Scalarane Sesterterpenoids with Antibacterial and Anti-Proliferative Activities from the Mushroom Neonothopanus nambi

Seven undescribed scalarane sesterterpenoids, nambiscalaranes B–H (1–7), together with two known compounds, nambiscalarane (8) and aurisin A (9) were isolated from the cultured mycelium of the luminescent mushroom Neonothopanus nambi. Their structures were elucidated by thorough analysis of their 1D and 2D NMR spectroscopic data. The absolute configurations of 1–8 were determined by electronic circular dichroism (ECD) calculations and optical rotation measurements. The isolated sesterterpenoids were evaluated against A549, HT29, HeLa, and HCT-116 cancer cell lines, and against five bacterial strains. Compounds 3, 5, and 7 showed strong cytotoxicity against HCT-116 cell line, with IC50 values ranging from 13.41 to 16.53 µM, and showed no cytotoxicity towards Vero cells. Moreover, compound 8 inhibited the growth of Bacillus subtilis with a MIC value of 8 µg/mL, which was equivalent to the MIC value of the standard kanamycin.


Introduction
The luminescent mushroom Neonothopanus nambi belongs to the Omphalotaceae family. It is known as 'Hed Ruang Sang Sirin-ratsami' or 'Sirin-ratsami mushroom' in Thai [1]. Previous agricultural studies have shown that N. nambi can induce systemic resistance against root-knot nematodes such as Meloidogyne incognita in tomato plants [2,3]. N. nambi produces diverse secondary metabolites that seemed to greatly vary in nature based on the conditions of its culture ( Figure S1 and Table S1). Our previous work showed that potato dextrose broth led to the production of sesquiterpenoids (and dimers) as well as terphenyls and benzoquinones [1,4]. Yeast malt agar conditions led to the isolation of more diverse secondary metabolites. One of them was a scalarane sesterterpenoid, named nambiscalarane (8) [5].
Sesterterpenoids are a relatively small family of terpenoids. Among this family, the carbocyclic core can greatly vary. Scalaranes are a rare group of sesterterpenoids with a 6/6/6/6 tetracyclic skeleton. Its first member was discovered from the marine sponge Cacospongia scalaris in 1972 [6]. Scalaranes display a wide range of biological The cultured mycelium of N. nambi (375.7 g) was ground to powder and then extracted at room temperature with EtOAc (3 × 3 L). Removal of solvent under reduced pressure gave the crude EtOAc (82.74 g) extract. The crude EtOAc extract led to the isolation of 36.97 g of 9 as yellow crystals after crystallization from EtOAc. The residue (45.7 g) was obtained from evaporation of the filtrate and chromatographed over silica gel flash column chromatography (FCC), eluting with a gradient system of hexanes:EtOAc and EtOAc:MeOH to afford six fractions, EF1-EF6. Fraction EF1 was separated over silica gel FCC, eluting with a gradient system of hexanes:EtOAc (95:5 to 80:20) to give three subfractions, EF1.1-EF1.3. Subfraction EF1.2 was purified by silica gel FCC, eluting with an isocratic system of hexanes:CH 2 Cl 2 (25:75) to give 1 (8.0 mg) as a yellow amorphous powder. Fraction EF3 was purified by silica gel FCC, using a gradient elution of hexanes:EtOAc to EtOAc, to give 8 (48.0 mg), 3 (8.5 mg), and 2 (15.0 mg) as pale yellow oils, as well as 6 (5.3 mg) and 5 (4.0 mg) as white viscous oils. Fraction EF4 was subjected to silica gel FCC, eluting with an isocratic system of hexanes:EtOAc (85:15) to obtain two subfractions, EF4.1 and EF4.2. EF4.1 was then purified by FCC, eluting with MeOH:CH 2 Cl 2 (5:95) to give 4 (

ECD Calculations
Preliminary conformational analyses were carried out using HyperChem software, Hypercube Inc., Gainesville, FL, USA. For theoretical ECD spectra, the possible configurations of compound 2 were established for both geometrical optimizations and electronic excited calculations. Geometrical optimizations of the structure were taken under density functional theory (DFT) calculations. These calculations were performed with hybrid density functional B3LYP, and using 6-311G(d,p) to diffuse basis set. In the single point energy calculations, the vertical transition energies to the valence excited-states were computed with the time-dependent density functional theory (TD-DFT) method using the long-range corrected functional CAM-B3LYP at the 6-311++G(d,p) level (σ = 0.40). The bulk solvent effects were evaluated using the Conductor-like Polarizable Continuum Model (C-PCM). All calculations were performed with the Gaussian09 program [17].

Antibacterial Assay
Five microorganism cultures (Methicillin resistant S. aureus DMST 20654, S. aureus ATCC 25923, S. sonnei ATCC 11060, B. subtilis ATCC 6633, and B. cereus ATCC 11778) were used. The experiments were performed at the Department of Microbiology, Faculty of Science, Khon Kaen University, Thailand. The antibacterial assay was performed as recommended by the Clinical and Laboratory Standards Institute [18]. The standard drugs kanamycin and chloramphenicol were used as positive controls.

Antiproliferative Activity Assay
The anti-proliferative effect on cancer cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells (8 × 10 3 cell/well) were seeded onto 96-well plates and incubated for 24 h to allow adherence. After 24 h, the cells were exposed to increasing concentrations (3.12, 6.25, 12.5, 25, 50, and 100 µg/mL) of pure metabolites in a mixture of DMSO and ethanol (1:1) for 72 h. Control groups were treated with a mixture of DMSO and ethanol (1:1). After the indicated time, the medium was replaced with 110 µL of fresh medium containing MTT (0.5 mg/mL in PBS) (Sigma Chemical Co., St Louis, MO, USA) and incubated for 2 h. The formazan formed after conversion of MTT was dissolved in DMSO. The absorbance of formazan was measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at the wavelength of 550 nm with a reference wavelength of 655 nm. The percentage of viable cells which corresponds to the production of formazan was calculated using the following formula [19].

Isolated Compounds from N. nambi
The dried cultured mycelium of N. nambi was extracted with ethyl acetate (EtOAc). Separation of the crude EtOAc extract via column chromatography (CC) yielded eight sesterterpenoids (1)(2)(3)(4)(5)(6)(7)(8) and aurisin A (9) [4]. Their structures were established by thorough analysis of spectroscopic evidence. They are depicted in Figure 1. The data obtained were compared with published values of similar compounds. The absolute configurations of 1-7 were determined by a combination of electronic circular dichroism (ECD) calculations and optical rotation ([α] D ) measurements. The absolute configuration of the known sesterterpenoid nambiscalarane (8), previously unspecified [5], was also determined in a similar manner.  (Table 2) revealed twenty-five carbon resonance peaks, consisting of five methyl, eight methylene, six methine, and six quaternary carbons. These data suggested the presence of a sesterterpenoid core. COSY and HMBC correlations ( Figure 2) established the presence of four spin systems, enabling the assignment of fragments C-1/C-2/C-3, C-5/C-6/C-7, C-9/C-11/C-12, and C-14/C-15/C-16. The key HMBC correlations from H-24 to C-17, C-18, and C-25, as well as H-25 to C-17, C-18, and C-24, indicated the presence of a disubstituted furan ring at these positions. The HMBC correlations of H 3 -19 (20) to C-3, C-4, C-5, and C-20 (19), of H-5 to C-6, and of H-7 to C-6 led to the location of the hydroxy group at C-6. In addition, correlations from H 3 -22 to C-1, C-5, C-9, and C-10, of H 3 -21 to C-7, C-8, C-9, and C-14, and of H 3 -23 to C-12, C-13, C-14, and C-18 confirmed the presence of a tetracyclic scalarane sesterterpenoid skeleton, which was similar to the known analogue 16-deacetoxy-12-epi-scalarafuranacetate isolated from Spongia officinalis in 1989 [20]. However, compound 1 contains a hydroxy group at C-6 instead of an acetyl group at C-12 in the known analogue. The relative configuration of compound 1 was determined by analysis of NOESY correlations and coupling constants (Figure 3) [22], suggested that the absolute configuration of compound 1 should be identical to the one of the known analogues. In order to confirm this assertion, the experimental ECD curve of compound 1 was compared with the ECD curve of compound 2, for which ECD calculations were undertaken (vide infra). Thus, the configuration of compound 1 was established as 5S, 6S, 8R, 9S, 10R, 13S, 14S.    The molecular ion peak could be observed but at a relatively low intensity due to rapid fragmentation via decarboxylation of the malonate. The 1 H and 13 C NMR data of compound 2 (Tables 1 and 2) were similar to those of compound 1.

Structural Characterization of the New Compounds
The strong deshielding of position 11 (δ H /δ C 5.49 (td, J = 11.2, 3.0 Hz)/72.3 ppm) suggested the presence of an ester group connected to this position through its oxygen atom. The moderate deshielding of the proton at position 6 (δ H /δ C 5.23 (td, J = 11.2, 3.3 Hz)/70.6 (C-6) ppm) suggested that the hydroxy group observed in compound 1 was esterified. Extra signals of an acetate (δ H /δ C 2.06, s/22.1 (C-2 ) and δ C 170.6 (C-1 ) ppm) and a malonate (δ H /δ C 3.43, s/41.4 (C-2 ); δ C 166.6 (C-1 ) and 170.0 (C-3 ) ppm) were also detected. HMBC correlations (Figure 2) between H-6 to C-1 and H-11 to C-1 located the acetate at C-6 and the malonate at C-11. In addition, the NMR data of 2 were very similar those of nambiscalarane (8), with slight differences arising from the substitution of the hydroxy group at C-11 by a malonate. NOESY correlations (Figure 3) 1 H-1 H coupling constants between H-6/H-7 (11.2 Hz) and H-11/H-12 (11.2 Hz) indicated that the relative configuration of compound 2 was identical to that of compound 1. The absolute configuration of compound 2 was established through comparison of calculated and experimental ECD curves. The conformational analysis of compound 2 showed one lowest energy conformer ( Figure S2). The ECD spectra of the two possible enantiomers (5S, 6S, 8S, 9S, 10S, 11R, 13S, 14S and 5R, 6R, 8R, 9R, 10R, 11S, 13R, 14R) based on the established relative configuration of compound 2 were initially optimized at the B3LYP level, using 6-311G(d,p). TDDFT was then utilized in MeOH to predict the rotational strengths of the transition states using the CAMB3LYP/6-311++G(d,p) level, and these calculated spectra were compared with the experimental ECD spectrum of compound 2.   (Tables 1 and 2) were very similar to those of compound 2, except for the malonyl group at C-11. Analysis of 2D NMR spectra (Figure 2) led to the conclusion that it was replaced by a 3-methylglutaconic acid unit, which was connected through the oxygen at its carbon n • 5 (  (Tables 1 and 2) were very similar to those of compound 3. The shielding of H-6 (from δ H 5.23 to 3.91 ppm) and the absence of signals of an acetate moiety in the proton and carbon NMR spectra suggested deacetylation at this position. The shielding of H-4 suggested a change in the nature of the ester group's tail (at C-11). Analysis of 2D NMR data ( Figure 2) indicated that the sidechain of C-11 was replaced by a 3-methylglutaconic acid unit, which was connected through the oxygen at its carbon n • 1 (Figure 1) (δ H /δ C 5.69, s/120.1 (C-2 ); 3.10, s/45.9 (C-4 ); 2.23, s/19.1 (C-6 ); 172.6 (C-5 ), 165.5 (C-1 ), and 152.0 (C-3 ) ppm). The NOESY (Figure 3

) correlations of H-11β/H-12β/H 3 -21/H 3 -22/H 3 -23 and H-6β/H-7β/H 3 -19/H 3 -21/H 3 -22,
as well as the large coupling constants of H-6 (10.8 Hz) and H-11 (11.2 Hz) indicated that the relative configuration of 4 was identical to that of compounds 2 and 3. The geometry of the double bond was identified as (E) due to the NOESY correlation observed between H-2 /H-4 ( Figure 3). Interestingly, a NOESY correlation between H 3 -6 /H-25 ( Figure S41) was observed when CDCl 3 with 2 drops of CD 3 OD was used as a solvent, indicating that an intramolecular π-π interaction was present between C-2 /C-3 and the furan ring ( Figure 5). This interaction was likely responsible for the splitting of the 13 Figure S43).   (Tables 1 and 2) were extremely similar to those of compound 5. Analysis of 2D NMR spectra (Figure 2) led to the conclusion that the ester unit at position 11 was altered at its tail. The sidechain of C-11 was replaced by a 3-methylglutaconic acid unit, which was connected through the oxygen at its carbon n • 5, as in compound 3 ( Figure 1 (Tables 1 and 2) were extremely similar to those of compound 5. Analysis of 2D NMR spectra (Figure 2) led to the conclusion that the butanolide was altered. Position C-25 was substituted by a hydroxy group, leading to a 1:1 mixture of hemiacetal epimers (δ H /δ C 5.84:5.82, s/98.8:98.7 ppm). Analysis of NOESY correlations and coupling constants showed that compound 7 shared the same relative configuration and double bond geometry as compound 5.
Compound 8, nambiscalarane, was isolated as a yellow viscous oil. Its NMR data were extremely similar to those of compound 4, except for the absence of an acetate group at position 6. Our spectroscopic data matched with reported values [5]. The relative and absolute configurations of this compound were not described at the time of its discovery. Therefore, NOESY and ECD spectra were recorded. Analysis of NOESY (Figure 3) correlations and coupling constants showed that compound 8 shared the same relative configuration as compounds 2-4. The double bond geometry was determined as (E) in a similar manner to compound 4.
The absolute configurations of compounds 3-8 were assigned by analogy with compound 2, as they showed similar ECD curves (Figures 4 and 6a,b) and optical rotations of identical sign, although the ECD curve of compound 7 proved inconclusive (Figure 6b), probably due to the presence of two epimers in a 1:1 ratio.

Cytotoxic Activities of Scalarane Sesterterpenoids 1-8
Compounds 1-8 were assessed for cytotoxic activity against A549 human lung carcinoma cell line, HT29 human colon cancer, HeLa human cervical cancer, and HCT-116 human colon cancer, cancer cell lines by the MTT assay (Table 3). Cis-platin was used as the positive control. Compound 7 was inactive against all the tested cell lines. Compounds 1, 3, 4, and 8 showed moderate cytotoxicity to A549 cell line, with IC 50 values ranging from 22.95 to 27.51 µM, while compounds 2, 5, and 6 were inactive. Compounds 1-4 and 8 showed moderate activities against HT29 cell line with IC 50 values ranging from 20.39 to 33.02 µM, while compound 5 showed weak activity (IC 50 54.46 µM) and compound 6 was inactive. Compounds 3, 4, and 8 showed moderate activities against HeLa cell line with IC 50 values ranging from 21.14 to 26.55 µM, compounds 1 and 6 showed weak activities (IC 50 40.81 and 45.94 µM, respectively), while compounds 2 and 5 were inactive. Compounds 2, 4, and 6 showed strong cytotoxicity against HCT-116 cell line with IC 50 values ranging from 13.41 to 16.53 µM, while compounds 1, 3, 5, and 8 showed moderate activities with IC 50 values ranging from 20.28 to 32.70 µM. No clear conclusion could be drawn in term of structure-activity relationship among the eight sesterterpenoids tested. In addition, the tested compounds proved inactive towards Vero cells.

Antibacterial Activities of Scalarane Sesterterpenoids 1-8
The isolated scalarane sesterterpenoids were evaluated for their antibacterial activities against four Gram-positive and one Gram-negative bacteria (Table 4). Compounds 5 and 8 showed moderate activities against Staphylococcus aureus with MIC values of 16 µg/mL. Compound 8 also showed strong antibacterial activity against Bacillus cereus with a MIC value of 16 µg/mL, which is equivalent to that of kanamycin. Compound 3 showed moderate antibacterial activity against Bacillus subtilis with a MIC value of 16 µg/mL. Compound 8 showed strong antibacterial activity against Bacillus subtilis with a MIC value of 8 µg/mL, which is equivalent to that of kanamycin. The other compounds showed either weak (MIC values in the range of 32−128 µg/mL) or no antibacterial activity against the tested bacterial strains.

Conclusions
Nambiscalaranes B-H (1-7), nambiscalarane (8), and aurisin A (9) were isolated from the cultured mycelium of the luminescent mushroom Neonothopanus nambi. The structures of compounds 1-9 were determined by thorough analysis of spectroscopic data (mostly NMR spectra) and ECD data. Compounds 1-8 were tested for anti-proliferative activities against A549, HT29, HeLa, and HCT-116 cancer cell lines. Remarkably, compounds 3, 5, and 7 showed strong cytotoxicity against HCT-116 cell line, with IC 50 values ranging from 13.41 to 16.53 µM, and showed no cytotoxicity towards Vero cells. In addition, compounds 1-8 were tested for antibacterial activities against selected Gram-positive and Gram-negative bacteria, with diverse results. Interestingly, compound 8 inhibited the growth of Bacillus subtilis with a MIC value of 8 µg/mL, which was equivalent to the MIC value of the standard kanamycin.