Design, synthesis, and cytotoxic activity of novel 2H-imidazo[1,2-c]pyrazolo[3,4-e]pyrimidine derivatives

Article abstract of DOI:10.1016/j.bioorg.2021.104711

In this study, a series of novel 2H-imidazo [1, 2-c] pyrazolo [3, 4-e] pyrimidine derivatives were designed, synthesized, and evaluated for their cytotoxic activities. The in vitro cell growth inhibition assay of the target compounds indicated their selectivity in inhibiting the proliferation of blood tumor cells (K562, U937) and solid tumor cells (HCT116, HT-29). Compound 9b exhibited the highest antiproliferative activities against K562 (IC₅₀ = 5.597 μM) and U937 (IC₅₀ = 3.512 μM). Based on the flow cytometry assays, compound 9b caused obvious induction of cell apoptosis and cell arrest at the S phase. Furthermore, western blot analysis revealed that compound 9b upregulated the expression of Bax, downregulated the levels of Bcl-2, and further activated caspase-3 in K562 cells. Therefore, compound 9b may be a potential anticancer agent that deserves further investigation.

Full text of DOI:10.1016/j.bioorg.2021.104711

Bioorganic Chemistry 109 (2021) 104711
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Short communication
Design, synthesis, and cytotoxic activity of novel 2H-imidazo[1,2-c]
pyrazolo[3,4-e]pyrimidine derivatives
a
,
b
,
*
,
1
a
,
b
,
1
c,
1
a
,
b
a
,
b
a,b
You-Guang Zheng
, Xin Pei
, De-Xin Xia , Yuan-Bo Wang , Ping Jiang , Lin An
a,b
,
a
,
b
Tong-Hui Huang , Yun-Sheng Xue
a
College of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China
b
c
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China
Department of Radiology, XuZhou Central Hospital, Xuzhou 221004, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, a series of novel 2H-imidazo [1, 2-c] pyrazolo [3, 4-e] pyrimidine derivatives were designed,
synthesized, and evaluated for their cytotoxic activities. The in vitro cell growth inhibition assay of the target
compounds indicated their selectivity in inhibiting the proliferation of blood tumor cells (K562, U937) and solid
tumor cells (HCT116, HT-29). Compound 9b exhibited the highest antiproliferative activities against K562 (IC50
Cytotoxic activities
Pyrazole derivatives
Imidazole derivatives
Antitumor agent
=
5.597 µM) and U937 (IC50 = 3.512 µM). Based on the flow cytometry assays, compound 9b caused obvious
induction of cell apoptosis and cell arrest at the S phase. Furthermore, western blot analysis revealed that
compound 9b upregulated the expression of Bax, downregulated the levels of Bcl-2, and further activated
caspase-3 in K562 cells. Therefore, compound 9b may be a potential anticancer agent that deserves further
investigation.
1
. Introduction
There has been remarkable progress in the application of nitrogen
as crizotinib (bearing a pyrazole skeleton) [28], osimertinib (bearing a
pyrimidine skeleton) [29], zoledronic acid (bearing an imidazole skel-
eton) [30], etc., have been approved in the market (Fig. 1). Since 2008,
our research group has been focusing on the discovery of nitrogen het-
erocyclic scaffolds to be used as anticancer agents. The previous studies
reported several pyrimidine and pyrazole-benzimidazole derivatives
[31–34] showing significant anticancer activities.
In view of the previous studies, novel 2H-imidazo[1,2-c]pyrazolo
[3,4-e]pyrimidine derivatives were designed by combining pyrazole,
pyrimidine, and imidazole skeletons (Fig. 2). In this paper, the target
compounds were synthesized, and their biological activities were
evaluated.
heterocyclic derivatives in medicinal chemistry for over a century [1–6].
Pyrazole motif has received much attention as nitrogen-containing
heterocyclic compounds possessing various biological activities, such
as anticancer [7], analgesic [8], antibacterial [9], anti-HIV [10], and
antiviral properties [2,11]. In addition, pyrimidine derivatives act as
important pharmacophores in drug discovery and exhibit a wide range
of biological activities, including antibacterial [12], anticonvulsant
[
13], antifungal [14], anti-HIV [15], antihypertensive [16], anti-
inflammatory [4], antimalarial[17] and anticancer activities
3,18,19]. Furthermore, the imidazole derivatives possess excellent
antibacterial [20], antifungal [21], anti-HIV [22], antihypertensive
[
2. Results and discussion
[
23], anti-inflammatory [24], and anticancer [25–27] activities.
A malignant tumor is one of the most common diseases that threaten
2.1. Chemistry
human life. Therefore, the discovery of anticancer drugs has always been
a subject of frontier research throughout the world. In the past 20 years,
several anticancer drugs containing nitrogen heterocyclic scaffolds, such
The overall synthetic route of compound (9) using 4-nitro-1H-pyra-
zole-3-carboxylic acid as the starting material is outlined in Scheme 1.
*
Corresponding author at: College of Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China; Jiangsu Key Laboratory of New Drug Research and
Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, PR China.
E-mail address: zhengyouguang@xzhmu.edu.cn (Y.-G. Zheng).
1
You-Guang Zheng, Xin Pei and De-Xin Xia contribute equally to the article.
https://doi.org/10.1016/j.bioorg.2021.104711
Received 26 November 2020; Received in revised form 25 January 2021; Accepted 30 January 2021
Available online 8 February 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

Y.-G. Zheng et al.
Bioorganic Chemistry 109 (2021) 104711
Fig. 1. nitrogen-containing heterocyclic derivatives as anticancer drugs.
compound (7), which was treated with phosphorus oxychloride to
obtain compound (8). Compound (9) was prepared by reacting com-
pound (8) with different amine derivatives.
2
2
.2. Biological activities
.2.1. Antiproliferation assay
The novel 2H-imidazo [1,2-c]pyrazolo [3, 4-e]pyrimidine de-
rivatives (9a-9n) were initially evaluated for their antiproliferative ac-
tivities against blood tumor cells (K562, U937) and solid tumor cells
(
HCT116, HT-29) at a single concentration of 10 µM. As shown in
Table 1, the target compounds offered selectivity in inhibiting the pro-
liferation of blood tumor cells and solid tumor cells. Most compounds
exhibited potential cytotoxicity against K562 and U937 cells, while they
display poor activities against HCT116 and HT-29 cells. It was found
that compound 9b showed > 80% growth inhibition against the two
blood tumor cells.
The IC50 values of the target compounds against the K562 and U937
cells were determined. The results of the antiproliferative activities are
given in Table 2. The selected compounds exhibited potent cytotoxicity
against these tumor cell lines. Most of the compounds possessing an
Fig. 2. Design of novel 2H-imidazo [1, 2-c] pyrazolo [3, 4-e] pyrimidine
derivatives.
2
electron withdrawing group substituted benzene ring at the R position
(
such as 9b, 9e, 9g, and 9k) showed a more potent antiproliferation
Compound (4) was obtained by sequential esterification, ammoniation,
von Braun amide dehydration, and reduction. Compound (5) was pre-
pared by treating compound (4) with thiophosgene. Compound (6) was
synthesized from various bromoacetophenone derivatives via the dele-
pine reaction. Compound (5) was reacted with compound (6) to form
activity (IC50 values of 3.512–13.945 µM) compared to the compounds
(9d, 9i, and 9m) containing a cycloalkyl group (growth inhibition ratio
< 35% at 10 µM). Compared to the antitumor drug 5-FU (IC
5
0 =
4.40
µM), the tested compounds (such as 9b with an IC value of 8.09 µM)
5
0
showed lower toxic effects on the normal peripheral blood mononuclear
Scheme 1. Synthesis of compounds 9a-9n. Reagents and conditions: (a) SOCl
2
, EtOH; (b) NH
4
OH, reflux; (c) POCl
3
; (d) SnCl
2
⋅2H
2 2
O, HCl; (e) CSCl , DCM; (f)
Hexamethylenetetramine; (g) Na
2
CO
3
, H
2
O, CH
2
Cl
2
, EtOH; (h) POCl
3
, DMF; (j) PTSA, n-Butanol.
2

Y.-G. Zheng et al.
Bioorganic Chemistry 109 (2021) 104711
Table 1
flow cytometry analysis was performed for the K562 cells treated with
different concentrations of the compound. A dose-dependent S phase
transition arrest was observed in the K562 cells treated with 9b, as
shown in Fig. 4 and Table S2. The treatment of K562 cells with 10 µM
concentration of compound 9b resulted in 53.99% of cell arrest at the S
phase compared to 33.27% in the untreated cultures.
The growth inhibition ratio of the target compounds (10 µM/72
h).
1
2
2.2.4. Western blot analysis of the expression of Bax, Bcl-2, and caspase-3
The Bcl-2 protein family (including anti-apoptotic protein Bcl-2 and
pro-apoptotic protein Bax) is a group of key apoptotic proteins that play
an important role in the regulation of apoptosis [35,36]. The caspase
family (such as caspase-3) is a large group of apoptotic regulatory genes
that mediate apoptosis [37,38]. The mechanism for the induction of
cellular apoptosis was proposed by evaluating the expression of proteins
related to the apoptotic pathway such as Bax, Bcl-2, and caspase-3
protein using the western blot analysis. The K562 cells were treated
with different concentrations of 9b (2, 4, 8 µM), and the expressions of
Bax, Bcl-2, and caspase-3 protein were detected. The addition of 8 µM of
compound 9b significantly increased the expression of Bax and
decreased the levels of Bcl-2, as shown in Fig. 5. Furthermore, the
treatment of the cells with 4 and 8 µM of 9b increased the expression
levels of caspase-3, indicating the induction of apoptosis in the K562
cells by increasing the activation of caspase-3.
Compd
R
R
Growth inhibition ratio (%)
K562
35.07
U937
33.40
HCT116
14.81
HT-29
17.04
9
9
a
b
Me
Me
83.94
85.07
34.25
35.80
9
9
c
Me
Me
29.95
11.71
29.44
11.39
14.42
1.57
10.85
6.69
d
9
9
9
e
f
Me
Br
61.34
36.77
58.97
67.85
15.28
32.50
33.70
31.82
32.05
30.93
7.51
g
Br
36.51
9
9
h
i
Br
Br
32.90
30.82
53.06
33.40
23.82
24.37
31.24
22.01
3
. Conclusions
In summary, novel 2H-imidazo[1,2-c]pyrazolo[3,4-e]pyrimidine
9
9
j
F
F
60.43
48.33
65.07
27.22
35.11
16.22
32.56
27.69
derivatives were designed and synthesized to be used as antitumor
agents. The in vitro cell growth inhibition assays indicated the potent
cytotoxicity of the synthesized compounds against K562 and U937 cells.
Among them, compound 9b exhibited the highest antiproliferative ac-
tivities against K562 (IC50 = 5.597 µM) and U937 (IC50 = 3.512 µM).
The flow cytometry assays revealed that compound 9b caused obvious
induction of cell apoptosis and cell arrest at the S phase. Furthermore,
compound 9b induced apoptosis in K562 cells by increasing the
expression of Bax, decreasing the levels of Bcl-2, and activating the
caspase-3 protein. Therefore, compound 9b may be used as a potential
anticancer agent that requires further research.
k
9
9
l
F
F
6.77
0.44
48.54
4.44
6.90
1.02
15.92
1.72
m
9
n
F
19.93
5.69
2.59
2.94
Table 2
Cytotoxic activities of the target compounds.
4
. Experimental
Compounds M)
IC50
(
μ
4.1. Chemistry
K562
U937
PBMC (mouse)
Unless otherwise noted, all reagents were purchased from commer-
cial sources and used without further purification. All compounds were
routinely monitored by thin-layer chromatography with silica gel GF-
9
9
9
9
9
9
5
b
5.597
3.512
11.24
>10
8.09
21.40
NT
e
11.724
8.081
>10
g
1
h
j
22.767
3.93
NT
254 glass plates and viewed under UV light at 254 nm. The H NMR
11.102
13.945
2.11
22.96
19.92
4.40
13
6
and C NMR spectra were determined in DMSO‑d on a JNM-ECZ400s/
k
>10
L spectrometer. Chemical shifts (δ) were reported in parts per million
ppm) relative to tetramethylsilane (TMS), which was used as an in-
-FU
1.38
(
ternal standard. The mass spectra (MS) were obtained from Agilent
1100LC/MS Spectrometry Services. HR-MS was obtained using a Q-tof
high resolution mass spectrometer. The purity was obtained using Agi-
lent 1260 HPLC instrument with ZORBAX SB-C18 chromatographic
column. Melting point was obtained using X-4A melting point analyzer.
The chemical synthesis and the spectrogram data of the final prod-
ucts were put in the supplementary material.
cells of the mouse (PBMC, mouse).
Compound 9b was found to exhibit the most potent inhibition to-
ward cancer cells and was selected for further study.
2
.2.2. Apoptosis analysis
To confirm the induction of apoptosis by 9b, the K562 cells were
treated with various concentrations of the compound (2.5, 5, 10 µM). On
treatment with 9b, the percentage of apoptotic K562 cells was increased
from 2.86% (control) to 49.65%, as shown in Fig. 3 and Table S1. This
suggested that compound 9b caused obvious induction of cell apoptosis
in a concentration-dependent manner.
4
.2. Antiproliferation assay
00
4
1
μL cell suspension (3.5 × 10 cells/mL) was added to each well
in 96-well plate and then treated with different concentrations of target
compounds after 24 h. CCK-8 staining was performed to determine the
OD value and calculate the inhibition rate after 72 h. IC50 was calculated
by LOGIT method.
2
.2.3. Cell cycle arrest
To determine the effect of compound 9b on cell cycle progression,
3

Y.-G. Zheng et al.
Bioorganic Chemistry 109 (2021) 104711
Fig. 3. Compound 9b induced cell apoptosis in a concentration-dependent manner.
Fig. 4. Compound 9b induced cell cycle arrest at S phase in K562.
4

Y.-G. Zheng et al.
Bioorganic Chemistry 109 (2021) 104711
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104711.
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