Introduction of mercaptoethyl at sorafenib pyridine‐2‐amide motif as a potentially effective chain to further get sorafenib‐PEg‐DGL

Article abstract of DOI:10.3390/molecules25030573

The crystal structure of the sorafenib and B‐RAF complex indicates that the binding cavity occupied by the pyridine‐2‐carboxamide in sorafenib has a large variable space, making it a reasonable modification site. In order to identify novel compounds with anti‐cancer activity, better safety and polar groups for further application, five sorafenib analogs with new pyridine‐2‐amide side chains were designed and synthesized. Preliminary pharmacologic studies showed that these compounds displayed much lower toxicities than that of sorafenib. Among them, compound 10b bearing mercaptoethyl group kept relevant antiproliferation potency compared to sorafenib in Huh7 and Hela cell lines with values of IC50 58.79 and 63.67 μM, respectively. As a small molecule inhibitor targeting protein tyrosine kinases, thiol in compound 10b would be an active group to react with maleimide in a mild condition for forming nanoparticles Sorafenib‐PEG‐DGL, which could be developed as a delivery vehicle to improve the concentration of anti‐tumor therapeutic agents in the target cancer tissue and reduce side effects in the next study.

Full text of DOI:10.3390/molecules25030573

molecules
Article
Introduction of Mercaptoethyl at Sorafenib
Pyridine-2-Amide Motif as a Potentially Effective
Chain to Further get Sorafenib-PEG-DGL
Ke Wang 1,2,†, Kudelaidi Kuerbana 3,†, Qi Wan 1, Zhihui Yu 1, Li Ye 3,* and Ying Chen 1,
*
1
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China;
16211030008@fudan.edu.cn (K.W.); kudelaidi@fudan.edu.cn (K.K.); 17211030068@fudan.edu.cn (Q.W.);
18211030008@fudan.edu.cn (Z.Y.)
Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison,
777 Highland Avenue, Madison, WI 53705-2222,USA
2
3
Department of Biological Medicines & Shanghai Engineering Research Center of ImmunoTherapeutics,
School of Pharmacy, Fudan University, Shanghai 201203, China
*
†
Correspondence: yelil@fudan.edu.cn (L.Y.); yingchen71@fudan.edu.cn (Y.C.)
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 December 2019; Accepted: 26 January 2020; Published: 28 January 2020
Abstract: The crystal structure of the sorafenib and B-RAF complex indicates that the binding cavity
occupied by the pyridine-2-carboxamide in sorafenib has a large variable space, making it a reasonable
modification site. In order to identify novel compounds with anti-cancer activity, better safety and
polar groups for further application, five sorafenib analogs with new pyridine-2-amide side chains
were designed and synthesized. Preliminary pharmacologic studies showed that these compounds
displayed much lower toxicities than that of sorafenib. Among them, compound 10b bearing
mercaptoethyl group kept relevant antiproliferation potency compared to sorafenib in Huh7 and Hela
cell lines with values of IC50 58.79 and 63.67 µM, respectively. As a small molecule inhibitor targeting
protein tyrosine kinases, thiol in compound 10b would be an active group to react with maleimide
in a mild condition for forming nanoparticles Sorafenib-PEG-DGL, which could be developed as a
delivery vehicle to improve the concentration of anti-tumor therapeutic agents in the target cancer
tissue and reduce side effects in the next study.
Keywords: sorafenib analogs; antiproliferation; nanoparticles (NPs); molecular modeling
Academic Editor: Fawaz Aldabbagh
1. Introduction
Protein tyrosine kinases (PTKs) are key enzymes in many signal transduction pathways and play
a crucial role in various cell regulatory processes [
have been developed as antitumor drugs in the past ten years [
and Drug Administration (FDA) in 2005, has shown to be effective against several solid tumors and a
standard treatment for HCC (hepatocellular carcinoma) [ ] and renal cancer. Sorafenib is a multikinase
inhibitor that blocks several targets including Fms-like tyrosine kinase-3 (FLT3), platelet-derived
growth factor (PDGF), vascular endothelial growth factor (VEGF), c-Kit and B-RAF signaling [ ] in
both tumor cells and the surrounding endothelial cells [ ]. The underlying mechanism is believed to
1,2]. Numerous small molecule inhibitors for PTKs
3]. Sorafenib, approved by the Food
4
5
6–8
involve competitive inhibition of the ATP binding to the catalytic domains of the various kinases [9].
The crystal structure of sorafenib and B-RAF kinase complex (PDB code: 1UWH) shows that
sorafenib spans the length of interfacial cleft of bilobal architecture and buries deep between C and
N loop [10]. As shown in Figure 1, the pyridyl ring of sorafenib occupies the ATP-binding pocket,
Molecules 2020, 25, 573; doi:10.3390/molecules25030573
www.mdpi.com/journal/molecules

Molecules 2020, 25, 573
2 of 12
forming a hydrogen bond with Cys531 and interacting with aromatic residues of Trp530 and Phe582.
On the other end of sorafenib, the lipophilic trifluoromethyl phenyl ring inserts into a hydrophobic
pocket formed between αC and αE helices and N-terminal regions of DFG (Asp-Phe-Gly) motif and
the catalytic loop. What is more, the urea group forms three hydrogen bonds with catalytic residue
Gln500 and the main chain nitrogen of Asp593. The binding mode implies that the pyridyl ring, urea
group and trifluoromethyl phenyl ring are key to binding affinity, and the methyl group of amide in
the pyridine-2-amide motif may allow to be further modified by other substituents.
Figure 1. The structure of sorafenib (top) and the binding mode (bottom, left) and binding cavity
(bottom, right) of sorafenib and B-RAF kinase.
In 2012, Feng’s group claimed a series of 2-picolinyl hydrazide-containing sorafenib analogs
exhibiting better antitumor activity against pancreatic cancer and HCC cell lines than that of
sorafenib [11]. The results suggested that the modification of the pyridine-2-amide motif was
reasonable, which encouraged us to replace the methyl group of pyridin-2-amide with several alkyl
side chains containing hydroxyl or thiol group and synthesize five new derivatives (Figure 2, 10a
and 15a ). The research work of this structure optimization aimed at maintaining their strong
antiproliferation activity as well as exploring an effective side chain containing OH or SH for preparing
NPs (nanoparticles) in the next study. After the structures of 10a and 15a were confirmed by
–c
–
b
–
c
–b
1H-NMR, 13C-NMR and ESI-HRMS spectra, they further were screened for cytotoxicity against seven
solid cancer cell lines and two nontumorigenesis cell lines with sorafenib as a control by MTT assays.
The results indicated that the length of side chains had moderate influence on the antitumor activity of
target compounds. In addition, compound 10b would be chosen for synthesizing Sorafenib-PEG-DGL
in the next study because of its potential antitumor activity and mercaptoethyl group, which could
react with maleimide (MAL) in a mild condition.
Figure 2. Design of sorafenib derivatives and nanoparticles (NPs).

Molecules 2020, 25, 573
3 of 12
2. Results and Discussion
2.1. Chemistry
Ethanolamine, 3-aminopropan-1-ol and 2-mercaptoethylamine as side chains were introduced
into sorafenib. Meanwhile, as Scheme 1 described, tertbutyldimethylsilane chloride (TBSCl) and
triphenylmethanol firstly reacted with OH or SH to get 2a
caused by OH or SH group over-reaction. Moreover, the key intermediate
starting material N-methyl-4-chloropyridine-2-carboxamide ( ), which reacted with 4-aminophenol to
–
b
and
4
for avoiding the by-products
7
was synthesized from
5
form compound 6 in the presence of potassium tert-butoxide and K2CO3, following demethylated in
2.5 mol/L KOH in 81% overall yield.
Scheme 1. Synthetic routes of 2a
imidazole, dichloromethane (DCM), rt, 1 h, 98%; (
–
b
,
4
and the key intermediate
) triphenylmethanol, trifluoroacetic acid (TFA), rt,
) 4-amino phenol, (CH3)3COK, K2CO3, 80 ◦C, 3 h, 90% and (
) KOH (2.5 mol/L), reflux, 2 h,
7. Reagents and conditions: (a) TBSCl,
b
2 h, 90%; (
90%.
c
d
The synthesis of target compounds 10a
–
c
and 15a
–
b
b
starting from the intermediate
7
was
in
depicted in Scheme 2. Compound was treated with 2a
7
–
and to provide intermediates 8a
4
–
c
the room temperature, using 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethyllaminopropyl)
carbodiimide hydrochloride(EDC.HCl) or diisopropyl ethyl amine (DIEA) as condensing agents.
Then, 8a
which were deprotected to give target compounds 10a
fluoride (TBAF)/THF or triisopropylsilane (TIS)/TFA. Moreover, the methylation of carboxyl group
of with methanol in thionyl chloride afforded ester compound 11, which was treated with
–
c
were reacted with 4-chloro-3-trifluoromethylphenyl isocyanate to give compounds 9a
–c,
–c
under the condition of tetrabutylammonium
7
4-chloro-3-trifluoromethylphenyl isocyanate to form compound 12 in high yield. Then, compound 12
was reacted with hydrazine hydrate to obtain compound 13, further condensed with hydroxyacetone
or 4-hydroxy-2-butanone using acetic acid (HAc) as a catalyst in EtOH to provide intermediates 14a–b.
Finally, the reduction of compounds 14a
compounds 15a–b.
–b with sodium cyanoborohydride (NaBH3CN) gave target

Molecules 2020, 25, 573
4 of 12
Scheme 2. Synthetic routes of target compounds 10a
or
, HOBt, EDC.HCl, DIEA, DCM, rt, 10 h, 86–88%; (
DCM, rt, 2 h, 85–93%; (
reflux, 8 h, 86%; (i) NH2NH2 H2O, EtOH, rt, 15 h, 95%; (j) Hydroxyacetone or 4-hydroxy-2-butanone,
HAc, EtOH, reflux, 8 h, 75% and (k) NaBH3CN, HAc, MeOH, rt, 8 h, 87%.
–
c
and 15a
–b. Reagents and conditions: (e) 2a–b
4
f) 4-chloro-3-trifluoromethylphenyl isocyanate,
g
) I: TBAF, THF, rt, 1 h, 95%; II: TIS, TFA, DCM, rt, 84%. (
h
) MeOH, SOCl2,
.
2.2. In Vitro Antitumor Activities
As shown in Table 1, five target compounds (10a
their effects on cell viability using the MTT assays. These compounds 10a
2-position of pyridine retained cytotoxic activity against two hepatocellular carcinoma cell lines HepG2
and Huh7. Compound 10b displayed the best activity against Huh7 with the IC50 value of 58.79 M.
–
c
and 15a
–
b) were tested in seven cancer cells for
–
c
bearing amide group at the
µ
What is more, thiol-containing 10b also exhibited better antitumor activity against MDA-MB-231
(triple negative breast cancer) and Hela (human cervical carcinoma) cell lines than that of 10a bearing
hydroxyl group. Compared to 10a–c, compounds 15a–b containing hydrazine side chain almost lost
the antiproliferation activity against Huh7, MCF-7, MDA-MB-231 and Hela cell lines. Especially,
compound 15b having the longest side chain hardly showed the cytotoxic effects on the viability in all
tested cancer cell lines. All newly synthesized compounds displayed much weaker activity against
mouse melanoma cell line B16F10 compared to sorafenib.
Table 1. Antiproliferation activities against seven cancer cell lines for 10a–c and 15a–b.
IC50 (µM)
Compound
HepG2
Huh7
MCF-7
MDA-MB-231
Hela
A549
B16F10
10a
10b
10c
15a
15b
70.67
282.2
90.1
80.14
NA
318.8
58.79
88.35
NA
471.2
159.3
268.1
NA
NAa
294.6
329.9
NA
NA
63.67
232.7
NA
287.3
446.6
NA
46.23
NA
NA
NA
449.3
NA
NA
NA
NA
NA
NA
Sorafenib
24.07
19.49
37.23
12.26
49.46
17.44
17.34
a: NA for IC50> 500 µM.

Molecules 2020, 25, 573
5 of 12
Notably, compounds 10a
–
c
and 15a
–
b
expressed far lower toxicities than sorafenib in HUVEC
(Human Umbilical Vein Endothelial Cells) and 293T (Human Adrenal microvascular Endothelial
Cells), indicating that these novel compounds have a noteworthy selectivity between tumor and
nontumorigenesis cell lines (Table 2).
Table 2. Antiproliferation activities for 10a–c and 15a–b in nontumorigenesis cells.
IC50 (µM)
10a
10b
10c
15a
15b
Sorafenib
HUVEC
293T
582.6
643.3
197.3
135.3
329.4
454.4
320.5
1576
1246
559.4
35.45
16.07
2.3. Molecular Modeling
Furthermore, we docked compound 10b into the B-RAF crystal structure (PDB: 1UWH) using
Autodock Vina 1.1.2 [12]. The docking mode showed that compound 10b also inserted into interfacial
cleft between the N and C lobes, binding to the same active site as sorafenib (Figure 3). The urea group
formed hydrogen bonds with a carboxyl of residue Gln500 and the main chain nitrogen of Asp593.
The central phenyl ring of 10b contacted with the residues Leu513 and Phe594, contributing to the van
der Waals interaction. However, the trifluoromethylphenyl ring in compound 10b distributed closely
to the αC helix instead of the hydrophobic pocket that formed between the αC and αE helices and
N-terminal regions of the DFG motif, which might explain the slightly decreased activity. Altogether,
the binding mode suggested that 10b could form a strong binding in the active site of B-RAF. Therefore,
the compound 10b bearing thiol group might be as an effective compound to react with maleimide
(MAL) in the mild condition for further application [13].
Figure 3. Predicted binding mode of sorafenib and compound 10b (affinity:
kinase. Sorafenib is in green and compound 10b in red.
−
11.2 kcal/mol) in B-RAF
3. Materials and Methods
3.1. General Information
Chemicals were purchased from the following suppliers: Sinopharm, Adamas and Sigma Aldrich.
Solvents were dried before use, if required. Air- and moisture-sensitive reactions were carried out
◦
under nitrogen atmosphere. Room temperature (rt) refers to 20–25 C. The progress of a reaction

Molecules 2020, 25, 573
6 of 12
was monitored by thin layer chromatography (TLC) using pre-coated TLC sheets purchased from
Sinopharm. Detected spots were observed under UV light at
λ
254 nm and 365 nm. Melting points
1
were measured on a SGW X-4 microscopy melting point apparatus without correction. H spectral data
were recorded with a Varian Mercury Plus 400 MHz spectrometer and 13C-NMR spectral data were
recorded with a Bruker DRX 600MHz spectrometer, both at 303 K using TMS as an internal standard.
All chemical shifts were reported in ppm (
δ) and coupling constants (J) were in hertz (Hz). Mass
spectra were recorded on Agilent Technologies 1260 infinity LC/MS instrument. The chromatograms
were conducted on silica gel (100
(Supplementary Materials).
−
200 mesh) and visualized under UV light at λ 254 and 365 nm
3.2. Synthesis
General Procedure for the Preparation of 2a–b. To dimethyl isopropyl chlorosilane (TBSCl,
3.15 g, 21.0 mmol) in dichloromethane (DCM, 10 mL) was added to a mixture of ethanolamine
or 3-aminopropan-1-ol (20.0 mmol) and imidazole (2.72 g, 40.0 mmol) in DCM (40 mL) in room
temperature. The mixture was reacted for 3 h and then poured into water (60 mL), extracted with
DCM (3
×
30 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered
and the filtrate was concentrated. The residue was dried to give 2a–b.
2-((Tert-butyldimethylsilyl)oxy)ethan-1-amine (2a): The title compound was obtained starting from
1
ethanolamine (1a). Analytical data for 2a (colorless liquid, 98% yield): H-NMR (400 MHz, CDCl3)
δ
3.68–3.58 (m, 2H, NH2CH2CH2O-), 2.78 (dd, 2H, NH2CH2CH2O-, J = 9.4, 4.6 Hz), 0.91 (s, 9H,
-SiC(CH3)3), 0.07 (s, 6H, -Si(CH3)2).
3-((Tert-butyldimethylsilyl)oxy)propan-1-amine (2b): The title compound was obtained starting from
1
3-aminopropan-1-ol (1b). Analytical data for 2b (colorless liquid, 99% yield): H-NMR (400 MHz,
CDCl3)
δ 3.70 (t, 2H, -CH2CH2O-, J = 6.0 Hz), 2.80 (t, 2H, NH2CH2-, J = 6.7 Hz), 1.71-1.61 (m, 2H,
-CH2CH2CH2O-), 0.89 (s, 9H, -SiC(CH3)3), 0.05 (s, 6H, -Si(CH3)2).
2-(Tritylthio)ethanamine ( ): Triphenylmethyl ( , Trt, 2.29 g, 8.8 mmol) was added to a solution of
4
3
2-mercaptoethylamine (1.0 g, 8.8 mmol) in trifluoroacetate (TFA, 10 mL) in room temperature. The
mixture was reacted for 1 h and turned to red. Then, it was poured into water (40 mL), extracted
with ethyl acetate (EA, 3
Na2SO4, filtered and the filtrate was concentrated. A white precipitate was filtered and recrystallized
from EA to produce
(white solid, 90% yield, mp 93–95 ◦C). ESI-MS m/z 320.2 [M + H]+; 1H-NMR
(400 MHz, CDCl3) 7.74 (brs, 2H, -NH2), 7.41 (d, 5H, ArH, J = 7.3 Hz), 7.27 (m, 6H, ArH), 7.22–7.17 (m,
4H, ArH), 2.59 (t, 2H, -CH2CH2S-, J = 6.6 Hz), 2.26-2.19 (m, 2H, NH2CH2CH2-).
×
20 mL). The combined organic layers were washed with brine, dried over
4
δ
N-methyl-4-(4-aminophenoxy)picolinamide ( ): Potassium tert-butoxide (1.4 g, 11.3 mmol) was added
to a solution of 4-aminophenol (1.0 g, 9.1 mmol) in DMF (15 mL) under nitrogen atmosphere. The
mixture was stirred for 3 h in room temperature and N-methyl-4-chloropyridine-2-carboxamide (
1.2 g, 7.1 mmol) and K2CO3 (0.6 g, 4.2 mmol) was added. Then, the suspension was stirred at 80 ◦C
for 3 h and poured into water (40 mL), extracted with ethyl acetate (EA, 3 30 mL). The combined
organic layers were washed with brine, dried over Na2SO4, filtered and the filtrate was concentrated
to afford 8.33 (d,
(yellow oil, 92% yield). ESI-MS m/z 244.2 [M + H]+; 1H-NMR (400 MHz, CDCl3)
1H, ArH, J = 5.5 Hz,), 8.01 (brs, 1H, -CONHCH3), 7.67 (d, 1H, ArH, J = 1.9 Hz), 6.92 (dd, 1H, ArH, J1 =
6
5,
×
6
δ
5.5 Hz, J2 = 2.4 Hz), 6.88 (d, 2H, ArH, J = 8.6 Hz), 6.71 (d, 2H, ArH, J = 8.6 Hz), 3.00 (d, 3H, -NHCH3
J = 5.1 Hz).
,
4-(4-Aminophenoxy)-2-pyridine carboxylic acid (7): To a stirred solution of K2CO3 (2.5 mol/L, 20 mL), 6
(1.70 g, 7.0 mmol) was added. After 5 h of refluxing reaction, the solution was carefully adjusted to
pH 5 by the addition of 2N HCl. The reaction mixture was evaporated and the residue was purified
by column chromatography on silica gel (100
−
200 mesh, and visualized under UV light at λ 254 and
365 nm; eluent, DCM/MeOH 5:1) to give
7
(white solid, 90% yield, mp 207–209 ◦C). ESI-MS m/z 231.1

Molecules 2020, 25, 573
7 of 12
[M + H]+; 1H-NMR (400 MHz, DMSO-d6)
δ
8.52 (d, 1H, ArH, J = 4.2 Hz), 7.36 (s, 1H, ArH), 7.12 (s, 1H,
ArH), 6.88 (d, 2H, ArH, J = 7.6 Hz), 6.64 (d, 2H, ArH, J = 7.5 Hz).
General Procedure for the Preparation of 8a . 1-Hydroxybenzotriazole (HOBt, 100 mg, 0.7 mmol),
3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC.HCl, 150 mg, 0.78 mmol) and
diisopropylamine (0.4 mL, 2.34 mmol) were combined in a stirred mixture of (100 mg, 0.43 mmol) in
DCM (15 mL). After adding compound 2a or and stirring reaction for 10 h at room temperature,
the mixture was poured into water (30 mL), extracted with DCM (3 15 mL). The combined organic
layers were washed with brine, dried over Na2SO4, filtered and the filtrate was concentrated. The
–
c
7
–b
4
×
residue was purified by column chromatography on silica gel (100 200 mesh, and visualized under
−
UV light at λ 254 and 365 nm; eluent, PE/EA) to give 8a–c.
N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4-aminophenoxy)picolinamide (8a): The title compound was
obtained starting from 2a and . Analytical data for 8a (yellow liquid, 86% yield): ESI-MS m/z 388.2
[M + H]+; 1H-NMR (400 MHz, CDCl3)
8.35 (d, 2H, ArH, J = 5.6 Hz), 7.66 (brs, 1H, -CONH-), 6.89
7
δ
(d, 2H, ArH, J = 8.8 Hz), 6.71 (d, 2H, ArH, J = 8.7 Hz), 3.77 (t, 2H, -CH2CH2O-, J = 5.3 Hz), 3.70 (s,
2H, -NH2), 3.56 (dd, 2H, -NHCH2CH2-, J1 = 10.3 Hz, J2 = 4.6 Hz), 0.91 (s, 9H, -SiC(CH3)3), 0.07 (s, 6H,
-Si(CH3)2).
N-(2-(tritylthio)ethyl)-4-(4-aminophenoxy)picolinamide (8b): The title compound was obtained starting
from
(400 MHz, CDCl3)
4 and 7
. Analytical data for 8b (yellow liquid, 88% yield): ESI-MS m/z 554.2 [M + H]+; 1H-NMR
δ
8.34 (d, 1H, ArH, J = 5.6 Hz), 8.13 (t, 1H, -CONH-, J = 5.7 Hz), 7.61 (d, 1H, ArH,
J = 1.3 Hz), 7.42 (d, 5H, ArH, J = 7.6 Hz), 7.28–7.17 (m, 10H, ArH), 6.93 (dd, 1H, ArH, J1 = 5.6 Hz,
J2 = 2.5 Hz), 6.88 (d, 2H, ArH, J = 9.4 Hz), 6.70 (d, 2H, ArH, J = 8.7 Hz), 3.70 (brs, 2H, -NH2), 3.28 (q,
2H, -NHCH2-, J = 6.6 Hz), 2.48 (t, 2H, -CH2CH2S-, J = 6.6 Hz).
N-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-(4-aminophenoxy)picolinamide (8c): The title compound was
obtained starting from 2b and
7
. Analytical data for 8c (yellow liquid, 84% yield): ESI-MS m/z 402.2
8.32 (d, 2H, ArH, J = 5.6 Hz), 7.65 (s, 1H, ArH), 6.88 (d, 2H,
[M + H]+; 1H-NMR (400 MHz, CDCl3)
δ
ArH, J = 8.9 Hz), 6.71 (d, 2H, ArH, J = 8.3 Hz), 4.68 (brs, 2H, -NH2), 3.77 (t, 2H, -CH2CH2O-, J = 5.7 Hz),
3.55 (q, 2H, -NHCH2CH2-, J = 6.0 Hz), 1.88–1.77 (m, 2H, -CH2CH2CH2-), 0.91 (s, 9H, -SiC(CH3)3), 0.08
(s, 6H, -Si(CH3)2).
General Procedure for the Preparation of 9a–c and 12. A mixture of compound 8a–c or 11 (1.5 mmol)
in DCM (10 mL) was stirred at room temperature, while a solution of 4-chloro-3-trifluoromethylphenyl
isocyanate (1.5 mmol) in DCM (5 mL) was slowly added in ice-bath condition. Afterwards, stirring was
continued for 2 h at room temperature. The reaction mixture was poured into water (30 mL), extracted
with DCM (3
×
15 mL). The combined organic layers were washed with brine, dried over Na2SO4,
filtered and the filtrate was concentrated. The residue was purified by column chromatography on
silica gel (eluent, PE/EA) to give 9a–c and 12.
N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)
picolinamide (9a): The title compound was obtained starting from 8a. Analytical data for 9a (white
solid, 86% yield, mp 133–135 ◦C): ESI-MS m/z 610.1 [M + H]+; 1H-NMR (400 MHz, CDCl3)
δ 8.75 (s,
1H, -CONH-), 8.48 (d, 1H, ArH, J = 5.7 Hz), 8.45 (s, 1H, -NHCONH-), 8.19 (s, 1H, -NHCONH-), 7.66 (d,
2H, ArH, J = 8.5 Hz), 7.52 (s, 1H, ArH), 7.34 (dd, 2H, ArH, J1 = 16.6 Hz, J2 =8.1 Hz), 7.27 (t, 1H, ArH,
J = 3.4 Hz), 7.18 (s, 1H, ArH), 6.97 (d, 2H, ArH, J = 7.7 Hz), 3.79 (t, 2H, -CH2CH2O-, J = 4.4 Hz), 3.57
(dd, 2H, -NHCH2-, J1 = 9.5 Hz, J2 = 4.5 Hz), 0.90 (s, 9H, -SiC(CH3)3), 0.06 (s, 6H, -Si(CH3)2).
N-(2-(tritylthio)ethyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinamide (9b): The
title compound was obtained starting from 8b. Analytical data for 9b (white solid, 93% yield, mp
107–109 ◦C): ESI-MS m/z 754.2 [M + H]+; 1H-NMR (400 MHz, CDCl3)
δ
8.48 (d, 2H, ArH, J = 1.6 Hz),
8.08 (s, 1H, -NHCONH-), 7.93 (s, 1H, -NHCONH-), 7.69 (s, 1H, -CONH-), 7.48 (dd, 3H, ArH, J1 =

Molecules 2020, 25, 573
8 of 12
12.3 Hz, J2 = 8.6 Hz), 7.35 (m, 5H, ArH), 7.29 (d, 2H, ArH, J = 2.6 Hz), 7.23–7.15 (m, 10H, ArH), 6.95 (d,
2H, ArH, J = 4.0 Hz), 3.28–3.23 (m, 2H, -NHCH2-), 2.48–2.43 (m, 2H, -CH2CH2S-).
N-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)
picolinamide (9c): The title compound was obtained starting from 8c. Analytical data for 9c (white solid,
90% yield, mp 118–120 ◦C): ESI-MS m/z 624.2 [M + H]+; 1H-NMR (400 MHz, CDCl3)
δ 8.83 (d, 1H,
-CONH-, J = 5.5 Hz), 8.53 (s, 1H, -NHCONH-), 8.44 (d, 1H, ArH, J = 5.3 Hz), 8.24 (s, 1H, -NHCONH-),
7.70 (d, 2H, ArH, J = 7.4 Hz), 7.54 (s, 1H, ArH), 7.41–7.34 (m, 3H, ArH), 7.15 (s, 1H, ArH), 6.99 (t,
2H, ArH, J = 7.0 Hz), 3.78 (dd, 2H, -CH2CH2O-, J1 = 12.1 Hz, J2 = 5.4 Hz), 3.58 (dd, 2H, -NHCH2-,
J1 = 12.8 Hz, J2 = 7.6 Hz), 1.87–1.81 (m, 2H, -CH2CH2CH2O-), 0.90 (s, 9H, -SiC(CH3)3), 0.08 (s, 6H,
-Si(CH3)2).
Methyl 4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinate (12): The title compound was
obtained starting from 11. Analytical data for 12 (white solid, 76% yield, mp 172–174 ◦C): ESI-MS m/z
466.2 [M + H]+; 1H-NMR (400 MHz, CDCl3)
δ
8.52–8.44 (m, 2H, ArH), 8.40 (s, 1H, ArH), 7.69 (s, 2H,
-
NHCONH-), 7.63 (d, 1H, ArH, J = 8.5 Hz), 7.45 (d, 2H, ArH, J = 8.6 Hz), 7.36 (d, 1H, ArH, J = 8.6 Hz),
7.00 (d, 2H, ArH, J = 8.7 Hz), 6.97 (dd, 1H, ArH, J1 = 4.9 Hz, J2 = 2.4 Hz), 4.02 (s, 3H, -COOCH3).
General Procedure for the Preparation of 10a and 10c. Tetrabutylammonium fluoride trihydrate
(416 mg, 1.3 mmol) was added to a solution of 9a or 9c (1.3 mmol) in tetrahydrofuran (10 mL). The
mixture was stirred at room temperature for 1 h and then poured into water (30 mL), extracted with
DCM (3
and the filtrate was concentrated. The residue was purified by column chromatography on silica gel
(100 200 mesh, and visualized under UV light at 254 and 365 nm; eluent, PE/EA) to offer 10a and 10c.
×
15 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered
−
λ
N-(2-hydroxyethyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinamide (10a): The title
compound was obtained starting from 9a. Analytical data for 10a (white solid, 96% yield, mp
193–195 ◦C): ESI-MS m/z 495.1 [M + H]+; 1H-NMR (400 MHz, DMSO-d6)
δ 9.24 (s, 1H, -NHCONH-),
9.03 (s, 1H, -NHCONH-), 8.71 (t, 1H, -CONH-, J = 5.9 Hz), 8.52 (d, 1H, ArH, J = 5.6 Hz), 8.13 (s, 1H,
ArH), 7.70-7.57 (m, 4H, ArH), 7.38 (d, 1H, ArH, J = 2.0 Hz), 7.18 (d, 3H, ArH, J = 8.6 Hz), 4.80 (t, 1H, -OH,
J = 5.4 Hz), 3.50 (q, 2H, -CH2CH2OH, J = 5.8 Hz), 3.38–3.31 (m, 2H, -CH2CH2OH). 13C-NMR (151 MHz,
DMSO-d6) δ 165.93, 163.10, 152.37, 152.17, 150.27, 147.75, 139.23, 136.96, 131.90, 126.72, 126.51, 123.62,
123.02, 122.24, 121.34, 120.42, 116.73, 114.09, 108.61, 59.48, 41.50. ESI-HRMS (m/z) [M + H]+ calcd for
C22H18ClF3N4O4 459.1041, obsd 459.1046, ppm error 0.9.
N-(3-hydroxypropyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinamide (10c): The title
compound was obtained starting from 9c. Analytical data for 10c (white solid, 96% yield, mp
103–105 ◦C): ESI-MS m/z 509.2 [M + H]+; 1H-NMR (400 MHz, DMSO-d6)
δ 9.98 (s, 1H, -NHCONH-),
9.64 (s, 1H, -NHCONH-), 8.83 (t, 1H, -CONH-, J = 4.4 Hz), 8.50 (d, 1H, ArH, J = 5.4 Hz), 8.12 (s, 1H,
ArH), 7.64 (d, 2H, ArH, J = 7.7 Hz), 7.59 (d, 2H, ArH, J = 8.2 Hz), 7.39 (s, 1H, ArH), 7.20–7.11 (m, 3H,
ArH), 4.55 (t, 1H, -OH, J = 4.7 Hz), 3.44 (dd, 2H, -CH2CH2OH, J1 = 11.0 Hz, J2 = 5.6 Hz), 3.32 (t, 2H,
-NHCH2-, J = 4.5 Hz), 1.70-1.60 (m, 2H, -CH2CH2OH). 13C-NMR (151 MHz, DMSO-d6)
δ 165.92, 163.08,
152.45, 152.35, 150.27, 147.70, 139.32, 137.04, 131.93, 126.74, 126.54, 123.64, 122.84, 122.14, 121.83, 121.36,
120.23, 116.54, 113.96, 108.68, 58.68, 36.44, 32.03. ESI-HRMS (m/z) [M + H]+ calcd for C23H20ClF3N4O4
509.1198, obsd 509.1203, ppm error 1.1.
N-(2-mercaptoethyl)-4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy) picolinamide
(10b):
Compound 9b (100 mg, 0.13 mmol) was dissolved in DCM (15 mL). Triisopropylsilane (95 mg, 0.6
mmol) and TFA (0.3 mmol) were added and the mixture was stirred at room temperature overnight.
The mixture was extracted with DCM (3
brine, dried over Na2SO4, filtered and the filtrate was concentrated. The residue was purified
×
20 mL). The combined organic layers were washed with
by column chromatography on silica gel (eluent, DCM/MeOH 10:1) to offer 10b (white solid, 84%

Molecules 2020, 25, 573
9 of 12
◦
1
yield, mp 129–131 C). ESI-MS m/z 511.1 [M + H]+; H NMR (400 MHz, DMSO-d6)
δ
10.41 (s, 1H,
-
NHCONH-), 9.99 (s, 1H, -NHCONH-), 9.02-8.99 (m, 1H, -CONH-), 8.52 (dd, 1H, ArH, J = 5.4, 2.4 Hz),
8.12 (d, 2H, ArH, J = 1.2 Hz), 7.65 (s, 2H, ArH), 7.59 (dd, 2H, ArH, J = 8.7, 2.4 Hz), 7.41 (t, 1H, ArH,
J = 2.7 Hz), 7.20-7.13 (m, 3H, ArH), 4.66 (t, 1H, -SH, J = 4.7 Hz), 3.49 (d, 2H, -CH2SH, J = 4.6 Hz), 2.63
(dd, 2H, -NHCH2-, J1 = 14.6 Hz, J2 = 5.7 Hz). 13C-NMR (151 MHz, DMSO-d6)
δ 165.94, 163.21, 158.03,
157.83, 152.57, 152.10, 150.31, 147.54, 139.51, 137.24, 131.95, 122.54, 121.92, 121.36, 119.89, 118.23, 116.24,
114.02, 108.90, 72.16, 60.13. ESI-HRMS (m/z) [M + H]+ calcd for C22H18ClF3N4O3S 511.0813, obsd
511.0824, ppm error 2.2.
Methyl 4-(4-aminophenoxy)picolinate (11): To compound 7 (1.0 g, 4.3 mmol) in methanol (30 mL) was
added dichlorosulfoxide (1.1 mL) slowly in an ice-bath. The reaction mixture was stirred and refluxed
overnight. The reaction mixture was concentrated under reduced pressure then extracted with ethyl
acetate (3
×
30 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure to afford 11 (white solid, 84% yield, mp 113–115 ◦C). ESI-MS
m/z 245.1 [M + H]+; H-NMR (400 MHz, CDCl3)
δ 8.53 (d, 1H, ArH, J = 5.7 Hz), 7.62 (d, 1H, ArH,
1
J = 2.2 Hz), 6.97 (dd, 1H, ArH, J1 = 5.6 Hz, J2 = 1.0 Hz), 6.89 (d, 2H, ArH, J = 8.7 Hz), 6.73 (d, 2H, ArH,
J = 8.7 Hz), 3.97 (s, 3H, -COOCH3).
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((2-(hydrazinecarbonyl)pyridin-4-yl)oxy)phenyl)urea
(13):
A mixture of 12 (500 mg, 1.1 mmol) and hydrazine hydrate (0.16 mL) in ethanol (10 mL) was stirred at
90 ◦C for 2 h. The reaction mixture was concentrated under reduced pressure then extracted with
ethyl acetate (3
×
30 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The solid residue was recrystallized from ethanol to
afford 13 (gray solid, 95% yield, mp 119-121 ◦C). ESI-MS m/z 466.1 [M + H]+; H-NMR (400 MHz,
1
DMSO-d6)
δ 9.93 (s, 1H, -CONHNH2), 9.25 (s, 1H, -NHCONH-), 9.04 (s, 1H, -NHCONH-), 8.49 (d, 1H,
ArH, J = 5.5 Hz), 8.13 (s, 1H, ArH), 7.68–7.58 (m, 4H, ArH), 7.35 (t, 1H, ArH, J = 5.0 Hz), 7.19 (d, 2H,
ArH, J = 8.8 Hz), 7.13 (dd, 1H, ArH, J1 = 5.3Hz, J2 = 2.4 Hz), 4.56 (brs, 2H, -CONHNH2).
General Procedure for the Preparation of 14a
–
b. A mixture of 13 (50 mg, 0.11 mmol) and
L, 0.15 mmol) in ethanol (8 mL) was
hydroxyacetone (10 L, 0.15 mmol) or 4-hydroxy-2-butanone (10
µ
µ
stirred at 90 ◦C for 24h, using acetic acid as a catalyst. The reaction mixture was concentrated under
reduced pressure. The residue was purified by recrystallization from ethanol or methanol to afford
14a–b.
(E)-1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((2-(2-(1-hydroxypropan-2-ylidene)hydrazine-1-
carbonyl)pyridin-4-yl)oxy)phenyl)urea (14a): The title compound was obtained starting from
hydroxyacetone. Analytical data for 14a (white solid, 75% yield, mp 198–200 ◦C): ESI-MS m/z 522.2
[M + H]+; 1H NMR (400 MHz, DMSO-d6, ratio 73:27, the minor signals are marked with an asterisk,
mixture signals are marked with double asterisks) δ 12.68 (s, 0.73H), 10.77* (s, 0.22H), 9.25** (s, 1H),
9.03** (s, 1H), 8.57* (d, 0.29H, J = 5.5 Hz), 8.51 (d, 0.62H, J = 5.6 Hz), 8.13** (s, 1H), 7.73–7.55** (m, 4H),
7.42** (s, 1H), 7.20** (d, 3H, J = 7.0 Hz), 6.11 (t, 0.68H, J = 4.9 Hz), 5.24* (t, 0.23H, J = 5.9 Hz), 4.34 (d,
1.49H, J = 4.8 Hz), 4.04* (d, 0.54H, J = 6.1 Hz), 1.98* (s, 0.86H), 1.91 (s, 2.14H).
(E)-1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((2-(2-(4-hydroxybutan-2-ylidene)hydrazine-1-
carbonyl)pyridin-4-yl)oxy)phenyl)urea (14b): The title compound was obtained starting from
4-hydroxy-2-butanone. Analytical data for 14b (white solid, 70% yield, mp 205–207 ◦C): ESI-MS m/z
1
536.2 [M + H]+; H-NMR (400 MHz, DMSO-d6, ratio 75:25, the minor signals are marked with an
asterisk, mixture signals are marked with double asterisks) δ 11.47 (s, 0.75H), 10.73* (s, 0.25H), 9.25** (s,
1H), 9.03** (s, 1H), 8.54** (dd, 1H, J = 13.9, 5.8 Hz), 8.13** (s, 1H), 7.64** (dt, 4H, J = 17.9, 8.8 Hz), 7.41**
(d, 1H, J = 2.5 Hz), 7.20** (d, 3H, J = 8.6 Hz), 5.36 (t, 0.75H, J = 4.4 Hz), 4.62* (t, 0.25H, J = 5.4 Hz),
3.76–3.60** (m, 2H), 2.50–2.45** (m, 2H), 2.04 (s, 2.33H), 1.98* (s, 0.54H). 13C-NMR (151 MHz, DMSO-d6)
δ
166.00, 160.49, 159.09*, 152.38, 151.96*, 150.49, 147.68, 139.24, 137.02, 131.91, 126.72, 126.52*, 123.63,

Molecules 2020, 25, 573
10 of 12
123.03*, 122.26, 121.82, 121.38*, 120.46, 116.74, 114.36, 108.85, 58.28*, 57.67, 41.71*, 34.30, 23.23, 15.67*.
ESI-HRMS (m/z) [M + H]+ calcd for C24H21ClF3N5O4 536.1307, obsd 536.1312, ppm error 1.0.
General Procedure for the Preparation of 15a
Methanol (5 mL) at room temperature was added sodium cyanoborohydride (7.6 mg, 0.11 mmol) and
acetic acid (3.3 L). The mixture was reacted at room temperature overnight and then poured into
water (10 mL), extracted with ethyl acetate (3 15 mL). The combined organic layers were washed
with brine, dried over Na2SO4, filtered and the filtrate was concentrated. The residue was purified
–b. To a stirred solution of 14a–b (0.057 mmol) in
µ
×
by column chromatography on silica gel (100 200 mesh, and visualized under UV light at λ 254 and
−
365 nm; eluent, DCM/MeOH) to give 15a–b.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((2-(2-(1-hydroxypropan-2-yl)hydrazine-1-carbonyl)pyridin-4-
yl)oxy)phenyl)urea (15a): The title compound was obtained starting from 14a. Analytical data for 15a
(white solid, 87% yield, mp 139–141 ◦C): ESI-MS m/z 524.2 [M + H]+; 1H-NMR (400 MHz, DMSO-d6)
δ
10.14 (brs, 1H, -CONH-), 9.49 (brs, 1H, -NHCONH-), 9.25 (brs, 1H, -NHCONH-), 8.50 (dd, 1H, ArH,
J1 = 5.4 Hz, J2 = 1.6 Hz), 8.13 (s, 1H, ArH), 7.69-7.56 (m, 4H, ArH), 7.35 (s, 1H, ArH), 7.18 (dd, 3H,
ArH, J1 = 9.1 Hz, J2 = 1.4 Hz), 5.13 (brs, 1H, -OH), 4.65 (t, 1H, -NHCH-, J = 5.3 Hz), 3.33–3.25 (m, 2H,
-CHCH2OH), 2.96 (d, 1H, -NHCH-, J = 4.3 Hz), 0.95 (d, 3H, =CHCH3, J = 5.0 Hz). 13C-NMR (151 MHz,
DMSO-d6) δ 165.83, 161.76, 152.46, 151.80, 150.45, 147.66, 139.35, 137.08, 131.91, 126.72, 123.63, 122.90,
122.13, 121.83, 121.35, 120.28, 116.60, 113.98, 108.83, 63.84, 56.44, 15.38. ESI-HRMS (m/z) [M + H]+ calcd
for C23H21ClF3N5O4 524.1307, obsd 524.1311, ppm error 0.8.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((2-(2-(4-hydroxybutan-2-yl)hydrazine-1-carbonyl)pyridin-4-
yl)oxy)phenyl)urea (15b): The title compound was obtained starting from 14b. Analytical data for 15b
(white solid, 87% yield, mp 89–92 ◦C): ESI-MS m/z 538.2 [M + H]+; 1H-NMR (400 MHz, DMSO-d6)
δ
10.06 (d, 1H, -CONH-, J = 7.0 Hz), 9.24 (s, 1H, -NHCONH-), 9.02 (s, 1H, -NHCONH-), 8.51 (d, 1H,
ArH, J = 5.6 Hz), 8.13 (s, 1H, ArH), 7.64 (d, 2H, ArH, J = 6.1 Hz), 7.60 (d, 2H, ArH, J = 8.7 Hz), 7.35 (s,
1H, ArH), 7.19 (d, 2H, ArH, J = 8.8 Hz), 7.16 (dd, 1H, ArH, J1 = 5.5 Hz, J2 = 2.1 Hz), 4.97 (t, 1H, -OH,
J = 6.0 Hz), 4.44 (t, 1H, -CH2CH2O-, J = 5.0 Hz), 3.58–3.38 (m, 1H, -CH2CH2O-), 3.09–2.95 (m, 1H,
-NHCHCH3), 1.63 (m, 1H, -CHCH2CH2-), 1.43–1.31 (m, 1H, -CHCH2CH2-), 0.98 (d, 3H, =CHCH3
,
J = 6.3 Hz). 13C-NMR (151 MHz, DMSO-d6)
δ
165.82, 161.81, 152.38, 151.92, 150.44, 147.73, 139.24,
136.98, 131.91, 126.73, 126.52, 123.63, 123.03, 122.25, 121.82, 121.37, 120.43, 116.76, 113.99, 108.77, 58.26,
52.86, 37.53, 18.70. ESI-HRMS (m/z) [M + H]+ calcd for C24H23ClF3N5O4 538.1463, obsd 538.1464, ppm
error 0.1.
3.3. In Vitro Anti-Proliferative Assay
The in vitro anti-proliferation of the chemical compounds was measured by the MTT reagent.
Briefly, 5
for 24 h at 37 ◦C, the cells were treated with different concentration of tested compound or DMSO (as
negative control) for 48 h. Then the medium with compound or DMSO was replaced with 200 L of
fresh medium containing 10% MTT (5 mg/mL in PBS) in each well and incubated at 37 ◦C for 4 h. Last,
the MTT-containing medium was discarded and 150 L of DMSO per well was added to dissolve the
×
103 cells in 100
µL of medium per well were plated in 96-well plates. After being incubated
µ
µ
formazan crystals newly formed. Absorbance of each well was determined by a microplate reader
(Synergy H4, Bio-Tek) at a 570 nm wavelength. The inhibition rates of proliferation were calculated
with the following equation:
Inhibition ratio (%) = (ODDMSO−ODcompd)/(ODDMSO−ODblank) × 100
(1)
The concentration of the compounds that inhibited cell growth by 50% (IC50) was calculated using
GraphPad Prism, version 6.0.

Molecules 2020, 25, 573
11 of 12
4. Conclusions
Based on sorafenib and B-ARF crystal complex, there is a space for the modification of the
2-pyridine motif of sorafenib using alkyl chains to develop new anti-cancer agents. In order to
introduce active side chains including thiol or hydroxyl for the synthesis of Sorafenib-PEG-DGL in the
next study, five novel sorafenib derivatives (10a–b, 15a–b) with oxygen or sulphur alkyl side-chain
on pyridine-2-amide scaffold were prepared and evaluated their anti-cancer activities in vitro. The
preliminary structure and activity relationships indicated that pyridine-2-amide was essential for
antitumor activity and pyridine-2-hydrazine led to the loss of activity. The length of side chain had
moderate influence on the antiproliferation activity against tumor cells. Among them, compound 10b
bearing thiol alkyl group showed a good antiproliferation activity, which provided us a promising
candidate for the future preparing nanoparticles. Moreover, the docking mode of 10b and B-RAF also
confirmed its interaction with the active sites of kinases. Taken together, 10b might be a prospective
compound to be developed as a promising Sorafenib-PEG-DGL.
Supplementary Materials: Supplementary Material includes NMR spectra of target compounds 10a–b and 15a–b.
Author Contributions: Y.C. and L.Y. oversaw all aspects of the experiments and manuscript preparation. K.W.
and K.K. designed new compounds and analyzed the data; K.W. performed chemical experiments and molecular
docking. K.K. tested in vitro antitumor activity of target compounds; K.W. wrote the draft manuscript; K.K., Q.W.
and Z.Y. revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: Many thanks to the grant from National Natural Science Foundation of China (No. 81572979), National
Science and Technology Major Project “Key New Drug Creation and Manufacturing Program”, China (Number:
2018ZX09711002) and Shanghai Biomedical Science and Technology Support Project (No. 18431902800) for
supporting this study.
Acknowledgments: The authors would like to thank Congmin Yuan for providing the facilities and advice for the
molecular modeling.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Hubbard, S.R.; Till, J.H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem. 2000, 69, 373–398.
[CrossRef] [PubMed]
2.
Whisler, R.L.; Bagenstose, S.E.; Newhouse, Y.G.; Carle, K.W. Expression and catalytic activities of protein
tyrosine kinases (PTKs) Fyn and Lck in peripheral blood T cells from elderly humans stimulated through the
T cell receptor (TCR)/CD3 complex. Mech. Ageing Dev. 1997, 98, 57–73. [CrossRef]
Al-Obeidi, F.A.; Lam, K.S. Development of inhibitors for protein tyrosine kinases. Oncogene 2000, 19,
5690–5701. [CrossRef] [PubMed]
Lowinger, T.B.; Riedl, B.; Dumas, J.; Smith, R.A. Design and discovery of small molecules targeting raf-1
kinase. Curr. Pharm. Des. 2002, 8, 2269–2278. [CrossRef] [PubMed]
Gril, B.; Palmieri, D.; Qian, Y.; Anwar, T.; Ileva, L.; Bernardo, M.; Choyke, P.; Liewehr, D.J.; Steinberg, S.M.;
Steeg, P.S. The B-Raf status of tumor cells may be a significant determinant of both antitumor and
anti-angiogenic effects of pazopanib in xenograft tumor models. Plos ONE 2011, 6, e25625. [CrossRef]
[PubMed]
3.
4.
5.
6.
7.
8.
Adnane, L.; Trail, P.A.; Taylor, I.; Wilhelm, S.M. Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that
targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature.
Methods Enzymol. 2006, 407, 597–612. [PubMed]
Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R.A.; Schwartz, B.; Simantov, R.; Kelley, S.
Discovery and development of sorafenib: A multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov.
2006, 5, 835–844. [CrossRef] [PubMed]
Wilhelm, S.M.; Carter, C.; Tang, L.; Wilkie, D.; McNabola, A.; Rong, H.; Chen, C.; Zhang, X.; Vincent, P.;
McHugh, M.; et al. BAY 43-9006 Exhibits Broad Spectrum Oral Antitumor Activity and Targets the
RAF/MEK/ERK Pathway and Receptor Tyrosine Kinases Involved in Tumor Progression and Angiogenesis.
Cancer Res. 2004, 64, 7099–7109. [CrossRef] [PubMed]

Molecules 2020, 25, 573
12 of 12
9.
Liu, L.; Cao, Y.; Chen, C.; Zhang, X.; McNabola, A.; Wilkie, D.; Wilhelm, S.; Lynch, M.; Carter, C. Sorafenib
blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in
hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006, 66, 11851–11858. [CrossRef] [PubMed]
10. Wan, P.T.; Garnett, M.J.; Roe, S.M.; Lee, S.; Niculescu-Duvaz, D.; Good, V.M.; Jones, C.M.; Marshall, C.J.;
Springer, C.J.; Barford, D.; et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic
mutations of B-RAF. Cell 2004, 116, 855–867. [CrossRef]
11. Qin, A.F.; Li, Y.; Song, H.R.; Chen, X.G.; Jin, X.F.; Wang, K.; Zhang, L.J.; Huo, L.C.; Feng, Z.Q. Design,
synthesis and antitumor activity of sorafenib analogues containing 2-picolinylhydrazide moiety. Acta Pharm.
Sin. 2012, 47, 1623–1629.
12. Trott, O.; Olson, A.J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading. J. Comput Chem 2010, 31, 455–461. [CrossRef] [PubMed]
13. Mutlu, H.; Ceper, E.B.; Li, X.; Yang, J.; Dong, W.; Ozmen, M.M.; Theato, P. Sulfur Chemistry in Polymer and
Materials Science. Macromol. Rapid Commun. 2019, 40, e1800650. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2020 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
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).