Design, synthesis and biological evaluation of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates containing sulfonamido as potential PI3Kα inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.04.021

A series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained sulfonamido were designed and synthesized, and their anticancer effects in vitro was evaluated to develop some new PI3Kα inhibitors. Most of desired compounds exhibited the better antiproliferative activities against four cancer cell lines than that of LY294002. Out of them, compound 4o displayed the potent antiproliferative activity and high selectivity against the PI3Kα protein and it can induce apoptosis of HCT116 in a dose-dependent manner. Western blot assay indicated that compound 4o obviously down-regulated expression of p-Akt (S473). Molecular docking was performed to clarify the possible binding mode between compound 4o and PI3Kα. All these results indicated that compound 4o could be a potential inhibitor of PI3Kα.

Full text of DOI:10.1016/j.bmc.2019.04.021

Accepted Manuscript
Design, synthesis and biological evaluation of novel chromeno[4,3-c]pyra-
zol-4(2H)-one derivates containing sulfonamido as potential PI3Kα inhibitors
Yong Yin, Jia-Qin Hu, Xu Wu, Shao Sha, She-Feng Wang, Fang Qiao, Zhong-
Cheng Song, Hai-Liang Zhu
PII:
S0968-0896(19)30423-7
https://doi.org/10.1016/j.bmc.2019.04.021
BMC 14875
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 March 2019
10 April 2019
14 April 2019
Please cite this article as: Yin, Y., Hu, J-Q., Wu, X., Sha, S., Wang, S-F., Qiao, F., Song, Z-C., Zhu, H-L., Design,
synthesis and biological evaluation of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates containing sulfonamido
as potential PI3Kα inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.
2019.04.021
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Design, synthesis and biological evaluation of novel
chromeno[4,3-c]pyrazol-4(2H)-one derivates containing sulfonamido
as potential PI3Kα inhibitors
Yong Yina,b,†, Jia-Qin Hub,d,†, Xu Wub, Shao Shab, She-Feng Wangb, Fang Qiaob,
Zhong-Cheng Song*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, People’ s Republic of
China.
d The Joint Research Centre of Gene Interference, Guangzhou University and Keele
University for Gene Interference and Application, School of Life Science, Guangzhou
University, 230 Waihuan West Road, Guangzhou 510006, People’ s Republic of
China.
† Both authors contributed equally to this work.
* 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
A series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained
sulfonamido were designed and synthesized, and their anticancer effects in vitro was
evaluated to develop some new PI3Kα inhibitors. Most of desired compounds
exhibited the better antiproliferative activities against four cancer cell lines than that

of LY294002. Out of them, compound 4o displayed the potent antiproliferative
activity and high selectivity against the PI3Kα protein and it can induce apoptosis of
HCT116 in a dose-dependent manner. Western blot assay indicated that compound 4o
obviously down-regulated expression of p-Akt (S473). Molecular docking was
performed to clarify the possible binding mode between compound 4o and PI3Kα. All
these results indicated that compound 4o could be a potential inhibitor of PI3Kα.
Keywords: chromeno[4,3-c]pyrazol-4(2H)-one derivates, anticancer, PI3Kα,
Molecular docking
1. Introduction
PI3K/Akt/mTOR signaling pathway is an important component in cellular
signaling pathways and it plays a critical role in occurrence, development, treatment
and transformation of tumors.1-3 As an upstream molecule, PI3K (phosphatidylinositol
3-kinases) involved in many cellular functions such as cell growth, survival,
proliferation, motility, differentiation and intracellular trafficking.4-9
As a family of lipid kinases, based on the sequence homology and substrate
specificity, PI3Ks are categorized into four different classes: class I, class II, class III
and Class IV. In particular, the class I is the most common studied and it is divided
into PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, which are heterodimers composed of a p110
catalytic subunit and a p85 regulatory subunit.10-13 Of these, PI3Ks α and β are
ubiquitously expressed, whereas the expression of δ and γ is more restricted, mostly to
leukocytes.14-17 The main function of class I PI3Ks is to phosphorylate
phosphatidylinositol at the 3-position hydroxyl group of the inositol ring, and the
important second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) which
regulate
many
cellular
functions
is
converted
from
4,5-phosphatidylinositolbisphosphate (PIP2) by phosphorylation. Meanwhile, the
process is strictly controlled and the high PIP3 levels resulting in negatively regulate
by phosphatase and tensin homologue (PTEN), which catalyzes the
dephosphorylation of PIP3 back to PIP2.18-21 The abnormalities which show in

functions of both the phosphatase and kinase are commonly discovered in tumors,
thus the PI3K pathway plays an important role in cancer and it becomes one of the
most promising target for cancer treatment. 22-24
In the past decades, many PI3K inhibitors have been discovered and identified,
such as Wortmannin, LY294002, BEZ235, GDC0941 and so on.25-27 Out of them,
Wortmannin and LY294002 were recognized as the first-generation PI3K inhibitors.
In our previous research, chromeno[4,3-c]pyrazol-4(2H)-one has been designed as the
so-called core region which based on the structure of these PI3K inhibitors, and as we
expected, chromeno[4,3-c]pyrazol-4(2H)-one derivates exhibited the potent
antiproliferative activity against cancer cell lines.28 Meanwhile, sulfonamido fragment
was observed in some PI3K inhibitors such as GDC0941, XL147, CH5132799,
GSK2126458, PIK75 and so on,27,29-32 sulfonamido fragment is used to employ in
drug design and it is a common strategy for structural modification. So it may be a
valid method that sulfonamido fragment is employed to optimize the
chromeno[4,3-c]pyrazol-4(2H)-one derivates (Figure 1).
In this study,
a
series of novel chromeno[4,3-c]pyrazol-4(2H)-one
Figure 1. Design novel 2-alkyl-chromeno[4,3-c]pyrazol-4(2H)-one containing sulfonyl
derivatives.
derivates contained sulfonamido were designed and synthesized, and their anticancer
effects in vitro was evaluated to develop some new PI3K inhibitors.

2. Results and discussion
2.1. Chemistry
The desired compounds (4a-4w) was synthesized followed the general pathway
outlined in Scheme 1. Chromeno[4,3-c]pyrazol-4(2H)-one was prepared according to
the previous research.28 Firstly, 3-formyl-4-chlorocoumarin (2) was prepared by
Vilsmeier-Haack reaction (POCl3/DMF) form 4-hydroxycoumarin (1). Subsequently,
cyclization reaction
using
85%
hydrazine
hydrate
to
obtain
chromeno[4,3-c]pyrazol-4(2H)-one (3) in high yields (87%). Lastly,
chromeno[4,3-c]pyrazol-4(2H)-one (3) and various sulfonyl chloride were reacted to
obtain the targeted compounds (4a–4w).
compound
R1
H
compound
R1
3-NO2
4a
4i
4j
4-CH3
4-OCH3
4-C(CH3)3
4-F
4-NO2
4b
4-CF3
4c
4k
4l
2,5-CH3
4d
4-Cl-3-NO2
2,4,6-CH3
2,4,6-CH(CH3)2
4e
4m
4n
4o
4-Cl
4f
4g
4-Br
2-NO2
R2
4h
compound
4p
compound
R2
CF3
CH2CH2CH2CH3
4t

CH3
cyclopropane
CH2CH2Cl
4q
4r
4s
4u
4v
CH2CH3
CH2CH2CH3
CH2CH2CH2Cl
4w
Scheme 1. Synthesis of compounds 4a-4w. Reagents and conditions: (i) DMF, POCl3, 60 °C, 6 h.
(ii) Ethanol, Et3N, 85% hydrazine hydrate, r.t.,14 h. (iii) benzenesulfonyl chloride/ alkylsulfonyl
chloride, DCM/TEA, 0 ℃, 6-8 h.
2.2. Antiproliferative activity
All the desired compounds (4a-4w) were evaluated for the anticancer activity in
vitro against four cell lines: A549, HCT116, HL60 and Huh7. The results are
summarized in Table 1. The results indicated that most desired compounds exhibit the
potent antiproliferative activity against the four cancer cell lines and they were better
than the positive control LY294002. In addition, the compounds containing the
phenylsulfonyl displayed the antiproliferative activity were superior to that of
compounds containing alkylsulfonyl. Out of them, compound 4o exhibited the most
potent antiproliferative activity against four cancer cell lines (IC50 = 0.07, 0.12, 0.09,
and 0.09 μM for HCT116, A549, Huh7 and HL60, respectively).
Table 1. Antiproliferative activities in vitro of target compounds 4a-4w against four cancer cell
lines.
IC50 ± SD(μM)
compound
HCT116
42.64±4.12
8.03±0.73
6.18±0.58
6.74±0.61
32.46±2.84
27.40±2.11
19.08±1.78
11.50±1.02
4.11±0.37
1.04±0.11
2.64±0.27
0.36±0.24
0.12±0.01
0.08±0.01
0.07±0.003
A549
Huh7
HL60
4a
4b
4c
4d
4e
4f
60.31±5.58
9.78±9.07
9.30±8.62
7.75±6.86
49.85±4.21
32.04±2.86
21.99±1.95
13.65±1.17
4.45±3.88
0.97±0.08
1.90±0.14
0.35±0.03
0.48±0.04
0.17±0.01
0.12±0.01
58.14±4.82
14.68±1.23
11.12±1.03
11.37±0.98
39.29±3.41
47.06±4.34
32.88±2.73
10.24±1.09
3.21±0.25
1.49±0.17
3.47±0.26
0.19±0.02
0.19±0.02
0.12±0.01
0.09±0.01
50.13±4.49
11.65±1.07
7.65±7.93
9.02±9.27
21.44±1.99
38.73±4.03
44.15±4.18
14.01±1.26
4.22±0.38
5.03±0.44
3.98±0.36
0.65±0.04
0.54±0.05
0.12±0.01
0.09±0.01
4g
4h
4i
4j
4k
4l
4m
4n
4o

4p
>100
>100
>100
>100
>100
89.64±8.15
>100±
>100
92.08±8.83
87.19±8.14
>100
4q
4r
>100
>100
4s
82.14±7.94
67.11±6.13
97.42±9.43
>100
89.45±8.28
78.66±7.23
>100
76.83±6.74
92.34±8.31
>100
4t
4u
>100
>100
4v
>100
>100
74.68±6.71
>100
4w
83.94±7.87
51.82 ±4.58
>100
96.44±8.97
67.18 ±5.64
LY294002
82.32 ±7.26
18.43 ±2.03
As showed in Table 1, the compounds containing phenylsulfonyl showed the
most antiproliferative activity, and there was a significant improvement in comparison
with LY294002. In particular, compound 4o exhibited an 200-740 fold improvement
versus the positive control LY294002 in the inhibition of four cancer cells
proliferation. It demonstrated that phenylsulfonyl fragment in desired compounds play
a critical role in the antiproliferative effect.
Among the compound 4a-4o, it was observed that the compounds with
trisubstituted benzene ring showed the interesting activities, such as compounds 4n
and 4o. What’s more, comparing the mono-substituted groups on the benzene ring, the
compounds containing the double substituent on the benzene ring displayed the better
antiproliferative activities. It was indicated that the more substituent on the phenyl
group, the better activity it exhibited. In addition, the compounds with
mono-substituted benzene ring exhibited the antiproliferative activities in the order of
electro-withdrawing substituents >electro-donating substituents >halogen substituents,
and the substituted position on the benzene ring exhibited the antiproliferative
activities in the order of para > meta > ortho. For the mono-substituted benzene ring,
electronic effects played relevant roles in the antiproliferative profiles, derivatives
with positive Hammett σ constants, displayed the best profiles, for example, σp=0.78
33
for -NO2; σp=0.54 for -CF3 and σp= -0.27 for -OCH3 . Among the compound 4p-4w
containing the alkylsulfonyl group, the poor activities were showed. As can be seen in
Table 1, The longer the alkyl chain, the less activity it showed. In a word, the result
indicated that the phenylsulfonyl group is necessary to show the potent
antiproliferative activity.
2.3. PI3K enzymatic activity
Due to the compounds (4a-4o) containing the phenylsulfonyl group show the

better antiproliferative activities, they were selected to evaluate for their PI3K
enzymatic activities. The results are displayed in Table 2. As a whole, most of
compounds exhibited the certain inhibitory activities against the four protein kinases
PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, and the tendency of inhibition was roughly similar
to their inhibition for cancer cells. Besides, compare to another three protein kinases,
the compounds showed a certain selectivity against protein PI3Kα. Specifically,
compound 4o exhibited the high selectivity to inhibit the protein PI3Kα with IC50
value up to 0.021 μM, which has an approximately 20-fold improvement for
LY294002. Meanwhile, it has an increased potency up to approximately 50-fold for
another three protein kinases. It was concluded that compound 4o has a great potential
to be a new PI3Kα inhibitor for antitumor.
Table 2. Enzymatic activities of compounds 4a-4o against PI3K.
IC50 ± SD (μM)
compound
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
4a
52.87±4.82
8.12±1.01
6.48±0.78
9.68±0.93
44.66±0.68
40.78±0.56
27.67±0.44
12.54±0.21
3.69±0.32
1.44±0.18
1.17±0.13
0.73±0.04
0.45±0.02
0.10±0.01
0.02±0.0003
0.48±0.17
32.65±2.32
4.64±0.22
22.65±1.77
20.84±1.91
67.92±3.78
54.87±3.44
56.74±2.98
22.84±1.71
18.66±1.13
9.58±0.89
11.32±0.97
1.68±0.12
2.49±0.17
1.13±0.093
0.90±0.014
0.98±0.012
38.29±2.34
10.53±1.03
18.24±1.46
17.81±1.52
42.57±2.47
24.64±1.83
32.18±2.14
19.03±1.07
9.47±0.95
5.10±0.44
3.48±0.41
1.90±0.11
1.87±0.12
0.80±0.02
0.28±0.01
0.94±0.01
56.19±3.12
23.68±1.99
44.74±2.76
23.68±2.37
67.81±4.43
38.16±2.11
47.92±2.83
28.61±1.98
11.16±0.94
8.14±0.77
9.49±0.82
3.91±.34
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4.62±0.29
1.78±0.11
0.99±0.04
1.36±0.07
4n
4o
LY294002

Figure 2. Compound 4o induced apoptosis in HCT116 cells with the concentrations of 0.25, 0.5
and 1 μM. HCT-116 cells were treated for 24 h. Values represent the mean ± S.D., n = 3. P < 0.05
versus the control. The percentage of cells in each part is indicated.
2.4. Cells apoptosis assay
In order to explore the mechanism of cancer cells death, apoptosis induced in
HCT116 cell was performed with Annexin V-FITC/PI FACS assay. The HCT116 cells
were treated with compound 4o in different concentration (0.25, 0.5 and 1μM) for 24
hours. The result was showed in Figure 2. The percentage of late apoptotic cells from
8.43 to 24.46% was significant influence in a dose-dependent manner by compound
4o. It was indicated that compound 4o killed the cancer cell by induction of apoptosis.
2.5. Western blot assay
As an upstream molecule, 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 determine whether compound 4o
affect phosphorylation of Akt after inhibition the PI3K, western blot was performed to
examine the related protein including Akt and p-Akt (S473) in HCT-116 cells by
treatment of compound 4o. As can be seen in Figure 3. compound 4o obviously
down-regulated expression of p-Akt (S473) in a dose-dependent manner, and the Akt
expression level unchanged. It was indicated that compound may be a promising

PI3K inhibitor.
Figure 3. Western blot analysis of compound 4o. The inhibition effects of compound 4o (1μM,
2μM and 4μM) on the expression of p-Akt, Akt in HCT116 cells are depicted. β-Actin was used as
internal control. Bar graphs showed the quantitative results of clonogenicity. Data are presented as
the mean for three independent experiments. *P < 0.05, **P < 0.01 compared with control.
2.6. Molecular docking
Molecular docking was performed to clarify the possible binding mode between
Ligands and proteins, compound 4o was fitted into the active center of the PI3Kα
protein. The results are showed in Figure 4(A and B). As can be seen, the binding
model of ligand and protein mainly focus on chroman-2-one moiety of compound and
PI3Kα protein. There are two hydrogen bonds, One hydrogen bond was formed
between the amino hydrogen of Lys802 and the carboxyl oxygen atoms of compound
4o (distance :1.83 Å, Angle: 160.47°), and the other hydrogen bond was caused by the
oxygen atom of chroman-2-one interaction with Lys776 (distance :2.02 Å, Angle:
145.59°). It was indicated that the two amino acids Lys802 and Lys776 play an
important in the pocket of protein. Besides, in the Figure 4B, the phenylsulfonyl
fragment was located into the hydrophobic pocket, it might be the reason that the
compound 4o has the high selectivity against the PI3Kα protein.

Figure 4. (A) 2D molecular docking model of compound 4o with 3HHM. (B) 3D interaction map
between compound 4o and active pocket of PI3Kα protein.
3. Conclusion
In summary, a series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates
contained sulfonamido were designed and synthesized, and their anticancer effects in
vitro was evaluated to develop some new PI3Kα inhibitors. Most of desired
compounds exhibited the better antiproliferative activities against four cancer cell
lines than that of LY294002. Out of them, compound 4o displayed the potent
antiproliferative activity and high selectivity against the PI3Kα protein and it can
induce apoptosis of HCT116 in a dose-dependent manner. Western blot assay
indicated that compound 4o obviously down-regulated expression of p-Akt (S473).
Molecular docking was performed to clarify the possible binding mode between
compound 4o and PI3Kα. In a word, it can be concluded that compound 4o was a
potential PI3K inhibitor for cancer treatment.

4. Experimental section
All chemicals and reagents used in the current study were of analytical grade.
The reactions were monitored by thin layer chromatography (TLC) on Merck
pre-coated silica GF254 plates. Melting points (uncorrected) were determined on an
XT4MP apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a
Mariner System 5304 mass spectrometer, 1H NMR spectra were collected on a Bruker
13
DPX400 spectrometer and C NMR spectra were collected on a Bruker Ascend 600
spectrometer at room temperature with TMS and solvent signals allotted as internal
standards. Chemical shifts are reported in ppm (δ). Elemental analyses were
performed on a CHN-O-Rapid instrument, and were within ±0.4% of the theoretical
values.
4.1. General procedure for the preparation of compound 3
Chromeno[4,3-c]pyrazol-4(2H)-one (3) was prepared according to the previous
research.28
4.2. General procedure for the preparation of compound 4a-4w
To the solution of 3 (1 mmol) and TEA (10 μL) in dry DCM (15 mL), various
sulfonyl chloride was added in the ice-bath and the reaction mixture was stirred at
0 °C for 6-8 h. After the reaction was complete, the mixture was concentrated in
vacuo. The residue was subjected to crystallization to afford the desired compounds
4a-4w.
4.2.1. 2-(Phenylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4a)
White power, yield: 49%. Mp: 213-214 ℃. 1H NMR (400 MHz, CDCl3): 8.90(s, 1H,
ArH), 8.14(d, J=7.92 Hz, 2H, ArH), 8.10(d, J=7.76 Hz, 1H, ArH), 7.73(t, J=7.38 Hz,
1H, ArH), 7.61(t, J=7.68 Hz, 2H, ArH), 7.51(t, J=7.78 Hz, 1H, ArH), 7.32(m, 2H,
ArH). MS(ESI): 327.18 (C16H11N2O4S, [M+H]+). Anal.Calcd for C16H10N2O4S: C,
58.89; H, 3.09; N, 8.58%. Found: C, 58.90; H, 3.11; N, 8.59%.
4.2.2. 2-Tosylchromeno[4,3-c]pyrazol-4(2H)-one (4b)
White power, yield: 51%. Mp: 221-223 ℃. 1H NMR (400 MHz, CDCl3): 8.88(s, 1H,

ArH), 8.09(m, 1H, ArH), 8.01(d, J=8.40 Hz, 2H, ArH), 7.50(m, 1H, ArH), 7.39(d,
J=8.12 Hz, 2H, ArH), 7.31(m, 2H, ArH), 2.44(s, 3H, CH3). 13C NMR (150 MHz,
CDCl3): 156.91, 153.34, 151.93, 147.34, 134.15, 132.53, 131.86, 130.45(2),
128.92(2), 124.83, 123.72, 117.72, 113.64, 109.74, 21.88. MS(ESI): 341.42
(C17H13N2O4S, [M+H]+). Anal.Calcd for C17H12N2O4S: C, 59.99; H, 3.55; N, 8.23%.
Found: C, 59.97; H, 3.56; N, 8.25%.
4.2.3. 2-((4-Methoxyphenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4c)
White power, yield: 58%. Mp: 207-208 ℃. 1H NMR (400 MHz, CDCl3): 8.87(s, 1H,
ArH), 8.08(t, J=8.92 Hz, 3H, ArH), 7.50(t, J=7.78 Hz, 1H, ArH), 7.31(m, 2H, ArH),
7.04(d, J=8.84 Hz, 2H, ArH), 3.88(s, 3H, OCH3). MS(ESI): 357.25 (C17H13N2O5S,
[M+H]+). Anal.Calcd for C17H12N2O5S: C, 57.30; H, 3.39; N, 7.86%. Found: C, 57.32;
H, 3.41; N, 7.85%.
4.2.4. 2-((4-(Tert-butyl)phenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4d)
White power, yield: 48%. Mp: 223-224 ℃. 1H NMR (400 MHz, CDCl3): 8.88(s, 1H,
ArH), 8.12(d, J=7.72 Hz, 1H, ArH), 8.05(d, J=8.24 Hz, 2H, ArH), 7.60(d, J=8.24 Hz,
13
2H, ArH), 7.50(t, J=7.84 Hz, 1H, ArH), 7.32(m, 2H, ArH), 1.32(s, 9H, 3CH3). C
NMR (150 MHz, CDCl3): 160.11, 153.36, 151.91, 134.16, 132.44, 131.85, 128.75(3),
126.91(2), 124.83, 123.74, 117.73, 113.68, 109.78, 35.57, 30.90. MS (ESI): 383.29
(C20H19N2O4S, [M+H]+). Anal.Calcd for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.33%.
Found: C, 62.83; H, 4.75; N, 7.35%.
4.2.5. 2-((4-Fluorophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4e)
1
Light yellow power, yield: 39%. Mp: 198-201 ℃. H NMR (400 MHz, CDCl3):
8.89(s, 1H, ArH), 8.10(d, J=7.68 Hz, 1H, ArH), 8.00(d, J=8.04 Hz, 2H, ArH), 7.69(d,
J=7.92 Hz, 2H, ArH), 7.55(t, J=7.72 Hz, 1H, ArH), 7.31(m, 2H, ArH). MS (ESI):
345.19 (C16H10FN2O4S, [M+H]+). Anal.Calcd for C16H9FN2O4S: C, 55.81; H, 2.63; N,
8.14%. Found: C, 55.83; H, 2.64; N, 8.13%.
4.2.6. 2-((4-Chlorophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4f)
1
Light yellow power, yield: 42%. Mp: 207-209 ℃. H NMR (400 MHz, CDCl3):
8.89(d, J=3.08 Hz, 1H, ArH), 8.08(t, J=4.20 Hz, 3H, ArH), 7.59(m, 2H, ArH), 7.52(d,
J=7.16 Hz, 1H, ArH), 7.33(m, 2H, ArH). 13C NMR (150 MHz, CDCl3): 156.67,
153.39, 152.27, 142.84, 134.32, 133.92, 132.09, 130.29(2), 130.23(2), 124.93, 123.72,

117.79, 113.43, 110.03. MS (ESI): 361.62 (C16H10ClN2O4S, [M+H]+). Anal.Calcd for
C16H9ClN2O4S: C, 53.27; H, 2.51; N, 7.76%. Found: C, 53.29; H, 2.52; N, 7.75%.
4.2.7. 2-((4-Bromophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4g)
1
Light yellow power, yield: 45%. Mp: 216-217 ℃. H NMR (400 MHz, CDCl3):
8.88(s, 1H, ArH), 8.08(d, J=7.76 Hz, 1H, ArH), 8.00(d, J=8.40 Hz, 2H, ArH), 7.75(d,
J=8.40 Hz, 2H, ArH), 7.52(t, J=7.86 Hz, 1H, ArH), 7.33(m, 2H, ArH). 13C NMR (150
MHz, CDCl3): 156.66, 153.78, 152.27, 134.47, 134.34, 133.23(2), 132.09, 131.57,
130.24(2), 124.93, 123.72, 117.78, 113.41, 110.03. MS (ESI): 406.37
(C16H10BrN2O4S, [M+H]+). Anal.Calcd for C16H9BrN2O4S: C, 47.42; H, 2.24; N,
6.91%. Found: C, 47.44; H, 2.23; N, 6.93%.
4.2.8. 2-((2-Nitrophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4h)
Yellow power, yield: 48%. Mp: 197-199 ℃. 1H NMR (400 MHz, CDCl3): 8.89(s, 1H,
ArH), 8.57(d, J=7.36 Hz, 1H, ArH), 8.06(m, 3H, ArH), 7.67(m, 1H, ArH), 7.54(m, 1H,
ArH), 7.30(m, 2H, ArH). MS (ESI): 372.28 (C16H10N3O6S, [M+H]+). Anal.Calcd for
C16H9N3O6S: C, 51.75; H, 2.44; N, 11.32%. Found: C, 51.78; H, 2.45; N, 11.34%.
4.2.9. 2-((3-Nitrophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4i)
Yellow power, yield: 44%. Mp: 224-227 ℃. 1H NMR (400 MHz, CDCl3): 8.92(s, 1H,
ArH), 8.81(s, 1H, ArH), 8.54(d, J=7.76 Hz, 1H, ArH), 8.24(m, 1H, ArH), 8.00(m, 2H,
ArH), 7.52(d, J=7.72 Hz, 1H, ArH), 7.30(m, 2H, ArH). MS (ESI): 372.28
(C16H10N3O6S, [M+H]+). Anal.Calcd for C16H9N3O6S: C, 51.75; H, 2.44; N, 11.32%.
Found: C, 51.76; H, 2.45; N, 11.30%.
4.2.10. 2-((4-Nitrophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4j)
Yellow power, yield: 42%. Mp: 237-239 ℃. 1H NMR (400 MHz, CDCl3): 8.90(s, 1H,
ArH), 8.35(d, J=7.76 Hz, 2H, ArH), 8.12(d, J=7.92 Hz, 2H, ArH), 8.00(m, 1H, ArH),
7.51(m, 1H, ArH), 7.32(m, 2H, ArH). MS (ESI): 372.28 (C16H10N3O6S, [M+H]+).
Anal.Calcd for C16H9N3O6S: C, 51.75; H, 2.44; N, 11.32%. Found: C, 51.77; H, 2.42;
N, 11.33%.
4.2.11.
2-((4-(Trifluoromethyl)phenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one
(4k)
1
Light yellow power, yield: 47%. Mp: 214-216 ℃. H NMR (400 MHz, CDCl3):

8.89(s, 1H, ArH), 8.08(d, J=8.04 Hz, 1H, ArH), 8.02(d, J=8.28 Hz, 2H, ArH), 7.79(d,
J=8.40 Hz, 2H, ArH), 7.51(t, J=7.92 Hz, 1H, ArH), 7.33(m, 2H, ArH). MS (ESI):
395.26 (C17H10F3N2O4S, [M+H]+). Anal.Calcd for C17H9F3N2O4S: C, 51.78; H, 2.30;
N, 7.10%. Found: C, 51.80; H, 2.31; N, 7.08%.
4.2.12. 2-((2,5-Dimethylphenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4l)
White power, yield: 52%. Mp: 221-223 ℃. 1H NMR (400 MHz, CDCl3): 8.94(s, 1H,
ArH), 8.05(d, J=7.92 Hz, 2H, ArH), 7.50(t, J=7.82 Hz, 1H, ArH), 7.40(d, J=7.68 Hz,
1H, ArH), 7.34(d, J=8.36 Hz, 1H, ArH), 7.29(t, J=7.60 Hz, 1H, ArH), 7.23(d, J=7.76
Hz, 1H, ArH), 2.62(s, 3H, CH3), 2.44(s, 3H, CH3). 13C NMR (150 MHz, CDCl3):
156.96, 153.35, 151.64, 137.41, 136.62, 134.29, 133.41, 133.29, 131.84, 131.52,
124.82, 123.72, 118.07, 117.73, 113.65, 109.38, 20.86, 20.24. MS (ESI): 355.24
(C18H15N2O4S, [M+H]+). Anal.Calcd for C18H14N2O4S: C, 61.01; H, 3.98; N, 7.90%.
Found: C, 61.03; H, 3.99; N, 3.97%.
4.2.13. 2-((4-Chloro-3-nitrophenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4m)
Yellow power, yield: 33%. Mp: 229-231 ℃. 1H NMR (400 MHz, CDCl3): 8.90(s, 1H,
ArH), 8.00(m, 1H, ArH), 7.87(d, J=8.08 Hz, 1H, ArH), 7.78(d, J=8.32 Hz, 1H, ArH),
7.52(m, 1H, ArH), 7.44(d, J=8.40 Hz, 1H, ArH), 7.33(m, 2H, ArH). MS (ESI): 406.67
(C16H9ClN3O6S, [M+H]+). Anal.Calcd for C16H8ClN3O6S: C, 47.36; H, 1.99; N,
10.36%. Found: C, 47.38; H, 2.01; N, 10.34%.
4.2.14. 2-(Mesitylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4n)
White power, yield: 51%. Mp: 242-244 ℃. 1H NMR (400 MHz, CDCl3): 8.92(s, 1H,
ArH), 8.00(dd, J1=7.80 Hz, J2=1.56 Hz, 1H, ArH), 7.48(m, 1H, ArH), 7.33(d, J=7.76
Hz, 1H, ArH), 7.28(d, J=6.72 Hz, 1H, ArH), 7.03(s, 2H, ArH), 2.73(s, 6H, 2CH3),
2.32(s, 3H, CH3). 13C NMR (150 MHz, CDCl3): 157.13, 153.30, 151.28, 146.11,
142.08(2), 133.46, 132.74(2), 131.68, 129.58, 124.74, 123.58, 117.68, 113.76, 108.77,
23.27(2), 21.27. MS (ESI): 369.34 (C19H17N2O4S, [M+H]+). Anal.Calcd for
C19H16N2O4S: C, 61.94; H, 4.38; N, 7.60%. Found: C, 61.97; H, 4.40; N, 7.58%.
4.2.15. 2-((2,4,6-Triisopropylphenyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4o)
White power, yield: 48%. Mp: 231-234 ℃. 1H NMR (400 MHz, CDCl3): 8.94(s, 1H,
ArH), 8.00(d, J=3.72 Hz, 1H, ArH), 7.50(t, J=7.82 Hz, 1H, ArH), 7.36(d, J=8.28 Hz,
1H, ArH), 7.30(t, J=7.60 Hz, 1H, ArH), 7.26(s, 2H, ArH), 4.29(m, 2H, 2-CH-),

2.93(m, 1H, -CH-), 1.26(t, J=6.02 Hz, 18H, 6CH3). 13C NMR (150 MHz, CDCl3):
157.10, 156.23, 153.35, 153.23(2), 151.01, 132.88, 131.66, 128.54, 124.82(3), 123.44,
117.74, 113.82, 108.99, 34.39, 30.27(2), 24.69(4), 23.40(2). MS (ESI): 453.36
(C25H29N2O4S, [M+H]+). Anal.Calcd for C25H28N2O4S: C, 66.35; H, 6.24; N, 6.19%.
Found: C, 66.36; H, 6.23; N, 6.21%.
4.2.16. 2-((Trifluoromethyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4p)
Yellow power, yield: 41%. Mp: 167-169 ℃. 1H NMR (400 MHz, CDCl3): 8.92(s, 1H,
ArH), 8.01(m, 1H, ArH), 7.50(m, 1H, ArH), 7.34(d, J=7.96 Hz, 1H, ArH), 7.26(d,
J=7.52 Hz, 1H, ArH). MS (ESI): 319.17 (C11H6F3N2O4S, [M+H]+). Anal.Calcd for
C11H5F3N2O4S: C, 41.52; H, 1.58; N, 8.80%. Found: C, 41.53; H, 1.60; N, 8.79%.
4.2.17. 2-(Methylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4q)
White power, yield: 43%. Mp: 173-174 ℃.1H NMR (400 MHz, CDCl3): 8.92(s, 1H,
ArH), 8.00(dd, J1=7.84 Hz, J2=1.60 Hz, 1H, ArH), 7.50(m, 1H, ArH), 7.35(d, J=7.84
Hz, 1H, ArH), 7.29(d, J=7.12 Hz, 1H, ArH), 2.97(s, 3H, CH3). MS (ESI): 265.21
(C11H9N2O4S, [M+H]+). Anal.Calcd for C11H8N2O4S: C, 50.00; H, 3.05; N, 10.60%.
Found: C, 50.02; H, 3.03; N, 10.58%.
4.2.18. 2-(Ethylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4r)
White power, yield: 40%. Mp: 179-181 ℃. 1H NMR (400 MHz, CDCl3): 8.94(s, 1H,
ArH), 8.00(m, 1H, ArH), 7.48(m, 1H, ArH), 7.36(d, J=8.04 Hz, 1H, ArH), 7.28(d,
J=7.44 Hz, 1H, ArH), 3.38(m, 2H, -CH2), 1.26(t, J=7.32 Hz, 3H, CH3). MS (ESI):
279.31 (C12H11N2O4S, [M+H]+). Anal.Calcd for C12H10N2O4S: C, 51.79; H, 3.62; N,
10.07%. Found: C, 51.82; H, 3.63; N, 10.06%.
4.2.19. 2-(Propylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4s)
White power, yield: 39%. Mp: 181-283 ℃. 1H NMR (400 MHz, CDCl3): 8.94(s, 1H,
ArH), 8.00(d, J=7.86 Hz, 1H, ArH), 7.49(m, 1H, ArH), 7.33(d, J=7.92 Hz, 1H, ArH),
7.28(d, J=7.48 Hz, 1H, ArH), 3.38(t, J=7.06 Hz, 2H, -CH2), 1.92(m, 2H, -CH2), 0.95(t,
J=7.38 Hz, 3H, CH3). MS (ESI): 293.21 (C13H13N2O4S, [M+H]+). Anal.Calcd for
C13H12N2O4S: C, 53.42; H, 4.14; N, 9.58%. Found: C, 53.44; H, 4.16; N, 9.57%.
4.2.20. 2-(Butylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4t)
White power, yield: 41%. Mp: 177-179 ℃. 1H NMR (400 MHz, CDCl3): 8.92(s, 1H,

ArH), 8.00(m, 1H, ArH), 7.49(m, 1H, ArH), 7.33(d, J=7.96 Hz, 1H, ArH), 7.28(d,
J=7.52 Hz, 1H, ArH), 3.41(t, J=7.28 Hz, 2H, -CH2), 1.94(m, 2H, -CH2), 1.73(m, 2H,
-CH2), 0.98(t, J=7.14 Hz, 3H, CH3). MS (ESI): 307.31 (C14H15N2O4S, [M+H]+).
Anal.Calcd for C14H14N2O4S: C, 54.89; H, 4.61; N, 9.14%. Found: C, 54.92; H, 4.60;
N, 9.15%.
4.2.21. 2-(Cyclopropylsulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4u)
White power, yield: 37%. Mp: 168-171 ℃. 1H NMR (400 MHz, CDCl3): 8.92(s, 1H,
ArH), 8.00(dd, J1=8.04 Hz, J2=1.56 Hz, 1H, ArH), 7.51(m, 1H, ArH), 7.38(d, J=7.92
Hz, 1H, ArH), 7.30(d, J=7.32 Hz, 1H, ArH), 3.42(m, 1H, -CH-), 1.97(m, 4H, 2-CH2).
MS (ESI): 291.32 (C13H11N2O4S, [M+H]+). Anal.Calcd for C13H10N2O4S: C, 53.79; H,
3.47; N, 9.65%. Found: C, 53.78; H, 3.48; N, 9.67%.
4.2.22. 2-((2-Chloroethyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4v)
1
Light yellow power, yield: 41%. Mp: 164-166 ℃. H NMR (400 MHz, CDCl3):
8.92(s, 1H, ArH), 8.00(m, 1H, ArH), 7.51(m, 1H, ArH), 7.36(d, J=7.84 Hz, 1H, ArH),
7.28(d, J=7.36 Hz, 1H, ArH), 3.48(t, J=7.14 Hz, 2H, -CH2), 3.42(t, J=7.18 Hz, 2H,
-CH2). MS (ESI): 313.67 (C12H10ClN2O4S, [M+H]+). Anal.Calcd for C12H9ClN2O4S:
C, 46.09; H, 2.90; N, 8.96%. Found: C, 46.11; H, 2.92; N, 8.95%.
4.2.23. 2-((3-Chloropropyl)sulfonyl)chromeno[4,3-c]pyrazol-4(2H)-one (4w)
1
Light yellow power, yield: 38%. Mp: 170-172 ℃. H NMR (400 MHz, CDCl3):
8.94(s, 1H, ArH), 8.00(m, 1H, ArH), 7.48(m, 1H, ArH), 7.34(d, J=7.92 Hz, 1H, ArH),
7.30(d, J=7.28 Hz, 1H, ArH), 3.44(t, J=7.22 Hz, 2H, -CH2), 3.41(t, J=7.08 Hz, 2H,
-CH2), 2.21(m, 2H, -CH2). MS (ESI): 327.71 (C13H12ClN2O4S, [M+H]+). Anal.Calcd
for C13H11ClN2O4S: C, 47.78; H, 3.39; N, 8.57%. Found: C, 47.80; H, 3.40; N, 8.55%.
4.3. Cell proliferation assay
Four human cell lines HCT116, A549, Huh7 and HL60 were cultured in
RPMI-1640 (Invitrogen Corp., Carlsbad, CA) medium with heat-inactivated 10% fetal
bovine serum, penicillin (100 units mL−1) and streptomycin (100 mg mL−1) and
incubated under an atmosphere with 20% O2, 5% CO2 at 37 °C.
The inhibitory activity in vitro was measured using the MTT assay. All the
compounds were dissolved in DMSO at a concentration of 100 μM and were then
diluted to appropriate concentrations. Cells were plated in 96-well plates for 24 h and

subsequently treated with different concentrations of all the tested compounds for 72
h. Viable cells were determined using
a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT, Sigma)
according to the manufacturer’s instructions. The concentration of drug causing 50%
inhibition of absorbance compared with control cells (IC50) was calculated using the
software of dose-effect analysis with microcomputers.
4.4. PI3K enzyme assay
The inhibition of PI3K activity was determined using a competitive fluorescence
polarization kinase activity assay based on the principle that PI3K phosphorylates
PI(3,4)P2 and converts it into PI(3,4,5)P3. PI3K isoforms were purchased from
Perkin-Elmer or were expressed and purified as heterodimeric recombinant proteins.
Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), diC8-PIP2, and PIP3 detection
reagents were purchased from Echelon Biosciences. PI3K reactions were performed
in 5 mM HEPES, pH 7, 2.5 mM MgCl2, 10 mM DTT and 50 mM ATP using
diC8-PI(4,5)P2 as the substrate, and the final reaction volumes were 10 μL. For the
evaluation of PI3K inhibitors, 50 ng of enzyme and 10 mM of substrate were used per
10 μL reaction volume with inhibitor concentrations ranging from 3.2 nM to 1 mM.
After incubating for 3 h at room temperature, reactions were quenched by the addition
of a chelator. A mixture of phosphoinositide binding protein was added and mixed,
followed by the addition of a fluorophorelabeled phosphoinositide tracer. Samples
were then mixed in 384-well black Corning nonbinding plates and incubated in the
dark for 1 h to equilibrate. Finally, polarization values were measured using red
fluorophores with appropriate filters to determine the extent of enzyme activity in the
reaction.34
4.5. Apoptosis analysis
5×105 HCT116 cells in exponential growth was seeded into each well of 6-well
plate. After incubation for 12 h, they were treated with various concentrations of the
test compounds for 24 h. Then the cells were collected totally, centrifuged and
resuspended in 500 mL of staining solution (containing 5 mL Annexin V-FITC and 5
mL PI in Binding Buffer) and incubated for 15 min on the ice with avoiding light.
Samples were analyzed on FACScalibur flow cytometer (BD, USA).
4.6. Western blotting

Total cell lysates were prepared in RIPA buffer (50mM Tris-HCl, Ph 8.0, 150mM
NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The protein concentration
was determined, and equal amounts of protein samples were subjected to 10%
SDSPAGE. Separated proteins were transferred to PVDF membranes (Millipore,
Bedford, MA, USA) and probed with the indicated antibodies. Exposures were
obtained using ChemiDoc Touch biomolecular imager (Bio-Rad, USA).
4.7. Molecular docking
The crystal structures of the protein complexes were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The three-dimensional
structures of the aforementioned compounds were constructed using Chem. 3D ultra
12.0 software [Chemical Structure Drawing Standard; Cambridge Soft Corporation,
USA (2010)]; then they were energetically minimized using MMFF94 with 5000
iterations and a minimum RMS gradient of 0.10. Molecular docking of compound 4o
into the three-dimensional PI3Kα complex structure (3HHM.pdb, downloaded from
the PDB) was carried out using the Discovery Studio (version 3.5) as implemented
through the graphical user interface DS-CDOCKER protocol, all bound water and
ligands were eliminated from the protein and the polar hydrogen was added to the
proteins.
Acknowledgment
This work was supported by National Natural Science Foundation of China (No.
81602985), the Fundamental Research Funds for the Central Universities (No.
2632018ZD19), a grant (No. 201505023) from Public Science, Technology research
Funds Project of Ocean and financed by National Natural Science Foundation of
China (No. 21602103). It was also supported by the National Undergraduate
Innovation Program and the National Natural Science Foundation of China (No.
J1210026).
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Captions
Scheme 1. Synthesis of compounds 4a-4w. Reagents and conditions:(i) DMF, POCl3,
60 °C, 6 h. (ii) Ethanol, Et3N, 85% hydrazine hydrate, r.t.,14 h. (iii) benzenesulfonyl
chloride/ alkylsulfonyl chloride, DCM/TEA, 0 ℃, 6-8 h.
Table 1. Antiproliferative activities in vitro of target compounds 4a-4w against four
cancer cell lines.
Table 2. Enzymatic activities of compounds 4a-4o against PI3K.
Figure 1. Design novel 2-alkyl-chromeno[4,3-c]pyrazol-4(2H)-one containing
sulfonyl derivatives.
Figure 2. Compound 4o induced apoptosis in HCT116 cells with the concentrations
of 0.25, 0.5 and 1 μM. HCT-116 cells were treated for 24 h. Values represent the mean
± S.D., n = 3. P < 0.05 versus the control. The percentage of cells in each part is
indicated.
Figure 3. Western blot analysis of compound 4o. The inhibition effects of compound
4o (1μM, 2μM and 4μM) on the expression of p-Akt, Akt in HCT116 cells are
depicted. β-Actin was used as internal control. Bar graphs showed the quantitative
results of clonogenicity. Data are presented as the mean for three independent
experiments. *P < 0.05, **P < 0.01 compared with control.
Figure 4. (A) 2D molecular docking model of compound 4o with 3HHM. (B) 3D
interaction map between compound 4o and 3HHM binding sites of PI3Kα protein.
Design, synthesis and biological evaluation of novel
chromeno[4,3-c]pyrazol-4(2H)-one derivates containing
sulfonamido as potential PI3Kα inhibitors
Yong Yina,b, Jia-Qin Hub,d, Xu Wub, Shao Shab, She-Feng Wangb, Fang
Qiaob, Zhong-Cheng Song*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, People’ s
Republic of China.
d
The Joint Research Centre of Gene Interference, Guangzhou University
and Keele University for Gene Interference and Application, School of
Life Science, Guangzhou University, 230 Waihuan West Road, Guangzhou
510006, People’ s Republic of China.