Studies on anti-HIV quinolones: New insights on the C-6 position

Article abstract of DOI:10.1016/j.bmc.2008.11.056

The 6-desfluoroquinolones which have been developed by our group represent a promising class of compounds for the treatment of HIV infection since they act on transcriptional regulation, a crucial step in the replication cycle that has not been clinically

Full text of DOI:10.1016/j.bmc.2008.11.056

Bioorganic & Medicinal Chemistry 17 (2009) 667–674
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Studies on anti-HIV quinolones: New insights on the C-6 position
Serena Massari a, Dirk Daelemans b, Giuseppe Manfroni a, Stefano Sabatini a, Oriana Tabarrini a,
,
*
Christophe Pannecouque b, Violetta Cecchetti a
a Dipartimento di Chimica e Tecnologia del Farmaco, Università degli studi di Perugia, Via del Liceo 1, 06123 Perugia, Italy
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2008
Revised 18 November 2008
Accepted 21 November 2008
Available online 3 December 2008
The 6-desfluoroquinolones which have been developed by our group represent a promising class of com-
pounds for the treatment of HIV infection since they act on transcriptional regulation, a crucial step in the
replication cycle that has not been clinically exploited, yet. Focussing attention on the N-1 and C-6 posi-
tions, a novel series of quinolones has been synthesized. New SAR insights have been obtained, in partic-
ular, the hydroxyl group emerged as a suitable C-6 substituent when coupled with the appropriate
arylpiperazine at the neighboring C-7 position.
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
6-Desfluoroquinolones (6-DFQs)
6-Hydroxyquinolones
Anti-HIV agents
Transcription inhibitors
1. Introduction
on the prototype of the 6-DFQs, WM51 (1, Fig. 1). In particular,
the omission of the N-1 substituent did not cause a loss of activity
One of the crucial steps in the HIV replication cycle that has not
been clinically exploited is the transcription of viral mRNA from
the HIV long terminal repeat (LTR) promoter.
in contrast with the well-established SAR for both antibacterial4
and antiviral quinolone agents,5 it rather gave derivatives showing
the same anti-HIV potency as the 1-methyl counterparts coupled
with a reduced cytotoxicity (from 3- to 20-fold).3 The real novelty
that emerged from our recent work,3 came from modifications at
the C-6 position where the lack of any substituent resulted in com-
pounds endowed with markedly reduced cytotoxicity, when the
appropriate 4-arylpiperazine was present at the C-7 position. In
fact, the 6-hydrogenquinolones HM12 and HM13 (2 and 3,
Fig. 1)3,6 were endowed with very pronounced activity and selec-
tivity on HIV-1 acutely infected MT-4, CEM and peripheral blood
mononuclear cells (PBMCs) as well as on chronically infected
HuT78 and human primary monocyte/macrophages (M/M), and
The regulation of HIV-1 gene expression could be a possible
intervention site for antiretroviral chemotherapy, because it gives
the possibility of controlling HIV-1 replication not only in acutely
but also in chronically infected cells. Inhibitors of HIV-1 transcrip-
tional regulation could have great potential in anti-HIV drug com-
bination therapy because they can slow down the virus replication
rate or even completely shut-off virus replication giving a lifetime
control of HIV infection. In addition, such inhibitors could disfavor
drug resistance development due to the complex interplay of viral
and cellular components involved in the transcriptional regulation
process.
It is precisely at this step of the replication cycle that the 6-des-
fluoroquinolones (6-DFQs), identified by our group,1–3 act, and
which are therefore a very promising class of compounds for the
control of the latent HIV-1 reservoir. However, to fulfill the expec-
tations of the transcription inhibitor quinolones in anti-HIV che-
motherapy field, the limitation caused by the low selectivity
index observed in some cell lines needs to be overcome.
With this main purpose, further structure modifications were
made in this study bearing in mind the recently published results3
regarding the structure–activity relationship (SAR) investigations
O
O
5
8
4
6
3
CO2H
H2N
N
CO2H
CF3
7
2
N
Me
N
N
Me
1
N
N
Ar
N
1 (WM5)
Ar =
Ar =
2 (HM12)
3 (HM13)
N
S
* Corresponding author. Tel.: +39 075 585 5139; fax: +39 075 585 5115.
E-mail address: oriana.tabarrini@unipg.it (O. Tabarrini).
Figure 1. Potent 6-DFQs.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.056

668
S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
Ar
OH
N
O
R6
N
CO2H
R6
N
CO2H
N
a:
b:
N
Me
CF3
N
N
Ar
Ar
R6 = H 4a-c
R6 = F 5a-c
R6 = CF3 6a-c
R6 = OMe 7a-c
N
c:
d:
R6 = OH 8a-e
S
N
N
N
e:
Figure 2. Structures of quinolones synthesized in this study.
latently HIV-1 infected M/M cells. This activity was also confirmed
in an in vivo mice model for HIV-1 latency which is encouraging
evidence for the use of quinolones in the control of HIV-1 infection
in patients.6
fluoro-4-(trifluoromethyl)aniline with diethylethoxymethylene-
malonate (EMME) gave compound 11 which was then cyclized
with polyphosphoric acid (PPA), to give the key intermediate 12.
Subsequent sequential steps were: N-1 methylation to give com-
pound 13, C-7 nucleophilic substitution with arylpiperazines a–c
to intermediates 14a–c, and basic hydrolysis to the target acids
6a–c.
Following an analogous procedure, 6-methoxyquinolone target
acids 7a–c were prepared starting from ethyl 7-fluoro-4-hydroxy-
6-methoxyquinoline-3-carboxylate 1513 (Scheme 2) through inter-
mediates 16 and 17a–c. The nucleophilic reaction was carried out
using N-methyl-2-pyrrolidone to make the 7-fluorine displace-
ment possible.
A first attempt to directly prepare the corresponding 6-hydroxy
acids 8a–c by treating the 6-methoxy esters 17a–c with 48% HBr
was disappointed since very low yield were obtained. On the other
hand, the de-O-methylation did not proceed when mild conditions
such as AlCl3 or PBr3, were used. In contrast, intermediate 16 was
easily de-O-methylated using 48% HBr which gave the acid 18
which was then C-7 functionalized with arylpiperazines a–c as
well as with 2-(1-piperazinyl)pyrazine (d),14 and 2-(1-piperazi-
nyl)quinoline (e),15 to afford the target acids 8a–e in good yield.
Focussing our attention on the N-1 and C-6 positions, new quin-
olone derivatives (compounds 4–8, Fig. 2) were designed and syn-
thesized in this study. The 4-arylpiperazines that conferred high
potency on the previous 6-DFQs were maintained at the C-7 posi-
tion. In particular, in the hope of obtaining a more pronounced
reduction of the cytotoxicity while conserving antiviral potency,
6-hydrogenquinolones 4a–c were prepared, together with 6-fluoro
analogs 5a–c, which incorporate a C-6 fluorine atom, the typical
substituent in antibacterial4 and some anti-HIV quinolones7–9
which is known to be responsible for the optimum pharmacoki-
netic characteristics, as well as for biological target recognition.
To further explore the role of the C-6 substituent in the antiviral
activity/cytotoxicity, groups with different chemical physical prop-
erties have been introduced at the C-6 position to give trifluoro-
methyl 6a–c, methoxy 7a–c, and hydroxy derivatives 8a–e. Until
now, these moieties have never been explored as C-6 substituent
in the anti-HIV quinolone field.
Herein we report the synthesis of these novel quinolone series
and their anti-HIV activity evaluation in MT-4 and PBMC cell cul-
tures which led to the identification of new antiviral compounds
and broadening of the SAR at the C-6 position.
3. Results and discussion
All of the new synthesized quinolones were initially evaluated
for anti-HIV-1 (IIIB) and anti-HIV-2 (ROD) activity in MT-4 cells,
using the MTT assay and expressed as the concentration of com-
pound required to protect 50% (EC50) of the MT-4 cells from HIV-
induced cytopathogenicity. The cytotoxicity of the compounds
was determined in parallel and expressed as the concentration of
compound that reduces the viability of mock-infected cells by
50% (CC50). For comparative purposes, compound WM5, nucleoside
reverse transcriptase inhibitor, AZT, and non-nucleoside reverse
transcriptase inhibitor, nevirapine, were assayed in the same cells.
The synthesis of the two series of N-1 unsubstituted quinolones
4a–c and 5a–c gave disappointing results. In fact, non-reproducible
biological data (not shown) were obtained assaying the com-
pounds in triplicate. This behavior is not attributable to their insta-
bility since this issue was tested. The low solubility in the DMSO/
H2O mixture used for the biological test, could instead account
for this behavior. Consequently, these compounds did not contrib-
ute to the SAR widening but only discouraged the synthesis of fur-
ther analogs.
2. Chemistry
The 6-hydrogenquinolones 4a–c and 6-fluoro analogs 5a–c
were synthesized according to Scheme 1 reacting 7-fluoro- and
6,7-difluoro-4-hydroxyquinoline-3-carboxilic acid (9)10 and
(10),11 respectively, with 1-(2-pyridinyl)piperazine (a), 1-[3-(tri-
fluoromethyl)phenyl]piperazine (b) and 2-(1-piperazinyl)-1,3-
benzothiazole (c)12 in DMSO, using Et3N or DIPEA as HF scavenger.
The nucleophilic reaction carried out on the corresponding ethyl
esters, did not proceed.
The usual Gould–Jacobs procedure was applied for the synthesis
of 6-trifluoromethylquinolones 6a–c (Scheme 2). The reaction of 3-
OH
OH
R6
F
CO2H
R6
N
CO2H
i
N
N
N
Ar
9 R6 = H
4a-c R6 = H
5a-c R6 = F
10 R6 = F
Thus, attention was then focused exclusively on the C-6 posi-
tion, keeping the methyl group at N-1 position because it gives
higher solubility. Regarding the C-6 modified quinolones (Table
Scheme 1. Reagents and conditions: (i) Ar-piperazine (for Ar, see Fig. 2), DIPEA or
Et3N, DMSO, 120 °C.

S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
669
EtO2C
CO2Et
F3C
F
F3C
F
i
NH2
N
H
11
OH
N
O
R6
F
CO2Et
R6
F
CO2Et
iii
ii
N
Me
12 R6 = CF3
15 R6 = OMe
13 R6 = CF3
16 R6 = OMe
vi
iv or v
O
O
N
Me
8a-e
O
HO
F
CO2H
HO
N
CO2H
R6
CO2Et
v
N
Me
18
N
N
N
Me
N
Ar
Ar
14a-c R6 = CF3
17a-c R6 = OMe
vii
O
R6
CO2H
N
N
N
Me
Ar
6a-c R6 = CF3
7a-c R6 = OMe
Scheme 2. Reagents and conditions: (i) EMME, 120 °C; (ii) PPA, 100 °C; (iii) MeI, K2CO3, DMF, 100 °C; (iv) Ar-piperazine, (for Ar, see Fig. 2), Et3N, DMSO, 120 °C; (v) N-methyl-
2-pyrrolidone, 105 °C; (vi) 48% HBr, reflux; (vii) 4% NaOH, reflux.
Table 1
Anti-HIV-1 and -HIV-2 activity and cytotoxicity of quinolones in MT-4 cells
Compound
HIV-1(IIIB) EC50
(l
g/mL)a,c
HIV-2(ROD) EC50
(l
g/mL)a,c
CC50
(
l
g/mL)b,c
SI (IIIB)d
SI (ROD)d
6a
6b
6c
7a
7c
8a
8b
8c
>13.93
>1.65
>0.54
0.47 0.02
>0.05
1.85 0.76
P1.13
>0.02
>13.93
>1.65
>0.54
0.40 0.09
>0.05
1.31 0.89
P0.133
>0.02
13.93 0.35
1.65 1.27
0.54 0.08
2.55 0.13
0.05 0.03
>125
1.13 0.90
0.02 0.00
>125
<1
<1
<1
5
<1
<1
<1
6.5
<1
>96
68
<1
<1
14
10
<1
>68
61
<1
<1
6
15
>176
1340
8d
>125
>125
8e
0.080 0.021
0.058 0.021
0.023 0.013
0.0015 0.0005
0.033 0.010
0.097 0.053
>4
0.47 0.02
0.84 0.40
>4
1e
Nevirapine
AZT
0.0008 0.0003
2.01 0.35
2475
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cells from HIV induced cytopathogenicity, as determined by the MTT method.
CC50: concentration of compound that reduces the viability of mock-infected cells by 50%, as determined by the MTT method.
All data represent mean values standard deviations for three separate experiments.
b
c
d
e
SI: ratio of CC50/EC50
.
Data from Ref. 3.
1), the 6-trifluoromethyl derivatives 6a–c showed variable cyto-
toxic effects, with 6a being the less cytotoxic. However, all of these
derivatives were completely devoid of any antiviral properties at
concentrations below the cytotoxic levels. On the contrary, in the
6-methoxyquinolone series 7a–c the presence of the 1-(2-pyridi-
nyl)piperazine at the C-7 position resulted in derivative 7a which
was endowed with good antiviral activity against both HIV-1 and
HIV-2 and showed reduced cytotoxicity compared to the lead com-
pound 1.1,3 The presence of a 7-benzothiazolpiperazine gave the
toxic compound 7c, while, the antiviral results were not reproduc-
ible for the 7-(trifluoromethyl)phenylpiperazinyl derivative 7b
(not shown).
8a was devoid of any cytoxicity (CC50 >125
coupled with the good anti-HIV activity led to SI values >68 and
>96 in MT-4 cells for HIV-1 and HIV-2, respectively.
l
g/mL) which when
Thus, in an attempt to increase the anti-HIV potency, while
maintaining the absence of toxicity, the 6-hydroxy series was en-
larged by synthesizing derivatives 8d and 8e, bearing the 2-(1-pip-
erazinyl)pyrazine and 2-(1-piperazinyl)quinoline at the C-7
position, respectively, which are strict analogs of the 1-(2-pyridi-
nyl)piperazine. Contrasting results were once again obtained.
Derivative 8d was neither toxic nor active while 8e recovered a
very potent anti-HIV activity against both HIV-1 and HIV-2 but also
showed a more pronounced level of toxicity.
Regarding the 6-hydroxy quinolone derivatives 8a–c, contrast-
ing biological results were obtained, but a new interesting com-
pound was obtained. In fact, the 7-pyridinylpiperazinyl derivative
When compound 8e was compared to clinically used drugs such
as non-nucleoside and nucleoside reverse transcriptase inhibitors,
nevirapine and AZT (Table 1), it was less active (particularly with

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S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
respect to AZT) and slightly more toxic. However, since 6-DFQs act
on an innovative step of the replicative cycle, even though they are
less potent, they could be promising candidates for use in associa-
tion with the currently used cocktail of drugs to fight resistance
and control HIV latency.
Molecules displaying a good profile in MT-4 cell lines, were also
evaluated in acutely infected PBMCs from healthy donors (Table 2).
Interestingly, all derivatives maintained good anti-HIV-1 activity
confirming 6-hydroxy derivatives 8a and 8e as the most selective
and potent quinolones obtained in this study.
5. Experimental
5.1. Chemistry
All reaction were routinely checked by thin-layer chromatogra-
phy (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 deter-
mined in capillary tubes (Büchi Electrothermal Mod. 9100) and
are uncorrected. Elemental analyses were performed on a Carlo
Erba elemental analyzer, Model 1106, 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 solvent such
as chloroform (d = 7.26) or dimethylsulfoxide (d = 2.48) as an inter-
nal standard. Chemical shifts are given in ppm (d) and the spectral
data are consistent with the assigned structures. Reagents and sol-
vents were purchased from commercial suppliers and were used as
such. Organic solvents were dried over anhydrous Na2SO4 and con-
centrated with a Büchi rotary evaporator at reduced pressure.
Yields are of purified product and were not optimized. All starting
materials were commercially available unless otherwise indicated.
4. Conclusion
In summary, starting from the novelties highlighted in the re-
cent SAR for the anti-HIV 6-DFQs, innovative quinolone structures
were designed and synthesized. While the N-1 unsubstituted quin-
olone series 4 and 5 was unproductive, new insights about the role
of the substituents at C-6 position, emerged. The trifluoromethyl
group is absolutely not suitable, while the methoxy group can oc-
cupy this position when coupled with an appropriate 4-arylpiper-
azine at the neighboring C-7 position. Similar observations as for
the 6-methoxy derivatives but more marked were apparent in
the 6-hydroxy quinolone series 8 where arylpiperazines a and d
gave compounds with no observed cytotoxicity, while arylpiper-
azine e produced a very potent derivative. These data showed a
strict interdependency between the C-6 and C-7 substituents in
modulating the biological property, in agreement with what has
been previously observed for other series of 6-DFQs.1–3 In an ef-
forts to understand the C-6/C-7 relationship, a molecular modeling
study will be performed to determine the extent to which the aryl-
piperazine conformation is influenced by the nature of the C-6
substituent.
5.2. General procedure for coupling reaction (method A)
A mixture of the appropriate synthone (9,15 1011 and 13)
(1 equiv), selected arylpiperazine (3 equiv) and DIPEA or Et3N
(4 equiv), in DMSO was heated at 120 °C until no starting material
could be detected by TLC (6–48 h). The reaction mixture was then
poured into ice/water and the precipitate was filtered, washed
with water and EtOH, and crystallized by EtOH/DMF.
5.2.1. 4-Hydroxy-7-(4-pyridin-2-ylpiperazin-1-yl)quinoline-3-
carboxylic acid (4a)
A
clear indication of structure/cytotoxicity relationship
The title compound was prepared starting from synthone 915
through the general procedure for coupling reaction (method A),
using 1-(2-pyridinyl)piperazine (a) and DIPEA (46 h) in 59% yield:
mp 267–268 °C; 1H NMR (DMSO-d6) d 3.50–3.55 and 3.65–3.75 (m,
each 4H, piperazine CH2), 6.65 (dd, J = 7.1 and 4.9 Hz, 1H, pyridine
CH), 6.90 (d, J = 8.5 Hz, 1H, pyridine CH), 6.95 (br s, 1H, H-8), 7.35
(d, J = 9.2 Hz, 1H, H-6), 7.50–7.60 (m, 1H, pyridine CH), 8.10 (d,
J = 9.2 Hz, 1H, H-5), 8.15–8.25 (m, 1H, pyridine CH), 8.70 (s, 1H,
H-2), 12.80 (br s, 1H, OH), 15.80 (s, 1H, COOH). Anal. Calcd for
C19H18N4O3: C, 65.13; H, 5.18; N, 15.99. Found: C, 65.23; H, 5.00;
N, 16.10.
emerged for the C-7 substituent itself, where, independently from
the C-6 substituent, the 1-(2-pyridinyl)piperazine (a) produced the
less toxic compounds, followed by the 1-[3-(trifluoro-
methyl)phenyl]piperazine (b) and finally by 2-(1-piperazinyl)-
1,3-benzothiazole (c), which characterizes the most toxic
derivatives.
Thus, in this study new interesting anti-HIV quinolones have
been disclosed (7a, 8a and 8e), and the hydroxyl group has been
identified as a new suitable C-6 substituent, together with the
known NH2 group and the H atom. However, it was not possible
to reduce the cytotoxicity while maintaining the high anti-HIV po-
tency. Perhaps this goal will only be reached when the mechanism
of action of the 6-DFQs is definitively clarified. Many papers have
reported that these compounds are Tat-mediated transcription
inhibitors,1,3,16–18 but their molecular target has not been fully
identified. Fishing for the target is, at the moment, one of our prin-
cipal interests, but it is not an easy task since there are so many
cellular and viral components involved in the regulation of
transcription.
5.2.2. 4-Hydroxy-7-{4-[3-(trifluoromethyl)phenyl]piperazin-1-
yl}quinoline-3-carboxylic acid (4b)
The title compound was prepared starting from synthone 915
through the general procedure for coupling reaction (method A),
using 1-[3-(trifluoromethyl)phenyl]piperazine (b) and DIPEA
(27 h) in 55% yield: mp 281–283 °C; 1H NMR (DMSO-d6) d 3.40–
3.50 and 3.50–3.60 (m, each 4H, piperazine CH2), 7.00 (s, 1H, H-
8), 7.10 (d, J = 7.6 Hz, 1H, aromatic CH), 7.25 (s, 1H, aromatic CH),
7.30 (d, J = 8.5 Hz, 1H, aromatic CH), 7.40 (dd, J = 9.2 and 2.0 Hz,
1H, H-6), 7.40–7.50 (m, 1H, aromatic CH), 8.10 (d, J = 9.2 Hz, 1H,
H-5), 8.75 (s, 1H, H-2), 12.80 (br s, 1H, OH), 15.80 (s, 1H, COOH).
Anal. Calcd for C21H18F3N3O3: C, 60.43; H, 4.35; N, 10.07. Found:
C, 60.05; H, 4.55; N, 10.31.
Table 2
Anti-HIV-1 activity and cytotoxicity of selected quinolones in PBMCs
Compound
HIV-1(IIIB) EC50
(l
g/mL)a,c
CC50
(l
g/mL)b,c
SId
7a
8a
8b
8e
1.78 0.17
0.39 0.05
0.40 0.06
0.26 0.12
8.46 5.61
P17.16
1.61 0.52
1.91 0.12
5
P44
4
7
a
5.2.3. 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-4-
EC50: concentration of compound required to achieve 50% inhibition of p24
production in PBMC.
hydroxyquinoline-3-carboxylic acid (4c)
b
The title compound was prepared starting from synthone 915
through the general procedure for coupling reaction (method A),
using 2-(1-piperazinyl)-1,3-benzothiazole (c)12 and DIPEA (48 h)
in 60% yield: mp 275–277 °C; 1H NMR (DMSO-d6) d 3.50–3.60
CC50: concentration of compound that reduces the viability of mock-infected
cells by 50%, as determined by the trypan blue exclusion method.
c
All data represent mean values standard deviations for at least two separate
experiments.
d
SI: ratio of CC50/EC50
.

S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
671
and 3.70–3.80 (m, each 4H, piperazine CH2), 7.00 (br s, 1H, H-8),
7.10 and 7.30 (t, J = 7.8 Hz, each 1H, benzothiazole CH), 7.35 (br
d, J = 9.2 Hz, 1H, H-6), 7.50 and 7.80 (d, J = 7.8 Hz, each 1H, benzo-
thiazole CH), 8.10 (d, J = 9.2 Hz, 1H, H-5), 8.70 (s, 1H, H-2), 12.50
(br s, 1H, OH), 15.80 (s, 1H, COOH). Anal. Calcd for C21H18N4O3S:
C, 62.05; H, 4.46; N, 13.78. Found: C, 62.10; H, 4.62; N, 13.45.
1H, aromatic CH), 7.45–7.55 (m, 2H, H-8 and aromatic CH), 7.60–
7.70 and 7.80–7.85 (m, each 1H, aromatic CH), 8.65 (s, 1H, H-5),
8.85 (s, 1H, H-2).
5.2.9. Ethyl 7-[4-(1,3-benzothiazol-2-yl)piperazin-1-yl]-1-
methyl-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-
carboxylate (14c)
5.2.4. 6-Fluoro-4-hydroxy-7-(4-pyridin-2-ylpiperazin-1-
yl)quinoline-3-carboxylic acid (5a)
The title compound was prepared starting from synthone 13
through general procedure for coupling reaction (method A), using
2-(1-piperazinyl)-1,3-benzothiazole (c)12 and Et3N (36 h). After
flash column chromatography purification (1% MeOH/CH2Cl2) the
title compound was obtained in 26% yield: mp 215–216 °C; 1H
NMR (CDCl3) d 1.45 (t, J = 7.2 Hz, 3H, CH2CH3), 3.15–3.25 (m, 4H,
piperazine CH2), 3.80 (s, 3H, CH3), 3.85–3.95 (m, 4H, piperazine
CH2), 4.45 (q, J = 7.2 Hz, 2H, CH2CH3), 7.10–7.20 (m, 2H, H-8 and
aromatic CH), 7.30–7.40, 7.55–7.65 and 7.70–7.75 (m, each 1H,
aromatic CH), 8.45 (s, 1H, H-5), 8.80 (s, 1H, H-2).
The title compound was prepared starting from synthone 1011
through the general procedure for coupling reaction (method A),
using 1-(2-pyridinyl)piperazine (a) and Et3N (11 h) in 58% yield:
mp 279–280 °C; 1H NMR (DMSO-d6) d 3.25–3.35 and 3.65–3.75
(m, each 4H, piperazine CH2), 6.65–6.75 (m, 1H, pyridine CH),
6.85 (d, J = 8.9 Hz, 1H, H-8), 7.30 (d, J = 7.0 Hz, 1H, pyridine CH),
7.50–7.65 (m, 1H, pyridine CH), 7.85 (d, J = 13.4 Hz, 1H, H-5),
8.10–8.20 (m, 1H, pyridine CH), 8.80 (s, 1H, H-2), 13.25 (br s, 1H,
OH), 15.40 (s, 1H, COOH). Anal. Calcd for C19H17FN4O3: C, 61.95;
H, 4.65; N, 15.21. Found: C, 62.15; H, 4.60; N, 15.14.
5.3. Diethyl 2-({[3-fluoro-4-
(trifluoromethyl)phenyl]amino}methylene)malonate (11)
5.2.5. 6-Fluoro-4-hydroxy-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}quinoline-3-carboxylic
acid (5b)
A
mixture of 3-fluoro-4-(trifluoromethyl)aniline (5.0 g,
27.9 mmol) and EMME (6.02 g, 27.9 mmol) was heated at 120 °C
for 2.5 h. The mixture was then evaporated to dryness to give a res-
idue which was triturated with cyclohexane to give a solid which
was filtered and dried to afford compound 11 (5.8 g, 59%): mp
88–89 °C; 1H NMR (CDCl3) d 1.30–1.45 (m, 6H, CH2CH3), 4.20–
4.40 (m, 4H, CH2CH3), 6.90–7.00 (m, 2H, H-2 and H-6), 7.55–7.65
(m, 1H, H-5), 8.45 (d, J = 13.2 Hz, 1H, vinyl-H), 11.10 (d,
J = 13.2 Hz, 1H, NH).
The title compound was prepared starting from synthone 1011
through the general procedure for coupling reaction (method A),
using 1-[3-(trifluoromethyl)phenyl]piperazine (b) and Et3N (6 h)
in 77% yield: mp 315–316 °C; 1H NMR (DMSO-d6) d 3.30–3.50
(m, 8H, piperazine CH2), 7.10 (d, J = 7.5 Hz, 1H, aromatic CH),
7.20–7.35 (m, 3H, H-8 and aromatic CH), 7.40–7.50 (m, 1H, aro-
matic CH), 7.85 (d, J = 13.4 Hz, 1H, H-5), 8.80 (s, 1H, H-2), 15.50
(s, 1H, COOH). Anal. Calcd for C21H17F4N3O3: C, 57.93; H, 3.94; N,
9.65. Found: C, 57.99; H, 4.12; N, 9.48.
5.4. Ethyl 7-fluoro-1-methyl-4-oxo-6-(trifluoromethyl)-1,4-
dihydroquinoline-3-carboxylate (13)
5.2.6. 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-6-fluoro-4-
hydroxyquinoline-3-carboxylic acid (5c)
A mixture of 11 (5.5 g, 15.7 mmol) and PPA was heated at
100 °C for 2 h, then poured into ice/water and neutralized with
10% NaOH. The obtained precipitate was filtered, washed with
water and with a mixture of CHCl3/EtOH and dried to give ethyl
7-fluoro-4-hydroxy-6-(trifluoromethyl)quinoline-3-carboxylate
(12) (1.5 g, 31%), that was used in the next step without further
characterization due to its low solubility in the common deuter-
ated solvent. To a mixture of synthone 12 (1 g, 32 mmol) and
K2CO3 (1.13 g, 82 mmol) in DMF (5 mL), a solution of MeI (0.93 g,
65 mmol) in DMF (2 mL) was added. The mixture was heated to
100 °C for 12 h and then poured into ice/water. The obtained pre-
cipitate was filtered, dried and purified by flash chromatography,
eluting with MeOH/CHCl3 (2%) to give 13 (0.5 g, 49%): mp 177–
178 °C; 1H NMR (CDCl3) d 1.48 (t, J = 7.1 Hz, 3H, CH2CH3), 3.80 (s,
3H, CH3), 4.40 (q, J = 7.1 Hz, 2H, CH2CH3), 7.25 (d, J = 11.2 Hz, 1H,
H-8), 8.50 (s, 1H, H-2), 8.80 (d, J = 7.9 Hz, 1H, H-5).
The title compound was prepared starting from synthone 1011
through the general procedure for coupling reaction (method A),
using 2-(1-piperazinyl)-1,3-benzothiazole (c)12 and Et3N (24 h) in
37% yield: mp 313–315 °C; 1H NMR (DMSO-d6) d 3.40–3.50 and
3.70–3.85 (m, each 4H, piperazine CH2), 7.05–7.15 (m, 1H, benzo-
thiazole CH), 7.25–7.35 (m, 2H, benzothiazole CH and H-8), 7.50 (d,
J = 7.8 Hz, 1H, benzothiazole CH), 7.65–7.75 (m, 1H, benzothiazole
CH), 7.85 (d, J = 13.4 Hz, 1H, H-5), 8.85 (s, 1H, H-2), 15.50 (s, 1H,
COOH). Anal. Calcd for C21H17FN4O3S: C, 59.42; H,4.04; N 13.20.
Found: C, 59.47; H, 4.36; N, 13.00.
5.2.7. Ethyl 1-methyl-4-oxo-7-(4-pyridin-2-ylpiperazin-1-yl)-6-
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate (14a)
The title compound was prepared starting from synthone 13
through the general procedure for coupling reaction (method A),
using 1-(2-pyridinyl)piperazine (a) and Et3N (24 h) in 58% yield:
mp 136–137 °C; 1H NMR (CDCl3) d 1.45 (t, J = 7.1 Hz, 3H, CH2CH3),
3.15–3.25 and 3.75–3.85 (m, each 4H, piperazine CH2), 3.90 (s, 3H,
CH3), 4.45 (q, J = 7.1 Hz, 2H, CH2CH3), 6.65–6.85 (m, 2H, pyridine
CH), 7.10 (s, 1H, H-8), 7.60–7.65 (m, 1H, pyridine CH), 8.15–8.25
(m, 1H, pyridine CH), 8.48 (s, 1H, H-5), 8.75 (s, 1H, H-2).
5.5. General procedure for basic hydrolysis
A suspension of selected ester (14a–c and 17a–c) (0.3 m equiv)
in 4% NaOH (5 mL) was refluxed until no starting material could be
detected by TLC (15 min–6 h) and worked and purified as defined
in the description of the compounds.
5.2.8. Ethyl 1-methyl-4-oxo-6-(trifluoromethyl)-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydroquinoline-
3-carboxylate (14b)
The title compound was prepared starting from synthone 13
through general procedure for coupling reaction (method A), using
1-[3-(trifluoromethyl)phenyl]piperazine (b) and Et3N (48 h) in 71%
yield: mp 177–178 °C; 1H NMR (DMSO-d6) d 1.50 (t, J = 7.0 Hz, 3H,
CH2CH3), 3.15–3.25 and 3.35–3.45 (each 4H, m, piperazine CH2),
4.20 (s, 3H, CH3), 4.40 (q, J = 7.0 Hz, 2H, CH2CH3), 7.25–7.35 (m,
5.5.1. 1-Methyl-4-oxo-7-(4-pyridin-2-ylpiperazin-1-yl)-6-
(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid
hydrochloride (6a)
The title compound was prepared starting from 14a through the
general procedure for basic hydrolysis (6 h): after cooling, the pre-
cipitate was filtered, suspended in 2 N HCl (2 mL) and refluxed for
1 h. After cooling, the hydrochloride was filtered, washed with
water, EtOH and finally recrystallized by EtOH/DMF to give 6a in

672
S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
40% yield: mp 287–288 °C; 1H NMR (DMSO-d6) d 3.20–3.30 and
3.80–3.90 (m, each 4H, piperazine CH2), 4.15 (s, 3H, CH3), 6.85–
6.95 and 7.30–7.40 (m, each 1H, pyridine CH), 7.75 (s, 1H, H-8),
7.85–8.00 and 8.10–8.20 (m, each 1H, pyridine CH), 8.60 (s, 1H,
H-5), 9.10 (s, 1H, H-2). Anal. Calcd for C21H20ClF3N4O3: C, 58.33;
H, 4.43; N, 12.96. Found: C, 58.18; H, 4.27; N, 12.99.
15.75 (s, 1H, COOH). Anal. Calcd for C23H22F3N3O4: C, 59.87; H,
4.81; N, 9.11. Found: C, 60.05; H, 4.83; N, 9.07.
5.5.6. 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-6-methoxy-
1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (7c)
The title compound was prepared starting from 17c through
general procedure for basic hydrolysis (15 min): after cooling, the
mixture was neutralized by adding 2 N HCl and the solid obtained
was collected by filtration, washed with water, EtOH and finally
recrystallized by EtOH/DMF to give 7c in 68% yield: mp 279–
280 °C; 1H NMR (DMSO-d6) d 3.40–3.50 and 3.75–3.85 (m, each
4H, piperazine CH2), 3.90–4.10 (m, each 3H, CH3 and OCH3),
7.10–7.20 (m, 2H, H-8 and aromatic CH), 7.30–7.40 (m, 1H, aro-
matic CH), 7.50–7.90 (m, 3H, H-5 and aromatic CH), 8.90 (s, 1H,
H-2). Anal. Calcd for C23H22N4O4S: C, 61.32; H, 4.92; N, 12.44.
Found: C, 61.70; H, 4.69; N, 12.54.
5.5.2. 1-Methyl-4-oxo-6-(trifluoromethyl)-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydroquinoline-
3-carboxylic acid (6b)
The title compound was prepared starting from 14b through
general procedure for basic hydrolysis, (2 h): after cooling, the
mixture was completely acidified by adding 2 N HCl and the solid
obtained was collected by filtration, washed with water, EtOH and
finally recrystallized by EtOH/DMF to give 6b in 4% yield: mp 233–
234 °C; 1H NMR (DMSO-d6) d 3.20–3.30 and 3.70–3.80 (m, each 4H,
piperazine CH2), 4.20 (s, 3H, CH3), 7.10–7.20 (m, 1H, aromatic CH),
7.25–7.35 (m, 2H, aromatic CH and H-8), 7.40–7.50 and 7.70–7.80
(m, each 1H, and aromatic CH), 8.60 (s, 1H, H-5), 9.10 (s, 1H, H-2),
14.80 (s, 1H, COOH). Anal. Calcd for C23H19F6N3O3: C, 55.31; H,
3.83; N, 8.41. Found: C, 55.55; H, 3.98; N, 8.21.
5.6. Ethyl 7-fluoro-6-methoxy-1-methyl-4-oxo-1,4-
dihydroquinoline-3-carboxylate (16)
Starting from intermediate 1513 and following the procedure as
used for the synthesis of compound 13 (75 °C, 1 h), the title com-
pound was obtained in 95% yield: mp 220–221 °C; 1H NMR
(DMSO-d6) d 1.25 (t, J = 6.7 Hz, 3H, CH2CH3), 3.85 (s, 3H, CH3),
3.95 (s, 3H, OCH3), 4.20 (t, J = 6.7 Hz, 2H, CH2CH3), 7.70–7.80 (m,
2H, H-5 and H-8), 8.60 (s, 1H, H-2).
5.5.3. 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-1-methyl-4-
oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic
acid (6c)
The title compound was prepared starting from 14c through
general procedure for basic hydrolysis (1 h): after cooling, the mix-
ture was completely acidified by adding 2 N HCl and the solid ob-
tained was collected by filtration, washed with water and EtOH
and finally recrystallized by EtOH/DMF to give 6c in 49% yield:
mp 326–327 °C; 1H NMR (DMSO-d6) d 3.20–3.30 and 3.35–3.45
(m, each 4H, piperazine CH2), 4.15 (s, 3H, CH3), 7.10–7.20, 7.25–
7.35 and 7.40–7.50 (m, each 1H, aromatic CH), 7.75–7.85 (m, 2H,
H-8 and aromatic CH), 8.60 (s, 1H, H-5), 9.10 (s, 1H, H-2), 14.75
(s, 1H, COOH). Anal. Calcd for C23H19F3N4O3S: C, 56.55; H, 3.92;
N, 11.47. Found: C, 83; H, 3.75; N, 11.59.
5.7. General procedure for coupling reaction (method B)
A mixture of the appropriate synthone (16 and 18) (1 equiv) and
selected arylpiperazine (6 equiv) in N-methyl-2-pyrrolidone, was
heated at 105 °C until no starting material could be detected by
TLC (15–24 h) and worked and purified as defined in the descrip-
tion of the compounds.
5.7.1. Ethyl 6-methoxy-1-methyl-4-oxo-7-(4-pyridin-2-
ylpiperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (17a)
The title compound was prepared starting from synthone 16
through general procedure for coupling reaction (method B), using
1-(2-pyridinyl)piperazine (a) (24 h). The reaction mixture was
poured into ice/water giving a solid which was filtered, washed
with water and Et2O to give 17a in 60% yield: mp 206–207 °C;
1H NMR (DMSO-d6) d 1.25 (t, J = 7.0 Hz, 3H, CH2CH3), 3.20–3.30
and 3.55–3.65 (m, each 4H, piperazine CH2), 3.90 (s, 6H, CH3 and
OCH3), 4.20 (q, J = 7.0 Hz, 2H, CH2CH3), 6.60–6.70 and 6.80–6.90
(m, each 1H, pyridine CH), 6.95 (s, 1H, H-8), 7.50–7.65 (m, 2H, pyr-
idine CH and H-5), 8.10–8.20 (m, 1H, pyridine CH), 8.55 (s, 1H, H-
2).
5.5.4. 6-Methoxy-1-methyl-4-oxo-7-(4-pyridin-2-ylpiperazin-
1-yl)-1,4-dihydroquinoline-3-carboxylic acid hydrochloride
(7a)
The title compound was prepared starting from 17a through
general procedure for basic hydrolysis (30 min): after cooling, the
precipitate was filtered, suspended in 2 N HCl and refluxed for
1 h. After cooling, the precipitate was filtered, washed with water,
EtOH and finally recrystallized by EtOH/DMF to give 7a in 98%
yield: mp 286–288 °C; 1H NMR (DMSO-d6) d 3.40–3.50 and 3.75–
3.85 (m, each 4H, piperazine CH2), 3.95 and 4.05 (s, each 3H, CH3
and OCH3), 6.80–6.90 (m, 1H, pyridine CH), 7.10 (s, 1H, H-8),
7.20–7.30 (m, 1H, pyridine CH), 7.65 (s, 1H, H-5), 7.80–7.90 and
8.10–8.20 (m, each 1H, pyridine CH), 8.90 (s, 1H, H-2), 15.75 (br
s, 1H, COOH). Anal. Calcd for C21H23ClN4O4: C, 63.95; H, 5.62; N,
14.20. Found: C, 64.09; H, 5.73; N, 14.02.
5.7.2. Ethyl 6-methoxy-1-methyl-4-oxo-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydroquinoline-
3-carboxylate (17b)
The title compound was prepared starting from synthone 16
through general procedure for coupling reaction (method B),
using 1-[3-(trifluoromethyl)phenyl]piperazine (b) (24 h). The
reaction mixture was poured into ice/water and extracted with
EtOAc; the organic layers were evaporated to give an oil which
by treatment with H2O/MeOH gave a solid which was filtered
and dried to give 17b in 55% yield: mp 127–128 °C; 1H NMR
5.5.5. 6-Methoxy-1-methyl-4-oxo-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydroquinoline-
3-carboxylic acid (7b)
The title compound was prepared starting from 17b through
general procedure for basic hydrolysis (15 min): after cooling, the
mixture was neutralized by adding 2 N HCl and the solid obtained
was collected by filtration, washed with water, EtOH and finally
recrystallized by EtOH/DMF to give 7b in 53% yield: mp 258–
259 °C; 1H NMR (DMSO-d6) d 3.30–3.45 (m, 8H, piperazine CH2),
3.95 and 4.05 (s, each 3H, CH3 and OCH3), 7.05–7.15 (m, 2H, H-8
and aromatic CH), 7.20–7.35 (m, 2H, H-5 and aromatic CH), 7.40–
7.50 and 7.65–7.75 (m, each 1H, aromatic CH), 8.85 (s, 1H, H-2),
(DMSO-d6)
d 1.25 (t, J = 7.0 Hz, 3H, CH2CH3), 3.30–3.40 and
3.70–3.80 (m, each 4H, piperazine CH2), 3.85–3.95 (br s, 6H,
CH3 and OCH3), 4.20 (q, J = 7.0 Hz, 2H, CH2CH3), 6.90–7.00 and
7.05–7.15 (m, each 1H, aromatic CH), 7.20–7.30 (m, 2H, aromatic
CH and H-8), 7.45–7.55 (m, 1H, aromatic CH), 7.65 (s, 1H, H-
5),8.55 (s, 1H, H-2).

S. Massari et al. / Bioorg. Med. Chem. 17 (2009) 667–674
673
5.7.3. Ethyl 7-[4-(1,3-benzothiazol-2-yl)piperazin-1-yl]-6-
methoxy-1-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate
(17c)
CH2), 4.10 (s, 3H, CH3), 7.10 (s, 1H, H-8), 7.70 (s, 1H, H-5), 7.90,
8.20 and 8.45 (s, each 1H, pyrazine CH), 8.80 (s, 1H, H-2), 10.70
(s, 1H, OH), 16.00 (s, 1H, COOH). Anal. Calcd for C19H19N5O4: C,
59.84; H, 5.02; N, 18.36. Found: C, 59.92; H, 5.00; N, 18.48.
The title compound was prepared starting from synthone 15
through general procedure for coupling reaction (method B), using
2-(1-piperazinyl)-1,3-benzothiazole (c)12 (24 h). The reaction mix-
ture was poured into ice/water and extracted with EtOAc; the or-
ganic layers were evaporated to give an oil which by treatment
with H2O/MeOH gave a solid which was filtered and dried to give
17c in 47% yield: mp 153–154 °C; 1H NMR (DMSO-d6) d 1.25 (t,
J = 6.6 Hz, 3H, CH2CH3), 3.25–3.40 and 3.70–3.80 (m, each 4H,
piperazine CH2), 3.90 (s, 6H, CH3 and OCH3), 4.20 (q, J = 6.6 Hz,
2H, CH2CH3), 6.95 (s, 1H, H-8), 7.05–7.15, 7.20–7.30 and 7.40–
7.50 (m, each 1H, aromatic CH),7.65 (s, 1H, H-5) 7.75–7.85 (m,
1H, aromatic CH), 8.55 (s, 1H, H-2).
5.7.8. 6-Hydroxy-1-methyl-4-oxo-7-(4-quinolin-2-ylpiperazin-
1-yl)-1,4-dihydroquinoline-3-carboxylic acid (8e)
The title compound was prepared starting from synthone 18
following the same procedure as used for the synthesis of com-
pound 8a, replacing the 1-(2-pyridinyl)piperazine (a) with the 2-
(1-piperazinyl)quinoline (e)15 in 71% yield: mp 297 °C (dec); 1H
NMR (DMSO-d6) d 3.40–3.50 and 3.90–4.00 (m, each 4H, piperazine
CH2), 4.10 (s, 3H, CH3), 7.10 (s, 1H, H-8), 7.30–7.50 (m, 2H, quino-
line CH), 7.60–7.80 (m, 4H, H-5 and quinoline CH), 8.15–8.20 (m,
1H, quinoline CH), 8.80 (s, 1H, H-2), 10.70 (s, 1H, OH), 16.00 (s,
1H, COOH). Anal. Calcd for C24H22N4O4: C, 66.97; H, 5.15; N,
13.02. Found: C, 66.96; H, 5.21; N, 12.98.
5.7.4. 6-Hydroxy-1-methyl-4-oxo-7-(4-pyridin-2-ylpiperazin-1-
yl)-1,4-dihydroquinoline-3-carboxylic acid (8a)
The title compound was prepared starting from synthone 18
through the general procedure for coupling reaction (method B),
using 1-(2-pyridinyl)piperazine (a) (15 h). After cooling, the mix-
ture was poured into ice/water and acidified with 2 N HCl (pH 6)
giving a solid which was filtered, washed with water, dried and
recrystallized by DMF to give 8a in 44% yield: mp 314–315 °C;
1H NMR (DMSO-d6) d 3.35–3.45 and 3.65–3.75 (m, each 4H, piper-
azine CH2), 4.10 (s, 3H, CH3), 6.65–6.75 and 6.90–7.00 (m, each 1H,
pyridine CH), 7.10 (s, 1H, H-8), 7.50–7.60 (m, 1H, pyridine CH), 7.75
(s, 1H, H-5), 8.10–8.20 (m, 1H, pyridine CH), 8.80 (s, 1H, H-2), 10.70
(s, 1H, OH), 16.00 (s, 1H, COOH). Anal. Calcd for C20H20N4O4: C,
63.15; H, 5.30; N, 14.73. Found: C, 63.28; H, 5.17; N, 14.92.
5.8. 7-Fluoro-6-hydroxy-1-methyl-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (18)
A mixture of compound 16 (0.1 g, 0.3 mmol) in 48% HBr
(10 mL) was refluxed for 5 h. After cooling, the hydrobromide
salt was filtered, solubilized in water and neutralized by adding
10% NaOH. The resulting precipitate was filtered, washed with
water and EtOH, and dried to give the acid 18 in 82% yield:
mp >330 °C; 1H NMR (CDCl3) d 4.00 (s, 3H, CH3), 7.90–8.00 (m,
2H, H-5 and H-8), 9.00 (s, 1H, H-2), 11.00 (s, 1H, OH), 15.40
(s, 1H, COOH).
5.9. In vitro anti-HIV assays
5.7.5. 6-Hydroxy-1-methyl-4-oxo-7-{4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}-1,4-dihydroquinoline-
3-carboxylic acid (8b)
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells was per-
formed using the MTT assay as previously described.19 Briefly,
stock solutions (10 Â final concentration) of test compounds were
The title compound was prepared starting from synthone 18
following the same procedure as used for the synthesis of com-
pound 8a, replacing the 1-(2-pyridinyl)piperazine (a) with the 1-
[3-(trifluoromethyl)phenyl]piperazine (b) in 56% yield: mp 318–
319 °C; 1H NMR (DMSO-d6) d 3.40–3.50 (m, 8H, piperazine CH2),
4.15 (s, 3H, CH3), 7.05–7.15 (m, 2H, H-8 and benzothiazole CH),
7.20–7.30 (m, 2H, benzothiazole CH), 7.40–7.50 (m, 1H, benzothi-
azole CH), 7.65 (m, 1H, H-5), 8.80 (s, 1H, H-2), 10.50 (br s, 1H,
OH), 16.00 (s, 1H, COOH). Anal. Calcd for C22H20F3N3O4: C, 59.06;
H, 4.51; N, 9.39. Found: C, 59.21; H, 4.38; N, 9.27.
added in 25 lL volumes to two series of triplicate wells so as to al-
low simultaneous evaluation of their effects on mock-and HIV-in-
fected 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
instruments, Fullerton, CA). Untreated control HIV-and mock-in-
fected cell samples were included for each sample.
HIV-1(IIIB)20 or HIV-2 (ROD)21 stock (50
lL) 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 com-
pound on uninfected cells in order to assess the cytotoxicity of
the test compound. Exponentially growing MT-4 cells22 were cen-
trifuged for 5 min at 1000 rpm and the supernatant was discarded.
5.7.6. 7-[4-(1,3-Benzothiazol-2-yl)piperazin-1-yl]-6-hydroxy-1-
methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8c)
The title compound was prepared starting from synthone 17
following the same procedure as used for the synthesis of com-
pound 8a, replacing the 1-(2-pyridinyl)piperazine (a) with the 2-
(1-piperazinyl)-1,3-benzothiazole (c)12 in 57% yield: mp >330 °C;
1H NMR (DMSO-d6) d 3.40–3.50 and 3.75–3.85 (m, each 4H, piper-
azine CH2), 4.10 (s, 3H, CH3), 7.05–7.15 (m, 2H, H-8 and aromatic
CH), 7.25–7.35 and 7.45–7.55 (m, each 1H, aromatic CH), 7.70 (s,
1H, H-5), 7.75–7.85 (m, 1H, aromatic CH), 8.80 (s, 1H, H-2), 10.70
(s, 1H, OH), 16.00 (s, 1H, COOH).
The MT-4 cells were resuspended at 6 Â 105 cells/mL and 50-
lL
volumes were transferred to the microtiter 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
Anal. Calcd for C22H20N4O4S: C, 60.54; H, 4.62; N, 12.84. Found:
C, 60.57; H, 4.62; N, 12.99.
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase 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 (Multi-
scan Ascent Reader, Labsystems, Helsinki, Finland), at two wave-
lengths (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-in-
fected control sample by 50%. The concentration achieving 50%
5.7.7. 6-Hydroxy-1-methyl-4-oxo-7-(4-pyrazin-2-ylpiperazin-1-
yl)-1,4-dihydroquinoline-3-carboxylic acid (8d)
The title compound was prepared starting from synthone 17
following the same procedure as used for the synthesis of com-
pound 8a, replacing the 1-(2-pyridinyl)piperazine (a) with the 2-
(1-piperazinyl)pyrazine (d)14 in 57% yield: mp >330 °C; 1H NMR
(DMSO-d6) d 3.40–3.50 and 3.80–3.90 (m, each 4H, piperazine

674
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protection from the cytopathic effect of the virus in infected cells
was defined as the 50% effective concentration (EC50).
Peripheral blood mononuclear cells (PBMCs) from healthy do-
nors were isolated by density centrifugation (Lymphoprep; Ny-
comed Pharma, AS Diagnostics, Oslo, Norway) and stimulated
with phytohemagglutin (PHA) (Sigma Chemical Co., Bornem Bel-
gium) for three days. The activated cells (PHA-stimulated blasts)
were washed with PBS and viral infections were done as described
by the AIDS clinical trial group protocols.23 Briefly, PBMCs (2 Â 105/
200
compound and were infected with HIV stocks at 1000 CCID50 per
mL. At day 4 post-infection, 125 L of the supernatant of the in-
fected cultures was removed and replaced with 150 L of fresh
lL) were plated in the presence of serial dilutions of the test
l
l
medium containing the test compound at the appropriate concen-
tration. At seven days after plating the cells, p24 antigen was de-
tected in the culture supernatant by an enzyme-linked
immunosorbent assay (Perkin Elmer, Brussels, Belgium).
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Acknowledgments
These investigations were supported in part by the MIUR (PRIN
2006, Roma, Italy). The authors thank Mr. Roberto Bianconi, Mrs.
Kristien Erven and Mr. Kris Uyttersprot for excellent technical
assistance.
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