Rapid tools to gain insights into the interaction dynamics of new 8-hydroxyquinolines with few fungal lines

Article abstract of DOI:10.1111/cbdd.13435

The combination of tools such as time-kill assay with subsequent application of mathematical modeling can clarify the potential of new antimicrobial compounds, since minimal inhibitory concentration (MIC) value does not provide a very detailed characterization of antimicrobial activity. Recently, our group has reported that the 8-hydroxy-5-quinolinesulfonic acid presents relevant antifungal activity. However, its intrinsic acidity could lead to an ionization process, decreasing fungal cell permeability. To overcome this potential problem and enhance activity, the purpose of this study was to synthesize and evaluate a novel series of hybrids between the 8-hydroxyquinoline core and sulfonamide and to prove their potential using broth microdilution method, obtaining the pharmacodynamic parameters of the most active derivatives combining time-kill studies and mathematical modeling and evaluating their toxicity. Compound 5a was the most potent, being active against all the fungal species tested, with low toxicity in normal cells. 5a and 5b have presented important antibacterial activity against Staphylococcus aureus strain. The EC₅₀ values obtained by combination of time-kill studies with mathematical model were similar to those of MIC, which confirms the potential of compounds. In addition, these derivatives are non-irritant molecules with the absence of topical toxicity. Finally, 5a and 5b are promising candidates for treatment of dermatomycosis and candidiasis.

Full text of DOI:10.1111/cbdd.13435

Received: 28 August 2018
DOI: 10.1111/cbdd.13435
Revised: 9 October 2018
Accepted: 28 October 2018
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S P E C I A L I S S U E A R T I C L E
Rapid tools to gain insights into the interaction dynamics of new
8
-hydroxyquinolines with few fungal lines
1
,2a
3a
3
1,2
Angélica Rocha Joaquim
|
Bruna Pippi
|
|
Maycon Antonio de Cesare
|
Débora
Thaís Carine
1,2
2
Assumpção Rocha
|
Roberta Taufer Boff
Keli Jaqueline Staudt
|
Ruaro2
Silveira
Santos4
Aline Rigon Zimmer2
Bibiana Verlindo de Araújo
2
Gustavo Pozza
Francisco Paulo dos
Saulo Fernandes de Andrade
|
|
|
4
1,3
5
|
Andreza Francisco Martins
Alexandre Meneghello Fuentefria
|
Mario Lettieri Teixeira
|
2,3
1,2,3
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|
1Pharmaceutical Synthesis Group
Abstract
(
PHARSG), Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil
The combination of tools such as time-kill assay with subsequent application of
mathematical modeling can clarify the potential of new antimicrobial compounds,
since minimal inhibitory concentration (MIC) value does not provide a very detailed
characterization of antimicrobial activity. Recently, our group has reported that the
2Programa de Pós-graduação em Ciências
Farmacêuticas, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil
3Programa de Pós-graduação
8
-hydroxy-5-quinolinesulfonic acid presents relevant antifungal activity. However,
em Microbiologia Agrícola e do
Ambiente, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil
4Programa de Pós-graduação em
Química, Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil
5Laboratório de Farmacologia, Instituto
Federal Catarinense, Campus Concórdia,
Concórdia, Brazil
its intrinsic acidity could lead to an ionization process, decreasing fungal cell perme-
ability. To overcome this potential problem and enhance activity, the purpose of this
study was to synthesize and evaluate a novel series of hybrids between the
8-hydroxyquinoline core and sulfonamide and to prove their potential using broth
microdilution method, obtaining the pharmacodynamic parameters of the most ac-
tive derivatives combining time-kill studies and mathematical modeling and evaluat-
ing their toxicity. Compound 5a was the most potent, being active against all the
fungal species tested, with low toxicity in normal cells. 5a and 5b have presented
important antibacterial activity against Staphylococcus aureus strain. The EC50 val-
ues obtained by combination of time-kill studies with mathematical model were
similar to those of MIC, which confirms the potential of compounds. In addition,
these derivatives are non-irritant molecules with the absence of topical toxicity.
Finally, 5a and 5b are promising candidates for treatment of dermatomycosis and
candidiasis.
Correspondence
Saulo Fernandes de Andrade,
Pharmaceutical Synthesis Group
(
PHARSG), Universidade Federal do Rio
Grande do Sul, Porto Alegre, Brazil.
Email: saulo.fernandes@ufrgs.br
Funding information
Fundação de Amparo à Pesquisa do
Estado do Rio Grande do Sul, Grant/
Award Number: EDITAL FAPERGS
0
4/2016 – PRONUPEQ 2016; Coordenação
K E Y W O R D S
de Aperfeiçoamento de Pessoal de Nível
Superior - Brasil (CAPES), Grant/Award
Number: Code 001
8
-hydroxyquinoline-5-sulfonamide, antimicrobial activity, candidiasis, dermatomycosis, synthesis
1
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INTRODUCTION
epidemics caused by bacterial infections were a major global
cause of mortality. In the late 1960s, there was a dramatic in-
crease in fungal infections because of the development of an-
tibiotic therapies (Vandeputte, Ferrari, & Coste, 2012), which
represents a serious problem today. However, the number of
antimicrobial medicines is limited, and it does not satisfy
Fungal and bacterial infections are of great importance in the
world public health scenario. In the early 20th century, the
aThese authors contributed equally to this work.
Chem Biol Drug Des. 2018;1–11.
wileyonlinelibrary.com/journal/cbdd
© 2018 John Wiley & Sons A/S
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1

2
JOAQUIM et Al.
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the need due to limitations related to the spectrum of action,
potency, pharmacokinetic properties, and the emergent re-
sistance of fungi and bacteria (Hipólito et al., 2018; Nett &
Andes, 2016; Patil et al., 2018; Tobudic, Kratzer, & Presterl,
& Hartinger, 2015; Mao et al., 2014; Nguyen, Hamby, &
Massa, 2005).
The antimicrobial activity of 8HQ derivatives seems
to be modulated by the nature of the substituent at the 5-
and 7-positions (Cherdtrakulkiat et al., 2016; Gershon,
Gershon, & Clarke, 2001; Zuo et al., 2016). In a recent
study of our group, we have evaluated the activity of the
8-hydroxy-5-quinolinesulfonic acid 2 (2, Figure 1) and 8-
hydroxy-7-iodo-5-quinolinesulfonic acid 3 (3, Figure 1)
against Candida albicans, Candida glabrata, Candida kru-
sei, Candida parapsilosis, Candida tropicalis, Microsporum
canis, Microsporum gypseum, Trichophyton mentagrophytes,
and Trichophyton rubrum isolates. These compounds have
presented relevant activity for these strains with MIC between
1 and 1,024 μg/ml (Pippi et al., 2017). Derivative 2 does not
possess an iodine atom at the seven-position that could be
an advantage because this atom could enhance the electro-
philicity of the compound and lead to toxicity (Gershon et al.,
2001; Pippi et al., 2017). However, the sulfonic acids may
have problems for penetrating the fungal cell, since it could
ionize in the biological pH range due to their acidic nature,
hindering their action. So, the conversion of the sulfonic acid
into a sulfonamide would be an interesting strategy, since
the sulfonamide is a very weak acid and does not ionize.
Besides, the extension of the structure by the addition of an
N-substituent at sulfonamide could lead to new interactions
and an improvement of the activity (Figure 2).
2
012). In this context, the search and development of new
antimicrobial therapies is imperative.
In the search of new antimicrobial agents, the minimal
inhibitory concentration (MIC) has been the major pharma-
codynamics parameter to check the susceptibility of micro-
organisms to compounds since it is a relatively simple test
(
Nielsen & Friberg, 2013). However, MIC value does not
provide a very detailed characterization of antimicrobial ac-
tivity because it is a measure evaluated at a single time-point
and does not report the rate of microbial kill. So, the dynamic
changes in growth and susceptibility over the studied time
period are not taken into account (Nielsen & Friberg, 2013;
Treyaprasert, Schmidt, Rand, Suvanakoot, & Derendorf,
2
007). On the other hand, time–kill curves are increasingly
used to offer detailed information about antimicrobial effi-
cacy as a function of both time and concentration (Ghannoum
et al., 2013; Gupta, Paquet, & Simpson, 2013). In addition,
pharmacokinetic/pharmacodynamic (PK/PD) modeling has
become a powerful tool to evaluate the antimicrobial effect
(
Bulitta et al., 2010; Nielsen et al., 2007; Treyaprasert et al.,
2
007). Thus, the combination of these two strategies, together
with MIC value, may improve the understanding of the inter-
action of new antimicrobial candidates with microorganisms.
In the last decades, the interest for derivatives of
Thus, the purpose of this study was to synthesize sulfon-
amides derived from 8HQ and to evaluate their antimicrobial
activity to select the most active compounds for a detailed an-
timicrobial evaluation using time-kill studies with subsequent
application of mathematical modeling, in order to determine
pharmacodynamic characteristics. We also studied the cell
viability and the potential for topical toxicity, resulting in the
selection of drug candidate to advance.
8
-hydroxyquinoline (8HQ) or 8-quinolinol (1, Figure 1) has
increased since they have presented several biological activ-
ities such as antibacterial, antifungal, antioxidant, antiviral,
and antiparasitic (Cherdtrakulkiat et al., 2016; Kadri, Crater,
Lee, Solomon, & Ananvoranich, 2014; Kassem et al., 2012;
Li et al., 2017; Oliveri & Vecchio, 2016; Zuo et al., 2016).
Recent studies also show that 8HQ derivatives are prom-
ising for the development of therapies for diseases such as
Alzheimer’s, Parkinson’s, Huntington’s, as well as neoplasms
(
Bareggi & Cornelli, 2012; Chen et al., 2007; Fu, Hsu,
2
2
METHODS AND MATERIALS
General
|
Liao, & Hu, 2016; Kubanik, Holtkamp, Söhnel, Jamieson,
.1
|
Melting points were determined on Buchi B-545 apparatus
1
13
(
Flawil, Switzerland). The H and C NMR spectra were ob-
tained on Bruker 400 (Billerica, USA) or Varian 400 (Palo
Alto, USA) nuclear magnetic resonance spectrometer. The
proton and carbon shifts (δ) are given with respect to TMS
(
(
tetramethylsinale). Fourier-transform infrared spectroscopy
IR) was performed on PerkinElmer Spectrum BX (Shelton,
USA). 8-hydroxyquinoline and other reactants were obtained
from commercial suppliers (Sigma-Aldrich, Saint Louis,
USA) and used without further purification. Column chroma-
tography was performed on silica gel Fluka (Sigma-Aldrich)
0.035–0.070 mm. The solvents have been distilled prior to use.
FIGURE 1 Structure of 8-hydroxyquinoline 1, 8-hydroxy-5-
quinolinesulfonic acid 2, and 8-hydroxy-7-iodo-5-quinolinesulfonic
acid 3

JOAQUIM et Al.
3
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FIGURE 2 Design of
8
-hydroxyquinoline-5-(N-substituted)-
sulfonamide by conversion of sulfonic
acid into sulfonamide and extension of the
structure of compound 2
(
d, 1H, J = 8.4 Hz), 6.83 (d, 2H, J = 9.0 Hz), 6.70 (d, 2H,
2
.2
Synthesis of 8-hydroxyquinoline-5-
|
13
J = 9.0 Hz), 3.60 (s, 3H). C NMR (100 MHz, DMSO-d )
6
sulfonyl chloride 4
δ (ppm): 158.3, 156.4, 148.9, 138.3, 133.1, 132.5, 129.9,
A
stirred solution of 8-hydroxyquinoline (0.500 g,
124.7, 123.5, 123.3, 122.9, 114.3, 109.6, 56.1. HRMS (m/z):
+
3
.44 mmol) in chlorosulfonic acid (2.5 ml, 37.61 mmol) was
[MH ] calc for C H N O S, 331.0753; found, 331.0752.
1
6
15
2
4
kept at room temperature. After 18 hr, the resulting solution
was added to a mixture of ice (50 g), brine (100 ml), and di-
chloromethane (150 ml). The organic layer was dried over
sodium sulfate anhydrous, filtered and concentrated to give
the 8-hydroxyquinoline-5-sulfonyl chloride that was used
immediately without further purification.
2
.3.3
8-Hydroxiquinoline-5-
|
(
N-4-cyanophenyl)sulfonamide 5c
Eluent hexanes: EtOAc (80:20). Yellow solid. 20% yield
(two steps): mp: 187–191°C. IR ν: 3,238, 2,221, 1,605, 1,572,
−1 1
1
,548, 1,505, 1,347, 1,288, 1,199, 1,156, 960, 896 cm . H
NMR (400 MHz, DMSO-d ) δ (ppm): 11.40 (s, 1H), 9.11
6
2
8
.3
General procedure 1—synthesis of
|
(
bd, 2H, J = 8.1 Hz), 8.99 (bd, 1H, J = 3.4 Hz), 8.30 (d,
-hydroxyquinoline-5-sulfonamides 5a–5f
1
H, J = 8.2 Hz), 7.86 (dd, 1H, J = 8.1 Hz, 3.4 Hz), 7.61 (d,
1
3
To a stirred solution of 8-hydroxyquinoline-5-sulfonyl chlo-
ride (0.2304 g, 0.945 mmol), a solution of appropriate amine
2H, J = 8.4 Hz), 7.23–7.16 (m, 3H). C NMR (100 MHz,
DMSO-d ) δ (ppm): 158.4, 148.7, 142.1, 137.2, 133.8,
6
(
0.4468 g, 3.78 mmol) in acetonitrile (5 ml) at 80°C was
133.7, 133.6, 124.6, 123.9, 122.7, 118.7, 117.8, 110.2, 105.0.
+
added. After 3 hr, the mixture was concentrated under re-
duced pressure, and the residue was purified by flash silica
gel column using Hexanes/EtOAc in gradient as eluent.
HRMS (m/z): [MH ] calc for C H N O S, 326.0599;
1
6
12
3
3
found, 326.0597.
2
.3.4
8-Hydroxiquinoline-5-
|
2
.3.1
8-Hydroxiquinoline-5-
(N-pyrimidinyl)sulfonamide 5d
|
(
N-4-chlorophenyl)sulfonamide 5a
Yellow solid. 15% yield (two steps): mp: 262–265°C. IR
ν: 3,369, 3,101, 3,048, 2,877, 2,822, 2,740, 1,670, 1,624,
1,571, 1,493, 1,444, 1,406, 1,375, 1,343, 1,275, 1,202,
Eluent hexanes: EtOAc (80:20). Yellow solid. 23% yield
(
two steps): mp: 160–163°C. IR ν: 3,303, 3,234, 1,570, 1,504,
−
1 1
−1 1
1
,490, 1,330, 1,284, 1,191, 1,150, 1,126, 1,016 cm . HNMR
1,157 cm . H NMR (400 MHz, DMSO-d ) δ (ppm): 9.11
6
(
(
(
(
400 MHz, CDCl ) δ (ppm): 8.95 (bd, 1H, J = 8.8 Hz), 8.87
(bd, 1H, J = 8,7 Hz), 8.93 (bd, 1H, J = 3.9 Hz), 8.39 (d,
2H, J = 4.7 Hz), 8.34 (d, 1H, J = 8.4 Hz), 7.77 (dd, 1H,
3
dd, 1H, J = 4.2 Hz, 1.6 Hz), 8.20 (d, 1H, J = 8.4 Hz), 7.60
dd, 1H, J = 8.8 Hz, 4.2 Hz), 7.16–7.12 (m, 3H), 6.91– 6.88
J = 8.7 Hz, 3.9 Hz), 7.21 (d, 1H, J = 8.4 Hz), 6.94 (t, 1H,
1
3
13
m, 2H). C NMR (100 MHz, CDCl ) δ (ppm): 157.5, 148.8,
J = 4.7 Hz). C NMR (100 MHz, DMSO-d ) δ (ppm): 158.4,
3
6
1
1
3
38.2, 134.8, 133.7, 133.6, 131.6, 129.6, 124.8, 124.0 123.7,
158.4, 156.9, 148.8, 138.0, 134.2, 133.2, 130.1, 124.8, 124.1,
+
+
23.7, 108.4. HRMS (m/z): [MH ] calc for C H ClN O S,
115.6, 109.7. HRMS (m/z): [MH ] calc for C H N O S,
1
5
12
2
3
13 11
4
3
35.0257; found, 335.0254.
303.0552; found, 303.0551.
2
.3.2
8-Hydroxiquinoline-5-
2.3.5
8-Hydroxiquinoline-5-(N-isopropyl)
|
|
(
N-4-methoxyphenyl)sulfonamide 5b
sulfonamide 5e
Eluent hexanes: EtOAc (80:20). Yellow solid. 15% yield
Eluent hexanes: EtOAc (60:40). White solid. 22% yield
(
two steps): mp: 175–177°C. IR ν: 3,294, 3,235, 1,606, 1,570,
(two steps): mp: 158–160°C. IR ν: 3,304, 2,972, 2,930,
−
1
−1 1
1
,503, 1,441, 1,413, 1,325, 1,296, 1,150, 1,125, 1,017 cm .
1,591, 1,506, 1,469, 1,326, 1,113 cm . H NMR (400 MHz,
1
H NMR (400 MHz, DMSO-d ) δ (ppm): 9.00 (dd, 1H, Ar,
CDCl ) δ (ppm): 8.99 (dd, 1H, J = 8.8 Hz, 1.2 Hz), 8.88
6
3
J = 8.8 Hz, 1.2 Hz), 8.95 (dd, 1H, J = 4.0 Hz, 1.2 Hz), 7.99
(
(dd, 1H, J = 4.4 Hz, 1.2 Hz), 8.28 (d, 1H, J = 8.0 Hz), 7.65
(dd, 1H, J = 8.8 Hz, 4.4 Hz), 7.20 (d, 1H, J = 8.0 Hz), 4.42
d, 1H, J = 8.4 Hz), 7.74 (dd, 1H, J = 8.8 Hz, 4.0 Hz), 7.06

4
JOAQUIM et Al.
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(
d, 1H, H–N, J = 8.0 Hz), 3.49–3.41 (m, 1H), 1.02 (d, 6H,
flasks using 0.25% trypsin/EDTA and plated in 96-well plates
at densities of 1.5 × 10 cells per well for 24 hr. Compounds
1
3
4
J = 6,4 Hz). C NMR (100 MHz, CDCl ) δ (ppm):156.9,
3
1
2
48.7, 138.4, 134.1,132.7, 125.6, 124.7, 123.8, 108.3, 46.3,
4.0. HRMS (m/z): [MH ] calc for C H N O S, 267.0803;
5a and 5b were tested at 3.0, 6.0, 12.0, 23.9, 47.8, 95.5,
191.0 μM and 12.1, 24.2, 48.5, 72.7, 97.0, 193.9, 387.9 μM,
respectively, for 48 hr. For the treatment of the cell culture,
the compounds were dissolved in culture medium contain-
ing DMSO 0.5%. Procedures were conducted as previously
described by Mosmann (1983). The results are expressed as
percentages relative to the control conditions. The experi-
ments are performed in triplicate from three independent as-
says. Data were statistically analyzed using one-way analysis
of variance (ANOVA), followed by a post-hoc Dunnett’s
test. Values of p < 0.05 were considered significant.
+
1
2
15
2
3
found, 267.0794.
2
.3.6
8-Hydroxyquinoline-5-(N,N-propyl)
|
sulfonamide 5f
Eluent hexanes: EtOAc (80:20). White solid. 10% yield (two
steps): mp: 161–164°C. IR ν: 3,299, 2,965, 2,935, 2,876,
1
1
,624, 1,571, 1,504, 1,469, 1,415, 1,372, 1,318, 1,283, 1,195,
,147, 1,114, 1,072 cm . H NMR (400 MHz, CDCl ) δ
−1
1
3
(
ppm): 9.01 (dd, 1H, J = 8.8 Hz, 1.6 Hz), 8.85 (dd, 1H,
J = 4.0 Hz, 1.6 Hz), 8.23 (d, 1H, J = 8.2 Hz), 7.61 (dd,
2
.6
Histopathological evaluation of pig ear
|
1
1
H, J = 8.8 Hz, 4.0 Hz), 7.17 (d, 1H, J = 8.2 Hz), 4.59 (s,
H), 2.86 (q, 1H, J = 7.2 Hz), 1.39 (sext, 2H), 0.75 (t, 3H,
skin exposed to 5a and 5b derivatives
1
3
J = 7.6 Hz). C NMR (100 MHz, CDCl ) δ (ppm): 157.0,
Tissue samples from adult male pigs, freshly slaughtered at
the Federal Institute of Santa Catarina—campus Concordia,
were used to evaluate the formation of tissue damage due to
the action of 5a and 5b. Pigs were slaughtered according to
the rules of the Brazilian Ministry of Agriculture, Livestock
and food supply, respecting animal welfare (BRASIL, 2000).
Ear skin of pigs was used in this study. Procedures were per-
formed according to our previous study (Pippi et al., 2017).
3
1
2
2
48.7, 138.4, 134.1, 132.8, 124.8, 124.7, 123.8, 108.2, 45.1,
+
3.1, 11.3. HRMS (m/z): [MH ] calc for C H N O S,
1
2
15
2
3
67.0803; found, 267.0794.
2
.4
Antimicrobial evaluation
|
The antimicrobial activity of prepared compounds was as-
sessed on Candida spp. and dermatophyte isolates, obtained
from the culture collections of the Laboratory of Applied
Mycology Research (Federal University of Rio Grande do
Sul, Porto Alegre, Brazil), as well as ATCC strains and
clinical isolates, obtained from the culture collections of the
Laboratory on Bacterial Resistance Research—LABRESIS
2
.7
Hens’ egg test on the chorioallantoic
|
membrane (HET-CAM)
Fresh white fertile eggs from Lohmann commercial breed
(Lohmann selected Leghorn, LSL, Brazil) were used in the
HET-CAM test. Procedures were performed according to our
previous study (Pippi et al., 2017). The experiment was per-
formed in triplicate. The result of the irritation score (IS) was
given according to Supporting Information Equation S1, on a
scale from 0 to 4.9 denoting non-irritation (or practically no
irritation) and 5.0–21 denoting irritation (moderate/severe or
extreme irritation) (ICCVMA, 2010).
(
Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil).
The MIC of the substances was determined by the broth mi-
crodilution method according to M27-A3 for yeasts, M38-
A2 for filamentous fungi, M-100 S26 for bacterial strains,
all documents published by the Clinical Laboratory and
Standards Institute, as described in our previous study (Pippi
et al., 2017). The MIC was defined as the lowest concentra-
tion of compound at which the microorganism tested did
not show visible growth. Fluconazole, ketoconazole, and ni-
troxoline were used as positive control against Candida spp.,
dermatophytes and bacterial strains, respectively.
2
.8
Time-kill assay
|
Time-kill assay was performed for 5a and 5b against C. al-
bicans ATCC 18804, M. canis MCA 01, and T. mentagro-
phytes TME 40. Procedures were conducted as previously
described by Ghannoum et al. (2013) and Klepser, Ernst,
Lewis, Ernst, and Pfaller (1998). For C. albicans, a yeast
cell suspension was prepared from 24 hr culture at 35°C on
SDA (Sabouraud dextrose agar; Himedia; India) with chlo-
ramphenicol and it was adjusted to a 0.5 McFarland turbid-
2
.5
Cytotoxicity
|
Cell viability was performed for 5a and 5b by the MTT (3
-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) reduc-
tion assay, using VERO cells obtained from BCRJ (Bank of
Cells of Rio de Janeiro). Cells were cultured in Dulbecco’s
Modified Eagle’s Medium supplemented with 20% of fetal
bovine serum, 0.5% penicillin/streptomycin and 0.1% am-
6
ity standard (1–5 × 10 colony-forming units [CFU]/ml). A
1:10 dilution of this suspension was made by adding 1 ml
of fungal suspension to 9 ml of RPMI 1640 with or with-
out (growth control) the desired amount of antifungal agents.
photericin B at 37°C in an atmosphere of 5% CO /95% air.
2
Exponentially growing cells were detached from the culture

JOAQUIM et Al.
5
|
SCHEME 1 Chlorosulfonation of the 8-hydroxyquinoline 1 and formation of derivatives 5a–5f. Reagents and conditions: (a) ClSO H, r. t.,
3
1
8 hr; (b) Appropriate amine, acetonitrile, 80°C, 3 hr
This dilution resulted in a solution with an inoculum of ap-
ꢁ
ꢂ
ꢁ
ꢂ
ꢀ
ꢅ
ꢃ
ꢄ
Kmax
C
5
dN
dt
N
-
xt
proximately 1–5 × 10 CFU/ml (Klepser et al., 1998). For
=
k0 1−
1−exp
−
N
(
(
3)
4)
Nmax
EC +C
50
the filamentous fungi (M. canis MCA 01 and T. mentagro-
phytes TME 40), a suspension of each fungus was prepared
from 7 days culture on potato dextrose agar (Himedia) and
ꢁ
ꢂ ꢁ
ꢂ
ꢀ
ꢅ
Kmax
C
ꢃ
ꢄ
dN
dt
N
-
zt
= k 1−
0
−
1−exp
N.
3
Nmax
EC +C
50
it was adjusted to 1–3 × 10 CFU/ml in RPMI 1640 with or
without (control) the desired amount of antifungal agents
Where dN/dt is the variation in number of fungal cells as
(
Ghannoum et al., 2013).
−
1
a function of time; k (hr ) is the fungal growth rate constant
The concentrations used for each compound were: MIC,
0
−
1
in the absence of the compound; K (hr ) is the maximum
MICx2, and MICx4. All test solutions and controls were
made in triplicate and were incubated at 35°C (Candida) or
max
killing rate constant (maximum effect); EC (μM) the con-
50
centration of compound necessary to produce 50% of maxi-
3
6
9
0°C (dermatophytes). At predetermined time points (0, 3,
, 12, 24, and 48 hr for Candida; and 0, 3, 6, 12, 24, 48, and
6 hr for dermatophytes), 100 μl samples were aseptically
mum effect; C (μM) the concentration of compound at any
time (t); N (log CFU/ml) the number of viable fungal cells
10
and N
is the maximum number of cells. The models ac-
removed from each tube and serially diluted 10-fold (until
max
−3
count for delays in growth (x) and onset of kill (z), and in-
corporate a Hill factor (h) that modifies the steepness of the
slopes and smoothes the curves.
1
0 ) with sterile water. Then, 15 μl directly from the test
solution and each dilution solution were spread onto a SDA
plate. Plates were incubated at 35°C/48 hr for Candida and
3
The mathematical modeling of the kill curve data was
performed with the non-linear regression software program
0°C/96 hr for dermatophytes. After this period, the colony
count was performed for determination of log CFU/ml.
1
0
®
Scientist 3.0 (Micromath, Salt Lake, UT, USA). Graphs
Fungicidal effect was considered when there was ≥99.9%
were visually inspected for quality of fit and model selection
decrease in the log of the number of CFU/ml compared to
the starting inoculum.
1
0
2
criterion, coefficient of determination (R ), and correlation
between measured and calculated data points.
2
.9
PK/PD modeling
|
3
3
RESULTS AND DISCUSSION
Chemistry
|
Kill curve data were used to apply the mathematical mod-
eling. Adapted E models described by Li et al. (2009)
max
.1
|
and Treyaprasert et al. (2007) were used for C. albicans
Equation (1)), M. canis (Equation (2)), and T. menta-
grophytes (Equation (3) for 5a; and Equation 4 for 5b)
Treyaprasert et al., 2007), modeling:
Sulfonyl chloride was prepared as previously re-
ported (Scheme 1) (Pearce, Jotterand, Carrico,
Imperiali, 2001). Pearce et al. (2001) report on the
-hydroxy-2-methyl-quinoline chlorosulfonation, followed
(
&
(
8
ꢅ
ꢆ
ꢁ
ꢂ
ꢀ
KmaxCh
ꢇ
ꢃ
ꢄ
by treatment using brine and ice and extraction with di-
chloromethane. The 8-hydroxyquinoline chlorosulfona-
tion in adapted conditions gave the sulfonyl chloride in
33% yield. Next, the appropriate amine was introduced,
in the previous conditions (Aik et al., 2013). Six differ-
ent amines were used, 4-chloroaniline, 4-methoxyaniline,
dN
N
-
xt
=
k0 1−
1−exp
−
N
(1)
hr
h
dt
Nmax
EC +C
5
0
ꢁ
ꢂ
ꢁ
ꢂ
ꢀ
ꢅ
ꢃ
ꢄ
Kmax
C
ꢃ
ꢄ
dN
dt
N
-xt
-zt
=
k0 1−
1−exp
−
1−exp
N
Nmax
EC50 +C
(2)

6
JOAQUIM et Al.
|

JOAQUIM et Al.
7
|
4
-aminobenzonitrile, 2-aminopyrimidine, isopropylamine,
showing activity against other tested Gram-positive cocci
ATCC isolates. Contrary to what was expected, the sulfadi-
azine hybrid (compound 5d) did not show antibacterial ac-
tivity. This suggests that the mechanism of action of these
hybrids could not be related to the mechanism of action of
sulfa drugs (reduction of folic acid by enzymatic inhibition)
(Sköld, 2000).
and n-propylamine, to give derivatives 5a–5f in 10%–23%
yield (two steps), Scheme 1. Aik et al. (2013) have re-
ported the purification of 8-hydroxyquinoline-derivatives,
such as 8-hydroxyquinoline-5-(methyl)sulfonamide, by
semipreparative reverse-phase HPLC. However, in our
case, the products were obtained in good purity using col-
1
13
umn chromatography, which was confirmed by H and
C
Some related sulfonamides derived from 8HQ are re-
ported in the literature. Ariyasu et al. (2014) reported many
compounds as radioprotective agents. Aik et al. (2013)
evaluated the activity of inhibitors of the fat mass and
obesity associated protein, aiming to obtain potential anti-
obesity agents. To the best of our knowledge, it is the first
time that sulfonamides derived from 8-hydroxyquinoline
have shown interesting antimicrobial activity. Previously,
Musiol et al. (2010) have evaluated a total of 14 analogues
against eight fungal species. The antifungal activity of
four 8HQ derivatives containing sulfonamides substituted
by bulky groups was virtually nil (Musiol et al., 2010). In
a more recent study, Kassem et al. (2012) evaluated the
microbiological activity of a series of substances derived
from 8-hydroxyquinoline-5-sulfonamide, with pyranopyra-
zole and pyranoimidazole substituents, using the diffusion
disc technique. Some substances tested showed relevant
activity against bacterial species. However, the only fungal
species tested was C. albicans, whose relevant activity of
compounds was not observed, since a quantity of 100 μg
was necessary to the inhibition (Kassem et al., 2012).
NMR spectra (Supporting Information). In order to exem-
plify the characterization of the synthesized compounds,
we have chosen the compound 5a because it was the most
potent compound in this series as will be highlighted in
the next section and has a representative structure. The
1
H NMR spectrum of 5a revealed the signals of the 8HQ
core at δ 8.95 (bd, 1H, H2), δ 8.87 (dd, 1H, H4), δ 8.20
(
d, 1H, H6), and δ 7.60 (dd, 1H, H3). The H7 signal of the
8
HQ core should appear as a doublet but it was detected
together with the hydrogens ortho to NH of sulfonamide
at δ 7.16–7.12, as a multiplet; the signal of the hydrogens
meta to NH appeared at δ 6.91–6.88, as a multiplet. In the
1
3
C NMR spectrum of 5a, the signals of the all aromatic
carbons were detected at δ 157.5–108.4 ppm and a peak
+
with m/z 335.0254 [MH ] in HRMS has confirmed the as-
signment. Similar analysis was carried out for the other de-
rivatives which have different substituents attached to the
nitrogen of the sulfonamide.
The first step yield was modest but relevant. The possible
cause for this could be due to quinoline protonation under
acid condition, and consequently, high affinity for the aque-
ous layer. However, the sulfonyl chloride treatment under al-
kaline condition is infeasible, since the hydroxy group and
the sulfonyl chloride could be deprotonated and hydrolyzed,
respectively.
3
.3
Cytotoxicity
|
As demonstrated in Supporting Information Figure S13,
compound 5a presented IC = 54.6 μM (4- to 16-fold the
5
0
antifungal MIC value depending on the fungal isolate), in-
dicating low cytotoxicity. However, compound 5b presented
toxicity (IC = 67.1 μM, which is around 3-fold the antifun-
3
.2
Antimicrobial evaluation
|
5
0
The results of the antifungal and antibacterial evaluation are
summarized in Table 1 and Supporting Information Table S1,
respectively, and are expressed as MIC in μM. Compound
gal MIC value).
3
.4
Histopathological evaluation of pig ear
|
5
a (X = p-ClPh) was the most potent derivative in this se-
skin exposed to 5a and 5b derivatives
ries being active against all fungal species tested, with MIC
values comparable to the positive control (fluconazole), and
it has kept the potency against control resistant isolates. The
activity of 5b (X = p-OMePh) was comparable to 5e, in gen-
eral, and lower than 5a. Compound 5e (X = isopropyl) was
more active than 5f (X = n-propyl). Finally, compounds 5c
The histopathological evaluation by microscopic analysis of
the pig ear skin exposed to 5a and 5b demonstrated no le-
sions (Figure 3) as well as PBS (negative control). This result
confirmed a low topical toxicity of 8-hydroxyquinoline-5-su
lfonamides. Topical treatment of fungal skin infections has
several benefits such as direct action at the site of infection,
increase of treatment efficacy, and minimized entering of
drugs into systemic circulation with reduction of side effects
(Güngör & Aksu, 2013). The low toxicity in histopathologi-
cal evaluation and the low level of irritability presented by 5a
and 5b are important properties for the development of new
drugs for topical treatment of skin mycoses.
(
X = p-CNPh) and 5d (X = 2-pyrimidinyl) have showed
poor activity; besides 5d was active against one C. glabrata
isolate.
Compounds 5a and 5b have presented important anti-
bacterial activity against the tested S. aureus strains, both
clinical isolates (Supporting Information Tables S2 and S3,
Supporting Information) and ATCC strain, in addition to

8
JOAQUIM et Al.
|
(
1a)
(1b)
(1c)
(
2a)
(2b)
(2c)
FIGURE 3 Histopathological evaluation of porcine cells treated with 8-hydroxyquinoline-5-sulfonamides and negative control at 100 and
00 times magnification. The concentration of each 8-hydroxyquinoline derivative used in the test was 1 mg/ml. (1a) Swine epidermal cells treated
4
with 5a at 100 times magnification; (1b) Swine epidermal cells treated with 5b at 100 times magnification; (1c) Swine epidermal cells treated with
PBS pH 7.0 at 100 times magnification; (2a) Swine epidermal cells treated with 5a at 400 times magnification; (2b) Swine epidermal cells treated
with 5b at 400 times magnification; (2c) Swine epidermal cells treated with PBS pH 7.0 at 400 times magnification
MICx4. The fitted curves resulting from modeling of 5a and
3
.5
Hens’ egg test on the chorioallantoic
|
5
b against the three different fungal species are shown in
membrane (HET-CAM)
Supporting Information Figures S14 and S15, respectively,
and the determined PD parameters are listed in Tables 2 and
3.
The IS of 8-hydroxyquinoline-5-sulfonamides was repre-
sented by Supporting Information Equation S1 (ICCVMA,
2
3
010) (Supporting Information). 5a and 5b showed IS of
With the use of time-kill assays we showed that by chang-
ing the substituent in the phenyl ring of 8-hydroxyquinolines
it is possible to obtain different inhibition patterns of fungal
cells. The methoxy presence in 5b appears to be important for
the fungicidal activity against dermatophytes. On the other
hand, the chlorine present in 5a favors the fungistatic activ-
ity. However, no substituent has led to the fungicidal activity
for C. albicans. In this regard, our data were in agreement
with previously reported in vitro results of the fungistatic and
fungicidal activity of other 8-hydroxyquinoline derivatives
against C. albicans and dermatophytes, respectively (Pippi
et al., 2017).
.15 and 2.91, respectively, showing statistically significant
difference (p ≤ 0.05) when compared to the positive control
(
IS = 19.83). 5b presents IS significantly lower than that of
a, however, both were classified as non-irritant according to
tested methodology.
5
3
.6
Time-kill assay and PK/PD modeling
|
5
a exhibited fungistatic activity for C. albicans and derma-
tophytes. 5b showed fungistatic effect for C. albicans and
exhibited fungicidal activity (≥99.9% reduction in CFU)
for M. canis and T. mentagrophytes. However, this effect
was concentration and time dependent. 5b showed fungi-
cidal effect at 96 hr in concentrations equal to MICx2 and
To model our time-kill data it was necessary to apply
different PK/PD models for each individual strain in order
to obtain a better description of the antifungal activity.

JOAQUIM et Al.
9
|
TABLE 2 Pharmacodynamic
parameters determined for 5a, and goodness
of fit criteria
Trichophyton
Candida albicans Microsporum
(ATCC 18804)
mentagrophytes (TME
Parameters
canis (MCA 01) 40)
Minimal inhibitory
concentration (μM)
6.0 ± 0
3.0 ± 0
3.0 ± 0
−1
k0 (hr
)
0.320
0.020
3.967
6.651
0.130
–
0.684
0.027
2.755
4.222
0.002
0.021
–
0.107
0.056
2.606
4.350
0.060
–
Kmax (hr−1)
EC50 (μM)
Nmax (log CFU/ml)
10
−
1
x (hr
)
z (hr−1)
H
0.612
–
Model selection criterion 2.580–3.938
range)
2.194–3.731
1.566–2.284
(
2
R (range)
0.999
0.999
0.998–0.999
TABLE 3 Pharmacodynamic
parameters determined for 5b, and goodness
of fit criteria
Trichophyton
mentagrophytes (TME
Candida albicans Microsporum
(ATCC 18804)
Parameters
canis (MCA 01) 40)
Minimal inhibitory
concentration (μM)
24.2 ± 0
24.2 ± 0
24.2 ± 0
−1
k0 (hr
)
0.153
0.097
22.061
7.945
0.742
–
1.042
0.039
26.864
4.234
0.001
0.010
–
0.028
2.786
21.530
4.310
–
Kmax (hr−1)
EC50 (μM)
Nmax (log CFU/ml)
10
−
1
x (hr
)
z (hr−1)
0.015
–
H
0.874
Model selection criterion 2.209–2.721
range)
2.289–2.282
1.065–3.380
(
2
R (range)
0.996–0.999
0.999
0.998–0.999
Summarizing, the data showed that 5a and 5b are effective
against C. albicans, M. canis, and T. mentagrophytes in low
concentrations. The EC ranged from 2.606 to 3.967 μM
Pharmacokinetic/PD modeling can be more efficiently
applied to optimize the use of currently available antimicro-
bial agents and to facilitate development of new antimicrobial
agents. Although experimental data of candidate drugs can be
sparse, the modeling still allow for the drug-effect parameters
to be successfully estimated and make drug development faster
at a lower cost. Furthermore, the model can be constantly up-
dated during drug development once the parameters can be re-
estimated to improve and extend the predictive capacity when
new findings are obtained (Nielsen & Friberg, 2013).
5
0
for 5a and 21.530 to 26.864 μM for 5b (concentrations sim-
ilar to MIC values). Although EC were higher for 5b, this
5
0
compound achieved maximum effect more rapidly than 5a
for all fungi studied. 5b reached the maximum effect against
C. albicans, M. canis, and T. mentagrophytes after 7, 17, and
0
.25 hr, respectively; while the estimated time for maximum
effect of 5a was 34, 25, and 12 hr. This approach of com-
bining in vitro time–kill data with PK/PD modeling might
serve to delineate future studies in animals in order to define
the dosage of the compounds to be applied to the animal.
However, in this case it is still necessary to measure the per-
meation profile in tissues of 5a and 5b to better delineate in
vivo studies.
4
CONCLUSIONS
|
In conclusion, we have described herein the synthesis and a
detailed antimicrobial evaluation of a novel series of hybrids

1
0
JOAQUIM et Al.
|
between the 8-hydroxyquinoline and sulfonamide, in 10%–
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3% yield overall. Moreover, this work has used tools to bet-
ter evaluate the interaction of the most active derivatives with
fungal cells. Compound 5a was the most active against all
fungal species tested, presenting an antifungal profile simi-
lar to fluconazole, an antifungal agent often used (Tobudic
et al., 2012), in addition to present low toxicity in normal
cells. Besides, 5a and 5b have presented an important an-
tibacterial activity against the tested S. aureus strains, both
clinical isolates and ATCC strain, in addition to showing ac-
tivity for other tested Gram-positive cocci ATCC. 5a exhib-
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5
b showed fungistatic effect for C. albicans and exhibited
fungicidal activity for M. canis and T. mentagrophytes (con-
centration and time dependent). The EC values obtained by
5
0
combination of time-kill studies with mathematical model
were similar to those of MIC, which clarifies the potential
of these compounds. Although EC were higher for 5b, this
5
0
compound achieved maximum effect more rapidly than 5a
for all fungi studied. 5a and 5b are highly effective, non-
irritant molecules, and do not exhibit topical toxicity. Thus,
these compounds are promising antifungal candidates for the
treatment of dermatomycosis and candidiasis.
ACKNOWLEDGMENTS
We are thankful the Fundação de Amparo à Pesquisa do
Estado do Rio Grande do Sul (FAPERGS – EDITAL
0
4/2016 – PRONUPEQ 2016) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico for the financial
support. This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior - Brasil
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Hardas, B. (2013). In vitro antifungal activity of naftifine hydrochlo-
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7, 4369–4372. https://doi.org/10.1128/AAC.01084-13
(
CAPES) - Finance Code 001.
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treatment of onychomycosis. Clinics in Dermatology, 31, 544–554.
https://doi.org/10.1016/j.clindermatol.2013.06.011
The authors have declared no conflict of interest.
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Additional supporting information may be found online in
the Supporting Information section at the end of the article.
1
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