Determination of the Degradation Chemistry of the Antitumor Agent Pemetrexed Disodium.
Stress-testing (forced degradation) studies have been conducted on pemetrexed disodium heptahydrate (1) (LY231514·2Na·7H2O) drug substance in order to identify its likely degradation products and establish its degradation pathways. Solid samples of the drug substance were stressed under various conditions of heat, humidity, and light, and solutions of the drug substance were stressed under various conditions of heat, light, oxidation, and over a wide pH range (1-13). The stressed samples were analyzed using a gradient elution reversed-phase HPLC method. The 7 major degradation products detected in the stress-testing studies were isolated, and the structures were elucidated via spectroscopic characterization. The structures of the degradation products and their proposed mechanisms of formation indicate that 1 degrades via 2 main pathways: oxidation and hydrolysis. Of the 7 identified degradation products, 6 are proposed to result from oxidation and 1 from hydrolysis.
Tryptophan oxidation by singlet molecular oxygen [O2(1Deltag)]: mechanistic studies using 18O-labeled hydroperoxides, mass spectrometry, and light emission measurements.
Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.
PEROXIDASE‐GENERATED TRIPLET INDOLE‐3‐ALDEHYDE ADDS TO URIDINE BASES AND EXCITES THE 4‐THIOURIDINE GROUP IN t‐RNAPhe
Abstract Triplet indole‐3‐aldehyde generated by the peroxidase catalyzed aerobic oxidation of indole‐3‐acetic acid adds to t‐RNAphe with an efficiency which can reach 20% of the theoretical value based upon the substrate. The reaction occurs with uridine groups and is of the Paterno‐Büchi type. Magnesium ions reduced addition and increased the sensitized emission from the 4‐thiouridine group. Our results, coupled with available biological data obtained with selectively labeled indole‐3‐acetic acid, support the hypothesis that formation of triplet indole‐3‐aldehyde is at the root of the biological effects of indole‐3‐acetic acid.