Synthesis and Antifungal Activity of Novel Triazole Compounds Containing Piperazine Moiety

Design and synthesis of triazole library antifungal agents having piperazine side chains, analogues to fluconazole were documented. The synthesis highlighted utilization of the click chemistry on the basis of the active site of the cytochrome P450 14α-demethylase (CYP51). Their structures were characterized by 1H-NMR, 13C-NMR, MS and IR. The influences of piperazine moiety on in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi.


Introduction
Triazole compounds have been specially paid increasing attention because of their extensively medicinal applications in antimicrobial agent's particularly antifungal therapy, and a large number of OPEN ACCESS predominant triazole drugs have been successfully developed and prevalently used in the treatment of various microbial infections for many years [1]. Azoles (fluconazole, itraconazole, voriconazole, and posaconazole, Figure 1) are important antifungal drugs for the treatment of invasive fungal infections (IFIs), which continue to be a major cause of morbidity and mortality in immunocompromised orseverely ill patients [2]. However, fluconazole is not effective against invasive aspergillosis and has suffered severe drug resistance [3,4]. The increasing frequency of fungal infections and development of resistance to the current treatment highlight the need for development of new triazole derivatives possessing broader antifungal spectra and higher therapeutic indexes. Azole antifungals act by competitive inhibition of CYP51, the enzyme that catalyzes the oxidative removal of the 14α-methyl group of lanosterol to give Δ 14,15 -desaturated intermediates in ergosterol biosynthesis [5]. In general, the active site of CYP51 for ligand binding can be divided into four subsites: a coordination bond with iron of the heme group, the hydrophilic H-bonding region, the hydrophobic region, and the narrow hydrophobic cleft formed by the residues in the helix B'-meander 1 loop and N-terminus of helix I [6]. These compounds target the biosynthesis of ergosterol by inhibiting the cytochrome P450-dependent lanosterol 14α-demethylase (Erg11p, CYP51), encoded by the ERG11 gene, resulting in accumulation of toxic methylsterols inmembranes that may culminate in fungi static effect or fungal death [7].
Literature precedents [8,9] revealed a pharmacophore of antifungal triazoles, which contained a triazole ring linking to a dihalophenyl ring through a two carbon chain. In addition, the carbon alpha to the phenyl ring bears a hydroxyl group. Itraconazole and posaconazole which containing the group of piperazine. We intended to alter the side chains to find potent systemic antifungal compounds with a broad antifungal spectrum and less potential to develop resistance. In our previous works [10][11][12][13][14][15][16], many studies on the structure-activity relationships (SAR) of antifungal azoles have been developed, and these studies have led to new compounds endowed with better biological and pharmacological properties.

Chemistry
Target compounds 1a-r were synthesized according to a very efficient and straightforward synthetic route outlined in Scheme 1. Compound 3 was synthesized by ring-open reaction of oxirane 2 with N-Boc-piperazine and simultaneous Boc-deprotection was accomplished by treatment with F 3 CCOOH to furnish compound 4. N-alkylation was effective in the presence of KI, K 2 CO 3 and propargyl bromide in acetonitrile at room temperature to secure compound 5. The target compounds were achieved by using click reaction [17] with variously substituted benzyl azides.

Pharmacology
The  The in vitro minimal inhibitory concentrations (MICs) of the compounds were determined by the micro-broth dilution method in 96-well microtest plates according to the methods defined by the National Committee for Clinical Laboratory Standards (NCCLS) [18]. The MIC 80 was defined as the first well with an approximate 80% reduction in growth compared to the growth of the drug-free well. For assays, the title compounds to be tested were dissolved in dimethyl sulfoxide (DMSO), serially diluted in growth medium, inoculated and incubated at 35 °C Growth MIC was determined at 24 h for C. alb. and at 72 h for C. neo. The in vitro antifungal activities are summarized in Table 1. The MIC values (in μg/mL) are presented against different pathogenic fungi, in comparison with ICZ, VCZ and FCZ.
The results of antifungal activities in vitro showed that all the 18 target compounds (1a-r) were active against nearly all fungi tested to some extent except against A. fum. and C. gla. The MIC 80 value of compound 1d and 1i is 4 times lower than that of FCZ against C. alb. 14053 in vitro (with the MIC 80 value of 0.25 μg/mL). The MIC 80 value of compound 1j, 1k, 1l and 1r is 128 times lower than that of FCZ against M. gyp. in vitro, and the same as VCZ against M. gyp. in vitro (with the MIC 80 value of 0.25 μg/mL). However, most of the target compounds' antifugal activities were not as good as the ICZ and VCZ.

General Procedures
1 H and spectra were recorded in CDCl 3 unless otherwise indicated with a Bruker AC-300P spectrometer, using TMS as internal standard. ESI mass spectra were performed on an API-3000 LC-MS spectrometer. The solvents and reagents were used as received or dried prior to use as needed. (1H-1,2,4-triazol-1-yl)propyl)piperazine-1-carboxylate (3): A mixture of compound 2 (16.65 g, 0.05 mol), CH 3 CH 2 OH (300 mL) and Et 3 N (25 mL), N-Boc-piperazine (13.96 g, 0.075 mol) was stirred and refluxed for 6 h. The reaction was monitored by TLC. After filtration, the filtrate was evaporated under reduced pressure. Water was added to the residue, extracted with ethylacetate twice, combinate the organic layer, washed with saturated NaHCO 3 and NaCl solution twice, dried over anhydrous Na 2 SO 4 and evaporated to get compound 3 (16.93 g, 80%). (4): A mixture of compound 3 (4.23 g, 0.01 mol), Trifluoroacetic acid (11.4 g, 0.1 mol) was stirred at room temperature for 30 min. The reaction was monitored by TLC. After reaction, most of the trifluoroacetic acid was removed in a vacuum. Column chromatography of the residue afforded compound 4 as yellow oil (DCM/CH 3 OH, 20:1-10:1, 2.81 g, 87%).

Conclusions
In summary, a novel series of antifungal agents have been designed and synthesized by chemical methods. In vitro antifungal activity assay indicated that most of the compounds showed antifungal activities against both systemic pathogenic fungi. Several compounds show high in vitro antifungal activity with a broad spectrum, which were valuable for further evaluation.