Design, synthesis, and biological evaluation of some novel 4-aminoquinazolines as Pan-PI3K inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.04.024

A series of 4-aminoquinazolines derivatives containing hydrophilic group were designed and identified as potent Pan-PI3K inhibitors in this study. The results of antiproliferative assays in vitro showed that this series of compounds had strong inhibition of tumor growth, especially compound 7b for MCF-7 cells but weak inhibition to normal cells. PI3K kinase assay showed that 7b had high activity for three PI3K isoforms with the IC₅₀ values of picomole. The western blot assay indicated that 7b could decrease the phospho-Akt (S473) in a dose-dependent manner. Further experiments showed that 7b could induce apoptosis in MCF-7 cells. Four key hydrogen bonding interactions were found in the docking of 7b with PI3K kinase. All these results suggested that 7b is a potent PI3K inhibitor and could be considered as a potential candidate for the development of anticancer agents.

Full text of DOI:10.1016/j.bmc.2019.04.024

Accepted Manuscript
Design, synthesis, and biological evaluation of some novel 4-aminoquinazolines
as Pan-PI3K inhibitors
Huai-Wei Ding, Shu Wang, Xiao-Chun Qin, Jian Wang, Hong-Rui Song, Qing-
Chun Zhao, Shao-Jiang Song
PII:
S0968-0896(18)31732-2
https://doi.org/10.1016/j.bmc.2019.04.024
BMC 14878
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 October 2018
9 April 2019
16 April 2019
Please cite this article as: Ding, H-W., Wang, S., Qin, X-C., Wang, J., Song, H-R., Zhao, Q-C., Song, S-J., Design,
synthesis, and biological evaluation of some novel 4-aminoquinazolines as Pan-PI3K inhibitors, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.024
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Design, synthesis, and biological evaluation of some novel 4-aminoquinazolines as Pan-
PI3K inhibitors
Huai-Wei Ding a, Shu Wang b, Xiao-Chun Qin b, Jian Wang a, Hong-Rui Song a, Qing-Chun Zhao b,*, Shao-
Jiang Song b,*
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
* Corresponding authors.
Email address: songsj99@163.com; zhaoqingchun1967@163.com.

Abstract：A series of 4-aminoquinazolines derivatives containing hydrophilic group were designed and
identified as potent Pan-PI3K inhibitors in this study. The results of antiproliferative assays in vitro showed
that this series of compounds had strong inhibition of tumor growth, especially compound 7b for MCF-7
cells but weak inhibition to normal cells. PI3K kinase assay showed that 7b had high activity for three PI3K
isoforms with the IC50 values of picomole. The western blot assay indicated that 7b could decrease the
phospho-Akt (S473) in a dose-dependent manner. Further experiments showed that 7b could induce
apoptosis in MCF-7 cells. Four key hydrogen bonding interactions were found in the docking of 7b with
PI3K kinase. All these results suggested that 7b is a potent PI3K inhibitor and could be considered as a
potential candidate for the development of anticancer agents.
Keywords: PI3K; 4-aminoquinazolines; anticancer
Abbreviations used
PI3K, phosphatidylinositol 3-kinases; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-
phosphate dehydrogenase; NMR, nuclear magnetic resonance; ESI-MS, electrospray ionization - mass
spectrum; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; Pd(dppf)2Cl2, [1,1'-
bis(diphenylphosphino)ferrocene]dichloropalladium(II); DMEM, dulbecco's modified eagle medium;
RPMI1640, Roswell Park Memorial Institute-1640; FBS, fatal bovine serun; ADP, adenosine diphosphate;
HEPES, N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid; ATP, adenosine triphosphate; EDTA,
ethylenediaminetetraacetic acid; RIPA, radio immunoprecipitation assay.

1. Introduction
The PI3K pathway regulates various cellular processes, such as cell growth, cell cycle, apoptosis and
survival 1. Based on the primary structure, regulation and lipid substrate specificity, PI3K families are
divided into four different classes: Class I, Class II, Class III and Class IV. The class I enzymes are further
divided into four subtypes: PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ 2. For the important role of PI3K played in
human tumors, lots of inhibitors targeting PI3K have been developed and are being evaluated in clinical
trials 3-4. Up to now, two PI3K inhibitors both of which have a nanomolar or picomolar activity have been
approved by FDA. Idelalisib as PI3Kδ inhibitor was approved for three types of blood cancers on July 23,
2014 5 and Copanlisib as Pan PI3K I type inhibitor was approved for the treatment of adult patients
experiencing relapsed follicular lymphoma on September 14, 2017 6-7. In addition, among PI3K inhibitors in
clinical trials, Omipalisib (GSK2126458) also exhibited potent and highly PI3K inhibitory activity with a
Ki50 of picomolar level 8.
In the development of anticancer agents, quinazoline moiety has been extensively used as a drug-like
9
scaffold in medicinal chemistry, especially in the development of anticancer agents . Lots of quinazoline
derivatives have been successfully developed, such as Gefitinib, Erlotinib, Lapatinib and many more are
potent and highly selective inhibitors of tyrosine kinase. Recently, 4-aminoquinazoline derivatives also have
been reported as PI3Kα and PI3Kδ inhibitors 10,12-15. Therefore, the aminoquinazoline derivatives still have a
very wide range of development prospects for anticancer agents.
Based on the reported corresponding X-ray structure of kinase domain and the literature on the analysis
of protein structure 8,16-17, we had a good understanding of the binding mode of Omipalisib. Omipalisib uses
the position-1 nitrogen as a hinge binder and has an aryl group at the position-4 at “Ribose pocket” and a
substituted pyridyl group at the position-6 at “Affinity pocket”, but there is no group in the “Phosphate
binding pocket” and there is no H-bond interaction with PI3K (Fig.1.A). These discoveries further
encourage the development of our work to design novel compounds by adjusting the substituent groups of
molecules.Based on the above points, we optimized a new series of 4-aminoquinazolines derivatives base on
our preliminary compound DHW-6i and Omipalisib (Fig.1.B). The biological activities evaluations suggest
that compound 7b could be as Pan-PI3K inhibitor.

Fig.1. The design strategy based on DHW-6i and Omipalisib.
2. Results and discussion
2.1. Chemistry
The synthetic routes for the target compounds are outlined in Schemes 1. 1a reacted with four
chlorinated organic compounds to afford 2a-d. Then 2a-d were reduced to amine which directly reacted
with 6-bromo-4-chloroquinazoline as soon as possible to get 3a-d because the amine are easily oxidized. 1b-
d as the starting material, 3e-j were synthesized using a similar method. Sulfonylation of 4 with three
benzenesulfonyl chlorides to yield 5a-c, which were then subjected to Suzuki coupling with
bis(pinacolato)diborane to afford arylboronic ester 6a-c which were coupled with 3a-j via Suzuki reaction to
afford the target compounds 7a-q.

Scheme 1. (a) chlorinated compounds, K2CO3, DMF, 60 ºC, 12 h; (b) i) ethanol, H2, Pd/C, 60 ºC, 12 h; ii) 6-bromo-4-
chloroquinazoline, isopropanol, 90 ºC, 4h; (c) i) chlorinated compounds or alcohols, K2CO3, DMF, 60 ºC, 12 h; (d) i)
ethanol, H2, Pd/C, 60 ºC, 12 h; ii) 6-bromo-4-chloroquinazoline, isopropanol, 90 ºC, 4h; (e) benzenesulfonyl chloride,
pyridine, rt, 24 h; (f) bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF, 100 ºC, 8 h; (g) Pd(dppf)2Cl2, Cs2CO3, DMF/
H2O, 90 ºC, 8 h.
2.2. Antiproliferative assays in vitro
It is reported that many kinds of tumor cells express PI3K kinase to different extent 18-21. So, all
synthesized compounds were evaluated for their cytotoxicities in vitro against three typically human cancer
cell lines including A549, HCT116 and MCF-7 cells. The results were summarized in Table 1.
As shown in Table 1, the results showed that most of the derivatives exhibited strong antiproliferative
effects, especially against MCF-7. As the representative, the structure-activity relationships (SARs) was
discussed with MCF-7 cell line. From the overall results, it showed that the activity of pyrazole group at

position-4 was better than that of phenyl and pyridine groups, this conclusion was manifested in the activity
of compounds 7a-7h (against MCF-7) reaching nanomolar level while the activity of compounds 7i-7q
(against MCF-7) being all more than 1 micromolar. Compound 7b (IC50 = 0.22 μM against MCF-7) was
more potent than compound 7d (IC50 = 1.31 μM against MCF-7), suggesting that the antiproliferation
activity could be decreased by the increased length of alkyl group at R1 and R2 position when R1 and R2
position contained the alcohol hydroxyl group, compound 7m (IC50 = 4.30 μM against MCF-7) and 7n (IC50
= 6.12 μM against MCF-7) also proved this conclusion. On the contrary, if R1 and R2 position contained
carbonyl groups whether the group is carboxyl or ester, the antiproliferation activity could be enhanced by
the increased length of alkyl group at R1 and R2 position, both compound 7e (IC50 = 0.67 μM against MCF-7)
vs. 7f (IC50 = 0.49 μM against MCF-7) and 7g (IC50 = 0.70 μM against MCF-7) vs. 7h (IC50 = 0.56 μM
against MCF-7) supported this conclusion. Moreover, compound 7o (IC50 = 2.26 μM against MCF-7), 7p
(IC50 = 3.74 μM against MCF-7) and 7q (IC50 = 4.82 μM against MCF-7) vs. 7j (IC50 = 1.65 μM against
MCF-7) suggesting that either introducing electron withdrawing group or electron donating group all could
lead to the decrease of activity. Among all the compounds, 7b and 7d not only showed potent inhibitory
potencies but also displayed favorable values of molecular parameters (ClogP, Table 1).
Not only that, we selected the representative compound 7b which with the best inhibitory activity to
investigate their cytotoxicity to normal cells and 7b displayed much lower cytotoxicity in HUVEC cells and
HL7702 cells. 7b showed an IC50 value of 19.6 μΜ and 60.0 μΜ against HUVEC and HL7702 respectively
(Table 2), which is at least 89 times and 272 times higher than that of MCF-7 respectively, implying the
possibility of 7b with minor effects on normal cells.
Table 1 Cytotoxicity in vitro of target compounds against three cancer cell lines (IC50 Valuesa in μM).
R4
S
R2
O
R4
S
R1
N
N
O
O
O
O
O
O
X
NH
NH
HN
N
HN
N
R3
N
N
N
N
7i-q
7a-h
Cells
A549
HCT116
MCF-7
cLogp
Comp.
R1
R2
R3
-
R4
X
-
OH
OH
-
-
8.22±0.86
3.10±0.02
1.49±0.09
0.33±0.02
0.77±0.06
0.22±0.01
2.00
4.02
7a
7b
F
F
-
-
F
F
OH
OH
-
-
-
-
-
-
3.60±0.03
4.83±0.03
1.42±0.12
0.48±0.03
0.73±0.04
1.31±0.04
2.26
4.28
7c
7d

F
F
F
F
O
-
-
-
-
-
-
-
-
-
-
-
-
7e
7f
4.28±0.04
2.99±0.02
5.20±0.22
3.84±0.12
1.26±0.08
0.97±0.04
1.54±0.14
1.29±0.12
0.67±0.03
0.49±0.02
0.70±0.04
0.56±0.02
4.68
4.92
4.32
4.46
F
F
F
F
O
O
O
O
7g
7h
OH
OH
O
OH
OH
7i
7j
-
-
H
H
CH
CH
5.20±0.21
2.69±0.10
4.86±0.24
1.06±0.10
3.27±0.12
1.65±0.09
3.59
5.61
F
F
OH
OH
OH
7k
7l
-
-
-
-
-
-
N
N
N
5.08±0.23
13.29±0.83
4.12±0.22
5.36±0.18
>20
3.08±0.11
>20
3.06
4.65
5.08
F
F
F
F
7m
2.34±0.22
4.30±0.20
F
F
F
F
OH
7n
7o
7p
-
-
-
-
N
9.36±0.24
3.76±0.13
5.98±0.23
3.68±0.26
5.72±0.32
7.32±0.33
6.12±0.25
2.26±0.10
3.74±0.21
5.47
5.23
5.27
OH
OH
OCH3
Cl
CH
CH
F
OH
7q
-
F
CH
6.64±0.52
0.86±0.08
9.16±0.64
4.82±0.26
4.87
4.07
F
0.14±0.08 0.03±0.002
Omipal
isib
a IC50 values are the mean of triplicate measurements.
b Calculated by ChemBioDraw Ultra 12.0.
Table 2 Cytotoxicity assay against HUVEC and HL7702 normal cells (IC50 Valuesa in μM).
Compound
7b
HUVEC
19.6±1.2
HL7702
60.0±5.3
a IC50 values are the mean of triplicate measurements.
2.3. PI3K and EGFR enzymatic activity assays
In order to elucidate the mechanism of antiproliferative activities of these active compounds, we
selected the most potent compound 7b for the further PI3K enzymatic activity assays. Omipalisib was
selected as the positive drugs. As shown in Table 3, kinase inhibition activity assay indicated that the
activity of 7b has reached the picomolar level. 7b showed a higher inhibitory activity against PI3Kα when
compared to that of other class I PI3Ks (PI3Kβ, PI3Kδ and PI3Kγ) and the PI3Kα, PI3Kγ, PI3Kδ inhibitory
activity of 7b was better to Omipalisib.
Because of the lead compound DHW-6i was an EGFR-PI3K dual target inhibitor, the EGFR activity of

compound 7b was also tested (Table 3) but the activity (>10000 nM) was lost unexpectedly. Analyzing the
reason, we suspected that the hydrophobicity of 2,4-difluorophenyl substituent of 7b was too strong
compared to the methyl for the reason of that it needs to be in “Ribose pocket”. The “Ribose pocket” should
have hydrophilic groups to benefit for EGFR activity. Not only that, it is beneficial to EGFR activity that the
amine group at position-4 of quinazoline is hydrophobic group 16. The EGFR activity of 7a (2520 nM)
confirmed this point when the 2,4-difluorophenyl of 7b changed into methyl. It is indicated that this kind of
compounds are not EGFR inhibitors or the activities are very weak for EGFR.
Table 3 Activities of 7b and Omipalisib against Class I PI3K and EGFR (IC50 Values in nM)
PI3Kα
0.20
ND
PI3Kβ
2.99
ND
PI3Kγ
0.58
ND
PI3Kδ
0.48
ND
EGFR
>10000
2520
7b
7a
Omipalisib
0.34
1.64
1.19
0.70
ND
ND: Not determined
2.4. Western blot assay
To further determine whether the PI3K signaling were affected by compound 7b, Western blot assay
was used to evaluate the effects of 7b on PI3K related protein levels including Akt and phospho-Akt (p-Akt,
S473) in MCF-7 cells. As shown in Fig.2, compound 7b decreased the phosphorylation levels of Akt in a
dose-dependent manner and the ratio of p-Akt/Akt was down-regulated. Therefore, the data suggested that
7b might inhibit the proliferation of MCF-7 cells though PI3K/Akt signaling pathway.
Fig.2.Western blot analysis of 7b. The inhibition effects of compound 7b (0.1 μM, 1 μM and 10 μM) on the expression of
Akt, p-Akt in MCF-7 cells are depicted. GAPDH was used as internal control.
2.5. Compound 7b induced growth inhibition in MCF-7 cells
The colony formation assay was used to further investigate the antiproliferation of 7b on MCF-7 cells.
As shown in Fig.3, the cell proliferation was inhibited by 7b in a dose-dependent manner. Compared with
the control group, 7b could significantly inhibit colony formation at a concertration of 0.8 μM. The results
of antiproliferative assay and colony formation assay suggested that 7b can inhibit the proliferation of MCF-
7 cells prominently.

Fig.3. The colony formation assay of compound 7b. Treatment with compound 7b, representative photographs of colony
formation are shown (scale bar = 200μm).
2.6. Compound 7b induced apoptosis in MCF-7 cells
Though 7b was proved antitumor effects, the mechanism was not clear. Annexin V-FITC/PI double-
staining method was used to detect cells apoptosis of 7b. As shown in Fig.4 (A), 7b can induce MCF-7 cells
apoptosis compared with control. The highest apoptosis percentage was 27.5% when cells was treated with
7b at a concentration of 0.8 μM.
To confirm the effects of 7b on apoptosis induction in MCF-7 cells, cells were staining with Hoechst
33342 and analyzing by fluorescence microscope. As shown in Fig.4 (B), the brighter fluorescence was seen
after being treated with different concentrations of 7b. Compared with control which had normal nuclear
morphology after Hoechst 33342 staining, the 7b treatment group marked with nuclear fragmentation and
condensation of chromatin.These results indicated that 7b was capable of inducing apoptosis in MCF-7 cells.
Fig.4. 7b induced apoptosis in MCF-7 cells. (A) AV-PI staining show early and late apoptosis of MCF-7 cells induced by
compound 7b. (B) The nuclei morphology changes of MCF-7 cells treated with 7b visualized by Hoechst 33342 (scale bar =
5μm).

2.7. Molecular docking
Molecular docking study was performed to elucidate the binding model of 7b in the binding site of
PI3K. The structure of PI3K kinase was obtained from protein data bank (PDB entry 3L08). The Fig.5 (A)
showed that the ligand 7b formed four hydrogen bonds. 7b reproduce three hydrogen bonds similar to
Omipalisib and 3L08, the nitrogen of quinazolyl group formed a hydrogen bond with the side chain of
Val882 in the hinge binder region of PI3K, the oxygen of sulfonamide group interaction with Lys833 and
nitrogen of pyridyl group formed a hydrogen bond with the conserved water molecule. The fourth hydrogen
bond is the oxygen of hydroxyl group interaction with Thr886 and this hydrogen bond will facilitate the
more stable combination of 7b with PI3K kinase. Binding of compound 7b to PI3K’s excellent activity, we
hypothesize that the fourth hydrogen bond may be beneficial to the improvement of activity. As showed in
Fig.5 (B), the folded form of compound 7b in the protein pocket was very similar to that of Omipalisib.
The docking analysis indicated that compound 7b could well fit into the binding site of PI3K kinases,
which also indicated that this compound may be a potent PI3K inhibitor.
Fig.5. Docking mode of 7b with protein crystal structure of PI3K. (A) Key interactions of compound 7b in the active site of
PI3K (PDB: 3L08). (B) The binding pose of 7b and Omipalisib in the active site of PI3K. Omipalisib was highlighted with
yellow.
3. Discussion and conclusion
In this study, a series of novel 4-aminoquinazolines were designed and synthesized. Their
antiproliferative activities against three cancer cell lines showed that the MCF-7 cells presented more
sensitive to these compounds. Discovered by the structure-activity relationship of these compounds that it is
very advantageous for the improvement of activity by introducing a group capable of forming a hydrogen
bond in the "Phosphate binding pocket", particularly a hydrophilic group. Eventually, compound 7b was
selected for further research. Antiproliferative activities against normal cells showed that 7b selectively

inhibits the proliferation of cancer cells compared to normal cells (Table 1 and Table 2). PI3K kinase
inhibition activity assay indicated that the activity of 7b had reached the picomolar level (Table 3). Though
the inhibitory activities for PI3K enzymatic activity of 7b would be potent, the cytotoxic activities of 7b are
weak compared to Omipalisib. The cause of this phenomenon is unknown, and this may be where the future
optimization of compounds needs to be improved. The Western blot experiment showed that 7b inhibited
the proliferation of MCF-7 cell though PI3K/Akt signaling pathway (Fig.2). The results of colony formation
assay suggested that 7b can inhibit the proliferation of MCF-7 cells prominently (Fig.3). Annexin V-
FITC/PI double-staining and Hoechst 33342 staining indicated that 7b was capable of inducing apoptosis in
MCF-7 cells (Fig.4). The docking analysis predicted four hydrogen bonds with PI3K indicated that
compound 7b could well fit into the binding site of PI3K kinases (Fig.5).
In summary, we designed and synthesized a series of compounds containing hydrophilic group in
position-4 of quinazoline and evaluated their antiproliferative activities against three cancer cell lines,
including HCT-116, A549 and MCF-7. Compound 7b, with most potent antiproliferative activity was
selected for further biological evaluation. Kinase inhibition activity assay indicated that 7b had low namol
activity. Further study showed that the suppression of cell survives and growths induced by 7b via PI3K/Akt
pathway inhibition and 7b can inhibit the proliferation and inducing apoptosis in MCF-7 cells. Base on these
results, compound 7b could be as a potential PI3K inhibitor with high activity and could be considered as a
promising lead compound for further biological evaluation.
4. Experimental section
4.1. Chemistry and chemical methods
All reagents and solvents were commercially available without further purification. 1H 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). 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.).
4.1.1. 2-(4-nitro-1H-pyrazol-1-yl)ethanol (2a)
To a solution of 4-nitro-1H-pyrazole (1.13 g, 10 mmol) in DMF (20 ml) was added K2CO3
(1.38 g, 10 mmol) and 2-Bromoethanol (1.50 g, 12 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 compound 2a as a white solid. Yield 98.6%,
mp 132-134 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.82 (s, 1H, Ar-H), 8.27 (s, 1H, Ar-H), 4.99 (t, J

= 5.4 Hz, 1H, OH), 4.22 (t, J = 5.4 Hz, 2H, CH2), 3.77 (q, J = 5.4 Hz, 2H, CH2). ESI-MS: m/z 158.1
[M+H]+.
Compounds 2b-d was synthesized according to the procedure described in 2a.
4.1.2. 3-(4-nitro-1H-pyrazol-1-yl)propan-1-ol (2b)
87.6% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 8.88 (d, J = 1.6 Hz, 1H, Ar-H), 8.25 (d, J = 1.8 Hz,
1H, Ar-H), 4.65 (t, J = 5.0 Hz, 1H, OH), 4.25 (t, J = 7.1 Hz, 2H, CH2), 3.40 (dd, J = 11.3, 5.9 Hz, 2H, CH2),
2.02 – 1.88 (m, 2H, CH2). ESI-MS: m/z 172.1 [M+H]+.
4.1.3. methyl 2-(4-nitro-1H-pyrazol-1-yl)acetate (2c)
95.8% yield. mp 98-100 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.89 (s, 1H, Ar-H), 8.32 (s, 1H, Ar-H),
5.21 (s, 2H, CH2), 3.72 (s, 3H, CH3). ESI-MS: m/z 186.0 [M+H]+.
4.1.4. methyl 3-(4-nitro-1H-pyrazol-1-yl)propanoate (2d)
90.2% yield. mp 50-52 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 1H, Ar-H), 8.26 (s, 1H, Ar-H),
4.43 (t, J = 6.7 Hz, 2H, CH2), 3.60 (s, 3H, CH3), 2.96 (t, J = 6.7 Hz, 2H, CH2). ESI-MS: m/z 200.1 [M+H]+.
4.1.5. 2-(4-nitrophenoxy)ethanol (2e)
To a solution of 4-nitrophenol (1.39 g, 10 mmol) in DMF (20 ml) was added K2CO3 (1.38 g, 10 mmol)
and 2-Bromoethanol (1.50 g, 12 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 compound 2e as a white solid. Yield 97.2%, mp 127-129 ºC. 1H NMR (400 MHz,
DMSO-d6) δ 8.20 (d, J = 9.2 Hz, 2H, Ar-H), 7.16 (d, J = 9.2 Hz, 2H, Ar-H), 4.96 (t, J = 5.5 Hz, 1H, OH),
4.15 (t, J = 4.8 Hz, 2H, CH2), 3.75 (dd, J = 9.9, 5.2 Hz, 2H, CH2). ESI-MS: m/z 184.1 [M+H]+.
Compound 2f was synthesized according to the procedure described in 2e.
4.1.6. 2-(2-methoxy-4-nitrophenoxy)ethanol (2f)
76.4% yield. mp 100-102 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.88 (dd, J = 9.0, 2.6 Hz, 1H, Ar-H),
7.74 (d, J = 2.5 Hz, 1H, Ar-H), 7.19 (d, J = 9.0 Hz, 1H, Ar-H), 4.95 (t, J = 5.4 Hz, 1H, OH), 4.14 (t, J = 4.9
Hz, 2H, CH2), 3.89 (s, 3H, CH3), 3.76 (dd, J = 9.9, 5.1 Hz, 2H, CH2). ESI-MS: m/z 214.1 [M+H]+.
4.1.7. 2-((5-nitropyridin-2-yl)oxy)ethanol (2g)
To a solution of 2-chloro-5-nitropyridine (1.58 g, 10 mmol) in DMF (20 ml) was added K2CO3 (1.38 g,
10 mmol) and ethane-1,2-diol (1.24 g, 20 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 compound 2g as a white solid. Yield 88.6%, mp 113-115 ºC. 1H NMR (400
MHz, DMSO-d6) δ 9.07 (d, J = 2.8 Hz, 1H, Ar-H), 8.48 (dd, J = 9.1, 2.9 Hz, 1H, Ar-H), 7.04 (d, J = 9.2 Hz,
1H, Ar-H), 4.92 (t, J = 5.5 Hz, 1H, OH), 4.47 – 4.39 (m, 2H, CH2), 3.75 (dd, J = 10.1, 5.3 Hz, 2H, CH2).
ESI-MS: m/z 185.0 [M+H]+.

Compound 2h-j was synthesized according to the procedure described in 2g.
4.1.8. 3-((5-nitropyridin-2-yl)oxy)propan-1-ol (2h)
62.7% yield. Oil. 1H NMR (400 MHz, DMSO-d6) δ 9.08 (d, J = 2.8 Hz, 1H, Ar-H), 8.47 (dd, J = 9.2,
2.9 Hz, 1H, Ar-H), 7.02 (d, J = 9.2 Hz, 1H, Ar-H), 4.59 (t, J = 5.0 Hz, 1H, OH), 4.46 (t, J = 6.5 Hz, 2H,
CH2), 3.56 (dd, J = 11.2, 6.0 Hz, 2H, CH2), 1.90 (p, J = 6.4 Hz, 2H, CH2). ESI-MS: m/z 199.1 [M+H]+.
4.1.9. 2-(2-chloro-4-nitrophenoxy)ethan-1-ol (2i)
78.4% yield. mp 112-114 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.30 (dd, J = 4.4 and 2.8 Hz, 1H, Ar-
H), 8.23-8.19 (m, 1H, Ar-H), 7.39 (dd, J = 9.2 and 4.4 Hz, 1H, Ar-H), 4.97 (m, 1H, OH), 4.25 (dd, J = 9.2
and 4.4 Hz, 2H, CH2), 3.78 (m, 2H, CH2). ESI-MS: m/z 218.1 [M+H]+.
4.1.10 2-(2-fluoro-4-nitrophenoxy)ethan-1-ol (2j)
72.2% yield. mp 93-95 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.17-8.09 (m, 2H, Ar-H), 7.42 (t, J = 9.2
Hz, 1H, Ar-H), 3.00 ((t, J = 5.2 Hz, 1H, OH), 4.24 (t, J = 4.8 Hz, 2H, CH2), 3.78 (dd, J = 5.2 and 7.6 Hz,
CH2). ESI-MS: m/z 202.0 [M+H]+.
4.1.11 2-(4-((6-bromoquinazolin-4-yl)amino)-1H-pyrazol-1-yl)ethanol (3a)
2a (0.32g, 0.002mol) was dissolved in 20 ml ethanol in a round-bottomed flask and Pd/C (10%, 0.1g)
was added which reacted at 60 °C for 12h under hydrogen atmosphere. The Pd/C was recycled by filtration
and the ethanol was recycled by evaporation to give the amine as purple oil which was retained in the round-
bottomed flask. The amine was used for next reaction without purification, because it was easily oxidized.
Then, added 6-bromo-4-chloroquinazoline (0.36g, 0.0015mol) and isopropanol (30 ml) to the round-
bottomed flask above and stirred at 90 ºC for 4 h. The isopropanol was removed under reduced pressure and
the residue was purified through a column chromatography on silica with chloroform/methanol (V:V 10:1)
as a white solid (0.43 g, 86.0% yield). mp 178-180 ºC. 1H NMR (600 MHz, DMSO-d6) δ 12.11 (d, J = 18.3
Hz, 1H, NH), 9.28 (d, J = 5.8 Hz, 1H, Ar-H), 8.97 (d, J = 4.9 Hz, 1H, Ar-H), 8.39 (d, J = 4.1 Hz, 1H, Ar-H),
8.18 (s, 1H, Ar-H), 8.04 (d, J = 3.9 Hz, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 4.22 (s, 2H, CH2), 3.77 (s, 2H, CH2).
13C NMR (150 MHz, DMSO-d6) δ 155.75, 152.06, 138.50, 132.79, 127.36, 124.55, 120.97, 120.48, 115.72,
60.51, 54.97. ESI-MS: m/z 334.0 [M+H]+.
Compounds 3b-k were synthesized according to the procedure described in 3a.
4.1.12. 3-(4-((6-bromoquinazolin-4-yl)amino)-1H-pyrazol-1-yl)propan-1-ol (3b)
72.8% yield. mp 188-190 ºC. 1H NMR (600 MHz, DMSO-d6) δ 12.09 (s, 1H, NH), 9.28 (s, 1H, Ar-H),
8.96 (s, 1H, Ar-H), 8.39 (s, 1H, Ar-H), 8.16 (d, J = 8.9 Hz, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 7.90 (d, J = 8.8
Hz, 1H, Ar-H), 4.23 (t, J = 7.0 Hz, 2H, CH2), 3.41 (t, J = 6.1 Hz, 2H, CH2), 2.24 – 1.45 (q, J = 6.6 Hz, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 155.78, 153.45, 137.67, 132.33, 126.75, 125.79, 123.42, 121.04,
120.22, 116.16, 58.09, 49.20, 33.61.ESI-MS: m/z 348.0 [M+H]+.

4.1.13. methyl 2-(4-((6-bromoquinazolin-4-yl)amino)-1H-pyrazol-1-yl)acetate (3c)
67.2% yield. mp 247-249 ºC. 1H NMR (600 MHz, DMSO-d6) δ 11.57 (s, 1H, NH), 9.09 (s, J = 2.1 Hz,
1H, Ar-H), 8.90 (s, 1H, Ar-H), 8.36 (s, 1H, Ar-H), 8.12 (d, J = 9.0, 2.0 Hz, 1H, Ar-H), 7.96 (s, 1H, Ar-H),
7.83 (d, J = 8.9 Hz, 1H, Ar-H), 4.42 (t, J = 6.6 Hz, 2H, CH2), 3.61 (s, 3H, CH3), 2.92 (t, J = 6.6 Hz, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) δ 171.63, 155.86, 152.99, 141.22, 138.00, 132.81, 126.87, 124.85,
123.84, 120.93, 120.53, 115.97, 52.06, 47.74, 34.51. ESI-MS: m/z 362.0 [M+H]+.
4.1.14. methyl 3-(4-((6-bromoquinazolin-4-yl)amino)-1H-pyrazol-1-yl)propanoate (3d)
58.9% yield. mp 187-189 ºC. 1H NMR (600 MHz, DMSO-d6) δ 11.45 (s, 1H, NH), 9.16 (s, 1H, Ar-H),
8.82 (s, 1H, Ar-H), 8.44 (d, J = 1.6 Hz, 1H, Ar-H), 8.08 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.00 (s, 1H, Ar-H),
7.84 (d, J = 9.0 Hz, 1H, Ar-H), 5.17 (s, 2H, CH2), 3.70 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ
168.68, 155.28, 153.10, 136.88, 132.61, 126.39, 125.88, 124.00, 121.34, 119.45, 115.78, 52.61, 52.12. ESI-
MS: m/z 376.1 [M+H]+.
4.1.15. 2-(4-((6-bromoquinazolin-4-yl)amino)phenoxy)ethan-1-ol (3e)
90.4% yield. mp 231-233 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.81 (s, 1H, NH), 9.05 (s, 1H, Ar-H),
8.74 (s, 1H, Ar-H), 8.10 (d, J = 8.9 Hz, 1H, Ar-H), 7.81 (d, J = 8.9 Hz, 1H, Ar-H), 7.67 (d, J = 8.8 Hz, 2H,
Ar-H), 7.03 (d, J = 9.0 Hz, 2H, Ar-H), 4.02 (t, J = 5.0 Hz, 2H, CH2), 3.74 (t, J = 5.0 Hz, 2H, CH2). 13C NMR
(150 MHz, DMSO-d6) δ 158.13, 156.84, 153.51, 143.37, 137.78, 130.77, 126.81, 126.24, 125.61, 120.18,
116.16, 114.83, 70.18, 60.02. ESI-MS: m/z 360.0 [M+H]+.
4.1.16. 2-(4-((6-bromoquinazolin-4-yl)amino)-2-methoxyphenoxy)ethan-1-ol (3f)
77.6% yield. mp 206-208 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.84 (s, 1H, NH), 9.11 (s, 1H, Ar-H),
8.76 (s, 1H, Ar-H), 8.11 (d, J = 8.9 Hz, 1H, Ar-H), 7.83 (d, J = 8.8 Hz, 1H, Ar-H), 7.44 (s, 1H, Ar-H), 7.38
(d, J = 6.3 Hz, 1H, Ar-H), 7.03 (d, J = 8.6 Hz, 1H, Ar-H), 4.00 (t, J = 5.1 Hz, 2H, CH2), 3.79 (s, 3H, CH3),
3.74 (t, J = 5.1 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 172.48, 158.07, 153.45, 149.06, 146.33,
137.77, 131.31, 126.84, 126.26, 120.20, 116.31, 116.21, 113.32, 108.94, 70.84, 60.03, 56.03. ESI-MS: m/z
390.0 [M+H]+.
4.1.17. 2-((5-((6-bromoquinazolin-4-yl)amino)pyridin-2-yl)oxy)ethan-1-ol (3g)
87.4% yield. mp 191-193 ºC. 1H NMR (600 MHz, DMSO-d6) δ 11.84 (s, 1H, NH), 9.26 (s, 1H, Ar-H),
8.93 (s, 1H, Ar-H), 8.47 (d, J = 2.7 Hz, 1H, Ar-H), 8.25 (dd, J = 8.9, 1.9 Hz, 1H, Ar-H), 8.04 (dd, J = 8.9,
2.7 Hz, 1H, Ar-H), 7.94 (d, J = 8.9 Hz, 1H, Ar-H), 6.97 (d, J = 8.8 Hz, 1H, Ar-H), 4.31 (t, J = 5.1 Hz, 2H,
CH2), 3.74 (t, J = 5.1 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 161.91 , 159.43 , 151.97 , 143.16 ,
139.19 , 136.63 , 127.92 , 127.65 , 122.99 , 121.29 , 115.57 , 110.96 , 68.17 , 59.78. ESI-MS: m/z 361.1
[M+H]+.
4.1.18. 3-((5-((6-bromoquinazolin-4-yl)amino)pyridin-2-yl)oxy)propan-1-ol (3h)

64.2% yield. mp 214-216 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.98 (s, 1H, NH), 9.09 (d, J = 2.1 Hz,
1H, Ar-H), 8.74 (s, 1H, Ar-H), 8.49 (d, J = 2.8 Hz, 1H, Ar-H), 8.11 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 8.07 (dd,
J = 8.8, 2.7 Hz, 1H, Ar-H), 7.83 (d, J = 8.9 Hz, 1H, Ar-H), 6.91 (d, J = 8.8 Hz, 1H, Ar-H), 4.34 (t, J = 6.5
Hz, 2H, CH2), 3.57 (t, J = 6.3 Hz, 2H, CH2), 1.88 (q, J = 6.4 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6)
δ 174.77, 160.75, 158.03, 153.15, 141.89, 137.35, 135.48, 128.39, 126.37, 126.18, 119.72, 115.68, 110.12,
62.99, 57.38, 31.93. ESI-MS: m/z 375.0 [M-H]+.
4.1.19. 2-(4-((6-bromoquinazolin-4-yl)amino)-2-chlorophenoxy)ethan-1-ol (3i)
74.9% yield. mp 227-229 ºC. 1H NMR (400 MHz, DMSO) δ 9.84 (s, 1H, NH), 8.82 (d, J = 2.0 Hz, 1H,
Ar-H), 8.62 (s, 1H, Ar-H), 8.00 (d, J = 2.4 Hz, 1H, Ar-H), 7.98 (dd, J = 8.9, 2.1 Hz, 1H, Ar-H), 7.73 (dd, J =
2.4, 8.8 Hz, 1H, Ar-H), 7.72 (d, J = 8.8 Hz, 1H, Ar-H), 7.22 (d, J = 8.8 Hz, 1H, Ar-H), 4.90 (s, 1H, OH),
4.10 (t, J = 5.1 Hz, 2H, CH2), 3.77 (d, J = 4.3 Hz, 2H, CH2).13C NMR (100 MHz, DMSO) δ 157.17, 155.35,
151.09, 148.89, 136.49, 132.94, 130.50, 125.82, 124.44, 122.69, 121.27, 119.23, 116.84, 114.44, 71.34,
59.99. ESI-MS: m/z 394.0 [M-H]+.
4.1.20. 2-(4-((6-bromoquinazolin-4-yl)amino)-2-fluorophenoxy)ethan-1-ol (3j)
75.6% yield. mp 220-222 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.41 (s, 1H, NH), 8.96 (d, J = 2.0Hz,
1H, Ar-H), 8.72 (s, 1H, Ar-H), 8.05 (dd, J = 2.0 and 8.8 Hz, 1H, Ar-H), 7.83 (dd, J = 2.4 and 13.6 Hz, 1H,
Ar-H), 7.77 (d, J = 9.2 Hz, 1H, Ar-H), 7.56-7.53 (m, 1H, Ar-H), 7.24 (t, J = 9.2 Hz, 1H, Ar-H), 4.10 (t, J =
4.8 Hz, 2H, CH2), 3.76 (t, J = 4.8 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO) δ 157.64, 154.31, 151.34 (d,
J = 240.7 Hz), 145.95 (d, J = 2.8 Hz), 144.01 (d, J = 10.7 Hz), 137.27, 131.93 (d, J = 9.6 Hz), 128.25,
126.37, 119.85, 119.46 (d, J = 3.3 Hz), 116.48, 115.37, 111.84 (d, J = 22.2 Hz), 71.39, 59.98. ESI-MS: m/z
378.1 [M-H]+.
4.1.21. N-(5-bromo-2-methoxypyridin-3-yl)methanesulfonamide (5a)
To a solution of 5-bromo-2-methoxypyridin-3-amine (3.02 g, 15 mmol) in pyridine (60 ml) at 0
ºC was added methanesulfonyl chloride (2.60 g, 17 mmol). Then the mixture was stirred at room
temperature for 24 h. Then the pyridine was removed at reduced pressure and added water (100 ml),
extracted with ethyl acetate (200ml) for two times, dried with anhydrous Na2SO4 and evaporated to
give compound 5a as a white solid. Yield 78.4%, mp 150-151 ºC. 1H NMR (400 MHz, DMSO-d6) 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, OCH3),
3.11(s, 3H, CH3). ESI-MS: m/z 281.1 [M+H]+.
Compounds 5b-c were synthesized according to the procedure described in 5a.
4.1.22. N-(5-bromo-2-methoxypyridin-3-yl)butane-1-sulfonamide (5b)
78.4% yield. mp 150-151 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H, NH), 8.09 (d, J = 2.4 Hz,
1H, Ar-H), 7.77 (d, J = 2.4Hz, 1H, Ar-H), 3.91 (s, 3H, OCH3), 3.16 (t, J = 7.6Hz, 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 323.1 [M+H]+.
4.1.23. N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (5c)
85.6% yield. mp 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), 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]+.
4.1.24. N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methanesulfonamide (6a)
A solution of the 5a (2.80 g, 10 mmol), bis(pinacolato)diborane (1.27 g, 5 mmol), Pd(dppf)2Cl2 (0.18 g,
0.25 mmol) and KOAc (1.47 g, 15 mmol) in anhydrous DMF (30 ml) under N2 was stirred at 100 ºC for 8 h.
Then DMF was removed under reduced pressure and add water (100 ml), extracted with ethyl acetate (200
ml) for two times, dried with anhydrous Na2SO4 and evaporated to get the residue which was purified
through a column chromatography on silica with petroleum ether / ethyl acetate (V:V 5:1) to give compound
6a as a white solid (1.98 g, 60.4% yield). mp 90-92 ºC. 1H NMR (400 MHz, DMSO-d6) 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, OCH3), 3.01 (s, 3H, CH3), 1.30
(s, 12H, CH3). ESI-MS: m/z 329.1 [M+H]+.
Compounds 6b-c were synthesized according to the procedure described in 6a.
4.1.25. N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)butane-1-sulfonamide (6b)
64.3% yield. mp 91-93 ºC.1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H, NH), 8.21 (d, J = 1.4 Hz, 1H,
Ar-H), 7.79 (d, J = 1.3 Hz, 1H, Ar-H), 3.94 (s, 3H, OCH3), 3.10 - 3.01 (m, 2H, CH2), 1.76 - 1.63 (m, 2H,
CH2), 1.38 (dt, J = 7.4, 14.8Hz, 2H, CH2), 1.30 (s, 12H, CH3), 0.86 (t, J = 7.3 Hz, 3H, CH3). ESI-MS: m/z
371.2 [M+H]+.
4.1.26. 2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-
yl)benzenesulfonamide (6c)
78.8% yield. mp 171-173 ºC. 1H NMR (400 MHz, DMSO-d6) 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), 7.20 (td, J =
2.0, 8.6 Hz, 1H, Ar-H), 3.62 (s, 3H, OCH3), 1.30 (s, 12H, CH3). ESI-MS: m/z 427.3 [M+H]+.
4.1.27.
N-(5-(4-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)methanesulfonamide (7a)
A solution of the 3a (0.167 g, 0.5 mmol), 6a (0.158 g, 0.5 mmol), Pd(dppf)2Cl2 (0.018 g, 0.025 mmol)
and Cs2CO3 (0.33 g, 0.56 mmol) in DMF (10 ml) under an atmosphere of N2 was stirred at 90 ºC for 4 h.
DMF was removed under reduced pressure and the residue was purified through a column chromatography
on silica with chloroform/methanol (V:V 50:1) as a light yellow solid (0.13g, 57.0% yield). mp 237-239 ºC.
1H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H, NH), 9.49 (s, 1H, NH), 8.90 (s, 1H, Ar-H), 8.80 (s, 1H, Ar-
H), 8.55 (d, J = 2.3 Hz, 1H, Ar-H), 8.35 (s, 1H, Ar-H), 8.26 – 8.23 (m, 1H, Ar-H), 8.09 (d, J = 2.2 Hz, 1H,

Ar-H), 7.90 (s, 1H, Ar-H), 7.88 (d, J = 8.7 Hz, 1H, Ar-H), 4.21 (t, J = 5.5 Hz, 2H, CH2), 4.01 (s, 3H, CH3),
3.77 (t, J = 5.4 Hz, 2H, CH2), 3.13 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 163.52, 157.43, 156.84,
153.69, 142.04, 135.84, 132.88, 132.24 (2C), 128.92, 125.35, 123.81, 121.82, 121.22, 121,12, 115.16, 60.62,
54.91, 54.39, 41.40. HRMS (ESI+) m/z calcd for C20H22N7O4S [M+H]+, 456.1448; found, 456.1441.
Compounds 7b-o was synthesized according to the procedure described in 7a.
4.1.28.
2,4-difluoro-N-(5-(4-((1-(2-hydroxyethyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-
methoxypyridin-3-yl)benzenesulfonamide (7b)
1
65.8% yield, mp 232-234 ºC. H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H, NH), 10.18 (d, J = 7.1
Hz, 1H, NH), 8.75 (s, 1H, Ar-H), 8.64 (d, J = 2.7 Hz, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.33 (d, J = 2.7 Hz, 1H,
Ar-H), 8.14-8.13 (m, 2H, Ar-H×2), 7.84 (d, J = 12.0 Hz, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 7.76 (q, J = 6.0 Hz,
1H, Ar-H), 7.62 -7.59 (m, 1H, Ar-H), 7.23 – 7.19 (m, 1H, Ar-H), 4.96 (t, J = 5.4 Hz, 1H, OH), 4.20 (t, J =
5.4 Hz, 2H, CH2), 3.78 (m, 2H, CH2), 3.64 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 165.52 (dd, J =
253.8, 11.6 Hz), 159.89 (dd, J = 257.4, 13.1 Hz), 158.40, 156.67, 155.49, 149.05, 143.49, 135.64, 134.31,
132.28 (d, J = 11.0 Hz), 131.60, 131.40, 129.35, 128.93, 125.58 (dd, J = 13.59, 3.0 Hz), 122.98, 122.01,
120.44, 120.00, 115.74, 112.21 (d, J = 22.2 Hz), 106.22 (t, J = 26.0 Hz), 60.71, 54.82, 53.84. HRMS (ESI+)
m/z calcd for C25H22F2N7O4S [M+H]+, 554.1417; found, 554.1436.
4.1.29. N-(5-(4-((1-(3-hydroxypropyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)methanesulfonamide (7c)
1
59.2% yield, mp 128-130 ºC. H NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H, NH), 9.48 (s, 1H, NH),
8.74 (s, 1H, Ar-H), 8.63 (s, 1H, Ar-H), 8.53 (d, J = 2.1 Hz, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 8.14 (d, J = 8.6
Hz, 1H, Ar-H), 8.09 (d, J = 2.1 Hz, 1H, Ar-H), 7.85 (d, J = 8.6 Hz, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 4.64 (t, J
= 6.0 Hz, 1H, OH), 4.21 (t, J = 7.0 Hz, 2H, CH2), 4.01 (s, 3H, CH3), 3.43 (dd, J = 10.5, 5.5 Hz, 2H, CH2),
13
3.12 (s, 3H, CH3), 1.98 – 1.92 (m, 2H, CH2). C NMR (150 MHz, DMSO-d6) δ 157.29, 156.73, 155.44,
148.97, 141.95, 134.77, 132.40, 131.65, 131.61, 129.46, 128.86, 122.54, 122.00, 121.72, 120.47, 115.73,
58.18, 54.34, 49.10, 41.33, 33.68. HRMS (ESI+) m/z calcd for C21H24N7O4S [M+H]+, 470.1605; found,
470.1631.
4.1.30.
2,4-difluoro-N-(5-(4-((1-(3-hydroxypropyl)-1H-pyrazol-4-yl)amino)quinazolin-6-yl)-2-
methoxypyridin-3-yl)benzenesulfonamide (7d)
61.8% yield, mp 197-199 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.32 (s, 1H, NH), 8.83 (s, 1H, Ar-H),
8.62 (s, 1H, Ar-H), 8.55 (d, J = 2.4 Hz, 1H, Ar-H), 8.34 – 8.29 (m, 1H, Ar-H), 8.13 – 8.07 (m, 2H, Ar-H×2),
7.87 – 7.81 (m, 2H, Ar-H×2), 7.76 (td, J = 8.5, 6.3 Hz, 1H, Ar-H), 7.61 – 7.53 (m, 1H, Ar-H), 7.20 (td, J =
8.4, 2.5 Hz, 1H, Ar-H), 4.65 (t, 1H, OH), 4.20 (t, J = 7.0 Hz, 2H, CH2), 3.65 (s, 3H, CH3), 3.42 (t, J = 6.1 Hz,
2H, CH2), 1.95 (p, J = 6.6 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.27 (dd, J = 253.9, 11.0 Hz),

163.81 , 159.83 (dd, J = 257.1, 13.5 Hz), 158.27, 156.71, 155.47, 149.08, 134.49, 132.22 (d, J = 10.4 Hz),
131.68 (2C), 131.33, 129.24, 128.90, 126.16 (dd, J = 14.10, 3.0 Hz), 122.42 (2C), 122.14, 120.61, 115.82,
112.09 (d, J = 22.9 Hz), 106.13 (t, J = 26.1 Hz), 58.16, 53.77, 49.08. HRMS (ESI+) m/z calcd for
C26H24F2N7O4S [M+H]+, 568.1573; found, 568.1593.
4.1.31.methyl
2-(4-((6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-
yl)amino)-1H-pyrazol-1-yl)acetate (7e)
53.7% yield, mp 181-183 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H, NH), 10.23 (s, 1H, NH),
8.74 (s, 1H, Ar-H), 8.64 (s, 1H, Ar-H), 8.54 (d, J = 2.2 Hz, 1H, Ar-H), 8.41 (s, 1H, Ar-H), 8.15 (dd, J = 6.9,
1.8 Hz, 2H, Ar-H×2), 7.85 (d, J = 8.6 Hz, 1H, Ar-H), 7.83 (s, 1H, Ar-H), 7.75 (dd, J = 14.8, 8.4 Hz, 1H, Ar-
H), 7.64 – 7.57 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.1 Hz, 1H, Ar-H), 5.16 (s, 2H, CH2), 3.71 (s, 3H, CH3),
3.64 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 169.35, 165.52 (dd, J = 254.6, 12.0 Hz), 159.89 (dd, J
= 257.8, 13.4 Hz), 158.42, 156.70, 155.43, 149.10, 143.51, 135.67, 134.39, 132.51, 132.28 (d, J = 10.6 Hz),
131.50, 129.35, 128.99, 125.58 (dd, J = 13.59, 3.0 Hz), 123.80, 122.60, 120.46, 120.00, 115.74, 112.22 (d, J
= 23.2 Hz), 106.23 (t, J = 26.2 Hz), 53.16, 52.66, 49.05. HRMS (ESI+) m/z calcd for C26H22F2N7O5S
[M+H]+, 582.1366; found, 582.1366.
4.1.32.
methyl
3-(4-((6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-
yl)amino)-1H-pyrazol-1-yl)propanoate (7f)
67.5% yield, mp 184-186 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H, NH), 10.17 (s, 1H, NH),
8.73 (d, J = 1.3 Hz, 1H, Ar-H), 8.63 (s, 1H, Ar-H), 8.53 (d, J = 2.3 Hz, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.13
(dd, J = 5.2, 2.1 Hz, 2H, Ar-H×2), 7.84 (d, J = 8.6 Hz, 1H, Ar-H), 7.79 (s, 1H, Ar-H), 7.75 (dd, J = 14.9, 8.5
Hz, 1H, Ar-H), 7.64 – 7.57 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 4.41 (t, J = 6.6 Hz, 2H,
CH2), 3.64 (s, 3H, CH3), 3.62 (s, 3H, CH3), 2.92 (t, J = 6.6 Hz, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ
171.70, 166.76 (dd, J = 624.6, 14.8 Hz), 159.90 (dd, J = 257.4, 13.5 Hz), 158.43, 156.74, 155.48, 149.13,
143.49, 135.67, 134.35, 132.29 (d, J = 11.1 Hz), 131.99, 131.45, 129.36, 128.99, 125.59 (dd, J = 14.5, 3.1
Hz), 122.68, 122.14, 120.44, 120.03, 115.74, 112.22 (d, J = 21.9 Hz), 106.23 (t, J = 26.0 Hz), 53.85, 52.04,
47.63, 34.63. HRMS (ESI+) m/z calcd for C27H24F2N7O5S [M+H]+, 596.1522; found, 596.1523.
4.1.33. 2-(4-((6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)-1H-
pyrazol-1-yl)acetic acid (7g)
1
54.4% yield, mp 195-197 ºC. H NMR (600 MHz, DMSO-d6) δ 13.19 (s, 1H, OH), 11.65 (s, 1H, NH),
10.43 (s, 1H, NH), 9.14 (s, 1H, Ar-H), 8.88 (s, 1H, Ar-H), 8.67 (d, J = 1.9 Hz, 1H, Ar-H), 8.45 (s, 1H, Ar-
H), 8.30 (d, J = 8.1 Hz, 1H, Ar-H), 8.16 (d, J = 2.2 Hz, 1H, Ar-H), 8.03 (s, 1H, Ar-H), 7.96 (d, J = 8.6 Hz,
1H, Ar-H), 7.75 (q, J = 8.3 Hz, 1H, Ar-H), 7.60 (d, J = 10.7 Hz, 1H, Ar-H), 7.23 – 7.19 (m, 1H, Ar-H), 5.06
(s, 2H, CH2), 3.65 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 170.11, 165.51 (dd, J = 254.0, 11.5 Hz),

160.11, 159.9 (dd, J = 256.7, 13.6 Hz), 158.64, 156.95, 152.67, 151.55, 143.94, 142.52, 135.81, 135.57,
133.18, 132.27 (d, J = 10.7 Hz), 128.51, 125.71 – 125.40 (m), 124.99, 121.56 (d, J = 26.8 Hz), 120.01,
118.95, 114.99, 112.26 (d, J = 21.6 Hz), 106.22 (t, J = 13.2 Hz), 53.92, 53.49. HRMS (ESI+) m/z calcd for
C25H20F2N7O5S [M+H]+, 568.1209; found, 568.1208.
4.1.34. 3-(4-((6-(5-((2,4-difluorophenyl)sulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)-1H-
pyrazol-1-yl)propanoic acid (7h)
1
57.6% yield, mp 153-155 ºC. H NMR (600 MHz, DMSO-d6) δ 12.40 (s, 1H, OH), 10.43 (s, 1H, NH),
9.39 (s, 1H, NH), 8.97 (s, 1H, Ar-H), 8.75 (s, 1H, Ar-H), 8.44 (s, 1H, Ar-H), 8.36 (d, J = 8.5 Hz, 1H, Ar-H),
8.17 (s, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 8.04 (d, J = 8.5 Hz, 1H, Ar-H), 7.75 (dd, J = 14.3, 7.6 Hz, 1H, Ar-H),
7.61 (t, J = 8.8 Hz, 1H, Ar-H), 7.21 (td, J = 8.2, 1.1 Hz, 1H, Ar-H), 4.40 (t, J = 5.9 Hz, 2H, CH2), 3.65 (s,
13
3H, CH3), 2.85 (t, J = 6.1 Hz, 2H, CH2). C NMR (150 MHz, DMSO-d6) δ 172.53, 165.50 (dd, J = 253.7,
12.1 Hz), 159.85 (dd, J = 257.9, 14.0 Hz), 158.75, 156.95, 151.31, 144.15, 138.52, 136.42, 135.57, 133.87,
133.34, 132.28 (d, J = 10.5 Hz), 128.13, 125.59 (dd, J = 13.6, 3.0 Hz), 124.41, 122.19, 121.24, 120.58,
120.00, 114.62, 112.27 (d, J = 21.6 Hz), 106.23 (t, J = 26.2 Hz), 53.95, 47.99, 34.82. HRMS (ESI+) m/z
calcd for C26H22F2N7O5S [M+H]+, 582.1366; found, 582.1366.
4.1.35. N-(5-(4-((4-(2-hydroxyethoxy)phenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)methanesulfonamid (7i)
51.7% yield, mp 179-181 ºC. 1H NMR (600 MHz, DMSO-d6) δ 9.92 (s, 1H, NH), 9.47 (s, 1H, NH), 8.81
(s, 1H, Ar-H), 8.55 (d, J = 2.2 Hz, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.16 (dd, J = 8.6, 1.6 Hz, 1H, Ar-H), 8.11
(d, J = 2.2 Hz, 1H, Ar-H), 7.85 (d, J = 8.6 Hz, 1H, Ar-H), 7.68 (d, J = 8.9 Hz, 2H, Ar-H), 7.01 (d, J = 8.9
Hz, 2H, Ar-H), 4.92 (s, 1H, OH), 4.03 (s, 2H, CH2), 4.01 (s, 3H, CH3), 3.82 – 3.64 (m, 2H, CH2), 3.12 (s,
13
3H, CH3). C NMR (150 MHz, DMSO-d6) δ 158.38, 157.27, 155.94, 155.16, 149.16, 141.97, 134.76,
132.40, 132.05, 131.76, 129.39, 128.77, 125.10 (2C), 121.69, 120.66, 115.74, 114.74 (2C), 70.14, 60.08,
54.33, 41.31. HRMS (ESI+) m/z calcd for C23H24N5O5S [M+H]+, 482.1493; found, 482.1492.
4.1.36.
2,4-difluoro-N-(5-(4-((4-(2-hydroxyethoxy)phenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)benzenesulfonamide (7j)
1
53.9% yield, mp 193-195 ºC. H NMR (600 MHz, DMSO-d6) δ 11.03 (s, 1H, NH), 10.44 (s, 1H, NH),
8.95 (s, 1H, Ar-H), 8.81 (s, 1H, Ar-H), 8.59 (d, J = 2.2 Hz, 1H, Ar-H), 8.36 (d, J = 8.3 Hz, 1H, Ar-H), 8.20
(d, J = 2.2 Hz, 1H, Ar-H), 7.90 (d, J = 8.6 Hz, 1H, Ar-H), 7.74 (dd, J = 14.9, 8.4 Hz, 1H, Ar-H), 7.64 (d, J =
8.9 Hz, 2H, Ar-H), 7.62 – 7.57 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.1 Hz, 1H, Ar-H), 7.08 (d, J = 8.9 Hz, 2H,
Ar-H), 4.04 (t, J = 4.9 Hz, 2H, CH2), 3.75 (t, J = 4.9 Hz, 2H, CH2), 3.64 (s, 3H, CH3). 13C NMR (150 MHz,
DMSO-d6) δ 165.54 (dd, J = 253.9, 11.6 Hz), 159.93 (dd, J = 258.2, 13.6 Hz), 159.45, 158.77, 157.31,
152.64, 143.74, 136.01, 135.76, 133.63, 132.24 (d, J = 11.0 Hz), 130.20, 128.46, 126.23 (2C), 125.54 (dd, J

= 14.0, 3.0 Hz), 123.39, 121.50, 120.11, 114.99 (2C), 114.77, 112.25 (d, J = 19.1 Hz), 106.25 (t, J = 26.1
Hz), 70.23, 60.02, 53.94. HRMS (ESI+) m/z calcd for C28H24F2N5O5S [M+H]+, 580.1461; found, 580.1471.
4.1.37.
N-(5-(4-((6-(2-hydroxyethoxy)pyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)methanesulfonamide (7k)
1
61.4% yield, mp 248-250 ºC. H NMR (600 MHz, DMSO-d6) δ 9.99 (s, 1H, NH), 9.47 (s, 1H, NH),
8.79 (d, J = 2.0 Hz, 1H, Ar-H), 8.55 (d, J = 2.5 Hz, 2H, Ar-H), 8.48 (d, J = 2.7 Hz, 1H, Ar-H), 8.18 (dd, J =
8.6, 1.9 Hz, 1H, Ar-H), 8.11 (d, J = 2.3 Hz, 1H, Ar-H), 8.09 (d, J = 2.7 Hz, 1H, Ar-H), 7.87 (d, J = 8.6 Hz,
1H, Ar-H), 6.91 (d, J = 8.8 Hz, 1H, Ar-H), 4.87 (t, J = 5.5 Hz, 1H, OH), 4.30 (t, J = 5.2 Hz, 2H, CH2), 4.01
13
(s, 3H, CH3), 3.77 – 3.72 (m, 2H, CH2), 3.11 (s, 3H, CH3). C NMR (150 MHz, DMSO-d6) δ 160.55,
158.64, 157.31, 155.10, 149.40, 141.96, 141.76, 135.84, 134.88, 132.41, 131.90, 129.98, 129.35, 128.99,
121.71, 120.63, 115.68, 110.61, 67.92, 59.90, 54.33, 41.31. HRMS (ESI+) m/z calcd for C22H23N6O5S
[M+H]+, 483.1445; found, 483.1465.
4.1.38. N-(5-(4-((6-(2-hydroxyethoxy)pyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)butane-1-
sulfonamide (7l)
1
62.1% yield, mp 220-222 ºC. H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H, NH), 9.49 (s, 1H, NH),
8.79 (s, 1H, Ar-H), 8.55 (s, 2H, Ar-H), 8.48 (d, J = 2.5 Hz, 1H, Ar-H), 8.17 (dd, J = 8.7, 1.5 Hz, 1H, Ar-H),
8.12 – 8.08 (m, 2H, Ar-H), 7.87 (d, J = 8.7 Hz, 1H, Ar-H), 6.91 (d, J = 8.8 Hz, 1H, Ar-H), 4.87 (t, J = 5.5
Hz, 1H, OH), 4.30 (t, J = 5.1 Hz, 2H, CH2), 4.00 (s, 3H, CH3), 3.74 (q, J = 5.2 Hz, 2H, CH2), 3.19 – 3.11 (m,
2H, CH2), 1.84 – 1.63 (m, 2H, CH2), 1.40 (q, J = 7.4 Hz, 2H, CH2), 0.89 (t, J = 7.4 Hz, 3H, CH3). 13C NMR
(150 MHz, DMSO-d6) δ 160.54, 158.64, 157.29, 155.12, 149.44, 142.02, 141.74, 135.83, 134.87, 132.82,
131.88, 129.99, 129.35, 129.04, 121.66, 120.65, 115.69, 110.61, 67.92, 59.90, 54.29, 52.56, 25.66, 21.29,
14.00. HRMS (ESI+) m/z calcd for C25H29N6O5S [M+H]+, 525.1842; found, 525.1948.
4.1.39. 2,4-difluoro-N-(5-(4-((6-(2-hydroxyethoxy)pyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-
yl)benzenesulfonamide (7m)
58.6% yield, mp 196-198 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H, NH), 10.00 (s, 1H, NH),
8.79 (d, J = 2.0 Hz, 1H, Ar-H), 8.57 – 8.54 (m, 2H, Ar-H), 8.48 (d, J = 2.7 Hz, 1H, Ar-H), 8.17 (d, J = 2.4
Hz, 1H, Ar-H), 8.10 (dd, J = 8.8, 2.7 Hz, 1H, Ar-H), 7.87 (d, J = 8.6 Hz, 1H, Ar-H), 7.77 – 7.72 (m, 1H, Ar-
H), 7.64 – 7.57 (m, 1H, Ar-H), 7.21 (td, J = 8.5, 2.5 Hz, 1H, Ar-H), 6.92 (d, J = 8.8 Hz, 1H, Ar-H), 4.87 (t, J
13
= 5.5 Hz, 1H, OH), 4.30 (t, J = 5.2 Hz, 2H, CH2), 3.74 (q, J = 5.3 Hz, 2H, CH2), 3.64 (s, 3H, CH3). C
NMR (150 MHz, DMSO-d6) δ 165.52 (dd, J = 254.6, 12.0 Hz), 160.57, 159.93 (dd, J = 257.4, 13.1 Hz),
158.66, 158.46, 155.16, 149.46, 143.51, 141.78, 135.87, 135.70, 134.46, 132.27 (d, J = 10.4 Hz), 131.70,
129.96, 129.24, 129.06, 125.55 (dd, J = 13.59, 3.0 Hz), 120.63, 120.02, 115.70, 112.21 (d, J = 21.9 Hz),
110.62, 106.3 (t, J = 26.2 Hz), 67.92, 59.90, 53.85. HRMS (ESI+) m/z calcd for C27H23F2N6O5S [M+H]+,

581.1413; found, 581.1410.
4.1.40. 2,4-difluoro-N-(5-(4-((6-(3-hydroxypropoxy)pyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-
3-yl)benzenesulfonamide (7n)
65.3% yield, mp 115-117 ºC. 1H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H, NH), 10.00 (s, 1H, NH),
8.80 (s, 1H, Ar-H), 8.57 (d, J = 2.2 Hz, 1H, Ar-H), 8.55 (s, 1H, Ar-H), 8.49 (d, J = 2.5 Hz, 1H, Ar-H), 8.20
– 8.17 (m, 2H, Ar-H), 8.10 (dd, J = 8.8, 2.6 Hz, 1H, Ar-H), 7.86 (d, J = 8.6 Hz, 1H, Ar-H), 7.77 – 7.73 (m,
1H, Ar-H), 7.61 (dd, J = 19.5, 2.2 Hz, 1H, Ar-H), 7.22 (d, J = 10.2 Hz, 1H, Ar-H), 6.91 (d, J = 8.8 Hz, 1H,
Ar-H), 4.59 (t, J = 4.8 Hz, 1H, OH), 4.34 (t, J = 6.5 Hz, 2H, CH2), 3.64 (s, 3H, CH3), 3.58 (d, J = 5.3 Hz, 2H,
CH2), 1.92 – 1.87 (m, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) δ 165.52 (dd, J = 253.8, 12.2 Hz), 160.65,
159.04 (dd, J = 257.7, 13.5 Hz), 158.66, 158.46, 155.16, 149.43, 143.53, 141.83, 135.80, 135.73, 134.45,
132.28 (d, J = 10.8 Hz), 131.70, 129.89, 129.24, 129.04, 125.57 (dd, J = 13.59, 3.0 Hz), 120.63, 119.99,
115.70, 112.22 (d, J = 22.1 Hz), 110.51, 106.22 (t, J = 26.1 Hz), 63.36, 57.93, 53.85, 32.48. HRMS (ESI+)
m/z calcd for C28H25F2N6O5S [M+H]+, 595.1570; found, 595.1575.
4.1.41.
2,4-difluoro-N-(5-(4-((4-(2-hydroxyethoxy)-3-methoxyphenyl)amino)quinazolin-6-yl)-2-
methoxypyridin-3-yl)benzenesulfonamide (7o)
1
56.8% yield, mp 123-125 ºC. H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H, NH), 9.85 (s, 1H, NH),
8.81 (s, 1H, Ar-H), 8.56 (s, 2H, Ar-H), 8.18 (d, J = 2.2 Hz, 1H, Ar-H), 8.16 (dd, J = 8.7, 1.4 Hz, 1H, Ar-H),
7.85 (d, J = 8.6 Hz, 1H, Ar-H), 7.75 (dd, J = 14.8, 8.4 Hz, 1H, Ar-H), 7.64 – 7.58 (m, 1H, Ar-H), 7.44 (d, J
= 2.2 Hz, 1H, Ar-H), 7.39 (dd, J = 8.6, 2.2 Hz, 1H, Ar-H), 7.21 (td, J = 8.5, 2.2 Hz, 1H, Ar-H), 7.03 (d, J =
8.7 Hz, 1H, Ar-H), 4.89 (t, J = 5.5 Hz, 1H, OH), 4.00 (t, J = 5.1 Hz, 2H, CH2), 3.82 (s, 3H, CH3), 3.74 (dd, J
= 10.3, 5.2 Hz, 2H, CH2), 3.64 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) δ 165.51 (dd, J = 253.9, 11.7
Hz), 159.89 (dd, J = 257.7, 13.5 Hz), 158.42, 158.31, 155.27, 149.45, 149.14, 145.40, 143.51, 135.71,
134.35, 132.70, 132.27 (d, J = 10.9 Hz), 131.53, 129.32, 129.02, 125.56 (dd, dd, J = 13.59, 3.0 Hz), 120.60,
120.02, 115.83, 115.68, 113.58, 112.20 (d, J = 24.9 Hz), 108.55, 106.21 (t, J = 26.2 Hz), 70.91, 60.11, 56.01,
53.83. HRMS (ESI+) m/z calcd for C29H26F2N5O6S [M+H]+, 610.1566; found, 610.1564.
4.1.42.
2,4-difluoro-N-(5-(4-((3-chloro-4-(2-hydroxyethoxy)phenyl)amino)quinazolin-6-yl)-2-
methoxypyridin-3-yl)benzenesulfonamide (7p)
1
61.6% yield, mp 166-168 ºC. H NMR (400 MHz, DMSO) δ 10.36 (s, 1H), 9.89 (s, 1H), 8.78 (d, J =
1.3 Hz, 1H), 8.60 (s, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.16 (dd, J = 6.1, 2.0 Hz, 2H), 7.99 (d, J = 2.6 Hz, 1H),
7.86 (d, J = 8.7 Hz, 1H), 7.80 – 7.71 (m, 2H), 7.63 – 7.53 (m, 1H), 7.24 (d, J = 9.0 Hz, 1H), 7.20 (dd, J =
8.6, 2.2 Hz, 1H), 4.91 (t, J = 5.3 Hz, 1H), 4.12 (t, J = 5.1 Hz, 2H), 3.78 (dd, J = 10.1, 5.1 Hz, 2H).13C NMR
(100 MHz, DMSO) δ 165.54 (dd, J = 252.3, 11.4Hz), 159.91 (dd, J = 242.7, 13.6 Hz), 158.44, 158.14,
155.16, 151.12, 149.48, 143.53, 135.59, 134.54, 133.02, 132.30 (d, J = 10.6 Hz), 131.72, 129.31, 129.01,

125.63 (dd, J = 14.3, 3.6 Hz), 124.75, 123.01, 121.31, 120.59, 120.08, 115.77, 114.44, 112.22 (dd, J = 21.6,
3.3 Hz), 106.23 (t, J = 22.9 Hz), 71.35, 60.01, 53.86. HRMS (ESI+) m/z calcd for C28H23F2N5O5S [M+H]+,
614.1071; found, 614.1040.
4.1.43.
2,4-difluoro-N-(5-(4-((3-fluoro-4-(2-hydroxyethoxy)phenyl)amino)quinazolin-6-yl)-2-
methoxypyridin-3-yl)benzenesulfonamide (7q)
56.8% yield, mp 163-165 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (brs, 1H, NH), 9.93 (s, 1H, NH),
8.80 (d, J = 1.6 Hz, 1H, Ar-H), 8.61 (s, 1H, Ar-H), 8.56 (d, J = 2.4 Hz, 1H, Ar-H), 8.17-8.15 (m, 2H, Ar-H),
7.96 (s, 1H, Ar-H), 7.89-7.87 (m, 2H, Ar-H), 7.79-7.73 (m, 1H, Ar-H), 7.82-7.53 (m, 2H, Ar-H), 7.27-7.19
(m, 2H, Ar-H), 4.93 (brs, 1H, OH), 4.10 (t, J = 4.8 Hz, 2H, CH2), 3.77 (brs, 2H, CH2), 3.66 (s, 3H, CH3). 13C
NMR (100 MHz, DMSO) δ 165.56 (dd, J = 252.0, 12.1 Hz), 163.48, 162.77, 159.92 (dd, J = 242.6, 13.3 Hz),
158.44, 158.14, 155.04, 152.67, 150.26, 149.35, 143.58, 143.51, 143.41, 135.82, 134.57, 132.78 (d, J = 9.8
Hz), 132.30 (d, J = 10.3 Hz), 131.75, 129.30, 129.10 (d, J = 9.0 Hz), 129.00, 125.62 (dd, J = 14.5, 3.6 Hz),
120.63, 120.03, 119.10 (d, J = 3.2 Hz), 115.77, 115.42 (d, J = 2.5 Hz), 112.23 (dd, J = 22.3, 3.5 Hz), 111.75,
111.52, 106.23 (t, J = 26.2 Hz), 73.89, 71.43, 60.03, 53.85, 36.24, 31.23, 25.42. HRMS (ESI+) m/z calcd for
C28H23F3N5O5S [M+H]+, 598.1367; found, 598.1370.
4.2. Biological assay methods
4.2.1. Cell culture
A549, MCF-7 and MCF-7 cells were purchased from the American Type Culture Collection (Manassas,
VA, USA). Cells were grown in DMEM (MCF-7 and MCF-7) or RPMI1640 (A549) 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 °C 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 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). Percent growth inhibition = 100 - 100
× (ODsample - ODDay0) / (ODneg control - ODDay0).
4.2.3. PI3K and EGFR 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 2.5-fold PI3K Reaction buffer with 2.5-fold Lipid Substrate working
solution).Transfer 8μl of prepared working kinase solution to the wells containing 1μl of test compound.
Incubate at room temperature for 15 min to allow inhibitor binding to kinase. Start reaction by adding 1 μl of
250 μM ATP. After incubation at room temperature for 60 min, 10uL of ADP-GloTM reagent was added
per well. The plates were incubated at room temperature for 40 min and then 20 μl of kinase detection
reagent was added. After incubation at room temperature for 40 min, 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).
A mobility shift assay was used to measure the potency of title compounds against EGFR. The kinase
base buffer was consist of 50 mM HEPES (pH 7.5), 10 mM MgCl2, 2 mM DTT and 0.0015% Brij35, while
the stop buffer contained a mixture of 100 mM HEPES (pH 7.5), 50 mM EDTA, 0.015% Brij-35 and 0.2%
Coating Reagent #3. Dilute the tested compounds to 50-fold of the final desired highest inhibitor
concentration in reaction by 100% DMSO. Subsequently, dilute the compounds by transferring 30 μl to 60
μl of 100% DMSO in the next well and so forth for a total of 10 concentrations. Add 100 μl of 100% DMSO
to two empty wells for no compound control and no enzyme control in the same 96-well plate. Mark the
plate as source plate. Transfer 10 μl of compound from source plate to a new 96-well plate as the
intermediate plate. Add 90 μl of 1-fold kinase buffer to each well of the intermediate plate. Mix the
compound in intermediate plate for 10 min on the shaker. The assay plate was prepared after transferring 5
μl of each well from the 96-well intermediate plate to a 384-well plate in duplicates. The prepared enzyme
solution was added to the assay plate, which was then incubated at room temperature for 10 min, followed
by the addition 10 μl of 2.5-fold peptide solution (FAM-labeled peptide and ATP in kinase base buffer). The
mixture was incubated at 28 °C for another 1 h, then add 25 μl of stop buffer to stop the reaction. The
convertion data was copied from Caliper program, and the values were converted to inhibition values.
Percent inhibition = (max - conversion)/(max - min) × 100. Fit the data in XLFit excel add-in version 4.3.1
to obtain IC50 values. Equation used is: Y = Bottom + (Top - Bottom)/(1 + (IC50/X)^HillSlope)
4.2.4. Western blot analysis
Total cell were collected and washed with cold PBS. Cells were then lysed with RIPA buffer
(BIOSESANG, Korea) containing protease and phosphatase inhibitor cocktails (GenDEPOT, Barker, TX).
The protein concentration was determined, and equal amounts of protein samples were subjected to 10%
SDS-PAGE followed by transferring to PVDF membranes, and incubated with primary antibodies specific
for total-Akt, phospho-Akt (Ser473) purchased from Abcam (Cambridge, United Kingdom), GADPH
purchased from Sigma–Aldrich (St Louis, MO, USA). Exposures were obtained using Image Quant LAS

4000 biomolecular imager (GE Healthcare).
4.2.5. Colony formation assay
MCF-7 cells were seeded in a 6-well plates with a density of 3×103 cells/well and then were cultured
with different concentrations (0, 0.2 0.4, 0.8 μM) of 7b for 72h. After being shifted into free-drug fresh
medium for one week, cells were stained with crystal violet. Colony images were obtained by a digital
camera.
4.2.6. Annexin V-FITC staining assay
MCF-7 cells were seeded in a 6-well plates (2×105 cells/well) and treated with 7b mentioned above
for 48h. At the end of treatments, the cells were harvested and washed by cold PBS. Then the resuspended
cells (1×106) were stained using Annexin V-FITC/PI staining kit (Nanjing KeyGen biotech, Nanjing,
China) at room temperature for 15 min in the dark and analyzed by flow cytometry (BD FACSCalibur, BD
Biosciences).
4.2.7. Hoechst33342 staining assay
MCF-7 cells were seeded in a 6-well plates (2×105 cells/well) and incubated for 12h. Cells were
treated with 7b as mentioned above for 48 h. The control group was incubated with culture medium. After
treatment the cells were stained with Hoechst 33342 for 30min. After washing off unbound dye, the cells
were photographed by a fluorescence microscope (Olympus, Tokyo, Japan).
4.2.8. Molecular docking studies
The crystal structure PI3K (PDB entry code: 3L08) in complex with Omipalisib was used for molecular
modeling. The AutoDock 4.2 was used to perform docking calculations. Polar hydrogens and partial charges
were added by Sybyl 6.9.1, and energy minimization was made employing both steepest descent and
conjugate gradients protocols. 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. Accelrys
Discovery Studio Visualizer 4.0 was used for graphic display.
Acknowledgments
Financial supported by Career Development Support Plan for Young and Middle-aged Teachers in
Shenyang Pharmaceutical University (Grant No. ZQN2015023) is gratefully acknowledged.
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A series of 4-aminoquinazolines derivatives containing 6-sulfonamide substituted pyridyl group were
designed.
4-Aminoquinazolines derivatives acted as potent Pan-PI3K inhibitors.
Compound 7b could be as potential PI3Kα inhibitor with an IC50 value of 0.20ꢀnM.
Compound 7b could decrease the phospho-Akt (T308) in a dose-dependent manner.