Ergostane-type sterols and sesquiterpenes with anti-neuroinflammatory activity from a Nigrograna species associated with Clematis shensiensis

Nigrograna sp. LY66, an endophytic fungus associated with the herbal medicinal plant Clematis shensiensis, produced four undescribed steroids, nigergostanes A−D (1−4), including an unusual ketal-containing nigergostane (1), and four undescribed sesquiterpenoids decorated with cyclohexanone motifs, nigbisabolanes A−D (7−10), along with three known compounds, 23R-hydroxy-(20Z,24R)-ergosta-4,6,8(14),20(22)-tetraen-3-one (5), ergosta-5,7,22-trien-3β-ol (6), and curculonone A (11). The structures and absolute configurations of these undescribed compounds were confirmed using spectroscopic data (NMR and HRESIMS), modified Mosher's method, and ECD experiments. Additionally, compounds 5 and 8 displayed significant inhibition of nitric oxide generation in lipopolysaccharide-induced BV-2 microglial cells with IC50 values of 2.8 and 2.7 μM, respectively, and is thus more potent than that of the positive control, quercetin (IC50 = 8.77 μM). A molecular docking study revealed that 23-OH of 5 binds to the Y347 residue of inducible nitric oxide synthase (iNOS), whereas the 2-OH and 9,10-diol moieties of 8 bind to R381 and W463 and haeme residues of iNOS, respectively, which has rarely been reported in previous studies. These findings provide a set of undescribed lead compounds that can be developed into anti-neuroinflammatory agents.