Optimization of rhodanine scaffold for the development of protein-protein interaction inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.04.072

Abstract Searching for novel protein-protein interactions inhibitors (PPIs) herein we describe the identification of a new series of rhodanine derivatives. The selection was performed by means virtual-screening, docking studies, Molecular Dynamic (MD) sim

Full text of DOI:10.1016/j.bmc.2015.04.072

Accepted Manuscript
Optimization of rhodanine scaffold for the development of protein-protein in-
teraction inhibitors
Stefania Ferro, Laura De Luca, Fatima Ezzahra Agharbaoui, Frauke Christ,
Zeger Debyser, Rosaria Gitto
PII:
S0968-0896(15)00380-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.072
BMC 12284
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 February 2015
23 April 2015
24 April 2015
Please cite this article as: Ferro, S., De Luca, L., Agharbaoui, F.E., Christ, F., Debyser, Z., Gitto, R., Optimization
of rhodanine scaffold for the development of protein-protein interaction inhibitors, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.072
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
Optimization of rhodanine scaffold for the development of
protein-protein interaction inhibitors.
Leave this area blank for abstract info.
Stefania Ferroa, Laura De Lucaa, Fatima E. Agharbaouia, Frauke Christb, Zeger Debyserb and Rosaria Gittoa
aDipartimento di Scienze del Farmaco e Prodotti per la Salute, Università di Messina, Viale Annunziata, 98168 Messina,
Italy, bMolecular Medicine, Katholieke Universiteit Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven,
Flanders, Belgium

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Optimization of rhodanine scaffold for the development of protein-protein interaction
inhibitors.
Stefania Ferroa,∗, Laura De Lucaa, Fatima Ezzahra Agharbaouia, Frauke Christb , Zeger Debyserb and
Rosaria Gittoa
aDipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Viale Annunziata I-98168 Messina, Italy
bMolecular Medicine, Katholieke Universiteit Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
Article history:
Received
Received in revised form
Accepted
Searching for novel protein-protein interactions inhibitors (PPIs) herein we describe the
identification of a new series of rhodanine derivatives. The selection was performed by means
virtual-screening, docking studies, Molecular Dynamic (MD) simulations and synthetic
approaches. All the new obtained compounds were tested in order to evaluate their ability to
inhibit the interaction between the HIV-1 integrase (IN) enzyme and the nuclear protein lens
epithelium growth factor LEDGF/p75.
Available online
Keywords:
Docking studies
Synthesis
2009 Elsevier Ltd. All rights reserved.
Protein-Protein Interactions
Antiviral activity
1. Introduction
Particularly, the disruption of association between HIV-1 IN
and the ubiquitous lens epithelium growth factor p75 (LEDGF/
Since the first description of the acquired immune deficiency
syndrome (AIDS), worldwide more than 25 million people have
fallen victim to HIV-1 infections. For the treatment of this
disease the Food and Drug Administration (FDA) has approved
several drugs, often used in combination regimens in the well-
known cART (combinatorial AntiRetroviral Therapy). They
belong to different categories: reverse transcriptase inhibitors
(RTIs), protease inhibitors (PIs); fusion inhibitors (FIs),
coreceptor inhibitors (CRIs) and integrase inhibitors (INIs).1-4
p75) is currently the most promising target for the design of
protein-protein inhibitors (PPIs) as potential anti-HIV drugs.10-16
LEDGF/p75, a transcriptional co-activator, is a co-factor of HIV-
integrase (IN) and is required for the tethering and correct
integration of the viral genome into the host chromatin. Several
studies highlighted that cells lacking of LEDGF/p75 showed a
severe defect in HIV-1 infection characterized by decreased
17-19
levels of integrated viral DNA.9,
Taking into account the
pivotal role of the IN-LEDGF/p75 interaction for the HIV-1
infection, in the past years we focused on this attractive and
innovative antiviral target. Our studies led us to the identification
of several small molecule protein-protein interaction inhibitors
(SMPPIIs) able to block the binding between the IN enzyme and
its cofactor.8, 14, 20-26
Although this current therapy decreases viral load and
provides a significant improvement in the life expectancy of
HIV-1/AIDS patients, the incidence of infection remains one of
the major problems in public health. Moreover, the long-term
treatment anti-AIDS drugs induces drug-resistant viral variants
and emergence of unwanted side effects.5-8 Therefore, the quest
of new more potent and less toxic anti-HIV agents, as a
complement for the existing treatment strategies, remains one of
the major goals in HIV drug discovery.
Recently, considering that natural products (NP) represent the
most productive source of leads for new drugs, we reported the
application of a structure-based virtual screening strategy for the
discovery of hit structures of natural origin useful in the
antiretroviral therapy. Nine compounds have been selected and
tested to evaluate their ability to prevent IN-LEDGF interaction.
Currently, small-molecule inhibitors of the direct protein-
protein interactions (PPIs), that play auxiliary roles in the HIV-1
life cycle mediating important biological processes, are an
emerging and interesting area in anti HIV-1 drug design.8, 9
Among them,
a
rhodanine derivative, the 2-(5-(4-
methoxybenzylidene)-2,4-dithioxothiazolidin-3-yl)-3-
methylbutanoic acid (A) (Figure 1) showed interesting efficacy,
displaying an IC50 value of 41.28 µM in AlphaScreen assay.25
———
∗ Corresponding author. Tel.: +39-090-676-6453; fax: +39-090-676-6402; e-mail: sferro@unime.it

The p-methoxyphenyl group is accommodated into the
binding pocket forming a panel of hydrophobic interactions with
the residues Ala128, Ala129, and Trp132, and Met178 of IN;
whereas the carboxylate group forms stable hydrogen bonds with
the crucial residues (occupancies of 96.00% with Glu170 NH
backbone; 91.60% with His171 NH backbone; 78.70% with
Thr174 side chain OH). Interactions between IN CCD and
compound A were examined using PyMOL and LIGPLUS.30
OH
O
O
N
S
S
We observed that the shape of the pocket occupied by the p-
methoxyphenyl group could house larger and lipophilic groups
and that there was absence of contact with the crucial Gln168
residue placed near the phenyl group. Furthermore it was not
clear the role of isopropyl group that seems not able to create
important contacts.
MeO
Figure
1:
2-(5-(4-Methoxybenzylidene)-2,4-dithioxothiazolidin-3-yl)-3-
methylbutanoic acid (A)
The analysis of these data prompted us to reconsider the class
of compound A keeping unchanged the rhodanine nucleus and
modifying, in this phase of our research, the other two portions of
the molecule that we named right hand side (3-methylbutanoic
acid moiety) and left hand side (4-methoxybenzylidenic moiety)
respectively (Figure 3).
As a continuation of these researches, herein we report
structural modifications and biological screening of new
rhodanine analogs designed and synthesized with the aim of
achieving further information about the IN-LEDGF/p75 protein
interaction. Further our studies are in progress to define the role
of the rhodanine scaffold in the antiviral activity also considering
that LJ001 rhodanine analogue is effective fighting multiple
deadly viruses including HIV-1 and Ebola. Moreover, in vitro
and in vivo assays showed that LJ001 acts on the virus and not on
the cells thus lacking of overt cytotoxicity.27, 28
The decision to change a portion at a time is determined by
the need to identify the essential pharmacophore of derivatives
establishing the structural features that are important for the
biological activity.
2. Results and discussion
With the aim to obtain useful insights for the development of
new small-molecules as inhibitors of the IN-LEDGF/p75
interaction (LEDGINs), we previously investigated the most
interactions between some PPI inhibitors and IN-LEDGF/p75
complex. In particular, by means of a combination of docking
and ultrashort MD, we generated a weighted ensemble of protein-
ligand configurations and estimated the binding affinity averaged
over snapshots taken from the MD trajectories, together with the
presence of fundamental hydrogen bonds.26 Using this
information in a structure-based virtual screening strategy we
identified the active compound A. The plausible binding mode of
this derivative is showed in Figure 2.
Figure 3: Structural portions of derivative A
2.1. Binding site analysis: left hand side modification
In order to identify some representative alternative groups,
ZINC database has been browsed to search for molecules that
varied only the left hand side portion. The subset of molecules
selected from ZINC consisted of 674 compounds. This subset
was filtered by Lipinki's rule, redundant structures were removed,
then appropriate ionization states and 3D structures were
generated. The obtained subset consisted of 64 molecules.
These compounds were docked into the LEDGF/p75 binding
site on HIV-1 integrase considering the protein retrieved by
complex 2B4J that was used for reference compound A (see
Experimental Section).25 After visual examination of the highest
scoring molecules, 13 protein-ligand complexes were retrieved,
minimized and rescored using MM-GBSA method. Based on
these results, ultrashort MD simulations (100 ps) were performed
obtaining the best results for the eight compounds (1-8) showed
in Figure 4.
Figure 2: Binding mode of reference compound A in complex with IN
CCD. Key residues of the pocket are presented. Hydrogen bonds are showed
by dotted lines as well as their occupancies during MD simulations in
percent. The figure was created using PyMOL.29

OH
OH
2.2. Binding site analysis: right hand side modification
O
OH
O
O
O
O
O
Starting from the reference compound A, several
modifications on the right hand side of the molecule were also
taken into account.
N
N
N
S
S
S
S
S
S
N
Considering the space of the pocket in which the isopropyl
group of reference compound A is located (Figure 2) and the
commercial availability and price of some amino acids we
selected 5 of them. The substitution of isopropyl moiety with
these groups generated compounds 11-15 that were submitted to
the same computational protocol to predict substitutions
influence on the inhibitory activity.
HO
3
2
1
MeO
OH
O
OH
OH
O
O
O
O
O
N
N
N
S
S
S
S
S
S
Based on computational results the five compounds were
selected and synthesized. (Figure 5)
CH3
O
5
O
O
CH3
O
HO
HO
CH3
6
H3C
4
O
OH
OH
O
OH
OH
O
OH
O
O
O
O
CH3
O
O
O
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
CH3
8
O
O
O
O
O
CH3
O
H3C
H3C
CH3
11
O
CH3
12
13
H3C
7
OH
H3C
OH
O
O
CH3
O
Figure 4: Designed left hand side modifications
N
O
N
S
To investigate the impact on the inhibitory effect of the left
hand side modifications derivatives 1-4 were initially purchased.
Successively, after we developed a suitable chemical pathway,
compounds 5-8 were synthesized to build a small SAR library for
SAR studies. The synthetic approach employed for analogues 5-8
is depicted in Scheme 1.
S
S
S
14
O
15
H3C
O
H3C
Figure 5: Designed right hand side modifications
The new designed compounds were obtained following the
synthetic procedure described in Scheme 2.
Scheme 1
O
CH3
O
CH3
Scheme 2
CH3
O
HO
O
HO
O
CH3
O
a,b,c
CH3
CH3
N
HO
d
O
N
S
HO
O
O
R
NH2
S
HO
O
S
R
R
S
9
5-8
N
10
a,b,c
d
HO
R
R'
S
N
NH2
16-20
S
5
6
7
8
OCH3
OCH3
OCH2CH3
OH
S
S
11-15
21-25
OCH2CH3 OCH3
OCH3 OCH3
R'
R
R
16-21-11 CH2COOH
17-22-12 CH2CH2COOH
18-23-13 CH(CH3)COOH
O
Reagents and conditions: a) NaOH, CS2, 23°C, overnight; b)
ClCH2CO2Na, 23 °C, 3h; c) HCl, reflux, 12h, d) suitable benzaldehyde,
NH4OAc, toluene, reflux, 1-2h.
19-24-14 CH(CH3)CH2COOH
20-25-15 CH(COOH)CH2CH3
CH3
Reagents and conditions: a) NaOH, CS2, MW: 5 min at continuous
temperature (100°C), 100 Watt; b) ClCH2CO2Na, MW: 5 min at continuous
temperature (100°C), 100 Watt; c) HCl, MW: 30 min at continuous
temperature (120°C), 100 Watt; d) p-methoxy benzaldehyde, NH4OAc,
toluene, reflux, 1-2h.
At first valine (9) was cyclized with CS2 and α-chloroacetate
to give the rhodanine nucleus (10). In the second step, following
Knoevenagel condensation of rhodanine with suitable substituted
benzaldehyde, the final desired analogues 5-8 were obtained. As
expected the condensation generated two isomers (E and Z); the
major product was the thermodynamically stable Z isomer as
characterized by the down-field shift of its methylene proton
when compared to that of the E isomer.
The commercially available amino acids 16-20 were cyclized
with CS2 and α-chloroacetate to form the substituted rhodanine
derivatives 21-25. In this step of the synthetic procedure
Microwave Assisted Organic Synthesis (MAOS) was employed
thus reducing reaction time (40min vs overnight) and usual
thermal reagents degradation.

Successively, intermediates 21-25 were transformed into the
final compounds 11-15 by reaction with p-methoxy
benzaldehyde. Also in this case the spectroscopic
characterization of the synthetized derivatives confirmed the
major thermodinamical stability of Z isomer.
2.3. Biological results
We tested all new compounds (1-8, 11-15 ) in AlphaScreen
assay to evaluate their inhibitory effects on the LEDGF/p75–IN
interaction. (Table 1)
Table 1. Inhibition of IN-LEDGF/p75 interaction of compounds 1-8 and
11-15
Compound
%
93
89
77
82
28
43
20
NA
36
66
47
29
54
88
IC50 (µM)
1
15.76 1.51
2
18.75 0.6
3
43.75 0.75
Figure 6: Binding mode of compound 1 in complex with IN CCD. Key
residues of the pocket are presented. Hydrogen bonds are showed by dotted
lines as well as their occupancies during MD simulations in percent. The
figure was created using PyMOL.29
4
24.99 0.8
5
-
-
-
6
Comparing the results of compound 1 with those obtained for
the prototype A (Figure 2), we highlighted that the overall
stability of hydrogen bonds is similar for both derivatives. The
methoxyquinoline moiety is accommodated into the hydrophobic
pocket formed by residues of, Ala128, Ala129, Trp131, Gln168,
Ala169. The carboxylate group is able to establish the same
contacts with residues of Glu170, His171 and Thr174 of chain A
of IN. Moreover, the sulphur atom of compound 1 forms a
hydrogen bond with residue Gln95 providing a better stability of
the complex thus suggesting a possible explanation to the higher
potency of this new derivative. Interactions between IN CCD and
compound 1 were examined using PyMOL and LIGPLUS.30
7
8
11
12
13
14
15
A
-
>100
-
-
>100
41.28
Concerning the right hand side modifications performed on
compound A we can observe generally a decrease of the activity,
for these compounds (11-15), when compared with left hand side
modified derivatives. This account suggest that the 3-
methylbutanoic acid portion, typical of the 2-(5-(4-
methoxybenzylidene)-2,4-dithioxothiazolidin-3-yl)-3-
methylbutanoic acid (A), is the most favorable moiety for the
contact with IN.
% inhibition at 100 µM
IC50: Concentration required to inhibit the HIV-1 IN-LEDGF/p75
interaction by 50%.
NA= Not Active
The biological results showed that all the new obtained
derivatives, with the exception of compound 8, exhibited
inhibitory effects at the fixed dose of 100 µM concentration with
a percentage ranging from 20% to 93%. Particularly, concerning
this new series of IN-LEDGF/p75 SMPPIIs we observed that the
best results were displayed for rhodanine-derivatives 1-4 in
which left hand side modifications were performed. Among them
compounds 1,2 and 4 present an improvement of the activity in
comparison with the reference compound A and are more active
than the derivatives characterized by different substitutions on
the the right hand side of the parent compound A.
Derivatives 1-8 and 11-15 were also tested in MT-4/MTT-
assay as anti-HIV agents and among them derivative 12 showed
antiviral activity (EC50=2.41µM) and
a very interesting
selectivity index (SI=61). According these results, it is reasonable
to think to another mechanism of action for this compound.
Further studies are in progress.
3. Conclusions
With the aim of improving the IN-LEDGF inhibition activity
of reference compound A, we identified and synthesized a new
series of small molecules, belonging to the family of rhodanine
derivatives. The selection was performed by virtual-screening,
docking studies and MD simulations.
In order to explain this enhancement of activity the binding
mode of compound 1, one of the most active rhodanine analog,
we show the computational results in Figure 6.

The biological results of the selected compounds showed for
some of them inhibitory effects at micromolar concentration.
Particularly, the best activity was obtained for derivatives in
which left hand side modifications of scaffold were performed,
thus affording novel SAR information that can be useful for
future studies
4.3. Chemicals
All commercially available reagents and solvents were used
without any further purification. The microwave-assisted
reactions were carried out in a CEM Focused Microwave
Synthesis System, Model Discover, working at the power
necessary for refluxing under atmospheric conditions. Melting
points were determined on a BUCHI Melting Point B-545
apparatus and are uncorrected. Elemental analyses (C, H, N)
were carried out on a Carlo Erba Model 1106 Elemental
Analyzer and the results are within 0.4% of the theoretical
values. Merck silica gel 60 F254 plates were used for analytical
TLC; column chromatography was performed on Merck silica
gel 60 (230-400 mesh) and Flash Chromatography (FC) on a
Biotage SP1 EXP. 1H-NMR spectra were recorded in CDCl3 with
TMS as internal standard or [D6]DMSO on a Varian Gemini-300
spectrometer. Chemical shifts were expressed in δ (ppm) and
coupling constants (J) in hertz (Hz). All the exchangeable
protons were confirmed by addition of D2O.
4. Experimental section
4.1. Docking studies
Docking studies were performed using the genetic
optimization for ligand docking (GOLD) software package
version 4.1.1 from the Cambridge Crystallographic Data Centre
(CCDC).31For our docking simulations, we used the crystal
structure of the dimeric CCD of HIV-1 IN complexed with the
IBD of LEDGF/p75 available from the RCSB Protein Data Bank
under the PDB ID: 2B4J.32 The LEDGF/p75 structure was
removed and hydrogen atoms were added to the IN protein in
Discovery Studio 2.5.5.33
4.4. Synthesis of 3-methyl-2-(4-oxo-2-thioxotetrahydrothiophene-
3-yl)butanoic acid (10)
For the prediction of ligand binding positions GoldScore
fitness function was used. Per each ligand 100 independent runs
and a maximum of 15000 genetic operations were performed
using the default operator weights and a population size of 100
chromosomes. Results differing by less than 1.00 Å in ligand-all
atom RMSD were clustered together. A 20.0 Å radius active site
was drawn on the original position of the LEDGF/p75 IBD
dipeptide Ile365-Asp366 and automated cavity detection was
used. Two hydrogen bond constraints were used to specify that
two protein atoms should be hydrogen-bonded to the ligand,
namely NH backbone of Glu170 and His171 with a constraint
weight of 5.
L-valine (9) (1 mmol, 117,14 mg) was dissolved with sodium
hydroxide (2 mmol, 80 mg) in water (10 ml). Then, carbon
disulfide (1 mmol, 60µl) was added to the reaction mixture and it
was stirred overnight. After the addition of an aqueous solution
(1M) of ClCH2CO2Na (1ml) the stirring was continued at room
temperature for 3h. Successively hydrochloric acid solution (3ml,
5.5N, 16.5mmol) was added ad the reaction mixture was refluxed
overnight. The mixture was neutralized with a saturated NaHCO3
aqueous solution, extracted with ethyl acetate (10 mL x 3) and
dried over Na2SO4. After removal of the solvent under reduced
pressure, the crude mixture was purified by flash chromatography
using a mixture of CH2Cl2/CH3OH (90:10) as eluent. Yield: 68%;
1
mp: 130-132°C. HNMR (CDCl3): δ = 0.82 (d, J=7.1, 3H, CH3),
1.25 (d, J=6.4, 3H, CH3), 2.75-2.90 (m, 1H, CH), 4.02 (s, 2H,
CH2), 5.27 (d, J=9.4, 1H, CH), 7.53 (bs, 1H, COOH). Anal. calcd
for C8H11NO3S2: C 41.19, H 4.75, N 6.00, found: C 41.32, H
4.88, N 5.91.
4.2. MM-GBSA rescoring and MD simulations
The starting model for simulations were prepared as described
in our previous paper.26 MD simulations were carried out using
34
the sander module of AMBER 11
and parm 99.dat and
frcmod.ff03 parameter files.35 General Amber force field (GAFF)
36 parameters were assigned to the designed ligands, while partial
charges were calculated using the AM1-BCC method as
implemented in the Antechamber suite of AMBER 11.
4.5. General procedure for the synthesis of 2-[(benzylidene)-4-
oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoic acids (5-8)
Suitable benzaldehyde (3 mmol) and ammonium acetate (3
mmol, 231,24 mg) were added to a solution of cyclized
rhodanine intermediated 10 (1 mmol, 233,31 mg) in toluene (20
ml). The reaction was refluxed overnight and a saturated
NaHCO3 solution was added. The mixture was extracted with
ethyl acetate (10 mL x 3), washed with BRINE (10 mL x 2), and
dried over Na2SO4. After removal of the solvent under reduced
pressure, the crude mixture was purified by flash chromatography
using a mixture of CH2Cl2/CH3OH (90:10) as eluent.
The geometry of the system was minimized in order to
remove any bad contact using the steepest descent algorithm for
the first 250 steps before switching to the conjugate gradient
algorithm for the remaining 250 steps.
Solvent effects were taken into account by using the
generalized Born implicit solvent model. The minimized
structure was the input for MD runs using constant-temperature
Langevin dynamics at 300 K for 100 ps with a time step of 1fs
and a distance cutoff of 12.0 Å for the nonbonded interactions.
2-[5-(4-ethoxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylbutanoic acid (5) Yield: 55%; mp: 182-
1
184°C. HNMR (DMSO-d6): δ = 0.67 (d, J=6.6, 3H, CH3), 1.21
Snapshots of the complexes during the simulations and the
average structures, were obtained with the Ptraj module of the
AMBER 11 suite. 34
(d, J=6.6, 3H, CH3), 1.32 (t, J=7.2, 3H, CH3), 2.65-2.85 (m, 1H,
CH), 3.81 (s, 3H, OCH3), 4.09 (q, J=7.2, 2H, CH2), 4.85-5.00
(m, 1H, CH), 7.09-7.21 (m, 3H, ArH), 7.68 (s, 1H, CH). Anal.
calcd for C18H21NO5S2: C 54.67, H 5.35, N 3.00, found: C 54.81,
H 5.12, N 3.21.
The hydrogen bonds were detected when the acceptor-donor
atom distance was lower than 3.5 Å and the acceptor-H-donor
angle was more than 120°. The MM-GBSA method
37
implemented in the AMBER program, was used to evaluate the
ligand-protein interaction free energies of both the minimized
complex and the 100 snapshots extracted at 1 ps intervals.
2-[5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylbutanoic acid (6) Yield: 61%; mp: 218-
220°C. HNMR (DMSO-d6): δ = 0.68 (d, J=6.4, 3H, CH3), 1.22
1
(d, J=6.5, 3H, CH3), 2.65-2.80 (m, 1H, CH), 3.81 (s, 3H, OCH3),

4.90-5.10 (m, 1H, CH), 6.92-7.16 (m, 3H, ArH), 7.65 (s, 1H,
CH). Anal. calcd for C16H17NO5S2: C 52.30, H 4.66, N 3.81,
found: C 52.18, H 4.78, N 3.95.
3-Ethyl-2-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)pentanedioic
1
acid (25) Yield: 61%; mp: 141-143°C. HNMR (DMSO-d6): δ =
0.79 (t, J=7.7, 3H, CH3), 2.02-2.14 (m, 2H, CH2), 4.35 (s, 2H,
CH2), 5.28-5.33 (m, 1H, CH). Anal. calcd for C7H9NO3S2: C
38.34, H 4.14, N 6.39, found: C 38.48, H 4.31, N 6.22.
2-[5-(3-ethoxy-4-methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylbutanoic acid (7) Yield: 68%; mp: 192-
1
4.7. General procedure for the synthesis of 2-[(5Z)-5-(4-
methoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]dioic
acids (11-15)
194°C. HNMR (DMSO-d6): δ = 0.67 (d, J=6.5, 3H, CH3), 1.24
(d, J=6.5, 3H, CH3), 1.34 (t, J=7.0, 3H, CH3), 2.68-2.82 (m, 1H,
CH), 3.83 (s, 3H, OCH3), 4.07 (q, J=7.0, 2H, CH2), 4.90-5.00 (m,
1H, CH), 7.12-7.23 (m, 3H, ArH), 7.66 (s, 1H, CH). Anal. calcd
for C18H21NO5S2: C 54.67, H 5.35, N 3.00, found: C 54.78, H
5.44, N 3.16.
The suitable cyclized rhodanine nucleus (1mmol) (21-25) was
reacted with p-methoxy benzaldehyde (3 mmol, 408,45 mg)
following the same synthetic approach employed to obtain
derivatives 5-8.
2-[5-(3,4-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylbutanoic acid (8) Yield: 59%; mp: 159-
161°C. 1HNMR (DMSO-d6): δ = 0.69 (d, J=7.00, 3H, CH3), 1.21
(d, J=6.4, 3H, CH3), 2.66-2.78 (m, 1H, CH), 3.82 (s, 3H, OCH3),
3.83 (s, 3H, OCH3), 4.95-5.09 (m, 1H, CH), 7.13-7.26 (m, 3H,
ArH), 7.73 (s, 1H, CH). Anal. calcd for C17H19NO5S2: C 53.53, H
5.02, N 3.67, found: C 53.44, H 4.86, N 3.72.
2-[(5Z)-5-(4-Methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]butanedioic acid (11) Yield: 64%; mp: 243-
1
245°C. HNMR (DMSO-d6): δ = 3.84 (s, 3H, OCH3), 2.02-2.14
(m, 2H, CH2), 4.72 (s, 2H, CH2), 7.12 (d, J= 9.1, 2H, ArH), 7.62
(d, J= 9.1, 2H, ArH) 7.85 (s, 1H, CH). Anal. calcd for
C13H11NO4S2: C 50.47, H 3.58, N 4.53, found: C 50.54, H 3.67,
N 4.89.
For compounds 1-4, purchased by means of
the
Chemical Sourcing Service of Sigma-Aldrich, all physical and
spectral data were in accordance with the literature.
2-[(5Z)-5-(4-Methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]pentanedioic acid (12) Yield: 64%; mp: 222-
224°C. HNMR (DMSO-d6): δ = 2.61 (t, J=7.9, 2H, CH2), 3.83
(s, 3H, OCH3), 4.21 (t, J=7.9, 2H, CH2), 7.12 (d, J= 9.0, 2H,
ArH), 7.61 (d, J= 9.0, 2H, ArH) 7.77 (s, 1H, CH). Anal. calcd for
C14H13NO4S2: C 52.00, H 4.05, N 4.33, found: C 51.84, H 4.18,
N 4.14.
1
4.6. General procedure for the synthesis of (4-oxo-2-thioxo-1,3-
thiazolidin-3-yl)dioic acids (21-25)
A suspension of suitable amino acid (16-20) (1 mmol) and
carbon disulfide (1 mmol, 60 µl) in a solution of NaOH (2mmol)
and water (10 ml) was reacted in a microwave reactor for 5 min
at 100°C. After automated cooling chloroacetic acid (1mmol)
was added and the mixture was reacted again at 100°C for 5 min.
After cooling HCl (3ml, 6N) was added and the reaction was
finished at 120 °C for 30 min. The mixture was neutralized with
a saturated NaHCO3 aqueous solution, extracted with ethyl
acetate (10 mL x 3) and dried over Na2SO4. After removal of the
solvent under reduced pressure, the crude mixture was purified
by flash chromatography using a mixture of CH2Cl2/CH3OH
(90:10) as euent.
2-[(5Z)-5-(4-Methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylpentanedioic acid (13) Yield: 59%;
1
mp: 130-132°C. HNMR (DMSO-d6): δ = 1.53 (d, J=7.0, 1H,
CH), 3.84 (s, 3H, OCH3), 5.60 (q, J=7.0, 3H, CH3), 7.12 (d, J=
8.5, 2H, ArH), 7.61 (d, J= 8.5, 2H, ArH) 7.79 (s, 1H, CH). Anal.
calcd for C14H13NO4S2: C 52.00, H 4.05, N 4.33, found: C 52.23,
H 4.21, N 4.19.
2-[(5Z)-5-(4-Methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]-3-methylpentanedioic acid (14) Yield: 65%;
1
mp: 176-178°C. HNMR (DMSO-d6): δ = 1.42 (d, J=7.0, 2H,
CH2), 2.90 (q, J=7.1, 3H, CH3), 3.83 (s, 3H, OCH3), 5.40-5.47
(m, 1H, CH), 7.12 (d, J= 8.8, 2H, ArH), 7.59 (d, J= 8.8, 2H, ArH)
7.70 (s, 1H, CH). Anal. calcd for C15H15NO4S2: C 53.40, H 4.48,
N 4.15, found: C 53.52, H 4.36, N 4.07.
2-(4-Oxo-2-thioxo-1,3-thiazolidin-3-yl)butanedioic acid (21)
Yield: 45%; mp: 148-150°C. HNMR (DMSO-d6): δ = 4.40 (s,
2H, CH2), 4.54 (s, 2H, CH2), 13.39 (bs, 1H, OH). Anal. calcd for
C5H5NO3S2: C 31.41, H 2.64, N 7.32, found: C 31.02, H 2.83, N
7.58.
1
3-Ethyl-2-[(5Z)-5-(4-methoxybenzylidene)-4-oxo-2-thioxo-1,3-
thiazolidin-3-yl]pentanedioic acid (15) Yield: 86%; mp: 164-
1
166°C. HNMR (DMSO-d6): δ = 0.81 (t, J=7.5, 3H, CH3), 2.13-
2-(4-Oxo-2-thioxo-1,3-thiazolidin-3-yl)pentanedioic acid (22)
1
2.23 (m, 2H, CH2), 3.83 (s, 3H, OCH3), 5.48 (t, J= 7.5, 1H, CH),
7.12 (d, J= 8.8, 2H, ArH), 7.62 (d, J= 8.8, 2H, ArH) 7.79 (s, 1H,
CH). Anal. calcd for C15H15NO4S2: C 53.40, H 4.48, N 4.15,
found: C 53.29, H 4.61, N 4.33.
Yield: 56%; mp: 152-154°C. HNMR (DMSO-d6): δ = 3.98-4.12
(m, 2H, CH2), 4.45 (d, J=7.0, 2H, CH2), 4.52 (s, 2H, CH2). Anal.
calcd for C6H7NO3S2: C 35.11, H 3.44, N 6.82, found: C 35.42, H
3.22, N 7.01.
2-Methyl-3-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)butanedioic
1
4.8. LEDGF/p75-HIV-1 Integrase interaction screening
acid (23) Yield: 67%; mp: 118-120°C. HNMR (DMSO-d6): δ =
1.43 (d, J=7.0, 3H, CH3), 4.30 (s, 2H, CH2), 5.42 (q, J=7.0, 1H,
CH). Anal. calcd for C6H7NO3S2: C 35.11, H 3.44, N 6.82, found:
C 35.34, H 3.12, N 6.68.
The AlphaScreen assay was performed as previously
describe.38 Reactions were performed in 25 µl final volume in
384-well Optiwell™ microtiter plates (Perkin–Elmer). The
reaction buffer contained 25 mM Tris–HCl (pH 7.4), 150 mM
NaCl, 1 mM MgCl2, 0.01% (v/v) Tween-20 and 0.1% (w/v)
bovine serum albumin. His6-tagged integrase (300 nM final
concentration) was incubated with the compounds at 4°C for 30
min. The compounds were added in varying concentrations from
1 up to 100 nM. Afterward 100 nM of recombinant flag-
LEDGF/p75 was added and incubation was extended by another
3-Methyl-2-(4-oxo-2-thioxo-1,3-thiazolidin-3-yl)pentanedioic
1
acid (24) Yield: 58%; mp: 125-127°C. HNMR (DMSO-d6): δ =
1.33 (d, J=7.0, 3H, CH3), 2.82-2.96 (m, 2H, CH2), 4.11 (s, 2H,
CH2), 5.25-5.31 (m, 1H, CH), 12.42 (bs, 1H, COOH). Anal. calcd
for C7H9NO3S2: C 38.34, H 4.14, N 6.39, found: C 38.52, H 4.27,
N 6.20.

15.
Gu, W. G.; Liu, B. N.; Yuan, J. F. Journal of drug
hour at 4°C. Subsequently, 5 µl of Ni-chelate-coated acceptor
beads and 5 µl of anti-flag donor beads were added to a final
concentration of 20 µg/ml of both beads. Proteins and beads were
incubated at 30°C for 1 h in order to allow association to occur.
Exposure of the reaction to direct light was prevented as much as
possible and the emission of light from the acceptor beads was
measured in the EnVision plate reader (Perkin–Elmer, Benelux)
and analyzed using the EnVision manager software.
targeting 2015, 23, 134.
16.
Desimmie, B. A.; Weydert, C.; Schrijvers, R.; Vets, S.;
Demeulemeester, J.; Proost, P.; Paron, I.; De Rijck, J.; Mast, J.;
Bannert, N.; Gijsbers, R.; Christ, F.; Debyser, Z. Retrovirology 2015,
12, 16.
17.
Busschots, K.; Voet, A.; De Maeyer, M.; Rain, J. C.;
Emiliani, S.; Benarous, R.; Desender, L.; Debyser, Z.; Christ, F.
Journal of molecular biology 2007, 365, 1480.
18.
Greene, W. C.; Debyser, Z.; Ikeda, Y.; Freed, E. O.;
4.9. In vitro anti-HIV and drug susceptibility assay
Stephens, E.; Yonemoto, W.; Buckheit, R. W.; Este, J. A.; Cihlar, T.
Antiviral research 2008, 80, 251.
19.
Schrijvers, R.; De Rijck, J.; Demeulemeester, J.; Adachi,
The inhibitory effect of antiviral drugs on the HIV-induced
cytopathic effect (CPE) in human lymphocyte MT-4 cell culture
was determined by the MT-4/MTT-assay.39 This assay is based
on the reduction of the yellow coloured 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial
dehydrogenase of metabolically active cells to a blue formazan
derivative, which can be measured spectrophotometrically. The
50% cell culture infective dose (CCID50) of the HIV(IIIB) strain
was determined by titration of the virus stock using MT-4 cells.
For the drug-susceptibility assays, MT-4 cells were infected with
100-300 CCID50 of the virus stock in the presence of five-fold
serial dilutions of the antiviral drugs. The concentration of the
various compounds that achieved 50% protection against the
CPE of the different HIV strains, which is defined as the EC50,
was determined. In parallel the 50% cytotoxic concentration
(CC50) was determined.
N.; Vets, S.; Ronen, K.; Christ, F.; Bushman, F. D.; Debyser, Z.;
Gijsbers, R. PLoS pathogens 2012, 8, e1002558.
20.
Gitto, R.; Monforte, A. M.; Debyser, Z.; Chimirri, A.
ChemMedChem 2009, 4, 1311.
21.
Z.; Chimirri, A.; Gitto, R. Journal of enzyme inhibition and
medicinal chemistry 2013, 28, 1002.
22.
Morreale, F.; Debyser, Z.; Chimirri, A. Antiviral research 2011, 92,
102.
De Luca, L.; Barreca, M. L.; Ferro, S.; Christ, F.; Iraci, N.;
De Luca, L.; Ferro, S.; Morreale, F.; Christ, F.; Debyser,
De Luca, L.; Gitto, R.; Christ, F.; Ferro, S.; De Grazia, S.;
23.
Ferro, S.; De Luca, L.; Morreale, F.; Christ, F.; Debyser,
Z.; Gitto, R.; Chimirri, A. Journal of enzyme inhibition and
medicinal chemistry 2014, 29, 237.
24.
Ferro, S.; De Luca, L.; Lo Surdo, G.; Morreale, F.; Christ,
F.; Debyser, Z.; Gitto, R.; Chimirri, A. Bioorganic & medicinal
chemistry 2014, 22, 2269.
25.
S.; Gitto, R. European journal of medicinal chemistry 2013, 68, 405.
26. De Luca, L.; Morreale, F.; Chimirri, A. Journal of
chemical information and modeling 2012, 52, 3245.
27. Vigant, F.; Lee, J.; Hollmann, A.; Tanner, L. B.; Akyol
Ataman, Z.; Yun, T.; Shui, G.; Aguilar, H. C.; Zhang, D.;
Meriwether, D.; Roman-Sosa, G.; Robinson, L. R.; Juelich, T. L.;
Buczkowski, H.; Chou, S.; Castanho, M. A.; Wolf, M. C.; Smith, J.
K.; Banyard, A.; Kielian, M.; Reddy, S.; Wenk, M. R.; Selke, M.;
Santos, N. C.; Freiberg, A. N.; Jung, M. E.; Lee, B. PLoS pathogens
2013, 9, e1003297.
De Luca, L.; Morreale, F.; Christ, F.; Debyser, Z.; Ferro,
Acknowledgment
This work was supported by THINC project (European
Commission HEALTH-F3-2008-201032). We thank Martine
Michiels, Nam Joo Van der Veken and Barbara Van Remoortel
for excellent technical assistance.
References
28.
Wolf, M. C.; Freiberg, A. N.; Zhang, T.; Akyol-Ataman,
1.
De Clercq, E. International journal of antimicrobial agents
Z.; Grock, A.; Hong, P. W.; Li, J.; Watson, N. F.; Fang, A. Q.;
Aguilar, H. C.; Porotto, M.; Honko, A. N.; Damoiseaux, R.; Miller,
J. P.; Woodson, S. E.; Chantasirivisal, S.; Fontanes, V.; Negrete, O.
A.; Krogstad, P.; Dasgupta, A.; Moscona, A.; Hensley, L. E.;
Whelan, S. P.; Faull, K. F.; Holbrook, M. R.; Jung, M. E.; Lee, B.
Proceedings of the National Academy of Sciences of the United
States of America 2010, 107, 3157.
2009, 33, 307.
2.
chemistry 2010, 53, 521.
3.
Mehellou, Y.; De Clercq, E. Journal of medicinal
Juday, T.; Gupta, S.; Grimm, K.; Wagner, S.; Kim, E. HIV
clinical trials 2011, 12, 71.
4.
Ammassari, A.; Trotta, M. P.; Shalev, N.; Marconi, P.;
Antinori, A. Antiviral therapy 2012, 17, 785.
29.
30.
http://www.pymol.org.
Laskowski, R. A.; Swindells, M. B. Journal of chemical
5.
Barreca, M. L.; Iraci, N.; De Luca, L.; Chimirri, A.
ChemMedChem 2009, 4, 1446.
information and modeling 2011, 51, 2778.
31. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Journal of molecular biology 1997, 267, 727.
32. Cherepanov, P.; Ambrosio, A. L.; Rahman, S.;
6.
Nikolenko, G. N.; Delviks-Frankenberry, K. A.; Pathak, V.
K. Journal of virology 2010, 84, 5238.
7.
Broglia, R.; Levy, Y.; Tiana, G. Current opinion in
structural biology 2008, 18, 60.
8.
S.; Christ, F.; Debyser, Z.; Chimirri, A. Bioorganic & medicinal
chemistry 2010, 18, 7515.
9.
Ellenberger, T.; Engelman, A. Proc. Natl. Acad. Sci. U S A 2005,
102, 17308.
33.
34.
De Luca, L.; Ferro, S.; Gitto, R.; Barreca, M. L.; Agnello,
Accelrys, Discovery Studio.
Case, D. A.; Darden, T. A.; Cheatham, T. E.; III;
Garcia-Rivera, J. A.; Bueno, M. T.; Morales, E.;
Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.;
Zhang, W.; Merz, K. M.; Roberts, B.; Wang, B.; Hayik, S.; Roitberg,
A.; Seabra, G.; Kolossváry, I.; Wong, K. F.; Paesani, F.; Vanicek, J.;
Liu, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.;
Ye, X.; Wang, J.; Hsieh, M.-J.; Cui, G.; Roe, D. R.; Mathews, D. H.;
Seetin, M. G.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.;
Kovalenko, A.; Kollman, P. A.; University of California: San
Francisco, AMBER, 2010.
Kugelman, J. R.; Rodriguez, D. F.; Llano, M. Journal of virology
2010, 84, 740.
10.
Al-Mawsawi, L. Q.; Neamati, N. ChemMedChem 2011, 6,
228.
11.
Debyser, Z.; Christ, F.; De Rijck, J.; Gijsbers, R. Trends in
biochemical sciences 2015, 40, 108.
12.
13.
Christ, F.; Debyser, Z. Virology 2013, 435, 102.
De Luca, L.; Ferro, S.; Morreale, F.; Chimirri, A. Mini
35.
Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.;
Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.;
reviews in medicinal chemistry 2011, 11, 714.
14. De Luca, L.; Ferro, S.; Morreale, F.; De Grazia, S.;
Chimirri, A. ChemMedChem 2011, 6, 1184.

Wang, J.; Kollman, P. Journal of computational chemistry 2003, 24,
1999.
36.
Case, D. A. J Comput Chem 2004, 25, 1157.
37. Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.;
Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.;
Chong, L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.; Donini, O.;
Cieplak, P.; Srinivasan, J.; Case, D. A.; Cheatham, T. E., 3rd.
Accounts of chemical research 2000, 33, 889.
38.
Neamati, N. FEBS letters 2008, 582, 1425.
39. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols,
Al-Mawsawi, L. Q.; Christ, F.; Dayam, R.; Debyser, Z.;
D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. Journal of virological
methods 1988, 20, 309.