Design, synthesis and biological evaluation of novel 4-aminoquinazolines as dual target inhibitors of EGFR-PI3Kα

Article abstract of DOI:10.1016/j.ejmech.2018.01.081

The overexpression of EGFR correlates with rapidly progressive disease, resistance to chemotherapy and poor prognosis. In certain human cancers, PI3K works synergistically with EGFR to promote proliferation, survival, invasion and metastasis. Development

Full text of DOI:10.1016/j.ejmech.2018.01.081

Accepted Manuscript
Design, synthesis and biological evaluation of novel 4-aminoquinazolines as dual
target inhibitors of EGFR-PI3Kα
Huai-Wei Ding, Cheng-Long Deng, Dan-Dan Li, Dan-Dan Liu, Shao-Meng Chai, Wei
Wang, Yan Zhang, Kai Chen, Xin Li, Jian Wang, Shao-Jiang Song, Hong-Rui Song
PII:
S0223-5234(18)30100-4
10.1016/j.ejmech.2018.01.081
EJMECH 10158
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 August 2017
Revised Date: 22 January 2018
Accepted Date: 24 January 2018
Please cite this article as: H.-W. Ding, C.-L. Deng, D.-D. Li, D.-D. Liu, S.-M. Chai, W. Wang, Y. Zhang,
K. Chen, X. Li, J. Wang, S.-J. Song, H.-R. Song, Design, synthesis and biological evaluation of novel 4-
aminoquinazolines as dual target inhibitors of EGFR-PI3Kα, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.01.081.
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ACCEPTED MANUSCRIPT

Design, synthesis and biological evaluation of novel 4-aminoquinazolines as dual target
ACCEPTED MANUSCRIPT
inhibitors of EGFR-PI3Kα
Huai-Wei Ding a, Cheng-Long Deng a, Dan-Dan Li b, Dan-Dan Liu a,Shao-Meng Chai c, Wei Wang b, Yan
Zhang b, Kai Chen a, Xin Li b, Jian Wang a, Shao-Jiang Song b, Hong-Rui Song a,*
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
c Key Laboratory for Molecular Enzymology & Engineering, Jilin University, China
* Corresponding authors.
Email address: hongruisong@163.com.

Abstract：The overexpression of EGFR correlates with rapidly progressive disease, resistance to
ACCEPTED MANUSCRIPT
chemotherapy and poor prognosis. In certain human cancers, PI3K works synergistically with EGFR to
promote proliferation, survival, invasion and metastasis. Development of dual-target drugs against EGFR
and PI3K has therapeutic advantage and was an attractive approach against tumors. In this work, based on
the molecular docking and previous studies, a series of 4-aminoquinazolines derivatives containing
6-sulfonamide substituted pyridyl group were rationally designed and identified as potent EGFR and PI3K
dual inhibitors. The cytotoxicity experiment results showed that this series of compounds could effectively
inhibit cell growth. The kinase assay demonstrated that 6c and 6i had high inhibition for EGFR and
selectivity for PI3Kα distinguished from other isoforms. Further experiments showed that 6c could induce
cell cycle arrest in G1 phase and apoptosis in BT549 cells. The western blot assay indicated that 6c inhibited
the proliferation of BT549 cell through EGFR and PI3Kα/Akt signaling pathway. Our study suggested that
compound 6c was a potential dual inhibitors of EGFR and PI3Kα.
Keywords: EGFR, PI3K, dual target, 4-aminoquinazolines, antiproliferative effects, anticancer agents

1. Introduction
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The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinase,
which includes four members: ErbB-1/EGFR, ErbB-2/HER-2/neu, ErbB-3/HER-3, and ErbB-4/HER-4 [1-2].
The overexpression of EGFR activates a series of downstream signals via several pathways and leads to
tumor growth and progression, including proliferation, differentiation, angiogenesis, inhibition of apoptosis
and invasiveness. The major pathways implicated in EGFR signaling include RAS/MAPK, PI3K/Akt, and
JAK/STAT [3-5]. A lot of inhibitors targeting EGFR have been successfully developed and contain six
launched drugs, Gefitinib [6], Lapatinib [7], Erlotinib [8], Vandetanib [9], Afatinib [10] and Icotinib [11]
(Fig.1).
The phosphatidylinositol 3-kinases (PI3Ks) are members of a unique group of intracellular lipid kinases
[12]. The PI3K family is divided into four different classes: Class I, Class II, Class III and Class IV. The
classifications are based on the primary structure, regulation, and in vitro lipid substrate specificity. Of these,
the most commonly studied are the class I enzymes that are activated directly by cell surface receptors. Class
I PI3Ks are further divided into PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ [13-14]. The abnormal activation of PI3K
signaling pathway leads to the occurrence of various cancers [15], suggesting that targeting inhibition of
PI3K may be a potential strategy agaist cancer. Several inhibitors targeting PI3K have been developed and
are being evaluated in preclinical studies and early clinical trials [16-17]. Among them, Omipalisib
(GSK2126458) and Dactolisib (NVP-BEZ235) has been identified as highly potent inhibitors of PI3K and
are currently under evaluation in clinical trials for oncology applications [18-19] (Fig.2).
Quinazoline derivatives, especially 4-anilinoquinazolines, have attracted interests over the years for
their multiple biological activities, notably as EGFR inhibitors [20]. In recent years, 4-aminoquinazoline
derivatives also have been reported as PI3K inhibitors [21-23]. We had a good understanding of the binding
mode of Gefitinib and Omipalisib based on the reported corresponding X-ray structure of kinase domain [18,
24]. Gefitinib and Omipalisib all using the position-1 nitrogen as a hinge binder and all having an aryl group
at the position-4, we hypothesized that we would gain dual target compounds by adjusting the substituent
groups of molecules. Coincidentally, we found that Dactolisib could be cut off the N-methyl formamide
group to obtain a “transition state” (Fig.2), which had structural similarity to Gefitinib and Omipalisib. This
discovery further encourages the development of our work to design dual inhibitors. In this paper, as an
attempt to pursue new antitumor agents potently inhibiting both EGFR and PI3Kα, we rationally designed
and synthesized a new series of 4-aminoquinazolines derivatives from Gefitinib, Omipalisib and Dactolisib
(Fig.2). The biological activities evaluation suggest that compound 6c and 6i could be as dual inhibitors of
EGFR and PI3Kα.
(Fig. 1. is here)

(Fig. 2. is here)
2. Results and discussion
2.1. Chemistry
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The synthetic routes for the target compounds are outlined in Schemes 1. Sulfonylation of 1 with three
benzenesulfonyl chlorides to yield 2a-c, which were then subjected to Suzuki coupling with
bis(pinacolato)diborane to afford arylboronic ester 3a-c. Intermediate 4 reacted with different substituted
anilines to abtain 5a-p, which were coupled with 3a-c via Suzuki reaction to afford the target compounds
6a-r.
(Scheme 1. is here)
2.2. Antiproliferative assays in vitro
All synthesized compounds were evaluated for their cytotoxicities in vitro against six human cancer
cell lines including A549, BT549, HCT-116, MCF-7, SK-HEP-1 and SNU638 cells. The antiproliferative
results of all the compounds were summarized in Table 1. As shown in Table 1, the results showed that most
of the derivatives exhibited potent antiproliferative effects. The structure-activity relationships (SARs)
suggested that all of compounds with methyl substituent on R2 position displayed better antiproliferative
activities than those substituted by N-butyl or phenyl group on the same position, including 6a-m vs.6n-p.
Especially from representative compounds 6i, 6o and 6p, gave the hints that the larger the volume of
sulfonamide, the worse the activity. Compounds 6b-d and 6f-l mainly showed better activities against six
cell lines than Gefitinib, but weaker than Dactolisib. In order to let target compounds closer to GSK2126458,
nitrogen atoms were introduced in the position-3’ of the 4-anilinoquinazolines to get the compound 6q and
6r. Interestingly, the activity was not increased but decreased, including 6b vs.6q and 6f vs.6r.
(Table 1 is here)
2.3. EGFR and PI3K enzymatic activity assays
To elucidate the mechanism of antiproliferative activities of these active compounds, we selected the
most potent compounds 6c and 6i for the further EGFR and PI3K enzymatic activity assays. Gefitinib and
Dactolisib were selected as the positive drugs. As shown in Table 2, kinase inhibition activity assay
indicated that the activity of 6c and 6i has reached the nanomolar level. Two compounds above showed a
higher inhibitory activity against PI3Kα when compared to that of other class I PI3Ks (PI3Kβ, PI3Kγ and
PI3Kδ). Coincidentally, the EGFR inhibitory activity of 6c was basically equal to Gefitinib.
(Table 2 is here)
Considering to the structure similarity to Gefitinib, the antiproliferative results and the BT549 cells
presenting more sensitive to this series of compounds, we selected compound 6c and BT549 cells for further
studies.

2.4. Effect of 6c on the growth of BT549 cells
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In order to further investigate the antiproliferation of 6c on BT549 cells, the colony formation assay
was used to measure the cell viability. As shown in Fig.3, the cell proliferation was inhibited by 6c in a
dose-dependent manner. Analyzing of the clonogenicity viability, the colony formation rate of 2.5 µM was
55.6% more than 25.7% of 5.0 µM. Compared with control group, 6c almost completely inhibited colony
formation at a concertration of 5.0 µM. The results of antiproliferative assay and colony formation assay
suggested that 6c can inhibit the proliferation of BT549 cells prominently.
(Fig. 3. is here)
2.5. Apoptosis induced by compound 6c
Though 6c was proved antitumor effects, the mechanism was not clear. Annexin V-PI double staining
assay was used to examine the effect of 6c on cells apoptosis. As shown in Fig.4 (A and B), 6c can induce
apoptosis compared with control. The percentage of late apoptotic at 5.0 µM was more than at 1.0 µM, it is
indicated that 6c induced the early apoptotic transform to late apoptotic following with the alterative
concentration.
In order to further evaluate 6c-induced apoptosis in BT549 cells, cells were staining with DAPI and
analyzing by fluorescence microscope. As shown in Fig.4 (C), treated with different concentrations of 6c,
the brighter fluorescence was seen. Compared with control which had normal nuclear morphology after
DAPI staining, 6c treatment group marked with nuclear fragmentation and condensation of chromatin.
Moreover, this situation was more obvious with the increased concentration. These results indicated that 6c
was capable of inducing apoptosis in BT549 cells.
(Fig. 4. is here)
2.6. Effect of compound 6c on cell cycle
To determine whether inhibitory effect of 6c on the cell survival was related to the cell cycle,
propidium iodide(PI) staining assay was used. After treatment with 6c for 24 h, as shown in Fig.4 (D and E),
the percentage of G1 phase was increased obviously and had a dose-dependent manner. The arrested G1
phase ratio increased from 55.9% to 80.7% with increasing 6c concentration from 0 µM to 5.0 µM. This
result showed that 6c induce cell cycle arrest in G1 phase.
2.7. Western blot assay
To further determine whether the EGFR and PI3K signaling were affected by compound 6c, Western
blot assay was used to evaluate the effects of 6c on EGFR and phospho-EGFR and the PI3K related protein
levels including Akt and phospho-Akt (p-Akt, S473) in BT549 cells. As shown in Fig.5 (A and B),
compound 6c decreased the phosphorylation levels of EGFR and Akt in a dose-dependent manner. The rate
of p-EGFR/EGFR and p-Akt/Akt was down-regulated by 6c. Therefore, the data suggested that 6c might

inhibit the proliferation of BT549 cell though EGFR and Akt signaling pathway.
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(Fig. 5. is here)
2.8. Molecular docking
Molecular docking study was performed to elucidate the binding model of 6c in the binding site of
EGFR and PI3K. As showed in Fig.6 (A), the nitrogen of quinazolyl group formed a hydrogen bond with the
side chain of Met793 in the hinge binder region of EGFR. Besides, the amidogen of sulfonamide group
forms extra two hydrogen bond interaction with Arg841 and Leu799 compare to Gefitinib. By Fig.6 (B), we
know that the folded form of compound 6c in the protein pocket was very similar to that of Gefitinib.
Compound 6c was also docked onto the PI3K-binding domain. The structure of PI3K kinase was obtained
from protein data bank (PDB entry 3L08). The Fig.6 (C) showed that the ligand 6c formed three strong
hydrogen bonds. The nitrogen of quinazolyl group formed a hydrogen bond with the side chain of Val882 in
the hinge binder region of PI3K. Another two hydrogen bonds were the oxygen of sulfonamide group
interaction with Lys833 and nitrogen of pyridyl group formed a hydrogen bond with the conserved water
molecule. As showed in Fig.6 (D), the folded form of compound 6c in the protein pocket was very similar to
that of Omipalisib.
The docking analysis indicated that compound 6c could fit into the binding site of EGFR and PI3K
kinases, which also indicated that this compound may be a potent EGFR and PI3K inhibitor.
(Fig. 6. is here)
3. Conclusion
In summary, a series of compounds containing 4-aminoquinazolines were designed and synthesized.
Their antiproliferative activities against six cancer cell lines showed that the BT549 cells presented more
sensitive to these compounds. Eventually, compound 6c and BT549 cells were selected for further research.
Thereafter, in order to identify the antiproliferation of 6c on BT549 cells, the colony formation assay and
apoptosis were evaluated in vitro. The results suggested that 6c could inhibit the proliferation of BT549 cells
prominently and induce the early apoptotic transform to late apoptotic following with the alterative
concentration. In order to verify the further mechanisms of 6c, the propidium iodide (PI) staining assay was
developed and the results showed that 6c arrested G1 phase of cell cycle. we also conducted Western-blot
experiment and the results showed that 6c inhibited the proliferation of BT549 cell though EGFR and
PI3Kα/Akt signaling pathway. According to these results, compound 6c could be as a potential
EGFR/PI3Kα dual target inhibitor and could be considered as a potential candidate for anticancer drug
development.
4. Experimental section
4.1. Chemistry and chemical methods

All reagents and solvents were commercially available without further purification. 1H NMR spectra
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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. N-(5-bromo-2-methoxypyridin-3-yl)butane-1-sulfonamide (2a)
To a solution of 5-bromo-2-methoxypyridin-3-amine (2.01 g, 10 mmol) in pyridine (50 ml) at 0
ºC was added methanesulfonyl chloride (1.72 g, 11 mmol). Then the mixture was stirred at room
temperature 24 h. Pyridine was removed at reduced pressure and add water (100 ml), extracted with
ethyl acetate (3 × 100 ml), the organic layer was washed with water (50 ml), dried with Na2SO4 and
1
evaporated to give compound 2a as a white solid. Yield 83.6%, mp 150-151 ºC. H NMR (400
DMSO-d6) δ 9.49 (s,1H, NH), 8.09 (d,
(s, 3H, OCH3), 3.11(s, 3H, CH3). ESI-MS: m/z 281.1 [M+H]+.
Compounds 2b-c was synthesized according to the procedure described in 2a
4.1.2. N-(5-bromo-2-methoxypyridin-3-yl)butane-1-sulfonamide (2b
J = 2.4 Hz, 1H, Ar-H), 7.79 (d, J = 2.0 Hz, 1H, Ar-H), 3.91
.
)
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]+.
4.1.3. N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (2c)
85.6% yield. mp 163-165 ºC. 1H 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,
OCH3). ESI-MS: m/z 376.9 [M-H]+.
4.1.4. 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)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.
DMF was removed under reduced pressure and add water (100 ml), extracted with ethyl acetate (3 × 100 ml),
the organic layer was washed with water (20 ml), dried with Na2SO4 and evaporated to give compound 3a as
a white solid (1.98 g, 60.4% yield). mp 90-92 ºC. 1H 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, OCH3), 3.01 (s, 3H, CH3), 1.30 (s, 12H,
CH3). ESI-MS: m/z 329.1 [M+H]+.
Compounds 3b-c were synthesized according to the procedure described in 3a.

4.1.5. N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)butane-1-sulfonamide (3b)
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64.3% yield. mp 91-93 ºC.1H NMR (400 MHz, DMSO) 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]+.
4.1.6.
2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide
(3c)
78.8% yield. mp 171-173 ºC. 1H 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), 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]+
4.1.7. 6-bromo-N-(3-fluorophenyl)quinazolin-4-amine (5a)
A solution of the 4 (0.48 g, 2 mmol), 3-fluoroaniline (0.24g, 2.2 mmol) in isopropanol (30 ml) were
stirred at 90 ºC for 4 h. Isopropanol 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 white solid (0.58 g,
91.8% yield). mp 167-169 ºC. 1H NMR (400 MHz, DMSO) δ 12.05 (s, 1H, NH), 9.45 (s, 1H, Ar-H), 9.03 (s,
1H, Ar-H), 8.26 (d, J = 8.9 Hz, 1H, Ar-H), 8.02 (d, J = 8.9 Hz, 1H, Ar-H), 7.78 (d, J = 10.9 Hz, 1H, Ar-H),
7.67 (d, J = 8.1 Hz, 1H, Ar-H), 7.53 (dd, J = 15.1, 8.0 Hz, 1H, Ar-H), 7.18 (td, J = 8.4, 1.8 Hz, 1H, Ar-H).
ESI-MS: m/z 315.9 [M-H]+.
Compounds 5b-p were synthesized according to the procedure described in 5a.
4.1.8. 6-bromo-N-(4-fluorophenyl)quinazolin-4-amine (5b)
90.6% yield. mp 228-230 ºC. 1H NMR (400 MHz, DMSO) δ 11.01 (s, 1H, NH), 9.15 (d, J = 1.5 Hz, 1H,
Ar-H), 8.77 (s, 1H, Ar-H), 8.10 (dd, J = 8.9, 1.7 Hz, 1H, Ar-H), 7.84 (dd, J = 8.7, 6.1 Hz, 3H, Ar-H), 7.29 (t,
J = 8.8 Hz, 2H, Ar-H). ESI-MS: m/z 315.9 [M-H]+.
4.1.9. 6-bromo-N-(2,4-difluorophenyl)quinazolin-4-amine (5c)
88.9% yield. mp 197-199 ºC. 1H NMR (400 MHz, DMSO) δ 10.55 (s, 1H, NH), 8.89 (d, J = 1.8 Hz, 1H,
Ar-H), 8.64 (s, 1H, Ar-H), 8.09 (dd, J = 8.9, 2.0 Hz, 1H, Ar-H), 7.80 (d, J = 8.9 Hz, 1H, Ar-H), 7.58 (td, J =
8.8, 6.3 Hz, 1H, Ar-H), 7.46 - 7.41(m, 1H, Ar-H), 7.25 - 7.15 (m, 1H, Ar-H). ESI-MS: m/z 333.9 [M+H]+.
4.1.10. 6-bromo-N-(3-chloro-4-fluorophenyl)quinazolin-4-amine (5d)
94.4% yield. mp 229-231 ºC. 1H NMR (400 MHz, DMSO) δ 11.92 (s, 1H, NH), 9.34 (d, J = 1.1 Hz, 1H,
Ar-H), 9.34 (d, J = 1.1 Hz, 1H, Ar-H), 9.00 (s, 1H, Ar-H), 8.25 (dd, J = 8.8, 1.4 Hz, 1H, Ar-H), 8.09 (dd, J =
6.7, 2.3 Hz, 1H, Ar-H), 7.97 (d, J = 8.9 Hz, 1H, Ar-H), 7.80 (ddd, J = 8.6, 4.0, 2.7 Hz, 1H, Ar-H), 7.56 (t, J
= 9.0 Hz, 1H, Ar-H). ESI-MS: m/z 351.9 [M+H]+.

4.1.11. 6-bromo-N-(4-(trifluoromethoxy)phenyl)quinazolin-4-amine (5e)
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96.1% yield. mp 178-180 ºC. 1H NMR (400 MHz, DMSO) δ 11.90 (s, 1H, NH), 9.34 (d, J = 1.7 Hz, 1H,
Ar-H), 8.97 (s, 1H, Ar-H), 8.26 (dd, J = 8.9, 1.8 Hz, 1H, Ar-H), 7.97 (d, J = 8.9 Hz, 1H, Ar-H), 7.90 (d, J =
9.0 Hz, 2H, Ar-H), 7.51 (d, J = 8.5 Hz, 2H, Ar-H). ESI-MS: m/z 381.8 [M-H]+.
4.1.12. 6-bromo-N-(4-methoxyphenyl)quinazolin-4-amine (5f)
86.2% yield. mp 195-197 ºC. 1H NMR (400 MHz, DMSO) δ 11.72 (s, 1H, NH), 9.27 (s, 1H, Ar-H),
8.89 (s, 1H, Ar-H), 8.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.95 (d, J = 8.9 Hz, 1H, Ar-H), 7.65 (d, J = 8.8 Hz, 2H,
Ar-H), 7.05 (d, J = 8.9 Hz, 2H, Ar-H), 3.81 (s, 3H, OCH3). ESI-MS: m/z 330.1 [M+H]+.
4.1.13. 6-bromo-N-(3,4-dimethoxyphenyl)quinazolin-4-amine (5g)
87.5% yield. mp 183-185 ºC. 1H NMR (400 MHz, DMSO) δ 11.67 (s, 1H, NH), 9.26 (d, J = 1.3 Hz, 1H,
Ar-H), 8.92 (s, 1H, Ar-H), 8.23 (dd, J = 8.8, 1.7 Hz, 1H, Ar-H), 7.94 (d, J = 8.9 Hz, 1H, Ar-H), 7.40 (d, J =
2.2 Hz, 1H, Ar-H), 7.33 (dd, J = 8.6, 2.2 Hz, 1H, Ar-H), 7.06 (d, J = 8.7 Hz, 1H, Ar-H), 3.81 (s, 3H, OCH3),
3.78 (s, 3H, OCH3). ESI-MS: m/z 360.1 [M+H]+.
4.1.14. 6-bromo-N-(3-chloro-4-methoxyphenyl)quinazolin-4-amine (5h)
91.4% yield. mp 260-262 ºC. 1H NMR (400 MHz, DMSO) δ 10.13 (s, 1H, NH), 8.91 (d, J = 1.7 Hz, 1H,
Ar-H), 8.66 (s, 1H, Ar-H), 8.00 (dd, J = 5.7, 2.2 Hz, 2H, Ar-H), 7.77 - 7.73 (m, 2H, Ar-H), 7.21 (d, J = 9.0
Hz, 1H, Ar-H), 3.88 (s, 3H, OCH3). ESI-MS: m/z 361.8 [M+H]+.
4.1.15. 6-bromo-N-(6-fluoropyridin-3-yl)quinazolin-4-amine (5i)
85.2% yield. mp 197-199 ºC. 1H NMR (400 MHz, DMSO) δ 10.65 (s, 1H, NH), 9.07 (dd, J = 7.8, 1.9
Hz, 1H, Ar-H), 8.69 (s, 2H, Ar-H), 8.48 - 8.44 (m, 1H, Ar-H), 8.05 (dd, J = 8.8, 1.8 Hz, 1H, Ar-H), 7.79 (d,
J = 8.9 Hz, 1H, Ar-H), 7.27 (dd, J = 8.7, 3.1 Hz, 1H, Ar-H). ESI-MS: m/z 316.8 [M-H]+.
4.1.16. 6-bromo-N-(6-methoxypyridin-3-yl)quinazolin-4-amine (5j)
87.3% yield. mp 180-182 ºC. 1H NMR (400 MHz, DMSO) δ 11.61 (s, 1H, NH), 9.19 (s, 1H, Ar-H),
8.89 (s, 1H, Ar-H), 8.50 (d, J = 2.5 Hz, 1H, Ar-H), 8.22 (dd, J = 8.9, 1.7 Hz, 1H, Ar-H), 8.05 (dd, J = 8.8, 2.6
Hz, 1H, Ar-H), 7.91 (d, J = 8.9 Hz, 1H, Ar-H), 6.96 (d, J = 8.9 Hz, 1H, Ar-H), 3.90 (s, 3H, OCH3). ESI-MS:
m/z 331.1 [M+H]+.
4.1.17. methyl 3-((6-bromoquinazolin-4-yl)amino)benzoate (5k)
95.6% yield. mp 218-220 ºC. 1H NMR (400 MHz, DMSO) δ 11.40 (s, 1H, NH), 9.19 (s, 1H, Ar-H),
8.93 (s, 1H, Ar-H), 8.39 (s, 1H, Ar-H), 8.21 (dd, J = 8.9, 1.7 Hz, 1H, Ar-H), 8.15 (dd, J = 8.0, 0.9 Hz, 1H,
Ar-H), 7.91 (d, J = 8.9 Hz, 1H, Ar-H), 7.87 (d, J = 7.8 Hz, 1H, Ar-H), 7.64 (t, J = 7.9 Hz, 1H, Ar-H), 3.90 (s,
3H, OCH3). ESI-MS: m/z 358.1 [M+H]+.
4.1.18. ethyl 3-((6-bromoquinazolin-4-yl)amino)benzoate (5l)

94.7% yield. mp 196-198 ºC. 1H NMR (400 MHz, DMSO) δ 12.04 (s, 1H, NH), 9.39 (d, J = 1.8 Hz, 1H,
ACCEPTED MANUSCRIPT
Ar-H), 9.01 (s, 1H, Ar-H), 8.34 (s, 1H, Ar-H), 8.26 (dd, J = 8.9, 1.9 Hz, 1H, Ar-H), 8.10 (dd, J = 8.1, 0.9 Hz,
1H, Ar-H), 8.02 (d, J = 8.9 Hz, 1H, Ar-H), 7.89 (d, J = 7.9 Hz, 1H, Ar-H), 7.64 (t, J = 7.9 Hz, 1H, Ar-H),
4.36 (q, J = 7.1 Hz, 2H, CH2), 1.35 (t, J = 7.1 Hz, 3H, CH3). ESI-MS: m/z 372.1 [M+H]+.
4.1.19. methyl 5-((6-bromoquinazolin-4-yl)amino)-2-fluorobenzoate (5m)
81.6% yield. mp 178-180 ºC. 1H NMR (400 MHz, DMSO) δ 11.23 (s, 1H, NH), 9.11 (d, J = 1.6 Hz, 1H,
Ar-H), 8.89 (s, 1H, Ar-H), 8.30 (dd, J = 6.5, 2.7 Hz, 1H, Ar-H), 8.17 (ddd, J = 8.8, 7.9, 2.8 Hz, 2H, Ar-H),
7.87 (d, J = 8.9 Hz, 1H, Ar-H), 7.54 - 7.44 (m, 1H, Ar-H), 3.91 (d, J = 8.0 Hz, 3H, OCH3). ESI-MS: m/z
376.1 [M+H]+.
4.1.20. methyl 5-((6-bromoquinazolin-4-yl)amino)-2-chlorobenzoate (5n)
84.3% yield. mp 185-187 ºC. 1H NMR (400 MHz, DMSO) δ 11.70 (s, 1H, NH), 9.25 (s, 1H, Ar-H),
9.00 (s, 1H, Ar-H), 8.32-8.24 (m, 2H, Ar-H), 8.08 (d, J = 8.8 Hz, 1H, Ar-H), 7.95 (d, J = 7.8 Hz, 1H, Ar-H),
7.72 (d, J = 8.3 Hz, 1H, Ar-H), 3.91 (s, 3H, OCH3). ESI-MS: m/z 392.1 [M+H]+.
4.1.21. methyl 4-((6-bromoquinazolin-4-yl)amino)-2-methoxybenzoate (5o)
93.5% yield. mp 165-167 ºC. 1H NMR (400 MHz, DMSO) δ 11.63 (s, 1H, NH), 9.31 (s, 1H, Ar-H),
9.03 (s, 1H, Ar-H), 8.25 (dd, J = 8.9, 1.9 Hz, 1H, Ar-H), 7.97 (d, J = 8.9 Hz, 1H, Ar-H), 7.79 (d, J = 8.5 Hz,
1H, Ar-H), 7.73 (d, J = 1.6 Hz, 1H, Ar-H), 7.61 (dd, J = 8.5, 1.8 Hz, 1H, Ar-H), 3.87 (s, 3H, OCH3), 3.81 (s,
3H, OCH3). ESI-MS: m/z 388.1 [M+H]+.
4.1.22. 6-bromo-N-(3-chloro-4-((4-fluorobenzyl)oxy)phenyl)quinazolin-4-amine (5p)
90.7% yield. mp 224-226 ºC. 1H NMR (400 MHz, DMSO) δ 11.53 (s, 1H, NH), 9.23 (s, 1H, Ar-H),
8.91 (s, 1H, Ar-H), 8.20 (dd, J = 8.9, 1.7 Hz, 1H, Ar-H), 7.95 (d, J = 2.4 Hz, 1H, Ar-H), 7.91 (d, J = 8.9 Hz,
1H, Ar-H), 7.70 (dd, J = 8.9, 2.4 Hz, 1H, Ar-H), 7.55 (dd, J = 8.3, 5.7 Hz, 2H, Ar-H), 7.35 (d, J = 9.0 Hz,
1H, Ar-H), 7.26 (t, J = 8.8 Hz, 2H, Ar-H), 5.25 (s, 2H, CH2). ESI-MS: m/z 458.0 [M+H]+.
4.1.23. N-(5-(4-((6-fluoropyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(6a)
A solution of the 3a (0.164 g, 0.5 mmol), 5a (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 white solid (0.12 g, 68.2% yield). mp 152-154 ºC. 1H
NMR (400 MHz, DMSO) δ 10.02 (s, 1H, NH), 9.53 (s, 1H, NH), 8.83 (s, 1H, Ar-H), 8.68 (s, 1H, Ar-H),
8.53 (s, 1H, Ar-H), 8.18 (d, J = 8.4 Hz, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 7.95 (d, J = 11.9 Hz, 1H, Ar-H), 7.90
(d, J = 8.6 Hz, 1H, Ar-H), 7.69 (d, J = 7.8 Hz, 1H, Ar-H), 7.52 - 7.36 (m, 1H, Ar-H), 6.98 (t, J = 7.2 Hz, 1H,
Ar-H), 4.01 (s, 3H, OCH3), 3.11 (s, 3H, CH3).13C NMR (100 MHz, DMSO) δ 162.5 (d, J = 239.4 Hz), 158.0,

157.3, 154.8, 149.6, 141.6, 141.4 (d, J = 5.6 Hz), 135.3, 132.2, 132.0, 130.5 (d, J = 9.1 Hz), 129.4, 129.2,
ACCEPTED MANUSCRIPT
122.3, 120.7, 118.3 (d, J = 2.3 Hz), 115.9, 110.5 (d, J = 20.6 Hz), 109,4 (d, J = 25.8 Hz), 54.3, 41.3. HRMS
(ESI+) m/z calcd for C21H19FN5O3S [M+H]+, 440.1187; found, 440.1191.
Compounds 6b-r was synthesized according to the procedure described in 6a.
4.1.24. N-(5-(4-((4-fluorophenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (6b)
64.3% yield, mp 157-159 ºC. 1H NMR (400 MHz, DMSO) δ 9.98 (s, 1H, NH), 9.46 (s, 1H, NH), 8.81
(s, 1H, Ar-H), 8.59 (s, 1H, Ar-H), 8.55 (d, J = 2.1 Hz, 1H, Ar-H), 8.17 (d, J = 8.7 Hz, 1H, Ar-H), 8.11 (d, J
= 2.1 Hz, 1H, Ar-H), 7.95 - 7.80 (m, 3H, Ar-H), 7.27 (t, J = 8.7 Hz, 2H, Ar-H), 4.02 (s, 3H, OCH3), 3.13 (s,
3H, CH3). 13C NMR (100 MHz, DMSO) δ 159.1 (d, J = 238.8 Hz), 158.3, 157.3, 155.0, 149.4, 142.0, 135.7,
135.0, 132.3, 131.9, 129.4, 129.0, 125.19 (d, J = 7.9 Hz, 2C), 121.7, 120.7, 115.7, 115.6 (d, J = 22.2 Hz, 2C),
54.3, 41.3. HRMS: m/z 440.1177 [M+H]+. HRMS (ESI+) m/z calcd for C21H19FN5O3S [M+H]+, 440.1187;
found, 440.1177.
4.1.25.
N-(5-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (6c)
61.2% yield, mp 257-259 ºC. 1H NMR (400 MHz, DMSO) δ 10.02 (s, 1H, NH), 9.45 (s, 1H, NH), 8.79
(s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 8.54 (d, J = 2.2 Hz, 1H, Ar-H), 8.21 - 8.17 (m, 2H, Ar-H), 8.10 (d, J = 2.2
Hz, 1H, Ar-H), 7.89 (d, J = 8.7 Hz, 1H, Ar-H), 7.85 (ddd, J = 8.9, 4.2, 2.9 Hz, 1H, Ar-H), 7.48 (t, J = 9.1 Hz,
1H, Ar-H), 4.02 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 158.0, 157.3, 154.8,
154.0 (d, J = 241.7 Hz), 149.5, 142.0, 136.8 (d, J = 2.6 Hz), 135.2, 132.3,132.1, 129.4, 129.1, 124.4, 123.2
(d, J = 6.8 Hz), 121.8, 120.6, 119.4 (d, J = 18.3 Hz), 117.1 (d, J = 21.8 Hz), 115.7, 54.3, 41.4. HRMS (ESI+)
m/z calcd for C21H18ClFN5O3S [M+H]+, 474.0797; found, 474.0799.
4.1.26. N-(5-(4-((2,4-difluorophenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(6d)
67.4% yield, mp 148-150 ºC. 1H NMR (400 MHz, DMSO) δ 10.00 (s, 1H, NH), 9.46 (s, 1H, NH), 8.79
(s, 1H, Ar-H), 8.61 - 8.48 (m, 2H, Ar-H), 8.22 (d, J = 8.6 Hz, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 7.90 (d, J = 8.7
Hz, 1H, Ar-H), 7.63 (dd, J = 15.0, 8.6 Hz, 1H, Ar-H), 7.43 (dd, J = 13.8, 5.6 Hz, 1H, Ar-H), 7.20 (t, J = 7.7
Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR (150 MHz, DMSO) δ 160.6 (dd, J = 11.6,
243.6 Hz), 159.3, 157.6 (dd, J = 8.6, 248.4 Hz), 157.4, 155.3, 149.4, 142.0, 134.9, 132.5, 132.0, 130.2 (dd, J
= 2.1, 9.8 Hz), 129.3, 129.0, 123.1 (dd, J = 3.5, 12.5 Hz), 121.7, 120.7, 115.4, 111.9 (dd, J = 3.2, 21.9 Hz),
105.2 (t, J = 26.0 Hz), 54.4, 41.3. HRMS (ESI+) m/z calcd for C21H18F2N5O3S [M+H]+, 458.1093; found,
458.1080.

4.1.27.
ACCEPTED MANUSCRIPT
N-(2-methoxy-5-(4-((4-(trifluoromethoxy)phenyl)amino)quinazolin-6-yl)pyridin-3-yl)methanesulfonamide
(6e)
65.8% yield, mp 156-158 ºC. 1H NMR (400 MHz, DMSO) δ 10.06 (s, 1H, NH), 9.45 (s, 1H, NH), 8.82 (s,
1H, Ar-H), 8.63 (s, 1H, Ar-H), 8.55 (d, J = 2.1 Hz, 1H, Ar-H), 8.19 (d, J = 8.6 Hz, 1H, Ar-H), 8.11 (d, J =
2.1 Hz, 1H, Ar-H), 7.99 (d, J = 9.0 Hz, 2H, Ar-H), 7.90 (d, J = 8.6 Hz, 1H, Ar-H), 7.44 (d, J = 8.7 Hz, 2H,
13
Ar-H), 4.02 (s, 3H, OCH3), 3.12 (s, 3H, CH3). C NMR (150 MHz, DMSO) δ162.8, 158.1, 157.3, 154.9,
149.5, 142.0, 138.7, 135.1, 132.4, 132.1, 129.4, 129.1, 124.3(2C), 121.8(2C), 121.7, 120.7, 120.6(q, J =
169.3 Hz ), 115.8, 54.3, 41.3. HRMS (ESI+) m/z calcd for C22H19F3N5O4S [M+H]+, 506.1104; found,
506.1127.
4.1.28. N-(2-methoxy-5-(4-((4-methoxyphenyl)amino)quinazolin-6-yl)pyridin-3-yl)methanesulfonamide (6f)
54.9% yield, mp 141-143 ºC. 1H NMR (400 MHz, DMSO) δ 9.93 (s, 1H, NH), 9.45 (s, 1H, NH), 8.81 (s, 1H,
Ar-H), 8.60 - 8.50 (m, 2H, Ar-H), 8.15 (d, J = 8.7 Hz, 1H, Ar-H), 8.11 (d, J = 2.0 Hz, 1H, Ar-H), 7.85 (d, J
= 8.6 Hz, 1H, Ar-H), 7.69 (d, J = 8.9 Hz, 2H, Ar-H), 7.01 (d, J = 8.9 Hz, 2H, Ar-H), 4.01 (s, 3H, OCH3),
3.79 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 158.4, 157.3, 156.5, 155.2, 149.2,
142.0, 134.8, 132.4, 132.2, 131.8, 129.4, 128.8, 125.1, 121.7, 120.7, 115.8, 114.2, 55.7, 54.3, 41.3. HRMS
(ESI+) m/z calcd for C22H22N5O4S [M+H]+, 452.1387; found, 452.1379.
4.1.29. N-(5-(4-((3,4-dimethoxyphenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(6g)
60.1% yield, mp 151-153 ºC. 1H NMR (400 MHz, DMSO) δ 9.90 (s, 1H, NH), 9.47 (s, 1H, NH), 8.84
(s, 1H, Ar-H), 8.57 (d, J = 2.1 Hz, 2H, Ar-H), 8.15 (d, J = 8.7 Hz, 1H, Ar-H), 8.12 (d, J = 2.1 Hz, 1H, Ar-H),
7.85 (d, J = 8.5 Hz, 1H, Ar-H), 7.46 (d, J = 1.9 Hz, 1H, Ar-H), 7.44 - 7.35 (m, 1H, Ar-H), 7.01 (d, J = 8.7
Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO) δ 158.4, 157.3, 155.2, 149.5, 148.9, 146.1, 142.0, 134.8, 132.7, 132.4, 131.7, 129.5,
129.0, 121.8, 120.7, 115.9, 115.7, 112.2, 108.6, 56.2, 56.1, 54.3, 41.4. HRMS (ESI+) m/z calcd for
C23H24N5O5S [M+H]+, 482.1493; found, 482.1488.
4.1.30.
N-(5-(4-((3-chloro-4-methoxyphenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(6h)
61.7% yield, mp 146-148 ºC. 1H NMR (400 MHz, DMSO) δ 9.90 (s, 1H, NH), 9.46 (s, 1H, NH), 8.78
(s, 1H, Ar-H), 8.67 - 8.51 (m, 2H, Ar-H), 8.16 (d, J = 8.5 Hz, 1H, Ar-H), 8.11 (d, J = 1.8 Hz, 1H, Ar-H),
8.00 (d, J = 2.1 Hz, 1H, Ar-H), 7.86 (d, J = 8.6 Hz, 1H, Ar-H), 7.76 (dd, J = 8.8, 2.1 Hz, 1H, Ar-H), 7.22 (d,
J = 9.0 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.13 (s, 3H, CH3). 13C NMR (100 MHz,

DMSO) δ 158.1, 157.3, 155.0, 151.6, 149.4, 142.0, 134.9, 133.0, 132.3, 131.9, 129.4, 129.0, 124.7, 123.0,
ACCEPTED MANUSCRIPT
121.7, 120.8, 120.6, 115.7, 113.1, 56.7, 54.3, 41.3. HRMS (ESI+) m/z calcd for C22H21ClN5O4S [M+H]+,
486.0997; found, 486.0992.
4.1.31. methyl 3-((6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)quinazolin-4-yl)amino)benzoate
(6i)
58.4% yield, mp 240-242 ºC. 1H NMR (400 MHz, DMSO) δ 10.09 (s, 1H, NH), 9.46 (s, 1H, NH), 8.85
(s, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H), 8.45 (s, 1H, Ar-H), 8.32 (d, J = 7.8 Hz, 1H, Ar-H),
8.19 (d, J = 8.6 Hz, 1H, Ar-H), 8.12 (s, 1H, Ar-H), 7.90 (d, J = 8.6 Hz, 1H, Ar-H), 7.75 (d, J = 7.6 Hz, 1H,
Ar-H), 7.58 (t, J = 7.9 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.13 (s, 3H, CH3). 13C NMR
(100 MHz, DMSO) δ 166.6, 158.1, 157.3, 154.9, 149.6, 142.0, 140.0, 135.2, 132.4, 132.1, 130.4, 129.4,
129.1, 127.3, 124.7, 123.1, 121.8, 120.7, 115.9, 54.3, 52.7, 41.3. HRMS (ESI+) m/z calcd for C23H22N5O5S
[M+H]+, 480.1336; found, 480.1323.
4.1.32. methyl
2-fluoro-5-((6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)quinazolin-4-yl)amino)benzoate
(6j)
63.6% yield, mp 246-248 ºC. 1H NMR (400 MHz, DMSO) δ 10.09 (s, 1H, NH), 9.45 (s, 1H, NH), 8.82
(s, 1H, Ar-H), 8.63 (s, 1H, Ar-H), 8.55 (d, J = 2.1 Hz, 1H, Ar-H), 8.34 (dd, J = 6.4, 2.7 Hz, 1H, Ar-H), 8.31
- 8.24 (m, 1H, Ar-H), 8.19 (d, J = 8.6 Hz, 1H, Ar-H), 8.10 (d, J = 2.1 Hz, 1H, Ar-H), 7.90 (d, J = 8.7 Hz, 1H,
Ar-H), 7.48 - 7.39 (m, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.12 (s, 3H, OCH3). 13C NMR
(100 MHz, DMSO) δ 164.4 (d, J = 3.7Hz), 158.8, 157.6 (d, J = 253.4Hz) , 157.3, 154.9, 149.5, 142.0, 135.9
(d, J = 2.9 Hz), 135.2, 132.2, 132.1, 129.4, 129.3 (d, J = 8.4Hz), 129.1, 125.3, 121.8, 120.7, 118.2 (d, J =
11.2 Hz), 117.6 (d, J = 23.3Hz), 115.7, 54.3, 52.9, 41.4. HRMS (ESI+) m/z calcd for C23H21FN5O5S [M+H]+,
498.1242; found, 498.1245.
4.1.33. methyl
2-chloro-5-((6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)quinazolin-4-yl)amino)benzoate (6k)
49.7% yield, mp 247-249 ºC. 1H NMR (400 MHz, DMSO) δ 10.11 (s, 1H, NH), 9.46 (s, 1H, NH), 8.82
(s, 1H, Ar-H), 8.67 (s, 1H, Ar-H), 8.55 (d, J = 1.6 Hz, 1H, Ar-H), 8.35 (d, J = 2.2 Hz, 1H, Ar-H), 8.26 (dd, J
= 8.8, 2.3 Hz, 1H, Ar-H), 8.19 (d, J = 8.5 Hz, 1H, Ar-H), 8.10 (d, J = 1.6 Hz, 1H, Ar-H), 7.90 (d, J = 8.6 Hz,
1H, Ar-H), 7.63 (d, J = 8.8 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C
NMR (100 MHz, DMSO) δ 165.8, 157.9, 157.3, 154.7, 149.6, 142.0, 138.8, 135.3, 132.3, 132.2, 131.4,
130.1, 129.4, 129.2, 126.8, 126.3, 124.5, 121.8, 120.7, 115.8, 54.4, 53.1, 41.4. HRMS (ESI+) m/z calcd for
C23H21ClN5O5S [M+H]+, 514.0946; found, 514.0947.
4.1.34. methyl

2-methoxy-4-((6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)quinazolin-4-yl)amino)benzoate
ACCEPTED MANUSCRIPT
(6l)
48.5% yield, mp 155-157 ºC. 1H NMR (400 MHz, DMSO) δ 10.07 (s, 1H, NH), 9.47 (s, 1H, NH), 8.84
(s, 1H, Ar-H), 8.73 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H), 8.20 (d, J = 8.7 Hz, 1H, Ar-H), 8.11 (s, 1H, Ar-H),
7.93 (d, J = 8.7 Hz, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.79 (d, J = 8.5 Hz, 1H, Ar-H), 7.72 (d, J = 8.5 Hz, 1H,
Ar-H), 4.02 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO) δ 166.0, 159.8, 158.0, 157.3, 154.7, 149.7, 144.9, 142.1, 135.3, 132.4, 132.3, 132.2, 129.4, 129.2,
121.8, 120.8, 116.0, 114.4, 113.4, 105.8, 56.3, 54.3, 52.1, 41.4. HRMS (ESI+) m/z calcd for C24H24N5O6S
[M+H]+, 510.1442; found, 510.1417.
4.1.35.
N-(5-(4-((3-chloro-4-((4-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesul
fonamide (6m)
60.9% yield, mp 168-170 ºC. 1H NMR (400 MHz, DMSO) δ 9.91 (s, 1H, NH), 9.45 (s, 1H, NH), 8.78
(s, 1H, Ar-H), 8.60 (s, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.17 (d, J = 8.7 Hz, 1H, Ar-H), 8.10 (d, J = 1.7 Hz, 1H,
Ar-H), 8.01 (d, J = 1.9 Hz, 1H, Ar-H), 7.87 (d, J = 8.7 Hz, 1H, Ar-H), 7.74 (dd, J = 8.9, 2.0 Hz, 1H, Ar-H),
7.56 (d, J = 6.0 Hz, 1H, Ar-H), 7.54 (d, J = 5.7 Hz, 1H, Ar-H), 7.32 - 7.24 (m, 3H, Ar-H), 5.22 (s, 2H, CH2),
4.01 (s, 3H, OCH3), 3.11 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 162.3 (d, J = 242.0 Hz), 158.1, 157.3,
155.0, 150.4, 149.6, 142.0, 135.0, 133.4, 133.3, 132.3, 131.9, 130.3 (d, J = 8.4 Hz, 2C), 129.4, 129.1, 124.7,
122.9, 121.8, 121.6, 120.6, 115.8 (d, J = 21.3 Hz, 2C), 115.7, 114.9, 70.1, 54.3, 41.3. HRMS (ESI+) m/z
calcd for C28H24ClFN5O4S [M+H]+, 580.1216; found, 580.1222.
4.1.36.
N-(5-(4-((3-chloro-4-fluorophenyl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)butane-1-sulfonamide
(6n)
65.8% yield, mp 255-257 ºC. 1H NMR (400 MHz, DMSO) δ 10.02 (s, 1H, NH), 9.51 (s, 1H, NH), 8.80
(s, 1H, Ar-H), 8.68 (s, 1H, Ar-H), 8.56 (d, J = 1.8 Hz, 1H, Ar-H), 8.22 (d, J = 2.3 Hz, 1H, Ar-H), 8.19 (d, J
= 9.2 Hz, 1H, Ar-H), 8.14 (d, J = 1.9 Hz, 1H, Ar-H), 7.91 (d, J = 8.7 Hz, 1H, Ar-H), 7.90 - 7.83 (m, 1H,
Ar-H), 7.50 (t, J = 9.1 Hz, 1H, Ar-H), 4.04 (s, 3H, OCH3), 3.26 - 3.15 (m, 2H, CH2), 1.79 (dt, J = 15.2, 7.7
Hz, 2H, CH2), 1.53 - 1.36 (m, 2H, CH2), 0.93 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO) δ
158.0, 157.3, 154.8, 153.9 (d, J = 241.9 Hz), 149.5, 142.0, 136.8 (d, J = 2.8 Hz), 135.1, 132.7, 132.1, 129.3,
129.1, 124.3, 123.1 (d, J = 6.5 Hz), 121.7, 120.6, 119.4 (d, J = 18.5 Hz), 117.1(d, J = 21.8 Hz), 115.7, 54.3,
52.6, 25.7, 21.3, 14.0. HRMS (ESI+) m/z calcd for C24H24ClFN5O3S [M+H]+, 516.1267; found, 516.1283.
4.1.37. ethyl 3-((6-(5-(butylsulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)benzoate (6o)

67.0% yield, mp 116-118 ºC. 1H NMR (400 MHz, DMSO) δ 10.11 (s, 1H, NH), 9.48 (s, 1H, NH), 8.86
ACCEPTED MANUSCRIPT
(s, 1H, Ar-H), 8.65 (s, 1H, Ar-H), 8.56 (s, 1H, Ar-H), 8.42 (s, 1H, Ar-H), 8.33 (d, J = 5.6 Hz, 1H, Ar-H),
8.18 (d, J = 7.5 Hz, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 7.90 (d, J = 6.9 Hz, 1H, Ar-H), 7.75 (d, J = 5.4 Hz, 1H,
Ar-H), 7.58 (s, 1H, Ar-H), 4.37 (d, J = 5.2 Hz, 2H, CH2), 4.02 (s, 3H, OCH3), 3.17 (s, 2H, CH2), 1.77 (s, 2H,
CH2), 1.41 (s, 2H, CH2), 1.36 (s, 3H, CH3), 0.90 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 166.1, 158.1,
157.3, 154.9, 149.6, 142.1, 134.0, 135.1, 132.7, 132.0, 130.7, 129.4, 129.3, 129.1, 127.3, 124.7, 123.1, 121.7,
120.7, 115.9, 61.3, 54.3, 52.6, 25.7, 21.3, 14.7, 14.0. HRMS (ESI+) m/z calcd for C27H30N5O5S [M+H]+,
536.1962; found, 536.1966.
4.1.38. methyl
3-((6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)quinazolin-4-yl)amino)benzoate (6p)
66.1% yield, mp 244-246 ºC. 1H NMR (400 MHz, DMSO) δ 10.40 (s, 1H, NH), 10.09 (s, 1H, NH),
8.85 (s, 1H, Ar-H), 8.66 (s, 1H, Ar-H), 8.58 (d, J = 2.2 Hz, 1H, Ar-H), 8.46 (s, 1H, Ar-H), 8.33 (d, J = 8.1
Hz, 1H, Ar-H), 8.20 (dd, J = 0.8, 9.2 Hz, 1H, Ar-H), 8.18 (s, 1H, Ar-H), 7.90 (d, J = 8.7 Hz, 1H, Ar-H), 7.80
- 7.74 (m, 2H, Ar-H), 7.63 - 7.57(m, 2H, Ar-H), 7.21 (td, J = 8.6, 1.9 Hz, 1H, Ar-H), 3.91 (s, 3H, OCH3),
3.66 (s, 3H, OCH3). 13C NMR (150 MHz, DMSO) δ 166.6, 165.5 (dd, J = 12.0, 253.1Hz), 162.8, 159.9 (dd,
J = 13.1, 255.9Hz),158.5, 158.1, 154.9, 149.6, 143.6, 140.0, 135.6, 134.7, 132.3 (d, J = 16.1 Hz), 131.9,
130.4, 129.5, 129.3, 129.2, 127.7, 125.6 (dd, J = 3.0, 21.5 Hz), 124.7, 123.1, 120.7, 120.0, 115.9, 112.2 (d, J
= 33.2 Hz), 106.2 (t, J = 38.9 Hz), 53.9, 52.7. HRMS (ESI+) m/z calcd for C22H22F2N5O5S [M+H]+,
578.1304; found, 578.1310.
4.1.39. N-(5-(4-((6-fluoropyridin-3-yl)amino)quinazolin-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide
(6q)
63.7% yield, mp 172-174 ºC. 1H NMR (400 MHz, DMSO) δ 10.13 (s, 1H, NH), 9.46 (s, 1H, NH), 8.79
(s, 1H, Ar-H), 8.64 (s, 1H, Ar-H), 8.62 (s, 1H, Ar-H), 8.54 (d, J = 1.9 Hz, 1H, Ar-H), 8.45 (dd, J = 11.1, 4.7
Hz, 1H, Ar-H), 8.20 (d, J = 8.5 Hz, 1H, Ar-H), 8.10 (d, J = 1.9 Hz, 1H, Ar-H), 7.90 (d, J = 8.6 Hz, 1H,
Ar-H), 7.28 (dd, J = 8.7, 2.9 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.12 (s, 3H, CH3). 13C NMR (101 MHz,
DMSO) δ 159.5 (d, J = 241.7 Hz), 158.3, 157.3, 154.9, 149.5, 142.0, 141.6 (d, J = 15.6 Hz), 136.9 (d, J =
8.1 Hz), 135.2, 134.5 (d, J = 4.1 Hz), 132.3, 132.2, 129.3, 129.1, 121.8, 120.7, 115.7, 109.5 (d, J = 39.1 Hz),
54.4, 41.3. HRMS (ESI+) m/z calcd for C20H18FN6O3S [M+H]+, 441.1140; found, 441.1145.
4.1.40. N-(2-methoxy-5-(4-((6-methoxypyridin-3-yl)amino)quinazolin-6-yl)pyridin-3-yl)methanesulfonamid
(6r)
63.7% yield, mp 143-145 ºC. 1H NMR (400 MHz, DMSO) δ 10.00 (s, 1H, NH), 9.47 (s, 1H, NH), 8.78
(s, 1H, Ar-H), 8.55 (s, 2H, Ar-H), 8.51 (s, 1H, Ar-H), 8.17 (d, J = 8.6 Hz, 1H, Ar-H), 8.10 (d, J = 7.6 Hz, 2H,
Ar-H), 7.86 (d, J = 8.6 Hz, 1H, Ar-H), 6.92 (d, J = 8.8 Hz, 1H, Ar-H), 4.02 (s, 3H, OCH3), 3.89 (s, 3H,

OCH3), 3.12 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) δ 160.8, 158.7, 157.3, 155.1, 149.4, 142.0, 141.8,
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135.8, 134.9, 132.4, 131.9, 130.1, 129.4, 129.0, 121.7, 120.7, 115.7, 110.4, 54.3, 53.7, 41.3. HRMS (ESI+)
m/z calcd for C21H21N6O4S [M+H]+, 453.1340; found, 453.1355.
4.2. Biological assay methods
4.2.1. Cell culture
A549, BT549, HCT-116, MCF-7, SK-HEP-1 and SNU638 cells were purchased from the American
Type Culture Collection (Manassas, VA, USA). Cells were grown in DMEM (SNU-638, BT549, MCF-7 and
SK-HEP-1) or RPMI1640 (HCT-116 and A549 cells) supplemented with 10% FBS and
antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 µg/mL streptomycin and 250 ng/mL
amphotericin B) in a humidified incubator containing 5% CO2 at 37 °C.
4.2.2. Antiproliferative activity
The cell viability was evaluated using the sulforhodamine B (SRB) cellular protein-staining method
with minor modifications. Briefly, cells were treated with various concentrations of compounds in 96-well
plates and incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 72 h. After treatment, the cells
were fixed with 10% TCA solution, and cell viability was determined with 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. EGFR and PI3K enzymatic activity assay
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.
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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)
The PI3K kinase assay was measured by PI3 Kinase Activity/Inhibitor Assay Kit (EMD Millipore,
#17-493) following the manufacturer’s protocols.
4.2.4. Colony formation assay
BT549 cells were seeded in a 6-well plates with a density of 3×103 cells/well and then were cultured
with different concentrations (0, 1.0, 2.5, 5.0 µM) of 6c about 2 weeks. Cells were fixed by ethanol and
stained with crystal violet.
4.2.5. Annexin V-FITC and propidium iodide (PI) double staining assay
BT549 cells were seeded in a 6-well plates (2×105 cells/well) and treated with 6c mentioned above. At
the end of treatments, the cells were harvested and washed by cold PBS. Then the resuspended cells (1×106)
were incubated with Annexin VFITC (5 µL) and PI (5 µL) at room temperature for 5 min in the dark and
examined with flow cytometry.
4.2.6. DAPI staining assay
Apoptotic feature of BT549 cells was assessed by DNA condensation using DAPI
(4’,6-diamidino-2-phenylindole). 2×104 BT549 cells were seeded in a 6-well plates and incubated with 6c
for 24 h. Then, cells were stained with DAPI for 10 min and observed under a fluorescence microscope.
4.2.7. Cell cycle analysis
Cell cycle was determined by the staining of DNA with propidium iodide (PI). 2 × 105 BT549 cells
were incubated with 6c for 24 h and fixed in 70% ethanol overnight. Then, cells were washed with PBS and
treated with RNase and PI for 1 h. The percentages of cells in different phase of cell cycle were measured by
flow cytometer.
4.2.8. Western blot analysis
Total cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS). The protein concentration was determined, and equal amounts of
protein samples were subjected to 10% SDS-PAGE followed by transferring to PVDF membranes, and
probed with were subsequently analyzed with EGFR, p-EGFR, AKT and p-AKT antibodies. Exposures were
obtained using ImageQuant LAS 4000 biomolecular imager (GE Healthcare).
4.2.9. Molecular docking studies
The crystal structure of EGFR (PDB entry code: 4WKQ) in complex with Gefitinib and the PI3K (PDB
entry code: 3L08) in complex with Omipalisib were 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
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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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Table(s)
Table 1 Cytotoxicity in vitro of target compounds against five cancer cell lines (IC50 Valuesa in µM).
A549
BT549
HCT-116
MCF-7
SK-HEP-1 SNU638
Cells
Comp.
6a
R1
3-F
R2
X
CH
CH
CH
CH
9.94±0.23
7.62±0.27
8.23±0.34
4.71±0.17
2.76±0.07
3.12±0.09
1.02±0.08
2.48±0.14
3.94±0.08
3.16±0.09
5.60±0.24
4.01±0.15
8.02±0.26 1.85±0.06 2.76±0.08
6.51±0.11 1.47±0.07 2.40±0.09
5.59±0.21 6.10±0.26 4.10±0.13
1.61±0.07 2.49±0.12 2.05±0.08
4-F
6b
3-Cl-4-F
2,4-diF
6c
6d
4-OCF3
CH 25.36±0.51 8.24±0.16 16.59±0.25 23.68±0.38 6.14±0.31 22.4±0.21
6e
4-OCH3
3,4-diOCH3
3-Cl-4-OCH3
3-COOCH3
3-COOCH3-4
-F
CH
CH
CH
CH
CH
3.58±0.16
5.82±0.34
5.24±0.33
1.10±0.05
3.42±0.13
1.88±0.10
2.59±0.22
2.47±0.14
1.08±0.09
2.74±0.25
3.49±0.09
4.36±0.22
5.62±0.22
0.40±0.02
2.36±0.12
1.79±0.09 1.61±0.06 3.97±0.15
2.43±0.15 2.84±0.15 1.68±0.06
3.32±0.12 2.81±0.17 3.62±0.10
10.1±0.09 2.40±0.09 1.12±0.03
1.15±0.09 5.18±0.16 2.54±0.08
6f
6g
6h
6i
6j
3-COOCH3-4
-Cl
CH
CH
CH
2.13±0.07
3.35±0.08
31.3±0.44
2.36±0.21
3.76±0.11
14.6±0.23
3.13±0.19
1.43±0.11 7.06±0.32 2.20±0.12
6k
6l
3-OCH3-4-C
OOCH3
3-Cl-4-(4-flu
orobenzyl)ox
y
1.69±0.16 10.94±0.14 0.83±0.03 3.44±0.16
5.41±0.31 28.36±0.54 1.95±0.07 2.36±0.14
6m
3-Cl-4-F
3-COOCH2C
H3
CH
CH
77.1±2.23
42.3±1.26
56.8±1.13
37.2±0.97
9.99±0.87
26.7±0.45
65.8±1.82 53.8±0.43 5.35±0.21
36.4±0.68 21.6±0.34 25.2±0.44
6n
6o

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3-COOCH3
CH 10.51±0.08 12.7±0.20
1.50±0.06 22.38±0.17 2.80±0.04 0.4±0.02
6p
6-F
N
N
6.72±0.21
5.56±0.23
8.27±0.42
4.78±0.16
3.49±0.18
6.56±0.35
7.62±0.34
6.91±0.23
5.98±0.72
5.39±0.22 5.24±0.17 6.52±0.26
3.77±0.13 3.44±0.11 4.28±0.18
26.7±1.02 10.1±0.32 7.56±0.24
6q
6r
6-OCH3
Gefitini
b
0.62±0.07
0.74±0.08
0.84±0.12
1.33±0.14 1.82±0.23 1.24±0.13
Dactolis
ib
aIC50 values are the mean of triplicate measurements.

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Table 2 Activities of 6c, 6i and Gefitinib, Dactolisib against EGFR and Class I PI3K (IC50 Values in nM)
EGFR
2.4
PI3Kα
317
PI3Kβ
9412
4936
ND
PI3Kγ
3560
1783
ND
PI3Kδ
8672
4364
ND
6c
6i
409
2.3
165
ND
Gefitinib
Dactolisib
ND
16.4
35.9
23.6
78.4
ND: Not determined

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Figure legends
Fig.1. The structures of some EGFR inhibitors.
Fig.2. The design strategy based on Dactolisib, Gefitinib and Omipalisib
Fig.3. The colony formation assay of compound 6c.(A) treatment with compound 6c, representative photographs of
colony formation are shown. (B)Bar graphs showed the quantitative results of clonogenicity. Data are presented as
the mean for three independent experiments. ** P < 0.01, ***P < 0.001 compared with control.
Fig.4. Apoptosis and the effect of compound 6c on cell cycle. (A) AV-PI staining show early and late apoptosis of
BT549 cells induced by compound 6c. (B) Quantification of early and late apoptosis. (C) Hoechst 33342 staining
and compound 6c induced morphological changes. (D) Cell cycle changes after compound 6c treatment. (E)
Quantification of the percentages of cells in different phase.
Fig.5. Western blot analysis of 6c. (A) The inhibition effects of compound 6c (0.1 µM, 0.5 µM and 2.5 µM) on the
expression of p-EGFR, EGFR in BT549 cells are depicted. β-Actin was used as internal control. (B) The inhibition
effects of compound 6c (5 µM, 10 µM and 20 µM) on the expression of p-Akt, Akt in BT549 cells are depicted.
β-Actin was used as internal control.
Fig.6. Docking mode of 6c with protein crystal structure of EGFR and PI3K. (A) Key interactions of compound 6c
in the active site of EGFR (PDB: 4WKQ). (B) The binding pose of 6c and Gefitinib in the active site of EGFR.
Gefitinib was highlighted with yellow. (C) Key interactions of compound 6c in the active site of PI3K (PDB: 3L08).
(D) The binding pose of 6c and Omipalisib in the active site of PI3K. Omipalisib was highlighted with yellow.
Scheme 1. (a) benzenesulfonyl chloride, pyridine, rt, 24 h; (b) bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF,
100 ºC, 8 h; (c) amines, isopropanol, 90 ºC, 4h; (d) Pd(dppf)2Cl2, Cs2CO3, DMF/ H2O, 90 ºC, 8 h.

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Figure 1

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