Synthesis, Biological Evaluation, and Structure–Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis

Abstract

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

Synthesis of novel benzylamine antimycotics and evaluation of their antimycotic potency

“…In contrast to earlier work, [ 12 ] the activity against Y. lipolytica showed not for all compounds a clear correlation to the activity of the pathogenic Candida strains. Compound 8i with high activity against Yarrowia showed no activity against Candida species and, on the other hand, compound 6d showed only low activity against Yarrowia but high activity against Candida strains.…”

“…Y. lipolytica was chosen because it is an apathogenic yeast [ 24–26 ] and in earlier work, we could find a good correlation between growth inhibition of Y. lipolytica and pathogenic Candida species. [ 12 ] The activity of the benzylamines against Y. lipolytica was compared with terbinafine and clotrimazole. Exemplarily the stability of 6g and 7a was checked by stirring both compounds for 5 days in all culture agar (AC‐agar).…”

“…Compounds 6c and 8e showed the highest cytotoxicity with IC 50 ‐values from 2 to 5 µM, but these values are in the same range as we found for systemically used posaconazole (5 µM). [ 12 ] So, the antimycotic activity might not be an unspecific cytotoxic effect but of course it needs to be evaluated in more detail, adding comparable information with terbinafine and other antifungals. The benzylamines might show a selective effect on fungi and show a mechanism of action as reported for butenafine or terbinafine, but further studies should evaluate this hypothesis.…”

“…Determination of MICs against Y. lipolytica was carried out according to Krauss et al [ 12 ] Y. lipolytica was cultivated in AC‐agar (Sigma Aldrich). Concentration of yeast cells was determined by photometer and adjusted to a turbitity of 0.5 according to McFarland Standard at 600 nm.…”

“…In continuation of our research on novel antimycotic structures, [ 12–14 ] we could identify the benzylamines 1 and 2 (Figure 2) with interesting activity against yeasts. [ 15 ] In the present work, we evaluated the influence of halogen substituents bromo, fluoro, and chloro atoms at the benzyl ether side chain on their activity and the difference between branched side chain and n ‐octyl side chain at the benzylamine core.…”