Design, synthesis, activity and docking study of sorafenib analogs bearing sulfonylurea unit

Article abstract of DOI:10.3390/molecules201019361

Two series of novel sorafenib analogs containing a sulfonylurea unit were synthesized and their chemical structures were confirmed by 1H-NMR, 13C-NMR, MS spectrum and elemental analysis. The synthesized compounds were evaluated for the cytotoxicity against A549, Hela, MCF-7, and PC-3 cancer cell lines. Some of the compounds showed moderate cytotoxic activity, especially compounds 1-(2,4-difluorophenylsulfonyl)-3-(4-(2- (methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6c) and 1-(4-bromophenylsulfonyl)-3-(4- (2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6f) with the IC50 values against four cancer cell lines ranging from 16.54 ± 1.22 to 63.92 ± 1.81 ?M, respectively. Inhibitory rates against vascular endothelial growth factor receptor-2 (VEGFR2/KDR) kinase at 10 ?M of target compounds were further carried out in this paper in order to investigate the target of these compounds. Structure-activity relationships (SARs) and docking studies indicated that the sulfonylurea unit was important to these kinds of compounds. None of the substitutions in the phenoxy group and small halogen atoms such as 2,4-difluoro substitution of the aryl group contributed to the activity. The results suggested that sulfonylurea sorafenib analogs are worthy of further study.

Full text of DOI:10.3390/molecules201019361

Molecules 2015, 20, 19361-19371; doi:10.3390/molecules201019361
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis, Activity and Docking Study of Sorafenib
Analogs Bearing Sulfonylurea Unit
Chunjiang Wu 1, Min Wang 1, Qidong Tang 1, Rong Luo 2, Le Chen 1, Pengwu Zheng 1
and Wufu Zhu 1,*
1
School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China;
E-Mails: wuchunjiang999@126.com (C.W.); wangmin6029@126.com (M.W.);
tangqidongcn@126.com (Q.T.); chenle182@163.com (L.C.); Zhengpw@126.com (P.Z.)
2
Jiangxi Province Institute of Materia Medica, Nanchang 330000, China; E-Mail: 6350173@163.com
* Author to whom correspondence should be addressed; E-Mail: zhuwf@jxstnu.edu.cn;
Tel./Fax: +86-0791-8380-2393.
Academic Editor: Jean Jacques Vanden Eynde
Received: 10 August 2015 / Accepted: 21 September 2015 / Published: 23 October 2015
Abstract: Two series of novel sorafenib analogs containing a sulfonylurea unit were
synthesized and their chemical structures were confirmed by 1H-NMR, 13C-NMR, MS spectrum
and elemental analysis. The synthesized compounds were evaluated for the cytotoxicity
against A549, Hela, MCF-7, and PC-3 cancer cell lines. Some of the compounds showed
moderate cytotoxic activity, especially compounds 1-(2,4-difluorophenylsulfonyl)-3-(4-(2-
(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6c) and 1-(4-bromophenylsulfonyl)-3-(4-
(2-(methylcarbamoyl)pyridin-4-yloxy)phenyl)urea (6f) with the IC50 values against four
cancer cell lines ranging from 16.54 ± 1.22 to 63.92 ± 1.81 μM, respectively. Inhibitory rates
against vascular endothelial growth factor receptor-2 (VEGFR2/KDR) kinase at 10 μM of
target compounds were further carried out in this paper in order to investigate the target of
these compounds. Structure-activity relationships (SARs) and docking studies indicated that
the sulfonylurea unit was important to these kinds of compounds. None of the substitutions
in the phenoxy group and small halogen atoms such as 2,4-difluoro substitution of the aryl
group contributed to the activity. The results suggested that sulfonylurea sorafenib analogs
are worthy of further study.
Keywords: sorafenib; sulfonylurea; VEGFR2/KDR kinase inhibitors; anticancer activity

Molecules 2015, 20
19362
1. Introduction
Cancer has been the second biggest killer of human beings, taking the lives of over 7 million people
a year [1]. Although a range of antitumor drugs have been discovered in the last decade [2–5], drug
resistance and adverse side effects are still serious problems. Therefore, it remains desirable to develop
new antitumor agents with improved tumor selectivity, efficiency, and safety.
In recent years, a number of new diarylurea derivatives as VEGFR signal pathway inhibitors have
been reported (Figure 1). The main modifications of these compounds were focused on the pyridine ring
or phenoxy group. In our previous study, we introduced a sulfonylurea unit instead of urea scaffolds, and
the resulting derivatives showed moderate activity as VEGFR2/KDR inhibitors [6].
Figure 1. Structures of small-molecule vascular endothelial growth factor (VEGFR2)
inhibitors based on the diarylurea scaffold and the target compounds 6a–f and 9a–e.
In order to find compounds with excellent in vitro/in vivo anti-tumor activity as well as improved
pharmacokinetic, further studies on analogous of sulfonylurea-based sorafenib analogs were carried
out in this research. Firstly, different substitutions were introduced to the aryl group to investigate the
substituents to the activity. Moreover, a fluorine atom was introduced to the phenoxy group, inspired by
Regorafenib, KI 8751 (Figure 1) and 6,7-disubstituted-4-phenoxyquinoline derivatives as c-Met kinase
inhibitors reported in our previous research [7,8]. The design strategy and structures of the target
compounds 6a–f and 9a–e was shown in Figure 1.
Herein, we report the newly synthesized target compounds and their biological activities against
four cancer cell lines A549, Hela, MCF-7, PC-3, and VEGFR2/KDR kinase.
2. Results and Discussion
The preparation of target compounds 6a–f and 9a–e is described in Scheme 1.
The synthesis of the key intermediate of 4-(4-aminophenoxy)-N-methylpicolinamide 4 was achieved
from 4-chloro-N-methylpicolinamide 3 as shown in Scheme 1, which has been illustrated in detail in
our previous study [6]. Etherification of 3 with 2-fluoro-4-nitrophenol afforded purified 7, which were
reduced using hydrazine hydrate and catalytic amounts of ferric chloride in ethanol to obtain amide 8.
Other key intermediate compounds 5a–h were prepared from commercially available substituted benzene
or thiophene via sulfonylation, ammonolysis and acylation reactions according to our previous method [9].

Molecules 2015, 20
19363
Condensation of 4 or 8 and 6a–f or 9a–e in toluene furnished the target compounds 6a–f and 9a–e in
good yield.
NH2
H
H
N
O
S
O
S
O
N
Ar
Ar
N
O
O
N
O
O
O
5a-h
H
O
N
a
H
b
N
H
N
Cl
O
4
6a-f
O
H
N
N
3
H
H
N
O
S
O
F
N
Ar
F
NO2
F
NH2
O
O
d
c
O
N
5a-h
b
O
O
N
H
H
H
N
N
N
N
O
O
O
9a-e
8
7
Scheme 1. Synthetic route of target compounds 6a–f and 9a–e. Reagents and conditions: (a)
t-BuOK, NaI, N,N-Dimethylformamide (DMF); (b) Toluene, reflux, 6 h; (c) Chlorobenzene,
reflux, 30 h; (d) EtOH, FeCl3·6H2O, N2H4·H2O, reflux, 8 h.
2.1. Biological Evaluation
Taking sorafenib as reference compound, the target compounds (6a–f and 9a–e) were evaluated for
the cytotoxicity against four cancer cell lines A549 (human lung cancer), Hela (human cervical cancer),
MCF-7 (human breast cancer), and PC-3 (human prostate cancer) by 3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide (MTT) cell proliferation assay. In addition, these compounds were
evaluated for the inhibitory activity against VEGFR2/KDR at 10 μM level in vitro by the mobility
shift assay together with reference compounds sorafenib and Staurosporine. The results expressed as
inhibition rates or IC50 values were summarized in Table 1 and the values are the average of at least
two independent experiments.
From Table 1, we could see that six target compounds showed moderate cytotoxicity against A549,
Hela, MCF-7, and PC-3 cancer cell lines. Among them, compounds 6c and 6f showed better activity with
the IC50 values against four cancer cell lines ranging from 16.54 μM to 63.92 μM, respectively. Moreover,
compound 6f showed good activity against all four cancer cell lines. Generally, the first series compounds
(6a–f) had more potent activities to cancer cells than the second series compounds (9a–e) which indicated
that the change in the substitution of the phenoxy group from H to F decrease the cytotoxicity of the
sulfonylurea derivatives. Different substitutions of the aryl group affected the cytotoxicity of target
compounds. Small halogen atoms (2,4-di F (6c) or 4-Br (6f)) substitutions of the aryl group contributed to
the activity of the first series, while there is no significant regularity of the second series.

Molecules 2015, 20
19364
Table 1. Structures and activity of target compounds 6a–f and 9a–e.
VEGFR2/KDR
Inhibitory
IC50(μM) a
Compounds No.
Ar
A549
>100
Hela
MCF-7
>100
PC-3
Rate@10μM (%)
6a
6b
6c
23.6% ± 12.9%
54.0% ± 2.7%
75.8% ± 5.5%
>100
>100
>100
>100
>100
65.86 ± 2.01
27.04 ± 1.43
72.43 ± 1.96
>100
25.35 ± 1.73
O
6d
6e
6f
61.3% ± 9.6%
31.4% ± 7.2%
46.6% ± 1.8%
86.91 ± 2.03
>100
>100
>100
80.56 ± 2.04
>100
>100
68.87 ± 2.14
Br
32.59 ± 1.51 63.92 ± 1.81 16.54 ± 1.22 17.97 ± 1.56
9a
9b
9c
<20.0%
<20.0%
<20.0%
>100
>100
42.43 ± 1.93 17.19 ± 1.54
ND c
ND
>100
>100
>100
>100
57.42 ± 1.89
ND
9d
9e
<20.0%
<20.0%
33.22 ± 1.82 24.65 ± 1.69
>100
ND
ND
Cl
>100
44.32 ± 1.75 69.25 ± 1.96
Sorafenib b
Staurosporine b
-
-
94.9% ± 1.1%
97.3% ± 2.82%
6.53 ± 0.82
ND
8.08 ± 0.91
ND
4.21 ± 0.62 11.05 ± 1.07
ND ND
a The values are an average of two separate determinations; b Used as a positive controls; c ND: Not determined.
Inhibitory rates against VEGFR2/KDR kinase at 10 μM of target compounds were further carried
out in this paper. Compounds 6b–d, 6f exhibit moderate inhibitory rates against VEGFR2/KDR kinase
at 10 μM. Compound 6c showed the best results, with the inhibitory rate of 75.8% ± 5.5%. It is also
suggested that small halogen atoms benefit the activity of target compounds.
Although all of the target compounds showed less activity than the positive compounds sorafenib
and staurosporine, it still showed that the replacement of urea with sulfonylurea unit is important to the
activity of this series of compounds. More compounds of sorafenib analogs bearing a sulfonylurea may
be screened by replacing the aryl groups with heterocyclic rings in further study.

Molecules 2015, 20
19365
2.2. Molecular Docking Study
To explore the binding modes of target compounds with VEGFR2, molecular docking simulation studies
were carried out by using SURFLEX-DOCK module of SYBYL package version. The co-crystal structure of
sorafenib with VEGFR2/KDR kinase was selected as the docking model (PDB ID code: 4ASD [10]). Based
on the in vitro inhibition results, we selected the best VEGFR2 inhibitor 6c as a ligand example.
The binding modes of compound 6c and lead compound were shown in Figure 2 and the docking
score of compound 6c and lead compound were 9.148 and 10.447. As depicted in Figure 2, compound 6c
and Sorafenib can nearly overlap in the binding model and amide group and urea group formed four
hydrogen bonds with residues CYS919 and ASP1046, respectively. The H-bond distances are 1.66 Å,
1.71 Å, 1.92 Å and 2.01 Å, respectively. The results showed that the four hydrogen bonds can be combined
with VEGFR protein residues. Analysis of compound 6c’s binding mode in the active binding site
demonstrated that the docking mode of the 6c is similar to the lead compound sorafenib with the same
H-bond between amide group and residues CYS919. The four hydrogen bonds play an important role
in increasing the inhibitory potency of sulfonylurea derivatives against VEGFR2/KDR kinase according
to the docking results and the activity. However, from the docking score of compound 6c and lead
compound, we could see why the activity of compound 6c was lower than lead compound. Furthermore,
the docking results also give us a new direction to design new VEGFR2/KDR inhibitors that can interact
with CYS919 and ASP1046. The above-mentioned results of SAR analysis and molecular docking study
may allow the rational design of more potent VEGFR2/KDR inhibitors.
Figure 2. Binding models of compound 6c ((a) shown in Capped Sticks) and parent
compound Sorafenib ((b) shown in Ball and Stick) target into the active site of VEGFR2.
Hydrogen bonds were showed in dashed lines (yellow).

Molecules 2015, 20
19366
3. Experimental Section
3.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik,
Flawil, Switzerland) and were uncorrected. NMR spectra were performed using Bruker 400 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Mass spectra
(MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). All the materials
were obtained from commercial suppliers and used without purification, unless otherwise specified.
Yields were not optimized. TLC analysis was carried out on silica gel plates GF254 (Qindao Haiyang
Chemical, Qingdao, China). Elemental analysis was determined on a Carlo-Erba1106 Elemental analysis
instrument (Carlo Erba, Milan, Italy).
General Procedure for Preparation of Compounds 5a–h.
Compounds 5a–h were synthesized according to the reported procedures [9,11,12].
4-(4-Aminophenoxy)-N-methylpicolinamide (4)
The synthesis of the key intermediates of 4-(4-aminophenoxy)-N-methylpicolinamide 4 has been
illustrated in detail in our previous study [6].
4-(2-Fluoro-4-nitrophenoxy)-N-methylpicolinamide (7)
A stirring mixture of an appropriate 4-chloro-N-methylpicolinamide (0.12 mol) and 2-fluoro-4-
nitrophenol (0.18 mol) in chlorobenzene (200 mL, 5 v/w) was refluxed for about 30 h. The resulting
mixture was cooled, and filtered. The filter cake was washed with saturated K2CO3 solution, and
recrystallized with ethanol to give the corresponding nitro compound 7.
4-(4-Amino-2-fluorophenoxy)-N-methylpicolinamide (8)
To a refluxing solution of an appropriate nitro compound (0.1 mol), FeCl3·6H2O and the activated
carbon in EtOH (200 mL, 10 v/w) was added hydrazine hydrate in batches. The mixture was kept at
this temperature for more 8 h. After completion of the reaction as indicated by TLC, the mixture was
filtered immediately, and the filtrate was cooled, filtered to obtain the corresponding aniline 8.
General Procedure for Preparation of Compounds 6a–f and 9a–e.
4-(4-Aminophenoxy)-N-methyl-pyridine carboxamide (0.41 mmol) or 4-(4-amino-2-fluorophenoxy)-N-
methylpicolinamide and compounds 5a–h (0.41 mmol) was added to the toluene (5 mL). Refluxed for
about 6 h to precipitate a brown solid, filtration and drying gave the title compounds 6a–f and 9a–e.
1
Compound 6a. Yield: 40.0%. ESI-MS [M + H]+ m/z: 443.1; m.p.: 187–190 °C; H-NMR (400 MHz,
DMSO) δ 9.09 (s, 1H), 8.77 (s, 1H), 8.49 (d, J = 5.0 Hz, 1H), 8.09–8.00 (m, 2H), 7.47 (d, J = 8.9 Hz,
13
3H), 7.34 (s, 1H), 7.15 (s, 1H), 7.13 (s, 1H), 7.12–7.08 (m, 1H), 2.77 (d, J = 4.1 Hz, 3H). C-NMR
(100 MHz, DMSO) δ 166.2(C), 164.3(C), 163.9(C), 152.9(C), 150.8(C), 150.0(C), 149.12(CH), 136.8(C),
136.2(C), 131.2(CH), 131.1(CH), 121.8(2CH), 121.5(2CH), 116.5(CH), 116.0(CH), 114.4(CH),
109.3(CH), 26.42(CH3). Anal. calcd. for C20H17FN4O5S (%): C, 54.05; H, 3.86; N, 12.61. Found (%):
C, 54.01; H, 3.83; N, 12.57.
1
Compound 6b. Yield: 45.4%. ESI-MS [M + H]+ m/z: 431.1; m.p.: 117–121 °C; H-NMR (400 MHz,
DMSO) δ 9.07 (s, 1H), 8.79 (d, J = 4.3 Hz, 1H), 8.49 (d, J = 5.6 Hz, 1H), 8.02 (dd, J = 5.0, 1.3 Hz,
1H), 7.80 (dd, J = 3.7, 1.3 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.35 (d, J = 2.4 Hz, 1H), 7.24–7.19 (m,

Molecules 2015, 20
19367
1H), 7.17 (s, 1H), 7.15 (s, 1H), 7.12 (dd, J = 5.6, 2.6 Hz, 1H), 2.77 (d, J = 4.8 Hz, 3H). Anal. calcd. for
C18H16N4O5S2 (%): C, 49.99; H, 3.73; N, 12.95. Found (%): C, 49.96; H, 3.71; N, 12.93.
1
Compound 6c. Yield: 33.6%. ESI-MS [M + H]+ m/z: 461.1; m.p.: 236–237 °C; H-NMR (400 MHz,
DMSO) δ 8.90 (s, 1H), 8.80 (d, J = 4.6 Hz, 1H), 8.51 (d, J = 5.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.38
(d, J = 2.2 Hz, 1H), 7.19 (s, 1H), 7.17 (s, 1H), 7.15 (d, J = 2.5 Hz, 1H), 2.79 (d, J = 4.7 Hz, 3H). Anal.
calcd. for C20H16F2N4O5S (%): C, 51.95; H, 3.49; N, 12.12. Found (%): C, 51.92; H, 3.46; N, 12.08.
1
Compound 6d. Yield: 41.7%. ESI-MS [M + H]+ m/z: 455.2; m.p.: 100–104 °C; H-NMR (400 MHz,
DMSO) δ 8.97 (s, 1H), 8.78 (s, 1H), 8.49 (d, J = 5.6 Hz, 1H), 7.90 (d, J = 8.9 Hz, 2H), 7.46 (d, J = 8.9 Hz,
2H), 7.34 (d, J = 2.2 Hz, 1H), 7.15 (s, 2H), 7.13 (s, 2H), 7.10 (d, J = 2.6 Hz, 1H), 3.85 (s, 3H), 2.78 (d,
J = 4.7 Hz, 3H). Anal. calcd. for C21H20N4O6S (%): C, 55.26; H, 4.42; N, 12.27. Found (%): C, 55.21;
H, 4.39; N,12.23.
Compound 6e. Yield: 32.8%. ESI-MS [M + H]+ m/z: 481.2; m.p.: 65–69 °C; 1H-NMR (400 MHz, DMSO)
δ 9.08 (s, 1H), 8.80 (d, J = 4.5 Hz, 1H), 8.49 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.76 (d,
J = 8.3 Hz, 3H), 7.48 (d, J = 8.8 Hz, 2H), 7.36 (s, 1H), 7.16 (s, 1H), 7.14 (s, 1H), 7.12 (d, J = 5.7 Hz, 1H),
2.78 (d, J = 4.7 Hz, 3H), 1.30 (s, 9H). Anal. calcd. for C24H26N4O5S (%): C, 59.74; H, 5.43; N, 11.61.
Found (%): C, 59.71; H, 5.39; N, 11.58.
1
Compound 6f. Yield: 39.2%. ESI-MS [M + H]+ m/z: 505.0; m.p.: 119–122 °C; H-NMR (400 MHz,
DMSO) δ 9.12 (s, 1H), 8.76 (s, 1H), 8.48 (s, 1H), 7.87–7.77 (m, 4H), 7.47 (s, 2H), 7.33 (s, 1H), 7.13
(s, 3H), 2.76 (s, 3H). Anal. calcd. for C20H17BrN4O5S (%): C, 47.53; H, 3.39; N, 11.09. Found (%): C,
47.51; H, 3.36; N, 11.05.
Compound 9a. Yield: 42.4%. ESI-MS [M + H]+ m/z: 463.1; m.p.: 186.5–187.7 °C; 1H-NMR (400 MHz,
DMSO) δ 8.68 (s, 1H), 8.45–8.38 (m, 1H), 7.96 (s, 1H), 7.79 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 13.1 Hz,
1H), 7.39 (t, J = 8.7 Hz, 1H), 7.33 (d, J = 9.9 Hz, 2H), 7.06 (d, J = 8.8 Hz, 1H), 6.94 (t, J = 8.9 Hz,
1H), 6.45 (d, J = 13.1 Hz, 1H), 6.37 (d, J = 8.3 Hz, 1H), 2.70 (d, J = 3.3 Hz, 3H). Anal. calcd. for
C20H16F2N4O5S (%): C, 51.95; H, 3.49; N, 12.12. Found (%): C, 51.92; H, 3.44; N, 12.09.
Compound 9b. Yield: 43.7%. ESI-MS [M + H]+ m/z: 451.2; m.p.: 182.5–183.6 °C; 1H-NMR (400 MHz,
DMSO) δ 8.68 (s, 1H), 8.46–8.36 (m, 1H), 7.74 (s, 1H), 7.56 (s, 1H), 7.47 (s, 1H), 7.27 (s, 1H), 7.21–7.09
(m, 1H), 7.05 (s, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.46 (d, J = 13.2 Hz, 1H), 6.37 (d, J = 7.9 Hz, 1H),
2.70 (s, 3H). 13C-NMR (100 MHz, DMSO) δ 166.6(C), 165.7(C), 164.1(C), 155.1(C), 150.9(C),
140.7(CH), 137.8(C), 135.4(C), 134.6(CH), 134.3(CH), 130.5(C), 127.7(CH), 124.4(CH), 116.5(CH),
113.6(CH), 110.8(CH), 108.6(CH), 26.4(CH3). Anal. calcd. for C18H15FN4O5S2 (%): C, 47.99; H, 3.36;
N, 12.44. Found (%): C, 47.95; H, 3.31; N, 12.40.
Compound 9c. Yield: 31.5%. ESI-MS [M + H]+ m/z: 481.2; m.p.: 126.7–128.3 °C; 1H-NMR (400 MHz,
DMSO) δ 8.76 (s, 1H), 8.53–8.46 (m, 1H), 7.86 (dd, J = 14.9, 8.5 Hz, 1H), 7.71 (s, 1H), 7.52 (dd,
J = 20.7, 9.9 Hz, 1H), 7.34 (d, J = 2.3 Hz, 1H), 7.25 (s, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.14–7.10 (m,
1H), 7.02 (t, J = 9.0 Hz, 1H), 6.54 (d, J = 13.2 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 2.78 (d, J = 3.8 Hz,

Molecules 2015, 20
19368
3H). Anal. calcd. for C20H15F3N4O5S (%): C, 50.00; H, 3.15; N, 11.66. Found (%): C, 49.96; H, 3.12;
N, 11.63.
1
Compound 9d. Yield: 45.3%. ESI-MS [M + H]+ m/z: 459.2; m.p.:93.5–95.6 °C; H-NMR (400 MHz,
DMSO) δ 8.68 (s, 1H), 8.40 (d, J = 5.3 Hz, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H),
7.35 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.1 Hz, 3H), 7.18 (s, 1H), 7.05 (s, 1H), 6.94 (t, J = 9.0 Hz, 1H),
13
6.45 (d, J = 13.0 Hz, 1H), 6.36 (d, J = 8.1 Hz, 1H), 2.70 (s, 3H), 2.41 (s, 3H). C-NMR (100 MHz,
DMSO) δ 166.6(C), 165.7(C), 164.1(C), 153.1(C), 150.9(C), 144.4(CH), 142.4(C), 137.9(C), 137.8(C),
137.5(C), 129.9(CH), 129.1(CH), 127.9(CH), 126.1(CH), 124.4(CH), 116.4(CH), 113.7(CH),
110.7(CH), 108.5(CH), 26.4(CH3), 21.5(CH3). Anal. calcd. for C21H19FN4O5S (%): C, 55.02; H, 4.18;
N, 12.22. Found (%): C, 54.98; H, 4.14; N, 12.18.
Compound 9e. Yield: 44.6%. ESI-MS [M + H]+ m/z: 479.2; m.p.: 118.5–120.2 °C; 1H-NMR (400 MHz,
DMSO) δ 8.68 (s, 1H), 8.40 (d, J = 5.4 Hz, 1H), 7.78 (dd, J = 15.1, 8.7 Hz, 1H), 7.63 (s, 1H), 7.47 (d,
J = 12.1 Hz, 1H), 7.41 (d, J = 10.2 Hz, 1H), 7.27 (s, 1H), 7.08 (d, J = 7.4 Hz, 2H), 7.05 (d, J = 5.6 Hz,
1H), 6.94 (t, J = 9.0 Hz, 1H), 6.46 (d, J = 13.2 Hz, 1H), 6.37 (d, J = 8.6 Hz, 1H), 2.70 (d, J = 3.5 Hz,
13
3H). C-NMR (100 MHz, DMSO) δ 166.6(C), 165.7(C), 164.1(C), 155.1(C), 150.9(C), 149.9(CH),
137.7(C), 137.6(C), 135.5(C), 133.7(C), 133.6(C), 129.3(CH), 128.6(CH), 125.7(CH), 124.3(CH),
116.6(CH), 113.6(CH), 110.9(CH), 108.5(CH), 26.4(CH3). Anal. calcd. for C20H16ClFN4O5S (%): C,
50.16; H, 3.37; N, 11.70. Found (%): C, 50.12; H, 3.34; N, 11.66.
3.2. VEGFR2/KDR Kinase Assay
The inhibitory activity against VEGFR2/KDR at 10 μM level in vitro was evaluated through the
mobility shift assay together with reference compounds sorafenib and Staurosporine[13]. All kinase
assays were performed in 96-well plates in a 50 μL reaction volume. The kinase buffer contains 50 mM
HEPES, pH 7.5, 10 mM MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer contains 100 mM
HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3 and 50 mM EDTA. Dilute the compounds to
500 μM by 100% DMSO, then transfer 10 μL of compound to a new 96-well plate as the intermediate
plate, add 90 μL kinase buffer to each well. Transfer 5 μL of each well of the intermediate plate 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 just the substrate without enzyme were used as low
control. Incubate at room temperature for 10 min. Add 10 μL peptide solution to each well. Incubate at
28 °C for specified period of time and stop reaction by 25 μL stop buffer. At last collect data on Caliper
program and convert conversion values to inhibition values. Percent inhibition = (max − conversion)/
(max − min) × 100. “max” stands for DMSO control; “min” stands for low control.
3.3. Cytotoxicity Assay in Vitro
The cytotoxic activities of compounds were evaluated with A549, Hela, MCF-7 and PC-3 cell lines
by the standard MTT assay in vitro, with compounds VEGFR inhibitor Sorafenib as positive control.
The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal

Molecules 2015, 20
19369
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 to the culture medium and the cell cultures were continued for 72 h. Fresh
MTT was added to each well at a terminal concentration of 5 mg/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 with the
ELISA reader. All of 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 (the Bacus Laboratories Inc. Slide Scanner (BLISS) system, Lombard, IL, USA).
3.4. Docking Studies
To further elucidate the binding mode of compounds, docking analysis was performed. The
three-dimensional structure of the VEGFR2 (PDB code: 4ASD) were obtained from RCSB Protein
Data Bank [14]. Hydrogen atoms were added to the structure allowing for appropriate ionization at
physiological pH. The protonated state of several important residues, such as CYS919, and ASP1046
were adjusted by using SYBYL6.9.1 (Tripos, St. Louis, MO, USA) in favor of forming reasonable
hydrogen bond with the ligand. Molecular docking analysis was carried out by the SURFLEX-DOCK
module of SYBYL 6.9.1 package (Tripos) to explore the binding model for the active site of VEGFR2
with its ligand. All atoms located within the range of 5.0 Å from any atom of the cofactor were selected
into the active site, and the corresponding amino acid residue was, therefore, involved into the active site
if only one of its atoms was selected. Other default parameters were adopted in the SURFLEX-DOCK
calculations. All calculations were performed on Silicon Graphics workstation (package version 6.9.1 on
silicon graphics origin300 workstation with IRIX 6.5 operating system, San Francisco, CA, USA).
4. Conclusions
In summary, two series of sorafenib derivatives bearing sulfonylurea scaffold were designed and
synthesized. All of the target compounds were evaluated the activity against four cancer cell lines and
VEGFR2/KDR kinase. Six of the target compounds showed moderate activity and compounds 6c and
6f were better. The first series with no substitution in the phenoxy group showed more activity than the
second series. Different substitutions of the aryl group affected the cytotoxicity of target compounds.
Small halogen atom substitutions of the aryl group contributed to the activity of the first series, while
there is no significant regularity of the second series. Although all of the target compounds showed
less activity than the positive compounds, structure-activity relationships (SARs) and docking studies
indicated that sulfonylurea unit is important to the activity of this kind of compounds. The results
suggested that the sulfonylurea sorafenib analogs are worthy of further study. More compounds of
sorafenib analogs bearing a sulfonylurea may be screened by replacing the aryl groups by heterocyclic
rings in our further study.

Molecules 2015, 20
19370
Acknowledgments
We gratefully acknowledge the generous support provided by the National Natural Science Foundation
of China (No. 81460527), Project supported by the Natural Science Foundation of Jiangxi Province
(No. 20142BAB215020) , Doctoral Scientific Research Foundation of Jiangxi Science & Technology
Normal University and Program of Key Laboratory of Drug Design and Optimization, Jiangxi Science
& Technology Normal University (300098010306) and College Students’ Science and Technology
Innovation Project of Jiangxi Province (201411318033).
Author Contributions
C. Wu, M. Wang, L. Chen and Q. Tang synthesized all of novel compounds, P. Zheng and R. Luo
run the bioassay evaluation and statistics analysis, W. Zhu started the project, designed the molecules
and wrote the paper.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Word Health Organization. Available online: http://www.who.int/mediacentre/factsheets/fs297/en/
(accessed on 26 March 2015).
2. Samadi, A.K.; Bazzill, J.; Zhang, X.; Gallagher, R.; Zhang, H.; Gollapudi, R.; Kindscher, K.;
Timmermann, B.; Cohen, M.S. Novel withanolides target medullary thyroid cancer through
inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway. Surgery
2012, 6, 1238–1247.
3. You, W.K.; Sennino, B.; Williamson, C.W.; Falcón, B.; Hashizume, H.; Yao, L.C.; Aftab, D.T.;
McDonald, D.M. VEGF and c-Met blockade amplify angiogenesis inhibition in pancreatic islet
cancer. Cancer. Res. 2011, 14, 4758–4768.
4. Dayyani, F.; Gallick, G.E.; Logothetis, C.J.; Corn, P.G. Novel therapies for metastatic castrate-resistant
prostate cancer. J. Natl. Cancer Inst. 2011, 22, 1665–1675.
5. Hoelder, S.; Clarke, P.A.; Workman, P. Discovery of small molecule cancer drugs: Successes,
challenges and opportunities. Mol. Oncol. 2012, 2, 155–176.
6. Zhu, W.; Wu, C.; Xu, S.; Li, W.; Fang, H.; Chen, Z.; Tu, X.; Zheng, P. Design, Synthesis, and
Biological Evaluation of Sorafenib Analogs Bearing a Sulfonylurea Unit as Novel VEGFR2 Kinase
Inhibitors. Adv. Mater. Res. 2014, 1048–1051, doi:10.4028/www.scientific.net/AMR.989-994.1048.
7. Tang, Q.; Zhao, Y.; Du, X.; Chong, L.; Gong, P.; Guo, C. Design, synthesis, and structure-activity
relationships of novel 6,7-disubstituted-4-phenoxyquinoline derivatives as potential antitumor agents.
Eur. J. Med. Chem. 2013, 77–89, doi:10.1016/j.ejmech.2013.08.019.
8. Tang, Q.; Zhang, G.; Du, X.; Zhu, W.; Li, R.; Lin, H.; Li, P.; Cheng, M.; Gong, P.; Zhao, Y.
Discovery of novel 6,7-disubstituted-4-phenoxyquinoline derivatives bearing 5-(aminomethylene)
pyrimidine-2,4,6-trione moiety as c-Met kinase inhibitors. Bioorg. Med. Chem. 2014, 4, 1236–1249.

Molecules 2015, 20
19371
9. Wu, C.; Chen, C.; Sun, C.; Lei, F.; Fang, H.; Zheng, P.; Luo, R.; Zhu, W. Synthesis of Substituted Ethyl
Argiosulfonylcarbamates. Adv. Mater. Res. 2014, doi:10.4028/www.scientific.net/AMR.1015.574.
10. Wang, C.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi, Y.; Zhang, J. Biphenyl derivatives
incorporating urea unit as novel VEGFR–2 inhibitors: Design, synthesis and biological evaluation.
Bioorg. Med. Chem. 2014, 1, 277–284.
11. Scarborough, R.M.; Huang, W.; Mehrotra, M.; Zhang, X.; Cannon, H.; Grant, C.M.
Preparation of [4-(6-halo-7-substituted-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl)-phenyl]-5-chloro-
thiophen-2-yl-sulfonylureas as ADP receptor inhibitors for treatment of cardiovascular diseases.
WO. 2007056219A2, 18 May 2007.
12. Tsai, H.J.; Chou, S.Y.; Chuang, S.H.; Chen, C.C.; Hsu, F.L. D-420720, a novel orally active
sulfonamide compound dipeptidylpeptidase inhibitor: Structure and activity relationship of
arylsulfonamide to dipeptidylpeptidase inhibitor. Drug Dev. Res. 2008, 8, 514–519.
13. Roper, J.; Richardson, M.P.; Wang, W.V.; Richard, L.G.; Chen, W.; Coffee, E.M.; Sinnamon, M.J.;
Lee, L.; Chen, P.C.; Bronson, R.T.; et al. The dual PI3K/mTOR inhibitor NVP-BEZ235 induces
tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer.
PLoS ONE 2011, 9, e25132.
14. Yang, H.; Rudge, D.G.; Koos, J.D.; Vaidialingam, B.; Yang, H.J.; Pavletich, N.P. mTOR kinase
structure, mechanism and regulation. Nature 2013, 7448, 217–223.
Sample Availability: Samples of the compounds 6a–f and 9a–e are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).