Inova 500 nb spectrometer

3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was obtained from MP Biomedicals Inc. (Santa Ana, CA, USA). RPMI 1640 was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS) were bought from Zhejiang Tianhang Biotechnology Co., Ltd. (Hangzhou, China). Anti-GAPDH antibodies were from Bioworld Technology Inc. (St. Louis Park, MN, USA), while anti-cytochrome c antibodies were acquired from Cell Signalling Technology Co. (Danvers, MA, USA). The caspase activity assay kit was a product of Beyotime Co. (Shanghai, China). Other routine laboratory reagents were obtained from commercial sources in analytical or HPLC grade. NMR data were recorded on a Inova-500 NB spectrometer (Varian, Palo Alto, CA, USA). CDCl₃ was used as solvent and TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the TMS peak. Mass spectra were acquired on an ultrahigh liquid chromatography instrument coupled with a quadrupole time-of-flight mass spectrometer (G6540A, UPLC-QTOF-MS, Agilent Technologies, Santa Clara, CA, USA). IR spectra were obtained on a 5DX-FTIR spectrophotometer (Nicolet, Madison, WI, USA). Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical, Qingdao, China). Fractions were monitored by TLC visualized by heating silica gel plates sprayed with 5% H₂SO₄ in EtOH.

All reagents were purchased from commercial suppliers and were dried and purified when necessary, and compounds 1a–d was prepared as previously described9 10 . Melting points were determined in capillary tubes on an electrothermal PIF YRT-3 apparatus and without correction. ESI-MS spectra were obtained from VG ZAB-HS spectrometer. ¹H NMR and ¹³C NMR spectra were recorded on a AVANCE III 400MHz spectrometer at 400 MHz and 100 MHz and a Varian INOVA 500NB spectrometer at 500 MHz and 125 MHz, respectively, using TMS as internal standard and CDCl₃ or DMSO-d₆ as solvent and chemical shifts (d) were expressed in ppm. HRMS were obtained from ESI-Q-TOF maxis 4G spectrometer. Silica gel F254 were used in analytical thin-layer chromatography (TLC) and silica gel were used in column chromatography respectively.