Pyrimido[1,2-a]quinoxaline 6-oxide and phenazine 5,10-dioxide derivatives and related compounds as growth inhibitors of Trypanosoma cruzi

Article abstract of DOI:10.1016/j.ejmech.2007.10.031

Two different families of N-oxide containing heterocycles were evaluated as in vitro growth inhibitors of T. cruzi. Both families of heterocycles were selected from our in-house library of compounds as analogues of active anti-T. cruzi N-oxide containing heterocycles. Derivatives from pyrimido[1,2-a]quinoxaline 6-oxide family were poorly active at the assayed doses. However, phenazine 5,10-dioxide derivatives displayed good to excellent anti-T. cruzi activities. The anti-T. cruzi activity of phenazine derivatives was related to substituent' electronic descriptors, σ_p^-. Derivatives 19, 20 and 23 were the most cytotoxic compounds against the protozoan and became excellent hit for further structural modifications.

Full text of DOI:10.1016/j.ejmech.2007.10.031

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1737e1741
http://www.elsevier.com/locate/ejmech
Short communication
Pyrimido[1,2-a]quinoxaline 6-oxide and phenazine 5,10-dioxide
derivatives and related compounds as growth
inhibitors of Trypanosoma cruzi
a
Marıa Laura Lavaggi , Gabriela Aguirre , Lucıa Boiani , Liliana Orelli ,
a
a
b
´
´
, Hugo Cerecetto , Mercedes Gonzalez
a,
b,**
*
a,**
´
Beatriz Garcıa
´
a Departamento de Qu´ımica Orga´nica, Facultad de Qu´ımica e Facultad de Ciencias, Universidad de la Repu´blica, Igua´ 4225, 11400 Montevideo, Uruguay
b Departamento de Qu´ımica Orga´nica, Facultad de Farmacia y Bioqu´ımica, Junin 956, 1113 Buenos Aires, Argentina
Received 5 October 2007; received in revised form 23 October 2007; accepted 24 October 2007
Available online 4 November 2007
Abstract
Two different families of N-oxide containing heterocycles were evaluated as in vitro growth inhibitors of T. cruzi. Both families of hetero-
cycles were selected from our in-house library of compounds as analogues of active anti-T. cruzi N-oxide containing heterocycles. Derivatives
from pyrimido[1,2-a]quinoxaline 6-oxide family were poorly active at the assayed doses. However, phenazine 5,10-dioxide derivatives displayed
good to excellent anti-T. cruzi activities. The anti-T. cruzi activity of phenazine derivatives was related to substituent’ electronic descriptors, sꢀp .
Derivatives 19, 20 and 23 were the most cytotoxic compounds against the protozoan and became excellent hit for further structural modifications.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Chagas disease; Pyrimido[1,2-a]quinoxaline 6-oxide; Phenazine 5,10-dioxide
1. Introduction
released as the non-dividing bloodstream trypomastigote form
that invades other tissues. The existence of the epimastigote
form as an obligate mammalian intracellular stage has been re-
visited [4,5] and confirmed recently [6]. The sequencing of the
T. cruzi genome has been recently completed [7] and large prog-
ress made in the study of T. cruzi biochemistry and physiology
[3], in which several crucial enzymes for parasite survival ab-
sent in the host have been identified as potential targets for
the design of new drugs [8e11]. In spite of these recent achieve-
ments, the chemotherapy to control this parasitic infection re-
mains undeveloped. In fact, although Nifurtimox (Nfx) and
Benznidazole (Bnz), the only two drugs currently in use for
clinical treatment of this disease, are able to wipe out parasite-
mia and reduce serological titres, they are not specific enough to
all T. cruzi strains to guarantee complete cure [12e14]. The re-
search and development of new active anti-T. cruzi compounds
have been based on different strategies, i.e. inhibition of specific
parasite enzymes, actions on parasite DNA, and oxidative stress
damage. In the first group a great number of enzymes have been
employed as targets for new inhibitors, especially trypanothione
Chagas’ disease or American trypanosomiasis is an impor-
tant health problem that affects around 20 million people in
Central and South America [1]. Around 2e3 million individuals
develop the typical symptoms of this disease that results in
50 000 yearly deaths [2]. The causative agent of this disease is
the haemoflagellate protozoan Trypanosoma cruzi (T. cruzi)
[3]. The parasite presents three main morphological forms in
a complex life cycle. It replicates within the crop and midgut
of Chagas’ disease vectors as the epimastigote form, it is re-
leased with the insect excrements as the non-dividing highly in-
fective metacyclic trypomastigotes that invade mammalian
tissues via wounds provoked by blood sucking action. The par-
asite multiplies intracellularly as amastigote form which is
* Corresponding author. Tel.: þ598 2 525 86 18x216; fax: þ598 2 525 07 49.
** Corresponding authors.
E-mail addresses: mbgarcia@ffyb.uba.ar (B. Garcıa), hcerecet@fq.edu.uy
´
´
(H. Cerecetto), megonzal@fq.edu.uy (M. Gonzalez).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.031

1738
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 43 (2008) 1737e1741
reductase inhibitors belonging to phenothiazine derivatives
(i.e. compound 1, Fig. 1) are specific competitive inhibitors
[15,16]. Agents based on DNA-damage include, between
others, camptothecin derivatives (i.e. 2, Fig. 1) [17,18] and ac-
ridine derivatives (i.e. 3, Fig. 1) [19]. In reference to oxidative
stress-producer agents the well known nitrofurans have been
the most studied compounds [20e22]. In this sense, we identi-
fied some N-oxide containing heterocycles with ability to pro-
duce oxidative stress intraparasitically (i.e. 4, Fig. 1) [23].
Recently, we have analyzed some quinoxaline dioxide deriva-
tives (i.e. 5, Fig. 1) as anti-T. cruzi agents identifying good in-
hibitors with a potential free radical production as mechanism
of action [24].
9 were methylated with CH3I to generate derivatives 16e18.
The phenazine 5,10-dioxide derivatives 19e28 were obtained
from the corresponding benzofuroxan reactants as inseparable
mixtures of 7- and 8-isomers which were evaluated without
further separation. In Table 1 are shown the main isomers
obtained in the synthetic procedures. Similar to pyrimidoqui-
noxaline 6-oxides deoxygenated derivatives, 29 and 30, were
included in this study in order to evaluate the relevance of
the N-oxide moiety in the bioactivity.
1
All compounds were identified by IR, MS, H NMR, 13C
NMR, HSQC and HMBC experiments, and their purity estab-
lished by TLC and microanalysis.
Looking for new N-oxides with anti-T. cruzi activity we
have selected, from our in-house chemical library of N-oxides,
derivatives from pyrimido[1,2-a]quinoxaline 6-oxide and
phenazine 5,10-dioxide systems with structural motives related
to compounds 1e5 (Fig. 1). These heterocyclic systems in-
clude planar backbone with two or three heteroatoms, nitro-
gens, distributed like in derivatives 1, 2 or 5. In order to
analyze structural requirements we have chosen different sub-
stituents (R1eR5, Fig. 1) with differential physicochemical be-
haviours. Consequently, we have synthesized and biologically
evaluated some selected pyrimido[1,2-a]quinoxaline 6-oxide
and phenazine 5,10-dioxide derivatives as anti-trypanosomal
agents. The 25 derivatives described in the present paper
were carefully selected from our near to 100 derivatives-library
in order to cover a wide structural spectrum, synthesized and
biologically evaluated. From the data generated here a new se-
ries of compounds will be selected.
3. Pharmacology
All the compounds were tested in vitro against T. cruzi, as
previously described [30]. The compounds were incorporated
into the media at 25 mM and its ability to inhibit the parasite
growth was evaluated in comparison to the control (no drug
added to the media). Nfx and Bnz were used as the reference
trypanocidal drugs. The percentage of growth inhibition (PGI)
at 25 mM is gathered in Table 1. The IC50 (50% inhibitory con-
centration) was assessed for compounds presenting higher try-
panocidal activity (Table 2). In the case of the most active
derivatives, phenazine 5,10-dioxides, three different T. cruzi
strains were studied, Tulahuen 2, CL Brener and Y strains.
4. Discussion
We reported the biological activity of 25 pyrimido[1,2-
a]quinoxaline 6-oxide and phenazine 5,10-dioxide derivatives
and related compounds, against the epimastigote form of
T. cruzi. The most active derivatives, belonging to phenazine
dioxide family, were evaluated against three different strains
of T. cruzi (Table 1). Derivatives 19, 20, and 23 were the
most active compounds against the studied strains. Conse-
quently, they were selected to determine its IC50 together
with Nfx and Bnz against the three strains (Table 2). The se-
lected derivatives have similar levels of bio-activities against
T. cruzi as the reference drugs. Derivatives belonging to
2. Chemistry
The studied compounds, 6e30 (Fig. 2, Table 1), were pre-
pared following synthetic procedures previously reported
[25e29]. The pyrimido[1,2-a]quinoxaline 6-oxides 6e14 were
obtained by the cyclization of the corresponding nitroaminoa-
mide reactants in presence of ethyl polyphosphate (EPP).
Compound 6 was then transformed into the deoxygenated
derivative 15 by reaction with NaBH4. Compounds 6, 7, and
R2
R1
N
Cl
S
N
N
O
O
O
N
N
Cl
N
Cl
Cl
HN
O
N+
Me
N
O
R3
Me
(CH2)4Me
O
HO
N
Studied
1
2
3
(CH2)4Me
compounds
O
N
Br
O
N
Cl
Cl
Me
S
N
O
R4
O
N
(CH2)3Me
R5
N
N
H
N
N
O
H
N
O
O
4
5
Fig. 1. Inhibitors of T. cruzi and structural related N-oxides studied for anti-T. cruzi activity.

M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 43 (2008) 1737e1741
1739
-R1= -H
N
N
NaBH4
R1
R1
R1
N
R1
N
N
15
Ph
NH HN
NO2
EPP
COCH2R
N
N
N+
R
N
R
Me
I-
CH3I
O
6-14
-R1= -H
O
16-18
O
OH
R3
N
R2 Na2S2O4
HCl/MeOH
N
N
R2
2
O
R
N
O
N
R3
R3
MeONa
MeOH/THF
N
O
Fig. 2. Synthetic procedures used for the preparation of the studied compounds.
pyrimido[1,2-a]quinoxaline 6-oxide family were less active
than phenazine 5,10-dioxides. It was not possible to establish
any clear relationship between structure of pyrimido[1,2-
a]quinoxaline 6-oxide derivatives and anti-T. cruzi activity.
Apparently, the 5-phenyl substituent is relevant for activity
(compare activity of derivative 6e13 and 14’s activities). Fur-
thermore, the absence of the N-oxide moiety (i.e. derivative
15) or the quaternization of nitrogen in 4 position (i.e. deriva-
tive 16) did not modify significantly the bioactivity. In
contrast, comparing anti-T. cruzi activity of derivatives 7 and
12, some steric requirements for the correct activity are
necessary.
For the phenazine 5,10-dioxide derivatives relevant struc-
tural information was amassed. For example, the 2-hydroxy
derivatives are, in general, less active than the 2-amino ana-
logues. In the 2-amino series, derivatives 19e23, a clear rela-
tionship between the eR3 electronic characteristic and activity
was observed (Fig. 3). Therefore, derivatives substituted by
Table 1
Structure and biological characterization of the studied compounds
R1
R1
O
N
m
R2
R3
N
N
(Me I-)n
N
O
N
O
R
m
m
Ref.
n
m
eR
eR1
eH
PGIa (%)b,c Ref.
Tul 2d
m
eR2
eR3
Ratio 7-isomer:8-isomere PGIa (%)b,c
Tul 2d CLBf Yg
6
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
ePh
36
36
14
9
19
20
21
22
23
24
25
26
27
28
29
30
1
1
1
1
1
1
1
1
1
1
0
0
eNH2
eNH2
eNH2
eNH2
eBr
eCl
eOMe
eMe
54:46
63:37
95:5
81
61
38
37
79
0
93
99
34
25
95
40
14
9
88
78
21
34
94
8
7
e( p-OMe)Ph eH
8
e( p-Cl)Ph
e( p-NO2)Ph
e(o-Cl)Ph
eH
eH
eH
eH
9
70:30
55:45
64:36
10
11
12
13
14
15
16
17
18
a
14
12
eNHCOCH2Cl e(1,3-Dioxolan-2-yl)
e(o-NO2)Ph
e( p-OMe)Ph eMe 13
eOH
eOH
eOH
eOH
eOH
eOH
eOH
eBr
eH
e
0
0
eCH2Ph
eMe
ePh
eH
eH
eH
eH
13
22
29
20
9
eOMe
eMe
100:0
76:24
35:65
70:30
56:44
0
0
43
0
6
9
e(1,3-Dioxolan-2-yl)
eBr
eMe
23
23
25
17
8
ePh
0
45
e( p-OMe)Ph eH
e( p-NO2)Ph eH
10
20
PGI: percentage of growth inhibition.
Inhibition of epimastigotes growth, dose ¼ 25 mM.
b
c
d
e
f
The results are the means of three different experiments with an SD less than 10% in all cases.
Tul 2: Tulahuen 2 strain.
Determined by 1H NMR from the reaction mixture [27e29]. The compounds were evaluated as mixture of isomers.
CLB: CL Brener strain.
Y: Y strain.
g

1740
M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 43 (2008) 1737e1741
Table 2
IC50 (mM) values for the most active derivatives
Furthermore, Lipinski has described desired ranges for cer-
tain properties thought to be important for pharmacokinetics
and drugs development. They are log P < 5, number of hydro-
gen bond donors ꢁ 5, number of hydrogen bond accept-
ors ꢁ 10 and molecular weight < 500 [31]. A compound that
fulfils at least three out of the four criteria is said to adhere
to Lipinski’s ‘rule of 5’. Table 3 lists the values of these prop-
erties for the best derivatives, 19, 20 and 23, and suggests that
these compounds are reasonable starting points for a drug dis-
covery effort. This information led us to re-design new struc-
tures with increased desired activity.
Ref.
IC50 (mM)a
Tul 2b
CLBc
Yd
19
20
23
Nfx
Bnz
a
9.0
5.3
8.0
13.0
6.5
8.2
7.7
4.5
6.5
3.8
14.2
7.7
8.5
4.5
7.4
The results are the means of three different experiments with an SD less
than 10% in all cases.
b
Tul 2: Tulahuen 2 strain.
CLB: CL Brener strain.
Y: Y strain.
c
d
5. Conclusions
The results presented above indicate, according to the in
vitro activity and adherence to Lipinski’s rules, that new phen-
azine 5,10-dioxide derivatives 19, 20 and 23 are excellent hits
for further in vivo studies in an animal model of Chagas’
disease.
electron withdrawing groups, like eBr and eCl, show better
bioactivity than derivatives substituted by electron donors,
like eOMe, eMe, 1,3-dioxolan-2-yl moiety. In the case of ac-
tivity against Y strain an excellent linear relationship
(r ¼ 0.9994) between R3 electronic descriptor, Hammett spꢀ
parameters, and activity was observed (Fig. 3). This phenom-
enon has been already observed previously for quinoxaline di-
oxide analogues [24]. The correlation with R3 electronic
property could indicate that a reductive metabolism, through-
out the N-oxide moiety, could be implicated in the mechanism
of action. However, derivative 23, with an electron donor moi-
ety, displays high activity against the three studied strains
(Table 2) maybe as consequence of the excellent electrophile
centre, chloroacetamide moiety, in the 2-amino position.
This structural motif could unselectively react with biological
nucleophiles (amino and phosphate groups from DNA, mer-
capto, hydroxyl and amino groups from proteins). Methyl de-
rivatives 27 and 30, 2-hydroxy dioxide and 2-hydroxy
deoxygenated derivatives, are medium active against Tulahuen
2 strain. Maybe another structural factor could play a role in
the activity of these compounds.
6. Experimental
6.1. Chemistry
All starting materials were commercially available re-
search-grade chemicals and used without further purification.
Compounds 6e30 and nitroaminoamide and benzofuroxans
reactants were prepared following literature procedures [25e
29]. All solvents were dried and distilled prior to use. All
the reactions were carried out in a nitrogen atmosphere. Melt-
ing points were determined using a Leitz Microscope Heating
Stage Model 350 apparatus and are uncorrected. Elemental
analyses were obtained from vacuum-dried samples (over
phosphorous pentoxide at 3e4 mm Hg, 24 h at room tempera-
ture) and performed on a Fisons EA 1108 CHNS-O analyser.
Infrared spectra were recorded on a Perkin Elmer 1310 appa-
ratus, using potassium bromide tablets. 1H NMR and 13C
NMR spectra were recorded on a Bruker DPX-400 (at 400
and 100 MHz) instrument, with tetramethylsilane as the inter-
nal reference. Mass spectra were recorded on a Shimadzu
GCeMS QP 1100 EX instrument at 70 eV.
100
CL Brener strain
r = 0.9589, s = 13.47, p = 0.0414
90
80
Y strain
6.2. Biology
r = 0.9994, s = 1.38, p = 0.0006
70
The Tulahuen 2, CL Brener and Y strains stocks of T. cruzi
were used in this study. Handling of live T. cruzi was done
60
50
Tul 2 strain
r = 0.9417, s = 8.65, p = 0.0583
Table 3
Physicochemical properties and Lipinski’s ‘rule of 5’
40
a
Ref.
log P
H-bond
donors
H-bond
acceptors
Molecular
weight
‘Rule of 5’
criteria met
Y
30
20
Tul2
CLBrener
Rule
<5
ꢁ5
ꢁ10
<500
At least 3
19
20
23
a
6.8
6.6
6.1
2
2
1
5
5
8
306
262
376
3
3
3
-30
-20
-10
0
10
20
30
-
p R3 x 100
Fig. 3. Substituent electronic descriptors ðsꢀpR3 ꢂ 100Þ vs percentages of try-
panosome growth inhibition at 25 mM (PGI). The curves show the tendency.
Calculated using GhoseeCrippen algorithm, implemented in Spartan’04
modeling package [32,33].

M.L. Lavaggi et al. / European Journal of Medicinal Chemistry 43 (2008) 1737e1741
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1741
according to established guidelines [34]. The epimastigote
form of the parasite was grown at 28 ꢃC in an axenic medium
(BHI-Tryptose), complemented with 5% foetal calf serum.
Cells from a 5-day-old culture were inoculated into 50 mL
of fresh culture medium to give an initial concentration of
1 ꢂ 106 cells/mL. Cell growth was followed by daily measur-
ing the absorbance of the culture at 600 nm for 11 days. Be-
fore inoculation, the media was supplemented with 25 mM
solutions of compounds from a stock DMSO solution. The fi-
nal DMSO concentration in the culture media never exceeded
0.4% (vol/vol) and had no effect by itself on the proliferation
of the parasites (no effect on epimastigote growth was ob-
served by the presence of up to 1% DMSO in the culture me-
dia). The compounds’ ability to inhibit growth of the parasite
was evaluated, in triplicate, in comparison to the control (no
drug added to the media). The control was run in the presence
of 0.4% DMSO and in the absence of any drug. The percent-
age of growth inhibition (PGI) was calculated as follows:
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È
ÂÀ
Áꢀ
ÃÉ
PGIð%Þ ¼ 1 ꢀ Ap ꢀ A0p ðAc ꢀ A0cÞ ꢂ 100;
´
[21] G. Aguirre, H. Cerecetto, M. Gonzalez, D. Gambino, L. Otero, C. Olea-
Azar, C. Rigol, A. Denicola, Bioorg. Med. Chem. 12 (2004) 4885e4893.
where Ap ¼ A600 of the culture containing the drug at day 5;
A0p ¼ A600 of the culture containing the drug just after addition
of the inocula (day 0); Ac ¼ A600 of the culture in the absence
of any drug (control) at day 5; and A0c ¼ A600 in the absence of
the drug at day 0. The 50% effective concentrations (IC50)
were obtained assaying five different points, 1.0, 5.0, 10.0,
15.0 and 25.0 mM. Each point was analyzed in three different
experiments with an SD less than 10%. Nifurtimox and Benz-
nidazole were used as the reference trypanocidal drug.
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´
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´
faro, A. Jaso, B. Zarranz, M.A.I. Ortega, I. Aldana, A. Monge-Vega, Bio-
org. Med. Chem. Lett. 14 (2004) 3835e3839.
´
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Acknowledgements
1703e1707.
[27] H. Cerecetto, M. Gonzalez, M.L. Lavaggi, A. Azqueta, O. Ezpeleta,
´
´
A. Lopez de Cerain, A. Monge-Vega, J. Med. Chem. 48 (2005) 21e23.
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[28] H. Cerecetto, M. Gonzalez, M.L. Lavaggi, W. Porcal, J. Braz. Chem.
Soc. 16 (2005) 1290e1296.
The authors would like to thank PEDECIBA and University
of Buenos Aires (grant B-105) for partial financial support.
´
[29] H. Cerecetto, M. Gonzalez, M.L. Lavaggi, M.A. Aravena, C. Rigol,
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