Synthesis and anti-proliferative activity evaluation of sorafenib derivatives with a 3-arylacryloyl hydrazide unit

Article abstract of DOI:10.1007/s00044-014-1249-9

A series of sorafenib derivatives with a 3-arylacryloyl hydrazide unit were designed and synthesized, and their anti-proliferative activity against human cancer cell lines (ACHN, HCT116, MDA-MB-231) were evaluated by MTT assay. Most of the synthesized compounds showed superior or similar cytotoxicity against the selected cell lines to the control sorafenib. Among these derivatives, compounds 8a, 8h, 8l, 8m, 11a and 11b showed potent anti-proliferative activity. Compounds 8h and 8m were selected for further evaluation of biological activity against more cancer cell lines. And oral administration of sorafenib analogue 8h at the same dose of sorafenib (30 mg/kg) in the pancreatic cancer Capan2 and Mia-PaCa2 xenograft models in nude mice showed tumour growth inhibition of 60.98 and 54.59 %, respectively, which is similar to the positive control sorafenib.

Full text of DOI:10.1007/s00044-014-1249-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1249-9
ORIGINAL RESEARCH
Synthesis and anti-proliferative activity evaluation of sorafenib
derivatives with a 3-arylacryloyl hydrazide unit
•
Lijing Zhang Yan Li Ke Wang Aifang Qin
•
•
•
•
Xiaoguang Chen Zhiqiang Feng
Received: 23 February 2014 / Accepted: 5 September 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of sorafenib derivatives with a 3-a-
rylacryloyl hydrazide unit were designed and synthesized,
and their anti-proliferative activity against human cancer
cell lines (ACHN, HCT116, MDA-MB-231) were evalu-
ated by MTT assay. Most of the synthesized compounds
showed superior or similar cytotoxicity against the selected
cell lines to the control sorafenib. Among these derivatives,
compounds 8a, 8h, 8l, 8m, 11a and 11b showed potent
anti-proliferative activity. Compounds 8h and 8m were
selected for further evaluation of biological activity against
more cancer cell lines. And oral administration of sorafenib
analogue 8h at the same dose of sorafenib (30 mg/kg) in
the pancreatic cancer Capan2 and Mia-PaCa2 xenograft
models in nude mice showed tumour growth inhibition of
60.98 and 54.59 %, respectively, which is similar to the
positive control sorafenib.
Keywords Sorafenib ꢀ Synthesis ꢀ
3-Arylacryloyl hydrazide ꢀ Anti-proliferative activity ꢀ
Xenograft model
Introduction
Cancer is a leading cause of death worldwide. According to
World Health Organization (WHO), there were 14.1 mil-
lion new cancer cases, 8.2 million cancer deaths and 32.6
million people living with cancer (within 5 years of diag-
nosis) in 2012 worldwide. 57 % (8 million) of new cancer
cases, 65 % (5.3 million) of the cancer deaths and 48 %
(15.6 million) of the 5-year prevalent cancer cases occurred
in the less developed regions (Globocan, 2012). Although
many drugs have been applied for cancer therapy, the use
of available antitumor agents are often limited mainly due
to the adverse side-effects and adaptive resistance (Ji and
Ding, 2008; Lin, 2009). Therefore, searching for new
anticancer agents with better activity remains important.
Recent decades have seen considerable research efforts
invested in the discovery and development of multi-target
therapeutics; it can overcome the limitations of many
monotherapies by attacking the disease system on multiple
fronts, and can be more efficacious and less vulnerable to
adaptive resistance (Bilanges et al., 2008; Giordano and
Petrelli, 2008; Zimmermann et al., 2007). There are several
multi-target drugs that have been in clinical trials or clin-
ical use, such as imatinib, sorafenib, sunitinib, vatalanib
and ABT-869 (Atkins et al., 2006; Capdeville et al., 2002;
Lijing Zhang and Yan Li have contributed equally to this paper.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1249-9) contains supplementary
material, which is available to authorized users.
L. Zhang ꢀ K. Wang ꢀ Z. Feng (&)
Beijing Key Laboratory of Active Substance Discovery and
Druggability Evaluation, Institute of Materia Medica, Chinese
Academy of Medical Sciences & Peking Union Medical College,
1 Xian Nong Tan Street, Beijing 100050, China
e-mail: fengzhq@imm.ac.cn
L. Zhang
e-mail: hltzlj@imm.ac.cn
Y. Li ꢀ X. Chen (&)
Department of Pharmacology, State Key Laboratory of Bioactive
Substances and Functions of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and
Peking Union Medical College, 1 Xian Nong Tan Street,
Beijing 100050, China
e-mail: chxg@imm.ac.cn
A. Qin
Shan Xi Institute for Food and Drug Control, 12 Taoyuan Road,
Taiyuan 030001, Shanxi, People’s Republic of China
123

Med Chem Res
CF3
O
O
OH
i
Cl
O
Cl
O
N
N
H
H
N
N
H2N
N
N
H
H
1
2
sorafenib
O
O
O
O
ii
OH
N
N
H
N
H2N
H2N
iii
O
H
4
3
O
N
R1
O
O
N
H
iv
O
R2
N
O
O
N
N
H
H
N
H2N
5
Fig. 1 Design of sorafenib derivatives with a 3-arylacryloyl hydra-
zide unit
CF3
CF3
CF3
O
v
Cl
Cl
Cl
O
O
O
N
N
N
N
N
H
H
6
Hernandez-Davies et al., 2011; Wang and Fu 2008; Wood
et al., 2000).
O
O
O
O
NH2
vi
Sorafenib (BAY 43-9006), the first oral multi-kinase
inhibitor, was co-developed by Bayer and Onyx, launched
in the U.S. in 2005 for the treatment of advanced metastatic
renal cell carcinoma (RCC) and approved in the U.S. for the
treatment of advanced hepatocellular carcinoma (HCC) as a
single agent in 2007 (Kane et al., 2006; Liu et al., 2006).
Sorafenib has a dual role as antitumor agent: blocking the
cell signalling pathways mediated by RAF/MEK/ERK to
inhibit the proliferation of tumour cells directly (Wan et al.,
2004), simultaneously, acting on the VEGFR to depress the
formation of new blood vessels and cut off the nutrient
supply of tumour cells to achieve the purpose that restrains
tumour growth. Because of the advantages of multi-mech-
anisms, broad-spectrum antitumor potency, safety and less
vulnerable to adaptive resistance, more and more attentions
are given to the optimization of sorafenib (Li et al., 2013;
Ramurthy et al., 2008; Wilhelm et al., 2011; Yao et al.,
2012; Zhao et al., 2013).
O
O
N
H
N
H
N
H
7
H
N
R1
N
H
O
N
H
N
H
8a-s
Scheme 1 Reagents and conditions: (i) t-BuOK, K2CO3, DMF,
100 °C; (ii) KOH, H2O, 100 °C; (iii) SOCl2, MeOH, reflux; (iv)
DCM, 0 °C to room temperature; (v) Hydrazine hydrate, MeOH, rt;
(vi) HATU, TEA, DMF, rt
substitution of N-methyl-4-chloropyridine-2-carboxamide
with 4-aminophenol in the presence of potassium tert-
butoxide under N2. Compound 3 was refluxed with aque-
ous potassium to afford carboxylic acid 4, and the esteri-
fication of compound 4 with SOCl2 to obtain compound 5.
Compound 6 was achieved by reacting compound 5 with
4-chloro-3-(trifluoro-methyl)phenyl isocyanate in dichlo-
romethane at room temperature. Compound 7 was syn-
thesized by hydrazinolysis of compound 6 in methanol
at room temperature. Finally, compounds 8a–s were
obtained by coupling compound 7 with the corresponding
3-arylacrylic acids in the presence of HATU and TEA in
DMF.
Our previous research showed that introduction of car-
boxhydrazide group could enhance the anti-proliferative
activity evidently while retaining the urea functional group
and replacing N-methylpyridine-2-carboxamide moiety
with 2-picolinyl hydrazide moiety (Qin et al., 2012). In this
paper, we designed and synthesized a new series of N0-((E)-
3-arylacryloyl)pyridine-2-carboxhydrazide analogues of
sorafenib, and the anti-proliferative activity of all the
synthesized compounds were evaluated against three cell
lines. Additionally, some of the most potent compounds
were selected for further evaluation (Fig. 1).
Synthesis of compounds 11a–h was shown in Scheme 2.
Compound 5 was treated with hydrazine hydrate in methanol
to give pyridine-2-carboxhydrazide 9. Compound 10 was
achieved by coupling compound 9 with 4-chlorocinnamic
acid in the presence of HATU and TEA in DMF. Then
compounds 11a–h were obtained by the formation of urea
functional group of compound 10 with the corresponding
phenyl isocyanates in dichloromethane.
Chemistry
The target compounds 8a–s were synthesized as shown in
Scheme 1. Compound 3 was obtained by the nucleophile
123

Med Chem Res
O
sorafenib. The IC50 values of compound 8h against ACHN,
HCT116 and MDA-MB-231 were 2.26, 2.07 and 1.29 lM,
respectively. As depicted, compound 8m was more sensi-
tive to ACHN and HCT116 cell lines than MDA-MB-231,
the IC50 values of compound 8m against ACHN and
HCT116 were 1.97 and 2.42 lM separately. Overall,
introduction of double substituents at the 3, 4- position or
2, 4-position of phenyl (compounds 8n–r) resulted in the
slight loss of anti-proliferative activity. Introduction of
multi-substituents of phenyl (compound 8s) led to a sharp
decrease in potency against ACHN (IC50 = 16.51 lM) and
MDA-MB-231 (IC50 = 23.44 lM), while the IC50 of
compound 8s against HCT116 was 4.43 lM, which might
be caused by their larger group space.
O
O
NH2
i
O
N
O
H
N
N
H2N
H2N
5
9
Cl
O
H
O
N
ii
N
H
N
O
H2N
10
Cl
O
H
iii
O
N
O
N
H
R2
N
O
N
N
H
H
11a-h
Scheme 2 Reagents and conditions: (i) Hydrazine hydrate, MeOH,
room temperature; (ii) 4-Chlorocinnamic acid, HATU, TEA, DMF,
room temperature; (iii) Corresponding phenyl isocyanates, CH2Cl2,
0 °C to room temperature
Keeping group R1 as 4-chlorophenyl to assess the
effect of R2 group on the antiproliferative activity: The
efficacy of R2 group with an electron-withdrawing group
(–Cl, –F or –CF3) at position 4 was evidently better than
having an electron-donating group (–EtO) viz compounds
11a–d. And compounds 11a–b were more sensitive to
ACHN and MDA-MB-231 than HCT116. Compound 11c
showed a similar sensitivity to all the three cancer cell
lines. As to compounds 11e–f having a substituent at
3-position, the compound having a Br was more potent
than that bearing a methyl. And introducing double sub-
stituents to R2 group resulted in loss of potency such as
compounds 11g–h.
Results and discussion
Anti-proliferative activity evaluation by MTT assay
The anti-proliferative activitities of the synthesized com-
pounds were evaluated against human cancer cell lines
ACHN (renal cancer cell lines), HCT116 (colon cancer cell
lines) and MDA-MB-231 (breast cancer cell lines) by the
MTT assay with sorafenib as the positive control, and the
IC50 values were calculated and summarized, as shown in
Table 1; most of the target compounds showed superior or
similar activity against the selected cell lines to sorafenib.
The structure–activity relationship of these derivatives was
preliminarily analyzed, and it is described in the following
paragraph.
Taking the above conclusions, the two most potent com-
pounds 8h and 8m were selected to estimate anti-prolifera-
tive activity against more human cancer cell lines including
pancreatic carcinoma cells JF305, Mia-PaCa2, PL45 and
SW1990, HCC cells HepG2 and SMMC7721 and colon
cancer cells HT29. The results are depicted in Table 2.
Compound 8h showed excellent activity against the Mia-
PaCa2 (IC50 = 0.25 lM), JF305 (IC50 = 0.77 lM) and
PL45 (IC50 = 0.77 lM), and compound 8m showed potent
activity against JF305 (IC50 = 0.72 lM) and Mia-PaCa2
(IC50 = 0.73 lM).
Keeping R2 group as 4-chloro-3-(trifluoromethyl) phe-
nyl to evaluate the influence of R1 group on the anti-pro-
liferative activity of these compounds: When R1 group is
bearing a heterocyclic ring, the potency of these com-
pounds, especially against human renal cancer cell lines
ACHN, was obviously lower than compound 8a with a
phenyl ring, except compound 8b (R1 = 2-furyl). It
encouraged us to choose phenyl ring for further research.
Regarding the substitution at the 4-position of phenyl,
compounds 8h–j having an electron-withdrawing group
(–Cl, –CF3, or –COOCH3) were generally more potent than
compounds 8f–g with an electron-donating group (–OH,
–OCH3), which can be attributed to their electron effect.
This relationship also existed in substitution at position 3
viz compounds 8k–m. And the potency of compounds 8h–
j decreased gradually, which might be ascribed to their
substituent space effect. Meanwhile, compound 8h with a
4-chlorophenyl moiety, showed superior activity to
Evaluation of pancreatic cancer xenograft models
in nude mice
We evaluated the biological activity of compound 8h in the
pancreatic cancer Capan2 and Mia-PaCa2 xenograft mod-
els in nude mice; the results are displayed in Table 3 and
Fig. 2. Oral administration of compound 8h at the same
dose (30 mg/kg) for 19 days showed tumour growth inhi-
bition with 35.02 % of T/C ratio at the end of the treatment
according to relative tumour volume (RTV), which is
slightly poorer than sorafenib with 23.11 % of T/C ratio in
the Capan2 model. This might be related to the poor sol-
ubility and large mass of compound 8h, which affects the
activity in vivo. In the Mia-PaCa2 model, repetitive
123

Med Chem Res
Table 1 Anti-proliferative activity of compounds 8a–s and 11a–h against three human cancer cell lines
O
H
O
N
R1
O
N
H
R2
N
O
N
H
N
H
Compound
R2
–
R1
IC50 (lM)
ACHN
HCT116
MDA-MB-231
Sorafenib
8a
–
20.48
3.07
20.53
3.13
4.67
5.2
9.24
2.52
2.73
5.55
4.34
4.34
5.92
4.16
1.29
3.98
2.97
7.75
3.79
7.33
3.83
5.32
6.45
2.84
7.43
23.44
2.21
1.68
3.58
[10.0
[10.0
4.36
4.74
[10.0
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chloro-3-trifluoromethylphenyl
4-Chlorophenyl
Phenyl
2-Furyl
3-Furyl
8b
3.94
8c
[10.0
5.29
8d
2-Thienyl
5.11
4.07
10.84
5.67
2.07
3.21
5.88
6.07
2.36
2.42
2.01
3.39
5.52
6.48
6.3
8e
Pyridine-3-yl
[10.0
8.13
8f
4-Hrdroxyphenyl
4-Methoxylphenyl
4-Chlorophenyl
8 g
8 h
8i
[10.0
2.26
4-Trifluoromethylphenyl
4-Carbomethoxyphenyl
3-Hydroxyphenyl
5.01
8j
10.73
2.52
8k
8l
3-Chlorophenyl
3.75
8m
8n
3-Trifluoromethylphenyl
1.97
4-Hydroxyl-3-methoxylphenyl
3,4-Bimethoxylphenyl
4-Acetoxy-3-methoxylphenyl
3,4-Bifluorophenyl
2,4-Bichlorophenyl
2,3,4,5,6-Pentafluorophenyl
4-Chlorophenyl
7.74
8o
4.52
8p
9.42
8q
1.28
8r
4.35
8 s
11a
11b
11c
11d
11e
11f
11g
11h
16.51
2.29
4.43
5.47
4.89
3.64
[10.0
7.01
4.96
6.99
8.27
4-Fluorophenyl
4-Chlorophenyl
2.74
4-Trifluoromethylphenyl
4-Chlorophenyl
4.2
4-Ethoxylphenyl
4-Chlorophenyl
5.65
3-Methylphenyl
4-Chlorophenyl
6.04
3-Bromophenyl
4-Chlorophenyl
4.45
3-Chloro-4-methylphenyl
4-Chlorophenyl
7.09
2,4-Bifluorophenyl
4-Chlorophenyl
[10.0
Table 2 The effect of compounds 8h and 8m on the proliferation of human cancer cells
Compound
IC50 (lM)
JF305
Mia-PaCa2
PL45
SW1990
HepG2
SMMC7721
HT29
8h
0.77
0.72
0.25
0.73
0.77
3.92
3.04
9.69
1.90
3.52
2.33
6.21
6.24
11.9
8m
administration of compound 8h at the same dose (30 mg/
kg) for 17 days showed 54.59 % inhibition according to
tumour weight, similar to sorafenib with a tumour growth
inhibition of 55.41 %.
Conclusion
We designed and synthesized a series of sorafenib deriva-
tives having a 3-arylacryloyl hydrazide unit, and evaluated
123

Med Chem Res
their anti-proliferative activities in vitro against human
cancer cell lines ACHN, HCT116 and MDA-MB-231 by the
MTT assay. Some compounds, for example, compounds 8a,
8h, 8l, 8m, 11a and 11b, showed potent antitumor activity
against the selected cancer cell lines. And compounds 8h
and 8m were chosen to investigate the activity against more
human cancer cell lines. Furthermore, the biological activity
of compound 8h in vivo was assessed. Compound 8h at the
dose of 30 mg/kg reduced the tumour growth of pancreatic
cancer in Mia-PaCa2 and Capan2 xenograft model with the
inhibition of 54.59 and 60.98 % (according to the tumour
weight), similar to the positive control sorafenib. All these
studies encouraged us to select compound 8h as a promising
candidate for further study.
tumour volume (length 9 width2/2) was calculated twice
every week. Tumours were excised, weighed and were
photographed.
Chemistry
All reagents and chemicals were obtained from commercial
suppliers and used without additional purification. Reaction
progress was monitored by thin-layer chromatography
(TLC) on silica gel 60 F254 plates (Qingdao) with UV
detection. Flash column chromatography was performed on
silica gel 60 (200–300 mesh) from Qingdao Haiyang
Chemical Factory. Melting points were determined on a
Yanaco micro melting point apparatus and were not cor-
1
rected. H NMR spectra were recorded on a Mercury-300
(300 MHz) spectrometer and Mercury-400 (400 MHz)
spectrometer with Chemical shifts in ppm (d) relative to the
solvent signal. Peak multiplicities are expressed as follows:
s singlet, d doublet, t triplet, q quartet, dd doublet of
doublet, dt doublet of triplet, td triplet of doublet, ddd
doublet of doublet of doublet, m multiplet, br s broad
singlet. Coupling constant (J) values are given in hertz
(Hz). HPLC–MS analysis was performed on a Thermo
Exactive plus-orbitrap mass spectrometer. Abbreviations
are used as follows: DMF, N, N-dimethylformamide; t-
BuOK, potassium tert-butanolate; EA ethyl acetate, PE
petroleum ether, TEA triethylamine.
Experimental
Cell viability assay
Cell lines ACHN, HCT116 and MDA-MB-231 (Table 1), or
JF305, Mia-PaCa2, PL45, SW1990, HepG2, SMMC7721
and HT29 (Table 2) cells were seeded in a 96-well plate.
After 24 h of incubation, cells were treated with several
different concentrations of sorafenib and the synthesized
compounds. After 144 h of incubation, the MTT assay was
performed to evaluate cell viability. Absorbance values were
normalized to the values obtained for vehicle-treated cells to
determine the percentage of surviving cells. The median
inhibitory concentration (IC50, defined as the drug concen-
tration at which cell growth was inhibited by 50 %) was
assessed from the dose–response curve.
Synthesis of 4-(4-aminophenoxy)-N-methylpyridine-2-
carboxamide (3)
To
a stirred solution of 4-aminophenol 1 (10.9 g,
Tumour implantation and growth in Capan2 and Mia-
PaCa2 xenografts
100.0 mmol, 1.0 equiv) in dry DMF (150 ml), potassium
tert-butanolate (14.6 g, 130 mmol, 1.3 equiv) was added in
portions, and the mixture was stirred at ambient temperature
for 2 h under nitrogen atmosphere, then N-methyl-4-chlor-
opyridine-2-carboxamide 2 (17.1 g, 100.0 mmol, 1.0 equiv)
and potassium carbonate (8.3 g, 60.0 mmol, 0.6 equiv) were
added, respectively. The mixture was heated to 100 °C and
the completion of the reaction was confirmed by TLC. The
mixture was then cooled to room temperature, poured into
300 ml water and extracted with ethyl acetate (EA). The
combined organic layers were washed with saturated sodium
carbonate solution and brine, dried over anhydrous sodium
sulphate and concentrated under reduced pressure to give the
crude product 3 22.8 g, yield 93.7 %.
All animal studies were in compliance with the policies of
the Institute of Materia Medica Animal Care and Use
Committee. Six-week-old female BALB/c/nu nude mice
(Vital River Laboratories [VRL]) were used. Ten million
Capan2 and Mia-PaCa2 cells were subcutaneously
implanted in the left flank of each mouse. When tumours
grew up to an average volume of 200–250 mm3, tumour-
bearing mice were randomly separated into four groups
(Capan2 xenograft model) of six animals or (Mia-PaCa2
xenograft model) of five animals. One group received oral
dose of 1 9 solution with cremophor EL/ethanol/water
(12.5:12.5:75) as a vehicle control; other groups received
oral dose of 30.0 mg/kg of sorafenib, 15.0 and 30.0 mg/kg
of 8h (Capan2 xenograft model) or 30.0 and 60.0 mg/kg of
8h (Mia-PaCa2 xenograft model) every day, respectively.
Mice were euthanized at the end of the treatment period.
Tumour size was measured with vernier callipers, and
Synthesis of 4-(4-aminophenoxy)pyridine-2-carboxylic
acid (4)
A mixture of 3 (22.8 g, 93.7 mmol, 1.0 equiv) and potas-
sium hydroxide (15.8 g, 281.1 mmol, 3.0 equiv) in 100 ml
123

Med Chem Res
water was refluxed overnight. After cooling to room tem-
perature, the mixture was extracted with EA twice and the
water layer was acidified with conc. HCl. The solvent was
concentrated under reduced pressure to precipitate solids,
and then filtered to give the target compound 4 18.9 g,
yield 87.6 %.
HATU (179 mg, 0.47 mmol, 1.1 equiv) and TEA (131 mg,
1.29 mmol, 3.0 equiv)were added, respectively. The solu-
tion was stirred at room temperature overnight, then excess
water was added and the precipitate formed. The solids
were filtrated, washed with water and dried under infrared
lamp to obtain the target compounds 8a–s.
Synthesis of methyl 4-(4-aminophenoxy) pyridine-2-
1-(4-(2-((N0-((E)-3-phenylacryloyl))hydrazinecarbonyl)
pyri-din-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)
phenyl)urea (8a) Yield 92 %, pale taupe solid. mp
155–156 °C. 1H NMR (300 MHz, DMSO-d6) d: 10.63
(br s, 2H, –NH), 9.35 (s, 1H, –NH), 9.12 (s, 1H, –NH), 8.57
(d, 1H, J = 5.1 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 7.66–7.53
(m, 7H, Ar–H, =CH), 7.42 (br s, 4H, Ar–H), 7.27–7.19 (m,
3H, Ar–H), 6.76–6.71 (d, 1H, J = 15.6 Hz, =CH); HR–MS
calcd. for C29H22ClF3N5O4 596.1306 [M?H]?, found 596.
1289.
carboxylate (5)
To an ice-cooled mixture of 4 (14.0 g, 60.8 mmol, 1.0
equiv) in dry MeOH (200 ml), thionyl chloride (21.7 g,
182.4 mmol, 3.0 equiv) was added dropwise with stirring.
Then the mixture was heated to reflux until the reaction
completed. After cooling, the solvent was evaporated, and
the residue was dissolved in water and pH of aqueous layer
was adjusted to about 8.0 with 2 N sodium hydroxide. The
product was filtered to give light-brown solid 5 (10.97 g)
without further purification, yield 73.9 %.
1-(4-(2-((N0-((E)-3-(furan-2-yl)acryloyl)) hydrazinecarbon-
yl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)
phenyl)urea (8b) Yield 85 %, pale taupe solid. mp
192–193 °C. 1H NMR (400 MHz, DMSO-d6) d: 10.60 (br s,
1H, –NH), 10.40 (br s, 1H, –NH), 9.41 (s, 1H, –NH), 9.18 (s,
1H, –NH), 8.57 (d, 1H, J = 5.2 Hz, Ar–H), 8.13 (s, 1H, Ar–
H), 7.85–7.81 (d, 1H, J = 15.2 Hz, Ar–H), 7.72–7.60 (m,
4H, Ar–H), 7.42–7.40 (d, 1H, J = 6.8 Hz, Ar–H), 7.37–7.33
(d, 1H, J = 15.6 Hz, Ar–H), 7.20–7.18 (m, 3H, Ar–H), 6.84
(s, 1H, Ar–H), 6.65–6.61 (d, 1H, J = 18.0 Hz, Ar–H), 6.
52–6.43 (d, J = 15.6 Hz, 1H,=CH); HR–MS calcd. for
C27H20ClF3N5O5 586.1099 [M?H]?, found 586.1085.
Synthesis of 1-(4-(2-carbomethoxypyridin-4-yloxy)
phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (6)
A solution of 4-chloro-3-(trifluoromethyl)phenyl isocya-
nate (5.0 g, 22.6 mmol, 1.05 equiv) in DCM was added
dropwise to a suspension of 5 (5.2 g, 21.4 mmol, 1.0
equiv) in DCM at 0 °C. The resulting mixture was stirred
at room temperature overnight. The resulting solid was
removed by filtration, then washed with small volume of
DCM several times and dried under infrared lamp to afford
6 (5.9 g) as off-white solid, yield 59.2 %.
1-(4-(2-((N0-((E)-3-(furan-3-yl)acryloyl)) hydrazinocarbonyl)
pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)
urea (8c) Yield 88 %, pale taupe solid. mp 176–178 °C. 1H
NMR (400 MHz, DMSO-d6) d: 10.57 (br s, 1H, –NH), 10.32
(br s, 1H, –NH), 9.44 (s, 1H, –NH), 9.21 (s, 1H, –NH), 8.57 (d,
1H, J = 4.8 Hz, Ar–H), 8.16–8.06 (m, 2H, Ar–H), 7.74–7.61
(m, 5H, Ar–H,=CH), 7.47–7.40 (m, 2H, Ar–H), 7.20–7.18 (m,
3H, Ar–H), 6.76 (s, 1H, Ar–H), 6.45–6.41 (d, 1H, J = 15.
6 Hz, =CH); HR–MS calcd. for C27H20ClF3N5O5 586.1099
[M?H]?, found 586.1087.
Synthesis of 1-(4-(2-(hydrazinocarbonyl)pyridin-4-
yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea
(7)
To a suspension of 6 (5.9 g, 12.7 mmol, 1.0 equiv) in 15 ml
MeOH, excess hydrazine hydrate (85 percent solution in
water) was added dropwise. The reaction mixture was stirred
at room temperature overnight and the resulting solid was
removed by filtration. The product was washed with small
amounts of MeOH and dried under infrared lamp to obtain
the key intermediate 7 (5.5 g) as pale-white solids, yield
1
1-(4-(2-((N0-((E)-3-(thiophen-2-yl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoro-
methyl)phenyl)urea (8d) Yield 81 %, light brown solid.
93.2 %. H NMR (400 MHz, DMSO-d6) d: 9.89 (s, 1H,
–NH), 9.22 (s, 1H, –NH), 9.00 (s, 1H, –NH), 8.49 (d, 1H,
J = 5.2 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 7.68–7.59 (m, 4H,
Ar–H), 7.36 (s, 1H, Ar–H), 7.19–7.12 (m, 3H, Ar–H), 4.55
(s, 2H, –NH2); ESI–MS [M?H]? 465.1
1
mp 183–184 °C. H NMR (400 MHz, DMSO-d6) d: 10.61
(br s, 1H, –NH), 10.38 (br s, 1H, –NH), 9.34 (s, 1H, –NH),
9.12 (s, 1H, –NH), 8.57 (d, 1H, J = 3.6 Hz, Ar–H), 8.12 (s,
1H, Ar–H), 7.72–7.60 (m, 6H, Ar–H, =CH), 7.44–7.40 (m,
2H, Ar–H), 7.21–7.13 (m, 4H, Ar–H), 6.49–6.45 (d, 1H,
J = 15.6 Hz, =CH); HR–MS calcd. for C27H20ClF3N5O4S
602.0871 [M?H]?, found 602.0851.
General procedure for the preparation of compounds 8a–s
To a solution of 7 (200 mg, 0.43 mmol) in DMF, corre-
sponding 3-arylacrylic acids (0.47 mmol, 1.1 equiv),
123

Med Chem Res
1-(4-(2-((N0-((E)-3-(pyridin-3-yl)acryloyl)) hydrazinocar-
bonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoromethyl)
phenyl)urea (8e) Yield 79 %, light brown solid. mp
(d, 1H, J = 5.2 Hz, Ar–H), 8.12 (s, 1H, Ar–H), 7.94–7.92
(d, 1H, J = 8.0 Hz, Ar–H), 7.82–7.78 (m, 4H, Ar–H), 7.
66–7.60 (m, 4H, Ar–H, =CH), 7.42 (s, 1H, Ar–H), 7.21–7.
19 (m, 3H, Ar–H), 6.88–6.84 (d, 1H, J = 15.6 Hz, =CH);
HR–MS calcd. for C30H21ClF6N5O4 664.1180 [M?H]?,
found 664.1160.
1
186–188 °C. H NMR (400 MHz, DMSO-d6) d: 10.66 (s,
1H, –NH), 10.48 (s, 1H, –NH), 9.23 (s, 1H, –NH), 9.02 (s,
1H, –NH), 8.87–8.81 (d, 1H, J = 27.2 Hz, Ar–H), 8.59–8.
57 (m, 2H, Ar–H), 8.16–8.12 (m, 1H, Ar–H), 8.04 (d, 1H,
J = 3.6 Hz, Ar–H), 7.75–7.58 (m, 5H, Ar–H, =CH), 7.
47–7.41 (m, 2H, Ar–H), 7.21–7.19 (m, 3H, Ar–H), 6.86–6.
82 (d, 1H, J = 16.0 Hz, =CH); HR–MS calcd. for
C28H21ClF3N6O4 597.1259 [M?H]?, found 597.1239.
1-(4-(2-((N0-((E)-3-(4-carbomethoxyphenyl)acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (8j) Yield 86 %, faint yel-
1
low solid. mp [ 250 °C. H NMR (300 MHz, DMSO-d6)
d: 10.67 (s, 1H, –NH), 10.51 (br s, 1H, –NH), 9.30 (s, 1H,
–NH), 9.08 (s, 1H, –NH), 8.58 (d, 1H, J = 5.4 Hz, Ar–H),
8.12 (d, 1H, J = 1.8 Hz, Ar–H), 7.99 (m, 2H, Ar–H), 7.76
(d, 2H, J = 8.7 Hz, Ar–H), 7.63 (m, 5H, Ar–H, =CH), 7.41
(d, 1H, J = 2.4 Hz, Ar–H), 7.22 (m, 3H, Ar–H), 6.86 (d,
1H, J = 15.9 Hz, =CH), 3.87 (s, 3H, –OCH3); HR–MS
calcd. for C31H24ClF3N5O6 654.1361 [M?H]?, found 654.
1339.
1-(4-(2-((N0-((E)-3-(4-hydroxylphenyl)acryloyl)) hydrazi-
nocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluo-
romethyl)phenyl)urea (8f) Yield 80 %, faint yellow solid.
1
mp 181–182 °C. H NMR (300 MHz, DMSO-d6) d: 10.56
(br s, 1H, –NH), 10.25 (br s, 1H, –NH), 9.89 (s, 1H, –NH),
9.33 (s, 1H, –NH), 9.14 (s, 1H, Ar–H), 8.57 (d, 1H, J = 5.
4 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 7.69–7.55 (m, 5H, Ar–H,
=CH), 7.48–.35 (m, 3H, Ar–H), 7.23–7.11 (m, 3H, Ar–H),
6.82 (d, 2H, J = 8.7 Hz, Ar–H), 6.54–6.49 (d, 1H, J = 15.
9 Hz, =CH); HR–MS calcd. for C29H22ClF3N5O5 612.1256
[M?H]?, found 612.1238.
1-(4-(2-((N0-((E)-3-(3-hydroxylphenyl)acryloyl)) hydrazi-
nocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluo-
romethyl) phenyl)urea (8k) Yield 83 %, pale taupe solid.
1
mp 160–161 °C. H NMR (300 MHz, DMSO-d6) d: 10.62
1-(4-(2-((N0-((E)-3-(4-methoxylphenyl)acryloyl)) hydrazi-
nocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoro-
methyl)phenyl)urea (8g) Yield 94 %, pale taupe solid. mp
(s, 1H, –NH), 9.66 (s, 1H, –NH), 9.42 (s, 1H, –NH), 9.19
(s, 1H, –NH), 8.57 (d, 1H, J = 5.7 Hz, Ar–H), 8.13 (s, 1H,
Ar–H), 7.69–7.41 (m, 6H, Ar–H, =CH), 7.24–7.18 (m, 4H,
Ar–H), 7.04–6.98 (m, 2H, Ar–H), 6.82–6.80 (d, 1H, J = 6.
3 Hz, Ar–H), 6.67–6.62 (d, 1H, J = 15.6 Hz, =CH); HR–
MS calcd. for C29H22ClF3N5O5 612.1256 [M?H]?, found
612.1237.
1
169–171 °C. H NMR (400 MHz, DMSO-d6) d: 10.58 (s,
1H, –NH), 10.36 (s, 1H, –NH), 9.47 (s, 1H, –NH), 9.23 (s,
1H, –NH), 8.57 (d, 1H, J = 5.6 Hz, Ar–H), 8.14 (s, 1H,
Ar–H), 7.69–7.48 (m, 7H, Ar–H, =CH), 7.41 (s, 1H, Ar–
H), 7.21–7.19 (m, 3H, Ar–H), 7.00–6.98 (d, 2H, J = 8.
0 Hz, Ar–H), 6.61–6.57 (d, 1H, J = 15.6 Hz, =CH), 3.80
(s, 3H, –OCH3); HR–MS calcd. for C30H24ClF3N5O5 626.
1412 [M?H]?, found 626.1390.
1-(4-(2-((N0-((E)-3-(3-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluoro-
methyl)phenyl)urea (8l) Yield 92 %, faint yellow solid.
1
mp 169–170 °C. H NMR (400 MHz, DMSO-d6) d: 10.57
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluo-
romethyl)phenyl)urea (8h) Yield 90 %, faint yellow
solid. mp [ 250 °C. 1H NMR (300 MHz, DMSO-d6) d: 10.
56 (br s, 2H, –NH), 9.36 (s, 1H, –NH), 9.14 (s, 1H, –NH),
8.58 (d, 1H, J = 5.4 Hz, Ar–H), 8.13 (br s, 1H, Ar–H), 7.
69–7.49 (m, 9H, Ar–H, =CH), 7.41 (d, 1H, J = 2.1 Hz,
Ar–H), 7.24–7.19 (m, 3H, Ar–H), 6.76–6.71 (d, 1H, J =
15.6 Hz, =CH); HR–MS calcd. for C29H21Cl2F3N5O4 630.
0917 [M?H]?, found 630.0898.
(br s, 2H, –NH), 9.30 (s, 1H, –NH), 9.08 (s, 1H, –NH), 8.57
(d, 1H, J = 5.2 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 7.70–7.33
(m, 9H, Ar–H, =CH), 7.27–7.19 (m, 4H, Ar–H), 6.79–6.75
(d, 1H, J = 15.6 Hz, =CH); HR–MS calcd. for
C29H21Cl2F3N5O4 630.0917 [M?H]?, found 630.0898.
1-(4-(2-((N0-((E)-3-(3-(trifluoromethyl)phenyl)acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (8m) Yield 89 %, faint
yellow solid. mp 147–148 °C. 1H NMR (400 MHz,
DMSO-d6) d: 10.65 (m, 2H, –NH), 9.39 (s, 1H, –NH), 9.16
(s, 1H, –NH), 8.57 (d, 1H, J = 4.8 Hz, Ar–H), 8.03 (m,
3H, Ar–H), 7.61 (m, 7H, Ar–H, =CH), 7.40 (s, 1H, Ar–H),
7.20 (m, 3H, Ar–H), 6.86 (d, 1H, J = 16.0 Hz, =CH); HR–
MS calcd. for C30H21ClF6N5O4 664.1180 [M?H]?, found
664.1157.
1-(4-(2-((N0-((E)-3-(4-(trifluoromethyl)phenyl)acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (8i) Yield 93 %, faint
yellow solid. mp 172–173 °C. 1H NMR (400 MHz,
DMSO-d6) d: 9.30 (s, 1H, –NH), 9.08 (s, 1H, –NH), 8.57
123

Med Chem Res
1-(4-(2-((N0-((E)-3-(4-hydroxyl-3-methoxylphenyl) acry-
loyl))hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (8n) Yield 87 %, light
brown solid. mp 188–189 °C. 1H NMR (300 MHz, DMSO-
d6) d: 9.45 (s, 1H, –NH), 9.22 (s, 1H, –NH), 8.56 (d, 1H,
J = 5.4 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 7.69–7.60 (m, 5H,
Ar–H, =CH), 7.46–7.36 (m, 2H, Ar–H), 7.21–7.16 (m, 4H,
Ar–H), 6.82–6.79 (d, 1H, J = 7.8 Hz, Ar–H), 6.56–6.51
(d, 1H, J = 15.9 Hz, =CH), 3.81 (s, 3H, –OCH3); HR–MS
calcd. for C30H24ClF3N5O6 642.1361 [M?H]?, found 642.
1342.
1-(4-(2-((N0-((E)-3-(3,4-dimethoxylphenyl)acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (8o) Yield 96 %, pale taupe
1
solid. mp 167–168 °C. H NMR (300 MHz, DMSO-d6) d:
10.58 (s, 1H, –NH), 10.32 (s, 1H, –NH), 9.51 (s, 1H, –NH),
9.27 (s, 1H, –NH), 8.57 (d, 1H, J = 5.7 Hz, Ar–H), 8.14 (s,
1H, Ar–H), 7.71–7.61 (m, 4H, Ar–H), 7.51–7.46 (s, 1H,
J = 15.6 Hz, =CH), 7.41 (d, 1H, J = 2.4 Hz, Ar–H), 7.
27–7.16 (m, 5H, Ar–H), 7.02–6.99 (s, 1H, J = 8.4 Hz, Ar–
H), 6.65–6.60 (d, 1H, J = 15.6 Hz, =CH), 3.81–3.77 (m,
6H, –OCH3); HR–MS calcd. for C31H26ClF3N5O6 656.
1518 [M?H]?, found 656.1500.
1-(4-(2-((N0-((E)-3-(4-acetoxy-3-methoxylphenyl) acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (8p) Yield 91 %, faint yel-
low solid. mp 169–170 °C. 1H NMR (300 MHz, DMSO-d6)
d: 10.61 (br s, 2H, –NH), 9.30 (s, 1H, –NH), 9.08 (s, 1H,
–NH), 8.58 (d, 1H, J = 5.4 Hz, Ar–H), 8.12 (br s, 1H, Ar–
H), 7.69–7.52 (m, 5H, Ar–H, =CH), 7.44–7.38 (m, 2H, Ar–
H), 7.24–7.13 (m, 5H, Ar–H), 6.76–6.71 (d, 1H, J = 15.
6 Hz, =CH), 3.83 (s, 3H, –OCH3), 2.27 (s, 3H, –CH3); HR–
MS calcd. for C32H26ClF3N5O7 684.1467 [M?H]?, found
684.1442.
1-(4-(2-((N0-((E)-3-(3,4-difluorophenyl)acryloyl)) hydrazi-
nocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluo-
romethyl)phenyl)urea (8q) Yield 93 %, pale taupe solid.
1
mp 175–177 °C. H NMR (300 MHz, DMSO-d6) d: 10.58
(br s, 2H, –NH), 9.34 (s, 1H, –NH), 9.12 (s, 1H, –NH), 8.57
(d, 1H, J = 5.4 Hz, Ar–H), 8.12 (s, 1H, Ar–H), 7.72–7.40
(m, 9H, Ar–H), 7.33–7.27 (s, 1H, J = 15.9 Hz, =CH), 7.
21–7.18 (d, 2H, J = 8.7 Hz, Ar–H), 6.73–6.68 (d, 1H, J =
15.9 Hz, =CH); HR–MS calcd. for C29H20ClF5N5O4 632.
1118 [M?H]?, found 632.1097.
1-(4-(2-((N0-((E)-3-(2,4-dichlorophenyl)acryloyl)) hydrazi-
nocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-(trifluo-
romethyl)phenyl)urea (8r) Yield 82 %, faint yellow
1
solid. mp 165–167 °C. H NMR (400 MHz, DMSO-d6) d:
10.64 (br s, 2H, –NH), 9.46 (s, 1H, –NH), 9.23 (s, 1H,
123

Med Chem Res
Fig. 2 Effects of 8h on the growth of pancreatic cancer Capan2
(a) and Mia-PaCa2 (b) xenograft models. Activity of 8h was
determined in Capan2 and Mia-PaCa2 xenograft in nude mice. The
animals were randomly divided into four groups when tumour volume
reached 250 mm3. The dosage of 8h is 15, 30 (Capan2) or 30, 60 mg/
kg (Mia-PaCa2) p.o. once daily continuously. Each treatment arm
involved six (Capan2) or five (Mia-PaCa2) independent tumour-
bearing mice representing the same xenograft line
Synthesis of 4-(4-aminophenoxy)-N0-((E)-3-(4-
–NH), 8.56 (d, 1H, J = 5.2 Hz, Ar–H), 8.12 (s, 1H, Ar–H),
7.87 (m, 1H, Ar–H), 7.72 (m, 3H, Ar–H, =CH), 7.40 (m,
3H, Ar–H), 6.78 (d, 1H, J = 16.0 Hz, =CH); HR–MS
calcd. for C29H20Cl3F3N5O4 664.0527 [M?H]?, found
664.0506.
chlorophenyl)acryloyl)pyridine-2-carboxhydrazide (10)
To a solution of compound 9 (1.8 g, 7.4 mmol, 1.0 equiv) in
DMF, 4-chlorocinnamic acid (1.5 g, 8.1 mmol, 1.1 equiv),
HATU (3.1 g, 8.1 mmol, 1.1 equiv) and TEA (2.2 g,
22.2 mmol, 3.0 equiv) were added, respectively, and was
stirred under room temperature overnight. To the solution,
water was added to get solids. The product was filtrated,
washed with excess water and dried under infrared lamp to
obtain compounds 10 (2.7 g), yield 89.4 %. 1H NMR
(400 MHz, DMSO-d6) d: 10.59 (br s, 1H, –NH), 10.48
(s, 1H, –NH), 8.52 (d, 1H, J = 5.6 Hz, Ar–H), 7.56
(m, 3H, Ar–H), 7.49 (m, 5H, Ar–H, =CH), 6.88 (d,
2H, J = 8.4 Hz, Ar–H), 6.73 (d, 1H, J = 16.0 Hz,
=CH), 6.65 (d, 1H, J = 8.8 Hz, Ar–H), 5.19 (s, 2H, –NH2),
ESI–MS [M?H]? 408.1.
1-(4-(2-((N0-((E)-3-(2,3,4,5,6-pentafluorophenyl) acryloyl))
hydrazinocarbonyl)pyridin-4-yloxy)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (8s) Yield 79 %, yellow
1
solid. mp 159–161 °C. H NMR (300 MHz, DMSO-d6) d:
10.73 (br s, 2H, –NH), 9.38 (s, 1H, –NH), 9.16 (s, 1H,
–NH), 8.58 (d, 1H, J = 5.4 Hz, Ar–H), 8.13 (d, 1H, J = 1.
8 Hz, Ar–H), 7.69–7.60 (m, 4H, Ar–H, =CH), 7.45–7.39
(m, 2H, Ar–H), 7.23–7.19 (m, 3H, Ar–H), 7.03–6.98
(d, 1H, J = 16.2 Hz, =CH); HR–MS calcd. for
C29H17ClF8N5O4 686.0835 [M?H]?, found 686.0820.
Synthesis of 4-(4-aminophenoxy)pyridine-2-
General procedure for the preparation of compounds
carboxhydrazide (9)
11a–h
To a suspension of 5 (2.4 g, 10.0 mmol, 1.0 equiv) in
10 ml MeOH, excess hydrazine hydrate was added (85
percent solution in water) dropwise. The reaction mixture
was stirred at room temperature overnight, and the result-
ing solid was removed by filtration. The product was
washed with small amounts of MeOH and dried under
infrared lamp to obtain compound 9 (1.8 g), yield 73.8 %.
1H NMR (300 MHz, DMSO-d6) d: 9.84 (s, 1H, –NH), 8.43
(d, 1H, J = 5.7 Hz, Ar–H), 7.30 (s, 1H, Ar–H), 7.04 (m,
1H, Ar–H), 6.88 (s, 1H, Ar–H), 6.85 (s, 1H, Ar–H), 6.66 (s,
1H, Ar–H), 6.63 (s, 1H, Ar–H), 5.18 (s, 2H, –NH2), 4.52
(br s, 2H, –NH2), ESI–MS [M?H]? 244.1.
To a suspension of 10 (1.0 equiv) in DCM, a DCM solution
of corresponding phenyl isocyanate (1.05 equiv) was added
dropwise at 0 °C. The mixture was stirred at room tem-
perature overnight. The resulting solid was removed by
filtration, then washed with small amounts of DCM and
dried under infrared lamp to afford compound 11a–h.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-chlorophenyl)urea
1
(11a) Yield 67 % aint yellow solid. mp 249–251 °C. H
NMR (300 MHz, DMSO-d6) d: 10.55 (br s, 2H, –NH), 8.97
123

Med Chem Res
(s, 2H, –NH), 8.57 (d, 1H, J = 5.1 Hz, Ar–H), 7.75–7.72
(d, 2H, J = 8.7 Hz, Ar–H), 7.66–7.58 (m, 3H, Ar–H), 7.
51–7.48(m, 4H, Ar–H, =CH), 7.41 (s, 1H, Ar–H), 7.34–7.
28 (m, 3H, Ar–H), 7.20–7.17 (d, 2H, J = 8.4 Hz, Ar–H), 6.
76–6.71 (d, 1H, J = 15.3 Hz, =CH); HR–MS calcd. for
C28H22Cl2N5O4 562.1043 [M?H]?, found 562.1029.
41 (br s, 1H, –NH), 8.93 (s, 2H, –NH), 8.57 (d, 1H, J = 5.
4 Hz, Ar–H), 7.86 (s, 1H, Ar–H), 7.64 (m, 7H, Ar–H, =
CH), 7.41 (s, 1H, Ar–H), 7.21 (m, 6H, Ar–H), 6.74 (d, 1H,
J = 15.3 Hz, =CH); HR–MS calcd. for C28H22BrClN5O4
606.0538 [M?H]?, found 606.0520.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(3-chloro-4-methyl-
phenyl)urea (11g) Yield 59 %, faint yellow solid.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-fluorophenyl)urea
(11b) Yield 61 %, light brown solid. mp 250 °C. 1H
NMR (300 MHz, DMSO-d6) d: 10.63 (br s, 1H, –NH), 10.
40 (br s, 1H, –NH), 8.84 (s, 1H, –NH), 8.76 (s, 1H, –NH),
8.57 (d, 1H, J = 4.8 Hz, Ar–H), 7.57 (m, 10H, Ar–H, =
CH), 7.17 (m, 5H, Ar–H), 6.68 (d, 1H, J = 12.0 Hz, =CH);
HR–MS calcd. for C28H22ClFN5O4 546.1338 [M?H]?,
found 546.1324.
1
mp [ 250 °C. H NMR (400 MHz, DMSO-d6) d: 8.96 (s,
1H, –NH), 8.91 (s, 1H, –NH), 8.57 (d, 1H, J = 5.6 Hz, Ar–
H), 7.74–7.70 (m, 2H, Ar–H), 7.65–7.63 (d, 1H, J = 8.
0 Hz, Ar–H), 7.60–7.58 (d, 2H, J = 8.4 Hz, Ar–H), 7.
51–7.49 (d, 2H, J = 8.4 Hz, Ar–H), 7.41 (s, 1H, Ar–H), 7.
33–7.17 (m, 6H, Ar–H, =CH), 6.75–6.71 (d, 1H, J = 16.
0 Hz, =CH), 2.26 (s, 3H, –CH3); HR–MS calcd. for
C29H24Cl2N5O4 576.1199 [M?H]?, found 576.1189.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-(trifluoromethyl)
phenyl)urea (11c) Yield 64 %, faint yellow solid.
mp [ 250 °C. 1H NMR (300 MHz, DMSO-d6) d: 10.62 (br
s, 2H, –NH), 9.29 (s, 1H, –NH), 9.10 (s, 1H, –NH), 8.57 (d,
1H, J = 4.8 Hz, Ar–H), 7.75–7.57 (m, 8H, Ar–H), 7.51–7.
41 (m, 3H, Ar–H), 7.34–7.29 (d, 1H, J = 15.6 Hz, =CH),
7.21–7.19 (m, 3H, Ar–H), 6.76–6.71 (d, 1H, J = 15.6 Hz,
=CH); HR–MS calcd. for C29H22ClF3N5O4 596.1306
[M?H]?, found 596.1290.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(2,4-difluorophenyl)
urea (11h) Yield 58 %, faint yellow solid. mp [ 250 °C.
1H NMR (400 MHz, DMSO-d6) d: 9.23 (s, 1H, –NH), 8.58
(br s, 2H, –NH), 8.08 (m, 1H, –NH), 7.74–7.72 (d, 1H,
J = 8.8 Hz, Ar–H), 7.65–7.58 (m, 4H, Ar–H), 7.51–7.49
(d, 2H, J = 8.4 Hz, Ar–H), 7.41 (s, 1H, Ar–H), 7.33–7.29
(m, 2H, Ar–H), 7.21–7.19 (d, 2H, J = 8.4 Hz, Ar–H), 7.
23–7.16 (m, 5H, Ar–H, =CH), 7.05 (t, 1H, J = 8.0 Hz,
Ar–H), 6.75–6.71 (d, 1H, J = 15.6 Hz, =CH); HR-MS
calcd. for C28H21ClF2N5O4 564.1244 [M?H]?, found 564.
1231.
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(4-ethoxylphenyl)urea
1
(11d) Yield 60 %, light brown solid. mp [ 250 °C. H
Acknowledgments This project was financially supported by
National S&T Major Special Project on ‘‘12ꢀ5 Major New Drug
Innovation’’ (No. 2012ZX09103-101-019).
NMR (300 MHz, DMSO-d6) d: 10.64 (br s, 1H, –NH), 10.
41 (br s, 1H, –NH), 8.76 (s, 1H, –NH), 8.57 (s, 1H, –NH),
8.51 (s, 1H, Ar–H), 8.36 (s, 1H, Ar–H), 7.52 (m, 12H, Ar–
H, =CH), 7.17 (m, 3H, Ar–H), 6.71 (m, 4H, Ar–H, =CH),
3.83 (br s, 2H, –OCH2), 1.15 (br s, 3H, –CH3); HR–MS
calcd. for C30H27ClN5O5 572.1695 [M?H]?, found 572.
1680.
References
Atkins M, Jones CA, Kirkpatrick P (2006) Sunitinib maleate. Nat Rev
Drug Discov 5:279–280
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(3-methylphenyl)urea
Bilanges B, Torbett N, Vanhaesebroeck B (2008) Killing two kinase
families with one stone. Nat Chem Biol 4:648–649
Capdeville R, Buchdunger E, Zimmermann J, Matter A (2002) Glivec
(STI571, imatinib), a rationally developed, targeted anticancer
drug. Nat Rev Drug Discov 1:493–502
Giordano S, Petrelli A (2008) From single- to multi-target drugs in
cancer therapy: when a specificity becomes an advantage. Curr
Med Chem 15:422–432
1
(11e) Yield 61 %, light brown solid. mp [ 250 °C. H
NMR (300 MHz, DMSO-d6) d: 10.64 (br s, 1H, –NH), 10.
41 (br s, 1H, –NH), 8.82 (s, 1H, –NH), 8.64 (s, 1H, –NH),
8.57 (s, 1H, Ar–H), 7.72 (m, 9H, Ar–H, =CH), 7.17 (m, 3H,
Ar–H), 6.71 (m, 4H, Ar–H, =CH), 2.28 (s, 3H, –CH3); HR–
MS calcd. for C29H25ClN5O4 542.1589 [M?H]?, found
542.1577.
Globocan 2012: Estimated Cancer Incidence, Mortality and Preva-
lence Worldwide in 2012. WHO website: http://globocan.iarc.fr/
Pages/fact_sheets_cancer.aspx
Hernandez-Davies JE, Zape JP, Landaw EM, Tan X, Presnell A,
Griffith D, Heinrich MC, Glaser KB, Sakamoto KM (2011) The
Multitargeted Receptor Tyrosine Kinase Inhibitor Linifanib
(ABT-869) Induces Apoptosis through an Akt and Glycogen
Synthase Kinase 3b–Dependent Pathway. Mol Cancer Ther
10:949–959
1-(4-(2-((N0-((E)-3-(4-chlorophenyl)acryloyl)) hydrazino-
carbonyl)pyridin-4-yloxy)phenyl)-3-(3-bromophenyl)urea
1
(11f) Yield 57 %, light brown solid. mp [ 250 °C. H
NMR (300 MHz, DMSO-d6) d: 10.64 (br s, 1H, –NH), 10.
123

Med Chem Res
Ji H, Ding C (2008) Secondary resistance of gefitinib in non-small-
lung cancer treatment. J Int Oncol 35:908–910
activation of the RAF-ERK signaling pathway by oncogenic
mutations of B-RAF. Cell Signal 116:855–867
Kane RC, Farrell AT, Saber H, Tang S, Williams G, Jee JM, Liang C,
Booth B, Chidambaram N, Morse D, Sridhara R, Garvey P,
Justice R, Pazdur R (2006) Sorafenib for the treatment of
advanced renal cell carcinoma. Clin Cancer Res 12:7271–7278
Li Y, Shi X, Xie N, Zhao Y, Li S (2013) 3,3-Dimethyl-1H-
pyrrolo[3,2-g]quinolin-2(3H)-one derivatives as novel Raf
kinase inhibitors. MedChemComm 4:367
Wang F, Fu L-W (2008) Research advances in sorafenib: a multiple
targeted anti-tumor agent. Chinese Pharmaco Bull 8:042
Wilhelm SM, Dumas J, Adnane L, Lynch M, Carter CA, Schu¨tz G,
Thierauch K-H, Zopf D (2011) Regorafenib (BAY 73-4506): a
new oral multikinase inhibitor of angiogenic, stromal and
oncogenic receptor tyrosine kinases with potent preclinical
antitumor activity. Int J Cancer 129:245–255
Lin L (2009) Mechanisms of drug resistance of gefitinib and erlotinib.
J Int Oncol 113:734–736
Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann
¨
F, Mestan J, Mett H, O’Reilly T, Persohn E, Rosel J, Schnell C,
Stover D, Theuer A, Towbin H, Wenger F, Woods-Cook K,
Menrad A, Siemeister G, Schirner M, Thierauch K-H, Schneider
Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, Wilhelm S,
Lynch M, Carter C (2006) Sorafenib blocks the RAF/MEK/ERK
pathway, inhibits tumor angiogenesis, and induces tumor cell
apoptosis in hepatocellular carcinoma model PLC/PRF/5. Can-
cer Res 66:11851–11858
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 (2012) Design, synthesis and antitumor
activity of sorafenib analogues containing 2-picolinylhydrazide
moiety. Acta Pharmaceutica Sinica 47:1623–1629
Ramurthy S, Subramanian S, Aikawa M, Amiri P, Costales A, Dove J,
Fong S, Jansen JM, Levine B, Ma S, McBride CM, Michaelian J,
Pick T, Poon DJ, Girish S, Shafer CM, Stuart D, Sung L,
Renhowe PA (2008) Design and synthesis of orally bioavailable
´
MR, Drevs J, Martiny-Baron G, Totzke F, Marme D (2000)
PTK787/ZK 222584, a novel and potent inhibitor of vascular
endothelial growth factor receptor tyrosine kinases, impairs
vascular endothelial growth factor-induced responses and tumor
growth after oral administration. Cancer Res 60:2178–2189
Yao J, Chen J, He Z, Sun W, Xu W (2012) Design, synthesis and
biological activities of thiourea containing sorafenib analogs as
antitumor agents. Bioorg Med Chem 20:2923–2929
Zhao C-R, Wang R-Q, Li G, Xue X-X, Sun C-J, Qu X-J, Li W-B
(2013) Synthesis of indazole based diarylurea derivatives and
their antiproliferative activity against tumor cell lines. Bioorg
Med Chem Lett 23:1989–1992
benzimidazoles as Raf kinase inhibitors.
51:7049–7052
J Med Chem
´
Zimmermann GR, Lehar J, Keith CT (2007) Multi-target therapeu-
Wan PTC, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good
VM, Project CG, Jones CM, Marshall CJ (2004) Mechanism of
tics: when the whole is greater than the sum of the parts. Drug
Discov Today 12:34–42
123