Structure-Activity Relationships of Natural C-9-Methyl-Substituted 10-Membered Lactones and Their Semisynthetic Derivatives.

Fungal 10-membered lactones (TMLs), such as stagonolide A, herbarumin I, pinolidoxin, and putaminoxin, are promising candidates for the development of nature-derived herbicides. The aim of this study was to analyze the structure-activity relationships (SAR) of C-9-methyl-substituted TMLs with a multitarget bioassay approach to reveal compounds with useful (phytotoxic, entomotoxic, antimicrobial) or undesirable (cytotoxic) bioactivities. A new TML, stagonolide L (1), along with five known compounds (stagonolides D (2) and E (3), curvulides A (4) and B1/B2 (5a,b), and pyrenolide C (6)), were purified from cultures of the phytopathogenic fungus Stagonospora cirsii, and five semisynthetic derivatives of 3 and 4 (7-11) were obtained. The absolute configuration of 4 was revised to 2Z, 4S, 5S, 6R, and 9R. The identity of 5a,b and stagonolide H is discussed. The phytotoxicity of compound 4, the entomotoxicity of 5a,b, and nonselective toxicity of compound 6 are demonstrated. The latter confirms the hypothesis that the α,ß-unsaturated carbonyl group is associated with the high general toxicity of TML, regardless of its position in the ring and other substituents. The epoxide in compound 4 is important for phytotoxicity. The revealed SAR patterns will be useful for further rational design of TML-based herbicides including curvulide A analogs with a 4,5-epoxy group.

Natural ten-membered lactones: sources, structural diversity, biological activity, and intriguing future.

Covering: 2012 to 2022Ten-membered lactones (TMLs) are an interesting and diverse group of natural polyketides that are abundant in fungi and, to a lesser extent, in bacteria, marine organisms, and insects. TMLs are known for their ability to exhibit a wide spectrum of biological activity, including phytotoxic, cytotoxic, antifungal, antibacterial, and others. However, the random discovery of these compounds by scientific groups with various interests worldwide has resulted in patchy information about their distribution among different organisms and their biological activity. Therefore, despite more than 60 years of research history, there is still no common understanding of the natural sources of TMLs, their structural type classification, and most characteristic biological activities. The controversial nomenclature, incorrect or erroneous structure elucidation, poor identification of producing organisms, and scattered information on the biological activity of compounds - all these factors have led to the problems with dereplication and the directed search for TMLs. This review consists of two parts: the first part (Section 2) covers 104 natural TMLs, published between 2012 and 2022 (after the publishing of the previous review), and the second part (Section 3) summarizes information about 214 TMLs described during 1964-2022 and as a result highlights the main problems and trends in the study of these intriguing natural products.

Tenuazonic acid alters immune and physiological reactions and susceptibility to pathogens in Galleria mellonella larvae.

Tenuazonic acid (TeA) is synthesized by phytopathogenic and opportunistic fungi and is detected in a broad range of foods. This natural compound is of interest in terms of toxicity to animals, but its mechanisms of action on insects are poorly understood. We administered TeA orally at different concentrations (0.2-5.0 mg/[gram of a growth medium]) to the model insect Galleria mellonella, with subsequent estimation of physiological, histological, and immunological parameters in different tissues (midgut, fat body, and hemolymph). Susceptibility of the TeA-treated larvae to pathogenic microorganisms Beauveria bassiana and Bacillus thuringiensis was also analyzed. The feeding of TeA to the larvae led to a substation delay of larval growth, apoptosis-like changes in midgut cells, and an increase in midgut bacterial load. A decrease in activities of detoxification enzymes and downregulation of genes Nox, lysozyme, and cecropin in the midgut and/or hemocoel tissues were detected. By contrast, genes gloverin, gallerimycin, and galiomycin and phenoloxidase activity proved to be upregulated in the studied tissues. Hemocyte density did not change under the influence of TeA. TeA administration increased susceptibility of the larvae to B. bassiana but diminished their susceptibility to B. thuringiensis. The results indicate that TeA disturbs wax moth gut physiology and immunity and also exerts a systemic action on this insect. Mechanisms underlying the observed changes in wax moth susceptibility to the pathogens are discussed.

Effects of Phytotoxic Nonenolides, Stagonolide A and Herbarumin I, on Physiological and Biochemical Processes in Leaves and Roots of Sensitive Plants.

Phytotoxic macrolides attract attention as prototypes of new herbicides. However, their mechanisms of action (MOA) on plants have not yet been elucidated. This study addresses the effects of two ten-membered lactones, stagonolide A (STA) and herbarumin I (HBI) produced by the fungus Stagonospora cirsii, on Cirsium arvense, Arabidopsis thaliana and Allium cepa. Bioassay of STA and HBI on punctured leaf discs of C. arvense and A. thaliana was conducted at a concentration of 2 mg/mL to evaluate phenotypic responses, the content of pigments, electrolyte leakage from leaf discs, the level of reactive oxygen species, Hill reaction rate, and the relative rise in chlorophyll a fluorescence. The toxin treatments resulted in necrotic and bleached leaf lesions in the dark and in the light, respectively. In the light, HBI treatment caused the drop of carotenoids content in leaves on both plants. The electrolyte leakage caused by HBI was light-dependent, in contrast with that caused by STA. Both compounds induced light-independent peroxide generation in leaf cells but did not affect photosynthesis 6 h after treatment. STA (10 µg/mL) caused strong disorders in root cells of A. thaliana leading to the complete dissipation of the mitochondrial membrane potential one hour post treatment, as well as DNA fragmentation and disappearance of acidic vesicles in the division zone after 8 h; the effects of HBI (50 µg/mL) were much milder. Furthermore, STA was found to inhibit mitosis but did not affect the cytoskeleton in cells of root tips of A. cepa and C. arvense, respectively. Finally, STA was supposed to inhibit the intracellular vesicular traffic from the endoplasmic reticulum to the Golgi apparatus, thus interfering with mitosis. HBI is likely to have another main MOA, probably inhibiting the biosynthesis of carotenoids.

Modern Approaches for the Development of New Herbicides Based on Natural Compounds.

Weeds are a permanent component of anthropogenic ecosystems. They require strict control to avoid the accumulation of their long-lasting seeds in the soil. With high crop infestation, many elements of crop production technologies (fertilization, productive varieties, growth stimulators, etc.) turn out to be practically meaningless due to high yield losses. Intensive use of chemical herbicides (CHs) has led to undesirable consequences: contamination of soil and wastewater, accumulation of their residues in the crop, and the emergence of CH-resistant populations of weeds. In this regard, the development of environmentally friendly CHs with new mechanisms of action is relevant. The natural phytotoxins of plant or microbial origin may be explored directly in herbicidal formulations (biorational CHs) or indirectly as scaffolds for nature-derived CHs. This review considers (1) the main current trends in the development of CHs that may be important for the enhancement of biorational herbicides; (2) the advances in the development and practical application of natural compounds for weed control; (3) the use of phytotoxins as prototypes of synthetic herbicides. Some modern approaches, such as computational methods of virtual screening and design of herbicidal molecules, development of modern formulations, and determination of molecular targets, are stressed as crucial to make the exploration of natural compounds more effective.

Structure-Activity Relationship of Phytotoxic Natural 10-Membered Lactones and Their Semisynthetic Derivatives.

Ten-membered lactones (nonenolides) demonstrate phytotoxic, antimicrobial, and fungicidal activity promising for the development of natural product-derived pesticides. The fungus Stagonospora cirsii is able to produce phytotoxic stagonolides A (1), J (2), K (3) and herbarumin I (4) with high yield. The aim of this study was to create a set of structurally related nonenolides and to reveal the structural features that affect their biological activity. Stagonolide A (1) and C-7 oxidized stagonolide K (11) showed the highest phytotoxicity in leaf puncture assay and agar seedlings assay. The oxidation of C-7 hydroxyl group (as in 1, acetylstagonolide A (10) and (11) led to the manifestation of toxicity to microalgae, Bacillus subtilis and Sf9 cells regardless of the configuration of C-9 propyl chains (R in 1 and 10, S in 11). C-7 non-oxidized nonenolides displayed none or little non-target activity. Notably, 7S compounds were more phytotoxic than their 7R analogues. Due to the high inhibitory activity against seedling growth and the lack of side toxicity, mono- and bis(acetyl)- derivatives of herbarumin I were shown to be potent for the development of pre-emergent herbicides. The identified structural features can be used for the rational design of new herbicides.

Entomotoxic Activity of the Extracts from the Fungus, Alternaria tenuissima and Its Major Metabolite, Tenuazonic Acid.

The study of fungal antibiotics in their competitive interactions with arthropods may lead to the development of novel biorational insecticides. Extracts of Alternaria tenuissima MFP253011 obtained using various methods showed a wide range of biological activities, including entomotoxic properties. Analysis of their composition and bioactivity allowed us to reveal several known mycotoxins and unidentified compounds that may be involved in the entomotoxic activity of the extracts. Among them, tenuazonic acid (TeA), which was the major component of the A. tenuissima extracts, was found the most likely to have larvicidal activity against Galleria mellonella. In the intrahaemocoel injection bioassay, TeA was toxic to G. mellonella and of Zophobas morio with an LT50 of 6 and 2 days, respectively, at the level of 50 µg/larva. Administered orally, TeA inhibited the growth of G. mellonella larvae and caused mortality of Acheta domesticus adults (LT50 7 days) at a concentration of 250 µg/g of feed. TeA showed weak contact intestinal activity against the two phytophages, Tetranychus urticae and Schizaphis graminum, causing 15% and 27% mortality at a concentration of 1 mg/mL, respectively. TeA was cytotoxic to the Sf9 cell line (IC50 25 µg/mL). Thus, model insects such as G. mellonella could be used for further toxicological characterization of TeA.

Destruxin A Interacts with Aminoacyl tRNA Synthases in Bombyx mori.

Destruxin A (DA), a hexa-cyclodepsipeptidic mycotoxin produced by the entomopathogenic fungus Metarhizium anisopliae, exhibits insecticidal activities in a wide range of pests and is known as an innate immunity inhibitor. However, its mechanism of action requires further investigation. In this research, the interactions of DA with the six aminoacyl tRNA synthetases (ARSs) of Bombyx mori, BmAlaRS, BmCysRS, BmMetRS, BmValRS, BmIleRS, and BmGluProRS, were analyzed. The six ARSs were expressed and purified. The BLI (biolayer interferometry) results indicated that DA binds these ARSs with the affinity indices (KD) of 10-4 to 10-5 M. The molecular docking suggested a similar interaction mode of DA with ARSs, whereby DA settled into a pocket through hydrogen bonds with Asn, Arg, His, Lys, and Tyr of ARSs. Furthermore, DA treatments decreased the contents of soluble protein and free amino acids in Bm12 cells, which suggested that DA impedes protein synthesis. Lastly, the ARSs in Bm12 cells were all downregulated by DA stress. This study sheds light on exploring and answering the molecular target of DA against target insects.

The Metarhizium anisopliae Toxin, Destruxin A, Interacts with the SEC23A and TEME214 Proteins of Bombyx mori.

Destruxin A (DA), a mycotoxin isolated from the entomopathogenic fungus Metarhizium anisopliae, has good insecticidal and immune-inhibitory activity, but the action mechanism has not yet been elucidated. In order to identify the DA-binding proteins, we conducted drug affinity responsive target stability (DARTS) experiments, which indicated that the silkworm's (Bombyx mori) transmembrane protein 214 (BmTEME214) and protein transport protein SEC23A isoform X2 (BmSEC23) are the potential DA-binding proteins. The current research was focused on validation of the interaction between DA and these two proteins via bio-layer interferometry (BLI) in vitro, insect two-hybrid (I2H) in Sf9 cells, and RNAi in the insect. The results of the BLI tests showed that DA has strong affinity to bind BmTEME214 and BmSEC23 proteins with a KD value of 0.286 and 0.291 µM, respectively. In the I2H experiments, DA inhibited (at 0.02 µg/mL) and activated (at 0.002-0.0002 µg/mL) the protein interactions of BmSEC23-BmSEC13, but it only inhibited the BmTMEM214-BmSEC13L interaction. Furthermore, in the RNAi tests, an apparent increase in the silkworm's mortality was recorded in the joint treatment of DA with dsBmSEC23 or dsBmTMEM214 when compared with the single treatment of DA (1.5 µg/g body), 40 µg/g body dsBmSEC23, or dsBmTMEM214. This research confirmed that BmSEC23 and BmTMEM214 are the DA-binding proteins and provided new insights to understand the action mechanism of DA.

The Chemical Ecology Approach to Reveal Fungal Metabolites for Arthropod Pest Management.

Biorational insecticides (for instance, avermectins, spinosins, azadirachtin, and afidopyropen) of natural origin are increasingly being used in agriculture. The review considers the chemical ecology approach for the search for new compounds with insecticidal properties (entomotoxic, antifeedant, and hormonal) produced by fungi of various ecological groups (entomopathogens, soil saprotrophs, endophytes, phytopathogens, and mushrooms). The literature survey revealed that insecticidal metabolites of entomopathogenic fungi have not been sufficiently studied, and most of the well-characterized compounds show moderate insecticidal activity. The greatest number of substances with insecticidal properties was found to be produced by soil fungi, mainly from the genera Aspergillus and Penicillium. Metabolites with insecticidal and antifeedant properties were also found in endophytic and phytopathogenic fungi. It was noted that insect pests of stored products are mostly low sensitive to mycotoxins. Mushrooms were found to be promising producers of antifeedant compounds as well as insecticidal proteins. The expansion of the number of substances with insecticidal properties detected in prospective fungal species is possible by mining fungal genomes for secondary metabolite gene clusters and secreted proteins with their subsequent activation by various methods. The efficacy of these studies can be increased with high-throughput techniques of extraction of fungal metabolites and their analysis by various methods of chromatography and mass spectrometry.

Effect of Adjuvants on Herbicidal Activity and Selectivity of Three Phytotoxins Produced by the Fungus, Stagonospora cirsii.

The use of many fungal phytotoxins as natural herbicides is still limited because they cannot penetrate leaf cuticle without injury and a little is known on their selectivity. In order to assess the herbicidal potential of phytotoxic 10-membered lactones (stagonolide A, stagonolide K, and herbarumin I), the selection of adjuvants, the evaluation of selectivity of the toxins and the efficacy of their formulations were performed. Among four adjuvants tested, Hasten™ (0.1%, v/v) increased phytotoxic activity of all the toxins assayed on non-punctured leaf discs of Sonchus arvensis. When assayed on intact leaf fragments of 18 plants species, 10 species were low to moderately sensitive to stagonolide A, while just five and three species were sensitive to stagonolide K and herbarumin I, respectively. Both leaf damage or addition of Hasten™ (0.1%) to the formulations of the compounds considerably increased or altered the sensitivity of plants to the toxins. Stagonolide A was shown to be non-selective phytotoxin. The selectivity profile of stagonolide K and herbarumin I depended on the leaf wounding or the adjuvant addition. Stagonolide A and herbarumin I formulated in 0.5% Hasten™ showed considerable herbicidal effect on S. arvensis aerial shoots. This study supported the potential of the oil-based adjuvant Hasten™ to increase the herbicidal efficacy of natural phytotoxins.

Isolation and Bioactivity of Secondary Metabolites from Solid Culture of the Fungus, Alternaria sonchi.

The fungus, Alternaria sonchi is considered to be a potential agent for the biocontrol of perennial sowthistle (Sonchus arvensis). A new chlorinated xanthone, methyl 8-hydroxy-3-methyl-4-chloro-9-oxo-9H-xanthene-1-carboxylate (1) and a new benzophenone derivative, 5-chloromoniliphenone (2), were isolated together with eleven structurally related compounds (3-13) from the solid culture of the fungus, which is used for the production of bioherbicidal inoculum of A. sonchi. Their structures were determined by spectroscopic (mostly by NMR and MS) methods. Alternethanoxins A and B, which were reported in A. sonchi earlier, were re-identified as moniliphenone and pinselin, respectively. The isolated compounds were tested for phytotoxic, antimicrobial, insecticidal, cytotoxic and esterase-inhibition activities. They did not demonstrate high phytotoxicity (lesions up to 2.5 mm in diameter/length at a concentration of 2 mg/mL) when tested on leaf disks/segments of perennial sowthistle (Sonchus arvensis) and couch grass (Elytrigia repens). They did not possess acute toxicity to Paramecium caudatum, and showed moderate to low cytotoxicity (IC50 > 25 µg/mL) for U937 and K562 tumor cell lines. However, chloromonilicin and methyl 3,8-dihydroxy-6-methyl-4-chloro-9-oxo-9H-xanthene-1-carboxylate (4) were shown to have antimicrobial properties with MIC 0.5-5 µg/disc. Compound 4 and chloromonilinic acid B were found to have contact insecticidal activity to wheat aphid (Schizaphis graminum) at 1 mg/mL. Compounds 2 and methyl 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate displayed selective carboxylesterase inhibition activity at concentration of 100 µg/mL. Therefore, the waste solid substrate for production of A. sonchi spores can be re-utilized for the isolation of a number of valuable natural products.

Stagonolides J and K and Stagochromene A, Two New Natural Substituted Nonenolides and a New Disubstituted Chromene-4,5-dione Isolated from Stagonospora cirsii S-47 Proposed for the Biocontrol of Sonchus arvensis.

Two new natural 10-membered macrolides (1, 2) and one chromene-4,5-dione derivative (3), named stagonolides J and K and stagochromene A, respectively, were isolated from the phytopathogenic fungus Stagonospora cirsii S-47, together with two known compounds, stagonolide A (4) and herbarumin I (5). Stagonolides J and K and stagochromene A were characterized as (5E,7R*,8S*,9R*)-7,8-dihydroxy-9-propyl-5-nonen-9-olide, (5E,7R,9S)-7-hydroxy-9-propyl-5-nonen-9-olide, and (2R*,3R*)-3-hydroxy-2-propyltetrahydro-2H-chromene-4,5(3H,4aH)-dione, respectively, by spectroscopic (mostly by NMR and ESIMS) data. Compounds 1-5 showed different rates of phytotoxic activity on punctured leaf discs of Sonchus arvensis. The antimicrobial, cytotoxic, and antiprotozoal activity of isolated compounds was also evaluated. Based on our data, stagonolide K and herbarumin I can be proposed as a potential scaffold for the development of a new natural herbicide and estimated as possible selection/quality markers of a bioherbicide based on S. cirsii, while stagonolide A can be considered as a mycotoxin.

Synthesis and Biological Evaluation of Phaeosphaeride A Derivatives as Antitumor Agents.

New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor activity studies were carried out on the HCT-116, PC3, MCF-7, A549, К562, NCI-Н929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All of the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 was potent against six cancer cell lines, HCT-116, PC-3, K562, NCI-H929, Jurkat, and RPMI8226, showing a 47, 13.5, 16, 4, 1.5, and 7-fold increase in anticancer activity comparative to those of etoposide, respectively. Compound 1 possessed selectivity toward the NCI-H929 cell line (IC50 = 1.35 ± 0.69 µM), while product 7 was selective against three cancer cell lines, HCT-116, MCF-7, and NCI-H929, each having IC50 values of 1.65 µM, 1.80 µM and 2.00 µM, respectively.

Curvulin and Phaeosphaeride A from Paraphoma sp. VIZR 1.46 Isolated from Cirsium arvense as Potential Herbicides.

Phoma-like fungi are known as producers of diverse spectrum of secondary metabolites, including phytotoxins. Our bioassays had shown that extracts of Paraphoma sp. VIZR 1.46, a pathogen of Cirsium arvense, are phytotoxic. In this study, two phytotoxically active metabolites were isolated from Paraphoma sp. VIZR 1.46 liquid and solid cultures and identified as curvulin and phaeosphaeride A, respectively. The latter is reported also for the first time as a fungal phytotoxic product with potential herbicidal activity. Both metabolites were assayed for phytotoxic, antimicrobial and zootoxic activities. Curvulin and phaeosphaeride A were tested on weedy and agrarian plants, fungi, Gram-positive and Gram-negative bacteria, and on paramecia. Curvulin was shown to be weakly phytotoxic, while phaeosphaeride A caused severe necrotic lesions on all the tested plants. To evaluate phaeosphaeride A's herbicidal efficacy, the phytotoxic activity of this compound in combination with five different adjuvants was studied. Hasten at 0.1% (v/v) was found to be the most potent and compatible adjuvant, and its combination with 0.5% (v/v) semi-purified extract of Paraphoma sp. VIZR 1.46 solid culture exhibited maximum damage to C. arvense plants. These findings may offer significant importance for further investigation of herbicidal potential of phaeosphaeride A and possibly in devising new herbicide of natural origin.

On the metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin A.

Ambrosia artemisiifolia L. is responsible for serious allergies induced on humans. Different approaches for its control were proposed during the COST Action FA1203 "Sustainable management of Ambrosia artemisiifolia in Europe" (SMARTER). Fungal secondary metabolites often show potential herbicidal activity. Three phytotoxins were purified from the fungal culture filtrates of Colletotrichum gloeosporioides, isolated from infected leaves of A. artemisiifolia. They were identified by spectroscopic and chemical methods as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute configuration 6'R to colletochlorin A was assigned for the first time applying the advanced Mosher's method. When assayed by leaf-puncture on A. artemisiifolia only 1 caused the appearance of large necrosis. The same symptoms were also induced by 1 on ambrosia plantlets associated with plant wilting. On Lemna minor, colletochlorin A caused a clear fronds browning, with a total reduction in chlorophyll content.

Biological evaluation and determination of the absolute configuration of chloromonilicin, a strong antimicrobial metabolite isolated from Alternaria sonchi.

Chloromonilicin was isolated for the first time from Alternaria sonchi, a mycoherbicide proposed for the control of the noxious weed Sonchus arvensis. The already known alternethanoxins A and B and the three recently isolated phytotoxic polycyclic ethanones named alternethanoxins C-E were also isolated from the same source. Chloromonilicin was identified by spectroscopic data (essentially one-dimensional NMR, 2-dimensional NMR and high-resolution ESI-MS) and its structure was confirmed by single X-ray analysis, which also allowed the assignment of the absolute configuration. This latter was independently confirmed by electronic CD calculations. When chloromonilicin was tested for its antimicrobial activity, it was active at concentrations 0.5-1 µg per disc against four bacterial species and a yeast fungus. The compound inhibited conidial germination of four plant pathogens at concentration of 1-10 µg ml(-1). No phytotoxic activity of this antibiotic by leaf-disc puncture bioassay was detected.

Synthesis of natural phaeosphaeride A derivatives and an in vitro evaluation of their anti-cancer potential.

Derivatives of phaeosphaeride A (PPA) were synthesised and characterised; then anti-cancer studies were carried out on the A549 cancer cell line. It was found that the acetyl derivative (compound 3) displayed comparable in vitro cytotoxicity to that of PPA (EC50=49±7 µM and EC50=46±5 µM, respectively), while chloroacetyl derivative 6 (EC50=33±7 µM) was found to have better efficacy towards the A549 cancer cell line.

Crystal structure of natural phaeosphaeride A.

The asymmetric unit of the title compound, C15H23NO5, contains two independent mol-ecules. Phaeosphaeride A contains two primary sections, an alkyl chain consisting of five C atoms and a cyclic system consisting of fused five- and six-membered rings with attached substituents. In the crystal, the mol-ecules form layered structures. Nearly planar sheets, parallel to the (001) plane, form bilayers of two-dimensional hydrogen-bonded networks with the hy-droxy groups located on the inter-ior of the bilayer sheets. The network is constructed primarily of four O-H⋯O hydrogen bonds, which form a zigzag pattern in the (001) plane. The butyl chains inter-digitate with the butyl chains on adjacent sheets. The crystal was twinned by a twofold rotation about the c axis, with refined major-minor occupancy fractions of 0.718 (6):0.282 (6).

Alternethanoxins C-E, Further Polycyclic Ethanones Produced by Alternaria sonchi, a Potential Mycoherbicide for Sonchus arvensis Biocontrol.

Three new polycyclic ethanones, named alternethanoxins C-E, were isolated together with the well-known and closely related alternethanoxins A and B, from the solid culture of Alternaria sonchi, a fungal pathogen proposed for perennial sowthistle (Sonchus arvensis L.) biocontrol. Alternethanoxins C-E were characterized by spectroscopic methods (essentially NMR and HRESI MS) as 2'-dihydroxymethyl-2,5,6,6'-tetrahydroxy-3'-methoxy-biphenyl-3-carboxylic acid methyl ester, 1,4,6,9,10-pentahydroxy-7-methoxy-6H-benzo[c]chromene-2-carboxylic acid methyl ester, and 7,9-dihydroxy-2-methoxy-9H-4,8-dioxa-cyclopenta[def]phenanthrene-5-carboxylic acid methyl ester. When assayed on leaf segments of weeds (Sonchus arvensis and Elytrigia repens), alternethanoxins A and C showed phytotoxic activity inducing notable necrotic lesions. Alternethanoxins C and D possess notable antimicrobial activity when tested against Bacillus subtilis (MIC 10 µg/disc) and Candida tropicalis (MIC 25 µg/disc). Alternethanoxins A and B had low activity against these microbes, while alternethanoxin E was inactive.