New Generation Modified Azole Antifungals against Multidrug-Resistant Candida auris

Abstract

The rise of antifungal resistance and limited treatment options highlight the urgent need for new drug classes. Candida auris is a serious global health threat with few effective therapies. In this study, novel azole-based compounds were developed by modifying the azole core with cyclic heteroaliphatic linkers connecting aromatic and heteroaromatic rings. Several compounds showed potent activity against C. auris, including azole-resistant strains, with MICs ranging from 0.016 to 4 μg/mL. The compounds also demonstrated strong activity against C. albicans, Nakaseomyces glabratus, C. tropicalis, and C. parapsilosis, with MICs mostly below 1 μg/mL. Compounds 7, 18, and 21 were more potent than fluconazole. Compound 7 inhibited CYP51, eradicated C. auris biofilms, and showed better intracellular accumulation than fluconazole. In vivo studies in Galleria mellonella and Drosophila melanogaster confirmed efficacy at 5 mg/kg and no toxicity up to 50 mg/kg, supporting further development of this scaffold against multidrug-resistant C. auris infections.

1

Chemical structures of fluconazole and modified azole compounds. (A) General structure of the modified azole compounds. (B) Compounds of library A containing a piperidine linker and different aromatic or heteroaromatic rings.

1. General Synthetic Routes for Library A where Different Aromatic/Heteroaromatic Fragments (A) were Connected to the Fluconazole Core via a Piperidine Linker (B). Conditions: (i) NaBH­(OAc)­3, AcOH, DCM/MeOH, Overnight; (ii) DIPEA, MeCN, DMA, 160–180 °C, 2–3 h or TEA, Ethylene Glycol, 200 °C, 15 min; (iii) rac-BINAP, Pd2­(dba)­3, KOtBu, Toluene, 100 °C, Overnight; (iv) 4 M HCl in 4-Dioxane, r.t., 1–2 h; (v) NaOH (aq.), Toluene, 80 °C, Microwave 50 min; (vi) TEA, EtOH, 80 °C, Overnight

2. General Synthetic Routes for Library B, where the Terminal Heteroaromatic Fragment of 7 was Connected to the Fluconazole Core via Different Linkers

2

Differences in 2D binding interactions observed for fluconazole (A) and compound 7 (B) with a lanosterol 14α-demethylase (LDM) enzyme in (PDB ID 5TZ1 was used as a template for homology modeling).

3

Intracellular accumulation of azole antifungals in strain TDG1912. Compound 7 showed significantly greater accumulation than fluconazole (p = < 0.0001).

4

Eradication of preformed TDG1912 biofilms by antifungal agents. Established biofilms were treated with serial dilutions of fluconazole (Fluc), voriconazole (Vori), amphotericin B (Amp B), or Compound 7 for 24 h. Residual biofilm biomass was quantified and normalized to the untreated control (% biomass). Data represent the mean ± SD of three independent experiments.

5

Time-kill analysis of compound 7, alongside two commercial reference antifungal drugs, fluconazole (Fluc) and voriconazole (Vori) against TDG1912. The limit of detection (LoD) is 1000 CFU/mL. MIC₅₀ results are given in μg/mL. NT represents no treatment.

6

Efficacy of modified fluconazole compound 7 and fluconazole (Fluc) against TDG1912 infection in the G. mellonella model.

7

Efficacy of compound 7 and fluconazole (Fluc) against TDG1912 infection in the model at an infection dose of 2 × 10³ yeast cells per fly.