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References and notes
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1
7. All compounds were characterized by H NMR spectros-
copy and LC-MS analysis. Purities were assessed via
analytical RP-HPLC and are >95%.
8. The logD for compound 6 was measured to be 3.5 and we
did not anticipate problems with membrane permeability
for this class given the body of work cited in Ref. 5.
9. Efavirenz (1) activity under these conditions (MT-4 cells):
EC50: 0.7 nM, SI > 6000. In biochemical assays of RdDp
compound 1 was more than 200 times more active than
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13. Resistance development was performed with analog 6 in
MT-2 cells, which display a slightly improved SI when
compared to the MT-4 cell line. In short, HIV-1 IIIB was
propagated in MT-2 cells using standard procedures. In
vitro selection of HIV-1 IIIB with compound 6 com-
menced at 0.5· EC50 (0.4 lM). Virus was harvested when
the cytopathic effect was evident in the majority of cells,
marking the completion of a passage. The drug concen-
tration was held at 0.5· EC50 for the first two passages,
and subsequently was increased 2-fold, finally reaching
51.2 lM. Reverse transcriptase V106I was the first muta-
tion detected, by population sequencing of a viral pool
maintained at 3.2 lM (4· EC50) and cultured over a total
of 35 days. Clonal sequencing of the terminal passage
revealed the presence of V106I (21/25 clones, 84%) and
F227L (15/25 clones, 60%). Consistent with the X-ray
crystallographic structural data, K103N transcriptase was
also present in a minority of clones (4/25, 16%). Viral
pools from passage 7 (12.8 lM) and the terminal passage
(51.2 lM) both demonstrated >75-fold resistance to 6
when compared to wild-type, and 4- and 10-fold reduc-
tions in susceptibility to nevirapine, respectively. No
change in susceptibility to a control compound, the NRTI
tenofovir, was observed for either viral pool.
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