Design, synthesis, and biological evaluation of novel 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives as potential anticancer agents via PI3K inhibition

Article abstract of DOI:10.1016/j.bmc.2021.116346

Abnormal activation of the PI3K/Akt pathway is demonstrated in most of human malignant tumors via regulation of proliferation, cell cycle, and apoptosis. Therefore, drug discovery and development of targeting the PI3K/Akt pathway has attracted great interest of researchers in the development of anticancer drugs. In this study, fifteen 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives were designed and synthesized. Anticancer activities of the synthetic compounds were evaluated and the potential mechanisms were explored. Several compounds showed certain proliferation inhibitory activity against the tested cancer cells including human non-small cell lung cancer (NSCLC) HCC827, human neuroblastoma SH-SY5Y and hepatocellular carcinoma LM3 cells. Among them, compound 7i and 7m showed the best inhibitory activity against all the cancer cell lines and more active against HCC827 cells with IC₅₀ values of 1.12 μM and 1.20 μM, respectively. In addition, 7i and 7m showed lower inhibitory activity against H7702 cells (human normal liver cells) with IC₅₀ values of 8.66 μM and 10.89 μM, respectively, nearly 8-fold lower than that in HCC827 cells. These results suggested that compounds 7i and 7m had certain selectivity to tumor cells, compared to human normal cells. Further biological studies indicated 7i induced G2/M phase arrests and cell apoptosis of HCC827 cells via PI3K/Akt and caspase dependent pathway. Together, these novel 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives such as compound 7i and 7m might be lead compounds for development of potential anti-cancer drugs.

Full text of DOI:10.1016/j.bmc.2021.116346

Bioorg. Med. Chem. 46 (2021) 116346
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of novel 6-(pyridin-3-yl)
quinazolin-4(3H)-one derivatives as potential anticancer agents via
PI3K inhibition
,
b
,
,
c
,
,
,
,
,c,**
Huarong Yanga 1, Qing Lia 1, Mingzhi Sua c, Fang Luoa c, Yahua Liub **, Daoping Wanga
,
,c,*
Yanhua Fana
a State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China
b School of Pharmacy, Guizhou University of Traditional Chinese Medicine, Guiyang 550002, PR China
c The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550014, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Abnormal activation of the PI3K/Akt pathway is demonstrated in most of human malignant tumors via regu-
lation of proliferation, cell cycle, and apoptosis. Therefore, drug discovery and development of targeting the
PI3K/Akt pathway has attracted great interest of researchers in the development of anticancer drugs. In this
study, fifteen 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives were designed and synthesized. Anticancer ac-
tivities of the synthetic compounds were evaluated and the potential mechanisms were explored. Several com-
pounds showed certain proliferation inhibitory activity against the tested cancer cells including human non-small
cell lung cancer (NSCLC) HCC827, human neuroblastoma SH-SY5Y and hepatocellular carcinoma LM3 cells.
Among them, compound 7i and 7m showed the best inhibitory activity against all the cancer cell lines and more
6-(pyridin-3-yl) quinazolin-4(3H)-one
derivatives
PI3K/Akt pathway
G2/M phase arrests
Cell apoptosis
active against HCC827 cells with IC50 values of 1.12
μ
M and 1.20
μ
M, respectively. In addition, 7i and 7m
M and
showed lower inhibitory activity against H7702 cells (human normal liver cells) with IC50 values of 8.66
μ
10.89 μM, respectively, nearly 8-fold lower than that in HCC827 cells. These results suggested that compounds 7i
and 7m had certain selectivity to tumor cells, compared to human normal cells. Further biological studies
indicated 7i induced G2/M phase arrests and cell apoptosis of HCC827 cells via PI3K/Akt and caspase dependent
pathway. Together, these novel 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives such as compound 7i and 7m
might be lead compounds for development of potential anti-cancer drugs.
1. Introduction
clinical trials for cancer therapy including PI3K inhibitors such as Aca-
lisib,8 Idelalisib9 and Tenalisib.10 For this purpose, Quinazolin-4(3H)-
Quinazolin-4(3H)-one is a promising pharmacophore with versatile
bioactivities for drug design and development, which is widely discov-
ered in natural products.1,2 Researchers have developed numerous novel
drugs on the market or candidates in the clinical trials based on Qui-
nazolin-4(3H)-one moiety.3–5 Halofuginone is a quinazolinone alkaloid
with potential anticancer activity due to its combined suppression of
invasiveness, vascularization, and cell proliferation via multi-molecular
mechanism including the mediation of PI3K/Akt.5–7 In addition to
Halofuginone, there are many small-molecule inhibitors in preclinical or
one derivatives are promising and useful PI3K inhibitors based on these
previous studies.
PI3K can be divided into three categories, among them, class I PI3K
have been extensively studied as one of the critical transducer signals of
receptor tyrosine kinases (RTKs).11–14 Abnormal activation of PI3K/Akt
pathway is identified in most of the human tumors via regulation of cell
cycle, proliferation, and apoptosis.11–12 Mutations in members of the
PI3K/Akt pathway commonly occurred in the PIK3CA, which encoded
p110
α
catalytic subunit of PI3K.11–12,14-17. Tumors harboring PIK3CA
* Corresponding author at: State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China.
** Corresponding author at: State Key Laboratory for Functions and Applications of Medicinal Plants, Guizhou Medical University, Guiyang 550014, PR China (D.
Wang).
E-mail addresses: liuyahu145@gzy.edu.cn (Y. Liu), wangdaoping@gzcnp.cn (D. Wang), yhfan@gzcnp.cn (Y. Fan).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2021.116346
Received 20 May 2021; Received in revised form 18 July 2021; Accepted 30 July 2021
Available online 8 August 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
mutations correspondingly remain active upon upstream targets inhib-
ition.14–17 In view of the key role of PI3K in tumorigenesis, development,
and drug resistance of RTK inhibitors, inhibitors targeting PI3K
signaling showed considerable anti-cancer activity and have achieved
great success in cancer therapy in clinical studies.18–20
ones was obtained in this study according to the reported conditions. As
shown in Scheme 2, critical intermediates 6 was prepared via a Suzuki-
Miyaura Cross-Coupling reaction. 6-bromoquinazolin-4(3H)-ones was
treated with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridin-2-
amine to afford intermediates 6, and Pd(dppf)Cl2 was chosen as a
highly efficient catalyst. To our surprise, the cross-coupling reactions
afforded the crucial intermediates 6 in good yields (above 80%). Finally,
treatment of intermediates 6 with various acyl chlorides provided 3,6-
disubstituted quinazolin-4(3H)-ones 7. Various substituents were
introduced at the N3– and C6- positions of the quinazolin-4(3H)-one
scaffold. Various amide derivatives were also synthesized to further
explore the structure–activity relationship. All compounds were shown
in Table 1.
Our research group has long been engaged in the development of
anti-cancer drugs. In our previous study, we designed and synthesized a
serial of quinazolinone derivatives and obtained several lead com-
pounds targeting PI3K and Aurora kinase.21–25 Quinazolinone has been
identified as a promising scaffold for the development of kinase in-
hibitors, especially PI3K inhibitors.8–10,26-28. To discover more potential
anti-cancer quinazolinone derivatives, we performed further structure
optimization based on our previous findings and other known quina-
zolinone PI3K inhibitors via conjugate some of the related pharmaco-
phore (Fig. 1). We also performed anticancer activity evaluation to
identify the most active compound and most sensitive cell lines for the
further investigation of possible mechanisms of action. As Fig. 1
2.2. Biological evaluation
2.2.1. Antiproliferation activity assay
described, PI3K inhibitors with Quinazolinone scaffold including PI3K
α
In order to investigate the anti-proliferation activity of the synthetic
compounds in cancer cell lines, cell viabilities of human non-small cell
lung cancer (NSCLC) HCC827, human red and white cell leukemia cell
HEL, human neuroblastoma SH-SY5Y and hepatocellular carcinoma
LM3 cells treated with the synthetic compounds at various concentra-
tions were measured by cell counting. As shown in Table 1 and Figs. S1-
S2, the 15 evaluated compounds showed diverse anti-proliferation ac-
tivity. Among them, compounds 7m and 7i significantly attenuated the
inhibitor DFX24 (IC50 is 6.5 nM against PI3Kα) and PI3Kδ inhibitor
Acalisib ((IC50 is 14 nM against PI3Kδ),23,24,29 and expected our target
compounds to maintain both PI3Kα and PI3Kδ kinase inhibitory activity.
In addition, the inhibitor of mTOR XL38830 was selected as the lead
compound and attempted to strengthen the suppression of mTOR.
2. Results and discussion
cell growth of all the tested cancer cells with IC50 values of 1.12 μM and
2.1. Chemistry
1.20 μM against HCC827 cells, respectively. We also evaluated the
cytotoxicity of the most potent compound 7i and 7m on human normal
liver HL-7702 cells to investigate their selectivity to tumor cell lines. As
shown in Table 1 and Fig. S1- S2, both compound 7i and 7m showed
lower inhibitory activities against HL-7702 cells (human normal liver
The substituted quinazolin-4(3H)-one intermediates was synthesized
from 2-amino-5-bromobenzoic acid. As shown in Scheme 1, two general
methods A and B were used to synthesize the substituted quinazolin-4
(3H)-one moiety. The alkylation of quinazolin-4(3H)-ones has been re-
ported to afforded N3-substituted products or a mixture of N3– and O4-
substituted products.31 However, only N3-substituted quinazolin-4(3H)-
cells) with an IC50 values of 8.66 μM and 10.89 μM, which was nearly 8-
fold lower than that in HCC827 cells. These results suggested that 7i and
7m had certain selectivity to tumor cells, compared to human normal
Figure 1. Diagram represents compounds design by molecular hybridization.
2

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Scheme 1. (i) formamide, 130 ◦C, 4 h; (ii) alkyl halides, NaH, DMF, ambient temperature, overnight; (iii) triethyl orthoformate, I2, EtOH, reflux, 6 h.
Scheme 2. (i) Pd(dppf)Cl2, K2CO3, Dioxane/H2O, 100 ◦C, 20 h; (ii) Acyl chloride, Et3N, THF, 0 ◦C to ambient temperature, overnight.
cells. Since compound 7i showed the best inhibitory activity against all
the tested cancer cells, hence it was selected for the further mechanism
study.
2.2.3. Molecular docking
To explore the potential binging mode of 7i and the catalytic subunit
of PI3K including PI3Kα (PDB entry code: 4ZOP) binding with its com-
plex ligand (PDB ID: 4Q2), PI3Kβ (PDB entry code: 4BFR) binding with
its complex ligand (PDB ID: J82), PI3Kγ (PDB entry code: 4ANW)
binding with its complex ligand (PDB ID: O92) and PI3Kδ (PDB entry
code: 4GB9) binding with its complex ligand (PDB ID: 0WR), we per-
formed molecular modeling studies. As depicted in Fig. 3 and Table S1,
compound 7i formed three hydrogen bonds with the critical residue
Lys802 in catalytic sites, Gln859 in the side chain and Arg770 residues in
2.2.2. Effects of 7i on PI3K/Akt pathway
Hyperactivation or mutation of the PI3K/Akt pathway has been
demonstrated as a characteristic feature of various cancers including
human non-small cell lung cancer (NSCLC),17 human neuroblastoma
and hepatocellular carcinoma, which have led to the clinical develop-
ment of PI3K kinase inhibitors for cancer therapy.32,33 In view of the
lead compounds with quinazolinone scaffold of 7i such as DFX24 and
Acalisib showed PI3K inhibitory activity,25–28,31 we analyzed the protein
alterations of the critical components of PI3K/Akt pathway including p-
Akt, p-GSK3β and p-mTOR using western blot to explore the effects of 7i
at various dose on PI3K/Akt pathway. As shown in Fig. 2, phosphory-
lated levels of downstream targets including Akt, GSK3β and mTOR
were completely suppressed, indicating the inhibition of PI3K/Akt
pathway by compound 7i.
the affinity pocket of PI3K
α
, consistent with other PI3K
α
inhibitors.34–36
Also, 7i formed hydrophobic interactions with residues in side chains of
PI3K
α
. All these hydrogen bonds and hydrophobic interactions would
stabilize the conformations and interactions between 7i and PI3K
α. In
addition, the potential binging mode of 7i and PI3Kβ was also predicted
by molecular docking study (Fig. 4). It was reported that the hydrogen
bonds with the nitrogen of Val848 in the hinge region and the hydro-
phobic interactions with residues Ile797 and Ile930 in the hydrophobic
region of ATP binding site was critical for the kinase inhibitory activity
of PI3Kβ inhibitors.37,38 Importantly, the previous mutagenesis study
showed that Try772 in the P-loop has played a pivotal role in selectivity
for PI3Kβ. As shown in Fig. 4 and Table S1, 7i formed hydrogen bonds or
hydrophobic interactions with residues mentioned above and other
different residues including Arg729, Lys771, Trp781 and Tyr833.
Although, there are various interactions were formed between 7i and
PI3Kβ, they bind in a very different manner to what has been reported
To confirm whether the effect of 7i on PI3K/Akt pathway was related
to the PI3K kinase activity, PI3K kinase assay was performed PI3K
phosphorylation evaluation in HCC827 cells using a cell PI3K assay kit.
As shown in Table 2, 7i markedly inhibited the total PI3K kinase ac-
tivities with an IC50 values of 0.072 μM. All these results showed 7i
might be a PI3K pathway inhibitor, at least in part due to its PI3K kinase
inhibitory activity.
3

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Table 1
a
Cytotoxic activity (IC50 , μM) against cancer cell lines.
Comp.
Structure
HCC827
LM3
SH-SY5Y
HEL
HL-7702
NT
7a
>40
>40
>40
>40
7b
7c
7d
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
NT
NT
NT
7e
7f
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
NT
NT
NT
7 g
7 h
7i
>40
>40
>40
>40
NT
1.12 ± 0.11
2.65 ± 0.23
2.13 ± 0.19
2.01 ± 0.19
8.66 ± 0.61
7j
32.17 ± 0.15
>40
>40
>40
35.38 ± 0.22
>40
18.84 ± 0.11
>40
NT
NT
7k
7l
29.23 ± 0.13
1.20 ± 0.17
>40
>40
>40
NT
7m
2.49 ± 0.19
3.72 ± 0.21
2.83 ± 0.36
10.89 ± 0.57
7n
7o
>40
>40
>40
>40
NT
NT
NT
28.96 ± 1.91
0.53 ± 0.11
>40
>40
>40
BEZ235
1.31 ± 0.25
0.79 ± 0.17
0.29 ± 0.09
a
Data are expressed as mean ± SD from three different experiments. NT: Not tested.
for PI3Kβ specific inhibitors.37,38 Given the molecular docking score of
7i (Scores = -7.94) is very close to that of its complex ligand J82
(Table S1), we guessed that the PI3Kβ kinase inhibitory activity of 7i
also contributed to its PI3K kinase inhibition. We further performed
capacity of 7i and PI3Kδ. As shown in Fig. 6, the nondifferentiated
binding mode of compound 7i and OWR suggesting their similar PI3Kδ
kinase inhibitory activity. In general, the data of molecular modeling
study implied compound 7i might showed PI3K kinase inhibitory ac-
molecular modeling study to investigate the potential binding capability
of 7i and PI3Kγ. Unfortunately, docking of 7i into the PI3Kγ binding
pocket PI3Kγ showed that the pocket does not cover the structure of 7i
and showed much lower docking score with -6.04 compared to that of its
complex ligand (Scores = -7.91), suggesting the less activity of 7i against
PI3Kγ (Fig. 5 and Table S1). Finally, we investigated the combining
tivity mainly due to the inhibition of PI3K
α, PI3Kβ and PI3Kδ.
2.2.4. Effects of 7i on cell cycle distribution
Mutation or abnormal levels of cell cycle related proteins and genes
usually led to uncontrolled tumor cell cycle and sustained proliferation
of tumor cells. There are also growing evidences that the PI3K pathway
4

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Figure 2. Compound 7i inhibited PI3K signaling pathway. (A) 7i decreased the phosphorylation levels of Akt at Ser473. (B) 7i decreased the phosphorylation levels
of GSK3β at Ser9. (C) 7i decreased the phosphorylation levels of mTOR at Ser2448. HCC827 cells were treated with different concentrations of 7i for 24 h to evaluate
the alteration of representative protein expression of PI3K pathway including p-Akt, p-GSKβ and p-mTOR. Data are expressed as mean ± SD from three parallel
experiments (*p < 0.05, **p < 0.01 vs DMSO control).
determined by western blot analysis. Reduced expressions levels of
Table 2
CHK1, p-CHK1, c-Myc and Cyclin B1 were observed after 7i treatment
Cell PI3 Kinase activity of compound 7i (IC50, μM).
(Fig. 7B-7D).
Name
PI3K inhibitory activity
2.2.5. Effects of 7i on cell apoptosis
7i
0.072 ± 0.012
0.018 ± 0.006
Wortmannin
We next assessed the efficacy of 7i on cell apoptosis in HCC827 cells.
The presence of 7i resulted in increased induction of cell apoptosis
compared to that achieved by control group or lower dose treatments
(Fig. 8A-B). To further clarify the molecular mechanisms of 7i involved
apoptosis, the effect of 7i on the expression levels of apoptosis-
associated proteins was examined by western blot analysis. In compar-
ison to the vehicle-treated control group, the pro-apoptotic proteins
cleaved-PRAP (Fig. 8C), cleaved-caspase 9 (Fig. 8D) and Bax (Fig. 8E)
were upregulated by 7i treatment in a concentration-dependent manner,
but the anti-apoptotic protein Bcl-2 was downregulated (Fig. 8F). Taken
together, these data suggested that 7i induced apoptotic cell death via
the mitochondrial (intrinsic) pathway in HCC827 cells.
IC50 values are the mean ± SD of three separate experiments.
Wortmannin is a known pan PI3K inhibitor.
plays a critical role in the regulation of the cell cycle. PI3Kα inhibitor HS-
173 has been found to induced G2 phase arrest. Therefore, we examined
the effects of 7i on cell cycle distribution. Consistent with our prior
studies, 7i also induced G2 cell cycle arrest in a concentration-
dependent manner, and G1 phase was relatively decreased compared
to that of the control groups (Fig. 7A-B). To further elucidate whether
cell cycle arrest was related to the regulation of cell cycle checkpoint
proteins, the expression of G2 cell cycle regulatory proteins was
Figure 3. Docking model of compound 7i in the PI3K
PI3K in the active site. (B) Compound 7i and 4Q2 docked into the active pocket. (C) 2D binding model of 4Q2 and PI3K
figure was generated using Accelrys Discovery Studio Visualizer 4.5.
α
(PDB entry code: 4ZOP, the complex ligand 4Q2). (A) The bound conformations of compound 7i and 4Q2 of
α
α
. (D) 2D binding model of 7i and PI3K .The
α
5

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Figure 4. Docking model of compound 7i in the PI3Kβ (PDB entry code: 4BFR, the complex ligand J82). (A) The bound conformations of compound 7i and J82 of
PI3Kβ in the active site. (B) Compound 7i and J82 docked into the active pocket. (C) 2D binding model of 7i and PI3Kβ. (D) 2D binding model of J82 and PI3Kβ. The
figure was generated using Accelrys Discovery Studio Visualizer 4.5.
Figure 5. Docking model of compound 7i in the PI3Kγ (PDB entry code: 4ANW, the complex ligand O92). (A) The bound conformations of compound 7i and O92 of
PI3Kγ in the active site. (B) Compound 7i and O92 docked into the active pocket. (C) 2D binding model of 7i and PI3Kγ. (D) 2D binding model of O92 and PI3Kγ. The
figure was generated using Accelrys Discovery Studio Visualizer 4.5.
3. Materials and methods
on a Beijing micro meltingpoint apparatus, and the values are uncor-
rected. High-resolution exact mass measurements were performed using
electrospray ionization (positive mode) on a quadrupole time-of-flight
(QTOF) mass spectrometer (micro TOF-Q, Bruker Inc.). The structures
of compounds 7a to 7o were confirmed based on 1H and 13C NMR
spectroscopy, HR-MS and element analysis.
3.1. Chemistry
3.1.1. Experimental general information
All reagents and solvents were commercially available and were used
without further purification. 1H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE NEO 600 MHz NMR spectrometer and the
NMR spectra was generated using Mestrenova 12.0 as processing soft-
ware. Spectra were recorded at 25 ◦C using dimethyl sulfoxide-d6 and
chloroform-d as solvents with tetramethylsilane (TMS) as an internal
standard. All chemical shifts are reported in ppm (d), and coupling
constants (J) are in hertz (Hz). All the melting points were determined
3.1.2. General procedure for the preparation of 7a-7o
3.1.2.1. Preparation of 6- bromo-N3-substituted quinazolin-4(3H)-ones,
method A. A mixture of 2-amino-5-bromobenzoic acid (2.16 g, 10
mmol) and formamide (1.80 g, 40 mmol) was heated at 130 ◦C. After the
mixture had been stirred for 4 h, water (30 mL) was added. The reaction
6

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Figure 6. Docking model of compound 7i in the PI3Kδ (PDB entry code: 4 GB9, the complex ligand OWR). (A) The bound conformations of compound 7i and OWR of
PI3Kδ in the active site. (B) Compound 7i and OWR docked into the active pocket. (C) 2D binding model of 7i and PI3Kδ. (D) 2D binding model of OWR and PI3Kδ.
The figure was generated using Accelrys Discovery Studio Visualizer 4.5.
Figure 7. The effects of compound 7i on cell cycle. (A) The effects of compound 7i on cell cycle distribution. Cells were treated with compound 7i for 24 h. The
treated cells were stained with propidium iodide mixed with RNase and incubated under dark conditions at room temperature for 30 min before being subjected to
flow cytometry analysis. (B-D) Cells were treated with the indicated concentrations of 7i for 24 h and then the protein expressions associated with G2/M phase were
analyzed by western blot. GAPDH was employed as the loading control. The changes of corresponding proteins were quantified using Image J. Each bar represents
mean ± SD (n = 3), *P < 0.05 or **P < 0.01 was considered statistically significant compared with corresponding control values.
mixture was cooled to 60 ◦C, and additional water (20 mL) was added to
the mixture. After stirring the mixture for another 30 min, the precipi-
tated product was separated by vacuum filtration. The crude products
were recrystallized from ethanol to give 6-bromoquinazolin-4(3H)-one 2
as a white solid (1.97 g, 8.76 mmol, 87.6% yield). ESI-MS: m/z 224.3,
226.3 [M + H]+. To a solution of compound 2 (1.13 g, 5 mmol) in
anhydrous DMF 15 mL under argon was added NaH (60% dispersion in
mineral oil, 0.24 g, 6 mmol) in one portion. The reaction mixture was
stirred at room temperature for 3 min, and then 2-chloro-1-morpholi-
noethan-1-one (1.15 g, 7 mmol) was added dropwise. The reaction
mixture was stirred at room temperature overnight and then partitioned
between EtOAc (100 mL) and brine (100 mL). The organic layer was
washed with more brine (100 mL). The combined aqueous washings
were extracted with EtOAc (3 × 75 mL). The combined AcOEt extracts
7

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
Figure 8. The effects of compound 7i on cell apoptosis. (A-B) Compound 7i induced apoptosis in HCC827 cells. Cells were treated with various concentrations of 7i
for 48 h and then analyzed the Annexin V-FITC/PI staining test by flow cytometry analysis. The number in the right quads of each panel means the percentage of
Annexin V positive cells. (C-F) Representative protein expression in compound 7i treated cells to investigated the effects of 7i on the alteration of mitochondrial
apoptosis. GAPDH was employed as the loading control. The changes of corresponding proteins were quantified using Image J. Each bar represents mean ± SD (n =
3), *P < 0.05 or **P < 0.01 was considered statistically significant compared with corresponding control values.
were washed with brine (2 × 100 mL), dried (MgSO4), and concentrated
in vacuo. Purification by column chromatography eluting with EtOAc/
PE, afforded 6-bromo-3-(2-morpholino-2-oxoethyl) quinazolin-4(3H)-one
4a as a white solid (1.21 g, 3.44 mmol, 68.8% yield). ESI-MS: m/z 352.0,
354.0 [M + H]+.
3.1.2.3. 6-(6-aminopyridin-3-yl)-3-(2-morpholino-2-oxoethyl) quinazolin-
4(3H)-one (6a). A solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl) pyridin-2-amine 5 (1.10 g, 5 mmol), 6-bromoquinazolin-4
(3H)-ones 4a (1.76 g, 5 mmol), and K2CO3 (2.07 g, 15 mmol), 1,4-
dioxane/water 25 mL [V(dioxane) : V(water) = 4 : 1] mixture as sol-
vent was heated to 100 ◦C and stirred for 15 min. Pd(dppf)Cl2 (0.18 g,
0.25 mmol) was added, and the mixture was continued stirred at this
temperature under argon for another 4–6 h. 1,4-dioxane and water was
removed under reduced pressure and the residue was purified through a
column chromatography on silica with dichloromethane/methanol to
give 6-(6-aminopyridin-3-yl)-3-(2-morpholino-2-oxoethyl) quinazolin-4
(3H)-one 6a as gray solid (1.611 g, 4.41 mmol, 88.1% yield). ESI-MS: m/
z 366.0 [M + H]+.
3.1.2.2. Preparation of 6-bromo-N3-substituted quinazolin-4(3H)-ones,
method B. The mixture of 2-amino-5-bromobenzoic acid (2.16 g, 10
mmol), triethyl orthoformate (1.93 g, 13 mmol), benzo[d] [1,3] dioxol-
5-ylmethanamine (1.97 g, 13 mmol), iodine (0.025 g, 0.1 mmol) and
anhydrous ethanol (30 mL) was refluxed under argon atmosphere for
4–6 h, then concentrated under vacuum to give a residue which was
dissolved in ethyl acetate (100 mL). The ethyl acetate solution was
washed with 1 N aqueous sodium hydroxide (50 mL × 3) and brine (50
mL × 3), dried over anhydrous sodium sulfate and concentrated to give
3-(benzo[d] [1,3] dioxol-5-ylmethyl)-6-bromoquinazolin-4(3H)-one 4b as
white solid (2.809 g, 7.82 mmol, 78.2% yield). ESI-MS: m/z 382.0, 384.0
[M + Na]+.
Compounds 6b-6f were synthesized according to the procedure
described in 6a. The ESI-MS information of compounds 6b-6f was listed
as below:
6-(6-aminopyridin-3-yl)-3-(benzo[d] [1,3] dioxol-5-ylmethyl) quina-
zolin-4(3H)-one (6b)
Compounds 4c-4f were synthesized according to the procedure
described in 4b. The ESI-MS information of compounds 4c-4f was listed
as below:
White solid, 83.2% yield. ESI-MS: m/z 395.1 [M + Na]+.
6-(6-aminopyridin-3-yl)-3-benzylquinazolin-4(3H)-one (6c)
White solid, 86.0% yield. ESI-MS: m/z 329.0 [M + H]+.
6-(6-aminopyridin-3-yl)-3-(4-methylbenzyl) quinazolin-4(3H)-one (6d)
White solid, 88.7% yield. ESI-MS: m/z 365.1 [M + Na]+.
6-(6-aminopyridin-3-yl)-3-butylquinazolin-4(3H)-one (6e)
White solid, 88.0% yield. ESI-MS: m/z 295.1 [M + H]+.
6-(6-aminopyridin-3-yl)-3-(2-morpholinoethyl) quinazolin-4(3H)-one
(6f)
3-benzyl-6-bromoquinazolin-4(3H)-one (4c)
White solid, 86.0% yield. ESI-MS m/z: 338.0, 340.0 [M + Na]+.
6-bromo-3-(4-methylbenzyl) quinazolin-4(3H)-one (4d)
White solid, 88.0% yield. ESI-MS: m/z 352.0, 354.0 [M + H]+.
6-bromo-3-butylquinazolin-4(3H)-one (4e)
White solid, 81.1% yield. ESI-MS: m/z 281.0, 283.0 [M + H]+.
6-bromo-3-(2-morpholinoethyl) quinazolin-4(3H)-one (4f)
White solid, 72.1% yield. ESI-MS: m/z 338.0, 340.0 [M + H]+.
White solid, 88.4% yield. ESI-MS: m/z 352.1 [M + H]+.
8

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
3.1.2.4. N-(5-(3-(2-morpholino-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-
6-yl) pyridin-2-yl) pro-pionamide (7a). A solution of 6a (0.365 g, 1
mmol) and triethylamine (0.202 g, 2 mmol) in anhydrous tetrahydro-
furan (THF, 20 mL) was stirred for 45 min. Subsequently, a solution of
propionyl chloride (0.139 g, 1.5 mmol) in THF (5 mL) was added drop
wisely to the reaction mixture under ice cold conditions using a drop-
ping funnel and the contents were stirred further for 2 h at ambient
temperature. Completion of the reaction was monitored by TLC. The
slurry was pulled into saturated salt water 50 mL and extracted with
dichloromethane (50 mL × 3), the combined organic fractions were
washed with saturated NaHCO3 aqueous solution three times, dried over
MgSO4 and the solvent was removed under reduced pressure to obtain a
solid residue which was purified through a column chromatography on
silica with dichloromethane/methanol to afford 7a, as a white solid
(0.229 g, 0.543 mmol, 54.3% yield), mp 189–191 ◦C, 1H NMR (600
MHz, DMSO‑d6) δ 10.60 (s, 1H), 8.74 (s, 1H), 8.37 (d, J = 6.6 Hz, 1H),
8.27 (s, 1H), 8.21 (d, J = 3.9 Hz, 2H), 8.20 (s, 1H), 7.79 (d, J = 8.4 Hz,
1H), 4.98 (s, 2H), 3.69 (s, 2H), 3.60 (s, 4H), 3.47 (s, 2H), 2.43 (q, J = 7.5
Hz, 2H), 1.09 (t, J = 7.5 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 173.1,
165.2, 160.1, 151.9, 148.7, 147.4, 146.0, 136.4, 132.6, 129.6, 128.1,
123.0, 121.9, 113.3, 66.0, 46.6, 45.6, 44.8, 42.1, 40.1, 29.4, 9.4. HRMS
(ESI): C22H24O4N5 [M + H]+ m/z: calcd. for 422.1823, found 422.1813.
Compounds 7bꢀ 7o were synthesized according to the procedure
described in 7a.
J = 2.1 Hz, 1H), 8.21–8.19 (m, 2H), 8.17 (d, J = 2.1 Hz, 1H), 7.78 (d, J =
8.5 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.28 (t, J
= 7.2 Hz, 1H), 5.23 (s, 2H), 2.41 (t, J = 7.4 Hz, 2H), 1.59–1.55 (m, 2H),
1.32 (d, J = 7.5 Hz, 1H), 1.30 (d, J = 7.4 Hz, 1H), 0.89 (t, J = 7.4 Hz,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 172.4, 160.1, 151.8, 148.2, 147.3,
146.0, 136.8, 136.4, 135.7, 132.5, 129.6, 128.7, 128.2, 127.7, 123.1,
122.1, 113.3, 49.0, 35.8, 27.1, 25.1, 21.8, 13.8. HRMS(ESI):
C
25H25O2N4 [M + H]+ m/z: calcd. for 413.1972, found 413.1960.
3.1.2.9. N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl) pyridin-2-yl)
pivalamide (7f). White solid, 55.8% yield, mp 144–146 ◦C, 1H NMR
(600 MHz, DMSO‑d6) δ 9.97 (s, 1H), 8.75 (d, J = 2.0 Hz, 1H), 8.61 (s,
1H), 8.40 (d, J = 1.7 Hz, 1H), 8.19 (d, J = 5.3 Hz, 2H), 8.17 (s, 1H), 7.79
(d, J = 8.5 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H),
7.29 (t, J = 7.1 Hz, 1H), 5.23 (s, 2H), 1.26 (s, 9H, 3 × CH3). 13C NMR
(151 MHz, DMSO‑d6) δ 177.3, 160.1, 152.0, 148.2, 147.3, 145.7, 136.8,
136.3, 135.6, 132.6, 129.7, 128.7, 128.2, 127.7, 123.1, 122.1, 121.7,
114.0, 108.7, 108.3, 101.1, 49.0, 40.1, 26.9, 25.0. HRMS(ESI):
C
25H25O2N4 [M + H]+ m/z: calcd. for 413.1972, found 413.1960.
3.1.2.10. N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl) pyridin-2-yl)
propionamide (7g). White solid, 54.7% yield, mp 156–158 ◦C, 1H NMR
(600 MHz, DMSO‑d6) δ 10.60 (s, 1H), 8.74 (t, J = 1.6 Hz, 1H), 8.61 (s,
1H), 8.40 (d, J = 2.2 Hz, 1H), 8.21 (t, J = 2.0 Hz, 3H), 7.79 (d, J = 8.5
Hz, 1H), 7.37 (dt, J = 15.1, 7.4 Hz, 6H), 5.23 (s, 2H), 1.08 (t, J = 7.5 Hz,
4H). 13C NMR (151 MHz, DMSO‑d6) δ 173.1, 160.1, 151.9, 148.1, 147.4,
147.3, 146.0, 136.9, 136.8, 136.4, 135.7, 132.6, 129.5, 128.7, 128.2,
127.7, 123.1, 122.1, 113.2, 49.0, 40.1, 29.4, 9.4. HRMS (ESI):
3.1.2.5. N-(5-(3-(benzo[d]
[1,3]
dioxol-5-ylmethyl)-4-oxo-3,4-
dihydroquinazolin-6-yl) pyridin-2-yl) propionamide (7b). White solid,
53.2% yield, mp 172–174 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 10.60 (s,
1H), 8.74 (s, 1H), 8.59 (s, 1H), 8.39 (s, 1H), 8.21 (s, 2H), 8.18 (d, J = 8.5
Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.04 (s, 1H), 6.92 (d, J = 8.1 Hz, 1H),
6.88 (d, J = 8.0 Hz, 1H), 5.99 (s, 2H), 5.12 (s, 2H), 2.43 (q, J = 7.6 Hz,
2H), 1.08 (t, J = 7.6 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 173.1,
160.1, 151.9, 148.0, 147.4, 147.2, 146.9, 146.0, 136.4, 135.6, 132.5,
130.5, 129.5, 128.1, 123.1, 122.1, 121.7, 113.2, 108.7, 108.3, 101.1,
48.8, 29.4, 9.4. HRMS(ESI): C24H21O4N4 [M + H]+ m/z: calcd. for
429.1557, found 429.1549.
C
23H21O2N4 [M + H]+ m/z: calcd. for 385.1659, found 385.1649.
3.1.2.11. N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl) pyridin-2-yl)-
2-methoxy-acetamide (7h). White solid, 53.8% yield, mp 147–149 ◦C, 1H
NMR (600 MHz, CDCl3) δ 10.18 (s, 1H), 8.75 (d, J = 2.3 Hz, 1H), 8.61 (s,
1H), 8.39 (d, J = 2.1 Hz, 1H), 8.24 (dd, J = 8.7, 2.4 Hz, 1H), 8.21–8.17
(m, 2H), 7.79 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.35 (t, J =
7.6 Hz, 2H), 7.29 (t, J = 7.1 Hz, 1H), 5.23 (s, 2H), 4.10 (s, 2H), 3.38 (s,
3H). 13C NMR (151 MHz, DMSO‑d6) δ 168.8, 160.1, 150.9, 148.2, 147.3,
146.1, 136.8, 136.6, 135.5, 132.6, 130.1, 128.7, 128.2, 127.7, 123.3,
122.1, 113.4, 71.2, 58.7, 49.0. HRMS(ESI): C23H21O3N4 [M + H]+ m/z:
calcd. for 401.1608, found 401.1600.
3.1.2.6. N-(5-(3-(benzo[d]
[1,3]
dioxol-5-ylmethyl)-4-oxo-3,4-
dihydroquinazolin-6-yl) pyridin-2-yl) pentanamide (7c). White solid,
54.1% yield, mp 191–193 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 10.61 (s,
1H), 8.73 (s, 1H), 8.59 (s, 1H), 8.36 (d, J = 8.7 Hz, 1H), 8.21 (d, J = 9.1
Hz, 1H), 8.20–8.19 (m, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.5 Hz,
1H), 7.04 (s, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H), 5.98
(s, 2H), 5.12 (s, 2H), 2.42 (t, J = 7.4 Hz, 2H), 1.61–1.54 (m, 2H), 1.32
(dq, J = 14.6, 7.3 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz,
DMSO‑d6) δ 172.4, 160.1, 151.8, 148.0, 147.4, 146.9, 146.0, 136.4,
135.6, 132.5, 130.5, 128.1, 123.1, 122.1, 121.7, 113.3, 108.7, 108.3,
101.1, 48.7, 45.4, 35.8, 27.1, 21.8, 13.7, 8.5. HRMS(ESI): C26H25O4N4
[M + H]+ m/z: calcd. for 457.1870, found 457.1854.
3.1.2.12. N-benzoyl-N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl)
pyridin-2-yl) benzamide (7i). White solid, 56.3% yield, mp 166–168 ◦C,
1H NMR (600 MHz, CDCl3) δ 8.74 (d, J = 2.3 Hz, 1H), 8.63 (s, 1H), 8.39
(d, J = 2.1 Hz, 1H), 8.36 (dd, J = 8.4, 2.5 Hz, 1H), 8.19 (dd, J = 8.5, 2.2
Hz, 1H), 7.81 (s, 2H), 7.80 (d, J = 1.2 Hz, 2H), 7.78 (d, J = 8.5 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.5 Hz, 2H), 7.47 (s, 1H), 7.46 (s,
2H), 7.44 (s, 1H), 7.38 (d, J = 7.2 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.29
(t, J = 7.1 Hz, 1H), 5.22 (s, 2H). 13C NMR (151 MHz, DMSO‑d6) δ 172.8,
160.0, 152.9, 148.5, 147.8, 146.7, 137.0, 136.7, 134.6, 134.3, 132.9,
132.8, 129.1, 128.8, 128.7, 128.2, 127.7, 124.1, 122.4, 122.1, 49.0.
HRMS(ESI): C34H25O3N4 [M + H]+ m/z: calcd. for 537.1921, found
537.1909.
3.1.2.7. N-(5-(3-(benzo[d]
[1,3]
dioxol-5-ylmethyl)-4-oxo-3,4-
dihydroquinazolin-6-yl) pyridin-2-yl) pivalamide (7d). White solid,
54.5% yield, mp 193–195 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 9.95 (s,
1H), 8.74 (s, 1H), 8.58 (s, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.18 (s, 2H),
8.16 (dd, J = 8.5, 2.0 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.03 (d, J = 1.1
Hz, 1H), 6.92 (dd, J = 8.0, 1.1 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 5.98 (s,
2H), 5.11 (s, 2H), 1.25 (s, 9H, 3 × CH3). 13C NMR (151 MHz, DMSO‑d6) δ
179.4, 177.3, 160.1, 152.0, 148.1, 147.3, 145.7, 136.8, 136.2, 135.6,
132.5, 129.7, 128.6, 128.1, 127.7, 123.1, 122.1, 121.7, 114.0, 108.7,
108.3, 101.1, 49.0, 37.7, 22.1, 13.9. HRMS(ESI): C26H25O4N4 [M + H]+
m/z: calcd. for 457.1870, found 457.1863.
3.1.2.13. N-(5-(3-(4-methylbenzyl)-4-oxo-3,4-dihydroquinazolin-6-yl)
pyridin-2-yl) pentanamide (7j). White solid, 56.6% yield, mp
213–215 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 10.68 (s, 1H), 8.81 (s, 1H),
8.66 (s, 1H), 8.47 (s, 1H), 8.30 (d, J = 8.5 Hz, 1H), 8.28 (s, 1H), 8.26 (s,
1H), 7.86 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 7.6 Hz, 2H), 7.24 (d, J = 7.5
Hz, 2H), 5.26 (s, 2H), 2.59 (s, 2H), 2.34 (s, 3H), 1.71–1.62 (m, 2H), 1.42
(d, J = 7.3 Hz, 1H), 1.40–1.36 (m, 1H), 0.98 (t, J = 7.2 Hz, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 172.5, 160.1, 151.9, 148.1, 147.3, 146.0, 137.1,
136.4, 135.7, 133.9, 132.6, 129.6, 129.3, 128.2, 127.8, 123.2, 122.2,
113.4, 48.8, 35.9, 27.2, 21.8, 20.7, 13.8. HRMS(ESI): C26H27O2N4 [M +
H]+ m/z: calcd. for 427.2129, found 427.2123.
3.1.2.8. N-(5-(3-benzyl-4-oxo-3,4-dihydroquinazolin-6-yl) pyridin-2-yl)
pentanamide (7e). White solid, 55.2% yield, mp 197–199 ◦C, 1H NMR
(600 MHz, DMSO‑d6) δ 10.61 (s, 1H), 8.73 (s, 1H), 8.62 (s, 1H), 8.38 (d,
9

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
3.1.2.14. N-(5-(3-(4-methylbenzyl)-4-oxo-3,4-dihydroquinazolin-6-yl)
pyridin-2-yl) propionamide (7k). White solid, 55.4% yield, mp
187–189 ◦C, 1H NMR (600 MHz, CDCl3) δ 10.59 (s, 1H), 8.72 (s, 1H),
8.57 (s, 1H), 8.37 (d, J = 1.7 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.19–8.14
(m, 2H), 7.76 (d, J = 8.5 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.15 (d, J =
7.8 Hz, 2H), 5.17 (s, 2H), 2.43 (q, J = 7.5 Hz, 2H), 2.25 (s, 3H), 1.08 (t, J
= 7.5 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 173.0, 160.0, 151.8,
148.0, 147.2, 145.9, 137.0, 136.3, 135.6, 133.8, 132.5, 129.5, 129.2,
128.1, 127.8, 123.1, 122.1, 113.2, 48.7, 29.4, 20.7, 9.4. HRMS(ESI):
entry code: 4ANW) and PI3Kδ (PDB entry code: 4GB9) in complex with
their ligands were used for molecular modeling. The protein optimiza-
tions were carried out using quickprep of MOE and the optimizations of
the geometry of the ligands and compound 7i were performed using the
field strengths in the MMFF94x implanted software MOE. The active site
was generated by using the site finder option of MOE (Molecular
Operating Environment) software based on the co-crystalline ligands.
Accelrys Discovery Studio Visualizer 4.5 was used for graphic display.
C
24H23O2N4 [M + H]+ m/z: calcd. for 399.1816, found 399.1807.
3.3. Anticancer assay
3.1.2.15. N-benzoyl-N-(5-(3-(4-methylbenzyl)-4-oxo-3,4-dihy-
3.3.1. Cell lines and culture medium
droquinazolin-6-yl) pyridin-2-yl) ben-zamide (7l). White solid, 56.3%
yield, mp 166–168 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 8.73 (s, 1H), 8.59
(s, 1H), 8.38 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H),
7.80 (d, J = 7.2 Hz, 4H), 7.77 (d, J = 8.7 Hz, 1H), 7.73 (d, J = 8.2 Hz,
1H), 7.55 (d, J = 6.9 Hz, 2H), 7.45 (t, J = 7.0 Hz, 4H), 7.27 (d, J = 7.3
Hz, 2H), 7.15 (d, J = 7.1 Hz, 2H), 5.17 (s, 2H), 2.25 (s, 3H). 13C NMR
(151 MHz, DMSO‑d6) δ 172.8, 160.0, 152.9, 148.5, 147.8, 146.7, 137.1,
137.1, 134.7, 134.4, 133.8, 133.0, 132.9, 132.8, 129.2, 129.1, 128.9,
128.3, 127.8, 124.1, 122.5, 122.2, 48.8, 20.7. HRMS(ESI): C35H27O3N4
[M + H]+ m/z: calcd. for 551.2078, found 551.2065.
The human non-small cell lung cancer (NSCLC) HCC827, human red
and white cell leukemia cell HEL, human neuroblastoma SH-SY5Y and
human normal liver HL-7702 cells were obtained from Cell Bank of the
Chinese Academy of Sciences (Shanghai, China). The hepatocellular
carcinoma LM3 cells were a kind gift of professor Qingchun Zhao.
HCC827, HEL, SH-SY5Y and HL-7702 cells were cultured in Roswell
Park Memorial Institute (RPMI, Gibco) 1640 medium with 10% fetal
bovine serum (FBS, BI) and antibiotics-antimycotics (100 units/mL
penicillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL ampho-
tericin B). LM3 cells were cultured in dulbecco’s modified eagle medium
(DMEM, Gibco) containing 10% FBS and antibiotics-antimycotics. Cells
were incubated with 5% CO2 in a humidified atmosphere at 37 ◦C.
3.1.2.16. N-(5-(3-butyl-4-oxo-3,4-dihydroquinazolin-6-yl) pyridin-2-yl)
propionamide (7m). White solid, 55.7% yield, mp 123–125 ◦C, 1H
NMR (600 MHz, DMSO‑d6) δ 10.59 (s, 1H), 8.73 (s, 1H), 8.41 (s, 1H),
8.38 (d, J = 1.9 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H), 8.19 (dd, J = 8.8, 2.2
Hz, 1H), 8.17 (dd, J = 8.5, 2.0 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 4.00 (t,
J = 7.2 Hz, 2H), 2.43 (q, J = 7.5 Hz, 2H), 1.71–1.67 (m, 2H), 1.33 (d, J =
7.5 Hz, 1H), 1.31 (d, J = 7.4 Hz, 1H), 1.09 (t, J = 7.5 Hz, 3H), 0.91 (t, J
= 7.4 Hz, 3H). 13C NMR (151 MHz, CDCl3) δ 172.6, 161.2, 151.2, 147.7,
146.9, 146.2, 137.1, 136.6, 132.6, 131.4, 128.5, 124.4, 122.8, 114.0,
47.1, 31.6, 31.0, 20.0, 13.8, 9.5. HRMS(ESI): C20H23O2N4 [M + H]+ m/
z: calcd. for 351.1816, found 351.1810.
3.3.2. Cell viability assay
Cell viability assay was performed using cell counting by flow
cytometry. Succinctly put, cells of 2.5–3 × 104/well cells were cultured
adherently with fresh medium in 12-well plates for 24 h before exposure
to various concentrations of compound 7a to 7o for 72 h. Then, the cell
numbers of living cells were measured by flow cytometry. Data were
analyzed using the SPSS, which calculated the half maximal inhibitory
concentration (IC50) for each drug.
3.4. Flow cytometry analysis
3.1.2.17. N-(5-(3-(2-morpholinoethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)
pyridin-2-yl) propionamide (7n). White solid, 56.3% yield, mp
110–112 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 10.58 (s, 1H), 8.73 (d, J =
2.1 Hz, 1H), 8.38 (d, J = 2.2 Hz, 1H), 8.34 (s, 1H), 8.21 (s, 1H), 8.19 (dd,
J = 8.8, 2.4 Hz, 1H), 8.17 (dd, J = 8.5, 2.2 Hz, 1H), 7.76 (d, J = 8.5 Hz,
1H), 4.11 (t, J = 6.0 Hz, 2H), 3.54–3.51 (m, 4H), 2.62 (t, J = 6.0 Hz, 2H),
2.44 (d, J = 3.3 Hz, 2H), 2.43 (s, 2H), 2.41 (d, J = 7.6 Hz, 2H), 1.09 (t, J
= 7.5 Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 173.1, 151.8, 147.3,
145.9, 136.4, 132.4, 129.6, 128.1, 123.0, 122.0, 113.3, 53.2, 45.3, 29.4,
9.4, 8.4. HRMS(ESI): C22H26O3N5 [M + H]+ m/z:calcd. for 408.2030,
found 408.2025.
Cells cycle analysis and Annexin FITC/PI assay were performed as
described previously.22,23 Briefly, cells were cultured with fresh medium
without FBS for 12 h before treatment with compound 7i for 24 h. The
treated cells were fixed with 70% ethanol overnight before staining with
propidium iodide mixed with RNase. Keep the dying cells under dark
conditions at room temperature for 30 min before being subjected to
flow cytometry analysis. Cell apoptosis was evaluated using Annexin V
FITC/PI apoptosis detection kit (BD Biosciences, CA, USA) according to
the manufacturer’s instruction. The stained cells were incubated for 15
min at room temperature in the dark before analyzing with flow
cytometry.
3.1.2.18. N-(5-(3-(2-morpholinoethyl)-4-oxo-3,4-dihydroquinazolin-6-yl)
pyridin-2-yl) pentanamide (7o). White solid, 56.7% yield, mp
162–164 ◦C, 1H NMR (600 MHz, DMSO‑d6) δ 10.60 (s, 1H), 8.73 (d, J =
1.5 Hz, 1H), 8.38 (d, J = 2.0 Hz, 1H), 8.35 (s, 1H), 8.22 (d, J = 8.7 Hz,
1H), 8.20–8.19 (m, 1H), 8.17 (dd, J = 8.6, 2.2 Hz, 1H), 7.76 (d, J = 8.5
Hz, 1H), 4.11 (t, J = 6.0 Hz, 2H), 3.54–3.51 (m, 4H), 2.62 (t, J = 6.0 Hz,
2H), 2.43 (d, J = 6.6 Hz, 4H), 2.41 (d, J = 7.5 Hz, 2H), 1.60–1.56 (m,
2H), 1.33 (d, J = 7.5 Hz, 1H), 1.31 (d, J = 7.4 Hz, 1H), 0.89 (t, J = 7.4
Hz, 3H). 13C NMR (151 MHz, DMSO‑d6) δ 172.4, 160.1, 151.8, 148.5,
147.3, 146.0, 136.4, 135.4, 132.4, 129.6, 128.0, 123.0, 122.0, 113.3,
66.3, 56.3, 53.2, 42.7, 40.1, 35.8, 27.1, 25.1, 21.8, 13.8. HRMS(ESI):
3.5. PI3 kinase inhibition assays
The PI3 kinase assay was measured by Cell PI3 Kinase Activity
Photometric Assay Kit (Shanghai Yiji Industrial Co., Ltd, China)
following the manufacturer’s protocols. In brief, cells were treated with
various concentration of compound 7i for 24 h. The treated cells were
washed with Reagent A. Regent B was used to lyse the cells and collected
the proteins. Protein concentration was measured by Braford reagent
(Bio-Rad, Hercules, Calif). 100 μg cell lysate protein for each group were
used to analyze the kinase activity.
C
24H30O3N5 [M + H]+ m/z: calcd. for 436.2343, found 436.2334.
3.6. Western blot
3.2. Molecular modeling
Cells were treated with various concentrations of compound 7i.
Western blot analysis was performed as described previously.22–25,39
Blots were imaged by ChemiDoc TM MP Imaging System (Bio-Rad,
USA).
To predict the binding mode of 7i and four subtypes of PI3K, MOE
(Molecular Operating Environment) and crystal structures of PI3K
α
(PDB entry code: 4ZOP), PI3Kβ (PDB entry code: 4BFR), PI3Kγ (PDB
10

H. Yang et al.
Bioorganic & Medicinal Chemistry 46 (2021) 116346
4. Conclusions
References
1
2
3
4
5
6
7
8
9
Mahato A, Shanthi N. Inventi Impact Med Chem. 2011;2011:1–6.
A series of novel 6-(pyridin-3-yl) quinazolin-4(3H)-one derivatives
were designed and synthesized as potential anticancer agents as in-
hibitors of PI3K/Akt pathway. Among them, compound 7i showed the
most antiproliferative activity against all the tested cells and inhibited
Jang CS, Fu FY, Huang KC, Wang CY. Nature. 2012;161:400–401.
˜
Castaner J, Sweetman AJ. Drugs Future. 1982;7, 539–539.
Gelbrich T, Haddow MF, Griesser UJ. Acta Crystallograph. 2011;67, 1343–1343.
Huo S, Yu H, Li C, et al. Int J Clin Exp Pathol. 2015;8:15863–15870.
Pines M. World J Gastroenterol. 2014;20:14778–14786.
the PI3K kinase activity with an IC50 values of 0.072 μM. Furthermore,
Cook JA, Choudhuri R, Degraff W, et al. Cancer Lett. 2010;289:119–126.
Kater AP, Tonino SH, Spiering M, et al. Blood Cancer J. 2018;8:16.
Yue D, Sun X. Cell Death Dis. 2018;9:935.
7i induced cell cycle arrest in G2/M phase and cell apoptosis in HCC827
cells. Collectively, the results suggested that 7i might be a hit compound
as inhibitor of PI3K/Akt pathway.
10 Huen A, Haverkos BM, Zain J, et al. Cancers (Basel). 2020;12:2293.
11 Thorpe LM, Yuzugullu H, Zhao JJ. Nat Rev Cancer. 2015;15:7–24.
12 Yang J, Nie J, Ma X, et al. Mol Cancer. 2019;18:26.
13 Fruman DA, Chiu H, Hopkins BD, et al. Cell. 2017;170:605–635.
14 Chen L, Yang L, Yao L, et al. Nat Commun. 2018;9:1357.
Declaration of Competing Interest
´
15 Martínez-Saez O, Chic N, Pascual T, et al. Breast Cancer Res. 2020;22:45.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
16 Alqahtani A, Ayesh HSK, Halawani H. Cancers (Basel). 2019;12:93.
17 Wang L, Hu H, Pan Y, et al. PLoS ONE. 2014;9:88291.
18 Hanker AB, Kaklamani V, Arteaga CL. Cancer Discov. 2019;9:482–491.
19 Mishra R, Patel H, Alanazi S, et al. Int J Mol Sci. 2021;22:3464.
20 Zhao HF, Wang J, Shao W, et al. Mol Cancer. 2017;16:100.
Acknowledgments
21 Fan Y, Ding H, Kim D, et al. Cancers (Basel). 2019;11:627.
22 Fan YH, Ding HW, Liu DD, et al. Bioorg Med Chem. 2018;26:1675–1685.
23 Fan YH, Li W, Liu DD, et al. Eur J Med Chem. 2017;139:95–106.
24 Fan YH, Ding HW, Kim D, et al. Pharmacol Res. 2020;160, 105147.
25 Fan CC, Zhong T, Yang HR, et al. Eur J Med Chem. 2020;190, 112108.
26 Marzaro G, Castagliuolo I, Schirato G, et al. Eur J Med Chem. 2016;115:416–425.
27 Ma CC, Zhang CM, Tang LQ, Liu ZP. Eur J Med Chem. 2018;151:9–17.
28 Roskoski R. Pharmacol Res. 2021;168, 105579.
Financial supported by the Natural Science Foundation of China,
grant number Grants 21907020, the Natural Science Foundation of
Guizhou Province, grant number QKH [2019]1215, Thousands of
Innovative Talents of Guizhou Province (Grant No. GZQ202006081) and
the project of Service Enterprise Action Plan of Guizhou Province (Grant
No. QKHFQ [2020]4013).
29 Kater AP, Tonino SH, Spiering M, et al. Blood Cancer J. 2018:16.
30 Takeuchi CS, Kim BG, Blazey CM, et al. J Med Chem. 201; 6: 2218-2234.
31 Bavetsias V, Skelton LA, Yafai F, et al. J Med Chem. 2002;45:3692–3702.
32 Chen Y, Tsai HW, Tsai YH, Tseng SH. J Pediatr Surg. 2020.
Appendix A. Supplementary material
33 Zhou Q, Lui VW, Yeo W. Future Oncol. 2011;7:1149–1167.
34 Jin RY, Tang T, Zhou S, et al. Bioorg Chem. 2020;98, 103737.
35 Furet P, Guagnano V, Fairhurst RA, et al. Bioorg Med Chem Lett. 2013;23:3741–3748.
36 Heffron TP, Heald RA, Ndubaku C, et al. J Med Chem. 2016;59:985–1002.
37 Certal V, Carry JC, Halley F, et al. J Med Chem. 2014;57:903–920.
38 Chandrasekhar J, Dick R, Van Veldhuizen J, et al. J Med Chem. 2018;61:6858–6868.
39 Lodola A, Bertolini S, Biagetti M, et al. J Med Chem. 2017;60:4304–4315.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116346.
11