Smardaesidins A-G, isopimarane and 20-nor-isopimarane diterpenoids from Smardaea sp., a fungal endophyte of the moss Ceratodon purpureus

Abstract

Five new isopimarane diterpenes, smardaesidins A-E (1- 5) and two new 20-nor-isopimarane diterpenes, smardaesidins F (6) and G (7), together with sphaeropsidins A (8) and C-F (10-13) were isolated from an endophytic fungal strain, Smardaea sp. AZ0432, occurring in living photosynthetic tissue of the moss Ceratodon purpureus . Of these, smardaesidins B (2) and C (3) were obtained as an inseparable mixture of isomers. Chemical reduction of sphaeropsidin A (8) afforded sphaeropsidin B (9), whereas catalytic hydrogenation of 8 yielded 7-O-15,16-tetrahydrosphaeropsidin A (14) and its new derivative, 7-hydroxy-6-oxoisopimara-7-en-20-oic acid (15). The acetylation and diazomethane reaction of sphaeropsidin A (8) afforded two of its known derivatives, 6-O-acetylsphaeropsidin A (16) and 8,14-methylenesphaeropsidin A methyl ester (17), respectively. Methylation of 10 yielded sphaeropsidin C methyl ester (18). The planar structures and relative configurations of the new compounds 1-7 and 15 were elucidated using MS and 1D and 2D NMR experiments, while the absolute configurations of the stereocenters of 4 and 6-8 were assigned using a modified Mosher's ester method, CD spectra, and comparison of specific rotation data with literature values. Compounds 1-18 were evaluated for their potential anticancer activity using several cancer cell lines and cells derived from normal human primary fibroblasts. Of these, compounds 8, 11, and 16 showed significant cytotoxic activity. More importantly, sphaeropsidin A (8) showed cell-type selectivity in the cytotoxicity assay and inhibited migration of metastatic breast adenocarcinoma (MDA-MB-231) cells at subcytotoxic concentrations.

Figure 1

Selected ¹H-¹H COSY ( ) and HMBC correlations (H→C) of 1.

Figure 2

Selected NOEs observed in the NOEDIFF experiments of 1.

Figure 3

Selected ¹H-¹H COSY ( ) and HMBC correlations (H→C) of 5.

Figure 4

Selected NOEs observed in the NOEDIFF experiments of 5.

Figure 5

Selected ¹H-¹H COSY ( ) and HMBC correlations (H→C) of 6.

Figure 6

Δδ_H values [Δδ (in ppm) = δ_S - δ_R] obtained for (S)- and (R)-MTPA esters (4a and 4b, respectively) of smardaesidin D (4) in pyridine-d₅.

Figure 7

Cell migration inhibition assay of compounds 8 and 11 in metastatic cancer cell line MDA-MB-231. (A) DMSO control (negative). (B) LY294002 (positive control) at 7.5 μM. (C) Compound 8 at 2.0 μM. (D) Compound 11 at 5.0 μM.