E. L. Grimm et al. / Bioorg. Med. Chem. 12 (2004) 845–851
851
1
compound 20 as colorless solid: H NMR (400 MHz,
6. (a) Similar approaches have appeared in the literature
while our work was in progress, see: Choong, I. C.; Lew, W.;
Lee, D.; Pham, P.; Burdett, M. T.; Lam, J. W.; Wiesmann,
C.; Luong, T. N.; Fahr, B.; DeLano, W. L.; McDowell,
R. S.; Allen, D. A.; Erlanson, D. A.; Gordon, E. M.;
O’Brien, T. J. Med. Chem. 2002, 45, 5005. (b) Siev, D. V.;
Semple, J. E. Org. Lett. 2000, 2, 19. (c) Lee, A.; Huang,
L.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 9907.
7. Pietta, P. G.; Marshall, G. R. J. Chem. Soc. D: Chem.
Comm 1970, 650.
8. The aminomethyl Merrifield resin was prepared from the
commercially available chloromethylpolystyrene-divi-
nylbenzene resin (Merrifield resin (100–200 mesh), 2%
DVB, 0.8 mmol/g, Novabiochem cat. no. 01-64-0104),
according to the procedure of Sparrow, J. T. J. Org.
Chem. 1976, 41, 1350.
CD3OD) d 8.14 (d, 1H, J=7.81 Hz), 7.75 (d, 1H,
J=7.24 Hz), 7.25–7.05 (m, 5H), 4.94 (t, 1H, J=6.59
Hz), 4.62 (t, 1H, J=6.20 Hz), 4.35–4.20 (m, 1H), 4.15–
4.05 (m, 1H), 3.78–3.70 (m, 1H), 3.48–3.38 (m, 1H), 3.29
(s, 3H), 2.88–2.80 (m, 1H), 2.75–2.45 (m, 5H), 2.15–2.03
(m, 1H), 1.97 (s, 3H), 1.90–1.80 (m, 2H), 1.37 (d, 3H,
J=7.26 Hz), 0.93 (d, 3H, J=6.77 Hz), 0.92 (d, 3H,
J=6.74 Hz); MS (–APCI) m/z 595 (MꢀH)ꢀ.
References and notes
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dett, M. T.; Lew, W.; DeLano, W. L.; Gordon, E. M.;
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mann, C.; Luong, T. N.; Simmons, R.; DeLano, W. L.;
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9. In order to demonstrate that racemization does not occur
during the reaction sequence, ketones 1a, 1d, and their d-
enantiomers were separately loaded onto solid support
and subsequently cleaved from the resin. As determined
by NMR and chiral HPLC analysis, no racemization
occurred during these procedures (<5%), see also refs 5,
6a, 6c. Prolonged standing in aqueous solvents (>24 h),
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14. Enzymatic activity was determined by a continuous
fluorometric assay, monitoring the release of AMC from
the substrates Ac-DEVD-AMC (for caspases-3,-7,-8) or
Ac-YVAD-AMC for caspase-1). Assays were performed
in 50 mM HEPES-KOH, pH 7.0, 2 mM EDTA, 10%
glycerol, 0.1% CHAPS, 5 mM DTT and 10 mM substrate
(for caspases-1 and-3), 50 mM substrate (for caspase-7),
and 7 mM substrate (for caspase-8).
15. Becker, J. W.; Rotonda, J.; Soisson, S. M.; Aspiotis, R.;
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Zamboni, R. J. Med. Chem., submitted for publication.
16. NT2 cells were plated into 96-well dishes at 5000 cells/well
in DMEM/10% FBS/2 mM l-glutamine/100 U/mL peni-
cillin/100 U/mL streptomycin (Gibco/BRL) the day
before the assay. On the day of the assay, the growth
media was replaced with 200 mL/well of Cyto-SF4 (sup-
plemented with 2 mM l-glutamine/100 U/mL penicillin/
100 U/mL streptomycin) serum free media (Kemp Bio-
technology) followed by the simultaneous addition 1 mL
of camptothecin (from 1 mg/mL in DMSO, Sigma) and 2
mL of inhibitor (in DMSO). After a 5–6 h incubation at
37 ꢁC and 6% CO2, the level of nucleosomal DNA frag-
mentation was quantified using the Cell Death Detection
ELISAPLUS kit (Boehringer Mannheim).
17. Xanthoudakis, S.; Tawa, P.; Tam, J. unpublished results.
18. Isabel, E.; Black, W. C.; Bayly, C. I.; Grimm, E. L.; Janes,
M. K.; McKay, D. J.; Nicholson, D. W.; Rasper, D. M.;
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dakis, S.; Vaillancourt, J. P.; Rasper, D. M.; Tam, J.;
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