Anti-HIV activity of stilbene-related heterocyclic compounds

Article abstract of DOI:10.1016/j.bmcl.2006.04.087

Viral transcription has not been routinely targeted in the development of new antiviral drugs. This crucial step of the viral cycle depends on the concerted action of cellular and viral proteins such as NF-κB and Tat. In the present study, stilbene-related heterocyclic compounds including benzalphthalide, phthalazinone, imidazoindole and pyrimidoisoindole derivatives are tested for their anti-HIV activity. Original assays based on recombinant viruses were used to evaluate HIV replication inhibition and stably transfected cell lines were used to evaluate inhibition of Tat and NF-κB proteins. Some of the stilbene-related heterocyclic compounds analysed displayed anti-HIV activity through interference with NF-κB and Tat function. Moreover, compounds inhibiting both targets displayed a stronger activity on viral replication.

Full text of DOI:10.1016/j.bmcl.2006.04.087

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4075–4079
Anti-HIV activity of stilbene-related heterocyclic compounds
a
b
c
b
´
Luis M. Bedoya, Esther del Olmo, Rocıo Sancho, Bianca Barboza,
a
b
a
´
´
˜
´
Manuela Beltran, Ana E. Garcıa-Cadenas, Sonsoles Sanchez-Palomino,
Jose L. Lopez-Perez, Eduardo Munoz, Arturo San Feliciano and Jose Alcamı
b
c
b
a,
*
´
´
´
´
´
aUnidad de Inmunopatalogı´a, Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, 28220-Majadahonda, Madrid, Spain
bDepartamento de Quı´mica Farmace´utica, Facultad de Farmacia, Universidad de Salamanca,
Campus Miguel de Unamuno, 37007-Salamanca, Spain
cDepartamento de Biologı´a Celular, Fisiologı´a e Inmunologı´a, Universidad de Co´rdoba,
Avda. Mene´ndez Pidal s/n, 14004-Co´rdoba, Spain
Received 16 March 2006; revised 27 April 2006; accepted 28 April 2006
Available online 18 May 2006
Abstract—Viral transcription has not been routinely targeted in the development of new antiviral drugs. This crucial step of the viral
cycle depends on the concerted action of cellular and viral proteins such as NF-jB and Tat. In the present study, stilbene-related
heterocyclic compounds including benzalphthalide, phthalazinone, imidazoindole and pyrimidoisoindole derivatives are tested for
their anti-HIV activity. Original assays based on recombinant viruses were used to evaluate HIV replication inhibition and stably
transfected cell lines were used to evaluate inhibition of Tat and NF-jB proteins. Some of the stilbene-related heterocyclic com-
pounds analysed displayed anti-HIV activity through interference with NF-jB and Tat function. Moreover, compounds inhibiting
both targets displayed a stronger activity on viral replication.
Ó 2006 Elsevier Ltd. All rights reserved.
Human immunodeficiency virus (HIV) is the etiological
agent of acquired immunodeficiency syndrome (AIDS).
This immunological disease remains the number one
cause of mortality produced by infectious agents.1 Drug
discovery efforts yield new compounds with anti-HIV
activity every year. However, these agents only interfere
with three viral proteins, reverse transcriptase, protease
and gp41 or fusion peptide. Moreover, emergence of
resistance makes currently available drugs insufficient
to maintain a safe therapeutic arsenal against HIV.
Effective synergy has been found when combinations
of drugs directed against different targets are used. Thus,
development of new compounds against diverse steps of
HIV life cycle is envisaged.
into the cell genome. As an integrated provirus HIV
can remain in a latent state from weeks to several years.
Transition from latency to HIV replication occurs main-
ly when cells are activated and requires the concerted
action of cellular transcription factors and regulatory
HIV proteins.2
Reactivation from latent state is mediated by viral and
cellular proteins such as Tat and NF-jB. Both could
be possible targets for anti-HIV chemotherapy. The
NF-jB family of transcription factors is involved in
different processes such as embryonic development,
apoptosis control, inflammatory response regulation
and immune system activation.3 In addition, NF-jB is
the major inducible regulatory element involved in Long
terminal repeats (LTR) transactivation and HIV-1 repli-
cation in CD4 lymphocytes.4,5
HIV viral cycle is divided into early and late events.
Early events begin with the viral entry into the host cell
and conclude with the integration of the HIV provirus
The virally encoded Tat protein is a potent activator of
HIV expression6,7 by, at least, two pathways.8 First
through binding the transactivation response element
(TAR), a stable RNA stem loop located downstream
of the transcriptional initiation site, leading to full
RNA elongation.9,6 Second through the interaction with
Keywords: NF-jB; Tat; HIV replication; Benzalphthalides; Phthalazi-
nones; Imidazoisoindoles; Pyrimidoisoindoles.
*
Corresponding author. Tel.: +34 918223943; e-mail: ppalcami@
isciii.es
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.087

4076
L. M. Bedoya et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4075–4079
cellular transcription factors that bind to the enhancer
region of the HIV-LTR, thus increasing HIV transcrip-
tion. In fact, functional relations between Tat and
NF-jB have been described.5,10 Moreover, Tat is also
able to modify the expression of genes involved in
apoptosis.11
contain the stilbene or dihydrostilbene skeletal basis
supporting a fused heterocyclic system and can be divided
in four main groups, benzalphthalides, phthalazinones,
imidazoisoindoles and pyrimidoisoindoles. Some
compounds belonging to these families displayed some
activity against protozoa19–21 and also promoted aorta
ring vasorelaxation22 and in vivo anxiolytic effects on
mice.23 The compounds evaluated in this research were
selected as part of a preliminary systematic screening,
and were prepared through chemical procedures already
reported by us21 that are summarized in Scheme 1. Aim-
ing to recognise the respective influence on the activity, a
number of natural-like and unnatural substituents, with
different electronic, lipophilic, polarity and size attributes,
Therefore, simultaneous targeting of both, Tat and
NF-jB,12,13 could provide better expectancy against
drug resistance than that attained in current AIDS
therapy.14,15
Stilbenoids have previously shown anti-HIV activity,16,17
in particular anti-Tat activity.18 Compounds tested here
R2
R2
COOH
O
R3NHNH2
O
NaOAc, Δ
R1
A
N
N
R1
R2
O
+
R1
12 - 95%
16 - 99%
R3
O
O
H2N
NH2
O
H2N
NH2
phthalazinones
2
3
R2
R2
HO
HO
R1
38 - 99%
R1
N
N
N
10 - 92%
pyrimido[2,1-a]isoindoles
N
imidazo[2,1-a]isoindoles
Scheme 1. The synthesis of fused heterocyclic compounds related to stilbene-2-carboxylic acid.
Table 1. Results of anti-HIV and cytotoxicity evaluations for some selected benzalphthalidesa
B
R2
3
5
R1
A
O
O
1
6
1k
O
O
Compound Yield (%)
Structure
R2
Antiviral activity
Cytotoxicity
Index
R1
H
5.1+TNF HeLa Tat-luc HeLa Tet-ON RVA IC50 Propidium iodide CC50/IC50
100 lM
100 lM
100 lM
lM
CC50 lM
1b
1c
1e
1h
1k
35
70
69
25
15
2-Cl
4-Cl
<0
76.8
84.6
89.7
87.2
6.5
S
24.4
23.8
23.4
12.2
>50
>195
>195
>187
138
>8.0
>8.2
>8.0
11.3
—
84.8
86.7
U
4-SCH3
U
3,4-OCH2O– 91.8
1-Naphthyl
U
ND
<0
ND
2a
2b
2c
2d
2f
65
95
90
59
54
5(6)-CH3
H
2-Cl
93.7
71.0
96.8
87.6
31.2
77.7
<0
U
22.3
133
ND
—
S
>185
ND
ND
>1.4
—
4-Cl
4-OCH3
2,4-Cl2
91.8
74.0
29.7
U
15.8
14.3
20.5
U
ND
—
>164
8.0
3c
4c
12
85
4-NO2
5,6-Cl2
4-Cl
4-Cl
<0
3.2
63.7
<0
U
ND
119
<20.7
>154
<0.2
>3.4
45.6
IC50, concentration that reduces luciferase activity, thus RV replication, by 50%. CC50, cytotoxic concentration that reduces viability of uninfected
cells by 50%. ND, not determined; S, specific; U, nonspecific.
Results for HeLa Tat-Luc and 5.1 assays expressed as percentage of inhibition relative to control. <0 values indicate enhancement of the corre-
sponding factor. Compounds 2a–g were evaluated as regioisomeric 1:1 mixtures.
a Complete data related to Table 1 can be found in Supplementary data.

L. M. Bedoya et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4075–4079
4077
were located at certain positions on the structure of each
group of compounds. Compounds submitted to
evaluation were obtained in pure form, either through
recrystallisation or preparative CC or TLC. All of them
the same reporter plasmid as 5.1 cells together with
the tat gene regulated by CMV promoter. Thus, LTR
is highly activated in this cell line secondarily to expres-
sion of intracellular Tat protein.
1
were characterised through their IR, H and 13C NMR
spectral data. Experimental CHN analytical data were
in agreement with the molecular formulae.
Compounds were considered active when percentage of
inhibition of NF-jB and/or Tat was higher than 50%
and 30%, respectively, as compared to an untreated con-
trol. In order to rule out nonspecific mechanism of ac-
tion, active compounds were evaluated through a
HeLa Tet-On-Luc assay, in which the luciferase gene
is under the control of an artificial promoter regulated
by doxycycline.25 Compounds lacking NF-jB and Tat
activities were not submitted to specificity evaluation.
To evaluate the antiviral activity of these compounds,
an original viral replication assay was performed using
HIV clones, in which luciferase reporter genes have been
cloned.24 In this model, inhibition of viral replication is
directly evaluated through measurement of luciferase
activity in cell lysates in the presence or the absence of
the different compounds. Inhibition of NF-jB was
evaluated using 5.1 LTR Luc stably transfected cells,
which contain a plasmid in which the reporter luciferase
gene is driven by HIV-1 LTR promoter and it is respon-
sive to the NF-jB activator cytokine TNFa. To perform
the screening of anti-Tat activity, HeLa Tat-Luc stably
transfected cell line was used. This cell line contains
Twenty-seven benzalphthalides were evaluated and the
results are partially shown in Table 1. Among them,
nine compounds were NF-jB inhibitors at 100 lM
and just compound 2b displayed a specific mode of
action. Tat inhibition was demonstrated for ten
compounds, but only 1b was specific.
Table 2. Results of anti-HIV and cytotoxicity evaluations for some selected phthalazinonesa
R3
B
m
6
N
N
N
N
N
N
R1
R1
R1
k
A
A
A
R2
7
R2
R2
O
O
O
Compound Yield
(%)
Structure
Antiviral activity
5.1+TNF HeLa HeLa
Cytotoxicity
Index
R1
R2
R3
RVA IC50
Propidium
iodide
CC50/IC50
100 lM
Tat-luc
100 lM
Tet-ON (lM)
100 lM
CC50 (lM)
5b
5j
93
97
64
61
92
99
99
90
79
39
23
16
62
H
H
H
2-Cl
3,4,5-(OCH3)3
2-Cl
67.3
96.5
73.0
97.2
100
29.8
<0
S
41.6
51.7
21.6
27.4
11.0
16.6
22.3
30.5
16.0
12.8
58.7
88.1
89.5
>185
135
>4.5
2.6
>8.2
4.8
5.7
3.5
4.0
2.3
4.4
5.6
1.2
1.0
1.1
S
6b
6h
6k
6m
7d
8d
9i
CH3
CH3
CH3
CH3
Et
18.3
25.5
43.2
11.8
14.2
2.9
S
>176
131
3,4-OCH2O–
1-Naphthyl
2-Naphthyl
4-OCH3
4-OCH3
3,4-(OCH3)2
4-Cl
S
U
U
S
62.9
57.2
89.5
68.6
70.3
72.1
67.7
91.8
96.3
99.9
93.5
50.0
83.0
82.1
86.4
<0
Allyl
n-Bu
t-Bu
Ph
S
6.7
U
S
10c
11.5
<0
11c
12c
13c
4-Cl
4-Cl
S
4-BrPh
4-NO2Ph
<0
29.6
ND
S
4-Cl
54.8
14c
14g
15f
74
84
81
6(7)-CH3
6,7-Cl2
H
4-Cl
3,4-Cl2
2,4-Cl2
76.6
14.9
<0
99.0
28.4
14.0
S
14.5
19.5
18.8
129
64.7
112
8.9
3.3
6.0
H
CH3
ND
ND
16b
16d
16f
17a
64
88
55
60
H
2-Cl
65.9
12.6
99.0
19.4
11.2
<0
S
22.0
15.7
96.2
47.5
>147
>149
58.0
>6.7
>9.5
0.6
H
4-OCH3
2,4-Cl2
H
ND
S
H
CH3
53.9
11.4
ND
>156
3.3
IC50, concentration that reduces luciferase activity by 50%. CC50, concentration that reduces viability of uninfected cells by 50%. ND, not deter-
mined; S, specific; U, nonspecific.
Results of HeLa Tat-Luc and 5.1 assays expressed as percentage of inhibition relative to control. <0 values indicate enhancement of the corre-
sponding factor. Compounds 5a–g and 14b–g were assayed as regioisomeric 1:1 mixtures.
a Complete data related to Table 2 can be found in Supplementary data.

4078
L. M. Bedoya et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4075–4079
Concerning antiviral activity, the recombinant virus as-
say (RVA) revealed low potency values except for com-
pounds 1b, 2f and 4c. Compound 2b showed a specific
mode of action, although percentage of NF-jB inhibi-
tion was in the medium range. The most interesting
compounds of this family were: 1b, which interferes
HIV replication by inhibiting Tat activity through an
NF-jB-independent mechanism, and 2f and 4c which
should act by a mechanism not related to those analysed
here. Interestingly, those benzalphthalides displaying
anti-HIV activity resulted less cytotoxic and it should
be noticed that they contain chlorine atoms at positions
2 or 4 of the phenyl substituent.
alkylation of the nitrogen atom at position 2 and the
presence of a chlorine atom at positions 2 or 4 on ring
B (compounds 5b and 5c, respectively) seem to be
important features for NF-jB inhibition and for the
specificity of antiviral activity. However, this apprecia-
2
tion clearly fails in the case of N-allyl (compounds 8)
or 2N-phenyl (compounds 11–13) derivatives. On the
other hand, the existence of methyl- or dichloro-substi-
tutions on ring A makes the absence of a substituent
on 2N preferred (compounds 14 vs 15), not only for
the above-mentioned aspects of antiviral activity, but
also for enhancing substantially the Tat inhibitory activ-
ity (99% at 100 lM, for compound 14c). Additionally,
and considering all of the evaluated phthalazinones,
three compounds merit to be mentioned especially.
Compound 14c was the only compound with a specific
and potent antiviral activity, inhibiting both targets
(NF-jB and Tat) and displaying a good RVA activity/
cytotoxicity ratio (index: 8.9; Table 2). Compound 10c
resulted more potent than 14c in the RVA, with an
IC50 value of 12.8 lM, being also specific, and display-
ing a fair NF-jB inhibitory activity. Compound 6b be-
haved similarly, it was less potent as antiviral than
10c, but also less cytotoxic. Finally, like benzalphtha-
lides, some phthalazinones (e.g., 8e, 14g, 15f and 16d)
showed relatively high potency in the RVA, without
notable inhibitory activities on NF-jB nor Tat, suggest-
ing that they would act through another mechanism.
The results of antiviral screening for phthalazinones are
partially shown in Table 2. Thirty-four compounds
(68%) inhibited HIV replication in the RV assay and
its mode of action was specific. The activity of these
compounds could be mainly due to NF-jB inhibition,
since twenty-two specific compounds (44% of the total)
inhibit TNFa activation of 5.1 LTR Luc cells. However,
some compounds that do not target neither NF-jB nor
Tat, remain active in the RVA, suggesting that some
compounds of this family could act on other targets.
Apart from the general observations about phthalazi-
nones (good NF-jB inhibitors, with medium potency
in the RVA and not too cytotoxic compounds), other
SAR considerations on the results shown in Table 2
could not be easily deduced. Nevertheless, it can be
observed that within the subfamily of phthalazinones
without substituent on ring A (compounds 5–13), the
The results for the two related families of imidazoisoin-
doles and pyrimidoisoindoles are partially shown in
Table 3. Inhibition of HIV replication was observed
Table 3. Results of anti-HIV and cytotoxicity evaluations for some selected imidazoisoindoles and pyrimidoisoindolesa
B
R2
B
R2
HO
HO
HO
HO
7
8
9
a - j
a - j
m
m
R1
R1
R1
R1
N
N
N
N
A
A
N
N
A
A
8
N
N
imidazo[2,1-a]isoindoles (18, 19, 20)
pyrimido[2,1-a]isoindoles (21)
Antiviral activity Cytotoxicity Index
5.1+TNF HeLa Tat-luc HeLa Tet-ON RVA IC50 Propidium
Compound Yield (%)
Structure
R2
R1
CC50/IC50
100 lM
100 lM
100 lM
(lM)
iodide
CC50 (lM)
18c
18f
19c
19g
20b
20d
88
99
96
38
82
65
H
H
4-Cl
2,4-Cl2
<0
<0
38.4
86.6
78.9
84.0
99.6
75.3
S
S
S
S
U
S
71.0
13.8
25.9
40.1
22.8
27.6
>168
87.5
34.4
37.8
30.1
44.1
2.4
6.3
1.3
0.9
1.3
1.6
7(8)-CH3 4-Cl
7(8)-CH3 3,4-Cl2
98.9
100
100
100
7,8-Cl2
7,8-Cl2
2-Cl
4-OCH3
21b
21d
21e
21g
21m
26
25
39
76
71
H
H
H
H
H
2-Cl
99.7
<0
<0
S
66.7
74.2
61.5
26.1
60.8
78.6
>162
142
1.2
>2.2
2.3
4-OCH3
4-SCH3
3,4-Cl2
3.8
ND
S
99.8
67.7
13.9
60.0
51.2
S
85.2
81.0
3.3
1.3
2-Naphthyl 100
S
IC50, concentration that reduces luciferase activity by 50%. CC50, concentration that reduces viability of uninfected cells by 50%. ND, not deter-
mined; S, specific; U, nonspecific.
Results of HeLa Tat-Luc and 5.1 assays expressed as percentage of inhibition relative to control. <0 values indicate enhancement of the corre-
sponding factor. Compounds 19a–c were evaluated as regioisomeric 1:1 mixtures.
a Complete data related to Table 3 can be found in Supplementary data. No attempts were made to optimise reactions and yields.

L. M. Bedoya et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4075–4079
4079
for almost all the compounds tested when RVA was
References and notes
used. After discarding for further studies nonspecific
and toxic compounds, imidazoisoindoles 18f, 19b, 19c
and 20d, and pyrimidoisoindoles 21d, 21e, 21g and
21m displayed HIV replication interference. Neverthe-
less, CC50 cytotoxicity values were close to the IC50 val-
ues observed in the RV assay, and the cytotoxicity/
activity indexes were too low, as seen in Table 3. This
toxicity could also justify the erratic changes of activity
observed for the series of compounds 18a–m. Among
these compounds, 18f showed the highest therapeutic in-
dex (6.3), but still modest, and its mode of action seems
to be related to Tat inhibition.
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thanks the fellowship from the EC ALFA program
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Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.bmcl.2006.
04.087.