Non deuterated DMSO solutions of validated hits were analyzed by HPLC(UV)-MS and NMR in order to determine the purity of the compounds.
High-performance liquid chromatography coupled to mass spectrometry (HPLC−MS) was performed with an Agilent 6520 QTOF LCMS system connected to an Agilent 1290 LC, equipped with a Zorbax Eclipse Plus C18 column (50 mm ×2.1 mm, particle size 1.8 μm). At a flow rate of 0.8 ml/min a stepwise gradient of Water (+ 0.01% HCOOH) (Eluent A) to Acetonitrile (+ 0.01% HCOOH) (Eluent B) was employed as follows: 0 min 5% B; 0.3 min 5% B; 4.5 min 99% B; 5 min 99% B; 5.1 min 5% B. Mass spectra were acquired in both ESI+ and ESI− mode, scanning from m/z 100 to 3200 Da and UV detection was done at 215 (or 220) and 265 (or 255) nm.
NMR Spectra were recorded on a Bruker Avance III 600 MHz spectrometer, equipped with a 5mm TCI Probe, at 25°C. Samples were diluted with DMSO-d6 to an end volume of 160 μl to be measured in 3mm NMR tubes. Double pre-saturation on the water and DMSO resonance frequency as well as 13C decoupling were employed in a gradient 1H-NMR experiment.
The purity level of the compounds was >95% as determined by both methods.
The syntheses of fragments 1 and 4 were described.[49 , 50 (link)] The syntheses of fragments 2 and 3 have not been disclosed to the public.
High-performance liquid chromatography coupled to mass spectrometry (HPLC−MS) was performed with an Agilent 6520 QTOF LCMS system connected to an Agilent 1290 LC, equipped with a Zorbax Eclipse Plus C18 column (50 mm ×2.1 mm, particle size 1.8 μm). At a flow rate of 0.8 ml/min a stepwise gradient of Water (+ 0.01% HCOOH) (Eluent A) to Acetonitrile (+ 0.01% HCOOH) (Eluent B) was employed as follows: 0 min 5% B; 0.3 min 5% B; 4.5 min 99% B; 5 min 99% B; 5.1 min 5% B. Mass spectra were acquired in both ESI+ and ESI− mode, scanning from m/z 100 to 3200 Da and UV detection was done at 215 (or 220) and 265 (or 255) nm.
NMR Spectra were recorded on a Bruker Avance III 600 MHz spectrometer, equipped with a 5mm TCI Probe, at 25°C. Samples were diluted with DMSO-d6 to an end volume of 160 μl to be measured in 3mm NMR tubes. Double pre-saturation on the water and DMSO resonance frequency as well as 13C decoupling were employed in a gradient 1H-NMR experiment.
The purity level of the compounds was >95% as determined by both methods.
The syntheses of fragments 1 and 4 were described.[49 , 50 (link)] The syntheses of fragments 2 and 3 have not been disclosed to the public.