3-trifluoromethylquinoxaline N, N′-dioxides as anti-Trypanosomatid agents. Identification of optimal anti- T. cruzi agents and mechanism of action studies

Article abstract of DOI:10.1021/jm2002469

For a fourth approach of quinoxaline N,N′-dioxides as anti-trypanosomatid agents against T. cruzi and Leishmania, we found extremely active derivatives. The present study allows us to state the correct requirements for obtaining optimal in vitro anti-T. cruzi activity. Derivatives possessing electron-withdrawing substituents in the 2-, 3-, 6-, and 7-positions were the most active compounds. With regard to these features and taking into account their mammal cytotoxicity, some trifluoromethylquinoxaline N,N′-dioxides have been proposed as candidates for further clinical studies. Consequently, mutagenicity and in vivo analyses were performed with the most promising derivatives. In addition, with regard to the mechanism of action studies, it was demonstrated that mitochondrial dehydrogenases are involved in the anti-T. cruzi activity of the most active derivatives.

Full text of DOI:10.1021/jm2002469

ARTICLE
pubs.acs.org/jmc
3-Trifluoromethylquinoxaline N,N0-Dioxides as Anti-Trypanosomatid
Agents. Identification of Optimal Anti-T. cruzi Agents and
Mechanism of Action Studies
Diego Benitez,† Mauricio Cabrera,† Paola Hernꢀandez,† Lucía Boiani,† María L. Lavaggi,† Rossanna Di Maio,†
Gloria Yaluff,‡ Elva Serna,‡ Susana Torres,‡ María E. Ferreira,‡ Ninfa Vera de Bilbao,‡ Enrique Torres,§
Silvia Pꢀerez-Silanes,§ Beatriz Solano,§ Elsa Moreno,§ Ignacio Aldana,§ Adela Lꢀopez de Cerꢀain,§
Hugo Cerecetto,† Mercedes Gonzꢀalez,*,† and Antonio Monge*,§
†Grupo de Química Medicinal, Laboratorio de Química Orgꢀanica, Facultad de Ciencias-Facultad de Química,
Universidad de la Repꢀublica, 11400 Montevideo, Uruguay
‡Departamento de Medicina Tropical, Instituto de Ciencias de la Salud, Universidad Nacional de Asunciꢀon, Paraguay
§Unidad de I þ D de Medicamentos, CIFA, University of Navarra, 31080 Pamplona, Spain
ABSTRACT: For a fourth approach of quinoxaline N,N0-
dioxides as anti-trypanosomatid agents against T. cruzi and
Leishmania, we found extremely active derivatives. The present
study allows us to state the correct requirements for obtaining
optimal in vitro anti-T. cruzi activity. Derivatives possessing
electron-withdrawing substituents in the 2-, 3-, 6-, and 7-posi-
tions were the most active compounds. With regard to these
features and taking into account their mammal cytotoxicity,
some trifluoromethylquinoxaline N,N0-dioxides have been pro-
posed as candidates for further clinical studies. Consequently,
mutagenicity and in vivo analyses were performed with the most
promising derivatives. In addition, with regard to the mechan-
ism of action studies, it was demonstrated that mitochondrial
dehydrogenases are involved in the anti-T. cruzi activity of the most active derivatives.
’ INTRODUCTION
However, new therapeutic alternatives should be found because
this drug is creating serious problems of resistance.3bꢀd
Parasitic diseases affect hundreds of millions of people
throughout the world, mainly in developing countries. Since
parasitic protozoa are eukaryotic, they share many common
features with their mammalian host, making the development
of effective and selective drugs a hard task. Diseases caused by
Trypanosomatidae, which share similar characteristics regarding
drug treatment, include Chagas’ disease (Trypanosoma cruzi) and
leishmaniasis (Leishmania spp.).1 These trypanosomatids alone
are responsible for an infected population of nearly 30 million,
and more than 400 million persons are at risk. The drugs
currently used in the treatment of Chagas’ disease are two
nitroaromatic heterocyles, nifurtimox (Nfx) and benznidazole
(Bnz), introduced empirically over 3 decades ago.2 Both drugs
are active in the acute phase of the disease, but efficacy is very low
in the established chronic phase. In addition, differences in drug
susceptibility among different T. cruzi strains lead to a variety of
parasitological cure rates depending upon the geographical area.
The drugs of choice for the treatment of leishmaniasis are
sodium stibogluconate, meglumine antimoniate, pentamidine,
and liposomal amphotericin B, but they sometimes meet with
failure.3 WHO/TDR is currently developing a research program
with miltefosine (Mtf), a very promising leishmanicidal drug.
The capability of the quinoxaline N,N0-dioxide system to act as
anti-infective agents toward a great number of microorganisms4
led us to evaluate some selected derivatives as anti-T. cruzi agents
from our quinoxaline library.5 This study allowed us to identify
excellent in vitro anti-T. cruzi agents against Tulahuen 2 strain
and CL Brener clone (derivatives 1 and 2, Figure 1). New
quinoxaline N,N0-dioxide derivatives were selected to analyze
some structural changes in parent compounds 1 and 2, and they
were biologically analyzed against different T. cruzi strains and
against Leishmania protozoa. In particular, we selected quinoxa-
line N,N0-dioxides in which substituents in positions 2, 3, 6, 7 of
parent compounds 1 and 2 were then modified (Figure 1). In
addition, some deoxygenated quinoxaline analogues were stu-
died to establish the importance of the N-oxide moiety. The
unspecific toxicity against mammalian cells was studied in order
to evaluate the quinoxaline selectivity to the parasites. In addi-
tion, in order to better understand the anti-T. cruzi-mechanism of
action, the changes in the parasite-excreted metabolites and the
Received: March 2, 2011
Published: April 20, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Biological Characterization. In Vitro Anti-T. cruzi Activity. The
new quinoxaline derivatives were initially tested in vitro against the
epimastigote form of T. cruzi, Tulahuen 2 strain. The existence of the
epimastigote form of T. cruzi as an obligate mammalian intracellular
stage has been reviewed and confirmed.7 The compounds were incor-
porated into the biological media at 25 μM, and their ability to inhibit
growth of the parasite was evaluated in comparison to that of the
control (no drug added to the media) on day 5. Nfx was used as the
trypanosomicidal reference drug. The percentage of growth inhibition
(PGI) was calculated as indicated in the Experimental Section (Table 1).
In addition, the ID50 concentrations (50% inhibitory dose) were
assessed for the most active derivatives and Nfx (Table 2). The parent
compound 2, derivatives 10, 21, 22, and 28, with a high anti-T. cruzi
activity against the Tulahuen 2 strain, and the less active derivatives 40,
and 41 were selected to study against the CL Brener clone and the in vivo
Nfx and Bnz partially resistant strains, Y and Colombiana (Table 3).8 In
these assays, viability of T. cruzi was colorimetrically assessed using MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide).9 For
each derivative, the percentage of cytotoxicity (PCyt) was initially
determined at 25 μM, as indicated in the Experimental Section, and
then the ID50 concentration was calculated in a dose-response assay,
between 1.0 and 50.0 μM (Table 3).
Figure 1. Quinoxaline N,N0-dioxide derivatives previously described as
T. cruzi growth inhibitors.5a
effects on the mitochondrion dehydrogenase activity promoted
by quinoxaline N,N0-dioxide derivatives were studied. The most
promising quinoxalines were studied for their mutagenicities and
in vivo activity on an acute murine model of Chagas disease.
’ METHODS AND RESULTS
Additionally, quinoxaline derivatives 21 and 44 were tested in vitro
against the bloodstream trypomastigote form of T. cruzi, CL Brener
clone. The compounds were incorporated into the biological media at
250 μg/mL and their ability to inhibit growth of the parasite was
evaluated, by microscopy, in comparison to that of the control (no drug
added to the media) after 24 h of incubation. Gentian violet (GV) was
used as the trypanosomicidal reference drug (Table 4).
In Vitro Leishmanicidal Activity. We also selected derivatives 2, 10,
21, 22, 28, 40, and 41 to assess the leishmanicidal activity. They were
tested in vitro against promastigote form of Leishmania braziliensis
(MHOM/BR/00/LTB300) strain. Viability of parasite was assessed
colorimetrically using the MTT assay.10 For each derivative, the
percentage of cytotoxicity was initially determined at 25 μM, as indicated
in the Experimental Section, and then the ID50 concentration
was calculated in a dose-response assay, between 1.0 and 50.0 μM
(Table 3). Mtf was used as the leishmanicidal reference drug.
Selected Compounds and Synthesis. Ten families of quinoxa-
line N,N0-dioxides were selected in order to analyze their in vitro anti-T.
cruzi activity (Table 1). Derivatives 3ꢀ8, belonging to the first family,
were included as derivatives in which modifications in position 2 of the
parent compounds 1 and 2 were then carried out. In this case, we
included moieties with different volumes, electronic behaviors, and
polarities. In the second family, made up of derivatives 9ꢀ15, we
maintained substitutions in positions 2 and 3 of parent compound 1
or active derivative 5, modifying the 6- and/or 7-benzo substitutions of
quinoxaline ring.
The third group of derivatives (16ꢀ19) involved modifications in
positions 6 and 7 together with ꢀCOꢀ or ꢀSꢀ substitution (ꢀXꢀ in
parent compounds 1 and 2, Figure 1) by a ꢀCH2ꢀ group. In the fourth
family, the 3-CH3 substituent, in parent compound 1, was changed by
the ꢀCF3 moiety, yielding derivative 20, and when 6- and 7-substituents
were concomitantly modified, the resulting derivatives were 21ꢀ26.
Other modifications involved the inclusion of a 3-Ph group as volumi-
nous moiety in this position, a nonvoluminous electrophilic moiety in
position 2, ꢀCO2Et group, and different electronic behavior substitu-
tions in the 6- and/or 7-benzo cycle (derivatives 27ꢀ33). In the sixth
and seventh families of compounds we modified derivatives 3ꢀ8 in the
benzo substitutions, yielding 6,7-dimethyl derivatives (34ꢀ39) and
benzo-nonsubstituted analogues (40ꢀ43). The ꢀCF3 substitution in
the 3-position, i.e., derivatives 20ꢀ26, resulted in very active com-
pounds. From here we designed the eighth group of studied compounds
(44ꢀ64), maintaining the ꢀCF3 at the 3-position, with modifications at
2-, 6-, and/or 7-positions. In the same way, the ninth group included
derivatives with ꢀCHF2 substitution in position 3 (derivatives 65ꢀ70).
Finally, the last group made up of quinoxalines 71 and 72, deoxygenated
analogues of 22 and 51, respectively, was included in our study to
investigate the relevance of the N-oxide moieties in the anti-T. cruzi
activities.
In Vitro Toxicity Studies. To explore the potential of these quinoxaline
N,N0-dioxides as drugs, we performed two different studies. First, we
evaluated their in vitro unspecific mammal cytotoxicity, using J-774
mouse macrophages. Second, we studied the mutagenicity capacity
using the Ames test.
In Vitro Unspecific Mammal Cytotoxicity. The 3-trifluoromethylqui-
noxaline N,N0-dioxides were selected according to their anti-T. cruzi and
leishmanicidal activity and trying to cover a wide range of structural
characteristics. The ID50 values for the studied compounds are shown in
Table 5. The selectivity indexes, SI, were expressed as the ratio of ID50 in
macrophages to ID50 in T. cruzi (Tulahuen 2 strain, Table 2).11
Mutagenicity Assay. The method of direct incubation in plate12 using
culture of Salmonella typhimurium TA98 strain was performed on
derivatives 21 and 44 and its 3-methyl analogue, 9, and Nfx. The
influence of metabolic activation was tested by adding S9 fraction of
mouse liver. Positive controls of 4-nitro-o-phenylendiamine and
2-aminofluorene were run in parallel. The revertant number was
manually counted and compared to the natural revertant (Table 6).
The compound is considered mutagenic when the number of
revertant colonies is at least 2-fold of the spontaneous revertant
frequencies for at least two consecutive dose levels.13 The maximum
assayed doses were determined according to the toxic effect on S.
typhimurium.
Most of the studied compounds were prepared following previously
reported5,6 synthetic procedures, and the new compounds 55, 56, and
63ꢀ70 were prepared using the Beirut expansion process as shown in
Scheme 1. Derivatives asymmetrically substituted in the benzo cycle
were obtained as a mixture of inseparable 6- and 7-isomers which were
evaluated without further separation. For simplicity, Table 1 only shows
one isomer. All the compounds were characterized by 1H NMR and IR.
The purity was established by TLC and microanalysis. Only compounds
with analytical results for C, H, and N within (0.4 of the theoretical
values were considered pure enough.
Studying the Mechanism of Action. It is well-known that some
quinoxaline N,N0-dioxides are species that suffer a bioreductive process
in hypoxic conditions to produce •OH and quinoxaline.14 This led us to
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Journal of Medicinal Chemistry
ARTICLE
Table 1. T. cruzi Antiproliferative Activity of Quinoxaline N,N0-Dioxide and Related Derivatives
compd
-R2
-R3
-R6
-Cl
-R7
-Cl
PGIa(%)
compd
-R2
-R3
-R6
-R7
PGIa(%)
1
-COPh
-SPh
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-Ph
91.0
93.0
66.0
61.0
60.0
49.0
32.0
28.0
100.0
92.0
33.0
19.0
9.0
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71c
72c
Nfx
-CO2CH2CH3
-CONH-t-Bu
-CO2CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CF3
-CHF2
-CHF2
-CHF2
-CHF2
-CHF2
-CHF2
-CF3
-CF3
-CH3
-CH3
-H
-CH3
-CH3
-H
-H
-H
-H
-F
1.0
0.0
2
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-F
3
-CO2CH3
-CONHPh
-COCH3
-Cl
24.0
4
-Cl
-CONHPh
-COCH3
-H
10.0
5
-Cl
-H
29.0
6
-CONHPh-o-CH3
-CO2CH2CH3
-CONH-t-Bu
-COCH3
-Cl
-CONHPh-o-CH3
-COCH3
-H
21.0
7
-Cl
-F
100.0
100.0
100.0
100.0
100.0
23.6
8
-Cl
-COCH2CH3
-COCH2CH3
-CO-i-Pr
-F
-F
9
-F
-Cl
-Cl
-H
-Cl
-Cl
-H
-F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
-COPh
-F
-F
-COPh
-Cl
-H
-CO-i-Pr
-COPh
-H
-H
-CO-i-Pr
-F
-COPh
-OCH3
-CH3
-CH3
-Cl
-H
-CO-t-Bu
-F
-F
100.0
26.0
-COPh
-H
8.0
-CO-t-Bu
-H
-H
-H
-H
-F
-COPh
-CH3
-Cl
-H
3.0
-COCH3
-H
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
50.8
-CH2Ph
24.0
0.0
-CO-1-naphthyl
-CO-1-naphthyl
-COPh-p-Cl
-COPh-p-Cl
-CO-2-furyl
-CO-2-furyl
-CO-2-furyl
-CO-2-furyl
-CO-2-thienyl
-CO-2-thienyl
-CO(4-Ph-Pi)b
-CO[4-(Ph-p-Cl)-Pi]
-COPh
-H
-CH2Ph
-Cl
-F
-CH2Ph
-H
-H
0.0
-F
-F
-CH2Ph
-CH3
-Cl
-CH3
-Cl
-F
0.0
-H
-H
-H
-F
-COPh
100.0
100.0
100.0
99.0
90.0
98.0
72.0
100.0
99.0
88.0
29.0
13.0
0.0
-H
-COPh
-F
-F
-COPh
-H
-H
-CF3
-H
-H
-CF3
-F
-COPh
-CH3
-Cl
-H
-COPh
-H
-F
-COPh
-OCH3
-CH3
-F
-H
-H
-H
-H
-H
-Cl
-H
-H
-H
-H
-H
-H
-H
-COPh
-CH3
-F
-H
-CO2CH2CH3
-CO2CH2CH3
-CO2CH2CH3
-CO2CH2CH3
-CO2CH2CH3
-CO2CH2CH3
-CO2CH2CH3
-CO2CH3
-CONHPh
-COCH3
-H
-Ph
-Cl
-Cl
-H
-Cl
-Cl
-F
-Ph
-Cl
-COPh
-Ph
-H
-H
-COPh
-Ph
-CH3
-OCH3
-CH3
-CH3
-CH3
-CH3
-CH3
-H
-COPh
-OCH3
-CH3
-H
-Ph
-H
-COPh
-Ph
-CH3
-CH3
-CH3
-CH3
-CH3
0.0
-COPh
35.8
-CH3
CH3
CH3
CH3
10.0
16.0
2.0
-COPh
-H
49.0
-CO-t-Bu-
-H
43.0
100.0
-CONHPh-o-CH3
12.0
a Percentage of growth inhibition. Inhibition of epimastigote growth of Tulahuen 2 strain, dose = 25 μM. The results are the mean of three independent
experiments with a SD less than 10% in all cases. b Pi = 1-piperazinyl. c Deoxygenated derivatives.
believe that these quinoxalines could produce parasitic damage through
the production of radical species affecting the redox metabolism.
Previously, we have studied a possible mechanism of action of
N-oxide containing heterocycles from a theoretical point of view.
We found that a bioreductive process could be involved.5a,c,15 In an
attempt to investigate the mode in which these quinoxaline N,
N0-dioxides act on parasites, we studied their effect on the mito-
chondrial dehydrogenase activities. We recently demonstrated that
these enzymes could be involved in the mechanism of action of
N-oxides containing heterocycles, such as furoxans and benzofuro-
xans.16 The percentage of mitochondrial dehydrogenase activities
(Pmdh) with respect to untreated control was assessed using the
colorimetric MTT assay performed at very short times, no more than
240 min of incubation, a procedure described for Leishmania
parasite.17 We compared the changes of the mitochondrial dehy-
drogenase activities of four active agents, parent compound 2 and
the three 3-trifluoromethyl derivatives 21, 22, and 44, together with
the reference drug Nfx (Figure 2a). Nfx does not affect the
mitochondrial dehydrogenase activities, while the quinoxalines
produce decrease in a time-dependent manner.
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthetic Procedures Used To Prepare the New Derivatives
Table 2. ID50 for Relevant Quinoxaline N,N0-Dioxides and
for Parent Compounds in Tulahuen 2 Strain
Table 3. Percentage of Cytotoxicity and ID50 for Relevant
Quinoxaline N,N0-Dioxides and for Parent Compound 2
against Different T. cruzi Strains and L. brazilensis LTB300
Strain
compd
ID50 (μM)a
compd
ID50 (μM)a
1
11800b
6500b
18400
400
48
50
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
>2500
4900
3000
1400
3900
2500
1600
2400
2500
5000
4800
2800
1100
760
ID50
(μM)b,d
2
PCyt (%)a,b/ID50 (μM)
5
9
CL
10
20
21
22
23
24
25
26
27
28
29
44
45
46
47
Nfx
4200
2400
700
Brener
clone
Colombiana
LTB300
Strain
compd
Y strain
strain
compd
2
98.0/<1.0 100.0/2.9
92.0/16.2 89.0/18.3
100.0/1.7
98.0/4.5
100.0
2
1.4
20.4
1.3
900
10
21
22
28
40
41
Nfxc
10
21
22
28
40
41
Mtf
1300
3000
3500
1400
3300
11300
12500
390
96.0/1.1
97.0/3.1
100.0/1.8
100.0/4.0
100.0
1.0
94.0/16.4 100.0/7.1
100.0
3.0
59.0
0.0
60.0
>50.0
>50.0
9.0
32.0
0.0
24.0
90.0/4.9
80.0/9.7
87.0/3.4
790
a Percentage of cytotoxicity. Dose = 25 μM. b The results are the means
of three independent experiments with a SD less than 10% in all cases.
c At 10 μM. d The results are the means of three independent experi-
ments with a SD less than 10% in all cases.
4700
6400
6300
10000
25000
500
780
1400
7700
of action elucidation.16a,18 We compared the spectra of the cell-free
medium of quinoxaline-treated parasites with those of the untreated
T. cruzi-free medium as control. We have mainly focused on the changes
of the excreted salts of the carboxylic acids lactate (Lac), acetate (Ace),
pyruvate (Pyr), and succinate (Suc) and the amino acids alanine (Ala)
and glycine (Gly), being the most relevant modified metabolites.
Figure 2b shows the changes in the excreted end-products without or
after treatment with the studied compounds, quinoxalines, with marked
a The results are the mean of three independent experiments with a SD
less than 10% in all cases. b From ref 5a.
Finally, in order to study the changes in the biochemical pathways
promoted by two of our active quinoxalines, we have studied the
1
modifications in the excreted metabolites by H NMR spectroscopy.
This type of studies has been proven to be a useful tool in the mechanism
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Journal of Medicinal Chemistry
ARTICLE
Table 4. In Vitro Trypanosomicidal Activity (against
Trypomastigote Form of CL Brener Clone)
Table 5. Biological Characterization of Quinoxaline
N,N0-Dioxides against Mammal Macrophages
compd
% lysisa
21
84 (63)b
80
44
GV
100
a % lysis = percentage of parasite lysis at 250 μg/mL compound. Results
are the mean of three different experiments with a SD less than 10% in
all cases. b Value in parentheses corresponds to the inhibition using 100
μg/mL compound.
compd
-R
-R6 -R7 ID50 (nM)a
SIb
21
44
49
50
51
52
53
56
57
62
63
64
Nfx
-Ph
-F
-F
8000
10600
11450
16250
8700
11.4
27.2
<0.5
<0.6
<0.3
2.8
-CH3
-F
-F
-i-Pr
-F
-F
effects on the mitochondrial dehydrogenase since the beginning of the
incubation, i.e., 2 and 21.
-t-Bu
-F
-F
-t-Bu
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
Preliminary in Vivo Anti-T. cruzi Studies. We evaluated
derivatives 21 and 44 in vivo in murine models of acute Chagas’ disease.
Bnz was used as the in vivo active reference drug. In one of the
preliminary studies (experiment 1), male Swiss albino mice were
infected with CL Brener trypomastigotes and treatment began 10 days
after infection with oral administration of each compound (10 or 50
(mg/kg bw)/day for 21 and 50 (mg/kg bw)/day for Bnz) during 10
days.20 In the second experiment (experiment 2), the only differences
were that the animals were infected with Y-trypomastigotes, compound
21 was administered at 10 or 30 (mg/kg bw)/day, and derivative 44 was
included in the study at 10 (mg/kg bw)/day. In the third experiment
(experiment 3), male BALB/c mice infected with CL Brener clone were
used and derivatives 21 (10 (mg/kg bw)/day) and 44 (10 (mg/kg bw)/
day) or Bnz (50 (mg/kg bw)/day) was administered orally. Three
different parameters were used to evaluate the in vivo activity, weekly
parasitemia (Figure 3), animal survival percentages (Figure 4a), and
anti-T. cruzi antibody levels at 30 and 60 or 90 days after infection
(Figure 4b).
-CH3
8550
-1-naphthyl
-Ph-p-Cl
-2-furyl
-2-thienyl
7920
5.5
8500
5.3
7130
3.0
4690
4.3
-4-Ph-1-piperazinyl
9510
12.5
10.4
41.0
-4-(Ph-p-Cl)-1-piperazinyl
8250
316000
a The results are the mean of three independent experiments with
a SD less than 10% in all cases. b SI: selectivity index, ID50,macrophage
ID50,T.cruzi(Tulahuen 2)
/
.
electron-withdrawing substituent in position 2, were completely
inactive at the assayed doses (Table 1), showing the significance
of this exigency at position 2 (compare activities of parent
compound 1 and derivative 11 to activities of derivatives 16,
and 17, respectively; Table 1). However, better electron-with-
drawing substituents in positions 6 and 7 (i.e., 6,7-difluoro in
derivative 10, Figure 5), increased the activity. Clearly, in some
cases the electronic character of 6- and 7-substituents also
improve the activities of the quinoxaline N,N0-dioxides (i.e.,
compare activity of parent compound 1 to activities of derivatives
10ꢀ15, Table 1). When a new electron-withdrawing substituent
was included, the biological behavior was better (compare 65 vs
1, 21 vs 65, or 21 vs 10). Finally, in the 3-CF3-substituted
derivatives 20ꢀ26, no relevant effects of the electronic character
of 6- and 7-substituents were observed. However, the lack of
N-oxide moieties, i.e., derivative 71, produced less active derivatives.
Other electrophilic centers different from ꢀCOPh, i.e., ꢀCOCH3,
ꢀCOCH2CH3, ꢀCO-i-Pr, ꢀCO-t-Bu, ꢀCO-1-naphthyl, ꢀCOPh-
p-Cl, ꢀCO-2-furyl, ꢀCO-2-thienyl, and ꢀCONRR0 (derivatives 5,
9, 44ꢀ48, 50, and 52ꢀ68), also produced good active derivatives.
The optimal activity was found in derivative 44 (Figure 5),
which contained all the structural exigencies observed for the rest
of the derivatives. Compound 44, the best anti-Tulahuen 2 of the
quinoxaline N,N0-dioxides studied, has a good electrophilic
center in position 2 and three excellent electron-withdrawing
moieties in positions 3, 6, and 7.
’ DISCUSSION
We reported the in vitro biological activity of nearly 70
quinoxaline derivatives against the epimastigote form of three
different strains and one clone of T. cruzi and the promasti-
gote form of one strain of L. braziliensis. Derivatives 9, 10,
20ꢀ25, 27ꢀ29, 44ꢀ48, 50, and 52ꢀ68 were the most active
ones against Tulahuen 2 strain (Table 1) with 9, 21, 22,
44ꢀ46, and 62ꢀ64 being at least 10 times more active than
the reference drug and the parent compounds, Nfx and 1 and
2, respectively (Table 2). The activity profiles against the
other studied clone and strains, CL Brener, Y, and Colombi-
ana, were similar to Tulahuen 2 activities, identifying deriva-
tives 21 and 22 with bioresponses similar to or higher than
those of the reference drug, Nfx, and parent compound 2
(Table 3).
Parent compound 2 and the new studied quinoxalines 21,
22, and 28 showed excellent in vitro activities against Nfx and
Bnz partially resistant strains, Y and Colombiana strains. The
same profiles were observed in the study with L. brasiliensis,
LTB300 strain, with compounds 2, 21, 22, and 28 being at
least 3 times more active than the reference drug Mtf
(Table 3). Concomitantly, derivatives 21 and 44 displayed
significant activity against the circulating form of T. cruzi,
trypomastigote form (Table 4).
Along with these findings, derivatives 21 and 44 were selective
against the parasite (Table 5), with the methylketone 44 having
the best selectivity index (macrophage/T. cruzi). Additionally,
these compounds were not mutagenic in the Ames test (Table 6),
making them excellent leads to further studies. Surprisingly, the
3-methyl analogue of compound 44, derivative 9, was mutagenic
in this test, showing the significance of the 3-trifluoromethyl
substituent in this property.
Optimal anti-T. cruzi quinoxaline N,N0-dioxides were identi-
fied from a structural point of view (Figure 5). Electrophilic
character changes in position 2 of parent compound 1 (Figure 1),
producing compounds with unmodifiable or decreasing
activity (derivatives 5 and 16). Derivatives 16ꢀ19, without an
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Table 6. Number of Revertants of Derivatives 21, 44, 9, and Nfx on TA98 S. typhimurium Strain
21
44
9
Nfx
Da
NRb,c
Md
Da
NRb,c
Md
Da
NRb,c
Md
Da
NRb,c
Md
ꢀS9
ꢀS9
ꢀS9
ꢀS9
ꢀS9
ꢀS9
ꢀS9
þS9
þS9
þS9
þS9
þS9
þS9
þS9
0.0
14 ( 6
10 ( 6
22 ( 8
22 ( 1
20 ( 4
27 ( 4
35 ( 14
19 ( 4
18 ( 1
20 ( 4
15 ( 1
20 ( 2
20 ( 4
21 ( 4
0.0
12 ( 1
9 ( 1
0.0
0.2
13 ( 3
0.0
0.5
21 ( 4
0.005
0.05
0.5
0.05
0.15
0.44
1.3
29 ( 7
29 ( 6
9 ( 1
0.6
97 ( 10
245 ( 10
348 ( 10
270 ( 4
1.0
43 ( 17
62 ( 2
14 ( 3
9 ( 1
1.7
(þ)e
3.0
1.5
5.0
10.0
30.0
144 ( 11
117 ( 17
(þ)e
5.0
4.0
18 ( 5
15.0
15.0
0.0
0.0
9 ( 2
0.0
0.2
12 ( 1
21 ( 3
34 ( 1
97 ( 2
178 ( 5
236 ( 14
0.0
0.5
31 ( 10
37 ( 5
0.005
0.05
0.5
0.05
0.15
0.44
1.3
12 ( 3
14 ( 1
14 ( 1
13 ( 3
15 ( 3
0.6
1.0
39 ( 18
53 ( 9
1.7
(þ)e
3.0
1.5
5.0
10.0
30.0
64 ( 6
5.0
4.0
15.0
139 ( 11
(þ)e
15.0
4-NPDf
AFg
D a
NRb,c
1223 ( 237
D a
NRb,c
-S9
20.0
þS9
10.0
801 ( 82
a D: dose in μg/plate. b NR: number of revertants. c The results are the mean of two independent experiments. d M: mutagenicity, according to ref 13 (see
text). e (þ): Response is considered positive because it is the second dose in which the revertant levels are at least twice the spontaneous frequencies. f 4-
NPD: 4-nitro-o-phenylendiamine. g AF: 2-aminofluorene.
With regard to the mechanism of action studies, the studied
quinoxalines, parent compounds 2 and 21, 22, 44, decreased
mitochondrial dehydrogenases activity, unlike what occurs with
the untreated or Nfx-treated parasite (Figure 2a). In general,
parent compound 2 promoted greater diminishing of the studied
end-metabolite concentrations than quinoxaline 21-treated para-
sites. The lower levels of excreted Ace and Suc in the case of
parasites treated with compound 2 compared to the excreted
levels obtained with compound 21 could be due to the fact that 2
is a better mitochondrial dehydrogenase inhibitor (Figure 2a).
The following three facts prove this statement:21 (1) Mitochon-
drial Suc dehydrogenase and fumarate reductase are very
homologous enzyme complexes, so most inhibitors of Suc
dehydrogenase could also act on fumarate reductase. (2) The
T. cruzi form with an active Krebs cycle, such as epimastigotes,
produce Suc, implying that fumarate reduction has to occur at the
same time as Suc oxidation. (3) Ace is the end-product of Pyr
oxidative decarboxylation by mitochondrial Pyr dehydrogenase.
The dehydrogenase inhibition does not correlate with the
potency of the studied compounds (compare activity of the less
potent parent compound 1 to activities of the most potent
derivatives 21, 22, and 44; Table 2), showing that other
phenomena are also involved in the mode of action of this family
of compound.
The in vivo analyzed 3-trifluoromethylquinoxaline dioxide
derivatives 21 and 44 displayed relevant behavior. In the three
experiments, the animals treated with derivative 21 showed the
best survival rates at the end of the experiment. For CL Brener
experiments, the blood-study findings showed that compound
21, at a dose 5 times lower than that used for Bnz, exhibited a
particular biological profile, shifting the parasitemia maximum
10 days with respect to the control and lacking the second
Figure 2. (a) Variation of the percentage of mitochondrial dehydro-
genase activities (Pmdh), produced by the compounds with respect to
time compared to the untreated control of T. cruzi epimastigote, Y strain
(for details see Experimental Section). (b) Percentage of the end-
products excreted to the medium in the different treatment, expressed
respect to DMF,19 by T. cruzi epimastigote, Y strain (for details see
Experimental Section).
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animals was lower than the parasite number in untreated mice.
According to experiment 3, treatment with derivative 44 exhib-
ited a better parasitemia profile than derivative 21 (Figure 3c).
These results were in agreement with the values of anti-T. cruzi
antibodies where derivative 44 showed lower findings than 21, at
30 and 90 days after infection (Figure 4b). For Y experiments
(experiment 2, Figure 3b), the blood-study findings showed that
the number of trypomastigotes in 21-treated animals was lower
than the parasite number in untreated mice for both doses, in a
nondose dependence, while 44-treated animals did not improve
the parasitemia profile. These results were completely in agree-
ment with the animals’ survival percentages in the Y-infected
mice (Figure 4a). On the other hand, both derivatives 21 and 44
avoided the second parasitemia maximum, evidenced in Bnz-treated
animals at day 55 of the assay. No significant differences were
observed regarding the antibody levels, at day 90 after infection,
between the four studied groups (quinoxalines and Bnz); however,
the antibody levels for 21, at both doses, and Bnz were lower than
the values for infected animals (upper line in Figure 4b).
These findings promote us further thorough studies, modify-
ing doses, administration routes, and combinations with
other drugs.
’ CONCLUSIONS
We have identified quinoxaline N,N0-dioxides as excellent anti-
trypanosomatid agents. In addition, we were able to establish that
mitochondria are affected when these derivatives are used.
Because of the lack of mutagenic properties of derivatives 21
and 44, the previously described lower acute systemic toxicity in
Wistar rats for 21,22 and the preliminary in vivo anti-trypanoso-
ma results, these compounds could be proposed as drug candi-
dates. Additional and more thorough in vivo studies are currently
being performed on animal models of Chagas disease and
leishmaniasis.
’ EXPERIMENTAL SECTION
Chemistry. All starting materials were purchased from Panreac
Química S.A. (Barcelona, Spain), Sigma-Aldrich Química, S.A.
(Alcobendas, Spain), Acros Organics (Janssen Pharmaceutical, Geel,
Belgium), and Lancaster (Bischheim-Strasbourg, France). All solvents
were dried and distilled prior to use. All the reactions were carried out in
a nitrogen atmosphere. All of the synthesized compounds were chemi-
cally characterized by thin layer chromatography (TLC), infrared (IR),
proton nuclear magnetic resonance (1H NMR), and elemental micro-
analyses (CHN). Alugram SIL G/UV254 (layer: 0.2 mm) (Macherey-
Nagel GmbH & Co. KG., D€uren, Germany) was used for TLC, and silica
gel 60 (0.040ꢀ0.063 mm, Merck) was used for flash column chroma-
tography. The 1H NMR spectra were recorded on a Bruker 400
Ultrashield instrument (400 MHz), using TMS as the internal standard
and with CDCl3-d6 as the solvent. The chemical shifts are reported in
ppm (δ), and coupling constants (J) values are given in hertz (Hz).
Signal multiplicities are represented by s (singlet), d (doublet), dd
(double doblet), t (triplet), tt (triple triplet), and m (multiplet). The IR
spectra were recorded on a Nicolet Nexus FTIR (Thermo, Madison, WI,
U.S.) in KBr pellets. To determine the purity of the compounds,
elemental microanalysis results obtained on a CHN-900 elemental
analyzer (Leco, Tres Cantos, Spain) from vacuum-dried samples were
used to confirm g95% purity. The analytical results for C, H, and N were
within (0.4 of the theoretical values. Compounds 3ꢀ54, 57ꢀ62, 71,
and 72 were prepared following synthetic procedures previously
reported.4,6 Compounds 55, 56, and 63ꢀ70 were prepared as
described below.
Figure 3. Parasitemia of mice treated with 50 (mg/kg bw)/day of Bnz
(O), group treated with 10 (mg/kg bw)/day of 21 (4), group treated
with 30 (mg/kg bw)/day of 21 (3), group treated with 10 (mg/kg bw)/
day of 44 (left-pointing triangle), and control group (9): (a) experiment
1 (CL-Brener clone); (b) experiment 2 (Y strain); (c) experiment 3
(CL-Brener clone).
parasitaemia maximum, evidenced in untreated and Bnz-treated
animals at day 53 of the assay (experiment 1, Figure 3a). On the
analysis days, the number of trypomastigotes on 21-treated
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Figure 4. (a) Animal survival percentages in the different treatment-schedules. (b) Examples of anti-T. cruzi antibody levels in the different treatment-
schedules. Upper lines represent the antibody levels for infected animals, and lower lines represent the antibody levels for healthy animals: (/) p < 0.05;
(//) p < 0.01.
General Procedure for the Synthesis of Compounds 55
and 56. 1-(4-Chlorophenyl)-4,4,4-trifluoromethyl-1,3-butanedione
(4.35 mmol) was added to a solution of the appropriate benzofuroxan
(2.90 mmol) in toluene (20 mL) in a microwave vessel. The mixture was
cooled, and triethylamine was added dropwise (1.5 mL). The solution
was stirred at room temperature for 30 min, and then it was put in the
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Figure 5. Structural-anti-T. cruzi activity for the studied quinoxalines. The ID50 refers to the Tulahuen 2 strain.
microwave reactor. The mixture was then subjected to microwave
irradiation at 70 W for 45 min, keeping the temperature at 70ꢀ80 °C.
After an important conversion as indicated by TLC, the reaction mixture
was cooled and the solvent was eliminated in vacuo. Brown oil was
obtained and purified by column chromatography on silica gel, eluting
with dichloromethane. The corresponding fractions were evaporated to
dryness in vacuo, and the yellow oil obtained was precipitated by adding
diethyl ether and filtered off.
piperazine in the presence of 2-hydroxypyridine as catalyst in a bath at
169 °C for 5 h under nitrogen atmosphere. The partly cooled mixture
was then stirred into hot water. The resulting suspension was extracted
with dichloromethane. The organic solvent was eliminated under
pressure, and a solid was obtained. The corresponding piperazinylamide
without purification (5.7 mmol) was added to a solution of the
appropriate benzofuroxan (2.8 mmol) in toluene (20 mL) in a micro-
wave vessel. The mixture was cooled, and triethylamine was added
dropwise (1.5 mL). The solution was stirred at room temperature for
10 min, and then it was put in the microwave reactor. The mixture was
then subjected to microwave irradiation at 50 W for 25ꢀ35 min, keeping
the temperature at 90 °C. After an important conversion as indicated by
TLC, the reaction mixture was cooled and the solvent was eliminated in
vacuo. A brown oil was obtained, and it was purified by column
chromatography on silica gel, eluting with toluene/dioxane (9:1). The
corresponding fractions evaporated to dryness in vacuo, and the yellow
oil obtained was precipitated by adding diethyl ether and filtered off.
2-(4-Phenylpiperazine-1-carbonyl)-3-(trifluoromethyl)quinoxaline
2-(4-Chlorobenzoyl)-6,7-difluoro-3-(trifluoromethyl)quinoxaline
1,4-dioxide, 55. Yield: 8.6%. 1H NMR (400 MHz, CDCl3-d6) δ ppm:
8.51 (dd, 1H, H8, J8-FC6 = 7.2 Hz, J8-FC7 = 9.1 Hz), 8.39 (dd, 1H, H5,
0
0
0
0
0
0
J5-FC7 = 7.1 Hz, J5-FC6 = 9.0 Hz); 7.83 (d, 2H, H2 þ H6 , J2 -3 = J6 -5 = 8.6
0
0
0
0
0
0
Hz); 7.55 (d, 2H, H3 þ H5 , J3 -2 = J5 -6 = 8.6 Hz). IR (KBr): 3065
(w, νCꢀH Ar), 1698 (s, νCdO), 1353 (s, νN-oxide), 1176 (s, νCꢀF),
910 (w, νArꢀCl) cmꢀ1. Calculated analysis for C16H6ClF5N2O3: C,
47.46; H, 1.48; N, 6.92. Found: C, 47.50; H,1.71; N, 6.72.
2-(4-Chlorobenzoyl)-3-(trifluoromethyl)quinoxaline 1,4-Dioxide, 56.
Yield: 6%. 1H NMR (400 MHz, CDCl3-d6) δ ppm: 8.73ꢀ8.70 (m, 1H,
H8); 8.61ꢀ8.58 (m, 1 H, H5); 8.04ꢀ8.02 (m, 2H, H6 þ H7); 7.86 (d, 2H,
1
1,4-Dioxide, 63. Yield: 7%. H NMR (400 MHz, CDCl3-d6) δ ppm:
0
0
0
0
0
0
0
0
0
0
0
0
H2 þ H6 , J2 -3 = J6 -5 = 8.7 Hz); 7.54 (d, 2H, H3 þ H5 , J3 -2 = J5 -6 = 8.7
Hz). IR (KBr): 3100 (w, νCꢀH Ar), 1689 (s, νCdO), 1351 (s, νN-oxide),
1162 (s, νCꢀF), 909 (w, νArꢀCl) cmꢀ1. Calculated analysis for C16H8ClF3
N2O3: C, 52.10; H, 2.17; N, 7.60. Found: C, 51.78; H, 2.08; N, 7.39.
General Procedure for the Synthesis of Compounds 63
and 64. The intermediates were prepared according to the synthetic
procedure for a similar compound N-(3-oxobutyryl)piperazine.4d
The trifluoromethyl acetoacetate was heated with the corresponding
8.68ꢀ8.66 (m, 1H, H8); 8.64ꢀ8.62 (m, 1H, H5); 8.04ꢀ7.96 (m, 2H, H6 þ
0
0
0
0
0
0
0
0
0
0
H7); 7.32 (dd, 2H, H3 þ H5 , J3 -4 = J5 -4 = 7.1 Hz, J3 -2 = J5 -6 = 8.9
0
0
0
Hz); 6.98ꢀ6.95 (m, 3H, H2 þ H4 þ H6 ); 4.04ꢀ4.00 (m, 2H,
CH2-piperazine); 3.60ꢀ3.15 (m, 6H, 3CH2-piperazine). IR (KBr): 3453
(w, νCꢀH Ar), 1655 (s, νCdO), 1444 (m, νCꢀN amide), 1356
(s, νN-oxide), 1236 (m, νCꢀN Ar-amine), 1153 (m, νCꢀF) cmꢀ1
.
Calculated analysis for C20H17F3N4O3: C, 57.41; H, 4.06; N, 13.39.
Found: C, 57.46; H,4.11; N, 13.36.
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0
0
0
0
0
0
2-[4-(4-Chlorophenyl)piperazine-1-carbonyl]-3-(trifluoromethyl)
quinoxaline 1,4-Dioxide, 64. Yield: 6%. 1H NMR (400 MHz, CDCl3-
d6) δ ppm: 8.63 (dd, 1H, H8, J8ꢀ6 =1.7 Hz, J8ꢀ7 = 8.1 Hz); 8.68 (dd, 1H,
H5, J5ꢀ7 = 1.4 Hz, J5ꢀ6 = 8.1 Hz); 8.04ꢀ7.97 (m, 2H, H6 þ H7); 7.26 (d,
J6 -4 = 1.2 Hz, J2 -3 = J6 -5 = 8.3 Hz); 7.81 (dd, 1H, H6, J6ꢀ8 = 1.46 Hz,
0
0
0
0
0
0
0
0
0
J6ꢀ5 = 8.8 Hz); 7.68 (dt, 1H, H4 , J4 -2 = J4 -6 = 1.2 Hz, J4 -3 = J4 -5 = 7.2
0
0
0
0
0
0
0
0
Hz); 7.54 (t, 2H, H3 þ H5 , J3 -2 = J5 -6 = 7.8 Hz, J3 -4 = 7.4 Hz); 7.35
(t, 1H, CHF2, JHꢀF = 52.4 Hz); 2.67 (s, 3H, CH3-C7). IR (KBr): 3064
(w, νCꢀH Ar), 1683 (s, νCdO), 1340 (s, νN-oxide), 1044 (m, νCꢀF)
cmꢀ1. Calculated analysis for C17H12F2N2O3: C, 61.81; H, 3.63; N, 8.48.
Found: C, 61.65; H, 4.00; N, 8.08.
0
0
0
0
0
0
0
0
0
0
2H, H2 þ H6 , J2 -3 = J6 -5 = 8.6 Hz); 6.88 (d, 2H, H3 þ H5 , J3 -2
=
0
0
J5 -6 = 8.6 Hz); 4.09ꢀ3.12 (m, 8H, 4 CH2-piperazine). IR (KBr): 3440 (w,
νCꢀH Ar), 1654 (s, νCdO), 1498 (m, νCꢀN amida), 1357 (s, νN-
oxide), 1236 (m, νCꢀN Ar-amine), 1157(m, νCꢀF) cmꢀ1. Calculated
analysis for C20H16ClF3N4O3: C, 53.05; H, 3.53; N, 12.37. Found: C,
52.90; H,3.4; N, 12.22.
2-Benzoyl-3-(difluoromethyl)quinoxaline 1,4-Dioxide, 70. Yield:
1
9%. H NMR (400 MHz, CDCl3-d6) δ ppm: 8.70 (m, 1H, H8); 8.62
0
0
0
0
0
(m, 1H, H5); 8.00 (m, 2H, H6 þ H7); 7.92 (dd, 2H, H2 þ H6 , J2 -4 = J6 -
0
0
0
0
0
0
0
0
40
= 1.2 Hz, J2 -3 = J6 -5 = 8.4 Hz); 7.69 (dt, 1H, H4 , J4 -2 = 1.2 Hz, J4 -3
=
General Procedure for the Synthesis of Compounds
65ꢀ70. 1-Phenyl-4,4-difluoro-1,3-butanedione (5.05 mmol) was
added to a solution of the appropriate benzofuroxan (5.05 mmol) and
KF (in alumina 40% supported) in acetone (40 mL). The mixture was
stirred at room temperature in darkness for 14ꢀ24 h. The mixture was
filtered to eliminate the KF in alumina. The organic phase was extracted
with dichloromethane/water. The organic phase was dried with anhy-
drous Na2SO4 and filtered. The solvent was removed in vacuo, and the
resulting oil was precipitated by adding n-hexane (20 mL) and methanol
(5 mL). The obtained yellow precipitate was washed with diethyl ether.
2-Benzoyl-3-(difluoromethyl)-6,7-dichloroquinoxaline 1,4-Dioxide,
0
0
0
0
0
0
0
0
0
0
J4 -5 = 7.2 Hz); 7.54 (dt, 2H, H3 þ H5 , J3 -5 = 1.5 Hz, J3 -4 = J5 -4 = 7.8
0
0
0
0
Hz, J3 -2 = J5 -6 = 7.7 Hz); 7.36 (t, 1H, CHF2, JHꢀF = 52.3 Hz). IR
(KBr): 3199 (w, νCꢀH Ar), 1638 (s, νCdO), 1340 (s, νN-oxide), 1044
(m, νCꢀF) cmꢀ1. Calculated analysis for C16H10F2N2O3: C, 60.75; H,
3.16; N, 8.86. Found: C,60.75; H, 3.60; N, 8.52.
Biology. Antitrypanosomatid in Vitro Evaluation. Anti-T.
cruzi in Vitro Test Using Tulahuen 2 Strain. Trypanosoma cruzi
epimastigotes (Tulahuen 2 strain) were grown at 28 °C in an axenic
medium (BHIꢀtryptose) as previously described,5,23 supplemented
with 5% fetal bovine serum (FBS). Cells from a 10-day-old culture
(stationary phase) were inoculated into 50 mL of fresh culture medium
to give an initial concentration of 1 ꢁ 106 cells/mL. Cell growth was
followed by measuring the absorbance of the culture at 600 nm every
day. Before inoculation, the medium was supplemented with the
indicated quantity of the drug from a stock solution in DMSO. The
final concentration of DMSO in the culture medium never exceeded
0.4%, and the control was run in the presence of 0.4% DMSO and in the
absence of drugs. No effect on epimastigote growth was observed
because of the presence of up to 1% DMSO in the culture medium.
The percentage of inhibition (PGI) was calculated as follows: PGI (%) =
{1 ꢀ [(Ap ꢀ A0p)/(Ac ꢀ A0c)]} ꢁ 100, where Ap = A600 of the culture
containing the drug on day 5, A0p = A600 of the culture containing the
drug just after addition of the inocula (day 0), Ac = A600 of the culture in
the absence of drugs (control) on day 5, and A0c = A600 in the absence of
the drug on day 0. In order to determine ID50, 50% inhibitory
concentration, parasite growth was followed in the absence (control)
and presence of increasing concentrations of the corresponding drug.
On day 5, the absorbance of the culture was measured and related to the
control. The ID50 was taken as the concentration of drug needed to
reduce the absorbance ratio to 50%.
1
65. Yield: 52%. H NMR (400 MHz, CDCl3-d6) δ ppm: 8.78 (s, 1H,
0
0
0
0
0
0
0
H8); 8.70 (s, 1H, H5); 7.89 (dd, 2H, H2 þ H6 , J2 -4 = J6 -4 = 1.2 Hz, J2 -
0
0
0
0
0
0
0
0
0
0
0
30
= J6 -5 = 8.3 Hz); 7.70 (dt, 1H, H4 , J4 -2 = J4 -6 = 1.2 Hz, J4 -3 = J4 -5
=
0
0
0
0
0
0
0
0
7.2 Hz); 7.55 (dt, 2H, H3 þ H5 , J3 -5 = 1.6 Hz, J3 -2 = J5 -6 = 8.1 Hz);
7.30 (t, 1H, CHF2, JHꢀF = 52.2 Hz). IR (KBr): 3090 (w, νCꢀH Ar),
1681 (s, νCdO), 1324 (s, νN-oxide), 1051(m, νCꢀF) cmꢀ1. Calcu-
lated analysis for C16H8Cl2F2N2O3: C, 49.87; H, 2.08; N, 7.27. Found:
C, 49.94; H,2.13; N, 6.98.
2-Benzoyl-3-(difluoromethyl)-7-chloroquinoxaline 1,4-Dioxide, 66.
Yield: 44%. 1H NMR (400 MHz, CDCl3-d6) δ ppm: 8.64 (d, 1H, H5,
0
J5ꢀ6 = 9.2 Hz); 8.60 (d, 1H, H8, J6ꢀ8 = 2.1 Hz); 7.93ꢀ7.90 (m, 2H, H2
0
þ H6 ); 7.91 (dd, 1H, H6, J6ꢀ8 = 2.1 Hz, J6ꢀ5 = 9.1 Hz); 7.70 (dt, 1H,
0
0
0
0
0
0
0
0
0
0
0
H4 , J4 -2 = 1.2 Hz, J4 -3 = J4 -5 = 7.2 Hz); 7.55 (dt, 2H, H3 þ H5 , J3 -5
=
0
0
0
0
0
0
0
0
0.5 Hz, J3 -4 = J5 -4 = 7.7 Hz, J3 -2 = J5 -6 = 7.9 Hz); 7.32 (t, 1H, CHF2,
JHꢀF = 52.2 Hz). IR (KBr): 3083 (w, νCꢀH Ar), 1679 (s, νCdO), 1333
(s, νN-oxide), 1045(m, νCꢀF) cmꢀ1. Calculated analysis for
C16H9ClF2N2O3: C, 54.78; H, 2.57; N, 7.99. Found: C, 54.54; H,
2.83; N, 7.96.
2-Benzoyl-3-(difluoromethyl)-7-fluoroquinoxaline 1,4-Dioxide, 67.
Yield: 8%. 1H NMR (400 MHz, CDCl3-d6) δ ppm: 8.73 (dd, 1H, H5, J5-
FC7 = 4.9 Hz, J5ꢀ6 = 9.52 Hz); 8.26 (dd, 1H, H8, J8ꢀ6 = 2.6 Hz, J8-FC7
=
Viability of CL Brener, Y, or Colombiana Strains of T. cruzi and
LTB300 Strain of L. braziliensis. Trypanosoma cruzi epimastigotes (CL
Brener, Y, or Colombiana strains) were grown as previously indicated for
Tulahuen 2 strain. Leishmania braziliensis promatigotes (MHOM/BR/
00/LTB300 strain) were grown at 28 °C in an axenic RPMI medium
supplemented with 5% FBS as previously described.23 Cell-culture plates
consisting of 24 wells were filled at 1 mL/well with the corresponding
strain of the corresponding parasite culture during its exponential
growth in the corresponding medium. BHIꢀtryptose medium was
supplemented with 5% fetal bovine serum (FBS). Different doses of
studied compounds dissolved in DMSO were added and maintained for
2 days. Afterward, the cells were washed twice with PBSꢀglucose
(5.5 mM) and resuspended in 200 μL of PBSꢀglucose. Then an amount
of 100 μL of suspension was incubated (28 °C) with 0.4 mg/mL MTT
(Sigma) for 4 h. Then the lysis of cells was carried out with
SDSꢀisoprapanol (1:5), and optical densities were measured at
610 nm. Each concentration was assayed three times, and six growth
controls were used in each test. Cytotoxicity percentages (PCyt (%))
were determined as follows: PCyt = [100 ꢀ (ODd ꢀ ODdm)/(ODc ꢀ
ODcm)] ꢁ 100, where ODd is the mean of OD595 of wells with
parasites and different concentrations of the compounds, ODdm is the
mean of OD595 of wells with different compound concentrations in the
medium, ODc is the growth control, and ODcm is the mean of OD595
0
0
0
0
0
0
0
0
0
0
8.1 Hz); 7.91 (dd, 2H, H2 þ H6 , J2 -4 = J6 -4 = 1.1 Hz, J2 -3 = J6 -5 = 8.2
Hz); 7.73 (ddd, 1H, H6, J6ꢀ8 = 2.8 Hz, J6-FC7 = 7.3 Hz, J6ꢀ5 = 9.9 Hz);
0
0
0
0
0
0
0
0
0
7.70 (dt, 1H, H4 , J4 -2 = J4 -6 = 0.8 Hz, J4 -3 = J4 -5 = 7.3 Hz); 7.55 (dt,
0
0
0
0
0
0
0
0
0
0
0
0
2H, H3 þ H5 , J3 -5 = 1.0 Hz, J3 -2 = J5 -6 = 7.7 Hz, J3 -4 = J5 -4 =7.5 Hz);
7.33 (t, 1H, CHF2, JHꢀF = 52.2 Hz). IR (KBr): 3077 (w, νCꢀH Ar),
1675 (s, νCdO), 1338 (s, νN-oxide), 1046 (m, νCꢀF) cmꢀ1. Calcu-
lated analysis for C16H9F3N2O3: C,57.48; H, 2.69; N,8.38. Found: C,
57.82; H,2.89; N, 8.18.
2-Benzoyl-3-(difluoromethyl)-7-methoxyquinoxaline 1,4-Dioxide,
1
68. Yield: 5%. H NMR (400 MHz, CDCl3-d6) δ ppm: 8.58 (d, 1H,
0
0
0
0
0
0
0
0
H5, J5ꢀ6 = 9.5 Hz); 7.92 (dd, 2H, H2 þ H6 , J2 -4 = J6 -4 = 1.2 Hz, J2 -3
=
=
0
0
0
0
0
J6 -5 = 7.9 Hz), 7.89 (d, 1H, H8, J8ꢀ6 = 2.6 Hz); 7.68 (dt, 1H, H4 , J4 -2
0
0
0
0
0
0
J4 -6 = 1.2 Hz, J4 -3 = J4 -5 = 7.0 Hz); 7.55 (dd, 1H, H6, J6ꢀ8 = 2.2 Hz,
0
0
0
0
0
0
0
0
J6ꢀ5 = 9.1 Hz); 7.53 (dd, 2H, H3 þ H5 , J3 -5 = 0.6 Hz, J3 -2 = J5 -6 = 7.2
0
0
0
0
Hz, J3 -4 = J5 -4 = 6.6 Hz); 7.35 (t, 1H, CHF2, JHꢀF =52.4 Hz); 4.03
(s, 3H, CH3O). IR (KBr): 3111 (w, νCꢀH Ar), 1685 (s, νCdO), 1343
(s, νN-oxide), 1042(m, νCꢀF) cmꢀ1. Calculated analysis for
C17H12F2N2O4: C, 58.95; H, 3.46; N, 8.09. Found: C, 58.78; H, 3.50;
N, 7.96.
2-Benzoyl-3-(difluoromethyl)-7-methylquinoxaline 1,4-Dioxide, 69.
Yield: 3%. 1H NMR (400 MHz, CDCl3-d6) δ ppm: 8.58 (d, 1H, H5, J5ꢀ6
=
=
0
0
0
0
8.8 Hz); 8.39 (d, 1H, H8, J8ꢀ6 = 1.0 Hz); 7.91 (dd, 2H, H2 þ H6 , J2 -4
3633
dx.doi.org/10.1021/jm2002469 |J. Med. Chem. 2011, 54, 3624–3636

Journal of Medicinal Chemistry
ARTICLE
of wells with medium only. Nfx (Lampit) and Mtf (Impavido) were used
as reference drugs for T. cruzi and L. braziliensis, respectively. For this
propose, Mtf was dissolved in ethanol. The ID50 was taken as the
concentration of drug needed to reduce the absorbance ratio to 50%.
Trypanosomicidal (Trypomastigotes) in Vitro Test20,24. Products
were evaluated in vitro on the trypomastigote form of T. cruzi (CL
Brener clone). BALB/c mice infected with T. cruzi were used 7 days after
infection. Blood was obtained by cardiac puncture using 3.8% sodium
citrate as anticoagulant in a 7:3 blood/anticoagulant ratio. The para-
sitemia in infected mice ranged from 1 ꢁ 105 to 5 ꢁ 105 parasites/mL.
The products were dissolved in a minimal quantity of DMSO and added
to PBS to give a final concentration of 100 and 250 μg/mL. Aliquots (10
μL) of each solution in triplicate were mixed in microtiter plates with
100 μL of infected blood containing parasites at a concentration near 106
parasites/mL. Infected blood and infected blood containing GV at 250
μg/mL were used as control. The plates were shaken for 10 min at room
temperature and kept at 4 °C for 24 h. Each solution was examined
microscopically (Olympus BH2) for parasite counting. The activity (%
of parasites reduction) was compared with that of the standard drug GV.
Mutagenicity Assay. The method of direct incubation in plate was
performed. A culture of S. typhimurium TA98 strain in the agar minimum
glucose medium (AMG) (agar solution, Vogel Bonner E(VB) 50ꢁ, and
40% glucose solution) was used. First, the direct toxicity of the
compounds under study against S. typhimurium TA98 strain was assayed.
DMSO solutions of 9, 21, 44, and Nfx at different doses (starting at the
highest doses without toxic effects, 15.0 and 30.0 μg/plate, respectively)
were assayed in triplicate. Positive controls of 4-nitro-o-phenylendia-
mine (20.0 μg/plate, in the run without S9 activation) and 2-amino-
fluorene (10.0 μg/plate, in the cases of S9 activation) and negative
control of DMSO were run in parallel. The influence of metabolic
activation was tested by adding 500 μL of S9 fraction of mouse liver
treated with Aroclor, obtained from Moltox, Inc. (Annapolis, MD, U.S.).
The revertant number was counted manually. The sample was con-
sidered mutagenic when the number of revertant colonies was at least
double that of the negative control for at least two consecutive dose
levels.
control solution with 4 μg/mL of each metabolite in buffer phosphate,
pH 7.4.
In Vivo Anti-T. cruzi Activity (Acute Model). BALB/c male or
Swiss albino male mice (30 days old, 25ꢀ30 g) bred under specific
pathogen-free (SPF) conditions were infected by intraperitoneal injec-
tion of 103 blood trypomastigotes (CL Brener or Y). One group of 10
animals was used as control (PBS), and three groups of eight animals
were treated with the two compounds and Bnz, respectively. First
parasitemia was carried out 5 days after infection (week 1), and the
treatment was begun 7 days later. Compounds were administered orally,
as aqueous solution in PBS, at 10, 30, or 50 (mg/kg bw)/day for
quinoxaline derivative 21, 10 (mg/kg bw)/day for quinoxaline derivative
44, and 50 (mg/kg bw)/day for Bnz, during 10 days. Parasitemia in the
control and treated mice was determined once a week after the first
administration, for 60 days, in tail-vein blood; the mortality rate was
recorded. All the sera obtained after centrifugation of the blood that was
extracted from infected mice were tested twice by ELISA (enzyme linked
immuno assay) at 30 and 60 or 90 days after infection. A locally
produced ELISA kit (Chagas test, IICS, Asunciꢀon, Paraguay) was used
following the procedure recommended by the manufacturer (IICS
Production Department, Asunciꢀon-Paraguay).24 The optical density
values were obtained in an ELISA plate reader (Titerek Unistan I).
Wilconxon test was used in order to compare the levels of anti-T. cruzi
antibodies between experimental groups. The experimental protocols
with animals were evaluated and supervised by the local Ethics Com-
mittee and the research adhered to the Principles of Laboratory Animal
Care.25
’ AUTHOR INFORMATION
Corresponding Author
*For M.G. (for requests for reprints): phone, 598 25258618
(extension 216); fax, 598 25250749; e-mail, megonzal@fq.edu.
uy. For A.M.: phone, 34 948 42 56 53; fax, 34 948 42 56 52,;
e-mail:, cifa@unav.es.
Studying the Mechanism of Action. Mitochondrial Dehydro-
genase Activities. Mitochondrial dehydrogenase activities were mea-
sured in 24-well plates. One million of T. cruzi epimastigotes (Y strain)
in 500 μL of medium were seeded in each well, and 20 μM of studied
compounds were added. Two wells with untreated parasites were
maintained as controls corresponding to the given time of treatment.
The cultures were incubated at 28 °C. At the different incubation times,
the epimastigotes were counted and the colorimetric MTT dye-reduc-
tion assay was performed, with the tetrazolium salt being converted into
purple formazan by living mitochondria. An amount of 50 μL of a
solution containing 5 mg/mL of MTT in PBS was added to each well,
and plates were incubated for an additional 4 h. The reaction was
stopped by the addition of 500 μL of acidic isopropanol (0.4 mL of HCl
10 N in 100 mL of isopropanol). The absorbance was measured at
570 nm. Under our conditions, compounds did not interfere with the
reaction mixture. The percentage of mitochondrial dehydrogenase
activity (Pmdh (%)) was determined using untreated parasite activity
as 100%.
’ ACKNOWLEDGMENT
The authors thank the CSIC-Universidad de la Repꢀublica,
Uruguay. Collaborative work was performed under the auspices
of the Iberoamerican Program for Science and Technology
(CYTED), Network RIDIMEDCHAG. M.C. thanks the
Fundaciꢀon Carolina for a fellowship. L.B. thanks PEDECIBA
(Uruguay), and M.C., P.H., and D.B. thank ANII (Uruguay) for
their scholarships. E.T. thanks the University of Navarra, Spain,
for his scholarship.
’ ABBREVIATIONS USED
Nfx, nifurtimox; Bnz, benznidazole; Mtf, miltefosine; PGI, per-
centage of growth inhibition; PCyt, percentage of cytotoxicity;
GV, gentian violet; SI, selectivity index; 4-NPD, 4-nitro-o-phe-
nylendiamine; AF, 2-aminofluorene; NR, number of revertants;
Pmdh, percentage of mitochondrial dehydrogenase activity; Lac,
lactate; Ace, acetate; Pyr, pyruvate; Suc, succinate; Ala, alanine;
Gly, glycine
1H NMR Study of the Excreted Metabolites. For the spectroscopic
studies, an amount of 5 mL of a 2-day-treated T. cruzi (Y strain) was
centrifuged with each studied compound (5 μM) at 1500g for 10 min at
4 °C. The pellet was discarded, and the parasite-free supernatant was
stored at ꢀ20 °C until use. Before measurement, approximately 0.1 mL
of DMF (10 mM) as internal standard and 0.1 mL of D2O were added to
0.3 mL of the supernatant. The spectra were registered with water
suppression in 5 mm NMR sample tubes. The chemical displacements
used to identify the respective metabolites were confirmed by adding
each analyzed metabolite to the studied supernatant and by the study of a
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