A new potential approach to block HIV-1 replication via protein-protein interaction and strand-transfer inhibition

Article abstract of DOI:10.1016/j.bmc.2014.02.012

Therapeutic treatment of AIDS is recently characterized by a crescent effort towards the identification of multiple ligands able to target different steps of HIV-1 life cycle. Taking into consideration our previously obtained SAR information and combining some important chemical structural features we report herein the synthesis of novel benzyl-indole derivatives as anti-HIV agents. Through this work we identified new dual target small molecules able to inhibit both IN-LEDGF/p75 interaction and the IN strand-transfer step considered as two crucial phases of viral life cycle.

Full text of DOI:10.1016/j.bmc.2014.02.012

Bioorganic & Medicinal Chemistry 22 (2014) 2269–2279
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A new potential approach to block HIV-1 replication via
protein–protein interaction and strand-transfer inhibition
a
a
a
b
Stefania Ferro a, , Laura De Luca , Giuseppa Lo Surdo , Francesca Morreale , Frauke Christ ,
⇑
Zeger Debyser b, Rosaria Gitto a, Alba Chimirri a
a Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Università di Messina, Viale Annunziata, I-98168 Messina, Italy
b Molecular Virology and Gene Therapy, KU 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:
Therapeutic treatment of AIDS is recently characterized by a crescent effort towards the identification of
multiple ligands able to target different steps of HIV-1 life cycle. Taking into consideration our previously
obtained SAR information and combining some important chemical structural features we report herein
the synthesis of novel benzyl-indole derivatives as anti-HIV agents. Through this work we identified new
dual target small molecules able to inhibit both IN-LEDGF/p75 interaction and the IN strand-transfer step
considered as two crucial phases of viral life cycle.
Received 22 October 2013
Revised 28 January 2014
Accepted 10 February 2014
Available online 20 February 2014
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Protein–protein interaction
Strand-transfer
Docking studies
Synthesis
1. Introduction
polypeptides by viral protease into single viral proteins during
new virion assembly.7
Although three decades of research against the human immu-
nodeficiency virus type 1 (HIV-1), acquired immunodeficiency syn-
drome (AIDS) pandemic and AIDS-related diseases remain among
the leading causes of deaths worldwide. The latest UNAIDS report
for the end of 2012 indicates that over 34 million individuals are
living with HIV and 2.5 million new infections occur every years.1
The current combination antiretroviral therapy (cART), based
on advances in the understanding of the viral life cycle, has
resulted in major advances in the longevity and quality of life for
infected patients, improving the clinical outcome of the disease
and transforming AIDS from a rapid and lethal infection into a
manageable chronic condition.2–4
However, the era of cART is not without problems: the emer-
gence of drug-resistant viral strains, persistent toxicities, the poor
ability of patients to adhere to the prescribed therapy and costs
make desirable to identify additional medications targeting simul-
taneously different steps in the HIV-1 viral life cycle.5,6
Throughout the years, all these steps were approached, and spe-
cific inhibitory molecules were developed and licensed to block
viral enzymes, thus preventing disease progression. Only in the last
decade the viral IN has emerged as a primary alternative target to
block HIV-1 replication. This enzyme is critical for retroviral
replication and is particularly interesting because no homolog is
present in human cells. IN performs three functions that lead to
viral integration into the host cell genome. The first is site-specific
endonucleolytic cleavage of the 30-ends of the viral DNA (30P).
Secondly, it participates in the assembly of the pre-integration
complex (PIC) on the ends of the viral DNA, which migrates into
the host nucleus. Lastly, in the strand-transfer step (ST) IN cataly-
ses the insertion of the viral DNA into host chromosomal DNA.
Figure 1 are shows some first generation integrase strand-trans-
fer inhibitors (INSTIs).
At the end of 2007 the first INSTI, raltegravir, clinically effective
against viruses resistant to other classes of antiretroviral agents,
was approved for the clinical use by the US Food and Drug Admin-
istration (FDA) and European Medicines Agency (EMA).8
Neverthless, raltegravir has a relatively low genetic barrier to
resistance9,10 and, in contrast to prior predictions based on
in vitro experiments, resistance evolves readily in the clinic.11
Elvitegravir12,13 is another first generation IN inhibitor, charac-
terized by virologic activity comparable with raltegravir, and
administered once daily combined with a pharmacological booster.
It has been reported that HIV-1 requires three main steps for
effective viral replication: reverse transcription of the RNA viral
genome into viral cDNA by viral reverse transcriptase (RT); inte-
gration of viral cDNA into the host cell genome using viral integr-
ase (IN); and, finally, cleavage of newly synthesized viral
⇑
Corresponding author. Tel.: +39 0906766453; fax: +39 0906766402.
E-mail address: sferro@unime.it (S. Ferro).
http://dx.doi.org/10.1016/j.bmc.2014.02.012
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
CHIBA series of indoles. They proved to inhibit the interaction be-
tween IN and LEDGF/p75,18,19 and CHIBA-3053 was the most active
OH
O
N
compound (Fig. 2)19 of the series.
O
N
O
OH
O
N
More recently, we found that one of the most promising indoles
developed as INSTIs (CHI-1043) showed also to be active against
the IN-LEDGF/p75 interaction at micromolar concentration20
(Fig. 2).
H
N
N
N
O
O
F
Cl
F
OH
O
HN
raltegravir
elvitegravir
F
As a continuation of our research studies in this field we de-
signed novel small molecules, containing the same scaffold of CHI-
BA and CHI analogues as potential inhibitors for the catalytic site of
IN as well as the LEDGF/p75–IN interaction. Herein, we report the
synthesis and the biological activity of this series of compounds, in
which further exploration with hydrophobic groups at N1 was per-
formed. The most active derivatives were subjected to molecular
docking studies in order to analyse the binding mode of these
new dual inhibitors.
CH3
O
OH
O
O
F
N
H
N
N
O
dolutegravir
Figure 1. First and second generation of integrase strand-transfer inibitors (INSTIs).
2. Results and discussion
Unfortunately, it is also cross-resistant with raltegravir and there-
fore it is no treatment option for patients failing on INSTIs.
The need to overcome these problems has driven the develop-
ment of a second generation of INSTIs such as dolutegravir
(Fig. 1) approved on August 2013.14
As the first-generation integrase inhibitors, dolutegravir blocks
the strand-transfer step of integration of the viral cDNA into the
host genome. It displays superior characteristics to raltegravir,
but partially it shares the resistance pathways.15,16
Therefore, alternative strategies and new targets, such as the
design of inhibitors of the interactions of IN with essential cellular
co-factors of integration, need to be identified to combat the grow-
ing AIDS pandemic.
The ubiquitous lens epithelium growth factor LEDGF/p75 is a
cellular cofactor of IN HIV-1, acting to tether the provirus to the
cellular genome and playing a crucial role in the transcriptional
regulation. It was demonstrated that IN-LEDGF/p75 interaction
inhibitors (LEDGINs) are active against a wide range of INSTIs-
resistant mutants and exhibit additive or synergistic activity when
combined with INSTIs.17
2.1. Design of new analogues
LEDGF/p75 is a cellular cofactor of HIV-1 integrase that inter-
acts with the enzyme via its C-terminal domain, also known as
the IN binding domain (LEDGFIBD) and promotes viral integration
by tethering the preintegration complex to the chromatin.
Several literature data support the notion that the LEDGF/p75–
IN interaction is a promising target for anti-HIV drug develop-
ment.21–24 Searching for HIV-1 inhibitors targeting protein–protein
interactions in our previous studies we reported the discovery of a
series of benzylindole derivatives able to block the IN-LEDGF/p75
interaction. The most potent compound was the 4-[1-(3,5-dimeth-
ylbenzyl)-4-hydroxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic
acid (CHIBA-3053), which showed an IC50 value of 3.5 lM (Fig. 2).
Moreover, considering that targeting different stages in the
integration phase represents a new attractive approach for future
inhibitor design,25–28 we focused our efforts towards the discovery
of new molecules with potential dual site binding ability.
Our goal was to identify small molecules able to inhibit both the
IN strand-transfer step and IN-LEDGF/p75 interaction.
Our previous studies led to the discovery of small molecule
protein-protein interaction inhibitors (SMPPIIs), belonging to the
CH3
N
CH3
Protein-Protein Interacꢀon Inhibitor
OH
O
O
O
H
OH
CHIBA-3053
F
N
Strand Transfer Inhibitor and
Protein-Protein Interacꢀon Inhibitor
MeO
O
O
O
H
OH
CHI-1043
Figure 2. Chemical structure of CHIBA-3053 and CHI-1043 and their major structural determinants for PPI and ST inhibition, respectively.

S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
2271
As a result of these preliminary studies, derivative CHI-1043
(Fig. 2), considered the most promising compound of our INSTIs
2.3. AlphaScreen assay for IN–LEDGF/p75 interaction inhibition
(IC50 = 0.14
l
M; EC50 = 0.59
l
M; SI = 70), proved to be able to inhi-
We tested all the new synthesized compounds in the Alpha-
Screen assay to evaluate their inhibitory effects on the LEDGF/
p75–IN interaction. The biological screening has been carried out
on the diketo acids 13–15 a–f as well as on the hydroxyl-furanone
16–18 a–f derivatives.
The obtained results are shown in Tables 1 and 2.
Among the compounds belonging to the diketo acid series
(Table 1) the best results were displayed for compounds 13b, 13c
bit the IN-LEDGF/p75 interaction with an IC50 value of 36.16
lM,
representing an interesting prototype of dual target agent.20
So, encouraged by these results and taking into consideration
the information already obtained by our previous researches we
decided to synthesize a new series of dual small molecules, com-
bining the chemical structural features of CHIBA and CHI analogues
(Fig. 2).
and 14b (IC50 = 22.9, 9.99 and 9.76 lM). All of them were charac-
2.2. Chemistry
terized by the presence of a halogen atom (Cl for 13b and 13c, F
for 14b) at 5 position of the indole nucleus and of some bulky
hydrophobic substituents on the benzyl moiety: 4-trifluoromethyl
(13b and 14b) and 4-tert-butyl (13c) group, thus confirming our
already reported studies.19
In fact, we have previously identified a new hydrophobic region
in IBD binding site and demonstrated the importance of lipophilic
chemical features for IN-LEDGF inhibition. Better biological results
in the Alpha screen assay were obtained when the diketo acid
chain has been turned into the hydroxyl-furanone cycle (Table 2).
For this series (16–18 a–f) the synthesized compounds generally
showed PPI inhibition at micromolar concentration thus highlight-
ing that the structural modification of diketoacid motif in hydro-
xyl-furanone is well tolerated for the inhibition of the IN-LEDGF
interaction.
Scheme 1 illustrates the general synthetic route for the prepara-
tion of a new series of 1H-benzyl indole derivatives (13–18 a–f). By
standard synthetic chemistry,19,29–31 the indole scaffold was easily
equipped with a functional chain at the C3 position and the intro-
duction of the benzyl moiety at N1, thus probing several substitu-
tions for rapid exploration of the SAR profile. The C3 position of the
indole nucleus was functionalized through the Vilsmeier–Haack
reaction (4–6). Then various aryl groups were attached to the N1
atom using dry sodium hydride and the suitable benzyl bromide,
and the obtained intermediates 7–9 a–f were reacted with diethyl
oxalate to provide the key ethyl ester precursors (10–12 a–f) of de-
sired compounds. Finally, the esters were hydrolyzed in basic med-
ium to give the final diketo acids (13–15 a–f), or converted into the
corresponding hydroxyl-furanones derivatives (16–18 a–f) by
treatment with an aqueous solution of formaldehyde.
Particularly compound 17f, in which the presence of fluorine
atom on the indole nucleus was associated to the introduction of
R'R''R'''
N
R
COOH
iv
R'R''R'''
O
OH
13-15 (a-f)
R'R''R'''
iii
H
N
i
N
N
ii
R
4-6
R
R
COOEt
OH
10-12 (a-f)
CH3
1-3
O
O
R'R''R'''
v
R
7-9 (a-f)
1,4,7,10,13,16
2,5,8,11,14,17
3,6,9,12,15,18
Cl
N
O
F
OCH3
R
O
O
OH
16-18 (a-f)
R’
R’’
R’’’
H
a
b
c
d
e
f
4-C6H5
4-CF3
4-tBut
4-CF3
H
H
H
H
H
H
2-CF3
3-CH3
2-CH3
H
5-CH3
6-CH3
4-CH3
Scheme 1. Synthetic route of benzyl-indole derivatives. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, RT, 12 h; (ii) benzyl bromide or chloride, NaH, DMF, mw: 10 min
at continuous temperature (50 °C), 100 Watt; (iii) diethyl oxalate, dry CH3ONa, THF, two separate steps under the same conditions mw: 2 min, at continuous temperature
(50 °C), 250 Watt; (iv) 2 N NaOH, MeOH, RT, 1.5 h; (v) CH2O, Et2O/H2O, RT, 2 h.

2272
S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
Table 1
onstrated better anti-HIV activity than of the closed analogues
(16–18).
The better anti-HIV activity of diketoacids series respect to the
hydroxyl-furanones (e.g. 17f) can be explained considering the sec-
ond series of compounds as pro-drugs of the open chain DKA
derivatives.32
Inhibition of IN–LEDGF/p75 interaction of diketo acid derivatives (13–15 a–f)
a
b
a
b
No.
%
IC50
NDd
(l
M)
No.
%
IC50 (lM)
13a
13b
13c
13d
13e
13f
14a
14b
31
80
88
80
NAc
59
72
77
33
14d
14e
14f
15a
15b32
15c
15d
15e
15f
NA
26
71
59
40
65
33
89
72
NDd
32.9 3.5
10.0 3.4
3.8 0.3
NDd
NDd
15.2 1.6
>100
NDd
2.5. Integrase strand-transfer inhibition
>100
91.7 6.2
8.1 2.9
17.8 7.4
ND
NDd
On the basis of the above reported results and taking into ac-
count that one of our objectives was to identify small molecules
able to simultaneously inhibit two different integration processes,
we focused our attention only on those diketoacids active as anti-
HIV agents for evaluating their potential activity also on the
strand-transfer step of IN HIV-1 integration. Thus derivatives
13b, 14b, 14f, 15e and 15f (Table 3) were selected and tested in
in vitro integration assays. The obtained results are reported in
Table 4.
53.9 4.9
33.1 0.1
14c
CHIBA-3053
CHI-1043
3.5
36.16
a
% Inhibition at 100
l
M.
b
IC50: concentration required to inhibit the HIV-1 IN–LEDGF/p75 interaction by
50%. The average of two IC50 determinations and the error is shown.
c
NA: not active.
ND: not detected.
d
As expected, all five derivatives showed relevant activity in the
strand-transfer step at very low micromolar range. Indeed our pre-
vious SAR findings and molecular modeling studies on benzylin-
dole diketo acids had suggested that the diketo acid portion
furnishes the triad oxygen lone-pairs to serve as chelating center
of the two divalent metals and that the 4-substituted benzyl group
provided the appropriate spatial relationship for the aromatic moi-
ety required for the inhibition of the integration strand-transfer
event. In particular compound 14b has an IC50 in the low micromo-
lar range for both IN-LEDGF/p75 interaction inhibition and strand
transfer step.
Table 2
Inhibition of IN–LEDGF/p75 interaction of hydroxyl-furanones derivatives (16–18 a–
f)
a
b
a
b
No.
%
IC50
(
lM)
No.
%
IC50
NDc
(lM)
16a
16b
16c
16d
16e
16f
17a
17b
80
92
83
86
96
85
89
65
96
21.8 2.9
12.3 1.1
52.5 1.8
21.5 0.8
23.1 0.5
23.6 2.2
15.8 4.4
12.2 6.5
36.2 3.4
3.50
17d
17e
17f
18a
18b
18c
18d
18e
18f
37
73
81
51
65
93
61
66
82
20.3 1.8
5.6 2.5
NDc
52.2 9.5
17.1 2.2
39.5 6.9
NDc
17c
CHIBA-3053
CHI-1043
33.2 1.9
2.6. Docking studies
36.16
Using Gold software and according to our previously applied
protocols20,33,34 we carried out docking experiments of the most
active compounds, in order to clarify their interaction with the
LEDGF binding site and the catalytic site on IN. Docking results
for the most promising dual inhibitor 14b are shown in Figure 3.
As regards the docking pose of 14b into the LEDGF binding site
(Fig. 3A), the key interactions include hydrogen bonds between the
a
% Inhibition at 100
l
M.
b
IC50: concentration required to inhibit the HIV-1 IN–LEDGF/p75 interaction by
50%. The average of two IC50 determinations and the error is shown.
c
ND: not detected.
trimethyl-substitution on the benzyl moiety, showed higher
potency (IC50 = 2.44) than our lead compound CHIBA-3053
(IC50 = 3.50).
Table 4
In general, by comparison of data reported in Tables 1 and 2 the
diketo acid derivatives 13–15 a–f are less active than the corre-
sponding hydroxyl-furanones 16–18 a–f.
Inhibition of HIV-1 integrase strand-transfer activity of diketo acid derivatives
No.
Strand-transfera
(lM)
13b
14b
14f
15e
15f
CHI-1043
0.26 0.01
1.55 0.24
2.51 0.56
1.09 0.15
0.42 0.15
0.14 0.11
2.4. In vitro anti-HIV and drug susceptibility assays
All the synthesized derivatives were also tested in MT-4/MTT-
assay as anti-HIV agents and Table 3 shows the results for only
active compounds.
The selected compounds proved to be active at micromolar
concentration; generally the diketoacid derivatives (13–15) dem-
a
Concentration required to inhibit by 50% the in vitro integrase activity assays as
M).
IC50 (l
Table 3
Antiviral activities of diketo acid derivatives (13–15 a–f) and hydroxyl-furanones (16–18 a–f)
a
b
a
b
No.
EC50
(l
M)
CC50
(l
M)
SIc
No.
EC50
(l
M)
CC50
53
(lM)
SIc
13b
13e
14b
14f
15e
15f
5.09 1.22
56.15 2.80
7.83 1.56
5.14 2.9
2.24 0.78
3.23 0.64
23 3.54
>125
15 1.75
104 53
51 4.68
23.5 2.86
5
>2
2
21
23
8
16c
16e
16f
17f
CHIBA-3053
CHI-1043
23.45 2.45
16.93 3.9
19.83 1.26
48.96 6.8
4.63
7
2
3
2
2
10
70
53.5 1.5
40.5 4.5
107 12
44
0.59
41.1
a
b
c
EC50: effective concentration required to reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells.
CC50: cytotoxic concentration to reduce MT-4 cell viability by 50%.
SI: ratio CC50/EC50
.

S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
2273
Figure 3. Docking of 14b (green) into (A) LEDGF/p75 binding pocket on IN core domain (PDB code 2B4J) and (B) PFV IN active site in its bound state with DNA (PDB code
3OYA). The divalent metal ions are shown as gray spheres, while the viral DNA is depicted in pink. This figure was produced with PyMol (DeLano Scientific LLC).
diketo acid moiety and the main chain nitrogens of residues
Glu170 and His171 as well as the side chain of residues Thr174
and Gln95. The N-benzyl substituent displays hydrophobic con-
tacts for the crucial Trp131, while the fused benzene ring of the in-
dole nucleus projects into the IN hydrophobic pocket with the
fluorine atom forming interactions with residue Leu102 and
Ala129 side chains.
The binding mode at the catalytic site during the strand-trans-
fer step was predicted by docking 14b on the structure of integrase
from PFV in complex with DNA and two Mg ions as previously
reported for CHI-1043 (Fig. 3B). The diketo acid moiety represents
the common metal-coordinating pharmacophore of INSTIs, and the
5-F indole group contacts Pro214, while the p-substituted benzyl
moiety (that represents the second conserved feature in the INSTIs)
interacts with viral DNA bases and residue Gln215.
spectrometer. Chemical shifts were expressed in d (ppm) and cou-
pling constants (J) in hertz (Hz). All the exchangeable protons were
confirmed by addition of D2O.
4.2. Synthesis of 3-acetyl-1H-indoles (4–6)
Synthesis of intermediates 4-6 was carried out following the
previously reported procedure. Phosphoryl chloride (0.92 ml,
10 mmol) was added to ice cold dimethylacetamide (2.79 ml,
30 mmol) under stirring and cooling in ice. The suitable starting
material (1–3) (1 mmol) was added and the reaction mixture was
stirred at room temperature for 12 h, then poured over ice and
basified with 4 N aqueous sodium hydroxide solution. The mixture
was extracted with ethyl acetate and dried over Na2SO4. After re-
moval of the solvent under reduced pressure, the residue was pow-
dered by treatment with diethyl ether and recrystallized from
dichloromethane.
In summary docking studies provide insights into the binding
mode of 14b to form the relevant key interactions in both sites
on IN.
4.2.1. 3-Acetyl-5-chloro-1H-indole (4)
Spectral data are in accordance with the literature.32,35
3. Conclusion
In conclusion we obtained novel anti-HIV agents some of which
are dual inhibitors of the integration process acting both in the IN
strand-transfer step and by disrupting the protein–protein interac-
tion between IN and its cofactor LEDGF/p75. Our approach was
based on molecular modification of the indole b-diketo acid scaf-
fold present in some inhibitors of IN–LEDGF interaction and de-
scribed as an important pharmacophoric feature for INSTIs.
4.2.2. 3-Acetyl-5-fluoro-1H-indole (5)
Mp 202–204 °C, yield 46%; 1H NMR (DMSO-d6): 2.43 (s, 3H,
CH3), 7.05–7.84 (m, 3H, ArH), 8.36 (s, 1H, ArH), 12.02 (br s, 1H,
NH). Anal. Calcd for C10H8FNO: C, 67.79; H, 4.55; N, 7.91. Found:
C, 67.94; H, 4.31; N, 7.72.
4.2.3. 3-Acetyl-5-methoxy-1H-indole (6)
Spectral data are in accordance with the literature.32,35
4. Experimental section
4.1. Chemistry
4.3. General procedure for the synthesis of 3-acetyl-1-benzyl-
1H-indoles (7–9 a–f)
All commercially available reagents and solvents were used
without any further purification. The microwave-assisted reactions
were carried out in a CEM Focused Microwave Synthesis System,
Model Discover, working at the power necessary for refluxing un-
der atmospheric conditions. Melting points were determined on a
BUCHI Melting Point B-545 apparatus and are uncorrected. Ele-
mental analyses (C, H and N) were carried out on a Carlo Erba Mod-
el 1106 Elemental Analyzer and the results are within 0.4% of the
theoretical values. Merck silica gel 60 F254 plates were used for
analytical TLC; column chromatography was performed on Merck
silica gel 60 (230–400 mesh) and Flash Chromatography (FC) on
a Biotage SP1 EXP. 1H NMR spectra were recorded in CDCl3 with
TMS as internal standard or [D6]DMSO on a Varian Gemini-300
Using the synthetic procedure previously reported by us18–
20,29,36
3-acetyl-1H-indole derivatives 4–6 were dissolved in DMF
(2 ml) at 0 °C and dry sodium hydride (120 mg, 5 mmol) was
added. After stirring for 2 min, the suitable benzyl bromide or chlo-
ride (1.5 mmol) was added in several portions. The resulting solu-
tion 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 ꢀ 3) and dried over Na2SO4. After removal of the solvent un-
der reduced pressure, the crude mixture was crystallized from
diethyl ether or purified by flash chromatography using a mixture
of cyclohexane/ethylacetate (60:40) as eluent.

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S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
4.3.1. 3-Acetyl-1-(4-phenylbenzyl)-5-chloro-1H-indole (7a)
Mp 154–56 °C, yield 90%; 1H NMR (d) (CDCl3): 2.52 (s, 3H, CH3),
5.38 (s, 2H, CH2), 7.20–7.24 (m, 3H, ArH), 7.36–7.47 (m, 4H, ArH),
7.55–7.59 (m, 4H, ArH), 7.80 (s, 1H, ArH), 8.43 (s, 1H, ArH). Anal.
Calcd for C23H18ClNO: C: 76.77; H: 5.04; N: 3.89. Found: C:
76.45; H: 5.23; N: 3.68.
H22FNO: C: 77.99; H: 6.86; N: 4.33. Found: C: 78.13; H: 6.99; N:
4.22.
4.3.10. 3-Acetyl-1-(2,4-bis-trifluoromethylbenzyl)-5-fluoro-1H-
indole (8d)
Mp 144–146 °C, yield 35%; 1H NMR (CDCl3): 2.55 (s, 3H, CH3),
5.63 (s, 2H, CH2), 6.73 (d, J = 8.2, 1H, ArH), 7.03 (d, J = 8.2, 2H,
ArH), 7.65 (d, J = 8.2, 1H, ArH), 7.82 (s, 1H, ArH), 8.02 (s, 1H,
ArH), 8.13 (d, J = 9.2, 1H, ArH). Anal. Calcd for C19H12F7NO: C:
58.92; H: 3.12; N: 3.62. Found: C: 59.21; H: 3.45; N: 3.96.
4.3.2. 3-Acetyl-1-(4-trifluoromethylbenzyl)-5-chloro-1H-indole
(7b)
Mp 163–165 °C, yield 50%; 1H NMR (CDCl3): 2.52 (s, 3H, CH3),
5.41 (s, 2H, CH2), 7.12 (d, J = 9.1, 1H, ArH), 7.21–7.26 (m, 3H,
ArH), 7.60 (d, J = 8.2, 2H, ArH), 7.77 (s, 1H, ArH), 8.43 (s, 1H,
ArH). Anal. Calcd for C18H13ClF3NO: C: 61.46; H: 3.73; N: 3.98.
Found: C: 61.72; H: 3.89; N: 4.03.
4.3.11. 3-Acetyl-1-(3,5-dimethylbenzyl)-5-fluoro-1H-indole (8e)
Mp 146–148 °C, yield 57%; 1H NMR (CDCl3): 2.28 (s, 6H, CH3),
2.51 (s, 3H, CH3), 5.25 (s, 2H, CH2), 6.76 (s, 2H, ArH), 6.96–7.02
(m, 2H, ArH), 7.20–7.24 (m, 1H, ArH), 7.76 (s, 1H, ArH), 8.08 (d,
J = 7.4, 1H, ArH). Anal. Calcd for C19H18FNO: C: 77.27; H: 6.14; N:
4.74. Found: C: 77.39; H: 6.28; N: 4.81.
4.3.3. 3-Acetyl-1-(4-tert-butylbenzyl)-5-chloro-1H-indole (7c)
Mp 158–160 °C, yield 44%; 1H NMR (CDCl3): 1.30 (s, 9H, CH3),
2.50 (s, 3H, CH3), 5.30 (s, 2H, CH2), 7.10 (d, J = 8.5, 2H, ArH), 7.22
(s, 1H, ArH), 7.36 (d, J = 8.5, 2H, ArH), 7.75 (s, 1H, ArH), 8.40 (s,
1H, ArH). Anal. Calcd for C21H22ClNO: C: 74.22; H: 6.52; N: 4.12.
Found: C: 74.34; H: 6.36; N: 4.31.
4.3.12. 3-Acetyl-1-(2,4,6-trimethylbenzyl)-5-fluoro-1H-indole
(8f)
Mp 135–137 °C, yield 42%; 1H NMR (CDCl3): 2.23 (s, 6H, CH3),
2.36 (s, 6H, CH3), 5.21 (s, 2H, CH2), 6.88 (s, 1H, ArH), 6.99 (s, 1H,
ArH), 7.10 (t, J = 8.8, 1H, ArH), 7.19 (s, 1H, ArH), 7.43 (d, J = 8.8,
1H, ArH), 8.07 (d, J = 9.6, 1H, ArH). Anal. Calcd for C20H20FNO: C:
77.64; H: 6.52; N: 4.53. Found: C: 77.87; H: 6.68; N: 4.31.
4.3.4. 3-Acetyl-1-(2,4-bis-trifluoromethylbenzyl)-5-chloro-1H-
indole (7d)
Mp 163–165 °C, yield 35%; 1H NMR (CDCl3): 2.55 (s, 3H, CH3),
5.63 (s, 2H, CH2), 6.71 (d, J = 7.6, 1H, ArH), 7.03 (d, J = 8.8, 1H,
ArH), 7.23 (d, J = 8.8, 1H, ArH), 7.64 (d, J = 7.6, 1H, ArH), 7.79 (s,
1H, ArH), 8.02 (s, 1H, ArH), 8.45 (s, 1H, ArH). Anal. Calcd for C19H12-
ClF6NO: C: 54.37; H: 2.88; N: 3.34. Found: C: 54.55; H: 3.09; N:
3.61.
4.3.13. 3-Acetyl-1-(4-phenylbenzyl)-5-methoxy-1H-indole (9a)
Mp 154–156 °C, yield 90%; 1H NMR (CDCl3): 2.52 (s, 3H, CH3),
3.91 (s, 3H, OCH3), 5.37 (s, 2H, CH2), 6.91 (d, J = 8.8, 1H, ArH),
7.20–7.24 (m, 3H, ArH), 7.37 (d, J = 7.2, 1H, ArH) 7.44 (t, J = 8.8,
2H, ArH), 7.55–7.58 (m, 4H, ArH), 7.76 (s, 1H, ArH), 7.94 (s, 1H,
ArH). Anal. Calcd for C24H21NO2: C: 81.10; H: 5.96; N: 3.94. Found:
C: 81.34; H, 5.72; N, 4.09.
4.3.5. 3-Acetyl-1-(3,5-dimethylbenzyl)-5-chloro-1H-indole (7e)
Mp 136–138 °C, yield 47%; 1H NMR (CDCl3): 2.27 (s, 3H, CH3),
2.50 (s, 3H, CH3), 5.24 (s, 2H, CH2), 6.75 (s, 2H, ArH), 6.95 (s, 1H,
ArH), 7.19–7.22 (m, 2H, ArH), 7.74 (s, 1H, ArH), 8.40 (s, 1H, ArH).
Anal. Calcd for C19H18ClNO: C: 73.19; H: 5.82; N: 4.49. Found: C:
73.34; H: 5.71; N: 4.56.
4.3.14. 3-Acetyl-1-(4-trifluoromethylbenzyl)-5-methoxy-1H-
indole (9b)
Mp 122–124 °C, yield 70%; 1H NMR (CDCl3): 2.53 (s, 3H, CH3),
3.90 (s, 3H, OCH3), 5.40 (s, 2H, CH2), 6.90 (d, J = 8.8, 1H, ArH),
7.09 (d, J = 8.8, 1H, ArH), 7.23 (d, J = 8, 2H, ArH), 7.60 (d, J = 8.2,
2H, ArH), 7.73 (s, 1H, ArH), 7.93 (s, 1H, ArH). Anal. Calcd for C19H16-
F3NO2: C: 65.70; H: 4.64; N: 4.03. Found: C: 65.87; H: 4.48; N: 4.21.
4.3.6. 3-Acetyl-1-(2,4,6-trimethylbenzyl)-5-chloro-1H-indole
(7f)
Mp 193–195 °C, yield 67%; 1H NMR (CDCl3): 2.22 (s, 6H, CH3),
2.35 (s, 3H, CH3), 2.36 (s, 3H, CH3), 5.20 (s, 2H, CH2), 6.87 (s, 1H,
ArH), 6.99 (s, 2H, ArH), 7.18 (s, 1H, ArH), 7.30 (d, J = 8.8, 1H,
ArH), 7.43 (d, J = 8.8, 1H, ArH). Anal. Calcd for C20H20ClNO: C:
73.72; H: 6.19; N: 4.30. Found: C: 73.59; H: 6.33; N: 4.48.
4.3.15. 3-Acetyl-1-(4-tert-butylbenzyl)-5-methoxy-1H-indole
(9c)
Mp 149–151 °C, yield 73%; 1H NMR (CDCl3): 1.29 (s, 9H, CH3),
2.68 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 5.25 (s, 2H, CH2), 6.69 (d,
J = 7.9, 1H, ArH), 6.96 (d, J = 7.9, 1H, ArH), 7.09 (d, J = 8.5, 2H,
ArH), 7.17 (d, J = 7.9, 1H, ArH), 7.34 (d, J = 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.
4.3.7. 3-Acetyl-1-(4-phenylbenzyl)-5-fluoro-1H-indole (8a)
Mp 81–83 °C, yield 48%; 1H NMR (CDCl3): 2.52 (s, 3H, CH3), 5.39
(s, 2H, CH2), 7.02 (t, J = 9, 1H, ArH), 7.21–7.25 (m, 3H, ArH), 7.37 (d,
J = 6.9, 1H, ArH), 7.44 (t, J = 8, 2H, ArH), 7.55–7.62 (m, 4H, ArH),
7.82 (s, 1H, ArH), 8.10 (d, J = 9.6, 1H, ArH). Anal. Calcd for C23H18-
FNO: C: 80.45; H: 5.28: N, 4.08. Found: C: 80.21; H: 5.45; N: 4.24.
4.3.16. 3-Acetyl-1-(2,4-bis-trifluoromethylbenzyl)-5-methoxy-
1H-indole (9d)
4.3.8. 3-Acetyl-1-(4-trifluoromethylbenzyl)-5-fluoro-1H-indole
(8b)
Mp 158–160 °C, yield 37%; 1H NMR (DMSO-d6): 2.49 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 5.80 (s, 2H, CH2), 6.80 (d, J = 8.4, 1H,
ArH), 6.92 (s, 2H, ArH), 7.25 (d, J = 8.8, 2H, ArH), 7.79 (s, 1H,
ArH), 7.95 (d, J = 8.4, 1H, ArH), 8.13 (s, 1H, ArH), 8.86 (s, 1H,
ArH). Anal. Calcd for C20H15F6NO2: C: 57.84; H: 3.64; N: 3.37.
Found: C: 57.70; H: 3.81; N: 3.52.
Mp 185–187 °C, yield 62%; 1H NMR (CDCl3): 2.53 (s, 3H, CH3),
5.42 (s, 2H, CH2), 7.01 (t, J = 9, 1H, ArH), 7.13 (d, J = 9, 1H, ArH),
7.23 (d, J = 7.9, 2H, ArH), 7.60 (d, J = 8.2, 2H, ArH), 7.80 (s, 1H,
ArH), 8.10 (d, J = 9.5, 1H, ArH). Anal. Calcd for C18H13F4NO: C:
64.48; H: 3.91; N: 4.18. Found: C: 64.32; H: 3.74; N: 4.29.
4.3.17. 3-Acetyl-1-(3,5-dimethylbenzyl)-5-methoxy-1H-indole
(9e)
4.3.9. 3-Acetyl-1-(4-tert-butylbenzyl)-5-fluoro-1H-indole (8c)
Mp 132–134 °C, yield 50%; 1H NMR (CDCl3): 1.32 (s, 9H, CH3),
2.52 (s, 3H, CH3), 5.33 (s, 2H, CH2), 7.02 (t, J = 8.8, 1H, ArH), 7.11
(d, J = 8.8, 2H, ArH), 7.23–7.28 (m, 1H, ArH), 7.39 (d, J = 8.8, 2H,
ArH), 7.79 (s, 1H, ArH), 8.09 (d, J = 9.9, 1H, ArH). Anal. Calcd for C21-
Mp 130–132 °C, yield 59%; 1H NMR (CDCl3): 2.27 (s, 6H, CH3),
2.50 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 5.22 (s, 2H, CH2), 6.77 (s,
2H, ArH), 6.91 (t, J = 8.8, 2H, ArH), 7.19 (d, J = 8.8, 1H, ArH), 7.69

S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
2275
(s, 1H, ArH), 7.92 (s, 1H, ArH). Anal. Calcd for C20H21NO2: C: 78.15;
H: 6.89; N: 4.56. Found: C: 78.36; H: 6.72; N: 4.68.
J = 8.8, 1H, ArH), 7.46 (d, J = 8.8, 1H, ArH), 8.13 (d, J = 6.1, 1H,
ArH), 8.44 (s, 1H, ArH). Anal. Calcd for C23H22ClNO4: C: 67.07; H:
5.38; N: 3.40. Found: C: 67.30; H: 5.41; N: 3.67.
4.3.18. 3-Acetyl-1-(2,4,6-trimethylbenzyl)-5-methoxy-1H-
indole (9f)
4.4.6. Ethyl 4-[1-(2,4,6-trimethylbenzyl)-5-chloro-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (10f)
Mp 150–152 °C, yield 28%; 1H NMR (CDCl3): 2.23 (s, 6H, CH3),
2.35 (s, 6H, CH3), 3.91 (s, 3H, OCH3), 5.19 (s, 2H, CH2), 6.97–7.01
(m, 2H, ArH), 7.12 (s, 1H, ArH), 7.40 (d, J = 8.5, 2H, ArH), 7.92 (s,
1H, ArH). Anal. Calcd for C21H23NO2: C: 78.47; H: 7.21; N: 4.36.
Found: C: 78.69; H: 7.04; N: 4.54.
Mp 150–152 °C, yield 97%; 1H NMR (DMSO-d6): 1.27 (t, J = 7.2,
3H, CH3), 2.16 (s, 6H, CH3), 2.26 (s, 3H, CH3), 4.26 (q, J = 7.2, 2H,
CH2), 5.44 (s, 2H, CH2), 6.81 (s, 1H, ArH), 6.95 (s, 2H, ArH and
CH), 7.35 (d, J = 8.5, 1H, ArH), 7.63 (d, J = 8.8, 1H, ArH), 8.17 (s,
1H, ArH), 8.24 (s, 1H, ArH). Anal. Calcd for C24H24ClNO4: C:
67.68; H: 5.68; N: 3.29. Found: C: 67.85; H: 5.79; N: 3.16.
4.4. General procedure for the synthesis of ethyl 4-[1-benzyl-
1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoates (10–12 a–f)
4.4.7. Ethyl 4-[1-(4-phenylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate (11a)
Intermediates 10–12 a–f were obtained adopting the synthetic
procedure previously reported by us.18,19,31,32 A mixture of suitable
3-acetyl-1-benzyl-1H-indole (7–9 a–f) (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 successive
time intervals under the same conditions (250 Watt, 2 min,
50 °C). The solvent was concentrated under reduced pressure and
collected yellow solid was crystallized from ethanol and diethyl
ether (1:4).
Mp 168 °C dec, yield 92%; 1H NMR (DMSO-d6) 1.14 (t, J = 7.3, 3H,
CH3), 3.94 (q, J = 7.3, 2H, CH2), 5.50 (s, 2H, CH2), 6.25 (br s, 1H, OH),
6.96–8.28 (m, 14H, ArH and CH). Anal. Calcd for C27H22FNO4: C,
73.13; H, 5.00; N, 3.16. Found: C, 73.38; H, 4.16; N 3.01.
4.4.8. Ethyl 4-{1-[4-(trifluoromethyl)benzyl]-5-fluoro-1H-indol-
3-yl}-2-hydroxy-4-oxobut-2-enoate (11b)
Mp 217 °C dec, yield 60%; 1H NMR (DMSO-d6): 1.23 (t, J = 7.3,
3H, CH3), 4.09 (q, J = 7.3, 2H, CH2), 5.57 (s, 2H, CH2), 6.17 (s, 1H,
OH), 6.96 (t, J = 9, 1H, ArH), 7.35–7.42 (m, 4H, 3ArH and CH),
7.68 (d, J = 8.1, 2H, ArH), 8.12 (s, 1H, ArH), 8.23 (s, 1H, ArH). Anal.
Calcd for C22H17F4NO4: C: 60.69; H: 3.94; N: 3.22. Found: C:
60.46; H: 4.11; N: 3.37.
4.4.1. Ethyl 4-[1-(4-phenylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate (10a)
Mp 198–200 °C, yield 95%; 1H NMR (DMSO-d6): 1.24 (t, J = 7.3,
3H, CH3), 4.12 (q, J = 7.3, 2H, CH2), 5.50 (s, 2H, CH2), 6.21 (br s,
1H, OH), 7.12 (d, J = 9, 1H, ArH), 7.31–7.45 (m, 6H, 5ArH and CH),
7.52 (d, J = 7.6, 1H, ArH), 7.58–7.61 (m, 4H, ArH), 8.25 (s, 1H,
ArH), 8.36 (s, 1H, ArH). (Anal. Calcd for C27H22ClNO4: C: 70.51; H:
4.82; N: 3.05. Found: C: 70.12; H: 4.99; N: 3.24.
4.4.9. Ethyl 4-[1-(4-tert-butylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate (11c)
Mp 227 °C dec, yield 88%; 1H NMR (DMSO-d6): 1.20 (s, 9H, CH3),
1.24 (t, J = 6.9, 3H, CH3), 4.07 (d, J = 6.9, 2H, CH2), 5.38 (s, 2H, CH2),
6.14 (s, 1H, ArH), 6.92–6.99 (m, 1H, ArH), 7.13–7.19 (m, 2H, ArH
and CH), 7.29–7.38 (m, 2H, ArH), 7.47 (d, J = 9, 1H, ArH), 8.09 (d,
J = 8.5, 1H, ArH), 8.51 (s, 1H, ArH). Anal. Calcd for C25H26FNO4: C:
70.91; H: 6.19; N: 3.31. Found: C: 71.04; H: 6.28; N: 3.51.
4.4.2. Ethyl 4-{1-[4-(trifluoromethyl)benzyl]-5-chloro-1H-
indol-3-yl}-2-hydroxy-4-oxobut-2-enoate (10b)
Mp 170–172 °C, yield 75%; 1H NMR (DMSO-d6): 1.24 (t, J = 7.3,
3H, CH3), 3.94 (q, J = 7.3, 2H, CH2), 5.59 (s, 2H, CH2), 6.35 (s, 1H,
OH), 7.14 (d, J = 8, 1H, ArH), 7.42 (d, J = 8, 2H, ArH and CH), 7.47
(d, J = 8.9, 2H, ArH), 7.68 (d, J = 8, 2H, ArH), 8.38 (s, 1H, ArH), 8.43
(s, 1H, ArH). Anal. Calcd for C22H17ClF3NO4: C: 58.48; H: 3.79; N:
3.10. Found: C: 58.61; H: 4.03; N: 3.17.
4.4.10. Ethyl 4-{1-[2,4-bis(trifluoromethyl)benzyl]-5-fluoro-1H-
indol-3-yl}-2-hydroxy-4-oxobut-2-enoate (11d)
Mp 269–271 °C, yield 99%; 1H NMR (DMSO-d6) 1.23 (t, J = 7.3,
3H, CH3), 4.10 (q, J = 7.3, 2H, CH2), 5.72 (s, 2H, CH2), 6.21 (s, 1H,
OH), 6.58–8.41 (m, 8H, ArH and CH). Anal. Calcd for C23H16F7NO4:
C, 54.88; H, 3.20; N, 2.78. Found: C, 54.96; H, 3.27; N, 3.35.
4.4.3. Ethyl 4-[1-(4-tert-butylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoate (10c)
4.4.11. Ethyl 4-[1-(3,5-dimethylbenzyl)-5-fluoro-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (11e)
Mp 280 °C dec, yield 85%; 1H NMR (DMSO-d6): 1.20 (s, 9H, CH3),
1.24 (t, J = 6.9, 3H, CH3), 4.13 (d, J = 7.2, 2H, CH2), 5.41 (s, 2H, CH2),
6.16 (s, 1H, ArH), 7.11–7.22 (m, 3H, 2ArH and CH), 7.33 (d, J = 7.7,
2H, ArH), 7.50 (d, J = 8.8, 1H, ArH), 8.17 (s, 1H, ArH), 8.43 (s, 1H,
ArH), 8.53 (s, 1H, ArH). Anal. Calcd for C25H26ClNO4: C: 68.25; H:
5.96; N: 3.18. Found: C: 68.34; H: 6.01; N: 3.25.
Mp 170–172 °C, yield 93%; 1H NMR (DMSO-d6): 1.23 (t, J = 7.4,
3H, CH3), 2.19 (s, 6H, CH3), 4.09 (q, J = 7.4, 2H, CH2), 5.34 (s, 2H,
CH2), 6.16 (s, 1H, ArH), 6.86–6.98 (m, 4H, 3ArH and CH), 7.41 (d,
J = 8.5, 1H, ArH), 7.93 (s, 1H, ArH), 8.11 (d, J = 6.4, 1H, ArH), 8.50
(s, 1H, ArH). Anal. Calcd for C23H22FNO4: C: 69.51; H: 6.09; N:
3.52. Found: C: 69.76; H: 6.24; N: 3.77.
4.4.4. Ethyl 4-{1-[2,4-bis(trifluoromethyl)benzyl]-5-chloro-1H-
indol-3-yl}-2-hydroxy-4-oxobut-2-enoate (10d)
4.4.12. Ethyl 4-[1-(2,4,6-trimethylbenzyl)-5-fluoro-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (11f)
Mp 223–225 °C, yield 68%; 1H NMR (DMSO-d6): 1.23 (t, J = 7.3,
3H, CH3), 4.10 (q, J = 7.3, 2H, CH2), 5.79 (s, 2H, CH2), 6.20 (s, 1H,
OH), 6.62 (d, J = 8.8, 1H, ArH), 7.11–7.23 (m, 3H, 2ArH and CH),
7.94 (d, J = 8.5, 1H, ArH), 8.11 (s, 1H, ArH), 8.20 (d, J = 7.2, 1H,
ArH), 8.49 (s, 1H, ArH). Anal. Calcd for C23H16ClF6NO4: C: 53.14;
H: 3.10; N: 2.69. Found: C: 53.29; H: 3.27; N: 2.83.
Mp 177–179 °C, yield 94%; 1H NMR (DMSO-d6): 1.20 (t, J = 7.3,
3H, CH3), 2.16 (s, 6H, CH3), 2.27 (s, 3H, CH3), 4.05 (q, J = 7.3, 2H,
CH2), 5.28 (s, 2H, CH2), 5.94 (d, J = 4.7, 1H, ArH), 6.19 (s, 1H, ArH),
6.96–7.03 (m, 2H, ArH and CH), 7.20 (s, 1H, ArH), 7.56 (t, J = 8.5,
1H, ArH), 8.06 (m, 1H, ArH). Anal. Calcd for C24H24FNO4: C:
70.40; H: 5.91; N: 3.42. Found: C: 70.23; H: 6.07; N: 3.24
4.4.5. Ethyl 4-[1-(3,5-dimethylbenzyl)-5-chloro-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (10e)
4.4.13. Ethyl 4-[1-(4-phenylbenzyl)-5-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoate (12a)
Mp 215–217 °C, yield 89%; 1H NMR (DMSO-d6): 1.25 (t, J = 7.2,
3H, CH3), 2.20 (s, 6H, CH3), 4.09 (q, J = 7.2, 2H, CH2), 5.37 (s, 2H,
CH2), 6.17 (s, 1H, ArH), 6.86–6.89 (m, 3H, 2ArH and CH), 7.13 (d,
Mp 164 °C dec, yield 95%; 1H NMR (DMSO-d6) 1.24 (t, J = 7.3, 3H,
CH3), 3.75 (s, 3H, OCH3) 3.93 (q, J = 7.3, 2H, CH2), 5.46 (s, 2H, CH2),

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S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
6.29 (br s, 1H, OH), 6.68-8.18 (m, 14H, ArH and CH). Anal. Calcd for
28H25FNO5: C, 73.83; H, 5.53; N, 3.08. Found: C, 73.55; H, 5.27; N
3.01.
and CH), 7.59–7.63 (m, 4H, ArH), 7.73 (d, J = 9, 2H, ArH), 8.21 (s,
1H, ArH), 9.18 (s, 1H, ArH). Anal. Calcd for C25H18ClNO4: C:
69.53; H: 4.20; N: 3.24. Found: C: 69.64; H: 4.42; N: 3.38.
C
4.4.14. Ethyl 4-{1-[4-(trifluoromethyl)benzyl]-5-methoxy-1H-
indol-3-yl}-2-hydroxy-4-oxobut-2-enoate (12b)
4.5.2. 4-{1-[4-(Trifluoromethyl)benzyl]-5-chloro-1H-indol-3-
yl}-2-hydroxy-4-oxobut-2-enoic acid (13b)
Mp 223 °C dec, yield 92%; 1H NMR (DMSO-d6): 1.23 (t, J = 7.3,
3H, CH3), 3.74 (s, 3H, OCH3), 4.09 (q, J = 7.3, 2H, CH2), 5.52 (s, 2H,
CH2), 6.19 (s, 1H, OH), 6.74 (d, J = 8.8, 1H, ArH), 7.27 (d, J = 9.1,
1H, ArH), 7.30–7.39 (m, 3H, 2ArH and CH), 7.68 (d, J = 7.9, 2H,
ArH), 7.92 (s, 1H, ArH), 8.09 (s, 1H, ArH). Anal. Calcd for C23H20F3-
NO5: C: 61.75; H: 4.51; N: 3.13. Found: C: 61.92; H: 4.38; N: 3.29.
Mp 176–178 °C, yield 30%; 1H NMR (DMSO-d6): 5.63 (s, 2H,
CH2), 6.98 (s, 1H, CH), 7.30 (d, J = 9, 1H, ArH), 7.49 (d, J = 8, 2H,
ArH), 7.62–7.72 (m, 3H, ArH), 8.21 (s, 1H, ArH), 9.07 (s, 1H, ArH).
Anal. Calcd for C20H13ClF3NO4: C: 56.69; H: 3.09; N: 3.31. Found:
C: 56.87; H: 3.32; N: 3.49.
4.5.3. 4-[1-(4-tert-Butylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (13c)
4.4.15. Ethyl 4-[1-(4-tert-butylbenzyl)-5-methoxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (12c)
Mp 109–111 °C, yield 51%; 1H NMR (DMSO-d6): 1.20 (s, 9H,
CH3), 5.46 (s, 2H, CH2), 6.98 (s, 1H, CH), 7.25–7.35 (m, 5H, ArH),
7.69 (d, J = 9.1, 1H, ArH), 8.20 (s, 1H, ArH), 9.06 (s, 1H, ArH). Anal.
Calcd for C23H22ClNO4: C: 67.07; H: 5.38, N: 3.40. Found: C:
67.24; H: 5.21, N: 3.29.
Mp 150–152 °C, yield 91%; 1H NMR (DMSO-d6): 1.16 (s, 9H,
CH3), 1.20 (t, J = 6.9, 3H, CH3), 3.69 (s, 3H, OCH3), 3.89 (d, J = 6.9,
2H, CH2), 5.30 (s, 2H, CH2), 6.22 (s, 1H, OH), 6.65 (d, J = 8.5, 1H,
ArH), 7.13 (d, J = 8.5, 2H, ArH), 7.28 (m, 4H, 3ArH and CH), 7.82
(s, 1H, ArH), 8.42 (s, 1H, ArH). Anal. Calcd for C25H27NO5: C:
71.70; H: 6.71; N: 3.22. Found: C: 71.97; H: 6.91; N: 3.45.
4.5.4. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-chloro-1H-indol-
3-yl}-2-hydroxy-4-oxobut-2-enoic acid (13d)
4.4.16. Ethyl 4-{1-[2,4-bis(trifluoromethyl)benzyl]-5-methoxy-
1H-indol-3-yl}-2-hydroxy-4-oxobut-2-enoate (12d)
Mp 365–367 °C, yield 38%; 1H NMR (DMSO-d6): 5.85 (s, 2H,
CH2), 6.79 (t, J = 8.5, 1H, ArH), 6.94 (s, 1H, CH), 7.32 (d, J = 9.6,
1H, ArH), 7.45 (d, J = 8.5, 1H, ArH), 7.95 (d, J = 7.5, 1H, ArH), 8.14
(s, 1H, ArH), 8.28 (s, 1H, ArH), 8.97 (s, 1H, ArH). Anal. Calcd for C21-
H12ClF6NO4: C: 51.29; H: 2.46; N: 2.85. Found: C: 51.42; H: 2.77; N,
3.03.
Mp 166–168 °C, yield 68%; 1H NMR (DMSO-d6): 1.29 (t, J = 7.3,
3H, CH3), 3.80 (s, 3H, OCH3), 3.95 (q, J = 7.3, 2H, CH2), 5.81 (s, 2H,
CH2), 6.76 (d, J = 8.1, 1H, ArH), 6.88–6.92 (m, 2H, ArH and CH),
7.24 (d, J = 8.9, 1H, ArH), 7.80 (s, 1H, ArH), 7.95 (d, J = 8.1, 1H,
ArH), 8.13 (s, 1H, ArH), 8.81 (s, 1H, ArH). Anal. Calcd for C24H19F6-
NO5: C: 55.93; H: 3.72; N: 2.72. Found: C: 56.11; H: 3.98; N: 2.89.
4.5.5. 4-[1-(3,5-Dimethylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (13e)
4.4.17. Ethyl 4-[1-(3,5-dimethylbenzyl)-5-methoxy-1H-indol-3-
yl]-2-hydroxy-4-oxobut-2-enoate (12e)
Mp 157–159 °C, yield 71%; 1H NMR (DMSO-d6): 2.21 (s, 6H,
CH3), 5.44 (s, 2H, CH2), 6.94 (d, J = 7.5, 3H, ArH), 6.98 (s, 1H, CH),
7.31 (d, J = 8.9, 1H, ArH), 7.65 (d, J = 8.9, 1H, ArH), 8.21 (s, 1H,
ArH), 9.05 (s, 1H, ArH). Anal. Calcd for C21H18ClNO4: C: 65.71; H:
4.73; N: 3.65. Found: C: 65.85; H: 4.68; N: 3.94.
Mp 240–242 °C, yield 89%; 1H NMR (DMSO-d6): 1.26 (t, J = 7.2,
3H, CH3), 2.20 (s, 6H, CH3), 3.76 (s, 3H, OCH3), 3.95 (q, J = 7.2, 2H,
CH2), 5.33 (s, 2H, CH2), 6.29 (s, 1H, ArH), 6.78 (d, J = 8.2, 1H, ArH),
6.86–6.98 (m, 2H, ArH and CH), 7.32 (d, J = 8.8, 1H, ArH), 7.89 (s,
1H, ArH), 8.10 (s, 1H, ArH), 8.48 (s, 1H, ArH). Anal. Calcd for
4.5.6. 4-[1-(2,4,6-Trimethylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (13f)
C24H25NO5: C: 70.75; H: 6.18; N: 3.44. Found: C: 70.67; H: 6.40;
N: 3.58.
Mp 238–240 °C, yield 59%; 1H NMR (DMSO-d6): 2.18 (s, 6H,
CH3), 2.24 (s, 3H, CH3), 5.42 (s, 2H, CH2), 6.82 (s, 1H, CH), 6.97 (s,
2H, ArH), 7.20–7.23 (m, 1H, ArH), 7.61–7.64 (m, 1H, ArH), 7.92–
7.95 (m, 1H, ArH), 8.15 (s, 1H, ArH). Anal. Calcd for C22H20ClNO4:
C: 66.42; H: 5.07; N: 3.52. Found: C: 66.55; H: 5.19; N: 3.41.
4.4.18. Ethyl 4-[1-(2,4,6-trimethylbenzyl)-5-methoxy-1H-indol-
3-yl]-2-hydroxy-4-oxobut-2-enoate (12f)
Mp 157–159 °C, yield 98%; 1H NMR (DMSO-d6): 1.27 (t, J = 7.1,
3H, CH3), 2.17 (s, 6H, CH3), 2.25 (s, 3H, CH3), 3.79 (s, 3H, OCH3),
4.26 (q, J = 7.1, 2H, CH2), 5.40 (s, 2H, CH2), 6.79 (s, 1H, ArH),
6.92–6.94 (m, 3H, 2ArH and CH), 7.48 (d, J = 8.8, 1H, ArH), 7.75
(s, 1H, ArH), 8.04 (s, 1H, ArH). Anal. Calcd for C25H27NO5: C:
71.24; H: 6.46; N: 3.32. Found: C: 71.51; H: 6.28; N: 3.46.
4.5.7. 4-[1-(4-Phenylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (14a)
Mp 173–175 °C, yield 42%; 1H NMR (DMSO-d6): 5.56 (s, 2H,
CH2), 6.98 (s, 1H, ArH), 7.15 (t, J = 8.1, 1H, ArH), 7.32 (d, J = 6.8,
1H, ArH), 7.35–7.45 (m, 4H, 3ArH and CH), 7.59–7.70 (m, 5H,
ArH), 9.08 (s, 1H, ArH). Anal. Calcd for C25H18FNO4: C: 72.28; H:
4.37; N: 3.37. Found: C: 72.44; H: 4.15; N: 3.21.
4.5. General procedure for the synthesis of 4-[1-benzyl-1H-
indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acids (13–15 a–f)
Following the synthetic procedure previously reported by
us,18,19,29,31 the appropriate 4-[1-benzyl-1H-indol-3-yl]-2-hydro-
xy-4-oxobut-2-enoate (10–12 a–f) (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 give the corresponding hydrolysed
derivative. The desired products were crystallized from a mixture
of ethanol and diethyl ether (1:4).
4.5.8. 4-{1-[4-(Trifluoromethyl)benzyl]-5-fluoro-1H-indol-3-
yl}-2-hydroxy-4-oxobut-2-enoic acid (14b)
Mp 174 °C dec, yield 48%; 1H NMR (DMSO-d6): 5.64 (s, 2H, CH2),
6.98 (s, 1H, CH), 7.16 (t, J = 9.4, 1H, ArH), 7.52 (d, J = 8.2, 2H, ArH),
7.63 (d, J = 8.9, 1H, ArH), 7.71 (d, J = 8.2, 1H, ArH), 7.91 (d, J = 9.4,
1H, ArH), 9.08 (s, 1H, ArH). Anal. Calcd for C20H13F4NO4: C:
58.98; H: 3.22; N: 3.44. Found: C: 59.14; H: 3.47; N: 3.24.
4.5.9. 4-[1-(4-tert-Butylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (14c)
4.5.1. 4-[1-(4-Phenylbenzyl)-5-chloro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (13a)
Mp 186–188 °C, yield 55%; 1H NMR (DMSO-d6): 1.22 (s, 9H,
CH3), 5.49 (s, 2H, CH2), 6.98 (s, 1H, CH), 7.11–7.20 (m, 1H, ArH),
7.29–7.37 (m, 4H, ArH), 7.68–7.72 (m, 1H, ArH), 7.99 (d, J = 9.4,
Mp 220 °C dec, yield 78%; 1H NMR (DMSO-d6): 5.59 (s, 2H, CH2),
7.03 (s, 1H, ArH), 7.33 (t, J = 9, 2H, ArH), 7.39–7.45 (m, 3H, 2ArH

S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
2277
1H, ArH), 9.08 (s, 1H, ArH). Anal. Calcd for C23H22FNO4: C: 69.86; H:
5.61; N: 3.54. Found: C: 69.99; H: 5.52; N: 3.59.
1H, ArH). Anal. Calcd for C22H21NO5: C: 69.65; H: 5.58; N: 3.69.
Found: C: 69.46; H: 5.81; N: 3.77.
4.5.10. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-fluoro-1H-indol-
3-yl}-2-hydroxy-4-oxobut-2-enoic acid (14d)
4.5.18. 4-[1-(2,4,6-Trimethylbenzyl)-5-methoxy-1H-indol-3-yl]-
2-hydroxy-4-oxobut-2-enoic acid (15f)
Mp 237–239 °C, yield 61%; 1H NMR (DMSO-d6): 5.82 (s, 2H,
CH2), 6.79 (d, J = 8.1, 1H, ArH), 6.95 (s, 1H, CH), 7.16 (t, J = 9.4,
1H, ArH), 7.39 (d, J = 8.9, 1H, ArH), 7.97 (d, J = 9.4, 1H, ArH), 8.25
(s, 1H, ArH), 8.97 (s, 1H, ArH). Anal. Calcd for C21H12F7NO4: C:
53.07; H: 2.54; N: 2.95. Found: C: 53.25; H: 2.32; N: 3.09.
Mp 204–206 °C, yield 49%; 1H NMR (DMSO-d6): 2.18 (s, 6H,
CH3), 2.27 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.40 (s, 2H, CH2), 6.76
(s, 1H, CH), 6.92–6.95 (m, 3H, ArH), 7.51 (d, J = 9, 1H, ArH), 7.76
(s, 1H, ArH), 8.00 (s, 1H, ArH). Anal. Calcd for C23H23NO5: C:
70.22; H: 5.89; N: 3.56. Found: C: 69.98; H: 5.97; N: 3.85.
4.5.11. 4-[1-(3,5-Dimethylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (14e)
4.6. General procedure for the synthesis of 4-[1-benzyl-1H-
indol-3-oyl)]-3-hydroxyfuran-2(5H)-ones (16–18 a–f)
Mp 231–233 °C, yield 98%; 1H NMR (DMSO-d6): 2.21 (s, 6H,
CH3), 5.43 (s, 2H, CH2), 6.92–6.99 (m, 4H, 3ArH and CH), 7.13–
7.20 (m, 1H, ArH), 7.61–7.65 (m, 1H, ArH), 7.88–7.92 (m, 1H,
ArH), 9.05 (s, 1H, ArH). Anal. Calcd for C21H18FNO4: C: 68.66; H:
4.94; N: 3.81. Found: C: 68.50; H: 5.18; N: 3.94.
Adopting the synthetic procedure previously reported by us32, a
solution of 40% aqueous formaldehyde in water (4 ml) was added
to a mixture of 13–15 a–f (1 mmol) in diethyl ether (5 ml). The stir-
ring was then continued until clear layers were formed (usually
within 1–2 h). 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 was collected, dried and recrystallized from ethanol.
4.5.12. 4-[1-(2,4,6-Trimethylbenzyl)-5-fluoro-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (14f)
Mp 110–112 °C, yield 14%; 1H NMR (DMSO-d6): 2.17 (s, 6H,
CH3), 2.26 (s, 3H, CH3), 5.44 (s, 2H, CH2), 6.80 (s, 1H, CH), 6.95 (s,
2H, ArH), 7.19–7.23 (m, 1H, ArH), 7.62–7.66 (m, 1H, ArH), 7.91–
7.95 (m, 1H, ArH), 8.13 (s, 1H, ArH). Anal. Calcd for C22H20FNO4:
C: 69.28; H: 5.29; N: 3.67. Found: C: 69.46; H: 5.15; N: 3.48.
4.6.1. 4-[1-(4-Phenylbenzyl)-5-chloro-1H-indol-3-yl)-3-
hydroxyfuran-2(5H)-one (16a)
Mp 199–201 °C, yield 69%; 1H NMR (DMSO-d6): 5.12 (s, 2H,
CH2), 5.60 (s, 2H, CH2), 7.29–8.81 (m, 13H, ArH). Anal. Calcd for C26-
H18ClNO4: C: 70.35; H: 4.09; N: 3.16. Found: C: 70.54; H: 4.29; N:
3.42.
4.5.13. 4-[1-(4-Phenylbenzyl)-5-methoxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (15a)
Mp 170–172 °C, yield 64%; 1H NMR (DMSO-d6): 3.78 (s, 3H,
OCH3), 5.52 (s, 2H, CH2), 6.90 (d, J = 8.5, 1H, ArH), 6.97 (s, 1H,
ArH), 7.34 (d, J = 7.3, 1H, ArH), 7.40–7.45 (m, 4H, 3ArH and CH),
7.55 (d, J = 8.9, 1H, ArH), 7.59–7.63 (m, 4H, ArH), 7.74 (s, 1H,
ArH), 8.97 (s, 1H, ArH). Anal. Calcd for C26H21NO5: C: 73.06; H:
4.95; N: 3.28. Found: C: 73.26; H: 4.81; N: 3.08.
4.6.2. 4-[1-(4-Trifluoromethylbenzyl)-5-chloro-1H-indol-3-yl)-
3-hydroxyfuran-2(5H)-one (16b)
Mp 212–214 °C, yield 32%; 1H NMR (DMSO-d6): 5.12 (s, 2H,
CH2), 5.59 (s, 2H, CH2), 7.10–7.85 (m, 7H, ArH), 8.79 (s, 1H, ArH).
Anal. Calcd for C21H13ClF3NO4: C: 57.88; H: 3.01; N: 3.21. Found:
C: 57.63; H: 3.22; N: 3.41.
4.5.14. 4-{1-[4-(Trifluoromethyl)benzyl]-5-methoxy-1H-indol-
3-yl}-2-hydroxy-4-oxobut-2-enoic acid (15b)
Mp 178–180 °C, yield 72%; 1H NMR (DMSO-d6) 3.80 (s, 3H,
OCH3), 5.61 (s, 2H, CH2), 6.97–7.78 (m, 8H, ArH and CH), 8.98 (s,
1H, ArH). Anal. Calcd for C21H16F3NO5: C, 60.15; H, 3.38; N, 3.34.
Found: C, 60.23; H, 3.57; N, 3.56.
4.6.3. 4-[1-(4-tert-Butylbenzyl)-5-chloro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (16c)
Mp 179–181 °C, yield 26%;1H NMR (DMSO-d6): 1.23 (s, 9H,
CH3), 5.13 (s, 2H, CH2), 5.51 (s, 2H, CH2), 7.26 (t, J = 8.3, 3H, ArH),
7.33 (t, J = 8.3, 3H, ArH), 7.65 (d, J = 8.8, 1H, ArH), 8.22 (s, 1H,
ArH), 8.77 (s, 1H, OH). Anal. Calcd for C24H22ClNO4:C: 68.00; H:
5.23; N: 3.30. Found: C: 68.11; H: 5.34; N: 3.21.
4.5.15. 4-[1-(4-tert-Butylbenzyl)-5-methoxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (15c)
Mp 177–179 °C, yield 26%; 1H NMR (DMSO-d6): 1.19 (s, 9H,
CH3), 3.74 (s, 3H, OCH3), 5.42 (s, 2H, CH2), 6.88 (d, J = 9, 1H, ArH),
6.95 (s, 1H, CH), 7.29 (d, J = 8.5, 4H, ArH), 7.52 (d, J = 9, 1H, ArH),
7.70 (s, 1H, ArH), 8.94 (s, 1H, ArH). Anal. Calcd for C24H25NO5: C:
70.74; H: 6.18; N: 3.44. Found: C: 70.92; H: 6.34; N: 3.70.
4.6.4. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-chloro-1H-indol-
3-yl}-3-hydroxyfuran-2(5H)-one (16d)
Mp 231–233 °C, yield 44%; 1H NMR (DMSO-d6): 5.10 (s, 2H,
CH2), 5.87 (s, 2H, CH2), 6.88–8.69 (m, 7H, ArH). Anal. Calcd for C22-
H12ClF6NO4: C, 52.45; H, 2.40; N, 2.78. Found: C, 52.39; H, 2.52; N,
2.89.
4.5.16. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-methoxy-1H-
indol-3-yl}-2-hydroxy-4-oxobut-2-enoic acid (15d)
4.6.5. 4-[1-(3,5-Dimethylbenzyl)-5-chloro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (16e)
Mp 198–200 °C, yield 72%; 1H NMR (DMSO-d6): 3.80 (s, 3H,
OCH3), 5.80 (s, 2H, CH2), 6.79 (d, J = 8.1, 1H, ArH), 6.89 (s, 1H,
ArH), 6.92 (s, 1H, CH), 7.26 (d, J = 8.9, 1H, ArH), 7.80 (s, 1H, ArH),
7.96 (d, J = 8.1, 1H, ArH), 8.13 (s, 1H, ArH), 8.86 (s, 1H, ArH). Anal.
Calcd for C22H15F6NO5: C: 54.22; H: 3.10; N: 2.87. Found: C:
54.35; H: 3.32; N: 3.04.
Mp 215–217 °C, yield 63%; 1H NMR (DMSO-d6): 2.19 (s, 6H,
CH3), 5.10 (s, 2H, CH2), 5.44 (s, 2H, CH2), 6.91 (s, 3H, ArH), 7.28
(d, J = 8.5, 1H, ArH), 7.58 (d, J = 8.5, 1H, ArH), 8.20 (s, 1H, ArH),
8.70 (s, 1H, ArH). Anal. Calcd for C22H18ClNO4: C: 66.75; H: 4.58;
N: 3.54. Found: C: 66.63; H: 4.75; N: 3.70.
4.5.17. 4-[1-(3,5-Dimethylbenzyl)-5-methoxy-1H-indol-3-yl]-2-
hydroxy-4-oxobut-2-enoic acid (15e)
4.6.6. 4-[1-(2,4,6-Trimethylbenzyl)-5-chloro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (16f)
Mp 180–182 °C, yield 45%; 1H NMR (DMSO-d6): 2.20 (s, 6H,
CH3), 3.78 (s, 3H, OCH3), 5.38 (s, 2H, CH2), 6.87–6.96 (m, 5H,
4ArH and CH), 7.47 (d, J = 9.1, 1H ArH), 7.74 (s, 1H, ArH), 8.87 (s,
Mp 228–230 °C, yield 48%; 1H NMR (DMSO-d6): 2.19 (s, 6H,
CH3), 2.25 (s, 3H, CH3), 4.94 (s, 2H, CH2), 5.38 (s, 2H, CH2), 6.94
(s, 2H, ArH), 7.37 (d, J = 8.5, 1H, ArH), 7.79 (d, J = 8.5, 1H, ArH),

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S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
8.24 (s, 2H, ArH). Anal. Calcd for C23H20ClNO4: C: 67.40; H: 4.92; N:
3.42. Found: C: 67.55; H: 5.02; N: 3.67.
(d, J = 9.0, 1H, ArH), 7.28 (dd, J = 8.5, 4H, ArH), 7.49 (d, J = 9.0, 1H,
ArH), 7.76 (s, 1H, ArH), 8.59 (s, 1H, ArH). Anal. Calcd for
C24H27NO5: C: 71.24; H: 6.46; N: 3.32. Found: C: 71.09; H: 6.37;
4.6.7. 4-[1-(4-Phenylbenzyl)-5-fluoro-1H-indol-3-yl)-3-
hydroxyfuran-2(5H)-one (17a)
N: 3.22.
Mp 165–167 °C, yield 41%; 1H NMR (CDCl3): 5.12 (s, 2H, CH2),
5.60 (s, 2H, CH2), 7.11-8.81 (m, 13H, ArH). Anal. Calcd for C26H18-
FNO4: C, 73.06; H, 4.24; N, 3.28. Found: C, 73.23; H, 4.43; N, 3.12.
4.6.16. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-methoxy-1H-
indol-3-yl}-3-hydroxyfuran-2(5H)-one (18d)
Mp 223–225 °C, yield 68%; 1H NMR (DMSO-d6): 3.79 (s, 3H,
OCH3), 5.12 (s, 2H, CH2), 5.83 (s, 2H, CH2), 6.83–8.13 (m, 6H,
ArH), 8.60 (s, 1H, ArH). Anal. Calcd for C23H15F6NO5: C, 55.32; H,
3.03; N, 2.80. Found: C, 55.29; H, 3.37; N, 2.89.
4.6.8. 4-[1-(4-Trifluoromethylbenzyl)-5-fluoro-1H-indol-3-yl)-
3-hydroxyfuran-2(5H)-one (17b)
Mp 209–211 °C, yield 42%; 1H NMR (DMSO-d6): 5.10 (s, 2H,
CH2), 5.67 (s, 2H, CH2), 7.08–7.95 (m, 7H, ArH), 8.86 (s, 1H, ArH).
Anal. Calcd for C21H13F4NO4: C, 61.15; H, 3.12; N, 3.34. Found: C,
61.35; H, 3.08; N, 3.27.
4.6.17. 4-[1-(3,5-Dimethylbenzyl)-5-methoxy-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (18e)
Mp 198–200 °C, yield 65%; 1H NMR (DMSO-d6): 2.21 (s, 6H,
CH3), 3.76 (s, 3H, OCH3), 5.15 (s, 2H, CH2), 5.41 (s, 2H, CH2),
6.88–6.93 (m, 4H, ArH), 7.45 (d, J = 8.8, 1H, ArH) 7.77 (s, 1H,
ArH), 8.54 (s, 1H, ArH). Anal. Calcd for C22H18NO5: C: 70.20; H:
4.82; N: 3.72. Found: C: 70.35; H: 4.99; N: 3.93.
4.6.9. 4-[1-(4-tert-Butylbenzyl)-5-fluoro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (17c)
Mp 229–231 °C, yield 29%; 1H NMR (DMSO-d6): 1.23 (s, 9H,
CH3), 4.87 (s, 2H, CH2), 5.42 (s, 2H, CH2),7.00 (t, J = 8.8, 1H, ArH),
7.17 (d, J = 8.3, 2H, ArH), 7.34 (d, J = 7.1, 2H, ArH), 7.51–7.55 (m,
1H, ArH), 8.12 (d, J = 9.4, 1H, ArH), 10.30 (s, 1H, ArH). Anal. Calcd
for C24H22FNO4:C: 70.75; H: 5.44; N: 3.44. Found: C: 70.89; H:
5.23; N: 3.55.
4.6.18. 4-[1-(2,4,6-Trimethylbenzyl)-5-methoxy-1H-indol-3-yl]-
3-hydroxyfuran-2(5H)-one (18f)
Mp 191–193 °C, yield 52%; 1H NMR (DMSO-d6): 2.19 (s, 6H,
CH3), 2.25 (s, 3H, CH3), 3.79 (s, 3H, OCH3),4.96 (s, 2H, CH2), 5.34
(s, 2H, CH2), 6.91–7.01 (m, 3H, ArH), 7.63 (d, J = 8.8, 1H, ArH),
7.79 (s, 1H, ArH), 7.94 (s, 1H, ArH). Anal. Calcd for C24H23NO5: C:
71.10; H: 5.72; N: 3.45. Found: C: 71.29; H: 5.91; N: 3.63.
4.6.10. 4-{1-[2,4-Bis(trifluoromethyl)benzyl]-5-fluoro-1H-indol-
3-yl}-3-hydroxyfuran-2(5H)-one (17d)
Mp 232–234 °C, yield 59%; 1H NMR (DMSO-d6): 5.19 (s, 2H,
CH2), 5.73 (s, 2H, CH2), 6.72–8.54 (m, 7H, ArH). Anal. Calcd for C22-
H12F7NO4: C, 54.22; H, 2.48; N, 2.87. Found: C, 54.46; H, 2.61; N,
2.74.
4.7. Docking studies
Docking calculations were carried out using the same protocol
that we successfully applied in our previous papers,20,34,37,38 using
Gold software39 and GoldScore scoring function.
4.6.11. 4-[1-(3,5-Dimethylbenzyl)-5-fluoro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (17e)
Regarding docking into the LEDGF/p75 binding pocket, the crys-
tal structure of the dimeric INCCD complexed with the LEDGF/
p75IBD was retrieved from the RCSB Protein Data Bank (entry code
2B4J).40 The LEDGF structure was removed and hydrogen atoms
were added to the IN protein in Discovery Studio 2.5.5.41 The stan-
dard default settings were used in all calculations. A 20.0 Å radius
active site was drawn on the original position of the LEDGFIBD
dipeptide Ile365-Asp366 and automated cavity detection was
used. Results differing by less than 0.75 Å in ligand-all atom RMSD
were clustered together. Two rotatable bonds of the diketo acid
moiety of the ligands were kept fixed during docking calculation.
The two molecules containing the diketo acid moiety were simu-
lated in the mono-ionized form.
Docking studies on IN catalytic site were performed using the
crystal structure of PFV–IN with DNA and two Mg ions complexed
with the inhibitor raltegravir, retrieved from the RCSB Protein Data
Bank (PDB code 3OYA).42 Hydrogen atoms were added using Dis-
covery Studio 2.5.5 and the structure of raltegravir was removed.
A 15 Å radius active site was defined considering the coordinates
of raltegravir in the X-ray position as the centre of the binding
pocket.
Mp 230–232 °C, yield 78%; 1H NMR (DMSO-d6): 2.21 (s, 6H,
CH3), 5.13 (s, 2H, CH2), 5.46 (s, 2H, CH2), 6.91–6.95 (m, 3H, ArH),
7.12–7.18 (m, 1H, ArH), 7.58–7.62 (m, 1H, ArH), 7.89–7.93 (m,
1H, ArH), 8.68 (s, 1H, ArH). Anal. Calcd for C22H18FNO4: C: 69.65;
H: 4.78; N: 3.69. Found: C: 69.81; H: 4.92; N: 3.89.
4.6.12. 4-[1-(2,4,6-Trimethylbenzyl)-5-fluoro-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (17f)
Mp 202–204 °C, yield 44%; 1H NMR (DMSO-d6): 2.20 (s, 6H,
CH3), 2.27 (s, 3H, CH3), 4.98 (s, 2H, CH2), 5.40 (s, 2H, CH2), 6.97
(s, 2H, ArH), 7.39 (d, J = 8.5, 1H, ArH), 7.76 (d, J = 8.5, 1H, ArH),
8.22 (s, 2H, ArH). Anal. Calcd for C23H20FNO4: C: 70.22; H: 5.12;
N: 3.56. Found: C: 70.50; H: 5.23; N: 3.69.
4.6.13. 4-[1-(4-Phenylbenzyl)-5-methoxy-1H-indol-3-yl)-3-
hydroxyfuran-2(5H)-one (18a)
Mp 209–211 °C, yield 49%; 1H NMR (DMSO-d6): 3.77 (s, 3H,
CH3), 5.15 (s, 2H, CH2), 5.55 (s, 2H, CH2), 6.86-8.60 (m, 13H, ArH).
Anal. Calcd for C27H21NO5:C, 73.79; H, 4.82; N, 3.19. Found: C,
73.52; H, 4.61; N, 3.34.
4.6.14. 4-[1-(4-Trifluoromethylbenzyl)-5-methoxy-1H-indol-3-
yl)-3-hydroxyfuran-2(5H)-one (18b)
4.8. Biological assays
Mp 213–215 °C, yield 38%; 1H NMR (DMSO-d6): 3.76 (s, 3H,
CH3), 5.10 (s, 2H, CH2), 5.62 (s, 2H, CH2), 6.85–7.80 (m, 7H, ArH),
8.79 (s, 1H, ArH). Anal. Calcd for C22H16F3NO5: C, 61.26; H, 3.74;
N, 3.25. Found: C, 61.44; H, 3.58; N, 3.39.
4.8.1. LEDGF/p75-HIV-1 integrase interaction screening
The AlphaScreen assay was performed as previously describe.43
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 compounds at 4 °C for 30 min. The compounds were
added in varying concentrations from 1 up to 100 nM. Afterward
4.6.15. 4-[1-(4-tert-Butylbenzyl)-5-methoxy-1H-indol-3-yl]-3-
hydroxyfuran-2(5H)-one (18c)
Mp 219–221 °C, yield 38%;1H NMR (DMSO-d6): 1.21 (s, 9H,
CH3), 3.76 (s, 3H, OCH3), 5.13 (s, 2H, CH2), 5.44 (s, 2H, CH2), 6.87

S. Ferro et al. / Bioorg. Med. Chem. 22 (2014) 2269–2279
8. listed, N. a. AIDS alert 2009, 24, 106.
2279
100 nM of recombinant flag-LEDGF/p75 was added and incubation
was extended by another hour at 4 °C. Subsequently, 5 l of
Ni-chelate-coated acceptor beads and 5 l of anti-flag donor beads
were added to a final concentration of 20 g/ml of both beads. Pro-
teins 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 (Per-
kin–Elmer, Benelux) and analyzed using the EnVision manager
software.
9. Cooper, D. A.; Steigbigel, R. T.; Gatell, J. M.; Rockstroh, J. K.; Katlama, C.; Yeni, P.;
Lazzarin, A.; Clotet, B.; Kumar, P. N.; Eron, J. E.; Schechter, M.; Markowitz, M.;
Loutfy, M. R.; Lennox, J. L.; Zhao, J.; Chen, J.; Ryan, D. M.; Rhodes, R. R.; Killar, J.
A.; Gilde, L. R.; Strohmaier, K. M.; Meibohm, A. R.; Miller, M. D.; Hazuda, D. J.;
Nessly, M. L.; DiNubile, M. J.; Isaacs, R. D.; Teppler, H.; Nguyen, B. Y. N. Eng. J.
Med. 2008, 359, 355.
10. Steigbigel, R. T.; Cooper, D. A.; Teppler, H.; Eron, J. J.; Gatell, J. M.; Kumar, P. N.;
Rockstroh, J. K.; Schechter, M.; Katlama, C.; Markowitz, M.; Yeni, P.; Loutfy, M.
R.; Lazzarin, A.; Lennox, J. L.; Clotet, B.; Zhao, J.; Wan, H.; Rhodes, R. R.;
Strohmaier, K. M.; Barnard, R. J.; Isaacs, R. D.; Nguyen, B. Y. Clin. Infect. Dis.
2010, 50, 605.
11. Malet, I.; Delelis, O.; Valantin, M. A.; Montes, B.; Soulie, C.; Wirden, M.;
Tchertanov, L.; Peytavin, G.; Reynes, J.; Mouscadet, J. F.; Katlama, C.; Calvez, V.;
Marcelin, A. G. Antimicrob. Agents Chemother. 2008, 52, 1351.
12. Shimura, K.; Kodama, E.; Sakagami, Y.; Matsuzaki, Y.; Watanabe, W.; Yamataka,
K.; Watanabe, Y.; Ohata, Y.; Doi, S.; Sato, M.; Kano, M.; Ikeda, S.; Matsuoka, M. J.
Virol. 2008, 82, 764.
l
l
l
4.8.2. 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
13. Zolopa, A. R.; Berger, D. S.; Lampiris, H.; Zhong, L.; Chuck, S. L.; Enejosa, J. V.;
Kearney, B. P.; Cheng, A. K. J. Infect. Dis. 2010, 201, 814.
oligonucleotide
(50-ACTGCTAGAGATTTTCCACACTGACTAAAAGG
14. Quashie, P. K.; Sloan, R. D.; Wainberg, M. A. BMC Med. 2012, 10, 34.
15. Garrido, C.; Soriano, V.; Geretti, A. M.; Zahonero, N.; Garcia, S.; Booth, C.;
Gutierrez, F.; Viciana, I.; de Mendoza, C. Antiviral Res. 2011, 90, 164.
16. Min, S.; Sloan, L.; DeJesus, E.; Hawkins, T.; McCurdy, L.; Song, I.; Stroder, R.;
Chen, S.; Underwood, M.; Fujiwara, T.; Piscitelli, S.; Lalezari, J. AIDS 2011, 25,
1737.
17. Saag, S. Top. Antiviral Med. 2012, 20, 162.
18. 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.
19. De Luca, L.; Ferro, S.; Gitto, R.; Barreca, M. L.; Agnello, S.; Christ, F.; Debyser, Z.;
Chimirri, A. Bioorg. Med. Chem. 2010, 18, 7515.
20. De Luca, L.; Gitto, R.; Christ, F.; Ferro, S.; De Grazia, S.; Morreale, F.; Debyser, Z.;
Chimirri, A. Antiviral Res. 2011, 92, 102.
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22. Al-Mawsawi, L. Q.; Neamati, N. ChemMedChem 2011, 6, 228.
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Llano, M. J. Virol. 2010, 84, 740.
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Buckheit, R. W.; Este, J. A.; Cihlar, T. Antiviral Res. 2008, 80, 251.
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GTC-30) is labeled with biotin at the 30 end and the other oligonucleo-
tide is labeled with digoxigenin at the 50 end. For the overall integra-
tion assay the second 50-digoxigenin labeled oligonucleotide is
(50-GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-30). For the
Strand Transfer assay the second oligonucleotide lacks GT at the
30end. The integrase enzyme was diluted in 750 mM NaCl, 10 mM
Tris pH 7.6, 10% glycerol and 1 mM b-mercapto ethanol. To perform
the reaction 4
of 1.6 M) and 4
in a final reaction volume of 40
DTT, 20 M HEPES pH 7.5, 5% PEG and 15% DMSO. The reaction
l
l diluted integrase (corresponding to a concentration
l of annealed oligonucleotides (7 nM) were added
l containing 10 M MgCl2, 5
l
l
l
l
lM
l
was carried out for 1 h at 37 °C. Reaction products were denatured
with 30 mM NaOH and detected by an immunosorbent assay on avi-
din coated plates.44
4.8.3. In vitro anti-HIV and drug susceptibility assays
The inhibitory effect of antiviral drugs on the HIV-induced cyto-
pathic effect (CPE) in human lymphocyte MT-4 cell culture was
determined by the MT-4/MTT-assay.45 This assay is based on the
reduction of the yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) by mitochondrial dehydroge-
nase of metabolically active cells to a blue formazan derivative,
which can be measured spectrophotometrically. The 50% cell cul-
ture infective dose (CCID50) of the HIV(IIIB) strain was determined
by titration of the virus stock using MT-4 cells. For the drug-sus-
ceptibility assays, MT-4 cells were infected with 100–300 CCID50
of the virus stock in the presence of fivefold serial dilutions of
the antiviral drugs. The concentration of the various compounds
that achieved 50% protection against the CPE of the different HIV
strains, which is defined as the EC50, was determined. In parallel
the 50% cytotoxic concentration (CC50) was determined.
26. Esposito, F.; Tramontano, E. Antiviral Chem. Chemother. 2014, 23(4), 129.
27. Di Santo, R. J. Med. Chem. 2014, 57(3), 539.
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A.; Scipione, L.; Tortorella, S.; Zinzula, L.; Novellino, E.; Pommier, Y.;
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Eur. J. Med. Chem. 2011, 46, 756.
30. Ferro, S.; De Luca, L.; Barreca, M. L.; De Grazia, S.; Christ, F.; Debyser, Z.;
Chimirri, A. Bioorg. Med. Chem. 2010, 18, 5510.
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Enzyme Inhib. Med. Chem. 2013, 28, 1002.
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35. Barreca, M. L.; Ferro, S.; Rao, A.; De Luca, L.; Zappala, M.; Monforte, A. M.;
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Acknowledgment
This work was supported by THINC Project (European Commis-
sion HEALTH-F3-2008-201032).
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