M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6049–6053
6053
antibody-coated, bead-based assay. For assay details, see
Fraley, M. E. et al. Bioorg. Med. Chem. Lett. 2006, 16,
1775, Ref. 16. The values of EC50 were measured with 10-
point, half-log dilution series and are reported as an
average of tetraplicate determinations.
In summary, we have described the effects of C6 sub-
stitution on Chek1 potency and selectivity for a series
of 3-(indol-2-yl)indazoles. We showed that selectivity
for Chek1 over Cdk7 was required for functional
activity in a cell-based checkpoint escape assay for this
series of compounds. The hydroxymethyl triazole
group provided enhanced binding affinity, apparently
through participation in a hydrogen bonding network
in HI following displacement of a conserved water
molecule.
10. Traxler, P.; Furet, P. Pharmacol. Ther. 1999, 82, 195.
11. Compound
3
displayed high passive permeability
(Papp = 30 · 10ꢀ6 cm/s) across LLC-PK1 cell monolayers.
For assay description, see Hochman, J. H.; Yamazaki, M.;
Ohe, T.; Lin, J. H. Curr. Drug Metab. 2002, 3, 257.
12. Of note, compound 3 showed relatively weak activity
against Cdc2 (IC50 = 4200 nM). Cdk7 and Cdc2 were
assayed using IMAP-FP kits from Molecular Devices at
0.05 lM and 0.03 lM ATP, respectively. Enzymes were
purchased from Upstate Biotechnology.
References and notes
13. Fisher, R. P.; Morgan, D. O. Cell 1994, 78, 713.
14. Harper, J. H.; Elledge, S. J. Genes Dev. 1998, 12, 285.
15. Chen, P.; Luo, C.; Deng, Y.; Ryan, K.; Register, J.;
Margosiak, S.; Tempczyk-Russell, A.; Nguyen, B.; Myers,
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1. Niida, H.; Nakanishi, M. Mutagenesis 2006, 21, 3.
2. Zhou, B.-B. S.; Bartek, J. Nat. Rev. Cancer 2004, 4, 1.
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4. For recent reviews on published Chek1 inhibitors, see: (a)
Prudhomme, M. Recent Patents Anti-Cancer Drug Discov.
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513.
16. Lolli, G.; Lowe, E. D.; Brown, N. R.; Johnson, L. N.
Structure 2004, 12, 2067.
17. Note that Chek1 inhibitory activity in the enzyme assay is
measured at Km for ATP (0.1 mM). In the cellular assays,
the ATP concentration is 2.0 mM resulting in an inherent
10-fold shift in potency between the enzyme and cellular
assays.
18. Functionally inactive compounds 6, 11, 13, and 14 were
cell permeable as indicated by their activity in the Phos
assay (IC50s 200, 2800, 700, and 2300 nM, respectively).
19. The NH of the triazole is arbitrarily assigned.
20. PDB coordinates for 21: 2HOG.
5. Hanney, B. A.; Kim, Y.; Hartman, G. D. Presented at the
229th National Meeting of the American Chemical Soci-
ety, San Diego, CA, March 2005; Abstract MEDI-121.
See also WO 2003024969.
6. Fraley, M. E.; Arrington, K. L.; Buser, C. A.; Ciecko, P.
A.; Coll, K. E.; Fernandes, C.; Hartman, G. D.; Hoffman,
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Chem. Lett. 2004, 14, 351.
7. Chek1 inhibitory activity was measured using a homoge-
neous time-resolved fluorescence assay that detects phos-
phorylation of a biotinylated GSK-3 peptide as described
by Barnett, S. F. et al. Biochem. J. 2005, 385, 399. The
assay was run at 0.5 nM enzyme and 0.1 mM ATP. IC50
values are reported as averages of at least two independent
determinations; standard deviations are within 25–50%
of IC50 values.
8. NCI-H1299 lung carcinoma cells were incubated with
Chek1 inhibitors for 2 h and then treated with campto-
thecin for an additional 4 h. Inhibition of Chek1 auto-
phosphorylation was measured by cyto-blot analysis using
an antibody against phospho-S296 Chek1. For assay
details, see WO2006086255. The values of IC50 were
measured with 10-point, 3-fold dilution series and are
reported as an average of triplicate determinations.
9. NCI-H1299 lung carcinoma cells were arrested with 16 h
treatment of camptothecin and then treated with Chek1
inhibitors for additional 8 h. Escape from arrest and
progression into mitosis were measured by quantifying the
mitosis-specific phosphorylation of nucleolin using an
21. Dunitz, J. D. Science 1994, 264, 670.
22. The substantial shift in cellular activity relative to enzyme
potency for 21–23 is believed to be due to suboptimal cell
permeability as a result of high polar surface area
2
˚
(PSA > 120 A , calculated by the method of Clark, D. E.
J. Pharm. Sci. 1999, 88, 807). For discussions on the
inverse relationship between PSA and cell permeability,
see Papageorgiou, C.; Camenisch, G.; Borer, X. Bioorg.
Med. Chem. Lett. 2001, 11, 1549, and references therein.
23. Foloppe, N.; Fisher, L. M.; Francis, G.; Howes, R.;
Kierstan, P.; Potter, A. Bioorg. Med. Chem. 2006, 14,
1792.
24. For full experimental detail on compounds described in
this paper, see WO2006086255.
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27. Journet, M.; Cai, D.; Kowal, J. J.; Larsen, R. D.
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