Original article
Design, synthesis and determination of antifungal activity of 5(6)-substituted benzotriazoles

https://doi.org/10.1016/j.ejmech.2010.01.062Get rights and content

Abstract

In an effort to find inhibitors that are effective against both Candida and Aspergillus spp., a series of 5(6)-(un)substituted benzotriazole analogs, represented by compounds 3a–3h and 3b′–3f′, were prepared using a crystalline oxirane intermediate 1 previously synthesized in our laboratory. All the compounds were evaluated for inhibitory activity against various species of Candida and Aspergillus. Compounds 3b′ (5,6-dimethylbenzotriazol-2-yl derivative), 3d (5-chlorobenzotriazol-1-yl derivative) and 3e′ (6-methylbenzotriazol-1-yl derivative) exhibited potent antifungal activity, with the MICs for Candida spp. and Aspergillus niger, ranging from 1.6 μg/mL to 25 μg/mL and 12.5 μg/mL to 25 μg/mL, respectively. The present work describes the design, synthesis, regioisomer characterization (through COSY and NOESY 2D-NMR spectroscopy and single molecule X-ray crystallography), antifungal evaluation, molecular docking, and structure-activity relationships of the various 5(6)-(un)substituted benzotriazole analogs.

Graphical abstract

A series of 5(6)-(un)substituted benzotriazole analogs were identified as broad spectrum antifungal agents.

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Introduction

The increasing incidence of fungal infection associated with unsatisfactory therapeutic treatment in immunocompromized patients and the emergence of azole-resistant fungal strains have stimulated the search for alternative antifungal drugs with higher potency and broader spectrum of activity against resistant fungal strains along with a greater metabolic stability. From a clinical stand point, candidiasis and aspergillosis are the most common fungal infections affecting immunocompromized individuals [1], [2]. Candida albicans is not only a common opportunistic pathogenic yeast of immunodeficient hosts but also a leading cause of life-threatening nocosomial infections with the mortality rate approaching 35% [3]. Among dimorphic fungi, C. albicans is probably the most common cause of disease among the female population with approximately 70% of all women experiencing at least one episode of Candida vaginitis, and among those with AIDS, in which oropharyngeal candidiasis occurs in approximately 70% of the patients [4]. On the other hand, invasive aspergillosis is the leading cause of death in leukemia and bone marrow transplant patients, and its infections are rather difficult to treat with the azole antifungal agents currently available. A four-fold increase in the number of cases of invasive aspergillosis by Aspergillus fumigatus and Aspergillus niger was reported during 1989–1998 [1]. In view of the limited number of therapeutic choices for controlling fungal infections there is at present an urgent need for novel antifungal compounds with a high potency and a broad spectrum of activity.

The triazole antifungal drugs fluconazole [5], [6], itraconazole [7], voriconazole [8], ravuconazole [9], [10], [11], and posaconazole [12], [13] form an important class of antifungal agents. These drugs act by displacing lanosterol from cytochrome P45014αDM and, in this manner, block the biosynthesis of ergosterol, an essential component of the fungal cell membrane [14], [15]. Cytochrome P45014αDM oxidatively removes the 14-α-methyl group of lanosterol by using oxygen and NADPH [16], [17]. Fluconazole is effective against candidiasis after both oral and parenteral administration but is ineffective against aspergillosis. The rising incidence of infections caused by non-albicans Candida spp. such as Candida glabrata and Candida krusei is of particular concern [18], because C. glabrata and C. krusei appear to be innately resistant to moderate (<64 μg/mL) and significant (>64 μg/mL) levels of fluconazole, respectively. It has been shown that fluconazole exposure can rapidly induce the expression of both the ABC transporter CgCdr1p and the drug target lanosterol 14α-demethylase in C. glabrata [19]. Itraconazole has a broader antifungal spectrum and is better tolerated by the patient; however, its use is hampered by a variable oral absorption and by a low oral bioavailability. Newer triazole agents such as voriconazole, ravuconazole, and posaconazole are found to be active against Aspergillus spp.

Our previous molecular modeling studies such as 3D QSAR [20] and docking [21] on azole antifungal agents led to the design and synthesis of several novel (un)substituted benzotriazole derivatives (see Fig. 1). 3D QSAR comparative molecular field analysis (CoMFA) steric/electrostatic contour maps revealed sterically favorable (green) and electropositive group favorable (blue) regions around one of the 1,2,4-triazole rings of fluconazole which in turn prompted us to modify the 1,2,4-triazole ring of fluconazole to a benzotriazole lead compound as shown in Fig. 1. In addition, docking studies showed that the 1,2,4-triazole ring of fluconazole binds in a hydrophobic (carbon affinity) pocket of cytochrome P45014αDM. Taking advantage of the hydrophobic pocket, we incorporated various substituents on the benzotriazole nucleus in an attempt to generate compounds with dual (against Candida and Aspergillus spp.) antifungal activity. From the structural information on the target enzyme and from the 3D QSAR models we were able to accelerate our efforts in developing new antifungal drugs. While this manuscript was nearing completion, two reports were published focusing on substituted indazole analogs [22] exhibiting broad spectrum antifungal activity (C. albicans ATCC 36082 and A. niger ATCC 9042) and on an unsubstituted benzotriazole analog [23] with antifungal activity against Candida albicans (CA98001) but was inactive against Aspergillus fumigatus (AF980003) based on MICs determined by fluorometric microdilution method. The present study focuses on the design, synthesis, regiochemistry, 2D NMR spectroscopic and X-ray crystallographic characterization, antifungal evaluation, molecular docking and SAR of 5(6)-(un)substituted benzotriazole analogs.

Section snippets

Chemistry

A key intermediate for the synthesis of the benzotriazole derivatives 3a–3h and 3b′–3f′, was the oxirane 1 (Scheme 1). This key intermediate was recently synthesized in our laboratory in crystalline form in two steps starting from 2,4-difluoro-α-chloroacetophenone [24]. The benzotriazoles needed for the preparation of target compounds 3a–3h and 3b′–3f′ were either purchased (2a, 2b, 2d, and 2e) or synthesized (2c and 2f–2h, Supplementary data) from an appropriately substituted ortho

Results and discussion

The in vitro antifungal activities of compounds 3a3h and 3b′3f′, using the macrodilution method in RPMI 1640 medium with minor modifications to the recommendation of National Committee for Clinical Laboratory Standards (NCCLS) [28], are presented in Table 1. The minimum inhibitory concentration (MIC) values (μg/mL) obtained from triplicate assay (three or two test tubes with identical results were taken as MICs) against several Candida spp., Saccharomyces cerevisiae and Aspergillus niger are

Conclusion

Based on information previously gathered through 3D QSAR and molecular docking studies on azole antifungal agents, we have designed, synthesized and biologically evaluated a series of substituted benzotriazole derivatives. Several of the new analogs were found to be effective against both Candida spp. and Aspergillus niger. Those compounds bearing hydrophobic groups at the 5- and/or 6-position of the benzotriazole ring demonstrated excellent antifungal profile against all of the fungal strains

General

Melting points (mp) were determined on a Thomas-Hoover capillary melting point apparatus and are uncorrected. All compounds were checked for their homogeneity by TLC using silica as the stationary phase. The 1H NMR spectra were recorded on a Bruker 400 Avance DPX spectrometer outfitted with a z-axis gradient probe. The chemical shifts are reported as parts per million (δ ppm) downfield from tetramethylsilane (TMS) serving as an internal standard. Data are reported as follows: chemical shift,

Acknowledgements

The financial support and resources provided by the College of Pharmacy of St. John's University are gratefully acknowledged. T.T. is thankful to Dr. Waykole from Novartis Pharma, East Hanover, NJ, for his insightful comments and suggestions on the synthetic aspects of this work and to Dr. Cesar A. Lau-Cam, College of Pharmacy, St. John's University, for his editorial help and valuable comments.

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