An A. nidulans extract library was prepared by adjusting 45 different culture conditions (variation of culture media, cultivation period, temperature and oxygen supply) and the metabolic profiles were screened by HPLC-DAD-MS. Investigation of the culture of A. nidulans AXB4A2 grown at elevated temperature (37 °C) and increased orbital shaking (200 r.p.m.) in malt medium and uridine/p-aminobenzoate supplementation revealed the production of two potentially new metabolites with a molecular weight of m/z 399 and m/z 385, respectively. Similar UV spectra and similar MSn fragmentation patterns suggested that both compounds are closely related. From an upscaled culture (14 l), the compounds were isolated and their structures elucidated by one- and two-dimensional NMR and MS measurements. For compound 1, a molecular formula of C24H33NO4 was established by HRESI-MS analysis. The 13C NMR spectrum displayed 24 C-atoms, which were assigned to four methyl, one methoxy, six methylene, four sp2-hybridized and nine quaternary carbons by DEPT measurements. The structure of the farnesyl side chain was deduced from characteristic H,H-COSY and HMBC correlations (Figure 1b). HMBC coupling of the methylene protons H-1′ to a quaternary carbon at 137.3 p.p.m. indicated O-prenylation of the aromatic system. H,H-COSY correlation of H-2 and H-3, HMBC couplings of H-3 and C-1, C-3a and C-4, as well as an HMBC coupling of 4-OH and C-3a, which could only be observed when measuring at decreased temperature (7 °C) revealed the phthalimidine substructure. HMBC coupling of H-1″ and C-6 established the position of the methoxy substituent. The structurally related compound 2 has a molecular formula of C23H31NO4 (deduced from HRESI-MS), which suggested the lack of a methyl group. One- and two-dimensional NMR data confirmed this assumption. Although a couple of isoindole derivatives have been isolated from fungal sources, the production of such metabolites has not yet been reported for A. nidulans. Zinnimidine (3) and porritoxin (4) were isolated from Alternaria porri,17, 18, 19 6-hydroxy-4-methoxy-5-methylphthalimidine (5) from cultures of Aspergillus silvaticus20 and duricaulic acid (6) from Aspergillus duricaulis.21 Several isoindoles are also produced by Stachybotrys spp.22 (Figure 1c). Notably, from Aspergillus rugulosus, a species closely related to A. nidulans, two aromatic dialdehydes, asperugins A (7) and B (8), were isolated, which show the same substitution pattern as the aromatic moiety of aspernidine A and B.23 The occurrence of these compounds in the same genus as well as the isolation of 9, an oxo analog of zinnimidine, and the hydroxy-methylene-substituted zinniol (10)24 suggest a common metabolic pathway for isoindole formation. It is well conceivable that the pathway is initiated by the formation of orsellinic acid or, more likely, the corresponding aldehyde by a nonreducing polyketide synthase. Ammonia nitrogen would be trapped from an intermediary aldehyde, and oxidoreductive and condensation steps would lead to the isoindolinone core. Hydroxylation, methylation and O-prenylation of the molecules represent the final steps of the biosynthetic process, the latter of which are often associated with increased biological activity. In a primary bioactivity screening, we found that 1 and 2 possess moderate antiproliferative activities (L-929 GI50 35.8 μM (1) and 39.5 μM (2); K-562 GI50 34.3 μM (1) and 39.5 μM (2), respectively) and even lower cytotoxicity (HeLa CC50 94.0 μM (1) and 65.5 μM (2)).

In conclusion, motivated by genomic data and through systematically varying the culture conditions, we have discovered two novel alkaloids from the model organism A. nidulans. Isolation of these metabolites and their characterization revealed the structures of two unusual prenylated isoindolinones, aspernidine A and B, which show moderate antiproliferative effects against various tumor cell lines. Our findings are thus an important addition to the body of knowledge on bioactive compounds from A. nidulans and further highlight the largely overlooked biosynthetic potential of this important model organism.

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