656
A. M. Venkatesan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 653–656
30
10
3
1
0.30
0
[µM]
probed with antibodies (Ab) specific to the protein and phospho-
protein components of the PI3 K/Akt/mTOR signaling pathway.
pAkt (T308)
cPARP
Abs obtained from Cell Signaling Technology were: anti( )-Akt,
a
29
a
-phospho(p)-Akt at T308, -Akt, -cleaved PARP, and a-actin.
a
a
Actin
Specific antigen/antibody interactions were identified by a horse-
radish peroxidase (HRP) conjugate secondary Ab that enabled
chemiluminescent signal detection.
Figure 4. Inhibition of pAkt at T308, and induction of cleaved PARP by 29.
Acknowledgment
in suppression of the phosphorylation of Akt particularly at the
threonine 308 site (T308, Fig. 4). As can be seen in Fig. 4, compound
29 inhibits Akt T308 phosphorylation in MDA361 breast tumor
cells, with an IC50 value of about ꢀ300 nM, after a 4 h exposure
The authors thank the members of the Wyeth Chemical Tech-
nologies group for analytical and spectral determination.
time. Compound 29 also induced cleaved PARP at 10 and 30 lM.
References and notes
Cleaved PARP is a marker for cell apoptosis (programmed cell
death). Actin (control protein) signal was unaffected by 29.
In this Letter, we have disclosed a series of novel 2-aryl or hetero-
aryl substituted-4-morpholino imidazolopyrimidine derivatives
14–34 possessing moderate to excellent PI3K/mTOR inhibitory
activity. Most interestingly, from the selectivity point of view
(against mTor) analogues such as 21, 22 and 29 are all of great inter-
est. Introduction of the 5-hydroxy-3-pyridyl appendage at the 2-po-
sition of the imidazolopyrimidine scaffold yielded potent analogues
such as 30 and 32. This could be due to an extra hydrogen bond inter-
action that could possibly occur between the pyridyl nitrogen and
Lys802. Analog 29 showed a good correlation between cell growth
inhibition and biomarker (pAkt at T308) suppression. Further stud-
ies concerning agents that target the PI3-K and mTor will be re-
ported in due course.
Cell growth inhibition assay: The MDA361 and PC3mm2 cell lines
were obtained from ATCC. Cell lines were grown at 37 oC in 5% CO2
incubators in growth media supplemented with penicillin/strepto-
mycin and 10% fetal calf serum. Cell growth inhibition was
determined using the CellTiter 96 aqueous non-radioactive cell
proliferation assay from Promega. This homogeneous colorimetric
method determined the number of viable cells in proliferation as-
says. The assay was carried out in 96 well format following manufac-
turer’s instructions, with cell number per well being adjusted based
on growth characteristics of the various cell lines used. Assay end-
point data was quantitated after 72 h compound exposure using a
Victor2 V (Wallac) model 1420 multilabel HTS counter.
Cell lysis and Western blotting: Cell lysis enabled biochemical
analysis of PI3 K/mTOR signaling pathway proteins after exposure
of cells to compounds. Cells (3 Â 105) were seeded onto 6-well
microtiter plates (Nunc) 24 h prior to being exposed to compound
in complete growth media. Cells were exposed to these compounds
for 4 h. After exposure to compounds cell growth media was re-
moved and cells were washed twice with cold (4 °C) PBS. Cell lysis
buffer (0.2 ml) was then added to each microtiter plate well with
sufficient mixing to insure complete cell lysis. Cell lysis buffer
consisted of: 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM
Na2EDTA 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate,
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1 mM b-glycerophosphate, 1 mM Na3VO4, and 1
Cell lysates were then spun for 30 s at 14,000 rpm. Supernatant
(75 l) was combined with 30
lg/ml leupeptin.
l
l
l of 3Â protein gel loading buffer
[187.5 mM Tris–HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 0.03%
(w/v) bromophenol blue, and 125 mM DTT]. Samples were boiled
(5 min) separated by SDS–PAGE, transferred to nitrocellulose and