S. Huang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5907–5912
Table 3. Pharmacokinetic profiles of indolylquinolinones
5911
Compoundc
R2
Cell EC50 (nM)
dog PK
CL (mL/min/kg)
a
Chek1 IC50 (nM)
2
PSA (A )
˚
t1/2 (h)
22
32
33
34
35
36b
0.65
1.0
97
120
89
89
30
22
34
27
23
4.4
2.5
2.0
1.5
2.0
4.1
0.83
2.5
170
102
111
123
98
870
2.1
1142
420
3.0
6.5
a Tested at 0.1 mM ATP.
b Pyrazole is N-methylated in 36.
c Compounds dosed at 0.25 mpk iv (DMSO) in cassette format.
1
11. All compounds were characterized by H NMR and high
resolution mass spectrometry. Experimental procedures
will be published in a submitted patent by these authors.
12. Kondo, Y.; Inamoto, K.; Sakamoto, T. J. Comb. Chem.
2000, 2, 232.
or high PSA, exhibited diminished cell potency.
Minimization of these parameters, resulted in Chek1
inhibitors with improved cell potency. Among these
potent Chek1 inhibitors, compounds 22 and 32 were
found to be leading compounds with moderate clear-
ance in dogs following intravenous dosing.
13. Marsais, F.; Godard, A.; Queguiner, G. J. Heterocyclic
Chem. 1989, 26, 1589.
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W. F.; Lynch, J. J.; McFall, R. C.; Rickert, K.; Singh, R.;
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15. Chek1 inhibitory activity was measured using a homoge-
neous time-resolved fluorescence assay which measures
phosphorylation of a biotinylated GSK-3 peptide as
described in Barnett et al., Biochem J. 2005, 385, 399.
For the construct, a naturally occurring exon 10 splice
variant of human Chek1 described in patent application
US20050266469(A1), containing primarily the kinase
domain, was expressed in baculovirus with a C-terminal
6-histidine tag. This protein was purified on a Ni affinity
column and used as it is for kinetic assays, or purified
further on Heparin and SEC columns for crystallography.
The Chek1 concentration was 0.5 nM and ATP was used
at 0.1 mM. IC50 values are reported as the averages of at
least two independent determinations; standard deviations
are within 25–50% of IC50 values.
16. NCI-H1299 lung carcinoma cells were arrested with 16-h
treatment of camptothecin, and then treated with Chek1
inhibitors for additional 8 h. Checkpoint escaped mitotic
index due to Chek1 inhibition was assessed by measuring
the mitotic-specific phosphorylation of nucleolin in Chek1
inhibitor-treated cells using an antibody-coated, bead-
based assay. In this assay, total nucleolin is captured on a
streptavidin-coated paramagnetic bead coupled with bio-
tinylated nucleolin monoclonal antibody 4E2 (Research
Diagnostics, Inc.). Phosphorylated nucleolin is detected by
an antibody complex consisting of a phospho-specific
nucleolin monoclonal antibody TG3 (Applied NeuroSo-
lutions, Inc.) and a ruthenylated goat anti-mouse IgM
antibody labeled with ruthenylation kit (BioVeris Corp).
5. Compounds 1, 2 or 22 were diffused into pre-formed apo
Chek1 crystals. The X-ray diffraction data were collected
from these Chek1 inhibitor complex crystals to 1.8, 2.0,
˚
and 1.7 A resolution with Rsym = 0.067, 0.093, 0.063 and
completeness = 96%, 94%, 98%, respectively. The complex
structures were refined to an R-factor of 0.24, 0.23, and
0.23, respectively. The detailed X-ray diffraction data and
refinement statistics are listed under PDB code 2HXL,
2HXQ, and 2HY0 at the protein data bank.
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