Synthesis and p38 inhibitory activity of some novel substituted N,N′-diarylurea derivatives

Article abstract of DOI:10.3390/molecules21050677

We have identified a novel series of substituted N,N′-diarylurea p38α inhibitors. The inhibitory activity of the target compounds against the enzyme p38α, MAPKAPK2 in BHK cells, TNF-α release in LPS-stimulated THP-1 cells and p38α binding experiments were

Full text of DOI:10.3390/molecules21050677

molecules
Article
Synthesis and p38 Inhibitory Activity of Some Novel
Substituted N,N1-Diarylurea Derivatives
Dianxi Zhu 1,2, Qifeng Xing 2, Ruiyuan Cao 2, Dongmei Zhao 1,* and Wu Zhong 2,
*
1
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, China; zhudianxi@126.com
Laboratory of Computer-Aided Drug Design and Discovery, Beijing Institute of Pharmacology
and Toxicology, 27 Taiping Rd., Beijing 100850, China; xingqf5522@163.com (Q.X.); 21cc@163.com (R.C.)
Correspondence: dongmeiz-67@163.com (D.Z.); zhongwu@bmi.ac.cn (W.Z.);
2
*
Tel.: +86-24-2398-6413 (D.Z.); +86-135-0133-4184 (W.Z.)
Academic Editor: James W. Leahy
Received: 11 April 2016; Accepted: 12 May 2016; Published: 23 May 2016
Abstract: We have identified a novel series of substituted N,N1-diarylurea p38
α inhibitors. The
inhibitory activity of the target compounds against the enzyme p38
TNF- release in LPS-stimulated THP-1 cells and p38 binding experiments were tested. Among
these compounds, 25a inhibited the p38 enzyme with an IC50 value of 0.47 nM and a KD value of
α, MAPKAPK2 in BHK cells,
α
α
α
1.54 ˆ 10´8 and appears to be the most promising one in the series.
Keywords: P38 MAPK; BIRB-796; SPR; TNF-α
1. Introduction
The mitogen-activated protein kinase p38 (p38 MAPK) belongs to the serine/threonine
kinase family and plays a major role in the regulation, biosynthesis and actions of key
proinflammatory mediators like tumour necrosis factor-alpha (TNF-
and mitogen-activated protein-kinase-activated protein kinase 2 (MAPKAPK2), etc. [
to the literature, there are four known distinct subunits of p38 MAPK (p38 and
investigations have indicated that p38 plays an important role in serious diseases such as chronic
obstructive pulmonary disease (COPD), rheumatoid arthritis (RA), psoriasis, and Crohn’s disease [ ].
In recent years, it has been shown that other diseases such as cancer, neuropathic pain, periodontal
diseases and lung inflammation caused by acute gas may also be treated and prevented using p38
inhibitors [ ]. However, although some select candidates are in clinical trials, including losmapimod
in phase III for treatment of acute coronary syndrome, LY2228820 in phase II for epithelial ovarian
cancer and PH-797804 in phase III for treatment of COPD [ 12], no p38 inhibitors are currently
α
), interleukin-1
β
(IL-1
]. According
). Previous
β), p53,
1
α
,
β,
γ
δ
α
2,3
α
3–8
9–
α
available on the market. Therefore, there is continuing need to identify novel bioavailable small
molecule inhibitors of p38α.
Based on their mode of action, p38
inhibitors (e.g., SB-203580) and non-competitive inhibitors (e.g., BIRB-796) that trap the p38
Asp-Phe-Gly-out (DFG-out) conformation (shown in Figure 1) [13 15]. ATP-competitive inhibitors
α
inhibitors can be divided into two categories: ATP-competitive
α
in an
–
use the ATP binding pocket and inhibit the kinase by directly competing with the binding of ATP [16].
Non-competitive inhibitors typically make use of the ATP binding site, but they also form unique
hydrogen bonding and hydrophobic interactions with p38
α. The non-competitive compounds inhibit
p38 by maintaining a kinase conformation that is not suitable for ATP binding [17]. Compared with
α
ATP competitive inhibitors, non-competitive inhibitors do not compete directly with the binding of ATP,
which could provide a kinetic advantage. In addition, the amino acid residues of the allosteric pocket
Molecules 2016, 21, 677; doi:10.3390/molecules21050677
www.mdpi.com/journal/molecules

Molecules 2016, 21, 677
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have very low conservation compared with that of the ATP binding region, therefore, non-competitive
inhibitors usually present higher kinase selectivity than ATP competitive inhibitors [18].
Figure 1. Structures of SB203580 and BIRB-796.
BIRB-796 is a classical allosteric p38 inhibitor with a N,N1-diarylurea scaffold. According to
the X-ray single crystal structure of p38
conformation by forming several hydrogen bonding and hydrophobic interactions [19]. TNF-
secretion assays showed that this material inhibits LPS-induced TNF- production with IC50 values of
α/BIRB-796, BIRB-796 matched very well with the DFG-out
α
α
18 nM and 780 nM for THP-1 cells in human whole blood and in mice in vivo, respectively (by 84%,
30 mg/kg p.o.) [20]. The major clinical problem of BIRB-796 is its hepatotoxicity, and the literature
has suggested that a naphthalene epoxide intermediate might be the cause. Therefore, some structure
modification and structure–activity relationship (SAR) investigations of BIRB-796 were carried out in
order to discover novel p38α inhibitors.
According to the SAR of BIRB-796, the “urea linkage” occupied a hydrogen-bonding site and
is important to maintain the activity [21]. Thus, this “urea linkage” is preserved in our compounds.
The naphthyl ring of BIRB-796 pushes quite deeply into the hydrophobic gatekeeper pocket and
hydrophobic interactions play a key role in ensuring high activity and selectivity for p38α inhibitors [22].
In order to increase the activity and selectivity and reduce or even eliminate the hepatotoxicity, the
naphthalene ring was replaced with other aromatic hydrophobic scaffolds, such as phenyl, benzyl
or chromene moieties, which allowed for synthetic flexibility and these structural modifications are
also the correct size for the small gatekeeper. According to the SAR information for kinase inhibitors,
a variety of functional groups are well tolerated by this adenine binding site, and introducing some
rigid groups in the site could improve p38α inhibitory activity according to the literature [23,24].
We therefore attempted to either replace this morpholinoethoxy moiety with an indazole moiety
or replace the morpholinoethoxy ring with a naphthalenol with the overall aim of increasing the
binding affinity. BIRB-796 also has a unique allosteric pocket that is created when the activation loop
adopts the “DFG-out” conformation. Two selectivity sites could be discovered in the allosteric pocket:
one is occupied by the t-butyl group, and the other by the p-tolyl group. It has been reported that
the t-butyl group offers a unique interaction with p38α that guarantees the inhibitory activity and
kinase selectivity [21]. According to the above information, the t-butyl group was retained in our
compounds, and we tried to replace the 4-tolyl group with other substituted phenyls to investigate
the SAR surrounding the phenyl ring unit. According to the information mentioned above, we now
report the design and synthesis of some series of new urea derivatives that were then evaluated for
their inhibitory activities against MAPKAPK2, TNF-α, and p38α.
2. Results and Discussion
2.1. Chemistry
The synthetic methods used to prepare our target compounds are summarized in Schemes 1–6. Firstly,
compounds 2a
in the presence of diluted hydrochloric acid. Treating 2a
in THF furnished intermediates 3a 3i (Scheme 1), while the intermediates 7a
–
2i were synthesized using substituted phenylhydrazines 1a
2i with 2,2,2-trichloro-ethyl carbonochloridate
7b were synthesized from
–1i and pivaloylacetonitrile
–
–
–

Molecules 2016, 21, 677
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substituted 2-fluorobenzonitriles 4a
and 4-amino-3-methylphenol in the presence of K2CO3. Intermediates 5a
–
4b (Scheme 1). The intermediates 5a
–
5b were prepared from 4a
5b were cyclized with isoamyl
nitrite in the presence of potassium acetate and acetic anhydride to generate 6a 6b. Finally, intermediates
7a–7b were synthesized from 6a–6b using LiAlH4 in THF at room temperature or H2 and Pd/C.
–4b
–
–
Scheme 1. Synthesis of intermediates 2a–2
pivaloylacetonitrile, HCl, 80 ˝C, 6–8 h; ( ) THF, 2,2,2-trichloroethyl carbonochloridate, NaHCO3, 0 ˝C,
) toluene, acetic anhydride, potassium
i, 3a–3i, and 4–7(a,b). Reagents and conditions: (a) ethanol,
b
12 h; (c d
) DMSO, 4-amino-3-methylphenol, K2CO3, 90 ˝C, 4 h; (
acetate, isopentyl nitrite, 80 ˝C, 8 h; (e) THF, LiAlH4, 65 ˝C, 30–40 min.
The chromene intermediate 14 was prepared from 2-nitro-5-fluorophenol (
compound 8 with propargyl bromide in the presence of potassium carbonate gave compound 9
8
, Scheme 2). Treating
, which
upon treatment with 4-amino-3-methylphenol in the presence of K2CO3 generated the intermediate 10
.
Compound 10 was cyclized with isoamyl nitrite in the presence of potassium acetate and acetic anhydride
to generate 11. The intermediate 12 was synthesized by reduction with SnCl2 in EtOH or by catalytic
hydrogenation under catalysis of Pd/C followed by (Boc)2O protection to obtain the compound 13.
Scheme 2. Syntheiss of compound 14. Reagents and conditions: (
12 h; (
) DMSO, 4-amino-3-methylphenol, K2CO3, 90 ˝C, 4 h; (
acetate, isopentyl nitrite, 80 ˝C, 8 h; ( ) ethanol, SnCl2, 80 ˝C, 2 h; (
(f) diphenyl ether, microwave, 220 ˝C, 25 min.
a
c
) DMF, 3-bromoprop-1-yne, K2CO3, rt,
) toluene, acetic anhydride, potassium
) DCM, (BOC)2O, Et3N, 0 ˝C, 48 h;
b
d
e

Molecules 2016, 21, 677
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Finally, the chromene compound 14 was cyclized using microwave irradiation under the following
˝
conditions: temperature: 220 C, pressure: 8.0 bar, time: 30 min, power: 250 W [25].
The morpholine intermediate 16 was prepared from 2-fluorobenzonitrile (4a, Scheme 3). Treating
4a with 2-morpholinoethanol in the presence of sodium hydride generated the intermediate 15. Finally,
the intermediate 16 was prepared using LiAlH4 in THF at room temperature [26].
Scheme 3. Synthesis of compound 16. Reagents and conditions: (
hydride, ´35 ˝C, 12 h; (b) THF, LiAlH4, 65 ˝C, 30–40 min.
a) DMF, 2-morpholinoethanol, sodium
The key intermediates 18a
2-fluorobenzonitriles 4a 4b by treatment with 6-hydroxy-1-tetralone to furnish the intermediates
17a–17b, followed by LiAlH4 reduction (Scheme 4).
–18b were synthesized from the corresponding substituted
–
Scheme 4. Synthesis of 18
(b) THF, LiAlH4, 65 ˝C, 30–40 min.
(a,b). Reagents and conditions: (a
) DMSO, 6-hydroxy-1-tetralone, 90 ˝C, 4 h;
Scheme 5. Synthesis of compounds 21 and 24. Reagents˝and conditions: (
a) DMF, 2-morpholinoethanol,
) DMSO, 4-amino-3-methylphenol,
˝
sodium hydride,
´
35 C, 12 h; (
b
) ethanol, SnCl2, 80 C, 2 h; (
c
K2CO3, 90 ˝C, 4 h; (
d
) toluene, acetic anhydride, potassium acetate, isopentyl nitrite, 80 ˝C, 8 h;
(e) ethanol, SnCl2, 80 ˝C, 2 h.

Molecules 2016, 21, 677
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The morpholine intermediate 21 was prepared from 1-fluoro-2-nitrobenzene (19, Scheme 5).
Treating 19 with 2-morpholinoethanol in the presence of sodium hydride generated the intermediate
20, which was reduced with LiAlH4 in THF to give the target compound.
H
N
N
O
O
7a-7b
a
N
N
N
N
H
H
R2
O
R1
25a-k
O
N
N
N
N
H
N
H
N
14
a
H
O
R1
26a-f
O
N
O
O
N
N
N
H
16
a
O
N
H
Cl
Cl
Cl
N
N
O
N
H
R1
OH
30a-d
O
R1
3a-3i
O
N
18a-18b
a
N
N
H
N
H
R2
R1
27a-e
O
N
N
N
H
21
a
N
H
O
N
O
R1
28a-i
O
N
N
N
H
24
a
N
H
O
N
N
H
R1
29a-i
Scheme 6. Synthesis of the target compounds 25a
–
25k
,
26a
–26f
,
27a
–27e
,
28a–
28i, 29a–291 and 30a–30d.
Reagents and conditions: (a) DMSO, Et3N, 90 ˝C, 2 h.
Another indazole intermediate 24 was also prepared from 1-fluoro-2-nitrobenzene (19, Scheme 5)
by treating 19 with 4-amino-3-methylphenol in the presence of K2CO3 to generate the intermediate 22
,
which was cyclized with isoamyl nitrite in the presence of potassium acetate and acetic anhydride to
generate 23. Finally, the intermediate 24 was prepared by LiAlH4 reduction of 23 in THF.
The products of interest 25a
in quite good yields by the reaction of 3a
the presence of DIEA in DMSO via a microwave-assisted method under the following conditions:
–
25k
,
26a
–
26f
,
27a
–
27e
,
28a
–
28i
,
29a
–
29i
,
30a
–
30d were obtained
–
3i with the intermediates
7,
14 16
,
,
18, 21 and 24 in

Molecules 2016, 21, 677
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temperature: 55–100 ˝C; power: 60–100 W; pressure: 8.0 bar; time: 10–20 min [27
,28]. We chose the
microwave-assisted pathway as a new method because it is easier and quicker and because yields are
improved to a certain degree.
2.2. Biology
2.2.1. MAPKAPK2 Inhibitory Activity
In order to study p38α inhibition in a cell system, we investigated the ability of all compounds
to inhibit the phosphorylation of mitogen-activated protein-kinase-activated protein kinase 2
(MAPKAPK2). Each compound was prepared at seven concentrations (1, 3, 10 and 30 nM, 0.1 and
0.3 µM), and the percentages of inhibition were calculated from the numbers of cells after treatment
with the different compounds. The results are displayed in Table 1.
Table 1. MAPKAPK2, p38α and TNF-α activity data for small molecule inhibitors.
Compounds
R1
R2
MAPKAPK2 IC50 (nM)
P38α IC50 (nM)
TNF-α IC50 (nM)
BIRB-796
SB203580
25a
25b
25c
25d
25e
25f
—
—
4-methyl
H
18.01
n.d.
13.74
6.96 ˘ 1.14
0.47 ˘ 0.02
0.80 ˘ 0.03
0.77 ˘ 0.08
1.22 ˘ 0.32
n.d.
n.d.a
800
—
H
1.36 ˘ 0.31
2.45 ˘ 0.59
6.60 ˘ 0.66
5.45 ˘ 0.43
9.92 ˘ 0.65
7.40 ˘ 0.87
3.16 ˘ 1.31
2.64 ˘ 0.56
3.83 ˘ 1.12
3.61 ˘ 2.43
38.98 ˘ 5.76
60.72 ˘ 2.31
22.32 ˘ 1.88
245.68 ˘ 2.09
69.83 ˘ 2.08
37.99 ˘ 1.24
41.20 ˘ 1.46
133.41 ˘ 0.07
98.52 ˘ 5.89
124.37 ˘ 6.64
n.d.
0.73 ˘ 0.13
1.00 ˘ 0.09
1.06 ˘ 0.05
1.43 ˘ 0.07
3.48 ˘ 0.46
1.82 ˘ 0.15
2.00 ˘ 0.01
1.79 ˘ 0.48
n.d.
H
3-methyl
4-methoxy
4-chloro
4-bromo
4-fluoro
4-nitro
H
H
H
H
0.73 ˘ 0.01
0.66 ˘ 0.09
0.82 ˘ 0.01
1.43 ˘ 0.08
1.53 ˘ 0.07
n.d.
25g
25h
25i
H
H
4-nitro
F
F
25j
4-methyl
4-sulfamoyl
4-methyl
H
n.d.
25k
26a
26b
26c
H
8.69 ˘ 0.01
—
—
—
—
—
—
H
n.d.
n.d.
5.14 ˘ 0.33
n.d.
4-sulfamoyl
4-methoxy
4-fluoro
4-nitro
4-methyl
4-nitro
n.d.
n.d.
26d
26e
26f
27a
27b
27c
27d
27e
28a
28b
28c
28d
28e
28f
28g
28h
28i
29a
29b
29c
29d
29e
29f
29g
29h
29i
n.d.
n.d.
5.96 ˘ 0.01
n.d.
n.d.
n.d.
3.59 ˘ 0.21
3.87 ˘ 0.43
3.73 ˘ 0.32
5.76 ˘ 0.24
5.00 ˘ 0.53
>10 µM
>10 µM
n.d.
15.31 ˘ 2.56
27.54 ˘ 1.21
18.37 ˘ 4.35
130.35 ˘ 3.33
182.37 ˘ 4.35
n.d.
H
H
F
F
H
4-nitro
4-methyl
4-methyl
H
3-methyl
4-methoxy
4-chloro
4-bromo
4-fluoro
4-nitro
4-sulfamoyl
4-methyl
H
3-methyl
4-methoxy
4-chloro
4-bromo
4-fluoro
4-nitro
n.d.
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
>10 µM
>10 µM
n.d.
>10 µM
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
>10 µM
>10 µM
n.d.
n.d.
>10 µM
n.d.
>10 µM
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
>10 µM
n.d.
n.d.
4-sulfamoyl
4-chloro
4-methyl
4-sulfamoyl
H
>10 µM
n.d.
n.d.
30a
30b
30c
>3 µM
n.d.
n.d.
>3 µM
n.d.
n.d.
>3 µM
n.d.
n.d.
30d
>3 µM
n.d.
n.d.
a
n.d.: not determined.
2.2.2. p38α Assay
To assess the p38α inhibition in an enzyme system, we selected the compounds that showed good
inhibitory activity against MAPKAPK2 and monitored their ability to inhibit the phosphorylation
of activation transcription factor (2ATF-2) in the enzyme system in an in vitro assay. The results are
shown in Table 1.

Molecules 2016, 21, 677
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2.2.3. Inhibition of TNF-α Production in THP-1 Cells
To assess the inhibition of TNF-
compounds that showed good inhibitory activity against the p38
monitored their ability to inhibit TNF-
also shown in Table 1.
α
release from THP-1 cells after LPS stimulation, we chose the
enzyme and MAPKAPK2 and
α release from THP-1 cells after LPS stimulation. The results are
α
2.2.4. Surface Plasmon Resonance (SPR)
To assess the binding ability between the compounds and p38
α
protein, we used SPR for kinase
inhibitor interaction studies. The results are shown in Table 2 and Figure 2.
Table 2. Detailed binding analysis of small molecule inhibitors with p38α mitogen-activated
protein kinase.
Compounds
KD (mol/L)
0.1 ˆ 10´9
6.15 ˆ 10´8
1.54 ˆ 10´8
3.82 ˆ 10´7
4.21 ˆ 10´7
2.21 ˆ 10´7
BIRB-796
SB203580
25a
25b
25c
25h
Figure 2. p38
Concentration series (0.003–1
α
affinity measurement and analysis chart for 25a (left); and SB203580 (right).
M) of inhibitor with different color lines enzyme kinetic characteristics
µ
were determined by surface plasmon resonance on the inactive form of the kinase using a
Biacore instrument.
2.3. Discussion
MAPKAPK2 is a well-known substrate of p38
signal is received, this protein complex is disrupted because of phosphorylation of p38
MAPKAPK2 could be used as an effector of p38 by phosphorylating further substrates. Certain
compounds showed good activities, and compounds 25a 25k and 27a 27e were also profiled for their
p38 inhibitory activity and ability to inhibit TNF- release in the lipopolysaccharide (LPS)-stimulated
α
. It forms an adduct with p38
α
, and when a stress
29]. Thus,
α
[
α
–
–
α
α
human acute monocytic leukaemia cell line (THP-1). The activities and structures of the compounds
revealed certain structure-activity relationship information. Overall, the R1 and R2 substituents
have little effect on the activity of compounds, but the inhibitory activity was reduced when R1
was substituted with a sulphamoyl group compared with R1 substituted with another group, as in
compounds 25a–25k, 26a–26f. The polarity of compounds with a sulphamoyl group at the R1 position

Molecules 2016, 21, 677
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is higher, therefore, the activities of 25k and 26c were lower than those of the other compounds owing
to their low transport through the cell membrane. Second, the activities of compounds 25i 25j 27d
and 27e were lower than those of 25h 25a 27b and 27a, probably because the fluorine atom can affect
the ability to interact with the small gatekeeper of the p38α protein.
In a comparison of the activity of compounds 25a 25k and BIRB-796 with those of 26a
and 28a 28i 29a 29i and 28a 28i in particular showed almost no inhibitory activity. These results
demonstrate that the naphthyl ring of BIRB-796 is important for maintaining the inhibitory activity.
We consider that the length of the naphthyl ring and chromene affects the anti-p38 activity of the
,
,
,
,
–
–26f, 29a–29i
–
,
–
–
α
corresponding derivatives. However, the activity of chromene urea compounds was poor compared
with that of the corresponding benzylurea analogues or BIRB-796, which indicates that the chromene
ring could not replace the naphthyl ring without reducing activity. Perhaps the chromene ring does
not offer a correct interaction with the p38
and 29a 29i are similar, compounds 29a
compounds 25a 25k, and similar activity as 28a
in 28a 28i and 29a 29i cannot replace the hydrophobic interactions of the naphthyl ring. A docking
model of the p38 25a complex was built using Discovery Studio (PDB code: 1KV2) and is shown in
α
protein. Although the structures of compounds 25a
29i showed almost no inhibitory activity compared with
28i. We consider that the phenyl ring substituents
–25k
–
–
–
–
–
–
α
/
Figure 3. Compound 25a fitted very well in the small gatekeeper position (Thr106) in Figure 3, while the
29a-linked phenyl ring system could not insert into hydrophobic region I and provide a complementary
interaction surface. According to the previous SAR discussion, compounds 30a
linked benzyl ring system should show good activity, but the results indicated that compounds
30a 30d also showed poor activity, similar to compounds 28a 28i. In analysing the structure activity
relationship of all compounds, we considered that 30a 30d could not bind with the p38 protein in
–30d with a directly
–
–
–
α
the correct configuration like BIRB-796 because of the flexibility of the molecules (ethoxymorpholine
and benzyl ring), while the rigidity of the indazole and 1,2,3,4-tetrahydronaphthalene moieties in
compounds 25a
In summary, compounds substituted with a rigid indazole instead of a morpholinoethoxy could show
good p38 inhibitory activity performance. The indazole also offers a hydrophobic interaction with
p38 . The benzyl ring system instead of a naphthyl ring effectively occupied the small gatekeeper
(Thr106) by forming hydrophobic interactions with the hydrophobic region I of the p38 protein,
25k, and especially 25a, stood out due to their
–25k and 27a–27e ensured that the active configuration was the same as in BIRB-796.
α
α
α
therefore, it was observed that compounds 25a
–
high activity.
Figure 3. Structure of compound 25a and a two-dimensional p38 MAPKα/25a interaction map.

Molecules 2016, 21, 677
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According to the information in Figure 3, the structure of 25a permits key interactions with p38
which are similar to those of BIRB-796. In addition, the important amino acids (Thr106, Lys53 and
Met109) that bind with p38 also preserve the activity. First, the nitrogen atom of the indazole in 25a
directly interacts with Met109 of p38 the same as pexmetinib, which could explain the previously
discussed high activity of 25a. In addition, the p38 inhibitory activity of 25a was better than that
of pexmetinib, which suggests that the unsubstituted indazole offers better binding ability with
p38 30 34]. Second, 25a occupied the small gatekeeper (Thr106) region by forming hydrophobic
interactions with the hydrophobic region I of the p38 protein. (Figure 3). Finally, the -cation
interaction between the pyrazole ring and the side chains of Lys53 also plays a role on the activity. The
above data also could explain why the binding capacity of 25a (KD = 1.54
10´8) was better than
α
α
α
α
α
[ –
α
π
ˆ
that of SB203580. The binding affinity of compound 25a is lower than literature value of the reference
BIRB-796, however it is more potent than BIRB-796 in other assays. We consider that SPR is a rough
binding assay method, as the data could only explain binding capacity with the protein instead of
binding capacity with the active site.
3. Materials and Methods
3.1. General Procedures
1
All reagents and solvents were used as received from commercial sources. H- and 13C-NMR
spectra were recorded at 400 MHz and 100 MHz, respectively, on a JNM-ECA-400 instrument (JEOL,
Tokyo, Japan) in CDCl3 or DMSO-d6, respectively. Proton and carbon chemical shifts are expressed in
parts per million (ppm) relative to internal tetramethylsilane (TMS), and coupling constants (J) are
expressed in Hertz (Hz). The splitting pattern abbreviations are described as follows: multiplicity
(s: singlet, d: doublet, dd: double doublet, ddd: double double doublet, dm: double multiplet, ds:
double single, dt: double triplet, t: triplet, td triple doublet, tm, triple multiplet, tt: triple triplet, q:
quartet, quint: quintuplet, m: multiplet, br: broad). The electronic information of compounds are
available in the Supplementary Materials. Infrared (IR) spectra were recorded with a Perkin-Elmer
1330 infrared spectrophotometer (PerkinElmer, Waltham, MA, USA) using KBr as solid matrix.
Low-resolution mass spectra were obtained using a 3000LC/MS instrument (API, Rockwell, MD,
USA) equipped with an ESI source or an 620BTOF LC/MS equipped with an ESI source (Agilent
Technologies, Santa Clara, CA, USA). Purities of the compounds were established by analytical HPLC,
which was carried out on an Agilent HPLC system. HPLC analysis conditions were as follows:
Diamonsil C18 column (250
mobile phase: 60% acetonitrile in water.
ˆ
4.6 mm, 5
µ
m); flow rate, 1.0 mL
min´1; DAD detection at 245 nm;
¨
3.2. Synthesis
3.2.1. General Method for the Synthesis of 2a–2i
A solution of the corresponding substituted phenylhydrazine hydrochloride (1 equiv.) and
pivaloylacetonitrile (1.2 equiv.) was stirred overnight at 80 ˝C in ethanol. The solution was extracted
with EtOAc. The organic layer was washed with water and brine, dried (MgSO4), filtered, and
evaporated to dryness.
3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-amine (2a). From p-tolylhydrazine hydrochloride (1.59 g, 10 mmol)
and pivaloylacetonitrile (1.5 g, 12 mmol), after work-up and without further purification, compound
2a was obtained as a white solid (1.8 g, 80%). IR (KBr): 3345, 1598, 1578 cm´1; 1H-NMR (CDCl3)
δ:
7.40 (d, 2H, J = 8.8 Hz), 7.25 (d, 2H, J = 8.8 Hz), 5.50 (s, 1H), 3.72 (brs, 2H, NH2), 2.37 (s, 3H), 1.32 (s,
˝
9H). MS (ESI): m/z 252.16 [M + Na]+. Mp 121.3–123.4 C.
3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-amine (2b). Yellow solid (1.74 g, 81%). IR (KBr): 3340, 1601,
1
1575 cm´1; H-NMR (CDCl3)
δ
: 7.60–7.69 (m, 2H), 7.50–7.57 (m, 2H), 7.36–7.41 (m, 1H), 5.56 (s,
1H), 3.79 (brs, 2H, NH2), 1.30 (s, 9H). MS (ESI): m/z 238.14 [M + Na]+. Mp 118.1–120.0 C.
˝

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3-(tert-Butyl)-1-(m-tolyl)-1H-pyrazol-5-amine (2c). White solid (2.06 g, 90%). IR (KBr): 3343, 1600,
1
1579 cm´1; H-NMR (CDCl3)
δ
: 7.60–7.68 (m, 1H), 7.40–7.48 (m, 1H), 7.35–7.38 (m, 1H), 7.29–7.33
(m, 1H), 5.56 (s,˝1H), 3.75 (brs, 2H, NH2), 2.37 (s, 3H), 1.30 (s, 9H). MS (ESI): m/z 252.16 [M + Na]+.
Mp 120.3–122.4 C.
3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-amine (2d). White solid (1.97 g, 80%). IR (KBr): 3341,
1593, 1574 cm´1; 1H-NMR (CDCl3)
δ: 7.41 (d, 2H), 6.97 (d, 2H), 5.43 (s, 1H), 3.83 (s, 3H), 3.72 (brs, 2H,
˝
NH2), 1.32 (s, 9H). MS (ESI): m/z 268.11 [M + Na]+. Mp 109.1–111.3 C.
3-(tert-Butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-amine (2e). White solid (1.87 g, 75%). IR (KBr): 3340, 1597,
1581 cm´1; 1H-NMR (CDCl3)
δ: 7.59 (d, 2H, J = 8.8 Hz), 7.10 (d, 2H, J = 8.8 Hz), 5.47 (s, 1H), 3.74 (brs,
˝
2H, NH2), 1.32 (s, 9H). MS (ESI): m/z 272.10 [M + Na]+. Mp 107.3–109.5 C.
1-(4-Bromophenyl)-3-(tert-butyl)-1H-pyrazol-5-amine (2f). White solid (2.50 g, 85%). IR (KBr): 3337, 1596,
1575 cm´1; 1H-NMR (CDCl3)
δ: 7.54 (d, 2H, J = 8.4 Hz), 7.14 (d, 2H, J = 8.4 Hz), 5.42 (s, 1H), 3.71 (brs,
˝
2H, NH2), 1.31 (s, 9H). MS (ESI): m/z 316.05 [M + Na]+. Mp 109.1–112.7 C.
3-(tert-Butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-amine (2g). White solid (1.86 g, 79%). IR (KBr): 3333, 1594,
1582 cm´1; 1H-NMR (CDCl3)
δ: 7.59 (d, 2H, J = 8.8 Hz), 7.10 (d, 2H, J = 8.8 Hz), 5.58 (s, 1H), 3.62 (brs,
˝
2H, NH2), 1.31 (s, 9H). MS (ESI): m/z 256.34 [M + Na]+. Mp 102.3–104.6 C.
3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-amine (2h). White solid (1.69 g, 65%). IR (KBr): 3333, 1594,
1582 cm´1; 1H-NMR (CDCl3)
δ: 8.28 (d, 2H, J = 8.8 Hz), 7.93 (d, 2H, J = 8.8 Hz), 5.55 (s, 1H), 3.69 (brs,
˝
2H, NH2), 1.30 (s, 9H). MS (ESI): m/z 283.13 [M + Na]+. Mp 105.1–107.3 C.
4-(5-Amino-3-(tert-butyl)-1H-pyrazol-1-yl)benzenesulfonamide (2i). White solid (2.35 g, 80%). IR (KBr):
3367, 3342, 1592, 1588 cm´1; 1H-NMR (DMSO-d6)
δ: 8.11 (d, 2H, J = 8.8 Hz), 7.83 (d, 2H, J = 8.8 Hz), 7.47
˝
(s, 2H), 5.51 (s, 1H), 3.73 (brs, 2H, NH2), 1.32 (s, 9H).MS (ESI): m/z 317.12 [M + Na]+. Mp 103.1–105.2 C.
3.2.2. General Method for the Synthesis of 3a–3i
A solution of the corresponding 2a–2i (1 equiv.) and 2,2,2-trichloroethyl carbonochloridate
˝
(1.1 equiv.) in THF was stirred 6 h at 0 C, then the mixture was filtered and extracted three times with
ethyl acetate. The solution was dried with MgSO4 and concentrated to dryness.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)carbamate (3a). From 2a (2.30 g, 10 mmol) and
2,2,2-trichloroethyl carbonochloridate (2.3 g, 11 mmol), after work-up and without further purification,
3a was obtained as a white solid (3.02 g, 75%). IR (KBr): 3169, 1597, 1580 cm´1; 1H-NMR (DMSO-d6)
δ
: 9.47 (s, 1H), 7.41 (d, 2H, J = 8.8 Hz), 7.23 (d, 2H, J = 8.8 Hz), 5.51 (s, 1H), 4.85(s, 2H), 1.32 (s, 9H).
MS (ESI): m/z 426.06 [M + Na]+. Mp 153.3–155.7 C.
˝
2,2,2-Trichloroethyl(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)carbamate (3b). White solid (3.50 g, 90%). IR
(KBr): 3170, 1593, 1577 cm´1; 1H-NMR (DMSO-d6)
δ: 9.47(s, 1H), 7.65 (m, 2H), 7.57 (m, 2H), 7.40 (m,
˝
1H), 5.52 (s, 1H), 4.85 (s, 2H), 1.32 (s, 9H). MS (ESI): m/z 412.11 [M + Na]+. Mp 151.1–153.2 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(m-tolyl)-1H-pyrazol-5-yl)carbamate (3c). White solid (3.62 g, 90%). IR
1
(KBr): 3168, 1601, 1582 cm´1; H-NMR (DMSO-d6)
δ
: 9.43 (s, 1H), 7.60–7.69 (m, 1H), 7.39–7.44 (m,
1H), 7.35–7.38 (m, 1H), 7.29–7.33 (m, 1H), 5.49 (s, 1H), 4.88 (s, 2H), 2.37 (s, 3H), 1.30 (s, 9H). MS (ESI):
˝
m/z 426.06 [M + Na]+. Mp 148.3–150.2 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)carbamate (3d). White solid (3.56 g,
85%). IR (KBr): 3170, 1600, 1578 cm´1; 1H-NMR (DMSO-d6)
δ: 9.40 (s, 1H), 7.41 (d, 2H, J = 8.8 Hz),
6.97 (d, 2H, J = 8.8 Hz), 5.44 (s, 1H), 4.81(s, 2H), 1.30 (s, 9H). MS (ESI): m/z 442.09 [M + Na]+.
˝
Mp 150.1–152.2 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)carbamate (3e). White solid (3.60 g,
85%). IR (KBr): 3166, 1600, 1577 cm´1; 1H-NMR (DMSO-d6)
δ: 9.42 (s, 1H), 7.57 (d, 2H, J = 8.8 Hz),

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7.12 (d, 2H, J =˝8.8 Hz), 5.41 (s, 1H), 4.80 (s, 2H), 1.32 (s, 9H). MS (ESI): m/z 446.01 [M + Na]+.
Mp 147.2–149.3 C.
2,2,2-Trichloroethyl(1-(4-bromophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)carbamate (3f). White solid (3.54 g,
76%). IR (KBr): 3169, 1602, 1579 cm´1; 1H-NMR (DMSO-d6)
δ: 9.42 (s, 1H), 7.54 (d, 2H, J = 8.8 Hz),
7.16 (d, 2H, J = 8.8 Hz), 5.37 (s, 1H), 4.78 (s, 2H), 1.30 (s, 9H). MS (ESI): m/z 490.00 [M + Na]+.
˝
Mp 145.1–147.6 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-yl)carbamate (3g). White solid (3.25 g,
80%). IR (KBr): 3166, 1593, 1570 cm´1; 1H-NMR (DMSO-d6)
δ: 9.40 (s, 1H), 7.48 (d, 2H, J = 8.8 Hz),
7.18 (d, 2H, J =˝8.8 Hz), 5.33 (s, 1H), 4.64 (s, 2H), 1.29 (s, 9H). MS (ESI): m/z 430.04 [M + Na]+.
Mp 148.7–150.1 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)carbamate (3h). Yellow solid (3.47 g,
80%). IR (KBr): 3165, 1595, 1573 cm´1; 1H-NMR (DMSO-d6)
δ: 9.39 (s, 1H), 8.28 (d, 2H, J = 8.8 Hz),
7.93 (d, 2H, J =˝8.8 Hz), 5.37 (s, 1H), 4.73 (s, 2H), 1.30 (s, 9H). MS (ESI): m/z 457.13 [M + Na]+.
Mp 149.3–151.2 C.
2,2,2-Trichloroethyl(3-(tert-butyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-5-yl)carbamate (3i). White solid (3.27 g,
70%). IR (KBr): 3367, 3160, 1599, 1580 cm´1; 1H-NMR (DMSO-d6)
δ: 9.39 (s, 1H), 8.15 (d, 2H, J = 8.8 Hz),
7.80 (d, 2H, J = 8.8 Hz), 7.45 (s, 2H), 5.35 (s, 1H), 4.70 (s, 2H), 1.29 (s, 9H); MS (ESI): m/z 491.02 [M + Na]+
Mp 143.4–145.6 C.
.
˝
3.2.3. General Method for the Synthesis of 5a–5b
A solution˝of the corresponding 4a
–4b (1 equiv.) and 4-amino-3-methylphenol (10 mmol) was
stirred 2 h at 90 C in the presence of K2CO3 (4.14g 30 mmol) in DMSO (50 mL). Then the solution was
extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO4), filtered
and evaporated to dryness. The product was separated by column chromatography using PE/EA (3:1)
as eluent.
2-(4-Amino-3-methylphenoxy)benzonitrile (5a). From 4a (1.21 g, 10 mmol), and 4-amino-3-methylphenol
(1.23 g, 10 mmol), after work-up and purification, 5a was obtained as a white solid. (1.56 g, 70%).
IR (KBr): 3275, 2250, 1595, 1575 cm´1; 1H-NMR (DMSO-d6)
δ: 8.10–8.16 (m, 1H), 8.00–8.06 (m, 1H),
7.77–7.81 (m, 1H), 7.39–7.42 (m, 1H), 7.29–7.32 (m, 1H), 7.22–7.27 (m, 1H), 7.09–7.11 (m, 1H), 4.39 (s, 2H,
˝
NH2), 2.55 (s, 3H); MS (ESI): m/z 447.09 [M + Na]+. Mp 191.3–193.7 C.
2-(4-Amino-3-methylphenoxy)-5-fluorobenzonitrile (5b). White solid (1.81 g, 75%). IR (KBr): 3274, 2250,
1
1593, 1574 cm´1; H-NMR (DMSO-d6)
δ: 8.10–8.16 (m, 1H), 8.00–8.06 (m, 1H), 7.74–7.78 (m, 1H),
7.39–7.42 (m, 1H), 7.19–7.24 (m, 1H), 7.10–7.14 (m, 1H), 4.38 (s, 2H, NH2), 2.54 (s, 3H); MS (ESI):
˝
m/z 465.25 [M + Na]+. Mp 191.3–193.7 C.
3.2.4. General Method for the Synthesis of 6a–6b
A solution of the corresponding 5a–5b (1 equiv.) and isoamyl nitrite (1.5 equiv.) was stirred 8 h at
˝
90 C in the presence of potassium acetate (0.98 g, 10 mmol) and acetic anhydride (1.75 g, 15 mmol) in
toluene (50 mL). The solution was extracted with EtOAc. The organic layer was washed with water
and brine, dried (MgSO4), filtered, and evaporated to dryness. The product was separated by column
chromatography using PE/EA (8:1) as eluent.
2-((1-Acetyl-1H-indazol-5-yl)oxy)benzonitrile (6a). From 5a (2.24 g, 10 mmol), acetic anhydride (3 g,
30 mmol), potassium acetate (0.98 g, 10 mmol) and isoamyl nitrite (1.75 g, 15 mmol), after work-up
and purification, 6a was obtained as a white solid. (1.66 g, 60%). IR (KBr): 2250, 1698, 1593,
1
1574 cm´1; H-NMR (DMSO-d6)
δ: 8.46 (s, 1H), 8.34–8.39 (m, 1H), 7.90–7.95 (m, 1H), 7.66–7.69 (m,
1H), 7.61–7.65 (m, 1H), 7.44–7.51 (m, 1H), 7.27–7.32 (m, 1H), 6.92–6.99 (m, 1H), 2.74 (s, 3H); MS (ESI):
˝
m/z 310.09 [M + Na]+. Mp 201.7–203.1 C.

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2-((1-Acetyl-1H-indazol-5-yl)oxy)-5-fluorobenzonitrile (6b). White solid (1.62 g, 55%). IR (KBr): 2250,
1
1696, 1599, 1579 cm´1; H-NMR (DMSO-d6)
δ
: 8.49 (s, 1H), 8.29–8.36 (m, 1H), 7.87–7.92 (m, 1H),
7.60–7.65 (m, 1H), 7.50–7.57 (m, 1H), 7.40–7.46 (m, 1H), 6.91–6.99 (m, 1H), 2.76 (s, 3H); MS (ESI):
˝
m/z 318.22 [M + Na]+. Mp 205.1–207.3 C.
3.2.5. General Method for the Synthesis of 7a–7b
A solution of the corresponding 6a or 6b (1 equiv.) and LiAlH4 (3 equiv.) in THF was stirred
1 h at 65 ˝C. Then the solution was extracted with EtOAc. The organic layer was washed with water
and brine, dried (MgSO4), filtered and evaporated to dryness. Products were purified by column
chromatography using DCM/MeOH (50:1) as eluent.
(2-((1H-Indazol-5-yl)oxy)phenyl)methanamine (7a). From 6a (2.77 g, 10 mmol) and LiAlH4 (1.14 g,
15 mmol), after work-up and purification, 7a was obtained as a solid a white solid. (1.20 g, 50%). IR
1
(KBr): 3277, 1603, 1579 cm´1; H-NMR (DMSO-d6)
δ
: 13.12 (s, 1H), 8.08 (s, 1H), 7.84–7.91 (m, 1H),
7.60–7.65 (m, 3H), 7.23–7.28 (m, 2H), 6.81–6.87 (m, 1H), 4.23–4.25 (t, 2H), 3.79–3.83 (m, 2H); MS (ESI):
˝
m/z 262.11 [M + Na]+. Mp 178.3–180.5 C.
(2-((1H-Indazol-5-yl)oxy)-5-fluorophenyl)methanamine (7b) White solid solid (1.41 g, 55%). IR (KBr):
; δ: 13.11 (s, 1H), 8.08 (s, 1H), 7.84–7.89 (m, 1H),
3279, 1599, 1576 cm´1 1H-NMR (DMSO-d6)
7.59–7.65 (m, 2H), 7.21–7.25 (m, 2H), 6.81–6.88 (m, 1H), 4.25–4.27 (t, 2H), 3.80–3.86 (m, 2H); MS (ESI):
˝
m/z 280.26 [M + Na]+. Mp 180.1–182.7 C.
3.2.6. Synthesis of 14
4-Fluoro-1-nitro-2-(prop-2-yn-1-yloxy)benzene (9). Compound 8 (6.7 g, 40 mmol) was dissolved in DMF
(60 mL), and K2CO3 (8.3 g, 60 mmol) was added while stirring vigorously. 3-Bromoprop-1-yne
(50 mmol, 5.9 g) was then added dropwise while the mixture was on ice, and the reaction was allowed
to proceed at room temperature for 12 h. The mixture was poured into ice water and kept in fridge for
a day. The yellow solid 9 that appeared was filtered off and dried (yield 97%). IR (KBr): 3309, 2160,
1593, 1573 cm´1; 1H-NMR (DMSO-d6)
δ
: 8.00–8.06 (m, 1H), 7.31–7.37˝(m, 1H), 7.01–7.07 (m, 1H), 5.06
(s, 2H), 3.75 (s, 1H); MS (ESI): m/z 218.15 [M + Na]+. Mp 110.4–112.7 C.
2-Methyl-1-nitro-4-(4-nitro-3-(prop-2-yn-1-yloxy)phenoxy)benzene (10). To DMF (40 mL) compound
9
(1.95 g, 10 mmol) and K2CO3 (4.0 g, 30 mmol) were added and the mixture was stirred at room
temperature for 4 h, followed by the addition of 4-amino-3-methylphenol (1.23 g, 10 mmol). The
suspension formed was stirred at 100 ˝C for 3 h. The reaction mixture was cooled, and poured into
ice water (100 mL), then the product was briefly placed in a fridge whereby a white solid appeared.
The product was recovered by suction filtration and the filter cake was dried to give 2.55 g (90%) of
compound 10. IR (KBr): 3309, 3267, 2160, 1593, 1573 cm´1; 1H-NMR (DMSO-d6)
δ: 8.00–8.08 (m, 1H),
7.30–7.37 (m, 1H), 7.20–7.26 (m, 1H), 7.10–7.15 (m, 1H), 7.00–7.07 (m, 1H), 6.08–6.12 (m, 1H), 5.06 (s,
˝
2H), 3.75 (s, 1H), 2.95 (s, 3H); MS (ESI): m/z 321.10 [M + Na]+. Mp 150.1–152.4 C.
1-(5-(3-(Ethynyloxy)-4-nitrophenoxy)-1H-indazol-1-yl)ethanone (11). Compound 10 (0.8 g, 2.7 mmol) and
acetic anhydride (1.14 g) were added to dry toluene (50 mL), followed by potassium acetate (0.3 g). The
˝
suspension was stirred at 80 C and then isopentyl nitrite (0.6 g, 5 mmol) was added. The reaction was
continued for 12 h, then the suspension was cooled down and filtered. Product 11 can be purified by
column chromatography using DCM/MeOH (100: 1) as eluent, yield (75%). IR (KBr): 3309, 2160, 1670,
1593, 1573 cm´1; 1H-NMR (DMSO-d6)
δ: 8.04 (s, 1H), 7.90–7.94 (m, 1H), 7.40–7.45 (m, 1H), 7.21–7.26
(m, 1H), 7.11–7.14 (m, 1H), 7.06–7.11 (m, 1H), 6.10–6.15 (m, 1H), 5.06 (s, 2H), 3.75 (s, 1H), 2.25 (s, 3H);
˝
MS (ESI): m/z 374.09 [M + Na]+. Mp 169.7–171.3 C.
4-((1H-Indazol-5-yl)oxy)-2-(ethynyloxy)aniline (12). Compound 11 (2.0 g, 6 mmol) was placed in a 100 mL
flask and then dissolved in ethyl acetate (40 mL). Ethanol (40 mL) was then added, followed by SnCl2
(4.5 g, 21 mmol). The mixture was heated to reflux for 6 h. Next saturated NaHCO3 solution was added

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until bubbles ceased to come out, and the mixture was filtered. The filtrate was extracted with ethyl
acetate and then dried and then separated by column chromatography using DCM/MeOH (100:1)
as eluent, yield (54%). IR (KBr): 3309, 3277, 2160, 1593, 1573 cm´1; 1H-NMR (DMSO-d6)
δ: 13.01 (s,
1H), 8.05 (s, 1H), 7.90–7.99 (m, 1H), 7.40–7.45 (m, 1H), 7.21–7.24 (m, 1H), 7.11–7.15 (m, 1H), 7.02–7.12
(m, 1H), 6.11–6.˝14 (m, 1H), 5.04 (s, 2H), 3.77 (s, 1H), 3.28 (s, 2H); MS (ESI): m/z 302.09 [M + Na]+
.
Mp 192.1–194.7 C.
tert-Butyl(4-((1H-indazol-5-yl)oxy)-2-(ethynyloxy)phenyl)carbamate (13). Compound 12 (2 g, 7 mmol) was
dissolved in CH2Cl2 (25 mL) in a 100 mL flask. The flask was placed in a low temperature reaction
˝
tank and Et3N (3 mL) was added while the temperature was kept under
´
10 C. Then (Boc)2O (3.9 g,
0.017 mol) was added to the flask in portions. The reaction was carried out for 48 h, then the solution
was washed with water, dried, then separated by column chromatography using DCM/MeOH (50:1)
1
as eluent, yield (63%). IR (KBr): 3309, 3250, 2160, 1690, 1593, 1573 cm´1; H-NMR (DMSO-d6)
δ:
13.01 (s, 1H), 8.01 (s, 1H), 7.92–7.99 (m, 1H), 7.52–7.57 (m, 2H), 7.30–7.35 (m, 1H), 7.11–7.15 (m, 2H),
6.83–6.88 (m, 1H), 6.48–6.53 (m, 1H), 4.80 (s, 2H), 3.61 (s, 1H), 2.30 (s, 1H), 1.45 (s, 9H); MS (ESI):
˝
m/z 412.15 [M + Na]+. Mp 177.4–179.1 C.
5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-amine (14). Compound 13 (1.6 g, 0.004 mol) was dissolved in
diphenyl ether (20 mL) in a 50 mL flask. The method using microwave was carried out as follows:
˝
temperature: 220 C, pressure: 8.0 bar, time: 30 min, power: 250 w. Then the oxydibenzene was
removed through vacuum distillation. Product 14 was then separated by column chromatography
using DCM/MeOH (80:1) as eluent, yield (25%). IR (KBr): 3310, 3245, 1680, 1593, 1573 cm´1; 1H-NMR
(DMSO-d6) δ: 12.99 (s, 1H), 7.92 (s, 1H), 7.48–7.52 (m, 1H), 7.05–7.09 (m, 1H), 6.98–7.02 (m, 1H),
6.45–6.49 (m, 1H), 6.40–6.43 (m, 1H), 6.30–6.34 (m, 1H), 5.80–5.83 (m, 1H), 4.73–4.76 (m, 2H), 4.56 (s,
˝
2H); MS (ESI): m/z 312.15 [M + Na]+. Mp 181.3–183.7 C.
3.2.7. Synthesis of 16
2-(2-Morpholinoethoxy)benzonitrile (15). 2-Morpholinoethanol (4.2 g) was added to DMF (50 mL) in
˝
a 250 mL flask, followed by addition of sodium hydride (3 g) at
´
35 C. An amount of 4a (3.6 g)
was dissolved in DMF (15 mL). This solution was added dropwise to the flask and the mixture was
stirred at
35 ˝C for 12 h. The mixture was poured into ice water (200 mL), and the solution was
extracted with ethyl acetate. A gray oil was collected after drying and concentrating; yield 64%. IR
(KBr): 2250, 1598, 1578 cm´1; 1H-NMR (DMSO-d6)
: 7.22–7.25 (m, 1H), 7.13–7.16 (m, 1H), 6.85–6.88
´
δ
(m, 1H), 6.70–6.74 (m, 1H), 4.07–4.09 (t, 2H, J = 5.6), 3.57–3.59 (t, 4H, J = 4.8), 2.72–2.74 (t, 2H, J = 5.6),
2.44–2.46 (t, 4H, J = 4.8); MS (ESI): m/z 255.12 [M + Na]+.
(2-(2-Morpholinoethoxy)phenyl)methanamine (16). LiAlH4 (1.5 g) was added to anhydrous THF (40 mL)
in a 150 mL flask, which was kept in an ice bath. Compound 15 (2.32 g, 10 mmol) was dissolved in
anhydrous THF (15 mL) and this solution was added dropwise to LiAlH4 suspension and the mixture
˝
then stirred at 60 C for 2 h. Ethanol was added to the mixture until gas evolution stopped, then
the suspension was filtered and concentrated. The product 16 was obtained as a solid by column
1
chromatography (DCM/MeOH (100:1)), yield (64%). IR (KBr): 3270, 1597, 1577 cm´1; H-NMR
(DMSO-d6) δ: 7.23–7.27 (m, 1H), 7.16–7.18 (m, 1H), 6.93–6.99 (m, 1H), 6.80–6.87 (m, 1H), 4.28–4.30 (t,
2H, J = 7.6), 4.08–4.10 (t, 2H, J = 5.6), 3.56–3.58 (t, 4H, J = 4.8), 2.70–2.72 (t, 2H, J = 5.6), 2.46–2.48 (t, 4H,
˝
J = 4.8); MS (ESI): m/z 259.12 [M + Na]+. Mp 181.3–183.7 C.
3.2.8. General Method for the Synthesis of 18a and 18b
2-((5-Oxo-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)benzonitrile (17a). 6-Hydroxy-1-tetralone (5 g, 30.86 mmol)
was placed in a 250 mL three-necked flask and dissolved in DMSO (100 mL). The flask was heated to
˝
90 C, and K2CO3 (12.78 g) was added under stirring. Compound 4a (4.48 g, 37.03 mmol) was added
˝
dropwise into the flask and the reaction mixture was heated to 90 C for 4 h. The mixture was poured

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into water (200 mL), and the water phase was extracted twice with ethyl acetate. The organic phases
were combined, washed with water and brine, dried with Na2SO4, and the solvent removed under
vacuum to yield the crude product, which was purified by column chromatography using petroleum
ether/ethyl acetate (5:1) as eluent to give compound 17a as a yellow solid (75.3%). IR (KBr): 2253,
1688, 1597, 1577 cm´1; 1H-NMR (CDCl3),
δ: 8.32–8.35 (m, 1H), 7.66–7.69 (m, 1H), 7.59–7.61 (m, 1H),
7.35–7.38 (m, 3H), 7.00–7.04 (m, 1H), 2.90–2.94 (m, 2H), 2.71–2.74 (m, 2H), 2.00–2.03 (m, 2H). MS (ESI):
˝
m/z 286.34 [M + Na]+. Mp 178.3–180.5 C.
5-Fluoro-2-((5-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)benzonitrile (17b). White solid, yield 71.1%. IR
(KBr): 2250, 1687, 1597, 1577 cm´1; 1H-NMR (CDCl3),
δ: 8.40–8.43 (m, 1H), 7.59–7.62 (m, 1H), 7.35–7.39
(m, 2H), 7.19–7.24 (m, 1H), 7.07–7.11 (m, 1H), 2.93–2.96 (m, 2H), 2.73–2.77 (m, 2H), 2.01–2.05 (m, 2H).
˝
MS (ESI): m/z 304.22 [M + Na]+. Mp 171.7–173.2 C.
6-(2-(Aminomethyl)phenoxy)-1,2,3,4-tetrahydronaphthalen-1-ol (18a). LiAlH4 (0.35 g, 9 mmol) was added
to anhydrous THF (15 mL) in a 50 mL flask, which was placed in an ice bath. Compound 17a (0.74 g,
3 mmol) was dissolved in anhydrous THF (15 mL) and this solution was added dropwise into the
˝
LiAlH4 suspension and the mixture then stirred at 65 C for 30–40 min. Ethanol was added until
bubble evolution ceased, then the suspension was filtered and concentrated. Compound 18a was
separated by column chromatography (dichloromethane/methanol 25:1), yield 77.7%. IR (KBr): 3350,
1597, 1577 cm´1; 1H-NMR (CDCl3),
δ: 7.20–7.25 (m, 1H), 7.14–7.17 (m, 1H), 7.05–7.09 (m, 2H), 7.00–7.02
(m, 1H), 6.87–6.93 (m, 1H), 6.86–6.90 (m, 1H), 4.55–4.60 (m, 1H), 4.20–4.22 (t, 2H, J = 7.6 Hz), 2.90–2.94
˝
(m, 2H), 2.73–2.76 (m, 2H), 2.01–2.04 (m, 2H). MS (ESI): m/z 292.01 [M + Na]+. Mp 165.2–167.1 C.
6-(2-(Aminomethyl)-4-fluorophenoxy)-1,2,3,4-tetrahydronaphthalen-1-ol (18b). White solid, yield 77.7%. IR
(KBr): 3347, 1595, 1575 cm´1; 1H-NMR (CDCl3), δ: 7.21–7.24 (m, 1H), 7.16–7.20 (m, 1H), 7.08–7.12 (m,
2H), 6.89–6.92 (m, 1H), 6.85–6.89 (m, 1H), 4.54–4.59 (m, 1H), 4.19–4.21 (t, 2H, J = 7.6 Hz), 2.95–2.99 (m,
˝
2H), 2.75–2.80 (m, 2H), 2.04–2.09 (m, 2H). MS (ESI): m/z 310.13 [M + Na]+. Mp 166.6–168.3 C.
3.2.9. Synthesis of 21
4-(2-(2-Nitrophenoxy)ethyl)morpholine (20). 2-Morpholinoethanol (1.3 g,10 mmol) was added to DMF
(30 mL) in a 50-mL flask, followed by addition of sodium hydride (0.48 g, 20 mmol) at
´
35 ˝C. An
amount of 1-fluoro-2-nitrobenzene (19, 3.26 g, 12 mmol) was dissolved in DMF (15 mL) and the solution
˝
was added dropwise to the flask and the reaction mixture stirred at
´
35 C for 12 h. The mixture was
poured into ice water (200 mL), and extracted with ethyl acetate. A gray oil was collected after drying
and concentrating; yield 64%. IR (KBr): 1598, 1578 cm´1; 1H-NMR (DMSO-d6)
δ: 8.29–8.32 (m, 1H),
7.87–7.89 (m, 1H), 7.57–7.61 (m, 1H), 7.43–7.48 (m, 1H), 4.07–4.09 (t, 2H, J = 5.6 Hz), 3.59–3.61 (t, 4H,
J = 4.8 Hz), 2.76–2.78 (t, 2H, J = 5.6 Hz), 2.46–2.48 (t, 4H, J = 4.8 Hz); MS (ESI): m/z 275.12 [M + Na]+.
2-(2-Morpholinoethoxy)aniline (21). Compound 20 (2.52 g, 10 mmol) was added to EtOH (50 mL) in
a 100-mL flask, followed by SnCl2
2H2O (11.28 g (50 mmol) and the mixture was heated at 80 ˝C
¨
for 2 h. The mixture was then poured into 10% aqueous NaOH solution (200 mL), and the solid
was filtered. The water phase was extracted twice with ethyl acetate, and the organic phases were
combined and washed with water and brine and dried with Na2SO4. The crude product was obtained
after concentration; yield 60%. IR (KBr): 3267, 1597, 1577 cm´1; 1H-NMR (DMSO-d6)
δ: 8.29–8.32 (m,
1H), 7.87–7.89 (m, 1H), 7.57–7.61 (m, 1H), 7.43–7.48 (m, 1H), 6.12 (s, 1H) 4.05–4.07 (t, 2H, J = 5.6 Hz),
3.58–3.60 (t, 4H, J = 4.8 Hz), 2.73–2.75 (t, 2H, J = 5.6 Hz), 2.49–2.51 (t, 4H, J = 4.8 Hz); MS (ESI):
˝
m/z 245.12 [M + Na]+. Mp 165.3–167.7 C.
3.2.10. Synthesis of 24
2-Methyl-4-(2-nitrophenoxy)aniline (22). Compound 19 (1.41 g, 10 mmol) and 4-amino-3-methylphenol
˝
(1.23 g, 10 mmol) were stirred 2 h at 90 C in the presence of K2CO3 (4.14g 30 mmol) in DMSO (50 mL).
The mixture was poured into ice water (200 mL), and the solution was extracted with ethyl acetate.

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The organic layer was washed with water and brine, dried (MgSO4), filtered, and evaporated to
dryness. After work-up and purification by column chromatography using PE/EA (3:1) as eluent,
intermediate 20 was obtained as a white solid; yield 74%. IR (KBr): 3275, 1595, 1575 cm´1; 1H-NMR
(DMSO-d6) δ: 8.17–8.21 (m, 1H), 8.01–8.04 (m, 1H), 7.72–7.76 (m, 1H), 7.44–7.47 (m, 1H), 7.33–7.36 (m,
1H), 7.27–7.31 (m, 1H), 7.12–7.15 (m, 1H), 4.41(s, 2H, NH2), 2.53(s, 3H); MS (ESI): m/z 267.08 [M + Na]+.
˝
Mp 153.1–155.5 C.
1-(5-(2-Nitrophenoxy)-1H-indazol-1-yl) ethanone (23). Compound 22 (2.44 g, 10 mmol, and acetic
anhydride (3.06 g, 30 mmol) were added to dry toluene (50 mL) followed by potassium acetate
(0.98 g, 10 mmol). The suspension was stirred at 80 ˝C, and isopentyl nitrite (1.76 g, 15 mmol) was
added. The reaction was continued for 18 h. The suspension was cooled and filtered. The product was
separated by column chromatography using PE/EA (10:1) as an eluent; yield 60%. IR (KBr): 1696, 1594,
1573 cm´1; 1H-NMR (DMSO-d6)
δ: 8.43–8.45 (s, 1H), 8.31–8.34 (m, 1H), 7.90–7.92 (m, 1H), 7.64–7.67
(m, 1H), 7.63–7.66 (m, 1H), 7.55–7.58 (m, 1H), 7.33–7.36 (m, 1H), 6.99 (m, 1H), 2.76 (s, 3H); MS (ESI):
˝
m/z 320.07 [M + Na]+. Mp 167.4–169.6 C.
2-((1H-Indazol-5-yl)oxy)aniline (24). Compound 23 (2.97 g, 10 mmol) was added to EtOH (50 mL) in
a 100-mL flask, followed by SnCl2
2H2O (11.30 g, 50 mmol) and the mixture was reacted at 80 ˝C
¨
for 2 h. The mixture was poured into 10% aqueous NaOH solution (200 mL), and the solid formed
was filtered. The water phase was extracted twice with ethyl acetate, and the organic phases were
combined, washed with water and brine, and dried with Na2SO4. A yellow solid was collected after
concentration; yield 57%. IR (KBr): 3272, 1600, 1578 cm´1; 1H-NMR (DMSO-d6)
δ: 13.11 (s, 1H), 8.04
(s, 1H), 7.84–7.89 (m, 1H), 7.63–7.66 (m, 3H), 7.20–7.24 (m, 2H), 6.86–6.89 (m, 1H), 3.80–3.82 (m, 2H);
˝
MS (ESI): m/z 248.09 [M + Na]+. Mp 180.1–182.4 C.
3.2.11. General Method for the Synthesis of 25a–25k
A solution of the corresponding 3a–3i (1.1 equiv.) and 7a–7b (1 equiv.) was stirred for 2 h at
90 ˝C in the presence of Et3N (1 mL) in DMSO (50 mL). The mixture was poured into ice water, and
the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried
(MgSO4), filtered and evaporated to dryness. The product was separated by column chromatography
using DCM/MeOH (100:1) as an eluent.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (25a). From 3a (4.03 g,
10 mmol), and 7a (2.39 g, 10 mmol), after work-up and purification 25a (3.46 g, 70%) was obtained as
a white solid. HPLC purity 98.1% (tR =11.97 min). IR (KBr): 3257, 1670, 1594, 1576 cm´1; 1H-NMR
(DMSO-d6)
(m, 3H), 7.06–7.10 (m, 4H), 6.95–6.97 (t, 1H, J = 5.6 Hz), 6.75(d, 1H, J = 8.0 Hz), 6.23(s, 1H), 4.31(d, 2H,
J = 5.6 Hz), 3.80 (s, 3H), 1.25(s, 9H). 13C-NMR (DMSO-d6)
: 160.4 (1C, pyrazole), 159.2 (1C, pyrazole),
δ: 13.13 (s, 1H), 8.22 (s, 1H), 8.01 (s, 1H), 7.58 (d, 1H, J = 8.8 Hz), 7.37–7.40 (m, 2H), 7.25–7.27
δ
156.8 (1C, CO), 154.6 (1C, pyrazole), 150.8, 137.6, 136.9, 136.5, 136.3, 133.4, 133.0, 132.9, 129.6, 124.1,
123.2, 119.6, 119.3, 115.0, 111.7, 107.6 (18C, Ar-C), 95.8 (1C, pyrazole), 38.1 (1C, CH2), 32.0 (1C, CH3-C),
˝
30.2 (3C, C-CH3), 20.6 (1C, Ar-CH3); MS (ESI): m/z 517.24 [M + Na]+. Mp 201.3–203.5 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)urea (25b). White solid (3.12 g,
65%). HPLC purity 97.2% (tR = 11.13 min). IR (KBr): 3254, 1670, 1596, 1578 cm´1; 1H-NMR (DMSO-d6)
δ
: 13.09 (s, 1H), 8.30 (s, 1H), 7.99 (s, 1H), 7.57 (d, 1H, J = 8.8 Hz), 7.48–7.51 (m, 4H), 7.39–7.43 (m, 1H),
7.26–7.29 (m, 3H), 7.10–7.12 (m, 2H), 6.94–6.96 (t, 1H, J = 5.6 Hz), 6.75 (d, 2H, J = 8.0 Hz), 4.32 (d, 2H,
J = 5.6 Hz), 1.27 (s, 9H). 13C-NMR (DMSO-d6)
: 160.6 (1C, pyrazole), 159.4 (1C, pyrazole), 157.1 (1C,
δ
CO), 154.8 (1C, pyrazole), 151.0, 153.2, 150.9, 138.0, 136.9, 136.8, 133.8, 133.4, 130.0, 124.5, 121.0, 120.7,
117.4, 115.3, 115.0, 111.1 (18C, Ar-C), 96.2 (1C, pyrazole),˝38.5 (1C, CH2), 32.6 (1C, CH3-C), 30.6 (3C,
C-CH3); MS (ESI): m/z 503.14 [M + Na]+. Mp 197.1–199.2 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(m-tolyl)-1H-pyrazol-5-yl)urea (25c). White solid
(3.70 g, 75%). HPLC purity 95.5% (tR = 11.82 min). IR (KBr): 3255, 1670, 1599, 1574 cm´1; NMR

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(DMSO-d6)
(m, 6H), 7.11–7.14 (m, 2H), 6.97–6.99 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.25 (s, 1H), 4.31 (d, 2H,
J = 5.6 Hz), 2.46 (s, 3H), 1.24 (s, 9H); 13C-NMR (DMSO-d6)
: 160.4 (1C, pyrazole), 154.4 (1C, pyrazole),
δ: 13.11 (s, 1H), 8.30 (s, 1H), 7.99 (s, 1H), 7.57 (d, 1H, J = 8.8 Hz), 7.36–7.39 (m, 1H), 7.25–7.28
δ
153.7 (1C, CO), 152.7 (1C, pyrazole), 152.1, 149.5, 146.5, 137.7, 136.5, 136.3, 130.9, 129.6, 129.1, 128.6,
124.2, 124.0, 122.5, 120.0, 119.1, 105.3 (18C, Ar-C), 95.1 (1C, pyrazole), 38.6 (1C, CH2), 31.0 (1C, CH3-C),
˝
30.2 (3C, C-CH3), 22.1 (1C, Ar-CH3); MS (ESI): m/z 517.24 [M + Na]+. Mp 200.4–201.9 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)urea (25d). White
solid (3.16 g, 62%). HPLC purity 96.3% (tR = 12.13 min). IR (KBr): 3259, 1670, 1600, 1579 cm´1
;
1H-NMR (DMSO-d6)
7.25–7.27 (m, 4H), 7.10–7.14 (m, 2H), 6.93–6.95 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.23 (s, 1H),
4.31 (d, 2H, J = 5.6 Hz), 2.35 (s, 3H), 1.25 (s, 9H); 13C-NMR (DMSO-d6)
: 161.6 (1C, pyrazole), 155.3 (1C,
δ: 13.09 (s, 1H), 8.23 (s, 1H), 7.99 (s, 1H), 7.57 (d, 1H, J = 8.8 Hz), 7.34–7.37 (m, 3H),
δ
pyrazole), 154.5 (1C, CO), 150.3 (1C, pyrazole), 142.0, 141.3, 138.2, 137.1, 133.4, 130.0, 128.7, 128.4, 126.8,
123.6, 123.2, 123.0, 119.8, 117.4, 111.7, 108.4 (18C, Ar–C), 97.0 (1C, pyrazole), 59.8 (1C, O-CH3), 37.5 (1C,
˝
CH2), 31.0 (1C, CH3-C), 30.1 (3C, C-CH3); MS (ESI): m/z 533.23 [M + Na]+. Mp 198.0–200.7 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)urea (25e). White
solid (2.83 g, 55%). HPLC purity 97.7% (tR = 12.18 min). IR (KBr): 3257, 1670, 1596, 1577 cm´1
;
1H-NMR (DMSO-d6)
7.25–7.28 (m, 3H), 7.10–7.13 (m, 2H), 6.91–6.93 (t, 1H, J = 5.6 Hz), 6.75 (d, 1H, J = 8.0 Hz), 6.25 (s, 1H),
4.31 (d, 2H, J = 5.6 Hz), 1.25 (s, 9H); 13C-NMR (DMSO-d6)
: 160.6 (1C, pyrazole), 159.4 (1C, pyrazole),
δ: 13.10 (s, 1H), 8.34 (s, 1H), 7.99(s, 1H), 7.57 (d, 1H, J = 8.8 Hz), 7.51–7.54 (m, 4H),
δ
157.0 (1C, CO), 154.8 (1C, pyrazole), 151.1, 137.7, 137.1, 136.6, 136.4, 133.6, 133.1, 133.0, 129.8, 124.3,
123.4, 119.8, 119.4, 115.2, 111.9, 107.7 (18C, Ar–C), 96.1 (1C, pyrazole), 38.3 (1C, CH2), 32.2 (1C, CH3-C),
˝
30.4 (3C, C-CH3); MS (ESI): m/z 537.19 [M + Na]+. Mp 199.6–201.4 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(1-(4-bromophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)urea (25f). White
solid (4.01 g, 72%). HPLC purity 97.5% (tR = 12.01 min). IR (KBr): 3256, 1670, 1593, 1575 cm´1
;
1H-NMR (DMSO-d6)
J = 8.8), 7.25–7.28 (m, 3H), 7.11–7.13 (m, 2H), 6.94–6.96 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.27
(s, 1H), 4.31 (d, 2H, J = 5.6 Hz), 1.25 (s, 9H); 13C-NMR (DMSO-d6)
: 161.6 (1C, pyrazole), 155.8 (1C,
δ: 13.13 (s, 1H), 8.38 (s, 1H), 8.00 (s, 1H), 7.66 (d, 2H, J = 8.4 Hz), 7.58 (d, 1H,
δ
pyrazole), 155.0 (1C, CO), 150.8 (1C, pyrazole), 138.5, 138.2, 137.5, 133.8, 131.8, 130.5, 129.6, 129.3, 128.8,
126.1, 123.7, 123.4, 120.3, 117.9, 112.1, 108.9 (18C, Ar-C), 96.8 (1C, pyrazole), 38.8 (1C, CH2), 32.6 (1C,
˝
CH3-C), 30.6 (3C, C-CH3); MS (ESI): m/z 581.14 [M + Na]+. Mp 203.6–205.7 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-yl)urea (25g). White
solid (3.23 g, 65%). HPLC purity 96.6% (tR = 12.13 min). IR (KBr): 3258, 1670, 1597, 1577 cm´1
;
1H-NMR (DMSO-d6)
7.25–7.28 (m, 5H), 7.10–7.13 (m, 2H), 6.93–6.95 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.25 (s, 1H),
4.31 (d, 2H, J = 5.6 Hz), 1.25 (s, 9H); 13C-NMR (DMSO-d6),
: 160.7 (1C, pyrazole), 155.2 (1C, pyrazole),
δ: 13.12 (s, 1H), 8.31 (s, 1H), 8.00 (s, 1H), 7.58 (d, 1H, J = 8.8 Hz), 7.51–7.54 (m, 2H),
δ
154.4 (1C, CO), 150.2 (1C, pyrazole), 138.0, 135.1, 133.3, 129.9, 128.7, 128.3, 126.5, 126.4, 123.1, 122.9,
119.7, 117.3, 116.0, 115.8, 111.5, 108.0 (18C, Ar-C), 95.5 (1C, pyrazole), 38.2 (1C, CH2), 32.0 (1C, CH3-C),
˝
30.1 (3C, C-CH3); MS (ESI): m/z 521.11 [M + Na]+. Mp 191.1–193.3 C.
1-(2-((1H-Indazol-5-yl)oxy)benzyl)-3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)urea (25h). White
solid (4.20 g, 80%). HPLC purity 97.3% (tR = 11.86 min). IR (KBr): 3256, 1670, 1595, 1578 cm´1
;
1H-NMR (DMSO-d6)
J = 8.8 Hz), 7.57 (d, 1H, J = 8.8 Hz), 7.26–7.30 (m, 3H), 7.08–7.11 (m, 3H), 6.74 (d, 1H, J = 8.0 Hz), 6.32
(s, 1H), 4.31 (d, 2H, J = 5.6 Hz), 1.26 (s, 9H); 13C-NMR (DMSO-d6),
: 161.6 (1C, pyrazole), 155.8 (1C,
δ: 13.12 (s, 1H), 8.62 (s, 1H), 8.31 (d, 2H, J = 8.8 Hz), 8.00 (s, 1H), 7.83 (d, 2H,
δ
pyrazole), 155.0 (1C, CO), 150.8 (1C, pyrazole), 138.6, 138.4, 137.5, 133.9, 132.5, 130.4, 129.3, 128.8, 126.4,
123.7, 123.4, 120.3, 120.1, 117.8, 112.1, 109.9 (18C, Ar-C), 96.9 (1C, pyrazole), 38.8 (1C, CH2), 32.6 (1C,
˝
CH3-C), 30.6 (3C, C-CH3); MS (ESI): m/z 548.21 [M + Na]+. Mp 177.3–179.9 C.
1-(2-((1H-Indazol-5-yl)oxy)-5-fluorobenzyl)-3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)urea (25i).
White solid (3.80 g, 70%). HPLC purity 98.7% (tR = 11.91 min). IR (KBr): 3260, 1670, 1599, 1578 cm´1
;

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1H-NMR (DMSO-d6)
δ: 13.11 (s, 1H), 8.61 (s, 1H), 8.30 (d, 2H, J = 8.8 Hz), 8.01(s, 1H), 7.81 (d, 2H,
J = 8.8 Hz), 7.51 (d, 1H, J = 8.8 Hz), 7.22–7.25 (m, 3H), 7.05–7.08 (m, 2H), 6.72 (d, 1H, J = 8.0 Hz), 6.31
(s, 1H), 4.30 (d, 2H, J = 5.6 Hz), 1.25 (s, 9H); 13C-NMR (DMSO-d6),
δ: 162.7 (1C, pyrazole), 156.9 (1C,
pyrazole), 156.1 (1C, CO), 152.0 (1C, pyrazole), 139.6, 139.3, 138.6, 135.0, 132.9, 131.6, 130.7, 130.4, 130.0,
127.2, 124.8, 124.5, 121.4, 119.0, 113.2, 110.0 (18C, Ar-C), 98.0 (1C, pyrazole), 39.9 (1C, CH2), 33.7 (1C,
˝
CH3-C), 31.5 (3C, C-CH3); MS (ESI): m/z 566.20 [M + Na]+. Mp 189.1–191.6 C.
1-(2-((1H-Indazol-5-yl)oxy)-5-fluorobenzyl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (25j). White
solid (3.84 g, 75%). HPLC purity 96.5% (tR = 11.84 min). IR (KBr): 3257, 1670, 1596, 1576 cm´1
;
1H-NMR (DMSO-d6)
7.26–7.30 (m, 3H), 7.04–7.07 (m, 4H), 6.93–6.95 (t, 1H, J = 5.6 Hz), 6.71 (d, 1H, J = 8.0 Hz), 6.20 (s, 1H),
4.30 (d, 2H, J = 5.6 Hz), 3.77(s, 3H), 1.24 (s, 9H). 13C-NMR (DMSO-d6),
: 160.4 (1C, pyrazole), 159.2 (1C,
δ: 13.11 (s, 1H) ,8.20 (s, 1H), 8.00 (s, 1H), 7.53 (d, 1H, J = 8.8 Hz), 7.33–7.37 (m, 1H),
δ
pyrazole), 156.8 (1C, CO), 154.6 (1C, pyrazole), 150.8, 137.6, 136.9, 136.5, 136.3, 133.4, 133.0, 129.9, 129.5,
124.1, 123.1, 119.6, 119.3, 115.0, 111.7, 107.6, 111.1 (18C, Ar-C), 95.8 (1C, pyrazole), 38.0 (1C, CH2), 32.0
˝
(1C, CH3-C), 30.2 (3C, C-CH3), 20.6 (1c, Ar-CH3); MS (ESI): m/z 535.23 [M + Na]+. Mp 188.7–190.3 C.
4-(5-(3-(2-((1H-Indazol-5-yl)oxy)benzyl)ureido)-3-(tert-butyl)-1H-pyrazol-1-yl)benzenesulfonamide (25k).
White solid (2.96 g, 53%). HPLC purity 97.3% (tR = 9.57 min). IR (KBr): 3257, 1670, 1595, 1575 cm´1
;
1H-NMR (DMSO-d6)
δ: 13.10 (s, 1H), 8.49 (s, 1H), 8.00 (s, 1H), 7.92 (d, 1H, J = 8.8 Hz), 7.71 (d, 2H,
J = 8.8 Hz), 7.59 (d, 1H, J = 8.8 Hz), 7.47 (s, 2H), 7.29–7.31 (m, 3H), 7.13–7.16 (m, 2H), 6.99–7.02 (m,
1H), 6.77 (d, 1H, J = 6.4 Hz), 6.36 (s, 1H), 4.70 (s, 1H), 4.34 (d, 2H, J = 5.6 Hz), 1.27 (s, 9H); 13C-NMR
(DMSO-d6) δ: 163.5 (1C, pyrazole), 157.7 (1C, pyrazole), 156.9 (1C, CO), 152.7 (1C, pyrazole), 140.5,
140.3, 139.4, 135.8, 134.4, 132.3, 131.2, 130.7, 128.3, 125.6, 125.3, 122.2, 122.0, 119.7, 114.0, 110.8 (18C,
Ar-C), 96.3 (1C, pyrazole), 38.6 (1C, CH2), 34.8 (1C, CH3-C), 32.4 (1C, C-CH3); MS (ESI):
˝
mboxemphm/z 582.20 [M + Na]+. Mp 194.7–196.8 C.
3.2.12. General Method for the Synthesis of 26a–26f
˝
A solution of the corresponding 3a–3i (1.1 equiv.) and 14 (1 equiv.) was stirred 2 h at 90 C in the
presence of Et3N (1 mL) in DMSO (50 mL). The mixture was poured into ice water, and the solution
was extracted with EtOAc. The organic layer was washed with water and brine, dried (MgSO4),
filtered, and evaporated to dryness. The product was separated by column chromatography using
DCM/MeOH (150:1) as eluent.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (26a). From
3a (4.03 g, 10 mmol), and 14 (2.79 g, 10 mmol), after work-up and purification the title compound
(3.73 g, 70%) was obtained as a white solid. HPLC purity 95.4% (tR = 10.32 min). IR (KBr): 3256,
1
1670, 1640, 1595, 1578 cm´1; H-NMR (DMSO-d6)
δ: 13.07 (s, 1H), 8.92 (s, 1H), 8.51 (s, 1H), 7.97 (s,
1H), 7.87 (d, 1H, J = 8.8 Hz), 7.54 (d, 2H, J = 8.8 Hz), 7.37–7.40 (m, 4H), 7.17 (d, 1H, J = 2.4 Hz), 7.11
(dd, 1H, J = 2.4, 8.8 Hz), 6.61–6.64 (m, 1H), 6.38(s, 1H), 6.34(d, 1H, J = 8.8 Hz), 5.93–5.95 (m, 1H), 4.85
(d, 2H, J = 1.6 Hz), 2.36 (s, 3H), 1.24 (s, 9H); 13C-NMR (DMSO-d6)
δ: 160.4 (1C, pyrazole), 159.8 (1C,
pyrazole), 157.5 (1C, CO), 154.6 (1C, pyrazole), 153.1, 149.5, 149.2, 146.5, 137.5, 136.5, 136.3, 134.7, 129.5,
124.0, 123.9, 122.5, 121.3, 120.1, 115.7, 115.0, 114.7, 104.5, 95.8 (1C, pyrazole), 73.5 (1C, O-CH2), 32.0 (1C,
˝
CH3-C), 30.2 (3C, C-CH3), 20.6 (1C, Ar-CH3); MS (ESI): m/z 557.20 [M + Na]+. Mp 210.3–212.7 C.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)urea (26b). White
solid (2.86 g, 55%). HPLC purity 95.9% (tR = 10.43 min). IR (KBr): 3259, 1670, 1644, 1599, 1579 cm´1
;
1H-NMR (DMSO-d6)
δ: 13.07 (s, 1H), 8.99 (s, 1H), 8.51 (s, 1H), 7.97 (s, 1H), 7.86 (d, 1H, J = 8.8 Hz), 7.53
(m, 5H), 7.41 (m, 1H), 7.16 (d, 1H, J = 2.4 Hz), 7.12 (dd, 1H, J = 2.4, 8.8 Hz), 6.61–6.64 (m, 1H), 6.39 (s,
1H), 6.36 (d, 1H, J = 8.8 Hz), 5.93–5.96 (m, 1H), 4.85 (d, 2H, J = 1.6 Hz), 1.24 (s, 9H); 13C-NMR (DMSO-d6)
δ
: 162.5 (1C, pyrazole), 161.2 (1C, pyrazole), 160.3 (1C, CO), 160.1 (1C, pyrazole), 157.9, 155.1, 153.5,
149.7, 149.3, 147.0, 138.2, 135.7, 134.4, 134.3, 126.8, 126.7, 123.1, 121.8, 116.5, 116.2, 115.4, 105.0, 96.6

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(1C, pyrazole), 74.3 (1C, O-CH2), 32.4 (1C, CH3-C), 30.6 (3C, C-CH3); MS (ESI): m/z 543.22 [M + Na]+
.
˝
Mp 209.9–212.1 C.
4-(5-(3-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)ureido)-3-(tert-butyl)-1H-pyrazol-1-yl)benzenesulfonamide
26c). White solid (2.99 g, 50%). HPLC purity 96.6% (tR = 8.33 min). IR (KBr): 3253, 1670, 1641, 1595,
1575 cm´1; 1H-NMR (DMSO-d6)
: 13.08 (s, 1H), 9.14 (s, 1H), 8.52 (s, 1H), 7.99–8.01 (m, 3H), 7.86–7.89
(
δ
(d, 1H, J = 8.8 Hz), 7.73–7.75 (d, 2H, J = 8.8 Hz), 7.54–7.57 (m, 3H), 7.17 (s, 1H), 7.10 (d, 1H, J = 8.8 Hz),
6.62 (d, 1H, J = 10.0 Hz), 6.41–6.44 (m, 2H), 5.93–5.95 (m, 1H), 4.87 (d, 2H, J = 1.6 Hz), 1.24 (s, 9H);
13C-NMR (DMSO-d6)
δ: 162.5 (1C, pyrazole), 161.2 (1C, pyrazole), 160.3 (1C, CO), 160.1 (1C, pyrazole),
158.0, 155.1, 153.6, 150.4, 149.6, 147.1, 138.2, 135.7, 134.5, 134.4, 126.8, 126.7, 124.6, 123.2, 121.9, 116.5,
116.2, 104.9, 96.7 (1C, pyrazole), 74.9 (1C, O-CH2), 32.5 (1C, CH3-C), 30.6 (3C, C-CH3); MS (ESI):
˝
m/z 622.20 [M + Na]+. Mp 213.4–215.2 C.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)urea
(
26d). White solid (2.47 g, 45%). HPLC purity 96.2% (tR = 10.04 min). IR (KBr): 3256, 1670, 1640, 1593,
1574 cm´1; 1H-NMR (DMSO-d6)
δ: 13.06 (s, 1H), 8.22 (s, 1H), 7.96 (s, 1H), 7.86 (d, 2H, J = 8.8 Hz), 7.71
(s, 1H), 7.53 (d, 1H, J = 8.8 Hz), 7.33 (d, 2H, J = 8.0 Hz), 7.25 (d, 3H, J = 8.0 Hz), 6.58–6.61 (m, 1H), 6.38
(d, 1H, J = 8.8 Hz), 5.88–5.93 (m, 1H), 4.84 (d, 2H, J = 1.6 Hz), 2.41 (s, 3H), 1.24 (s, 9H). 13C-NMR
(DMSO-d6) δ: 163.2 (1C, pyrazole), 161.9 (1C, pyrazole), 161.1 (1C, CO), 160.8 (1C, pyrazole), 158.7,
155.8, 154.2, 150.4, 150.0, 147.6, 138.9, 136.4, 135.1, 135.0, 127.5, 127.4, 123.8, 122.5, 117.2, 117.0, 116.1,
105.7, 97.4 (1C, pyrazole), 71.2 (1C, O-CH2), 51.2 (1C, O-CH3), 33.1 (1C, CH3-C), 31.3 (3C, C-CH3);
˝
MS (ESI): m/z 573.23 [M + Na]+. Mp 215.1–217.1 C.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-yl)urea (26e).
White solid (2.69 g, 45%). HPLC purity 98.3% (tR = 10.65 min). IR (KBr): 3253, 1670, 1637, 1599,
1
1579 cm´1; H-NMR (DMSO-d6)
δ: 13.07 (s, 1H), 8.95 (s, 1H), 8.46 (s, 1H), 7.97 (s, 1H), 7.86 (d, 1H,
J = 8.8 Hz), 7.53–7.56 (m, 3H), 7.38–7.41 (m, 2H), 7.17 (d, 1H, J = 2.4 Hz), 7.11 (dd, 1H, J = 2.4, 8.8 Hz),
6.61–6.65 (m, 1H), 6.39 (s, 1H), 6.35 (d, 1H, J = 8.8 Hz), 5.92–5.95 (m, 1H), 4.85 (d, 2H, J = 1.6 Hz), 1.24
(s, 9H); 13C-NMR (DMSO-d6)
δ: 162.8 (1C, pyrazole), 161.5 (1C, pyrazole), 160.6 (1C, CO), 160.4 (1C,
pyrazole), 158.2, 155.4, 153.8, 150.0, 149.6, 147.2, 138.5, 136.0, 134.7, 134.6, 127.1, 127.0, 123.4, 122.1,
116.7, 116.5, 115.7, 105.3, 96.9 (1C, pyrazole), 74.˝6 (1C, O-CH2), 32.8 (3C, CH3-C), 32.3 (1C, C-CH3);
MS (ESI): m/z 561.21 [M + Na]+. Mp 197.3–199.7 C.
1-(5-((1H-Indazol-5-yl)oxy)-2H-chromen-8-yl)-3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)urea (26f).
Yellow solid (3.50 g, 62%). HPLC purity 97.6% (tR = 10.54 min). IR (KBr): 3254, 1670, 1633, 1601,
1581 cm´1; 1H-NMR (DMSO-d6)
δ: 13.03 (s, 1H), 9.15 (s, 1H), 8.43 (s, 1H), 8.35 (dd, 1H, J = 2.0, 8.8 Hz),
7.93 (s, 1H), 7.81–7.84 (m, 3H), 7.50–7.53 (d, 1H, J = 8.8 Hz), 7.12 (d, 1H, J = 2.0 Hz), 7.06 (d, 1H, J = 2.0,
8.8 Hz), 6.58 (m, 1H), 6.40 (s, 1H), 6.34 (d, 1H, J = 8.8 Hz), 5.90–5.95 (m, 1H), 4.84 (d, 2H, J = 1.6 Hz),
1.24 (s, 9H); 13C-NMR (DMSO-d6)
δ: 162.0 (1C, pyrazole), 154.5 (1C, pyrazole), 153.0 (1C, CO), 149.2
(1C, pyrazole), 148.0, 146.2, 137.7, 136.9, 135.1, 133.8, 126.2, 126.1, 123.8, 122.1, 121.7, 121.1, 120.0, 115.9,
115.7, 115.4, 115.1, 104.5, 96.1 (1C,˝pyrazole), 74.4 (1C, O-CH2), 31.3 (1C, CH3-C), 30.1 (3C, C-CH3);
588.21 [M + Na]+. Mp 207.1–209.6 C.
3.2.13. General Method for the Synthesis of 27a–27e
˝
A solution of the corresponding 3a–3i (1.1 equiv.) and 18a–18b (1 equiv.) was stirred 2 h at 90 C
in the presence of Et3N (1 mL) in DMSO (50 mL). Then the mixture was poured into ice water and
the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried
(MgSO4), filtered and evaporated to dryness. The product was separated by column chromatography
using DCM/MeOH (80:1) as eluent.
1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-((5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)-benzyl)urea (27a)
From 3a (4.03 g, 10 mmol), and 18a (2.69 g, 10 mmol), after work-up and purification, 27a (3.66 g,
70%) was obtained as a white solid. HPLC purity 96.6% (tR = 8.46 min). IR (KBr): 3252, 1670, 1595,
.

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1
1575 cm´1; H-NMR (DMSO-d6)
δ: 8.25 (s, 1H), 7.38–7.41 (m, 2H), 7.35–7.38 (m, 2H), 7.11–7.15 (m,
3H), 7.15–7.18 (m, 1H), 6.91–6.93 (t, 1H, J = 8.0 Hz), 6.86–6.89 (m, 1H), 6.74–6.77 (m, 1H), 6.61 (d, 1H,
J = 8.8 Hz), 6.24 (s, 1H), 5.09–5.12 (m, 1H), 4.54 (s, 1H), 4.28 (d, 2H, J = 5.6 Hz), 2.65–2.68 (m, 2H),
2.37 (s, 3H), 1.87–1.91 (m, 2H), 1.63–1.67 (m, 2H), 1.29 (s, 9H). 13C-NMR (DMSO-d6)
δ: 160.9 (1C,
pyrazole), 156.0 (1C, CO), 154.9 (1C, pyrazole), 154.5, 139.0, 138.3, 137.0, 136.7, 135.9, 131.3, 130.7, 130.1,
129.3, 128.9, 124.7, 124.1, 119.2, 117.6, 116.0 (18C, Ar-C), 95.8 (1C, pyrazole), 66.4 (1C, HO-C), 38.6 (1C,
N-CH2), 32.9 (1C, CH2), 32.5 (1C, C), 31.5, (1C, CH2), 30.7 (3C, CH3), 21.3 (1C, Ar–CH3), 19.4 (1C, CH2).
˝
MS (ESI): m/z 547.20 [M + Na]+. Mp 201.6–203.7 C.
1-(3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-3-(2-((5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-oxy)benzyl)urea
(
27b). White solid (2.78 g, 50%). HPLC purity 96.7% (tR = 8.39 min). IR (KBr): 3251, 1667, 1593,
1
1573 cm´1; H-NMR (DMSO-d6)
δ: 8.59 (s, 1H), 8.33 (s, 2H), 7.83 (d, 2H, J = 8.8 Hz), 7.38 (d, 1H,
J = 8.8 Hz), 7.25–7.28 (m, 2H), 7.09–7.11 (m, 1H), 6.96–6.98 (t, 1H, J = 8.0 Hz), 6.81–6.85 (m, 1H),
6.74–6.77 (m, 1H), 6.61 (d, 1H, J = 8.8 Hz), 6.33 (s, 1H), 5.08–5.12 (m, 1H), 4.53 (s, 1H), 4.24 (d, 2H,
J = 5.6 Hz), 2.64–2.67 (m, 2H), 1.87–1.90 (m, 2H), 1.63–1.66 (m, 2H), 1.27 (s, 9H); 13C-NMR (DMSO-d6)
δ
: 162.9 (1C, pyrazole), 155.9 (1C, CO), 155.1 (1C, pyrazole), 154.4, 145.3, 144.8, 139.1, 139.0, 135.9, 131.2,
130.7, 129.2, 128.9, 125.3, 124.0, 123.6, 119.1, 117.6, 116.0 (18C, Ar-C), 99.1 (1C, pyrazole), 66.4 (1C,
HO-C), 38.7 (1C, N-CH2), 32.8 (1C, CH2), 32.7 (1C, C), 30.5 (3C, CH3), 29.5 (1C, CH2), 19.4 (1C, CH2);
˝
MS (ESI): m/z 578.05 [M + Na]+. Mp 202.1–204.3 C.
1-(3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-((5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)-benzyl)urea
(
27c). White solid (2.14 g, 42%). HPLC purity 98.8% (tR = 8.41 min). IR (KBr): 3250, 1667, 1593,
1
1573 cm´1; H-NMR (DMSO-d6)
δ: 8.31 (s, 1H), 7.49–7.52 (m, 4H), 7.39–7.42 (m, 2H), 7.26–7.28 (m,
2H), 7.11–7.14 (m, 1H), 6.92–6.94 (t, 1H, J = 8.0 Hz), 6.83–6.86 (m, 1H), 6.72–6.75 (m, 1H), 6.61 (d, 1H),
6.26 (s, 1H), 5.08–6.12 (m, 1H), 4.53 (s, 1H), 4.24 (d, 2H, J = 5.6 Hz), 2.64–2.68 (m, 2H), 1.87–1.91 (m,
2H), 1.67–1.71 (m, 2H), 1.25–1.29 (s, 9H); 13C-NMR (DMSO-d6)
δ: 160.7 (1C, pyrazole), 155.5 (1C,
CO), 154.5 (1C, pyrazole), 153.9, 138.8, 138.5, 137.8, 135.4, 130.8, 130.2, 129.2, 128.8, 128.4, 127.1, 124.2,
123.7, 118.7, 117.1, 115.5 (18C, Ar-C), 95.7 (1C, pyrazole), 65.9 (1C, HO-C), 38.1 (1C, N-CH2), 32.4 (1C,
CH2), 32.0 (1C, ˝C), 30.2 (3C, CH3), 29.0 (1C, CH2), 18.9 (1C, CH2); MS (ESI): m/z 533.07 [M + Na]+.
Mp 202.5–204.8 C.
1-(3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-3-(5-fluoro-2-((5-hydroxy-5,6,7,8-tetrahydro-naphthalen-
2-yl)oxy)benzyl)urea (27d). White solid (3.72 g, 65%). HPLC purity 97.4% (tR = 8.51 min). IR (KBr):
3251, 1668, 1593, 1573 cm´1; 1H-NMR (DMSO-d6)
δ: 8.59 (s, 1H), 8.33–8.36 (m, 2H), 7.83–7.88 (m, 2H),
7.38–7.41 (m, 1H), 7.25–7.29 (m, 1H), 7.09–7.13 (m, 1H), 6.96–6.98 (t, 1H, J = 8.0 Hz), 6.81–6.85 (m, 1H),
6.72–6.76 (m, 1H), 6.61 (d, 1H, J = 8.8 Hz), 6.31 (s, 1H), 5.05–5.07 (m, 1H), 4.53 (s, 1H), 4.23 (d, 2H,
J = 5.6 Hz), 2.64–2.68 (m, 2H), 1.86–1.89 (m, 2H), 1.63–1.66 (m, 2H), 1.25 (s, 9H). 13C-NMR (DMSO-d6)
δ
: 162.8 (1C, pyrazole), 160.0 (1C, CO), 157.7 (1C, pyrazole), 156.2, 155.3, 150.0, 145.3, 144.8, 139.0, 138.8,
135.8, 134.2, 130.7, 125.2, 123.5, 121.4, 116.9, 115.3, 115.0 (18C, Ar-C), 99.8 (1C, pyrazole), 66.4 (1C,
HO-C), 38.7 (1C, N-CH2), 32.8 (1C, CH2), 32.7 (1C, C), 30.4 (3C, CH3), 29.5 (1C, CH2), 19.4 (1C, CH2);
˝
MS (ESI): m/z 596.04 [M + Na]+. Mp 191.4–193.9 C.
1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(5-fluoro-2-((5-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-
oxy)benzyl)urea (27e). White solid (2.81 g, 52%). HPLC purity 97.3% (tR = 8.43 min). IR (KBr): 3251,
1
1667, 1597, 1576 cm´1; H-NMR (DMSO-d6)
δ
: 8.29 (s, 1H), 7.37–7.40 (m, 2H), 7.35–7.39 (m, 2H),
7.11–7.14 (m, 2H), 6.95–6.97 (t, 1H, J = 8.0 Hz), 6.86–6.91 (m, 1H), 6.98–7.01 (m, 1H), 6.70–6.77 (m, 1H),
6.58 (d, 1H, J = 8.8 Hz), 6.23 (s, 1H), 5.07–5.09 (m, 2H), 4.52 (s, 1H), 4.18 (d, 2H, J = 5.6 Hz), 2.61–2.65
(m, 2H), 2.35 (s, 3H), 1.84–1.87 (m, 2H), 1.63–1.66 (m, 2H), 1.21 (s, 9H). 13C-NMR (DMSO-d6)
δ: 160.9
(1C, pyrazole), 160.1 (1C, CO), 157.7 (1C, pyrazole), 156.3, 155.0, 150.0, 139.0, 138.0, 136.8, 135.8, 130.7,
130.1, 124.6, 121.5, 121.4, 117.0, 115.5, 115.4, 115.3 (18C, Ar-C), 96.2 (1C, pyrazole), 66.4 (1C, HO-C), 38.4
(1C, N-CH2), 32.9 (1C, CH2), 32.5 (1C, C), 30.7 (3C, CH3), 29.5 (1C, CH2), 21.1 (1C, Ar-CH3), 19.4 (1C,
˝
CH2).MS (ESI): m/z 565.09 [M + Na]+. Mp 200.1–203.8 C.

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3.2.14. General Method for the Synthesis of 28a–28i
˝
A solution of the corresponding 3a–3i (1.1 equiv.) and 21 (1 equiv.) was stirred 2 h at 90 C
in the presence of Et3N (1 mL) in DMSO (50 mL). Then the mixture was poured into ice water and
the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried
(MgSO4), filtered and evaporated to dryness. The product was separated by column chromatography
using DCM/MeOH (100:1) as eluent.
1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28a). From 3a (4.03 g,
10 mmol), and 21 (2.22 g, 10 mmol), 28a (3.43 g, 72%) was obtained as a white solid. HPLC purity
95.9% (tR = 8.02 min). IR (KBr): 3247, 1666, 1593, 1573cm´1; 1H-NMR (DMSO-d6)
δ: 9.04 (s, 1H), 8.29
(d, 1H, J = 8.0 Hz), 7.38–7.41 (m, 4H), 7.02 (t, 1H), 6.93–6.96 (m, 2H), 6.34 (s, 1H), 4.13–4.15 (t, 2H,
J = 5.6 Hz), 3.54–3.57 (m, 4H), 2.70–2.76˝(m, 2H), 2.44–2.48 (m, 4H), 2.36 (s, 3H), 1.25 (s, 9H); MS (ESI):
m/z 500.27 [M + Na]+. Mp 166.3–168.2 C.
1-(3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28b). White solid (2.31 g,
50%). HPLC purity 98.9% (tR = 8.11 min). IR (KBr) 3244, 1663, 1598, 1577 cm´1; 1H-NMR (DMSO-d6)
δ:
9.06 (s, 1H), 8.23 (d, 1H, J = 8.0 Hz), 7.35–7.39 (m, 4H), 7.00–7.04 (m, 2H), 6.91–6.95 (m, 2H), 6.30 (s, 1H),
4.13–4.15 (t, 2H, J = 5.6 Hz), 3.55–3.58 (m, 4H), 2.73–2.76 (m, 2H), 2.45–2.48 (m, 4H), 1.26 (s, 9H); MS
˝
(ESI): m/z 486.26 [M + Na]+. Mp 179.2–182.3 C.
1-(3-(tert-Butyl)-1-(m-tolyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28c). White solid
(3.00 g, 63%). HPLC purity 97.7% (tR = 8.05 min). IR (KBr): 3245, 1664, 1591, 1571 cm´1; 1H-NMR
(DMSO-d6) δ: 9.05 (s, 1H), 8.23 (d, 1H, J = 8.0 Hz), 7.36–7.40 (m, 4H), 7.03–7.07 (m, 1H), 6.91–6.94 (m,
2H), 6.36 (s, 1H), 4.13–4.15 (t, 2H, J = 5.6 Hz), 3.55–3.59 (m, 4H), 2.72–2.76˝(m, 2H), 2.42–2.46 (m, 4H),
2.33 (s, 3H), 1.26 (s, 9H); MS (ESI): m/z 500.27 [M + Na]+. Mp 191.6–193.7 C.
1-(3-(tert-Butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28d). White
solid (2.71 g, 55%). HPLC purity 96.6% (tR = 8.13 min). IR (KBr): 3246, 1659, 1594, 1575 cm´1; 1H-NMR
(DMSO-d6) δ: 9.06 (s, 1H), 8.25 (d, 1H, J = 8.0 Hz), 7.34–7.37 (m, 4H), 7.05–7.08 (m, 1H), 6.93–6.97 (m,
2H), 6.35 (s, 1H), 4.13–4.15 (t, 2H, J = 5.6 Hz), 3.84 (3H, O–CH3), 3.57–3.61 (m, 4H), 2.72–2.76 (m, 2H),
˝
2.42–2.48 (m, 4H), 1.25 (s, 9H); MS (ESI): m/z 516.24 [M + Na]+. Mp 154.2–156.4 C.
1-(3-(tert-Butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28e). White
solid (2.23 g, 45%). HPLC purity 99.1% (tR = 8.15 min). IR (KBr): 3243, 1657, 1597, 1577 cm´1; 1H-NMR
(DMSO-d6) δ: 9.12 (s, 1H), 8.23 (d, 1H, J = 8.0), 7.33–7.36 (m, 4H), 7.07–7.11 (m, 1H), 6.94–6.99 (m, 2H),
6.37 (s, 1H), 4.13–4.15 (t, 2H, J = 5.6), 3.59–3.62 (m, 4H), 2.74–2.78 (m, 2H), 2.44–2.48 (m, 4H), 1.26 (s,
˝
9H); MS (ESI): m/z 520.22 [M + Na]+. Mp 171.3–173.2 C.
1-(1-(4-Bromophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28f). White
solid (3.78 g, 70%). HPLC purity 96.4% (tR = 8.12 min). IR (KBr): 3243, 1657, 1597, 1577 cm´1; 1H-NMR
(DMSO-d6) δ: 9.13 (s, 1H), 8.20 (d, 1H, J = 8.0 Hz), 7.30–7.33 (m, 4H), 7.05–7.09 (m, 1H), 6.95–7.00 (m,
2H), 6.35 (s, 1H), 4.13–4.15 (t, 2H, J = 5.6 Hz), 3.57–4.02 (m, 4H), 2.76–2.81 (m, 2H), 2.42–2.45 (m, 4H),
˝
1.24 (s, 9H); MS (ESI): m/z 564.17 [M + Na]+. Mp 189.6–191.3 C.
1-(3-(tert-Butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28g). White
solid (2.84 g, yield 69%). HPLC purity 97.2% (tR = 8.13 min). IR (KBr): 3246, 1655, 1594, 1575 cm´1
;
1H-NMR (DMSO-d6),
δ: 9.13 (s, 1H), 8.23 (d, 1H, J = 8.0 Hz), 7.35–7.39 (m, 4H), 7.08–7.12 (m, 1H),
6.97–7.02 (m, 2H), 6.38 (s, 1H),4.16–4.18 (t, 2H, J = 5.6 Hz), 3.60–3.65 (m, 4H), 2.77–2.81 (m, 2H), 2.44–2.49
˝
(m, 4H), 1.25 (s, 9H); MS (ESI): m/z 504.17 [M + Na]+. Mp 170.3–172.0 C.
1-(3-(tert-Butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)phenyl)urea (28h). White solid
1
(2.54 g, 50%). HPLC purity 98.8% (tR =8.10 min). IR (KBr): 3244, 1656, 1600, 1578 cm´1; H-NMR
(DMSO-d6), δ: 9.10 (s, 1H), 8.21 (d, 1H, J = 8.0 Hz), 7.36–7.42 (m, 4H), 7.09–7.12 (m, 1H), 6.98–7.02 (m,

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2H), 6.39 (s, 1H), 4.17–4.19 (t, 2H, J = 5.6 Hz), 3.61–3.66 (m, 4H), 2.78–2.82 (m, 2H), 2.46–2.51 (m, 4H),
˝
1.26 (s, 9H); MS (ESI): m/z 531.24 [M + Na]+. Mp 163.3–165.6 C.
4-(3-(tert-Butyl)-5-(3-(2-(2-morpholinoethoxy)phenyl)ureido)-1H-pyrazol-1-yl)benzenesulfonamide
(28i).
White solid (2.81 g, 52%). HPLC purity 98.4% (tR = 7.53 min). IR (KBr): 3246, 1657, 1601, 1579 cm´1
;
1H-NMR (DMSO-d6)
δ: 9.09 (s, 1H), 8.23 (d, 1H, J = 8.0 Hz), 7.35–7.41 (m, 4H), 7.08–7.12 (m, 1H),
6.97–7.02 (m, 2H), 6.37 (s, 1H), 4.19–4.21 (t, 2H, J = 5.6 Hz), 3.63–3.67 (m, 4H), 2.75–2.77 (m, 2H),
˝
2.43–2.48 (m, 4H), 1.25 (s, 9H); MS (ESI): m/z 565.23 [M + Na]+. Mp 172.2–174.1 C.
3.2.15. General Method for the Synthesis of 29a–29i
˝
A solution of the corresponding 3a–3i (1.1 equiv.) and 24 (1 equiv.) was stirred 2 h at 90 C
in the presence of Et3N (1 mL) in DMSO (50 mL). Then the mixture was poured into ice water and
the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried
(MgSO4), filtered and evaporated to dryness. The product was separated by column chromatography
using DCM/MeOH (100:1) as eluent.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)urea (29a). From 3a (4.03 g,
10 mmol), and 24 (2.25 g, 10 mmol), after work-up and purification 29a (2.88 g, 60%) was obtained as
a white solid. HPLC purity 96.6% (tR = 11.32 min). IR (KBr): 3247, 1656, 1600, 1578 cm´1; 1H-NMR
(DMSO-d6) δ: 13.16 (s, 1H), 9.07 (s, 1H), 8.87 (s, 1H), 8.20 (d, 1H, J = 8.8 Hz), 8.04 (s, 1H), 7.53–7.59 (m,
3H), 7.40–7.44 (m, 3H), 7.37–7.39 (m, 1H), 7.35–7.37 (m, 1H), 7.30–7.34 (m, 1H), 7.13–7.16 (m, 1H), 6.39
˝
(s, 1H), 2.36 (s, 3H), 1.25 (s, 9H); MS (ESI): m/z 503.13 [M + Na]+. Mp 201.1–203.4 C.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)urea (29b). White solid
(1.95 g, 42%). HPLC purity 98.1% (tR = 11.41 min). IR (KBr): 3245, 1657, 1601, 1579 cm´1; 1H-NMR
(DMSO-d6) δ: 13.07 (s, 1H), 8.32 (s, 1H), 7.97 (s, 1H), 7.59 (d, 1H, J = 8.8 Hz), 7.49–7.55 (m, 4H), 7.37–7.42
(m, 1H), 7.27–7.32 (m, 3H), 7.12–7.15 (m, 2H), 6.95–6.97 (t, 1H, J = 5.6 Hz), 6.76 (d, 2H, J = 8.0 Hz), 1.25
˝
(s, 9H). MS (ESI): m/z 489.13 [M + Na]+. Mp 188.2–190.3 C.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(m-tolyl)-1H-pyrazol-5-yl)urea (29c). White solid
(2.64 g, 55%). HPLC purity 98.8% (tR =11.37 min). IR (KBr): 3246, 1652, 1594, 1574 cm´1; 1H-NMR
(DMSO-d6) δ: 13.08 (s, 1H), 8.31 (s, 1H), 7.97 (s, 1H), 7.54 (d, 1H, J = 8.8 Hz), 7.37–7.42 (m, 1H), 7.24–7.29
(m, 6H), 7.13–7.16 (m, 2H), 6.95–6.97 (t, 1H, J = 5.6 Hz), 6.73 (d, 1H, J = 8.0 Hz), 6.23 (s, 1H), 2.45 (s, 3H),
˝
1.26 (s, 9H); MS (ESI): m/z 503.56 [M + Na]+. Mp 194.3–196.5 C.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-yl)urea (29d). White
solid (2.13 g, 43%). HPLC purity 97.3% (tR = 11.11 min). IR (KBr): 3243, 1650, 1591, 1574 cm´1
;
1H-NMR (DMSO-d6)
δ:13.07 (s, 1H), 8.235 (s, 1H), 7.97 (s, 1H), 7.59 (d, 1H, J = 8.8 Hz), 7.36–7.42 (m,
3H), 7.27–7.31 (m, 4H), 7.13–7.16 (m, 2H), 6.95–6.97 (t, 1H, J = 5.6 Hz), 6.76 (d,˝1H, J = 8.0 Hz), 6.25 (s,
1H), 2.37(s, 3H), 1.27 (s, 9H); MS (ESI): m/z 519.23 [M + Na]+. Mp 197.1–199.6 C.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)urea
White solid (3.20 g, 63%). HPLC purity 95.9% (tR = 11.24 min). IR (KBr): 3252, 1671, 1600, 1580 cm´1
1H-NMR (DMSO-d6)
: 13.08 (s, 1H), 8.33 (s, 1H), 7.97 (s, 1H), 7.53 (d, 1H, J = 8.8 Hz), 7.47–7.50 (m,
(29e).
;
δ
4H), 7.26–7.31 (m, 3H), 7.12–7.16 (m, 2H), 6.92–6.94 (t, 1H, J = 5.6 Hz), 6.76 (d, 1H, J = 8.0 Hz), 6.23 (s,
1H), 1.26 (s, 9H); MS (ESI): m/z 523.17 [M + Na]+.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(1-(4-bromophenyl)-3-(tert-butyl)-1H-pyrazol-5-yl)urea (29f). White
solid (2.61 g, 48%). HPLC purity 97.7% (tR = 11.08 min). IR (KBr): 3250, 1667, 1602, 1579 cm´1
;
1H-NMR (DMSO-d6)
4H), 7.24–7.27 (m, 3H), 7.13–7.16 (m, 2H), 6.93–6.95 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.24 (s,
δ: 13.06 (s, 1H), 8.34 (s, 1H), 7.94 (s, 1H), 7.54 (d, 1H, J = 8.8 Hz), 7.46–7.52 (m,
˝
1H), 1.25 (s, 9H); MS (ESI): m/z 567.17 [M + Na]+. Mp 200.8.6–202.3 C.

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1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(4-fluorophenyl)-1H-pyrazol-5-yl)urea (29g). White
solid (2.66 g, yield 55%). HPLC purity 96.6% (tR = 11.14 min). IR (KBr): 3250, 1675, 1595, 1581 cm´1
1H-NMR (DMSO-d6)
: 13.05 (s, 1H), 8.35 (s, 1H), 7.99 (s, 1H), 7.54 (d, 1H, J = 8.8 Hz), 7.48–7.52 (m,
;
δ
4H), 7.27–7.32 (m, 3H), 7.13–7.16 (m, 2H), 6.95–6.97 (t, 1H, J = 5.6 Hz), 6.74 (d, 1H, J = 8.0 Hz), 6.25 (s,
˝
1H), 1.25(s, 9H); MS (ESI): m/z 507.20 [M + Na]+. Mp 189.6–191.7 C.
1-(2-((1H-Indazol-5-yl)oxy)phenyl)-3-(3-(tert-butyl)-1-(4-nitrophenyl)-1H-pyrazol-5-yl)urea (29h). White
solid (3.42 g, 67%). HPLC purity 98.9% (tR = 11.40 min). IR (KBr): 3247, 1673, 1591, 1579 cm´1
;
1H-NMR (DMSO-d6)
δ: 13.09 (s, 1H), 8.60 (s, 1H), 8.33 (d, 2H, J = 8.8 Hz), 8.03 (s, 1H), 7.85 (d, 2H,
J = 8.8 Hz), 7.56 (d, 1H, J = 8.8 Hz), 7.24–7.28 (m, 3H), 7.09–7.12 (m, 3H), 6.75 (d, 1H, J = 8.0 Hz), 6.33 (s,
˝
1H), 1.24 (s, 9H); MS (ESI): m/z 534.09 [M + Na]+. Mp 193.2–195.5 C.
4-(5-(3-(2-((1H-Indazol-5-yl)oxy)phenyl)ureido)-3-(tert-butyl)-1H-pyrazol-1-yl)benzenesulfonamide (29i).
White solid (3.59 g, yield 66%). HPLC purity 97.3% (tR = 9.96 min). IR (KBr): 3255, 1668, 1593,
1577 cm´1; 1H-NMR (DMSO-d6)
δ: 13.07 (s, 1H), 8.44 (s, 1H), 8.01 (s, 1H), 7.93 (d, 1H, J = 8.8 Hz), 7.73
(d, 2H, J = 8.8 Hz), 7.54 (d, 1H, J = 8.8 Hz), 7.48 (s, 2H), 7.28–7.31 (m, 3H), 7.15–7.18 (m, 2H), 6.96–7.01
(m, 1H), 6.75(d, ˝1H, J = 6.4 Hz), 6.34(s, 1H), 4.78 (s, 1H), 1.25(s, 9H); MS (ESI): m/z 568.18 [M + Na]+.
Mp 199.2–201.4 C.
3.2.16. General Method for the Synthesis of 30a–30d
A solution of the corresponding 3a–
3i (1.1 equiv.) and 16 (1 equiv.) was stirred 2 h at 90 ˝C in
the presence of Et3N (1 mL) and DMSO (50 mL). Then the mixture was poured into ice water and
the solution was extracted with EtOAc. The organic layer was washed with water and brine, dried
(MgSO4), filtered and evaporated to dryness. The product was separated by column chromatography
using DCM/MeOH (80:1) as eluent.
1-(3-(tert-Butyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)benzyl)urea (30a) From 3e
(4.23 g, 10 mmol), and 16 (2.36 g, 10 mmol), after work-up and purification 30a (3.83 g, 75%) was
obtained as a white solid. HPLC purity 98.4% (tR = 8.27 min). IR (KBr) 3254, 1669, 1596, 1578 cm´1
;
1H-NMR (DMSO-d6)
δ: 8.32 (s, 1H), 7.53–7.56 (m, 4H), 7.21–7.24 (m, 1H), 7.10 (dd, 1H, J = 1.6, 8.0 Hz),
6.98 (d, 1H, J = 8.0 Hz), 6.89–6.93 (m, 1H), 6.73–6.75 (t, 1H, J = 5.6 Hz), 6.27 (s, 1H), 4.19 (d, 2H, J = 5.6),
4.09–4.11 (t, 2H, J = 5.6 Hz), 3.55–3.57 (t, 4H, J = 4.8 Hz), 2.70–2.72 (t, 2H,˝J = 5.6 Hz), 2.50–2.52 (t, 4H,
J = 4.0 Hz), 1.25 (s, 9H); MS (ESI): m/z 534.24 [M + Na]+. Mp 163.6–165.7 C.
1-(3-(tert-Butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)benzyl)urea (30b). White solid
(2.94 g, 60%). HPLC purity 95.6% (tR = 8.16 min). IR (KBr): 3257, 1668, 1595, 1575 cm´1; 1H-NMR
(DMSO-d6) δ: 8.17 (s, 1H), 7.35 (d, 2H, J = 8.8 Hz), 7.21–7.26 (m, 1H), 7.11 (d, 1H, J = 8.0 Hz), 7.03 (d,
2H, J = 8.8 Hz), 6.98 (d, 1H, J = 8.0 Hz), 6.89–6.91 (t, 1H, J = 7.6 Hz), 6.74–6.76 (t, 1H, J = 5.6 Hz), 6.24
(s, 1H), 4.20 (d, 2H, J = 5.6 Hz), 4.09–4.11 (t, 2H, J = 5.6 Hz), 3.80 (s, 3H), 3.55–3.57 (t, 4H, J = 4.8 Hz),
2.71–2.73 (t, 2H,˝J = 5.6 Hz), 2.50–2.52 (t, 4H, J = 5.6 Hz), 1.24 (s, 9H). MS (ESI): m/z 514.24 [M + Na]+.
Mp 171.1–173.7 C.
4-(3-(tert-Butyl)-5-(3-(2-(2-morpholinoethoxy)benzyl)ureido)-1H-pyrazol-1-yl)benzenesulfonamide
White solid (3.05 g, 55%). HPLC purity 97.2% (tR = 7.49 min). IR (KBr) 3259, 1670, 1591, 1571 cm´1
1H-NMR (DMSO-d6)
: 8.46 (s, 1H), 7.90 (d, 2H, J = 8.0 Hz), 7.69 (d, 2H, J = 8.0 Hz), 7.48 (s, 2H),
(30c).
;
δ
7.22–7.25 (m, 1H), 7.12 (d, 1H, J = 6.4 Hz), 6.98 (d, 1H, J = 8.0 Hz), 6.91–6.93 (t, 1H, J = 8.0 Hz),
6.78–6.80 (t, 1H, J = 5.6 Hz), 4.20 (d, 2H, J = 5.6 Hz), 4.10 (t, 2H, J = 5.6 Hz), 3.55–3.57 (t, 4H, J = 4.8 Hz),
2.71–2.73(t, 2H, ˝J = 5.6 Hz), 2.50–2.52 (t, 4H, J = 5.6 Hz), 1.26 (s, 9H); MS (ESI): m/z 579.25 [M + Na]+.
Mp 159.5–161.9 C.

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1-(3-(tert-Butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(2-(2-morpholinoethoxy)benzyl)urea (30d). White solid (2.48 g,
52%). HPLC purity 97.2% (tR = 8.11 min). IR (KBr): 3261, 1671, 1593, 1574 cm´1; 1H-NMR (DMSO-d6)
δ
: 8.29 (s, 1H), 7.48–7.52 (m, 4H), 7.40–7.44 (m, 1H), 7.21–7.25 (m, 1H), 6.98 (d, 1H, J = 8.0 Hz), 6.89–6.92
(t, 1H, J = 7.6 Hz), 6.76–6.79 (t, 1H, J = 5.6 Hz), 6.28 (s, 1H), 4.20–4.22 (d, 2H, J = 5.6 Hz), 4.09–4.11 (t, 2H,
J = 5.6 Hz), 3.55–3.57 (t, 4H, J = 4.8 Hz), 2.70–2.72 (t, 2H, J = 5.6 Hz), 2.50–2.52 (t, 4H, J = 5.6 Hz), 1.25 (s,
˝
9H); MS (ESI): m/z 510.27 [M + Na]+. Mp 160.0–162.2 C.
3.3. IC50 Determination of Inhibition of MAPKAPK2 Release from the GFP-MAPKAPk2_BHK Cell Model
The GFP-MAPKAPk2_BHK cell model can differentially express the MAPKAPK2 molecule
and GFP information system so the activity can be derived from the contrast of the compound
BIRB-796 and the obtained compounds. GFP-MAPKAPk2_BHK cells were cultured in 96-well plates
(
2.0 ˆ 104/100 µL/well) containing 10% heat-inactivated foetal bovine serum (FBS) and 1 mg/mL
G418 at 37 ˝C in humidified air containing 5% CO2 for 18–24 h. The cells were washed with nutrient
solution (100 µL/well) and changed to the compound liquid (100 µL/well). Experiments were set
up with the control, and the compound was repeated at each concentration in three wells. The
cells were cultured in 96-well plates at 37 ˝C in humidified air containing 5% CO2 for 90 min. The
cells were washed with the dye solution (20 µL/well), and the dyed cells were held for 1 hour at
room temperature in the dye liquid. Analysis of nuclear translocation and calculation of the ability
of the compounds to inhibit nuclear translocation was performed using the IN Cell Analyzer 1000
Nuclear Trafficking Analysis Module System (GE, Madison, WI, USA). The cultured cells were treated
with the obtained compounds in seven concentration grads (1, 3, 10 and 30 nM, 0.1 and 0.3 µM).
The inhibitory percentage can be calculated using the amounts of cells after the disposition of the
different compounds.
3.4. IC50 Determination of Inhibition of TNF-α Release from THP-1 Cells after LPS Stimulation
THP-1 cells were cultured in RPMI medium 1640 with 10% (v/v) foetal bovine serum (FBS),
100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µ
M 2-mercaptoethanol at 37 ˝C and 5% CO2.
LPS stock was prepared at 1 mg/mL by adding 1 mL sterile endotoxin-free water to 1 mg LPS powder.
˝
The LPS stock was aliquoted and stored at
´
20 C. Test medium: RPMI Medium 1640 with 0.5%
(v/v) foetal bovine serum, 100 U/mL penicillin, 100
An amount of 100 K THP-1 cells were seeded into a 96-well cell culture plate in 100
The compounds were diluted at 1:3.5 in DMSO over 10 points (9 + 0) with a starting concentration of
333 M. An amount of 3.6 L of diluted compound was transferred to 96.4 L test medium, with a
starting concentration of 12 M. A total of 10 L compound was added to the plate with cells, and the
cells were incubated for 1 hour at 37 C. An amount of 10 L/well LPS was added to stimulate the cells.
µg/mL streptomycin, 50
µM 2-mercaptoethanol.
µL of test medium.
µ
µ
µ
µ
µ
˝
µ
The final LPS concentration was 300 ng/mL, and the DMSO concentration was 0.3%. Final compound
concentrations: 1000.00, 285.71, 81.63, 23.32, 6.67, 1.90, 0.54, 0.16, 0.04 and 0 nM. Two replicates were
˝
used for each dose. The plate was incubated for 5 hours at 37 C. The TNF-
were prepared with final concentrations of 2500, 1250, 625, 312, 156, 78, 39, 19.5, 9.75 and 0 pg/mL.
Three replicates were used for each dose. At the 5th hour, the TNF- in the supernatants and standard
curve samples was determined using the HTRF kit. The cells were centrifuged at 2000 rpm for 5 min,
and 10 L supernatant was added to a ProxiPlate-384 Plus assay plate (PerkinElmer). Amounts of
L Anti-TNF- -Cryptate (Invitrogen, Carlsbad, CA, USA) and 5 L Anti-TNF -XL665 (Invitrogen)
α standard curve samples
α
µ
5
µ
α
µ
α
were added, and the plate was incubated at RT for 15 h. The plate was read on an Envision instrument
(PerkinElmer) and the IC50 was calculated.

Molecules 2016, 21, 677
24 of 27
3.5. p38α MAP Kinase Activity Assessment Based on the Rate of Phosphorylation of ATF-2 (Activation
Transcription Factor 2) in an in Vitro Assay
Kinase reaction buffer was composed of 50 mM HEPES (pH 7.5), 0.01% BRIJ-35, 10 mM MgCl2
and 1 mM EGTA. Prepared MAPK14/p38
2-fold serial dilution was performed using 1
α
in 1
kinase buffer from 500 ng/mL using 16 dose points.
L) was added into the 384-well plate in triplicate and 0.8
M ATP in 1 kinase reaction buffer were prepared (1 mL). The
ˆ
kinase buffer with concentration at 500 ng/mL. A
ˆ
The serially diluted MAPK14/p38
substrate GFP-ATF2 (19–96) and 180
reaction was started by adding 5
well of the assay plate. The final starting concentration of MAPK14/p38
final GFP-ATF2 and ATP concentrations were 0.4 M and 90 M, respectively. The assay plate was
α
(5
µ
µM
µ
ˆ
µL of the GFP-ATF2(19–96) substrate and ATP solution into each
α
was 250ng/ml, and the
µ
µ
sealed and incubated for 1 hour at room temperature (RT). Antibody solution (1 mL) was prepared
from 20 mM EDTA and 4 nM Tb-antipATF2 (pThr71) antibody in TR-FRET dilution buffer. Antibody
solution (10 µL) was added into each well of the assay plate and mixed gently. The final EDTA
concentration was 10 mM, and the final Tb-antipATF2 (pThr71) concentration was 2 nM. The assay
plate was sealed and incubated for 30 minutes at RT. Finally, the TR-FRET signal was readed on the
Envision 2104 plate reader.
Inhibitor in 0.5% DMSO (2 µL/well) at 5-fold the final assay concentration was added into a
384-well assay plate. For the first inhibitor screening cycle, the final concentrations of inhibitors
were 3333, 1111, 370, 123, 41, 13.7, 4.57, 1.52, 0.51, 0.17 and 0.056 nM (3-fold dilution, 11 dose points,
two replicates for each dose). The inhibitor concentration was then adjusted according to the first cycle
result. MAPK14/p38
α
(4
µL/well) was added to each well of the 384-well assay plate and incubated
for 15 min at RT. To start the reaction, substrate GFP-ATF2 (19–96) and ATP (4
µL/well) were added
into kinase reaction buffer. The TR-FRET signal was read on the Envision 2104 plate reader. The
resulting TR-FRET emission ratio was plotted against the concentration of inhibitor, and the data fitted
to a sigmoidal dose-response curve with a variable slope. The IC50 concentration was calculated from
the curve.
3.6. Binding Experiments
The Biacore instrument uses a technique based on surface plasmon resonance to follow the
real-time binding of compounds to immobilized proteins on a carboxymethyl dextran surface in a
liquid milieu. Compounds were stored as 10 mM stock solutions in 100% dimethyl sulfoxide (DMSO).
All light-sensitive compounds were handled under yellow protective light. After dilution to 1 mM
with 100% DMSO, samples were mixed with 1.03- to 1.05-fold concentrated assay buffer and DMSO to
yield 10 µM compound in a final buffer composition of 50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM
MgCl2, 1 mM MnCl2, and 3% or 5% DMSO (assay buffer), which was also used as the instrument
running buffer and sample dilution buffer. ATP was dissolved directly in running buffer to make a
10 mM solution. The run was started with five start-up cycles in which running buffer was injected
(instead of a sample) followed by sample injection cycles. Zero concentration samples were used as
blanks. A typical analysis cycle consisted of a 60 s sample injection (30 µL/min), 120–200 s of buffer
flow (dissociation phase), followed by a needle and tubing wash with 50% DMSO and buffer, and
finally, a 30 s buffer injection to check for sample carry over. The flow cell temperature was 25 ˝C.
Between sample series, a solvent correction cycle was run according to the instrument manual to adjust
for referencing errors due to refractive index mismatches between running buffer and samples [35].
These data were used to calculate the KD for compounds.
4. Conclusions
In summary, we have designed and synthesized a novel series of substituted N,N1-diarylurea
compounds as potential p38
potencies. In order to obtain these target compounds, we designed a number of routes to synthesize the
targets. The target compounds were evaluated for the inhibitory activity against p38 , MAPKAPK2 in
α inhibitors and showed that some compounds possessed good inhibitory
α

Molecules 2016, 21, 677
25 of 27
BHK cells, TNF-
α
release in LPS-stimulated THP-1 cells and p38
α
binding experiments. A promising
overall profile led to the nomination of compound 25a as a p38
α
inhibitor development candidate. Further
information related to the research and development of compound 25a will be reported in due course.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
5/677/s1.
Acknowledgments: The authors gratefully acknowledge financial support from the National Nature Science
Foundation of China (Grant No. 81473139), the State Key Laboratory of Toxicology and Medical Countermeasures
and the program for innovative research team of the ministry of education and program for Liaoning innovative
research team at the university.
Author Contributions: W. Z. (Wu Zhong), D. Z. (Dongmei Zhao) and D.Z. (Dianxi Zhu) conceived and designed
the experiments; D.Z. (Dianxi Zhu) and Q.F. (Qifeng Xing) performed the experiments; D.Z. (Dianxi Zhu) wrote
the paper; All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).