V. W.-F. Tai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4554–4558
4557
demonstrated as shown in Figure 2A. PARP cleavage of
compound 1k-treated cells was demonstrated at 48 h
using Western blot analysis, whereas untreated control
did not show PARP cleavage. Figure 2B shows the
DNA laddering of compound 1k-treated cells after 48 h.
determined to be superior at this position. At position
D, a single methylene unit was determined to be the best
spacer between the aryl/heteroaryl group and the amide
group. Replacement of the 2-thienyl group with 2-fura-
nyl or substituted phenyl groups is tolerated. Compound
1k was further shown to cleave PARP and displayed
DNA laddering pattern, the hallmarks for apoptosis.
This compound showed tumor growth inhibition in hu-
man colorectal DLD-1 xenograft models. Additional
biological characterization of this class of compounds
will help to understand the mechanism of action of these
compounds and improve the efficacy in the future.
Based on its good caspase activation potency, prolifera-
tion assay, solubility, and pharmacokinetic profile, com-
pound 1k was evaluated in human tumor xenograft
models. At once a day dosing, compound 1k showed
21% (p = 0.096) and 38% (p = 0.002) tumor growth inhi-
bition of colorectal DLD-1 tumor xenografts at 50 mpk
and 100 mpk, respectively. At 50 mg/kg twice a day,
compound 1k achieved 63% (p < 0.001) tumor growth
inhibition. No significant weight loss was observed in
the above doses (Fig. 3).
Acknowledgments
The authors thank Drs. Michael C. Venuti, Michael J.
Green, Peter Young, and Sriram Balasubramanian for
helpful discussions in the in vitro evaluation of these
analogs and in preparation of the manuscript.
In summary, a new class of apoptosis inducers with a
2-phenyl-oxazole-4-carboxamide scaffold was identified
through a cell-based caspase HTS assay. SAR studies
at position A revealed an interesting correlation between
the van der Waals volume of the molecule with caspase
activation in one subgroup of analogs. A strong prefer-
ence for 6- and 7-membered cyclic amides at position A
was found and 3,3-difluoropiperidine-substitution was
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Figure 2. (A) Cleavage of PARP;16 The uncleaved PARP is approx-
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450
Vehicle, ip, qd;
50 mg/kg compound, 1k, ip, qd
100 mg/kg compound 1k, ip, qd
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0
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16. PARP cleavage—DLD-1 cells were seeded at 106 cells per
10-cm dish and 24 h later were treated with compound 1k
(1 lM) or vinblastine (400 nM) (final concentration of
DMSO was 0.2%). After 48 h, cells were harvested and
whole-cell lysates prepared using standard procedures.
Cell extract (30 lg) was electrophoretically separated on a
Days post dosing
Figure 3. Anti-tumor activity of compound 1k-treated athymic female
mice bearing DLD-1 human colon tumors.18 Twelve days post implant
of DLD-1 cells, mice were treated with various doses of compound 1k
on days 1–5, 8–12, and 15.