Design, synthesis and biological evaluation of novel 2-(4-(1H-indazol-6-yl)-1H-pyrazol-1-yl)acetamide derivatives as potent VEGFR-2 inhibitors

European Journal of Medicinal Chemistry 213 (2021) 1 13192

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design, synthesis and biological evaluation of novel 2-(4-(1H-indazol-

6-yl)-1H-pyrazol-1-yl)acetamide derivatives as potent VEGFR-2

inhibitors

Xing-Rong Wang, Shuai Wang, Wen-Bo Li, Kai-Yan Xu, Xue-Peng Qiao, Xue-Li Jing,

Zi-Xiao Wang, Chang-jiang Yang, Shi-Wu Chen^*

School of Pharmacy, Lanzhou University, Lanzhou, 730000, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Vascular endothelial growth factor-2 (VEGFR-2) plays a pivotal role in tumor angiogenesis. Herein, a

library of novel 2-(4-(1H-indazol-6-yl)-1H-pyrazol -1-yl)acetamide derivatives were designed and syn-

thesized as VEGFR-2 inhibitors based on scaffold hopping strategy. These compounds exhibited the

excellent inhibitory in both VEGFR-2 and tumor cells proliferation. Especially, compound W13 possessed

potent VEGFR-2 inhibition with IC₅₀¼ 1.6 nM and anti-proliferation against HGC-27 tumor cells with

Received 5 November 2020

Received in revised form

28 December 2020

Accepted 10 January 2021

Available online 18 January 2021

IC₅₀¼ 0.36 0.11

m

M, as well as less toxicity against normal GES-1 cells with IC₅₀¼ 187.46 10.13

mM.

Moreover, W13 obviously inhibited colony formation, migration and invasion of HGC-27 cells by

adjusting the expression of MMP-9 and E-cadherin, and induced HGC-27 cells apoptosis by increasing

ROS production and regulating the expression of apoptotic proteins. Furthermore, W13 blocked the PI3K-

Akt-mTOR signaling pathway in HGC-27 cells. In addition, anti-angiogenesis of W13 was proved by

inhibiting tube formation and the expression of p-VEGFR-2 in HUVEC cells. All the results demonstrated

that W13 could be developing as a promising anticancer agent for gastric cancer therapy.

Keywords:

VEGFR-2

Indazole

Anti-gastric cancer

Apoptosis

Anti-angiogenesis

1. Introduction

classiﬁed into type I and type II according to the trend of Asp-Phe-

Gly (DFG) conservative motif in the kinase [12]. Type I inhibitors

Angiogenesis plays an important role in tumor growth and

metastasis, and has been widely studied in a variety of clinical

pathological diseases including cancer, rheumatoid arthritis, dia-

betic neuropathy and psoriasis [1,2]. Vascular endothelial growth

factor-2 (VEGFR-2), as the most prominent angiogenic factor, be-

longs to type III receptor tyrosine kinase and is highly activated on

vascular endothelial cells after combining with VEGF secreted by

tumor cells [3]. Continuously active VEGFR-2 leads to the con-

struction of tumor blood vessel network, thereby providing nutri-

tion and oxygen to tumor tissue and enhancing tumor proliferation,

invasion and metastasis. More recent studies have conﬁrmed that

VEGFR-2 is over-expressed in liver, gastric, colon, lung and breast

cancer [4 ‒7].

bind to the ATP binding site with DFG-in “active” conformation,

such as Axitinib (I), Sunitinib (II) and Pazopanib (III) [13 ‒15]. In

contrast, type II inhibitors conduct a DFG-out “inactive” confor-

mation at the ATP binding site, including Linifanib (Ⅳ), Apatinib (Ⅴ)

and Sorafenib (Ⅵ) [16 ‒18] (Fig. 1). The latter can not only occupy

the hydrophobic “back pocket” of kinase, it also enhance the afﬁnity

and speciﬁcity of protein-ligand complex, resulting in better

selectivity and more extensive research on type II inhibitors [12].

Sorafenib (Nexavar) is an oral multi-target tyrosine kinase in-

hibitor with anti-angiogenic and anti-proliferative effects, having

been approved for the treatment of metastatic renal cell carcinoma

(mRCC) and hepatocellular carcinoma (HCC) in the ﬁrst-line setting

[19,20]. Multiple clinical trials have shown that sorafenib can

effectively improve the progression-free survival rate and overall

survival rate of cancer patients [21 ‒23]. However, the problems of

sorafenib have appeared in clinical medications, such as acquire

resistance, cardiovascular toxicity and acute elevated blood pres-

sure [24 ‒26]. Besides, sorafenib performs low oral bioavailability

(about 8.43%), poor water solubility, high protein binding rate

(99.5%) and a long half-life of 25e48 h [27]. Hence, developments of

Over the past decades, many effective small molecule VEGFR-2

tyrosine kinase inhibitors have been approved by the USA-FDA

and achieved satisfactory clinical results [8 ‒1 1], which can be

* Corresponding author.

E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).

https://doi.org/10.1016/j.ejmech.2021.113192

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 1. VEGFR-2/multitargeted inhibitors approved for clinical use.

more efﬁciency and less toxicity VEGFR-2 inhibitors basing sor-

afenib make it more meaningful for medicinal chemists.

docking found that they comprise ﬁve parts: the hinge binding,

solvent exposure area, linker, hydrogen binding and tail inserting

into hydrophobic back pocket. Additionally, the X-ray crystallo-

graphic structures of VEGFR-2 (PDB ID: 3EWH [30]) in DFG-out

inactive conformation was selected and downloaded from RCSB

Protein Data Bank. The bond angle formed by the oxygen atom in

the sorafenib extends the arylurea side chain into the hydrophobic

back pocket. This non-coplanar conformation of the pyridine and

benzene ring is the basis for maintaining activity.

Based on the observations described above, we regarded the

pyridine-oxy-phenyl of sorafenib as a whole, combined with the

glide-docking experiments, and found a 6-(1H-pyrazol-4-yl)-1H-

indazol-3-amine scaffold that could be basically consistent with the

three-dimensional conformation of sorafenib (Fig. 3A and B). In

addition, it was found that the hydrogen atom at N¹and the N²

atom of indazole core formed stable hydrogen bonding with Glu

917 and Cys 919, respectively. Besides, the N²atom of pyrazole

formed additional hydrogen bonding with Lys 868. Based on this

scaffold, we ﬁrst designed and synthesized compound W1 by

introducing the pyridine ring to solvent exposure area and insert-

ing a triﬂuoromethyl phenyl to hydrophobic back pocket through

the acetamide group, which formed key hydrogen bonding with

Indazoles are widely used in anticancer, antidiabetic, analgesic,

antidepressant and neurodegenerative disorders due to excellent

pharmacological activity [28]. Recently, Liu et al. reported an

indazole VEGFR-2 inhibitor based on the Axitinib which showed

excellent pharmacokinetic properties and safety [29]. Therefore, we

ﬁrst designed and synthesized 3-animo-1H-indazole W1 via the

scaffold hopping strategy basing three-dimensional conformation,

and the preliminary VEGFR-2 activity indicated that W1 showed a

moderate inhibition with an IC₅₀of 892 nM, consistent with the

docking result and could be carried out as a lead compound for later

structure optimization and structure-activity relationship (SAR)

discussion (Fig. 2). Moreover, the most potent compound W13 was

selected to identify the preliminary action mechanisms. The results

provided a new chemical tool or potent candidate for gastric cancer

therapy.

2. Results and discussions

2.1. Design strategy

the side chain of Glu 885 in the

aC helix and Asp 1046 in the

The molecular framework is not only the key to maintaining

activity, but also the basis for innovative drug design. Scaffold

hopping strategy basing three-dimensional conformation refers to

the use of an overall design strategy to ﬁnd a scaffold with the same

three-dimensional orientation and conformation as the active

compound, rather than a part of the chemical group. Structural

analysis of the clinical type II VEGFR-2 inhibitors by molecular

conserved DFG motif, stabilizing VEGFR-2 in DFG-out inactive

conformation and resembling to sorafenib (Fig. 3C). Furthermore,

replacing urea with acetamide could improve molecular water

solubility.

Fig. 2. The rationally design of target compounds.

2

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 3. Docking models of novel 1H-indazoles VEGFR-2 inhibitors. (A, B) Docking models of 6-(1H-pyrazol-4-yl)-1H-indazol-3-amine scaffold (yellow) and sorafenib scaffold (green),

and overlap of the proposed surface binding mode (PDB code: 3EWH); (C) Docking models of compound W1 (orange) and sorafenib (green) (PDB code: 3EWH); Yellow dashed lines

indicate the hydrogen bonds and important active site residues were labeled and colored with cyan and magenta.

2.2. Chemistry

dihydro-2H-pyran to provide N-protected indazole (7). Following,

through Buchwald-Hartwig reaction of 7 with different aromatic

amines, 3-arylamine substituted indazoles 8a‒e were obtained,

which then removed pyran protecting group by using triﬂuoro-

acetic acid to give 6-bromo-3-arylamine-1H-indazoles 9a‒e. Lastly,

Suzuki coupling reaction of 9a‒e and 3a under the catalysis of

Pd(dppf)Cl₂$CH₂Cl₂afforded W1‒5 [31].

The target compounds W6‒14 were synthesized as summarized

in Scheme 3. Coupling of 4-bromo-2-ﬂuorobenzonitrile (10) and

hydrazine gave 6-bromo-1H- indazole-3-amine (11), which was

then condensed with cyclopropane carbonyl chloride to give

cyclopropanecarboxamide (12) [32]. Intermediate 11 or 12 was

reacted with intermediates 3a‒d via palladium catalyzed Suzuki

Above all, the key intermediates 3a‒d were synthesized as

described in Scheme 1. Specially, commercially available 3-

triﬂuoromethylaniline derivatives 1a‒d were reacted with bro-

moacetyl bromide to obtain 2-bromo-N-(3-(triﬂuoromethyl)

phenyl) acetamide derivatives 2a‒d, which were underwent a

nucleophilic substitution with 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole to give 3a‒d.

The synthesis of target compounds W1‒5 was outlined in

Scheme 2. Firstly, coupling of 4-bromo-2-ﬂuorobenzaldehyde (4)

and hydrazine gave 6-bromo-1H- indazole (5), which followed a

nucleophilic substitution and an addition reaction with 3,4-

Scheme 1. Reagents and conditions: (i) Bromoacetyl bromide, Et₃N, CH₂Cl₂, rt, 3 h; (ii) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, KI, Cs₂CO₃, CH₃CN, reﬂux, 4 h.

3

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Scheme 2. Reagents and conditions: (i) 80% NH₂NH₂$H₂O, p-TsOH, EtOH, 100e130 ^ꢀC, 12 h; (ii) I₂, KOH, DMF, 0 ^ꢀC‒rt, 4 h; (ⅲ) 3,4-dihydro-2H-pyran, p-TsOH, THF, reﬂux, 7 h; (ⅳ)

relative amine, Pd(OAc)₂, xantphos, Cs₂CO₃, dry 1,4-dioxane, 100 ^ꢀC, 3 h; (ⅴ) TFA, CH₂Cl₂, 0 ^ꢀC‒rt, 4 h; (ⅵ) compound 3a, Pd(dppf)Cl₂$CH₂Cl₂, 2 M Na₂CO₃aq, 1,4-dioxane, 100 ^ꢀC, 3 h.

Scheme 3. Reagents and conditions: (i) 80% NH₂NH₂$H₂O, n-BuOH, 120 ^ꢀC, 4 h; (ii) cyclopropane carbonyl chloride, pyridine, 0 ^ꢀC, 4 h; (ⅲ) compounds 3a-3d, Pd(dppf)Cl₂$CH₂Cl₂,

2 M Na₂CO₃aq, 1,4-dioxane, 100 ^ꢀC, 3 h; (ⅳ) compound W8, acetyl chloride (for W13) or isobutyryl chloride (for W14), DIEA, THF, rt, 18 h; then 2 M K₂CO₃aq, MeOH-THF (1:1), rt,

1 h.

coupling reaction to obtain W6‒12. Furthermore, compound W8

was directly converted into W13‒14 by reacting with acetyl chlo-

ride or isobutyryl chloride in suitable 2 M K₂CO₃solution.

The target compounds W15‒20 were obtained according to the

methods summarized in Scheme 4. The N¹atom of 6-bromo-1H-

indazole-3-amine (11) was selectively protected with di-tert-butyl

pyrocarbonate (Boc₂O) to provide compound 13 [33]. On the one

hand, the exposed NH₂at C3 of 13 was coupled with various acid

chloride to give compounds 14a‒d [34]. On the other hand, it was

reacted with the corresponding aldehyde to form an alkyl

substituted intermediates 16a‒b [35] in the presence of NaB-

H(OAc)₃. Following, a Suzuki coupling reaction between 14a‒d or

16a‒b and compound 3c were conducted, and then removed N-Boc

group with triﬂuoroacetic acid to give target compounds W15‒20,

respectively.

method

summarized

in

Scheme

5.

Commercial

4-

bromomethylpyrazole derivatives 18a‒b were converted into

19a‒b by reacting with compound 2c, which underwent a Suzuki

coupling reaction to give 20a‒b, and then condensed with hydra-

zine to obtain 21a‒b. Ultimately, intermediates 21a‒b were directly

converted into W21‒22 by reacting with cyclopropane carbonyl

chloride.

The synthesis of target compounds W23‒25 was outlined in

Scheme 6. Commercially material 2-(5-bromothiophen-2-yl)acetic

acid 22 or bromophenylacetic acid 25a‒b were condensed with 4-

methyl-3-(triﬂuoromethyl) aniline using dicyclohexylcarbodiimide

(DCC) and 1-hydroxybenzotriazole (HOBT) as coupling agents to

afford compounds 23 or 26a‒b, which were converted into borate-

substituted derivatives 24 or 27a‒b [36] via Pd(dppf)Cl₂catalyzed.

Finally, it was reacted with intermediate 12 by Suzuki coupling

reaction to obtain W23‒25.

The target compounds W21‒22 were obtained according to the

4

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Scheme 4. Reagents and conditions: (i) (Boc)₂O, DMAP, THF, 0 ^ꢀC‒rt, 3 h; (ii) Acid chloride, pyridine, CH₂Cl₂, rt or reﬂux, 3 h; (ⅲ) compound 3c, Pd(dppf)Cl₂$CH₂Cl₂, 2 M Na₂CO₃aq,

1,4-dioxane, 100 ^ꢀC, 3 h; (ⅳ) TFA, CH₂Cl₂, 0 ^ꢀC‒rt, 4 h; (ⅴ) acetaldehyde (for 16a) or cyclopropane carboxaldehyde (for 16b), NaBH(OAc)₃, acetic acid, DME, rt, 18 h.

Scheme 5. Reagents and conditions: (i) compound 2c, KI, Cs₂CO₃, acetonitrile, reﬂux, 4 h; (ii) 2-ﬂuoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzonitrile, Pd(dppf)

Cl₂$CH₂Cl₂, 2 M Na₂CO₃aq, 1,4-dioxane, 100 ^ꢀC, 3 h; (ⅲ) 80% NH₂NH₂$H₂O, n-BuOH, 125 ^ꢀC, 3 h; (ⅳ) cyclopropane carbonyl chloride, DIEA, THF, rt, 18 h; then 2 M K₂CO₃aq, MeOH-

THF (1:1), rt, 1 h.

The target compound W26 was prepared as outlined in Scheme

7. 2-Bromo-N-(4-methyl-3-(triﬂuoromethyl)phenyl)acetamide 2c

was treated with sodium azide to give 28, which was coupled with

4-ethynyl-2-ﬂuorobenzonitrile [37] to obtain triazole derivative 29

via Click reaction [38], and reacted with hydrazine to obtain the

indazole core and further converted into W26 as described in

synthesis of W13.

2.3. Biological evaluation

2.3.1. In vitro VEGFR-2 inhibitory assay

All the synthesized target compounds W1‒26 were evaluated

for their inhibitory activities against VEGFR-2 by Caliper Mobility-

Shift Assay using sorafenib as a positive control. As shown in

Table 1, all the target compounds exhibited more than 53.8% inhi-

bition at 1

mM against VEGFR-2. Initially, based on the lead com-

pound W1, we replaced the 3-pyridine group with sulfonyl

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X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Scheme 6. Reagents and conditions: (i) compound 1c, DCC, HOBT, DMF, rt, 8 h; (ii) bis(pinacolato)diboron, Pd(dppf)Cl₂, KOAc, dry 1,4-dioxane, 100 ^ꢀC, 12 h; (ⅲ) compound 12,

Pd(dppf)Cl₂$CH₂Cl₂, 2 M Na₂CO₃aq, 1,4-dioxane, 100 ^ꢀC, 3 h.

Scheme 7. Reagents and conditions: (i) NaN₃, (n-Bu)₄N^þBr^ꢁ, DMSO, rt, overnight; (ii) 4-ethynyl-2-ﬂuorobenzonitrile, K₂CO₃, CuSO₄$5H₂O, VcNa, t-BuOH-H₂O (1:1), rt, 14 h; (ⅲ) 80%

NH₂NH₂$H₂O, n-BuOH, 125 ^ꢀC, 2 h; (ⅳ) cyclopropane carbonyl chloride, DIEA, THF, rt, 18 h; then 2 M K₂CO₃aq, MeOH-THF (1:1), rt, 1 h.

substituted phenyl, pyrazole and 2H-indazole ring to obtain W2‒5

with an increasing IC₅₀values of 143e407 nM. Surprisingly,

removing the substituent at the C3-amino of indazole obtained W6

leading to IC₅₀value increased to 12 nM. Simultaneously, the ac-

tivity of W7‒9 also improved upon the replacement of R²with H, 4-

Me and 2-F, and W10‒12 in which the C3-amino was acylated with

cyclopropylformyl exhibited more potent anti-VEGFR-2 activity.

Moreover, it could also be found that 4-Me substituted compounds

W8 and W11 showed improved inhibitory activities with the IC₅₀

values of 2.7 nM and 1.4 nM, respectively. It illustrated that in the

hinge region of VEGFR-2, the direct connection between C3-amino

group and a larger sterically hindered group affected the afﬁnity of

molecule and protein, causing relatively stronger molecular rigid-

ity. Furthermore, the suitable ClogP (2 < ClogP < 5) and ligand-

lipophilic efﬁciency values (LLE > 4.0) [39] are used to evaluate

the druggability of oral drugs for optimal physicochemical prop-

erties. W1‒5 showed an evident increase in ClogP values due to the

presence of aromatic heterocycles, resulting in low LLE values, and

W6‒12 exhibited relatively good values. Therefore, we selected

W11 with the most potent VEGFR-2 activity for subsequent struc-

tural optimization.

the solvent exposed area of VEGFR-2. Hence, exploring the sub-

stituents of C3-amino of indazole could further improve the ac-

tivity. We replaced the cyclopropylformyl group with an acetyl

(W13), isobutyryl (W14) and cyclopentanoyl (W15), and it was

found that W13 maintained potency (IC₅₀¼ 1.6 nM) with a suitable

ClogP. However, W14 and W15 resulted in a decrease in activity

about 20-fold and 11-fold compared with W11, respectively, while

the ClogP values exceeded 5. Moreover, substitution with the hy-

drophilic 1-methylpiperidine-4-formyl (W16) did not obviously

increase the kinase inhibitory activity. Compared with W1 and W4,

when substituted with nicotianoyl (W17) and 1-methyl-1H-pyr-

azole-4-formyl (W18), the VEGFR-2 inhibitory activity was signiﬁ-

cantly increased (IC₅₀

¼

2.3 vs 892 nM, 0.73 vs 220 nM,

respectively), and W18 was the most potent derivative in this se-

ries. It could be explained that the presence of the carbonyl group

changed the heteroatom ring extending to the solvent exposure

area, thereby reducing the steric hindrance of hinge region binding

and enhancing the kinase afﬁnity, while having better ClogP and

LLE values. When replacing C3-amino with simple alkyl groups,

such as ethyl (W19) and cyclopropylmethyl (W20), the VEGFR-2

inhibitory potency was greatly reduced about 27-fold and 43-fold

compared with W11.

The discovery of W11 was mainly attributed to the maintenance

of the 6-(1H-pyrazol-4-yl)-1H-indazol scaffold and the simpliﬁca-

tion of the substituent groups at 3-position of indazole extending to

To further explore the effects of 6-(1H-pyrazol-4-yl)-1H-indazol

skeleton to VEGFR-2 inhibitory activity, the SAR of 1H-pyrazol-4-yl

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X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Table 1

Table 2

The in vitro VEGFR-2 inhibitory activity of W21‒26.

The in vitro VEGFR-2 inhibitory activity of W1‒20.

Compound

R¹

R²

VEGFR-2 inhibition^a

ClogP^b

LLE^c

Compound

W21

A

VEGFR-2 inhibition^a

ClogP^b

4.78

LLE^c

2.79

1.76

% at 1

53.8

m

M

IC₅₀(nM)

892

% at 1

96.2

m

M

IC₅₀(nM)

27

W1

W2

4-Cl

5.65

4.90

0.40

1.94

77.7

77.0

143

249

W22

79.3

307

4.75

W23

W24

98.3

71.2

12

6.14

6.29

1.78

NT

W3

4-Cl

6.40

0.20

NT^d

W4

W5

4-Cl

80.6

69.6

220

407

5.06

6.18

1.60

0.21

W25

W26

92.4

68.5

NT

6.29

4.37

NT

W6

W7

W8

W9

W10

H

4-Cl

H

4-Me

2-F

98.7

99.2

98.4

99.0

99.5

12

7.4

2.7

22

14

4.15

3.55

4.05

3.18

4.91

3.77

4.58

4.52

4.48

2.94

Sorafenib

100.0

28

5.46

2.09

e

a

The VEGFR-2 inhibition% at

1 mM were the average of two independent

experiments.

b

c

4-Cl

ClogP values were calculated using ChemDraw 14.0.

Ligand-lipophilicity efﬁciency (LLE) [39] ¼ pIC₅₀ꢁ(ClogP).

NT means no test.

d

W11

W12

4-Me

2-F

100.3

98.9

1.4

12

4.81

3.94

4.04

3.98

reduced the LLE values. Secondly, about 8-fold decrease in activity

was observed upon replacement of the pyrazole ring with thio-

phene ring (W23). Thirdly, distinct reduction of VEGFR-2 inhibition

W13

W14

W15

Me

CHMe₂

4-Me

104.5

103.3

93.7

1.6

28

15

4.22

5.06

5.70

4.58

2.49

2.12

rate at 1 mM was found when substituted with meta- and para-

substituted phenyl, and the latter W25 was better than the former

W24. Moreover, we synthesized a triazole compound W26, which

displayed a decrease in VEGFR-2 activity due to its poor solubility.

The above discussion illustrated that the introduction of a lipophilic

thiophene, triazole or benzene ring reduced the receptor afﬁnity

and increased steric hindrance, which validated our original design

strategy that the pyrazole ring of scaffold was an important part of

maintaining activity and played a synergistic role with the indazole.

In summary, amidation of the 3-amino of indazole with acetyl,

cyclopropylformyl, nicotianoyl and 1-methyl-1H-pyrazole-4-

formyl obtained the effective VEGFR-2 inhibitors (e.g. W8, W11,

W13, W17 and W18) with IC₅₀values lower than 3 nM. In order to

further understand the mechanism of the C3-amino substituents in

VEGFR-2, following molecular docking studies were carried out

W16

W17

4-Me

98.6

11

4.28

4.54

3.68

4.10

102.9

2.3

W18

4-Me

100.4

0.73

4.05

4.81

W19

W20

CH₂Me

4-Me

99.6

100.3

38

60

5.37

5.81

2.05

1.41

Sorafenib

100.0

28

5.46

2.09

e

a

The VEGFR-2 inhibition% at

1 mM were the average of two independent

experiments.

b

c

ClogP values were calculated using ChemDraw 14.0.

Ligand-lipophilicity efﬁciency (LLE) [39] ¼ pIC₅₀ꢁ(ClogP).

€

using Schrodinger Suite and the SAR was summarized.

as linker moieties was discussed and substituted with various

functional group, such as 3-methyl or 3,5-dimethyl substituted 1H-

pyrazol-4-yl, thiophenyl, phenyl and triazolyl (Table 2). Firstly, we

introduced 3-methyl (W21) and 3,5-dimethyl (W22) to the pyr-

azole ring, and found that the inhibitory potency of VEGFR-2

decreased about 19-fold and 219-fold with an increasing number

of methyl substitutions compared with W11. It was obvious that the

introduction of sterically hindered substituents in the linker region

affected the molecules entering the hydrophobic back pocket and

2.3.2. Molecular docking studies

In this part, we selected W18 with the most potent VEGFR-2

inhibition activity (IC₅₀¼ 0.73 nM) as template control for dock-

ing study. As illustrated in Fig. 4A and B, the molecular docking

found that W18 inserted into the deep cleft of ATP binding site and

the hydrogen atom at N¹and the N²atom of indazole formed stable

hydrogen bonding with Glu 917 and Cys 919 in the hinge with the

distance of 2.2 Å and 2.0 Å, respectively. Besides, the terminal

amide formed two key hydrogen bonding between the Glu 885 and

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X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 4. Docking study of compounds W4, W18 and W20. (A) Docking models of compounds W4 (orange) and W18 (yellow) (PDB code: 3EWH), and (B) overlap of the proposed

binding mode; (C) Docking models of W18 (yellow) and W20 (magenta), and (D) overlap of the proposed binding modes; Yellow dashed lines indicate the hydrogen bonds and

important active site residues are labeled and colored with cyan and magenta; (E) The SAR summary of synthesized target compounds.

Asp 1046 with the distance of 2.1 Å and 2.4 Å, respectively, medi-

ating the insertion of 4-methyl-3-triﬂuoromethylphenyl into the

hydrophobic back pocket to stabilize the DFG-out inactive confor-

mation. Furthermore, additional hydrogen bonding was formed

between the N²atom of pyrazole and Lys 868 with the distance of

resulted in the pyrazole ring to ﬂip and could not form a hydrogen

bonding with Lys 868.

Taking in vitro VEGFR-2 inhibitory activities and docking results

into account, the preliminary SAR as shown in Fig. 4E were ob-

tained: (a) The 6-(1H-pyrazol-4-yl)-1H-indazol scaffold was

necessary to form key hydrogen bonding interaction with Glu 917

and Cys 919, as well as the acetamide interacting with Glu 885 and

Asp 1046; (b) An electron-rich ﬁve-membered ring containing ni-

trogen atom formed key hydrogen bonding with Lys 868; (c) 4-

methyl-3-triﬂuoromethylphenyl was advantageous to increase

the hydrophobic interaction in VEGFR-2 active site; (d) The amide

structure substitution at the 3-position of indazole extended to the

solvent exposure area and formed certain interaction with sur-

rounding amino acids. These docking results provided considerable

insights into the protein-ligand interactions and potential struc-

tural modiﬁcations for further drug design and activity

improvement.

2.5 Å, and pyrazole ring participated in a

with the Phe1047 residue of VEGFR-2.

p-p stacking interaction

To further explore the effects of different substituents at C3-

amino of indazole, docking models of compounds W4 and W20

were also carried out. As illustrated in Fig. 4A‒4D, all compounds

performed similar binding modes. The presence of 3-position

amide in W18 completely changed the direction of the pyrazole

ring stretching to the solvent exposed area composed by Lys 838,

Pro 839, Leu 840, Gly 841, Phe 918, Lys 920, Gly 922 and Asn 923,

showing better binding conformation than amino-linked W4.

However, the alkyl-substituted W20 showed a poor binding mode

due to its strong hydrophobic interaction with Phe 918, which

8

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

2.3.3. In vitro anti-proliferative evaluation

the compounds performed moderate to high inhibitory activity,

Based on the above discussion, the synthetic compounds were

evaluated for their in vitro anti-proliferative activity using the

standard MTT assay against four human cancer cells, including HT-

29 (colon), A549 (lung), HepG2 (liver) and HGC-27 (gastric) [5].

Additionally, human umbilical vein endothelial cells (HUVEC) were

selected as a critical in vitro model for antiangiogenic ability

research [40], and human gastric mucosal cells (GES-1) as normal

cells to evaluate the toxicity of tested compounds.

compounds W13‒14 and W25‒26 represented the lowest cyto-

toxicity with IC₅₀values more than 100 mM. However, W15 and

W18 exhibited obvious toxicity to normal GES-1 cells.

The results of enzyme potency and anti-proliferative activity in

some compounds were inconsistent, such as W1‒5, W9, W19 and

W20, which could be attributed to the increased ClogP values, poor

physicochemical properties, cell permeability and complicated

mechanism that acting on other cell signaling pathways to indi-

rectly inhibit tumor cells survival [41,42]. Ultimately, W13 exhibited

superior VEGFR-2 inhibitory potency (IC₅₀¼ 1.6 nM), moderate

As shown in Table 3, most of the synthetic compounds exhibited

comparable anti-proliferative effects in four types of tumor cells

compared with the positive control sorafenib (5.13e6.18

mM).

cytotoxicity on HUVEC cells (IC₅₀¼ 7.15 2.26

times selectivity of tumor HGC-27 cells than normal GES-1 cells

(IC₅₀: 0.36 0.11 M vs 187.46 10.13 M), and was selected for

further investigation.

mM), more than 500

Moreover, the cytotoxic activity of the test compounds against

gastric cancer were obviously better than those of lung cancer and

liver cancer. Compounds W5, W11, W13‒16, W18 and W21 man-

ifested excellent cytotoxic activity in HGC-27 cells with IC₅₀values

m

less than 1

compounds W11 and W18 exhibited broad spectrum anti-cancer

activity with all IC₅₀values below 5 M, and W15 showed the

m

M, especially W18 for 21

0.01 nM. Furthermore,

2.3.4. Compound W13 inhibited the colony formation of HGC-

27 cells

Colony formation assay is commonly used to assess the prolif-

eration potential and ability of adherent cells to form different

colonies from a single cell [43]. In this assay, HGC-27 cells were

grown in media containing different concentrations (0.1, 0.5 and

m

most potent activity in HT-29 with IC₅₀values of 190 0.02 nM.

The SAR of anti-proliferation could be summarized as follows.

Compounds bearing cyclopropylformyl (W10‒12) displayed

prominent growth inhibition properties on four cancer cell lines

whereas C3-amino (W6‒9) lead to a decrease of cytotoxic activity

that maybe the reduced cell membrane permeability in the pres-

ence of amino group, and the inhibitory activity of 2-ﬂuoro was

weaker than 4-chloro and 4-methyl substituted. Moreover, C3-

amino of indazole substituted with amide (W13‒18) showed rela-

tively stronger anti-cancer activity and better HUVEC cells inhibi-

tory activity than sorafenib except W16. Compounds substituted

with alkylamine (W19‒20) exhibited weak inhibitory effect due to

high lipophilicity. Besides, cytotoxicity could be maintained when

the pyrazole ring was substituted with monomethyl (W21),

dimethyl (W22) and thiophene (W23), while phenyl (W24‒25) and

triazole (W26) substitutions lost activity. Furthermore, in vitro GES-

1 anti-proliferation evaluation showed that although majority of

1

mM) of W13 over a period of 12 days to form colonies compared

with sorafenib (5 M). The cells were then stained with crystal

m

violet to visualise colonies. As shown in Fig. 5, the colony number of

W13 treated groups signiﬁcantly decreased in a concentration-

dependent manner, and exhibited better antiproliferation even at

less concentrations than sorafenib. It was worth noting that 1 mM of

W13 completely eliminated the colony formation, indicating strong

anti-proliferation effects on HGC-27 cells.

2.3.5. Compound W13 inhibited the migration and invasion of

HGC-27 cells

The process of tumor cells migration, invasion and metastasis

play an important role in tumor growth, which refers to individual

cells separated from the primary tumor and ﬁnally entering the

Table 3

The in vitro anti-proliferative evaluation of target compounds W1-26 against selected cell lines.

Compound

IC₅₀

(m

M)^a

HT-29

A549

HepG2

HGC-27

HUVEC

GES-1

W1

W2

W3

W4

W5

W6

W7

W8

5.21 0.82

3.69 0.08

6.11 1.12

9.17 1.08

3.27 0.59

3.57 1.04

3.97 0.75

1.54 0.15

7.83 2.54

1.37 0.73

1.01 0.03

5.06 0.77

2.97 0.90

0.48 0.41

0.19 0.02

1.79 0.66

7.78 1.22

4.92 1.01

8.32 2.86

10.96 1.93

7.03 0.62

14.7 0.4

8.09 1.29

>50

7.96 1.15

7.17 0.70

4.72 1.27

12.05 0.30

1.37 0.36

12.47 0.74

19.47 4.67

7.09 0.30

20.47 2.81

8.55 0.23

2.16 0.19

8.82 1.52

4.31 1.67

2.48 1.41

1.58 0.70

5.28 0.96

5.34 0.93

3.86 0.97

8.48 2.19

9.88 1.28

1.65 0.50

1.81 1.03

0.96 0.59

20.85 2.16

45.14 2.27

>50

5.63 1.00

6.54 1.37

4.32 1.49

10.44 0.76

3.83 0.29

7.71 0.32

11.53 0.36

4.18 0.24

13.34 1.03

6.03 0.003

2.51 0.13

5.61 0.56

15.02 2.88

13.66 2.40

8.34 1.31

15.27 1.22

9.08 0.63

2.61 0.26

15.11 2.15

22.29 1.26

20.35 1.97

27.39 3.37

38.12 1.24

25.51 2.78

>50

3.65 1.20

1.26 0.56

5.03 1.36

12.54 1.78

0.39 0.10

7.68 2.32

10.2 0.31

4.98 1.75

15.45 1.19

1.05 0.57

0.63 0.24

3.73 0.73

0.36 0.11

0.39 0.11

0.50 0.13

0.16 0.01

1.17 0.58

0.021 0.01

1.19 0.24

1.22 0.12

0.65 0.16

2.79 0.91

3.09 1.10

15.56 3.34

33.71 5.53

>50

8.04 0.88

7.22 1.84

6.40 0.59

4.03 0.41

1.45 0.59

6.84 1.28

7.25 1.20

5.24 1.52

23.85 0.89

1.070 0.72

0.95 0.88

5.26 1.21

7.15 2.26

2.43 0.10

1.49 0.21

53.88 1.30

1.75 0.24

4.41 2.71

>50

17.94 0.60

4.92 2.98

6.62 0.70

5.62 1.23

>50

>100

<1

8.33 3.55

12.83 1.07

17.68 2.94

10.02 0.50

13.30 2.08

3.37 0.22

11.97 0.79

22.26 2.02

6.96 0.73

7.64 0.55

29.83 3.03

4.24 1.61

25.03 2.73

187.46 10.13

>100

0.75 0.45

5.31 1.28

8.46 0.75

1.67 0.84

1.90 0.74

11.99 1.70

3.09 1.40

11.86 1.83

13.86 3.11

48.50 2.92

>100

W9

W10

W11

W12

W13

W14

W15

W16

W17

W18

W19

W20

W21

W22

W23

W24

W25

W26

Sorafenib

44.74 4.24

>50

5.57 0.53

>50

6.18 0.36

>100

9.59 2.48

5.13 1.06

5.22 0.96

a

Cells were treated with drugs for 72 h following MTT-based viability assay, and IC₅₀values presented as mean standard error (S.E.) of three independent experiments.

9

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 5. Compound W13 inhibited the colony formation of HGC-27 cells. HGC-27 cells were treated with indicated concentrations (0, 0.1, 0.5 and 1 mM) of W13 and 5 mM of sorafenib

for 12 days, and the colonies were stained by crystal violet.

lymphatic vessels, blood or other tissues [44]. At ﬁrst, a wound

healing assay was performed on HGC-27 cells to investigate the

inhibitory effect of W13 on migration ability, and the results were

shown in Fig. 6A. The control cells nearly migrated to ﬁll the initial

clear area after 24 h, while W13 treatment obviously reduced HGC-

27 cells migration in both dose- and time-dependent manner.

oxygen species (ROS) [48]. Therefore, monitoring the level of ROS in

tumor cells indirectly reﬂect the progress and mechanism of

apoptosis. In this assay, HGC-27 cells were incubated with indicated

concentrations of W13 for 24

h and stained with 2,7-

dichlorodihydroﬂuorescein diacetate (DCFH-DA), in which the

ﬂuorescence intensity gradually increased after the drug adminis-

tration using ﬂow cytometry. As shown in Fig. 8B, W13 could

Quantiﬁcation of relative closure of scratches displayed that 0.5 mM

of W13 suppressed HGC-27 migration by 9.5% and 28.0% at 6 and

24 h compared with the control (Fig. 6D).

effectively increase the expression of intracellular ROS in

concentration-dependent manner compared with the control, and

a

Moreover, we further evaluated the invasion and migration

ability of W13 on HGC-27 cells by Transwell assay. As shown in

Fig. 6B, it was clearly observed that W13 reduced the penetration of

HGC-27 cells through the chamber membrane under the increasing

concentrations for 24 h. The number of migration cells decreased to

212, 87 and 22 from 320, respectively, compared with control group

(Fig. 6E). Moreover, W13 also induced dose-dependent suppression

of the invasion of HGC-27 cells at the same concentration admin-

istration for 24 h (Fig. 6C). Quantitative statistical results indicated

that W13 signiﬁcantly suppressed HGC-27 cells from invasion and

reasoned its potent effect in tumor proliferation inhibition (Fig. 6F).

the ﬂuorescence intensity increased to 945 from 712 at 0.5 mM

concentration.

Furthermore, Western blot analysis explained the mechanism

that W13 treatment dose-dependently up-regulated the expression

of pro-apoptotic protein BAX and down-regulated the expression of

anti-apoptotic protein Bcl-xL (Fig. 8C and D).

2.3.8. Compound W13 blocked the PI3K/AKT/mTOR pathway of

HGC-27 cells

Angiogenesis is the basis for the invasion and metastasis of

gastric cancer cells, and the high expression of VEGF leads to the

increase of micro vessel density (MVD) and abnormal proliferation

of gastric cancer cells [49]. PI3K-Akt-mTOR, as one of the main

downstream signaling pathways of VEGFR-2, regulates angiogen-

esis and vascular permeability by strengthening the survival and

proliferation of endothelial cells, as well as over-activated in gastric

cancer cells [50,51]. As illustrated in Fig. 9A and B, W13 suppressed

VEGFR-2 phosphorylation with a concentration dependent manner

in HGC-27 cells. Effective downregulations of p-PI3K (Tyr607), p-

AKT (Thr308), p-mTOR (Ser2448) and p-VEGFR-2 (Tyr1175) were

2.3.6. Compound W13 regulated MMP-9 and E-cadherin in HGC-

27 cells

MMP-9 promotes tumor cells invasion and metastasis by

degrading extracellular matrix and basement membrane, regu-

lating cell adhesion and promoting tumor cell immune escape [45].

As an effective tumor suppressor, downregulation of E-cadherin is

often found in malignant epithelial cancers, which leads to meta-

static dissemination and activation of EMT transcription factors

[46]. As shown in Fig. 7A and B, it was clearly observed that W13

observed with 0.1, 0.5 and 1 mM of W13 treatment. These results

dose-dependently decreased the MMP-9 expression and 1

m

M of

demonstrated that W13 inhibited HGC-27 cells proliferation and

apoptosis by downregulating the PI3K-Akt-mTOR pathway.

Taken together, our data demonstrated that compound W13

displayed distinct antiangiogenic activity against tumor apoptosis

and migration by blocking the PI3K-Akt-mTOR pathway, down-

regulating Bcl-xL and MMP-9, and upregulating BAX and E-cad-

herin expression.

W13 signiﬁcantly increased the expression level of E-cadherin

protein, which could enhance cell adhesion ability and weaken cell

invasiveness.

2.3.7. Compound W13 induced the apoptosis of HGC-27 cells

Apoptosis involves activating, expressing and regulating a series

of internal pathway proteins, and leading to programmed cell death

to maintain tissue homeostasis [47]. To investigate the action

mechanism of W13 on HGC-27 cells, Annexin V-FITC/Propidium

iodide (PI) dual staining assay based ﬂow cytometry was per-

formed. As shown in Fig. 8A, after treatment of HGC-27 with W13

for 24 h, it was an evident enhancement of Annexin V-FITC/PI-

positive apoptotic cells, in which the percentage of total apoptotic

2.3.9. Compounds W13 inhibited the tube formation of HUVECs

In the early stage of neovascularization, endothelial cells are

recruited by neoplastic cells and polarized to form a lumen-like

structure [52]. To further investigate the antiangiogenic effects of

W13, matrigel tube formation assay of HUVECs was performed. As

shown in Fig. 10A, it was clearly that integrated tubes could be

found on Matrigel matrix in the control group. After adding 1, 2.5, 5

cells increased to 28.8% at 1

mM, and higher than sorafenib with

25.4% at 5 M. These above results indicated that W13 could

m

and 10 mM of W13 for 12 h, the HUVECs tube length, tube surface

concentration-dependently induce both early and late apoptosis in

HGC-27 cells.

When tumor cells undergo apoptosis, it often disrupts the redox

equilibration and causes a decrease in mitochondrial membrane

potential, leading to the generation and accumulation of reactive

area, and the number of branch points were signiﬁcantly sup-

pressed. The results of the matrigel assay conﬁrmed that W13

inhibited the tube formation in a dose-dependent manner and

similar effect was observed with the standard drug sorafenib. In

addition, W13 at 1, 2.5 and 5 mM alone had no obvious cytotoxicity

10

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 6. Compound W13 inhibited the colony formation, migration and invasion of HGC-27 cells. (A, D) HGC-27 cells were treated with indicated concentrations (0, 0.1, 0.5 and 1

mM)

of W13 and 1 M of sorafenib for 0, 6 and 24 h, and quantitative analysis of the number of relative closure of scratches in wound healing assay; (B, E) HGC-27 cells were treated with

m

indicated concentrations of W13 for 24 h and stained with 0.1% crystal violet solution, and quantitative analysis of the number of migrated cells in transwell migration assay; (C, F)

The effect of W13 in transwell invasion assay. All results performed above are presented as mean SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

compared to the control group.

on HUVECs for 72 h in MTT assay. The results revealed that the

inhibition of VEGF-induced tube formation in HUVEC cells was not

due to cytotoxicity. The statistical analysis results demonstrated

that compared with the control group, the tube formation ability

2.3.10. Compound W13 suppressed the p-VEGFR-2 expression in

HUVEC cells

It is well known that the excessive secretion of VEGF in tumor

tissues usually leads to VEGF/VEGFR-2 signaling pathway over-

activation during angiogenesis and rapid proliferation of endothe-

lial cells towards tumor cells forming new capillaries. Therefore,

was obviously inhibited by W13 at low concentrations of 2.5

and 5 M (Fig. 10B).

mM

m

11

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 7. Compound W13 inﬂuenced the expression of MMP-9 and E-cadherin in HGC-27 cells. (A) HGC-27 cells were treated with indicated concentrations (0, 0.1, 0.5 and 1

mM) of

W13 and 1 M of sorafenib for 24 h, and cell lysates were subjected to immunoblotting; (B) Quantitative evaluation of enzymatic activities after W13 treatment. All results

m

performed above are presented as mean SD from three independent experiments.

Fig. 8. The effect of compound W13 on apoptosis in HGC-27 cells. (A) The quantitative analysis of HGC-27 cells treated with indicated concentrations (0, 0.1, 0.5 and 1 mM) of W13

and 5

mM of sorafenib for 24 h, using Annexin V-FITC/PI double staining through ﬂow cytometry. (B) HGC-27 cells were treated with the same concentrations of W13 and sorafenib

for 24 h, stained with 10

mM of DCFH-DA, and analyzed by ﬂow cytometry; (C) Effects of W13 and sorafenib on BAX and Bcl-xL, and cell lysates were subjected to immunoblotting;

(D) Quantitative evaluation of enzymatic activities after W13 treatment. All data were expressed as the mean SEM.

12

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

Fig. 9. Compound W13 inhibited the VEGFR-2 and PI3K-Akt-mTOR signaling pathways in HGC-27 cells. (A) Effect of W13 on both signaling pathways protein; HGC-27 cells were

treated with indicated concentrations (0, 0.1, 0.5 and 1 M) of W13 and 5 M of sorafenib for 24 h, and cell lysates were subjected to immunoblotting; (B) Quantitative evaluation of

m

enzymatic activities after W13 treatment. All data were expressed as the mean SEM.

Fig. 10. Compound W13 inhibited tube formation and p-VEGFR-2 expression in HUVEC cells. (A) The representative image of HUVEC cells treated with W13 (0, 1, 2.5, 5 and 10 mM)

and sorafenib (10 mM) under 20 ng/mL VEGF induction for 12 h; (B) Quantitative analysis of W13 on tube formation in the bar graph; (C) The immunoblotting of HUVEC cells treated

with indicated concentrations of W13 and sorafenib for 24 h; (D) Quantitative evaluation of enzymatic expression after W13 treatment. All data were expressed as the mean SEM.

*P < 0.05, **P < 0.01, ***P < 0.001 compared to the control group.

assessing the expression of p-VEGFR-2 in VEGFR-2 overexpressed

HUVEC cells using Western blot analysis can intuitively reﬂect the

drug’s inhibitory effect on angiogenesis. In this assay, HUVEC cells

were seeded in 60 mm Prtri dishes (1.0 ꢂ 10⁶cells/dish) and

and 5 mM of sorafenib for 24 h. As illustrated in Fig. 10C and D, W13

dose-dependently blocked the phosphorylation of VEGFR-2

compared with control group, indicating that W13 inhibited

angiogenesis by directly targeting VEGFR-2 activation in HUVEC

cells.

incubated with increasing concentrations (0,1, 5 and 10 mM) of W13

13

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

3. Conclusion

(3a). Yellow solid powder; yield 61%; ¹H NMR (600 MHz, CDCl₃)

d

8.22 (s, 1 H), 7.86 (s, 1 H), 7.77 (d, J ¼ 8.4 Hz,1 H), 7.51 (d, J ¼ 8.4 Hz,

Gastric cancer is one of the malignant tumors with high inci-

dence and mortality, and it is difﬁcult to diagnose at the early stage.

In this work, a novel series of 2-(4-(1H-indazol-6-yl)-1H-pyrazol-1-

yl)acetamide derivatives were designed and synthesized as VEGFR-

2 inhibitors based on scaffold equivalent replacement strategy, and

assessed its potential in enzymatic assay, anti-proliferation and

anti-angiogenesis effect. The representative compound W13

revealed a moderate cytotoxicity on HUVEC cells and more than

500 times selectivity of tumor HGC-27 cells than normal GES-

1 cells indicating lowest toxicity. Moreover, the SAR and molecular

docking studies pointed that 6-(1H-pyrazol-4-yl)-1H-indazol was

essential scaffold for maintaining excellent activity.

1 H), 4.05 (s, 2 H).

4.1.2.2. 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyr-

azol-1-yl)-N-(3- (triﬂuoromethyl)phenyl)acetamide (3b). Yellow

solid powder; yield 73%; ¹H NMR (600 MHz, CDCl₃)

d 8.84 (s, 1 H),

7.99 (s,1 H), 7.90 (s, 1 H), 7.83 (s,1 H), 7.66 (d, J ¼ 7.8 Hz,1 H), 7.42 (d,

J ¼ 7.8 Hz, 1 H), 7.37 (d, J ¼ 7.8 Hz, 1 H), 4.96 (s, 2 H), 1.33 (s, 12 H).

4.1.2.3. N-(4-methyl-3-(triﬂuoromethyl)phenyl)-2-(4-(4,4,5,5-

tetramethyl-1,3,2-diox

(3c). Pale yellow solid powder; yield 94%; ¹H NMR (600 MHz,

CDCl₃) 8.63 (s, 1 H), 7.98 (s, 1 H), 7.82 (s, 1 H), 7.63 (s, 1 H), 7.58 (d,

aborolan-2-yl)-1H-pyrazol-1-yl)acetamide

d

It was found that W13 obviously inhibited colony formation,

transwell migration and invasion of HGC-27 cells in a dose-

dependent manner. Further mechanism investigation revealed

that W13 could induce apoptosis by increasing ROS production and

inﬂuencing apoptotic protein BAX and Bcl-xL expression in HGC-

27 cells. In addition, W13 not only blocked the PI3K-Akt-mTOR

signaling pathway of HGC-27 cells, it also signiﬁcantly reduced

the MMP-9 expression and increased the expression of E-cadherin

to prevent HGC-27 cells invasion and metastasis. Moreover, anti-

angiogenesis of W13 was evident by inhibiting tube formation

and the expression of p-VEGFR-2 in HUVEC cells. All the results

demonstrated that W13 could be as a promising lead candidate for

further research and structure optimization to facilitate the dis-

covery of effective VEGFR-2 inhibitors for gastric cancer therapy.

J ¼ 7.8 Hz, 1 H), 7.22 (d, J ¼ 8.4 Hz, 1 H), 4.95 (s, 2 H), 2.42 (s, 3 H),

1.33 (s, 12 H).

4.1.2.4. N-(2-ﬂuoro-5-(triﬂuoromethyl)phenyl)-2-(4-(4,4,5,5-

tetramethyl-1,3,2-diox

aborolan-2-yl)-1H-pyrazol-1-yl)acetamide

(3d). Reddish brown liquid; yield 34%; ¹H NMR (600 MHz, CDCl₃)

d

9.11 (s, 1 H), 8.65 (d, J ¼ 4.8 Hz, 1 H), 7.99 (s, 1 H), 7.82 (s, 1 H), 7.34

(s, 1 H), 7.18e7.15 (m, 1 H), 4.99 (s, 2 H), 1.33 (s, 12 H).

4.1.3. General procedure for the synthesis of target compounds W1‒

5

To a mixture of compound 9a‒e (0.7 mmol, 1.0 eq), compound

3a (1.3 eq) and Pd(dppf)Cl₂$CH₂Cl₂(0.1 eq) in 1,4-dioxane (4.0 mL)

was added 2 M Na₂CO₃aq (0.4 mL) and purged with argon. The

resulting mixture was reﬂuxed for 3 h. The reaction mixture was

ﬁltered and ﬁltrate was evaporated in vacuo, the crude product was

further puriﬁed by silica gel ﬂash chromatography (DCM:

MeOH ¼ 100:1e20:1) to obtain target compounds W1‒5.

4. Experimental

4.1. Chemistry

4.1.1. General

4.1.3.1. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(3-(pyridin-3-

All reagents and solvents were used as purchased from com-

mercial sources and used without further puriﬁed. Reactions were

monitored by thin-layer chromatography (TLC) analysis (0.25 mm

silica gel GF₂₅₄, Yantai Xinnuo Chemical Co., Ltd., china). Column

chromatography (CC) was performed on Silica Gel 60 (200e300

mesh, Yantai Xinnuo Chemical Co., Ltd., China). Melting points were

determined in Koﬂer apparatus and were uncorrected. ¹H NMR and

¹³C NMR spectra were recorded with Agilent NMR inova 600 or

Bruker AM 400 spectrometers with TMS as an internal standard, all

ylamino)-1H-ind

Pale yellow solid powder; yield 37%; m.p.: 205e206 ^ꢀC; ¹H NMR

(600 MHz, DMSO‑d₆) 12.10 (s, 1 H), 10.85 (s, 1 H), 9.13 (s, 1 H), 8.85

azol-6-yl)-1H-pyrazol-1-yl)acetamide

(W1).

d

(s, 1 H), 8.32 (s, 1 H), 8.21 (s, 1 H), 8.18 (d, J ¼ 7.8 Hz, 1 H), 8.04 (s,

2 H), 7.96 (d, J ¼ 8.4 Hz, 1 H), 7.85 (d, J ¼ 8.4 Hz, 1 H), 7.72 (d,

J ¼ 9.0 Hz, 1 H), 7.55 (s, 1 H), 7.35 (d, J ¼ 7.8 Hz, 1 H), 7.29 (s, 1 H), 5.11

(s, 2 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 166.2, 144.4, 141.3, 139.6,

138.0, 137.1, 132.3, 131.2, 129.3, 124.3, 124.0, 123.5, 122.4, 121.7,

120.3, 117.9, 117.2, 112.9, 104.9, 54.7. MS (ESI) m/z 512.2 for [MþH]^þ.

HRMS (ESI) m/z calculated for C₂₄H₁₈ClF₃N₇O [MþH]^þ512.1213,

found 512.1200. HPLC purity 96.4% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/

min, t_R¼ 5.69 min).

chemical shift values are reported as

d ppm. Mass spectra were

recorded on a Bruker Dalton APEXII49e and Esquire 6000 (ESI-ION

TRAP) spectrometer with ESI source as ionization, respectively. The

purities of all target compounds were estimated by Waters 1525

Binary HPLC Pump, and in each case, the major peak accounted for

ꢃ95% of the combined total peak area when monitored by a UV

detector. (The intermediate data of yield and ¹H NMR were pro-

vided in Supplementary Material, and only the ¹H NMR of the

boronic ester side chain intermediates were listed here).

4.1.3.2. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(3-((3-((meth-

ylsulfonyl)methyl) phenyl)amino)-1H-indazol-6-yl)-1H-pyrazol-1-yl)

acetamide (W2). Pale yellow solid powder; yield 47%; m.p.:

226e228 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

d 12.00 (s, 1 H), 10.83 (s,

1 H), 8.98 (s, 1 H), 8.30 (s, 1 H), 8.21 (s, 1 H), 8.02 (s, 1 H), 7.96 (d,

J ¼ 8.4 Hz, 1 H), 7.85 (d, J ¼ 8.4 Hz, 1 H), 7.75 (d, J ¼ 7.2 Hz, 1H), 7.72

(d, J ¼ 8.4 Hz, 1 H), 7.67 (s, 1 H), 7.53 (s, 1 H), 7.32e7.27 (m, 2 H), 6.86

(d, J ¼ 7.2 Hz, 1 H), 5.10 (s, 2 H), 4.43 (s, 2 H), 2.95 (s, 3 H); ¹³C NMR

4.1.2. General procedure for the synthesis of compounds 3a‒d

A mixture of compound 2a‒d (9.0 mmol, 1.1 eq), 4-(4,4,5,5-

tetramethyl-1,3,2- dioxaborolan-2-yl)-1H-pyrazole (8.2 mmol, 1.0

eq), KI (1.6 mmol, 0.2 eq) and Cs₂CO₃(16.4 mmol, 2.0 eq) in

acetonitrile was heated to reﬂux for 4 h under argon. The reaction

was monitored by TLC and ﬁltered through a pad of Celite, and the

ﬁltrate was concentrated in vacuo. The residue was puriﬁed by

column chromatography (DCM: acetone ¼ 100:1e5:1) to afford

compounds 3a‒d.

(150 MHz, DMSO‑d₆)

d 166.1, 144.7, 143.5, 143.3, 141.3, 138.0, 137.1,

132.2, 131.0, 129.2, 129.1, 128.8, 126.7, 126.5, 124.3, 124.0, 123.6,

122.5,121.3, 120.5,118.0, 117.0, 115.7, 113.1, 104.8, 60.0, 54.7. MS (ESI)

m/z 603.1 for [MþH]^þ. HRMS (ESI) m/z calculated for

C

₂₇H₂₃ClF₃N₆O₃S [MþH]^þ603.1193, found 603.1174. HPLC purity

96.5% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 5.12 min).

4.1.3.3. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(3-((2-methyl-

4.1.2.1. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(4,4,5,5-

2H-indazol-6-yl)

amino)-1H-indazol-6-yl)-1H-pyrazol-1-yl)acet-

tetramethyl-1,3,2-diox

aborolan-2-yl)-1H-pyrazol-1-yl)acetamide

amide (W3). Pale yellow solid powder; yield 48%; m.p.:

14

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

162e164 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

d

12.02 (s, 1 H), 10.85 (s,

yield 30%; m.p.: 243e245 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

d 11.30

1 H), 8.85 (s, 1 H), 8.31 (s, 1 H), 8.22 (d, J ¼ 1.8 Hz, 1 H), 8.17 (s, 1 H),

8.13 (s, 1 H), 8.03 (s, 1 H), 8.00 (d, J ¼ 9.0 Hz, 1 H), 7.86e7.84 (m, 1 H),

7.72 (d, J ¼ 8.4 Hz, 1 H), 7.55e7.54 (m, 2 H), 7.32 (d, J ¼ 9.0 Hz, 1 H),

7.14 (d, J ¼ 7.8 Hz,1 H), 5.11 (s, 2 H), 4.08 (s, 3 H); ¹³C NMR (150 MHz,

(s, 1 H), 10.68 (s, 1 H), 8.23 (s, 1 H), 8.10 (s, 1 H), 7.95 (s, 1 H), 7.79 (d,

J ¼ 7.8 Hz, 1 H), 7.67 (d, J ¼ 8.4 Hz, 1 H), 7.59 (d, J ¼ 8.4 Hz, 1 H), 7.45

(d, J ¼ 7.8 Hz, 1 H), 7.38 (s, 1 H), 7.17 (d, J ¼ 8.4 Hz, 1 H), 5.27 (s, 2 H),

5.08 (s, 2 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 166.0, 149.1, 142.2,

DMSO‑d₆)

d

166.1, 149.6, 145.1, 141.2, 140.1, 138.0, 137.1, 132.3, 131.0,

139.3, 136.9, 130.4, 130.1, 129.6, 129.4, 128.9, 124.9, 122.7, 120.6,

120.0, 116.0, 115.3, 112.7, 104.7, 54.7. MS (ESI) m/z 401.2 for [MþH]^þ.

HRMS (ESI) m/z calculated for C₁₉H₁₆F₃N₆O [MþH]^þ401.1338,

found 401.1323. HPLC purity 98.3% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/

min, t_R¼ 4.39 min).

129.2, 126.9, 124.3, 124.0, 123.5, 122.5, 121.9, 120.5, 120.0, 117.8,

116.8, 116.5, 116.0, 113.2, 104.8, 98.1, 54.7. MS (ESI) m/z 565.2 for

[MþH]^þ. HRMS (ESI) m/z calculated for C₂₇H₂₁ClF₃N₈O [MþH]^þ

565.1479, found 565.1462. HPLC purity 96.9% (MeOH: H₂O ¼ 80 :

20, 0.8 mL/min, t_R¼ 5.35 min).

4.1.4.3. 2-(4-(3-amino-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-(4-

methyl-3-(triﬂuoro methyl)phenyl)acetamide (W8). Pale yellow

solid powder; yield 30%; m.p.: 232e233 ^ꢀC; ¹H NMR (600 MHz,

4.1.3.4. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(3-((1-methyl-

1H-pyrazol-4-yl)

amide (W4). Green solid powder; yield 31%; m.p.: 155e157 ^ꢀC; ¹H

NMR (600 MHz, DMSO‑d₆) 11.65 (s,1 H),10.83 (s,1 H), 8.25 (s,1 H),

amino)-1H-indazol-6-yl)-1H-pyrazol-1-yl)acet-

DMSO‑d₆)

d 11.33 (s, 1 H), 10.60 (s, 1 H), 8.23 (s, 1 H), 8.03 (s, 1 H),

d

7.95 (s, 1 H), 7.71 (d, J ¼ 7.8 Hz, 1 H), 7.67 (d, J ¼ 8.4 Hz, 1 H), 7.42 (d,

8.19 (d, J ¼ 3.0 Hz, 1 H), 7.97 (s, 1 H), 7.85 (s, 1 H), 7.83e7.79 (m, 3 H),

J ¼ 7.8 Hz,1 H), 7.38 (s,1 H), 7.17 (d, J ¼ 9.0 Hz,1 H), 5.30 (s, 2 H), 5.06

7.69 (d, J ¼ 9.0 Hz, 1 H), 7.43e7.41 (m, 2 H), 7.23 (d, J ¼ 7.8 Hz, 1 H),

(s, 2 H), 2.39 (s, 3 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 165.7, 149.1,

5.07 (s, 2 H), 3.79 (s, 3 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 166.2,

142.2, 136.9, 136.7, 132.8, 130.7, 130.4, 128.9, 125.2, 123.4, 122.7,

122.6, 120.6, 116.2, 116.0, 112.7, 104.7, 54.6, 18.1. MS (ESI) m/z 415.2

for [MþH]^þ. HRMS (ESI) m/z calculated for C₂₀H₁₈F₃N₆O [MþH]^þ

415.1494, found 415.1480. HPLC purity 97.6% (MeOH: H₂O ¼ 80 : 20,

0.8 mL/min, t_R¼ 4.70 min).

147.1, 145.9, 142.0, 138.0, 137.0, 132.3, 130.9, 129.1, 128.3, 126.2,

124.2, 124.0, 122.6, 120.2, 118.6, 117.9, 116.4, 112.1, 104.7, 79.1, 54.7.

MS (ESI) m/z 515.2 for [MþH]^þ. HRMS (ESI) m/z calculated for

C

₂₃H₁₉ClF₃N₈O [MþH]^þ515.1322, found 515.1307. HPLC purity

96.1% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 5.15 min).

4.1.3.5. N-(4-chloro-3-(triﬂuoromethyl)phenyl)-2-(4-(3-((4-methyl-

3-(N-methylsulfa moyl)phenyl)amino)-1H-indazol-6-yl)-1H-pyrazol-

1-yl)acetamide (W5). White solid powder; yield 53%; m.p.:

4.1.4.4. 2-(4-(3-amino-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-(2-

ﬂuoro-5-(triﬂuoro methyl)phenyl)acetamide (W9). Pale yellow solid

powder; yield 20%; m.p.: 223e225 ^ꢀC; ¹H NMR (600 MHz,

210e212 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

d

12.04 (s, 1 H), 10.82 (s,

DMSO‑d₆)

d

11.33 (s, 1 H), 10.51 (s, 1 H), 8.46 (d, J ¼ 6.6 Hz, 1 H), 8.24

1 H), 9.15 (s, 1 H), 8.28 (s, 1 H), 8.19 (s, 2 H), 8.00 (s, 1 H), 7.93 (d,

J ¼ 8.4 Hz, 1 H), 7.87e7.86 (m, 1 H), 7.82 (dd, J ¼ 8.4 Hz, 1.8 Hz, 1H),

7.70 (d, J ¼ 9.6 Hz, 1 H), 7.51 (s, 1 H), 7.32e7.29 (m, 2 H), 7.27 (d,

(s, 1 H), 7.97 (s, 1 H), 7.67 (d, J ¼ 9.0 Hz, 1 H), 7.58e7.56 (m, 2 H), 7.38

(s, 1 H), 7.16 (d, J ¼ 8.4 Hz, 1 H), 5.30 (s, 2 H), 5.19 (s, 2 H); ¹³C NMR

(150 MHz, DMSO‑d₆)

d 166.7, 155.6, 149.1, 142.2, 136.9, 130.3, 129.0,

J ¼ 7.8 Hz,1 H), 5.08 (s, 2 H), 2.45 (s, 3 H), 2.43 (d, J ¼ 4.8 Hz, 3 H); ¹³

C

126.8, 126.7, 122.8, 122.3, 120.6, 119.8, 116.8, 116.7, 116.0, 112.7, 104.7,

NMR (150 MHz, DMSO‑d₆)

d

166.1, 144.6, 141.3, 141.0, 138.0, 137.4,

54.4. MS (ESI) m/z 419.1 for [MþH]^þ. HRMS (ESI) m/z calculated for

137.1,133.0,132.8,132.2,131.1,129.2,126.7,125.4,124.3,124.0,122.5,

120.3, 118.9, 117.8, 117.1, 116.3, 112.9, 104.8, 54.7, 28.3, 18.9. MS (ESI)

m/z 618.1 for [MþH]^þ. HRMS (ESI) m/z calculated for

C

₁₉H₁₅F₄N₆O [MþH]^þ419.1243, found 419.1230. HPLC purity 98.3%

(MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 4.35 min).

C

₂₇H₂₄ClF₃N₇O₃S [MþH]^þ618.1302, found 618.1284. HPLC purity

4.1.4.5. N-(6-(1-(2-((4-chloro-3-(triﬂuoromethyl)phenyl)amino)-2-

97.6% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 5.46 min).

oxoethyl)-1H-pyr

propanecarboxamide (W10). Gray solid powder; yield 31%; m.p.:

267e269 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.54 (s, 1 H), 10.79 (s,

azol-4-yl)-1H-indazol-3-yl)cyclo-

4.1.4. General procedure for the synthesis of target compounds W6‒

12

d

1 H), 10.60 (s, 1 H), 8.28 (s, 1 H), 8.20 (s, 1 H), 8.00 (s, 1 H), 7.85 (d,

J ¼ 8.4 Hz, 1 H), 7.78 (d, J ¼ 8.4 Hz, 1 H), 7.71 (d, J ¼ 8.4 Hz, 1 H), 7.56

(s, 1 H), 7.29 (d, J ¼ 8.4 Hz, 1 H), 5.09 (s, 2 H), 1.94 (s, 1 H), 0.85e0.83

To a mixture of compound 11 or 12 (1.0 mmol, 1.0 eq), com-

pounds 3a‒3d (1.3 eq) and Pd(dppf)Cl₂$CH₂Cl₂(0.1 eq) in 1,4-

dioxane (4.0 mL) was added 2 M Na₂CO₃aq (0.4 mL) and purged

with argon. The resulting mixture was reﬂuxed for 3 h before being

cooled to the room temperature. The reaction mixture was ﬁltered

and ﬁltrate was evaporated in vacuo, the crude product was further

(m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 171.6, 166.1, 141.8, 140.5,

138.0, 137.0, 132.2, 130.6, 129.2, 126.7, 124.3, 124.0, 123.0, 122.3,

121.7, 117.9, 114.7, 113.1, 105.0, 54.7, 13.6, 7.1. MS (ESI) m/z 503.1 for

[MþH]^þ. HRMS (ESI) m/z calculated for C₂₃H₁₉ClF₃N₆O₂[MþH]^þ

503.1210, found 503.1195. HPLC purity 97.8% (MeOH: H₂O ¼ 80 : 20,

0.8 mL/min, t_R¼ 5.32 min).

puriﬁed

by

silica

gel

ﬂash

chromatography

(DCM:

MeOH ¼ 100:1e20:1) to obtain target compounds W6‒12.

4.1.4.1. 2-(4-(3-amino-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-(4-

chloro-3-(triﬂuoro methyl)phenyl)acetamide (W6). Gray solid pow-

der; yield 46%; m.p.: 226e228 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

4.1.4.6. N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)-1H-pyr

propanecarboxamide (W11). Pale yellow solid powder; yield 46%;

m.p.: 247e249 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.57 (s, 1 H),

azol-4-yl)-1H-indazol-3-yl)cyclo-

d

11.37 (s, 1 H), 10.84 (s, 1 H), 8.25 (s, 1 H), 8.21 (s, 1 H), 7.97 (s, 1 H),

7.84 (d, J ¼ 8.4 Hz, 1 H), 7.72 (d, J ¼ 8.4 Hz, 1 H), 7.68 (d, J ¼ 9.0 Hz,

d

1 H), 7.39 (s, 1 H), 7.18 (d, J ¼ 8.4 Hz, 1 H), 5.42 (s, 2 H), 5.09 (s, 2 H);

10.64 (s, 1 H), 10.61 (s, 1 H), 8.29 (s, 1 H), 8.03 (d, J ¼ 1.8 Hz,1 H), 8.01

(s, 1 H), 7.78 (d, J ¼ 9.0 Hz, 1 H), 7.71 (d, J ¼ 9.0 Hz, 1 H), 7.56 (s, 1 H),

7.42 (d, J ¼ 7.8 Hz, 1 H), 7.29 (d, J ¼ 8.4 Hz, 1 H), 5.07 (s, 2 H), 2.39 (s,

3 H), 1.94 (s, 1 H), 0.86e0.82 (m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

¹³C NMR (150 MHz, DMSO‑d₆)

d 166.0, 137.9, 136.9, 132.1, 130.5,

128.9, 126.8, 126.6, 124.2, 123.9, 123.4, 122.6, 121.6, 117.8, 115.9,

104.8, 54.5. MS (ESI) m/z 435.1 for [MþH]^þ. HRMS (ESI) m/z calcu-

lated for C₁₉H₁₅ClF₃N₆O [MþH]^þ435.0948, found 435.0934. HPLC

purity 95.6% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 4.87 min).

d

171.6, 165.7, 141.8, 140.5, 136.9, 136.7, 132.8, 130.7, 129.2, 127.4,

125.2, 123.0, 122.5, 122.3, 117.9, 116.2, 114.7, 105.0, 54.7, 18.1, 13.6, 7.1.

MS (ESI) m/z 483.3 for [MþH]^þ. HRMS (ESI) m/z calculated for

4.1.4.2. 2-(4-(3-amino-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-(3-(tri-

ﬂuoromethyl)phe nyl)acetamide (W7). Pale yellow solid powder;

C

₂₄H₂₂F₃N₆O₂[MþH]^þ483.1756, found 483.1740. HPLC purity 97.5%

(MeOH: H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 6.07 min).

15

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

4.1.4.7. N-(6-(1-(2-((2-ﬂuoro-5-(triﬂuoromethyl)phenyl)amino)-2-

puriﬁed

by

silica

gel

ﬂash

chromatography

(DCM:

oxoethyl)-1H-pyr

propanecarboxamide (W12). Gray solid powder; yield 35%; m.p.:

258e260 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.56 (s, 1 H), 10.63 (s,

1 H), 10.51 (s, 1 H), 8.45 (d, J ¼ 6.6 Hz, 1 H), 8.29 (s, 1 H), 8.01 (s, 1 H),

7.78 (d, J ¼ 9.0 Hz, 1 H), 7.58e7.56 (m, 3 H), 7.29 (d, J ¼ 9.0 Hz, 1 H),

5.20 (s, 2 H), 1.94 (s, 1 H), 0.85e0.82 (m, 4 H); ¹³C NMR (150 MHz,

azol-4-yl)-1H-indazol-3-yl)cyclo-

MeOH ¼ 100:1e10:1) to obtain target compounds W15‒20.

d

4.1.6.1. N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)-1H-pyr azol-4-yl)-1H-indazol-3-yl)cyclo-

pentanecarboxamide (W15). Yellow solid powder; yield 67%; m.p.:

233e234 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.55 (s, 1 H), 10.61 (s,

d

DMSO‑d₆)

d

171.7, 166.1, 155.6, 141.8, 140.5, 137.1, 132.2, 130.7, 129.3,

1 H), 10.25 (s, 1 H), 8.29 (s, 1 H), 8.03e7.99 (m, 2 H), 7.76 (d,

J ¼ 8.4 Hz, 1 H), 7.71 (d, J ¼ 7.8 Hz, 1 H), 7.56 (s, 1 H), 7.42 (d,

J ¼ 7.8 Hz, 1 H), 7.30 (s, 1 H), 5.07 (s, 2 H), 2.91e2.90 (m, 1 H), 2.39 (s,

3 H), 1.89 (s, 2 H), 1.77 (s, 2 H), 1.70 (s, 2 H), 1.59 (s, 2 H); ¹³C NMR

126.9, 124.6, 123.2, 122.8, 122.4, 119.8, 117.9, 116.7, 114.7, 105.0, 54.4,

13.6, 7.2. MS (ESI) m/z 487.2 for [MþH]^þ. HRMS (ESI) m/z calculated

for C₂₃H₁₉F₄N₆O₂[MþH]^þ487.1506, found 487.1491. HPLC purity

97.6% (MeOH: H₂O ¼ 80 : 20, 0.8 mL/min, t_R¼ 4.63 min).

(150 MHz, DMSO‑d₆)

d 175.0, 166.3, 142.3, 141.0, 137.5, 137.3, 132.4,

130.8, 129.7, 127.9, 125.7, 123.9, 123.4, 123.1, 122.8, 118.4, 117.2, 115.3,

105.1, 54.7, 45.3, 30.7, 25.6, 18.6. MS (ESI) m/z 511.3 for [MþH]^þ.

HRMS (ESI) m/z calculated for C₂₆H₂₆F₃N₆O₂[MþH]^þ511.2069,

found 511.2056. HPLC purity 100% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/

min, t_R¼ 3.87 min).

4.1.5. General procedure for the synthesis of target compounds

W13‒14

To a suspension of compound W8 (0.4 mmol, 1.0 eq) in tetra-

hydrofuran (4 mL) was added acetyl chloride or isobutyryl chloride

(2.4 eq) followed by DIEA (4.8 eq). After the mixture was stirred for

18 h at room temperature, it was poured into water and extracted

with ethyl acetate. The combined organic layer was dried over

anhydrous Na₂SO₄and evaporated the solvent. The residue was

dissolved in tetrahydrofuran/methanol (2 mL/2 mL) and 2 N K₂CO₃

aq (0.25 mL) was added. After stirring for 1 h, appropriate water

was added and extracted with ethyl acetate. The combined organic

layer was dried over anhydrous Na₂SO₄, concentrated, and puriﬁed

by column chromatography (DCM: MeOH ¼ 100:1e10:1) to obtain

compounds W13‒14.

4.1.6.2. 1-Methyl-N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)

amino)-2-oxoeth yl)-1H-pyrazol-4-yl)-1H-indazol-3-yl)piperidine-4-

carboxamide (W16). White solid powder; yield 71%; m.p.:

178e180 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

d 12.59 (s, 1 H), 10.68 (s,

1 H), 10.29 (s, 1 H), 8.29 (s, 1 H), 8.04 (s, 1 H), 8.00 (s, 1 H), 7.75 (d,

J ¼ 8.4 Hz, 1 H), 7.72 (d, J ¼ 8.4 Hz, 1 H), 7.57 (s, 1 H), 7.42 (d,

J ¼ 8.4 Hz, 1 H), 7.31 (d, J ¼ 8.4 Hz, 1 H), 5.08 (s, 2 H), 2.91 (s, 2 H),

2.46 (s, 1 H), 2.39 (s, 3 H), 2.26 (s, 3 H), 2.06 (s, 2 H), 1.84 (s, 2 H),

1.75e1.73 (m, 2 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 174.2, 166.3,

142.3,140.8,137.5,137.3,133.3,131.2,129.7,127.9,125.7,123.9,123.3,

123.1, 122.8, 118.4, 116.7, 115.3, 105.5, 55.6, 46.4, 41.6, 37.0, 31.2, 28.8,

21.3, 18.6. MS (ESI) m/z 540.3 for [MþH]^þ. HRMS (ESI) m/z calcu-

lated for C₂₇H₂₉F₃N₇O₂[MþH]^þ540.2335, found 540.2315. HPLC

purity 95.4% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.89 min).

4.1.5.1. 2-(4-(3-acetamido-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-(4-

methyl-3-(triﬂu oromethyl)phenyl)acetamide (W13). White solid

powder; yield 46%; m.p.: 263e265 ^ꢀC; ¹H NMR (600 MHz,

DMSO‑d₆)

d 12.61 (s, 1 H), 10.61 (s, 1 H), 10.35 (s, 1 H), 8.30 (s, 1 H),

8.03 (s, 1 H), 8.01 (s, 1 H), 7.79 (d, J ¼ 8.4 Hz, 1 H), 7.71 (d, J ¼ 10.8 Hz,

1 H), 7.56 (s, 1 H), 7.42 (d, J ¼ 7.8 Hz, 1 H), 7.31 (d, J ¼ 8.4 Hz, 1 H),

5.07 (s, 2 H), 2.39 (s, 3 H), 2.11 (s, 1 H); ¹³C NMR (150 MHz,

4.1.6.3. N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)-1H- pyrazol-4-yl)-1H-indazol-3-yl)nicotinamide (W17).

Yellow solid powder; yield 90%; m.p.: 142e144 ^ꢀC; ¹H NMR

DMSO‑d₆) d 168.7, 166.2, 142.3, 140.9, 137.5, 137.3, 133.3, 131.2, 129.7,

127.9, 125.7, 123.9, 123.5, 123.1, 122.8, 118.5, 116.7, 115.2, 105.5, 55.2,

23.4, 18.6. MS (ESI) m/z 457.3 for [MþH]^þ. HRMS (ESI) m/z calcu-

lated for C₂₂H₂₀F₃N₆O₂[MþH]^þ457.1600, found 457.1585. HPLC

purity >99% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.75 min).

(600 MHz, DMSO‑d₆) d 12.81 (s, 1 H), 11.04 (s, 1 H), 10.65 (s, 1 H),

9.22 (s, 1 H), 8.79 (d, J ¼ 4.8 Hz, 1 H), 8.42 (d, J ¼ 7.8 Hz 1 H), 8.32 (s,

1 H), 8.03 (s, 2 H), 7.77 (d, J ¼ 8.4 Hz, 1 H), 7.72 (d, J ¼ 8.4 Hz, 1 H),

7.64 (s, 1 H), 7.60e7.58 (m, 1 H), 7.42 (d, J ¼ 8.4 Hz, 1 H), 7.37 (d,

J ¼ 8.4 Hz, 1 H), 5.08 (s, 2 H), 2.39 (s, 3 H); ¹³C NMR (150 MHz,

4.1.5.2. N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)-1H-pyr azol-4-yl)-1H-indazol-3-yl)isobutyramide (W14).

White solid powder; yield 59%; m.p.: 219e221 ^ꢀC; ¹H NMR

DMSO‑d₆) d 167.2, 164.6, 163.5, 153.4, 149.1, 142.4, 140.3, 137.5, 137.3,

136.1, 133.3, 131.3, 131.2, 129.8, 127.9, 125.7, 124.5, 123.1, 122.9, 122.7,

118.9, 117.1, 115.8, 105.7, 54.7, 18.6. MS (ESI) m/z 520.3 for [MþH]^þ.

HRMS (ESI) m/z calculated for C₂₆H₂₁F₃N₇O₂[MþH]^þ520.1709,

found 520.1696. HPLC purity 98.0% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/

min, t_R¼ 3.76 min).

(600 MHz, DMSO‑d₆)

d 12.57 (s, 1 H), 10.61 (s, 1 H), 10.25 (s, 1 H),

8.29 (s, 1 H), 8.03 (s, 1 H), 8.01 (s, 1 H), 7.75 (d, J ¼ 8.4 Hz, 1 H), 7.71

(d, J ¼ 6.0 Hz, 1 H), 7.57 (s, 1 H), 7.42 (d, J ¼ 8.4 Hz, 1 H), 7.31 (d,

J ¼ 8.4 Hz, 1 H), 5.07 (s, 2 H), 2.74e2.72 (m, 1 H), 2.39 (s, 3 H),

1.17e1.15 (m, 6 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d

175.8, 166.2,

4.1.6.4. 1-Methyl-N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)

142.3,141.0,137.5,137.3, 133.3,131.2,129.7,127.9,126.4,123.9,123.4,

123.1, 122.8, 117.9, 116.7, 115.4, 106.1, 54.4, 34.4, 20.1, 18.6. MS (ESI)

m/z 485.3 for [MþH]^þ. HRMS (ESI) m/z calculated for C₂₄H₂₄F₃N₆O₂

[MþH]^þ485.1913, found 485.1897. HPLC purity 99.7% (MeOH:

H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.73 min).

amino)-2-oxoeth

azole-4-carboxamide (W18). White solid powder; yield 84%; m.p.:

271e273 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.69 (s, 1 H), 10.63 (s,

1 H), 10.42 (s, 1 H), 8.36 (s, 1 H), 8.31 (s, 1 H), 8.09 (s, 1 H), 8.04 (s,

1 H), 8.02 (s, 1 H), 7.75 (d, J ¼ 8.4 Hz, 1 H), 7.72 (d, J ¼ 7.8 Hz, 1 H),

7.61 (s, 1 H), 7.42 (d, J ¼ 8.4 Hz, 1 H), 7.33 (d, J ¼ 9.0 Hz, 1 H), 5.08 (s,

2 H), 3.91 (s, 3 H), 2.39 (s, 3 H); ¹³C NMR (150 MHz, DMSO‑d₆)

yl)-1H-pyrazol-4-yl)-1H-indazol-3-yl)-1H-pyr-

d

4.1.6. General procedure for the synthesis of target compounds

W15‒20

d

166.3, 161.0, 142.4, 140.7, 139.7, 137.5, 137.3, 133.4, 133.3, 131.2,

To a solution of compounds 15a‒d or 17a‒b (0.8 mmol) with

dichloromethane (11 mL) was slowly added triﬂuoroacetate

(5.00 mL) dropwise under ice water bath. After 30 min, the reaction

was stirred at room temperature for 3.5 h. After the reaction was

completed, the solvent was evaporated and the residue was taken

up in ethyl acetate and saturated Na₂CO₃aq, and extracted with

ethyl acetate. The combined organic phase was washed with brine

and dried over Na₂SO₄. The solvent was concentrated in vacuo and

129.8, 128.1, 127.9, 125.7, 124.3, 123.3, 123.1, 122.8, 118.6, 118.3, 116.6,

115.9, 105.6, 55.2, 18.6. MS (ESI) m/z 523.3 for [MþH]^þ. HRMS (ESI)

m/z calculated for C₂₅H₂₂F₃N₈O₂[MþH]^þ523.1818, found 523.1802.

HPLC purity 99.2% (MeOH: H₂O

¼

80 : 20, 1.0 mL/min,

t_R¼ 3.33 min).

4.1.6.5. 2-(4-(3-(ethylamino)-1H-indazol-6-yl)-1H-pyrazol-1-yl)-N-

(4-methyl-3-(triﬂuoromethyl)phenyl)acetamide (W19). White solid

16

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

powder; yield 43%; m.p.: 238e240 ^ꢀC; ¹H NMR (600 MHz,

4.1.8. General procedure for the synthesis of compounds 24 and

27a‒b

DMSO‑d₆)

d 11.52 (s, 1 H), 10.70 (s, 1 H), 8.26 (s, 1 H), 8.03 (s, 1 H),

7.98 (s, 1 H), 7.72e7.69 (m, 2 H), 7.42e7.40 (m, 2 H), 7.21 (d,

J ¼ 8.4 Hz, 1 H), 5.07 (s, 2 H), 3.31 (q, J ¼ 7.2 Hz, 2 H), 2.39 (s, 3 H),

To a mixture of compounds 23 or 26a‒b (2.1 mmol, 1.0 eq),

bis(pinacolato)diboron (1.4 eq), Pd(dppf)Cl₂(0.1 eq) and potassium

acetate (3.0 eq) in anhydrous 1,4-dioxane (10 mL) was heated to

reﬂux for 12 h under argon. After reactant consumed completely,

the reaction solution was ﬁltered through a pad of Celite and rinsed

with ethyl acetate, and the ﬁltrate was concentrated in vacuo. The

residue was puriﬁed by column chromatography (PE:

EtOAc ¼ 50:1e6:1) to afford compounds 24 and 27a‒b.

1.25 (t, J ¼ 7.2 Hz, 3 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 166.3, 150.3,

143.1, 137.5, 137.3, 133.3, 131.6, 131.2, 129.6, 128.1, 127.9, 125.7, 123.9,

123.0, 121.2, 116.7, 112.7, 105.2, 55.8, 38.3, 18.6, 15.3. MS (ESI) m/z

443.3 for [MþH]^þ. HRMS (ESI) m/z calculated for C₂₂H₂₂F₃N₆O

[MþH]^þ443.1807, found 443.1792. HPLC purity 99.5% (MeOH:

H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.76 min).

4.1.8.1. N-(4-methyl-3-(triﬂuoromethyl)phenyl)-2-(5-(4,4,5,5-

tetramethyl-1,3,2-dio xaborolan-2-yl)thiophen-2-yl)acetamide (24).

4.1.6.6. 2-(4-(3-((cyclopropylmethyl)amino)-1H-indazol-6-yl)-1H-

Yellow liquid; yield 58%; ¹H NMR (600 MHz, CDCl₃)

d 7.60e7.59 (m,

pyrazol-1-yl)-N-

(W20). White solid powder; yield 48%; m.p.: 214e216 ^ꢀC; ¹H NMR

(600 MHz, DMSO‑d₆) 11.33 (s, 1 H), 10.60 (s, 1 H), 8.24 (s, 1 H), 8.03

(4-methyl-3-(triﬂuoromethyl)phenyl)acetamide

3 H), 7.31 (s, 1 H), 7.22 (d, J ¼ 7.8 Hz, 1 H), 7.12 (s, 1 H), 3.99 (s, 2 H),

d

2.42 (s, 3 H), 1.35 (s, 12 H).

(s, 1 H), 7.96 (s, 1 H), 7.73e7.69 (m, 2 H), 7.42 (d, J ¼ 8.4 Hz, 1 H), 7.38

(s, 1 H), 7.17 (d, J ¼ 8.4 Hz, 1 H), 5.06 (s, 2 H), 3.13 (d, J ¼ 7.2 Hz, 2 H),

2.38 (s, 3 H), 1.19e1.15 (m, 1 H), 0.47e0.45 (m, 2 H), 0.25e0.24 (m,

4.1.8.2. N-(4-methyl-3-(triﬂuoromethyl)phenyl)-2-(3-(4,4,5,5-

tetramethyl-1,3,2-dio

xaborolan-2-yl)phenyl)acetamide

(27a).

7.79

2 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d 166.3, 150.6, 143.0, 137.4,

White solid powder; yield >99%; ¹H NMR (600 MHz, CDCl₃)

d

137.3, 133.3, 131.2, 131.1, 129.5, 127.9, 125.7, 123.9, 123.2, 123.1, 121.1,

116.3, 112.9, 105.1, 55.2, 47.7, 18.6, 12.3, 4.0. MS (ESI) m/z 469.3 for

[MþH]^þ. HRMS (ESI) m/z calculated for C₂₄H₂₄F₃N₆O [MþH]^þ

469.1964, found 469.1947. HPLC purity 97.5% (MeOH: H₂O ¼ 80 : 20,

1.0 mL/min, t_R¼ 3.74 min).

(d, J ¼ 6.6 Hz, 1 H), 7.76 (s, 1 H), 7.60e7.58 (m, 2 H), 7.45e7.42 (m,

2 H), 7.32 (s, 1 H), 7.19 (d, J ¼ 8.4 Hz, 1 H), 3.74 (s, 2 H), 2.41 (s, 3 H),

1.36 (s, 12 H).

4.1.8.3. N-(4-methyl-3-(triﬂuoromethyl)phenyl)-2-(4-(4,4,5,5-

tetramethyl-1,3,2-dio

xaborolan-2-yl)phenyl)acetamide

(27b).

7.86

White solid powder; yield >99%; ¹H NMR (600 MHz, CDCl₃)

d

4.1.7. General procedure for the synthesis of target compounds

W21‒22

(d, J ¼ 7.8 Hz, 2 H), 7.56e7.55 (m, 2 H), 7.35 (d, J ¼ 7.8 Hz, 2 H), 7.19

(d, J ¼ 7.8 Hz, 1 H), 7.06 (s, 1 H), 3.77 (s, 2 H), 2.41 (s, 3 H), 1.36 (s,

12 H).

To a suspension of compound 21a or 21b (0.2 mmol, 1.0 eq) in

tetrahydrofuran (4 mL) was added cyclopropane carbonyl chloride

(2.5 eq) followed by DIEA (4.8 eq). The reaction condition and post-

treatment method were the same as compound W13. The ﬁnal

residue was puriﬁed by silica gel ﬂash chromatography (DCM:

MeOH ¼ 100:1e10:1) to obtain target compounds W21‒22.

4.1.9. General procedure for the synthesis of target compounds

W23‒25

To a mixture of compound 12 (1.0 mmol, 1.0 eq), compound 24

or 27a‒b (1.3 eq) and Pd(dppf)Cl₂$CH₂Cl₂(0.1 eq) in 1,4-dioxane

(4.0 mL) was added 2 M Na₂CO₃aq (0.4 mL) and purged with

argon. The resulting mixture was reﬂuxed for 3 h. The reaction

mixture was ﬁltered and ﬁltrate was evaporated in vacuo, the crude

product was further puriﬁed by column chromatography (DCM:

MeOH ¼ 100:1e20:1) to obtain target compounds W23‒25.

4.1.7.1. N-(6-(3-methyl-1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)

amino)-2-oxoeth

propanecarboxamide (W21). Pale pink solid powder; yield 47%;

m.p.: 240e242 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.54 (s, 1 H),

yl)-1H-pyrazol-4-yl)-1H-indazol-3-yl)cyclo-

d

10.65e10.63 (m, 2 H), 8.03 (s,1 H), 7.78 (d, J ¼ 8.4 Hz,1 H), 7.69e7.68

(m, 2 H), 7.40e7.39 (m, 2 H), 7.12 (s, 1 H), 5.06 (s, 1 H), 4.97 (s, 1 H),

2.38 (s, 3 H), 2.34 (s, 3 H), 1.92 (s, 1 H), 0.84 (s, 4 H); ¹³C NMR

4.1.9.1. N-(6-(5-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)thioph en-2-yl)-1H-indazol-3-yl)cyclopropanecarboxamide

(W23). Yellow solid powder; yield 32%; m.p.: 234e236 ^ꢀC; ¹H NMR

(150 MHz, DMSO‑d₆)

d 172.2, 166.4, 145.3, 142.2, 140.9, 138.2, 137.3,

(600 MHz, DMSO‑d₆)

d 12.65 (s, 1 H), 10.70 (s, 1 H), 10.50 (s, 1 H),

136.9, 133.3, 131.5, 131.2, 127.9, 125.7, 123.9, 123.4, 123.0, 120.4,

116.6, 107.4, 54.8, 18.6, 13.9, 10.8, 7.7. MS (ESI) m/z 497.3 for [MþH]^þ.

HRMS (ESI) m/z calculated for C₂₅H₂₄F₃N₆O₂[MþH]^þ497.1913,

found 497.1897. HPLC purity 99.7% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/

min, t_R¼ 3.82 min).

8.03 (s, 1 H), 7.81 (d, J ¼ 9.0 Hz, 1 H), 7.72 (d, J ¼ 8.4 Hz, 1 H), 7.56 (s,

1 H), 7.44 (d, J ¼ 3.6 Hz, 1 H), 7.40 (d, J ¼ 8.4 Hz, 1 H), 7.32 (d,

J ¼ 9.0 Hz, 1 H), 7.02 (d, J ¼ 3.6 Hz, 1 H), 3.92 (s, 2 H), 2.39 (s, 3 H),

1.93 (s, 1 H), 0.85e0.82 (m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d

172.2, 168.7, 143.2, 142.0, 141.1, 137.7, 137.2, 133.2, 132.5, 130.9,

128.4, 127.9, 125.8, 124.1, 123.9, 123.0, 118.2, 116.6, 105.9, 38.3, 18.6,

14.1, 7.7. MS (ESI) m/z 499.2 for [MþH]^þ. HRMS (ESI) m/z calculated

for C₂₅H₂₂F₃N₄O₂S [MþH]^þ499.1416, found 499.1401. HPLC purity

99.7% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.93 min).

4.1.7.2. N-(6-(3,5-dimethyl-1-(2-((4-methyl-3-(triﬂuoromethyl)

phenyl)amino)-2-oxo

cyclopropanecarboxamide (W22). Yellow solid powder; yield 64%;

m.p.: 237e238 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.57 (s, 1 H),

ethyl)-1H-pyrazol-4-yl)-1H-indazol-3-yl)

d

10.67 (s, 1 H), 10.62 (s, 1 H), 8.06 (s, 1 H), 7.80 (d, J ¼ 7.8 Hz, 1 H), 7.70

(d, J ¼ 9.0 Hz, 1 H), 7.42 (d, J ¼ 7.8 Hz, 1 H), 7.23 (s, 1 H), 6.97 (d,

J ¼ 8.4 Hz, 1 H), 4.97 (s, 2 H), 2.40 (s, 3 H), 2.27 (s, 3 H), 2.17 (s, 3 H),

1.94 (s, 1 H), 0.85e0.83 (m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

4.1.9.2. N-(6-(3-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)phenyl)- 1H-indazol-3-yl)cyclopropanecarboxamide (W24).

Pale yellow solid powder; yield 28%; m.p.: 163e165 ^ꢀC; ¹H NMR

(600 MHz, DMSO‑d₆)

d 12.69 (s, 1 H), 10.71 (s, 1 H), 10.45 (s, 1 H),

d

172.3, 166.6, 145.0, 142.0, 140.9, 137.9, 137.3, 133.3, 132.4, 131.2,

8.02 (s, 1 H), 7.87 (d, J ¼ 8.4 Hz, 1 H), 7.71e7.68 (m, 2 H), 7.59e7.54

(m, 2 H), 7.45e7.42 (m, 1 H), 7.36e7.31 (m, 3 H), 3.74 (s, 2 H), 2.36 (s,

3 H), 1.93 (s, 1 H), 0.86 (s, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

127.9, 125.7, 123.9, 123.0, 121.9, 119.0, 116.6, 115.1, 110.0, 52.5, 18.7,

14.1, 13.0, 10.5, 7.7. MS (ESI) m/z 511.3 for [MþH]^þ. HRMS (ESI) m/z

calculated for C₂₆H₂₆F₃N₆O₂[MþH]^þ511.2069, found 511.2057.

d

172.8, 169.9, 142.2, 141.0, 140.9, 139.0, 137.9, 136.8, 133.1, 130.7,

HPLC purity 99.6% (MeOH: H₂O

¼

80

:

20, 1.0 mL/min,

129.4, 128.8, 128.6, 128.0, 126.0, 123.7, 122.9, 120.1, 116.5, 115.9,

108.0, 43.8, 18.6, 14.1, 7.3. MS (ESI) m/z 493.3 for [MþH]þ. HRMS

t_R¼ 3.86 min).

17

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

(ESI) m/z calculated for C₂₇H₂₄F₃N₄O₂[MþH]^þ493.1851, found

493.1835. HPLC purity 99.7% (MeOH: H₂O ¼ 80 : 20, 1.0 mL/min,

t_R¼ 3.95 min).

Thereafter, cells were treated with different concentrations of

compounds or positive control Sorafenib (Aladdin, Shanghai,

CHINA) that dissolved in the medium and incubated at 37 ^ꢀC for

72 h. Then, 10

mL of MTT solution (5 mg/mL, Solarbio, Beijing,

4.1.9.3. N-(6-(4-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

oxoethyl)phenyl)- 1H-indazol-3-yl)cyclopropanecarboxamide (W25).

White solid powder; yield 45%; m.p.: 289e291 ^ꢀC; ¹H NMR

CHINA) was added to each well and the cells were further incu-

bated for 4 h in dark at 37 ^ꢀC. The unreacted MTT solution was

removed and 100 mL DMSO was added to each well to solubilise the

(600 MHz, DMSO‑d₆)

d

12.68 (s, 1 H), 10.70 (s, 1 H), 10.48 (s, 1 H),

produced formazan crystals. The absorbance of the solution at

490 nm was measured using a universal microplate reader (BIO-

RAD, iMerk™, Japan), and each experiment was performed at least

in triplicate. The IC₅₀values were determined from nonlinear

regression analysis of the sigmoidal dose-response curve generated

in Graph pad prism 5.0.

8.03 (s, 1 H), 7.84 (d, J ¼ 7.8 Hz, 1 H), 7.72 (d, J ¼ 7.8 Hz, 1 H), 7.67 (d,

J ¼ 7.2 Hz, 2 H), 7.58 (s, 1 H), 7.44 (d, J ¼ 7.8 Hz, 2 H), 7.37 (d,

J ¼ 9.0 Hz, 1 H), 7.32 (d, J ¼ 8.4 Hz, 1 H), 3.70 (s, 2 H), 2.36 (s, 3 H),

1.93 (s, 1 H), 0.84e0.81 (m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d

172.2, 169.9, 142.2, 140.9, 139.4, 138.9, 137.9, 135.5, 133.1, 130.7,

130.2, 128.0, 127.6, 125.8, 123.6, 122.9, 119.4, 116.5, 115.8, 107.8, 43.4,

18.6, 14.1, 7.7. MS (ESI) m/z 493.3 for [MþH]^þ. HRMS (ESI) m/z

calculated for C₂₇H₂₄F₃N₄O₂[MþH]^þ493.1851, found 493.1837.

4.2.3. In vitro VEGFR-2 inhibition assay

The synthesized compounds were evaluated for their in vitro

VEGFR-2 inhibitory activities using Caliper Mobility-Shift assay,

which were carried out by Sundia (Shanghai, CHINA) MediTech

Company, Ltd.. At ﬁrst, 1 ꢂ kinase buffer was prepared and com-

pounds were are diluted to different ﬁnal test concentrations and

transferred 250 nL of above solution to assay plate by Echo550.

Then 2.5 ꢂ protein solution using 1 ꢂ kinase buffer were prepared

HPLC purity 97.5% (MeOH: H₂O

¼

80 : 20, 1.0 mL/min,

t_R¼ 3.93 min).

4.1.10. Compound W26 was prepared following the synthetic

procedure of W13

To a suspension of compound 30 (0.36 mmol, 1.0 eq) in tetra-

hydrofuran (4 mL) was added cyclopropane carbonyl chloride (2.4

eq) followed by DIEA (4.8 eq). The reaction condition and post-

treatment method were the same as compound W13. The ﬁnal

residue was recrystallized from isopropanol and dried in vacuum to

obtain target compound W26.

and 10

mL of above solution was added to compound well and

positive control well with negative control well for 10

mL of

1 ꢂ kinase buffer, centrifuged at 1000 rpm for 30 s and incubated at

room temperature for 10 min. At the same time, 5 ꢂ ATP and kinase

substrate mixed solution with 1 ꢂ kinase buffer were prepared and

15 mL of above solution was added to each well of the reaction plate.

4.1.10.1. N-(6-(1-(2-((4-methyl-3-(triﬂuoromethyl)phenyl)amino)-2-

The mixture was centrifuged at 1000 rpm for 30 s and incubated at

room temperature for 30 min. Next, 30 L of detection solution was

added and the mixture was centrifuged at 1000 rpm for 30 s.

Finally, the conversion rate was read by microplate reader (Perki-

nElmer, Caliper EZ Reader). Curve ﬁtting was performed on

oxoethyl)-1H-1,

propanecarboxamide (W26). White solid powder; yield 83%; m.p.:

>300 ^ꢀC; ¹H NMR (600 MHz, DMSO‑d₆)

12.54 (s, 1 H), 10.80 (s,

2,3-triazol-4-yl)-1H-indazol-3-yl)cyclo-

m

d

1 H), 10.55 (s, 1 H), 8.69 (s, 1 H), 8.03 (s, 1 H), 7.92 (s, 1 H), 7.87 (d,

J ¼ 8.4 Hz, 1 H), 7.71 (d, J ¼ 8.4 Hz, 1 H), 7.55 (d, J ¼ 8.4 Hz, 1 H), 7.43

(d, J ¼ 8.4 Hz, 1 H), 5.42 (s, 2 H), 2.40 (s, 3 H), 1.93e1.91 (m, 1 H),

Graphpad Prism 5.0 software to calculate IC₅₀

.

0.88e0.82 (m, 4 H); ¹³C NMR (150 MHz, DMSO‑d₆)

d

171.6, 165.1,

4.2.4. Colony formation assay

147.0, 141.9, 140.5, 137.1, 133.4, 131.4, 129.8, 129.7, 128.2, 128.0, 125.7,

123.9, 123.1, 117.9, 116.7, 106.2, 52.8, 26.3, 18.6, 14.1, 7.7. MS (ESI) m/z

484.3 for [MþH]^þ. HRMS (ESI) m/z calculated for C₂₃H₂₁F₃N₇O₂

[MþH]^þ484.1709, found 484.1694. HPLC purity 95.2% (MeOH:

H₂O ¼ 80 : 20, 1.0 mL/min, t_R¼ 3.67 min).

HGC-27 cells in exponential growth phase were seeded into 6-

well plates (500 cells/well) and incubated at 37 ^ꢀC for 24 h. The

culture medium was replaced with medium containing increasing

concentrations (0.1, 0.5 and 1

with 0.1% DMSO (vehicle) and 5

m

M) of compound W13, compared

M of Sorafenib (positive control),

m

and incubated for 12 days. Then, the cells were washed with cold

PBS, ﬁxed with methanol and stained with 0.1% crystal violet so-

lution for 30 min, respectively. The colonies (greater than 50 cells)

were photographed.

4.2. Biological evaluation

4.2.1. Cell lines and cell culture

All the cell lines were obtained from the Shanghai Qishi

Biotechnology Co., Ltd. (Shanghai, CHINA). Colon (HT-29) cells were

grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM/F-12, Gibco)

whereas lung (A549), liver (HepG2) and gastric (HGC-27) cells were

cultured in RPMI-1640 medium (Gibco). Human umbilical vein

endothelial cells (HUVECs) and human gastric mucosal cells (GES-

1) were cultured in endothelial cell medium (ECM) (ScienCell, SC-

1001) with 1% endothelial cell growth supplement (ECGS) and

DMEM medium, respectively. All the mediums were supplemented

4.2.5. Wound healing assay

HGC-27 cells were cultured in 6-well culture plates until the

density reached to 80e90%. Cells were starved with serum-free

medium for 12 h and followed by wounding with 200 mL pipette

tips to obtain 1 mm wide lanes per well. The cell debris was

removed by washing with PBS and cells were supplied with 2 mL of

complete medium (controls), complete medium containing

different concentrations of compound W13 (0.1, 0.5 and 1

mM), and

with 10% FBS (HyClone, GE Healthcare, Australia), 2% -glutamine

sorafenib (5 M). Images were captured after 0, 6, and 24 h with an

m

L

and 1% ampicillin/streptomycin, and incubated at 37 ^ꢀC in a hu-

midiﬁed atmosphere containing 5% CO₂. All synthesized com-

pounds were prepared as 10 mM stock solutions for subsequent

assay.

inverted ﬂuorescence microscope (Zeiss, VERT1, USA) and analyzed

by Image J software.

4.2.6. Transwell migration assay

Initially, HGC-27 (1 ꢂ 10⁵cells) were suspended in 200

mL of an

4.2.2. In vitro cytotoxicity evaluation

FBS-free medium. The top chamber contained the vehicle and

The cytotoxicity of the target compounds was measured in vitro

using the MTT assay. Cells grown in the logarithmic phase were

counted and seeded in 96-well cell culture plates (5 ꢂ 10³cells per

well) for 24 h to allow attachment of cells to the wall of the plate.

various concentrations of compound W13 (0.1, 0.5 and 1

m

M) and

sorafenib (5

mM). The lower chamber was ﬁlled with 600 mL of

medium containing 10% FBS. After incubating at 37 ^ꢀC for 24 h, the

cells on the top side of the Transwell membrane were removed

18

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

with a cotton tip. The cells trapped on the bottom side of the

membrane were ﬁxed with methanol and stained with 0.1% crystal

violet solution for 30 min, respectively. The migrated cells were

then imaged with an inverted ﬂuorescence microscope (Zeiss,

VERT1, USA) and counted by Image J software for three indepen-

dent ﬁelds randomly.

5%

b

-mercaptoethanol (Beyotime, CHINA), and the mixture was

boiled at 100 ^ꢀC for 15 min. An equal amount of the proteins

(50 mg) were separated by 8e12% sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to

nitrocellulose membranes (Amersham Biosciences, Little Chalfont,

Buckinghamshire, UK). Then, the membranes were blocked with 5%

nonfat dried milk in TBS containing 1% Tween-20 for 2 h at room

temperature and were incubated overnight with speciﬁc primary

antibodies (CST, USA) at 4 ^ꢀC. After three washes in TBST, the

membranes were incubated with the appropriate HRP-conjugated

secondary antibodies at room temperature for 2 h. The blots were

developed with enhanced chemiluminescence (YEASEN, CHINA)

and were detected by an Tanon5200 imager (YPHBIO, CHINA). Each

experiment was performed at least in triplicate and analyzed by

Image J software.

4.2.7. Transwell invasion assay

The Transwell (12

coated with 50

L Matrigel for 5 h at 37 ^ꢀC to achieve solidiﬁca-

tion. HGC-27 cells were harvested and resuspended in serum-free

medium containing 0, 0.1, 0.5 and 1 M of compound W13

mm pore, Corning Incorporated) was pre-

m

compared with 5 mM of sorafenib, and added into the upper wells of

the Transwell chamber at the density of 5 ꢂ 10⁵cells/mL. While

600 mL of RPMI-1640 containing 10% FBS was added into the lower

chambers which had been coated with 50

mL of Matrigel (1:8

dilution in serum-free medium, Corning/BD Biosciences). After 24 h

of incubation at 37 ^ꢀC, the invasion cells were ﬁxed with methanol

and stained with 0.1% crystal violet for 30 min, respectively. Then,

the chambers were washed with PBS and left to dry. Images were

photographed using an inverted ﬂuorescence microscope (Zeiss,

VERT1, USA) and counted by Image J software for three indepen-

dent ﬁelds randomly.

4.2.11. HUVECs tube formation assay

BD Matrigel™ (BD Bio-science, Heidelberg Germany) was

thawed at 4 ^ꢀC overnight prior. The 96-well plate was placed on ice

prior to the coating and 50

mL of matrigel was slowly added to each

well with constant, gentle agitation for an even layer formation.

The Matrigel was polymerized for 1 h at 37 ^ꢀC and 100

mL of

HUVECs suspension (4 ꢂ 10⁵cells/mL) was added to each well.

Subsequently, the cells were cultured with 0.1% DMSO (vehicle),

4.2.8. Apoptosis analysis

10

5 and 10

m

M of Sorafenib (positive control) and various concentrations (1,

HGC-27 Cells were seeded in 60 mm Prtri dishes (1.0 ꢂ 10⁶cells/

mM) of compound W13, respectively, and added 20 ng/mL

dish), incubated with 0.1% DMSO (vehicle), 5

(positive control) and different concentrations (0.1, 0.5 and 1

compound W13 in the separately prepared medium for 24 h,

respectively. After incubation, cells were harvested and incubated

m

M of Sorafenib

VEGF to each well incubating at 37 ^ꢀC for 12 h. The tube formation

was observed with an inverted ﬂuorescence microscope (Zeiss,

VERT1, USA) and photographed using ZEN 2.3 software. The cor-

responding area was measured as the number of pixels using Image

J software.

m

M) of

with 5 mL of Annexin-V/FITC (YEASEN, CHINA) in binding buffer

(10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂at pH 7.4) at room

temperature for 15 min. PI solution was then added to the medium

for another 10 min incubation. Almost 10 000 events were collected

for each sample and analyzed by ﬂow cytometry (BD, LSRFortessa,

USA).

4.3. Molecular docking studies

Molecular docking simulation was performed using the Glide

€

module of Schrodinger. The ligands W18 and sorefenib were pre-

€

pared ﬁrstly using the Ligand Preparation module in Schrodinger,

4.2.9. Effect on ROS generation in HGC-27 cells

respectively. The X-ray crystallographic structures of VEGFR-2 (PDB

ID: 3EWH) in DFG-out inactive conformation was downloaded

from RCSB Protein Data Bank. The protein was ﬁlled in missing side

chain and loops using Prime, removed the water molecule and

minimized using the Protein Preparation Wizard module in the

The intracellular ROS levels in HGC-27 cells were determined by

CM-H₂DCFDA staining. In this assay, HGC-27 cells were seeded in 6-

well culture plates (5.0 ꢂ 10⁵cells/well) and incubated with various

concentrations of compound W13 (0.1, 0.5 and 1

m

M) compared

M of Sorafenib (positive control).

After incubation for 24 h, the cells were harvested and stained with

a 10

M solution of DCFH-DA in PBS for 20 min at 37 ^ꢀC. The in-

€

with 0.1% DMSO (vehicle) and 5

m

Schrodinger. Then, a grid was selected at the active site of the

prepared protein structure using the Grid Generation module of

€

m

Schrodinger and preserved the protein ﬁle. The Glide Docking

€

tensity of the green ﬂuorescence was analyzed using ﬂow

module of Schrodinger was used to investigate the interactions

cytometry.

between the synthesized compounds and VEGFR-2 after importing

ligand ﬁles. All the ﬁgures illustrating the molecular docking results

were visualized and generated using the Pymol software.

4.2.10. Western blot analysis

HUVECs and HGC-27 cells were seeded in 60 mm Prtri dishes

(1.0 ꢂ 10⁶cells/dish) and incubated with 0.1% DMSO (vehicle),

Declaration of competing interest

positive control Sorafenib (10

m

M for HUVEC; 5

m

M for HGC-27),

M for

M for HGC-27), respectively. After incuba-

and various concentrations of compound W13 (1, 5 and 10

HUVEC; 0.1, 0.5 and 1

m

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.

m

tion for 24 h, the cells were collected by centrifugation and washed

twice with PBS chilled to 0 ^ꢀC. Then, the cells were homogenized in

RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1%

(w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) SDS,

and 1 mM EDTA (Beyotime, CHINA) and 1% PMSF, 1% mixed phos-

phatase inhibitor (Solarbio, CHINA). The lysates were incubated on

ice for 30 min, intermittently vortexed every 5 min, and centrifuged

at 12 500 rpm for 15 min to harvest the supernatants. Next, the

protein concentrations were determined by a BCA Protein Assay Kit

(Solarbio, CHINA). The protein extracts were reconstituted in

loading buffer containing 62 mM Tris-HCl, 2% SDS, 10% glycerol and

Acknowledgments

This work was ﬁnancially supported by the National Natural

Science Foundation of China (NO. 21672093).

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113192.

19

X.-R. Wang, S. Wang, W.-B. Li et al.

European Journal of Medicinal Chemistry 213 (2021) 113192

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Design, synthesis and biological evaluation of novel 2-(4-(1H-indazol-6-yl)-1H-pyrazol-1-yl)acetamide derivatives as potent VEGFR-2 inhibitors

DOI: 10.1016/j.ejmech.2021.113192

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Article abstract of DOI:10.1016/j.ejmech.2021.113192

Full text of DOI:10.1016/j.ejmech.2021.113192

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