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tion was determined using previously described methods.13 The
RNase H, polymerase, and integrase strand-transfer biochemical
assays were conducted in the presence of 6 mM MgCl2. Potency
for inhibition of HIV-1 replication in HeLa P4-2 cells in a single-cy-
cle infectivity assay was determined as previously described.14
Infectious virus was produced by transfecting 293T cells with
HIV-1 proviral DNA isolated from an HXB2 strain. Cytotoxicity as-
says were conducted in HeLa P4-2 cells using the redox sensitive
indicator, Alamar Blue.15
Lead compound 1 had good potency in the RNase H cleavage as-
say (IC50 = 0.11 lM) and provided an attractive starting point for
optimization. Two magnesium-dependent viral enzymes, HIV-1
RT-polymerase and HIV-1 integrase, were used as selectivity
counterscreens. In these assays, 1 showed no measurable inhibi-
tors 12 and 13 showed a larger window between antiviral
efficacy and cellular toxicity (11- and 17-fold, respectively).
Compound 13 is twofold more potent than lead compound 1 in
the RNase H cleavage assay and 13-fold more potent than 1 in
the viral replication assay. The greater than proportional increase
in potency of 13 compared to 1 in the cellular assay suggests better
cell penetration of the former which may be related to its greater
lipophilicity (log P = 0.15 and ꢁ2.2 for 13 and 1, respectively).
Compound 13 did not exhibit a significant shift in antiviral potency
using virus pseudotyped with vesicular stomatitis virus glycopro-
tein (fold shift in IC50 value was 0.9), suggesting that the antiviral
activity of 13 is not related to inhibition of viral entry into the cell.
Compound 13 also did not show significant shifts in antiviral po-
tency using viruses containing mutations in the integrase enzyme
that confer resistance to several structural classes of integrase
inhibitors (fold shift in IC50 values for the N155H, Q148R, and
Y143R integrase mutants were 1.1, 1.9, and 0.84, respectively),
suggesting that the antiviral potency of 13 is not related to inhibi-
tion of integrase. In a multi-cycle viral replication assay in MT-4
cells,16 none of the compounds in Table 1 exhibited a significant
window between antiviral efficacy and cellular toxicity, for exam-
tion of the polymerase activity of RT (IC50 >50
inhibition of integrase strand-transfer activity (IC50 = 0.9
Compound 1 inhibited viral replication in a single-cycle infectivity
assay in HeLa P4-2 cells (IC50 = 2.5 M) and did not display cyto-
toxicity in these cells up to a maximum concentration of 50
lM) and modest
lM).
l
lM
as assessed using an Alamar Blue assay. Because of the highly polar
nature of 1 (log P = ꢁ2.2) and possible detrimental effect this might
have on cell penetration, one optimization strategy involved pur-
suit of analogs with greater lipophilicity. Enzyme kinetics studies
with 1 indicated that the inhibitor binds to an enzyme–substrate
or enzyme–product complex.9a A crystal structure of 1 with full
length RT9b showed the inhibitor binding to active site metals with
an open space above the inhibitor. From this we hypothesized that
substituents at positions 4 or 5 of the naphthyridinone core could
potentially access other parts of the enzyme or nucleic acid sub-
strate or product that would presumably be nearby. Toward this
end, triflate 4 (Scheme 1) served as an ideal intermediate for pre-
paring a variety of analogs containing lipophilic groups attached
at the 4-position of the naphthyridinone core. Structure–activity
results for several of these analogs are presented in Table 1.
The unsubstituted core 7 had modest potency in the RNase H
cleavage assay with >30-fold selectivity versus RT-Pol and ꢀ10-
fold selectivity versus integrase. Compound 7 also inhibited viral
ple, 13 had an antiviral IC95 value of 0.92
lM and the first observa-
tion of cellular toxicity was at a concentration of 1.5
l
M.
The aminomethylbiphenylmethyl group in 13 provided a 30-
fold increase in potency in the RNase H cleavage assay compared
to the unsubstituted analog 7. The exact role of the amin-
omethylbiphenyl motif for improving potency in the RNase H
cleavage assay is not known. Crystal soaking experiments using
conditions that provided density for 1 in full length RT were unsuc-
cessful using 13. Molecular dynamics simulations of inhibitor–
enzyme–substrate and inhibitor–enzyme–product complexes with
13 did not provide conclusive results; interactions of the biaryl
moiety with nucleic acid substrate/product or with protein were
possible.
In summary, 1-hydroxy-1,8-naphthyridin-2(1H)-one 1, an
HIV-1 RT RNase H active site inhibitor, was used as a starting point
for optimization. A series of more lipophilic analogs containing a
biaryl motif attached at the 4-position of the naphthyridinone core
was prepared. An optimal analog in this series, 13, was found to be
a potent RNase H inhibitor in biochemical assays (IC50 = 45 nM)
with good selectivity for inhibiting RNase H versus two other mag-
nesium-dependent enzymes, HIV RT-polymerase and HIV integr-
replication with an IC50 value of 4.5
concentration, there was evidence of cytotoxicity (Alamar Blue
CC50 = 20 M). Potency for inhibiting RNase H in the cleavage assay
l
M, but at ꢀfourfold higher
l
improved with the introduction of a meta-biphenyl group (8), how-
ever there was a proportionately greater increase in activity in the
RT-polymerase and integrase assays. The limited solubility of 8
made it difficult to accurately assess potency in the viral replica-
tion assay, and therefore more water soluble analogs of 8 were
sought. Introduction of an aminomethyl group on the distal phenyl
ring provided analogs with improved physical properties and inter-
esting structure–activity relationships. The ortho-aminomethyl
biphenyl analog 9 was equipotent to 8 in the RNase H cleavage as-
say, however improved selectivity versus both RT-polymerase and
integrase was obtained, as well as improved potency in the viral
replication assay. Further improvements were realized with the
meta- and para-aminomethyl analogs 10 and 11, respectively.
Homologation of 11 to the corresponding benzyl linked analog
12 produced little change in any of the assays. However, the para,
para analog 13 showed improvements in all assays. Compound 13
had good potency in the RNase H cleavage assay (IC50 = 45 nM),
selectivity versus both RT-polymerase and integrase (290- and
530-fold, respectively), and good potency in the single-cycle viral
replication assay (IC50 = 190 nM) with an improved window versus
cytotoxicity (17-fold). For the series of aminomethyl compounds
9–13, the antiviral potency tracked well with the potency in the
RNase H cleavage assay. The cytotoxic effects of 9–13 remained
ase (IC50 = 13 lM and 24 lM, respectively). In a single-cycle viral
replication assay in HeLa P4-2 cells, 13 inhibited viral growth at
concentrations 17-fold lower than its cytotoxic concentration
(antiviral IC50 = 0.19 lM; CC50 = 3.3 lM). Molecular dynamics sim-
ulations indicated that the biaryl group in 13 can interact with pro-
tein or nucleic acid substrate or product.
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relatively constant with CC50 values in the range of 2–9 lM. For
compounds 9–11, the window between antiviral IC50 and cytotox-
icity CC50 was small (3–5-fold). The more potent RNase H inhibi-