3800 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Brief Articles
Biologica l Eva lu a tion
We have identified three important features that
contribute to the enhancement of antiviral activity: (a)
the presence of the carboxylic acid at the C-3 position;
(b) the presence of a small polar group at the C-6
position and bulky substituents at the C-7 position, and
(c) the presence of small substituents at the N-1 position
of the quinolone moiety. As experimentally demon-
strated, there are some structural limitations at the N-1
and C-8 positions: bulky substituents are not well
tolerated at either N-1 and/or C-8, whereas no steric
restrictions are present around the C-7 position. RNA-
binding data confirm that the carboxylic function of
quinolones has to be free for efficient interaction. In
addition, to ensure a specific contact to TAR, appropri-
ate substituents must be located at positions 6 and 7 of
the quinolone moiety, as indicated by the lack of
response exhibited by 11a and ciprofloxacin. It is worth
observing that the latter two compounds are very poorly
active as anti-HIV-1 agents (Table 1), which supports
the hypothesis that RNA-quinolone interaction is
relevant to antiviral activity. Indeed, improved affinity
for the target seems to represent the basis for the
improved selectivity profile observed for 12a . In keeping
with this, preliminary data suggest that compounds
having a poorer SI (and activity), like 7a , also exhibit
reduced binding.
Since active 6-aminoquinolones are cytotoxic and
since the highest selectivity index obtained is 85, one
could assume that this class of molecules perturbs
physiological cellular functions or signals acting through
a nucleic acid intermediate as well. To address the issue
of antiviral versus cytotoxic activity, it will be now
crucial to further elucidate the mechanism of action of
the new quinolones at the molecular and biochemical
levels. In particular, the interactions of 6-aminoquino-
lones with other genomic viral sequences should be
investigated to assess their interference with the main
events of HIV-1 replication.
An tivir a l Activity. Anti-HIV-1 activity and toxicity
data, reported in Table 1, show that the most potent
drug is 7-[4-(2-pyridyl)-1-piperazinyl] derivative 12a ,
which bears a methyl group at the N-1 position. It
exhibits an EC50 in the submicromolar range, followed
by compounds 8a and 7a containing a tert-butyl and
cyclopropyl at the N-1 position, respectively. The corres-
ponding 6-fluoro congeners were 5-10-fold less potent
(compare 7a , 8a , and 12a vs 20a , 21a , and 22a ,
respectively). However, the N-1 substituent effect on
their relative activity was in the same order as that
found for 6-aminoquinolones: 22a (methyl) > 21a (cyclo-
propyl) > 20a (tert-butyl).
Alkylation of the C-6 amino group, as in the 6-meth-
ylamino derivative 14a , drastically decreased antiviral
response, pointing to the importance of an unsubstituted
amine at C-6. Comparison of compounds 12a and 16a
shows that the C-8 substituent is also crucial in terms
of interaction with the molecular target. Esterification
of the carboxyl group at the C-3 position dramatically
reduced antiviral activity and cytotoxic potential, thus
confirming the key role generally played by this portion
of the molecule in the biological activity of quinolones.
The test compounds appear to be selective for HIV,
since they were substantially less potent (the TC50
increases by almost 2 orders of magnitude) against an
HSV-1 wild-type strain, not withstanding their low level
of toxicity for Vero cells (Table 1).
In ter a ction w ith Vir a l Nu cleic Acid Sequ en ces.
Fluorometric titration experiments were carried out
with compounds 11a , 12a , and ciprofloxacin in the
presence of RNA and DNA sequences. The key results
are reported in Figure 1. Clearly, addition of TAR RNA
largely reduces the fluorescence quantum yield of 12a
(panel A), indicating the formation of a stable complex.
Conversely, derivative 11a was practically unaffected
by addition of the viral nucleic acid (panel B). The same
was true for a classical fluoroquinolone like ciprofloxacin
(not shown). Control experiments showed much lower
changes in fluorescence emission of 12a in the presence
of a TAR-unrelated t-RNA sequence or calf thymus
DNA, for both of which a slight increase, rather than a
decrease, in fluorescence emission is observed, even in
the presence of a 1000-fold excess of the nucleic acid
(panel C). Hence, specific recognition of the viral ribo-
nucleic acid depends on the structure of the test qui-
nolone and reflects HIV-1 inhibition properties. In
addition, complex formation between TAR and 12a is
quite effective, since we observed saturation of fluores-
cence emission at a nucleic acid concentration in the
submicromolar range.
Exp er im en ta l Section
An tivir a l a n d Cytotoxicity Assa ys. All compounds were
dissolved in dimethyl sulfoxide at 25 mM or higher concentra-
tion to exclude any cytotoxic effect of dimethyl sulfoxide after
dilution in the appropriate media. The anti-HIV-1 activities
and toxicities of compounds were assessed in C8166 with HIV-
1IIIB as described before.7 The anti-HSV-1 activity was
assayed in Vero cells by a plaque reduction method as
previously described.7 The inhibitory activity against HIV-1
was determined by examining cell cultures for syncytium
formation and by measuring reverse trancriptase (RT) activity
of culture supernatants.8 The antiviral activity was also
evaluated by measuring antigen gp120 production with an
ELISA method and by using a cytopathic effect reduction assay
as reported by Weinslow et al.9 The results shown in Table 1
represent the mean of at least three different experiments
performed in triplicate.
Nu cleic Acid s: TAR RNA. T7 polymerase was used for in
vitro transcription of pGEM7 zf(+) containing HIV-1 sequences
(HXBc2 strain) from nucleotide +454 to nucleotide +558,
where +1 represents the first nucleotide of the 5′-LTR.10 RNA
was precipitated by adding 1/10 volume of 3 M sodium acetate
and 2 volumes of ethanol at 0 °C.
t-RNA (from Wheat germ) and highly polymerized calf-
thymus DNA were purchased from Sigma Chemical Co. and
used following phenol extraction.
Discu ssion a n d Con clu sion s
The data presented here show that 6-aminoquinolones
represent useful novel leads for the development of new
and effective drugs for AIDS treatment. Indeed, 6-amino
derivatives appear to be more potent than the corres-
ponding fluorinated analogues as anti-HIV-1 agents.
Quite remarkably, 12a is the most potent compound of
the quinolone structural class so far described and also
the one with the most favorable selectivity index in
C8166 cells.
Sp ectr oscop ic Titr a tion s w ith RNA a n d DNA. Fluoro-
metric titrations were performed exploiting the drugs high
fluorescence yield upon excitation at 350 nm. Fluorescence
Cecchetti
