Exploring Long Arm Amide-Linked Side Chains in the Design of Antifungal Azole Inhibitors of Sterol 14α-Demethylase (CYP51)

Abstract

The rise in fungal drug resistance has exacerbated the treatment of invasive fungal infections, most commonly caused by Candida. This research describes the synthesis of extended "long-arm" azole antifungals that were evaluated against wild-type and resistant fungal species. Biphenyl derivative 22 was the most effective derivative, displaying potent inhibitory activity against Saccharomyces, Candida, and Cryptococcus CYP51 enzymes, including in resistant strains, in comparison with posaconazole. The X-ray crystal structure of S-22 complexed with S. cerevisiae CYP51 showed a hydrogen bond between the oxygen of the trifluoromethoxy group of S-22 and the His381 side chain of S. cerevisiae CYP51, which is postulated to contribute significantly to its binding, and stabilization in the presence of the S. cerevisiae CYP51 Y140F/H, C. parapsilosis and C. auris CYP51 Y132F mutations and the C. auris K143R mutation. Computational studies and IC₅₀ evaluation of compound 22 vs C. albicans wild-type, Y132F, and Y132H/K143 mutant strains supported MIC observations.

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Clinically used azole triazole and tetrazole antifungal agents.

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Design of triazole antifungal agents with long arm amide-linked extensions. (R = OH or H).

1. Reagents and Conditions: (i) TMSOI, 2M Aq. NaOH, Toluene, 60 °C, 6 h, 83% Crude (ii) NaN₃, NH₄Cl, DMF, 60 °C, 2 h, r.t., 60% (iii) H₂, Pd/C, EtOH, ∼40 psi, 5 h, 69% (iv) CDI, DMF, r.t., o/n 54–65% (v) Pyridine, r.t., o/n, 65%

2. Reagents and Conditions: (i) CDI, DMF, r.t., o/n 65–76% (ii) H₂ (Balloon), Pd/C, EtOH, r.t., 3 h, 100% (iii) 4-Chlorobenzoyl Chloride, Pyridine, r.t., o/n, 78%

3. Reagents and Conditions: (i) SOCl₂, MeOH, 60 °C, 3 h, 91%, (ii) NaOCH₃, (HCHO) _n , DMSO, r.t., 4 h, 78%, (iii) MsCl, Et₃N, CH₂Cl₂, r.t., o/n, 84%, (iv) (a) Triazole, K₂CO₃, CH₃CN, 45 °C, 1 h (b) 30, 70 °C, 4 h Then r.t. o/n, 85% (v) LiOH, THF, H₂O, r.t., 1 h, 73% (vi) CDI, DMF, r.t., o/n 40–66%

4. Reagents and Conditions: (i) B­(OCH₂CF₃)₃, CPME, 100 °C, o/n 67% (ii) H₂ (Balloon), Pd/C, CH₃OH, r.t., 3 h, 93% (iii) Acyl Chloride, Pyridine, r.t., o/n, 68–80%

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Susceptibilities of yeast constructs to exemplar compounds 14, 21, 22, 23, and 26. Y2411–azole sensitive control strain, Y2300 ScCYP51 overexpressed, Y2301 ScCYP51 Y140F mutant, Y2513 ScCYP51 Y140H mutant, Y525 MFS efflux pump CaMDR1a overexpressed, and Y570 ABC efflux pump CaCDR1B overexpressed. PCZ Posaconazole, MCF Micafungin.

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Binding of the Azole Antifungals to ScCYP51. (a) S-22 (white) bound in the active site of the enzyme (green cartoon, parts of the protein are hidden to aid visualization) (PDB ID: 8VK6). Water molecules are shown as red spheres, haem and the iron are shown as orange sticks. Hydrogen bonds are shown as dashed yellow lines. (b) FLC (cyan) complexed with ScCYP51 (PDB ID: 4WMZ) superimposed on the S-22 complex. Conserved waters from the FLC structure are shown as yellow spheres. (c) VCZ (magenta) (PDB ID: 5HS1) superimposed on the S-22 complex. (d) Oteseconazole (yellow) (PDB ID: 5UL0) superimposed on the S-22 complex. (e) PCZ (salmon) (PDB ID: 6E8Q) superimposed on the S-22 complex. (f) All 5 ligands in their ScCYP51-bound conformation. The four hydroxyl groups of S-22, FLC, VCZ, and oteseconazole along with the PCZ tetrahydrofuran oxygen are indicated with an asterisk.

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Viability analysis of compound 22 and Posaconazole incubated with MCF-10A cells for 48 h with staurosporine acting as a toxic control. Error bars represent SEM from three independent experiments.

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(a) Three-dimensional image of compound S-22 in CaCYP51 wild-type and Y132H + K143R double mutant [Haem in orange, H₂O shown as red spheres] (b) 2D ligand interactions (c) protein–ligand RMSD over 150 ns MD simulation time and (d) protein–ligand interactions over the course of the 150 ns MD simulation [Hydrophobic (purple), hydrogen bonds (green), ionic (pink), and water bridges (blue)].