T. M. Kirrane et al. / Bioorg. Med. Chem. Lett. 22 (2012) 738–742
741
O
H
N
N
NH
N
N
O
RSK2 1 nM
N
LRRK2
PRKD1
PRKD2
PRKD3
RET
16 nM
35 nM
139 nM
219 nM
161 nM
CLK1
CLK2
FGFR2
PDGFRa
FLT3
512 nM
112 nM
320 nM
956 nM
714 nM
Figure 2. Kinase selectivity profile of compound 24.
Figure 1. Docking of 3 into a crystal structure of RSK2 [30–350] (2.9 Å resolution,
Boehringer-Ingelheim unpublished results). The binding mode is based on an X-ray
structure for an analogous compound bound to MK2. Dashed yellow lines represent
H-bonds <2.5 Å.
At this point, we returned to the panel of 20 kinases used for
optimization to obtain a more accurate picture of kinase selectivity
for 24 (Fig. 2). Six kinases furnished an IC50 less than 1 lM: the five
that had shown greater than 80% inhibition in the selectivity count,
and PDGFRa which had shown 71% inhibition. Additionally, com-
pound 24 was more than 100-fold selective relative to RSK for
the kinase panel, with the exception of LRRK2 and PRKD1, for
which compound 24 showed 16- and 35-fold selectivity, respec-
tively. We then expanded the kinase panel to include over 200
additional unique kinases. We were pleased to find that 24 had
Table 4
Piperazinone and diazepinone SAR
H
N
Core
N
N
O
an IC50 of less than 1 lM for only four other family members
(PRKD2, PRKD3, RET, and FLT3). These results confirmed the power
of the selectivity screening methodology developed for this pro-
gram. By tracking a broader panel of potentially cross-reactive ki-
nases at a single concentration, we were able to identify a
compound with high selectivity vs. most of the readily testable
portion of the kinome.
N
Compound Core
RSK2
IC50
(nM)
HLR-
CREB
IC50
Selectivity
countb
Aqueous
solubility
(lg/mL)
a
10
(nM)
In summary, we followed up on the initial indole series leads
1–3 to further optimize potency, kinase selectivity and physical
properties. Based on its overall balanced profile, compound 24,
BIX 02565, is an attractive candidate for use in vitro and in vivo
to explore the role of RSK as a target for the treatment heart failure.
Further results will be reported in due course.10
O
24
1
20
5
5
26
NH
N
N
N
*
O
25
14
27
100
>45
NH
*
Acknowledgements
O
The authors would like to express their gratitude to Dr. John
Miglietta for determining the percent homology of the ATP pocket
for the kinase selectivity panel versus the N-terminal ATP pocket of
RSK2.
NH
26
290
3
>50
*
a
Values are means of at least two experiments, standard deviation is typically
50% reported value.
b
Selectivity count = # of kinases with >80% inhibition @ 3
lM.
References and notes
1. Karmazyn, M.; Sawyer, M.; Fliegel, L. Curr. Drug Targets Cardiovasc. Haematol.
Disord. 2005, 5, 323.
2. Nakamura, T. Y.; Iwata, Y.; Arai, Y.; Komamura, K.; Wakabayashi Circ. Res. 2008,
103, 891.
3. (a) Karmazyn, M.; Kilic, A.; Javadov, S. J. Mol. Cell Cardiol. 2008, 44, 647; (b)
Karmazyn, M.; Gan, X. T.; Humphreys, R. A.; Yoshida, H.; Kusumoto, K. Circ. Res.
1999, 85, 777.
4. (a) Moor, A. N.; Gan, X. T.; Karmazyn, M.; Fliegel, L. J. Biol. Chem. 2001, 276,
16113; (b) Maekawa, N.; Abe, J.-I.; Shishido, T.; Itoh, S.; Ding, B.; Sharma, V. K.;
Sheu, S.-S.; Vlaxall, B. C.; Berk, B. C. Circulation 2006, 113, 2516; (c) Avkiran, M.;
Cook, A. R.; Cuello, F. Curr. Opin. Pharmacol. 2008, 8, 133.
5. Boyer, S. J.; Burke, J.; Guo, X.; Kirrane, T. M.; Snow, R. J.; Zhang, Y.; Sarko, C.;
Soleymanzadeh, L.; Swinamer, A.; Westbrook, J.; DiCapua, F.; Padyana, A.;
Cogan, D.; Gao, A.; Xiong, Z.; Madwed, J. B.; Kashem, M.; Kugler, S.; O’Neill M.
6. Compounds were tested in the Kinase SelectScreenÓ Kinase Profiling service,
human proteins.
7. Vieth, M.; Higgs, R. E.; Robertson, D. H.; Shapiro, M.; Gragg, E. A.; Hemmerle, H.
Biochim. Biophys. Acta 2004, 1697, 243.
8. Bouillot, AMJ; Daugan, AC-M; Dean, AW; Fillmore, MC. WO2008074824.
LHS amide that substitution of the lactam ring could enhance
potency as well as selectivity. Therefore, we combined the amino-
propylbenzimidazole with a number of mono-, di-, and spiro-
substituted lactams.5 In general, the SAR trends for lactam substi-
tution did not differ from what was previously observed for the
pyridyl amide series: mono-methylation tended to boost potency
while gem-dimethylation, as in 26, was detrimental (Table 4).
We were pleased to see a 14-fold enantiomeric preference for
the predicted eutamer (R)-24 over the distamer (S)-25. This result
further supports the docking hypothesis, which suggested one
enantiomer of a methyl group adjacent to the indole nitrogen
would occupy a small indentation in the roof of the binding pocket.
Of the substituted lactam analogs containing the aminopropyl-
benzimidazole LHS, compound 24 demonstrated the best combina-
tion of potency, selectivity, and solubility and was chosen for
further profiling.