COMMUNICATIONS
heated up to 1508C for 30 min (Scheme 5) under microwave
irradiation. The reaction was cooled down and the reaction
solution was filtered through a pad of Celiteꢃ, which was
washed with three portions of ethyl acetate (10 mLꢂ3). The
combined organic layers were washed with brine (10 mL),
dried over anhydrous Na2SO4, filtered, and concentrated to
give the crude product, which was further purified by flash
column chromatography to yield compounds 5a–5c.
with phosphate-buffered saline (PBS) containing 0.1%
Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody
(100 mL; 1:500, diluted in 5 mgmLÀ1 BSA T-PBS) was then
added. After a 30-min incubation at 378C, the plate was
washed three times, and 100 mL horseradish peroxidase-con-
jugated goat anti-mouse IgG (1:2000, diluted in 5 mgmLÀ1
BSA T-PBS) was added. The plate was then incubated at
378C for 30 min and washed 3 times. A 100 mL aliquot of
a solution containing 0.03% H2O2 and 2 mgmLÀ1 o-phenyle-
nediamine in 0.1 molLÀ1 citrate buffer (pH 5.5) was added.
The reaction was terminated by the addition of 50 mL of
2 molLÀ1 H2SO4 as the color changed, and the plate was an-
alyzed using a multi-well spectrophotometer (SpectraMAX
190, Molecular Devices, and Sunnyvale, CA, USA) at
490 nm. The inhibition rate (%) was calculated using the fol-
lowing equation: [1À(A490/A490 control)]ꢂ100%. The IC50
values were calculated from the inhibition curves in two sep-
arate experiments.
Synthesis of Compound Yhhu-251
Compound 4i was obtained following the standard coupling
conditions. Afterwards, 4i (1.0 mmol) and LiOH·H2O
(1.5 mmol) were dissolved in a mixture of THF (10 mL) and
H2O (5 mL). The reaction solution was stirred at room tem-
perature for 0.5–1.0 hour until 4i was completely consumed
(monitored by TLC), then the reaction solution was concen-
trated under reduced pressure. The remaining aqueous solu-
tion was acidified by 1N HCl in an ice bath and white solid
precipitated out. The mixture was filtered to yield a filter
cake, which was dried and used directly for the next step
without further purification.
To a DMF solution (3 mL) of the dried residue obtained
from 4i (0.2 mmol), HATU (0.3 mmol), DIPEA (0.4 mmol),
and appropriately substituted arylamine (0.24 mmol) were
added accordingly. The mixture was stirred at room temper-
ature for 3 hours (monitored by TLC), followed by extrac-
tion with ethyl acetate (10 mLꢂ3). The combined organic
layers were washed with brine (10 mL), dried over anhy-
drous Na2SO4, filtered, and concentrated to yield the crude
product, which was further purified by flash column chroma-
tography to afford compound Yhhu-251.
Acknowledgements
This study was financially supported by the Science and
Technology Department of Zhejiang Province (2014C33181).
References
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ELISA Kinase Assay
The effects of the indicated compounds on the activities of
receptor tyrosine kinase RET were determined using
enzyme-linked immunosorbent assays (ELISAs) with puri-
fied recombinant proteins. Briefly, 20 mgmLÀ1 poly(Glu,-
Tyr)4:1 (Sigma, St. Louis, MO, USA) was pre-coated in 96-
well plates as a substrate. A 50-mL aliquot of 10 mmolLÀ1
ATP solution diluted in kinase reaction buffer (50 mmolLÀ1
HEPES [pH 7.4], 50 mmolLÀ1 MgCl2, 0.5 mmolLÀ1 MnCl2,
0.2 mmolLÀ1 Na3VO4, and 1 mmolLÀ1 DTT) was added to
each well; 1 mL of various concentrations of the indicated
compound diluted in 1% DMSO (v/v) (Sigma) was then
added to each reaction well. DMSO (1%, v/v) was used as
the negative control. The kinase reaction was initiated by
the addition of purified or commercial tyrosine kinase pro-
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Adv. Synth. Catal. 0000, 000, 0 – 0
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