Discovery of novel diaryl urea derivatives bearing a triazole moiety as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2016.02.071

Herein, we report a novel series of diaryl urea derivatives bearing a triazole moiety, from which potent antitumor agents have been identified. With a modified triazole, most compounds showed high level activity in both cellular and enzymatic assays, accompanied with a suitable ClogD_7.4 value. The most active compound, 13i, effectively suppressed proliferation of HT-29, H460 and MDA-MB-231 cancer cells, with IC₅₀ values of 0.90, 0.85 and 1.54 μM, respectively. Compound 13i also exhibited significant inhibition of tyrosine kinases including c-Kit, RET and FLT3. Furthermore, compound 13i could obviously induce apoptosis of HT-29 cells in a concentration-dependent manner. The study of structure-activity relationships also revealed that a hydrophilic tail at the 4-position of the triazole was crucial for high activity of the compound.

Full text of DOI:10.1016/j.ejmech.2016.02.071

Accepted Manuscript
Discovery of novel diaryl urea derivatives bearing a triazole moiety as potential
antitumor agents
Mingze Qin, Shuang Yan, Lei Wang, Haotian Zhang, Yanfang Zhao, Shasha Wu, Di
Wu, Ping Gong
PII:
S0223-5234(16)30171-4
10.1016/j.ejmech.2016.02.071
EJMECH 8422
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2016
Revised Date: 26 February 2016
Accepted Date: 27 February 2016
Please cite this article as: M. Qin, S. Yan, L. Wang, H. Zhang, Y. Zhao, S. Wu, D. Wu, P. Gong,
Discovery of novel diaryl urea derivatives bearing a triazole moiety as potential antitumor agents,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.02.071.
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ACCEPTED MANUSCRIPT

Please do not adjust margins
ACCEPTED MANUSCRIPT
Discovery of novel diaryl urea derivatives bearing a triazole moiety
as potential antitumor agents
Mingze Qina, Shuang Yana, Lei Wanga, Haotian Zhangb, Yanfang Zhaoa, Shasha Wua,
Di Wua, and Ping Gong*,a
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenyang 110016, PR China
bDepartment of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, PR China
Corresponding author.
Tel: +86 24 2398 6426; Fax: +86 24 2398 6429;
E-mail address: gongpinggp@126.com
Abstract
Herein, we report a novel series of diaryl urea derivatives bearing a triazole
moiety, from which potent antitumor agents have been identified. With a modified
triazole, most compounds showed high level activity in both cellular and enzymatic
assays, accompanied with a suitable ClogD7.4 value. The most active compound, 13i,
effectively suppressed proliferation of HT-29, H460 and MDA-MB-231 cancer cells,
with IC50 values of 0.90, 0.85 and 1.54 ꢀM, respectively. Compound 13i also
exhibited significant inhibition of tyrosine kinases including c-Kit, RET and FLT3.
Furthermore, compound 13i could obviously induce apoptosis of HT-29 cells in a
concentration-dependent manner. The study of structure-activity relationships also
revealed that a hydrophilic tail at the 4-position of the triazole was crucial for high
activity of the compound.
Keywords: Diaryl ureas; Organic synthesis; Antitumor activity; Structure-activity
relationship
Please do not adjust margins

ACCEPTED MANUSCRIPT
1. Introduction
Cancer is a serious worldwide public health concern. Most patients
suffering from cancer have a poor prognosis due to its high mortality rate and
incidence of relapse. Therefore, there is still an urgent need for new antitumor
agents with improved efficacy.
In the past two decades, multikinase inhibitors, such as sunitinib, imatinib,
and sorafenib have received great attention and have yielded great benefits in
numerous clinical cancer cases [1–4]. The simultaneous inhibition of multiple
targets can create a synergistic anti-cancer effect. This strategy is proven to
efficiently interrupt complex oncogenic pathways and reduce the possibility of
developing drug resistance [5]. Sorafenib, a well-known multikinase inhibitor,
potently suppresses several receptor tyrosine kinases, including PDGFR-β,
VEGFR-2, VEGFR-3, FLT3, Kit, and RET, as well as the downstream protein
Raf. In addition, it demonstrates significant inhibition across a broad spectrum
of tumour types [6–8]. Thus far, it has been approved by the U.S. Food and
Drug Administration (FDA) for the treatment of advanced renal carcinoma
(RCC), unresectable hepatocellular carcinoma (HCC), and differentiated
thyroid carcinoma (DTC). In addition, its applications in breast, lung, and
colorectal carcinomas are being evaluated in clinic, and the results are eagerly
awaited [9–12].
Despite its outstanding antitumor activity, the overall bioavailability of
sorafenib is limited, mainly because of its poor water-solubility which is
ascribed to the high hydrophobicity of its chemical structure [13,14]. We
considered rational optimization of sorafenib a feasible approach to develop
novel antitumor agents, placing equal attention and importance on their
antitumor potency as well as their hydrophilicity.
As reported previously, the diaryl urea framework plays a pivotal role in
the complexation of sorafenib with enzymes through hydrogen bonding and
hydrophobic interactions [15], which prompted us to retain this functional
template in the design of new compounds. In previous study, we have reported
a
series
of
compounds
based
on
a
2-(4-(2-(dimethylamino)ethyl)-4
H-1,2,4-triazol-3-yl)pyridine moiety as potent
antitumor agents [16]. Structure-activity relationships revealed that an
optimized triazole was crucial for high activity. Thus, in the current research,
we developed a strategy to investigate hybrids of diaryl urea and triazole for
application as antitumor agents.
Here, we reported the design, synthesis and biological evaluation of a
series of diaryl urea derivatives bearing a triazole moiety, to identify their
potential as antitumor agents (Fig. 1). The new compounds were screened in
HT-29, H460, and MDA-MB-231 cancer cells, and against a kinase panel
comprising c-Kit, RET, FLT3, B-Raf, and VEGFR-2. Additionally, cell
apoptosis induced by compound 13i was evaluated in HT-29 cells.
(Insert Fig. 1 here)

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2. Results and discussion
2.1 Chemistry
The general route to prepare compounds 8a and 8b is described in Scheme
1. The appropriate aniline was reacted with triphosgene in refluxing toluene to
give the corresponding isocyanate 1a–f as a solid or an oil. The synthesis of
intermediate
described in detail in our previous study [16]. A nucleophilic substitution of
with 2-fluoro-4-nitrophenol in refluxing chlorobenzene yielded the crude
product of , which was purified by recrystallization in absolute ethanol.
Aminolysis of with ammonia in acetone led to the desired intermediate
which then reacted with -dimethylformamide dimethyl acetal (DMF-DMA)
to generate in a high yield. Intermediate was obtained by cyclization of
with methylhydrazine under a modified condition, as previously reported [17]
.Then, intermediate was reduced using hydrazine hydrate, to efficiently yield
amine . Ureas 8a and 8b were obtained by reaction of intermediate with the
corresponding isocyanate.
2 was achieved in two steps from picolinic acid, which has been
2
3
3
4,
N,N
5
6
5
6
7
7
(Insert Scheme 1 here)
The synthetic procedure for compounds 13a–y is illustrated in Scheme 2.
As shown, these target compounds were synthesized via a five-step reaction
from intermediate
3. The amino intermediate 9 was afforded by the reduction of
intermediate in the presence of Pd/C, and was then reacted with the
3
appropriate isocyanates to generate urea intermediates 10a–f. A convenient
hydrazinolysis of 10a–f in MeCN resulted in 11a–f, respectively. The
condensation of 11a–f with DMF-DMA was accomplished under similar
conditions as described for intermediate 5, and generated 12a–f, respectively.
The proposed compounds 13a–y were generated by cyclization of 12a–f with
the appropriate amines in the presence of acetic acid [16]. All the target
compounds were purified by column chromatography on silica gel. The
chemical structures of the target compounds were confirmed by elemental
analysis, mass spectrometry and NMR.
(Insert Scheme 2 here)
2.2. Biological evaluation
2.2.1. In vitro cellular activity and structure-activity relationships
To get a preliminary insight into SARs, our strategy was to start by testing
compounds 8a, 8b, 13a, and 13b, which shared a diaryl urea template but were
distinct in the arrangement of the triazole and distal aryl moieties. The
antitumor activity of these compounds was screened in three cancer cell lines,
namely HT-29 (human colon cancer), H460 (human lung cancer), and
MDA-MB-231 (human breast cancer), as well as in a non-malignant cell line
WI-38 (human fetal lung fibroblasts), using standard MTT assays. Sorafenib
was used as a positive control. The biological data, expressed as IC50 values,
are listed in Table 1.

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(Insert Table 1 here)
Interestingly, the results of this screening revealed dramatic differences in
the activities of these compounds. Both compounds 8a and 8b, which possessed
a methyl group on 1-position of the triazole, were practically inactive against all
the tested cancer cell lines. However, shifting the methyl to 4-position led to a
significant improvement in potency, as shown with compounds 13a and 13b
.
The results indicated a clear SAR for the triazole moiety, as a suitable
substitution on 4-position was crucial for high potency. On the other hand, the
antitumor activity of the compounds was only fine-tuned by the pattern on the
distal aryl fragment. Compound 13b with a CF3 group at meta-position
possessed moderate activity, having an IC50 value of 3.02 ꢀM against HT-29
cells, and 1.27 ꢀM against H460 cells. Compound 13a, which contained an
additional chlorine atom (R2 = 3-CF3-4-Cl), exhibited a slightly increased
inhibition against HT-29 cells compared to 13b, but was less potent in H460
cells. Given these encouraging results, a more detailed investigation of SARs
was performed with compounds substituted on the 4-position of the triazole.
Meanwhile, to ameliorate the physicochemical properties of the new
compounds, diverse moieties with different hydrophilicities were introduced. In
addition, the distal aryl fragment was optimized with the aim of further
promoting the compounds’ potencies. Accordingly, compounds 13c–y were
prepared, and assessed for activity in HT-29, H460, MDA-MB-231, and WI-38
cell lines (Table 2).
(Insert Table 2 here)
The newly synthesized compounds exhibited significant inhibition of
cancer cell proliferation. Compounds 13a–d, 13g–j, and 13l–u were more
potent than sorafenib against one or more of the tested cancer cell lines. Among
them, compounds 13c and 13i effectively suppressed proliferation of all the
tested cancer cell lines, with IC50 values between 0.88–1.15 and 0.85–1.54 ꢀM,
respectively. On the other hand, most of the tested compounds were less toxic
to human fibroblasts WI-38 in comparison to their effects on cancer cells,
which indicated a favorable safety of the compounds.
Further SAR investigations into modifications on the 4-position of the
triazole revealed that a hydrophilic tail yielded the desired biological activity
and physicochemical properties. Compound 13a, which possessed a methyl
group on the triazole exhibited modest activity against HT-29 cells (IC50, 2.57
ꢀM). However, substitution for a cyclopropyl group, providing compound 13y
,
sharply reduced potency (IC50, 10.90 ꢀM). In contrast, an evident improvement
in inhibitory activity was observed when a hydrophilic tail was incorporated, as
shown by compounds 13c, 13i, and 13o (IC50: 0.88, 0.90, and 1.40 ꢀM,
respectively). Compounds 13c and 13i showed good activities against
MDA-MB-231 cells (IC50: 1.07 and 1.54 ꢀM, respectively), but compound 13o
was less effective (IC50: 7.88 ꢀM), which indicated that the length of chain
might be related to cellular selectivity. It was noted that with a morpholinyl
group, compound 13u, showed an unfavorable overall potency (IC50, 2.86–8.28
ꢀM), indicating that bulky moieties were not effective. Also noteworthy is that

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all the compounds bearing a hydrophilic tail possessed an attractive ClogD7.4,
which suggests a good drug distribution in blood serum.
The results regarding modifications of the distal aryl group revealed that
placing a CF3 group in the meta-position was most suitable. Compound 13c (R2
= 3-CF3-4-Cl) and 13d (R2 = 3-CF3) potently inhibited proliferation of HT-29
and H460 cells, showing IC50 values between 0.88–2.15 ꢀM. However,
decreased activity was observed for compounds 13f–h, which held a F or Cl
atom in R2. Moreover, shifting the CF3 group to the ortho-position (13e
)
resulted in a complete loss of inhibition in H460 cells, as well as a slight
decrease in the inhibition of HT-29 cells (IC50, 5.86 ꢀM). Similar results were
observed when 13i and 13j were compared with 13k–n
.
Regarding MDA-MB-231 cells, compound 13c, which bore a 3-CF3-4-Cl
group, displayed a high level of activity, exhibiting an IC50 value of 1.07 ꢀM.
However, any change of R2 led to a significant decline in potency. For example,
compounds 13d (R2 = 3-CF3), 13g (R2 = 3-Cl-4-F) and 13h (R2 = 2-4-(Cl)2)
exhibited only week inhibition of MDA-MB-231 cells (IC50, 16.35, 8.32 and
7.96 ꢀM, respectively), while compounds 13e (R2 = 2-CF3) and 13f (R2 =
3,4-(F)2) were totally inactive. Overall, the 3-CF3-4-Cl group was evaluated as
the optimal configuration for R2, which is consistent with the framework of
sorafenib.
2.2.2. In vitro enzymatic assay
To rationalize their appealing cell growth inhibition, compounds 13c–u
and 13x were screened against a kinase panel comprising c-Kit, RET, FLT3,
B-Raf and VEGFR-2. The enzymatic experiments were established using a
mobility shift assay, the results are summarized in Table 3.
(Insert Table 3 here)
It is interesting that several compounds (13c
13r 13s, and 13u) exhibited significant inhibition of the tested kinases at a
concentration of 10 ꢀM. Compounds 13c 13g 13i 13j 13o and 13p were
, 13d, 13g, 13i, 13j, 13m, 13o,
13p
,
,
,
,
,
,
evaluated as triple kinase inhibitors of c-Kit, RET and FLT3. Compound 13d
potently inhibited c-Kit and RET, while compound 13s effectively suppressed
c-Kit and FLT3. On the other hand, all the tested compounds showed poor
potency against B-Raf and VEGFR-2 except for 13p, indicating that these
compounds might have a different mechanism of action with sorafenib. Further
studies into SARs revealed that placing a 3-CF3-4-Cl group at R2 produced the
best kinase inhibition, which is consistent with the results obtained in the
cellular assay.
The optimal enzyme inhibition was exhibited by compound 13i, which
inhibited multiple kinases, c-Kit, RET and FLT3, by approximately 80%.
Nonetheless, it was still less active than sorafenib, which suggests that the
selected kinases may not be the precise biological targets of these new
compounds. Detailed investigations on their mechanisms of action are
underway.
2.2.3. Cell apoptosis analysis

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Cell apoptosis is the process of programmed cell death, and induction of
apoptosis is considered a major way that most antitumor drugs work [18]. To
determine the mode of cell death induced by compound 13i, a biparametric
cytofluorimetric analysis was performed using propidium iodide (PI) and
annexinV-FITC staining in HT-29 cells. After treatment with compound 13i for
48 h at different concentrations (0, 1, 2, 5 ꢀM) , the stained cells were analyzed
by a flow cytometer. As shown in Figure 2, the apoptotic rate of HT-29 cells
was obviously increased from 4.21% (control) to 48.10%, which suggested that
compound 13i caused a significant induction of cell apoptosis in a
concentration-dependent manner.
(Insert Fig. 2 here)
3. Conclusions
In summary, interesting diaryl ureas bearing a modified triazole have been
identified as potent antitumor agents. We provided a valuable rational design
strategy that aimed to improve both the antitumor activity as well as the
physicochemical properties of sorafenib. Detailed SAR studies on the triazole
and distal phenyl ring were undertaken, the efforts of which led to the discovery
of an optimal compound, 13i, which displayed high activity in both cellular and
enzymatic assays. Flow cytometry analysis demonstrated that compound 13i
inhibited the proliferation of HT-29 cells by inducing apoptosis. In addition,
compound 13i had a favourable ClogD7.4 value, which is crucial for its in vivo
bioavailability and activity. Taken together, compound 13i is a promising lead
compound for future antitumor drug development. Further investigations into
its pharmacokinetics and in vivo activity are in progress, the results will be
reported in due course.
4. Experimental section
4.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without
further purification. The purity of the synthesized compounds was measured by high
performance liquid chromatography (HPLC, Agilent, USA). Flash chromatography
was performed using silica gel (300–400 mesh). All reactions were monitored by TLC
on silica gel plates. Melting points were determined on a Büchi Melting Point B-540
1
apparatus (Büchi Labortechnik, Flawil, Switzerland). The H and 13C NMR were
recorded on Bruker ARX-400 or ARX-600 spectrometer (Bruker Bioscience,
Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were
measured in ESI mode on an Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA).
Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis
instrument (Carlo Erba, Milan, Italy).
4.1.1. General procedure for preparation of isocyanates (1a–f
)
To a stirred and cooled to 0 °C solution of triphosgene (0.1 mol) in
toluene, appropriate anilines (0.3 mol) was added slowly. The mixture was
stirred for 0.5 h at room temperature, and for another 7–10 h under reflux until
TLC showed the completion of the reaction. The solvent was evaporated, the
mixture was distilled under reduced pressure to afford intermediates 1a–f
.

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4.1.1.1. 1-Chloro-4-isocyanato-2-(trifluoromethyl)benzene (1a). White solid;
150–151 °C (15 mmHg).
4.1.1.2. 1-Isocyanato-3-(trifluoromethyl)benzene (1b). White solid; 172–174 °C
(15 mmHg).
4.1.1.3. 1-Isocyanato-2-(trifluoromethyl)benzene
(1c). Colorless oil;
165–167°C (15 mmHg).
4.1.1.4. 1,2-Difluoro-4-isocyanatobenzene (1d). Colorless oil; 177–179°C (15
mmHg).
4.1.1.5. 2-Chloro-1-fluoro-4-isocyanatobenzene (1e). Colorless oil; 159–161°C
(15 mmHg).
4.1.1.6. 2,4-Dichloro-1-isocyanatobenzene (1f). Colorless oil; 183–185°C (15
mmHg).
4.1.2.
).
3-Fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-5-yl)pyridin-4-yloxy)aniline
(
7
The synthesis of intermediate
7 has been described in detail in our
previous work [16], so the synthetic method is not listed here. Gray solid;
1
Yield: 63%; M.p. 150–153 °C; H NMR (300 MHz, DMSO-d6) δ 8.61 (d,
J =
5.7 Hz, 1H, ArH), 7.99 (s, 1H, ArH), 7.48 (d, = 2.4 Hz, 1H, ArH), 7.01–7.14
J
(m, 2H, ArH), 6.56 (dd,
J = 13.2, 2.4 Hz, 1H, ArH), 6.47 (dd, J = 8.7, 2.1 Hz,
1H, ArH), 5.56 (s, 2H, NH), 4.27 (s, 3H, CH3); ESI-MS m/z: 285.8 [M+H]+.
4.1.3. General procedure for preparation of the target compounds 8a and 8b
At room temperature, to a stirred solution of intermediate
7 (0.7 mmol) in
CH2Cl2, 1a–b (0.77 mmol) was added. The mixture was stirred for 5 h and
monitored by TLC. The precipitate was collected by filtration, washed with
Et2O, and dried to give the solids 8a and 8b, respectively.
4.1.3.1.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(2-(1-methyl-1H-1,2,4-tri
azol-5-yl)pyridin-4-yloxy)phenyl)urea (8a). HPLC purity: 98.2%; White solid;
1
Yield: 68%; M.p. 218.9–219.8 °C; H NMR (400 MHz, DMSO-d6) δ 9.35 (s,
1H, NH), 9.26 (s, 1H, NH), 8.65 (d,
7.99 (s, 1H, ArH), 7.74 (d, = 12.8 Hz, 1H, ArH), 7.62–7.70 (m, 2H, ArH),
7.52 (d, = 2.2 Hz, 1H, ArH), 7.40 (t, = 8.9 Hz, 1H, ArH), 7.31 (d, = 8.4
Hz, 1H, ArH), 7.17 (dd,
= 5.6, 2.5 Hz, 1H, ArH), 4.27 (s, 3H, CH3); 13C NMR
J = 5.7 Hz, 1H, ArH), 8.12 (s, 1H, ArH),
J
J
J
J
J
(101 MHz, DMSO-d6) δ 165.39, 153.98, 152.83, 151.80, 151.07, 150.53,
149.87, 139.56, 139.22, 134.38, 132.48, 127.19, 124.42, 123.77, 123.26,
123.09, 117.46, 115.92, 112.41, 109.66, 107.82, 39.04; ESI-MS m/z: 507.1
[M+H]+; Anal. calcd. for C22H15ClF4N6O2 (%): C, 52.13; H, 2.98; N, 16.58.
Found (%): C, 52.19; H, 3.07; N, 16.65.
4.1.3.2.
1-(3-Fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-5-yl)pyridin-4-yloxy)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (8b). HPLC purity: 98.7%; White solid; Yield:

ACCEPTED MANUSCRIPT
1
72%; M.p. 206.8–209.4 °C; H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H,
NH), 9.20 (s, 1H, NH), 8.65 (d,
(s, 1H, ArH), 7.76 (d, = 13.0 Hz, 1H, ArH), 7.62 (d,
7.52–7.56 (m, 2H, ArH), 7.32–7.42 (m, 3H, ArH), 7.18 (d,
J
= 5.5 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 7.99
= 6.7 Hz, 1H),
= 3.3 Hz, 1H,
J
J
J
13
ArH), 4.27 (s, 3H, CH3); C NMR (101 MHz, DMSO-d6) δ 165.42, 154.00,
152.91, 151.81, 151.08, 150.53, 149.87, 140.76, 139.34, 134.28, 130.43,
130.01, 124.66, 124.44, 122.57, 118.87, 115.79, 114.84, 112.43, 109.65,
107.70, 39.04; ESI-MS m/z: 473.1 [M+H]+; Anal. calcd. for C22H16F4N6O2 (%):
C, 55.94; H, 3.41; N, 17.79. Found (%): C, 55.86; H, 3.37; N, 18.13.
4.1.4. Methyl 4-(4-amino-2-fluorophenoxy)picolinate (
9)
To a solution of intermediate (5 g, 0.017 mol) in ethanol, 0.5 g palladium
3
on charcoal (10%) was added. The mixture was stirred for 4 h at room
temperature. After that time, the catalyst was filtered off and the filtrate was
evaporated. The residue was triturated with diethyl ether, filtered off, and dried
to give intermediate
°C; 1H NMR (400 MHz, DMSO-d6) δ 8.54 (dd,
(dd, = 9.5, 2.5 Hz, 1H, ArH), 7.14 (ddd, = 12.7, 5.6, 2.6 Hz, 1H, ArH), 7.02
(t, = 9.0 Hz, 1H, ArH), 6.51 (dd, = 13.2, 2.5 Hz, 1H, ArH), 6.43 (dd, = 8.7,
9
as a pale-yellow solid (2.72 g, 60.6%); M.p. 69.5–71.5
J
= 5.5, 3.2 Hz, 1H, ArH), 7.39
J
J
J
J
J
2.1 Hz, 1H, ArH), 5.52 (s, 2H, NH2), 3.83 (s, 3H, CH3); ESI-MS m/z: 263.0
[M+H]+.
4.1.5. General procedure for preparation of intermediates 10a–f
A mixture of intermediate
9 (2 mmol) and appropriate isocyanate (1a–e, 2
mmol) in CH2Cl2 was stirred at room temperature for 4–7 h until TLC showed
the completion of reaction. The precipitate was filtered off and washed to give
the solids 10a–f
.
4.1.5.1.
Methyl
4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-2-fluorophenoxy)picolinate
1
(
10a). White solid; Yield: 92%; M.p. 133.6–134.5 °C; H NMR (400 MHz,
DMSO-d6) δ 9.35 (s, 1H, NH), 9.27 (s, 1H, NH), 8.59 (d, = 5.6 Hz, 1H, ArH),
8.09 (d, = 2.1 Hz, 1H, ArH), 7.71 (dd, = 13.2, 2.2 Hz, 1H, ArH), 7.66 (dd,
= 8.9, 2.2 Hz, 1H, ArH), 7.62 (d, = 8.8 Hz, 1H, ArH), 7.45 (dd, = 7.4, 2.5
Hz, 1H, ArH), 7.36 (t, = 8.9 Hz, 1H, ArH), 7.28 (dd, = 8.9, 1.7 Hz, 1H,
J
J
J
J
J
J
J
J
ArH), 7.22 (dd,
J
= 5.6, 2.6 Hz, 1H, ArH), 3.84 (s, 3H, CH3); ESI-MS m/z:
484.5 [M+H]+.
4.1.5.2.
Methyl
4-(2-fluoro-4-(3-(3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinate
(
10b).
White solid; Yield: 86%; M.p. 169.7–171.1 °C; H NMR (400 MHz,
DMSO-d6) δ 9.20 (s, 1H, NH), 9.18 (s, 1H, NH), 8.59 (d, = 5.6 Hz, 1H, ArH),
8.00 (s, 1H, ArH), 7.73 (dd, = 13.2, 2.4 Hz, 1H, ArH), 7.59 (d, = 8.6 Hz,
1H, ArH), 7.52 (t, = 7.9 Hz, 1H, ArH), 7.45 (dd, = 6.9, 2.6 Hz, 1H, ArH),
1
J
J
J
J
J
7.31–7.39 (m, 2H, ArH), 7.27 (dd,
J = 8.9, 1.7 Hz, 1H, ArH), 7.22 (dd, J = 5.6,
2.6 Hz, 1H, ArH), 3.84 (s, 3H, CH3); ESI-MS m/z: 472.1 [M+Na]+.
4.1.5.3.
Methyl
10c).
4-(2-fluoro-4-(3-(2-(trifluoromethyl)phenyl)ureido)phenoxy)picolinate
White solid; Yield: 81%; M.p. 122.2–125.1 °C; H NMR (400 MHz,
(
1

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DMSO-d6) δ 9.68 (s, 1H, NH), 8.58 (dd,
J
= 5.6, 2.3 Hz, 1H, ArH), 8.19 (s, 1H,
NH), 7.89 (d,
J
= 8.2 Hz, 1H, ArH), 7.74 (dd,
J
= 13.3, 2.4 Hz, 1H, ArH),
= 15.5,
J = 8.5, 5.3, 2.4 Hz, 2H, ArH), 3.84 (s, 3H, CH3);
7.62–7.71 (m, 2H, ArH), 7.45 (dd, J = 7.7, 2.5 Hz, 1H, ArH), 7.33 (dt, J
8.3 Hz, 2H, ArH), 7.21 (ddd,
ESI-MS m/z: 472.2 [M+Na]+.
4.1.5.4. Methyl 4-(4-(3-(3,4-difluorophenyl)ureido)-2-fluorophenoxy)picolinate
1
(
10d). White solid; Yield: 86%; M.p. 213.0–214.7 °C; H NMR (400 MHz,
DMSO-d6) δ 9.13 (s, 1H, NH), 9.04 (s, 1H, NH), 8.59 (d, = 5.6 Hz, 1H, ArH),
7.70 (dd, = 13.3, 2.3 Hz, 1H, ArH), 7.65 (ddd, = 13.3, 7.5, 2.6 Hz, 1H,
ArH), 7.43 (d, = 2.3 Hz, 1H, ArH), 7.31–7.39 (m, 2H, ArH), 7.25 (dd, = 9.1,
J
J
J
J
J
1.3 Hz, 1H, ArH), 7.21 (dd, J = 5.6, 2.5 Hz, 1H, ArH), 7.12–7.16 (m, 1H, ArH),
3.83 (s, 3H, CH3); ESI-MS m/z: 418.1 [M+H]+.
4.1.5.5.
Methyl
10e).
4-(4-(3-(3-chloro-4-fluorophenyl)ureido)-2-fluorophenoxy)picolinate
White solid; Yield: 90%; M.p. 213.6–215.4 °C; H NMR (400 MHz,
(
1
DMSO-d6) δ 9.15 (s, 1H, NH), 9.03 (s, 1H, NH), 8.58 (d,
7.78 (dd, = 6.7, 1.6 Hz, 1H, ArH), 7.71 (dd, = 13.2, 2.4 Hz, 1H, ArH), 7.44
(dd, = 7.1, 2.5 Hz, 1H, ArH), 7.30–7.38 (m, 3H, ArH), 7.25 (dd, = 8.9, 1.5
= 5.6, 2.6 Hz, 1H, ArH), 3.83 (s, 3H, CH3); ESI-MS
J = 5.6 Hz, 1H, ArH),
J
J
J
J
Hz, 1H, ArH), 7.21 (dd,
J
m/z: 434.0 [M+H]+.
4.1.5.6. Methyl 4-(4-(3-(2,4-dichlorophenyl)ureido)-2-fluorophenoxy)picolinate
1
(
10f). White solid; Yield: 77%; M.p. 195.5–198.0 °C; H NMR (400 MHz,
DMSO-d6) δ 9.80 (s, 1H, NH), 8.61 (d, = 5.6 Hz, 1H, ArH), 8.52 (s, 1H, NH),
8.17 (d, = 9.0 Hz, 1H, ArH), 7.75 (dd, = 13.1, 2.3 Hz, 1H, ArH), 7.66 (d,
2.4 Hz, 1H, ArH), 7.47 (dd, = 7.2, 2.5 Hz, 1H, ArH), 7.37–7.43 (m, 2H, ArH),
= 5.7, 2.4 Hz, 2H, ArH), 3.86 (s, 3H, CH3); ESI-MS m/z: 450.1
J
J
J
J =
J
7.24 (dd,
[M+H]+.
J
4.1.6. General procedure for preparation of intermediates 11a–f
To a solution of 10a–f (1 mmol) in MeCN, 80% hydrazine hydrate (3
mmol) was added dropwise. The mixture was sirred under refulx and monitored
by TLC. The solvent was concentrated and cooled to room temperature. The
precipitate was collected by filtration and washed to yield the corresponding
compounds 11a–f
.
4.1.6.1.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-((2-(hydrazinecarbonyl)p
yridin-4-yl)oxy)phenyl)urea (11a). White solid; Yield: 72%; M.p. 173.5–175.0
1
°C; H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H, –NH–NH2), 9.37 (s, 1H,
NH), 9.27 (s, 1H, NH), 8.49 (br, 1H, ArH), 8.10 (s, 1H, ArH), 7.71 (d,
J = 12.4
Hz, 1H, ArH), 7.64 (dd, = 18.2, 9.5 Hz, 2H, ArH), 7.36 (m, 2H, ArH), 7.28
J
(dd,
J = 7.9, 0.8 Hz, 1H, ArH), 7.16 (d, J = 2.4 Hz, 1H, ArH), 4.54 (s, 2H,
–NH–NH2). ESI-MS m/z: 506.4 [M+Na]+.
4.1.6.2.
1-(3-Fluoro-4-((2-(hydrazinecarbonyl)pyridin-4-yl)oxy)phenyl)-3-(3-(trifluoro
1
methyl)phenyl)urea (11b). White solid; Yield: 69%; M.p. 183.4–185.7 °C; H

ACCEPTED MANUSCRIPT
NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H, –NH–NH2), 9.22 (s, 1H, NH), 9.19
(s, 1H, NH), 8.52 (d,
13.1, 2.3 Hz, 1H, ArH), 7.61 (d,
ArH), 7.38 (t, = 8.9 Hz, 1H, ArH), 7.35 (t,
J
= 5.6 Hz, 1H, ArH), 8.02 (s, 1H, ArH), 7.75 (dd,
= 8.2 Hz, 1H, ArH), 7.54 (t, = 7.9 Hz, 1H,
= 4.9 Hz, 2H, ArH), 7.29 (dd,
= 5.6, 2.6 Hz, 1H, ArH), 4.68 (s, 2H,
J =
J
J
J
J
J =
8.9, 1.4 Hz, 1H, ArH), 7.19 (dd,
J
–NH–NH2). ESI-MS m/z: 472.5 [M+Na]+.
4.1.6.3.
1-(3-Fluoro-4-((2-(hydrazinecarbonyl)pyridin-4-yl)oxy)phenyl)-3-(2-(trifluoro
1
methyl)phenyl)urea (11c). White solid; Yield: 76%; M.p. 202.3–205.0 °C; H
NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H, –NH–NH2), 9.68 (s, 1H, NH), 8.49
(d,
(dd,
Hz, 1H, ArH), 7.38 (d,
J
= 5.5 Hz, 1H, ArH), 8.19 (s, 1H, NH), 7.90 (d,
= 13.6, 0.9 Hz, 1H, ArH), 7.69 (d, = 7.8 Hz, 1H, ArH), 7.64 (d,
= 8.5 Hz, 1H, ArH), 7.30–7.35 (m, 2H), 7.22 (dd,
= 5.2, 1.8 Hz, 1H), 4.54 (s, 2H); ESI-MS m/z:
J
= 7.8 Hz, 1H, ArH), 7.74
J
J
J
= 8.4
J
J =
8.6, 0.8 Hz, 1H), 7.16 (dd,
J
472.1 [M+Na]+.
4.1.6.4.
1-(3,4-Difluorophenyl)-3-(3-fluoro-4-((2-(hydrazinecarbonyl)pyridin-4-yl)oxy)
1
phenyl)urea (11d). White solid; Yield: 81%; M.p. 200.1–203.5 °C; H NMR
(400 MHz, DMSO-d6) δ 9.92 (s, 1H, –NH–NH2), 9.24 (s, 1H, NH), 9.16 (s, 1H,
NH), 8.49 (d,
J
= 5.6 Hz, 1H, ArH), 7.70 (dd,
J
= 13.3, 2.2 Hz, 1H, ArH),
= 8.7, 0.9 Hz,
J = 5.6, 2.6 Hz, 2H, ArH), 4.54 (s, 2H, –NH–NH2);
7.62–7.68 (m, 1H, ArH), 7.30–7.38 (m, 3H, ArH), 7.26 (dd,
J
1H, ArH), 7.15 (dd,
ESI-MS m/z: 440.3 [M+Na]+.
4.1.6.5.
1-(3-Chloro-4-fluorophenyl)-3-(3-fluoro-4-((2-(hydrazinecarbonyl)pyridin-4-yl
1
)oxy)phenyl)urea (11e). White solid; Yield: 60%; M.p. 184.0–186.9 °C; H
NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H, –NH–NH2), 9.68 (s, 1H, NH), 9.57
(s, 1H, NH), 8.49 (d,
J
= 2.0 Hz, 1H, ArH), 7.79–7.83 (m, 1H, ArH), 7.72 (d,
J
= 13.0 Hz, 1H, ArH), 7.34 (m, 4H, ArH), 7.28 (d,
J
= 8.6 Hz, 1H, ArH), 7.15
(d,
J = 0.7 Hz, 1H, ArH), 4.54 (s, 2H, –NH–NH2); ESI-MS m/z: 456.4
[M+Na]+.
4.1.6.6.
1-(2,4-Dichlorophenyl)-3-(3-fluoro-4-((2-(hydrazinecarbonyl)pyridin-4-yl)oxy)
1
phenyl)urea (11f). White solid; Yield: 67%; M.p. 222.9–224.0 °C; H NMR
(600 MHz, DMSO) δ 10.18 (s, 1H, –NH–NH2), 9.79 (s, 1H, NH), 8.53 (d,
5.6 Hz, 1H, ArH), 8.51 (s, 1H, NH), 8.18 (d, = 8.9 Hz, 1H, ArH), 7.75 (dd,
= 13.1, 2.4 Hz, 1H, ArH), 7.66 (d, = 2.4 Hz, 1H, ArH), 7.42 (dd,
Hz, 1H, ArH), 7.39 (d, = 9.0 Hz, 1H, ArH), 7.37 (d, = 2.5 Hz, 1H, ArH),
7.24 (dd, = 8.8, 1.6 Hz, 1H, ArH), 7.20 (dd, = 5.6, 2.6 Hz, 1H, ArH), 5.66
(s, 2H, –NH–NH2); ESI-MS m/z: 450.3 [M+H]+.
J
=
J
J
J
J = 8.9, 2.4
J
J
J
J
4.1.7. General procedure for preparation of intermediates 12a–f
A mixture of an appropriate hydrazine derivative (11a–f, 1 mmol) and
DMF-DMA (5 mmol) in CH2Cl2 was heated under reflux for 4–6 h. The
completion of the reaction was determined by TLC, at which point the solvent

ACCEPTED MANUSCRIPT
was evaporated. Et2O was added to the mixture, the precipitate was filtered off
and washed to afford the solids 12a–f
.
4.1.7.1.
(E)-N'-(4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-2-fluorophenoxy)p
icolinoyl)-N,N-dimethylformohydrazonamide (12a). Light yellow solid; Yield:
1
52%; M.p. 208.6–209.6 °C; H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H,
–NH–N=CH), 9.33 (s, 1H, NH), 9.24 (s, 1H, NH), 8.49 (d,
ArH), 8.09 (d, = 2.2 Hz, 1H, ArH), 8.03 (s, 1H, –NH–N=CH), 7.71 (dd,
13.1, 2.3 Hz, 1H, ArH), 7.66 (dd, = 8.7, 2.1 Hz, 1H, ArH), 7.62 (d, = 8.8
Hz, 1H, ArH), 7.36 (m, 2H, ArH), 7.28 (dd, = 8.9, 1.7 Hz, 1H, ArH), 7.15
(dd,
= 5.6, 2.6 Hz, 1H, ArH), 2.80 (s, 6H, CH3); ESI-MS m/z: 539.1 [M+H]+.
J = 5.6 Hz, 1H,
J
J =
J
J
J
J
4.1.7.2.
(E)-N'-(4-(2-Fluoro-4-(3-(3-(trifluoromethyl)phenyl)ureido)phenoxy)picolinoyl
)-N,N-dimethylformohydrazonamide (12b). Light yellow solid; Yield: 66%;
1
M.p. 201.0–203.2 °C; H NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H,
–NH–N=CH), 9.23 (s, 1H, NH), 9.21 (s, 1H, NH), 8.49 (s, 1H, –NH–N=CH),
8.02 (d, J = 10.9 Hz, 2H, ArH), 7.73 (dd, J = 12.2, 1.1 Hz, 1H, ArH), 7.55 (d, J
= 30.9 Hz, 2H, ArH), 7.25–7.38 (m, 4H, ArH), 7.15 (s, 1H, ArH), 2.80 (s, 6H,
CH3); ESI-MS m/z: 505.1 [M+H]+.
4.1.7.3.
(E)-N'-(4-(2-Fluoro-4-(3-(2-(trifluoromethyl)phenyl)ureido)phenoxy)picolinoyl
)-N,N-dimethylformohydrazonamide (12c). Light yellow solid; Yield: 70%;
1
M.p. 220.3–225.1 °C; H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H,
–NH–N=CH), 9.68 (s, 1H, NH), 8.49 (d,
NH), 8.04 (s, 1H, –NH–N=CH), 7.90 (d,
J
J
= 5.6 Hz, 1H, ArH), 8.19 (s, 1H,
= 8.3 Hz, 1H, ArH), 7.73 (dd,
= 7.8 Hz, 1H,
= 7.6 Hz, 1H, ArH), 7.22 (dd, = 8.8,
J = 5.6, 2.6 Hz, 1H, ArH), 2.81 (s, 6H, CH3);
J =
13.2, 2.4 Hz, 1H, ArH), 7.69 (d,
J = 7.9 Hz, 1H, ArH), 7.65 (t, J
ArH), 7.34–7.39 (m, 2H, ArH), 7.30 (t,
J
J
1.5 Hz, 1H, ArH), 7.15 (dd,
ESI-MS m/z: 505.2 [M+H]+.
4.1.7.4.
(E)-N'-(4-(4-(3-(3,4-Difluorophenyl)ureido)-2-fluorophenoxy)picolinoyl)-N,N-d
imethylformohydrazonamide (12d). Light yellow solid; Yield: 69%; M.p.
191.3–193.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H, –NH–N=CH),
9.13 (s, 1H, NH), 9.05 (s, 1H, NH), 8.50 (d,
J = 5.6 Hz, 1H, ArH), 8.05 (s, 1H,
–NH–N=CH), 7.70 (dd, = 13.3, 2.4 Hz, 1H, ArH), 7.62–7.67 (m, 1H, ArH),
J
7.35 (m, 3H, ArH), 7.25 (dd,
J = 8.9, 1.4 Hz, 1H, ArH), 7.15 (dt, J = 9.5, 4.8
Hz, 2H, ArH), 2.82 (s, 6H, CH3); ESI-MS m/z: 473.1 [M+H]+.
4.1.7.5.
(E)-N'-(4-(4-(3-(3-Chloro-4-fluorophenyl)ureido)-2-fluorophenoxy)picolinoyl)-
N,N-dimethylformohydrazonamide (12e). Light yellow solid; Yield: 56%; M.p.
196.2–198.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H, –NH–N=CH),
9.17 (s, 1H, NH), 9.05 (s, 1H, NH), 8.50 (d, J = 5.7 Hz, 1H, ArH), 8.06 (s, 1H,
–NH–N=CH), 7.79 (dd,
1H, ArH), 7.31–7.36 (m, 4H, ArH), 7.25 (dd,
(dd,
= 5.4, 2.5 Hz, 1H, ArH), 2.83 (s, 6H, CH3); ESI-MS m/z: 489.1 [M+H]+.
J
= 6.9, 1.7 Hz, 1H, ArH), 7.71 (dd,
J = 13.5, 2.0 Hz,
J
= 7.8, 1.5 Hz, 1H, ArH), 7.16
J

ACCEPTED MANUSCRIPT
4.1.7.6.
(E)-N'-(4-(4-(3-(2,4-Dichlorophenyl)ureido)-2-fluorophenoxy)picolinoyl)-N,N-
dimethylformohydrazonamide (12f). Yellow solid; Yield: 77%; M.p.
172.7–175.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H, –NH–N=CH),
9.80 (s, 1H, NH), 8.52 (s, 1H, NH), 8.51 (br, 1H, ArH), 8.18 (d,
ArH), 8.05 (s, 1H, –NH–N=CH), 7.75 (dd, = 13.2, 2.2 Hz, 1H, ArH), 7.66 (d,
= 2.3 Hz, 1H, ArH), 7.39–7.44 (m, 2H, ArH), 7.37 (d, = 2.7 Hz,1H, ArH),
7.24 (dd, = 8.3, 1.3 Hz, 1H, ArH), 7.17 (dd, = 5.6, 2.6 Hz, 1H, ArH), 2.82
(s, 6H, CH3); ESI-MS m/z: 505.2 [M+H]+.
J = 8.9 Hz, 1H,
J
J
J
J
J
4.1.8. General procedure for preparation of compounds 13a–y
At room temperature, to a solution of an intermediate (12a–f, 0.3 mmol) in
glacial acetic acid, an appropriate amine (0.9 mmol) was added slowly. The
mixture was heated to 90 °C and stirred for 3 h. The solvent was evaporated
under reduced pressure, and the residue was poured into water. The mixture
was alkalized with aqueous NaOH to pH 8–9, at which time the precipitate was
filtered off to yield the crude product. The solids were purified by silica gel
column chromatography to generate the target compounds 13a–y
.
4.1.8.1.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-(2-(4-methyl-4H-1,2,4-tri
azol-3-yl)pyridin-4-yloxy)phenyl)urea (13a). Flash column chromatography
was performed using (dichloromethane:methanol, 100:1 to 25:1). HPLC purity:
1
98.7%; White solid; Yield: 51%; M.p. 179.8–181.3 °C; H NMR (400 MHz,
DMSO-d6) 9.79 (s, 1H, NH), 9.68 (s, 1H, NH), 8.62 (d,
8.11 (d, = 2.1 Hz, 1H, ArH), 7.75 (dd, = 13.2, 2.3 Hz, 1H, ArH), 7.68 (dd,
= 8.8, 2.1 Hz, 1H, ArH), 7.63 (d, = 8.8 Hz, 1H, ArH), 7.55 (d,
ArH), 7.40 (t,
J = 5.9 Hz, 2H, ArH),
J
J
J
J
J
= 2.4 Hz, 1H,
J
= 9.0 Hz, 1H, ArH), 7.29 (d,
J
= 8.8 Hz, 1H, ArH), 7.13 (dd,
J
= 5.7, 2.5 Hz, 1H, ArH), 4.00 (s, 3H, CH3); ESI-MS m/z: 507.2 [M+H]+; Anal.
calcd. for C22H15ClF4N6O2 (%): C, 52.13; H, 2.98; N, 16.58. Found (%): C,
51.37; H, 3.04; N, 16.72.
4.1.8.2.
1-(3-Fluoro-4-(2-(4-methyl-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)phenyl)-3-(3-
(trifluoromethyl)phenyl)urea (13b). Flash column chromatography was
performed using (dichloromethane:methanol, 150:1 to 25:1). HPLC purity:
1
99.2%; White solid; Yield: 37%; M.p. 231.0–232.3 °C; H NMR (400 MHz,
DMSO-d6) δ 9.20 (s, 2H, NH), 8.62 (s, 2H, NH), 8.03 (s, 1H, ArH), 7.76 (d, J =
10.9 Hz, 1H, ArH), 7.58 (m, 3H, ArH), 7.36 (m, 3H, ArH), 7.15 (s, 1H, ArH),
13
4.00 (s, 3H, CH3); C NMR (101 MHz, DMSO-d6) δ 165.29, 154.02, 152.90,
151.79, 151.18, 149.76, 147.84, 140.78, 139.38, 134.27, 130.43, 130.01,
124.66, 124.45, 122.54, 118.83, 115.74, 114.82, 112.35, 109.12, 107.65, 34.17;
ESI-MS m/z: 473.0 [M+H]+; ESI-MS m/z: 473.0 [M+H]+; Anal. calcd. for
C22H16F4N6O2 (%): C, 55.94; H, 3.41; N, 17.79. Found (%): C, 56.21; H, 3.25;
N, 17.62.
4.1.8.3.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(4-(2-(dimethylamino)ethyl)-4H
-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13c). Flash column

ACCEPTED MANUSCRIPT
chromatography was performed using (dichloromethane:methanol, 70:1 to
15:1). HPLC purity: 98.7%; White solid; Yield: 33%; M.p. 178.6–180.5 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H, NH), 9.85 (s, 1H, NH), 8.64 (s, 1H,
ArH), 8.60 (d,
1H, ArH), 7.65 (dd,
8.5 Hz, 1H, ArH), 7.28 (d,
ArH), 4.61 (t, = 5.9 Hz, 2H, –CH2–CH2–N(CH3)2), 2.57 (t,
J
= 5.2 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 7.75 (d,
= 15.6, 8.1 Hz, 2H, ArH), 7.56 (s, 1H, ArH), 7.40 (t,
= 8.0 Hz, 1H, ArH), 7.13 (dd, = 5.6, 2.6 Hz, 1H,
= 6.0 Hz, 2H,
J = 12.3 Hz,
J
J =
J
J
J
J
–CH2–CH2–N(CH3)2), 2.13 (s, 6H, –CH2–CH2–N(CH3)2); 13C NMR (101 MHz,
DMSO-d6) δ 165.30, 154.04, 152.94, 151.72, 150.62, 150.06, 147.45, 139.67,
139.30, 134.35, 132.54, 127.24, 124.47, 123.79, 123.48, 121.91, 117.17,
115.60, 112.19, 109.28, 107.48, 59.24, 45.51, 43.84; ESI-MS m/z: 586.1
[M+Na]+; Anal. calcd. for C25H22ClF4N7O2 (%): C, 53.25; H, 3.93; N, 17.39.
Found (%): C, 53.57; H, 4.15; N, 17.55.
4.1.8.4.
1-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fl
uorophenyl)-3-(3-(trifluoromethyl)phenyl)urea
(
13d).
Flash
column
chromatography was performed using (dichloromethane:methanol, 50:1 to
15:1). HPLC purity: 99.5%; White solid; Yield: 62%; M.p. 201.5–204.7 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H, NH), 9.24 (s, 1H, NH), 8.64 (s, 1H,
ArH), 8.60 (d,
1H, ArH), 7.62 (d,
8.9 Hz, 1H, ArH), 7.32 (m, 1H, ArH), 7.15 (dd,
(t, = 6.0 Hz, 2H, –CH2–CH2–N(CH3)2), 2.62 (t,
J
= 5.7 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 7.76 (d,
= 8.0 Hz, 1H, ArH), 7.49–7.56 (m, 2H, ArH), 7.40 (t,
= 5.7, 2.5 Hz, 1H, ArH), 4.63
= 6.2 Hz, 2H,
J = 13.2 Hz,
J
J =
J
J
J
–CH2–CH2–N(CH3)2), 2.17 (s, 6H, –CH2–CH2–N(CH3)2); 13C NMR (101 MHz,
DMSO-d6) δ 165.31, 154.02, 152.94, 151.72, 150.60, 150.05, 147.50, 140.82,
139.41, 134.27, 130.41, 139.99, 124.67, 124.45, 122.56, 118.82 115.76, 114.83,
112.29, 109.10, 107.65, 59.24, 45.53, 43.84; ESI-MS m/z: 530.2 [M+H]+; Anal.
calcd. for C25H23F4N7O2 (%): C, 56.71; H, 4.38; N, 18.52. Found (%): C, 56.67;
H, 4.65; N, 18.70.
4.1.8.5.
1-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fl
uorophenyl)-3-(2-(trifluoromethyl)phenyl)urea
(
13e).
Flash
column
chromatography was performed using (dichloromethane:methanol, 100:1 to
20:1). HPLC purity: 99.3%; White solid; Yield: 51%; M.p. 156.3–158.1 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H, NH), 8.64 (s, 1H, NH), 8.60 (s, 1H,
ArH), 8.34 (s, 1H, ArH), 7.90 (s, 1H, ArH), 7.73 (m, 3H, ArH), 7.55 (s, 1H,
ArH), 7.33 (m, 3H, ArH), 7.13 (s, 1H, ArH), 4.61 (br, 2H,
–CH2–CH2–N(CH3)2), 2.57 (br, 2H, –CH2–CH2–N(CH3)2), 2.13 (s, 6H,
–CH2–CH2–N(CH3)2); ESI-MS m/z: 552.1 [M+Na]+; Anal. calcd. for
C25H23F4N7O2 (%): C, 56.71; H, 4.38; N, 18.52. Found (%): C, 57.06; H, 4.17;
N, 19.03.
4.1.8.6.
1-(3,4-Difluorophenyl)-3-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-
yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13f). Flash column chromatography
was performed using (dichloromethane:methanol, 50:1 to 15:1). HPLC purity:
1
99.7%; White solid; Yield: 46%; M.p. 165.2–167.8 °C; H NMR (400 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ 9.13 (s, 1H, NH), 9.05 (s, 1H, NH), 8.59 (s, 1H, ArH), 8.56 (d, J =
5.8 Hz, 1H, ArH), 7.64 (m, 2H, ArH), 7.50 (d,
2H, ArH), 7.25 (m, 1H, ArH), 7.11 (m, 2H, ArH), 4.58 (t,
–CH2–CH2–N(CH3)2), 2.55 (t, = 6.4 Hz, 2H, –CH2–CH2–N(CH3)2), 2.10 (s,
J
= 2.6 Hz, 1H, ArH), 7.35 (m,
J
= 6.4 Hz, 2H,
J
13
6H, –CH2–CH2–N(CH3)2); C NMR (101 MHz, DMSO-d6) δ 165.33, 154.00,
153.06, 151.71, 150.60, 150.03, 147.48, 139.75, 137.36, 134.02, 124.38,
117.81, 115.53, 114.97, 112.20, 109.17, 107.82, 107.60, 107.37, 59.24, 45.53,
43.83; ESI-MS m/z: 498.1 [M+H]+; Anal. calcd. for C24H22F3N7O2 (%): C,
57.94; H, 4.46; N, 19.71. Found (%): C, 57.82; H, 4.57; N, 19.66.
4.1.8.7.
1-(3-Chloro-4-fluorophenyl)-3-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-tria
zol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea
(
13g).
Flash
column
chromatography was performed using (dichloromethane:methanol, 60:1 to
15:1). HPLC purity: 99.2%; White solid; Yield: 61%; M.p. 206.1–209.6 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H, NH), 9.11 (s, 1H, NH), 8.63 (s, 1H,
ArH), 8.59 (d,
12.5 Hz, 1H, ArH), 7.53 (d,
= 8.6 Hz, 1H, ArH), 7.14 (dd,
2H, –CH2–CH2–N(CH3)2), 2.57 (t,
J
= 5.6 Hz, 1H, ArH), 7.81 (d,
= 1.7 Hz, 1H, ArH), 7.37 (m, 3H, ArH), 7.29 (d,
= 5.3, 2.1 Hz, 1H, ArH), 4.61 (t,
= 6.1 Hz, 2H, –CH2–CH2–N(CH3)2), 2.12
J
= 6.5 Hz, 1H, ArH), 7.74 (d,
J
=
J
J
J
J = 5.9 Hz,
J
(s, 6H, –CH2–CH2–N(CH3)2); 13C NMR (101 MHz, DMSO-d6) δ 165.34,
154.15, 154.00, 152.91, 151.73, 150.59, 149.97, 147.49, 139.52, 137.26,
134.15, 124.40, 120.26, 119.61, 119.25, 117.33, 115.65, 112.26, 109.12,
107.54, 58.97, 45.29, 43.61; ESI-MS m/z: 514.1 [M+H]+; Anal. calcd. for
C24H22ClF2N7O2 (%): C, 56.09; H, 4.31; N, 19.08. Found (%): C, 56.36; H,
4.50; N, 19.37.
4.1.8.8.
1-(2,4-Dichlorophenyl)-3-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-
yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13h). Flash column chromatography
was performed using (dichloromethane:methanol, 50:1 to 20:1). HPLC purity:
1
98.9%; White solid; Yield: 52%; M.p. 183.6–185.8 °C; H NMR (400 MHz,
DMSO-d6) δ 10.06 (s, 1H, NH), 8.63 (s, 1H, NH), 8.60 (d,
ArH), 8.18 (d, = 8.9 Hz, 1H, ArH), 7.76 (d, = 11.5 Hz, 1H, ArH), 7.63 (s,
1H, ArH), 7.54 (s, 1H, ArH), 7.40 (m, 2H, ArH), 7.27 (d, = 8.8 Hz, 1H, ArH),
7.13 (dd, = 5.6, 2.5 Hz, 1H, ArH), 4.61 (t, = 6.0 Hz, 2H,
–CH2–CH2–N(CH3)2), 2.59 (t, = 6.1 Hz, 2H, –CH2–CH2–N(CH3)2), 2.13 (s,
J = 5.4 Hz, 2H,
J
J
J
J
J
J
13
6H, –CH2–CH2–N(CH3)2); C NMR (101 MHz, DMSO-d6) δ 165.32, 154.06,
152.53, 151.75, 150.59, 149.95, 147.48, 139.39, 135.45, 134.25, 129.08,
128.08, 126.93, 124.56, 123.66, 123.03, 115.37, 112.24, 109.17, 107.21, 58.88,
45.22, 43.50; ESI-MS m/z: 552.0 [M+Na]+; Anal. calcd. for C24H22Cl2FN7O2
(%): C, 54.35; H, 4.18; N, 18.49. Found (%): C, 55.15; H, 4.17; N, 18.63.
4.1.8.9.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(4-(2-(diethylamino)ethyl)-4H-
1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13i). Flash column
chromatography was performed using (dichloromethane:methanol, 100:1 to
20:1). HPLC purity: 99.0%; White solid; Yield: 41%; M.p. 202.2–205.6 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H, NH), 9.30 (s, 1H, NH), 8.62 (s, 1H,

ACCEPTED MANUSCRIPT
ArH), 8.59 (d,
J
= 4.5 Hz, 1H, ArH), 8.12 (s, 1H, ArH), 7.70 (m, 3H, ArH),
7.52 (s, 1H, ArH), 7.36 (m, 2H, ArH), 7.14 (dd,
(t, = 5.9 Hz, 2H, –CH2–CH2–N(CH2CH3)2), 2.66 (t,
–CH2–CH2–N(CH2CH3)2), 2.39 (quartet,
–CH2–CH2–N(CH2CH3)2), 0.76 (t, = 6.3 Hz, 6H, –CH2–CH2–N(CH2CH3)2);
J
= 5.5 2.4 Hz, 1H, ArH), 4.56
= 6.1 Hz, 2H,
6.3 Hz, 4H,
J
J
J
=
J
13C NMR (101 MHz, DMSO-d6) δ 165.27, 154.00, 152.83, 151.67, 150.79,
150.21, 147.70, 139.57, 139.22, 134.40, 132.50, 127.19, 124.44, 123.79,
123.08, 121.91, 117.48, 115.90, 112.24, 109.08, 107.81, 53.08, 47.03, 44.45,
12.25; ESI-MS m/z: 592.0 [M+H]+; Anal. calcd. for C27H26ClF4N7O2 (%): C,
54.78; H, 4.43; N, 16.56. Found (%): C, 54.56; H, 4.15; N, 17.03.
4.1.8.10.
1-(4-(2-(4-(2-(Diethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-flu
orophenyl)-3-(3-(trifluoromethyl)phenyl)urea
(
13j).
Flash
column
chromatography was performed using (dichloromethane:methanol, 100:1 to
25:1). HPLC purity: 99.3%; White solid; Yield: 36%; M.p. 181.3–183.5 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.72 (s, 2H, NH), 8.62 (s, 1H, ArH), 8.58 (d,
5.6 Hz, 1H, ArH), 8.01 (s, 1H, ArH), 7.76 (dd, = 13.0, 0.9 Hz, 1H, ArH), 7.62
(d, = 8.2 Hz, 1H, ArH), 7.52 (m, 2H, ArH), 7.39 (t, = 8.9 Hz, 1H, ArH),
7.30 (dd, = 21.4, 7.9 Hz, 2H, ArH), 7.12 (dd, = 5.1, 1.9 Hz, 1H, ArH), 4.55
(t, = 5.8 Hz, 2H, –CH2–CH2–N(CH2CH3)2), 2.66 (t, = 5.5 Hz, 2H,
–CH2–CH2–N(CH2CH3)2), 2.39 (quartet, 6.1 Hz, 4H,
–CH2–CH2–N(CH2CH3)2), 0.76 (t, = 6.9 Hz, 6H, –CH2–CH2–N(CH2CH3)2);
J =
J
J
J
J
J
J
J
J
=
J
13C NMR (101 MHz, DMSO-d6) δ 165.32, 154.05, 153.03, 151.68, 150.82,
150.22, 147.64, 140.89, 139.46, 134.24, 130.43, 130.04, 126.02, 124.44,
122.28, 118.71, 115.46, 114.59, 112.13, 109.29, 107.45, 53.12, 47.06, 44.46,
12.26; ESI-MS m/z: 580.7 [M+Na]+; Anal. calcd. for C27H27F4N7O2 (%): C,
58.16; H, 4.88; N, 17.59. Found (%): C, 58.37; H, 4.57; N, 18.03.
4.1.8.11.
1-(4-(2-(4-(2-(Diethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-flu
orophenyl)-3-(2-(trifluoromethyl)phenyl)urea
(
13k).
Flash
column
chromatography was performed using (dichloromethane:methanol, 100:1 to
1
30:1). HPLC purity: 98.7%; White solid; Yield: 49%; M.p. 72.5–74.6 °C; H
NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H, NH), 8.62 (s, 1H, NH), 8.58 (d,
5.6 Hz, 1H, ArH), 8.30 (s, 1H, ArH), 7.91 (d, = 8.0 Hz, 1H, ArH), 7.75 (m,
3H, ArH), 7.54 (s, 1H, ArH), 7.38 (d, = 9.1 Hz, 1H, ArH), 7.28 (m, 2H, ArH),
7.13 (dd, = 5.6 2.5 Hz, 1H, ArH), 4.56 (t, = 6.0 Hz, 2H,
–CH2–CH2–N(CH2CH3)2), 2.68 (t, = 5.9 Hz, 2H, –CH2–CH2–N(CH2CH3)2),
2.43 (quartet, = 6.1 Hz, 4H, –CH2–CH2–N(CH2CH3)2), 0.79 (t, = 6.3 Hz,
J =
J
J
J
J
J
J
J
6H, –CH2–CH2–N(CH2CH3)2); 13C NMR (101 MHz, DMSO-d6) δ 165.30,
154.08, 152.99, 151.67, 150.80, 150.20, 147.68, 139.46, 136.41, 134.23,
133.41, 126.69, 126.45, 125.77, 124.61, 123.05, 120.98, 115.37, 112.19,
109.13, 107.28, 53.07, 47.04, 44.42, 12.22; ESI-MS m/z: 558.2 [M+H]+; Anal.
calcd. for C27H27F4N7O2 (%): C, 58.16; H, 4.88; N, 17.59. Found (%): C, 58.22;
H, 4.73; N, 17.31.
4.1.8.12.
1-(4-(2-(4-(2-(Diethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-flu

ACCEPTED MANUSCRIPT
orophenyl)-3-(3,4-difluorophenyl)urea (13l). Flash column chromatography
was performed using (dichloromethane:methanol, 60:1 to 20:1). HPLC purity:
1
99.1%; White solid; Yield: 32%; M.p. 211.3–212.6 °C; H NMR (400 MHz,
DMSO-d6) δ 9.16 (s, 1H, NH), 9.08 (s, 1H, NH), 8.66 (s, 1H, ArH), 8.60 (d,
5.8 Hz, 1H, ArH), 7.73 (dd, = 13.3, 2.2 Hz, 1H, ArH), 7.67 (ddd, = 13.2,
7.6, 2.5 Hz, 1H, ArH), 7.54 (s, 1H, ArH), 7.37 (m, 2H, ArH), 7.29 (d, = 7.6
Hz, 1H, ArH), 7.16 (d, = 8.0 Hz, 2H, ArH), 4.64 (t, = 5.9 Hz, 2H,
–CH2–CH2–N(CH2CH3)2), 2.70 (t, = 6.0 Hz, 2H, –CH2–CH2–N(CH2CH3)2),
0.87 (t, = 6.5 Hz, 6H, –CH2–CH2–N(CH2CH3)2) (The protons of
J =
J
J
J
J
J
J
J
–CH2–CH2–N(CH2CH3)2 overlapped with the peak of the solvent); ESI-MS
m/z: 526.2 [M+H]+; Anal. calcd. for C26H26F3N7O2 (%): C, 59.42; H, 4.99; N,
18.66. Found (%): C, 59.36; H, 4.93; N, 18.30.
4.1.8.13.
1-(3-Chloro-4-fluorophenyl)-3-(4-(2-(4-(2-(diethylamino)ethyl)-4H-1,2,4-triazo
l-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea
(
13m).
Flash
column
chromatography was performed using (dichloromethane:methanol, 80:1 to
25:1). HPLC purity: 99.2%; White solid; Yield: 55%; M.p. 146.5–148.7 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H, NH), 9.34 (s, 1H, NH), 8.62 (s, 1H,
ArH), 8.58 (d,
7.74 (dd, = 13.2, 2.3 Hz, 1H, ArH), 7.52 (d,
9.0 Hz, 1H, ArH), 7.35–7.36 (m, 1H, ArH), 7.33 (m, 1H, ArH), 7.27 (dd,
J
= 5.7 Hz, 1H, ArH), 7.79–7.82 (dd,
J = 7.6, 2.0 Hz, 1H, ArH),
J
J
= 2.4 Hz, 1H, ArH), 7.39 (t,
J
J
=
=
8.8, 1.3 Hz, 1H, ArH), 7.13 (dd,
J
= 5.7, 2.5 Hz, 1H, ArH), 4.55 (t,
J
= 6.2 Hz,
2H,
–CH2–CH2–N(CH2CH3)2),
2.66
(quartet,
(t,
J
J
=
=
6.0
6.9
Hz,
Hz,
2H,
4H,
–CH2–CH2–N(CH2CH3)2),
2.39
–CH2–CH2–N(CH2CH3)2), 0.76 (t,
J = 7.0 Hz, 6H, –CH2–CH2–N(CH2CH3)2);
13C NMR (101 MHz, DMSO-d6) δ 165.31, 154.04, 152.97, 152.95, 151.67,
150.81, 150.22, 147.65, 139.48, 137.26, 134.22, 124.43, 120.14, 119.65,
119.13, 117.39, 115.52, 112.18, 109.21, 107.42, 53.11, 47.06, 44.45, 12.25;
ESI-MS m/z: 542.2 [M+H]+; Anal. calcd. for C26H26ClF2N7O2 (%): C, 57.62;
H, 4.84; N, 18.09. Found (%): C, 58.01; H, 4.67; N, 18.23.
4.1.8.14.
1-(2,4-Dichlorophenyl)-3-(4-(2-(4-(2-(diethylamino)ethyl)-4H-1,2,4-triazol-3-yl
)pyridin-4-yloxy)-3-fluorophenyl)urea (13n). Flash column chromatography
was performed using (dichloromethane:methanol, 100:1 to 30:1). HPLC purity:
1
99.5%; White solid; Yield: 37%; M.p. 139.1–142.1 °C; H NMR (400 MHz,
DMSO-d6) δ 9.87 (s, 1H, NH), 8.62 (s, 1H, NH), 8.59 (d,
8.53 (s, 1H, ArH), 8.18 (d, = 9.0 Hz, 1H, ArH), 7.76 (d,
ArH), 7.64 (d, = 1.9 Hz, 1H, ArH), 7.53 (d, = 1.4 Hz, 1H, ArH), 7.40 (m,
2H, ArH), 7.26 (d, = 9.1 Hz, 1H, ArH), 7.14 (dd, = 5.6, 2.4 Hz, 1H, ArH),
4.56 (t, = 6.0 Hz, 2H, –CH2–CH2–N(CH2CH3)2), 2.68 (t, = 6.1 Hz, 2H,
–CH2–CH2–N(CH2CH3)2), 2.42 (quartet, 5.9 Hz, 4H,
–CH2–CH2–N(CH2CH3)2), 0.78 (t, = 6.7 Hz, 6H, –CH2–CH2–N(CH2CH3)2);
J
= 5.7 Hz, 1H, ArH),
J
J
= 13.3 Hz, 1H,
J
J
J
J
J
J
J
=
J
13C NMR (101 MHz, DMSO-d6) δ 165.30, 154.07, 152.45, 151.70, 150.76,
150.12, 147.67, 139.22, 135.38, 134.35, 129.10, 128.16, 127.00, 124.60,
123.54, 122.93, 115.48, 112.27, 109.09, 107.36, 52.87, 47.10, 29.48, 12.05;
ESI-MS m/z: 558.0 [M+H]+; Anal. calcd. for C26H26Cl2FN7O2 (%): C, 55.92;
H, 4.69; N, 17.56. Found (%): C, 56.11; H, 4.53; N, 17.53.

ACCEPTED MANUSCRIPT
4.1.8.15.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(4-(3-(dimethylamino)propyl)-4
H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13o). Flash column
chromatography was performed using (dichloromethane:methanol, 50:1 to 8:1).
HPLC purity: 99.1%; White solid; Yield: 59%; M.p. 182.6–185.3 °C; 1H NMR
(400 MHz, DMSO-d6) δ 9.63 (s, 1H, NH), 9.53 (s, 1H, NH), 8.66 (s, 1H, ArH),
8.60 (d,
2.2 Hz, 1H, ArH), 7.68 (dd,
ArH), 7.56 (d, = 2.4 Hz, 1H, ArH), 7.40 (t,
J
= 5.7 Hz, 1H, ArH), 8.12 (d,
= 8.8, 2.1 Hz, 1H, ArH), 7.63 (d,
= 8.9 Hz, 1H, ArH), 7.30 (d,
= 5.7, 2.5 Hz, 1H, ArH), 4.52 (t, = 7.2 Hz, 2H,
2.20 (t, 6.7 Hz, 2H,
J
= 2.2 Hz, 1H, ArH), 7.75 (dd,
J = 13.1,
J
J
= 8.8 Hz, 1H,
J
J
J =
8.7 Hz, 1H, ArH), 7.13 (dd,
–CH2–CH2–CH2–N(CH3)2),
J
J
J
=
–CH2–CH2–CH2–N(CH3)2), 2.11 (s, 6H, –CH2–CH2–CH2–N(CH3)2), 1.88
(quintet,
J = 7.0 Hz, 2H, –CH2–CH2–CH2–N(CH3)2); ESI-MS m/z: 578.1
[M+H]+; Anal. calcd. for C26H24ClF4N7O2 (%): C, 54.03; H, 4.19; N, 16.96.
Found (%): C, 54.32; H, 4.22; N, 16.67.
4.1.8.16.
1-(4-(2-(4-(3-(Dimethylamino)propyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)-3-(3-(trifluoromethyl)phenyl)urea
(
13p).
Flash
column
chromatography was performed using (dichloromethane:methanol, 50:1 to
10:1). HPLC purity: 98.8%; White solid; Yield: 29%; M.p. 176.0–178.3 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.92 (s, 2H, NH), 8.65 (s, 1H, ArH), 8.59 (d,
5.7 Hz, 1H, ArH), 8.04 (s, 1H, ArH), 7.77 (d, = 13.2 Hz, 1H, ArH), 7.65 (d,
= 8.1 Hz, 1H, ArH), 7.55 (d, = 2.4 Hz, 1H, ArH), 7.51 (t,
ArH), 7.35 (m, 3H, ArH), 7.12 (dd, = 5.7, 2.5 Hz, 1H, ArH), 4.50 (t,
Hz, 2H, –CH2–CH2–CH2–N(CH3)2), 2.16 (t, = 6.7 Hz, 2H,
–CH2–CH2–CH2–N(CH3)2), 2.08 (s, 6H, –CH2–CH2–CH2–N(CH3)2), 1.85
J
=
J
J
J
J
= 7.9 Hz, 1H,
= 7.2
J
J
J
(quintet,
J = 6.9 Hz, 2H, –CH2–CH2–CH2–N(CH3)2); ESI-MS m/z: 544.2
[M+H]+; Anal. calcd. for C26H25F4N7O2 (%): C, 57.46; H, 4.64; N, 18.04.
Found (%): C, 57.62; H, 4.53; N, 18.38.
4.1.8.17.
1-(4-(2-(4-(3-(Dimethylamino)propyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)-3-(2-(trifluoromethyl)phenyl)urea
(
13q).
Flash
column
chromatography was performed using (dichloromethane:methanol, 30:1 to 7:1).
HPLC purity: 99.7%; White solid; Yield: 50%; M.p. 152.6–159.3 °C; 1H NMR
(400 MHz, DMSO-d6) δ 8.60 (s, 1H, ArH), 8.56 (d,
(d, = 8.2 Hz, 1H, ArH), 7.75 (dd, = 13.4, 2.3 Hz, 1H, ArH), 7.61 (dd,
17.7, 8.2 Hz, 2H, ArH), 7.52 (d, = 2.5 Hz, 1H, ArH), 7.34 (t, = 9.0 Hz, 1H,
ArH), 7.24 (m, 2H, ArH), 7.08 (dd, = 5.7, 2.5 Hz, 1H, ArH), 4.47 (t, = 7.2
Hz, 2H, –CH2–CH2–CH2–N(CH3)2), 2.12 (t, = 6.7 Hz, 2H,
–CH2–CH2–CH2–N(CH3)2), 2.04 (s, 6H, –CH2–CH2–CH2–N(CH3)2), 1.82
J = 5.8 Hz, 1H, ArH), 7.87
J
J
J =
J
J
J
J
J
(quintet,
J = 7.0 Hz, 2H, –CH2–CH2–CH2–N(CH3)2) (Protons of NH was
disappeared); 13C NMR (101 MHz, DMSO-d6) δ 165.30, 154.07, 153.08,
151.78, 150.73, 149.98, 147.35, 139.55, 136.48, 134.18, 133.41, 126.85,
126.47, 124.71, 124.56, 123.05, 121.16, 115.35, 112.24, 109.16, 107.27, 56.20,
45.42, 44.53, 28.64; ESI-MS m/z: 566.1 [M+Na]+; Anal. calcd. for
C26H25F4N7O2 (%): C, 57.46; H, 4.64; N, 18.04. Found (%): C, 57.57; H, 4.33;
N, 17.96.

ACCEPTED MANUSCRIPT
4.1.8.18.
1-(3,4-Difluorophenyl)-3-(4-(2-(4-(3-(dimethylamino)propyl)-4H-1,2,4-triazol-
3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13r). Flash column chromatography
was performed using (dichloromethane:methanol, 50:1 to 10:1). HPLC purity:
1
99.1%; White solid; Yield: 36%; M.p. 177.1–180.2 °C; H NMR (400 MHz,
DMSO-d6) δ 9.36 (s, 1H, NH), 9.28 (s, 1H, NH), 8.65 (s, 1H, ArH), 8.60 (d,
5.7 Hz, 1H, ArH), 7.74 (dd, = 13.2, 1.7 Hz, 1H, ArH), 7.68 (ddd, = 13.2,
7.5, 2.2 Hz, 1H, ArH), 7.55 (d, = 2.1 Hz, 1H, ArH), 7.38 (dd, = 18.0, 9.1
Hz, 2H, ArH), 7.30 (t, = 8.2 Hz, 1H, ArH), 7.17 (d, = 9.0 Hz, 1H, ArH),
7.13 (dd, = 5.6, 2.4 Hz, 1H, ArH), 4.51 (t, = 7.2 Hz, 2H,
–CH2–CH2–CH2–N(CH3)2), 2.16 (t, 6.6 Hz, 2H,
–CH2–CH2–CH2–N(CH3)2), 2.09 (s, 6H, –CH2–CH2–CH2–N(CH3)2), 1.85
J =
J
J
J
J
J
J
J
J
J
=
13
(quintet,
J = 7.2 Hz, 2H, –CH2–CH2–CH2–N(CH3)2); C NMR (101 MHz,
DMSO-d6) δ 165.29, 154.00, 152.87, 151.78, 150.72, 149.97, 147.35, 139.45,
137.00, 134.19, 124.45, 117.87, 115.66, 115.13, 112.28, 109.10, 108.01,
107.80, 107.55, 99.99, 56.14, 45.33, 44.50, 28.54; ESI-MS m/z: 512.1;
[M+H]+; Anal. calcd. for C25H24F3N7O2 (%): C, 58.70; H, 4.73; N, 19.17.
Found (%): C, 59.03; H, 4.59; N, 19.56.
4.1.8.19.
1-(3-Chloro-4-fluorophenyl)-3-(4-(2-(4-(3-(dimethylamino)propyl)-4H-1,2,4-tri
azol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea
(
13s).
Flash
column
chromatography was performed using (dichloromethane:methanol, 60:1 to 8:1).
HPLC purity: 99.5%; White solid; Yield: 52%; M.p. 173.5–176.3 °C; 1H NMR
(400 MHz, DMSO-d6) δ 9.32 (s, 1H, NH), 9.21 (s, 1H, NH), 8.66 (s, 1H, ArH),
8.60 (d,
2.0 Hz, 1H, ArH), 7.55 (d,
= 5.7, 2.5 Hz, 1H, ArH), 4.51 (t,
2.16 (t, = 6.7 Hz, 2H, –CH2–CH2–CH2–N(CH3)2), 2.09 (s, 6H,
–CH2–CH2–CH2–N(CH3)2), 2H,
J
= 5.7 Hz, 1H, ArH), 7.81 (d,
= 2.4 Hz, 1H, ArH), 7.34 (m, 4H, ArH), 7.13 (dd,
= 7.3 Hz, 2H, –CH2–CH2–CH2–N(CH3)2),
J = 7.2 Hz, 1H, ArH), 7.74 (dd, J = 13.2,
J
J
J
J
1.85
(quintet,
J
=
6.8
Hz,
–CH2–CH2–CH2–N(CH3)2); ESI-MS m/z: 528.3; [M+H]+; Anal. calcd. for
C25H24ClF2N7O2 (%): C, 56.87; H, 4.58; N, 18.57. Found (%): C, 58.63; H,
4.70; N, 18.30.
4.1.8.20.
1-(2,4-Dichlorophenyl)-3-(4-(2-(4-(3-(dimethylamino)propyl)-4H-1,2,4-triazol-
3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13t). Flash column chromatography
was performed using (dichloromethane:methanol, 30:1 to 7:1). HPLC purity:
1
99.2%; White solid; Yield: 43%; M.p. 181.9–184.2 °C; H NMR (400 MHz,
DMSO-d6) δ 9.79 (s, 1H, NH), 8.65 (s, 1H, NH), 8.61 (d,
8.50 (s, 1H, ArH), 8.18 (d, = 9.0 Hz, 1H, ArH), 7.76 (dd,
1H, ArH), 7.64 (d, = 2.4 Hz, 1H, ArH), 7.55 (d, = 2.5 Hz, 1H, ArH), 7.41
(m, 2H, ArH), 7.26 (d, = 8.9 Hz, 1H, ArH), 7.14 (dd, = 5.7, 2.6 Hz, 1H,
ArH), 4.52 (t, = 7.2 Hz, 2H, –CH2–CH2–CH2–N(CH3)2), 2.28 (t, = 6.7 Hz,
2H, –CH2–CH2–CH2–N(CH3)2), 2.17 (s, 6H, –CH2–CH2–CH2–N(CH3)2), 1.89
J
= 5.7 Hz, 1H, ArH),
J
J
= 13.2, 2.4 Hz,
J
J
J
J
J
J
13
(quintet,
J = 6.9 Hz, 2H, –CH2–CH2–CH2–N(CH3)2); C NMR (101 MHz,
DMSO-d6) δ 165.27, 154.08, 152.48, 151.79, 150.72, 149.98, 147.36, 139.24,
135.41, 134.35, 129.10, 128.15, 127.03, 124.60, 123.63, 123.03, 115.48,
112.28, 109.12, 107.39, 56.20, 45.42, 44.54, 28.64; ESI-MS m/z: 544.0

ACCEPTED MANUSCRIPT
[M+H]+; Anal. calcd. for C25H24Cl2FN7O2 (%): C, 55.15; H, 4.44; N, 18.01.
Found (%): C, 55.07; H, 4.52; N, 18.34.
4.1.8.21.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-((2-(4-(3-morpholinoprop
yl)-4H-1,2,4-triazol-3-yl)pyridin-4-yl)oxy)phenyl)urea (13u). Flash column
chromatography was performed using (dichloromethane:methanol, 60:1 to
20:1). HPLC purity: 99.3%; White solid; Yield: 38%; M.p. 150.5–152.9 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H, NH), 9.60 (s, 1H, NH), 8.66 (s, 1H,
ArH), 8.58 (d,
= 13.1, 1.5 Hz, 1H, ArH), 7.68 (dd,
Hz, 1H, ArH), 7.54 (d, = 2.0 Hz, 1H, ArH), 7.39 (t,
7.30 (d, = 8.7 Hz, 1H, ArH), 7.12 (dd, = 5.5, 2.3 Hz, 1H, ArH), 4.54 (t,
6.9 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)), 3.50 (br, 4H, morpholinyl),
J
= 5.7 Hz, 1H, ArH), 8.11 (d,
J
= 1.4 Hz, 1H, ArH), 7.74 (dd,
J
J
= 8.6, 0.9 Hz, 1H, ArH), 7.62 (d,
J
= 8.7
J
J = 8.9 Hz, 1H, ArH),
J
J
J =
2.19–2.25 (m, 6H, –CH2–CH2–CH2–(C4H8NO), morpholinyl), 1.89 (quintet, J =
7.1 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)); ESI-MS m/z: 642.1 [M+Na]+; Anal.
calcd. for C28H26ClF4N7O3 (%): C, 54.24; H, 4.23; N, 15.81. Found (%): C,
54.30; H, 4.16; N, 16.06.
4.1.8.22.
1-(3-Fluoro-4-((2-(4-(3-morpholinopropyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yl)o
xy)phenyl)-3-(2-(trifluoromethyl)phenyl)urea
(
13v).
Flash
column
chromatography was performed using (dichloromethane:methanol, 100:1 to
20:1). HPLC purity: 98.9%; White solid; Yield: 51%; M.p. 138.1–140.2 °C; 1H
NMR (300 MHz, DMSO-d6) δ 9.77 (s, 1H, NH), 8.63 (s, 1H, NH), 8.54 (d,
5.8 Hz, 1H, ArH), 8.21 (s, 1H, ArH), 7.88 (d, = 8.2 Hz, 1H, ArH), 7.72 (dd,
= 13.2, 2.2 Hz, 1H, ArH), 7.64 (dd, = 16.3, 8.0 Hz, 2H, ArH), 7.51 (d,
Hz, 1H, ArH), 7.36 (t, = 9.0 Hz, 1H, ArH), 7.28 (t, = 7.7 Hz, 1H, ArH), 7.22
(dd, = 8.9, 1.4 Hz, 1H, ArH), 7.10 (dd, = 5.6, 2.5 Hz, 1H, ArH), 4.51 (t,
7.0 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)), 3.47 (br, 4H, morpholinyl), 2.22 (m,
J
=
J
J
J
J = 2.4
J
J
J
J
J =
6H, –CH2–CH2–CH2–(C4H8NO), morpholinyl), 1.87 (quintet,
J = 6.8 Hz, 2H,
–CH2–CH2–CH2–(C4H8NO)); 13C NMR (101 MHz, DMSO-d6) δ 165.30,
154.08, 153.02, 151.73, 150.79, 150.04, 147.37, 139.49, 136.41, 134.20,
133.42, 126.76, 126.45, 125.76, 124.63, 123.05, 121.04, 115.36, 112.26,
109.18, 107.26, 66.55, 55.14, 53.51, 44.38, 27.30; ESI-MS m/z: 608.1
[M+Na]+; Anal. calcd. for C28H27F4N7O3 (%): C, 57.43; H, 4.65; N, 16.74.
Found (%): C, 57.50; H, 4.42; N, 16.67.
4.1.8.23.
1-(3-Chloro-4-fluorophenyl)-3-(3-fluoro-4-((2-(4-(3-morpholinopropyl)-4H-1,2
,4-triazol-3-yl)pyridin-4-yl)oxy)phenyl)urea
(
13w).
Flash
column
chromatography was performed using (dichloromethane:methanol, 60:1 to
20:1). HPLC purity: 99.2%; White solid; Yield: 47%; M.p. 162.6–165.2 °C; 1H
NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H, NH), 9.32 (s, 1H, NH), 8.65 (s, 1H,
ArH), 8.56 (d,
(dd, = 13.1, 1.9 Hz, 1H, ArH), 7.52 (d,
3H, ArH), 7.24 (dd, = 8.5, 0.7 Hz, 1H, ArH), 7.12 (dd,
ArH), 4.52 (t, = 6.9 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)), 3.49 (br, 4H,
morpholinyl), 2.23 (m, 6H, –CH2–CH2–CH2–(C4H8NO), morpholinyl), 1.87
J
= 5.7 Hz, 1H, ArH), 7.79 (dd,
= 1.7 Hz, 1H, ArH), 7.30–7.40 (m,
= 5.5, 2.4 Hz, 1H,
J = 6.1, 1.3 Hz, 1H, ArH), 7.72
J
J
J
J
J

ACCEPTED MANUSCRIPT
J = 7.0 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)); C NMR (101 MHz,
13
(quintet,
DMSO-d6) 165.30, 154.14, 154.03, 152.94, 151.74, 150.78, 150.02, 147.37,
139.49, 137.24, 134.14, 124.49, 120.08, 119.65, 119.10, 117.42, 115.50,
112.25, 109.17, 107.38, 66.50, 55.09, 53.47, 44.36, 27.25; ESI-MS m/z: 569.8
[M+H]+; Anal. calcd. for C28H27F4N7O3 (%): C, 56.89; H, 4.60; N, 17.20.
Found (%): C, 56.95; H, 4.55; N, 17.32.
4.1.8.24.
1-(2,4-dichlorophenyl)-3-(3-fluoro-4-((2-(4-(3-morpholinopropyl)-4H-1,2,4-tri
azol-3-yl)pyridin-4-yl)oxy)phenyl)urea (13x). Flash column chromatography
was performed using (dichloromethane:methanol, 100:1 to 30:1). HPLC purity:
1
99.4%; White solid; Yield: 66%; M.p. 156.8–160.2 °C; H NMR (400 MHz,
DMSO-d6) δ 9.97 (s, 1H, NH), 8.65 (s, 1H, NH), 8.55–8.58 (m, 2H, ArH), 8.16
(d,
Hz, 1H, ArH), 7.52 (d,
2H, ArH), 7.24 (dd, = 8.8, 1.0 Hz, 1H, ArH), 7.13 (dd,
ArH), 4.53 (t,
J
= 9.0 Hz, 1H, ArH), 7.75 (dd,
J = 13.2, 2.3 Hz, 1H, ArH), 7.63 (d, J = 2.4
J
= 2.3 Hz, 1H, ArH), 7.39 (ddd,
J
J
= 8.9, 5.7, 3.1 Hz,
= 5.7, 2.5 Hz, 1H,
J
J
= 7.0 Hz, 2H, –CH2–CH2–CH2–(C4H8NO)), 3.49 (br, 4H,
morpholinyl), 2.23 (m, 6H, –CH2–CH2–CH2–(C4H8NO), morpholinyl), 1.87
(br, 2H, –CH2–CH2–CH2–(C4H8NO)); 13C NMR (101 MHz, DMSO-d6) δ
165.29, 154.08, 152.47, 151.78, 150.73, 149.97, 147.35, 139.20, 135.39,
134.33, 129.11, 128.15, 127.01, 124.61, 123.58, 122.97, 115.49, 112.32,
109.12, 107.36, 66.14, 53.20, 44.25; ESI-MS m/z: 586.0 [M+H]+; Anal. calcd.
for C28H27F4N7O3 (%): C, 55.30; H, 4.47; N, 16.72. Found (%): C, 55.22; H,
4.68; N, 16.65.
4.1.8.25.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(2-(4-cyclopropyl-4H-1,2,4-triazol
-3-yl)pyridin-4-yloxy)-3-fluorophenyl)urea (13y). Flash column chromatography
was performed using (dichloromethane:methanol, 100:1 to 25:1). HPLC purity:
1
98.5%; White solid; Yield: 58%; M.p. 171.1–173.5 °C; H NMR (400 MHz,
DMSO-d6) δ 10.47 (s, 1H, NH), 10.33 (s, 1H, NH), 8.66 (s, 1H, ArH), 8.63 (d,
J
= 5.6 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 7.76 (dd,
7.65 (m, 2H, ArH), 7.50 (d, = 1.9 Hz, 1H, ArH), 7.40 (t,
ArH), 7.26 (d, = 8.0 Hz, 1H, ArH), 7.12 (dd, = 5.5, 2.2 Hz, 1H, ArH), 3.51
J
= 13.0, 1.9 Hz, 1H, ArH),
J
J
= 9.0 Hz, 1H,
J
J
(br, 1H, cyclopropyl), 0.96 (m, 2H, cyclopropyl), 0.84 (m, 2H, cyclopropyl);
ESI-MS m/z: 533.3 [M+H]+; Anal. calcd. for C24H17ClF4N6O2 (%): C, 54.09; H,
3.22; N, 15.77. Found (%): C, 53.89; H, 3.37; N, 15.65.
4.2. Pharmacology
4.2.1. MTT assay
MTT assay was carried out on H460, HT-29, MDA-MB-231, and WI-38
cells to evaluate the cytotoxicity of the newly synthesized compounds. The
cancer cell lines were cultured in minimum essential medium (MEM)
supplement with 10% foetal bovine serum (FBS).
Approximate 4 × 103 cells, suspended in MEM medium, were plated in a
96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The tested
compounds were added to the culture medium and the cell cultures were
incubated for another 72 h. Fresh MTT was added to each well at a terminal

ACCEPTED MANUSCRIPT
concentration of 5 ꢀg/mL, and incubated with cells at 37 °C for 4 h. The
formazan crystals in each well were dissolved in 100 ꢀL DMSO, and the
absorbency at 492 nm (absorbance of MTT formazan) and 630 nm (reference
wavelength) was measured with an ELISA reader. All of the compounds were
tested three times in each cell line. The results, expressed as inhibitory
concentration 50 % (IC50), are averages of at least three determinations and
calculated using the Bacus Laboratories Incorporated Slide Scanner (Bliss)
software.
4.2.2. Enzymatic activity assay
The tested compounds were diluted to 50-fold of the final highest desired
concentration by 100 % DMSO. Each well of a 96-well plate received 5 ꢀL of
compound and 95 ꢀL of 100 % DMSO. Two wells received only 100 ꢀL of
100% DMSO and served as negative controls (no compound and no enzyme).
This plate was labelled the “source plate.” Subsequently, 10 ꢀL of compound
from the “source plate” was transferred to a new 96-well plate named the
“intermediate plate,” and 90 ꢀL of kinase buffer was added. Then, 5 ꢀL from
each well of the “intermediate plate” was transferred in duplicate to a 384-well
plate. To each well, fresh enzyme and peptide solutions were added
successively. The mixture was incubated at 28 °C for 1 h, then 25 ꢀL of stop
buffer was added. The biological data was collected from Caliper program.
4.2.3. Apoptosis analysis
HT-29 cells (5×105 cells/mL) were seeded in six-well plates and treated
with compound 13i at different concentrations for 48 h. The cells were then
harvested by trypsinization and washed twice with cold PBS. After
centrifugation and removal of the supernatants, cells were resuspended in 400
ꢀL of 1 × binding buffer which was then added to 5 ꢀL of annexinV-FITC and
incubated at room temperature for 15 min. After adding 10 ꢀL of PI the cells
were incubated at room temperature for another 15 min in the dark. The stained
cells were analyzed by a flow cytometer (BD Accuri C6).
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative Research Team in
University. Also, we were supported by grants from the National Natural
Science Foundation of China (81502924), and PhD research start-up foundation
of Liaoning Province (201501052).
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ACCEPTED MANUSCRIPT
Legends
Figure 1. Design strategy of the target compounds.
Figure 2. Effect of compound 13i on cell apoptosis in HT-29
cells. FL1-H: AnnexinV-FITC; FL2-H: PI.
Scheme 1. Reagents and conditions: (a) triphosgene, toluene, reflux, 7–10 h; (b)
(i) SOCl2, NaBr, chlorobenzene, 50 °C, 30 min, 85 °C, 20 h; (ii) MeOH,
toluene, 0–15 °C, 1.5 h; (c) 2-fluoro-4-nitrophenol, chlorobenzene, reflux, 12 h;
(d) NH3·H2O, acetone, 50 °C, 3 h; (e) DMF-DMA, CH2Cl2, reflux, 2–3 h; (f)
CH3NHNH2, HOAC, 90 °C, 3 h; (g) NH2NH2·H2O, EtOH, reflux, 3–6h; (h)
1a–b, CH2Cl2, rt, 5 h.
Scheme 2 Reagents and conditions: (a) Pd/C, EtOH, rt, 4 h; (b) 1a–f, CH2Cl2, rt,
4–7 h; (c) NH2NH2·H2O, MeCN, reflux, 3–5 h; (d) DMF-DMA, CH2Cl2, reflux,
4–6 h; (e) appropriate amine, HOAc, 90 °C, 3h.
Table 1. Cellular antiproliferative activities of compounds 8a
13b
, 8b, 13a, and
.
Table 2. Cellular proliferative activities of compounds 13a–y.
Table 3. Enzyme activities of compounds 13c–u and 13x.

ACCEPTED MANUSCRIPT
Table 1. Cellular antiproliferative activities of compounds 8a, 8b, 13a, and 13b.
IC50 (µM)a
Compd
R2
HT-29
H460
MDA-MB-231
WI-38
8a
8b
3-CF3-4-Cl
3-CF3
12.37 ± 2.38
NA
NA
NA
NA
11.62 ± 1.36
2.31 ± 0.15
1.27 ± 0.22
2.25 ± 0.42
9.68 ± 0.52
5.32 ± 0.06
6.47 ± 0.37
3.08 ± 0.50
22.37 ± 2.31
9.85 ± 1.37
12.62 ± 1.03
8.42 ± 0.76
13a
3-CF3-4-Cl
3-CF3
2.57 ± 0.31
3.02 ± 0.08
3.37 ± 0.38
13b
sorafenib
aThe biological data are generated from at least three independent experiments.
NA: compound showing IC50 value > 50 µM.

ACCEPTED MANUSCRIPT
Table 2. Cellular proliferative activities of compounds 13a–y.
IC50 (µM)a
Compd
R1
R2
ClogDb
HT-29
H460
MDA-MB-231
WI-38
13a
13b
CH3
CH3
3-CF3-4-Cl
3-CF3
2.57 ± 0.31
3.02 ± 0.08
2.31 ± 0.15
1.27 ± 0.22
5.32 ± 0.06
6.47 ± 0.37
9.85 ± 1.37
4.58
3.97
12.62 ± 1.03
13c
3-CF3-4-Cl
0.88 ± 0.05
1.15 ± 0.10
1.07 ± 0.03
3.76 ± 0.88
2.99
T-1
T-1
T-1
T-1
T-1
T-1
13d
13e
13f
3-CF3
2-CF3
1.47 ± 0.06
5.86 ± 1.27
3.62 ± 0.66
0.72 ± 0.11
1.89 ± 0.32
2.15 ± 0.33
NA
16.35 ± 3.28
NA
11.22 ± 2.37
NA
2.38
2.38
1.79
2.25
2.71
3,4-(F)2
3-Cl-4-F
2-4-(Cl)2
9.05 ± 2.28
1.58 ± 0.55
4.35 ± 0.62
NA
NA
13g
13h
8.32 ± 1.43
7.96 ± 1.25
7.58 ± 1.29
10.36 ± 0.67
13i
3-CF3-4-Cl
0.90 ± 0.26
0.85 ± 0.32
1.54 ± 0.35
6.73 ± 0.79
3.18
T-2
T-2
T-2
T-2
T-2
T-2
13j
13k
13l
3-CF3
2-CF3
0.63 ± 0.03
3.41 ± 0.78
1.24 ± 0.53
0.50 ± 0.03
3.59 ± 0.76
9.87 ± 1.75
NA
0.72 ± 0.16
11.56 ± 2.58
41.32 ± 3.73
34.00 ± 3.68
1.79 ± 0.33
6.92 ± 1.34
23.52 ± 0.68
NA
2.57
2.57
1.98
2.44
2.90
3,4-(F)2
3-Cl-4-F
2-4-(Cl)2
14.85 ± 2.35
7.39 ± 1.30
5.38 ± 1.07
13m
13n
28.66 ± 3.53
4.28 ± 0.63
13o
3-CF3-4-Cl
1.40 ± 0.26
1.68 ± 0.07
7.88 ± 0.72
7.91 ± 1.06
2.42
T-3
T-3
T-3
T-3
T-3
T-3
13p
13q
13r
13s
13t
3-CF3
2-CF3
0.98 ± 0.19
3.13 ± 1.08
1.21 ± 0.37
0.89 ± 0.12
0.68 ± 0.09
1.79 ± 0.45
10.86 ± 1.66
1.08 ± 0.29
1.27 ± 0.06
3.68 ± 0.37
13.33 ± 2.38
40.52 ± 3.76
25.25 ± 1.96
14.15 ± 1.28
7.49 ± 1.65
11.55 ± 2.03
28.62 ± 2.37
15.32 ± 1.56
5.37 ± 0.45
3.26 ± 0.68
1.81
1.81
1.22
1.68
2.14
3,4-(F)2
3-Cl-4-F
2-4-(Cl)2
13u
3-CF3-4-Cl
2.86 ± 0.33
3.23 ± 0.17
8.28 ± 0.69
9.65 ± 1.33
4.09
T-4
T-4
T-4
13v
2-CF3
15.70 ± 1.72
NA
ND
ND
NA
ND
ND
3.49
3.36
13w
3-Cl-4-F
16.23 ± 2.28

ACCEPTED MANUSCRIPT
13x
13y
T-4
2-4-(Cl)2
3.56 ± 0.35
10.90 ± 2.32
3.37 ± 0.38
ND
NA
NA
ND
ND
3.82
5.04
4.34
3-CF3-4-Cl
NA
sorafenib
2.25 ± 0.42
3.08 ± 0.50
8.42 ± 0.76
aThe biological data are generated from at least three independent experiments.
bClogD values, predicted at pH = 7.4, calculated using JChem_For_Excel_6.0.0.865.
NA: compound showing IC50 value > 50 µM.
ND: not determined.

ACCEPTED MANUSCRIPT
Table 3. Enzyme activities of compounds 13c–u and 13x.
% inhibition at 10 µMa
Compd
c-Kit
RET
FLT3
B-Raf
VEGFR-2
13c
13d
13e
70.4
60.5
22.7
3.9
74.1
77.7
43.0
17.6
63.3
ND
66.3
42.2
17.6
13.5
52.2
ND
15.7
6.7
0
0
2.7
2.2
13f
ND
0
ND
26.9
6.0
13g
13h
13i
74.0
ND
0
79.9
66.5
ND
84.4
72.1
ND
84.9
61.0
ND
14.7
3.3
ND
1.2
0
10.0
11.5
ND
0
13j
13k
13l
27.7
40.4
ND
7.1
39.0
59.6
ND
13m
13n
13o
13p
13q
13r
1.9
2.7
ND
ND
0
ND
10.9
62.8
1.7
69.9
69.9
ND
79.3
85.6
ND
86.9
69.2
ND
11.8
24.8
11.7
27.4
0
63.1
71.0
ND
30.9
45.5
ND
44.3
72.1
ND
20.3
35.0
11.3
0
13s
13t
13u
13x
sorafenib
56.7
43.5
95.6
42.3
45.6
97.0
38.5
22.0
98.5
7.6
ND
100
ND
96.8
aThe biological data are means from at least two replicated experiments.
ND: not determined.

ACCEPTED MANUSCRIPT