Spirocyclic Drimanes from the Marine Fungus Stachybotrys sp. Strain MF347

A novel spirocyclic drimane coupled by two drimane fragment building blocks 2 and a new drimane 1 were identified in mycelia and culture broth of Stachybotrys sp. MF347. Their structures were established by spectroscopic means. This is the first example of spirocyclic drimane coupled by a spirodihydrobenzofuranlactam unit and a spirodihydroisobenzofuran unit; and the connecting position being N-C instead of an N and N connecting unit. Strain MF347 produced also the known spirocyclic drimanes stachybocin A (12) and stachybocin B (11) featured by two sesquiterpene-spirobenzofuran structural units connected by a lysine residue; the known spirocyclic drimanes chartarlactam O (5); chartarlactam K (6); F1839A (7); stachybotrylactam (8); stachybotramide (9); and 2α-acetoxystachybotrylactam acetate (10); as well as ilicicolin B (13), a known sesquiterpene. The relative configuration of two known spirobenzofuranlactams (3 and 4) was determined. All compounds were subjected to biological activity tests. The spirocyclic drimane 2, 11, and 12, as well as the sesquiterpene 13, exhibited antibacterial activity against the clinically relevant methicillin-resistant Staphylococcus aureus (MRSA).


Introduction
The fungal genus Stachybotrys (class: Sordariomycetes, order: Hypocreales) comprises approximately 100 species [1]. Members of Stachybotrys spp. are distributed worldwide and are commonly isolated from soil and various decaying plant substrates. Most species are able to decompose cellulose efficiently. Dangerous toxinogenic isolates of the Stachybotrys chartarum complex have gained importance, when colonizing cellulosic substrates in moist indoor environments [2]. S. chartarum is reported to be involved in animal and human toxicoses, which are associated with "sick building syndrome" in wet buildings [3,4].
During the course of a study of isolates of S. chartarum obtained from various areas around the world, spirocyclic drimanes were discovered as the major class of secondary metabolites produced by this fungus [3]. This type of compound has been reported to be produced by other species of Stachybotrys and members of this class are potent immunosuppressants, particularly the dialdehyde derivatives [13]. However, dialdehyde derivatives were rarely isolated from the genus Stachybotrys, since dialdehydes are relatively unstable and often undergo conversion to stachybotrylactones [14]. Although many spirocyclic drimanes were reported to be toxins, they showed various biological effects, such as immunosuppressive activity [13], endothelin receptor antagonistic activity [15,16], and tyrosine kinase inhibitory activity [17].
Fungi from marine habitats, living in a stressful habitat, are of great interest as new promising sources of biologically active products. As marine organisms live in a biologically competitive environment with unique conditions of pH, temperature, pressure, oxygen, light, nutrients, and salinity, the chemical diversity of the secondary metabolites from marine fungi is considerably high [18][19][20]. As far as is known, only two studies focused on biologically active compounds produced by Stachybotrys spp. strains originated from marine habitats and identified stachybotrin A and stachybotrin B, as well as a fibrinolytic active compound of unknown structure [8,12]. In order to find new natural products from marine microorganisms exhibiting, e.g., antibacterial and cytototoxic activities, Stachybotrys sp. strain MF347 was cultured and the secondary metabolites in the mycelia and the culture broth were investigated. A novel spirocyclic drimane coupled by two drimane fragment building blocks and a new drimane were identified. This is the first example of spirocyclic drimane coupled by a spirodihydrobenzofuranlactam unit and a spirodihydroisobenzofuran unit, and the connecting position being N-C instead of an N and N connecting unit (Chart 1).

Identification of Strain MF347
The fungus was isolated by Dr. Karsten Schaumann from a marine driftwood sample and could be taxonomically classified as a Stachybotrys species. Colonies growing on WSP30 agar attaining a diameter of 40 mm within 14 days of incubation at 26 °C. Colonies were greyish brown with a brown back side (Figure 1). Ellipsoidal conidia were produced by clusters of phialides in slimy masses at the top of conidiophores ( Figure 2). These morphological features are characteristic for the genus Stachybotrys, including the well-known species Stachybotrys chartarum.

Structural Elucidation
Compound 1 was isolated as a yellow powder. The molecular formula was determined to be C 25 H 33 NO 6 by analysis of the HR-TOF-MS ion peak at m/z 466.2181 [M + Na] + (calcd. 466.2200). The IR spectrum suggested the presence of an α,β-unsaturated γ-lactam (1673 cm −1 ) group. 1 H and 13 C NMR spectrum (Table 1) showed signals in close agreement with those of the known spirodihydrobenzofuranlactam, F 1839 A (7) [21], isolated from the same fungus, except that the acetoxyl group was added in the molecule of 1. Analysis of the 1D and 2D NMR data ( Figure 3) and comparison with those of F 1839 A led to the identification of the planar structure of 1 as drawn [21]. The acetoxyl group was positioned at C-17 from the observation of the HMBC cross peak from the oxygenated proton signal at δ H 5.21 (ddd, J = 12.6, 4.4, 2.6 Hz, H-17) to the acetoxyl carbon signal at δ C 172.6 (s, C-24). The 1,3-diaxial NOESY cross peaks of H-17β/Me-23, H-17β/Me-21β, Me-21β/H-11β revealed a 9.14-trans ring fusion between two cylcohexyl chair rings of the bicyclic decalin with a β-oriented methyl group at C-15 and α-oriented methyl group at C-11 ( Figure 4). The NOESY correlations from the axial methyl proton signals at δ H 1.    Compound 2 was obtained as a yellow powder. The HR-TOF-MS exhibited an ion peak at m/z 778.4319 [M + Na] + (calcd. 778.4289), corresponding to the molecular formula, C 46 H 61 NO 8 . The characteristic feature of the 13 C NMR spectrum of 2 is the presence of 15 pairs of identical or almost equivalent carbon signals, which are assigned to two sesquiterpene units. When comparing the NMR data of 1 ( Table 2) with those of stachybotrylactam (8), compound 2 was deduced to be the coupling of two units of spirodihydrobenzofuranlactam/lactone with one stachybotrylactam substructure. The remaining half of the molecule was proved by detailed analysis of 1D and 2D NMR spectra of 2 to have the same sesquiterpene part in its structure ( Figure 3). From the analysis of the molecule formula, C 46 H 61 NO 8 , the remaining unit was deduced to be a spirodihydrobenzofuranlactone or spirodihydrobenzofuran instead of spirodihydrobenzofuranlactam. The presence of the oxygenated methine (δ C 85.8, δ H 7.24) and oxygenated methylene (δ C 74.0, δ H 5.21, 5.00) implied that the structure of 2 possessed a dihydroisobenzofuran unit in one of the two spirocyclic drimane domains. Detailed analyzes of 2D NMR spectra (   spirodihydrobenzofuranlactam indicated that the linkage position of the two units was from N to C-8 as drawn. The relative configuration of the sesquiterpene parts of both monomers of 2 was deduced from the analysis of the NOESY spectrum to be the same as those in compounds 1 and the known stachybotrylactam (8) (see Supplementary Information). Therefore, the structure of this compound was elucidated as a new spirocyclic drimane coupled by two drimane fragment building blocks and given the trivial name stachyin B. Most dimeric spirodihydrobenzofuranlactams isolated from natural resources are those whose the monomers are connected by a N and N connecting unit, such as N-C(OOH)CH 2 CH 2 CH 2 CH 2 -N in compounds 11 and 12 [16]. The structure of 2 is the first example of spirocyclic drimane coupled by a spirodihydrobenzofuranlactam unit and a spirodihydroisobenzofuran unit, and the connecting position being an N-C instead of an N and N connecting unit. Compounds 3 and 4 were obtained both as yellow powder. From the detailed analyzes of their MS and NMR spectrum, the structures of 3 and 4 were deduced to be the same compounds patented [22]. No names have been given for these two compounds by the authors, but sum formula and a graphic presentation of the structures were shown. However, no relative configuration and detailed NMR data were reported [13]. Here, we reported the NMR data (Table 3), 2D NMR correlations ( Figure 3) and the relative configuration (Chart 1) of the two compounds.

Biological Activities
Among the isolates, stachyin B (2) inhibited the growth of the Gram-positive test strains Bacillus subtilis, Staphylococcus epidermidis and the methicillin-resistant Staphylococcus aureus (MRSA) with IC 50 values in the range of 1-1.7 µM (Table 4)  The Gram-negative test strain Klebsiella pneumoniae as well as the fungal strains Candida albicans, Trichophyton rubrum, and Septoria tritici were not inhibited by the compounds 1-13. All compounds exhibited no worth mentioning activities in the enzymatic assays. The IC 50 values are >50 µM for the acetylcholinesterase and >10 µM for the glycogen-synthase-kinase 3β and phosphodiesterase 4B2.

General Experimental Procedures
Optical rotations were recorded on a Perkin Elmer 241 polarimeter (PerkinElmer Inc., Rodgau, Germany). The IR spectra were run on a Perkin Elmer spectrometer (PerkinElmer Inc.) with an ATR unit. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra were measured at 25 °C on a Bruker AVANCE DRX 500 NMR spectrometer (Bruker Daltonics, Bremen, Germany) with TMS as internal standard. The signals of the residual solvent protons and the solvent carbons were used as internal references (δ H 3.31 ppm and δ C 49.0 ppm for methanol-d 4 ). High-resolution mass spectra were acquired on a benchtop time-of-flight spectrometer (micrOTOF II, Bruker Daltonics) with positive electrospray ionization (ESI).

Fermentation, Extraction and Isolation of the Compounds
Strain MF347 was cultured on WSP30 agar plates at 22 °C for 28 days. This pre-culture was used for the inoculation of 12 × 2 L Erlenmeyer flasks containing 750 mL WSP30TM medium (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 3% tropic marine salt (pH = 6.8)) each. After incubation for 22 days at 28 °C in the dark as static cultures, extracts of the cultures were obtained. The mycelium was separated from the culture broth. 150 mL ethanol were added to the mycelium of each flask and homogenized. After a centrifugation step at 10,000 rpm for 10 min, the ethanol was removed by evaporation. The remaining aquatic phases of all 12 flasks were combined and extracted twice with 100 mL ethyl acetate. The organic phase was used for evaporation. The resulting residue was dissolved in 20 mL methanol to get the extract of the mycelium. The fermentation broth was extracted with ethyl acetate (400 mL per each flask). 100 mL deionized water were added to the organic phase. The upper phases of all flasks were combined and used for evaporation. The residue was dissolved in 5 mL methanol to get the extract of the culture broth. Both extracts were subjected to analytical HPLC-UV/MS. For the purification of the compounds 1-13, both extracts were combined.
By preparative HPLC 14 fractions were collected. 10 fractions were purified using semi-preparative HPLC. The specification of the columns, solvents, and gradients as applied for the purification of the compounds 1-13 as well as the respective retention times and yields are listed in Table 5.

Conclusions
Although many compounds isolates from the genus Stachybotrys were reported to be toxins [3,4], they showed various biological effects, such as immunosuppressive activity [13] and antihyperlipidemic [23]. Antibiotic activities of spirodihydrobenzofuranlactam and spirodihydrobenzofuranlactone were rarely reported. The spirocyclic drimanes with two sesquiterpene-spirobenzofuran structural units 2, 11, and 12 showed antibacterial activity against the clinically relevant methicillin-resistant Staphylococcus aureus (MRSA), whereas spirocyclic drimanes with one sesquiterpene-spirobenzofuran structural unit 1, 3-10 exhibit no activities. It is tentatively implied that the structural feature of two sesquiterpene-spirobenzofuran units with either a N-C or a N-N linkage of spirocyclic drimanes is important for antibiotic activity. This is the first example of spirocyclic drimane coupled by a spirodihydrobenzofuranlactam unit and a spirodihydroisobenzofuran unit, and the connecting position being N-C. Stachyin A (1) and B (2) are structurally interesting, which would provide opportunities to design and synthesize new analogs that could improve the antibiotic and cytotoxic activities of these new compounds.