br Acknowledgements We thank the National Natural Science

Acknowledgements We thank the National Natural Science Foundation of China (41576156), Shandong Province Science and Technology Development Plan (2015GSF121045), Yantai Science and Technology Development Plan (2015ZH078), and the Public Science and Technology Research Funds Projects of Ocean (No. 201505022-3) for financial support of this work.
Introduction Fungal infections have been increasing dramatically and are currently estimated to directly affect approximately 1.2 billion people globally. The incidence of invasive fungal infections (IFIs) and the emergence of resistant fungal pathogens have increased markedly, leading to high morbidity and mortality in immunocompromised patients, such as patients receiving organ transplants, patients undergoing anticancer chemotherapy and patients with AIDS.2, 3 Clinically, the three fungal genera Aspergillus, Candida, and Cryptococcus account for most fungal infections. The common antifungal agents currently used in the clinic can be divided into four different prexasertib based on their mode of action: polyenes (e.g., amphotericin B and nystatin), echinocandins (e.g., caspofungin and micafungin), azoles(e.g., fluconazole, voriconazole and itraconazole), and antimetabolites (e.g., 5-fluorocytosine). Among these agents, azoles are most widely used as first-line antifungal therapy (see Fig. 1). Azole antifungal agents inhibit fungal lanosterol 14α-demethylase (CYP51), which is involved in the biosynthesis of ergosterol, a significant cellular membrane component. The inhibition of CYP51 would cause a reduction of the endogenous concentrations of ergosterol and an accumulation of lanosterol and other 14-methyl sterols, thus inhibiting the growth of fungal cells. Azole antifungal drugs, such as fluconazole, itraconazole, voriconazole and posaconazole, have had a significant impact on the treatment of systemic fungal infections.11, 12 However, many of the marketed azole drugs are limited in practical applications, due to their drug resistance, narrow antifungal spectrum, and low bioavailability.13, 14 Therefore, there is still an urgent need for developing novel azole antifungal agents with potent activity, broad spectrum, low toxicity, and low resistance. In our previous studies, a new series of benzothiazole derivatives were designed, synthesized and evaluated for their in vitro antifungal activity.15, 16 Most of the compounds showed excellent antifungal activity against Candida albicans and Cryptococcus neoformans, with MIC values in the range of 0.25 µg/mL to 2 µg/mL. Unfortunately, almost all of these target compounds were inactive against Aspergillus fumigatus. Therefore, this prompted us to continue studying the structural modification of our potent original compounds in search of novel compounds with improved anti-Aspergillus fumigatus efficacy. The structure of the voriconazole-A. fumigatus CYP51B complex (PDB ID:4UYM) (Fig. 2A) suggests that hydrogen bonds formed between the 5-fluoropyrimidine ring of voriconazole and A. fumigatus CYP51 Tyr122. This could explain why voriconazole has much higher antifungal potency against Aspergillus fumigatus than fluconazole does. To further explore the potent and broad antifungal spectrum of imidazole derivatives, compound 1 was docked into the active site of A. fumigatus CYP51B (PDB ID:4UYM, Fig. 2B). Based on the interactions between compound 1 and A. fumigatus CYP51, the benzothiazole ring was replaced by benzoheterocycles to facilitate the formation of hydrogen bond interactions with Tyr122 of CYP51. This might result in the development of more potent compounds with higher antifungal activities and a broader antifungal spectrum, especially with improved anti-Aspergillus efficacy (see Fig. 3).
Results and discussion
Conclusions To further enhance the anti-Aspergillus efficacy of our previous compounds, a novel class of benzoheterocycles ring derivatives were designed, synthesized and evaluated for their in vitro antifungal activity. Among these compounds, the 4,5-dihydronaphtho[2,1-d] isoxazole nucleus 13s and naphtho[2,1-d] isoxazole nucleus 14a exhibited the most remarkable in vitro activity against a variety of fungal pathogens, including Candida spp., C. neoformans and A. fumigatus and fluconazole-resistant strains of C. alb., that was superior or comparable to those of the reference drugs fluconazole and voriconazole. Further mechanistic investigations showed that the potent antifungal activity of novel compound 13s might act by inhibiting the CYP51 of Candida albicans. Notably, a CYP enzyme inhibition assay showed that compounds 13s and 14a had weak inhibition for various human cytochrome P450 isoforms, which indicated they had a low potential to cause DDI. In addition, compounds 13s and 14a exhibited excellent blood plasma stability. Further studies on the antifungal mechanism and structural optimization are in progress.