Design, synthesis, and biological evaluation of 4-benzoylamino-1H-pyrazole-3-carboxamide derivatives as potent CDK2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113281

Cyclin-dependent kinases play significant roles in cell cycle progression and are promising targets for cancer therapy. However, most potent CDK inhibitors lack the balance between efficacy and safety because of poor selectivity. Given the roles of CDK2 i

Full text of DOI:10.1016/j.ejmech.2021.113281

European Journal of Medicinal Chemistry 215 (2021) 113281
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis, and biological evaluation of 4-benzoylamino-1H-
pyrazole-3-carboxamide derivatives as potent CDK2 inhibitors
,
b
,
c
,
,
,
,
, b, d
Tingting Lin a
d, Jiacheng Li b d, Liping Liu b d, Yuanqing Li b d, Hualiang Jiang a
,
Kaixian Chen a d, Pan Xu b *, Cheng Luo a
**, Bing Zhou c
,
b
,
,
,
b,
d
,
, d, ***
a Shanghai Institute for Advanced Immunochemical Studies, and School of Life Science and Technology, ShanghaiTech University, Shanghai, 200031, China
b The Center for Chemical Biology, Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China
c Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zuchongzhi Road, Shanghai, 201203, China
d University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclin-dependent kinases play significant roles in cell cycle progression and are promising targets for
cancer therapy. However, most potent CDK inhibitors lack the balance between efficacy and safety
because of poor selectivity. Given the roles of CDK2 in tumorigenesis, selective CDK2 inhibition may
provide therapeutic benefits against certain cancer. In this study, a series of 4-benzoylamino-1H-pyr-
azole-3-carboxamide derivatives were designed, synthesized, and evaluated. The most selective com-
pound DC-K2in212 in this series exhibited high potency towards CDK2 and had effective anti-
proliferative activity against A2058 melanoma cell line and MV4-11 leukemia cell line while exhibiting
low toxic effect on human normal cell lines MRC5 and LX2. The molecular modeling illustrated that
compound DC-K2in212 had the similar binding mode with CDK2 as C-73, the most selective CDK2 in-
hibitor reported so far, which might account for selectivity against CDK2 over CDK1. Further biological
studies revealed that compound DC-K2in212 suppressed CDK2-associated downstream signaling
pathway, blocked cell cycle progression, and induced cellular apoptosis. Therefore, compound DC-
K2in212 could serve as a potential CDK2 inhibitor for further development.
Received 4 December 2020
Received in revised form
27 January 2021
Accepted 3 February 2021
Available online 11 February 2021
Keywords:
CDK2 inhibitor
Selectivity
Anti-proliferative potency
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
of CDKs is tightly regulated by its cyclin regulatory partners and
inhibitory proteins (CKIs).
Cyclin dependent kinases (CDKs) are serine/threonine protein
kinases that play significant roles in cell proliferation, apoptosis and
transcription [1]. There are 20 CDKs in human cells which can be
divided into two subfamilies represented as cell cycle-related
family (e.g. CDK1-6) and transcription-related family (e.g. CDK7-
13) according to their biological function [2]. The kinase activity
As an important member of CDK family, CDK2 plays a critical
role in regulating cell cycle progression. CDK2-cyclin E is involved
in late G1 to make the full phosphorylation of retinoblastoma (Rb)
to initiate S phase of cell cycle, whereas CDK2-cyclin A facilitates S/
G2 transition. Besides, CDK2 also plays a role in adaptive immune
response, apoptosis, cell differentiation, and normal DNA repair
[3e6]. CDK2 and its regulatory proteins are responsible for loss of
proliferation control, which might lead to cancer. For example,
CDK2 is highly expressed in glioblastoma, melanoma, and
lymphoid tumor tissues (Fig. S1), while its active regulator cyclin E
is frequently overexpressed in various cancer cells, and inhibitory
regulators p21 and p27 are often silenced during tumor progression
[7e9]. CDK2 is also a critical factor involved in metastasis of pros-
tate cancer [10], and inhibition of CDK2 could suppress prolifera-
tion of ovarian cancer cells with amplified CCNE1 expression [11],
induce apoptosis of MYCN-amplified neuroblastoma cells [12], and
* Corresponding author.
** Corresponding author. Shanghai Institute for Advanced Immunochemical
Studies, and School of Life Science and Technology, ShanghaiTech University,
Shanghai, 200031, China.
*** Corresponding author. Department of Medicinal Chemistry, State Key Labo-
ratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai, 201203, China.
E-mail addresses: xupan_322@simm.ac.cn, xvpan_322@simm.ac.cn (P. Xu),
cluo@simm.ac.cn (C. Luo), zhoubing@simm.ac.cn (B. Zhou).
https://doi.org/10.1016/j.ejmech.2021.113281
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
ATP-competitive inhibitors, which occupy the catalytic ATP binding
site. Flavopiridol is the first pan-CDK inhibitor entered in clinical
trials as a therapy for leukemia, breast cancer, colorectal cancer,
gastric cancer and etc., which inhibits CDKs and other protein ki-
nases [16,17]. Ongoing studies identified more selective CDK in-
hibitors and some have entered into clinical studies, such as
Seliciclib [18], Dinaciclib [19,20], AT7519 [21,22], BAY-1000394
[23,24], PHA-848125 [25,26] and etc [27,28]. However, most of
them lack the balance between efficacy and safety because of poor
selectivity, which might hinder further development. So far, three
selective CDK4/6 inhibitors (Palbociclib [29], Abemaciclib [30], and
Ribociclib [31]) have been approved by U.S. Food and Drug
Administration (FDA) for treatment of breast cancer, highlighting
the importance of selectivity in developing CDK inhibitor. However,
the most selective CDK2 inhibitor C-73 having been reported so far
had almost no anti-proliferative activity against cancer cells
[32,33]. Therefore, improving selectivity of CDK inhibitors towards
CDK2 with anti-proliferative activity remains an ongoing issue.
In this study, we chose a pan-CDK inhibitor AT7519 which is in
phase I clinical study of lymphocytic leukemia therapy as a starting
scaffold to design selective CDK2 inhibitors. AT7519 could effec-
tively inhibit kinase activity of CDK1, CDK2 and CDK9, and show
high anti-proliferation effect against various tumor cell lines, while
it also affects the viability of normal cell line MRC-5 with high
potency [34]. In order to optimize its selectivity towards CDK2, a
series of 4-benzoylamino-1H-pyrazole-3-carboxamide derivatives
were designed and synthesized as CDK2 inhibitors.
Abbreviations
CDK
CKI
Rb
cyclin-dependent kinase
cyclin-dependent kinase inhibitor
retinoblastoma protein
PI3K
BDR4
SSphos
phosphatidylinositol-3-kinase
bromodomain-containing protein 4
sodium 2’-(dicyclohexylphosphanyl)-2,6-
dimethoxy-[1,10-biphenyl]-3-sulfonate hydrate
1-ethyl-(3-dimethylaminopropyl)carbodiimide
hydrochiloride
EDCI
HOBt
Mcl-1
PI
1-Hydroxybenzotriazole
myeloid leukemia cell differentiation protein
propidium iodide
PARP
poly ADP-ribose polymerase
cause the death of BRCA1-deficient cancers [6]. Moreover, the
combination of CDK2 and phosphatidylinositol-3-kinase (PI3K)
inhibitors could induce cell death in colorectal cancer while dual
inhibition of CDK2 and bromodomain-containing protein 4 (BRD4)
could cause apoptosis in MYC-amplified medulloblastoma cells
[13,14]. Furthermore, inhibition of CDK2 might reverse acquired
resistance of CDK4/6 inhibitors [15]. Overall, the above studies all
suggest that CDK2 is a promising drug target for cancer therapy.
Over the past decades, a large number of CDK inhibitors with
various scaffolds have been discovered (Fig. 1). Most of them are
Fig. 1. Chemical structures of representative CDK inhibitors.
2

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
Table 1
2. Results and discussion
Enzymatic activity and in vitro anti-proliferative activity evaluated in melanoma cell
line A2058.
2.1. Drug design and cchemistry
Inspired by the discovery of selective CDK2 inhibitor C-73 [32],
we aligned AT7519 to the complex structure of CDK2-C-73. As
shown in Fig. 2, the 1H-pyrazole-3-carboxamide skeleton of
AT7519 forms three hydrogen bonds with the hinge region of CDK2
(residue Glu81 and Leu83) and the whole molecule occupies the
normal ATP binding site which is similar with C-73. Notably, the
piperidine of AT7519 overlaps with the sulfamoylphenyl part of C-
73. However, the 2, 6-dichlorophenyl of AT7519 shows a different
structure orientation with purine-proximal phenyl ring of C-73, and
AT7519 also lacks a group similar to the distal phenyl ring of C-73
which might promote selectivity.
Based on the structural information, exploration of the region
which is occupied by distal phenyl ring of C-73 could be a feasible
way to improve selectivity of AT7519 against CDK2. The 3-postion
in phenyl of AT7519 is closer to the above-mentioned region, thus
introducing groups at that position might help improve selectivity
towards CDK2. Moreover, fluorine has lower steric hindrance
relative to chlorine, which might make phenyl of AT7519 easier to
rotate to the similar conformation as purine-proximal phenyl ring
of C-73. However, the replacement of AT7519’s chlorine with
fluorine reduced the potency, while replacing piperidine with 4-
fluorophenyl at the same time increased the potency against
CDK2 (see Table 1). Compared to piperidine, the more rigid 4-
fluorophenyl may promote and stabilize the 1H-pyrazole-3-
carboxamide skeleton of compound anchoring to hinge region,
which may further lead to improved binding affinity with CDK2
protein and higher potency of compounds. Based on these con-
siderations, DC-K2in2 therefore represented a reasonable starting
point for further structure optimization.
Compd.
R1 R2
R3
IC50
(mM)
CDK1/cyclinB1 CDK2/cyclinA2 A2058
C-73
AT7519
e
e
e
>200
0.089
0.543
0.032
>100
0.166
Cl
H
DC-K2in1
DC-K2in2
F
F
H
H
0.594
0.014
0.250
0.006
NT
1.011
DC-K2in201
DC-K2in202
F
F
0.306
0.432
0.040
0.086
2.938
2.371
amide coupling of 4-nitro-1H-pyrazole-3-carboxylic acid with
corresponding amines provided compounds (2, 5), followed by
reduction of nitro with Pd/C, then acylation of compounds (3, 6)
with corresponding acids gave compounds (4, 7 and DC-K2in2).
Compound 4 was deprotected with 2 M HCl in dioxane to obtain
compound DC-K2in1. Compound 7 reacted with corresponding
boric acid or pinacol ester, catalyzed by [Pd (ally)Cl]2 and phos-
phorous ligand SSphos to give compounds (DC-K2in201 - DC-
Kin209, 8, and 9a-9c). Compound 8 was hydrolyzed by NaOH in
The synthesis schemes of compounds are depicted in Scheme 1,
Fig. 2. Comparison of the binding mode between AT7519 and C-73 within CDK2. Key residues of CDK2 are labeled, and hydrogen bonds in all panels are depicted by yellow dotted
lines. (A) Structure of C-73 and AT7519. (B) The binding mode of AT7519 in CDK2 (PDB code: 2VU3). (C) Alignment of AT7519 (green stick model) and C-73 (purple stick model)
within CDK2 (PDB code: 5NEV).
3

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
a
Scheme 1. Reagents and conditions: (i) Corresponding amine, EDCI, HOBt, DMF, r.t., 2h; (ii) Pd/C, MeOH: THF ¼ 3 : 1, H2, 40 ꢀC, 36h; (iii) Corresponding acid, EDCI, HOBt, DMF, rt,
2h; (iv) 2 M HCl in dioxane, r.t., DCM; (v) Corresponding boric acid or boric acid ester, [Pd (ally)Cl]2, SSphos, K3PO4, 1,4-dioxane: H2O ¼ 3 : 1, 100 ꢀC, 8h; (vi) NaOH, MeOH: H2O ¼ 1 :
1, 60 ꢀC, 2h; (vii) LiOH$H2O, MeOH: H2O ¼ 1 : 1, 40 ꢀC, 4h; (viii) Corresponding amine, EDCI, HOBt, DMF, r.t., 18h.
mixture of water and methanol at 60 ꢀC to give compound DC-
K2in212. Compounds (10a-10c) was obtained from 10a-10c by
hydrolysis, then reacted with corresponding amines to give com-
pounds (DC-K2in213 e DC-K2in217).
compounds DC-K2in201 and DC-K2in202 were more potent than
C-73 with IC50 of 40 nM and 86 nM against CDK2 while having
higher selectivity than AT7519 and DC-K2in2, which was consis-
tent with our expectation. Besides, compounds DC-K2in201 and
DC-K2in202 exhibited a bit lower anti-proliferative activity relative
to AT7519 with IC50 values of around 2 mM. Thus, further optimi-
2.2. Structure-activity relationships (SARs)
zation was performed based on DC-K2in201 and DC-K2in202.
According to the U-type conformation of C-73 in complex with
CDK2, we tried to modify the ortho-, meta- or para-postion in
pyridyl or phenyl of compound DC-K2in201 or DC-K2in202 to
produce the similar conformation as C-73. First, we introduced
different groups at R4 in pyridyl of DC-K2in201 or R5 in phenyl of
DC-K2in202. As show in Table 2, compared with DC-K2in201, DC-
K2in203 which had fluorine at R4 displayed similar potency in
in vitro enzymatic inhibition assay but the cellular activity dropped
At the beginning of our research, compound DC-K2in201 and
DC-K2in202, which contained pyridyl or phenyl at 3-position on
phenyl of compound DC-K2in2 were synthesized to validate our
design strategy. The kinase inhibition activity of compounds
against CDK1/cyclinB1 and CDK2/cyclinA2 was tested in vitro. CDK2
is highly expressed in melanoma cell lines. Moreover, it is reported
that in cells that are deficient in functional Rb but not p16INK4A, cell
cycle progression might dependent more on CDK2-mediated
phosphorylation of RBL2 [35], thus the anti-proliferative activity
of these compounds were evaluated in melanoma cell line A2058 in
which Rb protein is absent because of nonsense mutation of RB1
gene while p16INK4A is still functional. As shown in Table 1,
slightly. Compounds (DC-K2in204
e DC-K2in206) containing
electron-donating groups (methyl, methoxyl, and amino) at R4
reduced the potency against CDK1 and CDK2. Notably, DC-K2in212
with electron-withdraw group carbamoyl exhibited 17-fold
4

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
Table 2
Enzymatic activity and in vitro anti-proliferative activity evaluated in melanoma cell line A2058.
Compd.
X
R4
R5
R6
IC50 (mM)
CDK1/cycinB1
CDK2/cyclinA2
A2058
DC-K2in201
DC-K2in203
DC-K2in204
DC-K2in205
DC-K2in206
DC-K2in212
DC-K2in202
DC-K2in207
DC-K2in208
DC-K2in211
DC-K2in209
DC-K2in213
N
N
N
N
N
N
C
C
C
C
C
H
F
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
0.306
0.278
1.850
0.712
1.262
1.019
0.432
0.236
0.537
0.496
0.069
0.743
0.040
0.051
0.164
0.144
0.120
0.058
0.086
0.026
0.076
0.210
0.008
0.082
2.938
6.308
NT
Me
OMe
NH2
CONH2
H
H
H
H
H
NT
8.540
0.295
2.371
NT
NT
NT
CH2OH
COOMe
CONH2
1.989
3.340
C
H
DC-K2in214
DC-K2in215
DC-K2in216
DC-K2in217
C
C
C
C
H
H
H
H
H
0.350
0.723
1.299
3.689
0.067
0.073
0.169
0.485
NT
H
0.650
0.943
6.514
F
Me
NT (not test).
selectivity against CDK2 over CDK1 while showing effective anti-
proliferative activity in A2058 cell line with IC50 value of
0.295
K2in202, the inhibition activity of carbamoyl (DC-K2in209) sub-
stitution was more potent than any other substitutions including
hydroxyl (DC-K2in207), hydroxymethyl (DC-K2in208), and
methoxy formyl (DC-K2in211) when evaluated in vitro enzymatic
assay. DC-K2in209 showed over 8-fold selectivity against CDK2
over CDK1, while showing moderate anti-proliferative activity in
Table 3
Inhibitory profile of AT7519 and DC-K2in212 against different CDKs.
m
M. As for R5 substituents in phenyl of compound DC-
Kinase
AT7519 IC50 (nM)
DC-K2in212 IC50 (nM)
CDK1/cyclinB1
CDK2/cyclinA2
CDK3/cyclinE1
CDK4/cyclinD3
CDK6/cyclin D3
CDK7/cyclin H/MAT1
CDK9/cyclin T1
CDK12 wt/cyclin K
210a, 89b
47a,32b
360a
1019b
58b
113b
72b
3478b
>18000b
>18000b
1119b
>18000b
170a
2400a
<10a, 5b
NT
A2058 cell line with IC50 value of 1.989 mM. We then introduced N-
methylpiperidine methyl, 2-(N-methylpiperazine) ethyl and 3-
morpholinopropyl at amide of compound DC-K2in209 to obtain
compound DC-K2in213 e DC-K2in215. The activity of three de-
rivatives decreased, suggesting that large steric hindrance at R5
position were not benefit for enzymatic activity of compounds and
had little effect on the increase of selectivity. Interestingly, DC-
K2in215 showed relatively good anti-proliferative activity with IC50
NT (not test).
a
Reported by Matthew S. Squires et al. [34].
From this work.
b
As shown in Table 3, DC-K2in212 inhibited CDK1, CDK4, CDK6,
CDK7, CDK9, and CDK12 with IC50 values above 1 M, and CDK3
with IC50 value of 113 nM. Thus, DC-K2in212 was a potent CDK2
inhibitor with above 10-fold selectivity against CDK1, CDK4, CDK6,
CDK7, CDK9, and CDK12, while having around 2-fold selectivity
against CDK3.
m
value of 0.650 mM in A2058 cell line. We also added fluorine and
methyl at R6 in DC-K2in215, but it resulted in decreasing both
enzymatic activity and selectivity which might suggest steric hin-
drance at that position. Therefore, compound DC-K2in12 turned
out to be optimal in terms of selectivity and cellular activity in this
series.
2.4. Binding mechanism of DC-K2in212 to CDK2
2.3. CDKs selectivity of compound DC-K2in212
To better understand the mechanism of inhibition, we used
molecular docking to analyze the binding mode of DC-K2in212
with CDK2. The crystal structure of CDK2 in complex with C-73 was
selected for docking studies. As depicted in Fig. 3, DC-K2in212
exhibited U-type conformation similar to that of C-73 and
DC-K2in212 was then evaluated in a panel of structurally similar
CDK kinases including CDK3/cyclinE1, CDK4/cyclinD3, CDK6/cyclin
D3, CDK7/cyclin H/MAT1, CDK9/cyclin T1, and CDK12 wt/cyclin K.
5

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
Fig. 3. Binding mode analysis of DC-K2in212. The key residues of CDK2 are labeled, and hydrogen bonds in all panels are depicted by yellow dotted lines. (A) Docking conformation
of DC-K2in212 (orange stick model) with CDK2. (B) Comparison of binding mode of DC-K2in212 and C-73 (purple stick model) within CDK2.
Table 4
In vitro anti-proliferative activity of AT7519 and DC-K2in212 against cancer cells and normal cells.
Compd.
A2058 (IC50
mM)
MV4-11 (IC50 mM)
MRC5 (IC50 mM)
LX2 (Inhibition% at 100 mM)
AT7519
DC-K2in212
0.166
0.295
0.391
0.252
0.425
54.81
39.4
32.5
overlapped with C-73 at the same region. The pyrazole ring formed
three hydrogen bonds with Glu81 and Leu83 in hinge region of
CDK2. 4-(6-carbamoyl-3-pyridyl)-2, 6-difluorophenyl emulates the
position of biphenyl of C-73. The similar binding mode to C-73
might account for the selectivity of DC-K2in212.
phosphorylated by CDK4/6 and CDK2, the complete inhibition of Rb
phosphorylation might need block on both CDK2 and CDK4/6.
Therefore, treatment of DC-K2in212 in low concentrations for 24 h
exhibited mild inhibition on Rb phosphorylation, especially on
Ser780, phosphorylated mainly by CDK4/6 [37]. Besides, previous
study has indicated that CDK2 could regulate anti-apoptotic-
related protein myeloid leukemia cell differentiation protein
(Mcl-1) by phosphorylating to increase its stability [38]. Thus, the
effects of DC-K2in212 on Mcl-1 were analyzed. Treatment with DC-
K2in212 significantly downregulated Mcl-1 expression in A2058
and MV4-11 cells (Fig. 4C, D). Altogether, these results indicated
that DC-K2in212 could suppress CDK2-associated signaling
pathways.
2.5. In vitro anti-proliferative activity and toxicity evaluation of DC-
K2in212
In acute myeloid leukemia, CDK2 inhibition leads to reactivation
of differentiation pathway and arrest of tumor growth of AML [9].
Therefore, in addition to the human melanoma cell A2058, further
cellular anti-proliferative activity of DC-K2in212 was evaluated
against human myeloid leukemia cell MV4-11, meanwhile human
normal fibroblast cell MRC5 and human hepatic stellate cell LX2
were selected to evaluate toxicity of DC-K2in212 (Table 4). DC-
K2in212 significantly inhibited A2058 and MV4-11 cells growth
and displayed the similar high anti-proliferative activity as AT7519.
Furthermore, DC-K2in212 had lower toxic effect on human normal
cell lines MRC5 and LX2 than AT7519, which might be caused by
improved selectivity. Compared to AT7519, DC-K2in212 had
reduced activity against both CDK1 and CDK9 which played
essential roles in normal cell cycle and transcriptional regulation,
respectively [36]. This suggested that improved selectivity on CDK
family members might alleviate toxicity.
2.7. Effects of DC-K2in212 on cell cycle and apoptosis
In order to detect the effect of DC-K2in212 on cell cycle distri-
bution, A2058 cells were stained with PI probes and subjected to
flow cytometry analysis. As shown in Fig. 5A, treatment of
A2058 cells with DC-K2in212 at 0.2 mM induced accumulation of
cells in S phase with a corresponding loss of cells in G1 phase,
which was consistent with the effect of CDK2 knockdown using
siRNA in A2058 cells (Fig. S2). Besides, the percentage of
A2058 cells in S and G2/M phases were both increased at concen-
tration up to 0.4 mM, which might be related to the function of CDK2
in DNA damage response, accumulation of DNA damage may lead to
arrest in G2/M phase [6,39]. Overall, the data suggested that DC-
K2in212 might induce cell cycle arrest through different
mechanisms.
2.6. Effects of DC-K2in212 on CDK2-associated signaling pathway
To clarify effects of DC-K2in212 on signaling pathway regulated
by CDK2, Western blot analysis was used to detect the expression of
proteins modulated by CDK2 after treatment with DC-K2in212 at
different concentrations in MV4-11 and A2058 cells. Rb is the
phosphorylation target of CDK2 during late G1 phase, which is
absence in A2058 cells as the result of nonsense mutation of RB1, so
the level of phosphorylation of Rb was only detected in MV4-
11 cells. As shown in Fig. 4A and B, treatment of DC-K2in212
significantly inhibited Rb phosphorylation on Ser807/811 and
Ser780 in a dose and time dependent way, suggesting potent on-
target effects of DC-K2in212 in cells. Given that Rb is
CDK2 also regulates the process of cell apoptosis [4]. To deter-
mine the capacity of DC-K2in212 in inducing cell apoptosis, after
treating A2058 cells with DC-K2in212, Western blot and flow
cytometry were used to analyze. Fig. 4D suggested that cleaved
casepase-3 and cleaved PARP, two critical hallmarks of apoptosis,
were noticeably up-regulated by the treatment with DC-K2in212 in
a dose-dependent manner. As shown in Fig. 5B, the total proportion
of apoptotic cells were dependently increased from 9.72 to 68.6%
with concentrations, revealing that DC-K2in212 could induce
cellular apoptosis in a dose-dependent manner.
6

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
Fig. 4. DC-K2in212 inhibits the function of CDK2. (AeB) DC-K2in212 inhibits Rb phosphorylation. Immunoblot analysis of the indicated proteins in MV4-11 cells treated with DC-
K2in212 for 24 h (A) and 48 h (B). -Actin was used as the loading control. (C) Immunoblot analysis of Mcl-1 protein in MV4-11 cells treated with DC-K2in212 for 24 h, and GAPDH
b
was used as a loading control. (D) Immunoblot analysis of the indicated proteins in A2058 cells treated with DC-K2in212 for 24 h, and Tubulin was used as a loading control.
Fig. 5. Effects of DC-K2in212 on cell cycle and apoptosis of A2058 cells. (A) Cell cycle of A2058 cells treated with DC-K2in12. (B) Apoptosis of A2058 cells treated with DC-K2in212
at different concentrations after 48h.
7

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
3. Conclusions
5.0 Hz, 2H), 7.21 (t, J ¼ 8.8 Hz, 2H).
4-amino-N-(4-fluorophenyl)-1H-pyrazole-3-carboxamide (6).
Compound 6 (5.000g, 20.00 mmol) was dissolved in MeOH (30 ml)
and THF (30 ml), catalyzed with 10% Palladium on carbon (500 mg)
and then hydrogenated at 40 ꢀC for 36h. The reaction solution was
filtrated through Celite and concentrated to give 6 (3.301g, 75%) as
a brown foam without further purification.
4-(3-bromo-2,6-difluorobenzamido)-N-(4-fluorophenyl)-1H-
pyrazole-3-carboxamide (7). To a solution of compound 6 (3.000g,
13.63 mmol), EDCI (2.613g, 13.63 mmol), and HOBt (1.840g,
13.63 mmol) in DMF (20 ml) was added 3-bromo-2, 6-
difluorobenzoic acid (3.230g, 13.63 mmol). The mixture was stir-
red at ambient temperature for 2h and extracted with ethyl acetate
(40 ml ꢁ 3). The combined organic layers were dried over anhy-
drous sodium sulfate and concentrated in vacuum. The residue was
purified silica gel chromatography (2% DCM in MeOH) to give
compound 7 (4.188g, 70%) as white solid. 1H NMR (400 MHz,
CDK2 has emerged as a promising target for cancer therapy. Due
to toxicity caused by poor selectivity of CDK inhibitors, improving
selectivity against CDK2 has been an ongoing issue. In this study,
we designed, synthesized and evaluated 4-benzoylamino-1H-pyr-
azole-3-carboxamide derivatives as CDK2 inhibitors. These de-
rivatives showed potent inhibitory activity against CDK2 with IC50
values ranging from 6 nM to 485 nM, while having reduced activity
against CDK1 with IC50 values in the nanomolar to micromolar
ranges. Starting from pan-CDK inhibitor AT7519, several com-
pounds were designed and synthesized to validate our design
strategy that exploration of the region which is occupied by distal
phenyl ring of C-73 could be a feasible way to improve selectivity of
inhibitors against CDK2. Based on this idea further modification
was carried out, and finally DC-K2in212 was discovered as the most
selective CDK2 inhibitor in this series. DC-K2in212 exhibited
effective activity against CDK2 with IC50 value of 58 nM, meanwhile
it had weaker inhibitory activity against other CDK members
(CDK1, CDK3, CDK4, CDK6, CDK7, CDK9, and CDK12). Analysis of
binding mode illustrated that DC-K2in212 had the similar binding
mode with CDK2 as C-73, which might account for its selectivity.
Moreover, anti-proliferation assays showed that DC-K2in212
exhibited impressive activity against A2058 and MV4-11 cancer cell
lines and low toxic effect on human normal cell lines MRC5 and
LX2. Furthermore, DC-K2in212 suppressed CDK2-associated
downstream signaling pathway, arrested cell cycle, and induced
apoptosis. Accordingly, this study discovered a promising CDK2
inhibitor DC-K2in212 with good selectivity and high potency
which could be potential for further development.
DMSO‑d6)
d
13.60 (s,1H),10.44 (s, 2H), 8.44 (s,1H), 7.92 (dd, J ¼ 14.3,
8.3 Hz, 1H), 7.83 (dd, J ¼ 8.5, 5.1 Hz, 2H), 7.28 (t, J ¼ 8.9 Hz, 1H), 7.17
(t, J ¼ 8.7 Hz, 2H).
4-(2,6-difluorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-
carboxamide (DC-K2in1). White solid, 69% yield. 1H NMR
(400 MHz, MeOD-d4)
d 8.35 (s, 1H), 7.62e7.53 (m, 1H), 7.14 (t,
J ¼ 8.4 Hz, 2H), 4.20e4.10 (m, 1H), 3.50e3.42 (m, 2H), 3.13 (td,
J ¼ 12.6, 2.5 Hz, 2H), 2.17 (dd, J ¼ 13.8, 2.6 Hz, 2H), 1.93e1.82 (m,
2H).
4-(2,6-difluorobenzamido)-N-(4-fluorophenyl)-1H-pyrazole-
3-carboxamide (DC-K2in2). White solid, 75% yield. 1H NMR
(400 MHz, DMSO‑d6) d 13.57 (s, 1H), 10.43 (s, 1H), 10.31 (s, 1H), 8.43
(s, 1H), 7.85e7.77 (m, 2H), 7.67e7.57 (m, 1H), 7.26 (t, J ¼ 8.3 Hz, 2H),
7.16 (t, J ¼ 8.9 Hz, 2H).
4. Experimental
General procedure B for the synthesis of DC-K2in201 e DC-
K2in209, 8 and 9a-9c exemplified by DC-K2in202. To a solution
of compound 7 (50 mg, 0.114 mmol), phenylboronic acid (15 mg,
0.125 mmol) and potassium phosphate (36 mg, 0.171 mmol) in the
mixture of dioxane (3 ml) and water (1 ml) was added allylpalla-
dium chloride dimer (4 mg, 0.011 mmol) and SSPhos (12 mg,
0.023 mmol) under the protection of argon. The reaction was stir-
red at 100 ꢀC for 8h. After cooling, the mixture was extracted with
ethyl acetate (10 ml ꢁ 3) and the combine organic layers were dried
over anhydrous sodium sulfate then concentrated in vacuum. The
residue was purified by silica gel chromatography (2% DCM in
MeOH) to give compound DC-K2in202 (39 mg, 80%) as white solid.
4.1. Chemistry
General methods. All commercial reagents and solvents were
obtained from commercial suppliers and used without further
purification. Reactions were monitored by thin-layer chromatog-
raphy (visualized by UV fluorescence at
l
¼ 254 nm). All final
compounds were purified by silica gel chromatography with silica
gel 60H (200e300 mesh) manufacture by Qingdao Haiyang
chemical group Co. (China). 1H, 13C and 19F NMR spectral data were
recoded with BURKER AVANCE II 400 M, BURKER AVANCE III 500 M
or Varian MR-400 NMR spectrometer. High resolution ESI mass
analysis was recorded by Agilent G6520 Q-TOF high resolution
mass spectrometer. The purity of all final compounds was deter-
mined by Waters UPLC H-Class with ACQUITY UPLC BEH C18
1H NMR (400 MHz, DMSO‑d6)
d 13.57 (s, 1H), 10.40 (s, 1H), 10.35 (s,
1H), 8.45 (s, 1H), 7.81 (dd, J ¼ 8.7, 5.2 Hz, 2H), 7.71 (dd, J ¼ 15.2,
8.4 Hz, 1H), 7.59e7.43 (m, 5H), 7.35 (t, J ¼ 9.0 Hz, 1H), 7.15 (t,
J ¼ 8.9 Hz, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 162.01, 158.58 (dd,
JC-F ¼ 250.9, 6.5 Hz),158.57 (d, JC-F ¼ 240.9 Hz),156.81,156.02 (dd, JC-
reversed-phase column (2.1 mm ꢁ 50 mm, 1.7
m
m) and were
confirmed to be more than 95%. The analytical method was as
follows: flow rate, 0.5 ml/min; eluent A, 0.1% formic acid in water;
eluent B, neutral acetonitrile; gradient, 10% B to 100% B in 10min;
UV detection at 254 nm.
¼ 252.0, 7.0 Hz), 134.69 (d, JC-F ¼ 2.0 Hz), 133.93, 133.34, 132.94
F
(dd, JC-F ¼ 9.2, 4.3 Hz),128.90 (d, JC-F ¼ 1.4 Hz),128.72,128.18,125.51
(dd, JC-F ¼ 14.2, 3.4 Hz),122.61 (d, JC-F ¼ 7.7 Hz),122.13,121.61,115.14
(d, JC-F ¼ 22.1 Hz), 114.49 (t, JC-F ¼ 21.5 Hz), 112.49 (dd, JC-F ¼ 21.6,
2.9 Hz). HRMS (M þ H)þ calculated for C23H16F3N4Oþ2 437.1220,
found 437.1208.
5. Synthesis and characterization
4-(2,6-difluoro-3-(pyridin-3-yl)benzamido)-N-(4-
General procedure A for the synthesis of compounds DC-K2in1,
DC-K2in2 and 7 exemplified by compound 7.
fluorophenyl)-1H-pyrazole-3-carboxamide (DC-K2in201). White
solid, 48% yield. 1H NMR (400 MHz, Acetone-d6)
d 12.74 (s, 1H),
N-(4-fluorophenyl)-4-nitro-1H-pyrazole-3-carboxamide (5).A
mixture of 4-nitro-1H-pyrazole-3-carboxylic acid (10.000g,
63.69 mmol), 4-fluoroaniline (7.077g, 63.69 mmol), EDCI (12.209g,
63.69 mmol) and HOBt (8.605g, 63.69 mmol) in DMF (200 ml) was
stirred at ambient temperature for 2h and poured into water. A
precipitate was collected by filtration and concentrated to give the
title compound (13.056g, 82%) as brown-white solid. 1H NMR
10.27 (s,1H), 9.56 (s,1H), 8.81 (s,1H), 8.64 (d, J ¼ 4.8 Hz,1H), 8.54 (s,
1H), 8.01 (d, J ¼ 7.7 Hz, 1H), 7.92e7.85 (m, 2H), 7.79 (dd, J ¼ 15.0,
8.6 Hz, 1H), 7.51 (dd, J ¼ 7.9, 4.8 Hz, 1H), 7.33 (t, J ¼ 8.9 Hz, 1H), 7.13
(t, J ¼ 8.8 Hz, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 161.85, 159.00
(dd, J CeF ¼ 251.4, 6.8 Hz),158.48 (d, J CeF ¼ 240.7 Hz),156.57,156.19
(dd, J CeF ¼ 252.3, 7.1 Hz), 149.24 (d, J CeF ¼ 2.7 Hz), 149.17, 136.41,
134.63 (d, J CeF ¼ 2.0 Hz), 133.37, 133.04 (dd, J CeF ¼ 9.5, 4.1 Hz),
129.85, 123.73, 122.57 (d, J CeF ¼ 7.7 Hz), 122.28 (dd, J CeF ¼ 14.4,
(400 MHz, DMSO‑d6)
d
10.79 (s, 1H), 8.89 (s, 1H), 7.73 (dd, J ¼ 8.9,
8

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
3.3 Hz), 121.88, 121.67, 115.13 (d, J CeF ¼ 22.2 Hz), 114.60 (t, J
CeF ¼ 21.6 Hz), 112.78 (dd, J CeF ¼ 21.8, 2.9 Hz). HRMS (M þ H)þ
calculated for C22H15F3N5Oþ2 438.1172, found 438.1173.
DMSO‑d6)
d
161.87, 158.41 (dd, J CeF ¼ 251.3, 7.3 Hz), 157.67 (d, J
CeF ¼ 215.7 Hz), 157.56, 156.94e154.77 (m), 135.09, 134.64 (d, J
CeF ¼ 2.1 Hz), 133.36, 132.84 (dd, J CeF ¼ 9.0, 4.1 Hz), 129.82, 125.56
(dd, J CeF ¼ 14.4, 3.2 Hz), 122.59 (d, J CeF ¼ 7.8 Hz), 121.91, 121.61,
119.59, 115.80, 115.28, 115.15 (d, J CeF ¼ 22.2 Hz), 114.48 (t, J
CeF ¼ 21.8 Hz), 112.46 (dd, J CeF ¼ 22.0, 2.8 Hz). HRMS (M þ H)þ
calculated for C23H16F3N4Oþ3 453.1169, found 453.1164.
4-(2,6-difluoro-3-(6-fluoropyridin-3-yl)benzamido)-N-(4-
fluorophenyl)-1H-pyrazole-3-carboxamide(DC-K2in203). White
solid, 38% yield. 1H NMR (400 MHz, Acetone-d6)
d 12.70 (s, 1H),
10.25 (s, 1H), 9.55 (s, 1H), 8.54 (s, 1H), 8.46 (s, 1H), 8.25e8.17 (m,
1H), 7.90 (dd, J ¼ 9.1, 4.9 Hz, 2H), 7.80 (td, J ¼ 8.7, 6.2 Hz, 1H), 7.34
(td, J ¼ 8.9,1.3 Hz,1H), 7.25 (dd, J ¼ 8.6, 2.9 Hz,1H), 7.13 (t, J ¼ 8.9 Hz,
4-(2,4-difluoro-3’-(hydroxymethyl)-[1,10-biphenyl]-3-
carboxamido)-N-(4-fluorophenyl)-1H-pyrazole-3-
2H). 13C NMR (126 MHz, DMSO‑d6)
d
162.75 (d, J CeF ¼ 237.6 Hz),
carboxamide(DC-K2in208). White solid, 58%. 1H NMR (400 MHz,
161.90, 159.09 (dd,
J
CeF
¼
251.7, 6.7 Hz), 158.53 (d,
J
DMSO‑d6) d 13.58 (s,1H),10.43 (s,1H),10.37 (s,1H), 8.45 (s,1H), 7.81
CeF ¼ 240.8 Hz), 156.51, 156.18 (dd, J CeF ¼ 252.4, 7.0 Hz),147.33 (d, J
CeF ¼ 15.3 Hz), 142.44 (d, J CeF ¼ 6.4 Hz), 134.63 (d, J CeF ¼ 1.7 Hz),
133.33, 133.02 (d, J CeF ¼ 5.8 Hz), 128.20 (d, J CeF ¼ 3.6 Hz), 122.58
(d, J CeF ¼ 7.6 Hz), 121.95, 121.64, 121.18 (dd, J CeF ¼ 14.0, 2.3 Hz),
115.12 (d, J CeF ¼ 22.2 Hz), 114.57 (t, J CeF ¼ 21.5 Hz), 112.75 (dd, J
CeF ¼ 21.9, 2.4 Hz), 109.64 (d, J CeF ¼ 37.8 Hz). HRMS (M þ H)þ
calculated for C22H14F4N5Oþ2 456.1078, found 456.1079.
(dd, J ¼ 9.1, 5.1 Hz, 2H), 7.70 (td, J ¼ 8.7, 6.4 Hz, 1H), 7.50 (s, 1H),
7.48e7.32 (m, 4H), 7.15 (t, J ¼ 8.9 Hz, 2H), 5.28 (t, J ¼ 5.7 Hz, 1H),
4.57 (d, J ¼ 5.7 Hz, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 162.02,
158.60 (d, J CeF ¼ 240.8 Hz),159.66e157.51 (m),156.90,156.06 (dd, J
CeF ¼ 251.8, 6.9 Hz), 143.25, 134.71 (d, J CeF ¼ 1.7 Hz), 133.81, 133.41,
132.99 (dd, J CeF ¼ 8.5, 3.5 Hz),128.60,127.32,127.06,126.44,125.73
(dd, J CeF ¼ 14.3, 3.2 Hz), 122.68 (d, J CeF ¼ 7.7 Hz), 122.11, 121.69,
115.22 (d, J CeF ¼ 22.1 Hz), 114.53 (t, J CeF ¼ 21.5 Hz), 112.56 (dd, J
CeF ¼ 21.5, 2.1 Hz), 62.93. HRMS (M þ H)þ calculated for
4-(2,6-difluoro-3-(6-methylpyridin-3-yl)benzamido)-N-(4-
fluorophenyl)-1H-pyrazole-3-carboxamide (DC-K2in204). White
solid, 49% yield. 1H NMR (400 MHz, Acetone-d6)
d
12.70 (s, 1H),
C
24H18F3N4Oþ3 467.1326, found 467.1333.
10.25 (s, 1H), 9.54 (s, 1H), 8.73 (s, 1H), 8.53 (s, 1H), 7.94e7.84 (m,
3H), 7.76 (dd, J ¼ 15.0, 8.5 Hz, 1H), 7.39 (d, J ¼ 8.0 Hz, 1H), 7.32 (t,
J ¼ 8.8 Hz, 1H), 7.12 (t, J ¼ 8.8 Hz, 2H), 2.59 (s, 3H). 13C NMR
2,4-difluoro-N3-(3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)-[1,10-biphenyl]-3,30-dicarboxamide(DC-K2in209).
White
d 13.60 (s, 1H), 10.44 (s,
solid, 60%. 1H NMR (400 MHz, DMSO‑d6)
(126 MHz, DMSO‑d6)
d
161.94, 158.86 (dd, J CeF ¼ 251.6, 6.7 Hz),
1H), 10.39 (s, 1H), 8.46 (s, 1H), 8.10 (s, 1H), 8.06 (s, 1H), 7.94 (d,
J ¼ 7.7 Hz, 1H), 7.87e7.74 (m, 3H), 7.72 (d, J ¼ 7.7 Hz, 1H), 7.59 (t,
J ¼ 7.7 Hz, 1H), 7.48 (s, 1H), 7.39 (t, J ¼ 8.7 Hz, 1H), 7.15 (t, J ¼ 8.8 Hz,
158.54 (d, J CeF ¼ 240.9 Hz), 157.78, 156.64, 156.22 (dd, J
CeF ¼ 252.2, 6.9 Hz), 148.45 (d, J CeF ¼ 2.1 Hz), 136.50, 134.67,
133.36, 132.83 (d, J CeF ¼ 5.1 Hz), 126.92, 123.01, 122.59 (d, J
CeF ¼ 7.6 Hz), 122.43 (d, J CeF ¼ 14.9 Hz), 122.03, 121.65, 115.13 (d, J
CeF ¼ 22.2 Hz), 114.56 (t, J CeF ¼ 21.4 Hz), 112.70 (d, J CeF ¼ 20.1 Hz),
23.74. HRMS (M þ H)þ calculated for C23H17F3N5Oþ2 452.1329, found
452.1339.
2H). 13C NMR (126 MHz, DMSO‑d6)
d 167.69, 161.92, 158.70 (dd, J
CeF ¼ 250.9, 6.6 Hz), 158.53 (d, J CeF ¼ 240.1 Hz), 156.76, 156.03 (dd,
J
CeF ¼ 251.0, 5.8 Hz), 134.89, 134.63, 133.94, 133.39, 133.13 (d, J
CeF ¼ 8.2 Hz), 131.71, 128.79, 128.10, 127.39, 124.95 (dd, J CeF ¼ 14.0,
3.2 Hz), 121.93, 121.95 (d, J CeF ¼ 6.1 Hz), 121.68, 115.17 (d, J
CeF ¼ 22.2 Hz), 114.55 (t, J CeF ¼ 21.8 Hz), 112.64 (d, J CeF ¼ 21.5 Hz).
HRMS (M þ H)þ calculated for C24H17F3N5O3þ 480.1278, found
480.1289.
4-(2,6-difluoro-3-(6-methoxypyridin-3-yl)benzamido)-N-(4-
fluorophenyl)-1H-pyrazole-3-carboxamide(DC-K2in205). White
solid, 51% yield. 1H NMR (400 MHz, Acetone-d6)
d 12.68 (s, 1H),
10.25 (s, 1H), 9.56 (s, 1H), 8.54 (s, J ¼ 9.6 Hz, 1H), 8.38 (s, 1H),
4-(3-(6-cyanopyridin-3-yl)-2,6-difluorobenzamido)-N-(4-
fluorophenyl)-1H-pyrazole-3-carboxamide(8). White solid, 49%
7.94e7.87 (m, 3H), 7.73 (dd, J ¼ 15.1, 8.7 Hz, 1H), 7.29 (t, J ¼ 8.7 Hz,
1H), 7.12 (t, J ¼ 8.8 Hz, 2H), 6.90 (d, J ¼ 8.6 Hz, 1H), 3.94 (s, 3H). 13
C
J
yield. 1H NMR (400 MHz, DMSO‑d6)
d 13.58 (s, 1H), 10.39 (d,
NMR (126 MHz, DMSO‑d6)
d
163.36, 161.93, 158.59 (dd,
J ¼ 3.9 Hz, 2H), 8.97 (s, 1H), 8.45 (s, 1H), 8.29 (d, J ¼ 8.1 Hz, 1H), 8.19
(d, J ¼ 8.2 Hz, 1H), 7.90 (dd, J ¼ 15.0, 8.5 Hz, 1H), 7.81 (dd, J ¼ 8.4,
5.1 Hz, 2H), 7.46 (t, J ¼ 9.0 Hz, 1H), 7.15 (t, J ¼ 8.7 Hz, 2H).
Methyl 20,40-difluoro-3’-((3-((4-fluorophenyl)carbamoyl)-1H-
pyrazol-4-yl)carbamoyl)-[1,10-biphenyl]-3-carboxylate(DC-
K2in211(9a)). White solid, 81%. 1H NMR (400 MHz, DMSO‑d6)
CeF ¼ 251.8, 7.6 Hz), 158.51 (d, J CeF ¼ 240.6 Hz), 156.64, 156.08 (dd,
J
CeF ¼ 251.8, 7.0 Hz), 146.62(d, J CeF ¼ 2.4 Hz), 139.44, 134.64 (d, J
CeF ¼ 1.8 Hz), 133.30, 132.65 (d, J CeF ¼ 9.0 Hz), 123.19, 122.57 (d, J
CeF ¼ 7.7 Hz), 122.27 (dd, J CeF ¼ 14.3, 3.0 Hz), 122.00, 121.58, 115.12
(d, J CeF ¼ 22.1 Hz), 114.47 (t, J CeF ¼ 21.4 Hz), 112.60 (dd, J
CeF ¼ 21.8, 2.6 Hz), 110.52, 53.34. HRMS (M þ H)þ calculated for
d
13.60 (s, 1H), 10.45 (s, 1H), 10.40 (s, 1H), 8.46 (s, 1H), 8.12 (d,
C
23H17F3N5Oþ3 468.1278, found 468.1273.
J ¼ 1.4 Hz, 1H), 8.03 (dd, J ¼ 6.5, 1.4 Hz, 1H), 7.89e7.74 (m, 4H), 7.68
4-(3-(6-aminopyridin-3-yl)-2,6-difluorobenzamido)-N-(4-
(t, J ¼ 7.8 Hz, 1H), 7.38 (t, J ¼ 8.7 Hz, 1H), 7.20e7.11 (m, 2H), 3.88 (s,
fluorophenyl)-1H-pyrazole-3-carboxamide(DC-K2in206). White
3H). 13C NMR (126 MHz, DMSO‑d6)
d 165.94, 161.94, 158.87 (dd, J
solid, 36% yield. 1H NMR (400 MHz, MeOD-d4)
d
8.41 (s, 1H), 8.11 (s,
CeF ¼ 251.2, 6.4 Hz),158.53 (d, J CeF ¼ 241.0 Hz), 156.67, 156.12 (dd, J
1H), 7.75e7.67 (m, 3H), 7.62 (dd, J ¼ 15.0, 8.7 Hz, 1H), 7.19 (t,
CeF ¼ 252.5, 6.7 Hz), 134.70, 134.32, 133.51, 133.36, 132.97 (d, J
J ¼ 8.9 Hz, 1H), 7.07 (t, J ¼ 8.8 Hz, 2H), 6.70 (d, J ¼ 8.7 Hz, 1H). 13
C
CeF
¼
5.8 Hz), 130.18, 129.52, 129.24, 128.88, 124.38 (d, J
NMR (126 MHz, DMSO‑d6)
d
161.92, 158.53 (d, J CeF ¼ 240.9 Hz),
CeF ¼ 13.8 Hz), 122.55 (d, J CeF ¼ 7.3 Hz), 122.05, 121.65, 115.13 (d, J
CeF ¼ 22.1 Hz), 114.56 (t, J CeF ¼ 21.5 Hz), 112.66 (d, J CeF ¼ 21.2 Hz),
52.24. HRMS (M þ H)þ calculated for C25H18F3N4Oþ4 495.1275,
found 495.1277.
157.86 (dd, J CeF ¼ 256.2, 6.6 Hz), 156.92, 159.53, 155.85 (dd, J
CeF ¼ 250.6, 6.8 Hz), 147.70(d, J CeF ¼ 3.1 Hz), 137.41, 134.63 (d, J
CeF ¼ 2.2 Hz), 133.31, 131.96 (dd, J CeF ¼ 8.9, 4.8 Hz), 123.41 (dd, J
CeF ¼ 14.4, 3.3 Hz), 122.63 (d, J CeF ¼ 7.8 Hz), 122.01, 121.63, 117.74,
115.17 (d, J CeF ¼ 22.2 Hz), 114.43 (t, J CeF ¼ 21.6 Hz), 112.46 (dd, J
CeF ¼ 21.6, 2.7 Hz), 107.83. HRMS (M þ H)þ calculated for
Methyl 20, 40, 6-trifluoro-3’-((3-((4-fluorophenyl) carbamoyl)-
1H-pyrazol-4-yl)carbamoyl)-[1,10-biphenyl]-3-carboxylate (9b)
White solid, 68% yield. 1H NMR (400 MHz, MeOD-d4)
d 8.39 (s, 1H),
C
22H16F3N6Oþ2 453.1281, found 453.1274.
8.11e8.05 (m, 2H), 7.69e7.64 (m, 2H), 7.60 (dd, J ¼ 14.7, 8.4 Hz, 1H),
7.30 (t, J ¼ 9.4 Hz, 1H), 7.22 (t, J ¼ 8.5 Hz, 1H), 7.03 (t, J ¼ 8.8 Hz, 2H),
3.88 (s, 3H).
4-(2,4-difluoro-30-hydroxy-[1,10-biphenyl]-3-carboxamido)-N-
(4-fluorophenyl)-1H-pyrazole-3-carboxamide(DC-K2in207).
White solid, 54%. 1H NMR (400 MHz, DMSO‑d6)
d
13.58 (s, 1H),10.42
Methyl 20, 40-difluoro-3’-((3-((4-fluorophenyl)carbamoyl)-1H-
pyrazol-4-yl)carbamoyl)-6-methyl-[1,10-biphenyl]-3-carboxylate
(s,1H),10.36 (s,1H), 9.65 (s,1H), 8.45 (s,1H), 7.84e7.77 (m, 2H), 7.66
(td, J ¼ 8.8, 6.5 Hz, 1H), 7.35e7.28 (m, 2H), 7.19e7.12 (m, 2H),
6.97e6.93 (m, 2H), 6.85e6.82 (m, 1H). 13C NMR (126 MHz,
(9c) White solid, 72% yield. 1H NMR (400 MHz, MeOD-d4)
d
8.38 (s,
1H), 7.91 (dd, J ¼ 8.0, 1.7 Hz, 1H), 7.82 (d, J ¼ 1.6 Hz, 1H), 7.68e7.63
9

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
(m, 2H), 7.43 (td, J ¼ 8.4, 6.4 Hz, 1H), 7.37 (d, J ¼ 8.1 Hz, 1H), 7.18 (t,
J ¼ 8.8 Hz, 1H), 7.05e6.98 (m, 2H), 3.85 (s, 3H), 2.23 (d, J ¼ 7.4 Hz,
3H).
2.65 (s, 3H), 1.89e1.74 (m, 3H), 1.53e1.39 (m, 2H). 13C NMR
(126 MHz, DMSO‑d6)
d
166.31, 162.17, 159.04 (dd, J CeF ¼ 250.8,
6.7 Hz),158.87 (d, J CeF ¼ 240.6 Hz),157.11,156.34 (dd, J CeF ¼ 252.0,
7.3 Hz), 135.60, 135.04 (d, J CeF ¼ 2.0 Hz), 134.32, 133.60(m),
131.93,130.10, 129.21, 128.06, 127.55, 125.34 (dd, J CeF ¼ 14.0,
3.2 Hz), 122.98 (d, J CeF ¼ 7.8 Hz), 122.25, 115.57 (d, J CeF ¼ 22.2 Hz),
114.94 (t, J CeF ¼ 21.7 Hz), 113.07 (dd, J CeF ¼ 21.5, 2.8 Hz), 55.32,
5-(2,4-difluoro-3-((3-((4-fluorophenyl)carbamoyl)-1H-pyr-
azol-4-yl)carbamoyl)phenyl) picolinamide (DC-K2in212). To a
suspension of compound 8 (50 mg, 0.108 mmol) in the mixture of
H2O (3 ml) and MeOH (3 ml), and NaOH (18 mg, 0.432 mmol) was
added and stirred at 60 ꢀC for 2h. After cooling, the mixture was
treated with 2 M HCl to neutral and extracted with ethyl acetate
(5 ml ꢁ 3).The combine organic layers were dried over anhydrous
sodium sulfate then concentrated in vacuum. The residue was pu-
rified by silica gel chromatography (5% DCM:MeOH) to give com-
pound DC-K2in212 (18 mg, 35%) as white solid. 1H NMR (400 MHz,
46.19, 45.20, 35.33, 29.93. HRMS (M
31H30F3N6Oþ3 591.2326, found 591.2315.
þ
H)þ calculated for
C
2,4-difluoro-N3-(3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)-N3’-(2-(4-methylpiperazin-1-yl)ethyl)-[1,10-biphenyl]-3,30-
dicarboxamide (DC-K2in214). White solid, 30% yield. 1H NMR
(400 MHz, DMSO‑d6) d 13.61 (s, 1H), 10.43 (s, 1H), 10.39 (s, 1H), 8.52
DMSO‑d6)
d
13.60 (s, 1H), 10.42 (s, 1H), 10.40 (s, 1H), 8.82 (s, 1H),
(t, J ¼ 5.6 Hz, 1H), 8.46 (s, 1H), 8.01 (s, 1H), 7.89 (d, J ¼ 7.8 Hz, 1H),
7.85e7.74 (m, 3H), 7.71 (d, J ¼ 7.0 Hz,1H), 7.59 (t, J ¼ 7.7 Hz,1H), 7.38
(t, J ¼ 8.7 Hz, 1H), 7.19e7.10 (m, 2H), 3.40e3.35 (m, 2H), 2.48e2.24
8.45 (s, 1H), 8.24e8.13 (m, 3H), 7.88 (td, J ¼ 8.7, 6.5 Hz, 1H), 7.81 (dd,
J ¼ 9.1, 5.1 Hz, 2H), 7.72 (s, 1H), 7.44 (t, J ¼ 8.7 Hz, 1H), 7.15 (t,
J ¼ 8.9 Hz, 2H). 13C NMR (126 MHz, DMSO‑d6)
d
165.75, 161.81,
(m, 10H), 2.11 (s, 3H). 13C NMR (126 MHz, DMSO‑d6)
d 165.77,161.76,
159.27 (dd, J CeF ¼ 252.2, 6.6 Hz), 158.47 (d, J CeF ¼ 240.7 Hz),
156.49, 156.27 (dd, J CeF ¼ 252.7, 7.1 Hz), 149.74, 148.15, 137.82,
134.61 (d, J CeF ¼ 1.7 Hz), 133.39, 133.18 (d, J CeF ¼ 7.6 Hz), 132.21,
122.57 (d, J CeF ¼ 7.7 Hz), 121.96, 121.80, 121.74e121.51 (m), 115.14
(d, J CeF ¼ 22.1 Hz), 114.69 (t, J CeF ¼ 21.7 Hz), 112.92 (d, J
CeF ¼ 19.4 Hz).HRMS (M þ H)þ calculated for C23H16F3N6O3þ
481.1230, found 481.1239.
158.62 (dd, J CeF ¼ 250.4, 6.3 Hz), 158.44 (d, J CeF ¼ 240.7 Hz),
156.65, 155.92 (dd, J CeF ¼ 252.1, 7.3 Hz), 135.14, 134.59 (d, J
CeF ¼ 2.0 Hz), 133.88, 133.17, 133.08 (d, J CeF ¼ 6.1 Hz), 131.48,
128.82, 127.63, 127.03, 124.86 (dd, J CeF ¼ 14.1, 3.1 Hz), 122.55 (d, J
CeF ¼ 7.8 Hz), 121.85, 121.80, 115.12 (d, J CeF ¼ 22.2 Hz), 114.43 (d, J
CeF ¼ 21.5 Hz), 112.63 (dd, J CeF ¼ 21.8, 2.6 Hz), 56.90, 54.73, 52.67,
45.69, 36.97. HRMS (M
606.2435, found 606.2450.
þ
H)þ calculated for C31H31F3N7Oþ3
General procedure C for the synthesis of compounds 10a-10c.
To a suspension of compounds 9a-9c (1.0 eq) in the mixture of H2O
(5 ml) and MeOH (5 ml), and LiOH$H2O (4.0 eq) was added and
stirred at 40 ꢀC for 4h. After cooling, the mixture was treated with
2 M HCl. An amount of white solid was precipitated, filtered off,
washed with water and dried to afford compounds 10a-10c
without further purification.
2,4-difluoro-N3-(3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)-N3’-(3-morpholinopropyl)-[1,10-biphenyl]-3,30-dicarbox-
amide (DC-K2in215). White solid, 32% yield.1H NMR (400 MHz,
DMSO‑d6)
d 13.61 (s, 1H), 10.45 (s, 1H), 10.39 (s, 1H), 8.61 (t,
J ¼ 5.5 Hz, 1H), 8.45 (s, 1H), 8.01 (s, 1H), 7.90 (d, J ¼ 7.8 Hz, 1H),
7.84e7.75 (m, 3H), 7.71 (d, J ¼ 7.7 Hz,1H), 7.59 (t, J ¼ 7.7 Hz,1H), 7.40
(t, J ¼ 8.5 Hz, 1H), 7.19e7.12 (m, 2H), 3.55 (t, J ¼ 4.5 Hz, 4H), 3.31 (dd,
J ¼ 12.8, 6.8 Hz, 2H), 2.38e2.29 (m, 6H), 1.74e1.65 (m, 2H)$13C NMR
20,40-difluoro-3’-((3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)carbamoyl)-[1,10-biphenyl]-3-carboxylic acid (10a) White
solid, 89% yield. 1H NMR (400 MHz, DMSO)
d
13.60 (s, 1H), 13.18 (s,
(126 MHz, DMSO‑d6)
d
165.80, 161.82, 158.67 (dd, J CeF ¼ 251.2,
1H), 10.43 (s, 1H), 10.40 (s, 1H), 8.45 (s, 1H), 8.10 (s, 1H), 8.01 (d,
J ¼ 7.8 Hz, 1H), 7.85e7.74 (m, 4H), 7.65 (t, J ¼ 7.8 Hz, 1H), 7.38 (t,
J ¼ 8.6 Hz, 1H), 7.15 (t, J ¼ 8.9 Hz, 2H).
6.8 Hz), 158.49 (d, J CeF ¼ 240.7 Hz), 156.68, 155.98 (dd, J
CeF ¼ 252.2, 6.9 Hz),135.22,134.63 (d, J CeF ¼ 2.2 Hz),133.92,133.12
(dd, J CeF ¼ 9.6, 4.3 Hz), 131.48, 128.79, 127.65, 127.05, 124.93 (dd, J
CeF ¼ 14.2, 3.3 Hz), 122.58 (d, J CeF ¼ 7.8 Hz), 121.92, 121.77, 115.14
(d, J CeF ¼ 22.2 Hz), 114.51 (t, J CeF ¼ 21.6 Hz), 112.64 (dd, J
CeF ¼ 21.7, 2.5 Hz), 66.25, 56.17, 53.38, 37.91, 26.00. HRMS (M þ H)þ
calculated for C31H30F3N6Oþ4 607.2202, found 607.2253.
20,40,6-trifluoro-3’-((3-((4-fluorophenyl)carbamoyl)-1H-pyr-
azol-4-yl)carbamoyl)-[1,10-biphenyl]-3-carboxylic acid (10b)
White solid, 85% yield. 1H NMR (400 MHz, DMSO)
d 13.63 (s, 1H),
13.25 (s, 1H), 10.44 (s, 1H), 10.41 (s, 1H), 8.44 (s, 1H), 8.12e8.02 (m,
2H), 7.82 (dd, J ¼ 8.9, 5.0 Hz, 2H), 7.76 (dd, J ¼ 15.1, 8.6 Hz, 1H), 7.51
(t, J ¼ 9.2 Hz, 1H), 7.41 (t, J ¼ 8.8 Hz, 1H), 7.16 (t, J ¼ 8.9 Hz, 2H).
20,40-difluoro-3’-((3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)carbamoyl)-6-methyl-[1,10-biphenyl]-3-carboxylic acid (10c)
2,4,60-trifluoro-N3-(3-((4-fluorophenyl)carbamoyl)-1H-pyr-
azol-4-yl)-N3’-(3-morpholinopropyl)-[1,10-biphenyl]-3,30-dicar-
boxamide (DC-K2in216). White solid, 26% yield. 1H NMR (400 MHz,
MeOD-d4) d 8.41 (s, 1H), 7.99e7.93 (m, 2H), 7.72e7.63 (m, 3H), 7.35
White solid, 93% yield. 1H NMR (400 MHz, DMSO)
d
13.61 (s, 1H),
(t, J ¼ 9.1 Hz, 1H), 7.28 (t, J ¼ 9.0 Hz, 1H), 7.07 (dd, J ¼ 12.1, 5.5 Hz,
13.03 (s, 1H), 10.44 (s, 1H), 10.40 (s, 1H), 8.44 (s, 1H), 7.91 (dd, J ¼ 7.9,
1.5 Hz, 1H), 7.85e7.77 (m, 3H), 7.59 (dd, J ¼ 15.0, 8.5 Hz, 1H), 7.49 (d,
J ¼ 8.0 Hz, 1H), 7.36 (t, J ¼ 8.8 Hz, 1H), 7.16 (t, J ¼ 8.9 Hz, 2H), 2.24 (s,
3H).
General procedure D for the synthesis of compounds DC-
K2in213 e DC-K2in217 exemplified by DC-K2in213.
2H), 3.69 (t, J ¼ 4.6 Hz, 4H), 3.44 (t, J ¼ 6.9 Hz, 2H), 2.57e2.46 (m,
6H), 1.90e1.79 (m, 2H). HRMS (M
C
þ
H)þ calculated for
31H29F4N6Oþ4 625.2181, found 625.2183.
2,4-difluoro-N3-(3-((4-fluorophenyl)carbamoyl)-1H-pyrazol-
4-yl)-60-methyl-N3’-(3-morpholinopropyl)-[1,10-biphenyl]-3,30-
dicarboxamide (DC-K2in217). White solid, 28% yield.1H NMR
A mixture of compound 10a (50 mg, 0.104 mmol), (1-Methyl-4-
piperidyl)methanamine (13 mg, 0.104 mmol), EDCI (20 mg,
0.104 mmol) and HOBt (14 mg, 0.104 mmol) in DMF (0.6 ml) was
stirred at ambient temperature for 18h. Water (1.2 ml) was added to
the mixture, and extracted with ether acetate (6ml ꢁ 6).The com-
bined organic layers were dried over anhydrous sodium sulfate
then concentrated in vacuum. The residue was purified by silica gel
chromatography (10% MeOH in DCM) to afford compound DC-
(400 MHz, MeOD-d4)
d
8.40 (s, 1H), 7.80 (dd, J ¼ 8.0, 1.9 Hz, 1H),
7.71e7.66 (m, 3H), 7.50 (dd, J ¼ 14.8, 8.3 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz,
1H), 7.24 (t, J ¼ 8.8 Hz,1H), 7.10e7.03 (m, 2H), 3.66 (t, J ¼ 4.6 Hz, 4H),
3.42 (t, J ¼ 6.8 Hz, 2H), 2.56e2.46 (m, 6H), 2.26 (s, 3H), 1.88e1.77
(m, 2H). 13C NMR (126 MHz, DMSO‑d6)
d 166.15, 161.95, 158.88 (dd, J
CeF ¼ 250.8, 6.5 Hz), 158.59 (d, J CeF ¼ 240.8 Hz), 156.64, 155.96 (dd,
J
CeF ¼ 250.0, 7.0 Hz), 139.92, 134.64 (d, J CeF ¼ 1.7 Hz),
133.98e133.69 (m), 133.12, 132.16, 130.21, 129.03, 127.52, 124.98
(dd, J CeF ¼ 17.4, 3.0 Hz), 122.66 (d, J CeF ¼ 7.7 Hz), 122.04, 121.79,
115.15 (d, J CeF ¼ 22.2 Hz), 114.10 (t, J CeF ¼ 21.2 Hz), 112.42 (d, J
CeF ¼ 19.7 Hz), 63.48, 54.29, 51.23, 36.51, 23.75, 19.48. HRMS
(M þ H)þ calculated for C32H32F3N6O4þ 621.2432, found 621.2432.
K2in213 (21 mg, 35%). 1H NMR (400 MHz, DMSO‑d6)
d 13.68 (s, 1H),
10.45 (s, 1H), 10.38 (s, 1H), 8.73 (t, J ¼ 5.7 Hz, 1H), 8.43 (s, 1H), 8.03
(s, 1H), 7.92 (d, J ¼ 7.8 Hz, 1H), 7.85e7.76 (m, 3H), 7.72 (d, J ¼ 7.3 Hz,
1H), 7.60 (t, J ¼ 7.8 Hz, 1H), 7.39 (t, J ¼ 8.8 Hz, 1H), 7.15 (t, J ¼ 8.9 Hz,
2H), 3.34e3.27 (m, 2H), 3.26e3.18 (m, 2H), 2.82 (t, J ¼ 11.6 Hz, 2H),
10

T. Lin, J. Li, L. Liu et al.
European Journal of Medicinal Chemistry 215 (2021) 113281
5.1. Biology methods
culture medium was changed to Opti-MEM medium (Invitrogen,
Cat #11058021) before transfection. SiRNA-Lipofectamine mixture
was prepared using Lipofectamine RNAiMAX Transfection Reagent
(Invitrogen, Cat# 13,778,100) as manufacturer’s instructions. After
6 h, Opti-MEM medium was switched to culture medium and
incubated for 48 h. Western blot assays were used to detect the
expression of CDK2 protein. The sequence of the SiRNA duplex ol-
igonucleotides were as followed. Si-CDK2-1#: (Sense, 50-30)
5.1.1. Kinase assays
The kinase inhibition was detected by mobility shift assay.
Briefly, active kinases, FAM-labeled peptides and ATP were diluted
with kinase base buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35),
respectively. Then 10
of the 384-well assay plate, which already contains 5
diluted compound in 10% DMSO in each well, and incubated at
room temperature for 10 min. Then 5 l of peptide solution and 5 ml
m
l of kinase solution was added to each well
ml of serially
CCAGCUCUUCCGGAUCUUUTT,
AAAGAUCCGGAAGAGCUGGTT.
(Antisense,
50-30)
m
of ATP solution were added to each well of the 384-well assay plate.
Si-CDK2-2#: (Sense, 50-30) CCAGCUCUUCCGGAUCUUUTT,
(Antisense, 50-30)AAAGAUCCGGAAGAGCUGGTT.
After incubation at 28 ꢀC for specified time, kinase reactions were
stopped by 25 ml of stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-
35, 0.2% Coating Reagent #3 and 50 mM EDTA). Data were collected
on Caliper (PerkinElmer, Caliper EZ ReaderII). The source of the
active CDKs/cyclins proteins and FAM-labeled peptides were
shown in Table S1.
5.2. Molecular docking
Molecular docking was performed using Glide program imple-
€
mented in Schrodinger package. Crystal structure of CDK2 (PDB
code 5NEV) was downloaded from Protein Data Bank (PDB), then
protein was optimized by using Protein Preparation Wizard mod-
ule. Receptor grid file was generated using Receptor Grid Genera-
tion module. Compound DC-K2in202 was prepared using LigPrep
module to generate protonation states and stereoisomers. Standard
precision (SP) mode was adopted in molecular docking, the pose
with good hydrogen bond geometries and low energy conforma-
tions was considered for further analysis. Docking structures and
figures were analyzed and generated based on the PyMOL molec-
ular graphic system.
5.1.2. Cell culture and viability assay
A2058, LX2 and MRC5 cells were cultured in Dulbecco’s Modi-
fied Eagle’s Medium (DMEM, Invitrogen), while MV4-11 cells were
cultured in RPMI 1640 medium Medium (Invitrogen). Both medium
were supplemented with 10% fetal bovine, 1% penicillin and
streptomycin (Invitrogen). For cell viability assay, cells were plated
in 96-well plates in a volume of 100 ml and treated with compounds
in corresponding concentration (DMSO as control) for 96 h. Cell
viability was determined by CellTiter-Glo® Luminescent Cell
Viability assay kit (Promega).
Author contributions
5.1.3. Western blot
Total cell lysates were separated by 8% or 10% SDS-
polyacrylamide gels and transferred to nitrocellulose membranes.
The blots were blocked with blocking buffer (5% non-fat milk in
TBST) for 60 min at room temperature and incubated with primary
antibodies overnight at 4 ꢀC. Then the blots were washed three
times with TBST and incubated with 1:10,000 dilution of secondary
antibody (HRP conjugated) for 1 h. Following another three washes,
bands were detected in a ChemiScope3400 imaging system using
ECL substrate (Clinx). Primary antibodies used were as follows:
anti-Mcl-1 (Cell Signaling Technology, no.5453T), anti-Rb (Cell
Signaling Technology, no.9309T), anti-Phospho-Rb (Ser780) (Cell
Signaling Technology, no.8180T), anti-Phospho-Rb (Ser807/811)
(Cell Signaling Technology, no.8516T), anti-Cleaved Caspase-3 (Cell
Signaling Technology, no.9661S), anti-Cleaved PARP (Cell Signaling
Technology, no.5625T), anti- GAPDH (Cell Signaling Technology,
no.5174S), and anti-Tubulin (Cell Signaling Technology, no.9873S).
C.L. and B.Z. directed and supervised the project. T.L. prepared
and characterized compounds; J.L. and Y.L. performed biological
experiments; T.L., J.L., and P.X. performed computational study; H.J.,
C.L, and B.Z designed the study. All authors analyzed the data. T.L.,
J.L., P.X., C.L, and B.Z drafted the manuscript. All authors have given
approval to the final version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
We are grateful to National Centre for Protein Science Shanghai
(Protein Expression and Purification system) for their instrument
support and technical assistance. We also gratefully acknowledge
the financial supports from the National Natural Science Founda-
tion of China (91853205, 81625022, 81821005to C.L.), K. C. Wong
Education to C.L., the Science and Technology Commission of
Shanghai Municipality (19XD1404700 and 18431907100 to C.L.)
and National Science & Technology Major Project “Key New Drug
Creation and Manufacturing Program” (2018ZX09711002-008 to
C.L.)
5.1.4. Flow cytometric analysis
A2058 cells were plated in 6-cm dishes, and after 24 h, cells
were treated with compounds or DMSO control. For cell cycle
analysis, cells were harvested at 24 h and gently resuspended in
70% ethanol overnight at 4 ꢀC for fixation. Then samples were
washed with PBS twice and incubated with Propidium Iodide/
RNase Staining Buffer (BD Pharmingen) for 20 min at room tem-
perature. For cell apoptosis analysis, cells were harvested at 48 h
and were measured using Annexin V-FITC Apoptosis Detection Kit
(Vazyme Biotech) according to the manufacturer’s instructions.
Samples were detected by BD FACSCalibur (BD Pharmingen), and
data were analyzed by FlowJo V7.6.1.
Appendix B. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113281.
5.1.5. SiRNA transfection
SiRNA duplex oligonucleotides against human CDK2 and a non-
targeting negative control siRNA were synthesized by Genepharma.
A2058 cells were seeded in six-well plates (Corning) at a density of
5 ꢁ 105 cells per well and allowed to adhere overnight. The cell
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carboxamide (AT7519),
a novel cyclin dependent kinase inhibitor using
fragment-based X-ray crystallography and structure based drug design,
12