2-Aminothiazolones as anti-hiv agents that act as gp120-cd4 inhibitors

Article abstract of DOI:10.1128/AAC.02739-13

We report here the synthesis of 2-Aminothiazolones along with their biological properties as novel anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4 protein-protein interaction that occurs at the very early stage of the HIV-1 entry process. No cytotoxicity was found for these compounds, and broad antiviral activities against laboratory strains and pseudotyped viruses were documented. Docking simulations have also been applied to predict the mechanism, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity of HIV-1 gp120. Furthermore, a preliminary absorption, distribution, metabolism, and excretion (ADME) evaluation was performed. Overall, this study led the basis for the development of more potent HIV entry inhibitors.

Full text of DOI:10.1128/AAC.02739-13

AAC Accepts, published online ahead of print on 10 March 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02739-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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2-aminothiazolones as anti-HIV agents which act as
gp120-CD4 Inhibitors
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Running Title: 2-aminothiazolones as novel gp120-CD4 Inhibitors
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Marika Tiberi,1 Cristina Tintori,1 Elisa Rita Ceresola,2 Roberta Fazi, 1 Claudio Zamperini,1
Pierpaolo Calandro,1 Luigi Franchi,1 Manikandan Selvaraj,1 Lorenzo Botta,1 Michela Sampaolo, 2,3
Diego Saita,2,3 Roberto Ferrarese,3 Massimo Clementi, 2,3 Filippo Canducci3,4,# and Maurizio
Botta1,5,#
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1Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. De
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Gasperi 2, I-53100 Siena, Italy University Vita-Salute San Raffaele, Milan, Italy Ospedale San
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Raffale, Milan, Italy Department of Clinical and Experimental Medicine, Università degli Studi
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dell'Insubria, Varese, Italy Biotechnology College of Science and Technology, Temple University,
Biolife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, Pennsylvania 19122.
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To whom correspondence should be addressed. E-mail: botta.maurizio@gmail.com. Phone:
(+39)0577-234306. Fax: (+39)0577234333; E-mail: canducci.filippo@hsr.it. Phone: (+39)02-
26434284; Fax (+39)0226434288.
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Abstract
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We report here the synthesis of 2-aminothiazolones along with their biological properties as novel
anti-HIV agents. Such compounds have proven to act through the inhibition of the gp120-CD4
protein-protein interaction which occurs at the very early stage of the HIV-1 entry process. No
cytotoxicity was found for these compounds and broad antiviral activity was documented on
laboratory strains and pseudotyped viruses. Docking simulations have been also applied to predict
the way, at the molecular level, by which the inhibitors were able to interact within the Phe43 cavity
of HIV-1 gp120. Furthermore, a preliminary ADME evaluation was performed. Overall, this study
led the basis for the development of more potent HIV entry inhibitors.
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Introduction
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HIV-1 infection continues to be a major health threat worldwide. Approximately 40 million
individuals are living with HIV-1 and new infections occur every year. Drug discovery and
development have transformed HIV-1 infection into a chronic condition that can be controlled for
many years through combination therapies with different classes of antiretroviral drugs, known as
highly active antiretroviral therapy (HAART) (1). However, the lifelong duration of HAART and
the emergence of resistance to these drugs underscore the need to develop newer inhibitors with
reduced toxicity and improved activity and resistance profiles (2-4). The attachment of the human
immunodeficiency virus (HIV) to the host cells occurs via binding of the HIV envelope
glycoprotein gp120 to the host CD4 receptor, thus, the inhibition of this protein-protein interaction
offers an effective way for the development of new antiretroviral agents (5, 6). The crystal structure
of gp120 bound to CD4 and the fragment of an antibody (Fab) 17b was solved in 1998(7), revealing
that the CD4 binding site of HIV-1 gp120 envelope consists of a hydrophobic pocket, capped by the
CD4 Phe43, thus termed the Phe43 cavity. Research into gp120 inhibitors able to block the complex
formation between gp120 and CD4 has received increasing attention in recent years and has led to
the discovery of active small-molecules characterized by a high degree of chemical diversity. BMS-
378806 (BMS-806) and the related compounds #155 and BMS-488043 discovered through a cell-
based screening assay, are nanomolar inhibitors that prevent the binding of gp120 to CD4 receptors
(8-10). NBD-556 and NBD-557, first discovered by Zhao et al. by using an HIV syncytium
formation assay on a small library of 33,000 compounds, have been shown to compete with CD4
binding and possess low micromolar potency against several strains of HIV(11, 12). Interestingly,
the crystal structure of NBD-556 in complex with gp120 has been recently deposited in the Protein
Data Bank (PDB code: 3TGS) highlighting the binding mode of the compound within the Phe43
cavity of gp-120 (13). NBD-556 analogues were then developed to study the structure-activity
relationship (SAR) (14-17). Furthermore, molecular modeling techniques were successfully applied
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for the identification of new gp120-CD4 inhibitors (18). In this regard, our research group has
recently reported the successful application of different virtual screening approaches to the
discovery of the hit compounds 1-6 (19, 20). These compounds showed micromolar inhibition of
HIV-1 replication in cells infected by wild-type virus, but resulted totally inactive toward the
mutant Met475Ile, thus confirming that they target the CD4 binding site on HIV-1 gp120, as
residue 475 belongs to the Phe43 cavity. On the other hand, the 2-aminothiazolone derivatives
represent a versatile scaffold widely used in medicinal chemistry. Compounds containing the 2-
aminothiazolone nucleus have been found to exhibit a broad spectrum of biological activities, such
as antitumor (DBPT) (21), herbicidal (7) and αvβ3 receptor antagonist activities (8)(22). The 2-
aminothiazolone derivatives 9 were recently assayed by us as HIV-1 integrase inhibitors and they
resulted to be only moderately active (23).
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Herein, novel 2-aminothiazolones were synthesized and biologically tested in order to investigate
their potential in inhibiting the HIV infection. Compounds were found able to inhibit the HIV
replication at micromolar/submicromolar concentration in vitro, resulted to be non-toxic and
endowed with a high genetic barrier to the development of resistance at least in vitro. Experiments
demonstrated that the compounds acted as early inhibitors of the gp120–CD4 interaction. In line
with these results, docking studies were next performed to elucidate the binding mode of the 2-
aminothiazolones within the Phe43 cavity of gp120 and to identify amino acids involved in their
mechanism of action. To the best of our knowledge, this is the first time that 2-aminothiazolone
derivatives were investigated as antiretroviral agents targeting the gp120/CD4 interaction.
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MATERIALS AND METHODS
Chemistry
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General information. All commercially available reagents were used as purchased. Anhydrous
reactions were run under a positive pressure of dry N2. Thin-layer chromatography (TLC) was
carried out using Merck TLC plates: silica gel 60 F254. Chromatographic purifications were
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performed on columns packed with Merck 60 silica gel, 23-400 mesh, for the flash technique. H
and 13C NMR spectra were recorded at 400 MHz on a Bruker Avance DPX400 spectrometer.
Chemical shifts are reported relative to (CH3)4 Si at δ= 0.00 ppm (Supporting Information). Melting
points were measured using a Gallenkamp melting point apparatus and are uncorrected. Elemental
analyses were performed on a Perkin Elmer PE 2004 elemental analyzer, and the data for C, H, and
N are within 0.4% of theoretical values. T20 (enfuvirtide), AZT (azidothymidine) were obtained
from the NIH AIDS reagents repository (https://www.aidsreagent.org/), maraviroc and dolutegravir
were obtained from ViiV Healthcare.
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The final compounds 16 a-l were obtained by starting from the commercially available methyl-4-
iodosalycilate 10, which was used in a Suzuki coupling with the 5-formyl-2-furanylboronic acid 11
(Supporting material). The coupled product 12 was then hydrolyzed with an aqueous solution of
sodium hydroxide in MeOH/THF to give, after 12 h, the free acid analogue 13. Next, to generate
compounds with general structure 16 a-l, we adapted a recently published synthethic strategy that
describes a one-pot, multicomponent, microwave-assisted reaction that allows the desired final
compounds to be obtained directly. The methodology involves 4-(5-formylfuran-2-yl)-2-
hydroxybenzoic acid, rhodanine, and primary amines that catalyzed a Knoevenagel condensation
between the aldehyde and rhodanine, and then act as nucleophiles in the displacement of the
thiocarbonyl sulfur. This method allows to obtain, in very short reaction times and easy workup,
several final compounds by changing only the amines employed. We used 4-chloro-3-fluoro
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benzylamine, 4-chloro benzylamine, 3,4-dichloro benzylamine, 3-fluoro benzylamine, 4-fluoro
benzylamine, 3-chloro-4-fluoro aniline, 4-chloro-3-fluoro aniline, 4-fluorophenethylamine, 4-
ethylphenethyl amine, phenethylamine. After dissolving all reagents in EtOH, heating at 150°C for
20 minutes by microwave irradiation was required to effect completion of the reaction. After acidic
workup, the wishes products were collected by filtration from ethanol. (Figure 1A) Compound 16m
was obtained through Suzuki reaction from commercial reagents 17 and 11. Coupled compound 18
reacts with rhodanine and 4-fluorophenethylamine as seen above. (Figure 1B)
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HPLC and MS analysis. The purity of the tested compounds was assessed by reversed-phase liquid
chromatography and a mass spectrometer (Agilent series 1100 LC/MSD) equipped with a UV
detector (λ = 254 nm) and a electrospray ionization (ESI) source. The LC elution method (using a
Zorbax Eclipse XDB, 4.6 X 150 mm, 5µm C8 column) involved the following: (compounds 16a-m)
T = 25° C, mobile phase composed of A) 70% CH3CN and B) 30% H2O with 0.5% formic acid at a
flow rate of 1.0 mL min-1; (all solvents were HPLC grade, Sigma Aldrich). All analysed compounds
meet ≥ 95% purity criteria. MS data were obtained using an Agilent 1100 LC/MSD VL system
(G1946C) with a 0.4 mL min-1 flow rate using a binary solvent system of 95:5 CH3OH/H2O. UV
detection was monitored at λ = 254 nm. Mass spectra were acquired in negative mode scanning
over the mass range of 50-1500 Da. The following ion source parameters were used: drying gas
flow, 9 mL min-1 ; nebulizer pressure, 40 psig; drying gas temperature, 350 °C.
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Microwave irradiation experiments. Microwave reactions were conducted using a CEM Discover
Synthesis Unit (CEM Corp., Mathews, NC, USA). The instrument consists of a continuous focused
microwave power delivery system with operator-selectable power output from 0 to 300 W. The
temperature of the contents of the vessel was monitored with a calibrated IR temperature control
mounted under the reaction vessel. All experiments were performed using a stirring option,
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whereby the contents of the vessel are stirred by a rotating magnetic plate located below the floor of
the microwave cavity and a Teflon-coated magnetic stir bar in the vessel.
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Antiviral activity and cell toxicity
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Phenotypic analyses with fully replicating recombinant HIV-1 strains. The human TZM-bl
indicator cell line was obtained from American Type Culture Collection (Manassas, VA), and
maintained at 37°C in 5% CO2 in DMEM medium containing 10% fetal bovine serum, 50 μg/ml
penicillin, and 50 μg/ml streptomycin. HIV-1 viruses NL(AD8) and NL4.3 were titrated as follow:
serial fivefold dilutions of each virus were made in quadruplicate wells in 96-well culture plates in a
total volume of 100 μl of growth medium for a total of 8 dilution steps. Freshly trypsinized cells
(20,000 cells in 100 μl of growth medium containing 75 μg/ml DEAE-dextran) were added to each
well and the plates were incubated at 37°C in a humidified 5% CO2-95% air environment. After 48-
hour incubation, medium was removed and viral infection was quantified using CPRG assay
(Roche). Twenty thousand TZM-bl cells/well were seeded in 96 well plates in complete DMEM
supplemented with 30 ug/ml DEAE-dextran (Sigma-Aldrich). 300 TCID50/ml of each strain were
pre-treated for 1 hour at 37 °C with six serial dilutions (range 20.000 nM-6.4 nM) of each
compound and then added to the cells as described previously (24, 25). Vehicle (0.1% DMSO)
treated cells served as negative control. A CCR5 inhibitor (maraviroc) or an integrase inhibitor
(dolutegravir) were used as positive control drugs. After 2 days, viral infection was quantified using
CPRG assay (Roche). The inhibitory curves were fitted by non-linear regression, allowing IC50
calculation by using Prism software. To evaluate cell toxicity of the compounds the metabolic XTT
test (Sigma-Aldrich) was performed according to manufacturer’s instructions.
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Phenotypic analyses with pseudotyped viruses and compounds 16 and 16hl. Pseudoviruses were
prepared by transfecting exponentially dividing HEK 293T cells (5 × 106 cells in 15 ml growth
medium in a T-75 culture flask) with 5 μg of each env-expression plasmid (from the Tier2 panel:
QH0692.42, SC422661.8, TRO.11, AC10.0.29, RHPA4259.7, REJO4541.67, WITO4160.33,
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CAAN5342.A2; from the Tier3 panel: PVO.4 and TRJO4551.58) and 10 μg of an env-deficient
HIV-1 backbone vector (pSG3ΔEnv) obtained from the AIDS reagents repository, using FuGENE 6
reagent (Roche) as previously described (26, 27). All the envelopes were from subtype B viruses
and amplified from R5-tropic viruses during acute infection (28). Pseudovirus-containing culture
supernatants were harvested 2 days after transfection, filtered (0.45 μm), and stored at −80°C in 1-
ml aliquots. After pseudovirus titration on TZM-bl cells, 40000 TZM-bl cells/well were seeded in
96 well plates in complete DMEM supplemented with 30 µg/ml DEAE-dextran (Sigma-Aldrich).
300 TCID50/ml of each pseudovirus was pre-treated for 1 hour at 37 °C with six serial
dilutions (range 20.000 nM-6.4 nM) of compound 16l or 16h and then added to the cells. Vehicle
(0.1% DMSO) treated cells served as negative control. A CCR5 inhibitor (maraviroc) was used as
positive control drug. After 2 days, viral infection was quantified using CPRG assay (Roche). The
inhibitory curves were fitted by non-linear regression, allowing IC50 calculation by using Prism
software.
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Time-of-addition assay
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Time-of-addition experiment was performed as previously described with minor modifications (29)
by using a single cycle assay and the pseudotype virus REJO4541 clone 67. For the Time-of-
addition assay, 40.000 TZM-bl cells/well in a 96 multiwell plate were infected with 1500
TCID50/ml of the env-pseudotyped HIV-1 virus in complete medium supplemented with 30 µg/ml
DEAE dextran (Sigma-Aldrich). Virus was incubated with cells for 1 hour at 4° and unbound virus
was subsequently removed by extensive and repeated washing with PBS to synchronize the
replication (29). To analyse the very early steps of infection we performed a pre-incubation step
with the virus and a representative compound (16l) (using IgGb12 obtained from the AIDS reagents
repository as comparison) for 1h at 37 °C prior cell infection. For the following four hours,
antiretroviral compounds inhibiting distinct viral replication steps (IgGb12, maraviroc, T20, AZT,
dolutegravir) and compound 16l were added at the following time points: at time 0, after 60, 75, 90,
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120, 150, 180, 210, and 240 minutes. To ensure a completed inhibition of viral replication we used
a 40-fold IC50 concentration as previously evaluated for each compound on TZM- bl cells (IgGb12
7.5 μg/ml, Maraviroc 0.7 μM, T20 1.6 μM, AZT 3.2 μM, Dolutegravir 1 μM and 16l 5μM). β-
galactosidase expression in cell lysates 48h post-infection was used as a marker of HIV infection
and was normalized to untreated control cells.
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Gp120/CD4-HIS ELISA Binding Assay
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Competition between 2-aminothiazolone derivatives and CD4 was performed through a
Gp120/CD4-6HisTag ELISA binding assay as previously described with minor modifications (9,
12). Briefly, 96-well multiwell plates (Corning) were coated overnight at room temperature with
100 μL of sheep anti-gp120 antibody D7324 (Aalto Bio Reagents, Dublin) at 5 μg /ml in 100 mM
NaHCO3. Wells were blocked with 1% non-fat milk in PBS at 37 °C for 1 h. Coated plate were
incubated with 0.5 µg/ml recombinant gp120 (Abcam) in PBS, at 37 °C for 1 h and washed three
times with PBS-T 0.1%. Soluble CD4 6His-tagged (Life Technologies) at 0.20 μg/ml in PBS and an
equal volume of tested compound at six different concentration (50 µM-1.5 µM) were added to the
wells and incubated at 37 °C for 1 h. Vehicle (DMSO) was used as negative control and IgGb12
was used as positive control. Wells were washed five times with PBS-T 0.1% and incubated an
hour at 37 °C with a goat anti-His antibody (Roche) at a dilution of 1:1000 in PBS 0.5 % milk.
After five wash with PBS-T 0.1%, TMB chromogenic substrate for peroxidase (Pierce) was added
and absorbance was read at 450 nm.
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Computational studies
Protein Preparation. The HIV-1 gp120 three dimensional coordinates were extracted from the
crystallographic complex (PDB entry code 1G9M) (7) and were energy minimized to remove
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unfavorable contacts through the all-atom OPLS force field and Polak-Ribiere conjugate gradient
method. A continuum solvation method, with water as the solvent, was also applied. Extended
cutoffs were used and convergence was set to 0.05 kJ/mol‚Å. The homology models of HIV-1
gp120 for NL4.3 (CXCR4-tropic strain) and AD8 (CCR5-tropic strain) were performed with the
software PRIME, using two crystallographic complexes (PDB entry code 2B4C (30) and 1G9M) as
templates and the primary sequences of the two different strains as queries. The obtained models
were energy minimized by the software MacroModel, from the Schrödinger suite, using the Polak-
Ribiere conjugate gradient algorithm and OPLS_2005 force field (http://www.schrodinger.com/).
Docking Studies. Compounds were built using the Maestro 9.0 Graphical interface and modeled
using the Merk Molecular Force Field (MMFF) in Gibbs Born/Surface Area (GB/SA) water as
implemented in MacroModel (http://www.schrodinger.com/). Docking studies were performed
using the program GOLD (version 5.1) (31). The ChemScore was chosen as fitness function. The
GA parameter settings of Gold were employed using the Search efficiency set to 100%. Finally,
results differing less than 1.5 Å in ligand-all atom rmsd (root mean square deviation) were clustered
together. For each inhibitor, the first ranked solutions as well as the lowest energy conformation of
the most populated cluster were analyzed. Pictures of the modeled ligand/enzyme complexes
together with graphic manipulations were rendered with Pymol package (Version 1.2r3pre
http://www.pymol.org/).
Molecular Dynamics. Molecular Dynamic (MD) simulations have been performed through the use
of AMBER 12 suite of programs (http://ambermd.org/) and the ff03.r1 force field. An appropriate
number of counter-ions (7 Cl- ions for NL4.3 and 3 Cl- ions for AD8 complex) were added to
neutralize the system, and complexes were placed in a octagonal box of TIP3P water molecules.
The distance between the box walls and the protein was set to 10 Å. MD runs were carried out with
a protocol previously validated (32). Before MD simulation, two stage of energy minimization were
performed to remove bad contacts. In first stage, we kept the protein fixed with a constraint of 500
kcal/mol and we minimized the positions of the water molecules. Then, in the second stage, we
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minimize the entire system, applying a constraint of 10 kcal/mol on the α carbons. MD trajectories
were run using the minimized structure as starting input. Constant volume simulations were
performed for 50 ps, during which time temperature was raised from 0 to 300 K using the Langevin
dynamics method. Then 150 ps of constant-pressure MD simulations were performed at 300 K in
three steps of 50 ps each. During the three periods of this second stage, the α carbons were blocked
with harmonic force constants of 10, 5, and 1 kcal/mol·Å, respectively. Finally a 15 ns MD
simulation without restraint was run at a constant temperature of 300 K and a constant pressure of 1
atm.
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ADME assay
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Chemicals. All solvents, reagents, were from Sigma-Aldrich Srl (Milan,Italy). Dodecane was
purchased from Fluka (Milan, Italy). Pooled Male Donors 20 mg/mL HLM were from BD Gentest-
Biosciences (San Jose, California). Milli-Q quality water (Millipore, Milford, MA, USA) was used.
Hydrophobic filter plates (MultiScreen-IP, Clear Plates, 0.45 μm diameter pore size), 96-well
microplates, and 96-well UV-transparent microplates were obtained from Millipore (Bedford, MA,
USA).
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Parallel Artificial Membrane Permeability Assay (PAMPA). Donor solution (0.5 mM) was
prepared by diluting 1 mM dimethylsulfoxide (DMSO) compound stock solution using phosphate
buffer (pH 7.4, 0.025 M). Filters were coated with 5 µL of a 1% (w/v) dodecane solution of
phosphatidylcholine. Donor solution (150 µL) was added to each well of the filter plate. To each
well of the acceptor plate were added 300 µL of solution (50% DMSO in phosphate buffer). All
compounds were tested in three different plates on different days. The sandwich was incubated for 5
h at room temperature under gentle shaking. After the incubation time, the plates were separated,
and samples were taken from both receiver and donor sides and analyzed using LC with UV
detection at 280 nm. LC analysis was performed with a Perkin-Elmer (series 200) instrument
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equipped with an UV detector (Perkin-Elmer 785A, UV/vis Detector). Chromatographic separation
were conducted using a Polaris C18 column (150 - 4.6 mm, 5 µm particle size) at a flow rate of 0.8
mL min-1 with a mobile phase composed of 50% ACN/50% H2O-formic acid 0.1% for all
compounds. Permeability (Papp) for PAMPA was calculated according to the following equation,
obtained from Wohnsland and Faller and Sugano et al. equation with some modifications in order to
obtain permeability values in cm s-1,
V
D
V
A
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(
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P
app
=
− ln
1− r
(
V
D
+V At
A
)
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where VA is the volume in the acceptor well, VD is the volume in the donor well (cm3), A is the
“effective area” of the membrane (cm2), t is the incubation time (s) and r the ratio between drug
concentration in the acceptor and equilibrium concentration of the drug in the total volume (VD+VA).
Drug concentration is estimated by using the peak area integration. Membrane retentions (%) were
calculated according to the following equation:
[r − (D + A)]100
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%MR =
Eq
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where r is the ratio between drug concentration in the acceptor and equilibrium concentration,
while D, A, and Eq represent drug concentration in the donor, acceptor and equilibrium solution,
respectively.
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Water Solubility Assay. Each solid compound (1 mg) was added to 1 mL of water. The samples
were shaked in a shaker bath at room temperature for 24-36 h. The suspensions were filtered
through a 0.45-μm nylon filter (Acrodisc), and the solubilized compound determined by LC-MS-
MS assay. For each compounds the determination was performed in triplicate. For the
quantification was used an LC-MS system consisted of a Varian apparatus (Varian Inc) including a
vacuum solvent degassing unit, two pumps (212-LC), a Triple Quadrupole MSD (Mod. 320-LC)
mass spectrometer with ES interface and Varian MS Workstation System Control Vers. 6.9
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software. Chromatographic separation was obtained using a Pursuit C18 column (50 x 2.0 mm)
(Varian) with 3 μm particle size and gradient elution: eluent A being ACN and eluent B consisting
of an aqueous solution of formic acid (0.1%). The analysis started with 0% of eluent A, which was
linearly increased up to 70% in 10 min, then slowly increased up to 98% up to 15 min. The flow
rate was 0.3 ml/min and injection volume was 5 μL. The instrument operated in negative mode and
parameters were: detector 1850 V, drying gas pressure 25.0 psi, desolvation temperature 300.0 °C,
nebulizing gas 45.0 psi, needle 5000 V and shield 600 V. Air and nitrogen were used as nebulizer
and drying gas, respectively. Collision induced dissociation was performed using Argon as the
collision gas at a pressure of 1.8 mTorr in the collision cell. The transitions as well as the capillary
voltage and the collision energy used for compounds 16h and 16l are summarized in Table 1.
Quantification of the single compound was made by comparison with apposite calibration curves
realized with standard solutions in methanol.
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Microsomal Stability Assay. Each compound in DMSO solution was incubated at 37 °C for 60
min in 125 mM phosphate buffer (pH 7.4), 5 μL of human liver microsomal protein (0.2 mg mL-1),
in the presence of a NADPH-generating system at a final volume of 0.5 mL (compounds’ final
concentration, 50 μM); DMSO did not exceed 2% (final solution). The reaction was stopped by
cooling in ice and adding 1.0 mL of acetonitrile. The reaction mixtures were then centrifuged, and
the parent drug and metabolites were subsequently determined by LC-UV-MS. Chromatographic
analysis were performed with an Agilent 1100 LC/MSD VL system (G1946C) (Agilent
Technologies, Palo Alto, CA) constituted by a vacuum solvent degassing unit, a binary high-
pressure gradient pump, an 1100 series UV detector, and an 1100 MSD model VL benchtop mass
spectrometer. Chromatographic separation was obtained using a Varian Polaris C18-A column (150
- 4.6 mm, 5 μm particle size) and gradient elution: eluent A being ACN and eluent B consisting of
an aqueous solution of formic acid (0.1%). The analysis started with 2% of eluent A, which was
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rapidly increased up to 70% in 12 min, then slowly increased up to 98% in 20 min. The flow rate
was 0.8 mL min-1 and injection volume was 20 μL. The Agilent 1100 series mass spectra detection
(MSD) single-quadrupole instrument was equipped with the orthogonal spray API-ES (Agilent
Technologies, Palo Alto, CA). Nitrogen was used as nebulizing and drying gas. The pressure of the
nebulizing gas, the flow of the drying gas, the capillary voltage, the fragmentor voltage, and the
vaporization temperature were set at 40 psi, 9 L min-1, 3000 V, 70 V, and 350°C, respectively. UV
detection was monitored at 280 nm. The LC-ESI-MS determination was performed by operating the
MSD in the negative ion mode. Spectra were acquired over the scan range m/z 100-1500 using a
step size of 0.1 u. The percentage of not metabolized compound was calculated by comparison with
reference solutions.
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RESULTS
Inhibition of HIV-1 in cell culture
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All the synthesized compounds were tested in vitro to evaluate their ability to inhibit HIV
replication in human TZM-bl cells infected with HIV-1 NL4.3 (CXCR4-tropic strain) or AD8
(CCR5-tropic strain) and the biological results are listed in Table 2 together with toxicity data.
Remarkably, all compounds showed low micromolar/submicromolar activities, with the only
exception of compound 16m that was inactive. Being 16m the only compound in this series that not
contain the salicylic moiety, we can argue that such group is fundamental for the anti-HIV activity
of the 2-aminothiazolone derivatives under study. Furthermore, a substituent-dependent effect was
observed: the best results were obtained when phenylethylamines were introduced on the thiazolone
ring (16h-l), while the substitution with shorter chain such as benzylamines (16a-e) or
phenylamines (16f-g) determined a small reduction of the inhibitory potency against HIV-1.
When compounds 16h and 16l were tested against Tier2 and Tier3 pseudotyped viruses (all subtype
B strains), both compounds showed a similar but not fully overlapping spectrum of neutralization
(Table 3). A VSVG control pseudovirus was also used in parallel, but none of the compound
showed any inhibitory effect. Reference compound maraviroc showed a 19 ±5 nM IC50 with all R5-
strains (data not shown). Noteworthy, no toxicity was observed for the tested compounds (LD50
50µM). Further modifications of the most active compounds 16h-l are in progress aiming at
improving these results.
>
332
Mechanism-of-action studies: time of intervention&gp120/CD4 binding assay
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335
A time-of-addition experiment was carried out to exactly determine the target of inhibition of the 2-
aminothiazolones. This experiment determines how long the addition of an anti-HIV compound can
be postponed within the viral replication cycle before losing its antiviral activity. Reference
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compounds with a known mode of action were included. IgGb12 is an anti-gp120 antibody.
Maraviroc is a CCR5-receptor antagonist. T20 is a fusion inhibitor that acts by binding to the
envelope glycoprotein gp41. The nucleoside analogue AZT inhibits the reverse transcription
process while Dolutegravir is an inhibitor of integration process. The same profile of IgGb12 was
seen with 16l (Figure 2). These data confirmed gp120 as a target of interaction of 16l.
341
342
343
The gp120/CD4 binding assay, demonstrated a direct and dose-dependent inhibition by compounds
16h and 16l (IC50 =20 µM for both) of the interaction between a dual tropic gp120 that was used in
thee assay and soluble CD4 (Figure 3).
344
Molecular Modeling
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351
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353
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Compounds 16a-m were subjected to docking analysis into a refined gp120 core structure (pdb
code 1G9M) 26, by focusing calculations on the Phe43 cavity and its surrounding residues. Analysis
of docked complexes suggested that 2-aminothiazolone derivatives made some of the interactions
previously identified as crucial for the activity of gp120-CD4 small molecule inhibitors. In detail,
the aromatic side chains of the amines at the 2 position were deeply embedded at the bottom of the
cavity where established hydrophobic interactions with residues Phe382, Met475, Val255, Trp427,
Trp112, Ile424 and Tyr384 while an hydrogen bond was present between the NH and the backbone
carbonyl oxygen of Gly473. The thiazolone ring interacts with Ile371, Gly473 and Glu370 by
filling the region occupied by the CD4 residue Phe43 in the gp120-CD4 complex. Finally, the
salicylic ring was involved in polar contacts with arginine residue at position 476 of gp120 in a
solvent-exposed area. As an example, Figure 4A shows the predicted binding mode of compound
16h. The alignment between the pose predicted for compound 16h and the binding mode of the
NBD-based derivative DMJ-II-121 within the Phe43 cavity (PDB code: 4I54) highlights common
interactions between the two inhibitors such as the hydrogen bond with Gly473 and the
hydrophobic contacts with Trp112, Phe382, Tyr384 and Ile424. Vice versa, the interaction with
Arg476 is exclusively observed for compound 16h (Figure 4B). Noteworthy, compounds 16f and
16

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16g were found active against HIV-1 NL4.3 but not on AD8 infected cells. In order to investigate
this peculiar behavior, two homology models were built using the primary sequence of both NL4.3
and AD8 strains. A first analysis of the two 3D structures showed no differences into the Phe43
binding site. HIV-1 NL4.3 and AD8 strains differ in their ability to utilize either CCR5 or CXCR4
as coreceptor, and this specificity, is largely determined by the sequence of the V3 loop of the viral
envelope protein gp120(33). Accordingly, most of the mutations found in primary sequence are
located in the V3 and V4 loops, more than 5Å far from the Phe43 binding site residues into the 3D
structures. To evaluate the effects of distal amino acids mutations on the binding profile of 16f,
compound was docked into both the homology models and the two complexes were submitted to 15
ns Molecular Dynamic (MD) simulations. In agreement with biological results, compound 16f was
found more stable in complex with NL4.3 than AD8 and this could explain its different activity
profile against the two strains. Figure 5 shows the root mean square deviation calculated on ligand
heavy atoms during the time of simulation with respect to the starting pose in complexes AD8
(blue) and NL4.3 (red).
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382
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In vitro ADME
We initiated early preclinical in vitro ADME (adsorption, distribution, metabolism, excretion)
studies with compounds 16h and 16l to determine their aqueous solubility, parallel artificial
membrane permeability (PAMPA), and human liver microsomes (HLM) stability in order to early
assess the absorption/stability of these drug candidates (Table 2). Passive membrane permeability
was evaluated with the PAMPA assay while compound solubility was evaluated following the
method developed by Avdeef et al.(34), and results were expressed as LogS (log mol L-1).
Metabolic stability was finally evaluated by incubating the above-mentioned compounds with 5 μL
of man-pooled HLM for 1 h at 37 °C in order to simulate phase I metabolism. Compounds 16h and
16l showed low values of membrane permeation (permeability classification: high >20 x 10-6 cm/s,
17

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medium 10-20 x 10-6 cm/s, low < 10 x 10-6 cm/s). However, it has been previously reported that
salicylic acid derivatives could pass through cellular membranes by active transport, thus, further
experiments in this direction are necessary to better characterize the permeability of these
compounds (35). Furthermore, the ADME investigation highlighted good metabolic stability
(>99%) and low aqueous solubility (lower than -8 when the desired LogS for a drug candidate
should be between -4 and -6).
393
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395
18

396
Discussion
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404
405
406
407
408
Compared to other steps in the replication of HIV-1 that have been successfully inhibited by many
available antiretroviral agents, the entry process remains an elusive but extremely interesting target
for both therapeutic and preventive approaches against HIV-1 infection (36). Moreover, clinically
available entry inhibitors have still several limitations in term of neutralization spectra or
pharmacokinetic (PK) properties. In fact, maraviroc (MVC), which targets the interaction of gp120
with the CCR5 coreceptor (37) can only inhibit CCR5-using HIV strains and tropism prediction by
genotypic or phenotypic determination is currently mandatory to start MVC treatment (38);
enfuvirtide (ENF or T20) that prevents gp41-mediated fusion of the viral and host cell membranes
is an injectable peptide associated with still completely unsolved side-effects mainly due to its mode
of administration and antigenicity (39, 40). Only two small molecule compounds and their
derivatives have recently entered the preclinical and clinical development process (BMS-626529
and NBD-556) and show promising antiretroviral activity (8-12).
409
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411
412
413
414
415
416
In this paper, we showed that a novel class of small molecule compounds can inhibit the earliest
CD4-gp120 protein–protein interactions at the host cell viral interface without any cell toxicity in
vitro (Table 2). Moreover, we showed that these compounds were able to inhibit CXCR4 and/or
CCR5-using laboratory strains and that at least two of these compounds were active against some
Tier2 and Tier2 pseudotyped viruses suggesting a wider spectrum of neutralization (27). The tested
viruses are very different and have distinct coreceptor usage, however, neutralization assays with
more TIER 1 and TIER2 pseudovirus panels, with envelopes coming from non-B subtypes, or
antiviral assays on clinical isolates will be needed in order to further extend this observation.
417
418
419
420
We used the time of addition assay to indirectly demonstrate that the very early entry step is
inhibited by these novel molecules and indeed the addition of our representative compound could
not be postponed to virus seeding before losing completely its antiviral activity in cell culture.
These results were in agreement with the 120/CD4-His competition assay that directly demonstrated
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the interaction and inhibition of CD4 binding to gp120. These results suggest that these novel
compounds may be used either sequentially or concurrently with currently available HIV-1 entry
inhibitors. Although it is theoretically possible that resistance to these novel compounds may also
occur through selection of CD4-independent viruses, these variants are rarely selected in vivo and
have an increased sensitivity to host immune control compared to CD4-dependent viruses (41) and
may thus not be a clinically relevant hurdle to their usage. Thus, the need for novel small molecules
with broad neutralization profiles, with reduced toxicity and with favorable PK profiles and are still
needed to improve current life-long antiretroviral regimens. In fact, novel combinations of
antiretroviral drugs with distinct mechanism of action and with non-overlapping resistance profiles
will offer novel strategies to treat or prevent HIV-1 infection.
Next steps for the continuance of drug development of these compounds will be oriented to improve
their aqueous solubility. A series of chemical modifications will be performed to introduce polar
groups in the part of the molecule exposed to the solvent as well as to increase the binding affinity
towards the receptor in agreement with molecular modeling predictions. Furthermore, other
strategies to overcome solubility issue could be pursued such as the development of prodrugs or
liposome encapsulation (42).
Acknowledgements
This work was supported by the European Union collaborative project “CHAARM” (grant no.
HEALTH-F3-2009-242135) and by the Italian Ministero dell’Istruzione, dell’Università e della
Ricerca, Prin 2010 research project (grant no. 2010W2KM5L).
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594
26

Collision
595
596
597
598
599
600
601
602
Compounds Transition (m/z) Capillary voltage (V)
tR (min)
Energy (eV)
214.6
433.2
23.5
21.0
16h
-100
12.1
214.6
417.2
24.5
20.5
16l
-40
12.8
603
604
Table 1. Chromatographic and MS parameters (monitored transition, capillary voltage, collision
energy and retention time tR) of the selected compounds.
605
27

606
Table 2. Structure, anti-HIV activity and ADME properties of compounds 16a-m.
F
NHR
HN
S
N
S
N
O
HO
HO
O
O
O
16 a-l
16m
O
NO2
PAMPA a
Water Solub.
Papp × 10-6 (cm/s)
Metab.
Stability d
(%)
AD8
IC50 (µM)
NL4.3
IC50 (µM)
TZM-bl
LD50 (µM)
(LogS) c
(MR%) b
Cpds
16a
R
1.0±0.2
1.6±0.5
3.0±0.4
3.1±0.5
2.3±0.4
1.8±0.4
>50
>50
>50
>50
>50
F
F
16b
16c
16d
16e
1.3±0.3
4.3±0.3
3.0±0.6
2±0.4
Cl
F
Cl
Cl
Cl
Cl
16f
>10
>10
1±0.8
>50
>50
F
F
16g
1.8±0.4
Cl
0.96
(0.0)
-8.8
-8.2
99.9
99.9
16h
16i
0.6±0.3
1.5±0.3
0.85±0.2
>10
0.75±0.3
0.8±0.1
1±0.3
>50
>50
>50
>50
F
0.28
(0.0)
16l
16m
>10
F
607
608
a PAMPA see experimental section for details; b Membrane Retention; c LogS= log mol L-1; d Expressed as percentage of
609
unmodified parent. Values are expressed as mean +/- SD. Experiments were repeated three times in triplicate.
28

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611
Table 3. Activity of compounds 16h and 16l on laboratory strains (NL4-3 and AD8) and on Tier 2
and 3 pseudoviruses. IC50(μM±SD) of Dolutegravir on laboratory strains was 4nM +/-3; IC50 of
maraviroc on pseudoviruses was 19 ±5 nM IC50 . Experiments were repeated three times in
triplicate.
612
613
614
Pseudo viruses IC50 (µM)
Cpds
0.6±0.3
0.8±0.2
0.75±0.3
1±0.3
2.9±0.3
4.1±1.3
1.5±0.2
4.1±1.3
1.5±0.4
1.3±0.3
7.7±1.3
8.7±1.4
1±0.3
1±0.4
>10
>10
1.5±0.4
1.5±0.5
3.9±0.2
3.3±0.2
3.9±0.6
5±0.1
5.6±0.3
5.6±1.3
16h
16l
615
Values are expressed as mean ± SD.
616
29

617
618
619
620
621
622
623
624
625
626
627
628
629
Figure Legend
Figure 1. A) Synthesis of aminothiazolones 16 a-l: i) Pd(PPh3)2Cl2, Na2CO3, DMF/EtOH, RT,
1h; ii) 1N NaOH, MeOH/THF, reflux, overnight; iii) EtOH, 150 °C, MW, 20 min. B) Synthesis of
aminothiazolone 16m i) Pd(PPh3)2Cl2, Na2CO3, DMF/EtOH, RT, 1h; ii) EtOH, 150 °C, MW, 20
min.
Figure 2. The time-of-addition assay. The target of the antiviral compound 16l was identified by
comparing its activity in the time scale to that of reference drugs. In the assay a panel of reference
drugs sequentially targeting distinct replication steps of HIV-1 from entry to the integration into cell
chromosome were used: IgGb12 (an anti-gp120 antibody that binds the CD4-binding site),
maraviroc (a CCR5 coreceptor inhibitor), enfuvirtide (T20, a fusion inhibitor), azidothymidine
(AZT, an RT inhibitor) and dolutegravir (DTG, as integrase inhibitor). For compound 16l, efficacy
was maximum only when the virus is pre-treated as for IgGb12 and is ineffective once the virus has
attached the CD4 cell surface receptor.
630
631
632
633
634
635
636
637
638
639
Figure 3. Gp120/CD4-His tag binding assay. Two representative 2-aminothiazolone derivatives
molecules, 16h and 16l, were tested in a competitive ELISA to demonstrate that gp120 binding to
CD4 was the target of this class of compounds. Both compounds were able to displace soluble CD4
from a dual tropic gp120 in a dose-dependent manner (IC50 = 20 µM for both compounds). DMSO
at the same concentrations present at each compound dilution was also tested. Experiments were
repeated twice in triplicate.
Figure 4. A) Binding mode of compound 16h (blue) as predicted by computational studies. B)
Phe43 cavity occupied at the same time by 16h (blue) and DMJ-II-121 (green, PDB code: 4I54).
Figure. 5. Time evolution of root mean square deviation (RMSD, Å) calculated on heavy atoms of
ligand 16f in AD8 (blue line) and NL43 (red line).
640
30