Synthesis and biological evaluation of Hydrazone derivatives as antifungal agents

Article abstract of DOI:10.3390/molecules20059229

Emerging yeasts are among the most prevalent causes of systemic infections with high mortality rates and there is an urgent need to develop specific, effective and non-Toxic antifungal agents to respond to this issue. In this study 35 aldehydes, hydrazones and hydrazines were obtained and their antifungal activity was evaluated against Candida species (C. parapsilosis, C.Tropicalis, C. krusei, C. albicans, C. glabrata and C. lusitaneae) and Trichosporon asahii, in an in vitro screening. The minimum inhibitory concentrations (MICs) of the active compounds in the screening was determined against 10 clinical isolates of C. parapsilosis and 10 of T. asahii. The compounds 4-pyridin-2-ylbenzaldehyde] (13a) and tert-butyl-(2Z)-2-(3,4,5-Trihydroxybenzylidine)hydrazine carboxylate (7b) showed the most promising MIC values in the range of 16-32 μg/mL and 8-16 μg/mL, respectively. The compounds' action on the stability of the cell membrane and cell wall was evaluated, which suggested the action of the compounds on the fungal cell membrane. Cell viability of leukocytes and an alkaline comet assay were performed to evaluate the cytotoxicity. Compound 13a was not cytotoxic at the active concentrations. These results support the discovery of promising candidates for the development of new antifungal agents.

Full text of DOI:10.3390/molecules20059229

Molecules 2015, 20, 9229-9241; doi:10.3390/molecules20059229
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Hydrazone Derivatives as
Antifungal Agents
Bruna B. Casanova 1,*, Mauro N. Muniz 1, Thayse de Oliveira 2, Luís Flavio de Oliveira 3,
Michel M. Machado 3, Alexandre M. Fuentefria 2, Grace Gosmann 1 and Simone C. B. Gnoatto 1,*
1
Laboratório de Fitoquímica e Síntese Orgânica (LAFIS), Faculdade de Farmácia,
Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 90610-000, RS, Brazil;
E-Mails: mauro.neves.muniz@gmail.com (M.N.M.); grace.gosmann@ufrgs.br (G.G.)
2
Laboratório de Micologia Aplicada, Departamento de Analises, Faculdade de Farmácia,
Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre 90610-000, RS, Brazil;
E-Mails: thayseoliveira-@hotmail.com (T.O.); alexandre.fuentefria@ufrgs.br (A.M.F.)
3
Departamento de Farmácia, Universidade Federal do Pampa, Km 592, Uruguaiana 97500-970, RS,
Brazil; E-Mails: tcheluisoliveira-@gmail.com (L.F.O.);
michelmachado@unipampa.edu.br (M.M.M.)
* Authors to whom correspondence should be addressed;
E-Mails: bruna.casanova@ufrgs.br (B.B.C.); simone.gnoatto@ufrgs.br (S.C.B.G.);
Tel.: +55-51-3308-5451 (S.C.B.G).
Academic Editor: Derek J. McPhee
Received: 28 February 2015 / Accepted: 12 May 2015 / Published: 20 May 2015
Abstract: Emerging yeasts are among the most prevalent causes of systemic infections with
high mortality rates and there is an urgent need to develop specific, effective and non-toxic
antifungal agents to respond to this issue. In this study 35 aldehydes, hydrazones and
hydrazines were obtained and their antifungal activity was evaluated against Candida
species (C. parapsilosis, C. tropicalis, C. krusei, C. albicans, C. glabrata and C. lusitaneae)
and Trichosporon asahii, in an in vitro screening. The minimum inhibitory concentrations
(MICs) of the active compounds in the screening was determined against 10 clinical isolates
of C. parapsilosis and 10 of T. asahii. The compounds 4-pyridin-2-ylbenzaldehyde] (13a)
and tert-butyl-(2Z)-2-(3,4,5-trihydroxybenzylidine)hydrazine carboxylate (7b) showed the
most promising MIC values in the range of 16–32 μg/mL and 8–16 μg/mL, respectively. The
compounds’ action on the stability of the cell membrane and cell wall was evaluated, which
suggested the action of the compounds on the fungal cell membrane. Cell viability of

Molecules 2015, 20
leukocytes and an alkaline comet assay were performed to evaluate the cytotoxicity.
9230
Compound 13a was not cytotoxic at the active concentrations. These results support the
discovery of promising candidates for the development of new antifungal agents.
Keywords: aldehydes; hydrazones; emerging yeasts; antifungal activity
1. Introduction
Since the 1980s yeasts have emerged as some of the main agents of nosocomial systemic infections,
particularly in immunocompromised people or those who are undergoing intensive chemotherapy [1,2].
Approximately two decades ago Candida albicans represented 70%–80% of the clinical isolates from
fungaemia cases [3,4], but in the last years this has been changing. In 2011 for example the prevalence
of candidemia by non-albicans species of Candida in the world was about 50% [5]. Moreover, the most
frequent species of non-albicans Candida are C. glabrata, C. parapsilosis and C. tropicalis being the
C. parapsilosis the third most common species in clinical isolates [5–7]. Trichosporon is considered the
second most common genus cause of fungaemia in patients with hematologic malignant diseases, being
besides resistant to amphotericin and echinocandins.
Thereby, the emergent pathogenic species associated with the increase of antifungal resistance have
become a global problem for the success of the therapy against these microorganisms. In addition, most
of the antifungals used in fungaemia cases have several adverse effects and high costs [8].
In this context, compounds containing hydrazine and hydrazone functional groups are widely studied
due to their described tuberculostatic [9], anticonvulsant [10], analgesic, anti-inflammatory [11],
antiplatelet [12], antifungal [13], antiviral [14], antitumor [15] and antimalarial activities [16]. Thus,
according to Narang et al. [16], hydrazones constitute an important class of compounds for the
development of new chemical entities to treat various diseases of clinical importance and besides
presenting these interesting biological applications, they are easily synthesized in good yields.
Considering the clinical relevance of fungal infections and the broad biological activity demonstrated
for hydrazines and hydrazones, this study aimed to synthesize a series of compounds containing these
chemical functions and evaluate their antifungal activity against emerging species of C. parapsilosis and
T. asahii, their mechanism of action and in vitro cytotoxicity.
2. Results and Discussion
2.1. Synthesis
Twenty-eight compounds were synthesized: seven aldehydes, 14 hydrazones and seven hydrazines,
through a synthetic route that employs two steps from the aldehydes. All synthesized compounds are
shown in Table 1.

Molecules 2015, 20
9231
Table 1. All compounds tested in this work a.
R
R
R
1a b
H
6a b
3,5-OH
11a
12a
3,5-OMe
2,3,4-OMe
2a b 3-OH 7a b 2,3,4-OH
R
H
3a b 4-OH
4a b
4-Cl
8a
9a
3-OMe
4-OMe
13a 4-(2)pyridinyl)
14a 4-(4)NO2benzyl
O
5a b 2,4-OH 10a 2,4-OMe
1b
2b
3b
4b
H
6b c
3,5-OH
2,3,4-OH
3-OMe
11b
12b
3,5-OMe
3-OH
4-OH
4-Cl
7b
2,3,4-OMe
R
8b
13b 4-(2)pyridinyl)
14b c 4-(4)NO2benzyl
N
NHBoc
9b
4-OMe
5b 2,4-OH 10b 2,4-OMe
1c 10c 2,4-OMe
H
13c
4-(2)pyridinyl)
R
8c 3-Ome 11c 3,5-OMe
9c 4-Ome 12c 2,3,4-OMe
11
12
NH
NHBoc
a A total of 35 compounds were screened, seven commercially available ones purchased from Sigma-Aldrich
b
c
Co. (St. Louis, MO, USA) and 28 prepared by us. Unpublished compounds; the corresponding structural
elucidation data are presented in the Experimental Section. The other products are known compounds and were
identified by comparison with authentic samples.
The aldehydes 8a–12a were synthesized by O-methylation of the hydroxylated aldehydes 2a–3a,
5a–7a. Compounds 13a and 14a were obtained by Suzuki coupling between the appropriate boronic acid
and the brominated aryl compound. The aldehydes 1a–14a were subjected to coupling with
tert-butyl carbazate, forming the corresponding hydrazones. The final step was the reduction of these
hydrazones, which led to the hydrazines 1c and 8c–13c. The hydrazines produced from the hydrazones
2b–7b and 14b could not be analyzed due to the low yields.
2.2. Antifungal Activity
All the compounds shown in Table 1 were subjected to an antifungal activity screening at the
concentration of 500 μg/mL against seven clinical isolates: C. albicans, C. krusei, C. parapsilosis,
C. tropicalis, C. glabrata, C. lusitaneae and T. asahii. The active compounds of each class tested in the
screening are shown in Table 2.
Table 2. Active compounds of each class at the concentration of 500 μg/mL.
Column Heading
C. glabrata
C. parapsilosis
C. tropicalis
C. krusei
Aldehydes
Hydrazones
Hydrazines
2a, 5a, 7a, 13a, 14a
7b, 12b
12c
10c, 13c
13c
2a, 6a, 7a, 8a, 10a, 11a, 13a 2b, 5b, 6b, 7b, 11b, 13b
2a, 5a, 8a, 10a, 12a, 13a, 14a
2a, 5a, 12a, 13a, 14a
7b, 8b, 11b, 14b
7b, 9b, 12b
8c
C. lusitaneae
C. albicans
T. asahii
2a, 4a, 6a, 13a, 14a
2b, 6b, 7b, 9b, 11b
7b, 10b, 11b, 13b
13c
2a, 5a, 7a, 10a, 11a, 13a
1a, 2a, 5a, 6a, 8a, 13a
1c, 9c, 10c, 13c
3b, 5b, 6b, 7b, 11b, 13b 9c, 10c, 12c, 13c

Molecules 2015, 20
9232
Therefore, 30 compounds showed inhibitory activity against at least one species. The compounds 2a,
13a and 7b inhibited the growth of all species tested, while 3a, 9a, 11b, 4b and 11c did not inhibit the
growth of any. The species that showed the greatest susceptibility were T. asahii, C. parapsilosis and C.
albicans. C. parapsilosis is described as the third most frequently found species of non-albicans Candida
in clinical isolates [5–7] and Trichoporon as the second most important genus (only behind Candida) [5].
Therefore, the compounds that showed activity against C. parapsilosis (compounds 2a, 6a–8a, 10a, 11a,
13a, 2b, 5b–7b, 11b, 13b, 10c and 13b) and T. asahii (compounds 1a, 2a, 5a, 6a, 8a, 13a, 3b, 5b–7b,
11b, 13b, 9c, 10c, 12c and 13c) in the screening were selected for MIC determination. The MIC assay
was performed against 10 clinical isolates of each species (T. asahii and C. parapsilosis). The aldehyde
13a and the hydrazone 7b stood out for presenting MIC values <125 μg/mL against both species tested.
These results are shown in Table 3. The others compounds tested showed MIC values between 125 μg/mL
and 500 μg/mL. The variability of the responses can be due to the different susceptibility profiles the
isolates showed in previous studies [17]. Therefore, in such cases, mechanisms of resistance may be
constantly expressed, which results in these different profiles.
Table 3. Minimum inhibitory concentration (MIC) in µg/mL of the compounds 13a and 7b,
selected from the initial screening, against clinical isolates.
T. asahii
C. parapsilosis
Isolate
Isolate
13a
125
64
7b
16
16
16
8
Fluc a
Nt b
32
8
13a
7b
16
16
16
16
16
16
16
16
8
Fluc
TAH 05
TAH 06
TAH 07
TAH 09
TAH 10
TAH 11
TAH 12
TAH 13
TAH 14
TAH 15
RL 01
RL 05
RL 07
RL 13
RL 20
RL 27
RL 32
RL 33
RL 36
RL 38
32
32
32
64
16
32
32
32
32
32
64
≤1
≤1
≤1
4
64
250
32
Nt b
8
8
64
16
16
16
16
16
8
≤1
≤1
2
64
Nt b
4
64
32
4
2
32
4
8
4
a Fluconazole. b not tested. TAH: clinical isolate of T. asahii. RL clinical isolate of C. parapsilosis. The number
beside the acronym represents the identification in the culture collections of the Laboratory of Applied
Mycology Research of the Federal University of Rio Grande do Sul, Porto Alegre, Brazil.
An indication of the mechanism of action of the molecules could be observed when we assessed the
compounds’ activity against T. asahii TAH06 and C. parapsilosis RL01 isolates. Both isolates are
considered resistant to fluconazole according to the parameters established by the Clinical and Laboratory
Standards Institute (CLSI) [18], that determines that isolates presenting a MIC for fluconazole greater
than 8 μg/mL are considered resistant. Compound 7b showed a MIC of 16 μg/mL for both isolates, so
this activity may indicate differences in relation to the mechanism of action of fluconazole.
From the results of the MIC determination and the observation of distinct and diverse activities of
fluconazole our efforts were next focused on the investigation of the mechanism of action of the
active compounds.

Molecules 2015, 20
9233
2.3. Mechanism of Action
For the evaluated compounds, three tests were performed to investigate their mechanisms of action
as described below.
2.3.1. Evaluation of the Action on Cell Wall Stability: Sorbitol Protection Assay
The fungal cell wall serves as a protective barrier, preventing osmotic bursting of the cells. The cell
wall is, therefore, essential for growth and fungal viability in a hypotonic environment. This is
unnecessary if the fungi are protected with an osmotic support under specific conditions, so even if the
cells have their cell walls damaged by molecules that inhibit its synthesis, they will continue to grow.
Sorbitol was used in this experiment as osmotic protector [19]. The new MIC of these molecules was
determined with a YNB environment supplemented with glucose in parallel experiments with and
without the addition of 0.8 M sorbitol. The fungi treated with anidulafungin, an antifungal that acts on
the cell wall synthesis, started to grow after incubation for 7 days. As shown in Table 4, the derivatives
13a and 7b did not have activity on the cell wall of the isolates, because the MIC remained the same in
the reading at 2 and 7 days and in the environments with and without sorbitol.
Table 4. MIC in µg/mL of the compounds 13a and 7b, in the presence and absence of 0.8 M
of sorbitol in C. parapsilosis RL 33 and T. asahii TAH 10.
2 Days
7 Days
Compounds
−/Sorbitol +/Sorbitol −/Sorbitol +/Sorbitol
Anidulafungin
<1.0
16
<1.0
16
<1.0
16
>125
16
7b
13a
32
32
32
32
2.3.2. Evaluation of the Action on Cell Membrane Stability: Cellular Leakage Assay
When there is damage to the fungal cell membrane, cellular components such as nucleotides spill out
of the cell. These compounds exhibit a strong absorbance at 260 nm thus allowing its quantification, and
through these data it is possible to assess the extension of the damage to the fungal membrane, as shown
in Figure 1.
Figure 1. Release of intracellular components (absorbance read in 260 nm) of
C. parapsilosis RL 33 and T. asahii TAH 10 treated with the compounds 13a and 7b and
sodium dodecyl sulfate (SDS).

Molecules 2015, 20
9234
Taking the absorbance reading of 2% sodium dodecyl sulfate (SDS) as standard of 100% of cell lysis
we compared the results of the tested compounds. The compound 13a showed an absorbance of 90% of
the standard after 48 h, leading to the conclusion that it causes strong damage to the cell membrane
stability for both isolates. However, the compound 7b appears to cause damage to the membrane, but in
a more moderate manner when compared with the standard and with the former.
2.3.3. Evaluation of Action on Membrane Ergosterol: Ergosterol Effect Assay
To determine if the damage to cell membrane occurs by binding to sterols of the membrane, the MIC
of these compounds was determined again with and without the addition of ergosterol. In this test we
could evaluate if the activity of the derivative is due to binding to membrane ergosterol, once exogenous
ergosterol is added this prevents the binding of the compounds to endogenous ergosterol. As consequence,
in positive cases the MIC increases in the presence of exogenous ergosterol. The standard for this test
was amphotericin B, a drug that acts through this mechanism. As noted in Figure 2, none of the
derivatives showed a significant increase of the MIC, which led us to believe that they have a mechanism
of action that interferes with the cell membrane, but that does not involve an interaction with ergosterol.
Figure 2. Effect of exogenous ergosterol (50–250 μg/mL) in the MIC of compounds 13a,
7b and amphotericin B (AMP) against C. parasilosis RL33 and T. asahii TAH10.
2.4. Cell Viability and Genotoxicity
The cytotoxicity and genotoxicity were evaluated in order to elucidate the concentrations capable of
inducing cellular damage to both compounds. Our results demonstrate that compound 7b showed
toxicity at concentrations that had activity against yeasts and 13a exposure ranging from 8 to 128 µg/mL
had no effect on human leukocyte cell viability when compared to the negative control (p < 0.001)
(Figure 3).
Regarding the evaluation of damage to leukocyte DNA after exposure to compound 13a, the comet
assay demonstrated that the exposure concentrations tested were similar to the level of damage index
found in both negative and DMSO controls, which leads us to suggest that this compound is not able to
induce oxidative DNA damage in human leukocytes at the range of concentrations evaluated (Figure 4).

Molecules 2015, 20
9235
Figure 3. Percentage of cells in cell viability assay in human leukocytes exposed to different
concentrations of 13a. Data were analyzed by one-way ANOVA followed by Tukey test for
a, b, c
multiple comparisons and expressed average ± SEM, with p < 0.001;
the superscript
letters indicate statistical difference between groups; NC = negative control, DMSO C =
DMSO control; PC = positive control.
Figure 4. Index of DNA damage to human leukocytes exposed to different compound
concentrations of 13a. Data were analyzed by one-way ANOVA followed by Tukey test for
a, b
multiple comparisons and expressed average ± SEM, with p < 0.001.
the superscript
letters indicate statistical difference between groups; NC = negative control, DMSO C =
DMSO control; PC = positive control.
3. Experimental Section
3.1. General Information
All reactions requiring an inert atmosphere were carried out in a pre-dried apparatus under a nitrogen
atmosphere. The reactions were monitored by thin layer chromatography (TLC), performed on silica gel
60 F254 plates (Merck, Kenilworth, NJ, USA). Visualization was achieved using a UV lamp at a

Molecules 2015, 20
9236
wavelength of 254 nm. For the analysis of absorbance at 260 nm, a DU-600 spectrometer (Beckman,
Brea, CA, USA) was used.
1H- and 13C-NMR spectra were recorded with an Inova 400 and a VNMRS 400 spectrometer (Varian,
Palo Alto, CA, USA). Chemical shifts are shown in parts per million with tetramethylsilane (TMS) as a
reference. HR-EI-MS spectra were recorded with an UltrOTOF mass spectrometer (Bruker Daltonics,
São Paulo, Brazil).
The aldehydes 1a–7a and other reagents were purchased from a commercial supplier (Sigma-Aldrich
Co.). Compounds, 6b and 14b are unpublished and the corresponding structural elucidation data are
presented below. The other products are known compounds and were identified by comparison of their
physical and spectral data with those of authentic samples.
3.2. Chemistry
3.2.1. General Procedure for the Preparation of Derivatives 8a–12a
The O-methylation was performed by reaction of aldehydes 1a–7a with 4 equiv. of CH3I and 3 equiv.
of K2CO3 to protect each hydroxyl group in acetone with heating at 80 °C for 12 h in Schlenk flask [20].
Compounds were purified by column chromatography (cyclohexane/EtOAc, 9:1). Yields were 8a,
9a = 90%; 10a, 11a = 92% and 12a = 89%.
3.2.2. General Procedure for the Preparation of Derivatives 13a–14a
The brominated derivative (2-bromopyridine for 13a and 1-bromo-4-nitrobenzene for 14a) 1.1 equiv.
of 4-formylphenyl boronic acid, Pd(OAc)2 2 mol % and PPh3 4 mol % were added to a Schlenk flask.
Then, under an inert atmosphere methanol/THF 1:1 (2mL/mmol) and 2 equiv. of KOH were added. The
system was stirred for 24 h at 60 °C [21]. Compounds were purified by column chromatography
(cyclohexane/EtOAc, 9:1) in yields of 80% (13a) and 75% (14a).
3.2.3. General Procedure for the Preparation of Derivatives 1b–14b
The aldehydes 1a–14a were added to 1 equiv. of tert-butylcarbazate in a Schlenk flask under an inert
atmosphere in a 1:1 toluene/isopropanol mixture as solvent. The system was stirred for 2 h at 85 °C and
then stirring was continued for more 14 h at r.t. The precipitates were collected by filtration,
recrystallized and dried, yielding the products in 90%–95% yields [22].
Tert-Butyl(2Z)-2(3,5-dihydroxybenzylidene)hydrazinecarboxylate (6b). White powder; 91% yield;
1H-NMR (CD3OD) δ 7.7 (s, 1H), 6.6 (d, J = 2.2 Hz, 2H), 6.3 (t, J = 2.2 Hz, 1H), 1.5 (s, 9H); 13C-NMR
(CD3OD) δ 158.4, 154.1, 144.3, 136.3, 105.1, 103.8, 80.6, 80.5, 27.3. HRMS (ESI-MS, m/z),
(C12H16N2O4) calcd [M+H]+ 253.2664; found 253.2677.
Tert-Butyl(2Z)-2-[(4′nitrobiphenyl-4-l)methylene]hydrazinecarboxylate (14b). White powder; 90%
yield; 1H-NMR (CD3OD) δ 8.6 (s, 1H), 8.62 (d, J = 2.2 Hz, 2H), 7.8 (t, J = 2.2 Hz, 1H), 7.5 (s, 2H), 7.2
(s, 2H) 1.6 (s, 9H). 13C-NMR (CD3OD) δ 155.5, 148.1, 147.1, 146.9, 145.0, 128.8, 127.4, 123.9, 81.0,
28.4. (ESI-MS, m/z), (C18H19N3O4) calcd [M + H]+ 342.3613; found 342.3650.

Molecules 2015, 20
9237
3.2.4. General Procedure for the Preparation of Derivatives 1c, 8c–14c
The hydrazones 1b, 8b–14b were subjected to catalytic reduction with Pd/C 10% (0.2 equiv.) and
HCOONa (1.8 equiv.) dissolved in a 5:1 mixture of ethanol/water (1 mL/mmol). The mixture was stirred
for 1.5 h at 60 °C and then cooled down to a temperature below 40 °C and stirred for 12 h [22]. At the
end of the reaction, the solution was extracted with EtOAc, dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography (cyclohexane/EtOAc, 8:2) in yields of
70% (1c); 70% (8c, 9c, 12c) and 65% (11c, 13c).
3.3. Antifungal Activity
3.3.1. Isolates
The set of clinical isolates of Candida albicans, Candida krusei, Candida glabrata, Candida
tropicalis and Candida lusitaneae, Candida parapsilosis and Trichosporon asahii was obtained from
the culture collections of the Laboratory of Applied Mycology Research of the Federal University of Rio
Grande do Sul, Porto Alegre, Brazil.
3.3.2. Antifungal Activity Screening Assay
Antifungal activity screening assay was carried out using the broth microdilution method. In a
96-well plate samples were added to a final concentration of 500 µg/mL incubated at 35 °C for 48 h.
The molecules were considered active when there was no apparent fungal growth [23].
3.3.3. Minimal Inhibitory Concentration
Minimal inhibitory concentration (MIC) of the compounds was determined by the broth microdilution
method according to the M27-A3 documents published by the Clinical Laboratory and Standards
Institute [18] with RPMI-MOPS (RPMI 1640 medium containing L-glutamine without sodium
bicarbonate (Sigma-Aldrich Co.) buffered to pH 7.0 with 0.165 mol/L MOPS buffer (Sigma). The
concentrations of the extracts ranged from 0.5 to 256 µg/mL. The MIC was defined as the lowest
concentration of compounds at which the microorganism tested did not demonstrate visible growth.
3.4. Mechanisms of Action
3.4.1. Sorbitol Protection Assay
The MIC values were determined using the broth microdilution procedure according to the CLSI [18].
The cells were inoculated at a final concentration of 2 × 103 CFU/mL and cultured in Yeast Nitrogen
Base (Difco, Detroit, MI, USA) with 0.5% glucose and incubated at 30 °C. Duplicate plates containing
the test samples were prepared and in one of them 0.8 M sorbitol was added as an osmotic support
environment. The plates were read at 2 and 7 days [19,24,25]. Anidulafungin (Ecalta®) was purchased
from Pfizer® (New York City, NY, USA; each 1.0 g of injectable product contains 100 mg of
anidulafungin) and used as a control for this mechanism assay.

Molecules 2015, 20
9238
3.4.2. Cellular Leakage Assay
Cells were cultivated in MOPS tampon (Sigma-Aldrich Co., St. Louis, MO, USA), pH 6 to
107 CFU/mL and transferred to tubes. The compounds were added at a final concentration = MIC.
Sodium dodecyl sulfate, (SDS, 2%) was used as reference compound that produces 100% of cell loss.
During incubation at 30 °C, aliquots were taken at timed intervals (24, 48 h) [19,25].
3.4.3. Ergosterol Effect Assay
Performed by CLSI standard broth microdilution in duplicate, in the absence and presence of different
concentrations (50–250 µg/mL) of ergosterol (Sigma-Aldrich Co.) added to the environment.
Amphotericin B (AMP, 972 μg/mg kindly supplied by Cristália®, Itapira, Brazil) was used as a control
for this mechanism assay. The MIC was determined after 24 h of incubation [19,25].
3.5. Cytotoxicity
For the assays cell viability and alkaline comet samples were used in triplicate. The division of the
groups for the assays was designed as follows: Negative control, which consisted of adjusted leukocyte
suspension (LS, 130 cells/mm3) in PBS tampon pH 7.4; Positive control, consisting of the LS and the
addition of 4 mm H2O2; DMSO control, consisting of the LS and addition of DMSO 0.5%. In both
experiments the compounds were tested at 5 concentrations (8, 16, 32, 64, 128 µg/mL) starting from the
active concentrations in determining the MIC. All groups were incubated for an hour and subjected to
inversion every 10 min at an environment temperature of 25 °C.
3.5.1. Cell Viability
To evaluate the cytotoxicity the assay of cell viability was performed, considering the loss of
membrane integrity by the method of trypan blue [26]. Briefly, 100 µL of LS after incubation was mixed
with 100 µL of trypan blue 0.4% for 8 min. Cell viability was determined microscopically (magnification
400×) in a Neubauer chamber categorizing two scores: viable cells, which appear discolored or slightly
colored by blue; and dead cells which are colored by blue. For this test, 300 cells were counted for each
analyzed sample.
3.5.2. Genotoxicity
To evaluate the genotoxicity it was used the alkaline comet assay [27], and in accordance to the
guidelines for performing the comet assay [28]. Blades analysis was performed on microscope counting
100 cells per blade and classified in damage levels from 0 (no damage) to 4 (maximum damage). The
mean obtained from each treatment allowed the calculation of the damage rate, where the number of
cells was multiplied by the level of damage related, according to the scale of damage [28].
3.6. Statistical Analysis
All data were treated by analysis of variance (ANOVA) and complemented by Bartlett Test, when
relevant, accepting p < 0.05.

Molecules 2015, 20
9239
4. Conclusions
In this study thirty-five compounds, among them 28 synthesized in yields above 80%, were evaluated
for in vitro antifungal activity against seven species of yeasts. Two molecules, 13a and 7b, stood out,
with MIC values between 8 and 32 μg/mL, against clinical isolates of C. parapsilosis and T. asahii,
species of great epidemiological importance and of great severity in infections. It was further observed
that such compounds demonstrated great effect on cell membrane stability, but no interaction with
ergosterol. Compound 13a did not show cytotoxicity against leukocytes or genotoxic effects. It has been
shown, therefore, that this molecule presents promising activity against emerging yeasts, and could be
of great importance in the development of alternatives for the treatment of such infections.
Acknowledgments
This work was supported by the Brazilian agencies: National Council for Scientific and Technological
Development (CNPq) and Coordination of Improvement of Higher Education Personnel (CAPES). The
authors are also grateful to the Graduate Program in Pharmaceutical Sciences/UFRGS (Brazil).
Author Contributions
SCBG, GG, AMF and BBC designed and coordinated the research. BBC and MNM obtained the
compounds. BBC and TO performed the experiments for the antifungal activity and mechanisms of
action assays. LFO and MMM performed the cytotoxicity assays. BBC drafted the manuscript. SCBG,
GG and AMF corrected the manuscript. All authors approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Pappas, P.G.M.D. Opportunistic fungi: A view to the future. Am. J. Med. Sci. 2010, 340, 253–257.
2. Romani, L. Immunity to fungal infections. Nat. Rev. Immunol. 2011, 11, 275–288.
3. Barchiesi, F.; Arzeni, D.; Fothergill, A.W.; Di Francesco, L.F.; Caselli, F.; Rinaldi, M.G.; Scalise, G.
In vitro activities of the new antifungal triazole sch 56592 against common and emerging yeast
pathogens. Antimicrob. Agents Chemother. 2000, 44, 226–229.
4. Van Asbeck, E.C.; Clemons, K.V.; Stevens, D.A. Candida parapsilosis: A review of its epidemiology,
pathogenesis, clinical aspects, typing and antimicrobial susceptibility. Crit. Rev. Microbiol. 2009,
35, 283–309.
5. Miceli, M.H.; Díaz, J.A.; Lee, S.A. Emerging opportunistic yeast infections. Lancet Infect. Dis.
2011, 11, 142–151.
6. Chang, C.W.T.; Takemoto, J.Y. Antifungal amphiphilic aminoglycosides. Med. Chem. Commun.
2014, 5, 1048–1057.

Molecules 2015, 20
9240
7. Colombo, A.L.; Nucci, M.; Park, B.J.; Nouér, S.A.; Arthington-Skaggs, B.; da Matta, D.A.;
Warnock, D.; Morgan, J.; Brazilian Network Candidemia Study. Epidemiology of candidemia in
Brazil: A nationwide sentinel surveillance of candidemia in eleven medical centers. J. Clin. Microbiol.
2006, 44, 2816–2823.
8. Rajeshkumar, R.; Sundararaman, M. Emergence of Candida spp. and exploration of natural
bioactive molecules for anticandidal therapy—status quo. Mycoses 2012, 55, e60–e73.
9. Imramovský, A.; Polanc, S.; Vinšová, J.; Kočevar, M.; Jampílek, J.; Rečková, Z.; Kaustová, J. A
new modification of anti-tubercular active molecules. Bioorgan. Med. Chem. 2007, 15, 2551–2559.
10. Dimmock, J.R.; Vashishtha, S.C.; Stables, J.P. Anticonvulsant properties of various
acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and unsaturated
carbonyl compounds. Eur. J. Med. Chem. 2000, 35, 241–248.
11. Lima, P.C.; Lima, L.M.; da Silva, K.C.M.; Léda, P.H.O.; de Miranda, A.L.P.; Fraga, C.A.M.;
Barreiro, E.J. Synthesis and analgesic activity of novel n-acylarylhydrazones and isosters, derived
from natural safrole. Eur. J. Med. Chem. 2000, 35, 187–203.
12. Silva, G.A.; Costa, L.M.M.; Brito, F.C.F.; Miranda, A.L.P.; Barreiro, E.J.; Fraga, C.A.M. New class
of potent antinociceptive and antiplatelet 10h-phenothiazine-1-acylhydrazone derivatives.
Bioorgan. Med. Chem. 2004, 12, 3149–3158.
13. Loncle, C.; Brunel, J.M.; Vidal, N.; Dherbomez, M.; Letourneux, Y. Synthesis and antifungal
activity of cholesterol-hydrazone derivatives. Eur. J. Med. Chem. 2004, 39, 1067–1071.
14. Abdel-Aal, M.T.; El-Sayed, W.A.; El-Ashry, E.S.H. Synthesis and antiviral evaluation of some
sugar arylglycinoylhydrazones and their oxadiazoline derivatives. Arch. Pharm. 2006, 339, 656–663.
15. El-Hawash, S.A.M.; Abdel Wahab, A.E.; El-Demellawy, M.A. Cyanoacetic acid hydrazones of
3-(and 4-)acetylpyridine and some derived ring systems as potential antitumor and anti-HCV
agents. Arch. Pharm. 2006, 339, 14–23.
16. Walcourt, A.; Loyevsky, M.; Lovejoy, D.B.; Gordeuk, V.R.; Richardson, D.R. Novel
aroylhydrazone and thiosemicarbazone iron chelators with anti-malarial activity against
chloroquine-resistant and -sensitive parasites. Int. J. Biochem. Cell Biol. 2004, 36, 401–407.
17. Dalazen, D.; Zanrosso, D.; Wanderley, L.; da SIlva, N.L.; Fuentefria, A.M. Comparação do perfil
de suscetibilidade entre isolados clínicos de Candida spp. orais e vulvovaginais no sul do brasil.
J. Bras. Patol. Med. Lab. 2011, 47, 33–38.
18. Reference Method for Broth Dilution Antifungal, 3rd ed.; Document m27-a3; CLSI, Wayne, PA,
USA, 2008.
19. Escalante, A.; Gattuso, M.; Pérez, P.; Zacchino, S. Evidence for the mechanism of action of the
antifungal phytolaccoside b isolated from phytolacca tetramera hauman. J. Nat. Prod. 2008, 71,
1720–1725.
20. Khatib, S.; Nerya, O.; Musa, R.; Shmuel, M.; Tamir, S.; Vaya, J. Chalcones as potent tyrosinase
inhibitors: The importance of a 2,4-substituted resorcinol moiety. Bioorgan. Med. Chem. 2005, 13,
433–441.
21. Fan, X.; Song, Y.L.; Long, Y.Q. An efficient and practical synthesis of the hiv protease inhibitor
atazanavir via a highly diastereoselective reduction approach. Org. Proc. Res. Dev. 2008, 12, 69–75.

Molecules 2015, 20
9241
22. Xu, Z.; Singh, J.; Schwinden, M.D.; Zheng, B.; Kissick, T.P.; Patel, B.; Humora, M.J.; Quiroz, F.;
Dong, L.; Hsieh, D.M.; et al. Process research and development for an efficient synthesis of the
HIV protease inhibitor bms-232632. Org. Proc. Res. Dev. 2002, 6, 323–328.
23. Innocente, A.; Casanova, B.B.; Klein, F.; Lana, A.D.; Pereira, D.; Muniz, M.N.; Sonnet, P.;
Gosmann, G.; Fuentefria, A.M.; Gnoatto, S.C.B. Synthesis of isosteric triterpenoid derivatives and
antifungal activity. Chem. Biol. Drug Des. 2014, 83, 344–349.
24. Frost, D.J.; Brandt, K.D.; Cugier, D.; Goldman, R. A whole-cell Candida albicans assay for the
detection of inhibitors towards fungal cell wall synthesis and assembly. J. Antibiot. 1995, 48,
306–310.
25. Carrasco, H.; Raimondi, M.; Svetaz, L.; Liberto, M.D.; Rodriguez, M.V.; Espinoza, L.; Madrid, A.;
Zacchino, S. Antifungal activity of eugenol analogues. Influence of different substituents and
studies on mechanism of action. Molecules 2012, 17, 1002–1024.
26. Burow, M.E.; Weldon, C.B.; Tang, Y.; Navar, G.L.; Krajewski, S.; Reed, J.C.; Hammond, T.G.;
Clejan, S.; Beckman, B.S. Differences in susceptibility to tumor necrosis factor α-induced apoptosis
among mcf-7 breast cancer cell variants. Cancer Res. 1998, 58, 4940–4946.
27. Dos Santos Montagner, G.F.F.; Sagrillo, M.; Machado, M.M.; Almeida, R.C.; Mostardeiro, C.P.;
Duarte, M.M.M.F.; da Cruz, I.B.M. Toxicological effects of ultraviolet radiation on lymphocyte
cells with different manganese superoxide dismutase ala16val polymorphism genotypes.
Toxicol. In Vitro 2010, 24, 1410–1416.
28. Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. A simple technique for quantitation of low
levels of DNA damage in individual cells. Exp. Cell Res. 1988, 175, 184–191.
Sample Availability: All samples of the compounds are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).