Design and discovery of thioether and nicotinamide containing sorafenib analogues as multikinase inhibitors targeting B-Raf, B-RafV600E and VEGFR-2

Article abstract of DOI:10.1016/j.bmc.2018.03.039

New sorafenib derivatives containing thioether and nicotinamide moiety were designed and synthesized as B-Raf, B-Raf^V600E and VEGFR-2 multikinase inhibitors. Their in vitro enzymatic inhibitory activities against B-Raf, B-Raf^V600E and VEGFR-2 and their antiproliferative activities against HCT-116 and B16BL6 cell lines were evaluated and described. Most of the compounds showed potent activities against both cell lines and specific kinases. Compounds a1, b1 and c4, which exhibited the most potent inhibitory activities against B-Raf with IC₅₀ of 21 nM, 27 nM and 17 nM, B-Raf^V600E with IC₅₀ of 29 nM, 28 nM and 16 nM, VEGFR-2 with IC₅₀ of 84 nM, 46 nM and 63 nM, respectively, and good antiproliferative activities, also demonstrated competitive antiangiogenic activities to sorafenib in in vitro HUVEC tube formation assay.

Full text of DOI:10.1016/j.bmc.2018.03.039

Accepted Manuscript
Design and discovery of thioether and nicotinamide containing sorafenib ana-
logs as multikinase inhibitors targeting B-Raf, B-RafV600E and VEGFR-2
Shaofeng Sun, Zuopeng He, Mindong Huang, Ningning Wang, Zongzhong He,
Xiangkai Kong, Jianwen Yao
PII:
S0968-0896(18)30202-5
https://doi.org/10.1016/j.bmc.2018.03.039
BMC 14277
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
30 January 2018
23 March 2018
26 March 2018
Please cite this article as: Sun, S., He, Z., Huang, M., Wang, N., He, Z., Kong, X., Yao, J., Design and discovery of
thioether and nicotinamide containing sorafenib analogs as multikinase inhibitors targeting B-Raf, B-RafV600E and
VEGFR-2, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.03.039
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Bioorganic & Medicinal Chemistry
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Design and discovery of thioether and nicotinamide containing sorafenib analogs as
multikinase inhibitors targeting B-Raf, B-RafV600E and VEGFR-2
Shaofeng Suna, Zuopeng Hea, Mindong Huanga , Ningning Wanga, Zongzhong Heb，Xiangkai Konga and
Jianwen Yaoa,∗
a School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation(Yantai University), Ministry of Education, Collaborative Innovation
Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, 30 Qingquan Road, Yantai Shandong, 264005, PR
China
b Molecular Biology Division, Yantai Aillen Biological Technology Co.,Ltd.,39 Keji Road, Yantai Shandong, 264670, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
New sorafenib derivatives containing thioether and nicotinamide moiety were designed and
synthesized as B-Raf，B-RafV600E and VEGFR-2 multikinase inhibitors. Their in vitro
enzymatic inhibitory activities against B-Raf, B-RafV600E and VEGFR-2 and their
antiproliferative activities against HCT-116 and B16BL6 cell lines were evaluated and
described. Most of the compounds showed potent activities against both cell lines and
specific kinases. Compounds a1, b1 and c4, which exhibited the most potent inhibitory
activities against B-Raf with IC50 of 21 nM, 27 nM and 17 nM, B-RafV600E with IC50 of 29
nM, 28 nM and 16 nM, VEGFR-2 with IC50 of 84 nM, 46 nM and 63 nM, respectively, and
good antiproliferative activities, also demonstrated competitive antiangiogenic activities to
sorafenib in in vitro HUVEC tube formation assay.
Available online
Keywords:
Nicotinamide
B-Raf
VEGFR-2
Kinase inhibitor
Antitumor
2009 Elsevier Ltd. All rights reserved.
the patients with B-Raf mutant cancer.
1. Introduction
Vascular endothelial growth factor receptor (VEGFR-2), a
Tumor survival, growth and metastasis depend on tumor
member of the super family of protein tyrosine kinase receptors,
plays a crucial role in the regulation of tumour metastasis and
angiogenesis, and is closely related to solid tumour
development12. Signalling pathways are not strictly linear
cell proliferation, differentiation, apoptosis and angiogenesis,
which are regulated by numerous protein kinases and signal
transduction pathways1,2. The Ras-mitogen activated protein
kinase (MAPK) signaling pathway is the first signaling pathway
elucidated from the cell membrane to the nucleus, which consists
of the Ras/Raf/MEK/ERK signal transduction cascade. Raf is a
serine/threonine kinase that includes three isoforms (Raf-1 or c-
Raf, A-Raf, and B-Raf). Blockade of the Raf/MEK/ERK
pathway at the level of Raf kinases can effectively inhibit tumors
processes but rather involve
a complicated network of
interconnected circuits. When a single pathway is targeted alone,
redundancy and crosstalk between these pathways allow for
compensatory effects by alternative pathways13. Therefore,
multikinase inhibition targeting B-Raf, B-RafV600E and VEGFR-2
represent a promising approach to cancer treatment with a
synergistic effect.
driven by this pathway3-5. B-raf somatic mutation, particularly B-
V600E
raf
is a common oncogenic mutation across cancers, and
Sorafenib, a bisaryl ureas multikinase inhibitor, has been
developed and launched for the treatment of renal cell carcinoma
(RCC) and unresectable hepatocellular carcinoma (HCC). It can
not only inhibit Raf to block Raf/MEK/ERK signaling pathway,
but also inhibit other kinases involved in tumor proliferation and
these oncogenic B-Raf mutations are observed in malignant
melanomas, thyroid, ovarian, colorectal, and lung cancers6-9
.
Therefore, B-raf has been validated as an attractive target for the
design of anticancer agents.
angiogenesis, such as VEGFR-2, PDGFR, Flit-3 and c-KIT14-16
.
Despite selective B-Raf inhibitors have demonstrated
significant clinical benefit in melanoma patients harboring
oncogenic B-Raf mutations. However, the majority of patients,
especially with B-Raf mutant colorectal cancer, exhibit drug
resistance, and eventually relapse within 6 -12 months 10,11. Thus,
However, its weak inhibitory activity against tumors with
oncongenic B-Raf mutations, for example, melanoma and
colorectal cancer, may be the main reason causing the poor
therapeutic effect. To improve upon sorafenib in terms of both
kinase and cell growth inhibition, modifications on sorafenib
d—ru—g re—sistance continues to present a considerable challenge for
∗ Corresponding author. Tel.: +86 535 6706025; fax: +86 535 6706066; e-mail: jwy5610201@163.com

have been carried on for years17-23. In our previous work, a series
of sorafenib analogues containing thioether were synthesized and
evaluated24. The antiproliferative activities of most thioether
bearing analogues were better than sorafenib, which suggested
that the thioether moiety may improve the cytotoxicity of these
compounds (Figure 1). To improve the biological activities of
sorafenib analogues, the 2,4-substitution mode on the C ring was
changed with the 2,5-substitution mode, new sorafenib
analogues containing nicotinamide were also designed and
synthesized24. Interestingly, these compounds showed much
more significant antiangiogenic activities than sorafenib in both
in vitro HUVEC tube formation assay and ex vivo rat thoracic
aorta rings (TARs) assay. Base on the SAR described above,
several designed thioether and nicotinamide containing
analogues (a1-3) were described in this paper (Figure 2).
Based on bioisosteric replacement, several series of
thiourea containing sorafenib analogues have been designed and
synthesized (Figure 1)25-27. The thiourea moiety could enhance
the antiproliferative activities to some extent, and, with this in
mind, a series of thiourea containing sorafenib analogues derived
from series a (b1-8, Figure 2) were also synthesized.
Additionally, we found that the 1,3-substituted B-ring can
strengthen the antiangiogenic activities26. Thus a novel series of
compounds (c1-9, Figure 2) were prepared. Their enzymatic
inhibition and antiproliferative activities were evaluated.
Compounds a1, b1 and c4, which exhibited the potent inhibitory
activities against B-Raf (IC50 = 21 nM, 27 nM and 17 nM,
respectively), B-RafV600E (IC50 = 29 nM, 28 nM and 16 nM,
respectively), VEGFR-2 (IC50 = 84 nM, 46 nM and 63 nM,
respectively), and good antiproliferative activities, were chosen
to explored their antiangiogenic activities.
H
N
H
N
H
N
H
N
F3C
Cl
N
N
Ar
C
B
H
N
H
N
A
B
C
S
O
S
O
O
O
Sorafenib
Thiourea and thioether containing sorafenib analogues
H
N
H
N
O
O
N
C
H
N
H
N
Ar
O
H
N
B
N
H
Ar
S
B
C
S
N
O
Sorafenib analogues with 1,3-substituted on B ring
Thiourea and nicotinamide containing sorafenib
analogues with 1,3-substituted on B ring
Figure 1. Previously reported analogues of sorafenib.
H
H
O
H
N
H
N
F3C
Cl
N
N
N
H
N
Ar
N
H
A
B
C
O
B
C
N
O
O
S
O
Thioether and nicotinamide-containing sorafenib
derivatives (a1-3)
Sorafenib
H
N
H
N
O
S
N
H
N
H
N
Ar
H
N
C
Ar
N
H
B
S
B
C
N
S
S
O
Thiourea, thioether and nicotinamide-containing
sorafenib derivatives (b1-8)
Thiourea, thioether and nicotinamide-
containing sorafenib derivatives with 1,3-
substituted on B ring (c1-9)
Figure 2. Modifications of sorafenib.
The substituted phenyl isocyanates (5) were prepared by
treatment of corresponding anilines (4) and triphosgene (BTC),
which were reacted with compound 3 in DMF to afford target
compounds (a1-3) with the overall yield of 58.6 - 69.4%. Phenyl
isothiocyanates (6) can be easily obtained from substituted
anilines (4) by previously reported procedures26. These
isothiocyanates were reacted with compound 3 and compound 7
to give target compounds b1-8 and c1-9, , respectively, in
moderate to good yields.
2. Chemistry
The synthetic routes to the target compounds were
illustrated in Scheme 1. The key intermediates 6-(4-
aminophenylthio)-N-methylnicotinamide
(3)
and
6-(3-
aminophenylthio)-N-methylnicotinamide (7) were obtained with
6-chloronicotinic methyl ester (1) as the starting material,
respectively. 6-chloronicotinic methyl ester (1) was reacted with
methylamine to generate 6-chloronicotinic methyl amide (2),
which was furhter treated with 4-aminothiophenol to get
intermediate (3). Likewise, 6-chloronicotinic methyl amide (2)
was reacted with 3-aminothiophenol to produce another
intermediate (7).
The final products were purified by column
chromatography and their structures were identified by 1H NMR,
13C NMR and HRMS.

O
c
H
N
H
N
Ar NH2
4
Ar NCO
5
Ar
Ar
Ar
N
H
d
O
S
S
S
N
O
a1-3
O
O
NH2
O
a
b
N
H
N
H
N
Cl
O
N
Cl
N
S
H
N
H
N
2
3
1
N
H
g
S
S
N
N
h
e,f
NH2
Ar
Ar NCS
6
b1-8
4
H
N
H
N
i
O
H
N
N
H
O
c1-9
N
S
NH2
7
Scheme 1. Reagents and conditions: (a) methylamine, methanol, rt; (b) 4-amino thiophenol, t-BuOK, K2CO3, DMF, 85 ; (c) BTC,
dichloroethane, 0-5 and then reflux; (d) DMF, rt; (e) Dabco, CS2, toluene, 0-5 ; (f) BTC, DCM, reflux; (g) DMF, rt; (h) 3-amino thiophenol,
t-BuOK, K2CO3, DMF, 80 ; (i) DCM, rt.
further investigation.
3. Results and discussion
3.2 Cell inhibition
3.1 B-Raf, B-RafV600E and VEGFR-2 inhibitory assay
In vitro cell cytotoxicities of all the new compounds were
As shown in Table 1, in vitro enzymatic inhibition activity
initially evaluated against both HCT116 and B16BL6 cells lines
of all the title compounds were initially evaluated against B-Raf,
harboring oncogenic B-Raf mutations, by MTT assay using
B-RafV600E and VEGFR-2. B-Raf and B-RafV600E were assessed
by ELISA, and VEGFR-2 was tested in A Caliper mobility shift
assay format using sorafenib as a positive control. All these
sorafenib as a positive control. The structures and activities of
series a are shown in Table 1. All of these compounds showed
potent antiproliferative activities against HCT116 and B16BL6
compounds in series a exhibited potent inhibitory activities
cells lines, and compound a1 possessing the same terminal
against protein kinases. Among them, compound a1
phenyl ring with sorafenib demonstrated more potent activity
than sorafenib against both cell lines, which suggested that the
demonstrated competitive inhibitory activities against B-Raf
with IC50 of 21 nM (sorafenib, 22 nM), B-RafV600E with IC50 of
thioether and nicotinamide moiety did enhance the
29 nM (sorafenib, 24 nM), VEGFR-2 with IC50 of 84 nM
antiproliferative activities of sorafenib analogues.
(sorafenib, 68 nM).
As shown in Table 2, compounds b1, b6, b7 and b8 which
also have two strong electron-withdrawing groups (-F, -Cl or -
The results of sorafenib derivatives (b1-8) were
CF3) on terminal phenyl ring exhibited potent antiproliferative
activities against HCT116 and B16BL6 cell lines, and
compounds b1 and b6 having potent kinase inhibition showed
better antiproliferative activities than sorafenib, the reference
standard. The activities of compounds (b2 and b5) bearing only
one electron-withdrawing group were relatively weaker. While
the compounds b3 and b4, which have electron-donating groups
(-OCF3 and –CH3) on phenyl ring, showed the lowest
antiproliferative activities against both cell lines. As shown in
Table 3, most compounds in series c demonstrated moderate to
good antiproliferative activities, and compounds containing two
strong electron-withdrawing substitutes on the terminal phenyl
ring exhibited potent activities against both cell lines, which is
consistent with compounds in series b. For example, compound
c4 bearing 4-Cl and 3- CF3, which showed the best activities in
series c (IC50 = 5.18 and 19.75µM against HCT116 and B16BL6,
respectively), was comparable to compound b1 (IC50 = 4.90 and
23.51 µM against HCT116 and B16BL6, respectively). While
the compounds c3 and c1 demonstrated weaker activities than b6
and b7 which have the same substitutents with c3 and c1.
Generally, the antiproliferative activities of series b are better
than those of series c, which indicates that the 1,3-substitution on
the B-ring may not enhance the antiproliferative activities of
these analogues
summarized in Table 2. Compounds b1 and b6 having two
strong electron-withdrawing groups on terminal phenyl ring
exhibited potent activities against B-Raf (IC50 = 27 nM and 33
nM, respectively), B-RafV600E (IC50 = 28 nM and 45 nM,
respectively) and VEGFR-2 (IC50 = 46 nM and 74 nM,
respectively), whilst compounds b3 and b4 bearing electron-
donating groups were essentially inactive for B-RafV600E and
VEGFR-2. It can be inferred that the terminal electron-
withdrawing groups is of benefit to improve activity. As shown
in Table 3, most compounds showed potent inhibitory activities
against B-Raf, B-RafV600E and VEGFR-2. Compounds (c1, c3, c4,
c7 and c9), possessing two strong electron-withdrawing groups
on terminal aromatic ring, exhibited potent inhibitory activities
against B-Raf with IC50 values ranging from 17 to 442 nM, B-
RafV600E with IC50 values ranging from 16 to 539 nM, and against
VEGFR-2 with IC50 values ranging from 61 to 4016 nM. Among
these compounds, compound c4 bearing 4-Cl and 3-CF3 on the
terminal phenyl ring showed the most potent activity with IC50 of
17 nM against B-Raf, 16 nM against B-RafV600E and 63 nM
against VEGFR-2. Interestingly, compounds(c3 and c4) bearing
two strong electron-withdrawing groups at 3,4-position of phenyl
ring displayed potent inhibitory activities compared to
compound c9 having 3,5-CF3 group on terminal phenyl ring,
which indicated that these compounds might exist different
binding mode with target molecues. It is worthy to note that
compounds which showed strong suppression of B-Raf and B-
RafV600E can also inhibite VEGFR-2 significantly. Moreover, as
shown in Table 4, compounds a1, b1 and c4 exhibited selective
inhibition against B-Raf, B-RafV600E, VEGFR-2, and other
proliferation-related enzymes, such as Flt-3, c-Kit and EGFR,
but the detail enzyme selectivity and action mechanism still need
3.3 In vitro HUVEC tube formation assay
In view of their potent inhibitory activities against B-Raf,
B-RafV600E, VEGFR-2, and good antiproliferative activities,
compound a1, b1, and c4 which shared same substituents on the
terminal phenyl ring with sorafenib were chosen to test their
antiangiogenic activities by in vitro HUVEC tube formation

assay using sorafenib as positive control. As shown in Figure 3,
elongated and robust tube-like structures were well established in
the negative control group (DMSO). Treatment of HUVECs with
sorafenib at 0.05 µM can partly inhibit the tuber formation, and
the tube formation was completely inhibited at 0.1 µM. All three
compounds demonstrated better anti-angiogenic activities than
sorafenib and could entirely inhibit the tube formation even at
0.05 µM.
between O of amide moiety and residueTrp-531.
The terminal benzene ring of c4 located in a hydrophobic
pocket lined with Ile-513, Val-471, Asp-594 and Gly-464. Four
hydrogen bonds were formed strongly between NH of thiourea
moiety and O atoms of the Asp-594. Moreover, N atom of the
pyridine can simultaneously form two hydrogen bonds with the
Gly-596 and Lys-483.
Compound a1 could be accommodated to the DFG-out
conformation of VEGFR-2 (PDB ID: 2OH4). The terminal
benzene ring is located in a hydrophobic pocket lined with Phe-
1047, Phe-918, Ala-866 and Lys-868. One hydrogen bond was
formed between Arg-1027 and N of the pyridine, another
hydrogen bond was formed between O of urea moiety and
residue Lys-868. The nicotinamide moiety of c4 is located in a
hydrophobic pocket lined with Cys-1024, Leu-901 and Leu-889.
Hydrogen bonds were formed simultaneously between NH of
thiourea moiety and residue Asp-1046. Moreover, the O atom of
nicotinamide and residue Glu-885 formed another hydrogen
bond.
3.4 Molecular docking studies
In order to understand the interaction between the
synthesized compounds and kinases, molecular docking studies
on the two potent representative compounds a1 and c4 were
performed using the Tripos Sybyl-x2.0 software.
As shown in Figure 4, compound a1 could be
accommodated to the DFG-out conformation of B-Raf
(1)
comfortably (PDB ID: 1UWH). The terminal benzene ring is
located in a hydrophobic pocket lined with Asn-512, Leu-515
and His-585. Three hydrogen bonds were formed between F of
the CF3 and Asp-594, another hydrogen bond was formed
Table 1. Enzymatic and cellular results of series a
O
H
N
H
N
Ar
N
H
O
S
N
IC50 (µM)a
HCT-116
Comp
NO.
Subsituent
Ar
IC50 (nM)
B-Raf
21
B-RafV600E
VEGER-2
84
B16BL6
CF3
Cl
a1
a2
29
5.29 0.55
7.09 1.17
24.59 0.60
45.18 0.42
CF3
93
196
508
CF3
CF3
F
a3
48
22
65
24
102
68
5.80 0.78
8.41 0.19
39.62 1.76
36.48 3.52
sorafenib
___
a Assays were performed in triplicate (n≥3); values were shown as mean SD.
Table 2. Enzymatic and cellular results of series b
O
H
N
H
N
Ar
N
H
S
S
N
IC50 (µM)a
Comp
NO.
b1
Subsituent
Ar
IC50 (nM)
B-RafV600E
28
B-Raf
27
VEGER-2
46
HCT-116
B16BL6
CF3
Cl
4.90 0.45
23.51 1.53
CF3
503
571
> 10000
> 10000
> 10000
25.93 1.1
> 100
53.07 0.98
> 100
b2
b3
b4
OCF3
9866
> 10000
> 10000
> 10000
52.44 1.47
> 100

Cl
2064
33
1785
45
8663
74
34.46 0.89
6.37 0.48
69.29 0.52
29.32 1.31
b5
b6
CF3
F
F
810
892
> 10000
22.17 1.03
52.55 0.17
b7
F
CF3
CF3
N.D
22
N.D.
24
277
68
16.59 0.73
8.41 0.19
36.13 0.15
36.48 3.52
b8
sorafenib
___
a Assays were performed in triplicate (n≥3); values were shown as mean SD.
Table 3. Enzymatic and cellular results of series c
H
N
H
N
S
N
Ar
H
N
S
O
Comp
NO.
c1
Subsituent
Ar
IC50 (nM)
B-RafV600E
539
IC50 (µM)a
B-Raf
442
791
69
VEGER-2
3864
5986
61
HCT116
B16BL6
58.21 1.79
67.46 0.98
42.39 0.67
19.75 0.63
> 100
F
35.08 0.13
39.49 0.49
18.17 0.52
5.18 0.53
16.46 0.75
24.50 0.49
17.31 0.97
16.15 1.17
5.02 0.27
8.41 0.19
F
Cl
857
c2
CF3
F
77
c3
CF3
Cl
17
16
63
c4
CF3
Br
N.D
5851
280
973
158
22
N.D.
7485
392
133
c5
OCF3
> 10000
4016
6915
887
91.00 1.03
> 100
c6
Cl
F
c7
CF3
1267
169
19.22 1.62
54.16 2.69
36.48 3.52
c8
CF3
CF3
c9
sorafenib
___
24
68
a Assays were performed in triplicate (n≥3); values were shown as mean SD.
Table 4. Selective inhibition of kinases involved in tumor progression and angiogenesis

Enzymatic inhibition IC50 (nM)a
Comp
B-Raf
21
B-RafV600E
VEGER-2
FLT-3
67
c-KIT
535
EGFR
> 1000
> 1000
596
a1
b1
c4
29
28
16
84
46
63
27
17
949
781
701
> 1000
a Assays were performed at least two independent experiments.
sorafenib
DMSO
DMSO
0.05µM
0.1µM
0.05µM
0.1µM
a1
a1
b1
b1
0.05µM
0.05µM
0.1µM
0.1µM
c4
c4
0.05µM
0.1µM
Figure.3 Representative images of tubes formed on Matrigel after seeding with HUVEC and treating with DMSO, sorafenib, a1, b1 and c4 at
different concentrations.
(1)
(2)

(3)
(4)
Figure 4. Docking models of a1 and c4 with Kinases: (1) a1 in B-Raf active site; (2) c4 in B-Raf active site; (3) a1 in VEGFR-2 active site; (4)
c4 in VEGFR-2 active site. The hydrogen bonds are displayed as yellow dotted lines.
4. Conclusions
In summary, new thioether and nicotinamide containing
sorafenib analogs were designed and synthesized as B-Raf, B-
RafV600E and VEGFR-2 mutikinase inhibitors. Molecular docking
established the interaction of compounds a1 and c4 with the
DFG-out conformation of VEGFR-2 and B-Raf, and suggested
that compounds a1 and c4 could bind well with the active site of
B-Raf and VEGFR-2.
Compounds were diluted in DMSO and added to Raf-Mek
phosphorylation reactions with a final DMSO concentration of
10%. Mek was used at 5.6 nM in the reactions. Add 30 µl of
100% DMSO to two empty wells for no compound control and
no enzyme control in the same 96-well plate. Kinase reactions
were run in 30 µL volumes in 96-well plates for 2 hours at room
temperature. After incubation, the phosphorylation reactions
were quenched with 30 µL EDTA and 60 µL reactions volume is
transferred to a new 96 well plates. Capture was allowed to
proceed for 1 hour, and wells were then washed 4x with
DELFIA wash buffer. Wells were then probed with Phospho-
Mek1/2 (Ser217/221) antibody diluted 1:2000 in Delfia Assay
Buffer, and added for 1 hour followed by washing 4x with
DELFIA wash buffer. Signal detection is by Time Resolved
Flouresence utilizing a Europium labeled goat anti-rabbit
secondary antibody (Eu-N1, DAKO). Secondary antibody was
diluted 1:1000 in Delfia assay buffer and added for 1 hour
followed by washing 4x with DELFIA wash buffer. 100 µL
DELFIA enhancement solution is added for 5 minutes with
shaking followed by fluoremetry with the Victor reader
(PerkinElmer.)
Compared to sorafenib, most of the
compounds showed good inhibitory potencies
against B-Raf, B-RafV600E and VEGFR-2 as well as
competitive antiproliferative activities against
HCT-116 and B16BL6 cell lines, which suggests
that the thioether and nicotinamide moiety did
enhance the inhibitory activities of sorafenib
analogues. Notably, compounds possessing strong
electron-withdrawing groups (such as 4-Cl, 4-F or
3-CF3) at 3,4-position of phenyl ring exhibited
significant
enzymatic
inhibitory
and
VEGFR-2 kinase activity was valuated by a Caliper
mobility shift assay. Title compounds were dissolved in DMSO.
Dilute the compound to 50x of the final desired highest inhibitor
concentration in reaction by 100% DMSO. Transfer 100µl of this
compound dilution to a well in a 96-well plate and serially dilute
the compound by transferring 10µl to 90µl of 100% DMSO in
the next well. Add 100µl of 100% DMSO to two empty wells for
no compound control and no enzyme control in the same 96-well
plate. Mark the plate as source plate. Transfer 10µl of compound
from source plate to a new 96-well plate as the intermediate
plate. Add 90µl of 1x Kinase base buffer to each well of the
intermediate plate. Mix the compounds in intermediate plate for
10 min on the shakers. Transfer 5µl of each well from the 96-
well intermediate plate to a 384-well plate in duplicate.
antiproliferative activities, which indicated that the
introduction of electron-withdrawing groups on
terminal phenyl ring was more favorable for
enzyme inhibition and antiproliferative activities.
Moreover, compounds a1, b1 and c4 displayed
potent anti-angiogenic activities in vitro HUVEC
tube formation assay. Therefore, compounds a1, b1
and c4 could be developed as potent anticancer
agents, and the enzyme selectivity and
pharmacokinetic profile still need further
investigation.
Add kinase in 1x kinase base buffer. Add FAM-labeled
peptide and ATP in the 1x kinase base buffer. Assay plate
already contains 5µl of compound in 10% DMSO. Add 10µl of
2.5x enzyme solution to each well of the 384-well assay plate.
Incubate at room temperature for specified period of time, then
add 10µl of 2.5x peptide solution to each well of the 384-well
assay plate. The subsequent kinase reaction took place at room
5. Experimental
5.1. Kinase inhibition assays
The potency of title compounds against B-Raf and B-
RafV600E were assessed by ELISA with anti-pMek1 antibody
(Cell Signaling Technology) followed by a phosphorylation
assay using a bacterially purified Mek1 as the substrate.

temperature for 30 min. After incubation, the kinase reactions
were quenched with EDTA and the plates were washed.
Copy conversion data from Caliper program. Convert
conversion values to inhibition values according to percent
inhibition = (max-conversion)/(max-min)*100, in which “max”
stands for DMSO control; “min” stands for no enzyme activity
control. Fit the data in XLfit to obtain IC50 values.
6-chloronicotinic acid methyl ester (10.0 g, 58.3 mmol) was
dissolved in 10 mL methanol to get light yellow solution, the
methylamine (2.7 g, 87.5 mmol) in water solution was added
dropwise at 0-5℃ under stirring. The mixture was stirred at
room temperature for 4h, and then concentrated in vacuum.
Finally, ethyl acetate (100 mL) was added, and the resulting
mixture was stirred at room temperature. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give light yellow
5.2. Cell inhibition assays
1
crystalline (2) 9.4 g, yield: 99.1 %, mp: 145-146℃. H NMR
HCT-116 and B16BL6 cell lines were plated on 96-well
plates at a density of 5000 per well and incubated overnight. The
cells were treated with compounds and sorafenib at final
concentrations ranging from 0.5 to 200 µM, while control cells
were treated with equal volume DMSO. After 48 hr, 0.5% MTT
(Amresco, USA) solution was added to each well, and further
incubation for 4 h, then cells were centrifuged at 2,500 rpm for
15 min and removed from the culture medium. And add 150 µL
DMSO to dissolve the formazan. After mixing for 5 min, optical
density was detected at 570 nm on a microplate reader (Thermo,
USA).
(DMSO-d6, 400 MHz): δ 2.75 (d, J = 4.5 Hz, 3H), 6.89 (d, J =
8.8 Hz, 1H), 8.16 (dd, J = 8.7, 2.4 Hz, 1H), 8.46 (br d, J = 4.4
Hz, 1H), 8.57 (d, J = 2.3 Hz, 1H). ESI-MS m/z 171.1 [M+H]+ ;
Anal. Calcd for (C7H7ClN2O FW: 170.5); C, 49.28; H, 4.14; Cl,
20.78; N, 16.42. Found: C, 49.24; H, 4.16; Cl, 20.79; N, 16.43.
5.5.2. 6-(4-Aminophenylthio)-N-methylnicotinamide (3)
A solution of 4-aminothiophenol (1.6 g, 12.8 mmol) in dry
N,N-dimethylformamide (DMF, 30 mL) was treated with
potassium tert-butoxide (1.9 g, 16.8 mmol), then a solution of 6-
chloronicotinic methyl amide (2) ( 2.0 g, 11.8 mmol) in DMF
(15 mL) and potassium carbonate (0.7 g, 8.3 mmol) were added
respectively. Then the mixture was stirred at 85-88℃ for 12 h.
After cooling to room temperature, the mixture was diluted with
brine (200 mL) and extracted with EtOAc (3×100 mL). The
organic layer was washed with brine (3×100mL), dried over
anhydrous sodium sulfate, and concentrated under reduced
pressure to give brown solid (3) 2.1 g, yield: 68.6 %, mp: 146-
5.3 In vitro HUVEC tuber formation assay
Forty eight-well slide chambers were coated with 100 µL of
Matrigel (BD Biosciences, NJ) and allowed to gel at 37℃ and
5% CO2 for 30 min. HUVECs were then seeded at 40,000
cells/well in M199 (5% FBS) containing either the vehicle (0.5%
DMSO), sorafenib or synthesized compounds and incubated at
37℃ and 5% CO2 for 6 h. The morphological changes of the
cells and HUVEC tubes formation were observed under a phase-
contrast microscope and photographed at 200× magnification.
The corresponding area was measured as the number of pixels
using Adobe PhotoShop. The experiments were repeated three
times.
1
148℃. H NMR (400 MHz, CDCl3) δ 2.77 (d, J = 4.5 Hz, 3H),
5.64 (br s, 2H), 6.67, 7.23 ( AA’BB’, q, J = 8.4Hz; 4H ), 6.76 (
d, J = 8.4 Hz, 1H ), 7.94 (dd, J = 8.4, 2.1 Hz, 1H ), 8.48 ( br d, J
= 4.4 Hz, 1H ), 8.76 (d, J = 1.7Hz, 1H), ESI-MS m/z 260.2
[M+H]+; Anal. Calcd for (C13H13N3OS FW: 259.1); C, 60.21; H,
5.05; N, 16.20; O, 6.17; S, 12.36. Found: C, 60.41; H, 5.03; N,
16.21, O, 6.16; S, 12.37.
5.4. Molecular docking
5.5.3. Substituted phenyl isocyanates (5a-c)
Molecule docking was carried out using Sybyl/Surflex-dock
based on the crystal structures of B-Raf (PDB ID: 1UWH) and
VEGFR-2 (PDB ID: 2OH4). Hydrogen was added and
minimized using the Tripos force field and Pullman charges, and
the default settings were used. Compounds a1 and c4 were
depicted with the Sybyl/Sketch module (Tripos Inc.) and docked
into the defined binding site without constraint. Molecular
docking results were submitted and generated by Pymol.
Substituted phenyl isocyanates (5a-c) can be easily obtained
from substituted anilines (4) by previously reported procedures
in moderate to good yields28.
5.5.4. Substituted phenyl isocythiocyanates (6a-j)
Substituted phenyl isothiocyanates (6a-j) can be easily
obtained from substituted anilines (4) by previously reported
procedures in moderate to good yields27.
5.5. Chemistry: general procedures
5.5.5. 6-(3-aminophenylthio)-N-methylnicotinamide (7)
All the materials we used were purchased from commercial
suppliers and used directly without further purification. Solvents
were distilled before using and flash chromatography was
performed using silica gel (60 Å, 200–300 mesh). All reactions
were monitored by thin-layer chromatography (TLC) on 0.25
mm silica gel plates (60GF-254) and visualized with UV lamp
(254 and 365 nm). Melting points were determined on an
electrothermal melting point apparatus and were uncorrected.
Proton nuclear magnetic resonance (1H and 13C NMR) spectra
were obtained on a Brucker Avance 400/600 spectrometer using
TMS as an internal standard in DMSO-d6 or CDCl3 solutions.
Chemical shifts were reported in delta (δ) units, parts per million
(ppm) downfield from trimethylsilane. ESI-MS were determined
on an API 4000 spectrometer, High-resolution mass spectral
(HRMS) data were conducted by Shandong Analysis and Test
Center, and are reported as m/z (relative intensity).
A solution of 3-aminothiophenol (1.8 g, 14.4 mmol) in dry
N,N-dimethylformamide (DMF, 30 mL) was treated with
potassium tert-butoxide (1.9 g, 16.6 mmol), then a solution of 6-
chloronicotinic methyl amide (2) (2.0 g, 11.8 mmol) and
potassium carbonate (0.7 g, 8.3 mmol) were added respectively.
The mixture was stirred at 80-83℃ for 12 h. After cooling to
room temperature, the mixture was diluted with brine (200 mL)
and extracted with EtOAc (3×100 mL). The organic layer was
combined and washed with brine (3×100mL), dried over
anhydrous sodium sulfate, and concentrated under reduced
pressure to give brown solid (7) 2.3 g, yield: 75.1 %, mp: 169-
1
170℃. H NMR (400 MHz, CDCl3) δ 2.95 (d, J = 5.1 Hz, 3H),
4.10 (br s, 2H), 6.38 (m, 2H), 6.56 ( dd, J = 7.8, 1.5 Hz, 1H)，
6.94 (dd, J = 5.5, 2.5 Hz, 1H ), 7.10 ( t, J = 7.8Hz, 1H )，7.69
(d, J = 2.5 Hz, 1H), 8.11 (br d, J = 4.8 Hz, 1H), 8.33 (d, J = 5.4
Hz, 1H); ESI-MS m/z 260.2 [M+H]+; Anal. Calcd for
(C13H13N3OS FW: 259.3): C, 60.21; H, 5.05; N, 16.20; O, 6.17;
S, 12.36. Found: C, 60.23; H, 5.03; N, 16.21; O, 6.18; S, 12.36.
5.5.1. 6-Chloronicotinic acid methyl amide (2)

5.5.6.
phenylsulfanyl}-N-methylnicotinamide (a1)
6-{4-[3-(4-Chloro-3-trifluoromethylphenyl)ureido]
The crude product was purified by chromatography to afford a
off-white solid, anhydrous sodium sulfate, and concentrated
under reduced pressure to get the crude product. The crude
product was purified by chromatography to afford an off white
solid, yield: 67.5 %, mp: 161-163℃. 1H NMR (DMSO-d6, 400
MHz): δ 2.77 (d, J = 4.5 Hz, 3H), 7.00 (d, J = 8.4 Hz, 1H), 7.59
(d, J = 8.6 Hz, 2H), 7.63 – 7.74 (m, 3H), 7.82 (dd, J = 8.8, 2.5
Hz, 1H), 8.01 (dd, J = 8.4, 2.4 Hz, 1H), 8.09 (d, J = 2.6 Hz, 1H),
8.56 (d, J = 4.7 Hz, 1H), 8.79 (d, J = 2.5 Hz, 1H), 10.29 ( s, 1H),
10.37 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 26.09, 119.65,
122.23, 122.37, 124.01, 124.19, 124.36, 125.27, 125.38, 125.96,
126.36, 128.49, 131.59, 135.58, 135.86, 138.96, 140.49, 148.17,
164.59, 179.54; HRMS(ESI) m/z: calcd for C21H17ClF3N4OS2
[M+H]+ 497.0484, found 497.0479. HPLC purity = 99.41%.
Compound 3 (1.07 g, 4.12 mmol) was dissolved in 10 mL of
N,N-dimethylformamide (DMF) and the solution was cooled to
0–5 °C in ice bath under stirring, a solution of 5a (0.91 g, 4.12
mmol) in DMF were added dropwise at 0–5℃ under nitrogen
atmosphere. The mixture was stirred for 2 h at 0–5℃, and then
stirred for another 16 h at room temperature. The mixture was
poured into brine (200 mL) and extracted with EtOAc (3×200
mL). The organic layer was combined and washed with brine
(2×200 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give the crude product.
The crude product was purified by chromatography to afford a
light yellow solid, yield: 69.4 %, mp: 209-211℃. 1H NMR (400
MHz, DMSO-d6): δ 2.77 (d, J = 4.6 Hz, 3H), 6.90 (d, J = 8.5
Hz, 1H), 7.47 – 7.77 (m, 6H), 7.99 (dd, J = 8.5, 2.4 Hz, 1H),
8.12 (d, J = 2.5 Hz, 1H), 8.54 (q, J = 4.4 Hz, 1H), 8.79 (d, J =
The other compounds of series b (b2-b8) were synthesized
following the general procedure as described above
5.5.10. 6-{4-[3-(3-Trifluoromethylphenyl)thioureido]phenyl
sulfanyl}-N-methylnicotinamide (b2)
2.4 Hz, 1H), 9.19 (s, 1H), 9.30 (s, 1H)；13C NMR (100 MHz,
DMSO-d6): δ 26.07, 116.79, 116.85, 119.18, 119.60, 120.81,
122.50, 123.14, 124.10, 126.11, 126.61, 126.81, 131.98, 135.81,
136.29, 139.07, 140.96, 148.07, 152.21, 164.37, 164.62;
HRMS(ESI) m/z: calcd for C21H17ClF3N4O2S [M+H]+ 481.0713,
found 481.0699. HPLC purity = 98.32%.
Off white solid, yield: 51.8%, mp: 161-163℃. 1H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.5 Hz, 3H), 6.98 (d, J = 8.4 Hz,
1H), 7.49 (d, J = 7.8 Hz, 1H), 7.53 – 7.64 (m, 3H), 7.68 (d, J =
8.5 Hz, 2H), 7.73 – 7.85 (m, 1H), 7.86-8.14 (m, 2H), 8.56 (d, J =
4.7 Hz, 1H), 8.80 (d, J = 2.6 Hz, 1H), 10.25 ( s, 1H), 10.27 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ 26.08, 119.59, 119.75,
119.79, 119.83, 120.73, 120.78, 123.78, 124.08, 126.33, 127.31,
129.15, 129.54, 135.57, 135.82, 140.20, 140.67, 148.17, 163.59,
164.60, 179.57; HRMS(ESI) m/z: calcd for C21H18F3N4OS2
[M+H]+ 463.0874, found 463.0889. HPLC purity = 98.34%.
The other compounds of series a (a2-a3) were synthesized
following the general procedure as described above.
5.5.7. 6-{4-[3-(3,5-Bis-trifluoromethylphenyl)ureido]phenyl
sulfanyl}-N-methylnicotinamide (a2)
Light yellow solid, yield: 58.6 %, mp: 208-210℃. 1H NMR
(400 MHz, DMSO-d6) δ 2.77 (d, J = 4.5 Hz, 3H), 6.90 (d, J = 8.6
Hz, 1H), 7.73 – 7.50 (m, 5H), 7.99 (d, J = 8.8 Hz, 1H), 8.15 (s,
2H), 8.43 – 8.60 (m, 1H), 8.80 (d, J = 2.8 Hz, 1H), 9.33 ( s, 1H),
9.53 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 26.04, 114.48,
115.05, 118.03, 118.39, 119.18, 119.82, 121.12, 121.87, 126.11,
130.83, 135.55, 135.77, 136.23, 136.92, 140.78, 141.57, 147.88,
148.08, 152.24, 164.32, 164.63; HRMS(ESI) m/z: calcd for
C22H17F6N4O2S [M+H]+ 515.0976, found 515.0983. HPLC purity
= 98.03%.
5.5.11. 6-{4-[3-(4-trifluoromethoxyphenyl)thioureido]phenyl
sulfanyl}-N-methylnicotinamide (b3)
Light yellow solid, yield: 55.9 %, mp: 177-179℃. 1H NMR
(400 MHz, DMSO-d6) δ2.78 (d, J = 4.6 Hz, 3H), 6.98 (d, J = 8.7
Hz, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.42 – 7.87 (m, 6H), 7.86 -
8.12 (m, 1H), 8.55 (d, J = 5.8 Hz, 1H), 8.81 (br d, J = 2.8 Hz,
1H), 10.14 (s, 1H), 10.21 (s, 1H); 13C NMR (100 MHz, DMSO-
d6): δ 26.05, 118.79, 120.10, 121.15, 121.18, 123.15, 124.62,
125.21, 126.50 , 129.20, 129.61, 129.91, 130.39, 135.72, 138.43,
140.67, 144.65, 148.24, 162.93, 164.57, 179.97; HRMS(ESI) m/z:
calcd for C21H18F3N4O2S2 [M+H]+ 479.0823, found 479.0842.
HPLC purity = 98.73%.
5.5.8. 6-{4-[3-(4-Fluoro-3-trifluoromethylphenyl)ureido]
phenylsulfanyl}-N-methylnicotinamide (a3)
Light yellow solid, yield: 62.0 %, mp: 192-194℃.1H NMR
(400 MHz, DMSO-d6) δ 2.77 (d, J = 4.5 Hz, 3H), 6.89 (d, J = 8.4
Hz, 1H), 7.37 – 7.72 (m, 6H), 8.00 (m, 2H), 8.55 (d, J = 4.6 Hz,
1H), 8.79 (d, J = 2.8 Hz, 1H), 9.20 ( s, 1H), 9.23 (s, 1H); 13C
NMR (100 MHz, DMSO-d6): δ 26.06, 115.09, 115.94, 115.99,
116.12, 117.67, 120.30, 121.18, 124.27, 124.35, 126.09, 135.57,
135.79, 136.29, 136.93, 141.11, 148.07, 152.39, 152.44, 154.90,
164.44; HRMS(ESI) m/z: calcd for C21H17F4N4O2S [M+H]+
465.1008, found 465.0995. HPLC purity = 98.37%.
5.5.12. 4-(3-M-tolylthioureido)phenylsulfanyl-N-methyl
nicotinamide (b4)
Light yellow solid, yield: 73.7 %, mp: 144-146℃. 1H NMR
(600 MHz, DMSO-d6) δ 2.79 (d, J = 4.6 Hz, 3H), 3.38 (s, 3H),
6.97 (d, J = 8.0 Hz, 2H), 7.14 -7.36 (m, 3H), 7.56 ( d, J = 8.6 Hz,
2H), 7.70 ( d, J = 8.6 Hz, 2H), 7.90 -8.09 (m, 1H), 8.53 (d, J =
4.7 Hz, 1H), 8.81 (br d, J = 2.8 Hz, 1H), 9.93 (s, 1H), 9.99 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ 20.99, 26.10, 114.98,
119.53, 120.84, 121.49, 122.89, 123.30, 123.92, 124.14, 125.35,
126.31, 128.33, 135.41, 135.77, 139.05, 141.13, 148.17, 163.79,
164.64, 179.30; HRMS(ESI) m/z: calcd for C21H21N4OS2
[M+H]+ 409.1157, found 409.1171. HPLC purity = 98.31%.
5.5.9. 6-{4-[3-(4-Chloro-3-trifluoromethylphenyl)thioureido]
phenylsulfanyl}-N-methylnicotinamide (b1)
6-(4-aminophenylsulfanyl)-N-methylnicotinamide (3) (1.59
g, 6.12 mmol) was dissolved in 10 mL of N,N-
dimethylformamide (DMF) and the solution was cooled to 0–5
°C in ice bath under stirring, a solution of 1-chloro-4-
isothiocyanato-2-trifluoromethylbenzene (6a) (1.4g, 6.18 mmol)
in 10mL DMF were added dropwise at 0–5℃ under nitrogen
atmosphere. The mixture was stirred for 3 h at 0–5℃, and then
stirred for another 18 h at room temperature. The mixture was
poured into brine (200 mL) and extracted with EtOAc (3×200
mL). The organic layer was combined and washed with brine
(2×200 mL), dried over anhydrous sodium sulfate, and
concentrated under reduced pressure to give the crude product.
5.5.13. 6-{4-[3-(4-Chlorophenyl)thioureido]phenylsulfanyl}-
N-methylnicotinamide (b5)
1
Light yellow solid, yield: 77.8 %, mp: 157-159℃. H NMR
(600 MHz, DMSO-d6) δ 2.78 (d, J = 4.7 Hz, 3H), 6.98 (d, J = 8.5
Hz, 1H), 7.29 -7.45 (m, 2H), 7.46-7.61 (m, 4H), 7.63-7.76 (m,
2H ), 8.02 (dd, J = 8.5, 2.4 Hz, 1H), 8.55 (d, J = 4.6 Hz, 1H),
8.81 (d, J = 4.5 Hz, 1H), 10.08 (s, 1H), 10.14 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): δ 26.11, 119.54, 120.26, 120.36, 121.47,
122.59, 123.49, 123.96, 125.24, 126.29, 128.34, 128.44, 135.50,

135.82, 138.21, 140.91, 148.16, 163.71, 164.62, 179.39;
HRMS(ESI) m/z: calcd for C21H18ClN4OS2 [M+H]+ 429.0611,
found 429.0598. HPLC purity = 98.08%.
126.49, 129.26, 129.67, 129.91, 130.49, 135.80, 140.55, 148.25,
150.01, 162.92, 164.58, 179.94; HRMS(ESI) m/z: calcd for
C20H17F2N4OS2 [M+H]+ 431.0812, found 431.0785. HPLC purity
= 99.27%.
5.5.14. 6-{4-[3-(4-Fluoro-3-trifluoromethylphenyl)thioureido]
phenylsulfanyl}-N-methylnicotinamide (b6)
The other compounds of series c (c2-c9) were synthesized
following the general procedure as described above
1
Yellow solid, yield: 46.2%, mp: 137-139℃. H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.7 Hz, 3H), 7.00 (d, J = 8.4 Hz,
1H), 7.39 -7.75 (m, 5H), 7.81 (dd, J = 8.7, 2.1 Hz, 1H), 7.88-
8.11 (m, 2H), 8.53 (d, J = 5.0 Hz, 1H ), 8.80 (br d, J = 4.6 Hz,
1H), 10.13(s, 1H), 10.27(s, 1H); 13C NMR (100 MHz, DMSO-
d6): δ 26.16, 110.83, 116.32, 117.05, 117.27, 117.51, 119.96,
5.5.18. 6-{3-[3-(4-Chlorophenyl)thioureido]phenylsulfanyl}-
N-methylnicotinamide (c2)
Off white solid, yield: 75.7 %, mp: 131-132℃. 1H NMR (400
MHz, DMSO-d6) δ2.78 (d, J = 4.6 Hz, 3H), 7.00 (d, J = 8.1 Hz,
1H), 7.11 (d, J = 8.4 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.50 (t, J
= 7.9 Hz, 1H), 7.55-7.90 (m, 4H), 7.90-8.24 (m, 2H), 8.54 (d, J =
5.0 Hz, 1H), 8.82 (br d, J = 2.6 Hz, 1H), 10.20 (s, 1H), 10.23 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ 26.06, 118.53, 122.41,
122.46, 125.29, 126.54, 128.48, 129.38, 129.63, 129.95, 130.06,
130.70, 131.54, 135.83, 138.93, 140.31, 148.25, 162.81, 164.53,
179.89; HRMS(ESI) m/z: calcd for C21H18ClN4OS2 [M+H]+
429.0611, found 429.0620. HPLC purity = 98.36%.
1
3
122.48 (q, JFC = 270 Hz), 122.65 (q, JFC = 4.56 Hz), 125.66,
3
129.69, 130.67 (d, JFC = 8.38 Hz), 136.27, 139.14, 140.44,
1
147.04, 153.43, 155.53 (d, JFC = 250 Hz), 164.38, 164.56,
180.01; HRMS(ESI) m/z: calcd for C21H17F4N4OS2 [M+H]+
481.0780, found 481.0775. HPLC purity = 99.39%.
5.5.15. 6-{4-[3-(3,4-Difluoro-phenyl)-thioureido]-phenyl
sulfanyl}-N-methyl-nicotinamide (b7)
Light tan solid, yield: 84.0%, mp: 152-154℃. 1H NMR (400
MHz, DMSO-d6) δ2.77 (d, J = 4.4 Hz, 3H), 6.99 (d, J = 8.4 Hz,
1H), 7.20 – 7.29 (m, 1H), 7.36 – 7.49 (m, 1H), 7.52- 7.60 (m,
2H), 7.62 - 7.76 (m, 3H), 8.01 (dd, J = 8.5, 2.5 Hz, 1H), 8.53 (d,
J = 5.6 Hz, 1H), 8.79 (d, J = 2.8 Hz, 1H), 10.08 (s, 1H), 10.16 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ 26.07, 112.99, 122.57,
124.09, 126.57, 128.46, 129.36, 129.62, 130.07, 130.43, 130.71,
134.95, 135.09, 135.84, 139.39, 140.31, 148.24, 162.60, 164.43,
179.71; HRMS(ESI) m/z: calcd for C20H17F2N4OS2 [M+H]+
431.0812, found 431.0802. HPLC purity = 98.46%.
5.5.19.
phenyl)thioureido]
(c3)
6-{3-[3-(4-Fluoro-3-trifluoromethyl-
phenylsulfanyl}-N-methylnicotinamide
Off white solid, yield: 53.9 %, mp: 127-129℃. 1H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.6 Hz, 3H), 7.09 (dd, J = 7.6,
1.7 Hz, 1H), 7.30-7.64 (m, 3H), 7.65-7.90 (m, 3H), 7.95 -8.12
(m, 2H), 8.56 (d, J = 5.4 Hz, 1H), 8.81 (d, J = 2.3 Hz, 1H),
10.20 (s, 1H), 10.22 (s, 1H); 13C-NMR (100 MHz, DMSO-d6): δ
26.07, 112.99, 120.15, 122.63, 126.54, 128.46, 128.70, 129.36,
129.62, 130.07, 130.43, 130.71, 134.95, 135.09, 135.84, 139.39,
140.31, 148.24, 162.80, 164.53, 179.81; HRMS(ESI) m/z: calcd
for C21H17F4N4OS2 [M+H]+ 481.0780, found 481.0801. HPLC
purity = 98.73%.
5.5.16. 6-{4-[3-(3,5-Bis-trifluoromethyl-phenyl)-thioureido]-
phenylsulfanyl}-N-methyl-nicotinamide (b8)
Light yellow solid, yield: 53.6 %, mp: 170-172℃. 1H NMR
(400 MHz, DMSO-d6) δ 2.77 (d, J = 4.8 Hz, 3H), 7.02 (d, J =
8.6, 1H), 7.63 (q, J = 8.6 Hz, 4H), 7.84 (s, 1H), 8.02 (dd, J =
8.4, 2.3 Hz, 1H), 8.26 (s, 2H), 8.56 (d, J = 5.6 Hz, 1H ), 8.79 (br
d, J = 4.8 Hz, 1H), 10.43 (s, 1H), 10.53 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ 26.05, 116.99, 117.03, 120.10, 121.79,
123.52, 123.56, 124.50, 124.98, 126.51, 127.33, 129.49, 129.86,
130.18, 131.00, 135.83, 140.04, 141.59, 148.26, 162.80, 164.58,
180.08; HRMS(ESI) m/z: calcd for C22H17F6N4OS2 [M+H]+
531.0748, found 531.0764. HPLC purity = 99.73%.
5.5.20. 6-{3-[3-(4-Chloro-3-trifluoromethylphenyl)thioureido]
-phenylsulfanyl}-N-methylnicotinamide (c4)
Off white solid, yield: 70.4 %, mp: 135-137℃. 1H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.6 Hz, 3H), 7.11 (dd, J = 7.7,
1.9 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H),
7.59 (dd, J = 8.0, 2.1 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.72 –
7.87 (m, 2H), 7.95-8.14 (m, 2H), 8.54 (d, J = 4.6 Hz, 1H), 8.81
(d, J = 2.4 Hz, 1H), 10.19 (s, 1H), 10.22 (s, 1H); 13C NMR (100
MHz, DMSO-d6): 26.08, 120.15, 122.37, 122.43, 125.28, 126.54,
128.51, 129.36, 129.63, 130.08, 130.71, 131.56, 135.84, 138.93,
140.31, 148.24, 162.79, 164.52, 179.90; HRMS(ESI) m/z: calcd
for C21H17ClF3N4OS2 [M+H]+ 497.0484, found 497.0481. HPLC
purity = 98.65%.
5.5.17. 6-{3-[3-(3,4-Difluorophenyl)-thioureido]-phenyl
sulfanyl}-N-methylnicotinamide (c1)
6-(3-aminophenylsulfanyl)-N-methylnicotinamide (7) (1.07
g, 4.12 mmol) was dissolved in 10 mL of DMF and the solution
was cooled to 0–5 °C in ice bath under stirring, a solution of 1,2-
difluoro-4-isothiocyanato-benzene (6g) (0.71 g, 4.12 mmol) in
10mL DMF were added dropwise under nitrogen atmosphere.
The mixture was stirred for 3 h at 0–5℃, and then stirred for
another 18 h at room temperature. The mixture was poured into
brine (200 mL) and extracted with EtOAc (3×200 mL). The
organic layer was combined and washed with brine (2×200 mL),
dried over anhydrous sodium sulfate, and concentrated under
reduced pressure to give the crude product. which was purified
by chromatography to give an off white solid, yield: 55.7 %, mp:
150-152℃. 1H NMR (400 MHz, DMSO-d6) δ 2.79 (d, J = 4.4
Hz, 3H), 7.12 (d, J = 8.0Hz, 1H), 7.24 (dd, J = 8.4, 2.3 Hz, 1H),
7.31 -7.44 (m, 2H), 7.48 (s, 1H), 7.60 (dd, J = 8.2, 2.2 Hz, 1H),
7.69 (dd, J = 7.8, 2.4 Hz, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.92 -
8.15 (m, 1H), 8.54 (d, J = 4.7 Hz, 1H), 8.83 (d, J = 4.8 Hz, 1H),
10.02 (s, 1H), 10.04 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ
26.07, 113.12, 113.31, 116.85, 117.02, 120.11, 120.46, 124.75,
5.5.21. 6-{3-[3-(4-Bromo-3-trifluoromethylphenyl)thioureido]
phenylsulfanyl}-N-methylnicotinamide (c5)
Off white solid, yield: 53.6%, mp: 138-140℃. 1H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.5 Hz, 3H), 7.10 (dd, J = 7.6,
1.7 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.43-7.54 (m, 2H), 7.60
(dd, J = 8.2, 2.3 Hz, 1H), 7.79 (dt, J = 8.2, 2.4 Hz, 2H), 7.93 (dd,
J = 6.5, 2.7 Hz, 1H), 8.02 (dd, J = 8.5, 2.4 Hz, 1H), 8.57 (d, J =
5.0 Hz, 1H), 8.82 (d, J = 2.3 Hz, 1H), 10.11 (s, 1H), 10.18 (s,
1H); 13C NMR (100 MHz, DMSO-d6): δ 26.06, 117.24, 122.53,
122.58, 124.82, 126.47, 129.49, 129.71, 129.96, 130.05, 130.54,
130.62, 130.66, 135.83, 136.05, 136.08, 140.38, 148.24, 162.90,
164.53, 180.21; HRMS(ESI) m/z: calcd for C21H17BrF3N4OS2
[M+H]+ 540.9979, found 541.0002. HPLC purity = 98.21%.
5.5.22. 6-{3-[3-(4-trifluoromethoxyphenyl)thioureido]phenyl
sulfanyl}-N-methyl-nicotinamide (c6)

Off white solid, yield: 65.1 %, mp: 159-161℃. 1H NMR (400
MHz, DMSO-d6) δ 2.79 (d, J = 4.6 Hz, 3H), 7.12 (d, J = 8.5 Hz,
1H), 7.32 (dd, J = 5.3, 2.6 Hz, 1H), 7.33 (d, J = 9.0 Hz, 2H ),
7.48 (s, 1H), 7.54 -7.68 (m, 3H), 7.81 (s, 1H), 8.02 (dd, J = 8.4,
2.3 Hz, 1H), 8.55 (d, J = 5.3 Hz, 1H), 8.83 (br d, J = 4.2 Hz,1H),
10.05 (s, 1H), 10.06 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ
26.05, 55.20, 118.79, 120.10, 121.15, 121.33, 123.87, 124.62,
125.21, 126.50, 129.20, 129.51, 129.91, 130.39, 135.82, 138.43,
140.66, 144.67, 148.24, 162.94, 164.57, 179.91; HRMS(ESI) m/z:
calcd for C21H18F3N4O2S2 [M+H]+ 479.0823, found 479.0829.
HPLC purity = 98.33%.
3. Dumaz, N.; Marais, R. J. Biol. Chem. 2003, 278, 29819.
4. Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins,
S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.;
Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.;
Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.;
Menzies, A.; Mould, C.; Parker, A.; Stevens, C.;Watt, S.;
Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.;
Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.;
Maitland, N.; Chenevix-Trench, G.; Riggins, G. J.; Bigner,
D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson,
A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.;
Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.;
Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A.
Nature 2002, 417, 949.
5.5.23. 6-{3-[3-(3-Chloro-4-fluorophenyl)thioureido]phenyl
sulfanyl}-N-methylnicotinamide (c7)
5. Holderfield, M.; Merritt, H.; Chan, J.; Wallroth, M.;
Tandeske, L.; Zhai, H. Cancer Cell 2013, 23, 594.
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R.; Cox, C.; Stephens, P.; Edkins, S.; Tsui, W. W.; Chan,
A. S.; Futreal, P. A.; Stratton, M. R.; Wooster, R.; Leung,
S. Y. Cancer Res. 2002, 62, 6451.
7. Nazarian, R.; Shi, H.; Wang, Q.; Kong, X.; Koya, R. C.;
Lee, H.; Chen, Z.; Lee, M. K.; Attar, N.; Sazegar, H.;
Chodon, T.; Nelson, S. F.; McArthur, G.; Sosman, J. A.;
Ribas, A.; Lo, R. S. Nature. 2010, 468, 973.
8. Kimura, E. T.; Nikiforova, M. N.; Zhu, Z.; Knauf, J. A.;
Nikiforov, Y. E.; Fagin, J. Cancer Res. 2003, 63, 1454.
9. Brose, M. S.; Volpe, P.; Feldman, M.; Kumar, M.; Rishi,
I.; Gerrero, R.; Einhorn, E.; Herlyn, M.; Minna, J.;
Nicholson, A.; Roth, J. A.; Albelda, S. M.; Davies, H.;
Cox, C.; Brignell, G.; Stephens, P.; Futreal, P. A.; Wooster,
R.; Stratton, M. R. Cancer Res. 2002, 62, 6997.
10. Chapman, P. B.; Hauschild, A.; Robert, C.; Haanen, J. B.;
Ascierto, P.; Larkin, J. N Engl J Med. 2011, 364, 2507.
11. Mao, M.; Tian, F.; Mariadason, JM.; Tsao, CC.; Lemos,
RJr.; Dayyani, F.; Gopal, YN.; Jiang, Z.; Tang,
Off white solid, yield: 71.5 %, mp: 151-153℃. 1H NMR (400
MHz, DMSO-d6) δ 2.66 (d, J = 4.7 Hz, 3H), 6.74 (d, J = 8.2 Hz,
1H), 7.23 (m, 2H), 7.36 - 7.45 (m, 5H), 7.67 - 7.69 (m, 1H), 8.56
(d, J = 5.6 Hz, 1H), 8.80 (d, J = 4.7 Hz, 1H), 10.09 (s, 1H), 10.11
(s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 26.09, 116.34,
116.56, 118.69, 120.06, 124.71, 125.92, 129.19, 129.72, 129.95,
130.55, 135.82, 136.36, 140.53, 148.24, 153.05, 155.49, 162.97,
164.56,180.04; HRMS(ESI) m/z: calcd for C20H17ClFN4OS2
[M+H]+ 447.0516, found 447.0532. HPLC purity = 98.35%.
5.5.24. 6-{3-[3-(3-trifluoromethylphenyl)-thioureido]phenyl
sulfanyl}-N-methyl-nicotinamide (c8)
Off white solid, yield: 57.6 %, mp: 162-164℃. 1H NMR (400
MHz, DMSO-d6) δ 2.78 (d, J = 4.8 Hz, 3H), 7.11 (d, J = 8.5 Hz,
2H), 7.40 (d, J = 8.6 Hz, 1H), 7.48 - 7.52 (m, 1H), 7.58 (d, J=
1.9 Hz, 1H), 7.66 (d, J = 8.7 Hz, 1H), 7.74 – 7.83 (m, 2H), 7.79
– 8.11 (m, 2H), 8.57 (d, J = 4.6Hz, 1H), 8.81 (q, J = 2.8 Hz, 1H),
10.23 (s, 1H), 10.26 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ
26.08, 118.53, 120.08, 122.53, 122.68, 124.80, 126.48, 128.49,
129.31, 129.66, 129.96, 130.08, 130.73, 131.54, 135.84, 138.92,
140.30, 148.24, 162.86, 164.52, 179.86; HRMS(ESI) m/z: calcd
for C21H18F3N4OS2 [M+H]+ 463.0874, found 463.0867. HPLC
purity = 98.14%.
X.; Bornman. WG.; Bollag, G.; Mills, GB.; Powis,
G.; Desai, J.; Gallick, GE.; Davies, MA.; Kopetz, S. Clin
Cancer Res. 2013, 19, 657.
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Jr.; DiPietro, L.; Doerr, N.; Elbaum, D.; Estrada, J.;
Gallant, P.; Germain, J.; Gu, Y.; Harmange, J.-C.;
Kaufman, S. A.; Kendall, R.; Kim, J. L.; Kumar, G. N.;
Long, A. M.; Neervannan, S.; Patel, V. F.; Polverino, A.;
Rose, P.; Van der Plas, S.; Whittington, D.; Zanon, R.;
Zhao, H. J. Med. Chem. 2007, 50, 4351.
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Zhou, H. Med. Chem. Commun. 2013, 4, 979.
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Wilkie, D.; Wilhelm, S.; Lynch, M.; Carter, C. Cancer
Res. 2006, 66, 11851.
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Patruno, R.; Manqia, A.; Rizzo, A.; Sciorsci, RL.;
Gadaleta, CD. Curr. Med. Chem. 2012, 19, 938.
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Llovet, J. M.; Lynch, M. Mol. cancer ther. 2008, 7, 3129.
17. Niculescu-Duvaz, D.; Gaulon, C.; Dijkstra, H. P.;
Niculescu-Duvaz, I.; Zambon, A.; Menard, D.;
5.5.25. 6-{3-[3-(3,5-Bistrifluoromethylphenyl)thioureido]
phenylsulfanyl}-N-methylnicotinamide (c9)
1
Brown solid, yield: 67.6%, mp: 171-173℃. H NMR (400
MHz, DMSO-d6) δ 2.75 (d, J = 4.8 Hz, 3H), 6.96 (dd, J = 7.7,
1.8 Hz, 1H ), 7.16 (dd, J = 5.6, 2.4 Hz, 1H), 7.45 (d, J = 8.6 Hz,
1H), 7.46 - 7.54 (m, 3H), 7.55 (t, J= 7.7 Hz, 1H), 7.84 (d, J = 7.7
Hz, 1H), 8.27 (s, 2H), 8.31 (d, J = 5.4 Hz, 1H), 10.34 (s, 1H),
10.40 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 26.05, 116.99,
117.03, 120.10, 121.79, 123.52, 123.56, 124.50, 124.98, 126.51,
127.37, 129.49, 129.86, 130.18, 131.00, 135.83, 140.04, 141.59,
148.26, 162.80, 164.56, 180.02; HRMS(ESI) m/z: calcd for
C22H17F6N4OS2 [M+H]+ 531.0748, found 531.0759. HPLC purity
= 98.37%.
Acknowledgments
Suijkerbuijk, B. M.; Nourry, A.; Davies, L.; Manne, H.;
Friedlos, F.; Ogilvie, L.; Hedley, D.; Whittaker, S.; Kirk,
R.; Gill, A.; Taylor, R. D.; Raynaud, F. I.; Moreno-Farre,
J.; Marais, R.; Springer, C. J. J. Med. Chem. 2009, 52,
2255.
This work was supported by National Natural Science
Foundation of China (No. 81202038, 81728020), Key Research
Project of Yantai City (2017YT06000336, 2017ZH075) and
Ph.D. Programs Foundation of Yantai University (No.
YX13B03-2013).
18. Ramurthy, S.; Subramanian, S.; Aikawa, M.; Amiri, P.;
Costales, A.; Dove, J.; Fong, S.; Jansen, J. M.; Levine, B.;
Ma, S.; McBride, C. M.; Michaelian, J.; Pick, T.; Poon, D.
J.; Girish, S.; Shafer, C. M.; Stuart, D.; Sung, L.;
Renhowe, P. A. J. Med. Chem. 2008, 51, 7049.
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Graphical Abstract
Design and discovery of thioether and
nicotinamide containing sorafenib analogs as
multikinase inhibitors targeting B-Raf, B-
RafV600E and VEGFR-2
Leave this area blank for abstract info.
Shaofeng Suna, Zuopeng Hea, Mindong Huanga , Ningning Wanga, Zongzhong Heb，Xiangkai Konga and
Jianwen Yaoa,∗
a School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of
Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in
Universities of Shandong, Yantai University, 32 Qingquan Road, Yantai 264005, PR. China.
b Molecular Biology Division, Yantai Aillen Biological Technology Co.,Ltd.,39 Keji Road, Yantai Shandong,
264670, PR China
O
H
N
H
N
R1
H
H
F3C
Cl
N
N
N
N
H
H
N
O
X
O
S
N
R2
O
X=O/S
thioether
nicotinamide
Novel sorafenib derivatives
sorafenib
Thioether and nicotinamide-containing sorafenib analogues with better anti-proliferative and anti-angiogenic activities
than Sorafenib were well designed and synthesized as B-Raf, B-RafV600E and VEGFR-2 multikinase inhibitors.