Small molecules targeting the interaction between HIV-1 integrase and LEDGF/p75 cofactor

Article abstract of DOI:10.1016/j.bmc.2010.08.051

The search of small molecules as protein-protein interaction inhibitors represents a new attractive strategy to develop anti-HIV-1 agents. We previously reported a computational study that led to the discovery of new inhibitors of the interaction between enzyme HIV-1 integrase (IN) and the nuclear protein lens epithelium growth factor LEDGF/p75.¹ Herein, we describe new findings about the binding site of LEDGF/p75 on IN employing a different computational approach. In this way further structural requirements, helpful to disrupt LEDGF/p75-IN binding, have been identified. The main result of this work was the exploration of a relevant hydrophobic region. So we planned the introduction of suitable and simple chemical modifications on our previously reported 'hit' and the new synthesized compounds were subjected to biological tests. The results obtained demonstrate that the hydrophobic pocket could play a key role in improving inhibitory efficacy thus opening new suggestions to design active ligands.

Full text of DOI:10.1016/j.bmc.2010.08.051

Bioorganic & Medicinal Chemistry 18 (2010) 7515–7521
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecules targeting the interaction between HIV-1 integrase
and LEDGF/p75 cofactor
Laura De Luca a, , Stefania Ferro a, Rosaria Gitto a, Maria Letizia Barreca b, Stefano Agnello a,
⇑
Frauke Christ c, Zeger Debyser c, Alba Chimirri a
a Dipartimento Farmaco-Chimico, Università di Messina Viale Annunziata, I-98168 Messina, Italy
b Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia Via del Liceo 1, I-06123 Perugia, Italy
c 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:
The search of small molecules as protein–protein interaction inhibitors represents a new attractive strat-
egy to develop anti-HIV-1 agents. We previously reported a computational study that led to the discovery
of new inhibitors of the interaction between enzyme HIV-1 integrase (IN) and the nuclear protein lens
epithelium growth factor LEDGF/p75.1
Received 21 June 2010
Revised 24 August 2010
Accepted 27 August 2010
Available online 17 September 2010
Herein, we describe new findings about the binding site of LEDGF/p75 on IN employing a different com-
putational approach. In this way further structural requirements, helpful to disrupt LEDGF/p75-IN bind-
ing, have been identified. The main result of this work was the exploration of a relevant hydrophobic
region. So we planned the introduction of suitable and simple chemical modifications on our previously
reported ‘hit’ and the new synthesized compounds were subjected to biological tests.
The results obtained demonstrate that the hydrophobic pocket could play a key role in improving
inhibitory efficacy thus opening new suggestions to design active ligands.
Keywords:
HIV-1 integrase
LEDGF/p75
Protein–protein interactions
Molecular docking
GRID
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
regulation, as well as in cell survival and in prevention of apoptosis.
In particular, cells lacking of LEDGF/p75 showed a severe defect in
To date, 25 compounds for the treatment of HIV infections have
been formally licensed. They belong to different categories: reverse
transcriptase inhibitors (RTIs), protease inhibitors (PIs); fusion
inhibitors (FIs), coreceptor inhibitors (CRIs), and integrase inhibi-
tors (INIs).2 However, even the combination of these drugs is not
sufficient enough to stop the progression of AIDS partly due to
the emergence of drug-resistant HIV-1 mutants as well as the se-
vere side effects.3–5 Therefore, alternative strategies need to be
considered and new targets need to be identified to combat the
growing AIDS pandemic. Currently, small-molecule inhibitors of
the direct protein–protein interactions (PPIs), that mediate impor-
tant biological processes, are an emerging and interesting area in
drug design. In particular, there is evidence that various proteins
play auxiliary roles in the HIV-1 life cycle and that some host cel-
lular cofactors specifically control the integration process of viral
DNA. The disruption of these essential cofactor-IN protein–protein
interactions could represent a new frontier to design and develop
novel anti-HIV-1 agents for anti-AIDS therapy.6
HIV-1 infection characterized by decreased levels of integrated viral
DNA.7,8 This evidence pointed out the possibility that LEDGF/p75
could be a promising target to inhibit DNA integration of HIV-1 life
cycle. The LEDGF/p75 acts through a tethering mechanism as a po-
tent cofactor for HIV-1 integration with the N-terminal Pro-Trp-
Trp-Pro (PWWP) and A/T-hook elements binding to chromatin,
and a C-terminal integrase-binding domain (IBD) binding to IN.9,10
It is well known that these two regions of LEDGF/p75 are crucial
for cofactor activity.
The specific protein–protein interaction between HIV-1 IN
catalytic core domain (CCD) and IBD has been well described by
X-ray analysis (Protein Data Bank file 2B4J) as well as by site-direc-
ted mutation studies, and it is characterized by IBD residues Ile365,
Asp366, Phe406, and Val408.11 In a previous paper, we focused our
interest on key residues Ile365-Asp366 and used a computational
approach to describe the pharmacophoric requirements needed
to disrupt the interaction between HIV-1 IN CCD and LEDGF/p75
IBD.1 This study led to the identification of some small molecules
mimicking the Ile-Asp dipeptide; the most active molecule was
Amongst these cofactors, the ubiquitous lens epithelium growth
factor LEDGF/p75 seems to play a pivotal role in the transcriptional
the
acid (CHIBA-3003, I, Fig. 1) that inhibited the IN-LEDGF/p75 inter-
action showing IC50 value of 35 M in AlphaScreen assay.
2-hydroxy-4-(4-hydroxy-1H-indol-3-yl)-4-oxobut-2-enoic
l
We therefore considered that CHIBA-3003 was a promising ‘hit
compound’ for the development of new and more potent inhibitors
⇑
Corresponding author. Tel.: +39 0906766464; fax: +39 0906766402.
E-mail address: ldeluca@unime.it (L. De Luca).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.051

7516
L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
F
H
N
N
OH
O
OH
COOH
O
HO
COOH
I
II
HO
O
N
S
O
Cl
O
COOH
COOH
O
O
N
Br
IV
III
Figure 1. Chemical structures of CHIBA-3003 (I), CHIBA-3000 (II), D77 (III), and IV.
preventing IN-LEDGF/75 interaction. Considering that very few
inhibitors of this interaction are known and with the aim of achiev-
ing other information about the IN/LEDGF protein interaction and
identify new important chemical frames improving the inhibitory
potency of CHIBA-3003, in this study we have explored new
binding pocket regions through GRID molecular interaction fields
(MIFs) and synthesized several new small molecules targeting this
PPI. Furthermore, docking experiments furnished new suggestions
about the putative interactions within the binding site of these
inhibitors, which hopefully could advance into novel anti-HIV drug
targets.
corresponding to the IN residue Trp131.10 Furthermore it was
highlighted that the importance of IBD aminoacids Phe406 and
Val408 was correlated to their capability to contact and occlude
solvent from the exposed IN residue Trp131.11 In an effort to ex-
plore new regions of IN/LEDGF protein interaction, we studied
the HIV-1 INCCD surface using molecular interaction fields (MIFs)
calculated by the GRID program12 and on the basis of structural
information retrieved from IN/LEDGF crystal complex (PDB: 2
B4J).11 The GRID program initially builds a grid over the molecular
region of interest and then calculates the interaction energies be-
tween the protein and several molecular probes, at each grid point.
The GRID probes incorporate different physicochemical properties
and are able to mimic most of the atom types and small moieties
commonly found in ligands. The set of energies calculated for a gi-
ven probe constitutes the MIFs of the probe–target system. The
probes selected for this study were the DRY probe representing
steric and hydrophobic interactions; the N1 (amide nitrogen)
probe to represent hydrogen-bond-donor groups; and the O (sp2
carbonyl oxygen) probe to represent hydrogen-bond-acceptors.
The results of the MIF calculations are shown in Figure 2.
2. Results and discussion
2.1. Rational design
It is well known that a typical protein–protein interaction oc-
curs between the IBD of LEDGF/p75 and the integrase catalytic core
domain (INCCD). The IBD of LEDGF/p75 forms a specific interaction
with the binding pocket at the interface of two monomers of the
INCCD. The IBD residues, involved in the interaction, are located
in its interhelical loop regions and 4 relevant amino acid residues,
Ile365, Asp366, Phe406, and Val408, are considered essential for
the direct contact with INCCD residues.11 The Ile365 and Asp366
hot-spot residues are contained on an octapeptide that fits into a
HIV-IN binding pocket. Previous studies suggested that these two
residues provide a binding site for small molecules as inhibitors
of this protein–protein interaction. On this basis, we have recently
disclosed that some small molecules containing indole nucleus
could mimic the Ile365/Asp366 dipeptide thus inhibiting the inter-
action between IN and its cofactor LEDGF/p75.1 The most active
compound named CHIBA-3003 maps this binding site well, engag-
ing interactions with some crucial residues of HIV-1 IN. In particu-
lar, we found, by docking experiments, that CHIBA-3003 (I)
established H-bond interactions with Glu-170 and His-171 while
the fused benzene ring of the indole system occupied an hydropho-
bic region formed by Leu102, Ala128 and Ala129.1 Recently, other
researchers identified, by docking experiments and site-directed
mutagenesis, the benzoic acid derivative D77 as IN-LEDGF/p75
inhibitor (III, Fig. 1) and suggested that other hydrophobic regions
could play a role in the IN/IBD interaction, in particular the area
We compared these MIFs with the previous docking results of
our hit compound CHIBA-3003 in IN CCD. As shown in Figure 3A,
CHIBA-3003 well mapped the most large areas described by N1
(magenta) and O (green) probes.
The CHIBA-3003 carboxylate group was able to interact with
Glu-170 and His-171, as well as overlapping some areas described
by N1 and O probes. We also observed that the most significant N1
area was filled by diketoacid portion. Moreover, the hydroxyl
group at C-4 position of CHIBA-3003 formed a hydrogen-bond with
the backbone carbonyl group of Gln168 whereas the benzene
fused-ring of indole nucleus of CHIBA-3003 is placed into IN hydro-
phobic pocket well described by a DRY map. Nevertheless, we
found that the most relevant area calculated by DRY probe (cyan
and indicated by a red arrow in Fig. 3A) was vacant. This area cor-
responds to the hydrophobic region located near the residue
Trp131 in the IN B chain. The analysis of these data prompted us
to reconsider a class of CHIBA-3003 analogues containing hydro-
phobic moieties that could possibly occupy this large and relevant
area described by the DRY probe.
In fact for the low activity of some derivatives and the lack in
our pharmacophore model of this important hydrophobic area
discovered by GRID we had previously supposed that the benzyl

L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
7517
Figure 3. GRID MIFs calculated for the interaction between HIV-1 INCCD interface
and the docking pose of CHIBA-3003 (I, Fig. 3A) and CHIBA-3000 (II, Fig. 3B). The
magenta, green, and cyan contours correspond to the molecular interaction fields
calculated for the selected probes (N1, O, and DRY). This figure was prepared using
the PyMOL program.19
2.2. Chemistry and biological activity
In order to obtain more information on this hydrophobic por-
tion, we synthesized and tested a small library of new N-benzyl-
substituted indoles (4a–h, 5a–h, and 6a–h) in which we selected
the mono- and dimethylbenzyl fragment as lipophilic moiety.
The synthesis of the new compounds was accomplished following
our previous reported procedures,10 employed to prepare indole
derivatives (Scheme 1).
The experimental details are reported in Section 4. All synthe-
sized compounds were tested in an AlphaScreen assay. This is a ro-
bust biochemical assay for identification of HIV integrase
inhibitors, targeting the interaction between HIV-IN and the cellu-
lar cofactor LEDGF/p75 which has been developed for promoting
the identification of novel inhibitors specific for this protein–pro-
tein interaction effectively.12 The results of the screening are
shown in Table 1. We tested the precursor ethyl esters (4a–h) as
well as the corresponding diketoacid derivatives (5a–h); further-
more we evaluated compounds 6a–h in which the diketoacid
(DKA) portion was transformed into the hydroxyfuranone moiety
which can be considered as a closed form of DKA function. All
Figure 2. Molecular interaction fields MIFs calculated in the HIV-1 INCCD (surface,
yellow) obtained by running GRID with selected probes. (a) N1 (À6 kcal/mol,
magenta), (b) O (À6 kcal/mol, green), and (c) DRY (À0.5 kcal/mol, cyan) are viewed
together with the IDB LEDGF/p75 portion (green cartoon). The IDB residues
essential for the HIV-1 interaction (Ile365, Asp366, Phe406, Val408) are drawn in
stick to visualize that the interaction fields correspond to actual atomic positions.
This figure was prepared using the PyMOL program.19
group was not so important for the inhibition of PPIs.1 Now, our
docking experiments have allowed us to observe that the
molecules benzylsubstituted such as CHIBA-3000, II (Figs. 1, 3B
and Table 1) show a different binding mode when compared with
N-unsubstituted analogue CHIBA-3003 (Fig. 3A) and have sug-
gested the introduction of some hydrophobic groups (i.e.,
Me2C6H3) suitable to improve the potency of CHIBA derivatives.
tested compounds produced inhibitory effects at 100 lM concen-
tration with a percentage ranging from 16% to 96%. The analysis
of available IC50 values (see Table 1) pointed out that the ester moi-
ety negatively affected the efficacy of this series of PP inhibitors

7518
L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
Table 1
a
Compound
% Inhibition at 100
l
M
IC50
(
lM)
CHIBA-3003 (I)
CHIBA-3000
71
56
24
45
60
57
58
33
47
52
20
16
86
93
55
40
72
94
34
61
81
70
96
38
70
69
35.0
76
*
4a
4b
4c
4d
4e
4f
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
*
170.7
*
*
*
*
*
*
*
68.0
28.5
*
*
*
3.5
Figure 4. Docking pose of 5h after minimization procedures and GRID MIF
calculated for select probe DRY (cyan contours). This figure was prepared using
the PyMOL program.19
*
53.3
8.7
*
11.7
(b) the hydroxyl group of the diketoacid moiety interacts with
Gln95; (c) the benzene fused-ring of indole nucleus is placed into
IN hydrophobic pocket; (d) the 3,5-dimethylbenzyl N-substituent
occupies the most relevant area calculated by DRY probes.
The highest inhibitory efficacy of compound 5h could be ex-
plained for its ability to well map the DRY area related to the cru-
cial residue Trp131.
*
6g
6h
7.5
122.8
a
Concentration required to inhibit the HIV-1 IN-LEDGF/p75
interaction by 50%.
*
Not determined.
While our studies were in progress, the crystal structures of
other small-molecule inhibitors active as PPIs in LEDGF/p75-bind-
ing pocket were solved.13 The most active reported compound was
Ar
H
H
N
N
i
N
ii
Ar
derivative IV (Fig. 1) that showed a biological activity of 1.37
the comparison of the docking result of our most active compound
5h (3.5 M) and the X-ray position of compound IV is showed in
Figure 5.
lM:
a
b
c
2-MeC6H4
3-MeC6H4
4-MeC6H4
d 2,3-(Me)2C6H3
e 2,4-(Me)2C6H3
2,5-(Me)2C6H3
CH3
OH
OH
CH3
OH
l
O
3 a-h
O
1
2
These two compounds show comparable activity and present a
similar binding mode. In fact their carboxylate groups are in the
same position and the indole nucleus of 5h and the phenyl group
of IV occupy the same hydrophobic area. A different orientation
is observed between the dimethylphenyl moiety of 5h and the ben-
zene fused-ring containing a chlorine atom of IV which interacts
with two different hydrophobic spaces. These observations could
be particular useful for the optimization of these two important
series of new integrase-LEDGF/p75 interaction inhibitors.
Ar
f
iii
N
g 2,6-(Me)2C6H3
h 3,5-(Me)2C6H3
COOH
iv
OH
Ar
O
HO
5 a-h
N
Ar
v
COOEt
OH
N
O
HO
O
4 a-h
O
OH
O
HO
6 a-h
Scheme 1. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, 12 h, rt; (ii) appropri-
ate methylbenzyl chloride or bromide, K2CO3, DMF, 10 min. 100 °C, 100 W; (iii)
diethyl oxalate, dry CH3ONa, THF, two separate steps under the same conditions:
2 min 50 °C, 250 W; (iv) NaOH 2 N, MeOH, 90 min, rt; (v) CH2O, 1.5 h rt.
and that the most active molecules were the derivatives 5h, 6c, 6e,
and 6g, active at low micromolar concentration. In particular the
most potent derivative of the series (5h) was 10-fold more effica-
cious than CHIBA-3003 (3.5 lM vs 35.0 lM).
To investigate the recognition process we docked the synthe-
sized compounds into the region of protein–protein interaction be-
tween HIV-1 IN and cofactor LEDGF/p75. All obtained complexes
were subsequently optimized by a minimization procedure. Figure
4 shows a plausible binding mode of the most interesting deriva-
tive 5h which displays the following interactions: (a) the carboxyl-
ate group was able to interact with Glu-170, His-171, and Thr174;
Figure 5. Docking pose of compound 5h (magenta), compared to the crystallized
position of IV (yellow) into the LEDGF/p75 binding pocket of IN-core and GRID MIF
calculated for select probe DRY (cyan contours). This figure was prepared using the
PyMOL program.19

L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
7519
analyses (C, H, N) were carried out on a Carlo Erba Model 1106 Ele-
mental 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 DMSO-d6 on a Varian Gemini-300 spectrometer.
Chemical shifts are expressed in d (ppm) and coupling constants
(J) in hertz.
3. Conclusion
In conclusion the importance of an additional lipophilic feature
for IN-LEDGF/p75 inhibition has been suggested following the
identification of a new hydrophobic region in IBD binding site. No-
vel small molecules active as PPIs have been designed, synthesized
and tested. The obtained information could be helpful for the dis-
covery of new inhibitors of this emerging and promising PPI target
particularly important for anti-AIDS drug design.
Compound 2 was prepared following a previously reported
procedure and spectroscopic data are in accordance with the
literature.1
4. Experimental section
4.1. Molecular modeling
The synthesis of new compounds 3a–h, 4a–h, and 5a–h was
achieved using previously published procedures.1
Both the GRID analysis and docking were performed using IN
structure retrieved by X-ray crystallographic of the dimeric cata-
lytic core domain of HIV-1 IN complexed with LEDGF/p75 IBD
deposited in the RCSB Protein Data Bank (entry code 2 B4J).11
4.5.1. 3-Acetyl-1-(2-methyl-benzyl)-4-hydroxy-1H-indole (3a)
Compound 3a: (87%) mp 130 °C dec; 1H NMR (300 MHz, CDCl3):
d = 2.34 (s, 3H, CH3), 2.52 (s, 3H, CH3), 5.46 (s, 2H, CH2), 6.50–7.25
(m, 7H, ArH), 8.50 (s, 1H, ArH), 11.57 (s, 1H, OH). Anal. Calcd for
C
18H17FNO2: C, 77.40; H, 6.13; N, 5.01. Found: C, 77.64; H, 6.42;
4.2. GRID calculations
N, 5.13.
The calculations were performed with version 22 of the GRID
software.14 The GRID box dimensions were chosen to encompass
all residues able to interact with IBD; and the NPLA (Number of
Planes per Å for the grid box) was set to 2. The probes applied were
DRY probes to describe the potential hydrophobic interactions, sp2
carbonyl oxygen (O), and the neutral flat amino (N1) probes to
evaluate the hydrogen-bond-donor and acceptor capacity of the
target, respectively.
4.5.2. 3-Acetyl-1-(3-methyl-benzyl)-4-hydroxy-1H-indole (3b)
Compound 3b: (78%) mp 135–137 °C; 1H NMR (300 MHz,
CDCl3): d = 2.33 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.26 (s, 2H, CH2),
6.71–7.23 (m, 7H, ArH), 7.66 (s, 1H, ArH), 11.53 (s, 1H, OH). Anal.
Calcd for C18H17FNO2: C, 77.40; H, 6.13; N, 5.01. Found: C, 77.22;
H, 6.01; N, 5.32.
4.5.3. 3-Acetyl-1-(4-methyl-benzyl)-4-hydroxy-1H-indole (3c)
Compound 3c: (73%) mp 129–131 °C; 1H NMR (300 MHz,
CDCl3): d = 2.35 (s, 3H, CH3), 2.52 (s, 3H, CH3), 5.25 (s, 2H, CH2),
6.71–7.20 (m, 7H, ArH), 7.65 (s, 1H, ArH), 11.52 (s, 1H, OH). Anal.
Calcd for C18H17FNO2: C, 77.40; H, 6.13; N, 5.01. Found: C, 77.63;
H, 6.41; N, 5.31.
4.3. Molecular docking experiments
Hydrogen atoms were added to IN protein using the Biopolymer
module in SYBYL 8.0.15 The structures of the ligands were con-
structed using standard bond lengths and angles from the SYBYL
8.0 fragment library and were fully optimized by the semiempirical
quantum mechanical method AM1. Coordinates for the LEDGF
dipetide Ile365-Asp366 were taken directly from the C chain of
the PDB 2 B4J crystal structure and used as a control of program
performance. Docking studies were performed using CCDC’s GOLD
(Genetic Optimization for Ligand Docking) software package ver-
sion 3.1.1.16 ChemScore was chosen as a fitness function and the
standard default settings were used in all the calculations. For each
of the 100 independent genetic algorithm runs, a default maximum
of 100,000 genetic operations was performed, using the default
operator weights and a population size of 100 chromosomes. De-
fault cutoff values of 2.5 Å for hydrogen-bonds and 4.0 Å for VdW
were employed. All single bonds were treated as rotable. Results
differing by less than 1.5 Å in ligand-all atom RMSD, were clus-
tered together. A 15.0 Å radius active site was drawn on the origi-
nal position of the LEDGF/p75 IBD dipeptide I365-D366 and
automated cavity detection was used.
4.5.4. 3-Acetyl-1-(2,3-dimethyl-benzyl)-4-hydroxy-1H-indole (3d)
Compound 3d: (90%) mp 166–168 °C; 1H NMR (300 MHz,
CDCl3): d = 2.17 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.46 (s, 3H, CH3),
5.25 (s, 2H, CH2), 6.73–7.23 (m, 6H, ArH), 7.45 (s, 1H, ArH), 11.56
(s, 1H, OH). Anal. Calcd for C19H19FNO2: C, 77.79; H, 6.53; N,
4.77. Found: C, 77.96; H, 6.72; N, 5.02.
4.5.5. 3-Acetyl-1-(2,4-dimethyl-benzyl)-4-hydroxy-1H-indole
(3e)
Compound 3e: (74%) mp 139–141 °C; 1H NMR (300 MHz,
CDCl3): d = 2.19 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.52 (s, 3H, CH3),
5.21 (s, 2H, CH2), 6.68–7.19 (m, 6H, ArH), 7.54 (s, 1H, ArH), 11.13
(s, 1H, OH). Anal. Calcd for C19H19FNO2: C, 77.79; H, 6.53; N,
4.77. Found: C, 77.61; H, 6.69; N, 4.92.
4.5.6. 3-Acetyl-1-(2,5-dimethyl-benzyl)-4-hydroxy-1H-indole
(3f)
Compound 3f: (86%) mp 187–140 °C; 1H NMR (300 MHz,
CDCl3): d = 2.24 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.47 (s, 3H, CH3),
5.19 (s, 2H, CH2), 6.73–7.23 (m, 6H, ArH), 7.46 (s, 1H, ArH), 11.56
(s, 1H, OH). Anal. Calcd for C19H19FNO2: C, 77.79; H, 6.53; N,
4.77. Found: C, 78.02; H, 6.38; N, 4.51.
4.4. Minimization process
All resulting docked complexes were subjected to energy mini-
mization using 1000 steps of SD followed by 1000 steps of CG.
4.5. Chemistry
4.5.7. 3-Acetyl-1-(2,6-dimethyl-benzyl)-4-hydroxy-1H-indole
(3g)
Compound 3g: (61%) mp 170–172 °C; 1H NMR (300 MHz,
CDCl3): d = 2.28 (s, 6H, CH3), 2.35 (s, 3H, CH3), 5.21 (s, 2H, CH2),
6.76–7.31 (m, 7H, ArH), 11.60 (s, 1H, OH). Anal. Calcd for
All microwave-assisted reactions were carried out in a CEM Fo-
cused Microwave Synthesis System, Model Discover, working at the
power necessary for refluxing under atmospheric conditions (i.e.,
250–300 W). Melting points were determined on a BUCHI Melting
Point B-545 apparatus and have not been corrected. Elemental
C19H19FNO2: C, 77.79; H, 6.53; N, 4.77. Found: C, 77.62; H, 6.74;
N, 4.91.

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L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
4.5.8. 3-Acetyl-1-(3,5-dimethyl-benzyl)-4-hydroxy-1H-indole (3h)
Compound 3h: (89%) mp 167–169 °C; 1H NMR (300 MHz, CDCl3):
d = 2.29 (s, 6H, CH3), 2.53 (s, 3H, CH3), 5.21 (s, 2H, CH2), 6.71–7.20
(m, 6H, ArH), 7.65 (s, 1H, ArH), 11.54 (s, 1H, OH). Anal. Calcd for
2H, CH2), 5.22 (s, 2H, CH2), 5.28–8.13 (m, 8H, ArH and CH), 14.63 (s,
1H, OH). Anal. Calcd for C23H23NO5: C, 70.22; H, 5.89; N, 3.56.
Found: C, 70.39; H, 5.64; N, 3.44.
C
19H19FNO2: C, 77.79; H, 6.53; N, 4.77. Found: C, 77.53; H, 6.45; N, 4.96.
4.5.17. 4-[1-(2-Methylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-hyd-
roxy-4-oxobut-2-enoic acid (5a)
4.5.9. Ethyl 4-[1-(2-methyl-benzyl)-4-hydroxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (4a)
Compound 5a: (30%) mp 210 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 2.34 (s, 3H, CH3), 5.49 (s, 2H, CH2), 6.58–7.24 (m, 8H, ArH
and CH), 8.91 (s, 1H, ArH), 11.30 (s, 1H, OH). Anal. Calcd for
Compound 4a: (90%) mp 271 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.21 (t, J = 7.26, 3H, CH3), 2.24 (s, 3H, CH3), 4.08 (q, J = 7.26,
2H, CH2), 5.25 (s, 2H, CH2), 5.35–7.96 (m, 9H, ArH and CH), 14.33 (s,
1H, OH). Anal. Calcd for C22H21NO5: C, 69.65; H, 5.58; N, 3.69.
Found: C, 69.74; H, 5.71; N, 3.48.
C20H17NO5: C, 68.37; H, 4.88; N, 3.99. Found: C, 68.13; H, 4.59;
N, 4.09.
4.5.18. 4-[1-(3-Methylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-hyd-
roxy-4-oxobut-2-enoic acid (5b)
4.5.10. Ethyl 4-[1-(3-methyl-benzyl)-4-hydroxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (4b)
Compound 5b: (45%) mp 200 °C; 1H NMR (300 MHz, DMSO-d6):
d = 2.25 (s, 3H, CH3), 5.42 (s, 2H, CH2), 6.55–7.24 (m, 8H, ArH and
CH), 9.07 (s, 1H, ArH), 11.25 (s, 1H, OH). Anal. Calcd for C20H17NO5:
C, 68.37; H, 4.88; N, 3.99. Found: C, 68.62; H, 4.63; N, 3.81.
Compound 4b: (88%) mp 268 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.21 (t, J = 7.26, 3H, CH3), 2.24 (s, 3H, CH3), 4.08 (q, J = 7.26,
2H, CH2), 5.27 (s, 2H, CH2), 5.32–7.92 (m, 9H, ArH and CH), 14.21 (s,
1H, OH). Anal. Calcd for C22H21NO5: C, 69.65; H, 5.58; N, 3.69.
Found: C, 69.32; H, 5.41; N, 3.73.
4.5.19. 4-[1-(4-Methylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-hyd-
roxy-4-oxobut-2-enoic acid (5c)
Compound 5c: (55%) mp 216–218 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.24 (s, 3H, CH3), 5.40 (s, 2H, CH2), 6.54–7.27 (m,
8H, ArH and CH), 9.06 (s, 1H, ArH), 11.25 (s, 1H, OH). Anal. Calcd
for C20H17NO5: C, 68.37; H, 4.88; N, 3.99. Found: C, 68.54; H,
4.99; N, 3.73.
4.5.11. Ethyl 4-[1-(4-methyl-benzyl)-4-hydroxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (4c)
Compound 4c: (94%) mp 280 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.23 (t, J = 7.26, 3H, CH3), 2.23 (s, 3H, CH3), 4.09 (q, J = 7.26,
2H, CH2), 5.26 (s, 2H, CH2), 5.31–8.12 (m, 9H, ArH and CH), 14.54 (s,
1H, OH). Anal. Calcd for C22H21NO5: C, 69.65; H, 5.58; N, 3.69.
Found: C, 69.74; H, 5.47; N, 3.81.
4.5.20. 4-[1-(2,3-Dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (5d)
Compound 5d: (43%) mp 190 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.23 (s, 3H, CH3), 2.26 (s, 3H, CH3), 5.49 (s, 2H,
CH2), 6.47–7.11 (m, 7H, ArH and CH), 8.87 (s, 1H, ArH), 11.28
(s, 1H, OH). Anal. Calcd for C21H19NO5: C, 69.03; H, 5.24; N,
3.83. Found: C, 69.32; H, 5.46; N, 3.68.
4.5.12. Ethyl 4-[1-(2,3-dimethyl-benzyl)-4-hydroxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (4d)
Compound 4d: (82%) mp 205 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.22 (t, J = 7.26, 3H, CH3), 2.22 (s, 6H, CH3), 4.08 (q, J = 7.26,
2H, CH2), 5.33 (s, 2H, CH2), 5.38–7.99 (m, 8H, ArH and CH), 13.68 (s,
1H, OH). Anal. Calcd for C23H23NO5: C, 70.22; H, 5.89; N, 3.56.
Found: C, 70.39; H, 5.51; N, 3.86.
4.5.21. 4-[1-(2,4-Dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (5e)
Compound 5e: (56%) mp 235 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.20 (s, 3H, CH3), 2.23 (s, 3H, CH3), 5.29 (s, 2H,
CH2), 6.28–7.62 (m, 8H, ArH and CH), 10.14 (s, 1H, OH). Anal.
Calcd for C21H19NO5: C, 69.03; H, 5.24; N, 3.83. Found: C, 69.22;
H, 5.56; N, 3.71.
4.5.13. Ethyl 4-[1-(2,4-dimethyl-benzyl)-4-hydroxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (4e)
Compound 4e: (91%) mp 269 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.16 (t, J = 7.26, 3H, CH3), 2.21 (s, 6H, CH3), 4.08 (q, J = 7.26,
2H, CH2), 5.27 (s, 2H, CH2), 5.32–7.70 (m, 8H, ArH and CH), 14.56 (s,
1H, OH). Anal. Calcd for C23H23NO5: C, 70.22; H, 5.89; N, 3.56.
Found: C, 70.03; H, 5.71; N, 3.42.
4.5.22. 4-[1-(2,5-Dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (5f)
Compound 5f: (43%) mp 200 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.12 (s, 3H, CH3), 2.26 (s, 3H, CH3), 5.43 (s, 2H,
CH2), 6.56–7.12 (m, 7H, ArH and CH), 8.84 (s, 1H, ArH), 11.36
(s, 1H, OH). Anal. Calcd for C21H19NO5: C, 69.03; H, 5.24; N,
3.83. Found: C, 69.18; H, 5.08; N, 3.98.
4.5.14. Ethyl 4-[1-(2,5-dimethyl-benzyl)-4-hydroxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (4f)
Compound 4f: (83%) mp 298 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.16 (t, J = 7.26, 3H, CH3), 2.11 (s, 3H, CH3), 2.24 (s, 3H, CH3),
4.09 (q, J = 7.26, 2H, CH2), 5.28 (s, 2H, CH2), 5.34–7.71 (m, 8H, ArH
and CH), 14.56 (s, 1H, OH). Anal. Calcd for C23H23NO5: C, 70.22; H,
5.89; N, 3.56. Found: C, 70.41; H, 5.62; N, 3.47.
4.5.23. 4-[1-(2,6-Dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (5g)
Compound 5g: (43%) mp 199 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.21 (s, 6H, CH3), 5.43 (s, 2H, CH2), 6.58–7.21 (m,
7H, ArH and CH), 8.17 (s, 1H, ArH), 11.31 (s, 1H, OH). Anal. Calcd
for C21H19NO5: C, 69.03; H, 5.24; N, 3.83. Found: C, 68.92; H, 5.44;
N, 3.62.
4.5.15. Ethyl 4-[1-(2,6-dimethyl-benzyl)-4-hydroxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (4g)
Compound 4g: (70%) mp 199 °C; 1H NMR (300 MHz, DMSO-d6):
d = 1.19 (t, J = 7.26, 3H, CH3), 2.20 (s, 6H, CH3), 4.05 (q, J = 7.26, 2H,
CH2), 5.25 (s, 2H, CH2), 5.28–7.20 (m, 8H, ArH and CH), 14.56 (s, 1H,
OH). Anal. Calcd for C23H23NO5: C, 70.22; H, 5.89; N, 3.56. Found: C,
70.41; H, 5.62; N, 3.47.
4.5.24. 4-[1-(3,5-Dimethylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (5h)
Compound 5h: (40%) mp 170 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.20 (s, 6H, CH3), 5.37 (s, 2H, CH2), 6.55–7.22 (m,
7H, ArH and CH), 9.05 (s, 1H, ArH), 11.27 (s, 1H, OH). Anal. Calcd
for C21H19NO5: C, 69.03; H, 5.24; N, 3.83. Found: C, 68.89; H, 5.41;
N, 3.65.
4.5.16. Ethyl 4-[1-(3,5-dimethyl-benzyl)-4-hydroxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (4h)
Compound 4h: (93%) mp 202 °C dec; 1H NMR (300 MHz, DMSO-
d6): d = 1.25 (t, J = 7.26, 3H, CH3), 2.19 (s, 6H, CH3), 4.11 (q, J = 7.26,

L. De Luca et al. / Bioorg. Med. Chem. 18 (2010) 7515–7521
7521
4.6. General procedure for the synthesis of 4-[1-(methylbenzyl)-
6.58–7.22 (m, 6H, ArH), 8.18 (s, 1H, ArH), 11.61 (s, 1H, OH). Anal.
Calcd for C22H19NO5: C, 70.02; H, 5.07; N, 3.71. Found: C, 70.23;
H, 5.29; N, 3.94.
4-hydroxy-1H-indol-3-oyl)-3-hydroxyfuran-2(5H)-ones (6a–h) 17,18
A solution of 40% aqueous formaldehyde in water (4 ml) was
added to a mixture of 4a–h (1 mmol) in diethyl ether (5 ml). The
stirring was then continued until clear layers were formed (usually
within 1–2 h). Sometimes, an additional 4 ml of water was added if
the reaction was especially thick or when the solid appeared to re-
act slowly. The clear, aqueous bottom layer was removed and the
organic layer extracted twice with 5 ml of water. The combined
aqueous extracts were cooled followed by acidification with 3 ml
of concentrated hydrochloric acid. The corresponding furanone
precipitate and the solution was then cooled overnight to ensure
complete product formation. The resulting solid was collected,
dried, and recrystallized from ethanol.
4.6.8. 4-[1-(3,5-Dimethylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-
hydroxyfuran-2(5H)-one (6h)
Compound 6h: (31%) mp 202–204 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.21 (s, 6H, CH3), 5.11 (2H, CH2), 5.38 (s, 2H, CH2),
6.51–7.23 (m, 6H, ArH), 8.96 (s, 1H, ArH), 11.58 (s, 1H, OH). Anal.
Calcd for C22H19NO5: C, 70.02; H, 5.07; N, 3.71. Found: C, 70.19;
H, 5.19; N, 3.87.
4.7. LEDGF/p75-HIV-1 Integrase interaction screening
(AlphaScreen technology)
The AlphaScreen assay was performed as described previously.12
Reactions were performed in 25 ll final volume in 384-well Opti-
well™ microtiter plates (Perkin–Elmer). The reaction buffer con-
tained 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 com-
pounds at 4 °C for 30 min. The compounds were added in varying
4.6.1. 4-[1-(2-Methylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-hyd-
roxyfuran-2(5H)-one (6a)
Compound 6a: (33%) mp 200 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.34 (s, 3H, CH3), 4.96 (2H, CH2), 5.43 (s, 2H,
CH2), 6.43–7.21 (m, 7H, ArH), 10.42 (s, 1H, ArH), 13.88 (s, 1H,
OH). Anal. Calcd for C21H17NO5: C, 69.41; H, 4.72; N, 3.85. Found:
C, 69.57; H, 4.98; N, 3.71.
concentrations from 1 up to 100
nant flag-LEDGF/p75 was added and incubation was extended by
another hour at 4 °C. Subsequently, 5 l of Ni-chelate-coated accep-
tor beads and 5 l of anti-flag donor beads were added to a final con-
centration of 20 g/ml of both beads. Proteins and beads were
lM. Afterward 100 nM of recombi-
4.6.2. 4-[1-(3-Methylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-hyd-
roxyfuran-2(5H)-one (6b)
l
l
Compound 6b: (38%) mp 246–248 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.21 (s, 3H, CH3), 4.89 (2H, CH2), 5.35 (s, 2H, CH2),
6.33–7.22 (m, 7H, ArH), 10.50 (s, 1H, ArH), 13.90 (s, 1H, OH). Anal.
Calcd for C21H17NO5: C, 69.41; H, 4.72; N, 3.85. Found: C, 69.21; H,
4.61; N, 3.98.
l
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 mea-
sured in the EnVision plate reader (Perkin–Elmer, Benelux) and ana-
lyzed using the EnVision manager software.
4.6.3. 4-[1-(4-Methylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-hyd-
roxyfuran-2(5H)-one (6c)
Compound 6c: (26%) mp 186 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.24 (s, 3H, CH3), 5.00 (2H, CH2), 5.38 (s, 2H, CH2),
6.81–7.42 (m, 8H, ArH), 11.25 (s, 1H, OH). Anal. Calcd for
Acknowledgment
This work was supported by the European Commission
(HEALTH-F3-2008-201032) (THINC project).
C21H17NO5: C, 69.41; H, 4.72;N, 3.85. Found: C, 69.33; H, 4.59; N, 3.72.
4.6.4. 4-[1-(2,3-Dimethylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-
hydroxyfuran-2(5H)-one (6d)
References and notes
Compound 6d: (42%) mp 172 °C dec; 1H NMR (300 MHz,
DMSO-d6): d = 2.20 (s, 3H, CH3), 2.25 (s, 3H, CH3), 5.09 (2H,
CH2), 5.40 (s, 2H, CH2), 6.48–7.20 (m, 6H, ArH), 8.69 (s, 1H,
ArH), 11.39 (s, 1H, OH). Anal. Calcd for C22H19NO5: C, 70.02; H,
5.07; N, 3.71. Found: C, 70.21; H, 5.23; N, 3.42.
1. De Luca, L.; Barreca, M. L.; Ferro, S.; Christ, F.; Iraci, N.; Gitto, R.; Monforte, A.
M.; Debyser, Z.; Chimirri, A. ChemMedChem 2009, 4, 1311.
2. De Clercq, E. Med. Res. Rev. 2009.
3. Barreca, M. L.; Iraci, N.; De Luca, L.; Chimirri, A. ChemMedChem 2009, 4, 1446.
4. Nikolenko, G. N.; Delviks-Frankenberry, K. A.; Pathak, V. K. J. Virol. 2010, 84, 5238.
5. Broglia, R.; Levy, Y.; Tiana, G. Curr. Opin. Struct. Biol. 2008, 18, 60.
6. Garcia-Rivera, J. A.; Bueno, M. T.; Morales, E.; Kugelman, J. R.; Rodriguez, D. F.;
Llano, M. J. Virol. 2010, 84, 740.
4.6.5. 4-[1-(2,4-Dimethylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-
hydroxyfuran-2(5H)-one (6e)
7. Busschots, K.; Voet, A.; De Maeyer, M.; Rain, J. C.; Emiliani, S.; Benarous, R.;
Desender, L.; Debyser, Z.; Christ, F. J. Mol. Biol. 2007, 365, 1480.
8. Greene, W. C.; Debyser, Z.; Ikeda, Y.; Freed, E. O.; Stephens, E.; Yonemoto, W.;
Buckheit, R. W.; Este, J. A.; Cihlar, T. Antiviral. Res. 2008, 80, 251.
9. Hare, S.; Shun, M. C.; Gupta, S. S.; Valkov, E.; Engelman, A.; Cherepanov, P. PLoS
Pathog. 2009, 5, e1000259.
10. Du, L.; Zhao, Y.; Chen, J.; Yang, L.; Zheng, Y.; Tang, Y.; Shen, X.; Jiang, H. Biochem.
Biophys. Res. Commun. 2008, 375, 139.
11. Cherepanov, P.; Ambrosio, A. L.; Rahman, S.; Ellenberger, T.; Engelman, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 17308.
Compound 6e: (42%) mp 184–186 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.23 (s, 3H, CH3), 2.27 (s, 3H, CH3), 5.07 (2H, CH2),
5.42 (s, 2H, CH2), 6.53–7.21 (m, 6H, ArH), 8.75 (s, 1H, ArH), 11.54
(s, 1H, OH). Anal. Calcd for C22H19NO5: C, 70.02; H, 5.07; N, 3.71.
Found: C, 70.38; H, 5.16; N, 3.59.
12. Al-Mawsawi, L. Q.; Christ, F.; Dayam, R.; Debyser, Z.; Neamati, N. FEBS Lett.
2008, 582, 1425.
13. 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. Nat. Chem. Biol. 2010, 6, 442.
14. Grid 22, Molecular Discovery Ltd, Pinner, Middlesex, UK.
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17. Ferro, S.; Barreca, M. L.; De Luca, L.; Rao, A.; Monforte, A. M.; Debyser, Z.;
Witvrouw, M.; Chimirri, A. Arch. Pharm. (Weinheim) 2007, 340, 292.
18. Ferro, S.; De Grazia, S.; De Luca, L.; Barreca, M. L.; Debyser, Z.; Chimirri, A.
Heterocycles 2009, 78, 947.
4.6.6. 4-[1-(2,5-Dimethylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-
hydroxyfuran-2(5H)-one (6f)
Compound 6f: (26%) mp 219–221 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.15 (s, 3H, CH3), 2.26 (s, 3H, CH3), 5.07 (2H, CH2),
5.42 (s, 2H, CH2), 6.54–7.19 (m, 6H, ArH), 8.71 (s, 1H, ArH), 11.47
(s, 1H, OH). Anal. Calcd for C22H19NO5: C, 70.02; H, 5.07; N, 3.71.
Found: C, 70.24; H, 5.19; N, 3.84.
4.6.7. 4-[1-(2,6-Dimethylbenzyl)-4-hydroxy-1H-indol-3-oyl)-3-
hydroxyfuran-2(5H)-one (6g)
Compound 6g: (20%) mp 235–237 °C; 1H NMR (300 MHz,
DMSO-d6): d = 2.24 (s, 6H, CH3), 4.97 (2H, CH2), 5.34 (s, 2H, CH2),
19. The PyMOL Molecular Graphics System, W. L. DeLano, DeLano Scientific LLC,
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