Aethiopinolones A–E, New Pregnenolone Type Steroids from the East African Basidiomycete Fomitiporia aethiopica

A mycelial culture of the Kenyan basidiomycete Fomitiporia aethiopica was fermented on rice and the cultures were extracted with methanol. Subsequent HPLC profiling and preparative chromatography of its crude extract led to the isolation of five previously undescribed pregnenolone type triterpenes 1–5, for which we propose the trivial name aethiopinolones A–E. The chemical structures of the aethiopinolones were determined by extensive 1D- and 2D-NMR, and HRMS data analysis. The compounds exhibited moderate cytotoxic effects against various human cancer cell lines, but they were found devoid of significant nematicidal and antimicrobial activities.


Introduction
The fungal kingdom includes many species that produce various classes of structurally unique and biologically active metabolites [1,2]. Of interest to us are the largely neglected basidiomycetes and more so, those available from rich untapped sources like the African tropics. During the course of our studies on Kenya's tropical basidiomycetes we have encountered various interesting organisms that yielded new biologically active metabolites such as laetiporins, calocerins, 9-oxostrobilurins and laxitextines [3][4][5]. Another specimen collected in Kenya was identified as Fomitiporia aethiopica, a species that had been first reported from the Ethiopian highlands [6]. The mycelial culture showed an interesting secondary metabolite profile when studied by HPLC-MS. Although the taxonomy of the genus Fomitiporia in Africa has been reported, the secondary metabolites of these genus have so far not been studied extensively, even though one of its species is involved in the esca disease syndrome of grapevine [6][7][8]. The present paper is dedicated to the first investigation of the secondary metabolite production in mycelial cultures of Fomitiporia aethiopica.

Structure Elucidation
Solid phase fermentation on rice of the strain Fomitiporia aethiopica was carried out as described in the Materials and Methods section. In the antimicrobial assay the crude extracts initially showed activity against Bacillus subtilis but the activity was later attributed to fatty-acid like components of the extracts. However, we found some interesting peaks upon analysis of the HPLC-MS data. A subsequent search in the Dictionary of Natural Products database suggested the presence of hitherto undescribed metabolites [9]. Scale-up of fermentation and subsequent preparative chromatography yielded five new triterpenes 1-5, for which we propose the trivial names aethiopinolones A-E.
Aethiopinolone C (3) with a molecular formula C 21 H 28 O 4 and eight degrees of unsaturation deduced from HR mass spectrum was further isolated as yellow oil. Analysis of the 1 H-NMR revealed the absence of the methine proton at δ 2.71 (H-17) and the oxygenated methine proton δ 4.74 (H-16) which were observed in the 1 H-NMR of 1. Instead an olefinic doublet of doublet resonating at δ 6.91 (H-16) was recorded. In the 13 C spectrum new signals at δ 144.3 and δ 155.1 were identified (Table 2). H-16 showed HMBC correlations to C-13/C-14/C-20 and COSY correlations to H 2 -15 implying that 3 has similar planar structure as 1 with the difference being the double bond between C-16/C-17. Further, similar ROESY correlations patterns in compounds 1 and 3 were recorded. Derivatization of 3 with both S and R-MPA chloride gave similar results as 1 for protons neighboring C-3 hence the absolute stereochemistry was assigned as 3S, 5S, 9S, 10S, 13S and 14R (Table S2, SI).
Aethiopinolone D (4) was isolated as yellow oil with molecular formula C 21 H 28 O 4 and eight degrees of unsaturation established from the HRMS data. The 1D and 2D data of 4 indicated that 4 was analogous to 3 with the difference being the stereochemistry at C-3. The same ROESY correlations of H-3 to H 3 -19, H-3 multiplicity in the 1 NMR spectrum and the shielding effect reported in 13 C-NMR data of compound 2 were also observed in the 13 Table S3). Therefore the absolute stereochemistry of compound 4 was assigned as 3R, 5S, 9S, 10S, 13S and 14R.
Aethiopinolone E (5) was obtained as yellow oil. The molecular formula C 21 H 26 O 4 and nine degrees of unsaturation were deduced from the HRMS data. Analysis of the 13 C and DEPT NMR data of 5 indicated that it was similar to 3 with the difference being a keto group at position 3.

Biological Activities
Compounds 1-5 were tested for their cytotoxic effects against various mammalian cell lines (Table 3). 3-4 showed moderate activity against all the tested cell lines. All compounds apart from 2 showed significant activities against MCF-7 and A431 with IC 50 in the range 16-20 µg/mL and 14-27 µg/mL respectively. Compound 1 generally showed the strongest activity against the tested cell lines with the highest effects against PC-3 (8 µg/mL). Compound 2 showed moderate activity only against L929 and KB3.1 cells with IC 50 of 45 µg/mL and 39 µg/mL respectively. Aside from these cytotoxic activities, compounds 1-5 were found devoid of significant antimicrobial and nematicidal effects at concentrations ≤300 µg/mL and ≤100 µg/mL respectively.
Although steroids are rather common in the Basidiomycota, they have not been reported yet from the genus Fomitiporia. Studies on Fomitiporia ellipsoidea metabolites indicated that this fungus produced a large amount of common ergosterol and its derivatives but this species has since been moved to the genus Phellinus (currently valid name: Phellinus ellipsoidea) [11,12]. The close relationship between these two genera has seen the transfer of several other species previously assigned to the genus Phellinus to the genus Fomitiporia, examples being species like F. erecta, F. hartigii, F. robusta, F. punctata, F. hippophaeicola and F. pseudopunctata [13]. Styrylpyrones like the protein kinase C inhibitor, bihispidinyl and hypholomin B, which are common metabolites among the Hymenochaetales, have been reported to occur in some Fomitiporia species [14,15].

Fungal Material
The specimen MUCL 56047 was collected from Mount Elgon, located in the western part of Kenya (1 • 7 6 N, 34 • 31 30 E) by C. Decock in April 2016 (collection and isolation number KE/16-163). The dried herbarium specimen and culture are deposited at MUCL (Louvain-la-Neuve, Belgium) as MUCL 56047. The fungus was identified as Fomitiporia aethiopica by morphological studies and sequencing of the rDNA (5.8S gene region, the internal transcribed spacer ITS1 and ITS2). Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, ON, Canada). A Precellys 24 homogenizer (Bertin Technologies, Saint-Quentin-en-Yvelines, France) was used for cell disruption at a speed of 6000 rpm for 2 × 40 s. The gene regions were amplified with primers ITS 1f and ITS 4. Details are given in the Supplementary Material.

Fermentation
The mycelial culture of MUCL 56047 was subjected to solid state fermentation in rice according to [16] with slight modifications. The rice medium was prepared by weighing 90 g of rice into 500 mL Erlenmeyer flasks containing in 90 mL of distilled water and autoclaved twice. A well-grown YMG agar plate of the mycelial culture was cut into small pieces using a 7 mm cork borer and five plugs inoculated into each of the 21 flasks containing sterile rice media. The cultures were incubated in a dark room at 23 • C for 28 days.

Extraction
The cultures were diced into smaller pieces with a spatulum and each of the 21 flasks was soaked in 150 mL of methanol overnight. Repeated extraction and filtration in an ultrasonic bath at 40 • C for 30 min until an exhausted residue was yielded was carried out. The residue was discarded and the filtrate evaporated by means of a rotary evaporator. The resulting aqueous phase was suspended in equal amount of distilled water and extracted with equal amount of ethyl acetate four times. The aqueous phase was discarded and the organic phase filtered through anhydrous sodium sulphate. The resulting ethyl acetate extracts were evaporated to dryness by means of rotary evaporator to afford 800 mg of crude product.

Isolation and Physico-Chemical Characteristics of Compounds 1-5
The crude extract was fractionated using preparative reverse phase liquid chromatography (PLC 2020, Gilson, Middleton, MA, USA). A VP Nucleodur 100-5 C 18 ec column (250 × 40 mm, 7 µm: Macherey-Nagel, Schkeuditz, Germany) was used. Deionized water (Milli-Q, Millipore, Schwalbach, Germany) (solvent A) and acetonitrile (solvent B) were used as the mobile phase. The elution gradient used was 10-100% solvent B in 55 min and thereafter isocratic condition at 100% solvent B for 10 min. UV detection was carried out at 210, 254 and 350 nm. Seven fractions (F1-F7) were collected according to the observed peaks. Fraction F4 was purified by reverse phase LC (solvent A/solvent B), elution gradient 20-35% solvent B for 30 min, followed by a gradient shift from 35% to 100% in 3 min and finally isocratic condition at 100% solvent B for 5 min with a preparative (Kromasil, Mainz, Germany) 250 × 20 mm, 7 µL C-18 column as stationary phase to give compounds 1 (30 mg) and 3 (16 mg). Using the same column and elution gradient 25-40% solvent B for 35 min, fraction F6 was purified to afford 60 mg of compound 2, as well as 12 mg of 4 and 8 mg of 5. Hilden, Germany) and MCF-7 in RPMI (Lonza, Cologne, Germany) media, all supplemented with 10% of fetal bovine serum (Gibco) under 10% CO 2 at 37 • C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) method in 96-well microplates (ThermoFisher Scientific). Briefly 60 µL aliquots of serial dilutions from an initial stock of 1 mg/mL in MeOH of the test compounds were added to 120 µL aliquots of a cell suspension (5.0 × 10 4 cells/mL) in 96-well microplates. After 5 days incubation, a MTT assay was performed, and the absorbance measured at 590 nm using an ELISA plate reader (Victor, PerkinElmer, Überlingen, Germany). The concentration at which the growth of cells was inhibited to 50% of the control (IC 50 ) was obtained from the dose response curves. The negative control was methanol.

Nematicidal Assay
Compounds 1-5 were assessed for nematicidal activity against Caenorhabditis elegans according to [18] with slight modifications. Caenorhabditis elegans were inoculated monoxenically on nematode agar at room temperature for 4-5 days. Thereafter, nematodes were washed down from the plates with M9 buffer. The final nematodes concentration was adjusted to 500 nematodes/mL of M9 buffer. Assay was performed in 24-well microtiter plate at four different concentration (100, 50, 25 and 12.5 µ/mL) of each compound. Ivermectin was used as the positive control and methanol as a negative control. The plates were incubated at 20 • C in the shaker in the dark and nematicidal activity was recorded after 18 h of incubation and expressed as a LD 90 .

Conclusions
In our continuous search for novel and bioactive compounds from tropical basidiomycetes, we found five novel steroids from mycelial cultures of Fomitiporia aethiopica. The metabolites are the first steroids from the genus in the current circumscription, even though triterpenoids and steroids in particular are of widespread occurrence in Basidiomycota. The new metabolites were tested in various bioassays, but only moderate to weak cytotoxic activities were observed, and their biological functions remain obscure. Although closely related pregnane-type steroids have been reported before from Phellinus igniarius and the marine alga-derived fungus Phaeosphaeria spartinae, such pregnenolone-like compounds are unprecedented in fungal metabolism [19,20]. Accumulating evidence on triterpenoids broad spectrum pharmacological activities coupled with a low toxicity profile has sparked discussion with regard to their application, especially in cancer treatment.