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T. Takayama et al. / Bioorg. Med. Chem. Lett. 20 (2010) 112–116
Table 3
Kinase selectivity IC50 values (nM)
LcK
Src
0.6
Csk
1.7
ZAP-70
>1000
MEK1
PKA
PKB
a
PKC
a
CDK1
CaMKIV
>10,000
MAPK1
>10,000
25
0.72
>10,000
>10,000
>10,000
>10,000
>10,000
9. Sabat, M.; VanRens, J. C.; Brugel, T. A.; Maier, J.; Laufersweiler, M. J.;
Golebiowski, A.; De, B.; Easwaran, V.; Hsieh, L. C.; Rosegen, J.; Berberich, S.;
Suchanek, E.; Janusz, M. J. Bioorg. Med. Chem. Lett. 2006, 16, 4257.
10. Bamborough, P.; Angell, R. M.; Bhamra, I.; Brown, D.; Bull, J.; Christopher, J. A.;
Cooper, A. W. J.; Fazal, L. H.; Giordano, I.; Hind, L.; Patel, V. K.; Ranshaw, L. I.;
Sims, M. J.; Skone, P. A.; Smith, K. J.; Vickerstaff, E.; Washington, M. Bioorg. Med.
Chem. Lett. 2007, 17, 4363.
11. Martin, M. W.; Newcomb, J.; Nunes, J. J.; Boucher, C.; Chai, L.; Epstein, L. F.;
Faust, T.; Flores, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Kim, J. L.;
Middleton, S.; Morgenstern, K.; Oliveira-dos-Santos, A.; Patel, V. F.; Powers, D.;
Rose, P.; Tudor, Y.; Turci, S. M.; Welcher, A. A.; Zack, D.; Zhao, H.; Zhu, L.; Zhu,
X.; Ghiron, C.; Ermann, M.; Johnston, D.; Saluste, C.-G. P. J. Med. Chem. 2008, 51,
1637.
12. DiMauro, E. F.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; Boucher, C.; Chai, L.;
Chaffee, S. C.; Deak, H. L.; Epstein, L. F.; Faust, T.; Gallant, P.; Gore, A.; Gu, Y.;
Henkle, B.; Hsieh, F.; Huang, X.; Kim, J. L.; Lee, J. H.; Martin, M. W.; McGowan,
D. C.; Metz, D.; Mohn, D.; Morgenstern, K. A.; Oliveira-dos-Santos, A.; Patel, V.
F.; Powers, D.; Rose, P. E.; Schneider, S.; Tomlinson, S. A.; Tudor, Y.-Y.; Turci, S.
M.; Welcher, A. A.; Zhao, H.; Zhu, L.; Zhu, X. J. Med. Chem. 2008, 51, 1681.
13. Inhibition of Lck activity was measured using a recombinant human Lck kinase
domain, expressed as a GST fusion in insect cells. Lck (5 ng/well) was incubated
in kinase buffer (50 mM Hepes, 50 mM KCl, 25 mM MgCl2, 5 mM MnCl2,
Met319 main-chain NH. Furthermore, the phenyl ring of the pyra-
zolobenzodiazepine skeleton is presumed to be in contact with a
hydrophobic site consisting of Trp318, Leu251, and Gly322. In
addition, we think that the terminal N atom at the 7-position of
the pyrazolobenzodiazepine skeleton could be in H-bond contact
with the Glu320 side chain.
Compound 25, the most potent analog, was tested for selectivity
against several kinases, and these results are summarized in Table
3.19 Compound 25 exhibited >2-fold selectivity versus Csk, how-
ever, it exhibited non-selectivity versus the Src family member
Src. Compound 25 was inactive for other kinases, including ZAP-
70, MEK-1, PKA, PKBa, PKCa, CDK1, CaMKIV, and MAPK1.
In summary, we have described the synthesis, enzyme inhibi-
tory activity, cellular activity, SAR, and proposed binding mode of
a novel class of ring-fused pyrazole derivatives for Lck, as repre-
sented by compound 1. Optimization of compound 1 led to com-
pound 25 with excellent activity against Lck (IC50 = 0.72 nM) and
MLR (IC50 = 0.74 nM). Further work for an in vivo analysis of com-
pound 25 will be disclosed in due course.
100
concentration of 5
acid- -tyrosine, 4:1) were added and incubated at 25 °C for 60 min and stopped
with 100 L of 100 mM phosphoric acid. The mixture was transferred to a
l
M
Na3VO4, 0.01% CHAPS,1 mM DTT) with test compound. The final
l
M ATP, 0.1 Ci/well M poly ( -glutamic
l
c
33P-ATP, and 10
l
L
L
l
MultiScreen-HA mixed cellulose ester membrane plate and harvested by
filtration. Scintillation cocktail was added, and radioactivity was measured on a
Packard Topcount instrument.
References and notes
1. Molina, T. J.; Kishihara, K.; Siderovskid, D. P.; van Ewijk, W.; Narendran, A.;
Timms, E.; Wakeham, M. A.; Paige, C. J.; Hartmann, K.-U.; Veillette, A.;
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4. Goldberg, D. R.; Butz, T.; Cardozo, M. G.; Eckner, R. J.; Hammach, A.; Huang, J.;
Jakes, S.; Kapadia, S.; Kashem, M.; Lukas, S.; Morwick, T. M.; Panzenbeck, M.;
Patel, U.; Pav, S.; Peet, G. W.; Peterson, J. D.; Prokopowicz, A. S.; Snow, R. J.;
Sellati, R.; Takahashi, H.; Tan, J.; Tschantz, M. A.; Wang, X.-J.; Wang, Y.; Wolak,
J.; Xiong, P.; Moss, N. J. Med. Chem. 2003, 46, 1337.
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V.; Lin, J.; Wityak, J.; Iwanowicz, E. J.; McIntyre, K. W.; Shuster, D. J.; Behnia, K.;
Chong, S.; Fex, H.; Pang, S.; Pitt, S.; Shen, D. R.; Thrall, S.; Stanley, P.; Kocy, O. R.;
Witmer, M. R.; Kanner, S. B.; Schieven, G. L.; Barrish, J. C. J. Med. Chem. 2004, 47,
4517.
14. To isolate naïve T cells from C57BL/6 mice (Charles River Japan), spleen and
lymph node cell suspensions were passed through nylon wool columns.
Nonadherent T cells (3 Â 105 cells/well) were cocultured with mitomycin-C
treated spleen cells from BALB/c mice (Charles River Japan) (2 Â 105 cells/well).
These cultures were incubated at 37 °C in 5% CO2 for 72 h. Cell proliferation
was assayed by pulsing the cells with 3H-thymidine for the last 4 h. 3H-
Thymidine incorporation into DNA was measured by Topcount. Compounds
were added at the start of the culture.
15. The two regioisomers were successfully separated by column purification.
16. General procedure for preparation of ring-fused pyrazole derivative (12). To a
mixture of 6 (1 mmol) and K2CO3 (1.5 mmol) in DMF (3.5 ml) was added 1-
bromo-2-(2-bromoethyl)-4-methoxybenzene (1 mmol), stirred at 80 °C for 3 h,
then cooled to room temperature. The mixture was diluted with AcOEt, and
washed with brine. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column chromatography to
give 10. Compound 10 (1 mmol) was treated with diglyme (20 ml). Cs2CO3
(2 mmol), Pd2(dba)3 (6 mol %) and rac-BINAP (6 mol %) were added to the
mixture, heated at 160 °C for 1 h, cooled to room temperature. The mixture
was diluted with AcOEt, and washed with brine. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo. The residue was purified by
column chromatography to give the cyclization derivative. This compound
(1 mmol) was treated with DMSO (10 ml). K2CO3 (3 mmol) and H2O2 (30%
solution, 2.8 g) were added to the mixture, stirred at 40 °C for 2 h, then cooled
to room temperature. The mixture was diluted with AcOEt, and washed with
brine. The organic layer was dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography to give 12.
17. Jacobs, M. D.; Caron, P. R.; Hare, B. J. Proteins 2008, 70, 1451.
6. Borhani, D. W.; Calderwood, D. J.; Friedman, M. M.; Hirst, G. C.; Li, B.; Leung, A.
K. W.; McRae, B.; Ratnofsky, S.; Ritter, K.; Waegell, W. Bioorg. Med. Chem. Lett.
2004, 14, 2613.
7. Martin, M. W.; Newcomb, J.; Nunes, J. J.; McGowan, D. C.; Armistead, D. M.;
Boucher, C.; Buchanan, J. L.; Buckner, W.; Chai, L.; Elbaum, D.; Epstein, L. F.;
Faust, T.; Flynn, S.; Gallant, P.; Gore, A.; Gu, Y.; Hsieh, F.; Huang, X.; Lee, J. H.;
Metz, D.; Middleton, S.; Mohn, D.; Morgenstern, K.; Morrison, M. J.; Novak, P.
M.; Oliveira-dos-Santos, A.; Powers, D.; Rose, P.; Schneider, S.; Sell, S.; Tudor, Y.;
Turci, S. M.; Welcher, A. A.; White, R. D.; Zack, D.; Zhao, H.; Zhu, L.; Zhu, X.;
Ghiron, C.; Amouzegh, P.; Ermann, M.; Jenkins, J.; Johnston, D.; Napier, S.;
Power, E. J. Med. Chem. 2006, 49, 4981.
8. DiMauro, E. F.; Newcomb, J.; Nunes, J. J.; Bemis, J. E.; Boucher, C.; Buchanan, J.
L.; Buckner, W. H.; Cee, V. J.; Chai, L.; Deak, H. L.; Epstein, L. F.; Faust, T.; Gallant,
P.; Geuns-Meyer, S. D.; Gore, A.; Gu, Y.; Henkle, B.; Hodous, B. L.; Hsieh, F.;
Huang, X.; Kim, J. L.; Lee, J. H.; Martin, M. W.; Masse, C. E.; McGowan, D. C.;
Metz, D.; Mohn, D.; Morgenstern, K. A.; Oliveira-dos-Santos, A.; Patel, V. F.;
Powers, D.; Rose, P. E.; Schneider, S.; Tomlinson, S. A.; Tudor, Y.-Y.; Turci, S. M.;
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5671.
18. The binding model was examined and visualized using MOETM (Molecular Operating
Environment) Version 2007.09, Chemical Computing Group: Montreal, Canada.
19. Lck, Src, and Csk were purchased from Upstate (NY, USA). Kinase assays for
ZAP-70, MEK1, PKA, PKBa, PKCa, CDK1, CAMKIV and MAPK1 were run by
Upstate (NY, USA) using Kinase profiler service in according to the
manufacturer’s procedures.