In vitro tripanocidal effect of 1,8-dioxooctahydroxanthenes (xanthenodiones) and tetraketones and improvement of cardiac parameters in vivo

Article abstract of DOI:10.1016/j.jgar.2020.04.005

Objective: Trypanosoma cruzi infection affects millions of people worldwide, and the drugs available for its treatment have limited efficacy. 1,8-Dioxooctahydroxanthenes and tetraketones are compounds with important biological applications. The aim of thi

Full text of DOI:10.1016/j.jgar.2020.04.005

Journal Pre-proof
In vitro tripanocidal effect of 1,8-dioxooctahydroxanthenes
(xanthenodiones) and tetraketones and improvement of cardiac
parameters in vivo
Ana Paula de Jesus Menezes, Milene Lopes da Silva, Wagner Luiz
´
Pereira, Guilherme de Paula Costa, Aline Luciano Horta, Andrea
Aparecida Santos Mendonc¸a, Ana Claudia Alvarenga Carneiro,
ˆ
´
Debora Maria Soares de Souza, Romulo Novaes, Robson Ricardo
´
Teixeira, Andre Talvani
PII:
S2213-7165(20)30097-7
https://doi.org/10.1016/j.jgar.2020.04.005
JGAR 1221
DOI:
Reference:
To appear in:
Journal of Global Antimicrobial Resistance
Received Date:
Revised Date:
Accepted Date:
28 January 2020
2 April 2020
4 April 2020
Please cite this article as: Menezes APJ, Silva ML, Pereira WL, Costa GP, Horta AL,
Mendonc¸a AAS, Carneiro ACA, Souza DMS, Novaes R, Teixeira RR, Talvani A, In vitro
tripanocidal effect of 1,8-dioxooctahydroxanthenes (xanthenodiones) and tetraketones and
improvement of cardiac parameters in vivo, Journal of Global Antimicrobial Resistance
(2020), doi: https://doi.org/10.1016/j.jgar.2020.04.005

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Short title: Tripanocidal and inflammatory activities of xanthenodiones and tetraketones
In vitro tripanocidal effect of 1,8-dioxooctahydroxanthenes (xanthenodiones) and tetraketones and
improvement of cardiac parameters in vivo
Ana Paula de Jesus Menezes1, Milene Lopes da Silva2, Wagner Luiz Pereira2, Guilherme de Paula Costa1,
Aline Luciano Horta1, Andréa Aparecida Santos Mendonça3, Ana Claudia Alvarenga Carneiro1, Debora
Maria Soares de Souza1, Rômulo Novaes3, Róbson Ricardo Teixeira2*, André Talvani1*
1Immunobiology Laboratory of Inflammation, Department of Biological Sciences, Universidade Federal
2
de Ouro Preto, MG, Brazil; Chemistry Department, Universidade Federal de Viçosa, MG, Brazil;
3Department of Structural Biology, Universidade Federal de Alfenas, MG, Brazil.
*Corresponding authors:
André Talvani
Laboratory of Immunobiology of Inflammation
DECBI/ICEB, Campus Morro do Cruzeiro
Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil
35400-000 Phone: 55 31 3559-1767 talvani@ufop.edu.br
Róbson Ricardo Teixeira
Departamento de Química
Universidade Federal de Viçosa, Viçosa, MG, Brazil
36570-900 Phone: 55 31 3612-6645 robsonr.teixeira@ufv.br
Abstract

Objetive. Trypanosoma cruzi infection affects millions of people worldwide, and the drugs available for
its treatment have limited efficacy. 1,8-dioxooctahydroxanthenes and tetraketones are compounds with
important biological applications. The aim of this study was to assess the trypanocidal and inflammatory
activities of nine 1,8-dioxooctahydroxanthenes (1–9) and three tetraketones (10–12). Methods and
results. By in vitro killing assay, three compounds were able to eliminate CL TdTomato expressing strain
of T. cruzi, 9 (IC50 = 30.65 µM), 10 (IC50 = 14.11 µM), and 11 (IC50 = 26.43 µM). However, only 9 was not
toxic to Vero cells. Next, to evaluate the in vivo antitrypanossomal and immunological efficacy of 9,
Swiss mice were infected with the Y and CL strains of T. cruzi and treated for 10 days with 50 mg/kg of 9.
This compound reduced the cardiac inflammatory infiltration in animals infected with both strains.
RankL, CCL2, and IFN- were measured in the cardiac tissue homogenate of the Y-strain-infected
animals, and no interference of 9 was observed. However, compound 9 increased the RankL and IL-10
levels in CL-infected mice. No hepatic and renal toxicity was observed. Conclusion. Our findings showed
that 1,8-dioxooctahydroxanthene has anti-parasitic effect and ameliorates the cardiac inflammatory
parameters related to T. cruzi infection.
Keywords: Trypanosoma cruzi; xanthenodiones; tetraketones, inflammation; heart damage;
benznidazole.
1. Introduction
Trypanosoma cruzi is the causative agent of Chagas disease, and the World Health Organization
estimates that approximately eight million people worldwide are infected with this parasite [1]. The
infection in mammalian organisms induces intrinsic interactions between the parasite molecules and the
host immune defense system. From this interaction, systemic and focal inflammatory responses are
responsible for interrupting the parasitic replication [2-4]. Several investigations in experimental models
of T. cruzi infection have suggested that a Th1-like immune response, mediated by T CD4+ and T CD8+
cells, is important for parasitism control through the production of pro-inflammatory cytokines (e.g.,
IFN, TNF, and IL-18), nitric oxide, and oxidative stress components [5-8]. Furthermore, during T. cruzi
infection, IFN-γ also supports the activation of immune cells and the establishment of myocarditis by
leukocyte recruitment through chemokines such as CCL5, CCL2, CXCL10, and CXCL9 [9, 10]. These
chemokines are key proteins in the control of parasite replication as well as in the reinforcement of the
induction of nitric oxide production by macrophages and infected cardiomyocytes [11]. Thus,
therapeutic strategies that eliminate the parasites and ameliorate the tissue inflammatory response are
of significant importance, especially considering the limited availability of drugs against T. cruzi.
Currently, only the drugs nifurtimox and benznidazole are available. Benznidazole treatment presents
questionable efficacy because of its mutagenic and carcinogenic activity [12].

The intensity of the inflammatory response during T. cruzi infection is related to distinct factors,
such as the parasite strain, infection pathway, genetics, and immune response of the vertebrate host
[13]. Therefore, the development of drugs that result not only in parasite elimination but also promote
an equilibrium between the parasite and the host immune response is essential.
Within this context, several compounds have been evaluated for the treatment of American
trypanosomiasis [14]. Described for the first time by Merlin (1894), tetraketone is an important class of
compounds which is used as tyrosinase inhibitor [15], a laser dyes [16] and mainly as a lipoxygenase
inhibitor and antioxidant agent [17]. The 1,8-dioxooctahydroxanthenes (also known as xanthenodiones)
are heterocyclic compounds that possess a pyran nucleus fused on either side with cyclohex-2-enone
rings. They present different biological activities including antibacterial [18], antifungal [19], antimalarial
[20], leishmanicidal [21], and anti-inflammatory [22] effects. Considering that the laeishmanicidal and
anti-inflammatory activities of xanthenodiones are closely related to T. cruzi pathogenesis, the aim of
this study was to evaluate the trypanocidal action of 1,8-dioxooctahydroxanthenes and tetraketones in
vitro and in vivo, and to verify their role in the reduction of levels of the parameters of inflammation in
murine infection.
2. Material and methods
Synthesis
Generalities
All reagents were purchased from commercial sources (Sigma Aldrich; St. Louis, MO, USA and F
Maia; Charqueada, São Paulo, Brazil) and were employed as received. Solvents were procured from F
1
Maia and used as received. The H (300 MHz) and 13C nuclear magnetic resonance (NMR) (75 MHz)
spectra were recorded on a Varian Mercury 300 instrument (Varian, Palo Alto, California, USA), using
CDCl3 and DMSO-d6 as solvents. Hydrogen NMR data are presented as follows: chemical shift () in ppm,
number of hydrogen atoms, multiplicity, and J values in Hertz (Hz). Multiplicities are shown as the
following abbreviations: s (singlet), d (doublet), m (multiplet), and t (triplet). Infrared spectra (IR) were
obtained using the Agilent 660-IR (Santa Clara, California) with accessory GladiATR. Melting points are
uncorrected and were determined using the MQAPF-301 melting point apparatus (Microquimica,
Palhoça, Santa Catarina, Brazil). Mass spectra were obtained on a Shimadzu GCMS-QP5050A instrument
o
(Kyoto, Japan) by direct injection using the following temperature program: 40 C min-1 until the
o
o
o
temperature reached 60 C, and then 80 C min-1 until the temperature reached 300 C; the detector
o
temperature was 280 C. Analytical thin layer chromatography analyses were carried out on TLC plates
recovered with 60GF254 silica gel.
General procedure for the preparation of xanthenodiones 1-9
A round-bottomed flask (25 mL) was charged with a 1,3-diketone (2.00 mmol), aldehyde (1.00
o
mmol), and ZrOCl2∙8H2O (12.0 mg, 2 mol%). The mixture was stirred at 85 C, and the progress of the
reaction was monitored by TLC analysis. After completion of the reaction, the mixture was cooled down
o
to 20 C. Thereafter, 50 mL of dichloromethane was added, and the mixture was kept under stirring for

approximately 30 min. Then, the catalyst, which is insoluble in dichloromethane, was separated by
filtration. After that, 50 mL of ethanol was added to the filtrate and the system was kept undisturbed for
crystallization. The structures of xanthenodiones 1-9 (Fig. 1) are supported by the following data.
9-(4-hydroxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (1)
o
Brown solid (130.6 mg, 0.42 mmol, 42% yield). Mp 307 - 308 C, IR (ATR) max/cm−1: 3370, 2949, 2869,
1657, 1609, 1446, 1206, 1169 cm−1. 1H NMR (300 MHz, CDCl3) δ: 9.16 (1H, s), 6.94 (2H, d, J = 8.4 Hz), 6.57
(2H, d, J = 8.4 Hz), 4.46 (1H, s), 2.69 – 2.51 (4H, m), 2.31 – 2.17 (4H, m), 1.99 – 1.76 (4H, m). 13C NMR (75
MHz, CDCl3) δ: 196.8, 164.9, 156.1, 135.5, 129.3, 116.4, 115.1, 36.9, 30.2, 26.9, 20.3. MS, m/z (%): 310
(C19H18O4, M+, 93), 293 (60), 253 (17), 217 (100), 55 (24).
9-(3,4,5-trimethoxyphenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (2)
o
Yellow solid (358.8 mg, 0.93 mmol, 93% yield). Mp 188 - 189 C. IR (ATR) max/cm−1: 2963, 2882, 1659,
1
1619, 1453, 1230, 1170, 1123 cm−1. H NMR (300 MHz, CDCl3) δ: 6.51 (2H, s), 4.77 (1H, s), 3.80 (6H, s),
3.75 (3H, s), 2.70 – 2.53 (4H, m), 2.44 – 2.27 (4H, m), 2.08 – 1.93 (4H, m). 13C NMR (75 MHz, CDCl3) δ:
196.6, 164.0, 152.8, 139.9, 136.7, 116.7, 105.7, 60.6, 56.6, 37.0, 31.4, 27.1, 20.3. MS, m/z (%): 384
(C22H24O6, M+, 60), 369 (13), 353 (100), 217 (27), 55 (12).
9-(4-hydroxyphenyl)-3,6-dimethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (3)
o
Yellow solid (87.4 mg, 0.26 mmol, 52% yield). Mp 215 - 216 C. IR (ATR) max/cm−1: 3600, 3274, 2998,
1
2964, 2876, 1658, 1638, 1609, 1188 cm−1. H NMR (300 MHz, DMSO) δ: 9.16 (1H, s), 6.92 (2H, d, J = 8.3
Hz), 6.56 (2H, d, J = 8.3 Hz), 4.45 and 4.43 (1H, s), 2.66 – 2.55 (2H, m), 2.50 – 2.21 (6H, m), 2.08 – 2.02
(2H, m), 1.01 – 0.91 (6H, m). 13C NMR (75 MHz, DMSO) δ: 196.8, 196.7 and 196.6, 164.6, 164.5, 163.7
and 163.6, 156.1, 156.0, 135.5, 135.4, 135.2, 129.4, 129.3, 129.2, 116.1, 115.9, 115.7, 115.1, 115.0, 45.0,
44.9, 34.8, 34.3, 30.4, 30.2, 28.2, 28.1, 27.9, 20.8, 20.7. MS, m/z (%): 338 (C21H22O4, M+, 87), 321 (54),
268 (18), 245 (100), 203 (18), 69 (19).
9-(4-hydroxyphenyl)-3,6-diisopropyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (4)
o
Yellow solid (22.7 mg, 0.58 mmol, 58% yield). Mp 176 - 177 C. IR (ATR) max/cm−1: 3405, 3172, 2959,
1
2873, 1671, 1645, 1612, 1463, 1372, 1181 cm−1. H NMR (300 MHz, DMSO-d6) δ: 7.09 – 7.03 (2H, m),
6.61 – 6.55 (2H, m), 4.68 (1H, s), 2.66 – 2.32 (6H, m), 2.13 – 1.99 (2H, m), 1.96 – 1.79 (2H, m), 1.64 – 1.52
(2H, m), 0.94 - 0.89 (12H, m). 13C NMR (75 MHz, DMSO) δ: 198.0, 197.7, 164.5, 164.4, 163.9, 154.8,
135.8, 135.6, 129.3, 116.8, 116.7, 116.4, 115.4, 41.4, 41.0, 40.1, 38.7, 31.8, 31.0, 30.8, 19.6, 19.5, 19.4.
MS, m/z (%): 394 (C25H30O4, M+, 88), 377 (52), 351 (15), 301 (100), 296 (22), 41 (19).

9-(3,4-dimethoxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (5)
o
Yellow solid (406.1 mg, 0.99 mmol, 99% yield). Mp 181 – 182 C. IR (ATR) max/cm−1: 2959, 2873, 1656,
1
1621, 1361, 1260, 1198, 1167, 1136 cm−1. H NMR (300 MHz, CDCl3) δ: 6.89 (1H, s), 6.76 – 6.68 (2H, m),
4.68 (1H, s), 3.84 (3H, s), 3.78 (3H, s), 2.45 (4H, s), 2.23 (2H, d, J = 16.3 Hz), 2.16 (2H, d, J = 16.3 Hz), 1.09
(6H, s), 0.99 (6H, s). 13C NMR (75 MHz, CDCl3) δ: 196.5, 162.1, 148.4, 147.4, 136.9, 120.1, 115.7, 112.2,
110.8, 110.4, 55.8, 55.7, 50.7, 40.8, 32.2, 31.1, 29.3, 27.2. MS, m/z (%): 410 (C25H30O5, M+, 69), 379 (100),
273 (35), 217 (16), 83 (18).
3,3,6,6-tetramethyl-9-(4-nitrophenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (6)
o
White solid (343.9 mg, 0.87 mmol, 87% yield). Mp 221 - 222 C. IR (ATR) max/cm−1: 2959, 2871, 1654,
1620, 1515, 1359, 1342, 1199, 1164, 1138 cm−1. 1H NMR (300 MHz, CDCl3) δ: 8.08 (2H, d, J = 7.9 Hz), 7.46
(2H, d, J = 7.9 Hz), 4.81 (1H, s), 2.49 (4H, s), 2.24 (2H, d, J = 16.3 Hz), 2.15 (2H, d, J = 16.3 Hz), 1.10 (6H, s),
0.98 (6H, s). 13C NMR (75 MHz, CDCl3) δ: 196.3, 163.0, 151.5, 146.4, 129.3, 123.4, 114.5, 50.6, 40.8, 32.4,
32.2, 29.2, 27.2. MS, m/z (%): 395 (C23H26NO5, M+, 48), 378 (68), 348 (25), 273 (100), 217 (32), 161 (24),
152 (15), 77 (16), 55 (33), 43 (24), 41 (42).
4-(3,3,6,6-tetramethyl-1,8-dioxo-2,3,4,5,6,7,8,9-octahydro-1H-xanthen-9-yl)benzonitrile (7)
o
White solid (327.7 mg, 0.87 mmol, 87% yield). Mp 198 - 199 C. IR (ATR) max/cm−1: 2961, 2872, 2225,
1659, 1620, 1360, 1197, 1164, 1138 cm−1. 1H NMR (300 MHz, CDCl3) δ: 7.50 (2H, d, J = 7.6 Hz), 7.40 (2H,
d, J = 7.6 Hz), 4.75 (1H, s), 2.47 (4H, s), 2.23 (2H, d, J = 16.3 Hz), 2.14 (2H, d, J = 16.2 Hz), 1.10 (6H, s), 0.97
(6H, s). 13C NMR (75 MHz, CDCl3) δ: 196.3, 162.9, 149.5, 131.9, 129.3, 119.0, 114.5, 110.1, 50.6, 40.8,
32.5, 32.2, 29.2, 27.2. MS, m/z (%): 375 (C24H25NO3, M+, 41), 273 (100), 217 (21), 161 (14), 55 (22), 43
(17), 41 (33).
3,3,6,6-tetramethyl-9-phenyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (8)
o
Yellow solid (275.7 mg, 0.79 mmol, 79% yield). Mp 203 - 204 C. IR (ATR) max/cm−1: 2957, 2871, 1659,
1
1624, 1359, 1197, 1164, 1139 cm−1. H NMR (300 MHz, CDCl3) δ: 7.28 (2H, d, J = 7.5 Hz), 7.20 (2H, t, J =
7.5 Hz), 7.11 – 7.06 (1H, m), 4.74 (1H, s), 2.46 (4H, s), 2.23 (2H, d, J = 16.3 Hz), 2.15 (2H, d, J = 16.3 Hz),
1.09 (6H, s), 0.98 (6H, s). 13C NMR (75 MHz, CDCl3) δ: 196.4, 162.3, 144.1, 128.4, 128.0, 126.3, 115.6,
50.7, 40.9, 32.2, 31.8, 29.2, 27.3. MS, m/z (%): 350 (C23H26O3, M+, 49), 273 (100), 217 (23), 161 (8).

9-(4-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione(9)
o
White solid (355.5 mg, 0.91 mmol, 91% yield). Mp 250 - 251 C. IR (ATR) max/cm−1: 3401, 2961, 2872,
1660, 1613, 1358, 1199, 1165, 1133 cm−1. 1H NMR (300 MHz, CDCl3) δ: 7.05 (2H, d, J = 8.1 Hz), 6.54 (2H,
d, J = 8.1 Hz), 4.65 (1H, s), 2.45 (4H, s), 2.24 (2H, d, J = 16.5 Hz), 2.17 (2H, d, J = 16.5 Hz), 1.08 (6H, s), 0.99
(6H, s). 13C NMR (75 MHz, CDCl3) δ: 197.3, 162.5, 154.7, 135.5, 129.3, 115.8, 115.2, 50.7, 40.8, 32.3, 30.9,
29.3, 27.2. MS, m/z (%): 366 (C23H26O4, M+, 92), 349 (39), 282 (25), 273 (100), 217 (32), 161 (15), 83 (17).
General procedure for the preparation of tetraketones 10-12
The synthesis of compounds 10-12 (Fig. 2) has been previously described [23, 24]).
Biological Assays
Parasites and culture procedures in vitro
CL T. cruzi fluorescent parasites were generated by transfecting the parasite strain with pTREX-
Td-tomato plasmid as described by Canavaci, Bustamante [25]. The CL DNA allows a good and efficient
tagging by genetic manipulation and the transfection procedure and experimental conditions were
previously reported by Peng and Tarleton [26]. The Vero cell monolayer cultures were infected with
trypomastigote metacyclic forms of T. cruzi, and these cells were subsequently used in the amastigote
growth inhibition assays. Infected Vero cells were cultured in RPMI 1640 medium with 10% fetal bovine
serum in a humid atmosphere containing 5% CO2 at 37oC.
Amastigote growth inhibition assay
Vero cells were exposed to 2000 rad of gamma radiation for 10 min [27]. Then, 2.5 × 104 cells
were plated in 96-well plates (Black Greiner plates - Bio-One) overnight at 37 oC and under 5% CO2. Vero
cells were exposed to trypomastigotes of the CL tdTomato strain of T. cruzi for approximately 5 h at a
multiplicity of infection ratio 1:10 cell/trypomastigotes. After infection, cell cultures were washed to
remove non-internalized parasites. Then, fresh media with or without xanthenodiones (1-9),
tetraketones (10-12), or benznidazole were added in decreasing concentrations (50µM/mL to 0.5
µM/mL). The change in fluorescence intensity was determined as a measurement of growth over 3 days
of culture. The killing assays were performed by the average of the three independent experiments, in
duplicate. The 50% inhibitory concentration (IC50) and 50% cytotoxicity concentration (CC50) values were
determined by nonlinear regression analysis at day 3 using GraphPad Prism version 7. The selective
index (SI) indicates the number of times the compounds were more toxic to the parasites than the
mammalian cells, and it was calculated utilizing the CC50/IC50 ratio.
Cell toxicity

To evaluate the toxic effect of compounds 1-12 on the mammalian cells, Vero cells were treated
with decreasing concentrations of the compounds (100µM/mL to 1.5 µM/mL). The experiment was
performed using Alamar Blue® (Bio-Rad, California, USA), a colorimetric index of cell proliferation, as
described by Rampersad [28]. Vero cells were added (2.5 × 104 cells/well) on Black Greiner plates (Bio-
One), and after cell adhesion, the compounds were diluted with DMSO (0.01%) in each well and
maintained in a CO2 incubator at 37 oC for 72 h. Each plate contained a negative control (medium) and a
positive control (medium + cell). After 72 h of incubation, the culture medium was removed, and 10%
Alamar Blue® reagent was added. After 4 h of incubation, the fluorescence was measured on the
SynergyH4 plate reader (BioTek) at 560 and 590 nm wavelengths. The percentage of dye reduction
induced by the compounds was calculated considering the percent reduction of incubated cells in the
absence of compounds using the following equation:
% reduction = [A560-(A590 × R0) Treated/A560-(A590 × R0) + Control]
In this equation, A560 = fluorescence at 560 nm; A590 = fluorescence at 590 nm; control = well
containing cells, medium, and Alamar Blue in the absence of the compound; and R0 = the correction
factor, calculated from the fluorescence emission values of the negative control (-C), only culture
medium, and Alamar Blue® in the absence of cells [Ro = (A560/A590) C]. The results correspond to an
average of three independent experiments in duplicate.
In vivo drug testing
Swiss female mice aged 4–6 weeks, were bred and maintained at the Center of Animal Science
at the Federal University of Ouro Preto (UFOP), Brazil. The procedures adopted agreed with the ethical
principles of animal experimentation pre-established by the National Council for Control of Animal
Experimentation (CONCEA). This research was previously approved by the Ethics Committee on Animal
Research of UFOP-CEUA (protocol nº 2016/34).
Parasites and infection
Swiss mice (n = 10) were infected intraperitoneally with 5 × 103 blood trypomastigote forms of T. cruzi
strains CL and Y. Blood was stored in liquid nitrogen and maintained in vivo by successive passages in
Swiss mice. The CL strain was isolated from a Triatoma infestans collected in Rio Grande do Sul,
Encruzilhada, South Brazil, in 1963 [29]. For chemotherapy experiments, it presents a high susceptibility
to drugs clinically used in Chagas disease [30] and it is classified as a discrete typing units (DTUs) VI [31].
The Y strain belongs to DTU II [31] and it was obtained from a female child patient in 1953 in São Paulo,
Southeast Brazil [32]. Studies involving medical drugs against the T. cruzi, describes this strain as
partially susceptible to Bz and it evolves intense inflammatory response in mammalian hosts [32]. For all
this reason, both strains, CL and Y, might provide an adequate parasitological and inflammatory answers
related to the test of new and potential compounds.

The parasitemia was determined daily by optic microscopy analysis of 5 µL samples, after tail
bleeding [33].
Treatments and euthanasia
Treatments with xanthenodione 9 (50 mg/kg) and benznidazole (100 mg/kg) were initiated 4
days after infection with T. cruzi Y strain and 8 days after infection with the CL strain. After the
establishment of T. cruzi infection, the compounds were administered for 10 consecutive days orally
(gavage) at a volume of 0.2 mL of carboxymethylcellulose (CMC) 0.5% suspension plus Tween 80 (0.5%
solution). The untreated animal received a vehicle content (CMC) 0.5% suspension plus Tween 80 (0.5%
solution).
Animals (n = 10) were sorted into groups: (i) uninfected animals that received CMC + Tween 80;
(ii) infected untreated mice that received CMC + Tween 80; (iii) infected mice treated with 9; (iv)
infected mice treated with benznidazole; and (v) uninfected mice treated with 9. Animals in the acute
phase were euthanized after 14 and 18 days for Y and CL strains, respectively, according to euthanasia
practice guidelines regulated by the CONCEA. Blood and cardiac tissues were collected for biochemical
tests, immunoassays, and histopathological analysis.
Evaluation of cytokines and chemokines by Immunoassays
Immunoenzymatic assays were used to evaluate the concentrations of the inflammatory
cytokines IFN-gamma and RankL, the chemokine CCL2, and the regulatory cytokine IL-10. Cardiac tissues
were isolated and macerated, and the supernatant was used according to the manufacturer’s protocol
(PeproTech Inc., Rocky Hill, NJ, USA). At the end of the reaction, 100 μL/well of the chromogen substrate
ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); Sigma-Aldrich Inc., St. Louis, MO, USA) was
o
added for 60 min, followed by incubation in the dark at 25 C. Finally, samples were read in a
spectrophotometer (Emax Molecular Devices, Sunnyvale, CA, USA) using wavelengths of 405 and 630
nm.
Heart histopathology and stereology
Heart samples of all animals were fixed in 10% buffered formaldehyde for 48 h. The samples were
embedded in glycol methacrylate histological resin and cut in a rotary microtome into 3-μm-thick
sections using glass knives (Leica Biosystems, Wetzlar, Germany). Five histological sections were
collected in semi-series, using one out of every 50 sections, which were stained with hematoxylin and
eosin. Histopathological analysis was performed by evaluating the distribution and organization of the
organ parenchyma and connective stroma, tissue necrosis and inflammatory infiltrate, morphology and
distribution of myocytes, blood vessels, and interstitial cells, and the presence of T. cruzi amastigote
nests [34].

The intensity of cellularity or heart inflammation was assessed by comparing the
distribution of interstitial cells in the myocardium of all groups [35]. For this purpose, tissue
cellularity per histological area was determined as the number of interstitial cell nuclei in a
standardized test area (At = 25 × 103 μm2). Tissue cellularity was evaluated from 20 randomly
sampled histological fields of heart sections from each animal using a bright field microscope
with a ×40 objective lens (400× magnification; Axioscope A1, Carl Zeiss, Germany). A total
tissue area of 25.0 × 105 μm2 was analyzed for each group. Total interstitial cellularity was
analyzed using the image analysis software, Image-Pro Plus® (version 4.5; Media Cybernetics
Inc., Silver Spring, MA, USA). The results are expressed as an octagonal field diagram
organized in domains that represents tissue cellularity as follows: (-) minimum, (- - +) mild, (+
+) moderate or (+ + +) intense cellularity/inflammatory infiltrate [36].
The amplitude of the cardiac microstructural reorganization was analyzed using the
stereological method [35]. Volume density (Vv, %) of heart parenchyma (cardiomyocytes
[cmy]), stroma (connective tissue [cnt]), and blood vessels (bvs) were estimated as Vv= ΣPt/P,
where ΣPt is the number of test points that hit on the structure of interest, and P is the total
number of points in the test system. We used a test system with 72 test points in a standard test
area (At = 25 × 103 μm2) at the tissue level. The relationship between blood vessels and
cardiomyocytes (Vv[bvs]/Vv[cmy]) was used as a morphological index of myocardium
vascularization. The relationship between structural and functional heart compartments was
estimated as Vv[cnt]/Vv[cmy]. Volume density was estimated from 20 randomly sampled
histological fields of heart sections from each animal using a ×40 objective lens (400×
magnification; Axioscope A1, Carl Zeiss, Germany). A total tissue area of 25.0 × 105 μm2 was
analyzed for each group.

The number density of mononuclear and polymorphonuclear interstitial cells per
histological area was estimated as QA = ΣQ/At, where ΣQ is the number of mononuclear or
polymorphonuclear in the microscopic focal plane, and At is the dimension of test area (At =
8.56 × 103 μm2). Volume density was estimated from 20 randomly sampled histological fields of
heart sections from each animal using a ×100 objective lens (1000× magnification; Axioscope
A1, Carl Zeiss, Germany). A total tissue area of 85.6 × 104 μm2 was analyzed for each group. All
counts were performed using the image analysis software AxionVision (Carl Zeiss, Germany).
Hepatic and renal toxicity assays
Hepatic toxicity was assessed in order to detect the plasma levels of the enzymes aspartate
aminotransferase and alanine aminotransferase. Urea and creatinine clearance were also detected in
the plasma to identify renal damage. All the assays were performed using commercial enzymatic kits
from Bioclin Labtest kits (Brazil).
Statistical analysis
The data obtained in the present investigation are expressed as the mean ± standard deviation.
The GraphPad Prism v.6 (GraphPad Software, San Diego, CA, USA) program was used, and data were
analyzed using the Kolmogorov-Smirnov test to confirm normality patterns. Depending on the nature of
the data, one-way analysis of variance with post-test Bonferroni correction and the Kruskal-Wallis test
with Tukey-Kramer and Dunn’s post-tests were used for multiple comparisons. For all analyses, a
significance level of p ≤ 0.05 was adopted.
3. Results
Preparation of xanthenodiones and tetraketones
The xanthenodiones 1-9 herein investigated were synthesized via solvent-free zirconium-
catalyzed Knoevenagel condensation between 1,3-diketones and appropriate aldehydes [23], as
depicted in Fig. 1. The compounds were obtained with yields ranging from 42% to 99% and fully
characterized via spectroscopic (infrared and nuclear magnetic resonance) and spectrometric (mass
spectrometry) methods.

In addition to xanthenodiones, our research group has also been interested in the structural
features (Silva et al., 2018) and biological activities of tetraketones. Thus, we undertook the task of
evaluating the tripanocidal activity of the tetraketones 10-12 (Fig. 2).
To the best of our knowledge, this is the first time that xanthenodiones and tetraketones have
been evaluated for their tripanocidal activity.
In vitro bioassays
Vero cells with and without infection with CLtdTomato parasites were treated or not treated
with xanthenodiones 1-9, tetraketones 10-12, and benznidazole. The fluorescent intensity was
measured on the third day of infection as an indicator of parasite growth. Among the twelve tested
compounds, three showed anti-T. cruzi activities, namely, 9 (IC50 = 30.65 ± 1.0), 10 (IC50 = 14.11 ± 1.9),
and 11 (IC50 = 26.43 ± 2.3) (Fig. 3A & 3B). However, tetraketones 10 and 11 showed high levels of toxicity
to mammalian cells (Fig. 3A and Table 1).
Moreover, the xanthenodione 9 displayed a great SI (3.1) and no toxicity to Vero cells even at
the highest tested concentration (Table 1). Therefore, compound 9 was selected to verify its capability
of reducing the parasitemia and to alter the inflammatory parameters in T. cruzi-infected mice.
In vivo bioassays
Initially, we proceeded to determine a dose and route standardization for the treatment. Based
on the number of surviving animals and parasitemia reduction, we selected an oral dosage of 50 mg/kg
to start the experiment (data not shown). After 10 days of therapy using compound 9, on the 14th and
18th days of infection with the Y and CL strains, respectively, we did not observe quantitative differences
in the circulating levels of parasites in animals treated or untreated with 9, the benznidazole treated
group demonstrated a significant reduction (Fig. 4A and B). The treatment reduced the peak of
parasitemia in the infected animals, which was responsible for a reduction in their mortality when
compared to that in the untreated ones (Fig. 4C and D). Benznidazole treatment also prevented mice
mortality for both tested strains.
By analyzing the levels of the inflammatory mediators on the myocardium, we observed an
increase in RankL in animals infected with the CL strain and treated with 9 (Fig. 5A) and an increase of
CCL2 (Fig. 5B) in the untreated animals when compared to uninfected mice. However, compound 9 was
able to increase the plasma production of IFN- as well as IL-10 in the CL+9 group (Fig. 5C and D).
Benznidazole administration was able to maintain the inflammatory mediators at basal levels close to
the uninfected/treated and uninfected mice. A ratio of these inflammatory markers and the regulatory
IL-10 is pointed out in Table 2.
Regarding the present investigation, the Y and CL strains of T. cruzi were associated with the
prevalence of a moderate mononuclear cell influx, which was reduced upon treatment with 9 (Fig. 6A &
6B). The cardiac amastigote nests of the parasite were only observed in the infected and untreated mice
(Fig. 6C). Furthermore, xanthenodione 9 treatment could reduce inflammatory infiltration for both

tested strains (Fig. 6D). In addition, cardiac damage caused by T. cruzi infection was prevented in groups
treated with benznidazole, and no differences were observed between the uninfected and treated mice.
Compound 9 did not induce renal or liver toxicity. The levels of urea and creatinine did not
increase in the plasma of any of the groups (Table 3). We verified an increase in aspartate
aminotransferase and alanine aminotransferase levels in infected mice associated with the parasite
infection (Table 3). In addition, there were no differences in the levels of markers between the
uninfected and uninfected/treated mice, confirming the previous results.
4. Discussion
American trypanosomiasis still presents great impact in the population in tropical countries. The
parasite-host interaction remains partially responsible for the absence of a therapeutic strategy against the
T. cruzi [37]. In this sense, researchers have been struggling to find alternative compounds from different
molecular scaffolds [38, 39]. It is outlined in Fig. 7 the trials carried out in the present investigation.
From twelve compounds evaluated in this study, three presented trypanocidal activity. The
technique used to generate xanthenodiones and tetraketones is called "green chemistry” since it is not
harmful to the environment and environmentally acceptable [40, 41]. Low SI is a common problem faced by
researchers who work with these compounds, mainly for the high cytotoxicity [42-44]. The same profile
was also observed by Abe et al. (2004) who found unsatisfactory SIs, even when using T. cruzi strains Bz-
susceptible. To reduce the harmful effects of xanthenodiones and tetraketones on mammalian cells and,
consequently, to reduce their toxicity, authors have suggested small conformational changes in the
molecule to guarantee the optimization of the compounds [45-47]. Although several potential molecules
anti-T. cruzi are described annually, there is a failure in the in vitro and in vivo tests, which exclude the
possibility of the compounds to move forward as efficient drugs [13]. Herein, we verified consistent results
between the compounds activities in cellular and animal model. Our aim was not only to evaluate the anti-
T.cruzi action, but also the action of the compounds on the inflammatory response. The choice of strains of
the parasite was based on the protocols for the discovery of new drugs for Chagas disease [30]. In addition,
the strains proposed in this assay allowed the rapid identification (less than 40 days) of an ideal dose
required for subsequent steps in the screening process [38, 48-50]. In the animals infected with the Y strain
of the T. cruzi, ten days of daily treatment were able to reduce the number of circulating parasites in fresh
blood analysis in 40%, besides preventing the mortality of the animals. Parasitemia and mortality are basic
criteria in the test of any new anti-T. cruzi compound [50]. For the CL strain, the pre-patent period occurs
between the 6th and 7th days after infection and there is an increase in parasitemia from the 16th day,
reaching the peak on the 22nd day [51]. Our experiments were finished at the 18th day post-infection, so
that the same 10 days of treatment was maintained for both strains. Therefore, it was not possible to verify
the parasitemia peak related to this strain.
During the acute infection driven by the T. cruzi, the exacerbated immune response is often
observed systemically; in the myocardium, this results in lesions that, in extreme cases, can lead to death
[52, 53]. Our results indicate the existence of a cardiac damage associated to the parasite strain and,
although without parasitological cure, compound 9 proved to be effective in preventing cardiac injury in
animals infected with both strains.

The trigger and the maintenance of the inflammatory response are directly related to the
activation of cell signaling pathways, such as NF-kB [54-57]. In this context, we assessed the production of
Rank's ligand (RankL), characterized as an activator receptor to the NF-kb pathway. There was no change in
this inflammatory mediator in mice infected with the Y neither CL strain of T. cruzi, reinforcing the intrinsic
strains features involved in the inflammatory context [30, 58-60].
Interestingly, the cardiac protection induced by 9 treatment in T. cruzi-infected mice (with Y and CL
strains) was not followed by reduction of plasma levels of IFN- and CCL2. However, the balance between
low IFN- and high IL-10 levels may represent a cardioprotective effect using different strains of the
parasite [9, 11, 61-63], which could be a suggestive mechanism to explain our findings. Besides, the
production of CCL2 is related to both parasitic growth and morphogenesis of trypomastigotes as well as
increased activation and recruitment of inflammatory infiltration into the heart [62, 64, 65].
Finally, the treatment using compound 9 did not induce detectable adverse reactions in the mice.
The increase in hepatic enzymes in the infected animals was possible caused by the metabolites generated
from the T. cruzi infection [66]. Other studies shown that treatment with benznidazole was not able to raise
the enzymes of liver damage, possibly for the short treatment period [67].
Thus, our in vitro findings showed that 1,8-dioxooctahydroxanthene (xanthenodione) has an
anti-parasitic effect. In the acute murine infection, 10-days treatment with 50 mg/kg of xanthenodione 9
resulted in an inflammatory reduction associated to the T. cruzi infection. Notwithstanding, further
investigations and new structural changes in the structure of 9 remain necessary to potentiate its
trypanocidal and anti-inflammatory effects. However, in face to a scenario of failures in the field of anti-
T. cruzi chemotherapy, the 1,8-dioxooctahydroxanthenes appear as new plastic and promising scaffold.
Acknowledgments
We thank our colleagues from Rick L. Tarleton’s lab, University of Georgia, Athens/GA/EUA who
provided insight and expertise that greatly assisted the in vitro research. AT (Process # 305634/2017-8)
is grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the
research productivity fellowship.
Funding
This research was supported by CAPES, CNPQ, and FAPEMIG funding agencies in Brazil.
Competing Interests
None declared.
Ethical approval
This research was previously approved by the Ethics Committee on Animal Research of UFOP-CEUA
(protocol nº 2016/34).

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Legend of figures

Figure 1. Synthesis of xanthenodiones 1-9 catalyzed by ZrOCl2∙8H2O. The compounds were obtained
from the reaction of 1,3-diketones and aldehydes. Reaction conditions: 1,3-diketone (2.00 mmol),
aldehyde (1.00 mmol), ZrOCl2∙8H2O (2 mol%), 85oC.
Figure 2. Structures of tetraketones 10-12.
Figure 3. The effects of xanthenodiones inhibitors on T. cruzi amastigotes after 72 h of treatment. (A)
Vero cell cultures were infected with Tdtomato-expressing trypomastigotes of CL strains (10:1 ratio
parasites:cells). (B) Dose-response curve showing the inhibitory effect of 10, 11 and 9 on T. cruzi
amastigotes CLtdTomato strain on 3st day post-infection. Results are representative of three
independent experiments with tree replicates.
Figure 4. Parasitemia and survival of Swiss mice infected with T. cruzi and treated with 9 and/or Bz.
Mice were infected with T. cruzi (Y and CL strain 5×103 parasites/animal) and treated orally with 50
mg/kg of 9 for 10 consecutive days, as well as, another group of mice received 100mg/kg of Bz and/or
only CMC (control group). (a) Parasitemia levels; (b) survival curves. The data are representative of the
group average (10 mice per group) * means data statistically significant in relation to Bz-treated animals.
Bz = benznidazole; NI = uninfected animals; NTI = uninfected mice treated with 9; CMC
(carboxymethylcellulose).
Figure 5. Inflammatory mediators in the cardiac tissue during acute phase of T. cruzi infection. (A)
RankL, (B) CCL2, (C) IFN and (D) IL-10 were measured in by enzyme-linked immunosorbent assay in Swiss
mice infected with Y and CL strains of T. cruzi (under treatment with 9 or Bz). In parallel, uninfected mice
treated with 9 or untreated animals (control group) were also evaluated and all data shown as a mean
of 10 animals ± SEM, and differences represented by p<0.05; Bz = benznidazole; NI = uninfected mice;
NIT = uninfected mice treated with 9; SEM = standard error of the mean.
Figure 6. Quantification of the acute inflammatory process in the cardiac tissue of mice infected with
T. cruzi Y and CL strains. Analysis of inflammatory infiltration in cardiac tissue was performed after the
mice treatment with 9 (50 mg/kg) or Bz (100 mg). Analysis of the inflammatory infiltration highlighting
the polymorphonuclear (A) and mononuclear cells (B) Photomicrography of histological sections was
performed on the cardiac tissue (C). Octagonal field diagram was organized in domains representing
tissue cellularity as follows: (-) minimal cellularity / (+ -) mild, (+ +) moderate or (+ + +) intense /
inflammatory infiltrate (D). (*) p<0.05 compared to the uninfected (NI) and uninfected/treated (NI+9)

control group; (#) p<0.05 compared to the animals treated with Bz and (&) p<0.05 compared to the
animals treated with 9. Bz = benznidazole; NI = uninfected mice; NIT = uninfected mice treated with 9.
Figure 7. Illustrative panel of the trials carried out in this work Figure 7. Illustrative panel of the trials
carried out in this work. (A) The first step was to test twelve xanthenodiones against CLtdTomado T.
cruzi amastigotes. Irradiated Vero cell was platted (2,5x104/well) and infected by T. cruzi (10:1 ratio
parasite/cell) and 72 h later the fluorescence was read as a measure of the growth (IC50 calculated). In
parallel, the compounds toxicity was measured by AlamarBlue® reagent in Vero cells and CC50 was
calculated. We obtained the SI through CC50/IC50 ratio and selected the best xanthenodione, named
compound 9, in the in vitro step, (B) Moving to the in vivo step, we tested 5 different doses 800 to
50mg/kg (1:2) and the treatment route (oral and intraperitoneal) and we assessment the parasitemia
and survival levels in mice (n=5). (C) Then, we infected Swiss mice female by Y or CL T. cruzi strain and
treated the animals by gavage, only, for 10 consecutive days(n=10) and, again, evaluated the
parasitemia and survival levels. After the treatment period, animals were euthanized, and half section of
heart used for histology. Another heart fragment was used for measurement of chemokines and
cytokines by immunoassays (ELISA). We also analyzed the AST and ALT hepatic enzymes, urea and
creatinine in the plasm to check the hepatic/renal compound toxicity.
Highlights
(i) The Xanthenediones and Tetraketones presented antitrypanosomal activity against
ClTdtomato amastigotes in vitro.
(ii) In the mice infected with the Y strain of T. cruzi, the compound 9 reduced the parasitemia
(40%) and prevented the mortality.
(iii) The compound 9 reduced the inflammatory cardiac infiltration in mice infected with Y and
CL strains of T. cruzi.
(iv) The compound 9 increased the IL-10 in those animals infected with the CL strain of T. cruzi
with no hepatic and renal toxicity.

Table 1. Vero cells toxicity, trypanocidal activity and
xanthenodiones selectivity index
SD = Standard Error. (*) p<0.05 compared to benznidazol
group. CC50 = 50 % Cytotoxicity concentrations. IC50 =
50% Inhibitory concentration. SI = Selective Index

Table 2. Ratio of inflammatory mediators/IL-10 in mice infected with Y and
CL strains of T. cruzi under different treatments.

Table 3. Biochemical analysis (mean ± SD values) performed in infected mice after 14 (Y strain)
and 18 (CL strain) days post T. cruzi infection