Malfilanol C, a new sesquiterpenoid isolated from the deep-sea-derived Aspergillus puniceus A2 fungus

Abstract The chemical examination of the rice solid fermentation products of the deep-sea-derived fungus Aspergillus puniceus A2 resulted in the isolation of one new sesquiterpenoid malfilanol C (1), together with a rare analogue malfilanol B (2). The planar structure of 1 was resolved on the basis of the extensive analyses of the spectroscopic data (HRESIMS and NMR spectra), and its absolute configuration was assigned by quantum chemical calculation of the ECD data. Compound 1, featuring a bicyclo[5.4.0]-undecane nucleus skeleton, was the third example of this subclass sesquiterpenoids found from nature. Additionally, the subclass sesquiterpenoids 1 and 2 were discovered from marine-derived-fungi for the first time. All the isolated metabolites were evaluated the antibacterial activities against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922. Compound 1 exhibited weak antibacterial activity against S. aureus ATCC 29213. Graphical Abstract


Introduction
Marine-derived-fungi, inhabiting extreme environments such as high pressure, high salinity, absence light, and low oxygen, etc, have been proved to be promising sources for structurally unique and biologically significant secondary metabolites (Daletos et al. 2018;Carroll et al. 2019;Khalifa et al. 2019;Carroll et al. 2022).More than 5000 marine natural products, including alkaloids, polyketides, and terpenoids, etc, have been reported from the marine-derived-fungi (Rateb and Ebel 2011;Carroll et al. 2020Carroll et al. , 2022;;Chen et al. 2022).Some of the new metabolites displayed a broad range of pharmacological activities, such as cytotoxicity (Liu et al. 2020), anti-inflammatory (Guo et al. 2021), antibacterial (Chi et al. 2020), and antiviral (Wu et al. 2014).Recently, marinederived fungal species Aspergillus puniceus has been attracted considerable attention from natural product chemists and pharmacologists (Liang et al. 2019(Liang et al. , 2021;;Liu et al. 2022).For example, puniceloids C and D, novel quinazolinone alkaloids isolated from deep-sea-derived fungus A. puniceus SCSIO z021, exhibited potent liver X receptor a transcriptional activation with EC 50 values of 1.7 mM, respectively (Liang et al. 2019); and austocystins J-N, five new xanthone-type mycotoxins, showed inhibitory effects against phosphatases and toxicity against brine shrimps or Vero cell (Liang et al. 2021).In our over a long period of time search for new bioactive metabolites from marinederived fungi, the fungus Aspergillus puniceus A2 isolated from the Pacific Ocean deep sea sediment (4841 meters) drawn our attentions.Chemical investigation of the EtOAc extract of fermented cultures led to the isolation of two rare sesquiterpenoids (Figure 1), including one new metabolite namely malfilanol C (1), together with a known analogue malfilanol B (2). Malfilanol C was the third representative of this subclass sesquiterpenoids found from nature, except that only two analogues malfilanols A and B were discovered from fungus Malbranchea filamentosa in 2009 (Wakana et al. 2009).Interestingly, the isolated sesquiterpenoids 1 and 2 were discovered from the Aspergillus genus for the first time.Herein, we report the isolation, structure identification, and antibacterial activity of 1 and 2.

Results and discussion
Compound 1 was purified as a white solid and assigned to have the molecular formula C 15 H 26 O 3 on the basis of the HRESIMS spectrum at ion peak of m/z 277.1778  20.4, 27.3, 33.4, 33.9, 37.0, and 40.7), three methyls (d C 30.4, 31.1, and 32.5), as well as two nonprotonated sp 3 carbons (d C 32.6 and 72.2) (one oxygenated).Compound 1 was deduced to be a bicyclic molecule on the basis of presence of only one carbonyl group to account for one out of three degrees of unsaturation.In the COSY spectrum, two isolated spin systems were assigned to be H 2 -2 (d H 1.  S1).The HMBC interactions from H 3 -12 (d H 1.02) to C-1 (d C 72.2), C-2 (d C 33.9), and C-11 (d C 51.1), as well as from both H 3 -13 (d H 0.78) and H 3 -14 (d H 0.84) to C-3 (d C 33.4)/C-4 (d C 32.6)/C-5 (d C 40.7), in association with the above two spin systems, established the presence of a bicyclo[5.4.0]-undecane nucleus skeleton with one methyl and hydroxy groups at C-1 and two methyl groups at C-4 (Figure S1).Additional HMBC interactions between C-15 (d C 177.8) and H 2 -7, H-8, and H 2 -9, in association with the deshielded exchangeable hydrogen at d H 12.00, revealed the presence of a carboxylic acid group to be attached at C-8 (d C 36.9).Therefore, the gross structure of 1 was elucidated to be a rare sesquiterpenoid featuring a bicyclo[5.4.0]-undecane framework as shown in Figure 1.
The relative configuration of 1 was resolved on the basis of the analysis of NOESY correlations and coupling constants (J).The J H-6/H-11 value (2.9 Hz) of H-6 and H-11 revealed the cis-relationship of both protons and arbitrarily assigned to be a orientation.The deduction was further corroborated by the NOESY cross-peak between H-6 and H-11.Additional NOESY correlations from H-6 to H-3a (d H 1.79), H 3 -14, and 1-OH (d H 3.96), from H-11 to H-7b (d H 1.68) and H-9b (d H 1.31), and from H-8 to H-7b and H-9b indicated that 1-OH, H 3 -14, and H-8 were oriented in the same orientation as H-6 and H-11 (Figure S1).Thus, the relative configuration of 1 was elucidated to be 1S Ã , 6S Ã , 8 R Ã , and 11S Ã , respectively.In order to assign the absolute configuration, we turned to apply quantum chemical ECD calculation by the Gaussian 09 software (Frisch et al. 2009).The experimental ECD data of 1, exhibiting a negative Cotton effect at 218 nm, was similar to that of the calculated ECD curve of (1S,6S,8R,11S)-1 (Figure S2), indicating the S configurations for C-1, C-6, and C-11, while R for C-8.Therefore, the structure of 1 was assigned and given the trivial name malfilanol C.
In addition, one additional rare congener was determined to be malfilanol B (2) (Wakana et al. 2009), on the basis of the comparisons of the 1 H and 13 C NMR spectroscopic data as well as specific rotation data with those reported in the literature.
Two isolated metabolites were tested their antibacterial effects against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 using the paper disk method (Kim et al. 1995).As a result, compound 1 exhibited weak antibacterial activity against S. aureus ATCC 29213 with the 8 mm diameters of the inhibition circles at 200 mg/disc.However, none of the tested compounds displayed inhibitory effects against E. coli ATCC 25922, indicating that 1 had the selective inhibitory activity.

General experimental procedures
Anton Paar MCP 500 automatic polarimeter was used to measure the optical rotation data.Chirascan CD spectrometer was used to record the ECD data.Xevo G2 Q-TOF mass spectrometer was used to record the HRESIMS data.Bruker AV-600 MHz spectrometer was used to measure the 1 D ( 1 H and 13 C) and 2 D (HSQC, COSY, HMBC, and NOESY spectra) NMR data.Chemical shifts (d) were expressed on parts per million (ppm) scale reference to the solvent signals of DMSO-d 6 at 2.50 and 39.5 ppm for 1 H and 13 C, respectively.Coupling constants (J) were expressed in Hz.Semipreparative HPLC was performed by Alltech LS class pump equipped with UV/Vis detector, and Cosmosil packed column (5 lm, ODS-A, 250 Â 10 mm) was used to isolation.Column chromatography (CC) was carried out on the basis of silica gel, ODS, and Sephadex LH-20, respectively.The TLC analysis was carried out by the precoated GF-254 silica gel plates.All solvents used for CC were analytical grade.

Fungal material and identification
The fungal material Aspergillus puniceus A2 was isolated from deep sea sediment (4841 meters) sampling in the Pacific Ocean.The producing fungus was identified to be species A. puniceus on the basis of the internal transcribed spacer (ITS) gene sequence, which showed 99.83% homology to those of the A. puniceus SRRC 2155.The ITS sequence information was submitted to the GenBank and assigned the accession no.OM063154.The voucher specimen is deposited in the Technology Innovation Center for Exploitation of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen, China.

Fermentation, isolation, and purification
The producing fungus was cultivated on potato dextrose agar (PDA) medium at 28 C for 4 days.The fresh mycelia and spores were inoculated into 300 mL potato dextrose broth (PDB) in 500 mL Erlenmeyer flasks and incubated on a rotary shaker (25 C, 100 rpm) for 3 days to prepare seed cultures.Fermentation was carried out on sterile rice solid medium (sterilization at 121 C for 25 min) in 1 L Erlenmeyer flasks (each containing 130 g of rice, 170 mL of H 2 O, and 5.6 g of sea salt) after inoculating the seed cultures.After static incubation at 25 C for 50 days, the fermented solid mash was extracted four times with ethyl acetate (EtOAc), and the combined EtOAc extracts were evaporated under reduced pressure to obtain dark oily extracts.Then the organic extracts were subjected to silica gel column CC by eluting with a stepwise gradient of petroleum ether (PE) and EtOAc (1:0 $ 3:1) to produce five fractions (A -E).Fraction B was fractionated on the basis of ODS CC eluting with MeOH/H 2 O (from 9:11 to 1:0) to yield 9 subfractions (B1 -B9).Subfraction B5 was further purified by the CC over silica gel eluting with PE/EtOAc (10:1 À 2:1) gradient elution and then with CH 2 Cl 2 /MeOH (150:1 À 50:1) to furnish 1 (24.0 mg).Subfraction B7 was chromatographed by silica gel CC (PE/EtOAc, 3:1) and then further isolated with CH 2 Cl 2 /Me 2 CO (50:1) to yield 6 subfractions (B7-1 -B7-6).Subfraction B7-2 was subjected to semipreparative HPLC with a mobile phase of CH 3 CN/H 2 O (7:3) to get 2 (9.5 mg).

Antibacterial test
Antibacterial activity was measured against Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 by the paper disk method.In brief, the test strains were inoculated into LB medium and were cultured in a rotary shaker at 37 C for 14 h.The cultures were diluted with sterile LB medium to achieve an optical absorbance of 0.1-0.5 at 600 nm, and then 300 mL bacterial fluid was spread evenly on LB agar plates by a glass rod spreader.The plates were remained in clean bench for 30 min to allow the agar surface dryness.Sterilized filter paper disks (6 mm in diameter) were placed on the plates, and a 5 mL of tested compounds (40 mg/mL) were added on the disks.Chloroamphenicol and DMSO were used as positive and negative controls, respectively.After 37 C incubation for 18 h, the diameter of the clear zones were recorded using a metric ruler.

ECD calculation of compound 1
Conformational search of (1 R,6R,8S,11R)-1 was carried out by the Maestro 10.2 software with the OPLS3 molecular mechanics force field in an energy window of 5.0 kcal/ mol.The acquired 48 conformers were further optimized by the 6-31 þ G(d,p) level in gas phase using Gaussian 09 software.The conformers with the Boltzmann population over 1% were chosen for the ECD calculations.The theoretically calculated ECD data was performed by the time-dependent density functional theory (TDDFT) method at the B3LYP/6-311 þ G(2d, p) level with the CPCM model in methanol.The calculated ECD data were simulated by the SpecDis software (Bruhn et al. 2013), and the finally calculated ECD data were weighted and summed up of each stable conformers according to the Boltzmann population.The theoretical ECD spectrum of corresponding enantiomer was furnished by directly inverting the ECD spectrum of (1 R,6R,8S,11R)-1.

Conclusions
One new sesquiterpenoid malfilanol C (1) as well as a rare analogue malfilanol B (2) were isolated from the fermented broths of the deep-sea-derived fungus Aspergillus puniceus A2.The structure of malfilanol C was resolved on the basis of extensive analyses of the spectroscopic data (HRESIMS and NMR), in addition to the experimental and calculated ECD spectra for the assignment of the absolute configuration.Malfilanol C was the third example of this subclass sesquiterpenoids found from nature.Additionally, the subclass sesquiterpenoids 1 and 2 were first discovered from marine-derived-fungi, which provides additional evidence to support deep-sea-derived fungi as a new source of the structurally fascinating compounds.Additionally, malfilanol C exhibited weak antibacterial activity against S. aureus ATCC 29213.