M. P. Dwyer et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6216–6219
6219
C. L.; Powles, J.; Robertson, A.; Surgenor, A. E.;
Torrance, C. J. Bioorg. Med. Chem. Lett. 2005, 15, 863.
12. Cyclin A/CDK2 kinase assay. Recombinant baculoviruses
were purified from Sf9 cells engineered to express cyclin A
and CDK2. Cyclin A/CDK2 enzyme was diluted to a final
concentration of 50 lg/mL in kinase buffer containing
50 mM Tris, pH 8.0, 10 mM MgCl2, 1 mM DTT, and
0.1 mM sodium orthovanadate. For each kinase reaction,
1 lg of enzyme and 20 lL of 2 lM substrate solution (a
biotinylated peptide derived from Histone H1; Amersham,
UK) were mixed and combined with 10 lL of diluted
compound. The reaction was started by addition of 50 lL
of 2 lM ATP and 0.1 lCi of 33P-ATP (Amersham, UK).
Kinase reactions ran for 1 h at room temperature and were
stopped by the addition of 0.1% Triton X-100, 1 mM ATP,
5 mM EDTA, and 5 mg/mL streptavidin-coated SPA
beads (Amersham, UK). SPA beads were captured using
a 96-well GF/B filter plate (Packard/Perkin Elmer Life
Sciences) and a Filtermate universal harvester (Packard/
Perkin-Elmer Life Sciences.) Beads were washed twice with
2 M NaCl and twice with 2 M NaCl with 1% phosphoric
acid. Signal was then assayed using a TopCount 96-well
liquid scintillation counter (Packard/Perkin-Elmer Life
Sciences). Dose–response curves were generated from
duplicate 8 point serial dilutions of inhibitory compounds.
IC50 values were derived by nonlinear regression analysis.
13. Senga, K.; Novinson, T.; Wilson, H. R.; Robins, R. K.
J. Med. Chem. 1981, 24, 610.
dramatic potency differences between the core structures
9–12 will be reported in due course.22 The two most
potent analogs (9 and 10) were further evaluated for
their ability to alter uptake and incorporation of radio-
actively labeled thymidine by living cells. In line with the
observed in-vitro potency trends in Table 1, pyrazolo-
[1,5-a]pyrimidine adduct 9 possessed superior activity
in the thymidine incorporation assay with an
IC50 = 0.6 lM versus the comparable imidazo[1,2-a]pyr-
azine adduct 10. As summarized in Table 1, pyrazol-
o[1,5-a]pyrimidine adduct 9 displayed comparable
in-vitro potency in the cyclin A/CDK2 assay to the known
CDK inhibitor flavopiridol (1) shown in Figure 1.
In summary, four bicyclic cores were designed and pre-
pared bearing identical functionality based upon early
screening hits. Based upon both in-vitro and cell-based
data, the pyrazolo[1,5-a]pyrimidine core (represented
by 9) emerged from these efforts as the preferred bicyclic
motif for our CDK2 program. Further optimization of
the pyrazolo[1,5-a]pyrimidine series as CDK2 inhibitors
appears in the accompanying paper.23
References and notes
1. Murray, A. W. Cell 2004, 116, 221.
2. (a) Sherr, C. J. Cancer Res. 2000, 60, 3689; (b) Nevins, J.
R. Hum. Mol. Genet. 2001, 10, 699.
3. Webster, K. R.; Kimball, D. Emerging Drugs 2000, 5, 45.
4. (a) Thomas, M. P.; McInnes, C. IDrugs 2006, 9, 273; (b)
Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770; (c) Fischer, P.
M. Drugs Future 2005, 30, 911; (d) Fischer, P. M. Cell
Cycle 2004, 3, 742.
14. Davey, D. D. J. Org. Chem. 1987, 57, 4379.
15. Larson, S. D.; Spilman, C. H. WO 1993/25553.
16. Lober, S.; Huber, H.; Utz, W.; Gmeiner, P. J. Med. Chem.
2001, 44, 2941.
17. Aboul-Fadl, T.; Lober, S.; Gmeiner, P. Synthesis 2000, 1727.
18. Cai, S. X.; Huang, J.-C.; Espitia, S. A.; Tran, M.; Ilyin, V.
I.; Hawkinson, J. E.; Woodward, R. M.; Weber, E.;
Keana, J. F. W. J. Med. Chem. 1997, 40, 3679.
5. Bible, K. C.; Lensing, J. L.; Nelson, S. A.; Lee, Y. K.;
Reid, J. M.; Ames, M. M.; Isham, C. R.; Piens, J.; Rubin,
S. L.; Rubin, J.; Kaufmann, S. H.; Atherton, P. J.; Sloan,
J. A.; Daiss, M. K.; Adjei, A. A.; Erlichman, C. Clin.
Cancer Res. 2005, 11, 5935.
6. McClue, S. J.; Blake, D.; Clarke, R.; Cowan, A.; Cum-
mings, L.; Fischer, P. M.; MacKenzie, M.; Melville, J.;
Stewart, K.; Wang, S.; Zhelev, N.; Zheleva, D.; Lane, D.
P. Int. J. Cancer 2002, 102, 463.
7. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.-C.;
Barbosa, S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.;
Ahmed, S. Z.; Qian, L.; Chen, B.-C.; Zhao, R.; Bednarz, M.
S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta,
U.; Kamath, A.; Marathe, P.; Ranadive, S. A.; Sack, J. S.;
Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.; Webster, K.
R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
8. Chu, X.-J.; DePinto, W.; Bartkovitz, D.; Sung-Sau, S.; Vu,
B. T.; Packman, K.; Luckacs, C.; Ding, Q.; Jiang, N.; Wang,
K.; Goelzer, P.; Yin, X.; Smith, M. A.; Higgins, B. X.; Chen,
Y.; Xiang, Q.; Moliterni, J.; Kaplan, G.; Graves, B.; Lovey,
A.; Fotouhi, N. J. Med. Chem. 2006, 49, 6549.
9. Toogood, P. L.; Harvey, P. J.; Repine, J. T.; Sheehan, D.
J.; VanderWel, S. N.; Zhou, H.; Keller, P. R.; McNamara,
D. J.; Fry, D. W. J. Med. Chem. 2005, 48, 2388.
10. (a) Hirai, H.; Kawanishi, N.; Iwasawa, Y. Curr. Top. Med.
Chem. 2005, 5, 167; (b) McInnes, C.; Fischer, P. M. Curr.
Pharm. Des. 2005, 11, 1845; (c) Fischer, P. M. Curr. Med.
Chem. 2004, 11, 1563.
19. Hand, E. S.; Paudler, W. W. J. Org. Chem. 1978, 43, 2906.
20. Thymidine uptake growth inhibition assay. The thymidine
incorporation assay was used to measure inhibition of
asynchronously growing A2780 ovarian carcinoma cells
exposed to test compound. These cells were maintained in
DMEM (Cellgro) plus 10% fetal bovine serum (HyClone)
and passaged twice a week by detaching the monolayer with
Trypsin-EDTA (Gibco). One hundred microliters of A2780
cells (5000 cells/well) was addedper wellto a 96-wellCytostar
T plate(Amersham, UK) andincubated for 16–24 h at 37 °C.
Compounds were serially diluted in complete media plus 2%
14C-labeled thymidine (Amersham, UK). Media were
removed from Cytostar T plate, 200 lL of compound
dilution was added in quadruplicate and incubated for 24 h
at 37 °C. Accumulated incorporation of radiolabel was
assayed using scintillation proximity and measured on
TopCount (Packard/Perkin-Elmer Life Sciences). Percentage
inhibitions, relative to vehicle controls, were calculated and
plotted on log-linear plots to allow derivation of IC50 values.
21. The X-ray coordinates for compound 9 bound to CDK2
have been deposited in the ProteinDataBank (PDB ID
code: 2R3R).
22. Duca, J.; Fischmann, T. O.; Hruza, A.; Madison, V.
Unpublished results.
23. Paruch, K.; Dwyer, M. P.; Alvarez, C.; Brown, C.; Chan,
T.-Y.; Doll, R. J.; Keertikar, K.; Knutson, C.; McKittrick,
B.; Rivera, J.; Rossman, R.; Tucker, G.; Fischmann, T.
O.; Hruza, A.; Madison, V.; Nomeir, A. A.; Wang, Y.;
Lees, E.; Parry, D.; Sgambellone, N.; Seghezzi, W.;
Schultz, L.; Shanahan, F.; Wiswell, D.; Xu, X.; Zhou,
Q.; James, R. A.; Paradkar, V. M.; Park, H.; Rokosz, L.
R.; Stauffer, T. M.; Guzi, T. J. Bioorg. Med. Chem. Lett.
11. For a recent report on pyrazolo[1,5-a]pyrimidine-based
CDK2 inhibitors, see: Williamson, D. S.; Parratt, M. J.;
Bower, J. F.; Moore, J. D.; Richardson, C. M.; Dokurno,
P.; Cansfield, A. D.; Francis, G. L.; Hebdon, R. J.; Howes,
R.; Jackson, P. S.; Lockie, A. M.; Murray, J. B.; Nunns,