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J. Bussenius et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2283–2286
kinases, particularly Rsk2 (68 nM) and PKA (42 nM) were observed.
For the two Akt isoforms Akt1 and Akt2 only weak activity was
measured (371 and 3021 nM, respectively). Compound 14 on the
other hand had stronger activity against AGC kinases (Akt1:
53 nM, Akt2: 311 nM, Rsk2: 248 nM, and PKA: 25 nM). Addition-
ally, this compound showed some cross reactivity with IC50s
<500 nM against several other targets outside the AGC kinase fam-
ily (e.g., PIM1). Further, the trifluoromethyl-analog 15 was also ac-
tive against AGC kinases (Akt1: 74 nM, Akt2: 477 nM, Rsk2: 24 nM,
and particularly PKA with 6 nM).
On the basis of these in vitro data we decided to take compound
13c forward into in vivo studies. First, the pharmacokinetic profile
of 13c was determined in the rat and the results are summarized in
Table 4. Compared to compound 5b, the absorption and oral bio-
availability were significantly improved. This result in combination
with the high potency led to the advancement of 13c into a mouse
pharmacodynamic study in which the inhibition of S6 phosphory-
lation was measured. In this study 13c demonstrated >60% inhibi-
tion at 100 mg/kg PO dosing in the PC3 prostate carcinoma model.
Compound 13c was subsequently evaluated in a PC3 xenograft effi-
cacy experiment and dosed orally at 30, 100, and 300 mg/kg twice
a day for 14 days. The compound was also active in this study and
showed a good dose–response relationship with 21%, 49%, and 69%
tumor growth inhibition, respectively
executing cell based assays. Julie Ren and Yeping Zhao generated
the PK data. Paul Keast, Wendy Abulafia, Brian Sutton, and Teresa
Carabeo performed the PD experiment and Jason Chen and F. Mi-
chael Yakes the efficacy study.
References and notes
1. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
2. Hennessy, B. T.; Smith, D. L.; Ram, P. T.; Lu, Y.; Mills, G. B. Nat. Rev. Drug
Discovery 2005, 4, 988.
3. Engelman, J. A. Nat. Rev. Cancer 2009, 9, 550.
4. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Discovery 2009, 8, 627.
5. Courtney, K. D.; Corcoran, R. B.; Engelman, J. A. J. Clin. Oncol. 2010, 28, 1075.
6. Matthews, D. J.; Gerritsen, M. E. Targeting Protein Kinases for Cancer Therapy;
John Wiley & Sons: Hoboken, NJ, 2010. 274–275 and 282–283.
7. Thomas, G. Biol. Res. 2002, 35, 305.
8. (a) Bandarage, U.; Hare, B.; Parsons, J.; Pham, L.; Marhefka, C.; Bemis, G.; Tang,
Q.; Moody, C. S.; Rodems, S.; Shah, S.; Adams, C.; Bravo, E.; Savic, V.; Come, J. H.;
Green, J. Bioorg. Med. Chem. Lett. 2009, 19, 5191; (b) Ye, P.; Kuhn, C.; Juan, M.;
Sharma, R.; Connolly, B.; Alton, G.; Liu, H.; Stanton, R.; Kablaoui, N. M. Bioorg.
Med. Chem. Lett. 2011, 21, 849.
9. p70S6K assay: kinase activity was measured as the percent of ATP consumed
following
the
kinase
reaction
using
luciferase–luciferin-coupled
chemiluminescence. Reactions were conducted in 384-well white, medium
binding microtiter plates (Greiner). P70S6 Kinase reactions were initiated by
combining test compounds, 500 nM ATP,
12 nM Human p70S6K (residues 1–421, contains D18H and T412E mutations,
Upstate Bio-technology) in 20 L of reaction buffer (20 mM Hepes pH 7.5,
10 mM MgCl2, 0.03% NP40, 0.03% BSA, 1 mM DTT). The reaction mixture was
incubated at ambient temperature for 3 h after which 20 aliquot of
3 lM RRRLSSLRA (Synpep), and
l
lL
Scheme 3 describes the synthesis of highly functionalized phe-
nylpiperazines needed for the preparation of the products shown
in Table 3. Reaction of aniline 16 with bis(2-chloroethyl)amine fol-
lowed by protection with di-tert-butyl dicarbonate yielded iod-
ophenylpiperazine 17. Palladium mediated conversion of 17 into
pinacolester 18 and oxidation with hydrogen peroxide gave phenol
19, which was then alkylated with chloroethylamines followed by
deprotection to afford the phenylpiperazines 20a–c, where the ba-
sic amino group is attached to the phenyl ring via oxygen. The
preparation of the corresponding compounds 22a–c, in which the
basic amine was tethered to the phenyl ring through a propyl lin-
ker, was carried out by Heck reaction of iodophenylpiperazine 17
with allyl alcohol,12 followed by reductive amination and depro-
tection. Finally, the nitrogen linked analogs 23a–c were synthe-
sized by Buchwald–Hartwig amination of 17 followed by
deprotection of the Boc group. The coupling of these phenylpiper-
azines with the respective chloropyrazolopyrimidines was then
carried out again as described in Scheme 1.
luciferase–luciferin mix (50 mM HEPES, pH 7.8, 67 mM oxalic acid (pH 7.8), 5
(or 50) mM DTT, 0.4% Triton X-100, 0.25 mg/mL coenzyme A, 63 mM AMP,
28 mg/mL luciferin and 40,000 units/mL luciferase) was added and the
chemiluminescence signal measured using a Victor2 plate reader (Perkin–
Elmer).
10. Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Curr. Drug Meta. 2006, 7, 715.
11. A549 cell assay: cells were seeded in 96-well plates (all plates and dishes are
NUNC unless otherwise noted) in DMEM (Cellgro) containing 10% FBS (all FBS
is heat-inactivated and from Hyclone unless otherwise noted), 1% penicillin–
streptomycin (Cellgro), and 1% non-essential amino acids (Cellgro). A549 cells
were seeded at 7.5 ꢀ 103cells/well. Cells were incubated at 37 °C, 5% CO2 for
48 h. Test compound in DMSO was diluted in DMEM. The DMSO concentration
in cell culture medium was maintained at 0.2% in all wells. Cells were treated
with diluted compound and each concentration assayed in triplicate at
concentrations of 1.2–300 nM, 0.004–1 nM rapamycin (Cell Signaling
Technology, 9904) and 0.12–30 nM staurosporine (Calbiochem, 569396)
served as positive controls. Negative control wells were treated with DMSO.
Cells were incubated for 3 h at 37 °C, 5% CO2 for 3 h in the presence of
compound. Post compound treatment cells were fixed as follows: medium was
removed and 100
TBS (Pierce, 28376) was added to each well at room temperature (rt) for
20 min. Cells were quenched with 100 0.6% H2O2 (VWR International,
43038308) in TBS (Pierce, 28376) +0.1%Tween20 (Sigma, P-7949) (TBST) for
l TBS and blocked with 100
ll/well of 4% formaldehyde (Sigma–Aldrich, 08920AD) in
ll
20 min at rt. Plates were washed 3ꢀ with 200
l
ll
In summary, we have outlined the successful optimization of
novel and highly potent p70S6K inhibitors based on the pyrazolo-
pyrimidine structural motif. The resulting lead compound 13c is
selective over other kinases, has a reasonable ADME profile, and
is efficacious in the PC3 xenograft model. Further optimization of
13c leading to clinical candidate XL418 will be reported in due
course.
5% BSA (Jackson ImmunoResearch Laboratory, 001-000-162) in TBST for 1 h at
rt. Anti-phospho-S6 ribosomal protein antibody (Cell Signaling Technology, Ser
240/244 2212L) and anti-total-S6 ribosomal protein antibody (Cell Signaling
Technology, 2215L) were diluted 1:200 in 5% BSA in TBST. Fifty microlitre
primary antibody solution was added to each well and incubated overnight at
4 °C. Plates were washed 3ꢀ with 200
antibody (Chemicon International, 24070101) was diluted at 1:20000 in 5%
non-fat dry milk in TBST. 50 l of antibody solution was added to each well and
l TBST and 2 ꢀ 200
ll TBST. Goat anti-rabbit secondary
l
incubated for 1 h at rt. Plates were washed 3ꢀ with 200
l
ll
TBS. Chemiluminescent substrate (Super Signal Elisa Femto Chemiluminescent
Substrate; Pierce, 37075) was prepared at rt. Hundred microlitre of
chemiluminescent substrate per well was added and then the plate was
shaken for 1 min. Luminescence was read immediately on a Wallac plate
reader.
Acknowledgments
The authors appreciate the contributions of the following
persons. Steven Richards for help with the manuscript. Tracy Lou
for conducting the biochemical assays and Edith Ubannwa for
12. Melpolder, J. B.; Heck, R. F. J. Org. Chem. 1976, 41, 265.