Bioactive Polyketides from the Natural Complex of the Sea Urchin-Associated Fungi Penicillium sajarovii KMM 4718 and Aspergillus protuberus KMM 4747

The marine-derived fungal strains KMM 4718 and KMM 4747 isolated from sea urchin Scaphechinus mirabilis as a natural fungal complex were identified as Penicillium sajarovii and Aspergillus protuberus based on Internal Transcribed Spacer (ITS), partial β-tubulin (BenA), and calmodulin (CaM) molecular markers as well as an ribosomal polymerase two, subunit two (RPB2) region for KMM 4747. From the ethyl acetate extract of the co-culture, two new polyketides, sajaroketides A (1) and B (2), together with (2′S)-7-hydroxy-2-(2′-hydroxypropyl)-5-methylchromone (3), altechromone A (4), norlichexanthone (5), griseoxanthone C (6), 1,3,5,6-tetrahydroxy-8-methylxanthone (7), griseofulvin (8), 6-O-desmethylgriseofulvin (9), dechlorogriseofulvin (10), and 5,6-dihydro-4-methyl-2H-pyran-2-one (11) were identified. The structures of the compounds were elucidated using spectroscopic analyses. The absolute configurations of the chiral centers of sajaroketides A and B were determined using time-dependent density functional theory (TDDFT)-based calculations of the Electronic Circular Dichroism (ECD) spectra. The inhibitory effects of these compounds on urease activity and the growth of Staphylococcus aureus, Escherichia coli, and Candida albicans were observed. Sajaroketide A, altechromone A, and griseofulvin showed significant cardioprotective effects in an in vitro model of S. aureus-induced infectious myocarditis.


Introduction
Marine microbial ecosystems are characterized by an uneven ratio between prokaryotes and eukaryotes.For example, an investigation of the microbial community during a natural Noctiluca scintillans algal bloom in the coastal area of Dongchong (Shenzhen, China) showed that, in addition to dinoflagellates, Bacteria and Archaea predominate in this community, and Fungi are represented by only four phyla in very small quantities [1].This leads to considerable competition within the community and the production of exolites to control the competitors.A number of reports have confirmed that the produced secondary metabolites may be extensively involved in a variety of communication events among microorganisms when a bacteria-fungus co-culture produces bacteriostatic and fungicidal exolites [2].From 2009 to 2019, various alkaloids, anthraquinones, cyclopeptides, macrolides, phenylpropanoids, polyketides, steroids, terpenoids, and others (153 compounds in total) with antimicrobial, cytotoxic, hemolytic, and anti-proliferative activities were isolated from marine microbial co-cultures [3].The latest report by Li et al. described 194 compounds with cytotoxic, antibacterial, antifungal, antimalarial, and antifouling properties isolated from marine microbial co-cultures in 2012-2022 [4].Our study of various marine fungus-fungus co-cultures resulted in the isolation of new cytotoxic and hemolytic metabolites [5][6][7].
In the course of our investigation of the fungal community of various marine substrates in the Sea of Japan, a natural fungal complex was isolated from the aboral surface of the sea urchin Scaphechinus mirabilis collected from Troitsa Bay and formed by only two strains, KMM 4718 and KMM 4747.The fungal species Penicillium sajarovii is one of three representatives of the Raistrickiorum series (Ramosa) and is phylogenetically close to the species P. raistrickii [8].Various compounds have been reported to be isolated from P. raistrickii.Among them, there are benzo-fused 2,8-dioxabicyclo[3.3.1]nonane-containedspiroketals [9]; several p-terphenyl and xanthone derivatives together with griseofulvin-related metabolites [10]; indole diketopiperazine alkaloids and benzodiazepine derivatives [11]; radical scavenging phenetyltetrahydrofuranes [12]; and cytotoxic spiroditetrahydropyran polyketides [13].The fungal species Aspergillus protuberus belongs to the Versicolores series (Nidulantes), which currently includes 17 species, and is phylogenetically close to the species A. versicolor, A. amoenus, A. tabacinus, and A. austroafricanus [8].These fungi are well-known sources of polyketides, diketopiperazines, sterols, terpenoids, and meroterpenoids [14][15][16].Penicillium and Aspergillus species are two of the most widespread fungal organisms on Earth, growing on many different substrates, and are permanent components of marine microbial communities [17,18].Each of these studied fungi is a promising producer of various metabolites with a wide range of biological activities; therefore, studying their natural complex can yield interesting results.
Here, we report the isolation and structure elucidation, including absolute configuration determination, of 11 polyketides (Figure 1) from the natural complex of the sea urchin-associated fungi P. sajarovii KMM 4718 and A. protuberus KMM 4747.The antimicrobial and cytotoxic activities of the isolated compounds are also evaluated.protuberus KMM 4747 isolated from the sea urchin S. mirabilis.

Molecular Identification of the Fungal Strains
In this study, to clarify the taxonomic position of the strains KMM 4718 and KMM 4747, we sequenced molecular markers, such as ITS, partial BenA, and CaM regions.Ap-  A BLAST search showed that the ITS region was 100% identical to the sequence of the ex-type strain Aspergillus protuberus CBS 602.74 T , whereas the partial BenA, CaM, and RPB2 gene sequences were more than 99% identical.The phylogenetic ML tree of the concatenated ITS-BenA-CaM-RPB2 gene sequences clearly showed that the strain KMM 4747 clustered with ex-type strain Aspergillus protuberus CBS 602.74 T (Figure 3).A BLAST search showed that the ITS region was 100% identical to the sequence of the ex-type strain Aspergillus protuberus CBS 602.74 T , whereas the partial BenA, CaM, and RPB2 gene sequences were more than 99% identical.The phylogenetic ML tree of the concatenated ITS-BenA-CaM-RPB2 gene sequences clearly showed that the strain KMM 4747 clustered with ex-type strain Aspergillus protuberus CBS 602.74 T (Figure 3).
The molecular formula of 2 was established to be C 13 H 14 O 5 by HRESIMS (m/z 273.0735 [M + Na] + ) (Figure S1) and was confirmed by the 13 C NMR spectrum.The 1 H and 13 C NMR data (Table 1, Figures S9-S14) observed for compound 2 closely resembled those obtained for 1, except for the additional methoxy group (δ H 3.87, δ C 55.9).The location of this group at C-6 was established based on the HMBC correlation from 6-OMe to C-6 (δ C 166.1).Thus, the planar structure of 2 was determined to be the 6-O-methyl derivative of sajaroketide A, and this compound was named sajaroketide B. Unfortunately, correlations in the ROESY spectrum were not informative and could not be used to establish the relative configurations of the stereocenters of 1 and 2. Thus, theoretical and experimental ECD spectra were compared to determine the absolute configurations of the chiral centers of sajaroketides A (1) and B (2). UV and ECD spectra were calculated for the most stable conformations of 1 and 2 using the time-dependent density functional theory (TDDFT_B3LYP).All calculations were performed with the Gaussian 16 suite of programs [20] using ultra-fine integration grids and very tight optimization convergence criteria.
The performed preliminary modeling showed that the signs and intensities of individual bands in their ECD spectra strongly depended on the large-amplitude motions (LAMs) occurring in these compounds-the internal rotations of hydroxy and methoxy groups around C(3)-O(3), C(4)-O(4), and C(6)-O(6) bonds, the tautomeric rearrangement, and the inversion-type motion of cycle "A".For λ ≤ 240 nm, these dependencies complicated the task of the ECD spectral interpretation.The rigorous accounting of contributions from these LAMs to spectral properties must be performed based on some kind of intramolecular dynamics modeling-the Schrödinger equations for the motions along these LAMs' degrees of freedom must be constructed and solved.This approach is far from the present standard theoretical scheme, which is generally used in a stereochemical analysis.An alternative to this is the approach where efforts are focused on the investigation of those parts of ECD spectra where individual bands are well separated and the influence of LAMs may be accounted for qualitatively and correctly, even based on the standard theoretical scheme with moderate improvements.The latter approach was used in this study, and the characteristic energy region chosen for the investigation was λ ≥ 230 nm.Calculations, performed at the "PCM level", showed that conformation R-1_c1 was the most thermodynamically stable-its Gibbs free energy was minimal (Figure S42).The conformation 4R-1_c2 is less stable then 4R-1_c1 for about ∆G ≈ 0.45 kcal/mol.Figures 4 and 5 demonstrate the UV and ECD spectra, respectively, for these two most stable conformations of 4R-1, calculated using TDDFT_B3LYP/cc-pvTz_PCM//B3LYP/cc-pvTz_PCM methods at the "PCM level" of theory.While the UV spectra of 4R-1_c1 and 4R-1_c2 are nearly similar, their ECD spectra differ qualitatively in the 220 ≤ λ ≤ 270 nm region.The UV shift, obtained from a comparison of the theoretical statistically averaged and experimental UV spectra, was Δλ ≈ +7 nm.This UV shift value was used to construct the average ECD spectrum (Figure 5).The theoretically averaged ECD spectrum of 4R-1 satisfactorily reproduced the experimental ECD spectrum of 1, except for the 200 ≤ λ ≤ 250 nm region.At the same time, the ECD spectrum of the "minor" conformer 4R-1_c2 was in very good agreement with the experiment at λ ≥ 230 nm.This may provide evidence that the PCM approach and standard theoretical scheme alone fail to account for the full measure of the compound-  While the UV spectra of 4R-1_c1 and 4R-1_c2 are nearly similar, their ECD spectra differ qualitatively in the 220 ≤ λ ≤ 270 nm region.The UV shift, obtained from a comparison of the theoretical statistically averaged and experimental UV spectra, was Δλ ≈ +7 nm.This UV shift value was used to construct the average ECD spectrum (Figure 5).The theoretically averaged ECD spectrum of 4R-1 satisfactorily reproduced the experimental ECD spectrum of 1, except for the 200 ≤ λ ≤ 250 nm region.At the same time, the ECD spectrum of the "minor" conformer 4R-1_c2 was in very good agreement with the experiment at λ ≥ 230 nm.This may provide evidence that the PCM approach and standard theoretical scheme alone fail to account for the full measure of the compound- Experimental (black) and theoretical ECD spectra, calculated for 4R-1 at the TDDFT_B3LYP/cc-pvTZ_PCM//B3LYP/cc-pvTZ_PCM level of theory: calculated for 4R-1_c1 (red), 4R-1_c2 (green), and calculated averaged for 4R-1 (blue).
While the UV spectra of 4R-1_c1 and 4R-1_c2 are nearly similar, their ECD spectra differ qualitatively in the 220 ≤ λ ≤ 270 nm region.The UV shift, obtained from a comparison of the theoretical statistically averaged and experimental UV spectra, was ∆λ ≈ +7 nm.This UV shift value was used to construct the average ECD spectrum (Figure 5).
The theoretically averaged ECD spectrum of 4R-1 satisfactorily reproduced the experimental ECD spectrum of 1, except for the 200 ≤ λ ≤ 250 nm region.At the same time, the ECD spectrum of the "minor" conformer 4R-1_c2 was in very good agreement with the experiment at λ ≥ 230 nm.This may provide evidence that the PCM approach and standard theoretical scheme alone fail to account for the full measure of the compound-solvent interaction, and hence to correctly describe the thermodynamics of the conformational rearrangement process.To overcome these discrepancies, we used a more complex theoretical model, in which the direct modeling of sajaroketide A (1) interacting with two methanol molecules (4R-1&(CH 3 OH) × 2 and 4R-2&(CH 3 OH) × 2) was performed.The different variants of the intermolecular hydrogen-bond formation (IMHB) were modeled using the B3LYP/cc-pvTz_PCM approach.The optimized structures of the seven most stable conformations are shown in Figure S43.
We found that the thermodynamic equilibrium in cluster 4R-1&(CH 3 OH) × 2 was dislocated to the conformations of the 4R-1_c2 type (structures Direct_R-1_c-Direct_R-1_c5), and this prevalence governed the existence in the experimental ECD spectrum of the negative band at λ ≈ 230-240 nm.The relative intensities of the positive bands in the λ ≥ 255 nm diapason were also reproduced properly at the "direct level" than at the "PCM level" of theory (Figure 6).Therefore, the absolute structure of 1 is 4R (for demonstration, the theoretical ECD spectrum of 4S-1 is plotted in Figure 6 using a dashed line).
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 8 of 22 solvent interaction, and hence to correctly describe the thermodynamics of the conformational rearrangement process.To overcome these discrepancies, we used a more complex theoretical model, in which the direct modeling of sajaroketide A (1) interacting with two methanol molecules (4R-1&(CH3OH) × 2 and 4R-2&(CH3OH) × 2) was performed.The different variants of the intermolecular hydrogen-bond formation (IMHB) were modeled using the B3LYP/cc-pvTz_PCM approach.The optimized structures of the seven most stable conformations are shown in Figure S43.We found that the thermodynamic equilibrium in cluster 4R-1&(CH3OH) × 2 was dislocated to the conformations of the 4R-1_c2 type (structures Direct_R-1_c2−Direct_R-1_c5), and this prevalence governed the existence in the experimental ECD spectrum of the negative band at λ ≈ 230−240 nm.The relative intensities of the positive bands in the λ ≥ 255 nm diapason were also reproduced properly at the "direct level" than at the "PCM level" of theory (Figure 6).Therefore, the absolute structure of 1 is 4R (for demonstration, the theoretical ECD spectrum of 4S-1 is plotted in Figure 6   The experimental ECD and UV spectra of 2 were similar to those of 4R-1.A comparison of the theoretical ECD spectra of 4R-2 and 4S-2 calculated at the "direct level" of theory with the experimental one is shown in Figure 7. Spectra Δεexp and Δεcalc(4R-2) are in good qualitative agreement, while Δεcalc(4S-2) "fails" to reproduce experimental data throughout the diapason.Thus, the absolute structure of 2 is 4R (for demonstration, the theoretical ECD spectrum of 4S-2 is plotted in Figure 6   The experimental ECD and UV spectra of 2 were similar to those of 4R-1.A comparison of the theoretical ECD spectra of 4R-2 and 4S-2 calculated at the "direct level" of theory with the experimental one is shown in Figure 7. Spectra ∆ε exp and ∆ε calc (4R-2) are in good qualitative agreement, while ∆ε calc (4S-2) "fails" to reproduce experimental data throughout the diapason.Thus, the absolute structure of 2 is 4R (for demonstration, the theoretical ECD spectrum of 4S-2 is plotted in Figure 6 using a dashed line).
The molecular formula of compound 3 was determined to be C 13 H 14 O 4 by HRESIMS (m/z 257.0788 [M + Na] + ) (Figure S15), which corresponded to seven degrees of unsaturation.These data and a careful inspection of the 13 C and 1 H NMR spectra (Figures S16 and S17  The molecular formula of compound 3 was determined to be C13H14O4 by HRESIMS (m/z 257.0788 [M + Na] + ) (Figure S15), which corresponded to seven degrees of unsaturation.These data and a careful inspection of the 13 C and 1 H NMR spectra (Figures S16 and S17  [22,24]) proved the absolute configuration of the stereo center in 3 as 2′S.

Cytotoxic Activity
The cytotoxic activities of compounds 1-9 and 11 toward human hepatocarcinoma HepG2 and normal rat cardiomyocyte H9c2 cells were evaluated.Cell viability and the half-maximal concentration of the cytotoxic effects are presented in Table 3. Sajaroketide A (1) at 100 µM decreased H9c2 cell viability by 34.6%.Sajaroketide B (2) at 100 µM decreased HepG2 and H9c2 cell viability values by 23.7% and 21.0%, respectively.Nevertheless, sajaroketides A (1) and B (2) were nontoxic to both cell lines at a concentration of 10 µM.
Chromone derivative 3 was non-toxic to HepG2 cells and decreased H9c2 cell viability by 25.7% at 100 µM.Altechromone A (4) was non-toxic to both cell lines at concentrations up to 100 µM.

The Effect of Compounds against S. aureus Infection Damage of H9c2
The following step of the study was to determine the cytoprotective effects of compounds 1-9 and 11 in an in vitro model of infectious myocarditis when H9c2 cardiomy-ocytes were co-cultured with an S. aureus suspension.We treated S. aureus-infected H9c2 cells with the compounds 1-9 and 11 at concentrations of 1 and 10 µM and measured the viability of the cells by MTT as described in the Section 4 (Figure 8).

The Effect of Compounds against S. aureus Infection Damage of H9c2
The following step of the study was to determine the cytoprotective effects of compounds 1-9 and 11 in an in vitro model of infectious myocarditis when H9c2 cardiomyocytes were co-cultured with an S. aureus suspension.We treated S. aureus-infected H9c2 cells with the compounds 1-9 and 11 at concentrations of 1 and 10 µM and measured the viability of the cells by MTT as described in the Materials and Methods Section (Figure 8).New sajaroketide A (1) increased the viability of S. aureus-infected H9c2 cardiomyocytes by 50.8% and 49.2% at 1 and 10 µM, respectively.Chromone derivative 3 at 10 µM increased the viability of these cells by 24.9%.Altechromone A (4) increased the viability of these cells by 17.4% and 39.9% at 1 and 10 µM, respectively.Norlichexanthone (5), xanthone derivative 7, griseofulvin (8), and 6-O-desmethylgriseofulvin (9) at 10 µM increased the viability of these cells by 23.9%, 33.9%, 47.7%, and 28.9%, respectively.Compounds 2, 6, and 11 did not show any statistical effect on the viability of S. aureus-infected H9c2 cardiomyocytes.New sajaroketide A (1) increased the viability of S. aureus-infected H9c2 cardiomyocytes by 50.8% and 49.2% at 1 and 10 µM, respectively.Chromone derivative 3 at 10 µM increased the viability of these cells by 24.9%.Altechromone A (4) increased the viability of these cells by 17.4% and 39.9% at 1 and 10 µM, respectively.Norlichexanthone (5), xanthone derivative 7, griseofulvin (8), and 6-O-desmethylgriseofulvin (9) at 10 µM increased the viability of these cells by 23.9%, 33.9%, 47.7%, and 28.9%, respectively.Compounds 2, 6, and 11 did not show any statistical effect on the viability of S. aureus-infected H9c2 cardiomyocytes.

Discussion
Thus, 11 polyketides, including two new sajaroketides, A and B, were isolated from the natural complex of P. sajarovii KMM 4718 and A. protuberus KMM 4747 associated with sea urchin S. mirabilis.
The anti-infection effect of norlichexanthone (5) and griseofulvin (8) correlated with their influence on S. aureus growth and urease activity.Chromone derivative 3 and griseofulvin-related compound 9 did not show any effect on urease activity, but inhibited S. aureus growth and, so may have inhibited bacterial growth in the in vitro model of myocarditis.Xanthone derivative 7 did not show any effect on S. aureus growth, but inhibited the activity of urease, which might be of relevance to this case.
Griseofulvin is a known antifungal polyketide produced by various fungi [30].It was approved by the Food and Drug Administration (FDA) in 1959 as an anti-dermatophyte drug with an anti-inflammatory effect, as well as improving coronary blood flow and decreasing blood pressure [31].Moreover, the inhibition of tumor growth and several forms of cancer cell proliferation were found [32].Griseofulvin, currently used per os for the treatment of scalp dermatophytosis, achieving smooth skin, and improving the nails, and our new data concerning its possibility to inhibit urease activity and its cardioprotective effect on in vitro infectious myocarditis are interesting subjects.
Sajaroketide A (1) and altechromone A (4) showed greater cytoprotective effects in these experiments.It has previously been published in the literature that altechromone A (4) was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens, and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 µg/mL, respectively [33].In our experiments, altechromone A (4) not only inhibited the growth of S. aureus, but was also effective against the staphylococcal infection of cardiomyocytes H9c2.Sajaroketide A (1) inhibited the growth of S. aureus similar to altechromone A ( 4), but its effect on S. aureus-infected H9c2 cells was more significant at a concentration of 1 µM.The influences of 1 and 4 on urease activity were very poor, which indicated their effects on S. aureus defined by other mechanisms.
According to the Global Burden of Disease project, in 2019, 0.428 million cases of endocarditis were recorded worldwide and 0.0663 million of them led to the deaths of patients.Over 10 years (2010-2019), the number of cases increased by 29% and the number of deaths by 21.8%.In 2019, 4.06 million cases of cardiomyopathy and myocarditis of various origins were recorded worldwide, and almost 10% (0.34 million) of the cases resulted in the deaths of patients.Over 10 years (2010-2019), the number of cases increased by 20% and the number of deaths by 3.3%.It should be noted that a significant proportion (30%) of myocarditis was caused by alcohol intoxication, but drug damage and infectious effects (viral and bacterial) played a dominant role [34].Thus, the discovery of new compounds effective against staphylococcal infections is a potential future study of sajaroketide A.

General Experimental Procedures
Optical rotations were measured on a PerkinElmer 343 polarimeter (PerkinElmer, Waltham, MA, USA) in MeOH.UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in MeOH.ECD spectra were measured using a Chirascan-Plus CD Spectrometer (Leatherhead, UK) in MeOH. 1 H and 13 C NMR spectra were recorded in aceton-d 6 on Bruker Avance-500 and Avance III-700 spectrometers (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 500 and 125 MHz and 700 and 176 MHz, respectively, using TMS as an internal standard.HRESIMS spectra were obtained using a Bruker maXis Impact II mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany).

Fungal Strains
The fungal culture used in this study was likely a natural fungal complex isolated from the aboral surface of the sea urchin S. mirabilis collected from the Sea of Japan (Troitsa bay).This complex is a co-culture of the filamentous fungi P. sajarovii and A. protuberus.
Initially, in the course of studying the metabolic profile of the fungal isolate, an isolate was selected that visually presented signs of a monoculture of P. sajarovii, both when growing on agar media for analytical cultivation (wort agar) and diagnostics (Czapek's medium with yeast extract), and during further preparative cultivation on a medium with rice.During the microscopy of the culture, fragments of mycelium were found that did not belong to P. sajarovii and indicated the presence of a co-culture of a fungus of an unknown taxonomic affiliation, which was confirmed by a molecular genetic analysis.Subsequently, the components of the fungal complex were dispersed and their monocultures were obtained.The molecular genetic analysis of the second component of the fungal complex showed that it belonged to the species A. protuberus.
The resulting fungal strains were stored in the Collection of Marine Microorganisms (PIBOC FEB RAS, Vladivostok, Russia) as P. sajarovii KMM 4718 and A. protuberus KMM 4747.

Phylogenetic Analysis
The ITS region, partial BenA and CaM gene sequences, fungal strain KMM 4718, and members of genus Penicillium (Ramosum), series Lanosa, Raistrickiorum, Scabrosa, Soppiorum, and Virgata were aligned by MEGA X software version 11.0.9 [37] using the Clustal W algorithm.The ex-type homologs were searched in the GenBank database (http://ncbi.nlm.nih.gov) 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 [37].The ITS region and partial BenA and CaM gene sequences were concatenated into one alignment.A phylogenetic tree was constructed according to the maximum likelihood (ML) algorithm based on a general time-reversible model [38].The tree topology was evaluated by 1000 bootstrap replicates.The Talaromyces marneffei CBS 388.87 T strain was used in the phylogenetic analysis as an outgroup (Table 4).
The ITS region, partial BenA, CaM, and RPB2 gene sequences, fungal strain KMM 4747, and members of genus Aspergillus, series Versicolores, were aligned by MEGA X software version 11.0.9 [37] using the Clustal W algorithm.The search for the ex-type homologs and phylogenetic analysis were performed as described above.The ITS region and partial BenA, CaM, and RPB2 gene sequences were concatenated into one alignment.A phylogenetic tree was constructed according to the maximum likelihood algorithm based on the Kimura 2-parameter model [39].The tree topology was evaluated by 1000 bootstrap replicates.The Talaromyces marneffei CBS 388.87 T strain was used in the phylogenetic analysis as the outgroup (Table 4).

Cultivation of Penicillium sajarovii KMM 4718 and Aspergillus protuberus KMM 4747
The fungi were grown stationary at 22 • C for 21 days 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 sea water (Marine Experimental Station of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Troitsa (Trinity) Bay, Sea of Japan).

Extraction and Isolation
At the end of the incubation period, the mycelia and medium were homogenized and extracted with EtOAc (3 L).The obtained extract was dried in vacuo.The residue was dissolved in H 2 O−EtOH (4:1) (500 mL) and was extracted with n-hexane (0.3 L × 4) and EtOAc (0.3 L × 10).After the evaporation of the EtOAc layer, the residual material (60.182 g) was passed over a silica column (4 × 55 cm), which was eluted followed by a step gradient from 100% n-hexane to EtOAc (total volume: 50 L).Fractions of 250 mL were collected and combined based on the TLC results (Si gel, toluene-isopropanol, 6:1 and 3:1).As a result, 2 fractions were obtained: Pr-4718-6 (1.735 g) and Pr-4718-9 (4.398 g).

Quantum-Chemical Modeling
The quantum-chemical modeling of the geometry and spectroscopic properties of compounds 1 and 2 were performed using the Gaussian 16 package of programs [20].Geometry optimizations and calculations of the IR spectra were conducted with B3LYP exchangecorrelation functional, the polarization continuum model (PCM), and 6-311+G(d,p) and cc-pvTz split-valence basis sets.
The statistical weights (gim) of conformations were calculated via Gibbs free energies: where index "m" denotes the most stable conformation and ∆Gim = Gi − Gm are the relative Gibbs free energies.The ECD spectra were calculated using the time-dependent density functional theory (TDDFT), B3LYP functional, PCM model, and cc-pvTz basis set.Thirty electronic transitions were calculated for each conformation of 1 and 2. The individual bands in the theoretical spectra were simulated as a Gauss-type functions with the bandwidth ζ = 0.28 eV.The UV shift ∆λ = +7 nm was used for the best correspondence between the experimental and calculated spectra for 1 and 2.

Urease Inhibition Assay
A reaction mixture consisting of 25 µL of enzyme solution (urease from Canavalia ensiformis, Sigma, 1U final concentration) and 5 µL of test compounds dissolved in water (10-300.0µM final concentration) was preincubated at 37 • C for 60 min in 96-well plates.Then, 55 µL of phosphate-buffered solution with 100 µM of urea was added to each well and incubated at 37 • C for 10 min.The urease inhibitory activity was estimated by determining ammonia production using the indophenol method.Briefly, 45 µL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaClO) were added to each well.The absorbance was measured after 50 min at 630 nm using a MultiskanFS microplate reader (Thermo Scientific Inc., Beverly, MA, USA).All reactions were performed in triplicate in a final volume of 200 µL.The pH was maintained at 7.3-7.5 in all assays.DMSO 5% was used as a positive control.

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 a yeast-like fungi suspension (10 9 CFU/mL).Then, 10 µL of a compound diluted at concentrations ranging from 1.5 to 100.0 µM using a 2-fold dilution was added (DMSO concentration < 1%).Culture plates were incubated overnight at 37 • C, and the OD 620 was measured using a Multiskan FS spectrophotometer (Thermo Scientific Inc., Beverly, MA, USA).The antibiotic gentamicin and antifungal agent nitrofungin were used as positive controls at 1 mg/mL; 1% DMSO in PBS served as a negative control.

Cell Culture
The rat cardiomyocyte H9c2 cells were kindly provided by Prof. Dr. Gunhild von Amsberg from the Martini-Klinik Prostate Cancer Center, University Hospital Hamburg-Eppendorf, Hamburg, Germany.The human hepatocarcinoma cell HepG2 was obtained from ATCC.

Cell Viability Assay
The HepG2 and H9c2 cells were seeded at concentrations of 5× 10 3 and 3×10 3 cell/well, respectively, 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 by 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

22 Figure 1 .
Figure 1.Metabolites isolated from the natural complex of the fungi P. sajarovii KMM 4718 and A. protuberus KMM 4747 isolated from the sea urchin S. mirabilis.

Figure 1 .
Figure 1.Metabolites isolated from the natural complex of the fungi P. sajarovii KMM 4718 and A. protuberus KMM 4747 isolated from the sea urchin S. mirabilis.

Figure 2 .
Figure 2. ML tree based on concatenated ITS-BenA-CaM gene sequences showing the phylogenetic position of strain KMM 4718 among members of the genus Penicillium (Ramosum), series Lanosa, Raistrickiorum, Scabrosa, Soppiorum, and Virgata.Bootstrap values (%) of 1000 replications.Nodes with confidence values higher than 50% are indicated.The scale bars represent 0.05 substitutions per site.

Figure 2 .
Figure 2. ML tree based on concatenated ITS-BenA-CaM gene sequences showing the phylogenetic position of strain KMM 4718 among members of the genus Penicillium (Ramosum), series Lanosa, Raistrickiorum, Scabrosa, Soppiorum, and Virgata.Bootstrap values (%) of 1000 replications.Nodes with confidence values higher than 50% are indicated.The scale bars represent 0.05 substitutions per site.

Figure 3 .
Figure 3. ML tree based on concatenated ITS-BenA-CaM-RPB2 gene sequences showing the phylogenetic position of strain KMM 4747 among the members of the genus Aspergillus series Versicolores.Bootstrap values (%) of 1000 replications.Nodes with confidence values higher than 50% are indicated.The scale bars represent 0.1 substitutions per site.

Figure 3 .
Figure 3. ML tree based on concatenated ITS-BenA-CaM-RPB2 gene sequences showing the phylogenetic position of strain KMM 4747 among the members of the genus Aspergillus series Versicolores.Bootstrap values (%) of 1000 replications.Nodes with confidence values higher than 50% are indicated.The scale bars represent 0.1 substitutions per site.
using a dashed line).
using a dashed line).

Figure 8 .
Figure 8.The viability of S. aureus-infected H9c2 cardiomyocytes treated with compounds 1-9 and 11.The compounds at concentrations of 1 and 10 µM were added after 1 h of S. aureus infection.All the data are presented as means ± standard errors of means.The experiments are conducted in three independent replicates.Asterisk * indicates the significant differences at p ≤ 0.05.

Figure 8 .
Figure 8.The viability of S. aureus-infected H9c2 cardiomyocytes treated with compounds 1-9 and 11.The compounds at concentrations of 1 and 10 µM were added after 1 h of S. aureus infection.All the data are presented as means ± standard errors of means.The experiments are conducted in three independent replicates.Asterisk * indicates the significant differences at p ≤ 0.05.

Table 1 .
13C and 1 H NMR spectroscopic data for compounds 1

Table 2 .
Inhibition of urease and antimicrobial activities of the compounds.

Table 2 .
Inhibition of urease and antimicrobial activities of the compounds.

Compounds Inhibition of Urease Activity IC 50 , µM Inhibition of Microbial Growth 1 , %
1Compounds are tested at a concentration of 100 µM.

Table 3 .
Cytotoxic activities of the compounds.

Table 4 .
The strains of the species used in the multi-locus phylogenetic analysis and GenBank accession numbers.
Note: ex-type strains Penicillium lusitanum MUM 18.49 (the sequence for the CaM gene is not established) and Aspergillus pepii CBS 142028 (the sequence for the RPB2 gene i not been established) are not used for the phylogenetic analysis.