Design, synthesis and biological study of potent and covalent HER-2 tyrosine kinase inhibitors with low cytotoxicity in vitro

Article abstract of DOI:10.1007/s11696-019-00686-0

The discovery and development of a novel HER-2 tyrosine kinase inhibitor for the treatment of HER2-positive breast cancer are presented in this article. EGFR family has been recognized as a crucial meditator in the cancer progression; HER-2 tyrosine kinase was one of the members among them. In the effort to explore potent HER-2 inhibitors, a novel series of 4-anilino-3-cyanoquinoline derivatives have been designed, synthesized and evaluated. Most compounds possessed modest proliferation inhibition on SK-BR-3 cell line and HER-2 kinase. Compound 16 appeared to be the most potent compound (HER-2 kinase IC₅₀: 19.4?nM, SK-BR-3 IC₅₀: 94?nM). In the experiment of cellular cytotoxicity assay, compound 16 shows a much lower cytotoxicity than neratinib on Beas-2b cell line (Human bronchial epithelial cells). In conclusion, compound 16 would be a promising lead compound for further anti-breast cancer drug discovery.

Full text of DOI:10.1007/s11696-019-00686-0

Chemical Papers
https://doi.org/10.1007/s11696-019-00686-0
ORIGINAL PAPER
Design, synthesis and biological study of potent and covalent HER‑2
tyrosine kinase inhibitors with low cytotoxicity in vitro
Shuyu Jin1,2 · Xiuyun Sun2,3 · Dan Liu1 · Hua Xie4 · Yu Rao2
Received: 30 April 2018 / Accepted: 9 January 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
The discovery and development of a novel HER-2 tyrosine kinase inhibitor for the treatment of HER2-positive breast can-
cer are presented in this article. EGFR family has been recognized as a crucial meditator in the cancer progression; HER-2
tyrosine kinase was one of the members among them. In the efort to explore potent HER-2 inhibitors, a novel series of
4-anilino-3-cyanoquinoline derivatives have been designed, synthesized and evaluated. Most compounds possessed modest
proliferation inhibition on SK-BR-3 cell line and HER-2 kinase. Compound 16 appeared to be the most potent compound
(HER-2 kinase IC50: 19.4 nM, SK-BR-3 IC50: 94 nM). In the experiment of cellular cytotoxicity assay, compound 16 shows
a much lower cytotoxicity than neratinib on Beas-2b cell line (Human bronchial epithelial cells). In conclusion, compound
16 would be a promising lead compound for further anti-breast cancer drug discovery.
Keywords HER-2 inhibitors · 4-Anilino-3-cyanoquinoline · Covalent · Anticancer activity
Introduction
plays signifcant roles in cell proliferation, survival, adhe-
sion, migration and diferentiation through downstream sign-
aling pathway activation during breast cancer development
(Li et al. 2017). Overexpression of HER-2 now serves as
a biomarker for poor prognosis in many kinds of human
cancer which was considered to be responsible for around
30% breast cancer occurrence (Rabindran et al. 2004; Eroglu
et al. 2014). So far, breast cancer was the second leading
type cancer of women worldwide, accounting for 25% in all
cases (Dzimitrowicz et al. 2016). HER-2-positive cancer was
even more aggressive than negative (Awada et al. 2012). Due
to the circumstance, developing small molecule inhibitors
on HER-2 kinase as anti-breast cancer drugs would provide
advantages for the cure of breast cancer.
Human epidermal growth factor receptor (EGFR) family
consists of four members: EGFR (ErbB-1; human epider-
mal growth factor receptor 1), HER-2 (c-ErbB-2), HER-3
(c-ErbB-3), and HER-4 (c-ErbB-4). Among them, HER-2
* Dan Liu
sammyld@163.com
* Hua Xie
yrao@mail.tsinghua.edu.cn
* Yu Rao
hxie@simm.ac.cn
1
Key Laboratory of Structure-Based Drugs Design
Research endeavor targeting on HER-2 has already
brought about some signifcant biological and therapeutic
advances (Tsou et al. 2005). Here represents four represent-
ative EGFR/ HER-2 dual inhibitors in Fig. 1 launched in
recent years for the cure of breast cancer. As an irreversible
EGFR/HER-2 dual inhibitor, lapatinib (Tykerb) was the frst
inhibitor for HER-2-positive breast cancer approved by FDA
in 2007 (Rabindran et al. 2004). In 2013, afatinib (Giotrif)
was launched to serve as a treatment for NSCLC and HER-
2-positive late-stage breast cancer (Yoshioka et al. 2018;
Cortés JD 2015). In terms of pyrotinib, another highly potent
EGFR/HER-2 dual inhibitor, phase III clinical trial started
and Discovery of Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road,
Shenyang 110016, Liaoning, People’s Republic of China
2
MOE Key Laboratory of Protein Sciences, School
of Pharmaceutical Science, MOE Key Laboratory
of Bioorganic Phosphorus Chemistry and Chemical
Biology, Tsinghua University, Beijing 100084,
People’s Republic of China
3
Tsinghua-Peking Center for Life Sciences, Haidian Dist.,
Beijing 100084, People’s Republic of China
4
Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, 555 Zuchongzhi Road, Shanghai 201203,
People’s Republic of China
Vol.:(0123456789)
1 3

Chemical Papers
Fig. 1 Representative EGFR/HER-2 inhibitors
since 2016 and just proved by CFDA recently (August 14,
2018).
and mechanism of covalent binding, a series of 4-anilino-
3-cyanoquinoline derivatives were designed and synthesized
(Jackson et al. 2017).
Neratinib, developed by Puma Genetech, was launched as
the treatment for breast cancer (Ito et al. 2012). Neratinib,
a pan-ErbB inhibitor, could irreversibly binds to the ErbB-
1, ErbB-2 and ErbB-4 developing a covalent complex with
a cysteine residue located within the ErbB kinase domain
(Martin et al. 2017). The formation of this complex leads to
the inhibition of tyrosine kinase activity (Tiwari et al. 2016).
Although the strong inhibition of the tumor cell proliferation
and decrease in tumor burden, neratinib shows severe side
efects in clinical trials(Ma et al. 2017). Those side efects
happens commonly among clinical trial, like diarrhea (88%),
nausea (64%), fatigue (63%) and vomiting (50%) (Burstein
et al. 2010; Wong et al. 2009). Despite those unexpected
side efects, neratinib was approved by FDA as a therapy for
late-stage breast cancer.
The purpose of this work was trying to explore novel
compounds as potent HER-2 inhibitors. In this present work,
19 substituted 4-anilino-3-cyanoquinoline derivatives have
been designed, synthesized and evaluated (Table 1). Anti-
proliferation activity, kinase inhibition and cellular cyto-
toxicity assay were conducted separately to evaluate the
potency. Compound 16 turned out to be the most potent
HER-2 inhibitor among 19 analogs, and bores a much lower
cytotoxicity than neratinib on Base-2b cell line. To get a bet-
ter understanding of compound 16, the docking experiment
was also conducted.
Experimental
Based on the structure of neratinib, chemical screen-
ing shows that retention of the upper right warhead of the
scafold was necessary for the potency, and changes of the
down left warhead would be crucial for the activities (Zhang
et al. 2013). Thereby, at the beginning of design, we kept the
4-anilino-3-cyanoquinoline as the core structure and focused
on the modifcation of left part (Mao et al. 2013). Consider-
ing the length between the main scafold to cysteine residue
Materials
All commercial materials (Alfa Aesar, Aladdin, J&K
Chemical LTD.) were used without further purifcation. All
solvents were of analytical grade. The H-NMR and 13C-
1
NMR spectra were recorded on a Bruker 400-MHz spec-
trometer in CDCl3 using TMS or solvent peak as a standard.
1 3

Chemical Papers
Table 1 Anti-proliferation activity of compound 1–19 on SK-BR-3 cell line.
1 3

Chemical Papers
All 13C-NMR spectra were recorded with complete proton
decoupling. Low-resolution mass spectral analyzes were per-
formed with a Waters AQUITY UPLCTM/MS. All reactions
were carried out in oven-dried sealed tube. Analytical TLC
was performed on Yantai Chemical Industry Research Insti-
tute silica gel 60 F254 plates and fash column chromatogra-
phy was performed on Qingdao Haiyang Chemical Co. Ltd
silica gel 60 (200–300 mesh). The rota vapor was BUCHI’s
Rota vapor R-3 (Scheme 1).
washed with heptane twice, and dried in an oven to give
5.1-g crude product (22).
To a stirred suspension of 4.00 g (20.4 mmol) of 22 and
7.90 g (57.2 mmol) of K2CO3 in 30 ml DMF at 60 °C, 2.94 g
(27.0 mmol) of EtBr was added slowly. The reaction mixture
was stirred at 60 °C for 2 h. Then, another 1.00-ml EtBr was
added until the reaction completed. An amount of 400 ml of
H2O was added to the reaction mixture after it was cooled
to 25 °C. The suspension was stirred in H2O for 30 min,
fltered, washed with warm (50 °C) H2O (maintain pH<8)
and heptane to give 6.1-g crude product 23.
Chemicals
An amount of 4.10 g (18.3 mmol) of compound 23 in
42 ml THF was reduced to 3-(4-acetamido-3-ethoxyaniline)-
2-cyanopropenoicacidethyl ester under hydrogenation (at
28–30 °C, 50 psi) with 350 mg of 10% Pd–C for 53 h. The
reaction mixture was fltered and rinsed with THF. The fl-
trate was concentrated to give the aniline and dissolved in
150 ml toluene was added 4.75 g (28 mmol) of ethyl (eth-
oxymethylene) cyanoacetate (25) and 350 ml of toluene. The
suspension was stirred and refuxed at 90°C for 16 h. The
reaction mixture was cooled to room temperature over 1 h
Synthesis of intermediate 30
To a stirred suspension of 4.00 g (26 mmol) of 2-amino-
5-nitrophenol (21) in 30 ml of HOAc at 60 °C, 6.00-ml Ac2O
was added slowly. The reaction mixture was stirred at 60 °C
for 1 h. An amount of 75.0 ml H2O was added to the reac-
tion mixture after it was cooled to 25 °C. The suspension
was stirred in H2O for 1 h, fltered, washed with H2O twice,
Scheme 1 Synthesis route of intermediate 30
1 3

Chemical Papers
and stirred at room temperature for 30 min. The light-yellow
solid was fltered, and washed with toluene to give 3.45-g
crude product 26 as a mixture of E and Z isomers.
General procedure B
The relevant acid was treated with oxalyl chloride for under
room temperature. Excess thionyl chloride was removed by
evaporation. Then, the compound 30 was dissolved in NMP,
trimethylamine was added, and the solution was cooled to
0 °C. To this stirred suspension, previously prepared acyl
chloride was added and stirred at ambient temperature
overnight.
An amount of 2.10 g (6.64 mmol) of 26 was stirred in
120 ml of Dowtherm A at 250–255 °C for 20 h. After the
mixture was cooled to room temperature, the solid was fl-
tered and washed with toluene. The solid crude material was
stirred in THF at refux temperature (66 °C) for 30 min and
cooled. The dark-brown solid was fltered, and washed with
THF to give 27 (1.00 g).
To a dark-brown suspension of 0.80 g (2.96 mmol) of
27 in 16.0 ml of diethylene glycol dimethyl ether, 0.96 ml
(10.4 mmol) of phosphorus oxychloride in one portion was
added. The reaction mixture was stirred at 100–102 °C for
45 min. The reaction mixture was cooled to 80–85 °C, and
2.50 g of Celite was added. The reaction mixture was fltered
through a Celite pad with diethylene glycol dimethyl ether.
The fltrate was concentrated and stirred in an aqueous solu-
tion of 7.50 g of K2CO3 (maintaining temperature less than
50 °C). The yellow solid was fltered, washed with warm
H2O and toluene to give crude 28 (0.66 g) (Scheme 2).
A mixture of 28 (0.66 g), 34 (0.50 g) and 2.21-g pyridine
hydrochloride in 6.00 ml of i-PrOH was refuxed for 16 h.
The solid was fltered and washed with H2O and ether to give
intermediate 29. Then, 3 ml of H2O and 7.00 ml of 12M HCl
were added to compound 29 and stirred and refuxed for 2 h.
When the reaction completed monitoring by TLC, 50.0 ml
of saturated aqueous NaHCO3, and then fltered. This solid
was dissolved in methanol, concentrated and purifed by
chromatography to give intermediate 30 in a yield of 68%.
Starting from intermediate 30, a series of neratinib ana-
logs could be synthesized from corresponding chlorides.
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phe‑
nyl) amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)
cyclohex‑1‑ene‑1‑carboxamide (1)
Cyclohex-1-ene-1-carboxylic acyl chloride was prepared fol-
lowing the general procedure A. Compound 30 (67.0 mg,
0.15 mmol, 1.0 mol e.q.) was dissolved in 2-ml NMP, fol-
lowing the addition of trimethylamine (23.0 µL, 0.18 mmol,
1.2 mol e.q.) and cooled to 0 °C. Newly prepared chloride
(1.00 mmol, 6.0 mol e.q.) was added to the above mixture
dropwise and then stirred at ambient temperature overnight.
The resulting brown precipitate was collected and purifed
by silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 75-mg compound 1 was obtained in 89.9% yield. 1H
NMR (400 MHz, CDCl3) δ (ppm) 9.17 (s, 1H), 8.59 (s, 1H),
8.58 (s, 1H), 8.45(s, 1H), 7.75(t, J = 1.2 Hz, 1H), 7.69(s,
1H), 7.64 (d, J=8.0 Hz, 1H), 7.26~7.24 (m, 2H), 7.20 (d,
J = 2.4 Hz, 1H), 6.96 (d, J = 2.4 Hz 1H), 6.89 ~ 6.87 (m,
2H), 5.27 (s, 2H), 4.25~4.24 (m, 2H), 2.30~2.23 (m, 2H),
2.03~1.99 (m, 2H), 1.74~1.61 (m, 4H), 1.54 (t, J=6.9 Hz,
3H). LRMS (ESI+) calcd for C31H28ClN5O3 [M+H]+:
554.20, found 554.09.
General procedure A
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl) cyclo‑
pent‑1‑ene‑1‑carboxamide (2)
The relevant acid was treated with thionyl chloride for 6 h
under 70 ℃. Excess thionyl chloride was removed by evapo-
ration. Then, compound 30 was dissolved in NMP, trimeth-
ylamine was added, and the solution was cooled to 0 °C. To
this stirred suspension, previous prepared acyl chloride was
added and stirred at ambient temperature overnight.
Cyclopent-1-ene-1-carboxylic acyl chloride was prepared
following the general procedure A. Compound 30 (55.0 mg,
0.12 mmol, 1.00 mol eq.) was dissolved in 2-ml NMP, fol-
lowing the addition of trimethylamine (0.18 mmol, 23 µl,
N
N
O
O
O
R2
O
R1
Cl
R-hydroxylamine (3.5 mol eq.),
O
HN
N
Cl
N
HN
N
Cl
H
H
sodium acetate (4.5 mol eq. )
O
N
CN
N
CN
30
R1
R1
EtOH : H2O = 1:1, 40oC
trimethylamine (1.5 mol eq.),
NMP,0oC~r.t., overnight
O
O
EtO
EtO
4
5: R1=Methyl R2=Methyl
6: R1=Methyl R2=Isopropyl
7: R1=H R2=Methyl
Scheme 2 Synthesis of compound 4–7
1 3

Chemical Papers
1.20 mol eq.) and cooled to 0 °C. Newly prepared chloride
(6.0 mol eq.) was added dropwise to the above mixture and
then stirred at ambient temperature overnight. After comple-
tion, the reaction mixture was poured into saturated aqueous
sodium bicarbonate to quench the reaction; then, precipitate
was observed and collected. The resulting brown precipitate
was collected and purifed by silica-gel column chromatogra-
phy (DCM/MeOH = 50:1). Finally, 62-mg compound 2 was
obtained in 83% yield. 1H NMR (400 MHz, CDCl3) δ (ppm)
9.17 (s, 1H), 8.57 (d, J=4.5 Hz, 1H), 8.41 (s, 1H), 8.18 (s,
1H), 7.91 (s, 1H), 7.75~7.73 (m, 1H), 7.63 (d, J=8.0 Hz,
1H), 7.25~7.20 (m, 2H), 7.16 (d, J=2.4 Hz, 1H), 6.90 (dd,
J1 =8.8 Hz, J2 =2.4 Hz, 1H), 6.82 (d, J=8.8 Hz, 1H), 6.75
(s, 1H), 5.23 (s, 2H), 4.20 (q, J1 =13.8 Hz, J2 =6.9 Hz, 2H),
2.59~2.52 (m, 4H), 1.52 (t, J=6.9 Hz, 3H). LRMS (ESI+),
m/z: Calcd for C31H21ClF3N5O3 [M+H]+, 540.18; found,
540.09.
completion, the reaction mixture was poured into saturated
aqueous sodium bicarbonate to quench the reaction; then,
precipitate was observed and collected. The resulting brown
precipitate was collected and purifed by silica-gel column
chromatography (DCM/MeOH = 50:1). Finally, 37-mg com-
pound 4 was obtained in 61% yield. 1H NMR (400 MHz,
CDCl3) δ (ppm) 9.49 (s, 1H), 9.10 (s, 1H), 9.58 (d,
J=3.6 Hz, 1H), 8.50 (s, 1H), 8.35 (s, 1H), 7.75 (t, J=7.2 Hz,
1H), 7.64 (d, J=7.6 Hz, 1H), 7.33 (s, 1H), 7.27~7.22 (m,
2H), 7.15 (d, J = 15.2, 1H), 7.06 (d, J = 2.0 Hz, 1H), 6.96
(d, J=8.8 Hz, 1H), 6.89 (d, J=15.2 Hz, 1H), 5.28 (s, 2H),
4.32 (q, J1 =13.9 Hz, J2 =6.9 Hz, 2H), 1.57 (t, J=6.9 Hz,
3H), 1.46 (t, J=7.2 Hz, 3H). LRMS (ESI+), m/z: Calcd for
C29H24ClN5O4 [M+H]+, 542.16; found, 542.30.
(2E,4Z)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4‑(methoxyimino)
pent‑2‑enamide (5)
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl) acrylamide (3)
Compound 4 (15.3 mg, 0.03 mmol, 1.0 mol eq.) was dis-
solved in 2-ml solvent (EtOH: H2O = 1:1), methoxyamine
hydrochloride (13.3 mg, 0.16 mmol, 1.0 mol eq.) and sodium
acetate were added to this solution. The mixture was heated
to 40 ℃, reaction solvent was removed by evaporation. The
resulting yellow precipitate was collected and purifed by
silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 10.3-mg compound 5 was obtained in 63.8% yield.
LRMS (ESI+), m/z: Calcd for C30H26ClN6O4 [M+H]+,
570.18; found, 570.34.
Compound 30 (40.0 mg, 0.09 mmol, 1.0 mol eq.) was dis-
solved in 2-ml NMP, following the addition of trimethyl-
amine (17.4 µl, 0.13 mmol, 1.5 mol eq.) and cooled the
solution to 0 °C, commercial available acryloyl chloride
(44.0 µl, 0.54 mmol, 6.0 mol eq.) was added dropwise to the
above mixture and then stirred at ambient temperature over-
night. After completion, the reaction mixture was poured
into saturated aqueous sodium bicarbonate to quench the
reaction; then, precipitate was observed and collected. The
resulting brown precipitate was collected and purifed by
silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 62-mg compound 3 was obtained in 83% yield. 1H
NMR (400 MHz, DMSO) δ (ppm) 9.65 (s, 1H), 8.62 (s, 1H),
8.59 (d, J=4.4 Hz, 1H), 8.47 (s, 1H), 7.89~7.85 (m, 1H),
7.58 (d, J = 8.0 Hz, 1H), 7.38 ~ 7.35 (m, 3H), 7.27 ~ 7.19
(m, 2H), 6.76 (q, J1 = 16.8 Hz, J2 = 10.0 Hz, 1H), 6.28
(dd, J1 =16.8 Hz, J2 =1.6 Hz, 1H), 5.80 (dd, J1 =10.0 Hz,
J2 = 2.0 Hz, 1H), 5.28 (s, 2H), 1.46 (t, J = 6.8 Hz, 3H).
LRMS (ESI+), m/z: Calcd for C27H22ClN5O3 [M+H]+,
500.15; found, 500.09.
(2E,4Z)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmeth‑
oxy)phenyl)amino)‑3‑cyano‑7‑ethoxyquino‑
lin‑6‑yl)‑4‑(isopropoxyimino)pent‑2‑enamide (6)
Compound 4 (38.0 mg, 0.07 mmol, 1.0 mol eq.) was dis-
solved in 2-ml solvent (EtOH: H2O = 1:1), o-isopropylhy-
droxylamine (28.0 mg, 0.25 mmol, 3.5 mol eq.) and sodium
acetate (26.4 mg, 0.32 mmol, 26.2 mg) were added to this
solution. The mixture was heated to 40 ℃, reaction solvent
was removed by evaporation. The resulting yellow precipi-
tate was collected and purifed by silica-gel column chroma-
tography (DCM/MeOH = 50:1). Finally, 33-mg compound
6 was obtained in 78.6% yield.
(E)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4‑oxopent‑2‑ena‑
mide (4)
(E)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4‑oxobut‑2‑enam‑
ide (7a)
(E)-4-oxopent-2-enoic chloride was prepared following the
procedure A. Compound 30 (50.0 mg, 0.12 mmol, 1.0 mol
eq.) was dissolved in 2-ml NMP, following the addition
of trimethylamine (0.17 mmol, 22 microL, 1.5 mol eq.)
and cooled the solution to 0 °C. Newly prepared chloride
(6.0 mol eq.) was added dropwise to the above mixture
and then stirred at ambient temperature overnight. After
(E)-4-oxobut-2-enoyl chloride was prepared following the
procedure B. Compound 30 (30.0 mg, 0.07 mmol, 1.0 mol
eq.) was dissolved in 2-ml NMP, following the addition of
trimethylamine (0.11 mmol, 14.0 µl, 1.5 mol eq.) and cooled
the solution to 0 °C. Newly prepared chloride (5.0 mol eq.)
1 3

Chemical Papers
was added dropwise to the above mixture and then stirred
at ambient temperature overnight. After completion, the
reaction mixture was poured into saturated aqueous sodium
bicarbonate to quench the reaction; then, precipitate was
observed and collected. The resulting brown precipitate was
collected and purifed by silica-gel column chromatography
(DCM/MeOH = 50:1). Finally, 11-mg compound 7a was
obtained in 30.1% yield.
0.09 mmol) was dissolved in 2-ml DCM, following the addi-
tion of trimethylamine (16.0 µl, 0.12 mmol, 1.5 mol eq.) and
cooled to 0 °C. Newly prepared chloride (5.0 mol eq.) was
added dropwise and stirred at ambient temperature over-
night. After completion, the reaction mixture was poured
into saturated aqueous sodium bicarbonate to quench the
reaction; then, precipitate was observed and collected. The
resulting brown precipitate was collected and purifed by
silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 19.8-mg compound 10 was obtained in 48.9% yield.
1H NMR (400 MHz, DMSO) δ (ppm) 9.82 (s, 1H), 9.74 (s,
1H), 9.07 (d, J = 1.6 Hz, 1H), 8.85 (s, 1H), 8.77 (m, 1H),
8.59 (d, J=4.0 Hz, 1H), 8.52 (s, 1H), 8.47 (d, J=8.0 Hz,
1H), 8.42 (s, 1H), 7.89~7.85 (m, 1H), 7.68~7.65 (m, 1H),
7.58 (d, J=8.0 Hz, 1H), 7.46 (s, 1H), 7.43 (d, J=2.0 Hz,
1H), 7.38~7.28 (m, 1H), 7.261~7.19 (m, 2H), 5.29 (s, 3H),
4.33 (q, J1 =13.6 Hz, J2 =6.8 Hz, 2H), 1.47 (t, J=6.8 Hz,
3H). LRMS (ESI+), m/z: Calcd for C33H24ClN7O3 [M+H]+,
602.17; found, 602.3.
(2E,4Z)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy)phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4‑(methoxyimino)
but‑2‑enamide (7)
Compound 7a (41.0 mg, 0.08 mmol, 1.0 mol eq.) was dis-
solved in 2-ml solvent (EtOH: H2O = 1:1), methoxyamine
hydrochloride (13.0 mg, 0.26 mmol, 3.5 mol eq.) and sodium
acetate were added to this solution. The mixture was heated
to 40 ℃; the solvent was removed by evaporation. The result-
ing yellow precipitate was collected and purifed by silica-
gel column chromatography (DCM/MeOH = 50:1). Finally,
37.1-mg compound 7 was obtained in 85.5% yield. 1H NMR
(400 MHz, CDCl3) δ (ppm) 9.59 (s, 2H), 8.96 (s, 1H), 8.59
(d, J = 8.6 Hz, 1H), 8.47 (s, 1H), 7.87 (t, J= 7.6 Hz, 1H),
7.58 (d, J = 7.8 Hz, 1H), 7.39 ~ 7.35 (m, 3H), 7.26 ~ 7.19
(m, 2H), 6.76 (q, J1 =16.8 Hz, J2 =10.2 Hz, 1H), 6.29 (d,
J = 16.9 Hz, 1H), 5.80 (d, J = 10.3 Hz, 1H), 5.29 (s, 2H),
4.31 (d, J = 6.9 Hz, 2H), 3.35 (s, 3H), 1.46 (t, J = 6.8 Hz,
3H). LRMS (ESI+), m/z: Calcd for C29H25ClN6O4 [M+H]+,
557.17; found, 559.23.
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑2‑cyanoacetamide
(11)
2-Cyanoacetyl chloride was prepared following the General
Procedure B. Compound 30 (35.0 mg, 0.08 mmol) was dis-
solved in 2-ml DCM, following the addition of trimethyl-
amine (16.0 µl, 0.12 mmol, 1.5 mol eq.) and cooled to 0 °C.
Newly prepared chloride (5.0 mol eq.) was added dropwise
and stirred at ambient temperature overnight. After comple-
tion, the reaction mixture was poured into saturated aqueous
sodium bicarbonate to quench the reaction; then, precipitate
was observed and collected. The resulting brown precipi-
tate was collected and purifed by silica-gel column chro-
matography (DCM/MeOH = 50:1). Finally, 12.7-mg com-
pound 11 was obtained in 36.8% yield. 1H NMR (400 MHz,
CDCl3) δ (ppm) 9.93 (s, 1H), 9.64 (s, 1H), 8.81 (s, 1H),
8.52 (d, J=3.6 Hz, 1H), 8.43 (s, 1H), 7.82~7.78 (m, 1H),
7.51 (d, J = 7.6 Hz, 1H), 7.36 ~ 7.28 (m, 3H), 7.19 ~ 7.15
(m, 2H), 5.22 (s, 2H), 4.26 (d, J = 6.8 Hz, 2H), 2.43 (s,
2H), 1.38 (t, J=6.8 Hz, 3H). LRMS(ESI+), m/z: Calcd for
C27H20BrClN6O3 [M+H]+, 513.14; found, 513.18.
(E)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑3‑(pyridin‑2‑yl)
acrylamide (8)
Compound 30 (50.0 mg, 0.12 mmol, 1.0 mol eq.) was dis-
solved in 2-ml NMP, following the addition of trimethyl-
amine (0.17 mmol, 22 µl, 1.5 mol eq.) and cooled the solu-
tion to 0 °C. Newly prepared chloride (6.0 mol eq.) was
added dropwise to the above mixture and then stirred at
ambient temperature overnight. After completion, the reac-
tion mixture was poured into saturated aqueous sodium
bicarbonate to quench the reaction; then, precipitate was
observed and collected. The resulting brown precipitate was
collected and purifed by silica-gel column chromatography
(DCM/MeOH = 50:1). Finally, 37-mg compound 8 was
obtained in 61% yield.
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑2,4,6‑trifuoroben‑
zamide (12)
(E)‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phe‑
nyl) amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑2‑cy‑
ano‑3‑(pyridin‑3‑yl) acrylamide (10)
2,4,6-Trifuorobenzoic acyl chloride was prepared follow-
ing the general procedure A. Compound 30 (89.0 mg, 0.2
mmogt l0.0 mol eq.) was dissolved in 2-ml NMP, follow-
ing the addition of trimethylamine (29.0 µl, 0.22 mmol,
1.1 mol eq.) and cooled to 0 °C. Newly prepared chloride
(5.0 mol eq.) was added dropwise to the above mixture
(E)-2-cyano-3-(pyridin-3-yl) acryloyl chloride was prepared
following the General procedure A. Compound 30 (35.0 mg,
1 3

Chemical Papers
and then stirred at ambient temperature overnight. After
completion, the reaction mixture was poured into satu-
rated aqueous sodium bicarbonate to quench the reaction;
then, precipitate was observed and collected. The resulting
brown precipitate was collected and purifed by silica-gel
column chromatography (DCM/MeOH = 50:1). Finally,
27-mg compound 12 was obtained in 22.3% yield. 1H NMR
(400 MHz, CDCl3) δ (ppm) 9.16 (s, 1H), 8.69 (s, 1H), 8.56
(s, 1H), 8.49 (s, 1H), 7.95 (s, 1H), 7.74 ~ 7.70 (m, 1H),
7.63 (d, J=7.6 Hz, 1H), 7.36 (s, 1H), 7.32 (d, J=2.4 Hz,
1H), 7.23 (d, J=6.0 Hz, 1H), 7.11 (t, J=2.4 Hz, 1H), 7.11
(dd, J1 =2.8 Hz, J2 =8.8 Hz, 1H), 6.97 (d, J=8.8 Hz, 1H),
6.80 (t, 2H, J=8.4 Hz), 5.27 (s, 1H), 4.28 (q, J1 =7.2 Hz,
J2 =6.8 Hz, 2H), 1.51 (t, J=6.9 Hz, 3H). LRMS (ESI+), m/z:
Calcd for C31H21ClF3N5O3 [M+H]+, 604.14; found, 604.26.
(s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 8.49 (s, 1H), 8.87 (t,
J = 6.8 Hz, 1H), 7.58 (d, 1H, J = 8.0 Hz), 7.41 ~ 7.35 (m,
3H), 7.27 ~ 7.19 (m, 2H), 5.29 (s, 2H), 7.47 (s, 2H), 4.32
(q, 2H, J1 =13.6 Hz, J2 =6.8 Hz), 1.46 (t, 3H, J=6.8 Hz).
LRMS (ESI+), m/z: Calcd for C26H21Cl2N5O3 [M+H]+,
522.11; found, 522.20.
2‑Bromo‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl) acetamide (15)
2-bromoacetic acid was treated with SOCl2 under room tem-
perature; the reaction mixture was stirred at the ambient tem-
perature. After complement, the excess SOCl2 was removed
by evaporation. Compound 30 (30.0 mg, 0.07 mmol) was
dissolved in 2-ml NMP, following the addition of trimeth-
ylamine (14.0 µl, 0.10 mmol, 1.5 mol eq.) and cooled to
0 °C. Newly prepared chloride (6.0 mol eq.) was added;
ropwise to the above mixture and then stirred at ambient
temperature overnight. After completion, the reaction mix-
ture was poured into saturated aqueous sodium bicarbonate
to quench the reaction, then, precipitate was observed and
collected. The resulting brown precipitate was collected
and purifed by silica-gel column chromatography (DCM/
MeOH = 50:1). 27.8-mg compound 15 was obtained in
4‑((3‑Chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl) amino)‑7‑eth‑
oxy‑6‑((5‑fuoro‑2,4‑dinitrophenyl) amino) quinoline‑3‑car‑
bonitrile (13)
Compound 30 (89 mg, 0.2 mmol, 1.0 mol eq.) in 2 ml THF
was added to a solution of DFDNB (45 mg, 0.22 mmol,
1.1 mol eq.) and DIPEA (15 µl, 0.22 mmol, 1.1 mol eq.) in
2-ml THF under stirring at 0 °C, then elevated to ambient
temperature, stirred for 8 h. After completion, the reaction
mixture was poured into saturated aqueous sodium bicar-
bonate and fltered. The resulting brown precipitate was
collected and purifed by silica-gel column chromatography
(DCM/MeOH = 50:1). Finally, 27-mg compound 13 was
1
72.8% yield. H NMR (400 MHz, DMSO) δ (ppm) 9.83
(s, 1H), 8.90 (s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 8.54 (s,
1H), 7.89~7.85 (m, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.43 (d,
J = 2.4 Hz, 1H), 7.39 ~ 7.35 (m, 2H), 7.28 ~ 7.23 (m, 2H),
5.29 (s, 2H), 4.31 ~4.29 (m, 5H), 1.47 (t, J =7.2 Hz, 3H).
LRMS (ESI+), m/z: Calcd for C26H21BrClN5O3 [M+H]+,
566.84; found, 568.13 (Scheme 3).
1
obtained in 22.3% yield. H NMR (400 MHz, CDCl3) δ
(ppm) 10.31 (s, 1H), 9.75 (s, 1H), 8.96 (d, J=7.2 Hz, 1H),
8.59~8.50 (m, 3H), 7.82 (t, J=7.2 Hz, 1H), 7.58~7.46 (m,
3H), 77.37~7.27 (m, 3H), 7.07 (d, J=12.0 Hz, 1H), 5.29 (s,
2H), 4.30 (d, J=4.3 Hz, 2H), 1.34 (s, 3H). LRMS (ESI+),
m/z: Calcd for C30H21ClFN7O6 [M+H]+, 630.13; found,
630.20.
3‑Bromo‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4,5‑dihydroisoxa‑
zole‑5‑carboxamide (16)
Hydroxycarbonimidic dibromide (8.90 mg, 0.04 mmol,
1.2 mol eq.) and compound 7 (18.0 mg, 0.036 mmol, 1.0 mol
eq.) were dissolved in 2-ml DMF at 0℃. An aqueous solu-
tion of KHCO3 was added to this solution dropwise during
1 h. Then the resulting mixture was warmed to room tem-
perature and stirred for 1 h. After addition of EA and wani-
haiter, the aqueous layer was separated and extracted three
times with EA. Then, it was purifed by silica-gel column
chromatography (DCM/MeOH = 50:1). 7-mg compound 16
was obtained in 31.2% yield. 1H NMR (400 MHz, CDCl3)
δ (ppm) 9.33 (s, 1H), 8.90 (s, 1H), 8.58 (d, J = 4.4 Hz,
1H), 8.51 (s, 1H), 7.76 (m, 1H), 7.64 (d, J= 8.0 Hz, 1H),
7.47 (s, 1H), 7.33 (s, 1H), 7.27 ~ 7.22 (m, 2H), 7.07 (dd,
J1 =6.0 Hz, J2 =2.8 Hz, 1H), 6.97 (d, J=8.0 Hz, 1H), 5.28
(s, 2H), 5.20~5.17 (m, 1H), 4.27~4.24 (m, 2H), 3.64~3.57
(m, 2H), 1.54 (t, J=6.8 Hz, 3H). LRMS (ESI+), m/z: Calcd
for C28H22BrClN6O4 [M+H]+, 621.06; found, 623.66.
2‑Chloro‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl) acetamide (14)
2-Chloroacetyl chloride was prepared following the general
procedure A. Compound 30 (45 mg, 0.1 mmol) was dis-
solved in 2-ml NMP, following the addition of trimethyl-
amine (20 µl, 0.15 mmol, 1.5 mol eq.) and cooled to 0 °C.
Newly prepared chloride (6.0 mol eq.) was added dropwise
to the above mixture and then stirred at ambient tempera-
ture overnight. After completion, the reaction mixture was
poured into saturated aqueous sodium bicarbonate to quench
the reaction; then, precipitate was observed and collected.
The resulting brown precipitate was collected and purifed
by silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 27-mg compound 14 was obtained in 46.1% yield.
1H NMR (400 MHz, DMSO) δ (ppm) 9.71 (s, 2H), 8.89
1 3

Chemical Papers
Scheme 3 Synthesis of compound 16–19
N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl) ‑3‑methoxy‑4,5‑di‑
hydroisoxazole‑5‑carboxamide (17)
stirred at 80 ℃ for 3 h. After completion, HCl and MeOH
were removed by evaporation. Without further purifcation,
33-mg crude methyl 3-chloro-4,5-dihydroisoxazole-5-car-
boxylate was obtained in 82.9% yield.
Tert‑butyl
3‑bromo‑4,5‑dihydroisoxazole‑5‑carboxylate
(17.1) Dibromoformaldoxine (407 mg, 2.07 mmol) and
methyl propiolate (311 mg, 3.17 mmol) were dissolved in
10 ml of 50% aqueous EtOH; while stirring, a solution of
243-mg KHCO3 in 5 ml of H2O was added dropwise over
1 h. The resulting solution was stirred for additional 4 h,
then diluted with H2O and extracted with DCM. The com-
bined organic layer was dried with Na2SO4 and concentrated
as a mixture. After purifed by column, 67-mg tert-butyl
3-bromo-4,5-dihydroisoxazole-5-carboxylate was obtained
in 33.9% yield.
3‑Methoxy‑4,5‑dihydroisoxazole‑5‑carboxylic
acid
(17.3) Methyl 3-chloro-4,5-dihydroisoxazole-5-carboxy-
late (80.0 mg, 0.49 mmol, 1.00 e.q.) in 5 ml MeOH was
treated with LiOH (117 mg, 4.90 mmol, 10.0 e.q.) under
room temperature. After completion, adjust the pH until
pH=3 and extracted with DCM. 62.5-mg pure product was
obtained in 88% yield.
3-Chloro-4,5-dihydroisoxazole-5-carbonyl chloride was
prepared. Compound 30 (30.0 mg, 0.07 mmol, 1.00 e.q.) was
dissolved in 1 ml NMP, following the addition of trimethyl-
amine (12.9 µl, 0.10 mmol, 1.50 mol eq.) and cooled to 0 °C.
Newly prepared chloride (6.0 mol eq.) was added dropwise
to the above mixture and then stirred at ambient tempera-
ture overnight. After completion, the reaction mixture was
Methyl
3‑chloro‑4,5‑dihydroisoxazole‑5‑carboxylate
(17.2) Tert-butyl 3-bromo-4,5-dihydroisoxazole-5-carbox-
ylate was treated with 6M HCl in MeOH. The solution was
1 3

Chemical Papers
poured into saturated aqueous sodium bicarbonate to quench
the reaction, precipitate was observed and collected. The
resulting brown precipitate was collected and purifed by
silica-gel column chromatography (DCM/MeOH = 50:1).
Finally, 18.0-mg compound 17 was obtained in 42% yield.
1H NMR (400 MHz, CDCl3) δ (ppm) 9.48 (s, 1H), 8.97 (s,
1H), 8.59 (d, J=3.2 Hz, 1H), 8.54 (s, 1H), 7.78~7.74 (m,
1H), 7.66 (d, J=7.6 Hz, 1H),7.33~7.32 (m, 3H), 7.20~7.24
(m, 1H) 7.14 ~ 7.11 (m, 1H), 7.0 (d, J= 8.8 Hz, 1H), 5.31
(s, 2H), 5.12~5. 09 (m, 1H), 4.27~4.18 (m, 2H), 3.88 (s,
3H), 3.48 ~ 3.24 (m, 2H), 1.52 (t, J= 6.8 Hz, 3H). LRMS
(ESI+), m/z: Calcd for C29H25ClN6O5 [M+H]+,573.16;
found,573.09.
was observed and collected. The resulting brown precipitate
was collected and purifed by silica-gel column chromatog-
raphy (DCM/MeOH = 50:1). Finally, 6.5-mg compound 18
was obtained in 25.1% yield. 1H NMR (400 MHz, CDCl3)
δ (ppm) 10.34(s, 1H), 9.75 (s, 1H), 8.71 (s, 1H), 8.59 (d,
J=4.4 Hz, 1H), 8.52 (s, 1H), 7.89~7,84 (m, 1H), 7.64~7.57
(m, 2H), 7.44 (s, 2H), 7.36 (q, J1 = 5.2 Hz, J2 = 1.6 Hz,
1H), 7.26~7.25 (m, 2H), 5.29 (s, 2H), 4.30 (q, J1 =6.8 Hz,
J2 =6.8 Hz, 2H), 1.42 (t, J=6.8 Hz, 3H). LRMS (ESI+), m/z:
Calcd for C28H22Cl2N6O4 [M+H]+, 577.12; found, 577.30.
3‑Chloro‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4,5‑dihydroisoxa‑
zole‑5‑carboxamide (19)
3‑Chloro‑N‑(4‑((3‑chloro‑4‑(pyridin‑2‑ylmethoxy) phenyl)
amino)‑3‑cyano‑7‑ethoxyquinolin‑6‑yl)‑4,5‑dihydroisoxa‑
zole‑5‑carboxamide (18)
Ethyl 3‑bromoisoxazole‑5‑carboxylate (19.1) Dibromo-
formaldoxine (117 mg, 0.6 mmol) and ethyl propiolate
(145.3 mg, 1.27mmol, 1.5 mmol) were dissolved in 2 ml of
50% aqueous EtOH; while stirring, a solution of 243-mg
KHCO3 in 5 ml of H2O was added dropwise over 1 h. The
resulting solution was stirred for additional 4 h, then diluted
with H2O and extracted with DCM. The combined organic
layer was dried with Na2SO4 and concentrated by evapora-
tion. After purifed by column, 57.6-mg ethyl 3-bromoisox-
azole-5-carboxylate was obtained in 48.6% yield.
Tert‑butyl
3‑bromo‑4,5‑dihydroisoxazole‑5‑carboxylate
(18.1) Dibromoformaldoxine (407 mg, 2.07 mmol) and
methyl propiolate (311 mg, 3.17 mmol) were dissolved in
10 ml of 50% aqueous EtOH; while stirring, a solution of
243-mg KHCO3 in 5 ml of H2O was added dropwise over
1 h. The resulting solution was stirred for additional 4 h,
then diluted with H2O and extracted with DCM. The com-
bined organic layer was dried with Na2SO4 and concentrated
as a mixture. After purifed by column, 67-mg tert-butyl
3-bromo-4,5-dihydroisoxazole-5-carboxylate was obtained
in 33.9% yield.
3‑Bromoisoxazole‑5‑carboxylic acid (19.2) A solution
of methyl 3-bromoisoxazole-5-carboxylate (57.6 mg
0.26 mmol) in THF was added 1M LiOH and stirred at
room temperature for 5 h; then, the pH was adjusted with
1M HCl until pH=3, and extracted with EA. Then, it was
purifed by silica-gel column chromatography; 28.5 mg of
3-bromoisoxazole-5-carboxylic acid was got in 57.7% yield.
3-Bromoisoxazole-5-carbonyl chloride was prepared fol-
lowing the general procedure B. Compound 30 (30.0 mg,
0.07 mmol) was dissolved in 2-ml NMP, following the addi-
tion of trimethylamine (14.0 µl, 0.10 mmol, 1.5 mol eq.)
and cooled to 0 °C. Newly prepared chloride (6.0 mol eq.)
was added dropwise to the above mixture and then stirred
at ambient temperature overnight. After completion, the
reaction mixture was poured into saturated aqueous sodium
bicarbonate to quench the reaction; then, precipitate was
observed and collected. The resulting brown precipitate
was collected and purifed by silica-gel column chroma-
tography (DCM/MeOH = 50:1). 24.1-mg compound 19
was obtained in 57.8% yield. 1H NMR (400 MHz, CDCl3)
δ (ppm) 10.34(s, 1H), 9.75 (s, 1H), 8.71 (s, 1H), 8.59 (d,
J=4.4Hz, 1H), 8.52 (s, 1H), 7.89~7,84 (m, 1H), 7.64~7.57
(m, 2H), 7.44 (s, 2H), 7.36 (q, J1 = 5.2 Hz, J2 = 1.6 Hz,
1H), 7.26~7.25 (m, 2H), 5.29 (s, 2H), 4.30 (q, J1 =6.8 Hz,
J2 = 6.8 Hz, 2H), 1.42 (t, J = 6.8 Hz, 3H). LRMS (ESI+),
m/z: Calcd for C28H20BrClN6O4 [M+H]+619; found, 621.67.
Methyl
3‑chloro‑4,5‑dihydroisoxazole‑5‑carboxylate
(18.2) Tert-butyl 3-bromo-4,5-dihydroisoxazole-5-carbox-
ylate was treated with 6M HCl in MeOH. The solution was
stirred at 80 ℃ for 3 h. After completion, HCl and MeOH
were removed by evaporation. Without further purifcation,
33-mg crude methyl 3-chloro-4,5-dihydroisoxazole-5-car-
boxylate was obtained in 82.9% yield.
3‑Chloro‑4,5‑dihydroisoxazole‑5‑carboxylic acid (18.3) To
a solution of methyl 3-chloro-4,5-dihydroisoxazole-5-car-
boxylate in THF was added LiOH (3.0 e.q.) and stirred at
70 ℃ for 4 h. After completion, adjust pH to pH=2–3, 27.0-
mg 3-chloro-4,5-dihydroisoxazole-5-carboxylic acid was
obtained in 87.3% yield.
Following the general procedure A, 3-chloro-4,5-dihy-
droisoxazole-5-carbonyl chloride was prepared. Compound
30 (20.0 mg, 0.045 mmol) was dissolved in 1-ml NMP, fol-
lowing the addition of trimethylamine (9.80 µl, 0.07 mmol,
1.5 mol eq.) and cooled to 0 °C. Newly prepared chloride
(6.0 mol eq.) was added dropwise to the above mixture and
then stirred at ambient temperature overnight. After comple-
tion, the reaction mixture was poured into saturated aqueous
sodium bicarbonate to quench the reaction; then, precipitate
1 3

Chemical Papers
them, compound 16 exhibited the best inhibition among
them with 67% inhibition at 100 nM.
Results and discussion
With the preliminary cell inhibition, compounds 6, 7, 9,
10, 11 and 16 show better cell inhibition, and their IC50 of
HER-2 kinase inhibition were further evaluated (Fig. 3a).
Although all of them showed weaker inhibition than ner-
atinib, compound 16 showed the best potency, the IC50 on
HER-2 kinase and on SK-Br-3 cell line are 19.6 nM and
94 nM, respectively.
Among all the derivatives, cell inhibition of compounds
1–15 was evaluated at 100 nM on SK-Br-3 cell line
(Table 1). Compounds 3 and 6 showed 75% and 71% inhi-
bition, respectively. Compounds 5 and 10 show worse,
even no, inhibition at all. Due to potential toxicity of
1,3-dicarbonyl compounds, compound 6 was not studied
further.
Considering the side efects of neratinib, further cel-
lular cytotoxicity assay was performed to determine the
cytotoxicity of compound 16. It was tested over Beas-2b
cell line (normal lung epithelial cells) using MTT assay. As
illustrated in Fig. 3b under three diferent concentrations,
compound 16 exhibited a much lower cytotoxicity against
Beas-2b cell in a dose-dependent manner compared to ner-
atinib. This result could suggest compound 16 could be a
lead compound in the drug discovery against breast cancer.
This cellular cytotoxicity assay was repeated for three times
separately.
Consequently, compounds 16–19 were designed with
the inspiration from natural product acivicin (Fig. 2). Aci-
vicin, derived from natural product, is a potent inhibitor of
γ-glutamyl transferase. Due to its unique structure of the
chlorodihydroisoxazoles, it could target on the cysteine
side chain in γ-glutamyltranspeptidase by nucleophilic
replacement of the chloride in the scafold [17]. It was
proved that halogen dihydroisoxazole substituted of com-
pounds could also target on the cysteine in peptidases and
form a covalent bond (Fig. 2). Enlightened by this fact,
compounds 16–20 with substituted dihydroisoxazole as
novel warheads were synthesized and evaluated. Among
To clarify the structure–activity relationship of com-
pound 16 and HER-2 kinase, a docking model of the HER-2/
Fig. 2 Structure of acivicin and proposed mechanism of dihydroisoxazole
A
B
Neratinib
100
50
0
16
uM
uM
uM
uM
uM
uM
0
0
5
10
10
5
100
100
Concentration
Fig. 3 a IC50 of compounds 6, 7, 9, 10, 11, 16 and neratinib over HER-2 kinase. b Cellular cytotoxicity of compound 16 and Neratinib under
10 µM, 50 µM, 100 µM
1 3

Chemical Papers
B
A
CYS 805
LYS 753
Fig. 4 Docking model of HER-2 and compound 16 (PDB code: 3RCD). a 2D model of compound 16 interact with HER-2. b Docking model
of 16 with HER-2 (the dark-green dash represents Pi–cation interaction, and covalent bond between compound 16 and Cys 805 colored yellow)
compound 16 complex was built with the Schrödinger soft-
ware (Schrödinger Suites 2016-1, PDB code: 3RCD). Since
the mechanism of bromodihydroisoxazole was proposed as
nucleophilic substitution, the covalent docking was con-
ducted under nucleophilic substitution (Fig. 4).
All these data indicated that compound 16 could be a prom-
ising lead for further anti-breast cancer drug discovery. This
fnding could serve as the following basis for the future
research in the feld of HER-2-positive breast cancer.
Acknowledgements This work was supported by National Natural Sci-
ence Foundation of China (#81573277, 81622042, 81773567), National
Major Scientifc and Technological Special Project for “Signifcant
New Drugs Development” (#SQ2017ZX095003) and Tsinghua Uni-
versity Initiative Scientifc Research Program.
The cysteine residue (Cys 805) could form a covalent
bond with bromodihydroisoxazole with bromine leaving
through SNi reaction. Additionally, the afnity was further
enhanced by Pi–cation interaction between amino residue
Lys 753 and pyridine ring. Furthermore, there was another
Pi–cation interaction between amino residue Lys 753 and
quinoline ring which could also help in improving the bind-
ing activity. This docking model might indicate a novel
mechanism of compound 16 interacting with HER-2 through
a covalent complex to inhibit the function of HER-2.
References
Awada A, Bozovic-Spasojevic I, Chow L (2012) New therapies in
HER2-positive breast cancer: a major step towards a cure of the
disease? Cancer Treat Rev 38:494–504. https://doi.org/10.1016/j.
ctrv.2012.01.001
Burstein HJ, Sun Y, Dirix LY, Jiang Z, Paridaens R, Tan AR, Awada
A, Ranade A, Jiao S, Schwartz G, Abbas R, Powell C, Turnbull
K, Vermette J, Zacharchuk C, Badwe R (2010) Neratinib, an irre-
versible ErbB receptor tyrosine kinase inhibitor, in patients with
advanced ErbB2-positive breast cancer. J Clin Oncol 28:1301–
1307. https://doi.org/10.1200/JCO.2009.25.8707
Conclusions
In summary, a series of 4-anilino-3-cyanoquinoline deriva-
tives with novel warheads were designed, synthesized and
evaluated for anti-breast cancer activity. According to the
anti-proliferation activity data of dihydroisoxazolecarboxa-
mide derivatives (compound 16>18>17), it may lead us to
the conclusion that the leaving ability of substitution of war-
head shows remarkable infuence on the activity. Compound
16 with bromodihydroisoxazole as warhead turned out to be
the most potent compound among 19 analogs. The proposed
mechanism is nucleophilic replacement of the bromine in a
diferent mechanism with classical warheads. Importantly,
compound 16 showed a much acceptable potency (HER-2
kinase IC50: 19.4, SK-BR-3 IC50: 94) and lower cytotoxicity.
Cortés JD, Dieras VM, Ro JM, Barriere JMM, Bachelot TM, Hurvitz
SM, Le Rhun EM, Espié MM, Kim SM, Schneeweiss AM, Sohn
JHM, Nabholtz JP, Kellokumpu-Lehtinen PP, Taguchi JM, Pia-
centini FM, Ciruelos EM, Bono PM, Ould-Kaci MM, Roux FM,
Joensuu HP (2015) Afatinib alone or afatinib plus vinorelbine
versus investigator’s choice of treatment for HER2-positive breast
cancer with progressive brain metastases after trastuzumab, lapat-
inib, or both (LUX-Breast 3): a randomised, open-label, multicen-
tre, phase 2 trial. 16:1700–1710. https://doi.org/10.1016/S1470
-2045(15)00373-3
Dzimitrowicz H, Berger M, Vargo C, Hood A, Abdelghany O, Raghav-
endra AS, Tripathy D, Valero V, Hatzis C, Pusztai L, Murthy R
(2016) T-DM1 activity in metastatic human epidermal growth
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factor receptor 2-positive breast cancers that received prior ther-
Martin M, Holmes FA, Ejlertsen B, Delaloge S, Moy B, Iwata H, von
Minckwitz G, Chia SKL, Mansi J, Barrios CH, Gnant M, Toma
Evi Z, Denduluri N, Eparovi R, Gokmen E, Bashford A, Ruiz Bor-
rego M, Kim S, Jakobsen EH, Ciceniene A, Inoue K, Overkamp
F, Heijns JB, Armstrong AC, Link JS, Joy AA, Bryce R, Wong
A, Moran S, Yao B, Xu F, Auerbach A, Buyse M, Chan A (2017)
Neratinib after trastuzumab-based adjuvant therapy in HER2-pos-
itive breast cancer (ExteNET): 5-year analysis of a randomised,
double-blind, placebo-controlled, phase 3 trial. Lancet Oncol
18:1688–1700. https://doi.org/10.1016/S1470-2045(17)30717-9
Tiwari SR, Mishra P, Abraham J (2016) Neratinib, a novel HER2-
targeted tyrosine kinase inhibitor. Clin Breast Cancer 16:344–348.
https://doi.org/10.1016/j.clbc.2016.05.016
apy with trastuzumab and pertuzumab. J Clin Oncol 34:3511–
3517. https://doi.org/10.1200/JCO.2016.67.3624
Eroglu Z, Tagawa T, Somlo G (2014) Human epidermal growth fac-
tor receptor family-targeted therapies in the treatment of HER2-
overexpressing breast cancer. Oncologist 19:135–150. https://doi.
org/10.1634/theoncologist.2013-0283
Ito Y, Suenaga M, Hatake K, Takahashi S, Yokoyama M, Onozawa Y,
Yamazaki K, Hironaka S, Hashigami K, Hasegawa H, Takenaka
N, Boku N (2012) Safety, efcacy and pharmacokinetics of ner-
atinib (HKI-272) in Japanese patients with advanced solid tumors:
a phase 1 dose-escalation study. Jpn J Clin Oncol 42:278–286.
https://doi.org/10.1093/jjco/hys012
Jackson PA, Widen JC, Harki DA, Brummond KM (2017) Cova-
lent modifers: a chemical perspective on the reactivity of α,
β-unsaturated carbonyls with thiols via hetero-michael addition
reactions. J Med Chem 60:839–885. https://doi.org/10.1021/acs.
jmedchem.6b00788
Tsou H, Overbeek-Klumpers EG, Hallett WA, Reich MF, Floyd MB,
Johnson BD, Michalak RS, Nilakantan R, Discafani C, Golas J,
Rabindran SK, Shen R, Shi X, Wang Y, Upeslacis J, Wissner A
(2005) Optimization of 6,7-disubstituted-4-(arylamino)quinoline-
3-carbonitriles as orally active, irreversible inhibitors of human
epidermal growth factor receptor-2 kinase activity. J Med Chem
48:1107–1131. https://doi.org/10.1021/jm040159c
Li X, Yang C, Wan H, Zhang G, Feng J, Zhang L, Chen X, Zhong D,
Lou L, Tao W, Zhang L (2017a) Discovery and development of
pyrotinib: a novel irreversible EGFR/HER2 dual tyrosine kinase
inhibitor with favorable safety profles for the treatment of breast
cancer. Eur J Pharm Sci 110:51–61. https://doi.org/10.1016/j.
ejps.2017.01.021
Wong KK, Fracasso PM, Bukowski RM, Lynch TJ, Munster PN, Sha-
piro GI, Janne PA, Eder JP, Naughton MJ, Ellis MJ, Jones SF,
Mekhail T, Zacharchuk C, Vermette J, Abbas R, Quinn S, Powell
C, Burris HA (2009) A phase I study with neratinib (HKI-272),
an irreversible pan ErbB receptor tyrosine kinase inhibitor, in
patients with solid tumors. Clin Cancer Res 15:2552–2558. https
://doi.org/10.1158/1078-0432
Ma CX, Bose R, Gao F, Freedman RA, Telli ML, Kimmick G, Winer
E, Naughton M, Goetz MP, Russell C, Tripathy D, Cobleigh M,
Forero A, Pluard TJ, Anders C, Niravath PA, Thomas S, Ander-
son J, Bumb C, Banks KC, Lanman RB, Bryce R, Lalani AS,
Pfeifer J, Hayes DF, Pegram M, Blackwell K, Bedard PL, Al-
Kateb H, Ellis MJC (2017) Neratinib efcacy and circulating
tumor DNA detection of HER2 mutations in HER2 nonamplifed
metastatic breast cancer. Clin Cancer Res 23:5687–5695. https://
doi.org/10.1158/1078-0432.CCR-17-0900
Yoshioka T, Shien K, Namba K, Torigoe H, Sato H, Tomida S, Yama-
moto H, Asano H, Soh J, Tsukuda K, Nagasaka T, Fujiwara T,
Toyooka S (2018) Antitumor activity of pan-HER inhibitors in
HER2-positive gastric cancer. Cancer Sci 109:1166–1176. https
://doi.org/10.1111/cas.13546
Zhang X, Peng T, Ji X, Li J, Tong L, Li Z, Yang W, Xu Y, Li M, Ding
J, Jiang H, Xie H, Liu H (2013) Design, synthesis and biological
evaluation of novel 4-anilinoquinazolines with C-6 urea-linked
side chains as inhibitors of the epidermal growth factor recep-
tor. Bioorg Med Chem 21:7988–7998. https://doi.org/10.1016/j.
bmc.2013.09.049
Mao Y, Zhu W, Kong X, Wang Z, Xie H, Ding J, Terrett NK, Shen J,
Shen J (2013) Design, synthesis and biological evaluation of novel
pyrimidine, 3-cyanopyridine and m-amino-N-phenylbenzamide
based monocyclic EGFR tyrosine kinase inhibitors. Bioorg Med
Chem 21:3090–3104. https://doi.org/10.1016/j.bmc.2013.03.053
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