6732
V. J. Huber et al. / Tetrahedron Letters 43 (2002) 6729–6733
solved in ethyl acetate and washed with saturated aq.
C14H15N3O5·0.1H2O: C, 54.76; H, 4.99; N, 13.68. Found:
C, 55.04; H, 5.23; N, 13.31%.
14. (a) Lane, D. P.; Hall, P. A. Trends Biochem. Sci. 1997,
10, 372–374; (b) Cho, Y.; Gorina, S.; Jeffrey, P. D.;
Pavletich, N. P. Science 1994, 265, 346–355; (c) Prives, C.
Cell 1994, 78, 543–546.
NaHCO3 (2×) then brine. The resulting organic solution
was then dried over MgSO4, filtered and concentrated in
vacuo giving an orange-red oil. The crude product was
purified by column chromatography (silica gel, 1%
MeOH/DCM), giving 6 as a yellow solid (6.7 g, 87%
yield). Mp=78–79°C (dec.). Proton, carbon and IR spec-
tra were consistent with the desired compound. Anal.
calcd for C19H21N3O5: C, 58.91; H, 5.46; N, 10.85.
Found: C, 58.95; H, 5.32; N, 10.84%.
15. (a) Uesugi, M.; Verdine, G. L. Proc. Natl. Acad. Sci.
USA 1999, 96, 14801–14806; (b) Bo¨ttger, A.; Bo¨ttger, V.;
Garcia-Echeverria, C.; Che`ne, P.; Hochkeppel, H.-K.;
Sampson, W.; Ang, K.; Howard, S. F.; Picksley, S. M.;
Lane, D. P. J. Mol. Bio. 1997, 269, 744–756; (c) Kussie,
P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau,
J.; Levine, A. J.; Pavletich, N. P. Science 1996, 274,
948–953.
16. (a) Kanovsky, M.; Raffo, A.; Drew, L.; Rosal, R.; Do,
T.; Friedman, F. K.; Rubinstein, P.; Visser, J.; Robinson,
R.; Brandt-Rauf, P. W.; Michl, J.; Fine, R. L.; Pincus,
M. R. Proc. Natl. Acad. Sci. USA 2001, 98, 12438–12443;
(b) Bo¨ttger, A.; Bo¨ttger, V.; Sparks, A.; Liu, W.-L.;
Howard, S. F.; Lane, D. P. Curr. Bio. 1997, 7, 860–869;
(c) Bo¨ttger, A.; Bo¨ttger, V.; Howard, S. F.; Picksley, S.
M.; Che`ne, P.; Garcia-Echeverria, C.; Hochkeppel, H.-
K.; Lane, D. P. Oncogene 1996, 13, 2141–2147; (d) Picks-
ley, S. M.; Vojtesek, B.; Sparks, A.; Lane, D. P. Oncogene
1994, 9, 2523–2529.
17. Standard Fmoc-Xaa-Wang solid-phase peptide coupling
procedures were used for the synthesis of 9–13. All
reactions were carried out on an 80 mM scale in 10 mL
polyethylene syringes fitted with a fritted polyethylene
disk. The peptides were cleaved from the resin using a
15:4:1 solution of TFA/DCM/H2O (8 mL). All peptides
were purified by preparative HPLC following standard
procedures. Peptide 9 was isolated after purification in
29% yield (31.1 mg). Anal. calcd for C68H93F9N12-
10. Compound 6 (1.5 g, 3.9 mmol) was dissolved in 50%
THF/CH3OH (50 mL) to which moist 5% Pd/C (0.50 g)
was added. The reaction vessel was sealed, evacuated
then back-filled with H2 at ambient pressure, and the
resulting mixture was stirred for 3 h. The resulting mix-
ture was filtered through a pad of Celite giving a reddish
solution. The solvent was removed in vacuo giving the
crude triamine as a viscous red oil. The crude residue was
dissolved in THF (10 mL), then added to a mixture of
TEA (2.2 mL, 16 mmol), NHS (0.91 g, 7.9 mmol) and
allyl chloroformate (0.56 mL, 4.3 mmol) that had been
stirred in THF for 1 h. The resulting mixture was stirred
for 4 h then diluted with EtOAc and washed with satu-
rated aq. NaHCO3 (2×) and brine. The resulting organic
solution was dried over MgSO4, filtered, and concen-
trated in vacuo giving a yellow oily residue. The residue
was dissolved in 33% EtOAc/hexanes then filtered
through a pad of silica-gel giving an amber oil. This
residue was crystallized from ether giving 7 as an off-
white solid (1.0 g, 85% yield). Mp=75–75.4°C. Proton,
carbon and IR spectra were consistent with the desired
compound. Anal. calcd for C15H21N3O4: C, 58.62; H,
6.89; N, 13.67. Found: C, 58.57; H, 6.86; N, 13.55%.
11. Wei, G. P.; Phillips, G. B. Tetrahedron Lett. 1998, 39,
179–182.
12. Diamine 7 (3.6 g, 12 mmol) was dissolved in dry THF (30
mL) followed by the addition of pyridine (7.5 mL, 93
mmol). The resulting solution was cooled to 0°C and a
20% solution of phosgene (6.1 mL, 12 mmol) in toluene
was slowly added by syringe. The resulting mixture was
stirred for 1 h. The reaction was then diluted with EtOAc
and washed with 1N HCl (2×) then brine. The resulting
organic solution was dried over MgSO4, filtered and
concentrated in vacuo giving a tan solid. The crude
product was crystallized from Et2O giving the desired
product as an off-white solid (2.7 g, 70% yield). Mp=
135–136°C. Proton, carbon and IR spectra were consis-
tent with the desired compound. Anal. calcd for
C16H19N3O5: C, 57.65; H, 5.75; N, 12.61. Found: C,
57.44; H, 5.57; N, 12.55%.
O23·4H2O: C, 47.59; H, 6.11; N, 10.09. Found: C, 47.72;
2+
H, 5.78; N, 10.05%. MS (ES) calcd for C60H92N12O17 :
m/z, 626.7. Found: m/z, 626.8 (100%). Peptide 10 was
isolated after purification in 38% yield (42.3 mg). Anal.
calcd for C68H94N11O23F9·2H2O: C, 49.78; H, 6.02; N,
9.39. Found: C, 49.62; H, 5.76; N, 9.53%. MS (ES) calcd
for C62H93N11O172+: m/z, 632.2. Found: m/z, 632.3
(100%). Peptide 11 was isolated after purification in 35%
yield (38.5 mg). Anal. calcd for C68H94F9N11O23·2H2O:
C, 49.78; H, 6.02; N, 9.39. Found: C, 49.77; H, 5.91; N,
9.59%. MS (ES) calcd for C62H93N11O172+: m/z, 632.2.
Found: m/z, 632.2 (100%). Peptide 12 was isolated after
purification in 25% yield (27.6 mg). Anal. calcd for
C65H92F9N13O24·2H2O: C, 47.42; H, 5.88; N, 11.05.
Found: C, 47.15; H, 5.68; N, 11.31%. MS (ES) calcd for
C59H91N13O182+: m/z, 635.2. Found: m/z, 635.3 (100%).
Peptide 13 was isolated after purification in 23% yield
(23.2 mg). Anal. calcd for C58H88F9N11O23·0.5H2O: C,
46.84; H, 6.03; N, 10.36. Found: C, 46.50; H, 6.11; N,
10.65%. MS (ES) calcd for C52H87N11O172+: m/z, 569.2.
Found: m/z, 569.2 (100%).
13. The methyl ester (2.4 g, 7.2 mmol) was suspended in 50%
aqueous MeOH (100 mL), to which NaOH (0.64 g, 16
mmol) was added. The resulting solution was stirred for
3 h at room temperature. The reaction mixture was dried
in vacuo giving a viscous residue. The crude residue was
partitioned between water and 50% EtOAc/Hex. The
aqueous phase was separated and acidified with 6N HCl.
The immediately formed white precipitate was collected
by filtration and washed with excess H2O and Et2O then
dried in vacuo, giving 8 as a white solid (2.0 g, 93%
yield). Mp=237.7–238°C (dec.). [h]2D7=−3.5 (c 0.25, 25%
AcOH/CH3OH). Proton, carbon and IR spectra were
consistent with the desired compound. Anal. calcd for
18. Recombinant HDM2 was expressed in E. coli utilizing
the GST gene fusion vector pGEX-4T-3 (Amersham
Pharmacia). A DNA fragment coding for amino acids
1–188 of HDM2 was generated by PCR amplification of
human colon cDNA (Clontec). This DNA fragment was
ligated into a pGEX-4T-3 vector modified by addition of
a DNA sequence coding for six histidine residues. The
resulting recombinant plasmid was transfected into E.