Synthesis and characterization of a series of N,N′‐substituted urea derivatives by using electrospray ionization tandem mass spectrometry: Differentiation of positional isomers

Rationale Characterization of N,N′‐substituted ureas was found to be challenging by nuclear magnetic resonance (NMR) spectroscopy, particularly N‐di‐ and tri‐alkylated ureas because of the absence of adjacent protons. In the present study, electrospray ionization tandem mass spectrometry has been used to differentiate positional isomeric pairs and to characterize a series of N,N′‐substituted ureas, as these compounds have significant importance for drug discovery. Additionally, urea is an essential functionality in several bioactive compounds as well as a variety of clinically approved therapies. Methods High‐resolution electrospray ionization tandem mass spectrometry (ESI‐HR‐MS/MS) has been used to characterize a series of N,N′‐substituted urea derivatives and differentiate two pairs of positional isomers. The data was acquired by Xcaliber application in positive ionization mode. Results ESI‐HR‐MS/MS spectra of [M + H]+ ions of the positional isomeric urea derivatives 8a and 8b show distinct fragmentation patterns. For example, the MS/MS spectrum of the [M + H]+ ion of isomer 8a displays the abundant fragment ion at m/z 285.1595, which was totally absent in isomer 8b. This would be plausibly formed by the cleavage of the C–N bond of the urea group with the elimination of the isocyanate moiety. In contrast, the MS/MS spectrum of the [M + H]+ ion of isomer 8b shows an intense ion at m/z 311.1389 which is completely absent in isomer 8a which would be formed by the cleavage of the C–N bond attached to the ring nitrogen. Similarly, another pair of positional isomers, 8c and 8d, have been clearly distinguished by their fragmentation behaviour. In addition, a series of N,N′‐substituted urea derivatives were studied to investigate the impact of different substitution on the fragmentation behaviour. Conclusions The present study demonstrates that ESI‐HR‐MS/MS can be used to differentiate pairs of N,N′‐substituted urea positional isomers and characterize a series of derivatives. It was observed that a characteristic fragment ion was formed by the C–N bond cleavage with the elimination of an isocyanate moiety. The proposed mechanism of fragmentation was supported by the change in the fragmentation pathway upon alkylation of the NH. In order to generalize this fragmentation pattern, a series of N‐alkylated ureas was synthesized and studied by MS/MS.