110.38, 103.39, 100.10, 62.00, 41.83, 40.95.
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4.1.6.2.3
1-(2-(2-chloroacetoxy)ethyl)-8-((3-chloro-4-(3-(trifluoromethyl)phenoxy)phenyl)amin
o)oxazolo[4,5-g]quinazolin-2(1H)-one (12c)
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.24. Light
f
yellow solid, 208 mg, yield, 87.8%; mp: 255-256 °C; HPLC purity: 97%; HRMS,
+
+
1
ESI , m/z: Calcd for C H Cl F N O (M+H) , 593.0601; found, 593.0603; H NMR
2
6
18
2
3
4
5
(300 MHz, DMSO-d ) δ 11.82 (1H, br s), 9.23 (1H, s), 8.98 (1H, s), 8.22 (1H, d, J =
6
1.9 Hz), 7.94-7.89 (2H, m), 7.66 (1H, t, J = 7.9 Hz), 7.53 (1H, d, J = 7.4 Hz), 7.40
(1H, d, J = 8.9 Hz), 7.34-7.23 (2H, m), 4.60 (2H, t, J = 4.6 Hz), 4.32 (2H, s), 4.23 (2H,
13
t, J = 4.6 Hz); C NMR (75 MHz, DMSO-d ) δ 167.29, 159.01, 157.11, 153.17,
6
150.08, 148.08, 147.86 (d, J = 16.5 Hz), 134.90, 132.55, 131.62, 126.28, 124.83,
122.12, 120.85, 120.03 (d, J = 4.1 Hz), 113.47 (d, J = 3.8 Hz), 110.53, 103.18, 100.43,
62.07, 41.87, 40.99.
4.1.6.2.4
1-(2-(2-chloroacetoxy)ethyl)-8-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)oxazo
lo[4,5-g]quinazolin-2(1H)-one (12d)
Purified by silica-gel column chromatography (DCM/MeOH, 100/1), R = 0.25. Light
f
yellow solid, 191 mg, yield, 85.7%; mp: 251-252 °C; HPLC purity: 98%; HRMS,
+
1
ESI+, m/z: Calcd for C H Cl FN O (M+H) , 557.0789; found, 557.0797; H NMR
2
6
20
2
4
5
(300 MHz, DMSO-d ) δ 11.88 (1H, br s), 9.23 (1H, s), 8.94 (1H, s), 7.97 (1H, d, J =
6
2.4 Hz), 7.89 (1H, s), 7.75-7.71(1H, m), 7.52-7.43 (1H, m), 7.37-7.30 (2H, m),
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7.22-7.15 (1H, m), 4.60 (2H, t, J = 4.6 Hz), 4.33 (2H, s), 4.24 (2H, t, J = 4.6 Hz); C
NMR (75 MHz, DMSO-d ) δ 167.26, 163.81, 160.60, 158.92, 153.12, 151.54, 149.82,
6
147.57, 136.28, 132.40, 130.61 (d, J = 7.5 Hz), 126.11, 124.37, 121.10, 114.75 (d, J =
17.3 Hz), 114.14 (d, J = 8.3 Hz), 113.93, 110.29, 103.46, 99.92, 69.40, 62.04, 41.86,
40.98.
4.1.6.2.5
1-(2-(2-chloroacetoxy)ethyl)-8-((3-chloro-4-(4-methylpiperazin-1-yl)phenyl)amino)ox
azolo[4,5-g]quinazolin-2(1H)-one (12e)
Purified by silica-gel column chromatography (DCM/MeOH, 30/1), R = 0.24. Yellow
f
+
solid, 176 mg, yield, 82.6%; mp: 265-266 °C; HPLC purity: 98%; HRMS, ESI , m/z:
+
1
Calcd for C H Cl N O (M+H) , 531.1309; found, 531.1314; H NMR (300 MHz,
2
4
25
2
6
4
DMSO-d ) δ 9.87 (1H, br s), 8.59 (1H, s), 8.54 (1H, s), 8.09 (1H, d, J = 2.4 Hz), 7.86
6
(1H,dd, J = 8.8, 2.4 Hz), 7.69(1H, s), 7.28 (1H, d, J = 8.8 Hz), 4.62 (2H, t, J = 4.6 Hz),
4.33 (2H, s), 4.20 (2H, t, J = 4.6 Hz), 3.21-3.06 (4H, m), 2.95 (3H, s), 2.57-2.54 (4H,
1
3
m); C NMR (126 MHz, DMSO-d ) δ 167.24, 159.00, 153.13, 149.72, 147.67,
6
132.51, 127.04, 126.29, 124.19, 120.58, 110.36, 103.61, 99.76, 62.08, 57.04, 52.31,
46.63, 41.92, 41.02.
4.1.6.2.6
1-(2-(2-chloroacetoxy)ethyl)-8-((3-chloro-4-morpholinophenyl)amino)oxazolo[4,5-g]
quinazolin-2(1H)-one (12f)
Purified by silica-gel column chromatography (DCM/MeOH, 40/1), R = 0.22. Yellow
f
solid, 167 mg, yield, 80.7%; mp: 269-271 °C; HPLC purity: 98%; HRMS, ESI+, m/z:
2
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Calcd for C H Cl N O (M+H) , 518.0993; found, 518.0990; H NMR (300 MHz,
2
3
22
2
5
5
DMSO-d ) δ 11.86 (1H, br s), 9.25 (1H, s), 8.94 (1H, s), 7.94 (1H, d, J = 2.2 Hz), 7.88
6
(1H, s), 7.75 (1H,dd, J = 8.6, 2.2 Hz), 7.27 (1H, d, J = 8.6 Hz), 4.60 (2H, t, J = 4.6
Hz), 4.33 (2H, s), 4.24 (2H, t, J = 4.6 Hz), 3.79-3.73 (4H, m), 3.05-2.2.99 (4H, m);
1
3
C NMR (126 MHz, DMSO-d ) δ 167.28, 159.02, 153.15, 149.74, 147.69, 132.53,
6
127.06, 126.31, 124.21, 120.60, 110.38, 103.63, 99.77, 66.35, 62.10, 54.93, 41.92,
41.02.
4.1.6.2.7
1-(2-(2-chloroacetoxy)ethyl)-8-((3-chloro-4-(piperidin-1-yl)phenyl)amino)oxazolo[4,5
-g]quinazolin-2(1H)-one (12g)
Purified by silica-gel column chromatography (DCM/MeOH, 35/1), R = 0.21. Yellow
f
solid, 185 mg, yield, 89.4%; mp: 270-272 °C; HPLC purity: 98%; HRMS, ESI+, m/z:
1
Calcd for C H Cl N O (M+H)+,516.1200; found, 516.1206; H NMR (300 MHz,
2
4
24
2
5
4
DMSO-d ) δ 9.64 (1H, br s), 8.54 (1H, s), 8.26 (1H, s), 7.95 (1H, d, J = 2.4 Hz), 7.74
6
(1H,dd, J = 8.8, 2.4 Hz), 7.66 (1H, s), 7.20 (1H, d, J = 8.8 Hz), 4.60 (2H, t, J = 4.6
Hz), 4.33 (2H, s), 4.24 (2H, t, J = 4.6 Hz), 2.97-2.88 (4H, m), 1.75-1.62 (4H, m),
1
3
1.61-1.50 (2H, m); C NMR (126 MHz, DMSO-d ) δ 167.28, 159.02, 153.15, 149.74,
6
147.69, 132.53, 127.06, 126.31, 124.21, 120.60, 110.38, 103.63, 99.77, 66.35, 62.10,
49.52, 41.92, 41.02, 26.13, 24.74.
5
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92 4.2 Biological evaluation
93
4.2.1 In Vitro Enzymatic Activity Assay.
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EGFR , HER2 and the Z’-Lyte Kinase Kit were purchased from Invitrogen. The
experiments were performed according to the instructions of the manufacturer. Briefly,
the respective final concentrations of different kinases were determined as follows:
EGFR (PV3872, Invitrogen), 0.200 ꢀg/mL; HER2 (PV3366, Invitrogen), 0.192
ꢀg/mL. Six concentration (0.0001-10 ꢀM) gradients were set for all the tested
compounds in DMSO, and a 4× compound solution was prepared. An ATP solution
and a kinase/peptide mixture were prepared right before use. The 10 ꢀL kinase
reaction consisted of 2.5 ꢀL of compound solution, 5 ꢀL of kinase/peptide mixture,
and 2.5 ꢀL of ATP solution. A 100% phosphorylation control was provided by 5 ꢀL
of phosphopeptide solution instead of the kinase/peptide mixture. A 100% inhibition
control was prepared using 2.5 ꢀL of kinase buffer instead of the ATP solution, and
2.5 ꢀL of 4% DMSO instead of the compound solution was used as a 0% inhibition
control. The solutions on the plate were mixed thoroughly, and the plate was
incubated for 1 h at room temperature. After 5 ꢀL of development solution was added
to each well, the plate was incubated for 1 h at room temperature; the
non-phosphopeptides were cleaved in this time. Finally, 5 ꢀL of stop reagent was
added to stop the reaction. The activity was measured with an Infinite M100 Pro
multilabel reader. Curve fitting and data presentations were performed using Graph
Pad Prism 5.0. Every experiment was repeated at least three times. Data represented
as means ± SD from three independent experiments.
4.2.2 Cancer Cell Proliferation Inhibition and Normal Cell Cytotoxicity Assay.
Human lung adenocarcinoma cell line (A549), human breast cancer cell line (SK-Br3),
2
8