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S. Li et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5279–5282
ABL kinase; IC50 = 2180.3 nM against K562 cell). It may be that
pyrimidinyl substitution was more conducive to the compounds
to form hydrogen bonds with the surrounding amino acid residues.
In addition, the modification of the substituent in C ring had a great
effect on their bioactivities. When the group R1 or R2 in C ring were
replaced by fluoro-, chloro- or bromo- groups, the compounds
induced loss in activities towards ABL kinase and K562 cells (such
as 12c, 12e, 13c and 13e). To our surprise, while the substituent
in C ring was the trifluoromethyl group, the compounds had even
higher potency to antiproliferative activities. In particular, the
compound 13f exhibited an IC50 value as low as 20.6 nM, 10.5-fold
lower than that of Imatinib in inhibiting ABL kinase, and, with an
IC50 value of 32.3 nM, was about 12-fold more active in antiprolif-
eratiion than Imatinib. It was clear that the trifluoromethyl group
at R1 or R2 position promoted conformation of the compound to
fit into the ATP binding site of ABL protein. In addition, when the
group R1 or R2 in C ring was replaced by a big group such as 1-
ethyl-4-methylpiperazine (13h: IC50 = 575.2 nM against ABL
kinase, IC50 = 579.3 nM against K562 cell), the inhibitory activity
of a compound was as good as Imatinib. It was proved again that
our newly designed and synthesized compounds were new BCR–
ABL inhibitors from Imatinib and Nilotinib.
In summary, a series of acrylamide derivatives were synthe-
sized and evaluated for their inhibitory activity against ABL kinase
and K562 cells. The majority of the compounds strongly sup-
pressed the activity of ABL kinase and potently inhibited the prolif-
eration of K562 leukemia cancer cells. We found the
trifluoromethyl group at R1 or R2 position to be much more potent
than the original structure in Imatinib. Furthermore, the SAR for
this class of compounds were defined clearly, and the compound
13f was proved to be a promising candidate for development, judg-
ing from its overall characteristics, including its physico-chemical
property and its main pharmacodynamics.
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20. Enzyme assay: One unit of recombinant BCR–ABL protein was incubated with
various concentrations of Imatinib, 12a–12h and 13a–13h, or DMSO and
reaction buffer (50 mM Tris–HCl, pH 7.5/10 mM MgCl2/1 mM EDTA) in a total
volume of 15
reactions, which were added 2
and 1 L (1 g) of EAIYAAPFAKKK, were performed at 30 °C for 15 min. The
lL. After incubation at room temperature for 30 min, the kinase
l
L of 1 mM ATP, 10 L of [ Ci)
l
c
-7P] ATP (40
l
l
l
reaction mixtures were spotted onto strips of P81 phosphocellulose paper,
washed, and counted. Nonspecific binding was determined by conducting the
assay in the absence of enzyme and then subtracting the value from each of the
experimental values.
21. Cellular assay: The BCR–ABL positive K562 cells were cultured in RPMI-1640
medium, supplemented with 10% fetal calf serum (FCS), and maintained in a 5%
CO2, 95% humidity atmosphere at 37 °C. 3 Â 103 K562 cells were seeded into
each well of a 96-well plate and incubated for 12 h. The cells were then
Acknowledgment
cultured in medium that contained 10, 1
13a–13h and Imatinib for 48 or 72 h. Then 100
was added to each well, and the cells were incubated for 4 h at 37 °C to allow
the MTT to form formazan crystals by reacting with metabolically active cells.
l
g/mL, 0.10, 0.01
lg/mL of 12a–12h,
This work was financially supported by the National Natural Sci-
ence Foundation of China (Grant No. 30973616 and No. 20872095).
lL of MTT solution (5 mg/mL)
The formazan crystals were solubilized in 200 lL of DMSO after 5 min
incubation at 37 °C. The absorbance of each well was measured at 570 nm.
The drug IC50 was determined from the cell survival plots.
Supplementary data
Supplementary data associated with this article can be found, in