Design, synthesis, and biological evaluation of indazole derivatives as selective and potent FGFR4 inhibitors for the treatment of FGF19-driven hepatocellular cancer

Article abstract of DOI:10.1016/j.ejmech.2021.113219

Fibroblast growth factor receptor 4 (FGFR4) is a member of the fibroblast growth factor receptor family, which is closely related to the occurrence and development of hepatocellular carcinoma (HCC). In this article, a series of indazole derivatives were designed and synthesized by using computer-aided drug design (CADD) and structure-based design strategies, and then they were evaluated for their inhibition of FGFR4 kinase and antitumor activity. F-30 was subtly selective for FGFR4 compared to FGFR1; it affected cell growth and migration by inhibiting FGFR4 pathways in HCC cell lines in a dose-dependent manner.

Full text of DOI:10.1016/j.ejmech.2021.113219

European Journal of Medicinal Chemistry 214 (2021) 113219
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, and biological evaluation of indazole derivatives as
selective and potent FGFR4 inhibitors for the treatment of FGF19-
driven hepatocellular cancer
Xiaolu Chen a, 1, Yanan Liu , Liting Zhang a , Daoxing Chen , Zhaojun Dong ,
a
,
1
,
1
a
a
a
a
a
a
,
**
b
Chengguang Zhao , Zhiguo Liu , Qinqin Xia , Jianzhang Wu
, Yongheng Chen ,
a,
***
a, *
Xiaohui Zheng
a
, Yuepiao Cai
School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, 325035, China
Department of Oncology, NHC Key Laboratory of Cancer Proteomics and Laboratory of Structural Biology, Key Laboratory of Medical Genetics and College
of Life Science, and National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 October 2020
Received in revised form
Fibroblast growth factor receptor 4 (FGFR4) is a member of the fibroblast growth factor receptor family,
which is closely related to the occurrence and development of hepatocellular carcinoma (HCC). In this
article, a series of indazole derivatives were designed and synthesized by using computer-aided drug
design (CADD) and structure-based design strategies, and then they were evaluated for their inhibition of
FGFR4 kinase and antitumor activity. F-30 was subtly selective for FGFR4 compared to FGFR1; it affected
cell growth and migration by inhibiting FGFR4 pathways in HCC cell lines in a dose-dependent manner.
15 January 2021
Accepted 17 January 2021
Available online 29 January 2021
©
2021 Elsevier Masson SAS. All rights reserved.
Keywords:
Indazole derivatives
Hepatocellular carcinoma
Fibroblast growth factor receptor 4
Covalent inhibitor
Antitumor activity
1
. Introduction
Human health is seriously threatened by the complexity and het-
erogeneity of HCC [5]. FGFR4 selectively binds FGF19 to stimulate
autophosphorylation in trans and mediates cellular effects via
downstream signaling pathways, which are involved in prolifera-
tion, anti-apoptosis, angiogenesis, drug resistance, invasion, and
epithelial-to-mesenchymal transition in HCC cells [6]. Therefore,
FGFR4 is considered an important target for the treatment of HCC.
Small-molecule inhibitors targeting FGFR4 are a promising thera-
peutic approach for the treatment of HCC.
As high affinity receptors for fibroblast growth factors (FGFs),
fibroblast growth factor receptors (FGFR1-4) are receptor tyrosine
kinases (RTKS), which consist of an extracellular region, a trans-
membrane domain, and an intracellular tyrosine kinase region [1].
FGFR4 has been verified to play important roles in a variety of cell
functions, such as cell proliferation, cell migration, differentiation,
and biological processes, during the normal metabolic processes
[
2]. However, abnormal activation of the FGFR4 signaling pathway
Great efforts have been made to develop effective FGFR4 in-
hibitors for anticancer treatment. Several multitargeting tyrosine
kinase inhibitors (TKIs), such as ponatinib [7,8], dovitinib [9], luci-
tanib [10], have been studied for the suppression of FGFR4
signaling. Although these inhibitors have good kinase inhibitory
activity against FGFR4, the toxicity profiles of those inhibitors
relative to on-target activity against other kinases have restricted
their further development for the treatment of HCC [11]. Non-
covalent FGFR inhibitors, such as AZD4547 [12], NVP-BGJ398 [13],
LY2874455 [14], and JNJ-42756493 [15,16], have been reported.
Analysis of the ATP-binding domain of the FGFR kinase family has
has been found to be closely associated with malignancy [3,4].
Aberrant activation of the FGFR4-FGF19 signaling pathway has
been confirmed to be one of the carcinogenic factors in HCC.
*
Corresponding author.
*
*
* Corresponding author.
** Corresponding author.
E-mail addresses: wjzwzmu@163.com (J. Wu), zhengxh@wmu.edu.cn
(
X. Zheng), ypcai@wmu.edu.cn (Y. Cai).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113219
223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
0

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
found a unique cysteine for which it is possible to design covalent
inhibitors on the P-loop ring. TAS-120 [17], FIIN-1 [18], FIIN-2
FGFR4 [28]. X-ray crystallography showed that a covalent bond was
formed between the 2-formyl quinoline group and Cys552 by an
addition reaction to generate a hemithioacetal adduct [29]. FGF401
displayed at least 1000-fold selectivity for FGFR4 over FGFR1/2/3
and other kinases. On account of its excellent antitumor activity
and pharmacokinetics and pharmacodynamics, FGF401 has
entered a phase I/II clinical trial to evaluate its safety and efficacy in
HCC [30]. Although several inhibitors targeting FGFR4 have been
reported, the chemical structural diversity of FGFR4 inhibitors still
needs to be enriched. Therefore, our group used a compound-aided
drug design strategy to analyze and explore the structure-activity
relationship of reported FGFR4 inhibitors. We found an excellent
combination of indazole scaffold with FGFR4 protein by studying
the Protein Data Bank (PDB) reported structure. Then, a structure-
based design strategy was used to explore the possibility of inda-
zole derivatives as FGFR4 selective inhibitors. Herein, we report the
design, synthesis, and evaluation of indazole derivatives as a novel
class of potent, selective, and irreversible FGFR4 inhibitors.
[
19,20], FIIN-3 [20], FIIN-4 [21], and PRN1371 [22] were succes-
sively reported as FGFR covalent inhibitors (see Fig. 1). They func-
tion by introducing an acrylamide group to target Cys477. The
acrylamide moiety undergoes a Michael addition reaction with the
thiol group of cysteine to form a covalent bond. However, when
these pan-inhibitors were used for treatment, patients had the
severe toxic side effect of hyperphosphatemia due to on-target
systemic inhibition of FGFR1 and FGFR3 [21]. Therefore, inhibitors
selectively targeting FGFR4 may have better therapeutic effects
with lower toxicity in HCC, and it is urgent to develop selective
FGFR4 inhibitors.
Given the fact that the FGFR family proteins share significant
sequence homology in their kinase domains, it has been a consid-
erable challenge to develop inhibitors selective for FGFR4 over the
other family members. In 2015, Hagel et al. reported a selective
FGFR4 inhibitor, BLU9931, by targeting the conserved Cys552 in the
hinge region of the FGFR4 protein sequence with an acrylamide
moiety. An irreversible reaction of BLU9931 could occur with the
sulfhydryl group of Cys552 by a Michael addition [23]. BLU9931
provided selective activity against FGFR4, which was more than
2. Results and discussion
2.1. Design and SAR (structure-activity relationship) exploration
150-fold greater than its activity against FGFR1. Further optimiza-
tion of BLU9931, with the aim of improving its physicochemical
properties, afforded the clinical candidate BLU554 [5,24]. Disas-
sembly and analysis of the quinoline scaffold was performed to
obtain compounds 1 [25] and 2 [6,26] which had pyrimidine scaf-
folds. More recently, H3 Biomedicine developed the FGFR4-
selective irreversible inhibitor H3B-6527, based on the structure
of NVP-BGJ398, by adding an acrylamide moiety on the ortho po-
sition of the aniline group to target the Cys552 residue of FGFR4
As shown in Fig. 2, a series of amino pyrazoles and their de-
rivatives, which showed high FGFR kinase activities, have been
reported.These include AZD4547, LY-2874455, 3 [31], 4 [32]. When
we studied the reported pan-FGFR inhibitors, we discovered the
significant role that aminopyrazole and aminoindazole scaffolds
had played in designing FGFR inhibitors. Analyzing the binding
pattern of the first reported aminopyrazole pan-FGFR inhibitor
AZD4547 to FGFR4, we speculated that the aminopyrazole ring can
be replaced with an aminoindazole ring [33]. Molecular docking
was used to verify our conjecture. Compared to AZD4547, the
benzamide portion of the new compound was closer to the hinge
region. We wondered if the new compound could be designed as an
FGFR4 inhibitor by introducing an acrylamide moiety.
[
27]. The success of BLU9931 and H3B-6527 indicated that covalent
modification based on pan-inhibitors was an effective tactic for
designing FGFR4 inhibitors. Most recently, FGF401 was improved
by Novartis as a highly-selective, reversible, covalent inhibitor of
As shown in Fig. 3B, the result of molecular docking showed that
the acrylamide part could form a covalent bond with Cys552. The
aminoindazole part could form two intramolecular hydrogen bonds
with Ala553 and Glu551 on the hinge region. The 3,5-
dimethoxyphenyl part extended to the hydrophobic pocket,
which was in agreement with our design hypothesis. Hence, F-1
was synthesized (Fig. 3A). A kinase assay showed that the activity of
Fig. 1. Chemical structures of reported FGFR4 inhibitors.
Fig. 2. Amino pyrazol Fibroblast Growth Factor Receptor (FGFR) Inhibitors.
2

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
The binding mode of the lead compound F-1 predicted that the
, 5-dimethoxyphenyl was adjacent to the gatekeeper Asp630. For
3
initial structure-activity relationship exploration, we assumed that
modification of this position might bring about a good effect on the
selectivity of compounds. Therefore, a series of compounds with a
ring substituent of
R
1
were studied. When the 3, 5-
dimethoxyphenyl was replaced by 3-methoxyphenyl (F-2), the
potency against FGFR4 decreased a little. However, changing of the
substituent group of 3-methoxyl to 4-methoxyl (F-3) and 3, 5-
dimethylphenyl (F-4) led to a 3-fold decrease of potency against
FGFR4. Introducing a trifluoromethoxy (F-9, F-10) almost abolished
the FGFR4 activities of the compound. After substitution of the
benzene ring with a pyridine ring (F-5), benzofuran (F-8) and
thiophene (F-6, F-7) showed decreased inhibitory activity against
FGFR4. Previous data indicate that the introduction of a fluorine
atom in the benzene ring can improve potency. In drug develop-
ment, researchers found the fluorine atom had the dual property of
hydrogen bond acceptor and hydrophobic moiety [34]. Therefore,
compounds F-11, F-12, F-13, and F-14 were synthesized. Surpris-
ingly, F-11 and F-14 had higher IC50 and selectivity than F-1. And
then, the focus of previous studies had been transferred to the N-
substituted indazole ring, to explore the potency of different scaf-
folds. Therefore, compounds F-15dF-19 were synthesized by uti-
lizing a similar protocol (Scheme S1). Kinase inhibitory activity
suggested that the indazole ring was suitable for further
exploration (see Table 1).
Because compound F-1 had higher potential for inhibiting
FGFR4 than FGFR1, there was an assumption that an acrylamide
moiety could target the Cys552 residue, which was crucial to the
design of FGFR4 inhibitors. In order to improve the potency of
FGFR4 further, we investigated this. Compounds F-20eF-27, with
different “warheads” adjacent to acid amides, were synthesized.
Surprisingly, none of these compounds showed good IC50 value (see
Table 2). It is likely that the length of the warheads and steric
hindrance contributed to this result. We asked what conformation
would be seen if the warhead was put in the meta-position of the
acid amides. Surprisingly, compound F-30, with a halogen warhead,
showed good FGFR4 kinase activity with an IC50 value of 8.6 nM as
well as remarkable selectivity against FGFR1.
Fig. 3. (A) Design of our compound. (B) Covalent docking of compound F-1 with FGFR4
(
PDB 4TYI).
the compound against FGFR4 was observable at 160 nM. This ac-
tivity was more than 10-fold greater than its activity against FGFR1.
This revealed the potential of compound F-1 to be an FGFR4 in-
hibitor. Therefore, F-1 was taken as the lead compound for further
optimization.
2.2. Chemistry
The designed inhibitors were readily prepared by a Suzuki
coupling as the key step (Scheme 1). Briefly, reaction of the
commercially available 4-bromo-2-fluorobenzonitrile with hydra-
zine hydrate produced intermediate 5, and then commercially
available di-tert-butyl pyrocarbonate reacted with intermediate 5
in the presence of 4-dimethylaminopyridine (DMAP) to provide the
intermediate 6. Next, 7ae7b were obtained by a nucleophilic sub-
stitution reaction between intermediate 6 and ortho or meta-
position nitro-substituted benzoyl chloride. Compounds 7ae7b
were coupled with various substituted boronic acids or boronic acid
esters to afford the Suzuki-coupling product 8. The catalytic hy-
drogenation of the nitro-group with Pd/C provided the desired
aniline 9 in high yield. Intermediate 9 was reacted with different
acyl chlorides and deprotected to produce the designed molecule
10 with good yields. The other designed inhibitors were synthe-
sized by utilizing a similar protocol (Scheme S1).
Reagents and conditions:(a) hydrazine hydrate, 2-amyl alcohol,
ꢀ
1
20 C, 3 h, 81%e95%; (b) (Boc)
2
O, DMAP, THF, rt, 0.5 h, 77%e84%;
(
c) 2-nitro-benzoyl chloride or 3-nitro-benzoyl chloride, DIPEA,1,4-
ꢀ
dioxane, 0 C to rt, 68%e89%; (d) Pd (pph
ester, 1,4-dioxane, 120 C, 1 h, 60%e82%; (e) Pd/C, H
3
)
4
, Cs
2
CO
3
, boronic acid/
, CH OH,
Combining the best fragments above, F-30, F1-1, and F1-2 were
synthesized for the purpose of obtaining FGFR4 inhibitors with
better kinase activity and selectivity. As shown in Table 3, all three
compounds showed excellent FGFR4 kinase activity. Unexpectedly,
F-30, F1-1, F1-2 also showed good potency against FGFR2 and
FGFR3, but they had less inhibition effect on FGFR1. Thus, com-
pounds F-30 and F1-2 were chosen as representatives for further
investigation due to their strong FGFR4 inhibitory potency and
good selectivity against FGFR1.
ꢀ
2
3
overnight, 70%e92%; (f) substituted acetyl chloride, DIPEA, 1,4-
ꢀ
dioxane, 0 C to rt, 2 h, 66%e78%; (g) TFA, DCM, rt, 3 h, 45%e56%.
2.3. F-30 blocking FGFR4 signaling in HCC cells
The Cancer Genome Atlas (TCGA) is a large cancer genomics
program, molecularly characterizing over 20,000 primary cancers
and matched normal samples spanning 33 cancer types [35].
Exploring from the TCGA datasets, we validated that liver hepato-
cellular carcinoma (LIHC) patients in the TCGA database express
much higher mRNA in tumor tissue than in normal tissue (Fig. 4A).
In addition, patients expressing higher FGFR4 showed lower sur-
vival rates compared to patients with low FGFR4 expression
(Fig. 4B).
Therefore, we selected hepatocellular carcinoma cell lines to
validate further the FGFR4 inhibition effect of compound F-30.
Western blot analysis was performed to determine which related
signaling pathway was involved in HUH7, HepG2, and SMMC-
7721 cell lines (Fig. 4C, 4D, 4E, and S1). Compound F-30 inhibited
Scheme 1. Synthesis of Compounds F-1dF-14, F-20dF-33 and F1-1dF1-2.
3

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
Table 1
Table 2
Structure-activity relationships in the modifications of Warheads.
Structure-activity relationships in the modification of hydrophobic Region.
X
C
R1
Kinase inhibitor activity
(IC50, nM)
Compounds
F-1
o/ma
R
2
Kinase inhibitor
activity (IC50, nM)
FGFR1
FGFR4
120
Compounds
>1500
FGFR1
FGFR4
F-1
o
o
o
o
o
>1500
160
F-2
F-3
C
C
>1500
>1500
273
423
F-20
F-21
F-22
F-23
>1500
>1500
>1500
>1500
>1500
>1500
>1500
>1500
F-4
F-5
C
C
>1500
>1500
632
>1500
F-6
F-7
C
C
>1500
>1500
258
>1500
F-24
F-25
F-26
F-27
o
o
o
o
>1500
>1500
>1500
>1500
>1500
>1500
>1500
>1500
F-8
C
C
C
C
>1500
>1500
>1500
>1500
>1500
1137
>1500
60
F-9
F-10
F-11
F-12
F-13
F-14
C
C
C
>1500
>1500
>1500
>1500
304
F-28
F-29
F-30
F-31
m
m
m
m
>1500
>1500
>1500
>1500
>1500
>1500
8.6
100
F-15
N
>1500
449
>1500
F-16
F-17
N
N
>1500
>1500
726
374
F-32
F-33
m
m
>1500
>1500
>1500
>1500
F-18
F-19
N
N
>1500
>1500
>1500
a
446
“o”: ortho-substituted of the benzamide; “m”: meta-substituted of the benza-
mide (see Table 2).
2
.4. F-30 inhibits cell invasion in HCC
FGF19-induced FGFR4 phosphorylation and the phosphorylation of
downstream kinases MAPK in a concentration-dependent manner,
while the total amounts of their target proteins remain unchanged.
The MAPKs are abnormally expressed in a series of cancers and are
involved in the regulation of cell proliferation, migration, survival,
and apoptosis [36]. Phosphorylation of FGFR4 and MAPK was
FGFR4-dependent signaling pathways involve cell invasion,
migration, and proliferation [37]. We observed obvious morpho-
logical changes of F-30-treated HCC cells. Therefore, we speculate
that F-30 might affect the expression and function of cytoskeletal
proteins of HUH7 cells. Focal adhesion kinase (FAK) is a non-
receptor tyrosine kinase that regulates adhesion spot integrity,
which plays an important role in maintaining cell morphology, cell
division, cell mobility, and other activities which play an important
role in tumorigenesis [38]. To explore whether F-30 treatment
almost undetectable in HUH7 cells treated with 0.625 mM of com-
pound F-30. Together, these results confirmed that F-30 could block
the FGFR4 signaling pathway by inhibiting the phosphorylation of
FGFR4.
4

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
Table 3
Structure-activity relationships in the modification of hydrophobic region and
warhead.
Compounds
F-30
R
1
R
2
Kinase inhibitor activity (IC50, nM)
FGFR1
FGFR2
15
FGFR3
146
FGFR4
8.6
>1500
F1-1
106
528
15
27
86
5.6
6.8
F1-2
208
BLU9931
staurosporine
e
e
e
e
e
e
e
6.3
240
21
7.4
16
affected the expression and distribution of FAK, an immunofluo-
rescence (IF) assay was performed using anti-FAK antibody. The
results indicated that the area of fluorescence decreases and the
intensity of fluorescence weakens at different concentrations
(
Fig. 5A and 5B). Additionally, Western blot assay showed that F-30
inhibited the expression of p-FAK and FAK in HUH7 cell lines in a
concentration-dependent manner (Fig. 5C).
Adhesion of metastatic cancer cells to the vascular endothelium
of the target organs is the key occurrence in transendothelial
migration [39]. In this study, an adhesion assay was performed to
test whether F-30 could reduce cell adhesion. As shown in Fig. 5D,
F-30 significantly reduced the adhesion ability of the HUH7 cell
lines compared to the control. There was a dose-effect relationship
between the adhesion rate and F-30, and the adhesion rate of
Fig. 5. F-30 induced cancer cell migration. (A), (B) Cells were incubated with the F-30
at different concentrations for 12 h. The expression of FAK (green) in HUH7 cells was
determined by immunofluorescence assay. Phalloidine-stained actin red, DAPI-stained
nuclei blue. The area of FAK surface was calculated. (C) Western-blot assay was per-
formed to analysis phosphorylation and total FAK levels in HUH7 treated with F-30 in
different concentration. (D) Cell adhesion assay and invasion assay was performed to
evaluate the migration impact of F-30 on HUH7. The results were representative of
three replicate experiments. (E) HUH7 cells were treated with F-30 at different con-
centrations for 12 h. The migratory capacity of cells was then assessed. (F) Wound
healing assays examined the effects of F-30 on HUH7 cell migration, and migration
area was calculated (G).
5
.0 mM F-30 treated cells was only 50% of that of the control. Similar
results were obtained in HepG2 and SMMC-7721 cells. (Figure S2.
A). Furthermore, transwell invasion assays were used to explore the
effect of F-30 on the invasion ability of HCC cells. Compared to
control, F-30 at different concentrations (0, 1.25, 2.5, and 5
significantly inhibited the invasion of HUH7 (Fig. 5E) and SMMC-
721 (Figure S2$B) cells. The wound healing assay indicated that
mM)
2.5. F-30 inhibits HCC cells proliferation
7
different concentrations of F-30 markedly inhibited migration of
HUH7 (Fig. 5F and 5G) and SMMC-721 cell lines (Figure S2$C).
To explore the antitumor activity of F-30 further, an MTT assay
was carried out. The quantitative data demonstrated that F-30
efficiently decreases the viability of HCC cells in a concentration-
dependent manner, with an IC50 of 3e5
mM. Compared to the
positive control BLU9931, a potent, selective, and irreversible FGFR4
inhibitor, F-30 was more cytotoxic to HCC cells (Fig. 6A, 6B, 6C).
Furthermore, F-30 significantly reduced colony formation of three
HCC cell lines in a dose-dependent manner (Fig. 6D).
Next, we treated the three human HCC cell lines with F-30 at
different concentrations (0, 1.25, 2.5, and 5.0 mM) for 48 h, and then
observed apoptosis by flow cytometry (FCM). Treatment with F-30
increased the proportion of apoptotic cells in a dose-dependent
manner (Fig. 6E and 6F, and S3$B). Similar apoptotic effects were
shown by morphological changes in cell nuclei by Hoechst staining.
Overall, these results suggested that F-30 significantly inhibits cell
survival and increases apoptosis in HCC cells (Fig. 6G).
Further studies were carried out to see how F-30 induces
apoptosis. Genomic stability relies on an effective DNA damage
repair pathway to retain the chromosomes intact [40]. Introducing
DNA damage not only causes genome mutations but also accu-
mulates cell cycle arrest and finally apoptosis. We hypothesized
that apoptosis might be caused by F-30 induced DNA damage. To
confirm this hypothesis, we performed an immunofluorescence
assay with antibody against p53-binding protein 1 (53BP1), an
important regulator in the response to DNA damage. The amount of
Fig. 4. F-30 inhibited the phosphorylation of FGFR4 and its downstream pathways. (A)
Plots were based on the expression values of FGFR4 in liver cancer samples compared
to normal tissues (n ¼ 423). (B) KaplaneMeier analysis showed the association be-
tween FGFR4 expression and disease-free survival or overall survival of liver cancer
patients. Data was acquired from The Cancer Genome Atlas (TCGA). (C) Compound F-
30 dose-dependently inhibited the activation of FGFR4 and downstream proteins in
HUH7 cells. HUH7 were incubated with the F-30 at different concentrations for 6 h and
then added FGF19 before cell lysis. Western blot analysis was used to determine the
protein expression of p-FGFR, FGFR4, p-MAPK, MAPK. GAPDH was used as loading
control. The results were representative of three replicated experiments. (D), (E) The
intensity of p-FGFR4 and p-MAPK were calculated.
5

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
Fig. 8. (A) Predicted binding model of F-30 and FGFR4 (PDB 4TYI). (B) Predicted
binding model of F-30 and FGFR4 (PDB 5NWZ). (C) MALDI-TOF MS determination of
FGFR4. (D) MALDI-TOF MS determination of FGFR4 and FGFR4/F-30 complex.
scaffold was adjacent to the hinge region of the FGFR4 protein
and interacted effectively with Glu551 and Ala553 through three H
bonds. The 3, 5-dimethoxybenzene motif was anchored to the hy-
drophobic region by adopting the parallel orientation with respect
to the plane of the 1H-indazol-3-amine core; an H bond was pre-
sent between the oxygen atom of the 3-methoxy and the amine of
Lys 503. Benzamide-linked 2-acetbromamide was poised in a
suitable position to engage Cys552 in a covalent bond. This
reasonable combination mode indicates a rational design of our
inhibitor, which convinced us of our design approach. To discover
more possible binding modes, we docked F-30 with another FGFR4
protein (PDB 5NWZ). What we found interesting was that F-30 also
covalently bound to Cys477. The 1H-indazol-3-amine scaffold
interacted effectively with Glu551 and Ala553 and formed two H
bonds. The 3, 5-dimethoxybenzene motif vertically inserted into
the hydrophobic pocket and form H bond with the residue of
Asp630 (Fig. 8B). Combining the kinase activities of F-30, F1-1, F1-2,
and the selectivity theory of FGFR4 inhibitors reported by Lin’s
group [41], we predicated that F-30 was covalently bound to
Cys477/Cys 522 which lead to the selectivity against FGFR1. On
account of the high homologous of FGFR1-4 kinase sequence, the p-
loop cysteine is conserved across FGFR1-4. Hence, the covalent
binding mode of the p-loop cysteine of FGFR4 could occur to a
greater or lesser extent across the FGFR1/2/3. It means that F-30
may covalently bind to the cysteine on the p-loop ring of FGFR1/2/3
protein and eventually lead to the different selectivity against
FGFR1/2/3.
Fig. 6. F-30 showed promising efficacy to suppress HCC cells proliferation. (A) IC50
values of F-30 in HCC. Three HCC lines were seeded in 96-well plates and treated with
various concentrations of F-30. Proliferation was measured after 48 h of treatment by
the MTT assay. Data were expressed as the mean ± SD of 3 independent experiments.
(
B) Colony-forming assay of the indicated cell lines. The cell was incubated with F-30
for 24 h. On day 7, colonies were fixed and photographed. Representative images were
displayed. (C) HUH7 cells were treated with the indicated concentrations of F-30 and
incubated for 48 h. Cells were stained with Annexin V and propidium iodide (PI) and
then analyzed by flow cytometry. (E) Cell stained with Hoechst staining was observed
in HUH7, HepG2, and SMMC-7721 with F-30 for 12 h.
Fig. 7. F-30 induced tumor cell apotosis. (A) HUH7 cells were incubated with the F-30
at different concentrations for 12 h 53BP1 (green) immunofluorescence assay was
performed, DAPI-stained nuclei blue. (B) Green focis were counted to make statistics.
5
7
5
3BP1 represents the level of DNA damage. As shown in Fig. 7A and
B, with increasing concentrations of drug, increasing numbers of
3BP1 foci gathered in the nuclei in HUH7 cell lines. In the un-
2
.7. F-30 covalently binds to FGFR4 protein
treated group, there were almost no foci, but the average number of
foci per cell at the highest concentration was six times higher than
in the control group. F-30 showed the same effect in the SMMC-
To verify whether compound F-30 inhibited FGFR4 kinase via
irreversible binding, MALDI-TOF MS assay was performed to
confirm the covalent binding. In this assay, FGFR4 kinase was
preincubated with compound F-30 at 4 C for 24 h. As shown in
7
721 cell line (Figure S3. A).
ꢀ
2
.6. Molecular docking of compound F-30 to FGFR4
Fig. 8C and 8D, the molecular weight of FGFR4 was 51,654.966 Da,
and the molecular weight of FGFR4 incubated with F-30 was
52,096.322 Da, consistent with the sum of the molecular weights of
the kinase and compound within the margin of error. This assay
suggested that F-30 could irreversibly bind to FGFR4.
To investigate the binding mode of the inhibitor we had
designed, F-30 was docked into the ATP-binding pocket of FGFR4
PDB: 4TYI). As portrayed in Fig. 8A, the 1H-indazol-3-amine
(
6

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
3
. Conclusion
Tert-butyl 3-amino-6-bromo-1H-indazole-1-carboxylate (6). To
a mixture of compound 5 (1 eq, 5 g), DMAP (0.2 eq, 435 mg), THF
Uniting the structure-based design and computer-aided drug
2
(50 mL) was added (Boc) O (1.2 eq, 6.175 g) dropwise, the solution
design strategies, a series of indazole derivatives were designed
and synthesized as novel potent FGFR4 inhibitors. After three
rounds of structure-activity relationship exploration, kinase activ-
ities indicated that compounds F-30, F1-1, and F1-2 had better
inhibitory activity to FGFR4 kinase with different FGFR1 selectivity.
It was found that F-30 and F1-2 showed excellent FGFR4 selectivity
against FGFR1, compound F-30 had the best selectivity against
FGFR1. Moreover, all three compounds showed better antitumor
activity than BLU9931 in a dose-dependent manner in three FGFR4
high-expression HCC cell lines. In addition, F-30 exhibited a potent
antiproliferative effect against FGFR4 high-expression HCC cell
lines HUH7, HepG2, and SMMC-7721 through potent inhibition of
the MAPK signaling pathways, suppression of cell cycle progres-
sion, and induction of apoptosis. Further study confirmed that
apoptosis was induced by DNA damage. Additionally, F-30 inhibited
the expression of p-FAK and FAK, adhesion, and invasion ability of
HCC cell lines in a dose-dependent manner. Mass spectrometry
experiments showed that F-30 was bound to FGFR4 protein via
covalent combination manner. The expected aim to design covalent
inhibitor has been achieved. Although we expected the compounds
to target the hinge Cys552 of FGFR4 at the beginning of the
experiment, molecular docking of compound F-30 with FGFR4
protein suggested that F-30 could bind to Cys552 or Cys477.
Considering that the cysteine of the p-loop ring is conservatively
present in FGFR1-4, we suspected that the preferred compound F-
was stirred at room temperature for 0.5 h. Then the solution was
concentrated with a rotary evaporator, the mixture was poured into
water and extracted with CH
washed with saturated aqueous NaCl, dried over anhydrous
Na SO , concentrated in vacuo, purified by silica gel chromatog-
2
Cl
2
(3 ꢂ 15 mL). The organic layer was
2
4
raphy (petroleum ether/ethyl acetate/dichloromethane ¼ 4:1:1, v/
1
v/v) to give the product 6 as a white solid (6.183 g, 84%). H NMR
(400 MHz, DMSO-d
6
)
d
8.11 (s, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.44 (dd,
J ¼ 8.4, 1.5 Hz, 1H), 6.42 (s, 2H), 1.57 (s, 9H).
Tert-butyl
6-bromo-3-(2-nitrobenzamido)-1H-indazole-1-
carboxylate (7). To a solution of compound 6 (1 eq, 100 mg) in 1,
4-dioxane (20 mL) was added acryloyl chloride (2.5 eq, 149 mg)
ꢀ
dissolved in DCM (0.5 mL) at 0 C. The reaction solution was stirred
at room temperature for 3 h. The mixture was poured into water
and extracted with CH
washed with saturated aqueous NaCl, dried over anhydrous
Na SO , and concentrated in vacuo. The residue was purified by
2
Cl
2
(3 ꢂ 20 mL). The organic layer was
2
4
silica gel chromatography (petroleum ether/ethyl acetate/
dichloromethane ¼ 3:1:1, v/v/v) to give the title product 7 as a
1
white solid (126 mg, 85%). H NMR (400 MHz, DMSO-d
6
)
d
11.78 (s,
1H), 8.29 (s, 1H), 8.18 (d, J ¼ 8.1 Hz, 1H), 7.99 (d, J ¼ 8.7 Hz, 1H), 7.88
(d, J ¼ 6.9 Hz, 2H), 7.78 (t, J ¼ 7.1 Hz,1H), 7.59 (dd, J ¼ 8.7, 1.4 Hz,1H),
1.63 (s, 9H).
Tert-butyl 6-(3,5-dimethoxyphenyl)-3-(2-nitrobenzamido)-1H-
indazole-1-carboxylate (8). To a mixture of compound 7 (1 eq,
3
0 could bind to the cysteine of the p-loop ring of FGFR1/2/3
2 3
400 mg), Cs CO (2 eq, 566 mg), (3, 5-dimethoxyphenyl)boronic
covalently to different extent. That may explain the difference in
biochemical IC50 for the F-30 across FGFR1/2/3. A deep mechanism
of the binding model is underway, and we will give it further
exploration. Altogether, this target compound showed promise for
more exploration as a selective FGFR4 inhibitor for the treatment of
HCC. Further studies on structural optimization and biological ac-
tivities of these derivatives are still in progress in our laboratory.
And we are supposed to find highly effective FGFR4 inhibitors in the
future.
acid (2 eq, 316 mg) in 1,4-dioxane (20 mL) was added Pd (pph
3
)
4
ꢀ
(0.1 eq, 100 mg), the solution was stirred at 110 C under N
2
at-
mosphere for 1 h. Then the solution was concentrated with a rotary
evaporator. The mixture was poured into water and extracted with
CH
2
Cl
2
(3 ꢂ 15 mL). The organic layer was washed with saturated
aqueous NaCl, dried over anhydrous Na
2
SO , and concentrated in
4
vacuo. The residue was purified by silica gel chromatography (pe-
troleum ether/ethyl acetate/dichloromethane ¼ 2:1:1, v/v/v) to give
1
the product 8 as a white solid (400 mg, 77%). H NMR (400 MHz,
6
DMSO-d ) d 11.72 (s, 1H), 11.72 (s, 1H), 11.72 (s, 1H), 8.31 (s, 1H), 8.31
4
. Experimental section
(s, 1H), 8.31 (s, 1H), 8.18 (t, J ¼ 10.2 Hz, 1H), 8.09 (d, J ¼ 8.5 Hz, 1H),
7
.89 (s, 2H), 7.80 (d, J ¼ 8.1 Hz, 1H), 7.73 (d, J ¼ 8.5 Hz, 1H), 6.87 (d,
4.1. Chemistry
J ¼ 1.3 Hz, 2H), 6.58 (s, 1H), 3.83 (s, 6H), 1.66 (s, 9H).
Tert-butyl 3-(2-aminobenzamido)-6-(3,5-dimethoxyphenyl)-
1H-indazole-1-carboxylate (9). To a solution of compound 8
All starting materials and regents were acquired from com-
mercial suppliers or prepared according to known procedures. All
starting materials and solvents were used without further purifi-
cation. Chemical reactions were monitored by thin-layer chroma-
tography (TLC), using silica gel plates with fluorescence F254 and
visualized under UV light. Flash chromatography was conducted
3
(400 mg) in CH OH (15 mL) was added catalytic Pd/C, then the
reaction was stirred overnight in hydrogen environment. Then the
mixture was filted and the filtrate was concentrated with a rotary
evaporator. The residue was purified by silica gel chromatography
(petroleum ether/ethyl acetate/dichloromethane ¼ 5:1:1, v/v/v) to
1
13
1
using silica gel (200e300 mesh). H NMR and C NMR were
generated in DMSO‑d on Bruker 400 or 500 NMR spectrometers.
give the product 9 as a white amorphous solid (343 mg, 91%). H
6
6
NMR (500 MHz, DMSO-d ) d 11.00 (s,1H), 8.30 (s,1H), 7.94e7.75 (m,
All tested compounds were purified to ꢁ95% purity by the Agilent
2H), 7.66 (d, J ¼ 8.5 Hz, 1H), 7.24 (t, J ¼ 7.6 Hz, 1H), 6.86 (s, 2H), 6.78
infinity 1260 HPLC system.
(d, J ¼ 8.3 Hz, 1H), 6.62 (s, 2H), 6.57 (t, J ¼ 7.4 Hz, 2H), 3.82 (s, 6H),
1.67 (s, 9H).
4.2. General procedure for synthesis of compounds (take F-1 as
Tert-butyl
3-(2-acrylamidobenzamido)-6-(3,5-
example)
dimethoxyphenyl)-1H-indazole-1-carboxylate. To a solution of
compound 9 (1 eq, 100 mg) in 1, 4-dioxane (5 mL) was added DIPEA
ꢀ
6
-bromo-1H-indazol-3-amine (5). To a solution of 4-bromo-2-
(3 eq, 96 mg) and acryloyl chloride (2.5 eq, 62 mg) at 0 C. The
fluorobenzonitrile (1 eq, 100 mg) in 2-amyl alcohol (4 mL) was
reaction solution was stirred at room temperature for 2 h. The
added hydrazine hydrate (5 eq, 125 mg), the mixture was stirred at
mixture was poured into water and extracted with CH
(3 ꢂ 20 mL). The organic layer was washed with saturated aqueous
NaCl, dried over anhydrous Na SO , and concentrated in vacuo to
get the crude product. H NMR (400 MHz, DMSO-d 11.43 (s, 1H),
2 2
Cl
ꢀ
120 C for 3 h. Then the solution was cooled to the room temper-
ature, filtered and washed by ethyl alcohol to give the title com-
2
4
1
1
pound 5 as white crystalline solid (86 mg, 81%). H NMR (400 MHz,
6
) d
DMSO-d
6
)
d
11.51 (s, 1H), 7.66 (d, J ¼ 8.5 Hz, 1H), 7.46 (d, J ¼ 0.9 Hz,
10.77 (s, 1H), 8.31 (s, 1H), 8.13 (d, J ¼ 8.2 Hz, 1H), 8.00 (d, J ¼ 8.4 Hz,
1
H), 7.02 (dd, J ¼ 8.5, 1.3 Hz, 1H), 5.48 (s, 2H).
1H), 7.93 (d, J ¼ 7.5 Hz, 1H), 7.67 (d, J ¼ 8.5 Hz, 1H), 7.59 (t, J ¼ 7.7 Hz,
7

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
1
(
H), 7.28 (t, J ¼ 7.5 Hz, 1H), 6.86 (d, J ¼ 1.9 Hz, 2H), 6.58 (s, 1H), 6.39
dd, J ¼ 17.0, 10.2 Hz, 1H), 6.22 (d, J ¼ 16.9 Hz, 1H), 5.75 (d,
J ¼ 10.2 Hz, 1H), 3.83 (s, 6H), 1.67 (s, 9H).
-acrylamido-N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)
4.2.4. 2-Acrylamido-N-(6-(2-methoxypyridin-4-yl)-1H-indazol-3-
yl)benzamide (F-5)
White powder, 56.8% yield. HPLC analysis: purity ¼ 98.1307%,
1
2
retention time ¼ 12.679 min H NMR (500 MHz, DMSO‑d
6
): d 13.07
benzamide (10). To a solution of crude product in the step above in
DCM (4 mL) was added TFA (2 mL), the mixture was stirred at room
temperature for 2 h. Then the solution was concentrated with a
rotary evaporator, extracted with saturated aqueous NaHCO and
3
DCM, The organic layer was collected and dried. Then the residue
was washed by 2 mL DCM twice to get purity target product 10 (F-
(s, 1H), 11.10 (s, 1H), 11.06 (s, 1H), 8.33 (d, J ¼ 8.2 Hz, 1H), 8.25 (d,
J ¼ 5.3 Hz,1H), 8.02 (d, J ¼ 7.4 Hz,1H), 7.91 (d, J ¼ 8.5 Hz,1H), 7.85 (s,
1H), 7.59 (t, J ¼ 7.7 Hz,1H), 7.49 (d, J ¼ 8.5 Hz,1H), 7.39 (d, J ¼ 5.2 Hz,
1H), 7.28 (t, J ¼ 7.5 Hz, 1H), 7.19 (s, 1H), 6.40 (dd, J ¼ 17.0, 10.2 Hz,
13
1H), 6.24 (d, J ¼ 16.9 Hz, 1H), 5.77 (d, J ¼ 10.3 Hz, 1H). C NMR
6
(126 MHz, DMSO‑d ): d 167.52, 164.45, 163.28, 150.69, 147.42,
1
) as a white compound (43 mg, 47%). White powder, 54.5% yield.
141.43, 139.68, 138.41, 135.59, 132.39, 132.19, 129.11, 127.01, 123.38,
1
HPLC analysis: purity ¼ 95.1980%, retention time ¼ 13.058 min H
NMR (400 MHz, DMSO‑d ): 12.91 (s, 1H), 11.11 (s, 1H), 11.02 (s, 1H),
.34 (d, J ¼ 8.3 Hz, 1H), 8.01 (d, J ¼ 7.7 Hz, 1H), 7.84 (d, J ¼ 8.5 Hz,
H), 7.70 (s, 1H), 7.60 (t, J ¼ 7.8 Hz, 1H), 7.41 (dd, J ¼ 8.6, 1.2 Hz, 1H),
122.65, 122.44, 121.65, 119.08, 117.27, 115.56, 108.52, 107.95, 53.22.
þ
6
d
HRMS (ACPI) m/z calcd. for C23
414.1568.
H
19
N
5
O
3
[M þ H] : 414.1561. Found:
8
1
7
6
.28 (t, J ¼ 7.6 Hz, 1H), 6.86 (d, J ¼ 2.2 Hz, 2H), 6.54 (t, J ¼ 2.1 Hz, 1H),
4.2.5. 2-Acrylamido-N-(6-(thiophen-2-yl)-1H-indazol-3-yl)
benzamide (F-6)
.39 (dd, J ¼ 17.0,10.2 Hz,1H), 6.24 (dd, J ¼ 17.0,1.5 Hz,1H), 5.78 (dd,
13
J ¼ 10.2, 1.5 Hz, 1H), 3.83 (s, 6H). C NMR (126 MHz, DMSO‑d
167.50, 163.27, 160.86, 142.74, 141.60, 139.55, 138.71, 138.45,
32.41, 132.20, 129.09, 127.02, 123.36, 122.34, 122.16, 121.59, 119.74,
6
)
White powder, 61.2% yield. HPLC analysis: purity ¼ 96.4612%,
1
d
1
retention time ¼ 12.863 min H NMR (400 MHz, DMSO‑d
6
): d 12.90
(s, 1H), 11.07 (s, 1H), 11.02 (s, 1H), 8.31 (d, J ¼ 8.3 Hz, 1H), 7.99 (d,
J ¼ 7.5 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 1H), 7.69 (s, 1H), 7.65e7.54 (m,
3H), 7.43 (dd, J ¼ 8.6, 1.2 Hz, 1H), 7.27 (t, J ¼ 7.5 Hz, 1H), 7.17 (dd,
J ¼ 5.0, 3.7 Hz, 1H), 6.39 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.23 (dd, J ¼ 17.0,
116.48, 107.95, 105.35, 99.50, 55.30. HRMS (ACPI) m/z calcd. for
þ
C
25
22
H N
4
O
4
[M þ H] : 443.1714. Found: 443.1715.
13
1
.5 Hz, 1H), 5.77 (dd, J ¼ 10.2, 1.4 Hz, 1H). C NMR (126 MHz,
4.2.1. 2-Acrylamido-N-(6-(3-methoxyphenyl)-1H-indazol-3-yl)
DMSO‑d
6
): d 167.48, 163.28, 143.60, 141.53, 139.75, 138.40, 132.41,
benzamide (F-2)
132.19, 132.07, 129.09, 128.55, 127.02, 125.84, 124.24, 123.37, 122.63,
White powder, 47.5% yield. HPLC analysis: purity ¼ 96.3415%,
1
122.43, 121.63, 118.54, 116.32, 106.11. HRMS (ACPI) m/z calcd. for
retention time ¼ 13.108 min H NMR (400 MHz, DMSO‑d
6
): d 12.93
þ
C
21
H
16
N
4
O
2
S [M þ H] : 389.1067. Found: 389.1069.
(
s, 1H), 11.10 (s, 1H), 11.03 (s, 1H), 8.32 (d, J ¼ 8.0 Hz, 1H), 8.01 (d,
J ¼ 7.3 Hz, 1H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.69 (s, 1H), 7.59 (t, J ¼ 8.0 Hz,
4.2.6. 2-Acrylamido-N-(6-(thiophen-3-yl)-1H-indazol-3-yl)
1
H), 7.48e7.36 (m, 2H), 7.23e7.34 (m 3H), 6.97 (d, J ¼ 8.0 Hz, 1H),
.39 (dd, J ¼ 16.9, 10.1 Hz, 1H), 6.23 (d, J ¼ 16.9 Hz, 1H), 5.77 (d,
benzamide (F-7)
6
13
White powder, 51.7% yield. HPLC analysis: purity ¼ 96.5045%,
J ¼ 10.8 Hz, 1H), 3.85 (s, 3H). C NMR (126 MHz, DMSO‑d
6
):
1
retention time ¼ 12.763 min H NMR (500 MHz, DMSO‑d
6
): d 12.88
d
1
167.53, 163.28, 159.76, 142.05, 141.68, 139.57, 138.66, 138.47,
32.42, 132.20, 129.98, 129.10, 127.01, 123.36, 122.33, 122.24, 121.60,
(
s, 1H), 11.09 (s, 1H), 10.99 (s, 1H), 8.33 (d, J ¼ 8.1 Hz, 1H), 8.00 (d,
J ¼ 7.1 Hz, 1H), 7.97 (s, 1H), 7.80 (d, J ¼ 8.5 Hz, 1H), 7.75 (s, 1H),
119.71, 119.51, 116.41, 113.14, 112.68, 107.88, 55.16. HRMS (ACPI) m/z
þ
7.71e7.63 (m, 2H), 7.59 (t, J ¼ 7.6 Hz,1H), 7.49 (d, J ¼ 8.5 Hz,1H), 7.28
calcd. for C24
H
20
N
4
O
3
[M þ H] : 413.1608. Found: 413.1607.
(
1
t, J ¼ 7.4 Hz, 1H), 6.39 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.23 (d, J ¼ 17.0 Hz,
13
H), 5.78 (d, J ¼ 10.3 Hz, 1H). C NMR (126 MHz, DMSO‑d
167.49, 163.28, 141.72, 141.61, 139.61, 138.44, 133.53, 132.42,
32.19, 129.09, 127.08, 127.02, 126.48, 123.36, 122.38, 122.22, 121.60,
6
):
4
.2.2. 2-Acrylamido-N-(6-(4-methoxyphenyl)-1H-indazol-3-yl)
benzamide (F-3)
White powder, 46.3% yield. HPLC analysis: purity ¼ 98.7086%,
d
1
1
C
21.48, 119.22, 116.09, 106.84. HRMS (ACPI) m/z calcd. For
1
retention time ¼ 12.818 min H NMR (400 MHz, DMSO‑d
6
):
d
12.93
þ
21
H
16
N
4
O
2
S [M þ H] : 389.1067. Found: 389.1067.
(
s, 1H), 11.10 (s, 1H), 11.03 (s, 1H), 8.32 (d, J ¼ 8.0 Hz, 1H), 8.01 (d,
J ¼ 7.3 Hz, 1H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.69 (s, 1H), 7.59 (t, J ¼ 8.0 Hz,
4.2.7. 2-Acrylamido-N-(6-(2,3-dihydrobenzofuran-5-yl)-1H-
1
H), 7.48e7.36 (m, 2H), 7.23e7.34 (m 3H), 6.97 (d, J ¼ 8.0 Hz, 1H),
.39 (dd, J ¼ 16.9, 10.1 Hz, 1H), 6.23 (d, J ¼ 16.9 Hz, 1H), 5.77 (d,
indazol-3-yl)benzamide (F-8)
6
White powder, 52.4% yield. HPLC analysis: purity ¼ 97.9621%,
13
J ¼ 10.8 Hz, 1H), 3.85 (s, 3H). C NMR (126 MHz, DMSO‑d
6
):
1
retention time ¼ 12.746 min H NMR (400 MHz, DMSO‑d
6
): d 13.02
d
1
167.51, 163.28, 158.99, 141.87, 139.71, 138.54, 138.39, 132.84,
32.49, 132.08, 129.12, 128.19, 126.95, 123.29, 122.39, 122.18, 121.52,
(
s, 1H), 11.09 (s, 2H), 8.32 (d, J ¼ 8.1 Hz, 1H), 8.03 (d, J ¼ 7.3 Hz, 1H),
7
7
6
.80 (d, J ¼ 8.5 Hz, 1H), 7.66e7.51 (m, 3H), 7.45 (d, J ¼ 8.3 Hz, 1H),
119.34, 115.87, 114.41, 106.95, 55.19. HRMS (ACPI) m/z calcd. for
.32 (d, J ¼ 8.4 Hz,1H), 7.26 (t, J ¼ 7.2 Hz,1H), 6.86 (d, J ¼ 8.3 Hz,1H),
þ
C
24 20
H N
4
O
3
[M þ H] : 413.1608. Found: 413.1609.
.41 (dd, J ¼ 17.0, 10.0 Hz, 1H), 6.23 (d, J ¼ 16.6 Hz, 1H), 5.77 (d,
13
J ¼ 6.1 Hz, 1H), 4.58 (t, J ¼ 8.7 Hz, 2H), 3.25 (t, J ¼ 8.6 Hz, 2H).
C
4.2.3. 2-Acrylamido-N-(6-(3,5-dimethylphenyl)-1H-indazol-3-yl)
6
NMR (126 MHz, DMSO‑d ): d 167.49, 163.30, 159.50, 141.87, 139.70,
benzamide (F-4)
138.90, 138.52, 133.04, 132.53, 132.03, 129.19, 128.19, 126.86, 123.89,
White powder, 47.2% yield. HPLC analysis: purity ¼ 99.1762%,
123.28, 122.66, 122.04, 121.55, 119.39, 115.79, 109.16, 106.95, 71.18,
1
þ
retention time ¼ 13.886 min H NMR (400 MHz, DMSO‑d
6
):
d
12.93
29.09. HRMS (ACPI) m/z calcd. for C25
Found: 425.1608.
H
20
N
4
O
3
[M þ H] : 425.1608.
(
s, 1H), 11.10 (s, 1H), 11.03 (s, 1H), 8.32 (d, J ¼ 8.0 Hz, 1H), 8.01 (d,
J ¼ 7.3 Hz, 1H), 7.85 (d, J ¼ 8.4 Hz, 1H), 7.69 (s, 1H), 7.59 (t, J ¼ 8.0 Hz,
1
6
H), 7.48e7.36 (m, 2H), 7.23e7.34 (m 3H), 6.97 (d, J ¼ 8.0 Hz, 1H),
.39 (dd, J ¼ 16.9, 10.1 Hz, 1H), 6.23 (d, J ¼ 16.9 Hz, 1H), 5.77 (d,
4.2.8. 2-Acrylamido-N-(6-(3-(trifluoromethoxy)phenyl)-1H-
indazol-3-yl)benzamide (F-9)
1
3
J ¼ 10.8 Hz, 1H), 3.85 (s, 3H). C NMR (126 MHz, DMSO‑d
6
):
White powder, 48.1% yield. HPLC analysis: purity ¼ 96.4298%,
1
d
1
1
C
167.53, 163.27, 141.75, 140.43, 139.54, 139.00, 138.47, 137.91,
32.42, 132.19, 129.10, 128.91, 127.00, 124.96, 123.35, 122.32, 122.12,
retention time ¼ 12.856 min H NMR (400 MHz, DMSO‑d
6
): d 13.03
(s, 1H), 11.11 (s, 1H), 11.07 (s, 1H), 8.33 (d, J ¼ 8.2 Hz, 1H), 8.02 (d,
J ¼ 7.6 Hz,1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.77 (s,
1H), 7.74 (s, 1H), 7.68e7.55 (m, 2H), 7.45 (d, J ¼ 8.5 Hz, 1H), 7.40 (d,
21.58, 119.68, 116.26, 107.58, 20.98. HRMS (ACPI) m/z calcd. for
þ
25
22
H N
4
O
2
[M þ H] : 411.1816. Found: 411.1814.
8

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
þ
J ¼ 8.1 Hz, 1H), 7.28 (t, J ¼ 7.5 Hz, 1H), 6.40 (dd, J ¼ 17.0, 10.2 Hz, 1H),
(ACPI) m/z calcd. for C24
431.1512.
H
19FN
4
O
3
[M þ H] : 431.1514. Found:
13
6
.24 (d, J ¼ 16.8 Hz, 1H), 5.77 (d, J ¼ 11.9 Hz, 1H). C NMR (126 MHz,
6
DMSO‑d ): d 167.54, 163.28, 148.98, 142.91, 141.59, 139.64, 138.44,
136.92, 132.41, 132.18, 130.84, 129.11, 126.98, 126.28, 123.36, 123.22,
4.2.13. 2-Acrylamido-N-(6-(2-fluoro-5-methoxyphenyl)-1H-
1
22.56, 122.40, 121.62, 121.18, 119.75, 119.65, 119.46, 119.14, 117.10,
indazol-3-yl)benzamide (F-14)
þ
116.71, 108.33. HRMS (ACPI) m/z calcd. for C24
H
17
F
3
N
4
O
3
[M þ H] :
White powder, 44.4% yield. HPLC analysis: purity ¼ 98.4778%,
1
4
67.1325. Found: 467.1324.
retention time ¼ 12.955 min H NMR (400 MHz, DMSO‑d
6
): d 12.96
(
s, 1H), 11.10 (s, 1H), 11.03 (s, 1H), 8.33 (d, J ¼ 8.2 Hz, 1H), 8.02 (d,
4.2.9. 2-Acrylamido-N-(6-(2-fluoro-5-(trifluoromethoxy)phenyl)-
J ¼ 7.8 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.64 (s, 1H), 7.59 (t, J ¼ 7.8 Hz,
1H), 7.27 (dd, J ¼ 11.1, 8.3 Hz, 3H), 7.12 (dd, J ¼ 6.4, 3.1 Hz, 1H),
6.94e7.02 (m, 1H), 6.39 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.23 (dd, J ¼ 17.0,
1H-indazol-3-yl)benzamide (F-10)
White powder, 46.5% yield. HPLC analysis: purity ¼ 98.8326%,
1
13
retention time ¼ 13.480 min H NMR (500 MHz, DMSO‑d
6
):
d
13.05
1.5 Hz, 1H), 5.77 (dd, J ¼ 10.2, 1.5 Hz, 1H), 3.81 (s, 3H). C NMR
(
s, 1H), 11.11 (s, 1H), 11.07 (s, 1H), 8.34 (d, J ¼ 8.2 Hz, 1H), 8.03 (d,
6
(126 MHz, DMSO‑d ): d 167.54, 163.28, 155.69, 154.44, 152.55,
J ¼ 7.6 Hz, 1H), 7.91 (d, J ¼ 8.5 Hz, 1H), 7.70 (s, 1H), 7.66 (dd, J ¼ 6.0,
141.17, 139.61, 138.44, 133.29, 132.41, 132.21, 129.10, 128.99, 127.02,
123.38, 122.37, 121.91, 121.62, 121.13, 116.88, 116.69, 116.41, 115.59,
2
2
5
d
1
1
.3 Hz, 1H), 7.59 (t, J ¼ 7.8 Hz, 1H), 7.55e7.40 (m, 2H), 7.35e7.20 (m,
H), 6.40 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.24 (dd, J ¼ 17.0, 1.2 Hz, 1H),
115.56, 114.54, 114.47, 110.31, 110.29, 55.71. HRMS (ACPI) m/z calcd.
13
þ
.77 (dd, J ¼ 10.3, 1.1 Hz, 1H). C NMR (126 MHz, DMSO‑d
6
):
for C24
H
19FN
4
O
3
[M þ H] : 431.1514. Found: 431.1512.
167.57, 163.28, 158.53, 156.57, 144.54, 141.07, 139.68, 138.43,
32.40, 132.20, 131.65, 130.34, 130.21, 129.11, 126.98, 123.76, 123.73,
23.37, 123.14, 122.40, 122.19, 122.15, 121.63, 121.10, 120.88, 120.87,
4.2.14. 2-Acrylamido-N-(6-(3,5-dimethoxyphenyl)-1H-pyrazolo
[3,4-b]pyridin-3-yl)benzamide (F-15)
1
19.06, 118.02, 117.81, 117.02, 116.70, 110.68. HRMS (ACPI) m/z calcd.
White powder, 45.6% yield. HPLC analysis: purity ¼ 97.4189%,
þ
1
for C24
H
16
F
4
N
4
O
3
[M þ H] : 485.1231. Found: 485.1232.
retention time ¼ 13.061 min H NMR (400 MHz, DMSO‑d
6
): d 13.17
(
s, 1H), 10.81 (s, 2H), 8.42 (d, J ¼ 8.5 Hz, 1H), 8.20 (d, J ¼ 8.1 Hz, 1H),
4.2.10. 2-Acrylamido-N-(6-(2-fluoro-3-methoxyphenyl)-1H-
7.96 (d, J ¼ 7.5 Hz, 1H), 7.79 (d, J ¼ 8.6 Hz, 1H), 7.57 (t, J ¼ 7.6 Hz, 1H),
7.32 (d, J ¼ 2.0 Hz, 2H), 7.31e7.16 (m, 1H), 6.62 (s, 1H), 6.44 (dd,
indazol-3-yl)benzamide (F-11)
White powder, 51.5% yield. HPLC analysis: purity ¼ 98.8745%,
J ¼ 17.0, 10.2 Hz, 1H), 6.23 (d, J ¼ 16.9 Hz, 1H), 5.76 (d, J ¼ 11.2 Hz,
1
13
retention time ¼ 12.749 min H NMR (400 MHz, DMSO‑d
6
):
d
12.98
1H), 3.84 (s, 6H). C NMR (126 MHz, DMSO‑d
6
):
d
167.18, 163.33,
(
s, 1H), 11.10 (s, 1H), 11.06 (s, 1H), 8.33 (d, J ¼ 8.3 Hz, 1H), 8.01 (d,
160.82, 155.45, 151.99, 140.81, 139.22, 138.01, 133.02, 132.36, 131.97,
J ¼ 7.6 Hz, 1H), 7.86 (d, J ¼ 8.5 Hz, 1H), 7.67e7.54 (m, 2H), 7.35e7.17
129.20, 126.95, 123.50, 123.44, 121.95, 113.80, 107.89, 105.16, 101.57,
þ
(
6
m, 4H), 7.14 (dd, J ¼ 9.6, 4.5 Hz, 1H), 6.40 (dd, J ¼ 17.0, 10.1 Hz, 1H),
55.39. HRMS (ACPI) m/z calcd. for C24
Found: 444.1665.
H
21
N
5
O
4
[M þ H] : 444.1666.
13
.24 (d, J ¼ 15.9 Hz, 1H), 5.77 (d, J ¼ 11.2 Hz, 1H), 3.89 (s, 3H).
): 167.55, 163.28, 149.80, 147.86, 147.85,
47.78, 141.17, 139.62, 138.44, 133.18, 132.41, 132.20, 129.26, 129.18,
29.10, 127.01, 124.53, 124.50, 123.37, 122.37, 121.92, 121.62, 121.17,
C
NMR (126 MHz, DMSO‑d
1
1
6
d
4.2.15. 2-Acrylamido-N-(6-(3-methoxyphenyl)-1H-pyrazolo[3,4-b]
pyridin-3yl)benzamide (F-16)
1
21.16, 116.40, 113.06, 110.27, 110.25, 56.15. HRMS (ACPI) m/z calcd.
White powder, 54.3% yield. HPLC analysis: purity ¼ 96.8679%,
þ
1
for C24
H19FN
4
O
3
[M þ H] : 431.1514. Found: 431.1513.
retention time ¼ 12.939 min H NMR (400 MHz, DMSO‑d
6
): d 13.46
(
s, 1H), 11.24 (s, 1H), 10.94 (s, 1H), 8.45 (d, J ¼ 8.5 Hz, 1H), 8.25 (d,
4.2.11. 2-Acrylamido-N-(6-(4-fluoro-3-methoxyphenyl)-1H-
J ¼ 8.2 Hz,1H), 7.97 (d, J ¼ 7.6 Hz,1H), 7.80 (d, J ¼ 8.6 Hz,1H), 7.75 (d,
J ¼ 7.4 Hz, 2H), 7.59 (t, J ¼ 7.7 Hz, 1H), 7.45 (t, J ¼ 7.9 Hz, 1H), 7.28 (t,
J ¼ 7.6 Hz,1H), 7.13e7.01 (m,1H), 6.43 (dd, J ¼ 17.0,10.2 Hz,1H), 6.25
indazol-3-yl)benzamide (F-12)
White powder, 53.3% yield. HPLC analysis: purity ¼ 99.0780%,
1
13
retention time ¼ 12.805 min H NMR (500 MHz, DMSO‑d
6
):
d
12.93
(d, J ¼ 16.1 Hz, 1H), 5.84e5.71 (m, 1H), 3.86 (s, 3H). C NMR
(
s, 1H), 11.11 (s, 1H), 11.03 (s, 1H), 8.34 (d, J ¼ 6.9 Hz, 1H), 8.02 (d,
6
(126 MHz, DMSO‑d ): d 172.43, 168.54, 164.98, 160.86, 157.34,
J ¼ 5.5 Hz, 1H), 7.86 (dd, J ¼ 8.4, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t,
145.40, 144.39, 143.36, 138.42, 137.59, 137.35, 135.13, 134.46, 132.29,
J ¼ 7.7 Hz, 1H), 7.48 (d, J ¼ 7.8 Hz, 1H), 7.42 (d, J ¼ 8.5 Hz, 1H),
128.73, 128.29, 127.09, 124.82, 120.54, 119.00, 117.48, 113.02, 60.45.
þ
7
1
(
1
.36e7.08 (m, 3H), 6.39 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.24 (dd, J ¼ 17.0,
HRMS (ACPI) m/z calcd. for C23
444.1560.
H
19
N
5
O
3
[M þ H] : 414.1561. Found:
13
.3 Hz, 1H), 5.78 (dd, J ¼ 10.2, 1.3 Hz, 1H), 3.97 (s, 3H). C NMR
126 MHz, DMSO‑d ): 167.51, 163.27, 152.34, 150.39, 147.41, 147.32,
41.64, 139.60, 138.45, 138.02, 137.56, 137.53, 132.42, 132.18, 129.10,
27.00, 123.36, 122.38, 122.24, 121.60, 119.72, 119.51, 119.46, 116.31,
6
d
4.2.16. 2-Acrylamido-N-(6-(4-fluoro-3-methoxyphenyl)-1H-
pyrazolo[3,4-b]pyridin-3yl)benzamide (F-17)
1
1
C
16.21, 116.06, 112.87, 107.93, 56.11. HRMS (ACPI) m/z calcd. for
White powder, 44.8% yield. HPLC analysis: purity ¼ 95.4518%,
þ
1
24
H19FN
4
O
3
[M þ H] : 431.1514. Found: 431.1505.
retention time ¼ 12.558 min H NMR (400 MHz, DMSO‑d
6
): d 13.45
(
s, 1H), 11.22 (s, 1H), 10.93 (s, 1H), 8.44 (d, J ¼ 8.5 Hz, 1H), 8.24 (d,
4.2.12. 2-Acrylamido-N-(6-(3-fluoro-5-methoxyphenyl)-1H-
J ¼ 8.2 Hz, 1H), 7.95 (dd, J ¼ 11.5, 4.7 Hz, 2H), 7.83 (d, J ¼ 8.6 Hz, 1H),
7.79e7.72 (m, 1H), 7.58 (t, J ¼ 7.8 Hz, 1H), 7.36 (dd, J ¼ 11.1, 8.6 Hz,
1H), 7.28 (t, J ¼ 7.5 Hz, 1H), 6.43 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.24 (dd,
indazol-3-yl)benzamide (F-13)
White powder, 39.8% yield. HPLC analysis: purity ¼ 97.7365%,
1
13
retention time ¼ 13.105 min H NMR (400 MHz, DMSO‑d
6
):
d
12.99
J ¼ 17.0, 1.1 Hz, 1H), 5.77 (d, J ¼ 11.4 Hz, 1H), 3.98 (s, 3H). C NMR
(
s, 1H), 11.10 (s, 1H), 11.06 (s, 1H), 8.33 (d, J ¼ 8.2 Hz, 1H), 8.01 (d,
6
(126 MHz, DMSO‑d ): d 167.17,163.28,154.84,153.56,152.03,151.59,
J ¼ 7.7 Hz, 1H), 7.87 (d, J ¼ 8.5 Hz, 1H), 7.75 (s, 1H), 7.60 (t, J ¼ 7.7 Hz,
147.45, 147.36, 139.16, 138.08, 135.59, 135.56, 133.19, 132.34, 132.08,
129.16, 127.00, 123.47, 123.08, 121.90, 119.96, 119.91, 116.19, 116.05,
1
1
1
1
1
1
1
H), 7.43 (d, J ¼ 9.1 Hz, 1H), 7.28 (t, J ¼ 7.5 Hz, 1H), 7.18 (d, J ¼ 9.8 Hz,
H), 7.15 (s, 1H), 6.87 (dd, J ¼ 10.9, 1.9 Hz, 1H), 6.40 (dd, J ¼ 17.0,
113.57, 112.39, 107.66, 56.07. HRMS (ACPI) m/z calcd. for
þ
0.1 Hz, 1H), 6.24 (dd, J ¼ 17.0, 1.1 Hz, 1H), 5.78 (dd, J ¼ 10.3, 1.1 Hz,
C
23
H18FN
5
O
3
[M þ H] : 432.1466. Found: 432.1467.
H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO‑d
63.28, 162.42, 161.11, 161.01, 143.48, 143.39, 141.52, 139.60, 138.43,
37.39, 132.40, 132.19, 129.10, 127.01, 123.37, 122.36, 121.63, 119.54,
16.70, 109.16, 108.20, 106.21, 106.03, 100.67, 100.47, 55.74. HRMS
6
): d 167.51, 164.34,
4.2.17. 2-Acrylamido-N-(6-(3-fluoro-5-methoxyphenyl)-1H-
pyrazolo[3,4-b]pyridin-3yl)benzamide (F-18)
White powder, 51.1% yield. HPLC analysis: purity ¼ 96.3863%,
9

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
1
þ
retention time ¼ 12.702 min H NMR (500 MHz, DMSO‑d
s, 1H), 11.17 (s, 2H), 8.47 (d, J ¼ 8.5 Hz, 1H), 8.25 (d, J ¼ 8.2 Hz, 1H),
.97 (d, J ¼ 7.6 Hz, 1H), 7.83 (d, J ¼ 8.6 Hz, 1H), 7.65e7.52 (m, 3H),
.27 (t, J ¼ 7.5 Hz, 1H), 6.95 (d, J ¼ 10.7 Hz, 1H), 6.43 (dd, J ¼ 17.0,
0.2 Hz,1H), 6.25 (d, J ¼ 17.0 Hz,1H), 5.84e5.71 (m,1H), 3.88 (s, 3H).
6
):
d
13.44
[M þ H] : 471.2027. Found: 471.2027.
(
7
7
4.2.22. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-2-
methacrylamidobenzamide (F-23)
1
White powder, 59.6% yield. HPLC analysis: purity ¼ 96.3535%,
1
3
1
C NMR (126 MHz, DMSO‑d
61.11, 161.02, 154.20, 154.18, 151.94, 141.58, 141.50, 139.32, 138.11,
33.34, 132.36, 132.04, 129.19, 126.97, 123.43, 123.14, 121.87, 113.72,
6
):
d
167.20, 164.34, 163.29, 162.42,
retention time ¼ 13.463 min H NMR (400 MHz, DMSO‑d
6
): d 12.98
1
1
(s, 1H), 11.67 (s, 1H), 11.13 (s, 1H), 8.56 (d, J ¼ 8.3 Hz, 1H), 8.11 (d,
J ¼ 7.7 Hz, 1H), 7.79 (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.62 (t, J ¼ 7.8 Hz,
1H), 7.42 (d, J ¼ 9.2 Hz,1H), 7.26 (t, J ¼ 7.7 Hz,1H), 6.87 (d, J ¼ 2.0 Hz,
2H), 6.54 (s, 1H), 5.86 (s, 1H), 5.54 (s, 1H), 3.83 (s, 7H), 1.98 (s, 3H).
1
08.89, 108.14, 106.08, 105.89, 102.62, 102.42, 55.79. HRMS (ACPI)
þ
m/z calcd. for C23
H18FN
5
O
3
[M þ H] : 432.1466. Found: 432.1466.
13
6
C NMR (126 MHz, DMSO‑d ): d 168.02, 165.60, 160.86, 142.71,
4.2.18. 2-Acrylamido-N-(6-(2-fluoro-5-methoxyphenyl)-1H-
141.63, 140.27, 139.39, 139.34, 138.79, 132.66, 129.19, 122.87, 121.83,
pyrazolo[3,4-b]pyridin3yl)benzamide (F-19)
120.83, 120.54, 120.28, 119.91, 116.56, 108.07, 105.36, 99.54, 55.30,
þ
White powder, 53.9% yield. HPLC analysis: purity ¼ 96.8993%,
18.15. HRMS (ACPI) m/z calcd. for C26
Found: 457.1867.
H N
24 4
O
4
[M þ H] : 457.1870.
1
retention time ¼ 13.085 min H NMR (400 MHz, DMSO‑d
6
):
d
13.54
(
s, 1H), 11.26 (s, 1H), 10.92 (s, 1H), 8.46 (d, J ¼ 8.5 Hz, 1H), 8.23 (d,
J ¼ 8.0 Hz, 1H), 7.96 (d, J ¼ 7.6 Hz, 1H), 7.59 (dd, J ¼ 10.1, 4.0 Hz, 2H),
.46 (dd, J ¼ 6.1, 3.2 Hz, 1H), 7.36e7.23 (m, 2H), 7.13e7.03 (m, 1H),
.43 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.24 (d, J ¼ 15.8 Hz, 1H), 5.77 (d,
4.2.23. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-2-
isobutyramidobenzamide (F-24)
7
6
White powder, 58.7% yield. HPLC analysis: purity ¼ 99.0815%,
13
1
J ¼ 11.0 Hz, 1H), 3.83 (s, 3H). C NMR (126 MHz, DMSO‑d
167.23, 163.28, 155.62, 155.28, 153.36, 152.25, 152.23, 151.86,
39.18, 138.11, 132.83, 132.35, 132.12, 129.17, 127.68, 127.57, 127.03,
23.47, 123.00, 121.87, 117.34, 117.14, 116.95, 116.89, 116.61, 116.54,
6
):
retention time ¼ 13.267 min H NMR (400 MHz, DMSO‑d
6
): d 12.95
d
1
1
(s, 1H), 11.02 (s, 2H), 8.36 (d, J ¼ 8.3 Hz, 1H), 8.02 (d, J ¼ 7.7 Hz, 1H),
7.83 (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.57 (t, J ¼ 7.7 Hz, 1H), 7.42 (d,
J ¼ 8.5 Hz, 1H), 7.23 (t, J ¼ 7.5 Hz, 1H), 6.87 (d, J ¼ 1.6 Hz, 2H), 6.54 (s,
1
C
14.95, 114.93, 107.74, 55.73. HRMS (ACPI) m/z calcd. for
1H), 3.83 (s, 6H), 2.55 (dd, J ¼ 13.8, 6.9 Hz, 1H), 1.13 (d, J ¼ 6.9 Hz,
þ
13
23
H18FN
5
O
3
[M þ H] : 432.1466. Found: 432.1464.
6H). C NMR (126 MHz, DMSO‑d
6
):
d
174.84, 167.70, 160.86, 142.74,
141.63, 139.54, 139.11, 138.75, 132.28, 129.06, 122.75, 122.05, 121.35,
4
.2.19. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-2-
121.07, 119.77, 116.50, 108.01, 105.36, 99.53, 55.30, 36.01, 19.23.
þ
propionamidobenzamide (F-20)
HRMS (ACPI) m/z calcd. for C26
459.2022.
26
H N
4
O
4
[M þ H] : 459.2027. Found:
White powder, 62.2% yield. HPLC analysis: purity ¼ 98.3956%,
1
retention time ¼ 12.982 min H NMR (400 MHz, DMSO‑d
6
): d 12.93
(
s, 1H), 11.01 (s, 1H), 10.88 (s, 1H), 8.33 (d, J ¼ 8.4 Hz, 1H), 7.99 (d,
J ¼ 8.0 Hz, 1H), 7.83 (d, J ¼ 8.5 Hz, 1H), 7.70 (s, 1H), 7.56 (t, J ¼ 7.5 Hz,
H), 7.41 (dd, J ¼ 8.6, 0.6 Hz, 1H), 7.23 (t, J ¼ 7.7 Hz, 1H), 6.86 (d,
J ¼ 2.1 Hz, 2H), 6.54 (t, J ¼ 1.9 Hz,1H), 3.83 (s, 6H), 2.36 (q, J ¼ 7.5 Hz,
4.2.24. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-2-
pivalamidobenzamide (F-25)
1
White powder, 57.6% yield. HPLC analysis: purity ¼ 98.2992%,
1
retention time ¼ 13.656min. H NMR (400 MHz, DMSO‑d
6
): d 12.98
13
2
1
1
H), 1.08 (t, J ¼ 7.5 Hz, 3H). C NMR (126 MHz, DMSO‑d
67.62, 160.87, 142.75, 141.63, 139.59, 138.94, 138.74, 132.21, 129.04,
22.74, 122.12, 121.54, 121.14, 119.75, 116.48, 107.98, 105.36, 99.50,
6
):
d
171.81,
(s, 1H), 11.38 (s, 1H), 11.08 (s, 1H), 8.52 (d, J ¼ 8.3 Hz, 1H), 8.07 (d,
J ¼ 7.6 Hz, 1H), 7.77 (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.58 (t, J ¼ 7.5 Hz,
1H), 7.42 (d, J ¼ 8.5 Hz,1H), 7.22 (t, J ¼ 7.5 Hz,1H), 6.86 (d, J ¼ 2.1 Hz,
13
5
5.29, 30.30, 9.39. HRMS (ACPI) m/z calcd. for C25
H
24
N
4
O
4
[M þ
2H), 6.54 (t, J ¼ 2.0 Hz, 1H), 3.83 (s, 6H), 1.21 (s, 9H). C NMR
þ
H] : 445.1870. Found: 445.1867.
(126 MHz, DMSO‑d
6
): d 176.46, 168.05, 160.86, 142.70, 141.65,
139.75, 139.36, 138.80, 132.58, 129.09, 122.51, 121.77, 120.47, 120.06,
4
.2.20. 2-(2-chloroacetamido)-N-(6-(3,5-dimethoxyphenyl)-1H-
119.90, 116.56, 108.11, 105.36, 99.56, 55.31, 27.19. HRMS (ACPI) m/z
þ
indazol-3-yl)benzamide (F-21)
28
calcd. for C27H N
4
O
4
[M þ H] : 473.2183. Found: 473.2187.
White powder, 59.7% yield. HPLC analysis: purity ¼ 96.4727%,
1
retention time ¼ 12.933 min H NMR (400 MHz, DMSO‑d
6
):
d
12.97
4.2.25. 2-(2-chloropropanamido)-N-(6-(3,5-dimethoxyphenyl)-
1H-indazol-3yl)benzamide (F-26)
(
s, 1H), 11.54 (s, 1H), 11.13 (s, 1H), 8.40 (d, J ¼ 8.3 Hz, 1H), 8.05 (d,
J ¼ 7.3 Hz, 1H), 7.83 (d, J ¼ 8.5 Hz, 1H), 7.72 (s, 1H), 7.62 (t, J ¼ 7.7 Hz,
White powder, 55.7% yield. HPLC analysis: purity ¼ 98.0673%,
1
1
2
H), 7.42 (d, J ¼ 8.6 Hz,1H), 7.31 (t, J ¼ 7.5 Hz,1H), 6.87 (d, J ¼ 2.0 Hz,
retention time ¼ 13.196 min H NMR (400 MHz, DMSO‑d
6
): d 12.96
13
H), 6.54 (s, 1H), 4.42 (s, 2H), 3.83 (s, 6H). C NMR (126 MHz,
): 167.35, 165.00, 160.86, 142.72, 141.63, 139.45, 138.78,
37.95, 132.38, 129.14, 123.65, 122.10, 121.84, 121.02, 119.80, 116.43,
(s, 1H), 11.51 (s, 1H), 11.11 (s, 1H), 8.34 (d, J ¼ 8.4 Hz, 1H), 8.05 (d,
J ¼ 7.5 Hz, 1H), 7.84 (d, J ¼ 8.5 Hz, 1H), 7.71 (s, 1H), 7.62 (t, J ¼ 7.8 Hz,
1H), 7.41 (d, J ¼ 8.6 Hz,1H), 7.31 (t, J ¼ 7.5 Hz, 1H), 6.87 (d, J ¼ 1.5 Hz,
DMSO‑d
1
6
d
108.02, 105.37, 99.53, 55.31, 43.38. HRMS (ACPI) m/z calcd. for
2H), 6.54 (d, J ¼ 1.5 Hz, 1H), 4.82 (q, J ¼ 6.7 Hz, 1H), 3.83 (s, 7H), 1.65
þ
13
C
24
H
21ClN
4
O
4
[M þ H] : 465.1324. Found: 465.1320.
(d, J ¼ 6.8 Hz, 3H). C NMR (126 MHz, DMSO‑d
6
): d 167.74, 167.35,
1
60.86, 142.72, 141.62, 139.45, 138.78, 137.99, 132.36, 129.16, 123.70,
4
.2.21. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-2-(3-
122.12, 121.16, 119.78, 116.43, 108.02, 105.36, 99.52, 55.66, 55.30,
þ
methylbut-2enamido) benzamide (F-22)
21.65. HRMS (ACPI) m/z calcd. for C25
Found: 479.1481.
H23ClN
4
O
4
[M þ H] : 479.1481.
White powder, 60.7% yield. HPLC analysis: purity ¼ 98.5531%,
1
retention time ¼ 13.613 min H NMR (400 MHz, DMSO‑d
6
): d 12.93
(
s, 1H), 11.01 (s, 1H), 10.90 (s, 1H), 8.40 (d, J ¼ 8.3 Hz, 1H), 8.01 (d,
J ¼ 7.7 Hz, 1H), 7.82 (d, J ¼ 8.5 Hz, 1H), 7.71 (s, 1H), 7.56 (t, J ¼ 7.8 Hz,
H), 7.41 (dd, J ¼ 8.6, 1.1 Hz, 1H), 7.22 (t, J ¼ 7.5 Hz, 1H), 6.86 (d,
J ¼ 2.2 Hz, 2H), 6.54 (t, J ¼ 2.1 Hz, 1H), 5.79 (s, 1H), 3.83 (s, 6H), 2.14
4.2.26. 2-(cyclopropanecarboxamido)-N-(6-(3,5-
dimethoxyphenyl)-1H-indazol-3yl)benzamide (F-27)
1
White powder, 63.5% yield. HPLC analysis: purity ¼ 98.7522%,
1
retention time ¼ 13.130 min H NMR (500 MHz, DMSO‑d
6
): d 12.91
13
(
1
1
9
s, 3H), 1.84 (s, 3H). C NMR (126 MHz, DMSO‑d
60.87, 152.51, 142.74, 141.61, 139.55, 139.23, 138.73, 132.22, 129.04,
22.63, 122.05, 121.20, 120.98, 119.76, 119.29, 116.51, 107.98, 105.37,
9.48, 55.30, 26.76, 19.58. HRMS (ACPI) m/z calcd. for C27
6
):
d
167.77, 164.38,
(s, 1H), 11.10 (s, 1H), 10.99 (s, 1H), 8.26 (d, J ¼ 8.2 Hz, 1H), 7.99 (d,
J ¼ 7.5 Hz, 1H), 7.85 (d, J ¼ 8.5 Hz, 1H), 7.71 (s, 1H), 7.55 (t, J ¼ 7.8 Hz,
1H), 7.42 (dd, J ¼ 8.5, 1.0 Hz, 1H), 7.23 (t, J ¼ 7.5 Hz, 1H), 6.87 (d,
J ¼ 2.1 Hz, 2H), 6.54 (t, J ¼ 2.0 Hz, 1H), 3.83 (s, 6H), 1.79e1.58 (m,
26 4 4
H N O
10

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
1
1
H), 0.94e0.67 (m, 4H). 13C NMR (126 MHz, DMSO‑d
67.58, 160.86, 142.74, 141.60, 139.60, 138.73, 138.71, 132.15, 129.01,
6
):
d
171.58,
1H), 7.79 (d, J ¼ 8.5 Hz, 1H), 7.76 (d, J ¼ 7.7 Hz, 1H), 7.69 (s, 1H), 7.45
(t, J ¼ 7.9 Hz,1H), 7.40 (d, J ¼ 8.6 Hz,1H), 6.86 (d, J ¼ 2.1 Hz, 2H), 6.54
122.82, 122.20, 121.85, 121.83, 119.71, 116.46, 107.95, 105.35, 99.49,
(t, J ¼ 1.9 Hz, 1H), 5.91 (s, 1H), 3.83 (s, 6H), 2.18 (s, 3H), 1.88 (s, 3H).
þ
13
5
4
5.30, 15.51, 7.41. HRMS (ACPI) m/z calcd. for C26
57.1870. Found: 457.1874.
H N
24 4
O
4
[M þ H] :
6
C NMR (126 MHz, DMSO‑d ): d 165.70, 164.74, 160.85, 151.74,
142.78, 141.63, 140.16, 139.71, 138.64, 134.52, 128.64, 122.22, 122.02,
1
19.57, 119.01, 118.94, 116.47, 107.95, 105.33, 99.48, 55.30, 27.03,
þ
4.2.27. 3-Acrylamido-N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-
19.51. HRMS (ACPI) m/z calcd. for C27
Found: 471.2025.
H
26
N
4
O
4
[M þ H] : 471.2027.
yl)benzamide (F-28)
White powder, 57.4% yield. HPLC analysis: purity ¼ 98.9477%,
1
retention time ¼ 12.561 min H NMR (400 MHz, DMSO‑d
6
):
d
12.95
4.2.32. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-3-
methacrylamidobenzamide (F-33)
(
s, 1H),10.85 (s,1H),10.61 (s,1H), 8.34 (s,1H), 7.97 (d, J ¼ 7.9 Hz,1H),
7
.80 (t, J ¼ 8.0 Hz, 2H), 7.70 (s, 1H), 7.49 (t, J ¼ 7.9 Hz, 1H), 7.39 (d,
White powder, 56.5% yield. HPLC analysis: purity ¼ 99.5540%,
1
J ¼ 8.6 Hz, 1H), 6.85 (d, J ¼ 1.8 Hz, 2H), 6.63e6.53 (m, 1H), 6.53 (d,
J ¼ 1.9 Hz, 1H), 6.29 (d, J ¼ 17.0 Hz, 1H), 5.78 (d, J ¼ 10.4 Hz, 1H), 3.83
retention time ¼ 12.940 min H NMR (400 MHz, DMSO‑d
6
): d 12.89
(s, 1H), 10.86 (d, J ¼ 9.1 Hz, 1H), 10.01 (s, 1H), 8.34 (s, 1H), 7.97 (dd,
J ¼ 8.1, 1.3 Hz, 1H), 7.81 (dd, J ¼ 11.9, 8.3 Hz, 2H), 7.70 (s, 1H), 7.49 (t,
J ¼ 7.9 Hz, 1H), 7.41 (dd, J ¼ 8.6, 1.1 Hz, 1H), 6.87 (d, J ¼ 2.2 Hz, 2H),
(
1
1
5
s, 6H). 13C NMR (126 MHz, DMSO‑d
): d 165.62, 163.40, 160.85,
6
42.79, 141.63, 140.08, 139.26, 138.61, 134.63, 131.86, 128.76, 126.94,
22.62, 122.54, 122.17, 119.56, 119.27, 116.44, 107.98, 105.34, 99.49,
6.55 (t, J ¼ 2.1 Hz, 1H), 5.88 (s, 1H), 5.56 (s, 1H), 3.84 (s, 6H), 1.98 (s,
þ
13
5.31. HRMS (ACPI) m/z calcd. for C25
Found: 443.1708.
H
22
N
4
O
4
[M þ H] : 443.1714.
3H). C NMR (126 MHz, DMSO‑d
6
):
d
166.85, 165.61, 160.86, 142.78,
141.64, 140.18, 140.15, 139.22, 138.66, 134.41, 128.55, 123.38, 122.64,
1
22.19, 120.24, 120.22, 119.59, 116.48, 107.95, 105.35, 99.49, 55.30,
þ
4.2.28. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-3-
18.65. HRMS (ACPI) m/z calcd. for C26
Found: 457.1872.
H N
24 4
O
4
[M þ H] : 457.1870.
propionamidobenzamide (F-29)
White powder, 56.8% yield. HPLC analysis: purity ¼ 98.9258%,
1
retention time ¼ 12.586 min H NMR (500 MHz, DMSO‑d
6
):
d
12.86
4.2.33. 3-(2-chloroacetamido)-N-(6-(2-fluoro-3-methoxyphenyl)-
1H-indazol-3yl)benzamide (F1-1)
(
s,1H),10.80 (s,1H),10.07 (s,1H), 8.22 (s,1H), 7.87 (d, J ¼ 7.9 Hz,1H),
7
.78 (t, J ¼ 8.0 Hz, 2H), 7.69 (s, 1H), 7.46 (t, J ¼ 7.9 Hz, 1H), 7.40 (d,
White powder, 60.7% yield. HPLC analysis: purity ¼ 97.0521%,
1
J ¼ 8.6 Hz, 1H), 6.86 (d, J ¼ 1.9 Hz, 2H), 6.54 (s, 1H), 3.83 (s, 6H), 2.36
retention time ¼ 12.351 min H NMR (400 MHz, DMSO‑d
6
): d 12.93
13
(
q, J ¼ 7.5 Hz, 2H), 1.11 (t, J ¼ 7.5 Hz, 3H). C NMR (126 MHz,
(s, 1H), 10.90 (s, 1H), 10.53 (s, 1H), 8.24 (s, 1H), 7.85 (d, J ¼ 7.8 Hz,
2H), 7.81 (d, J ¼ 8.5 Hz, 1H), 7.60 (s, 1H), 7.52 (t, J ¼ 7.9 Hz, 1H),
7.28e7.22 (m, 2H), 7.22e7.17 (m, 1H), 7.14 (dd, J ¼ 9.6, 4.5 Hz, 1H),
6
DMSO‑d ): d 172.18, 165.65, 160.85, 142.77, 141.62, 140.14, 139.52,
1
1
C
38.65, 135.12, 134.53, 128.68, 122.19, 122.07, 119.59, 118.95, 116.48,
13
07.95, 105.34, 99.49, 55.30, 29.51, 9.59. HRMS (ACPI) m/z calcd. for
6
4.30 (s, 2H), 3.89 (s, 3H). C NMR (126 MHz, DMSO‑d ): d 165.48,
þ
25
24
H N
4
O
4
[M þ H] : 445.1870. Found: 445.1873.
164.86, 149.80, 147.85, 147.78, 141.20, 140.15, 138.64, 134.66, 133.13,
29.27, 129.19, 128.93, 124.53, 124.49, 123.03, 122.60, 121.95, 121.91,
1
4
.2.29. 3-(2-chloroacetamido)-N-(6-(3,5-dimethoxyphenyl)-1H-
121.04, 119.32, 116.36, 113.06, 110.26, 56.16, 43.51. HRMS (ACPI) m/z
þ
indazol-3-yl)benzamide (F-30)
calcd. for C23
H18ClFN
4
O
3
[M þ H] : 453.1124. Found: 453.1123.
White powder, 61.7% yield. HPLC analysis: purity ¼ 98.4646%,
1
retention time ¼ 12.533 min H NMR (400 MHz, DMSO‑d
6
):
d
12.88
4.2.34. 3-(2-chloroacetamido)-N-(6-(2-fluoro-5-methoxyphenyl)-
1H-indazol-3yl)benzamide (F1-2)
(
s, 1H), 10.88 (s, 1H), 10.53 (s, 1H), 8.24 (s, 1H), 7.92e7.82 (m, 2H),
7
.79 (d, J ¼ 8.5 Hz, 1H), 7.70 (s, 1H), 7.51 (t, J ¼ 7.9 Hz, 1H), 7.40 (d,
White powder, 57.9% yield. HPLC analysis: purity ¼ 98.8851%,
1
J ¼ 8.6 Hz, 1H), 6.86 (d, J ¼ 2.1 Hz, 2H), 6.54 (d, J ¼ 2.0 Hz, 1H), 4.30
retention time ¼ 12.514 min H NMR (500 MHz, DMSO‑d
6
): d 12.94
13
(
s, 2H), 3.83 (s, 6H). C NMR (126 MHz, DMSO‑d
6
):
d
165.45, 164.87,
(s, 1H), 10.91 (s, 1H), 10.54 (s, 1H), 8.25 (s, 1H), 7.86 (d, J ¼ 6.1 Hz,
160.86, 142.77, 141.64, 140.10, 138.68, 138.63, 134.70, 128.93, 123.04,
2H), 7.82 (d, J ¼ 8.4 Hz, 1H), 7.64 (s, 1H), 7.52 (t, J ¼ 7.7 Hz, 1H), 7.27
122.60, 122.19, 119.61, 119.33, 116.45, 107.96, 105.36, 99.50, 55.30,
(t, J ¼ 9.4 Hz, 2H), 7.12 (d, J ¼ 2.5 Hz,1H), 6.98 (d, J ¼ 8.6 Hz,1H), 4.31
þ
13
4
4
3.50. HRMS (ACPI) m/z calcd. for C24
H21ClN
4
O
4
[M þ H] :
(s, 2H), 3.82 (s, 3H). C NMR (126 MHz, DMSO‑d
6
):
d
165.48, 164.87,
65.1324. Found: 465.1340.
155.67, 154.43, 152.54, 141.20, 140.15, 138.64, 134.65, 133.24, 129.12,
29.00, 128.94, 123.03, 122.60, 121.94, 121.02, 119.32, 116.87, 116.68,
1
4
1
.2.30. 3-(2-chloropropanamido)-N-(6-(3,5-dimethoxyphenyl)-
H-indazol-3yl)benzamide (F-31)
116.37, 115.57, 115.55, 114.51, 114.44, 110.31, 110.29, 55.69, 43.51.
þ
HRMS (ACPI) m/z calcd. for C23
Found: 453.1122.
H18ClFN
4
O
3
[M þ H] : 453.1124.
White powder, 63.4% yield. HPLC analysis: purity ¼ 99.1385%,
1
retention time ¼ 12.698 min H NMR (400 MHz, DMSO‑d
6
): d 12.89
(
7
s, 1H),10.89 (s, 1H),10.56 (s, 1H), 8.27 (s, 1H), 7.86 (t, J ¼ 9.6 Hz, 2H),
Declaration of competing interest
.79 (d, J ¼ 8.5 Hz, 1H), 7.70 (s, 1H), 7.52 (t, J ¼ 7.9 Hz, 1H), 7.40 (d,
J ¼ 8.6 Hz,1H), 6.86 (d, J ¼ 2.1 Hz, 2H), 6.54 (t, J ¼ 2.0 Hz,1H), 4.71 (q,
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.
13
J ¼ 6.6 Hz, 1H), 3.83 (s, 6H), 1.64 (d, J ¼ 6.6 Hz, 3H). C NMR
(
126 MHz, DMSO‑d
6
): d 167.52, 165.45, 160.86, 142.77, 141.64,
140.09, 138.67, 138.64, 134.69, 128.92, 123.07, 122.66, 122.19, 119.61,
1
19.44, 116.45, 107.96, 105.35, 99.49, 55.30, 54.75, 21.00. HRMS
Acknowledgements
þ
(
ACPI) m/z calcd. for C25
H23ClN
4
O
4
[M þ H] : 479.1480. Found:
479.1488.
This study was supported by the National Natural Science
Foundation of China (81473242, 81773579, 21602159, 21877085).
4.2.31. N-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-3-(3-
methylbut-2enamido)benzamide (F-32)
Appendix A. Supplementary data
White powder, 49.6% yield. HPLC analysis: purity ¼ 99.3849%,
1
retention time ¼ 12.690 min H NMR (500 MHz, DMSO‑d
6
):
d
12.87
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113219.
(
s, 1H), 10.81 (s, 1H), 10.06 (s, 1H), 8.28 (s, 1H), 7.88 (d, J ¼ 8.0 Hz,
11

X. Chen, Y. Liu, L. Zhang et al.
European Journal of Medicinal Chemistry 214 (2021) 113219
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