HIV-1 X4 activities of polycationic Viologen based dendrimers by interaction with the chemokine receptor CXCR4: Study of structure-activity relationship

Article abstract of DOI:10.1021/jm301337y

A series of viologen based dendrimers with polycationic scaffold carrying 10, 18, 26, 42, and 90 charges per molecule were used to determine the structure-activity relationship (SAR) with regard to HIV-1 inhibitory activity. The studies involved five comp

Full text of DOI:10.1021/jm301337y

Article
pubs.acs.org/jmc
HIV‑1 X4 Activities of Polycationic “Viologen” Based Dendrimers by
Interaction with the Chemokine Receptor CXCR4: Study of
Structure−Activity Relationship
Simona Asaftei,*,† Dana Huskens, and Dominique Schols‡
†Institute of Chemistry, University of Osnabruck, Barbarastrasse 7, D-49069 Osnabruck, Germany
̈
̈
‡Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
S
* Supporting Information
ABSTRACT: A series of “viologen” based dendrimers with polycationic scaffold carrying 10, 18, 26, 42, and 90 charges per
molecule were used to determine the structure−activity relationship (SAR) with regard to HIV-1 inhibitory activity. The studies
involved five compounds with a high activity against HIV-1 already utilized in our previous study1 and five new dendrimers. Such
dendrimers block HIV-1 entry into the cell, indicating that they bind to HIV-1 surface proteins and/or on the host cell receptors
required for entry. The increasing positive character of dendrimers leads to more cytotoxicity. The 10 charges dendrimers (1, 6)
have less influence on the cell viability but low inhibition of the binding of the CXCR4 mAb clone 1D9. Thus, dendrimers with
18 charges (2, 7) are the most promising CXCR4 imaging probes. We report the design, synthesis, and biological activity of new
HIV-1 inhibitors that are conceptually distinct from those of the existing HIV-1 inhibitors.
in peripheral blood mononuclear cells (PBMCs) and these cells
have a low expression of HS.1 This observation initiated the
search for the exact mechanism of action of polycationic
dendrimers.
INTRODUCTION
■
Dendrimers (dendri = tree, mer = part) are a class of
macromolecules characterized by highly branched, well-defined,
three-dimensional structures that are being developed as drug
delivery vehicles and as therapeutic agents. In fact, their branches
can be capped with different surface groups that can impart
distinct biological and pharmacological properties.2−4 In
contrast, with small molecule drugs that tend to make
monovalent contacts, dendrimers can bind to their target in a
multivalent manner.5
The entry of HIV into the host cell starts with the binding of
the envelope glycoprotein gp120 to its primary host cell receptor
CD4. Subsequently, HIV has to bind to a co-receptor, the
chemokine receptor CCR5 for R5 viruses or the chemokine
receptor CXCR4 for X4 viruses.16,17 CXCR4 is the receptor for
the CXC-chemokine CXCL12 or stromal-cell-derived factor 1α
(SDF-1α),18,19 whereas CCR5 is recognized by the CC-
chemokines CCL3 or macrophage inflammatory protein 1α
(MIP-1α), CCL4 or MIP-1β and CCL5 or regulated upon
activation normal T-cell expressed and secreted (RANTES).16,20
These chemokines can block HIV-1 infection by binding to the
co-receptor and inducing internalization of the receptor from the
cell surface.16,18,19 For the treatment of HIV-1-infected patients,
one CCR5 antagonist, maraviroc, was approved by the Food and
Drug Administration (FDA)21,22 However, until now, no
clinically useful drug is available for the treatment of HIV-1
infected patients, although several CXCR4 antagonists have been
previously reported. In particular 1,1′-[1,4-phenylenebis-
N-Alkylated 4,4′-bipyridinium units, so-called “viologens”,6
are classic organic building blocks to build polycationic
dendrimers that are utilized in a variety of fields, such as
electrochemistry,7 photochemistry,8 electrical conductivity,6,9
solar-energy conversion,10 electron transfer,11 charge transfer,12
ligands in metallosupramolecular assemblies,13 molecular
wires,14 and biocide.15 In addition, we have previously shown
that some polycationic “viologen” based dendrimers carrying
between 10 and 90 charges per molecule were active against the
human immunodeficiency virus type 1 (HIV-1) in T cell lines
and primary cell cultures.1 In the beginning we assumed that
these polycationic compounds bind by electrostatic interactions
to the polysaccharide heparan sulfate (HS), a negatively charged
structure expressed on the surface of the cells of the T cell lines.
However, one polycationic compound was active against HIV-1
Received: June 24, 2012
Published: November 19, 2012
© 2012 American Chemical Society
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a
Chart 1. Structures of Ethyl/Thymine “Viologen” Based Dendrimers 1−10
a
(A) Model representation of “comb-branched” dendrimer structure showing central core and branches G1 and G2 with 10 and 26 charges,
respectively, and surface groups of 4 and 8 ethyl or thymine units, respectively, denoted as red spheres. (B) “spheroidal” “viologen” based dendrimers
showing central core and branches G1, G2, and G3 with 18, 42, and 90 charges, respectively, and surface groups of 6, 12, and 24 ethyl or thymine
units, respectively. (C) Structures of the cores, branches, and capping groups of the “comb-branched” and “spheroidal” dendrimers.
(methylene)]bis-1,4,8,11-tetraazacyclotetradecane (AMD 3100)
has clinical use for patients with non-Hodgkin’s lymphoma and
multiple meloma.23 The key players in the interaction of HIV-1
with CXCR4 are the V3 loop of gp120 together with the N-
terminal region and extracellular domains of CXCR4.24,25 It is
known that the overall positive charge of the V3 loop is
correlated to the usage of CXCR4 and CCR5 (a high positive
charge promotes CXCR4 usages and a low positive charge
promotes CCR5 usages).26 More specifically, if the glycosylation
motif (N6X7T8|S8X9, where X ≠ Pro and N is the glycosylation
site) is absent from the V3 loop sequence, the virus will show
preference toward CXCR4 as co-receptor.27 If the N6X7T8|S8X9
motif is present, the co-receptor selection will be influenced by
the amino acids at positions 11, 24, and 25 (of the “11/24/25”
rule). If any of these amino acids are not positively charged, the
virus will show a preference toward CCR5.28 De Victoria et al.
proposed that if the N6X7T8|S8X9 glycosylation motif is present
and any of the amino acids at positions 11, 24, and 25 are
positively charged, the co-receptor preference will be governed
by the net charge of the V3 loop sequence. If the net charge of the
V3 loop is >5, the virus will show a preference toward CXCR4.27
The chemokine ligand/receptor pair SDF-1/CXCR4 appears
to play a central role in the metastasis of several types of cancer
and certain inflammatory autoimmune disorders such as
rheumatoid arthritis.29−33 Therefore, chemokine receptor
antagonists may be attractive drug candidates not only for anti-
HIV therapy. AMD3100, the lead compound of the bicyclams,
was developed as a stem cell mobilizing agent that culminated in
the approval of plerixafor in 2008 for the mobilization of
hematopoietic stem cells in patients with non-Hodgkin’s
lymphoma and multiple myeloma.34,35 However, initially
AMD3100 was described as the first low-molecular-weight
agent that inhibits virus fusion and infectivity through interaction
with the HIV-1 co-receptor CXCR4.36 Mutational studies have
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a
Scheme 1. Synthesis of the Dendrimers 1−10
a
Reagents and conditions: (i) bipyridine, 1 h, 110 °C, nitrobenzene; (ii) ion exchange chromatography with 10% aq NH4PF6; (iii) 48 h, under reflux
with HBr/AcOH; (iv) N-[3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)propyl]-4,4′-bipyridinium hexafluorophosphate (m1), MeCN
under reflux; (v) N-(3,5-di(hydroxymethyl)-benzyl)-4,4′-bipyridinium hexafluorophosphate (m2), MeCN under reflux.
revealed that the amino acid residues Asp171, Asp262, and Glu288
are key interaction points for AMD3100. It suggested that Asp171
in transmembrane domain 4 (TM-IV) interacts with one of the
bicyclam rings and the other ring is sandwiched between Asp262
and Glu288 in TM-VI and TM-VII, respectively.37,38
In the context of systematic studies on “viologen” based
dendrimers with polycationic scaffold carrying 10 (1, 6), 18 (2,
7), 26 (3, 8), 42 (4, 9), and 90 charges (5, 10) per molecule, we
have determined the structure−activity relationship (SAR) of
dendrimers with regard to HIV-1 inhibitory activity. We have
extended our studies from the five compounds (1−5) with only
4,4′-bipyridinium units as an integral part of the polycationic
scaffold, with high activity against HIV-1 (IIIB) already utilized in
our previously study15 to another five new dendrimers (6−10)
with a recognition function on the surface. We report the
synthesis, characterization, and inhibitory activities of five new
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a
Table 1. Inhibitory Effects of 1−10 on Replication of HIV-1 X4 NL4.3 Virus
compds, ethyl units
compds, thymine units
ref AMD3100
1
2
3
4
5
6
7
8
9
10
IC50 (μg/mL), HIV-1 NL 4.3
CC50 (μg/mL), tox MT-4
0.017
>10
1.1
0.9
34
0.8
11
1.4
9
1.3
9
2.4
1.5
19
1.8
10
1.5
9
1.2
10
>100
>100
a
All data represent mean values and standard deviations for at least two separate experiments. IC50: concentration of compound to inhibit 50% HIV-
1 (NL4.3) replication in MT4 cells (average of three independent experiments). CC50: concentration of compound to inhibit 50% the viability in
MT-4 cells.
“viologen” based dendrimers with polycationic scaffold and 4, 6,
8, 12, and 24 thymine units as a recognition function on the
surface (Chart 1). Such dendrimers have a dual advantage. We
expect that the polycationic scaffold interacts with the heparan
sulfate and the thymine units are capable of hydrogen-bonding to
the carboxylate group of the residues Asp262, Glu288, and Asp171 of
the HIV co-receptor CXCR4. The compounds were evaluated
for their ability to inhibit HIV-1 replication and for their
interactions with the CXCR4 HIV co-receptor and compared to
the specific CXCR4 inhibitor AMD3100.
The tetrahydroxy derivative P1a was converted to tetrabromide
P1b, using hydrobromic acid/acetic acid, followed by ion
−
exchange of the PF6 salt in good yield (78%). From the
−
intemediate product P1b (PF6 ), the desired dendrimer 6 was
obtained by reaction of monomer m1 followed by ion exchange
(route IIa, Scheme 1).
The dendrimer 8 phenyl core was obtained by reaction with
monomer N-(3,5-di(hydroxymethyl)benzyl)-4,4′-bipyridinium
hexafluorophosphate (m2) synthesized according to the
literature,46 with the intermediate P1b, followed by substitution
of −OH by −Br in compound P2a with 5.7 M HBr/acetic acid at
room temperature to obtain precursor P2b. This reaction step was
repeated once to obtain the precursors P3a and P3b, respectively.
In the last reaction step, the precursor P3b was reacted with
monomer m1 in MeCN to obtain the compound 8 in 60% yield
(route IIb, Scheme 1).
The dendrimers 7, 9, and 10 containing 6, 12, and 24 thymine
units, respectively, as capping groups on the peripheries were
built from precursor core 1,1′,1″-{[(benzene-1,3,5-triyltris-
(methylene)][tris(3,5-di(bromomethyl)benzene]-(4,4′-bipiridi-
nium) }hexafluorophosphate P4b, reported in the litera-
ture.1,40−42 (scheme 1, route III).
RESULTS AND DISCUSSION
■
Chemistry. The main aim of our chemical design was to
obtain compounds with double function able to minimize the
adhesion of the virus envelope on the host cell by the interaction
of polycationic scaffold and HS expressed from the host cell and
increasing the binding affinity of dendrimers via hydrogen-
bonding between thymine units and the carboxylate group of the
residues Asp262, Glu288, and Asp171 of HIV co-receptor CXCR4.
To achieve this, we prepared polycationic “viologen” based
dendrimers, where the periphery labels consist of thymine units
(Chart 1). The synthesis of the dendrimers 1−5 was previously
published.1 The synthesis of dendrimers 6−10 is outlined in
Scheme 1 and is described in detail in the Experimental Section
and Supporting Information.
The trifunctional cross-linker 1,3,5-tris[(-4,4′-bipiridinium)-
methyl]benzene trihexafluorophosphate consisting of a mesithyl
derivative linked to three 4,4′-bipyridine units was prepared by
reaction of 1,3,5-tris(bromomethyl)benzene with an excess of
4,4′-bipyridine in MeCN and further alkylated with 1,3-
di(hydroxymethyl)benzyl bromide to the hexavalent alcohol
P4a. This was succeeded by substitution of −OH for −Br,
yielding P4a with 5.7 M HBr/acetic acid and a subsequent ion
exchange to the intermediate P4b as PF6− salt. The compound 7
dendrimer with 18 charges per molecule was available from
intermediate P4b as tetrabromide via alkylation with monomer
m1 in 61% yield (route IIIa, Scheme 1). Dendrimers of 9 and 10
were prepared by reaction of precursor p4b with monomer m2, to
give the intemediate P5a 18PF6− (routes IIIb and IIIc, Scheme 1).
The resulting dodecahydroxyalcohol P5a was converted into
dodecabromide P5b by treatment with 5.7 M HBr/acetic acid.
This reaction step was repeated once to obtain the intermediates
P6a and P6b, respectively. In the latter step the corresponding
polybromide intemediates P5b and P6b were closed to 9 and 10,
respectively, by reaction with monomer m1 in MeCN in 50% and
63% yields respectively.
First, the monomer N-[3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidine-1(2H)-yl)propyl]-4,4′-bipyridinium hexafluoro-
phosphate (m1), used as a capping group, was prepared by
nucleophilic substitution of 1-(3-bromopropyl)-5-methylpyrimi-
dine-2,4(1H,3H)-dione obtained following a known procedure39
and 4,4′-bipyridine in nitrobenzene in 60% yield (Scheme 1,
route I). To synthesize the dendrimers core, we used our
established method previously published by Asaftei and De
Clercq1 starting from the corresponding initiator core and
following the “divergent dendrimers synthesis method” reported
previously by Anroin et al.40 and Heinen et. al.41,42
The conjugated 4-fold nucleophilic phenyl precursors P1a,b
have been prepared according to route II, Scheme 1. First 4,4′-
bipyridine was reacted with the intemediate compound N,N′-
bis[2,4-bis(dinitophenyl)-4,4′-bipyridium dichloride by nucleo-
philic substitution with 1-chloro-2,4-dinitobenzene.43 The N,N′-
bis[2,4-bis(dinitophenyl)-4,4′-bipyridium dichloride reacted
with 3,5-di(hydroxymethyl)aniline, synthesized according to
the literature44 to obtain P1a.
1
All the target compounds were characterized by H and 13C
NMR, IR, and UV−vis spectroscopy as well as by elemental
The reaction of 1-(2,4-dinitrophenyl)pyridinium chloride with
aniline forming an intermediate pentamethine salt “Zincke salt”
and 2,4-dinitroaniline is known as the “Zincke reaction”. This
process is now the standard route to N-arylpyridinium salts that
cannot be formed by the direct reaction of electrophilics and
pyridine.45 The mechanism consists of two stages, ring opening
and ring closing, and the excess amine plays an important role in
assisting proton transfer in the conversion of Zincke salts to
Zincke product.
analysis.
Antiviral Activity in Vitro. The anti-HIV-1 activity of
dendrimers 1−10 was determined with respect to the number of
charges in the scaffold and the ethyl (1−5) or thymine units (6−
10) on the periphery (Chart 1). Dendrimers were evaluated for
their capacity to inhibit replication of NL4.3, a HIV-1 strain that
utilizes the chemokine co-receptor CXCR4 (X4 strains) for
entry, in MT-4 cells (Table 1) and in human peripheral blood
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Table 2. Effect of Charges and Number of Thymine Units on Surfaces of 1−10 and Inhibitory Effects (IC50) on Replication of HIV-
a
1 X4 NL4.3
compds, ethyl units
compds, thymine units
ref AMD3100
1
2
3
4
5
6
7
8
9
10
IC50 (μg/mL), HIV-1 NL 4.3
CC50 (μg/mL), tox PBMC
0.002
>10
5.6
0.12
100
0.34
20
1.07
20
1.79
20
4.62
0.41
20
0.89
20
2.32
20
>4
20
>100
>100
a
All data represent the mean values and standard deviations for at least three separate experiments. IC50: concentration of compound to inhibit 50%
HIV-1 (NL4.3) replication in human PBMC cells (average of two independent experiments). CC50: concentration of compound to inhibit 50% the
viability in human PMBC cells.
Table 3. Inhibitory Effects of 1−10 on Chemokine Induced Calcium Signaling in U87.CD4.CXCR4 and U87.CD4.CCR5 Cells and
a
Inhibition of CXCL-12AF647 Binding to CXCR4+ SupT1 Cells
IC50 (μg/mL)
compds, ethyl units
compds, thymine units
ref AMD3100 maravioc
1
2
3
4
5
6
7
8
9
10
b
CXCL12-induced calcium signaling
0.17
nd
nd
61.15
>100
14.81
3.61
80.92
1.91
1.37
22.64
1.44
0.97
5.9
1.19
5.9
65.26
>100
10.57
2.13
28.6
1.62
1.53
10.25
1.27
0.48
21.95
1.55
0.48
5.57
1.06
b
CCL3L1-induced calcium signaling
0.0024
nd
c
CXCL12AF647 binding
0.057
1.10
1.17
a
b
All data represent mean values and standard deviations for at least three separate experiments. Concentration of compound to inhibit 50%
c
CXCL12-induced or CCL3L1-induced calcium signaling in U87.CD4.CXCR4 cells or U87.CD4.CCR5 cells, respectively. Concentration of
compound to inhibit for 50% CXCL-12AF647 binding in SupT1 cells.
a
Table 4. Inhibitory Effects of 1-10 on Anti-CXCR4 mAbs Binding in SupT1 Cells
IC50 (μg/mL) anti-CXCR4 mAb binding inhibition
compds, ethyl units
compds, thymine units
ref AMD3100
1
2
3
4
5
6
7
8
9
10
>4
clone 12G5
clone 173
clone 1D9
0.02
0.02
>1
1.96
1.39
8.59
2.31
1.36
2.99
1.21
1.76
2.16
1.21
1.18
1.96
1.56
1.74
2.57
2.25
2.05
9.56
1.84
1.64
2.55
1.93
2.21
2.83
2.23
2.40
2.92
2.06
>10
a
All data represent mean values and standard deviations for at least three separate experiments. Concentration of compound to inhibit 50% anti-
CXCR4 mAb binding in SupT1 cells.
mononuclear cells (PBMC) (Table 2). The cytotoxicity of
dendrimers was evaluated in the same assay (see Experimental
Section).
investigate the CCR5 antagonistic activity of these dendrimers,
calcium signaling was induced in U87.CD4.CCR5 cells by
CCL3L1. Again, a dose-dependent decrease of the Ca2+ flux was
measured; however, overall, the IC50 values increased and
compounds 1 and 6 at 100 μg/mL could not inhibit the Ca2+ flux
with 50% (Table 3). No differences were detected between
thymine dendrimers and dendrimers without thymine. As
controls, the antagonists AMD3100 and maraviroc were used
for CXCR4 and CCR5, respectively. Next, SupT1 cells were
All dendrimers showed comparable antiviral activity against
HIV-1 NL4.3 with IC50 values between 0.8 and 2.4 μg/mL.
However, differences in cytotoxicity of the dendrimers in MT-4
cells were observed. The dendrimers 1 and 6 were not toxic at
100 μg/mL, while the CC50 of the other dendrimers ranged
between 9 and 34 μg/mL. In addition, no significant differences
were observed between the dendrimers with or without thymine
units as surface groups (Table 2). In fact, there is a trend toward a
decrease in antiviral activity when the thymine units are present.
To identify the mode of action of the different dendrimers, we
investigated their interaction with the HIV-1 co-receptors
CXCR4 and CCR5 by using calcium signaling assays. After
binding to the receptor, chemokines trigger an intracellular signal
transduction cascade comprising a transient cytosolic calcium
mobilization. The chemokine receptors CCR5 and CXCR4 were
transfected into human atroglioma U87.CD4 cells (see
Experimental Section), and these cells were used to investigate
the agonistic and antagonistic activity of the different dendrimers.
None of the compounds could induce calcium signaling by itself,
and these compounds thus have no detectable agonistic
properties (data not shown). When CXCL12-induced calcium
mobilization was measured in U87.CD4.CXCR4 cells, the
dendrimers showed a dose-dependent decrease of the Ca2+ flux
with IC50 values between 0.4 and 65 μg/mL (Table 3). To
incubated with Alexa-fluor647-labeled CXCL12 (CXCL12AF647
)
in the presence of the dendrimers. Here again, they all inhibited
the binding of CXCL12AF647 equally (IC50 ranging between 1 and
2 μg/mL) except for dendrimers 1 and 6, which were less potent
in inhibiting CXCL12AF647 binding (IC50 ranging between 10 and
15 μg/mL) (Table 3).
To further investigate the interaction of the “viologen”
dendrimers with the CXCR4 receptor, we examined the
inhibition of the binding of anti-CXCR4 mAbs directed against
different epitopes of CXCR4: clone 1D9 recognizing the N
terminus, clone 173 recognizing mainly ECL2, and clones 12G5
recognizing ECL1 and ECL2. All dendrimers dose-dependently
inhibited the binding of these three mAbs, and there were no
differences observed between thymine dendrimers and den-
drimers without thymine (Table 4). Only the “comb-branched”
dendrimers 1 and 6 were not as effective in inhibiting the binding
of the CXCR4 mAb clone 1D9, and the complex “spheroidal”
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dendrimer 10 could not inhibit the binding of the anti-CXCR4
mAbs clone 12G5 and 1D9 (Table 4).
potent inhibitors, which are conceptually distinct from any other
existing HIV-1 inhibitors.
CONCLUSIONS
EXPERIMENTAL SECTION
■
■
In this study, we have carried out the first structure−activity
relationship (SAR) study for a novel class of compounds,
polycationic “viologen” based dendrimers that inhibit HIV-1
(strain IIIB) replication in MT-4 cells by interactions with the
CXCR4 HIV co-receptor. We previously demonstrated that the
specificity of the inhibition clearly indicates that the mechanism
of action of such compounds is more refined than electrostatic
interaction between cell surface and virus without inhibitory
activity of the enzyme’s reverse transcriptase (RT). Furthermore,
we synthesized and evaluated the capacity to inhibit replication of
NL4.3, a HIV-1 strain that utilizes the chemokine co-receptor
CXCR4 (X4 strains) for entry, in MT-4 cells and in human
peripheral blood mononuclear cells (PBMC) of 10 compounds,
5 with ethyl as capping group, 1−5, used in a previous study. In
addition five new compounds with thymine as capping group, 6−
10, have been established. The cytotoxicity of dendrimers was
evaluated in the same assay. The CXCR4 receptor affinity was
evaluated and compared to the specific CXCR4 inhibitor
AMD3100 at different concentrations. In vitro studies regarding
the binding affinity are the most reliable point of comparison for
the polycationic “viologen”-based dendrimers.
The trend that the increasing positive character of the
dendrimers 3−5 and 8−10 leads to more cytotoxicity than has
previously been observed by us and is most likely to be a charge-
related effect.1 The dendrimers 1 and 6 are +10 charged, and the
dendrimers 2 and 7 are +18 charged. They were not toxic at 100
and 34 μg/mL, while the CC50 of the other dendrimers ranged
between 9 and 11 μg/mL. We reason that this increase in positive
charge may result in charge-driven nonspecific membrane
binding and causes cytotoxicity, similar to that reported for
positively charged PAMAM dendrimers.47 In contrast in the
calcium signaling assays low charged denrimers 1 and 6, +10
charges, were 10-fold less potent in inhibiting CXCL12AF647
binding (IC50 ranging between 10 and 15 μg/mL) compared
with dendrimers 3−5 and 8−10 (IC50 ranging between 1 and 2
μg/mL). Only the dendrimers 1 and 6, +10 charges, were not as
effective in inhibiting the binding of the CXCR4 mAb clone 1D9,
and the complex “spheroidal” dendrimer 10 with thymine
capping group could not inhibit the binding of the anti-CXCR4
mAbs clones 12G5 and 1D9.
The CXCR4 affinity of dendrimers 1−5 ethyl capping and 6−
10 thymine capping is comparable. A bivalent effect based on
electrostatic interaction between polycationic scaffold and
heparan sulfate corroborates hydrogen bonding between
thymine units, and that of carboxylate group residues of HIV
co-receptor CXCR4 for dendrimers 6−10 was not observed but
rather a slight decrease in the affinity of 10.
Our results clearly demonstrate that with the use of highly
positively charged compounds some polycationic “viologen”
based dendrimers should be avoided to suppress the cytotoxicity
and the nonspecific cell and tissue binding. However, the
dendrimers 1 and 6, with +10 charges, and 2 and 7, with +18
charges, have less influence on the cell viability. The 1 and 6, +10
charges, were not so effective in inhibiting the binding of the
CXCR4 mAb clone 1D9. Thus, dendrimers 2 and 7, with +18
charges, are the most promising CXCR4 imaging probes. These
compounds have potential for development as inhibitors of other
nonviral pathogens such as human tumor cells. This study
provides an important framework for the rational design of more
Chemistry. Materials and Methods. The chemicals were
analytical grade from Aldrich, Merck, or Fluka and used as received
except ethyl acetate, diethyl ether, THF, methanol, and petroleum ether,
which were distilled before use. Organic solutions were dried over
anhydrous Na2SO4 or MgSO4·2H2O and concentrated with a Heidolph
rotary evaporator 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). H NMR, DEPT135, and 13C
1
NMR spectra were recorded in CD3CN (δ = 1.93), Me2SO-d6 (δ =
2.50), CD3OD-d6 (δ = 4.87) (internal Me4Si), respectively, at 500 and at
125 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 FT-IR spectropho-
tometer. UV−vis spectra were obtained with an 8453 UV−visible
spectophotometer (Agilent, Germany), with λmax in nm (ε in M−1
cm−1). The UV−vis spectra from dendrimers in their oxidized (V+2) and
reduced (V•+) state have been recorded spectroelectrochemically in
MeCN/NaClO4 (0.1), and the corresponding λmax values and extinction
coefficients (ε) are reported. The reference for UV/vis spectra of
dendrimers was the pure solvent/electrolyte. The purity of tested
compounds was verified by elemental analyses on a vario Micro cube
analyzer, and the data for C, H, and N are within 0.4% of the
theoretical values.
General Procedure for the Synthesis of 10 and 20 6-Fold
Charged “Comb-Branched” Dendrimers (6, 8). A mixture of the
phenylic dendrimer precursor P1b and the headgroup 1-[3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-4yl)-
pyridinium hexafluorophosphate (m1) or the cross-linker N-(3,5-
di(hydroxymethyl)-benzyl)-4,4′-bipyridinium hexafluorophosphate
(m2) (Scheme 1, route II) in acetonitrile 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-, 42-, and 90-Fold
Charge “Spheroidal” Dendrimers (7, 9, 10). A solution of the
benzylic dendrimer precursor P4b in acetonitrile and 1-[3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-4-yl)-
pyridinium hexafluorophosphate (m1) as capping group was refluxed for
1 week (Scheme 1, route IIIa) to obtain 7. The cross-linker N-(3,5-
di(hydroxymethyl)benzyl)-4,4′-bipyridinium hexafluorophosphate
(m2) was used for the preparation of the intermediates P5b and P6b by
repetitive application followed in the last reaction step by coupling m1
capping group monomer to close the 9 and 10, respectively . The
resulting compounds were worked up and purified as reported in the
description of the single compounds. The purity checks by mass
spectrometry using the MALDI-TOF method were not very successful
because highly charged molecules are very difficult to measure (see
Supporting Information). Heinen et al. also had no success in MALDI-
TOF measurements of similar compounds.42 Stoddart and Balzani also
synthesized dendrimers on bipyridine basis (between 6 and 42 charges
per molecule) and could obtain MS analysis results for low charged
molecules.48
Synthesis of Dendrimer 6. Amounts of 80 mg (0.082 mmol) of
tetrabromide precursor P1b and 230 mg (0.49 mmol) of 1-[3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-4-yl)-
pyridinium hexafluorophosphate (m1) were dissolved in 20 mL of
MeCN. The mixture was refluxed at 70 °C for 5 days. The cooled
reaction mixture was filtered, washed with MeCN (10 mL) and ether,
successively, and dried. The mother liquor was evaporated, and the
residue was dried. The residue was dissolved in 7 mL of nitromethane
and extracted with 3 mL of a NH4PF6 solution (10% in water). The
organic phase was washed one more time with water and evaporated to
dryness. The residue was put in water, filtered, washed several times with
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water, and dried in a vacuum for 12 h. The product was purified by
chromatography (Sephadex LH-20, column 2.5 cm × 25 cm, MeCN/
MeOH, 1:1, as eluent), giving 160 mg of dendrimer 6 (0.051 mmol, 62%
yield) after drying in a vacuum at 40 °C for 24 h. 1H NMR (500 MHz,
CD3CN): δ = 9.15−9.10 (m, 8H), 8.98 (d, 8H, 3J[H−H] = 5.5 Hz), 8.94
(d, 8H, 3J[H−H] = 6.0 Hz), 8.63 (d, 4H, 3J[H−H] = 4.0 Hz), 8.41 (d,
8H, 3J[H−H] = 5.5 Hz), 8.37 (d, 8H, 3J[H−H] = 5.5 Hz), 7.97 (s, 2H),
7.90 (s, 4H), 7.20 (s, 4H), 5.98 (s, 8H). 4.65 (t, 8H, 3J[H−H] = 7.2 Hz),
3.77 (t, 8H, 3J[H−H] = 6.2 Hz), 2.36 (t, 8H, 3J[H−H] = 6.7 Hz), 1.82
(s, 12H). 13C NMR (125 MHz, CD3CN): δ = 165.2, 152.6, 152.0, 151.0,
147.1, 147.0, 146.8, 144.5, 141.7, 137.3, 135.0, 128.7, 128.3, 128.1,111.6,
64.2, 60.4, 45.3, 31.4, 12.4. IR (cm−1): 3650.5, 3073.0, 2977.6, 1674.8,
1637.4, 1452.4, 1366.1, 1223.2, 820.1, 552.9. UV−vis (MeCN, partial
reduced): V+2 λmax, 264 (644 65), 422 (4352); V•+ at −0.5 V λmax, 402
( 7 0 2 5 3 ) , 6 0 8 ( 2 7 7 4 0 ) . A n a l . C a l c d f o r
C98H98F60N18O8P10·4.4CH4O·1.85C2H3N (3105.5 + 216): C 38.36,
H 3.68, N 8.37. Found: C 38.32, H 3.70, N 8.40.
Synthesis of Dendrimer 7. Amounts of 106 mg (0.046 mmol) of
hexabromide precursor P4b and 230 mg (0.49 mmol) of 1-[3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-4-yl)-
pyridinium hexafluorophosphate (m1) were suspended in 20 mL of
MeCN. The mixture, protected from light, was refluxed at 75 °C for 6
days. The cooled reaction mixture was filtered and washed with ether,
successively, and dried. The mother liquor was evaporated, and the
residue was dried. The combined solids were dissolved in 10 mL of
nitromethane and extracted with 4 mL of a NH4PF6 solution (10% in
water). The organic phase was washed one more time with water and
evaporated to dryness. The residue was put in water, filtered, washed
several times with water, and dried in a vacuum for 12 h. For purification
the product was further dissolved in MeNO2 and extracted with water
for 1 week in a Ludwig apparatus. The MeNO2 phase was evaporated
and dried in HV for 12 h. The residue was washed with water, filtered,
and dried in a vacuum to obtain 156 mg of 7 (0.028 mmol, 61% yield).
1H NMR (500 MHz, DMSO-d6): δ = 9.29 (s, 6H), 8.98−8.92 (m, 36H),
8.32−8.38 (m, 36H), 7.69 (s, 9H), 7.65 (s, 3H), 7.24 (s, 6H), 5.85 (s,
24H), 4.69 (t, 12H, 3J[H−H] = 7.5 Hz), 3.81 (t, 12H, 3J[H−H] = 8.7
Hz), 2.39 (t, 12H, 3J[H−H] = 6.7 Hz), 1.85 (s, 18H). 13C NMR (125
MHz, CD3CN): δ = 165.6, 153.1, 152.3, 151.7, 147.4, 142.2, 136.5,
136.5, 133.5, 133.4, 129.1, 129.1, 128.8, 112.1, 65.3, 61.0, 45.8, 31.8,
12.0. IR (cm−1): 3651.4, 3072.2, 2977.9, 1674.8, 1637.9, 1450.9, 1362.6,
1223.4, 1167.7, 813.0, 552.3. UV−vis (MeCN): V+2 λmax, 268 (159365),
422 (20177); V•+ at −0.5 V λmax, 402 (121 785), 608 (37 488). Anal.
Calcd for C174H174F108N30O12P18·5H2O (5486.8 + 90): C 37.47, H 3.33,
N 7.53. Found: C 37.2, H 3.35, N 7.39.
Synthesis of Dendrimer 8. Amounts of 100 mg (0.028 mmol) of
octabromide precursor P3b and 150 mg (0.31 mmol) of 1-[3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-4-yl)-
pyridinium hexafluorophosphate (m1) were dissolved in 20 mL of
MeCN. The mixture, protected from light, was refluxed at 75 °C for 1
week. The cooled reaction mixture was filtered and washed with ether,
successively, and dried. The mother liquor was evaporated, and the
residue was dried. The residue was dissolved in 7 mL of nitromethane
and extracted with 3 mL of a NH4PF6 solution (10% in water). The
organic phase was washed one more time with water and evaporated to
dryness. The residue was put in water, filtered, washed several times with
water, and dried in a vacuum for 12 h. The product was purified by
chromatography (Sephadex LH-20, column 2.5 cm × 25 cm, MeCN/
MeOH, 1:1, as eluent), giving 140 mg of 8 (0.017 mmol, 60% yield) after
drying in a vacuum at 40 °C for 24 h. 1H NMR (500 MHz, CD3CN): δ =
9.19−9.01 (m, 8H), 8.97−8.92 (m, 48H), 8.68 (s, 4H), 8.44−8.37 (m,
48H), 8.01 (s, 4H), 7.68 (s, 12H), 7.66 (s, 2H), 7.23 (s, 8H), 5.98 (s,
8H), 5.84 (s, 24H), 4.67 (t, 16H, 3J[H−H] = 7.2 Hz), 4.30 (s, 4H), 3.79
(t, 16H, 3J[H−H] = 6.0 Hz), 2.38 (qu, 16H, 3J[H−H] = 6.6 Hz), 1.83 (s,
24H). 13C NMR (125 MHz, CD3CN): δ = 165.3, 152.7, 151.7, 151.1,
147.1, 146.9, 146.8, 141.8, 137.4, 137.2, 128.6, 128.5, 128.3, 111.6, 64.8,
64.2, 45.3, 31.4, 12.4. IR (cm−1): 3650.0, 3071.9, 2977.7, 1674.0, 1636.9,
1451.6, 1367.3, 1223.1, 1172.2, 811.8, 551.3. UV−vis (MeCN, partial
reduced): V+2 λmax, 269 (250 850), 424 (16 487); V•+ at −0.5 V λmax, 402
( 2 0 6 6 6 5 ) , 6 0 8 ( 7 8 8 2 5 ) . A n a l . C a l c d f o r
C
246H242F156N42O16P26·13CH4O·6C2H3N (7811.8 + 662): C 37.41, H
3.71, N 7.93. Found: C 38.11, H 4.09, N 7.79.
Synthesis of Dendrimer 9. Amounts of 140 mg (0.022 mmol) of
dodecabromide precursor P5b and 150 mg (0.32 mmol) of 11-[3-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)propyl]-4-(pyridin-
4-yl)pyridinium hexafluorophosphate (m1) were suspended in 20 mL of
MeCN. The mixture, protected from light, was refluxed at 75 °C for 4
days. The cooled reaction mixture was filtered and washed with ether,
successively, and dried. The mother liquor was evaporated, and the
residue was dried. The combined solids were dissolved in 10 mL of
nitromethane and extracted with 4 mL of a NH4PF6 solution (10% in
water). The organic phase was washed one more time with water and
evaporated to dryness. The residue was put in water, filtered, washed
several times with water, and dried in a vacuum for 12 h. For purification
the product was further dissolved in MeNO2 and extracted with water
for 1 week in a Ludwig apparatus. The MeNO2 phase was evaporated
and dried in HV for 12 h. The residue was washed with water, filtered,
and dried in a vacuum at 40 °C for 24 h to obtain 140 mg of 9 (0.011
mmol, 50% yield). 1H NMR (500 MHz, CD3CN): δ = 11.24 (s, 12H),
9.37 (s, 84H), 9.14 (m, 84H), 7.79 (b, 30H), 7.49 (s, 12H), 5.92 (s,
60H), 4.74 (s, 24H), 3.78 (s, 24H), 2.35 (s, 24H), 1.77 (s, 36H). 13C
NMR (125 MHz, CD3CN): δ = 164.2, 151.1, 149.1, 148.3, 146.0, 140.8,
135.3, 130.0, 126.8, 126.3, 109.0, 62.7, 58.6, 43.9, 30.2, 11.9. IR (cm−1):
3648.6, 3073.0, 2980.5, 1673.3, 1637.7, 1450.9, 1363.5, 1223.6, 1167.7,
817.0, 553.3. UV−vis (MeCN): V+2 λmax, 267 (436 080), 422 (20 119);
V•+ at −0.5 V λmax, 402 (358 640), 608 (145 420). Anal. Calcd for
C396H390F252N66O24P42·72.5CH4O·31C2H3N·12H2O (12546.3 +
3807): C 38.95, H 4.91, N 8.31. Found: C 38.55, H 5.28, N 8.70.
Synthesis of Dendrimer 10. Amounts of 230 mg (0.016 mmol) of
tetraeicosabromid precursor P6b and 225 mg (0.48 mmol) of 1-[3-(6-
hydroxy-5-methyl-2-oxo-1,2,3,6-tetrahydropyridin-3-yl)propyl]-4-(pyr-
idin-4-yl)pyridinium were suspended in 15 mL of MeCN. The mixture
was refluxed at 75 °C for 1 week. The cooled reaction mixture was
filtered and washed with ether, successively, and dried. The mother
liquor was evaporated, and the residue was dried. The combined solids
were dissolved in 10 mL of nitromethane and extracted with 4 mL of a
NH4PF6 solution (10% in water). The organic phase was washed one
more time with water and evaporated to dryness. The residue was put in
water, filtered, washed several times with water, and dried in a vacuum
for 12 h. For purification the product was further dissolved in MeNO2
and extracted with water for 1 week in a Ludwig apparatus. The MeNO2
phase was evaporated and dried in HV for 12 h. The residue was washed
with water, filtered, and dried in a vacuum at 40 °C for 24 h to obtain 270
mg of 10 (0.010 mmol, 63% yield). 1H NMR (500 MHz, CD3CN): δ =
9.40 (b signal, 24H), 8.98−8.93 (m, 180H), 8.40−8.39 (m, 180H), 7.68
(b, 66H), 7.25 (s, 24H), 5.86 (s, 132H), 4.68 (t, 48H, 3J[H−H] = 7.0
Hz), 3.80 (t, 48H, J[H−H] = 5.5 Hz), 2.39 (t, 48H, J[H−H] = 6.0
Hz), 1.84 (s, 72H). 13C NMR (125 MHz, CD3CN): δ = 165.1, 152.4,
151.5, 150.8, 146.6, 135.7, 132.6, 128.4, 128.3, 128.3, 111.35, 64.5, 60.1,
45.0, 31.2, 12.1. IR (cm−1): 3650.7, 3138.63, 2977.7, 1676.3, 1638.2,
1451.1, 1380.2, 1165.0, 820.7, 553.7. UV−vis (MeCN): V+2 λmax, 268
(517 700), 416 (28 088); V•+ at −0.5 V λmax, 402 (418 805), 608 (170
175). Anal. Calcd for C841H824F540N138O48P90·106CH4O·30C2-
H3N·18H2O (26679.4 + 4946): C 38.24, H 4.38, N 7.64. Found: C
37.94, H 4.76, N 7.72.
Materials and Measurements of Biological Activities. Anti-
HIV-1 (X4) NL4.3 Activity of the Dendrimers 1−10. The
antiretroviral assays in MT-4 cells have been described in detail earlier
by Vermeire et al.49 Briefly, MT-4 cells (50 μL, 1 × 106 cells/mL) were
preincubated for 30 min at 37 °C with test compounds (100 μL) in a 96-
well plate (Falcon). Then HIV-1 X4 NL4.3 was added according to the
TCID50 of the viral stock. Cytopathic effect (CPE) was scored
microscopically 5 days postinfection, and EC50 values were determined
using the MTS/PES method by Vermeire et al.50 (Table 1).
3
3
Calcium Signaling Assays. U87.CD4.CXCR4 cells or
U87.CD4.CCR5 cells were digested by trypsin and seeded in gelatin-
coated (0.2%) black-wall 96-well microplates (Costar, Cambridge, MA)
at 2 × 104 cells per well the day before the experiment. The next day, the
cells were loaded with the fluorescent calcium indicator Fluo-3-
acetoxymethyl (Molecular Probes, Leiden, The Netherlands) at 4 μM
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for 45 min at 37 °C. Then cells were washed with assay buffer (see
above) and incubated with 150 μL of 1−10 and AMD3100 or maraviroc
at different concentrations and diluted in assay buffer for 10 min at 37
°C. The intracellular calcium signaling in response to 25 ng/mL CXCL-
12 or 10 ng/mL LD78β for U87.CD4.CXCR4 cells or U87.CD4.CCR5
cells, respectively, was measured in all 96 wells simultaneously as a
function in time, using the fluorometric imaging plate reader (FLIPR)
(Molecular Devices, Sunnyvale, CA) as described previously by Princen
et al.51 (Table 3).
normal T-cell expressed and secreted; AMD3100, 1,1′-[1,4-
phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetracene;
TM-IV, transmembrane domain 4; TM-VII, transmembrane
domain 7; MeCN, acetonitrile; U87.CD4, human atroglioma;
FDA, Food and Drug Administration
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of monomers and precursors, spectroscopic details,
and analytical data of target compounds 6−10. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49-541-969 2802. Fax: + 49-541-969 2370. E-mail:
sasaftei@uos.de.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express our thanks to Prof. Jef Rozinski (Rega Institute for
Medical Research, University of Leuven, Belgium) for perform-
ing the mass spectroscopic analyses. Mass spectrometry was
made possible by the support of the Hercule Foundation of the
Flemish Government (Grant 20100225-7). Many thanks are also
extended to our co-workers Ana Maria Lepadatu and Marius
Ciobanu (University of Osnabrueck, Germany) for their support
in the laboratory.
ABBREVIATIONS USED
■
HS, polysaccharide heparin sulfate; PBMC, peripheral blood
mononuclear cell; SDF-1α, stromal-cell-derived factor 1α; MIP-
1α, macrophage inflammatory protein 1α; MIP-1β, macrophage
inflammatory protein 1β; RANTES, regulated upon activation
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