Small molecules restore azole activity against drug-tolerant and drug-resistant Candida isolates

ABSTRACT Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100–1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.


Synthetic protocols
Analytical thin-layer chromatography (TLC) was performed with silica gel GF254 plates.
Column chromatography was performed with silica gel (300-400 mesh) eluting with solvent mixtures (ethyl acetate (EtOAc)/hexane, acetone/hexane). Nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry (HRMS, with relevant soft ionization techniques) were used to characterize the synthesized analogs and intermediates. 1 H NMR and 13 C NMR spectra were recorded at 400 MHz and 600 MHz (Bruker) with CDCl3 as solvent.

Synthetic scheme for accessing the 1,4-BZD analogs
The scheme begins with substituted acetanilides 1, purchased or synthesized from aniline (2).

Synthesis of acetanilides (1) from anilines (0)
Acetyl chloride (1.1eq) was added to a solution of substituted aniline 0 (4 mM), Pyridine (1.1eq) in dry CH2Cl2 (20 mL) at 0 °C, the mixture was allowed to cool to ambient temperature and stirred until the consumption of starting material was observed (monitored by TLC). After completion of reaction, the crude reaction mixture was sequentially washed and extracted with 2 N solution of HCl, brine and subsequently dried over Na2SO4. After filtration, the crude solution (organic layer) was concentrated in vacuo and purified by flash column chromatography (using EtOAc/hexane) to afford substituted acetanilide 1. The yield was between 70-95% (2).

Synthesis of N-acetylaminoketones (1a) from acetanilides (1)
Acetanilide (1eq), H2O (0.5 M solution of acetanilide 1), TFA (0.3eq), substituted benzylic alcohols or substituted benzaldehydes (2eq) and TBHP (4eq. using a 70 % solution in H2O) were added to a 50 mL Pd(OAc)2 charged round bottom flask (0.1eq). A rubber septum was used to seal the reaction vessel and the mixture was stirred at 40 o C. After completion (monitored disappearance of acetanilide by TLC, usually ~16 h for most reactions), the reaction mixture was dissolved in EtOAc and the organic layer was extracted with water, brine and dried over sodium sulfate. The organic layer was concentrated in vacuo. The concentrated crude was purified by flash column chromatography using EtOAc and hexane solvent mixtures. The yield of products 1a was 25-85 %. Higher yields were observed for Deshaloacetanilides.

Synthesis of aminoketones (18) from N-acetyl-aminoketones (17)
Concentrated HCl (1 mL of HCl per mM of 1a) was added to a solution of the keto acetamide 1a in ethanol (EtOH, 2 mL/mM) and the mixture was stirred at 75 °C for 16 h, the reaction crude was cooled to room temperature and the solution was subsequently brought to a pH of 8 using saturated NaHCO3 solution at room temperature. EtOAc was added to the aqueous solution followed by extraction of the biphasic mixture. The organic layer was washed with water, brine and dried over Na2SO4 to yield a crude reaction solution. The solution was concentrated in vacuo and purified using flash column chromatography (5-8% EtOAc/hexane as solvent mixture) to afford mostly yellowish solids, aminoketones 2 (the yield was 75-93%) (3).
The reaction was heated to 60 o C for 1 h. We ensured the reaction was properly vented to allow the release of CO2 during this period, usually done through a nitrogen/argon inlet and an air needle running through the septum. Next, neat triethylamine (Et3N, 2eq.) was added to the reaction mixture, the reaction was heated to 80 o C, allowed to stir for another 1 h and monitored by TLC/MS for complete conversion to the benzodiazepine-imine product. On completion, the reaction was cooled to room temperature, followed by evaporation of the toluene and addition of EtOAc to the reaction mixture, this crude solution of EtOAc was extracted with water, brine and dried over sodium sulphate. The crude solution was then concentrated in vacuo. Methanol (MeOH, 0.2 M) was then added to the crude mixture and excess acetic acid (3eq.), the mixture was cooled to 0 o C. Sodium cyanoborohydride (4eq.) was then added to the cooled mixture, allowed to warm slowly to room temperature and stirred overnight (16 h) at room temperature.
After completion of reaction, sodium bicarbonate was added to the crude mixture to bring the pH to 8, and then extracted with EtOAc. The organic layer was further extracted with water, brine, dried over sodium sulphate and filtered. The filtrate was subsequently concentrated in vacuo and purified by flash column chromatography (using a 20% and 50% solution of EtOAc/hexane) to yield the product 3 (the yield was 40-82%).

benzo[e][1,4]diazepin-1-yl)acetamide 4 from amino ketones via tandem C-H activation and reduction
The synthesis was adapted from (5) atmosphere. Next, 3eq. of sodium bicarbonate was added to the solution followed by the slow addition of 1.3eq. of 2-thionyl chloride and allowed to stir for 16 h. On completion of the reaction, the residue was filtered off through celite and the filtrate was concentrated in vacuo to afford the crude product. The crude product was purified by flash column chromatography (usually with 15-20% EtOAc/hexane solvent mixture). The yield was 84%. (3, 4) to afford the tertiary amines (6,

7)
The reductive aminations were performed using either of the two different protocols: 1.
1.5eq of the suitable aldehyde was added to a 0.33 mM solution of the secondary amine TLC plates (on silica), using a 40% acetone/hexane solvent mixture. The isolated product yield was 72% (11).

Characterization data of final analogs: 1 H NMR, 13 C NMR, HRMS
FLC-Cy5 was synthesized and characterized using previously described synthetic protocols (1).