Design, synthesis and evaluation of novel diaryl urea derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.03.020

A novel series of diaryl ureas containing different linker groups were designed and synthesized. Their in vitro antitumor activity against MX-1, A375, HepG2, Ketr3 and HT-29 was evaluated using the standard MTT assay. Compounds having a rigid linker group such as vinyl, ethynyl and phenyl showed significant inhibitory activity against a variety of cancer cell lines. Specifically, compound 23 with a phenyl linker group demonstrated broad-spectrum antitumor activity with IC₅₀ values of 5.17-6.46 μM against five tested tumor cell lines. Compound 23 is more potent than reference drug sorafenib (8.27-15.2 μM), representing a promising lead for further optimization.

Full text of DOI:10.1016/j.ejmech.2014.03.020

European Journal of Medicinal Chemistry 77 (2014) 351e360
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of novel diaryl urea derivatives as
potential antitumor agents
*
Chenshu Lu, Ke Tang, Yan Li, Peng Li, Ziyun Lin, Dali Yin, Xiaoguang Chen ,
**
Haihong Huang
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Peking Union Medical College & Chinese
Academy of Medical Sciences, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of diaryl ureas containing different linker groups were designed and synthesized. Their
in vitro antitumor activity against MX-1, A375, HepG2, Ketr3 and HT-29 was evaluated using the standard
MTT assay. Compounds having a rigid linker group such as vinyl, ethynyl and phenyl showed significant
inhibitory activity against a variety of cancer cell lines. Specifically, compound 23 with a phenyl linker
group demonstrated broad-spectrum antitumor activity with IC50 values of 5.17e6.46 mM against five
tested tumor cell lines. Compound 23 is more potent than reference drug sorafenib (8.27e15.2 mM),
Received 25 August 2013
Received in revised form
4 February 2014
Accepted 8 March 2014
Available online 12 March 2014
representing a promising lead for further optimization.
Keywords:
Ó 2014 Elsevier Masson SAS. All rights reserved.
Diaryl ureas
Sorafenib
Linker
Antitumor activity
1. Introduction
portion of the molecule is highly conserved and shared by most
type II kinase inhibitors. The N-methyl-4-picolinamide moiety that
binds to the hinge region of the kinase is highly mobile. The pyridyl
ring nitrogen atom in sorafenib forms hinge hydrogen bonding, but
it is known to be less critical compared with that in other kinase
inhibitors [14,15].
The interest in designing novel diaryl ureas with improved
antitumor activity has been growing following the successful
launch of sorafenib [16e20]. Linifanib is another potent kinase in-
hibitor in clinical development. As compared to the structure of
Cellular signaling network in tumor cells is complex and
redundant, therefore broad-acting and multi-targeted kinase in-
hibitors may be more advantageous than selective agents because
of their ability to block multiple signaling pathways associated with
tumor survival [1e3]. A number of multi-targeted kinase inhibitors
based on different scaffolds, such as sorafenib [4], linifanib [5,6] and
curcumin [7], have reached market or have been in late-stage
clinical trials. These agents have demonstrated clinical benefits
with manageable side effects (Fig. 1).
sorafenib, linifanib has
a more rigid structure with a 3-
The antitumor activity of kinase inhibitors containing a diaryl
urea scaffold has been described in literature [8e10]. These mole-
cules possess a unique binding mode and kinase inhibition profile
[11]. For example, sorafenib, a diaryl urea multi-targeted inhibitor
of several kinases including Raf, VEGFR and PDGFR, was approved
by Food and Drug Administration (FDA) for the treatment of
advanced renal cell carcinoma (RCC) in 2005 and unresectable
hepatocellular carcinoma (HCC) in 2007 [4,12].
aminoindazole moiety at the hinge region. It has been demon-
strated that a large group at the hinge region can be well tolerated
[15,21e24] and there is growing interest in finding an optimal
hinge binding scaffold.
To develop a new generation of more potent multi-targeted
kinase inhibitors, we took advantage of the previous structuree
activity relationships (SAR) of sorafenib analogs. The diaryl urea
and amide groups, as key pharmacophores, were kept intact. At the
same time, we planned to preserve the chloro and trifluoromethyl
substituents on the distal phenyl ring, while replacing the N-methyl
Crystal structure of B-Raf in complex with sorafenib revealed a
distinctive mode of proteineligand interaction [13]. The diaryl urea
group by
a-methylbenzyl and cyclic alkyl groups in order to
investigate their effects on antitumor activity. We focused our main
modifications on the oxygen atom and pyridyl ring of sorafenib,
exploring a variety of linker groups such as vinyl, ethynyl and
phenyl to connect the diaryl urea and amide moieties. A total of
* Corresponding author. Tel./fax: þ86 010 63165207.
** Corresponding author. Tel./fax: þ86 010 63165244.
E-mail addresses: chxg@imm.ac.cn (X. Chen), joyce@imm.ac.cn (H. Huang).
http://dx.doi.org/10.1016/j.ejmech.2014.03.020
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

352
C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
H
N
N
H2N
O
O
Cl
O
N
H
O
N
N
H
N
H
N
H
CF3
N
H
F
Sorafenib
Linifanib
O
O
L
NR3R4
O
O
O
R1 R2
O
N
H
N
H
OH
HO
Curcumin
Target compounds
Fig. 1. Structures of known multi-targeted kinase inhibitors and target compounds.
twenty seven novel diaryl ureas were designed and synthesized.
The inhibitory activity of these compounds 1e27 against five cancer
cell lines, including human breast cancer (MX-1), human mela-
noma (A375), human liver cancer (HepG2), human kidney cancer
(Ketr3) and human colon cancer (HT-29) cell lines, was evaluated
reported procedure [29], followed by ester saponification and
amidation using BOP reagent gave amide intermediate. Herein, an
improved approach for selective reduction of nitro group in
compounds containing an ethynyl group was investigated. We
reduced the nitro group with Fe powder in ethanol under acidic
condition, which gave intermediates A6(aeb) in high yield (about
80%).
using
the
standard
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Herein, we wish to
report the synthesis and SAR of the target compounds.
2.1.5. General synthesis of intermediate A7eA8
2. Results and discussion
Suzuki coupling between 4-bromonitrobenzene and 3-
carboxybenzeneboronic acid with Pd(PPh3)4 as a catalyst in the
presence of K2CO3 afforded 4-nitrobiphenyl-3-carboxylic acid
quantitatively [30]. 4-Biphenylcarboxylic acid was treated with ice-
cold nitric acid to provide 4-nitrobiphenyl-4-carboxylic acid [31].
Condensation the two acids with the corresponding amines, fol-
lowed by reduction of nitro group with 5% Pd/C gave intermediates
A7(aeb) and A8(bec), respectively.
2.1. Chemistry
The intermediate amines A1eA8 were synthesized following
known synthetic procedures as outlined in Scheme 1. Some
improvements were made, especially for A6. The general syn-
thetic procedures of target compounds 1e27 were illustrated in
Scheme 2.
2.1.6. General synthesis of target compounds 1e27
2.1.1. General synthesis of intermediate A1eA3
Finally, condensation of A1eA8 with corresponding aryl iso-
cyanates in anhydrous dichloromethane (DCM) afforded the
desired diaryl urea derivatives 1e27 in 79%e91% yields [32].
4-Nitrobenzaldehyde was submitted to the Knoevenagel reac-
tion with malonic acid or methylmalonic acid [25]. Condensation
with corresponding amines, followed by reduction of nitro group
with SnCl2 gave intermediates A1(aed) or A3(aec). The subsequent
catalytic hydrogenation of nitro group and double bond simulta-
neously using 5% Pd/C gave intermediates A2(aed).
2.2. Biological evaluation
The in vitro inhibitory activity of target compounds against MX-
1, A375, HepG2, Ketr3 and HT-29 cell lines was evaluated using the
MTT-based assay. The IC50 results of various analogs were sum-
marized in Table 1 with sorafenib as the reference compound.
As illustrated in Table 1, most compounds bearing a rigid linker
group such as vinyl (compounds 2e4), ethynyl (compounds 20e21)
and phenyl (compounds 23e25) exhibited moderate to potent ac-
tivities against the tested cancer cell lines, while flexible linker
group such as saturated ethyl chain (compounds 6e9) and bulky
linker group such as branched vinyl (compounds 10e15) were all
detrimental for activities. The results were consistent with several
2.1.2. General synthesis of intermediate A4
4-Nitroacetophenone was treated with trimethyl phosphonoa-
cetate in the presence of N-sodiumhexamethyldisilazane
(NaHMDS) [26]. Ester saponification and amidation, followed
by reduction of nitro group with SnCl2 provided intermediates
A4(aec).
2.1.3. General synthesis of intermediate A5
Treatment of N-Boc-4-aminophenol with methyl propiolate in
the presence of a catalytic amount of 1,4-diazabicyclo[2.2.2]octane
(DABCO) [27], followed by ester saponification and amidation using
(benzotriazol-1-yloxy)-tris-(dimethylamino)phosphonium hexa-
fluorophosphate (BOP) as a condensation reagent [28], and Boc
deprotection afforded intermediates A5(aeb).
recently published articles [33e35] which suggested that a,b-un-
saturated ketone may be the pharmacophore of many antitumor
agents like curcumin. It was also noted that a moderate to dramatic
decrease in potency was found if the vinyl group was replaced with
a more electron-deficient eOCH]CH group (compounds 2 versus
17, 3 versus 18). These observations may be due to a combination of
electronic and steric effects of the linker groups. Interestingly, most
compounds that contain a rigid linker group demonstrated potent
activities against both MX-1 and HT-29, especially with an
2.1.4. General synthesis of intermediate A6
The conversion of 4-iodonitrobenzene to methyl 3-(4-
nitropheny1)propiolate was accomplished according to the

C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
353
Br
O
OH
O2N
A7
A8
1)n, 2)b, 3)c, 4)e
1)m, 2)b, 3)c, 4)e
1)a, 2)b, 3)c, 4)d
O
L
NR3R4
1)k, 2)g, 3)i, 4)l
I
A1 or A3
H
A6
O
1)a, 2)b, 3)c, 4)e
H2N
O2N
O2N
A2
A1-A8
A4
A5
O
OH
1)h, 2)g, 3)i, 4)j
1)f, 2)g, 3)b
4)c, 5)d
BOCHN
O2N
N
O
*
NH
*
NH
*
NH
*
NR3R4:
(a),
(b),
(c),
(d).
A3(a-c) L:
A7(a-b) L:
A4(a-c) L:
A8(b-c) L:
A2(a-d) L:
A6(a-b) L:
A1(a-d) L:
A5(a-b) L:
O
Scheme 1. General synthetic scheme for amines A1eA8 with various designed linker groups. Reagents and conditions: a. malonic acid or methylmalonic acid, piperidine, pyridine,
90 ꢀC, 4e8 h, 72%e83%; b. SOCl2, reflux, 2 h; c. R3R4NH, TEA, DCM, rt, 4e8 h, 82%e92%; d. SnCl2, 6 mol/L HCl, EtOHeH2O, reflux, 4 h, 50%e68%; e. H2, 5% Pd/C, THF, 6 h, 83%e94%; f.
trimethyl phosphonoacetate, 1 mol/L NaHMDS/THF, toluene, rt, 10 h, 70%; g. NaOH, THFeH2O, rt, 12 h, 87%e93%; h. methyl propiolate, DABCO, DCM, rt, 1.5 h, 89%; i. R3R4NH, BOP,
TEA, THF, rt, 3 h, 59%e78%; j. (i) HCl/EtOAc, rt, 1 h, (ii) NaHCO3, DCM, 80%e84%; k. methyl propiolate, Pd(PPh3)2Cl2, CuI, K2CO3, THF, reflux, 5 h, 78%; l. Fe, 2 mol/L HCl, EtOHeH2O,
70 ꢀC, 4 h, 78%e83%; m. 3-carboxybenzeneboronic acid, Pd(PPh3)4, K2CO3, EtOHeH2Oetoluene, reflux, 48 h, 96%; n. HNO3, 1 h, 55%.
outstanding activity on HT-29 cell line with IC50 lying in the
micromolar range, such as compounds 2e4, 20e21, 23e27. In
particular, in the phenyl series, there was a similar trend for
different linking patterns of amide groups, either on meta-position
or para-position of the biphenyl substructure. Compounds 26 and
27 that contain the linker as 1,4-disubstituted phenyl group
exhibited superior selective anticancer activity against HT-29 with
ring did not have a significant impact on activities, as exemplified
by compound 2 versus compound 3.
It is worth noting that compound 23 with a 1,3-disubstituted
phenyl linker, demonstrated superior broad-spectrum antitumor
activities with IC50 values in the micromolar range (5.17
6.46
compound sorafenib with IC50 values in the range of 8.27
15.2
m
Me
m
M) against all tested five cell lines compared to the reference
mMe
IC50 values of 2.05
mM and 1.90
mM, respectively, compared to
m
M. Compound 23 could become a valuable lead for further
sorafenib with IC50 value of 15.2
mM. This information could be
optimization.
useful in designing potent inhibitors against colon cancer.
Additionally, we found that introduction of a large substituent
like N-methylbenzyl compared to N-methyl in amide moiety led to
more potent inhibitors, as evidenced by compound 2 versus com-
pound 1, compound 20 versus compound 19, compound 23 versus
compound 22. It was also consistent with the results that many
researches have demonstrated that the large groups are well
tolerated in this region to be beneficial to the activity and physi-
cochemical properties [18,21]. Finally, the effect of the position of
the electron-withdrawing substituents on the distal phenyl ring
such as 4-Cle3-CF3 versus 2-Cle5-CF3 was investigated. The pre-
liminary result showed that the position of Cl and CF3 on the phenyl
2.3. Docking analysis
In order to understand the mode of binding for these novel
diaryl ureas, the most promising compound 23 was docked into
the crystal structure published for B-Raf (1UWH) [36] using
Discovery Studio 2.0 software (Accelrys, Inc.) (Fig. 2). The dock-
ing model suggested a mode of binding with matching hydrogen
bonds to the backbone NH and C]O of Cys531 in the hinge re-
gion through the amide moiety, and to the Asp593/Glu500 pair
in the catalytic region through the urea moiety. Furthermore, a
p
-stacking interaction of the phenyl ring of
a
-methylbenzyl with
NR3R4
L
NCO
L
NR3R4
O
a
R1 R2
+
R1 R2
O
O
N
H
N
H
H2N
Target compounds
A1-A8
Scheme 2. General synthetic route of target compounds 1e27. Reagent and condition: a. anhydrous DCM, Ar, rt, 12 h.

354
C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
Table 1
Structures and inhibitory activity of target compounds against MX-1, A375, HepG2, Ketr3 and HT-29 cancer cell lines in vitro.
Compd.
R1R2
NR3R4
L
IC50 (mmol/L)
MX-1
13.8
A375
HepG2
Ketr3
HT-29
NH
*
1
2
4-Cle3-CF3
4-Cle3-CF3
20.9
20.8
20.4
12.2
21.2
21.2
11.7
1.06
3.13
NH
*
3
2-Cle5-CF3
1.89
4.53
40.9
35.9
14.3
4.19
NH
NH
N
*
4
5
4-Cle3-CF3
4-Cle3-CF3
15.4
7.51
45.9
17.3
4.43
9.10
*
12.8
>50
>50
O
*
NH
6
7
4-Cle3-CF3
4-Cle3-CF3
20.4
25.2
21.0
24.0
20.6
26.5
*
>50
>50
>50
>50
NH
NH
N
*
8
9
4-Cle3-CF3
4-Cle3-CF3
24.0
>50
>50
>50
>50
>50
>50
21.3
*
>50
>50
O
O
O
*
NH
10
11
4-Cle3-CF3
4-Cle3-CF3
>50
>50
>50
>50
44.3
>50
>50
>50
>50
*
>50
NH
N
*
*
12
4-Cle3-CF3
>50
>50
>50
>50
>50
NH
13
14
4-Cle3-CF3
4-Cle3-CF3
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
*
NH
N
*
*
15
4-Cle3-CF3
>50
46.2
>50
>50
26.2
NH
NH
16
17
4-Cle3-CF3
4-Cle3-CF3
18.8
36.8
20.7
24.2
14.1
>50
>50
>50
*
*
O
O
13.7
14.5

C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
355
Table 1 (continued )
Compd.
R1R2
NR3R4
L
IC50 (mmol/L)
MX-1
A375
16.0
HepG2
11.9
Ketr3
16.4
HT-29
18
2-Cle5-CF3
16.1
>50
O
NH
NH
*
19
20
4-Cle3-CF3
4-Cle3-CF3
26.4
12.8
>50
23.3
20.5
24.7
21.4
14.0
4.92
*
16.8
NH
*
21
22
23
24
2-Cle5-CF3
4-Cle3-CF3
4-Cle3-CF3
4-Cle3-CF3
16.1
30.5
46.5
19.0
37.1
5.67
16.8
23.2
39.3
3.17
NH
NH
*
>50
29.3
*
5.24
5.17
6.46
6.32
5.96
NH
NH
*
*
4.63
12.8
16.9
19.3
16.8
41.9
25
26
2-Cle5-CF3
4-Cle3-CF3
17.7
18.4
31.1
6.57
2.05
NH
N
*
*
>50
>50
O
O
27
2-Cle5-CF3
>50
>50
>50
>50
1.90
N
*
Sorafenib
8.27
9.17
8.67
18.8
15.2
Trp530 can be observed, which could provide additional binding
affinity.
2.4. Kinase inhibitory activity
The docking model indicates that an appropriate linker group
such as 1,3-disubstituted phenyl would allow each fragment of the
molecule to nicely fit into the desired pockets, which may explain
the potent antitumor activity of compound 23 observed.
To investigate the possible mechanism of action responsible
for the observed antitumor activities and to verify the
docking analysis above, compound 23 was selected to be
initially screened for B-Raf and C-Raf inhibitory activities
H
N
Cl
O
O
CF3
N
H
N
H
Compound 23
Fig. 2. Docking model of compound 23 (yellow) in B-Raf. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

356
C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
in vitro by employing the standardized ADP-Glo assay
methodology.
As shown in Table 2, compound 23 is very weak against B-Raf
and C-Raf kinases with IC50 more than 50 mM. The inconsistency
between the enzyme inhibitory activity and cellular efficiency
against five tested cell lines suggested that this compound may
exert antitumor activity by inhibiting other receptor tyrosine ki-
nases rather than Raf kinase. This structural modification might
have switched the binding affinity to other biomolecular targets
involved in cellular signaling pathways. It is worthy of further
research to explore the exact target(s) and details on the mecha-
nism of action for this novel target compounds in the future.
at 0 ꢀC, then a DCM (1 mL) solution of corresponding aryl isocya-
nate (0.55 mmol) was added drop wise with constant stirring under
argon atmosphere. The reaction mixture was stirred at room tem-
perature for 12 h and then filtered. The resulting solid was washed
with ethyl acetate (EtOAc) and dried under vacuum for 4 h to afford
diaryl ureas 1e27.
4.1.1. 1-(4-((E)-2-(methylcarbamoyl)vinyl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (1)
A mixture of 4-nitrobenzaldehyde (23 mmol), malonic acid
(25 mmol) and catalytic amounts of piperidine in anhydrous pyri-
dine (12 mL) was stirred at 90 ꢀC for 4 h. After cooling, the resulting
solution was poured into ice water, acidified to pH 1e2 with
concentrated HCl and the solid formed was filtered and dried to
obtain 3-(4-nitrophenyl)acrylic acid (82.6%). Then the acid
(10 mmol) was slowly added to thionyl chloride (10 mL) at 0 ꢀC, the
mixture was stirred at refluxing temperature for 2 h. After cooled to
room temperature, the reaction mixture was diluted with toluene
(30 mL), and concentrated to near dryness in vacuo to obtain oily
residue. To a solution of corresponding amine (11 mmol) in DCM
(40 mL) containing triethylamine (TEA) (22 mmol), a DCM (10 mL)
solution of the oily residue was added at 0 ꢀC and then the mixture
was stirred at room temperature for 8 h. After removal of the sol-
vent under reduced pressure, the resulting residue was recrystal-
lized from EtOAc and dried to give (E)-N-substituted-3-(4-
nitrophenyl)acrylamide (82.3%e92.3%). To a solution of the amide
(1.5 mmol) in 6 mL ethanol (EtOH) was added SnCl2 (4.5 mmol) in
6 mol/L HCl (6 mL), and the mixture was heated to reflux for 4 h.
The reaction mixture was poured into saturated solution of NaHCO3
and extracted with DCM (2 ꢁ 30 mL), after which the organic layer
was washed with saturated brine, dried over anhydrous Na2SO4,
and evaporated in vacuo. The residue was recrystallized from EtOAc
and dried to give intermediates A1(aed) in yields of 64.4%, 56.4%,
54.8%, 63.6% respectively. Finally, following the general procedure
for the preparation of target compounds to give compounds 1e5.
Off white solid, yield: 83.5%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
3. Conclusion
We herein described the design and synthesis of a novel series
of diaryl ureas with the goal to develop a new generation of potent
multi-targeted antitumor agents. All target compounds were
evaluated for their inhibitory activities on a series of human cancer
cell lines including MX-1, A375, HepG2, Ketr3 and HT-29. Our
modifications were focused on the variation of the linker groups
between diaryl urea and amide moieties. We concluded that most
compounds containing a rigid linker group such as vinyl, ethynyl
and phenyl showed potent activities against both MX-1 and HT-29
cell lines with IC50 values at the micromolar level. Compound 23
with a 1,3-disubstituted phenyl linker group exhibited potent
broad-spectrum activities against tumor cell lines with IC50 values
less or equal to 6 mM. It was also noteworthy that 1,4-disubstituted
phenyl analogs 26 and 27 bearing a large polar amide displayed
selective inhibition against HT-29 cell line, approximately eight-
fold more potent than sorafenib.
Finally, the lack of Raf kinase inhibitory activity of compound 23
suggested that other mechanism of action accounts for its signifi-
cant broad-spectrum antitumor activity. Efforts to identify molec-
ular targets of compound 23 and further optimization are ongoing
and will be reported in due course.
300 MHz)
d
: 2.70 (d, 3H, J ¼ 4.2 Hz, CH3), 6.51 (d, 1H, J ¼ 16.2 Hz,
CH]CH), 7.36 (d, 1H, J ¼ 15.3 Hz, CH]CH), 7.50 (brs, 4H, ArH),
7.62e7.64 (m, 2H, ArH), 7.97 (d, 1H, J ¼ 4.8 Hz, ArH), 8.11 (s, 1H,
CONH), 9.03 (s, 1H, NHCONH), 9.20 (s, 1H, NHCONH). HRMS (ESI):
m/z [M þ H]þ calcd for C18H16ClF3N3O2 398.0801, found 398.0812.
4. Experimental protocols
Melting points were determined with a Yanaco MP-J3 micro
melting point apparatus and were uncorrected. The NMR spectra
were obtained on Mercury-300, 400 MHz and Bruker AV500-III
spectrometers with tetramethylsilane (TMS) as the internal stan-
dard. High-resolution mass spectra (HRMS) were taken in electro-
spray ionization (ESI) mode on an Agilent 1100 series LC/MSD mass
spectrometer. IR spectra were run on Nicolet 5700 spectrometer
(Thermo). All reagents and solvents were purchased from com-
mercial sources unless otherwise indicated. TLC was carried out on
silica gel plates (GF254) with visualization of components by UV
light (254 nm) or exposure to I2. Column chromatography was
carried out on silica gel (200e300 mesh).
4.1.2. 1-(4-((E)-2-(1-phenylethylcarbamoyl)vinyl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (2)
Off white solid, yield: 86.9%, mp > 250 ꢀC. IR (KBr, cmꢂ1): 3293.7,
3064.7, 2979.5, 2931.3, 1704.5, 1653.0, 1592.3, 1484.1, 1418.7, 1178.4,
1032.5, 827.9, 698.7. 1H NMR (DMSO-d6, 300 MHz)
d: 1.39 (d, 3H,
J ¼ 7.2 Hz, CH3), 5.01e5.06 (m, 1H, NHCH), 6.58 (d, 1H, J ¼ 15.6 Hz,
CH]CH), 7.21e7.37 (m, 6H, ArH, CH]CH), 7.46e7.53 (m, 4H, ArH),
7.62 (brs, 2H, ArH), 8.11 (s, 1H, CONH), 8.47 (d, 1H, J ¼ 7.8 Hz, ArH),
9.03 (s, 1H, NHCONH), 9.20 (s, 1H, NHCONH). 13C NMR (DMSO-d6,
100 MHz) d: 22.47, 47.89, 116.78, 116.84, 118.55, 120.17, 121.42,
122.41, 123.12, 124.13, 126.61, 126.84, 128.23, 128.87, 131.99, 138.43,
139.17, 140.40, 144.64, 152.19, 164.23. HRMS (ESI): m/z [M þ H]þ
calcd for C25H22ClF3N3O2 488.1239, found 488.1262.
4.1. General procedure for the preparation of target compounds 1e
27
The intermediate amine with designed linker (0.5 mmol) was
dissolved in anhydrous DCM (3 mL). Temperature was maintained
4.1.3. 1-(4-((E)-2-(1-phenylethylcarbamoyl)vinyl)phenyl)-3-(2-
chloro-5-(trifluoromethyl)phenyl)urea (3)
Off white solid, yield: 90.9%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
Table 2
Raf kinase inhibitory activity of compound 23 in vitro.
300 MHz)
d
: 1.40 (d, 3H, J ¼ 6.9 Hz, CH3), 5.01e5.06 (m, 1H, NHCH),
6.59 (d, 1H, J ¼ 15.6 Hz, CH]CH), 7.21e7.38 (m, 7H, Ar, CH]CH),
7.48e7.54 (m, 4H, ArH), 7.72 (d, 1H, J ¼ 8.7 Hz, ArH), 8.48 (d, 1H,
J ¼ 8.1 Hz, ArH), 8.63 (s, 1H, CONH), 8.66 (s, 1H, NHCONH), 9.73 (s,
1H, NHCONH). HRMS (ESI): m/z [M þ H]þ calcd for C25H22ClF3N3O2
488.1239, found 488.1265.
Compd.
IC50 (nM)
B-Raf
C-Raf
23
>50,000
>50,000
Sorafenib
43.04
21.98

C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
357
4.1.4. 1-(4-((E)-2-(cyclobutylcarbamoyl)vinyl)phenyl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (4)
1H, NHCH), 7.10 (d, 2H, J ¼ 8.1 Hz, ArH), 7.34 (d, 2H, J ¼ 8.1 Hz, ArH),
7.57e7.60 (m, 2H, ArH), 8.02 (d, 1H, J ¼ 7.5 Hz, ArH), 8.09 (s, 1H,
CONH), 8.76 (s, 1H, NHCONH), 9.14 (s, 1H, NHCONH). 13C NMR
Off white solid, yield: 87.3%, mp 239e241 ꢀC. 1H NMR (DMSO-d6,
400 MHz)
d: 1.63e1.64 (m, 2H, cyclobutyl-CH2), 1.88e2.00 (m, 2H,
(DMSO-d6, 100 MHz) d: 14.62, 30.26, 30.40, 37.09, 43.74, 116.57,
cyclobutyl-CH2), 2.17e2.20 (m, 2H, cyclobutyl-CH2), 4.26e4.32 (m,
1H, NHCH), 6.45 (d,1H, J ¼ 15.6 Hz, CH]CH), 7.33 (d,1H, J ¼ 15.6 Hz,
CH]CH), 7.46e7.52 (m, 4H, ArH), 7.59e7.62 (m, 2H, ArH), 8.10 (s,
1H, CONH), 8.27 (d, 1H, J ¼ 7.6 Hz, ArH), 9.05 (s, 1H, NHCONH), 9.23
116.63, 118.65, 122.09, 122.91, 128.47, 131.95, 135.28, 136.99, 139.44,
152.39, 170.15. HRMS (ESI): m/z [M þ H]þ calcd for C21H22ClF3N3O2
440.1347, found 440.1335.
(s, 1H, NHCONH). HRMS (ESI): m/z [M
21H20ClF3N3O2 438.1191, found 438.1199.
þ
H]þ calcd for
C
4.1.9. 1-(4-(2-(2,6-Dimethylmorpholinocarbamoyl)ethyl)phenyl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (9)
4.1.5. 1-(4-((E)-2-(2,6-dimethylmorpholinocarbamoyl)vinyl)
phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (5)
Off white solid, yield: 90.0%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
Off white solid, yield: 80.3%, mp 202e203 ꢀC. 1H NMR (DMSO-
d6, 300 MHz)
d
: 1.03e1.07 (m, 6H, 2 ꢁ CH3), 2.13e2.21 (m, 1H,
CHCH3), 2.55e2.74 (m, 6H, CH2CH2, NCH2), 3.18e3.22 (m, 1H,
CHCH3), 3.70e4.25 (m, 2H, NCH2), 7.14 (d, 2H, J ¼ 8.4 Hz, ArH), 7.34
(d, 2H, J ¼ 8.4 Hz, ArH), 7.57e7.64 (m, 2H, ArH), 8.08 (s, 1H, ArH),
8.75 (s, 1H, NHCONH), 9.12 (s, 1H, NHCONH). 13C NMR (DMSO-d6,
300 MHz)
d
: 1.11 (d, 6H, J ¼ 5.4 Hz, 2 ꢁ CH3), 2.27e2.35 (m, 1H,
CHCH3), 2.68e2.76 (m, 1H, CHCH3), 3.46 (brs, 2H, NCH2), 4.21e4.37
(m, 2H, NCH2), 7.14 (d, 1H, J ¼ 15.6 Hz, CH]CH), 7.45 (d, 1H,
J ¼ 15.6 Hz, CH]CH), 7.50 (d, 2H, J ¼ 8.7 Hz, ArH), 7.59e7.67 (m, 4H,
ArH), 8.08 (d, 1H, J ¼ 1.8 Hz, ArH), 9.04 (s, 1H, NHCONH), 9.19 (s, 1H,
100 MHz)
d: 18.36, 50.32, 71.31, 116.59, 118.65, 121.48, 122.13,
122.96, 124.19, 126.53, 126.83, 128.78, 131.99, 135.24, 137.07, 139.47,
152.42, 170.39. HRMS (ESI): m/z [M þ H]þ calcd for C23H26ClF3N3O3
484.1609, found 484.1618.
NHCONH). 13C NMR (DMSO-d6, 100 MHz)
d: 18.37, 50.39, 71.66,
115.81, 116.87, 118.28, 121.93, 123.18, 124.13, 126.84, 128.92, 129.12,
131.99, 139.16, 140.56, 141.49, 152.18, 164.39. HRMS (ESI): m/z
[M þ H]þ calcd for C23H24ClF3N3O3 482.1453, found 482.1442.
4.1.10. 1-(4-((E)-2-(methylcarbamoyl)propen-1-yl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (10)
4.1.6. 1-(4-(2-(Methylcarbamoyl)ethyl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (6)
Intermediates A3(aec) were prepared using methylmalonic acid
instead of malonic acid as the staring material in a series of re-
actions as described for intermediate A1, with the yields of 61.3%,
53.2%, 67.9% respectively. Finally, following the general procedure
for the preparation of target compounds to give compounds 10e12.
Off white solid, yield: 82.7%, mp 224e225 ꢀC. 1H NMR (DMSO-
To a tetrahydrofuran (THF) (20 mL) solution of the (E)-N-
substituted-3-(4-nitrophenyl)acrylamide (5 mmol) which was ob-
tained in the synthesis of intermediate A1, was added 5% Pd/C
(50 mg), and the mixture was vigorously stirred under hydrogen
atmosphere for 6 h. The reaction mixture was filtered and the
filtrate was evaporated in vacuo, the resulting solid formed was
washed with diethyl ether and dried to give intermediates A2(aed)
in yields of 89.0%, 83.9%, 82.5%, 88.8% respectively. Finally,
following the general procedure for the preparation of target
compounds to give compounds 6e9.
d6, 300 MHz)
d
: 2.01 (d, 3H, J ¼ 1.2 Hz, CH]CCH3), 2.67 (d, 3H,
J ¼ 4.5 Hz, NHCH3), 7.12 (s, 1H, CH]CCH3), 7.35 (d, 2H, J ¼ 8.7 Hz,
ArH), 7.50 (d, 2H, J ¼ 8.7 Hz, ArH), 7.61e7.63 (m, 2H, ArH), 7.92 (d,
1H, J ¼ 4.2 Hz, ArH), 8.10 (s, 1H, CONH), 8.98 (s, 1H, NHCONH), 9.19
(s, 1H, NHCONH). 13C NMR (DMSO-d6, 125 MHz)
d: 14.27, 26.22,
Off white solid, yield: 81.2%, mp 185e186 ꢀC. 1H NMR (DMSO-d6,
116.80, 116.84, 118.31, 123.11, 130.02, 130.08, 130.80, 131.76, 131.98,
300 MHz)
d
: 2.31 (t, 2H, J ¼ 7.2 Hz, CH2CH2), 2.55 (d, 3H, J ¼ 4.5 Hz,
138.70, 139.23, 152.28, 169.36. HRMS (ESI): m/z [M þ H]þ calcd for
CH3), 2.74 (t, 2H, J ¼ 7.2 Hz, CH2CH2), 7.10 (d, 2H, J ¼ 8.4 Hz, ArH),
7.34 (d, 2H, J ¼ 8.4 Hz, ArH), 7.59e7.60 (m, 2H, ArH), 7.71 (brs, 1H,
ArH), 8.09 (s, 1H, CONH), 8.73 (s, 1H, NHCONH), 9.11 (s, 1H,
NHCONH). HRMS (ESI): m/z [M þ H]þ calcd for C18H18ClF3N3O2
400.2520, found 400.2515.
C19H18ClF3N3O2 412.1034, found 412.1023.
4.1.11. 1-(4-((E)-2-(1-phenylethylcarbamoyl)propen-1-yl)phenyl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (11)
Off white solid, yield: 86.0%, mp 179e180 ꢀC. IR (KBr, cmꢂ1):
3325.4, 3125.3, 3064.8, 2976.8, 1703.0, 1643.3, 1593.0, 1541.4,
1482.2, 1420.2, 1181.8, 1030.5, 829.6, 699.5. 1H NMR (DMSO-d6,
4.1.7. 1-(4-(2-(1-Phenylethylcarbamoyl)ethyl)phenyl)-3-(4-chloro-
3-(trifluoromethyl)phenyl)urea (7)
Off white solid, yield: 88.9%, mp 218e219 ꢀC. IR (KBr, cmꢂ1):
3321.9, 3086.1, 2968.8, 2929.0, 1708.4,1632.3, 1549.1, 1484.8,1420.1,
300 MHz)
d
: 1.43 (d, 3H, J ¼ 7.2 Hz, CHCH3), 2.04 (d, 3H,
J ¼ 0.9 Hz, CH]CCH3), 5.03e5.08 (m, 1H, NHCH), 7.18e7.24 (m,
2H, ArH, CH]CCH3), 7.30e7.38 (m, 6H, ArH), 7.52 (d, 2H,
J ¼ 8.4 Hz, ArH), 7.60e7.63 (m, 2H, ArH), 8.11 (s, 1H, CONH), 8.34
(d, 1H, J ¼ 8.1 Hz, ArH), 8.99 (s, 1H, NHCONH), 9.20 (s, 1H,
NHCONH). HRMS (ESI): m/z [M þ H]þ calcd for C26H24ClF3N3O2
502.1331, found 502.1368.
1174.7, 1030.8, 824.9, 698.4. 1H NMR (DMSO-d6, 300 MHz)
d: 1.29 (d,
3H, J ¼ 6.9 Hz, CH3), 2.38 (t, 2H, J ¼ 7.2 Hz, CH2CH2), 2.75 (t, 2H,
J ¼ 7.2 Hz, CH2CH2), 4.87e4.92 (m,1H, NHCH), 7.09 (d, 2H, J ¼ 8.1 Hz,
ArH), 7.20e7.30 (m, 5H, ArH), 7.34 (d, 2H, J ¼ 8.4 Hz, ArH), 7.61e7.62
(m, 2H, ArH), 8.09 (s,1H, CONH), 8.22 (d,1H, J ¼ 7.8 Hz, ArH), 8.75 (s,
1H, NHCONH), 9.12 (s, 1H, NHCONH). 13C NMR (DMSO-d6, 100 MHz)
d: 22.39, 30.48, 37.09, 47.54, 116.59, 118.62, 121.44, 122.09, 122.92,
124.16, 125.88, 126.42, 126.81, 128.10, 131.95, 135.17, 137.02, 139.44,
4.1.12. 1-(4-((E)-2-(2,6-dimethylmorpholinocarbamoyl)propen-1-
yl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (12)
Off white solid, yield: 90.3%, mp 230e232 ꢀC. 1H NMR (DMSO-
144.65, 152.39, 170.39. HRMS (ESI): m/z [M þ H]þ calcd for
C
25H24ClF3N3O2 490.1504, found 490.1488.
d6, 300 MHz)
d
: 1.09 (d, 6H, J ¼ 6.3 Hz, 2 ꢁ CH3), 2.00 (d, 3H,
4.1.8. 1-(4-(2-(Cyclobutylcarbamoyl)ethyl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (8)
J ¼ 0.9 Hz, CH]CCH3), 2.48e2.50 (m,1H, CHCH3), 3.33e3.45 (m,1H,
CHCH3), 3.46e3.52 (m, 2H, NCH2), 3.95e4.05 (m, 2H, NCH2), 6.42 (s,
1H, CH]CCH3), 7.33 (d, 2H, J ¼ 8.4 Hz, ArH), 7.48 (d, 2H, J ¼ 8.7 Hz,
ArH), 7.60e7.62 (m, 2H, ArH), 8.09 (d, 1H, J ¼ 1.8 Hz, ArH), 8.96 (s,
1H, NHCONH), 9.17 (s, 1H, NHCONH). HRMS (ESI): m/z [M þ H]þ
calcd for C24H26ClF3N3O3 496.1609, found 496.1585.
Off white solid, yield: 88.3%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
300 MHz) d: 1.55e1.63 (m, 2H, cyclobutyl-CH2), 1.74e1.84 (m, 2H,
cyclobutyl-CH2), 2.09e2.11 (m, 2H, cyclobutyl-CH2), 2.27 (t, 2H,
J ¼ 7.2 Hz, CH2CH2), 2.72 (t, 2H, J ¼ 7.2 Hz, CH2CH2), 4.11e4.20 (m,

358
C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
4.1.13. 1-(4-((E)-1-(methylcarbamoyl)propen-2-yl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (13)
(4-nitrophenoxy)acrylate (89.3%), then treated it (3 mmol) as
described for the synthesis of the acid in intermediate A4 to give
(E)-3-(4-nitrophenoxy)acrylic acid (82.4%). A mixture of the acid
(1 mmol), BOP (1.1 mmol) and TEA (2.2 mmol) in THF (6 mL) was
stirred at room temperature for 5 min, then a THF (1 mL) solution of
corresponding amine (1.1 mmol) was added and the mixture was
stirred for 3 h. After removal of the solvent under reduced pressure,
the resulting residue was poured into water and extracted with
DCM (2 ꢁ 20 mL), then the organic layer was washed with satu-
rated brine, dried over anhydrous Na2SO4, and evaporated in vacuo.
The residue was purified by column chromatography (light petro-
leum/EtOAc ¼ 1:1) on silica gel to give (E)-N-substituted-3-(4-
nitrophenoxy)acrylamide as a white solid (59.4%e78.0%). The
amide (1 mmol) was deprotected by stirring in 7 mol/L HCl/EtOAc
(3 mL) at room temperature for 1 h. The solid formed was filtrated
and put into DCM (20 mL) immediately, then washed with satu-
rated solution of NaHCO3 to pH 8e9, concentrated to give in-
termediates A5(aeb) in yields of 84.3% and 80.3% respectively.
Finally, following the general procedure for the preparation of
target compounds to give compounds 16e18.
18-Crown-6 (5 mmol) was added to a solution of trimethyl
phosphonoacetate (11 mmol) in toluene (20 mL) under argon at-
mosphere, then a 1 mol/L NaHMDS/THF solution (20 mL) was added
and the obtained mixture was stirred at room temperature for 1 h.
Under ice-cooling, 4-nitroacetophenone (10 mmol) was added and
the mixturewas stirred for 10 h. After the reactionwas complete, the
solid formed was filtered, washed with cold water and dried to give
(E)-methyl-3-(4-nitrophenyl)-2-butenoate (69.7%). To a THF (6 mL)
solution of the intermediate methyl ester (2 mmol) was added
1 mol/L NaOH (10 mL). The reaction mixture was stirred at room
temperature for 12 h, then chilled in ice and acidified with
concentrated HCl to pH 1e2. The solid formed was filtered, washed
with water and dried to afford (E)-3-(4-nitrophenyl)-2-butenoic
acid as a white solid (91.1%). Then, amidation of carboxyl group
and reduction of nitro group following the synthetic methods as
described for A1 to give intermediates A4(aec) in yields of 50.0%,
67.9%, 58.2% respectively. Finally, following the general procedure
for the preparation of target compounds to give compounds 13e15.
Off white solid, yield: 84.6%, mp 224e225 ꢀC. 1H NMR (DMSO-
Off white solid, yield: 84.6%, mp 218e219 ꢀC. 1H NMR (DMSO-d6,
d6, 300 MHz)
d
: 2.46 (s, 3H, NHCH3), 2.65 (d, 3H, J ¼ 4.2 Hz, CH3C]
300 MHz)
d
: 2.62 (d, 3H, J ¼ 4.8 Hz, CH3), 5.58 (d, 1H, J ¼ 12.0 Hz,
CH), 6.17 (d, 1H, J ¼ 0.6 Hz, CH3C]CH), 7.45 (d, 2H, J ¼ 8.7 Hz, ArH),
7.50 (d, 2H, J ¼ 8.7 Hz, ArH), 7.62e7.70 (m, 2H, ArH), 7.90 (d, 1H,
J ¼ 4.8 Hz, ArH), 8.11 (s, 1H, CONH), 8.99 (s, 1H, NHCONH), 9.21 (s,
OCH]CH), 7.08 (d, 2H, J ¼ 8.7 Hz, ArH), 7.46 (d, 2H, J ¼ 9.0 Hz, ArH),
7.53 (d, 1H, J ¼ 12.0 Hz, OCH]CH), 7.60e7.62 (m, 2H, ArH), 7.76 (d,
1H, J ¼ 7.2 Hz, ArH), 8.09 (s, 1H, CONH), 8.88 (s, 1H, NHCONH), 9.15
1H, NHCONH). 13C NMR (DMSO-d6, 125 MHz)
d: 16.25, 25.34, 116.77,
(s, 1H, NHCONH). 13C NMR (DMSO-d6, 125 MHz)
d: 25.30, 104.88,
116.81, 118.28, 119.00, 123.09, 126.39, 131.96, 135.78, 139.22, 139.49,
116.72, 116.76, 118.16, 120.23, 123.01, 131.90, 135.71, 139.31, 150.65,
146.80, 152.25, 166.69. HRMS (ESI): m/z [M þ H]þ calcd for
152.40, 154.50, 165.32. HRMS (ESI): m/z [M þ H]þ calcd for
C
19H18ClF3N3O2 412.1034, found 412.1011.
C18H16ClF3N3O3 414.0827, found 414.0808.
4.1.14. 1-(4-((E)-1-(1-phenylethylcarbamoyl)propen-2-yl)phenyl)-
3-(4-chloro-3-(trifluoromethyl)phenyl)urea (14)
4.1.17. 1-(4-((E)-2-(1-phenylethylcarbamoyl)vinyloxy)phenyl)-3-
(4-chloro-3-(trifluoromethyl)phenyl)urea (17)
Off white solid, yield: 82.0%, mp 212e214 ꢀC. IR (KBr, cmꢂ1):
3324.4, 3063.3, 2979.0,1698.8,1638.7,1592.1,1539.6,1483.4,1419.2,
Off white solid, yield: 90.6%, mp 196e197 ꢀC. IR (KBr, cmꢂ1):
3295.1, 3067.0, 2975.7, 1695.1, 1664.6, 1602.7, 1551.9, 1428.1, 1421.4,
1172.5, 1030.5, 835.0, 698.5. 1H NMR (DMSO-d6, 300 MHz)
d
: 1.39
1174.0, 1031.4, 834.1, 698.8. 1H NMR (DMSO-d6, 300 MHz)
d: 1.34 (d,
(d, 3H, J ¼ 6.9 Hz, NHCH3), 2.45 (s, 3H, CH3C]CH), 4.97e5.02 (m,
1H, NHCH), 6.29 (d, 1H, J ¼ 1.2 Hz, CH3C]CH), 7.21e7.33 (m, 5H,
ArH), 7.46 (d, 2H, J ¼ 9.3 Hz, ArH), 7.50 (d, 2H, J ¼ 9.3 Hz, ArH), 7.60e
7.63 (m, 2H, ArH), 8.12 (s, 1H, CONH), 8.40 (d, 1H, J ¼ 7.8 Hz, ArH),
9.00 (s, 1H, NHCONH), 9.21 (s, 1H, NHCONH). 13C NMR (DMSO-d6,
3H, J ¼ 7.2 Hz, CH3), 4.94e4.99 (m, 1H, NHCH), 5.64 (d, 1H,
J ¼ 12.0 Hz, OCH]CH), 7.08 (d, 2H, J ¼ 8.7 Hz, ArH), 7.21e7.30 (m,
5H, ArH), 7.46 (d, 2H, J ¼ 9.0 Hz, ArH), 7.57 (d,1H, J ¼ 12.0 Hz, OCH]
CH), 7.60e7.62 (m, 2H, ArH), 8.09 (d, 1H, J ¼ 1.8 Hz, NHCH), 8.25 (d,
1H, J ¼ 8.1 Hz, ArH), 8.90 (s,1H, NHCONH), 9.18 (s,1H, NHCONH). 13
C
125 MHz)
d: 16.22, 22.54, 47.60, 116.78, 117.62, 118.25, 119.03,
NMR (DMSO-d6, 125 MHz) d: 22.37, 47.57, 104.68, 116.74, 118.43,
123.13, 125.94, 126.01, 126.45, 126.58, 128.07, 128.24, 132.00, 135.76,
139.24, 139.59, 144.86, 147.49, 152.27, 165.23. HRMS (ESI): m/z
[M þ H]þ calcd for C26H24ClF3N3O2 502.1454, found 502.1464.
120.20, 122.99, 125.91, 126.52, 128.15, 131.90, 135.83, 139.31, 144.64,
150.50, 152.39, 155.22, 163.97. HRMS (ESI): m/z [M þ H]þ calcd for
C25H22ClF3N3O3 504.1296, found 504.1277.
4.1.15. 1-(4-((E)-1-(2,6-dimethylmorpholinocarbamoyl)propen-2-
yl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (15)
Off white solid, yield: 90.8%, mp 240e241 ꢀC. 1H NMR (DMSO-d6,
4.1.18. 1-(4-((E)-2-(1-phenylethylcarbamoyl)vinyloxy)phenyl)-3-
(2-chloro-5-(trifluoromethyl)phenyl)urea (18)
Off white solid, yield: 87.1%, mp 200e201 ꢀC. 1H NMR (DMSO-d6,
300 MHz)
d
: 1.07e1.12 (m, 6H, 2 ꢁ CH3), 2.16 (s, 3H, CH3C]CH),
300 MHz)
d
: 1.34 (d, 3H, J ¼ 7.2 Hz, CH3), 4.95e4.99 (m, 1H, NHCH),
2.26e2.34 (m, 1H, CHCH3), 2.68e2.76 (m, 1H, CHCH3), 3.45e3.46
(m, 2H, NCH2), 3.79e4.34 (m, 2H, NCH2), 6.43 (s, 1H, CH3C]CH),
7.46e7.53 (m, 4H, ArH), 7.62e7.63 (m, 2H, ArH), 8.09 (s, 1H, ArH),
9.00 (s, 1H, NHCONH), 9.20 (s, 1H, NHCONH). 13C NMR (DMSO-d6,
5.66 (d, 1H, J ¼ 12.3 Hz, OCH]CH), 7.10 (d, 2H, J ¼ 9.0 Hz, ArH),
7.21e7.30 (m, 6H, ArH), 7.40e7.51 (m, 3H, ArH, OCH]CH), 7.70 (d,
1H, J ¼ 8.4 Hz, ArH), 8.26 (d, 1H, J ¼ 8.4 Hz, ArH), 8.58 (s, 1H,
NHCONH), 8.62 (d, 1H, J ¼ 1.8 Hz, NHCH), 9.60 (s, 1H, NHCONH). 13
C
125 MHz)
d
: 18.12, 51.14, 71.49, 116.78, 117.98, 118.23, 119.93, 122.36,
NMR (DMSO-d6, 125 MHz) d: 22.38, 47.59, 104.75, 118.57, 119.96,
123.10, 126.46, 127.58, 131.96, 134.69, 139.02, 141.21, 143.81, 152.25,
166.03, 166.27. HRMS (ESI): m/z [M þ H]þ calcd for C24H26ClF3N3O3
496.1589, found 496.1586.
125.92, 126.53, 128.16, 130.31, 135.62, 136.89, 144.64, 150.62, 152.01,
155.18, 163.98. HRMS (ESI): m/z [M þ H]þ calcd for C25H22ClF3N3O3
504.1296, found 504.1273.
4.1.16. 1-(4-((E)-2-(methylcarbamoyl)vinyloxy)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (16)
4.1.19. 1-(4-(2-(Methylcarbamoyl)ethynyl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (19)
Methyl propiolate (7 mmol), N-Boc-4-aminophenol (7 mmol)
and DABCO (0.7 mmol) were dissolved in DCM (10 mL). The reac-
tion mixture was stirred at room temperature for 1.5 h, and then
the solvent was concentrated under reduced pressure. The oily
residue was recrystallized from diethyl ether to give (E)-methyl-3-
To a THF (30 mL) solution of 4-iodonitrobenzene (10 mmol)
and methyl propiolate (40 mmol) were added Pd(PPh3)2Cl2
(140 mg), CuI (76 mg) and K2CO3 (20 mmol). The obtained mixture
was heated to reflux for 5 h. Subsequently, the solvent was
concentrated under reduced pressure, the residue was poured into

C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
359
water and extracted with DCM (2 ꢁ 30 mL), then the organic layer
was washed with saturated brine, dried over anhydrous Na2SO4,
and evaporated in vacuo. The residue was purified by column
chromatography (light petroleum/EtOAc ¼ 6:1) on silica gel to give
methyl 3-(4-nitrophenyl)propiolate (78.1%), then treated it
(1.5 mmol) as described for the synthesis of the amide in inter-
mediate A5 to provide N-substituted-3-(4-nitrophenyl)propiola-
mide as a white solid (69.3%e74.1%). To a solution of the amide
(0.5 mmol) in EtOH (5 mL) and 2 mol/L HCl (2 mL) was added Fe
(3.5 mmol), then the mixture was stirred at 70 ꢀC for 4 h. The
reaction mixture was concentrated to near dryness under reduced
pressure, the oily residue was poured into water and extracted
with DCM (2 ꢁ 20 mL), then the organic layer was washed with
saturated brine, dried over anhydrous Na2SO4, and evaporated in
vacuo. The residue was purified by column chromatography (light
petroleum/EtOAc ¼ 1:1) on silica gel to give intermediates A6(ae
b) in yields of 78.2% and 83.3% respectively. Finally, following the
general procedure for the preparation of target compounds to give
compounds 19e21.
the synthetic methods as described for A2 to give intermediates
A7(aeb) in yields of 87.6% and 93.9% respectively. Finally, following
the general procedure for the preparation of target compounds to
give compounds 22 and 23.
Off white solid, yield: 80.2%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
300 MHz)
d
: 2.81 (d, 3H, J ¼ 4.2 Hz, CH3), 7.48e7.66 (m, 7H, ArH),
7.69e7.76 (m, 2H, ArH), 8.09 (d, 2H, J ¼ 10.8 Hz, ArH), 8.53 (s, 1H,
CONH), 9.03 (s, 1H, NHCONH), 9.26 (s, 1H, NHCONH). 13C NMR
(DMSO-d6, 125 MHz) d: 26.26, 116.69, 118.90, 121.47, 122.32, 123.09,
124.18, 124.66, 125.69, 127.19, 128.64, 128.94, 132.01, 133.35, 135.13,
139.05, 139.32, 139.75, 152.36, 166.56. HRMS (ESI): m/z [M þ H]þ
calcd for C22H18ClF3N3O2 448.1034, found 448.1045.
4.1.23. 1-(4-(3-(1-Phenylethylcarbamoyl)phenyl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (23)
Off white solid, yield: 78.8%, mp 240e242 ꢀC. IR (KBr, cmꢂ1):
3226.5, 3180.9, 3109.3, 3059.3, 1730.1, 1613.5, 1554.6, 1486.8,1427.4,
1322.2, 1139.0, 1034.4, 877.1, 836.3, 777.7, 698.5. 1H NMR (DMSO-d6,
300 MHz)
d
: 1.51 (d, 3H, J ¼ 6.9 Hz, CH3), 5.15e5.21 (m, 1H, NHCH),
Off white solid, yield: 80.4%, mp 221e223 ꢀC. 1H NMR (DMSO-d6,
7.22e7.24 (m, 1H, ArH), 7.30e7.39 (m, 3H, ArH), 7.52e7.67 (m, 5H,
ArH), 7.81e7.83 (m, 2H, ArH), 7.95 (d, 1H, J ¼ 8.7 Hz, ArH), 8.12e8.35
(m, 3H, ArH), 8.90 (d, 1H, J ¼ 8.1 Hz, ArH), 9.12 (s, 1H, CONH), 9.37 (s,
1H, NHCONH), 9.51 (s, 1H, NHCONH). HRMS (ESI): m/z [M þ H]þ
calcd for C29H24ClF3N3O2 538.1589, found 538.1559.
300 MHz)
d
: 2.56 (d, 3H, J ¼ 4.5 Hz, CH3), 7.18 (d, 2H, J ¼ 8.4 Hz,
ArH), 7.34e7.37 (m, 3H, ArH), 7.69 (d, 1H, J ¼ 8.7 Hz, ArH), 7.88e7.95
(m, 1H, ArH), 8.57 (s, 1H, CONH), 8.64 (s, 1H, NHCONH), 9.51 (s, 1H,
NHCONH). HRMS (ESI): m/z [M þ H]þ calcd for C18H14ClF3N3O2 396.
1288, found 396.1298.
4.1.24. 1-(4-(4-(1-Phenylethylcarbamoyl)phenyl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (24)
4.1.20. 1-(4-(2-(1-Phenylethylcarbamoyl)ethynyl)phenyl)-3-(4-
chloro-3-(trifluoromethyl)phenyl)urea (20)
To ice-cold nitric acid (15 mL) was added 4-biphenylcarboxylic
acid (20 mmol) slowly, the reaction mixture was stirred under ice
cooling for 1 h. The mixture was poured into ice water and then
filtered. The collected solid was suspended in EtOH and heated to
reflux for 2 h. The mixture was filtered at a high temperature, then
the solid was washed with EtOH and dried to give 4-
nitrodiphenyl-4-carboxylic acid (55.1%). Then, amidation of
carboxyl group and reduction of nitro group following the syn-
thetic methods as described for A2 to give intermediates A8(bec)
in yields of 89.2% and 85.2% respectively. Finally, following the
general procedure for the preparation of target compounds to give
compounds 24e27.
Off white solid, yield: 84.8%, mp 217e218 ꢀC. IR (KBr, cmꢂ1):
3349.3, 3123.9, 2977.9, 2932.2, 2204.0, 1718.6, 1634.9, 1528.6,
1483.6, 1420.7, 1176.2, 1030.9, 833.0, 698.6. 1H NMR (DMSO-d6,
300 MHz)
d
: 1.37 (d, 3H, J ¼ 6.9 Hz, CH3), 4.95e5.00 (m, 1H, NHCH),
7.21e7.33 (m, 5H, ArH), 7.50 (d, 2H, J ¼ 8.7 Hz, ArH), 7.52 (d, 2H,
J ¼ 8.7 Hz, ArH), 7.63 (s, 2H, ArH), 8.10 (s, 1H, CONH), 9.19 (s, 1H,
NHCONH), 9.23 (d, 1H, J ¼ 8.4 Hz, ArH), 9.29 (s, 1H, NHCONH). 13
C
NMR (DMSO-d6, 100 MHz) d: 22.06, 48.39, 83.39, 83.73, 112.85,
116.95, 118.38, 121.41, 122.60, 123.23, 124.12, 126.02, 126.55, 128.29,
132.02, 133.07, 139.03, 141.08, 143.98, 151.61, 152.12. HRMS (ESI): m/
z [M þ H]þ calcd for C25H20ClF3N3O2 486.1411, found 486.1400.
Off white solid, yield: 79.7%, mp 203e204 ꢀC. IR (KBr, cmꢂ1):
3320.5, 3062.6, 3031.1, 2977.1, 1651.7, 1633.7, 1598.5, 1547.5, 1484.3,
1419.8, 1317.6, 1128.4, 1033.5, 829.2, 697.6. 1H NMR (DMSO-d6,
4.1.21. 1-(4-(2-(1-Phenylethylcarbamoyl)ethynyl)phenyl)-3-(2-
chloro-5-(trifluoromethyl)phenyl)urea (21)
Off white solid, yield: 82.1%, mp 215e216 ꢀC. 1H NMR (DMSO-d6,
400 MHz)
d
: 1.49 (d, 3H, J ¼ 6.8 Hz, CH3), 5.17e5.20 (m, 1H, NHCH),
300 MHz)
d
: 1.38 (d, 3H, J ¼ 7.2 Hz, CH3), 4.95e5.00 (m, 1H, NHCH),
7.20e7.23 (m, 1H, ArH), 7.30e7.32 (m, 2H, ArH), 7.40 (d, 2H,
J ¼ 7.2 Hz, ArH), 7.58 (d, 2H, J ¼ 8.4 Hz, ArH), 7.62e7.64 (m, 2H, ArH),
7.68 (d, 2H, J ¼ 8.8 Hz, ArH), 7.73 (d, 2H, J ¼ 8.0 Hz, ArH), 7.95 (d, 2H,
J ¼ 8.0 Hz, ArH), 8.11 (s, 1H, CONH), 8.21 (d, 1H, J ¼ 8.0 Hz, ArH), 9.01
(s, 1H, NHCONH), 9.20 (s, 1H, NHCONH). HRMS (ESI): m/z [M þ H]þ
calcd for C29H24ClF3N3O2 538.1589, found 538.1559.
7.22e7.40 (m, 6H, ArH), 7.52 (d, 2H, J ¼ 8.7 Hz, ArH), 7.57 (d, 2H,
J ¼ 8.7 Hz, ArH), 7.72 (d, 1H, J ¼ 8.4 Hz, ArH), 8.61 (s, 1H, CONH), 8.71
(s,1H, NHCONH), 9.23 (d,1H, J ¼ 8.4 Hz, ArH), 9.85 (s,1H, NHCONH).
13C NMR (DMSO-d6, 100 MHz)
d: 22.06, 48.39, 83.45, 83.64, 113.09,
116.95, 118.26, 119.63, 122.41, 125.11, 125.63, 126.02, 128.04, 128.29,
130.43, 133.19, 136.64, 140.82, 143.97, 151.59, 151.82. HRMS (ESI): m/
z [M þ H]þ calcd for C25H20ClF3N3O2 486.1411, found 486.1412.
4.1.25. 1-(4-(4-(1-Phenylethylcarbamoyl)phenyl)phenyl)-3-(2-
chloro-5-(trifluoromethyl)phenyl)urea (25)
4.1.22. 1-(4-(3-(Methylcarbamoyl)phenyl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (22)
Off white solid, yield: 79.6%, mp > 250 ꢀC. 1H NMR (DMSO-d6,
400 MHz)
d
: 1.50 (d, 3H, J ¼ 7.2 Hz, CH3), 5.18e5.22 (m, 1H, NHCH),
4-Bromonitrobenzene (9.6 mmol) and 3-carboxybenzeneboronic
acid (10.8 mmol) were dissolved in a solution of toluene (40 mL),
EtOH (40 mL) and water (5 mL), then K2CO3 (29.3 mmol) and
Pd(PPh3)4 (200 mg) were added. The reaction mixture was heated to
reflux for 48 h under argon atmosphere. After cooled to room tem-
perature, the resulting solution was diluted with EtOAc (100 mL),
then extracted with water (3 ꢁ 50 mL). The aqueous extracts were
combined and acidified to pH 1e2 with concentrated HCl to afford an
off white precipitate, which was washed with water and dried to give
4-nitrobiphenyl-3-carboxylic acid as a white solid (96.1%). Then,
amidation of carboxyl group and reduction of nitro group following
7.21e7.24 (m, 1H, ArH), 7.32e7.37 (m, 3H, ArH), 7.42 (d, 2H,
J ¼ 8.0 Hz, ArH), 7.60 (d, 2H, J ¼ 8.4 Hz, ArH), 7.71e7.77 (m, 5H, ArH),
7.98 (d, 2H, J ¼ 8.0 Hz, ArH), 8.66 (s, 2H, CONH, NHCONH), 8.83 (d,
1H, J ¼ 8.0 Hz, ArH), 9.73 (s, 1H, NHCONH). HRMS (ESI): m/z
[M þ H]þ calcd for C29H24ClF3N3O2 538.1589, found 538.1571.
4.1.26. 1-(4-(4-(2,6-Dimethylmorpholinocarbamoyl)phenyl)
phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea (26)
Off white solid, yield: 86.2%, mp 236e237 ꢀC. 1H NMR (DMSO-
d6, 400 MHz)
d
: 1.05e1.12 (m, 6H, 2 ꢁ CH3), 2.87e2.90 (m, 1H,
CHCH3), 3.54e3.56 (m, 4H, 2 ꢁ NCH2), 4.27e4.29 (m, 1H, CHCH3),

360
C. Lu et al. / European Journal of Medicinal Chemistry 77 (2014) 351e360
7.47 (d, 2H, J ¼ 8.0 Hz, ArH), 7.59 (d, 2H, J ¼ 8.4 Hz, ArH), 7.62e7.65
(m, 2H, ArH), 7.67 (d, 2H, J ¼ 8.4 Hz, ArH), 7.72 (d, 2H, J ¼ 8.4 Hz,
ArH), 8.13 (d, 1H, J ¼ 2.0 Hz, ArH), 9.01 (s, 1H, NHCONH), 9.23 (s, 1H,
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.020.