Design, synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase

Article abstract of DOI:10.1016/j.bmc.2018.08.013

Inspired by that the multi-target inhibitors against receptor tyrosine kinases (RTKs) have significantly improved the effect of clinical treatment for cancer, and based on the chemical structure of Linifanib (ABT-869, Abbott), two series of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure were designed and synthesized as multi-target inhibitors against RTKs. The preliminary biological evaluation showed that several compounds exhibited comparable potency with Linifanib. Compound S21 was identified as the most potent inhibitor against Fms-like tyrosine kinase 3 (FLT-3), kinase insert domain containing receptor (KDR) and platelet-derived growth factor receptor β (PDGFR-β) with its IC₅₀ values were 4 nM, 3 nM and 8 nM respectively, it also showed potent inhibitory activities against several cancer cells.

Full text of DOI:10.1016/j.bmc.2018.08.013

Accepted Manuscript
Design, synthesis and structure-activity relationship of diaryl-ureas with novel
isoxazol[3,4-b]pyridine-3-amino-structure as multi-target inhibitors against re-
ceptor tyrosine kinase
Zhi-Hao Shi, Feng-Tao Liu, Hao-Zhong Tian, Yan-Min Zhang, Nian-Guang Li,
Tao Lu
PII:
S0968-0896(18)31350-6
https://doi.org/10.1016/j.bmc.2018.08.013
BMC 14500
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 July 2018
9 August 2018
10 August 2018
Please cite this article as: Shi, Z-H., Liu, F-T., Tian, H-Z., Zhang, Y-M., Li, N-G., Lu, T., Design, synthesis and
structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-amino-structure as multi-target
inhibitors against receptor tyrosine kinase, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.bmc.2018.08.013
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Design, synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-
b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase
Zhi-Hao Shia, Feng-Tao Liua, Hao-Zhong Tiana, Yan-Min Zhanga, Nian-Guang Lib,*, Tao Lua,*
aSchool of Science, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu
211198, China
bNational and Local Collaborative Engineering Center of Chinese Medicinal Resources
Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine,
138 Xianlin Road, Nanjing, Jiangsu 210023, China
* Corresponding author.
E-mail addresses: linianguang@njucm.edu.cn (N.-G. Li), lutao@cpu.edu.cn (T. L)
ABSTRACT
Inspired by that the multi-target inhibitors against receptor tyrosine kinases (RTKs) have
significantly improved the effect of clinical treatment for cancer, and based on the chemical
structure of Linifanib (ABT-869, Abbott), two series of diaryl-ureas with novel isoxazol[3,4-
b]pyridine-3-amino-structure were designed and synthesized as multi-target inhibitors against
RTKs. The preliminary biological evaluation showed that several compounds exhibited
comparable potency with Linifanib. Compound S21 was identified as the most potent inhibitor
against Fms-like tyrosine kinase 3 (FLT-3), kinase insert domain containing receptor (KDR) and
platelet-derived growth factor receptor β (PDGFR-β) with its IC50 values were 4 nM, 3 nM and 8
nM respectively, it also showed potent inhibitory activities against several caner cells.
Keywords: Multi-target inhibitors, receptor tyrosine kinase, FLT3, KDR, PDGFR-β,
antiangiogenesis
1. Introduction
Cancer is one of challenging field for medicinal chemists to discover effective yet safer
chemotherapeutic agents targeting various biochemical processes involved in progression of
cancers.1 Angiogenesis, the formation of new capillaries from the endothelium of an existing
vascular network, plays a crucial role in tumor growth. Angiogenesis is involved in metastasis
(uncontrolled spread of tumor cells) by supplying oxygen, nutrients, and related growth factors to
small tumors and removing the waste products of metabolism. Solid tumors cannot grow beyond
several cubic millimeters until they establish a blood supply because cells must be within 100–200
μm of a blood vessel to survive.2
Receptor tyrosine kinases (RTKs) have been shown not only to be key regulators of normal
cellular processes, but also to have a critical role in the development and progress of cancers.
RTKs play fundamental roles in transformation, proliferation, migration, differentiation and
metastasis of cancer cells.3 At least 19 RTK subfamilies have been identified. One example is the
platelet-derived growth factor receptor (PDGFR) subfamily. Members of this family include
PDGFR-α, PDGFR-β, colony-stimulating factor-1 receptor (CSF-1R), Fms-like tyrosine kinase 3
(FLT-3) and c-KIT. These kinases are believed to promote angiogenesis and tumor cell growth.
1

All RTKs share a similar molecular architecture, including a ligand binding extracellular region, a
single transmembrane helix, an intracellular regulatory domain, and a cytoplasmic tyrosine kinase
domain.4 The ATP-binding site of RTK shares remarkable sequence homology and structural
resemblance with each other such as epidermal growth factor receptor (EGFR), kinase insert
domain containing receptor (KDR) and fibroblast growth factor receptor 1 (FGFR1). All these
information provides a possibility for the design of multi-target inhibitors against RTKs. The
clinical application of multi-target RTK inhibitors has been validated as a therapeutic strategy by
positive results with Sorafenib (BAY-43-9006, Bayer), Vandetanib (ZD6474, AstraZeneca),
Sunitinib (SU-11248, Pfizer) and Linifanib (ABT-869, Abbott) (Fig. 1).5
NEt2
Br
O
Me
MeHN
O
N
H
H
H
N
NH2
H
H
N
Me
HN
N
HN
N
N
CF3
Cl
N
MeO
O
F
Me
N
O
N
F
H
O
F
Sunitinib
O
O
Linifanib
N
Sorafenib
N
Vandetanib
H
N
Me
Figure 1. Structures of multi-target receptor tyrosine kinase inhibitors
Sorafenib (BAY-43-9006, Bayer) is a small molecular inhibitor of several RTKs
(VEGFR/PDGFR/ERK) and Raf kinases, approved for the treatment of primary kidney cancer
(advanced renal cell carcinoma), advanced primary liver cancer (hepatocellular carcinoma), and
radioactive iodine resistant advanced thyroid carcinoma.6 Vandetanib (ZD6474, AstraZeneca) is
an anti-cancer drug that is used for the treatment of certain tumors of the thyroid gland, it acts as a
kinase inhibitor of a number of cell receptors (VEGFR/EGFR/RET-tyrosine kinase).7 Sunitinib
(SU11248, Pfizer) is an oral, small-molecule, multi-targeted RTK inhibitor of PDGF-
Rs/VEGFRs/c-KIT for the treatment of renal cell carcinoma (RCC) and imatinib-resistant
gastrointestinal stromal tumor (GIST).8 Linifanib (ABT-869, Abbott) is an oral active multi-target
RTK inhibitor of KDR, PDGFR-β and CSF-1R, the phase II trial studies shows that Linifanib
works well in treating patients with advanced, refractory colorectal cancer expressing k-Ras
mutations.9
Recently, many researches have highlighted diarylureas as potential antiproliferative
agents,10 and Linifanib has attracted considerable attention. In this study, we selected N,N'-
diarylurea as core structure to obtain novel multi-target RTK inhibitors,11 and we took Linifanib as
our lead compound because it could inhibit FLT-3, VEGFR-2 and PDGFR-β with its IC50s were 4
nM, 4 nM and 66 nM respectively.12
The structure–activity relationship (SAR) of Linifanib (Fig. 2) indicated that 3-
aminoindazole13 could serve as an efficient hinge-binding template for kinase inhibitors, and 3-
methyl-pyrazolo[3,4-b] pyridine core scaffold14 was also a good pharmacophore of the c-
KIT/PDGFRα dual inhibitor. By incorporating the N,N'-diaryl urea moiety at the above
pharmacophore, the RTK inhibitors were generated, which potently inhibited the tyrosine kinase
activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor
receptor families. The urea linker with lone linear alkyl group and terminal N-substituents, e.g.,
large fused heteroaryl group, could substantially improve the potency. Unfortunately, the amide,
sulfonamide and thiourea linker obviously lost activity against all the kinases (PDGFRα, VEGFR2
and FGFR1).14
2

N
N
N
HN
HN
HN
N
N
N
N
H
H
N
H
H
N
N
N
OH
potency (especially PDGFRα) was increased
OH
S
O
O
S
O
lost activity
CH2OH
CF3
N
N
HN
the potency against c-KIT/PDGFRα was increased
large fused aryl groups increased c-KIT/PDGFRα inhibition
HN
H
H
N
NH2
N
Me
N
N
N
HN
maintained fairly
potent c-KIT inhibition
but lost PDGFRα inhibition
HN
N
complete loss of
all kinase activities
O
S
N
N
F
heteroaryl groups and cyclopentyl or a cyclohexyl group reduced kinase potency
Linifanib
NH2
Introduction of substitution at R1 positon
greatly reduced KDR potency.
Incorporation of a fluoro or a methyl group was tolerated at meta-, ortho-, and para-positions.
Substitution with an m-Br or m-OH is also tolerated.
N
N
R1
R
Very potent KDR inhibitory activity was achieved by introduction of an m-Et, an m-Cl, or an m-CF3
Incorporation of an additional fluoro group at the meta-position to the CF3 group
caused a reduction in KDR affinity.
.
Substituents at R2 such as Me, MeO, F,
and Br as well as various polar groups
that were attached to the position via a
three-atom ether link (CH2CH2O) were
well tolerated. When the link was
R2
shortened to a methylene unit, RTK
inhibitory activity was reduced.
Figure 2. The structure activity relationship of Linifanib.
In order to expand the structural diversity of Linifanib, our design strategy firstly
incorporated 3-amino-isoxazol[3,4-b]pyridine to form interaction with ATP-bind site (Fig. 3).
Based on the docking studies between Linifanib and S27 with FLT3 (PDB ID: 4RT7), the 3-
aminoindazole in Linifanib could interact with the active sites in FLT3 as the same as 3-amino-
isoxazol[3,4-b]pyridine in S27. The two nitrogen atoms in 3-aminoindazole in Linifanib, the
nitrogen atom and oxygen atom in isoxazole ring of S27 could form two hydrogen bonds with Cys
694, and the amino group in the two compounds could form a hydrogen bond to Glu 692 in the
active hinge region of FLT3. The carbonyl group in urea moiety in Linifanib and S27 could form
one hydrogen bond with Asp 829, and one NH of the urea moiety could form one hydrogen bond
with Glu 661. Secondly, we investigated the influence of the terminal aniline which was replaced
by other substituted benzene rings, considering its broadly biological activity in anticancer drugs.
Furthermore, the substituted acrylamide was also introduced to the target compounds, hoping our
target compounds could form irreversible bonds with the kinase, because the irreversible kinase
inhibitors show enhanced potency when compared to structurally similar conventional inhibitors,
and there is also the expectation that an irreversible inhibitor need not exhibit prolonged
circulating blood levels to achieve a desired biological effect.15 Based on the docking studies, we
found that the substituted acrylamide at R1 position could be in the large hydrophobic pockets
formed by Lys644, Met664, Leu668, Val808 and His809. The distance between the β-carbon in
the unsaturated amide and the Cys807 was only 4.4Å, it was fit to form a covalent bond.
H
N
H
N
NH2
O
O
N
O
S27
N
H
N
Docking model of Linifanib and S27 in FLT3
The action between S27 and FLT3
R= CH3,
R3
H
H
H
N
H
N
NH2
NH2
N
N
R2
R1
Me
O
O
O
O
N
N
R1= H, F, Cl, CH3, OCH3,
,
HN
,
O
O
N
H
N
N
F
H
H
R2= H, F, Cl, CH3, CF3
R3= H, F, Cl
N
Linifanib
N
O
R
Target compounds S11-S110 and S21-S28
Figure 3. The design of expanding the structural diversity of Linifanib.
2. Results and discussion
2.1. Synthesis
3

The target compounds were synthesized through two different routes. The series S11–S110 in
which the phenyl group was substituted at the isoxazol[3,4-b]pyridine were synthesized as shown
in Scheme 1. The Aldol reaction between acetophenone (1) and 4-nitro-benzaldehyde (2) afforded
3 in 86% yield,16 and the Michael addition of propanedinitrile to 3 produced 4.17 Subsequently, we
focused our attention to optimize the reaction condition between 4 and hydroxylamine
hydrochloride to produce the important intermediate 5,18 when MeOH was selected as solvent, and
the molar ration of compound 4:hydroxylamine hydrochloride:KOH was 1:2:3.12, compound 5
was obtained in 77% yield. After the nitro-group in 5 was reduced to amino by stannous chloride
dehydrate, the reaction between 6 and isocyanates (14a–14i, 18a) afforded the target compounds
S11–S110 in good yield.
NO2
O
CN
NO2
NO2
+
a
c
b
NC
O
OHC
1
2
4
3
O
R1
H
H
NH2
NH2
NH2
NH2
e
NO2
N
N
R2
R3
O
N
O
N
O
N
O
d
N
N
N
S11-S110
6
5
Scheme 1. Reagents and conditions: (a) NaOH, EtOH, 25°C, 24h, 86%; (b) CH2(CN)2, Bu3P, DCM, N2, reflux, 2h,
.
78%; (c) HO-NH2 HCl, KOH, MeOH, 0–5°C, 1h, 25°C, 24h, 77%; (d) SnCl2·2H2O, EtOH, reflux, 89%; (e)
Isocyanates (14a–14i, 18a), Et3N, EtOH, 25°C, 2h, 73%–88%.
The series S21–S28 in which the methyl group was substituted at the isoxazol[3,4-b]pyridine
were obtained as shown in Scheme 2. The Aldol reaction between 4-nitro-benzaldehyde (7) and
acetone afforded 8 in 85% yield,16 then the cyclization between 8 and cyanoacetamide produced 9
in 83% yield.19 The carbonyl group in 9 was transferred into chlorine by POCl3 to give 10, then
the reduction of nitro group to amino group by iron powder afforded 11. The second cyclization
using 11 and hydroxylamine hydrochloride produced 12 in 69%. Finally, the amino group in 12
reacted with isocyanates (14a, 14d, 14g, 14k, 14l, 18b, 18c, 25) afforded the target compounds
S21–S28 in good yield.
NO2
NO2
CN
CN
NO2
NO2
c
a
b
Cl
O
O
N
HN
9
7
10
8
O
R1
H
H
NH2
NH2
N
N
R2
R3
NH2
NH2
O
N
O
N
CN
O
e
Cl
f
d
N
N
N
12
S21-S28
11
Scheme 2. Reagents and conditions: (a) Acetone, K2CO3, 12h, then HCl, 6h, 85%; (b) t-BuOK, cyanoacetamide,
DMSO, 1h, then t-BuOK, air, 2h, 83%; (c) POCl3, reflux, 2h, 77%; (d) Fe, acetic acid, EtOH, reflux, 2h, 89%; (e)
.
HO-NH2 HCl, t-BuOH, reflux, 2h, then t-BuOK, reflux, 0.5h, 69%; (f) Isocyanates (14a, 14d, 14g, 14k, 14l, 18b,
18c, 25), Et3N, EtOH, 25°C, 2h, 65%–82%.
4

R1
14a: R1=H, R2=CH3, R3=H.
14b: R1=H, R2=H, R3=F.
14c: R1=H, R2=H, R3=H.
14d: R1=H, R2=Cl, R3=H.
14e: R1=H, R2=H, R3=CH3.
14f: R1=H, R2=H, R3=OCH3.
14g: R1=H, R2=CF3, R3=Cl.
14h: R1=H, R2=F, R3=H.
14i: R1=F, R2=H, R3=H.
14j: R1=H, R2=H, R3=H.
14k: R1=H, R2=CF3, R3=H.
14l: R1=Cl, R2=H, R3=H.
R1
OCN
R2
R3
H2N
R2
R3
a
14a~14l
13a-13l
O
R1
H2N
R1
OCN
R1
O2N
R1
O2N
R2
R2
d
R2
Cl
R2
c
b
NH2
N
H
O
N
H
O
N
H
O
15
18a: R1=H, R2=H
17a: R1=H, R2=H
16a: R1=H, R2=H
18b: R1=H, R2=CH3
18c: R1=CH3, R2=CH3
17b: R1=H, R2=CH3
17c: R1=CH3, R2=CH3
16b: R1=H, R2=CH3
16c: R1=CH3, R2=CH3
O
O2N
O2N
O2N
O2N
OH
Br
g
f
Cl
N
H
O
N
O
NH2
N
H
O
e
H
CH3
CH3
O2N
CH3
CH3
N
22
20
21
19
H2N
OCN
N
N
j
i
h
O
O
O
N
H
O
N
O
N
O
H
H
CH3
CH3
CH3
23
25
24
Scheme 3: Reagents and conditions: (a) Triphosgene, EtOAc, 25°C, 1h, then reflux, 4–6h; (b) DIPEA, EtOAc,
25°C, 3h, 85–93%; (c) Fe, EtOH, acetic acid, reflux, 2h, 82–91%; (d) Triphosgene, EtOAc, 0°C, 0.5h, 25°C, 1h,
reflux, 4–6h; (e) DIPEA, EtOAc, 25°C, 3h, 85%; (f) paraformaldehyde, DABCO, 1,4-dioxane:H2O=5:3, 85°C, 8h,
88%; (g) PBr3, DCM, 25°C, 15min, 84%; (h) morpholine, DCM, 25°C, 15min, 85%; (i) Fe, EtOH, acetic acid,
reflux, 93%; (j) Triphosgene, EtOAc, 0°C, 0.5h, 25°C, 1h, reflux, 4h.
The side chains 14a–14l, 18a–18c and 25 were synthesized as shown in Scheme 3. The
substituted aniline 13a–13l reacted with triphosgene afforded the isocyanate 14a–14l.20,21 The
reaction between 15a–15b and acroleyl chloride produced the amide derivatives 16a–16c,22,23
reduction of the nitro group in 16a–16c followed by reaction with triphosgene yielded the
substituted isocyanate 18a–18c. The side chain 25 was synthesized from 2-methyl-4-
nitrobenzenamine (19), reaction of 19 with acroleyl chloride afforded 20 in 85% yield, then the
aldol condensation between 20 and paraformaldehyde produced 21. After the hydroxyl group in
21 was transformed into bromine by PBr3, the alkylation of 22 to morpholine afforded 23 in 85%
yield. At last, the nitro group in 23 was reduced using iron powder, the obtained aniline 24 reacted
with triphosgene produced the side chain 25.
2.2. Biological investigations
2.2.1. Receptor tyrosine kinases inhibitory activities
Table 1 Chemical structures and biological activities of title compounds (IC50, nM)
R1
H
H
NH2
N
N
R2
R3
O
N
A
O
N
R
Compd.
R
R1
H
R2
R3
H
FLT3
191
KDR
130
PDGFR-β
S11
CH3
H
1230
S12
S13
H
H
F
449
696
NA
3780
3520
H
H
1010
5

S14
S15
S16
S17
S18
S19
H
H
H
H
H
F
Cl
H
H
CH3
OCH3
Cl
128
311
177
24
259
NA
NA
685
105
NA
2230
1260
NA
H
CF3
F
290
H
565
NA
3250
NA
H
H
O
S110
H
H
NA
NA
NA
N
H
S21
S22
S23
S24
S25
CH3
CH3
CH3
CH3
CH3
H
H
H
H
Cl
CH3
Cl
H
H
Cl
H
H
4
5
3
8
40
20
20
820
25
CF3
CF3
H
3
177
11
9
51
600
O
S26
S27
CH3
CH3
H
H
H
438
39
NA
NA
NA
NA
N
H
O
CH3
N
H
O
S28
CH3
H
CH3
N
N
40
7
167
28
510
60
H
O
Linifanib
All the title compounds were evaluated for their enzymatic inhibition against FLT3, KDR
and PDGFR-β. The tyrosine kinase inhibitory potency was assayed according to our previous
report.24 The enzymatic inhibitory activity of two series of diarylureas was summarized in Table 1.
For the phenyl substituted isoxazol[3,4-b]pyridine derivatives, compound S19 with fluorine
substituted at the ortho position in the A ring showed no inhibitory activity against the three
enzymes. When the substitution was introduced at para position in the A ring, only S12 showed
inhibitory activity with its IC50s against FLT3, KDR and PDGFR-β were 449 nM, 696 nM and
3780 nM respectively. Furthermore, when the substitution was introduced at meta position, three
compounds S11, S14 and S18 showed potent inhibitory activities against FLT3, KDR and
PDGFR-β. Interestingly, when the meta position and the para position were both substituted, the
obtained compound S17 showed the most potent inhibitory activities in this series derivatives,
with its IC50s against FLT3 and PDGFR-β were 24 nM and 290 nM. This result suggested that
introduction of substitution at meta position and the para position in ring A of the derivatives was
6

favorable for their kinases inhibitory activities.
Compounds incorporated with methyl group substituted at the isoxazol[3,4-b]pyridine were
generally more potent that those bearing phenyl group. For example, the IC50s against the three
enzymes for compound S22 were 5 nM, 25 nM and 40 nM respectively, while the IC50s for
compound S14 were 128 nM, 259 nM and 2230 nM respectively. When the substitution was
introduced at the para position in the A ring, compound S26 lost its inhibitory activity, although
its IC50 for FLT3 was 438 nM. When the chlorine was introduced at the ortho position in the A
ring, the obtained compound S25 showed potent inhibitory activity against the three enzymes,
with its IC50s against FLT3, KDR and PDGFR-β were 51 nM, 600 nM and 820 nM respectively.
When the chlorine was introduced at the meta position in the A ring, compound S22 showed more
potent inhibitory activity than S25. The most potent compound was S21, with its IC50s against
FLT3, KDR and PDGFR-β were 4 nM, 3 nM and 8 nM respectively, while the IC50s of Linifanib
against the three enzymes were only 7 nM, 28 nM and 60 nM. Although the compound lost its
inhibitory activity when the substitution was introduced at the para position at the A ring, when
the para and the meta positions were both substituted, compound S28 still showed potent
inhibitory activity against the three enzymes, with its IC50s were 40 nM, 167 nM and 510 nM
respectively. This result confirmed that introduction of substitution at meta position in ring A was
favorable for the activity.
Unfortunately, compounds S110, S26, S27 and S28 with substituted acrylamide were less
potent activities than the other derivatives against the three enzymes, this might be that these
compounds did not form irreversible bonds with the kinase.
2.2.2. Antiproliferative activities
Table 2 Antiproliferative activities of title compounds (IC50, μM)
Compd.
S21
HUVEC
11.67
15.18
12.64
7.95
MCF-7
10.49
39.48
28.79
37.44
17.03
MV4-11
0.12
S22
0.54
S23
0.34
S24
0.61
Linifanib
40.11
0.69
Because the four compounds S21–S24 showed potent inhibitory activities against the three
enzymes, so we selected these four compounds to evaluate their antiproliferative activity against
human umbilical vein endothelial cells (HUVEC), human breast adenocarcinoma cell line (MCF-7)
and human leukemia cell lines (MV4-11) through the CellTiter-Glo (CTG) cell growth inhibition
test module.24 HUVECs, derived from the endothelium of veins from the umbilical cord, are the
most commonly studied human endothelial cell type in angiogenesis.25 The ability of these
derivatives to inhibit the cell growth was summarized in Table 2 with Linifanib as positive control.
All the four compounds showed potent antiproliferative activity against HUVEC with their IC50
values were 11.67 μM, 15.18 μM, 12.64 μM and 7.95 μM respectively, and the IC50 value of
Linifanib against HUVEC was only 40.11 μM. They also showed more potent inhibitory activities
than Linifanib against MV4-11 with their IC50 values were 0.12 μM, 0.54 μM, 0.34 μM and 0.61
μM respectively. For the MCF-7, only S21 was more potent than Linifanib with its IC50 value of
10.49 μM.
7

2.2.3. Docking studies
Because compound S21 was very potent to inhibit the three kinases with its IC50s against
FLT3, KDR and PDGFR-β were 4 nM, 3 nM and 8 nM respectively, and S21 also showed potent
antiproliferative activities against HUVEC, MCF-7 and MV4-11. So in order to further investigate
the action mechanism of the S21, we performed molecular docking studies to investigate the
binding modes and rationalize the efficiency. Compound S21 was docked into the ATP-bind site
of FLT3 (PDB ID: 4RT7), KDR (PDB ID: 1YWN) and PDGFR-β (PDB ID: 3MJG) which were
obtained from the Protein Data Bank (RCSB PDB).26 The binding mode of S21 into the active site
of three RTKs was shown in Figure 4.
A
B
C
Figure 4. Interaction between compound S21 and FLT3 (PDB ID: 4RT7) (A), KDR (PDB ID: 1YWN) (B) and
PDGFR-β (PDB ID: 3MJG) (C). Hydrogen bonds are shown as yellow lines.
Compound S21 bound across the hydrophobic pocket and ATP binding site of FLT3 through
five hydrogen bonds (Fig. 4A). The nitrogen atom and oxygen atom in isoxazole ring formed two
hydrogen bonds with Cys 694 with the length were 2.1 Å and 1.8 Å respectively. The amino group
formed a hydrogen bond to Glu 692 with length of 1.9 Å. The carbonyl group in urea moity
formed one hydrogen bond with Asp 829 with the length of 1.8 Å, and one NH of the urea moiety
formed one hydrogen bond with Glu 661 with distance of 1.7 Å. For complex of compound S21
with KDR (Fig. 4B), there were also five hydrogen bonds consisting the binding. The nitrogen
atom and oxygen atom in isoxazole ring formed two hydrogen bonds with Cys 917 with the length
were 2.6 Å and 1.9 Å respectively. The carbonyl group in urea moity formed one hydrogen bond
with the length of 1.8 Å, and two NH of the urea moiety formed two hydrogen bonds with Glu
8

883 with distances were both 1.9 Å. For complex of compound S21 with PDGFR-β (Fig. 4C),
only the two NH of the urea moiety formed two hydrogen bonds with Glu 241. This information
confirmed that the urea moiety was very important to form hydrogen bonds with various RTKs.
Furthermore, the isoxazole could serve as a favorable pharmacophore, it played an essential role in
binding with the ATP-bind site of FLT3 and KDR.
3. Conclusion
In this study, we designed and synthesized diaryl-ureas with novel isoxazol[3,4-b]pyridine-3-
amino-structure using Linifanib as a lead compound, considering that the multi-target inhibitors
against RTKs are highly useful anticancer agents with improved clinical efficacies. The kinase
inhibitory and anticancer activities showed that some diaryl-ureas exhibited comparable potency
with Linifanib. Fortunately, compound S21 was identified as the most potent inhibitor against
FLT3, KDR and PDGFR-β with its IC50 values were 4 nM, 3 nM and 8 nM respectively, it also
showed potent inhibitory activities against several caner cells, docking studies showed that the
urea moiety and isoxazole ring could form hydrogen bonds with the ATP-bind sites in RTKs. S21
could be used as novel lead compound to further develop multi-target inhibitors against RTKs.
4. Experimental
4.1. Chemical synthesis
Reagents and solvents were purchased from commercial sources and used without further
purification unless otherwise specified. Air- and moisture-sensitive liquids and solutions were
transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary
evaporation below 45 °C at approximately 20 mmHg. All non-aqueous reactions were carried out
under anhydrous conditions using flame-dried glassware in a nitrogen atmosphere in dry, freshly
distilled solvents, unless otherwise noted. Yields refer to chromatographically and
spectroscopically (1H NMR) homogeneous materials, unless otherwise stated. Reactions were
monitored by thin-layer chromatography (TLC) carried out on 0.15–0.20 mm Yantai silica gel
plates (RSGF 254) using UV light as the visualizing agent. Chromatography was performed on
Qingdao silica gel (160–200 mesh) with petroleum ether (60–90) and ethyl acetate mixtures as
1
eluant. Melting points (Mp) were measured on a WRS-1B apparatus and were uncorrected. H
NMR spectra were obtained with a Bruker AV-300 (300 MHz). Chemical shifts are recorded in
ppm downfield from tetramethylsilane. J values are given in Hz. Abbreviations used are s (singlet),
d (doublet), t (triplet), q (quartet), b (broad) and m (multiplet). ESI-MS spectra were recorded on a
Waters Synapt HDMS spectrometer.
4.1.1 The synthesis of S11–S110
(E)-3-(4-Nitrophenyl)-1-phenylprop-2-en-1-one (3)
A mixture of acetophenone (1) (6 g, 0.05 mol) and 4-nitro-benzaldehyde (2) (7.5 g, 0.05 mol)
was stirred in EtOH (120 mL) at 0–5 °C, then 100 mL solution of NaOH (1 N) in water was added
dropwise into the reaction mixture under stirring. After the mixture was allowed to stir for 24 h at
25°C, the ph value of the solution was adjusted to 7 by dilute hydrochloric acid (1 N), the yellow
solid obtained was collected by filtration, washed with water, and dried under vacuum to produce
3 (10.88 g, 86%) as a yellow solid. Mp 167–169 °C.16 1H NMR (300 MHz, DMSO-d6) δ 8.21 (d, J
= 8.8 Hz, 2H), 8.11 (d, J = 15.8 Hz, 1H), 7.93 (d, J = 15.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.62-
7.66 (m, 2H), 7.47-7.53 (m, 3H). ESI-MS m/z: 252 [M-H]-, 254 [M+H]-, 276 [M+Na]-.
2-(1-(4-Nitrophenyl)-3-oxo-3-phenylpropyl)malononitrile (4)
9

Bu3P (280 μl, 0.001 mmol) was added to a solution of 3 (8 g, 0.032 mol) and malononitrile
(6.6 g, 0.1 mol) in anhydrous CH2Cl2 (25 mL), after the mixture was refluxed under N2 at room
temperature for 2h, the white solid appeared was obtained by filtration to produce 4 (7.96 g, 78%).
Mp 150–152 °C.17 1H NMR (300 MHz, DMSO-d6) δ 8.21 (d, J = 8.5 Hz, 2H), 8.01 (d, J = 7.5Hz,
2H), 7.8 (d, J = 8.5Hz, 2H), 7.59-7.62 (m, 1H), 7.51-7.54 (m, 2H), 5.32 (d, J = 10.4 Hz, 1H), 4.01
(d, J =10.4 Hz, 2H), 5.29-5.32 (m, 1H). ESI-MS m/z: 318 [M-H]-, 320 [M+H]+, 342 [M+Na]+.
4-(4-Nitrophenyl)-6-phenylisoxazolo[3,4-b]pyridin-3-amine (5)
A solution of KOH (0.42 g, 7.5 mmol) in MeOH (60 ml) was added dropwise into the
reaction mixture of 4 (0.77 g, 2.4 mmol) and hydroxylamine hydrochloride (0.33 g, 4.8 mmol) in
MeOH (40 ml) in 1 h at 0–5 °C, then the reaction mixture was warmed to 25 °C and stirred for 24
h, the solid appeared was filtered to afford 5 (0.61 g, 77%) as a yellow solid. Mp > 300 °C.18 1
H
NMR (300 MHz, DMSO-d6) δ 8.38 (d, J = 8.7 Hz, 2H), 8.20-8.24 (m, 2H), 8.03 (d, J = 8.7Hz,
2H), 7.89 (s, 1H), 7.47-7.51 (m, 3H), 5.62 (s, 2H). ESI-MS m/z: 331 [M-H]-, 333 [M+H]+, 355
[M+Na]+.
4-(4-Aminophenyl)-6-phenylisoxazolo[3,4-b]pyridin-3-amine (6)
Stannous chloride dehydrate (1.2 g 0.53 mmol) was added to a solution of compound 5 (0.8 g,
2.4 mmol) dissolved in 95% ethanol (50 ml), after the mixture was refluxed under N2 for 3 h, the
reaction mixture was partitioned between 200 ml dichloromethane and 200 ml water. The
dichloromethane layer was washed with brine, dried over MgSO4, filtered and concentrated. The
crude material was purified by column chromatography (2% methanol in dichloromethane) to
yield 6 (645 mg, 89%) as a yellow solid. Mp 229–231 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.19
(d, J = 8.4 Hz, 2H), 7.72 (s, 1H), 7.47-7.53 (m, 5H), 6.73 (d, J = 8.4 Hz, 2H), 5.7 (s, 2H), 5.5 (s,
2H). ESI-MS m/z: 301 [M-H]-, 303 [M+H]+, 325 [M+Na]+.
The substituted isocyanates (0.6~0.8mmol) was added into a solution of compound 6 (60 mg,
0.2 mmol) dissolved in anhydrous EtOH (13 mL) at 25 °C, followed by
catalytic amount of triethylamine, after stirring at 25 °C for 2 h, the solid obtained was purified by
column chromatography on silica gel using 30% ethyl acetate in petroleum ether as eluent to
afford S11–S10 as yellow solids.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-m-tolylurea (S11)
1
Synthesized from 14a. Yield 76%. Mp 228–230 C. H-NMR (300 MHz, DMSO-d6) δ 9.00
(s, 1H), 8.73 (s, 1H), 8.25 (d, J = 8.7 Hz, 2H), 7.86 (s, 1H), 7.68-7.71 (m, 4H), 7.53-7.57 (m, 3H),
7.34 (s, 1H), 7.26-7.29 (m, 1H), 7.16-7.18 (m, 1H), 6.82 (d, J = 7.41 Hz, 1H), 5.60 (s, 2H), 2.30 (s,
3H). ESI-MS m/z: 434 [M-H]-, [M+H]+ 436, [M+Na]+ 458.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-fluorophenyl)urea (S12)
1
Synthesized from 14b. Yield 73%. Mp 229–231 C. H-NMR (300 MHz, DMSO-d6) δ 9.01
(s, 1H), 8.79 (s, 1H), 8.23 (m, 2H), 7.85 (s, 1H), 7.68-7.71 (m, 4H), 7.48-7.55 (m, 5H), 7.12-7.18
(m, 2H), 5.58 (s, 2H). ESI-MS m/z: 438 [M-H]-, 440 [M+H]+, 462 [M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-phenylurea (S13)
1
Synthesized from 14c. Yield 79%. Mp 198–200 C. H-NMR (300 MHz, DMSO-d6) δ 9.01
(s, 1H), 8.79 (s, 1H), 8.23-8.26 (m, 2H), 7.85 (s, 1H), 7.71-7.74 (m, 4H), 7.48-7.55 (m, 5H), 7.28-
7.34 (t, 2H), 6.97-7.03 (t, 1H), 5.58 (s, 2H). ESI-MS m/z: 420 [M-H]-, 422 [M+H]+, 444 [M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(3-chlorophenyl)urea (S14)
1
Synthesized from 14d. Yield 85%. Mp 240–242 C. H-NMR (300 MHz, DMSO-d6) δ 9.10
(s, 1H), 9.02 (s, 1H), 8.19-8.21 (m, 2H), 7.80 (s, 1H), 7.68-7.72 (m, 5H), 7.49-7.53 (m, 3H), 7.31-
10

7.34 (m, 2H), 7.01-7.04 (m, 1H), 5.5 (s, 2H). ESI-MS m/z: 454 [M-H]-, 456 [M+H]+, 478
[M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-p-tolylurea (S15)
1
Synthesized from 14e. Yield 84%. Mp 244–245 C. H-NMR (300 MHz, DMSO-d6) δ 8.97
(s, 1H), 8.69 (s, 1H), 8.23-8.26 (m, 2H), 7.85 (s, 1H), 7.68-7.72 (m, 4H), 7.50-7.58 (m, 3H), 7.36-
7.39 (m, 2H), 7.10-7.13 (m, 2H), 5.60 (s, 2H), 2.26 (s, 3H). ESI-MS m/z: 434 [M-H]-, 436 [M+H]+,
458 [M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-methoxyphenyl)urea (S16)
Synthesized from 14f. Yield 88%. Mp 189–190 C. 1H-NMR (300 MHz, DMSO-d6) δ 8.99 (s,
1H), 8.62 (s, 1H), 8.24-8.26 (m, 2H), 7.85 (s, 1H), 7.73-7.77 (m, 4H), 7.53-7.57 (m, 3H), 7.29-
7.33 (m, 2H), 6.84-6.91 (m, 2H), 5.60 (s, 2H), 3.71 (s, 3H). ESI-MS m/z: 450 [M-H]-, 452 [M+H]+,
474 [M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (S17)
1
Synthesized from 14g. Yield 74%. Mp 263–265 C. H-NMR (300 MHz, DMSO-d6) δ 9.30
(s, 1H), 9.20 (s, 1H), 8.23-8.26 (m, 2H), 8.15 (d, J = 2.01 Hz, 1H), 7.86 (s, 1H), 7.71-7.75 (m, 4H),
7.66-7.68 (m, 2H), 7.53-7.56 (m, 3H), 5.59 (s, 2H). ESI-MS m/z: 522 [M-H]-, 524 [M+H]+, 546
[M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(3-fluorophenyl)urea (S18)
Synthesized from 14h. Yield 83%. Mp 237–239 C. 1H-NMR(300 MHz, DMSO-d6) δ 9.17 (s,
1H), 9.12 (s, 1H), 8.23-8.26 (m, 2H), 7.86 (s, 1H), 7.71-7.74 (m, 4H), 7.52-7.56 (m, 4H), 7.30-
7.37 (m, 1H), 7.12-7.23 (m, 1H), 6.75-6.85 (m, 1H), 5.60 (s, 2H). ESI-MS m/z: 438 [M-H]-, 440
[M+H]+, 462 [M+Na]+.
1-(4-(3-Amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(2-fluorophenyl)urea (S19)
Synthesized from 14i. Yield 81%. Mp 238–240 C. 1H-NMR (300 MHz, DMSO-d6) δ 9.40 (s,
1H), 8.68 (s, 1H), 8.19-8.26 (m, 3H), 7.86 (s, 1H), 7.68-7.72 (m, 4H), 7.51-7.54 (m, 3H), 7.20-
7.23 (m, 2H), 7.08 (s, 1H), 5.61 (s, 2H). ESI-MS m/z: 438 [M-H]-, 440 [M+H]+, 462 [M+Na]+.
1-(4-Acrylamidophenyl)-3-(4-(3-amino-6-phenylisoxazolo[3,4-b]pyridin-4-yl)phenyl)urea (S110)
Synthesized from 18a. Yield 82%. Mp > 300 C. 1H-NMR (300 MHz, DMSO-d6) δ 10.10 (s,
1H), 9.05 (s, 1H), 8.81 (s, 1H), 8.24-8.26 (m, 2H), 7.86 (s, 1H), 7.65-7.70 (m, 2H, 7.43-7.63(m,
9H), 6.40-6.48 (m, 1H), 6.21-6.26 (m, 1H), 5.72-5.76 (m, 1H), 5.53 (s, 2H). ESI-MS m/z: 489 [M-
H]-, 491 [M+H]+, 513 [M+Na]+.
4.1.2 The synthesis of S21–S28
(E)-4-(4-Nitrophenyl)but-3-en-2-one (8)
K2CO3 in water (0.02 mol, 20 ml) was added into a solution of p-nitrobenzaldehyde (15 g,
0.1 mol) in acetone (250 ml), after the mixture stirred at 25 °C for 24 h, concentrated hydrochloric
acid (40 ml) was added into the solution and the reaction was stirred for another 8 h, at last, 200
ml water was added and the solid appeared was collected by filtration and purified by column
chromatography (20% ethyl acetate in petroleum ether) to yield 8 (16.2 g, 85%) as a yellow solid.
Mp 250–252C.17 1H NMR (300 MHz, DMSO-d6) δ 8.24 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.8 Hz,
2H), 7.51 (d, J = 15.8 Hz, 1H), 6.84 (d, J = 15.8 Hz, 1H), 2.41 (s, 3H). ESI-MS m/z: 190 [M-H]-,
192 [M+H]+, 214 [M+Na]+.
6-Methyl-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (9)
t-BuOK (4 g, 35.6 mmol) was added into a solution of 8 (4 g, 20.9 mmol) and
11

cyanoacetamide (3.3 g, 39.2 mmol) in DMSO (40 ml), after the mixture stirred at 25 °C for 1h, t-
BuOK (8g, 71.2mmol) was added and the mixture stirred for another 2h, then 200 ml water was
added and dilute hydrochloric acid solution (4 N, 150 ml) was added to adjust the pH to be 2, the
solid appeared was obtained by filtration and dried under vacuum to afford 9 (4.4 g, 83%). Mp
183–185 °C.19 1H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.26 (d, J = 8.8 Hz, 2H), 7.63 (d, J
= 8.8 Hz, 2H), 5.58 (s, 1H), 1.84 (s, 3H). ESI-MS m/z: 254 [M-H]-, 256 [M+H]+, 278 [M+Na]+.
2-Chloro-6-methyl-4-(4-nitrophenyl)nicotinonitrile (10)
9 (6.2 g, 24.3 mmol) was added into a solution of POCl3 (40 ml), after the reaction mixture
was refluxed for 2 h, 500 ml ice water was added, the solid obtained by filtration was purified by
column chromatography (20% ethyl acetate in petroleum ether) to yield 10 (5.1 g, 77%) as a
yellow solid. Mp 168–170 °C. 1H-NMR (300 MHz, DMSO-d6) δ 8.39 (d, J = 8.7Hz, 2H), 7.82 (d,
J = 8.7Hz, 2H), 7.31 (s, 1H), 2.53 (s, 3H). ESI-MS m/z: 272 [M-H]-, [M+H]+ 274, 296 [M+Na]+.
4-(4-Aminophenyl)-2-chloro-6-methylnicotinonitrile (11)
The iron powder (1.51 g, 27 mmol) and glacial acetic acid (3 ml) were added into a solution
of 10 (1.91 g, 7 mmol) in ethanol (120 ml), after the mixture was refluxed for 2 h, the reaction
mixture was partitioned between 100 ml EtOAc and 504 ml water. The EtOAc layer was washed
with brine, dried over MgSO4, filtered, concentrated and purified by by column chromatography
on silica gel using 25% ethyl acetate in petroleum ether as eluent to afford 11 (1.51g, 89%) as a
yellow solid. Mp 122–124 °C. 1H-NMR (300 MHz, DMSO-d6) δ 7.49 (s, 1H), 7.41 (d, J = 8.5Hz,
2H), 6.7 (d, J = 8.5Hz, 2H), 5.8 (s, 2H), 2.5 (s, 3H). ESI-MS m/z: 242 [M-H]-, 244 [M+H]+, 266
[M+Na]+.
4-(4-Aminophenyl)-6-methylisoxazolo[3,4-b]pyridin-3-amine (12)
Hydroxylamine hydrochloride (530 mg, 7.6 mmol) was added into a solution of 11 (340 mg,
1.4 mmol) in t-BuOH (50 ml), after the mixture was refluxed for 2 h, t-BuOK (896 mg, 8 mmol)
was added and the mixture was refluxed for another 0.5 h, 200 ml methanol was added and the
mixture was filtrated, dried over anhydrous Na2SO4, filtered and concentrated. The crude material
was purified by column chromatography (2% methanol in dichloromethane) to yield 12 (232 mg,
1
69%) as a yellow solid. Mp 205–207 °C. H-NMR (300 MHz, DMSO-d6) δ 7.39 (d, J = 8.4Hz,
2H), 7.01 (s, 1H), 6.72 (d, J = 8.4Hz, 2H), 5.81 (s, 2H), 5.38 (s, 2H), 2.52 (s, 3H). ESI-MS m/z:
239 [M-H]-, [M+H]+ 241, 263 [M+Na]+.
The substituted isocyanate (0.9~1.2 mmol) was added into a solution of compound 12 (72 mg,
0.3 mmol) dissolved in anhydrous EtOH (15 mL) at 25 °C, followed by
catalytic amount of triethylamine, after stirring at 25 °C for 2 h, the solid obtained was purified by
column chromatography on silica gel using 2% MeOH in CH2Cl2 as eluent to afford S21–S28 as
yellow solids.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-m-tolylurea (S21)
1
Synthesized from 14a. Yield 67%. Mp 144–146 C. H-NMR (300 MHz, DMSO-d6) δ 8.96
(s, 1H), 8.71 (s, 1H), 7.67 (d, J = 8.56, 2H), 7.56 (d, J = 8.56, 2H), 7.33 (s, 1H), 7.25-7.28 (m, 1H),
7.12-7.19 (m, 2H), 6.81 (d, J = 7.41, 1H), 5.60 (s, 2H), 2.59 (s, 3H), 2.29 (s, 3H). ESI-MS: m/z
372 [M-H]-, 374 [M+H]+, 396 [M+Na]+.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(3-chlorophenyl)urea (S22)
Synthesized from 14d. Yield 81%. Mp 165–167 C. 1H-NMR(300 MHz, DMSO-d6) δ 9.09 (s,
12

1H), 9.03 (s, 1H), 7.74 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.28-7.36 (m,
2H), 7.20 (s, 1H), 7.03-7.06 (m, 1H), 5.50 (s, 2H), 2.51 (s, 3H). ESI-MS m/z: 392 [M-H]-, [M+H]+
394, [M+Na]+ 416.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-chloro-3-
(trifluoromethyl)phenyl)urea (S23)
1
Synthesized from 14g. Yield 69%. Mp 150–151 C. H-NMR (300 MHz, DMSO-d6) δ 9.30
(s, 1H), 9.18 (s, 1H), 8.15 (s, 1H), 7.67-7.76 (m, 4H), 7.57-7.62 (m, 2H), 7.20 (s, 1H), 5.52 (s, 2H),
2.60 (s, 3H). ESI-MS m/z: 460 [M-H]-, 462 [M+H]+, 484 [M+Na]+.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(3-(trifluoromethyl)phenyl)urea
(S24)
1
Synthesized from 14k. Yield 65%. Mp 135–137 C. H-NMR (300 MHz, DMSO-d6) δ 9.19
(s, 1H), 9.13 (s, 1H), 8.05 (s, 1H), 7.59-7.67(m, 2H), 7.51-7.56 (m, 4H), 7.33-7.35 (m, 1H), 7.19 (s,
1H), 5.50 (s, 2H), 2.67 (s, 3H). ESI-MS m/z: 426 [M-H]-, 428 [M+H]+, 450 [M+Na]+.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(2-chlorophenyl)urea (S25)
Synthesized from 14l. Yield 82%. Mp 238–240 C. 1H-NMR (300 MHz, DMSO-d6) δ 9.71 (s,
1H), 8.43 (s, 1H), 8.18 (d, J = 7.53Hz, 1H), 7.67-7.70 (m, 2H), 7.57-7.60 (m, 2H), 7.47-7.50 (m,
1H), 7.30-7.35 (t, J = 7.5 Hz, 1H), 7.20 (s, 1H), 7.04-7.09 (t, J = 7.05Hz, 1H), 5.51 (s, 2H), 2.60 (s,
3H). ESI-MS m/z: 392 [M-H]-, 394 [M+H]+, 416 [M+Na]+.
(Z)-1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-but-2-enamidophenyl)urea
(S26)
1
Synthesized from 18b. Yield 66%. Mp 281–283 C. H-NMR (300 MHz, DMSO-d6) δ 9.89
(s, 1H), 8.97 (s, 1H), 8.75 (s, 1H), 7.65-7.68 (m, 2H), 7.54-7.59 (m, 4H), 7.40-7.46 (m, 2H), 7.19
(s, 1H), 6.73-6.83 (m, 1H), 6.08-6.14 (dd, J1 = 1.4 Hz, J2 = 15.1 Hz, 1H), 5.50 (s, 2H), 2.60 (s,
3H), 1.86 (d, J = 5.9 Hz, 3H). ESI-MS m/z: 441 [M-H]-, 443 [M+H]+, 465 [M+Na]+.
(Z)-1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(4-but-2-enamido-3-
methylphenyl)urea (S27)
1
Synthesized from 18c. Yield 79%. Mp 266–268 C. H-NMR (300 MHz, DMSO-d6) δ 9.44
(s, 1H), 9.24 (s, 1H), 9.14 (s, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.25-7.34 (m,
3H), 7.20 (s, 1H), 6.75 (dd, J1 = 6.9 Hz, J2 = 15.2 Hz, 1H), 6.20 (d, J = 14.6 Hz, 1H), 5.48 (s, 2H),
2.59 (s, 3H), 2.17 (s, 3H), 1.86 (d, J = 6.5 Hz, 3H). ESI-MS m/z: 455 [M-H]-, 457 [M+H]+, 479
[M+Na]+.
1-(4-(3-Amino-6-methylisoxazolo[3,4-b]pyridin-4-yl)phenyl)-3-(3-methyl-4-(2-
(morpholinomethyl)acrylamido)phenyl)urea (S28)
1
Synthesized from 25. Yield 76%. Mp 144–146 C. H-NMR (300 MHz, DMSO-d6) δ 10.39
(s, 1H), 10.24 (s, 1H), 10.01 (s, 1H), 7.83 (d, J = 8.7 Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.54 (d, J =
8.6 Hz, 2H), 7.38 (s, 1H), 7.30-7.34 (m, 1H), 7.20 (s, 1H), 6.7(m, 1H), 6.14 (d, J = 1.5 Hz, 1H),
5.61 (d, J = 1.5 Hz, 1H), 5.50 (s, 2H), 4.17-4.22 (t, 2H), 3.64-3.67 (t, 4H), 3.15-3.18 (t, 4H), 2.59
(s, 3H), 2.29 (s, 3H). ESI-MS m/z: 540 [M-H]-, 542 [M+H]+, 564 [M+Na]+.
4.1.3 The synthesis of side chains
The synthesis of isocyanates 14a–14l
A solution of substituted aniline (0.01 mol) in ethyl acetate (20 ml) was added dropwise to a
solution of triphosgene (1.5 g, 0.005 mol) dissolved in ethyl acetate (20 ml) at 0 °C, after the
mixture reacted at 0 °C for 0.5 h, then it was warmed to room temperature and reacted for 1 h. At
13

last, the mixture refluxed for another 4–6 h. The solvent was distilled at reduced pressure to afford
the side chain 14a~14l which could be used directly for the next reaction without further
purification.20,21
The synthesis of isocyanate 18a–18c
Substituted acryloyl chloride (29 mmol) and DIPEA (5 ml, 29 mmol) were added into a
solution of substituted aniline (15) (13 mmol) in EtOAc (100 ml), after the mixture was stirred at
25°C for 3h, the reaction mixture was partitioned between 100 ml EtOAc and 200 ml water. The
EtOAc layer was washed with brine, dried over MgSO4, filtered and concentrated to yield 16 as
yellow solids.22,23
1
16a, yield 88%. H-NMR (300 MHz, DMSO-d6) δ 8.18 (d, J = 9.2 Hz, 2H), 7.93 (s, 1H),
7.70 (d, J = 9.2 Hz, 2H), 6.43-6.51 (m, 1H), 6.18-6.23 (m, 1H), 5.74-5.78 (m, 1H). ESI-MS m/z:
191 [M-H]-, 193 [M+H]+, 215 [M+Na]+.
1
16b, yield 85%. H-NMR (300 MHz, DMSO-d6) δ 8.20 (d, J = 9.1 Hz, 2H), 7.91 (s, 1H),
7.68 (d, J = 9.1 Hz, 2H), 5.78-5.83 (m, 1H), 5.58 (d, J = 15.1 Hz, 1H), 2.01 (d, J = 6.4 Hz, 3H).
ESI-MS m/z: 205 [M-H]-, 207 [M+H]+, 229 [M+Na]+.
1
16c, yield 93%. H-NMR (300 MHz, DMSO-d6) δ 8.41 (d, J = 8.5 Hz, 1H), 8.11-8.14 (m,
2H), 7.28 (s, 1H), 7.00-7.04 (m, 1H), 6.02 (d, J = 15.1 Hz, 1H), 2.38 (s, 3H), 2.01 (d, J = 6.4 Hz,
3H). ESI-MS m/z: 219 [M-H]-, 221 [M+H]+, 243 [M+Na]+.
The iron powder (1.52 g, 27 mmol) and glacial acetic acid (3 ml) were added into a solution
of 16 (7 mmol) in ethanol (120 ml), after the mixture was refluxed for 2 h, the reaction mixture
was partitioned between 100 ml EtOAc and 504 ml water. The EtOAc layer was washed with
brine, dried over MgSO4, filtered, concentrated and purified by by column chromatography on
silica gel using 25% ethyl acetate in petroleum ether as eluent to afford 17 as yellow oil.27,28
1
17a, yield 91%. H-NMR (300 MHz, DMSO-d6) δ 9.65 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H),
6.53 (d, J = 8.8 Hz, 2H), 6.28-6.32 (m, 1H), 6.15-6.19 (m, 1H), 5.66-5.71 (m, 1H), 4.85 (s, 2H).
ESI-MS m/z: 161 [M-H]-, 163 [M+H]+, 185 [M+Na]+.
1
17b, yield 82%. H-NMR (300 MHz, DMSO-d6) δ 9.71 (s, 1H), 7.28 (d, J = 8.7 Hz, 2H),
6.56 (d, J = 8.7 Hz, 2H), 5.69-5.73 (m, 1H), 5.42 (d, J = 14.8 Hz, 1H), 4.91 (s, 2H), 2.01 (d, J =
6.2 Hz, 3H). ESI-MS m/z: 175 [M-H]-, 177 [M+H]+, 199 [M+Na]+.
17c, yield 88%. 1H-NMR (300 MHz, DMSO-d6) δ 7.31 (s, 1H), 6.98-7.03 (m, 3H), 6.47-6.52
(m, 1H), 5.88 (d, J = 15.1 Hz, 1H), 3.6 (s, 2H), 2.22 (s, 3H), 1.91 (d, J = 5.9 Hz, 3H). ESI-MS m/z:
189 [M-H]-, 191 [M+H]+, 213 [M+Na]+.
Triphosgene (1.5 g, 0.005 mol) in ethyl acetate (20 ml) was added dropwise to a solution of
17 (0.01 mol) in ethyl acetate (20 ml) at 0 °C, after the mixture reacted at 0 °C for 0.5 h, then it
was warmed to room temperature and reacted for 1h. At last, the mixture refluxed for another 4–6
h. The solvent was distilled at reduced pressure to afford the side chain 18a–18c which could be
used directly for the next reaction without further purification.
The synthesis of isocyanate 25
Acryloyl chloride (2.4 ml, 29 mmol) and DIPEA (5 ml, 29 mmol) were added into a solution
of 2-methyl-4-nitroaniline (19) (2 g, 13 mmol) in EtOAc (100 ml), after the mixture was stirred at
25 °C for 3 h, the reaction mixture was partitioned between 100 ml EtOAc and 200 ml water. The
EtOAc layer was washed with brine, dried over MgSO4, filtered and concentrated to yield 20 (2.28
g, 85%) as a yellow solid. 1H-NMR (300 MHz, DMSO-d6) δ 9.27 (s, 1H), 7.80 (s, 1H), 7.72-7.76
(m, 1H), 7.58-7.62 (m, 1H), 6.41-6.48 (m, 1H), 6.20-6.24 (m, 1H), 5.71-5.75 (m, 1H), 2.52 (s, 3H).
14

ESI-MS m/z: 205 [M-H]-, 207 [M+H]+, 229 [M+Na]+.
DABCO (1 g, 9 mmol) and paraformaldehyde (0.45 g, 15 mmol) were added into a solution
of 20 (0.62 g, 3 mmol) in 1,4-dioxane:H2O (5:3), after the mixture was reacted for 8 h at 80 °C,
the reaction mixture was partitioned between 100 ml EtOAc and 200 ml water. The EtOAc layer
was washed with brine, dried over MgSO4, filtered and concentrated to yield 21 (0.62 g, 88%) as a
white solid. 1H-NMR (300 MHz, DMSO-d6) δ 9.22 (s, 1H), 7.84 (s, 1H), 7.76-7.80 (m, 1H), 7.62-
7.66 (m, 1H), 6.73 (s, 1H), 6.37 (s, 1H), 4.12 (s, 2H), 3.32 (s, 1H), 2.54 (s, 3H). ESI-MS m/z: 235
[M-H]-, 237 [M+H]+, 259 [M+Na]+.
PBr3 (0.34 g, 1.2 mmol) was added into a solution of 21 (0.24 g, 1 mmol) in DCM (30 ml),
after the mixture was reacted for 30 min at 25 °C, the reaction mixture was partitioned between 80
ml DCM and 503 ml water. The DCM layer was washed with brine, dried over MgSO4, filtered
1
and concentrated to yield 22 (0.25 g, 84%) as a white solid. H-NMR (300 MHz, DMSO-d6) δ
9.24 (s, 1H), 7.83 (s, 1H), 7.78-7.81 (m, 1H), 7.68-7.71 (m, 1H), 6.68 (s, 1H), 6.32 (s, 1H), 3.85 (s,
2H), 2.52 (s, 3H). ESI-MS m/z: 297 [M-H]-, 299 [M+H]+, 321 [M+Na]+.
Morpholine (0.27 g, 2.5 mmol) was added into a solution of 22 (0.15 g, 0.5 mmol) in DCM
(30 ml), after the mixture was reacted for 30 min at 25 °C, the reaction mixture was partitioned
between 80 ml DCM and 503 ml water. The DCM layer was washed with brine, dried over
MgSO4, filtered and concentrated to yield 23 (0.13 g, 85%) as a white solid. 1H-NMR (300 MHz,
DMSO-d6) δ 9.21 (s, 1H), 7.80 (s, 1H), 7.70-7.73 (m, 2H), 6.70 (s, 1H), 6.11 (s, 1H), 3.59-3.62 (m,
4H), 3.31 (s, 2H), 2.57-2.59 (m, 4H), 2.54 (s, 3H). ESI-MS m/z: [M-H]- 304, [M+H]+ 306,
[M+Na]+ 328.
The iron powder (1.52 g, 27 mmol) and glacial acetic acid (3 ml) were added into a solution
of 23 (2.1 g, 7 mmol) in ethanol (120 ml), after the mixture was refluxed for 2 h, the reaction
mixture was partitioned between 100 ml EtOAc and 504 ml water. The EtOAc layer was washed
with brine, dried over MgSO4, filtered, concentrated and purified by by column chromatography
on silica gel using 25% ethyl acetate in petroleum ether as eluent to afford 24 (1.79 g, 93%) as
1
yellow solid. H-NMR (300 MHz, DMSO-d6) δ 10.12 (s, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.45 (s,
1H), 7.38-7.42 (m, 1H), 6.68 (s, 1H), 6.16 (s, 1H), 3.89 (s, 2H), 3.61-3.65 (t, 4H), 3.28 (s, 2H),
3.11-3.15 (t, 4H), 2.28 (s, 3H). ESI-MS m/z: [M-H]- 274, [M+H]+ 276, [M+Na]+ 298.
Triphosgene (1.5 g, 0.005 mol) in ethyl acetate (20 ml) was added dropwise to a solution of
24 (0.01 mol) in ethyl acetate (20 ml) at 0°C, after the mixture reacted at 0 °C for 0.5 h, then it
was warmed to room temperature and reacted for 1h. At last, the mixture refluxed for another 4–
6h. The solvent was distilled at reduced pressure to afford the 25 which could be used directly for
the next reaction.
4.2 Biological evaluation
4.2.1 In vitro kinase FLT3, KDR and PDGFR-β assay
Inhibition ratio determination and IC50 testing at a single concentration (10 μM) were
entrusted to Reaction Biology Corporation. Cisbio’s HTRF KinEASE Kit was used to test the
enzyme inhibitory activity. This method utilizes a unique substrate containing a single
phosphorylation site recognized by a europium cryptate (Eu(K))-labeled antibody to
phosphotyrosine. Based on homogeneous time-resolved fluorescence (HTRF), all KinEASE
assays involve two steps: the enzymatic step and the detection step with HTRF reagents. In the
kinase reaction step, 2 μL of kinase (FLT3, KDR and PDGFR-β) solution, 2 μL of biotin substrate,
and 4 μL of compound (SEB-supplemented kinase buffer) were added to each well for incubation.
15

Then, 2 μL of ATP was added at room temperature (18−22 °C) to initiate the reaction, which was
run for 1 h. In the second step, detection reagents including 5 μL of streptavidin-XL665 (SA-
XL665) in EDTA and 5 μL tyrosine kinase antibody-Eu(K) in EDTA were added to each well and
incubated for 1 h at room temperature. The Beckman Coulter platform HTRF detection module
was used to detect the signal. The detection reagents catch the phosphorylated substrate and the
resulting HTRF signal is proportional to the amount of phosphorylation.29 GraphPad Prism 5.0
software
was
used
to
calculate
the
IC50
values
for
each
compound
(https://www.graphpad.com/scientific-software/prism/). Each test was repeated three times.
4.2.2 Antiproliferative assay
All cell activity tests were entrusted to Crown Bioscience Inc. The luciferase in the CTG
reagent uses luciferin, ATP and oxygen as substrates to produce oxidized luciferin and release
energy in the form of light. The amount of light produced is proportional to the total amount of
ATP, which can reflect the total number of viable cells (HUVECs, MCF-7, and MV4-11). The
anticell proliferation rate can be calculated by the fluorescence intensity. This method included
several steps. The first step was cell planking, where the cells in the exponential growth phase
were collected and the viable cells were counted with Vi-Cell XR cell counting instrument.
According to the density in the cell culture medium, the cell suspension was adjusted and 90 μL of
medium was added to each well of a 96-well cell culture plate. The final cell concentration was
approximately 2000−4000 cells per well (the specific cell density was adjusted according to cell
growth). The next step was compound dispensation, where the target compound was dissolved
from 10 μM stock solutions in DMSO, and then these solutions were diluted 10-fold with the
medium solution. A total of 10 μL of the 10-fold compound dilution was added per well to each
cell line, leading to a final drug concentration of 10 μM and a final DMSO concentration of 0.1%.
The plate was placed in an incubator containing 5% CO2 at 37 °C for 72 h. Next was the plate
detection step, where according to the manufacturer instructions, 50 μL of CTG solution that was
previously thawed and equilibrated to room temperature was added to each well after 72 h of drug
treatment. A microplate oscillator was used to mix the solution for 2 min. After a 10 min
incubation at room temperature, the fluorescence signal value was measured by an Envision2104
plate reader. Each compound was submitted for a 10 concentrations test (from 1 nM to 100 μM).
Data processing: inhibition ratio = 1 − Vsample/Vvehicle control × 100%. Vsample is for drug treatment
group while Vvehicle control is for solvent control group. The GraphPad Prism 5.0 software was used
to draw nonlinear regression model and S type dose survival rate curve, and then calculate IC50
values. Each test was repeated three times.
4.3 Molecular dock modeling30
The binding modes of compound S21 were investigated using molecular docking modeling in
the AutoDock 4.2 software. Before starting the docking process, the protein structure was
subjected to optimization step in order to minimize the crystallographic induced bond clashes.
The Kollman united atom charges and polar hydrogen was added to the receptor and the
crystallographic waters were removed. PyMol was used to display the 3D structure of the
compound S21 complexed with crystal structures. Charges of the Gasteiger type were assigned to
the new constructed structures in AutoDock. Non-polar hydrogen atoms were merged and
rotatable bonds were defined. The grid maps of the protein were calculated using AutoGrid
16

module embedded in AutoDock software. The grid was set in a way to include not only the active
site amino acids but also the considerable portions of the surrounding surface. Hence, a grid size
of 60 60 60 Å points and 0.375 Å spacing were generated based on the binding position of the
cognate ligand in the protein. Docking simulations were performed using the autodock module of
the software. Every docking program was taken out in 250,000 times energy evaluation with 10
conformations kept and the most favorable pose of each compound was exhibited.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81302634
and 21302225), China Scholarship Council (No. 201407060046), Natural Science Foundation of
Jiangsu Province (BK20151563 and BK20130662), Six Talents Project Funded by Jiangsu
Province (2013-YY-010), Jiangsu Collaborative Innovation Center of Chinese Medicinal
Resources Industrialization (ZDXMHT-1-13), Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions and Project Funded by the Flagship Major
Development of Jiangsu Higher Education Institutions (PPZY2015A070).
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Design, synthesis and structure-activity relationship of diaryl-ureas with novel isoxazol[3,4-
b]pyridine-3-amino-structure as multi-target inhibitors against receptor tyrosine kinase
Zhi-Hao Shia, Feng-Tao Liua, Hao-Zhong Tiana, Yan-Min Zhanga, Nian-Guang Lib,*, Tao Lua,*
aSchool of Science, China Pharmaceutical University, 639 Longmian Avenue, Nanjing, Jiangsu
211198, China
bNational and Local Collaborative Engineering Center of Chinese Medicinal Resources
Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine,
138 Xianlin Road, Nanjing, Jiangsu 210023, China
* Corresponding author.
E-mail addresses: linianguang@njutcm.edu.cn (N.-G. Li), lutao@cpu.edu.cn (T. L)
Graphical abstract
18

R3
H
H
H
H
H
N
H
N
NH2
NH2
NH2
N
N
R2
R1
N
N
CH3
Me
O
N
O
N
N
HN
O
O
O
F
S21
N
N
Linifanib
Target compounds
IC50 = 4nM, FLT3
IC50 = 3nM, KDR
IC50 = 8nM, PDGFR-β
R
CH3
19