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11. Norman, B. H. Drugs Future 1998, 23, 1001.
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Winter, M. A.; Sluka, J. P.; Startling, J. J. Bioorg. Med. Chem.
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13. Compounds were tested against the HeLa-T5 cell line in
vitro using an MTT-like assay. A fixed concentration of
doxorubicin (0.5 mg/mL) and varying concentrations of test
compounds, starting at 10 mM, were added to triplicate wells
of a 96-well plate and incubated for 72 h at 37 ꢁC. EC50 values
were calculated from level of inhibition of cell growth using
curve fitting software. Each compound was also tested for
inherent toxicity in the same assay in the absence of doxo-
rubicin. Test compounds showed <20% growth inhibition at
10 mM.
14. Ether isostere 11 was prepared using the following condi-
tions: (a) 3,4,5-trimethoxyphenol, 3-nitrophenethyl alcohol,
DEAD, PPh3, toluene, 1 h (75%); (b) H2/Pd on C, methanol,
30 psi, 2 h (98%); see Scheme 2 (c) and (d).
15. Reduced amide 12 was prepared according to Scheme 2,
beginning with BOC-protected 3-nitrobenzylamine. Upon
formation of the tricyclic moiety, the BOC group was removed
(TFA, 100%) and reductive amination with 3,4,5-trimethoxy-
benzaldehyde (NaCNBH3, methanol, 87%) gave the product.
16. The N-methyl analogue 13 was prepared according to
Scheme 2 and was methylated after the first acylation (NaH,
MeI, DMF, 1 h, 90%).
Figure 2. In vivo activity of LY 402913.
iv) showed the effect of drug without inhibitor. A final
control was performed where the animals were dosed
with inhibitor alone (same regimen). This curve, which
overlaps the vehicle control curve (data not shown),
indicates that the inhibitor has no antitumor activity of
its own. As shown in Figure 2, the combination treat-
ment of vincristine and LY 402913 gave a statistically
significant improvement in efficacy, relative to the con-
trols. We believe that this is the first report of a selective
MRP1 modulator with in vivo efficacy in an MRP1-
dependent animal model.
In summary, these studies show that the tricyclic isox-
azoles are a promising series of selective MRP1 inhibitors.
As more is learned on the tissue specificity of ABC
transporters, and their relationship to clinical drug
resistance, we feel that it is essential that selective drug
resistance modulators be developed. Due to the fact that
most of the known MRP1 modulators also inhibit the
related transporter, P-gp, this series is particularly
exciting.
17. Dantzig, A. H.; Shepard, R. L.; Tabas, L.; Pratt, S.; Gil-
lesppie, J. I.; Binkley, S.; Wrighton, S. A. J. Pharmacol. Exp.
Ther. 1999, 290, 854.
18. McGrath, T.; Latoud, C.; Arnold, S. T.; Safa, A. R.;
Felsted, R. L.; Center, M. S. Biochem. Pharmacol. 1989, 38,
3611.
19. Modulator EC50 in HL60/Adr and HL60/Vinc cells were
determined by assessing the concentration-dependent ability of
the modulator to enhance the antiproliferative response of the
cells to an IC20 concentration of doxorubicin using alamar-
Blue1 reduction as a surrogate measure for cell number. The
selectivity ratio was calculated by dividing the average EC50
for HL60/Vinc cells by the average EC50 for HL60/Adr cells.
20. The ability of the compounds to increase the antitumor
activity of co-administered vincristine in the HeLa-T5 xeno-
graft model was tested. Test compounds were orally adminis-
tered for 5 consecutive days to female nude mice with and
without intravenous administration of vincristine sulfate.
Caliper measurements of tumor growth were taken at various
time points and plotted to indicate antitumor response.
References and Notes
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