Scaffold Simplification Strategy Leads to a Novel Generation of Dual Human Immunodeficiency Virus and Enterovirus-A71 Entry Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01737

Currently, there are only three FDA-approved drugs that inhibit human immunodeficiency virus (HIV) entry-fusion into host cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71. These compounds contain in their structure three or four tryptophan (Trp) residues linked to a central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings, bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and the fivefold axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis, and great opportunities for chemical optimization make them useful prototypes for future medicinal chemistry studies.

Full text of DOI:10.1021/acs.jmedchem.9b01737

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Article
A SCAFFOLD SIMPLIFICATION STRATEGY LEADS TO A
NOVEL GENERATION OF DUAL HUMAN IMMUNODEFICIENCY
VIRUS AND ENTEROVIRUS-A71 ENTRY INHIBITORS
Belén Martínez-Gualda, Liang Sun, Olaia Martí-Marí, Sam Noppen, Rana Abdelnabi, Carol M.
Bator, Ernesto Quesada, Leen Delang, Carmen Mirabelli, Hyunwook Lee, Dominique Schols,
Johan Neyts, Susan Hafenstein, María-José Camarasa, Federico Gago, and ANA SAN-FELIX
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01737 • Publication Date (Web): 06 Dec 2019
Downloaded from pubs.acs.org on December 7, 2019
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of their duties.

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A SCAFFOLD SIMPLIFICATION STRATEGY
LEADS TO A NOVEL GENERATION OF DUAL
HUMAN IMMUNODEFICIENCY VIRUS AND
ENTEROVIRUS-A71 ENTRY INHIBITORS
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1a¥
2b¥
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Belén Martínez-Gualda , Liang Sun , Olaia Martí-Marí , Sam Noppen , Rana Abdelnabi ,
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2c
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Carol M. Bator , Ernesto Quesada , Leen Delang , Carmen Mirabelli , Hyunwook Lee ,
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,4,5
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Dominique Schols , Johan Neyts , Susan Hafenstein , María-José Camarasa ,
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*1
Federico Gago , Ana San-Félix
1
Instituto de Química Médica (IQM-CSIC), 28006 Madrid, Spain
2University of Leuven, Department of Microbiology and Immunology, Rega Institute for
Medical Research, Laboratory of Virology and Chemotherapy, B-3000 Leuven, Belgium
3
Departamento de Ciencias Biomédicas y Unidad Asociada IQM-UAH, Universidad de Alcalá,
E-28805 Alcalá de Henares, Madrid, Spain
4
Department of Biochemistry and Molecular Biology, Huck Institutes of the Life Sciences, the
Pennsylvania State University, University Park, 16802 Pennsylvania, USA
5
Department of Medicine, the Pennsylvania State University College of Medicine, Hershey,
1
7033 Pennsylvania, USA
Keywords: Antivirals; HIV; EV71; Tryptophan; indole; palladium-catalyzed C-H activation
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ABSTRACT
Currently there are only three FDA-approved drugs that inhibit HIV entry-fusion into host
cells. The situation is even worse for enterovirus EV71 infection for which no antiviral therapies
are available. We describe here the discovery of potent entry dual inhibitors of HIV and EV71.
These compounds contain in their structure three or four tryptophan (Trp) residues linked to a
central scaffold. Critical for anti-HIV/EV71 activity is the presence of extra phenyl rings,
bearing one or two carboxylates, at the C2 position of the indole ring of each Trp residue. The
most potent derivatives, 22 and 30, inhibit early steps of the replicative cycles of HIV-1 and EV-
A71 by interacting with their respective viral surfaces (glycoprotein gp120 of HIV and 5-fold
axis of the EV-A71 capsid). The high potency, low toxicity, facile chemical synthesis and great
opportunities for chemical optimization make them useful prototypes for future medicinal
chemistry studies.
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INTRODUCTION
Human immunodeficiency virus (HIV) is a retrovirus that infects immune cells within the
body. Without treatment the infection can result in the development of Acquired Immune
Deficiency Syndrome (AIDS) and death. HIV infection affects more than 30 million people
1
worldwide and caused about 1 million deaths in 2018. Current antiretroviral therapy consists on
more than 30 approved drugs or regimens. Although this therapy has significantly improved the
quality of life of infected people, there is still no cure for the HIV infection.2-7 Moreover, the
combined therapy is lifelong, expensive, associated with significant side-effects/resistance, and
not universally available in all regions of the world.1,8-12 These limitations highlight the need for
new lead compounds and/or novel therapeutic approaches to fight HIV infection.13-17 In this
regard, the initial steps in the HIV replicative cycle (entry and/or fusion with the host cells) are
very attractive targets.18-23 Despite their intrinsic interest there are only three entry inhibitors
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approved by FDA, namely enfuvirtide24,25 which targets the envelope glycoprotein gp41,
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maraviroc, which targets the host cell receptor CCR5 and ibalizumab, an anti-CD4 humanized
IgG4 monoclonal antibody.27
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We have previously reported a family of tryptophan (Trp) dendrimers, exemplified by 1 (Fig.
1
), that inhibit an early step in the replicative cycle of HIV by interacting with the gp120
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glycoprotein of the viral envelope. Interestingly, these compounds also proved to be potent
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inhibitors of the replication of the unrelated enterovirus 71 (EV71), the virus responsible for
most of the severe forms of hand, foot and mouth disease (HFMD). This virus is associated with
neurological/cardiopulmonary complications and mortality in children below 5 years old.30-34 In
the last two decades, the increasing number of EV71 cases and the spread of the virus across
Asia, followed by case reports in Spain, France, Germany and other European countries, have
raised major concerns about its pandemic potential.35-37
Although a sub-genogroup C4 EV71-inactivated vaccine has been recently approved in China
no antiviral therapies are so far, available for the prevention or treatment of EV71 infection.38,39
Therefore, the development of effective and specific antiviral drugs is urgently needed.40,41
Remarkably, dendrimer 1 inhibits not only the laboratory BrCr strain of EV-A71 but also a
large panel of clinical isolates (belonging to each of the genogroups) in the low-nanomolar/high-
2
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picomolar range. Recently, it was demonstrated that 1 inhibits an early step in the replicative
cycle of EV-A71 by targeting residues of the structural viral protein VP1, particularly the region
that forms the 5-fold axis of the viral capsid, and thereby blocking the binding of the virus to the
host cell.42
With respect to the SAR studies, modifications have been made in the central core, arms and
periphery of the prototype 1.28,29,43,44 All of these modifications led to the conclusion that a
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multivalent presentation of Trps, with their respective indole side chain and free carboxylates, is
required to preserve the antiviral activity against both HIV and EV-A71.
The prototype 1 has a too high molecular weight (MW: 3576 Da) for drug development
purposes. The aim of the present work is to develop compounds with smaller size retaining the
same level of antiviral potency.
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N1
N1
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TrpOH
O
HOTrp
O
O
O
HOTrp
O
C2
TrpOH
O
C2
H
H
H
H
N
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HOTrp
HN
N
TrpOH
H
O
O
O
O
O
NO2
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O
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N
N
H
N
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H
HOTrp
O
OH H
HO
OH
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OH
O
TrpOH
O
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NH
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NH
H
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HN
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HO
O
OH
O
OH
HOTrp
TrpOH
O
O
TrpOH
O
HN
HOTrp
N
N
H
O
I
H
II
HIV: EC50 > 250 M
EV71: EC50 > 100 M
HN
=
OH
MADAL-385 (1)
HIV: EC50 > 100 M
EV71:EC50 > 120 M
EC50 : 2.3 ± 0.3 M
CC50 > 100 M
TrpOH
HIV
N
H
EC50 : 0.3 ± 0.07 M
EV71 CC50 : 30.0 ± 2.5 M
Clinical isolates EC50 : 0.0002-0.001 M
Figure 1. Structures of the prototype pentaerythritol derivative MADAL-385 (1) and “truncated”
analogues I and II.
With this aim, a scaffold simplification strategy was applied and “truncated” analogues (I and
II) of 1, with only 3 or 4 Trps respectively (Figure 1), were firstly synthesized. However, as it
will be later discussed in the Antiviral Evaluation section, both compounds resulted inactive
against HIV and EV-A71.
Next, we decided to “decorate” I and II with moieties that could potentially participate in extra
interactions with protein residues in the corresponding binding site. Phenyl rings functionalized
with carboxylic groups were preferentially chosen because according to previous SAR studies,
both aromaticity and negatively charged moieties are beneficial for anti-HIV and anti-EV-A71
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activity. Moreover, these functionalized phenyl rings could potentially participate in hydrogen
bonding and/or hydrophobic/aromatic interactions with protein residues of the corresponding
binding site. To introduce these substituents, we exploited the particular reactivity of the indole
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ring of Trp. Thus, the C2 and N1 positions of the indole moiety were chosen as attachment
points (Figure 1). Hereafter, we describe the synthesis, antiviral activities against HIV and EV71,
and structure-activity relationships of this new generation of compounds.
RESULTS AND DISCUSSION
Chemistry
As mentioned, we designed truncated analogues of the prototype with extra functionalized
aromatic rings attached to the C2 and N1 positions of the indole ring. We started with the
synthesis of the tripodal derivatives 1422 (Scheme 1), with functionalized phenyl rings on the
C2 position of the indole ring of the three Trp residues. The synthesis of these compounds takes
advantage of the methodology developed by Prof. Lavilla´s group which allows the direct C2
arylation of Trp and proteins containing this amino acid.46,47 The reaction involves the
simultaneous formation of new C-C bonds through a metal-catalyzed (Pd II) cross-coupling
reaction.
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O
O
NO2
R
R
t
2
, R =O Bu
HCOOH
3
, R = OH (quant)
O
R
H-TrpOMe.HCl
HATU, DIPEA
DMF
H
H
N
N
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R
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O
H
R
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I
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NH
NH
O
O
NO2
O
R
1. LiOH
THF/H O
O
R
O
O
NO2
NO2
O
N
N
O
O
O
O
R
N
N
H
OMeH
H
N
N
2
OMe
MeO
H
H
OMe
HO
H
OH
O
NH
O
Pd(OAc)2,
AgBF4, TFA
DMF, MW
2. HCl 1M
OMe
O
NH
NH
OMe
OH
O
O
4 (97%) HN
HN
HN
1. LiOH, THF/H O
5, R = H (57%)
14, R = H (quant)
2
2. HCl 1M
6, R = 2-F (29%)
15, R = 2-F (quant)
7
, R = 4-NO (47%)
16, R = 4-NO2 (quant)
17, R = 4-Br (quant)
2
H
N
H
N
8, R = 4-Br (37%)
, R = 4-CF3 (33%)
9
18, R = 4-CF3 (quant)
19, R = 4-OMe (quant)
20, R = 3-COOH (quant)
21, R = 4-COOH (quant)
22, R = 3,5-COOH (quant)
O
O
NO2
O
N
O
10, R = 4-OMe (38%)
N
H
OH H
11, R = 3-COOMe (38%)
OH
12, R = 4-COOMe (37%)
O
NH
OH
13, R = 3,5-COOMe (35%)
O
HN
I (quant)
Scheme 1. Synthesis of the tripodal derivatives I and 14-22.
The synthesis started with the commercially available scaffold 2. Removal of the t-butyl
protecting groups of 2, followed by condensation of the resulting triacid (3)48 with OMe
protected Trp (H-TrpOMe·HCl) in the presence of HATU, as the coupling reagent, and DIPEA
as the base, gave intermediate 4 (97%). The simultaneous chemoselective arylation on the C2
position of the three indole moieties of 4 was first attempted with phenyl iodide following
Lavilla’s conditions.46,47 Overall, a mixture of 5 mol% Pd(OAc) , AgBF (3 eq), TFA (3 eq),
2
4
phenyl iodide (3 eq) in N,N’-dimethylformamide (DMF) was microwave (MW) irradiated at 80
ºC for 15 min. However, HPLC analysis revealed that only 5% of the desired compound 5 was
present in the reaction mixture. The less lipophilic peaks were attributed to partial reaction in
only one or two of the three Trps present in the starting compound 4. However, the use of higher
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temperatures (120 ºC), longer reaction times (30 min), excess of aryl iodide and AgBF (2 eq for
4
each Trp), allowed to finally obtain the desired methyl ester intermediate 5 with 57% yield.
Saponification of the protecting ester moieties using LiOH·H O afforded the final compound 14
2
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in quantitative yield (Scheme 1).
Next, and based on the good results obtained with phenyl iodide, the same synthetic
methodology was applied with the other aryl iodides to afford compounds 6 (29%), 7 (47%), 8
(37%), 9 (33%), 10 (38%), 11 (38%), 12 (37%) and 13 (35%). These reactions proceed in
remarkably short times (30 minutes), are completely selective (only reaction at C2 was observed)
and compatible with ambient air/moisture. Methyl ester deprotection (LiOH/H O) of 613
2
afforded the final tripodal derivatives 1522 in quantitative yields. In addition, the unsubstituted
Trp derivative I was also prepared by deprotection of 4.
The synthesis of the tetrapodal derivatives 2830 was accomplished following a similar
methodology as described for the tripodal series (Scheme 2). Again, the key step of the reaction
involves the simultaneous formation of new C-C bonds through a metal-catalyzed (Pd II) cross-
coupling reaction. In this case, the tetrapodal Trp derivative 24 was used as the starting material.
In turn, this compound was obtained by condensation of the tetraacid 2349,50 with OMe-protected
Trp (H-TrpOMe·HCl) in the presence of HATU as the coupling reagent and DIPEA as the base.
Reaction of 24 with the corresponding aryl iodide, under the previously mentioned optimized
conditions, afforded intermediates 25 (39%), 26 (32%) and 27 (26%), the subsequent
deprotection (LiOH/H O) gave the desired final compounds 2830 in quantitative yields. The C2
2
unsubstituted Trp derivative II was also prepared by deprotection (LiOH/H O) of 24.
2
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O
O
O
O
HO
HO
OH
OH
O
O
O
O
23
H-TrpOMe.HCl
HATU, DIPEA
DMF
H
H
N
H
H
H
H
0
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0
N
N
N
N
N
R
R
R
R
I
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
1. LiOH
THF/H2O
N
N
H
MeO
OMe
N
H
N
H
H
R
N
H
N
H
HO
OH
O
O
O
MeO
O
O
OMe
O
O
O
O
O
O
O
HO
O
H
N
H
N
H
N
H
N
MeO
O
H
N
H
N
OH
O
MeO
O
OMe
O
Pd(OAc)2,
AgBF4, TFA
DMF, MW
OMe
O
2. HCl 1M
2
4 (82%)
N
N
H
N
N
N
H
R
N
H
H
H
R
R
H
R
1
2
. LiOH, THF/H2O
. HCl 1 M
2
5, R = 3-COOMe (39%)
26, R = 4-COOMe (32%)
7, R = 3,5-COOMe (26%)
28, R = 3-COOH (quant)
29, R = 4-COOH (quant)
30, R = 3,5-COOH (quant)
H
N
H
N
2
O
O
O
O
O
N
H
N
HO
OH
H
O
O
O
O
H
N
H
HO
O
N
OH
O
O
N
N
H
II (quant)
H
Scheme 2. Synthesis of the tetrapodal derivatives II and 28-30.
Finally, to complete the SAR studies functionalized aromatic substituents at the N1 position of
the indole moiety were introduced. Starting from 4, the N1-substituted analogues 3436 were
synthesized by reaction with the corresponding benzyl bromides, in the presence of Cs CO at 80
2
3
ºC followed by subsequent deprotection (LiOH/H O) of intermediates 3133 (Scheme 3). The
2
same synthetic methodology was applied to the synthesis of the tetrapodal analogue 38 which
was obtained in 31% overall yield (Scheme 3).
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R
R
R
H
R
H
N
Br
R
N
N
N
N
N
O
O
NO2
O
O
1. LiOH
THF/H2O
O
O
NO2
O
O
N
N
O
O
NO2
MeO
H
H
OMe
N
N
O
O
H
H
OMe
N
N
H
MeO
HO
H
OH
O
NH
O
Cs2CO3,
CH3CN, 80 ºC
2. HCl 1M
OMe
O
NH
O
O
NH
O
OMe
OH
HN
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
R
4
R
31, R = H ( 33%)
2, R = 4-F (42%)
3, R = 4-COOMe (33%)
34, R = H (quant)
3
3
35, R = 4-F (quant)
36, R = 4-COOH (quant)
R
R
H
N
H
N
N
N
Br
O
O
O
O
O
N
O
O
O
O
O
N
H
N
MeO
OMe
H
R1
N
H
R1
O
O
O
O
R
H
O
O
O
O
R1
H
N
H
N
H
N
H
N
R1
O
MeO
O
OMe
O
2 3,
Cs CO
CH CN, 80 ºC
3
O
O
O
24
N
N
H
N
N
H
R
R
1
3
3
7, R = 4-COOMe; R = OMe (31%)
8, R = 4-COOH; R1 = OH (quant)
1. LiOH,THF/H O
2
2. HCl 1M
Scheme 3. Synthesis of the N1 substituted tripodal (3436) and tetrapodal (38) derivatives.
As will be discussed later, compounds 22 and 30, with isophthaloyl moieties at C2, were
promising as anti-HIV and EV71 agents. To have access to higher amount of these compounds
for further biological studies and structural modifications we explored a novel convergent
synthetic strategy (Scheme 4) in which a C-2 arylated Trp derivative 41, with a free amino
group, was used as a key common intermediate.
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CO2Me
CO2Me
CO2Me
CbzNH
H2N
CbzNH
MeO2
C
CO2Me
CO Me
2
CO Me
2
Pd(OAc)2, AgBF4
TFA, DMF, MW, 90ºC, 30'
HCO2NH4, Pd/C (10%)
DMF, r.t
I
N
H
N
H
N
H
2
CO Me
2
CO Me
39
40 (71%)
41 (89%)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CO2H
CO2H
O
O
HO2C
NO2
O
HO
OH
HATU/DIPEA
DMF
HO2C
HATU/DIPEA
DMF
3
23
OH
1
3, (85%)
2, (quant %)
27 (75%)
1. LiOH, THF/H2O
1. LiOH, THF/H2O
2. HCl 1M
2
2. HCl 1M
3
0 (quant %)
Scheme 4. Alternative synthetic route for the synthesis of 22 and 30.
α
The synthesis of 41 started with the commercially available N -CBz tryptophan methyl ester
(
39) that was C-2 arylated with dimethyl 5-iodoisophthlate. The reaction was carried out in DMF
as before, 5 mol% Pd(OAc) , AgBF TFA, microwave irradiation during 30 min at 120 ºC, to
2
4,
afford intermediate 40 in good yield (71%). Subsequent carboxybenzyl (CBz) deprotection, was
attempted first using hydrogen and 10% Pd/C. However, the starting compound was recovered
intact. Instead, catalytic transfer hydrogenation using dry ammonium formate and 10% Pd/C51
afforded the amino intermediate 41 in very good yield (89%). Further attachment of 41, through
the formation of amide bonds, to the tripodal (3) or tetrapodal (23) central scaffols afforded
intermediates 13 (85%) and 27 (75%) respectively. Finally, methyl esther deprotection
(LiOH/H O) afforded the desired final compounds 22 and 30 (structure in Scheme 1) in
2
quantitative yields.
Compared to the divergent approach (Scheme 1), this convergent methodology enables de
synthesis of 22 and 30 in a very simple and straightforward form. Moreover, the global yields of
2
2 and 30 improved considerably (54% versus 34% for 22) and (47% versus 21% for 30). Based
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on these results we consider this second synthetic strategy as a general and more efficient
approach for the synthesis of this type of tricyclic and tetracyclic Trp derivatives.
1
13
All the synthesized compounds were characterized by H and C NMR spectroscopies and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
High Resolution Mass Spectrometry (HRMS).
Antiviral evaluation
Antiviral activity against HIV
Compounds substituted at C2 (1422 and 2830) and N1 (3436 and 38) positions of the
indole moiety, together with the unsubstituted trimer I and tetramer II, were first evaluated for
+
their inhibitory effects against HIV-1 and HIV-2 replication in cultured CD4 T cells (Table 1).
The antiviral data of the prototype 1, with 12 unsubstituted Trp residues on the periphery, is also
included as a reference compound, as well as that of dextran sulfate-5000 (DS-5000) and
5
2
pradimicin A (PRM-A). DS-5000 is a negatively charged HIV adsorption inhibitor and PRM-A
represents a gp120 carbohydrate-binding entry inhibitor.53 These control compounds showed
antiviral activities in the range previously reported.52,53
As shown in Table 1, the unsubstituted dendrimers I and II did not show any anti-HIV activity
up to 100 µM. Besides, compounds 17 and 19, with 4-Br-phenyl and 4-OMe-phenyl moieties,
respectively, at C2 were also inactive at sub-toxic concentrations. However, compounds 14-16
and 18, bearing phenyl, 2-F-phenyl, 4-NO -phenyl and 4-CF -phenyl moieties, respectively, at
2
3
C2 showed modest but significant anti-HIV activity. Although the activity of these compounds
was only moderate these data were important because they represented a first hint of activity in
this series of compounds. Moreover, and taking into consideration that I and II did not exhibit
1
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2
2
2
2
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3
3
3
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3
4
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4
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5
5
5
5
6
any antiviral activity against HIV, this result suggests that the C2 substituent may play an
important role in the eventual antiviral activity.
Interestingly, the tripodal derivative 21, with one COOH substituent in the para position of the
phenyl ring (EC = 2.4 µM), was as active as the prototype 1 (EC = 2.3 µM) while the tripodal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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5
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7
8
9
0
1
2
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9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
derivative 22, with 2 COOHs at positions 3 and 5 of the phenyl ring (isophthaloyl moiety) was
even better (EC = 0.27 µM). These data clearly demonstrate that the phenyl rings with free
50
carboxylic acids on C2 play an important role in the anti-HIV activity.
Shifting the position of the substituted aromatic ring on the indole side-chain to N1
(compounds 3436 and 38) led to a substantial decrease of antiviral activity. This effect was
especially pronounced when comparing the activity of the pentaerythritol derivatives 29 and 38,
with para-COOH arylated Trp residues, in C2 (compound 29) or in N1 (compound 38) positions.
Compound 29 (EC = 0.73 µM) was 3-fold more active than the prototype (EC = 2.3 µM)
50
50
while 38 was devoid of antiviral activity. This data indicates that the position of the aromatic
substituent is also crucial for anti-HIV activity.
Within the series of tetramers (2830), the most active compound was 30 (EC = 0.07 µM),
50
with two COOHs at positions 3 and 5 of the phenyl ring, followed by 29 (EC = 0.73 µM) and
5
0
2
8 (EC50 = 2.0 µM), with one COOH substituent in either the para- or meta- position,
respectively, of the phenyl ring.
It should be emphasized that the anti-HIV-1 activity (EC = 0.07–2.4 µM) of the trimers (21,
5
0
2
2) and tetramers (2830), bearing at the C2 position of the indole ring phenyl substituents with
one or two COOHs, was above that of the prototype 1 (EC = 2.3 µM) (Table 1), although their
50
molecular weight is considerably smaller (approximately 1500 Da versus 3576 Da). Moreover,
the activities displayed by some of these compounds (22, 29 and 30) are far below their toxicity
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threshold (CC50 > 100 µM), resulting in compounds with improved selectivity indices
(
CC /EC ratios SI > 370, 137 and 1429, respectively) with respect to the prototype (SI > 43).
50 50 ,
Interestingly, tetramer 30 also shows a 330-fold improvement of activity against HIV-2 (EC =
5
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
.02 µM) with respect to the prototype (EC = 6.6 µM).
50
Table 1. Anti-HIV-1 and anti-HIV-2 activity of the selected group of compounds
a
EC50a (µM)
HIV-2
EC5 (µM)
CC50b (µM)
0
SIc
d
Compound
MW (Da)
HIV-1
19 ± 10
35 ± 10
41 ± 3.8
˃30
1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
4
5
6
7
8
9
0
1
2
8
9
0
4
5
6
8
>83
>100
83 ± 4
>100
>100
30 ± 4.3
>100
9.3 ± 0.29
>100
71
4.4
>3
1064.14
1118.12
1199.14
1300.83
1268.14
1154.22
1196.17
1196.17
1328.20
1649.66
1649.66
1825.69
1106.24
1160.22
1238.27
1705.76
835.86
43 ± 11
˃30
>2
ND
>4
26 ± 10
˃9.3
68 ± 6.0
˃9.3
ND
>6
16 ± 5.7
2.4 ± 1.3
0.27 ± 0.12
2.0 ± 0.56
0.73 ± 0.51
0.07 ± 0.04
49
64 ± 28
>71
29.6
>370
>50
>137
>1429
>2
0.63 ± 0.83
0.76 ± 0.53
0.75 ± 0.83
0.02 ± 0.09
38
>100
>100
>100
>100
>100
>100
>100
76
43
34
>2.3
>2.4
ND
ND
41
47
>76
>76
I
>100
>100
>100
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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5
5
5
5
5
5
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II
1
>250
>250
>250
>100
>20
ND
>43
1169.24
3575.84
~5000
2.3 ± 0.30
0.07 ± 0.02
3.3 ± 1.2
6.6 ± 7.7
0.03 ± 0.01
5.9 ± 3.7
DS-5000
PRM-A
>285.7
>30.3
>100
842.80
0
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9
0
1
2
3
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5
6
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9
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1
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9
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3
4
5
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9
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Data are the mean ± S.D. of at least 2 to 3 independent experiments
ND: No Determined
EC50: 50% Effective concentration, or the concentration required to inhibit HIV-induced cytopathicity
by 50%
CC50: 50% Cytostatic concentration, or the compound concentration required to inhibit CD4 T cell
proliferation by 50%
SI: Selectivity Index (CC50 / EC50
MW: Molecular weight of the molecules expressed as Da
a
b
+
c
)
d
Mode of action in the context of HIV infection
Time-of-drug-addition (TOA) experiment
To determine at which stage of the HIV replication cycle the compounds act, a time-of-drug-
addition study54,55 was performed with the most potent derivatives 22 and 30. The HIV
entry/fusion inhibitor DS5000 (DS) and the HIV reverse transcriptase inhibitor AZT were used
as references. Similar to DS, HIV-1 replication, as measured by p24 antigen production, could be
inhibited only if 22 and 30 were added to the cells prior to or at the moment of infection.
However, addition of AZT, an inhibitor of the reverse transcription step, could be delayed up to
3
h after infection without loss of the inhibitory activity. These experiments clearly show that the
compounds need to be present in T cell cultures at the start of the virus infection process
(binding/adsorption period). Thus, it can be concluded that compounds 22 and 30 interfere with
early event(s) of the replicative HIV cycle, very likely by inhibiting the entry of HIV into its
+
susceptible human CD4 T cells.
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Figure 2. Results of the time-of-drug-addition experiment for compounds 22 and 30. CD4 T
cells were treated with the compounds at selected times before, during or after infection with
HIV-1. 8h post-infection, cells were harvested and p24 was isolated and quantified by means of
RT-qPCR. Data for DS-5000 and AZT are given as reference.
Surface plasmon resonance (SPR) analysis (HIV)
As mentioned, the prototype compound 1 inhibits an early step in the replicative cycle of HIV by
2
8
interacting with the gp120 glycoprotein of the HIV envelope. To investigate if this glycoprotein
is also the potential target of 22 and 30, a Surface Plasmon Resonance (SPR) experiment was
carried out. First, a direct binding experiment, where gp120 was immobilized to the sensorchip,
was used. Both compounds dose-dependently bound to immobilized gp120 but we could not fit
the experimental data with an existing binding model, nor could steady-state affinity be
measured (data not shown).
Because the sensorgrams obtained from these first direct binding experiments were
inconclusive, we turned to an indirect experiment in which the inhibitory effects of 22 and 30 on
the heparin-gp120 interaction, were determined. In this experiment heparin, as mimic of the host
5
6
cell membrane glycosaminoglycans (GAGs), was bound to the sensorchip. Both 22 and 30
were able to block the binding of gp120 onto immobilized heparin in a dose-dependent manner
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2
2
2
2
2
2
2
2
3
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3
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4
4
4
4
4
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5
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(
figs 3A, B). Compound 30 was the most potent inhibitor of gp120-heparin binding with an IC50
of 210.9 ± 30.2 nM (fig 3 C). Compound 22 was approximately 5 times less active than 30 (IC50
= 981.7 ± 197.0 nM), which correlates with the weaker anti-HIV-1 activity that was observed in
the antiviral assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
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5
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Overall, these data indicate that the observed in vitro anti-HIV activity of the Trp derivatives
may be explained by their ability to specifically block the interaction between gp120 and the
polysulfated polysaccharides on the host cell membranes. The tetramer 30 resulted more potent
than the trimer 22.
Figure 3. Inhibition of gp120-heparin binding by 22 and 30 as measured by SPR. Biotinylated
heparin was captured on a Streptavidin Sensor Chip. Gp120 (10 nM) was mixed with a
concentration range of compound 22 (A) or 30 (B). One representative sensorgram per
compound out of three independent experiments is shown. Binding levels (RU) were converted
into percentage inhibition relative to gp120 binding without compound. Mean (+SEM) and
nonlinear fit of three independent experiments are shown (C).
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Antiviral activity against EV71
The newly synthesized compounds were also evaluated for their in vitro inhibitory effects
against EV-A71 (Table 2). Tryptophan prototype 1 and pirodavir, a potent EV-A71 capsid
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
binder, were used as reference compounds.57,58
First, we performed an antiviral activity assay against EV-A71 BrCr, a laboratory-adapted
strain, on rhabdomyosarcoma (RD) cells, which are known for their high susceptibility to EV-
5
9
A71 infection. For each analogue, the EC value was determined as the concentration of the
5
0
drug protecting 50% of cells from EV-A71-induced cell death. Cell viability (CC ) was also
5
0
assessed on compound-treated, uninfected cells.
Interestingly, the SAR studies for EV-A71 revealed a similar profile to that observed for HIV.
Compound 14, bearing a phenyl ring at C2 position, and compounds 15, 16, 17, 18 and 19,
bearing phenyl rings substituents different than COOH (2-F, 4-NO , 4-Br, 4-CF , 4-OMe,
2
3
respectively), were inactive or more than 80-fold less active than the prototype at sub-toxic
concentrations. Similar to HIV, the unsubstituted tripodal and tetrapodal derivatives I and II,
also proved inactive. However, the C2-arylated tryptophan derivatives 20, 22, 2830, with
phenyl rings substituted with one or two COOHs were as active or even more active than the
prototype 1, indicating the importance of the carboxylic acids on the aromatic ring as a necessary
requisite for antiviral activity. Notably, compound 30 resulted approximately eight-fold more
potent than the prototype (EC : 0.04 µM vs 0.3 µM, respectively).
5
0
Interestingly, the antiviral potency of 20 (EC : 0.99 µM), bearing a COOH substituent at the
5
0
meta-position of the phenyl ring, was 12-fold that of 21 (EC : 12.2 µM), which carried the same
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substituent in the para position. These activity data suggest that the position of the COOH
substituent on the phenyl ring is crucial for antiviral activity. Furthermore, similarly to HIV, no
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anti-EV-A71 activity was observed with compound 38, possessing the aromatic substituent at
N1, while the closely related analog 29 (EC : 0.37 µM), with the aromatic substituent at C2,
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was as active as the prototype (EC : 0.3 µM). This result clearly demonstrates that the position
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0
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of the aromatic substituent also plays an important role in the anti-EV-A71 and anti-HIV
activity.
Several compounds (i.e., 22, 28, 29) showed selectivity indices higher (SI >203, >336, 161)
than those found for the prototype 1 (SI: 83) and comparable to those found for the reference
compound, pirodavir (i.e., 28: SI >336; pirodavir: SI >333). This increase in the selectivity index
is particularly due to a lower cytotoxicity.
Notably, compound 30 is not toxic for the host cell. This compound turned out to have the
highest selectivity index yet reported from the Trp class of compounds (SI >2500 for 30 vs SI: 83
for the prototype 1).
Table 2. Antiviral activity of dendrimers against the BrCr lab strain of EV-A71 virus in RD
cells.
EC50a (µM) EV-
A71
EC90b (µM)
EV-A71
e
Compound
CC50c (µM)
SId
MW (Da)
1
1
1
1
1
1
2
4
5
6
7
8
9
0
>100
>89.4
ND
ND
ND
ND
ND
ND
1064.14
1118.12
1199.14
1300.83
1268.14
1154.22
1196.17
28.8 ± 4.9
65.2
58.2 ± 16.3
ND
>83.4
ND
ND
ND
>78.9
ND
ND
ND
>86.6
ND
ND
ND
0.99 ± 0.01
1.35 ± 0.1
>83.6
>73.4
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0.35 ± 0.03
0.19 ± 0.01
0.37 ± 0.08
0.04 ± 0.01
>19.5
ND
0.55 ± 0.02
0.30 ± 0.01
0.63 ± 0.1
0.16 ± 0.01
ND
>33
>75.3
>60.6
56.4
>100
ND
ND
>203
>336
161
1196.17
1328.20
1649.66
1649.66
1825.69
1705.76
835.86
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1
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>2500
ND
38
I
>120
ND
>120
ND
ND
II
1
>100
ND
ND
1169.24
3573.84
369.47
0.3 ± 0.1
0.3 ± 0.1
0.5 ± 0.06
0.6 ± 0.2
24.9
>100
83
Pirodavir
>333
All values are in micromolar (μM) and are a summary of multiple dose-response curves (>2) in multiple
independent (>1) experiments.
ND: Not Determined
EC50: concentration of compound at which the virus-induced cytopathic effect is reduced by 50%
EC90: concentration of compound at which the virus-induced cytopathic effect is reduced by 90%
CC50: concentration of compound at which a 50% reduction in cell viability is observed
SI: selectivity index (CC50/EC50
MW: Molecular weight of the molecules expressed as Da
a
b
c
d
)
e
The compounds with the best activity/toxicity profile, 22, 28 and 30, were evaluated in virus-
cell-based assays against a panel of clinical isolates representatives of the different
sub)genogroups of EV71 (B2, B5, C2 and C4) (Table 3). The prototype 1 was included as a
(
reference. As previously observed for the prototype, the activity of compounds 22, 28 and 30 on
the clinical isolates was improved with respect to those found for the BrCr lab strain (sub-
genogroup A). In particular, the greatest enhancement in antiviral activity was observed against
sub-genogroup C4: 9-fold, 88-fold and 78-fold higher than the BrCr strain, respectively; whereas
the lowest improvement was detected in the context of the infection with sub-genogroup B2
(
only 2-fold, 4-fold and 6-fold improvement, respectively). It should be highlighted that the
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improvement was higher for the prototype 1 than for the new compounds 22, 28, 30 and among
them for tetramers 28, 30 than for the trimer 22.
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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7
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Table 3. Evaluation of the broad-spectrum antiviral effect of compounds 22, 28 and 30 against a
representative panel of clinical EV71 isolates in RD cells.
a
EC (nM)
5
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EV71
Genogroup
Virus strain
1
22
28
30
A
BrCr
285 ± 70
0.4 ± 0.0
0.2 ± 0.1
1.1 ± 0.3
0.2 ± 0.2
353 ± 31
179 ± 11
81 ± 13
57 ± 0.2
38 ± 3
175 ± 22
52 ± 1
109 ± 34
19 ± 0.6
5.9 ± 0.7
2.6 ± 0.6
1.4 ± 0.7
B2
B5
C2
C4
11316
TW/70902/08
H08300 461#812
TW/1956/05
18 ± 1
7.3 ± 0.8
2.0 ± 0.2
aAll 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.
Mode of action in the context of EV-A71 infection
Time-of-drug-addition (TOA) experiment (EV-A71)
To determine whether the reduced size of the newly synthetized analogues affects their
mechanism of inhibition of EV-A71 replication, we performed a TOA assay with a
representative member, compound 22. In order to confirm the activity of compound 22 as an
early stage inhibitor, we included the capsid binder pirodavir as a control (Figure 4).57,60
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Figure 4. Time-of-drug-addition for compound 22. RD cells were treated with the compounds at
selected times before, during or after infection with EV-A71. 8h post-infection, cells were
harvested and intracellular viral RNA was isolated and quantified by means of RT-qPCR.
Similar to pirodavir, only the addition of compound 22, prior to or at the moment of infection
resulted in a complete inhibition of viral replication whereas the addition after infection was
associated with a complete loss of protection. This experiment clearly shows that 22, as
29
previously observed for prototype 1, inhibits early step(s) of the replicative cycle of EV-A71.
Cross-resistance studies
Two of the most active anti-EV-A71 compounds 22 and 30, were also evaluated for their
inhibitory activity against single mutant EV-A71 strains VP1_S184T and VP1_P246S and the
double mutant VP1_S184T_ P246S that confer resistance to the prototype 1 (Table 4).
Interestingly, 22 and 30 retain some antiviral activity (EC : 1.96-2.35 µM) against these mutant
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virus strains, although this activity is at least 5.6 to 6.7-fold lower than against the wild-type
virus. These data point to the fact that the resistant viruses emerging under the selective pressure
of 1 are also cross-resistant to 22 and 30 suggesting that these compounds are likely to share an
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overall similar mechanism of antiviral activity (and resistance) and a binding site with the parent
prototype 1, namely the five-fold axis of the capsid. However, it cannot be excluded that 22 and
30, which show some residual activity against the mutant viral strains and are the most active
Trp derivatives ever reported, may give rise to additional interactions with amino acids at the
five-fold axis, thus accounting for the residual activity against EV-A71 mutant viruses.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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Table 4. Susceptibility of reverse-engineered EV-A71 variants to prototype 1 and
compounds 22 and 30
1
22
30
Virus
EC50a (μM)
EC50a (μM)
EC50a (μM)
EV-A71 BrCr
VP1_S184T
VP1_P246S
0.28 ± 0.01
0.35 ± 0.03
0.1 ± 0.03
1.99 ± 0.10 (7.1) 2.35 ± 0.60 (6.7)
4.71 ± 0.18 (16.8) 1.96 ± 0.04 (5.6)
0.9 ± 0.05 (8.5)
0.2 ± 0.09 (1.8)
0.3 ± 0.04 (3.1)
VP1_S184T_ P246S 8.93 ± 0.53 (31.9) 2.18 ± 0.08 (6.2)
Averages and SD (standard deviation) were calculated from data obtained from three independent
antiviral assays; between brackets, in red color, fold resistance.
Cryo-electron microscopy studies
To identify the binding sites of 22 and 30, purified capsids of EV-A71 strain 11319 were
incubated with each drug and vitrified for cryo-electron microscopy (cryo-EM) reconstructions.
Refinement with icosahedral symmetry averaging imposed generated 2.98 Å and 2.87 Å
resolution maps for 22 and 30 complexes respectively (Figure 5 and S1 Table). Sharpened maps
of the two EV-A71-22 and 30 complexes were almost identical with the map of the EV-A71
strain 11319 42 (Figure 5A) and no extra densities on the 5-fold vertexes, where the drugs are
predicted to bind, were found. However, unsharpened maps of both virus-drug complexes
revealed thin extra densities covering the 5-fold axis (Fig 5B and C). This finding indicates that
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the drugs bound asymmetrically on the vertex and their densities were averaged together by the
imposed icosahedral symmetry, impeding the precise interpretation of the drug densities.
1
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Figure 5. Cryo-EM 3D maps identified the MADAL compounds on the capsid 5-fold axis.
(A) Surface rendering of the sharpened 3D maps for EV-A71 incubated with 22 (left) and 30
(right) showed canonical features of the capsid. Icosahedral symmetry axes were marked with
arrows. (B) Comparison of the unsharpened maps (gray) of 22 and 30 complexes with EV-A71
showed extra densities (yellow) on the 5-fold axis. The surface rendered at 1 and 1.9 (insets)
for EV-A71 (left), 22 (middle) and 30 (right). (C) Central sections of the three cryo-EM maps
showed extra densities on the 5-fold-vertex covering the hole at the symmetry axis. Icosahedral
symmetry axes were marked.
Compound 30 showed a stronger drug density compared to 22 (Fig 5B inlets), suggesting
higher affinity of the former with respect to the latter since both compounds were incubated at
the same concentration. The significant density for 30 appeared to cover the 5-fold axis like a lid
and did not penetrate into the pore (Figure 5C) although it cannot be excluded the possibility that
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a part of the drug might have lodged into it but its density was averaged out. The density
intensity of the bound molecule in an icosahedral map is directly related to the occupancy and/or
structural heterogeneity (multiple conformations) of the molecules. As shown in our previous
study using prototype 1, multiple conformations of the bound drug would decrease its density
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intensity. This may be a common observation for drugs bound at the 5-fold axis.
Atomic models of the viral capsids were refined using the two complex maps (Figure 6A).
Densities attributable to residue backbones and side chains were recognizable in the maps
(Figure 6B). The capsid proteins VP1-4 did not show any significant conformational differences
or rigid body movements from the atomic model of EV-A71 (RMSD 0.719 Å and 0.838 Å for 22
and 30, respectively). Cryo-EM densities corresponding to the pocket factor were also observed
in both maps (Figure 6B and data not shown). The capsid interactions with RNA genome also
did not change (data not shown). Thus, binding of 22 and 30 on EV-A71 did not induce any
significant conformational changes on the capsids, including pocket factor release.
Figure 6. Atomic model built into the cryo-EM map. (A) The atomic models for EV-A71 VP1
(blue), VP2 (green), VP3 (red), VP4 (yellow), and the pocket factor (orange) were built into the
cryo-EM map of the virus-30 complex (gray mesh). (B) Representative residues of each VP and
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the pocket-factor are shown within the corresponding cryo-EM density envelopes to illustrate the
quality of the 3D map.
The superimposition of the atomic model in the unsharpened maps showed that the extra
densities are strongly connected to Lys244 (K244) and Tyr245 (Y245) residues of VP1 (Figure
1
1
1
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A,B).
Figure 7. The drug densities are connected to two VP1 residues. (A) The icosahedrally
averaged drug density (yellow, 30) was connected mostly with Lys244 and Tyr245 on the VP1
surface (grey). The atomic model of VP1 is depicted as a blue ribbon and the side-chains are
shown as sticks. The surface is rendered at 1.9. Neighboring residues are also labeled. (B) Side-
view of the interaction site shows the thin drug density connected to the two VP1 residues. (C)
Binding pose proposed by the automated docking program for the substituted Trp residue in 30
at the interface between chains E (cyan) and Q (pink) using the coordinates deposited in PDB
entry 6UH7.
Further atomistic detail about the complex formed between 30 and the VP1 pentamer was
gained by performing automated docking calculations with the decorated Trp moiety attached to
each of its four ‘legs’ (Scheme 2). In the best-scored and most populated pose (Figure 7C and
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Supporting Information, Figure 1) the Trp is lodged at the interface between two VP1 monomers
in a cavity lined by the adjoined 141TPTGQVVP148 and 242QSKYP246 stretches from one subunit
and the 242QSKYP246 stretch from a neighboring subunit. In this orientation, (i) the indole ring of
Trp and the phenyl ring of the isophthalate moiety stack on Pro246 and Tyr245, respectively, the
latter in an edge-to-face arrangement; (ii) the Trp carboxylate establishes a hydrogen bond with
the side-chain hydroxyl of Thr143 and a strong ionic interaction with the charged amino group of
Lys244 from a neighboring subunit; (iii) the carboxamide nitrogen of Gln145 hydrogen bonds to
one of the isophthalate carboxylates; and (iv) the other isophthalate carboxylate points to the
positively charged Lys244 that is vicinal to the Tyr243 on which the phenyl ring stacks.
Importantly, this pose is compatible not only with the electron density (Figure 7) but also with
the fact that the ethylene linker connecting this decorated Trp to the pentaerythritol core points
towards the central pore. Thus, one can figure out that one of the tetrapod’s legs fits snugly into
the proposed intersubunit cavity whereas the other three, which cannot bind in a similar fashion
simultaneously, establish less specific van der Waals and electrostatic interactions with the
residues lining the pore. Since all five VP1 subunits are identical, as are all of the dendrimer’s
protrusions, the overall binding mode is an average of all five combinations that is also in
consonance with the average experimental electron density (Figure 8 and Supporting
Information, Figure 2). Thus, it can be assumed that binding of 30 within this region prevents the
interaction of the virus with its cellular (co-)receptors P-selectin glycoprotein ligand-1 (PSGL-1)
and heparan sulfate thereby blocking attachment of EV-A71 to the host cells.
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Figure 8. Proposed binding mode of the decorated Trp moiety of 30 around the 5-fold
axis of the EV-A71 capsid, as deposited in PDB entry 6UH7. (A) A semitransparent surface
envelops the five VP1 chains making up the pore. C atoms in chains A, E, I, M, and Q are
colored in green, cyan, magenta, yellow and pink, respectively. Note that the ethylene linker
points towards the central pore. (B) Solvent-accessible surface of the VP1 pentamer color-coded
according to the molecular electrostatic potential calculated with APBS (red, -3.0 kT/e; blue,
+3.0 kT/e). The yellow surface filling the subunit interfaces corresponds to the van der Waals
surface of the five docked fragments shown in (A).
CONCLUSIONS
Using a scaffold simplification strategy, a novel generation of dual HIV and enterovirus-A71
entry inhibitors has been synthesized. These compounds contain three or four tryptophan (Trp)
residues bearing at the C2 position of their indole side-chains a substituted phenyl ring. A highly
efficient and scalable method has been developed for the synthesis of these compounds.
SAR studies showed that the presence of one or two carboxylates on the C2 extra phenyl rings
is critical for anti-HIV and EV-A71 activity. The most potent, 22 and 30, showed enhanced
activity (~10 and 100-fold) against HIV-1 compared to the former dendrimer prototype
discovered in our group. Notably, 30 is the only congener within this series that showed potent
anti-HIV-2 activity. Time-of-drug-addition (TOA) experiments, followed by SPR studies
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revealed that 22 and 30 act at early stages of the viral replication cycle of HIV by binding to the
viral envelope glycoprotein gp120. Moreover, they also proved to be potent inhibitors of EV71,
inhibiting not only the laboratory BrCr strain of EV-A71 but also clinical isolates of
enteroviruses A, B and C. By means of cross-resistance studies, followed by cryo-EM, we
mapped the binding region of 22 and 30 to the 5-fold axis of the EV-A71 capsid. Further details
about the binding mode were provided by computer-assisted modeling studies that included
docking of the common isophthaloyl-decorated Trp residue and molecular dynamics simulations.
All in all, the structures presented herein provide a very consistent picture about the mode of
binding of these compounds to the VP1 pentamer making up the pore region.
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Compounds 22 and 30 are non-toxic for different types of host cells and show the highest
selectivity indices yet reported for the Trp class of compounds. Moreover, these compounds
specifically inhibit replication of HIV (HIV-1 and HIV-2) and EV71 (laboratory-adapted strain
BrCr and different EV71 clinical isolates from different genogroups and geographic origins).
Taken together, these results show that this new type of Trp derivatives are important leads as
dual HIV and EV71 entry inhibitors. The higher potency, lower toxicity, half-size, easy of
chemical synthesis and greater opportunities for chemical optimization make them more useful
than the former Trp dendrimers discovered in our group for future medicinal chemistry studies.
EXPERIMENTAL SECTION
Synthesis
General Chemistry Procedures. Commercial reagents and solvents were used as received
from the suppliers without further purification unless otherwise stated. The solvents used in some
reactions were dried prior to use. DMF dry was commercially available (Aldrich).
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A microwave reactor EmrysTM Synthesizer (Biotage AB) was used for the reactions which
needed microwave irradiation.
Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-
coated with silica gel 60 (F , 0.20 mm). Products were visualized using an ultraviolet lamp
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(
254 nm and 365 nm) or by heating after treatment with a 5% solution of phosphomolybdic acid
PMA) or vanillin in ethanol.
(
The compounds were purified by: a) high performance flash chromatography (HPFC) with
a system “Isolera One” (Biotage) in reverse phase using water/acetonitrile (100:0 to 0:100) as
eluent, b) flash column chromatography on silica gel (60 Merck 230-400 mesh), c) preparative
®
centrifugal circular thin layer chromatography (CCTLC) on a chromatotron (Kiesegel 60 PF254
gipshaltig, Merck) layer thickness 1 mm, flow rate 2–4 mL/min.
The purity of final compounds was at least 95% as determined by microanalyses with a
Heraeus CHN-O-RAPID instrument. Analyses indicated by the symbols of the elements or
functions were within ±0.4% of the theoric value.
For HPLC analysis an Agilent Technologies 1120 Compact LC with a reverse phase
column ACE 5 C18-300 (4.6 mm  150 mm, 3.5 μm) equipped with a PDA (Photo Diode Array)
detector was used. Acetonitrile was used as mobile phase A, and water 0.05% of TFA was used
-1
as mobile phase B with at a flow rate of 1 mL·min . All retention times are quoted in minutes
and the gradients are specified for each compound in the experimental data.
For high resolution mass spectrometry (HRMS) was used an Agilent 6520 Accurate Mass
QTOF (quadrupole time of flight) coupled with LC/MS using an electrospray interface (ESI)
+
-
working in the positive-ion (ESI ) and negative-ion (ESI ) mode.
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