Design, synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.06.022

The abnormal activation of PI3K signaling pathway leads to the occurrence of various cancers. The PI3Kα is frequently mutated and overexpressed in many human cancers. Therefore, the PI3Kα was considered as a promising target in therapeutic treatment of cancer. In this study, two series of compounds containing 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold were synthesized and evaluated antiproliferative activities against three cancer cell lines, including HCT-116, MDA-MB-231 and SNU638. Compound 7f with the most potent antiproliferative activity was selected for further evaluation on normal cells and PI3K kinase. Studies indicated that compound 7f could decrease the phospho-Akt (T308) in a dose-dependent manner. Four key hydrogen bonding interactions were found in the docking of 7f with PI3K enzyme. All the results suggested that 7f was a potent PI3Kα inhibitor.

Full text of DOI:10.1016/j.bmc.2018.06.022

Accepted Manuscript
Design, synthesis and biological evaluation of novel series of 2H-benzo[b]
[1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold derivatives as
PI3Kα inhibitors
Fu-Dan Dong, Dan-Dan Liu, Cheng-Long Deng, Xiao-chun Qin, Kai Chen, Jian
Wang, Hong-Rui Song, Huai-Wei Ding
PII:
S0968-0896(18)30962-3
https://doi.org/10.1016/j.bmc.2018.06.022
BMC 14414
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 May 2018
13 June 2018
15 June 2018
Please cite this article as: Dong, F-D., Liu, D-D., Deng, C-L., Qin, X-c., Chen, K., Wang, J., Song, H-R., Ding, H-
W., Design, synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-
benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors, Bioorganic & Medicinal Chemistry (2018), doi:
https://doi.org/10.1016/j.bmc.2018.06.022
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Design, synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one
and 2H-benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors
Fu-Dan Donga,c, Dan-Dan Liua, Cheng-Long Denga, Xiao-chun Qinb, Kai Chena, Jian Wanga,
Hong-Rui Songa, Huai-Wei Dinga,*
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
c School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China
* Corresponding authors.
Email address: dinghuaiwei627@163.com.

Abstract: The abnormal activation of PI3K signaling pathway leads to the occurrence of various
cancers. The PI3Kα is frequently mutated and overexpressed in many human cancers. Therefore, the
PI3Kα was considered as a promising target in therapeutic treatment of cancer. In this study, two
series of compounds containing 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine
scaffold were synthesized and evaluated antiproliferative activities against three cancer cell lines,
including HCT-116, MDA-MB-231 and SNU638. Compound 7f with the most potent
antiproliferative activity was selected for further evaluation on normal cells and PI3K kinase. Studies
indicated that compound 7f could decrease the phospho-Akt (T308) in a dose-dependent manner.
Four key hydrogen bonding interactions were found in the docking of 7f with PI3K enzyme. All the
results suggested that 7f was a potent PI3Kα inhibitor.
Keywords: PI3Kα, 2H-benzo[b][1,4]oxazin-3(4H)-one; 2H-benzo[b][1,4]oxazine; anticancer.

1. Introduction
The phosphatidylinositol 3-kinases (PI3Ks) are members of a unique group of intracellular lipid
kinases 1. Based on the primary structure, regulation and in vitro lipid substrate specificity, the PI3K
family is divided into four different classes: Class I, Class II, Class III, and Class IV. Of these, the
most commonly studied is the Class I which is further divided into PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ
2-3. The PI3K/Akt signaling pathway is frequently activated, amplified and mutated in human cancers
4-7 and regulates various cellular processes, such as proliferation, growth and apoptosis 8.
Given the important role of PI3K/Akt signaling pathway in cancers and inhibition of this
pathway is a promising approach for novel chemotherapeutic agents 9-11. Several inhibitors targeting
12-14
PI3K have been developed and are being evaluated in preclinical studies and early clinical trials
.
Among them, there are some PI3K inhibitors which containing morpholine group being developed,
15
18
21
such as BKM120 , GDC0941 16-17, GDC0980 , GSK2636771 19-20, LY294002 (Fig.1) . What’s
more, Omipalisib (GSK2126458) has been identified as a highly potent PI3K inhibitor with
22
picomolar activity and is currently under evaluation in clinical trials for oncology applications .
Based on the reported corresponding X-ray structure, Omipalisib uses the position-1 nitrogen as
a hinge binder 22. The research has shown that a tremendous number of PI3K inhibitors which
containing morpholine group using the oxidation of morpholine interact with the hinge region of the
23
ATP . Thus, we try to replace the pyridine ring of quinoline with the morpholine ring to get
compounds with new skeleton on the basis of Omipalisib. Considering to reduce the difficulty of
synthesis, we replace the quinoline moiety with 2H-benzo[b][1,4]oxazin-3(4H)-one which also has
the morpholine ring scaffold. Not only that, similar to the cyano group of Dactolisib (NVP-BEZ235)
as a hydrophilic group 24, benzoxazine can easily introduce hydrophilic substituents on the position-4
nitrogen atom or position-3 to increase the solubility of the molecules. As a result, we designed two
series of derivatives containing benzoxazine moiety. They were prepared and evaluated for their
anticancer effects and PI3K inhibitory activities in vitro. The results suggested that compound 7f
could be as a PI3Kα inhibitor.

Fig.1. The structures of some PI3K inhibitors which have the morpholine ring.
Fig.2. The design strategy based on Omipalisib, PI3K inhibitors which containing morpholine group and
Dactolisib.
2. Results and discussion
2.1 Chemistry
The general synthetic routes for the target compounds are summarized in Scheme 1. Coupling
reaction of 1 with ten chlorinated compounds to give 2a-j. Compound 1 was treated with P2S5 and
piperazine to get 3. Coupling reaction of 3 with four chlorinated compounds to give 4a-d.
Sulfonylation of 5 with two sulfonyl chlorides to give 6a-b, which were then subjected to Suzuki
coupling with bis(pinacolato)diborane to get arylboronic esters. Then, the arylboronic esters which

were not purified coupling with 2a-j and 4a-d via adding the catalyst required for the Suzuki
coupling reaction to yield the target compounds 7a-s. The structure of the new analogs were
characterized by 1H NMR, 13C NMR and HRMS.
Scheme 1. (a) chlorinated compounds, K2CO3, DMF, 60 ºC, 6 h; (b) i) THF, P2S5, 90 ºC, 8 h ii) THF,
Piperazine, 90 ºC, 6 h; (c) chlorinated compounds, K2CO3, DMF, 60 ºC, 6 h; (d) sulfonyl chlorides, pyrid
ine, 25 ºC, 24 h; (e) Pd(dppf)2Cl2, AcOK, bis(pinacolato)diborane, DMF, 100 ºC, 8 h; (f) Pd(dppf)2Cl2, C
s2CO3, DMF, 90 ºC, 4 h.
2.2 Antiproliferative assays in vitro
All synthesized compounds were evaluated for their cytotoxicities in vitro against three human
cancer cell lines including HCT-116, MDA-MB-231 and SNU638 cells. The antiproliferative results
of all the compounds were summarized in Table 1. The results of antiproliferative effect assay
showed that most of the derivatives exhibited high antiproliferative effects, especially against
HCT-116. Compound 7a (IC50 = 2.40 μM against HCT-116) was more potent than compound 7l (IC50
= 15.8 μM against HCT-116) and compound 7f (IC50 = 0.58 μM against HCT-116) was more potent
than compound 7k (IC50 = 4.62 μM against HCT-116) suggesting that 2,4-difluorophenyl compared
to N-butyl at position of R3 can significantly increase the antiproliferative activity. This conclusion
was also verified by 7c vs. 7n and 7e vs. 7o. Compound 7f was more potent than compound 7a also
suggesting that the activity could be increased with the increased length of alkyl group and this
conclusion could also be confirmed by compounds 7k vs. 7l. From compounds 7i (IC50 = 33.6 μM
against HCT-116), 7k, 7m (IC50 = 32.9 μM against HCT-116) and 7o (IC50 = 42.6 μM against

HCT-116) suggesting that the activity of the compounds with unsaturated group was weaker than the
compounds with saturated group on R1 position. Among them, compound 7f showed the best
anticancer activities with IC50 value of about 0.5 μΜ against three cancer cell lines and the activity
was better than the positive control Dactolisib.
To develop more core structures, we synthesized another series of compounds 7p to 7s which
substituted at position-3 of benzoxazine scaffold. However, the activities of this series were no
significant improvement as good compared to the first series derivatives. However, very similar to
7a-o, the introduction of hydrophilic groups at position 3 could also improve their biological activity
which could be verified by 7p vs. 7r and 7q vs. 7s.
We also test the cytotoxic effects of 7f on normal human cells. Compound 7f showed much less
inhibitory activity against MRC5 (IC50 = 32.8 μM against MRC5 and IC50 = 15.6 μM against
HUVEC) (Table 2). These indicated that our compounds could inhibit the growth of cancer cell in
effective concentration in vitro and no effect on normal cells.
Table 1 Cytotoxicity in vitro of target compounds against three cancer cell lines (IC50 Valuesa in μM).
R1
R2
R3
HCT-116
2.40±0.12
MDA-MB-231
1.82±0.06
SNU638
Compd
7a
2.38±0.11
1.24±0.04
3.18±0.22
2.22±0.09
4.23±0.32
4.98±0.26
3.26±0.13
5.49±0.15
12.4±0.58
7.84±0.32
5.77±0.16
6.96±0.12
6.76±0.31
7b
7c
7d
7e

0.58±0.03
0.49±0.02
0.52±0.04
7f
14.6±0.62
41.4±3.13
13.8±0.71
37.9±2.71
17.6±0.89
46.5±1.42
7g
7h
33.6±1.12
8.43±0.82
37.9±2.06
9.35±0.74
36.2±3.46
8.97±0.66
7i
7j
4.62±0.42
15.8±0.76
4.23±0.36
19.5±0.92
5.01±0.60
20.4±1.14
7k
7l
32.9±2.30
23.5±1.72
42.6±1.42
40.4±3.16
43.8±2.86
47.9±3.18
32.7±2.42
41.9±1.96
44.3±2.54
7m
7n
7o
6.18±0.32
3.26±0.22
14.5±0.62
8.26±0.60
4.78±0.16
`17.2±0.72
7.39±0.54
4.97±0.22
21.4±0.68
7p
7q
7r
18.6±0.60
0.84±0.12
22.8±0.48
0.56±0.09
24.9±0.94
1.24±0.13
7s
Dactolisib
aIC50 values are the mean of triplicate measurements.
Table 2 Cytotoxicity of 7f to normal human cells (IC50 Values in μM)
Cells MRC5
32.8
HUVEC
15.6
7f
2.3 PI3K enzymatic activity assay

To elucidate the mechanism of antiproliferative activities of these active compounds, we
selected the most potent compound 7f according to the antiproliferative results for the further PI3K
enzymatic activity assays. Dactolisib was selected as the positive drug. As shown in Table 3, the
inhibitory activity of 7f was 26.7 nM for PI3Kα. The activity against PI3Kα is seven-fold higher than
the other class I PI3Ks (PI3Kβ, PI3Kγ and PI3Kδ). These results suggest that compound 7f was a
selective PI3Kα inhibitor.
Table 3 Activities of 7f and Dactolisib against Class I PI3K (IC50 Values in nM)
PI3Kα
26.7
PI3Kβ
382.3
35.9
PI3Kγ
184.7
23.6
PI3Kδ
425.2
78.4
7f
16.4
Dactolisib
2.4 Western blot assay
Activation of the PI3K pathway leads to phosphorylation of Akt and because of compound 7f
suppressed PI3Kα activity, therefore, we evaluated the effects of 7f on the related protein levels of
Akt by western blot analysis. As shown in Fig.3, compound 7f could decrease the phospho-Akt
(T308) in a dose-dependent manner, indicating that 7f might act as a potential PI3K inhibitor.
Fig.3. Western blot analysis of 7f. (A) The inhibition effects of compound 7f (1 μM, 2 μM and 4 μM) on the
expression of p-Akt, Akt in HCT-116 cells are depicted. β-actin was used as internal control. (B) Bar graphs
showed the quantitative results of clonogenicity. Data are presented as the mean for three independent experiments.
** P < 0.01, *** P < 0.001 compared with control.
2.5 Molecular docking
Molecular docking study was performed to elucidate the binding model of 7f in the binding site
of PI3K. As showed in Fig 4 (A), the oxygen atom of morpholine ring of 7f forms hydrogen bond
interaction with Val882 in the hinge binder region of PI3K. Besides, the oxygen of sulfonamide

group interaction with Lys833 and nitrogen of pyridyl group formed a hydrogen bond with the
conserved water molecule. In addition, the morpholinyl formed an additional hydrogen bond with
Tyr887. As showed in Fig 4 (B), the folded form of compound 7f in the protein pocket was very
similar to that of Omipalisib. The formation of hydrogen bonds suggested that 7f can exactly interact
with the catalytic domain of PI3K and also indicated that 7f might be a potent PI3K inhibitor.
A
B
Fig.4. Docking mode of 7f with protein crystal structure of PI3K. (A) Key interactions of compound 5f in the
active site of PI3K (PDB: 3L08). (B) The binding pose of 7f and Omipalisib in the active site of PI3K. Omipalisib
was highlighted with yellow.
3. Conclusion
In summary, two series of 2H-benzo[b][1,4]oxazin derivatives containing sulfonamide
substituted pyridyl group were designed and synthesized based on Omipalisib, PI3K inhibitors which
containing morpholine group and Dactolisib. Their antiproliferative activities against three cancer
cell lines, two normal human cell lines were evaluated in vitro which indicating that 7f selectively
inhibits the proliferation of cancer cells compared to normal cells. The structure activity relationships
(SARs) of the title compounds was discussed. The PI3K inhibitory activities and Western blot assay
proved that 7f could be considered as a PI3Kα inhibitor. According to these results, it can be
concluded that 2H-benzo[b][1,4]oxazin core via adding a hydrophilic group was a feasible way to
get PI3K inhibitor with high activities.
4. Experimental section
4.1 Chemistry and chemical methods
1
All reagents and solvents were commercially available without further purification. H NMR
spectra and 13C NMR spectra were recorded on 400 and 600 Bruker NMR spectrometer with

tetramethylsilane (TMS) as an internal standard. All chemical shifts are reported in ppm (δ) and
coupling constants (J) are in hertz (Hz). High-resolution exact mass measurements were performed
using electrospray ionization (positive mode) on a quadrupole time-of-flight (QTOF) mass
spectrometer (microTOF-Q, Bruker Inc).
4.1.1 6-bromo-4-(2-morpholinoethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2a)
To a 25 mL flask was added 6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one (0.46 g, 2 mmol),
4-(2-chloroethyl)morpholine (0.30 g, 2 mmol), potassium carbonate (0.42 g, 3 mmol) and DMF (8
mL), the reaction mixture was heated at 60 ºC and stirred for 6 h. After the reaction was completed,
the DMF was removed at reduced pressure and the residue was purified on silica gel with
chloroform/methanol (V:V 100:1) to give the title compound as a white powder. Yield 94.1%, m.p.
80-82 ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.51 (d, J = 2.2 Hz, 1H, Ar-H), 7.17 (dd, J = 8.5, 2.2 Hz,
1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H, Ar-H), 4.65 (s, 2H, Ar-H), 4.02 (t, J = 6.4 Hz, 2H, CH2), 3.53 (s,
4H, CH2×2), 2.47 (t, J = 6.4 Hz, 2H, CH2), 2.43 (s, 4H, CH2×2). ESI-MS: m/z 341.0 [M+H]+.
Compounds 2b-j were synthesized according to the procedure described in 2a.
4.1.2 4-(3-(1H-pyrazol-1-yl)propyl)-6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one (2b)
Yield 80.2%, m.p. 99-102 ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.75 (d, J = 2.1 Hz, 1H, Ar-H),
7.50-7.47 (m, 1H, Ar-H), 7.25 (d, J = 2.2 Hz, 1H, Ar-H), 7.16 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 6.96 (d,
J = 8.5 Hz, 1H, Ar-H), 6.26 (t, J = 2.0 Hz, 1H, Ar-H), 4.65 (s, 2H, CH2), 4.20 (t, J = 6.7 Hz, 2H,
CH2), 3.87-3.83 (m, 2H, CH2), 2.07-2.03 (m, 2H, CH2). ESI-MS: m/z 336.0 [M+H]+.
4.1.3 4-(3-(1H-imidazol-1-yl)propyl)-6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one (2c)
Yield 75.6%, m.p. 116-120 ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.71 (s, 1H, Ar-H), 7.29 (d, J
= 2.2 Hz, 1H, Ar-H), 7.24 (s, 1H, Ar-H), 7.18 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H,
Ar-H), 6.94 (s, 1H, Ar-H), 4.66 (s, 2H, CH2), 4.06 (t, J = 7.0 Hz, 2H, CH2), 3.89-3.85 (m, 2H, CH2),
2.02-1.96 (m, 2H, CH2). ESI-MS: m/z 336.0 [M+H]+.
4.1.4 6-bromo-4-(4-fluorobenzyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2d)
1
Yield 95.1%, m.p. 118-120 ºC. H NMR (600 MHz, DMSO-d6) δ 7.36-7.31 (m, 2H, Ar-H×2),
7.21 (d, J = 2.2 Hz, 1H, Ar-H), 7.18 (t, J = 8.9 Hz, 2H, Ar-H×2), 7.15 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H),
6.98 (d, J = 8.5 Hz, 1H, Ar-H), 5.17 (s, 2H, CH2), 4.82 (s, 2H, CH2). ESI-MS: m/z 336.0 [M+H]+.
4.1.5 6-bromo-4-(pyridin-4-ylmethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2e)

1
Yield 93.3%, m.p. 120-124 ºC. H NMR (600 MHz, DMSO-d6) δ 8.53 (d, J = 6.0 Hz, 2H,
Ar-H×2), 7.28 (d, J = 6.0 Hz, 2H, Ar-H×2), 7.17 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.11 (d, J = 2.2 Hz,
1H, Ar-H), 7.01 (d, J = 8.5 Hz, 1H, Ar-H), 5.21 (s, 2H, CH2), 4.86 (s, 2H, CH2). ESI-MS: m/z 319.0
[M+H]+.
4.1.6 6-bromo-4-(3-morpholino-3-oxopropyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2f)
Yield 81.5%, m.p. 118-120ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.42 (d, J = 2.2 Hz, 1H, Ar-H),
7.17 (dd, J = 8.5, 2.2 Hz, 1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H, Ar-H), 4.64 (s, 2H, CH2), 4.10-4.07 (m,
2H, CH2), 3.54 (q, J = 5.0, 4.6 Hz, 4H, CH2×2), 3.40 (dt, J = 22.1, 4.9 Hz, 4H, CH2×2), 2.65-2.62 (m,
2H, CH2). ESI-MS: m/z 369.0 [M+H]+.
4.1.7 6-bromo-4-(3-morpholinopropyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2g)
Yield 78.4%, m.p. 136-140 ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.47 (d, J = 2.1 Hz, 1H, Ar-H),
7.17 (dd, J = 8.5, 2.1 Hz, 1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H, Ar-H), 4.65 (s, 2H, CH2), 3.92 (t, J =
7.0 Hz, 2H, CH2), 3.60 (s, 4H, CH2×2), 2.31 (s, 6H, CH2×3), 1.74-1.67 (m, 2H, CH2). ESI-MS: m/z
355.1 [M+H]+.
4.1.8 6-bromo-4-(3-(diethylamino)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2h)
1
Yield 76.6%, m.p. 76-78 ºC. H NMR (600 MHz, DMSO-d6) δ 7.44 (d, J = 2.1 Hz, 1H, Ar-H),
7.17 (dd, J = 8.5, 2.1 Hz, 1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H, Ar-H), 4.65 (s, 2H, CH2), 3.94-3.90 (m,
2H, CH2), 2.56 (dq, J = 13.7, 6.9 Hz, 6H, CH2×3), 1.68 (p, J = 7.1 Hz, 2H, CH2), 0.99 (t, J = 7.1 Hz,
6H, CH2×3). ESI-MS: m/z 341.1 [M+H]+.
4.1.9 6-bromo-4-(3-(2-oxopyridin-1(2H)-yl)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2i)
Yield 80.2%, m.p. 132-135 ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.72 (dd, J = 6.7, 1.8 Hz, 1H,
Ar-H), 7.41 (ddd, J = 8.9, 6.6, 2.0 Hz, 1H, Ar-H), 7.30 (d, J = 2.0 Hz, 1H, Ar-H), 7.17 (dd, J = 8.5,
2.1 Hz, 1H, Ar-H), 6.97 (d, J = 8.5 Hz, 1H, Ar-H), 6.37 (d, J = 9.1 Hz, 1H, Ar-H), 6.23 (t, J = 6.6 Hz,
1H, Ar-H), 4.65 (s, 2H, CH2), 3.93 (dt, J = 16.2, 7.3 Hz, 4H, CH2×2), 1.91 (q, J = 7.3 Hz, 2H, CH2).
ESI-MS: m/z 363.0 [M+H]+.
4.1.10 6-bromo-4-(3-(dimethylamino)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (2j)
Yield 80.2%, m.p. 172-178ºC. 1H NMR (600 MHz, DMSO-d6) δ 7.44 (d, J = 1.7 Hz, 1H, Ar-H),
7.20 (dd, J = 8.5, 1.8 Hz, 1H, Ar- H), 6.99 (d, J = 8.5 Hz, 1H, Ar-H), 4.69 (s, 2H, CH2), 3.96 (t, J =
7.1 Hz, 2H, CH2), 3.13 (s, 2H, CH2), 2.77 (s, 6H, CH2×3), 1.95-1.90 (m, 2H, CH2). ESI-MS: m/z
313.0 [M+H]+.

4.1.11 6-bromo-3-(piperazin-1-yl)-2H-benzo[b][1,4]oxazine (3)
To a 25 mL flask was added 6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one (0.46 g, 2 mmol),
P2S5 (0.89 g, 4 mmol) and THF (8 mL), the reaction mixture was heated at 90 ºC and stirred for 8 h.
After the reaction was completed, the THF was removed at reduced pressure and pour into water,
filtered dry and directly reacted with piperazine (0.34g, 4 mmol) at 90 ºC in THF without
purification. Then the THF was removed at reduced pressure and the residue was purified on silica
gel with chloroform/methanol (V:V 10:1) to give the title compound as a white powder. Yield 56.2%,
oil. 1H NMR (400 MHz, DMSO-d6) δ 6.99 (d, J = 2.4 Hz, 1H, Ar-H), 6.92 (dd, J = 8.4, 2.4 Hz, 1H,
Ar-H), 6.74 (d, J = 8.4 Hz, 1H, Ar-H), 4.76 (s, 2H, Ar-H), 3.50 – 3.39 (m, 4H, CH2×2), 3.19 (m, 4H,
CH2×2). ESI-MS: m/z 296.0 [M+H]+.
4.1.12 2-(4-(6-bromo-2H-benzo[b][1,4]oxazin-3-yl)piperazin-1-yl)ethanol (4a)
To a solution of 4-nitro-1H-pyrazole (0.29 g, 1 mmol) in DMF (10 mL) was added K2CO3 (0.14 g, 1
mmol) and 2-Bromoethanol (0.15 g, 1.2 mmol). Then the mixture was stirred at 60 ºC for 12 h. DMF
was removed at reduced pressure and add water (100 mL), extracted with ethyl acetate (3 × 100 mL),
dried with Na2SO4 and evaporated to give and the residue which was purified on silica gel with
chloroform/methanol (V:V 10:1) to give the title compound as a white powder. Yield 82.6%, oil. 1H
NMR (400 MHz, DMSO-d6) δ 7.01 (d, J = 2.4 Hz, 1H, Ar-H), 6.93 (dd, J = 8.4, 2.4 Hz, 1H, Ar-H),
6.75 (d, J = 8.4 Hz, 1H, Ar-H), 4.77 (s, 2H, CH2), 4.45 (s, 1H, OH), 3.56 – 3.50 (m, 4H, CH2×2),
3.38 (d, J = 15.4 Hz, 2H, CH2), 2.47 – 2.44 (m, 4H, CH2×2), 2.42 (t, J = 6.2 Hz, 2H, CH2). ESI-MS:
m/z 340.1 [M+H]+.
Compounds 4b-d was synthesized according to the procedure described in 4a.
4.1.13 3-(4-(6-bromo-2H-benzo[b][1,4]oxazin-3-yl)piperazin-1-yl)propan-1-ol (4b)
Yield 81.6%, oil. 1H NMR (400 MHz, DMSO-d6) δ 7.01 (d, J = 2.4 Hz, 1H, Ar-H), 6.93 (dd, J
= 8.4, 2.4 Hz, 1H, Ar-H), 6.75 (d, J = 8.4 Hz, 1H, Ar-H), 4.77 (s, 2H, CH2), 3.52 (s, 2H, CH2), 3.45
(t, J = 6.3 Hz, 4H, CH2×2), 2.42 – 2.38 (m, 4H, CH2×2), 2.36 (d, J = 7.4 Hz, 2H, CH2), 1.62 – 1.57
(m, 2H, CH2). ESI-MS: m/z 354.1 [M-H]+.
4.1.14 ethyl 2-(4-(6-bromo-2H-benzo[b][1,4]oxazin-3-yl)piperazin-1-yl)acetate (4c)
Yield 78.3%, m.p. 133-105 ºC. 1H NMR (400 MHz, DMSO-d6) δ 7.01 (d, J = 2.3 Hz, 1H, Ar-H),
6.94 (dd, J = 8.4, 2.4 Hz, 1H, Ar-H), 6.75 (d, J = 8.4 Hz, 1H, Ar-H), 4.78 (s, 2H, CH2), 4.11 – 4.06

(m, 2H, CH2), 3.53 (s, 4H, CH2×2), 3.28 (s, 2H, CH2), 2.60 – 2.55 (m, 4H, CH2×2), 1.19 (d, J = 7.2
Hz, 3H, CH3). ESI-MS: m/z 382.1 [M-H]+.
4.1.15 methyl 3-(4-(6-bromo-2H-benzo[b][1,4]oxazin-3-yl)piperazin-1-yl)propanoate (4d)
Yield 71.7%, m.p. oil. 1H NMR (400 MHz, DMSO-d6) δ 7.02 (d, J = 2.4 Hz, 1H, Ar-H), 6.93
(dd, J = 8.4, 2.4 Hz, 1H, Ar-H), 6.74 (d, J = 8.4 Hz, 1H, Ar-H), 4.77 (s, 2H, CH2), 3.61 (s, 4H,
CH2×2), 3.51 (s, 3H, CH3), 2.61 (t, J = 6.9 Hz, 2H, CH2), 2.51 (d, J = 6.6 Hz, 2H, CH2), 2.44 – 2.40
(m, 4H, CH2×2). ESI-MS: m/z 382.1 [M-H]+.
4.1.16 N-(5-bromo-2-methoxypyridin-3-yl)butane-1-sulfonamide (6a)
To a solution of 5-bromo-2-methoxypyridin-3-amine (2.01 g, 10 mmol) in pyridine (50 mL) at 0
ºC was added methanesulfonyl chloride (1.72 g, 11 mmol). Then the mixture was stirred at 25 ºC for
24 h. Pyridine was removed at reduced pressure and add water (100 mL), extracted with ethyl acetate
(3×100 mL), the organic layer was washed with water (50 mL), dried with Na2SO4 and evaporated to
1
give compound 6a as a white solid. Yield 78.4%, m.p. 150-151 ºC. H NMR (400 MHz, DMSO-d6)
9.51 (s, 1H, NH), 8.09 (d, J = 2.4 Hz, 1H, Ar-H), 7.77 (d, J = 2.4 Hz, 1H, Ar-H), 3.91 (s, 3H, OCH3),
3.16 (t, J = 7.6 Hz, 2H, CH2), 1.73-1.65 (m, 2H, CH2), 1.42-1.33 (m, 2H, CH2), 0.87 (t, J = 7.2 Hz,
3H, CH3). ESI-MS: m/z 384.1 [M+H]+.
Compounds 6b was synthesized according to the procedure described in 6a.
4.1.17 N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (6b)
Yield 85.6%, m.p. 163-165 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H, NH), 8.13 (d, J =
2.2 Hz, 1H, Ar-H), 7.83-7.74 (m, 2H, Ar-H×2), 7.62-7.52 (m, 1H, Ar-H), 7.24 (td, J = 2.0, 8.5 Hz,
1H, Ar-H), 3.62 (s, 3H, OCH3). ESI-MS: m/z 376.9 [M-H]+.
4.1.18 2,4-difluoro-N-(2-methoxy-5-(4-(2-morpholinoethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]ox-
azin-6-yl)pyridin-3-yl)benzenesulfonamide (7a)
A solution of the N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (0.19 g,
0.5 mmol), bis(pinacolato)diboron (0.13 g, 0.5 mmol), potassium acetate (0.15 g, 1.5 mmol),
Pd(dppf)2Cl2 (18 mg, 0.025 mmol) and anhydrous DMF (30 mL) was reflux under an atmosphere of
N2 atmosphere for 4 h. To the resulted residue was added Pd(dppf)2Cl2 (0.018 g, 0.025 mmol),
6-bromo-4-(2-morpholinoethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (0.14 g, 0.4 mmol), Cs2CO3
(0.33 g, 0.56 mmol) and water (0.5 mL). The obtained mixture was refluxed under an atmosphere of
N2 at 90 ºC for another 4 h. DMF was removed at reduced pressure and the residue was purified

through a column chromatography on silica gel with chloroform/methanol (V:V 50:1) as a white
solid. Yield 50.5%, m.p. 185-188 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.29 (s, 1H, NH), 8.32 (d, J
= 2.3 Hz, 1H, Ar-H), 7.89 (d, J = 2.3 Hz, 1H, Ar-H), 7.76 (dd, J = 8.3, 6.5 Hz, 1H, Ar-H), 7.61-7.55
(m, 1H, Ar-H), 7.44 (d, J = 1.7 Hz, 1H, Ar-H), 7.26-7.23 (m, 1H, Ar-H), 7.23-7.19 (m, 1H, Ar-H),
7.10 (d, J = 7.5 Hz, 1H, Ar-H), 4.68 (s, 2H, CH2), 4.15 (t, J = 6.7 Hz, 2H, CH2), 3.64 (s, 3H, OCH3),
3.53 (t, J = 4.2 Hz, 4H, CH2×2), 2.53 (t, J = 6.8 Hz, 2H, CH2), 2.47 (s, 4H, CH2×2). 13C NMR (150
MHz, DMSO-d6) δ 164.40 (dd, J = 253.9, 11.8 Hz), 163.33, 158.77 (dd, J = 257.6, 13.5 Hz), 156.63,
144.16, 141.43, 133.43, 131.16 (d, J = 10.7 Hz), 130.45, 128.65, 128.47, 124.65 (dd, J =15, 4.5 Hz),
120.97, 119.00, 116.64, 113.02, 111.18 (dd, J = 22.2, 3.2 Hz), 105.17 (t, J = 26.1 Hz), 66.49, 65.55,
54.43, 52.83, 52.70, 37.22. HRMS m/z calc for C26H26F2N4O6S [M+Na]+: 583.1541; found:
583.1433.
Compounds 7b-s were synthesized according to the procedure described in 7a.
4.1.19 2,4-difluoro-N-(5-(4-(4-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-me-
thoxypyridin-3-yl)benzenesulfonamide (7b)
Yield 53.2%, m.p. 177-179 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.27 (s, 1H, NH), 8.17 (d, J =
2.3 Hz, 1H, Ar-H), 7.79 (d, J = 2.3 Hz, 1H, Ar-H), 7.71 (td, J = 8.5, 6.2 Hz, 1H, Ar-H), 7.56 (ddd, J =
10.4, 9.1, 2.5 Hz, 1H, Ar-H), 7.41 (dd, J = 8.6, 5.5 Hz, 2H, Ar-H×2), 7.30 (d, J = 1.9 Hz, 1H, Ar-H),
7.22 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.20 (dd, J = 8.5, 2.4 Hz, 1H, Ar-H), 7.19-7.15 (m, 2H, Ar-H×2),
13
7.11 (s, 1H, Ar-H), 5.29 (s, 2H, CH2), 4.84 (s, 2H, CH2), 3.62 (s, 3H, OCH3). C NMR (150 MHz,
DMSO-d6) δ 164.5 (dd, J = 252.0, 10.5 Hz), 161.51 (d, J = 3.0 Hz), 159.90, 158.76 (dd, J = 257.7,
13.4 Hz), 156.59, 144.17, 141.27, 133.39, 132.07 (d, J = 2.9 Hz), 131.15 (d, J = 10.8 Hz), 130.15,
129.41 (t, J = 4.5 Hz), 129.11, 124.46 (dd, J = 13.5, 3.0 Hz), 121.09, 118.85, 116.65, 114.97, 114.83,
113.23, 111.17 (dd, J = 22.2, 3.1 Hz), 105.17 (t, J = 26.0 Hz), 66.50, 52.73, 41.91. HRMS m/z calc
for C27H20F3N3O5S [M+H]+: 556.1076; found: 556.1149.
4.1.20 2,4-difluoro-N-(2-methoxy-5-(4-(3-morpholino-3-oxopropyl)-3-oxo-3,4-dihydro-2H-benzo[b]
[1,4]oxazin-6-yl)pyridin-3-yl)benzenesulfonamide (7c)
Yield 52.4%, m.p. 154-156 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.28 (s, 1H, NH), 8.35 (d, J =
2.3 Hz, 1H, Ar-H), 7.89 (d, J = 2.3 Hz, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.65-7.55 (m, 1H, Ar-H), 7.43
(d, J = 2.0 Hz, 1H, Ar-H), 7.24 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.23-7.18 (m, 1H, Ar-H), 7.10 (d, J =
8.2 Hz, 1H, Ar-H), 4.67 (s, 2H, CH2), 4.27-4.21 (m, 2H, CH2), 3.63 (s, 3H, OCH3), 3.51 (dt, J = 7.9,

5.0 Hz, 4H, CH2×2), 3.45-3.41 (m, 2H, CH2), 3.41-3.37 (m, 2H, CH2), 2.72-2.67 (m, 2H, CH2). 13C
NMR (150 MHz, DMSO-d6) δ 168.27, 164.44 (dd, J = 253.9, 11.8 Hz), 163.32, 158.77(dd, J = 257.6,
13.4 Hz), 156.62, 144.03, 141.74, 133.64, 131.20 (d, J = 10.7 Hz), 130.60, 128.68, 128.33, 124.51
(dd, J = 14.4, 3.5 Hz), 121.07, 118.75, 116.65, 112.80, 111.19 (dd, J = 22.2, 3.2 Hz), 105.18 (t, J =
26.1 Hz), 66.45, 65.34, 52.69, 44.68, 40.78, 29.39. HRMS m/z calc for C27H26F2N4O7S [M+H]+:
589.1490; found: 589.1563.
4.1.21 2,4-difluoro-N-(2-methoxy-5-(3-oxo-4-(pyridin-4-ylmethyl)-3,4-dihydro-2H-benzo[b][1,4]ox
azin-6-yl)pyridin-3-yl)benzenesulfonamide (7d)
Yield 50.4%, m.p. 102-105 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.28 (s, 1H, NH), 8.53 (d, J =
5.0 Hz, 2H, Ar-H×2), 8.14 (d, J = 2.3 Hz, 1H, Ar-H), 7.78 (d, J = 3.1 Hz, 1H, Ar-H), 7.73-7.67 (m,
1H, Ar-H), 7.60-7.53 (m, 1H, Ar-H), 7.34 (d, J = 6.3 Hz, 2H, Ar-H×2), 7.27-7.22 (m, 1H, Ar-H),
7.22-7.16 (m, 2H, Ar-H×2), 7.14 (d, J = 8.3 Hz, 1H, Ar-H), 5.34 (s, 2H, CH2), 4.88 (s, 2H, CH2),
13
3.61 (s, 3H, OCH3). C NMR (150 MHz, DMSO-d6) δ 164.44 (dd, J = 253.9, 11.8 Hz), 163.89,
158.75 (dd, J = 257.7, 13.5 Hz), 156.61, 149.27, 145.07, 144.09, 141.31, 133.36, 131.14 (d, J = 10.8
Hz), 130.27, 128.37, 128.19, 124.47 (dd, J = 14.4, 3.5 Hz), 121.27, 121.22, 118.85, 116.75, 112.97,
111.16 (dd, J = 22.1, 3.2 Hz), 105.16 (t, J = 26.1 Hz), 66.48, 52.71, 42.05. HRMS m/z calc for
C26H20F2N4O5S [M+H]+: 539.1122; found: 539.1195.
4.1.22 2,4-difluoro-N-(2-methoxy-5-(3-oxo-4-(3-(2-oxopyridin-1(2H)-yl)propyl)-3,4-dihydro-2H-ben
zo[b][1,4]oxazin-6-yl)pyridin-3-yl)benzenesulfonamide (7e)
1
Yield 53.1%, m.p. 83-85 ºC. H NMR (600 MHz, DMSO-d6) δ 10.30 (s, 1H, NH), 8.31 (d, J =
2.3 Hz, 1H, Ar-H), 7.86 (d, J = 2.3 Hz, 1H, Ar-H), 7.73 (dd, J = 6.7, 2.0 Hz, 2H, Ar-H×2), 7.57 (s,
1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.20 (s, 2H, Ar-H×2), 7.10 (d, J = 8.3 Hz, 1H,
Ar-H), 6.37 (d, J = 9.1 Hz, 1H, Ar-H), 6.22-6.19 (m, 1H, Ar-H), 4.68 (s, 2H, CH2), 4.07-3.96 (m, 4H,
13
CH2×2), 3.64 (s, 3H, OCH3), 1.99 (s, 2H, CH2). C NMR (150 MHz, DMSO-d6) δ 164.42 (dd, J =
254.0, 11.8 Hz), 163.31, 160.82, 158.78 (dd, J = 257.5, 13.5 Hz), 156.70, 144.09, 141.74, 139.30,
138.42, 133.76, 131.19 (d, J = 10.8 Hz), 130.56, 128.62, 128.12, 124.56 (dd, J = 14.4, 3.5 Hz),
121.10, 119.01, 118.76, 116.74, 112.37, 111.17 (dd, J = 22.1, 3.1 Hz), 105.17 (t, J = 25.5 Hz), 104.72,
72.90, 66.37, 52.70, 45.84, 24.36. HRMS m/z calc for C28H24F2N4O6S [M+H]+: 583.1385; found:
583.1457.
4.1.23 2,4-difluoro-N-(2-methoxy-5-(4-(3-morpholinopropyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]o

xazin-6-yl)pyridin-3-yl)benzenesulfonamide (7f)
Yield 54.6%, m.p. 151-153 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.28 (s, 1H, NH), 8.29 (d, J =
2.3 Hz, 1H, Ar-H), 7.85 (d, J = 2.3 Hz, 1H, Ar-H), 7.76 (dd, J = 8.4, 6.4 Hz, 1H, Ar-H), 7.63-7.50 (m,
1H, Ar-H), 7.39 (d, J = 1.3 Hz, 1H, Ar-H), 7.24-7.21 (m, 1H, Ar-H), 7.21-7.18 (m, 1H, Ar-H), 7.10 (d,
J = 8.2 Hz, 1H, Ar-H), 4.67 (s, 2H, CH2), 4.05 (t, J = 7.1 Hz, 2H, CH2), 3.64 (s, 3H, OCH3), 3.49 (s,
4H, CH2×2), 2.36 (d, J = 6.8 Hz, 2H, CH2), 2.33 (d, J = 11.5 Hz, 4H, CH2×2), 1.79-1.73 (m, 2H,
13
CH2). C NMR (150 MHz, DMSO-d6) δ 164.36 (dd, J = 253.7, 11.7 Hz), 163.22, 158.76 (dd, J =
257.5, 13.4 Hz), 156.58, 144.09, 141.21, 133.12, 131.17 (d, J = 10.7 Hz), 130.62, 128.74, 128.46,
124.72 (dd, J = 14.5, 3.3 Hz), 120.99, 119.33, 116.63, 112.81, 111.13 (dd, J = 22.1, 3.2 Hz), 105.15 (t,
J = 26.1 Hz), 66.46, 65.39, 54.67, 52.68, 52.62, 37.93, 22.93. HRMS m/z calc for C27H28F2N4O6S
[M+H]+: 575.1698; found: 575.1770.
4.1.24 N-(5-(4-(3-(diethylamino)propyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-methox
ypyridin-3-yl)butane-1-sulfonamide (7g)
Yield 49.5%, m.p. 68-70 ºC. 1H NMR (600 MHz, DMSO-d6) δ 8.33 (d, J = 2.3 Hz, 1H, Ar-H),
7.87 (d, J = 2.3 Hz, 1H, Ar-H), 7.44 (d, J = 2.0 Hz, 1H, Ar-H), 7.26 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H),
7.12 (d, J = 8.3 Hz, 1H, Ar-H), 4.70 (s, 2H, CH2), 4.08 (t, J = 7.3 Hz, 2H, CH2), 3.96 (s, 3H, OCH3),
3.15-3.08 (m, 2H, CH2), 3.02-2.89 (m, 4H, CH2×2), 2.85 (s, 2H, CH2), 1.88 (d, J = 10.3 Hz, 2H,
CH2), 1.77-1.69 (m, 2H, CH2), 1.39 (h, J = 7.4 Hz, 2H, CH2), 1.09 (t, J = 6.8 Hz, 6H, CH3×2), 0.88 (t,
13
J = 7.4 Hz, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 163.46, 155.54, 144.06, 140.21, 131.06,
130.99, 128.74, 128.12, 121.26, 120.44, 116.70, 112.77, 66.43, 53.11, 51.43, 48.03, 45.61, 37.33,
25.73, 24.58, 20.48, 20.21, 12.93. HRMS m/z calc for C25H36N4O5S [M+H]+: 505.2406; found:
505.2479.
4.1.25 N-(2-methoxy-5-(3-oxo-4-(pyridin-4-ylmethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyri
din-3-yl)butane-1-sulfonamide (7h)
Yield 48.5%, m.p. 65-68 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H, NH), 8.64 (d, J = 6.1
Hz, 2H, Ar-H×2), 8.14 (d, J = 2.3 Hz, 1H, Ar-H), 7.72 (d, J = 2.3 Hz, 1H, Ar-H), 7.55 (d, J = 5.4 Hz,
2H, Ar-H×2), 7.26 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.17-7.13 (m, 2H, Ar-H×2), 5.41 (s, 2H, CH2),
4.89 (s, 2H, CH2), 3.92 (s, 3H, OCH3), 3.62 (t, J = 6.6 Hz, 2H, CH2), 1.72-1.66 (m, 2H, CH2), 1.37 (q,
13
J = 7.4 Hz, 2H, CH2), 0.86 (t, J = 7.4 Hz, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 164.00,
155.36, 144.01, 139.71, 130.73, 130.32, 128.39, 128.11, 122.15, 121.44, 120.53, 117.24, 116.75,

115.26, 113.00, 66.49, 58.94, 53.08, 45.01, 20.18, 12.92, 7.94. HRMS m/z calc for C24H26N4O5S
[M+H]+: 483.1624; found: 483.1679.
4.1.26 N-(5-(4-(3-(1H-pyrazol-1-yl)propyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-meth
oxypyridin-3-yl)butane-1-sulfonamide (7i)
Yield 51.1%, m.p. 63-65 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.40 (s, 1H, NH), 8.29 (d, J = 2.3
Hz, 1H, Ar-H), 7.83 (d, J = 2.2 Hz, 1H, Ar-H), 7.76 (d, J = 1.9 Hz, 1H, Ar-H), 7.45 (d, J = 1.3 Hz,
1H, Ar-H), 7.26 (d, J = 1.7 Hz, 1H, Ar-H), 7.24 (d, J = 1.9 Hz, 1H, Ar-H), 7.10 (d, J = 8.2 Hz, 1H,
Ar-H), 6.23 (t, J = 2.0 Hz, 1H, Ar-H), 4.68 (s, 2H, CH2), 4.23 (t, J = 6.6 Hz, 2H, CH2), 3.97 (s, 3H,
OCH3), 3.97-3.93 (m, 2H, CH2), 3.16-3.09 (m, 2H, CH2), 2.13 (p, J = 6.8 Hz, 2H, CH2), 1.73 (p, J =
7.7 Hz, 2H, CH2), 1.39 (q, J = 7.5 Hz, 2H, CH2), 0.88 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (150 MHz,
DMSO-d6) δ 164.40, 156.70, 145.04, 141.34, 139.26, 132.22, 132.00, 130.56, 129.73, 129.22,
122.20, 121.45, 117.74, 113.51, 105.46, 67.46, 54.18, 52.55, 49.01, 38.45, 27.68, 25.66, 21.28, 14.00.
HRMS m/z calc for C24H29N5O5S [M+H]+: 500.1889; found: 500.1902.
4.1.27 N-(5-(4-(3-(dimethylamino)propyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-metho
xypyridin-3-yl)butane-1-sulfonamide (7j)
1
Yield 50.5%, m.p. 79-85 ºC. H NMR (600 MHz, DMSO-d6) δ 8.34 (d, J = 2.1 Hz, 1H, Ar-H),
7.87 (s, 1H, Ar-H), 7.44 (s, 1H, Ar-H), 7.25 (s, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 4.69 (s, 2H, CH2), 4.07
(s, 2H, CH2), 3.96 (s, 3H, OCH3), 3.14-3.10 (m, 2H, CH2), 2.89 (s, 6H, CH3×2), 1.94 (s, 2H, CH2),
1.91 (s, 2H, CH2), 1.72 (p, J = 7.7 Hz, 2H, CH2), 1.40 (t, J = 7.4 Hz, 2H, CH2), 0.88 (t, J = 7.4 Hz,
3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 163.41, 155.51, 143.98, 140.17, 130.96, 128.71,
128.16, 125.78, 121.20, 120.43, 116.67, 112.73, 66.39, 54.22, 53.09, 51.42, 42.51, 37.36, 25.73,
24.58, 20.20, 12.93. HRMS m/z calc for C23H32N4O5S [M+H]+: 477.2093; found: 477.2166.
4.1.28 N-(2-methoxy-5-(4-(3-morpholinopropyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)py
ridin-3-yl)butane-1-sulfonamide (7k)
Yield 50.6%, m.p. 54-58 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.38 (s, 1H, NH), 8.29 (d, J = 2.3
Hz, 1H, Ar-H), 7.84 (d, J = 2.3 Hz, 1H, Ar-H), 7.40 (d, J = 1.9 Hz, 1H, Ar-H), 7.23 (dd, J = 8.3, 1.9
Hz, 1H, Ar-H), 7.10 (d, J = 8.3 Hz, 1H, Ar-H), 4.67 (s, 2H, CH2), 4.04 (t, J = 7.2 Hz, 2H, CH2), 3.96
(s, 3H, OCH3), 3.50-3.46 (m, 4H, CH2×2), 3.13-3.10 (m, 2H, CH2), 2.33 (d, J = 6.8 Hz, 2H, CH2),
2.32 (s, 4H, CH2×2), 1.76 (d, J = 7.2 Hz, 2H, CH2), 1.72 (s, 2H, CH2), 1.40-1.36 (m, 2H, CH2), 0.87
(t, J = 7.4 Hz, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 163.20, 155.40, 144.04, 140.02, 130.91,

130.72, 127.79, 126.47, 121.07, 120.48, 116.59, 112.90, 66.45, 65.44, 54.70, 53.09, 52.66, 51.37,
37.98, 24.56, 22.96, 20.19, 12.91. HRMS m/z calc for C25H34N4O6S [M+H]+: 519.2199; found:
519.2272.
4.1.29 N-(2-methoxy-5-(4-(2-morpholinoethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)pyri
din-3-yl)butane-1-sulfonamide (7l)
Yield 50.6%, m.p. 115-118 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.40 (s, 1H, NH), 8.38 (d, J =
2.3 Hz, 1H, Ar-H), 7.90 (d, J = 2.3 Hz, 1H, Ar-H), 7.44 (d, J = 1.8 Hz, 1H, Ar-H), 7.29 (d, J = 1.9 Hz,
1H, Ar-H), 7.14 (d, J = 8.3 Hz, 1H, Ar-H), 4.73 (s, 2H, CH2), 4.44 (t, J = 6.7 Hz, 2H, CH2), 3.96 (s,
3H, OCH3), 3.67 (s, 4H, CH2×2), 3.43 (t, J = 6.7 Hz, 2H, CH2), 3.22-3.10 (m, 4H, CH2×2), 1.77-1.70
13
(m, 2H, CH2), 1.41-1.37 (m, 2H, CH2), 1.27-1.16 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3). C
NMR (150 MHz, DMSO-d6) δ 165.25, 162.76, 156.67, 145.00, 141.54, 132.31, 132.10, 129.72,
128.57, 121.44, 117.90, 113.68, 67.40, 63.80, 54.15, 52.88, 52.53, 51.84, 46.09, 25.65, 21.29, 14.00.
HRMS m/z calc for C24H32N4O6S [M+Na]+: 527.2043; found: 527.1935.
4.1.30 N-(5-(4-(3-(1H-imidazol-1-yl)propyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-2-met
hoxypyridin-3-yl)butane-1-sulfonamide (7m)
Yield 49.2%, m.p. 69-71 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.43 (s, 1H, NH), 8.30 (d, J = 2.2
Hz, 1H, Ar-H), 7.83 (d, J = 2.2 Hz, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.27 (d, J = 8.3 Hz, 2H, Ar-H×2),
7.24 (dd, J = 8.1, 2.0 Hz, 1H, Ar-H), 7.11 (d, J = 8.3 Hz, 1H, Ar-H), 6.93 (s, 1H, Ar-H), 4.68 (s, 2H,
CH2), 4.09 (t, J = 7.0 Hz, 2H, CH2), 3.96 (s, 3H, OCH3), 3.37 (q, J = 7.2 Hz, 2H, CH2), 3.16-3.10 (m,
2H, CH2), 2.06 (p, J = 7.1 Hz, 2H, CH2), 1.73 (p, J = 7.8 Hz, 2H, CH2), 1.39 (q, J = 7.5 Hz, 2H,
13
CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 164.48, 156.58, 145.09,
141.22, 137.69, 132.03, 131.98, 129.74, 129.17, 128.55, 122.27, 121.52, 119.80, 117.77, 113.61,
67.47, 54.17, 52.51, 52.45, 44.27, 38.21, 25.66, 21.28, 14.00. HRMS m/z calc for C24H29N5O5S
[M+Na]+: 522.1889; found: 522.1815.
4.1.31 N-(2-methoxy-5-(4-(3-morpholino-3-oxopropyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-
6-yl)pyridin-3-yl)butane-1-sulfonamide (7n)
Yield 51.1%, m.p. 56-59 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.36 (s, 1H, NH), 8.33 (d, J = 2.3
Hz, 1H, Ar-H), 7.86 (d, J = 2.3 Hz, 1H, Ar-H), 7.43 (d, J = 1.8 Hz, 1H, Ar-H), 7.25 (d, J = 1.9 Hz,
1H, Ar-H), 7.11 (d, J = 8.3 Hz, 1H, Ar-H), 4.67 (s, 2H, CH2), 4.22 (t, J = 7.5 Hz, 2H, CH2), 3.95 (s,
3H, OCH3), 3.50 (t, J = 8.4 Hz, 4H, CH2×2), 3.44-3.40 (m, 2H, CH2), 3.40-3.36 (m, 2H, CH2),

3.15-3.10 (m, 2H, CH2), 2.69 (t, J = 7.5 Hz, 2H, CH2), 1.72 (d, J = 7.6 Hz, 2H), 1.44-1.34 (m, 2H),
13
0.88 (t, J = 7.4 Hz, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 169.31, 164.39, 156.56, 145.04,
141.26, 132.06, 131.97, 129.84, 129.35, 122.23, 121.50, 117.69, 113.95, 67.53, 66.41, 54.16, 52.51,
45.75, 41.84, 30.44, 25.66, 21.28, 14.00. HRMS m/z calc for C25H32N4O7S [M+Na]+: 555.1992;
found: 555.1884.
4.1.32 N-(2-methoxy-5-(3-oxo-4-(3-(2-oxopyridin-1(2H)-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]ox
azin-6-yl)pyridin-3-yl)butane-1-sulfonamide (7o)
Yield 50.6%, m.p. 80-83 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.39 (s, 1H, NH), 8.30 (d, J = 2.3
Hz, 1H, Ar-H), 7.84 (d, J = 2.3 Hz, 1H, Ar-H), 7.72 (dd, J = 6.9, 2.0 Hz, 1H,Ar-H), 7.42-7.36 (m, 1H,
Ar-H), 7.30 (d, J = 1.9 Hz, 1H, Ar-H), 7.24 (dd, J = 8.3, 2.0 Hz, 1H, Ar-H), 7.11 (d, J = 8.3 Hz, 1H,
Ar-H), 6.36 (ddd, J = 9.1, 1.4, 0.6 Hz, 1H, Ar-H), 6.20 (td, J = 6.7, 1.4 Hz, 1H, Ar-H), 4.68 (s, 2H,
CH2), 4.03 (t, J = 7.5 Hz, 2H, CH2), 3.98 (s, 2H, CH2), 3.97 (s, 3H, OCH3), 3.16-3.09 (m, 2H, CH2),
2.00 (d, J = 7.3 Hz, 2H, CH2), 1.72 (tt, J = 8.7, 6.9 Hz, 2H, CH2), 1.42-1.35 (m, 2H, CH2), 0.87 (t, J
13
= 7.4 Hz, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 164.39, 161.88, 156.60, 145.10, 141.22,
140.38, 139.49, 132.03, 129.78, 129.15, 122.24, 121.49, 120.07, 117.77, 113.54, 105.79, 67.45,
54.17, 52.52, 46.91, 38.19, 26.89, 25.66, 21.28, 14.00. HRMS m/z calc for C26H30N4O6S [M+H]+:
527.1886; found: 527.1959.
4.1.33
2,4-difluoro-N-(5-(3-(4-(2-hydroxyethyl)piperazin-1-yl)-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxypyr
idin-3-yl)benzenesulfonamide (7p)
Yield 54.3%, oil. 1H NMR (600 MHz, DMSO-d6) δ 8.22 (d, J = 2.3 Hz, 1H, Ar-H), 7.76 (d, J =
6.5 Hz, 1H, Ar-H), 7.75 (d, J = 6.0 Hz, 1H, Ar-H), 7.58 – 7.53 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.2
Hz, 1H), 7.09 (d, J = 2.2 Hz, 1H, Ar-H), 7.05 (dd, J = 8.2, 2.2 Hz, 1H, Ar-H), 6.89 (d, J = 8.1 Hz, 1H,
Ar-H), 4.80 (s, 2H, CH2), 4.03 (t, J = 7.1 Hz, 1H, OH), 3.65 (s, 3H, CH3), 3.56 (t, J = 6.0 Hz, 4H,
CH2×2), 2.55 – 2.53 (m, 4H, CH2×2), 2.49 (t, J = 6.1 Hz, 2H, CH2), 2.28 (s, 2H, CH2). 13C NMR
(150 MHz, DMSO-d6) δ 165.43 (dd, J = 253.7, 11.9 Hz), 162.77, 159.79 (dd, J = 257.5, 13.3 Hz),
157.21, 155.75, 144.73, 141.54, 136.21, 133.46, 132.27 (d, J = 10.5 Hz), 130.74, 130.24, 125.68 (dd,
J = 15.0, 3.0 Hz), 121.53, 121.03, 120.30, 115.67, 112.24 (d, J = 20.1 Hz), 106.21 (t, J = 26.0 Hz),
60.65, 60.44, 58.70, 53.72 (2C), 53.16 (2C). HRMS m/z calc for C26H28F2N5O5S [M+H]+: 560.1774;
found: 560.1748.

4.1.33
2,4-difluoro-N-(5-(3-(4-(3-hydroxypropyl)piperazin-1-yl)-2H-benzo[b][1,4]oxazin-6-yl)-2-methoxyp
yridin-3-yl)benzenesulfonamide (7q)
Yield 56.4%, oil. 1H NMR (600 MHz, DMSO-d6) δ 8.22 (d, J = 2.3 Hz, 1H, Ar-H), 7.76 (d, J =
10.9 Hz, 1H, Ar-H), 7.73 (d, J = 10.1 Hz, 1H, Ar-H), 7.58 – 7.54 (m, 1H, Ar-H), 7.20 (td, J = 8.5, 2.2
Hz, 1H, Ar-H), 7.09 (d, J = 2.2 Hz, 1H, Ar-H), 7.04 (dd, J = 8.2, 2.3 Hz, 1H, Ar-H), 6.88 (d, J = 8.1
Hz, 1H, Ar-H), 4.80 (s, 2H, CH2), 4.31 (t, J = 4.8 Hz, 4H, CH2×2), 3.64 (s, 3H, CH3), 3.56 (s, 2H,
CH2), 3.39 (d, J = 6.5 Hz, 1H, OH), 2.42 (brs, 2H, CH2), 2.47 – 2.35 (m, 4H, CH2×2), 1.64 – 1.61 (m,
2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.47(dd, J = 252.3, 12.0 Hz), 163.51, 159.19(dd, J =
242.0, 11.9 Hz), 157.23, 155.74, 144.75, 141.71, 136.20, 133.62, 132.28(d, J = 10.5 Hz), 130.71,
130.24, 125.59 (d, J = 15.0 Hz), 121.54, 121.06, 120.10, 115.68, 112.27 (d, J = 24.2 Hz), 106.24 (t, J
= 25.5 Hz), 67.65, 60.66, 59.58, 55.34, 53.74, 52.84 (2C), 43.85(2C). HRMS m/z calc for
C27H30F2N5O5S [M+H]+: 574.1930; found: 574.1907.
4.1.33 ethyl
2-(4-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-2H-benzo[b][1,4]oxazin-3-yl)pi
perazin-1-yl)acetate (7r)
Yield 48.2%, oil. 1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1H, NH), 8.24 (d, J = 2.3 Hz, 1H,
Ar-H), 7.77 (d, J = 5.8 Hz, 1H, Ar-H), 7.76 (d, J = 6.6 Hz, 1H, Ar-H), 7.61 – 7.54 (m, 1H, Ar-H),
7.21 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.10 (d, J = 2.2 Hz, 1H, Ar-H), 7.07 – 7.04 (m, 1H, Ar-H), 6.89
(d, J = 8.1 Hz, 1H, Ar-H), 4.10 (q, J = 7.1 Hz, 2H, CH2), 3.65 (s, 3H, CH3), 3.56 (s, 4H, CH2×2),
3.35 (s, 2H, CH2), 3.30 (s, 2H, CH2),2.62 – 2.58 (m, 4H, CH2×2), 1.20 (t, J = 7.1 Hz, 3H, CH3). 13C
NMR (150 MHz, DMSO-d6) δ 170.27, 165.50 (dd, J = 254.2, 11.7 Hz), 159.80 (dd, J = 257.8, 13.3
Hz), 157.24, 155.75, 144.75, 141.93, 136.17, 133.81, 132.30 (d, J = 10.5 Hz), 130.67, 130.26, 125.48
(dd, J = 14.0, 2.7 Hz), 121.53, 121.07, 119.83, 115.68, 112.28 (d, J = 21.8 Hz), 106.24 (t, J = 26.2
Hz), 60.66, 60.35, 58.61, 53.74 (2C), 52.00, 44.02(2C), 14.58. HRMS m/z calc for C28H30 F2N5O6S
[M+H]+: 602.1807; found: 602.1854.
4.1.33 methyl
3-(4-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-2H-benzo[b][1,4]oxazin-3-yl)pi
perazin-1-yl)propanoate (7s)

Yield 48.2%, oil. 1H NMR (600 MHz, DMSO-d6) δ 10.22 (s, 1H, NH), 8.23 (d, J = 2.3 Hz, 1H,
Ar-H), 7.77 (d, J = 8.5 Hz, 1H, Ar-H), 7.76 – 7.73 (m, 1H, Ar-H), 7.57 (t, J = 8.6 Hz, 1H, Ar-H),
7.21 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.09 (d, J = 2.2 Hz, 1H, Ar-H), 7.05 (dd, J = 8.2, 2.3 Hz, 1H,
Ar-H), 6.88 (d, J = 8.1 Hz, 1H, Ar-H), 4.80 (s, 2H, CH2), 3.94 (s, 2H, CH2), 3.65 (s, 3H, CH3), 3.61
(s, 3H, CH3), 3.53 (s, 4H, CH2×2), 2.63 (t, J = 7.1 Hz, 2H, CH2), 2.47 – 2.44 (m, 4H, CH2×2). 13C
NMR (150 MHz, DMSO-d6) δ 172.77, 165.48 (dd, J = 254.0, 11.9 Hz), 159.80 (dd, J = 257.5, 13.4
Hz), 157.23, 155.71, 144.74, 141.82, 136.22, 133.72, 132.29 (d, J = 11.1 Hz), 130.68, 130.26, 125.54
(dd, J = 15.0, 3.0 Hz), 121.54, 121.03, 119.96, 115.66, 112.28 (d, J = 22.1 Hz), 106.23 (t, J = 26.2
Hz), 60.65 (2C), 53.73, 53.48, 52.58, 51.77 (2C), 43.92 (2C), 31.88. HRMS m/z calc for C28H30
F2N5O6S [M+H]+: 602.1807; found: 602.1819.
4.2 Biological assay methods
4.2.1 Cell culture
Cancer cells were grown in DMEM (SNU-638) or RPMI 1640 (HCT-116 and MDA-MB-231
cells) supplemented with 10% FBS and antibiotics-antimycotics (PSF; 100 units/mL penicillin G
sodium, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B) in a humidified incubator
containing 5% CO2 at 37 °C.
4.2.2 Antiproliferative activity
The cell viability was evaluated using the sulforhodamine B (SRB) cellular protein-staining
method with minor modifications. Briefly, cells were treated with various concentrations of
compounds in 96-well plates and incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 72
h. After treatment, the cells were fixed with 10% TCA solution, and cell viability was determined
with a sulforhodamine B (SRB) assay. The percentage of cell-growth inhibition was calculated using
the formulae below. The IC50 values were calculated using a non-linear regression analysis (percent
growth versus concentration).
4.2.3 PI3K enzymatic activity assay
The PI3K kinase assay was determined by PI3K-GloTM Class I Profiling Kit (Promega, #V1690)
measuring the amount of ADP produced during the kinase reaction. Transfer 1 μL of the test
compound serial dilutions to the 384 well low volume assay plate. Dilute Lipid Kinase Enzyme
((PI3K (p110α/p85α), PI3K (p110β/p85α), PI3K (p120γ), PI3K (p110δ/p85α)) into prepared PI3K
Reaction Buffer/Lipid Substrate (combining equal volumes of 2.5X PI3K Reaction buffer with 2.5X

Lipid Substrate working solution).Transfer 8 μL of prepared working kinase solution to the wells
containing 1 μL of test compound. Incubate at room temperature for 15 minutes to allow inhibitor
binding to kinase. Start reaction by adding 1 μL of 250 μM ATP. After incubation at room
temperature for 1 h, 10 μL of ADP-GloTM reagent was added per well. The plates were incubated at
room temperature for 40 minutes and then 20uL of kinase detection reagent was added. After
incubation at room temperature for 40 minutes, the luminescence was recorded using Bio-Tek
SYENRGY2. The IC50 values were calculated using nonlinear regression with normalized
dose-response fitting using Prism software (GraphPad Software, San Diego, CA, USA).
4.2.4 Western blot assay
Total cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The protein concentration was determined, and
equal amounts of protein samples were subjected to 10% SDSPAGE. Separated proteins were
transferred to PVDF membranes (Millipore, Bedford, MA, USA) and probed with the indicated
antibodies. Exposures were obtained using ImageQuant LAS 4000 biomolecular imager (GE
Healthcare).
4.2.5 Molecular docking studies
The crystal structure of PI3K (PDB entry code: 3L08) in complex with Omipalisib was used for
molecular modeling. The molecular docking procedure was performed by using C-DOCKER
protocol within Accelrys Discovery Studio Visualizer 4.0. For enzyme preparation, the hydrogen
atoms were added. The whole PI3K enzyme was defined as a receptor and the site sphere was
selected on the basis of the ligand binding location of Omipalisib. Compound Omipalisib was
removed and compound 7f was placed. Accelrys Discovery Studio Visualizer 4.0 was used for
graphic display.
Acknowledgments
Financial supported by the project for national basic science personnel training fund (Grant No.
J1103606) is gratefully acknowledged.
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Highlights
 Two series of novel 2H-benzo[b][1,4]oxazin derivatives were synthesized and
characterized.
 Their antiproliferative activities against cancer cell lines were evaluated.
 7f exhibited PI3Kα inhibitory activity with an IC50 value of 26.7 nM.
 7f exhibited potent antiproliferative effects via PI3K/Akt signaling pathway
inhibition.