C. D. Cox et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3175–3179
3179
6. Yan, Y.; Sardana, V.; Xu, B.; Homnick, C.; Halczenko,
W.; Buser, C. A.; Schaber, M.; Hartman, G. D.; Huber, H.
E.; Kuo, L. C. J. Mol. Biol. 2004, 335, 547.
7. In our recently disclosed structure, the KSPi occupies an
˚
‘induced fit’ pocket 12 A from the nucleotide binding site.
This pocket, not present in the apo structure of KSP, has
been termed the allosteric site. Compound 2 was chosen
for docking because it was the first sub-micromolar lead
compound we discovered in this series, and these studies
were initiated before 1 was fully characterized. Addition-
ally, the in silico predicted pose of 2 was later confirmed
by X-ray crystallography (Fig. 3a), affording more confi-
dence to the predictions made from our modeling efforts.
8. Cox, C. D.; Breslin, M. J.; Mariano, B. J. Tetrahedron
Lett. 2004, 45, 1489.
half-life. Finally, unlike in a related series of inhibitors
where the introduction of a basic amine resulted in
greater binding affinity for the potassium channel hERG
(human Ether-a-go-go Related Gene),1b,c the hERG
binding of 12 is not significantly increased with respect
to 1.14
In conclusion, we have described how the introduction
of an alkylamino group at the C5 position of the 3,5-dia-
ryl-4,5-dihydropyrazole core provided access to a series
of KSP inhibitors that displayed improved potency,
pharmacokinetics, and water solubility relative to the
first generation compounds. Of special note is how
molecular modeling and X-ray crystallography were em-
ployed to identify an unoccupied region of space in the
binding site that is effectively utilized in the second gen-
eration compounds described herein.
9. Intermediate 6 could also be treated with acyl chlorides to
provide the analogous dihydropyrazole amides (see Ref.
8); however, for the purposes of this Communication, we
will focus only on dihydropyrazole ureas 7.
10. The phenol functionality on the ‘eastern’ phenyl group of
1 and 2 is not present in subsequent analogs because we
generally see only a modest increase (<10-fold) in potency
due to its presence (Ref. 1c), and also because of the
known drawbacks of phenol-containing drugs, namely
propensity for electrophilic activation; see: Zhou, S.;
Chan, E.; Duan, W.; Huang, M.; Chen, Y.-Z. Drug
Metab. Rev. 2005, 37, 41.
11. KSP inhibitory activity was measured using a standard
ATPase assay; see: Breslin, M. J.; Coleman, P. J.; Cox, C.
D.; Culberson, C. J.; Hartman, G. D.; Mariano, B. J.;
Torrent, M. PCT WO 079973 A2, 2003.
Acknowledgments
We thank Dr. David Dubost for determining the solu-
bility of 12, Dr. Chuck Ross and Ms. Joan Murphy
for high resolution mass spectral analyses, and Drs.
Robert Garbaccio and Mark Fraley for helpful discus-
sions and careful reading of the manuscript.
References and notes
12. The experimental data for the KSP-2-ADP structure have
been reported previously (Ref. 1a). KSP-8h-ADP struc-
ture: co-crystals of the ternary complex of KSP-
ADP(Mg2+)-monastrol were first formed with the vapor
diffusion method (see Ref. 6). The KSP monastrol ternary
crystals then soaked in the harvest solution (28%
PEG3350, 0.2 M K2HPO4 at pH 8.0) containing 2 mM
8h for 3 days to replace monastrol at the inhibitor binding
site. The X-ray diffraction data were collected at 100 K to
1. For the previous papers in this series, see: Part 1: (a) Cox,
C. D.; Breslin, M. J.; Mariano, B. J.; Coleman, P. J.;
Buser, C. A.; Walsh, E. S.; Hamilton, K.; Huber, H. E.;
Kohl, N. E.; Torrent, M.; Yan, Y.; Kuo, L. C.; Hartman,
G. D. Bioorg. Med. Chem. Lett. 2005, 15, 2041; Part 2: (b)
Fraley, M. E.; Garbaccio, R. M.; Arrington, K. L.;
Hoffman, W. F.; Tasber, E. S.; Coleman, P. J.; Buser, C.
A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R.
B.; Tao, W.; South, V. J.; Yan, Y.; Kuo, L. C.;
Prueksaritanont, T.; Shu, C.; Torrent, M.; Heimbrook,
D. C.; Kohl, N. E.; Huber, H. E.; Hartman, G. D. Bioorg.
Med. Chem. Lett. 2006, 16, 1775; Part 3: (c) Garbaccio, R.
M.; Fraley, M. E.; Tasber, E. S.; Olson, C. M.; Hoffman,
W. F.; Torrent, M.; Buser, C. A.; Walsh, E. S.; Hamilton,
K.; Schaber, M. D.; Lobell, R. B.; Tao, W.; South, V. J.;
Yan, Y.; Kuo, L. C.; Prueksaritanont, T.; Slaughter, D.
E.; Shu, C.; Heimbrook, D. C.; Kohl, N. E.; Huber, H. E.;
Hartman, G. D. Bioorg. Med. Chem. Lett. 2006, 16, 1780.
2. Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Curr. Opin.
Pharmacol. 2001, 370.
˚
2.51 A resolution in the space group P212121 with cell
˚
˚
˚
dimensions of a = 68.7 A, b = 79.6 A, and c = 158.7 A
(Rsym = 0.047 and completeness = 99%). The ternary com-
plex structure of KSP-8h-ADP(Mg2+) was determined by
the use of the difference Fourier method and refined to an
R-factor of 0.234 (Rfree = 0.279). The coordinates have
been deposited with RCSB Protein Data Bank under the
accession code 2G1Q.
13. Compound 11 was synthesized by the general procedure
described in Ref. 8. Separation of the enatiomers of 11 was
carried out on a 5 cm Chiralpak AD column at a flow rate
of 80 mL/min with an eluent of 15% 2-propanol in hexanes
containing 0.1% diethylamine as a modifier. Under these
conditions, the first enantiomer to elute was more potent
in the ATPase assay (190 vs. 8200 nM for the second
eluting isomer) and was carried on to provide 12 as
described in Scheme 2. The (S)-stereochemistry is assigned
by analogy to our previous work (Ref. 1a).
3. (a) Heald, R. Cell 2000, 102, 399; (b) Sharp, D. J.; Rogers,
G. C.; Scholey, J. M. Nature 2000, 407, 41; (c) Mandel-
kow, E.; Mandelkow, E.-M. Trends Cell Biol. 2002, 12,
585; (d) Endow, S. A.; Barker, D. S. Annu. Rev. Physiol.
2003, 65, 161.
4. (a) Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R.
W.; Schreiber, S. L.; Mitchison, T. J. Science 1999, 286,
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A.; Lewis, E.; Fritsch, A.; Lee, Y.; Mak, J.; Moody, R.;
Turincio, R.; Chabala, J. C.; Gonzales, P.; Roth, S.;
Weitman, S.; Wood, K. W. Cancer Res. 2004, 64, 3276.
5. (a) Coleman, P. J.; Fraley, M. E. Expert Opin. Ther.
Patents 2004, 14, 1659; (b) Duhl, D. M.; Renhowe, P. A.
Curr. Opin. Drug Discov. Dev. 2005, 8, 431.
14. Blockade of the hERG channel has been implicated in
drug-induced prolongation of the QTc interval of the
EEG, an observation that has been linked to potentially
fatal ventricular arrhythmias. The hERG IC50 values were
determined by radioligand competition experiments using
membrane preparations from human embryonic kidney
cells that stably express hERG. For assay details, see:
Bilodeau, M. T. et al. J. Med. Chem. 2004, 47, 6363, and
references therein.