944
M. Marastoni et al. / Bioorg. Med. Chem. 9 (2001) 939±945
0
Fmoc-Phe- [CO±N(OH)]-Gly-Phe-NHtBu (10). HPLCK
Cell culture activity against HIV-1 IIIB
21
D
MeOH); H NMR (CDCl3) 1.25 (s, 9H); 3.13±3.22 (m,
5.55 (a), 7.03 (b); mp 151±154 ꢁC; ꢀ À13.9 (c 1.0,
1
HIV-1 IIIB was obtained from HIV-1 IIIB chronically
infected Molt-4 cells as a supernatant ¯uid. The 50%
tissue culture infection dose (TC ID50) was determined
by an endpoint titration procedure.35 CEM cells (5000/
mL) were exposed to HIV-1 IIIB ¯uid at a multiplicity
of infection (m.o.i.) 0.001 TC ID50 (mL). Aliquots
(0.2 mL) of cells were placed in 96well microtitre plates
with 2 mL of the appropriate concentrations of inhibi-
tors dissolved in DMSO. After incubation for 6days in
RPMI-1640 medium containing 10% fetal calf serum,
the p24 antigen of HIV in the supernatant was deter-
mined by an ELISA assay kit (RETRO-TEK, Cellular
Products Inc., Bualo, USA). The ED50 (50% dose)
values were calculated as the dose of the inhibitor that
resulted in a 50% reduction in p24 levels as compared to
those in control wells.
4H); 4.05 (s, 2H); 4.68±4.94 (m, 4H); 5.15 (s, 1H); 7.08±7.90
(m, 21H); 8.39 (sbr, 1H). MS (M+H) 662.8 (calcd 662.8).
0
Hmba-Phe- [CO±N(OH)]-Gly-Phe-NHtBu (11). HPLCK
21
D
MeOH); H NMR (CDCl3) 1.35 (s, 9H); 2.41 (s, 3H);
4.04 (a), 4.55 (b); mp 173±176 ꢁC; ꢀ À19.5 (c 1.0,
1
3.08±3.19 (m, 4H); 4.11 (s, 2H); 4.83±4.91 (m, 2H); 5.23
(s, 1H); 6.88±7.50 (m, 16H); 8.79 (sbr, 1H). MS (M+H)
574.7 (calcd 574.7).
0
Hmba- [CO±N(OH)]-Gly-Phe-NHtBu (12). HPLCK
21
D
MeOH); H NMR (CDCl3) 1.30 (s, 9H); 2.35 (s, 3H);
3.14 (a), 3.66 (b); mp 185±187 ꢁC; ꢀ À25.3 (c 1.0,
1
3.12 (dd, J=16.6 and 6.8 Hz, 2H); 4.07 (s, 2H); 4.85 (m,
1H); 5.23 (s, 1H); 6.93±7.48 (m, 10H); 8.69 (sbr, 1H).
MS (M+H) 427.5 (calcd 427.5).
Acknowledgements
Metabolic stability assay
The kinetics of new inhibitors degradation were studied
in culture medium (RPMI) and human plasma. 0.1 mL
of a solution of each compound (10 mg/mL in acetoni-
trile/H2O 1:1) was added to 1 mL of RPMI containing
20% fetal calf serum. Alternatively, test compound were
incubated with plasma (0.6mL) in a total volume of
1.5 mL of 10 mM Tris±HCl buer, pH 7.5. Incubation
were performed at 37 ꢁC for dierent times: up to
360 min in the case of human plasma and up to 4 days
in the case of RPMI containing 20% FCS. The incuba-
tion was terminated by addition of ethanol (0.2 mL), the
mixture poured at 21 ꢁC and after centrifugation
(5000 rpm for 10 min), aliquots (20 mL) of the clear
supernatant were injected into RP-HPLC column.
HPLC was performed as described above (see Experi-
mental procedures, general).
Financial support of this work by University of Ferrara
and by Ministero dell'Universita e della Ricerca
Scienti®ca
acknowledged.
e
Tecnologica (MURST) is gratefully
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protease (Bachem Bioscience), 1.1 nM ®nal concentra-
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