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Compounds 7a, 7c, 7e, and 7h were tested in a TEL-JAK2 prolifera-
tion assay in Ba/F3 cells13 or EPOR-STAT5 assay.22 In addition, their
selectivity versus Aurora B, CDK2 and TRKA enzymes was evalu-
ated. As it can be seen in Table 1, the introduction of the Me group
on the pyrazine ring results in improved selectivity versus CDK2
while the other kinase activities evaluated were much weaker than
the corresponding JAK2 activity. Compounds 7c and 7h were used
as examples in order to assess the in vivo pharmacokinetic proper-
ties in pre-clinical species (Table 2). Both compounds had low
intrinsic clearance in rat microsomes and hepatocytes (7c: 18 ml/
min/mg and 3 ml/min/10ꢀ6 cells and 7h:12 ml/min/mg and 3 ml/
min/10ꢀ6 cells, respectively) that translated into low in vivo clear-
ance. Compound 7c was therefore chosen to progress to dog phar-
macokinetic studies. The compound showed excellent plasma
stability when it was dosed at 2.5 mg/kg (iv) with reasonable expo-
sure and long half-life.
19. Burk, M. J.; Feaster, J. E.; Nugent, W. A.; Harlow, R. L. J. Am. Chem. Soc. 1993, 115,
10125.
20. Liu, G.; Cogan, D. A.; Owens, T. D.; Tang, T. P.; Ellman, J. A. J. Org. Chem. 1999, 64,
1278.
21. To measure JAK2 kinase activity at 5 mM ATP, 150 pM JAK2 enzyme (Upstate
Biotechnology MA) was incubated with 80 nM biotinylated TYK2 peptide
substrate (Cell Signaling Technology, MA) and adenosine triphosphate (ATP
5 mM) for 60 min at room temperature in reaction buffer containing 10 mM
Based upon the favorable pharmacokinetic properties com-
pounds 7c and 7h were profiled in our pharmacodynamic model.
In nude mice implanted with TEL-JAK2 transfected Ba/F3 cells
the compounds demonstrated a dose-dependent effect, as indi-
cated by the reduced levels of STAT5 phosphorylation in splenic
infiltrates of leukemia cells (Table 3).23
In conclusion, we have demonstrated that a series of 6-pyrazol-
3-ylamino pyrazines are potent and selective JAK2 inhibitors.
These compounds possess good pharmacokinetic properties and
several lead compounds were identified as suitable candidates to
further explore the hypothesis that modulation of JAK2 activity
could be beneficial in the treatment of MPNs and other cancers.
Further evaluation of such compounds will be reported in due
course.
MgCl2
, 1 mM DTT, 50 mM HEPES, and 0.025% Tween20. Similarly, JAK3 kinase
activity was assayed using 600 pM JAK3 enzyme (Upstate Biotechnology MA),
5 nM biotinylated poly-EY peptide substrate (HTRF CisBio) and 5 mM ATP in
reaction buffer for 60 min at room temperature. Reactions were stopped by
addition of Detection mix consisting of 20 mM HEPES, 102 mM
ethylenediamine tetraacetic acid, 1.65 mg/ml BSA, 136 mM NaCl, 40
lg/ml
Streptavidin donor beads (Perkin Elmer, MA), and 40 g/ml phosphotyrosine-
l
specific antibody coated acceptor beads (Perkin Elmer, MA). Phosphorylated
substrate was detected after overnight incubation in an EnVision plate reader
(Perkin Elmer, MA) 680 nm excitation, 520–620 nm emission. Data was
graphed and IC50s calculated using Xlfit4 4.2.2 (Microsoft).
22. EPOR-STAT5 assay was used as a mode of action assay, which measured the
relocalization of STAT5 upon the stimulation of Erythropoietin (EPO). U2OS/
EPOR-STAT5-zsGreen stable cell line was generated in house. The cells were
seeded at 17,500 cells/well in 96 well plates and incubated at 37 oC/5% CO2 for
24 h. Cells were dosed with compounds for 30 min and then EPO (0.5 U/ml
final concentration, R&D System) for 1.5 h. Cells were then fixed with
paraformaldehyde (4% final concentration, Sigma) and the nuclei were
stained with Hoechst 33,342 (Invitrogen). The relocalization of STAT5/
zsGreen were measured by ImagXpress 5000.
Acknowledgments
The authors would like to thank Ethan Hoffmann for PK studies,
Nancy DeGrace and Kanayochukwu Azogu for chiral purification.
Special thanks to Susan Ashwell and Timothy Pontz for useful dis-
cussions during the preparation of this Letter.
23. Female NCr mice (Taconic Farms, Germantown, NY), aged 5–6 weeks were
implanted intravenously with Ba/F3 TEL-JAK2 cells. Ten days post-
implantation animals received a single oral dose of vehicle, reference or test
compound. Spleens were harvested and snap frozen in liquid nitrogen at
specified time points post-compound administration for analysis of
pharmacodynamic markers. In addition, blood samples were collected via
cardiac puncture from the same animals for pharmacokinetic analysis. Lysates
of each spleen sample were prepared by homogenizing tissue samples in lysis
buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40 substitution, and 1 mM
Sodium Vanadate (phosphates inhibitor set II, CALBIOCHEM #524625)) using a
Qiagen Tissue Lyser. Protein values were quantitated using the BCA Protein
Assay Kit (Pierce, cat #23227). Lysates were prepared at equivalent protein
concentrations in Laemmli Sample Buffer and loaded onto Novex 4–12% Tris–
Glycine gels (Invitrogen) for SDS PAGE. Gels were transferred to Nitrocellulose
(Invitrogen) and blocked in Blocking Buffer (Licor). Western blots were
processed with primary antibodies that recognize phospho-STAT5 (BD
Transduction Labs) or total-STAT5 (Epitomics) and then treated with
appropriate secondary antibodies (Licor). Phospho-signal was quantitated
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