Melting point m 560 apparatus

All melting points were taken on a Büchi melting point M-560 apparatus. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. ¹H and ¹³C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d₆ or CDCl₃ solution using a Bruker Avance II series 200 MHz spectrometer. Column chromatography was performed with Merck silica gel (230 − 400 mesh ASTM) or a Büchi Sepacor chromatography module (prepacked cartridge system). Elemental analyses (C, H, N) were within ± 0.4% of theoretical values and were performed with a VARIO EL III elemental analyzer. The purity of all the tested compounds was > 95%, determined by HPLC (Agilent 1100 series).
Compounds 9a–e were prepared according to published procedure (Barreca et al. 2022a (link)).

All melting points were taken on a Büchi melting point M-560 apparatus and were uncorrected. IR spectra were recorded in Bromoform with a JASCO FT-IR spectrophotometer. 1H and 13C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d₆ solution and TMS as an internal standard, using a Bruker Avance II Series 200 MHz spectrometer or at 300 and 75 MHz (APT) with a Bruker AC-E spectrometer. Column chromatography was performed with Merck silica gel (230–400 mesh ASTM). Elemental analyses (C, H, and N) were within ±0.4% of theoretical values. The substituted benzylamine derivatives were commercially available and were used without further purification. For all the compounds already cited in the literature, the IR and NMR spectra results were identical to those reported (1A–E). The 1H and 13C NMR spectra of new synthesized compounds (1F,G and 2A–C) are reported in the Supplementary Materials.

All solvents and reagents were from Sigma Aldrich and used without further purification. All melting points are uncorrected and were determined on a Büchi Melting Point M-560 apparatus. FTIR spectra were recorded by a Perkin Elmer FTIR Spectrum One by using ATR system (4000–650 cm⁻¹). The ¹H and ¹³C NMR spectra were recorded at 298 K on a JEOL ECA 400 MHz or Bruker Advance 500 MHz spectrometer equipped with a z-gradient, triple-resonance (¹H, ¹³C, ¹⁵N) cryoprobe, using DMSO-d6 as solvent. The ¹⁹F-NMR spectra were acquired on an Oxford Instruments Pulsar benchtop NMR 60 MHz Spectrometer. Chemical shifts are expressed in ppm with tetramethylsilane (TMS, δ = 0 ppm) as an internal reference for protons and trifluoroacetic acid (TFA, δ = − 75.39 ppm) for fluorine. Accurate mass data were obtained using a Waters (Waltham, MA) model LCT Premiere time-of-flight (TOF) mass spectrometer. Reactions were monitored by TLC on silica gel using ethyl acetate/hexane mixtures as a solvent and compounds visualized by UV lamp. The reported yields are for the purified material and are not optimized.

All solvents (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Chemical (Pittsburgh, PA, USA). Streptomycin, penicillin G, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from AppliChem (Darmstadt, Germany). The cell medium (RPMI, acquired from Sigma-Aldrich, USA) used in the experiments consisted of RPMI 1640 with 20 mM HEPES and l-glutamine, without sodium bicarbonate, containing 10% (v/v) fetal bovine serum, 200 mg mL⁻¹ Streptomycin, and 200 IU mL⁻¹ penicillin. Silica gel 60, particle size distribution 0.02–0.045 mm (Carl Roth, Karlsruhe, Germany), was used for column chromatography. Thin-layer chromatography (TLC) was performed on Merck plates (Darmstadt, Germany), layer thickness 0.2 mm with Silica gel 60 and fluorescence indicator F₂₅₄. Visualization was accomplished by spraying the plates with a mixture of sulfuric and nitric acids (1:1) followed by short, gentle heating. Melting point was determined on a Büchi (Flawil, Switzerland) M-560 melting point apparatus. Optical rotation was measured in chloroform on an Autopol IV (Rudolph Research Analytical, Flanders, NJ, USA) polarimeter equipped with a sodium lamp (589 nm) and a 1 mL cell with a 1 dm path length.

Chemical reagents were purchased from commercial sources (Sigma-Aldrich, Tokyo Chemical Industry, and Duksan Pure Chemical, Korea) and used without further purification. Human serum was purchased from Sigma-Aldrich. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded using a JEOL 400 MHz NMR spectrometer. High-resolution mass spectra (HRMS) were recorded using a Bruker Impact II quadruple time-of-flight (QToF) mass spectrometer with an electrospray ionization (ESI) source. Melting point analysis was performed using a Büchi M-560 melting point apparatus. The fluorescence spectra were recorded using an Agilent Cary Eclipse fluorescence spectrophotometer, and absorbance spectra were recorded on a JASCO V-630 UV-Vis spectrophotometer. Fourier-transform infrared (FTIR) spectra were recorded on a Thermo Scientific NICOLET iS10 spectrometer using a KBr disc (Thermo Scientific, 25 × 4 mm).