An unexpected nitrosamine product of oclacitinib: Synthesis, structural elucidation, ADMET profiling and preliminary toxicological assessment-Pub

N-nitrosamines Chemistry
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https://www.sciencedirect.com/science/article/abs/pii/S0022286026021216

Abstract

The control of N-nitrosamine drug substance-related impurities (NDSRIs) has become an important issue in pharmaceutical quality assessment because of their potential mutagenic and carcinogenic risks. Oclacitinib maleate, a Janus kinase inhibitor used in veterinary medicine, contains two potentially nitrosatable nitrogen sites. In this study, its nitrosation behavior was investigated under acidic nitrite conditions and tert-butyl nitrite conditions, and only one N-nitroso product was selectively formed. Structural elucidation by LC-MS/MS, FT-IR, 1D/2D-NMR, and single-crystal X-ray diffraction confirmed that the nitrosation site of the product was located at the sulfonamide nitrogen of oclacitinib. In contrast, the heteroaromatic N-nitroso product was not detected. Crystallographic analysis of both oclacitinib maleate and the synthesized product further supported this regioselective assignment. In silico ADMET prediction suggested that N19-nitroso-oclacitinib exhibited higher toxicity. In HepG2 MTT assays, it showed stronger cytotoxicity than oclacitinib maleate under the tested experimental conditions. The IC50 values of the N19-nitroso-oclacitinib were 201.0 μM (95% CI: 195.6–206.7 μM) in the absence and 245.0 μM (95% CI: 234.0–256.9 μM) in the presence of hamster liver S9 fraction, whereas the IC50 of oclacitinib maleate > 400 μM under the same experimental conditions. These findings provide structural and preliminary toxicological evidence for an oclacitinib-related NDSRI and indicate that this N-nitroso impurity may present a higher cytotoxicity concern than the parent drug.

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The issue here is that the nitrosation has not given rise to a “nitrosamine” persay. It is an N-nitrososulfonamide, which is structurally very differnt and acts very differently from a nitrosamine. I feel that organic chemistry is dead when people give such blatantly wrong headings and peers who review it do not even know what they are doing or saying.

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Hi Yosukemino, Tert-butyl nitrite is mild and selective for the nitrosation of sulfonamides and carboxamides. Then, N19-nitroso-oclacitinib is the expected product only. The authors of the reference are confusing the readers by saying unexpected nitrosamine product.

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Thank you for your insightful comment. We agree that formation of the N19-nitroso product is not unexpected, as the sulfonamide nitrogen would generally be anticipated to be more reactive toward nitrosation than the heteroaromatic nitrogen. The heteroaromatic nitrogen was likely considered a potential nitrosation site because heteroaromatic nitrogen atoms can, depending on their structural context, undergo nitrosation. However, in the case of N1, the lone pair is delocalized within the aromatic conjugated system, which is expected to reduce its nucleophilicity and consequently its susceptibility to nitrosation. Therefore, the selective nitrosation of N19 observed under all three reaction conditions appears chemically reasonable. We agree that the term “unexpected” is not appropriate in this context and will revise the manuscript accordingly.

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