During the terminal heat sterilization of the lipid emulsion final dose formulation of the photodynamic therapeutic (PDT) agent tin ethyl etiopurpurin (SnET2), a new degradant was observed at very low levels. The degradant, which was prone to photo-instability, was isolated by preparative chromatography and subsequently characterized by mass spectrometry and NMR methods. Reproducible parent ion clusters were only observable via negative ion APCI methods. Because of the limited isolate sample, NMR characterization was done using 1.7 min SMIDG (SubMicro Inverse-Detection Gradient) NMR probe technology in conjunction with the accordion-optimized IMPEACH-MBC long-range heteronuclear shift correlation experiment. The "static" 8 Hz optimization of the GHMBC experiment failed to allow the observation of a number of long-range correlations that were of critical importance to the determination of the structure of the impurity. In contrast, all of the correlations required to assemble the structure were obtained from an IMPEACH-MBC experiment optimized for long-range heteronuclear couplings in the range from 2-10 Hz.

Identification of degradants of pharmaceuticals is a necessary challenge of the drug development process following the subjection of candidate molecules to a variety of physico-chemical stresses. It would be desirable to be able to conduct such studies on a minimal amount of material. As a prototypical study, the isolation and identification of degradants of a sample of the complex indoloquinoline alkaloid, cryptospirolepine, was undertaken after prolonged storage in DMSO solution using a combination of cryogenic NMR probe technology and CASE (Computer-Assisted Structure Elucidation) programs. None of the starting alkaloid remained after storage; a chromatogram of the DMSO solution demonstrated the presence of >25 components in the mixture. The two most abundant degradation products were identified as the known alkaloid cryptolepinone (similar to35%) and an unprecedented rearrangement product, DP-2, (similar to16%).

The structures of the major thermal degradation products of linezolid (Zyvox(R), PNU-100766) are reported. Following hydrolytic decarboxylation, the remaining oxazolidinone-derived skeleton of the antibiotic underwent a variety of acetyl migrations prior to deacetylation. Three of the degradation products are structural isomers. Structural characterization of the degradants was accomplished via the concerted analysis of 2D NMR, mass spectrometric, and infrared data. Direct and long-range H-1-N-15 heteronuclear shift correlation experiments at natural abundance were performed on linezolid as well as each of the isolated products of the degradation cascade. N-15 chemical shifts for linezolid and each of the degradants, as well as the observed long-range H-1-N-15 coupling pathways are reported.

Parenteral formulations of linezolid (PNU-100766), a novel (S)-3-(4'-fluorophenyl)-5-N-acetamidomethyloxazolidin-2-one antibiotic, were subjected to photo-stability testing as required by ICH guidelines. Direct, reversed phase chromatographic evaluation of the photo-irradiated solutions revealed two chromatographically broad degradants. Following preparative chromatographic isolation, the structures of both degradants were determined by mass spectrometric, nmr, and vibrational spectroscopic methods. The degradant structures formed by decomposition involving photo-oxidation of the morpholine, followed by a carbon-carbon bond scission to a formyloxyethylformamide-containing degradant. Facile hydrolytic cleavage of the formate ester in acidic media gave the second chromatographically broad degradant containing a N-2-hydroxyethylformamide group. The formamide substituent underwent further hydrolysis in acidic media to the corresponding secondary amine.

The effects of N-oxidation on the N-15 chemical shifts of the Strychnos alkaloids strychnine and brucine are discussed. The N-15 shifts were determined using the inverse-detected, long-range GHMBC experiment at natural abundance. Following N-oxidation, the N-19 resonance shifted downfield from 35.0 ppm in strychnine to 136.3 ppm in the N-19-oxide. The N-19 resonance of brucine shifted downfield from 35.9 to 135.5 ppm in the N-19-oxide, Small upheld shifts were observed for the N-9 resonances of both N-oxides. The H-1 and C-13 shifts of both N-oxides were assigned using inverse-detected 2D NMR methods to ensure proper assignments of the long-range H-1-N-15 couplings. Copyright (C) 1999 John Wiley & Sons, Ltd.

TipranavirTM (PNU-140690) is a protease inhibitor under clinical investigation for the treatment of human acquired immunodeficiency syndrome (AIDS). During scale-up synthesis of clinical quantities of the bulk drug, a colored, transient by-product of the final coupling reaction was observed. Quantities of this colored, transient chemical species were too low (<<0.1%) for characterization by conventional spectroscopic methods. It was, however, possible to isolate sufficient material for characterization based on mass spectrometry and submicro inverse-detection gradient (SMIDG) nmr methods by methanol stripping of silica gel that had been used in purification of bulk drug. This process afforded an enriched feedstock from which small quantities of this highly colored and unstable (halflife < 18 hours in methanol and < 10 minutes in acetone) trace contaminant could be isolated by semi preparative reversed phase hplc. The impurity was identified as an unstable Zincke salt formed by the condensation of two molecules of the anilino precursor and the pyridine used as a base in the final step of the synthetic process. Following identification of this impurity, efforts were undertaken to engineer it out of the synthetic process.

The rigorous structural characterization of novobiocin, is reported using mass spectrometric, infrared and nmr spectroscopic analysis. Complete nmr assignments are reported. Previous reports in the literature had left some quaternary carbon resonances unassigned. Isonovobiocin and decarbamylnovobiocin, although known in the literature for a number of years, have never been completely characterized. Mass spectrometric fragmentation pathways and complete H-1 and C-13 nmr assignments are reported for these congeners for the first time. The mass spectral fragmentation pathway and nmr assignments are also reported for 2 "-(O-carbamyl)novobiocin although the nmr assignments at lower field were reported previously. The structural characterization of novobiocin-2 ",3 "-carbonate observed in the Test Assay procedure used for Novobiocin is reported for the first time.

Early efforts to utilize N-15 NMR spectroscopy in structure elucidation studies were often frustrated by the low gyromagnetic ratio (gamma(N)) and the low natural abundance (0.37%) of the nuclide. The advent of H-1 and inverse-detected 2D NMR methods has eliminated many of the difficulties inherent to the use of N-15 as a structural probe. This paper reports the partial assignment of the N-15 NMR resonances of the thiopeptide antibiotic sulfomycin-I produced by Streptomyces viridochromogenes. With the exception of two tertiary nitrogen resonances that had no two- or three-bond coupling pathways, assignments were made either through direct correlation H-1-N-15 GHSQC or one-bond optimized H-1-N-15 GHNMQC or via two or three bonds using H-1-N-15 GHNMQC spectra. Assignments are also reported for the heterocyclic nitrogen resonances of two thiazole and one oxazole moiety contained in the structure of the antibiotic via (3)J(N,H) coupling from the heterocyclic ring protons. Despite the suggestion that these coupling pathways, suspected to be ca. 2 Hz, might be difficult to observe since they are comparable to the linewidths of the thiazole and oxazole protons in question, they were still exploitable for assignment purposes. (C) 1998 John Wiley & Sons Ltd.

The major degradation products occurring in pH 2–3.75 solutions of a 21-aminosteroid (tirilazad) are described. Degradation products were identified using two methods. In the first method, molecular ion information obtained from LC/MS of degraded samples implied an identity. The proposed structures of I, II, V, and VI were confirmed by comparing the mass spectra and chromatographic retention time of synthetically prepared samples to those obtained for the degraded sample by LC/MS. In the second method, degradation products (III and VI) isolated by preparative HPLC were identified by spectroscopic techniques. No structure can be assigned to compound IV. In acidic solutions, tirilazad degrades via oxidative and acid-catalyzed reactions. Oxidative degradation produces the C20 carboxylic acid and the amine side-chain while acid catalyzed degradation includes hydrolysis of the pyrimidine-piperidine bond and dienon-phenol rearrangement of the steroid A-ring. Quantitative data on degradation products were obtained with a gradient HPLC method.