Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents targeting reverse transcriptase enzyme

Article abstract of DOI:10.1016/j.ejmech.2019.111987

Infection by human immunodeficiency virus still represents a continuous serious concern and a global threat to human health. Due to appearance of multi-resistant virus strains and the serious adverse side effects of the antiretroviral therapy administered, there is an urgent need for the development of new treatment agents, more active, less toxic and with increased tolerability to mutations. Quinoxaline derivatives are an emergent class of heterocyclic compounds with a wide spectrum of biological activities and therapeutic applications. These types of compounds have also shown high potency in the inhibition of HIV reverse transcriptase and HIV replication in cell culture. For these reasons we propose, in this work, the design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV reverse transcriptase enzyme. For this, we first carried out a structure-based development of target-specific compound virtual chemical library of quinoxaline derivatives. The rational construction of the virtual chemical library was based on previously assigned pharmacophore features. This library was processed by a virtual screening protocol employing molecular docking and 3D-QSAR. Twenty-five quinoxaline compounds were selected for synthesis in the basis of their docking and 3D-QSAR scores and chemical synthetic simplicity. They were evaluated as inhibitors of the recombinant wild-type reverse transcriptase enzyme. Finally, the anti-HIV activity and cytotoxicity of the synthesized quinoxaline compounds with highest reverse transcriptase inhibitory capabilities was evaluated. This simple screening strategy led to the discovery of two selective and potent quinoxaline reverse transcriptase inhibitors with high selectivity index.

Full text of DOI:10.1016/j.ejmech.2019.111987

Journal Pre-proof
Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV
agents targeting reverse transcriptase enzyme
Lucas Fabian, Marisa Taverna Porro, Natalia Gomez, Melina Salvatori, Gabriela Turk,
Dario Estrin, Albertina Moglioni
PII:
S0223-5234(19)31139-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.111987
EJMECH 111987
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 September 2019
Revised Date: 21 November 2019
Accepted Date: 17 December 2019
Please cite this article as: L. Fabian, M. Taverna Porro, N. Gomez, M. Salvatori, G. Turk, D. Estrin, A.
Moglioni, Design, synthesis and biological evaluation of quinoxaline compounds as anti-HIV agents
targeting reverse transcriptase enzyme, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2019.111987.
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Design, synthesis and biological evaluation of quinoxaline compounds as anti-
HIV agents targeting reverse transcriptase enzyme
Lucas Fabiana,b,§, Marisa Taverna Porrob,§, Natalia Gomezc, Melina Salvatorid, Gabriela Turkd, Dario
Estrine, Albertina Moglionia,b*
a Cátedra de Química Medicinal, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CABA, 1113,
Argentina
b Instituto de la Química y Metabolismo del Fármaco (IQUIMEFA), CONICET-Universidad de Buenos Aires, CABA,
1113, Argentina
c
Instituto de Investigaciones Farmacológicas (ININFA)
,CONICET-Universidad de Buenos Aires, CABA, 1113,
Argentina
d Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS), CONICET-Universidad de Buenos Aires,
CABA, 1113, Argentina
e
Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE), Facultad de Ciencias
Exactas y Naturales, CONICET-Universidad de Buenos Aires, CABA,1428 , Argentina
§ Both authors contributed equally
Keywords
Quinoxaline synthesis, reverse transcriptase, virtual chemical library, anti-HIV agents
Highlights
•
•
•
•
•
A quinoxaline library was constructed and evaluated using docking and 3D-QSAR.
Twenty-five quinoxaline derivatives were selected and synthesized.
All compounds were screened for their in vitro RT inhibitory activity.
Four compounds were evaluated as anti-HIV agents in infected MT2 cells.
Compounds 12 and 3 showed promising anti-HIV activities.
Abstract
Infection by human immunodeficiency virus still represents a continuous serious concern and a global
threat to human health. Due to appearance of multi-resistant virus strains and the serious adverse side
effects of the antiretroviral therapy administered, there is an urgent need for the development of new
treatment agents, more active, less toxic and with increased tolerability to mutations. Quinoxaline
derivatives are an emergent class of heterocyclic compounds with a wide spectrum of biological activities
and therapeutic applications. These types of compounds have also shown high potency in the inhibition of
HIV reverse transcriptase and HIV replication in cell culture. For these reasons we propose, in this work,
the design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV reverse
transcriptase enzyme. For this, we first carried out a structure-based development of target-specific
compound virtual chemical library of quinoxaline derivatives. The rational construction of the virtual
chemical library was based on previously assigned pharmacophore features. This library was processed
by a virtual screening protocol employing molecular docking and 3D-QSAR. Twenty-five quinoxaline

compounds were selected for synthesis in the basis of their docking and 3D-QSAR scores and chemical
synthetic simplicity. They were evaluated as inhibitors of the recombinant wild-type reverse transcriptase
enzyme. Finally, the anti-HIV activity and cytotoxicity of the synthesized quinoxaline compounds with
highest reverse transcriptase inhibitory capabilities was evaluated. This simple screening strategy led to
the discovery of two selective and potent quinoxaline reverse transcriptase inhibitors with high selectivity
index.
1. Introduction
Infection by human immunodeficiency virus (HIV) still represents a continuous serious concern and a
global threat to human health and has important social and economic consequences all over the world.
Nowadays, 37 million people are living with HIV and still around 1 million people annually dies from AIDS
related illness [1]. Despite the outstanding advances in the treatment of HIV infection, AIDS remains an
incurable disease. The side effects due to daily use and elevated drug toxicity promotes reduced patient
adherence. This, in addition to the high replication rate of HIV, favors the appearance of multi-resistant
HIV strains [2]. For these reasons AIDS treatment is still chemotherapeutically challenging and,
therefore, there is an urgent need for the development of new agents that are more active, less toxic and
with increased tolerability to mutations.
Combination antiretroviral therapy (cART) is required for durable virologic suppression. One of the most
frequent targets in cART is the HIV reverse transcriptase enzyme (RT), since its reverse transcription
step is essential in viral replication and it is not present in humans. Non-nucleoside RT inhibitors
(NNRTIs) have become an important ingredient of the cART schemes, which includes nevirapine,
delavirdine, efavirenz, etravirine, and rilpivirine approved for clinical use [3]. However, their efficiency has
been undermined by the appearance of drug resistant variants of HIV and therefore it is necessary to
design novel drugs to cope with resistance [4]. Quinoxaline derivatives are a versatile class of
heterocyclic compounds with a wide spectrum of biological activities and therapeutic applications [5].
Compounds containing a quinoxaline scaffold have shown high potency as NNRTIs and in the inhibition
of HIV-1 replication in cell culture [6]. Derivatives such as HBY [7], HBQ [8] and S-2720 [9], efavirenz
(EFV) [10] and quinoline U-78036 [11] and UC38 [12] (Figure 1) are examples of quinoxaline and related
compounds with RT inhibitory action.

Figure 1. Quinoxaline structures and structurally related compounds with RT inhibitory activity.
The development of novel NNRTIs using Computer Aided Drug Design (CADD) is well documented in
literature [13-16]. Approaches employing a combination of virtual screening and de-novo design based on
QSAR studies, docking and molecular dynamics have been successful in the discovery of new and active
NNRTIs [17-19]. Particularly, Patel et al [20] have designed novel quinoxaline derivative inhibitors of HIV
integrase by a two-ligand based drug design approach using pharmacophore modelling and 3D-QSAR.
These compounds were chosen by their docking scores, synthesized and biologically evaluated, resulting
in two derivatives with IC50 in the micromolar range. In this context, we developed a virtual screening
protocol employing molecular docking and 3D-QSAR in order to predict the NNRTI activity of different
families of compounds [21, 22]. This methodology allowed us to successfully predict the RT inhibitory
activity of 58 quinoxaline compounds, whose IC50 had previously been described in the literature.
In the present study, based on the previously developed computational approach, we carried out the
design, synthesis and biological evaluation of quinoxaline derivatives targeting HIV RT. For this aim, we
first carried out a structure-based development of target-specific compound virtual chemical library of
quinoxaline derivatives. The screening of the virtual chemical library using the protocol previously
described (docking and 3D-QSAR) [21, 22] resulted in the preparation of twenty-five quinoxaline with
potential as RT inhibitors. These were selected on the basis of their calculated IC50 values and their
chemical synthetic simplicity, and then evaluated as RT inhibitors and as anti-HIV agents in infected cells.
2.
Results and discussion
2.1
Virtual chemical library construction and screening
To construct a virtual chemical library of quinoxaline (VCL) compounds with potential RT inhibitory
activity, we needed first to define the most important structural requirements for their interaction with the
enzyme allosteric site. Structural knowledge was obtained from the two-dimensional pharmacophore
model developed previously by Wang [13], and more specifically, from ligand–protein complexes of
related compounds, as shown in Figure 2. For the latter, we took into account quinoxaline and related

compounds (Figure 1) that were equally represented into the three-featured pharmacophore. This
analysis revealed that the important chemical features to identify novel RT inhibitors were: a five- or six-
membered aromatic ring; a five- to seven-membered ring which can either be aromatic or aliphatic and a
hydrophilic center that can be nitrogen, oxygen, or sulfur.
A.
B.
HBY
EFV
C.
D.
HBQ
UC38
Figure 2. Different structures of the RT ligand binding domain bound to different NNRTIs containing a
quinoxaline or quinoxaline related scaffold: A. HBY, B.EFV, C. HBQ and D. UC38. The RT structure is
reported in blue and the residues relevant to ligand binding are labeled on top.
Thus, the quinoxaline VCL was constructed by combining different substituents, namely R3, R3´, R4, R6 y
R7 (contemplating also both stereochemistries at position 3 of the heterocyclic ring), with tQNX and dQNX
structures as scaffolds (Table 1). Alkyl or aromatic chains in positions 3 and 4, small substituents in
positions 6 and 7, and a hydrogen forming bond group at position 1 and 2 were, as previously mentioned,
the most important features taken into account. The residues selected as possible substituents at
positions 3, 4, 6 and 7 are shown in Table 1. The VCL was finally constructed by combining the SMILES
strings of the different substituents with those of tQNX and dQNX scaffolds (Table 1). All the possible
combinations of the proposed residues gave rise to a VCL composed by a total of 45804 quinoxaline or
related compounds that were further evaluated in the screening step.

Table 1. Substituents at positions 3, 4, 6 and 7.
H
R7
R6
N
O
H
N
O
N
R3
N
R3´
O
R4
1,2,3,4-tetrahydroquinoxalin-2-one (tQXN
R3
)
1,2-dihydroquinoxalin-2-one (dQNX)
R3’
H
methyl
isobutyl
hydroxyl
(methylthio)ethyl
2-carboxy ethyl
2-butyl
2-carboxy ethyl,
ethyl ester
2-carboxy ethyl,
isopropyl ester
H
methyl
2-butyl
isobutyl
(methylthio)ethyl
isopropyl
isopropyl
benzyl
2-carboxy ethyl,
methyl ester
R4
methyl
ethyl
pent-3-in-1-yl
pent-4-in-1-yl
1-methyl-2-en-
3,3-dimethyl-pentyl
4-chloro-2-butenyl
4-fluor-2-butenyl
3-phenyl-propyl
benzyl
2,2-dimethyl-pentyl
2-propen-1-yl
propyl
butyl
2-cyclopropyl-ethyl
cyclopentyl
Isopropyl
2-buten-1-yl
pentyl
3-buten-1-yl
2-amine-1-ethyl
2-cyano-1-ethyl
phenacyl
hexyl
2-methyl-3-buten-1-yl
(Z)-penta-2,4-dien-1-yl
3-cyclopenten-1-yl
cyclobutyl-methyl
cyclopropyl-methyl
cyclohexyl-methyl
2-chloro-ethyl
heptyl
2-butyl
octyl
Isobutyl
4-methoxy-phenacyl
4-nitro-phenacyl
4-chloro-phenacyl
4-methyl-phenacyl
4-bromo-phenacyl
4-hydroxy-phenacyl
2-nitro-phenacyl
2-chloro-phenacyl
2-methyl-phenacyl
2-methoxy-phenacyl
benzhydryl
nonyl
terbutyl
decyl
2-pentyl
cyclopropyl
cyclobutyl
cyclopentyl
cyclohexyl
2,4-dien-cyclopentyl
2,5-dien-cyclohexyl
propen-2-in-1-yl
buten-2-in-1-yl
buten-3-in-1-yl
penta-2-in-1-yl
3-pentyl
isopentyl
neopentyl
3-chloro-propyl
2-chloro-propyl
4-chloro-butyl
2-hexyl
3-hexyl
3,3-dimethyl-butyl
2,2-dimethyl-butyl
2,3-dimethyl-butyl
3,4-dimethyl-pentyl
4,4-dimethyl-pentyl
3-chloro-butyl
2-trifluoromethyl-ethyl
3-bromo-propyl
4-bromo-butyl
2-thienyl
3-bromo-butyl
2-thienyl-methyl
4-chloro-
thiophenesulfonyl
formyl
isopropyloxycarbonyl
phenylacetyl
acetyl
difluoroacetyl
4-methoxy-phenylacetyl
thiophenesulfonyl
propionyl
4-methoxybenzoyl
4-nitrophenylacetyl
3-chloro-thiophene-2-
carbonyl
1-naphtalenesulfonyl
4-amine-benzoyl
butanoyl
phenylpyruvate
4-chloro-thiophene-2-
carbonyl
(furan-2-yl)-acetyl
(3-chloro-furan-2-yl)-
acetyl
chloroacetyl
3-chloro-propanoyl
benzoyl
2-nitrobenzoyl
3-nitrobenzoyl
4-nitrobenzoyl
2-hydroxy-benzoyl
isonicotinohydrazide-N'-yl
3-isopropylurea-1-yl
benzenesulfonyl
2-butyl-oxy-carbonyl
bromoacetyl
2-chlorobenzoyl
3-chlorobenzoyl
4-chlorobenzoyl
(E)-3-(furan-3-yl) acryloyl
acetamide-N-yl
aniline-N-yl
nicotinate
isonicotinate
dihydrocinnamoyl
3-methoxy-
benzenesulfonyl
2-methyl-acryloyl
benzyloxycarbonyl
acryloyl
4-methyl-benzenesulfonyl
3-chloro-benzenesulfonyl
thiophene-2-carbonyl
thiophene-3-carbonyl

R6 / R7
H
chloro
fluor
methyl
acetylamine
amine
The chemical library screening was performed using two stages: docking and 3D-QSAR, as previously
described [21, 22]. Briefly, the 3D structures of all the VCL members were generated from their SMILES
isomeric notations followed by geometry optimization. Subsequently, molecular docking was used to
generate the ligand conformers that served as input for WHIM descriptors calculation, using the PDB ID
2B6A RT structure as template. This protein was selected as the ligand conformer generating template
from a previous cross-docking calibration experiment and showed to be the most efficient for
crystallographic pose prediction among a strucurally diverse set of compounds. Only the best scoring
docking pose of those VCL compounds with the lowest ∆Gdock values with a cutoff of ∆Gdock ≤ -8.0
Kcal/mol were selected as conformers for further analysis. This cutoff was selected as it was the average
value found for active compounds in the calibration steps.This constraint allowed us the selection of a
total of 14792 compounds (approximately 30% of the VCL). Afterwards, these structures were evaluated
by means of the 3D-QSAR model shown in equation 1, together with WHIM descriptors obtained in the
previous step. In this way, the half maximal inhibitory concentration (IC50) values were calculated.
pIC50= 3.18 * E3m + 5.06 * E2u + 4.4 * Dv – 0.72
Equation 1
From the 14792 compounds enlisted in the VCL, a total of 2970 showed a pIC50 higher than 5 (6.5% of
the initial VCL). Structural analysis of the 2970 compounds that met both docking and 3D-QSAR criteria
showed that the chemical nature of substituents at position 6 and 7 didn’t play a significant role in the
calculated inhibitory effect. For this reason, quinoxaline derivatives not bearing substituents at these
positions were chosen to be synthesized from commercial and inexpensive o-halonitrobenzene and/or o-
phenylenediamine. Chemical variability was achieved by introduction of a wide variety of substituents at
positions 3 and 4 of the heterocyclic system. Their synthetic simplicity was also considered, as some
compounds with the highest predicted pIC50 showed to be synthetically challenging. Taking all these into
account, twenty-five quinoxaline and/or quinoxaline related compounds (1-25, Figure 3) with predicted
pIC50 values within the range of actual active compounds (4.93-8.65) were chosen to be chemically
prepared and evaluated as RT inhibitors. The ∆Gdock, WHIM coefficient descriptors and predicted pIC50
values determined for the synthesized compounds are shown in Table 2.

1
2
3
4
5
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
N
N
N
N
N
O
CH3
O
O
O
O
Cl
Cl
6
7
8
9
10
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
N
N
N
N
CH3
N
CH3
O S O
O
O
O
O
O
Cl
O
Cl
11
12
13
14
15
H
N
H
N
H
N
H
N
H
N
O
O
O
O
O
CH3
CH3
CH3
CH3
N
O
CH3
N
CH3
N
N
N
Cl
O
O
O
Cl
O
O
O
16
17
18
19
20
H
H
H
H
H
N
O
O
N
O
O
N
O
N
O
N
O
N
O
N
H
N
N
N
Cl
21
22
23
24
25
H
H
H
H
H
N
N
O
N
O
N
O
N
O
N
N
O
N
O
N
O
N
O
OH
O
O
O
Figure 3. Synthesized quinoxaline and related compounds.
Table 2. ∆Gdock, predicted pIC50 and WHIM coefficient descriptors determined for compounds 1-25.
Compound #
∆Gdock
-11.7
-8.5
pIC50cal
6.11
6.12
5.68
8.18
8.01
8.65
7.23
6.44
8.05
6.21
5.77
6.46
E3m
0.060
0.136
0.063
0.372
0.401
0.400
0.212
0.102
0.330
0.071
0.088
0.146
Dv
E2u
1
2
0.897
0.952
0.882
1.118
1.004
1.220
1.100
1.058
1.111
1.041
0.897
1.048
0.533
0.439
0.458
0.552
0.600
0.540
0.482
0.430
0.560
0.420
0.448
0.416
3
-8.2
4
-9.3
5
-9.1
6
-10.6
-10.4
-10.6
-8.3
7
8
9
10
11
12
-10.5
-8.9
-8.9

13
14
15
16
17
18
19
20
21
22
23
24
25
-11.4
-10
7.16
6.99
7.30
7.33
7.00
4.93
5.19
5.26
5.82
6.52
8.25
7.40
7.24
0.216
0.245
0.331
0.274
0.148
0.002
0.037
0.052
0.041
0.230
0.175
0.221
0.099
1.165
1.048
1.107
1.120
0.989
0.694
0.811
0.849
0.878
0.934
1.220
1.049
1.086
0.409
0.458
0.414
0.444
0.573
0.512
0.440
0.410
0.504
0.475
0.601
0.553
0.566
-11.6
-9.8
-10.2
-11.2
-9.6
-9.3
-10.8
-10.2
-10.9
-10.5
-9.4
∆Gdock: Gibbs free energy calculated with AutodockVina 1.0 program.
pIC50cal: biological activity calculated using model 1 expressed as log 1/IC50
where IC50 is in Molar unit. WHIM descriptors: E2u, component eta 2 weighted
at connectivity; E3m, component eta 3 weighted at mass; Dv, non-directional
global descriptor weighted for molecular volume
.
2.2. Synthesis of quinoxaline and quinoxaline related compounds (1-25)
2.2.1. 1,2,3,4-tetrahydroquinoxalin-2-ones (tQNXs)synthesis(1-16)
The 1,2,3,4-tetrahydroquinoxalin-2-ones (tQNXs) derivatives 1-16 (Figure 3) bearing different substituents
at positions 3 and 4 were prepared according to general Scheme 1. In general, commercially available o-
halonitrobenzene was reacted with the corresponding α-amino acid (bearing the substituents at position
3) through an aromatic nucleophilic substitution reaction. Then, by nitro reduction and further cyclization,
the acyclic intermediate (a, Scheme 1) gave rise to the corresponding heterocyclic secondary amines (b,
Scheme 1). Finally, the different 4-substituted tQNXs derivatives were prepared by N-acylation of the
heterocyclic amines with the corresponding acyl chlorides.
Scheme 1. General synthetic scheme for the preparation of N4-acylated tQNXs.
Microwave (MW) assisted methods were developed for the first synthetic step. MW assisted synthesis
provides cheaper and cleaner chemistry, enhanced reaction rates and higher yields. Optimum reaction
conditions were initially determined by using α-amino acid methyl esthers as nucleophiles and two
halonitrobenzenes: 1-fluoro-2-nitrobenzene (FNB) and 1-chloro-2-nitrobenzene (ClNB) [23]. The
nucleophilic displacement reaction gave higher reaction rates yields using as substrate FNB than ClNB

(Table 3). Due to the fact that other authors have described the obtention of these derivatives by means
of a modification of the Sanger reaction [24], in which they directly use the free amino acid as starting
material, we decided to evaluate these MW-assisted nucleophilic displacement reactions using as
nucleophiles the potassium salts of the α-amino acids. The yields and rates obtained were similar
independently of the amino acid starting material employed. For this reason, we decided to continue with
the N-arylsubstituted intermediates preparation using directly the potassium salt of the corresponding α-
amino acids, giving rise to the different products with yields around 60 %.
Recently, graphene has shown great potential as an alternative solid carbocatalyst for a variety of MW-
assisted catalyzed reactions [25-28]. Therefore, in order to increase the observed yields and reaction
rates, we decided to evaluate the incorporation of graphene to the displacement reaction. The
heterogeneous system was reacted as a suspension of the solid catalyst and halonitrobenzene in a
potassium carbonate solution of the free α-amino acids. The adittion of graphene as catalyst in this MW-
assisted reaction greatly increased the reaction rate and yields, obtaining the N-arylsubtituted
intermediates with around 90 % yield in only 15 minutes reaction time, while products were obtained in
much lower yields without the addition of this catalyst (for comparison reactions with glycine see Table 4).
This optimized one-pot methodology allowed us to obtain with high yields (approx. 90%) the nitro
derivatives ND1-ND8 (Figure 4), by means of reaction between amino acids such as glycine, (S)-alanine,
(S)-valine and (S)-leucine and their corresponding methyl esters with commercial FNB.
ND1
ND2
ND3
ND4
ND5
ND6
ND7
ND8
Figure 4. N-nitrophenyl derivatives (ND) obtained from amino acids and their methyl esters.
Table 3. Yields and reaction times for the preparation of the acyclic synthesis intermediates of TQNXs.
2-ClNB
2-FNB
2-FNB + graphene
Amino acid derivative
Potassium glycinate
Time Yield Time Yield
(min) (%) (min) (%)
Time
Yield
(min)
15
(%)
90
40
45
50
30
30
75
80
Glycine methyl ester 40
15
90

The preparation of the tQNXs by reduction of the NDs (ND1-8, Figure 4) has already been reported in
literature [29-30] using different reducing agents such as HCOONH4/Pd-C; Na2S2O3/K2CO3; SnCl2/HCl or
Zn0/HCl. These works showed that while reducing the nitro group, partial cyclization of the intermediate
aniline was also simultaneously taking place and that the complete conversion of the acyclic intermediate
to the tQNXs could be achieved by means of heating [24]. In this context, the reduction of the nitro group
followed by the subsequent cyclization reaction was evaluated using: HCOONH4/Pd-C and SnCl2/HCl.
The first reducing agent produced a mixture of products including cyclized compounds with different
oxidation degrees. On the other hand, reduction with SnCl2 in acidic medium and subsequent heating led
to the cyclized hydrochlorides tQNXs as major products. Using the latter experimental conditions,
compounds tQNX1, tQNX2, tQNX3 and tQNX4 were prepared (Figure 5).
tQNX 1
tQNX 2
tQNX 3
tQNX 4
Figure 5.Chemical structures of prepared tQNXs.
N-acylation of tQNX1 (Figure 5) with different acylating agents such as acetyl and propionyl chloride, β-
chloro propanoyl chloride, isobutyl chloroformate, among others, led to derivatives 1-8 (Figure 3) with
very good yields. The general reaction conditions involved the use of the corresponding acyl chloride in a
slight stequiometric excess, using ethyl acetate or acetonitrile as solvents and hexamethylenetetramine or
triethylamine as base. Analogously, the acylation of the derivatives tQNX 2, 3 and 4 led to derivatives 9-
16 (Figure 3) with high yields.
2.2.2 Quinoxalin-2-ones (dQNXs) synthesis (17-25)
The preparation of the quinoxalin-2-ones (dQNXs) derivatives with a variety of substituents at position 3
was carried out according to different synthetic approaches.
Compounds 17, 18, 21 and 22 (Figure 3) were prepared as shown in Scheme 2, by condensation of o-
phenylenediamine with the corresponding α-keto acids. The optimization of this synthetic approach using
MW-assisted methodologies has already been reported by us [31]. Using the previously optimized
conditions, high yields (approx. 90 %) were obtained using equimolar amounts of the reactants and a
minimum amount of solvent (ethanol).

Scheme 2. General synthetic scheme for the preparation dQNXs.
Compounds 23, 24 and 25 (Figure 3) were obtained by MW-assisted esterification of compound 22 with
the corresponding alcohol using methanesulfonic acid supported on alumina as catalyst, also as
previoulsy reported by us [23].
Finally, dQNXs 19 and 20 (Figure 3) were prepared by hydrogen peroxidation of tQNX3 and tQNX4
(Figure 5), respectively.
2.3
Evaluation of the RT inhibitory activity of compounds 1-25
In order to determine if the synthesized quinoxaline compounds 1-25 (Figure 3) acted as RT inhibitors,
we performed an initial screening of their in-vitro RT inhibitory activity at two concentrations: 10 µM and
100 µM. The activity of the expressed wild-type enzyme was optimized for RT activity by measuring
dTTP incorporation on a poly(rA)-oligo(dT) template primer duplex. HIV-1 RT was presented in the
reaction at a final concentration of 50 nM. The commercial NNRTI nevirapine (NVP) was included as a
positive control. The inhibition percentages relative to the wild-type enzymatic activity of the twenty-five
quinoxaline derivatives evaluated were calculated are summarized in Table 5. Most compounds
evaluated showed inhibitory capabilities at the highest concentration tested in this assay (100 µM). Also,
six of the evaluated quinoxalines presenting the higher inhibitory activity values were considered as hit
compounds (1, 3, 8, 12, 15 and 20, Figure 3). Compound 3 (Figure 3) was a particularly interesting
derivative, since it exhibited a potent inhibitory activity at both of the concentrations evaluated, with
values comparable to the commercial compound NVP.
The results presented in Table 4 show that introduction of isobutyl substituents on the quinoxaline amide
nitrogen atom (R4) seems to cause an increase in the RT inhibitory potency (compounds 8, 12 and 15,
Figure 3) regardless of the identity of the susbtituent at position 3, while with other subtituents at this
position (R4), the presence of diverse residues at position 3 were detrimental for the inhibitory activity.
For instance, at 100 µM, the potent inhibitory activity of compound 3 (99% of inhibition) was greatly
diminished by the introduction of a methyl residue at position 3 (compound 11, 23 % of inhibition). The
same behaviour was observed when comparing the inhibition percentages between unsubstituted
compound 5 (32 % of inhibition) with the corresponding values of compounds with alkyl subtituents at
position 3, compounds 10 (8 % inhibition) and 13 (12 % of inhibition). All of the observed results are in
agreement with the theoretical pharmacophore characterization proposed by Wang and also by us (see
2.1).

Table 4. Percentage RT inhibition by compounds 1-25.
% RT Inhibition
% RT Inhibition
% RT Inhibition
Comp. # 100 µM
10 µM
91
Comp. # 100 µM
10 µM
Comp. # 100 µM
10 µM
NVP
98
56
33
99
26
32
12
19
46
9
28
8
15
8
18
19
20
21
22
23
24
25
1
3
7
1
2
3
4
5
6
7
8
10
11
12
13
14
15
16
17
17
73
26
15
23
12
25
12
15
10
20
8
14
23
64
12
7
7
91
37
5
12
24
7
51
20
1
22
10
8
14
15
21
20
20
The four most active compounds (3, 12, 15 and 20, Table 4, Figure 3) were chosen for further analysis.
Assesment of the inhibition of RT activity was repeated using a wider range of concentrations ranging
from 0.01 ꢀM to 200 ꢀM (Table 5, Figure 6). The IC50 was then calculated through concentration-
response curves together with NVP as control. Compound 3 was the most active whereas compounds
12, 15 and 20 were less active. As depicted in Figure 6, the most active compound 3 exhibited an
inhibitory activity comparable to that of the commercial compound, having an IC50 of 0.63 ꢀM (Table 5),
only slighlty higher that the IC50 calculated for NVP (IC50 = 0.13 ꢀM, Table 5). This indicated that
compound 3 has high affinity and inhibitory potency towards RT enzyme and that it could be a
promising therapeutic agent for further development. Conversely, compounds 12, 15 and 20 shared IC50
values in the micromolar range. As evidenced by the confidence intervals observed, the estimation of
IC50 values of these compounds (12, 15 and 20, Table 5) was poor. This was due to their low solubility
which limited the evaluation of the inhibitory activity at high concentrations, thus being unable to reach
higher inhibition percentage values for these compounds. Moreover, IC50 of compound 8 could not be
determined as inhibition percentages greater than 50 % could not reached at maximal solubility.
As can be inferred from Tables 2 and 4, there isn’t a strong correlation between the predicted pIC50
values and the actual inhibitory data. These differences are probably due to the use of heterogeneous
sources of biological data for the construction of the computational model. However, the main purpose
of this work was to build a selection criteria in order to roughly estimate the inhibitory capabilities of the
different compounds and, in this way, as a guide to direct their synthesis. The use of this virtual
screening protocol resulted in the preparation of twenty-five quinoxaline derivatives with RT inhibitory
activity from a library composing more than 45000 molecules, among which, one of them showed high
affinity and inhibitory potency towards this enzyme.

Figure 6. Inhibition of RT activity (IC50) of compound 3 and NVP. IC50 values were calculated according
to the equation for sigmoidal concentration-response using Prism 6.0 (GraphPad Software, San Diego,
USA).
Table 5. IC50 of NVP and compounds 3,12, 15 and 20.
Comp. #
NVP
3
IC50 (µM)a
0,13 (0,09-0,17)
0,63 (0,53-0,76)
64 (38-103)
12
15
67 (54-87)
20
23 (21-25)
*[a] IC50 values were determined by measuring
fluorescene following exposure to different
concentrations of the compounds. IC50 values
were calculated according to the equation for
sigmoidal concentration-response using Prism 6.0
(GraphPad Software, San Diego, USA). Data
represent the mean of two independent
experiments; the 95% confidence interval is given
in parentheses.
It is known that NVP has a low genetic barrier for resistance and that its efficacy is extremely susceptible
to mutations in the non-nucleoside inhibitor binding pocket of RT. Particularly, K103N mutation is among
the most prevalent mutation in RT and it is associated with resistance to this antiretroviral drug. For this
reason, the most potent RT inhibitors in this study, compounds 3, 12, 15 and 20, were tested in HIV-RT
enzyme assays using the K103N mutant. To roughly estimate the inhibitors potency towards the mutant
enzyme, a single concentration of 50 µM was evaluated for these compounds, and their efficacy was
compared against that of the wild-type RT.
Table 6. Percentage K103N RT inhibition by NVP and compounds 3, 12, 15 and 20.
Comp. #
NVP
K103N RT inhibition (%)a
Fold changeb
50 ± 7
47 ± 7
2
2
3

12
15
20
13 ± 1
20 ± 7
5 ± 1
5
3
16
[a] Percentage of K103N inhibition were determined at 50 µM concentration. [b] Fold
change was approximate by comparing the percentage of inhibition of the K103N
mutant at a concentration of inhibitor of 50 ꢀM versus the percentage of inhibition of
the wild-type RT at the same concentration of the inhibitors.
The inhibitory efficacy of the evaluated quinoxaline derivatives shows a 2–16 fold reduction in efficacy
when tested against the K103N mutant (Table 6). The effect of the mutation is more pronounced for
compound 20 where there is a 16-fold reduction in efficacy compared to wild-type RT. The assay shows
that compound 3 is the most potent of the four towards the K103N mutant with an inhibitory behavior
close to that of NVP at the single concentration evaluated. Because of the low inhibitory potency of all of
the compounds evaluated towards the mutant enzyme, IC50 values were not determined.
2.4 Cell toxicity and antiviral activity
The anti-HIV activity and cell toxicity of the synthesized quinoxaline compounds that had showed the
highest in-vitro RT inhibitory capabilities (3, 8, 12 and 20, Figure 3) was finally evaluated in cell cultures.
Thus, anti-HIV activity (EC50), cytoxocity (CC50) and selectivity index (SI) were determined as described
in the experimental section using NVP as reference.
A.
B.
Figure 7. A. % Viral replication and B. Cell viability versus concentration of NVP and compounds 3 and
12.
Table 7. EC50, CC50 and SI of NVP and compounds 3, 8, 12 and 20.
Compd. #
EC50 (nM)a
CC50 (nM)b
SI
NVP
3
6.7 (4.0-11.3)
3.1 (1.5-6,2)
> 10000
96171 (154165-170754)
98576 (25435-382061)
262001 (180020-343981)
116818 (94390-144575)
133520 (72634-194405)
14353
31798
ncc
8
12
20
1576 (931-2667)
> 10000
74
ncc
*[a] EC50 values were determined by measuring the reduction of syncytia formation following exposure to different
concentrations of the compounds in MT2 cells. [b] CC50 values were assessed by Trypan Blue exclusion assays. *[c] not
calculated. CC50, EC50 values were calculated according to the equation for sigmoidal concentration-response using Prism

6.0 (GraphPad Software, San Diego, USA). Data represent the mean of two independent experiments; the 95%
confidence interval is given in parentheses.
To evaluate the effect of the quinoxaline derivatives on viral replication, infected MT2 cells were treated
with the different compounds from 0.001 µM to 10 µM and incubated for 4 days at 37°C. In each case,
the reduction in the production of p24 antigen was correlated with the reduction in the formation of
syncytia. The cell assays indicate a similar inhibitory activity for NVP and compound 3 with an EC50 of
6.7 nM and 3.1 nM, respectively (Figure 7, Table 7). Compound 12 also showed good inhibitory
properties (EC50 of 1576 nM, Table 7) whereas compounds 8 and 20 were not effective as anti-HIV
agents (Table 7).
To confirm that the antiviral activity observed with the evaluated compounds at the cellular level was not
due to the cellular toxicity associated with their use, the cytotoxicity at different concentrations of the
inhibitors was thereof evaluated. In these experiments, uninfected MT2 cells were treated with
compounds from 0.01 µM to 1000 µM and were also incubated at 37 °C for 4 days. Cell viability was
measured for each concentration. All of the evaluated compounds were well tolerated, with CC50 at
approximately 100-200 µM. Both active compounds (3 and 12, Figure 3) showed high selectivity as it is
shown by the SI (Table 7).
As shown in Table 7, compound 3 has higher EC50 and SI than commercial NVP, with an inhibitory
potency in the nanomolar range. Thus, this molecule may be considered a promising lead compound
and further studies should be performed to complete the path from lead to clinical drug candidate.
Compound 12 also shows good anti-HIV properties in infected cells and SI (hit compound). A hit-to-lead
process will be conducted in order to optimize its inhibitory potency.
3.
Conclusion
With the aim to find novel HIV RT inhibitors containing a quinoxaline scaffold, we first carried out the
construction of a virtual chemical library based on previously described pharmacophore features. The
virtual library screening was performed using two complementary computational tools: molecular
docking and 3D-QSAR. Based on these results, twenty-five quinoxaline compounds were selected for
synthesis. They were then prepared and evaluated as inhibitors of the recombinant wild-type RT
enzyme, and the most promising compounds were also evaluated against the K103N RT mutant.
Compound 3 demonstrated potent in-vitro enzymatic inhibitory activity towards HIV wild-type RT, only
slightly weaker than commercial NVP, currently in used in the clinic. When these compounds were
evaluated in HIV infected cells, although the two compounds 3 and 12 showed promising anti-HIV
activity, compound 12 was the less active. Overall, compound 3 had similar anti-HIV and cytotoxicity
profiles than commercial NVP. Taking into account the findings in this study regarding the structural
requirements and biological activity profiles of the tested compounds, further development of 3 (lead
compound) and 12 (hit compound) will likely provide novel and more potent RT inhibitors for HIV
treatment.
4.
Experimental

4.1. General
All reactions were carried out using reagent-grade materials under nitrogen atmosphere. Solvents were
reagent-grade or better. Silica gel column chromatography was performed using silica gel (Kieselgel 60,
230-400 mesh, Merck). Thin layer chromatography (TLC) was performed on silica gel plates (Kieselgel 60
1
F254, Merck) having layer thickness of 0.25 mm. H and 13C NMR spectra were recorded on a Bruker
300 MHz NMR using CDCl3 and DMSO-d6 as solvent, and tetramethylsilane (TMS) as internal standard.
All the chemical shifts values (d) were recorded in parts per million (ppm) and coupling constants (J) in
hertz (Hz). IR spectra were recorded using a Perkin Elmer Spectrum One FT-IR spectrophotometer and
are expressed in cm-1. High resolution mass spectra were obtained on Bruker micrOTOF-Q II
spectrometer. Microwave-assisted reactions were carried out using an Anton Paar Monowave 300 as
microwave reactor, stirring at 500 rpm.
4.2. Procedure for the virtual chemistry library construction and virtual screening protocol
4.2.1 Virtual chemistry library construction
The virtual chemistry library (VCL) was constructed using a script that combines the SMILES strings
corresponding to the structural scaffolds and the selected substituents (Table 1) as building blocks. The
selection of the scaffolds and substituents was based in previously described structural knowledge,
obtained from ligand–protein complexes of related compounds and from the two-dimensional
pharmacophore model developed by Wang [13]. Also, non-favorable substituents, such as bulky groups,
were also included in the library in order to test the accuracy of the virtual screening method. All the
possible combinations of the proposed residues gave rise to a VCL composed by a total of 45804
compounds derived from 1,2,3,4-tetrahydroquinoxalin-2-one (tQXN) and from 1,2-dihydroquinoxalin-2-
one (dQNX) as scaffolds. Three-dimensional models were built from the isomeric SMILES notations of
the assembled VCL compounds and geometry was optimized using the MMFF94 force field [32]
(conjugated gradient, convergence criteria of 1x10-6 or a maximum of 5000 iterations) with OpenBabel
2.3.2 software [33]. Also, non-polar hydrogens were removed from the ligands and Gasteiger charges
were assigned employing OpenBabel 2.3.2 software. These models were further evaluated in the
screening step.
4.2.2 Virtual screening protocol
The VCL was screened applying a previously developed virtual screening protocol for the search of
NNRTI candidates from a compound collection. Briefly, this protocol consists in a combination of
docking and 3D-QSAR methods; the first, as a conformer generator and the latter, for compound activity
prediction. Both methods were developed, calibrated and validated using RT crystallographic data
together with biological activity values extracted from literature.
Initially, docking experiments were used in order to obtain conformers of VCL compounds for further
analysis by 3D-QSAR. The experiments were performed using AutodockVina 1.0 software [34] with the
settings previously described [21-22] using the RT structure that was extracted from PDBID: 2B6A using
USCF Chimera 1.9 software. Only the conformer corresponding to the best scoring docking pose of

those compounds from the VCL that achieved a ∆Gdocking ≤ -8.0 Kcal/mol were selected for further 3D-
QSAR analysis. E3m, E2u and Dv WHIM descriptors [35] were then calculated using PaDEL 2.21
software [36], and pIC50 estimated using the 3D-QSAR model previously developed (equation 1).
4.3. Chemistry
4.3.1 General method for the preparation of N-(2-nitrophenyl)-amino acids (ND1-8)
A. N-(2-nitrophenyl)- aminoacids methyl esters (ND1-4):In a typical reaction, the amino acid methyl ester
(10 mmol), the corresponding halonitrobenzene (10 mmol), diisopropylamine (22 mmol) and graphene
(10 mg) were dissolved and mixed in 10 ml acetonitrile in the provided reaction glass tube equipped with
a screw cap and magnetic agitation. The reaction mixture was irradiated with microwaves (Anton
PaarMonowave 300 reactor) at 100 °C for around 30-4 0 min. On cooling, the reaction mixture was
concentrated, and purified by silica-gel chromatography (hexane/chloroform1:1) to give oily reddish
products.
Methyl [(2-nitrophenyl)amino]acetate (ND1)
Amino acid employed: Glycine methyl ester. Reaction time: 35 min. Yield: 90 % (yellow oil). MS (m/z):
1
210.0, H-RMN (300 MHz) δ (ppm) (CDCl3): 3.83 (s; 3H; OCH3), 4.11 (d; J=4.40; 2H; CH2), 6,73 (t; J=
8.07 Hz; 1H; ArH), 6.73 (d; J=8.10 Hz; 1H; ArH), 7.48 (t; J= 8.10 Hz; 1H; ArH), 7.48 (d; J= 8.80 Hz; 1H;
ArH), 8.41 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 44.7 (CH2), 52.6 (OCH3), 113.6, 116.3,
127.0, 132.8, 136.3, 144.1, 169.8 (C=O).
Methyl-(2S)-2-[(2-nitrophenyl)amino]propanoate (ND2)
Amino acid employed: (S)-Alanine methyl ester. Reaction time: 35 min. Yield: 89 % (yellow oil). MS
1
(m/z): 224.0. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.67 (d; J= 6.70 Hz; 3H; CH3), 3.79 (s; 3H; OCH3),
4.28-4.36 (m; 1H; CH), 6.27 (sa; 1H; NH); 6.70-6.76 (m; 2H; ArH), 7.42-7.48 (m; 1H; ArH), 8.22 (dd; J=
8.80 Hz, J’= 2.00 Hz ; 1H; ArH), 8.32 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 18.5 (CH3),
51.1, 52.5, 113.7, 116.1, 126.9, 132.4, 136.2, 146.8, 173.0 (C=O).
Methyl-(2S)-3-methyl-2-[(2-nitrophenyl)amino]butanoate (ND3)
Amino acid employed: (S)-Valine methyl ester. Reaction time: 38 min. Yield: 91 % (orange oil). MS (m/z)
1
= 252.0. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.08 (d; J= 6.70 Hz; 3H; CH3), 1.13 (d; J= 7.10 Hz; 3H;
CH3), 2.26-2.49 (m; 1H; CH), 3.77 (s; 3H; OCH3), 4.07 (t; J= 8.27 Hz; 1H; CH), 6.72 (m; 2H; ArH); 7.44
(t; J= 7.54Hz; 1H; ArH), 8.21 (d; J= 8.55 Hz; 1H; ArH), 8.40 (d; J= 7.00 Hz; 1H; NH). 13C-RMN (75 MHz)
δ (ppm) (CDCl3): 18.5 (CH3), 19.3 (CH3), 31.4 (CH), 52.3 (OCH3), 61.7 (CH), 113.7, 116.2, 127.1, 136.3,
136.4, 144.6, 172.2 (C=O).
Methyl-(2S)- 4-methyl-2-[(2-nitrophenyl)amino]pentanoate (ND4)
Amino acid employed: (S)-Leucine methyl ester. Reaction time: 40 min. Yield: 90 % (orange oil). MS
(m/z): 266.1. 1H-RMN (300 MHz) δ (ppm) (CDCl3): 0.96 (d; J=5.90 Hz; 3H, CH3), 1.03 (d; J=5.90 Hz; 3H;

CH3), 1.78-1.88 (m; 3H; CHCH2), 3.76 (s; 3H; OCH3), 4.25 (m; 1H; NCH), 6.68-6.78 (m; 2H; ArH), 7.44
(t; J= 7.90 Hz; 1H; ArH), 8.20 (dd; J= 8.42 Hz, J’= 1.35 Hz; 1H; ArH), 8.20 (d; J=8.42 Hz; 1H; NH).13C-
RMN (75 MHz) δ (ppm) (CDCl3): 21.9 (CH3), 22.7 (CH3), 25.0 (CH2), 41.7 (CH), 52.5 (OCH3), 54.5 (CH),
113.6, 116.3, 127.0, 132.7, 136.4, 144.3, 173.2 (C=O).
B. N-(2-nitrophenyl)- amino acids (ND5-8): In a typical reaction, the corresponding amino acid (10
mmol), FNB (10 mmol), and graphene (10 mg) were dissolved and mixed in 20 ml of a 20 % K2CO3
solution in the provided reaction glass tube equipped with a screw cap and magnetic agitation. The
reaction mixture was irradiated with microwaves (Anton Parr Monowave 300 reactor) at 120 °C for
around 15-20 min. After cooling, 30 ml of water was added to the reaction vessel and the aqueous
solution was washed with EtOAc. The aqueous layer was acidified using HCl 5 M and extracted with
EtOAc. The organic phase was dried over Na2SO4 and concentrated to give yellow/orange solids.
Methyl [(2-nitrophenyl)amino]acetate (ND5)
Amino acid employed: Glycine. Reaction time: 16 min. Yield: 90 % (yellow solid). Mp: 192-194°C. MS
(m/z): 196.0; 1H-RMN (300 MHz) δ (ppm) (DMSO-d6): 4.17 (sa; 2H; CH2), 6.73 (t; J= 7.47 Hz; 1H; ArH),
6.91 (d; J= 8.50 Hz; 1H; ArH), 7.54 (t; J= 6.88 Hz; 1H; ArH), 8.09 (d; J= 8.60 Hz; 1H; ArH), 8.37 (sa; 1H;
NH); 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 44.6 (CH2), 115.4, 116.2, 126.6, 131.8. 137.0, 144.9,
171.5 (C=O).
2-[(2-Nitrophenyl)amino]propanoic acid (ND6)
Amino acid employed: (S)-Alanine. Reaction time: 15 min. Yield: 92 % (yellow solid). Mp: 144º C. MS
1
(m/z): 210.0. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.66 (d; J= 7.16 Hz; 3H; CH3), 4.28 (m; 1H; CH),;
6.73 (m; 2H; ArH), 7.45 (t; J= 7.60 Hz; 1H; ArH), 8.19 (d; J=7.90 Hz; 1H; ArH), 8.32 (sa; 1H; NH). 13C-
RMN (75 MHz) δ (ppm) (CDCl3): 18.5 (CH3), 51.2 (CH), 113.8, 116.2, 126.9, 132.4, 136.5, 143.8, 177.5
(C=O).
3-Methyl-2-[(2-nitrophenyl)amino]butanoic acid (ND7)
Amino acid employed: (S)-Valine. Reaction time: 18 min. Yield: 88 % (yellow solid). Mp: 83º C. MS
1
(m/z): 238.0. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.14 (d; J= 7.34 Hz; 3H; CH3), 1.17 (d; J= 6.25 Hz;
3H; CH3), 2.34-2.45 (m, 1H, CH), 4.12 (t; J= 8.27 Hz, 1H, CH), 6.75 (m; 2H; ArH), 7.45 (t; J= 7.70 Hz;
1H; ArH), 8.22 (dd; J= 8.53 Hz, J’= 1.10 Hz; 1H; ArH), 8.41 (d; J=7.54 Hz; 1H; NH), 9.10-10.10 (1H; sa;
OH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 18.2 (CH3), 19.2 (CH3), 31.2 (CH), 61.2 (CH), 113.7, 116.4,
127.1 132.8, 136.4, 144.4, 177.8 (C=O).
4-Methyl-2-[(2-nitrophenyl)amino]pentanoic acid (ND8)
Amino acid employed: (S)-Leucine. Reaction time: 20 min. Yield: 90 % (orange solid). Mp: decomposes.
1
MS (m/z): 252.1. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 0.89 (d; J= 6.56 Hz; 3H; CH3), 0.97 (d; J=
6.56 Hz; 3H, CH3), 1.70-1.82 (m, 3H, CHCH2), 4.40 (m; 1H; CH), 6.76 (m, 1H; ArH), 6.98 (d; J= 8.25 Hz;
1H; ArH), 7.57 (m; 1H; ArH), 8.10 (dd; J= 8.55 Hz, J= 1.56 Hz; 1H; ArH), 8.17 (d; J= 7.70 Hz; 1H;

NH).13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 22.5 (CH3); 23.1 (CH3), 25.1 (CH2), 40.9 (CH), 54.1 (CH),
115.2, 116.6, 126.8, 132.0, 137.3, 144.6, 174.2 (C=O).
4.3.2. General method for the preparation of tQNXs from N-(2-nitrophenyl)-aminoacids (tQNX1-4)
In a typical reaction, the corresponding N-(2-nitrophenyl)-amino acid (10 mmol) was dissolved and
refluxed in ethanol. SnCl2 (36 mmol) in a 5 M solution of HCl (26 ml) was added to the stirred reaction
mixture during 1 h, and the mixture was stirred for an additional 1 h. After cooling, the solvent was
partially evaporated, and the concentrated solution was left at 5º C for a period of 12 h. The quinoxaline
chlorhydrate product precipitated as a red colored solid. The solid product was washed with cold
ethanol, vacuum dried and stored as the chlorhydrate stable salt. For product characterization and
subsequent reactions, the solid was dissolved in 10% K2CO3 solution and the aqueous solution was
extracted with EtOAc. The organic phase was dried over Na2SO4 and evaporated. In this way, the free
quinoxaline bases were obtained.
3,4-Dihydroquinoxalin-2(1H)-one(tQNX1)
Starting material: ND5. Yield: 90%. Mp: 138-140°C. IR (cm -1): 3371, 3183, 1668, 1602, 1504. MS (m/z):
148.2. 1H-RMN (300 MHz) δ (ppm) (DMSO-d6): 2.49 (sa; 1H; NH), 3.75 (sa; 2H; CH2), 6.68-6.71 (m; 1H;
ArH), 6.75-6.81 (m; 3H; ArH), 10.33 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 46.7 (CH2),
115.1, 115.4, 119.9, 123.0, 127.5, 132.6, 166.6 (C=O).
(3S)-3-Methyl-3,4-dihydroquinoxalin-2(1H)-one (tQNX2)
Starting material: ND6. Yield: 90%. Mp: 110° C. [ α]: +8.30 (c 0.6, CHCl3) IR (cm-1): 3370, 3180, 1680,
1
1600, 1500. MS (m/z): 162.1. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.49 (d; J= 6.71 Hz; 3H; CH3); 2.64
(sa; 1H; NH); 4.05 (c; J= 6.80 Hz; 1H; CH); 6.71 (d; J= 7.83 Hz; 1H; ArH), 6.76-6.82 (m; 2H; ArH), 6.86-
6.96 (m; 1H; ArH), 8.89 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 17.9 (CH3), 52.0 (CH), 114.1,
115.5, 119.6, 123.8, 125.5, 133.4, 169.6 (C=O).
(3S)-3-(Propan-2-yl)-3,4-dihydroquinoxalin-2(1H)-one (tQNX3)
Starting material: ND7. Yield: 75%.Mp: 98-99°C. [ α]: +21.0 (c 0.6, CHCl3). IR (cm-1): 3369, 3188, 1688,
1
1600, 1501. MS (m/z): 190.1. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.00 (d; J= 6.67 Hz; 3H; CH3), 1.07
(d; J= 6.90 Hz; 3H; CH3), 2.20-2.37 (m; 1H; CH), 3.80 (d; J= 5.65 Hz; 1H; NCH), 4.09 (sa; 1H; NH), 6.65-
6.80 (m; 3H; ArH), 6.89 (td; J= 7.55 Hz, J’=1.10 Hz; 1H; ArH), 9.52 (sa; 1H; NH).13C-RMN (75 MHz) δ
(ppm) (CDCl3): 17.5 (CH3), 19.0 (CH3), 30.9 (CH), 61.8 (CH), 113.4, 115.5, 118.8, 123.9, 124.9, 133.3,
168.7 (C=O).
3-(2-Methylpropyl)-3,4-dihydroquinoxalin-2(1H)-one (tQNX4)
Starting material: ND8. Yield: 80 %.Mp: decomposes. [α]: +32.0 (c 0.3, CHCl3). IR (cm-1): 3300, 3100,
1670, 1601, 1501. MS (m/z): 204.4. 1H-RMN (300 MHz) δ (ppm) (CDCl3): 0.97 (d; J= 6.67 Hz; 3H; CH3),
1.00 (d; J= 6.67 Hz; 3H; CH3), 1.60-1.73 (m; 2H; CH2), 1.75-1.87 (m; 1H; CH), 3.96-4.03 (dd; J=8.70 Hz,
J’=5.07; 1H; NCH), 4.10 (sa; 1H; NH), 6.66-6.80 (m; 2H; ArH), 6.82-6.94 (m; 2H; ArH), 9.89 (sa; 1H;

NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 21.5 (CH3), 23.2 (CH3), 24.2 (CH), 40.3 (CH2), 54.6 (CH),
114.3, 115.7, 119.4, 123.8, 125.6, 132.9, 170.1 (C=O).
4.3.3. General method for the preparation of N-acylated tQNXs (1-16)
The tQNX1-4 stored as chlorhydrate salts were converted to their free bases as described above. In a
typical acylation reaction, the corresponding acylating agent (1.2 mmol) was added to a solution of tQNX
(1mmol) in EtAcO (30 ml) and triethylamine (1.2 mmol) and heated to reflux for 1 h. After cooling, 50 ml
of a 10 % solution of K2CO3 was added and the mixture was stirred for additional 15 min. The solution
was left still, and the organic phase was separated. The organic phase was then sequentially washed
with HCl 0.5 M, 10 % K2CO3 and water, dried with Na2SO4 and concentrated. The product was
characterized without further purification.
4-Acetyl-3,4-dihydroquinoxalin-2(1H)-one (1)
Starting material: tQNX1. Acylating agent: acetyl chloride. Yield: 90%. Mp: 162-163°C. IR (cm -1): 3190,
1702, 1666, 1503. MS (m/z): 190.0. 1H-RMN (300 MHz) δ (ppm) (CDCl3): 2.25 (sa; 4H; CH3, NH), 4.52 (s;
2H; CH2), 7.04-7.05 (d; J= 7.80 Hz; 1H; ArH), 7.12 (td; J= 7.80 Hz, J’= 1.20 Hz; 1H; ArH), 7.18-7.28 (m;
2H; ArH), 9.74 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 22.2 (CH3), 45.5 (CH2), 116.9, 123.3,
124.0, 127.0, 127.3, 131.2, 167.7 (C=O), 169.8 (C=O).
4-Benzoyl-3,4-dihydroquinoxalin-2(1H)-one(2)
Starting material: tQNX1. Acylating agent: benzoyl chloride. Yield: 90%. Mp: 207° C. IR (cm -1): 3266,
1708, 1666, 1500. MS (m/z): 252.0. 1H-RMN (300 MHz) δ (ppm) (CDCl3): 4.63 (s; 2H; CH2); 6.70 (sa, 1H,
ArH); 6.80 (t; J= 7.80 Hz ; 1H; ArH); 7.00 (dd; J= 7.80 Hz, J’= 1.40 Hz; 1H; ArH); 7.11 (td; J= 8.40 Hz;
J’=1.50 Hz ; 1H; ArH); 7.30-7.40 (m; 2H; ArH); 7.40-7.50 (m; 3H; ArH); 9.34 (sa; 1H; NH). 13C-RMN (75
MHz) δ (ppm) (CDCl3): 47.7 (CH2), 116.5; 123.0; 124.7; 126.2; 128.4; 128.9; 131.2; 134.1; 141.1; 141.4;
168.6 (C=O); 169.3 (C=O).
4-Propanoyl-3,4-dihydroquinoxalin-2(1H)-one(3)
Starting material: tQNX1. Acylating agent: propanoyl chloride. Yield: 90%. Mp: 162°C. IR (cm -1): 3084,
1
1698, 1650, 1497. MS (m/z): 204.2. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.19 (t; J=7.32 Hz; 3H; CH3),
2.57 (c J=7.32 Hz; 2H; CH2), 4.53 (s; 2H; NCH2), 7.02 (d; J= 7.80 Hz; 1H; ArH), 7.11 (m; 1H; ArH), 7.20-
7.30 (m; 2H; ArH), 9.46 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 9.70 (CH3), 27.2 (CH2), 46.2
(NCH2); 116.8 (para 2 C); 123.3; 124.2; 126.8; 127.1, 169.4 (C=O), 173.3 (C=O).
4-(3-Chloropropanoyl)-3,4-dihydroquinoxalin-2(1H)-one(4)
Starting material: tQNX1. Acylating agent: 3-chloropropanoyl chloride. Yield: 95%. Mp: 143-144° C. IR
1
(cm-1): 3084, 1698, 1651, 1496, 747. MS (m/z): 238.7. H-RMN (300 MHz) δ (ppm) (CDCl3): 3.04 (t; J=
6.60 Hz; 2H, CH2), 3.87 (t J=6,60 Hz; 2H; ClCH2), 4.57 (s; 2H; NCH2), 7.05 (d; J= 7.60 Hz; 1H; ArH),
7.15 (t; J=7.80; 1H; ArH), 7.20-7.40 (m; 2H; ArH), 9.59 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm)

(CDCl3): 36.6 (CH2); 39.8 (ClCH2); 45.9 (NCH2); 116.9; 123.3; 124.0; 127.0; 127.3; 131.2; 169.3 (C=O);
169.5 (C=O).
4-(Chloroacetyl)-3,4-dihydroquinoxalin-2(1H)-one (5)
Starting material: tQNX1. Acylating agent: chloroacetyl chloride. Yield: 95%. Mp :168-170°C. IR (cm -1):
1
3140, 1702, 1656, 1497, 759. MS (m/z): 224.6. H-RMN (300 MHz) δ (ppm) (CDCl3): 4.27 (s; 2H; CH2),
4.56 (sa; 2H, CH2); 7.02 (d; J= 7.80 Hz; 1H; ArH); 7.15 (t; J= 7.80 Hz; 1H; ArH); 7.25-7.48 (m; 2H; ArH);
8,90 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 40.6 (ClCH2); 46.6 (CH2); 116,9; 117.0; 123.8;
126.1; 127.8; 131.2; 165.9 (C=O); 166.6(C=O).
4-(Phenylsulfonyl)-3,4-dihydroquinoxalin-2(1H)-one (6)
Starting material: tQNX1. Acylating agent: benzensulfonyl chloride. Yield: 70%. Mp: 183-185°C. IR (cm -
1
1): 3218, 1702, 1693, 1604, 1492. MS (m/z): 288.1. H-RMN (300 MHz) δ (ppm) (CDCl3): 4.35 (sa; 2H;
CH2); 6.71 (dd; J= 7,80 Hz, J’=1,10 Hz; 1H; ArH); 7.18 (td; J= 1.30, J’= 7.80; 1H; ArH); 7.22-7.37 (m; 3H;
ArH); 7.42-7.55 (m; 3H; ArH); 7.75 (d; J= 7.80 Hz ; 1H; ArH); 8.56 (sa; 1H; NH). 13C-RMN (75 MHz) δ
(ppm) (CDCl3): 49.2 (CH2); 116.2; 124.0; 124.4; 127.0; 127.7; 128.4; 129.0; 132.1; 133.5; 136.8; 166.3
(C=O).
Propan-2-yl 3-oxo-3,4-dihydroquinoxaline-1(2H)-carboxylate (7)
Starting material: tQNX1. Acylating agent: isopropyl chloroformate. Yield: 80%. Mp: 160-161°C. IR (cm -
1
1): 3200, 1712, 1682, 1504. MS (m/z): 234.3. H-RMN (300 MHz) δ (ppm) (CDCl3): 1,34 (d; J= 6.03Hz,
3H, CH3); 1.36 (d; J=6.03Hz; 3H; CH3); 4.46 (sa; 2H; NCH2); 5.09 (m; 1H; CH); 6.93 (m; 1H; ArH); 7.11
(m; 2H; ArH); 7.69 (sa; 1H; ArH); 9,05 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 22.0 (2 x CH3);
47.4 (CH2); 70.8 (CH); 116.2; 123.4; 123.9; 125.4; 126.6; 129.8; 153.1 (NC(=O)O); 168.2 (C=O).
2-Methylpropyl 3-oxo-3,4-dihydroquinoxaline-1(2H)-carboxylate (8)
Starting material: tQNX1. Acylating agent: isobutyl chloroformate. Yield: 95 %. Mp: 142-144°C. IR (cm -
1): 3199, 1720, 1692, 1600, 1503. MS (m/z): 248.3 1H-RMN (300 MHz) δ (ppm) (CDCl3): 0.99 (d; J=6,90
Hz; 6H; CH3); 2,03 (m; 1H; CH); 4,03 (d; J=6,60 Hz; 2H; CH2); 4.48 (sa; 2H; NCH2); 6,94 (d; J=7.50 Hz;
1H; ArH); 7.12 (m; 2H; ArH); 7,67 (sa; 1H; ArH); 9,19 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3):
19.1 (2 x CH3); 27.9 (CH); 47.5 (N-CH2); 72.9 (O-CH2); 116.3; 123.4; 124.0; 125.6; 126.5; 129.8; 153.7
(N-C(O)-O); 168.2 (C=O).
(3S)-4-(3-Chloropropanoyl)-3-methyl-3,4-dihydroquinoxalin-2(1H)-one (9)
Starting material: tQNX2. Acylating agent: 3-chloropropanoyl chloride. Yield: 95 %. Mp:155-156° C. IR
1
(cm-1): 3200, 1681, 1657, 1503. MS (m/z): 252.7 H-RMN (300 MHz) δ (ppm) (CDCl3): 1.27 (d J=6.57;
3H; CH3); 2.77-2.97 (m, 1H, ClCH2CHH); 3.09-3.26 (m; 1H; ClCH2CHH); 3.73-3.96 (m; 2H; ClCH2); 5.60
(sa; 1H; NCH); 7.04 (d; J=7.42; 1H; ArH); 7.14 (td; J= 7.50 Hz, J’=1.10 Hz; 1H; ArH); 7.20-7.35 (m; 2H;
ArH); 9.51 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 15.3 (CH3); 37.0 (CH2); 39.8 (ClCH2); 51.1

(NCH); 116.7; 123.5; 124.1; 125.1; 127.2; 130.9; 169.0 (C=O); 171.9 (C=O). Anal. calc. for
C12H13ClN2O2: C, 57.04; H, 5.19; N, 11.09. Found: C, 57.00; H, 5.25; N, 11.00.
(S)-2-Chloro-1-(2-methyl-3,4-dihydroquinoxalin-1(2H)-yl)ethanone(10)
Starting material: tQNX2. Acylating agent: chloroacetyl chloride. Yield: 80 %. Mp: 198-200°C. IR (cm -1):
1
3183, 1692, 1650, 1502, 757. MS (m/z): 238.7. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.31 (d; J= 7.32
Hz; 3H; CH3); 4.19 (d J= 14.30 Hz; 1H; ClCHH); 4.33 (d; J= 14.30 Hz; 1H; ClCHH); 5.52 (sa; 1H; NCH);
7.05 (d; J=7.14; 1H; ArH); 7.17 (t; J= 7.40 Hz; 1H; ArH); 7.22-7.35 (m; 1H; ArH); 7.35-7.47 (m; 1H; ArH);
9.41 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 15.2 (CH3); 41.4 (ClCH2); 52.0 (NCHCO); 116,8;
123.8; 123.9; 127.7; 130.7; 130.9; 165.8 (C=O); 171.3 (C=O). Anal. calc. for C11H11ClN2O2: C, 55.36; H,
4.65; N, 11.74. Found: C, 55.30; H, 4.68; N, 11.70.
(3S)-3-Methyl-4-propanoyl-3,4-dihydroquinoxalin-2(1H)-one (11)
Starting material: tQNX2. Acylating agent: propanoyl chloride. Yield: 90 %. Mp: 176-177° C. IR (cm -1):
1
3183, 1702, 1650, 1500. MS (m/z): 218.3. H-RMN (300 MHz) δ (ppm) (CDCl3): 1.20 (t; J= 7.13 Hz; 3H;
CH3); 1.26 (d J= 7.22 Hz; 3H; CH3); 2.38-2.56 (m; 1H, ;CHH); 2.57-2.75 (m; 1H; CHH); 5.59 (sa; 1H;
NCHCO); 6.98 (d; J=7.23; 1H; ArH); 7.12 (t; J= 7.61 Hz; 1H; ArH); 7.23 (t J=7.61; 1H; ArH); 7.27-7.34
(m; 1H; ArH); 8.98 (sa; 1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 15.2 (CH3); 27.5 (CH2); 52.0
(NCH); 116,3; 123.3; 124.8; 125.2; 126.6; 130.5; 168.0 (C=O); 173.0 (C=O). Anal. calc. for C12H14N2O2:
C, 66.04; H, 6.47; N, 12.84. Found: C, 66.00; H, 6.49; N, 12.70.
2-Methylpropyl (2S)-2-methyl-3-oxo-3,4-dihydroquinoxaline-1(2H)-carboxylate(12)
Starting material: tQNX2. Acylating agent: isobutyl chloroformate. Yield: 90 %. Mp: 165-167°C. IR (cm -
1
1): 3193, 1720, 1681, 1600, 1503. MS (m/z): 262.2. H-RMN (300 MHz) δ (ppm) (CDCl3): 0.99 (d;
J=6.25Hz; 6H; 2xCH3); 1.31 (d; J= 7.22 Hz, 3H, CH3); 1.92-2.14 (m; 1H; CH); 4,05 (ac; 2H; CH2); 5.23
(ac; J= 6.27 Hz; 1H; NCH); 6,89-6.99 (m; 1H; ArH); 7.03-7.20 (m; 2H; ArH); 7.69 (sa; 1H; ArH); 9.12 (sa;
1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 16.0 (CH3), 19.1 (2 x CH3); 27.9 (CH); 53.2 (NCH); 72.7
(OCH2); 115.9; 123.5; 124.3; 124.8; 125.4; 129.1; 153.5 (NC(=O)O); 170.7 (C=O). Anal. calc. for
C14H18N2O3: C, 64.10; H, 6.92; N, 10.68. Found: C, 64.00; H, 6.98; N, 10.62.
(3S)-4-(Chloroacetyl)-3-(propan-2-yl)-3,4-dihydroquinoxalin-2(1H)-one(13)
Starting material: tQNX3. Acylating agent: Chloroacetyl chloride. Yield: 75 %. Mp: 215-216°C. IR (cm -1):
1
3190, 1674, 1603, 1503. MS (m/z): 266.7. H-RMN (300 MHz) δ (ppm) (CDCl3): 0.91 (sa; 3H; CH3);
1.09 (d; J= 6.81 Hz; 3H; CH3); 1.65-1.75 (m; 1H; CH); 4.16 (d; J= 12.32 Hz; 1H; ClCHH); 4.34 (d; J=
12.32 Hz; 1H; ClCHH); 5.09 (m; 1H; NCH); 7.00 (d; J=7.42; 1H; ArH); 6.89 (td; J= 7.80 Hz, J= 1.35 Hz;
1H; ArH); 7.22-7.32 (m; 1H; ArH); 7.35-7.45 (m; 1H; ArH); 8.81 (sa; 1H; NH). 13C-RMN (75 MHz) δ
(ppm) (CDCl3): 19.1 (2 x CH3); 28.5 (CH); 40.4 (ClCH2); 61.1 (NCH); 116,7; 123.6; 123.8; 127.7; 131.0;
131.1; 166.3 (C=O); 169.3 (C=O). Anal. calc. for C13H15ClN2O2: C, 58.54; H, 5.67; N, 10.50. Found: C,
58.50; H, 5.70; N, 10.48.

(3S)-4-(3-Chloropropanoyl)-3-(propan-2-yl)-3,4-dihydroquinoxalin-2(1H)-one(14)
Starting material: tQNX3. Acylating agent: 3-Chloropropionyl chloride. Yield: 80 %. Mp: 179-180°C. IR
1
(cm-1): 3200, 1687, 1657, 1600, 1503, 748. MS (m/z): 280.8. H-RMN (300 MHz) δ (ppm) (CDCl3): 0.90
(d; J=6.51; 3H; CH3); 1.08 (d; J= 6.51 Hz; 3H; CH3); 1.69 (m; 1H; CH); 2.77-2.86 (m; 1H; ClCH2CHH);
3.19-3.28 (m; 1H; ClCH2CHH); 3.76-3.84 (m; 1H; ClCHH); 3.88-3.97 (m; 1H; ClCHH); 5.14 (d; J=8.05;
1H; NCH); 7.00 (d; J=7.42; 1H; ArH); 6.89 (td J=1.35 Hz, J’= 7.80 Hz; 1H; ArH); 7.22-7.32 (m; 1H; ArH);
7.35-7.45 (m; 1H; ArH); 8.81 (sa; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 18.9 (2 x CH3); 28.3
(CH); 36.7 (CH2); 40.1 (ClCH2); 60.7 (NCH); 116,5; 123.4; 124.9; 125.2; 127.2; 131.2; 169.5 (C=O);
171.7 (C=O). Anal. calc. for C14H17ClN2O2: C, 59.89; H, 6.10; N, 9.98; Found: C, 59.80; H, 6.15; N, 9.95.
2-Methylpropyl (2S)-2-(2-methylpropyl)-3-oxo-3,4-dihydroquinoxaline-1(2H)-carboxylate(15)
Starting material: tQNX4. Acylating agent: isobutyl chloroformate. Yield: 85 %. Mp: 149-151°C. IR (cm -
1
1): 3185, 1704, 1685, 1603, 1503. MS (m/z): 304.4. H-RMN (300 MHz) δ (ppm) (CDCl3): 0.92 (d; J=
6.73 Hz; 3H; CH3); 0.94-1.01 (sa; 6H; 2xCH3); 1.02(d; J= 6.73 Hz; 3H; CH3); 1.36-1.46 (m; 2H; CH2);
1.65-1.77 (m; 1H; CH); 2.02 (sa; 1H; CH); 3.90-4.20 (sa; 2H; OCH2); 5.19 (ac;1H; NCH); 6.92 (d; J=7.41
Hz; 1H; ArH); 7.11 (t; J=7.16 Hz; 1H; ArH); 7.16 (t; J=7.41 Hz; 1H; ArH); 7.60 (sa; 1H; ArH); 8.80 (sa;
1H; NH).13C-RMN (75 MHz) δ (ppm) (CDCl3): 19.1 (CH3); 19.2 (CH3); 21.9 (CH3); 22.9 (CH3); 24.4 (CH);
27.9 (CH); 38.5 (CH2); 55.7 (NCH); 72.9 (OCH2); 115.8; 123.8, 125.7; 126.1; 129.8; 154.1 (C=O), 171.5
(C=O). Anal. calc. for C17H24N2O3: C, 67.08; H, 7.95; N, 9.20. Found: C, 67.00; H, 7.99; N, 9.18.
(3S)-4-(3-Chloropropanoyl)-3-(2-methylpropyl)-3,4-dihydroquinoxalin-2(1H)-one(16)
Starting material: tQNX4. Acylating agent: 3-Chloropropionyl chloride. Yield: 85 %. Mp: 150-151°C. IR
1
(cm-1): 3203, 1681, 1651, 1502, 746. MS (m/z): 294.7. H-RMN (300 MHz) δ (ppm) (DCCl3): 0.89 (d; J=
6.19 Hz; 3H; CH3); 1.03 (d; J= 6.19 Hz; 3H; CH3); 1.29-1.37 (m; 1H; CHH); 1.38-1.46 (m; 1H; CHH); 1.57
(m; 1H; CH); 2.81 (dt; J=16.1 Hz, J’=6.59 Hz; 1H; CHH); 3.19 (dt; J=15.9 Hz, J’=6.60 Hz; 1H; CHH);
3.77-3.85 (m; 1H; ClCHH); 3.87-3.94 (m; 1H; ClCHH); 5.57 (dd; J=11.3 Hz, J’=4.23 Hz; 1H; NCH); 7.01
(d; J=7.29 Hz; 1H; ArH); 7.15 (td; J=7.60 Hz, J’=1.02 Hz; 1H; ArH); 7.25-7.30 (m; 2H; ArH); 8.99 (sa; 1H;
NH). 13C-RMN (75 MHz) δ (ppm) (CDCl3): 21.7 (CH3); 23.0 (CH3); 24.6 (CH); 36.9 (CH2); 37.6 (CH2);
39.9 (CH2); 53.9 (NCH); 116.6; 123.5; 124.4; 125.4; 127.5; 131.3; 169.5 (C=O), 171.5 (C=O). Anal. calc.
For C15H19ClN2O2: C, 61.12; H, 6.50; N, 9.50;. Found: C, 61.10; H, 6.52; N, 9.48.
4.3.4. General method for the preparation of dQNXs17-18-21-22
In a typical reaction, o-phenyldiamine (10 mmol) and the corresponding dicarbonylic compound (10
mmol) were dissolved in ethanol (5 ml) in a reaction glass tube equipped with a screw cap and magnetic
agitation. The reaction mixture was irradiated with microwaves (Anton Parr Monowave 300 reactor) at 30
W for around 5-13 min. After cooling, 10 ml of ethanol was added to the reaction vessel. The product
was recrystallized from ethanol, precipitating as a white solid.
3-Methylquinoxalin-2(1H)-one(17)

Dicarbonyl compound: pyruvic acid. Reaction time: 8.5 min. Yield: 98 %. Mp: 243-245°. IR (cm -1): 3045,
1
1670, 1640. MS (m/z): 160.2. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 2,39 (s; 3H; CH3); 7.22-7.35 (sa;
2H; ArH); 7.41-7.54 (sa; 1H; ArH); 7.64-7.74 (sa; 1H; ArH); 12,33 (sa; 1H; NH). 13C-RMN (75 MHz) δ
(ppm) (CDCl3): 21.0 (CH3); 115.7; 123.5; 128.3; 129.8; 132.1; 132,3; 155.4 (C=N); 159.9 (C=N).
1,4-Dihydroquinoxaline-2,3-dione (18)
Dicarbonyl compound: oxalic acid. Reaction time: 6 min. Yield: 90 %. Mp >320°C. IR (cm -1): 3043, 1681,
1
1613, 1500. MS (m/z): 162.1. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 7.02-7.20 (m; 4H; ArH); 11.93
(s.a.; 2H; NH). 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 115.6; 123.5; 126.0; 155.7 (C=N).
3-Benzylquinoxalin-2(1H)-one(21)
Dicarbonyl compound: Phenylpyruvic acid. Reaction time: 13 min. Yield: 95 %. Mp: 198 - 200°C. IR (cm -
1): 3061, 1655, 1597, 1500. MS (m/z): 236.2. 1H-RMN (300 MHz) δ (ppm) (DMSO-d6): 4.13 (s; 2H; CH2);
7.16-7.38 (m; 7H; ArH); 7.49 (t; J=7.24 Hz; 1H; ArH); 7.73 (d; J= 8.11 Hz; 1H; ArH); 12,39 (s.a.; 1H; NH).
13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 39.9 (CH2); 115.7; 123.6; 126.7; 128.7; 128.8; 129.6; 130.2;
132.1; 132.4; 137.9; 155.5 (C=N); 160.8 (C=N).
3-(3-Oxo-3,4-dihydroquinoxalin-2-yl)propanoicacid (22)
Dicarbonyl compound: alpha-ketoglutaric acid. Reaction time: 5 min. Yield: 98 %. Mp: 256 - 258°C. IR
(cm-1): 3435, 3102, 1730, 1666, 1608, 1502. MS (m/z): 218.11H-RMN (300 MHz) δ (ppm) (DMSO-d6):
2.72 (t; J=6.30 Hz; 2H; CH2); 3.02 (t; J=6.30 Hz; 2H; CH2); 7.21-7.35 (m; 2H; ArH); 7.46 (t; J= 7.65 Hz;
1H; ArH), 7.69 (d; J= 8.06, 1H, ArH), 12.10 (s.a.; 1H; OH); 12.30 (s.a.; 1H; NH). 13C-RMN (75 MHz) δ
(ppm) (DMSO-d6): 28.1; 30.4; 115.7; 123.5; 128.5; 129.9; 131.9, 132.1; 155.0 (C=N); 160.7 (C=O);
174.4 (COOH).
4.3.5. General method for the preparation of dQNXs 19-20
Typically, 0.5 ml of H2O2 (100 vol) were added to a 10% K2CO3 solution (50 ml) of the corresponding
tetrahydroquinixalinic derivatives (10 mmol). The reaction mixture was heated to reflux for 1 h. After
cooling, the reaction mixture was extracted with EtAcO. The organic phase was dried over Na2SO4 and
concentrated. The residue was recrystallized from ethanol precipitating as a white solid.
3-(Propan-2-yl)quinoxalin-2(1H)-one(19)
1
Yield: 70%. Mp: 190° C.IR (cm -1): 3120, 1660, 1490. MS (m/z): 188.1. H-RMN (300 MHz) δ (ppm)
(DMSO-d6): 1.39 (d; J= 6.83 Hz; 6H, CH3); 3.60-3.75 (m, 1H, CH); 7.30-7.40 (m; 2H; ArH); 7.50 (t; J=
7.94 Hz; 1H; ArH); 7.87 (d; J= 7.94 Hz; 1H; ArH); 11.78 (s.a.; 1H; NH).13C-RMN (75 MHz) δ (ppm)
(DMSO-d6): 23.2 (2 x CH3); 30.7 (CH); 115.3; 124.0; 128.9; 129.5; 130.8; 132.9; 156.0 (C=N); 165.6
(C=O).
3-(2-Methylpropyl)quinoxalin-2(1H)-one(20)

1
Yield: 70%. Mp: 184-185°C. IR (cm -1): 3308, 1661, 1600, 1556. MS (m/z): 202.3. H-RMN (300 MHz) δ
(ppm) (DMSO-d6): 0.94 (d; J= 6.79 Hz; 6H; CH3); 2.19-2.31 (m; 1H; CH); 2.67 (d; J= 6.40 Hz; 2H; CH2);
7.23-7.33 (m; 2H; ArH); 7.48 (t; J= 7.56 Hz; 1H; ArH); 7.73 (d; J= 8.31 Hz; 1H; ArH); 12,32 (s.a.; 1H;
NH). 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 23.0 (2 x CH3); 26.6 (CH); 42.0 (CH2); 115.7; 123.5;
128.6; 129.9; 132.1; 132.2; 155.3 (C=N); 161.7 (C=O).
4.3.6. General method for the preparation of dQNXs (23-25) from compound 22.
Methanesulfonic acid supported on Al2O3 (AMA 2:3) was prepared mixing Al2O3 (3 mol), previously
activated in an oven at 150 °C for 72 h, with CH 3SO3H (2 mol) in a mortar until homogeneity.
In a typical reaction, AMA 2:3 (10 mmol), the corresponding carboxylic acid (1 mmol) and the alcohol (2
ml) were mixed in the provided reaction glass tube equipped with a screw cap and magnetic agitation
until a wet mixture was achieved. The reaction mixture was irradiated with microwaves (Anton Parr
Monowave 300 reactor) at 120 °C for 10-25 min. On c ooling, the mixture was diluted with DCM (41 mL),
and filtered over celite. Then the filtrate was washed with Na2CO3 (ss) and water. The organic layer was
dried over Na2SO4, filtered, and concentrated under reduced pressure to give the ester.
Methyl 3-(3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (23)
Alcohol: Methanol. Reaction time: 10 min. Yield: 95%. Mp: 208-209° C. IR (cm -1): 1730, 1655, 1615,
1
1510, 1490, 1565. MS (m/z): 232.3. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 2.79 (t; J=7.01 Hz; 2H;
CH2); 3.06 (t; J=6.83 Hz; 2H; CH2); 3.60 (s; 3H; OCH3); 7.23-7.33 (m; 2H; ArH); 7.48 (t; J= 7.99 Hz; 1H;
ArH), 7.68 (d; J= 8.40; 1H; ArH), 12.36 (s.a.; 1H; NH).13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 28.0
(CH2); 29.8 (CH2); 51.8 (OCH3); 115.7; 123.6; 128.6; 130.0; 131.9; 132.2; 155.0 (C=N); 160.4 (C=O);
173.4 (CO).
Ethyl 3-(3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (24)
Alcohol: Ethanol. Reaction time: 20 min.Yield: 95%. Mp: 186-188°C. IR (cm -1): 1727, 1677, 1668, 1617,
1
1510, 1490, 1572. MS (m/z): 246.3. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 1.17 (t; J=7.01 Hz; 2H;
CH3); 2.77 (t; J=6.95 Hz; 2H; CH2); 3.05 (t; J=6.95 Hz; 2H; CH2); 4.06 (c; J= 7.25 Hz; 2H, OCH2); 7.23-
7.33 (m; 2H; ArH); 7.43-7.51 (t; J= 7.74 Hz; 1H; ArH), 7.67 (d; J= 7.55 Hz; 1H; ArH), 12.36 (s.a.; 1H;
NH). 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 14.6 (CH3); 28.1 (CH2); 30.1 (CH2); 60.2 (OCH2); 115.7;
123.6; 128.5; 130.0; 131.9; 132.2; 155.0 (C=N); 160.4 (C=O); 172.8 (CO).
Propan-2-yl 3-(3-oxo-3,4-dihydroquinoxalin-2-yl)propanoate (25)
Alcohol: Isopropyl alcohol. Reaction time: 25 min.Yield: 90%. Mp: 178-179° C. IR (cm -1): 1719, 1666,
1
1651, 1562, 1427, 1186. MS (m/z): 260.2. H-RMN (300 MHz) δ (ppm) (DMSO-d6): 1.17 (d; J=7.01 Hz;
3H; CH3); 1.18 (d; J=7.01 Hz; 3H; CH3); 2.73 (t; J=6.80 Hz; 2H; CH2); 3.04 (t; J=6.80 Hz; 2H; CH2); 4.88
(m; 1H; CH); 7.23-7.33 (m; 2H; ArH); 7.48 (t; J= 7.74 Hz; 1H; ArH), 7.65 (d; J= 7.14 Hz; 1H, ArH), 12.35
(s.a.; 1H; NH). 13C-RMN (75 MHz) δ (ppm) (DMSO-d6): 22.1 (2 x CH3); 28.2 (CH2); 30.4 (CH2); 67.4
(OCH); 115.7; 123.6; 128.4; 129.9; 131.9, 132.1; 155.0 (C=N); 160.5 (C=O); 172.3 (CO).

4.4. Biology
4.4.1. HIV-1 RT inhibition enzyme assay
The p66/p51 HIV-1 wild-type and K103N reverse transcriptase heterodimer overexpression and
purification were carried out by means of the plasmids gently donated by Dr. Stephen Hughes according
to the procedures previously described [37]. The RT recombinant enzyme is from subtype B HIV, strain
BH10 (Genebank HIVBH102). RT activity was measured using the commercial kit EnzChek® Reverse
Transcriptase Assay Kit (Invitrogen Co., USA) according to the manufacturer's instructions, with minor
modifications. Briefly, 2 µl of a solution of the different inhibitors were incubated with 10 µl of the
recombinant RT enzyme (approximately 50 nM) in reaction buffer in a 96-well solid black plate for
fluorescence reading. After 2 min, the previously incubated templates poly (A) and oligo-dT were added.
The reaction mixture was then incubated for 2 h at room temperature. The resulting DNA heteroduplexes
generated in each well were then detected and quantitatively measured using the PicoGreen reagent
(173 ꢀl) provided in the kit using a FlexStation 3 microplate reader (Molecular Devices Inc., San Jose,
CA, United States).at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.
Nevirapine was used as positive control. All experiments were performed in triplicate wells for each
condition and repeated at least twice. The results are expressed as percentage (%) of inhibition,
comparing the activity of the enzyme incubated with the inhibitor with the activity without any additives.
The half maximal inhibitory concentration (IC50) was determined by concentration-responses curves
varying the amount of inhibitor in each well. The values were calculated according to the equation for
sigmoidal concentration-response using Prism 6.0 (GraphPad Software, San Diego, USA).
4.4.2. Cells based assays
Cells: MT2 cell line was cultured in RPMI-1640 medium (HyClone USA) supplemented with 10% fetal
bovine serum (Sigma-Aldrich USA), 2 mML-glutamine (Sigma-Aldrich), 100 U/ml penicillin (Sigma-
Aldrich), 100 mg/ml streptomycin (Sigma-Aldrich), and 10 mM HEPES (Sigma-Aldrich).
Virus: Viral stock HTLV-IIIB (laboratory strain of the HIV-1 virus provided by the McGill AIDS Center,
Montreal, Canada.) Produced from chronically infected H9 cells and propagated in MT2 cells. Title of
viral stock used: 1.58 x 106 TCID50/ml.
4.4.2.1 Cytotoxicity tests: The toxicity of the compounds was tested by performing 10-fold serial dilutions
of both compounds, from 0.01 µM to 1000 µM, and subsequently incubated with the MT2 cells without
infecting. The toxic effect was evaluated at 4 days by Trypan Blue dye and subsequent counting of
viable cells in Neubauer chamber. All experiments were performed in duplicate for each condition and
repeated at least twice.
4.4.2.2. Inhibition assays of viral activity: First, MT2 cells were incubated with a 1/5 dilution of de viral
stock in complete culture medium (at a concentration of 1 M cell / ml virus dilution) for 2 hours at 37 °C.
The cells were washed and resuspended in fresh medium. Infected cells were incubated with the 10-fold
serial dilutions of the compounds, from 0.001 µM to 10 µM. The supernatants were collected after 4

days. The production of p24 antigen was evaluated by Elisa (Fujirebio Japan). All experiments were
performed in duplicate for each condition and repeated at least twice.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
This work was financially supported by AgenciaNacional de Promoción Científica y Tecnológica,
Universidad de Buenos Aires and Consejo Nacional de Investigaciones Científicas y Tecnológicas
(Argentina) grants. Special thanks to Emiliano Barrionuevo and Gabriel Jasinsky for their assistance on
this publication.
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