S500 spectrometer

All reagents were of reagent grade quality obtained from Sigma-Aldrich (Prague, Czech Republic). Solvents for synthesis were obtained from Penta Chemicals Co. (Chrudim, Czech Republic). All experiments were carried out under nitrogen atmospheres. Thin layer chromatography (TLC) was performed on aluminium sheets precoated with silica gel 60 F254 (Merck, Prague, Czech Republic) and then visualized by UV 254 nm and 366 nm. Column chromatography was performed at normal pressure on silica gel 100 (particle size 0.063–0.200 mm, 70–230 mesh ASTM, Fluka, Prague, Czech Republic). Mass spectra were recorded using a combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS). The Dionex Ultimate 3000 LC-MS analytical system was connected with an Orbitrap Q Exactive Plus hybrid spectrometer (Thermo Fisher Scientific, Bremen, Germany). ¹H-NMR and ¹³C-NMR spectra were recorded with a Varian Mercury VX BB 300 (operating at 300 MHz for ¹H and 75 MHz for ¹³C) or on a Varian S500 spectrometer operating at 500 MHz for ¹H and 126 MHz for ¹³C (Varian Inc. Palo Alto, CA, USA). Chemical shifts are reported in parts per million (ppm, δ) relative to TMS. The assignment of chemical shifts is based in standard NMR experiments (¹H, ¹³C, ¹H-¹H COSY, ¹H-¹³C HSQC, HMBC, DEPT). Melting points were measured using a M-565 automated melting point recorder (Büchi, Flawil, Switzerland).

The chemicals were purchased from Sigma-Aldrich Co., LLC (Prague, Czech Republic) and were used without additional purification. Analytical thin-layer chromatography was carried out using plates coated with silica gel 60 with the fluorescent indicator F254 (Merck, Prague, Czech Republic). The thin-layer chromatography (TLC) plates were visualized by exposure to ultraviolet light (254 nm) or by the detection reagent ninhydrin. Column chromatography was performed using silica gel 100 at atmospheric pressure (70–230-mesh ASTM, Fluka, Prague, Czech Republic). The NMR spectra were all recorded on a Varian S500 spectrometer (500 MHz for ¹H and 126 MHz for ¹³C). Chemical shifts are reported in δ ppm referenced to residual solvent signals (for ¹H NMR and ¹³C NMR: chloroform-d (CDCl₃; 7.26 (D) or 77.16 (C) ppm). A CEM Explorer SP 12 S was used for the MW-assisted reaction. The final compounds were analyzed by LC-MS with a Dionex Ultimate 3000 RS UHPLC system coupled with a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) to obtain high-resolution mass spectra. Gradient LC analysis with UV detection (254 nm) confirmed >95% purity.

All commercial reagents and solvents used were of the highest available purity from Sigma-Aldrich (Prague, Czech Republic). For flash column chromatography on silica gel, Kieselgel 60 (0.063–0.200 mm, 70–230 mesh, Fluka) was used. Solvents for flash column chromatography were purchased from Penta Chemicals Unlimited (Prague, Czech Republic). Thin-layer chromatography was run on Merck silica gel 60 F₂₅₄ analytical plates; detection was carried out with ultraviolet light (254 nm). Melting points were recorded on a Melting Point Apparatus – Stuart SMP30 and are uncorrected. The ¹H NMR and ¹³C NMR spectra were recorded in DMSO-d₆ and CD₃OD-d₄ solution at ambient temperature on a Varian S500 spectrometer (499.87 MHz for ¹H and 125.71 MHz for ¹³C). Chemical shifts, δ, are given in parts per million (ppm), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), or m (multiplet). Coupling constants, J, are expressed in hertz (Hz). For ¹H δ relative to DMSO-d₆ (δ = 2.50) or CD₃OD-d₄ (δ = 3.31) and for ¹³C relative to DMSO-d₆ (δ = 39.43) or CD₃OD-d₄ (δ = 49.05). High Resolution Mass Spectrometry (HRMS) was determined by Q Exactive Plus hybrid quadrupole-orbitrap spectrometer.