The structures of compounds 1–11.

(a) Zebrafish larvae treated with increasing concentrations (from 5 to 50 μM) of α-costic and cyclopaldic acid up to 72 h. (b) Embryo viability at different times after treatment with α-costic and cyclopaldic acid, respectively. (c) Some of the surviving embryos (10%) showed body malformations after treatment with 25 μM α-costic acid. Values shown in the plot are means ± SD of triplicate determinations. Means and standard deviations were calculated on biological triplicates using GraphPad Prism8 software.

Effect of treatments on larval length following treatment with cyclopaldic (a) and α-costic acid (b). Surviving zebrafish larvae were measured after hatching and compared to controls. Measurements were made using Image J software. Values shown in the plot are means ± SD of triplicate determinations. Means and standard deviations were calculated on biological triplicates using GraphPad Prism8 software.

Effect of cyclopaldic acid on mammalian cell lines treated with different concentrations for up to 72 h. Hacat, A431, NIH3T3 and transformed SVT2 fibroblasts were plated in DMEM with serum. The seeding medium was changed and supplemented with increasing concentrations of cyclopaldic acid in dimethyl sulfoxide. Cell viability was measured by MTT assays. Values shown in the plot are mean ± SD of triplicate determinations. *** p ≤ 0.005, ** p ≤ 0.01, * p ≤ 0.05.

Immunofluorescence microscopy showing γ-H2AX foci formation (green) in nuclei of Hacat (a) or SVT2 cells (b) treated for 72 h with dimethyl sulfoxide alone or 10, 25 or 50 μM cyclopaldic acid. Nuclei were stained with DAPI (blue). Images from five fields per each experimental point were collected. Quantitation of γ-H2AX foci fluorescence was performed by Image J software and shown as mean ± SD in graph bars of panels (c) (Hacat) and (d) (SVT2) cells. ** p ≤ 0.01, * p ≤ 0.05.

The effect of cyclopaldic acid on the induction of reactive oxygen species (ROS). Hacat and SVT2 cells grown in 96-well plates were treated with increasing amounts of cyclopaldic acid for 48 h, followed by ROS assay using 2′−7′ dichlorofluorescein diacetate (DCFDA). The measurement of ROS was obtained using a Sinergy H4 microplate reader (Gen5 2.07). The positive control of the experiment was carried out with 1 mM hydrogen peroxide. Values shown in the plot are mean ± SD of triplicate determinations. Means and standard deviations were calculated on biological triplicates using GraphPad Prism8 software. *** p ≤ 0.005, ** p ≤ 0.01.

Effect of cyclopaldic acid on the p21WAF and PARP protein levels in SVT2 cells. Representative immunoblots showing the effects of cyclopaldic acid on p21WAF expression in SVT2 cells. Cells were incubated for 72 h with the indicated concentrations (1-50 μM). Proteins were separated on SDS-polyacrylamide gel (25 μg/lane) and transferred to PVDF membranes. The level of proteins was analyzed via Western blotting with monoclonal antibodies. The blots were then re-probed with anti-GAPDH antibody to confirm an equal amount of protein loading. The signal intensities, indicated by numbers, were quantitated by ImageLab software and expressed as the rate between p21WAF or PARP and GAPDH. *** p ≤ 0.005, ** p ≤ 0.01.

Effect of α-costic and cyclopaldic acids on a zebrafish embryo-derived fibroblast cell line. PAC2 cells were seeded in Leibovitz’s L-15 medium. After 24 h cells were treated with different concentrations of α-costic and cyclopaldic acid for up to 72 h. Cell viability was measured by MTT assays. Values shown in the plot are mean ± SD of triplicate determinations.

Cyclopaldic and α-costic acids block medaka embryo development. Stereo-microscopic images of Medaka embryos treated with dimethyl sulfoxide, 5 μM, 7 μM, or 10 μM of cyclopaldic acid, or 50 μM, 25 μM or 5 μM of α-costic acid at stage 24 and 32 (a). The graph reports the death percentage of medaka embryos treated with dimethyl sulfoxide, 5 μM, 7 μM, or 10 μM of cyclopaldic acid, or 50 μM, 25 μM or 5 μM of α-costic acid treatment at stage 24 and 32. (t-test: *** p ≤ 0.005, ** p ≤ 0.01, * p ≤ 0.05) (b).

Effect of α-costic acid on mammalian cell lines treated with different concentrations of up to 72 h. Hacat, A431, NIH3T3 and transformed SVT2 fibroblasts were plated in DMEM with serum. The seeding medium was changed and supplemented with increasing concentrations of α-costic acid in dimethyl sulfoxide. Cell viability was measured by MTT assays. Values shown in the plot are mean ± SD of triplicate determinations. ** p ≤ 0.01, *** p ≤ 0.05.

Effect of cyclopaldic acid on the p21WAF and PARP protein levels. Representative immunoblots showing the effects of cyclopaldic acid on p21WAF expression in SVT2 cells. Cells were incubated for 72 h with the indicated concentrations (1–50 μM). Proteins were separated on SDS-polyacrylamide gel (25 μg/lane) and transferred to PVDF membranes. The level of proteins was analyzed via Western blotting with monoclonal antibodies. The blots were then re-probed with anti-GAPDH antibody to confirm an equal amount of protein loading. The signal intensities, indicated by numbers, were quantitated by ImageLab software and expressed as the rate between p21WAF or PARP and GAPDH.

ORTEP view of cyclopaldic acid derivative (11) with ellipsoids drawn at the 30% probability level.