Marco Masi

C1,C2-ether derivatives of the Amaryllidaceae alkaloid lycorine: retention of activity of highly lipophilic analogues against cancer cells.

As a continuation of the studies aimed at the development of new anticancer agents derived from the Amaryllidaceae alkaloid lycorine, 35 C1,C2-ether analogues of this natural product were synthesized. The compounds were evaluated for antiproliferative activities in vitro in a panel of tumor cell lines with varied levels of apoptosis resistance. A strong correlation between the compound lipophilicity and anticancer activity was observed, indicating that cell permeability properties must be an important determinant in the design of lycorine-based anticancer agents. A theoretical docking model, consistent with the experimental observations, is presented.

In vitro antibacterial activity of sphaeropsidins and chemical derivatives toward Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight.

Sphaeropsidin A, the main phytotoxin produced by Diplodia cupressi, as well as the two natural analogues sphaeropsidins B and C and 14 derivatives obtained by chemical modifications were assayed for antibacterial activity against Xanthomonas oryzae pv. oryzae, Pseudomonas fuscovaginae, and Burkholderia glumae, the causal agents of severe bacterial rice diseases. The results showed a strong and specific activity of sphaeropsidin A against X. oryzae pv. oryzae, while no activity was observed against the other two pathogens. The results of structure-activity relationship studies showed that structural features important to impart this antibacterial activity are the presence of the C-7 carbonyl group and the hemiketalic lactone functionality. The C-13 vinyl group, the double bond of ring C, and/or the tertiary C-9 hydroxy group, as well as the pimarane arrangement of the tricylic carbon skeleton, were also important for the antibacterial activity. These findings may be useful in designing novel compounds for practical applications in agriculture.

Effect of strain and cultural conditions on the production of cytochalasin B by the potential mycoherbicide Pyrenophora semeniperda (Pleosporaceae, Pleosporales)

The seed pathogen Pyrenophora semeniperda has demonstrated potential as a mycoherbicidal biocontrol for eliminating persistent seed banks of annual bromes on western North American rangelands. This pathogen exhibits variation in virulence that is related to mycelial growth rate, but direct laboratory tests of virulence on seeds often have low repeatability. We developed a rapid and sensitive high pressure liquid chromatography method for quantification of the phytotoxin cytochalasin B in complex mixtures in order to evaluate its use in virulence screening. All 10 strains tested produced large quantities of this metabolite in solid wheat seed culture, with production varying over a fourfold range (535–2256 mg kg−1). No cytochalasin B was produced in liquid potato dextrose broth culture, showing that its synthesis is strongly dependent on cultural conditions. In a Bromus tectorum coleoptile bioassay, solid culture extracts showed mild toxicity similar to the cytochalasin B standard at a concentration equivalent to 10−4 M cytochalasin B (72–95% of control), whereas at 10−3 M equivalent, the extracts exhibited significantly higher toxicity (8–18% of control) than the cytochalasin B standard (34% of control). This suggests the possible presence of other phytotoxic metabolites. Cytochalasin B production in solid wheat seed culture exhibited the predicted significant negative correlation with mycelial growth rate on potato dextrose agar, but the correlation was not very strong, possibly because cytochalasin B production and growth rate were measured under different cultural conditions.

Insights on the susceptibility of plant pathogenic fungi to phenazine-1-carboxylic acid and its chemical derivatives

Pseudomonas chlororaphis subsp. aureofaciens strain M71 produced two phenazine compounds as main secondary metabolites. These metabolites were identified as phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH P). In this study, the spectrum of the activity of PCA and 2-OH P was evaluated against a group of crop and forestal plant pathogenic fungi by an agar plate bioassay. PCA was active against most of the tested plant pathogens, while 2-OH P slightly inhibited a few fungal species. Furthermore, four semisynthesised derivatives of PCA (phenazine-1-carboxymethyl, phenazine-1-carboxamide, phenazine-1-hydroxymethyl and phenazine-1-acetoxymethyl) were assayed for their antifungal activity against 11 phytopathogenic species. Results showed that the carboxyl group is a structural feature important for the antifungal activity of PCA. Since the activity of phenazine-1-carboxymethyl and phenazine-1-carboxamide, the two more lipophilic and reversible PCA derivatives remained substantially unaltered compared with PCA.

Pyrenophoric Acids B and C, Two New Phytotoxic Sesquiterpenoids Produced by Pyrenophora semeniperda

Two new phytotoxic sesquiterpenoid acids, named pyrenophoric acids B and C, were isolated together with the related pyrenophoric and abscisic acids from solid Bromus tectorum (cheatgrass) seed culture of the seed pathogen Pyrenophora semeniperda. This fungus has been proposed as a mycoherbicide for biocontrol of cheatgrass (Bromus tectorum), a Eurasian annual grass that has become invasive in rangelands and is also a serious agricultural weed in the western U.S. Pyrenophoric acids B and C were characterized by spectroscopic methods (NMR and HR ESIMS) as (2Z,4E)-5-[(1R*,4R*,6R*)-1,4-dihydroxy-2,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dienoic and (2Z,4E)-5-[(1S*,3S*,4R*,6S*)-3,4-dihydroxy-2,2,6-trimethylcyclohexyl]-3-methylpenta-2,4-dienoic acids, respectively. Cytochalasins A, B, F, and Z3, as well as deoxaphomin and pyrenophoric acid, all previously isolated from P. semeniperda grown on wheat seed, were also isolated from cheatgrass seed culture. In a cheatgrass seedling bioassay at 10(-3) M, pyrenophoric acid B showed higher coleoptile toxicity than pyrenophoric acid, while pyrenophoric acid C showed lower phytotoxicity. Abscisic acid was by far the most active compound.

Afritoxinones A and B, dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea

Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(-)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR(*),6R(*),7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed.

Fungal metabolite ophiobolin A as a promising anti-glioma agent: In vivo evaluation, structure-activity relationship and unique pyrrolylation of primary amines.

Glioblastoma, the most common form of malignant primary brain tumor, is characterized by resistance to apoptosis, which is largely responsible for the low effectiveness of the classical chemotherapeutic approaches based on apoptosis induction in cancer cells. Previously, a fungal secondary metabolite ophiobolin A was found to have significant activity against apoptosis-resistant glioblastoma cells through the induction of a non-apoptotic cell death, thus, offering an innovative strategy to combat this type of cancer. The current work describes the results of a preliminary evaluation of ophiobolin A in an in vivo glioblastoma model and its chemical derivatization to establish first synthetically generated structure-activity relationship. The synthetic work has also led to the discovery of a unique reaction of ophiobolin A with primary amines suggesting the possibility of pyrrolylation of lysine residues on its intracellular target protein(s).

Pyrenophoric Acid, a Phytotoxic Sesquiterpenoid Penta-2,4-dienoic Acid Produced by a Potential Mycoherbicide, Pyrenophora semeniperda

A new phytotoxic sesquiterpenoid penta-2,4-dienoic acid, named pyrenophoric acid, was isolated from solid wheat seed culture of Pyrenophora semeniperda, a fungal pathogen proposed as a mycoherbicide for biocontrol of cheatgrass (Bromus tectorum) and other annual bromes. These bromes are serious weeds in winter cereals and also on temperate semiarid rangelands. Pyrenophoric acid was characterized as (2Z,4E)-5-[(7S,9S,10R,12R)-3,4-dihydroxy-2,2,6-trimethylcyclohexyl)]-3-methylpenta-2,4-dienoic acid by spectroscopic and chemical methods. The relative stereochemistry of pyrenophoric acid was assigned using 1H,1H couplings and NOESY experiments, while its absolute configuration was determined by applying the advanced Moshers method. Pyrenophoric acid is structurally quite closely related to the plant growth regulator abscisic acid. When bioassayed in a cheatgrass coleoptile elongation test at 10(-3) M, pyrenophoric acid showed strong phytotoxicity, reducing coleoptile elongation by 51% relative to the control. In a mixture at 10(-4) M, its negative effect on coleoptile elongation was additive with that of cytochalasin B, another phytotoxic compound found in the wheat seed culture extract of this fungus, demonstrating that the extract toxicity observed in earlier studies was due to the combined action of multiple phytotoxic compounds.

Sphaeropsidones, phytotoxic dimedone methyl ethers produced by diplodia cupressi : a structure−activity relationship study

Sphaeropsidone and episphaeropsidone are two phytotoxic dimedone methyl ethers produced by Diplodia cupressi, the causal agent of a canker disease of cypress in the Mediterranean area. In this study, eight derivatives obtained by chemical modifications and two natural analogues were assayed for phytotoxic and antifungal activities, and a structure-activity relationship was examined. Each compound was tested on nonhost plants and on five fungal pathogenic species belonging to the genus Phytophthora. The results provide insights into structure-activity relationships within these compounds. It was found that the hydroxy group at C-5, the absolute C-5 configuration, the epoxy group, and the C-2 carbonyl group appear to be structural features important in conferring biological activity. The conversion of sphaeropsidone into the corresponding 1,4-dione derivative led to a compound showing greater antifungal activity than its precursor. This finding could be useful in devising new natural fungicides for practical application in agriculture.

Higginsianins A and B, Two Diterpenoid α-Pyrones Produced by Colletotrichum higginsianum, with in Vitro Cytostatic Activity.

Two new diterpenoid α-pyrones, named higginsianins A (1) and B (2), were isolated from the mycelium of the fungus Colletotrichum higginsianum grown in liquid culture. They were characterized as 3-[5a,9b-dimethyl-7-methylene-2-(2-methylpropenyl)dodecahydronaphtho[2,1-b]furan-6-ylmethyl]-4-hydroxy-5,6-dimethylpyran-2-one and 4-hydroxy-3-[6-hydroxy-5,8a-dimethyl-2-methylene-5-(4-methylpent-3-enyl)decahydronaphthalen-1-ylmethyl]-5,6-dimethylpyran-2-one, respectively, by using NMR, HRESIMS, and chemical methods. The structure and relative configuration of higginsianin A (1) were confirmed by X-ray diffractometric analysis, while its absolute configuration was assigned by electronic circular dichroism (ECD) experiments and calculations using a solid-state ECD/TDDFT method. The relative and absolute configuration of higginsianin B (2), which did not afford crystals suitable for X-ray analysis, were determined by NMR analysis and by ECD in comparison with higginsianin A. 1 and 2 were the C-8 epimers of subglutinol A and diterpenoid BR-050, respectively. The evaluation of 1 and 2 for antiproliferative activity against a panel of six cancer cell lines revealed that the IC50 values, obtained with cells reported to be sensitive to pro-apoptotic stimuli, are by more than 1 order of magnitude lower than their apoptosis-resistant counterparts (1 vs >80 μM). Finally, three hemisynthetic derivatives of 1 were prepared and evaluated for antiproliferative activity. Two of these possessed IC50 values and differential sensitivity profiles similar to those of 1.