Biologically active drimane derivatives isolated from submerged cultures of the wood-inhabiting basidiomycete Dentipellis fragilis

Abstract

Four previously undescribed drimane sesquiterpenoids were isolated from submerged cultures of the wood-inhabiting basidiomycete Dentipellis fragilis along with two compounds that were previously reported as synthetic or biotransformation compounds but not as natural products. The constitution and relative configuration of these compounds was determined based on high-resolution electrospray ionization mass spectrometry as well as by 1D and 2D nuclear magnetic resonance spectroscopy. The absolute configurations were established based on exemplary calculation of circular dichroism spectra and comparison with measured data as well as on biogenetic considerations. The biological activities of the isolated compounds were assessed in antimicrobial, cytotoxicity and neurotrophic assays. 10-Methoxycarbonyl-10-norisodrimenin (3) exhibited weak activity against the Gram-positive bacterium Staphylococcus aureus and the zygomycete Mucor hiemalis with minimal inhibitory concentrations of 66.7 μg mL^-1. In addition, compound 3 showed weak inhibition of the mammalian cell line KB3.1 (human endocervical adenocarcinoma) with a half maximal inhibitory concentration of 21.2 μM. The neurotrophic activities of 15-hydroxyisodrimenin (1) and 10-carboxy-10-norisodrimenin (5) were assed in neurite outgrowth and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. When supplemented with 5 ng mL^-1 nerve growth factor (NGF), the drimanes 1 and 5 induced neurite outgrowth in PC-12 (rat pheochromocytoma) cells compared to cells solely treated with NGF. As evaluated by RT-qPCR, compounds 1 and 5 also increased NGF and brain-derived neurotrophic factor expression levels in 1321N1 astrocytoma cells. Interestingly, the current study only represents the second report on neurotrophic activities of this widespread class of terpenoids. The only other available study deals with Cyathus africanus, another basidiomycete that can produce drimanes and cyathanes, but is only distantly related to Dentipellis and the Hericiaceae.

Fig. 1. Structures of novel drimane sesquiterpenoids 1–4 and of the known drimanes 5 and 6 isolated from submerged cultures of D. fragilis.

Fig. 2. Key NMR correlations of 15-hydroxyisodrimenin (1). Bold bonds: COSY correlations; red arrows: ¹H–¹³C HMBC correlations; blue arrows: ROESY correlations, indicating β-orientation; green arrows: ROESY correlations, indicating α-orientation and grey: arrows: ROESY correlations between H-7α/β and H-12α/β that secure the position of CH₂-12.

Fig. 3. Calculated (dotted green and orange lines) and experimental (solid blue line) ECD spectra of (A) 15-hydroxyisodrimenin (1), (B) 1α-hydroxymarasmene (4) and (C) 3β-hydroxyisodrimenin (6).

Fig. 4. Key NMR correlations of 1α-hydroxymarasmene (4). Bold bonds: COSY correlations; red arrows: ¹H–¹³C HMBC correlations; blue arrows: ROESY correlations, indicating β-orientation and green arrows: ROESY correlations, indicating α-orientation.

Fig. 5. (A–C): Phase contrast images of PC-12 cells supplemented with NGF (5 ng mL⁻¹) and treated with either DMSO (A, 5 μg mL⁻¹, control), drimane 1 (B, 5 μg mL⁻¹) or drimane 5 (C, 5 μg mL⁻¹) after 24 h of incubation. (D): bar chart displaying the normalized neurite outgrowth of PC-12 cells supplemented with NGF (5 ng mL⁻¹) treated with either DMSO, compound 1 (5 μg mL⁻¹) or compound 5 (5 μg mL⁻¹) after 24 h of incubation. Neurite outgrowths were normalized with respect to the neurite outgrowths detected after 0 h (t₀) and are given in fold changes. Data are means ± S.E.M from three independent experiments. **p < 0.05, *p < 0.10 in unpaired t-test.

Fig. 6. Quantification of NGF and BDNF mRNA levels by RT-qPCR in 1312N1 astrocytoma cells treated with either DMSO (negative control, 5 μg mL⁻¹), compound 1 (5 μg mL⁻¹), or compound 5 (5 μg mL⁻¹) for 48 h. Expression levels are given in fold changes with respect to the DMSO control. Data are means ± S.E.M from three independent experiments. **p < 0.05, *p < 0.10 in unpaired t-test.