Microwave assisted organic synthesis (MAOS) of small molecules as potential HIV-1 integrase inhibitors

Article abstract of DOI:10.3390/molecules16086858

Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). In recent years our research group has been engaged in the stucture-function study of this enzyme and in the development of some three-dimensional pharmacophore models which have led to the identification of a large series of potent HIV-1 integrase strand-transfer inhibitors (INSTIs) bearing an indole core. To gain a better understanding of the structure-activity relationships (SARs), herein we report the design and microwave-assisted synthesis of a novel series of 1-H-benzylindole derivatives.

Full text of DOI:10.3390/molecules16086858

Molecules 2011, 16, 6858-6870; doi:10.3390/molecules16086858
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave Assisted Organic Synthesis (MAOS) of Small
Molecules as Potential HIV-1 Integrase Inhibitors
Stefania Ferro 1,*, Sara De Grazia 1, Laura De Luca 1, Rosaria Gitto 1, Caterina Elisa Faliti 1,
Zeger Debyzer 2 and Alba Chimirri 1
1
Department of Medicinal Chemistry, University of Messina, Viale Annunziata, I-98168 Messina,
Italy; E-Mails: sdegrazia@pharma.unime.it (S.D.G.); ldeluca@unime.it (L.D.L.);
rgitto@unime.it (R.G.); katia_chem@tiscali.it (C.E.F.); achimirri@unime.it (A.C.)
2
Molecular Medicine, Katholieke Universiteit Leuven and IRC KULAK, Kapucijnenvoer 33,
B-3000 Leuven, Flanders, Belgium; E-Mail: zeger.debyser@med.kuleuven.be (Z.D.)
* Author to whom correspondence should be addressed; E-Mail: sferro@unime.it;
Tel.: +39-090-676-6465; Fax: +39-090-676-6402.
Received: 18 July 2011; in revised form: 4 August 2011 / Accepted: 8 August 2011 /
Published: 11 August 2011
Abstract: Integrase (IN) represents a clinically validated target for the development of
antivirals against human immunodeficiency virus (HIV). In recent years our research group
has been engaged in the stucture-function study of this enzyme and in the development of
some three-dimensional pharmacophore models which have led to the identification of a
large series of potent HIV-1 integrase strand-transfer inhibitors (INSTIs) bearing an indole
core. To gain a better understanding of the structure-activity relationships (SARs),
herein we report the design and microwave-assisted synthesis of a novel series of
1-H-benzylindole derivatives.
Keywords: AIDS; integrase inhibitors; small molecules; MAOS
1. Introduction
Human Immunodeficiency Virus 1 (HIV-1) is the etiological agent of Acquired Immune Deficiency
Syndrome (AIDS) which has caused a large number of deaths for over 20 years. During this period,
many efforts have been made to discover antiretroviral drugs that are able to block HIV-1 replication
by targeting different but crucial steps of its life cycle.

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To date, the U.S. Food and Drug Administration (FDA) has approved drugs that are often used in
combination regimens in the well-known Highly Active Antiretroviral Therapy (HAART). These can
be divided into seven groups: Nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse
transcriptase inhibitors (NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease
inhibitors (PIs), fusion inhibitors (FIs), co-receptor inhibitors (CRIs), and integrase inhibitors (INIs) [1].
Although this current therapy has been successful in substantially decreasing in morbidity and
mortality rates, the use of this arsenal of drugs is limited by toxicity, adverse effects, drug resistance,
and more worryingly by the fact that some newly HIV-infected patients carry viruses that are already
resistant to currently approved AIDS treatments [2,3]. These issues make it apparent that the discovery
and the development of new more potent and less toxic anti-HIV agents are urgently required.
Considering that the stability of the infection depends critically on the insertion of viral DNA into
the host genome, the integration process mediated by HIV-1 Integrase (IN) has become an attractive
and a promising target. HIV-1 IN is a 32 kDa protein that belongs to the superfamily of polynucleotidyl
transferases, comprising three structural domains: (1) the amino-terminal domain (NTD); (2) a catalytic
core domain (CCD); and (3) the carboxyl-terminal domain (CTD) [4,5].
The insertion of a double-stranded DNA copy into the chromosomes of an infected cell comprises a
multistep process that involves two separate reactions: 3’-processing (3’-P) and strand-transfer (ST). In
the first step, IN leads to the formation of new 3’-CA-OH while the ST step is a trans-esterification
reaction involving the direct nucleophilic attack of the 3’-hydroxy group of the two newly processed
viral 3’-DNA ends on the phosphodiester backbone of the host target DNA. Finally, the two
overhanging 5’-end nucleotides are cleaved, and the target DNA is then integrated into cellular DNA.
No energy source (e.g., ATP) is needed for the integration reaction, and only divalent cations are
required for catalytic activity. In particular, the two metal cofactors of HIV-IN are both thought to be
Mg2+ ions under physiological conditions [6-10].
To date, all of the small-molecules that have claimed potent antiviral activity as integrase strand-
transfer inhibitors (INSTIs) contain a structural motif able to coordinate the two divalent metal ions in
the active site of the enzyme. Although we now have a good collection of potential INSTIs, there is
only one FDA-approved HIV-IN inhibitor, raltegravir (1, IC50 = 0.0068 μM), marketed as Isentress
and one more, elvitegravir (2, IC50 = 0.002 μM) which is undergoing phase III clinical trials [11,12]
(Figure 1).
Figure 1. Chemical structures of INSTIs: Raltegravir (1) and elvitegravir (2).
O
OH
OH
O
N
O
O
N
H
N
N
O
F
N
O
N
Cl
O
HN
F
OH
2
1
On these bases and taking into account the structural requirements necessary to form a ligand-Mg2+-IN
complex [13,14], in recent years our research group has been engaged in the structure-function study

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of HIV-IN and in the development of new inhibitors able to selectively inhibit the ST step of the
integration. Previously, we reported ligand-based approaches that are useful to generate three-dimensional
pharmacophore models which led to the identification of a large series of indole DKA analogs as novel
and potent INST inhibitors [15-20]. In particular, we discovered the potent derivative 3, CHI-1043
(Figure 2), that displayed the best anti-HIV activity, thus holding promise for further development [20,21].
Figure 2. Chemical structure of CHI-1043 (3); Major structural domains are highlighted.
hydrophobic
aromatic domain
F
hydrophobic
aliphatic domain
N
COOH
OMe
O
HO
chelating domain
We herein report the rational design and synthesis of new methyl-benzylindoles CHI-1043
analogues with the aim of expanding the SARs (structure-activity relationships) of this class of
compounds and of improving both their activity and selective index (SI). The use of high-speed
technology MAOS (Microwave-Assisted-Organic-Synthesis) provided the following advantages:
(i) controlled heating conditions; (ii) reaction time and solvent reduction; (iii) faster exploration of SARs.
2. Results and Discussion
Our ligand- and structure-based drug design studies highlighted the importance of the hydrophobic
feature of a p-fluorobenzyl ring on CHI-1043 derivatives. Taking into account these findings in which
we previously reported a large series of halogen-substituted derivatives [17,18], we went on to plan the
synthesis of some new 1H-benzylindoles bearing one or more methyl groups on the different positions
of the benzyl moiety, with the aim of gaining a better understanding of the SAR of these series of
compounds. In this work we focus on the use of an efficient and practical method for the rapid
preparation of 1-H-benzyl indole derivatives as potential INSTIs under microwave conditions.
This innovative and eco-friendly technology has the potential to influence medicinal chemistry
work in the main phases of the drug discovery process such as the generation of chemical libraries of
“small molecules” or lead optimization. Unlike conventional conditions in which heat is transferred by
conductance, microwave irradiation produces efficient internal heating by the direct coupling of
microwave energy with the polar molecules present in the reaction mixture. Simple, quick, and
efficient MAOS reactions frequently occur much faster than under conventional heating, often

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providing higher yields of the desired products which can be more easily and rapidly purified [22]. The
chemical synthesis of the designed compounds was carried out as depicted in Scheme1.
Scheme 1. Schematic representation for the synthesis of compounds 7a–i.
RR'R''
H
O
O
O
O
H
N
N
N
ii
i
CH3
CH3
OMe
OMe
OMe
O
O
4
5a-i
RR'R''
COOH
RR'R''
RR'R''
N
N
iv
N
iii
COOEt
COOEt
OH
OMe
OMe
OMe
O
OH
O
HO
O
6a-i
7a-i
a
b
c
d
e
R = 2-CH3, R' = H, R'' = H
f
R = 2-CH3, R' = 5-CH3, R'' = H
R = 2-CH3, R' = 6-CH3, R'' = H
R = 3-CH3, R' = 5-CH3, R'' = H
R = 3-CH3, R' = 5-CH3, R'' = 4-F
R = 3-CH3, R' = H, R'' = H
R = 4-CH3, R' = H, R'' = H
g
h
i
R = 2-CH3, R' = 3-CH3, R'' = H
R = 2-CH3, R' = 4-CH3, R'' = H
Reagents and conditions: (i) POCl3, CH3CON(CH3)2, rt, 12 h; (ii) suitable benzyl bromide or
chloride, NaH, DMF, mw: 5 min at continuous temperature (50 °C), 100 Watt; (iii) 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.
Commercially available 4-methoxy-1H-indole was first 3-acetylated by a Vilsmeier Haack reaction,
in which the formylating agent was generated in situ from dimethylacetamide and phosphorus
oxychloride. Reaction of the obtained intermediate 4 with the suitable benzylbromide or chloride gave
the corresponding N-alkyl derivatives 5a–i. This step of the synthetic pathway was carried out by the
addition of sodium hydride in dimethylformamide at 50 °C for 5 min under 100 W by the application
of Microwave-Assisted-Organic-Synthesis (MAOS).
To synthesize diketoester derivatives 6a–i we employed the following two-step sequence under the
same conditions: 50 °C for 2 min under 250 W of microwave irradiation. Benzyl derivatives 5a–i
reacted with diethyl oxalate providing a mixture of tautomers which without separation were converted
into the corresponding diketo acids 7a–i by hydrolysis in basic medium. All the desired products were
fully characterized by elemental analysis, 1H-NMR, and 13C-NMR.
In order to determine the susceptibility of the HIV-1 integrase enzyme towards all of the
synthesized diketo esters and diketo acids, they were tested by enzyme-linked immunosorbent assays.

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Their inhibitory effect on the HIV-induced cytopathic effect (CPE) in human lymphocyte MT-4 cell
cultures was also determined using the MT-4/MTT-assay. The results obtained were compared with
those of our “lead compound” CHI-1043 (3).
The analysis of the enzymatic inhibition confirmed our previously reported results, showing also in
this case major activity for the diketo acids 7a–i in comparison to the corresponding diketo esters 6a–i
(1.08 < µM < 55.85 vs. 17.02 < µM < 79.65 on ST step). The mono-methyl substitution on the benzyl
moiety seems to positively influence the IN binding (compounds 7a–c), of these the ortho- and
meta-methyl substituted compounds 7a and 7b proved to have the best IC50 values: 1.07 μΜ and
1.36 μΜ, respectively (Table 1).
Table 1. Inhibition of HIV-1 integrase enzymatic activity.
IN enzymatic activity (IC50-µM)
Compound
a Overall
b ST
7a
1.74 ± 1.32
0.42 ± 0.29
5.76 ± 1.82
2.61 ± 1.17
51.76 ± 33.81
3.83 ± 2.21
7.34 ± 1.32
7.69 ± 5.59
2.17 ± 1.65
0.08 ± 0.003
1.07 ± 0.76
1.36 ± 0.64
8.84 ± 5.60
6.79 ± 0.64
55.85 ± 43.83
9.47 ± 1.06
23.54 ± 17.78
5.49 ± 2.67
5.24 ± 1.34
0.14 ± 0.11
7b
7c
7d
7e
7f
7g
7h
7i
CHI-1043
a
b
Concentration required to inhibit the in vitro overall integrase activity by 50%; Concentration
required to inhibit the in vitro strand transfer step by 50%.
By comparison of these data with those of our 4-fluorosubstituted lead compound CHI-1043 we
hypothesized that the substituent in the para position of the benzyl moiety would positively influence
the activity due not to its lipophilicity (i.e., Me), but more probably due to its electronic effect and/or
ability to form an additional hydrogen bond.
Association of 2-substitution with another methyl group provided a gradual decrease of IN
inhibition showing the most interesting result for derivative 7d (IC50 = 6.79 μΜ), which is
characterized by the presence of 2,3-dimethyl substitution on the benzyl moiety, and the worst for
2,4- and 2,6-dimethyl substitution (7e and 7g, Table 1). A comparable IC50 value (5.49 μΜ) was
obtained for derivative 7h, bearing the 3,5-dimethyl substitution, while an additional fluorination at the
4-position (compound 7i) led to only a marginal improvement in activity (IC50 = 5.24 μΜ).
When the synthesized compounds were tested against HIV-1 replication in cell cultures, the 2- and
3-methyl substituted derivatives showed good EC50 values: 1.74 μΜ (7a) and 2.14 μΜ (7b). Notably,
compound 7a displayed the best selectivity index (SI = 43), measured as a ratio of EC50 and CC50 values.
Considering the disubstituted derivatives, the results obtained for compound 7d (EC50 = 1.74 μΜ)
further confirmed the positive effect of the methyl group when it occupies the 2- and 3- positions of
the benzyl moiety.

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3. Experimental
3.1. Chemistry
3.1.1. General
All the microwave-assisted reactions were carried out in a CEM Focused Microwave Synthesis
System, Model Discover, working at the power required for refluxing under atmospheric conditions.
Melting points were determined on a Buchi Melting Point B-545 apparatus and are uncorrected.
Elemental analyses (C, H, N) were carried out on a Carlo Erba Model 1106 Elemental Analyzer and
the results were 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
1
Flash Chromatography (FC) on a Biotage SP1 EXP. H-NMR spectra were recorded in CDCl3 with
TMS as internal standard or DMSO-d6 on a Varian Gemini-300 spectrometer. Chemical shifts were
expressed in δ (ppm) and coupling constants (J) in hertz (Hz). All the exchangeable protons were
confirmed by addition of D2O. 3-Acetyl-4-methoxy-1H-indole (4) was synthesized following the
previously reported procedure [18]. Briefly, phosphoryl chloride (0.92 mL, 10 mmol) was added to ice
cold dimethylacetamide (2.79 mL, 30 mmol) under stirring and cooling in ice. 4-Methoxy-1H-indole 5
(147.18 mg, 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 removal of the solvent under reduced
pressure, the residue was crystallized by treatment with diethyl ether and recrystallized from
1
dichloromethane. mp 113–115 °C, yield 89%. H-NMR (δ) 2.70 (s, 3H, CH3), 3.97 (s, 3H, OCH3),
6.68–7.79 (m, 4H, ArH), 8.69 (br s, 1H, NH). Anal. Calcd for C11H11NO2: C, 69.83; H, 5.86; N, 7.40.
Found: C, 69.62; H, 5.63; N, 7.51.
3.1.2. General procedure for the synthesis of 3-acetyl-4-methoxy-1-benzyl-1H-indoles 5a–i
Using the synthetic procedure previously reported by us [17,18], 3-acetyl-4-methoxy-1H-indole
(189 mg, 1 mmol) was dissolved in DMF (1 mL) at 0 °C and dry sodium hydride (120 mg, 5 mmol)
was added. After stirring for 2 min, the appropriate 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 5 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 under reduced pressure, the crude mixture was purified by flash
chromatography using a mixture of cyclohexane/ethylacetate (6:4).
1
3-Acetyl-1-(2-methylbenzyl)-4-methoxy-1H-indole (5a). Yield: 64%; mp: 138–140 °C. H-NMR
(CDCl3): δ = 2.29 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 5.24 (s, 2H, CH2), 6.61 (d, 1H,
J = 7.9, ArH), 6.83 (d, 1H, J = 7.4, ArH), 6.95 (d, 1H, J = 8.2, ArH), 7.12–7.25 (m, 3H, ArH), 7.56 ppm
(s, 1H, ArH); Anal. calcd for C19H19NO2: C 77.79, H 6.53, N 4.77, found: C 77.83, H 6.49, N 4.73.
1
3-Acetyl-1-(3-methylbenzyl)-4-methoxy-1H-indole (5b). Yield: 73%; mp: 117–119 °C. H-NMR
(CDCl3): δ = 2.31 (s, 3H, CH3), 2.70 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 5.26 (s, 2H, CH2), 6.70 (d, J = 7.9,

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1H, ArH), 6.93–6.96 (m, 3H, ArH), 7.12–7.25 (m, 3H, ArH), 7.73 ppm (s, 1H, ArH); Anal. calcd for
C19H19NO2: C 77.79, H 6.53, N 4.77, found: C 77.62, H 6.38, N 4.62.
1
3-Acetyl-1-(4-methylbenzyl)-4-methoxy-1H-indole (5c). Yield: 61%; mp: 100–102 °C. H-NMR
(CDCl3): δ = 2.31 (s, 3H, CH3), 2.67 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 5.25 (s, 2H, CH2), 6.68 (d,
J = 7.7, 1H, ArH), 6.95–7.20 (m, 6H, ArH), 7.71 ppm (s, 1H, ArH); Anal. calcd for C19H19NO2: C
77.79, H 6.53, N 4.77, found: 77.56, H 6.39, N 4.93.
1
3-Acetyl-1-(2,3-dimethylbenzyl)-4-methoxy-1H-indole (5d). Yield: 99%; mp: 145–147 °C. H-NMR
(CDCl3): δ = 2.16 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 5.25 (s, 2H,
CH2), 6.70–6.74 (m, 2H, ArH), 6.96–7.14 (m, 2H, ArH), 7.17–7.26 (m, 2H, ArH), 7.52 (s, 1H, ArH);
Anal. calcd for C20H21NO2: C 78.15, H 6.89, N 4.56, found: C 78.23, H 6.71, N 4.38.
1
3-Acetyl-1-(2,4-dimethylbenzyl)-4-methoxy-1H-indole (5e). Yield: 60%; mp: 114–116 °C. H-NMR
(CDCl3): δ = 2.23 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.66 (s, 3H, CH3), 3.99 (s, 3H, OCH3), 5.20 (s, 2H,
CH2), 6.71 (d, J = 7.9, 1H, ArH), 6.78 (d, J = 7.9, 1H, ArH), 6.94–6.98 (m, 2H, ArH), 7.05 (s, 1H,
ArH), 7.18–7.26 (m, 1H, ArH), 7.54 (s, 1H, ArH); Anal. calcd for C20H21NO2: C 78.15, H 6.89, N
4.56, found: C 78.41, H 6.72, N 4.71.
1
3-Acetyl-1-(2,5-dimethylbenzyl)-4-methoxy-1H-indole (5f). Yield: 63%; mp: 130–132 °C. H-NMR
(CDCl3): δ = 2.23 (s, 6H, CH3), 2.67 (s, 3H, CH3), 3.99 (s, 3H, OCH3), 5.19 (s, 2H, CH2), 6.71–6.73
(m, 2H, ArH), 6.98–7.21 (m, 4H, ArH), 7.54 (s, 1H, ArH); Anal. calcd for C20H21NO2: C 78.15, H
6.89, N 4.56, found: C 78.39, H 7.01, N 4.21.
1
3-Acetyl-1-(2,6-dimethylbenzyl)-4-methoxy-1H-indole (5g). Yield: 78%; mp: 140–142 °C. H-NMR
(CDCl3): δ = 2.24 (s, 6H, CH3), 2.60 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 5.20 (s, 2H, CH2), 6.75 (d,
J = 7.7, 1H, ArH), 7.12–7.32 (m, 6H, ArH); Anal. calcd for C20H21NO2: C 78.15, H 6.89, N 4.56,
found: C 78.46, H 6.69, N 4.68.
1
3-Acetyl-1-(3,5-dimethylbenzyl)-4-methoxy-1H-indole (5h). Yield: 73%; mp: 110–112 °C. H-NMR
(CDCl3): δ = 2.26 (s, 6H, CH3), 2.69 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 5.20 (s, 2H, CH2), 6.69 (d,
J = 7.9, 1H, ArH), 6.77 (s, 1H, ArH), 6.93–6.97 (m, 2H, ArH), 7.19 (t, J = 7.97, 1H, ArH), 7.72 (s, 1H,
ArH); Anal. calcd for C20H21NO2: C 78.15, H 6.89, N 4.56, found: C 78.23, H 6.71, N 4.38.
3-Acetyl-1-(4-fluoro-3,5-dimethylbenzyl)-4-methoxy-1H-indole (5i). Yield: 61%; mp: 128–130 °C.
1H-NMR (CDCl3): δ = 2.20 (s, 6H, CH3), 2.69 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 5.16 (s, 2H, CH2), 6.69
(d, J =7.9, 1H, ArH), 6.80 (d, J = 6.6, 2H, ArH), 6.93 (d, J = 8.2, 1H, ArH), 7.16–7.21 (m, 1H, ArH),
7.69 (s, 1H, ArH); Anal. calcd for C20H20FNO2: C 73.83, H 6.20, N 4.30, found: C 73.43, H 6.24, N 4.46.
3.1.3. General procedure for the synthesis of 4-[1-benzyl-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-
oxobut-2-enoates 6a–i
Adopting the synthetic procedure previously reported by us [17,18,20], a mixture of 3-acetyl-4-
methoxy-1-benzyl-1H-indole (1 mmol), diethyl oxalate (219 mg, 1.5 mmol) and a catalytic amount of

Molecules 2011, 16
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NaOCH3 was suspended 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).
4-[1-(2-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6a). Yield: 96%; mp:
1
167 °C dec. H-NMR (DMSO-d6): δ = 1.22 (t, J = 7.1, 3H, CH3), 2.24 (s, 3H, CH3), 3.84 (s, 3H,
OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.36 (s, 2H, CH2), 6.59–7.44 (m, 9H ArH and CH); Anal. calcd for
C23H23NO5: C 70.21, H 5.89, N 3.56, found C 70.32, H 5.76, N 3.69.
4-[1-(3-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6b). Yield: 98%; mp:
1
151–153 °C. H-NMR (DMSO-d6): δ = 1.22 (t, J = 7.1, 3H, CH3), 2.23 (s, 3H, CH3), 3.82 (s, 3H,
OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.32 (s, 2H, CH2), 6.54–7.64 (m, 9H, ArH and CH); Anal. calcd for
C23H23NO5: C 70.21, H 5.89, N 3.56, found C 70.39, H 5.96, N 3.41.
4-[1-(4-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6c). Yield: 99%; mp:
1
198 °C dec. H-NMR (DMSO-d6): δ = 1.22 (t, J = 7.1, 3H, CH3), 2.23 (s, 3H, CH3), 3.82 (s, 3H,
OCH3), 4.12 (q, J = 7.1, 2H, CH2), 5.32 (s, 2H, CH2), 6.55–7.64 (m, 9H, ArH and CH); Anal. calcd for
C23H23NO5: C 70.21, H 5.89, N 3.56, found C 70.09, H 6.02, N 3.41.
4-[1-(2,3-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6d). Yield: 97%;
mp: 172–174 °C. 1H-NMR (DMSO-d6): δ = 1.23 (t, J = 7.1, 3H, CH3), 2.11 (s, 3H, CH3), 2.24 (s, 3H,
CH3), 3.85 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2) 5.36 (s, 2H, CH2), 6.53–7.40 (m, 8H, ArH and
CH); Anal. calcd for C24H25NO5: C 70.75, H 6.18, N 3.44, found C 70.71, H 6.22, N 3.40.
4-[1-(2,4-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6e). Yield: 98%;
mp: 200–202 °C. 1H-NMR (DMSO-d6): δ = 1.25 (t, J = 7.1, 3H, CH3), 2.17 (s, 3H, CH3), 2.22 (s, 3H,
CH3), 3.85 (s, 3H, OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.31 (s, 2H, CH2), 6.62–7.43 (m, 8H, ArH and
CH); Anal. calcd for C24H25NO5: C 70.75, H 6.18, N 3.44, found C 70.58, H 6.34, N 3.28.
4-[1-(2,5-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6f). Yield: 94%,
mp: 180–182 °C. 1H-NMR (DMSO-d6): δ = 1.23 (t, J = 7.1, 3H, CH3), 2.15 (s, 6H, CH3), 3.85 (s, 3H,
OCH3), 4.10 (q, J = 7.1, 2H, CH2), 5.30 (s, 2H, CH2), 6.64–7.41 (m, 8H, ArH and CH); Anal. calcd for
C24H25NO5: C 70.75, H 6.18, N 3.44, found C 70.61, H 6.13, N 3.38.
4-[1-(2,6-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6g). Yield: 91%,
mp: 195–197 °C. 1H-NMR (DMSO-d6): δ = 1.18 (t, J = 7.1, 3H, CH3), 1.91 (s, 6H, CH3), 3.57 (s, 3H,
OCH3), 4.02 (q, J = 6.8, 2H, CH2), 5.30 (s, 2H, CH2), 6.62–7.25 (m, 8H, ArH and CH); Anal. calcd for
C24H25NO5: C 70.75, H 6.18, N 3.44, found C 70.82, H 6.27, N 3.31.
4-[1-(3,5-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6h). Yield: 96%,
mp: 270 °C dec. 1H-NMR (DMSO-d6): δ = 1.23 (t, J = 7.1, 3H, CH3), 2.18 (s, 6H, CH3), 3.82 (s, 3H,

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OCH3), 4.12 (q, J = 7.1, 2H, CH2), 5.28 (s, 2H, CH2), 6.58–7.65 (m, 8H, ArH and CH); Anal. calcd for
C24H25NO5:C 70.75, H 6.18, N 3.44, found C 70.82, H 6.09, N 3.51.
4-[1-(4-Fluoro-3,5-dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (6i). Yield:
1
98%, mp 255–257 °C. H-NMR (DMSO-d6): δ = 1.22 (t, J = 7.1, 3H, CH3), 2.13 (s, 6H, CH3), 3.82
(s, 3H, OCH3), 4.09 (q, J = 7.1, 2H, CH2), 5.27 (s, 2H, CH2), 6.60–7.65 (m, 7H, ArH and CH); Anal.
calcd for C24H24FNO5: C 67.75, H 5.69, N 3.29, found C 67.79, H 5.29, N 3.25.
3.1.4. General procedure for the synthesis of 4-[1-benzyl-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-
oxobut-2-enoic acids 7a–i
Following the synthetic procedure previously reported by us [17,18,20], the appropriate 4-[1-
benzyl-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoate (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 hydrolyzed
derivative. The desired products were crystallized from a mixture of ethanol and diethyl ether (1:4).
4-[1-(2-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7a). Yield: 60%,
mp 164–166 °C. 1H-NMR (DMSO-d6): δ = 2.30 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 5.51 (s, 2H, CH2),
13
6.60–7.60 (m, 8H, ArH and CH), 8.26 (s, 1H, ArH), 13.7 (bs, 1H, OH), 15.7 (bs, 1H, OH); C-NMR
([D6]DMSO): δ = 21.8, 56.2, 56.6, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 125.7, 128.7, 134.8, 136.8,
152.1, 164.5, 187.6, 189.7 ppm; Anal. calcd for C21H19NO5: C 69.03, H 5.24, N 3.83, found C 69.07,
H 5.28, N 3.87.
4-[1-(3-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7b). Yield: 45%,
mp 150–152 °C. 1H-NMR (DMSO-d6): δ = 2.24 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.46 (s, 2H, CH2),
13
6.78–7.58 (m, 8H, ArH and CH), 8.50 (s, 1H, ArH); C-NMR ([D6]DMSO): δ = 24.6, 56.2, 59.4,
103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 126.0, 128.6, 130.9, 134.8, 136.1, 137.5, 138.3, 152.1, 164.5,
187.6, 189.7 ppm; Anal. calcd for C21H19NO5: C 69.03, H 5.24, N 3.83, found C 69.11, H 5.17, N 3.94.
4-[1-(4-Methylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7c). Yield: 58%,
mp 141–143 °C. 1H-NMR (DMSO-d6): δ = 2.24 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 5.44 (s, 2H, CH2),
13
6.77–7.56 (m, 8H, ArH and CH), 8.48 (s, 1H, ArH); C-NMR ([D6]DMSO): δ = 24.3, 56.2, 59.1,
103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 128.9, 133.2, 134.8, 135.4, 137.5, 152.1, 164.5, 187.6, 189.7
ppm; Anal. calcd for C21H19NO5: C 69.03, H 5.24, N 3.83, found C 68.97, H 5.11, N 3.73.
4-[1-(2,3-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7d). Yield:
1
66%, mp 160–162 °C. H-NMR (DMSO-d6): δ = 2.18 (s, 3H, CH3), 2.26 (s, 3H, CH3), 3.91 (s, 3H,
OCH3), 5.52 (s, 2H, CH2), 6.46–7.61 (m, 7H, ArH and CH), 8.20 (s, 1H, ArH); 13C-NMR
([D6]DMSO): δ = 19.3, 22.1, 56.2, 56.9, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 125.6, 125.9, 134.8,
135.8, 136.7, 137.3, 137.5, 152.1, 164.5, 187.6, 189.7 ppm; Anal. calcd for C22H21NO5: C 69.65, H
5.58, N 3.69, found C 69.69, H 5.54, N 3.65.

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4-[1-(2,4-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7e). Yield:
1
60%, mp 143–145 °C. H-NMR (DMSO-d6): δ = 2.22 (s, 3H, CH3), 2.23 (s, 3H, CH3), 3.89 (s, 3H,
OCH3), 5.42 (s, 2H, CH2), 6.58–7.93 (m, 7H, ArH e CH), 8.14 (s, 1H, ArH); 13C-NMR ([D6]DMSO):
δ = 22.1, 24.6, 56.2, 56.6, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 125.9, 128.8, 130.8, 134.4, 134.8,
135.3, 136.7, 137.5, 152.1, 164.5, 187.6, 189.7 ppm; Anal. calcd for C22H21NO5: C 69.65, H 5.58, N
3.69, found C 69.71, H 5.43, N 3.51.
4-[1-(2,5-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7f). Yield:
1
59%, mp 163–165 °C. H-NMR (DMSO-d6): δ = 2.13 (s, 3H, CH3), 2.22 (s, 3H, CH3), 3.93 (s, 3H,
OCH3), 5.46 (s, 2H, CH2), 6.56–7.63 (m, 7H, ArH e CH), 8.23 ppm (s, 1H, ArH); 13C-NMR (DMSO-d6):
δ = 21.8, 24.6, 56.2, 56.6, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 125.9, 128.8, 130.8, 133.8, 134.8,
135.3, 137.3, 137.5, 152.1, 164.5, 187.6, 189.7 ppm; Anal. calcd for C22H21NO5: C 69.65, H 5.58, N
3.69, found C 69.82, H 5.41, N 3.78.
4-[1-(2,6-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7g). Yield:
1
76%, mp 173–175 °C. H-NMR (DMSO-d6): δ = 2.19 (s, 6H, CH3), 3.92 (s, 3H, OCH3), 5.40 (s, 2H,
13
CH2), 6.85–7.44 (m, 7H, ArH e CH), 7.63 ppm (s, 1H, ArH); C-NMR (DMSO-d6): δ = 22.1, 54.1,
56.2, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 125.6, 125.9, 134.8, 136.7, 137.5, 141.1, 152.1, 164.5,
187.6, 189.7 ppm; Anal. calcd for C22H21NO5: C 69.65, H 5.58, N 3.69, found C 69.49, H 5.72, N 3.48.
4-[1-(3,5-Dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7h). Yield:
52%; mp: 120–122 °C. 1H-NMR (DMSO-d6): δ = 2.19 (s, 6H, CH3), 3.89 (s, 3H, OCH3), 5.41 (s, 2H,
CH2), 6.78–7.59 (m, 7H, 6ArH and CH), 8.48 ppm (s, 1H, ArH); 13C-NMR DMSO-d6): δ = 24.1, 56.2,
59.7, 103.4, 111.1, 111.7, 113.9, 121.1, 125.2, 127.9, 134.8, 136.0, 137.5, 138.2, 152.1, 164.5, 187.6,
189.7 ppm; Anal. calcd for C22H21NO5: C 69.65, H 5.58, N 3.69, found: C 69.45, H 5. 41, N 3.75.
4-[1-(4-Fluoro-3,5-dimethylbenzyl)-4-methoxy-1H-indol-3-yl]-2-hydroxy-4-oxobut-2-enoic acid (7i).
1
Yield: 40%; mp: 153–155 °C. H-NMR (DMSO-d6): δ = 2.15 (s, 6H, CH3), 3.89 (s, 3H, OCH3), 5.39
13
(s, 2H, CH2), 6.78–7.21 (m, 5H, ArH e CH), 7.57 (s, 1H, ArH), 8.47 ppm (s, 1H, ArH); C-NMR
([D6]DMSO): δ = 18.1, 56.2, 59.7, 103.4, 111.1, 111.7, 113.9, 121.1, 123.7, 125.2, 129.5, 131.6, 134.8,
137.5, 152.1, 160.5, 164.5, 187.6, 189.7 ppm; Anal. calcd for C22H20FNO5: C 66.49, H 5.07, N 3.52,
found: C 66.36, H 5.28, N 3.39.
3.2. Biological Assays
Overall integrase assay using an enzyme-linked immunosorbent assay (ELISA): To determine the
susceptibility of the HIV-1 integrase enzyme towards different compounds we used enzyme-linked
immunosorbent assays. These assays use an oligonucleotide substrate of which one oligonucleotide
(5’-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-3’) is labeled with biotin at the 3’ end
and the other oligonucleotide is labeled with digoxigenin at the 5’ end. For the overall integration
assay the second 5’-digoxigenin labeled oligonucleotide is (5’-GACCCTTTTAGTCAGTGTGGAAA
ATCTCTAGCAGT-3’). For the Strand Transfer assay the second oligonucleotide lacks GT at the 3’
end. The integrase enzyme was diluted in 750 mM NaCl, 10 mM Tris pH 7.6, 10% glycerol, and 1 mM

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β-mercaptoethanol. To perform the reaction, diluted integrase (4 µL, corresponding to a concentration
of 1.6 µM) and annealed oligonucleotides (4 µL, 7 nM) were added in a final reaction volume of 40 µL
containing 10 mM MgCl2, 5 mM DTT, 20 mM HEPES pH 7.5, 5% PEG, and 15% DMSO. The
reaction was carried out for 1 h at 37 °C. Reaction products were denatured with 30 mM NaOH and
detected by an immunosorbent assay on avidin-coated plates [23].
In vitro anti-HIV and drug susceptibility assays: The inhibitory effect of antiviral drugs on the
HIV-induced cytopathic effect (CPE) in human lymphocyte MT-4 cell culture was determined by the
MT-4/MTT-assay [24]. This assay is based on the reduction of the yellow 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active
cells to a blue formazan derivative, which can be measured spectrophotometrically. The 50% cell
culture infective dose (CCID50) of the HIV(IIIB) strain was determined by titration of the virus stock
using MT-4 cells. For the drug-susceptibility assays, MT-4 cells were infected with 100–300 CCID50
of the virus stock in the presence of 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.
4. Conclusions
A novel series of indole derivatives was synthesized by means of an eco-friendly method which
afforded the expected molecules quickly, in very high yields and using a very small amount of solvent.
Although their biological data show that the modifications did not improve the biological profile of our
lead compound “CHI-1043”, some HIV-1 INSTIs with an IC50 in the low micromolar range were
obtained, thus affording further SAR information that can be considered useful for future studies.
Acknowledgments
This work was supported by THINC project (European Commission HEALTH-F3-2008-201032).
Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 7a–i are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).