NMR and mass spectrometric characterization of vinblastine, vincristine and some new related impurities-Part II

In the course of developing a new, improved process at Gedeon Richter for the production of the “bisindole” alkaloids vinblastine (VLB) and vincristine (VCR), some novel VLB/VCR-related trace impurities were detected by analytical HPLC at the production site. Repeated attempts to isolate and purify these unknown impurities by preparative liquid chromatography yielded small amounts of materials whose main components were the unknown impurities, but were still contaminated with other VLB/VCR-related compounds. In spite of these difficulties, by using a combination of high-resolution (LC-)MS/MS and off-line 1D and 2D ultra high-field NMR techniques and leaning on the relevant spectroscopic data for VLB and VCR as discussed in Part 1 [1], we could unambiguously solve the structures of, and could give a complete spectral characterization for, the trace impurities. Among these, although “cyclo-VCR” (impurity-2), “[VCR]-C(16)-COOEt” (impurity-4) and “[VLB]-C(16)-COOEt” (impurity-5) are known synthetic VLB/VCR-derivatives, and “[VLB]-C(14′)-OH(α)” is a known natural alkaloid (leurocolombine), they are new VLB/VCR impurities, and “[VCR]-N(4′)-C(21′)-iminium-salt” (impurity-3) is also a new chemical structure which provides direct proof of a hypothetic metabolic pathway of VLB/VCR. The structure determination of impurity-4 and impurity-5, and the rationalization of their origin was a particularly challenging task: since VCR is produced by the oxidation of VLB, it may be assumed that [VCR]-C(16)-COOEt (impurity-4) originates from the oxidization of [VLB]-C(16)-COOEt (impurity-5). This is consistent with the finding that [VLB]-C(16)-COOEt (impurity-5) could be detected by LC-MS/MS in the raw VLB samples in similar amounts as [VCR]-C(16)-COOEt (impurity-4) in the final VCR product. Our investigations indicate that [VLB]-C(16)-COOEt (impurity-5) does not form directly from VLB during extraction or chromatographic separation, suggesting that it may be a new natural product.