¹H, ¹³C and ¹⁵N NMR assignments of phenazopyridine derivatives

Phenazopyridine hydrochloride (1), a drug in clinical use for many decades, and some derivatives were studied by one- and two-dimensional ¹H, ¹³C and ¹⁵N NMR methodology. The assignments, combined with DFT calculations, reveal that the preferred protonation site of the drug is the pyridine ring nitrogen atom. The chemoselective acetylation of phenazopyridine (2) and its influence on the polarization of the azo nitrogen atoms were evidenced by the ¹⁵N NMR spectra. Molecular calculations of the phenazopyridines 2-4 show that the pyridine and phenyl groups are oriented in an antiperiplanar conformation with intramolecular hydrogen bonding between the N-b atom and the C-2 amino group preserving the E-azo stereochemistry.