Design, synthesis, and biological evaluation of novel 3-substituted imidazo[1,2-a]pyridine and quinazolin-4(3H)-one derivatives as PI3Kα inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.07.074

Phosphatidylinositol 3-kinase (PI3K) is a pivotal regulator of intracellular signaling pathways and considered as a promising target in the development of a therapeutic treatment of cancer. Among the different PI3K subtypes, the PIK3CA gene encoding PI3K

Full text of DOI:10.1016/j.ejmech.2017.07.074

Accepted Manuscript
Design, synthesis, and biological evaluation of novel 3-substituted
imidazo[1,2-a]pyridine and quinazolin-4(3H)-one derivatives as PI3Kα inhibitors
Yan-Hua Fan, Wei Li, Dan-Dan Liu, Meng-Xuan Bai, Hong-Rui Song, Yong-Nan Xu,
SangKook Lee, Zhi-Peng Zhou, Jian Wang, Huai-Wei Ding
PII:
S0223-5234(17)30596-2
10.1016/j.ejmech.2017.07.074
EJMECH 9637
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2017
Revised Date: 27 July 2017
Accepted Date: 30 July 2017
Please cite this article as: Y.-H. Fan, W. Li, D.-D. Liu, M.-X. Bai, H.-R. Song, Y.-N. Xu, S. Lee, Z.-
P. Zhou, J. Wang, H.-W. Ding, Design, synthesis, and biological evaluation of novel 3-substituted
imidazo[1,2-a]pyridine and quinazolin-4(3H)-one derivatives as PI3Kα inhibitors, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.074.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of novel 3-substituted imidazo[1,2-a]pyridine and
quinazolin-4(3H)-one derivatives as PI3Kα inhibitors
Yan-Hua Fan b, Wei Li a, Dan-Dan Liu a, Meng-Xuan Bai a, Hong-Rui Song a, Yong-Nan Xu a, SangKook Lee c,
Zhi-Peng Zhou b, Jian Wanga, Huai-Wei Dinga,*
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China
b School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
c College of Pharmacy, Seoul National University, Seoul 151-742, Korea
* Corresponding authors.
Email address: dinghuaiwei627@163.com.

ACCEPTED MANUSCRIPT
Abstract：Phosphatidylinositol 3-kinase (PI3K) is a pivotal regulator of intracellular signaling pathways and
considered as a promising target in the development of a therapeutic treatment of cancer. Among the different
PI3K subtypes, the PIK3CA gene encoding PI3K p110α is frequently mutated and overexpressed in majority
of human cancers. Therefore, the inhibition of PI3Kα has been considered to be an efficient approach for the
treatment of cancer. In this study, two series compounds containing hydrophilic group in
imidazo[1,2-a]pyridine and quinazolin-4(3H)-one were synthesized and their antiproliferative activities against
five cancer cell lines, including HCT-116, SK-HEP-1, MDA-MB-231, SNU638 and A549, were evaluated.
Compound 1i with most potent antiproliferative activity was selected for further biological evaluation. PI3K
kinase assay showed that 1i has selectivity for PI3Kα distinguished from other isoforms. The western blot
assay indicated that 1i is more effective than HS-173, an imidazopyridine-based PI3Ka inhibitor, in reducing
the levels of phospho-Akt. All these results suggested that 1i is a potent PI3Kα inhibitor and could be
considered as a potential candidate for the development of anticancer agents.
Keywords: Phosphatidylinositol 3-kinase, imidazo [1, 2-a] pyridine, quinazolin-4(3H)-one, PIK3CA gene,
anticancer agents
Abbreviations: PI3K, Phosphatidylinositol 3-kinase; RTK, Receptor tyrosine kinases;

ACCEPTED MANUSCRIPT
1. Introduction
The phosphatidylinositol-3-kinase (PI3K) signaling pathway is crucial to multiple cellular functions
including cell growth, cell cycle, cell survival, differentiation and invasion. The PI3K pathway is frequently
activated, amplified and mutated in human cancers [1-5]. Aberrant PI3K activity is often involved in malignant
transformation, suggesting that the inhibition of this pathway is a promising approach for novel
chemotherapeutic agents [5-8]. PI3K-AKT signaling is activated in cancers by several different mechanisms
(Fig. 1). Receptor tyrosine kinase (RTKs) activation or mutation, Ras mutation, PTEN loss and PIK3CA
mutation can lead to PI3K signaling activation. PIK3CA, which encodes the p110 catalytic subunit, is the only
gene with somatic mutations and is the most frequently mutated gene in human cancers [9]. In recent years, it is
evident that many human tumors carry somatic missense mutations in PIK3CA at high frequency [10-12].
Fig.1. RTK/PI3K/Akt signaling cascade in cancer.
Several inhibitors targeting PI3K have been developed, and are being evaluated in preclinical studies and
early clinical trials [13-15]. These include pan-PI3K and isoform-specific PI3K inhibitors that block the
catalytic site of p110 isoforms. Among them, GSK2126458 has been identified as highly potent, orally
bioavailable inhibitor of PI3K with picomolar activity and is currently under evaluation in clinical trials for

ACCEPTED MANUSCRIPT
oncology applications [16-17] (Fig.2). In addition, there are a number of potential PI3K inhibitors have been
developed and under preclinical tests [18-21]. HS-173, exhibiting a potent and highly selective PI3K
α
inhibitory activity with an IC50 of 0.8 nM, showed remarkable antitumor activity in vitro and in vivo [18, 22-23]
(Fig.2).
Previous study indicates that difluorophenyl and 2-methyloxy group of GSK2126458 are vital to its PI3K
inhibitory activity [16]. Hence we introduce these two groups into HS-173. However, HS-173 is somewhat
unstable in an aqueous phosphate buffer with pH 6.7 and this instability is probably due to the hydrolysis of the
ester linkage [19]. It is known that amide is more stable than ester in the buffer solution having a pH ranging
from 4.5 to 7.4. Therefore, the stability of HS-173 might be increased if the ester linkage is replaced with amide
group. In addition, we also tried to improve its physical and chemical properties via introducing a substituent on
amide nitrogen. Many drugs approved for marketing, such as Gefitinib [24], Lapatinib [25] and Bosutinib [26],
contain a hydrophilic group extending into the solvent region, , to increase their water solubility and
bioavailability. Therefore, we proposed replacing the ester linkage with amide group containing hydrophilic
substituent, to eliminate the inherent hydrolysis of ester as well as providing a potential means to enhance the
solubility of the molecules. In addition, we also tried to replace the imidazopyridine moiety with indole ring,
however, this decreased the PI3K
position to C2 position led to a decrease in PI3K
imidazopyridine moiety is essential for its PI3K
α inhibitory activity. Moreover, the removal of the ester linkage from C3
α
inhibitory activity. Thus it suggested that the 3-formyl
inhibitory activity.
α
Separately, the design strategy of quinazolin-4(3H)-one derivative was derived from Gefitinib and several
other clinical EGFR-TKIs owing to their quinazoline scaffold, whose nitrogen atom in 1 position is very similar
to the quinoline’s nitrogen atom of GSK2126458. As the quinoline nitrogen of GSK2126458 forms a hydrogen
bond with the hinge of PI3K, we hypothesize that nitrogen atom in position 1 of quinazoline ring may also
form a hydrogen bond in the binding site of PI3K. In addition, it just so happened that position3 of
quinazolin-4(3H)-one moiety can introduce an ethyl acetate group similar to that of HS-173to afford 10a. 10a
showed modest specificity profiles against five cancer cell lines. According to the above-mentioned findings,
we further introduced different hydrophilic group to quinazoline scaffold and compounds 10b-10g were
obtained. As expected, the activity was improved to some extent compared to that of 10a.
In this study, we synthesized two series of compounds containing hydrophilic group at position 3 in
imidazo[1,2-a]pyridine and quinazolin-4(3H)-one (Fig.3), with the goal of developing some new PI3K
inhibitors. These compounds were prepared and evaluated for their PI3K inhibitory activities and anticancer
effects in vitro
.

ACCEPTED MANUSCRIPT
Fig.2. The structures of PI3K inhibitors.
Fig.3. The design strategy based on HS-173 and GSK2126458
2. Results and discussion
2.1. Chemistry
The synthetic routes for the target compounds are outlined in Schemes 1-2. Sulfonylation of 1 reacted with
two benzenesulfonyl chlorides to yield 2a and 2b, which were then subjected to Suzuki coupling with
bis(pinacolato)diborane to afford arylboronic ester 3a and 3b. Coupling 3a with three intermediates containing
five-membered rings of benzo derivatives via Suzuki reaction to yield corresponding target compounds 1a-c.
Using the same synthetic method as 3a, intermediate 5 was obtained by the reaction of 4. Intermediate 5 was
coupled with ethyl 6-bromoimidazo[1,2-a]pyridine-3-carboxylate via Suzuki reaction to yield 6. Subsequently,
the nitro group was reduced to produce amide 7. Sulfonylation of 7 with different sulfonyl chlorides gave the
title compounds 1d-e and the compound 1c could also be obtained by this method. The compound 1e was
refluxed in EtOH-H2O-NaOH (1.5 mol/L), acidified by HCl (2 mol/L) to afford 1f, which was condensed with
different amines to afford the target compounds 1g-m.
The quinazolin-4(3H)-one derivatives were synthesized according to the Scheme 2. Commercial agent 8
reacted with different types of chlorinated compounds to afford 9a-g, which were coupled with 3b via Suzuki
reaction to get 10a-g.

ACCEPTED MANUSCRIPT
Scheme 1. (a) Benzenesulfonyl chloride, pyridine, rt, 24 h; (b) Bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF, 100 ºC, 8 h;
(c) Pd(dppf)2Cl2, Cs2CO3, DMF/ H2O, 90 ºC, 8 h; (d) Bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF, 100 ºC, 8 h; (e)
Pd(dppf)2Cl2, Cs2CO3, DMF, 90ºC, 8h; (f) Fe, HCl/H2O, 80 ºC , 4 h; (g) Sulfonyl chloride, pyridine, rt, 24 h; (h)
NaOH/Ethanol/ H2O, 80 ºC, 0.5 h, then HCl (2 mol/L); (i) DIPEA, HOBt, EDCI, THF,12 h, rt.
Scheme 2. (a) Chlorinated compounds, K2CO3, DMF, 60 ºC, 12 h; (b) 3b, Pd(dppf)2Cl2, Cs2CO3, DMF/ H2O, 90ºC, 8h.
2.2. Antiproliferative assays in vitro
All synthesized compounds were evaluated for their cytotoxicities in vitro against five human cancer cell
lines including HCT-116, A549, SK-HEP-1, SNU638 and MDA-MB-231. PI3K/AKT signaling is activated in
all these five cancer cell lines [27-31]. In addition, we also examined the cytotoxicity of three selected
compounds against HUVEC, which is a human normal cell.
The antiproliferative results of all the compounds are summarized in Table 1 and Table 2. The results of
antiproliferative effect assay showed most of the derivatives exhibited stronger antiproliferative effects,
especially against HCT-116 and SNU638. Compound 1c (IC50 = 0.64
than compound 1a (IC50 = 1.3 M against HCT-116) and 1b (IC50 >20
imidazo[1,2-a]pyridine group, especially substitution at its position 3, can improve the antiproliferative activity.
Compound 1f (IC50 = 0.09 M against HCT-116) was more potent than 1c, 1d (0.32 M against HCT-116) and
µM against HCT-116) was more potent
µ
µM against HCT-116), suggesting that
µ
µ

ACCEPTED MANUSCRIPT
1e (4.94
µ
M against HCT-116), indicating that aromatic group at position of R2 is more beneficial to activity
than alkyl group. Moreover, compared to 4-fluorophenyl, 2,4-difluorophenyl at R2 position can improve
antiproliferative activity. To confirm our conjecture that hydrophilic group at R3 position can enhance activity,
we synthesized compounds 1h to 1m by changing substituent R3. As we had expected, almost all the activities
of compounds 1h to 1m were improved and basically reached the nanomolar level. Among them, compound 1i
showed the best anticancer activity with an IC50 value of 0.01 µΜ against HCT-116 and SNU638. Besides,
compound 1j also showed significant antiproliferative effects against HCT-116. Previous studies have reported
that HCT116 cells contain a PIK3CA kinase domain mutation and Met amplification has been reported in
SNU638 cells [31-33]. Considering that both of PIK3CA mutation and Met amplification could activate
PI3K/Akt pathway, it is speculated this contribute to the stronger antiproliferative effects of our compounds
against HCT116 and SNU638. Moreover, compound 1i also showed better antiproliferative effects in A549 and
MDA-MB-231 cells with Ras mutation [34-35] than those in SK-Hep-1 cells, which is a wild type for PIK3CA,
PTEN and Ras. All the results indicated that our compounds inhibit the cell growth more effective in PI3K
signal activated cells.
To develop more core structure, we synthesized another series of compounds 10a to 10g with
quinazolin-4(3H)-one scaffold. However, the activities of this series were not as good as those of the
imidazo[1,2-a]pyridine derivatives. Thus we confirmed the imidazopyridine moiety is essential to its PI3K
α
inhibitory activity. However, very similar to 1a-1m, the introduction of hydrophilic groups at position 3 of
quinazolin could also improve their biological activity. Nevertheless, the antiproliferation activity could be
decreased by the increased length of alkyl group. Among 10a to 10g, compound 10b showed significant
antiproliferative effects against all these five human cancer cell lines.
Not only that, we selected three representative compounds 1i, 1j, 10b with better inhibitory activity to
investigate their cytotoxicity to normal cells. All of the three compounds displayed much lower cytotoxicity in
HUVEC. 1i showed an IC50 value of 2.93 µΜ against HUVEC, which is at least 290 times higher than that of
HCT-116 and MDA-MB-231 cells (Table 2). These indicated that our compounds could inhibit the growth of
cancer cell in effective concentration in vitro without damaging the normal cells.
Table 1 Cytotoxicity in vitro of target compounds against five cancer cell lines (IC50 Valuesa in µM).

ACCEPTED MANUSCRIPT
Cells
Comp.
1a
HCT-116
SK-HEP-1 MDA-MB-231
SNU638
A549
>20
R1
R2
R3
R4
1.3±0.25
0.3±0.02
>20
0.15±0.05
>20
15.9±0.94
>20
>20
>20
>20
1b
1c
0.64±0.29
>20
0.53±0.06
0.51±0.05
0.32±0.03
4.94±0.65
0.17±0.02
0.83±0.07
0.19±0.02
2.32±0.12
0.09±0.01
0.51±0.05
0.23±0.04
3.56±0.15
1d
1e
0.09±0.01
1.31±0.19
0.14±0.01
0.01±0.003
0.1±0.04
0.3±0.05
0.75±0.11
>20
0.12±0.02
0.37±0.07
1.8±0.58
0.36±0.04
1.46±0.25
0.51±0.04
0.3±0.02
1.15±0.09
0.4±0.3
1f
1g
1h
1i
H
0.11±0.04
0.4±0.08
0.04±0.002
0.3±0.07
0.01±0.005 0.04±0.004
0.3±0.03
0.5±0.08
1j
1.23±0.29
0.49±0.03
2.05±0.31
0.79±0.04
1.45±0.29
0.63±0.03
1.39±0.26
1.62±0.31
1k
1l
0.27±0.02
0.48±0.05
0.31±0.07
0.82±0.12
0.98±0.11
1.4±0.14
0.42±0.03
0.12±0.01
0.49±0.06
0.3±0.03
0.5±0.04
0.1±0.02
1m
10a
0.2±0.01
0.6±0.09
0.5±0.11
0.28±0.06
0.1±0.02
10b

ACCEPTED MANUSCRIPT
4.0±0.78
>20
>20
0.36±0.04
0.25±0.03
0.2±0.05
0.2±0.03
10c
10d
0.8±0.07
>20
11.7±0.87
1.36±0.18
1.11±0.09
0.81±0.11
2.25±0.18
0.15±0.04
1.83±0.11
2.26±0.23
>20
0.57±0.06
2.54±0.13
10e
10f
2.23±0.09
0.54±0.13
11.49±1.27
1.09±0.16
2.15±0.89
0.76±0.16
19.72±1.72
0.68±0.11
7.6±1.18
10g
HS-173
0.26±0.04
aIC50 values are the mean of triplicate measurements.
Table 2 Cytotoxicity assay against HUVE cells (HUVEC) (IC50 Valuesa in µM).
Compound
HUVE cells
2.94±0.77
3.56±0.95
8.97±1.02
1i
1j
10b
aIC50 values are the mean of triplicate measurements.
2.3. PI3K enzymatic activity assays
To elucidate the mechanism of antiproliferative activities of these active compounds, we selected the most
potent compounds 1i, 1j and 10b according to the antiproliferative results for the further PI3K enzymatic
activity assays. HS-173 was selected as the positive drug. As shown in Table 3, kinase inhibition activity assay
indicated that these three compounds above showed a highly potent inhibitory activity against PI3K
α
when
compared to that of other class I PI3Ks (PI3Kβ, PI3Kγ and PI3Kδ). Most important, the kinase inhibitory
activity of 1i is better than that of HS-173.
Table 3 Activities of 1i, 1j, 10b and HS-173 against Class I PI3K (IC50 Values in nM)
PI3Kα
0.5
PI3Kβ
89
PI3Kγ
45
PI3Kδ
93
1i
1j
3.8
106
108
59
37
101
69
6.5
116
104
10b
HS-173
1.1
87

ACCEPTED MANUSCRIPT
2.4. Molecular docking
Molecular docking study was performed to elucidate the binding model of 1i in the binding site of PI3K
As showed in Fig.4, the amidogen of sulfonamide group forms three hydrogen bond interaction with Val859 in
the hinge binder region of PI3K . In addition, the morpholinyl formed an additional hydrogen bond with
α
.
α
Lys802 and the nitrogen of pyridyl group formed another hydrogen bond with the side chain of Ser854. All
these binding interaction were further stabilized in the binding site via the hydrophobic interactions with
residues including Lys802, Tyr836, Ile848, Val851 and Ile932 in the back pocket of the enzyme.
Fig.4. Docking mode of 1i and HS-173 into protein crystal structure of PI3Kα. (A) Key interactions of the crystal ligand in the
active site of PI3Kα (PDB: 4ZOP). (B) Key interactions of HS-173 in the active site of PI3Kα. (C) Key interactions of
compound 1i in the active site of PI3Kα. (D) The binding pose of 1i and HS-173 in the active site of PI3Kα. 1i was highlighted
with yellow. (E) Binding modes of 1i and HS-173 in the binding pocket of PI3Kα. The ligands are shown in stick model, while
the proteins are shown in surface model for better visualization in the three dimensional combination models. 1i was highlighted
with yellow.
2.5. Western blot assay
Activation of the PI3K pathway leads to phosphorylation of Akt and subsequently regulated the
downstream targets. PI3K inhibitors show significant tumor growth inhibition via suppression of Akt
phosphorylation and then inhibit phosphorylation of other proteins downstream of PI3K [24-26]. To further
determine whether these compounds affect the PI3K/Akt signaling pathway, we evaluated the effects of 1i on
the related protein levels including Akt and phospho-Akt (p-Akt, S473) in HCT-116 cells by Western blot. As
shown in Fig.5, compound 1i obviously decreased the phospho-Akt (S473) in a dose-dependent manner,

ACCEPTED MANUSCRIPT
indicating that 1i might act as a potential PI3K inhibitor (Fig. 5A). Moreover, compound 1i is more effective
than HS-173 in reducing the phospho-Akt (S473) levels at the concentration of 0.5 µM (Fig.5B).
Fig. 5. Effect of compound 1i on PI3K pathway in HCT-116 cells. (A) Compound 1i decreased phospho-Akt (S473) in a
dose-dependent manner. (B) Compound 1i is more effective than HS-173 in reducing the levels of phospho-Akt (S473). Cells
were treated with 0.1% DMSO, 0.5µM compound 1i or HS-173 for 24 h.
3. Conclusion
In summary, two series of compounds containing hydrophilic group in imidazo[1,2-a]pyridine and
quinazolin-4(3H)-one were designed and synthesized. Their PI3K
α
inhibitory activities and antiproliferative
inhibitor
activities against various cancer cell lines were evaluated in vitro. Compound 1i as a potential PI3K
α
significantly inhibited the PI3K/Akt/mTOR pathway and could be considered as a potential candidate for the
development of anticancer agents. According to these results, it can be concluded that derivatization at the C-3
position of the imidazo[1,2-a]pyridine core via adding a hydrophilic group is a feasible way to enhance the
enzymatic and anti-proliferative activities.
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 a 400 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)-4-fluorobenzenesulfonamide (2a)
To a solution of 5-bromo-2-methoxypyridin-3-amine (1) (2.01 g, 10 mmol) in pyridine (50 ml) at 0 ºC,
4-fluorophenylsulfonyl chloride (2.14 g, 11 mmol) was added. Then the mixture was stirred at room
temperature for 24 h. Pyridine was removed under reduced pressure and adding water (100 ml), extracted with

ACCEPTED MANUSCRIPT
ethyl acetate (3 × 100 ml), the organic layer was washed with water (50 ml), dried with Na2SO4 and evaporated
to give compound 2a as a white solid (3.22 g, 89.4% yield). mp152-154 ºC. 1H NMR (400 MHz, DMSO)
10.22 (s, 1H, NH), 8.08 (d, = 2.2 Hz, 1H, Ar-H), 7.83 (dd, = 8.8, 5.2 Hz, 2H, Ar-H), 7.72 (d, = 2.2 Hz, 1H,
Ar-H), 7.43 (t,
= 8.8 Hz, 2H, Ar-H), 3.64 (s, 3H, OCH3). ESI-MS: m/z 385.1 [M+H+Na]+.
δ
J
J
J
J
4.1.2. N-(5-bromo-2-methoxypyridin-3-yl)-2,4-difluorobenzenesulfonamide (2b)
This compound was synthesized according to the procedure described in 2a.
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,
OCH3). ESI-MS: m/z 376.9 [M-H]+.
J
= 2.0, 8.5 Hz, 1H, Ar-H), 3.62 (s, 3H,
4.1.3. 4-fluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide
(3a)
A solution of the 2a (1.80 g, 5 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 (1.52 g, 74.5% yield). mp 149-151 ºC. 1H NMR (400 MHz, DMSO)
δ
9.92 (s, 1H, NH), 8.18 (s, 1H,
Ar-H), 7.73 (m, 3H, Ar-H), 7.40 (t,
m/z 409.3 [M+H]+.
4.1.4.
J = 8.7 Hz, 2H, Ar-H), 3.62 (s, 3H, OCH3), 1.29 (s, 12H, CH3). ESI-MS:
2,4-difluoro-N-(2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)benzenesulfonamide
(3b)
This compound was synthesized according to the procedure described in 3a.
78.8% yield. mp 171-173 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 7.72 (d, = 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,
δ
10.19 (s, 1H, NH), 8.21 (d,
J
= 1.5 Hz, 1H,
= 2.0, 8.6
J
J
Hz, 1H, Ar-H), 3.62 (s, 3H, OCH3), 1.30 (s, 12H, CH3). ESI-MS: m/z 427.3 [M+H]+
4.1.5. 2-methoxy-3-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (5)
This compound was synthesized according to the procedure described in 3a.
72.4% yield, mp107-109 ºC. 1H NMR (400 MHz, DMSO)
δ 8.64 (d, J = 1.6 Hz, 1H, Ar-H), 8.45 (d, J =
1.6 Hz, 1H, Ar-H), 4.07 (s, 3H, OCH3), 1.33 (s, 12H, CH3). ESI-MS: m/z 281.3 [M+H]+.
4.1.6. ethyl 6-(6-methoxy-5-nitropyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate (6)
A solution of the 5 (1.40 g, 5 mmol), ethyl 6-bromoimidazo[1,2-a]pyridine-3-carboxylate (1.34 g, 5 mmol),
Pd(dppf)2Cl2 (0.18 g, 0.25 mmol) andCs2CO3 (3.26 g, 10 mmol) in DMF (30 ml) under an atmosphere of N2
was stirred at 90 ºC for 8 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 (3.06 g, 89.5% yield).
mp 217-219 ºC.1H NMR (400 MHz, CDCl3)
δ 9.60 (s, 1H, Ar-H), 8.68 (d, J = 2.4 Hz, 1H, Ar-H), 8.52 (d, J =

ACCEPTED MANUSCRIPT
2.4 Hz, 1H, Ar-H), 8.37 (s, 1H, Ar-H), 7.92 (d,
J
= 9.3 Hz, 1H, Ar-H), 7.64 (dd,
J = 9.3, 1.8 Hz, 1H, Ar-H),
4.46 (q, = 7.1 Hz, 2H, CH2), 4.20 (s, 3H, CH3), 1.45 (t,
J
J
= 7.1 Hz, 3H, CH3). ESI-MS: m/z 343.2 [M+H]+.
4.1.7. ethyl 6-(5-amino-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate (7)
A solution of the 6 (2.57 g, 7.5 mmol), iron dust (4.2 g, 75 mmol) and HCl/H2O (50 ml, V:V 1:5) was
stirred at 80 ºC for 4 h. Add water (100 ml), the pH of the mixture was adjusted to 8 using saturated aqueous
NaHCO3. Extracted with ethyl acetate (3 × 200 ml), the organic layer was washed with water (50 ml), dried
with Na2SO4 and evaporated to give compound 2 as a white solid (1.76 g, 75.4% yield). mp 113-115 ºC.1H
NMR (400 MHz, DMSO)
9.3, 1.6 Hz, 1H, Ar-H), 7.72 (d,
(q,
= 7.1 Hz, 2H, CH2), 3.93 (s, 3H, CH3), 1.36 (t,
4.1.8. ethyl 2-(6-bromo-4-oxoquinazolin-3(4H)-yl)acetate (9a)
δ
9.36 (s, 1H, Ar-H), 8.31 (s, 1H, Ar-H), 7.88 (d,
= 2.1 Hz, 1H, Ar-H), 7.20 (d, = 2.2 Hz, 1H, Ar-H), 5.25 (s, 2H, NH2), 4.38
= 7.1 Hz, 3H, CH3). ESI-MS: m/z 313.3 [M+H]+.
J = 9.3 Hz, 1H, Ar-H), 7.83 (dd, J =
J
J
J
J
A solution of the 6-bromoquinazolin-4(3H)-one (0.44 g, 2 mmol), ethyl chloroacetate (0.147 g, 2.4 mmol),
K2CO3(0.28 g, 4 mmol) and DMF(10 ml) was stirred at 6ºC for 12 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 (0.59 g, 95.2% yield). mp 137-139ºC.1H NMR (400 MHz, DMSO)
Ar-H), 8.24 (d, = 2.3 Hz, 1H, Ar-H), 8.03 (dd, = 8.7, 2.3 Hz, 1H, Ar-H), 7.68 (d, = 8.7 Hz, 1H, Ar-H), 4.84
(s, 2H, CH2), 4.18 (q, = 7.1 Hz, 2H, CH2), 1.22 (t,
= 7.1 Hz, 3H, CH3). ESI-MS: m/z 308.9 [M-H]+.
δ 8.43 (s, 1H,
J
J
J
J
J
Compounds 9b-g were synthesized according to the procedure described in 9a.
4.1.9. 6-bromo-3-(3-morpholinopropyl)quinazolin-4(3H)-one (9b)
92.4% yield, mp 84-86 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 7.97 (dd, = 8.7, 2.3 Hz, 1H, Ar-H), 7.64 (d, = 8.7 Hz, 1H, Ar-H), 4.03 (t,
= 6.6 Hz, 2H, CH2), 2.27 (s, 4H, CH2), 1.87 (p, = 6.6 Hz, 2H, CH2). ESI-MS: m/z
δ
8.44 (s, 1H, Ar-H), 8.23 (d,
= 6.8 Hz, 2H, CH2), 3.45
J = 2.3 Hz, 1H,
J
J
J
(s, 4H, CH2), 2.32 (t,
352.2 [M+H]+.
J
J
4.1.10. 3-(3-(1H-pyrazol-1-yl)propyl)-6-bromoquinazolin-4(3H)-one (9c)
83.6% yield, mp 181-183ºC. 1H NMR (400 MHz, DMSO)
8.40 (s, 1H, Ar-H), 8.22 (d,
Ar-H), 7.97 (dd, = 8.7, 2.4 Hz, 1H, Ar-H), 7.75 (d, = 2.0 Hz, 1H, Ar-H), 7.69 (d, = 2.0 Hz, 1H, Ar-H), 7.63
(d, = 8.7 Hz, 1H, Ar-H), 7.43 (dd, = 3.9, 1.4 Hz, 2H, Ar-H), 6.22 (dt, = 4.0, 2.0 Hz, 2H, Ar-H), 4.20 (t,
7.3 Hz, 2H, CH2), 3.99 (t, = 7.0 Hz, 2H, CH2), 2.23 (p,
= 6.9 Hz, 2H, CH2). ESI-MS: m/z 355.2 [M+Na]+.
4.1.11. 3-(3-(1H-imidazol-1-yl)propyl)-6-bromoquinazolin-4(3H)-one (9d)
78.8% yield, mp 151-153 ºC. 1H NMR (400 MHz, DMSO)
8.39 (s, 1H, Ar-H), 8.23 (d,
Ar-H), 7.98 (dd, = 8.7, 2.3 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.64 (d, = 8.7 Hz, 1H, Ar-H), 7.23 (s, 1H,
Ar-H), 6.90 (s, 1H, Ar-H), 4.06 (t, = 7.2 Hz, 2H, Ar-H), 4.00 (dd, = 13.4, 6.4 Hz, 2H, CH2), 2.18 (p, = 7.1
Hz, 2H, CH2). ESI-MS: m/z 333.1 [M+H]+.
4.1.12. 6-bromo-3-(4-morpholino-4-oxobutyl)quinazolin-4(3H)-one (9e)
91.8% yield, mp 50-52 ºC. 1H NMR (400 MHz, DMSO)
8.40 (s, 1H, Ar-H), 8.23 (d,
δ
J = 2.3 Hz, 1H,
J
J
J
J
J
J
J =
J
J
δ
J = 2.2 Hz, 1H,
J
J
J
J
J
δ
J = 2.2 Hz, 1H,

ACCEPTED MANUSCRIPT
Ar-H), 7.97 (dd,
(dd, = 12.7, 8.3 Hz, 4H, CH2), 3.40 (dd,
CH2). ESI-MS: m/z 380.2 [M+H]+.
4.1.13. 6-bromo-3-(5-morpholino-5-oxopentyl)quinazolin-4(3H)-one (9f)
90.3% yield, mp 52-54 ºC. 1H NMR (400 MHz, DMSO)
8.46 (s, 1H, Ar-H), 8.23 (d,
Ar-H), 7.97 (dd, = 8.7, 2.3 Hz, 1H, Ar-H), 7.64 (d, = 8.7 Hz, 1H, Ar-H), 4.00 (t, = 7.1 Hz, 2H, Ar-H), 3.52
(dd, = 9.4, 4.8 Hz, 4H, CH2), 3.42 (brs, 4H, CH2), 2.35 (t, = 7.4 Hz, 2H, CH2), 1.81 – 1.65 (m, 2H, CH2),
1.52 (dd,
= 15.0, 7.5 Hz, 2H, CH2). ESI-MS: m/z 394.2 [M+H]+.
4.1.14. 6-bromo-3-(3-(2-oxopyridin-1(2H)-yl)propyl)quinazolin-4(3H)-one (9g)
82.5% yield, mp148-150 ºC. 1H NMR (400 MHz, DMSO)
8.47 (s, 1H, Ar-H), 8.23 (d,
Ar-H), 7.99 - 7.97 (m, 1H, Ar-H), 7.70 (d, = 5.5 Hz, 1H, Ar-H), 7.64 (d, = 8.6 Hz, 1H, Ar-H), 7.41-7.37 (m,
1H, Ar-H), 6.36 (d, J = 9.0 Hz, 1H, Ar-H), 6.21 (t, J = 6.3 Hz, 1H, Ar-H), 4.02 (t, = 6.7 Hz, 2H, CH2), 3.95 (t,
= 6.9 Hz, 2H, CH2), 2.11- 2.04 (m, 2H, CH2). ESI-MS: m/z 382.1 [M+Na]+.
J
= 8.7, 2.3 Hz, 1H, Ar-H), 7.63 (d,
J
= 8.7 Hz, 1H, Ar-H), 4.01 (t,
J
= 6.8 Hz, 2H, Ar-H), 3.55
J
J
= 10.3, 5.0 Hz, 4H, CH2), 2.39 (m, 2H, CH2), 1.95 (t,
J
= 6.8 Hz, 2H,
δ
J = 2.3 Hz, 1H,
J
J
J
J
J
J
δ
J = 1.5 Hz, 1H,
J
J
J
J
4.1.15. ethyl 5-(5-(4-fluorophenylsulfonamido)-6-methoxypyridin-3-yl)-1H-indole-2-carboxylate (1a)
A solution of the 3a (0.204 g, 0.5 mmol), ethyl 5-bromo-1H-indole-2-carboxylate (0.134 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 20:1) as a white solid (0.12 g, 51.2% yield).
mp 108-110 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 7.87 – 7.80 (m, 4H, Ar-H), 7.55 (d, = 8.6 Hz, 1H, Ar-H), 7.49 (dd,
= 1.5 Hz, 1H, Ar-H), 4.36 (q, = 7.1 Hz, 2H, CH2), 3.66 (s, 3H, CH3), 1.36 (t,
= 7.1 Hz, 3H, CH3).13C NMR (100 MHz, DMSO)
164.8 ( = 249.7 Hz), 161.7, 156.4, 141.6, 137.4, 137.1,
= 9.6 Hz), 129.2, 128.7, 127.8, 124.2, 120.6, 120.2, 116.7 (d, = 22.8 Hz),
δ
12.00 (s, 1H, NH), 10.02 (s, 1H, NH), 8.27 (d,
J = 2.2 Hz, 1H,
J
J
= 8.6, 1.4 Hz, 1H, Ar-H), 7.43 (t,
J
J
= 8.8 Hz, 2H, Ar-H), 7.22 (d,
J
J
δ
J
137.0, 132.3, 131.1, 130.3 (d,
J
J
113.8, 108.5, 61.0, 53.8, 14.8. HRMS: m/z 470.1181 [M+H]+.
Compounds 1b-f was synthesized according to the procedure described in 1a.
4.1.16. ethyl 6-(5-(4-fluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-2-carboxylate
(1b)
72.6% yield, mp 222-224 ºC. 1H NMR (400 MHz, DMSO)
1H, Ar-H), 8.32 (d, = 2.1 Hz, 1H, Ar-H), 7.94 (d, = 2.1 Hz, 1H, Ar-H), 7.85 (dd,
7.73 (d, = 9.5 Hz, 1H, Ar-H), 7.65 (dd, = 1.3, 9.5Hz, 1H, Ar-H), 7.43 (t, = 8.8 Hz, 2H, Ar-H), 4.34 (q,
7.1 Hz, 2H, CH2), 3.66 (s, 3H, CH3), 1.34 (t, 164.8 (d,
= 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO)
= 249.6 Hz), 163.0, 157.2, 144.2, 141.8, 137.1, 136.5, 132.0, 130.3 (d,
121.1, 118.9, 118.6, 116.7 (d,
= 22.7 Hz), 60.75, 53.94, 14.71. HRMS: m/z 471.1126 [M+H]+.
δ
10.11 (s, 1H, NH), 8.90 (s, 1H, Ar-H), 8.57 (s,
=5.2, 8.7 Hz, 2H, Ar-H),
J
J
J
J
J
J
J
=
J
δ
J
J
= 9.7 Hz), 126.7, 126.3, 124.9, 123.3,
J
4.1.17. ethyl 6-(5-(4-fluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate
(1c)

ACCEPTED MANUSCRIPT
83.1% yield, mp168-170 ºC. 1H NMR (400 MHz, DMSO)
δ
10.17 (s, 1H, NH), 9.34 (s, 1H, Ar-H), 8.33 (d,
J
= 2.3 Hz, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 7.93 – 7.83 (m, 5H, Ar-H), 7.44 (t,
J
= 8.8 Hz, 2H, Ar-H), 4.41 (q,
164.9 (d,
= 9.6 Hz), 128.0, 126.3, 124.7, 124.6,
J
= 22.7 Hz), 116.2, 60.80, 54.04, 14.75. HRMS: m/z 471.1131 [M+H]+.
J
= 7.1 Hz, 2H, CH2), 3.72 (s, 3H, CH2), 1.39 (t,
J
= 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO)
= 250.1 Hz), 160.4, 157.1, 147.5, 141.9, 141.7, 136.9, 131.2, 130.3 (d,
δ
J
J
121.3, 118.3, 116.8( d,
4.1.18. ethyl 6-(6-methoxy-5-(methylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate (1d)
65.9% yield, mp 208-210 ºC. 1H NMR (400 MHz, DMSO)
9.45 (s, 1H, NH), 9.38 (s, 1H, Ar-H), 8.36 (d,
= 2.2 Hz, 1H, Ar-H), 8.33 (s, 1H, Ar-H), 7.94 (d, = 2.3 Hz, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 7.90 (d, = 1.6
Hz, 1H, Ar-H), 4.39 (q, = 7.1 Hz, 2H, CH2), 4.00 (s, 3H, CH3), 3.12 (s, 3H, CH3), 1.37 (t, = 7.1 Hz, 3H,
CH3). 13C NMR (100 MHz, DMSO)
160.3, 156.9, 147.5, 142.0, 141.1, 130.6, 128.3, 126.5, 125.0, 124.7,
122.2, 118.2, 116.2, 60.8, 54.4, 41.2, 14.7. HRMS: m/z 391.1066 [M+H]+.
4.1.19. ethyl 6-(5-(butylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate (1e)
78.4% yield, mp 147-149 ºC. 1H NMR (400 MHz, DMSO)
9.48 (s, 1H, NH), 9.36 (s, 1H, Ar-H), 8.35 (s,
1H, Ar-H), 8.31 (s, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 7.88 (q, = 9.5 Hz, 2H, Ar-H), 4.39 (q, = 7.0 Hz, 2H, CH2),
4.00 (s, 3H, CH3), 3.25 – 3.11 (m, 2H, CH2), 1.83 – 1.68 (m, 2H, CH2), 1.46 – 1.36 (m, 5H, CH2 and CH3), 0.89
(t, 160.3 , 156.9, 147.4, 141.9, 141.1, 130.9, 128.1, 126.4,
= 7.3 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO)
δ
J
J
J
J
J
δ
δ
J
J
J
δ
124.9, 124.5, 122.1, 118.2, 116.2, 60.8, 54.3, 52.5, 25.6, 21.3, 14.7, 14.0. HRMS: m/z 433.1560 [M+H]+.
4.1.20 ethyl 6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate
(1f)
76.3% yield, mp 159-161 ºC. 1H NMR (400 MHz, DMSO)
= 2.3 Hz, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 7.97 – 7.74 (m, 4H, Ar-H), 7.65 – 7.54 (m, 1H, Ar-H), 7.24 (td,
1.9, 8.5 Hz, 1H, Ar-H), 4.40 (q, = 7.1 Hz, 2H, CH2), 3.70 (s, 3H, CH3), 1.38 (t,
= 7.1 Hz, 3H, CH3). 13C
NMR (100 MHz, DMSO) 165.6 (dd, = 12.2, 252.9 Hz), 160.3, 159.8 (dd, = 13.3, 256.1 Hz), 158.1, 147.4,
142.7, 141.9, 133.7, 132.3 (d, = 10.6 Hz), 128.0, 126.4, 125.4 (dd, = 3.5, 14.2 Hz), 124.6, 124.5, 120.5,
= 22.4, 3.5 Hz), 106.3 (t, = 26.1 Hz), 60.8, 54.0, 14.7. HRMS: m/z 489.1022
δ 10.43 (s, 1H, NH), 9.35 (s, 1H, Ar-H), 8.38 (d,
J
J =
J
J
δ
J
J
J
J
118.2, 116.2, 112.4 (dd,
[M+H]+.
J
J
4.1.21. 6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylic acid
(1g)
1f was dissolved in EtOH-H2O-NaOH (1.5 mol/L) and refluxed for 0.5 h, then acidified to pH 4-5 with
HCl (2 mol/L) to afford white solid precipitate, following by wash and dry, to afford 1g. 94.0% yield, mp
217-219 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.29 (s, 1H, Ar-H), 7.91-7.69 (m, 4H, Ar-H), 7.59 (t,
Ar-H), 3.69 (s, 3H, CH3).13C NMR (100 MHz, DMSO)
167.4, 165.6 (dd, J = 11.6, 252.5 Hz), 159.9 (dd,
13.4, 270.9 Hz), 158.1, 147.4, 142.7, 141.9, 133.7, 132.4 (d, = 10.8 Hz), 127.8, 126.6, 125.4 (dd, = 3.5, 14.2
Hz), 124.7, 124.3, 120.5, 118.2, 116.9, 112.4 (dd, = 3.5, 22.4 Hz), 106.3(t, J = 25.8 Hz), 54.0. HRMS: m/z
δ
13.20 (s, 1H), 10.42 (s, 1H, NH), 9.42 (s, 1H, Ar-H), 8.38 (s, 1H,
= 9.2 Hz, 1H, Ar-H), 7.23 (t, = 8.4 Hz, 1H,
J
J
δ
J =
J
J
J

ACCEPTED MANUSCRIPT
461.0727 [M+H]+.
4.1.22.6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-N-(2-(dimethylamino)ethyl)imidazo[1,2-a]
pyridine-3-carboxamide (1h)
A solution of the 1f (0.23 g, 0.5 mmol), N,N-dimethylethane-1,2-diamine (0.044 g, 0.5 mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.14 g, 0.75mmol),
N-hydroxybenzotrizole (HOBt, 0.21 g, 0.5 mmol), and N-diisopropylethylamine (DIPEA, 0.26ml, 1.5 mmol) in
anhydrous THF (10 ml) was stirred for 24 h. THF was removed under reduced pressure and the residue was
purified through a column chromatography on silica with chloroform/methanol (V:V 20:1) as a white solid.
78.9% yield, mp 256-258 ºC. 1H NMR (400 MHz, DMSO)
8.36 (s, 1H, Ar-H), 8.11 (s, 1H, Ar-H), 7.86 (dd, = 8.3, 15.1 Hz, 1H, Ar-H), 7.79 (d,
7.77 – 7.66 (m, 2H, Ar-H), 7.46 (t, = 9.0 Hz, 1H, Ar-H), 7.21 (t, = 7.7 Hz, 1H, Ar-H), 3.72 (s, 3H, CH3),
3.52 (dd, = 5.9, 11.9 Hz, 2H, CH2), 2.75 (t,
= 6.3 Hz, 2H, CH2), 2.44 (s, 6H, CH3×2). 13C NMR (100 MHz,
DMSO) 165.6 (dd, = 12.0, 252.8 Hz), 160.8, 159.7 (dd, = 12.7, 269.2 Hz), 158.2, 145.2, 142.8, 135.8,
133.7, 132.3(d, = 10.7 Hz), 128.4, 126.3 , 125.4 (dd, = 13.5, 4.4 Hz), 125.0 , 124.5, 120.5, 119.0, 117.1,
112.5 (dd, = 3.7, 21.7 Hz), 106.4 (t,
= 25.7 Hz), 56.4, 54.0, 43.0, 34.4. HRMS: m/z 531.1631 [M+H]+.
δ
9.62 (s, 1H, NH), 8.60 (t,
J
= 5.3 Hz, 1H, Ar-H),
J
J = 9.3 Hz, 1H, Ar-H),
J
J
J
J
δ
J
J
J
J
J
J
Compounds 1i-m were synthesized according to the procedure described in 1h.
4.1.23.6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-N-(2-morpholinoethyl)imidazo[1,2-a]pyri
dine-3-carboxamide (1i)
74.5% yield, mp 114-116 ºC. 1H NMR (400 MHz, DMSO)
1H, Ar-H), 8.32 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.85 – 7.79 (m, 2H, Ar-H), 7.75 (d,
7.57 (t, = 9.1 Hz, 1H, Ar-H), 7.24 (t, = 7.9 Hz, 1H, Ar-H), 3.70 (s, 3H, CH3), 3.61 (s, 4H, CH2×2), 3.48 (d,
= 6.1 Hz, 2H, CH2), 2.51 (s, 4H, CH2×2). 13C NMR (100 MHz, DMSO)
165.5 (dd, = 11.3, 252.0 Hz), 160.6,
159.8 (dd, = 13.3, 255.6 Hz), 157.9, 146.4, 142.0, 137.6, 133.0, 132.4 (d, = 10.6 Hz), 126.7(2C), 125.7(dd,
= 3.3, 22.0 Hz), 106.3 (t, = 26.1 Hz), 66.5,
δ
9.67 (s, 1H, NH), 8.56 (s, 1H, Ar-H), 8.37 (s,
= 9.3 Hz, 1H, Ar-H),
J
J
J
J
δ
J
J
J
J
= 3.4, 14.7 Hz), 124.8, 123.7, 121.1, 119.1, 117.9, 112.4 (dd,
57.9, 53.9, 53.7, 36.2. HRMS: m/z 573.1727 [M+H]+.
4.1.24.
J
J
6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-N-(3-morpholinopropyl)imidazo[1,2-a]pyridine-3
-carboxamide (1j)
76.0% yield, mp 100-102 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.35 (d, = 8.2 Hz, 1H, Ar-H), 8.31 (d, = 1.9 Hz, 1H, Ar-H), 7.88 – 7.79 (m, 2H, Ar-H), 7.77 –7.68 (m,
1H, Ar-H), 7.62 – 7.52 (m, 1H, Ar-H), 7.28 – 7.20 (m, 1H, Ar-H), 3.69 (s, 3H, CH3), 3.60 (s, 4H, CH2×2), 3.35
(dt, = 6.5, 15.4 Hz, 4H, CH2×2), 2.44 (d,
= 6.4 Hz, 4H, CH2×2), 1.83 - 1.68 (m, 2H, CH2). 13C NMR (100
MHz, DMSO) 165.4 (dd, = 11.9, 252.1 Hz), 160.6, 159.8 (dd, = 12.9, 255.3 Hz), 157.9, 146.3, 141.7,
137.5, 132.7, 132.4 (d, = 10.6 Hz), 126.7, 126.6, 125.8 (dd, = 4.1, 15.1 Hz), 124.7, 123.7, 121.5, 119.1,
δ 9.67 (s, 1H, NH), 8.58 (t, J = 5.2 Hz, 1H,
J
J
J
J
δ
J
J
J
J

ACCEPTED MANUSCRIPT
117.9, 112.3 (dd, J = 3.3, 22.2 Hz), 106.3 (t,
587.1865 [M+H]+.
J = 26.2 Hz), 66.4, 56.2, 53.9, 53.6, 37.3, 26.4. HRMS: m/z
4.1.25.1-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carbonyl)pipe
ridin-4-yl benzoate (1k)
67.2% yield, mp 129-131 ºC. 1H NMR (400 MHz, DMSO)
= 2.3 Hz, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H), 8.03 (d,
2.3 Hz, 1H, Ar-H), 7.82 (dt, = 6.9, 14.8 Hz, 2H, Ar-H), 7.75 (dd, = 1.7, 9.4 Hz, 1H, Ar-H), 7.68 (t,
Hz, 1H, Ar-H), 7.58 (dt, = 5.0, 15.3 Hz, 3H, Ar-H), 7.24 (td, = 2.1, 8.5 Hz, 1H, Ar-H), 5.38 – 5.24 (m, 1H,
CH), 4.12 – 3.98 (m, 2H, CH2), 3.87 – 3.73 (m, 2H, CH2), 3.69 (s, 3H, CH3), 2.20 – 2.06 (m, 2H, CH2), 1.88
(ddd, 165.6 (dd, = 11.5, 252.6 Hz), 165.5,
= 5.2, 8.7, 11.4 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO)
160.5, 159.8 (dd, = 12.9, 255.9 Hz), 158.1, 146.1, 142.7, 137.6, 134.0, 133.8, 132.4 (d, = 10.5 Hz), 130.4,
129.7, 129.2, 126.8, 125.5 (dd, = 14.2, 3.4 Hz), 125.3, 123.1, 120.4, 118.5, 117.8, 112.4 (dd, = 3.2, 22.2 Hz),
= 25.8 Hz), 70.3, 53.9, 30.9, 25.4. HRMS: m/z 648.1742 [M+H]+.
δ
10.42 (s, 1H, NH), 9.06 (s, 1H, Ar-H), 8.37
= 1.2 Hz, 1H, Ar-H), 7.91 (d,
= 7.4
(d,
J
J
J =
J
J
J
J
J
J
δ
J
J
J
J
J
106.32 (t,
J
4.1.26.
1-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)imidazo[1,2-a]pyridine-3-carbonyl)piperidin-4
-yl benzoate (1l)
73.2% yield, mp 89-91 ºC. 1H NMR (400 MHz, DMSO)
= 2.3 Hz, 1H, Ar-H), 8.02 (s, 1H, Ar-H), 7.89 (d, = 2.3 Hz, 1H, Ar-H), 7.84 – 7.76 (m, 2H, Ar-H), 7.73 (dd,
= 1.7, 9.4 Hz, 1H, Ar-H), 7.63 – 7.54 (m, 1H, Ar-H), 7.23 (td, = 2.1, 8.5 Hz, 1H, Ar-H), 4.85 (d, = 3.5 Hz,
1H, Ar-H), 4.08 (dt, = 3.9, 9.1 Hz, 2H, CH2), 3.82 (d, = 3.5 Hz, 1H, CH), 3.67 (s, 3H, CH3), 3.52 – 3.37 (m,
2H, CH2), 1.91 – 1.80 (m, 2H, CH2), 1.57 – 1.42 (m, 2H, CH2). 13C NMR (100 MHz, DMSO)
165.6 (dd,
11.2, 252.2 Hz), 160.2, 159.8 (dd, = 13.3, 256.0 Hz), 158.0, 146.0, 143.3, 137.4, 134.0 , 132.4 (d, = 10.4 Hz),
126.8, 126.7, 125.5 (dd, = 3.9, 14.6 Hz), 125.2, 123.0, 120.4, 118.6, 117.8, 112.4 (dd, = 2.9, 22.2 Hz),
= 26.3 Hz), 66.0, 53.9, 34.7. HRMS: m/z 544.1457 [M+H]+.
δ 10.39 (s, 1H, NH), 9.02 (s, 1H, Ar-H), 8.36 (d, J
J
J
J
J
J
J
δ
J =
J
J
J
J
106.32 (t,
J
4.1.27.
6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyridine-3-
carboxamide (1m)
69.5% yield, mp 91-93 ºC. 1H NMR (400 MHz, DMSO)
= 5.8 Hz, 1H, Ar-H), 8.84 (d, = 6.1 Hz, 2H, Ar-H), 8.60 (s, 1H, Ar-H), 8.36 (d,
= 3.9, 11.5 Hz, 4H, Ar-H), 7.87 (d, = 2.3 Hz, 1H, Ar-H), 7.79 (dt, = 7.5, 14.9 Hz, 1H, Ar-H), 7.64 – 7.53
= 5.3, 11.6 Hz, 1H, Ar-H), 4.81 (d,
= 5.6 Hz, 2H, CH2), 3.68 (s, 3H, CH3). 13C
165.6 (dd, = 11.3, 252.0 Hz), 160.6, 159.8 (dd, = 13.8, 255.6 Hz), 158.1, 144.8,
143.1 (2C), 142.7, 135.1, 133.5, 132.4 (d, = 3.7 Hz), 125.3, 125.1 (2C),
= 25.7 Hz), 54.0, 42.2. HRMS: m/z
δ
10.43 (s, 1H, NH), 9.64 (s, 1H, Ar-H), 9.54 (t,
J
J
J
= 2.3 Hz, 1H, Ar-H), 7.93 (td,
J
J
J
(m, 1H, Ar-H), 7.19 (dd,
J
J
NMR (100 MHz, DMSO)
δ
J
J
J
= 10.8 Hz), 129.2, 126.1, 125.4 (d, J
124.9, 120.5, 118.8, 116.7, 112.4 (dd,
551.1317 [M+H]+.
J
= 22.3, 3.2 Hz), 106.6, 106.3 (t, J

ACCEPTED MANUSCRIPT
Compounds 10a-g were synthesized according to the procedure described in 1a.
4.1.28. ethyl 2-(6-(5-(2,4-difluorophenylsulfonamido)-6-methoxypyridin-3-yl)-4-oxoquinazolin-3(4H)-yl)acetate
(10a)
81.2% yield, mp 208-210 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.42 (s, 1H, Ar-H), 8.28 (s, 1H, Ar-H), 8.15 (dd, = 8.4, 1.4 Hz, 1H, Ar-H), 7.98 (d,
Ar-H), 7.82 (d,
(s, 2H, CH2), 4.20 (q,
MHz, DMSO) 168.36, 165.6 (dd,
147.8, 143.0, 135.6, 134.3, 133.3, 132.3 (d,
δ
10.37 (s, 1H, H), 8.45 (d,
J
J
= 1.8 Hz, 1H,
= 1.7 Hz, 1H,
J
J
= 8.5 Hz, 1H, Ar-H), 7.79 – 7.75 (m, 1H, Ar-H), 7.59 (m, 1H, Ar-H), 7.22 (m, 1H, Ar-H), 4.87
= 7.0 Hz, 2H, CH2), 3.68 (s, 3H, CH3), 1.24 (t,
= 7.1 Hz, 3H, CH3). 13C NMR (100
= 11.6, 252.6 Hz), 160.5, 159.8 (dd, = 13.5, 256.0 Hz), 158.1, 148.6,
= 10.8 Hz), 128.8, 125.5 (dd, = 3.8, 14.3 Hz), 123.5, 122.2,
= 25.9 Hz), 61.8, 54.0, 47.8, 14.5. HRMS: m/z 531.1159
J
J
δ
J
J
J
J
120.3, 112.4 (dd,
[M+H]+.
J = 3.2, 22.3 Hz), 106.3 (t, J
4.1.29.2,4-difluoro-N-(2-methoxy-5-(3-(3-morpholinopropyl)-4-oxo-3,4-dihydroquinazolin-6-yl)pyridin-3-yl)be
nzenesulfonamide (10b)
77.8% yield, mp 179-181 ºC. 1H NMR (400 MHz, DMSO)
8.37 (m, 2H, Ar-H), 8.09 (d, = 2.1 Hz, 1H, Ar-H), 8.06 (d, = 8.7 Hz, 1H, Ar-H), 7.83 – 7.68 (m, 2H, Ar-H),
7.64 – 7.50 (m, 1H, Ar-H), 7.20 (dd, = 5.2, 11.9 Hz, 1H, Ar-H), 3.63 (s, 3H, CH3), 3.62 – 3.53 (m, 6H,
CH2×3), 2.48 – 2.32 (m, 6H, CH2×3), 1.86 (p, 165.4 (dd,
= 6.8Hz, 2H, CH2). 13C NMR (100 MHz, DMSO)
= 11.8, 252.2 Hz), 159.9, 159.8 (dd, = 13.3, 255.9 Hz), 158.3, 155.8, 148.9, 142.9, 135.2, 133.8, 132.3 (d,
10.1 Hz), 131.1, 129.4, 128.8, 125.8 (dd, = 3.1, 14.5 Hz), 120.5 (2C), 115.7, 112.4 (dd, = 3.0, 22.0 Hz),
106.2 (t,
= 25.7 Hz), 66.5, 56.4, 53.8, 53.7, 26.8, 25.8. HRMS: m/z 571.1905 [M]+.
δ 10.37(s, 1H, NH), 8.55 (s, 1H, Ar-H), 8.52 –
J
J
J
J
δ
J
J
J =
J
J
J
4.1.30.N-(5-(3-(3-(1H-pyrazol-1-yl)propyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-2-methoxypyridin-3-yl)-2,4-diflu
orobenzenesulfonamide (10c)
80.2% yield, mp 182-184 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.39 (s, 1H, Ar-H), 8.28 (d, = 2.0 Hz, 1H, Ar-H), 8.10 (dd,
Hz, 1H, Ar-H), 7.84 – 7.74 (m, 3H, Ar-H), 7.64 – 7.54 (m, 1H, Ar-H), 7.45 (d,
= 2.0, 8.5 Hz, 1H, Ar-H), 6.24 (t, = 1.9 Hz, 1H, Ar-H), 4.22 (t, = 6.9 Hz, 2H, CH2), 4.03 (t,
CH2), 3.68 (s, 3H, CH3), 2.26 (p, 165.6 (dd,
= 6.9 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO)
252.3 Hz), 160.7, 159.8 (dd, = 13.4, 256.0 Hz), 158.0, 148.7, 147.8, 142.8, 139.1, 135.2, 134.2, 132.8, 132.3
(d, = 11.4 Hz), 130.3, 128.9, 128.6, 125.5 (dd, = 3.5, 14.4 Hz), 123.5, 122.5, 120.3, 112.4 (dd, = 3.3, 21.9
Hz), 106.3 (d,
= 26.2 Hz), 105.6, 54.0, 49.0, 44.4, 30.0. HRMS: m/z 553.1464 [M+H]+.
δ
10.37 (s, 1H, H), 8.43 (d,
= 2.1, 8.5 Hz, 1H, Ar-H), 7.97 (d,
= 1.3 Hz, 1H, Ar-H), 7.23 (td,
= 6.9 Hz, 2H,
= 11.1,
J
= 2.2 Hz, 1H,
J
J
J
= 2.2
J
J
J
J
J
J
δ
J
J
J
J
J
J
4.1.31.N-(5-(3-(3-(1H-imidazol-1-yl)propyl)-4-oxo-3,4-dihydroquinazolin-6-yl)-2-methoxypyridin-3-yl)-2,4-difl
uorobenzenesulfonamide (10d)
75.8% yield, mp 191-193 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.28 (d, = 2.1 Hz, 1H, Ar-H), 8.10 (dd, = 8.5, 2.2 Hz, 1H, Ar-H), 7.95 (d,
– 7.71 (m, 3H, Ar-H), 7.61 – 7.52 (m, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.22 (td, = 2.2, 8.6 Hz, 1H, Ar-H), 6.93
δ
8.40 (d,
J
= 2.3 Hz, 1H, Ar-H), 8.38 (s, 1H,
= 2.3 Hz, 1H, Ar-H), 7.83
J
J
J
J

ACCEPTED MANUSCRIPT
(s, 1H, Ar-H), 4.08 (t,
2H, CH2). 13C NMR (100 MHz, DMSO)
158.0, 148.5, 147.8, 142.3, 137.6, 135.4, 133.6, 132.8, 132.3 (d,
J
= 7.1 Hz, 2H, CH2), 4.02 (t,
J
= 6.9 Hz, 2H, CH2), 3.68 (s, 3H, CH3), 2.30 – 2.13 (m,
= 10.9, 251.7 Hz), 160.7, 159.8 (d, = 10.5, 253.5 Hz),
= 11.2 Hz), 128.9, 128.6, 128.5, 125.7 (dd,
= 3.3, 21.9 Hz), 106.3 (t, = 26.5 Hz), 53.9, 44.2, 44.1,
δ
165.4 (dd,
J
J
J
J
= 2.9, 14.6 Hz), 123.5, 122.5, 121.0, 119.8, 112.3 (dd,
30.6. HRMS: m/z 553.1474 [M+H]+.
J
J
4.1.32.2,4-difluoro-N-(2-methoxy-5-(4-oxo-3-(3-(2-oxopyridin-1(2H)-yl)propyl)-3,4-dihydroquinazolin-6-yl)pyr
idin-3-yl)benzenesulfonamide (10e)
74.2% yield, mp 118-120 ºC. 1H NMR (400 MHz, DMSO)
= 2.2 Hz, 1H, Ar-H), 8.28 (d, = 1.9 Hz, 1H, Ar-H), 8.10 (dd,
Hz, 1H, Ar-H), 7.83 – 7.75 (m, 2H, Ar-H), 7.73 (dd, = 1.5, 6.7 Hz, 1H, Ar-H), 7.63 – 7.54 (m, 1H, Ar-H),
7.40 (ddd, = 1.9, 6.6, 8.8 Hz, 1H, Ar-H), 7.22 (td, = 1.9, 8.6 Hz, 1H, Ar-H), 6.38 (d, = 9.0 Hz, 1H, Ar-H),
6.22 (t, = 6.2 Hz, 1H, Ar-H), 4.06 (t, = 6.8 Hz, 2H, CH2), 3.98 (t, = 7.1 Hz, 2H, CH2), 3.68 (s, 3H, CH3),
2.19 – 2.05 (m, 2H, CH2). 13C NMR (100 MHz, DMSO)
165.6 (dd, = 11.8, 252.6 Hz), 162.8, 161.9, 160.6,
159.8 (d, = 13.9, 252.3 Hz), 158.0, 148.6, 147.8, 142.7, 140.3, 139.4, 135.2, 134.1, 132.7, 132.3 (d, = 11.0
Hz), 128.9, 128.6, 125.5 (dd, = 3.7, 14.1 Hz), 123.5, 122.5, 120.3, 120.1, 112.3 (dd, = 3.5, 22.2 Hz), 106.3 (t,
= 26.1 Hz), 105.9, 53.9, 46.5, 44.3, 29.1. HRMS: m/z 580.1477 [M+H]+.
δ
10.39 (s, 1H, NH), 8.46 (s, 1H, Ar-H), 8.42
= 8.5, 2.1 Hz, 1H, Ar-H), 7.96 (d, = 2.2
(d,
J
J
J
J
J
J
J
J
J
J
J
δ
J
J
J
J
J
J
4.1.33. 4-difluoro-N-(2-methoxy-5-(3-(4-morpholino-4-oxobutyl)-4-oxo-3,4-dihydroquinazolin-6-yl)pyridin-3-yl)
benzenesulfonamide (10f)
80.1% yield, mp 113-115 ºC. 1H NMR (400 MHz, DMSO)
Ar-H), 8.39 (s, 1H, Ar-H), 8.28 (d, = 2.1 Hz, 1H, Ar-H), 8.10 (dd,
Hz, 1H, Ar-H), 7.83 – 7.73 (m, 2H, Ar-H), 7.64 – 7.54 (m, 1H, Ar-H), 7.22 (td,
(t, = 6.8 Hz, 2H, CH2), 3.68 (s, 3H, CH3), 3.60 – 3.53 (m, 2H, CH2), 3.53 – 3.48 (m, 2H, CH2), 3.41 (dd,
4.9, 11.4 Hz, 4H, CH2×2), 2.41 (t, = 7.2 Hz, 2H, CH2), 1.98 (p,
= 7.0 Hz, 2H, CH2). 13C NMR (100 MHz,
DMSO) 170.4, 165.6 (dd, = 11.5, 252.4 Hz), 160.6, 159.8 (dd, = 13.6, 256.3 Hz), 158.0, 148.7, 147.8,
142.8, 135.2, 134.1, 132.7, 132.3 (d, = 11.0 Hz), 128.9, 128.6, 125.4 (dd, = 3.4, 14.6 Hz), 123.6, 122.5,
120.3, 112.3 (dd, = 3.2, 22.3 Hz), 106.3 (t, = 25.8 Hz), 55.4, 53.9, 46.3, 45.8, 41.9, 29.5. HRMS: m/z
600.1741 [M+H]+.
δ
10.36 (s, 1H, NH), 8.43 (d,
= 2.2, 8.5 Hz, 1H, Ar-H), 7.96 (d,
= 2.0, 8.4 Hz, 1H, Ar-H), 4.04
J
= 2.3 Hz, 1H,
J
J
J
= 2.3
J
J
J =
J
J
δ
J
J
J
J
J
J
4.1.34.4-difluoro-N-(2-methoxy-5-(3-(5-morpholino-5-oxopentyl)-4-oxo-3,4-dihydroquinazolin-6-yl)pyridin-3-y
l) benzenesulfonamide (10g)
72.7% yield, mp 136-138 ºC. 1H NMR (400 MHz, DMSO)
= 2.2 Hz, 1H, Ar-H), 8.28 (d, = 1.8 Hz, 1H, Ar-H), 8.10 (dd,
Hz, 1H, Ar-H), 7.85 – 7.73 (m, 2H, Ar-H), 7.65 – 7.52 (m, 1H, Ar-H), 7.31 – 7.15 (m, 1H, Ar-H), 4.03 (t,
6.8 Hz, 2H, CH2), 3.68 (s, 3H, CH3), 3.52 (m, 4H, CH2×2), 3.42 (m, 4H, CH2×2), 2.36 (t, = 7.3 Hz, 2H, CH2),
1.81 – 1.66 (m, 2H, CH2), 1.54 (dd, 171.0, 165.6
= 7.5, 14.7 Hz, 2H, CH2). 13C NMR (100 MHz, DMSO)
(dd, = 12.5, 252.6 Hz), 160.5, 159.8 (dd, = 14.6, 255.9 Hz), 158.0, 148.7, 147.8, 142.8, 135.3, 134.1, 132.8,
δ
10.36 (s, 1H, NH), 8.44 (s, 1H, Ar-H), 8.43
= 2.0, 8.5 Hz, 1H, Ar-H), 7.97 (d, = 2.1
(d,
J
J
J
J
J
=
J
J
δ
J
J

ACCEPTED MANUSCRIPT
132.3 (d, J = 10.6 Hz), 128.9, 128.6, 125.4 (dd, J = 3.8, 14.4 Hz), 123.5, 122.4, 120.3, 112.3 (dd, J = 3.3, 22.1
Hz), 106.2 (t, J = 26.0 Hz), 53.9, 46.3, 45.8, 41.9, 31.9, 28.8, 22.1. HRMS: m/z 614.1896 [M+H]+.
4.2 Biological assay methods
4.2.1. Cell culture
HCT-116, A549, SK-HEP-1, SNU638, MDA-MB-231 cells and HUVEC were purchased from the
American Type Culture Collection (Manassas, VA, USA). Cells were grown in DMEM (SNU-638, HUVEC and
SK-HEP-1) or RPMI1640 (HCT-116, A549 and MDA-MB-231 cells) supplemented with 10% FBS and
antibiotics-antimycotics (PSF; 100 units/mL penicillin G sodium, 100 µg/mL streptomycin, and 250 ng/mL
amphotericin B) in a humidified incubator containing 5% CO2 at 37 °C.
4.2.2. Antiproliferative activity
The cell viability was evaluated using the sulforhodamine B (SRB) cellular protein-staining method with
minor modifications. Briefly, cells were treated with various concentrations of compounds in 96-well plates and
incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 72 h. After treatment, the cells were fixed with
10% TCA solution, and cell viability was determined with a sulforhodamine B (SRB) assay. The percentage of
cell-growth inhibition was calculated using the formulae below. The IC50 values were calculated using a
non-linear regression analysis (percent growth versus concentration).
4.2.3. PI3K enzymatic activity assay
The PI3K kinase assay was measured by PI3 Kinase Activity/Inhibitor Assay Kit (EMD Millipore,
#17-493) following the manufacturer’s protocols.
4.2.4. Molecular docking studies
The crystal structure of PI3Kα (PDB entry code: 4ZOP) in complex with 4Q2 was used for molecular
modeling. The AutoDock 4.2 was used to perform docking calculations. Polar hydrogens and partial charges
were added for PI3Kα protein using the Kollman United Atom charges with Sybyl 6.9.1, and energy
minimization was made employing both steepest descent and conjugate gradients protocols. Atomic solvation
parameters and fragmental volumes for the proteins were assigned using the addsol utility in the AutoDock 4.2
program. A 60×60×60 Å grid box with a grid spacing of 0.375 Å was generated for the receptor. Affinity grid
fields were generated using the auxiliary program AutoGrid 4.0. The Lamarckian genetic algorithm (LGA) was
used to find the appropriate binding positions, orientations, and conformations of the ligands. The optimized
AutoDocking parameters are as follows: the maximum number of energy evaluations was increased to
25,000,000 per run; the iterations of Solis & Wets local search was 3000; the number of individuals in
population was 300 and the number of generations was 100. Results differing by less than 2 Å in a positional
root mean square deviation (RMSD) were clustered together. In each group, the lowest binding energy

ACCEPTED MANUSCRIPT
configuration with the highest % frequency was selected as the group representative. All other parameters were
maintained as default. Accelrys Discovery Studio Visualizer 4.0 was used for graphic display.
4.2.5. Western blot analysis
Preparation of whole-cell protein lysates and western blot analysis were described previously [36]. Total
cell lysates were prepared in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS). The protein concentration was determined, and equal amounts of protein samples
were subjected to 10% SDSPAGE. Separated proteins were transferred to PVDF membranes (Millipore,
Bedford, MA, USA) and probed with the indicated antibodies. Exposures were obtained
using ImageQuant LAS 4000 biomolecular imager (GE Healthcare).
Acknowledgments
Financial supported by Career Development Support Plan for Young and Middle-aged Teachers in
Shenyang Pharmaceutical University (Grant No. ZQN2015023) is gratefully acknowledged.
References
[1] M. Martini, M.C.D. Santis, L. Braccini, F. Gulluni, E. Hirsch, PI3K/AKT signaling pathway and cancer: an updated review,
Ann Med, 46 (2014) 372-383.
[2] L.M. Thorpe, H. Yuzugullu, J.J. Zhao, PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic
targeting, Nature Reviews Cancer, 15 (2015) 7-24.
[3] P.J. Jr, F. Janku, Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway, Pharmacology & Therapeutics,
142 (2014) 164-175.
[4] I.A. Mayer, C.L. Arteaga, The PI3K/AKT Pathway as a Target for Cancer Treatment, Annu Rev Med, 67 (2016) 11-28.
[5] S. Volinia, I. Hiles, E. Ormondroyd, D. Nizetic, R. Antonacci, M. Rocchi, M.O. Waterfield, Molecular Cloning, cDNA
Sequence, and Chromosomal Localization of the Human Phosphatidylinositol 3-Kinase p110α (PIK3CA) Gene, Genomics,
24 (1994) 472-477.
[6] Y.L. Lai, B.L. Mau, W.H. Cheng, H.M. Chen, H.H. Chiu, C.Y. Tzen, PIK3CA Exon 20 Mutation is Independently
Associated with a Poor Prognosis in Breast Cancer Patients, Annals of Surgical Oncology, 15 (2008) 1064-1069.
[7] Y. Baohua, Z. Xiaoyan, Z. Tiecheng, Q. Tao, S. Daren, Mutations of the PIK3CA gene in diffuse large B cell lymphoma,
Diagnostic Molecular Pathology the American Journal of Surgical Pathology Part B, 17 (2008) 159-165.
[8] E. Dirican, M. Akkiprik, Ö. A, Mutation distributions and clinical correlations of PIK3CA gene mutations in breast cancer,
Tumor Biology, 37 (2016) 1-13.
[9] I. Vivanco, C.L. Sawyers, The phosphatidylinositol 3-Kinase|[ndash]|AKT pathway in human cancer, Nature Reviews
Cancer, 2 (2002) 489-501.
[10] F. Mericbernstam, A. Akcakanat, H. Chen, K.A. Do, T. Sangai, F. Adkins, A.M. Gonzalezangulo, A. Rashid, K. Crosby, M.
Dong, PIK3CA/PTEN Mutations and Akt Activation As Markers of Sensitivity to Allosteric mTOR Inhibitors, Journal of
Molecular Biology, 18 (2012) 1777-1789.
[11] L.M. Thorpe, H. Yuzugullu, J.J. Zhao, PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic

ACCEPTED MANUSCRIPT
targeting, Nature Reviews Cancer, 15 (2015) 7-24.
[12] J.A. Engelman, Targeting PI3K signalling in cancer: opportunities, challenges and limitations, Nature Reviews Cancer, 9
(2009) 550-562.
[13] P. Liu, H. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-kinase pathway in cancer, Nature Reviews
Drug Discovery, 8 (2009) 627-644.
[14] C.D. Britten, PI3K and MEK inhibitor combinations: examining the evidence in selected tumor types, Cancer
Chemotherapy and Pharmacology, 71 (2013) 1395-1409.
[15] Sharma, S., Singh, J., Ojha, R., Singh, H., Kaur, M., Bedi, P. M. S., & Nepali, K., Design strategies, structure activity
relationship and mechanistic insights for purines as kinase inhibitors. Eur J Med Chem112 (2016) 298-346.
[16] S.D. Knight, N.D. Adams, J.L. Burgess, A.M. Chaudhari, M.G. Darcy, C.A. Donatelli, J.I. Luengo, K.A. Newlander, C.A.
Parrish, L.H. Ridgers, Discovery of GSK2126458, a Highly Potent Inhibitor of PI3K and the Mammalian Target of
Rapamycin, Acs Med Chem Lett, 1 (2010) 39-43.
[17] P. Munster, R. Aggarwal, D. Hong, J.H. Schellens, d.N.R. Van, J. Specht, P.O. Witteveen, T.L. Werner, E.C. Dees, E.
Bergsland, First-in-Human Phase I Study of GSK2126458, an Oral Pan-Class I Phosphatidylinositol-3-Kinase Inhibitor, in
Patients with Advanced Solid Tumor Malignancies, Clinical Cancer Research, 22 (2016) 1932-1939.
[18] H. Lee, K.H. Jung, Y. Jeong, S. Hong, S.S. Hong, HS-173, a novel phosphatidylinositol 3-kinase (PI3K) inhibitor, has
anti-tumor activity through promoting apoptosis and inhibiting angiogenesis, Cancer Letters, 328 (2013) 152-159.
[19] R. Morrison, J.M. Al-Rawi, I.G. Jennings, P.E. Thompson, M.J. Angove, Synthesis, structure elucidation, DNA-PK and
PI3K and anti-cancer activity of 8-and 6-aryl-substituted-1-3-benzoxazines, European journal of medicinal chemistry 110
(2016) 326-339.
[20] R.R. Yadav, S.K. Guru, P. Joshi, G. Mahajan, M.J. Mintoo, V. Kumar, S.S. Bharate, D.M. Mondhe, R.A. Vishwakarma, S.
Bhushan, 6-Aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines as a new class of isoform-selective PI3K-alpha
inhibitors, European Journal of Medicinal Chemistry 122 (2016) 731-743.
[21] J. Han, Y. Chen, C. Yang, T. Liu, M. Wang, H. Xu, L. Zhang, C. Zheng, Y. Song, J. Zhu, Structure-based optimization leads
to the discovery of NSC765844, a highly potent, less toxic and orally efficacious dual PI3K/mTOR inhibitor, European
Journal of Medicinal Chemistry 122 (2016) 684-701.
[22] O. Kim, Y. Jeong, H. Lee, S.S. Hong, S. Hong, Design and synthesis of imidazopyridine analogues as inhibitors of
phosphoinositide 3-kinase signaling and angiogenesis, Journal of Medicinal Chemistry, 54 (2011) 2455-2466.
[23] [19] H. Lee, S.J. Kim, K.H. Jung, M.K. Son, H.H. Yan, S. Hong, S.S. Hong, A novel imidazopyridine PI3K inhibitor with
anticancer activity in non-small cell lung cancer cells, Oncology Reports, 30 (2013) 863-869.
[24] M.M. Moasser, A. Basso, S.D. Averbuch, N. Rosen, The tyrosine kinase inhibitor ZD1839 ("Iressa") inhibits HER2-driven
signaling and suppresses the growth of HER2-overexpressing tumor cells, Cancer Research, 61 (2001) 7184-7188.
[25] S.R. Johnston, A. Leary, Lapatinib: a novel EGFR/HER2 tyrosine kinase inhibitor for cancer, Drugs of Today, 42 (2006)
441-453.
[26] J.M. Golas, K. Arndt, C. Etienne, J. Lucas, D. Nardin, J. Gibbons, P. Frost, F. Ye, D.H. Boschelli, F. Boschelli, SKI-606, a
4-anilino-3-quinolinecarbonitrile dual inhibitor of Src and Abl kinases, is a potent antiproliferative agent against chronic
myelogenous leukemia cells in culture and causes regression of K562 xenografts in nude mice, Cancer Research, 63 (2003)
375-381.
[27] S.-C. Tsai, J.-S. Yang, S.-F. Peng, C.-C. Lu, J.-H. Chiang, J.-G. Chung, M.-W. Lin, J.-K. Lin, S. Amagaya, C. Wai-Shan
Chung, Bufalin increases sensitivity to AKT/mTOR-induced autophagic cell death in SK-HEP-1 human hepatocellular

ACCEPTED MANUSCRIPT
carcinoma cells, International journal of oncology 41(4) (2012) 1431-1442.
[28] Y. Lin, H. Cui, H. Xu, L. Yue, H. Xu, L. Jiang, J. Liu, Jolkinolide B induces apoptosis in MDA-MB-231 cells through
inhibition of the PI3K/Akt signaling pathway, Oncology Reports 27(6) (2012) 1976.
[29] Z.H. Xie, F.X. Meng, A.M. Yu, PI3K signaling pathway regulates the proliferation of lung cancer cell lines H446 and A549
through SKP2 and p27, Journal of China Medical University 38(9) (2009) 663-665.
[30] R.W. Carón, A. Yacoub, M. Li, X. Zhu, C. Mitchell, Y. Hong, W. Hawkins, T. Sasazuki, S. Shirasawa, A.P. Kozikowski,
Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the
effect of therapeutic kinase inhibitors on cell survival, Molecular Cancer Therapeutics 4(2) (2005) 257.
[31] E.J. Haagensen, S. Kyle, G.S. Beale, R.J. Maxwell, D.R. Newell, The synergistic interaction of MEK and PI3K inhibitors
is modulated by mTOR inhibition, British Journal of Cancer 106(8) (2012) 1386-94.
[32] Qi, A. Rogers, J. Christensen, P. Janne, J. Engelman, Abstract #3401: Mechanisms of resistance to MET inhibitors in
MET-amplified gastric cancer cells, Cancer Research 69 (2009).
[33] V. Asati, D.K. Mahapatra, S.K. Bharti, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as
anticancer agents: structural and pharmacological perspectives, European journal of medicinal chemistry 109 (2016)
314-341.
[34] M. Toulany, U. Kastenpisula, I. Brammer, S. Wang, J. Chen, K. Dittmann, M. Baumann, E. Dikomey, H.P. Rodemann,
Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of
K-RAS mutated human tumor cells in vitro by affecting DNA repair, Clinical Cancer Research An Official Journal of the
American Association for Cancer Research 12(13) (2006) 4119-26.
[35] J.Y. Pillé, C. Denoyelle, J. Varet, J.R. Bertrand, J. Soria, P. Opolon, H. Lu, L.L. Pritchard, J.P. Vannier, C. Malvy,
Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro
and in vivo, Molecular Therapy the Journal of the American Society of Gene Therapy 11(2) (2005) 267.
[36] Y. Fan, J. Liu, D. Liu, Z. Zhou, Y. Bao, J. Wang, Q. Zhao, Y. Xu, NSCA-1-a novel N-substituted coumalamide
derivative-increases Adriamycin sensitivity in HepG2/adriamycin cells through modulating Akt/GSK-3β signaling and
p53-dependant apoptotic pathway, Environmental Toxicology and Pharmacology 49 (2017) 1-7.

ACCEPTED MANUSCRIPT
Figure legends
Fig.1. RTK/PI3K/Akt signaling cascade in cancer.
Fig.2. The structures of PI3K inhibitors.
Fig.3. The design strategy based on HS-173 and GSK2126458
Fig.4. Docking mode of 1i and HS-173 into protein crystal structure of PI3Kα. (A) Key interactions of HS-173 in the active site
of PI3Kα. (B) Key interactions of compound 1i in the active site of PI3Kα. (C) The binding pose of 1i and HS-173 in the active
site of PI3Kα. 1i was highlighted with yellow. (D) Binding modes of 1i and HS-173 in the binding pocket of PI3Kα. The
ligands are shown in stick model, while the proteins are shown in surface model for better visualization in the three dimensional
combination models. 1i was highlighted with yellow.
Fig.5. Effect of compound 1i on PI3K pathway in HCT-116 cells. (A) Compound 1i decreased phospho-Akt (S473) in a
dose-dependent manner. (B) Compound 1i is more effective than HS-173 in reducing the levels of phospho-Akt (S473). Cells
were treated with 0.1% DMSO, 0.5µM compound 1i or HS-173 for 24 h.
Scheme 1. (a) benzenesulfonyl chloride, pyridine, rt, 24 h; (b) bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF, 100 ºC, 8 h;
(c) Pd(dppf)2Cl2, Cs2CO3, DMF/ H2O, 90 ºC, 8 h; (d) bis(pinacolato)diborane, Pd(dppf)2Cl2, AcOK, DMF, 100 ºC, 8 h; (e)
Pd(dppf)2Cl2, Cs2CO3, DMF, 90ºC, 8h; (f) Fe, HCl/H2O, 80 ºC , 4 h; (g) sulfonyl chloride, pyridine, rt, 24 h; (h) NaOH/Ethanol/
H2O, 80 ºC, 0.5 h, then HCl (2 mol/L); (i) DIPEA, HOBt, EDCI, THF,12 h, rt.
Scheme 2. (a) chlorinated compounds, K2CO3, DMF, 60 ºC, 12 h; (b) 3b, Pd(dppf)2Cl2, Cs2CO3, DMF/ H2O, 90ºC, 8h.

ACCEPTED MANUSCRIPT
Highlights
ò Imidazo [1, 2-a] pyridine and quinazolin-4(3H)-one derivatives were synthesized.
ò Their antiproliferative activities against cancer cell lines were evaluated.
ò 1i exhibited an IC50 value of 10nM against HCT116 and SNU638 cells.
ò The compounds 1b, 1i and 10b were evaluated for their PI3K inhibitory activity
and selectivity.
ò Compound 1i is considered as a potent PI3Kα inhibitor with IC50 of 0.5nM.