Exploring the thiazole scaffold for the identification of new agents for the treatment of fluconazole resistant Candida

Article abstract of DOI:10.3109/14756366.2015.1113171

Cyclohexyliden- and 2-methylcyclohexyliden-hydrazo-4-arylthiazoles were synthesized and tested as antifungal agents. All compounds exhibited minimal inhibitory concentration (MIC) values comparable with those of fluconazole (FLC). Moreover, some compounds

Full text of DOI:10.3109/14756366.2015.1113171

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Exploring the thiazole scaffold for the
identification of new agents for the treatment of
fluconazole resistant Candida
Rita Meleddu, Simona Distinto, Angela Corona, Elias Maccioni, Antonella
Arridu, Claudia Melis, Giulia Bianco, Peter Matyus, Filippo Cottiglia, Adriana
Sanna & Alessandro De Logu
To cite this article: Rita Meleddu, Simona Distinto, Angela Corona, Elias Maccioni, Antonella
Arridu, Claudia Melis, Giulia Bianco, Peter Matyus, Filippo Cottiglia, Adriana Sanna &
Alessandro De Logu (2016): Exploring the thiazole scaffold for the identification of new agents
for the treatment of fluconazole resistant Candida, Journal of Enzyme Inhibition and Medicinal
Chemistry, DOI: 10.3109/14756366.2015.1113171
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1113171
Published online: 08 Jan 2016.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ienz20
Download by: [Gazi University]
Date: 11 January 2016, At: 05:50

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2016 Taylor & Francis. DOI: 10.3109/14756366.2015.1113171
!
SHORT COMMUNICATION
Exploring the thiazole scaffold for the identification of new agents for
the treatment of fluconazole resistant Candida
Rita Meleddu1, Simona Distinto1, Angela Corona1, Elias Maccioni1, Antonella Arridu1, Claudia Melis1, Giulia Bianco1,
Peter Matyus2, Filippo Cottiglia1, Adriana Sanna3, and Alessandro De Logu1
2
1Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy, Department of Organic Chemistry, Semmelweis University
3
Hogyes Endre U, Budapest, Hungary, and Department of Public Health, Clinical and Molecular Medicine, University of Cagliari, Cittadella
Universitaria, Monserrato, Italy
Abstract
Keywords
Cyclohexyliden- and 2-methylcyclohexyliden-hydrazo-4-arylthiazoles were synthesized and
tested as antifungal agents. All compounds exhibited minimal inhibitory concentration (MIC)
values comparable with those of fluconazole (FLC). Moreover, some compounds showed
fungicidal activity at low concentration. Worth noting five out of nine compounds were
active towards Candida albicans 25 FLC resistant isolated from clinical specimens. The cellular
toxicity was evaluated and none of the compounds is toxic at the MIC. On the basis of our data
we can conclude that these derivatives are promising agents for the treatment of resistant
C. albicans.
Fungicidal activity, heterocyclic antifungal
agents, iron (III) chelators
History
Received 29 September 2015
Revised 20 October 2015
Accepted 21 October 2015
Published online 7 January 2016
Introduction
In order to contrast this tendency, different efforts have been
made to overcome the emergence of resistant fungi by using multi
drugs therapy. Unfortunately, high costs and serious side effects
Over the past 20 years the incidence of invasive fungal infections
and associated mortality have increased significantly as a
consequence of the rising number of immunocompromised
patients, the wide development of organ transplantation, tracheal
intubation and endoscopic techniques, and the extensive applica-
tion of broad-spectrum antibiotics, immunosuppressant drugs and
corticosteroids1,2. In this respect, Candida albicans accounts for a
large proportion of invasive fungal infections, placing itself at the
first place as the major fungal pathogen in humans3.
have put limitation on the combinations of antifungal drugs12,13
.
In addition, contradictory results of the synergistic or antagonistic
action of various antifungals combination have been
reported9,14,15
.
FLC-like drugs inhibit the cytochrome P450 sterol 14a-
demethylase (14DM, CYP51) by coordinating the heme iron atom
in the catalytic site with the azole nitrogen atom (N-3 and N-4 in
the case of imidazole and triazole respectively). Inhibition of the
cytochrome P450 3 A-dependent lanosterol 14-a-demethylase
leads to accumulation of 14-a-methylsterol and depletion of
ergosterol16 resulting in plasma membrane structure disruption11.
The efficacy and selectivity of azoles depends on the strength of
the binding to heme iron as well as the affinity of the N-1
substituent for the CYP5117.
It has been reported that the presence of iron chelators
influences the growth of C. albicans18,19. In this respect,
thiosemicarbazones iron chelators have been reported as anti-
fungal agents and their activity compared with that of FLC20.
On the basis of the above, the focus of our research is the
design of a new class of anti-Candida agents characterized by a
different scaffold and mechanism of action, with respect to the
azole derivatives, that might be active towards FLC resistant
Candida species. Indeed, iron chelators could represent a valuable
therapeutic approach to Candida infection.
This organism, together with related Candida species, has
become one of the commonest agents of hospital-acquired
infection and in AIDS patients2–4
.
So far there are four classes of antifungal agents, (polyenes, 5-
fluorocytosine, azoles and echinocandins) that can be used for the
treatment of systemic infections5–8. Nevertheless, these antifungal
agents have achieved limited success in term of severe resistance,
limited efficacy and spectrum, drug-related toxicity, non-optimal
pharmacokinetics and serious drug–drug interactions. Azoles, and
in particular fluconazole (FLC), have been extensively used in
clinical practice because of their great efficacy and low toxicity3,9
However, due to the excessive exposure to FLC, FLC-resistant
C. albicans species have emerged2,10,11
.
.
Address for correspondence: Elias Maccioni, Department of Life and
Environmental Sciences, University of Cagliari, via Ospedale 72,
Cagliari, 09124, Italy. Tel: +39-070-675-8744. Fax: +39-070-675-8553.
E-mail: maccione@unica.it
Our research group has already studied similar scaffolds as
antifungal and antibacterial agents and some of these compounds
exhibited potent activity towards several species of Candida21–24
.

2
R. Meleddu et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Table 1. Chemical, analytical and physical data of derivatives EMAC2098–2100–2101–2103–2104–2105–2107–2109–2110.
R
S
R'
N
N
N
H
CHN
ꢀ
Compound
R
R0
Mp C
% Yield
Calc.
Found
EMAC2098
EMAC2100
EMAC2101
EMAC2103
EMAC2104
EMAC2105
EMAC2107
EMAC2109
EMAC2110
H
H
H
4-Br
4-NO2
4-CH3
4-CN
H
4-Br
4-NO2
4-CN
4-F
179–180
172–173
183–185
206–207
162–164
130–131
163–164
200–201
178–180
79
83
76
83
79
77
85
79
79
51.43, 4.60, 12.00
56.94, 5.10, 17.71
67.33, 6.71, 14.72
64.10, 5.38, 18.69
67.33, 6.71, 14.72
52.75, 4.98, 11.53
58.16, 5.49, 16.96
65.78, 5.84, 18.05
63.34, 5.98, 13.85
51.65, 4.61, 12.05
57.16, 5.12, 17.65
67.58, 6.72, 14.76
64.35, 5.40, 18.76
67.58, 6.72, 14.77
52.96, 4.99, 11.57
58.39, 5.51, 17.01
66.04, 5.85, 18.12
63.58, 6.00, 13.90
H
CH3
CH3
CH3
CH3
CH3
Moreover, very similar compounds were reported as potent and were subcultured on Potato Dextrose Agar plates (Difco, BD
selective monoamine oxidase inhibitors25,26
.
Diagnostics, Sparks, MD).
Methods
Testing compounds
Materials and apparatus
Compounds
EMAC2098–2100–2101–2103–2104–2105–2107–
2109–2110 were dissolved in dimethyl sulfoxide (Sigma-Aldrich,
Saint Louis, MO) at concentrations ranging between 2 and 25 mg/
ml depending on their specific solubility. FLC (Sigma-Aldrich),
employed as reference drug, was dissolved in dimethyl sulfoxide at
50 mg/ml. Solutions of the testing compounds and FLC were stored
at ꢁ20 ꢀC until use.
Starting materials and reagents were obtained from commercial
suppliers and were used without purification. All melting points
were determined on a Stuart SMP11 melting points apparatus and
are uncorrected. Electron ionization mass spectra were obtained by
a Fisons QMD 1000 mass spectrometer (70 eV, 200 mA, ion source
temperature 200 ꢀC). Samples were directly introduced into the ion
source. Found mass values are in agreement with theoretical ones.
Melting points, yield of reactions and the analytical and descriptive
data of derivatives are reported in Tables 1 and 2.
Antifungal susceptibility assays
Minimal inhibitory concentrations (MICs) were determined by the
broth microdilution method according to the Clinical and
Laboratory Standard Institute reference document M27-A3. RPMI
1640 medium (Sigma-Aldrich) without sodium bicarbonate, sup-
plemented with L-glutamine (Gibco, Life Technologies, Grand
Island, NY) and buffered with 0.165M MOPS (Sigma-Aldrich) at
pH 7.0 was used as test medium. Two-fold dilutions of the testing
compounds at concentration ranging between 0.048 and 200mg/ml
were obtained in RPMI 1640 and 100ml of the obtained solutions
were dispensed into the wells of microdilutions trays. 100 ml
inoculum containing 2 ꢂ 104 cells/ml of C. albicans was added to
each well to obtain final concentrations of the testing compounds
ranging between 0.024 and 100 mg/ml and final inocula of 1 ꢂ 104
cells/ml. Plates were incubated at 35ꢀC for 24–48h and then read
visually. MICs were determined as the lowest concentration at
which a 100% inhibition of growth compared with drug-free
control was observed. Minimal fungicidal concentrations (MFCs)
were determined by seeding a 100 -ml sample obtained from the
wells for the MIC determination on plates of Sabouraud Dextrose
agar. Plates were incubated at 35 ꢀC for 72h. MCF was defined as
the lowest as the minimum concentration of compound that resulted
in the growth of less than two colonies representing the killing of
499% of the original inoculum.
1H NMR were registered on a Bruker AVANCE III 500 MHz
spectrometer. All samples were measured in CDCl3. Chemical
shifts are reported referenced to the solvent in which they were
measured. Coupling constants J are expressed in Hertz (Hz).
Elemental analyses were obtained on a Perkin–Elmer 240 B
microanalyser. Analytical data of the synthesized compounds are
in agreement within 0.4% of the theoretical values. TLC
chromatography was performed using silica gel plates (Merck F
254), spots were visualized by UV light.
General procedure for the synthesis of compound
derivatives EMAC2098–2100-2101–2103–2104–
2105–2107–2109–2110
In a typical experiment, 2 mmol of cycloalkyl-thiosemicarbazone
were reacted at room temperature (rt) in 20 ml of tap water with
2 mmol of a-halogen ketone The mixture was stirred for a period
ranging between 24 and 32 h and the formation of a forming
product is observed. The stirring was stopped and the solid filtered.
The crude product was crystallized from ethanol, water/ethanol.
Biological assay
Strains
Cytotoxicity studies
Antifungal susceptibility studies. The in vitro activity of com-
pounds EMAC2098–2100–2101–2103–2104–2105–2107–2109–
2110 was assessed against C. albicans ATTC 10231 and against
a strain of C. albicans FLC resistant isolated from clinical
specimen identified by standard methods. Prior to testing, strains
Vero cells lines (African green monkey kidney, BS CL 86) used in
this study were obtained from IZSLER (Brescia, Italy). Cells were
grown on RPMI 1540 medium supplemented with 2 Mm
L-glutamine, penicillin 50 IU/ml, streptomycin 50 mg/l and 10%

DOI: 10.3109/14756366.2015.1113171
Identification of new agents for the treatment of FLC resistant Candida
3
Table 2. Chemical and descriptive data of derivatives EMAC 2098–2100–2101–2103–2104–2105–2107–2109–2110.
Compound
Reaction solvent
Crystallization solvent
Crystals aspect
Reaction time (h)
EMAC2098
EMAC2100
EMAC2101
EMAC2103
EMAC2104
EMAC2105
EMAC2107
EMAC2109
EMAC2110
Water
Water
Water
Water
Water
Water
Water
Water
n-propanol
Ethanol
Ethanol
Ethanol
Crystalline pale yellow
Crystalline orange
32
26
26
26
26
48
24
28
28
Crystalline pale brown
Crystalline pale red
Crystalline pale brown
Crystalline pale orange
Crystalline yellow
Ethanol
Water/ethanol
Hexane
Water/ethanol
Water/ethanol
Ethanol
Crystalline bright red
Crystalline white
H N
S
2
R
C
R
C
HN
C
NH
N
NH
O
2
C
S
i.
H N
2
O
ii.
C
R'
H C
X
2
R'
S
R
C
N
C
N
NH
Scheme 1. Synthesis of EMAC compounds. Reagents and conditions: (i) n-propanol, acetic acid; (ii) water.
fetal bovine serum. Cells were seeded in 96-well plastic microtiter them toward FLC resistant C. albicans. Thus, to achieve more
plates (Falcon, BD Biosciences, Bedford, MD) at a density of information on the SARs of these compounds and to evaluate their
1 ꢂ 104 cells/ml in RPMI 1640 containing no antibiotics and the activity toward FLC resistant Candida species, we have synthesized
testing compounds at concentrations ranging between 0.78 and differently substituted cycloalkylidenhydrazo-4-arylthiazoles.
500 mg/ml depending on their solubility in DMSO. To avoid
Desired compounds were synthesized starting from different
interference by the solvent, the maximum concentration of DMSO cyclic ketones (1.2 mol) with thiosemicarbazide (1 mol) in n-
was 2%. Cells were incubated at 37 ꢀC in 5% CO2 and observed propanol with catalytic amounts of acetic acid at reflux condition
for morphological changes at 24, 48 and 72 h of incubation. After for 12–24 h to obtain the corresponding thiosemicarbazones that
72 h the effects on the proliferation of Vero cells were determined were crystallized from ethanol. 4-Substituted thiazole derivatives
by the tetrazolium-based colorimetric MTT (3-[4,5-dimethylthia- were obtained by reaction of differently substituted a-halogen-
zol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The 50% cell- oketones with the previously obtained thiosemicarbazones as
inhibitory concentration (IC50) reduced by 50% the optical density shown in Scheme 1.
values (OD540 690) with respect to control untreated cells.
The reaction was performed using water as a solvent. Indeed
water proved to be a more efficient, cheaper, and green solvent in
comparison with both ethanol and isopropanol. The reaction
was performed at rt and the formation of a foamy precipitate
is observed. All derivatives were crystallized from ethanol or
water/ethanol.
The structures were confirmed by means of analytical and
spectroscopic methods. 1H NMR chemical shifts of all the
synthesized compounds are reported in Table 3.
Coordination studies
The interaction between EMAC 2104 and the Fe2+ or Fe3+ ions
was checked by recording the UV/vis absorbance spectra of
EMAC2104 in absence and in presence either of FeCl3 or FeSO4
using FLC as positive control. Compounds were solubilized in
DMSO then diluted to 100 mM in water. The UV/vis spectrum of
the solution was recorded before and after addition of 10 mM final
concentration either of FeCl3 or FeSO4. The spectra were
recorded with a Ultrospec 2100 pro (Amersham Biosciences)
and analyzed with SWIFT II–METHOD software (GE
Healthcare).
Compounds
EMAC2098–2100–2101–2103–2104–2105–
2107–2109–2110 were assayed for their inhibitory activity
toward C. albicans (Table 4). All tested compounds exhibit a
remarkable activity towards C. albicans ATCC 10231.
Noteworthy two of the synthesized compounds exhibit a very
interesting activity towards C. albicans 25 FLC-resistant.
Results and discussion
Compounds EMAC2098, EMAC2100, EMAC2101 and
As a continuation of our previous work on the discovery of new anti- EMAC2103 exhibit an interesting activity toward C. albicans
Candida agents, we decided to focus our attention on the synthesis ATCC 10231. MIC values range from 0.19 to 3.12 mg/ml.
and biological evaluation of thiazole derivatives and test screen Interestingly, all compounds, with the exception of EMAC2100,

4
R. Meleddu et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Table 3. 1H NMR data of derivatives EMAC2098, EMAC2100, EMAC2101, EMAC2103, EMAC2104, EMAC2105, EMAC2107, EMAC2109,
EMAC2110.
Compound
EMAC2098
1H NMRd (ppm)
1H NMR: (500 MHz, CDCl3) dH 1.67 (m, 2H, CH2, cyclohexyl), 1.78 (m, 4H, CH2, cyclohexyl), 2.40 (t, 2H, J: 6.3, CH2,
cyclohexyl), 2.63 (t, 2H, J: 6.5, CH2, cyclohexyl), 6.69 (s, 1H, CH, thiazole), 7.60 (m, 4H, CH, phenyl), 12.45 (br.s, 1H, NH, D2O
exch.)
EMAC2100
EMAC2101
1H-NMR: (500 MHz, CDCl3) dH 1.70 (m, 6H, CH2, cyclohexyl), 2.31 (m, 2H, CH2, cyclohexyl), 2.38 (m, 2H, CH2, cyclohexyl),
7.07 (s, 1H, CH, thiazole), 7.93 (d, 2H, J: 9.0, CH, phenyl), 8.24 (d, 2H, J: 8.9, CH, phenyl), 8.40 (s, 1H, NH, D2O exch.)
1H-NMR: (500 MHz, CDCl3) dH 1.67 (m, 2H, CH2, cyclohexyl), 1.78 (m, 4H, CH2, cyclohexyl), 2.39 (m, 5H, CH2, cyclohexyl +
CH3), 2.64 (m, 2H, CH2, cyclohexyl), 6.60 (s, 1H, CH, thiazole), 7.28 (d, 2H, J: 7.8, CH, phenyl), 7.60 (d, 2H, J: 8.2, CH, phenyl),
12.49 (br.s, 1H, NH, D2O exch)
EMAC2103
EMAC2104
1H NMR: (500 MHz, CDCl3) dH 1.65 (m, 4H, CH2, cyclohexyl), 1.74 (m, 2H, CH2, cyclohexyl), 2.28 (m, 2H, CH2, cyclohexyl),
2.37 (m, 2H, CH2, cyclohexyl), 7.00 (s, 1H, CH, thiazole), 7.65 (d, 2H, J: 8.3, CH, phenyl), 7.87 (d, 2H, J: 8.4, CH, phenyl), 8.54
(s, 1H, NH)
1H NMR: (500 MHz, CDCl3) dH 1.19 (s, 3H, CH3), 1.33 (m, 1H, CH, cyclohexyl), 1.51 (m, 2H, CH2, cyclohexyl), 1.79 (m, 1H,
CH, cyclohexyl), 1.86 (m, 1H, CH, cyclohexyl), 1.95 (m, 2H, CH2, cyclohexyl), 2.37 (m, 1H, CH, cyclohexyl), 2.60 (m, 1H, CH,
cyclohexyl), 6.84 (s, 1H, CH, thiazole), 7.29 (m, 1H, CH, phenyl), 7.38 (d, 2H, J: 7.7, CH, phenyl), 7.79 (d, 2H, J: 6.6, CH,
phenyl), 8.44 (br. s, 1H, NH)
EMAC2105
EMAC2107
EMAC2109
EMAC2110
1H NMR: (500 MHz, CDCl3) dH 1.18 (s, 3H, CH3), 1.32 (m, 1H, CH, cyclohexyl), 1.51 (m, 2H, CH2, cyclohexyl), 1.90 (m, 4H,
CH2, cyclohexyl), 2.37 (m, 1H, CH, cyclohexyl), 2.58 (m, 1H, CH, cyclohexyl), 6.84 (s, 1H, CH, thiazole), 7.49 (d, 2H, J: 8.5, CH,
phenyl), 7.66 (d, 2H, J: 8.5, CH, phenyl), 8.46 (br. s, 1H, NH, D2O exch.)
1H NMR: (500 MHz, CDCl3) dH 1.19 (s, 3H, CH3), 1.33 (m, 1H, CH, cyclohexyl), 1.55 (m, 2H, CH2, cyclohexyl), 1.81 (m, 1H,
CH, cyclohexyl), 1.91 (m, 1H, CH, cyclohexyl), 1.98 (m, 2H, CH2, cyclohexyl), 2.39 (m, 1H, CH, cyclohexyl), 2.62 (m, 1H, CH,
cyclohexy), 7.07 (s, 1H, CH, thiazole), 7.94 (d, 2H, J: 8.9, CH, phenyl), 8.24 (d, 2H, J: 8.9, CH, phenyl), 8.41 (br. s, 1H, NH)
1H NMR: (500 MHz, CDCl3) dH 1.18 (s 3H, CH3), 1.29 (m, 1H, CH, cyclohexyl), 1.52 (m, 2H, CH2, cyclohexyl), 1.88 (m, 4H,
CH2, cyclohexyl), 2.37 (m, 1H, CH, cyclohexyl), 2.60 (m, 1H, CH, cyclohexyl), 7.00 (s, 1H, CH, thiazole), 7.65 (d, 2H, J: 8.3, CH,
phenyl), 7.88 (d, 2H, J: 8.3, CH, phenyl), 8.50 (br. s, 1H, NH)
1H NMR: (500 MHz, CDCl3) dH 1.16 (s 3H, CH3), 1.35 (m, 1H, CH, cyclohexyl), 1.58 (m, 2H, CH2, cyclohexyl), 1.81 (m, 1H,
CH, cyclohexyl), 1.99 (m, 2H, CH2, cyclohexyl), 2.20 (m, 1H, CH, cyclohexyl), 2.44 (m, 1H, CH, cyclohexyl), 3.01 (m, 1H, CH,
cyclohexyl), 6.62 (s, 1H, CH, thiazole), 7.18 (m, 2H, CH, phenyl), 7.72 (dd, 2H, J: 4.9, J: 8.8, CH, phenyl), 12.45 (br. s, 1H, NH)
Table 4. MIC, MCF and vero cells toxicity of EMAC compounds against C. albicans ATCC 10231 and C. Albicans 25 FLC resistant.
R
S
R'
N
N
N
H
C. albicans ATCC 10231
C. albicans 25 FLC resistant
Vero cells
CC50
Compound
R
R0
MIC
MCF
MIC
MCF
EMAC2098
EMAC2100
EMAC2101
EMAC2103
EMAC2104
EMAC2105
EMAC2107
EMAC2109
EMAC2110
FLC
H
H
H
4-Br
4-NO2
4-CH3
4-CN
H
4-Br
4-NO2
4-CN
4-F
0.19
0.78
0.39
3.12
0.19
0.78
0.78
1.56
0.78
0.78
0.39
100
12.5
25
25
25
4100
425
3.12
4100
0.39
0.78
425
425
0.19
425
3.12
425
1.56
4100
50
100
4125.00
129.22
229.66
4500.00
98.142
199.32
4250.00
320.54
462.50
41000.00
425
425
1.56
425
50
425
3.12
4100
H
CH3
CH3
CH3
CH3
CH3
–
–
exhibit fungicidal activity when tested on C. albicans ATCC
The introduction of a methyl substituent in the position 2 of the
cyclohexylidene moiety leads to a different behavior: compound
different behavior was observed when compounds EMAC2105, bearing a 4-bromophenyl moiety in the position 4 of
10231.
A
EMAC2098, EMAC2100, EMAC2101 and EMAC2103 were the thiazole ring is active only toward C. albicans ATCC 10231.
tested against C. albicans 25 FLC-resistant. In this case, only Although its activity is comparable with that of FLC, but less
compounds EMAC2098 and EMAC2100 show remarkable MIC potent than its homologous EMAC2098, it has neither fungicidal
values (0.39 and 0.78 mg/ml, respectively), while almost no activity on C. albicans ATCC 10231, nor efficacy against
activity is observed for EMAC2101 and EMAC2103. C. albicans 25 FLC-resistant.
Unfortunately, none of the tested compounds have interesting On the contrary, the 4-nitro-substituted derivative EMAC2107
fungicidal activity when tested on C. albicans 25 FLC resistant, exhibits a similar activity profile with respect to its homologous
with the exception of EMAC2104 and EMAC2110 which show EMAC2100. Both compounds only exhibit fungi-static activity on
values of MCF at 1.56 and 3.12 mg/ml, respectively.
both Candida species.

DOI: 10.3109/14756366.2015.1113171
Identification of new agents for the treatment of FLC resistant Candida
5
Figure 1. Effect of Fe2+ or Fe3+ ions on the spectrum of absorbance of EMAC2104 and FLC compounds. UV/vis spectrum was measured with 100 mM
of compound alone (unbroken line) or in the presence of 10 mM FeCl3 or FeSO4 (dotted line).
On the contrary, compounds EMAC2104 and EMAC2110
exhibit a very potent activity with respect to FLC.
These data corroborate the hypothesis that EMAC derivatives
might inhibit the growth of C. albicans either by chelating the
The 4-phenylthiazole derivative EMAC2104 is particularly Fe3+ ions inside the 14a-demethylase catalytic pocket or by Fe3+
active against C. albicans 25 FLC resistant (MIC 0.19 mg/ml; withdrawal in the cell cytosol. Further studies are in progress to
MFC 1.56 mg/ml). better understand the mode of action of cycloalkylidenhydrazo-4-
Although slightly less potent than EMAC2104, compound arylthiazoles.
EMAC2110 is active toward both the tested strains. Moreover, its
Conclusions
fungicidal activity is comparable to that of EMAC2104.
The cellular toxicity of all compounds was evaluated. CC50
values were generally lower than that of the reference FLC. The size
of the substituent in the position 4 of the phenyl ring appeared as
relevant in determining the toxicity. Thus, either the introduction of
a nitrile (EMAC2103 and 2109) or a nitro group (EMAC2107 and
2100) leaded to a reduction of the toxicity with respect to fluorine
(EMAC2110) and un-substituted (EMAC2104) derivatives.
In order to achieve a preliminary elucidation of the possible
mechanism of action of such compounds, we verify the possibility
that EMAC2104 could chelate the divalent and trivalent iron ions.
Therefore, we determined its spectrum of absorbance in the
absence and the presence of both Fe2+ and Fe3+ and compared
this behavior with that exhibited by FLC (Figure 1).
The purpose of this study was the synthesis of a series of
cycloalkylidenhydrazo-4-arylthiazoles to examine whether molecu-
lar modification on the thiazole core might lead to the identification
of new potential C. albicans FLC resistant inhibitors. All the
compounds were obtained in good yields. With respect to FLC
EMAC compounds exhibited comparable and, in some cases
(EMAC 2098, 2101 and 2104), lower MIC values. Moreover, most
of the compounds exhibited fungicidal activity. When tested against
FLC resistant C. albicans isolated from clinical specimens, EMAC
2098, 2100, 21042107 and 2110 retained their antifungal activity.
The cellular toxicity was evaluated and none of the compounds is
toxic at the MIC. All together these data indicated that the thiazole
scaffold could be relevant date have been obtained and will
constitute the basis for the future work.
Interestingly neither EMAC2104 nor FLC maximum absorb-
ance was modified by the addition of Fe2+. On the contrary, when
Fe3+ was added either to EMAC2104 or to FLC a notable increase
of maximum absorbance was observed, indicating that both
compounds interact with ferric ions.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.

6
R. Meleddu et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
15. Chaturvedi V, Ramani R, Andes D, et al. Multilaboratory testing of
two-drug combinations of antifungals against Candida albicans,
Candida glabrata, and Candida parapsilosis. Antimicrob Agents
Chemother 2011;55:1543–8.
16. Aperis G, Mylonakis E. Newer triazole antifungal agents: pharma-
cology, spectrum, clinical efficacy and limitations. Expert Opin
Invest Drugs 2006;15:579–602.
17. Monk BC, Tomasiak TM, Keniya MV, et al. Architecture of a single
membrane spanning cytochrome P450 suggests constraints that
orient the catalytic domain relative to a bilayer. Proc Natl Acad Sci
U S A 2014;111:3865–70.
18. Holbein BE, Mira de Ordun˜a R. Effect of trace iron levels and iron
withdrawal by chelation on the growth of Candida albicans and
Candida vini. FEMS Microbiol Lett 2010;307:19–24.
19. Santos ALS, Sodre CL, Valle RS, et al. Antimicrobial action
of chelating agents: repercussions on the microorganism develop-
ment, virulence and pathogenesis. Curr Med Chem 2012;19:
2715–37.
References
1. Kriengkauykiat J, Ito JI, Dadwal SS. Epidemiology and treatment
approaches in management of invasive fungal infections. J Clin
Epidemiol 2011;3:175–91.
2. Mulu A, Kassu A, Anagaw B, et al. Frequent detection of ‘azole’
resistant Candida species among late presenting AIDS patients in
northwest Ethiopia. BMC Infect Dis 2013;13:82.
3. Pfaller MA, Diekema DJ, Gibbs DL, et al. Results from the
ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to
2007: a 10.5-year analysis of susceptibilities of Candida species to
fluconazole and voriconazole as determined by CLSI standardized
disk diffusion. J Clin Microbiol 2010;48:1366–77.
4. Fridkin SK, Jarvis WR. Epidemiology of nosocomial fungal
infections. Clin Microbiol Rev 1996;9:499–511.
5. Roemer T, Krysan DJ. Antifungal drug development: challenges,
unmet clinical needs, and new approaches. Cold Spring Harb
Perspect Med 2014;4:a019703.
6. Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of
clinical experience. Rev Infect Dis 1990;12:308–29.
7. Sheehan DJ, Hitchcock CA, Sibley CM. Current and emerging azole
antifungal agents. Clin Microbiol Rev 1999;12:40–79.
8. Denning DW. Echinocandins:
J Antimicrob Chemother 2002;49:889–91.
¨
9. Lignell A, Lowdin E, Cars O, et al. Voriconazole-induced inhibition of
the fungicidal activity of amphotericin in Candida strains
with reduced susceptibility to voriconazole: an effect not predicted by
the MIC value alone. Antimicrob Agents Chemother 2011;55:1629–37.
10. Arendrup MC, Dzajic E, Jensen RH, et al. Epidemiological
changes with potential implication for antifungal prescription
recommendations for fungaemia: data from a nationwide fungaemia
surveillance programme. Clin Microbiol Infect 2013;19:e343–53.
11. Lupetti A, Danesi R, Campa M, et al. Molecular basis of resistance
to azole antifungals. Trends Mol Med 2002;8:76–81.
´
´
20. Opletalova V, Kalinowski DS, Vejsova M, et al. Identification and
characterization of thiosemicarbazones with antifungal and anti-
tumor effects: cellular iron chelation mediating cytotoxic activity.
Chem Res Toxicol 2008;21:1878–89.
a new class of antifungal.
21. De Logu A, Saddi M, Cardia MC, et al. In vitro activity of
2-cyclohexylidenhydrazo-4-phenyl-thiazole compared with those of
amphotericin B and fluconazole against clinical isolates of Candida
spp. and fluconazole-resistant Candida albicans. J Antimicrob
Chemother 2005;55:692–8.
22. Cardia MC, Begala M, Delogu A, et al. Synthesis and antimicrobial
activity of novel arylideneisothiosemicarbazones. Farmaco 2000;55:
93–8.
B
23. Maccioni E, Cardia MC, Bonsignore L, et al. Synthesis and anti-
microbial activity of isothiosemicarbazones and cyclic analogues.
Farmaco 2002;57:809–17.
24. Chimenti F, Bizzarri B, Maccioni E, et al. Synthesis and in vitro
activity of 2-thiazolylhydrazone derivatives compared with the
activity of clotrimazole against clinical isolates of Candida spp.
Bioorg Med Chem Lett 2007;17:4635–40.
25. Chimenti F, Maccioni E, Secci D, et al. Synthesis, stereochemical
identification, and selective inhibitory activity against human
monoamine oxidase-B of 2-methylcyclohexylidene-(4-arylthiazol-
2-yl)hydrazones. J Med Chem 2008;51:4874–80.
12. Marchetti O, Moreillon P, Entenza JM, et al. Fungicidal synergism
of fluconazole and cyclosporine in Candida albicans is not
dependent on multidrug efflux transporters encoded by the CDR1,
CDR2, CaMDR1, and FLU1 genes. Antimicrob Agents Chemother
2003;47:1565–70.
13. Tragiannidis A, Tsoulas C, Kerl K, Groll AH. Invasive candidiasis:
update on current pharmacotherapy options and future perspectives.
Expert Opin Pharmacother 2013;14:1515–28.
26. Chimenti F, Secci D, Bolasco A, et al. Synthesis, semipreparative
HPLC separation, biological evaluation, and 3D-QSAR of hydra-
zothiazole derivatives as human monoamine oxidase B inhibitors.
Bioorg Med Chem 2010;18:5063–70.
14. Karlowsky JA, Hoban DJ, Zhanel GG, Goldstein BP. In vitro
interactions of anidulafungin with azole antifungals, amphotericin B
and 5-fluorocytosine against Candida species. Int J Antimicrob
Agents 2006;27:174–7.