Synthesis and biological evaluation of novel antiviral agents as protein-protein interaction inhibitors

Article abstract of DOI:10.3109/14756366.2013.766609

In continuation of our research efforts toward the identification and optimization for novel inhibitors of interaction between human immunodeficiency virus type 1 integrase and cellular cofactor LEDGF/p75, we designed and synthesized a new series of 4-ben

Full text of DOI:10.3109/14756366.2013.766609

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(2): 237–242
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.766609
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ORIGINAL ARTICLE
Synthesis and biological evaluation of novel antiviral agents as
protein–protein interaction inhibitors
Stefania Ferro1, Laura De Luca1, Francesca Morreale1, Frauke Christ2, Zeger Debyser2, Rosaria Gitto1,
and Alba Chimirri1
1
2
Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universita di Messina, Messina, Italy and Molecular Virology and Gene Therapy
`
KU Leuven and IRC KULAK, Leuven, Flanders, Belgium
Abstract
Keywords
In continuation of our research efforts toward the identification and optimization for novel
inhibitors of interaction between human immunodeficiency virus type 1 integrase and cellular
cofactor LEDGF/p75, we designed and synthesized a new series of 4-benzylindole derivatives.
Most of the title compounds proved to be able to block this protein–protein interaction (PPI),
with a percentage ranging from 30% to 90% at 100 mM. The most promising derivative
was compound 10b showing IC50 value of 6.41 mM. The main structure–activity relationships
(SAR) are discussed and rationalized by docking studies.
Docking studies, HIV-1, indoles, LEDGF/p75,
synthesis
History
Received 20 December 2012
Revised 10 January 2013
Accepted 10 January 2013
Published online 15 July 2013
Introduction
Despite HAART remarkably decreases viral load and provides
a significant improvement in the life expectancy of HIV/AIDS
patients, several factors, including the emergence of drug-resistant
viral strains, drug toxicity, the poor ability of patients to adhere to
the prescribed therapy and costs, require to refine the current
therapy and develop new therapeutic paradigms targeting other
In the last years, the global community has made great progress
in responding to the acquired immunodeficiency syndrome
(AIDS) epidemic but there are yet about 40 million individuals
living with the human immunodeficiency virus type 1 (HIV-1)
and every day thousands more people are newly infected with
HIV1. United Nations Member States set clear targets for
significantly reducing HIV infection and AIDS death and for
scaling up treatment by 2015.
The goal is to strengthen and sustain a valid response to AIDS
including the prevention of transmission of HIV as well as
providing support and care to those already living with the
virus, to prevent the HIV/AIDS epidemic from becoming a severe
pandemic. Particularly, it is necessary to ensure that 15 million
people can be receiving the antiretroviral therapy, thus reducing
the transmission of HIV at least among children.
The overall number of people living with HIV has increased
as result of new infections and the beneficial effects of the
more widely available highly active anti-retroviral therapy
(HAART), which changed AIDS from a rapidly lethal disease
into a chronic manageable condition; nevertheless, new efforts are
needed.
The currently FDA approved anti-HIV drugs belong to several
different groups such as nucleoside reverse transcriptase inhibitors
(NRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs),
nonnucleoside reverse transcriptase inhibitors (NNRTIs), protease
inhibitors (PIs), fusion inhibitors (FIs), co-receptor inhibitors
(CRIs) and integrase inhibitors (INIs)2.
steps in the viral replication process3,4
.
In this context, HIV-1 integrase (IN), the enzyme which
catalyzes the integration of viral cDNA into the host genome,
represents a validated target for the development of new drugs
against HIV-1 infection5–9. Furthermore, discovery of the keto-
enol acid class, often referred as diketoacid (DKA) class, and their
analogues and bioisosters as IN inhibitors was an additional
advance in the validation of this enzyme as a therapeutically
viable antiretroviral drug target, representing major leads in the
development of anti-HIV-1 IN drugs.
More recently, it was demonstrated that the multifunctional IN
interacts with viral DNA and its key cellular cofactor lens
epithelium-derived growth factor (LEDGF/p75) to effectively
integrate the reverse transcript into a host cell chromosome10,11
.
LEDGF/p75 binds HIV-1 IN via a small IN-binding domain
(LEDGFIBD, residues 347–429) within its C-terminal region. IBD
is both necessary and sufficient for interaction with HIV-1 IN12.
Taking into account that the association between IN and the
cellular cofactor LEDGF/p75 can be considered as one of the
most promising IN interaction for the design of protein–protein
disrupting therapeutics13, we have directed our research toward
the identification of new small molecules that are able to inhibit
the protein–protein interaction (PPI) between the enzyme and its
cofactor.
In previous research from our laboratory, it was found that
N-benzyl-indoles, bearing diketo acid moiety, were able to exert
a specific blockade of the IN/LEDGF interaction14–18 and among
Address for correspondence: Stefania Ferro, Dipartimento di Scienze
`
del Farmaco e dei Prodotti per la Salute, Universita di Messina, Viale
Annunziata, I-98168 Messina, Italy. E-mail: sferro@unime.it.

238 S. Ferro et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 237–242
Figure 1. Chemical structure of CHI-1043,
CHIBA-3000, CHIBA-3017 and
CHIBA-3053.
them we found CHI-1043, CHIBA-3000 and some methylbenzyl of the ligands were kept fixed during docking calculation. The
derivatives such as CHIBA-3017 and CHIBA-3053 (Figure 1) molecules containing the diketo acid moiety were simulated
as interesting PPI inhibitors14,19
.
in the mono-ionized form24.
Encouraged by these promising results, which could represent
an interesting starting point for the development of novel dual
drugs, and with the aim of achieving further information about the
Chemistry
IN/LEDGF protein interaction, we focused our efforts to synthe- All commercially available reagents and solvents were used
size new indole analogues. Hence, several N-benzylsubstituents without any further purification. The microwave-assisted reac-
were inserted and structural modification of diketo acid portion tions were carried out in a CEM Focused Microwave Synthesis
was performed, replacing this critical moiety in a corresponding System, Model Discover (CEM, Matthews, NC), working at the
closed system which was able to maintain the important polar power necessary for refluxing under atmospheric conditions.
contacts with IBD. Moreover, docking simulations were carried Melting points were determined on a BUCHI Melting Point B-
out to investigate the possible binding mode of title compounds. 545 apparatus (Buchi, Milano, Italy) and are uncorrected.
Elemental analyses (C, H, N) were carried out on a Carlo Erba
Material and methods
Model 1106 Elemental Analyzer (Carlo Erba, Arese (MI), Italy)
and the results are within ꢀ0.4% of the theoretical values. Merck
silica gel 60 F254 (Darmstadt, Germany) plates were used for
Computational studies
Docking studies were performed using GOLD software package20 analytical TLC; column chromatography was performed on
version 4.1.1 (Gold, UK) and GoldScore scoring function as Merck silica gel 60 (230–400 mesh) and Flash Chromatography
described in our previous paper21.
(FC) on a Biotage SP1 EXP (Biotage, Uppsala, Sweden). 1H-
The crystal structure of the dimeric INCCD complexed with the NMR spectra were recorded in CDCl3 with TMS as internal
LEDGF/p75IBD was retrieved from the RCSB Protein Data Bank standard or [D6]DMSO on a Varian Gemini-300 spectrometer
(entry code 2B4J)22 and used for our docking simulations. The (Varia, Gemini, USA). Chemical shifts were expressed in ꢀ (ppm)
LEDGF structure was removed and hydrogen atoms were added and coupling constants (J) in hertz (Hz). All the exchangeable
to the IN protein in Discovery Studio 2.5.523. The standard default protons were confirmed by the addition of D2O.
˚
settings were used in all calculations. A 20.0 A radius active site Synthesis of 3-acetyl-4-methoxy-1H-indole (2a) and 3-acetyl-
was drawn on the original position of the LEDGFIBD dipeptide 4-hydroxy-1H-indole (2b) was carried out following the previ-
Ile365-Asp366 and automated cavity detection was used. Results ously reported procedure14,15,25. Phosphoryl chloride (0.92 mL,
˚
differing by less than 0.75 A in ligand-all atom RMSD were 10 mmol) was added to ice cold dimethylacetamide (2.79 mL,
clustered together. Two rotatable bonds of the diketo acid moiety 30 mmol) under stirring and cooling in ice. 4-Methoxy-1H-indole

DOI: 10.3109/14756366.2013.766609
Synthesis and biological evaluation of novel antiviral agents as PPI inhibitors 239
1a (147.18 mg, 1 mmol) or 4-hydroxy-1H-indole 1b (133.15 mg, tube (diam. 2 cm), stirred and irradiated at continuous temperature
1 mmol) was added and the reaction mixture was stirred at room in a microwave oven for two successive time intervals under
temperature for 12 h, and then poured over ice and basified with the same conditions (250 W, 2 min, 50 ꢁC). The solvent was
a 4N aqueous sodium hydroxide solution. The mixture was concentrated under reduced pressure and collected yellow solid
extracted with ethyl acetate and dried over Na2SO4. After the was crystallized from ethanol and diethyl ether (1:4).
removal of solvent under reduced pressure, the residue was
Ethyl
4-{1-(4-methyl-benzyl)-4-methoxy-1H-indol-3-yl}-2-
powdered by treating with diethyl ether and recrystallized from
dichloromethane.
For the obtained derivatives 2a and 2b, spectral data are in
hydroxy-4-oxobut-2-enoate (6a). Spectral data are in accordance
with the literature26.
accordance with the literature14,25
.
Ethyl 4-{1-[4-(trifluoromethyl)benzyl]-4-methoxy-1H-indol-3-yl}-
2-hydroxy-4-oxobut-2-enoate (7a). m.p. 150 ꢁC dec., yield 89%;
1H-NMR (ꢀ) 1.25 (t, J ¼ 7.2, 3H, CH3), 3.86 (s, 3H, CH3), 4.15
(d, J ¼ 5.5, 2H, CH2), 5.55 (s, 2H, CH2), 6.64–7.70 (m, 9H, ArH
and CH). Anal. Calcd for C23H20F3NO5 – C: 61.75; H: 4.51;
N: 3.13. Found – C: 61.99; H: 4.40; N: 3.37.
General procedure for the synthesis of 3-acetyl-1-benzyl-
1H-indoles (3a, 4–5a,b)
Using the synthetic procedure previously reported by us14,15,25–
27, 3-acetyl-4-methoxy-1H-indole (2a) (189 mg, 1 mmol) or 3-
acetyl-4-hydroxy-1H-indole (2b) (159 mg, 0.001 mol) was
dissolved in DMF (1 mL) at 0 ꢁC and dry sodium hydride
(120 mg, 5 mmol) or K2CO3 (138 mg, 1 mmol) was added.
After stirring for 2 min, the suitable benzyl bromide
(1.5 mmol) was added dropwise. The resulting solution was
placed in a cylindrical quartz tube (diam. 2 cm), stirred and
irradiated in a microwave oven at 100 W and at continuous
temperature (50 ꢁC) for 10 min. A saturated NaHCO3 solution
was added. The reaction mixture was extracted with ethyl
acetate (10 mL ꢂ 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 cyclo-
hexane:ethyl acetate (60:40) as eluent.
Ethyl 4-{1-[4-(trifluoromethyl)benzyl]-4-hydroxy-1H-indol-3-yl}-
2-hydroxy-4-oxobut-2-enoate (7b). m.p. 263–265 ꢁC, yield 96%;
1H-NMR (ꢀ) 1.21 (t, J ¼ 7.3, 3H, CH3), 4.09 (q, J ¼ 7.3, 2H,
CH2), 5.45 (s, 2H, CH2), 6.25 (s, 1H, OH), 6.64–8.20 (m, 9H, ArH
and CH), 13.52 (s, 1H, OH). Anal. Calcd for C22H18F3NO5 – C:
60.97; H: 4.19; N: 3.23. Found – C: 61.06; H: 4.33; N: 3.40.
Ethyl
4-{1-[(4-tert-butyl)benzyl]-4-methoxy-1H-indol-3-yl}-2-
hydroxy-4-oxobut-2-enoate (8a). m.p. 253 ꢁC dec., yield 96%;
1H-NMR (ꢀ) 1.20 (s, 9H, CH3), 1.24 (t, J ¼ 7.3, 3H, CH3), 3.81
(s, 3H, CH3), 4.08 (d, J ¼ 7.3, 2H, CH2), 5.23 (s, 2H, CH2), 6.54–
8.50 (m, 9H, ArH and CH). Anal. Calcd for C25H27NO5 – C:
71.70; H: 6.71; N: 3.22. Found – C: 71.95; H: 6.89; N: 3.41.
3-Acetyl-1-(4-methyl benzyl)-4-methoxy-1H-indole (3a). Spectral
data are in accordance with the literature26.
Ethyl
4-{1-[(4-tert-butyl)benzyl]-4-hydroxy-1H-indol-3-yl}-2-
hydroxy-4-oxobut-2-enoate (8b). m.p. 219–221 ꢁC, yield 92%;
1H-NMR (ꢀ) 1.20 (t, J ¼ 7.3, 3H, CH3), 1.21 (s, 9H, CH3), 4.09 (q,
J ¼ 7.3, 2H, CH2), 5.27 (s, 2H, CH2), 5.36–8.13 (m, 10H, ArH,
OH and CH), 13.71 (s, 1H, OH). Anal. calcd for C25H27NO5 – C:
71.24; H: 6.46; N: 3.32. Found – C: 71.33; H: 6.31; N: 3.53.
3-Acetyl-1-(4-trifluoromethyl benzyl)-4-methoxy-1H-indole (4a).
m.p. 152–154 ꢁC, yield 85%; 1H-NMR (ꢀ) 2.70 (s, 3H, CH3), 3.98
(s, 3H, CH3), 5.36 (s, 2H, CH2), 6.71 (d, J ¼ 8.0, 1H, ArH), 6.84
(d, J ¼ 8.0, 1H, ArH), 7.15–7.23 (m, 3H, ArH), 7.57 (d, J ¼ 8.5,
2H, ArH), 7.73 (s, 1H, ArH). Anal. Calcd for C19H16F3NO2 – C:
65.70; H: 4.64; N: 4.03. Found – C: 65.87; H: 4.91; N: 4.33.
General procedure for the synthesis of 4-[1-benzyl-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoic acids (9–10a,b)
3-Acetyl-1-(4-trifluoromethyl benzyl)-4-hydroxy-1H-indole (4b).
m.p. 190–192 ꢁC, yield 51%; 1H-NMR (ꢀ) 2.54 (s, 3H, CH3), 5.38
(s, 2H, CH2), 6.66–6.75 (m, 2H, ArH), 7.17 (t, J ¼ 7.9, 1H, ArH),
7.25 (d, J ¼ 7.1, 2H, ArH), 7.62 (d, J ¼ 7.4, 2H, ArH), 7.70 (s, 1H,
ArH). Anal. Calcd for C18H14F3NO2 – C: 64.86; H: 4.23; N: 4.20.
Found – C: 64.99; H: 4.54; N: 4.35.
Following the synthetic procedure previously reported by
us14,15,19,26,27, the appropriate ethyl 4-[1-benzyl-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (7–8a,b) (1 mmol) was dissolved in
methanol (5 mL) and treated with 2N NaOH (5 mL). The reaction
mixture was stirred at room temperature for 1.5 h and then
acidified with conc. HCl to give corresponding acids (9–10a,b).
The desired products were crystallized from a mixture of ethanol
and diethyl ether (1:4).
3-Acetyl-1-(4-tert-butyl benzyl)-4-methoxy-1H-indole (5a). m.p.
1
143–145 ꢁC, yield 73%; H-NMR (ꢀ) 1.29 (s, 9H, CH3), 2.68 (s,
3H, CH3), 3.97 (s, 3H, CH3), 5.25 (s, 2H, CH2), 6.69 (d, J ¼ 7.9,
1H, ArH), 6.96 (d, J ¼ 7.9, 1H, ArH), 7.09 (d, J ¼ 8.5, 2H, ArH),
7.17 (d, J ¼ 7.9, 1H, ArH), 7.34 (d, J ¼ 8.5, 2H, ArH), 7.72 (s, 1H,
ArH). Anal. Calcd for C22H25NO2–C: 78.77; H: 7.51; N: 4.18.
Found – C: 78.95; H: 7.74; N: 4.51.
4-{1-[4-(Trifluoromethyl)benzyl]-4-methoxy-1H-indol-3-yl}-2-
hydroxy-4-oxobut-2-enoic acid (9a). m.p. 280 ꢁC dec, yield
72%; 1H-NMR (ꢀ) 3.90 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.79–7.71
(m, 8H, ArH and CH), 8.57 (s, 1H, ArH). Anal. Calcd for
C21H16F3NO5 – C: 60.15; H: 3.85; N: 3.34. Found – C: 60.44;
H: 4.05; N: 3.56.
3-Acetyl-1-(4-tert-butyl benzyl)-4-hydroxy-1H-indole (5b). m.p.
184–186 ꢁC, yield 75%; 1H-NMR (ꢀ) 1.30 (s, 9H, CH3), 2.52
(s, 3H, CH3), 5.27 (s, 2H, CH2), 6.71–7.67 (m, 8H, ArH), 11.53
(s, 1H, OH). Anal. Calcd for C21H23NO2 – C: 78.47; H: 7.21;
N: 4.36. Found – C: 78.28; H: 7.43; N: 4.21.
4-{1-[4-(Trifluoromethyl)benzyl]-4-hydroxy-1H-indol-3-yl}-2-
hydroxy-4-oxobut-2-enoic acid (9b). m.p. 298–300 ꢁC, yield 80%;
1H-NMR (ꢀ) 5.50 (s, 2H, CH2), 6.27–8.02 (m, 9H, ArH and CH),
14.56 (s, 1H, OH). Anal. Calcd for C20H14F3NO5 – C: 60.44; H:
3.38; N: 3.36. Found – C: 60.61; H: 3.57; N: 3.49.
General procedure for the synthesis of ethyl 4-[1-benzyl-1H-
indol-3-yl]-2-hydroxy-4-oxobut-2-enoates (6a, 7–8a,b).
4-{1-[4-(tert-Butyl)benzyl]-4-methoxy-1H-indol-3-yl}-2-hydroxy-
4-oxobut-2-enoic acid (10a). m.p. 215–217 ꢁC, yield 15%;
1H-NMR (ꢀ) 1.19 (s, 9H, CH3), 3.75 (s, 3H, CH3), 5.28 (s, 2H,
CH2), 6.51–9.06 (m, 9H, ArH and CH). Anal. Calcd for
C24H25NO5 – C: 70.74; H: 6.18; N: 3.44. Found – C: 70.98; H:
6.31; N: 3.72.
Adopting the synthetic procedure previously reported by
us14,15,19,26,27, a mixture of suitable 3-acetyl-1-benzyl-1H-indole
(3a, 4–5a,b) (1 mmol), diethyl oxalate (219 mg, 1.5 mmol) and a
catalytic amount of NaOCH3 was suspended in anhydrous THF
(2 mL). The reaction mixture was placed in a cylindrical quartz

240 S. Ferro et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 237–242
4-{1-[4-(tert-Butyl)benzyl]-4-hydroxy-1H-indol-3-yl}-2-hydroxy- Subsequently, 5 mL of Ni-chelate-coated acceptor beads and 5 mL
4-oxobut-2-enoic acid (10b). m.p. 196–198 ꢁC, yield 74%; of anti-flag donor beads were added to a final concentration
1H-NMR (ꢀ): 1.21 (s, 9H, CH3), 5.41 (s, 2H, CH2), 6.55–9.06 of 20 mg/mL of both beads. Proteins and beads were incubated at
(m, 9H, ArH and CH), 11.33 (s, 1H, OH). Anal. Calcd for 30 ꢁC for 1 h in order to allow association to occur. Exposure
C23H23NO5 – C: 70.21; H: 5.89; N: 3.56. Found – C: 70.37; of the reaction to direct light was prevented as much as possible
H: 5.61; N: 3.28.
and the emission of light from the acceptor beads was measured
in the EnVision plate reader (PerkinElmer, Benelux) and analyzed
General procedure for the synthesis of 4-[1-benzyl-1H-indol-3- using the EnVision manager software (Envision, Wichita, KS).
oyl)-3-hydroxyfuran-2(5H)-ones (11a, 12–13a,b)28
A solution of 40% aqueous formaldehyde in water (4 mL) Results and discussion
was added to a mixture of appropriate diketoester derivative
The most interesting previously reported compounds were benzyl-
(6a, 7–8a,b) (1 mmol) in diethyl ether (5 mL). The stirring was
indole derivatives CHI-1043, CHIBA-3000, CHIBA-3017 and
then continued until clear layers were formed (usually within
CHIBA-3053 (Figure 1) which proved to be active showing an
1–2 h). Sometimes, an additional 4 mL of water was added if the
IC50 value in the low-micromolar range (36.16, 76.44, 67.96 and
reaction was especially thick or when the solid appeared to react
3.5 mM, respectively)14,15,19,25
.
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 and acidified with 3 mL of
concentrated hydrochloric acid. Then the saturated solution was
cooled overnight thus ensuring complete product formation.
The resulting solid was collected, dried and recrystallized from
ethanol.
First, in this study we focused our attention on the benzyl
moiety as a relevant lipophilic feature regulating the IN-LEDGF/
p75 inhibitory process. Particularly, by means of DRY probe
in GRID software, we previously disclosed the hydrophobic
region in the LEDGFIBD binding site, corresponding to IN residue
Trp131 (15). This approach is common to other studies and
applied also for the analysis of several classical HIV target30,31
.
Thus, the high inhibitory efficacy of the most efficacious
inhibitors could be explained for their ability to map well the
DRY area related to the crucial residue Trp131. On this basis,
we have explored the effect of bulkier hydrophobic substituents
on the benzyl group, such as tert-butyl and trifluoromethyl,
that could increase the ability to cover the hydrophobic area on
LEDGFIBD binding site as well as the lipophilicity of our
compounds.
4-{1-[4-(Methyl)benzyl]-4-methoxy-1H-indol-3-oyl}-3-hydroxyfuran-
1
2(5H)-one (11a). m.p. 120–122 ꢁC, yield 71%; H-NMR (ꢀ) 2.26
(s, 3H, CH3), 3.88 (s, 3H, OCH3), 5.16 (s, 2H, CH2), 5.44 (s, 2H,
CH2), 6.64–8.21 (m, 8H, ArH). Anal. Calcd for C23H21NO4 – C,
73.58; H, 5.64; N, 3.73. Found – C, 73.78; H, 5.42; N, 3.91.
4-{1-[4-(Trifluoromethyl)benzyl]-4-methoxy-1H-indol-3-oyl}-3-
hydroxyfuran-2(5H)-one (12a). m.p. 170–172 ꢁC, yield 63%;
1H-NMR (ꢀ): 3.77 (s, 3H, CH3), 5.05 (s, 2H, CH2), 5.59 (s, 2H,
CH2), 6.68–7.71 (m, 7H, ArH), 8.21 (s, 1H, ArH). Anal. Calcd
for C22H18F3NO5 – C: 60.97; H: 4.19; N: 3.23. Found – C: 61.13;
H, 4.36; N, 3.45.
In addition, we incorporated the 1,3-diketoacid motif into a
closed system characterized by the same chemical functionalities,
in order to explore the influence of this chemical manipulation on
binding inhibitory effect. Following an earlier reported method,
the synthesis of the target compounds was accomplished using the
4-{1-[4-(Trifluoromethyl)benzyl]-4-hydroxy-1H-indol-3-oyl}-3-
hydroxyfuran-2(5H)-one (12b). m.p. 249–251 ꢁC, yield 61%;
1H-NMR (ꢀ) 5.00 (s, 2H, CH2), 5.58 (s, 2H, CH2), 6.44–7.71
(m, 7H, ArH), 9.77 (s, 1H, ArH), 12.79 (s, 1H, OH). Anal. Calcd
for C21H14F3NO5 – C: 60.44; H: 3.38; N: 3.36. Found – C: 60.63;
H, 3.56; N, 3.59.
synthetic pathways shown in Scheme 115,27,28
.
Suitable 4-substituted indoles (1a,b) were converted into the
corresponding 3-acetyl derivatives (2a,b) under Vilsmeier–Haack
conditions, in the presence of DMF, acid chloride, such as
phosphoryl chloride, and an excess of N,N-dimethylacetamide.
The obtained intermediates 2a,b were N-alkylated by reaction
with the suitable benzyl bromide. This synthetic step was carried
out by the addition of sodium hydride for the methoxy substituted
derivatives (a), and potassium carbonate for the hydroxyl
substituted derivatives (b). In both cases, the synthesis was
performed under microwave (MW) irradiation conditions, thus
reducing reaction time, usual thermal reagents degradation and
increasing the yields. Reaction of benzyl derivatives (3a and
4–5a,b) with diethyl oxalate under the same conditions: 50 ꢁC for
2 min under 250 W (MW) provided key diketoester intermediates
(6a and 7–8a,b), which were converted into the corresponding
diketoacids 9–10a,b, by hydrolysis in basic medium, or cyclized
in the hydroxyl-furanone derivatives (11a and 12–13a,b), by
treatment with formaldehyde aqueous solution. In order to profile
inhibitory effects we tested all new synthesized compounds,
4-{1-[4-(Tert-butyl)benzyl]-4-methoxy-1H-indol-3-oyl}-3-hydro-
1
xyfuran-2(5H)-one (13a). m.p. 179–181 ꢁC, yield 19%; H-NMR
(ꢀ) 1.22 (s, 9H, CH3), 3.76 (s, 3H, CH3), 5.04 (s, 2H, CH2), 5.41
(s, 2H, CH2), 6.68–7.34 (m, 8H, ArH), 8.19 (s, 1H, OH). Anal.
Calcd for C24H27NO5 – C: 71.24; H: 6.46; N: 3.32. Found – C:
71.37; H: 6.58; N: 3.55.
4-{1-[4-(Tert-butyl)benzyl]-4-hydroxy-1H-indol-3-oyl}-3-hydro-
1
xyfuran-2(5H)-one (13b). m.p. 212–214 ꢁC, yield 71%; H-NMR
(ꢀ) 1.22 (s, 9H, CH3), 5.12 (s, 2H, CH2), 5.44 (s, 2H, CH2), 6.53–
7.35 (m, 8H, ArH), 8.81 (s, 1H, OH). Anal. Calcd for C24H23NO5
– C: 71.10; H: 5.72; N: 3.45. Found – C: 71.32; H: 5.56; N: 3.64.
LEDGF/p75-HIV-1 integrase interaction screening
The AlphaScreen assay was performed as previously described29. CHI-1310 and CHI-1164 in AlphaScreen assay (Table 1).
Reactions were performed in 25 mL final volume in 384-well The obtained results showed that derivatives exhibited inhibi-
OptiwellÔ microtiter plates (Perkin-Elmer, Benelux). The reac- tory effects at 100 mM concentration with a percentage ranging
tion buffer contained 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, from 30% to 99%. Analysis of IC50 values pointed out the
1 mM MgCl2, 0.01% (v/v) Tween-20 and 0.1% (w/v) bovine relevance of the introduction of hydrophobic substituents on
serum albumin. His6-tagged integrase (300 nM final concentra- benzyl group as previously predicted by our molecular modeling
tion) was incubated with the compounds at 4 ꢁC for 30 min. studies15.
The compounds were added in varying concentrations from 1 up
In this series of molecules, both for the diketo acids derivatives
to 100 nM. Later, 100 nM of recombinant flag-LEDGF/p75 was and 3-hydroxyfuran-2(5H)-ones, generally the presence of
added and incubation was extended by another hour at 4 ꢁC. hydroxyl group at C4 of benzene fused ring positively influences

DOI: 10.3109/14756366.2013.766609
Synthesis and biological evaluation of novel antiviral agents as PPI inhibitors 241
Scheme 1. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, RT, 12 h; (ii) benzyl bromide, K2CO3 or NaH, DMF, MW: 10 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; (v) CH2O, Et2O/H2O, RT, 2 h.
Table 1. Inhibition of IN-LEDGF/p75 interaction of new synthesized compounds, CHI and CHIBA derivatives.
I
II
R
R0
%
IC50(mM)
R
R0
%
IC50(mM)
CHI-1043*
CHIBA-3000*
CHIBA-3017
CHIBA-3053*
9a
9b
10a
10b
OMe
OH
OMe
OH
OMe
OH
OMe
OH
F
F
CH3
CH3
CF3
CF3
C(CH3)3
C(CH3)3
75
56
82
86
66
30
57
86
36.16
76.44
–
67.96
–
CHI-1164
CHIBA-3004*
11a
CHIBA-3048*
12a
12b
13a
13b
OMe
OH
OMe
OH
OMe
OH
OMe
OH
F
F
CH3
CH3
CF3
CF3
C(CH3)3
C(CH3)3
44
75
52
81
73
65
38
99
–
22.28
–
8.7
51.3
28.28
–
–
64.00
6.41
23.71
% Inhibition at 100 mM; IC50, concentration required to inhibit the HIV-1 IN-LEDGF/p75 interaction by 50%.
*Data from references14,15,19
.
the inhibitory effects. Overall, the contemporary presence of the active compound of the series (10b) into the LEDGFIBD binding
alkyl substitution on the benzyl moiety and the hydroxyl group pocket on INCCD is reported in Figure 2 and shows nearly the
on the indole system provided the most interesting results.
same binding mode of previously reported diketo acid derivatives
The best activity was displayed when the hydroxyl group of the CHI-1043 and CHIBA-305321. In particular, the diketo acid
indole nucleus was associated to the t-butyl substituent of the moiety forms hydrogen bonds with the main chain nitrogens of
benzyl moiety (10b IC50 ¼ 6.41 mM). Docking result for the most residues Glu170 and His171 and the side chain of residues Thr174

242 S. Ferro et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 237–242
13. Al-Mawsawi LQ, Neamati N. Allosteric inhibitor development
targeting HIV-1 integrase. ChemMedChem 2011;6:228–41.
14. De Luca L, Barreca ML, Ferro S, et al. Pharmacophore-based
discovery of small-molecule inhibitors of protein–protein inter-
actions between HIV-1 integrase and cellular cofactor LEDGF/p75.
ChemMedChem 2009;4:1311–6.
15. De Luca L, Ferro S, Gitto R, et al. Small molecules targeting the
interaction between HIV-1 integrase and LEDGF/p75 cofactor.
Bioorg Med Chem 2010;18:7515–21.
16. De Luca L, Ferro S, Morreale F, Chimirri A. Inhibition of the
interaction between HIV-1 integrase and its cofactor LEDGF/p75:
a promising approach in anti-retroviral therapy. Mini Rev Med
Chem 2011;11:714–27.
17. De Luca L, Ferro S, Morreale F, et al. Inhibitors of the interactions
between HIV-1 IN and the cofactor LEDGF/p75. ChemMedChem
2011;6:1184–91.
18. De Luca L, Ferro S, Morreale F, et al. Fragment hopping approach
directed at design of HIV IN-LEDGF/p75 interaction inhibitors.
J
doi:10.3109/14756366.2012.703184.
19. De Luca L, Gitto R, Christ F, et al. 4-[1-(4-Fluorobenzyl)-
4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid as
Enzyme Inhib Med Chem 2012. [Epub ahead of print].
Figure 2. Binding mode of compound 10b on INCCD. Hydrogen bonds
are shown as dotted lines. This figure was produced with pymol32
.
a
prototype to develop dual inhibitors of HIV-1 integration process.
Antiviral Res 2011;92:102–7.
and Gln95. The fused benzene ring of the indole nucleus projects
into the IN hydrophobic pocket, and the N-benzyl substituent
presents hydrophobic contacts for the crucial Trp131 residue of
chain B. Overall, this new work supports our previous findings on
the main structural requirements for IN-LEDGF/p75 inhibition.
20. Jones G, Willett P, Glen RC, et al. Development and validation of a
genetic algorithm for flexible docking. J Mol Biol 1997;267:727–48.
21. De Luca L, Morreale F, Chimirri A. Insight into the fundamental
interactions between LEDGF binding site inhibitors and integrase
combining docking and molecular dynamics simulations. J Chem Inf
Model 2012;52:3245–54.
22. Cherepanov P, Ambrosio AL, Rahman S, et al. Structural basis for
the recognition between HIV-1 integrase and transcriptional
coactivator p75. Proc Natl Acad Sci USA 2005;102:17308–13.
23. Accelrys, Catalyst: http://www.accelrys.com
Declaration of interest
This work was supported by the European Commission (HEALTH-
F3-2008-201032) (THINC project). The authors declare no conflict of
interest.
24. Sechi M, Bacchi A, Carcelli M, et al. From ligand to complexes:
inhibition of human immunodeficiency virus type 1 integrase by
beta-diketo acid metal complexes. J Med Chem 2006;49:4248–60.
25. De Luca L, Barreca ML, Ferro S, et al. A refined pharmacophore
model for HIV-1 integrase inhibitors: optimization of potency in the
1H-benzylindole series. Bioorg Med Chem Lett 2008;18:2891–5.
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