6,7-Dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine derivatives as selective inhibitors of PI3Kα

Article abstract of DOI:10.1016/j.bmc.2015.01.052

Twenty eight 6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine derivatives were synthesized and evaluated their biological activities as PI3K inhibitors. Biological evaluation against four human tumor cell lines revealed that most target compounds

Full text of DOI:10.1016/j.bmc.2015.01.052

Bioorganic & Medicinal Chemistry 23 (2015) 1231–1240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
6,7-Dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine
derivatives as selective inhibitors of PI3K
a
Yong Yin a, Yan-Qing Zhang a, Biao Jin a, Shao Sha a, Xun Wu a, Chetan B. Sangani a, She-Feng Wang a,
Fang Qiao a, Ai-Min Lu b, Peng-Cheng Lv a, , Hai-Liang Zhu a,
⇑
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b College of Science, Nanjing Agriculture University, Nanjing 210095, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty eight 6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine derivatives were synthesized
and evaluated their biological activities as PI3K inhibitors. Biological evaluation against four human
tumor cell lines revealed that most target compounds showed impressively better antiproliferative activ-
ities than that of LY294002. Among these compounds, compound 25 exhibited the most potent and selec-
Received 8 December 2014
Revised 24 January 2015
Accepted 28 January 2015
Available online 7 February 2015
tive activity for PI3Ka, with the IC50 value of 0.016 lM, an approximately 30-fold increase in comparison
with LY294002, it also has an increased potency of approximately 11-fold for PI3Kb. It indicated the
potential of developing 6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine derivatives as the
new PI3Ka selective inhibitors for tumor treatment.
Keywords:
6,7-Dihydrobenzo[f]benzo[4,5]imidazo[1,2-
d][1,4]oxazepine derivatives
Ó 2015 Elsevier Ltd. All rights reserved.
PI3K
a
Antitumor
1. Introduction
ing BEZ-235, GSK-2126458 and PF-04691502, which are dual
inhibitors of PI3K/mTOR.8,10
Phosphoinositide 3-kinases (PI3Ks) are key components of the
PI3K/Akt/mTOR pathway which play essential roles in various cellu-
lar activities, they are lipid kinases that responsible for the conver-
sion of membrane-bound 4,5-phosphatidylinositolbisphosphate
(PIP2) into 3,4,5-phosphatidylinositoltrisphosphate (PIP3).1,2
Phosphorylation to PIP3 in turn promotes downstream activation
of Akt and mTOR, which influence several functions important to
cellular integrity including proliferation, evasion of apoptosis,
metabolism and angiogenesis.3,4 Not surprisingly, perturbations of
this crucial pathway are highly associated with cancer.5 There are
Recently, a set of novel compounds with potent activity against
PI3Ka were reported such as GEN-614, ZSTK474, GDC0032 and so
on (Fig. 1).5,14–17 As can be seen in their structures, there is a ben-
zoxepin scaffold as main core. Meanwhile, benzimidazole scaffold
also plays a critical role in pharmacological activity. Based on the
benzoxepin core and benzimidazole scaffold which are favored
lead structures for antitumor agents,5,15 a series of novel 6,7-dihy-
drobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine
derivatives
was designed and synthesized, their bioactivities against the four
human tumor cells and PI3Ks were evaluated in detail. Docking
simulations were also performed using the X-ray crystallographic
structure of PI3Ka (PDB code: 3R7R, retrieved from the RCSB Pro-
tein Data Bank) in complex with an inhibitor to explore the binding
four class I PI3K isoforms (
cascade, the amplification or activating mutation of PI3K
most common in many cancers.6,7 As a result, small molecule inhib-
itors that target the PI3Ks, especially PI3K , have been intensely
a, b, d, and
c) implicated in the signaling
a
is the
a
modes of these compounds at the active site.
pursued as potential treatments for various types of cancers.8–11
Wortmannin, a natural product, as the first PI3K inhibitor was
discovered.12 In the years which followed LY294002 as the first
generation of designed small molecule PI3K inhibitors was found
to be the most potent.13 Since then, many kinds of PI3K inhibitors
were developed, and some of these have recently advanced into
the clinic such as BKM-120, CAL101, GDC-0941 and so on, includ-
2. Results and discussion
2.1. Chemistry
The synthesis of compounds (4–31) followed the general path-
way outlined in Scheme 1. Compounds (4–31) were prepared in
two steps. Firstly, 2-(1H-benzo[d]imidazol-2-yl)phenol (2) was pre-
pared from salicylaldehyde and o-phenylenediamine, using sodium
metabisulfite as catalyst for 6–8 h, the yield is up to 92%. Secondly,
2-(1H-benzo[d]imidazol-2-yl)phenol (2) and 1,2-dibromoethane
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
http://dx.doi.org/10.1016/j.bmc.2015.01.052
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

1232
Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
O
O
O
N
N
Me
O
N
H
HO
S
S
N
O
Me
N
Me
H3C
N
Me
N
N
Cl
GEN-614
N
H3C
S1
O
N
H2N
H3C
O
N
N
CHF2
CH3
N
N
N
N
N
N
H3C
H3C
O
N
N
N
N
N
CH3
O
GDC-0032
ZSTK474
O
R1
N
N
R2
Figure 1. Various PI3K inhibitors and the design solution of novel 6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine derivatives.
were reacted to get the desired compounds (4–31). All of the syn-
thesised compounds gave satisfactory analytical and spectroscopic
data in accordance with their depicted structures.
2.2.2. Druglikeness analysis
Druglike properties were analyzed and the data were showed in
Table 2. These properties include octanol–water partitioning coef-
ficient (AlogP) based on Ghose and Crippen’s method,18 molecular
weight (MW), the number of hydrogen bond donors (HBD), the
number of hydrogen bond acceptors (HBA), and molecular frac-
tional polar surface area (MFPSA). All the properties were calcu-
lated using the Discovery Studio molecular simulation package.19
As summarized in Table 2, all the desired compounds had AlogP
value less than 5, showed favorable values for the involved molec-
ular parameters that fit well with the Lipinski’s Rule of Five.20
2.2. Bioassay
2.2.1. Antiproliferative activity
To test the antiproliferative activity of the synthesized com-
pounds, we evaluated the anti-proliferative activity of compounds
4–31 against four cell lines: HCT-116, A549, Huh7 and HL60. The
results are summarized in Table 1. The results revealed that some
of the synthetic compounds displayed potent antiproliferative
effects against the four cell lines, and they were superior to the
positive control LY294002. Out of the given compounds, it was
observed that compound 25 showed the most potent antiprolifer-
2.2.3. PI3K enzymatic activity
Compounds 9, 13, 14, 15, 18, 19, 21, 23, 25 and 29 were selected
to evaluate their PI3Ka, PI3Kb, PI3Kc and PI3Kd enzymatic activity
ative activity (IC50 = 0.08, 0.11, 0.12, 0.10
A549, Huh7 and HL60, respectively).
lM against HCT-116,
using a competitive fluorescence polarization kinase activity
assay.21 The results are presented in Table 3.
As shown in Table 1, a significant improvement in antiprolifer-
ative against four tested cell lines comparing with that of
LY294002 was observed for some target compounds. In order to
gain the SARs (structure–activity relationships) of the desired com-
pounds intuitionally, the substituent on the salicylaldehyde (R1)
was setted as abscissa. As can be seen in Figure 2, it was found that
the substituents on the salicylaldehyde play an important role in
anti-proliferative activity, and the order of influence is CH3 >
Br > Cl > H. Meanwhile, the different substituents (R2) at the 4-
position of the benzimidazole ring led to different degrees of vari-
ation in anti-proliferative activity, and the order of potency is
F > Cl > Br > NO2 > CF3 > CH3 > H.
As shown in Table 3, all the tested compounds showed inac-
tive for PI3Kc and PI3Kd, but exhibited selectivity for PI3Ka,
and the tendency of inhibition was roughly similar to their inhi-
bition for cancer cell. The most potent compound 25 exhibited
PI3K
for PI3Kb. Meanwhile, two compounds (19 and 25) exhibited
greatly increased inhibition against PI3K in comparison with
LY294002. In particular, compound 25 (PI3K , IC50: 0.016 M)
has an increased potency of approximately 30-fold for
LY294002 (PI3K , IC50: 0.481 M). It was concluded that com-
a inhibitory activity of 0.016 lM, which is almost 11-fold
a
a
l
a
l
pound 25 has a great opportunity as the new PI3K
a selective
inhibitor for cancer therapy.

Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
1233
OH
H
O
OH
H2N
H2N
a
b
N
N
R1
R1
N
N
R1
CHO
R2
R2
R2
compounds
R1
H
R2
H
H
H
H
compounds
R1
Cl
R2
Cl
4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
5
CH3
Cl
Br
H
Cl
6
Br
7
Br
CH3
Cl
Br
8
H
CH3
CH3
CH3
CH3
F
Br
9
CH3
Cl
Br
H
Br
10
11
12
13
14
15
16
17
NO2
NO2
NO2
NO2
CF3
CF3
CF3
CF3
Br
CH3
Cl
H
CH3
Cl
F
Br
H
F
Br
F
CH3
Cl
H
Cl
CH3
Cl
Br
Scheme 1. Synthesis of compounds 4–31. Reagents and conditions: (a) Sodium metabisulfite, DMF, 110 °C, 6–8 h. (b) 1,2-Dibromoethane, DMF, Cs2CO3, 100 °C, 10–12 h.
Table 1
Antiproliferative activities in vitro of target compounds 4–31 against four cancer cell lines
Compounds
IC50 SDa
(lM)
HCT-116
A549
Huh7
HL60
4
5
6
7
8
9
101.42 9.82
112.32 10.11
87.65 8.43
98.51 8.26
73.48 6.91
0.59 0.04
58.17 4.97
80.11 7.56
76.55 7.33
2.46 0.28
1.83 0.19
0.74 0.05
71.55 6.81
46.44 4.38
0.52 0.04
0.12 0.01
43.86 3.94
7.38 0.71
64.38 7.02
0.47 0.05
91.00 8.89
0.08 0.006
28.44 3.02
19.00 2.14
13.81 1.41
4.45 0.44
34.72 2.78
60.61 5.68
51.82 4.76
133.48 14.18
147.55 14.47
108.31 9.21
128.09 13.03
83.67 8.53
0.61 0.06
61.58 5.98
92.81 9.04
89.41 7.99
2.85 0.31
1.71 0.21
0.70 0.04
77.33 6.45
55.65 4.38
0.56 0.04
0.17 0.02
49.52 4.42
7.88 0.73
74.08 7.64
0.48 0.03
121.64 11.73
0.11 0.01
28.32 2.14
22.03 2.21
11.47 1.12
4.95 0.47
38.29 3.22
68.47 6.48
82.32 7.61
124.06 12.37
144.31 11.67
92.55 9.33
132.48 12.68
82.64 8.53
0.67 0.05
63.41 6.21
97.42 9.46
91.88 8.39
3.86 0.40
1.35 0.12
0.74 0.06
80.32 7.66
64.52 6.08
0.45 0.04
0.18 0.01
58.45 5.43
4.91 4.12
72.07 7.07
0.51 0.04
104.53 9.88
0.12 0.007
20.12 1.89
24.34 2.23
18.66 2.07
6.47 0.61
42.34 4.04
69.65 7.16
67.18 6.54
107.64 9.73
121.54 10.06
91.31 6.48
102.66 7.49
75.15 7.09
0.58 0.04
57.43 3.28
88.64 9.44
82.55 7.83
4.56 0.47
1.89 0.13
0.78 0.05
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
LY294002
73.82 6.77
51.63 4.76
0.53 0.03
0.15 0.01
44.34 3.84
7.68 0.78
66.67 6.39
0.47 0.04
94.52 6.79
0.10 0.006
32.85 2.93
17.48 1.68
10.11 0.93
4.86 0.44
37.99 3.22
66.42 6.08
18.43 1.43
a
The averaged values determined by three separate determinations.

1234
Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
Table 2
Molecular parameters of 6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4]oxazepine
derivatives
Compound
Molecular parameters
MW
AlogPa
HBAb
HBDc
MFPSAa
4
5
6
7
8
9
236.27
250.30
270.71
315.17
250.30
264.32
284.74
329.19
254.26
268.29
288.70
333.16
270.71
284.74
305.16
349.61
315.17
329.19
349.61
394.06
281.27
295.29
315.71
360.16
304.27
318.29
338.71
383.16
3.28
3.76
3.94
4.03
3.76
4.25
4.43
4.51
3.48
3.97
4.15
4.23
3.94
4.43
4.61
4.69
4.03
4.51
4.69
4.78
3.17
3.66
3.84
3.92
4.22
4.71
4.89
4.97
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.13
0.12
0.11
0.11
0.12
0.11
0.11
0.10
0.12
0.11
0.11
0.11
0.11
0.11
0.10
0.10
0.11
0.10
0.10
0.10
0.29
0.27
0.26
0.25
0.11
0.10
0.10
0.09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
a
b
c
Calculated by Discovery Studio 3.5.
Counted as the sum of Ns and Os.
Counted as the sum of NHs and OHs.
Figure 2. Analysis of relationships between activity and R1, R2. R1 was setted as
abscissa, different colors represent different R2.
pound 25 and amidogen of Asp964 (bondlength: O@NÁ Á ÁH = 1.51 Å),
The second charge interaction between the oxygen atom of nitrogen
in compound 25 and amidogen of Lys833 (bond length: N–
OÁ Á ÁH = 2.07 Å, bond angle: N@OÁ Á ÁH = 135.48°). The 3D model of
the interaction between compound 25 and 3R7R was showed in Fig-
ure 5, it was found that the active pockets of 3R7R were occupied by
compound 25. The enzyme surface model was showed in Figure 5B,
which revealed that compound 25 was well inserted into the active
pocket of 3R7R. Overall, the binding model suggested that com-
2.2.4. Analysis of the apoptosis induced in cells
To evaluate the biological ability of the compounds, we per-
formed an apoptosis experiment of compound 25 in the HCT-116
cell line using Annexin-V and propidium iodide (PI) double stain-
ing by flow cytometry and treating the HCT-116 cells with 0.5, 1
and 2 lM of compound 25 for 24 h. The results can be seen in Fig-
ure 3, and showed compound 25 is very effective in the induction
of apoptosis in a dose-dependent manner. Moreover, the percent-
age of early apoptotic cells from 15.1% to 27.3% was markedly ele-
vated in a dose-dependent manner, as compared to 1.93% of
apoptotic cells in the untreated control.
pound 25 is a potential inhibitor of PI3Ka.
3. Conclusion
In summary, a series of novel 6,7-dihydrobenzo[f] benzo[4,5]imi-
dazo[1,2-d][1,4]oxazepine derivatives were synthesized and evalu-
ated their biological activities as PI3Ka inhibitors. In vitro biological
2.3. Molecular docking
evaluation against four human tumor cell lines revealed that most
target compounds showed impressively better antiproliferative
activities than that of LY294002. Compound 25 exhibited the most
To help understanding the SARs observed at the PI3Ka and guide
further SAR studies, a docking analysis utilizing the C-DOCKER pro-
gram within Discovery Studio 3.5 software package was performed
potent activity, which showed inhibitory activity of 0.016
lM
using cocrystallography of the more amenable PI3K
c as a surrogate
against PI3K , an approximately 30-fold increase in comparison
a
since the co-crystal structure of PI3K with a small ligand inhibitor
a
has not been resolved yet.22–24 The amino acid residue which had
interaction with 3R7R was labeled. As can be seen in Figure 4, in
the binding mode, compound 25 was nicely bound to the 3R7R via
hydrophobic interaction and the binding was stabilized by two
hydrogen bond interactions and two charge interactions. One
hydrogen bond was formed between Lys833 and nitro of compound
25 (bond length: N@OÁ Á ÁH = 2.18 Å, bond angle: N@OÁ Á ÁH =
126.79°). Another one hydrogen bond was formed by the oxygen
atom of compound 25 and the amino hydrogen of Val882 (bond
length: OÁ Á ÁHN = 2.14 Å, bond angle: N–HÁ Á ÁO = 164.56°). The first
charge interaction between the nitrogen atom of nitrogen in com-
with LY294002, it also has an increased potency of approximately
11-fold for PI3Kb. This study validated the potential of developing
6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d][1,4] oxazepine series
as the new PI3Ka selective inhibitors for cancer treatment.
4. Experiments
4.1. Materials and measurements
All chemicals and reagents used in the current study were of
analytical grade. The reactions were monitored by thin layer chro-

Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
1235
Table 3
4.2. General procedure for the preparation of compound 3
Enzymatic activities of compounds 4–31 against PI3K
a
To a stirring solution of o-phenylenediamine (1 mmol) and
benzaldehyde (1 mmol) in DMF (20 mL), sodium metabisulfite
(0.2 mmol) was added. The mixture was stirred for 6–8 h at
110 °C. After the reaction was completed, the mixture was poured
into cold water under stirring. The resulting solid was filtered,
dried and recrystallized from ethanol.
Compounds
IC50
(
l
M)
IC50 (PI3Kb)/ Fold
IC50 (PI3K
a
)
improvementb
PI3K
a
PI3Kb PI3K
c
PI3Kd
9
13
14
15
18
19
21
23
0.118 0.394 17.087 >50
0.474 0.907 34.624 >50
0.283 0.488 21.668 42.618
0.181 0.473 27.916 >50
0.127 0.363 24.342 >50
0.031 0.188 11.812 >50
0.662 1.032 44.117 47.763
0.092 0.284 19.479 39.842
3.34
1.91
1.72
2.61
2.86
6.06
1.56
3.09
4.08
1.01
1.70
2.66
3.79
15.52
0.73
5.23
30.06
0.70
—
4.3. General procedure for synthesis of compounds 4–31
To a mixture of compound 3 (0.5 mmol) and cesium carbon-
ate (0.65 g, 2 mmol) in DMF (15 mL), 1,2-dibromoethane
(2 mmol) was added. The completion of the reaction was moni-
tored by TLC, and the reaction mixture was heated at 100 °C for
10–12 h. After completed, the mixture was poured into water
(200 mL) under vigorous stirring and the solid was formed and
collected by filtration. The crude products were purified by
recrystallization with anhydrous ethanol to afford the desired
compounds 4–31.
25
29
0.016 0.184
9.443 34.624 11.50
0.685 1.213 41.626 >50
1.77
—
LY294002
0.481 0.983
0.947 1.362
a
IC50 values are averaged values determined by at least two independent
experiments. Variation was generally 5%.
b
Value for LY294002 divided by the value obtained for tested compounds.
matography (TLC) on Merck pre-coated silica GF254 plates. Melt-
ing points (uncorrected) were determined on a XT4MP apparatus
(Taike Corp., Beijing, China). ESI mass spectra were obtained on a
Mariner System 5304 mass spectrometer, and 1H NMR spectra
were collected on a Bruker DPX400 spectrometer at room temper-
ature with TMS and solvent signals allotted as internal standards.
Chemical shifts are reported in ppm (d). Elemental analyses were
performed on a CHN-O-Rapid instrument, and were within 0.4%
of the theoretical values.
4.3.1. 6,7-Dihydrobenzo[f]benzo[4,5]imidazo[1,2-
d][1,4]oxazepine (4)
Yield 48%, mp 208–209 °C. 1H NMR (400 MHz, DMSO-d6): 8.67
(dd, J1 = 8.08 Hz, J2 = 1.68, 1H, ArH), 7.69 (m, 1H, ArH), 7.58 (m,
1H, ArH), 7.43–7.39 (m, 1H, ArH), 7.29–7.24 (m, 2H, ArH), 7.21–
7.17 (m, 1H, ArH), 7.11 (m, 1H, ArH), 4.60 (s, 4H, –CH2CH2–). 13C
NMR (100 MHz, CDCl3): 157.52, 152.04, 143.24, 136.32, 132.18,
Figure 3. Compound 25 induced apoptosis in HCT-116 cells with the concentration of 0. 5, 1 and 2
n = 3. P <0.05 versus the control. The percentage of cells in each part is indicated.
lM. HTC-116 cells were treated for 24 h. Values represent the mean S.D,

1236
Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
Figure 4. 2D molecular docking model of compound 25 with 3R7R. Blue dashed arrow and green dashed arrow represent the H-bond interactions with amino acids; Pink
dashed arrow represents the charge interactions.
132.06, 123.52, 123.41, 121.04 (2), 120.74, 111.71, 111.34, 70.34,
48.12. MS (ESI): 237.18 (C15H13N2O, [M+H]+). Anal. Calcd for
4.3.5. 11-Methyl-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (8)
C15H12N2O: C, 76.25; H, 5.12; N, 11.86. Found: C, 76.28; H, 5.13;
Yield 61%, mp 189–192 °C. 1H NMR (400 MHz, DMSO-d6): 8.64
(dd, J1 = 8.04 Hz, J2 = 1.60, 1H, ArH), 7.53 (m, 1H, ArH), 7.55 (m,
1H, ArH), 7.50–7.45 (m, 2H, ArH), 7.21 (m, 1H, ArH), 7.13 (m, 1H,
ArH), 4.66 (m, 4H, –CH2CH2–), 2.41 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3): 158.02, 152.68, 139.28, 134.32, 133.67, 132.31,
132.11, 126.78, 123.34, 123.28, 118.32, 117.12, 111.68, 70.44,
48.24, 23.28. MS (ESI): 251.27 (C16H15N2O, [M+H]+). Anal. Calcd
for C16H14N2O: C, 76.78; H, 5.64; N, 11.19. Found: C, 76.77; H,
5.66; N, 11.21.
N, 11.88.
4.3.2. 2-Methyl-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (5)
Yield 53%, mp 213–215 °C. 1H NMR (400 MHz, DMSO-d6): 8.71
(d, J = 7.92 Hz, 1H, ArH), 7.62 (m, 1H, ArH), 7.55 (m, 1H, ArH),
7.31 (m, 1H, ArH), 7.26 (m, 1H, ArH), 7.03 (m, 1H, ArH), 6.98 (m,
1H, ArH), 4.62 (m, 4H, –CH2CH2–), 2.44 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3): 154.28, 152.21, 143.45, 136.57, 134.47, 132.31,
132.02, 121.27 (2), 121.03, 119.23, 111.53, 111.24, 69.91, 48.31,
24.03. MS (ESI): 251.44 (C16H15N2O, [M+H]+). Anal. Calcd for
4.3.6. 2,11-Dimethyl-6,7-dihydrobenzo[f]benzo[4,5]imidazo
[1,2-d][1,4]oxazepine (9)
Yield 43%, mp 181–182 °C. 1H NMR (400 MHz, DMSO-d6): 8.68
(d, J = 7.92 Hz, 1H, ArH), 7.58 (m, 1H, ArH), 7.56 (m, 1H, ArH),
7.38 (m, H, ArH), 7.18 (m, 1H, ArH), 7.02 (m, 1H, ArH), 4.64 (m,
4H, –CH2CH2–), 2.41 (s, 3H, CH3), 2.44 (s, 3H, CH3). 13C NMR
(100 MHz, CDCl3): 154.61, 152.74, 139.46, 135.07, 134.58, 133.74,
132.46, 131.94, 126.45, 120.12, 118.62, 117.43, 112.02, 70.12,
48.03, 24.32, 23.62. MS (ESI): 265.31 (C17H17N2O, [M+H]+). Anal.
Calcd for C17H16N2O: C, 77.25; H, 6.10; N, 10.60. Found: C, 77.28;
H, 6.11; N, 10.58.
C16H14N2O: C, 76.78; H, 5.64; N, 11.19. Found: C, 76.80; H, 5.65;
N, 11.21.
4.3.3. 2-Chloro-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (6)
Yield 57%, mp 201–203 °C. 1H NMR (400 MHz, DMSO-d6): 8.76
(d, J = 7.96 Hz, 1H, ArH), 7.65 (m, 1H, ArH), 7.59 (m, 1H, ArH), 7.43
(m, 1H, ArH), 7.33 (m, 1H, ArH), 7.29 (m, 1H, ArH), 6.98 (m, 1H,
ArH), 4.61 (m, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3):
155.41, 152.34, 143.18, 136.26, 132.28, 128.96, 128.21, 121.47
(2), 121.18, 121.09, 114.36, 111.89, 70.56, 48.23. MS (ESI):
271.67 (C15H12ClN2O, [M+H]+). Anal. Calcd for C15H11ClN2O: C,
66.55; H, 4.10; N, 10.35. Found: C, 66.57; H, 4.09; N, 10.38.
4.3.7. 2-Chloro-11-methyl-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (10)
Yield 45%, mp 191–194 °C. 1H NMR (400 MHz, DMSO-d6): 8.76
(m, 1H, ArH), 7.59 (m, 1H, ArH), 7.54 (m, 1H, ArH), 7.42 (m, 1H,
ArH), 7.31 (m, H, ArH), 7.04 (m, 1H, ArH), 4.66 (m, 4H, –CH2CH2–
), 2.45 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): 156.07, 152.34,
139.38, 134.13, 133.37, 132.54, 129.32, 128.37, 126.62, 121.74,
4.3.4. 2-Bromo-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (7)
Yield 52%, mp 210–213 °C. 1H NMR (400 MHz, DMSO-d6): 8.42
(s, 1H, ArH), 7.65 (m, 1H, ArH), 7.61 (m, 1H, ArH), 7.47 (m, 1H,
ArH), 7.26 (m, 1H, ArH), 7.17 (m, 1H, ArH), 6.91 (m, 1H, ArH),
4.67 (s, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 156.48,
151.89, 142.74, 136.65, 134.62, 134.19, 122.13, 121.68 (2),
121.08, 117.08, 114.57, 111.68, 69.89, 48.11. MS (ESI): 316.22
(C15H12BrN2O, [M+H]+). Anal. Calcd for C15H11BrN2O: C, 57.16; H,
3.52; N, 8.89. Found: C, 57.14; H, 3.54; N, 8.91.
118.12, 117.63, 114.57, 70.08, 48.11, 23.74. MS (ESI): 285.67 (C16
H
N, 9.84. Found: C, 67.51; H, 4.61; N, 9.83.
-
14ClN2O, [M+H]+). Anal. Calcd for C16H13ClN2O: C, 67.49; H, 4.60;
4.3.8. 2-Bromo-11-methyl-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (11)
Yield 55%, mp 179–181 °C. 1H NMR (400 MHz, DMSO-d6): 8.22
(m, 1H, ArH), 7.55 (m, 1H, ArH), 7.51 (m, H, ArH), 7.49 (m, 1H,

Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
1237
Figure 5. (A) 3D interaction map between compound 25 and 3R7R. (B) 3D conformation position of the compound 25 in the binding pocket.
ArH), 7.39 (m, 1H, ArH), 6.94 (m, 1H, ArH), 4.62 (m, 4H, –CH2CH2–),
2.42 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): 156.51, 152.17,
139.42, 135.14, 134.41, 134.36, 133.28, 126.37, 122.32, 118.43,
4.3.10. 11-Fluoro-2-methyl-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (13)
Yield 49%, mp 238–240 °C. 1H NMR (400 MHz, DMSO-d6): 8.68
(m, 1H, ArH), 7.75 (m, 1H, ArH), 7.59 (m, 1H, ArH), 7.27 (m, 1H,
ArH), 7.12–7.04 (m, 2H, ArH), 4.64 (m, 4H, –CH2CH2–), 2.46 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3): 159.68, 154.89, 151.84,
140.64, 134.48, 134.28, 133.04, 132.27, 120.34, 117.42, 111.91,
108.21, 105.48, 71.24, 48.17, 24.71. MS (ESI): 269.24 (C16H14FN2O,
[M+H]+). Anal. Calcd for C16H13FN2O: C, 71.63; H, 4.88; F, 7.08; N,
10.44. Found: C, 71.66; H, 4.87; N, 10.45.
117.53, 117.34, 115.01, 70.01, 47.92, 24.02. MS (ESI): 330.28 (C16
H
-
14BrN2O, [M+H]+). Anal. Calcd for C16H13BrN2O: C, 58.38; H, 3.98;
N, 8.51. Found: C, 58.41; H, 3.97; N, 8.53.
4.3.9. 11-Fluoro-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (12)
Yield 56%, mp 221–224 °C. 1H NMR (400 MHz, DMSO-d6): 8.66
(dd, J1 = 8.04 Hz, J2 = 1.60, 1H, ArH), 7.78 (m, 1H, ArH), 7.61 (d,
J = 1.96 Hz, 1H, ArH), 7.39 (m, 1H, ArH), 7.23 (m, 1H, ArH),
7.13–7.05 (m, 2H, ArH), 4.61 (m, 4H, –CH2CH2–). 13C NMR
(100 MHz, CDCl3): 159.50, 158.24, 151.76, 140.83, 134.11, 132.46,
131.84, 123.56, 123.51, 112.04, 117.78, 108.44, 105.61, 71.39,
48.24. MS (ESI): 255.18 (C15H12FN2O, [M+H]+). Anal. Calcd for
4.3.11. 2-Chloro-11-fluoro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (14)
Yield 61%, mp 208–209 °C. 1H NMR (400 MHz, DMSO-d6): 8.77
(m, 1H, ArH), 7.72 (m, 1H, ArH), 7.60 (m, 1H, ArH), 7.46 (m, 1H,
ArH), 7.10–7.02 (m, 2H, ArH), 4.66 (m, 4H, –CH2CH2–). 13C NMR
(100 MHz, CDCl3): 159.74, 155.74, 151.58, 140.57, 134.32, 132.25,
129.34, 129.07, 121.26, 117.61, 114.13, 108.27, 105.73, 71.02,
C15H11FN2O: C, 70.86; H, 4.36; N, 11.02. Found: C, 70.88; H, 4.36;
N, 11.01.

1238
Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
48.11. MS (ESI): 289.68 (C15H11ClFN2O, [M+H]+). Anal. Calcd for
15H10ClFN2O: C, 62.40; H, 3.49; N, 9.70. Found: C, 64.42; H,
3.50; N, 9.71.
1H, ArH), 7.19 (m, 1H, ArH), 7.12 (d, J = 8.16 Hz, 1H, ArH), 4.60
(m, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 157.41, 151.18,
141.39, 137.53, 132.11, 131.68, 127.23, 123.11, 123.00, 121.27,
120.54, 118.26, 111.04, 69.32, 47.38. MS (ESI): 316.19 (C15H12BrN2-
O, [M+H]+). Anal. Calcd for C15H11BrN2O: C, 57.16; H, 3.52; N, 8.89.
Found: C, 57.19; H, 3.54; N, 8.87.
C
4.3.12. 2-Bromo-11-fluoro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (15)
Yield 66%, mp 232–225 °C. 1H NMR (400 MHz, DMSO-d6): 8.21
(m, 1H, ArH), 7.69 (m, 1H, ArH), 7.59 (m, 1H, ArH), 7.53 (m, 1H,
ArH), 7.04 (m, 1H, ArH), 6.95 (m, 1H, ArH), 4.63 (m, 4H, –CH2CH2–
). 13C NMR (100 MHz, CDCl3): 159.42, 156.32, 152.02, 141.12,
134.87, 134.48, 134.28, 122.10, 117.92, 116.89, 115.21, 108.63,
105.54, 71.13, 48.21. MS (ESI): 334.18 (C15H11BrFN2O, [M+H]+).
Anal. Calcd for C15H10BrFN2O: C, 54.08; H, 3.03; N, 8.41. Found:
C, 54.10; H, 3.04; N, 8.40.
4.3.18. 11-Bromo-2-methyl-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (21)
Yield 48%, mp 188–190 °C. 1H NMR (400 MHz, DMSO-d6): 8.64
(m, 1H, ArH), 7.72 (d, J = 1.56 Hz, 1H, ArH), 7.66 (d, J = 8.04 Hz,
1H, ArH), 7.45 (m, 1H, ArH), 7.22 (m, 1H, ArH), 7.06 (d,
J = 8.04 Hz, 1H, ArH), 4.62 (m, 4H, –CH2CH2–), 2.44 (s, 3H, CH3).
13C NMR (100 MHz, CDCl3): 154.34, 151.42, 142.02, 138.13,
134.52, 132.23, 131.79, 126.93, 121.72, 121.14, 119.14, 118.06,
111.48, 68.97, 47.83, 23.56. MS (ESI): 330.24 (C16H14BrN2O,
[M+H]+). Anal. Calcd for C16H13BrN2O: C, 58.38; H, 3.98; N, 8.51.
Found: C, 58.41; H, 3.99; N, 8.53.
4.3.13. 11-Chloro-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (16)
Yield 44%, mp 171–174 °C. 1H NMR (400 MHz, DMSO-d6): 8.62
(dd, J1 = 8.12 Hz, J2 = 1.76 Hz, 1H, ArH), 8.19 (d, J = 1.84 Hz, 1H,
ArH), 7.63 (d, J = 8.48 Hz, 1H, ArH), 7.46 (m, 1H, ArH), 7.24 (m,
1H, ArH), 7.18 (m, 1H, ArH), 7.10 (d, J = 8.16 Hz, 1H, ArH), 4.62 (s,
4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 157.44, 151.82,
140.63, 136.38, 132.28, 132.21, 131.69, 125.24, 123.74, 123.57,
118.85, 117.76, 111.47, 70.23, 47.94. MS (ESI): 271.73 (C15H12ClN2-
O, [M+H]+). Anal. Calcd for C15H11ClN2O: C, 66.55; H, 4.10; N, 10.35.
Found: C, 66.57; H, 4.09; N, 10.36.
4.3.19. 11-Bromo-2-chloro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (22)
Yield 42%, mp 202–204 °C 1H NMR (400 MHz, DMSO-d6): 8.78
(d, J = 8.12 Hz, 1H, ArH), 7.78 (d, J = 1.48 Hz, 1H, ArH), 7.66 (m,
1H, ArH), 7.49 (m, 1H, ArH), 7.41 (m, 1H, ArH), 7.11 (d,
J = 7.94 Hz, 1H, ArH), 4.65 (s, 4H, –CH2CH2–). 13C NMR (100 MHz,
CDCl3): 155.43, 151.34, 141.61, 137.83, 132.65, 129.12, 128.42,
127.27, 121.32, 121.22, 120.76, 118.34, 114.31, 70.02, 47.44. MS
4.3.14. 11-Chloro-2-methyl-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (17)
(ESI): 350.70 (C15H11BrClN2O, [M+H]+). Anal. Calcd for C15H10
BrClN2O: C, 51.53; H, 2.88; N, 8.01. Found: C, 51.55; H, 2.90; N,
8.02.
-
Yield 49%, mp 162–164 °C. 1H NMR (400 MHz, DMSO-d6): 8.62
(m, 1H, ArH), 8.17 (d, J = 1.92 Hz, 1H, ArH), 7.62 (d, J = 8.52 Hz,
1H, ArH), 7.28 (m, 1H, ArH), 7.19 (m, 1H, ArH), 7.09 (d,
J = 8.04 Hz, 1H, ArH), 4.60 (m, 4H, –CH2CH2–), 2.46 (3, 3H, CH3).
13C NMR (100 MHz, CDCl3): 154.32, 151.24, 140.49, 136.24,
134.32, 132.47, 132.21, 131.72, 125.38, 119.52, 119.10, 117.89,
111.49, 70.82, 48.03, 24.56. MS (ESI): 285.71 (C16H14ClN2O,
[M+H]+). Anal. Calcd for C16H13ClN2O: C, 67.49; H, 4.60; N, 9.84.
Found: C, 67.51; H, 4.61; N, 9.82.
4.3.20. 2,11-Dibromo-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (23)
Yield 53%, mp 193–195 °C. 1H NMR (400 MHz, DMSO-d6): 8.21
(d, J = 7.84, 1H, ArH), 7.76 (d, J = 1.64 Hz, 1H, ArH), 7.64–7.55 (m,
3H, ArH), 6.98 (m, 1H, ArH), 4.68 (s, 4H, –CH2CH2–). 13C NMR
(100 MHz, CDCl3): 156.52, 151.26, 141.28, 137.41, 134.87, 134.22,
127.32, 122.04, 121.43, 121.02, 118.18, 117.18, 114.63, 69.46
47.24. MS (ESI): 395.11 (C15H11Br2N2O, [M+H]+). Anal. Calcd for
4.3.15. 2,11-Dichloro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (18)
C15H10Br2N2O: C, 45.72; H, 2.56; N, 7.11. Found: C, 45.71; H,
Yield 61%, mp 167–169 °C. 1H NMR (400 MHz, DMSO-d6): 8.78
(m, 1H, ArH), 8.21 (m, 1H, ArH), 7.64 (d, J = 8.36 Hz, 1H, ArH),
7.41 (m, 1H, ArH), 7.18 (m, 1H, ArH), 7.07 (d, J = 7.96 Hz, 1H,
ArH), 4.63 (s, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 155.63,
152.12, 140.71, 136.43, 132.36, 131.81, 129.38, 128.74, 125.41,
2.58; N, 7.12.
4.3.21. 11-Nitro-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (24)
Yield 34%, mp 198–201 °C. 1H NMR (400 MHz, DMSO-d6): 8.69
(dd, J1 = 7.96 Hz, J2 = 1.60, 1H, ArH), 8.52 (m, 1H, ArH), 8.22 (m,
1H, ArH), 8.01 (m, 1H, ArH), 7.44 (m, H, ArH), 7.16 (m, 1H, ArH),
7.08 (m, 1H, ArH), 4.63 (m, 4H, –CH2CH2–). 13C NMR (100 MHz,
CDCl3): 158.13, 152.37, 148.23, 144.38, 141.24, 132.56, 132.44,
124.11, 124.02, 119.56, 114.46, 112.21, 111.97, 70.54, 48.61. MS
(ESI): 282.31 (C15H12N3O3, [M+H]+). Anal. Calcd for C15H11N3O3:
C, 64.05; H, 3.94; N, 14.94. Found: C, 64.08; H, 3.95; N, 14.93.
121.54, 119.03, 117.56, 114.97, 71.04, 48.17. MS (ESI): 306.19 (C15
H
-
11Cl2N2O, [M+H]+). Anal. Calcd for C15H10Cl2N2O: C, 59.04; H,
3.30; N, 9.18. Found: C, 59.07; H, 3.29; N, 9.19.
4.3.16. 2-Bromo-11-chloro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (19)
Yield 71%, mp 208–209 °C. 1H NMR (400 MHz, DMSO-d6): 8.29–
8.21 (m, 2H, ArH), 7.62 (m, 1H, ArH), 7.58 (m, 1H, ArH), 7.21 (m, 1H,
ArH), 6.98 (m, 1H, ArH), 4.64 (m, 4H, –CH2CH2–). 13C NMR
(100 MHz, CDCl3): 157.04, 151.74, 141.02, 136.14, 135.03, 134.41,
131.69, 125.32, 122.24, 119.11, 118.02, 117.28, 115.12, 70.94,
48.21. MS (ESI): 350.63 (C15H11BrClN2O, [M+H]+). Anal. Calcd for
4.3.22. 2-Methyl-11-nitro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (25)
Yield 32%, mp 211–213 °C. 1H NMR (400 MHz, DMSO-d6):
8.62 (m, 1H, ArH), 8.54 (m, 1H, ArH), 8.26 (m, 1H, ArH), 8.05
(m, 1H, ArH), 7.21 (m, 1H, ArH), 7.06 (m, 1H, ArH), 4.65 (m,
4H, –CH2CH2–), 2.43 (s, 1H, CH3). 13C NMR (100 MHz, CDCl3):
155.21, 152.14, 148.62, 144.12, 141.48, 134.55, 132.83, 132.34,
120.31, 119.81, 114.74, 112.54, 112.13, 69.92, 48.17, 25.02.
MS (ESI): 296.24 (C16H14N3O3, [M+H]+). Anal. Calcd for
C15H10BrClN2O: C, 51.53; H, 2.88; N, 8.01. Found: C, 51.55; H,
2.90; N, 8.02.
4.3.17. 11-Bromo-6,7-dihydrobenzo[f]benzo[4,5]imidazo[1,2-d]
[1,4]oxazepine (20)
Yield 58%, mp 169–171 °C. 1H NMR (400 MHz, DMSO-d6): 8.62
(dd, J1 = 8.08 Hz, J2 = 1.60 Hz, 1H, ArH), 7.75 (d, J = 1.68 Hz, 1H,
ArH), 7.69 (d, J = 8.56 Hz, 1H, ArH), 7.43 (m, 1H, ArH), 7.27 (m,
C16H13N3O3: C, 65.08; H, 4.44; N, 14.23. Found: C, 65.11; H,
4.42; N, 14.25.

Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
1239
4.3.23. 2-Chloro-11-nitro-6,7-dihydrobenzo[f]benzo[4,5]
4.4. Cell proliferation assay
imidazo[1,2-d][1,4]oxazepine (26)
Yield 41%, mp 208–209 °C. 1H NMR (400 MHz, DMSO-d6): 8.79
(d, J = 8.04 Hz, 1H, ArH), 8.53 (m, 1H, ArH), 8.24 (m, 1H, ArH),
8.03 (m, 1H, ArH), 7.46 (m, H, ArH), 7.14 (m, 1H, ArH), 4.63 (m,
4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 156.23, 151.68,
148.37, 144.47, 141.03, 132.63, 129.68, 129.44, 121.33, 119.49,
114.58, 114.47, 112.73, 69.47, 47.92. MS (ESI): 316.59 (C15H11ClN3-
O3, [M+H]+). Anal. Calcd for C15H10ClN3O3: C, 57.06; H, 3.19; N,
13.31. Found: C, 57.08; H, 3.20; N, 13.30.
Four human cell lines HCT-116, A549, Huh7 and HL60 were cul-
tured in RPMI-1640 (Invitrogen Corp., Carlsbad, CA) medium with
heat-inactivated 10% fetal bovine serum, penicillin (100 units/mL)
and streptomycin (100 mg/mL) and incubated in atmosphere with
20% O2, 5% CO2 at 37 °C.
The inhibitory activity in vitro was measured using the MTT
assay. All the compounds were dissolved in DMSO at the concen-
tration of 100 lM and were then diluted to appropriate concentra-
tions. Cells were plated in 96-well plates for 24 h and subsequently
treated with different concentrations of all tested compounds for
72 h. Viable cells were determined using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT, Sigma)
according to the manufacturer’s instructions. The concentration
of drug causing 50% inhibition in absorbance compared with con-
trol cells (IC50) was calculated using the software of dose-effect
analysis with microcomputers.
4.3.24. 2-Bromo-11-nitro-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (27)
Yield 37%, mp 219–222 °C. 1H NMR (400 MHz, DMSO-d6): 8.53
(m, 1H, ArH), 8.29-8.24 (m, 2H, ArH), 8.01 (m, 1H, ArH), 7.57 (m,
H, ArH), 6.99 (m, 1H, ArH), 4.61 (m, 4H, –CH2CH2–). 13C NMR
(100 MHz, CDCl3): 156.92, 152.41, 148.51, 144.28, 141.29, 134.94,
134.38, 122.04, 119.23, 117.32, 114.86, 114.16, 112.43, 70.32,
48.03, MS (ESI): 361.31 (C15H11BrN3O3, [M+H]+). Anal. Calcd for
C15H10BrN3O3: C, 50.02; H, 2.80; N, 11.67. Found: C, 50.06; H,
4.5. PI3K enzyme assay
2.81; N, 11.66.
The inhibition of PI3K activity was determined using a compet-
itive fluorescence polarization kinase activity assay based on the
principle that PI3K phosphorylates PI(3,4)P2 and converts it to
PI(3,4,5)P3.21 PI3K isoforms were purchased from Perkin–Elmer
or were expressed and purified as heterodimeric recombinant pro-
teins. Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), diC8-
PIP2, and PIP3 detection reagents were purchased from Echelon
Biosciences. PI3K reactions were performed in 5 mM HEPES, pH
7, 2.5 mM MgCl2, 10 mM DTT and 50 mM ATP, using diC8-
PI(4,5)P2 as the substrate, and the final reaction volumes were
4.3.25. 11-(Trifluoromethyl)-6,7-dihydrobenzo[f]benzo[4,5]
imidazo[1,2-d][1,4]oxazepine (28)
Yield 41%, mp 204–206 °C. 1H NMR (400 MHz, DMSO-d6): 8.68
(d, J = 7.44 Hz, 1H, ArH), 8.07 (s, 1H, ArH), 7.88–7.80 (m, 1H,
ArH), 7.58 (t, J = 11.10 Hz, 1H, ArH), 7.47 (t, J = 7.10 Hz, 1H, ArH),
7.21 (t, J = 7.30 Hz, 1H, ArH), 7.14 (d, J = 7.84 Hz, 1H, ArH), 4.68 (t,
J = 17.42 Hz, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3): 157.39,
151.93, 140.69, 140.12, 132.34, 132.11, 129.54, 127.96, 123.74,
123.36, 123.12, 116.65, 114.34, 111.74, 69.54, 48.22. MS (ESI):
305.17 (C16H12F3N2O, [M+H]+). Anal. Calcd for C16H11F3N2O: C,
63.16; H, 3.64; N, 9.21. Found: C, 63.19; H, 3.66; N, 9.23.
10
l
L. For evaluation of PI3K inhibitors, 50 ng of enzyme and
L reaction volume with
10 mM of substrate were used per 10
l
inhibitors concentrations ranging from 3.2 nM to 1 mM. After incu-
bating at room temperature for 3 h at room temperature, reactions
were quenched by the addition of chelator. A mixture of phospho-
inositide binding protein was added and mixed, followed by the
addition of a fluorophore-labeled phosphoinositide tracer. Samples
were then mixed in 384-well black Corning nonbinding plates and
incubated in a dark location for 1 h to equilibrate. Finally, polariza-
tion values were measured using red fluorophores with appropri-
ate filters to determine the extent of enzyme activity in the
reaction.25
4.3.26. 2-Methyl-11-(trifluoromethyl)-6,7-dihydrobenzo[f]
benzo[4,5]imidazo[1,2-d][1,4]oxazepine (29)
Yield 47%, mp 210–213 °C. 1H NMR (400 MHz, DMSO-d6): 8.65
(m, 1H, ArH), 8.09 (s, 1H, ArH), 7.86–7.79 (m, 1H, ArH), 7.61 (m,
1H, ArH), 7.24 (m, 1H, ArH), 7.08 (d, J = 7.92 Hz, 1H, ArH), 4.66 (m,
4H, –CH2CH2–). 2.43 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3):
155.03, 152.17, 140.48, 140.44, 134.53, 132.64, 132.21, 129.23,
128.14, 123.61, 120.43, 116.21, 114.62, 112.08, 70.13, 48.74,
24.78. MS (ESI): 319.21 (C17H14F3N2O, [M+H]+). Anal. Calcd for C17
13F3N2O: C, 64.15; H, 4.12; N, 8.80. Found: C, 64.17; H, 4.13; N, 8.81.
-
H
4.6. Flow cytometry
4.3.27. 2-Chloro-11-(trifluoromethyl)-6,7-dihydrobenzo[f]
benzo[4,5]imidazo[1,2-d][1,4]oxazepine (30)
The HCT-116 cells were seeded in 6-well plates at a seeding
density of 105 cells per mL. Twelve hours later, various concentra-
tions of compound 25 were added. Cells were treated with com-
pound 25 for 24 h. Then cells were transferred to EP tubes and
washed three times with PBS buffer. Then the procedures accord-
ing to the operating instructions of the kit were followed. Ulti-
mately, cell apoptosis was analyzed using Annexin-V and
propidium iodide (PI) double staining by flow cytometry. Early
apoptotic cells were defined as Annexin-V positive/PI negative, late
apoptotic cells as Annexin-V/PI-double positive and necrotic cells
as Annexin-V positive/PI positive.
Yield 44%, mp 212–214 °C. 1H NMR (400 MHz, DMSO-d6): 8.79
(d, J = 7.96 Hz, 1H, ArH), 8.12 (m, 1H, ArH), 7.83 (m, 1H, ArH),
7.65 (m, 1H, ArH), 7.42 (m, 1H, ArH), 7.11 (d, J = 8.08 Hz, 1H,
ArH), 4.67 (m, 4H, –CH2CH2–). 13C NMR (100 MHz, CDCl3):
155.87, 152.23, 140.34, 140.23, 132.41, 129.68, 129.12, 128.93,
127.84, 123.43, 121.73, 116.71, 115.10, 114.46, 70.21, 48.36. MS
(ESI): 339.62 (C16H11ClF3N2O, [M+H]+). Anal. Calcd for C16H10ClF3-
N2O: C, 56.74; H, 2.98; N, 8.27. Found: C, 56.76; H, 2.99; N, 8.28.
4.3.28. 2-Bromo-11-(trifluoromethyl)-6,7-dihydrobenzo[f]
benzo[4,5]imidazo[1,2-d][1,4]oxazepine (31)
Yield 46%, mp 207–210 °C. 1H NMR (400 MHz, DMSO): 8.27 (m,
1H, ArH), 8.16 (s, 1H, ArH), 7.85–7.78 (m, 1H, ArH), 7.66 (m, 1H,
ArH), 7.57 (m, 1H, ArH), 6.96 (m, 1H, ArH), 4.62 (m, 4H, –CH2CH2–).
13C NMR (100 MHz, CDCl3): 156.89, 151.98, 140.71, 140.18,
135.62, 134.78, 129.02, 127.33, 123.40, 122.73, 117.46, 116.82,
114.94, 114.54, 70.01, 47.81. MS (ESI): 384.27 (C16H11BrF3N2O,
[M+H]+). Anal. Calcd for C16H10BrF3N2O: C, 50.15; H, 2.63; N, 7.31.
Found: C, 50.17; H, 2.62; N, 7.33.
4.7. Molecular docking
The crystal structures of the proteins complex were retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/
home/home.do). The three-dimensional structures of the afore-
mentioned compounds were constructed using Chem. 3D ultra
12.0 software [Chemical Structure Drawing Standard; Cambridge
Soft corporation, USA (2010)], then they were energetically mini-

1240
Y. Yin et al. / Bioorg. Med. Chem. 23 (2015) 1231–1240
8. Shuttleworth, S. J.; Silva, F. A.; Cecil, A.; Tomassi, C. D.; Hill, T. J.; Raynaud, F. I.;
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9. Jarvis, L. M. Chem. Eng. News 2011, 89, 15.
mized by using MMFF94 with 5000 iterations and a minimum RMS
gradient of 0.10. Molecular docking of compound 25 into the three-
dimensional PI3Kc complex structure (3R7R.pdb, downloaded
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from the PDB) was carried out using the Discovery Studio (version
3.5) as implemented through the graphical user interface DS-
CDOCKER protocol, all bound water and ligands were eliminated
from the protein and the polar hydrogen was added to the proteins.
Acknowledgments
16. Heffron, T. P.; Wei, B.; Olivero, A.; Staben, S. T.; Tsui, V.; Do, S.; Dotson, J.;
Folkes, A. J.; Goldsmith, P.; Goldsmith, R. J. Med. Chem. 2011, 54, 7815.
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3762.
This work was supported by Natural Science Foundation of
Jiangsu Province (No. BK20130554) and supported by Major Pro-
jects on Control and Rectification of Water Body Pollution (No.
2011ZX07204-001-2214 004).
19. Discovery Studio 3.5 Guide, Accelrys Inc., San Diego, http://www.accelrys.com.
20. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
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