C. D. Cox et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2041–2045
2045
2. (a) Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King,
R. W.; Schreiber, S. L.; Mitchison, T. J. Science 1999, 286,
971; (b) Kapoor, T. M.; Mayer, T. U.; Coughlin, M. L.;
Mitchison, T. J. J. Cell Biol. 2000, 150, 975.
Kif3A, Kif1B, uKHC, nKHC, KIF14, and MCAK) were
greater than 50 lM in ATPase assays similar to that
described for KSP. The selection of these counter-screens
was based on considerations of homology and function.
12. The effect of 15 on the polymerization of tubulin was
measured by turbidity (optical density at 340 nm) in 96-
well plates at 37 ꢁC in a SpectroMax Ultra (modified from
Lopes, N. M.; Miller, H. P.; Young, N. D.; Bhuyan, B. K.
Cancer Chemother. Pharmacol. 1997, 41, 37). KSPi 15 and
control compounds (taxol and colchicine) were resus-
pended in DMSO and diluted to 20 lM in 1· buffer
(80 mM K-PIPES, 1 mM EGTA, 1 mM MgCl2, pH 6.8)
containing 1 mM GTP. The assay was initiated by adding
cold tubulin at 2.5 mg/mL to each well (to a total volume
of 200 lL; 1% final DMSO).
3. Sakowicz, R.; Finer, J. T.; Beraud, C.; Crompton, 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.
4. For an overview of the mitotic spindle as target for cancer
therapeutics, see: (a) Wood, K. W.; Cornwell, W. D.;
Jackson, J. R. Curr. Opin. Pharmacol. 2001, 0, 370; For
kinesin-targeted therapeutics, see: (b) Wood, K. W.;
Bergnes, G. Ann. Rep. Med. Chem. 2004, 39, 173.
5. The biological assays utilized herein have been described;
see: (a) Breslin, M. J.; Coleman, P. J.; Cox, C. D.;
Culberson, C. J.; Hartman, G. D.; Mariano, B. J.;
Torrent, M. PCT WO 03/079973 A2, 2003; (b) Ref. 13.
6. For example synthetic procedures, see: (a) Safak, C.;
Tayhan, A.; Sarac, S.; Yulug, N. J. Indian Chem. Soc.
1990, 67, 571; (b) Ref. 5a.
7. Asselin, A. A.; Humber, L. G.; Crosilla, D.; Oshiro, G.;
Wojdan, A.; Grimes, D.; Heaslip, R. J.; Rimele, T. J.;
Shaw, C.-C. J. Med. Chem. 1986, 29, 1009.
8. A similar synthetic approach has previously been taken in
a parallel solution phase library synthesis; see: Bauer, U.;
Egner, B. J.; Nilsson, I.; Berghult, M. Tetrahedron Lett.
2000, 41, 2713.
13. 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.
14. Co-crystals of the ternary complex of KSP-ADP(Mg2+)-
monastrol were first formed with the vapor diffusion
method (see Ref. 13). The KSP monastrol ternary crystals
then soaked in the harvest solution (28% PEG3350, 0.2 M
K2HPO4 at pH 8.0) containing 2 mM 4 for 2 days to
replace monastrol at the inhibitor binding site. The X-ray
diffraction data were collected at 100 Kat synchrotron
beamline 17-ID of the Advanced Photon Source at
˚
9. Dihydropyrazole 13f can be easily accessed by heating
chalcone in the presence of methylhydrazine in EtOH at
80 ꢁC for 2 h.
10. Separation of 500 mg of 7 was carried out on a 250 · 5 cm
20 lM Chiralpak AD column with 80% hexanes (modified
with 0.1% TFA) and 20% EtOH at 75 mL/min. The first
Argonne National Laboratory to 2.5 A resolution in the
˚
space group P212121 with cell dimensions of a = 69.3 A,
˚
˚
b = 79.8 A, and c = 159.6 A (Rsym = 0.066 and complete-
ness = 94%). The ternary complex structure of KSP-4-
ADP(Mg2+) was determined by the use of the difference
Fourier method and refined to an R-factor of 0.25. The
coordinates have been deposited with RCSB Protein Data
Bank under the accession code 1YRS.
isomer to elute, the (+) antipode, was ÔinactiveÕ (IC50
=
4.1 lM). The second isomer to elute, the (ꢀ) antipode, was
active (IC50 = 26 nM). Each isomer was P99% ee by
analytical HPLC under the same conditions. Data for 15:
15. We have solved the crystal structure of the ternary
complex of KSP, ADP and over 20 structurally-related
inhibitors. In each case, only the S-antipode was found to
bind in the active site. The absolute stereochemistry of 14
was assigned by analogy.
16. Caspase-3 activity in cell lysates was determined using the
ApoAlert Caspase-3 fluorescent assay kit (Clontech, Palo
Alto, CA), which measures the release of the 7-amino-4-
trifluoromethylcoumarin (AFC) fluorophore from the
substrate DEVD-AFC.
20
½aꢁD ꢀ88.9 (c 3.5, MeOH). NMR (500 MHz, DMSO-d6): d
9.4 (s, 1H), 7.7 (m, 1H), 7.4 (m, 2H), 7.1 (m, 1H), 6.7–6.5
(m, 3H), 5.4 (m, 1H), 3.9 (m, 1H), 3.1 (m, 1H), 2.3 (s, 3H)
ppm. HRMS (ES) calcd M+H for C17H14F2N2O2:
317.1096. Found 317.1105. See Ref. 15 for assignment of
absolute stereochemistry as S.
11. The IC50Õs of KSPi 15 when tested against the motor
domains of eight additional kinesins (CENP-E, MKLP-1,