New Eremophilane-Type Sesquiterpenes from the Marine Sediment-Derived Fungus Emericellopsis maritima BC17 and Their Cytotoxic and Antimicrobial Activities

The fungal strain BC17 was isolated from sediments collected in the intertidal zone of the inner Bay of Cadiz and characterized as Emericellopsis maritima. On the basis of the one strain–many compounds (OSMAC) approach, four new eremophilane-type sesquiterpenes (1–4), together with thirteen known derivatives (5–17) and two reported diketopiperazines (18, 19), were isolated from this strain. The chemical structures and absolute configurations of the new compounds were determined through extensive NMR and HRESIMS spectroscopic studies and ECD calculation. Thirteen of the isolated eremophilanes were examined for cytotoxic and antimicrobial activities. PR toxin (16) exhibited cytotoxic activity against HepG2, MCF-7, A549, A2058, and Mia PaCa-2 human cancer cell lines with IC50 values ranging from 3.75 to 33.44 µM. (+)-Aristolochene (10) exhibited selective activity against the fungal strains Aspergillus fumigatus ATCC46645 and Candida albicans ATCC64124 at 471 µM.


Introduction
Marine fungi are recognized as rich producers of structurally interesting and biologically active natural products [1,2].Recently, the sequence data of fungal genomes have demonstrated the presence of cryptic biosynthetic pathways, which are not always expressed under standard laboratory conditions [3].As a result, modification of growth conditions may be of great value for the discovery of novel and useful bioactive metabolites.
The one strain-many compounds (OSMAC) approach, conceptualized by Bode et al. [4], has been successfully applied to increase the chemical diversity and yield of new natural products from a single microbial strain.According to this approach, each microbial strain is cultured in a variety of media and/or under different culture conditions to induce the production of cryptic metabolites [4,5].For example, cultivation of the marine-derived fungus Penicillium adametzioides AS-53 on rice medium produced acorane sesquiterpenes, while growth on potato dextrose broth (PDB) gave rise to a new spiroquinazoline derivative, N-formyllapatin A, along with two new bisthiodiketopiperazine derivatives, adametizines A and B [6,7].Recently, nine meroterpenoids, peniciacetal A-I, and five analogues were isolated from the mangrove endophytic fungus Penicillium sp.HLLG-122 by altering the composition of the culture medium according to the OSMAC approach [8].
As part of our ongoing research to search for new bioactive compounds from marinederived fungi, this work includes the isolation of the fungal strain BC17 from sediment samples from the intertidal zone of the inner Bay of Cadiz (Cádiz, Spain) and its identification as Emericellopsis maritima.In recent studies, we evaluated the antitumor and antioxidant activity of fractions derived from the extract of the culture of this strain in rice medium.Three of these fractions displayed potent cytotoxic activity against two colorectal cancer (CRC) cell lines, T84, and SW480, with no activity in the non-tumour line.In addition, one of them demonstrated a strong antioxidant capacity, suggesting a potential role of marine fungi in combating oxidative stress, a factor contributing to CRC development and progression [9].
Neighbour-joining phylogenetic analysis was conducted using the Kimura two-parameter model and a bootstrap test with 5000 runs (MegAlign, DNASTAR ® Lasergene package).Sequences of related fungal species/genera were downloaded from the GenBank database, from the class Hypocreales, to which Emericellopsis belongs.The phylogenetic tree shown in Figure 1 was constructed using thirty-eight sequences, including twelve genera and twentyfour species, and the ribosomal DNA region comprising the intergenic spaces ITS1 and ITS2, including the 5.8S rRNA.Strain BC17 is clearly grouped with the species E. maritima.The phylogenetic tree shown in Figure 2 was constructed using thirty-nine sequences, including seven genera and twenty-seven species, of the β-tubulin (tub2) partial gene.Amplification and subsequent partial sequencing of the 28S rRNA (LSU) gene was also carried out, but this region does not allow resolution at the species level within the Emericellopsis clade.Based on all these studies, it was determined that isolate BC17 is clearly grouped with the species E. maritima (Figures 1 and 2).
Based on the OSMAC approach, a systematic manipulation of nutritional factors, altering cultivation parameters and media composition was carried out to induce the expression of silent biosynthetic genes and the production of cryptic metabolites by the marine-derived fungus E. maritima BC17.For this purpose, the strain BC17 was cultivated on different solid culture media [malt agar (MA), potato dextrose agar (PDA), and rice medium] and incubated for 28 days.Broths were extracted with ethyl acetate and the extracts were fractionated through column chromatography.Final HPLC purification of the fractions of interest from the three extracts led to four new eremophilane-type sesquiterpenes (1-4), thirteen known eremophilane derivatives (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and two known diketopiperazines (18,19)-all isolated for the first time from the marine-derived fungus E. maritima.Specifically, the MA fermentation broth yielded compounds 1-11 and 13-19; the PDA fermentation broth, compounds 8, 10, and 12; and the rice medium fermentation broth, compounds 5-6 and 8 (Figure 3).Therefore, the alteration of the fermentation medium affected the chemical profile of this fungus, with the MA fermentation broth showing the highest chemodiversity.
Compound 1 was isolated as a white solid with a molecular formula C 15 H 20 O 2 , according to the ion peak at m/z 233.1557 [M+H] + (calcd.for C 15 H 21 O 2 , 233.1542) observed in its HRESIMS.Its 1 H NMR and 13 C NMR data (Tables 1 and 2) showed a characteristic pattern of signals of an eremophilane-type sesquiterpene, resembling the structures of compounds 5-17.The 13 C NMR spectrum of 1 (Figure S2) exhibited the presence of four methines, two of them bounded to oxygen, two methylenes, four methyl groups, and five quaternary carbons, one of which corresponded to a carbonyl group (δ C 190.6 ppm) (Table 2).The 1 H NMR spectrum (Figure S1) showed, in addition to the signals for four methyl groups at δ H 0.90 (3H, d, J = 6.7 Hz), 1.01 (3H, d, J = 0.9 Hz), 1.84 (3H, d, J = 1.9 Hz), and 2.13 (3H, d, J = 1.9 Hz), the signal characteristic of one proton on a trisubstituted double bond at δ H 6.17 (1H, s, H-9) (Table 1).The spectrum of 1 was very similar to that of compound 8 [15] except for the shielding of the signal for H-1 from δ H 4.33 to 3.38 ppm and the presence of a signal at δ H 3.52 ppm.These signals, which are coupled to each other, were assigned to the protons of an oxirane ring due to the absence, in the IR spectrum, of the band characteristic of free hydroxyl groups in the 3200-3600 cm −1 region.The HMBC correlations from H-1 to C-2, C-5, C-9, and C-10 and from H-2 to C-3 and C-4 located the oxirane ring between C-1 and C-2 (Figure S5).Consequently, the structure for compound 1 is proposed as 1-epoxyeremophil-7(11),9-dien-8-one.The relative configuration of 1 was elucidated on the basis of correlations observed in the 1D NOESY experiments (Figure S6a-g) and comparisons with those described for previously reported natural eremophilanes [11][12][13][14][15][16].Compounds 5-9 showed β-orientations for H 3 -14 and H 3 -15.Since compounds 1 and 5-9 were cometabolites, 1 should have identical orientations of methyl groups to those of 5-9.NOESY correlations observed for H-1α/H-9, H-2α/H-4α, and H-4α/H-6α indicated that these protons were on the same side of the ring, whereas the correlations between H 3 -15β/H-3β, H 3 -15β/H-6β, H 3 -15β/H 3 -14β, and H 3 -14β/H-6β were used to place these protons on the opposite face of the skeleton (Figure 4).All of these observations implied that the methyl groups H 3 -14 and H 3 -15 and the 1,2-epoxy ring are β-oriented.13 22.5, CH3 The relative configuration of 1 was elucidated on the basis of correlations observed in the 1D NOESY experiments (Figure S6a-g) and comparisons with those described for previously reported natural eremophilanes [11][12][13][14][15][16].Compounds 5-9 showed β-orientations for H3-14 and H3-15.Since compounds 1 and 5-9 were cometabolites, 1 should have identical orientations of methyl groups to those of 5-9.NOESY correlations observed for H-1α/H-9, H-2α/H-4α, and H-4α/H-6α indicated that these protons were on the same side of the ring, whereas the correlations between H3-15β/H-3β, H3-15β/H-6β, H3-15β/H3-14β, and H3-14β/H-6β were used to place these protons on the opposite face of the skeleton (Figure 4).All of these observations implied that the methyl groups H3-14 and H3-15 and the 1,2-epoxy ring are β-oriented.Absolute stereochemistry was established by comparing the experimental electronic circular dichroism (ECD) spectrum of compound 1 with the ECD spectrum predicted from quantum mechanical time-dependent density functional theory (TD-DFT) calculations [29].In the 200-400 nm region, the theoretically calculated ECD spectrum of 1 was in good agreement with the experimental ECD spectrum (Figure 5).Consequently, the structure for compound 1 was assigned as (1S,2R,4S,5R)-1-epoxyeremophil-7(11),9-dien-8-one.Absolute stereochemistry was established by comparing the experimental electronic circular dichroism (ECD) spectrum of compound 1 with the ECD spectrum predicted from quantum mechanical time-dependent density functional theory (TD-DFT) calculations [29].In the 200-400 nm region, the theoretically calculated ECD spectrum of 1 was in good agreement with the experimental ECD spectrum (Figure 5).Consequently, the structure for compound 1 was assigned as (1S,2R,4S,5R)-1-epoxyeremophil-7(11),9-dien-8-one.
Compound 2 was isolated as a yellow oil whose molecular formula was established as The pattern of signals in its 1 H NMR and 13 C NMR spectra (Figures S8 and S9) revealed the presence of two carbonyl groups (δ C 208.0 and 190.9 ppm), two double bonds (δ C/H 129.1/6.09,161.9, 146.9, and 126.6), and four methyl groups (δ C/H 23.0/2.16,22.7/1.89,20.2/1.11, and 7.4/1.12).Its spectroscopic data (Tables 1 and 2) were in accord with a structure similar to that proposed for isopetasone (5).The main difference between the 1 H NMR spectrum of 2 and that of the previously described eremophilene 5 was the presence of a geminal proton to a hydroxyl group at δ H 4.83 (q), which correlated to a methine group at δ C 73.2 in the HSQC experiment.The hydroxyl group was located at C-1 on the basis of the gHMBC correlations (Figure S12) of the protons H-9, H-2α, and H-2β to C-1.Analysis of gCOSY, gHSQC, and gHMBC spectra led to the assignment of all the proton and carbon signals, confirming the structure assigned to compound 2. Compound 2 was isolated as a yellow oil whose molecular formula was established as C15H20O3 through HRESIMS (m/z 247.1342 [M-H] -, calcd.for C15H19O3, 247.1334).The pattern of signals in its 1 H NMR and 13 C NMR spectra (Figures S8 and S9) revealed the presence of two carbonyl groups (δC 208.0 and 190.9 ppm), two double bonds (δC/H 129.1/6.09,161.9, 146.9, and 126.6), and four methyl groups (δC/H 23.0/2.16,22.7/1.89,20.2/1.11, and 7.4/1.12).Its spectroscopic data (Tables 1 and 2) were in accord with a structure similar to that proposed for isopetasone (5).The main difference between the 1 H NMR spectrum of 2 and that of the previously described eremophilene 5 was the presence of a geminal proton to a hydroxyl group at δH 4.83 (q), which correlated to a methine group at δC 73.2 in the HSQC experiment.The hydroxyl group was located at C-1 on the basis of the gHMBC correlations (Figure S12) of the protons H-9, H-2α, and H-2β to C-1.Analysis of gCOSY, gHSQC, and gHMBC spectra led to the assignment of all the proton and carbon signals, confirming the structure assigned to compound 2.
The relative configuration of the molecule was determined from the 1D and 2D NO-ESY experiments (Figures S13 and S14a-f) and comparisons with those described for the previously reported eremophilanes [11,12].Thus, the correlations of H-1α with H-9 and H-4α, H-2α with H-4α, and H-4α with H-6α indicated that these protons were on the same face of the molecule.On the other face were H-2β, H-6β, and methyl groups H3-14 and H3-15, as supported by the NOESY correlations between H-2β and H3-14β, and H-6β and H3-15β (Figure 4).
Its absolute configuration was studied using Mosher's method and through a comparison of the calculated ECD spectrum with the experimental one.Compound 2 was treated with (R)-α-methoxy phenyl acetic acid (R(−)-MPA) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).Unfortunately, the MPA ester could not be achieved from the hydroxyl group at C-1.Instead, the elimination product 9 was obtained, confirming the β-orientation of the methyl groups H3-14 and H3-15.The ECD curve for the (1R,4R,5R) stereoisomer, calculated with the TD-DFT theoretical method, matched well The relative configuration of the molecule was determined from the 1D and 2D NOESY experiments (Figures S13 and S14a-f) and comparisons with those described for the previously reported eremophilanes [11,12].Thus, the correlations of H-1α with H-9 and H-4α, H-2α with H-4α, and H-4α with H-6α indicated that these protons were on the same face of the molecule.On the other face were H-2β, H-6β, and methyl groups H 3 -14 and H 3 -15, as supported by the NOESY correlations between H-2β and H 3 -14β, and H-6β and H 3 -15β (Figure 4).
Compound 3 was isolated as an amorphous solid.The molecular formula was established as C The relative configuration of 3 was elucidated on the basis of correlations observed in the 1D and 2D NOESY experiments (Figures S21 and S22a-f) and comparisons with those described for previously reported natural eremophilanes [14].As depicted in Figure 4, the NOESY correlations observed for H-9/H-1α, H-3α/H-1α, and H-4α/H-2α suggested that these protons are α-oriented, whereas the correlations of H-1β with H 3 -14β and H 3 -15β with H 3 -14β placed these protons on the β face of the skeleton.The 7,11-epoxy ring was assigned β-oriented on the basis of the NOESY cross-peak between H-4α and H 3 -12.
This compound possesses an α,β-unsaturated ketone chromophore.As a result, its absolute configuration could be determined from the Cotton effects observed in its ECD.The positive Cotton effect at 332 nm and the negative Cotton effects at 223 and 247 nm observed in the experimental ECD spectrum of 3 were consistent with those observed in the spectrum predicted from TD-DFT calculations (Figure 5).Accordingly, the structure of compound 3 was assigned as (3S,4R,5R,7R)-3-acetoxy-7(11)-epoxyeremophil-9-en-8-one.
Compound 4 was assigned a molecular formula of C 15 H 28 O 2 on the basis of the observed sodium adduct ion in its HRESIMS (m/z 263.1994 [M+Na] + , calcd.for C 15 H 28 O 2 Na, 263.1987), which was consistent with two degrees of unsaturation.The IR spectrum exhibited an absorption band at 3394 cm −1 , characteristic of hydroxyl groups.The 13 C NMR spectrum (Figure S25) revealed the presence of 15 carbon resonances encompassing four methyl groups, six methylene groups, two methine groups, and three non-protonated carbons, two of which bore an oxygen function (Table 2).The 1 H NMR spectrum showed four singlet methyl groups at δ H 1.17 (H 3 -12), 1.16 (H 3 -13), 1.08 (H 3 -15), and 0.88 (H 3 -14) (Table 1), characteristic of a C-4 and C-11 tetrasubstituted eremophilane derivative.The decaline unit was deduced through a comprehensive analysis of its 2D NMR spectra.Selective 1D TOCSY spectra (Figure S30a-d The relative configuration of 4 was deduced from the 1D NOESY spectra (Figure S29a-e) and that described for the previously reported eremophilanes [25].As depicted in Figure 4, the NOESY correlations observed between H 3 -14/H 3 -15, H 3 -14/H-6β, H 3 -14/H-7β, H 3 -14/H-9β, H-1β/H 3 -15, and H-9β/H 3 -15 indicated that the methyl groups at C-4 and C-5 were cofacial and β-oriented.The configuration at C-7 was assigned as S based on NOESY correlations between H 3 -14β and H-7β, and between H-9β and H-7β.On the other hand, H-10 showed NOESY correlations with H-6α and H-8α.Based on these correlations and the α-orientation of H-10 in its cometabolite 17 [27], the trans junction of the decaline ring was established.These assignments were consistent with the absolute configurations of the reported eremophilanes and allowed compound 4 to be proposed as (4R,5S,7S,10S)-eremophilane-4,11-diol.

Fungal Material and Identification
The marine-derived fungus E. maritima BC17 was isolated from intertidal sediments collected in the inner Bay of Cadiz (Cádiz, Spain) within a Spartina spp.bed with the permission of the national competent authority (ABSCH-CNA-ES-240784-3, reference number ESNC84).Surface sediment samples were collected aseptically in the field, stored in sterile packaging, kept on ice, brought to the laboratory, and immediately processed.Sediment was diluted with sterile seawater water (SSW) and aliquots were grown on PDA plates and marine agar plates (Condalab S.L.) and incubated at 25 • C for 5-10 days.Fungal colonies were selected and streaked on PDA plates under axenic conditions.The isolates were maintained on PDA at 25 • C for routine experiments and spores were stored in 60% (v/v) glycerol at −20 • C for later studies.
The BC17 fungal strain isolated was identified as E. maritima using the service of the Spanish Type Culture Collection (CECT, https://www.uv.es/cect, accessed on 5 December 2023) based on both phenotypic and molecular techniques.Three regions of the fungal genome were amplified through conventional PCR: (a) amplification and sequencing (with readings in both directions) of the ribosomal DNA region comprising the intergenic spaces ITS1 and ITS2, including the 5.8S rRNA (ITS5 5 -GGAAGTAAAAGTCGTAACAAGG-3 ; ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ); (b) partial amplification and sequencing (with readings in both directions) of the 28S rRNA gene (LR0R 5 -GTACCCGCTGAACTTAAGC-3 ; LR7 5 -TACTACCACCAAGATCT-3 ); and (c) partial amplification and sequencing of the β-tubulin gene (with readings in both directions) (Bt2a 5 -GGTAACCAAATCGGTGCTGCT TTC-3 ; Bt2b 5 -ACCCTCAGTGTAGTGACCCTTGGC-3 ).The sequencing of these regions was compared with those in NCBI databases.Sequences were submitted to the NCBI database with the accession number OR815285 for the ITS region; OR835190 for the 28S rRNA gene; and OR832338 for the β-tubulin gene.To study the phylogenetic relationship of our isolate, other sequences of related genera and species from the class Hypocreales were downloaded from the GenBank database and included in the phylogenetic trees.
Culture of E. maritima BC17 has been deposited at the University of Cádiz, Mycological Herbarium Collection (UCA).Spore suspensions of this strain are maintained viable in 80% glycerol at −40 • C.

OSMAC-Based Cultivation Procedures
Using the OSMAC approach, E maritima BC17 was grown on three different solid culture media: MA (20 g glucose, 20 g malt extract, 20 g agar, and 1 g peptone, per litre of water, pH 6.5−7), PDA (Condalab, Madrid, Spain), and rice medium (80 g white rice per 100 mL of water).All the media were Rice medium ingredients were soaked overnight before autoclaving.
For MA and PDA media, strain BC17 was grown on Petri dishes, each containing 100 mL of the corresponding medium.Each plate was inoculated with one mycelium plug of 0.9 cm diameter from a seven-day culture on MA or PDA, and then incubated for 28 days at 24-26 • C under continuous white light (daylight lamp) for metabolite production.
Rice medium was also used to culture E. maritima BC17.Sixteen Erlenmeyers flasks (500 mL), containing 100 mL of rice medium, were inoculated with 750 µL of a conidial suspension obtained by adding 15 mL of sterile water to three Petri dishes (9 cm diameter) cultured with E. maritima BC17.The flasks were then incubated for 28 days under the conditions described above for metabolite production.

Extraction, Isolation, and Characterization of Eremophilane-Type Sesquiterpenes
The MA (3.6 L), PDA (3 L), and rice (1 L) fermentation broths were separately sonicated with ethyl acetate (×2) for 15 min at a frequency of 40 kHz using an AU-65 ultrasonic system (Argo Lab, Bogotá, Colombia) and then filtered.Before solid-liquid extraction, the MA and PDA media were cut into small pieces and the rice media was crushed.The organic extracts were dried over dry Na 2 SO 4 and the solvent evaporated at reduced pressure to yield crude extracts of 1.597 g for MA, 0.599 g for PDA, and 4.196 g for rice medium.
The crude extracts were fractionated through silica gel column chromatography using an increasing gradient of ethyl acetate in n-hexane and methanol as eluents.Final purification of the different fractions was carried out using semipreparative HPLC.

Computational Details of ECD Calculations
The conformational analysis of compounds 1-3 was conducted through the application of the semiempirical PM6 method [34].Quantum mechanical computations were executed utilizing the Gaussian 16 package [35].A comprehensive geometric optimization was undertaken emploing density functional theory (DFT) within the framework of B3LYP functionals [36,37] and the 6−311+G(2d,p) basis set.Subsequently, calculations were performed to determine the energies, oscillator strengths, and rotational strengths associated with the initial 20 electronic excitations, employing the TD-DFT methodology [38,39].The solvent's influence (methanol) was considered within the calculations, incorporating the polarizable continuum model (PCM) with the implementation of the implicit solvation energy (IEF) approach [40][41][42].To mimic the ECD spectrum of the conformer, a Gaussian function was used, featuring a half-bandwidth of 0.33 eV.

In Vitro Antimicrobial Assay
The antimicrobial activities of compounds (1, 3, 5-6, 8-11, 13-17) were evaluated against six bacterial and two fungal human pathogens.Antibacterial susceptibility of the compounds was tested against A. baumannii ATCC19606, P. aeruginosa PAO-1, K. pneumoniae ATCC700603, E. coli ATCC25922, methicillin-resistant S. aureus MB5393, and sensitive S. aureus ATCC29213, while antifungal activity was tested against C. albicans ATCC64124 and A. fumigatus ATCC46645 following previously described methodologies [43].Briefly, each compound was serially diluted in 100% DMSO with a dilution factor of 2 to provide 10 final assay concentrations in all antimicrobial assays.The MIC was defined as the lowest concentration of the compound that inhibited ≥90% of the growth of a microorganism after overnight incubation.Genedata Screener software, version 18.0.4-Standard(Genedata, Inc., Basel, Switzerland) was used to process and analyze the data and to calculate the RZ factor, which predicts the robustness of an assay [43].In all experiments performed in this work, the RZ factor obtained was between 0.89 and 0.98.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra for compounds 1-3.Calculations were performed with the conformers shown in Figure 4.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra for compounds 1-3.Calculations were performed with the conformers shown in Figure 4.
17 H 24 O 4 on the basis of an HRESIMS peak at m/z 315.1546 [M+Na] + (calcd.for C 17 H 24 O 4 Na, 315.1572), consistent with six degrees of unsaturation.Its 1 H NMR and 13 C NMR spectra (Figures S16 and S17) also displayed characteristic resonances of an eremophilane-type sesquiterpene.In particular, the 13 C NMR spectrum of compound 3 showed signals in close agreement with the known eremophilane derivative 7 [14], except for the lack of the characteristic signals of the ∆ 7(11) double bond [δ C 143.6 (C-11) and 127.6 (C-7) in 7].Instead, two quaternary carbons at δ C 64.8 and 65.4 ppm (Table 2) attached to an oxygen function were observed, pointing to the presence of a 7,11-epoxy.The position of the oxirane ring was confirmed by the correlations observed in the gHMBC experiment (Figure S20) from H-6α/H-6β to C-4, C-7, C-8, C-10, and C-14; H-9 to C-1, C-5 and C-7; H 3 -12 to C-7 and C-13; and from H 3 -13 to C-7, C-11, and C-12.As a result, the structure for compound 3 was proposed as 3-acetoxy-7(11)-epoxyeremophil-9-en-8-one.