D. K. Neff et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6467–6471
6471
Table 4. Amide analogs
O
2
N
1
H2N
3
O
n
6
N
H
N
O
4
R4
5
R4b
nb
N locationb
SIF (mM)
SGF (mM)
a
Compound
Chk2 IC50
39
40
41
42
43
44
45
46
39 18
52 51
Ph
Ph
1
2
2
0
1
1
2
2
4
2
4
3
3
4
2
4
ND
ND
0.018
0.003
ND
0.028
0.003
ND
23
3
Ph
100 20
140 31
78 16
292 40
86 10
CH3
CH3
CH3
CH3
CH3
ND
ND
ND
ND
0.193
0.002
>0.400
0.087
a IC50 values expressed in nM SEM, all values are means of at least three replicated experiments.
b As described in Scheme 4.
9. McClure, K. J.; Huang, L.; Arienti, K. L.; Axe, F. U.;
Brunmark, A.; Blevitt, J.; Breitenbucher, G. J. Bioorg.
Med. Chem. Lett. 2006, 16, 1924.
References and notes
˚
1. Falck, J.; Mailand, N.; Syljuasen, R. G.; Bartek, J.; Lukas,
J. Nature 2001, 410, 842.
10. Equilibrium solubility: Solubility samples were prepared
by mixing 400 lmol of compounds (solids dissolved in
DMSO) with either 1 mL of SGF (0.1N HCl, 0.2% NaCl,
pH 1.2) or FasSIF (Galia et al. Pharm. Res. 15(5), 698–
705, 1998) for at least 3 days at room temperature. The
resulting mixtures were then filtered and the supernatants
were analyzed by UV-HPLC against external standards.
11. Activity of inhibitors was determined by incubating
inhibitory compounds with recombinant full-length
Chk2: 5 nM recombinant human Chk2, 50 mM Hepes
(pH 7.4), 100 mM NaCl, 10 mM MgCl2, 25 lM synthetic
peptide substrate (Biotin-SGLYRSPSMPENLNRPR,
1 lM ATP, 50 lCi/mL [g-33P]) ATP, and a protease
inhibitor mixture. The reaction mixtures were incubated at
37 °C for 3 h, and the peptide substrate was captured on
streptavidin conjugated to agarose beads. The agarose
beads were washed repeatedly with a 0.1% solution of
Tween-20 in phosphate-buffered saline, pH 7.4. Enzyme
activity at different Chk2 inhibitory compound concen-
trations was determined by measuring the amount of
radioactive phosphate bound to the substrate peptide by
scintillation counting.
2. Takai, H.; Naka, K.; Okada, Y.; Watanabe, M.; Harada,
N.; Saito, S.; Anderson, C. W.; Appella, E.; Nakanishi,
M.; Suzuki, H.; Nagashima, K.; Sawa, H.; Ikeda, K.;
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Sakai, T.; de Stanchina, E.; Bristow, R. G.; Suda, T.;
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