Structure-activity relationship study on anti-HIV 6-desfluoroquinolones

Article abstract of DOI:10.1021/jm701585h

On the basis of our recent findings that 6-aminoquinolones inhibit the HIV Tat-mediated transactivation, we have designed a broad series of derivatives identifying novel potent agents such as the 6-desfluoroquinolones 24 (HM12) and 27 (HM13), which showed pronounced anti-HIV activity in acutely, chronically, and latently HIV-1 infected cell cultures. We demonstrate here that highly potent molecules can be obtained by optimizing the substituent in the various positions of the quinolone nucleus.

Full text of DOI:10.1021/jm701585h

5454
J. Med. Chem. 2008, 51, 5454–5458
Structure-Activity Relationship Study on Anti-HIV 6-Desfluoroquinolones
Oriana Tabarrini,*,† Serena Massari,† Dirk Daelemans,‡ Miguel Stevens,‡ Giuseppe Manfroni,† Stefano Sabatini,† Jan Balzarini,‡
Violetta Cecchetti,† Christophe Pannecouque,‡ and Arnaldo Fravolini†
Dipartimento di Chimica e Tecnologia del Farmaco, UniVersita` di Perugia, Via del Liceo 1, 06123 Perugia, Italy, Rega Institute for Medical
Research, Katholieke UniVersiteit LeuVen, B-3000 LeuVen, Belgium
ReceiVed December 18, 2007
On the basis of our recent findings that 6-aminoquinolones inhibit the HIV Tat-mediated transactivation,
we have designed a broad series of derivatives identifying novel potent agents such as the 6-desfluoroqui-
nolones 24 (HM12) and 27 (HM13), which showed pronounced anti-HIV activity in acutely, chronically,
and latently HIV-1 infected cell cultures. We demonstrate here that highly potent molecules can be obtained
by optimizing the substituent in the various positions of the quinolone nucleus.
Introduction
this compound owes its anti-HIV activity exclusively to the
inhibition of the viral enzyme integrase.12
The HIV pandemic remains one of the most serious threats
to public health. While the currently available drugs effectively
reduce the levels of the rapidly replicating virus, the benefit is
only short-term due to the emergence of resistant strains1 and
the inability of the current anti-HIV chemotherapy to completely
eradicate viral infection2 caused by the occurrence of stable
latent reservoirs mainly represented by latently infected resting
CD4+ lymphocytes3 and monocyte/macrophages (M/Ma).4
Therefore, there is an urgent need to develop anti-HIV agents
with a new mechanism of action able to overcome these
problems.
Starting from 1 (WM5),8 the prototype of the 6-aminoqui-
nolone series, and in order to determine the optimal substitution
pattern, we have designed new derivatives challenging all the
features thought to be essential for the biological activity (Chart
1). The carboxylic function was replaced by a primary amide
(2), secondary amides (3 and 4), a keto group (5), or oxime
moieties (6 and 7). In a more drastic structural modification,
the C-3 carboxylic function was banned, synthesizing the C-3
decarboxylate quinolinone derivatives 8-10. Concerning the
N-1 position, earlier structure-activity relationship (SAR)
studies showed that the highest antiretroviral activity was
achieved with a small substituent, with a methyl group being
the most optimal. In this study, we synthesized derivatives
11-14 to explore the effect of the absence of a substituent at
this position. We also turned our attention to the C-7 position,
modifying the 4-(2-pyridinyl)-1-piperazinyl moiety character-
izing the lead 1, in both the piperazine and N-4 aryl rings,
synthesizing derivatives 15-22. In a previous study, we showed
that the elimination of the 6-amino group in 1 resulted in the
6-hydrogen analogue 239 (Chart 1), which had a weak antiviral
activity in MT-4 cells but a markedly lower cytotoxicity (about
100 times less toxic than 1). In this study, we aimed at
maintaining low cytotoxicity while increasing antiviral potency,
and therefore we have prepared new 6-hydrogenquinolones,
24-28 (Chart 1), by introducing those 4-arylpiperazines which
conferred high potency on previous 6-aminoquinolone series
at the C-7 position.
An innovative intervention site could be the transcription from
the 5′ LTR (long terminal repeat) promoter, a crucial step in
the HIV replication cycle. Inhibitors of this process will be able
to suppress HIV replication not only in acutely but also in
chronically infected cells.5 Various attempts have been made
to discover suitable selective inhibitors of HIV-1 gene expression
and transcription,6,7 but to date, none of the compounds have
led to a successful clinical development due to low bioavail-
ability and/or high toxicity.
In this scenario, the 6-aminoquinolones8,9 have emerged as
a promising new class of HIV expression inhibitors, interfering
with the Tat-mediated transactivation of the HIV-1 LTR
promoter.10 The advent of quinolones as antiviral agents is
particularly attractive because quinolones are small extremely
versatile molecules, easily synthesized at low cost on a large
scale and endowed with well-known biochemical properties that
make them very suitable pharmacophore structures. Therefore,
it is extremely important to explore this class of compounds in
an effort to design even more selective anti-HIV inhibitors. In
fact, the first quinolone-based structure (GS-9137/JTK-303)11
with very strong antiretroviral properties is currently in advanced
clinical trials. Interestingly, in contrast to 6-aminoquinolones,
Chemistry
The C-3 amide derivatives 2-4 were prepared in good yield
by reacting the ethyl 6-amino-1-methyl-4-oxo-7-[4-(2-pyridinyl)-
1-piperazinyl]-1,4-dihydro-3-quinolinecarboxylate8 with a satu-
rated ammonia solution in EtOH in a closed vessel or with the
selected benzylamines in neat. The synthesis of C-3 keto (5)
and oxime derivatives (6 and 7) (Scheme 1) was accomplished
by starting from 3-chloro-4-nitroaniline, which was elaborated
to synthone 31 by reaction with methyl 2-acetyl-3-ethoxy-2-
propenoate13 to give the acrylate 29, which was successively
thermically cyclized to derivative 30 and finally N-1 methylated.
The nucleophilic reaction with 1-(2-pyridinyl)piperazine fol-
lowed by catalytic reduction, gave the 6-amino-3-acetyl deriva-
tive 5, which was further elaborated to oxime derivatives 6 and
7 by reaction with hydroxylamine and methoxylamine, respec-
* To whom correspondence should be addressed. Phone: + 39 075 585
5139. Fax: +39 075 585 5115. E-mail: oriana.tabarrini@unipg.it.
†
Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di
Perugia.
‡
Rega Institute for Medical Research, Katholieke Universiteit Leuven.
a Abbreviations: 6-DFQs, 6-desfluoroquinolones; GFP, green fluorescent
protein; HCMV, human cytomegalovirus; HIV, human immunodeficiency
virus; LTR, long terminal repeat; M/M, human primary monocyte/
macrophages; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; PBMCs, peripheral blood mononuclear cells; SAR, structure-activity
relationship.
10.1021/jm701585h CCC: $40.75
2008 American Chemical Society
Published on Web 08/19/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5455
Chart 1. Structure of the 6-DFQs Synthesized in this Study and Reference Compounds
tively. To obtain the C-3 decarboxylate quinolones 8-10
(Scheme 2), carboxylic acid 3214 was decarboxylated, even if
in very low yield, to quinolinone 33 by using Ph2O at reflux
for 15 h. The successive nucleophilic reaction with selected
4-arylpiperazines gave the nitro derivatives 34-36, which were
then catalytically reduced to the 6-amino target derivatives. The
route followed to synthesize 4-hydroxyquinolinyl derivatives
11-14 (Scheme 3) parallels the cycloaracylation procedure used
to synthesize similar compounds,8,9 which starts with acrylate
3715 and proceeds through quinolone 3816 and C-7 functional-
ized intermediates 39-42. The 6-aminoquinolones 15-22 were
prepared by conventional route (Scheme 4) starting from
synthone 43,8 which was reacted with various side chains to
give intermediates 44-51 and in turn hydrogenated and finally
hydrolyzed. The 6-hydrogen derivatives 24-28 were prepared
as previously reported for 239 by nucleophilic reaction of
7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
borine complex14 with selected 4-arylpiperazines in DMSO and
Et3N.
Results and Discussion
All the synthesized compounds were initially evaluated for
anti-HIV-1 (IIIB) and anti-HIV-2 (ROD) activity in MT-4 cells.
The cytotoxicity of the compounds was determined in parallel.
The results of the active compounds are shown in Table 1,
together with reference compounds 18 and 23,9 for comparative
purposes (the results of inactive compounds are reported in Table
S1 of Supporting Information). The anti-HIV activity of some
selected compounds was also evaluated in the human lympho-

5456 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Brief Articles
Scheme 1a
and co-workers for anti-HIV fluoroquinolones.17 For the C-7
position, the piperazine ring was confirmed as the best linker
between quinolone and pyridine rings; it cannot be replaced by
either its piperidine bioisoster (15) or ring constrained pyrro-
lidine (16) without a loss of antiviral activity. The opening of
the piperazine ring was also detrimental for activity in that
compound 17 was not active at subtoxic concentrations. When
we eliminated the aromatic portion on the N-4 position of the
piperazine ring (18) or introduced one methylenic unit in
the aromatic portion (19), the antiviral activity disappeared. On
the contrary, the 7-quinolinylpiperazinyl derivative 20 and
7-quinoxalinylpiperazinyl derivative 21 showed good anti-HIV
activity. Both compounds also showed high anti-HIV-1 and
HIV-2 activity in CEM cells (20, EC50 ) 0.036 and 0.075 µg/
mL; 21, EC50 ) 0.05 and 0.28 µg/mL), while they are less toxic
resulting in higher SI values (115 and 55 on HIV-1 and HIV-2,
respectively, for 20).
a Reagents: (i) EtOCHdC(COMe)CO2Me, 120 °C; (ii) Dowtherm A,
200 °C; (iii) MeI, K2CO3, DMF, 90 °C; (iv) 1-(2-pyridinyl)piperazine, DMF,
80 °C; (v) H2, Raney Ni, DMF; (vi) H2NOR·HCl, pyridine, 65 °C.
The most interesting results came from the SAR investigations
on the N-1 and C-6 positions. In particular, the absence of a
substituent at the N-1 position, did not cause a loss of activity
in contrast with the well-established SAR for both antibacterial18
and antiviral quinolone agents.19 In fact, derivatives 11-14,
generally maintain good antiviral activity in both MT-4 and
CEM cell cultures, and more importantly they were also active
in PBMCs with EC50 values ranging from 0.094 to 0.21 µg/
mL. It is worth noting that the absence of the substituent at the
N-1 position gives rise to the keto/enol tautomers, and the
relative equilibrium, under physiological or assay conditions,
is not easily predictable.20 Considering that the 4-keto-3-
carboxylic arrangement was previously reported as indispensable
for target recognition, through a Mg2+ bridge, in the antibacterial
quinolones21 as well as for the anti-HIV 6-aminoquinolones,8
fluorimetric titration experiments were carried out for the
derivative 1122 to check its ability to form complexes with this
divalent ion. The experiments showed that it was still able to
complex Mg2+ ions even if with a lower constant (KMg ) 2530
M-1) compared to its N-1 methyl counterpart 1 (KMg ) 6650
Scheme 2a
a Reagents: (i) Ph2O, reflux; (ii) R7H, DMF, 90 °C; (iii) H2, Raney Ni,
DMF or HCO2NH4, 10% Pd/C, DMF.
Scheme 3a
M-1 23
but superior to that of the antibacterial ciprofloxacin
)
(KMg ) 790 M-1).23
The synthesis of the new quinolones lacking the C-6
substituent resulted in very interesting compounds. In particular,
at concentrations lower than 125 µg/mL in MT-4 and 100 µg/
mL in CEM cells, the 6-hydrogenquinolones 25, 26, and 28
were not toxic but the anti-HIV activity was compromised. In
contrast, a balanced activity was displayed by the m-(trifluo-
romethyl)phenylpiperazinyl derivative 24 (HM12), which in
a Reagents: (i) NH3/EtOH/Et2O; (ii) K2CO3, DMF, 100 °C; (iii) R7H,
DMF, 80-110 °C; (iv) H2, Raney Ni, DMF or HCO2NH4, 10% Pd/C, DMF;
(v) 6N HCl/EtOH, reflux, or 1N NaOH, reflux.
Scheme 4a
MT-4 cells showed EC50 ) 0.07 and 0.13 µg/mL and CC50
)
2.61 µg/mL. Very good anti-HIV activity was also observed
with the benzothiazolpiperazinyl derivative 27 (HM13), which
was the most interesting compound when assayed in acutely
infected CEM cell cultures because it displayed the same anti-
HIV-1 and HIV-2 activity as observed in MT-4 but was 10 times
less toxic, resulting in SI values of 281 and 104, respectively.
Compounds 24 and 27 were also tested in PBMCs, where they
maintained good anti-HIV-1 activity with EC50 ) 0.28 and 0.15
µg/mL, respectively.
a Reagents: (i) R7H, DMF, 70-110 °C or K2CO3, CH3CN, 90 °C; (ii)
H2, DMF, Raney Ni or 5% Pd/C; (iii) 6N HCl/EtOH, reflux or 1N NaOH,
reflux.
cytic CEM cell line and PBMCs (see Table S2 in Supporting
Information).
Whereas other quinolones also blocked the in vitro replication
of HCMV (human cytomegalovirus), compounds 24 and 27
have an antiviral activity that is limited to HIV-1 and -2.
SAR studies confirmed that the C-3 carboxylic moiety is
indispensable for the biological activity. In fact, its replacement
with an amide function, as well as with keton or oxime groups,
resulted in derivatives 2-7 that were completely devoid of
cytotoxicity and antiviral properties. The same was also observed
with 8-10, which lacked the C-3 carboxylic moiety; this is in
agreement with what has been previously observed by Baba
To study the molecular mechanism of action of the new
6-desfluoroquinolones (6-DFQs), time-of-addition experiments
were carried out for derivative 27, including reference com-
pounds with a known mode of action. This experiment
determines how long the addition of an HIV replication inhibitor

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5457
Table 1. Anti-HIV-1 and -HIV-2 Activity and Cytotoxicity of Selected Quinolones in MT-4 cells.
compd
HIV-1 (IIIB) EC50 (µg/mL)a,c
HIV-2 (ROD) EC50 (µg/mL)a,c
CC50 (µg/mL)b,c
SId (IIIB)
SId (ROD)
18
0.058 ( 0.021
0.45 ( 0.27
0.38 ( 0.04
1.21 ( 0.55
0.23 ( 0.09
0.035 ( 0.004
0.18 ( 0.04
19.20 ( 3.53
0.07 ( 0.01
10.03 ( 1.80
58.25 ( 31.32
0.03 ( 0.01
5.76 ( 4.84
0.097 ( 0.053
0.16 ( 0.02
0.46 ( 0.08
1.92 ( 0.17
0.28 ( 0.04
0.022 ( 0.017
0.19 ( 0.10
>65.53
0.13 ( 0.02
12.70 ( 0.57
16.35 ( 0.49
0.04 ( 0.03
11.86 ( 6.99
0.84 ( 0.40
2.16 ( 0.18
1.64 ( 0.24
7.61 ( 2.07
1.95 ( 0.45
0.21 ( 0.10
3.20 ( 1.02
65.53 ( 26.39
2.61 ( 0.66
>125
15
5
4
7
11
10
14
4
5
7
10
17
<1
21
>10
8
11
12
13
14
20
21
239
24
25
26
27
28
6
18
3.4
38
>12
>2
14.7
>22
>125
0.44 ( 0.12
>125
5.5
>11
a EC50: concentration of compound required to achieve 50% protection 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 least two separate experiments. d SI: ratio of CC50/EC50.
can be postponed before losing its antiviral activity. Compound
27 kept its antiviral activity when added at least up to 10 h
postinfection, suggesting that it inhibits the viral replication at
a late stage (details and Figure S1 in Supporting Information).
To study if these compounds act on a postintegrational target
of the HIV replication, compounds 24 and 27 were tested for
their effect on virus expression from persistently infected human
T lymphoma cell line HuT78IIIB. These cells contain integrated
provirus and constitutively release virus in the supernatant. In
this assay, compounds 24 and 27 efficiently prevent virus
production at nontoxic concentrations, suggesting a postint-
egrational mechanism of action (details and Figure S2 in
Supporting Information). Subsequently, the effect on Tat-
mediated gene expression was investigated using a Tat-assay
in Jurkat cells. In this assay, compounds 27 and 28 (selected
due to its nontoxic profile) inhibited the Tat-mediated green
fluorescent protein (GFP) gene expression from the HIV-1 LTR
promoter in a dose-dependent manner, suggesting a target of
interaction related to HIV-1 gene expression (details and Figure
S3 in Supporting Information).
HIV-1 replication cycle, principally a target involving HIV-1
gene expression.
Experimental Section
General Procedure for Coupling Reaction (Method A). A
mixture of the appropriate synthone (1 equiv) and selected side
chains (3-5 equiv) in dry DMF was heated at 70-110 °C until no
starting material could be detected by TLC (1-36 h). The reaction
mixture was worked up and purified as reported in the description
of the single compounds.
General Procedure for Reduction of 6-Nitro Group (Method
B). A stirred solution of the selected 6-nitro derivative in DMF
was hydrogenated over a catalytic amount of Raney nickel at room
temperature and atmospheric pressure until no starting material
could be detected by TLC (1-30 h). The mixture was then filtered
over celite, and the filtrate was evaporated to dryness to afford a
residue which, by treatment with EtOH/EtOAc, gave a solid that
was filtered and dried to give the corresponding 6-amino derivative.
General Procedure for Acid Hydrolysis (Method C). A
mixture of the selected ester (1 mequiv) in EtOH (6 mL) and 6N
HCl (6 mL) was refluxed until no starting material could be detected
by TLC (2-72 h). It was worked up and purified as reported in
the description of the single compounds.
In conclusion, this study provided new insights in the SAR
of anti-HIV quinolones and led to the identification of new
potent and selective derivatives. While the presence of a
carboxylic function and the 4-arylpiperazine ring at the C-3 and
C-7 positions, respectively, turned out to be crucial to preserve
antiviral activity, the expansion of the heteroaryl system at the
N-4 piperazine side chain increased the activity as shown by
derivatives 20 and 21. The real novelty of this study, however,
emerged from the elimination of any substituent at the N-1 or
C-6 positions of the quinolone nucleus. The preserved anti-HIV
activity of the N-1 unsubstituted derivatives 11-14 challenges
the well-established quinolone SAR. This unexpected structure-
activity feature should be taken into account in the design of
additional selective anti-HIV quinolone analogues. The most
significant result was definitely achieved with the synthesis of
a variety of new 6-hydrogenquinolones. In fact, when the
appropriate 4-arylpiperazine was introduced at the C-7 position,
very active and selective derivatives were obtained such as the
6-hydrogenquinolones 24 and 27. These compounds are en-
dowed with very strong activity on HIV-1 acutely infected MT-
4, CEM, and PBMCs cells as well as on chronically infected
HuT78. A potent antiviral activity was also observed in latently
HIV-1 infected M/M cells at drug concentrations as low as 40
ng/mL.24 This activity was further confirmed in an in vivo model
for HIV-1 latency, which provides encouraging evidence for
the use of quinolones in the control of HIV-1 infection in vivo.24
The mechanism of action studies clearly demonstrated that the
new 6-DFQs interfere with a postintegrational target of the
6-Amino-4-hydroxy-7-[4-(2-pyridinyl)-1-piperazinyl]-3-quino-
linecarboxylic acid (11). The title compound was prepared starting
from synthone 3816 through general coupling procedure (method
A), using 1-(2-pyridinyl)piperazine (110 °C, 30 h); after cooling,
the precipitate solid was filtered, washed with EtOH, and dried to
give compound 39. It was then reduced by general reduction
procedure (method B) (3 h) and hydrolyzed by general hydrolysis
procedure (method C) (30 h): after cooling, the mixture was filtered
and the filtrate was neutralized by adding 10% NaOH. The solid
obtained was collected by filtration, washed with water and EtOH
and finally recrystallized by EtOH/DMF to give acid 11 in 15%
overall yield.
1-Methyl-4-oxo-7-{4-[3-(trifluoromethyl)phenyl]-1-piperazi-
nyl}-1,4-dihydro-3-quinolinecarboxylic acid (24). A mixture of
7-chloro-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
BF2 chelate9 (0.80 g, 2.80 mmol), 1-[3-(trifluoromethyl)phenyl]pip-
erazine (3.22 g, 14 mmol), and Et3N (1.4 g, 14 mmol) in dry DMSO
(10 mL) was heated at 90 °C for 30 min. After cooling, the
precipitate was filtered, washed with ice/water, dried, and then
recrystallized from DMSO to give 24 (0.30 g, 25%).
Acknowledgment. We acknowledge Prof. Manlio Palumbo
and Prof. Barbara Gatto (University of Padova, Italy) for the
fluorimetric titration experiments carried out for 11. These
investigations were supported in part by the MIUR (PRIN 2006,
Roma, Italy) and in part by grants from the K.U. Leuven (GOA-
2005/19) and the FWO (1.5.104.07). We are grateful to Roberto
Bianconi, Kristien Erven, Cindy Heens, Liesbet De Dier, and
Kris Uyttersprot for excellent technical assistance.

5458 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Brief Articles
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Supporting Information Available: Experimental details cor-
responding to the synthesis and analytical data of compounds
described in this paper. A table containing elemental analysis data
for target compounds 2-22 and 24-28. Complete descriptions of
biological protocols. This material is available free of charge via
the Internet at http://pubs.acs.org.
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