Synthesis of 1,2,3-Triazole Derivatives and in Vitro Antifungal Evaluation on Candida Strains

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 thein 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.


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].
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.

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 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 triazolesubstituted 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 (LD 50 ) is above 2,000 mg·kg −1 . According to Lorke [28], substances presenting a LD 50 higher than 2,000 mg·kg −1 can be considered low toxicity 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.

General
All commercially available reagents were used without any further purification and the reactions were monitored by TLC analysis (TLC plates GF 254 E. Merck). Melting points were determined on a Büchi apparatus and are uncorrected. Column chromatography was performed on Silica Gel 60

Synthesis of the Aromatic Azides 2a-j
To a solution of the corresponding aniline 1a-j (4.1 mmol) dissolved in CH 2 Cl 2 (30 mL), was added 6 N HCl (30 mL) at 0 °C. To this biphasic system was added dropwise a saturated aqueous solution of NaNO 2 (10 mL). After stirring for 30 min at 0 °C, NaN 3 (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 CH 2 Cl 2 (3 × 20 mL). The combined organic layers were washed with aqueous solution of NaHCO 3 , then brine, dried (Na 2 SO 4 ) 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.

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.

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 × 10 6 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 × 10 3 and 5 × 10 3 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 CO 2 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.

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 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 n o 23076.003830). Studies of acute toxicity were performed according to "Up and down" method with slight modifications, as described by OECD 425 [32].

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-1,2,3triazol-4-yl) series can be done in order to obtain more potent prototypes.