Identification of neomacrophorins isolated from Trichoderma sp. 1212-03 as proteasome inhibitors.

Neomacrophorins I-III (1-3) and X have previously been isolated from Trichoderma sp. 1212-03. Their mode of action against cancer cells and the mechanism of biosynthesis of the characteristic [4.4.3] propellane framework in neomacrophorin X have not been reported. The isolation and characterization of neomacrophorins IV (4), V (5), and VI (6) is reported. Epoxyquinones 1, 4, and 6 potently induced apoptotic cell death in human acute promyelocytic leukemia HL60 cells, while epoxysemiquinols 2, 3, and 5 showed weak activity. This indicates that the epoxyquinone moiety is crucial for apoptosis-inducing activities of neomacrophorins. We also found that neomacrophorins inhibit proteasome in vitro, and 1, 4, and 6 induced significant accumulation of ubiquitinated proteins in HL60 cells. These activities were completely suppressed by a nucleophile, N-acetyl-l-cysteine (NAC). The analysis of reaction mechanisms using LC-MS suggested that C2' and C7' of neomacrophorins could be Michael acceptors in the reaction with NAC methyl ester (NACM). These findings indicated that the electrophilic properties of neomacrophorins are responsible for both their potent biological effects and the biosynthesis of unique [4.4.3] propellane framework in neomacrophorin X.

Neomacrophorin X, a [4.4.3]Propellane-Type Meroterpenoid from Trichoderma sp. 1212-03.

Neomacrophorin X (1) was isolated from Trichoderma sp. 1212-03. Heteronuclear multiple bond correlation (HMBC) spectral analysis indicated a unique [4.4.3]propellane framework, which was verified by the 1H and 13C chemical shift calculations based on density functional theory (DFT) and subsequent comparison with experimental data obtained in CDCl3. The DFT-based electronic circular dichroism (ECD) calculations were effective in not only determining the absolute configuration but also confirming the relative structure. The predominant conformation of 1 was found to be solvent-dependent, with different conformations presenting different NMR and ECD profiles. Introduction of J-based analysis with a J-resolved HMBC aided in this investigation. This conformational alternation was reproduced by considering the solvation with the SM5.4 model in the calculation, although it was not sufficiently quantitative. Although the calculations without solvent effects suggested a conformer that satisfies the spectral profiles in CDCl3, postcalculations with the SM5.4 solvation protocol stabilized the second major conformer, which reproduces the NMR and ECD profiles in polar solvents. Neomacrophorin X (1) is assumed to be biosynthesized by a coupling between the reduced form of anthraquinone and a neomacrophorin derivative. This hypothesis was supported experimentally by the isolation of pachybasin and chrysophanol, as well as acyclic premacrophorin (2), from the same fungus. Some biological properties of 1 are described.