Synthesis of 1,2,3-triazole derivatives and in Vitro antifungal evaluation on Candida strains

Article abstract of DOI:10.3390/molecules17055882

1,2,3-Triazoles have been extensively studied as compounds possessing important biological activities. In this work, we describe the synthesis of ten 2-(1-aryl-1H- 1,2,3-triazol-4-yl)propan-2-ols via copper catalyzed azide alkyne cycloaddition (CuAAc or click chemistry). Next the in vitro antifungal activity of these ten compounds was evaluated using the microdilution broth method against 42 isolates of four different Candida species. Among all tested compounds, the halogen substituted triazole 2-[1-(4-chlorophenyl)-1H- (1,2,3)triazol-4-yl]propan-2-ol, revealed the best antifungal profile, showing that further modifications could be done in the structure to obtain a better drug candidate in the future.

Full text of DOI:10.3390/molecules17055882

Molecules 2012, 17, 5882-5892; doi:10.3390/molecules17055882
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of 1,2,3-Triazole Derivatives and in Vitro Antifungal
Evaluation on Candida Strains
Reginaldo G. Lima-Neto 1, Nery N. M. Cavalcante 2, Rajendra M. Srivastava 2,
Francisco J. B. Mendonça Junior 3, Almir G. Wanderley 4, Rejane P. Neves 1 and
Janaína V. dos Anjos 2,*
1
Laboratório de Micologia Médica, Centro de Ciências Biológicas,
Universidade Federal de Pernambuco (UFPE), 50670-901, Recife, PE, Brazil
2
Laboratório de Síntese Orgânica, Departamento de Química Fundamental,
Universidade Federal de Pernambuco (UFPE), 50740-560, Recife, PE, Brazil
3
Laboratório de Síntese e Vetorização de Substâncias Bioativas,
Universidade Estadual da Paraíba (UEPB), 58058-420, João Pessoa, PB, Brazil
4
Laboratório de Farmacologia Pré-Clínica e Toxicologia de Produtos Bioativos,
Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco (UFPE),
50670-901, Recife, PE, Brazil
* Author to whom correspondence should be addressed; E-Mail: janaina.anjos@ufpe.br;
Tel.: +55-81-2126-7411; Fax: +55-81-2126-8440.
Received: 12 April 2012; in revised form: 3 May 2012 / Accepted: 5 May 2012 /
Published: 16 May 2012
Abstract: 1,2,3-Triazoles have been extensively studied as compounds possessing
important biological activities. In this work, we describe the synthesis of ten 2-(1-aryl-1H-
1,2,3-triazol-4-yl)propan-2-ols via copper catalyzed azide alkyne cycloaddition (CuAAc or
click chemistry). Next the in vitro antifungal activity of these ten compounds was evaluated
using the microdilution broth method against 42 isolates of four different Candida species.
Among all tested compounds, the halogen substituted triazole 2-[1-(4-chlorophenyl)-1H-
(1,2,3)triazol-4-yl]propan-2-ol, revealed the best antifungal profile, showing that further
modifications could be done in the structure to obtain a better drug candidate in the future.
Keywords: click chemistry; 1,2,3-triazoles; Candida spp.; antifungal activity

Molecules 2012, 17
5883
1. Introduction
Deep and superficial fungal infections have increased significantly over the past few decades.
Control of fungal disease has proved to be difficult because of several risk factors. The number of
patients at highest risk for these infections has been steadily increasing, especially among patients
immunocompromised due to AIDS, organ transplantation, chemotherapy or other invasive procedures [1].
Because of this, there is a clear need for the development of effective antimycotic therapeutic agents
for the treatment of fungal infections, since the major classes of antifungal drugs available have
encountered resistance in clinical use [2,3]. Among these classes, azoles are the most used because of
their broad spectrum, high potency and low toxicity [4].
Azoles are competitive inhibitors of lanosterol 14 α-demethylase (a cytochrome P-450 enzyme),
leading to a decrease in the fungal biosynthesis of ergosterol, which is a key compound of fungal cell
membranes, thereby preventing fungal growth [5,6]. Beyond the antifungal properties [7–9], triazoles
possess a variety of interesting biological activities, forming part of the scaffolds of antibacterial and
antituberculosis agents [10–14], neuraminidase inhibitors [15], anticancer compounds [16], antiviral
agents [17], analgesic compounds [18], herbicides [19] and plant growth regulators [20].
Considering the above mentioned advantages of triazole-containing antifungal drugs and the
increasing drug resistance mechanisms in these type of microorganisms, we decided to synthesize
2-(1-aryl-1H-1,2,3-triazol-4-yl)propan-2-ols capable of inhibiting cell growth of some Candida species
with clinical relevance and testing their activity using the microdilution broth method.
2. Results and Discussion
2.1. Chemistry
Prior to the synthesis of the 2-(1-aryl-1H-1,2,3-triazol-4-yl)propan-2-ols, the aromatic azides 2a–j
were prepared from the corresponding anilines 1a–j following the Sandmeyer conditions [21]. The
aromatic azides were then reacted with 2-methylbut-3-yn-2-ol (3) using Cu(OAc)2 and sodium ascorbate
as catalyst in 1:1 dichloromethane:water [22,23] to give the products 4a–j in good yields (Scheme 1).
Scheme 1. Synthesis of triazoles 4a–j.
HCl, NaNO2,
NaN3
Ar
NH2
Ar
N3
CH2Cl2/H2O
0°C - r.t.
1a-j
2a-j
Cu(OAc)2 5 mol %
Na-Ascorbate 15 mol %
N
N
+
Ar
N3
N
Ar
CH2Cl2/H2O
OH
31-96%
4a-j
2a-j
OH
3
f: m-NO2-Phenyl
g: p-NO2-Phenyl
a: Phenyl
b: p-Tolyl
h: 3,4-di-CH3-Phenyl
i: alpha-Naphthyl
j: beta-Naphthyl
c: p-Cl-Phenyl
d: p-Br-Phenyl
e: p-OCH3-Phenyl

Molecules 2012, 17
5884
1
For all synthesized molecules, only one regioisomer could be detected by H-NMR. Only one
1
singlet was observed in the H-NMR spectrum for the triazole ring ( 7.19–8.16 ppm), which can be
attributed to the proton in the C-5 position of the triazole nucleus. According to earlier literature on
copper catalyzed cycloadditions [24,25], it is believed that the obtained products are 1,4-regioisomers.
2.2. Biology
For each experiment, inocula controls produced clearly detectable growth after the chosen
incubation period, indicating that all isolates were viable and that the conditions used were suitable for
fungal growth. The antifungal screening results by MIC measurements are summarized in Table 1.
Most of the synthesized 1,2,3-triazoles showed weak (4a,b,d,e,f) or no activity (4g,h,i,j) against the
Candida species used herein. However, for 2-[1-(4-chlorophenyl)-1H-(1,2,3)triazol-4-yl]propan-2-ol
(4c) and for the reference drug (fluconazole), it was possible to determine a MIC for Candida growth.
As it can be seen, fluconazole showed fungistatic activity in concentrations ranging from 0.5 to
64 μg·mL−1. Eight isolates (4987, 4986, 4984, 4970, 4790, 4608, 1059 and 109) had their growth
inhibited in a dose-dependent pattern, presenting MIC values ranging from 16 to 32 μg·mL−1.
In contrast to these findings, nine isolates (4990, 4802, 4388, 4263, 4261, 4124, 3719, 1150 and 934)
were resistant to the reference azole showing MIC values above 64 μg·mL−1. It can be also observed
that the triazole 4c showed good antifungal activity, presenting MIC values ranging from 64 to
256 μg·mL−1 against all the tested strains.
Table 1. Antifungal activity of triazoles 4a–j and fluconazole against the studied Candida strains.
Compounds (MICs in μg·mL−1) a
Tested strain
4a
4b
4c
4d
4e
4f
4g
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4h
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4i
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4j Flu b
n° URM
4990
4987
4986
4820
4819
4817
4609
4606
4388
4387
4386
4385
4384
4260
4127
4126
4125
4124
2,048 1,024 256 2,048 1,024 2,048
2,048 1,024 128 2,048 1,024 2,048
2,048 1,024 128 2,048 1,024 2,048
2,048 1,024 256 2,048 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 2,048 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 128 1,024 1,024 2,048
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
>64
16
16
4
4
4
4
2
>64
2
1
2
2
2
0.5
0.5
0.5
>64
R
1,024 128 1,024 2,048 2,048
2,048 1,024 128 1,024 1,024 2,048
2,048 1,024 256 1,024 1,024 2,048
2,048 1,024 256 2,048 1,024 2,048

Molecules 2012, 17
5885
Table 1. Cont.
Compounds (MICs in μg·mL−1) a
Tested strain
n° URM
4a
4b
4c
4d
4e
4f
4g
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
4h
4i
4j Flu b
3719
3716
4802
4263
1059
934
R
R
1,024 256 2,048 1,024 2,048
1,024 256 2,048 1,024 2,048
R
R
R
R
R
R
R
64
0.5
64
64
16
64
16
16
16
2
4
8
16
4
>64
0.5
0.5
0.5
8
32
4
>64
4
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
2,048 2,048 256 2,048 1,024
2,048 2,048 256 2,048 1,024
2,048 1,024 64
2,048 2,048 256 2,048 1,024
2,048 1,024 128 1,024 1,024 2,048
R
R
R
R
R
R
R
R
2,048 1,024 64 1,024 1,024 2,048
2,048 1,024 64 1,024 1,024 2,048
2,048 1,024 64 1,024 1,024 2,048
R
R
R
R
R
R
R
R
R
R
512
1,024 2,048
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
109
4984
4970
4889
4818
4804
4608
4607
4261
3627
3624
3621
22019 c
4790
4262
1150
933
2,048 128 1,024 1,024 2,048
2,048 128 1,024 512 2,048
1,024 64 1,024 1,024 2,048
2,048 128 1,024 1,024 2,048
2,048 128 1,024 1,024 2,048
1,024 128 1,024 1,024 2,048
1,024 128 1,024 1,024 2,048
2,048 256 1,024 1,024
R
2,048 256 2,048 1,024 2,048
2,048 128 2,048 1,024 2,048
1,024 128 2,048 1,024 2,048
2,048 128 2,048 1,024 2,048
2,048 128 2,048 1,024 2,048
2,048 128 2,048 1,024 2,048
916
a
4
The MIC value was defined as the lowest concentration of the antifungal agent and were read
after two days at 37 °C. Inocula sizes contained approximately 2.5 × 103 cells·mL−1. Culture media
tested were the RPMI 1640 (Sigma Chemical Co., St. Louis, MO, USA). The final concentration of
triazoles was between 4–2,048 μg·mL−1 and 0.125–64 μg·mL−1 for fluconazole; Fluconazole;
b
c Candida parapsilosis ATCC 22019 was used as reference strain. R = Resistance.
Observing the drug structure, it can be noticed that 4c possesses a chlorine atom substituted in the
para position of the phenyl ring present at the N-1 position of the triazole nucleus. Chlorine-substituted
rings were found to be good antifungal tools, as reported by Wang and colleagues [26]. In their study,
sixteen N-methyl-substituted phenoxybutan-1-amine chloro-substituted derivatives exhibited strong
in vitro antifungal activity, being more active against the tested microorganisms than the used
reference drug, voriconazole. Later, Wang et al. [27] decided to synthesize fourteen novel triazole-
substituted compounds containing a phenoxyalkyl group. They also observed that the best antifungal
drugs were those with halogen atoms as substituents in the phenyl rings.
The acute preliminary toxicological tests in rats showed that the oral administration of triazole 4c at
the 2,000 mg·kg−1 dose did not produce any signs of toxicity or mortality, indicating that the lethal
dose for 50% of the animal population in this study (LD50) is above 2,000 mg·kg−1. According to
Lorke [28], substances presenting a LD50 higher than 2,000 mg·kg−1 can be considered low toxicity

Molecules 2012, 17
5886
drugs. Since our pharmacological studies have shown that 4c is active in 64 to 256 μg·mL−1
concentrations, those toxicological findings demonstrate that this drug candidate is quite safe for
further in vivo studies and can be considered an an antifungal lead for this class of compounds.
3. Experimental
3.1. General
All commercially available reagents were used without any further purification and the reactions
were monitored by TLC analysis (TLC plates GF254 E. Merck). Melting points were determined on a
Büchi apparatus and are uncorrected. Column chromatography was performed on Silica Gel 60
(70–230 mesh, Merck Chemicals International). NMR spectra were recorded with a Bruker AC-200
1
MHz spectrometer (Billerica, MA, USA) and referenced as follows: H (200 MHz), internal SiMe4 at
13
δ = 0.00 ppm, C (50 MHz), internal standard at  = 77.23 ppm. Exact mass measurements of the
molecular ions were obtained on a Shimadzu LC/MS-IT-TOF Eletrospray.
3.2. Synthesis of the Aromatic Azides 2a–j
To a solution of the corresponding aniline 1a–j (4.1 mmol) dissolved in CH2Cl2 (30 mL), was added
6 N HCl (30 mL) at 0 °C. To this biphasic system was added dropwise a saturated aqueous solution
of NaNO2 (10 mL). After stirring for 30 min at 0 °C, NaN3 (0.53 g, 8.2 mmol) was added at
0 °C. Stirring was maintained for 30 min, and the mixture was allowed to warm to room temperature.
The two phases were separated, and the aqueous phase was extracted with CH2Cl2 (3 × 20 mL). The
combined organic layers were washed with aqueous solution of NaHCO3, then brine, dried (Na2SO4)
and filtered from active charcoal. Evaporation of the solvent in vacuo gave the crude azides 2a–j that
were used in the next step without further purification.
3.3. Synthesis of 2-(1-Aryl-1H-1,2,3triazol-4-yl)propan-2-ols 4a–j
2-Methylbut-3-yn-2-ol (3, 1,1 mmol) and the azido compound 2a–j (1 mmol) were suspended in a
1:1 mixture of CH2Cl2 and water (10 mL). To this solution was added a mixture of Cu(OAc)2 (36 mg,
0.2 mmol) and sodium ascorbate (79 mg, 0.4 mmol). The resulting mixture was stirred at room
temperature until TLC analysis indicated complete consumption of the azide. The mixture was diluted
with CH2Cl2 (5 mL) and water (5 mL). The organic layer was separated, and the water phase was
extracted again with CH2Cl2 (5 mL). The combined organic layers were dried over Na2SO4. Removal
of the solvent in vacuo gave a residue that was recrystallized from chloroform-hexanes to afford the
corresponding triazoles 4a–j.
2-(1-Phenyl-1H-1,2,3-triazol-4-yl)propan-2-ol (4a): White crystals; yield 86%; m.p.: 95–96 °C; Rf
1
0,60 (ethyl acetate-chloroform 9:1, v/v). H-NMR (CDCl3): δ 0.97 (6H, s); 2.92 (1H, bs); 6.73
(3H, m); 6.93 (1H, dd, J = 8 Hz, 2 Hz); 6.97 (1H, dd, J = 8 Hz, 2 Hz); 7.24 (1H, s). 13C-NMR (CDCl3):
δ 30.3; 68.5; 117.7; 120.4; 128.5; 129.6; 136.9; 156.4. ESI–HRMS m/z: 226.0911 (calcd. for
C11H13N3ONa [M+Na]+: 226.0956).

Molecules 2012, 17
5887
2-[1-(4-Tolyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4b): White crystals; yield 78%; m.p.: 120–121 °C;
1
Rf 0,65 (ethyl acetate-chloroform 9:1, v/v). H-NMR (CDCl3):  0.99 (6H, s); 1.68 (3H, s); 3.13 (1H,
bs); 6.54 (2H, bd, J = 8.0 Hz); 6.85 (2H, bd, J = 8.0 Hz); 7.24 (1H, s). 13C-NMR (CDCl3):  20.9; 30.4;
68.4; 117.7; 120.2; 130.0; 134.6; 138.5; 156.2. ESI–HRMS m/z: 240.1049 (calcd. for C12H15N3ONa
[M+Na]+: 240.1113).
2-[1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4c): White crystals; yield 67%; m.p.:
93–94 °C; Rf 0,67 (ethyl acetate-chloroform 9:1, v/v). 1H-NMR (CDCl3):  0.98 (6H, s); 2.90 (1H, bs);
6.75 (2H, dd, J = 8.0 Hz, 2 Hz); 6.93 (2H, dd, J = 8.0 Hz, 2.0 Hz); 7.24 (1H, s). 13C-NMR (CDCl3): 
30.3; 68.5; 107.1; 117.6; 121.5; 129.7; 134.2; 135.4; 156.6. ESI–HRMS m/z: 260.0518 (calcd. for
C11H12ClN3ONa [M+Na]+: 260,0567).
2-[1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4d): White crystals; yield 96%; m.p.:
95–96 °C; Rf 0,68 (ethyl acetate-chloroform 9:1, v/v). 1H-NMR (CDCl3):  1.70 (6H, s); 3.68 (1H, bs);
7.59 (4H, m); 7.97 (1H, s). 13C-NMR (CDCl3):  30.3; 68.4; 107.0; 121.7; 122.1; 132.7; 135.8; 156.7.
ESI–HRMS m/z: 305.9989 (calcd. for C11H12BrN3ONa [M+Na]+: 304.0061).
2-[1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4e): Red crystals; yield 69%; m.p.:
1
106–107 °C; Rf 0,65 (ethyl acetate-chloroform 9:1, v/v). H-NMR (CDCl3): δ 1.67 (6H, s); 3.00 (1H,
bs); 3.82 (3H, s); 6.96 (2H, dd, J = 6.0 Hz, 4.0 Hz); 7.57 (2H, dd J = 6.0 Hz, 4.0 Hz); 7.81 (1H, s).
13C-NMR (CDCl3): 30.4; 55.5; 68.5; 114.6; 122.1; 130.5; 159.6. ESI–HRMS m/z: 238.0757 (calcd.
for C12H13ClNO2 [M+H]+: 238.0635).
2-[1-(3-Nitrophenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4f): White crystals; yield 75%; m.p.:
1
98–100 °C; Rf 0,60 (ethyl acetate-chloroform 9:1, v/v). H-NMR (CDCl3): δ 1.63 (6H, s); 3.96 (1H,
bs); 7.65 (1H, bd, J = 8.0 Hz); 8.12 (2H, m); 8.16 (1H, s); 7.65 (1H, d, J = 2.1 Hz). 13C-NMR (CDCl3):
δ 30.2; 68.4; 114.9; 122.9; 125.8; 130.8; 137.5; 148.6; 157.1. ESI–HRMS m/z: 271.0764 (calcd. for
C11H12N4O3Na [M+Na]+: 271.0807).
2-[1-(4-Nitrophenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4g): Yellow crystals; yield 58%; m.p.:
1
123–124 °C; Rf 0,63 (ethyl acetate-chloroform 9:1, v/v). H-NMR (CDCl3): δ 1.69 (6H, s); 2.82 (1H,
13
bs); 7.95 (2H, dd, J = 6.0 Hz, 4.0 Hz); 8.03 (1H, s); 8.37 (2H, dd, J = 6.0 Hz, 2.0 Hz). C-NMR
(CDCl3): δ 30.4; 68.7; 107.1; 120.3; 125.5; 141.2; 147.0; 157.2. ESI–HRMS m/z: 265.1469 (calcd. for
C11H13N4O4 [M+H2O−H]+: 265.2453).
2-[1-(3,4-Dimethylphenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4h): yellow crystals; yield 31%; m.p.:
1
128–129 °C; Rf 0,80 (ethyl acetate:chloroform 9:1, v/v). H-NMR (CDCl3):  1.00 (6H, s); 1.60
(6H, s); 2.69 (1H, bs); 6.53 (1H, m); 6.72 (2H, dd, J = 8.1 Hz, 2.0 Hz); 7.19 (1H, s). 13C-NMR
(CDCl3): δ 19.3; 19.8; 30.4; 68.5; 107.1; 117.7; 121.5; 130.4; 134.9; 137.2; 138.1; 156.0. ESI–HRMS
m/z: 254.1229 (Calcd for C13H17N3ONa [M+Na]+: 254.1269).
2-[1-(-Naphthyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4i): Red crystals; yield 62%; m.p.: 152–153 °C;
1
Rf 0,65 (ethyl acetate:chloroform 9:1, v/v). H-NMR (CDCl3):  1.75 (6H, s); 3.00 (1H, bs); 7.54
(4H, m); 7.82 (1H, s); 7.95 (3H, m). 13C-NMR (CDCl3):  30.5; 68.6; 122.3; 123.5; 127.0; 128.2;

Molecules 2012, 17
5888
128.5; 130.3; 133.7; 134.0; 155.5. ESI–HRMS m/z: 276.1070 (calcd. for C15H15N3ONa [M+Na]+:
276.1113).
2-[1-(-Naphthyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4j): Yellow crystals; yield 60%; m.p.:
1
143–144 °C; Rf 0,68 (ethyl acetate:chloroform 9:1, v/v). H-NMR (CDCl3): δ 1.72 (6H, s); 2.97 (1H,
bs); 7.53 (2H, d, J = 8.1 Hz); 7.89 (4H, m); 8.03 (1H, s); 8.12 (1H, d, J = 2.1 Hz). 13C-NMR (CDCl3):
δ 30.4; 68.6; 118.3; 118.9; 126.9; 127.3; 127.8; 128.2; 129.9; 132.7; 133.1; 134.4; 155.6. ESI–HRMS
m/z: 276.1071 (calcd. for C15H15N3ONa [M+Na]+: 276.1113).
3.4. Strains and Growth Cultures
Twenty strains of Candida albicans, five of Candida krusei, eleven of Candida parapsilosis and
five of Candida tropicalis were supplied by the URM Culture Collection of the Department of
Mycology, Biological Sciences Centre of the Federal University of Pernambuco, Recife, Brazil.
Strains have been stocked in mineral oil at 18 °C [28]. Viability tests and subsequent taxonomic
confirmation of their morphological, biochemical and physiological characteristics were carried out [29].
Species, accession numbers, stock time and isolation substratum are summarized in Table 2.
Table 2. Samples of Candida species preserved in the Mycotheca Culture Collection—
University of Recife Mycology (URM).
Species
Accession nº (URM) Storage (years)
Substratum
Vaginal secretion
Vaginal secretion
Vaginal secretion
Ungual scrap
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. krusei
4990
4987
4986
4820
4819
4817
4609
4606
4388
4387
4386
4385
4384
4260
4127
4126
4125
4124
3719
3716
4802
4263
1059
934
01
01
01
02
02
02
03
03
05
05
05
05
05
05
07
07
07
07
10
10
02
05
48
49
Ungual scrap
Ungual scrap
Blood
Blood
Oropharyngeal secretion
Oropharyngeal secretion
Oropharyngeal secretion
Oropharyngeal secretion
Oropharyngeal secretion
Oropharyngeal secretion
Inguinal area
Urine
Spittle
Oropharyngeal secretion
Tooth scrap
Tooth scrap
*
C. krusei
C. krusei
C. krusei
Oropharyngeal secretion
*
Appendix biopsy

Molecules 2012, 17
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Table 2. Cont.
Species
Accession nº (URM) Storage (years)
Substratum
C. krusei
109
4984
4970
4889
4818
4804
4608
4607
4261
3627
3624
3621
ATCC22019
4790
4262
1150
933
52
01
01
02
02
02
03
03
05
12
12
12
-
*
Vaginal secretion
Vaginal secretion
Blood
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. parapsilosis
C. tropicalis
Ungual scrap
IFM
Blood
Blood
Oropharyngeal secretion
Spittle
Spittle
Spittle
-
02
06
46
49
49
Cassava powdery
Oropharyngeal secretion
Tongue
C. tropicalis
C. tropicalis
C. tropicalis
C. tropicalis
Vaginal secretion
Feces
916
* Substratum not identified.
3.5. In vitro Antifungal Susceptibility
Reference microdilution trays, containing serial drug dilutions were prepared by following the CLSI
M27-A3 guidelines [30]. The triazoles were dissolved in dimethylsulfoxide (DMSO) and then these
stock solutions were stored at −80 °C. The concentrations tested ranged from 2 to 2,048 μg·mL−1.
Fluconazole was used as reference drug at concentrations from 0.125 to 64 μg·mL−1. In order to obtain
a fungal inoculum containing 1–5 × 106 CFU·mL−1, each strain was cultured on a tube containing
20 mL of Sabouraud Dextrose Agar (SDA) plus yeast extract at 35 °C for two days. After this time,
yeast suspensions were prepared in sterile physiological solution (0.85%) and maintained at 28 ± 2 C
and then were adjusted to 90% transmittance at 530 nm. Two serial dilutions from 1:100 and 1:20
sequentially were made to obtain a final inoculum containing 1.0 × 103 and 5 × 103 CFU·mL−1. The
microdilution wells containing 100 µL of the twofold serial dilutions of the test and reference drugs in
standard RPMI 1640 medium (Sigma Chemical Co., St. Louis, MO, USA), buffered to pH 7.0 with
0.165 mol·L−1 of morpholinopropanesulphonic acid (MOPS, Sigma), were inoculated with 100 µL of
inoculum. After inoculation, the microplates were incubated at 35 °C in a non CO2 incubator and were
read visually 48 h after the incubation. MICs corresponded to the lowest drug dilution that showed
growth inhibition compared to untreated yeasts. C. parapsilosis ATCC 22019 was used as reference
strain. All tests were performed in triplicate.
3.6. Animals and Preliminary Toxicological Tests
Adult male Wistar rats (Rattus norvegicus), aged 2–3 months, weighing 220–260 g, were obtained
from the Pound of the Department of Physiology and Pharmacology at the Federal University of

Molecules 2012, 17
5890
Pernambuco. They were kept under standard environmental conditions (23 ± 2 °C; 12:12 h dark/light
cycle) and water and animal food (Labina®, Purina, Brazil) were made available ad libitum. The
animals were randomly divided into two groups (n = 3–4/group) and deprived of feed for 12 h with
access to water ad libitum. Further, group 1 received vehicle (solution of 2.5% tween 80) and group 2
received 2-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl]propan-2-ol (4c) in a single oral dose of
2,000 mg·kg−1. The observations were performed at 30, 60, 120, 180 and 240 min after the oral
treatments and then, daily for 14 days. Behavioral changes, weight, consumption of food and water,
clinical signs of toxicity, and mortality were recorded daily [31]. The experimental protocol was
approved by the Federal University of Pernambuco’s Ethics Committee for Animal Experimentation
(Process no 23076.003830). Studies of acute toxicity were performed according to “Up and down”
method with slight modifications, as described by OECD 425 [32].
4. Conclusions
In conclusion, a series of analogs of 1,2,3-triazoles with ten distinct substituents at the N-1 of the
triazole ring were synthesized and assessed for their antifungal activity. All compounds were tested
against 42 pathogenic strains of four different Candida species. Modification of substituents has a
great impact on the minimal inhibitory concentration values, since we could obtain triazole derivatives
showing no antimycotic activity, with moderate antifungal activity and one compound with promising
activity. The antifungal tests data show that the chloro-substituted triazole derivative exhibited, in
particular, good fungal growth inhibition, showing that further modifications in the 2-(1-aryl-1H-1,2,3-
triazol-4-yl) series can be done in order to obtain more potent prototypes.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgments
The authors gratefully acknowledge CAPES, CNPq, PPP/FACEPE and PRONEX/FACEPE for
fellowships and financial support.
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Sample Availability: Samples of the compounds 4a–j are available from the authors.
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