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D. E. Levy et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2395–2398
Table 4. SAR related to the incorporation of rigid cyclic amines
directly connected to the indole
amines, rigid amine-tether combinations exhibited equi-
potent activity to the previously identified leads. In one
specific example, hydroxylation of the tether indicated
that enhanced activity over the parent may be
achieved.
H
N
O
O
References and notes
N
N
R
CH3
1. Thorneloe, K. S.; Nelson, M. T. Can. J. Physiol.
Pharmacol. 2005, 83, 215.
Compound
R
IC50 (lM)
2. Rossier, M. F. Recent Res. Dev. Biochem. 2003, 4, 13.
3. Takano, H.; Zou, Y.; Akazawa, H.; Nagai, T.; Mizukami,
M.; Toko, H.; Komuro, I. Prog. Exp. Cardiol. 2003, 7, 85.
4. Verkhratsky, A. Physiol. Rev. 2005, 85, 201.
5. Putney, J. W. Cell Calcium 2003, 34, 339.
7m13
0.31 (n = 1)
NH2
6. Hook, S. S.; Means, A. R. Annu. Rev. Pharmacol. Toxicol.
2001, 41, 471.
7. Hudmon, A.; Schulman, H. Biochem. J. 2002, 364, 593.
8. Edman, C. F.; Schulman, H. Biochim. Biophys. Acta 1994,
1221, 89.
9. Levy, D. E.; Wang, D.-X.; Lu, Q.; Chen, Z.; Perumattam,
J.; Xu, Y.-J.; Liclican, A.; Higaki, J.; Dong, H.; Laney,
M.; Mavunkel, B.; Dugar, S. Bioorg. Med. Chem. Lett.
2008, 18, 2390.
10. An homology model of autoinhibited CaMKIId was built
based on crystal structure 1A06 of autoinhibited rat
CaMKI. Because rat CaMKI shows high sequence
homology with CaMKIId, this model was used to study
inhibitors that were not ATP competitive. Due to the lack
of availability of a crystal structure of activated CaMKIId,
homology models were built based on crystal structures
1CDK, 1PHK, and 1KOB. From these homology models,
we selected the one that best explained the SAR. The
accuracy of this model was validated using point mutation
studies.
7n
7o
0.30 (n = 1)
0.64 (n = 1)
NH
N
H
7p
1.87 0.18 (n = 2)
N
11. Faul, M. M.; Winneroski, L. L.; Krumrich, C. A. J. Org.
Chem. 1998, 63, 6053.
Table 5. Effect of hydroxyl substitution on tether
12. Assays were performed with inhibitor or suitable control
solvent added 10 ll per well in a 96-well microtiter plate
(Corning, NY). CaMKIId was diluted in enzyme buffer
(50 mM PIPES pH 7, 0.2 mg/ml BSA, 1 mM DTT) and
added 10 ll per well. Reactions were initiated with 30 ll
reaction buffer (62.5 mM PIPES pH 7, 0.25 mg/ml BSA,
33.3 mM MgCl2, 83 lM ATP, 0.4 mM CaCl2, 8.3 lg/ml
calmodulin, 25 lM [His 5] autocamtide-2, 120 nM [g-
33P]ATP) and incubated at rt for 3 min. Reactions were
terminated by transferring 25 ll to a UNIFILTER 96-well
P81microplate (Whatman, UK), pre-wet with 15 ll 1%
phosphoric acid. After 10 min, the plate was washed three
times with 1% phosphoric acid and one time with 95%
ethanol on a BiomekFX (Beckman Coulter, CA) equipped
with a vacuum manifold. Plates were dried for approxi-
mately 60 min, scintillant was added to the wells, and the
plates were read on a TopCount NXT Microplate
Scintillation and Luminescence Counter (Perkin-Elmer,
MA).
H
N
O
O
N
R
Compound
R
IC50 (lM)
7c
1.35 0.64 (n = 2)
NH2
NH2
7q
0.45 (n = 1)
OH
13. The following compounds were obtained from Calbio-
chem–Novabiochem: 7a (cat # 203294), 7g (cat # 203297),
7l (cat # 203292) and 7m (cat # 557508).
14. The following compounds were obtained from Sigma: 7j
(cat # B3681) and 7k (cat # B3556).
observing the influence of the amine tether on potency.
While primary acyclic amines generally demonstrated
better activity than cyclic secondary or cyclic tertiary