A 1,8-naphthyridone derivative targets the HIV-1 Tat-mediated transcription and potently inhibits the HIV-1 replication

Article abstract of DOI:10.1021/jm901211d

The emergence of multidrug resistant HIV-1 strains and the inability of the HAART to eradicate HIV-1 virus from infected patients demand new drugs able to interfere with an alternative step of the replicative cycle. The naphthyridone 3 (HM13N), described in the present study, is a promising anti-HIV agent due to its ability to inhibit the HIV-1 Tat-mediated transcription and the potent antiviral activity observed in acutely, chronically, and latently infected cells. The absence of any tendency to select for resistance mutations in vitro adds to the potential clinical value of this type of compounds, especially as these compounds are drug-like and obey the Lipinski rules.

Full text of DOI:10.1021/jm901211d

J. Med. Chem. 2010, 53, 641–648 641
DOI: 10.1021/jm901211d
A 1,8-Naphthyridone Derivative Targets the HIV-1 Tat-Mediated Transcription and Potently Inhibits
the HIV-1 Replication
Serena Massari,† Dirk Daelemans,‡ Maria Letizia Barreca,† Anna Knezevich,§ Stefano Sabatini,† Violetta Cecchetti,†
Alessandro Marcello,§ Christophe Pannecouque,*,‡ and Oriana Tabarrini*,†
†Dipartimento di Chimica e Tecnologia del Farmaco, Universitaꢀ di Perugia, Via del Liceo 1, 06123 Perugia, Italy,
‡Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, and §International Centre for
Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy
Received August 14, 2009
The emergence of multidrug resistant HIV-1 strains and the inability of the HAART to eradicate HIV-1
virus from infected patients demand new drugs able to interfere with an alternative step of the replicative
cycle. The naphthyridone 3 (HM13N), described in the present study, is a promising anti-HIV agent due
to its ability to inhibit the HIV-1 Tat-mediated transcription and the potent antiviral activity observed in
acutely, chronically, and latently infected cells. The absence of any tendency to select for resistance
mutations in vitro adds to the potential clinical value of this type of compounds, especially as these
compounds are drug-like and obey the Lipinski rules.
Introduction
Quinolone derivatives, whose potent antiviral activity is
related to the inhibition of the Tat-mediated HIV-1 tran-
scription,4-8 are a class of molecules which may contribute
to the control of the latent reservoirs. Other different classes of
compounds were found to inhibit the HIV-1 transcription,9-14
but unfortunately, none of them have still led to a successful
clinical development. Inhibitors of the HIV-1 transcriptional
regulation may have great potential in anti-HIV-1 drug com-
bination therapy because they can slow down the viral replica-
tion rate or even shut it off.9 The potent in vitro antiviral ac-
tivity of these quinolones in many acutely, latently, and
chronically HIV-1 infected cells, was recently confirmed in
an in vivo model for HIV-1 latency where 6-hydrogenquino-
lone derivatives prevented virus reactivation from a human
viral reservoir in mice.6,15
In an ongoing effort to develop analogues combining
optimal antiviral and limited cytotoxic properties for clinical
application, we have found that the bioisosteric replacement
of the quinolone nucleus by an 1,8-naphthyridone is beneficial
when a suitable arrangement of the functional groups is
present at the C-6, N-1, and C-7 positions. Here we report
the synthesis, the anti-HIV activity, and the mechanism of
actionstudy ofthe firstseries of1,8-naphthyridone derivatives
1-4 characterized by a C-6 hydrogen atom and a N-1 methyl
group coupled with a few selected 4-arylpiperazines at the C-7
position (Figure 1).
The recent approval of the entry inhibitor maraviroc1 and
the first integrase inhibitor raltegravir,2 which differ from
existing agents with regard to their mechanism of action and
resistance profiles, could revitalize the treatment regimens of
multidrug-resistant HIV-1 patients. However, previous ex-
perience with the currently used anti-HIV-1 agents would
presage an inevitable emergence of HIV-1 resistant strains
against these new drugs once they are widely used. Resistance
selection is expected to occur with any newly developed agent,
particularly when it is targeting a viral protein. While the
addition of these new drugs to the highly active antiretroviral
therapy (HAARTa) will allow a better management of the
HIV-1 infection, it is unlikely that the virus in HIV-1-infected
individuals will be completely eradicated using this new drug
regimen. Some major obstacles to the eradication of HIV-1
are the occurrence of latent reservoirs, mainly in the resting
CD4þ lymphocytes and monocyte/macrophages (M/M),
along with the residual low-level virus production that con-
tinues even in patientson optimal therapy. Thesecells carry an
integrated provirus that is transcriptionally silent but will
produce infectious particles after cellular activation that
will lead to a rebound of the viral load after HAART is
interrupted.3
*To whom correspondence should be addressed. For O.T.: phone, þ
39 075 585 5139; fax, þ39 075 585 5115; e-mail, oriana.tabarrini@unipg.
it. For C.P.: phone, þ32 016 332171; fax, þ32 016 332131; e-mail,
christophe.pannecouque@rega.kuleuven.be.
Chemistry
The 1,8-naphthyridones 1-4 were prepared through a
cycloaracylation procedure, as shown in Scheme 1. Acrylate
a
Abbreviations: HAART, highly active antiretroviral therapy; HIV,
5
16 was reacted with methylamine in Et2O/EtOH mixture at
human immunodeficiency virus; IFD, induced fit docking; INSTI;
strand transfer integrase inhibitor; LTR, long terminal repeat; CDK,
cyclin-dependent kinase; CTD, carboxy-terminal domain; M/M, human
primary monocyte/macrophages; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; PBMCs, peripheral blood mononuc-
lear cells; PMA, phorbol 12-myristate 13-acetate; P-TEFb, positive
transcription elongation factor b; TAR, transactivating region; TNF-
R, tumor necrosis factor alpha; TOA, time of addition.
room temperature to give intermediate 6, which was then
cyclized to synthone 7 by using K2CO3 in DMF at 60 °C. This
key intermediate was then reacted with selected 4-arylpiper-
azines to generate compounds 8-11, which were hydrolyzed
in basic condition furnishing target acids 1-4.
r
2009 American Chemical Society
Published on Web 12/03/2009
pubs.acs.org/jmc

642 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Massari et al.
Table 2. Anti-HIV-1 Activity and Cytotoxicity of 3 on Jurkat Cells and
PBMCs
Jurkat
PBMCs
EC50
(μg/mL)a,e
CC50
(μg/mL)b,e
EC50
(μg/mL)c,e
CC50
(μg/mL)d,e
SIf
44
SIf
17
0.34
15.13
0.60 ( 0.49
10.31 ( 3.09
aEC50: concentration required to inhibit 50% of the level of GFP
expression obtained in absence of compound. CC50: concentration of
b
compound that reduces the viability of mock-infected cells by 50%, as
determined by the MTT method. cEC50: concentration of compound
required to achieve 50% inhibition of p24 production in PBMC. dCC50
:
Figure 1. Structure of 1,8-naphthyridones synthesized in this
study.
concentration of compound that reduces the viability of mock-infected
cells by 50%, as determined by the trypan blue exclusion method. eAll
data represent mean values ( standard deviations for at least two
Scheme 1a
separate experiments. fSI: ratio of CC50/EC50
.
a Reagents and conditions: (i) MeNH2, Et2O/EtOH; (ii) K2CO3,
DMF, 60 °C; (iii) Ar-piperazine (for Ar, see Figure 1), DMF, 70 °C;
(iii) 4% NaOH, reflux.
Figure 2. Time of addition experiment. MT-4 cells were infected
with HIV-1 (IIIB) at an moi of 0.5, and the test compounds were
added at different times postinfection. Viral p24 production was
determined at 31 h p.i. and is expressed in pg/mL.
Table 1. Anti-HIV-1 and HIV-2 Activity and Cytotoxicity of
1,8-Naphthyridone Derivatives on MT-4 Cells
HIV-1(IIIB)
HIV-2(ROD)
CC50
SI
SI
compd EC50(μg/mL)a,c EC50 (μg/mL)a,c (μg/mL)b,c (IIIB)d (ROD)d
corresponding previously reported 6-desfluoroquinolone
series,5,8 when placed in the naphtyridone nucleus, the cyto-
toxicity also markedly decreased resulting in a double benefit.
The presence of the other C-7 side chains yielded divergent
results. Good activity and encouraging SI values were ob-
tained by introducing the 1-(2-pyridinyl)piperazine moiety
(compound 1), whereas the 1-[3-(trifluoromethyl)phenyl]-
piperazine moiety (compound 2) resulted in a compound that
was cytotoxic and devoid of any antiviral activity at lower
concentrations. Finally, the absence of cytotoxicity, coupled
to a good anti-HIV-2 activity, was achieved with the intro-
duction of a 1-(1,3-thiazolyl)piperazine, as in compound 4.
To better define the antiviral profile of naphthyridone
derivative 3, its anti-HIV-1 activity was also assayed on
acutely infected Jurkat cells and in human PBMC (isolated
from healthy HIV-1 seronegative donors). Compound 3 was
about 10 times less active in these cells as compared to MT-4
cells but maintained the same low cytotoxicity (Table 2).
It has previously been reported that quinolones inhibit the
HIV-1 replication by interfering with a postintegrational
target of the HIV-1 replicative cycle.17 Therefore, we aimed
at pinpointing the target of action of our new naphthyridone
3. To do so, time-of-addition (TOA) experiments were per-
formed and the results were compared to reference com-
pounds with a known mode of action. In contrast to the
reference inhibitors, dextran sulfate 8000, AZT, and ritonavir
for which addition can be delayed until about 0, 5, and
approximately 18 h postinfection, addition of compound 3
could be postponed up to 10 h post infection before it lost its
antiviral activity (Figure 2). In this experiment, the last time-
point at which diketo acid strand transfer integrase inhibitors
(INSTIs), that inhibit the very last step of the integration
process, can be added for optimal antiviral activity, is 7-8 h
postinfection.18 Because addition of 3 can be postponed even
1
2
3
4
0.62 ( 0.19
>0.41
0.55 ( 0.42
>0.41
9.93 ( 0.94
0.41 ( 0.04 <1
12.06 ( 3.11 457
16
18
<1
522
0.03 ( 0.00
3.15 ( 0.54
0.02 ( 0.00
0.922 ( 0.322
>125
>40 >136
a EC50: concentration of compound required to achieve 50% protec-
tion of MT-4 cells from HIV induced cytopathogenicity, as determined
b
by the MTT method. CC50: concentration of compound that reduces
the viability of mock-infected cells by 50%, as determined by the MTT
c
method. All data represent mean values ( standard deviations for at
least two separate experiments. dSI: ratio of CC50/EC50
.
Results and Discussion
The poor solubility in aqueous medium is a typical draw-
back that sometimes hampers the biological evaluation of
quinolones as anti-HIV agents. We therefore aimed at repla-
cing the benzo moiety in our compounds with a pyrido
moiety. As expected, the bioisosteric substitution of the
phenyl ring with a pyridine increased the solubility of the
new derivatives.
The newly synthesized compounds were initially evaluated
for their anti-HIV-1 (IIIB) and anti-HIV-2 (ROD) activity in
MT-4cellsand cytotoxicity of the compounds was determined
in parallel. The results reported in Table 1 show that the
naphthyridone nucleus allowed a very potent and selective
derivative to be obtained when a 1-(1,3-benzothiazol-2-
yl)piperazine is present at the C-7 position. In fact, derivative
3 (HM13N) inhibited both HIV-1 and HIV-2 replication with
EC50 values of 0.03 and 0.02 μg/mL, yielding selectivity
indexes of 457 and 522, respectively. In MT-4 cells, these
values are the best obtained to date among the HIV-inhibitory
quinolone class oftranscriptioninhibitors. The 1,3-benzothia-
zole is confirmed as an optimal aryl substituent at posi-
tion N-4 of the piperazine ring in giving high anti-HIV
potency. Moreover, in contrast to what was observed in the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 643
Figure 3. HIV-1 LTR transactivation assay. (A) HeLa LTR-luc were activated with Tat at suboptimal doses and treated with increasing
concentration of compound 3. Luciferase assay was performed on cell lysates 24 h after treatment. DMSO, control with compound solubilizing
agent. Mock, cells not treated. FVP, flavopiridol at 10 μM. (B) HeLa LTR-luc were treated without Tat treatment to measure the basal activity
of the promoter. Assay performed like in Figure 3A.
beyond 8 h before loss of activity, it is obvious that 3 acts at a
postintegrational level, pointing toward a mechanism of ac-
tion coinciding with the viral transcription process. Indeed, in
a Tat-mediated transcription assay, performed in HeLa cell
clone carrying an LTR-driven luciferase reporter, compound
3 inhibited the transcription in a dose-dependent manner in
the μM range (Figure 3A); cytotoxicity was not observed in
the same range (not shown). The inhibition is restricted to the
Tat-mediated transcription as shown by the lack of activity in
the basal expression (Figure 3B).
compound appears to be too rigid to simultaneously allow the
metal chelation and the occupation of the pocket defined by
the catalytic loop and the viral DNA (Figure 5). The lack of
such a strong and critical interaction may explain why the
compound 3 cannot inhibit HIV-1 integrase.
Unlike the compounds that target an early event of the
HIV-1 replication cycle (viral entry, reverse transcription, or
integration), transcription inhibitors are expected to inhibit
the viralproductionofchronicallyand latently infectedcellsin
which HIV-1 proviral DNA has been integrated into the host-
cell genomes.
In highlighting the structural features of naphthyridone 3,
one notices that this compound displays striking similar
features to those of diketo acid-based integrase inhibitors19
and in particular to that of quinolone derivative GS-9137
(elvitegravir).20 Thus, to undeniably ascertain that the anti-
HIV activity of compound 3 is mainly based on the Tat-
mediated transcription inhibition and to rule out any anti-
integrase activity, the effect of our compound on the prolif-
eration of wild-type versus integrase deficient HIV-121 was
studied (Figure 4). Compound 3 efficiently inhibited the
replication of the wild-type NL4.3 as well as the integrase
deficient N/NTag.oriT virus, while the well-characterized
integrase inhibitor L-870,81022 was inactive against the inte-
grase deficient virus. These data clearly demonstrate that
compound 3 does not inhibit the HIV-1 integration process.
An in-depth theoretical investigation of the interaction
between INSTIs and an HIV-1 IN-Mg-DNA model has
recently been carried out.23 This study provided new insights
into the possible mechanism of action and binding conforma-
tions of INSTIs. In particular, induced fit docking (IFD)
solutions revealed that all the inhibitors (a) have a pharma-
cophore that can establish interactions with one/two Mg2þ
ions, (b) interact with the donor DNA, and (c) can occupy a
deep region defined by the catalytic loop and the viral DNA
through their substituted aromatic tail. The same IFD proto-
col has now been applied to naphthyridone 3. Only one pose
came out from the IFD study, showing one-metal binding
model. (Figure S1 and relative comments in Supporting
Information). A comparison between the IFD conforma-
tions obtained for known INSTIs and 3 highlights that our
Therefore, compound 3 was first evaluated on persistently
infected HuT-78(IIIB) cells. These cells continuously pro-
duced HIV-1(IIIB) from the integrated proviral DNA. After
having removed the already-produced viruses, the cells were
exposed to a serial dilution of the compound and the quantity
of newly produced virus in the presence of the inhibitor is
compared with a control condition. As shown in Table 3,
compound 3 had a pronounced inhibitory effect on the virus
production from persistently infected HuT-78(IIIB) cells, far
below the cytotoxicity level, and comparable to that observed
in MT-4 cells (SI values >313). Latently HIV-1 infected
promyelocytic cells (OM-10.1) and promonocytic cells (U1),
in which the virus remains in a dormant state, were then used.
In contrast to the HuT-78(IIIB) cells, these cell lines have to be
stimulated in order to produce virus. Upon exposure to TNF-
R or PMA, a dramatic (∼1000-fold) increase in HIV-1
expression could be detected. As the concentrations of com-
pound 3 were increased, a dose-dependent inhibition of p24
production in both HIV-1 infected cell lines was observed
(Table 3). This inhibition took place at compound concentra-
tion levels that were far below the cytotoxicity level and was
similar under both (TNF-R and PMA) experimental stimula-
tion conditions. In this experimental setup, compound 3
showed very strong antiviral activities with IC50 values in
the 0.002-0.004 μg/mL range; these values were 10 times
lower thanthose observedonMT-4 andthe SI valueof1333 in
U1 cells stimulated with PMA.
Encouraged by the promising inhibitory activity on HIV-
production, we engaged in developing resistance to this new

644 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Massari et al.
Figure 5. Comparison among IFD conformations of L-870,810
(orange), elvitegravir (black), and 3 (white). Molecular surfaces
are shown for IN (gray), catalytic loop (residues 140-149; cyan),
metal ions (magenta), 30 DNA strand (green), and 50 DNA strand
(yellow). This figure was prepared using PyMol.41
Table 3. Antiviral Activity and Cytotoxicity of 3 in Chronically (HuT-
78) and Latently (U1 and OM10.1) HIV-1 Infected Cells
EC50
(μg/mL)a,c
CC50
(μg/mL)b,c
cell line
stimulator
SId
HuT-78(IIIB)
U1
none
TNF-R
PMA
0.032
0.003
0.002
0.003
0.004
>10
0.99
2.67
1.27
0.75
>313
330
1333
422
OM10.1
TNF-R
PMA
189
aEC50: concentration of compound required to achieve 50% reduc-
tion of p24 production in HIV-1 infected cells. bCC50: concentration of
compound that reduces the viability of cells by 50%, as determined by
the MTT method. cAll data represent mean values ( standard deviations
for at least three separate experiments. dSI: ratio of CC50/EC50
.
Table 4. Sensitivity of Mutant HIV-1 (IIIB) Strains towards 3
selected straine
Figure 4. Concentration-dependent anti-HIV-1 effects of nevira-
pine, L-870,810 and compound 3. C8166 T-cells infected with HIV-
1 NL4.3 (red circles) or N/N.Tag.oriT (blue squares) were treated
with the indicated compound concentrations. The definition of
100% is the level of p24 in the supernatants in control, nondrug-
treated samples. Toxicity of the compounds was measured using the
MTT-method (green triangles).
IIIB
EC50
(μg/mL)a,c
CC50
(μg/mL)b,c
EC50
(μg/mL)a,c
fold
resistance
SId
SId
0.019
24.7
1280
0.017
1419
0.9
a EC50: concentration of compound required to achieve 50% protec-
tion of MT-4 cells from HIV induced cytopathogenicity, as determined
by the MTT method. b CC50: concentration of compound that reduces
the viability of mock-infected cells by 50%, as determined by the MTT
method. c All data represent mean values ( standard deviations for at
compound. MT-4 cells infected with HIV-1 (IIIB) were sub-
jected to serial passages (i.e., every third or fourth day of
cultivation) in the presence of different concentrations of
naphthyridone 3. After 60 passages (30 weeks of selective
pressure), we were unsuccessful in selecting resistance against
the naphthyridone derivative 3 (Table 4).
Stimulated by the interesting profile of 3, an in-depth
investigation of its mechanism of action at the molecular level
was carried out. The fishing of the target of anti-HIV quino-
lones is of great interest because once found it would provide
the structural basis for the discovery of improved transcrip-
tion inhibitors.
As previously reported, WM5,24 the lead of the 6-desfluor-
oquinolones, interferes with the formation of the Tat/TAR
complex by selectively binding to the bulge of TAR. The same
effect was not seen with other potent 6-desfluoroquinolone
analogues, some of which bind the TAR RNA in a nonselec-
tive way,25 while others do not recognize any nucleic acids
least two separate experiments. d SI: ratio of CC50/EC50 e HIV strain
.
that emerged upon prolonged exposure to compound 3.
(unpublished data). Consequently, the inhibitory activity of
this class of compounds is not restricted to the inhibition of
Tat/TAR complex formation but can also involve other
cellular factors that are implicated in the Tat-mediated tran-
scription.
In the present study, the effect of 3 on two key steps of
HIV-1 transcriptional transactivation3 that involve either the
recruitment of the positive transcription elongation factor b
(P-TEFb) or the recruitment of the coactivator p300 were
investigated. P-TEFb, composed of Cyclin T1 and CDK9 can
phosphorylate the carboxy-terminal domain (CTD) of RNA
polymerase II promoting transcriptional elongation. The
p300 and highly homologous cyclic adenosine monopho-
sphate (cAMP)-responsive binding protein (CREB)-binding

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 645
protein (CBP) are acetyltransferases that act on histones to
relieve the chromatin-imposed inhibition on the viral promo-
ter, and on other proteins, including Tat itself.26 Compound 3
did not show any effect on the kinase activity of CDK9
(measured in vitro on a recombinant CTD substrate) or on
the acetyltransferase activity of p300/CBP on histones (not
shown). It is concluded that 3 may act on a different step in the
transactivation reaction and further experiments will be re-
quired to address this question.
reaction was concentrated in vacuo, obtaining a residue which
was treated with cyclohexane, to give a solid which was filtered
and dried to give 6 (0.9 g, 94%): mp 156-157 °C. H NMR
1
(DMSO-d6) δ 0.75 and 0.95 (each t, J = 7.1 Hz, 3H, CH2CH3),
3.15-3.20 (m, 3H, CH3), 3.85-3.95 (m, 2H, CH2CH3), 7.55 (d,
J = 7.9 Hz, 1H, aromatic CH), 7.75 and 7.78 (each d, J = 7.9
Hz, 1H, aromatic CH), 8.20 and 8.25 (each d, J = 15.0 Hz, 1H,
CHNH), 9.50-9.55 and 10.70-10.75 (each m, 1H, NH).
Ethyl
7-Chloro-1-methyl-4-oxo-1,4-dihydro-1,8-naphthyri-
dine-3-carboxylate (7). A mixture of acrylate 6 (0.9 g, 2.96 mmol)
and K2CO3 (1.22 g, 8.90 mmol) in DMF (7 mL) was heated at
60 °C for 2 h. After cooling the reaction mixture was poured into
ice/water, obtaining a precipitate which was filtered, washed
with water, and Et2O and dried to give 7 (0.67 g, 85%): mp
199-200 °C. 1H NMR (CDCl3) δ 1.40 (t, J = 7.1 Hz, 3H,
CH2CH3), 3.95 (s, 3H, CH3) 4.40 (q, J = 7.1 Hz, 2H, CH2CH3),
7.40 (d, J = 8.2 Hz, 1H, H-6), 8.60 (s, 1H, H-2), 8.70 (d, J = 8.2
Hz, 1H, H-5).
General Procedure for Coupling Reaction. Ethyl 1-Methyl-4-
oxo-7-(4-pyridin-2-ylpiperazin-1-yl)-1,4-dihydro-1,8-naphthyr-
idine-3-carboxylate (8). A mixture of 7 (0.1 g, 0.37 mmol) and
1-(2-pyridinyl)piperazine (0.18 g, 1.12 mmol) in DMF (3 mL),
was heated at 70 °C for 4 h. The reaction mixture was then
poured into ice/water giving a precipitate which was filtered, and
washed with water and EtOH, to give 8 (0.14 g, 98%): mp
169-170 °C. 1H NMR (DMSO-d6) δ 1.25 (t, J = 7.1 Hz, 3H,
CH2CH3), 3.70-3.75 (m, 4H, piperazine CH2), 3.80-3.90 (m,
7H, piperazine CH2 and CH3), 4.25 (q, J = 7.1 Hz, 2H, CH2-
CH3), 6.75 (t, J = 5.9 Hz, 1H, pyridine CH), 6.90 (d, J = 8.4 Hz,
1H, pyridine CH), 7.10 (d, J = 9.0 Hz, 1H, H-6), 7.60 (t, J = 7.4
Hz, 1H, pyridine CH), 8.20-8.30 (m, 2H, H-5 and pyridine CH),
8.65 (s, 1H, H-2).
General Procedure for Hydrolysis Reaction. 1-Methyl-4-oxo-
7-(4-pyridin-2-ylpiperazin-1-yl)-1,4-dihydro-1,8-naphthyridine-
3-carboxylic Acid (1). A suspension of 8 (0.1 g, 0.25 mmol) in 4%
NaOH (2 mL) was refluxed for 4 h. After cooling, the mixture
reaction was treated with 2 N HCl to obtain a precipitate
(∼pH = 6). The solid was filtered and washed with water and
EtOH, to give 1 (0.08 g, 90%): mp 270-271 °C. 1H NMR
(DMSO-d6) δ 3.80-3.85 (m, 4H, piperazine CH2), 3.90 (s, 3H,
CH3), 3.95-4.00 (m, 4H, piperazine CH2), 6.80 (t, J = 5.4 Hz,
1H, pyridine CH), 7.10-7.20 (m, 2H, H-6 and pyridine CH),
7.75-7.85 (m, 1H, pyridine CH), 8.10 (d, J = 5.3 Hz, 1H,
pyridine CH), 8.30 (d, J = 9.0 Hz, 1H, H-5), 8.95 (s, 1H, H-2),
15.80 (bs, 1H, COOH). Anal. (C19H19N5O3) C, H, N.
Conclusions
Remarkable progress has been made in the treatment of
HIV-1 infection/AIDS. However, critical problems such as
the emergence of resistant strains and the inability of the
HAART to eradicate the virus necessitate the identification of
new compounds that can interfere with an alternative step of
the replicative cycle. In this context, quinolone derivatives are
very promising due to their ability to inhibit the Tat-mediated
transcription. The results of the present work have shown that
the bioisosteric substitution of the quinolone ring with the
1,8-naphthyridone was advantageous when a 1-(1,3-benzo-
thiazol-2-yl)piperazine is present at the C-7 position. In addi-
tion to the improved solubility, compound 3 displayed very
potent and selective anti-HIV activity in acutely, chronically,
and latently infected cells. It did not show any tendency to
select for resistance mutations after 60 passages. Mechanism
of action studies, demonstrate that the anti-HIV activity of
naphthyridone 3 is mainly due to the inhibition of the Tat-
mediated transcription. In silico studies give some indications
why naphthyridone 3 cannot efficiently interact with integrase
enzyme.
In conclusion, the quinolone structure is confirmed as
“privileged” to obtain potent anti-HIV agents, capable of
inhibiting different steps of the viral replicative cycle such as
the integration (i.e., GS-9137) or transactivation (i.e., com-
pound 3) based on the different quinolone nucleus substitu-
tion pattern.
Taking into account the promising anti-HIV results and
drug-like nature of the compounds, it is certainly worthwhile
to spend efforts on establishing the activity of compound 3 in
the SCID-mice model.
1-Methyl-4-oxo-7-{4-[3-(trifluoromethyl)phenyl]piperazin-1-
yl}-1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid (2). The ti-
tle compound was prepared through the general procedure for
coupling reaction replacing 1-(2-pyridinyl)piperazine with
1,3-(trifluoromethyl)phenyl]piperazine to give intermediate 9 in
78% yield, followed by the general procedure for hydrolysis to
Experimental Section
All reactions were routinely checked by thin-layer chromatog-
raphy (TLC) on silica gel 60F254 (Merck) and visualized by using
UV. Flash column chromatography separations were carried out
on Merck silica gel 60 (mesh 230-400). Melting points were
1
give 2 in 70% yield: mp 279-281 °C. H NMR (DMSO-d6) δ
€
determined in capillary tubes (Buchi Electrothermal model 9100)
3.35-3.45 (m, 4H, piperazine CH2), 3.85 (s, 3H, CH3), 3.90-4.00
(m, 4H, piperazine CH2), 6.90-7.00 and 7.05-7.10 (m, each 1H,
aromatic CH), 7.20-7.30 (m, 2H, H-6 and aromatic CH),
7.40-7.50 (m, 1H, aromatic CH), 8.25 (d, J = 9.0 Hz, 1H,
H-5), 8.60 (s, 1H, H-2). Anal. (C21H19F3N4O3) C, H, N.
and are uncorrected. Elemental analyses were performed on a
Fisons elemental analyzer, model EA1108CHN, and the data for
C, H, and N are within (0.4% of the theoretical values. 1H NMR
spectra were recorded at 200 MHz (Bruker DPX 200) using
residual solvents such as chloroform (δ = 7.26) or dimethylsulf-
oxide (δ=2.48) asaninternalstandard. Chemical shiftsaregiven
in ppm (δ), and the spectral data are consistent with the assigned
structures. Reagents and solvents were purchased from common
commercial suppliers and were used as such. Organic solutions
were dried over anhydrous Na2SO4, filtered, and concentrated
7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-methyl-4-oxo-
1,4-dihydro-1,8-naphthyridine-3-carboxylic Acid Hydrochloride
(3). The title compound was prepared through the general
procedure for coupling reaction replacing 1-(2-pyridinyl)-
piperazine with 1-(1,3-benzothiazol-2-yl)piperazine27 to give
intermediate 10 in 53% yield, followed by the general procedure
for hydrolysis to give 3 in 99% yield: mp 338-339 °C. 1H NMR
(DMSO-d6) δ 3.65-3.75 (m, 4H, piperazine CH2), 3.95 (s, 3H,
CH3), 3.95-4.05 (m, 4H, piperazine CH2), 7.10 (t, J=7.3 Hz,
1H, benzothiazole CH), 7.25 (d, J = 9.1 Hz, 1H, H-6), 7.30 (t,
J=7.3 Hz, 1H, benzothiazole CH), 7.50 and 7.80 (d, J=8.0 Hz,
each 1H, benzothiazole CH), 8.30 (d, J = 9.1 Hz, 1H, H-5) 8.90
(s, 1H, H-2). Anal. (C21H20ClN5O3S) C, H, N.
€
with a Buchi rotary evaporator at reduced pressure. Yields are of
purified product and were not optimized. All starting materials
were commercially available unless otherwise indicated.
Ethyl 2-[(2,6-Dichloro-3-pyridinyl)carbonyl]-3-(methylamino)-
2-propenoate (6). A mixture of 516 (1.0 g, 3.15 mmol) in EtOH/
Et2O (1:4, 20 mL) was treated with a 33% solution of methyla-
mine in EtOH (0.44 mL, 3.78 mmol). After 30 min, the mixture

646 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Massari et al.
1-Methyl-4-oxo-7-[4-(1,3-thiazol-2-yl)piperazin-1-yl]-1,4-di-
hydro-1,8-naphthyridine-3-carboxylic Acid (4). The title com-
pound was prepared through the general procedure for
coupling reaction replacing 1-(2-pyridinyl)piperazine with
1-(1,3-thiazol-2-yl)piperazine28 to give intermediate 11 in 87%
yield, followed by the general procedure for hydrolysis to give 4
in 99% yield: mp 292-293 °C (d). 1H NMR (DMSO-d6) δ
3.70-3.80 (m, 4H, piperazine CH2), 3.95-4.05 (m, 7H, piper-
azine CH2 and CH3), 7.10 (d, J = 3.9 Hz, 1H, thiazole CH), 7.25
(d, J = 9.1 Hz, 1H, H-6), 7.35 (d, J = 3.9 Hz, 1H, thiazole CH),
8.30 (d, J = 9.1 Hz, 1H, H-5) 8.90 (s, 1H, H-2). Anal.
(C17H17N5O3S) C, H, N.
In Vitro Antiviral Assays. Evaluation of the antiviral acti-
vity of the compounds against HIV-1 strain IIIB and HIV-2
strain (ROD) in MT-4 cells was performed using the MTT
assay as previously described.29,30 Stock solutions (10ꢀ
final concentration) of test compounds were added in 25 μL
volumes to two series of triplicate wells so as to allow simulta-
neous evaluation of their effects on mock-and HIV-infected cells
at the beginning of each experiment. Serial 5-fold dilutions of
test compounds were made directly in flat-bottomed 96-well
microtiter trays using a Biomek 3000 robot (Beckman Instru-
ments, Fullerton, CA). Untreated control HIV-and mock-in-
fected cell samples were included for each sample.
After 5 days incubation, cells were harvested, washed, and fixed
in 3% paraformaldehyde solution. Evaluation of the antiviral
activity of the compounds was performed using flow cytometry
using a FACSCanto II flow cytometry system (Becton-
Dickinson) equipped with a 488 nm argon-ion laser and a 530/
30 bandpass filter. Acquisition was stopped when 10000 events
were counted. Cytotoxicity was monitored using the MTT-
method as described above.
Latently Infected OM-10.1 and U1 Cells. The activity values
of the naphtyridones against chronic HIV-1 infection were based
on the inhibition of p24 antigen production in OM-10.1 an U1
cells after stimulation with TNF-R (Roche Diagnostics, Belgium)
and PMA (Sigma Chemical Co., Bornem Belgium). Briefly, OM-
10.1 and U1 cells (5 ꢀ 105 cells/mL) were incubated in the presence
or absence of the compounds for 2 h in 48 wells plates. After this
short incubation period, the cells were stimulated with 1 ng/mL of
TNF-R or 0.02 μM of PMA, followed by transfer of 2 ꢀ 200 μL
into a 96-well plate for toxicity evaluation. After a 2-day incuba-
tion at 37 °C, the cell culture supernatants were collected from the
48-well plates and examined for their p24 antigen levels with the
HIV-1 p24 ELISA kit (Perkin-Elmer, Boston, MA). Cytotoxicity
of the compounds for both latently HIV-1-infected OM-10.1 and
U1 cell lines in the 96-well plates were based on the MTT cell
viability staining as described previously.29
HIV-1(IIIB)31 or HIV-2 (ROD)32 stock (50 μL) at 100-300
CCID50 (50% cell culture infectious dose) or culture medium
was added to either the infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the
effect of test compound on uninfected cells in order to assess the
cytotoxicity of the test compound. Exponentially growing MT-4
cells33 were centrifuged for 5 min at 1000 rpm and the super-
natant was discarded. The MT-4 cells were resuspended at 6 ꢀ
105 cells/mL and 50 μL volumes were transferred to the micro-
titer tray wells. Five days after infection, the viability of mock-
and HIV-infected cells was examined spectrophotometrically by
the MTT assay.
The MTT assay is based on the reduction of yellow-colored
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial
dehydrogenase of metabolically active cells to a blue-purple
formazan that can be measured spectrophotometrically.
The absorbances were read in an eight-channel computer-
controlled photometer (Multiscan Ascent Reader, Labsystems,
Helsinki, Finland) at two wavelengths (540 and 690 nm). All
data were calculated using the median OD (optical density)
value of tree wells. The 50% cytotoxic concentration (CC50) was
defined as the concentration of the test compound that reduced
the absorbance (OD540) of the mock-infected control sample by
50%. The concentration achieving 50% protection from the
cytopathic effect of the virus in infected cells was defined as the
50% effective concentration (EC50).
Persistently Infected HuT-78 Cells. Chronically infected
HuT-78 cells were washed with PBS and pelleted. The super-
natant was discarded. This manipulation was repeated 3 times.
The test compounds at required concentrations were transferred
into the cups of a 48-well plate. Then 200000 cells were added to
each cup (final volume per cup is 1 mL). The cultures were
allowed to grow for 44 h. Supernatant was collected and
p24 concentrations were determined by ELISA. Nevirapine
(NNRTI) and ritonavir (PI) were included as negative and a
positive control, respectively.
Time-of-Addition Experiment. MT-4 cells were infected with
HIV-1 (IIIB) at a multiplicity of infection (moi) of 0.5 to
synchronize all the steps of the viral replication. The test
compounds were added at different times after infection (from
0 to 25 h).36 Viral p24 Ag production was determined at 31 h
post infection by ELISA (Perkin-Elmer, Boston, MA). Refer-
ence compounds with a known mode of action were included:
AZT, which inhibits the reverse transcription process, and
ritonavir, an inhibitor of the proteolytic cleavage of precursor
proteins, were added at 100 times their 50% inhibitory concen-
tration (IC50) obtained in the MT-4 cells/MTT assay. Addition
of these compounds can be delayed for 5 and 16 h postinfection,
respectively.
Transactivation Assay. The inhibition of basal and Tat-
mediated transactivation of the HIV-1 long terminal repeat
(LTR) in a HeLa cell clone carrying an LTR-driven luciferase
reporter was performed as described previously.37 Cells were
either mock transfected or treated with subsaturating concen-
trations of Tat to activate the viral promoter and incubated with
increasing amounts of the compound.
In Vitro Resistance Selection. An attempt to generate an HIV-
1 strain resistant to compound 3 in MT-4 cells was undertaken
by passaging the virus in the presence increasing concentrations
of compound 3 during 60 passages (= 30 weeks).
Peripheral Blood Mononuclear Cells (PBMCs). PBMCs from
healthy donors were isolated by density centrifugation
(Lymphoprep; Axis-Shield, PoC AS, Oslo, Norway) and stimu-
lated with phytohemagglutin (PHA) (Sigma Chemical Co.,
Bornem, Belgium) for 3 days. The activated cells (PHA-stimu-
lated blasts) were washed with PBS, and viral infections were
done as described by the AIDS clinical trial group protocols.34
Briefly, PBMCs (2 ꢀ 105/200 μL) were plated in the presence of
serial dilutions of the test compound and were infected with HIV
stocks at 1000 CCID50 per mL. At day 4 postinfection, 125 μL of
the supernatant of the infected cultures was removed and
replaced with 150 μL of fresh medium containing the test
compound at the appropriate concentration. At 7 days after
plating the cells, p24 antigen was detected in the culture super-
natant by an enzyme-linked immunosorbent assay (Perkin-
Elmer, Boston, MA).
Kinase Assay. The CDK9 kinase activity was measured in
vitro on a recombinant GST-CTD substrate as described pre-
viously.38
Histone Acetyltransferase (HAT) Activity of p300/CBP. The
HAT activity of p300/CBP was detected using a recombinant
GST-HAT fragment on a histone substrate as described pre-
viously.39,40
Cellular Integrase Assay. This assay was carried out as pre-
viously reported.21
Induced Fit Docking Studies. The IFD protocol used in this
study was carried out as previously reported.23 The carboxylic
acid of compound 3 was modeled as carboxylate.
J-Lat GFP Clone A72 Jurkat Cells.35 J-Lat GFP clone
A72 Jurkat cells (30000 cells per 96-well) were infected with
respective viruses in the absence or presence of test compound.

Article
Acknowledgment. These investigations were supported in
part by the MIUR (PRIN 2006, Roma, Italy), in part by
grants from the “Fonds voor Wetensschappelijk Onderzoek
(FWO)” number 1.5.104.07, and from the EU FP6 “hidden
HIV challenge” (ICGEB). We are grateful to Roberto
Bianconi, Kristien Erven, Kris Uyttersprot, Liesbet De Dier,
Lotte Bral, and Cindy Heens for excellent technical assistance.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 647
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