Cyclohexene-fused 1,3-oxazines with selective antibacterial and antiparasitic action and low cytotoxic effects

Article abstract of DOI:10.1016/j.tiv.2017.07.021

Oxazine derivatives, a class of heterocyclic compounds, exhibit a variety of biological properties, such as anticonvulsant and antitumor activities. In this study, we evaluated the effect of two cyclohexene-fused 1,3-oxazines (cis?1-benzyl-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (1) and trans?N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (2)) in cultures of Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Serratia marcescens, Shigella flexneri and Staphylococcus aureus by the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Additionally, the ex vivo antiparasitic activity of oxazines was assessed against Schistosoma mansoni, a helminth that is one of the major agents of the disease schistosomiasis Also, oxazines were evaluated on three tumor cell lines, NCI-H292 (human lung carcinoma), MCF-7 (human breast adenocarcinoma) and HEp-2 (human cervix carcinoma), and two normal cell lines (Vero and red blood cells). Bioassays revealed that oxazine 2 is more effective against bacteria than oxazine 1, with the lowest MIC and MBC values of 3.91 and 32.5 μg/mL, respectively. Similarly, compound 2 demonstrated higher antiparasitic activity than 1, and scanning electron microscopy analysis showed several morphological alterations in the tegument of worms in a concentration-dependent manner. In contrast, both oxazines exhibited low cytotoxic effects on cancer and normal cell lines. These results indicated that oxazines exerted direct effects on bacteria and parasite schistosomes. More importantly, since schistosomiasis control programs rely on one drug, praziquantel, oxazines may have the potential to become new antischistosomal agents.

Full text of DOI:10.1016/j.tiv.2017.07.021

Accepted Manuscript
Cyclohexene-fused 1,3-oxazines with selective antibacterial and
antiparasitic action and low cytotoxic effects
Maria R.M. de Brito, Walter J. Peláez, Martín S. Faillace,
Gardenia C.G. Militão, Jackson R.G.S. Almeida, Gustavo A.
Argüello, Zsolt Szakonyi, Ferenc Fülöp, Maria C. Salvadori,
Fernanda S. Teixeira, Rivelilson M. Freitas, Pedro L.S. Pinto, Ana
C. Mengarda, Marcos P.N. Silva, Ademar A. Da Silva Filho, Josué
de Moraes
PII:
S0887-2333(17)30207-2
doi: 10.1016/j.tiv.2017.07.021
TIV 4070
DOI:
Reference:
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
20 April 2017
20 July 2017
22 July 2017
Please cite this article as: Maria R.M. de Brito, Walter J. Peláez, Martín S. Faillace,
Gardenia C.G. Militão, Jackson R.G.S. Almeida, Gustavo A. Argüello, Zsolt Szakonyi,
Ferenc Fülöp, Maria C. Salvadori, Fernanda S. Teixeira, Rivelilson M. Freitas, Pedro L.S.
Pinto, Ana C. Mengarda, Marcos P.N. Silva, Ademar A. Da Silva Filho, Josué de Moraes
,
Cyclohexene-fused 1,3-oxazines with selective antibacterial and antiparasitic action and
low cytotoxic effects, Toxicology in Vitro (2017), doi: 10.1016/j.tiv.2017.07.021
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ACCEPTED MANUSCRIPT
Cyclohexene-fused 1,3-oxazines with selective antibacterial and antiparasitic action and low
cytotoxic effects
a
b
b
c
Maria R. M. de Brito , Walter J. Peláez , Martín S. Faillace , Gardenia C. G. Militão , Jackson
d
b
e
e
f
R. G. S. Almeida , Gustavo A. Argüello , Zsolt Szakonyi , Ferenc Fülöp , Maria C. Salvadori ,
f
a
g
h
Fernanda S. Teixeira , Rivelilson M. Freitas , Pedro L.S. Pinto , Ana C. Mengarda , Marcos P.
h
i
h,*
N. Silva , Ademar A. Da Silva Filho , and Josué de Moraes
a
Research Laboratory of Experimental Neurochemistry, Program in Pharmaceutical Sciences,
Federal University of Piauí, Teresina, PI, Brazil
b
INFIQC-CONICET. Departamento de Fisicoquímica, Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba, Argentina
c
Laboratory of Bioassays for Drug Research, Federal University of Pernambuco, Recife, PE,
Brazil
d
Department of Pharmacy, Federal University of San Francisco Valley, Petrolina, PE, Brazil
e
Institute of Pharmaceutical Chemistry, University of Szeged, Hungary
f
Institute of Physics, University of São Paulo, São Paulo, SP, Brazil
g
Department of Parasitology, Adolf Lutz Institute, São Paulo, SP, Brazil
h
Research Center for Neglected Diseases, Guarulhos University, Guarulhos, SP, Brazil
i
Faculty of Pharmacy, Department of Pharmaceutical Sciences, Federal University of Juiz de
Fora, Juiz de Fora, MG, Brazil
*
Corresponding author at: Núcleo de Pesquisa em Doenças Negligenciadas, Centro de Pós-
Graduação e Pesquisa, Universidade Guarulhos, Praça Teresa Cristina Praça Tereza Cristina, 229,
Centro, 07023-070, Guarulhos, SP, Brazil.
E-mail address: josuem@usp.br; moraesnpdn@gmail.com (J. de Moraes)
1

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Highlights
-
-
-
-
Oxazine 2 exhibited antischistosomal activity against Schistosoma mansoni
Oxazine 2 displayed activity against Staphylococcus aureus and Escherichia coli
Oxazine 2 caused death and tegumental damage of adult S. mansoni worms
Oxazines exhibited low cytotoxic effects on human cancer cell lines.
2

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Abstract
Oxazine derivatives, a class of heterocyclic compounds, exhibit a variety of biological properties,
such as anticonvulsant and antitumor activities. In this study, we evaluated the effect of two
cyclohexene-fused 1,3-oxazines (cis-1-benzyl-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-
2
-imine (1) and trans-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (2)) in cultures
of Bacillus cereus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Salmonella
enterica, Serratia marcescens, Shigella flexneri and Staphylococcus aureus by the Minimum
Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Additionally,
the ex vivo antiparasitic activity of oxazines was assessed against Schistosoma mansoni, a
helminth that is one of the major agents of the disease schistosomiasis Also, oxazines were
evaluated on three tumor cell lines, NCI-H292 (human lung carcinoma), MCF-7 (human breast
adenocarcinoma) and HEp-2 (human cervix carcinoma), and two normal cell lines (Vero and red
blood cells). Bioassays revealed that oxazine 2 is more effective against bacteria than oxazine 1,
with the lowest MIC and MBC values of 3.91 and 32.5 μg/mL, respectively. Similarly, compound
2
demonstrated higher antiparasitic activity than 1, and scanning electron microscopy analysis
showed several morphological alterations in the tegument of worms in a concentration-dependent
manner. In contrast, both oxazines exhibited low cytotoxic effects on cancer and normal cell lines.
These results indicated that oxazines exerted direct effects on bacteria and parasite schistosomes.
More importantly, since schistosomiasis control programs rely on one drug, praziquantel, oxazines
may have the potential to become new antischistosomal agents.
Keywords: Cyclohexene-fused 1,3-Oxazines; Anthelmintic activity; Antimicrobial activity;
Schistossoma mansoni.
3

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1
. Introduction
Chronic diseases such as schistosomiasis and cancer reach a large proportion of the
world’s population, which may significantly affect the quality of life and even cause death. Cancer
affects approximately 15 million people per year and, more than half die. Only in 2013, there were
1
4.9 million incident cancer cases worldwide and 8.2 million cancer deaths (Global Burden of
Disease Cancer Collaboration et al., 2015). On the other hand, schistosomiasis, caused by blood-
dwelling flatworms of the genus Schistosoma, affects more than 250 million people worldwide,
with mortality around 280,000 ( Inobaya et al., 2014; WHO, 2016). Schistosomiasis is considered
the most important helminthic disease of humanity in terms of morbidity and mortality, and is one
of the major neglected diseases (Colley et al., 2014). However, praziquantel (PZQ) is
unfortunately the only drug available for treatment of this disease (Cioli et al., 2014). Thus, the
development of new antischistosomal compounds to curb the concern about the development of
PZQ resistance should be a high priority (de Moraes., 2015; Mafud et al., 2016a). Regarding PZQ
resistance, recent review has been published indicating that mass drug administration, especially
in Africa, has led to appearance of reduced efficacy of PZQ, which portends the selection of drug
resistant forms of schistosomes (Vale et al., 2017).
Microbial infections are the cause of other serious impacts on human health, especially due
to multi-drug resistance. So too are opportunistic mycoses, which have become increasingly
common and are identified as a major cause of morbidity and mortality. The rapid emergence and
spread of multidrug-resistant bacteria increases the need for identification and control of the
pathogens involved (Luepke et al., 2017). Bacterial resistance is very dangerous because of its
high prevalence in hospitals as well as its relationship with the failure in treatment with
antimicrobials agents. These factors increase the mortality and the costs related to the treatment of
patients suffering from those diseases (Sartelli et al., 2016).
In recent years, an increasing number of studies have shown that oxazine derivatives, a
class of heterocyclic compounds that contain one nitrogen and one oxygen atom, exhibit a variety
4

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of biological properties, such as sedative, anticonvulsant, analgesic, antipyretic, anticoagulant, and
antimalarial activities (Sindhu et al., 2013). Due to the biological significance of these
compounds, they could be used for the development of new chemical entities to combat various
diseases. On the other hand, the antibacterial, antitumor, and mainly anthelmintic properties of
oxazine derivatives have been little studied.
The present study investigated the antimicrobial and antischistosomal properties of two
partially saturated derivatives of oxazines, named cis-1-benzyl-N-phenyl-1,4,4a,5,8,8a-hexahydro-
3
,1-benzoxazin-2-imine (1) and trans-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine
2). Additionally, the cytotoxicity of oxazines was evaluated on three tumor and two normal cell
lines.
(
2
. Material and Methods
2
.1. Drugs and instrumentation
The oxazine derivatives cis-1-benzyl-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-
imine (1) and trans-N-phenyl-1,4,4a,5,8,8a-hexahydro-3,1-benzoxazin-2-imine (2) were prepared
by modification according to previously reported procedures (Peláez et al., 2008; Fülöp et al.,
1
987). The synthesis of both oxazines started from commercial cis-1,2,3,6-tetrahydrophtalic
anhydride, which was used as received, to prepare the cis derivative (1), and turned into its trans
isomer to start the synthetic path ending in compound (2). After some synthetic steps already
reported, (1) there were new reactions under new conditions, and these included the esterification
reaction of cis-6-aminocyclohex-3-ene-1-carboxylic acid to afford cis-methyl-6-aminocyclohex-3-
ene-1-carboxylate, while the trans- derivative was prepared from the ester trans-methyl-6-
aminocyclohex-3-ene-1-carboxylate generated, in turn, from the trans-6-aminocyclohex-3-ene-1-
carboxylic acid.
Melting points were determined in open glass capillaries with a Scientific Electrothermal
Digital apparatus (model IA9100). NMRs were recorded in CDCl with a Bruker Avance II 400
3
5

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1
13
MHz spectrometer (BBI probe, z gradient) ( H at 400 MHz and C at 100 MHz). Chemical shifts
◦
are reported in parts per million (ppm) downfield from TMS. The spectra were measured at 22 C.
The infrared spectra were recorded at room temperature, with a Bruker IFS-28 spectrometer, in the
-1
range 4000-400 cm . The IR spectra were recorded from solid samples in KBr pellets (spectral
-1
resolution 2 cm ), at room temperature.
2
.2. Bacteria and determination of the minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC)
Bacterial strains used in this study were obtained from the National Institute for Quality
Control in Health (INCQS/FIOCRUZ, Brazil). The microorganisms used were: Bacillus cereus
(
ATCC 11778), Enterococcus faecalis (ATCC 19433), Escherichia coli (ATCC 25922),
Klebsiella pneumoniae (ATCC 13883), Salmonela enterica (ATCC 10708), Serratia marcescens
ATCC 13880), Shigella flexneri (ATCC 12022) and Staphylococcus aureus (ATCC 25923). The
(
antibacterial effect was evaluated by microdilution methods, as recommended by Clinical and
Laboratory Standards Institute (CLSI). Stock solutions of oxazine derivatives (1 and 2) were
prepared using as a solvent a 20% DMSO aqueous solution. After this procedure, 100 µL of this
dilution was transferred to a microplate with 100 µL of Muller-Hinton broth, and eight serial
dilutions were performed, which led to concentrations from 500 to 3.91 µg/mL of both oxazines.
5
The inoculum with 5 x 10 CFC/mL was added to each well and Muller-Hinton broth and
gentamicin were used, respectively, as a sterility control and an antimicrobial reference control,
the latter at concentrations from 0.8 to 0.0125 µg/mL. The microplates were incubated under
aerobic conditions for 24 h at 37 °C, and 2,3,5-triphenyltetrazolium chloride (TTC, 10 µL, 2 %)
was added to each tissue well to detect active bacterial metabolism (the colorless solution changed
to red). The MIC was defined as the lowest concentration of the oxazine (1 and 2) that inhibited
bacterial growth. To determine the MBC, aliquots of these microplates (10 µL) were transferred to
6

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Petri dishes with Muller-Hinton agar and were incubated for 24 h at 37 °C. The appearance of
colonies of bacteria indicates that some solutions of oxazines were not able to kill 99.9 % (or
more) of the bacterial inoculum used. The assays were performed in triplicate.
2
.3. Parasite and determination of schistosomicidal activity
Schistosoma mansoni (BH strain Belo Horizonte, Brazil) worms are maintained in
Biomphalaria glabrata snails as intermediate hosts and Mesocricetus auratus hamsters as
definitive host at the Adolfo Lutz Institute (São Paulo, Brazil), according to standard procedures
previously described (Moraes, 2012). All experiments were authorized by the Committee for
Ethics in Animal Care of Institute Adolf Lutz, in accordance with nationally and internationally
accepted principles for laboratory animal use and care (CEUA, 11.794/08). The study was
conducted in adherence to the institution’s guidelines for animal husbandry. Female hamsters,
weighting 20–22 g, were infected by subcutaneous injection of 150 cercariae. After 7 weeks, the
S. mansoni adults were recovered from a hamster previously infected with 150 cercariae, by
infusion in RPMI 1640 medium with heparin. For preparations and culture of S. mansoni, the
parasites were washed in RPMI 1640 medium maintained at pH 7.5 with 20 mM HEPES buffer
(N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) supplemented with 10% fetal bovine
serum, 200 µg/mL of streptomycin and 200 IU/mL of penicillin. After washing, a pair of adult
parasites were transferred to each well (24 pairs in total) with the same medium at 37 °C in an
atmosphere of CO 5 %, as previously described (de Moraes et al.,2011; de Moraes et al., 2013).
2
Compounds 1 and 2 were dissolved in 0.5% DMSO and tested at concentrations of 100, 50, 25,
and 10 µM. The control groups were performed with incubation of parasites in RPMI 1640 as a
negative control group and 5 µM of PZQ in the positive control group. The parasites were
maintained for 120 h and monitored every 24 h. The effect of both oxazines and the standard drug
(PZQ) was evaluated using microscopes, with emphasis on motor activity, alteration in the
tegument, and mortality rate (Veras et al., 2012; de Almeida et al., 2016).
7

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2
.4. Scanning electron microscopy studies
To show morphological alterations in adult schistosomes caused by oxazine 2, the most
active compound, worms were rinsed twice in PBS, and fixed in 1 mL 2.5% glutaraldehyde for 3–
2
2
4 h at room temperature. Samples were prepared as previously described (Guimarães et al.,
015). Briefly, specimens were air-dried, mounted on stubs and metalized with gold using a
Sputter Coater. Samples were then visualized using a high-resolution SEM accelerating voltage of
2
2
7
0 kV (Jeol-JSM-6460LV).
.5. Cell cultures and determination of the effective inhibition concentration against cell growth
The human lung carcinoma cells (NCI-H292), human breast adenocarcinoma cells (MCF-
), and human cervix carcinoma cells (HEp-2) were obtained from the Cell Bank of Rio de Janeiro
(BCRJ, Brazil). Vero, a monkey kidney cell line, was obtained from the American Type Culture
Collection (ATCC CCL-81; Manassas, VA). All cell lines were cultured in RPMI 1640 or DMEM
media supplemented with 10% fetal bovine serum and 1% antibiotic, and then incubated at 37 C
in an atmosphere containing 5 % CO ( Silva et al., 2015; de Moraes et al., 2015). Oxazines 1 and
2
2
were solubilized in sterile dimethylsulfoxide (DMSO) and diluted with culture medium to
achieve a final concentration of 1% DMSO. Compounds were serially diluted in RPMI 1640
5
medium to obtain the final concentration of 100 µM and added to cells that were seeded at 1 × 10
cells/mL in 96-well plates. The plates were incubated for 72 h at 5 % of CO and 37 C. Then, 3-
2
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added (25 L, 5 mg/mL)
and the plates were further incubated for 3 h. After dissolving the precipitate in DMSO, the
absorbance was read in a plate spectrophotometer at 595 nm (Berridge et al., 1996).
2
.6. Hemolytic activity
8

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The hemolytic assay was performed using sheep red blood cells (Marani et al., 2015; Wei
et al., 2016). Erythrocytes were separated from plasma by centrifugation and washed three times
(3,000 x g, 5 min) with 150 mM NaCl. Red blood cells suspension was added to an equal volume
of each compound solution at 100 μM. The mixtures were incubated for 1 h at 37 °C and
centrifuged at 3,000 g for 2 min. The value of absorbance of the supernatant was measured at 550
nm. NaCl 150 mM and Triton-X (0.1% v/v) were used as negative and positive controls,
respectively. Experiments were performed in triplicate.
2
.7. Statistical analysis
Statistical tests were performed with GRAPHPAD PRISM (version 6.0) software. The
results were expressed as mean ± SEM (standard error of mean) and statistical evaluation was
performed using analysis of variance (ANOVA) followed by Student-Newman-Keuls test with
post hoc test. The results of the statistical analysis among the groups were considered significant
when p < 0.05.
3
. Results
3
.1. Synthesis of oxazine derivatives
Oxazines 1 and 2 were obtained through the synthetic pathways described in Scheme 1.
When cis-methyl-6-aminocyclohex-3-ene-1-carboxylate (3) was treated with benzoyl chloride
step A), cis-methyl-6-benzamidocyclohex-3-ene-1-carboxylate was formed in good yield.
Simultaneous LAH reduction of the ester and carboxamide function resulted in cis-(6-
benzylamino)cyclohex-3-en-1-yl)methanol (step B), which after treatment with phenyl
isothiocyanate afforded cis-1-benzyl-1-(-6-(hydroxymethyl)cyclohex-3-en-1-yl)-3-phenylthiourea
4) (step C). Starting from trans-methyl-6-aminocyclohex-3-ene-1-carboxylate (5) and following
(
(
(
steps B and C, trans-1-(6-(hydroxymethyl)cyclohex-3-en-1-yl)-3-phenylthiourea (6) was obtained.
Reacting thioureas 4 and 6 with methyl iodide, thiomethyl ethers were formed which were
9

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transformed without isolation in alkaline medium to oxazine derivatives 1 and 2 with methyl
mercaptol elimination (70-80 % yield, step D).
The synthesis of compounds 1 and 2 followed literature procedures (Peláez et al., 2008;
Fülöp et al.,1987). All chemical and physical properties of 1 and 2 were similar to those reported
by Vainiotalo et al. (Vainiotalo e al., 1991) for 1 and Fülöp et al. (Fülöp et al.,1987) for 2.
Compound 1 was synthesized in 79% yield and obtained with 97.3% purity, while compound 2
was produced in 80% yield and 99.8% purity. Characterization of synthesized compounds:
1
Compound 1: mp (°C): 86-88 (Lit.: 89-91), Purity: 97.3% Yield: 79 %. H-NMR (400 MHz,
CDCl ) δ (ppm): 1.86 (1H, d, J= 18.3 Hz); 2.15 (1H, m); 2.15 (1H, m); 2.36 (1H, dm, J=16.4 Hz);
3
2
.43 (1H, m); 2.47 (1H, m); 3.42 (1H, m); 4.06 (1H, dd, J = 10.3 Hz, J = 5.4 Hz, J = 1.4 Hz); 4.14
1 2 3
(
1H, dd, J = 12.4 Hz, J = 10.8 Hz); 4.32 (1H, d, J= 15.4 Hz); 5.30 (1H, d, J= 15.3 Hz); 5.55 (2H, s
1 2
coalesced signals); 6.91 (1H, tt, J =7.4 Hz, J = 1.2 Hz); 6.98 (2H, dd, J = 8,5 Hz, J = 1.2 Hz);
1
2
1
2
1
3
7
.22 (2H, t, 8.3 Hz); 7.27 (1H, d, J= 7.2 Hz); 7.33 (2H, t, J=7.3 Hz); 7.4 (2H, d, J= 7.4 Hz). C-
NMR (100 MHz, CDCl ) δ (ppm): 26.0, 27.2, 30.5, 50.5, 51.5, 67.6, 121.3, 123.2, 123.8, 124.2,
3
-
1
1
1
27.3, 128.1, 128.5, 128.6, 138.6, 148.5, 148.8. IR (KBr, cm ): 3057, 3028, 2922, 2908, 2895,
616, 1575, 1487, 1450, 1440, 1261, 1224, 1114, 1035, 773, 732, 696.
1
Compound 2: mp (°C): 190-191 (Lit.: 192-193). Purity: 99.8%. Yield: 80%. H-NMR
(
400 MHz, CDCl ) δ (ppm): 1.76 (1H, tm, J= 14.1 Hz), 1.94 (2H, m), 2.17 (1H, dm, J= 16.8 Hz),
3
2
1
.45 (1H, d, J= 15.3 Hz), 2.28 (1H, td, J = 10.8 Hz, J = 5.4 Hz), 3.90 (1H, dd, J = 10.9 Hz, J =
1 2 1 2
0.9 Hz), 4.27 (1H, dd, J = 10.3 Hz, J = 4.3 Hz), 5.69 (2H, s colesced signals), 6.94 (1H, t, J= 7.2
1
2
1
3
Hz), 7.26 (5H, m). C-NMR (100 MHz, CDCl ) δ (ppm): 27.3, 34.2 (x2), 52.7, 70.2, 119.5,
3
-
1
1
2
21.8, 125.0, 126.7, 128.9. IR (KBr, cm ): 3449, 3225, 3132, 3107, 3020, 2916, 2893, 2860,
829, 1618, 1585, 1485, 1489, 1468, 1294, 1202, 1184, 1157, 1082, 854, 781, 704, 669.
3
.2. Antibacterial activities
1
0

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In this study, the MIC and the MBC of oxazines 1 and 2 were investigated against the
Gram-positive bacteria and Gram-negative bacteria and the results are summarized in Table 1. In
general, oxazine 2 was more effective against bacteria than oxazine 1. Interestingly, oxazine 2 was
able to inhibit both Gram-positive and Gram-negative strains, especially for S. aureus and S.
flexneri, which had the same MIC values of 3.91 µg/mL (about 10 M). Oxazine 2 also showed
moderate action against E. coli with MIC values of 62.5 μg/mL, and relatively weak activity
against B. cereus, E. faecalis, S. enterica, and S. marcescens, with the same MIC values of 500
μg/mL. All tested strains presented the predicted susceptibility to the standard antibiotic
gentamicin (MIC ˂ 0.5 µg/mL). In order to address the bacteriostatic and bactericidal properties of
the oxazines, the MBC values were also evaluated. As shown in Table 1, the MBC values ranged
from 31.25-500 μg/mL.
3
.3. Antischistosomal properties
The effect of incubation with different concentrations of oxazines 1 and 2 on the motor
activity and viability of both male and female S. mansoni adult worms for up to 72 h is shown in
Table 2. Both 1 and 2 exhibited anthelmintic activity, but 2 had considerably higher activity than
1
against schistosomes, reducing motility and causing death at concentration 25 M, while 1 was
inactive at concentrations less than 100 M. Interestingly, male worms were more susceptible to
oxazines than females. For example, when tested at concentrations of 50 M of oxazine 2, all S.
mansoni males showed reduced motor activity and died after 24 h, whereas no mortality was
observed in the female worms. Positive control (PZQ, 5 M) resulted in the death of all parasites
within 24 h, whereas no mortality was observed in schistosomes of the negative (RPMI medium)
and solvent control (RPMI medium plus 0.5% DMSO) groups.
In addition to the in vitro antiparasitic activity against S. mansoni described above (Table
2
), we also analyzed the effects of oxazine 2 on the tegument of schistosomes using a scanning
electron microscope to examine the surface of male worms exposed to different concentrations of
1
1

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2
. As shown in Figure 1, morphological alterations of the tegument on the S. mansoni surface
were detected after exposure to a medium containing oxazine 2 at concentrations of 25 M, 50
M, and 100 M, whereas no tegumental surface alterations were observed in the negative control
group. More specifically, the teguments of schistosome incubated with 2 showed massive
sloughing, as well as shrinking and disintegration of the tubercles in a concentration-dependent
manner.
3
.4. Cytotoxicity and hemolytic activities
The inhibitory activity against three tumour cell lines (NCI-H292, MCF-7 and HEp-2) and
normal cell line (Vero) was evaluated by MTS assay. Any compound should present, at least, 50%
of cell growth inhibition to be considered as cytotoxic. As shown in Table 3, oxazine 1 and 2 had
a low cytotoxic effect on tumor cells lines when tested at concentration of 100 M. Indeed, both
compounds affected less than 30% of inhibition of cell growth, whereas positive control
(doxorubicin) caused 62 to 88% of inhibition. In addition, oxazines at 100 M did not exhibit any
hemolytic effect on sheep red blood cells.
4
. Discussion
Recently, a large number of 1,3-oxazines have been synthesized and subsequently
demonstrated to possess biological and pharmacological activities, such as antimicrobial,
anticancer, analgesic, and anti-inflammatory properties (Sindhu et al., 2013). In this study, we
evaluated, for the first time, the effect of two cyclohexene-fused 1,3-oxazines (1) and (2) against
Gram-positive and Gram-negative bacteria, S. mansoni adult worms, and three tumor cell lines.
Results showed, in general, that oxazine 2 was more active than oxazine 1 when exposed to
bacteria and parasites, and both compounds had a low cytotoxic effect.
In order to perform the tests of antibacterial activity for oxazines, strains with different
patterns of pathogenicity and susceptibility to antimicrobial agents were used. The MIC procedure
1
2

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was important to evaluate the activity of compounds. Based on this, our results showed that
oxazine 2 was capable of inhibiting the bacterial growth of both Gram-positive and Gram-negative
bacteria. The most susceptible bacteria strains were Staphylococcus aureus and Shigella flexneri
with a MIC value of 3.91ꢀμg/mL (about 17 M). Despite lower than gentamicin, the antibacterial
activities of cyclohexene-fused 1,3-oxazine 2 against S. aureus are similar to some of the best 1,2-
or 1,3-oxazine derivatives previously synthesized (D’Andrea et al., 2005; Verma et al., 2012),
which displayed activity with MIC values ranging from 2-8ꢀμg/mL. However, it is difficult to
compare the antibacterial activities of 2 with other previous synthesized oxazine derivatives, since
they are structurally different. Also, it is important to point out that no previous antibacterial
studies with cyclohexene-fused 1,3-oxazine derivatives has been reported. No significant
difference in susceptibility between Gram-positive bacteria and Gram-negative bacteria was
found, similarly to studies with other oxazine derivatives which revealed inhibition of both Gram-
positive and Gram-negative strains (Mathew et al., 2010; Mayekar et al., 2011; Didwagh et al.,
2
013; Kamala et al., 2016).
With respect to the anthelmintic tests, both oxazines 1 and 2 exhibited anthelmintic
activity, but 2 was more potent than 1 in reducing motility and causing death of the S. mansoni
adult worms. Also, despite the schistosomicidal activity of 2 is lower than PZQ at the same
comparative concentration, it could be considered a promising lead schistosomicidal compound,
since there are little no cytotoxic compounds able to cause death in adult schistosomes. The results
also showed that oxazines acted preferentially against male rather than female worms. These
differences in the mortality between male and female parasites have been observed with several
antischistosomal compounds. For example, studies with the terpene phytol showed that females
were more susceptible than males (de Moraes et al., 2014), whereas studies with the terpene
nerolidol showed that male worms were more susceptible than females (Silva et al., 2014). Similar
results described here, which showed differences between male and female schistosomes, also
1
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ACCEPTED MANUSCRIPT
suggest that the mechanism of action in relation the sexes of schistosomes remains to be
investigated.
The schistosome’s tegument, which is the interface between host and parasite, is an
important target for antischistosomal drugs, and alterations in the surface topography of worms
have been described in several in vitro and in vivo studies (Guimarães et al.,2015; de Moraes.,
2
014; Silva et al., 2014; Mafud et al., 2014b; Quelemes et al., 2015). Because oxazine 2 was
active against adult schistosomes, we further analyzed its effects on S. mansoni tegument. To
better understand the effects of oxazine 2 on the worm’s tegument, we used a scanning electron
microscope to examine the surface of worms under different concentrations. Oxazine 2 induced
severe tegumental damage in male schistosomes and pronounced changes in the aspect of
tubercles, which often appeared collapsed and disrupted. These effects were similar to those
reported in studies with other compounds, such as piplartine (Moraes et al., 2011), (+)-limonene
epoxide (Moraes et al., 2013), cardamonin (de Castro et al., 2015), and licoflavone B (Aleixo de
Carvalho et al., 2015). Likewise, it is known that PZQ exhibits potent in vitro effects against
schistosomes and it causes extensive tegumental alterations in a concentration-dependent manner
(Cioli et al., 1995; Pica-Mattoccia and Cioli, 2004).
Also, the tegument of schistosomes is usually considered for its key role in nutrient uptake,
secretory functions and parasite protection against the host immune system. Also, in case of severe
damages on the tegument, the host’s immune response can affect the reparation process (Veras et
al., 2012). The tegument destruction induced by oxazine 2 will probably not allow reparation,
since the damages lead to extensive peeling of the tegument surface, as well as formation and
collapsing of tubercles, indicating straight damage to the cells in a direct dose-dependent effect.
Since schistosomiasis control programs rely on one drug, PZQ, these results suggest that oxazine 2
may have the potential to become a new antischistosomal agent.
During recent years, despite lack of interest from the pharmaceutical industry, considerable
efforts have been made in the development of novel schistosomicidal agents. As a result, many
1
4

ACCEPTED MANUSCRIPT
compounds with promising antischistosomal properties have been identified by researchers.
Among these, piplartine (piperlongumine) and curcumin have also been described as promising
anticancer agents (Bar-Sela et al., 2010; Bezerra et al., 2013). Additionally, artemisinin and its
derivatives, some of the most interesting antischistosomal compounds (Keiser and Utzinger,
2
012), have also been shown to exhibit anticancer properties (Hooft van Huijsduijnen et al., 2013).
Furthermore, the antitumor activity of 1,3-oxazine derivatives has been described in different
tumor cell lines (Ouberai et al., 2006; Narita et al 2014). In this study, we assessed the toxicity
properties of oxazines 1 and 2 on three human tumor cell lines (NCI-H292, MCF-7, and HEp-2)
and two normal cell lines (Vero and erythrocytes). Nevertheless, our results show that both
compounds show no cytotoxicity toward the tested cancer and normal cell lines when tested at 100
M. Thus, oxazine 2 exhibited antiparasitic activity without cytotoxicity to mammalian cells.
Considering only the chemical structures of oxazines 1 and 2 and their influence on the
activity of both compounds, it can be speculated that the -NH- moiety in 2 appears to have a vital
role in the activity of this molecule, since in the oxazine 1 N-H bound is not present. Then, we
hypothesized about the capability of oxazine 2 acting as a hydrogen bond donor, allowing better
interactions than the bulky benzyl substituent (Ph-CH -) in 1. Nevertheless, this observation is
2
based only on two compounds and should be confirmed. Regarding this and due to the complexity
of drug mechanisms and their mode of action, future biological experiments are necessary to
clarify mechanisms of action, as well as the structure-activity relationship.
In conclusion, hexahydro-benzoxazin imine derivatives have displayed selective
antibacterial and schistosomicidal actions, and low cytotoxic effects. Both Gram-positive and
Gram-negative bacteria were susceptible to oxazine 2. Additionally, a remarkable reduction in the
motor mobility of the S. mansoni parasite, followed by mortality and tegumental alterations, was
detected in the presence of 2. Considering the obtained results, oxazine 2 is a promising
compound that could be evaluated in additional chemotherapeutic investigations.
1
5

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Conflict of interest
We declare no conflict of interest.
Acknowledgment
This work was partially supported by grants from the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP grant number 2016/488-3), the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), and the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), Brazil. The work at the Instituto de Investigaciones en
Fisicoquímica de Córdoba (INFIQC) was supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Fondo para la Investigación Científica y Tecnológica
(FONCyT) and Secretaría de Ciencia y Tecnología (SeCyT - UNC). M.S. Faillace wants to thank
CONICET for the fellowship. M.P. Silva is the recipient of a postgraduate fellowship from
CAPES, Brazil. A. A. Da Silva Filho is grateful to FAPEMIG (Grant number # APQ 02015/14)
for financial support and CNPq for fellowship. The authors also thank Silvia G. Chiodelli,
Anderson B. Gonçalves, and Yone C. Xavier for technical help with S. mansoni life-cycle
maintenance at the Adolfo Lutz Institute (São Paulo, SP, Brazil).
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Scheme 1. Synthesis of oxazines 1 and 2. A: Benzoyl chloride / TEA. B: LiAlH (LAH) /THF. C:
4
Phenyl isothiocyante / Toluene. D: CH I /MeOH then KOH / MeOH.
3
2
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Table 1. Antimicrobial activities of oxazine 1 and 2.
Strains
Gentamicin
Oxazine 1
MIC
Oxazine 2
MIC
MIC
MCB
MCB
MCB
(
µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
(µg/mL)
Gram +
B. cereus
E. faecalis
S. aureus
Gram -
0.40
0.40
0.025
0.40
0.40
0.025
500
500
500
500
3.91
>500
500
>500
250
>500
500
31.25
E. coli
0.025
0.05
0.40
500
>500
125
250
500
500
62.5
>500
500
125
K. pneumoniae
S. enterica
S. marcescens
S. flexneri
0.05
>500
>500
500
>500
>500
>500
125
0.05
0.05
0.025
0.025
0.025
0.025
500
500
3.91
Minimum Inhibitory Concentration (MIC); Minimum Bactericidal Concentration (MCB).
2
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Table 2. Effect of oxazine 1 and 2 against S. mansoni adult worms.
a
Groups
Period of
incubation
Mortality(%)
Reduction in motor
activity (%)
a
(
h)
male
0
female
0
male
0
female
0
b
Control
24
4
7
8
2
0
0
0
0
0
0
0
0
DMSO 0.5%
Praziquantel
24
0
0
0
100
100
100
0
0
0
100
100
100
0
0
0
100
100
100
0
0
0
100
100
100
4
7
8
2
24
48
5
µM
7
2
Oxazine 1
1
5
00 µM
0 µM
24
20
50
50
0
0
0
0
0
0
0
0
0
20
100
100
0
0
0
0
0
0
0
0
0
4
7
8
2
24
4
7
8
2
Oxazine 2
1
5
2
1
00 µM
0 µM
5 µM
0 µM
24
100
100
100
100
100
100
0
0
40
0
50
50
50
0
0
40
0
0
10
0
100
100
100
100
100
100
0
20
50
0
60
70
0
0
0
40
0
0
10
0
4
7
8
2
24
4
7
8
2
24
4
7
8
2
24
4
7
8
2
0
0
0
0
0
0
0
0
a
Percentages relative to 20 worms investigated.
b
RPMI 1640.
2
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ACCEPTED MANUSCRIPT
Figure 1. Scanning electron microscopy observations of ex vivo S. mansoni male worms after
incubation with oxazine 2. Adult worms were incubated in 24-well culture plates containing RPMI
1
640 medium with 0.5% DMSO and treated with oxazine 2 at different concentrations. Control
after 72h, showing tubercles with numerous spines (white arrow) (A). Worm treated with oxazine
at concentrations of 25 µM (B), 50 µM (C), and 100 µM (D).
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ACCEPTED MANUSCRIPT
Table 3. Cell growth inhibition and hemolytic activity of oxazine 1 and 2.
Compounds NCI-H292
inhibition
MCF-7
HEp-2
Vero inhibition
(%)
Hemolytic
activity
(%)
inhibition
(%)
inhibition
(%)
(
%)
Dox
1
88.4 ± 11.5
13.2 ± 1.4
18.4 ± 0.4
62.2 ± 0.7
21.8 ± 8.9
28.4 ± 4.9
80.0 ± 0.4
28.0 ± 1.4
19.3 ± 3.1
n.d.
n.d.
10.7 ± 3.1
12.4 ± 3.6
0.9 ± 0.5
1.1 ± 0.8
2
Compounds were tested at 100 µM.
Doxorubicin (DOX). Human lung carcinoma (NCI-H292). Human breast carcinoma (MCF-7). Human
cervix carcinoma (HEp-2). Animal kidney cells (Vero). n.d: Not determined.
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