Avance 600 instrument

All reagents were purchased from Sigma-Aldrich and used without further purification. All solvents were anhydrous or analytical grade (Sigma-Aldrich) and used without further purification. The reaction progress was monitored by GC–MS (using the procedure given in Section 4.4). Solvents were removed under reduced pressure using a Büchi Rotavapor R-200 and Büchi B-490 heating bath set to 40 °C. Mixtures were further dried under high vacuum using an Alcatel Pascal 2005SD vacuum pump. NMR spectra were recorded on a Bruker AVANCE-400 instrument (¹H NMR: 400 MHz, ¹³C NMR: 101 MHz) or a Bruker AVANCE-600 instrument equipped with a cryoprobe (¹H NMR: 600 MHz, ¹³C NMR: 150 MHz) using CDCl₃ and C₆D₆. The ¹H NMR chemical shifts were referenced to the residual protonated solvent peaks at δH 7.26 for chloroform-d and 7.15 for benzene-d₆. ¹³C NMR chemical shifts were referenced to the central solvent peaks of bulk solvent at δC 77.16 for chloroform-d and 127.68 for benzene-d₆. J values are given in Hz.

Product 3a was characterized by mass spectrometry (MS) using HPLC-MS (HPLC: Agilent 1100, Waldbronn, Germany; MS: Bruker Daltonics microTOF ESI-TOF-HRMS Massenspektrometer, Bremen, Germany). The separation of the substances and products was achieved by a Zorbax SB-C18 (2.1 × 50 mm, 1.8 μm) column (Agilent, Waldbronn, Germany) at a flow rate of 0.5 mL/min. A solvent system consisting of acetonitrile (eluent A) and 0.1% formic acid (eluent B), starting from an initial ratio of 10% A and 90% B and reaching 100% acetonitrile within 14 min, was used.
Products 3b, 3c and 3d were characterized by MS using electrospray ionization under atmospheric conditions (API-ES) (drying and nebulizing gas: nitrogen) on a Bruker-Daltonics microTOF instrument (Bremen, Germany).
The NMR spectra were recorded on a Bruker Avance 600 instrument (Rheinstetten, Germany) at 600 MHz. The solvents used were H₂O-d2 and DMSO-d6. Chemical shifts are expressed in δ (ppm) calibrated on the resonances of the residual non-deuterated solvent. J values are given in Hz.

For the structural elucidation of the active compound, one and two-dimensional nuclear magnetic resonance spectra (¹H NMR—Proton (H) Nuclear Magnetic Resonance, HSQC—Heteronuclear Single-Quantum Correlation, HMBC—Heteronuclear Multiple Bond Correlation, ¹³C NMR—Carbon-13 (C13) Nuclear Magnetic Resonance, and ¹H¹H COSY—Proton (H) Proton (H) homonuclear correlation spectra) were recorded. Fraction #5 was recorded on a Bruker Avance 600 instrument (Rheinstetten, Germany) at 600 MHz. The solvents of the different measurements were DMSO-d₆ and D₂O. Chemical shifts are expressed in δ (ppm) calibrated on the resonances of the residual non-deuterated solvent. J values are given in Hz.