Chrysosporazines Revisited: Regioisomeric Phenylpropanoid Piperazine P-Glycoprotein Inhibitors from Australian Marine Fish-Derived Fungi.

A library of fungi previously recovered from the gastrointestinal tract (GIT) of several fresh, commercially sourced Australian mullet fish was re-profiled for production of a rare class of phenylpropanoid piperazine alkaloids (chrysosporazines) using an integrated platform of; (i) miniaturized 24-well plate cultivation profiling (MATRIX), (ii) UPLC-DAD and UPLC-QTOF-MS/MS (GNPS) chemical profiling, and; (iii) precursor directed biosynthesis to manipulate in situ biosynthetic performance and outputs; to detect two new fungal producers of chrysosporazines. Chemical analysis of an optimized PDA solid phase cultivation of Aspergillus sp. CMB-F661 yielded the new regioisomeric chrysosporazine T (1) and U (2), while precursor directed cultivation amplified production and yielded the very minor new natural products azachrysosporazine T1 (3) and U1 (4), and the new unnatural analogues neochrysosporazine R (5) and S (6). Likewise, chemical analysis of an optimized M1 solid phase cultivation of Spiromastix sp. CMB-F455 lead to the GNPS detection of multiple chrysosporazines and brasiliamides, and the isolation and structure elucidation of chrysosporazine D (7) and brasiliamide A (8). Access to new chrysosporazine regioisomers facilitated structure activity relationship investigations to better define the chrysosporazine P-glycoprotein (P-gp) inhibitory pharmacophore, which is exceptionally potent at reversing doxorubrin resistance in P-gp over expressing colon carcinoma cells (SW600 Ad300).

Precursor-Directed Biosynthesis Mediated Amplification of Minor Aza Phenylpropanoid Piperazines in an Australian Marine Fish-Gut-Derived Fungus, Chrysosporium sp. CMB-F214.

Chemical analysis of an M1 agar plate cultivation of a marine fish-gut-derived fungus, Chrysosporium sp. CMB-F214, revealed the known chrysosporazines A-D (11-14) in addition to a suite of very minor aza analogues 1-6. A microbioreactor (MATRIX) cultivation profiling analysis failed to deliver cultivation conditions that significantly improved the yields of 1-6; however, it did reveal that M2 agar cultivation produced the new natural product 15. A precursor-directed biosynthesis strategy adopting supplementation of a CMB-F214 M1 solid agar culture with sodium nicotinate enhanced production of otherwise inaccessible azachrysposorazines A1 (1), A2 (2), B1 (3), C1 (4), C2 (5) and D1 (6), in addition to four new chrysosporazines; chrysosporazines N-P (7-9) and spirochrysosporazine A (10). Structures inclusive of absolute configurations were assigned to 1-15 based on detailed spectroscopic and chemical analyses, and biosynthetic considerations. Non-cytotoxic to human carcinoma cells, azachrysosporazies 1-5 were capable of reversing doxorubicin resistance in P-glycoprotein (P-gp)-overexpressing human colon carcinoma cells (SW620 Ad300), with optimum activity exhibited by the C-2' substituted analogues 3-5.