D. R. Goldberg et al. / Bioorg. Med. Chem. Lett. 18 (2008) 938–941
941
Table 5. Cellular efficacy studies of 32a
Overall, we have been able to improve the profile of our
initially disclosed carboline based MK2 inhibitors. We
have demonstrated a better balance of physicochemical
properties and cellular efficacy. Furthermore, we have
demonstrated this potency is due to inhibition of MK2
via examining both upstream and downstream effects
of our inhibitors in the MK2 pathway.
O
H
O
N
N
N
NH
N
S
O
N
H
MK2 IC50 (nM)
2
LPS stimulation of THP-1 cells IC50 (nM)
TNFa
300
Anisomycin-stimulated HeLa cells IC50 (nM)
P-p38a
References and notes
>30,000
710
1. Imajo, M.; Tsuchiya, Y.; Nishida, E. IUBMB Life 2006,
58, 312.
P-HSP27 cellular efficacy IC50 (nM)
THP-1
2. (a) Wagner, G.; Laufer, S. Med. Res. Rev. 2005, 26, 1; (b)
Calcagni, F.; Elenkov, I. Ann. N.Y. Acad. Sci. 2006, 62.
3. Kotlyarov, A.; Neininger, A.; Schubert, C.; Eckert, R.;
Birchmeier, C.; Volk, H.-D.; Gaestel, M. Nat. Cell Biol.
1999, 1, 94.
4. Winzen, R.; Kracht, M.; Ritter, B.; Wilhelm, A.; Chen, C.-
Y. A.; Shyu, A.-B.; Muller, M.; Gaestel, M.; Resch, K.;
Holtmann, H. EMBO J. 1999, 18, 4969.
5. Gaestel, M. Nat. Rev. Mol. Cell Biol. 2006, 7, 120.
6. Anderson, D. R.; Mahoney, M. W.; Phillion, D. P.;
Rogers, T. E.; Meyers, M. J.; Poda, G.; Hegde, S. G.;
Singh, M.; Reitz, D. B.; Wu, K. K.; Buchler, I. P.; Xie, J.;
Vernier, W. F. WO 2004/058762 A1, 2004.
a Values are means of P2 experiments, standard deviation typically
50% of reported value.
Table 6. BalbC mouse pharmacokinetics
Compound
1
2
32
IV dose 0.5 mg/kg (70:30 PEG, 400:H2O)
18
Cl (%Q)
MRT (h)
18
2.0 1.0
535 165
30
6.1 0.7
165 34
0.8 0.07
525 140
AUC0–1 (ng h/mL)
PO dose 1.0 mg/kg (PEG 400)
Cmax (lM)
AUC0–1 (ng h/mL)
%F
0.06 0.01
88 1.8
0.2
0.08 0.05
115 17
0.06 0.01
446 56
>100 17
7. (a) Rousseau, S.; Peggie, M.; Campbell, D. G.; Nebreda,
A. R.; Cohen, P. Biochem. J. 2005, 391, 433; (b) Adams,
R. H.; Porras, A.; Alonso, G.; Jones, M.; Vintersten, K.;
Panelli, S.; Valladares, A.; Perez, L.; Klein, R.; Nebreda,
A. R. Mol. Cell 2000, 6, 109.
8
11
2
the upstream and downstream effects of our inhibitors in
the p38 MAPK pathway. This was assessed in two dif-
ferent ways (Table 5). First, inhibitor treated HeLa cells
that were stimulated with anisomycin to maximally acti-
vate the p38/MK2 pathway did not show any inhibition
of p38 phosphorylation by flow cytometry ruling out
compound effects on signaling events upstream of
MK2. Second, 32 inhibited phosphorylation of HSP27,
a direct downstream substrate of MK2, to a similar level
of potency to that observed in the THP-1 cell assay.13,14
Taken together, this supports that the cellular efficacy
we are observing is driven by MK2 inhibition.
8. Wu, J.-P.; Wang, J.; Abeywardane, A.; Andersen, D.;
Emmanuel, M.; Gautschi, E.; Goldberg, D. R.; Kas-
hem, M. A.; Lukas, S.; Mao, W.; Martin, L.; Morwick,
T.; Moss, N.; Pargellis, C.; Patel, U. R.; Patnaude, L.;
Peet, G. W.; Skow, D.; Snow, R. J.; Ward, Y.;
Werneburg, B.; White, A. Bioorg. Med. Chem. Lett.
2007, 17, 46649.
9. Kermack, W. O. J. Chem. Soc., Trans. 1924, 2285.
10. Bellemin, R.; Decerprit, J.; Festal, D. Eur. J. Med. Chem.
1996, 31, 123.
11. Heinzman, S. W.; Ganem, B. J. Am. Chem. Soc. 1982, 104,
6801.
12. Levin, J. I.; Du, M. T. Drug Des. Discov. 2003, 18, 123.
13. Anderson et al., hypothesize in their MK2 paper that a
potential reason for a large shift between molecular and
cellular potency may be due to the affinity of MK2 for
ATP in which they calculated a theoretical shift in potency
based on the measured in vitro Km of ATP for MK2 to be
30 lM, see: Anderson, D. R.; Meyers, M. J.; Vernier, W.
F.; Mahoney, M. W.; Kurumbail, R. G.; Caspers, N.;
Poda, G. I.; Schindler, J. F.; Reitz, D. B.; Mourey, R. J. J.
Med. Chem. 2007, 50, 2647, and references therein.
14. Stokoe, D.; Engel, K.; Campbell, D. G.; Cohen, P.;
Gaestel, M. FEBS Lett. 1992, 313, 307.
15. Goldberg, D. R.; Hao, M. H.; Qian, K. C.; Swinamer, A.
D.; Gao, D. A.; Xiong, Z.; Sarko, C.; Berry, A.; Lord, J.;
Magolda, R. L.; Fadra, T.; Kroe, R. R.; Kukulka, A.;
Madwed, J. B.; Martin, L.; Pargellis, C.; Skow, D.; Song,
J. J.; Tan, Z.; Torcellini, C. A.; Zimmitti, C. S.; Yee, N.
K.; Moss, N. J. Med. Chem. 2007, 50, 4016.
The mouse pharmacokinetic properties for this class of
compounds are described in Table 6 and indicate that
overall, 32 is better than either compounds 1 and 2. This
may in part be again due to the poor aqueous solubility
of 1 and 2 (pH 7.4 <0.1 lg/mL) while 32 is considerably
more soluble (pH 7.4 >96 lg/mL).
In order to assess the activity of our compounds in vivo,
a close analog of 32 (compound 26) was tested in a
mouse model of LPS-stimulated TNFa production.15
However, even at an oral dose of 60 mg/kg, we did not
observe any inhibition of TNFa. This is not surprising
given the low plasma levels which are barely above the
THP-1 cell potency (90 min post-dose = 690 nM; THP-
1 IC50 = 735 nM).