4592
T. R. Rheault et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4587–4592
Table 3 (continued)
Compd
X
R
PLK1 IC50
nMa
HCT116 IC50
nMa,b
CYP3A4 IC50
CYP2C9 IC50
FCA
Solubility
mg/mLc,f
Mouse iv Cl
mL/min/Kgf
DEF/7BQ
l
Md,f
l
Md
N
N
N
N
43
Cl
2
1
15
15
0.9/5.0
0.4
5.83
3.12
44
44
N
N
44
Cl
1.0/4.5
2.4
N
a
b
c
d
e
f
Values are means of at least two experiments.
Cell assay data obtained using the CTG assay format.
Solubility was determined in 20% SBE b-cyclodextrin at pH 6 (equilibrium).
P450 determination using Cypex recombinant human enzyme unless otherwise stated.
P450 determination using Gentest recombinant human enzyme and PPR as fluorescent substrate instead of 7BQ.
(—) indicates data not determined.
Davis-Ward, R. G.; Dickson, H. D.; Hassler, D. F.; Hornberger, K. R.; Jackson, J. R.;
Kuntz, K. W.; Lansing, T. J.; Mook, R. A., Jr.; Nailor, K. E.; Pobanz, M. A.; Smith, S.
C.; Sung, C.-M.; Cheung, M. Bioorg. Med. Chem. Lett. 2009, 19, 1694.
Pyridines 38 and 39 and pyrazole 44 met all of these criteria and
were advanced into further biological studies.
12. Hornberger, K. H.; Badiang, J. G.; Salovich, J. M.; Kuntz, K. W.; Emmitte, K. A.;
Cheung, M. Tetrahedron Lett. 2008, 49, 6348.
Acknowledgements
13. Rheault, T. R.; Donaldson, K. H.; Cheung, M. Tetrahedron Lett. 2009, 50, 1399.
14. The PLK1 enzyme assay was conducted using the kinase domain only. The PLK3
assay was conducted using full length enzyme. Both were in an SPA format. For
a more detailed description see: Lansing, T. J.; McConnell, R. T.; Duckett, D. R.;
Spehar, G. M.; Knick, V. B.; Hassler, D. F.; Noro, N.; Furuta, M.; Emmitte, K. A.;
Gilmer, T. M.; Mook, R. A., Jr.; Cheung, M. Mol. Cancer Ther. 2007, 6, 450.
15. Cell proliferation assays: The HCT116 cell line is a rapidly dividing human
The authors thank Chiu-Mei Sung and Dan Price for assistance
in the preparation of this manuscript.
References and notes
colorectal carcinoma cell line. Cell-based assays were run using either
a
1. Pecorino, L. Molecular Biology of Cancer; Oxford: New York, 2006. pp 1–19.
2. Chabner, B. A.; Ryan, D. P.; Paz-Ares, L.; Garcia-Carbonero, R.; Calabresi, P. In
Goodman s the Pharmacological Basis of Therapeutics; Hardman, J. G., Limbird, L.
E., Gilman, A. G., Eds., 10th ed.; McGraw-Hill: New York, 2001; pp 1417–1425.
3. (a) Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429; (b)
Fisher, R. A. H.; Ferris, D. K. Curr. Med. Chem. Immune, Endocr. & Metabol. Agents
2002, 2, 125; (c) Glover, D. M.; Hagan, I. M.; Tavares, A. A. M. Genes Dev. 1998,
12, 3777; (d) Mundt, K. E.; Golsteyn, R. M.; Lane, H. A.; Nigg, E. A. Biochem.
Biophys. Res. Commun. 1997, 239, 377.
4. The PLK family also includes PLK2 (SNK), PLK3 (PRK/FNK), and PLK4 (SAK),
although it is unknown whether selectivity versus these family members is
either desirable or necessary for an anticancer treatment (a) Ma, S.; Charron, J.;
Erikson, R. L. Mol. Cell. Biol. 2003, 19, 6936; (b) Ouyang, B. Et al. Jopurnal Biol.
Chem. 1997, 272, 28646; (c) Fode, C.; Motro, B.; Yousefi, S.; Heffernan, M.;
Dennis, J. W. Proc. Natl Acad. Sci. USA 1994, 91, 6388.
CellTiter-Glo (CTG) or methylene blue (MEB) assay format. Either proliferation
assay format gives an IC50 within a twofold range for this chemical series. CTG
assay: Cells were seeded in 96-well dishes, incubated overnight at 37 °C, and
treated with various concentrations of inhibitors for 72 h. Cell proliferation
was quantified using the CellTiter-Glo Luminescent Cell Viability Assay
(Promega, Madison, WI), following the manufacturer’s recommendations.
IC50 values were determined from using a 4-parameter curve fit software
package (XLfit4). For a description of the MEB assay see: Rusnak, D. W.; Lackey,
K.; Affleck, K.; Wood, E. R.; Alligood, K. J.; Rhodes, N.; Keith, B. R.; Murray, D. M.;
Knight, W. B.; Mullin, R. J.; Gilmer, T. M. Mol. Cancer Ther. 2001, 1, 85–94.
16. The inhibition of P450 activity was assessed using fluorescent probe substrates
in a 96-well plate based assay format. The Gentest P450 assay format is
described in the following reference: Crespi, C. L.; Miller, V. P.; Penman, B. W.
Anal. Biochem. 1997, 248, 188–190. The Cypex assay format: Incubations with
recombinant enzyme (Cypex Bactosomes) and test compound were performed
5. (a) Liu, X.; Erikson, R. L. Proc. Natl. Acad. Sci. USA 2003, 100, 5789; (b) Yim, H.;
Erikson, R. L. Mol. Cell. Biol. 2009, 29, 2609; (c) Spänkuch, B.; Kurunci-Csacsko,
E.; Kaufmann, M.; Strebhardt, K. Oncogene 2007, 26, 5793.
6. Lez, R.; Piper, A.; Kronenberger, B.; Kock, M.; Brendel, M.; Hermann, E.; Pliquett,
U.; Neumann, E.; Zeuzem, S. Oncogene 2003, 22, 69.
in black 96-well clear bottom microtiter plates with a final volume of 250
approximately 37 °C. Each incubation contained 220 L of incubation mix
(containing 50 mM potassium phosphate buffer pH 7.4, recombinant enzyme,
and probe substrate) and 5 L of test compound solution. Incubations for each
isoform were performed separately. Samples were pre-warmed at 37 °C for
10 min before the reactions were initiated with 25 of an NADPH
lL at
l
l
7. Liu, X.; Lei, M.; Erikson, R. L. Mol. Cell. Biol. 2006, 26, 2093.
lL
8. (a) Takai, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene 2005, 24,
287; (b) Kanaji, S.; Saito, H.; Tsujitani, S., et al Oncology 2006, 126; (c) Weichert,
W.; Kristiansen, G.; Schmidt, M., et al World J. Gastroenterol. 2005, 11, 5644; (d)
Yamada, S.; Ohira, M.; Horie, H., et al Oncogene 2004, 23, 5901.
9. Strebhardt, K.; Ullrich, A. Nat. Rev. Cancer 2006, 6, 321.
10. Rowinsky, E. K.; Tolcher, A. W. Pharmacology of Cancer Chemotherapy:
Antimicrotubule Agents. In Cancer: Principles & Practice of Oncology; DeVita,
regenerating system (containing 1.7 mg NADP, 7.8 mg glucose-6-phosphate,
and 6 units of glucose-6-phosphate dehydrogenase per mL). Final incubation
concentrations of test compound were 0.033, 0.1, 0.33, 1, 3.3, 10, and 33 lM.
Miconazole was used as positive control. Enzyme activity in the presence of
test compound was normalized for the enzyme activity in absence of test
compound and expressed as percent control activity. Control incubations with
no inhibitor were prepared by adding 5 lL of vehicle solvent (DMSO) in place
V. T., Hellman, S., Rosenberg, S. A., Eds.; Lippincott Williams
Philadelphia (PA), 2005; pp 390–416.
& Wilkins:
of the inhibitor. Incubations were analyzed using a fluorescence plate reader.
17. The PLK1 homology model was based on the active confirmation of the protein
PKA C-alpha.
18. (a) For detailed synthetic procedures, see: Rheault, T. R.; Cheung, M.; Badiang, J.
G. S.; Donaldson, K. H. PCT Int. Appl. 2008, WO 2008070354.; (b) Cheung, M.;
Badiang, J. G.; Donaldson, K. H.; Rheault, T. R. PCT Int. Appl. 2007, WO
2007030359.
11. (a) Emmitte, K. A.; Andrews, C. W.; Badiang, J. G.; Davis-Ward, R. G.; Dickson, H.
D.; Drewry, D. H.; Emerson, H. K.; Hassler, D. F.; Knick, V. B.; Kuntz, K. W.;
Lansing, T. J.; Linn, J. A.; Mook, R. A., Jr.; Nailor, K. E.; Salovich, J. M.; Spehar, G.
M.; Cheung, M. Bioorg. Med. Chem. Lett. 2009, 19, 1018; (b) Emmitte, K. A.;
Adjabeng, G. M.; Andrews, C. W.; Badiang, J. G.; Bambal, R.; Chamberlain, S. D.;