Novel 4-aminoquinazoline derivatives induce growth inhibition, cell cycle arrest and apoptosis via PI3Kα inhibition

Article abstract of DOI:10.1016/j.bmc.2018.02.015

Phosphatidylinositol 3-kinase (PI3K) signaling pathway has diverse functions, including the regulation of cellular survival, proliferation, cell cycle, migration, angiogenesis and apoptosis. Among class I PI3Ks (PI3Kα β γ δ), the PIK3CA gene encoding PI3K

Full text of DOI:10.1016/j.bmc.2018.02.015

Accepted Manuscript
Novel 4-aminoquinazoline derivatives induce growth inhibition, cell cycle ar-
rest and apoptosis via PI3Kα inhibition
Yan-Hua Fan, Huai-Wei Ding, Dan-Dan Liu, Hong-Rui Song, Yong-Nan Xu,
Jian Wang
PII:
S0968-0896(17)32441-0
https://doi.org/10.1016/j.bmc.2018.02.015
BMC 14201
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 December 2017
3 February 2018
12 February 2018
Please cite this article as: Fan, Y-H., Ding, H-W., Liu, D-D., Song, H-R., Xu, Y-N., Wang, J., Novel 4-
aminoquinazoline derivatives induce growth inhibition, cell cycle arrest and apoptosis via PI3Kα inhibition,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.02.015
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Novel 4-aminoquinazoline derivatives induce growth inhibition, cell cycle arrest and
apoptosis via PI3Kα inhibition
b
a,
a
a
a
a
Yan-Hua Fan , Huai-Wei Ding *, Dan-Dan Liu , Hong-Rui Song , Yong-Nan Xu , Jian Wang
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b
Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
*
Corresponding authors.
Email address: dinghuaiwei627@163.com.

Abstract：Phosphatidylinositol 3-kinase (PI3K) signaling pathway has diverse functions, including
the regulation of cellular survival, proliferation, cell cycle, migration, angiogenesis and apoptosis.
Among class I PI3Ks (PI3Kα, β, γ, δ), the PIK3CA gene encoding PI3K p110α is frequently mutated
and overexpressed in a large portion of human cancers. Therefore, the inhibition of PI3Kα has been
considered as a promising target for the development of a therapeutic treatment of cancer. In this
study, we designed and synthesized a series of 4-aminoquinazoline derivatives and evaluated their
antiproliferative activities against six cancer cell lines, including HCT-116, SK-HEP-1,
MDA-MB-231, SNU638, A549 and MCF-7. Compound 6b with the most potent antiproliferative
activity and without obvious cytotoxicity to human normal cells was selected for further biological
evaluation. PI3K kinase assay showed that 6b has selectivity for PI3Kα distinguished from other
isoforms. The western blot assay and PI3K kinase assay indicated that 6b effectively inhibited cell
proliferation via suppression of PI3Kα kinase activity with an IC50 of 13.6 nM and subsequently
blocked PI3K/Akt pathway activation in HCT116 cells. In addition, 6b caused G1 cell cycle arrest
owing to the inhibition of PI3K signaling and induced apoptosis via mitochondrial dependent
apoptotic pathway. Our findings suggested that 6b has a therapeutic value as an anticancer agent via
PI3Kα inhibition.
Keywords: PI3K inhibitor; 4-aminoquinazoline; antiproliferative effects; cell cycle arrest; apoptosis

1. Introduction
The phosphatidylinositol 3-kinases (PI3K) families are divided into four different classes: Class
I, Class II, Class III and Class IV. The most studied are the class I enzymes which are further divided
1
-2
into PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ . PI3K pathway regulates various cellular processes, such as
3
-6
proliferation, growth, cell cycle, autophagy and apoptosis . Hyperactivation of the PI3K/Akt
pathway has been confirmed as an essential step in the initiation and maintenance of human tumors
5
-8
.
An accumulation of evidence indicates that PI3K pathway plays a key role in the regulation of
9
-10
cell cycle and apoptosis
. The PI3K pathway becomes activated during the G1/S transition of the
Cip1
cell cycle and regulates several key cell cycle regulators, including p21 , CyclinD, and
Kip1
10
p27
protein stability . It is known that hyperactive PI3K/Akt signaling prevents apoptosis. PI3K
11-12
increases Mdm2 protein levels and causes a reduction of p53
. Bcl-2 family plays an instrumental
1
3
role in the regulation of apoptosis . PI3K/Akt pathway mediates cell viability and apoptosis via
Bcl-2 family proteins including Bad and Bcl-2, which is a crucial regulator of the mitochondrial
1
4
apoptosis pathway . Therefore, PI3K inhibition will cause cell apoptosis via p53 and mitochondrial
apoptosis pathway.
Given the important role of PI3K played in the occurrence and development of human tumors
1
5-18
,
a lot of PI3K inhibitors have been developed and are being evaluated in preclinical studies and
1
9-21
early clinical trials
. Among them, BEZ235 has been identified as highly potent inhibitor of PI3K
22
and is currently under evaluation in clinical trials for oncology applications . The other two
2
3
24
BEZ235 analogues BGT226 and PF-04979064 were also being evaluated in preclinical studies
and early clinical trials. In addition, GSK2126458 and HS-173 which all have sulfonamide pyridyl
both exhibited potent and highly selective PI3Kα inhibitory activity with an IC50 of picomolar level

2
5-27
.
Quinazoline derivatives, especially 4-anilinoquinazolines, have attracted interests over the years
2
8
for their multiple biological activities, notably as EGFR inhibitors . Numerous drugs containing
quinazoline moiety have been successfully developed, such as Gefitinib, Lapatinib, Erlotinib,
Vandetanib and Afatinib. Thereby, we designed and synthesized a new series of 4-aminoquinazoline
derivatives containing 6-sulfonamide substituted pyridyl group. In addition, we evaluated the
anti-tumor effects of all these prepared compounds and investigated their anti-tumor
pharmacologic mechanism in vitro.
2. Results and discussion
2.1 Chemistry
Fig.1. The design strategy based on NVP-BEZ235 and GSK2292767.
We had a good understanding of the binding mode of PI3K inhibitors based on the analysis of
1
9, 25, 27
the reported crystal structure complexes and reviews
. Most of the PI3K inhibitors have very
similar binding modes and the active sites of PI3K kinase are mainly composed of three regions: the

hinge binder domain, the affinity pocket domain and the ribose pocket domain (Fig.1).
Quinazoline is an important scaffold in drug discovery and 4-aminoquinazoline derivatives had
2
9-32
been reported as PI3K inhibitors in recent years
. So, we believe that the quinazoline group still
has good prospects for development. In order to develop new PI3K inhibitors containing quinazoline
structure, we opened the five ring of BEZ235 and the quinazoline ring was used instead of the
quinoline ring. Next, we introduce hydrophilic groups on the position-4 of quinazoline nucleus and
the “affinity pocket” was filled by sulfonamide pyridine instead of the quinoline ring.
2.2 Antiproliferative assays in vitro
All synthesized compounds were evaluated for their cytotoxicity in vitro against six human
cancer cell lines including HCT116, A549, SK-HEP-1, SNU638, MDA-MB-231 and MCF-7 cells.
The antiproliferative results of all the compounds were summarized in Table 1. The results of
antiproliferative effect assay showed most of the derivatives exhibited potent antiproliferative effects,
especially against HCT-116. Notably, 6b showed the most significant antiproliferative activity
against all these cancer cell lines. From compounds 6a-6d (IC50 = 13.5, 0.16, 3.20 and 1.44 µM
against HCT-116 separately), which all have a common 4-N, N-dimethylethane-1,2-diamine-
quinazoline moiety with different sulfonamide groups. Compared to 4-fluorophenyl,
2 2
2,4-difluorophenyl at R position markedly improved antiproliferative activity and N-butyl at R
position showed the best antiproliferative activities. The structure-activity relationships (SARs)
indicated that butyl sulfonamide and 2,4-difluorosulfonamide were beneficial to the enhancement of
anti-tumor activity, so the subsequent synthesis retained both sulfonamides and only changed the
ammonia on the position-4 of quinazoline. Compound 6b and 6e (IC50 = 0.49 µM against HCT-116),
6
d and 6f (IC50 = 2.20 µM against HCT-116), 6g (IC50 = 0.59 µM against HCT-116) and 6i (IC50 =

1
.74 µM against HCT-116), 6h (IC50 = 2.74 µM against HCT-116) and 6j (IC50 = 18.8 µM against
HCT-116), suggesting that two carbon atoms in the R group was better than that of the three carbon
atoms. Compound 6a - 6j more potent than 6k (IC50 = 5.72 µM against HCT-116) and 6l (IC50 = 17.6
1
µM against HCT-116), indicating that alkyl group at position of R
1
is more beneficial to activity than
aromatic group.
a
Table 1 Cytotoxicity in vitro of target compounds against five cancer cell lines (IC50 Values in µM).
IC50 (µM)
Comp.
R
1
R
2
HCT-116
SK-HEP1
MDA-MB SNU638
231
A549
MCF-7
-
6
6
a
13.5±0.27
0.16±0.01
12.6±0.22
0.28±0.02
2.32±0.07 0.41±0.02 4.12±0.15 14.7±0.34
0.28±0.03 0.48±0.04 1.32±0.05 3.24±0.08
b
6
c
3.20±0.06
1.44±0.06
0.49±0.05
9.50±0.14
4.72±0.09
0.86±0.06
1.22±0.04 1.21±0.05 2.42±0.08 1.23±0.04
0.71±0.04 0.62±0.03 0.94±0.06 1.02±0.07
0.88±0.07 1.26±0.08 3.52±0.12 4.73±0.14
6
d
6
e
6
f
2.20±0.04
4.68±0.07
1.54±0.03 1.26±0.02 2.78±0.06 3.66±0.07

6
6
g
0.59±0.01
2.74±0.06
1.74±0.04
18.8±0.26
0.44±0.01
0.84±0.06
1.14±0.03
13.4±0.15
0.42±0.02 0.61±0.03 1.56±0.04 10.8±0.16
0.38±0.04 3.96±0.09 0.62±0.08 28.9±0.24
2.58±0.07 0.98±0.03 3.14±0.08 4.59±0.07
8.96±0.10 4.52±0.08 9.82±0.12 13.5±0.14
h
6
i
6
j
6
k
5.72±0.08
17.6±0.18
0.84±0.12
8.98±0.11
12.4±0.14
1.82±0.23
4.32±0.08 6.66±0.12 9.42±0.14 18.7±0.22
9.71±0.09 6.21±0.04 11.4±0.08 22.5±0.34
0.18±0.02 1.24±0.13 0.62±0.07 1.33±0.14
6
l
BEZ235
a
IC50 values are the mean of triplicate measurements.
We also test the cytotoxic effects of 6b on normal human cells MRC5 and HEK293. We
obserbed 6b showed much less inhibitory activity against human normal cells (IC50 = 18.5 µM
against MRC5 and IC50 = 12.6 µM against HEK293), implying the possibility of inhibition of cancer
cells without any effects on normal cells (Table 2).
Table 2 Cytotoxicity of 6b to normal human cells (IC50 Values in µM)
Cells
MRC5
18.5
HEK293
12.6
6
b
2.3 6b inhibits PI3Kα and blocks the PI3K-Akt pathway in HCT116 cells
6
b was found to inhibit PI3Kα with an IC50 of 13.6 nM (Table 3). Docking simulations were

conducted to predict the binding mode of 6b and PI3Kα using Autodock 4.2 (Fig. 2). On the basis of
the docking model results, there is a hydrogen bond formed between the nitrogen atom of N,
N-dimethylethanamine group and the NH group of the catalytic lysine (Lys802). Moreover, the
sulfonamide formed two additional hydrogen interactions with Gln859 and Thr856. In addition to
these, the nitrogen atoms of quinazolin moiety form two key hydrogen bond with the conserved
water molecule. These multiple hydrogen bonds and some other interaction including van
der Waals forces, aromatic stacking interaction, electrostatic interaction and hydrophobic interaction
appeared to be the most significant binding force stabilizing 6b in the active site of PI3Ka. To
analyze the binding affinity of 6b and other PI3K isoforms, we also conducted docking simulations
to predict their binding mode. As shown in Fig. S1, unlike PI3Kα, no such interactions between the
Alkyl sidechain at the 4-quinoline position and 6b were formed in the active site of other three PI3K
isoforms, indicating this Alky sidechain played a key role on enhancement of PI3Ka inhibitory
potency of 6b and may offer a selective inhibition to PI3Kα.
Table 3 Activities of 6b and BEZ235 against Class I PI3K (IC50 Values in nM)
PI3Kα
13.6
PI3Kβ
396.2
35.9
PI3Kγ
117.5
23.6
PI3Kδ
101.8
78.4
6
b
BEZ235
16.4

Fig.2. The binding model of 6b bound to PI3Kα. Autodock 4.2 programs were used to perform this molecular
docking simulation. (A) 2D binding model of 6b and PI3Kα. (B-C) 3D binding model of 6b and PI3Kα.
Because 6b suppressed PI3Kα activity, we concluded that downstream of PI3K signaling that
mediates cell growth and blocks cell apoptosis might also be inhibited by 6b. Therefore, we assessed
whether 6b changed the expression of the PI3K/Akt signaling pathways and their downstream
signaling molecules by western blot analysis. Compared to the control group, the levels of p-PI3K,
p-Akt (Ser473) and p-GSK3β were significantly decreased by 6b in HCT116 cells. In addition, the
suppressive effect of 6b on p-Akt expression was also confirmed by immunofluorescence analysis
under a confocal microscope after treatment of 6b for 8 h.

Fig.3. 6b suppresses PI3K signaling pathway. (A) The effects of 6b on the expressions of PI3K-associated
signaling proteins in HCT116 cells. (B) The effects of 6b on the expression level of p-Akt (Ser473) in cells as
determined by immunofluorescence analysis. Cells were treated with the indicated concentrations of 6b for 24 h
and then the protein expressions were determined by using Western blot. The alternations of related proteins were
quantified using Image J program. *P<0.05 or **P<0.01 was considered statistically significant compared with
corresponding control values.
2.4 6b induce G1 phase arrest in HCT116 and NCI-H1975 cells
To better elucidate the antitumor mechanism responsible for antiproliferation and induction of
apoptosis of 6b, the cell cycle distribution was evaluated by flow cytometric analysis. As shown in
the cell cycle data showed that 6b induced an obvious accumulation of cells in G1 phase and reduced
the percentage of cells at the S phase compared with the control group in HCT116 cells. These
results are consistent with cell cycle arrest due to the inhibition of PI3K kinase activity.

Fig.4. 6b induced G1 cell cycle arrest in HCT116 cells. (A) Cells were treated with the indicated concentrations of
b for 24 h. The DNA content of the cells was then analyzed by flow cytometry analysis as described in Materials
6
and Methods. (B) Cells were treated with the indicated concentrations of 6b for 24 h and then analyzed the protein
expressions associated with the G1 phase cell cycle arrest in HCT116 cells. The changes of corresponding proteins
were quantified using Image J. Each bar represents mean ± SEM (n = 3), *P<0.05 or **P<0.01 was considered
statistically significant compared with corresponding control values.
2.5 6b induced cell apoptosis via the mitochondrial pathway
Since PI3K/Akt pathway is well known as a pro-survival mechanism in many cancer cells, we
want to know whether 6b induced apoptosis in HCT116 cells. Flow cytometric analysis data showed
the percentage of Annexin V-positive cells was significantly increased after exposure to increasing
dose of 6b, compared with medium control (5.15% vs 15.71% for HCT116, P < 0.01). These results

suggested that compound 6b can induce apoptosis in vitro and this is one of mechanisms by which
compound 6b exerted its antiproliferative activity.
Fig.5. 6b induced apoptosis in HCT116 cells. Cells were treated with various concentrations of 6b for 48 h and
then analyzed the Annexin V-FITC/PI staining test by flow cytometry analysis. The number in the right quads of
each panel means the percentage of Annexin V positive cells.
To confirm the effects of 6b on apoptosis induction, we examined the changes in apoptotic
related proteins in HCT116 after treatment with 6b for 48 h. In comparison to the control, the
cleaved caspase-9 and cleaved caspase-3 levels were remarkably boosted when treated with 6b at 4
µM. In addition, the level of cleaved PARP was also elevated by 6b treatment for 48 h in HCT116
cells. Taken together, these results indicated that 6b induced apoptosis in HCT116 cells via the
mitochondrial pathway.

Fig.6. (A) The effects of 6b on the induction of apoptosis were also analyzed by Western blot in HCT116. The
changes of corresponding proteins were quantified using Image J. Each bars represent mean ± SE (n = 3). *P<0.05
or **P<0.01 was considered statistically significant compared with corresponding control values.
3. Discussion and conclusion
PI3K/Akt signaling pathway is continuously active in many cancers promoting cell viability and
3
3
results in resistance to chemotherapy . Numerous studies have indicated that somatic mutations in
genes associate with PI3K/Akt/mTOR pathway increase Akt activity and show an antagonism to
3
4
chemotherapy and radiation induced apoptosis . High frequency of somatic mutations in PIK3CA
has been reported in many kinds of human cancer, suggesting that mutated PIK3CA gene as an
3
5
oncogene in cancers . Therefore, PI3Kα has been extensively studied and explored as a target for
the development of cancer drug. In theory, PI3Kα-selective inhibitors should provide more specific
inhibition of PI3Kα while reducing the side effects caused by non-selective blockade of other PI3K
isoforms. Recently, lots of PI3Kα inhibitors were designed and synthesized from all kinds of
structural scaffolds including quinazoline derivatives.
An increasing number of studies showed that quinazoline derivatives are of considerable

3
6
pharmacological chemicals as anticancer agents . Quinazoline moiety has been extensively used as
3
7
a drug-like scaffold in medicinal chemistry, especially in the development of anticancer agents
.
Several quinazoline derivatives are potent and highly selective inhibitors of tyrosine kinase, such as
gefitinib, erlotinib, lapatinib, and afatinib, have been successfully developed and used in clinical
1
7-19
trials in the past few years
. Therefore, in recent years, the synthesis of 4-aminoquinazolines has
attracted much attention.
In this study, we designed and synthesized a serial of 4-aminoquinazoline derivatives based on
NVP-BEZ235 and GSK2292767. The in vitro antiproliferative activities of all the prepared
chemicals were evaluated against six human cancer cell lines and two normal human cells. Although
a potential growth inhibitory activity of 6b was found in cultured human lung cancer cell lines, 6b
did not exhibit a considerable anti-proliferative activity against MRC-5 human lung normal epithelial
cells (IC50 of 18.5 µΜ) and HEK293 human embryonic kidney cells (IC50 of 12.6 µΜ) indicating that
6
b selectively inhibits the proliferation of cancer cells compared to normal cells (Table 2). Among of
these compounds, compound 6b effectively inhibited the PI3K kinase activity, in particularly PI3Kα
Table 3) and markedly induced a dose dependent suppression of the phosphorylation of Akt and its
(
downstream factor GSK3β (Fig.3). In addition, 6b also induced G1 phase cell cycle arrest through
regulation of G1 phase related targets, which is consistent with the effects of some PI3K inhibitors
(
Fig.4). We next determined if 6b caused apoptosis in HCT116 cells. Annexin V-PI staining showed
6
6
b enhanced the cell apoptosis after treatment for 48 h (Fig.5). Western blot analysis indicated that
b strongly reduced Bcl-2 levels and increased the Bax levels. In addition, 6b induced cell apoptosis
in HCT116 cells via increasing cytochrome C and the activated caspase 3 and caspase 9 (Fig.6).
In summary, we designed and synthesized a series of compounds containing hydrophilic group

in position-4 of quinazoline and evaluated their antiproliferative activities against six cancer cell
lines, including HCT-116, A549, SK-HEP-1, SNU638, MDA-MB-231 and MCF-7. Compound 6b,
with the most potent antiproliferative activity was selected for further biological evaluation. Our
further study showed that the suppression of cell survives and growths induced by 6b via
PI3K/Akt/mTOR pathway inhibition. In addition, inhibition of PI3K signaling by 6b further resulted
in G1 cell cycle arrest and cell apoptosis. These findings suggest that 6b may be used as a potential
PI3K inhibitor to treat human cancers.
4
4
4
. Material and methods
.1 Chemistry
.1.1 Instruments & chemicals
1
All reagents and solvents were commercially available without further purification. H NMR
1
3
spectra and C 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). All the melting points were determined on a Beijing
micromelting-point apparatus and thermometer was uncorrected. 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.).
4.1.2 General experimental protocol for preparation of compounds
The synthetic routes for the target compounds are outlined in Schemes 1. Sulfonylation of 1 with
four benzenesulfonyl chlorides to yield 2a-d, which were then subjected to Suzuki coupling with
bis(pinacolato)diborane to afford arylboronic ester 3a-d. Intermediate 4 was reacted with five amines

to abtain 5a-e, which were coupled with 3a-d via Suzuki reaction to afford the target compounds
a-l.
6
2 2
Scheme 1. (a) benzenesulfonyl chloride, pyridine, rt, 24 h; (b) bis(pinacolato)diborane, Pd(dppf) Cl , AcOK, DMF,
1
2 2 2 3 2
00 ºC, 8 h; (c) amines, isopropanol, 50 ºC, 1h; (d) Pd(dppf) Cl , Cs CO , DMF/ H O, 90 ºC, 8 h.
4.1.2.1 N-(5-bromo-2-methoxypyridin-3-yl)methanesulfonamide (2a)
To a solution of 5-bromo-2-methoxypyridin-3-amine (1) (2.01 g, 10 mmol) in pyridine (50 ml)
at 0 ºC was added methanesulfonyl chloride (1.25 g, 11 mmol). Then the mixture was stirred at room
temperature for 24 h. Pyridine was removed under reduced pressure before adding water (100 ml),
then extracted with ethyl acetate (3 × 100 ml), the organic layer was washed with water (50 ml),
dried with Na
2
SO and evaporated to give compound 2a as a white solid (2.42 g, 86.5%). mp
4
1
1
36-138 ºC. H NMR(400 MHz, DMSO-d
6
) δ 9.49 (s, 1H, NH), 8.09 (d, J = 2.4 Hz, 1H, Ar-H), 7.79
+
(
d, J = 2.0 Hz, 1H, Ar-H), 3.91(s, 3H, OCH
3
), 3.11(s, 3H, CH
3
). ESI-MS: m/z 280.1 [M+H] .

Compounds 2b-d were synthesized according to the procedure described in 2a.
.1.2.2 N-(5-bromo-2-methoxypyridin-3-yl)butane-1-sulfonamide (2b)
4
7
8.4% yield. mp 150-151 ºC.9.51 (s, 1H, NH), 8.09 (d, J = 2.4 Hz, 1H, Ar-H), 7.77 (d, J =
.4Hz, 1H, Ar-H), 3.91 (s, 3H, OCH ), 3.16 (t, J = 7.6Hz, 2H, CH ), 1.73 - 1.65 (m, 2H, CH ), 1.42 -
.33 (m, 2H, CH ), 0.87 (t, J = 7.2 Hz, 3H, CH ). ESI-MS: m/z 323.1 [M+H] .
2
1
4
3
2
2
+
2
3
.1.2.3 N-(5-bromo-2-methoxypyridin-3-yl)-4-fluorobenzenesulfonamide (2c)
1
8
9.4% yield. mp 152-154 ºC. H NMR (400 MHz, DMSO) δ 10.22 (s, 1H, NH), 8.08 (d, J = 2.2
Hz, 1H, Ar-H), 7.83 (dd, J = 8.8, 5.2 Hz, 2H, Ar-H), 7.72 (d, J = 2.2 Hz, 1H, Ar-H), 7.43 (t, J = 8.8
+
Hz, 2H, Ar-H), 3.64 (s, 3H, OCH
3
). ESI-MS: m/z 385.1 [M+H+Na] .
4.1.2.4 N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (2d)
1
8
5.6% yield. mp 163-165 ºC. H NMR (400 MHz, DMSO) δ 10.46 (s, 1H, NH), 8.13 (d, J = 2.2
Hz, 1H, Ar-H), 7.83-7.74 (m, 2H, Ar-H), 7.62-7.52 (m, 1H, Ar-H), 7.24 (td, J = 2.0, 8.5 Hz, 1H,
+
Ar-H), 3.62 (s, 3H, OCH
.1.2.5
N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide (3a)
A solution of the 2a (2.80 g, 10 mmol), bis(pinacolato)diborane (1.27 g, 5 mmol), Pd(dppf) Cl
0.18 g, 0.25 mmol) and KOAc (1.47 g, 15 mmol) in anhydrous DMF (30 ml) under N was stirred at
00 ºC for 8 h. DMF was removed under reduced pressure and adding water (100 ml), extracted with
3
). ESI-MS: m/z 376.9 [M-H] .
4
2
2
(
2
1
ethyl acetate (3 × 100 ml), the organic layer was washed with water (20 ml), dried with Na
2
SO and
4
1
evaporated to give compound 3a as a white solid (1.98 g, 60.4% yield). mp 90-92 ºC. H NMR (400
MHz, DMSO) δ 9.25 (s, 1H, NH), 8.21 (d, J = 1.6 Hz, 1H, Ar-H), 7.78 (d, J = 1.6 Hz, 1H, Ar-H),
+
3
.94 (s, 3H, OCH
3
), 3.01 (s, 3H, CH
3
), 1.30 (s, 12H, CH
3
). ESI-MS: m/z 329.1 [M+H] .

Compounds 3b-d were synthesized according to the procedure described in 3a.
.1.2.6
N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)butane-1-sulfonamide
3b)
4
(
1
6
4.3% yield. mp 91-92 ºC. H NMR (400 MHz, DMSO) δ 9.26 (s, 1H, NH), 8.21 (d, J = 1.4 Hz,
H, Ar-H), 7.79 (d, J = 1.3 Hz, 1H, Ar-H), 3.94 (s, 3H, OCH ), 3.10 - 3.01 (m, 2H, CH ), 1.76 - 1.63
m, 2H, CH ), 1.38 (dt, J = 7.4, 14.8Hz, 2H, CH ), 1.30 (s, 12H, CH ), 0.86 (t, J = 7.3 Hz, 3H, CH ).
ESI-MS: m/z 371.2 [M+H] .
1
3
2
(
2
2
3
3
+
4
.1.2.7
-fluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonam
4
ide (3c)
4.5% yield. mp 149-151 ºC. H NMR (400 MHz, DMSO) δ 9.92 (s, 1H, NH), 8.18 (s, 1H,
3
Ar-H), 7.73 (m, 3H, Ar-H), 7.40 (t, J = 8.7 Hz, 2H, Ar-H), 3.62 (s, 3H, OCH ), 1.29 (s, 12H, CH ).
1
7
3
+
ESI-MS: m/z 409.3 [M+H] .
4
.1.2.8
,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfo
2
namide (3d)
8.8% yield. mp 171-173 ºC. H NMR (400 MHz, DMSO) δ 10.19 (s, 1H, NH), 8.21 (d, J = 1.5
Hz, 1H, Ar-H), 7.72 (d, J = 1.5 Hz, 1H, Ar-H), 7.71 - 7.67 (m, 1H, Ar-H), 7.59 - 7.54 (m, 1H, Ar-H),
.20 (td, J = 2.0, 8.6 Hz, 1H, Ar-H), 3.62 (s, 3H, OCH ), 1.30 (s, 12H, CH ). ESI-MS: m/z 427.3
1
7
7
3
3
+
[
M+H] .
4.1.2.9 N-(6-bromoquinazolin-4-yl)-N,N-dimethylethane-1,2-diamine (5a)

A solution of the 4 (0.24 g, 1 mmol), N,N-dimethylethane-1,2-diamine (0.13g, 1.5 mmol) in
isopropanol (20 ml) were stirred at 50 ºC for 1 h. Isopropanol was removed under reduced pressure
and the residue was purified through a column chromatography on silica with chloroform/methanol
1
(
V:V 20:1) as a white solid (0.27 g, 92.0% yield). mp 132-134 ºC. H NMR (600 MHz, DMSO) δ
8.56 (d, J = 2.1 Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.34 (t, J = 5.3 Hz, 1H, NH), 7.88 (dd, J = 8.8, 2.2
Hz, 1H, Ar-H), 7.62 (d, J = 8.8 Hz, 1H, Ar-H), 3.63 (dd, J = 12.3, 6.6 Hz, 2H, CH ), 2.52 (t, J = 6.6
2
+
2 3
Hz, 2H, CH ), 2.20 (s, 6H, CH ). ESI-MS: m/z 295.2 [M+H] .
Compounds 5b-e were synthesized according to the procedure described in 5a.
4.1.2.10 N-(6-bromoquinazolin-4-yl)-N,N-dimethylpropane-1,3-diamine (5b)
1
9
3.8% yield. mp 95-97 ºC. H NMR (600 MHz, DMSO) δ 8.54 (d, J = 2.2 Hz, 1H, Ar-H), 8.49
(
s, 1H, Ar-H), 8.46 (t, J = 5.4 Hz, 1H, NH), 7.88 (dd, J = 8.8, 2.2 Hz, 1H, Ar-H), 7.62 (d, J = 8.8 Hz,
1
1
4
H, Ar-H), 3.54 (dd, J = 12.6, 7.0 Hz, 2H, CH
2 2 3
), 2.41 (t, J = 7.1 Hz, 2H, CH ), 2.23 (s, 6H, CH ),
+
.85 -1.76 (m, 2H, CH ). ESI-MS: m/z 309.2 [M+H] .
2
.1.2.11 6-bromo-N-(2-morpholinoethyl)quinazolin-4-amine (5c)
1
9
4.3% yield. mp 118-120 ºC. H NMR (600 MHz, DMSO) δ 8.53 (d, J = 2.1 Hz, 1H, Ar-H),
8
.48 (s, 1H, Ar-H), 8.39 (t, J = 5.1 Hz, 1H, NH), 7.88 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.62 (d, J =
.8 Hz, 1H, Ar-H), 3.56 (m, 6H, CH ), 2.38 (d, J = 6.8 Hz, 4H, CH ), 1.86 - 1.76 (m, 2H, CH ).
8
2
2
2
+
ESI-MS: m/z 337.2 [M+H] .
4.1.2.12 6-bromo-N-(3-morpholinopropyl)quinazolin-4-amine (5d)
1
9
1.7% yield. mp 127-129 ºC. H NMR (600 MHz, DMSO) δ 8.55 (d, J = 2.0 Hz, 1H, Ar-H),
8.49 (s, 1H, Ar-H), 8.38 (t, J = 4.7 Hz, 1H, NH), 7.89 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.62 (d, J = 8.8
Hz, 1H, Ar-H), 3.67 (dd, J = 12.5, 6.5 Hz, 2H, CH
2
), 3.62 - 3.54 (m, 4H, CH ), 2.60 (s, 2H, CH ),
2
2

+
2
2 2
.47 (s, 4H, CH ), 1.92 (s, 2H, CH ). ESI-MS: m/z 351.1 [M+H] .
4.1.2.13 6-bromo-N-(pyridin-4-ylmethyl)quinazolin-4-amine (5e)
1
8
9.1% yield. mp 175-177 ºC. H NMR (600 MHz, DMSO) δ 9.03 (t, J = 5.6 Hz, 1H, NH), 8.53
(
d, J = 1.9 Hz, 1H, Ar-H), 8.50 (d, J = 4.8 Hz, 2H, Ar-H), 8.42 (s, 1H, Ar-H), 7.89 (dd, J = 8.9, 2.0
Hz, 1H, Ar-H), 7.62 (d, J = 8.9 Hz, 1H, Ar-H), 7.35 (d, J = 5.3 Hz, 2H, Ar-H), 4.80 (d, J = 5.7 Hz,
+
2
H, CH
2
). ESI-MS: m/z 315.1 [M+H] .
4.1.2.14
N-(5-(4-((2-(dimethylamino)ethyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamid
e (6a)
·
. DMF was removed under reduced pressure and the residue was purified through a column
chromatography on silica with chloroform/methanol (V:V 20:1) as a white solid (0.15 g, 58.3%
1
yield). mp 256-258 ºC. H NMR (600 MHz, DMSO) δ 9.26 (s, 1H, NH), 8.92 (s, 1H, Ar-H), 8.61 (s,
1
H, Ar-H), 8.51 (s, 1H, Ar-H), 8.26 (s, 1H, NH), 8.08 (s, 2H, Ar-H), 7.79 (s, 1H, Ar-H), 3.99 (s, 3H,
13
OCH
3
), 3.92 (brs, 2H, CH
2
), 3.31 (brs, 2H, CH
2
), 3.17 (s, 3H, CH
3
), 2.76 (s, 6H, CH
3
). C NMR
(
150 MHz, DMSO) δ 164.0, 159.4, 156.5, 154.9, 148.3, 141.2, 133.7, 131.2, 130.9, 128.7, 128.0,
+
1
21.2, 120.7, 115.3, 55.6, 53.7, 42.6, 41.0, 36.0. HRMS: m/z 417.1716 [M+H] .
Compounds 6b-l was synthesized according to the procedure described in 6a.
.1.2.15
4
N-(5-(4-((2-(dimethylamino)ethyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)butane-1-sulfonami
de (6b)
1
6
7.4% yield. mp 108-110 ºC. H NMR (600 MHz, DMSO) δ 8.57 (d, J = 1.5 Hz, 1H, Ar-H),
8.50 (d, J = 2.3 Hz, 2H, Ar-H), 8.48 (s, 1H, NH), 8.07 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H), 8.05 (d, J =

2
.3 Hz, 1H, Ar-H), 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 3.99 (s, 3H, OCH
.18 - 3.10 (m, 2H, CH ), 2.73 (brs, 2H, CH ), 2.36 (brs, 6H, CH ), 1.74 (dt, J = 15.3, 7.7 Hz, 2H,
), 1.40 (dq, J = 14.7, 7.4 Hz, 2H, CH ), 0.89 (t, J = 7.4 Hz, 3H, CH
3 2
), 3.78 - 3.70 (m, 2H, CH ),
3
2
2
3
13
CH
2
2
3
). C NMR (150 MHz,
DMSO) δ162.2, 159.4, 156.6, 155.1, 148.4, 141.3, 133.6, 132.2, 130.8, 128.9, 128.2, 121.1, 120.0,
+
1
15.2, 57.2, 53.7, 52.0, 44.7, 25.1, 20.7, 13.4. HRMS: m/z 459.2147 [M+H] .
4.1.2.16
N-(5-(4-((2-(dimethylamino)ethyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)-4-fluorobenzenesul
fonamide (6c)
1
5
6.9% yield. mp 215-218 ºC. H NMR (600 MHz, DMSO) δ 10.05 (s, 1H, NH), 8.53 (d, J = 1.6
Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.43 (t, J = 5.2 Hz, 1H, NH), 8.40 (d, J = 2.3 Hz, 1H, Ar-H), 8.04
d, J = 2.4 Hz, 1H, Ar-H), 8.02 (dd, J = 8.6, 1.8 Hz, 1H, Ar-H), 7.83 - 7.78 (m, 2H, Ar-H), 7.76 (d, J
8.6 Hz, 1H, Ar-H), 7.40 (t, J = 8.8 Hz, 2H, Ar-H), 3.71 (dd, J = 12.2, 6.4 Hz, 2H, CH ), 3.64 (s, 3H,
). C NMR (150 MHz, DMSO) δ 164.1 (d, J
249.3 Hz), 159.4, 156.9, 155.1, 148.4, 141.4, 137.2, 133.6, 132.2, 130.6, 129.58 (d, J = 9.5 Hz),
(
=
2
1
3
3 2 3
OCH ), 2.66 (t, J = 6.7 Hz, 2H, CH ), 2.31 (s, 6H, CH
=
1
4
4
28.7, 128.2, 121.4, 120.0, 115.9 (d, J = 22.5 Hz), 115.2, 57.4, 53.2, 45.0, 38.2. HRMS: m/z
+
97.1738 [M+H] .
.1.2.17
N-(5-(4-((2-(dimethylamino)ethyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenzen
esulfonamide (6d)
1
6
4.3% yield. mp 197-199 ºC. H NMR (600 MHz, DMSO) δ 8.57 - 8.43 (m, 3H, Ar-H and NH),
8
.35 (d, J = 1.7 Hz, 1H, Ar-H), 7.99 (t, J = 16.3 Hz, 2H, Ar-H), 7.77 (dd, J = 15.1, 8.4 Hz, 2H, Ar-H),
.51 (dd, J = 13.8, 5.2 Hz, 1H, Ar-H), 7.18 (dd, J = 11.7, 4.9 Hz, 1H, Ar-H), 3.76 (d, J = 5.0 Hz, 2H,
7

1
3
CH
2 3 2 3
), 3.66 (s, 3H, CH ), 2.81 (t, J = 6.1 Hz, 2H, CH ), 2.42 (s, 6H, CH ). C NMR (150 MHz,
DMSO) δ 164.5 (dd, J = 11.4, 251.1 Hz), 162.2, 159.4, 159.3 (dd, J = 13.2, 255.2 Hz), 157.6, 155.0,
148.3, 140.4, 133.8, 132.5, 131.6 (d, J = 10.4 Hz), 130.6, 128.7, 128.2, 126.4 (dd, J = 3.6, 14.9 Hz),
122.5, 119.8, 115.2, 111.3 (dd, J = 2.7, 21.5 Hz), 105.5 (t, J = 26.0 Hz), 57.1, 53.1, 44.5, 37.8.
+
HRMS: m/z 515.1632 [M+H] .
4.1.2.18
N-(5-(4-((3-(dimethylamino)propyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)butane-1-sulfona
mide (6e)
1
5
8.6% yield. mp 45-47 ºC. H NMR (600 MHz, DMSO) δ 8.66 (brs, 1H, NH), 8.59 (s, 1H,
Ar-H), 8.51 (d, J = 2.1 Hz, 1H, Ar-H), 8.49 (s, 1H, Ar-H), 8.08 (dd, J = 8.6, 1.3 Hz, 1H, Ar-H), 8.06
d, J = 2.1 Hz, 1H, Ar-H), 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 3.99 (s, 3H, OCH ), 3.63 (dd, J = 11.8, 6.2
Hz, 2H, CH ), 3.19 - 3.11 (m, 2H, CH ), 2.92 - 2.88 (m, 2H, CH ), 2.58 (s, 6H, CH ), 2.02 - 1.94 (m,
H, CH ), 1.74 (dt, J = 15.3, 7.7 Hz, 2H, CH ), 1.46 - 1.34 (m, 2H, CH ), 0.89 (t, J = 7.4 Hz, 3H,
). C NMR (150 MHz, DMSO) δ 162.2, 159.4, 156.6, 155.1, 148.4, 141.3, 132.1, 130.8, 128.9,
(
3
2
2
2
3
2
2
2
2
1
3
CH
3
128.2, 121.1, 120.1, 115.2, 55.3, 53.7, 52.0, 43.1, 37.9, 25.1, 21.0, 20.7, 13.5. HRMS: m/z 473.2307
+
[
M+H] .
4.1.2.19
N-(5-(4-((3-(dimethylamino)propyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)-2,4-difluorobenz
enesulfonamide (6f)
1
5
6.9% yield. mp 48-50 ºC. H NMR (600 MHz, DMSO) δ 8.61 (s, 1H, NH), 8.54 (d, J = 1.8 Hz,
1
2
H, Ar-H), 8.48 (d, J = 4.6 Hz, 1H, Ar-H), 8.38 (d, J = 2.3 Hz, 1H, Ar-H), 8.02 (dd, J = 8.0, 2.1 Hz,
H, Ar-H), 7.80 - 7.73 (m, 2H, Ar-H), 7.55 - 7.49 (m, 1H, Ar-H), 7.18 (td, J = 8.5, 2.4 Hz, 1H, Ar-H),

3
.66 (s, 3H, OCH
CH ), 2.02 - 1.95 (m, 2H, CH
59.4, 159.3 (dd, J = 13.2, 255.3 Hz), 157.6, 155.0, 148.3, 140.7, 133.8, 132.8 (d, J = 1.7 Hz), 131.6
3
), 3.63 (dd, J = 12.0, 6.6 Hz, 2H, CH
2
), 2.92 - 2.85 (m, 2H, CH
2
), 2.57 (s, 6H,
1
3
3
2
). C NMR (150 MHz, DMSO) δ 164.5 (dd, J = 11.7, 251.4 Hz),
1
(
d, J = 10.8 Hz), 130.6, 128.7, 128.2, 126.2 (dd, J = 1.7, 14.0 Hz), 122.2, 120.0, 111.4 (dd, J = 3.2,
+
2
1.8 Hz), 105.5 (t, J = 26.0 Hz), 55.3, 53.1, 43.1, 38.0, 24.7. HRMS: m/z 529.1734 [M+H] .
4.1.2.20
N-(2-methoxy-5-(4-((2-morpholinoethyl)amino)quinazolin-6-yl)pyridin-3-yl)butane-1-sulfonamide
6g)
(
1
5
8.1% yield. mp 113-116 ºC. H NMR (600 MHz, DMSO) δ 9.46 (s, 1H, NH), 8.54 (d, J = 1.2
Hz, 1H, Ar-H), 8.49 (d, J = 2.2 Hz, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.44 (t, J = 5.1 Hz, 1H, NH), 8.07
dd, J = 9.4, 1.8 Hz, 2H, Ar-H), 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 4.00 (s, 3H, OCH ), 3.72 (dd, J =
2.4, 6.4 Hz, 2H, CH ), 3.66 - 3.52 (m, 4H, CH ), 3.19 - 3.09 (m, 2H, CH ), 2.64 (t, J = 6.8 Hz, 2H,
CH ), 2.50 (d, J = 9.7 Hz, 4H, CH ), 1.75 (dt, J = 15.3, 7.7 Hz, 2H, CH ), 1.46 - 1.35 (m, 2H, CH ),
). C NMR (150 MHz, DMSO) δ 159.4, 156.6, 155.2, 148.4, 141.3,
(
3
1
2
2
2
2
2
2
2
1
3
0
1
1
4
2
.89 (t, J = 7.4 Hz, 3H, CH
3
33.6, 132.2, 130.7, 128.9, 128.2, 121.1, 119.9, 115.1, 66.0, 56.8, 53.7, 53.3, 52.0, 39.7, 25.1, 20.7,
+
3.4. HRMS: m/z 501.2233 [M+H] .
.1.2.21
,4-difluoro-N-(2-methoxy-5-(4-((2-morpholinoethyl)amino)quinazolin-6-yl)pyridin-3-yl)benzenesulf
onamide (6h)
9.4% yield. mp 234-236 ºC. H NMR (600 MHz, DMSO) δ 10.40 (s, 1H, NH), 8.55 (d, J = 1.6
1
5
Hz, 1H, Ar-H), 8.49 (d, J = 2.4 Hz, 2H, Ar-H), 8.44 (t, J = 5.2 Hz, 1H, NH), 8.21 - 8.02 (m, 2H,
Ar-H), 7.75 (dd, J = 13.0, 8.6 Hz, 2H, Ar-H), 7.68 - 7.54 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.2 Hz, 1H,

Ar-H), 3.73 (dd, J = 12.5, 6.5 Hz, 2H, CH
t, J = 6.9 Hz, 2H, CH ), 2.52 - 2.51 (m, 4H, CH
1.6, 252.3 Hz), 159.4, 159.3 (dd, J = 13.4, 256.1 Hz), 157.8, 155.2, 148.4, 142.5, 134.7, 133.3,
31.7 (d, J = 10.7 Hz), 130.5, 128.8, 128.2, 125.2 (dd, J = 3.5, 14.6 Hz), 120.0, 119.8, 115.2, 111.6
2
), 3.64 (s, 3H, OCH
3
), 3.60 (t, J = 4.4 Hz, 4H, CH
2
), 2.64
1
3
(
2
2
). C NMR (150 MHz, DMSO) δ 164.9 (dd, J =
1
1
(
[
dd, J = 3.2, 22.1 Hz), 105.6 (t, J = 26.0 Hz), 66.0, 56.9, 53.3, 48.5, 37.7. HRMS: m/z 557.1753
+
M+H] .
4
.1.2.22
N-(2-methoxy-5-(4-((3-morpholinopropyl)amino)quinazolin-6-yl)pyridin-3-yl)butane-1-sulfonamide
6i)
(
1
6
7.5% yield. mp 140-142 ºC. H NMR (600 MHz, DMSO) δ 9.47 (s, 1H, NH), 8.56 (s, 1H,
Ar-H), 8.49 (d, J = 2.2 Hz, 1H, Ar-H), 8.47 (d, J = 4.4 Hz, 1H, Ar-H), 8.07 (d, J = 1.7 Hz, 1H, Ar-H),
8
.05 (d, J = 1.9 Hz, 1H, Ar-H), 7.76 (d, J = 8.6 Hz, 1H, Ar-H), 3.99 (s, 3H, OCH
H, CH ), 3.16 - 3.09 (m, 2H, CH ), 2.41 (d, J = 7.3 Hz, 6H, CH ), 1.85 (dt, J = 14.3, 7.1 Hz, 2H,
), 1.79 - 1.69 (m, 2H, CH ), 1.40 (dq, J = 14.8, 7.4 Hz, 2H, CH ), 0.89 (t, J = 7.4 Hz, 3H, CH ).
3
), 3.67 - 3.55 (m,
6
2
2
2
CH
2
2
2
3
1
3
C NMR (150 MHz, DMSO) δ 159.3, 156.6, 155.2, 148.4, 141.3, 133.6, 132.2, 130.7, 128.9, 128.2,
121.2, 120.0, 115.2, 66.0, 55.9, 53.7, 53.2, 52.0, 25.3, 25.1, 21.0, 20.7, 13.4. HRMS: m/z 515.2407
+
[
M+H] .
4
2
.1.2.23
,4-difluoro-N-(2-methoxy-5-(4-((3-morpholinopropyl)amino)quinazolin-6-yl)pyridin-3-yl)benzenesu
lfonamide (6j)
1.9% yield. mp 178-180 ºC. H NMR (600 MHz, DMSO) δ 10.55 (s, 1H, NH), 8.56 (d, J = 1.5
Hz, 1H, Ar-H), 8.50 - 8.47 (m, 2H, Ar-H and NH), 8.46 (d, J = 2.2 Hz, 1H, Ar-H), 8.07 (d, J = 2.2
1
6

Hz, 1H, Ar-H), 8.04 (dd, J = 8.6, 1.7 Hz, 1H, Ar-H), 7.80 - 7.71 (m, 2H, Ar-H), 7.59 - 7.52 (m, 1H,
Ar-H), 7.19 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 3.64 (s, 3H, OCH ), 3.62 - 3.56 (m, 6H, CH ), 2.43 (m,
). C NMR (150 MHz, DMSO) δ164.8 (dd, J = 11.3, 252.0 Hz),
59.3, 159.2 (dd, J = 13.5, 255.9 Hz), 157.7, 155.2, 148.4, 141.9, 134.1, 133.3, 131.7 (d, J = 10.8
3
2
1
3
6
1
2 2
H, CH ), 1.90 - 1.82 (m, 2H, CH
Hz), 130.5, 128.8, 128.2, 125.5 (dd, J = 1.4, 13.7 Hz), 120.6, 119.9, 111.5 (dd, J = 3.2, 22.1 Hz),
+
1
05.6 (t, J = 26.0 Hz), 66.0, 55.8, 53.2, 53.1, 38.8, 25.2. HRMS: m/z 571.1905 [M+H] .
4.1.2.24
N-(2-methoxy-5-(4-((pyridin-4-ylmethyl)amino)quinazolin-6-yl)pyridin-3-yl)butane-1-sulfonamide
6k)
(
1
6
6.7% yield. mp 57-59 ºC. H NMR (600 MHz, DMSO) δ 9.47 (s, 1H, NH), 9.09 (t, J = 5.7 Hz,
H), 8.66 (s, 1H, Ar-H), 8.51 (d, J = 6.4 Hz, 3H, Ar-H), 8.46 (s, 1H, NH), 8.13 (d, J = 8.7 Hz, 1H,
Ar-H), 8.09 (d, J = 1.8 Hz, 1H, Ar-H), 7.81 (d, J = 8.6 Hz, 1H, Ar-H), 7.38 (d, J = 5.2 Hz, 2H, Ar-H),
.85 (d, J = 5.6 Hz, 2H, CH ), 3.99 (s, 3H, CH ), 3.14 (t, J = 7.8 Hz, 2H, CH ), 1.77 - 1.72 (m, 2H,
CH ), 1.43 - 1.37 (m, 2H, CH ), 0.89 (t, J = 7.4 Hz, 3H, CH ). C NMR (150 MHz, DMSO) δ 159.4,
1
4
2
3
2
13
2
2
3
1
1
4
2
56.7, 155.0, 149.5, 148.4, 148.3, 141.4, 133.9, 132.3, 131.0, 128.8, 128.3, 122.1, 121.1, 120.0,
+
15.0, 53.7, 52.0, 42.6, 25.1, 20.7, 13.4. HRMS: m/z 479.1847 [M+H] .
.1.2.25
,4-difluoro-N-(2-methoxy-5-(4-((pyridin-4-ylmethyl)amino)quinazolin-6-yl)pyridin-3-yl)benzenesulf
onamide (6l)
2.4% yield. mp 121-123 ºC. H NMR (600 MHz, DMSO) δ 10.41 (s, 1H, NH), 9.10 (t, J = 5.8
1
6
Hz, 1H, NH), 8.68 (d, J = 1.7 Hz, 1H, Ar-H), 8.54 (d, J = 2.3 Hz, 1H, Ar-H), 8.52 (d, J = 5.5 Hz, 2H,
Ar-H), 8.47 (s, 1H, Ar-H), 8.14 (dd, J = 10.6, 2.1 Hz, 2H, Ar-H), 7.81 (d, J = 8.7 Hz, 1H, Ar-H), 7.76

(
td, J = 8.5, 6.6 Hz, 1H, Ar-H), 7.64 - 7.56 (m, 1H, Ar-H), 7.39 (d, J = 5.8 Hz, 2H, Ar-H), 7.21 (td, J
1
3
=
2 3
8.5, 2.3 Hz, 1H, Ar-H), 4.87 (d, J = 5.6 Hz, 2H, CH ), 3.64 (s, 3H, OCH ). C NMR (150 MHz,
DMSO) δ 164.96 (dd, J = 11.7, 253.9Hz), 159.5, 159.4 (dd, J = 13.4, 256.1Hz), 157.9, 155.0, 149.5,
48.4 (d, J = 18.7 Hz), 142.8, 135.1, 133.1, 131.7 (d, J = 10.8 Hz), 130.8, 128.7, 128.3, 125.1 (dd, J
1
=
3.5, 14. 5 Hz), 122.1, 120.0, 119.5, 115.1, 111.6 (dd, J = 3.1, 22.1 Hz), 105.7 (t, J = 25.8 Hz),
+
5
4
4
3.27, 42.65. HRMS: m/z 535.1348 [M+H] .
.2 Biological (pharmacological) evaluation
.2.1 Cell culture
HCT-116, A549, SK-HEP-1, SNU638, MDA-MB-231, MCF7, HEK293 and MRC-5 were
purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in
DMEM (SNU-638, MCF7, HEK293, MRC-5 and SK-HEP-1) or RPMI1640 (HCT-116, A549 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% CO
.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 °C in a humidified atmosphere with 5% CO for 72
2
at 37 °C.
4
2
h. After treatment, the cells were fixed with 10% TCA solution, and cell viability was determined
with the 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).

%
growth inhibition = 100 - 100×(ODsample - ODDay0) / (ODNeg control - ODDay0)
4
.2.3 PI3K enzymatic activity assay
The PI3K kinase assay was measured by PI3 Kinase Activity/Inhibitor Assay Kit (EMD
Millipore) following the manufacturer’s protocols.
4.2.4 Molecular docking studies
The crystal structure of PI3Kα (PDB entry code: 4ZOP/ 5ITD) in complex with 4Q2 was used
for molecular modeling. The AutoDock 4.2 was used to perform docking calculations. Polar
hydrogens and partial charges were added for PI3Kα protein using the Kollman United Atom charges
with Sybyl 6.9.1, and energy minimization was made employing both steepest descent and conjugate
gradients protocols. Atomic solvation parameters and fragmental volumes for the proteins were
assigned using the addsol utility in the AutoDock 4.2 program. A 60 × 60 × 60 Å grid box with a grid
spacing of 0.375 Å was generated for the receptor. Affinity grid fields were generated using the
auxiliary program AutoGrid 4.0. The Lamarckian genetic algorithm (LGA) was used to find the
appropriate binding positions, orientations, and conformations of the ligands. The optimized
AutoDocking parameters are as follows: the maximum number of energy evaluations was increased
to 25,000,000 per run; the iterations of Solis & Wets local search was 3000; the number of
individuals in population was 300 and the number of generations was 100. Results differing by less
than 2 Å in a positional root mean square deviation (RMSD) were clustered together. In each group,
the lowest binding energy configuration with the highest % frequency was selected as the group
representative. All other parameters were maintained as default. Accelrys Discovery Studio
Visualizer 4.0 was used for graphic display.
4.2.5 Cell cycle analysis

3
9
Cells cycle analysis was carried out as described previously . Cells were plated in
100mm-diameter culture dishes and cultured overnight. Then cells were treated with indicated
concentration of 6b for 24 h. The floating and adherent cells were collected and fixed in cold 70%
ethanol at -20 °C overnight. After washing with PBS for 3 times, cells were treated with 100 µg/mL
RNase A at room temperature for 30 min and then stained with 50 µg/mL propidium iodide (PI) for
another 30 min in the dark before subjected to flow cytometric analysis.
4.2.6 Annexin V assay
Cell apoptosis was evaluated using Annexin V FITC/PI apoptosis detection kit (BD Biosciences,
CA, USA) according to the manufacture’s instruction.
.2.7 Western blot analysis
Cells were treated with various concentration of 6b. Western blot analysis was carried out as
4
3
5
described previously . Blots were imaged by Image Quant LAS 4000 (GE Healthcare Life
Sciences, USA). Antibodies for PI3 Kinase p110α, Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199),
Phospho-Akt (Ser473), Phospho-GSK-3 alpha/beta (Ser21/9), GSK-3β, Cyclin D1, Cleaved
Caspase-3 (Asp175) and Cleaved Caspase-9 were purchased from Cell Signaling Technology
(
Danvers, MA, USA). Antibodies for CDK2, p27, Bax, Cytochrome c and Bcl-2 were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
4.2.8 Immunofluorescence microscopy
Cells were cultured on a confocal dish coated with 0.2% gelatin. After 6b treatment (0.5µM) for
h, the cells were fixed with 4% paraformaldehyde for 20 min and rinsed with washing buffer 3
8
times before blocking with 1% BSA. The cells were incubated with the p-Akt (Ser473) antibody at

4
°C overnight and then incubated with the second antibody before DAPI staining. The cells were
visualized under a fluorescence reverse microscope (Leica TCS SP8, Germany). The images were
recorded with a Leica TCS SP8 confocal microscope using a 40x silicone oil objective (40x/1.30
NA).
Conflicts of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
Financial supported by Liaoning Provincial Department of Education (Grant No. L2015514)
and Career Development Support Plan for Young and Middle-aged Teachers in Shenyang
Pharmaceutical University (Grant No. ZQN2015023).
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