Synthesis and biological evaluation of novel 2,3-disubstituted quinoxaline derivatives as antileishmanial and antitrypanosomal agents

Article abstract of DOI:10.1016/j.ejmech.2014.11.018

Quinoxalines belong to the N-containing heterocyclic compounds that stand out as having promising biological activity due to their privileged scaffold. In this work, we report the synthesis, antileishmanial, and antitrypanosomal properties of 46 new 2,3-disubstituted quinoxaline and 40 previously reported derivatives. Among all of the compounds screened for in vitro activity against epimastigotes and trypomastigotes of Trypanosoma cruzi and promastigotes of Leishmania amazonensis as well as mammalian toxicity on LLCMK₂ cells and J774 macrophages, analogues from series 5, 6, 7, 9, 12, and 13 displayed high activity at micromolar IC₅₀ and EC₅₀ concentrations. Sixteen quinoxaline derivatives were selected and evaluated on T. cruzi and/or L. amazonensis amastigotes. The most active compounds were 6a-b and 7d-e, on all evolutive forms of L. amazonensis and T. cruzi evaluated with IC₅₀ values 0.1-0.8 μM on promastigotes and epimastigotes 1.4-8.6 on amastigotes. Compounds 5k, 12b and 13a were the most selective (SI = 19.5-38.4) on amastigotes of T. cruzi. In general their activity was directly related to the methylsulfoxyl, methylsulfonyl, and amine groups as well as the presence of chorine or bromine in the molecules. The current results indicate that these quinoxaline derivatives are novel and promising agents for further development towards a treatment for Chagasg € disease and leishmaniasis.

Full text of DOI:10.1016/j.ejmech.2014.11.018

Accepted Manuscript
Synthesis and biological evaluation of novel 2,3-disubstituted quinoxaline derivatives
as antileishmanial and antitrypanosomal agents
Juliana Cogo , Vanessa Kaplum , Diego Pereira Sangi , Tânia Ueda-Nakamura ,
Arlene Gonçalves Corrêa , Celso Vataru Nakamura
PII:
S0223-5234(14)01040-X
10.1016/j.ejmech.2014.11.018
EJMECH 7511
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2013
Revised Date: 29 August 2014
Accepted Date: 8 November 2014
Please cite this article as: J. Cogo, V. Kaplum, D.P. Sangi, T. Ueda-Nakamura, A.G. Corrêa, C.V.
Nakamura, Synthesis and biological evaluation of novel 2,3-disubstituted quinoxaline derivatives as
antileishmanial and antitrypanosomal agents, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.11.018.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel 2,3-disubstituted
quinoxaline derivatives as antileishmanial and antitrypanosomal
agents
a
a
b,c
a
Juliana Cogo , Vanessa Kaplum , Diego Pereira Sangi , Tânia Ueda-Nakamura , Arlene
b
a
Gonçalves Corrêa , Celso Vataru Nakamura *
a
Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá,
Avenida Colombo 5790, 87020-900 Maringá, PR, Brazil.
b
Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luis Km
2
35, 13565-905 São Carlos, SP, Brazil
c
Instituto de Ciências Exatas, Universidade Federal Fluminense, 27213-145 Volta Redonda, RJ,
Brazil.
∗
Corresponding author: Programa de Pós graduação em Ciências Farmacêuticas, Laboratório de
Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Bloco-B-08,
Universidade Estadual de Maringá, Av. Colombo 5790, CEP 87.020-900, Maringá, Paraná,
Brazil. Tel.: +55 44 3011 5012; fax: +55 44 3011 5046. E-mail addresses: cvnakamura@uem.br
Abbreviations
T. cruzi, Trypanosoma cruzi; L. amazonensis, Leishmania amazonensis; IC, inhibitory
concentration; EC, effetive concentration; SI, selective index; CC, cytotoxic concentration;
LLCMK , Epithelial cells from the kidney from Macaca mulatta.
2

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ABSTRACT
Quinoxalines belong to the N-containing heterocyclic compounds that stand out as having
promising biological activity due to their privileged scaffold. In this work, we report the
synthesis, antileishmanial, and antitrypanosomal properties of 46 new 2,3-disubstituted
quinoxaline and 40 previously reported derivatives. Among all of the compounds screened for in
vitro activity against epimastigotes and trypomastigotes of T. cruzi and promastigotes of L.
amazonensis as well as mammalian toxicity on LLCMK cells and J774 macrophages, analogues
2
from series 5, 6, 7, 9, 12, and 13 displayed high activity at micromolar IC50 and EC50
concentrations. Sixteen quinoxaline derivatives were selected and evaluated on T. cruzi and/or L.
amazonensis amastigotes. The most active compounds were 6a-b and 7d-e, on all evolutive
forms of L. amazonensis and T. cruzi evaluated with IC values 0.1-0.8 µM on promastigotes
5
0
and epimastigotes 1.4-8.6 on amastigotes. Compounds 5k, 12b and 13a were the most selective
SI = 19.5-38.4) on amastigotes of T. cruzi. In general their activity was directly related to the
(
methylsulfoxyl, methylsulfonyl, and amine groups as well as the presence of chorine or bromine
in the molecules. The current results indicate that these quinoxaline derivatives are novel and
promising agents for further development towards a treatment for Chagas’ disease and
leishmaniasis.
KEYWORDS: Quinoxaline derivatives; Leishmania amazonensis; Trypanosoma cruzi; Anti-
trypanosomatid agents

ACCEPTED MANUSCRIPT
Introduction
Neglected tropical diseases are significant public health problems and have been attracting
increasing worldwide attention [1]. Chagas’ disease is caused by the protozoan Trypanosoma
cruzi, which is found in 21 countries and affects approximately 8 million people, with
approximately 50,000 new cases per year [2]. Latin America has most of the cases of Chagas’
disease, which has become a global health problem as a result of migration to non-endemic
regions, such as Australia, Europe, the United States, and Canada, resulting in annual treatment
costs of approximately USD$ 600 million [3,4]. Its pathogenesis is subdivided into an acute
phase characterized by nonspecific inflammation, an asymptomatic indeterminate phase, and a
chronic phase, during which approximately 30-40% of the cases develop irreversible
cardiovascular, gastrointestinal, and neurological lesions [5,6]. The transmission of Chagas’
disease generally occurs through the bite and infection with contaminated feces of insects of the
subfamily Triatominae (Hemiptera, Reduviidae). It may also be transmitted through blood
transfusion or congenitally and orally, such as through contaminated food [7,8].
Leishmaniasis is endemic in 98 countries worldwide, with approximately 350 million people at
risk of infection and 12 million currently infected. The disease may be caused by more than 20
different species of Leishamania sp, which are responsible for clinical manifestations that can be
classified as cutaneous, mucocutaneous, and visceral. Cutaneous leishmaniasis is the most
common clinical form of the disease, with 1.5 million new cases per year. The species that stand
out in the New World are L. amazonensis, L. braziliensis, and L. guyanensis [2,9,10]. Among the
cutaneous leishmaniasis cases worldwide, 70-75% occur in just 10 countries, including Brazil,
which experienced approximately 30,000 new cases in 2010 [11,12]. Transmission occurs
through the bite of the female phlebotomine sandfly. The vector in the New World is Lutzomyia,

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and the vector in the Old World is Phlebotomus [13]. Clinical manifestations are characterized by
single or multiple lesions that are usually located on the legs, arms, and head. They initially
appear as papules and progress into nodules and finally ulcerative lesions [14,15].
Nitroderivative compounds, such as benznidazole and nifurtimox, are currently the primary
treatment options for Chagas’ disease, despite their reduced efficacy in the chronic phase and
adverse reactions in approximately 40% of patients [16-18]. For antileishmania chemotherapy,
pentavalent antimonials, such as meglumine antimoniate and sodium stibogluconate, are the first-
line treatment, whereas amphotericin B, pentamidine, paromomycin, and miltefosine are the
second-line treatment [19-21]. However, the available drugs for both diseases have severe side
effects, long-term treatment, and variable efficacy, factors that encourage the search for new
therapeutic alternatives [22].
Quinoxalines belong to the N-containing heterocyclic compounds that stand out as having
promising biological activity because of their privileged scaffold [23,24]. They have numerous
reported biological activities, including anticancer [25-27], beneficial effects for sleep disorders
[28], antimycobacterial [29-30], antibacterial [31], antifungal [32], antiviral [33], anti-
inflammatory, and antioxidant activities [34-35]. The antiprotozoal activity of quinoxalines is
relevant, especially their antitrypanosomatid activity, which has been reported for quinoxaline
1
,4-di-N-oxide [36,37], 3-trifluoromethylquinoxaline
N,N’-dioxide [38], and
3-
aminoquinoxaline-2-carbonitrile 1,4-dioxide derivatives [39]. Quinoxalines also exhibit
antileishmanial activity, which has been reported for 4-substituted pyrrolo[1,2-a]quinoxalines
[40], 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one [41-42], and 1,4-di-N-oxide
quinoxaline derivatives [43].

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Recently, we have reported the activity of 3-chloro-7-methoxy-2-(methylsulfonyl)quinoxaline,
against T. cruzi [44]. A synergistic effect between this quinoxaline and benznidazole was
observed against epimastigotes and trypomastigotes, accompanied by an antagonistic interaction
against LLCMK cells. Based on the above considerations, novel 2,3-disubstituted quinoxaline
2
derivatives were synthesized to evaluate their in vitro antitrypanosomal and antileishmanial
activity.
Results and Discussion
The discovery and development of new drugs for the treatment of neglected diseases, such as
leishmaniasis and Chagas’ disease, is necessary and urgent. Their current treatments have several
limitations, including limited effectiveness, parenteral administration, long courses of treatment,
severe side effects, toxicity, and high cost, making them unaffordable for most patients
[22,45,46]. Many studies have reported that quinoxaline derivatives are promising
chemotherapeutic agents against Leishmania sp and T. cruzi [36-43].
Several methods have been reported for the synthesis of quinoxalines [47]. In the present study,
we have focused on straight forward synthetic routes, especially those based on green chemistry
principles, Thus, we synthesized 46 new 2,3-disubstituted quinoxaline derivatives (3d; 5e, 5fa-fb,
5
g, 5ka; 6a-b; 7d; 8a; 10a; 11a-d, 11f-p; 12a-p; 13a-b; 14a-c) and 40 previously reported
compounds (1; 2a-p; 3a-c, 3e; 4a-b; 5a-d, 5f, 5h-k; 7a-c, 7e; 9a-c; 10b-c; 11e) with the goal of
discovering new drugs for the treatment of Chagas’ disease and leishmaniasis.
Initially, quinoxaline derivatives were prepared using a procedure described by Venkatesh et al.
[48]. The first step was improved by using microwave (MW) irradiation [49] and the nitroketene

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N,S-acetal derivatives 2, obtained by vinylic substitution, were cyclized to produce quinoxaline 3.
By using quinoxaline 3 as starting material, we also synthesized quinoxalines 4a-b and 5a-b
through cross-coupling reactions (Scheme 1).
Scheme 1. Synthesis of quinoxalines 3, 4 and 5a-b
Employing a series of sulfur oxidations with m-chloroperbenzoic acid, and solvent-free
nucleophilic substitutions, we synthesized quinoxalines 6, 7, 9, 10, 11, 12 and 13, using
quinoxalines 3 as starting material (Scheme 2) [48].

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Scheme 2. Synthesis of quinoxalines 6, 7, 9, 10, 11, 12 and 13
Quinoxalines 8a and 14a-c were synthesized using 4a as starting material through oxidation
followed by nucleophylic substitution (Scheme 3). We have tried to synthesize
aryilaminoquinoxalines 14 at room temperature without success, thus microwave irradiation was
applied under the same conditions used to obtain aminosulfonylquinoxalines 11 reaching good
results.
Scheme 3. Synthesis of quinoxalines 8a and 14a-c

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Quinoxalines 5c-k were synthesized through the condensation of 1,2-diarylethanediones with O-
phenylenediamine by using ultrasound irradiation as energy source (Scheme 4) [50]. Compounds
5
fa, 5fb, and 5ka were prepared from 5f or 5k by deprotection of the methoxyl group followed
by O-alkylation.
Scheme 4: Synthesis of 2,3-aryldisubstituted quinoxalines 5
The screening for antichagasic and antileishmanial activity was performed on the epimastigote
and trypomastigote forms of T. cruzi and promastigote form of L. amazonensis. Epimastigotes
and promastigotes are the extracellular replicative forms inside the insect vectors of T. cruzi and
L. amazonensis, respectively. The easily cultivable and drug-sensitive epimastigote and
promastigote forms make these models an excellent choice for preliminary in vitro screening.
Trypomastigotes are an extracellular non-replicative stage of T. cruzi found in the bloodstream of
infected vertebrate hosts. Selective toxicity is an important principle of antiparasitic therapy,
therefore the cell viability was also carried out to verify their cytotoxic effects on mammalian
cells (LLCMK and J774-A1 macrophages).
2

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Compound l,l-bis(methylthio)-2-nitroethene 1 that was used in the synthesis of quinoxaline
derivatives was also assayed against both protozoa (Table 1). Notably, compound 1 presented
activity against the epimastigote and promastigote forms. Thirteen nitroketene N,S-acetal
derivatives 2, which were synthesized through the reaction of compound 1 with primary or
secondary amines (Scheme 1) [49], showed antileishmanial and antitrypanosomal activity at
different levels (Table 1). The presence of benzenamine and 2-[nitroethenyl]benzenamine rings
in compounds 2b and 2k, respectively, and the presence of fluorine and chlorine on the benzene
ring (2o and 2p, respectively) caused an increase in biological activity, with IC values < 26 µM
5
0
for both protozoa. The presence of the amine group in 2n made it the only active compound in
this group against trypomastigotes.
Table 1. In vitro antileishmanial and antitrypanosomal activities of 1,1-bis(methylthio)-2-
nitroethene (1) and nitroketene N,S-acetals (2)
µM
Comp
1
Promastigote
Epimastigote
IC50/96h
Trypomastigote
1
2
3
4
R
R
R
R
IC50/72h
SI
SI
EC50/96h
>50.0
NT
SI
29.6 ± 2.1
27.9 ± 5.9
24.1 ± 0.6
28.7 ± 3.5
42.3 ± 0.8
41.5 ± 7.3
33.0 ± 3.2
21.9 ± 8.2
19.7 ± 1.2
6.0
ND
ND
ND
ND
ND
ND
ND
ND
10.8 ± 0.6
32.0 ± 8.3
11.4 ± 6.6
39.0 ± 12.4
61.9 ± 11.0
67.3 ± 15.6
20.4 ± 4.3
70.1 ± 13.6
19.7 ± 6.6
18.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2a
H
H
H
H
OMe
H
H
H
H
H
H
H
2
b
NT
2c
H OMe
H OCH
H
NT
2
d
2
CH
2
O
NT
2f
H
H
OH
Br
H
NT
2
h
H
NT
2
i
NT
2
k
H
F
H
H
NT

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µM
Comp
Promastigote
Epimastigote
IC50/96h
Trypomastigote
1
2
3
4
R
R
R
R
IC50/72h
SI
SI
ND
9.6
6.6
EC50/96h
>50.0
SI
2
l
73.6 ± 4.8 11.1
>100.0
ND
ND
6.0
2
m
>100.0
ND
90.1 ± 3.1
31.8 ± 3.8
>50.0
2
n
o
p
H NMe
2
H
H
H
H
H
H
52.8 ± 4.2 18.9
35.0 ± 6.1
>50.0
2
H
H
F
17.2 ± 0.4
21.3 ± 0.2
8.4
6.9
18.1 ± 1.4
25.8 ± 1.8
10.5
7.3
ND
ND
2
Cl
>50.0
IC: inhibitory concentration; EC: effective concentration; SI: selective index; NT: not tested; ND:
not determined
Using nitroketene N,S-acetals 2, five 3-chloro-2-methylthioquinoxalines 3 were synthesized
(
Scheme 1). The changes in the pyrazine ring were not able to increase the activity levels in
relation to compound 2 but made it more selective against protozoa than against mammalian cells
Table 2). Compound 3a was 19.3-times more selective for epimastigotes, whereas 3e was 22.1-
and 16.1-times more selective for promastigotes and epimastigotes, respectively.
(

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Table 2. In vitro antileishmanial and antitrypanosomal activities of 3-chloro-2-
methylthioquinoxalies (3) and 3-aryl-2-methylthioquinoxalines (4)
µM
Comp
Promastigote
IC50/72h
Epimastigote
Trypomastigote
2
3
R
R
SI
IC50/96h
38.7 ± 2.9
52.9 ± 5.4
>100.0
SI
EC50/96h
>50.0
>50.0
>50.0
>50.0
>50.0
NT
SI
3
3
a
H
H
OMe
H
93.6 ±2.6
9.8
19.3
8.1
ND
ND
ND
ND
ND
ND
ND
b
74.1 ± 7.2
>100.0
13.5
ND
7.4
3
c
OMe
Br
H
ND
9.2
3
d*
H
57.9 ± 10.6
45.3 ± 1.0
>100.0
47.8 ± 2.6
39.4 ± 1.6
>100.0
3
e
a
b
Cl
H
22.1
ND
ND
16.1
ND
ND
4
OMe
H
4
>100.0
>100.0
NT
IC: inhibitory concentration; EC: effective concentration; SI: selective index; NT: not tested; ND:
not determined. *Gray highlights the newly synthesized quinoxaline derivatives
Two 3-aryl-2-methylthioquinoxalines 4 were prepared from compound 3a (Scheme 2). The
chloride replacement by phenyl group in the pyrazine ring in compounds 4a and 4b caused a
reduction of potency, with no activity at the highest concentration evaluated against all evolutive
forms (Table 2).
Fourteen 2,3-diarylsubstituted quinoxalines 5 were obtained as described in Schemes 2 and 3.
The addition of an extra phenyl group in the pyrazine ring in these compounds improved the
biological activity against L. amazonensis and T. cruzi (Table 3) in relation to quinoxaline
derivatives 4a and 4b. The additional methoxyl groups in the phenyl rings in compound 5c gave
rise to compound 5k, and this change was responsible for an increase in the activity against
epimastigotes. Although 5k was moderately active against promastigotes and epimastigotes, its
low toxicity to host cells revealed selectivity indices of 19.7 and 9.0, respectively. Structural

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changes in 5k gave rise to compound 5ka, which showed an increase in activity (IC = 5.7 µM)
5
0
against the promastigote form of L. amazonensis. However, higher toxicity against the host cell
was observed compared with its precursor 5k, thereby causing a reduction of the selectivity index
5
to 8.2. Compound 5f, which has only one methoxyphenyl group on R , was more active against
both protozoa, especially against promastigotes, with an IC of 12.8 µM.
5
0
Table 3. In vitro antileishmanial and antitrypanosomal activities of 2,3-diarylsubstituted
quinoxalines (5)
µM
Comp
Promastigote
Epimastigote
Trypomastigote
2
3
5
6
R
R
R
R
IC50/72h
SI
ND
IC50/96h
NT
SI
EC50/96h
SI
5
a
H OMe
H OMe
H
H
H
OMe
H
NT
ND
ND
ND
NT
NT
ND
ND
5b
40.5 ± 17.7 ND
21.1 ± 0.3 ND
>100.0
>100.0
5
c
H
H
H
H
H
H
H
H
H
Cl
H
NT
NT
NT
NT
ND
ND
ND
ND
5d
Me
Me
OMe
H
H
8.9 ± 1.2 ND 35.7 ± 17.6 ND
24.3 ± 1.9 ND 36.0 ± 11.1 ND
12.8 ± 0.0 ND 21.5 ± 0.8 ND
5
e* Cl
H
5
f
H
H
H
5fa*
OCH
OCH
2
CH
2
NC
5
H
10
1.9 ± 0.2 29.6 21.4 ± 1.1 1.8 20.3 ± 2.3 1.8
5
fb* H
g* Cl
H
2
CH
NC
2
4
H
8
O
6.2 ± 0.6 8.7 40.3 ± 4.5 4.6
31.5 ± 1.6 9.5 43.8 ± 3.0 6.5
28.1 ± 1.4 6.6 42.7 ± 2.2 6.1
5.3 ± 0.7 38.5 54.0 ± 1.8 8.7
22.2 ± 1.0 3.2 83.4 ± 6.2 1.3
30.0 ± 0.6 19.7 36.6 ± 3.0 9.0
5.7 ± 0.4 8.2 39.2 ± 4.7 1.0
>50.0
>50.0
>50.0
>50.0
>50.0
>100.0
>50.0
ND
ND
ND
ND
ND
ND
ND
5
H
OMe
H
5h
H
H
5
i
Cl Cl
Cl Cl
H
H
5
j
H
Me
OMe
5
k
H
H
H
OMe
OH
5
ka* H
OCH
2
CH
2
NC
H
5
10
IC: inhibitory concentration; EC: effective concentration; SI: selective index; NT: not tested; ND:
not determined. *Gray highlights the newly synthesized quinoxaline derivatives

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Piperidine and morpholine derivatives 5fa and 5fb, respectively, were synthesized. Similar to
compound 5ka, these structural changes led to a significant increase in activity, mainly against
promastigotes, with IC50 values of 1.9 µM for 5fa and 6.2 µM for 5fb. Such changes increased
their potency, resulting in increased selectivity of 29.6 and 8.7, respectively.
Furthermore, compound 5fa was the only one that showed activity against trypomastigotes of T.
cruzi, with an EC of 20.3 µM. All of the chlorine containing compounds in this group were
5
0
active against both protozoa. In quinoxaline 5i, two chlorines at the benzene ring were
responsible for increased activity against the promastigote form (IC50 = 5.3 µM), with low levels
of toxicity and a high selectivity index of 38.5. Halogeneted molecules have been described by
other researchers to improve the antiprotozoal properties of compounds [51-52].
To investigate the influence of methylsulfoxyl and methylsulfonyl groups on biological activity,
3
-chloro-2-methylthioquinoxalines 3 underwent oxidation reactions led to a series of 3-chloro-2-
methylsulfoxylquinoxalines 6 and 3-chloro-2-methylsulfonylquinoxalines 7 (Scheme 1). The
assays performed with these two groups of quinoxaline derivatives confirmed the influence of
halogens on activity (Table 4). Compound 6b, which has two chlorines in its structure, was the
most potent quinoxaline derivative against all evolutive forms, with an IC50 of 0.1 µM for
promastigotes and epimastigotes and an EC of 1.7 µM for trypomastigotes. Moreover, it was
5
0
the most selective for L. amazonensis (selectivity index = 107.6). Compound 6a was among the
most active (IC50 > 0.8 µM) and selective (selectivity index = 39.2 and 27.8) compounds against
T. cruzi and L. amazonensis.

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Table 4. In vitro antileishmanial and antitrypanosomal activities of 3-chloro-2-
methylsulfoxylquinoxalines (6) and 3-chloro-2-methylsulfonylquinoxalines (7)
µM
Comp
Promastigote
Epimastigote
Trypomastigote
EC50/96h SI
2
3
R
R
IC50/72h
0.8 ± 0.2
0.1 ± 0.0
1.6 ± 0.6
2.9 ± 0.7
0.2 ± 0.1
0.2 ± 0.1
SI
IC50/96h
0.5 ± 0.1
0.1 ± 0.0
0.6 ± 0.1
3.1 ± 0.4
0.3 ± 0.1
0.3 ± 0.0
SI
6
a*
b*
H
Cl
OMe
H
27.8
107.6
14.4
7.6
39.2
54.2
70.1
7.5
4.2 ± 1.2
1.7 ± 0.1
6.4 ± 0.3
9.8 ± 1.4
1.8 ± 0.1
6.9 ± 1.0
4.4
2.9
7.3
2.4
7.0
4.1
6
7b
H
H
7c
OMe
Br
H
7
d*
H
71.8
66.0
49.6
25.9
7e
Cl
H
IC: inhibitory concentration; EC: effective concentration; SI: selective index; *Gray highlights
the newly synthesized quinoxaline derivatives
Compounds 7 with a methylsulfonyl group showed excellent activity, similar to compounds 6.
The most active (IC50 > 0.3 µM) and selective (SI = 25.9-71.8) were compounds 7d and 7e,
which have bromine and fluorine in the structure, respectively. The addition of a methoxyl group
in the benzene ring in compound 7c resulted in decreased activity and selectivity against T. cruzi
and L. amazonensis. Despite the cytotoxicity observed in host cells, high levels of selectivity
were obtained. This increase in the antiprotozoal activity of compounds in groups 6 and 7 was
directly linked to the introduction of methylsulfoxyl and methylsulfonyl groups in the
quinoxaline ring.
Compound 4a oxidation gave rise to compound 2-phenyl-3-methylsulfonyl-6-
methoxyquinoxaline (8a). Chlorine replacement by phenyl ring resulted in decreased activity and
selectivity against T. cruzi and L. amazonensis. It showed an IC values of 24.7 and 28.4 µM and
5
0

ACCEPTED MANUSCRIPT
an EC50 value of 48.1 µM for the promastigote, epimastigote, and trypomastigote forms,
respectively (Table 5).
Table 5. In vitro antileishmanial and antitrypanosomal activities of 2-phenyl-3-methylsulfonyl-6-
methoxyquinoxaline (8), 3-chloro-2-aminoquinoxalines (9) and 2,3-diaminoquinoxalines (10)
µM
Comp
Promastigote
IC50/72h SI
24.7 ± 2.3 4.1 28.4 ± 1.9 9.3 48.1 ± 0.9 5.5
Epimastigote Trypomastigote
5
6
R
R
IC50/96h SI EC50/96h SI
8
9
a
a
n
Bu
36.2 ± 1.0 4.4 49.5 ± 6.4 7.9
24.5 ± 1.7 5.4 15.9 ± 1.6 22.2
20.9 ± 0.9 4.3 29.4 ± 5.0 3.2
13.6 ± 0.8 3.6 38.4 ± 2.9 0.6
> 50.0
>50.0
> 50.0
> 50.0
ND
ND
ND
ND
9
b
Bn
n
n
n
1
0a*
0b
0c
Bu
Bu
Bu
Bn
Ph
1
1
Ph
6.5 ± 0.0 4.4 45.6 ± 3.4 1.0 37.9 ± 2.3 1.2
IC: inhibitory concentration; EC: effective concentration; SI: selective index; ND: not
determined. *Gray highlights the newly synthesized quinoxaline derivatives
3
-Chloro-2-methylsulfonylquinoxalines 7 were submitted to nucleophilic substitution reactions
that provided 3-chloro-2-aminoquinoxalines 9 and 2,3-diaminoquinoxalines 10 (Scheme 1).
Phenyl ring addition in the amine group made compound 9b more active and selective than
compound 9a. Compound 9b had IC values of 24.5 and 15.9 µM against the promastigote and
5
0
epimastigote forms, respectively, which was up to 22-times more selective for the protozoa than
for the host cells (Table 5). As well as other quinoxaline derivatives, 2,3-diaminoquinoxalines 10
were considered more active against the promastigote form of L. amazonensis (IC50 = 6.5-20.9
µM) than for T. cruzi epimastigotes ( IC50 = 29.4-45.6 µM, EC50 > 50 µM) (Table 5). However,

ACCEPTED MANUSCRIPT
when evaluated against the trypomastigote form of T. cruzi, these two classes of compounds
showed no activity, with the exception of compound 10c that had an EC50 value of 37.9 µM. This
loss of antiprotozoal activity was further enhanced by the substitution of the methylsulfoxyl and
methylsulfonyl groups for an amine group in the quinoxaline ring.
A series with sixteen 3-amino-2-methylthioquinoxalines 11 was synthesized using 3-
chloroquinoxalines 3 as starting material through substitution reactions (Scheme 1). These
compounds showed different levels of activity (IC50 between 21.5 and > 100 µM) (Table 6).
Despite the large number of compounds obtained and evaluated, no high selectivity index was
found. The best selectivity was obtained with compound 11f against the epimastigote form of T.
cruzi, which was 8.5-times more selective for protozoa than for host cells.

ACCEPTED MANUSCRIPT
Table 6. In vitro antileishmanial and antitrypanosomal activities of 3-amino-2-
methylthioquinoxalines (11)
µM
Comp
Promastigote
IC50/72h SI
Epimastigote
IC50/96h SI
Trypomastigote
SI
2
3
5
R
R
R
EC50/96h
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
1
1a*
H
H
H
H
H
H
OMe
OMe
OMe
H
Me
2
42.8 ± 1.3 5.9 83.6 ± 1.5 6.2
35.2 ± 4.0 2.1 86.7 ± 1.5 3.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
n
1
1b
Bu
1
1c*
CH
2
CH
2
OH
82.9 ± 1.4 5.0
75.9 ± 1.7 1.9 93.1 ± 1.0 1.2
96.2 ± 2.2 2.9 >100.0 ND
>100.0
ND
n
1
1d*
Bu
1
1e
H
EtOH
1
1f*
OMe
H
Cyclohexyl
29.8 ± 4.4 1.2 30.5 ± 5.8 8.5
30.2 ± 1.7 3.5 69.2 ± 2.1 1.1
n
1
1g* OMe
1h* OMe
Bu
1
H
CH
2
CH
2
OH
>100.0
ND
>100.0
ND
1
1i* OMe
1j* OMe
H
Cyclohexyl
21.5 ± 1.3 2.4 41.0 ± 8.7 1.8
26.9 ± 0.5 4.7 95.7 ± 1.0 0.7
27.1 ± 2.1 3.3 79.6 ± 1.7 0.7
25.2 ± 2.7 5.1 41.8 ± 3.5 0.9
i
1
H
Bu
1
1k* OMe
H
Isopentyl
n
1
1l*
1m*
1n*
Br
Br
H
H
Bu
1
H
CH
2
CH
2
OH
>100.0
ND
>100.0
ND
i
1
OMe
H
Bu
27.6 ± 4.2 6.7 69.9 ± 2.7 1.0
24.4 ± 2.5 4.6 63.6 ± 4.5 1.0
n
1
1o*
Cl
Cl
Bu
1
1p*
H
Cyclohexyl
>100.0
ND 52.3 ± 2.3 4.8
IC: inhibitory concentration; EC: effective concentration; SI: selective index; ND: not
determined. *Gray highlights the newly synthesized quinoxaline derivatives
The methylthio groups in quinoxaline derivatives 11 were subsequently oxidized to sulfone and
sulfoxide, giving rise to 3-amino-2-methylsulfonylquinoxalines 12 and 3-amino-2-
methylsulfoxylquinoxalines 13, respectively (Scheme 1).

ACCEPTED MANUSCRIPT
Sixteen 3-amino-2-methylsulfonylquinoxalines 12 were synthesized. The replacement for a
methylsulfonyl group was responsible for an increase in the antileishmanial and antitrypanosomal
activity, similar to the observations with the compounds 7 (Table 7). Among the products of this
synthesis, derivatives 12b, 12c, and 12f differ only in the amine group. The similarities between
these compounds were also seen with regard to biological activity, with high activity against the
promastigote and epimastigote forms (IC50 = 2.2-3.6 µM) and moderate activity against the
trypomastigote form (EC = 27.7 and 39.2 µM). Notably, the butylamine group in 12b caused a
5
0
significant reduction of toxicity in host cells compared with the other compounds, with selectivity
index ratios of 108.5 and 10.0 for epimastigotes and trypomastigotes, respectively.

ACCEPTED MANUSCRIPT
Table 7. In vitro antileishmanial and antitrypanosomal activities of 3-amino-2-
methylsulfonylquinoxalines (12), 3-amino-2-methylsulfoxylquinoxalines (13) and 3-aryl-2-
aminoquinoxalines (14)
µM
Comp
Promastigote
Epimastigote
SI
6.8
Trypomastigote
2
3
5
6
R
R
R
R
IC50/72h
35.9 ± 1.0
2.5 ± 0.3
2.9 ±0.4
SI
IC50/96h
25.2 ± 3.7
3.6 ± 0.3
2.9 ± 0.8
4.4 ± 1.0
4.2 ± 1.0
2.2 ± 0.4
55.3 ± 1.0
90.9 ± 4.7
29.1 ± 4.8
55.4 ± 1.7
38.0 ± 2.5
2.3 ± 0.1
1.6 ± 0.0
3.1 ± 0.3
2.3 ± 0.1
2.0 ± 0.0
2.5 ± 0.3
1.8 ± 0.0
66.8 ± 5.0
>100.0
EC50/96h
SI
1
2a*
2b*
2c*
2d*
2e*
2f*
H
H
H
H
H
H
OMe
OMe
OMe
H
Me
2
4.5
>50.0
ND
n
1
Bu
17.1
13.3
11.4
13.0
18.1
5.5
108.5 39.2 ± 1.8 10.0
25.2 35.7 ± 4.1 2.0
1
C
C
2
H
n
OH
4
1
Bu
2.9 ± 0.5
2.9 ± 0.8
2.9 ± 0.1
32.2 ± 5.7
69.2 ± 6.1
14.7 ± 1.5
27.8 ± 2.4
21.2 ± 2.1
1.6 ± 0.5
0.8 ± 0.4
2.6 ± 0.3
1.4 ± 0.3
2.2 ± 0.1
2.5 ± 0.4
1.9 ± 0.0
>100.0
5.5
4.4
15.7 ± 1.2 1.5
22.6 ± 3.9 0.8
1
H
2
H OH
4
1
OMe Cyclohexyl
20.8 27.7 ± 2.5 1.6
n
1
2g* OMe
2h* OMe
H
H
Bu
OH
1.6
2.6
2.7
1.5
2.6
43.7
3.4
41.2 ± 1.2 2.1
>50.0 ND
1
C
2
H
4
3.7
1
2i* OMe
H
Cyclohexyl
6.5
23.6 ± 2.6 3.3
24.8 ± 4.1 3.3
22.9 ± 1.9 4.2
5.6 ± 0.5 17.8
i
1
2j* OMe
H
Bu
3.6
1
2k* OMe
H
Isopentyl
5.0
n
1
2l*
2m*
2n*
Br
Br
H
H
Bu
28.3
40.3
22.8
50.6
15.0
8.0
1
H
C
2
H
4
OH
8.2 ± 2.7
0.7
i
1
OMe
H
Bu
22.6 11.7 ± 1.7 6.0
n
1
2o*
Cl
Cl
H
Bu
Cyclohexyl
93.4
38.3
9.0 ± 1.8 23.8
3.9 ± 1.4 19.6
1
2p*
H
1
3a*
3b*
4a*
4b*
4c*
OMe
H
72.8 25.4 ± 1.5 7.1
1
Cl
33.8
ND
4.8
21.8
12.5
ND
3.8
6.3 ± 1.4
>50.0
6.2
ND
ND
ND
n
1
Bu
1
C
2
H
4
OH 72.8 ± 4.3
>50.0
1
C
2
H
4
NC
H
5
10 50.3 ± 1.9
0.9
47.6 ± 2.4
>50.0
IC: inhibitory concentration; EC: effective concentration; SI: selective index; ND: not
determined. *Gray highlights the newly synthesized quinoxaline derivatives

ACCEPTED MANUSCRIPT
The presence of bromine or chlorine in compounds 12l, 12m, 12o, and 12p caused a significant
increase activity compared with the other compounds that belong to this group, especially against
trypomastigotes, with EC50 < 9.0 µM. The introduction of a halogen and a butylamine group in
1
2l and 12o was responsible for the improvement in activity and consequently more selectivity,
which was 17.8- to 92.0-times more selective for the protozoa.
-amino-2-methylsulfoxylquinoxalines 13 were obtained through oxidation of amino-
3
methylthioquinoxalines 11. Compounds 13a and 13b showed high activity against promastigotes
and epimastigotes, with IC50 values < 2.5 µM and low levels of cytotoxicity (Table 7).
Furthermore, 13a was almost 73-times more selective for epimastigotes, whereas 13b was 33.8-
and 21.8-times more selective for promastigotes and epimastigotes, respectively. Compound 13b
was more active against the trypomastigote form than 13a, with an EC50 of 6.3 and 25.4,
respectively, and a selectivity index > 7.
From a mixture of 3-phenyl-7-methylsulfonyl-2-methoxyquinoxaline (8a) and amine derivatives,
three 3-aryl-2-aminoquinoxalines 14 were obtained. The changes in the quinoxaline ring, such as
the addition of aryl and an amine group, were responsible for a decrease in the antiprotozoal
activity (Table 7). This low activity against these protozoa was also seen in analogues 4 and 8,
which have an aryl group at the same position and an amine group in compounds 9a and 9b.
The results obtained with the quinoxaline derivatives were compared to the ones obtained with
amphotericin B and benznidazol, antileishmanial and antitrypanosomal reference-drugs,
respectively. Benznidazole exhibited an IC of 8.1 µM on epimastigotes and an EC 3.4 µM on
5
0
50
trypomastigotes [53] while amphotericin B showed an IC 0,75 µM on promastigotes.
5
0

ACCEPTED MANUSCRIPT
Analogues from series 5, 6, 7, 9, 12, and 13 with high activity and selectivity on promastigotes,
and/or epimastigotes and trypomastigotes may be considered equally or more potent than the
reference-drugs and were selected to be evaluated on intracellular amastigotes.
T. cruzi and L. amazonensis are obligate intracellular protozoan parasites. Amastigotes are the
replicative stage inside mammalian host cells and are the clinically important stage of these
parasites. The in vitro antiproliferative activity on intracellular amastigotes were performed on L.
amazonensis amastigotes infecting macrophages and T. cruzi amastigotes infecting LLCMK
2
cells. The results are presented in Table 8.
Table 8. In vitro activity of quinoxaline derivatives against intracellular amastigotes of L.
amazoznensis and T. cruzi
Comp
Amastigote
Amastigote
L. amazonensis
T. cruzi
IC50/72h (µM)
SI
IC50/96h (µM)
SI
38.4
ND
ND
ND
1.1
5
k
NT
4.6 ± 1.0
1.5 ± 0.1
NT
ND
4.9
9.0
ND
9.9
4.3
5.3
3.3
2.2
2.6
ND
ND
ND
ND
2.8
ND
8.6 ± 1.9
NT
6
a*
b*
6
NT
7b
> 15.0 ± 0.0
13.5 ± 1,8
8.6 ± 3.2
>20.0 ± 0.0
14.3 ± 1.4
40.5 ± 1.0
>25.0 ± 0.0
>25.0 ± 0.0
7.4 ± 0.3
14.9 ± 0.4
9.6 ± 0.7
9.3 ± 0.6
7.7 ± 0.2
7
d*
1.4 ± 0.0
3.1 ± 0.6
25.0 ± 0.0
12.9 ± 2.5
17.3 ± 1.4
20.2 ± 2.3
NT
7
e
0.9
9b
ND
27.3
1.8
1
2b*
2c*
2f*
2l*
2m*
1
1
ND
ND
0.8
1
1
NT
1
2n*
2p*
NT
4.7
1
NT
7.9
13a*
7.3 ± 0.3
NT
19.5
5.1
1
3b*
IC: inhibitory concentration; SI: selective index; NT: not tested; ND: not determined: *Gray
highlights the newly synthesized quinoxaline derivatives

ACCEPTED MANUSCRIPT
Although all compounds evaluated on L. amazonensis amastigotes showed some level of
antiparasite activity, with IC50 lower than 25 µM the most active were 6a, 6b, 7d and 7e with
IC50 values less than 4.6 µM. As in the case of antiproliferative activity on promastigotes, 2,7-
dichloro-3-(methylsulfinyl)quinoxaline
(6b)
and
6-bromo-3-chloro-2-(methylsulfonyl)
quinoxaline (7d) were the most potent (IC values of 1.5 and 1.4 µM, respectively) and selective
5
0
(SI = 9.0 and 9.9, respectively) compounds on amastigotes of L. amazonensis values very similar
to obtained with Amphotericin B (IC of 0.4 µM).
5
0
On amastigotes of T. cruzi the most active compounds were 5k, 7e, 12m, 12p, 13a and 13b with
IC values less than 9.6 µM while benznidazole presents IC of 26.1 µM [54]. Despite the slight
5
0
50
decrease in the activity on T. cruzi amastigotes, some of them showed a high selective index. We
have to highlight compound 5k, a quinoxaline 2,3-diarylsubstituted, although not the most active
(
IC : 8.6 µM), it becomes the most selective (SI: 38.4) against the amastigotes of T. cruzi due to
50
its low cytotoxicity on mammalian cells (LLCMK cells).
2
Other compounds that deserve attention for its high activity and selectivity on T. cruzi
amastigotes are 2-butylamino-3-methylsulfonyl-6-methoxyquinoxaline (12b) and 2-cycloexyl-3-
methylsulfinyl-6-methoxyquinoxaline (13a), with IC values of 14.3 and 9.3 µM an SI ratio of
5
0
2
7.3 and 19.5 respectively.
Evaluation of the antileishmanial and antitrypanosomal activity led to the identification of a
number of structure activity relationships, which showed that the new compounds can be equally
or more potent than reference-drugs.

ACCEPTED MANUSCRIPT
Fig. 1 General SAR scheme of quinoxaline derivatives against T. cruzi and L. amazonensis
Although clearly defining structure-activity relationships (SAR) is difficult, the comparisons of
the activities of quinoxaline derivatives against T. cruzi and L. amazonensis allowed us to
6
conclude that the methylsulfoxyl and methylsulfonyl groups at the R in the quinoxaline ring
(compounds 6, 7, 12, and 13) were mainly responsible for the antileishmanial and
antitrypanosomal activity (Fig. 1). These characteristics of compounds 6 and 7, coupled with
5
halogens at R position, were generally responsible for the increase in activity. Unfortunately,
however, they were also responsible for high cytotoxicity and consequently low selectivity.
Replacement by an amino group at the same position in compounds 12 and 13 caused a reduction
of cytotoxicity in mammalian cells and an increase in the selective index. The methylthio groups
6
at the R position of the quinoxalines (compounds 2, 3, 4, and 11) resulted in dramatically
reduced activity. Importantly, the addition of two aryl groups in the quinoxaline ring in analogues
5
was responsible for higher activity against L. amazonensis than against T. cruzi with low
toxicity levels.

ACCEPTED MANUSCRIPT
The 2,3-diarylsubstituted quinoxalines (5) presented a moderate to high antitrypanosomal and
antileishmanial activity with low levels of toxicity and appeared to be more selective than the
other quinoxalines derivatives evaluated. The analogs 4a-b, 8a, and 14a-c including only an aryl
5
group at R did not displayed significant activity and indicate that this group alone was not
sufficient for providing antitrypanosomal and antileishmanial activity. Recently, 4-
trichloromethylpyrrolo[1,2-a]quinoxalines where evaluated against Plasmodium falciparum, aryl
addition on the same position increase the potency and was able to reduced the toxicity [55].
2
Compounds with halogen elements at positions R in the quinoxaline ring, resulted in improved
activity while methoxyl group slightly decrease it. Its improvements in the activity against L.
infantum, L. amazonensis, and P. falciparum by the halogens and methoxyl groups have been
3
described [43]. Hidrogen substitution by methoxyl, OCH
2
and chloride at R are tolered and not
showed great activity interference.
Conclusion
In conclusion, among the various quinoxaline derivatives synthesized and evaluated in the
present study, series 5, 6, 7, 12, and 13 exhibited potent antileishmanial and antitrypanosomal
activity. Methylsulfoxyl, methylsulfonyl, and amine were the main groups responsible for this
activity. In summary, the present results revealed high in vitro antiprotozoal activity against T.
cruzi and L. amazonensis, which encourages further investigations to identify potential targets
and delineate putative mechanisms of action involved the antileishmanial and antitrypanosomal
properties of these compounds.

ACCEPTED MANUSCRIPT
Experimental section
Unless otherwise noted, all commercially available reagents were purchased from Aldrich
Chemical Co. Reagents and solvents were purified when necessary according to the usual
procedures described in the literature. The IR spectra refer to films and were measured on a
1
13
Bomem M102 spectrometer. H and C nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker ARX-400 (400 and 100 MHz, respectively). Mass spectra were recorded on
a Shimadzu GCMS-QP5000. Direct-infusion Ultrahigh Resolution and Accurate Mass
Spectrometry (orbitrap ESI-FT-MS) was performed with an LTQ Orbitrap Velos FT-MS
instrument (Thermo Fischer Scientific, Bremen, Germany) equipped with an electrospray source
(HESI-II) that operated in full-scan negative-ionization mode. The elemental analyses were
performed on a Fisons EA 1108 CHNS-O. Analytical thin-layer chromatography (TLC) was
performed on a 0.25 µm film of silica gel containing fluorescent indicator UV254 supported on an
aluminum sheet (Sigma-Aldrich). Flash column chromatography was performed using silica gel
(
Kieselgel 60, 230-400 mesh, E. Merck). Reactions were conducted in an ultrasound bath
Branson mod. 1510 or in a CEM Discovery focused microwave oven. The synthetic compounds
showed purity rates above to 99% in gas chromatography.
General procedure to synthesize 2-chloro-3-methylthioquinoxalines 3a-e [48]: To a
3 3
suspension of N,S-acetals 2o-p [49] (0.208 mmol) in CH CN (1 mL), POCl (0.625 mmol) was
added dropwise at 0°C over a period of 15 min with constant stirring. After completion of the
addition, the reaction mixture was heated at 80°C for 3-4 h and monitored by TLC. It was then
3 3
cooled and neutralized with ice-cold saturated NaHCO solution (2.5 mL), extracted with CHCl

ACCEPTED MANUSCRIPT
(
3 × 2 mL), washed with H
2
O (2 × 2 mL) followed by brine (1 × 2 mL), and dried over anhydrous
Na SO . The solvent was evaporated under vacuum to give quinoxalines 3a-k and 5, which were
2
4
purified by column chromatography over silica gel using hexane:EtOAc (9:1) as eluent.
-Chloro-6-methoxy-3-methylthioquinoxaline (3a) [48]: 54% yield. MP : 109 – 111 ºC. H
NMR (400 MHz, CDCl ) δ : 7.83 - 7.80 (m, 1H), 7.28 – 7.26 (m, 2H), 3.96 (s, 3H), 2.67 (s, 3H).
1
2
3
1
3
C NMR (100 MHz, CDCl ) δ : 161.08, 157.10, 142.94, 134.68, 129.08, 121.18, 105.95, 99.99,
3
-1
+
5
1
5.80, 13.76. IR (νmax, KBr): 3006, 1616, 1494, 1213 cm . MS (m/z) : 240 (M , 100), 205 (88),
90 (35), 159 (40), 63 (29).
1
2
7
1
-Chloro-3-methylthioquinoxaline (3b) [48]: 36% yield. H NMR (400 MHz, CDCl ) δ :7.99 –
3
.93 (m, 2H), 7.73 -7.69 (m,1H), 7.66 – 7.62 (m, 1H), 2.69 (s, 3H). IR (νmax, KBr): 2925, 2850,
-1
+
527, 1267, 1118, 999, 765 cm . MS (m/z): 210 (M , 100), 177 (47), 175 (80), 160 (52), 129
(55), 102 (60), 75 (35), 50 (30).
1
2
-Chloro-7-methoxy-3-methylthioquinoxaline (3c) [48]: 31% yield. H NMR (400 MHz,
3
CDCl ) δ: 7.84 (d, J = 9.18 Hz, 1H), 7.33 (dd, J = 9.18, 2.84 Hz,1H), 7.23 (d, J = 2.84 Hz, 1H),
1
3
3
1
.93 (s, 3H), 2.65 (s, 3H). C NMR (100 MHz, CDCl ) δ: 159.59, 152.58, 145.50, 140.44,
3
+
37.45, 128.41, 122.68, 106.32, 55.79, 13.76. MS (m/z): 240 (M , 88), 207 (100), 190 (25), 63
(20).
1
6
-Bromo-3-chloro-2-methylthioquinoxaline (3d): 30% yield. H NMR (400 MHz, CDCl ) δ:
3
8
.10 (d, J = 2.00 Hz, 1H), 7.83 (d, J = 9.07, 1H), 7.77 (dd, J = 9.07, 2.00 Hz, 1H), 2.66 (s, 3H).
1
3
C NMR (100 MHz, CDCl ) δ: 158.71, 146.23, 140.13, 139.53, 133.68, 130.53, 128.74, 122.23,
3
+
1
3.95. MS (m/z): 290 (M , 100), 255 (80), 100 (60), 75 (65).

ACCEPTED MANUSCRIPT
1
2
,7-Dichloro-3-methylthioquinoxaline (3e) [48]: 32% yield. H NMR (400 MHz, CDCl
3
) δ:
7
.91 (d, J = 2.19 Hz, 1H), 7.88 (d, J = 8.98, 1H), 7.63 (dd, J = 8.98, 2.19 Hz, 1H), 2.66 (s, 3H).
1
3
C NMR (100 MHz, CDCl ) δ: 156.50, 146.32, 139.21, 134.29, 131.07, 128.60, 127.21, 123.41,
3
+
1
3.95. MS (m/z): 244 (M , 100), 211 (88), 194 (60), 100 (51), 75 (39).
General procedure for iron catalyzed cross-coupling reactions: synthesis of 4a-b [48]: A
flame-dried 100 mL two-necked flask was charged with quinoxalines 3a or 3b (1.0 mmol),
Fe(acac) (0.10 mmol), and THF (5 mL), and the mixture was cooled to -30°C. A solution of
3
phenylmagnesium bromide or 4-methoxyphenylmagnesium bromide (2.2 mmol) was added via
syringe to the resulting red solution, causing an immediate color change to dark brown-black.
The reaction mixture was further stirred for 10-15 min and quenched with brine solution. It was
then extracted with CHCl (3 × 5 mL), washed with H O (2 × 10 mL) followed by brine (20 mL),
3
2
and dried over anhydrous Na SO . Standard purification by column chromatography using
2
4
hexane:EtOAc (9:1) as eluent provided the products 4a and 4b.
-Phenyl-7-methoxy-2-methylthioquinoxaline (4a) [48]: 31% yield. H NMR (400 MHz,
CDCl ) δ: 7.93 (d, J = 8.81Hz, 1H), 7.76 – 7.74 (m, 2H), 7.51 – 7.48 (m, 3H), 7.30 – 7.24 (m,
1
3
3
1
3
2
1
2
H), 3.98 (s, 3H), 2.63 (s, 3H). C NMR (100 MHz, CDCl
3
) δ: 160.86, 155.89, 150.93, 143.13,
37.57, 135.06, 130.19, 129.40, 129.02, 128.39, 120.65, 105.83, 55.75, 13.70. IR (νmax, KBr):
-1
+
952, 2923, 2852, 1616, 1222, 1097, 829, 692 cm . MS (m/z) : 282 (M , 100), 267 (32), 249
(50), 235 (18), 192 (15), 141 (18), 77 (35), 63 (30).
1
3
-Phenyl-2-methylthioquinoxaline (4b) [56]: 36% yield. H NMR (400 MHz, CDCl ) δ: 8.06 –
3
8
.05 (m, 1H), 7.99 - 7.97 (m, 1H), 7.79 – 7.76 (m, 2H), 7.70 – 7.66 (m, 1H), 7.64 – 7.59 (m, 1H),

ACCEPTED MANUSCRIPT
1
3
7
1
2
.52 – 7.51 (m, 3H), 2.64 (s, 3H). C NMR (100 MHz, CDCl
3
) δ: 155.94, 153.50, 141.68,
39.32, 137.45, 129.75, 129.70, 129.27, 129.01, 128.46, 128.10, 127.49, 13.77. IR (νmax, KBr):
-1
920, 2852, 1328, 1272, 1128, 1089, 759, 690 cm . MS (m/z): 252 (M+, 100), 237 (75), 219
(68), 205 (25), 134 (20), 102 (23), 77 (50), 51 (38).
General procedure for nickel-catalyzed cross-coupling reactions of 4a: synthesis of 5a and
b quinoxalines [48]: A solution of the respective Grignard reagent (0.018 mmol) in Et O was
added dropwise to a stirring suspension of (PPh P) NiCl (30 mol%, 0.027 mmol) in dry benzene
5
2
3
2
2
(3 mL) in an argon atmosphere, and the mixture was refluxed for 15 min. After the catalyst
reduction, the Grignard reagent (PhMgBr or 4-MeOC H MgBr; 0.16 mmol) and a solution of 2-
6
4
methylthio-3-phenylquinoxaline 4a (0.089 mmol) in dry benzene (2 mL) were added to the
reaction mixture and refluxed for 12 h. It was then cooled, poured into a saturated solution of
4 3
NH Cl (5 mL), and extracted with CHCl (3 × 50 mL). The organic layer was dried over
anhydrous Na SO and evaporated to give the crude products 5a or 5b, which were purified by
2
4
column chromatography using hexane:EtOAc (19:1) as the eluent.
1
2
,3-diphenyl-7-methoxyquinoxaline (5a) [57]: 69% yield. H NMR (400 MHz, CDCl ) δ: 8.05
3
(d, J = 9.16 Hz, 1H), 7.53 – 7.47 (m, 5H), 7.42 (dd, J = 9.16 , 2.85 Hz, 1H), 7.35-7.29 (m, 6H),
1
3
3
1
.99 (s, 3H). C NMR (100 MHz, CDCl
3
) δ: 160.91, 152.17, 150.95, 139.24, 137.41, 130.16,
+
29.80, 128.65, 128.44, 128.21, 123.32, 106.52, 55.84. MS (m/z): 312 (M , 100), 297 (25), 269
(23), 209 (10), 156 (20), 134 (30), 106 (57), 63 (64).
1
3
-phenyl-7-methoxy-2-(4-methoxyphenyl)quinoxaline (5b) [48]: 31% yield. H NMR (400
3
MHz, CDCl ) δ: 8.03 (d, J = 9.28 Hz, 1H), 7.52 – 7.33 (m, 9H), 6.86 – 6.84 (m, 1H), 3.98 (s,