New Cyclopiane Diterpenes and Polyketide Derivatives from Marine Sediment-Derived Fungus Penicillium antarcticum KMM 4670 and Their Biological Activities

Two new cyclopiane diterpenes and a new cladosporin precursor, together with four known related compounds, were isolated from the marine sediment-derived fungus Penicillium antarcticum KMM 4670, which was re-identified based on phylogenetic inference from ITS, BenA, CaM, and RPB2 gene regions. The absolute stereostructures of the isolated cyclopianes were determined using modified Mosher’s method and quantum chemical calculations of the ECD spectra. The isolation from the natural source of two biosynthetic precursors of cladosporin from a natural source has been reported for the first time. The antimicrobial activities of the isolated compounds against Staphylococcus aureus, Escherichia coli, and Candida albicans as well as the inhibition of staphylococcal sortase A activity were investigated. Moreover, the cytotoxicity of the compounds to mammalian cardiomyocytes H9c2 was studied. As a result, new cyclopiane diterpene 13-epi-conidiogenone F was found to be a sortase A inhibitor and a promising anti-staphylococcal agent.


Introduction
Marine fungi are a significant source of compounds with antimicrobial properties [1], among which sediment-derived fungi can be one of the promising sources of novel secondary metabolites [2].
The section Canescentia of the genus Penicillium, besides species P. canescens and P. janczewskii, includes such widespread species as P. antarcticum, P. atrovenetum, and P. novae-zeelandiae which are predominantly isolated from soil and leaf litter [3].Difficulties in using morphological characters to identify strains make identification of this group continually problematic and induce a re-evaluation using phylogenetic data.Earlier Penicillium attenuatum KMM 4671, P. ochotense KMM 4670, and P. piltunense KMM 4668 were isolated from the Sea of Okhotsk and reported as new species [3], but recently Visagie and co-authors reduced these fungi to synonymy with P. antarcticum from series Atroveneta [4].
The assumption about several frameshift mutations in coding regions at sequence ends, which may imply low-quality sequence reads, needs to be verified.Thus, the new thorough investigation confirmed earlier conclusions that the strain KMM 4670 should be identified as Penicillium antarcticum.
The absolute stereostructure of 1 was established as 4R,5S,6S,9R,11S,15S based on the comparison of experimental and calculated ECD spectra (Figure 5).Compound 1 was named 4-hydroxyleptosphin C.  The absolute stereostructure of 1 was established as 4R,5S,6S,9R,11S,15S based on the comparison of experimental and calculated ECD spectra (Figure 5).Compound 1 was named 4-hydroxyleptosphin C. The molecular formula of 2 was determined as C20H30O2 based on the (+)-HRESIMS spectrum (Figure S39) containing the peak of the cationized molecule [M+Na] + (m/z 325.2142) and was confirmed by the 13 C NMR data.The main features of 1 H and 13 C NMR spectra (Table 1, Figures S3 and S4) were close to those of known cyclopiane conidiogenone F (3) [19] with significant differences in some signals    The absolute stereostructure of 1 was established as 4R,5S,6S,9R,11S,15S based on the comparison of experimental and calculated ECD spectra (Figure 5).Compound 1 was named 4-hydroxyleptosphin C. The molecular formula of 2 was determined as C20H30O2 based on the (+)-HRESIMS spectrum (Figure S39) containing the peak of the cationized molecule [M+Na] + (m/z 325.2142) and was confirmed by the 13 C NMR data.The main features of 1 H and 13 C NMR spectra (Table 1, Figures S3 and S4) were close to those of known cyclopiane conidiogenone F (3) [19] with significant differences in some signals  The molecular formula of 2 was determined as C 20 H 30 O 2 based on the (+)-HRESIMS spectrum (Figure S39) containing the peak of the cationized molecule [M+Na] + (m/z 325.2142) and was confirmed by the 13 C NMR data.The main features of 1 H and 13 C NMR spectra (Table 1, Figures S3 and S4) were close to those of known cyclopiane conidiogenone F (3) [19] with significant differences in some signals.The HMBC correlations (Figures 3 and S12) confirmed a planar structure of 2 to be the same as conidiogenone F (3).
The ROESY correlations (Figure 4 and Figure S14) H-  The absolute configurations of the chiral centers of 2 could not be determined with a modified Mosher's method [23] due to an insufficient amount of this compound.Therefore, this method was used for compound 3.The esterification of 3 with (S)-and (R)-MTPA chloride occurred at the C-13 hydroxy group to yield the (R)-and (S)-MTPA esters 3a and 3b, respectively.The observed chemical shift differences ∆δ(δ S -δ R ) (Figures 6 and S45-S48) indicated the 13S configuration, and therefore, the absolute configurations other chiral centers of 3 were established as 4S,5S,6S,9R,11S,15S.Due to the identity of relative stereochemistry in the diterpene core, the absolute configurations of 2 were determined as 4S,5S,6S,9R,11S,13R,15S. Compound 2 was named 13-epi-conidiogenone F.
To establish absolute stereo configurations, an attempt was made to apply a modified Mosher's method.Unfortunately, in the preparation of MTPA esters at 3-OH, compound 5 was destroyed.Nevertheless, the absolute configurations of all stereocenters in 5 were suggested based on obvious biogenetic relationships between 5, 6, and 7 [17].
To the best of our knowledge, the established structure of compound 5 was new, and this compound was named antaketide A.

The Influence on S. aureus, E. coli, and C. albicans
The influence of compounds 1-6 on the growth and biofilm formation of S. aureus, E. coli, and C. albicans was investigated.Cladosporin (7) is a naturally occurring fungal metabolite with potent antibacterial, antifungal, insecticidal, and anti-inflammatory activities as well as plant growth regulatory effects [16].Therefore, its biological activity in the present work was not studied.
The effects of compounds 1-6 on the growth and biofilm formation of Gram-positive bacteria Staphylococcus aureus are presented in Figure 8.To the best of our knowledge, the established structure of compound 5 was new, and this compound was named antaketide A. The influence of compounds 1-6 on the growth and biofilm formation of S. aureus, E. coli, and C. albicans was investigated.Cladosporin (7) is a naturally occurring fungal metabolite with potent antibacterial, antifungal, insecticidal, and anti-inflammatory activities as well as plant growth regulatory effects [16].Therefore, its biological activity in the present work was not studied.
The effects of compounds 1-6 on the growth and biofilm formation of Gram-positive bacteria Staphylococcus aureus are presented in Figure 8.The NOESY correlations (Figure S25) between H-5 and H3-10 (δH 1.23) revealed the same relative stereostructure of the tetrahydropyrane moiety of 5.
To establish absolute stereo configurations, an attempt was made to apply a modified Mosher's method.Unfortunately, in the preparation of MTPA esters at 3-OH, compound 5 was destroyed.Nevertheless, the absolute configurations of all stereocenters in 5 were suggested based on obvious biogenetic relationships between 5, 6, and 7 [17].
To the best of our knowledge, the established structure of compound 5 was new, and this compound was named antaketide A.

The Influence on S. aureus, E. coli, and C. albicans
The influence of compounds 1-6 on the growth and biofilm formation of S. aureus, E. coli, and C. albicans was investigated.Cladosporin (7) is a naturally occurring fungal metabolite with potent antibacterial, antifungal, insecticidal, and anti-inflammatory activities as well as plant growth regulatory effects [16].Therefore, its biological activity in the present work was not studied.
The effects of compounds 1-6 on the growth and biofilm formation of Gram-positive bacteria Staphylococcus aureus are presented in Figure 8.  4-Hydroxyleptosphin C (1), at concentrations of 12.5 µM and 100 µM, inhibited S. aureus growth by 15.3% and 29.3%, respectively.The prevention of the biofilm formation by 15.9% and 34.5% was observed when the concentrations of 1 were 12.5 µM and 100 µM, respectively.
13-epi-conidiogenone F (2) showed a weak inhibition of S. aureus growth by 19.1% at 100 µM and no effect at 12.5 µM, but this one significantly prevented S. aureus biofilm formation from 37.9% at 12.5 µM to 52.6% at 100 µM.The half-maximal concentration (IC 50 ) of the biofilm formation inhibition was calculated as 76.1 µM.
Conidiogenone F (3) inhibited S. aureus growth by 17.6% when it was used at 100 µM.Antaketide A ( 5) and tetraketide derivative 6 showed similar inhibition effects on S. aureus growth and biofilm formation.Antaketide A (5) inhibited S. aureus growth by 48.5% at 100 µM, and this one did not show any influence on S. aureus growth at 12.5 µM.Compound 6 inhibited S. aureus growth by 46.5% and 25.6% at 100 µM and 12.5 µM, respectively.In addition, 5 and 6 prevented the biofilm formation of S. aureus by nearly 30-40%.
The effects of compounds 1-6 on the growth and biofilm formation of Gram-negative bacteria Escherichia coli are presented in Figure 9.
100 µM and no effect at 12.5 µM, but this one significantly prevented S. aureus biofilm formation from 37.9% at 12.5 µM to 52.6% at 100 µM.The half-maximal concentration (IC50) of the biofilm formation inhibition was calculated as 76.1 µM.
Antaketide A ( 5) and tetraketide derivative 6 showed similar inhibition effects on S. aureus growth and biofilm formation.Antaketide A (5) inhibited S. aureus growth by 48.5% at 100 µM, and this one did not show any influence on S. aureus growth at 12.5 µM.Compound 6 inhibited S. aureus growth by 46.5% and 25.6% at 100 µM and 12.5 µM, respectively.In addition, 5 and 6 prevented the biofilm formation of S. aureus by nearly 30-40%.
The effects of compounds 1-6 on the growth and biofilm formation of Gram-negative bacteria Escherichia coli are presented in Figure 9.All investigated compounds inhibited the growth of E. coli culture but only compounds 4 and 5 affect the biofilm formation of E. coli.4-Hydroxyleptosphin C (1) and 13epi-conidiogenone F (2) at a concentration of 100 µM inhibited E. coli growth by 35.9% and 27.3%, respectively.The growth inhibitory effect of these two compounds was not observed when its concentration was 12.5 µM.
The effects of compounds 1-6 on the growth and biofilm formation of Candida albicans are presented in Figure 10.The effects of compounds 1-6 on the growth and biofilm formation of Candida albicans are presented in Figure 10.

The Influence on Sortase A Activity
Sortase A enzyme is key for biofilm formation and virulence of S. aureus and sortase A' inhibitors can prevent the biofilm formation [24].Therefore, a natural inhibitor of sortase A activity rhodionin decreases the adhesion of S. aureus to fibrinogen via reducing the capacity of protein A on the bacterial surface and biofilm formation without affecting the survival and growth of bacteria [25].
The investigated compounds 1-6 significantly reduced the formation of S. aureus biofilm; therefore, the influence of compounds 1-6 on the activity of sortase A studied in a cell-free test using SensoLyte 520 Sortase A Activity Assay Kit Fluorometric and data are presented in Figure 11.

The Influence on Sortase A Activity
Sortase A enzyme is key for biofilm formation and virulence of S. aureus and sortase A' inhibitors can prevent the biofilm formation [24].Therefore, a natural inhibitor of sortase A activity rhodionin decreases the adhesion of S. aureus to fibrinogen via reducing the capacity of protein A on the bacterial surface and biofilm formation without affecting the survival and growth of bacteria [25].
The investigated compounds 1-6 significantly reduced the formation of S. aureus biofilm; therefore, the influence of compounds 1-6 on the activity of sortase A studied in a cell-free test using SensoLyte 520 Sortase A Activity Assay Kit Fluorometric and data are presented in Figure 11.All compounds at 50 µM statistically significantly inhibited the activity of sortase A (Figure 11a).4-Hydroxyleptosphin C (1) and 13-epi-conidiogenone F (2) inhibited sortase A activity by 28.2% and 36.9%, and this effect continued during all experiments (Figure 11b).Compounds 3-6 inhibited the sortase A activity by 13.1%, 20.0%, 25.5%, and 16.8%, respectively.
13-epi-Conidiogenone F (2) is a stereo isomer of conidiogenone F (3) at C-13, and the β-position of OH-group at C-13 in the structure of 2 is possibly better for interaction with sortase A that caused higher inhibition of sortase A activity.To confirm or refute this assumption as well as determine the contribution of an additional OH-group to the activity of 1, we carried out the molecular docking of compounds 1-4 with sortase A structure (PDB ID 1T2P) using SwissDock [26].
In the apo structure of sortase A (PDB ID 1T2P), a V-shaped pocket is formed by the β4, β7, and β8 strands on one side of the β barrel, together with three surrounding loops.The left side of the pocket is a hydrophobic tunnel formed by Ala92, Ala104, Ala118, Val161, Pro163, Val166, Val 168, Ile182, Val193, Trp194, Ile199, and Val201, along with two putative catalytic residues: Cys184 and Arg197.The right side of the pocket consists of several polar residues: Glu105, Asn114, Ser116, and Thr180 [27].
In our calculations, the most active sortase A inhibitor, 13-epi-conidiogenone F (2), can form a pose (∆G − −7.16274 kcal/mol) with the hydrogen-bonding interaction between Glu105 and its OH-group at C-13, and hydrophobic interaction between the keto-group at C1 and Gly192 as well as another hydrophobic interaction with Ala92, Thr93, Thr187, Trp194, and Ala104 (Table 3).Conidiogenone F (3) can form a pose (∆G − −6.2875967 kcal/mol) with the hydrogenbonding interaction between Arg197 and its OH-group at C-13 and hydrophobic interactions with only Ala104 and Ile182.
Thus, the α-OH-group at C-13 in the structure of 2 allows 13-epi-conidiogenone F (2) to form the interactions with the amino acid residue of sortase A from both sides of diterpene moiety (Figure 12b).At the time, the β-OH-group at C-13 in the structure of 3 allows for the interaction on only one side of the molecule (Figure 12c).The additional hydroxy group in the structure of 1 also allows this compound to contract with 4 forms the interactions with the amino acid residue of sortase A from both sides of the diterpene moiety (Figure 12a,d).
to form the interactions with the amino acid residue of sortase A from both sides of diterpene moiety (Figure 12b).At the time, the β-OH-group at C-13 in the structure of 3 allows for the interaction on only one side of the molecule (Figure 12c).The additional hydroxy group in the structure of 1 also allows this compound to contract with 4 forms the interactions with the amino acid residue of sortase A from both sides of the diterpene moiety (Figure 12a,d).

The Cardiotoxicity of Compounds 1-6
Therefore, secondary metabolites from marine fungus P. antarcticum KMM 4670 as inhibitors of S. aureus sortase A and the biofilm formation can be promising for future investigations, but its toxicity toward mammalian cells should be tested before such conclusions.Cardiovascular toxicity remains a major cause of concern during preclinical and clinical development, as well as contributing to a post-approval withdrawal of medicines [28].Therefore, macrolides erythromycin, azithromycin, and clarithromycin cause QT prolongation, torsades de pointes, and arrhythmia-affected cardiomyocyte mitochondria

The Cardiotoxicity of Compounds 1-6
Therefore, secondary metabolites from marine fungus P. antarcticum KMM 4670 as inhibitors of S. aureus sortase A and the biofilm formation can be promising for future investigations, but its toxicity toward mammalian cells should be tested before such conclusions.Cardiovascular toxicity remains a major cause of concern during preclinical and clinical development, as well as contributing to a post-approval withdrawal of medicines [28].Therefore, macrolides erythromycin, azithromycin, and clarithromycin cause QT prolongation, torsades de pointes, and arrhythmia-affected cardiomyocyte mitochondria in isolated rat heart mitochondria [29].The cardiotoxicity of fluoroquinolones was discussed in the review [30].Thus, the investigation of the cardiotoxicity of leading molecules and drug candidates is actually in the pre-clinical stage.At this time, only three cultivating cardiomyocyte cultures, murine HL-1, rat H9c2, and human AC-16 cell lines, are available.Despite the limitations, the H9c2 cell line is the most used in various experiments [31], including the study of antibiotics' cardiotoxicity [32].
The effect of compounds 1-6 on the viability of H9c2 cardiomyocytes is presented in Table 4. Therefore, polyketides 5 and 6 showed significant toxicity toward normal cardiomyocytes at the concentrations in which they affected the growth of microorganisms indicates their non-specific action against living cells.
Both compounds 1 and 2 caused the H9c2 viability to decrease by nearly 30% at a concentration of 100 µM.Moreover, compound 1 caused the decrease in H9c2 cell viability, near 30%, at a concentration of 10 µM, while compound 2-4 was non-toxic.

Cyclopiane Derivatives from P. antarcticum KMM 4670
Cyclopiane-type diterpenes were discovered 20 years ago, and since then, about 25 related compounds have been described.These compounds are usually produced by a number of fungi of the subgenus Penicillium, except one report of an isolation from a fungus of the genus Leptosphaeria.Structurally, cyclopianes are characterized by a conserved 5/5/5/6 tetracyclic core with methyl groups at C-4, C-9, C-11, and C-14 as well as an oxygenated C-1 atom.The biogenesis of cyclopiane diterpenes was proposed from geranyl geraniol pyrophosphate via hypothetical deoxyconidiogenol [21].Unfortunately, the absolute stereoconfigurations of these compounds were not established in most of the published works, and in a number of other works, the absolute stereochemistry is questionable.This has led to high confusion about stereostructures of cyclopianes.This study is the very first proof of absolute stereoconfigurations of known conidiogenone F (3) and leptosphin C; (4) using a modified Mosher's method.Despite the identity of the ECD data of compound 3 with conidiogenone F, the absolute stereostructure of 3 established by us is a mirror structure of that proposed earlier for conidiogenone F [19].It should be noted that, in recent papers [33,34], conidiogenone F was depicted identically to that established by us.However, no evidence for such an absolute stereostructure has been previously published.The absolute stereostructure of leptosphin C (4), determined by us from the obvious biogenetic relationship with conidiogenone F (3), is also enantiomeric in comparison with the previously published [21].
Earlier it was reported that conidiogenone F (3) at 50 µg/mL inhibited bacteria Escherichia coli growth and did not inhibit the growth of Staphylococcus aureus when this one was used at concentrations up to 100 µg/mL [21].Our own experiments confirm that conidiogenone F (3) is more active against Gram-negative E. coli growth.
In the same work, it was reported that leptosphin C (4) showed antibacterial activity against Staphylococcus aureus (100 µg/mL) and Escherichia coli (>100 µg/mL) as well as another bacterial strain [21].In this work, authors used bacterial suspension at a concentration of 10 6 CFU per milliliter and did not use any instrumental equipment for the detection of bacterial growth inhibition.This probably explains why the authors recorded the activity of 3 and 4 at concentrations 166 µM and 333 µM (calculated from 50 and 100 µg/mL) only, whereas in our experiments the activity was detected at significantly lower concentrations.

Polyketides from P. antarcticum KMM 4670
As was shown recently, tetraketide 5 and pentaketide 6 are intermediates in the biosynthesis of cladosporin (7).Thus, 6 is formed from a linear tetraketide precursor by its cyclization, and 5 is the product of the elongation of 6, with one unit of malonyl-CoA.Both compounds are biosynthesized by the highly reducing I type polyketide synthase.Further formation of cladosporin is controlled by another non-reducing I type polyketide synthase [35].It proves the absolute stereostructures of both compounds to be the same with cladosporin (7).It should be noted that 6 was isolated earlier as a product of the synthetic degradation of cladosporin [36], and its epimer was reported as a civet constituent [22].Synthetic N-acetylcysteamine thioester derivative of 5 was used for the in vitro biosynthesis of cladosporin by non-reducing I type polyketide synthase [35].Thus, the current research is the very first report of isolation of cladosporin precursors 5 and 6 as individual natural compounds.
Earlier the antimicrobial activity for 6 was not reported.In our experiments, this one and antaketide A (5) showed the highest inhibition activity against the growth of all used test-strains.However, they significantly inhibited biofilm formation in the S. aureus culture only, and this effect was supported by the inhibition of sortase A activity.However, we observed the cytotoxicity of the cladosporin precursors 5 and 6 to H9c2 cardiomyocytes, which makes these compounds not as promising leading molecules as we would like.

Biological Activity of Cyclopiane Diterpenes
Cyclopiane diterpenes 1-4 have a little difference in their chemical structures, but these differences result in differences in their bioactivity.According to their cytotoxic activity, substances can be arranged in the following order: 1 > 2 > 3 = 4.According to their ability to inhibit the microbial growth, it will be the order: 4 > 1 > 2 = 3 (S.aureus), 3 > 4 > 1 = 2 (E.coli), and 1 = 2 = 3 = 4 (C.albicans), but the differences are not so big.According to their ability to inhibit biofilm formation, it will be the order: Finally, the order of compounds according to their ability to inhibit the sortase A activity is 2 > 1 > 4 > 3.
Obviously, that the presence of an additional OH-group at C-4 in the structure of the compound new cyclopiane diterpene 4-hydroxyleptosphin C (1), in contrast with leptosphin C (4), resulted in its higher cytotoxic and sortase A inhibition activities.
It is believed that antibiotics aimed at the vital functions of bacteria cause the development of resistance much faster.The appearance of penicillin-resistant S. aureus strains was registered in 1949, while penicillin was put into practice in the early 40s.The appearance of methicillin and vancomycin-resistant strains was also not long in coming [37].The identification and analysis of the virulence factors used by pathogens to colonize, invade, and persist within a susceptible host resulted in a new strategy, suggesting that drugs blocking these factors without killing the bacteria create less evolutionary pressure and diminish the chances of resistant genes to emerge.This strategy offers the targeting of the inhibition of the quench pathogen quorum sensing systems or the inhibition of the biofilm formation in the discovery of new antibiotics [38].
Bacteria can survive in two forms: planktonic cells and biofilm, and the formation of biofilm depends on many factors, including the attachment of cell wall components to the cell wall [39].Staphylococcal sortase A (EC 3.4.22.70) is the cysteine transpeptidase and mainly acts as an anchor surface protein [40].About 25 years have passed since the beginning of the study of sortase A inhibitors [41], and during this time, a large amount of experimental data has accumulated [42].Experiments involving laboratory animals confirm that sortase A inhibitors are effective not only in in vitro, but also in in vivo studies [43].For example, flavonol glycoside hibifolin inhibited sortase A activity, decreased the adhesion of S. aureus bacteria to the host cells and the biofilm formation, and, in combination with cefotaxime, protected mice from S. aureus infection-induced pneumonia [44].
The natural compounds of various classes, such as alkaloids, polyketides, quinones, and others, were described as sortase A inhibitors.In our experiments, first, it was shown that cyclopiane diterpenes can affect sortase A activity and biofilm formation.The compounds 13-epi-conidiogenone F (2) and 4-hydroxyleptosphin C (1) showed a more effective inhibition of sortase A activity, but 4-hydroxyleptosphin C (1) had more cardiotoxicity.
3 ), was used.The reaction profile was 95 • C for 300 s; 35 cycles of 95 • C for 20 s, 65 • C for 30 s, and 72 • C for 90 s; and, finally, 72 • C for 300 s.For amplification of the partial RPB2 gene, the degenerate primer pair, rpb2_Pen_F (5 -GAGACYAAYCGBGARATYTA-3 ) and rpb2_Pen_R (5 -GTCATSACAATCATRATDGT-3 ), was used.The reaction profile was an initial denaturation at 95 • C for 300 s, followed by 5 cycles of 95  C for 420 s.The amplified ITS, BenA, CaM, and RPB2 genes were purified with the ExoSAP-IT™ PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA).Sequencing was bidirectionally performed with the same primers on an Applied Biosystems SeqStudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) using the Big Dye Terminator reagent kit, version 3.1.Gene sequences were deposited in GenBank under accession numbers KU358553.2for ITS, KU358556.2for the partial BenA, KU358559.2for the partial CaM, and OR271597 for the partial RPB2 (Table 1).

Phylogenetic Analysis
The ITS region, the partial BenA, CaM, and RPB2 gene sequences of the fungal strain KMM 4670 and other members from the section Canescentia, series Atroveneta, were aligned using MEGA X software version 11.0.9 [47] using Clustal W algorithm.The ex-type homologs were searched in the GenBank database (http://ncbi.nlm.nih.gov,accessed on 8 November 2023) using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/BLAST,accessed on 20 July 2023).The phylogenetic analysis was conducted using MEGA X software [47].The ITS region and partial BenA, CaM, and RPB2 gene sequences were concatenated into one alignment.Phylogenetic tree was constructed according to the Maximum Likelihood (ML) algorithm based on the Tamura-Nei model [48].The tree topology was evaluated with 1000 bootstrap replicates.The Aspergillus glaucus NRRL 116 T strain was used in the phylogenetic analysis as outgroup (Table 5).T -ex-type strain.

Cultivation of Fungus for Metabolite Isolation
The fungal strain KMM 4670 was cultured on a rice medium at 22 • C for three weeks in 100 Erlenmeyer flasks (500 mL), each containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH 2 PO 4 , and 40 mL of natural seawater from the Marine Experimental Station of PIBOC, Troitsa (Trinity) Bay, the Sea of Japan.

Extraction and Isolation
At the end of cultivation, the mycelium of the fungal strain KMM 4670, together with the medium, was homogenized and extracted with EtOAc (1 L).The obtained extract was concentrated in vacuo.The dry residue (57.5 g) was dissolved in H 2 O−EtOH (4:1) (300 mL) and extracted successively with n-hexane (3 × 0.2 L), EtOAc (3 × 0.2 L), and butanol-1 (3 × 0.2 L).The ethyl acetate extract was evaporated to dryness (2.6 g) and chromatographed on a silica gel column (3 × 14 cm), which was first eluted with n-hexane-EtOAc from 100% n-hexane with a stepwise gradient of 10% to 100% EtOAc (total volume 20 L).Fractions of 250 mL were collected and combined based on TLC data.

The Quantum Chemical Calculations
The quantum chemical modeling of geometry and spectroscopic properties of compounds 1 and 4 was performed using Gaussian 16 package of programs [49].Geometry optimizations and calculations IR spectra were performed with B3LYP exchange-correlation functional, the polarization continuum model (PCM), and 6-311G(d) split-valence basis set using default algorithms.
Two important conformations of 1 were found; their statistical weights (g im ) were calculated via Gibbs free energies: where index "m" denotes the most stable conformation and ∆G im = G i − G m are the relative Gibbs free energies.The contributions of translational, rotational, vibrational, and electronic motions to G im were calculated at temperature T = 298.15K.The ECD spectra were calculated using time-dependent density functional theory (TDDFT), cam-B3LYP functional, PCM model, and 6-311G(d) basis set.Twenty-five electronic transitions were calculated for each conformation of 1 and 4. The individual bands in theoretical spectra were simulated as Gauss-type functions with the bandwidths ζ = 0.44 eV.The UV shifts ∆λ = 17 nm and 14 nm were used for best correspondence between experimental and calculated spectra for compounds 1 and 4, respectively.

Antimicrobial Activity
The yeast-like fungi of Candida albicans KMM 455 and bacterial strains Staphylococcus aureus ATCC 21027 and Escherichia coli VKPM (B-7935) (Collection of Marine Microorganisms PIBOC FEB RAS) were cultured on solid medium Mueller Hinton broth with agar (16.0 g/L) in a Petri dish at 37 • C for 24 h.
The assays were performed in 96-well microplates in appropriate Mueller Hinton broth.Each well contained 90 µL of bacterial or of yeast-like fungi suspension (10 9 CFU/mL).Then, 10 µL of a compound diluted at concentrations from 1.5 µM to 100.0 µM using two-fold dilution were added (DMSO concentration <1%).Culture plates were incubated for 18 h at 37 • C, and the OD 620 was measured using a Multiskan FS spectrophotometer (Thermo Scientific Inc., Beverly, MA, USA).The antibiotic gentamicin and the antifungal agent nitrofungin were used as positive controls at 1 mg/mL; 1% DMSO in PBS served as a negative control [50].

Biofilm Formation Assay
The inhibition reducing biofilm formation and growth was assessed using the crystal violet biofilm assay as described [24].Overnight cultures of bacteria S. aureus and E. coli and yeast-like fungi C. albicans were inoculated into Mueller-Hinton broth at a concentration of 10 9 CFU/mL.A total of 90µL of this cell suspension was then dispensed into 96-well microtiter plates containing 10 µL of different concentrations of compounds 1-6.After 24 h growth at 37 • C, the plates were washed with PBS to remove unbound cells.Next, the wells were stained with 0.1% crystal violet solution for 10 min at 37 • C. At the completion of the incubation, plates were washed 3 times with PBS and dried.Then, the crystal violet dye from the biofilm was solubilized with 100 µL of ethanol.A total of 100 µL of this solution was then moved to a new microtiter plate for absorbance measurement at λ = 570 nm.The results were reported as percent inhibition normalized to the wild-type control.

Sortase A Activity Inhibition Assay
The enzymatic activity of sortase A from S. aureus was determined using SensoLyte 520 Sortase A Activity Assay Kit Fluorimetric (AnaSpec AS-72229, AnaSpec, San Jose, CA, USA) in accordance with the manufacturer's instructions.The compounds were dissolved in DMSO and diluted with reaction buffer to obtain a final concentration of 0.8% DMSO, which did not affect enzyme activity.DMSO at a concentration of 0.8% was used as a control.4-(Hydroxymercuri)benzoic acid (PCMB) was used as sortase A enzyme activity inhibitor.Fluorescence was measured with a plate reader PHERAStar FS (BMG Labtech, Offenburg, Germany) for 60 min, with a time interval of 5 min.The data were processed with MARS Data Analysis v. 3.01R2 (BMG Labtech, Offenburg, Germany).The results were presented as relative fluorescent units (RFUs) and percentage of the control data, calculated using STATISTICA 10.0 software.

Cell Culture
The rat cardiomyocytes H9c2 cells were kindly provided by Prof. Dr. Gunhild von Amsberg from Martini-Klinik Prostate Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

Cell Viability Assay
The H9c2 cells were seeded at concentrations of 3 × 10 3 cell/well, and the experiments were started after 24 h.The compounds at concentrations up to 100 µM were added into the wells for 24 h, and the viability of the cells was measured with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was performed according to the manufacturer's instructions (Sigma-Aldrich, Munich, Germany).All compounds were dissolved with DMSO so that the final concentration of DMSO in the cell culture was not more than 1%.Moreover, DMSO was used as a control.The results were presented as a percent of the control data and calculated IC 50 .

Molecular Docking
The pdb file of sortase A (PDB ID 1T2P) was obtained from the RCSB Protein Data Bank (https://www.rcsb.org,accessed on 8 November 2023) and prepared for docking with the PrepDock package of UCFS Chimera 1.16 software.The chemical structures of ligands were prepared for docking with ChemOffice and checked with the PrepDock package of UCFS Chimera 1.16 software.
Docking was conducted on the SwissDock online server (http://www.swissdock.ch, accessed on 8 November 2023) based on EADock DSS docking software [51].The algorithm implies the generation of many binding modes in the vicinity of all target cavities (blind docking) and estimation of their CHARMM energies via the Chemistry at HARvard Macromolecular Mechanics (CHARMM) algorithm [52] for evaluation of the binding modes with the most favorable energies with FACTS (Fast Analytical Continuum Treatment of Solvation) [53] and, finally, clustering of these binding modes [54].
The predicted building models for each target/ligand pair were visualized and analyzed with UCFS Chimera 1.16 software.Docking parameters, such as Gibb's free energy (∆G, kcal/mol), FullFitness score (FF, kcal/mol), and hydrogen-bonding and hydrophobic interactions, were used for analysis of target/ligand complexes.

Statistical Data Evaluation
All data were obtained in three independent replicates, and calculated values were expressed as a mean ± standard error mean (SEM).Student's t-test was performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to determine statistical significance.Differences were considered statistically significant at p < 0.05.

Conclusions
Two new cyclopiane diterpenes and a new cladosporin precursor, together with four known related compounds, were isolated from marine sediment-derived fungus P. antarcticum KMM 4670.The absolute stereostructures of conidiogenone F and leptosphin

Figure 1 .
Figure 1.ML tree based on concatenated ITS-BenA-CaM-RPB2 nucleotide sequences showing the phylogenetic position of the strain KMM 4670 among members of genus Penicillium section Canescentia, series Atroveneta.Bootstrap values (%) of 1000 replications.The scale bars represent 0.05 substitutions per site.

Figure 1 .
Figure 1.ML tree based on concatenated ITS-BenA-CaM-RPB2 nucleotide sequences showing the phylogenetic position of the strain KMM 4670 among members of genus Penicillium section Canescentia, series Atroveneta.Bootstrap values (%) of 1000 replications.The scale bars represent 0.05 substitutions per site.

Mar. Drugs 2023, x 3 of 22 Figure 1 .
Figure 1.ML tree based on concatenated ITS-BenA-CaM-RPB2 nucleotide sequences showing the phylogenetic position of the strain KMM 4670 among members of genus Penicillium section Canescentia, series Atroveneta.Bootstrap values (%) of 1000 replications.The scale bars represent 0.05 substitutions per site.
H 3 -18/H-10β established the relative configurations of all stereocenters, which was the same as those for conidiogenone F (3) with the exception of C-13.

Figure 8 .
Figure 8.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on Staphylococcus aureus growth.Gentamicin was used as a positive control at a concentration of 1 mg/mL.(c) The effect of compounds 1-6 on the biofilm formation.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.

Figure 8 .
Figure 8.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on Staphylococcus aureus growth.Gentamicin was used as a positive control at a concentration of 1 mg/mL.(c) The effect of compounds 1-6 on the biofilm formation.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.

Figure 8 .
Figure 8.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on Staphylococcus aureus growth.Gentamicin was used as a positive control at a concentration of 1 mg/mL.(c) The effect of compounds 1-6 on the biofilm formation.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.

Figure 9 .
Figure 9.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on E. coli growth.Gentamicin was used as a positive control at a concentration of 1 mg/mL.(c) The effect of compounds 1-6 on the biofilm formation.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.

Figure 9 .
Figure 9.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on E. coli growth.Gentamicin was used as a positive control at a concentration of 1 mg/mL.(c) The effect of compounds 1-6 on the biofilm formation.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.All investigated compounds inhibited the growth of E. coli culture but only compounds 4 and 5 affect the biofilm formation of E. coli.4-Hydroxyleptosphin C (1) and 13-epiconidiogenone F (2) at a concentration of 100 µM inhibited E. coli growth by 35.9% and 27.3%, respectively.The growth inhibitory effect of these two compounds was not observed when its concentration was 12.5 µM.Conidiogenone F (3) and leptosphin C; (4) at a concentration of 100 µM inhibited E. coli growth by 36.4% and 41.7%, respectively.At 12.5 µM, conidiogenone F (3) inhibited E. coli growth by 24.3% while leptosphin C; (4) inhibited it by only 7.2%.Nevertheless, 4, at concentrations of 25-100 µM, prevented E. coli biofilm formation by nearly 30-40%.Antaketide A (5) at 100 µM and 12.5 µM inhibited E. coli growth by 40.9% and 12.5%, respectively.Moreover, 5 prevented the biofilm formation by 21-45% at concentrations of 12.5-100 µM, respectively.Finally, tetraketide derivative 6 at 100 µM and 12.5 µM inhibited E. coli growth by 56.9% and 27.7%, respectively, and IC 50 was calculated as 84.9 µM.The effects of compounds 1-6 on the growth and biofilm formation of Candida albicans are presented in Figure10.

Figure 10 .Figure 10 .
Figure 10.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on C. albicans growth.The effect of compounds 1-6 on the biofilm formation.Nitrofungin was used as a positive control at a concentration of 1 mg/mL.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.All compounds inhibited C. albicans growth by nearly 30-40%, and compounds 4 and 6 also affected the biofilm formation by C. albicans.Compounds 1-3 at a 12.5 µM inhibited C. albicans growth by 30.4%, 27.9%, and 26.5%, respectively.Compound 4 inhibited C. albicans growth by 38.7-44.8% and the biofilm formation by 30.6-36.4% at concentrations

Figure 10 .
Figure 10.The effect of compounds 1-6 at concentrations of 12.5 µM (a) and 100.0 µM (b) on C. albicans growth.The effect of compounds 1-6 on the biofilm formation.Nitrofungin was used as a positive control at a concentration of 1 mg/mL.The data are presented as a mean ± standard mean error.All experiments were carried out in independent triplicates.All compounds inhibited C. albicans growth by nearly 30-40%, and compounds 4 and 6 also affected the biofilm formation by C. albicans.Compounds 1-3 at a 12.5 µM inhibited C. albicans growth by 30.4%, 27.9%, and 26.5%, respectively.Compound 4 inhibited C. albicans growth by 38.7-44.8% and the biofilm formation by 30.6-36.4% at concentrations from 12.5 to 100 µM.Compound 5 inhibited C. albicans growth by 36.2-48.4% and did not affect the biofilm formation.Compound 6 inhibited C. albicans growth by 34.7-52.1% and the biofilm formation by 14.7-23.1% at concentrations from 12.5 to 100 µM.Its IC50 for C. albicans growth was calculated as 89.9 µM.

Figure 11 .
Figure 11.The effects of compounds 1-6 on sortase A activity after 10 min of incubation (a) and time-dependent graph of the inhibitory effect of 4-hydroxyleptosphin C (1) and 13-epi-conidiogenone F (2).(b) 4-(Hydroxymercuri)benzoic acid (PCMB) was used as a control.All experiments were carried out in triplicate.The data are presented as a mean ± standard error of the mean (SEM).* indicates significant differences between the control (DMSO 0.8%) and compounds (p value ≤ 0.05).

Table 3 .
The results of molecular docking of compounds 1-4 with sortase A structure (PDB ID 1T2P).

Table 4 .
The effect of compounds 1-6 on the viability of H9c2 cardiomyocytes.
• C for 20 s, 48 • C for 30 s, and 72 • C for 90 s; 5 cycles of 95 • C for 20 s, 50 • C for 30 s, and 72 • C for 90 s; 25 cycles of 95 • C for 20 s, 52 • C for 30 s, and 72 • C for 90 s; and, finally, 72

Table 5 .
The strains of the species used in multi-locus phylogenetic analysis and GenBank accession numbers.