Anti-Candida, anti-enzyme activity and cytotoxicity of 3,5-diaryl-4,5- dihydro-1H-pyrazole-1-carboximidamides

Article abstract of DOI:10.3390/molecules19055806

Because of the need for more effective and less harmful antifungal therapies, and interest in the synthesis of new carboximidamides, the goal of this study was to determine the antifungal and anti-enzyme activities of some new pyrazole carboximidamides and their cytotoxicity. For this purpose, tests were performed to evaluate: minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC); production of proteinases and phospholipase, and cytotoxicity of the extracts. Data were analyzed by ANOVA and Tukey Tests (a = 5%). The results were: MIC and MFC ≥ 62.5 μg/mL (C. albicans, C. parapsilosis, C. famata, C. glabrata, and Rhodotorula mucillaginosa) and MIC and MFC ≥ 15.6 μg/mL (C. lipolytica). The values of proteinase and phospholipase (Pz) of C. albicans before and after exposure to the compounds were: 0.6 (±0.024) and 0.2 (±0.022) and 0.9 (±0.074) and 0.3 (±0.04), respectively. These proteinase results were not significant (p = 0.69), but those of phospholipase were (p = 0.01), and 15.6 μg/mL was the most effective concentration. The cytotoxicity means were similar among the tests (p = 0.32). These compounds could be useful as templates for further development through modification or derivatization to design more potent antifungal agents. Data from this study provide evidence that these new pyrazole formulations could be an alternative source for the treatment of fungal infections caused by Candida. However, a specific study on the safety and efficacy of these in vivo and clinical trials is still needed, in order to evaluate the practical relevance of the in vitro results.

Full text of DOI:10.3390/molecules19055806

Molecules 2014, 19, 5806-5820; doi:10.3390/molecules19055806
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Anti-Candida, Anti-Enzyme Activity and Cytotoxicity of
3,5-Diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides
Simone Oliveira 1, Lucas Pizzuti 2, Frank Quina 3, Alex Flores 4, Rafael Lund 1,*,
Claiton Lencina 5, Bruna S. Pacheco 5, Claudio M. P. de Pereira 5 and Evandro Piva 6
1
Laboratory of Microbiology, Postgraduate Program in Dentistry, School of Dentistry,
Federal University of Pelotas (UFPel)—Rua Gonçalves Chaves, 457/504,
Pelotas, RS 96015-000, Brazil; E-Mail: sisi_mone@hotmail.com
2
Faculty of Exact Sciences and Technology, Federal University of Grande Dourados,
Dourados, MS 79825-070, Brazil; E-Mail: lucas.pizzuti@gmail.com
3
Department of Chemistry and the Consortium for Photochemical Technology (NAP-PhotoTech),
University of São Paulo, São Paulo, SP 05508-000, Brazil; E-Mail: quina@usp.br
4
School of Chemistry and Food, Federal University of Rio Grande, Rio Grande, RS 95500-000, Brazil;
E-Mail: alex@pesquisador.cnpq.br
5
Laboratory of Bioactive Heterocycles and Bioprospection (LAHBBio), Center for Chemical,
Pharmaceutical and Food Sciences, Federal University of Pelotas, Pelotas, RS 96010-610, Brazil;
E-Mails: leonetti.lencina@gmail.com (C.L.); bruna.spacheco@hotmail.com (B.S.P.);
claudiochemistry@gmail.com (C.M.P.P.)
6
Department of Restorative Dentistry, School of Dentistry, Federal University of Pelotas (UFPel),
Pelotas, RS 96015-000, Brazil; E-Mail: evpiva@gmail.com
* Author to whom correspondence should be addressed; E-Mail: rglund@ufpel.edu.br;
Tel./Fax: +55-53-3222-6690.
Received: 6 February 2014; in revised form: 14 April 2014 / Accepted: 16 April 2014 /
Published: 6 May 2014
Abstract: Because of the need for more effective and less harmful antifungal therapies,
and interest in the synthesis of new carboximidamides, the goal of this study was to
determine the antifungal and anti-enzyme activities of some new pyrazole carboximidamides
and their cytotoxicity. For this purpose, tests were performed to evaluate: minimum
inhibitory concentration (MIC) and minimum fungicidal concentration (MFC); production
of proteinases and phospholipase, and cytotoxicity of the extracts. Data were analyzed by
ANOVA and Tukey Tests (α = 5%). The results were: MIC and MFC ≥ 62.5 μg/mL
(C. albicans, C. parapsilosis, C. famata, C. glabrata, and Rhodotorula mucillaginosa) and

Molecules 2014, 19
MIC and MFC ≥ 15.6 μg/mL (C. lipolytica). The values of proteinase and phospholipase
5807
(Pz) of C. albicans before and after exposure to the compounds were: 0.6 (±0.024) and
0.2 (±0.022) and 0.9 (±0.074) and 0.3 (±0.04), respectively. These proteinase results were
not significant (p = 0.69), but those of phospholipase were (p = 0.01), and 15.6 μg/mL was
the most effective concentration. The cytotoxicity means were similar among the tests
(p = 0.32). These compounds could be useful as templates for further development through
modification or derivatization to design more potent antifungal agents. Data from this study
provide evidence that these new pyrazole formulations could be an alternative source for
the treatment of fungal infections caused by Candida. However, a specific study on the
safety and efficacy of these in vivo and clinical trials is still needed, in order to evaluate the
practical relevance of the in vitro results.
Keywords: pyrazoles; Candida albicans; antifungal activity; anti-enzyme activity
1. Introduction
Candida species are important pathogens correlated with 8% to 10% of the opportunistic infections
capable of affecting the skin, mouth, esophagus, gastrointestinal tract, vagina and vascular system [1].
These infections have increased over the past several years and represent a challenge, especially to
immunocompromised patients [2]. C. albicans is regarded as being the main causative agent of these
infections, which are often related to predisposing local and systemic factors. This species has become
a more pathogenic and tough genre because of its ability to coalesce and form heterogeneous biofilms,
and irrespective of the mechanism involved, may have an impact on the selection of antimicrobial
therapy and the response to infections associated with antimicrobial therapy [3]. However, other
Candida species have been correlated with increased fungal infections, such as C. tropicalis,
C. parapsilosis, C. stellatoidea, C. glabrata and C. krusei [4]. Some evidence indicates that increased
use of fluconazole or other antifungal agents may be responsible for fungal infections (other than
C. albicans) with reduced antifungal susceptibility [5].
Even with this important overview of fungal infections and increase in the number of antifungal
agents on the market, there is still a wide gap in the effectiveness of treatment of these infections, in
comparison with the results obtained with antibacterial therapy [2]. Furthermore, the biochemical and
physiological similarities between fungal and human cells provide many substances with deleterious
actions and nonspecific antifungal properties, which can causing a number of side effects during
therapy [6]. Furthermore there is also concern about the increasing pathogenicity of the less virulent
Candida albicans species [7]. Considering the aspects of diagnosis and therapy, these infections are a
significant challenge to medicine [8]. Thus, there is a need to encourage research and innovative
efforts with regard to new antifungal compounds.
Heterocyclic compounds containing the pyrazole unit have a broad spectrum of biological activities,
such as monoamine oxidase inhibitor [9], anticonvulsant [10], antibacterial [11], hypotensive [12],
antipyretic [13] and anti-inflammatory [14] activity. In addition, 1-carboxamidino has replaced pyrazole,

Molecules 2014, 19
5808
and its derivatives have been used for the synthesis of several bi-heterocycles [13] and as amidinating
reagents to convert primary and secondary amines into the corresponding guanidines [15].
There were recent reports of the regiospecific synthesis of many pyrazole derivatives under mild
conditions via (3+2) cyclocondensations between 1,3-dielectrophiles and hydrazines [16–18].
In addition, there have been reports of the synthesis of a series of 5-aryl-1-carboxamidino-3-styryl-4,5-
dihydro-1H-pyrazole derivatives from the cyclocondensation of 1,5-diaryl-1,4-pentadien-3-ones and
aminoguanidine hydrochloride and their application in the synthesis of new 2-(5-aryl-3-styryl-4,5-
dihydro-1H-pyrazol-1-yl)-4-(trifluoromethyl)pyrimidines [19]. Furthermore, the synthesis under mild
conditions of novel 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides by the reaction of
chalcones with aminoguanidine hydrochloride in KOH has been described [20]. In addition to the
antioxidant capacity of pyrazoles [16,17] several potential applications should be investigated.
Because of the need for more effective and less harmful antifungal therapies, and the availability of
novel syntheses of new carboximidamides, the goal of this study was to determine the antifungal and
anti-enzyme activities of some new pyrazole carboximidamides and their cytotoxicity. The hypothesis
to be tested was that 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides would show antifungal
activity against Candida and low cytotoxicity.
2. Results and Discussion
2.1. Chemistry
The tested pyrazoles 1–11 were obtained by the reaction of chalcones with aminoguanidine
hydrochloride in ethanol under ultrasound irradiation (Scheme 1).
Scheme 1. General synthesis of the 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides 1–11 [18].
1
13
The preparation, yields and the H- and C-nuclear magnetic resonance (NMR) data of compounds
1–11 are reported in the literature [18,20]. In this work, the structures of compounds 1–11 were
confirmed by High Resolution Mass Spectrometry (HRMS). The representative spectrum of compound
1 is shown in Figure 1 and selected experimental High Resolution Mass Spectrometry data for
compounds 1–11 are given in Table 1.

Molecules 2014, 19
Figure 1. Representative HRMS spectrum of 5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-
5809
1H-pyrazole-1-carboximidamide (1).
Intens.
x10
+MS, 1.3-1.6min #(38-48)
5
295.1557
3
N
2
N
MeO
H2N
NH
1
0
EM: 294,15
296.1593
294.0
294.5
295.0
295.5
296.0
296.5
297.0
m/z
Table 1. Experimental High Resolution Mass Spectrometry data for compounds 1–11.
Pyrazole a
Molecular Formula
HRMS m/z [MH]+
Calculated
Found
1
2
C17H19N4O
C16H17N4
295.1559
265.1453
299.1063
343.0558
295.1559
333.0674
279.1610
325.1664
310.1304
343.0558
279.1610
295.1557
265.1452
299.1047
343.0547
295.1550
333.0685
279.1610
325.1661
310.1318
343.0546
279.1602
3
C16H16ClN4
C16H16BrN4
C17H19N4O
C16H15Cl2N4
C17H19N4
4
5
6
7
8
C18H21N4O2
C16H16N5O2
C16H16BrN4
C17H19N4
9
10
11
a
(1) 5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide; (2) 3,5-Diphenyl-4,5-
dihydro-1H-pyrazole-1-carboximidamide; (3) 5-(4-Chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboximidamide; (4) 5-(2-Bromophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide; (5) 5-(2-
Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide; (6) 5-(2,4-Dichlorophenyl)-3-phenyl-
4,5-dihydro-1H-pyrazole-1-carboximidamide; (7) 5-(4-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboximidamide; (8) 5-(3,4-Dimethoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide; (9) 5-(3-
Nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamide; (10) 5-(4-Bromophenyl)-3-phenyl-4,5-
dihydro-1H-pyrazole-1-carboximidamide; (11) 5-(2-Methylphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carboximidamide.
2.2. Biological Activity
The choice of the broth dilution test to determine the sensitivity of the yeasts to antifungal therapy,
as recommended by The National Committee for Clinical Laboratory Standards (NCCLS, M27-A2
protocol), was based on the advantages of easy reproducibility [21], low cost and sensitivity.
In addition, this method requires only a small amount of each sample, which can be used for various
tests [22]. The standard M27-A2 methodology recommends reading the results for Candida after 48 h.
In our study, we chose to read the results at two time intervals, 24 and 48 h, due to the lack of
knowledge about the possible activity and behavior of these novel compounds. The two readouts

Molecules 2014, 19
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showed the occurrence of trailing and partial inhibition of growth over a wide range of concentrations
of the antifungal agent.
The research hypothesis was confirmed since data from this study provide evidence that these new
formulations of pyrazoles could be an alternative source for the treatment of fungal infections caused
by C. albicans and non- C. albicans. The fungal species chosen for this study were Candida albicans,
Candida non-albicans (C.parapsilosis; C famata; C. glabrata; C. lipolytica) and Rhodotorula mucillaginosa.
This choice reflects the fact that Candida spp. are responsible for the majority of fungal infections [23].
On the other hand, the widespread use of antifungal therapies and prophylaxis has resulted in an
increasing number and severity of the infections caused by other non-albicans species of Candida,
mainly C. glabrata and C. parapsilosis [24], making it is essential to study treatment alternatives
for these other Candida species as well. Table 2 shows the results for the minimum inhibitory
concentration (MIC) and minimum fungicidal concentration (MFC) of the pyrazole derivatives 1–11
dissolved in dimethyl sulfoxide (DMSO), or in 70% ethanol, respectively, as assessed against strains of
C. albicans. For most of the compounds, the MIC and MFC values were similar and indicated that
several of the compounds were potential fungicidal agents. Three pyrazole derivatives, compounds 8, 9
and 11, failed to exhibit any antifungal activity in this study.
It was shown that most of the pyrazole derivatives had a similar effect at 125–250 μg/mL. However,
compound 5 showed a MIC and a MFC of 15.6 μg/mL against one of the strains of Candida albicans.
This result can be compared with other studies that used the same methodology to test promising novel
antifungal compounds, for example, Tamoxifen and Clomiphene [25].
Table 2 shows the results of the MIC and MFC of the pyrazole derivatives assessed against strains
of non-C. albicans species. In recent times, there has been a greater recognition of the importance of
non-Candida albicans species in human disease. C. glabrata recently received special attention
because of its greater resistance to certain antifungal agents, arousing pharmacological interest [26].
However, inhibition of fungal growth alone may not be sufficient to prevent the spread of Candida
in immunosuppressed patients [27]. Until recently, fungal infections were generally correlated with
opportunism in debilitated or otherwise weakened patients. Although debilitated patients comprise a
large percentage of those infected by Candida spp., it is recognized today that candidiasis may also
develop in otherwise healthy individuals [28].
The interaction between yeast and human cells is essential for the colonization and invasion of
tissues, and thus, for the onset and establishment of the disease. C. albicans uses three strategies
for pathogenicity and invasion: (a) escape from the immune responses [29]; (b) changes in the
morphogenic shapes of the yeast hyphae, which enhance the ability of yeast to adhere to and invade
host cells; and (c) the invasion of host cells, supported by factors associated with the hyphae, such as
the secretion of hydrolytic enzymes [30]. Aspartyl proteinases (Saps) and phospholipases are among
the major hydrolytic enzymes secreted. The proteases are responsible for degrading various
physiologically important substrates [30] and the phospholipases are directly associated with adhesion
to the epithelial tissue of the yeast host [31]. Thus, the study of the activity of these enzymes and of
substances capable of inhibiting their release is important for the development of new alternatives for
the treatment of candidiasis.

Molecules 2014, 19
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Table 2. In vitro antifungal activity against Candida albicans and non- C.albicans of the pyrazole derivatives dissolved in DMSO or in 70% ethanol.
C. albicans (n = 33)
R. mucillaginosa
C. parapsilosis
(n = 2)
C. famata
C. glabrata
C. lypolytica
Median (range)
(n = 2)
(n = 2)
(n = 2)
(n = 2)
Chemical
Formula
70%
DMSO
70%
DMSO
70%
70%
70%
70%
Compound
Structural Formula
DMSO
DMSO
DMSO
DMSO
ethanol
ethanol
ethanol
MFC
ethanol
MFC
ethanol
MFC
ethanol
MFC
MIC
MFC
MIC
MFC
MIC
MIC
MIC
MIC
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
Ph
N
N
C17H18N4O
250
250
125
250
>250
250
125
250
250
125
250
250
250
125
250
250
>62.5
125
>62.5
125
>62.5
>62.5
62.5
>62.5
>62.5
62.5
125
62.5
62.5
62.5
125
62.5
62.5
62.5
15.6
31.2
15.6
31.2
1
2
3
4
OMe
HN
Ph
NH2
N
N
C16H16N4
HN
Ph
NH2
N
C16H15ClN4
C16H15BrN4
62.5
125
62.5
125
31.2
31.2
N
Cl
HN
Ph
NH2
Br
N
>62.5
>62.5
>31.2
>31.3
N
N
HN
Ph
NH2
MeO
N
C17H18N4O
15.6
250
15.6
250
62.5
250
62.5
250
>62.5
125
>62.5
125
62.5
125
62.5
125
125
125
125
125
15.6
15.6
5
6
HN
NH2
Cl
Ph
N
C16H14Cl2N4
>15.6
>15.6
N
Cl
HN
NH2

Molecules 2014, 19
5812
Table 2. Cont.
C. albicans (n = 33)
R. mucillaginosa
(n = 2)
C. parapsilosis
C. famata
C. glabrata
C. lypolytica
Median (range)
(n = 2)
(n = 2)
(n = 2)
(n = 2)
Chemical
Formula
70%
DMSO
70%
70%
DMSO
70%
70%
70%
Compound
Structural Formula
DMSO
DMSO
DMSO
DMSO
ethanol
ethanol
MFC
ethanol
ethanol
MFC
ethanol
MFC
ethanol
MFC
MIC
MFC
MIC
MIC
MFC
MIC
MIC
MIC
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
µg/mL
Ph
N
C17H18N4
C18H20N4O2
C16H15N5O2
C16H15BrN4
250
-
>250
250
-
250
-
125
-
125
-
125
-
125
-
125
125
62.5
62.5
7
8
N
Me
HN
Ph
NH2
OMe
OMe
N
-
-
-
-
-
-
-
-
-
-
N
HN
Ph
NH2
NO2
N
-
-
-
-
-
-
-
9
N
N
HN
Ph
NH2
N
125
125
250
250
125
125
125
125
>125
>125
<62.5
<62.5
10
Br
HN
NH2
Ph
Me
N
C17H18N4
-
-
-
-
-
-
-
-
-
-
-
-
11
N
HN
NH2
No differences were observed in the MIC and MFC values for compounds dissolved in DMSO or 70% ethanol. Thus, it is preferable for the compounds derived
from pyrazoles with antifungal activity in vitro to be dissolved in 70% ethanol, which is less toxic and is compatible with pharmaceutical preparations for the treatment
of candidiasis.

Molecules 2014, 19
5813
The precipitation zone (Pz) values for proteinase and phospholipase of C. albicans before and after
exposure to the compounds were: 0.2 (±0.022) and 0.6 (±0.024) and 0.3 (±0.04) and 0.9 (±0.074),
respectively. These proteinase results were not significant. The phospholipase results were (p = 0.01)
and 15.6 mg/mL was the most effective concentration. For both tests, the addition of 3,5-diaryl-4,5-
dihydro-1H-pyrazole-1-carboximidamides resulted in decreased secretion of phospholipase and proteinase
in the strains tested (p < 0.001—Spearman Correlation). Other studies have tested compounds capable
of inhibiting phospholipase, such as chlorhexidine [32]. Clearly there is a need for further studies in
order to clarify the mechanism of action of the new substances tested here, in order to eventually
establish them as a feasible alternative treatment for the various diseases caused by Candida spp.
With regard to cytotoxicity, the present findings showed that the cytotoxic effect of compounds
were not significantly different from those obtained for the control group (Figure 2), reinforcing the
potential for therapeutic use of these antifungal agents. Among antifungal agents, the therapeutic
agents most frequently used for the topical treatment of oral candidiasis are polyenes (nystatin and
amphotericin B) and azoles (itraconazole, miconazole and clotrimazole) [33,34]. Although they are
widely used, they have certain limitations due to side effects such as toxicity and the emergence of
resistant strains [35], thus reaffirming the need for the development of other compounds with low
cytotoxicity and the potential for treatment of mycoses [36].
Figure 2. Cytotoxicity of compounds dissolved in DMSO after 24 h of cell exposure.
Compounds showed statistically similar cytotoxicity levels.
500 µg/mL
250 µg/mL
In view of the difficulties encountered in the treatment of fungal infections, some authors have
recommended the isolation of the causative agent of infection, and determination of the minimum
inhibitory concentration and minimum fungicidal concentration of the drugs that might potentially be
used. A minimum inhibitory concentration of the chosen antimicrobial should be established by a
standardized laboratory method to ensure susceptibility [37]. However, establishing the antifungal
activity of a new drug in vitro provides only part of the information needed to predict the outcome of
treatment with a new drug. This reflects the fact that the interaction between antifungal drugs and
fungi, the fungus and the host, and the host and the antifungal drugs is very complex. Nonetheless, at
present, in vitro assays and susceptibility tests still continue to be indispensable tools for selecting the

Molecules 2014, 19
5814
most appropriate antifungal agent [38]. In particular, protocols for monitoring patients with Acquired
Immunodeficiency Syndrome (AIDS) or other immunocompromising diseases, who have an increased
frequency of fungal infections, should routinely be included in a rapid laboratory confirmation of the
presence of fungal species and their susceptibility profile. New antifungal drugs for the treatment of
oral candidiasis [39] should be a particular area of interest to the pharmaceutical industry.
The remarkable antifungal effects of some of the pyrazole compounds assessed in this study could
be a useful lead for the development of new and promising antifungal agents against oral candidiasis,
mainly caused by C. albicans. However, further pharmacological and toxicological studies will be
necessary to confirm this hypothesis.
Compound 5, bearing a methoxy group at the ortho position, was the most active of all of the
compounds tested. It is the only one that has a hydrogen bond acceptor group at this position,
suggesting that this feature might be decisive for its greater activity. Compounds 4 and 10 have similar
oil-water partition coefficients, but different activities, indicating that although it promotes greater cell
penetration, lipophilicity is not the determining factor for activity. These assumptions are reinforced by
a comparison of the MIC of compounds 3 and 6, for which an inverse relationship between biological
activity and lipophilicity was observed. Analogs 3 and 6 and 4 and 10 have similar hydrophobicities
and show that the presence of a halogen atom at the ortho position decreases the activity. Compounds
5 and 1 provide additional evidence for the importance of the substituent position on the aromatic ring,
where the presence of the methoxy group at the para position greatly reduces the activity. Derivatives
bearing the meta substituent were inactive. This study underlines the importance of the ortho position
for activity and certainly will guide us in the design of the next generation of antifungal compounds. In
the future work, we will be testing compounds with different groups at the ortho position with distinct
steric, electronic, hydrophobic or hydrophilic features and evaluate the impact of these structural
variations on the antifungal activity.
The largest class of antimycotics available today, the azoles, act by inhibiting ergosterol
biosynthesis through inhibition of 14-α-demethylase, a key heme enzyme in the biosynthetic pathway.
This inhibition results in the accumulation in the fungal cell membrane of anomalous steroids with
different shapes and physical properties from the normal membrane sterol ergosterol. The effects
arising from this modification lead to fungal cell death. The N-3 atom of the heterocyclic aromatic
ring of azoles (imidazole or triazole) is essential for the activity of these compounds. Based on this
fact, our further research will verify whether aromatization of the heterocyclic ring increases the
anti-Candida activity.
3. Experimental
3.1. General Information
The 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides 1–11 were prepared according to the
1
literature and characterized by H, 13C-NMR [18,20], and confirmed by HRMS. All solvents and
reagents were obtained from Aldrich (Hamburg, Germany) and used without further purification.
NMR spectra were recorded on a Bruker DPX 300 spectrometer (Bremen, Germany) at 300.13 MHz
for 1H and 75.48 MHz for 13C) at 300 K. The progress of reactions was monitored on a Thermo Trace

Molecules 2014, 19
5815
GC Ultra chromatograph (Ramsey, MN, USA) equipped with a Varian FactorFour™ capillary column
(95% dimethyl- and 5% diphenylpolysiloxane; 0.25 mm × 30 m; column head pressure, 14 psi). The
mass spectra were obtained on an Applied Biosystems LC/MS/MS Hybrid Triple Quadrupole Linear
Ion Trap System, Model 3200 QTRAP (Cole-Parmer, Vernon Hills, IL, USA). The parameters were:
Curtain Gas (CUR) = 10, Ion Spray Voltage (IS) = 5500, Temperature (TEM) = 25 °C, Ion Source Gas
(GS1) = 15, Ion Source Gas (GS2) = 0, Declustering Potential (DP) = 70, Entrance Potential (EP) = 10
and Scan and Product Ion (MS2). The reactions were carried out with a microtip probe connected to a
500 W Sonics Vibra-cell ultrasonic processor (Newtown, Connecticut, USA) operating at 20 KHz with
25% of the maximum power output.
3.2. Strains
C. albicans (33), C. parapsilosis (2), C. famata (2), C. glabrata (2), C. lipolytica (2) and
R. mucillaginosa (2) strains were used for determining the in vitro anti-Candida activity of eleven
3,5-diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides. These strains of C. albicans were collected
from denture user patients with Chronic Atrophic Candidiasis (CAC), enrolled in the Center for
Diagnosis of Diseases of the Mouth (CDDB), School of Dentistry, Federal University of Pelotas
(FOP-UFPel). The diagnosis of CAC was clinical and microbiological, as previously described [40].
3.3. In Vitro Antifungal Activity
3.3.1. Inoculum
The strains of C. albicans and non- C. albicans were sub-cultured on Sabouraud Dextrose Agar
with 0.2 mg/mL of chloramphenicol at 36 °C for 24 h. After incubation, these strains were individually
inoculated into tubes containing 5 mL of a sterile 0.85% saline solution and the yeast suspension was
adjusted to a 0.5 McFarland standard (which is approximately 106 colony forming units CFU/mL).
The inoculum was then resuspended to obtain a final concentration in Roswell Park Memorial Institute
(RPMI) 1640 medium (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with 165 mmol L−1 of
morpholinepropanesulfonic acid (MOPS; Vetec, Duque de Caxias, Rio de Janeiro, Brazil).
3.3.2. Determination of the MIC and MFC
MICs and MFCs of the new pyrazole derivatives were determined by using the broth microdilution
techniques described by the Clinical and Laboratory Standards Institute for yeasts (CLSI, M27-A2
protocol). Stock solutions of these eleven new pyrazole derivatives were prepared in DMSO and
in 70% ethanol solution at the concentration of 500 μg/mL. The solutions were diluted in Roswell
Park Memorial Institute (RPMI) 1640 medium (Sigma) and the final drug concentrations ranged from
0.49 to 500 μg/mL. Two replicates were performed for each concentration of the compounds tested
against the strains of Candida albicans and non- C. albicans.
For the test, 20 µL of each concentration of each compound were collected, and 1980 µL of
Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) were added. For the test, 20 µL of
each concentration of each compound were collected, and 1980 µL of Roswell Park Memorial Institute
(RPMI) 1640 medium (Sigma) were added. After preparation and dissolution of the above-mentioned

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compounds, 100 mL of each compound were pipetted into each well of the test plate, to which another
100 mL of the innoculum were added. For the positive control test plate, we added 100 mL of the
innoculum and did not include the compounds to be tested. For the negative control, we added 100 mL
of the compound and did not add the innoculum.
After 48 h of incubation at 35 °C, the MIC was determined visually by comparison with the
drug-free growth control well. Each inoculum from the previous test that did not show growth was
subcultured on agar plates. After 24 h of incubation, the readout was determined by the visible growth
of strains. The MFC was considered the lowest concentration that prevented visible growth.
3.4. In Vitro Anti-Enzyme Activity
3.4.1. Phospholipase
To conduct the tests, all samples were cultured on Sabouraud Dextrose Agar 4% for 24 h. After this
period, samples were diluted in sterile distilled water at a 0.5 McFarland scale. After preparing the
dilution, 20 mL of each sample was placed in the test medium. Reduced egg yolk agar (Reya) was
used and the plates were inoculated (four per isolate) with a 5 μL of inoculum suspension was plated
on equidistant points of the surface of Reduced egg yolk agar (Reya) and left to dry at room
temperature. The plates were then incubated aerobically at 37 °C for 48 h and the diameters of the
precipitation zones around the colonies were measured. The enzyme activity was expressed as the
Precipitation Zone (Pz) value, determined from the diameter of colony/(diameter of the hyaline zone)
and scaled as: (Pz = 1, Negative; 1 > Pz ≥ 0.64, Semi-positive; Pz ≤ 0.63, Positive). Each experiment
was repeated twice [32].
3.4.2. Proteinase
To conduct the tests, all samples were cultured on Sabouraud Dextrose Agar 4% for 24 h. After this
period, samples were diluted in sterile distilled water at a 0.5 McFarland scale. After preparing the
dilution, 20 mL of each sample was placed in the test medium. Clinical isolates were inoculated in
tubes containing 5 mL of Yeast Extract-Peptone-Dextrose (YPD) medium and incubated at 37 °C for
18 h. After the incubation, 1.5 mL aliquots of the culture were transferred to Eppendorf tubes at 4 °C
and centrifuged at 3000 rpm for 5 min. The cell pellets were resuspended in saline solution (0.9% NaCl)
and centrifuged twice more under the same conditions to remove cultivation debris. The concentrations
of the suspensions of the strains were standardized by using the index range of 0.5 MacFarland
(1 × 106 CFU/mL) and 20 µL volumes were inoculated at equidistant points in the middle of Proteinase
Agar plates. The plates with different inocula were incubated at 37 °C for four days. The tests were
performed in duplicate. The Precipitation Zone (Pz) values were determined from the radius of the
diameter of colony/hyaline zone (Pz = 1, Negative; 1 > Pz ≥ 0.64, Semi-positive; Pz ≤ 0.63, Positive.) [33].
3.5. Cytotoxicity Assay
The cell culture medium was Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 2% L-glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL).

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Mouse fibroblasts of the 3T3/NIH immortalized cell line were maintained as a stock culture in DMEM
and incubated at 37 °C in a humidified atmosphere of 5% CO2 in air until subconfluency.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess
cell metabolic function according to mitochondrial dehydrogenase activity. Mouse fibroblasts
(3T3/NIH; 2 × 104/cells) were maintained in DMEM in 96 well plates for 24 h. DMEM was removed
and replaced with 200 µL of extract from different groups with 10% FBS. Cytotoxicity produced by
the different extracts was assessed at 24 h of cell exposure time. After removing the extracts, the cells
were washed with phosphate-buffered saline (PBS) and then 200 µL of medium in 20 µL of MTT solution
(5 mg of MTT/mL DMEM) were added to each well. After 5 h of incubation at 37 °C in the dark, the
blue formazan precipitate was extracted from the mitochondria using 200 µL/well of DMSO on a
shaker at 150 rpm for 5 min. The absorption at 540 nm was determined spectrophotometrically. [41]
The cell viability was analyzed using SigmaStat 3.5 (Systat Software Inc., San Jose, CA, USA)
statistical analysis software program. The statistical analysis was performed by one-way ANOVA with
the level of significance set at p < 0.05.
4. Conclusions
3,5-Diaryl-4,5-dihydro-1H-pyrazole-1-carboximidamides could be useful as templates for further
development through modification or derivatization to design more potent antifungal agents. Data from
this study provide evidence that these new formulations of pyrazoles could be an alternative source for
the treatment of fungal infections caused by Candida. It was clear that many of the synthesized
compounds exhibited good antifungal activity for C. albicans and low cytotoxicity. However,
a specific study on the safety and efficacy of these in vivo and clinical trials is still needed to evaluate
the practical relevance of in vitro results.
Acknowledgments
The authors are grateful to INCT de Estudos do Meio Ambiente (573.667/2008-0), CEPEMA-USP
(Centro de Capacitação e Pesquisa em Meio Ambiente), CNPq (310472/2007-5, 475575/2008-3,
308087/2011-9), FAPERGS and CAPES (grant 23.038.019120/2009-3) for financial and fellowship
support. FHQ is affiliated with the INCT for Catalysis in Molecular and Nanostructured Systems
(INCT-Catalysis).
Author Contributions
Simone Oliveira, Rafael Lund and Evandro Piva were responsible for the biological assays and
writing and revision of the final scientific paper. Lucas Pizzuti, Frank Quina, Alex Flores were
responsible for the synthesis, purification and identification of new pyrazoles. Claiton Lencina,
Bruna Pacheco and Claudio de Pereira were responsible for the chemical and pharmacological analysis
of new pyrazoles.

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Conflicts of Interest
The authors declare no conflict of interest. All our affiliations, corporate or institutional, and all
sources of financial support for this research are properly acknowledged. We certify that do not have
any commercial or associate interest that represents a conflict of interest in connection with the
manuscript submitted.
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