4-[1-(4-Fluorobenzyl)-4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid as a prototype to develop dual inhibitors of HIV-1 integration process

Article abstract of DOI:10.1016/j.antiviral.2011.07.005

In recent years several potent HIV-1 integrase (IN) inhibitors have been identified and after the successful clinical use of raltegravir, they have gained a definitive place in the treatment of HIV-1 infection. Yet, there is a continuous effort to design

Full text of DOI:10.1016/j.antiviral.2011.07.005

Antiviral Research 92 (2011) 102–107
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4-[1-(4-Fluorobenzyl)-4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic
acid as a prototype to develop dual inhibitors of HIV-1 integration process
Laura De Luca a, Rosaria Gitto a, Frauke Christ b, Stefania Ferro a, Sara De Grazia a, Francesca Morreale a,
Zeger Debyser b, Alba Chimirri a,
⇑
a Dipartimento Farmaco-Chimico, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Molecular Virology and Gene Therapy Molecular Medicine, Katholieke Universiteit Leuven and IRC, KULAK Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In recent years several potent HIV-1 integrase (IN) inhibitors have been identified and after the successful
clinical use of raltegravir, they have gained a definitive place in the treatment of HIV-1 infection. Yet,
there is a continuous effort to design newer inhibitors that target different steps in the integration pro-
cess. Furthermore, the increased understanding of IN structural biology has opened novel approaches to
inhibit IN, such as targeting its multimerization or interaction with cellular cofactors. On these bases, we
have concentrated our research on the identification of small molecules able to inhibit two different
stages of the integration process: the IN strand-transfer phase and the IN–LEDGF/p75 interaction. We
found that the 4-[1-(4-fluorobenzyl)-4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (CHI-
1043) is an interesting anti-HIV agent exhibiting dual inhibitory effects. This work has suggested the pos-
sibility of also constructing an integration dual inhibitor using a design-in strategy.
Received 11 March 2011
Revised 16 June 2011
Accepted 5 July 2011
Available online 13 July 2011
Keywords:
HIV-1 integrase
INSTI
IN–LEDGF/p75
Dual inhibitor
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
HIV-1 IN is an essential viral enzyme that binds to the double-
stranded viral DNA generated by reverse transcription and medi-
The current treatment regimen for HIV-1-infected individuals
involves highly active anti-retroviral therapy (HAART) which typi-
cally consists of a cocktail of several potent drugs that includes
inhibitors of reverse transcriptase, protease and recently, integrase
and viral entry (Ramkumar et al., 2010). Its success is limited by se-
vere side effects, drug–drug/food interactions, and by the develop-
ment of multidrug resistant viral strains. Therefore, alternative
strategies and new targets need to be considered to combat the
growing AIDS pandemic.
In recent years the enzyme HIV-1 integrase (IN) has increas-
ingly become an attractive target from the viewpoint of drug de-
sign because it has no known human homolog (Barbaro et al.,
2005; De Clercq, 2002, 2005; Sechi et al., 2009) and therefore its
inhibition has a lower risk of disrupting normal cellular processes.
However, because of the rapid emergence of resistance mutations
observed with the use of raltegravir (MK-0518, IsentressÒ, Merck
Inc.), the first and only IN inhibitor approved by FDA (Summa
et al., 2008), the design of inhibitors against IN can be challenging
and therefore there is a continuous effort to identify newer inhib-
itors that belong to different structural classes or that target differ-
ent steps in the integration process.
ates its integration into the cellular genomic DNA of the infected
host to produce a functional provirus (McColl and Chen, 2010).
Based on partial proteolysis experiments, this enzyme can be di-
vided into three domains. The N-terminal domain (residues 1–50)
contains two histidines and two cysteines that bind Zn2+. The cata-
lytic core domain (INCCD, residues 50–212) contains the endonucle-
aseandpolynucleotidyl transferasesitewithitsthree acidicresidues
(Asp64, Asp116 and Glu152) which are highly conserved in the
integrase superfamily and polynucleotide transferases. They are
commonly referred as the ‘DDE motif’ and bind two divalent metal
physiological cofactors of HIV-1 IN, Mn2+ or Mg2+, required for cata-
lytic activity. The C-terminus domain (residues 213–288) contains
basic amino acid residues and is involved in host DNA binding (Ben-
khelifa-Ziyyat et al., 2010; Dyda et al., 1994; Pommier et al., 2000).
The INCCD is essential for two key steps of the integration reac-
tion. In the first step, termed 30 end processing (30-EP), IN removes
two nucleotides from each viral cDNA end adjacent to a conserved
30-CA sequence and leads to the formation of new 30-CA–OH. The
second step, named strand transfer (ST), is also a trans-esterifica-
tion reaction involving a direct nucleophilic attack of the 30-hydro-
xyl group of the two newly processed viral 30-DNA ends on the
phosphodiester backbone of the host target DNA (McColl and Chen,
2010; Nair et al., 2006).
Multimeric species of IN are thought to play a role in the inte-
⇑
Corresponding author. Tel.: +39 090 6766412; fax: +39 090 6766402.
E-mail address: achimirri@unime.it (A. Chimirri).
gration process. IN dimers appear to be involved in 30-EP, whereas
0166-3542/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2011.07.005

L. De Luca et al. / Antiviral Research 92 (2011) 102–107
103
tetrameric structures participate in the ST step (Ramkumar et al.,
2010).
IN is supposed to be in equilibrium between its monomeric, di-
meric, tetrameric and high order oligomeric states. Only the dimer
is able to bind the viral DNA, and thus it represents the active form
of the enzyme (Faure et al., 2005; Guiot et al., 2006).
The IN–DNA complex is then transported into the nucleus
where two DNA-bound dimers form a tetramer in the presence
of the cellular protein LEDGF/p75 (Lens Epithelium Derived
Growth Factor) prior to the strand-transfer step (Tintori et al.,
2010).
LEDGF/p75 is a cellular protein that has been identified and val-
idated as a cellular cofactor of HIV integration and replication
(Busschots et al., 2007; Greene et al., 2008). It binds HIV-1 IN via
a small (ꢀ80 residues) IN-binding domain (LEDGFIBD, residues
347–429) within its C-terminal region. IBD is both necessary and
sufficient for interaction with HIV-1 IN (Cherepanov et al., 2005).
The targeted disruption of direct cellular IN cofactors required
for viral replication has become a heavily sought-after approach
for the development of novel first-in-class allosteric IN inhibitors
for clinical development. In particular the association between IN
and the cellular cofactor LEDGF/p75 is currently the most promis-
ing IN interaction for the design of protein–protein disrupting ther-
apeutics (Al-Mawsawi and Neamati, 2011).
1000 steps of the Steepest Descent method were followed by
1000 steps of the Conjugate Gradient method for faster conver-
gence toward a local minimum. The two Mg ions within the active
site were kept fixed during the minimization process.
2.3. Experimental chemistry
All 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 chromatog-
raphy 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 d (ppm) and coupling constants (J) in hertz
(Hz). All the exchangeable protons were confirmed by addition of
D2O.
This information suggests that the HIV-1 integration is a multi-
step process and that compounds that target different stages in the
integration phase would be attractive candidates for future inhib-
itor design, as they could have a lesser likelihood for cross-resis-
tance and a possible synergistic effect when combined with the
current IN inhibitors and/or with other anti-HIV drugs (Ramkumar
et al., 2010). On this basis we have directed our research towards
the discovery of candidates with dual site binding ability, i.e. able
to inhibit both the IN strand-transfer step and IN–LEDGF/p75
interaction.
2.3.1. Synthesis of 3-acetyl-4-methoxy-1H-indole (a)
See synthetic procedure reported by Ferro et al. (2010).
2.3.2. Synthesis of 3-acetyl-4-methoxy-1-(3,5-dimethylbenzyl)-1H-
indole (b)
According to the synthetic procedure previously reported by us
(Ferro et al., 2009a,b), 3-acetyl-4-methoxy-1H-indole (189 mg,
1 mmol) was dissolved in DMF (1 mL) at 0 °C and dry sodium hy-
dride (120 mg, 5 mmol) was added. After stirring for 2 min, 3,5-
dimethylbenzyl bromide (298 mg, 1.5 mmol) was added dropwise
and the resulting solution was placed in a cylindrical quartz tube
(diam. 2 cm), stirred at r.t., and irradiated in a microwave oven at
the following conditions: 100 W, 50 °C, 5 min. After addition of a
saturated NaHCO3 solution, the reaction mixture was extracted
with ethyl acetate (10 mL ꢁ 3) and dried over Na2SO4. The solvent
was removed under reduced pressure and the crude purified by
flash chromatography using a mixture of cyclohexane/ethylacetate
(6:4) to give a white solid. Yield: 73%; mp: 110–112 °C. 1HNMR
(CDCl3): d = 2.26 (s, 6H, 2 ꢁ CH3), 2.69 (s, 3H, CH3), 3.98 (s, 3H,
OCH3), 5.20 (s, 2H, CH2), 6.69 (d, J = 7.9, 1H, ArH), 6.77 (s, 1H,
ArH), 6.93–6.97 (m, 2H, ArH), 7.19 (t, J = 7.97, 1H, ArH), 7.72 ppm
(s, 1H, ArH); Anal. calcd for C20H21NO2: C 78.15, H 6.89, N 4.56,
found: C 78.23, H 6.71, N 4.38.
2. Materials and methods
2.1. Molecular docking experiments
All inhibitors used for these studies were constructed using Dis-
covery Studio 2.5 (Accelrys, San Diego, CA) and energy minimized
using the Smart Minimizer protocol (1000 steps) which combines
the Steepest Descent and the Conjugate Gradient methods.
Docking calculations into the LEDGF/p75IBD binding pocket on
INCCD were carried out using the same protocol that we success-
fully applied in our previous papers (De Luca et al., 2009, 2010).
Docking studies for INSTIs were performed using for the first
time the crystal structure of PFV–IN with DNA and two Mg ions
complexed with the inhibitor raltegravir. This structural informa-
tion was retrieved from the Protein Data Bank (PDB code 3OYA)
(Hare et al., 2010a,b). Hydrogen atoms were added using Discovery
Studio 2.5. The structure of raltegravir was extracted from X-ray
complex and used as a control of program performance. Inhibitors
were docked using GOLD 4.1.2 (Jones et al., 1997). A 15 Å radius ac-
tive site was defined considering the coordinates of raltegravir in
the X-ray position as the center of the binding pocket. CHEMPLP
scoring function was chosen as the fitness function, default param-
eters were used and the ligands were submitted to 100 genetic
algorithm runs (De Luca et al., 2011).
2.3.3. Synthesis of 4-[1-(3,5-dimethylbenzyl)-4-methoxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (c)
According to the synthetic procedure previously reported by us
(Ferro et al., 2009a,b), a mixture of 3-acetyl-4-methoxy-1-(3,5-
dimethylbenzyl)-1H-indole (307 mg, 1 mmol) diethyl oxalate
(219 mg, 1.5 mmol) and a catalytic amount of NaOCH3 was sus-
pended in anhydrous THF (2 mL). The reaction mixture was placed
in a cylindrical quartz tube (diam. 2 cm), stirred and irradiated at
continuous temperature in a microwave oven for two subsequent
periods at the same conditions (250 W, 2 min, 50 °C). The solvent
was concentrated under reduced pressure and the collected yellow
solid was crystallized from ethanol/diethyl ether. Yield: 89%; mp:
270 °C (dec). 1HNMR (DMSO): d = 1.23 (t, J = 7.26, 3H, CH3), 2.18
(s, 6H, 2 ꢁ CH3), 3.82 (s, 3H, OCH3), 4.11 (q, J = 7.26, 2H, CH2),
5.28 (s, 2H, CH2), 6.58–7.65 ppm (m, 8H, 7ArH and CH); Anal. calcd
for C24H25NO5: C 70.75, H 6.18, N 3.44, found: C 70.63, H 6.24, N
3.53.
2.2. Minimization process
All of the protein–ligand complexes obtained by docking stud-
ies were minimized using the CHARMM force field in Discovery
Studio 2.5 with the Generalized Born implicit solvent model.

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L. De Luca et al. / Antiviral Research 92 (2011) 102–107
2.3.4. Synthesis of 4-[1-(3,5-dimethylbenzyl)-4-methoxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoic acid (2)
chondrial dehydrogenase of metabolically active cells to a blue
formazan derivative, which can be measured spectrophotometri-
cally. 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 fivefold se-
rial dilutions of the antiviral drugs. The concentration of the various
compounds that achieved 50% protection against the CPE of the dif-
ferent HIV strains, which is defined as the EC50, was determined. In
parallel, the 50% cytotoxic concentration (CC50) was determined.
According to the synthetic procedure previously reported by us,
(Ferro et al., 2009a,b) 4-[1-(3,5-dimethylbenzyl)-4-methoxy-1H-
indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (407 mg, 1 mmol) was
dissolved in methanol (5 mL) and treated with 2 N NaOH (5 mL,
50 mmol). The reaction mixture was stirred at room temperature
for 1.5 h and then acidified with conc. HCl to afford the hydrolyzed
derivative as a yellow precipitate. Recrystallization from ethanol/
diethyl ether afforded the pure compound 2 as a yellow solid.
Yield: 52%; mp: 120–122 °C. 1HNMR (DMSO): d = 2.19 (s, 6H,
2 ꢁ CH3), 3.89 (s, 3H, OCH3), 5.41 (s, 2H, CH2), 6.78–7.59 (m, 7H,
6ArH and CH), 8.48 ppm (s, 1H, ArH); Anal. calcd for C22H21NO5:
C 69.65, H 5.58, N 3.69, found: C 69.45, H 5. 41, N 3.75.
3. Results and discussion
In previous papers we reported ligand-based approaches useful
to generate three-dimensional pharmacophore models which led
to the identification of a series of indole DKA analogs (Barreca
et al., 2005; De Luca et al., 2008, 2011; Ferro et al., 2004,
2007a,b, 2009a,b, 2010) able to selectively inhibit the ST step of
the integration process which represents the major target in the
development of anti-HIV-1 IN drugs.
More recently we focused our attention on the interaction be-
tween IN and its cellular cofactor LEDGF/p75, the inhibition of
which is increasingly recognized as an emerging potential thera-
peutic strategy. In fact LEDGF/p75 is an IN interacting cellular pro-
tein (Van Maele et al., 2006) which plays an important role in
chromatin tethering. It interacts with IN via its C-terminal domain,
also known as the IN binding domain (LEDGFIBD). The biochemical
test for the identification of HIV integrase inhibitors, targeting the
interaction between HIV–IN and the cellular cofactor LEDGF/p75 is
the AlphaScreen assay which has been developed for facilitating
the identification of novel inhibitors specific for this protein–pro-
tein interaction (De Luca et al., 2010).
2.4. LEDGF/p75–HIV-1 Integrase interaction screening (AlphaScreen
technology)
The AlphaScreen assay was performed as described previously
(Christ et al., 2010). Reactions were performed in 25 ll final vol-
ume 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 at varying concentrations from 1 up to 100 lM. After-
wards 100 nM of recombinant flag-LEDGF/p75 was added and
incubation was extended by another hour at 4 °C. Subsequently,
5
ll of Ni-chelate-coated acceptor beads and 5
ll of anti-flag donor
beads were added to a final concentration of 20
l
g/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.
Our studies led to the discovery of a series of benzylindoles
derivatives active as inhibitors against IN–LEDGF/p75 interaction
evaluated in the AlphaScreen assay (De Luca et al., 2009). After a
number of rational modifications both on the indole system and
the benzyl moiety, suggested by molecular modeling studies (De
Luca et al., 2009, 2010), the most potent compound was the 4-
[1-(3,5-dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-
2.5. Integrase inhibition
To determine the susceptibility of the HIV-1 integrase enzyme
towards our compounds we used enzyme-linked immunosorbent
assays. These assays use an oligonucleotide substrate of which
one oligonucleotide (50-ACTGCTAGAGATTTTCCACACTGACTAAAA-
GGGTC-30) is labeled with biotin at the 30 end and the other
oligonucleotide is labeled with digoxigenin at the 50 end. For the
overall integration assay the second 50-digoxigenin labeled oligo-
nucleotide is (50- GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-
30). For the Strand Transfer assay the second oligonucleotide lacks
GT at the 30 end. The integrase enzyme was diluted in 750 mM
NaCl, 10 mM Tris pH 7.6, 10% glycerol and 1 mM b-mercapto eth-
oxobut-2-enoic acid (1) (Fig. 1) showing an IC50 value of 3.5 lM. So
we considered this derivative as a promising lead compound to
identify small molecules able to exert a specific blockade of this
protein–protein interaction.
The results of the biological assay were in agreement with our
computational studies that suggested the positioning as well as
the interactions between the inhibitor 1 and IN. Docking studies
suggested the ability of compound 1 to interact with the portion
of IN dimer (A and B chains) in contact with LEDGFIBD engaging
the following contacts (Fig. 2): (a) the carboxylate group interacted
with Glu170, His171 and Thr174 of IN A-chain; (b) the hydroxyl
group of the diketoacid moiety bound Gln95 of B-chain; (c) the
benzene fused-ring of indole nucleus was placed into the IN hydro-
anol. To perform the reaction, 4
ing to concentration of 1.6
oligonucleotides (7 nM) were added in a final reaction volume of
40 l containing 10 mM MgCl2, 5 mM DTT, 20 mM HEPES pH 7.5,
ll diluted integrase (correspond-
a
l
M) and of annealed
4 ll
l
5% PEG and 15% DMSO. The reaction was carried out for 1 h at
37 °C. Reaction products were denatured with 30 mM NaOH and
detected by an immunosorbent assay on avidin coated plates
(Christ et al., 2010).
F
N
N
N
2.6. In vitro anti-HIV and drug susceptibility assays
OH
OMe
OMe
O
O
O
COOH
H
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 (Pauwels et al., 1988). This
assay is based on the reduction of the yellow colored 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mito-
COOH
H
COOH
H
O
O
O
1
CHI-1043
2
Fig. 1. Chemical structures of compounds 1, 2 and CHI-1043.

L. De Luca et al. / Antiviral Research 92 (2011) 102–107
105
Table 1
Number of hydrophobic contacts between the derivatives 1 and CHI-1043 and the
aminoacids of the LEDGF/p75 binding hydrophobic pocket of INCCD calculated by
Ligplot 4.5.3 program (Wallace et al., 1995) from docked ligand–target complexes.
IN, chain B
Derivative 1
CHI-1043
Ala 128
Ala 129
Trp 131
Trp 132
4
3
6
6
2
2
5
5
our developed INSTIs presenting a good trade-off between the
anti-IN and anti-HIV-1 activities (IC50 = 0.14 M; EC50 = 0.59 M;
l
l
SI = 70) (De Luca et al., 2008). CHI-1043 interferes with the viral
replication cycle at the time of retroviral integration and also
shows efficacy against HIV-2, SIV and viral strains resistant to
other ST inhibitors (Hombrouck et al., 2008).
Firstly, we carried out docking experiments to explore the abil-
ity of CHI-1043 to establish those contacts considered necessary to
block the interaction between the targeted proteins IN and LEDGF/
p75. Docking studies were performed on the LEDGF/p75 binding
pocket of INCCD using the GOLD program (Jones et al., 1997) and
the docking protocol that we successfully applied in our previous
papers (De Luca et al., 2009, 2010). The result of this study is
shown in Fig. 3A, revealing that CHI-1043 occupies the same bind-
ing pocket of derivative 1 thus implying its potential ability to in-
hibit the IN–LEDGF/p75 interaction.
Fig. 2. Docking of compound 1 (magenta), into the LEDGF/p75IBD binding pocket of
INCCD. This figure as well as Fig. 3 were produced with PyMol (DeLano and DeLano
Scientific LLC).
phobic pocket; (d) the 3,5-dimethylbenzyl N-substituent occupied
a large hydrophobic area consisting of Ala128, Ala129, Trp132 and
the important residue Trp131.
Unfortunately, when compound 1 was tested in MT4 cells as
anti-HIV agent it showed low antiviral activity (EC50 = 44 lM)
Once selected, CHI-1043 was tested in the AlphaScreen assay
and proved to be able to inhibit the IN–LEDGF/p75 interaction with
and displayed cytotoxic effects. Obviously, our goal was not only
to obtain potent inhibitors of IN–LEDGF/p75 interaction, but also
potential antiretroviral candidates and the results in cells were
disappointing.
an IC50 value of 36.16
cule inhibits both the IN ST step (IC50 0.14
interaction (IC50 36 M), thus affording, to our knowledge, the first
l
M. These results suggested that this mole-
lM) and IN–LEDGF/p75
l
dual inhibitor of HIV-1 integration process.
At this point, considering that compound 1 presented a chemi-
cal structure similar to that of a series of our indole derivatives ac-
tive against the IN strand-transfer step (Barreca et al., 2005; De
Luca et al., 2008; De Luca et al., 2011; Ferro et al., 2007a,b, 2010,
2009a,b), we chose to select among our INSTIs, some derivatives
highly active in cells with low toxicity and to verify their ability
to bind not only to the portion of the INCCD containing the DDE mo-
tif but also to the LEDGFIBD. Following this idea, our objective was
to identify molecules able to inhibit two different integration pro-
cesses: the ST step and the IN–LEDGF/p75 interaction.
In particular the inhibitory activity of CHI-1043 on the IN–
LEDGF/p75 interaction could be due, as expected, to its ability to
engage a set of interactions similar to those of 1, which had proved
to be a potent IN–LEDGF/p75 inhibitor. (Fig. 3A).
Docking studies highlighted that both the carboxylate group
and the hydroxyl group of the diketoacid moiety of CHI-1043 inter-
acted with the same aminoacids of compound 1 and also the ben-
zene-fused ring of indole system was placed into the same IN
hydrophobic pocket. Moreover, the 4-methoxy group of CHI-1043
could create an accessory hydrophobic contact with residue
Leu102 chain B. However, the presence of the 4-fluorobenzyl sub-
stituent, instead of the 3,5-methylbenzyl one, reduced the possibil-
With these thoughts in mind we addressed our attention to
CHI-1043 (Fig. 1), considered the most promising compound of
Fig. 3. Docking of CHI-1043 (green) into (A) LEDGF/p75IBD binding pocket of INCCD and (B) IN–DNA active site. The divalent metal ions are shown as gray spheres, while the
viral DNA is depicted in violet(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

106
L. De Luca et al. / Antiviral Research 92 (2011) 102–107
H
N
H
N
N
iii
ii
CH3
N
iv
i
2
CH3
COOEt
OMe
OMe
OMe
O
OMe
O
O
OH
a
b
c
Scheme 1. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, RT, 12 h; (ii) 3,5-dimethylbenzyl bromide, NaH, DMF, mw: 5 min at continuous temperature (50 °C), 100 W;
(iii) diethyl oxalate, dry CH3ONa, THF, two separate steps under the same conditions mw: 2 min, at continuous temperature (50 °C), 250 W; (iv) 2 N NaOH, MeOH, RT, 1.5 h.
ity to create some important contacts in the hydrophobic area, as
described above for derivative 1 (Table 1).
This last observation could explain the lower potency of CHI-
1043 as protein–protein interaction inhibitor when compared with
In conclusion, this study suggests that CHI-1043 could be con-
sidered a good starting point towards our goal of identifying dual
inhibitors of the HIV-1 integration processes with antiretroviral
activity and safety profile. The design of inhibitors that can accom-
modate binding to two distinct biological targets or two different
sites remains an intriguing yet challenging scientific endeavor.
We are working in this direction and suitable modifications are
in progress; the results of our efforts together with mutation stud-
ies will be reported in due course.
1 (36.16 lM versus 3.5 lM) in the AlphaScreen assay.
Even though the 3,5-dimethylbenzyl substituent seems impor-
tant for inhibition of IN–LEDGF/p75 interaction, the 4-fluorobenzyl
one, according to our experience, appears crucial for the inhibition
of the INST step.
This hypothesis is in accordance with the evidence that highly
potent INSTIs present a halogen-substituted aromatic functional-
ity. Recently Hare et al. (2010a) reported cocrystal structures con-
taining prototype foamy virus (PFV) intasome complexed with an
expanded set of first and second-generation INSTIs evidencing that
an important common and conserved structural feature of INSTIs is
a halogenated benzyl group.
Acknowledgment
This work was supported by THINC project (European Commis-
sion HEALTH-F3-2008-201032).
In fact this moiety is able to displace the 3’ adenine of bound
viral DNA, inserting between the base of the penultimate cytidine,
References
with which it forms a face-to-face p–p interaction, and Pro214 of
Al-Mawsawi, L.Q., Neamati, N., 2011. Allosteric inhibitor development targeting
HIV-1 Integrase. Chem. Med. Chem. 6, 228–241.
Barbaro, G., Scozzafava, A., Mastrolorenzo, A., Supuran, C.T., 2005. Highly active
antiretroviral therapy: current state of the art, new agents and their
pharmacological interactions useful for improving therapeutic outcome. Curr.
Pharm. Des. 11, 1805–1843.
Barreca, M.L., Ferro, S., Rao, A., De Luca, L., Zappala, M., Monforte, A.M., Debyser, Z.,
Witvrouw, M., Chimirri, A., 2005. Pharmacophore-based design of HIV-1
integrase strand-transfer inhibitors. J. Med. Chem. 48, 7084–7088.
Benkhelifa-Ziyyat, S., Bucher, S., Zanta-Boussif, M.A., Pasquet, J., Danos, O., 2010.
Changes in the accessibility of the HIV-1 Integrase C-terminus in the presence
of cellular proteins. Retrovirology 7, 27.
Busschots, K., Voet, A., De Maeyer, M., Rain, J.C., Emiliani, S., Benarous, R., Desender,
L., Debyser, Z., Christ, F., 2007. Identification of the LEDGF/p75 binding site in
HIV-1 integrase. J. Mol. Biol. 365, 1480–1492.
Cherepanov, P., Ambrosio, A.L., Rahman, S., Ellenberger, T., Engelman, A., 2005.
Structural basis for the recognition between HIV-1 integrase and transcriptional
coactivator p75. Proc. Natl. Acad. Sci. USA 102, 17308–17313.
Christ, F., Voet, A., Marchand, A., Nicolet, S., Desimmie, B.A., Marchand, D., Bardiot,
D., Van der Veken, N.J., Van Remoortel, B., Strelkov, S.V., De Maeyer, M., Chaltin,
P., Debyser, Z., 2010. Rational design of small-molecule inhibitors of the LEDGF/
p75-integrase interaction and HIV replication. Nat. Chem. Biol. 6, 442–448.
De Clercq, E., 2002. Strategies in the design of antiviral drugs. Nat. Rev. Drug. Discov.
1, 13–25.
De Clercq, E., 2005. New approaches toward anti-HIV chemotherapy. J. Med. Chem.
48, 1297–1313.
De Luca, L., Barreca, M.L., Ferro, S., Iraci, N., Michiels, M., Christ, F., Debyser, Z.,
Witvrouw, M., Chimirri, A., 2008. A refined pharmacophore model for HIV-1
integrase inhibitors: optimization of potency in the 1H-benzylindole series.
Bioorg. Med. Chem. Lett. 18, 2891–2895.
IN, thus contributing to the inhibition of strand-transfer step.
On this basis, and while realizing that the structure of integrase
with DNA and two Mg ions from PFV–IN can be considered realis-
tic, we used the complex with raltegravir (PDB code 3OYA) to per-
form docking studies for CHI-1043 in order to explore its binding
mode (see Section 2).
After the validation of the docking protocol, CHI-1043 was
docked in the same binding site of raltegravir. The results revealed
that CHI-1043 showed a similar binding mode compared to other
INSTI-bound crystal structures. (Fig. 3B) (Hare et al., 2010b).
In particular, the common metal-coordinating pharmacophore
of the INSTIs, that usually comprises three specifically positioned
heteroatoms (oxygen or, less commonly, nitrogen), is represented
by the diketo acid moiety coordinating the two metal cofactors
within the active site of IN. The second conserved structural fea-
ture of INSTIs is a halogenated benzyl group, in our case the 4-flu-
orobenzyl group which interacts with the residues C16 of viral
DNA and Pro214. Furthermore the fluorine atom seems to establish
van der Waals interactions with Gln 215, thus confirming the key
role of the halogen atoms in INSTIs.
In order to confirm that the elimination of the halogenated ben-
zyl group negatively influenced the inhibition of IN ST step, we
synthesized. A new CHI-1043 analog (2) in which the 4-fluoroben-
zyl portion at N-1 has been replaced with a 3,5-dimethylbenzyl
moiety.
For the synthesis of derivative 2 we followed our previous syn-
thetic procedures (Ferro et al., 2010, 2009a,b) outlined in Scheme
1.
De Luca, L., Barreca, M.L., Ferro, S., Christ, F., Iraci, N., Gitto, R., Monforte, A.M.,
Debyser, Z., Chimirri, A., 2009. Pharmacophore-based discovery of small-
molecule inhibitors of protein–protein interactions between HIV-1 integrase
and cellular cofactor LEDGF/p75. Chem. Med. Chem. 4, 1311–1316.
De Luca, L., Ferro, S., Gitto, R., Barreca, M.L., Agnello, S., Christ, F., Debyser, Z.,
Chimirri, A., 2010. Small molecules targeting the interaction between HIV-1
integrase and LEDGF/p75 cofactor. Bioorg. Med. Chem. 18, 7515–7521.
De Luca, L., De Grazia, S., Ferro, S., Gitto, R., Christ, F., Debyser, Z., Chimirri, A., 2011.
HIV-1 integrase strand-transfer inhibitors: design, synthesis and molecular
modeling investigation. Eur. J. Med. Chem. 46, 756–764.
Derivative 2 was then evaluated as INSTI and, as expected, this
modification drastically reduced the ST inhibitory effects with re-
DeLano, W.L., DeLano Scientific LLC, The PyMOL Molecular Graphics System, San
Carlos, CA, USA, 2008 <http://www.pymol.org>.
Dyda, F., Hickman, A.B., Jenkins, T.M., Engelman, A., Craigie, R., Davies, D.R., 1994.
Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other
polynucleotidyl transferases. Science 266, 1981–1986.
spect to CHI-1043 (IC50 5.49
compound 2 showed a poorer anti-HIV activity (EC50 4.63
sus EC50 0.59
concentration.
l
M versus IC50 0.14
l
M). Moreover,
M ver-
lM) in MT4 cells and cytotoxicity at the same
l

L. De Luca et al. / Antiviral Research 92 (2011) 102–107
107
Faure, A., Calmels, C., Desjobert, C., Castroviejo, M., Caumont-Sarcos, A., Tarrago-
Litvak, L., Litvak, S., Parissi, V., 2005. HIV-1 integrase crosslinked oligomers are
active in vitro. Nucleic Acids Res. 33, 977–986.
Ferro, S., Rao, A., Zappala, M., Chimirri, A., Barreca, M.L., Witvrouw, M., Debyser, Z.,
Monforte, P., 2004. Synthesis of new potential HIV-1 integrase inhibitors.
Heterocycles 2727, 27234.
Hombrouck, A., Van Remoortel, B., Michiels, M., Noppe, W., Christ, F., Eneroth, A.,
Sahlberg, B.L., Benkestock, K., Vrang, L., Johansson, N.G., Barreca, M.L., De Luca,
L., Ferro, S., Chimirri, A., Debyser, Z., Witvrouw, M., 2008. Preclinical evaluation
of 1H-benzylindole derivatives as novel human immunodeficiency virus
integrase strand transfer inhibitors. Antimicrob. Agents Chemother. 52, 2861–
2869.
Ferro, S., Barreca, M.L., De Luca, L., Rao, A., Monforte, A.M., Debyser, Z., Witvrouw,
M., Chimirri, A., 2007a. New 4-[(1-benzyl-1H-indol-3-yl)carbonyl]-3-
hydroxyfuran-2(5H)-ones, beta-diketo acid analogs as HIV-1 integrase
inhibitors. Arch. Pharm. (Weinheim) 340, 292–298.
Ferro, S., Barreca, M.L., De Luca, L., Rao, A., Monforte, A.M., Michiels, M., Witvrouw,
M., Debyser, Z., Chimirri, A., 2007. HIV-1 Integrase strand transfer inhibitors
(INSTIS): design, synthesis and biological evaluation. Reports from the 5th Joint
Meeting on Medicinal Chemistry, Portoroz (Slovenia) June 16–21, 2007,
Medimond International Proceedings, 11–15.
Jones, G., Willett, P., Glen, R.C., Leach, A.R., Taylor, R., 1997. Development and
validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267, 727–748.
McColl, D.J., Chen, X., 2010. Strand transfer inhibitors of HIV-1 integrase: bringing
IN a new era of antiretroviral therapy. Antiviral Res. 85, 101–118.
Nair, V., Chi, G., Ptak, R., Neamati, N., 2006. HIV integrase inhibitors with nucleobase
scaffolds: discovery of a highly potent anti-HIV agent. J. Med. Chem. 49, 445–
447.
Pauwels, R., Balzarini, J., Baba, M., Snoeck, R., Schols, D., Herdewijn, P., Desmyter, J.,
De Clercq, E., 1988. Rapid and automated tetrazolium-based colorimetric assay
for the detection of anti-HIV compounds. J. Virol. Methods 20, 309–321.
Pommier, Y., Marchand, C., Neamati, N., 2000. Retroviral integrase inhibitors year
2000: update and perspectives. Antiviral Res. 47, 139–148.
Ferro, S., De Grazia, S., De Luca, L., Barreca, M.L., Debyser, Z., Chimirri, A., 2009a.
Structural modification of diketo acid portion in 1H-benzylindole derivatives
HIV-1 integrase inhibitors. Heterocycles 78, 947–959.
Ferro, S., De Luca, L., Barreca, M.L., Iraci, N., De Grazia, S., Christ, F., Witvrouw, M.,
Ramkumar, K., Serrao, E., Odde, S., Neamati, N., 2010. HIV-1 integrase inhibitors:
2007–2008 update. Med. Res. Rev. 30, 890–954.
Debyser, Z., Chimirri, A., 2009b. Docking studies on
a new human
immunodeficiency virus integrase-Mg–DNA complex: phenyl ring exploration
and synthesis of 1H-benzylindole derivatives through fluorine substitutions. J.
Med. Chem. 52, 569–573.
Sechi, M., Carta, F., Sannia, L., Dallocchio, R., Dessi, A., Al-Safi, R.I., Neamati, N., 2009.
Design, synthesis, molecular modeling, and anti-HIV-1 integrase activity of a
series of photoactivatable diketo acid-containing inhibitors as affinity probes.
Antiviral Res. 81, 267–276.
Summa, V., Petrocchi, A., Bonelli, F., Crescenzi, B., Donghi, M., Ferrara, M., Fiore, F.,
Gardelli, C., Gonzalez Paz, O., Hazuda, D.J., Jones, P., Kinzel, O., Laufer, R.,
Monteagudo, E., Muraglia, E., Nizi, E., Orvieto, F., Pace, P., Pescatore, G., Scarpelli,
R., Stillmock, K., Witmer, M.V., Rowley, M., 2008. Discovery of raltegravir, a
potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of
HIV-AIDS infection. J. Med. Chem. 51, 5843–5855.
Tintori, C., Veljkovic, N., Veljkovic, V., Botta, M., 2010. Computational studies of the
interaction between the HIV-1 integrase tetramer and the cofactor LEDGF/p75:
Insights from molecular dynamics simulations and the Informational spectrum
method. Proteins 78, 3396–3408.
Van Maele, B., Busschots, K., Vandekerckhove, L., Christ, F., Debyser, Z., 2006.
Cellular co-factors of HIV-1 integration. Trends Biochem. Sci. 31, 98–105.
Wallace, A.C., Laskowski, R.A., Thornton, J.M., 1995. LIGPLOT: a program to generate
schematic diagrams of protein–ligand interactions. Protein Eng. 8, 127–134.
Ferro, S., De Luca, L., Barreca, M.L., De Grazia, S., Christ, F., Debyser, Z., Chimirri, A.,
2010. New chloro, fluorobenzylindole derivatives as integrase strand-transfer
inhibitors (INSTIs) and their mode of action. Bioorg. Med. Chem. 18, 5510–5518.
Greene, W.C., Debyser, Z., Ikeda, Y., Freed, E.O., Stephens, E., Yonemoto, W.,
Buckheit, R.W., Esté, J.A., Cihlar, T., 2008. Novel targets for HIV therapy. Antiviral
Res. 80, 251–265.
Guiot, E., Carayon, K., Delelis, O., Simon, F., Tauc, P., Zubin, E., Gottikh, M.,
Mouscadet, J.F., Brochon, J.C., Deprez, E., 2006. Relationship between the
oligomeric status of HIV-1 integrase on DNA and enzymatic activity. J. Biol.
Chem. 281, 22707–22719.
Hare, S., Gupta, S.S., Valkov, E., Engelman, A., Cherepanov, P., 2010a. Retroviral
intasome assembly and inhibition of DNA strand transfer. Nature 464, 232–236.
Hare, S., Vos, A.M., Clayton, R.F., Thuring, J.W., Cummings, M.D., Cherepanov, P.,
2010b. Molecular mechanisms of retroviral integrase inhibition and the
evolution of viral resistance. Proc. Natl. Acad. Sci. USA 107, 20057–20062.