G 60 230

Nuclear magnetic resonance (NMR) measurements were conducted on Varian INOVA-600 (600 MHz and 150 MHz for ¹H and ¹³C, respectively). Chemical shifts (δ) are expressed in ppm and coupling constants (J) are in Hz. A Bruker microTOF mass spectrometer was used for high-resolution mass measurement. ECD spectra were recorded at room temperature on a Jasco J815 spectrophotometer in 1 cm cuvettes. A PerkinElmer Model 343 polarimeter was used for recording the optical rotation. Column chromatography was performed using silica gel G 60-230 (Merck, Germany). The thin-layer chromatographic screening was made with Merck precoated silica gel F254 plates.

For column chromatography, silica gel G60-230 (Merck, Germany) and Sephadex LH-20 (Sigma–Aldrich, USA) were used. Analytical thin layer chromatography (TLC) was performed on a pre-coated silica gel 60 GF₂₅₄ (Merck or Machery-Nagel, Germany). The 1D and 2D NMR spectra were performed on Bruker-400 Ascend^TM spectrometer using CDCl₃ or dimethyl sulfoxide deuterated (DMSO-d₆) as solvents. The electrospray mass spectrometry (ESMS) experiments were conducted with the 3200 Q-trap LC/MS/MS system (Applied Biosystems, Foster City, CA, USA) Analyst version 1.4.1 software (MDS Sciex; Toronto, CA, USA).
The TLC chromatogram of the obtained EtOAc extract [CH₂Cl₂–MeOH (95: 5 v/v)] revealed the presence of three major spots on visualization with 10% H₂SO₄ spray reagent and heating at 110°C for 1 min. The first spot (R_f 0.65) had no response both under UV₂₅₄ and under UV₃₆₆ lights; on visualization, however, it gave a pale orange color. The other two spots (R_f 0.25 and 0.39) quenched UV₂₅₄ light and gave a brown color.
The EtOAc extract (700 mg) was applied to a silica gel chromatographic column (35 g), packed in CH₂Cl₂ 100% and eluted with CH₂Cl₂–MeOH mixtures with different polarities to afford compounds 1 (R_f 0.65, 18 mg), 2 (R_f 0.39, 7 mg), and 3 (R_f 0.25, 10 mg). A detailed isolation procedure is presented in Supplementary Materials.