Design, synthesis and biological evaluation of thiourea and nicotinamide-containing sorafenib analogs as antitumor agents

Article abstract of DOI:10.1039/c4md00536h

A series of thiourea and nicotinamide-containing sorafenib analogs (7a-n) were designed and synthesized and their antiproliferative activities were tested against HCT116, MDA-MB-231, PC-3 and HepG2 cell lines. Most of the compounds showed potent activities against the four cell lines, compound 7h showed better activities than sorafenib against all four cell lines, and compounds 7a and 7e showed better activities against HCT116 and MDA-MB-231 cell lines. The anti-angiogenic activities of 7e and 7h were also better than that of sorafenib in both in vitro HUVEC tube formation assay and ex vivo rat thoracic aorta ring assay.

Full text of DOI:10.1039/c4md00536h

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Design, synthesis and biological evaluation of thiourea and
nicotinamide-containing sorafenib analogs as antitumor
agents
www.rsc.org/medchemcomm
Xiangkai Kong,a Zeyu Yao,a Zuopeng He,a Wenfang Xub and Jianwen Yao a*
aDepartment of Medicinal Chemistry, School of Pharmacy, Yantai University, Yantai, Shandong, 264005,
PR China. E-mail: jwy5610201@vip.sina.com
bDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, Jinan, Shandong 250012, PR China
A series of thiourea and nicotinamide-containing sorafenib analogs (7a-n) were designed and
synthesized and their antiproliferative activities were tested against HCT116, MDA-MB-231, PC-3 and
HepG2 cell lines. Most compounds showed potent activities against four cell lines, compound 7h
showed better activities than sorafenib against all four cell lines, and compound 7a, 7e showed better
activities against HCT116 and MDA-MB-231 cell lines. The anti-angiogenic activities of 7e and 7h are
also better than that of sorafenib in both in vitro HUVEC tuber formation assay and ex vivo rat thoracic
aorta rings assay.
Introduction
In our previous work, a primary structure and activity
relationships (SARs) of diaryl thiourea-containing sorafenib
It is well known that inhibition of two or more tumor-related
enzymes is effective in the treatment of cancers. More and more
attentions are being paid to multi-target drugs, because they can
impact multiple targets simultaneously, and are better at controlling
complex disease systems and are less prone to drug resistance1. One
of the most famous multiple-targeted drugs, sorafenib, shows many
derivatives can be summarized: 1) Substitution of urea with
thiourea can enhanced the antiproliferative activities of target
compounds. 2) Compounds with a methyl group on the terminal
amide have the best antiproliferative activities. 3) Compounds with
1,3-substitution on the B ring showed stronger anti-angiogenic
activities than those with 1,4-substitution.
advantages in the treatment of primary kidney cancer (advanced
2,3
renal cell carcinoma)
and advanced primary liver cancer4,5
Based the above mentioned structure and activity relationships
of diaryl thiourea-containing sorafenib derivatives, in this report,
we describe a novel series of thiourea-containing derivatives of
sorafenib (as shown in Fig. 1D). To enhance their biological
activities, the thiourea group and formamide terminal are reserved,
and 1,3-substitution mode is chosen on the B ring. Especially the
2,4-substitution mode on C ring is changed with 2,5-substitution
mode for the following reasons: a) Modification on this part may
change the binding mode and get better antitumor agents. b) 2,5-
substitution mode could increase the water solubility of target
compounds because there is no intramolecular hydrogen bond
between the amide and the nitrogen atoms on the pyridine. c) The
2,5-substitution mode (nicotinamide) can increase the drug-likeness
of target compounds because nicotinamide is already used
clinically.
because it can inhibit several kinases involved in tumor
proliferation and angiogenesis including Raf, VEGFR, PDGFR and
KIT3,6,7. It has been considered as a lead compound for further
optimization in the past several years8.
Hitherto, many efforts have been undertaken in modification
of sorafenib to find better multiple-targeted drugs9-11. And most of
the reported sorafenib derivatives are based on diaryl urea
skeleton12-13. Inspired by the classic bioisosteric paradigm of
thiopental replacing urea in pentobarbital with thiourea, a series of
diaryl thiourea-containing sorafenib derivatives (as shown in Fig.
1B and 1C) were synthesized in our group 14-16, and showed
stronger antiproliferative activities against HCT-116 or MDA-MB-
231 cells than sorafenib. And some of them also showed inhibitory
activities against the phosphorylation of VEGFR and the anti-
angiogenic activities to rat aortic ring.
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Fig.1 Modifications of sorafenib
O
NH
COOCH
N
3
H N
O
2
a
b
H
N
N
N
H
H
R
N
N
O
Cl
1
Cl
O
e
2
3
H
S
N
N
H
NH
2
N
SH
NCS
c
O
d
7a-i
R
R
R
S
5a-i
4a-i
6a-i
Scheme 1 Reagents and conditions: (a) CH3NH2 (b) 3-aminophenol, DMF, t-BuOK (c) CS2, toluene (d) BTC, DCM (e) DMF
Biological evaluation
Results and discussions:
In vitro antiproliferative activity
Chemistry:
In vitro cell cytotoxicity of the new nicotinamide-containing
The synthetic routes to the target compounds were illustrated as
derivatives was initially evaluated against HCT116, MDA-MB-231,
outlined in Scheme 1.
PC-3 and HepG2 cells lines by MTT assay using sorafenib and
BMCL9o16 as positive controls. As show in table 1, most
compounds showed potent activities against all four cell lines.
6-chloronicotinic acid methyl ester (1) reacts with
methylamine to generate 6-chloronicotinic acid methyl amide (2),
Compound 7a, 7e, 7h showed better antiproliferative activities than
which was treated with 3-aminophenol to get diaromatic ether (3).
sorafenib and compound 7d, 7i, 7l, 7m, 7n showed relative weaker
Furthermore, substituted anilines 4 were treated with CS2 to
generate 5, and then treated with triphosgene (BTC) to produce
various isothiocyanates (6). Finally, these isothiocyanates were
reacted with 3 in DMF to afford 7 in the total yield of 19–48%. The
activities than others. This suggested that compounds which had
two strong electron-withdrawing groups (such as F, Cl, Br, CF3) on
their terminal phenyl ring have better activities than those with only
one substitution. Compound 7h showed better antiproliferative
activities than BMCL9o, this indicated that the nicotinamide
moiety may enhance the cell cytotoxicity of this series of
compounds than pyridine moiety. Compared to the thiourea and
final products were purified by column chromatography and their
1
structures were characterized by H NMR，13C NMR, MS and
elemental analysis.
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pyridine-2-carboxamide containing compounds in our previous
work15, the inhibitory activities against MDA-MB-231 cells of the
target compounds in this paper were much better, which indicated
that the different aromatic C ring may change the binding mode of
compounds to their specific targets .
sorafenib at 0.05 μM and completely inhibited at 0.1 μM.
Surprisingly, both 7e and 7h can inhibit tuber formation completely
at 0.05 μM.
Ex vivo rat thoracic aorta rings (TARs) assay
Just as compound 7e and 7h can well inhibit the formation of
HUVECs tubular structure, ex vivo TARs assay was also employed
to further study the anti-angiogenic activity of compound 7e and 7h
because of TARs model involving multiple steps in angiogenesis:
sprouting, proliferation, migration and differentiation and being
more close to conditions in vivo. As shown in Fig. 3, Compound 7e
and 7h have better inhibitory activities than sorafenib at 0.1 μM,
and have comparable inhibitory activities at 0.05 μM.
In vitro HUVEC tuber formation assay
In view of their significant antiproliferative activities to tumor cells,
compound 7e and 7h were chosen to test their anti-angiogenic
activities by in vitro HUVEC tuber formation assay using sorafenib
as positive controls. As shown in Fig. 2, elongated and robust tube-
like structures were well established in the negative control
(DMSO); The HUVEC tuber formation was partially inhibited by
a
Table 1 Structures and IC50 values of target compounds
H
H
N
N
O
Ar
H
S
N
N
O
Compounds
NO.
Substituent
IC50 (µM)a
Ar-
CF3
Br
HCT116
MDA-MB-231
PC-3
HepG2
3.12 ± 0.55
3.02 ± 0.42
2.67 ± 0.02
8.53 ± 0.56
7a
7b
7c
7d
CF3
F
19.01 ± 1.03
25.41 ± 1.19
36.65 ± 3.87
13.29 ± 0.09
28.59 ± 1.87
38.08 ± 2.21
7.95 ± 0.05
18.35 ± 0.73
15.36 ± 2.37
20.20 ± 0.21
> 100
Cl
F
CF3
23.32 ± 2.82
CF3
2.50 ± 0.35
2.67 ± 0.15
5.86 ± 0.25
4.55 ± 0.34
7e
CF3
Cl
42.51 ± 0.38
38.19 ± 0.37
66.48 ± 3.27
69.34 ± 3.22
28.36 ± 0.46
23.14 ± 0.63
29.37 ± 1.08
26.72 ± 1.53
7f
F
7g
F
CF3
Cl
2.21 ± 0.09
54.68 ± 0.53
20.89 ± 0.58
53.72 ± 0.93
2.33 ± 0.22
55.95 ± 0.57
20.67 ± 0.78
29.46± 0.84
1.98 ± 0.23
> 100
5.35 ± 0.21
> 100
7h
7i
OCF3
Cl
19.34 ± 0.96
> 100
21.56 ± 1.62
65.38 ±0.64
7j
Cl
CH
3
7k
CH
3
48.23 ± 0.36
38.56 ± 0.89
67.38 ± 1.32
> 100
7l
CH
3
F
39.37 ± 0.45
49.84 ± 0.24
21.65 ± 0.57
31.54 ± 0.63
32.67 ± 0.48
> 100
42.54 ± 0.69
85.34 ± 1.43
7m
7n
OCH
3
H
N
H
N
F
C
3
S
Cl
O
BMCL9o
14.58 ± 0.34
42.43 ± 0.68
16.38 ± 0.38
24.89 ± 1.45
O
N
H
N
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sorafenib
___
8.41 ± 0.19
17.84 ± 1.93
10.98 ± 0.67
10.48 ± 1.52
a Assays were performed in triplicate (n≥3); values were shown as mean ± SD.
sorafenib
sorafenib
DMSO
DMSO
0.05μM
0.1μM
7e
7h
7e
7h
0.05μM
0.1μM
0.05μM
0.1μM
Fig.2 Representative images of tubes formed on Matrigel after seeding with HUVEC and treating with DMSO, sorafenib, 7e and 7h at
different concentrations.
sorafenib
sorafenib
DMSO
DMSO
0.1μM
0.05μM
7e
7e
7h
7h
0.05μM
0.05μM
0.1μM
0.1μM
Fig.3 Microvessle growth ex vivo. Aortic segments isolated from Sprague-Dawley rats were placed in growth factor-reduced Matrigel-
covered wells in the absence (DMSO) or presence of the compounds (sorafenib, 7e, 7h) for 6 days at different concentrations.
Conclusion
Acknowledgements
In summary, a novel series of sorafenib analogs were
designed and synthesized as antitumor agents. Compared to
sorafenib, most of the compounds showed potent inhibitory
activities against four cell lines and compound 7a, 7e, 7h with
This work was supported by Ph.D. Programs Foundation of
Ministry of Education of China (No. 20110131110037) and
Ph.D. Programs Foundation of Yantai University (No.
YX13B03-2013).
two strong electron-withdrawing groups on their terminal
phenyl ring exhibited better inhibitory activities. Compared to
the reported compound BMCL9o, compound 7h showed
better antiproliferative activities against all four cell lines. It
indicated that the nicotinamide moiety may enhance the cell
cytotoxicity of this series of compounds. It can also be
inferred that the nicotinamide-containing derivates may lead a
new binding mode to specific target in MDA-MB-231 cells
because of the dramatic changes of the antiproliferative
activities against MDA-MB-231 cell lines compared to the
diaryl thiourea-containing sorafenib derivatives previously
reported16. Furthermore, As shown in vitro HUVEC tuber
formation assay and ex vivo rat thoracic aorta rings assay, 7e
and 7h have excellent anti-angiogenic activities, and may be
developed as potent anticancer agents in the future. And their
mechanisms is now under investigated.
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