"viologen"dendrimers as antiviral agents: The effect of charge number and distance

Article abstract of DOI:10.1021/jm100093p

A series of "viologen"derivatives (4,4′-bipyridinium salts) carrying between 1 and 90 charges per molecule have been prepared and investigated for their activity against human immunodeficiency virus (HIV), herpes simplex virus (HSV), vesicular stomatitis, Punta Toro virus, Sindbis virus, Reovirus, and respiratory syncytial viruses. In general, most of the compounds showed good activities against HIV-1 (strain III_B). In particular, compound 36 exhibited the highest in vitro activity and selectivity index against HIV-1 (strain III_B) (EC₅₀ = 0.26 ± 0.08 μM, SI = 75.7) in MT-4 cells. The results imply that the antiviral activity requires an optimal number and distance of the positive charges.

Full text of DOI:10.1021/jm100093p

3480 J. Med. Chem. 2010, 53, 3480–3488
DOI: 10.1021/jm100093p
“Viologen” Dendrimers as Antiviral Agents: The Effect of Charge Number and Distance†
Simona Asaftei*,‡ and Erik De Clercq*,§
‡Institute of Chemistry, University of Osnabru€ck, Barbarastrasse 7, D-49069 Osnabru€ck, Germany, and
§Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received November 13, 2009
A series of “viologen” derivatives (4,40-bipyridinium salts) carrying between 1 and 90 charges per mole-
cule have been prepared and investigated for their activity against human immunodeficiency virus (HIV),
herpes simplex virus (HSV), vesicular stomatitis, Punta Toro virus, Sindbis virus, Reovirus, and respira-
tory syncytial viruses. In general, most of the compounds showed good activities against HIV-1 (strain
IIIB). In particular, compound 36 exhibited the highest in vitro activity and selectivity index against
HIV-1 (strain IIIB) (EC50=0.26 ( 0.08 μM, SI=75.7) in MT-4 cells. The results imply that the antiviral
activity requires an optimal number and distance of the positive charges.
Introduction
potency (Chart 2). The highest activity against HIV-1 exhibits
36; it inhibits the replication of HIV-1(IIIB) in MT-4 cell at a
50% effective concentration (EC50) 0.26μM. In this context, it is
interesting to note that poly(propylene imine) dendrimers
with 16 dimethyldodecylammonium end groups exhibited pro-
nounced activity against gram-negative bacteria (4 μg/mL) 2
orders of magnitude higher than their monofunctional ana-
logues.1 In our study are included: (i) the activity of the
compounds in an MT-4 cell line against HIV, (ii) anti-HIV
activity in peripheral blood mononuclear cells (PBMC), (iii)
the virucidal effect of the compounds and inhibition of virus
binding. Furthermore, the novel compounds were tested
against vesicular stomatitis, Punta Toro virus, Sindbis virus,
Reovirus, and respiratory syncytial viruses.
The dendrimers’ architecture offers a high local concentra-
tion of a given functionality, cooperative effects, and poly-
valent effects, and sometimes a polycationic structure can be
utilized to design effective antimicrobial agents and efficient
microbiocide delivery systems.1 In general, antiviral dendri-
mers function as artificial mimics of the anionic cell surfaces,
thus dendrimers are generally designed having anionic surface
groups such as acid- or sulfonate residues (heparan sulfate,
sulphated glycosaminoglycans, etc.), which are exposed on
eukaryotic cell surfaces and extracellular matrix (ECMa).
Several viruses such as human immunodeficiency virus, her-
pes virus, and cytomegalovirus interact with host cells by
binding to sulphated residues on cell surfaces and ECM.2 In
other words, the dendritic drug competes with the cellular
surface for binding of the virus, leading to a lower cell-virus
infections probability.3 Polylysine dendrimers are known as
viral inhibitors for Herpes Simplex virus in vitro,4 and PA-
MAM dendrimers show also antiviral activity against HIV.5,6
The dendrimers work as an inhibitor for virus entry and in the
late stages of virus replication, but the exact mechanism of
action has not been described in detail.
Results and Discussion
Chemistry. The synthesis of the biologically active com-
pounds 35-38 is described in Schemes 1 and 2. The com-
pounds 35 and 37 with a phenyl core were synthesized,
following the divergent dendrimers’ synthesis method, accor-
ding to the method of Ardoin and Astruc7 and Heinen et al.8
from the corresponding initiator core 3 (Scheme 1).
4,40-Bipyridine was converted to the intermediarily for-
med compound 1,10-bis(2,4-dinitrophenyl)4,40-bipyridinium
salt (28) by a nucleophilic substitution reaction with 1-chloro-
2,4-dinitrobenzene.9 The resulting dinitro intermediate is a
well-known starting material for the synthesis of diphenylbi-
pyridinium salts, and was further reacted with 3,5-di(hydroxy-
methyl) aniline, synthesized according to the literature10 by
reaction of 5-amino-isophthalic acid dimethylester and lithium-
Through a systematic analysis of structure-activity rela-
tionships of a series of “viologen” derivatives (4,40-bipyridi-
nium salts) carrying between 1 and 90 charges per molecule
(Charts 1 and 2), we report here a new biological aspect of
polycationic “viologen”-based dendrimers as novel HIV-1
replication inhibitors.
We found four polycationic “viologen”-based dendrimers
(out of 38 derivatives) which significantly enhanced antiviral
-
aluminum hydride. The intermediate PF6 salt of compound
26 is highly soluble in hydrobromic acid/acetic acid and can be
further converted to the tetra-bromide 27, followed by ion
†
€
Dedicated to Professor Dr. Lorenz Walder, University Osnabruck,
on the occasion of his 60th birthday.
-
*To whom correspondence should be addressed. For S.A.: phone, þ49-
541-969 2802; fax, þ 49-541-969 2370; E-mail: sasaftei@uos.de. For
E.D.: phone, 32-16-33.73.67; fax, 32-16-33.73.40; E-mail: erik.declercq@
rega.kuleuven.be.
exchange of the PF6 salt in good yield (78%). From the
intermediary product 27 2PF6-, the desired compound 35,
3
a dendrimer with phenyl core, was obtained by reaction of
N-ethyl-4,40-bipyridinium hexafluorophosphate (10)11 follo-
wed by ion exchange (route Ia, Scheme 1).
a
Abbreviation: ECM, extracellular matrix ; HIV, human immunodefi-
ciency virus; HSV, herpes simplex virus; PBMCs, peripheral blood mono-
nuclear cells; THF, tetrahydrofuran; MeCN, acetonitrile; DS5000, dextran
sulfate 5000; AZT, 30-azido-30-deoxythymidine; MTT, 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; GFP, green fluorescent
protein; CPE, cytopathic effect.
The first generation phenyl core dendrimer was obtained
by reaction with N-(3,5-di(hydroxymethyl)-benzyl)-4,40-bi-
pyridinium hexafluorophosphate, prepared by reaction of
r
pubs.acs.org/jmc
Published on Web 04/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3481
Chart 1. “Viologen” Monomers Carrying One to Six Charges Used in This Study
3,5-di(hydroxymethyl)-benzylbromide with 4,40-bipyridine
in freshly distilled tetrahydrofuran (THF),12 followed by
substitution of -OH by -Br in compound 27a with 5.7 M
HBr in conc acetic acid at room temperature and alkyla-
tion with N-ethyl-4,40-bipyridinium hexafluorophosphate
(10) (route Ib, Scheme 1).
The synthetic pathways for the dendrimers of first and third
generation with a benzyl core were performed according to
the method of Heinen et al.13 (Scheme 2). The three-charge
initiator core 1,3,5-tris[(4,40-bipiridinium)methyl]benzene tri-
hexafluorophosphate (30) was prepared by reaction of 1,3,5-
tris bromomethyl benzene14 with an excess of 4,40-bipyridine
in MeCN according to the literature.15
Antiviral Activity in Vitro. The synthesized compounds
were initially evaluated for anti-HIV-1 (IIIB) and anti-HIV
(ROD) activity in MT-4 cells, and cytotoxicity was estimated
in parallel by an MTT assay.16,17 The results, expressed as
CC50 (50% cytotoxicity), EC50 (50% effective concent-
ration), and SI (selectivity index =CC50/EC50) values, were
then calculated from the cell viability from each concentra-
tion of the compounds and are presented in Table 1. The
“viologen” derivatives from 1 to 9 (monoalkylated 4,40-
bipyridinium units with ethylphosphate-residues) showed
no significant antiviral activity. The compounds from 10 to
34 exhibited moderate activity against HIV-1 (IIIB) and HIV
(ROD) but no good selectivity. It is worth noting that
polycationic “viologen” 35, 36, 37, and 38 dendrimers were
found to be the most active compounds against wild-type
HIV-1. Good activity and encouraging SI values were obta-
ined by both the first generation of dendrimers with a phenyl-
or benzyl-core, respectively. In particular, compound 36
with 18 charges per molecule and a spheroidal struc-
ture was the most potent (EC50 value of 1.01 ( 0.09 μM
and SI=75.76), inhibiting HIV-1 replication in MT-4 cells
more effectively than 35 (comb-branched structure).
To better define the antiviral profile of derivative 35-38, its
anti-HIV-1 activity was determined in human PBMC cells.
Compound 35 showed better activity and selectivity in the
PMBC cells as compared to MT-4 cells but also higher
cytotoxicity (Table 2 PBMC) compared with compound 36.
The HIV-activity of compound 36 was also assayed in
acutely infected Jurkat cells (Table 3). In these cells, com-
pound 36 showed potent and selective activity against dif-
ferent strains of HIV-1.
The three peripheral nitrogens can be alkylated quantita-
tively with primary alkylhalides such as 1,3-di(hydroxymethyl)-
benzyl bromide to the hexavalent alcohol (30a). This suc-
ceeded by substitution of -OH for -Br, yielding 30a with
5.7 M HBr in conc acetic acid and subsequent ion exchange
-
to the intermediate 30b as PF6 salt. The compound 36
dendrimer with 18 charges per molecule was available
from intermediate 30b as tetrabromide via alkylation with
N-ethyl-4,40-bipyridinium hexafluorophosphate (10) in MeCN
(route Ia, Scheme 2). The dendrimers of the third generation
-
were prepared by reaction of the precursor 30b 6PF6 with
3
N-(3,5-di(hydroxymethyl)-benzyl)-4,40-bipyridinium
hexa-
fluorophosphate to give the intermediate 30d 18PF6-. The
3
resulting polyalcohol was activated by conversion to the poly-
bromide. This reaction step was repeated once to obtain the
intermediate 30f 42PF6-. For solubility reasons, an ion ex-
change was carried out after each reaction step. In the last
3
-
reaction step, the intermediate 30f 42PF6 reacted with N-
3
ethyl-4,40-bipyridinium hexafluorophosphate (10) in MeCN to
afford the compound 38 in good yields (route Ib, Scheme 2).
All the target compounds were characterized by
1H-, 13 C-NMR, and IR spectroscopy as well as by elemental
analysis.
To clarify the viral replication process inhibited by 36, a
time-of-addition assay was performed and the results were
compared to reference compounds with a known mode
of action. In contrast to the reference inhibitors, dextran
sulfate 5000 (DS5000), 30-azido-30-deoxythymidine (AZT),

3482 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Asaftei and De Clercq
Chart 2. Chemical Structure of the Biologically Active “Viologen”-Based Dendrimers
and nevirapine, for which addition can be delayed until 0, 5,
and approximately 10 h postinfection, addition of com-
pound 36 could be postponed for only 1 h postinfection
before it lost its antiviral activity (Figure 1).
attachment to target cells in the presence of different con-
centrations of compound 36 and DS-5000. After a 6 h
incubation period with a viral input of 15 ng p24 Gag,
approximately 4% of CD4-expressing human T cells con-
tained HIV-1 virions. Interestingly, the presence of 36 had no
effect on cell-associated GFP fluorescence. In contrast, DS-
5000 completely prevented appearance of cell-associated GFP
fluorescence (0.03%), pointing to an efficient inhibition of virus
adsorption to the cells. The decrease of the antiviral activity and
viral selectivity suggest that the π-π stacking could play a role
as well as the dendrimers with aromatic units in the structure.18
Compounds 35-38 were also evaluated for their activity
against HSV-1 (KOS), Herpes simplex virus-2, vaccinia virus,
vesicular stomatitis virus (VSV), herpes simplex virus-1 TK-,
respiratory syncytial virus (RSV), parainfluenza-3 virus, reo-
virus-1, Sindbis virus, and Punta Toro virus in E6SM, Vero,
and HeLa cells. Table 4 clearly shows that all polycationic
viologen dendrimers exhibit almost no activity against the
viruses mentioned above and that these compounds are only
selective against HIV-1.
To further investigate the effect of 36 on the HIV-1 entry
process VSV-G-pseudotyped (env-deficient) GFP-encoding
HIV-1 particles were exposed to C8166 cells in the absence
(control) or presence of different concentrations of the test
compounds (Figure 2). After 48 hours of postinfection, super-
natants were analyzed for GFP production. The reverse tran-
scriptase inhibitor nevirapine suppressed dose-dependently GFP
expression by the pseudotyped virus-infected cells, while the
CXCR4 antagonist AMD3100 and 36 completely lost their
inhibitory effect, pointing to a specific interaction of the HIV-1
envelope with susceptible cells as target of antiviral intervention
of 36.
To study in more detail the mechanism of action of 36 on
the HIV-1 specific entry, C8166 cell cultures were exposed
to HIV-1/NL4.3 virions labeled with GFP-Vpr fusion
proteins in the intact virion particles to monitor virion

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3483
Scheme 1. Synthetic Scheme of the 10 and 26-Fold Charged Monomers; Core Type Phenyl- (35; 37)a
a Reagents: (i) MeCN, 24 h, reflux and ion exchanged with NH4PF6; (ii) MeOH, 23 h, reflux; HBr/AcOH, 3 days, rt; (iii) ion exchanged with NH4PF6;
(iv) N-ethyl-4,40-bipyridinium hexafluorophosphate (10), MeCN, reflux, 1 week, extracted with NH4PF6, purified by CC, Sephadex LH-20, MeOH:
MeCN 1:1; (v) N-(3,5-di(hydroxymethyl)-benzyl)-4,40-bipyridinium hexafluorophosphate, MeCN, 10 days, reflux, extracted with NH4PF6; purified by
CC, Sephadex LH-20, MeOH: MeCN 1:1.
Scheme 2. Synthetic Scheme of the 18- and 90-Fold Charged Monomers; Core Type Benzyl- (36; 38)a
a Reagents: (i) MeCN, 45 h, reflux and HBr/AcOH, 3 days, rt (ii); ion exchanged with NH4PF6; (iii) N-ethyl-4,40-bipyridinium hexafluorophosphate
(10), MeCN, reflux, 1 week, purified by extraction from MeNO2/H2O 1 week; (iv) N-(3,5-di(hydroxymethyl)-benzyl)-4,40-bipyridinium hexafluorophosphate,
MeCN, 1 week, reflux.
Future Aspects. Further experiments will be focused on
the preparation of dendritic compounds built up from units
which contain positive charges in a larger distance as well as of
those which are furnished with heterocyclic nucleobases. Such
compounds and those described in this manuscript should also
be evaluated as CXCR4 antagonists and as possible inhibitors
of X4 HIV-1 strains and as stem cell mobilizers which depend on
their antagonization with CXCR4 to mobilize stem cells.

3484 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Asaftei and De Clercq
Table 1. Antiretroviral Activity and Cytotoxicity of HIV Inhibitors in MT-4a
EC50b [μM]
compd
HIV-1IIIB
HIV-2ROD
SIVMAC251
SO6141
NT
NT
CC50c [μM]
SI
<1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
>281.8
>281.8
>363.4
>346.3
>79.2
>15.9
>25.2
>3.3
NT
NT
281. ( 11.98
>363.4
>346.4
>363.4
>346.3
>79.2
<1
<1
<1
<1
<1
<1
<1
NT
NT
NT
NT
79.2 ( 18.0
15.9 ( 2.7
25.2 ( 1.2
3.3 ( 0.4
12.1 ( 1.0
20.6 ( 3.1
5.1 ( 0.4
26.1 ( 1.3
2.7 ( 1.0
0.9 ( 0.1
0.9 ( 0.2
0.4 ( 0.4
0.2 ( 0.0
2.7 ( 0.2
>148.5
>15.9
>25.2
NT
NT
NT
NT
>3.3
>12.1
NT
NT
NT
NT
>12.1
>20.6
>5.1
5.88 ( 3.08
>5.1
>26.1
>20.6
NT
NT
NT
NT
3.5
<1
<1
>26.1
>2.7
>0.9
NT
NT
>2.7
>0.9
NT
NT
<1
<1
NT
NT
>0.9
>0.4
>0.9
>0.4
NT
NT
<1
<1
NT
NT
>0.2
>2.7
>0.2
>2.7
NT
NT
<1
<1
NT
NT
>148.5
g42.2
>148.5
>107.4
>124.0
>151.5
>10.6
>81.1
>33.4
>104.2
>49.0
>19.7
9.82 ( 1.30
>0.490
NT
NT
<1
e2.54
<1
NT
NT
107.4 ( 10.8
124.0 ( 4.1
>151.5
>124.0
58.8 ( 4.6
>10.6
NT
NT
>151.5
NT
NT
2.57
<1
<1
NT
NT
10.6 ( 0.6
>81.1
>81.1
>33.4
NT
NT
33.4 ( 12.9
>104.2
>49.0
<1
33.3 ( 3.7
1.01 ( 0.09
0.26 ( 0.08
1.40 ( 0.50
0.066 ( 0.002
37.7 ( 13.2
>49.0
0.46 ( 0.37
g0.49
g0.052
>104.2
>49.0
g1.55
1.23 ( 0.99
g0.075
3.11
48.51
75.76
13.28
7.42
19.7 ( 2.1
>18.6
0.490 ( 0.002
a All data represent mean values and standard deviations for at least two separate experiments. b Concentration required to inhibit 50% of virus-
induced cytopathic effect. c Concentration that reduced cellular viability by 50%
than just a general block of the electrostatic interaction between
cell surface and virus.
Table 2. Anti-HIV Activity and Cytotoxicity of Selected Viologens
in PBMCa
HIV-1 IIIB
We assume that these polycationic compounds bind to
heparan sulfate structures expressed on the surface of the host
cells. This is corroborated by the fact that the positive
compd
EC50 (μg/mL)b
CC50 (μg/mL)c
SI
35
36
37
38
0.79 ( 0.09
1.62 ( 0.33
>0.52
26.81 ( 3.33
4.04 ( 0.10
0.52 ( 0.05
0.61 ( 0.02
34
0
˚
charge distance of the 4,4 -bipyridine moiety (∼7.6 A) fits
2.5
<1
2.5
˚
roughly to the negative charge distance (∼6 A) calculated
fromanenergy-minimizedheparane sulfate disaccharideunits
(3D-optimized structure of a heparane sulfate disaccharide
unit using ChemSkech, 3D viewer version 12.0 Advanced
Chemistry Developments, Inc., Toronto, Canada, http://www.
acdlabs.com).
0.25 ( 0.03
aAll data represent mean values and standard deviations for at least
two separate experiments. b Concentration required to inhibit 50% of
the level of the HIV-1 p24 produced in the absence of drugs. c Concen-
tration that reduced cellular viability by 50%
We have tested two types of compounds: spheroidal den-
drimers 36, 38, and comb-branched dendrimers 35, 37. The
spheroidal dendrimers are effective in inhibiting virus replica-
tion and the adhesion on the virus envelope is better than for
the comb-branched dendrimers 35, 37. These showed only
moderate antiviral activities. The main intention of this paper
is to show that there is a correlation between the number of
charges as well as of their distance with the antiviral activity.
To corroborate this point, we included the data of other
similar compounds (Charts 1 and 2 in the Introduction) show-
ing no activity because lack of charges.
Conclusion
Dendrimers consisting of viologen units such as 35-38
inhibit HIV-1 (strain IIIB) replication in MT-4 cells. These
compounds also inhibit the SIV (strain MAC251) replication
but to a lesser extent. No inhibitory activity was observed on
HIV-2 (strain ROD) replication. The reason why we asked for
this specific structure is that we expected these polycationic
compounds to bind to heparan sulfate structures expressed on
the surface of the host cells. Logically, one might expect that if
heparan sulfate would be the target for blocking HIV replica-
tion, the active compounds should also block herpes simplex
virus replication in vitro, as heparan sulfate also plays an
important role in the cellular entry of this virus. When assay-
ing the anti-HIV activity of the most active compounds (35-38)
in human PBMC, we observed no inhibitory activity, which can
be explained by the low expression of heparan sulfate on these
cells. The specificity of the inhibition clearly indicates that the
mechanism of action of polycationic compounds is more refined
Experimental Section
4.1. Chemistry. Materials and Methods. Without further
mention, the chemicals were analytical grade from Aldrich,
Merck, or Fluka and used as received. Ethyl acetate, diethy-
lether, THF, methanol, and petrol ether were distilled before
use. Organic solutions were dried over anhydrous Na2SO4 or
MgSO4 2H2O and concentrated with a Heidolph rotary evaporator
3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3485
Table 3. Antiretroviral Activity and Cytotoxicity of Compound 36 in A72 Jurkat Cellsa
EC50b[μg/mL]
HIV strain
compd
HIV-1IIIB
HIV-2ROD
NL4.3
AZTR
S056
<2
S067
<0.2
S07
<0.2
CC50c[μg/mL]
SI
36
Nevi-
rapine
0.685 ( 0.8
0.008 ( 0.002 >2
21.53 ( 6.9 <0.2
5.9 ( 1.9
102 ( 30
>2
148.9
0.008 ( 0.0003 0.005 ( 0.0002 <0.2 0.016 ( 0.0008 0.012 ( 0.007
<250
a All data represent mean values and standard deviations for at least two separate experiments. b Concentration required to inhibit 50% of the level of
GFP expression obtained in the absence of drug. c Concentration that reduced cellular viability by 50%.
General Procedure for the Synthesis of 10 and 26-Fold Charged
“Comb-Branched” Dendrimers (35-37). A mixture of the pheny-
lic dendrimer precursor 27 and the headgroup N-ethyl-4,40-
bipyridinium hexafluorophosphate (10) (Scheme 1, route Ia)
or the cross-linker N-(3,5-di(hydroxymethyl)-benzyl)-4,40-bipyri-
dinium hexafluorophosphate (Scheme 1, route Ib) in acetoni-
trile was heated at 70 °C for 1 week. The reaction mixture was
worked up and purified as reported in the description of the
single compounds.
General Procedure for Synthesis of 18- and 90-Fold Charge
“Spheroidal” Dendrimers (36, 38). A solution of the benzylic
dendrimer precursor 30b in acetonitrile and N-ethyl-4,40-bipyr-
idinium hexafluorophosphate (10) as headgroup (Scheme 2,
route Ia) or the cross-linker N-(3,5-di(hydroxymethyl)-benzyl)-
4,40-bipyridinium hexafluorophosphate (Scheme 2, route Ib) was
Figure 1. Time-of-addition experiment. MT-4 cells were infected
refluxed for 1 week. The cross-linker equipped with a reac-
tive pyrimidine nitrogen and two benzylic alcohols was used for
the preparation of the intermediate 30d 18PF-6 and 30f 42PF-6,
with HIV-1 IIIb at an moi of 0.5 and test compounds were added at
different times post infection. Viral p24 Ag production was deter-
mined at 31 h post infection and is expressed as the log10 of the p24
Ag content in pg/mL.
3
3
respectively, by repetitive application of the reaction steps v and iii
(Scheme 2, route Ib). The resulting compound 38 was worked up
and purified as reported in the description of the single compound
in supporting materials. Compound 36 was obtained according to
a procedure described as follows.
Benzyl Core Dendrimer 18 PF- (36). The synthesis was
6
3
performed according to a literature procedure.13 1,10,100-[Benzene-
1,3,5-triyl-tris(methylene)tris](4-pyridin-4-yl-pyridinium trihexa-
fluorophosphate (30) (1 g, 0.98 mmol) and 900 mg (3.9 mmol) of
3,5-di(hydroxymethyl)benzylbromide were dissolved in MeCN
(50 mL) and stirred for 44 h at 70 °C under reflux. After this
period, the precipitate was filtered, washed with ether, and dried
in vacuo to yield 1.24 g (0.72 mmol, 73%) as yellow powder of
hexahydroxy precursor (30a). 1H NMR (250 MHz, CD3CN): δ
8.99 (d, J (H,H)=6.6 Hz, 6 H), 8.93 (d, J (H,H)=6.7 Hz, 6H),
8.42-8.37 (m, 12H), 7.68 (s, 3H), 7.44 (s,3H), 7.41 (s, 6H), 5.85
(s, 12H), 4.64 (d, J (H,H) = 5.1 Hz, 12 H), 3.44 (t, J (H,H) =
5.3 Hz, 6 H).
Figure 2. Compound 36 is inactive against VSV-G pseudotyped
HIV. C8166 cells were infected in vitro with GFP-encoding HIV-1
pseudotyped with VSV-G and treated with the respective compounds at
different concentrations as indicated. Infection was assayed for GFP
expression 48 h postinfection. One hundred percent of GFP expres-
sion was defined for the infection control (no compound). Toxicity
of the compounds was measured in parallel using the MTT-based
method.
The hexahydroxy precursor (30a) (1.063 g, 0.10 mmol) was
dissolved in 320 mL of HBr (5.7 M) in acetic acid under argon
and stirred for 3 days at rt. The reaction mixture was evapo-
rated, and the residue was dissolved in 2 mL of water, and 3 mL
of a 10% aq NH4PF6 solution were added. The brown pre-
cipitate was filtered, washed with water, and dried in HV. The
hexabromide precursor (30b) was obtained as a white powder
1.23 g (yield: 0.54 mmol, 87%). 1H NMR (250 MHz, CD3CN): δ
8.97 (d, J (H,H)=6.5 Hz, 6 H), 8.95 (d, J (H,H)=6.5 Hz, 6H),
8.40 (d, J(H,H)=6.5 Hz, 12 H), 7.68 (s, 3H), 7.49 (s, 6H), 5.83 (s,
6H), 5.80 (s, 6H), 4.58 (s, 12H).
at reduced pressure. Yields are of purified products and were not
optimized. All reactions were routinely monitored by thin-layer
chromatography (TLC) on silica gel 60F254 (Merck) plated and
visualized by using UV erasing. Flash column chromatographic
separations were carried out on silica gel Baker 60 (mesh 30-60)
or Sephadex LH-20, Fluka (25-100 μm). 1H NMR, DEPT135,
and 13C NMR spectra were recorded in CD3CN (δ = 1.93),
Me2SO-d6 (δ = 2.50), CD3OD-d6 (δ = 4.87) (internal Me4Si),
respectively, at 250 MHz and at 63 MHz (Bruker Avance DPX-
250). Chemical shifts are given in ppm (δ), and the spectral data
are consistent with the assigned structures. IR spectra were
recorded with a Bruker Vector 22 FTIR spectrophotometer.
Elemental analyses were performed on a Vario Micro Cube
analyzer, and the data for C, H, and N are within (0.32% of the
theoretical values.
The hexabromide precursor (30b) (300 mg, 0.131 mmol) and
330 mg (1 mmol) of N-ethyl-4,40-bipyridinium-hexapfluorophos-
phate (10) were dissolved in MeCN (25 mL) and stirred for
4 days at 70 °C under reflux. The resulting precipitate was filte-
red off, and the mother liquor was evaporated. The crude pro-
duct was dissolved in MeNO2 and extracted with an aq NH4PF6
solution. The product formed (36) was dried in vacuo to yield
1
285 mg (0.061 mmol, 47%) as brown powder H NMR (250
MHz, CD3CN): δ 8.93-8.90 (m, 36H), 8.42-8.36 (m, 36H), 7.66
(s, 9H), 7.64 (s, 3 H), 5.83 (s, 24H), 4.67 (q, J (H,H) = 7.3 Hz,
12 H), 1.65 (t, J (H,H) = 7.3 Hz, 18 H). 13C NMR (63 MHz,

3486 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Asaftei and De Clercq
Table 4. Antiviral Activity and Cytotoxicity Evaluation of Compounds 35-38
EC50a [μg/mL]
E6SM
HeLa
MCC b[μg/mL]
compd
HSV-1 (KOS)
HSV-2(G)
vaccinia virus
VSV
TK- KOS ACVr
VSV
RSV
E6SM ; HeLa
35
36
37
38
c
6
50
10
50
4
>50
> 50
>50
>50
> 50
>50
>4
30
10
>50
> 50
>50
>20
>400
400
50
50
>50
> 50
>50
20
2
6
30
4
>400
50
4
0.384
>4 (12)
9.6
>20
>400
>400
16
80
>400
>400
>400
>400
d
e
>400
>400
48
>400
>400
48
EC50a [μg/mL]
Vero
compd
para-influenza-3
Reo-virus
Sindbis virus
Punta Toro virus
MCC b[μg/mL]
Vero
35
36
37
38
c
>50
>50
>50
>4
>50
>50
>50
>4
>50
>50
>50
>50
>50
>4
>50
> 50
>50
20
>50
>4
>400
400
48
>400
>400
80
>400
>400
>400
>400
>400
48
>400
>400
>400
d
e
a Effective concentration required to inhibit virus-induced cytopathicity by 50%. b Minimal cytotoxic concentration required to elicit a micro-
scopically visible alteration of cell morphology. HSV: Herpes simplex virus, VSV: vesicular stomatitis virus; TK-: thymidine kinase deficient; RSV:
respiratory syncytial virus, ACVr: Acyclovir-resistant, E6SM embryonic skin-muscle cells, c Brivudin. d (S)-2,3-Dihydroxypropyladenine(S)-DHPA.
e Ribavirin.
CD3CN): δ 151.1, 151.0, 150.0, 146.3, 146.2, 145.8, 135.3, 135.2,
132.3, 132.2, 127.9, 127.8, 127.6, 64.0, 58.2, 16.0. IR (KBr) 3140,
1640, 1562, 1508, 1453, 1224, 1176, 834, 558 cm-1, Anal. calcd
Briefly, 50 μL HIV-1(IIIB) and HIV-2 ROD at 100-300
CCID50 (50% cell culture infective doses) were added to either
the infected or mock-infected wells of the microtiter tray. Mock-
infected cells were used to evaluate the effects of test compounds
on uninfected cells in order to assess the cytotoxicity of the test
compounds. The MT-4 cells were added at a final concentration
of 6 ꢀ 105 cells/mL, and 50 μL volumes were transferred to the
microtiter tray wells. Five days after infection, the viability of
mock-and HIV-infected cells was examined spectrophotometri-
cally using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT). The absorbances were read in an eight-channel
computer-controlled spectrophotometer (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 three wells. The 50% cytotoxic concentration
(CC50) was defined as the concentration of the test compound
that reduced the absorbance (OD540) of the mock-infected con-
trol sample by 50%. The 50% cytotoxic concentration (CC50),
the 50% effective concentration (EC50), and the selectivity index
(SI = CC50/EC50) were then calculated from the cell viability
from each concentration of the compound. Anti-HIV activity
was also investigated in PBMCs infected with HIV-1IIIB and
cultured with various concentrations of the test compounds.
The activity was evaluated by the level of inhibition of the p24
core antigen in the culture supernatant, assessed with the HIV-1
p24 core antigen in the culture supernatant, assessed with the
HIV-1 p24 core profile enzyme-linked immunosorbent assay
(NEN, Paris, France).
for C138H138N18P18F108 3H2O (4658.10 þ 54) C 35.18, H 3.08,
3
N 5.35; found C 35.13, H 3.21, N 5.32.
4.2. Biological Activity. Dextran sulfate [DS, average mole-
cular weight (MW) 5000] was purchased from Sigma (Bornem,
Belgium). Nevirapine was obtained from Boehringer Ingelheim
(Ridgefield, CT). 30-Azido-30-deoxythymidine (AZT) was syn-
thesized according to the method described by Horwitz et al.19
Ritonavir (ABT538) was obtained from Abbott Laboratories
(Abbott Park, IL).
Cells and Virus. MT-420 and C816621 cells were grown and
maintained in RPMI 1640 supplemented with 10% heat-inactivated
fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate,
and 20 μg gentamicyne per mL.
The HIV-1(IIIB) strain was provided by R. C. Gallo and M.
Popovic.22 The NL4-3.GFP11 strain expressing an enhanced
version of GFP23 instead of Nef has been described previously.24
The HIV-1 G89 V, NL4-3A92E, and NL4-3P90A-A924E mutants
were a kind gift of Dr J. Luban.25,26 For all tests described, NL4-
3.GFP11 virus was obtained from transfection of 293T cells with
the molecular clone. Then, 1 mL of virus-containing super-
natant was used to infect 8 ꢀ 106 MT-4 cells in 40 mL of culture
medium. Three days after infection, the supernatant was collec-
ted and used as inoculum in the respective assays.
The vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped
HIV-1 was generated by transient cotransfection of recombi-
nant pNL4-3-ΔE-GFP (Zhang et al., 2004)27 (a kind gift of
Dr R. Siliciano, Johns Hopkins University, Baltimore, MD) or
pNL4-3.Nef-GFP-G89 V (a kind gift of Dr J. Luban) and
pVSV-G using calcium phosphate in 293T cells. Supernatants
containing VSV-G-pseudotyped HIV-1 were collected 60 h after
transfection. Cell debris was removed from the supernatant by
centrifugation (450g for 10 min), and the supernatant was used
for infection or stored at -80 °C.
Evaluation of the antiviral activity of the compounds
against NL4-3.GFP11 in C8166 cells was performed using
flow cytometry (see below) and was described earlier.28 HIV-1
core antigen (p24 Ag) in the supernatant was quantified by the
p24 Ag enzyme-linked immunosorbent assay (PE, Brussels,
Belgium).
Evaluation of the antiviral activity of the compounds against
VSV-G pseudotyped viruses in C8166 cells was performed using
flow cytometry. Briefly, 3 ꢀ 104 C8166 in 200 μL medium were
infected with VSV-G pseudotyped NL4.3 or NL4.3G89 V in the
presence of different concentrations of the test compound.
Inhibitory effects of the compounds on HIV-1 and HIV-2
replication were monitored by inhibition of virus-induced cyto-
pathic effect in MT-4 cells and were estimated by MTT assay.16,17

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3487
Forty-eight hours later, the infection was monitored by measuring
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Toxicity of the compounds was assessed using an MTT-based
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flow cytometer equipped with a 488 nm argon-ion laser and a
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was used as inoculum to infect new MT-4 cells at the same
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After serial passaging, we were able to culture resistant viruses in
the presence of 10.7 μM 36.
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MT-4 cells were infected with HIV-1 (IIIB) at an moi of 0.5.
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compounds were added at different times (0, 1, 2, 3, 4, 5, 6, 7,
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compounds were used at the following concentrations: dextran
sulfate: 100 μg/mL; AZT: 0.5 μg/mL; nevirapine: 2 μg/mL. 36
was used at 100 μg/mL.
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Acknowledgment. We express our thanks to Prof. Christophe
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(Rega Institute for Medical Research) for the experiments
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Supporting Information Available: Experimental details cor-
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