Synthesis, biological evaluation and structure-activity relationship of a novel class of PI3Kα H1047R mutant inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.09.002

Phosphatidylinositol 3-kinase α (PI3Kα) is one of the most attractive therapeutic targets for cancer treatment. As our continuing endeavor to discover isoform and/or mutant selective class of PI3K inhibitors, herein we report the optimization of a structu

Full text of DOI:10.1016/j.ejmech.2018.09.002

Accepted Manuscript
Synthesis, biological evaluation and structure-activity relationship of a novel class of
PI3Kα H1047R mutant inhibitors
Ning Zhang, Zhimei Yu, Xiaohong Yang, Yan Zhou, Jia Wang, Shao-Lin Zhang, Ming-
Wei Wang, Yun He
PII:
S0223-5234(18)30774-8
10.1016/j.ejmech.2018.09.002
EJMECH 10713
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 July 2018
Revised Date: 31 August 2018
Accepted Date: 2 September 2018
Please cite this article as: N. Zhang, Z. Yu, X. Yang, Y. Zhou, J. Wang, S.-L. Zhang, M.-W. Wang, Y.
He, Synthesis, biological evaluation and structure-activity relationship of a novel class of PI3Kα H1047R
mutant inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.002.
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Graphical Abstract
EC50 (µM)
IC50 (µM)
HCT116
Improved potency
(From Hit-02 to 7h)
Comp.
PI3Kα H1047R
PI3Kα WT
mutant
115.3
NT
> 100
10.9
115.3 / 0.55 > 200 (enzymatic level)
100 / 10.9 > 9 (cellular level)
Hit-02
7h
0.55
0.57

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Synthesis, biological evaluation and structure-activity relationship of a novel
class of PI3Kα H1047R mutant inhibitors
Ning Zhang1, #, Zhimei Yu1, #, Xiaohong Yang1, Yan Zhou2, Jia Wang2, Shao-Lin
Zhang1*, Ming-Wei Wang2*, and Yun He1*
1
School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product
Synthesis and Drug Research, Chongqing University, Chongqing, 401331, P. R.
China
2
The National Center for Drug Screening and the CAS Key Laboratory of Receptor
Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS),
189 Guo Shou Jing Road, Shanghai, 201203, China
* Corresponding address:
Tel.: +86-23-65678465; E-mail: zhangsl@cqu.edu.cn; mwwang@simm.ac.cn;
yun.he@cqu.edu.cn
#
:
These authors contributed equally.
Abstract
Phosphatidylinositol 3-kinase α (PI3Kα) is one of the most attractive therapeutic
targets for cancer treatment. As our continuing endeavor to discover isoform and / or
mutant selective class of PI3K inhibitors, herein we report the optimization of a
structurally novel PI3Kα H1047R mutant inhibitor Hit-02 (EC50 = 115.3 µM), which
was identified from a high-throughput screening campaign. Structure-activity
relationship analysis enabled us to discover compound 7h, which strongly inhibited
PI3Kα H1047R mutant with an EC50 value of 0.55 µM, over 200-fold more potent
than Hit-02, while having little effect on other PI3K isoforms. Western blotting assay
suggested that 7h decreased the phosphorylation level of AKT, another proof that 7h
inhibited PI3Kα H1047R mutant function. Cell viability assay revealed that 7h
inhibited HCT-116 cancer cell growth with an IC50 value of 10.9 µM. In addition, 7h
was found to arrest cell cycle at G2 phase but did not show any cell apoptosis effect.
Furthermore, 7h obviously induced cell autophagy, which might contribute to its
anti-proliferation effect in cancer cell. Collectively, all these data demonstrated that
7h could be a promising lead for the development of structurally novel PI3Kα
inhibitor.
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Keywords:
Anticancer drug; Phosphatidylinositol 3-kinase; Structure-activity relationship
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1. Introduction
Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid kinases that play
crucial roles in cell growth and proliferation, differentiation, motility, invasion and
death [1, 2]. The PI3Ks family could be categorized into three main classes (class I, II
and III) and various subgroups according to their structural characteristics and
substrate specificity [2, 3]. Among which, class I PI3Ks (PI3Kα, β, γ) normally exist
as heterodimers and comprise a p110 catalytic subunit and a p85 regulatory subunit,
which can be activated by recruitment to the cell membrane via receptor tyrosine
kinases [4]. The activation of PI3Ks results in the generation of the second messenger,
phosphatidylinositol-3,4,5-trisphosphate
(PIP3)
from
phosphatidylinositol-4,5-bisphosphate (PIP2) [5-7]. PIP3 acts as an anchor for the
AKT serine / threonine kinase (PKB) and phosphoinositide dependent protein
kinase-1 (PDK1), both of which bind to PIP3 via their pleckstrin-homology (PH)
domains [8, 9]. Activates AKT and phosphorylates PDK1 can regulate a number of
cellular functions including those involved in tumor development and progression [10,
11].
Recently, PIK3CA (PI3K, catalytic, alpha polypeptide) encoding for the p110α
subunit of PI3K was found to frequently mutate in many different human cancers
[12-15]. Notably, colorectal, brain and gastric cancer cells were found to have a high
rate of PIK3CA gene mutations with frequencies of 32, 27 and 25%, respectively [12,
16, 17]. These somatic missense mutations were scattered across most of the exons,
but were predominantly found in the kinase (H1047R) and helical domains (E542K,
E545K) of the PIK3CA subunit [18, 19]. Increasing evidences demonstrated that
mutation in the catalytic domain of PIK3CA resulted in kinase activation [12, 20, 21].
Although the mechanism for this occurrence is unclear, it could be similar to the
oncogenic activation of protein kinase by mutation in their catalytic domain [22].
Collectively, inhibition of PIK3CA provides a potential opportunity to develop
therapeutic agents to treat cancer. For examples, BYL-719 and ZSTK474, both
exhibited strong antitumor activities in vitro and tumor xenograft models in vivo, and
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are particularly effective against cancer cell lines with mutant or amplified PIK3CA
[23-25].
PIK3CA gene mutations are important in various tumors, but the discovery of such
mutant specific inhibitors remains a great challenge [26-28]. Via a high-throughput
screening campaign using time-resolved fluorescence assay, an in-house library with
diverse chemical structures was screened against the PI3Kα H1047R mutant protein,
from which Hit-02 (Figure 1) with weak inhibitory effect for the enzyme was
identified [29]. Herein, we report the synthesis, biological evaluations and
structure−activity relationship (SAR) analysis of a series of Hit-02 analogues as novel
PI3Kα H1047R mutant inhibitors with greatly improved potency.
Figure 1 Chemical structures of the reported PI3K inhibitors and Hit-02
2. Results and discussion
2.1 Chemistry
Hit-02 contains a trihydroxyacetophenone core with a phenyl substitution. As
shown in Scheme 1, we synthesized Hit-02 and its analogues (3-5) via the Friedel–
Crafts reaction catalyzed by BF3·Et2O using various phenylacetic acids. To explore
whether the trihydroxyl group is essential for biological activities, we prepared 3a-b,
with dihydroxyl groups, and 4a, with 1,2,3-trihydroxyl moiety attaching to the
acetophenone core, respectively. Among these analogues, 4a exhibited the best
inhibitory activity against PI3Kα H1047R mutant, and was chosen for further
modifications.
In addition, to identify the optimal substitutions on the benzene ring, analogues
with various halo-substitutions (4a-e, 4g-i) and bis-methoxy (4f) were synthesized via
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the same protocols as for Hit-02.
Scheme 1 Reagents and conditions: BF3·Et2O, reflux, overnight, yields: 42-79%.
For further modifications, 4a-i reacted with DMF-DMA in toluene/DMF to afford
isoflavone analogues 6a-i (Scheme 2) via a cyclization reaction, which efficiently
reacted with hydrazine hydrate to provide the corresponding pyrazole analogues 7a-i
in excellent yields ranging from 82 to 93%.
Scheme 2 Reagents and conditions: (a) DMF-DMA, DMF / toluene (v / v: 2 / 15), reflux, 55-83%;
(b) NH2-NH2·H2O, THF, reflux, 82-93%.
2.2 Inhibition of PI3Kα H1047R mutant and cell proliferation
All the synthesized compounds were evaluated for their antiproliferative activities
on a PI3Kα H1047R mutated cell line HCT-116 and a PI3Kα H1047R non-mutation
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cancer cell line MCF-7, as well as their inhibitory potency against PI3Kα H1047R
mutant. ZSTK474 and DOX were used as positive controls in the cell viability assay,
and the results of these biological evaluations are summarized in Table 1.
For the first series of compounds 3a-b, and 4a, the trihydroxyl group played
important roles for exerting their enzymatic activities. Compared with Hit-02,
compound 3a with a 1,2-dihydroxyl group, and compound 3b with a 1,3-dihydroxyl
group attaching to the benzene ring showed decreased inhibitory activities against the
enzyme, while 4a with a 1,2,3-trihydroxyl group exhibited an improved enzymatic
inhibition. Therefore, 4a was chosen for further modifications.
Compound 4b or 4c with a chlorine atom relocated from position 4 to 3 or 2 of the
benzene ring displayed an decreased activity or totally lost its activity in the
biochemical assay. When the chlorine atom was replaced by a fluorine atom, the
corresponding compounds 4d and 4e showed inferior activities as compared with
Hit-02. To identify the optimal substitution on the benzene ring, different groups were
introduced.
Comparison of 4f (EC50 = 242.0 µM) and 4g (EC50 = 58.4 µM) suggests that the
introduction of an electron with-drawing group is beneficial for the improving
inhibitory activity. Among this series compounds, 3,4-bis-substituted compounds 4h
and 4i showed the best inhibitory activities, with EC50 values of 8.9 and 9.7 µM
respectively, clearly outperformed Hit-02. At last, the benzene ring was replaced by
polar heterocyclic groups, however, the resulting compounds 5a and 5b lost all
potencies.
Table 1 Inhibitory activities of the prepared compounds against two cancer cell lines and
PI3Kα H1047R mutated kinase
EC50 (µM)
PI3Kα (H1047R)
115.3 ± 1.3
> 500
IC50 (µM) a
MCF-7
Compound
HCT-116
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
Hit-02
3a
196 ± 3.5
3b
69.3 ± 1.8
98.2 ± 2.5
> 500
4a
4b
4c
170.4 ± 3.8
4d
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>500
> 50
> 50
> 50
> 50
> 50
> 50
4e
4f
4g
4h
4i
242 ± 4.7
58.4 ± 2.4
8.9 ± 0.35
9.7 ± 0.12
> 500
35.5 ± 1.28
47.3 ± 2.15
> 50
48.9 ± 2.45
> 50
> 50
5a
> 500
> 50
> 50
5b
40.2 ± 1.6
36.8 ± 1.1
> 500
> 50
> 50
> 50
> 50
> 50
> 50
6a
6b
6c
139.6 ± 2.5
> 500
> 50
> 50
> 50
> 50
6d
6e
335.5 ± 3.25
42.7 ± 1.25
6.3 ± 0.20
7.1 ± 0.18
1.6 ± 0.12
6.37 ± 0.87
15.1 ± 1.39
2.1 ± 0.102
39.3 ± 1.21
242.3 ± 4.8
5.1 ± 0.41
0.55 ± 0.015
0.92 ± 0.027
0.05 ± 0.01
ND
> 50
> 50
> 50
> 50
> 50
> 50
6f
6g
6h
6i
7a
7b
7c
7d
7e
7f
7g
7h
44.2 ± 1.25
45.3 ± 1.31
24.2 ± 1.17
> 50
49.3 ± 2.24
32.0 ± 1.35
> 50
> 50
41.8 ± 2.59
> 50
39.8 ± 1.77
> 50
> 50
> 50
28.0 ± 1.02
10.9 ± 0.52
14.9 ± 0.21
1.31 ± 0.24
1.96 ± 0.09
42.4 ± 1.26
12.7 ± 0.59
22.5 ± 0.96
5.27 ± 0.73
3.28 ± 0.52
7i
ZSTK474
DOX
a
Values are average of three independent experimental measurements and the values were
expressed as Mean ± SD. ND: not determined. DOX: Doxorubicin.
Furthermore, a new series of isoflavone analogues 6a-i were synthesized via a
cyclization reaction and then evaluated for their enzymatic inhibitory activities. It was
interesting that 6a-i exhibited almost the same level of activities as compared to their
corresponding precursors (4a-i).
Finally, 6a-i reacted with hydrazine hydrate to give their corresponding pyrazole
analogues 7a-i, which displayed unexpected inhibitory activities. As shown in Table 1,
compounds 7a-d and 7g exhibited not only improved EC50 values for inhibiting the
mutated kinase function, but also enhanced anti-proliferative activities against cancer
cells. Among this series of analogues, compound 7h showed the best inhibitory
activity, with an EC50 of 0.55 µM against PI3Kα H1047R mutant. Moreover, 7h
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displayed promising antiproliferative activities for HCT-116 (IC50 = 10.9 µM) and
MCF-7 (IC50 = 12.7 µM) cells, respectively. Besides, compound 7i (EC50 = 0.92 µM
for PI3Kα H1047R mutant) also showed a similar inhibitory effect as 7h. Collectively,
we conducted a series of modifications on Hit-02 and identified two potent
compounds 7h and 7i, which strongly inhibited PI3Kα H1047R mutant and cancer
cell proliferation. Compounds 7h and 7i displayed similar anti-proliferative activities
on both PI3Kα H1047R mutated cell line HCT-116 and PI3Kα non-mutation cancer
cell line MCF-7, suggesting they may also inhibit PI3Kα wild-type protein.
Table 2. Inhibitory activity (EC50, µM) against PI3Kα wild type and H1047R mutant.
Compounds
PI3Kα wild-type
EC50 (µM) a
79.3 ± 2.9
PI3Kα H1047R
EC50 (µM) a
96.0 ± 1.3
SI b
1.21
1.98
2.25
1.37
0.97
1.02
Hit-02
4a
35.2 ± 1.55
17.8 ± 0.72
1.2 ± 0.05
69.3 ± 1.8
40.2 ± 1.6
6a
1.6 ± 0.12
7a
0.57 ± 0.02
0.90 ± 0.41
0.55 ± 0.015
0.92 ± 0.027
7h
7i
a Values are average of three determinations and the EC50 values were expressed as Mean ± value.
b
SI stands for selectivity index, which was calculated by comparing the EC50 values of
compounds inhibiting PI3Kα H1047R mutant against the EC50 values of the same compounds
inhibiting PI3Kα wild-type in a biochemical assay.
To verify our hypothesis, some of the synthesized compounds were selected and
measured for their inhibitory activity against PI3Kα wild-type. As expected, all the
selected compounds showed similar inhibitory activities against PI3Kα wild-type and
H1047R mutant (Table 2). Of note, compounds 7h and 7i displayed the best
inhibitory potencies against these two proteins, with EC50 values of 0.57 and 0.90 µM
against PI3Kα wild type, and 0.55 and 0.92 µM against PI3Kα H1047R mutant,
respectively. Although compounds 7h and 7i did not exhibit selectivity on the PI3Kα
wild type and mutant, which could be used as novel and potent PI3Kα inhibitors.
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Then 7h was selected to compare the selectivity index on other PI3K isoforms and
mTORC1. As shown in Table 3, compound 7h exhibited a significant inhibitory
activity against PI3Kα, is over 10-fold more potent than other PI3K isoforms, and
showed little effect on mTORC1 even with the tested concentration up to 30 µM. All
these results suggested that 7h is a novel PI3Kα inhibitor and deserves further
biological evaluations.
Table 3. Enzymatic activities of compound 7h against PI3K and mTORC1 (EC50, µM a).
Kinases
PI3Kα
BEZ-235
ZSTK474
7h
0.57 ± 0.02
5.7 ± 0.12
10.0 ± 0.86
19.1 ± 0.73
> 30
0.040 ± 0.010
0.015 ± 0.003
0.067 ± 0.021
0.093 ± 0.032
0.079 ± 0.024
0.050 ± 0.017
0.044 ± 0.014
0.051 ± 0.018
0.010 ± 0.004
ND
PI3Kβ
PI3Kγ
PI3Kδ
mTORC1
a Values are average of three independent experimental determinations and the EC50 values were
expressed as Mean ± SD. ND: not determined.
2.3 Cell cycle arrest
Since 7h exhibited the best potency, we conducted additional studies to investigate
its biological roles on HCT-116 cancer cell. The effect of 7h on cell cycle distribution
was examined using the propidium iodide (PI) staining kit. HCT-116 cells were
treated with compound 7h (5, 10, 20 µM) or control for 24 h, stained with PI and then
analyzed on a flow cytometer.
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Figure 2 Compound 7h induced cell cycle arrest in HCT-116 cell. A: Cells were treated with 7h of
5, 10 and 20 µM for 24 h, which were then harvested, washed with PBS, and fixed in 70%
cold-ice ethanol at 4ꢀ°C. After incubation overnight, the cell pellets were collected by
centrifugation, resuspended in 50ꢀ µg/mL of RNase A in PBS, and incubated at 37ꢀ°C for 1 h. Next,
PI dye (50 µg/mL) was added, and the mixture was incubated at 37ꢀ°C for 15ꢀ min. Cell cycle
analysis was performed via PI fluorescence and flow cytometer. B: Bar graph represents the
values of cells in G1, S, and G2 phases, which were expressed as Means ± SD. *p < 0.05 and **p
< 0.01 vs. the control group.
As shown in Figure 2, compound 7h led to a significant accumulation of cells at
G2 phase from 8.58% to 10.78% (5 µM), 18.97% (10 µM) and 31.54% (20 µM),
respectively. At the same time, it reduced the cells at G1 phase from 58.0% to 39.9%,
and S phase from 33.37% to 28.56%, respectively. Meanwhile, we found that cell
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cycle arrest at G2 phase induced by compound 7h was in a concentration-dependent
manner, which might be one of the possible mechanisms for its cytotoxicity.
2.4 Cell autophagy
To explore the mechanism of cancer cell death, 7h was used to induce HCT-116
cell apoptosis and was examined with Annexin V-FITC/PI FACS assay. As shown in
Figure 3, the percentages of apoptotic population in HCT-116 cells treated with 7b at
5, 10, 20 and 40 µM for 24 h were almost unchanged, suggesting that no apoptosis
effect was induced by 7h.
Figure 3 Pro-apoptotic effect of compound 7h on HCT-116 cell. (A) Apoptotic assay by flow
cytometry. HCT-116 cell was treated with compound 7h at 0, 5, 10, 20 and 40 µM for 24 h. Cells
were then stained with Annexin V-FITC/PI and detected by flow cytometry analysis. Cells in the
upper right quadrant indicate PI positive/Annexin V positive, late apoptotic, or necrotic cells, and
cells in the lower right quadrant indicate early apoptotic cells. (B) Bar graph represents statistics
of total apoptotic cell percentages from three dependent experiments.
Autophagy is known to be another pathway to induce programmed cell death,
pharmacological inhibition of PI3Kα might also induce cell autophagy. Then, we
determined the effect of compound 7h on HCT-116 cells autophagy by using various
of biological assays. As a well-known PI3K inhibitor, ZSTK474 was reported to
induce autophagy activity in cancer cell, which therefore was used as a positive
control in our experiments.
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Firstly, monodansylcadaverine (MDC) incorporation assay was carried out to verify
our hypothesis. MDC is an auto-fluorescent substance, which selectively accumulates
in acidic vesicular organelles (AVOs), and therefore is used as a marker to detect cell
autophagy. After staining with MDC, HCT-116 cells treated with or without 7h were
observed under a fluorescence microscope. As shown in Figure 4, the number of
autophagic vacuoles in HCT-116 cells dose-dependently increased after treating with
7h, suggesting that 7h induced autophagy in HCT-116 cells. As expected, ZSTK474
also exhibited an increased number of autophagic vacuoles, which was in line with
previous report [30].
Figure 4 Effect of compound 7h on HCT-116 cells autophagy. HCT-116 cells were incubated on
coverslips in 6-well plates, then treated with various concentrations of compound 7h (0, 5, 10 µM)
or ZSTK474 (5 µM) for 6 h, and stained with 0.05 mM MDC for 1 h at 37 °C. After washing with
PBS, the stained MCF-7 cells were immediately examined by Olympus BX51 fluorescence
microscope. Organelles stained with bright blue fluorescence indicates the autophagic vacuoles.
To evaluate the influence of 7h on the process of autophagosome formation, fusion
with lysosome and degradation, we transfected HCT-116 cells with a plasmid, which
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stably expresses GFP (green fluorescent protein) / mRFP (monomeric red fluorescent
protein)-tagged LC3 (tfLC3), an autophagic flux reporter containing LC3 protein
fused with GFP and mRFP [31]. At the early stage of autophagy, the tfLC3 localizes
to autophagosomes, develops both mRFP (red) and GFP (green) signals. The GFP
protein is acid sensitive, and could be quenched quickly following the fusion of
autophagosomes with lysosomes, whereas mRFP is relatively stable in the acidic
environment of the autolysosome [32]. Therefore, yellow puncta (merge of green GFP
signal and red mRFP signal) indicates early autophagosomes while red puncta (red
mRFP signal alone) represents late autolysosomes. As shown in Figure 5, only weak
signals of GFP and mRFP before treatment of 7h, indicating diffuse LC3 protein exist
in the cytoplasm. While after treatment of 7h (0, 5, 10 µM) or ZSTK474 (5 µM) for
12 h, the mRFP and GFP signals accumulated dose-dependently in cytoplasm,
suggesting the gradual increase of the autophagosomes in HCT-116 cells.
Figure 5 Effect of compound 7h on HCT-116 cells autophagy. HCT-116 cells were transfected
with a plasmid containing GFP/mRFP-tagged LC3 (tfLC3) reporter gene for 12 h by use of
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lipofectamine 2000, then treated with compound 7h (0, 5, 10 µM) and 5 µM of ZSTK474,
respectively. The treated cells were fixed for microscopy assay. The GFP/mRFP images were
acquired using Olympus FV1000 laser scanning confocal microscope. Arrows indicate
autophagosomes.
To further study the autophagy mechanism of 7h on HCT-116 cells, we examined
expression of autophagy biomarker LC3B proteins, which are known to bind with
autophagosome, therefore conversion of LC3B was considered to be a reliable marker
of autophagy. As shown in Figure 6, the expression of LC3BⅠ and LC3BⅡ was
increased in a dose-dependent manner after treatment with 7h, suggesting that the
LC3B protein activation pathway was involved in the induction of autophagy in
HCT-116 cancer cells by 7h.
Figure 6 Effects of compound 7h on LC3BⅠand LC3BⅡ expressions. Cells were treated with
compound 7h for 72 h, then total protein was extracted and subjected to Western blot analysis.
β-Actin was used as an internal control. *p < 0.05 and **p < 0.01, vs. the control.
2.5 AKT Phosphorylation
Since 7h exhibited the most potent PI3Kα H1047R mutant inhibitory activity in our
biochemical assay, we therefore docked 7h into the PI3Kα H1047R protein active
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binding pocket (PDB code: 3hhm) to rationalize its potency. As shown in Figure 7,
compound 7h formed strong hydrogen bond interactions with the two important
amino acids Val851 and Asp933, with the total binding scores is 6.03. While for the
ligand in the PDB code (3hhm), the total binding is 6.31, which slightly outperformed
7h. As both Val851 and Asp933 play crucial roles in PI3Kα phosphorylation function,
these interactions could potentially explain the inhibitory activity of 7h on PI3Kα
function. Moreover, both trihydroxyl group and pyrazole moiety formed several
strong hydrogen bond interactions with amino acids Asp933, Tyr 836, Val851 and
Glu849 in PI3Kα protein, the lacking of pyrazole group (3-6) or trihydroxyl group (3a
and 3b) displayed decreased potencies, which is in line with our observed SAR.
Figure 7 Molecular docking of 7h with the PI3Kα H1047R mutant (PDB code: 3hhm). Docking
model of 7h in the binding pocket (left); Stereo view of 7h in the binding pocket (right). Amino
acid residues and compound 7h are shown as stick models, H-bonds are shown as yellow dashed
lines. The 3D graphical presentations were drawn by PyMol.
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Figure 8 Effects of compound 7h on the AKT phosphorylation. Cells were treated with compound
7h for 24 h, total protein was extracted and subjected to Western blot analysis. β-Actin was used
as an internal control. Data were expressed as mean ± SD (n= 3). *p < 0.05 and **p < 0.01 vs. the
control.
Inhibition of PI3Kα protein should lead to a decrease of phosphorylation level in
AKT signal pathway. Hence, we assessed the in vitro PI3Kα inhibitory activity of 7h
in cultured HCT-116 cells. The cells were treated with 7h at 5, 10 and 15 µM,
respectively, and subjected to Western blotting analysis. As expected, 7h caused a
decrease in p-AKT level (Figure 8), demonstrating that 7h is capable of inhibiting
PI3Kα H1047R activities.
3. Conclusion
In conclusion, several series of novel PI3Kα inhibitors containing a
1,2,3-trihydroxyacetophenone core were synthesized and evaluated for their inhibitory
effects on PI3Kα H1047R mutant. Systematic SAR studies enabled us to discover
compound 7h, which strongly inhibited both PI3Kα H1047R mutant and wild-type
with EC50 values of 0.55, and 0.57 µM, respectively, while exerting little effect on
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other PI3K isoforms. Western blotting assay indicated that 7h reduced the AKT
phosphorylation level, demonstrating its inhibitory activity on the PI3Kα H1047R
mutant. Compound 7h was docked well into the PI3Kα H1047R mutant active site
and could form strong hydrogen bonds with the protein. In addition, 7h reduced
HCT-116 cancer cell proliferation with an IC50 of 10.9 µM. Furthermore, studies on
potential cytotoxic mechanisms indicated that compound 7h could arrest cell cycle at
G2 phase. Annexin V-FITC assay revealed that compound 7h did not induce any
apoptosis effect. Compound 7b significantly induced cell autophagy, which may
contribute to its anti-proliferation effect in cancer cell. Collectively, our results
suggest that 7h is a selective PI3Kα inhibitor, which could serve as a promising lead
for developing potent PI3Kα inhibitors for cancer treatment.
4. Experimental section
4.1 Materials and methods
All chemicals were purchased from commercial sources and used as received.
Reactions were monitored by thin-layer chromatography (TLC) and carried out on
Merck Kieselgel 60 F254 plates, which could be visualized under UV light at 254 nm.
1
Column chromatography was performed on silica gel (60−120 mesh). All H NMR
spectra were obtained on an Agilent 400 MR spectrometer at ambient temperature and
13
reported in ppm downfield from TMS (0 ppm). All C NMR spectra were obtained
with proton decoupling on an Agilent 400 MR DD2 (100 MHz) or 600 MR DD2
spectrometer and reported in ppm relative to CDCl3 (77.16 ppm). Coupling constants
were reported as Hz with multiplicity denoted as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and br (broad). The NMR data was processed by software
Mest Re-Nova (Ver. 9.0.0.12821, Mestrelab Research S.L.). High resolution mass
spectra (HRMS) were obtained on a Bruker SolariX 7.0T spectrometer. Melting point
was recorded on a WRS-2A digital melting point apparatus. Compounds exhibited
purities of more than 95% as determined by peak area integration in reverse phase
chromatography. The mobile phase is the mixtures of A and B, where A is 0.05%
TFA water and B is acetonitrile, with a flow rate of 7 mL/min.
17

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4.2 Chemical synthesis
4.2.1 General procedure for the preparation of Hit-02
1,3,5-Trihydroxybenzene (10.0 mmol, 1261 mg) and 4-chlorophenylacetic acid
(10.0 mmol, 1695 mg) were dissolved in 25 mL BF3·Et2O. The mixture was heated to
reflux for 24 h and cooled to room temperature. Ethyl acetate (50 mL) was added, the
organic phase was washed with NaHCO3 saturated solution (3 × 50 mL), dried over
MgSO4, and concentrated. The crude products were purified by column
chromatography, affording Hit-02 (1529 mg, 55% yield) as yellow solid. M.p.:
220−221°C. 1H NMR (400 MHz, DMSO-d6) (ppm) 12.28 (s, 2H), 10.41 (s, 1H), 7.35
(d, J = 8.2, 2H), 7.24 (d, J = 8.2, 2H), 5.83 (s, 2H), 4.35 (s, 2H). The data is in line
with the literature reported previously [33].
4.2.2 General procedure for the preparation of 3a-b
Compounds 3a-b were synthesized according to the protocol as preparing of Hit-02.
Pyrocatechol (10.0 mmol, 1100 mg) or resorcinol (10.0 mmol, 1100 mg) were
dissolved in 25 mL BF3·Et2O. The mixture was then heated to reflux for 24 h and
cooled to room temperature. Ethyl acetate (50 mL) was added, the organic phase was
washed with NaHCO3 saturated solution (3 × 50 mL), dried over MgSO4, and
concentrated. The crude products were purified by column chromatography
Compound 3a (2-(4-chlorophenyl)-1-(2,3-dihydroxyphenyl)ethanone) was obtained
1
as white solid (1179 mg, 45% yield). M.p.: 155−157°C. H NMR (400 MHz,
DMSO-d6) δ (ppm) 9.88 (s, 1H), 9.36 (s, 1H), 7.45 (dd, J = 8.3, 2.2 Hz, 1H), 7.43–
7.29 (m, 3H), 7.28–7.19 (m, 2H), 6.81 (d, J = 8.2 Hz, 1H), 4.23 (s, 2H). 13C NMR
(100 MHz, DMSO-d6) δ 195.90, 151.29, 145.71, 135.25, 131.99, 131.48, 128.60,
128.53, 122.31, 115.59, 115.51, 43.79. HRMS (m/z): calcd for C14H12ClO3 263.0316;
found 263.0324.
Compound 3b (2-(4-chlorophenyl)-1-(2,4-dihydroxyphenyl)ethanone) was
obtained as white solid (1493 mg, 57% yield). M.p.: 139−140°C. 1H NMR (400 MHz,
acetone-d6) δ (ppm) 12.62 (s, 1H), 9.62 (s, 1H), 6.34–7.89 (m, 7H) and 4.35 (s, 2H).
The data is in line with the literature reported previously [34].
4.2.3 General procedures for preparation of 4a-i
18

ACCEPTED MANUSCRIPT
1,2,3-Trihydroxyl benzene (10.0 mmol, 1260 mg) and various of
substituted-phenylacetic acid (10.0 mmol) were dissolved in 25 mL BF3·Et2O. The
mixture was heated to reflux for 24 h and cooled to room temperature. Ethyl acetate
(50 mL) was added, the organic phase was washed with NaHCO3 saturated solution (3
× 50 mL), dried over MgSO4, and concentrated. The crude products were purified by
column chromatography.
Compound 4a (2-(4-chlorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1807 mg, 65% yield). M.p.: 147−148°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 10.14 (s, 1H), 8.65 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.38 (d, J =
8.6 Hz, 2H), 7.30(d, J = 8.6 Hz, 2H), 6.43 (d, J = 8.6 Hz, 1H), 4.31(s, 1H). The data is
in line with the literature reported previously [35].
Compound 4b (2-(3-chlorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1668 mg, 60% yield). M.p.: 138−139°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 12.31 (s, 1H), 10.13 (s, 1H), 8.63 (s, 1H), 7.49 (d, J = 9.2 Hz, 1H),
7.35 (d, J = 7.4 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 725 (d, J = 7.4 Hz, 1H), 6.42 (d, J =
9.2 Hz, 1H), 4.31 (s, 1H). The data is in line with the literature reported previously
[35].
Compound 4c (2-(2-chlorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1306 mg, 47% yield). M.p.: 133−134°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 12.21(s, 1H), 10.12(s, 1H), 8.65 (s, 1H), 7.52 (d, J = 8.6Hz, 1H),
7.42-7.44 (m, 1H), 7.36-7.38 (m, 1H), 7.29-7.31 (m, 2H), 6.44 (d, J = 8.6Hz, 1H),
4.51 (s, 2H). The data is in line with the literature reported previously [36].
Compound 4d (2-(4-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1781 mg, 68% yield). M.p.: 140−141°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 12.42 (s, 1H), 10.11 (s, 1H), 8.62 (s, 1H), 7.48 (d, J = 8.9 Hz, 1H),
7.38–7.21 (m, 2H), 7.20–7.02 (m, 2H), 6.41 (d, J = 8.9 Hz, 1H), 4.29 (s, 2H). The
data is in line with the literature reported previously [37].
Compound 4e (2-(2-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1781 mg, 68% yield). M.p.: 149−150°C. 1H NMR (400 MHz,
methanol-d4) δ 7.45 (d, J = 8.9 Hz, 1H), 7.39–7.19 (m, 2H), 7.17–6.98 (m, 2H), 6.44
19

ACCEPTED MANUSCRIPT
(d, J = 8.9 Hz, 1H), 4.31 (d, J = 1.1 Hz, 2H). 13C NMR (100 MHz, methanol-d4) δ
201.47, 162.33, 159.90, 152.11, 132.24, 131.63, 128.63, 123.83, 122.24, 114.74,
112.65, 109.99, 107.28, 37.48. HRMS (m/z): calcd for C14H11FO4 330.9963 [M+H]+;
found 330.9971.
Compound 4f (2-(3,4-dimethoxyphenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (2158 mg, 71% yield). M.p.: 152−153°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 12.74 (s, 1H), 8.78 (br, 1H), 7.96 (br, 1H), 7.55 (d, J = 8.8 Hz,
1H), 6.96 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.84 (dd, J = 2.0, 8.4 Hz, 2H),
6.48 (d, J = 8.8 Hz, 1H), 4.20 (s, 2H), 3.77 (s, 3H), 3.76(s, 3H). The data is in line
with the literature reported previously [38].
Compound 4g (2-(4-(trifluoromethyl)phenyl)-1-(2,3,4-trihydroxyphenyl)ethanone)
1
was obtained as white solid (2158 mg, 71% yield). M.p.: 142−143°C. H NMR (400
MHz, methanol-d4) δ (ppm) 7.59 (d, J = 8.1 Hz, 2H), 7.45 (dd, J = 8.5, 2.7 Hz, 3H),
6.43 (d, J = 8.9 Hz, 1H), 4.35 (s, 2H). The data is in line with the literature reported
previously [38].
Compound 4h (2-(3,4-dichlorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone) was
obtained as white solid (1964mg, 63% yield). M.p.: 158−159°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 12.26 (s, 1H), 10.15 (s, 1H), 8.66 (s, 1H), 7.56 (dd, J = 5.1, 3.1
Hz, 2H), 7.46 (d, J = 9.0 Hz, 1H), 7.26 (dd, J = 8.3, 2.0 Hz, 1H), 6.43 (d, J = 8.9 Hz,
1H), 4.37 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 202.07, 153.15, 152.72, 136.83,
132.81, 132.35, 131.09, 130.80, 130.70, 129.71, 123.13, 112.95, 108.33, 43.25.
HRMS (m/z): calcd for C14H11Cl2O4 313.0129 [M+H]+; found 313.0121.
Compound 4i (2-(3-chloro-4-fluorophenyl)-1-(2,3,4-trihydroxyphenyl)ethanone)
1
was obtained as white solid (1924 mg, 65% yield). M.p.: 146−147°C. H NMR (400
MHz, DMSO-d6) δ (ppm) 12.28 (s, 1H), 10.13 (s, 1H), 8.64 (s, 1H), 7.53–7.45 (m,
2H), 7.34 (t, J = 8.9 Hz, 1H), 7.26 (dd, J = 8.5, 4.9 Hz, 1H), 6.43 (d, J = 8.9 Hz, 1H),
4.34 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 202.31, 153.12, 152.75, 133.51,
133.48, 132.82, 132.24, 130.99, 123.12, 119.47, 117.07, 112.94, 108.31, 43.08.
HRMS (m/z): calcd for C14H11ClFO4 297.0317 [M+H]+; found 297.0319.
4.2.4 General procedures for the preparation of 5a-b
20

ACCEPTED MANUSCRIPT
1,2,3-Trihydroxyl benzene (10.0 mmol, 1260 mg) and pyridinyl acetic acid (10.0
mmol) were dissolved in 25 mL BF3·Et2O. The mixture was heated to reflux for 24 h
and cooled to room temperature. Ethyl acetate (50 mL) was added, the organic phase
was washed with NaHCO3 saturated solution (3 × 50 mL), dried over MgSO4, and
concentrated. The crude products were purified by column chromatography.
Compound 5a (2-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)ethanone) was obtained
as white solid (980 mg, 40% yield). M.p.: 140−141°C. 1H NMR (400 MHz, DMSO-d6)
δ (ppm) 11.78 (s, 1H), 10.41 (s, 1H), 9.13–8.73 (m, 2H), 8.23–7.88 (m, 2H), 7.44 (d, J
= 8.9 Hz, 1H), 6.53 (d, J = 8.9 Hz, 1H), 4.78 (s, 2H). 13C NMR (100 MHz, DMSO-d6)
δ (ppm) 199.45, 156.32, 153.35, 152.42, 141.43, 132.90, 129.19, 122.93, 113.28,
108.63, 44.82. HRMS (m/z): calcd for C13H12NO4 246.0703 [M+H]+; found
246.0704.
Compound 5b (2-(pyridin-4-yl)-1-(2,3,4-trihydroxyphenyl)ethanone) was obtained
1
as white solid (1029 mg, 42% yield). M.p.: 185−186°C. H NMR (400 MHz,
DMSO-d6) δ (ppm) 11.86 (s, 1H), 10.31 (s, 1H), 8.96–8.79 (m, 2H), 8.44 (dt, J = 8.1,
1.7 Hz, 1H), 8.13–7.81 (m, 1H), 7.46 (d, J = 9.0 Hz, 1H), 6.51 (d, J = 8.9 Hz, 1H),
4.68 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm) 200.15, 153.25, 152.40, 147.48,
143.60, 140.98, 135.51, 132.88, 126.74, 122.78, 113.12, 108.53, 41.46. HRMS (m/z):
calcd for C13H12NO4 246.0703 [M+H]+; found 246.0704.
4.2.5 General procedure for the preparation of 6a-i
Compounds 4a-i (2 mmol) was dissolved in 15 mL toluene, then DMF-DMA (5
mmol) and 2 mL DMF was added to the mixture, respectively. The reaction was
heated to reflux for 6 h and cooled to room temperature. After the solvent was
evaporated, ethyl acetate (30 mL) was added, the organic phase was washed with
NaHCO3 saturated solution (3 × 30 mL), dried over MgSO4, and concentrated. The
crude products were purified by column chromatography.
Compound 6a (3-(4-chlorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
obtained as white solid (403 mg, 70% yield). M.p.: 255−256°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 10.35 (s, 1 H), 9.47 (s, 1 H), 8.47 (s, 1 H), 7.61 (d, J = 8.6 Hz,
2H), 7.48 (d, J = 8.7 Hz, 1H,), 7.47 (d, J = 8.59 Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H).
21

ACCEPTED MANUSCRIPT
HRMS (m/z): calcd for C15H11ClO4 289.0108 [M+H]+; found 289.0116.
Compound 6b (3-(3-chlorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
obtained as white solid (391 mg, 68% yield). M.p.: 250−252°C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm) 9.89 (s, 2H), 8.42 (s, 1H), 7.46 (d, J = 9.0 Hz, 1H), 7.42-7.68 (m,
4H), 7.01 (d, J = 9.0 Hz, 1H). HRMS (m/z): calcd for C15H11ClO4 289.0108 [M+H]+;
found 289.0116.
Compound 6c (3-(2-chlorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
1
obtained as white solid (259 mg, 45% yield). M.p.: 238−239°C. H NMR (400 MHz,
methanol-d4) δ 8.14 (d, J = 1.3 Hz, 1H), 7.96 (s, 1H), 7.56 (dd, J = 8.8, 1.4 Hz, 1H),
7.50 (s, 1H), 7.44–7.30 (m, 3H), 6.97 (dd, J = 8.8, 1.4 Hz, 1H). 13C NMR (100 MHz,
methanol-d4) δ 176.17, 154.62, 150.65, 147.14, 134.59, 132.97, 132.11, 131.33,
129.62, 129.16, 126.58, 123.17, 117.15, 115.85, 114.18. HRMS (m/z): calcd for
C15H11ClO4 289.0108 [M+H]+; found 289.0116.
Compound 6d (3-(4-fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
obtained as white solid (348 mg, 64% yield). M.p.: 246−247°C. 1H NMR (400 MHz,
DMSO-d6) δ 9.89 (s, 3H), 8.43 (s, 1H), 7.66–7.55 (m, 2H), 7.46 (d, J = 8.7 Hz, 1H),
7.28–7.20 (m, 2H), 6.95 (d, J = 8.7 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
175.24, 154.11, 150.61, 147.14, 133.40, 131.49, 129.03, 122.43, 117.78, 116.10,
115.48, 115.26, 114.73. HRMS (m/z): calcd for C15H10FO4 273.0463 [M+H]+; found
273.0669.
Compound 6e (3-(2-fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was obtained
1
as white solid (310 mg, 57% yield). M.p.: 253−254°C. H NMR (400 MHz,
methanol-d4) δ 8.19 (d, J = 1.5 Hz, 1H), 7.94 (s, 1H), 7.56 (dd, J = 8.8, 1.6 Hz, 1H),
7.43–7.32 (m, 2H), 7.28–7.10 (m, 2H), 6.96 (dd, J = 8.8, 1.6 Hz, 1H). 13C NMR (101
MHz, methanol-d4) δ 176.10, 157.02, 150.54, 147.01, 132.88, 131.85, 131.82, 130.04,
123.85, 119.48, 117.13, 115.88, 115.25, 115.02, 114.21. HRMS (m/z): calcd for
C15H10FO4 273.0463 [M+H]+; found 273.0669.
Compound 6f (3-(3,4-dimethoxyphenyl)-7,8-dihydroxy-4H-chromen-4-one) was
1
obtained as white solid (420 mg, 67% yield). M.p.: 249−250°C. H NMR (400 MHz,
DMSO-d6) δ 9.87 (s, 3H), 8.39 (s, 1H), 7.46 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 2.0 Hz,
22

ACCEPTED MANUSCRIPT
1H), 7.11 (dd, J = 8.3, 2.0 Hz, 1H), 6.96 (dd, J = 14.2, 8.5 Hz, 2H), 3.76 (s, 7H).13C
NMR (100 MHz, DMSO-d6) δ 175.46, 153.61, 150.43, 148.97, 148.68, 147.08,
133.35, 125.14, 123.11, 121.69, 117.87, 116.08, 114.63, 113.32, 111.97, 55.97. HRMS
(m/z): calcd for C17H15O6 315.0709 [M+H]+; found 315.0717.
Compound 6g (7,8-dihydroxy-3-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one)
was obtained as white solid (457 mg, 71% yield). M.p.: 243−244°C. 1H NMR (400
MHz, DMSO-d6) δ 10.39 (s, 1H), 9.57 (s, 1H), 8.55 (s, 1H), 7.79 (q, J = 8.4 Hz, 4H),
7.48 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 8.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
174.96, 155.05, 150.82, 147.10, 137.10, 133.48, 130.11, 128.63, 128.31, 126.12,
125.31, 123.41, 122.13, 117.74, 116.16, 114.87. HRMS (m/z): calcd for C16H10F3O4
323.0468 [M+H]+; found 323.0473.
Compound 6h (3-(3,4-dichlorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
1
obtained as white solid (204 mg, 61% yield). M.p.: 248−250°C. H NMR (400 MHz,
DMSO-d6) δ 8.55 (s, 1H), 7.88 (d, J = 2.0 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.59 (dd,
J = 8.4, 2.1 Hz, 1H), 7.46 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H). 13C NMR (150
MHz, methanol-d4) δ 175.68, 155.83, 151.62, 147.84, 134.27, 134.26, 131.98, 131.84,
131.48, 131.44, 130.35, 121.77, 118.45, 116.94, 115.68. HRMS (m/z): calcd for
C15H9Cl2O4 322.9785 [M+H]+; found 322.9791.
Compound 6i (3-(3-chloro-4-fluorophenyl)-7,8-dihydroxy-4H-chromen-4-one) was
obtained as white solid (385 mg, 63% yield). M.p.: 247−248°C. 1H NMR (400 MHz,
DMSO-d6) δ 9.85 (s, 2H), 8.51 (s, 1H), 7.82 (dd, J = 7.3, 2.2 Hz, 1H), 7.60 (dd, J =
7.5, 4.9 Hz, 1H), 7.46 (dt, J = 8.9, 4.7 Hz, 2H), 6.96 (d, J = 8.7 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) δ 174.99, 155.94, 154.75, 150.77, 147.08, 133.44, 131.29,
130.50, 130.11, 121.19, 119.41, 117.67, 116.87, 116.12, 114.85. HRMS (m/z): calcd
for C15H9ClFO4 306.9987 [M+H]+; found 306.9995.
4.2.5 General procedure for the preparation of 7a-i
Compounds 6a-i (1 mmol) was dissolved in 10 mL THF, then NH2-NH2·H2O (2
mmol) was added to the mixture. The reaction was heating to reflux for 6 h and
cooled to room temperature. After the solvent was evaporated, ethyl acetate (20 mL)
was added, the organic phase was washed with NaHCO3 saturated solution (3 × 20
23

ACCEPTED MANUSCRIPT
mL), dried over MgSO4, and concentrated. The crude products were purified by
column chromatography.
Compound 7a (4-(4-(4-chlorophenyl)-1H-pyrazol-3-yl)benzene-1,2,3-triol) was
1
obtained as white solid (232 mg, 77% yield). M.p.: 295−295°C. H NMR (400 MHz,
methanol-d4) δ 7.74 (s, 1H), 7.26 (q, J = 8.6 Hz, 4H), 6.50 (d, J = 8.5 Hz, 1H), 6.30 (d,
J = 8.5 Hz, 1H), 4.60 (s, 1H). 13C NMR (151 MHz, methanol-d4) δ 146.13, 144.57,
133.11, 132.57, 131.61, 129.01, 128.02, 120.01, 118.45, 109.62, 106.62. HRMS (m/z):
calcd for C15H12ClN2O3 303.0374 [M+H]+; found 303.0382.
Compound 7b (4-(4-(3-chlorophenyl)-1H-pyrazol-3-yl)benzene-1,2,3-triol) was
1
obtained as white solid (220 mg, 73% yield). M.p.: 288−289°C. H NMR (400 MHz,
methanol-d4) δ 7.76 (s, 1H), 7.31 (q, J = 1.3 Hz, 1H), 7.26–7.12 (m, 3H), 6.50 (d, J =
8.5 Hz, 1H), 6.30 (d, J = 8.5 Hz, 1H). 13C NMR (100 MHz, methanol-d4) δ 146.23,
144.60, 135.95, 133.74, 133.12, 129.37, 127.25, 125.83, 125.71, 120.01, 118.30,
109.48, 106.58. HRMS (m/z): calcd for C15H12ClN2O3 303.0374 [M+H]+; found
303.0382.
Compound 7c (4-(4-(2-chlorophenyl)-1H-pyrazol-3-yl)benzene-1,2,3-triol) was
obtained as white solid (187 mg, 62% yield). M.p.: 263−264°C. 1H NMR (400 MHz,
DMSO-d6) δ 7.85 (s, 1H), 7.39–7.07 (m, 4H), 6.24–6.00 (m, 2H). 13C NMR (100
MHz, DMSO-d6) δ 161.16, 158.73, 145.00, 143.74, 132.06, 129.43, 124.82, 124.51,
122.17, 122.02, 116.13, 115.91, 111.91, 109.92, 106.74. HRMS (m/z): calcd for
C15H12ClN2O3 303.0374 [M+H]+; found 303.0382.
Compound 7d (4-(4-(4-fluorophenyl)-1H-pyrazol-3-yl)benzene-1,2,3-triol) was
1
obtained as white solid (214 mg, 75% yield). M.p.: 291−292°C. H NMR (400 MHz,
methanol-d4) δ 7.71 (s, 1H), 7.41–7.26 (m, 2H), 7.00 (t, J = 8.6 Hz, 2H), 6.49 (d, J =
8.5 Hz, 1H), 6.27 (dd, J = 8.5, 4.4 Hz, 1H). 13C NMR (151 MHz, methanol-d4) δ
162.43, 160.81, 145.97, 144.62, 133.05, 130.10, 130.08, 129.47, 119.77, 118.62,
114.64, 114.50, 109.19, 106.41. HRMS (m/z): calcd for C15H12FN2O3 286.0672
[M+H]+; found 286.0680.
Compound 7e (4-(4-(2-fluorophenyl)-1H-pyrazol-3-yl)benzene-1,2,3-triol) was
1
obtained as white solid (200 mg, 70% yield). M.p.: 282−283°C. H NMR (400 MHz,
24

ACCEPTED MANUSCRIPT
methanol-d4) δ 7.75 (d, J = 1.2 Hz, 1H), 7.33–7.22 (m, 2H), 7.17–7.06 (m, 2H), 6.38
(d, J = 8.5 Hz, 1H), 6.16 (d, J = 8.5 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
160.15, 157.43, 144.95, 143.52, 131.96, 129.40, 124.62, 124.23, 121.87, 121.92,
115.73, 114.91, 111.21, 109.03, 105.33. HRMS (m/z): calcd for C15H12FN2O3
286.0672 [M+H]+; found 286.0680.
Compound 7f (4-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-4-yl)benzene-1,2,3-triol)
1
was obtained as white solid (229 mg, 70% yield). M.p.: 306−307°C. H NMR (400
MHz, methanol-d4) δ 7.70 (s, 1H), 7.00–6.81 (m, 3H), 6.55 (d, J = 8.5 Hz, 1H), 6.28
(d, J = 8.5 Hz, 1H), 3.81 (s, 3H), 3.64 (s, 3H). 13C NMR (101 MHz, methanol-d4) δ
148.73, 147.70, 144.69, 133.03, 126.80, 120.17, 119.97, 119.38, 111.84, 111.66,
106.33, 55.07, 54.77. HRMS (m/z): calcd for C17H18N2O5 329.0978 [M+H]+; found
329.0986.
Compound
7g
(4-(3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)benzene-1,2,3-triol) was obtained
1
as white solid (218 mg, 65% yield). M.p.: 299−300°C. H NMR (400 MHz,
methanol-d4) δ 7.85 (s, 1H), 7.52 (q, J = 8.6 Hz, 5H), 6.51 (d, J = 8.4 Hz, 1H), 6.33 (d,
J = 8.5 Hz, 1H). 13C NMR (100 MHz, methanol-d4) δ 146.42, 144.58, 138.09, 134.09,
133.18, 129.72, 127.52, 125.83, 124.77, 123.13, 120.16, 118.32, 109.49, 106.68.
HRMS (m/z): calcd for C16H12F3N2O3 337.0658 [M+H]+; found 337.0649.
Compound 7h (4-(3-(3,4-dichlorophenyl)-1H-pyrazol-4-yl)benzene-1,2,3-triol) was
obtained as white solid (258 mg, 77% yield). M.p.: 267−268°C. 1H NMR (400 MHz,
methanol-d4) δ 7.79 (s, 1H), 7.44 (d, J = 2.1 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.21
(dd, J = 8.3, 2.1 Hz, 1H), 6.52 (d, J = 8.5 Hz, 1H), 6.35 (d, J = 8.4 Hz, 1H).13C NMR
(100 MHz, methanol-d4) δ 146.45, 144.56, 134.51, 133.22, 131.69, 129.92, 129.25,
128.85, 126.96, 120.11, 117.37, 106.75. HRMS (m/z): calcd for C15H11Cl2N2O3
336.9985 [M+H]+; found 336.9995.
Compound 7i (4-(3-(3-chloro-4-fluorophenyl)-1H-pyrazol-4-yl)benzene-1,2,3-triol)
was obtained as white solid (233 mg, 73% yield). M.p.: 282−283°C. 1H NMR (400
MHz, methanol-d4) δ 7.77 (s, 1H), 7.39 (dd, J = 7.2, 2.2 Hz, 1H), 7.24 (dd, J = 8.6,
4.6 Hz, 1H), 7.11 (t, J = 8.9 Hz, 1H), 6.50 (d, J = 8.4 Hz, 1H), 6.32 (d, J = 8.5 Hz,
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1H). 13C NMR (150 MHz, methanol-d4) δ 157.34, 155.71, 146.31, 144.58, 133.17,
131.59, 129.23, 127.55, 120.06, 119.96, 117.52, 116.02, 115.88, 109.05, 106.62.
HRMS (m/z): calcd for C15H11ClFN2O3 321.0280 [M+H]+; found 321.0291.
4.3 Cell culture
HCT-116 and MCF-7 cancer cell lines were purchased from Shanghai Cell Bank of
Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM
supplemented with 10% FBS, 1% penicillin and streptomycin at 37 °C in a 5% CO2
humidified atmosphere. Cells grown at exponential stage were used for all the
biological experiments.
4.4 Cell viability
Cell viability was assessed by Cell Counting Kit-8 (CCK-8, Beyotime, Jiangsu,
China) following manufacturer’s instructions. Compounds were dissolved in DMSO
and diluted with culture medium. Cells at a density of 5 × 103 per well were seeded in
96-well plates and cultured overnight, which were then treated with either vehicle (1%
DMSO PBS buffer) or desired concentrations (50, 25, 12.5, 6.25, 3.12, 1.6, 0.8, and
0.4 µM) of compounds for 72 h at 37 °C. Cell viability was measured by the CCK-8
kit and the absorbance was measured using a microplate reader (Bio-Rad Laboratories)
at 450 nm. At last, resultant OD450 nm values were expressed as IC50 values, which
were the mean values derived from three independent experiments.
4.5 Cell cycle
Cells were seeded in 6-well plates at density of 1 × 105 cells/well and treated with
different concentrations of compound 7h (5, 10 and 20 µM) for 24 h at 37 °C. Then,
cells were harvested and fixed in 70% precooled ethanol. Finally, the cells were then
treated with RNase A, stained by PI (Product #: C1052, Beyotime, Jiangsu, China).
Finally, the suspended cells were analyzed with a flow cytometer (Accuri C6, BD
Biosciences).
4.6 Cell apoptosis
HCT-116 cells were seeded at a density of 1 × 105 cells/well on each well of 6-well
plates and allowed to grow overnight. Then the cells were treated with 7h (5, 10, 20
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and 40 µM) and ZSTK474 (5 µM) for 24 h, cells without treatment were used as
control group. Then the cells were trypsinized, washed with cold PBS for three times,
centrifuged at 1200 rpm for 5 min, and the supernatants were discarded. Then cells
were stained by an Annexin-V-fluorescein isothiocyanate (FITC) kit (Beyotime,
Jiangsu, China) in the binding buffer for 15 min at room temperature. Subsequently,
the cells were labeled by PI and the apoptotic cells were measured by a flow
cytometer (Accuri C6).
4.7 Western blotting
After treating HCT-116 cells with 7h (5, 10 and 20 µM) for 24 h, the total proteins
were extracted and their concentrations were balanced to the same level using BCA
Protein Assay Reagent (Beyotime, Jiangsu, China), followed by 8 min protein
denaturation with SDS loading buffer at 100 °C. Proteins (30 µg) were separated by
sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) electrophoresis and transferred
onto polyvinylidene fluoride (PVDF) membranes, which were blocked with 5%
fat-free dry milk in 1 × Tris-buffered saline (TBS) containing 0.05% Tween 20 for 2 h
at room temperature. Then membranes were then incubated with AKT, p-AKT, LC3B
Ⅰ, LC3BⅡ and β-actin antibodies at 4 °C overnight, followed by treatment with
secondary horseradish-peroxidase-conjugated anti-rabbitIgG for 2 h. Membranes were
finally scanned in a ChemiDoc MP Imaging System (Bio-Rad) after 2 min incubation
in Clarity Western ECL Substrate (Bio-Rad).
4.8 Kinase activity EC50
EC50 values of the selected compounds against PI3Kα H1047R mutant kinase were
determined using the HTRF assay as described in the literature [29]. Compounds were
dissolved in DMSO as stocking solution, and serially diluted in PBS to a final
concentration of DMSO as 2.5%. The results were obtained using an EnSpire
MultimodePlate Reader (PerkinElmer) and analyzed using GraphPad Prism 6
nonlinear regression (curve fit), log (inhibitor) vs. response variable slope (four
parameters) with constraints added at 0 and 100.
4.9 EC50 of other PI3K isoforms activity
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The assay was carried out as described previously [39]. All of the enzymatic
reactions were conducted at 30℃ for 40 min. First, we prepared 50 µL reaction
mixtures containing 40 mM Tris (pH = 7.4), 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM
DTT, 0.2 ug/mL PI3Ks and 100 µM lipid substrate. Compounds were diluted in 10%
DMSO, then 5 µL of the dilution with 100 µM ATP was added to the 50 µL reaction
mixtures so that the final concentration of DMSO is 1% in all of reactions. The assay
was then performed by using Kinase-Glo Plus luminescence kinase assay kit
(Promega, no: V3771) to measure the kinase activity by quantitating the amount of
ATP remaining in solution after kinase reaction. The luminescent signal is correlated
with the amount of ATP remaining in the solution, and is inversely correlated with the
amount of kinases activity. The EC50 values were calculated using nonlinear
regression with normalized dose−response fit using Prism GraphPad software.
4.10 MDC incorporation
MDC staining assay was used to confirm the existence of autophagic vacuoles as
reported previously [40]. Briefly, HCT-116 cells were plated on coverslips in 6-well
plates (4 × 105 cells/well) overnight at 37 °C under a 5% CO2 atmosphere, then
treated with various concentrations of 7h (0, 5, 10 µM) or ZSTK474 (5 µM) for 6 h,
cells were incubated with fresh medium containing 0.05 mM MDC for 1 h at 37 °C.
After washing with PBS, the stained HCT-116 cells on coverslips were immediately
examined by a fluorescence microscope (BX51, Olympus, Tokyo, Japan).
4.11 mRFP-GFP-LC3 fluorescence microscopy
HCT-116 cells were transfected for 5 h with a plasmid (Addgene, Cambridge, MA,
USA) containing mRFP-GFP-tagged LC3 (tfLC3) reporter gene which expresses LC3
protein fused with GFP and mRFP by use of Lipofectamine 2000 according to the
instruction of the manufacturer. Then, the transfected (HCT-116-tfLC3) cells were
seeded on glass coverslips in 24-well plates and incubated overnight. The next day,
cells were washed and treated with 7h (0, 5, 10 µM) and 5 µM of ZSTK474 for 12 h,
respectively. The treated cells were fixed for 15 min in 4% paraformaldehyde and
washed with PBS. Finally, the coverslips were removed and mounted onto glass slides
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