Gliorosein

The new polyketides lopouzanones A and B, as well as the new 1-O-acetyl and 2-O-acetyl derivatives of dendrodochol B, were isolated from the sponge-derived marine fungusLopadostoma pouzariistrain 168CLC-57.3. Moreover, six known polyketides, gliorosein, balticolid, dendrodolide G, dihydroisocoumarine, (-)-5-methylmellein, and dendrodochol B, were identified. The structures of the isolated compounds were determined by a combination of NMR and ESIMS techniques. The absolute configurations of the lopouzanones A and B were determined using the Mosher's method. The cytotoxicity of the isolated compounds against human prostate cancer cells PC-3 and normal rat cardiomyocytes H9c2 was investigated. Gliorosein showed weak DPPH radical-scavenging activity and in vitro cardioprotective effects toward rotenone toxicity and CoCl2-mimic hypoxia.

1. Gliorosein was obtained in excellent yield (150mg./200ml. of Raulin-Thom medium) from surface cultures of Gliocladium roseum. Its nuclear-magnetic-resonance spectrum showed conclusively that it is 1,6-dihydro-3,4-dimethoxy-6-methyltoluquinone. 2. Sodium [2-(14)C]acetate was incorporated into gliorosein and the related products (3.3% conversion). The specific activities of these substances increased in the order gliorosein, 3,4-dimethoxy-6-methyltoluquinol, the related quinhydrone and quinone, indicating that gliorosein was the actual metabolite that was secreted and that the other compounds were derived from it in the medium. 3. 6-Methylsalicylic acid was not taken up by the mycelium and could be recovered unchanged. Orsellinic acid was decarboxylated by G. roseum and an equivalent amount of orcinol was secreted into the medium. The methyl esters of 6-methylsalicylic acid and orsellinic acid were both hydrolysed by an esterase present in the mycelium. Some of the 6-methylsalicylic acid thus produced was secreted into the medium and the orsellinic acid was decarboxylated. 4. Washed mycelium of G. roseum converted aurantiogliocladin and 3,4-dimethoxy-6-methyltoluquinol quantitatively into gliorosein within 18hr. More critical experiments with (14)C-labelled substrates demonstrated that 3-hydroxy-4-methoxy-6-methyltoluquinol and 3,4-dimethoxy-6-methyltoluquinol, and their respective quinones, were effectively incorporated into gliorosein and related products (49, 68, 30 and 57% respectively). 5. The following sequence of reactions is proposed for the biosynthesis of gliorosein: acetyl-CoA+3 malonyl-CoA+S-adenosyl-methionine --> 5-methylorsellinic acid --> 3-hydroxy-4-methoxy-6-methyltoluquinol --> 3,4-dimethoxy-6-methyltoluquinol --> gliorosein. 6. Since gliorosein is optically active (dextrorotatory), the final tautomerization reaction leading to its formation must be enzyme-catalysed.

The plant-parasitic nematode, Neotylenchus linlordi, congregated around colonies or filtrates from mycelia of Gliocladium roseum, Rhizoctonia solani, Pyrenochaeta terrestris and Chaetomium indicum. The average time required for the nematodes to reach the fungal colonies ranged from less than 4 hr for G. roseum to 20 hr for R. solani. Nematodes first circled near the point of introduction, then moved toward the fungus or filtrate. Several methods of measuring the response of N. linfordi to G. roseum culture filtrate were evaluated. The response was strongest when the test materials were assayed on an agar disk submerged in water agar and the introduced nematodes suspended in agar in a center well midway between the test materials. Filtrates obtained from cultures of G. roseum incubated between 12 and 21 days in potato dextrose broth, were most active. The attractants were small thermostable molecules, soluble in methyl alcohol and unaffected by pH. A yellow pigment with properties similar to a mixture of aurantiogliocladin, rubrogliocladin, and gliorosein was shown to be one of the active materials. The response of N. linfordi to the G. roseum filtrate was not associated with any nutritive factors which would result in reproduction.