Design, synthesis and antitumor activity of Novel Sorafenib derivatives bearing pyrazole scaffold

Article abstract of DOI:10.1016/j.bmc.2017.09.003

Four series of Sorafenib derivatives bearing pyrazole scaffold (8a–m, 9a–c, 10a–e and 11a) were synthesized and characterized by NMR and MS. All of the target compounds were evaluated for the cytotoxicity against A549, HepG2, MCF-7, and PC-3 cancer cell lines and some selected compounds were further evaluated for the activity against VEGFR-2/KDR, BRAF, CRAF, c-Met, EGFR and Flt-3 kinases. Compounds 8b and 8i were more active than that of compounds 8h, 9a, especially the IC₅₀ value of compounds 8b on VEGFR-2 kinase was 0.56 μM. And compound 8b exhibited moderate to good activity toward c-Met and showed moderate to no activity against CRAF, c-Met, EGFR, Flt-3 kinases. Eleven of the target compounds exhibited moderate to good antitumor activities. The most promising compound 8b showed strong antitumor activities against A549, HepG2 and MCF-7 cell lines with IC₅₀ values of 2.84 ± 0.78 μM, 1.85 ± 0.03 μM and 1.96 ± 0.28 μM, which were equivalent to Sorafenib (2.92 ± 0.68 μM, 3.44 ± 0.50 μM and 3.18 ± 0.18 μM). Structure–activity relationships (SARs) and docking studies indicated that the pyrazole scaffolds exerted key effect on antitumor activities of target compounds. Substitutions of aryl group at C-3 positions had a significant impact on the antitumor activities, and 3-Br substitution produced the best potency.

Full text of DOI:10.1016/j.bmc.2017.09.003

Accepted Manuscript
Design, Synthesis and Antitumor Activity of Novel Sorafenib Derivatives Bear-
ing Pyrazole Scaffold
Min Wang, Shan Xu, Huajun Lei, Caolin Wang, Zhen Xiao, Shuang Jia, Jia Zhi,
Pengwu Zheng, Wufu Zhu
PII:
S0968-0896(17)31277-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.003
BMC 13963
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 June 2017
29 August 2017
4 September 2017
Please cite this article as: Wang, M., Xu, S., Lei, H., Wang, C., Xiao, Z., Jia, S., Zhi, J., Zheng, P., Zhu, W., Design,
Synthesis and Antitumor Activity of Novel Sorafenib Derivatives Bearing Pyrazole Scaffold, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.003
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Design, Synthesis and Antitumor Activity of Novel Sorafenib Derivatives Bearing Pyrazole
Scaffold
Min Wang†, Shan Xu †, Huajun Lei†, Caolin Wang, Zhen Xiao, Shuang Jia, Jia Zhi, Pengwu Zheng*, Wufu Zhu*1
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation，School of Pharmacy, Jiangxi Science &
Technology Normal University,Nanchang 330013,P.R. China
Abstract: Four series of Sorafenib derivatives bearing pyrazole scaffold (8a–m, 9a–c, 10a–e and
11a) were synthesized and characterized by NMR and MS. All of the target compounds were
evaluated for the cytotoxicity against A549, HepG2, MCF-7, and PC-3 cancer cell lines and some
selected compounds were further evaluated for the activity against VEGFR-2/KDR, BRAF, CRAF,
c-Met, EGFR and Flt-3 kinases. Compounds 8b and 8i were more active than that of compounds 8h, 9a,
especially the IC50 value of compounds 8b on VEGFR-2 kinase was 0.56 µM. And compound 8b exhibited
moderate to good activity toward c-Met and showed moderate to no activity against CRAF，c-Met，EGFR，Flt-3
kinases. Eleven of the target compounds exhibited moderate to good antitumor activities. The most
promising compound 8b showed strong antitumor activities against A549, HepG2 and MCF-7 cell
lines with IC50 values of 2.84 0.78µM, 1.85 0.03µM and 1.96 0.28 µM, which were equivalent to
sorafenib (2.92 0.68 µM, 3.44 0.50 µM and 3.18 0.18 µM). Structure–activity relationships (SARs)
and docking studies indicated that the pyrazole scaffolds exerted key effect on antitumor activities of
target compounds. Substitutions of aryl group at C-3 positions had a significant impact on the
antitumor activities, and 3-Br substitution produced the best potency.
Key words: Sorafenib derivatives; Pyrazole, VEGFR-2/KDR kinase inhibitors; Anticancer activity
† These authors contribute equally to this work
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: zhengpw@126.com, zhuwf@jxstnu.edu.cn, zhuwufu-1122@163.com (W. Zhu).
1

1. Introduction
Cancer is a widespread, complex, and lethal disease. And its burden is increasing across the
world dramatically. It is considered as the first leading cause of deaths in economically developed
countries and the second leading cause of deaths in developing countries [1]. Despite the rapid
progress in medicine, the commitment to the laborious task of discovering new anticancer agents
remains critically important [2]. Transformed cells secrete a cocktail of pro-angiogenic proteins
including vascular endothelial growth factors (VEGFs) and fibroblast growth factors (FGFs) in the
process of tumor growth. So in recent years, VEGF has been the key anti-tumor angiogenesis therapy
Targets. The family members of VEGFR mainly contain VEGFR-1, VEGFRR-2 and VEGFR-3.
Among them, VEGFR2 is a key mediator of pro-angiogenic signalling in the endothelium, which can
adjust the lymphatic endothelial cells and vascular endothelial cells, and promote the formation of
lymphatic and blood vessels, and adjust the migration of lymphocytes, and so on. Small-molecule
VEGFR2 inhibitors (ATP analogues) were some of the first treatments to show anti-angiogenic
efficacy with clinical benefits for cancer patients. The Ras/RAF/MEK/ERK mitogen-activated protein
kinase (MAPK) signaling pathway plays an important role in the transduction of signals from cell surface receptors
to the nucleus, like regulating cell growth, survival, differentiation and proliferation in response to external stimuli
(growth factors, cytokines or hormones)[3-4]. Sorafenib (BAY 43–9006; Nexavar) is a bis-aryl urea, which
can inhibit the Ras / Raf / MEK signal transduction pathway and also can block the formation of new
blood vessels by suppressing the receptor protein of VEGFR and PDGFR. Thus, it inhibits the
growth of tumor cells [5-9]
.
The preliminary study showed that the urea structure was very important for its biological
activity, and NH atoms can play an important role in the formation of hydrogen bonds with amino
[10-12]
acid residues. In recent years, compounds containing pyrazole are widely used in biology
,
pharmacology [13-14], food[15], agrochemical fields[16] and so on. And many pyrazole derivatives were
[17-21]
reported, such as compound I, II, III, IV and V (The structures were shown in Figure 1)
and
these compounds elicited strong cytotoxic activity. The results showed that the pyrazole scaffold
played a key role to the activity. Encouraged by this, we replaced the urea with pyrazole yielding
compounds 8a–m. In order to investigate the effect of the position of carbon-nitrogen double bond to
the activity of the compounds, we designed compounds 9a–c. It is proposed to introduce thioamide
at carbon-nitrogen single bond and got two series compounds (10a–e, 11a) for the purpose of making
2

the sorafenib derivatives binding with receptor more closly, which was effected by active
pharmacophore of urea. Then we disclosed the synthesis and antitumor activity against A549, PC-3,
MCF-7, and HepG2 cancer cell lines, and VEGFR-2 kinase of target compounds (see Fig. 1).
Moreover, docking studies were presented in this paper as well.
Figure 1. Structures of small-molecule antitumor agents and target compounds
(8a–m, 9a–c, 10a–e and 11a).
2. Chemistry
The preparation of target compounds 8a–m, 9a–c, 10a–e and 11a was described in Scheme 1.
Compounds 2 and 3 were synthesized according to the reported procedures [22]. Subsequently,
1-(4-hydroxyphenyl)ethanone, 4-chloro-N-methylpicolinamide (3) and K2CO3 were added in
DMSO, which was refluxed for about 5 h in three neck flask and yielded the key compound
4-(4-acetylphenoxy)-N-methylpicolinamide(4).
The
other
intermediate
4-(4-formylphenoxy)-N-methylpicolinamide (6) was achieved from 4-chloro-N-methylpicolinamide
(3) via substitution reaction with 4-hydroxybenzaldehyde and potassium tert-butoxide.
4-(4-acetylphenoxy)-N-methylpicolinamide(4)[intermediate4-(4-formylphenoxy)
-N-methylpicolinamide (6)] was reacted with substituted benzaldehyde (substituted acetophenone)
through aldol condensation to get the compound 5a–m (7a–c). Finally, the compound 5a–m (7a–c)
with hydrazine hydrate(80%) in acetic acid at 108 °C for 1 h through addition reaction yielded target
compounds 8a–m and 9a–c respectively. And we were used compound 5a–e (7a) in an addition
reaction with thiosemicarbazide to generate target compounds 10a–e and 11a.
3

Reagents and conditions: (a) SOCl2, NaBr, chlorobenzene, 85 °C, 20 h; (b) 30% MeNH2, toluene,
20 °C, 6 h; (c) K2CO3,DMSO, reflux, 5h; (d) KOH, methanol, r.t 6 h.(e) tert-BuOK,DMF, reflux, 15
h; (f) NaH, THF, r.t 8 h; (g) hydrazine hydrate, acetic acid, reflux, 2.5 h; (h) thiosemicarbazide, KOH,
ethanol, microwave, 100 W ,60 °C,15 min.
Scheme 1. Synthetic route of target compounds.
3. Results and discussion
3.1. Biological evaluation
Selecting A549 (human lung cancer), PC-3 (human prostatic cancer), MCF-7(human breast
cancer) and HepG2 (human liver cancer) with high expression of VEGFR-2 kinase as tested cancer
lines, the target compounds (8a–m, 9a–c, 10a–e and 11a) were evaluated for the cytotoxicity against
four cancer cell lines by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell
proliferation assay, using Sorafenib as lead compound. In addition, some selected compounds were
evaluated for the IC50 values against VEGFR-2 kinase in vitro by the mobility shift assay, together
with reference compound Sorafenib. The results expressed as IC50 values were summarized in Tables
1–3 and the values are the average of at least two independent experiments.
As shown in Tables 1–2, the activity of the first and the second series(8a–m, 9a–c) is much
better than that of the third and fourth series (10a–e, 11a). And twelve of the compounds exhibited
4

excellent cytotoxicity activity against different cancer cells with potency from the single-digit
nanomole to µM range. Among them, compounds 8b, 8i and 10d showed superior activity to positive
control Sorafenib against one or more cancer cell lines. The most promising compound 8b exhibits
the best activity against A549, HepG2 and MCF-7 cell lines with IC50 values of 2.84 0.78 µM,
1.85 0.03 µM and 1.96 0.28 µM, which were equivalent to Sorafenib (2.92 0.68 µM, 3.44 0.50 µM,
3.18 0.18 µM). The results suggested that the replacement of the urea fragment with pyrazole scaffold
was benefit for the activity. It also told us that the application of pyrazole scaffold was feasible.
Furthermore, different substitutions of aryl group affected the cytotoxicity of target compounds.
The latter two series (10a–e and 11a) of compounds had lower cytotoxicity than the first two series
(8a–m and 9a–c) of compounds. It was seem to be that the group Br and Cl at C-3 position or none
substituents increased the activity of the target compounds, such as compounds 8b, 8i and 10d. In
another way, the strong electron-withdrawing groups such as –CN, NO2 at C-4 position reduced the
activity of the target compounds. This dramatic boost of activity told us that the group Br at C-3 had
an obviously impact on the activity.
Activity against VEGFR-2, B-RAF, C-RAF,
c-Met, EGFR and Flt-3 kinases of four target
compounds were further carried out in this paper to investigate the target of these compounds. In
Table 3, obviously, compounds 8b and 8i were more active than that of compounds 8h, 9a, especially
the IC50 value of compounds 8b on VEGFR-2 kinase was 0.56 µM. And compound 8b exhibited
moderate to good activity toward c-Met and showed moderate to no activity against CRAF，c-Met，
EGFR，Flt-3 kinases. The results prompted us that this compound may act through other mechanism.
In order to examine the relationship between the antitumor activity of the compound and the
concentrations, we used the method of MTT and selected seven different concentrations. The
inhibition rate was measured after 72 h. Though Figure 2, the phenomenon of dose-dependent was
observed of representative compound 8b.
Table 1 Structures and activity of target compounds 8a-m and 9a–c
IC50(µM) a
Compounds No.
Ar
A549
HepG2
ND
MCF-7
ND
PC-3
6.93 0.89
7.3 0.26
8a
5

2.84 0.78
37.14 0.44
17.36 1.44
13.69 1.09
19.53 0.84
ND
3.54 0.61
28.28 1.27
9.91 1.81
24.25 2.48
NA
1.85 0.03
24.26 1.32
14.97 1.33
11.46 0.2
NA
1.96 0.28
ND
8b
8c
8d
8e
8f
ND
ND
ND
26.46 0.91 35.37 1.37
ND
8g
8h
8i
13.14 1.27
8.60 0.09
10.71 1.06
15.14 0.81
21.04 0.12
10.97 1.32
7.74 0.72
4.52 0.34
36.22 1.44
6.77 0.34
NA
9.64 1.01
1.50 0.38
14.43 0.99
5.28 0.22
57.56 0.99
15.65 0.23
1.24 0.02
ND
8j
9.48 0.23
ND
8k
8l
Br
ND
19.0 1.3
9.94 0.17
8m
8n
9a
9b
9c
12.12 0.21
ND
ND
ND
ND
4.99 0.08
28.87 1.75
8.03 0.10
16.89 0.94 15.30 1.10
ND
ND
ND
Cl
15.28 0.66
10.88 1.27
20.47 0.86
6

Sorafenibb
2.92 0.68
3.44 0.50
3.18 0.18
3.24 0.45
a The values are an average of two separate determinations;
b Used as positive controls
c NA: Not active (IC50 > 50 µM)
d Not determined.
Table 2 Structures and activity of target compounds 10a-e and 11a
IC50(µM) a
Compounds No.
R
A549
HepG2
PC-3
NAc
NA
NA
NA
NA
10a
10b
10c
Cl
19.41 0.24
NA
NA
NA
10d
NA
15.9 1.33
1.89 0.28
10e
11a
NA
NA
NA
NA
NA
NA
Sorafenibb
-
2.92 0.68
3.44 0. 5
3.18 0.18
a The values are an average of two separate determinations;
b Used as positive controls
c NA: Not active (IC50 > 50 µM)
Table 3. Activity against six kinases of four compounds
IC50(µM) a
Comp.No.
VEGFR-2/KDR
0.56
BRAF
3.4
CRAF
7.9
c-Met
0.54
EGFR
>10
Flt-3
5.2
8b
8h
NAc
2.3
NA
3.9
ND
ND
ND
0.12
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8i
9a
NA
NA
0.31
NDd
Sorafenib b
Staurosporine b
0.17
0.072
a The values are an average of two separate determinations;
b Used as positive controls
7

c NA: Not active (IC50 > 50 µM)
d Not determined.
120
100
80
Compound 8b
Inhibitory
Rate(%)
A549
60
HepG2
MCF-7
PC-3
40
20
0
0.137
0.41
1.23
3.7
11.1
33.3
100
Concentration(µg/mL)
Figure 2 Relationship between activity and concentration of selected compounds 8b against our
cancer cell lines
3.2 Compound 8b inhibited the growth of HepG2 cells
As shown in Figure 3, HepG2 cells were treated with compound 8b (1.04~3.29 µg/mL) for 24,
48 and 72 h, and cell growth inhibition were determined by MTT assay. Results indicated that
compound 8b inhibited the proliferation of HepG2 cells in a time and concentration-dependent
manner in figure 3.
Figure 3 Compound 8b inhibited the growth of HepG2 cells
8

3.3 Morphologic changes of A549 cells under inverted microscopy and fluorescence Microscopy
We used acridine orange (AO) single staining by MTT method, in the control group [figure
4(a)], we observed the nucleus of A549 cells were stained into green from fluorescence microscopy.
And the cells grew well, were full, the edge were clear and the refraction was good. While 1.85 µM
concentration of compound 8b acted on the A549 cell lines after 12 h, the cells shrink, chromatin
condensation, the nucleus was dyed bright green, some cells apoptotic bodies and DNA fragments in
figure 4(b).
Figure 4 Morphologic changes of A549 cells under inverted microscopy and fluorescence
Microscopy
3.4 Apoptosis result by flow cytometry method of operation
According to the apoptosis expression by flow cytometry, the target compounds show cell
proliferation activity in another way. To examine whether compound 8b can induce apoptosis of
A549, we adopted flow cytometry method of operation. An obvious reduction in 3µM, 6µM and 12
µM concentration was observed respectively. In contrast, no significant change was seen in control
group. When the concentration increased to 12 µM, apoptosis emerges more evidently in Figure 5.
A549
3µM
Control
12µM
6µM
Annexin V-FITC
Figure 5 Apoptotic cell death analysis in A549 cells treated with compound 8b
9

3.5 Molecular docking study
To explore the binding modes of target compounds with the active site of VEGFR-2 and c-Met,
molecular docking simulation studies were carried out by using SURFLEX-DOCK module of
SYBYL package version. Based on the in vitro inhibition results, we selected compound 8b, as the
ligand example, and the structures of VEGFR-2(PDB ID code: 4ASD) and c-Met (PDB ID code:
3LQ8) were selected as the docking model. The binding modes of compound 8b and lead
compounds were shown in Figure. 6a-d. Analysis of compound 8b and Sorafenib’s binding mode in
the active binding site demonstrated that the docking mode of 8b produced a new H-bond with
residues ASN923 of N-methylpicolinamide’s carbonyl in Figure. 6a, b. The new hydrogen bond
really play an important role in increasing the inhibitory potency of pyrazole derivatives against
VEGFR-2 kinase according to the docking results. As depicted in Figure. 6c, d, we can see
compound 8b formed three hydrogen bonds with residue MET1160 and LYS1110. Compared with
the lead compound, it also produced a new H-bond with residues LYS1110 of Pyrazole. Similarly,
The new hydrogen bond really play an important role in increasing the inhibitory potency of
pyrazole derivatives against c-Met kinase according to the docking results. Furthermore, the docking
results also give us a new direction to design new inhibitors. The above-mentioned results of SARs
analysis and molecular docking study may allow the rational design of more potent VEGFR-2 or
c-Met inhibitors.
10

(c)
(d)
Figure 6. a-d. Binding modes of compound 8b with VEGFR-2 and c-Met. 8b (shown in cyan
Sticks) and lead compound (shown in orange Sticks) with VEGFR-2/KDR (Figure a and b). 8b
(shown in orange Sticks) and lead compound (shown in black blue Sticks) with c-Met (Figure c
and d) .H-bonding interactions between the 8b, lead compound ,VEGFR-2/KDR and c-Met are
denoted by dashed lines in yellow.
4. Conclusions
In summary, we designed and synthesized four series of Sorafenib derivatives of 4,
5-dihydro-1H-pyrazole(8a-m,9a-c,10a-e and 11a), VEGFR-2/KDR, B-Raf, CRAF, c-Met, EGFR
and Flt-3 kinases and four human cancer cell lines (HepG2, A549, MCF-7, and PC-3) were used to
evaluate the target compounds. Twelve of the compounds are equal to or more active than positive
control Sorafenib against one or more cell lines. The most promising compound 8b showed strong
activity, with the IC50 values of 2.84 0.78 µM, 1.85 0.03 µM and 1.96 0.28 µM, which were 1.02,
1.86 and 1.62 times more active than Sorafenib (2.92 0.68 µM, 3.44 0.50 µM, 3.18 0.18 µM),
respectively. Structure–activity relationships (SARs) and docking studies indicated that the
replacement of pyrazole scaffold with urea fragment of the target compounds was benefit for the
activity. From the result of flow cytometry, more and more cancer cell gradually encountered
11

apoptosis or died with the dose of compound 8b increasing, which indicated the compound 8b could
induce remarkable apoptosis of A549 cells. Further study will be carried out to identify the exact
action mechanism in near future.
5. Experimental
5.1. Chemistry
All melting points are obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and are unchanged. NMR spectra are performed by using Bruker 400 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass
spectra (MS) are taken into ESI mode on Agilent 1100 LCMS (Agilent, Palo Alto, CA, USA). All the
materials are obtained from commercial suppliers and used without purification, unless otherwise
specification. Yields are not optimized. TLC analysis is carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China). All the materials are obtained from commercial suppliers and
used without purification, unless otherwise specification. Yields are not optimized.
5.2 General procedure for preparation of compounds 2 and 3
Compounds 2 and 3 were synthesized according to the reported procedures [22]
5.3 4-(4-acetylphenoxy)-N-methylpicolinamide 4
.
A stirring mixture of 1-(4-hydroxyphenyl)ethanone (22 mmol) ,
4-chloro-N-methylpicolinamide(14 mmol) and K2CO3 (35 mmol) in DMSO(100 mL) was refluxed
for about 5 h in three neck flask. Then the reaction mixture was cooled and cold water was added.
The precipitate of 4-(4-acetylphenoxy)-N-methylpicolinamide (4) formed was filtered. Yiled:32%.
5.4 General procedure for the preparation of compounds 5a–m
A mixture of 4-(4-acetylphenoxy)-N-methylpicolinamide (0.67 mmol) and substituted
benzaldehyde (0.67 mmol) were dissolved in methanol (10 mL) and to it 3 mmol of 20%KOH was
added. The mixture was kept at room temperature for 6 hours. The solid formed was filtered and
washed with isopropanol. No further purification was needed and products were used such as were
obtained.
5.5 General procedure for the preparation of compounds 8a–m
Compounds 5a–m (0.26 mmol) and hydrazine hydrate (2.4 mmol) were dissolved in acetic
acid (10 mL). The mixture was refluxed for 2.5 h with constant stirring. The progress of the reaction
12

was monitored through TLC. After completion of reaction, mixture was cooled and poured into water,
and it was neutralized with potassium carbonate, then it was filtered through vacuum by washing
with water.
4-(4-(5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8a)
1
Yield: 75.3%; ESI-MS[M+H]m/z:442.3; m.p.: 129.1-131.0 °C; H NMR (400 MHz, DMSO-d6) δ
8.80 (d, J = 4.9 Hz, 1H), 8.54 (d, J = 5.6 Hz, 1H), 7.92 (s, 1H), 7.90 (s, 1H), 7.58 (d, J = 6.8 Hz, 1H),
7.42 (d, J = 2.5 Hz, 1H), 7.33 (dd, J = 10.0, 5.8 Hz, 2H), 7.30 (s, 1H), 7.22 (dd, J = 5.6, 2.6 Hz, 1H),
7.07 (d, J = 7.5 Hz, 1H), 5.80 (dd, J = 12.1, 5.0 Hz, 1H), 4.02 (d, J = 12.0 Hz, 1H), 3.97 (d, J = 11.9
Hz, 1H), 2.78 (d, J = 4.8 Hz, 3H).
4-(4-(5-(3-bromophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8b)
1
Yield: 78.5%; ESI-MS[M+H]m/z:452.3; m.p.: 116.3-117.8 °C; m.p.: 123.3-124.5; H NMR (400
MHz, DMSO-d6) δ 8.80 (d, J = 4.7 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 7.92 (d, J = 8.6 Hz, 1H), 7.45
(dd, J = 12.6, 5.3 Hz, 1H), 7.31 (dd, J = 12.3, 8.3 Hz, 1H), 7.22 (dd, J = 8.8, 5.6 Hz, 1H), 5.56 (dd, J
= 11.8, 5.0 Hz, 1H), 3.89 (dd, J = 15.2, 12.3 Hz, 1H), 3.24 (dd, J = 15.3, 5.0 Hz, 1H), 2.78 (d, J = 4.8
Hz, 1H).
N-methyl-4-(4-(5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)picolinamide(8c)
Yield: 74.1%; ESI-MS[M+H]m/z:464.2; m.p.: 123.3-124.5 °C; 1H NMR (400 MHz, DMSO-d6) δ
8.80 (d, J = 4.4 Hz, 1H), 8.55 (d, J = 5.5 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 2.5 Hz, 1H),
7.33 (s, 1H), 7.31 (s, 1H), 7.23 (dd, J = 5.6, 2.5 Hz, 1H), 6.48 (s, 2H), 5.49 (dd, J = 12.2, 5.1 Hz, 1H),
3.86 (q, J = 12.0 Hz, 2H), 3.66 (dd, J = 15.5, 15.3 Hz, 9H), 2.78 (d, J = 4.8 Hz, 3H).
4-(4-(5-(4-cyanophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8d)
1
Yield: 79.3%; ESI-MS[M+H]m/z:398.3; m.p.: 122.3-124.1 °C; H NMR (400 MHz, DMSO-d6) δ
8.82 (d, J = 4.7 Hz, 1H), 8.55 (d, J = 5.5 Hz, 1H), 7.92 (s, 1H), 7.90 (s, 1H), 7.82 (d, J = 7.3 Hz, 1H),
7.78 (s, 1H), 7.43 (d, J = 4.5 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.32 (t, J = 9.2 Hz, 2H), 7.23 (dd, J = 5.5,
2.5 Hz, 1H), 5.64 (dd, J = 11.9, 4.8 Hz, 1H), 3.95 (d, J = 12.1 Hz, 1H), 3.91 (d, J = 12.0 Hz, 1H),
3.20 (d, J = 4.8 Hz, 1H), 2.78 (d, J = 4.7 Hz, 3H).
4-(4-(5-(2-chloro-4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8e)
Yield: 80.1%; m.p.: 112.6-113.7 °C; ESI-MS[M+H]m/z: 426.2;1H NMR (400 MHz, DMSO-d6) δ
8.80 (d, J = 4.9 Hz, 1H), 8.54 (d, J = 5.5 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.50 (dd, J = 8.8, 2.4 Hz,
13

1H), 7.42 (d, J = 2.5 Hz, 1H), 7.32 (d, J = 8.7 Hz, 2H), 7.22 (dd, J = 5.6, 2.6 Hz, 1H), 7.17 (dd, J =
8.0, 2.1 Hz, 1H), 7.14 (s, 1H), 5.74 (dd, J = 12.0, 5.2 Hz, 1H), 3.97 (dd, J = 15.0, 11.9 Hz, 1H), 3.14
(dd, J = 15.2, 5.1 Hz, 1H), 2.78 (d, J = 4.8 Hz, 3H)
N-methyl-4-(4-(5-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)picolinamide(8f)
Yield: 78.2%; m.p.: 122.3-124.3 °C; ESI-MS[M+H]m/z:418.4;1H NMR (400 MHz, DMSO-d6) δ
8.81 (d, J = 4.7 Hz, 1H), 8.55 (d, J = 5.5 Hz, 1H), 8.27 – 8.16 (m, 2H), 7.92 (d, J = 8.5 Hz, 2H), 7.50
(t, J = 10.6 Hz, 2H), 7.42 (d, J = 2.3 Hz, 1H), 7.33 (t, J = 9.1 Hz, 2H), 7.23 (dd, J = 5.5, 2.5 Hz, 1H),
5.70 (dd, J = 12.0, 5.0 Hz, 1H), 4.02 – 3.95 (m, 1H), 3.93 (d, J = 12.0 Hz, 1H), 3.23 (d, J = 5.0 Hz,
1H), 2.78 (d, J = 4.7 Hz, 3H).
N-methyl-4-(4-(5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)picolinamide(8g)
Yield: 79.2%; ESI-MS[M+H]m/z:418.7; m.p.: 159.7-161.2 °C; 1H NMR (400 MHz, DMSO-d6) δ
8.80 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.14 (d, J = 6.9 Hz, 1H), 8.09 (s, 1H), 7.93 (d, J = 8.3 Hz, 2H),
7.72 – 7.60 (m, 2H), 7.43 (s, 1H), 7.34 (d, J = 8.3 Hz,2H), 7.24 (s, 1H), 5.74 (d, J = 6.9 Hz, 1H), 3.95
(dd, J = 15.2, 12.4 Hz, 1H), 3.29 – 3.24 (m, 1H), 2.79 (d, J = 4.7 Hz,3H).
4-(4-(5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide
(8h)
1
Yield: 74.5%; ESI-MS[M+H]m/z:403.5; m.p.: 134.7-135.6 °C; H NMR (400 MHz, DMSO-d6) δ
8.80 (d, J = 4.6 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.44 (s, 1H), 7.33 (d, J =
8.6 Hz, 2H), 7.25 – 7.19 (m, 1H), 7.13 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H), 5.51 (dd, J =
11.7, 3.8 Hz, 1H), 3.91 – 3.81 (m, 1H), 3.72 (s, 3H), 3.17 (dd, J = 15.2, 4.5 Hz, 1H), 2.79 (d, J = 4.7
Hz, 3H).
N-methyl-4-(4-(5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)picolinamide(8i)
1
Yield: 79.1%; ESI-MS[M+H]m/z:373.1; m.p.: 133.5-134.7 °C; H NMR (400 MHz, DMSO-d6) δ
8.80 (s, 1H), 8.55 (d, J = 5.5 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.44 (s, 1H), 7.34 (s, 2H), 7.32 (s,
2H), 7.29 – 7.24 (m, 1H), 7.23 (d, J = 5.7 Hz, 2H), 7.20 (s, 1H), 5.56 (d, J = 7.7 Hz, 1H), 3.90 (dd, J
= 15.0, 12.0 Hz, 1H), 3.19 (d, J = 15.1 Hz, 1H), 2.79 (d, J = 4.7 Hz, 3H).
4-(4-(5-(3,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8j)
14

Yield: 79.1%; ESI-MS[M+H]m/z:442.5; m.p.: 93.7-94.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.80
(d, J = 5.3 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.2 Hz, 1H), 7.52
(s, 1H), 7.43 (s, 1H), 7.34 (d, J = 8.2 Hz, 2H), 7.22 (t, J = 8.1 Hz, 2H), 5.57 (dd, J = 11.9, 4.8 Hz, 1H),
3.89 (dd, J = 14.1, 11.9 Hz, 1H), 3.27 (dd, J = 15.2, 4.7 Hz, 1H), 2.79 (d, J = 4.6 Hz, 3H)
4-(4-(5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8k)
1
Yield:72.8%; ESI-MS[M+H]m/z:391.3; m.p.: 201.1-202.3 °C; H NMR (400 MHz, DMSO-d6) δ
8.81 (s, 1H), 8.55 (d, J = 5.5 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.43 (s, 1H), 7.33 (d, J = 8.2 Hz, 2H),
7.30 – 7.25 (m, 2H), 7.24 (d, J = 6.9 Hz, 1H), 7.16 (t, J = 8.6 Hz, 2H), 5.57 (d, J = 7.6 Hz, 1H), 3.93
– 3.83 (m, 1H), 3.20 (dd, J = 15.1, 4.3 Hz, 1H), 2.79 (d, J = 4.5 Hz, 3H).
4-(4-(5-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide(8i)
Yield:75.6%; ESI-MS[M+H]m/z:452.3; m.p.:138.3-138.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.80
(s, 1H), 8.55 (d, J = 5.5 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 2.5
Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.22 (dd, J = 5.5, 2.6 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 5.54 (dd, J
= 11.6, 4.6 Hz, 1H), 3.89 (dd, J = 15.2, 11.9 Hz, 1H), 3.20 (dd, J = 15.0, 4.5 Hz, 1H), 2.78 (d, J = 4.8
Hz, 3H).
N-methyl-4-(4-(5-(2,3,4-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)picolinamide
(8m)
Yield:73.6%; ESI-MS[M+H]m/z:463.4; m.p.:140.0-142.1oC; 1H NMR (400 MHz, DMSO-d6) δ 8.80
(s, 1H), 8.54 (d, J = 5.1 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.44 (s, 1H), 7.31 (d, J = 7.5 Hz, 2H), 7.19
(t, J = 16.8 Hz, 1H), 6.78 – 6.65 (m, 2H), 5.57 (dd, J = 14.7, 13.9 Hz, 1H), 3.78 (d, J = 13.3 Hz,
11H), 2.77 (t, J = 13.9 Hz, 3H).
4-(4-(5-(2,3-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinamide (8n)
Yield:71.3%; ESI-MS[M+H]m/z:442.1;m.p.:142.5-143.6°C; 1H NMR (400 MHz, CDCl3) δ 9.83 (s,
1H), 8.57 (d, J = 5.9 Hz, 1H), 7.99 (d, J = 13.8 Hz, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.38 (dd, J = 15.6,
7.0 Hz, 2H), 7.22 (d, J = 8.2 Hz, 2H), 7.19 – 7.14 (m, 1H), 6.99 (d, J = 7.6 Hz, 1H), 5.98 (dd, J =
11.9, 4.7 Hz, 1H), 3.95 – 3.81 (m, 1H), 2.49 (d, J = 7.7 Hz, 3H).
5.5 4-(4-formylphenoxy)-N-methylpicolinamide(6)
Benzaldehyde(10 mmol) and potassium tert-butoxide(20 mmol) were dissolved in 30 ml DMF .
Then 4-(4-acetylphenoxy)-N-methylpicolinamide(10 mmol) was added. The mixture was refluxed
for 15h. The reaction mixture was cooled and cold water was added. The precipitate of
15

4-(4-formylphenoxy)-N-methylpicolinamide(6) formed was filtered and recrystallized from
isopropanol. Yiled: 38%.
5.6 General procedure for the preparation of compounds 7a–c
A mixture of substituted acetophenone (0.67 mmol), NaH (1.34 mmol) and THF (10 ml) was
stirred at room temperature for 1h. At the same time 4-(4-formylphenoxy)-N-methylpicolinamide
(0.67 mmol) was dissolved in methanol(10 mL) .One hour later the latter mixture was added slowly
with vigorous stirring in the former one. The mixture was kept for 8 hours. The solid formed was
filtered. No further purification was needed and products were used such as were obtained.
5.7 General procedure for the preparation of compounds 9a–c
Compounds 7a–c (0.24 mmol) and hydrazine hydrate (2.2 mmol) were dissolved in acetic
acid (10mL). The mixture was refluxed for 2 h with constant stirring. The progress of the reaction
was monitored through TLC. After completion of reaction, mixture was cooled and poured into water,
and it was neutralized with potassium carbonate, then it was filtered through vacuum by washing
with water.
N-methyl-4-(4-(3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy) picolinamide (9a)
Yield:73.2%; ESI-MS[M+H]m/z:418.5; m.p.:151.2-152 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.78
(s, 1H), 8.51 (d, J = 5.5 Hz, 1H), 8.31 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.5 Hz, 2H), 7.41 – 7.35 (m,
2H), 7.34 – 7.31 (m, 1H), 7.22 – 7.17 (m, 2H), 7.16 – 7.11 (m, 1H), 5.67 (d, J = 7.1 Hz, 1H), 4.36 (t,
J = 4.8 Hz, 2H), 3.96 (dd, J = 17.7, 12.0 Hz, 1H), 2.78 (d, J = 4.5 Hz, 3H).
4-(4-(3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-methylpicolinamide(9b)
Yield:75.2%; ESI-MS[M+H]m/z:452.5; m.p.:155.8-156.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 8.77
(d, J = 4.5 Hz, 1H), 8.51 (d, J = 5.5 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.39
(s, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.18 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 5.3 Hz, 1H), 5.62 (dd, J = 11.9,
4.4 Hz, 1H), 4.37 (s, 2H), 3.88 (dd, J = 17.9, 12.0 Hz, 1H), 2.78 (d, J = 4.7 Hz, 3H).
4-(4-(3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-methylpicolinamide(9c)
Yield:76.9%; ESI-MS[M+H]m/z:408.5; m.p.:159.3-160.6°C; 1H NMR (400 MHz, DMSO-d6) δ 8.77
(s, 1H), 8.51 (s, 1H), 7.81 (t, J = 9.4 Hz, 3H), 7.54 (d, J = 6.9 Hz, 3H), 7.39 (s, 1H), 7.33 (d, J = 7.5
Hz, 2H), 7.22 – 7.12 (m, 3H), 6.97 (d, J = 8.3 Hz, 1H), 5.62 (s, 1H), 4.36 (s, 2H), 3.94 – 3.79 (m,
1H), 2.78 (s, 3H)
16

5.8 General procedure for the preparation of compounds 10a–e, 11a
A mixture of compounds 5a–e or 7a (0.26 mmol), thiosemicarbazide (0.29 mmol) and
potassium hydroxide (0.31 mmol) in ethanol (6mL) was submitted to microwave irradiation for 15
min at a power of 100 W and a temperature of 60 °C. The progress of the reaction was monitored
through TLC. The reaction mixture was cooled and cold water was added. The compound was
filtered and recrystallized from ethanol.
4-(4-(1-carbamothioyl-5-(3-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolinami
de (10a)
Yield:69.9%; ESI-MS[M+H]m/z:477.5; m.p.:108.5-110.6°C; 1H NMR (400 MHz, DMSO-d6) δ 8.78
(d, J = 4.3 Hz, 1H), 8.54 (d, J = 5.4 Hz, 1H), 8.19 (s, 1H), 8.16 – 8.07 (m, 2H), 8.02 (d, J = 8.8 Hz,
2H), 7.64 (d, J = 6.0 Hz, 2H), 7.43 (s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.22 (d, J = 5.6 Hz, 1H), 6.10 (d,
J = 10.6 Hz, 1H), 4.00 (dd, J = 15.4, 11.8 Hz, 1H), 3.28 (s, 1H), 2.78 (d, J = 4.6 Hz, 3H).
4-(4-(1-carbamothioyl-5-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolina
mide (10b)
Yield:65.9%; ESI-MS[M+H]m/z:450.7; m.p.:102.4-103.9°C; 1H NMR (400 MHz, DMSO-d6) δ 8.80
(d, J = 4.8 Hz, 1H), 8.54 (d, J = 5.6 Hz, 1H), 8.07 (s, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.97 (s, 1H), 7.44
(t, J = 7.3 Hz, 1H), 7.32 (d, J = 8.7 Hz, 2H), 7.22 (dd, J = 5.7, 2.7 Hz, 1H), 7.20 – 7.16 (m, 2H), 7.13
(d, J = 8.9 Hz, 1H), 5.93 (dd, J = 11.4, 3.3 Hz, 1H), 4.12 (dd, J = 10.6, 5.3 Hz, 1H), 3.93 (dd, J =
15.3, 11.7 Hz, 1H), 3.22 (d, J = 3.6 Hz, 1H), 2.78 (d, J = 4.8 Hz, 2H).
4-(4-(1-carbamothioyl-5-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolina
mide(10c)
Yield:63.6%; ESI-MS[M+H]m/z:466.3; m.p.:105.1-106.4°C; 1H NMR (400 MHz, DMSO-d6) δ 8.77
(d, J = 4.6 Hz, 1H), 8.51 (d, J = 5.5 Hz, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.94 (t, J = 8.3 Hz, 2H), 7.53
(d, J = 8.2 Hz, 2H), 7.40 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 7.16 – 7.10 (m,
1H), 5.98 (d, J = 11.5 Hz, 1H), 3.93 (dd, J = 15.0, 11.7 Hz, 1H), 3.27 (s, 1H), 2.77 (d, J = 4.7 Hz,
3H).
4-(4-(1-carbamothioyl-5-(2,3-dichlorophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicol
inamide(10d)
Yield:70.3%; ESI-MS[M+H]m/z:500.4; m.p.:107.5-108.8°C; 1H NMR (400 MHz, DMSO-d6) δ 8.79
(d, J = 4.5 Hz, 1H), 8.52 (t, J = 12.6 Hz, 1H), 8.23 (s, 1H), 8.09 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H),
7.55 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 18.4 Hz, 1H), 7.34 (d, J = 6.0 Hz, 1H), 7.32 (s, 1H), 7.30 (s,
1H), 7.26 – 7.15 (m, 1H), 6.93 (s, 1H), 6.18 (d, J = 7.8 Hz, 1H), 4.04 (dd, J = 15.1, 12.1 Hz, 1H),
3.20 (d, J = 14.0 Hz, 1H), 2.78 (d, J = 4.5 Hz, 3H).
17

4-(4-(1-carbamothioyl-5-(4-cyanophenyl)-4,5-dihydro-1H-pyrazol-3-yl)phenoxy)-N-methylpicolina
mide(10e)
Yield:71.2%; ESI-MS[M+H]m/z:457.2; m.p.:98.7-100.2°C; 1H NMR (400 MHz, DMSO-d6) δ 8.79
(d, J = 4.6 Hz, 1H), 8.54 (d, J = 5.6 Hz, 1H), 8.16 (s, 1H), 8.01 (d, J = 8.6 Hz, 2H), 7.80 (d, J = 8.2
Hz, 2H), 7.43 (t, J = 8.7 Hz, 1H), 7.35 (s, 1H), 7.34 – 7.29 (m, 2H), 7.24 – 7.17 (m, 1H), 6.07 – 5.93
(m, 1H), 4.12 (d, J = 5.2 Hz, 1H), 3.97 (dd, J = 15.1, 11.8 Hz, 1H), 3.27 – 3.19 (m, 1H), 2.78 (d, J =
4.8 Hz, 3H).
4-(4-(1-carbamothioyl-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-methylpicolinami
de(11a)
Yield:65.8%; ESI-MS[M+H]m/z:447.2; m.p.:118.4-120.1°C; 1H NMR (400 MHz, DMSO-d6) δ 8.78
(s, 1H), 8.64 – 8.47 (m, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.16 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 9.5 Hz,
1H), 7.95 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 8.9 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 7.5 Hz,
1H), 7.13 (d, J = 3.0 Hz, 1H), 6.03 (d, J = 11.5 Hz, 1H), 4.20 – 4.08 (m, 1H), 3.99 (dd, J = 15.0, 11.4
Hz, 1H), 2.77 (d, J = 5.0 Hz, 3H).
5. 9 Cytotoxicity assay in vitro
The cytotoxic activities of compounds were evaluated byA549, PC-3, HepG2 and MCF-7 cell
lines by the standard MTT assay in vitro, with VEGFR2/KDR inhibitors sorafenib as positive control.
The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10%
fetal bovine serum (FBS). Approximately 4×103 cells, suspended in MEM medium, were plated onto
each well of a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The test compounds at
indicated final concentrations were added into the culture medium and the cell cultures were
continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5µg/mL and
incubated with cells at 37°C for 4 h. The formazan crystals were dissolved in 100 µL DMSO each
well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the
reference wavelength) was measured by the ELISA reader. The initial concentration of the compound
was 100 ug/mL , which was three times dilution and diluted six times. That is to say, we tested seven
concentrations of each compound. And all the compounds were tested three times in each of the cell
lines. The results expressed as inhibition rates or IC50 (half-maximal inhibitory concentration) were
the averages of two determinations and calculated by using the Bacus Laboratories Incorporated
Slide Scanner (Bliss) software.
5.10 Tyrosine kinases assay in vitro
18

The selected compounds (8b, 8h, 8i and 9a) are tested for their activity against one or several
tyrosine kinases (VEGFR-2/KDR, BRAF, CRAF, c-Met, EGFR and Flt-3 kinases) through the
mobility shift assay together with reference compounds Sorafenib and staurosporine. All kinase
assays were performed on 384-well plates in a 50 µL reaction volume. The kinase base buffer
contains 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer
contains 100mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50 mM EDTA. The
compounds were diluted to 500 µM by 100% DMSO, then 10 µL of compound was transferred to a
new 96-well plate as the intermediate plate, and 90 µL kinase buffer was added to each well. 5 µL of
each well of the intermediate plate was transferred to 384-well plates. The following amounts of
enzyme and substrate were used per well: kinase base buffer, FAM-labeled peptide, ATP and enzyme
solution. Wells containing the substrate, enzyme, DMSO without compound were used as DMSO
control. Wells containing substrate without enzyme were used as low control and then incubated at
room temperature for 10 min. 10 µL peptide solution was added to each well. Incubated at 28℃ for
specified period of time and stopped reaction by 25 µL stop buffer. At last, collected data on Caliper
program and converted conversion values to inhibition values. Percent inhibition =
(max-conversion)/ (max-min)*100. “max” denotes DMSO control; “min” denotes low control. It was
tested by Shanghai ChemPartners.
5.11 Cell apoptosis assay by flow cytometry
6
A549 cells were seeded in 16-well plates at a density of 1 × 10 cells/well in RPMI 1640 medium
and treated with 3, 6 and 12 µM concentration of 8b for 48 h. Cultured cells were stained with
Annexin VFITC and propidium iodide (PI) in the dark at 4 °C for 30 min and analyzed by FACS
Calibur flow cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software[23].
5.12 Docking studies
For docking purposes, the three-dimensional structure of the VEGFR2/KDR (PDB code: 4ASD)
and c-Met (PDB code: 3LQ8) were obtained from RCSB Protein Data Bank. Autodock Vina 1.12,
Discover Studio 3.5 visualizer and Open Babel were used for the docking study. Firstly, using the
Discover Studio 3.5 visualizer to prepare ligands (8b) and acceptor protein (PDB code: 4ASD and
3LQ8), then saved as pdb format after energy minimization for ligand and clean protein, defined the
activity site for acceptor protein. Secondly, the pdb file of ligand and acceptor protein were
converted to the pdbqt format by the Open Babel tool. Thirdly, docking was carried out by uploading
19

the pdbqt file to the Autodock vina 1.1.2, and the result was named out.pdbq. Moreover, out.pdbpt
file was uploaded to the Discover Studio 3.5 visualizer for analyzing the result.
Acknowledgments
We gratefully acknowledge the generous support provided by The National Natural Science
Funds，China (No. 21662014), Outstanding Youth Foundation of Jiangxi, Natural Science
Foundation of Jiangxi, China (20171BCB23078), Natural Science Foundation of Jiangxi, China
(20171ACB21052 & 20171BAB215073)，Science and Technology Project Founded by the
Education Department of Jiangxi Province (GJJ160787)
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Research Highlights
► Four series of Sorafenib derivatives bearing pyrazole scaffold (8a–m, 9a–c, 10a–e and 11a) were
synthesized and characterized.
► Most of the synthesized compounds showed moderate to significant antitumor activity.
► 8b showed excellent cytotoxicity activity and selectivity with the IC50 values were equivalent to
Sorafenib.
► Docking study was investigated to explore the binding modes of compounds with VEGFR and
c-Met.
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