Structure modifications of 6-aminoquinolones with potent anti-HIV activity

Article abstract of DOI:10.1021/jm049721p

We have recently discovered that 6-aminoquinolone derivatives could be valid leads for the development of new anti-HIV agents because of their new and diversified mode of action. In fact, studies carried out on the lead WM5 showed that this derivative is

Full text of DOI:10.1021/jm049721p

J . Med. Chem. 2004, 47, 5567-5578
5567
Str u ctu r e Mod ifica tion s of 6-Am in oqu in olon es w ith P oten t An ti-HIV Activity1
Oriana Tabarrini,§ Miguel Stevens,† Violetta Cecchetti,§ Stefano Sabatini,§ Micaela Dell’Uomo,§
Giuseppe Manfroni,§ Manlio Palumbo,# Christophe Pannecouque,† Erik De Clercq,† and Arnaldo Fravolini*,§
Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di Perugia, Via del Liceo 1, 06123 Perugia, Italy,
Dipartimento di Scienze Farmaceutiche, Universita` di Padova, Via Marzolo 5, 35131 Padova, Italy, and Rega Institute for
Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received April 13, 2004
We have recently discovered that 6-aminoquinolone derivatives could be valid leads for the
development of new anti-HIV agents because of their new and diversified mode of action. In
fact, studies carried out on the lead WM5 showed that this derivative is able to inhibit the
Tat-mediated long terminal repeat driven transcription, an essential step in the HIV-1
replication cycle. Thus, starting from lead WM5, we performed the design and synthesis of an
enlarged series of 6-aminoquinolones, which permitted some very potent anti-HIV 6-amino
derivatives to be obtained and the structure-activity relationship to be delineated. Some
derivatives, 26c, 26e, 26i, and 26j, proved to be highly effective in inhibiting HIV replication
at 50% inhibitory concentration in the range of 0.0087-0.7 µg/mL in MT-4, PBMCs and CEM
cell lines coupled with positive selectivity indexes that reach values higher than 1000 on CEM
cell lines for compounds 26e and 26i. Time-of-addition experiments clearly confirm that the
new, potent 6-aminoquinolones interact at a postintegration step in the replication cycle of
HIV.
In tr od u ction
The rate of disease progression in AIDS patients has
been significantly reduced through early and aggressive
intervention with highly active antiretroviral therapy
(HAART), which involves a combination of reverse
transcriptase inhibitors and protease inhibitors. How-
ever, several serious problems still remain including
F igu r e 1. Structure of the lead compound WM5.
multidrug resistance,2 toxicity,3 and high cost.4 More-
over, the current antiretroviral regimens are unable to
completely suppress viral replication, thereby allowing
a latent reservoir of HIV-1 to persist which is the major
documented hurdle to virus eradication5 although other
viral sanctuaries may exist.6
DFQs), in which we found that the 6-amino-1-tert-butyl-
7-[4-(2-pyridinyl)-1-piperazinyl]-4-oxo-1,4-dihydroquin-
oline-3-carboxylicacid exhibited good anti-HIV-1 activity.15
To pinpoint the structural features responsible for its
antiviral activity and to explore the structural variations
of this lead compound, we first prepared a series of
6-aminoquinolones variously substituted in the different
positions of the quinolone nucleus.14 The antiviral
activity of this first series of compounds confirmed that
the 6-aminoquinolones could provide valid leads for the
research of new drugs for the treatment of AIDS. From
this series, compound WM5 (Figure 1) was the most
potent, inhibiting HIV-1 replication on de novo infected
C8166 human lymphoblastoid cells, with an EC50 value
of about 0.1 µM and a CC50 of 7 µM (SI ) 70).14 The
same good antiviral activity was also observed in human
T-lymphoid J urkat cells and in chronically infected H9
cells, where WM5 was notably less cytotoxic with a CC50
value of 56 µM in J urkat and no cytotoxicity was
observed in H9 cells.16,17 From the preliminarly struc-
ture-activity relationship (SAR) study of 6-aminoqui-
nolones the structural features that grant the antiviral
activity were determined: the presence of carboxylic
acid at C-3, a small polar group at C-6, a bulky
substituent at C-7, and a small substituent at N-1 of
the quinolone moiety.
In the search for new anti-HIV agents, efforts to
discover compounds with new and diversified modes of
action are still a challenging task. Transcription of the
viral genome (integrated proviral DNA) into its mRNA
is an essential step in the HIV-1 replication cycle and
is considered to be a good potential target for chemo-
therapeutic intervention7 because it could allow the
control of HIV-1 replication not only in acutely infected
cells but also in chronically infected cells. Moreover,
compounds that could interfere with this replication
step would result in a lower incidence of drug resistance
because HIV gene regulation requires the interplay of
both viral and cellular components.8
In this context, quinolones have been pursued as new
potential candidates for the treatment of AIDS.9-14 Our
studies in this area began with the random screening
of our quinolone chemical library, including 6-fluoro-
quinolonones (6-FQs) and 6-desfluoroquinolonones (6-
* To whom correspondence should be addressed. Phone: +39 075
5855130. Fax: +39 075 585 5115. E-mail: fravo@unipg.it.
§ Universita` di Perugia.
Regarding the mechanism of action, studies carried
out on WM5 showed that this compound does not impair
† Katholieke Universiteit Leuven.
#
Universita` di Padova.
10.1021/jm049721p CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/28/2004

5568 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tabarrini et al.
the activities of the reverse transcriptase (RT), HIV
integrase (I), and HIV protease (P).17 Initially the
transcomplementation assay used to examine the activ-
ity of WM5 on the replicative potential of HIV-1 in a
single round of infection showed a sustained inhibition
of Tat mediated long terminal repeat (LTR) driven
transcription.17 Moreover, preliminary binding studies
using fluorimetric titration experiments and conducted
in the presence of different RNA and DNA sequences
indicated that WM5 selectively and efficiently complexes
the transactivation responsive (TAR) element with a
dissociation constant in the nanomolar range (19 ( 0.6
nM), suggesting a nucleic acid targeted mechanism of
action.14,17
substituent was always maintained at the N-1 position
except for compound 27a which had a ethyl group,
compounds 28b, 28c, and 28d which had a cyclopropyl
group, and 29a , 29d , 30a , 31a , 32a , and 33d character-
ized by a bulky substituent (Table 1).
All the synthesized quinolones were tested for their
ability to inhibit both HIV-1 and HIV-2 replication in
MT-4 cells and were also evaluated for cytotoxicity
(Table 3). The anti-HIV activity of the most active
compounds was also evaluated in the human lympho-
cytic CEM cell line (Table 4) and pheripheral blood
mononuclear cells (PBMCs) (Table 5). Time-of-addition
(TOA) experiments were also carried out to further
investigate the step of the replicative cycle that was
inhibited by the new 6-aminoquinolones (Figure 2).
The indication that WM5 acts through an innovative
mechanism of action validates the 6-aminoquinolones
as attractive candidates for combination with the avail-
able antiretroviral drugs.
Ch em istr y
The 6-aminoquinolone derivatives 26-33 were pre-
pared according to the synthetic sequence outlined in
Scheme 1. The key step of the synthesis involved the
usual intramolecular cycloaracylation to the quinolone
moiety. Thus, the reaction of acrylate 119 with the
appropiate amine (R1NH2), followed by basic cyclization,
gave the crucial key intermediates 2,14 3,20 4,21 5,14 6,21
and 7-9 variously substituted at the N-1 position with
a small or bulky group.
Although the 6-aminoquinolone WM5 and 6-FQs had
high anti-HIV activities on all cell lines used, both
acutely and chronically HIV-infected, they always dis-
played modest SI values that are strongly dependent
on the used cell lines. Thus, in the synthesis of new
quinolones, one of the main goals is to lower their
cytotoxicity.
To rationalize the SAR for this promising class of anti-
HIV agents, we previously performed a 3D-QSAR
study18 on a library of 6-FQs and 6-DFQs, including
those taken from the literature as well as those syn-
thesized in our laboratory. The results confirmed that
high antiviral activity could be ensured by the hydro-
philic region around the 4-keto-3-carboxylic moiety and
a suitable hydrophobic region around the substituent
at the C-7 position. Moreover, high antiviral activity
seems to be related to the presence of a hydrophobic
substituent at the N-1 position, which could also be a
bulky group. Taking into account the indications from
both our SAR and QSAR studies, further modifications
at the N-1, C-6, C-7, and C-8 positions were planned in
an effort to identify the structural groups capable of
optimizing the antiviral activity and to reduce the
cytotoxicity.
Subsequent sequential steps were nucleophilic sub-
stitution at C-7 with selected piperazines to give 6-nitro
ester intermediates 10-17, reduction to corresponding
6-amino ester derivatives 18-25, and finally hydrolysis
to the target acids 26-33. The aryl or heteroarylpip-
erazines used were commercial products or were pre-
pared according to literature procedures as for 1-(1,3-
benzothiazol-2-yl)piperazine (d ),22 1-(2-thiazolyl)pipera-
zine (e),23 1-(2-benzimidazolyl)piperazine (h ),24 1-(2-
pyrazinyl)piperazine (i),25 and 1-(1,3-benzoxazol-2-
yl)piperazine (j).23 On the other hand, 1-(5-chloro-1,3-
benzothiazol-2-yl)piperazine (g) and 1-(5-methyl-1,3,4-
thiadiazol-2-yl)piperazine (l) are new heteroarylpipera-
zines that were synthesized, as depicted in Scheme 2,
starting from the corresponding 2-mercapto-5-chloro-
1,3-benzothiazole and 2-mercapto-5-methyl-1,3,4-thia-
diazole derivatives, which were converted into the
thiomethyl derivatives and then reacted with pipera-
zine.
For the synthesis of derivative 10n (Scheme 1), it was
impossible to directly introduce the base 1-(1,4-ben-
zothiazin-3-yl)piperazine by nucleophilic displacement
of the C-7 chlorine atom, under various conditions.
Thus, it was prepared in two steps by reacting synthone
2 with piperazine to give derivative 10m , followed by a
reaction with 3-methylthiobenzothiazine.26
The 6-nitrocarboxylic acid 34a (Table 2) was obtained
by direct acid hydrolysis of the corresponding ethyl
6-nitrocarboxylate 10a .14
An informative survey of 6-aminoquinolone SAR
showed that potency was fundamentally sensitive to
substitution at the C-7 position where the 4-(2-pyridi-
nyl)-1-piperazinyl substituent was shown to be the best.
Therefore, in this study we turned our attention mainly
toward the C-7 substituent where the pyridinylpipera-
zine was modified by replacing the pyridine ring at the
N-4 piperazine core with aromatic heterocyclic or ben-
zoheterocyclic groups such as thiazole (compound 26e),
pyrazine (compound 26i), thiadiazole (compound 26l),
benzothiazoles (compounds 26d and 26g), benzoxazole
(compound 26j), benzimidazole (compound 26h ), and
benzothiazine (compound 26n ) or substituted phenyl
nucleus (compounds 26b, 26c, 26f, and 26k ) (Table 1).
On the other hand, with the C-7 4-(2-pyridinyl)-1-
piperazinyl maintained as the side chain, the NH2 group
at the C-6 position was replaced with an H atom, NO2
group, and endocyclic N, as in compounds 45a , 34a and
49a , respectively (Table 2). Three derivatives were
supplemented at the C-8 position with an OMe as in
compound 41a and a fluorine atom as in compound 39a
and its nitro analogue 38a (Table 2). The methyl
The 6-amino-8-fluoro acid 39a was obtained by start-
ing with acrylate 3527 (Scheme 3) and following the
general synthetic route reported above through the
intermediates 36, 37a , and 38a . Intermediate 38a was
also converted to 6-amino-8-methoxycarboxylic acid 41a
by reaction with MeONa and successive catalytic reduc-
tion.
The synthesis of the two examples of 6-hydrogenqui-
nolone derivatives, 45a and 45b, was accomplished
starting from acrylate 4219 and converted in the usual

Anti-HIV 6-Aminoquinolones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5569
Ta ble 1. Physical Properties for the 6-Aminoquinolones Tested in This Study

5570 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tabarrini et al.
Ta ble 1 (Continued)
a
b
See General Procedure for Coupling Reaction in the Experimental Section. See General Procedure for Reduction of 6-Nitro Group
in the Experimental Section. c See General Procedure for Acidic Hydrolysis Reaction in the Experimental Section. (A) The mixture was
d
poured into ice/water, and the precipitate was filtered, washed with water and EtOH, and then dried. (B) After the mixture was cooled,
the precipitate solid was filterd off, washed with EtOH, and dried. (C) After it was cooled, the excess side chain was filtered off and the
filtrate, after concentration, gave a solid that was filtered, washed with EtOH, and dried. (D) The mixture was evaporated to dryness to
give a residue that was purified by flash chromatography, eluting with a gradient of CH2Cl2/CH3OH (100:0 to 90:10). (E) The mixture
was concentrated to give a precipitate solid that was filtered, washed with EtOH, and dried. (F) The mixture was poured into ice/water
and extracted with CH2Cl2. The organic solvent was evaporated to dryness and the residue was treated with EtOH to give a solid that
was filtered and dried. e (G) The filtrate was concentrated, and after the mixture was cooled, the solid obtained was filtered, washed with
EtOH, and dried. (H) The filtrate was concentrated to dryness to give a residue that was triturated with a mixture of EtOH/EtOAc to give
a solid compound that was filtered and dried. (I) The filtrate was concentrated to give a residue that was purified by flash chromatography,
eluting with a gradient of CH2Cl2/CH3OH (100:0 to 97:3). f (J ) After the mixture was cooled, the crystalline precipitated solid was filterd,
washed with small amount of EtOH and 6 N HCl, dried, and then crystallized by MeOH/DMF. (K) After cooling at room temperature, the
solution was filtered and neutralized by adding a solution of 10% NaOH. The resulting precipitate was filtered and washed with water
h
and EtOH and then crystallized by EtOH/DMF. g Referenced to three steps (coupling, reduction, and hydrolysis reactions). All compounds
had elemental analyses within (0.4% of the theoretical values. i See General Procedure for Basic Hydrolysis Reaction. j See in Experimental
k
l
Section the reduction of 10e to 18e. See the preparation of 10n in the Experimental Section. Referenced to the last two steps.
Ta ble 2. Structures of Various Quinolones Tested in This
determined in parallel. The results are shown in Table
3. For comparative purposes, compound WM5 was
Studya
assayed in the same cells.
Among all the synthesized compounds, derivatives
26c, 26e, 26j, and 28c were the most potent inhibitors
of HIV replication of the series against both HIV-1 and
HIV-2, coupled with the highest selectivity index. The
C-7 thiazolpiperazinyl derivative 26e and C-7 benzox-
azolpiperazinyl derivative 26j proved to be more potent
than reference compound WM5. In fact, compound 26e
showed EC50 values of 0.13 and 0.080 µg/mL on HIV-1
and HIV-2, respectively, and compound 26j showed
values of 0.015 and 0.0087 µg/mL, respectively. Pyrazi-
nylpiperazine derivative 26i also showed good antiviral
activity, particularly against HIV-1 (EC50 ) 0.70 µg/mL).
The biological data of the synthesized compounds led
to a series of considerations that permitted a more
defined SAR to be delineated for the antiviral 6-ami-
noquinolone class. None of the modifications at the N-1
position of the lead WM5 proved successful in improving
the biological properties of the compound. The introduc-
tion of either the ethyl group 27a or a bulky substituent,
such as p-fluorophenyl 30a , 1-benzyl-4-piperidinyl 31a ,
and 2-benzothiazolyl gave compounds that were devoid
of anti-HIV activity at an EC50 lower than the CC50.
Only compound 29a , bearing a 4-(2-pyridinyl)-1-piper-
azinyl substituent at the N-1 position, was characterized
a
For the physical properties, see the Experimental Section.
by modest antiviral activity against both HIV-1 and
HIV-2 coupled with low cytoxicity. Thus, within the
6-aminoquinolone series, the best activity was provided
by the N-methyl substituent.
way to 43 (Scheme 4). The key intermediate 43, con-
verted into the borine complex 44, was then reacted with
the selected arylpiperazines in DMSO and Et3N as
scavenger. The treatment with water during the workup
Variations at the C-7 position play a crucial role, as
previously observed, and allow compounds with very
high antiviral activity to be obtained. The introduction
of a 4-(1,3-benzothiazol-2-yl)-1-piperazinyl moiety at the
C-7 position gave compound 26d , which displayed the
highest anti-HIV-2 activity but which had a high level
of cytotoxicity. In an attempt to reduce its cytotoxicity,
the benzothiazole nucleus at the N-4 piperazine ring
was replaced by thiazole 26e, benzimidazole 26h , pyra-
zine 26i, benzoxazole 26j, thiadiazole 26l, and benzothi-
azine 26n . Moreover, the benzothiazole was function-
alized with a chlorine atom at the C-6 position, as in
compound 26g. Among these serial modifications, the
replacement of the sulfur atom with an oxygen atom as
in the benzoxazole derivative 26j, as well as the
elimination of the benzene ring in the thiazole derivative
26e, gave very active compounds characterized by SI
of the reaction directly cleaved the boron ester chelate
to produce the target free acids 45a and 45b.
Scheme 5 reports the synthesis of the 1,6-naphthy-
ridinecarboxylic acid 49a . Thus, the reaction of the acid
chloride of 2,4-dichloropyridine-5-carboxylic acid 4628
with ethyl (dimethylamino)acrylate gave the adduct 47,
which was reacted at room temperature with MeNH2
and successively cyclized in the presence of NaH at 0
°C to afford quinolone 48. Nucleophilic substitution of
the C-7 chlorine atom with 1-(2-pyridinyl)piperazine and
successive basic hydrolysis gave the target acid 49a .
Resu lts a n d Discu ssion
The synthesized compounds were initially evaluated
for anti-HIV activity by determining their ability to
inhibit the replication of HIV-1 (IIIB) and HIV-2 (ROD)
in MT-4 cells. The cytotoxicity of the compounds was

Anti-HIV 6-Aminoquinolones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5571
Ta ble 3. Anti-HIV-1 and HIV-2 Activity (EC50 in µg/mL) and Cytotoxicity of Quinolone Derivatives in MT-4 Cells
HIV-1 (IIIB) HIV-2 (ROD)
compd
EC50 (µg/mL)a,c
max % prot
EC50 (µg/mL)a,c
max % prot
CC50 (µg/mL)b,c
SI (IIIB)
SI (ROD)
WM5
26b
26c
26d
26e
26f
26g
26h
26i
g0.29
>7.45
0.31 ( 0.14
>0.0054
0.13 ( 0.04
>125
>0.0021
>125
0.70 ( 0.042
0.015 ( 0.0013
>0.157
>125
>2.14
>2.20
>10.33
0.27 ( 0.14
>0.076
g17.4
(35-59)
(16-21)
(57-110)
(25-30)
(55-118)
(7-30)
(4-8)
0.19 ( 0.099
>7.45
(60-64)
(17-27)
(43-78)
(51-81)
(58-83)
(17-31)
(3)
0.71 ( 0.20
7.45 ( 2.77
2.18 ( 0.28
0.0054 ( 0.0018
1.17 ( 0.44
>125
e2.4
<1
3.7
<1
g0.31
7.0
e7.0
5.1
0.0011
<1
9.0
0.080 ( 0.067
14.6
>125
>0.0021
>125
0.0021 ( 0.0016
>125
<1
<1
(0-1)
(0)
(60-91)
(65-86)
(5-9)
1.54 ( 0.44
0.0087 ( 0.0034
>0.157
g10.30
>2.14
(51-55)
(15-21)
(13)
(29-63)
(15)
(26-27)
(10)
(71-71)
(42)
(55-58)
(0)
(22-24)
(5)
(21)
(1)
(5)
(4)
3.98 ( 0.98
0.098 ( 0.023
0.157 ( 0.030
>125
5.7
6.5
<1
<1
<1
<1
<1
14.2
<1
e3.4
<1
<1
<1
<1
<1
<1
<1
<1
<1
3.4
2.6
11.3
<1
26j
26k
26l
(29-40)
(10-27)
(11-24)
(9-24)
(88-115)
(16-36)
(32-62)
(2-18)
(34-37)
(2-14)
(14-20)
(5-6)
e12.1
<1
26n
27a
28b
28c
28d
29a
29d
30a
31a
32a
33d
34a
38a
39a
41a
45a
45b
49a
2.14 ( 0.098
2.20 ( 0.29
10.33 ( 5.42
3.83 ( 0.98
0.076 ( 0.052
58.46 ( 19.90
0.24 ( 0.20
22.03 ( 4.64
2.36 ( 0.27
9.13 ( 5.10
0.67 ( 0.19
10.07 ( 2.48
29.57 ( 9.64
6.08 ( 1.01
4.28 ( 1.95)
65.53 ( 26.39
>125
>2.20
<1
<1
>10.33
0.38 ( 0.18
>0.076
19.20 ( 0.57
>0.24
10.1
<1
3.0
>0.24
<1
>22.03
>2.36
>22.03
>2.36
<1
<1
>9.13
>9.13
<1
>0.67
>0.67
<1
>10.07
>29.57
>6.08
(4-13)
(3-8)
>10.07
>29.57
>6.08
<1
<1
(29-37)
(16-49)
(53-59)
(8-9)
(40-42)
(21-26)
(40-43)
(6)
<1
>4.28
>4.28
<1
19.20 ( 3.53
>125
>65.53
>125
<1
>67.37
(32-39)
>67.37
(27-33)
67.37 ( 30.79
<1
<1
a
EC50: concentration of compound required to achieve 50% protection 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 method. c All data represent mean values ( standard deviations for at least two separate experiments.
Ta ble 4. Anti-HIV-1 and HIV-2 Activity and Cytotoxicity of Selected Quinolones in CEM Cells
HIV-1 (IIIB)
HIV-2 (ROD)
compd
EC50 (µg/mL)a,c
EC50 (µg/mL)a,c
CC50 (µg/mL)b,c
SI (IIIB)
SI (ROD)
WM5
26c
26e
26i
0.03 ( 0.01
0.09 ( 0.01
0.06 ( 0.00
0.08 ( 0.02
0.01 ( 0.00
0.80 ( 0.17
9.40 ( 1.22
1.20 ( 0.42
0.06 ( 0.01
0.20 ( 0.16
0.08 ( 0.00
0.20 ( 0.11
0.02 ( 0.01
0.80 ( 0.58
12.00 ( 0.92
1.80 ( 0.17
>100
>3333
89
g1667
>1250
220
11
>11
>83
>1667
40
g1250
>500
110
11
>8
>56
8.00 ( 3.51
g100
>100
26j
2.20 ( 0.25
9.00 ( 0.31
>100
28c
29a
45a
>100
a
EC50: concentration of compound required to achieve 50% protection of CEM cells from HIV induced cytopathogenicity, as determined
b
microscopically. 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 three separate experiments.
Ta ble 5. Anti-HIV-1 Activity and Cytotoxicity of Selected
Quinolones in PBMCs
this time with a marked decrease in cytotoxicity. For
the C-7 benzimidazole derivative 26h and the thiadia-
zole derivative 26l, the toxicity was eliminated but so
HIV-1 (IIIB)
compd
EC50 (µg/mL)a,c
CC50 (µg/mL)b,c
SI
28
28
41
81
26
was the antiviral activity. The broadening of the thiazole
ring to thiazine produced benzothiazine derivative 26n
characterized by both low cytotoxicity and antiviral
activity. The insertion of a chlorine atom in the ben-
zothiazole ring as in 26g did not affect activity or
cytotoxicity. Finally, benzothiazole derivative 26d was
modified by introducing a cyclopropyl (compound 28d )
and bulky groups (compounds 29d and 33d ) at the N-1
position; these modifications did not lead to pronounced
anti-HIV activity.
WM5
26c
26e
26i
0.051 ( 0.05
0.056 ( 0.040
0.032 ( 0.010
0.045 ( 0.00
0.043 ( 0.020
1.09 ( 0.80
1.45 ( 0.07
1.55 ( 0.88
1.30 ( 1.27
3.65 ( 2.33
1.12 ( 0.21
4.63 ( 0.47
g43.30
26j
28c
29a
45a
4
2.24 ( 1.59
g19
>73
1.37 ( 1.11
>100
a
EC50
: concentration of compound required to achieve 50%
reduction of p24 production in HIV-1 (IIIB) infected PBMCs.
b
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.
The C-7 position was further modified by introducing
an m-trifluoromethylphenyl ring at the N-4 piperazine
moiety as in compound 26c, which displayed good
activity with EC50 values of 0.31 µg/mL against both
HIV-1 and HIV-2 coupled with positive SI values. The
values ranging from 6.5 to 14.6. The replacement of the
benzothiazole with a pyrazine ring gave compound 26i,
which was characterized by a lower activity but coupled

5572 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tabarrini et al.
Molecules displaying good activity on MT-4, 26c, 26e,
26i, 26j, 28c, and two molecules that are less active but
with positive SI, 29a and 45a , were also evaluated for
their anti-HIV-1 and anti-HIV-2 activity on CEM cells
(Table 4), as well as against HIV-1 in PBMCs (Table
5). These two cell lines appear to be more sensitive to
all the tested compounds. In fact, with the exception of
28c, the compounds proved to be approximately 2- to
16-fold more active in PBMCs and CEM cells than in
MT-4 cells; a 36-fold activity increase against HIV-2 on
CEM cells was obtained with compound 45a .
The increased antiviral activity was also coupled with
a markedly lower cytotoxicity in CEM cells. In particu-
lar, derivatives 26e, 26i, 29a , and 45a , as well as the
lead WM5, were devoid of any cytotoxicity (CC50 >100
µg/mL); thus, very high SI values were obtained par-
ticularly on HIV-1. The highest SI value was observed
for reference compound WM5 (>3333), but it was also
particularly good for derivatives 26e (g1667), 26i
(>1250), and 26j (220). The comparison between N-1
methyl derivative 26c and its N-1 cyclopropyl analogue
28c showed that, contrary to their behavior on MT-4
cells, derivative 26c was 10 and 4 times more active
against HIV-1 and HIV-2, respectively, in CEM cells.
On PBMCs the compounds showed a cytotoxicity that
was on the same order as that observed on MT-4 cells
(Table 5). The SI values ranged from 4 for compound
28c to 81 for compound 26i. The 6-hydrogenquinolone
45a was the only derivative that was not toxic (CC50
g
100 µg/mL) in this cell line according to its behavior in
the other tested cell lines. This observation appears to
be very interesting and should be considered for further
development of anti-HIV 6-hydrogenquinolones.
To investigate which step of the replicative cycle was
inhibited by the new 6-aminoquinolones, time-of-addi-
tion experiments were carried out for derivatives 26e,
26j, and 28c (Figure 2). Briefly, this experiment deter-
mines how long the addition of an anti-HIV compound
can be postponed before losing its antiviral activity in
the viral replication cycle. The virus was added at high
multiplicity of infection (moi ) 0.5) to synchronize all
the steps of the viral replication. Reference compounds
with a known mode of action were included: dextran
sulfate, which interferes with the binding of the virus
to the cells; AZT, which inhibits the reverse transcrip-
tion process; and ritonavir, which is an inhibitor of the
proteolytic cleavage. Addition of these compounds can
be delayed for 0, 5, and 16 h postinfection, respectively
(Figure 2). In the same experiment, 6-aminoquinolones
lost their antiviral activities when added at 9-16 h
postinfection, which clearly indicates an inhibition at a
postintegrational level.
F igu r e 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 postinfection and is
expressed in pg/mL. Symbols represent the following: (9)
control; (×) dextran sulfate; (0) AZT; (2) 28c; ([) 26e; (+) 26j;
(/) ritonavir.
same profile was also shown by the N-1 cyclopropyl
analogue 28c.
The absence of a substituent at the C-6 position
(compound 45a ), as well as the insertion of an NO2
group (compound 34a ) or endocyclic N (compound 49a ),
gave inactive or slighly active compounds confirming
that the 6-NH2 substituent was more suitable. This
behavior was also confirmed by the inactivity of 6-nitro
derivative 38a and 6-hydrogen derivative 45b. However,
it is noted that the 6-hydrogen atom lowered the
cytotoxicity (45a and 45b).
The insertion of a fluorine atom and a methoxy group
at the C-8 position gave compounds 39a and 41a , which
did not inhibit HIV-1 and HIV-2 replication at EC50
values lower than the CC50. This confirms what was
already reported for the C-8 methyl derivative14 and
seems to indicate that the antiviral 6-aminoquinolones
do not tolerate any substituent at this position in
contrast to what was observed for the fluoroquinolones
reported to date.9-13
In conclusion, the structural modification around the
lead WM5 permitted new, potent anti-HIV-1 and HIV-2
6-aminoquinolones 26c, 26e, 26i, 26j, and 28c to be
obtained. In general, they show the same high antiviral
potency on MT-4, CEM, and PBMCs coupled with
cytotoxicity that is strongly determined by the used cell.
In the CEM cell line, some derivatives such as 26e do
not appear to be toxic at all. The SAR study based on
MT-4 antiviral data shows that modifications of the
6-NH2 group are detrimental for the antiviral activity;
however, a hydrogen atom seems to be suitable for
lowering the cytotoxicity. The suitability of a small

Anti-HIV 6-Aminoquinolones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5573
Sch em e 1a
a
Reagents: (i) R1NH2, EtOH/Et2O; (ii) K2CO3, DMF, 100 °C; (iii) R7H (for R7, see Chart 1), DMF; (iv) H2, Raney Ni, DMF or DMF/
EtOH; (v) HCOONH4, 10% Pd/C, MeOH/DMF; (vi) 6 N HCl, EtOH, reflux; (vii) 1 N NaOH, reflux.
Sch em e 2a
model 1106, and the data for C, H, and N are within (0.4% of
1
the theoretical values. H NMR spectra were recorded at 200
MHz (Bruker DPX 200) with Me4Si as the internal standard.
Chemical shifts are given 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. Flash column chromatography separa-
tions were carried out on Merck silica gel 60 (mesh 230-400).
Organic solutions were dried over anhydrous Na2SO4 and
concentrated with a Bu¨chi rotary evaporator at low pressure.
Yields are of purified product and were not optimized. All
starting materials were commercially available unless other-
wise indicated.
a
Reagents: (i) MeI, KOH, TBAB, THF; (ii) piperazine, 110 °C,
closed vessel.
Gen er a l P r oced u r e for Cycloa r a cyla tion Rea ction . A
stirred solution of the appropiate acrylate (1, 35, 42) (1 equiv)
in EtOH and Et2O (3:1) was treated dropwise with a selected
R1NH2 group (1.1 equiv), and the mixture was reacted at room
temperature for 8 h. The solution was then evaporated to
dryness to give an oil that was washed with a mixture of
cyclohexane/MeOH and then solubilized in DMF. To this
solution K2CO3 (2 equiv) was added, and the mixture was
heated at 100 °C for 1 h. After cooling, the reaction mixture
was poured into ice/water, giving a precipitate that was
filtered, dried, and purified by flash chromatography, eluting
with a gradient of CH2Cl2/MeOH (100:0 to 98:2).
group such as CH3 at the N-1 position was confirmed,
which can be replaced only by a cyclopropyl; generally,
bulky groups led to a decrease in activity. The C-8
position does not appear to tolerate substituents, in
contrast to what was reported for known antiviral
6-fluoroquinolones. Moreover, it was confirmed that
substitution at C-7 is still the best way to influence the
anti-HIV activity; besides 2-pyridinylpiperazine, other
aryl/heteroaryl piperazines produced potent quinolones.
Further studies are needed to better delineate the
structure-activity relationships in order to meet the
important challenge of recognizing the structural fea-
tures that are responsible for the antiviral activity and/
or cytotoxicity.
E t h yl 1-(1-Ben zyl-4-p ip er id in yl)-7-ch lor o-6-n it r o-4-
oxo-1,4-d ih yd r oqu in olin -3-ca r boxyla te (7). Following the
general procedure for cycloaracylation reaction, starting from
ethyl 2-(2,4-dichloro-5-nitrobenzoyl)-3-etoxyacrylate 119 and
using 4-amino-1-benzylpiperidine, the title compound was
1
obtained in 35% yield: mp 170-172 °C; H NMR (CDCl3) δ
1.35 (3 H, t, J ) 7.0 Hz, CH3), 2.10-2.50 (6 H, m, piperidine
CH2), 3.15-3.35 (2 H, m, piperidine CH2), 3.70 (2 H, s, benzyl
CH2), 4.25-4.50 (3 H, m, CH2CH3 and piperidine CH), 7.30-
7.45 (5 H, m, benzyl CH), 7.75 (1 H, s, H-8), 8.75 (1 H, s, H-5),
9.05 (1 H, s, H-2).
Exp er im en ta l Section
Melting points were determined in capillary tubes (Bu¨chi
melting point apparatus) and are uncorrected. Elemental
analyses were performed on a Carlo Erba elemental analyzer,

5574 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tabarrini et al.
Sch em e 3a
a
Reagents: (i) MeNH2, EtOH/Et2O; (ii) K2CO3, DMF, 100 °C; (iii) 1-(2-pyridinyl)piperazine, DMF; (iv) 6 N HCl, EtOH, reflux; (v) H2,
Raney Ni, DMF; (vi) MeONa, DMF/MeOH.
Sch em e 4a
Eth yl 1-(1,1′-Bip h en yl-4-yl)-7-ch lor o-6-n itr o-4-oxo-1,4-
d ih yd r oqu in olin -3-ca r boxyla te (9). Following the general
procedure for cycloaracylation reaction, starting from ethyl
2-(2,4-dichloro-5-nitrobenzoyl)-3-etoxyacrylate 119 and using
1,1′-biphenyl-4-amine, the title compound was obtained in 35%
1
yield: mp 189-191 °C; H NMR (CDCl3) δ 1.40 (3 H, t, J )
7.0 Hz, CH3), 4.40 (2 H, q, J ) 7.0 Hz, CH2), 7.00-7.10 and
7.25-7.35 (each 3 H, m, aromatic CH), 7.50-7.55 (1 H, m,
aromatic CH), 7.60-7.85 (3 H, m, aromatic CH and H-8), 8.75
(1 H, s, H-5), 9.00 (1 H, s, H-2).
Eth yl 7-Ch lor o-8-flu or o-1-m eth yl-6-n itr o-4-oxo-1,4-d i-
h yd r oqu in olin -3-ca r boxyla te (36). Following the general
procedure for cycloaracylation reaction, starting from ethyl
2-(2,4-dichloro-3-fluoro-5-nitrobenzoyl)-3-(dimethylamino)acry-
late 3527 and using MeNH2, the title compound was prepared
1
in 52% yield: mp 174-176 °C; H NMR (DMSO-d6) δ 1.30 (3
H, t, J ) 7 Hz, CH2CH3), 4.15 (3 H, d, J ) 9 Hz, CH3), 4.25 (2
H, q, J ) 7 Hz, CH2CH3), 8.65 (1 H, d, J ) 2.0 Hz, H-5), 8.75
a
Reagents: (i) MeNH2, EtOH/Et2O; (ii) K2CO3, DMF, 100 °C;
(1 H, s, H-2).
(iii) 48% HBF4, 90-100 °C; (iv) R7H (for R7, see Chart 1), DMSO;
(v) H2O.
Eth yl 7-Ch lor o-1-m eth yl-4-oxo-1,4-d ih yd r oqu in olin -3-
ca r boxyla te (43). Following the general procedure for cy-
cloaracylation reaction, starting from ethyl 2-(2,4-dichloroben-
Sch em e 5a
zoyl)-3-etoxyacrylate 4219 and using MeNH2, the title compound
was prepared in 57% yield: mp 178-180 °C; 1H NMR (DMSO-
d6) δ 1.45 (3 H, t, J ) 7 Hz, CH2CH3), 3.90 (3 H, s, CH3), 4.45
(2 H, q, J ) 7 Hz, CH2CH3), 7.45 (1 H, dd, J ) 1.8 and 7.0 Hz,
H-6), 7.45 (1 H, bs, H-8), 8.45-8.55 (2 H, m, H-5 and H-2).
Eth yl 7-Ch lor o-1-m eth yl-4-oxo-1,4-d ih yd r o-1,6-n a p h -
th yr id in e-3-ca r boxyla te (48). A mixture of acid 4628 (1.3 g,
6.8 mmol) and thionyl chloride (6 mL) was refluxed for 2 h.
The excess thionyl chloride was removed by distillation under
reduced pressure to give an oil residue that was dissolved in
dry toluene (15 mL) and added to ethyl 3-(dimethylamino)-
acrylate29 (1.35 g, 9.47 mol). The resulting solution was heated
at 120 °C for 4 h. After cooling, the insoluble material was
filtered and the solvent was evaporated to dryness. The residue
was purified by flash chromatography, eluting with a mixture
of petroleum ether/EtOAc (70:30) to give 47 (0.8 g, 37%).
a
Reagents: (i) (a) SOCl2; (b) Me2NCHdCHCO2Et, Et3N, tolu-
ene; (ii) MeNH2, EtOH/Et2O; (iii) 60% NaH, THF; (iv) 1-(2-
pyridinyl)piperazine, DMF; (v) 4% NaOH reflux.
Thus, a stirred solution of 47 (0.7 g, 2.2 mmol) in EtOH (20
mL) and Et2O (8 mL) was treated dropwise with MeNH2 (33%
solution in EtOH) (0.476 mL, 3.53 mmol). After 1.5 h at room
temperature the solvent was evaporated to dryness and
washed with Et2O to give 0.6 g of methylamine acrylate as a
white solid that was solubilized in THF (30 mL). To this
solution maintained at 0 °C, NaH (60% in an oil suspension)
(0.09 g, 2.2 mmol) was added portionwise. After 1 h at 0 °C,
the mixture was evaporated to dryness and the residue was
treated with water to give a solid that was filtered and dried
Eth yl 1-(1,3-Ben zoth ia zol-2-yl)-7-ch lor o-6-n itr o-4-oxo-
1,4-d ih yd r oqu in olin -3-ca r boxyla te (8). Following the gen-
eral procedure for cycloaracylation reaction, starting from ethyl
2-(2,4-dichloro-5-nitrobenzoyl)-3-etoxyacrylate 119 and using
2-aminobenzothiazole, the title compound was obtined in 32%
yield. In this case, the reaction mixture with amine was heated
1
at 60 °C for 2 h: mp 249-250 °C; H NMR (CDCl3) δ 1.45 (3
1
H, t, J ) 7.0 Hz, CH3), 4.50 (2 H, q, J ) 7.0 Hz, CH2), 7.55-
7.80 (3 H, m, benzothiazole CH), 8.00 (1 H, s, H-8), 8.20-8.25
(1 H, m, benzothiazole CH), 8.80 (1 H, s, H-5), 9.05 (1 H, s,
H-2).
to give 48 (0.3 g, 51%): mp 165-168 °C; H NMR (CDCl3) δ
1.40 (3 H, t, J ) 7 Hz, CH3), 3.80 (3 H, s, NCH3), 4.40 (2 H, q,
J ) 7 Hz, CH2), 7.30 (1 H, s, H-8), 8. 45 (1 H, s, H-5), 9.40 (1
H, s, H-2).

Anti-HIV 6-Aminoquinolones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5575
7.20-7.30 (2 H, m, thiazole CH and H-8), 7.50 (1 H, s, H-5),
Gen er a l P r oced u r e for Cou p lin g Rea ction . A mixture
of the appropiate synthone (2-9, 36, 48) (1 equiv) and selected
arylpiperazine (3 equiv) in dry DMF was heated at 90-120
°C until no starting material could be detected by TLC (usually
2-15 h). The reaction mixture was then worked up and
purified as defined in footnote d in Table 1.
8.75 (1 H, s, H-2), 16.00 (1 H, bs, COOH). Anal. (C18
C, H, N.
H19N5O3S)
Com p ou n d 26f: 1H NMR (DMSO-d6) δ 3.20-3.35 (8 H, m,
piperazine CH2), 4.10 (3 H, s, CH3), 6.90-7.25 (4 H, m,
aromatic CH), 7.35 (1H, s, H-8), 7.75 (1 H, s, H-5), 8.90 (1 H,
s, H-2). Anal. (C21H22ClFN4O3) C, H, N.
Eth yl 7-[4-(2H-1,4-Ben zoth ia zin -3-yl)-1-p ip er a zin yl]-1-
m et h yl-6-n it r o-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxy-
la te (10n ). Following the general procedure for coupling
reaction, synthone 2 was reacted with piperazine for 4 h at
80 °C. After the mixture was cooled, the precipitate was
filtered, washed with EtOH, and dried to give 10m in 80%
yield. A mixture of compound 10m (0.4 g, 1,1 mmol) and
3-(methylthio)-2H-1,4-benzothiazine26 (1.3 g, 6.6 mmoli) in dry
DMF (10 mL) was refluxed for 16 h. The reaction mixture was
then evaporated to dryness, put into water, and extracted
several times with EtOAc. The organic layers were concen-
trated to give a solid that was filtered and dried to afford
compound 10n (0.250 g, 45%): mp 297-298 °C; 1H NMR
(DMSO-d6) δ 1.25 (3 H, t, J ) 7.0 Hz, CH2CH3), 3.25-3.50 (6
H, m, piperazine CH2 and SCH2), 3.75-4.00 (7 H, m, pipera-
zine CH2 and NCH3), 4.25 (2 H, q, J ) 7.0 Hz, CH2CH3), 6.90-
7.20 (5 H, m, aromatic H and H-8), 8.55 (1 H, s, H-5), 8.70 (1
H, s, H-2).
Com p ou n d 26g: 1H NMR (DMSO-d6) δ 3.00-3.10 and
3.70-3.80 (each 4 H, m, piperazine CH2), 4.00 (3 H, s, CH3),
7.05 (1 H, dd, J ) 8.6 and 2.0 Hz, H-6′), 7.20 (1 H, s, H-8),
7.40 (1 H, d, J ) 2.0 Hz, H-4′), 7.50 (1 H, s, H-5), 7.75 (1 H, d,
J ) 8.6 Hz, H-7′), 8.70 (1 H, s, H-2), 16.00 (1 H, bs, COOH).
Anal. (C22H21Cl2N5O3S) C, H, N.
Com p ou n d 26h : 1H NMR (DMSO-d6) δ 3.20-3.35 and
4.00-4.15 (each 4 H, m, piperazine CH2), 4.00 (3 H, s, CH3),
7.25-7.35 (3 H, m, aromatic H and H-8), 7.40-7.50 (2 H, m,
aromatic H), 7.05 (1 H, s, H-5), 8.75 (1 H, s, H-2). Anal. (C22H23
ClN6O3) C, H, N.
-
Com p ou n d 26i: 1H NMR (DMSO-d6) δ 3.10-3.25 and
3.80-3.95 (each 4 H, m, piperazine CH2), 4.05 (3 H, s, CH3),
7.30 (1 H, s, H-8), 7.75 (1 H, s, H-5), 7.90, 8.20 and 8.50 (each
1 H, s, pyrazine CH), 8.80 (1 H, s, H-2). Anal. (C19H21ClN6O3)
C, H, N.
Com p ou n d 26j: 1H NMR (DMSO-d6) δ 3.10-3.30 (4 H, m,
piperazine CH2), 3.80-4.15 (7 H, m, piperazine CH2 and CH3),
7.10-7.50 (5 H, m, aromatic H and H-8), 7.65 (1 H, s, H-5),
8.75 (1 H, s, H-2). Anal. (C22H22ClN5O4) C, H, N.
Gen er a l P r oced u r e for Red u ction of 6-Nitr o Gr ou p .
A stirred solution of a selected 6-nitro derivate in DMF or in
a mixture of DMF/EtOH was hydrogenated over a catalytic
amount of Raney nickel at room temperature and atmospheric
pressure until no starting material could be detected by TLC
(usually 2-10 h). The mixture was then filtered over Celite,
and the filtrate was worked up and purified as described in
footnote e in Table 1.
Com p ou n d 26k : 1H NMR (DMSO-d6) δ 2.45 (3 H, s,
COCH3), 3.15-3.25 and 3.55-3.65 (each 4 H, m, piperazine
CH2), 4.00 (3 H, s, NCH3), 7.00 (2 H, d, J ) 7.0 Hz, aromatic
CH), 7.25 (1 H, s, H-8), 7.60 (1 H, s, H-5), 7.80-7.90 (2 H, m,
aromatic CH), 8.80 (1 H, s, H-2). Anal. (C23H24N4O4) C, H, N.
1
Com p ou n d 26l: H NMR (DMSO-d6) δ 2.45 (3 H, s, CH3),
Eth yl 6-Am in o-1-m eth yl-7-[4-(1,3-th ia zol-2-yl)-1-p ip er -
azin yl]-4-oxo-1,4-dih ydr oqu in olin -3-car boxylate (18e). Am-
monium formate (0.49 g, 7.9 mmol) and 10% Pd/C (0.7 g) were
added to a solution of nitro derivate 10e (0.7 g, 1.58 mmol) in
dry MeOH (20 mL) and dry DMF (10 mL). The mixture was
stirred for 12 h at room temperature and then filtered over
Celite, washing several times with hot DMF. The filtrate was
evaporated to dryness, and the residue was crystallized by
DMF to give derivate 18e (0.080 g, 13%): mp 297-298 °C; 1H
NMR (DMSO-d6) δ 1.20 (3 H, t, J ) 7.0 Hz, CH2CH3), 2.95-
3.05 and 3.50-3.60 (each 4 H, m, piperazine CH2), 3.75 (3 H,
s, CH3), 4.10 (2 H, q, J ) 7.0 Hz, CH2), 5.20 (2 H, bs, NH2),
6.75 (1 H, d, J ) 9.4 Hz, thiazole CH), 7.00 (1 H, s, H-8), 7.10
(1 H, d, J ) 9.4 Hz, thiazole CH), 7.40 (1 H, s, H-5), 8.40 (1 H,
s, H-2).
3.00-3.10 and 3.50-3.60 (each 4 H, m, piperazine CH2), 3.95
(3 H, s, NCH3), 7.20 (1 H, s, H-8), 7.60 (1 H, s, H-5), 8.70 (1 H,
s, H-2). Anal. (C18H21ClN6O3S) C, H, N.
Com p ou n d 26n : 1H NMR (DMSO-d6) δ 3.10-3.40 (4 H, m,
piperazine CH2), 4.00 (3 H, s, NCH3), 4.10-4.30 (6 H, m,
piperazine CH2 and CH2), 7.15-7.45 (5 H, m, aromatic H and
H-8), 7.50 (1 H, s, H-5), 8.70 (1 H, s, H-2), 12.10 (1 H, bs,
COOH). Anal. (C23H23N5O3S) C, H, N.
1
Com p ou n d 27a : H NMR (DMSO-d6) δ 1.40 (3 H, t, J )
7.0 Hz, CH3), 3.00-3.15 and 3.60-3.75 (each 4 H, m, pipera-
zine CH2), 4.55 (2 H, q, J ) 7.0 Hz, CH2CH3), 5.50 (2 H, bs,
NH2), 6.50-6.65 (1 H, m, pyridine CH), 6.90 (1 H, d, J ) 8.5
Hz, pyridine CH), 7.25 (1 H, s, H-8), 7.50-7.65 (2 H, m,
pyridine CH and H-5), 8.20 (1 H, d, J ) 4.5 Hz, pyridine CH),
8.90 (1 H, s, H-2), 16.00 (1 H, bs, COOH). Anal. (C21H23N5O3)
C, H, N.
Gen er a l P r oced u r e for Acid ic Hyd r olysis Rea ction . A
mixture of selected 6-amino ester (0.3 mequiv) in EtOH (2 mL)
and 6 N HCl (2 mL) was refluxed until no starting material
could be detected by TLC (usually 7-72 h) and worked up and
purified as defined in footnote f in Table 1.
Com p ou n d 28c: 1H NMR (DMSO-d6) δ 1.05-1.40 (4 H, m,
cyclopropyl CH2), 3.15-3.30 and 3.45-3.65 (each 4 H, m,
piperazine CH2), 3.80-3.90 (1 H, m, cyclopropyl CH), 7.00-
7.50 (4 H, m, aromatic CH), 7.70 (2 H, s, H-8 and H-5), 8.60 (1
H, s, H-2). Anal. (C24H24ClF3N4O3) C, H, N.
Com p ou n d 28d : 1H NMR (DMSO-d6) δ 1.05-1.20 and
1.30-1.45 (each 2 H, m, cyclopropyl CH2), 3.20-3.30 (4 H, m,
piperazine CH2), 3.75-4.00 (5 H, m, piperazine CH2 and
cyclopropyl CH), 7.05-7.20 and 7.30-7.40 (each 1 H, m,
aromatic H), 7.45-7.55 (2 H, m, aromatic H and H-8), 7.65 (1
H, s, H-5), 7.70-7.80 (1H, m, aromatic H), 8.50 (1 H, s, H-2).
Anal. (C24H25Cl2N5O3S) C, H, N.
Com p ou n d 29a : 1H NMR (CDCl3) δ 3.10-3.50 (8 H, m,
piperazine CH2), 3.70-3.90 and 4.30-4.60 (each 4 H, pipera-
zine CH2), 6.60-6.90 (4 H, m, pyridine CH), 7.50-7.70 (2 H,
m, pyridine CH), 7.80 (1 H, s, H-8), 7.90 (1 H, s, H-5), 8.20-
8.40 (2 H, m, pyridine CH), 8.90 (1 H, s, H-2), 15.50 (1 H, bs,
COOH). Anal. (C22H21F3N4O3) C, H, N. Anal. (C28H30N8O3) C,
H, N.
Com p ou n d 29d : 1H NMR (CDCl3) δ 3.10-3.45 (8 H, m,
piperazine CH2), 3.80-4.00 and 4.25-4.60 (each 4 H, pipera-
zine CH2), 6.75-6.90 (2 H, m, aromatic CH), 7.10-7.40 (4 H,
m, aromatic CH), 7.50 (1 H, s, H-8), 7.60-7.70 (2 H, m,
aromatic CH), 7.85 (1 H, s, H-5), 8.20-8.30 (1 H, m, aromatic
By use of this procedure, the target acids 26c, 26d , 26e,
26f, 26g, 26h , 26i, 26j, 26l, 26n , 27a , 28c, 28d , 29a , 29d ,
30a , 31a , 32a , and 33d were obtained. Further experimental
details are given in Table 1, while their spectral data are listed
below.
Com p ou n d 26c: 1H NMR (DMSO-d6) δ 3.20-3.30 and
3.50-3.60 (4 H, m, CH piperazine), 4.15 (3 H, s, CH3), 7.15 (1
H, d, J ) 8.0 Hz, aromatic CH), 7.25-7.40 (3 H, m, aromatic
CH and H-8), 7.60 (1 H, s, H-5), 8.80 (1 H, s, H-2). Anal.
(C22H21F3N4O3) C, H, N.
Com p ou n d 26d : 1H NMR (DMSO-d6) δ 3.10-3.25 and
3.75-3.90 (each 4 H, m, piperazine CH2), 4.00 (3 H, s, CH3),
7.05-7.15 (1 H, m, benzothiazole CH), 7.25-7.40 (2 H, m,
benzothiazole CH and H-8), 7.45-7.55 (1 H, m, benzothiazole
CH), 7.60 (1 H, s, H-5), 7.75-7.85 (1 H, m, benzothiazole CH),
8.75 (1 H, s, H-2), 16.00 (1 H, bs, COOH). Anal. (C22H22
ClN5O3S) C, H, N.
-
Com p ou n d 26e: 1H NMR (DMSO-d6) δ 3.10-3.25 and
3.60-3.70 (each 4 H, m, piperazine CH2), 4.00 (3 H, s, CH3),
5.50 (2 H, bs, NH2), 6.80 (1 H, d, J ) 3,4 Hz, thiazole CH),

5576 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22
Tabarrini et al.
CH), 8.90 (1 H, s, H-2), 15.50 (1 H, bs, COOH). Anal.
(C30H30N8O3S) C, H, N.
6-Am in o-8-flu or o-1-m et h yl-7-[4-(2-p yr id in yl)-1-p ip er -
a zin yl]-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r boxylic Acid
(39a ). By use of the general procedure for reduction of the
6-nitro group (purification method H), the title compound 39a
was obtained starting from nitroacid 38a . After crystallization
Com p ou n d 30a : 1H NMR (DMSO-d6) δ 2.90-3.00 and
3.90-4.00 (each 4 H, m, piperazine CH2), 6.50 (1 H, s, H-8),
6.90 (1 H, t, J ) 7.0 Hz, pyridine CH), 7.35-7.60 (3 H, m,
aromatic H and pyridine CH), 7.70-7.80 (3 H, m, aromatic H
and H-5), 7.95-8.05 (2 H, m, pyridine H), 8.50 (1 H, s, H-2).
Anal. (C25H23ClFN5O3) C, H, N.
1
by EtOH, it was obtained in 22% yield: mp 288-289 °C; H
NMR (DMSO-d6) δ 3.10-3.40 (8 H, m, piperazine CH2), 4.15
(3 H, d, J ) 9.0 Hz, CH3), 5.95 (2 H, bs, NH2), 6.70 (1 H, t, J
) 7.0 Hz, pyridine CH), 6.80 (1 H, d, J ) 8.5, pyridine CH),
7.35 (1 H, s, H-5), 7.60 (1 H, t, J ) 7.0 Hz, pyridine CH), 8.10
(1 H, d, J ) 4.0 Hz, pyridine CH), 8.70 (1 H, s, H-2). Anal.
(C20H20FN5O3) C, H, N.
Com p ou n d 31a : 1H NMR (DMSO-d6) δ 1.75-2.00 (4 H,
m, piperidine CH2), 2.90-3.20 (6 H, m, piperidine CH2 and
piperazine CH2), 3.60-3.90 (6 H, m, piperazine CH2 and
piperidine CH2), 4.70-4.80 (1 H, m, piperidine CH), 5.50 (2
H, s, benzyl CH2), 6.55 (1 H, t, J ) 7.0 Hz, H-5′), 6.80 (1 H, d,
J ) 8.5 Hz, H-3′), 7.20-7.40 (6 H, m, aromatic CH and H-8),
7.45-7.55 (2 H, m, H-4′ and H-5), 8.10 (1 H, d, J ) 4.0 Hz,
H-6′), 8.50 (1 H, s, H-2). Anal. (C31H34N6O3) C, H, N.
6-Am in o-8-m eth oxy-1-m eth yl-7-[4-(2-p yr id in yl)-1-p ip -
er a zin yl]-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r boxylic Acid
(41a ). A solution of MeONa (0.36 g, 6.67 mmol) in dry DMF
(5 mL) and dry MeOH (3 mL) was added to a suspension of
8-fluoro derivate 38a (0.5 g, 1.17 mmol) in dry DMF (10 mL).
The mixture was heated to 50 °C for 3 h and then poured into
ice/water. The solution was acidified to pH 6 with 2 N HCl,
and the solid so obtained was filtered and dried to give 0.44 g
(92%) of 8-methoxy-6-nitro derivative 40a : mp 210-211 °C;1H
NMR (DMSO-d6) δ 3.20-3.30 and 3.60-3.75 (each 4 H, m,
piperazine CH2), 3.80 (3 H, s, CH3), 4.25 (3 H, s, CH3), 6.70 (1
H, t, J ) 7.0 Hz, H-5′), 6.90 (1 H, d, J ) 8.5 Hz, H-3′), 7.60 (1
H, t, J ) 7.0 Hz, H-4′), 8.15 (1 H, d, J ) 4 Hz, H-6′), 8.40 (1 H,
s, H-5), 8.90 (1 H, s, H-2), 14.55 (1 H, bs, COOH).
Compound 40a was then converted to 6-amino acid 41a
following the general procedure for reduction (purification
method H) to give a 95% yield: mp 272-274 °C; 1H NMR
(DMSO-d6) δ 3.10-3.40 (8 H, m, piperazine CH2), 3.50 (3 H,
s, CH3), 4.05 (3 H, s, CH3), 5.50 (2 H, bs, NH2), 6.70 (1 H, t, J
) 7.0 Hz, H-5′), 6.90 (1 H, d, J ) 8.5 Hz, H-3′), 7.45 (1 H, s,
H-5), 7.60 (1 H, t, J ) 7.0 Hz, H-4′), 8.10 (1 H, d, J ) 4 Hz,
H-6′), 8.60 (1 H, s, H-2), 15.80 (1 H, bs, COOH) Anal.
(C21H23N5O4) C, H, N.
1
Com p ou n d 32a : H NMR (DMSO-d6/CDCl3) δ 2.90-3.10
and 3.35-3.55 (each 4 H, m, piperazine CH2), 4.60 (2 H, bs,
NH2), 6.10-6.35 (2 H, m, aromatic H), 6.90 (1 H, s, H-8), 7.10-
7.40 (5 H, m, aromatic H and H-5), 7.50-7.70 (4 H, m, aromatic
H), 8.50 (1 H, s, H-2), 14.50 (1 H, bs, COOH). Anal.
(C26H22N6O3S) C, H, N.
Com p ou n d 33d : 1H NMR (DMSO-d6) δ 2.75-2.95 and
3.75-3.95 (each 4 H, m, piperazine CH2), 6.30 (1 H, s, H-8),
7.00-7.50 (10 H, m, aromatic CH and H-5), 7.60-7.90 (4 H,
m, aromatic CH), 8.60 (1 H, s,H-2). Anal. (C33H27N5O3S) C, H,
N.
Gen er a l P r oced u r e for Ba sic Hyd r olysis Rea ction . A
suspension of selected 6-amino ester (0.3 mequiv) in 1 N NaOH
(5 mL) was refluxed until the suspension became a solution
(usually 2-3 h). After the mixture was cooled to room
temperature, the precipitate obtained was filtered and solu-
bilized in water and the solution was brought to pH 6 by
adding a 2 N HCl solution. The resulting precipitate was
filtered and washed with water to give the corresponding acid.
By use of this general procedure, the target acids 26b and 28b
were obtained. Further experimental details are given in Table
1, while their spectral data are listed below.
Com p ou n d 26b: 1H NMR (DMSO-d6) δ 3.10-3.50 (8 H,
m, piperazine CH2), 3.75 and 4.10 (each 3 H, s, CH3), 5.50 (2
H, bs, NH2), 6.80-7.00 (4 H, m, aromatic CH), 7.25 (1 H, s,
H-8), 7.50 (1 H, s, H-5), 8.75 (1 H, s, H-2), 16.00 (1 H, bs,
COOH). Anal. (C22H24N4O4) C, H, N.
Com p ou n d 28b: 1H NMR (CDCl3) δ 1.10-1.40 (4 H, m,
cyclopropyl CH2), 3.10-3.25 (7 H, m, piperazine CH2 and
OCH3), 3.75-3.85 (5 H, m, piperazine CH2 and cyclopropyl
CH), 5.50 (2 H, bs, NH2), 6.90-7.00 (4 H, m, aromatic CH),
7.50 (1 H, s, H-8), 7.70 (1 H, s, H-5), 8.50 (1 H, s, H-2). Anal.
(C24H26N4O4) C, H, N.
1-Met h yl-6-n it r o-7-[4-(2-p yr id in yl)-1-p ip er a zin yl]-4-
oxo-1,4-d ih yd r oqu in olin e-3-ca r boxylic Acid (34a ). Start-
ing from nitro ester 10a 14 and using the above general
procedure for acidic hydrolysis (4 h at reflux, purification
method J as reported in footnotes of Table 1), the title
compound was obtained in 80% yield: mp 295-300 °C; 1H
NMR (DMSO-d6) δ 3.50-3.60 and 3.80-3.90 (each 4 H, m,
piperazine CH2), 4.10 (3 H, s, CH3), 6.90 (1 H, t, J ) 7.0 Hz,
H-5′), 7.20 (1 H, d, J ) 7.5 Hz, H-3′), 7.30 (1 H, s, H-8), 7.85-
8.15 (2 H, m, H-4′ and H-6′), 8.75 (1 H, s, H-5), 9.00 (1 H, s,
H-2). Anal. (C20H19N5O5) C, H, N.
8-F lu or o-1-m et h yl-6-n it r o-7-[4-(2-p yr id in yl)-1-p ip er -
a zin yl]-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r b oxylic Acid
(38a ). The title compound was obtained starting from synthone
36 and using the general procedure for the coupling reaction
(8 h at 70-80 °C, purification method C in Table 1) to give
compound 37a (86% yield) that was then acid-hydrolyzed to
38a (general method for acidic hydrolysis, 2 h at reflux,
purification method K, 82% yield): mp 248-250 °C; 1H NMR
(DMSO-d6) δ 3.25-3.40 and 3.75-3.90 (each 4 H, m, pipera-
zine CH2), 4.25 (3 H, d, J ) 9.0 Hz, CH3), 6.95 (1 H, t, J ) 7.0
Hz, H-3′), 7.40 (1 H, d, J ) 9.0 Hz, H-5′), 8.00 (1 H, t, J ) 7.0
Hz, H-4′), 8.10 (1 H, d, J ) 4.0 Hz, H-6′), 8.50 (1 H, d, J ) 1.5
Hz, H-5), 9.00 (1 H, s, H-2). Anal. (C20H18FN5O5) C, H, N.
7-Ch lor o-1-m et h yl-4-oxo-1,4-d ih yd r oq u in olin e-3-ca r -
boxylic Acid BF 2 Ch ela te (44). A mixture of 43 (0.4 g, 1.5
mmol) and 48% HBF4 in water was heated at 90-100 °C for
30 min. After the mixture was cooled, the white solid was
filtered, washed with water, and dried to give borine chelate
44 (0.4 g, 93%): 1H NMR. (DMSO-d6) δ 4.40 (3 H, s, CH3),
8.00 (1 H, dd, J ) 2.0 and 8.9 Hz, H-6), 8.50 (1 H, d, J ) 2.0
Hz, H-8), 8.55 (1 H, d, J ) 8.9, H-5), 9.60 (1 H, s, H-2).
7-[4-(2-Meth oxyp h en yl)-1-p ip er a zin yl]-1-m eth yl-4-oxo-
1,4-d ih yd r oqu in olin e-3-ca r boxylic Acid (45b). A mixture
of borine complex 44 (0.65 g, 2.3 mmol), 1-(2′-methoxyphenyl)-
piperazine (2.18 g, 11.4 mmol), and Et3N (0.69 g, 6.8 mmol) in
dry DMSO (30 mL) was heated to 100 °C for 4 h. The mixture
was then evaporated to dryness to give a residue that was
treated with ice/water. The obtained solid was filtered, dried,
and purified by flash chromatography, eluting with MeOH/
CHCl3 (2:98) to give acid 45b (0.18 g, 20%): mp 290-310 °C;
1
H NMR (DMSO-d6) δ 3.10-3.20 (4 H, m, piperazine CH2),
3.70-3.90 (7 H, m, piperazine CH2 and CH3), 4.25 (3 H, s,
CH3), 6.80-7.10 (4 H, m, aromatic CH), 7.15 (1 H, d, J ) 2.0
Hz, H-8), 7.70 (1 H, dd, J ) 2.0 and 8.9 Hz, H-6), 8.25 (1 H, d,
J ) 8.9 Hz, H-5), 9.25 (1 H, s, H-2). Anal. (C22H23N3O4) C, H,
N.
1-Meth yl-7-[4-(2-p yr id in yl)-1-p ip er a zin yl]-4-oxo-1,4-d i-
h yd r oqu in olin e-3-ca r boxylic Acid (45a ). Following the
procedure of 45b, starting from borine complex 44 and
substituting 1-(2-methoxyphenyl)piperazine with 1-(2-pyridi-
nyl)piperazine, the title compound was prepared (26%): mp
1
302-303 °C;
H NMR(DMSO-d6) δ 3.60-3.90 (8 H, m,
piperazine CH2), 4.25 (3 H, s, NCH3), 6.70 (1 H, t, J ) 7.0 Hz,
pyridine CH), 6.90 (1 H, d, J ) 8.5 Hz, pyridine CH), 7.10 (1
H, bs, H-8), 7.50-7.70 (2 H, m, pyridine CH), 8.15 (1 H, d, J
) 4 Hz, pyridine CH), 8.25 (1 H, d, J ) 8.9 Hz, H-5), 9.30 (1
H, s, H-2). Anal. (C20H20N4O3) C, H, N.
1-Met h yl-7-[4-(2-p yr id in yl)-1-p ip er a zin yl)]-4-oxo-1,4-
d ih ydr o-1,6-n a p h th yr idin e-3-ca r boxylic Acid (49a ). Start-
ing from synthone 48 and with the general procedure for
coupling (100 °C for 72 h, purification method D in Table 1)
followed by hydrolysis as reported in the general procedure

Anti-HIV 6-Aminoquinolones
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 22 5577
for basic hydrolysis, the title compound was obtained in 50%
yield: 319-320 °C; 1H NMR (DMSO-d6/CDCl3) δ 3.50-3.70
(4 H, m, piperazine CH2), 3.80-4.00 (7 H, m, piperazine CH2
and CH3), 6.50-6.65 (2 H, m, pyridine CH and H-8), 6.80 (1
H, d, J ) 8.5 Hz, pyridine CH), 7.40-7.55 (1 H, m, pyridine
CH), 8.10-8.20 (1 H, m, pyridine CH), 8.85 (1 H, s, H-5), 9.10
(1 H, s, H-2), 15.00 (1 H, bs, COOH). Anal. (C19H19N5O3) C, H,
N.
1-(5-Ch lor o-1,3-ben zoth ia zol-2-yl)p ip er a zin e (g). A mix-
ture of 2-mercapto-5-chloro-1,3-benzothiazole, (2 g, 9.93 mmol),
MeI (1.41 g, 9.93 mmol), KOH (0.056 g, 9.93 mmol), and TBAB
(0.30 g, 1.0 mmol) in dry THF was reacted at room tempera-
ture for 3 h. The mixture was then poured into water to give
a white solid that was filtered, dried, and recrystallized from
MeOH, affording 2-methylmercapto-5-chloro-1,3- benzothiazole
(1.7 g, 79%).
The mixture of the above compound (0.6 g, 2.78 mmol) and
piperazine (1.436 g, 16.7 mmol) was heated in a closed vessel
for 5 h at 110 °C. After cooling, the mixture was treated with
MeOH to give a solid that was filtered to afford base g (0.7 g,
94%); mp 128-132 °C; 1H NMR (CDCl3) δ 3.00-3.20 and 3.60-
3.70 (each 4 H, m, piperazine CH2), 7.05 (1 H, dd, J ) 13.2
Hz, H-6), 7.50-7.65 (2 H, m, H 4 and H-7).
collected by centrifugation and suspended in fresh tissue
culture medium at 250 000 cells per mL. The cells were
infected with 100 50% cell culture infective doses (CCID50) of
HIV-1(IIIB) or HIV-2 (ROD). Then, 100 µL of the infected cell
suspensions was added to 200 µL microtiter plate wells
containing 100 µL of an appropriate dilution of the test
compounds. The inhibitory effects of the test compounds on
HIV-induced syncytium formation in CEM cells were examined
on day 4 postinfection, as previously described.36,37
Peripheral blood mononuclear cells (PBMCs) from healthy
donors were isolated by density centrifugation (Lymphoprep;
Nycomed Pharma, AS Diagnostics, Oslo, Norway) and stimu-
lated with phytohemagglutin (PHA) (Sigma Chemical Co.,
Bornem Belgium) for 3 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.38 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 supernatant by an enzyme-linked
immunosorbent assay (NEN, Paris, France).
1-(5-Meth yl-1,3,4-th ia d ia zol-2-yl)p ip er a zin e (l). By use
of the above procedure, replacing 2-mercapto-5-chloro-1,3-
Tim e-of-Ad d ition Exp er im en t. MT-4 cells were infected
with HIV-1 (IIIB) at a multiplicity of infection (moi) of 0.5. The
test compounds were added at different times after infection
(from 0 to 25 h).39 Viral p24 Ag production was determined at
31 h postinfection by ELISA (NEN, Brussels, Belgium). The
reference compounds were added at 100 times their 50%
inhibitory concentration (IC50) obtained in the MT-4 cells/MTT
assay.
benzothiazole with 2-mercapto-5-methyl-1,3,4-thiadiazole, the
1
title compound was obtained in 88% yield: mp 115 °C;
H
NMR (CDCl3) δ 2.60 (3 H, s, CH3), 3.00-3.10 and 3.50-3.60
(each 4 H, m, piperazine CH2), 4.20 (1 H, bs, NH).
In Vitr o An tivir a l Assa ys. Evaluation of the antiviral
activity 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.30 Stock solutions (10× final
concentration) of test compounds were added in 25 µL volumes
to two series of triplicate wells to allow simultaneous evalu-
ation 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 mi-
crotiter trays using a Biomek 2000 robot (Beckman Instru-
ments, Fullerton, CA). Untreated control HIV- and mock-
infected cell samples were included for each sample.
HIV-1(IIIB)31 or HIV-2 (ROD)32 stock (50µL) at 100-300
CCID50 (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
supernatant was discarded. The MT-4 cells were resuspended
at 6 × 105 cells/mL, and an amount of 50 µL volumes was
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.
Ack n ow led gm en t. These investigations were sup-
ported in part by Ministero dell’Istruzione, dell’Universita`
e della Ricerca (Grant PRIN 2002, Rome, Italy), and in
part by grants from the European Commission (Credit
No. QLRT200-30291 and the Rene Descartes Prize 2001
No. HPAW-2002-90001), the Belgian “Geconcerteerde
Onderzoeksacties” (Project NoGOA-00/12), the “Fonds
voor Wetenschappelijk Onderzoek Vlaanderen” (Grants
G.0104.98 and G.0267.04), and FORTIS/ISEP. We are
grateful to Roberto Bianconi, Liesbet De Dier, Kristien
Erven, and Cindy Heens for excellent technical as-
sistance.
Su p p or tin g In for m a tion Ava ila ble: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
The MTT assay is based on the reduction of yellow 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Acros Organics, Geel, Belgium) by mitochondrial dehydroge-
nase 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 concen-
tration 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).
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Cecchetti, V.; Sabatini, S.; Gatto, B.; De Clercq, E.; Fravolini,
A. New 6-Aminoquinolones as Antiviral Agents. Presented at
the First Magna Graecia Medicinal Chemistry Workshop on New
Perspectives in Drug Research, Copanello, Italy, 2001; Abstract
PC 39.
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effect in CEM cells was performed using the MTS assay as
previously described.34 This cell-based microtiter assay quan-
titates the ability of a compound to inhibit the HIV-induced
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