Structure-activity relationship studies on a Trp dendrimer with dual activities against HIV and enterovirus A71. Modifications on the amino acid

Article abstract of DOI:10.1016/j.antiviral.2016.12.010

We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship studies of this family to identify key features that might be significant for the antiviral activity. With this aim, novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a “decarboxylated” analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71 clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the divergent approach previously reported.

Full text of DOI:10.1016/j.antiviral.2016.12.010

Accepted Manuscript
Structure-activity relationship studies on a Trp dendrimer with dual activities against
HIV and enterovirus A71. Modifications on the amino acid
Belén Martínez-Gualda, Liang Sun, Eva Rivero-Buceta, Aida Flores, Ernesto
Quesada, Jan Balzarini, Sam Noppen, Sandra Liekens, Dominique Schols, Johan
Neyts, Pieter Leyssen, Carmen Mirabelli, María-José Camarasa, Ana San-Félix
PII:
S0166-3542(16)30622-2
10.1016/j.antiviral.2016.12.010
AVR 3964
DOI:
Reference:
To appear in: Antiviral Research
Received Date: 19 October 2016
Revised Date: 13 December 2016
Accepted Date: 19 December 2016
Please cite this article as: Martínez-Gualda, B., Sun, L., Rivero-Buceta, E., Flores, A., Quesada, E.,
Balzarini, J., Noppen, S., Liekens, S., Schols, D., Neyts, J., Leyssen, P., Mirabelli, C., Camarasa, M.-
J., San-Félix, A., Structure-activity relationship studies on a Trp dendrimer with dual activities against
HIV and enterovirus A71. Modifications on the amino acid, Antiviral Research (2017), doi: 10.1016/
j.antiviral.2016.12.010.
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STRUCTURE-ACTIVITY RELATIONSHIP STUDIES ON A TRP DENDRIMER WITH DUAL
ACTIVITIES AGAINST HIV AND ENTEROVIRUS A71. MODIFICATIONS ON THE AMINO ACID
Belén Martínez-Gualda1, Liang Sun2, Eva Rivero-Buceta1,3, Aida Flores1,4, Ernesto Quesada1, Jan Balzarini2,
Sam Noppen2, Sandra Liekens2, Dominique Schols2, Johan Neyts2, Pieter Leyssen2, Carmen Mirabelli,2 María-
José Camarasa1, Ana San-Félix*1
1Instituto de Química Médica (IQM-CSIC), Madrid.
2KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical
Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium
3Present address: Instituto de Tecnología Química (UPV-CSIC), Valencia, Spain
4Present
address:
Departamento
de
Farmacia,
Biotecnología,
Óptica
y
Nutrición.
Universidad Europea de Madrid
Abstract
We have recently described a new class of dendrimers with tryptophan (Trp) on the surface that show dual
antiviral activities against HIV and EV71 enterovirus. The prototype compound of this family is a
pentaerythritol derivative with 12 Trps on the periphery. Here we complete the structure-activity relationship
studies of this family to identify key features that might be significant for the antiviral activity. With this aim,
novel dendrimers containing different amino acids (aromatic and non-aromatic), tryptamine (a
“decarboxylated” analogue of Trp) and N-methyl Trp on the periphery have been prepared. Dendrimer with N-
Methyl Trp was the most active against HIV-1 and HIV-2 while dendrimer with tyrosine was endowed with the
most potent antiviral activity against EV71. This tyrosine dendrimer proved to inhibit a large panel of EV71
clinical isolates (belonging to different clusters) in the low nanomolar/high picomolar range. In addition, a new
synthetic procedure (convergent approach) has been developed for the synthesis of the prototype and some
other dendrimers. This convergent approach proved more efficient (higher yields, easier purification) than the
divergent approach previously reported.
Keywords: Antiviral agents; AIDS; HFMD; HIV; EV71; Tryptophan

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1. Introduction
Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of Acquired Immunodeficiency
Syndrome (AIDS), is an enveloped retrovirus with glycoproteins gp120 and gp41 on its surface (Dumas et al.,
2014; Terwilliger et al., 1990) while enterovirus A71 (EV71), the etiologic agent of hand-, foot- and mouth-
disease (HFMD), is a (+) RNA virus (genus Enterovirus, family Picornaviridae) without envelope (Ng and
Kwang, 2015; Pourianfar and Grollo, 2015).
At present, more than 30 approved drugs or regimens exist for the treatment of AIDS. These compounds,
which are administrated in the so-called Highly Active Anti-Retroviral Therapy (HAART) (Butera, 2005; De
Clercq, 2009; Pomerantz and Horn, 2003; Volderding and Deeks, 2010), are primarily focused on the viral
enzymes integrase, reverse transcriptase and protease. HAART keeps the disease under control, however is
often associated with the emergence of cross-resistant HIV strains and side-effects (Dube and Sattler, 2010;
Gaardbo et al., 2012; Hawkins, 2010; Margolis et al., 2014; Rojas and Holguín, 2014). These limitations
highlight the need for new lead compounds and/or novel therapeutic approaches to fight against HIV
transmission and infection (Barré-Sinoussi et al., 2013; Flexner, 2007; Kumari and Singh, 2013; Tintori et al.,
2014). In this respect, the entry of HIV into its target cells represents an attractive target for the development of
anti-AIDS therapy (Esté, 2003; Haqqani and Tilton, 2013; Hertje et al., 2010; Kuritzkes, 2009; Singh and
Chauthe, 2011; Tilton and Doms, 2010; Wilen et al., 2012). In fact, drugs that interfere with this early event
may represent an advantage over other existing therapeutic approaches that target the viral enzymes such as
reverse transcriptase or protease, as they may show remarkable efficacy against viruses resistant to these anti-
HIV inhibitors and prevent the uptake of the virus by uninfected CD4-positive cells. To date, only two drugs
among this category have been approved by the FDA, enfuvirtide (T20) (Fletcher, 2003; Williams, 2003), a 36-
amino acid peptide that binds viral glycoprotein gp41, and maraviroc (Gulick et al., 2008), a small-molecule
CCR5-antagonist.
On the other hand, the therapeutic or prophylactic panorama for EV71 infection is more limited as no
approved drugs are available for EV71 treatment to date. As already mentioned, this virus is the etiologic agent
of hand-, foot- and mouth-disease (HFMD), a mild syndrome that affects mostly children below 6 years old
(Chan, 2011). Unlike other HFMD-related enteroviruses, EV71 also may cause severe neurological problems
like aseptic meningitis and brainstem encephalitis, which may lead to cardiopulmonary failure and death
(Alexander, 1994; Luan Yin et al., 1999; McMinn, 2002). Patients with neurological syndromes often have
permanent neurological sequelae, with delayed neurodevelopment, reduced cognitive function and polio-like
paralysis. Large EV71 outbreaks have been reported throughout the world but have been especially severe in
the Pacific region of Asia (Chang et al., 2007; Chen et al., 2001). A very recent outbreak in Catalonia, Spain,

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with 73 cases of enterovirus infection with neurological complications suggests that the epidemiological pattern
of EV71 is going through a change also in Europe (ProMED, 2016; ECDC, 2016). Therefore, effective
antivirals are urgently needed for prophylaxis or treatment of patients with severe EV71 infections.
We have recently reported that a new family of dendrimers containing different central scaffolds and
multiple (9 to 18) tryptophans (Trp) on the periphery, whose prototype is the pentaerythritol derivative 1 (Fig.
1), inhibit HIV infection (Rivero-Buceta et al., 2015) and also proved to be potent, specific and selective
inhibitors of EV71 (Rivero-Buceta et al., 2016). These compounds may inhibit an early step of the replicative
cycles of both HIV and EV71, presumably virus entry into its respective target cells. In the case of HIV, the
inhibition is probably due to the interaction with the glycoproteins gp120 and gp41 of the viral surface while
for EV71, the mechanism of action is still under investigation (Rivero-Buceta et al., 2015, 2016).
Preliminary structure-activity relationship studies for EV71 and HIV on this family of dendrimers are very
similar and revealed that the absence of Trps on the periphery is detrimental for antiviral activity and that a
multivalent presentation of Trp is important for both activities, being 9-12 Trps sufficient for activity.
Here we decided to extend the structure-activity relationship studies of this class of compounds. In
particular, three aspects have now been studied. The first one is the nature of the amino acid residues on the
periphery. This was assessed by replacing the Trp residue of the prototype by aromatic (phenylalanine and
tyrosine) and non-aromatic (alanine) amino acids. The second one is the importance of the carboxylic acid on
the periphery. This was elucidated by preparing the corresponding tryptamine derivative, a “decarboxylated”
analogue of the prototype. The third one is the importance of the NH of the indol moiety on the activity. This
was explored by using a N-Me analogue of Trp.
Finally, we were also interested in the development of an optimized synthetic procedure that would allow
an efficient multi-gram synthesis of the prototype 1, needed for additional biological studies or mass
production. With this aim, an alternative synthetic procedure, based on a convergent approach, has been
explored.
2. Materials and methods
2.1.
Synthesis
Commercial reagents and solvents were used as received from the suppliers without further purification
unless otherwise stated. Dichloromethane was dried prior to use by distillation from CaH2 and stored over
Linde type activated 4Å molecular sieves.

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Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-coated with silica gel
60 (F254, 0.25 mm). Products were visualized using an ultraviolet lamp (254 nm) or by heating on a hot plate
(approx. 200 ºC), directly or after treatment with a 5% solution of phosphomolybdic acid or vanillin in ethanol.
For reversed phase purification, a Biotage HPFC (High Performance Flash Chromatography) purification
system using water/acetonitrile (100:0 to 0:100) as eluent was used.
For HPLC analysis an Alliance 2695 (Waters) equipped with a PDA (Photo Diode Array) detector Waters
2996 was used. Acetonitrile was used as mobile phase A with 0.08% of formic acid, and water was used as
mobile phase B with 0.1% of formic acid at a flow rate of 1 mL·min-1. Two different methods were used, one
on a XBridge C18 (2.1 x 100 mm, 3.5 µm) column with 5-80% of A, that will be noted as tR(X), and the other on
a SunFire C18 (4.6 x 50 mm, 3.5 µm) column with 0-100% of A, that will be noted as tR(S). All retention times
are quoted in minutes.
Melting points were measured on a Mettler Toledo and are uncorrected.
NMR spectra (1H, 13C NMR) were recorded on a 300 MHz (Varian Inova 300) and 500MHz (Varian System
500) spectrometers, using (CD3)2SO and CD3OD as solvents. Chemical shift (δ) values are reported in parts per
million (ppm) relative to tetramethylsilane (TMS) in 1H and CDCl3 (δ = 77.0) in 13C NMR. Coupling constant
(J values) are reported in hertz (Hz), and multiplicities of signals are indicated by the following symbol: s
(singlet), d (doublet), m (multiplet), bs (broad singlet).
Mass spectra (MS) were registered in a quadrupole mass spectrometer 1100 Hewlett Packard equipped with
an electrospray source. HRMS was registered in an Agilent 1200 Series LC system (equipped with a binary
pump, an autosampler, and a column oven) coupled to a 6520 quadrupole-time of flight (QTOF) mass
spectrometer using an ESI interface working in the positive-ion and negative-ion mode. The instrument was
from Agilent Technologies (Santa Clara, CA). The electrospray voltage was set at 4.5 kV, the fragmentor
voltage at 150 V and the drying gas temperature at 300 °C. Nitrogen (99.5% purity) was used as nebulizer
(207 kPa) and drying gas (6 L min−1), while nitrogen of higher purity (99.999%) was used as the collision gas.
Data acquisition and processing were performed using Agilent Mass Hunter Workstation Acquisition Rev.
B.02.00 software.
Microanalyses were obtained with a Heraeus CHN-O-RAPID instrument.
The purity of final compounds was at least 95%. The purity has been determined by high resolution mass
spectrometry (HRMS, “exact mass”) and reverse phase-HPLC.
Details regarding the synthesis and characterization of the compounds can be found in the Supplementary
material.

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2.2. Biological methods
2.2.1. Anti-HIV Activity Assay
The compounds were evaluated for their inhibitory activity against HIV-1 (NL4.3) and HIV-2 (ROD)
infection in MT-4 cell cultures. Briefly, MT-4 cells (1ꢀ×ꢀ106 cells/mL) were pre-incubated for 30 min at 37°C
with the test compounds in a 200 µl-96-well plate. Next, NL4.3 virus was added at 100 CCID50 of the viral
stock. The cytopathic effect was scored microscopically 5 days post-infection, and the 50% effective
concentration (EC50) values were determined using the MTS method.
2.2.2. Surface Plasmon Resonance Experiments for evaluation of the binding of the compounds to gp120 and
gp41
Recombinant gp120 protein from the HIV-1 IIIB strain (ImmunoDiagnostics Inc., Woburn, MA) (produced
in cell cultures of chinese hamster ovary cell cultures) and recombinant gp41 HIV-1(HxB2) (Acris Antibodies
GmbH, Herford, Germany) were covalently immobilized on the carboxymethylated dextran matrix of a CM5
sensor chip in 10 mM sodium acetate, pH 4.0, using standard amine coupling chemistry up to a final density of,
respectively, 700 RUs and 580 RUs. All interaction studies were performed at 25 °C on a Biacore T200
instrument (GE Healthcare, Uppsala, Sweden). The compounds 10 and 24 were diluted in HBS-P (10 mM
HEPES, 150 mM NaCl and 0,05% surfactant P20; pH 7.4), and supplemented with 5% dimethyl sulfoxide
(DMSO, Merck) and 10 mM Ca2+ at a concentration of 50 µM. Samples were injected for 2 minutes at a flow
rate of 30 µl/min followed by a dissociation phase of 2 minutes. The sensor chip surface was regenerated with
an injection of 50 mM NaOH. A reference flow cell was used as a control for non-specific binding and
refractive index changes. Several buffer blanks were used for double referencing. A DMSO concentration
series was included to eliminate the contribution of DMSO to the measured response. The binding affinity (KD)
was calculated for 24 based on the data obtained in the sensorgram.
2.2.3. Antiviral Activity against EV71
EV71 BrCr laboratory adapted strain and clinical isolates representative of B genogroup (B2 sub-
genogroup: 11316; B5 sub-genogroup: TW/96016/08 and TW/70902/08) and C genogroup (C2 sub-genogroup:
H08300 461#812; C4 subegenogroup: TW/1956/05 and TW/2429/04) were used at a low multiplicity of
infection (MOI) in a standardized antiviral assay. Briefly, a serial dilution of the compounds was prepared in
assay medium that was added to an empty microtiter 96 well-plate, after which the virus inoculum was added
first, followed by a suspension of freshly harvested rhabdosarcoma (RD) cells (2*10^4 cells/well). While the

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cells were settling to the bottom, the virus was allowed to infect them in the presence of compound. The assay
plates were incubated at 37 ºC, 5% CO2 with virus inoculum and compounds until full virus –induced cell death
was observed in the untreated, infected controls (3-4 days post-infection). Subsequently, the antiviral effect was
quantified using a colorimetric readout with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium/phenazine methosulfate (MTS/PMS method) and the concentration of compound
at which 50% inhibition of virus-induced cell death would be observed (EC50) was calculated from the antiviral
dose-response curves. A similar assay setup was used to determine the adverse effect of the compound on
uninfected, treated cells for calculation of the CC50 (concentration of compound that reduces overall cell health
with 50% as determined by the MTS/PMS method). Selectivity index (SI) was calculated as the ration of CC50
to EC50.
3. Results and discussion
3.1. Chemistry
Firstly, the novel amino acid dendrimers were obtained following the divergent approach previously
described for 1 (Rivero-Buceta et al , 2016). In this approach, dendrimers were built starting from the central
core and growing towards the periphery. The commercially available pentaerythritol scaffold 2, that
incorporates four aminotriester (Behera’s amine) branched arms, has been chosen as central core (Scheme 1).
Deprotection of the t-butyl protecting groups of 2, followed by condensation of the resulting poly acid 3 with
the corresponding (OMe or OBn) protected amino acids gave intermediates 4 (25%), 6 (15%) and 8 (23%).
Saponification (OMe esters) or hydrogenation (OBn esters) of the protecting ester moieties of these
intermediates afforded the final compounds 5, 7 and 10, bearing twelve amino acids on the periphery, in
quantitative yields. This divergent approach represents a short route in terms of synthetic steps. However, the
high number of covalent bonds that should be simultaneously formed in the final coupling step (12) leads to
incomplete reactions and mixtures of dendrimers with very similar chromatographic properties that were very
difficult to purify. Particularly arduous was the synthesis and purification of the triptamine derivative 11 that
was obtained in a very low yield (5%). In addition, the N-Me analogue of Trp could not be obtained by this
method. An alternative convergent approach was then explored. In this convergent approach, building blocks,
called dendrons, are constructed first and then attached to the central core in the final step of the synthesis.
For the dendron construction, the commercially available aminotriester 12 (Behera’s amine) was selected as
the starting material (Scheme 2). First, the amino group in 12 was protected with the fluorenylmethyl
oxycarbonyl group (Fmoc) to afford intermediate 13 (Carpino and Han, 1972; Karasugi et al., 2012) (73%).

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Deprotection of the t-butyl protecting groups of 13 afforded dendron 14 (Carpino and Han, 1972; Karasugi et
al., 2012) in quantitative yield. Reaction of 14 with H-Trp-OMeꢁHCl in the presence of HATU as coupling
reagent and DIPEA as base gave intermediate 15 (95%) whose subsequent Fmoc deprotection, using
piperidine, afforded dendron 18 (95%), with a reactive amino group as focal point. This free amino group
enables further attachment of 18, through the formation of amide bonds, to the central pentaerythritol scaffold
21 (Flores et al., 2014; Newkome and Weis, 1996) to afford intermediate 22. Finally, methyl ester deprotection
(LiOH/H2O) of 22 afforded compound 1 in quantitative yield. Compared to the divergent approach, the
convergent methodology considerably improves the global yield of dendrimer 1 (20% versus 4%).
Next, and based on the good results of the convergent approach, the same synthetic methodology was
applied for the synthesis of the tryptamine 11 and (NMe)Trp 24 dendrimers, for which the divergent approach
gave poor results (in the case of 11) or even failed (in the case of 24). In both cases the key dendron
intermediate 14 was reacted with Trp(NMe)-OMe or triptamine to give dendrons 16 (53%) and 17 (67%),
respectively. Fmoc deprotection, followed by coupling of the resulting intermediates 19 and 20 with the
pentaerytritol derivative 21 gave compounds 23 (27%) and 11 (16%). Methyl ester deprotection of 23, as
described above (LiOH/H2O) gave 24 in quantitative yield.
Thus, compared to the divergent approach, the convergent methodology improved considerably the global
yield of the tryptamine dendrimer 11 (16% versus 5%) and allowed the synthesis of 24 that could not be
obtained by the divergent approach.
All the synthesized compounds were characterized by 1H and 13C NMR spectroscopies and HRMS.
3.2. Biological results
3.2.1. Anti-HIV activity of the test compounds
Dendrimers 5, 7, 10, 11 and 24 were first evaluated for their inhibitory effects against HIV-1 and HIV-2
replication in CD4 cell culture. Table 1 summarizes the results of this evaluation. The antiviral data of the
prototype 1, with Trps on the periphery, is also included as a reference, as well as dextran sulfate-5000 (DS-
5000), and pradimicin A. DS-5000 is a negatively charged HIV adsorption inhibitor (Baba et al., 1988) and
pradimicin A represents a gp120 carbohydrate-binding entry inhibitor (Balzarini et al., 2007). These control
compounds showed antiviral activities in the range as previously reported (Baba et al., 1988; Balzarini et al.,
2007).
Alanine (5) and phenylalanine (7) dendrimers, with methyl and phenyl side chains, respectively, were
inactive. However, tyrosine derivative 10, with a phenolic OH side chain, showed significant anti-HIV activity.

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It was three-fold less active against HIV-1, and equally active against HIV-2 than the prototype 1, with an indol
side chain. This result suggests that the nature of the side chain of the amino acid is very important for the
activity and in particular, the indol moiety was preferred over the phenolic moiety. Tryptamine derivative 11
was 19-fold less active than the prototype 1 against HIV-1 showing the importance of the concomitant presence
of the free COOHs for optimal activity.
Interestingly, the replacement of the indole moiety by an N-methyl indole moiety improved the activity of
compound 24 versus the prototype 1. This result suggests that the free indole-NH is not crucial for activity.
Dendrimer 24 was also active against HIV-2 at subtoxic concentrations, but the inhibitory potential proved
inferior to HIV-1 inhibition, as also observed for dendrimer 10.
It should be emphasized that the activity showed by our compounds (EC50 = 1.7 - 6.4 µM), in the low
micromolar range, is very similar to that of pradimicin A (EC50 = 3.3 µM), the gp120 carbohydrate-binding
entry inhibitor (Balzarini et al., 2007) used as control (Table 1). However, pradimicin A is a structurally
complex molecule difficult to synthesize that has to be obtained by fermentation. By contrast, our dendrimers
are synthetic molecules easy to prepare and suitable to modification by conventional synthetic procedures.
In addition, our compounds have a precise structure and a well-defined composition because they have been
synthesized with complete control over the size, shape and surface functionalization. This is in clear contrast
with those happen with other negatively charged HIV entry inhibitors such as sulfated polysaccharides of
which dextran sulfate-5000, that has been used as control, is a representative example (Baba et al., 1988). The
antiviral activities of these compounds vary depending on the size (molecular weight) and density (charge
distribution) of the anionic groups (-OSO3) present on the molecules. For this reason, it is very difficult to
obtain standarized preparations of dextran sulfates or other sulfated polymers (De Clercq, 1995). In addition,
in certain systems, the sulfate groups (-OSO3) present in these compounds can be subjected to hydrolysis
(release) by sulfatases (Balzarini and Van Damme, 2007) resulting in inactivation of the compounds.
For all of these reasons and based on the synthetic accessibility, well defined molecular structure, presence
of COOH instead of –OSO3 and relatively moderate Molecular Weight ( ̴ 3000 Da vs 5000 to 500,000 Da of
dextran sulfates) the therapeutic possibilities of our compounds might be broader than those of dextran sulfate
or pradimicin, that have been used as control compounds in the present study.
3.2.2. SPR interaction experiments for 10 and 24 with HIV-1 gp120 and gp41
SPR experiments performed with the most active compounds, 10 and 24, revealed that they dose-dependently
interact with the glycoproteins gp120 and gp41 of the HIV envelope (Fig. 2). In this respect, the 4-fold better

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anti-HIV-1 activity of compound 24 versus 10 correlated with the 7-fold higher binding amplitude of 24 to
HIV-1 gp120 and gp41 than 10.
The KD value (affinity constant) of the most potent HIV-1 inhibitor 24 (EC50: 1.7 µM) was determined by
SPR analysis and compared with the KD value of the prototype compound 1 (EC50: 2.2 µM) that had earlier
been determined (Rivero-Buceta, personal communication). Whereas 1 had a KD of 6.7 µM for HIV-1 gp120
and 18 µM for HIV-1 gp41, 24 proved somewhat superior (KD: 1.53 µM for HIV-1 gp120 versus 3.40 µM for
HIV-1 gp41) (Fig. 3). Thus, both 1 and 24 were endowed with comparable anti-HIV-1 activity in cell culture,
and with pronounced affinities against glycosylated HIV-1 gp120 and gp41 proteins. In this respect, they were
equally inhibitory against HIV-1 as the non-peptidic carbohydrate-binding antibiotic pradimicin S, although
pradimicin S has a 5-fold higher affinity for HIV-1 gp120 than 24 (Balzarini et al., 2007). It should be kept in
mind that different affinities of 1 and 24 for HIV-1 gp120 can be explained by differential interactions of these
compounds with the different glycan types that are present on HIV-1 gp120.
3.2.3. Anti-EV71activity of the test compounds
The new synthesized compounds were also evaluated for their inhibitory effects against EV71 in vitro.
Tryptophan prototype 1 was included in the assay together with pirodavir, a potent EV71 capsid binder, as
reference compounds (Tijsma et al., 2014; Andries et al., 1992).
In particular, dendrimers 5, 7, 10, 11 and 24 were evaluated for selective antiviral activity (EC50) against the
BrCr laboratory-adapted strain of EV71 in a cell-based assay on rhabdomyosarcoma (RD) cells, which are
known for their high susceptibility to EV71-induced cell death (Yamayoshi et al., 2009). Cell viability (CC50)
was also assessed in a similar setup with compound-treated, uninfected cells. Overall, with a range of activity
0.4-7.2 µM, the dendrimers showed a better antiviral activity against EV71 BrCr than HIV (range of activity:
1.7-45 µM) (Table 2).
Interestingly, the SAR for EV71 revealed a similar profile to that observed for HIV. The tyrosine derivative
10 and the N-Me indole 24 showed the best antiviral activity against EV71. However, compound 24 was three-
fold less active than the prototype whereas it was as active as the prototype against HIV. The tryptamine
derivative 11 proved inactive similarly to HIV, indicating the importance of the negative (carboxyl) charges in
the molecule as a concomitant prerequisite for antiviral activity.

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The most active compounds, 10 and 24, were evaluated in virus-cell-based assays against a panel of EV71
clinical isolates representative for the different (sub)genogroups (A, B2, B5, C2 and C4) (Table 3). Compound
1 was included as reference.
Similar to the results found for the Trp prototype 1 (EC50 values ranging between 0.2 nM and 1.1 nM)
(Rivero-Buceta el al., 2016), the tyrosine analogue 10 inhibited the replication of all EV71 strains, with 50%
effective concentrations (EC50s) in the low-nanomolar/high-picomolar range (EC50s ranging between 0.7 nM
and 6.7 nM). A remarkably lower activity was observed for the N-Me indole compound 24. Nonetheless, it
achieved full inhibition of all three genogroups with EC50 values ranging between 51 nM and 180 nM. This
result clearly demonstrated that the presence of hydrophilic groups (NH, OH) on the aromatic ring of the side
chain of the amino acid is beneficial for anti-EV71 activity.
Interestingly, as previously reported (Rivero-Buceta et al., 2016), the selected dendrimer compounds were
more potent inhibitor of clinical EV71 isolates than the BrCr laboratory strain (Table 3). Such a difference was
not observed when cells were treated with Pirodavir (data not shown). This finding suggests that dendrimers
may act with a different mechanism than Pirodavir to block EV71 entry (Tijsma et al., 2014). Further
investigations are currently ongoing to determine the mode of action of dendrimers.
To determine at which stage of the virus replication cycle the compounds act, a time-of-drug-addition study
was performed with the most active, 10. In this experiment the capsid binder pirodavir and the 3C protease
inhibitor rupintrivir were used as reference compounds (Fig. 4) (Thibaut et al., 2011; Tijsma et al., 2014).
Similar to the results found for pirodavir and the prototype 1 (Rivero-Buceta el al., 2016), clear inhibition of
virus replication was observed only when the drug was added during or before infection. Compound 10, like 1
and pirodavir, lost its activity when added after the infection period. This correlates with the observation that
the compounds inhibit HIV entry. The viral 3C protease inhibitor, rupintrivir, lost its activity when added 6 or 7
h after infection, which is in line with its mechanism of action.
4. Conclusions
Starting from 1 as prototype, novel dendrimers containing different aromatic and non-aromatic amino acids,
tryptamine (a “decarboxylated” analogue of Trp) and N-Methyl Trp on the periphery have been synthesized. A
new synthetic procedure (convergent approach) has been developed for the obtention of these compounds and
the prototype 1. This convergent approach proved to be more efficient (higher yields, easier purifications) than
the divergent approach and was crucial for the synthesis of dendrimers whose synthesis either proved
inefficient (triptamine) or failed (N-Methyl Trp) when the divergent approach was used.

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Aromatic amino acids Trp (present on the prototype, 1) and Tyr (present on 10), with their respective indol
and phenol moieties and free COOHs, are required for anti-HIV and anti-EV71 activity. Tryptamine derivative
11 was 19-fold less active against HIV than the prototype 1 and inactive against EV71, thus showing the
importance of the COOHs for activity. Dendrimer 24, with N- Methyl Trp on the periphery, was slightly more
active against HIV-1 and HIV-2 than the prototype, which suggests that the NH of the indole moiety is not
crucial for HIV activity. However, dendrimer 24 was three-fold less active than the prototype against EV71,
suggesting that for the anti-enteroviral activity, this NH is important since it may probably be involved in a
hydrogen bond with its corresponding binding site. Dendrimer 10, with tyrosine, was endowed with the most
potent antiviral activity against EV71. This compound represents a novel low-nanomolar inhibitor of the
clinical isolates of enterovirus A71 while dendrimer 24, with N-Methyl Trp, was the most active against HIV-1.
In conclusion we report herein a family of dendrimers with dual action against HIV and EV71 that deserve
further studies and development. The ability of these compounds to act early in the HIV replicative cycle,
before HIV enters the host cell, makes them ideal candidates for use as microbicides, compounds in which exist
a great interest, especially in developing countries, where live more than 90% of HIV-infected people, whose
access to the conventional anti-HIV therapies of the developed world are very limited (WHO, 2016; UNAIDS,
2016). On the other hand, and because this class of molecules show extremely high potency against clinical
isolates of EV71, they may be of significant interest for future therapeutic/prophylactic strategies against this
virus.
Acknowledgements
This work has been supported by the Spanish MINECO [projects SAF2012-39760-C02 and SAF2015-64629-
C2-1-R (MINECO/FEDER)], “The Centers of Excellence” of the KU Leuven (EF-05/15 and PF-10/18), EU
FP7 (FP7/2007-2013) Project EUVIRNA (Grant 408 Agreement 264286), EU FP7 SILVER (Contract
HEALTH-F3-2010-260644), a grant from the Belgian Interuniversity Attraction Poles (IAP) Phase VII–P7/45
(BELVIR) and the EU FP7 Industry-Academia Partnerships and Pathways Project AIROPICO. The Spanish
MEC/MINECO is also acknowledged for a grant to B.M.G and E.R. and the China Scholarship Council (CSC)
(Grant 201403250056) for a grant to L.S. We also thank Charlotte Vanderheydt and Evelyne Van Kerckhove
for help with the processing of the antiviral data.
Appendix. Supplementary data
Details regarding the synthesis and characterization of the compounds are included.

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12
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LIST OF CAPTIONS
Fig. 1. Structure of the prototype pentaerythritol derivative 1
Fig. 2. SPR analysis of the interaction of 10 and 24 with HIV-1 gp120 and gp41
Fig. 3. Apparent KD for 10-gp120 and 10-gp41
Fig. 4. Effects of pirodavir (capsid binder), rupintrivir (3C protease inhibitor), and compound 10 in a time-of-
drug-addition assay

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Scheme 1. Synthesis of dendrimers 4-11 by the divergent approach. Reagents and conditions: (i) HOOCH,
40ºC, 48 h, (ii) HATU/DIPEA (iii) H2, Pd/C (iv) LiOH.H2O
Scheme 2. Synthesis of compounds 1, 11 and 24 by the divergent approach. Reagents and conditions: (i)
FmocCl, Na2CO3 (ii) HOOCH, 40ºC, 48 h (iii) H-aa, HATU/DIPEA (iv) piperidine (v) HATU/DIPEA (vi)
LiOH·H2O
Table 1. Anti-HIV activity of the selected group of compounds
Table 2. Antiviral activity of dendrimers against the BrCr lab strain of EV71 in RD cells
Table 3. Evaluation of the broad-spectrum antiviral effect of compounds 10 and 24 against a representative
panel of clinical EV71 isolates in RD cells
LIST OF FIGURES
Fig. 1. Structure of the prototype pentaerythritol derivative 1

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Fig. 2. SPR analysis of the interaction of 10 and 24 with HIV-1 gp120 and gp41
Fig. 3. Apparent KD for 10-gp120 and 10-gp41

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Fig. 4. Effects of pirodavir (capsid binder), rupintrivir (3C protease inhibitor), and compound 10 in a time-of-
drug-addition assay

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Scheme 1. Synthesis of dendrimers 4-11 by the divergent approach. Reagents and conditions: (i) HOOCH,
40ºC, 48 h, (ii) HATU/DIPEA (iii) H2, Pd/C (iv) LiOH.H2O

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COR
COR
COR
COR
COR
COR
COR
COR
COR
H-aa (OMe) or Triptamine
FmocCl, Na2CO3
Dioxane/H2O
FmocHN
H2N
FmocHN
HATU, DIPEA
HCOOH
15, R = Trp-OMe (95%)
16, R = Trp(NMe)-OMe (53%)
17, R = Triptamine (67%)
13, R = OtBu (73%)
14, R = OH (quant)
12, R = OtBu
R
R
O
O
Piperidine
DMF
O
O
R
R
NH
HOOC
COOH
O
R
O
R
O
O
O
O
O
NH
O
O
COR
O
HATU, DIPEA
O
HOOC
COOH
21
H2N
COR
COR
O
O
O
DMF
HN
O
R
R
O
HN
O
18, R = Trp-OMe (95%)
19, R = Trp(NMe)-OMe (94%)
20, R = Triptamine (90%)
O
R
R
O
R
O
R
22, R = Trp-OMe (30%)
1, R = Trp-OH (quant)
LiOH/H2O
23, R = Trp(NMe)-OMe (27%)
24, R = Trp(NMe)-OH (quant)
11, R = Triptamine (16%)
LiOH/H2O
O
O
HN
OH
HN
Trp-OMe =
OMe
LiOH/H2O
Trp-OH
=
N
N
H
H
O
O
HN
OH
HN
Trp(NMe)-OMe =
OMe
R =
LiOH/H2O
HN
Trp(NMe)-OH
=
N
Me
N
Me
Triptamine =
N
H
Scheme 2. Synthesis of compounds 1, 11 and 24 by the divergent approach. Reagents and conditions: (i)
FmocCl, Na2CO3 (ii) HOOCH, 40ºC, 48 h (iii) H-aa, HATU/DIPEA (iv) piperidine (v) HATU/DIPEA (vi)
LiOH·H2O

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Table 1. Anti-HIV activity of the selected group of compounds
EC50a (µM)
HIV-1
EC50a (µM)
HIV-2
MWc
(g/mol)
Compound
5
CC50b (µM)
>100
>100
>28
>100
>28
2194.25
3287.58
3299.40
3047.72
3744.15
7
28
10
11
24
6.4 ± 1.4
45
20 ± 15
77
>100
>100
1.7 ± 0.0
7.3 ± 2.8
>100
2.3 ± 0.3
0.07 ± 0.02
3.3 ± 1.2
22 ± 2.8
0.03 ± 0.01
5.9 ± 3.7
>100
>20
>100
3575.84
~ 5000
842.80
1
DS-5000
Pradimicin A
Data are the mean ± S.D. of at least 2 to 3 independent experiments
a 50% Effective concentration, or the concentration required to inhibit HIV-induced cytopathicity by 50%
b 50% Cytostatic concentration, or the compound concentration required to inhibit CD4 T cell proliferation by 50%
c Molecular weight of the molecules expressed as g/mol
Table 2. Antiviral activity of dendrimers against the BrCr lab strain of EV71 in RD cells
EC50a (µM)
EC90a (µM)
CC50b (µM)
SI
Compound
5
EV71
EV71
>46
>46
ND
>32
>30
ND
>27
ND
>4
7
7.2 ± 0.2
0.4 ± 0.1
>33
>10
10
11
24
0.7 ± 0.4
>33
78
ND
7.5
3.6 ± 1.1
5.3 ± 0.9
0.3 ± 0.1
0.3 ± 0.1
0.5 ± 0.1
0.6 ± 0.2
>100
>54
1
Pirodavir
ND = Not Determined
All values are in micromolar (ꢂM) and are a summary of multiple dose-response curves (>2) in multiple independent (>1)
experiments.
CC50 = concentration of compound at which a 50% reduction in cell viability is observed; EC50 = concentration of compound at
which the virus-induced cytopathic effect is reduced by 50%; SI = selectivity index (CC50/EC50).

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Table 3. Evaluation of the broad-spectrum antiviral effect of compounds 10 and 24 against a representative
panel of clinical EV71 isolates in RD cells
EC50 (nM)a
EV71
Virus strain
Genogroup
1
10
24
BrCr
A
285 ± 70
0.4 ± 0.0
0.2 ± 0.0
0.2 ± 0.1
1.1 ± 0.3
0.2 ± 0.2
0.3 ± 0.2
390 ± 140
6.7 ± 3.2
0.7 ± 0.1
1.0 ± 0.3
2.0 ± 0.4
0.3 ± 0.2
0.4 ± 0.1
3560 ± 1620
160 ± 10
51 ± 18
11316
B2
B5
TW/96016/08
TW/70902/08
H08300 461#812
TW/1956/05
TW/2429/04
66 ± 11
C2
C4
180 ± 10
58 ± 6
59 ± 18
a All values are in nanomolar (nM) and are obtained in multiple (>2) independent (>1) experiments.
Following microscopic quality control, at least at one concentration of compound, no virus-induced cell death was
observed and the compound did not cause an adverse effect on the host cell or monolayer morphology.
SUPPLEMENTARY MATERIAL
Supplementary material related to the article

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Novel dendrimers containing different amino acids on the periphery have been
synthesized.
•
•
These compounds have dual action against HIV and EV71.
Tyrosine dendrimer is the most potent against EV71 while dendrimer with N-methyl
Trp is the most potent against HIV-1.
•
•
Dendrimer with tyrosine is an extremely highly potent inhibitor of clinical EV71 isolates
(nanoMolar-picoMolar potency).
Presence of hydrophilic groups (NH, OH) on the aromatic ring of the amino acid is
beneficial for anti-EV71 activity.