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O
N
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X
a
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Compound
R
X
IC50 (lM)
12
13
14
15
16
17
18
19
20
21
22
23
Thiophene
Furan
4-Cl-Ph
O
O
O
O
O
O
O
S
0.46
0.87
0.92
1.23
>20
20.0
>20
1.49
4.79
20.0
>20
>20
6-Cl-Ph
4-OMe-Ph
4-Pyridine
2-Pyridine
Thiophene
3-Pyridine
2-Cl-Ph
S
S
4-F-Ph
4-Pyridine
S
CH2
O
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N
O
O
24
>20
O
O
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a Values are means of three experiments.
In summary, a series of substituted pyridone molecules
was identified from a high throughput screen as potent
inhibitors of Pim-1 kinase. Because of its compelling
in vitro efficacy, a complex structure and mechanism
of action were defined for compound 1. The pyridone
carbonyl group was critically positioned within the
Pim-1 ATP binding site effecting kinase inhibition. Pre-
liminary data further suggested that 1 lacked in vitro
inhibitory activity toward related serine/threonine ki-
nases Pim-2 and MEK1/2 (IC50 > 20 lM; data not
shown). Hence, small molecules similar to compound 1
may serve as useful starting scaffolds for the develop-
ment of other improved yet selective Pim-1 inhibitors.
Acknowledgment
25. Bullock, A. N.; Debreczeni, J. E.; Federov, O. Y.; Nelson,
A.; Marsden, B. D.; Knapp, S. J. Med. Chem. 2005, 48,
7604.
The authors thank Dr. J. M. Vernier for insightful com-
ments pertaining to this manuscript.
26. High throughput screening assay: A purified recombinant
GST–Pim-1 fusion protein together with histone proteins
(Sigma) as substrate was used for the Kinase-Glo assay
(Promega). Briefly, reaction conditions were 25 mM Hepes
(pH 7.5), 10 mM MgCl2, 5 mM DTT, 4% DMSO, 15 lM
ATP, 1 lg GST-Pim-1, 5 lg calf thymus histones plus the
test compound in a 20 ll total volume. Reactions were
performed in a 384-well plate for 1 h at room temperature
and were terminated by the addition of the Kinase-Glo
reagent then counted on the LJL Analyst plate reader. The
Z0 for the assay was 0.8.
References and notes
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Robanus-Manndag, E.; Boelens, W.; van Wezenbeek, P.;
Berns, A. Cell 1984, 37, 141.
2. van der Lugt, N. M.; Domen, J.; Verhoeven, E.; Linders,
K.; van der Gulden, H.; Allen, J.; Berns, A. EMBO
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Marcheselli, V. L.; Bazan, N. G.; Baudry, M.; Herschman,
H. R. J. Biol. Chem. 1998, 273, 16535.
27. Radioactive filter plate assay: Pim-1 kinase reaction con-
ditions included 25 mM Hepes (pH 7.5), 10 mM MgCl2,