N. Gommermann et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3628–3631
3631
% inhibition
(+/-St. Dev.)
significance
(PEG/H2O/HCl)
0
+/-21
Control
4c
0.6089
0.0104
-7 +/-28
10 mg/kg p.o.
30 mg/kg p.o.
21 +/-9
0.0001
100 mg/kg p.o.
15 mg/kg p.o.
57 +/-18
82 +/-6
0.00000001
Cyclosporin A
Background
100 +/-0
0
2000
4000
6000
8000
10000
12000
14000
mIL-2 [pg/ml]
Figure 6. Effect of 4c on anti-CD3-induced IL-2 response in BALB/c mice.
4. Strauss, B.; Weiss, A. Cell 1992, 70, 585.
16e) destroyed Lck activity. Although the solubilizing group is sol-
vent exposed and forms only limited contacts with the protein, we
reasoned that the lower Lck activity of 16b and 16e is due to repul-
sive electrostatic interactions between the sulfonamide group
(16b) or the morpholine (16e) and acidic residues located on the
surface of Lck (Glu249, Glu320 and Asp326).
The NH-piperazine 16a showed increased metabolic stability
over its N-Me analogue 4c, but a substantial decrease in permeabil-
ity was also observed. Compound 4c was selected for further
profiling since it displayed promising PK-properties in rat
(VD = 6421 mL/kg; Cl = 1928 mL/h/kg; t1/2 = 5.7 h; BAV 95%). The
broader selectivity profile of 4c (Table 5) showed good selectivity
against the major anti-targets cSrc, Hck, KDR and Tie-2, and
showed crossreactivity on only 3 out of 26 kinases tested, namely
cAbl, cKit and PDGFRa.
Lck inhibitors have been previously reported to inhibit the anti-
CD3-mediated IL-2 production in whole blood as well as the conca-
navalin A-induced IL-2-release in vivo.20 Compound 4c was there-
fore tested in vivo for the inhibition of T-cell activation, the anti-
CD3 antibody-induced IL-2 response in BALB/c mice.21 A dose-
dependent inhibition of IL-2 production was observed, with 57%
( 18%) inhibition at 100 mg/kg po of 4c (Fig. 6).
In summary, a promising new structural class of potent Lck
inhibitors was discovered. Selective Lck inhibitors were obtained
through structure-based drug design and SAR-guided optimiza-
tion. It was demonstrated that pyrazolopyrimidine Lck inhibitors
show an effect in an in vivo model of T-cell activation.
5. Weil, R.; Veilette, A. Curr. Top. Microbiol. Immunol. 1996, 205, 63.
6. Van Oers, N. S.; Kileen, N.; Weiss, A. J. Exp. Med. 1996, 183, 1053.
7. Trobridge, P. A.; Levin, S. D. Eur. J. Immunol. 2001, 31, 3567.
8. Molina, T. J.; Kishihara, K.; Siderovskid, D. P.; van Ewijk, W.; Narendran, A.;
Timms, E.; Wakeham, A.; Paige, C. J.; Hartmann, K. U.; Veillatte, A.; Davidson,
D.; Mak, T. W. Nature 1992, 357, 161.
9. Kamens, J. S.; Ratnofsky, S. E.; Hirst, G. C. Curr. Opin. Invest. Drugs 2001, 2, 1213.
10. Burchat, A.; Borhani, D. W.; Calderwood, D. J.; Hirst, G. C.; Li, B.; Stachlewitz, R.
F. Bioorg. Med. Chem. Lett. 2006, 16, 118.
11. Five microliters of compound in dilution buffer (in 50 mM Tris/HCl, pH 7.6,
0.05% Tween 20) was added into a black 384-well plate. Then 10
buffer (50 mM Tris/HCl, pH 7.6, 0.05% Tween 20, 20 mM MnCl2, 2 mM DTT,
20 Na3VO4, 20 mM ATP) containing peptide substrate (biotin-
KVEKIGEGTYGVVYK) was added. The reaction was started by the addition of
L Lck (80 ng/mL) in 50 mM Tris/HCl, pH 7.6, 0.05% Tween 20, 1% BSA. After
2 h at room temperature the reaction was stopped by the addition of 10
22 mM EDTA followed by the addition of 45 L detection mix (0.6 nM Eu-PT66
lL of assay
lM
1 lM
5
l
lL
l
and 367 nM SA-APC) in detection buffer (20 mM Tris/HCl, pH 7.6, 0.05% Tween
20, 150 mM NaCl, 0.1% BSA). The plates were incubated for 60 min in the dark
und then read on an EnVision 2102 Multilabel reader.
12. Furet, P., manuscript in preparation.
13. Scientists at Abbotthave independently discovered the pyrazolo[1,5a]pyrimidines
as kinase inhibitors: Frey, R. R.; Curtin, M. L.; Albert, D. H.; Glaser, K. B.; Pease, L. J.;
soni, N. B.; Bouska, J. J.; Reuter, D.; Stewart, K. D.; Marcotte, P.; Bukofzer, G.; Li, J.;
Davidsen, S. K.; Michaelides, M. R. J. Med. Chem. 2008, 51, 3777.
14. Crystallographic coordinates and structure factors of 1a in complex with Lck
have been deposited with the Protein Data Bank (Entry No. 3MPM).
15. Bold, G.; Floersheimer, A.; Furet, P.; Imbach, P.; Masuya, K.; Schoepfer, J.;
Martiny-Baron, G. WO2005/070431.
16. Zucotto, F.; Ardini, E.; Casale, E.; Angiolini, M. J. Med. Chem. 2010, 53, 2681.
17. Substituents binding to the back pocket or to the hydrophobic channel were
selected based on docking results. Compounds were docked in the ATP site of
the crystal structure of the Lck-1a complex, using GOLD and the GOLDSCORE
scoring function. A protein H-bond constraint with the backbone NH of Met319
(hinge) was set. Default values were considered for other parameters. For each
compound under evaluation, 25 docking poses were generated. We selected
docking poses with the following features: (1) the same H-bond pattern with
the hinge and gate keeper as observed for 1a; (2) no bump with the protein.
18. Haas, A.; Weckbecker, G.; Welzenbach, K. Cytometry A 2008, 73A, 799.
19. Trunzer, M.; Faller, B.; Zimmerlin, A. J. Med. Chem. 2009, 52, 329; Cruciani, G.;
Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Hove, T.; Vianello, R. J. Med.
Chem. 2005, 48, 6970.
Acknowledgments
The authors thank K. Welzenbach and M. Streiff for generating
biological data and C. Burkhart for performing the in vivo model as
well as G. Rummel for crystallization and data collection, J.-M. Flo-
rent, P. Ramage, S. Rieffel, A. Strauss, R. Cebe and M. Geiser for pro-
tein production for crystallography.
20. Stachlewitz, R. F.; Hart, M. A.; Bettencourt, B.; Kebede, T.; Schwartz, A.;
Ratnofsky, S. E.; Calderwood, D. J.; Waegell, W. O.; Hirst, G. C. J. Pharm. Exp.
Ther. 2005, 315, 36.
21. Groups of six mice were treated with 10, 30 and 100 mg/kg of the test
compound per os. One hour later, mice were injected intravenously with
References and notes
10
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