Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing sulfonylpiperazine as antitumor inhibitors targeting PI3Kα

Article abstract of DOI:10.1016/j.ejmech.2019.111630

PI3K signal pathway plays a vital role in cellular functions and becomes an attractive approach for cancer therapy. Herein, a new series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivatives bearing sulfonylpiperazine based on the PI3K inhibitors and our previous research. They were screened for their PI3K inhibitory activities and anticancer effects in vitro. Biological studies indicated that compound 7m revealed the remarkable antiproliferative activity (IC₅₀ ranging from 0.03 to 0.09 μM) against four cancer cell lines (A549, Huh7, HL60 and HCT-116). Besides, compound 7m displayed a certain selective for PI3Kα (IC₅₀ = 0.009 μM) over PI3Kβ, γ and δ, and meanwhile, it can remarkable decreased the expression level of p-Akt (Ser473) and p-S6K. In addition, compound 7m could not only induce HCT-116 cell arrest at G1 phase in a dose-dependent manner, but also induce cell apoptosis via upregulation of Bax and cleaved-caspase 3/9, and downregulation of Bcl-2. Besides, compound 7m can remarkably inhibit the growth of tumor in vivo. The above results suggested that compound 7m could be considered as a promising PI3Kα inhibitor.

Full text of DOI:10.1016/j.ejmech.2019.111630

Journal Pre-proof
Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing
sulfonylpiperazine as antitumor inhibitors targeting PI3Kα
Yong Yin, Shao Sha, Xun Wu, She-Feng Wang, Fang Qiao, Zhong-Cheng Song, Hai-
Liang Zhu
PII:
S0223-5234(19)30764-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.111630
EJMECH 111630
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 June 2019
Revised Date: 14 August 2019
Accepted Date: 16 August 2019
Please cite this article as: Y. Yin, S. Sha, X. Wu, S.-F. Wang, F. Qiao, Z.-C. Song, H.-L. Zhu,
Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing sulfonylpiperazine as
antitumor inhibitors targeting PI3Kα, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111630.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Masson SAS.

Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing
sulfonylpiperazine as antitumor inhibitors targeting PI3Kα
Yong Yina,b, Shao Shab, Xun Wub, She-Feng Wangb, Fang Qiaob, Zhong-Cheng
Song*b,c, Hai-Liang Zhu*b
a
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry,
School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong
Jia Xiang, Nanjing 210009, People’ s Republic of China.
b State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210093, People’s Republic of China.
c
School of Chemistry & Environmental Engineering, Jiangsu University of
Technology, 1801 Zhongwu Rd., Changzhou, Jiangsu, 213001 China
* Corresponding authors.
Fax:+86-25-83592672; Tel:+86-25-83592572;
E-mail: zhuhl@nju.edu.cn (Hai-Liang Zhu);
songzhongcheng@jsut.edu.cn(Zhong-Cheng Song)

Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing
sulfonylpiperazine as antitumor inhibitors targeting PI3Kα
Yong Yina,b, Shao Shab, Xun Wub, She-Feng Wangb, Fang Qiaob, Zhong-Cheng
Song*b,c, Hai-Liang Zhu*b
a
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry,
School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong
Jia Xiang, Nanjing 210009, People’ s Republic of China.
b
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, People’s Republic of China.
c
School of Chemistry & Environmental Engineering, Jiangsu University of
Technology, 1801 Zhongwu Rd., Changzhou, Jiangsu, 213001 China
* Corresponding authors.
Fax:+86-25-83592672; Tel:+86-25-83592572;
E-mail: zhuhl@nju.edu.cn (Hai-Liang Zhu);
songzhongcheng@jsut.edu.cn(Zhong-Cheng Song)
Abstract
PI3K signal pathway plays a vital role in cellular functions and becomes an
attractive approach for cancer therapy. Herein, a new series of novel
chromeno[4,3-c]pyrazol-4(2H)-one derivatives bearing sulfonylpiperazine based on
the PI3K inhibitors and our previous research. They were screened for their PI3K
inhibitory activities and anticancer effects in vitro. Biological studies indicated that
compound 7m revealed the remarkable antiproliferative activity (IC50 ranging from
0.03 to 0.09µM) against four cancer cell lines (A549, Huh7, HL60 and HCT-116).
Besides, compound 7m displayed a certain selective for PI3Kα (IC50 = 0.009µM) over
PI3Kβ, γ and δ, and meanwhile, it can remarkable decreased the expression level of
p-Akt (Ser473) and p-S6K. In addition, compound 7m could not only induce
HCT-116 cell arrest at G1 phase in a dose-dependent manner, but also induce cell
apoptosis via upregulation of Bax and cleaved-caspase 3/9, and downregulation of
Bcl-2. Besides, compound 7m can remarkably inhibit the growth of tumor in vivo.
The above results suggested that compound 7m could be considered as a promising
PI3Kα inhibitor.
Keywords: chromeno[4,3-c]pyrazol-4(2H)-one derivates; sulfonylpiperazine;
antitumor; PI3Kα inhibitor

1. Introduction
As an important signaling pathway, phosphatidylinositol 3-kinases (PI3Ks) play
an extremely pivotal role in a large number of cellular functions such as cell motility,
differentiation, growth, proliferation, intracellular trafficking and so on [1-3]. The
PI3K family is categorized into three classes (classes I, II and III) in terms of their
structural features and substrate specificity [4-6]. Out of them, Class I PI3Ks are
studied most extensively and have been an attractive approach for cancer therapy over
the past decades, and they comprise four different isoforms (PI3Kα, β, γ and δ) and
consist of heterodimers between a p110 catalytic subunit and a p85 regulatory subunit,
which commonly used to be activated by receptor tyrosine kinases (RTKs) and
G-protein coupled receptors [7-10]. Caused by activation of PI3Ks,
phosphatidylinositol(4,5)diphosphate (PIP2) is converted into phosphatidylinositol
3,4,5-triphosphate (PIP3), which as one of second messenger, regulating many
physiological roles including generation and development of tumor [11-12].
Meanwhile, PIP3 levels are strictly controlled by phosphatase and tensin homologue
(PTEN), which convert PIP3 into PIP2 [13-14]. In addition, PI3Kα and PI3Kβ are
expressed commonly, nevertheless PI3Kδ and PI3Kγ are present in the central
nervous system, epithelial cells and the hematopoietic system [15-18]. Due to the
importance in cell functions, PI3K pathway becomes an attractive approach for cancer
therapy.
Nowadays many pharmaceutical chemists try to develop PI3K inhibitors cancer
therapy, and several drug candidates are in preclinical study [19-22].
Chromeno[4,3-c]pyrazol-4(2H)-one has been discovered and identified to design new
PI3Kα
inhibitors
in
our
previous
work
[23-25].
In
fact,
chromeno[4,3-c]pyrazol-4(2H)-one skeleton was designed based on the structural
characteristics of LY294002, Wortmannin, GDC-0941 and BEZ235 (Fig. 1), which
are all inhibitors of PI3K [26-30]. Subsequently, we designed a novel
chromeno[4,3-c]pyrazol-4(2H)-one derivates and some interesting results were
obtained[23-25]. Encouraged by these, continuous research is meaningful to further
explore. Based on the molecular structure of PI3K inhibitors, it was observed that
piperazine and sulfonamide fragment are employed such as GDC-0941, PI-3065,
CH5132799 (Fig. 1) and so on [29, 31-33]. Sulfonylpiperazine fragment is introduced
may result in increased binding affinity of derivates and improved biology activity or
selectivity.
On
the
basis
of
this
assumption,
a
novel
chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing sulfonylpiperazine fragment

were designed and synthesized, and biological evaluation was performed to verify
their PI3K inhibitory activities and antitumor effects.
2. Results and discussion
2.1. Chemistry
The targeted compounds 7a-7w were prepared as outlined in Scheme 1. The
main core chromeno[4,3-c]pyrazol-4(2H)-one (3) was synthesized by the method in
our previous study [23]. Chromeno[4,3-c]pyrazol-4(2H)-one (3) was produced via a
Vilsmeier-Haack reaction of 4-hydroxycoumarin (1) in the presence of
dimethylformamide and phosphoryl chloride followed by cyclization with 85%
hydrazine hydrate. The yield was up to 85%.
Compounds 7a-7w were synthesized as outlined in Scheme 1. Compounds
6a-6w were produced by sulfonylation reaction between piperazine and sulfonyl
chloride
Subsequently,
Compounds
6a-6w
reacted
with
Chromeno[4,3-c]pyrazol-4(2H)-one (3) in the presence of 40% formaldehyde to get
the targeted compounds 7a-7w.
2.2. Antiproliferative assays in vitro
All synthesized compounds were assessed in terms of anti-proliferative activity
against four cancer cell lines (A549, Huh7, HL60 and HCT-116) using MTT assay,
and LY294002 and BEZ235 were assigned as positive control. The results were
displayed in Table 1. The results manifested that most the designed compounds reveal
the better anti-proliferative effects than that of the positive control LY294002. Notably,
three tested compounds (7m, 7n and 7o) showed the significant activities against
these four cancer cell lines. Particularly, compound 7m exhibited the remarkable
anti-proliferative effect with the IC50 values of 0.03, 0.06, 0.06 and 0.09µM against
HCT-116, HL60, A549 and Huh7, respectively.
Anti-proliferative activities of the first series of compounds (7a-7o) were
compared. As presented in Table 1, most of the compounds showed the potent
anti-proliferative activities, indicating that phenylsulfonyl piperazine fragment is
beneficial to the improvement of anti-proliferative activity. Compounds 7m-7o
displayed the comparable activities against these cancer cell lines, and it was found
that these compounds have two or three substituent group. As can be seen in Table 1,
the more substituent groups on the benzene ring, the better anti-proliferative activities
they showed. However, compound 7m (two substituents on the benzene ring)

revealed the best anti-proliferative activity, there may be a great possibility caused by
the NO2 group. Furthermore, for the compounds with mono-substituted on the
benzene ring, it was observed that compounds show the anti-proliferative effects with
the tendency of the order is Cl > CF3 > NO2 > Br > F > H > CH3 > OCH3. Obviously,
electron-withdrawing group plays an important role for their anti-proliferative effects
against these cancer cell lines.
Meanwhile, compounds (7p-7w) with alkylsulfonyl piperazine fragment were
synthesized and evaluated their anti-proliferative activities to verify the importance of
phenylsulfonyl piperazine fragment. The results were summarized in Table 1,
compare to compounds 7a-7o, compounds 7p-7w revealed the much poorer
anti-proliferative activities against these cancer cell lines. It was further verified that
phenylsulfonyl piperazine is the necessary fragment in term of anti-proliferative
effects who exhibited.
Furthermore, the top twelve compounds were selected to examine the cytotoxic
effects on human normal cells 293T, and the results were shown in Table 2. It was
observed that all the tested compounds exhibit the poor anti-proliferative activities
against the human normal cells 293T, indicating that there is almost no any effect on
human normal cells.
2.3. Inhibition of PI3K kinase
The twelve designed compounds were evaluated for their biological activities
against four isoforms of PI3K (α, β, γ and δ) using a competitive fluorescence
polarization kinase activity assay (FPA). The results were revealed in Table 2. Notly,
compounds 7l-7o exhibited the potent activities against these four isoforms of PI3K.
Interestingly, compound 7m revealed the prominent inhibition activity comparing to
the other compounds, and it displayed an IC50 value of 0.009 µM against PI3Kα, with
a certain selectivity of 13-fold over β-isozyme, 9-fold over γ-isozyme and 13-fold
over δ-isozyme. It was also observed that compound 7m display an almost 53-fold
improvement against PI3Kα comparing to that of LY294002, and comparable activity
with BEZ235 to PI3Kα. Furthermore, these compounds were also evaluated for their
biological activities against mTOR, and they displayed the poor activities, indicating
compound 7m exhibit the selectivity for PI3Kα. These results suggested compound
7m could be a promising PI3Kα inhibitor.
2.4. Western blot analysis

The above results indicated that compound 7m could be a PI3Kα inhibitor, and
activation of the PI3K can phosphorylation of Akt directly and then regulated the
downstream molecule, inhibition of PI3K leads to suppression of Akt phosphorylation.
To further investigate the mechanism of action, and find out the effects on
downstream factors of PI3K pathway, the representative proteins phosphorylated Akt
(p-Akt) and phosphorylated S6K (p-S6K) were examined by western blot. The results
were displayed in Fig. 2. After treatment by compound 7m with different
concentrations at 0.25, 0.5 and 1 µM in HCT-116 cells, both p-Akt (Ser473) and
p-S6K were down-regulated, and the expression level were a remarkably decreased in
a dose-dependent manner. These results suggested compound 7m could also inhibit
the PI3K pathway, and it further proved that compound 7m could act as an antitumor
inhibitor targeting PI3Kα.
2.5. Cell cycle arrest
In order to further investigate the mechanism of anti-proliferative effects in
cancer cell lines, compound 7m was selected to test the effect on cell cycle in
HCT-116 cells distribution due to its best potency. After treatment by compound 7m
with different concentrations at 0.25, 0.5 and 1 µM for 24 h, the results were
displayed in Fig. 3, compound 7m could induce a remarkable accumulation of
HCT-116 cells at G1 phase from 46.87 to 58.13% with a dose-dependent manner.
Furthermore, compound 7m decreased the percentage of cells in S phase (from 42.40
to 33.78%). Besides, there was almost no change in G2 phase (from 10.73 to 8.09%).
These results revealed induction of HCT-116 cells arrest at G1 phase may be one of
the possible mechanisms of cancer cells death.
2.6. Cell apoptosis
Induce cell apoptosis in HCT-116 was also performed to investigate the
mechanism of anti-proliferative effects, and it was examined using Annexin
V-FITC/PI FACS assay. HCT-116 cells were treated by compound 7m at 0.25, 0.5 and
1 µM four different concentrations for 24 h. The results were displayed in Fig. 4A, the
percentages of late apoptotic population remarkably increased with
the concentration increasing, and the percentages ranged from 9.61 to 36.9%.
Similarly, the percentages of early apoptotic population ranged from 10.0 to 25.5%
with a dose-dependent manner.
To further explore the apoptosis mechanism by compound 7m in HCT-116 cell

line, apoptotic related proteins was examined in HCT-116 after treatment by 7m at
concentrations of 0.25, 0.5 and 1µM for 24 h. As shown in Fig.4B, the proapoptotic
protein Bax level was were remarkably upregulated in a dose-dependent manner, in
contrast, Bcl-2 level was decreased. What’s more, the cleaved caspase-3 and cleaved
caspase-9 levels which are considered as bio-markers for cell apoptosis, were
increased in a dose-dependent manner. These results indicated that compound 7m
induce cell apoptosis of HCT-116 cells in a dose-dependent manner.
2.7. Molecular modeling
To elucidate the binding model of compound 7m with PI3Kα, molecular docking
was performed by docking 7m into the active pocket of PI3Kα (PDB: 3HHM). As
shown in Fig. 5, compound 7m could bind well to the active site of 3HHM via three
hydrogen bonds and three π-cation interactions. It was observed that
chromeno[4,3-c]pyrazol-4(2H)-one part formed three π-cation interactions with
Lys776, one hydrogen bond (distance: 2.05Å) was formed between carbonyl and
Ser774. Meanwhile, the sulfonyl formed another hydrogen bond(distance: 2.03Å)
with Val851, the third hydrogen bond (distance: 2.36Å) was formed by nitro and
Gln859. All these interactions make compound 7m bind well to the active site of
PI3Kα, indicating that compound 7m is a promising PI3Kα inhibitor.
2.9. In vivo antitumor activity evaluations
Due to the potent activity in vitro, antitumor activities in vivo of compound 7m
were performed. we established the HCT-116 cells xenograft model by subcutaneous
inoculation. The mice were divided into three groups once the tumor volume was up
to about 100 mm3, LY294002 was selected as positive drug, and they were treated
with compound 7m (10 mg/kg), LY294002 (10 mg/kg) and control (CMC-Na) by
intragastric administration. The results were presented in Fig. 6, it was revealed a
distinct effect in inhibiting tumor growth after treatment by compound 7m at a dose of
10ꢀmg/kg once every three days, what’s more, it can decrease the weight of tumor up
to 66.4%. By comparison, it showed about 35.2% growth inhibitory rate after treated
with LY294002 (10 mg/kg). Overall, these indicated that compound 7m display the
remarkable effect in inhibiting tumor growth and it could be a promising antitumor
agent.
3. Conclusion
In summary, we designed and synthesized
a
series of novel

chromeno[4,3-c]pyrazol-4(2H)-one derivates bearing sulfonylpiperazine fragment,
and their biological evaluation was performed. Most designed compounds revealed
the anti-proliferative activities for cancer cell lines (Huh7, A549, HCT-116 and HL60),
particularly, compound 7m displayed the remarkable anti-proliferative effect (IC50
ranging from 0.03 to 0.09µM), with the significant improvement in comparison to
LY294002. Meanwhile, compound 7m exhibited the selective activity against PI3Kα
(IC50 = 0.009µM) which compared to the other three PI3K isoforms (β, γ and δ), and
decreased the expression level of p-Akt (Ser473) and p-S6K. Further research
indicated that compound 7m could induce HCT-116 cell arrest at G1 phase in a
dose-dependent manner. Not only that, compound 7m could significantly induce cell
apoptosis, and increased the level of cleaved caspase 3, cleaved caspase 9 and Bax,
down-regulated the level of Bcl-2, which as one of anti-apoptotic protein. The
efficiency in vivo of compound 7m was also performed on HCT-116 Xenograft nude
mice by intragastric administration, indicating that compound 7m exhibited
remarkable antitumor activity in vivo, which compared to the positive group
(LY294002). All above results suggested compound 7m may be a promising PI3Kα
inhibitor for cancer therapy.
4. Experiments
All chemicals and reagents were purchased from commercial company (J&K,
China; Sigma-Aldrich, China). Melting points were measured by a melting point
apparatus (Taike Corp., China). All reactions were monitored by thin layer
1
chromatography (TLC; silica GF254 plates) which purchased from Merck. H NMR
and 13C NMR spectra were recorded using Bruker DPX400 spectrometer, and TMS as
an internal standard. Chemical shifts are described in ppm (δ). ESI mass spectra were
determined on a Mariner System 5304 mass spectrometer.
4.1. Synthesis of chromeno[4,3-c]pyrazol-4(2H)-one (3)
Chromeno[4,3-c]pyrazol-4(2H)-one was synthesized by described as the reported
methods of our previous study [23].
4.2. General procedure for the preparation of designed compounds 7a-7w
Compounds 6a-6w were synthesized by described in the literature [34].
40% formaldehyde (300 µL) was added slowly into the ethanol solution of
6a-6w (2 mmol) and 3 (2 mmol), the mixtures were stirred at room temperature for

5-7 h. A large quantity of solid was precipitated and then filtered, washed by cold
ethanol, and crystallized with ethanol to get the designed compounds 7a-7w.
4.2.1.
2-((4-(Phenylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one (7a)
White power, yield: 63%. Mp: 199-200 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.04(d, J=7.60 Hz, 1H, ArH), 7.72(d, J=7.28 Hz, 2H, ArH), 7.57-7.47(m, 4H,
ArH), 7.38(d, J=8.24 Hz, 1H, ArH), 7.32(t, J=7.48 Hz, 1H, ArH), 5.05(s, 2H, CH2),
13
3.07(s, 4H, -CH2CH2), 2.74(s, 4H, -CH2CH2). C NMR (100 MHz, CDCl3): 157.97,
152.96, 149.35, 135.87, 132.98, 132.33, 130.54, 129.13(2), 127.70(2), 124.57, 122.81,
117.62, 114.79, 108.05, 73.80, 49.26(2), 45.79(2). MS (ESI): 425.33 [M+H]+ (Calcd
for 425.48, C21H21N4O4S).
4.2.2. 2-((4-Tosylpiperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one (7b)
White power, yield: 57%. Mp: 176-177 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(d, J=7.72 Hz, 1H, ArH), 7.61(d, J=7.92 Hz, 2H, ArH), 7.49(t, J=7.70 Hz,
1H, ArH), 7.38(d, J=8.28 Hz, 1H, ArH), 7.31(t, J=9.66 Hz, 3H, ArH), 5.04(s, 2H,
-CH2), 3.05(s, 4H, -CH2CH2), 2.75(t, J=4.38 Hz, 4H, -CH2CH2), 2.40(s, 3H, CH3). 13C
NMR (100 MHz, CDCl3): 157.98, 152.97, 149.37, 143.90, 132.70, 132.27, 130.53,
129.74(2), 127.80(2), 124.58, 122.79, 117.64, 114.78, 108.07, 73.83, 49.37(2),
45.80(2), 21.49. MS (ESI): 439.32 [M+H]+ (Calcd for 439.50, C22H23N4O4S).
4.2.3.
2-((4-((4-Methoxyphenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2
H)-one (7c)
White power, yield: 54%. Mp: 182-184 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(d, J=7.64 Hz, 1H, ArH), 7.66(d, J=8.36 Hz, 2H, ArH), 7.48(t, J=5.76 Hz,
1H, ArH), 7.38(d, J=8.24 Hz, 1H, ArH), 7.32(t, J=7.52 Hz, 1H, ArH), 6.96(d, J=8.52
Hz, 2H, ArH), 5.05(s, 2H, -CH2), 3.85(s, 3H, OCH3), 3.05(s, 4H, -CH2CH2), 2.74(d,
J=4.32 Hz, 4H, -CH2CH2). 13C NMR (100 MHz, CDCl3): 163.22, 158.01, 152.98,
149.37, 132.23, 130.54, 129.88(2), 127.31, 124.58, 122.78, 117.64, 114.78, 114.31(2),
108.08, 73.84, 55.64, 49.31(2), 45.79(2). MS (ESI): 455.37 [M+H]+ (Calcd for 455.50,
C22H23N4O5S).
4.2.4.

2-((4-((4-(Tert-butyl)phenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4
(2H)-one (7d)
White power, yield: 58%. Mp: 264-266 ℃. 1H NMR (400 MHz, CDCl3): 8.22(s, 1H,
ArH), 8.05(d, J=7.76 Hz, 1H, ArH), 7.65(d, J=8.44 Hz, 2H, ArH), 7.52(d, J=8.32 Hz,
2H, ArH), 7.47(d, J=7.28 Hz, 1H, ArH), 7.37(d, J=8.24 Hz, 1H, ArH), 7.32(t, J=7.50
Hz, 1H, ArH), 5.05(s, 2H, -CH2), 3.06(s, 4H, -CH2CH2), 2.77(t, J=4.66 Hz, 4H,
-CH2CH2), 1.33(s, 9H, 3CH3). 13C NMR (100 MHz, CDCl3): 157.98, 156.87, 152.98,
149.41, 132.52, 132.30, 130.54, 127.70(2), 126.11(2), 124.57, 122.80, 117.64, 114.78,
108.10, 73.82, 49.47(2), 45.80(2), 35.20, 31.09(3). MS (ESI): 481.44 [M+H]+ (Calcd
for 481.58, C25H29N4O4S).
4.2.5.
2-((4-((4-Fluorophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)
-one (7e)
Yellow power, yield: 55%. Mp: 202-205 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.78(d, J=8.04 Hz, 2H, ArH), 7.31(d, J=8.44 Hz, 2H, ArH),
7.49(m, 1H, ArH), 7.37(d, J=7.88 Hz, 1H, ArH), 7.33(t, J=7.54 Hz, 1H, ArH), 7.31(d,
J=8.44 Hz, 2H, ArH), 5.04(s, 2H, CH2), 3.08(s, 4H, -CH2CH2), 2.76(t, J=4.56 Hz, 4H,
-CH2CH2). 13C NMR (100 MHz, CDCl3): 161.21(d, J=252.0 Hz), 157.86, 152.91,
149.42, 133.36, 132.34, 130.51, 128.27(d, J=6.8 Hz)(2),124.54, 122.81, 117.56,
114.83, 113.18(d, J=21.6 Hz)(2), 108.01, 73.82, 49.11(2), 45.96(2). MS (ESI): 443.35
[M+H]+ (Calcd for 443.47, C21H20FN4O4S).
4.2.6.
2-((4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H
)-one (7f)
1
Light yellow power, yield: 61%. Mp: 229-230 ℃. H NMR (400 MHz, CDCl3):
8.20(s, 1H, ArH), 8.05(d, J=7.72 Hz, 1H, ArH), 7.67(d, J=8.44 Hz, 2H, ArH), 7.48(m,
3H, ArH), 7.38(d, J=8.32 Hz, 1H, ArH), 7.32(t, J=7.48 Hz, 1H, ArH), 5.06(s, 2H,
-CH2), 3.07(s, 4H, -CH2CH2), 2.76(t, J=4.52 Hz, 4H, -CH2CH2). 13C NMR (100 MHz,
CDCl3): 157.98, 152.98, 149.43, 139.71, 134.28, 132.30, 130.58, 129.50(2),
129.14(2), 124.60, 122.77, 117.67, 114.75, 108.11, 73.76, 49.29(2), 45.78(2). MS
(ESI): 459.81 [M+H]+ (Calcd for 459.92, C21H20ClN4O4S).
4.2.7.

2-((4-((4-Bromophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)
-one (7g)
1
Light yellow power, yield: 57%. Mp: 250-251 ℃. H NMR (400 MHz, CDCl3):
8.19(s, 1H, ArH), 8.05(m, 1H, ArH), 7.67(d, J=8.16 Hz, 2H, ArH), 7.59(d, J=8.56 Hz,
2H, ArH), 7.49(m, 1H, ArH), 7.38(d, J=7.92 Hz, 1H, ArH), 7.33(t, J=7.50 Hz, 1H,
ArH), 5.06(s, 2H, CH2), 3.07(s, 4H, -CH2CH2), 2.76(t, J=4.84 Hz, 4H, -CH2CH2). 13C
NMR (100 MHz, DMSO-d6): 157.54, 152.87, 148.43, 135.13, 134.90, 132.92(2),
131.02, 129.90(2), 127.71, 125.19, 122.86, 117.81, 115.00, 107.01, 72.97, 48.66(2),
46.12(2). MS (ESI): 504.27 [M+H]+ (Calcd for 504.37, C21H20BrN4O4S).
4.2.8.
2-((4-((4-Nitrophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-
one (7h)
Yellow power, yield: 59%. Mp: 252-254 ℃. 1H NMR (400 MHz, DMSO): 8.80(s, 1H,
ArH), 8.43(d, J=8.76 Hz, 1H, ArH), 8.35(d, J=8.76 Hz, 2H, ArH), 7.97(d, J=8.76 Hz,
2H, ArH), 7.56(m, 1H, ArH), 7.43(d, J=8.12 Hz, 1H, ArH), 7.38(t, J=7.50 Hz, 1H,
ArH), 5.19(s, 2H, -CH2), 3.04(s, 4H, -CH2CH2), 2.66(d, J=4.28 Hz, 4H, -CH2CH2).
13C NMR (100 MHz, CDCl3): 157.99, 152.88, 152.24, 149.41, 144.98, 132.32, 130.51,
128.86(2),124.56, 123.87(2), 122.81, 117.59, 114.78, 108.02, 73.78, 48.87(2),
46.12(2). MS (ESI): 470.38 [M+H]+ (Calcd for 470.47, C21H20N5O6S).
4.2.9.
2-((4-((3-Nitrophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-
one (7i)
Yellow power, yield: 43%. Mp: 231-233 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.11(s, 1H, ArH), 8.05(d, J=7.52 Hz, 1H, ArH), 7.79(m, 2H, ArH), 7.68(d,
J=8.48 Hz, 1H, ArH), 7.51(t, J=7.88 Hz, 1H, ArH), 7.37(d, J=8.40 Hz, 1H, ArH),
7.33(t, J=7.42 Hz, 1H, ArH), 5.11(s, 2H, -CH2), 3.12(s, 4H, -CH2CH2), 2.78(s, 4H,
-CH2CH2). 13C NMR (100 MHz, CDCl3): 157.94, 152.86, 149.39, 146.14, 133.68,
132.30, 131.41, 130.52, 128.32, 125.87, 124.52, 122.83, 121.94, 117.61, 114.81,
108.06, 73.81, 49.04(2), 45.93(2). MS (ESI): 470.38 [M+H]+ (Calcd for 470.47,
C21H20N5O6S).
4.2.10.
2-((4-((2-Nitrophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-

one (7j)
Yellow power, yield: 37%. Mp: 218-219 ℃. 1H NMR (400 MHz, CDCl3): 8.22(s, 1H,
ArH), 8.14(m, 1H, ArH), 8.04(d, J=7.60 Hz, 1H, ArH), 7.72(m, 2H, ArH), 7.50(t,
J=7.76 Hz, 1H, ArH), 7.42(m, 1H, ArH), 7.39(d, J=8.44 Hz, 1H, ArH), 7.33(t, J=7.46
13
Hz, 1H, ArH), 5.09(s, 2H, -CH2), 3.10(s, 4H, -CH2CH2), 2.77(s, 4H, -CH2CH2). C
NMR (100 MHz, CDCl3): 157.96, 152.82, 149.44, 145.32, 132.97, 132.34, 131.14,
130.56, 130.12, 127.21, 124.54, 122.93, 122.81, 117.58, 114.79, 108.02, 73.86,
49.11(2), 45.99(2). MS (ESI): 470.38 [M+H]+ (Calcd for 470.47, C21H20N5O6S).
4.2.11.
2-((4-((4-(Trifluoromethyl)phenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyr
azol-4(2H)-one (7k)
1
Light yellow power, yield: 46%. Mp: 189-291 ℃. H NMR (400 MHz, CDCl3):
8.20(s, 1H, ArH), 8.06(s, 1H, ArH), 7.86(d, J=8.20 Hz, 2H, ArH), 7.81(d, J=8.32 Hz,
2H, ArH), 7.49(s, 1H, ArH), 7.37(t, J=7.96 Hz, 2H, ArH), 5.07(s, 2H, CH2), 3.11(s,
4H, -CH2CH2), 2.79(s, 4H, -CH2CH2). 13C NMR (100 MHz, DMSO-d6): 157.52,
152.84, 148.43, 139.84, 134.88, 133.28(q, J=24.4 Hz), 130.99, 128.91(2), 127.04(q,
J=4.5 Hz)(2), 125.14, 123.82(q, J=270 Hz), 122.83, 117.75, 114.96, 107.01, 72.95,
48.75(2), 46.13(2). MS (ESI): 493.31 [M+H]+ (Calcd for 493.47, C22H20F3N4O4S).
4.2.12.
2-((4-((2,5-Dimethylphenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4
(2H)-one (7l)
White power, yield: 58%. Mp: 223-225 ℃. 1H NMR (400 MHz, CDCl3): 8.19(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.56(d, J=8.44 Hz, 1H, ArH), 7.47(m, 1H, ArH), 7.36(d,
J=7.92 Hz, 1H, ArH), 7.33(t, J=7.56 Hz, 1H, ArH), 7.23(m, 2H, ArH), 5.08(s, 2H,
CH2), 3.07(s, 4H, -CH2CH2), 2.78(t, J=4.56 Hz, 4H, -CH2CH2) 2.60(s, 3H, CH3),
2.45(s, 3H, CH3). 13C NMR (100 MHz, CDCl3): 157.87, 152.85, 149.38, 135.82,
134.79, 132.31, 131.92, 131.21, 130.52, 128.12, 124.71, 124.57, 122.83, 117.55,
114.81, 108.04, 73.79, 49.12(2), 45.93(2), 23.36, 21.53. MS (ESI): 453.41 [M+H]+
(Calcd for 453.53, C23H25N4O4S).
4.2.13.
2-((4-((4-Chloro-3-nitrophenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyraz
ol-4(2H)-one (7m)

1
Light yellow power, yield: 39%. Mp: 247-248 ℃. H NMR (400 MHz, CDCl3):
8.20(s, 2H, ArH), 8.05(d, J=7.56 Hz, 1H, ArH), 7.85(d, J=8.12 Hz, 1H, ArH), 7.74(d,
J=8.48 Hz, 1H, ArH), 7.50(t, J=7.76 Hz, 1H, ArH), 7.39(d, J=8.44 Hz, 1H, ArH),
7.33(t, J=7.46 Hz, 1H, ArH), 5.09(s, 2H, -CH2), 3.14(s, 4H, -CH2CH2), 2.80(s, 4H,
-CH2CH2). 13C NMR (100 MHz, DMSO-d6): 157.52, 152.88, 148.44, 148.21, 136.59,
134.91, 133.53, 132.49, 131.03, 130.45, 125.19, 125.05, 122.84, 117.79, 114.97,
107.00, 72.93, 48.60(2), 46.07(2). MS (ESI): 504.73 [M+H]+ (Calcd for 504.91,
C21H19ClN5O6S).
4.2.14.
2-((4-(Mesitylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one (7n)
White power, yield: 67%. Mp: 217-219 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.48(m, 1H, ArH), 7.38(d, J=7.92 Hz, 1H, ArH), 7.32(t,
J=7.52 Hz, 1H, ArH), 7.04(m, 2H, ArH), 5.06(s, 2H, CH2), 3.07(t, J=4.58 Hz, 4H,
-CH2CH2), 2.76(t, J=4.82 Hz, 4H, -CH2CH2), 2.71(s, 6H, 2CH3), 2.29(s, 3H, CH3).
13C NMR (100 MHz, CDCl3): 157.92, 152.88, 149.36, 144.13, 139.21(2), 135.92,
132.34, 130.50, 128.94(2), 124.54, 122.81, 117.59, 114.84, 108.02, 73.83, 49.08(2),
45.89(2), 21.48(2), 20.29. MS (ESI): 467.39 (C24H27N4O4S, [M+H]+). (Calcd for
467.56, C24H27N4O4S).
4.2.15.
2-((4-((2,4,6-Triisopropylphenyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyra
zol-4(2H)-one (7o)
White power, yield: 65%. Mp: 238-240 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.03(d, J=7.64 Hz, 1H, ArH), 7.48(t, J=7.70 Hz, 1H, ArH), 7.38(d, J=8.36 Hz,
1H, ArH), 7.31(t, J=7.48 Hz, 1H, ArH), 7.09(s, 2H, ArH), 5.12(s, 2H, CH2), 4.05(m,
2H, 2-CH), 3.24(s, 4H, -CH2CH2), 2.85(m, 1H, -CH), 2.71(s, 4H, -CH2CH2), 1.21(d,
J=6.96 Hz, 6H, 2CH3), 1.14(d, J=6.68 Hz, 12H, 4CH3). 13C NMR (100 MHz, CDCl3):
157.99, 153.45, 152.96, 151.76(2), 149.28, 132.33, 130.49, 129.47, 124.53, 123.96(2),
122.74, 117.63, 114.78, 107.96, 74.03, 49.48(2), 43.91(2), 34.16, 29.25(2), 24.80(4),
23.52(2). MS (ESI): 551.42 [M+H]+ (Calcd for 551.72, C30H39N4O4S).
4.2.16.
2-((4-((Trifluoromethyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)
-one (7p)

1
Light yellow power, yield: 41%. Mp: 167-169 ℃. H NMR (400 MHz, CDCl3):
8.20(s, 1H, ArH), 8.05(d, J=7.76 Hz, 1H, ArH), 7.50(t, J=7.64 Hz, 1H, ArH), 7.37(d,
J=8.56 Hz, 1H, ArH), 7.33(t, J=7.64 Hz, 1H, ArH), 5.10(s, 2H, -CH2), 2.78(t, J=4.18
Hz, 4H, -CH2CH2), 2.57(s, 4H, -CH2CH2). 13C NMR (100 MHz, CDCl3): 157.94,
152.93, 149.42, 132.31, 130.48, 124.52, 122.76, 119.94(q, J=274 Hz), 117.61, 114.78,
107.98, 73.82, 49.12(2), 45.86(2). MS (ESI): 417.28 [M+H]+ (Calcd for 417.38,
C16H16F3N4O4S).
4.2.17.
2-((4-(Methylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one (7q)
White power, yield: 59%. Mp: 191-194 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.04(d, J=7.88 Hz, 1H, ArH), 7.51(t, J=7.60 Hz, 1H, ArH), 7.38(d, J=8.52 Hz,
1H, ArH), 7.33(t, J=7.52 Hz, 1H, ArH), 5.10(s, 2H, -CH2), 2.97(s, 3H, CH3), 2.79(t,
J=4.22 Hz, 4H, -CH2CH2), 2.61(s, 4H, -CH2CH2). 13C NMR (100 MHz, CDCl3):
157.91, 152.89, 149.38, 132.33, 130.54, 124.56, 122.78, 117.63, 114.81, 107.94,
73.80, 49.08(2), 45.82(2), 38.22. MS (ESI): 363.32 [M+H]+ (Calcd for 363.40,
C16H19N4O4S).
4.2.18. 2-((4-(Ethylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one
(7r)
White power, yield: 52%. Mp: 201-203 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.50(t, J=7.68 Hz, 1H, ArH), 7.38(d, J=8.44 Hz, 1H, ArH),
7.32(t, J=7.58 Hz, 1H, ArH), 5.11(s, 2H, -CH2), 3.36(m, 2H, -CH2), 2.76(t, J=4.30 Hz,
13
4H, -CH2CH2), 2.57(d, J=4.12 Hz, 4H, -CH2CH2), 1.22(t, J=7.48 Hz, 3H, CH3). C
NMR (100 MHz, CDCl3): 157.92, 152.86, 149.41, 132.34, 130.51, 124.54, 122.81,
117.61, 114.82, 108.02, 73.78, 49.14, 49.08(2), 45.82(2), 6.94. MS (ESI): 377.29
[M+H]+ (Calcd for 377.43, C17H21N4O4S,).
4.2.19.
2-((4-(Propylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one (7s)
White power, yield: 56%. Mp: 208-210 ℃. 1H NMR (400 MHz, CDCl3): 8.21(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.51(t, J=7.42 Hz, 1H, ArH), 7.37(d, J=8.32 Hz, 1H, ArH),
7.33(t, J=7.46 Hz, 1H, ArH), 5.08(s, 2H, -CH2), 3.26(t, J=7.62 Hz, 2H, -CH2), 2.77(t,
J=4.34 Hz, 4H, -CH2CH2), 2.54(d, J=4.08 Hz, 4H, -CH2CH2), 2.12(m, 2H,
-CH2),1.01(t, J=7.48 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3): 157.96, 152.91,

149.42, 132.33, 130.57, 124.53, 122.84, 117.66, 114.80, 107.98, 73.79, 58.34,
49.11(2), 45.88(2), 14.53, 12.94. MS (ESI): 391.24 [M+H]+ (Calcd for 391.46,
C18H23N4O4S).
4.2.20. 2-((4-(Butylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one
(7t)
White power, yield: 51%. Mp: 211-213 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.04(m, 1H, ArH), 7.49(t, J=7.54 Hz, 1H, ArH), 7.38(d, J=8.40 Hz, 1H, ArH),
7.32(t, J=7.34 Hz, 1H, ArH), 5.09(s, 2H, -CH2), 3.29(t, J=7.16 Hz, 2H, -CH2), 2.79(t,
J=4.28 Hz, 4H, -CH2CH2), 2.55(d, J=4.16 Hz, 4H, -CH2CH2), 1.98(m, 2H, -CH2),
1.34(m, 2H, -CH2), 0.97(m, 3H, CH3). 13C NMR (100 MHz, CDCl3): 157.98, 152.94,
149.41, 132.32, 130.54, 124.54, 122.87, 117.61, 114.79, 107.97, 73.74, 55.93,
49.08(2), 45.82(2), 20.19, 19.86, 11.89. MS (ESI): 405.26 [M+H]+ (Calcd for 405.49,
C19H25N4O4S).
4.2.21.
2-((4-(Cyclopropylsulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-one
(7u)
White power, yield: 54%. Mp: 194-196 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.51(t, J=7.48 Hz, 1H, ArH), 7.37(d, J=8.36 Hz, 1H, ArH),
7.34(t, J=7.72 Hz, 1H, ArH), 5.11(s, 2H, -CH2), 3.34(m, 2H, -CH2), 2.81(t, J=4.12 Hz,
4H, -CH2CH2), 2.57(d, J=4.08 Hz, 4H, -CH2CH2), 1.94(m, 4H, 2-CH2). 13C NMR
(100 MHz, CDCl3): 158.01, 152.98, 149.38, 132.34, 130.51, 124.59, 122.82, 117.58,
114.74, 107.99, 73.82, 49.12(2), 45.87(2), 36.15, 3.87(2). MS (ESI): 389.31 [M+H]+
(Calcd for 389.44, C18H21N4O4S).
4.2.22.
2-((4-((2-Chloroethyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-
one (7v)
White power, yield: 49%. Mp: 187-189 ℃. 1H NMR (400 MHz, CDCl3): 8.19(s, 1H,
ArH), 8.05(m, 1H, ArH), 7.50(t, J=7.56 Hz, 1H, ArH), 7.38(d, J=8.24 Hz, 1H, ArH),
7.32(t, J=7.86 Hz, 1H, ArH), 5.10(s, 2H, -CH2), 3.47(t, J=7.86 Hz, 2H, -CH2), 3.38(t,
J=7.78 Hz, 4H, 2-CH2), 2.79(t, J=4.18 Hz, 4H, -CH2CH2), 2.58(d, J=4.10 Hz, 4H,
-CH2CH2). 13C NMR (100 MHz, CDCl3): 157.98, 152.91, 149.42, 132.31, 130.53,
124.56, 122.81, 117.54, 114.76, 107.96, 73.81, 59.11, 49.10(2), 45.83(2), 31.82. MS

(ESI): 411.81 [M+H]+ (Calcd for 411.87, C17H20ClN4O4S).
4.2.23.
2-((4-((3-Chloropropyl)sulfonyl)piperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)
-one (7w)
White power, yield: 53%. Mp: 189-192 ℃. 1H NMR (400 MHz, CDCl3): 8.20(s, 1H,
ArH), 8.04(m, 1H, ArH), 7.49(t, J=7.44 Hz, 1H, ArH), 7.38(d, J=8.36 Hz, 1H, ArH),
7.33(t, J=7.74 Hz, 1H, ArH), 5.09(s, 2H, -CH2), 3.42(t, J=7.70 Hz, 2H, -CH2), 3.36(t,
J=7.46 Hz, 4H, 2-CH2), 2.78(t, J=4.34 Hz, 4H, -CH2CH2), 2.55(d, J=4.24 Hz, 4H,
-CH2CH2), 2.11(m, 2H, -CH2). 13C NMR (100 MHz, CDCl3): 157.94, 152.89, 149.36,
132.36, 130.55, 124.52, 122.81, 117.58, 114.81, 107.99, 73.87, 54.31, 49.08(2),
45.80(2), 41.89, 21.04. MS (ESI): 425.79 (C18H22ClN4O4S, [M+H]+). (Calcd for
425.90, C18H22ClN4O4S).
4.3. Cell proliferation assay
Four human cell lines HCT-116, Huh7, HL60 and A549 were cultured growth
medium (RPMI-1640 medium with 10% FBS, 100 U/mL penicillin and 100 mg/mL
streptomycin, and incubated at 37 °C under the atmosphere of 5% CO2 and 20% O2.
The antiproliferative activity in vitro was determined by the MTT assay. The
tested compounds were dissolved in DMSO and diluted to the different concentrations.
Cells in logarithmic phase were harvested and divided into 96-well plates (0.5×104
each well), culture medium containing the test compounds were added to each well at
different concentrations and incubated for 48 h. Viable cells were examined using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT, Sigma)
according to the manufacturer’s instructions. Triplicate wells were used for each
concentration and each assay was carried out at least three times. The cytotoxic
activity was expressed as the IC50 values.
4.4. PI3K enzyme assay
The inhibition of PI3K activity was examined by competitive fluorescence
polarization kinase activity assay as our previous research described [24]. Briefly, 10
µL PI3K (Perkin-Elmer) reactions were performed in pH 7, 2.5 mM MgCl2, 5 mM
HEPES, 50 mM ATP and 10 mM DTT using diC8-PI(4,5)P2 (Echelon Biosciences) as
the substrate. Firstly, tested compounds (ranging from 3.2 nM to 1 mM) were added
into per 10 µL reaction volume (prepared with 50 ng of enzyme and 10 mM of
substrate). After incubating for 3 h at room temperature, a chelator was added to

quench the reactions. And then a mixture of phosphoinositide binding protein was
added and mixed, subsequently, a fluorophorelabeled phosphoinositide tracer was
added. Samples were incubated in the dark for 1 h to equilibrate after mixing in
384-well black Corning nonbinding plates. Finally, polarization values were recorded
using red fluorophores with appropriate filters to determine the extent of enzyme
activity in the reaction.
4.5. Apoptosis analysis
6×105 HCT-116 cells in exponential growth was averagely seeded into each well
of 6-well plate, and treated with compound 7m at different concentrations for 24 h,
the cells were collected, followed by centrifuged and resuspended in 500 ml
AnnexinV binding buffer, and incubated for 15 min in the darkness under the ice-bath.
Samples were analyzed using a FACS Calibur flow cytometer (Bectone Dickinson,
San Jose, CA, USA).
4.6. Western blotting
HCT-116 cells were incubated in the presence of 7m for 24 h. Subsequently,
trypsinized the cells and collected, the prepared 1 × RIPA lysis buffer (1% NP-40,50
mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.25% deoxycholic acid, 1 mM EDTA
containing protease inhibitors PMSF) (Amresco, Solon, USA) was added to extract
the total proteins. The proteins was separated by sodium dodecyl sulfate (8% or 10%)
polyacrylamide gel electrophoresis (SDS-PAGE, BioRad Laboratories, Hercules, CA),
and transferred from the gel onto to PVDF membrane (BioRad Laboratories, Hercules,
CA), blotted with primary antibodies, probed with secondary isotype specific
antibodies tagged with horseradish peroxidase (Cell Signaling Technology). Bound
immunocomplexes were detected using a ChemiDOC™ XRS+system (BioRad
Laboratories, Hercules, CA).
4.7. Molecular docking
Docking study was performed by Discovery Studio 3.5 and the PI3Kα protein
(PDB:3HHM) was downloaded from RCSB Protein Date Bank (www.rcsb.org). The
protein and all ligands were prepared by minimization with CHARMM force field.
Molecular docking was carried out using DS-CDOCKER protocol without constraint,
all bound water and ligands were eliminated from the protein and the polar hydrogen
was added to the proteins.

4.8. Antitumor effect in vivo
A mouse tumor xenograft model was established by injecting the HCT-116 cells
(5 ×106 cells per animal) into the right flank of nude mice. After the tumor size reach
approximately 100 mm3, the mice were randomly divided into three groups (6 mice
per group), they were respectively treated with 5% CMC-Na (control), LY294002 (10
mg/kg, dissolved in 5% CMC-Na) and compound 7m (10 mg/kg, dissolved in 5%
CMC-Na) by intragastric administration every three days in three weeks. The body
weight and tumor size were recorded every three days, the tumor volumes were
calculated by a formula: tumor volume (mm3)ꢀ= 0.5 × length × width2. After treatment
for three weeks, the mice were sacrificed and the tumors were separated and
weighted.
Acknowledgments
This work was supported by National Natural Science Foundation of China (No.
81602985 and 21202153), the Fundamental Research Funds for the Central
Universities (2632018ZD19), It was also supported by Technology research Funds
Project of Ocean and financed by National Natural Science Foundation of China (No.
21602103).
References
[1] L.C. Cantley, The phosphoinositide 3-kinase pathway, Science 296 (2002)
1655-1657.
[2] V. Igor, C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human
cancer, Nat Rev Cancer 2 (2002) 489-501.
[3] F. Janku, T.A. Yap, F. Mericbernstam, Targeting the PI3K pathway in cancer: are
we making headway?, Nature Reviews Clinical Oncology 15 (2018) 273-291.
[4] C.L. Carpenter, L.C. Cantley, Phosphoinositide kinases, Biochemistry-Us 29
(1990) 11147-11156.
[5] J.E. Burke, R.L. Williams, Synergy in activating class I PI3Ks, Trends Biochem.
Sci. 40 (2015) 88-100.
[6] P. Liu, H. Cheng, T.M. Roberts, J.J. Zhao, Targeting the phosphoinositide 3-kinase
pathway in cancer, Nat. Rev. Drug Discov. 8 (2009) 627-644.
[7] W. Zhao, Y. Qiu, D. Kong, Class I phosphatidylinositol 3-kinase inhibitors for
cancer therapy, Acta Pharmacol. Sin. B 7 (2017) 27-37.
[8] M.P. Wymann, L. Pirola, Structure and function of phosphoinositide 3-kinases,

Biochim Biophys Acta 1436 (1998) 127-150.
[9] F.I. Raynaud, S. Eccles, P.A. Clarke, A. Hayes, B. Nutley, S. Alix, A. Henley, F.
DiStefano, Z. Ahmad, S. Guillard, L.M. Bjerke, L. Kelland, M. Valenti, L. Patterson,
S. Gowan, A.d.H. Brandon, M. Hayakawa, H. Kaizawa, T. Koizumi, T. Ohishi, S.
Patel, N. Saghir, P. Parker, M. Waterfield, P. Workman, Pharmacologic
characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases, Canc.
Res. 67 (2007) 5840-5850.
[10] P. Workman, P.A. Clarke, F.I. Raynaud, R.L.M. van Montfort, Drugging the
PI3kinome: from chemical tools to drugs in the clinic, Canc. Res. 70 (2010)
2146-2157.
[11] M.P. Czech, PIP2 and PIP3: complex roles at the cell surface, Cell 100 (2000)
603-606.
[12] C.-H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V.E. Velculescu,
K.W. Kinzler, B. Vogelstein, S.B. Gabelli, L.M. Amzel, The structure of a human
p110 alpha/p85 alpha complex elucidates the effects of oncogenic PI3K alpha
mutations, Science 318 (2007) 1744-1748.
[13] N.R. Leslie, C.P. Downes, PTEN function: how normal cells control it and tumor
cells lose it, Biochem. J 382 (2004) 1-11.
[14] N. Chalhoub, S.J. Baker, PTEN and the PI3-kinase pathway in cancer, Annu. Rev.
Pathol. 4 (2009) 127-150.
[15] Y. Liu, W.Z. Wan, Y. Li, G.L. Zhou, X.G. Liu, Recent development of
ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as
anticancer agents, Oncotarget 8 (2016) 7181-7200.
[16] K.D. Courtney, R.B. Corcoran, J.A. Engelman, The PI3K pathway as drug target
in human cancer, J. Clin. Oncol 28 (2010) 1075-1083.
[17] I.G. Campbell, S.E. Russell, D.Y.H. Choong, K.G. Montgomery, M.L. Ciavarella,
C.S.F. Hooi, B.E. Cristiano, R.B. Pearson, W.A. Phillips, Mutation of the PIK3CA
gene in ovarian and breast cancer, Cancer Res 64 (2004) 7678-7681.
[18] Y. Samuels, Z.H. Wang, A. Bardelli, N. Silliman, J. Ptak, S. Szabo, H. Yan, A.
Gazdar, D.M. Powell, G.J. Riggins, J.K.V. Willson, S. Markowitz, K.W. Kinzler, B.
Vogelstein, V.E. Velculescu, High frequency of mutations of the PIK3CA gene in
human cancers, Science 304 (2004) 554-554.
[19] A. E Garces, M. Stocks, Class 1 PI3K Clinical Candidates and Recent Inhibitor
Design Strategies: A Medicinal Chemistry Perspective, J Med Chem 62 (2018)
4815-4850

[20] M. Falasca, J. Hamilton, M. Selvadurai, K. Sundaram, A. Adamska, P. Thompson,
Class II Phosphoinositide 3-Kinases as Novel Drug Targets, J Med Chem 60 (2017)
47-65.
[21] V. Asati, D.K. Mahapatra, S.K. Bharti, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK
signaling pathways inhibitors as anticancer agents: Structural and pharmacological
perspectives, Eur J Med Chem 109 (2016) 314-341.
[22] T. Jian-Feng, W. Qing, S. Jian, Z. Wei-Ming, Z. Hai-Liang, Advances in the
researches on the biological activities and inhibitors of phosphatidylinositol 3-kinase,
Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry
- Anti-Cancer Agents) 14 (2014) 673-687.
[23] Y. Yong, W. Xun, H. Hong-Wei, S. Shao, W. She-Feng, Q. Fang, L. Ai-Min, L.
Peng-Cheng, Z. Hai-Liang, Discovery and synthesis of a novel series of potent,
selective inhibitors of the PI3Kα: 2-alkyl-chromeno[4,3-c]pyrazol-4(2H)-one
derivatives, Organic & Biomolecular Chemistry 12 (2014) 9157-9165.
[24] Y. Yin, J. Hu, X. Wu, S. Sha, S. Wang, F. Qiao, Z. Song, H. Zhu, Design,
synthesis and biological evaluation of novel chromeno[4,3-c]pyrazol-4(2H)-one
derivates containing sulfonamido as potential PI3Kα inhibitors, Bioorg Med Chem
11(2019) 2261-2267.
[25] L. Lu, S. Sha, K. Wang, Y.H. Zhang, Y.D. Liu, G.D. Ju, B. Wang, H.L. Zhu,
Discovery of chromeno[4,3-c]pyrazol-4(2H)-one containing carbonyl or oxime
derivatives as potential, selective inhibitors PI3Kα, Chem Pharm Bull 64 (2016)
1576-1581.
[26] A. Arcaro, M. Wymann, Wortmannin is a potent phosphatidylinositol 3-kinase
inhibitor: The role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses,
Biochem J 296 (1994) 297-301.
[27] G. Powis, R. Bonjouklian, M.M. Berggren, A. Gallegos, R. Abraham, C.
Ashendel, L. Zalkow, W. F Matter, J. Dodge, G. Grindey, Wortmannin, a potent and
selective inhibitor of phosphatidylinositol-3-kinase, Cancer Research 54 (1994)
2419-2423
[28] S. Shuho, I. Nanami, I. Masafumi, H. Masaru, Y. Mitsunori, The in vitro and in
vivo effects of 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), a specific
inhibitor of phosphatidylinositol 3'-kinase, in human colon cancer cells, Clinical
Cancer Research An Official Journal of the American Association for Cancer
Research 8 (2002) 1957-1963.
[29] A.J. Folkes, K. Ahmadi, W.K. Alderton, S. Alix, S.J. Baker, G. Box, I.S.

Chuckowree, P.A. Clarke, P. Depledge, S.A. Eccles, L.S. Friedman, A. Hayes, T.C.
Hancox, A. Kugendradas, L. Lensun, P. Moore, A.G. Olivero, J. Pang, S. Patel, G.H.
Pergl-Wilson, F.I. Raynaud, A. Robson, N. Saghir, L. Salphati, S. Sohal, M.H. Ultsch,
M. Valenti, H.J.A. Wallweber, N.C. Wan, C. Wiesmann, P. Workman, A. Zhyvoloup,
M.J.
Zvelebil,
S.J.
Shuttleworth,
The
Identification
of
2-(1H-Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-th
ieno[3,2-d]pyrimidine (GDC-0941) as a Potent, Selective, Orally Bioavailable
Inhibitor of Class I PI3 Kinase for the Treatment of Cancer, J Med Chem 51 (2008)
5522-5532.
[30] S. Maira, F. Stauffer, J. Brueggen, P. Furet, C. Schnell, C. Fritsch, S. Brachmann,
P. Chene, A. De Pover, K. Schoemaker, D. Fabbro, D. Gabriel, M. Götte, L. Murphy, P.
Finan, W. Sellers, C. García-Echeverría, Identification and characterization of
NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian
target of rapamycin inhibitor with potent in vivo antitumor activity, Mol Cancer Ther
7 (2008) 1851-1863.
[31] A. Bilancio, B. Rinaldi, M. Antonietta Oliviero, M. Donniacuo, M. Monti, A.
Boscaino, I. Marino, L. Friedman, F. Rossi, B. Vanhaesebroeck, A. Migliaccio,
Inhibition of p110? PI3K prevents inflammatory response and restenosis after artery
injury, Bioscience Reports, 37 (2017) BSR20171112.
[32] C. Garcia-Echeverria, W. Sellers, Drug design approaches targeting the PI3K/Akt
pathway in cancer, Oncogene 27(2008) 5511-5526.
[33] O. Mirzoeva, B. Hann, Y. K Hom, J. Debnath, D. Aftab, K. Shokat, W. Michael
Korn, Autophagy suppression promotes apoptotic cell death in response to inhibition
of the PI3K-mTOR pathway in pancreatic adenocarcinoma, Journal of Molecular
Medicine 89 (2011) 877-889.
[34] J. R. Shroff, N4-Substituted N1-tolysulfonylpiperazines, J.med.chem, 11 (1968)
196-197.

Captions
Scheme 1. Synthesis of compounds 7a~7w. Reagents and conditions: (i) DCM/TEA,
0 ℃, 2-4 h. (ii) formaldehyde, EtOH, r.t., 4-6 h.
Table 1. Antiproliferative activities in vitro of target compounds 7a-7w against four
cancer cell lines.
Table 2. Enzymatic activities of selected compounds against PI3K and their
cytotoxicity on 293T cell line.
Fig. 1. Various PI3K inhibitors and the design compounds.
Fig. 2. Effects of compound 7m on the Akt phosphorylation and S6K phosphorylation.
HCT-116 cells were treated with compound 7m for 24 h, total protein was extracted
and subjected to Western blot analysis, β-actin was used as an internal control. Bar
graphs represent the expression level of Akt, p-Akt and p-S6K proteins. Data were
expressed as Mean±SEM (n = 3). ***p < 0.005 vs. the control.
Fig. 3. Compound 7m induced cell cycle arrest in the HCT-116 cell. Cells were
treated with 0.25, 0.5 and 1 µM of 7m for 24 h. Cell cycle analysis was performed via
PI fluorescence and flow cytometer. Data were expressed as Mean ± SEM (n=3). *p <
0.05 vs. the control.
Fig. 4. Induced apoptotic effect of compound 7m on HCT-116 cell. (A) Apoptotic
assay by flow cytometry. HCT-116 cells were treated with compound 7m at 0, 0.25,
0.5, and 1 µM for 24 h. The cells were stained with Annexin V-FITC/PI, then detected
by a flow cytometer. (B) Effects of compound 7m on Bax, Bcl-2, Cleaved caspase 3
and Cleaved caspase 9 expression. Cells were treated with compound 7m for 24 h,
then total protein was extracted and subjected to Western blot analysis, β-actin was
used as an internal control. Bar graphs represent the expression level of Caspase
proteins. Data were expressed as Means±SEM (n =3). *p < 0.05, **p < 0.01 and ***p
< 0.005 vs. the control.
Fig. 5. (A)Molecular docking model of compound 7m with 3HHM. (B) 3D
conformation position of the compound 7m in the binding pocket.
Fig. 6. Compound 7m inhibited the growth of HCT-116 xenograft tumors in vivo. (A)
Picture of dissected nude mice's tumor tissues untreated or treated with LY294002 (10
mg/kg) and 7m (10 mg/kg). (B) The growing curves of mice's body weight. (C) The
growing curves of mice's tumor volume. (D) Tumor weight results, data were
expressed as Means ±SD (n=6). *p < 0.05 and **p < 0.01 vs. the control.

Compounds
R1
H
Compounds
R1
3-NO2
7a
7i
7j
4-CH3
4-OCH3
4-C(CH3)3
4-F
2-NO2
7b
4-CF3
7c
7k
7l
2,5-CH3
7d
4-Cl-3-NO2
2,4,6-CH3
2,4,6-CH(CH3)2
7e
7m
7n
7o
4-Cl
7f
4-Br
7g
4-NO2
R2
7h
Compounds
Compounds
R2
CF3
CH2CH2CH2CH3
cyclopropane
CH2CH2Cl
7p
7q
7r
7s
7t
7u
7v
7w
CH3
CH2CH3
CH2CH2CH3
CH2CH2CH2Cl
Scheme 1. Synthesis of compounds 7a~7w. Reagents and conditions: (i) DCM/TEA,
0 ℃, 2-4 h. (ii) formaldehyde, EtOH, r.t., 4-6 h.

Table 1. Antiproliferative activities in vitro of target compounds 7a-7w against four
cancer cell lines.
IC50±SD a(µM)
compound
HCT-116
7.53±0.68
11.07±1.23
12.14±1.31
0.47±0.04
2.45±0.19
0.81±0.07
1.89±0.15
0.92±0.08
1.29±0.11
4.87±0.37
0.84±0.06
0.13±0.01
0.03±0.002
0.09±0.008
0.08±0.006
>100
A549
Huh7
HL60
13.91±1.24
21.52±2.38
27.36±3.05
0.74±0.05
5.11±0.42
1.21±0.11
3.09±0.31
2.10±0.23
3.67±0.31
7.66±0.77
1.15±0.10
0.48±0.05
0.06±0.005
0.11±0.01
0.09±0.006
>100
11.35±1.04
28.52±3.09
34.02±2.95
0.90±0.07
6.44±0.52
1.61±0.17
5.08±0.51
2.82±0.29
3.65±0.33
7.92±0.78
2.34±0.13
0.66±0.04
0.09±0.007
0.30±0.02
0.14±0.01
>100
22.07±1.91
16.92±1.48
24.51±2.14
0.84±0.07
6.33±0.56
0.64±0.06
4.78±0.43
2.67±0.21
2.01±0.19
8.23±0.78
1.10±0.10
0.22±0.01
0.06±0.005
0.11±0.01
0.09±0.008
>100
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
>100
>100
97.13±10.06
>100
>100
7q
>100
>100
>100
7r
57.64±5.23
87.93±9.01
69.34±7.22
45.77±4.87
38.04±4.12
51.82±4.58
79.71±8.83
>100
87.64±9.17
>100
89.38±9.29
>100
7s
7t
82.01±8.91
68.74±7.23
52.09±5.14
82.32±7.26
78.99±8.63
62.85±6.78
48.51±4.53
67.18±5.64
71.42±7.28
78.58±6.87
55.10±6.33
18.43±2.03
7u
7v
7w
LY294002
0.24±0.012
0.38±0.017
0.53±0.022
0.91±0.038
BEZ235
a
Values are average of three independent experimental measurements and expressed as Mean ±
SD.
Table 2. Enzymatic activities of selected compounds against PI3K and their
cytotoxicity on 293T cell line.
IC50±SD a(µM)
compound
CC50±SD b(µM)
PI3Kα
PI3Kβ
2.65±0.32
>10
PI3Kγ
PI3Kδ
mTOR
>10
7d
7e
0.95±0.061
8.91±0.78
1.32±0.12
7.51±0.73
0.75±0.078
9.76±0.89
231.42±18.94
298.13±22.17
9.12±0.46

1.55±0.13
8.30±0.76
2.63±0.21
6.18±0.54
>10
2.19±0.21
9.71±0.87
4.14±0.43
>10
2.65±0.21
>10
4.72±0.47
9.31±0.91
5.45±0.52
>10
>10
>10
277.62±26.46
387.43±31.36
346.58±29.74
412.08±37.81
372.38±39.35
334.77±36.27
397.15±40.12
386.07±32.44
354.24±34.81
331.53±27.56
-
7f
7g
7h
7i
4.01±0.38
9.34±0.81
8.42±0.78
2.30±0.22
>10
>10
9.14±0.89
5.75±0.56
6.15±0.54
7.51±0.73
1.42±0.12
8.37±0.74
8.12±0.43
>10
7j
7k
7l
3.84±0.31
0.30±0.022 0.85±0.079 0.81±0.075
7m
7n
7o
0.009±0.001 0.12±0.010 0.081±0.006 0.12±0.011
0.12±0.011 0.41±0.031 0.14±0.012 0.23±0.018
0.042±0.003 0.21±0.015 0.11±0.009 0.42±0.039
9.38±0.86
7.44
>10
0.98±0.012 0.95±0.082
1.36±0.13
>10
LY294002 0.48±0.07
BEZ235 0.011±0.002 0.064±0.004 0.027±0.003 0.021±0.003 0.012±0.001
-
a
Values are average of three independent experimental measurements and expressed as Mean ±
SD.
b
Cytotoxicity on 293T cell line, Values are average of three independent experimental
measurements and expressed as Mean ± SD.

Fig. 1. Various PI3K inhibitors and the design compounds.
Fig. 2. Effects of compound 7m on the Akt phosphorylation and S6K phosphorylation.
HCT-116 cells were treated with compound 7m for 24 h, total protein was extracted
and subjected to Western blot analysis, β-actin was used as an internal control. Bar
graphs represent the expression level of Akt, p-Akt and p-S6K proteins. Data were
expressed as Mean±SEM (n = 3). ***p < 0.005 vs. the control.

Fig. 3. Compound 7m induced cell cycle arrest in the HCT-116 cell. Cells were
treated with 0.25, 0.5 and 1 µM of 7m for 24 h. Cell cycle analysis was performed via
PI fluorescence and flow cytometer. Data were expressed as Mean ± SEM (n=3). *p <
0.05 vs. the control.

Fig. 4. Induced apoptotic effect of compound 7m on HCT-116 cell. (A) Apoptotic
assay by flow cytometry. HCT-116 cells were treated with compound 7m at 0, 0.25,
0.5, and 1 µM for 24 h. The cells were stained with Annexin V-FITC/PI, then detected
by a flow cytometer. (B) Effects of compound 7m on Bax, Bcl-2, Cleaved caspase 3
and Cleaved caspase 9 expression. Cells were treated with compound 7m for 24 h,
then total protein was extracted and subjected to Western blot analysis, β-actin was
used as an internal control. Bar graphs represent the expression level of Caspase
proteins. Data were expressed as Means±SEM (n =3). *p < 0.05, **p < 0.01 and ***p
< 0.005 vs. the control.

Fig. 5. (A)Molecular docking model of compound 7m with 3HHM. (B) 3D
conformation position of the compound 7m in the binding pocket.