Annotation of the results obtained in 2021
 Screening of antimicrobial activity of 33 strains from three samples of sea soil of the Arctic waters of the Arctic Ocean, as well as the Pacific Ocean, was carried out against five opportunistic test cultures. Antibacterial activity was shown by extracts of 13 strains of fungi; no antimycotic activity was found. Six new polyketides of acrucipentyns A-F (five of which are chlorine-containing), three new anthraquinones 9,13-isodendriol A, 3,9,13-isodendriol D, acruciciquinone, and eleven known metabolites of a predominantly polyketide nature were isolated from an extract of a culture of the obligate marine fungus Asteromyces cruciatus KMM 4696. The relative configurations of the new compounds were established on the basis of ROESY data, the values of spin-spin coupling constants and some chemical experiments. The absolute configuration of acrucipentyn A was determined using the Mosher’s method. The absolute configurations of the new anthraquinones were established based on a comparison of the experimental and theoretical ECD spectra. It has been shown that acruccipentyns do not exhibit cytotoxic activity against normal human cells, as well as against tumor cells of human prostate cancer. However, all new compounds have a pronounced antibiotic effect. Acrucipentyns A and F have the most pronounced antimicrobial activity against gram-positive bacteria S. aureus. Acrucipentyn F almost completely inhibits the growth of S. aureus at a concentration of 100.0 mkM. Acrucipentyn A at a concentration of 100.0 mkM reduced bacterial growth by 60%. Acrucipentyn C at a concentration of 100.0 mkM reduced the growth of S. aureus by 50%. The highest antibacterial activity against E. coli was found in acruciquinone (42%), quadricinctapyran A (39%), acrucipentyn D (37%), dendriol B (33%), acrucipentyn B (33%), 3,9,13- isodendriol D (32%), 9,13-isodendriol A (30%). It was noted that most of the secondary metabolites of this fungus inhibited the growth of yeast (C. albicans) by more than 50% at the maximum studied concentration of 100.00 mkM. Acrucipentyn D, the most active in this test, showed an MIC50 of 25 mkM; acruccipentyns B, C, E, 9,13-isodendriol A, and coniothyrinones B and D had an MIC50 of 50.0 mkM. It has been established that some substances from the fungus Asteromyces cruciatus KMM 4696 have a pronounced cytoprotective effect in an in vitro model of bacterial skin lesions. Thus, acrucipentyns A-F at a concentration of 10 mkM reduced the yield of the LDH enzyme from HaCaT cells incubated with S. aureus by 17.4%, 18.6%, 8.9%, 18.8%, 24.1%, and 9.1%, respectively. The anthraquinones 9,10-anthracenedione, coniothyrinone D, and dendriol B decreased the LDH yield by 18.7%, 13.7%, and 12.3%, respectively. It was shown that acrucipentyns A, C and E at a concentration of 50 mkM showed a sufficiently high inhibitory activity against the sortase A enzyme: 18.23 ± 0.32, 29.95 ± 0.57 and 20.72 ± 0.72%, respectively, which also indicates their antimicrobial activity. Three new peptides, asterripeptides A-C, were isolated from an ethyl acetate extract of the fungus Aspergillus terreus (leaves of an unidentified mangrove tree). For asterripeptides, the absolute configuration of all stereocenters was established using the Murfey’s method. Compounds isolated from a strain of the marine fungus Aspergillus terreus have shown themselves to be broad-spectrum antimicrobial agents. The highest activity against gram-negative and gram-positive bacteria was shown by asterripeptide A, which inhibited the growth of bacteria at a concentration of 100.0 mkM by 52.8 and 57.3%, respectively. Also, asterripeptides inhibited the growth of yeast (C. albicans) by more than 50% at concentrations of 50.0 and 100.0 µM. It was shown that asterripeptides B and C at a concentration of 50 mkM showed a sufficiently high inhibitory activity against the sortase A enzyme: 20.67 ± 0.18 and 22.36 ± 0.21%, respectively, which also indicates their antimicrobial activity. It was shown that asterripeptides B and C, in contrast to asterripeptide A, had a positive effect on the state of HaCaT keratinocyte cells incubated with a suspension of S. aureus. Under the action of these substances at a concentration of 10 µM, the release of LDH from HaCaT cells decreased by 20.2% and 22.3%, respectively. Three new meroterpenoids of the andrastin-type of meroantarctines A-C containing three unique 6/5/6/6, 6/5/6/5/6 and 6/5/6/5 polycyclic systems, seven new derivatives of ß-resorcylic acid, a new 5-(2-Hydroxyethyl)-4-(hydroxymethyl)dihydrofuran-2(3H)-one was isolated from the fungus Penicillium antarcticum KMM 4685 (brown seaweed Sargassum miyabei, Sea of Japan). The structures of all compounds were established based on the analysis of high-resolution mass spectra HRESIMS, data from 1D and 2D NMR spectra, and analysis of literature data. The relative configuration of the meroantarctine A was established on the basis of X-ray diffraction data, and the absolute configuration, on the basis of quantum-chemical calculations of ECD spectra. For the novel cladosporin analogue 14-hydroxyasperentin B, the absolute configuration of the chiral centers was determined using the Mosher’s method. Three derivatives of ß-resorcylic acid were shown to exhibit moderate cytotoxic activity against LNCaP, DU145, and 22Rv1 cells. At the same time, one of these derivatives 2,3,4-trihydroxybutyl-2,4-dihydroxy-6-(6-oxoheptyl)benzoate and meroantarctine A showed significant inhibitory activity of p-glycoprotein already at concentrations of 5 and 1.56 µM, respectively, which determines their ability to resensitize drug-resistant PC3-DR cells to the action of docetaxel. The new meroterpenoid meroantartartine A can be considered as a promising candidate for combination therapy of drug-resistant prostate cancer. Secondary metabolites isolated from the marine fungus Penicillium antarcticum are promising antimicrobial agents with a wide spectrum of biological activity. The greatest inhibition of the growth of gram-positive bacteria was noted for the compound citreohybriddione B (39% at a concentration of 100.0 mkM). The most pronounced antibacterial effect against gram-negative bacteria was also shown for citreohybriddionone B, which completely inhibits the growth of E. coli at a concentration of 100.0 mkM. The compounds citreohybriddion B and 5'-hydroxyasperentin inhibited the growth of C. albicans by 58% at the highest concentration tested. Cladosporin inhibited yeast growth by 54% at a concentration of 100.0 mkM. It was shown that cladosporin isolated from this strain at a concentration of 10 mkM reduced the LDH output from HaCaT cells incubated with S. aureus by 27.7%, while at the same concentration meroantartartine A reduced the LDH output only by 18.5%. Cladasporin at a concentration of 50 mkM showed a fairly high inhibitory activity against the sortase A enzyme (29.78 ± 0.28%), which also indicates its antimicrobial activity against S. aureus. Secondary metabolites 5'-hydroxyasperentine, ethyl 3-(6,8-dihydroxy-1-oxoisochroman-3-yl)propanoate and methyl 3-(6,8-dihydroxy-1-oxoisochroman-3-yl)propanoate inhibited the activity urease enzyme by 50% at concentrations of 2.97, 8.58 and 10.99 mkM, respectively. These compounds showed a high inhibitory activity against the urease enzyme and, undoubtedly, are promising for further study as urease inhibitors. Urease activity is an important pathogenic factor, and ten of the twelve antibiotic-resistant pathogens designated by the WHO as "priority pathogens" are in fact ureolytic. Approaches to the targeted chemical modification of cladosporin isolated from the fungus Penicillium attenuatum KMM 4671 were studied, six new individual compounds were obtained, five of which were tested for antimicrobial, radical-binding, and enzyme-inhibiting activities. It has been shown that semi-synthetic bromine-containing compounds obtained by chemical modifications from cladosporin have a sufficiently high activity against S. aureus and E. coli.

 

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Annotation of the results obtained in 2019
During ourongoing investigation for the project"Marine micromycetes - a promising source of bioactive molecules" No. 19-74-10014from on 9 July2019 to 15 May 2020 the following studies were carried out: Antimicrobial activity of 42 marine fungal strains from the Collection of Marine Microorganisms (КMM) of PIBOC FEB RAS and the collection of Nha Trang Institute for Scientific Research and Applied Technologies (Nha Trang, Vietnam) was tested for five opportunistic test cultures. Seven fungal strains, promising sources of substances with high antibiotic activity, were identified. The extracts of the fungi were also analyzed by TLC. The conditions for preparative cultivation ofAsteromycescruciatus (brown alga Sargassum pallidum, Sea of Japan),Penicillium attenuatum KMM 4671 (sediment, Sea of Okhotsk) and Aspergillus flaschentraegeri (unidentified sponge, South China Sea) were selectedfor further work in 2020-2021 years. Ten new diterpene glycosides virescenosides Z9-Z18 together with three known analogues and aglycon of virescenoside A were isolated from the marine-derived fungus Acremonium striatisporum KMM 4401. These compounds were obtained by cultivating fungus on wort agar medium with the addition of potassium bromide. Structures of the isolated metabolites were established based on spectroscopic methods. Virescenoside Z9 was structurally identified as altroside having isopimaradiene-type aglycon with additional oxidation patterns. Virescenosides Z12-Z16 contain unique methyl esters of altruronic acid 4C1 and 1C4 conformations as their carbohydrate components. Virescenoside Z18 exhibit an unusual methyl ester of mannuronic acid as sugar moiety. Virescenoside Z10 at a concentration of 10 µM decreased the ROS content in macrophages by 45%.Virescenoside Z13 and aglycone of virescenoside C at a concentration of 1 µM induced moderate inhibition of NO production in LPS-stimulated macrophages by 20% and 23%, respectively. Six carotane sesquiterpenoids piltunines A‒F together with known compounds were isolated from the fungus Penicillium piltunense KMM 4668 obtained from marine subaqueous soils. The absolute configurations of compounds were determined using a combined CD and NOESY data as well as biogenetic considerations. Piltunine C was established as a 13-hydroxy derivative of aspterric acid. Piltunines E and F contain the 9-en-11-one and 6-en-11-one enone chromophores in structure of molecules. Piltunine A is reported here for first time as a natural product. Some of the isolated compounds exhibited cytotoxic activity in human drug resistant prostate cancer cells as well as were able to inhibit ROS and NO production in LPS-stimulated macrophages.Piltunine E and 5'-hydroxyasperentin were shown to cause significant inhibition of ROS production by 35% and 40%, respectively. The 5'-hydroxyasperentin compound inhibited the production of NO in LPS-stimulated macrophages at a non-toxic concentration of 1 µM. The NO level in these cells decreased by 24.1 ± 0.7% (p <0.05, Student's test) compared with the control. From an ethyl acetate extract of Aspergillus flocculosus fungus isolated from a sediment sample collected in Nha Trang Bay (South China Sea, Vietnam), the structures of two new drimane sesquiterpenoids were established, as well as two well-known related metabolites were identified.

 

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Annotation of the results obtained in 2020
In the period from May 16, 2020 to May 15, 2021, within the framework of the project "Marine fungi-micromycetes - a promising source of bioactive molecules" No. 19-74-10014, the following research works were carried out: Screening of the antimicrobial activity of 137 strains of fungi of marine habitats from the Collection of Marine Microorganisms (KMM) of the TIBOC FEB RAS and the collection of the Nha Trang Institute of Scientific Research and Applied Technologies (Nha Trang, Vietnam) in relation to five opportunistic test cultures was carried out. Four strains were selected for analytical cultivation, showing activity against Staphylococcus aureus and Pseudomonas aeruginosa, their extracts were analyzed by thin layer chromatography. The conditions for preparative cultivation of micromycete fungi Penicillium ochotense KMM 4670 (ground, Sea of Okhotsk) Penicillium dimorphosporum KMM 4689 (unidentified soft coral, South China Sea) and Aspergillus melleus KMM 4687 (unidentified sponge, South China Sea) were selected. Preparative cultivation of five strains of marine micromycete fungi was carried out: Penicillium attenuatum KMM 4671, Asteromyces cruciatus KMM 4696 and Aspergillus flaschentraegeri 1901NT-1.2.2, Penicillium dimorphosporum KMM 4689 and Aspergillus melleus KMM 4687. Two new chlorine-containing polyketides acrucipentins A and B, a new anthraquinone 1,2-isodendriol A, and eight known metabolites of a predominantly polyketid nature were isolated from an extract of the culture of the obligate marine fungus micromycete Asteromyces cruciatus KMM 4696. The compounds were obtained by growing the culture of the fungus on the medium containing potassium bromide. The structures of the compounds were established by one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry. The new anthraquinone 1,2-isodendriol A exhibited antimicrobial activity against gram-positive bacteria S. aureus with a MIC100 of 25 µM. New chlorine-containing acrucepentins A and B at a concentration of 100 µM inhibited the growth of gram-positive bacteria S. aureus by about 20 and 35%, respectively. The presence of chlorine in the structures of acrucipentin A and B molecules makes the fungus strain Asteromyces cruciatus KMM 4696 a potential target for rehalogenation. Seven new alkaloids belonging to the family of deoxyisoaustamide, as well as three known ones, were isolated from the micromycete fungus Penicillium dimorphosporum KMM 4689 associated with an unidentified soft coral (South China Sea, Vietnam). The structures of the compounds were established by one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry. The absolute stereoconfigurations of the six compounds were established by comparing the experimental CD spectra, as well as on the basis of biogenetic considerations. The protective effect of the isolated compounds against the acute toxicity of paraquat (PQ) on the cells of the mouse neuroblastoma Neuro-2a, which are usually used for neuroprotective studies, was studied. Compounds 16,17-dihydroxydeoxy-dihydroisoaustamide and 16β, 17α-dihydroxydeoxydihydroisoaustamide at a concentration of 1 μM increased the viability of cells treated with paraquat by 38.6% and 30.3%, respectively. Compound 16α, 17α-dihydroxydeoxydihydroisoaustamide increased the viability of these cells by 36.5% and 39.4% at concentrations of 1 μM and 10 μM, respectively. It was shown that this type of activity is observed for alkaloids of this series in the presence of two hydroxyl groups in the proline ring. These compounds did not exhibit radical-binding activity with respect to 2,2-diphenyl-1-picrylhydrazyl (DPPH), which makes them very promising subjects for further study of the mechanism of their neuroprotective activity. For 16β-hydroxy-17α-methoxydeoxydihydroisoaustamide at 100 μM, a statistically significant decrease in the viability of HeLa tumor cells to 70.8% was observed after 48 h of cell treatment with substances. It was also shown that this compound at a non-cytotoxic concentration of 20 μM can reduce the ability on formation of colonies HeLa tumor cells by 60%. Thus, it can be assumed that this compound may also have antimetastatic activity against at least some types of cancer. Three new tripeptide compounds, asterripeptides A-C, were isolated from the ethyl acetate extract of the fungus Aspergillus terreus (leaves of an unidentified mangrove tree). For all three compounds, a flat structure was established using 1D and 2D NMR methods and high-resolution mass spectrometry. For asterripeptide A, the absolute configuration of all stereocenters was established using the Marfey’s method with the addition of L-FDAA-derived α-amino acid standards. The biological activity of the compounds is established.

 

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