1778
M. E. Fraley et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1775–1779
Figure 5. Crystal structures of 6, 13 and 19 bound to the allosteric site of KSP. Protein shown in solid ribbon. Green patch indicates location of a
hydrophobic region in the binding site.
´
2. Blangy, A.; Lane, H. A.; d’Herin, P.; Harper, M.; Kress,
M.; Nigg, E. A. Cell 1995, 83, 1159.
Table 3. Pharmacokinetic profile of 19 following iv dosing to animals
F
3. Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R.
W.; Schreiber, S. L.; Mitchison, T. J. Science 1999, 286,
971.
F
H
4. Marcus, A. I.; Peters, U.; Thomas, S. L.; Garrett, S.;
Zelnak, A.; Kapoor, T. M.; Giannakakou, P. J. Biol.
Chem. 2005, 280, 11569.
5. Tao, W.; South, V. J.; Zhang, Y.; Davide, J. P.; Farrell, L.;
Kohl, N. E.; Sepp-Lorenzino, L.; Lobell, R. B. Cancer
Cell 2005, 8, 49.
N
O
NH2
19
Species
Dose
(mg/kg)
Cl
(mL/min/kg)
Vdss
t1/2
6. For reviews, see: (a) Duhl, D. M.; Renhowe, P. A. Curr.
Opin. Drug Discov. Dev. 2005, 8, 431; (b) Coleman, P. J.;
Fraley, M. E. Expert Opin. Ther. Patents 2004, 14, 1659.
7. 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.
8. Carpes, M. J. S.; Correia, C. R. D. Synlett 2000, 9, 1037.
(L/kg)
(h)
Rat (n = 4)
Dog (4)
Monkey (3)
1.0
0.4
0.4
40 5.8
23 4.5
21 2.5
4.4 1.5
3.8 0.3
7.4 0.8
1.4 0.4
2.3 0.3
4.5 0.8
the S stereochemical configuration of the a-amino
amides for optimal potency.
1
9. All compounds were characterized by H NMR and high
resolution mass spectrometry. For detailed experimental
procedures, see WO03105855.
The pharmacokinetic profile of compound 19 in rat, dog,
and monkey following iv administration is presented in
Table 3 and was representative of the series. Plasma clear-
ance was moderate in these species with half-lives ranging
from 1 to 4 h, respectively.
10. KSP inhibitory activity was assessed by measuring the
release of inorganic phosphate from ATP hydrolysis
through absorbance detection of a quinaldine red-phos-
phate complex. For assay details, see: Cogan, E. B.; Birell,
G. B.; Griffith, O. H. Anal. Biochem. 1999, 271, 29, IC50
values are reported as the averages of at least two
independent determinations; standard deviations are with-
in 25-50% of IC50 values.
In summary, we have described the development of
dihydropyrroles as novel and potent KSP inhibitors
through core modification of dihydropyrazole-based
lead structures. Potency and aqueous solubility were
gained simultaneously through the introduction of basic
amides and ureas, a key finding from our optimization
efforts. In addition, it was discovered that the moderate
level of hERG binding demonstrated by the basic inhib-
itors could be attenuated through the use of charge-
neutral acyl groups.
11. The KSP IC50 for the (+, R) enantiomer of 6 was
determined to be >50,000 nM.
12. Compound 8 was prepared from (S)-epichlorohydrin by
modification of the method of Sassaman, M. B.; Prakash,
G. K. S.; Olah, G. A. J. Org. Chem. 1990, 55, 2016.
13. Maeda, K.; Yamamoto, Y.; Tomimoto, K.; Mase, T.
Synlett 2001, 1808.
14. Interestingly, use of LiHMDS under the same reaction
conditions produced a 2:1 mixture of enol triflates,
favoring 11.
15. As with the dihydropyrazoles, the 2,5-difluorophenyl ring
was found to be optimal in the dihydropyrrole series (data
not shown).
Acknowledgments
16. Mitotic arrest was measured by assessing the mitosis-
specific phosphorylation of nucleolin using an antibody-
coated, bead-based assay. In this assay, total nucleolin
We thank Dr. Harry Ramjit for high resolution mass
spectral analysis, Carl Homnick for chiral separation
to give 6, and Dr. Laszlo Kiss for cellular electrophysi-
ological evaluation of 19 on hERG.
is captured on
a streptavidin-coated parameagnetic
bead coupled with biotinylated nucleolin monoclonal
IgG1 antibody 4E2 (Research Diagnostics, Inc.). Nucle-
olin phosphorylation is detected by an antibody com-
plex consisting of a phospho-specific nucleolin IgM
monoclonal antibody, TG3 (Applied NeuroSolutions,
References and notes
Inc.) and
a goat anti-mouse IgM labeled with a
1. For reviews, see: (a) Heald, R. Cell 2000, 102, 399; (b)
Sharp, D. J.; Rogers, G. C.; Scholey, J. M. Nature 2000,
407, 41; (c) Endow, S. A. Eur. J. Biochem. 1999, 262, 12.
ruthenium Tris-bipyridyl complex (BV-TAG Technolo-
gy, BioVeris Corp.). The complex is quantitated via