Synthesis, stability studies, and antifungal evaluation of substituted α- and β-2,3-dihydrofuranaphthoquinones against sporothrix brasiliensis and sporothrix schenckii

Article abstract of DOI:10.3390/molecules24050930

Sporotrichosis is a neglected fungal infection caused by Sporothrix spp., which have a worldwide distribution. The standard antifungal itraconazole has been recommended as a first-line therapy. However, failure cases in human and feline treatment have been reported in recent years. This study aimed to synthesize several α- and β-2,3-dihydrofuranaphthoquinones and evaluate them against Sporothrix schenckii and Sporothrix brasiliensis—the main etiological agents of sporotrichosis in Brazil. The stability of these compounds was also investigated under different storage conditions for 3 months. The samples were removed at 0, 60, and 90 days and assessed by¹H-NMR, and their in vitro antifungal susceptibility was tested. Furthermore, we evaluated the superficial changes caused by the most effective and stable compounds using scanning electron microscopy and determined their effects when combined with itraconazole. Nine dihydrofuranaphthoquinones showed good antifungal activity and stability, with MIC values of 2–32 μM. Compounds 6 and 10 were the most active dihydrofuranaphthoquinones in vitro for both species; in fungi, these compounds induced yeast–hyphae conversion and alteration in the hyphae and conidia structures. Compound 10 also exhibited a synergistic activity with itraconazole against S. schenckii, with a ΣFIC index value of 0.3. Our results indicate that Compounds 6 and 10 are potential candidates for the development of new antifungal agents for the treatment of sporotrichosis.

Full text of DOI:10.3390/molecules24050930

molecules
Article
Synthesis, Stability Studies, and Antifungal
Evaluation of Substituted α- and
β-2,3-Dihydrofuranaphthoquinones against
Sporothrix brasiliensis and Sporothrix schenckii
Patricia Garcia Ferreira 1, Luana Pereira Borba-Santos 2 , Leticia Lorena Noronha 1,
Caroline Deckman Nicoletti 1, Marcella de Sá Haddad Queiroz 1,
Fernando de Carvalho da Silva 3 , Sônia Rozental 2, Débora Omena Futuro 1 and
Vitor Francisco Ferreira 1,
*
1
Departamento de Tecnologia Farmacêutica, Faculdade de Farmácia, Universidade Federal Fluminense,
Niterói-RJ 24241-000, Brazil; patricia.pharma@yahoo.com.br (P.G.F.); leticianoronha95@gmail.com (L.L.N.);
caroline_nicoletti@yahoo.com.br (C.D.N.); marcellahaddad@id.uff.br (M.d.S.H.Q.);
dfuturo@id.uff.br (D.O.F.)
Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Filho,
Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, RJ-Brazil;
luana.p.borba@gmail.com (L.P.B.-S.); rozental@biof.ufrj.br (S.R.)
2
3
Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense,
Niterói-RJ 24210-141, Brazil; gqofernando@vm.uff.br
*
Correspondence: vitorferreira@id.uff.br; Tel.: +55-21-998578148
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Ericsson Coy-Barrera
Received: 11 February 2019; Accepted: 4 March 2019; Published: 7 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Sporotrichosis is a neglected fungal infection caused by Sporothrix spp., which have a
worldwide distribution. The standard antifungal itraconazole has been recommended as a first-line
therapy. However, failure cases in human and feline treatment have been reported in recent years.
This study aimed to synthesize several α- and β-2,3-dihydrofuranaphthoquinones and evaluate them
against Sporothrix schenckii and Sporothrix brasiliensis—the main etiological agents of sporotrichosis in
Brazil. The stability of these compounds was also investigated under different storage conditions for
3 months. The samples were removed at 0, 60, and 90 days and assessed by 1H-NMR, and their in vitro
antifungal susceptibility was tested. Furthermore, we evaluated the superficial changes caused
by the most effective and stable compounds using scanning electron microscopy and determined
their effects when combined with itraconazole. Nine dihydrofuranaphthoquinones showed good
antifungal activity and stability, with MIC values of 2–32 µM. Compounds 6 and 10 were the most
active dihydrofuranaphthoquinones in vitro for both species; in fungi, these compounds induced
yeast–hyphae conversion and alteration in the hyphae and conidia structures. Compound 10 also
exhibited a synergistic activity with itraconazole against S. schenckii, with a
ΣFIC index value of 0.3.
Our results indicate that Compounds and 10 are potential candidates for the development of new
6
antifungal agents for the treatment of sporotrichosis.
Keywords: furanophthoquinones; antifungal; Sporothrix brasiliensis; Sporothrix schenckii; stability study
1. Introduction
Fungal infections are opportunistic and neglected diseases that are difficult to treat and are
therefore a serious global public health problem for humans, plants, and animals. It is estimated
that there are approximately 1.5 million species of fungi, of which 8000 are known to cause diseases
Molecules 2019, 24, 930; doi:10.3390/molecules24050930
www.mdpi.com/journal/molecules

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in plants and more than 300 affect humans [
1
]. These infections affect more than one billion people
worldwide and can cause more than 1.5 million deaths annually [2,3]. Candida, Aspergillus, Pneumocystis,
and Cryptococcus are the most common fungi species that cause serious diseases in humans and lead to
morbidity and mortality, especially among severely ill or immunocompromised patients or those with
complicated medical conditions [4,5]. Resistance of fungi to antifungal drugs poses another threat to
public health, food biosafety, and biodiversity.
The development of new drugs, diagnostic kits, and antifungal therapies has received little
attention from development agencies and pharmaceutical industries, particularly when compared
with bacterial and viral infections.
Among neglected mycoses, sporotrichosis is a subacute or chronic disease that affects humans
and several animals, especially felines [6]. Sporotrichosis is caused by several thermodimorphic fungi
species of the Sporothrix genus, such as Sporothrix brasiliensis, Sporothrix schenckii, Sporothrix globosa,
Sporothrix mexicana, and Sporothrix luriei [7].
The S. schenckii is the main etiological agent of sporotrichosis in the world [
8], but in Brazil,
S. brasiliensis prevails as the main etiological agent of human and animal sporotrichosis [
9
–
11].
Currently, S. brasiliensis has become the most frequent species associated with feline and human cases
of sporotrichosis in the country [12]. The infection is usually acquired by the traumatic inoculation of
this dimorphic fungus by suppurating subcutaneous nodules that slowly progress along the lymphatic
channels [13].
In the past, cutaneous and lymphocutaneous sporotrichosis infections have been treated
with saturated solutions of potassium iodide, which have low effectiveness and present adverse
effects [14]. Currently, some more effective antifungal drugs, such as amphotericin B (
), itraconazole ( ), and fluconazole ( ) [15] are in the therapeutic arsenal (Figure 1) [16
regard, itraconazole ( ) is the most common and effective drug used for sporotrichosis infection in
clinical treatments [ ]. However, it is important to note that several failure cases have already been
reported in both human and feline treatment [20,21].
1), terbinafine
(2
3
4
–
19]. In this
3
8
Figure 1. Some drugs currently used in the treatment of sporotrichosis.
The discovery of novel, effective, and stable antifungal agents is a long-term process with high
cost. Therefore, the search for new antifungal agents is extremely necessary and urgent. Regarding the
Sporothrix infection, few synthetic compounds [22–24], natural products [25], and crude extracts [26,27]
have been investigated.
Natural products have been an important source of bioactive substances and have been an
inspiration for the synthesis of new drug prototypes. Among these products, naphthoquinones
are widely distributed in plants, lichens, bacteria, and higher animals, and they perform functions

Molecules 2019, 24, 930
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in vital biochemical processes. Over the last few years, this class of compounds has gained
remarkable attention due to their diversified biological activities, including their bactericidal [28],
leishmanicidal [29], antimalarial [30], tripanocidal [31], antitubercular [32], anticancer, and antifungal [33
]
effects. Their mechanism of action is related to redox cycles, arylation of the thiol groups of proteins,
intercalation, the induction of breaks in the DNA chain, the generation of free radicals and other reactive
oxygen species (ROS), and bioreductive alkylation via the formation of quinone methide. A number
of studies have demonstrated that substituted pyranaphthoquinones [34] show a particular activity
against Candida species and dermatophytes fungi [35
and synthetic naphthoquinones against the Sporothrix genus [24
,
36]. However, there are few studies about natural
37 38] as well as their sensitivity to
,
,
temperature [39]—an important information that would allow their manufacture, formulation, storage,
and administration.
Tandon and coworkers reported the synthesis of some hetero-1,4-naphthoquinone derivatives
and evaluated their antifungal and antibacterial activities, including those against S. schenckii.
The results indicated that several derivatives had potent antifungal activity compared to clinically
prevalent antifungal drugs against S. schenckii, reaffirming the importance of quinones as a drug
candidate [24,40–45].
Considering our preliminary studies involving
against various biological targets as well as our research program on the synthesis of biologically
active naphthoquinones [35 46], we became interested in the synthesis of substituted - and
-2,3-dihydrofuranaphthoquinones and their in vitro antifungal activities against S. brasiliensis and
α- and β-pyran and α- and β-furanaphthoquinones
,
α
β
S. schenckii. For this purpose, we synthesized 17 naphthoquinones and evaluated their chemical
stability. We determined the inhibitory activities of these compounds and compared them with
itraconazole (3). The most effective and stable naphthoquinones were selected, and we evaluated their
effect on fungal morphology, their interaction with itraconazole (3), and their hemolytic activity.
It is well established that, in addition to the search of new molecules, it is important to investigate
the safety and effectiveness of these compounds at different temperatures, which can compromise a
possible candidate drug. Therefore, the evaluation of the stability of drug products is an essential stage
in the formulation development process of the pharmaceutical industry and is an important tool for
obtaining information for clinical trials for the evaluation of new drug registration. The purpose of
the stability test is to provide evidence of how the quality of a product changes with time under the
influence of various environmental factors such as temperature, humidity, and light [47].
2. Experimental Methods
2.1. Materials
Itraconazole (Sigma Chemical, Co., St. Louis, MO, USA) and α- and β-2,3-dihydrofuranaphthoquinones
were diluted in dimethyl sulfoxide (DMSO) to obtain stock solutions of 1 mM and 50 mM, respectively.
◦
Compounds were kept at
−
20 C until testing, when they were diluted in RPMI 1640 medium
supplemented with 2% glucose, and buffered with 0.165 M 3-(N-morpholino)propanesulfonic acid at
pH 7.2.
2.2. Synthesis of α- and β-2,3-Dihydrofuranaphthoquinones 5–21
All the dihydrofuranaphthoquinones used for antifungal screening against S. brasiliensis and
S. schenckii were prepared from lawsone by different methods (Scheme 1) with a yield of 23–76%.
The
literature [35
spectrometer using chloroform-D as a solvent and tetramethylsilane as an internal standard. The
and -2,3-dihydrofuranaphthoquinones 19 21 were prepared from nor-lapachol, as previously
described [49,50].
α
- and
β
-2,3-dihydrofuranaphthoquinones
5–18 were prepared as previously described in the
,
48] and were confirmed by 1H-NMR spectra recorded on a Varian VNMR (500 MHz)
α
-
β
–

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Scheme 1. Synthetic routes for preparing α- and β-2,3-dihydrofuranaphthoquinones 5–21.
2.3. Yeast Sources and Manipulation
S. brasiliensis CBS 133,006 and S. schenckii ATCC 3228 yeast strains were used in this study and
◦
were stored in a saline solution with 10% glycerol and 10% glucose at
−
20 C until use. The conversion
of the isolate to the yeast (pathogenic form) was performed from the◦filamentous form (saprophytic
form). Mycelium forms were cultivated on Sabouraud broth at 36 C for 7 days. The conversion
to the yeast phase was achieved by adding 105 conidia/mL to brain heart infusion (Difco) broth
supplemented with 2% glucose, pH 7.8 at 36 ◦C with orbital agitation (150 rpm) for 7 days.
2.4. Stability Study
A stability study of α- and β-2,3-dihydrofuranaphthoquinones was conducted for 3 months under
different storage conditions. The samples were placed into 2 mL Eppendorf and then stored at ambient
room temperature (25 ◦C) and under refrigeration (
and assessed by 1H-NMR and antifungal activity.
−
20 ◦C). Samples were taken at 0, 60, and 90 days

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2.5. Susceptibility Assays
2.5.1. Antifungal Assay of Compounds 5–21
α
- and β-2,3-Dihydrofuranaphthoquinones and itraconazole (standard) were diluted in
RPMI 1640 medium (supplemented with 2% glucose and buffered at pH 7.2, and 0.165 M
3-(N-morpholino)propanesulfonic acid (MOPS)) and added into flat-bottom 96-well microplates to
obtain a final concentration of 32
yeast concentration ranging from 0.5–1
in a dark 5% CO2 chamber. Fungal growth was evaluated by visual inspection in an inverted light
microscope and quantified by spectrophotometric readings at 492 nm in a microtiter plate reader
(Emax ELISA plate reader Molecular Devices, Sunnyvale, CA, USA).
µ
M. Sporothrix yeasts were added into a microtiter plate at a final
×
105 ufc/mL; treatments were performed for 48 h at 35 ◦C
2.5.2. Minimum Inhibitory Concentration Determination
The minimum inhibitory concentrations (MIC) of
α- and β-2,3-dihydrofuranaphthoquinones
5
–21 were determined by the broth microdilution test as described in the Clinical and Laboratory
Standards Institute (CLSI) protocols M27-A3 [51], with minor adaptations for the use of Sporothrix spp.
yeasts. The compounds were diluted in RPMI 1640 medium buffered at pH 7.2 with 0.165 M MOPS
with 2% glucose into 96-well plates to obtain final concentrations of 0.06–32
and 0.002–1 M for itraconazole ( ). Then, 100 L of the fungal suspension was dispensed into each
well for a final yeast cell density of 0.5–1.5
105 colony forming units (CFU)/mL. The samples were
µM for naphthoquinones
µ
3
µ
×
incubated for 48 h in the dark in a humid chamber with 5% CO2. The fungal growth inhibition was
observed visually with an inverted optical microscope. Subsequently, the samples were homogenized,
and the OD was measured using a microplate reader (Emax ELISA plate reader Molecular Devices,
Sunnyvale, CA, USA) at 492 nm. MIC corresponds to the lowest drug concentration that inhibits 50%
of fungal growth relative to untreated controls.
2.5.3. Combination Assay
The effects of interactions between the most effective
10 and itraconazole ( ) were evaluated using the checkerboard microdilution method [52]. Concentrations
tested ranged from 0.25 to 16
itraconazole, respectively. Yeasts (0.5–1
α- and β-2,3-dihydrofuranaphthoquinones 6 and
3
µ
M or from 0.001 to 1
µ
M for the dihydrofuranaphthoquinones and
◦
×
105 CFU/mL) were treated for 48 h at 35 C in a 5% CO2
chamber. The MIC values for individual compounds and for their combinations were determined,
and a better combination was defined as the lowest value of the fractional inhibitory concentration
index (ΣFIC index), which is calculated using the equation: ΣFIC index = (MICnaphthoquinone in
combination/MICnaphthoquinone alone) + (MICitraconazole in combination/MICitraconazole alone) [53].
Interactions were considered to be synergistic if FICI ≤ 0.5 [52].
2.6. Scanning Electron Microscopy
Yeasts were incubated with a sub-inhibitory concentration (1/2 MIC) of the most effective
naphthoquinones at 36 ◦C with orbital agitation for 48 h. Untreated and treated cells were washed
three times in phosphate buffered saline (PBS) and fixed in a solution of 2.5% glutaraldehyde
◦
and 4% formaldehyde in a 0.1 M cacodylate buffer for 24 h at 4 C. The cells were plated on
poly-L-lysine-covered glass coverslips and postfixed in 1% osmium tetroxide in a 0.1 M cacodylate
buffer containing 1.25% potassium ferrocyanide and 5 mM CaCl2 for 30 min. Samples were washed,
dehydrated in increasing ethanol concentrations (30% to ultradry), critical-point-dried in CO2,
and coated with gold. The images were obtained using a Quanta FEC 250 scanning electron microscope
(Thermo Scientific, Hillsboro, OR, USA).

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2.7. Hemolytic Assay
Human red blood cells (RBCs) were collected in accordance with an approved protocol from the
Human Research Ethics Committee of Hospital Universitário Clementino Fraga Filho, UFRJ (protocol
number # 50765015.5.0000.5257). Solutions of the most effective naphthoquinones were prepared
in DMSO to obtain stock solutions of 100 mM and were diluted in a saline solution to different
concentrations. Naphthoquinones were added to microplate wells to obtain final concentrations
ranging from 0.5 to 1000 µM. RBC suspensions were then prepared in a saline solution (0.9% NaCl
in ultra-pure water, pH 7.2) and added to microplate wells at a final density of 0.5%. RBCs were
incubated in the presence of naphthoquinones for 1 h at 37 ◦C before centrifuging at 3000 rpm for
15 min and transferring the supernatant to a new microplate. The absorbance of hemoglobin released
from RBCs was measured at 562 nm using a microtiter plate reader (EMax Plus, Molecular Devices),
and the concentration that lysed 50% of RBCs (HA50) was determined [35].
2.8. Thermal Analyses
Differential scanning calorimetry (DSC) analyses were performed using a SHIMADZU differential
scanning calorimeter DSC-60A (SHIMADZU, Columbia, SC, USA.) thermal analyzer in a nitrogen
dynam◦ic atmosphere with a flow rate of 50 mL. min−1 at a heating rate of 10 ◦C min−1 over the range of
25–300 C; for DSC analyses, 3.0 mg of sample was evaluated in a closed aluminum cell. Thermogravimetric
(TG) analyses were performed using a NETZSCH STA 409 PC/PG (Netzsch-Gerätebau GmbH, Selb,
Germany) in a nitrogen dynamic atmosphere with a flow rate of 60 mL min−1 and a heating rate
of 10 ◦C min−1 over the range of 30–300 ◦C; for TG analyses, 6 mg of sample was evaluated in an
aluminum cell. An empty aluminum cell was used as a reference for both analyses.
3. Results and Discussion
Dihydrofuranaphthoquinones
screening against S. brasiliensis and S. schenckii growth at 32
5
–
21 were prepared and evaluated in a preliminary inhibitory
M, as shown in Figures 2 and 3,
µ
respectively. All compounds evaluated were able to inhibit the growth of both fungi. From these
preliminary tests, it was observed that only one compound (12) had activity below 50%, two
compounds (
9
and 19) had activity between 60 and 70%, three compounds (11 15, and 16) had
,
activity of approximately 70%, and eleven compounds (
6
,
7,
8,
10 13 14 15 17 18, 20, and 21) were
,
,
,
,
,
able to inhibit the fungi growth by more than 70% and were similar in activity to itraconazole (p > 0.05)
against both species. Among the most active compounds, three were -dihydrofuranaphthoquinones
13, and 17), but eight were the -dihydrofuranaphthoquinone isomers ( 10 14 15 18 20, and
21), indicating that these isomers were the most active series as shown in Figure 1, Figure 2, and Table 1,
respectively. The concentration of 32 M was selected for this preliminary test due to the precipitation
of naphthoquinones in a more concentrated medium.
α
(7
,
β
6,
8,
,
,
,
,
µ
S. brasiliensis
100
90
80
70
60
50
40
30
20
10
0
Figure 2. Evaluation of 5–21 against S. brasiliensis growth at 32 µM.

Molecules 2019, 24, 930
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S. schenckii
100
90
80
70
60
50
40
30
20
10
0
Figure 3. Evaluation of 5–21 against S. schenckii growth at 32 µM.
Table 1. % Inhibition at 32 µM of naphthoquinones 5–20.
Compounds
%Inhibition at 32 µM a
S. brasiliensis
S. schenckii
Itraconazole
87.5 ± 12.5
95.7 ± 3.1
93.8 ± 2.6
100.0 ± 0.0
98.2 ± 2.4
5
6
7
8
88.3 ± 12.8
90.5 ± 7.5
80.3 ± 22.4
91.0 ± 12.5
67.8 ± 19.5 *
96.7 ± 4.7
93.3 ± 11.2
63.3 ± 16.0 *
98.3 ± 1.9
9
10
11
12
13
14
15
16
17
18
19
20
21
69.5 ± 13.4
49.5 ± 21.8 ***
94,2 ± 11.4
96.3 ± 5.7
61.2 ± 18.1 **
56.7 ± 6.8 ***
76.5 ± 19.9
91.2 ± 9.8
70.7 ± 17.0
69.8 ± 14.5
92.3 ± 6.1
54.0 ± 7.5 ***
46.7 ± 8.2 ***
86.7 ± 10.6
97.2 ± 2.2
100.0 ± 0.0
58.3 ± 18.5 **
87.2 ± 19.2
78.5 ± 13.7
49.3 ± 24.6 ***
85.7 ± 15.5
76.3 ± 24.0
a Mean
±
Standard Deviation of two independent experiments, made in triplicate. * p < 0.05, ** p < 0.001, *** p < 0.0001
vs. treatment with itraconazole (by one-way ANOVA and Dunnett’s test).
After preliminary evaluation as reported in Figures 2 and 3, the active derivatives were submitted
to a broth microdilution assay to determine the lowest concentration capable of inhibiting fungi
growth by 50% (MIC—minimum inhibitory concentration). Additionally, a stability study of the
dihydrofuranaphthoquinones vs. antifungal activity was performed for different time periods and at
different storage temperatures. Samples were also analyzed by 1H-NMR spectroscopy (Table 2).
The MIC values obtained for selected compounds at T0 indicate that
isomers have higher antifungal activities against both Sporothrix species when compared to their
-dihydrofuranaphthoquinone isomers. This is most evident when the MIC values of the compounds
, and 18 are compared with those of their respective isomers -dihydrofuranaphthoquinones
and 17.
β-dihydrofuranaphthoquinone
α
6
,
8
α
5, 7,

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Table 2. Antifungal activity of dihydrofuranaphthoquinones over time and at different storage
temperatures against S. brasiliensis and S. schenckii fungi.
Time and Condition
MIC (µM)
DihydrofuraNa-Phthoquinone
Time (day)
S. brasiliensis
S. schenckii
0
16
8
16
16
8
32
16
16
16
4
60, refrigerator
60, room temperature
90, refrigerator
5
90, room temperature
0
4
4
4
4
4
4
4
4
4
4
60, refrigerator
60, room temperature
90, refrigerator
6
7
90, room temperature
0
32
8
8
16
16
32
16
16
16
16
60, refrigerator
60, room temperature
90, refrigerator
90, room temperature
0
4
8
60, refrigerator
8
8
60, room temperature
90, refrigerator
90, room temperature
16
16
16
32
16
16
8
0
2
4
4
4
16
4
4
4
4
8
60, refrigerator
60, room temperature
90, refrigerator
10
17
18
20
90, room temperature
0
16
16
16
>32
>32
32
16
16
>32
>32
60, refrigerator
60, room temperature
90, refrigerator
90, room temperature
0
4
4
4
8
8
8
8
8
8
8
60, refrigerator
60, room temperature
90, refrigerator
90, room temperature
0
16
16
16
>32
>32
16
16
16
32
32
60, refrigerator
60, room temperature
90, refrigerator
90, room temperature
0
32
4
16
8
60, refrigerator
60, room temperature
90, refrigerator
4
4
8
8
21
90, room temperature
4
8
Itraconazole
0.25
0.125
MIC: minimum inhibitory concentration. The results represent two independent experiments in duplicate. T0 = 0
days; T60 = 60 days; T90 = 90 days.
The samples were submitted to a stability study and were analyzed by 1H-NMR at T0, T60, and
T90; their spectra showed no changes. The biological activity of samples stored at room temperature

Molecules 2019, 24, 930
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and under refrigeration showed some variations. One of the most distinct features observed in Table 2
is that Compounds and 10 exhibited the lowest MIC values against S. brasiliensis and S. schenckii after
90 days under both storage conditions. The 1H-NMR spectra of compounds
and 10 were used in the
study of their stabilities and are available in the supplementary materials. However, their antifungal
6
6
activity was not higher than itraconazole, which displayed MIC values of 0.25 and 0.125
S. brasiliensis and S. schenckii, respectively.
µM for
We also analyzed the superficial changes in the Sporothrix cells induced by
β-2,3-
dihydrofuranaphthoquinones
6 and 10. The fungi were treated with sub-inhibitory concentrations
of these compounds for 48 h, and cells were visualized by scanning electron microscopy. Untreated
S. brasiliensis exhibited the most cells in yeast form, with oval or elongated cells showing multiple
budding and some hyphae (Figure 4A); the untreated S. schenckii showed mainly filamentous
morphology, with thin hyphae and conidia adhered (Figure 4B). S. brasiliensis treated with 1
of Compound 10 showed cells mainly in a filamentous form with an altered morphology (Figure 4C).
S. schenckii treated with 2 M of Compound 10 exhibited altered hyphae with a ruptured cell wall
(Figure 4D). Treatment of both species with 2 M of Compound led to the alteration in hyphae
µM
µ
µ
6
morphology and conidia structure, mainly S. brasiliensis cells (Figure 4E,F). These results indicate that,
even at sub-inhibitory concentrations, the compounds were able to induce damage in Sporothrix spp.
Figure 4. Scanning electron microscopy images of S. brasiliensis (
A,
C,E
) and S. schenckii (
B,D,
F) after
exposure to sub-inhibitory concentrations of Compound 10
compared to untreated cells (A and B) (bars = 10 µm).
(C
and
D) and Compound
6
(E
and F)

Molecules 2019, 24, 930
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In view of the high effectiveness and stability of
we decided to test whether these compounds could improve itraconazole (
with the FIC index <0.5 were categorized as synergistic, and those with FIC index values from >0.5
to 4 were categorized as having no interaction [53]. As shown in Table 3, Compound 10 exhibited a
synergistic activity with itraconazole against S. schenckii with a FIC index value of 0.3 i. Importantly,
β-2,3-dihydrofuranaphthoquinones
6 and 10,
3
) activity. Interactions
Σ
Σ
Σ
it should be noted that the MIC of itraconazole was reduced when S. schenckii was coincubated with
Compound 6.
Table 3. Associations of itraconazole and Compounds
yeast cells. MIC values are in µM.
6
and 10 against S. schenckii and S. brasiliensis
MIC b Drugs in Combination
MIC Drugs Tested Alone
ΣFIC Index a
Itraconazole
0.25
6
4
4
Itraconazole/6
0.25/0.5
S. brasiliensis
S. schenckii
1.13
0.54
0.125
0.06/0.25
Itraconazole
10
Itraconazole/10
S. brasiliensis
S. schenckii
0.25
0.125
2
4
0.25/0.25
0.03/0.25
1.13
0.30
a
Fractional inhibitory concentration index;
ΣFIC index = (MICa in combination/MICa tested alone) + (MICb
in combination/MICb tested alone), where a and b are itraconazole and naphthoquinone (10 or
Combinations with the lowest ΣFIC index. MIC was the lowest concentration able to inhibit 50% of fungal growth
6
), respectively.
b
(mean of two independent experiments).
Finally, the hemolytic activities of Compounds
effect toward RBCs was observed for either compound at concentrations up to 1000
6
and 10 were studied, and no hemolytic
M. Thus,
these compounds were more selective for the fungi because much smaller concentrations affected
the fungi when compared to the RBCs. These results also confirm that Compounds and 10 do
µ
6
not affect the plasma membrane of eukaryotic cells; if this happened, we would see hemolysis at
low concentrations.
Thermogravimetry (TG) and differential scanning calorimetry (DSC) are techniques with potential
for application in pharmaceutical research to determine the physicochemical properties of compounds
such as polymorphism, purity, and thermal stability. In addition, they are essential for preformulation
studies because they provide important information about the physical characteristics or chemical
interactions between drugs and excipients [54,55]. Therefore, it is important to study thermal
degradation and, consequently, to evaluate the quality control of Compounds 6 and 10 using techniques
such as TG and DSC. The difference between the two techniques is that TG measures mass variation,
whereas DSC allows for the measurement of changes in enthalpy. Both analyses are performed using a
heating program [56].
Figure 5 shows the DSC curves obtained for quinones
Compound
show that the naphthoquinone is thermally stable up to 200 ◦C. According to the TG
curve of Compound (Figure 6), in the first step, the quinone lost approximately 6.2% of mass between
6 and 10. The TGA and DTG curves of
6
6
100 and 120◦◦C and continued to lose mass. This initial mass loss is associated with an endothermic
peak at 110 C in the DSC curve (Figure 5), corresponding to the melting of the compound. The fusion
is characterized as a physical phenomenon, which can be detected through DSC or DTA curves,
and presents itself as an endothermic event [57]. The additional mass loss of approximately 20%
associated with a set of successive exothermic and endothermic peaks at 200 and 225 ◦C, respectively,
in the DSC curves may be due to the decomposition of the compound. At higher temperatures (above
225 ◦C), the peaks may be attributed to the pyrolysis of the compound.
Compound 10 also shows continual mass loss between 200 and 350 ◦C (Figure 7). This mass loss
is associated with an endothermic peak at 150 ◦C in the DSC curve (Figure 5), which is also related to
the fusion of the compound. In summary, according to TG and DSC curves, Compound
6 is thermally
stable only until approximately 150 ◦C. Above this temperature, the naphthoquinone is decomposed.

Molecules 2019, 24, 930
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An exothermic peak at 200 ◦C may be due to the decomposition of the compound, which is followed
by another exothermic peak at 250 ◦C that is likely attributed to the pyrolysis of the compound.
Figure 5. DSC curves of dihydrofuranaphthoquinones 6 and 10.
Figure 6. TG curves of dihydrofuranaphthoquinones 6.
Figure 7. TG curves of dihydrofuranaphthoquinones 10.

Molecules 2019, 24, 930
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4. Conclusions
In conclusion, we synthesized a series of substituted
α
- and
β-2,3-dihydrofuranaptoquinones,
which were confirmed by 1H-NMR data and are consistent with those reported in the literature.
The antifungal profile indicated that Compounds and 10 presented potent antifungal activity against
6
the main etiological agents of sporotrichosis in Brazil—S. brasiliensis and S. schenckii. Studies indicate
the existence of several strains of S. brasiliensis and S. schenckii that are resistant to itraconazole, and this
fact led us to evaluate the effect of Compounds
6 and 10 in combination with itraconazole. Compound
10 showed a synergistic action against itraconazole against S. schenckii resulting in a reduction of
MIC, showing that this is a promising strategy for the treatment of Sporotrichosis with the use of
itraconazole and naphthoquinone combinations. In addition, it was possible to highlight the main
thermal events of these compounds, which is very important for the quality control of a candidate
drug or drug combination. This work opens perspectives for future studies in the attempt to discover
new antifungal agents against S. brasiliensis and S. schenckii based on naphthoquinone framework.
Supplementary Materials: Supplementary Materials are available online.
Author Contributions: P.G.F., L.L.N., C.D.N. and M.d.S.H.Q. performed the synthesis of the compounds, literature
search and drafted the manuscript. L.P.B.-S. performed the antifungal essays, literature search and drafted the
manuscript. F.d.C.d.S., S.R., D.O.F. and V.F.F. contributed with the development of concept, data analysis,
and writing the final manuscript.
Funding: This research was funded by CAPES that granted the scholarships, CNPq (303713/2014-3), CNPq
(305915/2017-7), FAPERJ (E-26/2003.085/2016) and FAPERJ (E-26/2002.800/2017, E-26/200.930/2017) for
financial support.
Acknowledgments: Authors wish to thank Juliana Silva of the Laboratório de Biologia Celular de Fungos for her
help with susceptibility tests. The authors are grateful to professor Wanderley de Souza and colleagues from the
Laboratory of Cellular Ultrastructure Hertha Meyer for providing support for preparation of scanning electron
microscopy samples, and the Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO, UFRJ, Rio de
Janeiro, Brazil) for facilities and support for the use of scanning electron microscopy equipment and FIOCRUZ for
the HRMS.
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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