Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates containing piperazine as inhibitors of PI3Kα

Article abstract of DOI:10.1016/j.bioorg.2019.103238

PI3K pathway has been heavily studied and is one of the most potential targets for various cancer treatment. Herein, we designed and synthesized a series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained piperazine based on our previous resear

Full text of DOI:10.1016/j.bioorg.2019.103238

Bioorganic Chemistry 92 (2019) 103238
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates
containing piperazine as inhibitors of PI3Kα
a,b,1
b,d,1
b
b
b
b
Yong Yin
, Yang Zhou
, Shao Sha , Xun Wu , She-Feng Wang , Fang Qiao ,
b,c,⁎
b,⁎
Zhong-Cheng Song
, Hai-Liang Zhu
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
c
d
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
School of Chemistry & Environmental Engineering, Jiangsu University of Technology, 1801 Zhongwu Rd., Changzhou, Jiangsu 213001, People’s Republic of China
Polymers and Composites Division, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, 1219 Zhongguan West Rd, Zhenhai District,
Ningbo 315201, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
PI3K pathway has been heavily studied and is one of the most potential targets for various cancer treatment.
Herein, we designed and synthesized a series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained
piperazine based on our previous research. They were evaluated for their PI3Kα wild-type and H1047R mutant
inhibitory activities and anticancer effects in vitro. Most of these compounds displayed the potential anti-
proliferative activities against four cancer cell lines (HCT-116, A549, Huh7 and HL60). Among them, Compound
Chromeno[4,3-c]pyrazol-4(2H)-one derivates
Selectivity
Antitumor
PI3Kα
4
p revealed the remarkable antiproliferative activity and was selected for further biological evaluation.
Compound 4p displayed the potent activity against both PI3Kα wild-type and H1047R mutant, and a certain
degree of selectivity for PI3Kα over PI3Kβ, γ and δ, and meanwhile it can remarkable down-regulate the
phosphorylation of Akt. In addition, compound 4p was found to induce cell apoptosis via upregulation of Bax
and cleaved-caspase 3/9, and downregulation of Bcl-2. The above results suggested that compound 4p could be
considered as a promising PI3Kα inhibitor.
1
. Introduction
PI3Kγ are present in the central nervous system, epithelial cells and the
hematopoietic system [17–21]. As one of the most frequently dysre-
As we all known, phosphatidylinositol 3-kinases (PI3Ks) play an
gulated signaling pathways in oncogenesis and other human patholo-
gies, PI3K pathway have been heavily studied and is one of the most
potential approaches for various cancer treatment.
extremely important role in many cellular functions such as cell moti-
lity, differentiation, proliferation, growth and intracellular trafficking
[
1–3]. These physiological roles are regulated by phosphatidylinositol
Nowadays several PI3K inhibitors have been identified and devel-
oped, and they are in preclinical studies or early clinical trials, such as
Wortmannin, LY294002, BEZ235, GDC-0941, GDC-0980, GSK2269557,
PI-3065 and so on [22–27]. In our previous research, chromeno[4,3-c]
pyrazol-4(2H)-one has been explored according to the structural feature
of Wortmannin, LY294002, BEZ235 and GDC-0941. Subsequently,
chromeno[4,3-c]pyrazol-4(2H)-one derivates have been identified as
PI3Kα inhibitors [28,29]. However, the selectivity was unsatisfactory.
Piperazine fragment is commonly found in many active molecules and
used to design drug [30]. In addition, it contributes to improve the
flexibility and solubility of molecule, and it is observed in the structure
of GDC-0980, GSK2269557 and PI-3065 (Fig. 1). On the basis of our
3
,4,5-triphosphate (PIP3), a second messenger who converted from
phosphatidylinositol(4,5)diphosphate (PIP2) catalyzed by PI3Ks [4–7].
Meanwhile, PIP3 levels are strictly controlled by phosphatase and
tensin homologue (PTEN), which converts PIP3 into PIP2 [8,9]. Based
on structural features and substrate specificity, the PI3K family is ca-
tegorized into three classes (classes I, II, and III) [10–12]. Class I PI3Ks
comprise four different isoforms (PI3Kα, β, γ and δ) and consist of
heterodimers between a p110 catalytic subunit and a p85 regulatory
subunit, they are studied most extensively and have been an attractive
approach for cancer therapy over the past decades [13–16]. In addition,
PI3Kα and PI3Kβ are expressed commonly, nevertheless PI3Kδ and
⁎
Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China (Z.-C. Song).
E-mail addresses: songzhongcheng@jsut.edu.cn (Z.-C. Song), zhuhl@nju.edu.cn (H.-L. Zhu).
Both authors contributed equally to this work.
1
https://doi.org/10.1016/j.bioorg.2019.103238
Received 19 July 2019; Received in revised form 29 August 2019; Accepted 30 August 2019
Available online 30 August 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
Fig. 1. Various PI3K inhibitors and the design compounds.
previous work and the function of piperazine, a series of chromeno[4,3-
c]pyrazol-4(2H)-one derivates containing piperazine fragment was de-
signed and synthesized, and some other nitrogen heterocycles were also
introduced to chromeno[4,3-c]pyrazol-4(2H)-one to testify the im-
portance of fragment.
and HCT-116, and the results were shown in Table 1. Most of the de-
signed compounds showed the good antiproliferative activities against
four cancer cell lines. Compared to the positive drug LY294002, a great
improvement of their activities was performed. Out of them, com-
pounds 4p, 4q and 4r exhibited the potent and top-3 antiproliferative
activities, particularly, compound 4p revealed the potent anti-
proliferative activity against four cancer cell lines with the IC50 value of
0.08 μM, 0.08 μM, and 0.07 μM, 0.05 μM for Huh7, A549, HL60 and
HCT-116, respectively.
In this research, we designed and synthesized a series of chromeno
[
4,3-c]pyrazol-4(2H)-one derivates containing piperazine, and they
were evaluated for their PI3K inhibitory activities and anticancer effects
in vitro.
As can be seen in Table 1, an interesting point was observed that the
compounds containing the piperazine fragment (4a-4r, 4v) exhibited
the better antiproliferative activities than that of other compounds
without piperazine fragment (4s, 4t and 4w). What’ more, the com-
pounds containing the phenylpiperazine or benzylpiperazine moiety
revealed the potent antiproliferative activities against cancer cell lines.
It was indicated that phenylpiperazine or benzylpiperazine moiety play
a crucial role for the designed compounds in the effect of anti-
proliferative activities.
2
. Results and discussion
2.1. Chemistry
The synthetic routes for the desired compounds followed the gen-
eral pathway outlined in Scheme 1. Firstly, synthesis of the so-called
core region described as our previous research [28]. Briefly, Chromeno
[
4,3-c]pyrazol-4(2H)-one (3) was produced via a Vilsmeier-Haack re-
Obviously, the compounds bearing diphenylmethylpiperazine frag-
ment exhibited the better antiproliferative activities than that of com-
pounds containing phenylpiperazine and other compounds, such as
compounds 4p, 4q and 4r, and it was observed that the more sub-
stituents on the benzene ring, the poorer antiproliferative activities.
Among the compounds 4a-4m which contained benzylpiperazine
moiety, compounds with disubstituted groups on the benzene ring
showed antiproliferative activities were superior to that of mono-sub-
stituted, e.g. compound 4k, 4l and 4m. For the compounds which
contained disubstituted groups on the benzene ring, different sub-
stituents bring the different effect on compounds, and they exhibited
action of 4-hydroxycoumarin (1) in the presence of dimethylformamide
and phosphoryl chloride followed by cyclization with 85% hydrazine
hydrate, and the yield was up to 85%. It was purified by crystallization
to afford the satisfactory product. And then, chromeno[4,3-c]pyrazol-
4
(2H)-one (3) and various amines were reacted under 40% for-
maldehyde condition to get the designed compounds (4a-4w).
2.2. Antiproliferative assays in vitro
All the designed compounds were evaluated for their anti-
proliferative activities against four cancer cell lines: HL60, Huh7, A549
2

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
Compounds
R
1
Compounds
R
1
4
a
4j
4
b
4k
4
c
4l
4m
4n
4
d
4
e
4
f
4o
Scheme 1. Synthesis of compounds 4a-4w. Reagents and conditions: (i) DMF, POCl
maldehyde/ various N-heterocyclic compounds, EtOH, r.t., 4–6 h.
3
, 60 °C, 6 h. (ii) Ethanol, Et N, 85% hydrazine hydrate, r.t., 14 h. (iii) for-
3
antiproliferative activities with the trend of order was
diphenylmethylpiperazine group, exerting the remarkable efficacy.
OCH
3
> NO
2
> F > CF > Cl. Meanwhile, it was observed that the
3
substituted position on the benzene ring displayed the antiproliferative
2.3. PI3K enzymatic activity
activities with the trend of ortho > para > meta, such as compounds
4
b, 4c and 4d. Besides, for other compounds, hydrophilic group played
To validate whether the antiproliferative effect of compounds was
a positive role for the activity of these compounds, e.g. compound 4k-
w. In brief, the aboved results indicated that N-substituted piperazine
group is an essential fragment for the designed compounds to displayed
the potent antiproliferative activities, and especially
exerted by targeting PI3K protein, the twelve compounds which re-
vealed the highest activities were selected to test against PI3K enzy-
matic activity, and the results are shown in Table 2. Among these
compounds, most compounds displayed the inhibition activities against
4
3

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
4
g
4p
4q
4r
4
h
4
i
Compounds
R
1
Compounds
R
2
R
3
4
s
4u
4
t
4v
4
w
Scheme 1. (continued)
Table 1
Table 2
Antiproliferative activities in vitro of targeted compounds 4a-4w against four
Enzymatic activities of selected compounds against PI3K.
cancer cell lines.
Compound
a
Compounds IC50
±
SD (μM)
IC50
±
SDa (μM)
PI3Kα
PI3Kβ
PI3Kγ
PI3Kδ
HCT-116
A549
Huh7
HL60
4
b
2.45 ± 0.18
> 10
5.82 ± 0.67
9.43 ± 0.84
5.09 ± 0.37
> 10
4.13 ± 0.37
> 10
8.17 ± 0.74
> 10 ±
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
22.03 ± 1.92
0.76 ± 0.06
3.40 ± 0.24
1.30 ± 0.12
2.84 ± 0.24
3.27 ± 0.31
9.36 ± 0.86
7.18 ± 0.62
1.95 ± 0.16
5.79 ± 0.57
0.26 ± 0.02
0.33 ± 0.02
0.17 ± 0.01
11.39 ± 1.27
14.63 ± 1.45
0.05 ± 0.004
0.08 ± 0.006
0.10 ± 0.007
62.80 ± 6.43
47.57 ± 4.36
> 100
28.47 ± 2.18
1.04 ± 0.12
6.15 ± 0.59
1.14 ± 0.13
4.68 ± 0.45
7.04 ± 0.68
14.67 ± 1.22
10.81 ± 0.94
2.49 ± 0.23
8.66 ± 0.71
0.38 ± 0.03
0.61 ± 0.04
0.43 ± 0.03
27.10 ± 3.22
19.56 ± 2.03
0.08 ± 0.006
0.11 ± 0.01
0.12 ± 0.01
> 100
27.08 ± 2.23
0.79 ± 0.07
7.81 ± 0.79
1.37 ± 0.14
2.97 ± 0.21
5.31 ± 0.48
16.41 ± 1.39
11.68 ± 1.14
3.15 ± 0.28
12.51 ± 1.14
0.67 ± 0.06
0.82 ± 0.07
0.31 ± 0.02
21.31 ± 2.27
32.60 ± 2.94
0.08 ± 0.005
0.12 ± 0.01
0.17 ± 0.01
89.04 ± 9.12
67.46 ± 6.33
92.80 ± 9.31
79.13 ± 9.28
64.31 ± 5.81
67.18 ± 5.64
27.10 ± 2.23
0.92 ± 0.07
3.91 ± 0.25
1.85 ± 0.14
3.12 ± 0.21
4.09 ± 0.37
14.44 ± 1.26
12.61 ± 1.18
3.65 ± 0.39
8.74 ± 0.81
0.51 ± 0.04
0.49 ± 0.04
0.32 ± 0.02
19.73 ± 1.44
22.71 ± 2.08
0.07 ± 0.006
0.11 ± 0.01
0.14 ± 0.01
> 100
4c
4d
5.84 ± 0.32
7.61 ± 0.31
9.37 ± 0.28
4.37 ± 0.38
0.84 ± 0.02
1.74 ± 0.11
0.32 ± 0.02
3.97 ± 0.24
7.82 ± 0.28
> 10
7.52 ± 0.66
> 10
4e
4f
8.01 ± 0.12
7.74 ± 0.37
0.42 ± 0.41
1.12 ± 0.22
0.65 ± 0.04
9.74 ± 0.78
7.52 ± 0.54
1.58 ± 0.12
1.44 ± 0.11
0.66 ± 0.051
4i
8.17 ± 0.56
0.73 ± 0.037
0.40 ± 0.021
0.46 ± 0.023
4k
g
h
i
4l
4m
4p
0.012 ± 0.001 0.12 ± 0.003 0.068 ± 0.004 0.19 ± 0.009
j
4q
0.058 ± 0.002 0.20 ± 0.006 0.13 ± 0.008
0.75 ± 0.042
0.16 ± 0.011
1.36 ± 0.13
k
l
4r
0.10 ± 0.002
0.48 ± 0.07
0.19 ± 0.006 0.11 ± 0.012
0.98 ± 0.012 0.95 ± 0.082
LY294002
m
n
o
p
q
r
a
Values are average of three independent experimental measurements and
expressed as Mean ± SD.
Compared to other three PI3K isoforms, compound 4p selectively in-
hibited PI3Kα, and it was better than that of LY294002.
s
t
81.04 ± 8.37
> 100
84.93 ± 8.74
89.04 ± 8.21
56.84 ± 5.25
68.74 ± 7.02
18.43 ± 2.03
What’s more, compounds 4k-4m and 4p-4r were selected to test for
their inhibitory activity against PI3Kα H1047R mutant, and the results
were showed in Table 3. it was observed that all the selected com-
pounds exhibit the compared inhibitory activities against PI3Kα wild-
type and H1047R mutant. Interestingly, compound 4p also exhibited
the potent inhibitory activity against PI3Kα H1047R mutant
(IC50 = 0.01 μM). All the results indicated that compound 4p may be a
new PI3Kα inhibitor.
u
v
w
74.30 ± 6.92
> 100
78.64 ± 7.62
58.33 ± 5.44
82.32 ± 7.26
LY294002
51.82 ± 4.58
a
Values are average of three independent experimental measurements and
expressed as Mean ± SD.
PI3Kα, β, γ and δ to a certain degree. Particularly, compounds 4p-4r
showed greatly improved inhibition activities against PI3Ks comparing
to LY294002. Additionally, compound 4p exhibited the prominent ac-
tivity against PI3Kα with IC50 values of 0.012 μM, 0.12 μM, 0.068 μM
and 0.19 μM for PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ, respectively.
2.4. Western blot assay
Once the PI3Kα activity was suppressed, downstream signal factor
of PI3K which mediates cell growth and blocks cell apoptosis might also
4

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
Table 3
(PDB ID: 3HHM) using Discovery Studio 3.5. As shown in Fig. 4, the
compound 4p was well filled in the active pocket via one hydrogen
bond, three π-cation interactions and one π-Sigma interaction. the
carboxyl oxygen atoms of compound 4p formed one hydrogen bond
(distance:1.79 Å) with the amino of Lys802; Two π-cation interactions
was formed by Lys776 and the coumarin ring, while another π-cation
interaction was formed between Tyr836 and nitrogen atom of piper-
azine; The benzene ring of compound 4p formed one π-sigma interac-
tion with Met922. Interestingly, the four amino acid residues play a
vital role in the active pocket of PI3Kα, and they are the key binding
site of most PI3Kα inhibitors. These interactions resulting in compound
4p binding well to the active pocket of PI3Kα protein, indicating that
compound 4p is a promising PI3Kα inhibitor.
Inhibitory activity against PI3Kα wild type and H1047R mutant.
a
Compounds
IC50
±
SD (μM)
PI3Kα (wild-type)
PI3Kα (H1047R mutant)
4
4
4
4
4
4
k
l
0.84 ± 0.02
1.74 ± 0.11
0.32 ± 0.02
0.012 ± 0.001
0.058 ± 0.002
0.10 ± 0.002
0.76 ± 0.08
1.46 ± 0.11
0.21 ± 0.03
0.010 ± 0.002
0.076 ± 0.004
0.088 ± 0.005
m
p
q
r
a
Values are average of three independent experimental measurements and
expressed as Mean ± SD.
be inhibited, and as we known, activation of PI3K leads to the Akt
phosphorylated. Based on these, western blot was performed to assess
whether effect on the expression levels of Akt and p-Akt in HCT-116 cell
line after treated with compound 4p at the concentration of 0.25 μM,
3. Conclusion
In summary, twenty-three chromeno[4,3-c]pyrazol-4(2H)-one deri-
vates containing piperazine fragment as PI3K inhibitors have been de-
signed and synthesized, and their antiproliferative activities against
HCT-116, Huh7, A549 and HL60 four cancer cell lines were evaluated,
and most desired compounds showed better antiproliferative activities.
Among of them, compound 4p exhibited the remarkable anti-
proliferative activity (IC50 ranging from 0.05 to 0.08 μM), which was
superior to that of reference drug LY294002. In addition, compound 4p
revealed the potent activity against both PI3Kα wild-type and H1047R
mutant with IC50 values of 0.012, and 0.01 μM, respectively, com-
paring to another three PI3K isoforms (PI3Kβ, PI3Kγ and PI3Kδ), and an
about 40-fold increase in comparison with LY294002. Western blotting
assay showed that 4p decrease the p-AKT phosphorylation level, in-
dicating compound 4p could inhibit PI3Kα activity. What’s more,
compound 4p remarkably induced apoptosis in HCT-116 cell in a dose-
dependent manner, up-regulated Bax, cleaved caspase-3 and cleaved
caspase-9 expressions, and reduced the anti-apoptotic protein Bcl-2
level. Molecular docking indicating compound 4p was docked well into
the active pocket of PI3Kα and strong bind to the protein. These results
suggested that compound 4p could be a promising PI3Kα inhibitor for
cancer therapy.
0
.5 μM and 1 μM. As shown in Fig. 2, compound 4p caused a decrease in
p-Akt (S473) level obviously in a dose-dependent manner, indicating
that 4p might be a potential PI3K inhibitor.
2.5. Cell apoptosis assay
To identify whether compound 4p could induce cell apoptosis,
compound 4p was used to induce cell apoptosis in HCT-116 cell line
using Annexin V-FITC/PI FACS assay. After treated with 4p at different
concentration (0.25, 0.5 and 1 μM) in HCT-116 cell line for 24 h, the
percentages of late apoptotic population from 17.6 to 30.1%, and the
percentages of early apoptotic population from 18.4 to 27.0% (Fig. 3),
which was remarkable influence in a dose-dependent manner by com-
pound 4p.
To further investigate the apoptosis mechanism by compound 4p in
HCT-116 cell line, apoptotic related proteins were examined in HCT-
1
2
16 after treatment with 4p at concentrations of 0.25, 0.5 and 1 μM for
4 h. As shown in Fig. 3, the proapoptotic protein Bax level was re-
markably increased in a dose-dependent manner, in contrast, Bcl-2 level
was decreased. What’s more, the cleaved caspase-3 and cleaved cas-
pase-9 levels which are recognized as bio-markers for cell apoptosis,
were increased in a dose-dependent manner. These results indicated
that compound 4p induce cell apoptosis of HCT-116 cells in a dose-
dependent manner.
4. Experimental section
All chemicals and reagents were of analytical grade and purchased
from commercial company. Melting points were determined by a
melting point apparatus (Taike Corp., China). All reactions were mon-
itored by thin layer chromatography (TLC; silica GF254 plates) which
2.6. Molecular docking study
1
purchased from Merck. H NMR spectra were recorded using Bruker
1
3
To study a possible interaction mode between compound 4p and
DPX400 spectrometer and C NMR spectra were determined by Bruker
Ascend 600 spectrometer, and TMS as an internal standard. Chemical
shifts are described in ppm (δ). ESI mass spectra were determined on a
PI3Kα H1047R mutant, molecular docking was carried out to fit com-
pound 4p into the active pocket of the PI3Kα H1047R mutant protein
Fig. 2. Effects of compound 4p on the AKT phosphorylation. HCT-116 cells were treated with compound 4p 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 and p-AKT proteins. Data were expressed as
Mean ± SD (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.005 vs. the control.
5

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
Fig. 3. Induced apoptotic effect of compound 4p on HCT-116 cell. (A) Apoptotic assay by flow cytometry. HCT-116 cells were treated with compound 4p 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 and C) Effects of compound 4p on Bcl-2, Bax, cleaved-
caspase-3 and cleaved-caspase-9 expression. Cells were treated with compound 4p 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 ± SD (n = 3). *p < 0.05,
**p < 0.01 and ***p < 0.005 vs. the control.
6

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
Fig. 4. (A) 2D molecular docking model of compound 4p with 3HHM (PI3Kα H1047R mutant). (B) 3D conformation position of the compound 4p in the binding
pocket.
Mariner System 5304 mass spectrometer.
4.2. General procedure for the preparation of compounds 4a-4w
4
0% formaldehyde (300 μL) was added slowly into the ethanol so-
4
.1. General procedure for the preparation of compound 3
lution of 3 (2 mmol) and various heterocyclic amine (2 mmol), the
mixtures were stirred at room temperature for 5–7 h. A large quantity of
solid was precipitated and then filtered, washed with cold ethanol, and
crystallized with ethanol to get the designed compounds 4a-4w.
Chromeno[4,3-c]pyrazol-4(2H)-one (3) was synthesized described
in the previous research [28]. Briefly, POCl (0.15 mol) was added
3
partly to a mixture of 4-hydroxycoumarin (0.05 mol) in anhydrous DMF
30 ml) under the ice-bath. The mixture was stirred for 0.5–1 h, and
then heated to 65 °C and reacted for 6 h. Subsequently, ice water
200 ml) was poured into the reaction mixture and stirred vigorously,
the yellow solid was precipitated out and then filtered, washed suc-
cessively with aqueous Na CO (5%) and water, crystallized with
acetone to obtain the compound 2.
5% Hydrazine hydrate (3 mmol) and TEA (6 mmol) were mixed in
(
4
.2.1. 2-((4-Phenylpiperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-
one (4a)
White power, yield: 48%. Mp: 212–214 °C. H NMR (400 MHz,
): 8.29 (s, 1H, ArH), 8.09(m, 1H, ArH), 7.49–7.45(m, 1H, ArH),
.38(t, J = 4.10 Hz, 1H, ArH), 7.32(m, 1H, ArH), 7.24(m, 2H, ArH),
.89–6.84(m, 3H, ArH), 5.18(s, 2H, eCH ), 3.21(t, J = 4.96, 4H,
CH e), 2.83(t, J = 4.94 Hz, 4H, eCH
): 158.71, 152.96, 151.11, 149.21, 132.43, 130.42,
(
1
CDCl
3
2
3
7
6
2
8
13
eCH
2
2
2
CH
2
e).
C
NMR
6
5–70% ethanol (15 ml), the mixture was slowly added dropwise to the
(
125 MHz, CDCl
3
solution of compound 2 (3 mmol) in ethanol (15 ml) under the tem-
perature did not exceed 25 °C. The mixture was stirred for 12 h at room
temperature, and then removed ethanol in vacuo. The residue was
purified by recrystallization to afford Chromeno[4,3-c]pyrazol-4(2H)-
one (3).
1
7
3
29.17(2), 124.54, 122.80, 120.25, 117.63, 116.52(2), 114.94, 107.98,
+
4.30, 50.11(2), 49.33(2). MS(ESI) m/z: 361.38 [M+H] (Calcd for
61.42, C21
H
21
N
4
O ).
2
4
.2.2. 2-((4-(2-Methoxyphenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
pyrazol-4(2H)-one (4b)
White power, yield: 42%. Mp: 190–193 °C. 1H NMR (400 MHz,
7

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
CDCl3): 8.27(s, 1H, ArH), 8.08(d, J = 7.70 Hz, 1H, ArH), 7.49–7.45(m,
ArH), 7.38(d, J = 8.28 Hz, 1H, ArH), 7.32(m, 2H, ArH), 7.21(m, 1H,
1
1
H, ArH), 7.39(d, J = 8.40 Hz, 1H, ArH), 7.32(m, 1H, ArH), 7.00(m,
H, ArH), 6.91(m, 2H, ArH), 6.83(d, J = 7.68 Hz, 1H, ArH), 5.19(s, 2H,
ArH), 7.02(m, 1H, ArH), 6.96(m, 1H, ArH), 5.18(s, 2H, eCH
2
), 3.09(d,
J = 4.32 Hz, 4H, eCH
2
CH
2
3
e), 2.86(t, J = 4.58 Hz, 4H, eCH
2
CH e).
2
1
3
eCH
2
), 3.80(s, 3H, eOCH
3
), 3.10(s, 4H, eCH
2
CH
2
e), 2.87(t,
): 158.23,
C NMR (125 MHz, CDCl ): 158.23, 152.98, 149.23, 148.90, 132.32,
1
3
J = 4.76 Hz, 4H, eCH
2
CH
2
e). C NMR (125 MHz, CDCl
3
130.72, 130.39, 128.85, 127.61, 124.52, 124.00, 122.83, 120.43,
1
1
5
52.95, 152.24, 149.20, 140.91, 132.39, 130.36, 124.49, 123.27,
117.64, 114.99, 108.05, 74.43, 51.08(2), 50.33(2). MS(ESI) m/z:
22.80, 120.97, 118.30, 117.64, 114.98, 111.18, 107.98, 74.46, 55.35,
395.71 [M+H]+ (Calcd for 395.86, C21
H20ClN
4
O ).
2
+
0.51(2), 50.28(2). MS(ESI) m/z: 391.27 [M+H] (Calcd for 391.44,
C
22
H
23
N
4
O
3
).
4.2.8. 2-((4-(3-Chlorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
pyrazol-4(2H)-one (4h)
1
4
.2.3. 2-((4-(3-Methoxyphenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
pyrazol-4(2H)-one (4c)
White power, yield: 49%. Mp: 168–169 °C. H NMR (400 MHz,
CDCl ): 8.28(s, 1H, ArH), 8.08(d, J = 7.71 Hz, 1H, ArH), 7.47(t,
Light yellow power, yield: 51%. Mp: 193–195 °C.
H NMR
(400 MHz, CDCl ): 8.28(s, 1H, ArH), 8.08(m, 1H, ArH), 7.47(m, 1H,
3
1
ArH), 7.38(d, J = 4.15 Hz, 1H, ArH), 7.31(m, 1H, ArH), 7.13(t,
J = 8.05 Hz, 1H, ArH), 6.82(m, 2H, ArH), 6.74(m, 1H, ArH), 5.17(s,
3
J = 7.71 Hz, 1H, ArH), 7.39–7.30(m, 2H, ArH), 714(t, J = 8.49 Hz Hz,
2H, eCH
4H, eCH
2
), 3.21(t, J = 4.98 Hz, 4H, eCH
2
CH e), 2.81(t, J = 4.98 Hz,
2
1
3
1
5
H, ArH), 6.49(d, J = 9.0 Hz, 1H, ArH), 6.41(d, J = 6.27 Hz, 2H, ArH),
.17(s, 2H, eCH ), 3.76(s, 3H, eOCH ), 3.21(t, J = 4.77 Hz, 4H,
CH e), 2.82(t, J = 4.77 Hz, 4H, eCH
2
CH
2
e). C NMR (125 MHz, CDCl ): 158.16, 152.96, 152.11,
3
2
3
149.26, 134.99, 132.42, 130.47, 130.09, 124.56, 122.80, 119.83,
117.64, 116.18, 114.90, 114.36, 108.02, 74.21, 49.90(2), 48.83(2). MS
1
3
eCH
2
2
2
CH
2
e).
C
NMR
(
125 MHz, CDCl
3
): 160.59, 158.17, 152.96, 152.49, 149.23, 132.39,
(ESI) m/z: 395.71 [M+H]+ (Calcd for 395.86, C21
H20ClN
4
2
O ).
1
1
30.43, 129.85, 124.54, 122.79, 117.64, 114.93, 109.25, 108.00,
04.93, 103.00, 74.30, 55.21, 50.07(2), 49.24(2). MS(ESI) m/z: 391.27
4.2.9. 2-((4-(4-Nitrophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
+
[
M+H] (Calcd for 391.44, C22
H
23
N
4
O
3
).
pyrazol-4(2H)-one (4i)
1
Yellow power, yield: 40%. Mp: 221–224 °C. H NMR (400 MHz,
4
.2.4. 2-((4-(4-Methoxyphenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
CDCl ): 8.27(s, 1H, ArH), 8.07(m, 3H, ArH), 7.47(m, 1H, ArH), 7.37(d,
3
pyrazol-4(2H)-one (4d)
J = 8.24 Hz, 1H.ArH), 7.30(m, 1H, ArH), 6.78(d, J = 9.44 Hz, 2H,
ArH), 5.19(s, 2H, eCH eCH ), 3.45(t, J = 5.04 Hz, 4H, eCH CH e),
2.82(t, J = 5.02 Hz, 4H, eCH ):
1
White power, yield: 43%. Mp: 193–194 °C. H NMR (400 MHz,
CDCl ): 8.28(s, 1H, ArH), 8.09(m, 1H, ArH), 7.47(m, 1H, ArH), 7.38(d,
J = 8.08 Hz, 1H, ArH), 7.32(m, 1H, ArH), 6.86(d, J = 8.88 Hz, 2H,
ArH), 6.81(d, J = 9.08 Hz, 2H, ArH), 5.17(s, 2H, eCH ), 3.75(s, 3H,
2
2
2
2
1
3
3
2
CH
2
e). C NMR (125 MHz, CDCl
3
158.06, 154.59, 152.97, 149.38, 138.91, 132.42, 130.57, 125.94(2),
124.59, 122.77, 117.67, 114.81, 113.07(2), 108.11, 74.03, 49.58(2),
47.11(2). MS(ESI) m/z: 406.28 [M+H]+ (Calcd for 406.41,
2
1
3
eOCH
3
), 3.10(s, 4H, eCH
2
CH
2
e), 2.84(s, 4H, eCH
2
CH e). C NMR
2
(
125 MHz, CDCl
3
): 158.18, 152.96, 149.23, 145.45, 132.37, 130.42,
C
21
H
20
N
5
O ).
4
1
5
3
24.53, 122.79, 118.71, 117.65, 114.94, 114.49(2), 107.98, 74.32,
+
5.58, 50.79(2), 50.21(2). MS(ESI) m/z: 391.27 [M+H] (Calcd for
4.2.10. 2-((4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)methyl)chromeno
91.44, C22
H
23
4
N O
3
).
[4,3-c]pyrazol-4(2H)-one (4j)
1
Yellow power, yield: 47%. Mp: 174–176 °C. H NMR (400 MHz,
4
.2.5. 2-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
CDCl ): 8.28(s, 1H, ArH), 8.08(m, 1H, ArH), 7.48(m, 1H, ArH), 7.38(d,
3
pyrazol-4(2H)-one (4e)
J = 8.24 Hz, 1H.ArH), 7.32(m, 2H, ArH), 7.08(d, J = 5.96 Hz, 2H.ArH),
7.02(m, 1H, ArH), 5.19(s, 2H, eCH ), 3.26(t, J = 4.97 Hz, 4H,
eCH CH e), 2.84(m, J = 4.97 Hz, 4H, eCH
(125 MHz, CDCl ): 158.13, 152.98, 151.19, 149.30, 132.39, 130.51(d,
1
Light yellow power, yield: 39%. Mp: 197–199 °C.
H
NMR
2
1
3
(
400 MHz, CDCl
3
): 8.29(s, 1H, ArH), 8.09(d, J = 6.20 Hz, 1H, ArH),
2
2
2
CH
2
e).
C NMR
7
7
1
4
.47(m, 1H, ArH), 7.38(d, J = 7.64 Hz, 1H, ArH), 7.32(m, 1H, ArH),
3
.04(m, 1H, ArH), 7.01(m, 1H, ArH), 6.99–6.91(m, 2H, ArH), 5.18(s,
J = 264 Hz), 130.48, 124.56, 122.79, 119.22, 117.66, 116.42(d,
H, eCH
H, eCH
2
), 3.14(d, J = 3.24 Hz, 4H, eCH
2
CH e), 2.86(t, J = 3.60 Hz,
2
J = 4.5 Hz), 114.89, 112.60(d, J = 9 Hz), 112.59, 108.05, 74.20,
1
3
+
2
CH
2
e).
C NMR (125 MHz, CDCl
3
): 158.19, 155.74(d,
49.92(2), 48.85(2), 29.73. MS(ESI) m/z: 429.31 [M+H] (Calcd for
J = 294 Hz), 152.97, 149.25, 139.78(d, J = 9 Hz), 132.36, 130.41,
429.42, C22
H
F
20 3
N
4
2
O ).
1
1
5
24.53, 124.50(d, J = 4.5 Hz), 122.88, 122.82, 119.11(d, J = 4.5 Hz),
17.64, 116.21(d, J = 25.5 Hz), 114.95, 108.01, 74.38, 50.41(2),
4.2.11. 2-((4-(2,4-Dimethylphenyl)piperazin-1-yl)methyl)chromeno[4,3-
+
0.19(2). MS(ESI) m/z: 379.33 [M+H]
(Calcd for 379.41,
c]pyrazol-4(2H)-one (4k)
1
C
21
H
20FN
4
O
2
).
White power, yield: 43%. Mp: 226–228 °C. H NMR (400 MHz,
CDCl ): 8.28(s, 1H, ArH), 8.11(m, 1H, ArH), 7.48(m, 1H, ArH), 7.39(d,
3
4
.2.6. 2-((4-(4-Fluorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
J = 7.52 Hz, 1H, ArH), 7.32(m, 1H, ArH), 6.96(d, J = 6.32 Hz, 2H,
ArH), 6.91(d, J = 8.72, 1H, ArH), 5.19(s, 2H, eCH ), 2.92(t,
J = 4.64 Hz, 4H, eCH CH e), 2.82(d, J = 4.44 Hz, 4H, eCH CH e),
), 2.18(s, 3H, eCH ):
pyrazol-4(2H)-one (4f)
2
1
Light yellow power, yield: 42%. Mp: 197–200 °C.
H
NMR
2
2
2
2
1
3
(
400 MHz, CDCl
3
): 8.28(s, 1H, ArH), 8.08(m, 1H, ArH), 7.48(m, 1H,
2.25(s, 3H, eCH
3
3
). C NMR (125 MHz, CDCl
3
ArH), 7.38(t, J = 4.14 Hz, 1H, ArH), 7.31(m, 1H, ArH), 6.93(m, 2H,
ArH), 6.83(m, 2H, ArH), 5.17(s, 2H, eCH ), 3.12(t, J = 4.92 Hz, 4H,
158.26, 152.99, 149.16, 148.69, 132.96, 132.66, 132.30, 131.87,
130.38, 127.05, 124.52, 122.84, 119.02, 117.64, 115.04, 108.01,
2
1
3
+
eCH
2
CH
2
e), 2.83(t, J = 4.94 Hz, 4H, eCH
2
CH
2
e).
C
NMR
74.54, 51.79(2), 50.65(2), 20.70, 17.67. MS(ESI) m/z: 389.32 [M+H]
(
125 MHz, CDCl
3
): 158.61, 157.46(d, J = 285 Hz), 152.96, 149.23,
(Calcd for 389.47, C23
H
25
N
4
O ).
2
1
1
5
47.76, 132.41, 130.44, 124.54, 122.78, 118.35(d, J = 9 Hz)(2),
17.64, 115.61(d, J = 25.5 Hz)(2), 114.92, 107.99, 74.25, 50.32(2),
4.2.12. 2-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
+
0.09(2). MS(ESI) m/z: 379.33 [M+H]
(Calcd for 379.41,
pyrazol-4(2H)-one (4l)
1
C
21
H
20FN
4
O
2
).
Light yellow power, yield: 56%. Mp: 220–222 °C.
H NMR
(
400 MHz, CDCl ): 8.30(s, 1H, ArH), 8.08(m, 1H, ArH), 7.47(m, 1H,
3
4
.2.7. 2-((4-(2-Chlorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
ArH), 7.38(d, J = 8.12 Hz, 1H, ArH), 7.31(t, J = 7.48 Hz, 1H, ArH),
pyrazol-4(2H)-one (4g)
7.14(m, 2H, ArH), 6.92(m, 1H, ArH), 5.18(s, 2H, eCH
eCH CH e), 2.86(t, J = 4.38 Hz, 4H, eCH CH e).
(125 MHz, CDCl
2
), 3.07(s, 4H,
1
13
Light yellow power, yield: 45%. Mp: 211–213 °C.
H
NMR
2
2
2
2
C
NMR
(
400 MHz, CDCl
3
): 8.29(s, 1H, ArH), 8.09(m, 1H, ArH), 7.47(m, 1H,
3
): 158.21, 152.97, 150.87, 149.24, 134.13, 132.34,
8

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
1
1
30.42, 127.62, 127.48, 124.93, 124.54, 122.84, 118.67, 117.63,
4.2.18. 2-((4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)methyl)chromeno
14.98, 108.07, 74.36, 51.18(2), 50.26(2). MS(ESI) m/z: 430.18 [M
[4,3-c]pyrazol-4(2H)-one (4r)
+
1
+
H] (Calcd for 430.30, C21
H
19Cl
2
N
4
O
2
).
Light yellow power, yield: 34%. Mp: 201–203 °C.
H NMR
(
400 MHz, CDCl
ArH), 7.39(d, J = 2.70 Hz, 1H, ArH),7.31(m, 1H, ArH), 7.27(m, 4H,
ArH),6.93(m, 4H, ArH), 5.13(s, 2H, eCH ), 4.20(s, 1H, eCH), 2.67(s,
3
): 8.22(s, 1H, ArH), 8.09(m, 1H, ArH), 7.49(m, 1H,
4
.2.13. 2-((4-(3,4-Dichlorophenyl)piperazin-1-yl)methyl)chromeno[4,3-c]
2
pyrazol-4(2H)-one (4m)
1
3
1
2
4H, eCH CH
2
e), 2.40(s, 4H, eCH
2
CH
2
e). C NMR (125 MHz, CDCl
3
):
Light yellow power, yield: 53%. Mp: 216–219 °C.
H NMR
1
61.87(J = 292.5 Hz)(2), 158.24, 152.98, 149.10, 137.96(J = 3 Hz)
(
400 MHz, CDCl ): 8.28(s, 1H, ArH), 8.07(m, 1H, ArH), 7.47(m, 1H,
3
(
2), 132.32, 130.42, 129.18(J = 9 Hz)(4), 124.54, 122.82, 117.66,
ArH), 7.37(d, J = 8.20 Hz, 1H, ArH), 7.31(m, 1H, ArH), 7.24(d,
1
5
15.49(J = 25.5 Hz)(4), 115.03, 107.94, 74.31, 74.19, 51.46(2),
J = 9.00 Hz, 1H, ArH), 6.90(d, J = 2.80 Hz, 1H, ArH), 6.67 (m, 1H,
0.13(2). MS(ESI) m/z: 487.26 [M+H]+ (Calcd for 487.52,
ArH), 5.17(s, 1H, eCH
2
2
), 3.17(t, J = 4.96 Hz, 4H, eCH
2
CH
2
3
e), 2.80(t,
1
3
C
28
H
25
F
2
N
4
O ).
2
J = 4.96 Hz, 4H, eCH
CH
2
e). C NMR (125 MHz, CDCl
): 158.13,
1
1
4
52.95, 150.43, 149.27, 132.84, 132.43, 130.51, 130.49, 124.57,
22.79, 122.71, 117.64, 117.62, 115.75, 114.88, 108.02, 74.14,
4
.2.19. 2-((4-Ethylpiperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-4(2H)-
one (4s)
White power, yield: 44%. Mp: 185–189 °C. H NMR (400 MHz,
CDCl ): 8.24(s, 1H, ArH), 8.06(d, J = 7.79 Hz, 1H, ArH), 7.45 (m, 1H,
ArH), 7.36(d, J = 7.88 Hz, 1H, ArH), 7.29 (m, 1H, ArH), 5.10(s, 2H,
+
9.76(2), 48.80(2). MS(ESI) m/z: 430.18 [M+H] (Calcd for 430.30,
C
21
H
19Cl
2
4
N O
2
).
1
3
4
.2.14. 2-((4-(Pyridin-2-yl)piperazin-1-yl)methyl)chromeno[4,3-c]
eCH
2
), 2.71(s, 4H, eCH
2
CH
2
e), 2.49(s, 4H, eCH
2
CH
2
e), 2.39(m, 2H,
pyrazol-4(2H)-one (4n)
1
3
1
eCH ), 1.03(s, 3H, eCH ).
C NMR (125 MHz, CDCl ): 158.18,
Light yellow power, yield: 49%. Mp: 192–194 °C.
H
NMR
2
3
3
1
1
52.91, 149.13, 132.36, 130.34, 124.49, 122.76, 117.59, 114.92,
(
400 MHz, CDCl ): 8.28(s, 1H, ArH), 8.13(m, 1H, ArH), 8.06 (t,
3
07.84, 74.28, 52.49, 52.19(2), 49.94(2), 11.94. MS (ESI): 313.17 [M
J = 6.20 Hz, 1H, ArH), 7.45(m, 2H, ArH), 7.37–7.28(m, 2H, ArH),
+
+
H] (Calcd for 313.37, C17
H
21
N
4
O ).
2
6
.60(t, J = 6.20 Hz, 2H, ArH), 5.17(s, 2H, eCH ), 3.58(t, J = 5.02 Hz,
13
2
4
2
H, eCH CH
2
e), 2.77(t, J = 5.05 Hz, 4H, eCH
2
CH
2
e).
C NMR
(
125 MHz, CDCl
3
): 159.11, 158.15, 152.94, 149.20, 147.93, 137.58,
4
.2.20. 2-((4-(2-Hydroxyethyl)piperazin-1-yl)methyl)chromeno[4,3-c]
pyrazol-4(2H)-one (4t)
White power, yield: 36%. Mp: 200–202 °C. H NMR (400 MHz,
CDCl ): 8.25(s, 1H, ArH), 8.07(m, 1H, ArH), 7.47(m, 1H, ArH), 7.36(d,
J = 8.20 Hz, 1H, ArH), 7.31(m, 1H, ArH), 5.11(s, 2H, eCH ), 3.57(t,
J = 5.30 Hz, 2H, eCH ), 2.71(s, 4H, eCH CH e), 2.56(s, 4H,
eCH CH e), 2.53(t, J = 5.35 Hz, 2H, eCH ). C NMR (125 MHz,
CDCl ): 158.18, 152.96, 149.19, 132.33, 130.41, 124.53, 122.79,
1
1
32.39, 130.41, 124.52, 122.79, 117.61, 114.91, 113.62, 107.97,
+
07.21, 74.39, 49.92(2), 45.11(2). MS(ESI) m/z: 362.18 [M+H]
1
(
Calcd for 361.41, C20
H
20
N
5
2
O ).
3
2
4
.2.15. 2-((4-Benzylpiperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-
2
2
2
1
3
4
(2H)-one (4o)
2
2
2
1
White power, yield: 41%. Mp: 169–172 °C. H NMR (400 MHz,
3
DMSO): 8.23(s, 1H, ArH), 8.05(d, J = 7.70 Hz, 1H, ArH), 7.46(m, 1H,
ArH), 7.38(d, J = 8.12 Hz, 1H, ArH), 7.30(m, 1H, ArH), 7.28–7.21(m,
117.62, 114.94, 107.93, 74.24, 59.19, 57.78, 52.62(2), 50.01(2). MS
(ESI): 329.24 (C17
H
21
N
4
O
3
, [M+H]+ (Calcd for 329.37, C17
H
21
N
4
O ).
3
5
H, ArH), 5.11(s, 2H, eCH
2
), 3.48(s, 2H, eCH ), 2.69(s, 4H,
2
1
3
eCH
2
CH
2
e), 2.49(s, 4H, eCH
2
CH
2
e). C NMR (125 MHz, CDCl ):
3
4
.2.21. 2-(Piperidin-1-ylmethyl)chromeno[4,3-c]pyrazol-4(2H)-one (4u)
1
1
4
58.20, 152.94(2), 149.11, 132.29, 130.35, 129.13, 128.29(2), 127.20,
1
White power, yield: 49%. Mp: 187–190 °C. H NMR (400 MHz,
CDCl ): 8.23(s, 1H, ArH), 8.09(d, J = 7.64 Hz, 1H, ArH), 7.47(t,
J = 5.21 Hz, 1H, ArH), 7.38(d, J = 8.36 Hz, 1H, ArH), 7.31(m, 1H,
ArH), 5.10(s, 2H, eCH ), 2.60(s, 4H, eCH CH e), 1.60(d, J = 3.80 Hz,
H, eCH CH e), 1.38(d, J = 3.92 Hz, 2H, eCH
): 158.17, 152.94, 149.22, 132.18, 130.28, 124.48, 122.77,
24.50, 122.78(2), 117.62, 114.98, 107.89, 74.35, 62.88, 52.77(2),
+
3
9.99(2). MS(ESI) m/z: 375.33 [M+H]
(Calcd for 375.44,
C
22
H
23
N
4
O ).
2
2
2
2
1
3
4
2
2
2
). C NMR (125 MHz,
4
.2.16. 2-((4-Benzhydrylpiperazin-1-yl)methyl)chromeno[4,3-c]pyrazol-
CDCl
3
4
(2H)-one (4p)
1
2
17.61, 114.79, 107.94, 75.42, 51.40(2), 25.87(2), 23.60. MS (ESI):
1
White power, yield: 36%. Mp: 191–194 °C. H NMR (400 MHz,
, [M+H]+ (Calcd for 285.32, C16
85.14 (C16
H
17
N
4
O
2
H
17
N
4
2
O ).
CDCl ): 8.23(s, 1H, ArH), 8.09(t, J = 7.62 Hz, 1H, ArH), 7.48(t,
3
J = 7.71 Hz, 1H, ArH), 7.39(d, J = 8.40 Hz, 1H, ArH), 7.34(m, 5H,
ArH), 7.23(m, 4H, ArH), 7.15(m, 2H, ArH), 5.12(s, 2H, eCH ), 4.21(s,
4
.2.22. 2-(Piperazin-1-ylmethyl)chromeno[4,3-c]pyrazol-4(2H)-one (4v)
2
1
1
3
White power, yield: 52%. Mp: 265–268 °C. H NMR (400 MHz,
1
H, eCH), 2.68(s, 4H, eCH
2
CH
2
e), 2.44(s, 4H, eCH
2
CH e). C NMR
2
DMSO): 8.82(s, 1H, ArH), 8.00(d, J = 6.30 Hz, 1H, ArH), 7.52–7.58(m,
(
125 MHz, CDCl
3
): 158.27, 152.98, 149.06, 142.50(2), 132.38, 130.37,
1
H, ArH), 7.41–7.44(d, J = 8.28 Hz, 1H, ArH), 7.34–7.39(t,
1
28.56(4), 127.86(2), 127.05(2), 124.53, 122.84, 117.64, 115.08,
J = 7.50 Hz, 1H, Ar), 5.15(s, 2H, eCH
2
), 3.31(s, 4H, eCH
2
CH e),
2
1
07.90, 76.06, 74.26, 51.64(2), 50.20(2). MS(ESI) m/z: 451.21 [M
1
3
+
2.61(s, 4H, eCH CH e), 1.03–1.07(t, J = 6.99 Hz, 1H, eNH). C NMR
+
H] (Calcd for 451.54, C28
H
27
4
N O
2
).
2
2
(
125 MHz, CDCl
3
): 158.13, 152.94, 149.11, 132.29, 130.33, 124.46,
1
22.79, 117.64, 114.94, 107.88, 74.32, 51.14(2), 50.91(2). MS(ESI) m/
4
.2.17. 2-((4-((4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl)methyl)
+
z: 284.23 [M+H] (Calcd for 284.33, C16
H
18
N
3
O ).
2
chromeno[4,3-c]pyrazol-4(2H)-one (4q)
1
Light yellow power, yield: 39%. Mp: 228–230 °C.
H NMR
(
400 MHz, CDCl
3
): 8.22(s, 1H, ArH), 8.09(d, J = 7.65 Hz, 1H, ArH),
4.2.23. 2-(Morpholinomethyl)chromeno[4,3-c]pyrazol-4(2H)-one (4w)
White power, yield: 58%. Mp: 135–138 °C. 1H NMR (400 MHz,
CDCl3): 8.26(s, 1H, ArH), 8.08(m, 1H, ArH), 7.48(m, 1H, ArH), 7.38(d,
7
7
4
1
1
1
4
.48(m, 1H, ArH), 7.39(d, J = 7.77 Hz, 1H, ArH), 7.33 (m, 5H, ArH),
.25–7.15(m, 5H, ArH), 5.12(s, 2H.eCH ), 4.19(s, 1H, eCH), 2.67(s,
2
1
3
H, eCH
2
CH
2
e), 2.42(s, 4H, eCH
2
CH
2
e). C NMR (125 MHz, CDCl
3
):
J = 7.76 Hz, 1H, ArH), 7.32(m, 1H, ArH), 5.09(s, 2H, eCH
2
), 3.73(t,
CH e).
): 158.14, 152.98, 149.26, 132.33, 130.46,
58.25, 152.98, 149.09, 141.90, 141.08, 132.69, 132.31, 130.40,
29.11(2), 128.74(2), 128.68(2), 127.76(2), 127.29, 124.53, 122.82,
J = 4.66 Hz, 4H, eCH
2
CH
2
e), 2.67(t, J = 4.66 Hz, 4H, eCH
2
2
1
3
C NMR (125 MHz, CDCl
3
17.65, 115.04, 107.94, 75.28, 74.22, 51.54(2), 50.14(2). MS(ESI) m/z:
124.55, 122.78, 117.66, 114.90, 108.04, 74.51, 66.71(2), 50.32(2). MS
+
(ESI) m/z: 286.18 [M+H]+ (Calcd for 286.30, C15
85.78 [M+H] (Calcd for 485.98, C28
H26ClN
4
O
2
).
H
16
N
3
O ).
3
9

Y. Yin, et al.
Bioorganic Chemistry 92 (2019) 103238
4
.3. Cell proliferation assay
prepared by minimization with CHARMM force field. Homology
Modeling in Discovery Studio was used to treat with the missing re-
sidues in protein, which mainly based on MODELER program.
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.
Four human cell lines HCT-116, A549, HL60 and Huh7 were cul-
tured growth medium (RPMI-1640 medium with 100 mg/mL strepto-
mycin, 100 U/mL penicillin and 10% FBS, and incubated at 37 °C under
the atmosphere of 5% CO
2
and 20% O .
2
The antiproliferative activity in vitro was measured using the MTT
assay. The tested compounds were dissolved in DMSO and diluted to
the different concentrations. Cells in logarithmic phase were harvested
Acknowledgments
4
and divided into 96-well plates (0.5 × 10 each well), culture medium
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).
containing the test compounds at different concentrations were added
to each well and incubated for 48 h. Viable cells were determined by 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.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103238.
4.4. PI3K enzyme assay
The inhibition of PI3K activity was determined by competitive
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