Oxoquinoline acyclonucleoside phosphonate analogues as a new class of specific inhibitors of human immunodeficiency virus type 1

Article abstract of DOI:10.1016/j.bmcl.2012.06.020

The emergence of a multidrug-resistant HIV-1 strain and the toxicity of anti-HIV-1 compounds approved for clinical use are the most significant problems facing antiretroviral therapies. Therefore, it is crucial to find new agents to overcome these issues. In this study, we synthesized a series of new oxoquinoline acyclonucleoside phosphonate analogues (ethyl 1- [(diisopropoxyphosphoryl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxylates 3a-3k), which contained different substituents at the C6 or C7 positions of the oxoquinoline nucleus and an N1-bonded phosphonate group. We subsequently investigated these compounds' in vitro inhibitory effects against HIV-1-infected peripheral blood mononuclear cells (PBMCs). The most active compounds were the fluoro-substituted derivatives 3f and 3g, which presented excellent EC ₅₀ values of 0.4 ± 0.2 μM (3f) and 0.2 ± 0.005 μM (3g) and selectivity index values (SI) of 6240 and 14675, respectively.

Full text of DOI:10.1016/j.bmcl.2012.06.020

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5055–5058
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Oxoquinoline acyclonucleoside phosphonate analogues as a new class
of specific inhibitors of human immunodeficiency virus type 1
Letícia V. Faro a, Jéssica M. de Almeida a, Cláudio C. Cirne-Santos b,c, Viveca A. Giongo b,
Luís R. Castello-Branco c, Ingrid de B. Oliveira b, Juliana E. F. Barbosa d, Anna C. Cunha a, Vítor F. Ferreira a,
Marcos C. de Souza a, Izabel C. N. P. Paixão b, Maria Cecília B. V. de Souza a,
⇑
a Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, Outeiro de São João Batista s/n, centro, Niterói, Programa de Pós-graduação em
Química, Rio de Janeiro 24020-150, Brazil
b Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de São João Batista s/n, centro, Niterói, Programa de Pós-graduação em
Biologia das Interações, Rio de Janeiro 24020-150, Brazil
c Laboratório de Imunologia Clínica, IOC/Instituto Oswaldo Cruz e Fundação, Fundação Ataulpho de Paiva IOC/FIOCRUZ, Brazil
d Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Outeiro de São João Batista s/n, centro, Niterói, Programa de Pós-graduação em Biologia
das Interações, Rio de Janeiro 24020-150, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence of a multidrug-resistant HIV-1 strain and the toxicity of anti-HIV-1 compounds approved
for clinical use are the most significant problems facing antiretroviral therapies. Therefore, it is crucial to
find new agents to overcome these issues. In this study, we synthesized a series of new oxoquinoline
acyclonucleoside phosphonate analogues (ethyl 1-[(diisopropoxyphosphoryl)methyl]-4-oxo-1,4-dihy-
droquinoline-3-carboxylates 3a–3k), which contained different substituents at the C6 or C7 positions
of the oxoquinoline nucleus and an N1-bonded phosphonate group. We subsequently investigated these
compounds’ in vitro inhibitory effects against HIV-1-infected peripheral blood mononuclear cells
(PBMCs). The most active compounds were the fluoro-substituted derivatives 3f and 3g, which presented
Received 18 April 2012
Revised 2 June 2012
Accepted 4 June 2012
Available online 15 June 2012
Keywords:
Oxoquinoline
Acyclonucleoside phosphonate
Antiviral
excellent EC50 values of 0.4 0.2
and 14675, respectively.
l
M (3f) and 0.2 0.005
lM (3g) and selectivity index values (SI) of 6240
Anti-HIV-1
Ó 2012 Published by Elsevier Ltd.
Since human immunodeficiency virus type 1 (HIV-1) was first
identified as the etiological agent of acquired immunodeficiency
syndrome (AIDS),1,2 several steps of the viral growth cycle have
been studied as potential targets for therapeutic intervention.
Combination antiretroviral therapy (cART) has led to a major
reduction in HIV-related mortality and morbidity. However, the
treatment of HIV infections has been limited somewhat by the sub-
stantial toxicity of clinically approved anti-HIV drugs3 and by the
development of drug resistance.4,5
Oxoquinoline derivatives are a class of compounds that are
well-known for their antibacterial properties, yet many such com-
pounds have several other biological activities that have been
described in the literature.6 These activities include antimalarial,7
anti-ischemic,8 antitumoral9 and antiviral10 activities. Many oxo-
quinoline derivatives have been shown to inhibit in vitro HIV-1
replication in acutely and chronically infected cells.11–22
(ANPs): cidofovir, adefovir dipivoxil and tenofovir disoproxil fuma-
rate.24–26 GS-8374 is a novel HIV-1 protease inhibitor that contains
a diethylphosphonate moiety. This compound exhibits a resistant
profile compared to clinically approved protease inhibitors
(PIs).27 Phosphonate compounds are also described as antibacte-
rial,28,29 anticancer30 and anti-parasitic agents.31–35
The antiviral activity of oxoquinoline compounds has been a
subject of interest in our group for the past several years.36–41
We demonstrated that oxoquinoline ribonucleoside 1 (Fig. 1)
inhibits HIV-1 replication in human primary cells with an EC50
value of 1.5 0.5 lM and a selectivity index value of 1134. This
substance was also shown to inhibit virus replication in macro-
phage cells (EC50 = 4.9 M), which are reservoirs of HIV-1 and may
remain infected even during antiretroviral therapy. Kinetic assays
have determined that compound 1 inhibits the HIV-1 enzyme
reverse transcriptase (RT) in a dose-dependent manner with a Ki
The phosphonate functionality is present in a variety of clini-
cally useful drugs.23 Of the few compounds marketed for the treat-
ment of HIV-1 infection, three are acyclonucleoside phosphonates
value of 0.5 0.04 lM. Using a panel of HIV-1 isolates harbouring
non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance
mutations, we found a low degree of cross-resistance for both com-
pound 1 and clinically available NNRTI anti-HIV-1 substances.
Ribonucleoside 1 also exhibited additive effects with the RT
inhibitors AZT and nevirapine and synergistic effects with the pro-
tease inhibitor atazanavir.40
⇑
Corresponding author. Tel.: +55 21 26 29 23 49.
E-mail address: gqocica@vm.uff.br (M.C.B.V. de Souza).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.06.020

5056
L. V. Faro et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5055–5058
Figure 1. Chemical structures of chloroxoquinoline ribonucleoside 1 and oxoquinoline acyclonucleosides 2a, 2b, 2c and 2d.
Scheme 1. Synthesis of oxoquinoline acyclonucleoside phophonate analogues 3a–3k. Reagents and conditions: (i) EtOH, diethyl ethoxymethylenemalonate, reflux; (ii)
Dowtherm A, reflux; (iii) dry DMF/potassium carbonate, 80 °C.
We also described the synthesis and anti-HIV-1 activity of
oxoquinoline acyclovir analogues, such as 2a–d, containing
different substituents at carbons C6 or C7 and C3 of the oxoquin-
oline nucleus. Compounds 2a, 2b and 2c showed excellent in vitro
anti-HIV-1 activity with cytotoxicity concentration (CC50) values of
Compounds 3a–3k were synthesized by N-alkylation of 4-oxoquin-
olines 5a–5k49,50 with diisopropyl(tosylmethoxy)phosphonate 4.
This reagent was synthesized by condensation of diisopropyl phos-
phonate with formaldehyde followed by tosylation51 (Scheme 1).
Experimental procedures, reaction yields and analytical data are
available in the Supplementary data.
2400
l
M (2a), 3200
lM (2b) and 2800
lM (2c). These values are
higher than that observed for AZT (CC50 = 126
l
M) under the same
For biological assays (see Supplementary data), PBMCs were
obtained from healthy human donors. The cells were stimulated
with phytohemagglutinin for two to three days and further main-
tained in culture medium containing recombinant human interleu-
kin-2. The HIV-1 Ba-L isolate (R5-tropic, subtype B)40 was used to
infect PBMC cells.
For cytotoxic activity assays, confluent cells were exposed to
different concentrations of compounds 3a–3k for seven days. After
incubation, cell viability was assessed by the XTT method.52
The screening of in vitro anti-HIV-1 activity followed previously
described protocols40 with minor modifications. PBMCs were in-
fected with viral suspensions and treated with different concentra-
tions (lM) of compounds 3a–3k. After seven days, viral replication
was assessed by measuring the presence of HIV p-24 Ag in culture
supernatants by an ELISA capture assay.
The cytotoxicity values of compounds 3a–3k, as well as their
respective abilities to inhibit HIV-1 replication in PBMCs, are re-
ported in Table 1.
assay conditions. The SI values were determined to be 960, 711,
and 1000, respectively. Although these values are lower than the
AZT value (SI = 2520), they are greater than the values found for
commercial anti-HIV-1 compounds DDC (SI = 110) and DDI
(SI = 10).41 Conversely, compound 2d, containing a carboxyethyl
group at carbon C3, was found to be a much less active anti-
HIV-1 agent (CC50 and SI values of 1500
lM
and 18,
respectively).41
As part of an ongoing research program on the synthesis of new
bioactive oxoquinoline derivatives, we report the synthesis and
anti-HIV-1 evaluation of new oxoquinoline acyclonucleoside ana-
logues 3a–3k in which the acyclic chain of 2d was replaced with
a diisopropylphosphonate group (Scheme 1). In addition, we inves-
tigated the influence of substituents at C6 and C7 positions on the
biological activity of the compounds.
Ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate derivatives
5a–5k were prepared according to published methods.42–48

L. V. Faro et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5055–5058
5057
Table 1
analogues, we cannot say at this stage of our research if they act
as HIV-1 nucleoside inhibitors or which phase of the HIV-1 replica-
tion cycle is inhibited by these compounds. These results also indi-
cate that the presence of the fluoro substituent in these structures
is crucial to the increased anti-HIV-1 activity of compounds 3f and
3g compared to other derivatives (R = H, C-6 or C-7 = Cl, Br, NO2,
CH3).
These results also indicate that oxoquinolinephosphonate 3g
(7-F) is a notably promising compound for the development of a
new, more potent and selective anti-HIV-1 drug and consequently
represent a significant advance in the field of anti-HIV-1 oxoquin-
oline derivatives.
Cytotoxicity (CC50), anti-HIV-1 profile (EC50) and selectivity index (SI) of oxoquinoline
acyclonucleoside phosphonate analogues 3a–3k
SIc
a
b
Compound
Substituent
CC50
(l
M)
EC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
2310 18
1235 16
828 9.0
802 8.2
731 8.8
2496 22
2935 17
1315 12
1115 13
931 19
812 10
128 2.8
7.3 0.7
8.1 0.2
21 0.4
316.4
152.4
39.4
14.3
11.9
6240
14,675
61.3
123.9
116.35
36.9
6-Cl
7-Cl
6-Br
7-Br
6-F
56 1.4
61.2 1.8
0.4 0.02
0.2 0.005
21.3 0.8
9.0 0.9
38 2.1
7-F
6-CH3
7-CH3
6-NO2
7-NO2
3j
3k
22 1.8
0.16 0.01
AZTd
2520
Acknowledgments
The mean values standard deviations are representative of three independent
experiments.
This work was supported by the Brazilian agencies FAPERJ and
CNPq. Fellowships granted to UFF by CAPES, CNPq-PIBIC and FA-
PERJ are gratefully acknowledged. The authors have declared no
conflict of interest.
a
Concentration that reduced 50% cytotoxic concentration when compared to
untreated controls.
b
Concentration that reduced 50% of HIV-1 replication when compared to infec-
ted controls.
c
Selectivity index was defined as the ratio between CC50 and EC50 and represents
Supplementary data
the safety for in vitro assays.
d
AZT was used as a positive control.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.06.
020.
Replacement of the N-bonded acyclic chain in compound 2d
(EC50 = 83.3 2.3
group in 3f led to a marked increase in anti-HIV-1 activity
(EC50 = 0.4 0.02 M; SI = 6240).
lM; SI = 18) with the diisopropylphosphonate
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