Inês L. Martins

Synthesis and Biological Activity of 6-Selenocaffeine: Potential Modulator of Chemotherapeutic Drugs in Breast Cancer Cells

We report the development of a new microwave-based synthetic methodology mediated by Woollins’ reagent that allowed an efficient conversion of caffeine into 6-selenocaffeine. A preliminary evaluation on the modulation of antioxidant activity upon selenation of caffeine, using the DPPH assay, indicated a mild antioxidant activity for 6-selenocaffeine, contrasting with caffeine, that exhibited no antioxidant activity under the same experimental conditions. Interestingly, whereas 6-selenocaffeine has revealed to have a low cytotoxic potential in both MCF10A and MCF-7 breast cells (24 h, up to 100 µM, MTT assay), a differential effect was observed when used in combination with the anticancer agents doxorubicin and oxaliplatin in MCF-7 breast cancer cells. The co-treatment of doxorubicin (1 µM) and 6-selenocaffeine (100 µM) resulted in a slight decrease in cellular viability when compared to doxorubicin (1 µM) alone. Conversely, the seleno-caffeine derivative at the same concentration markedly increased the viability of oxaliplatin (100 µM)-treated cells (p < 0.01). Overall, this work highlights an emerging methodology to synthesize organoselenium compounds and points out the differential roles of 6-selenocaffeine in the modulation of the cytotoxicity of anticancer agents.

An insight into dapsone co-crystals: sulfones as participants in supramolecular interactions

Herein we disclose a new pathway for the design of dapsone co-crystals exploring the formation of N–H⋯O/N interactions using amide and pyridinic derivatives as potential co-formers. Two new co-crystals of dapsone, a sulfonamide antibiotic, with e-caprolactam and 4,4′-bipyridine have been synthesized preferentially by traditional solution techniques, but mechanochemistry has also been addressed. The full structural characterization of these forms is discussed and shows that: (a) in the co-crystal with e-caprolactam the typical NNH2⋯OSO2 interactions of dapsone molecules and the cages formed between them are disrupted by a new NNH2⋯OCONH interaction, in which e-caprolactam molecules further form amide⋯amide R22(8) synthons and (b) in the co-crystal with 4,4′-bipyridine, the NNH2⋯OSO2 interactions between dapsone molecules are maintained and additional NNH2⋯Npyridine interactions are responsible for the formation of 4,4′-bipyridine channels between dapsone cages. Moreover, the thermal stability of these co-crystals is also discussed, showing that the co-formers leave the structure and hence the reported melting corresponds to the melting of pure dapsone.

The first-line antiepileptic drug carbamazepine: Reaction with biologically relevant free radicals

&NA; Carbamazepine (CBZ) is one of the most widely used antiepileptic drugs by both adults and children. Despite its widespread use, CBZ is associated with central nervous system toxicity and severe hypersensitivity reactions, which raise concerns about its chronic use. While the precise mechanisms of CBZ‐induced adverse events are still unclear, metabolic activation to the epoxide (CBZ‐EP) has been thought to play a significant role. This work reports first‐hand evidence that CBZ reacts readily with biologically relevant thiyl radicals with no need for bioactivation. Using liquid chromatography coupled with high resolution mass spectrometry, multiple products from direct reaction of CBZ with glutathione (GSH) and N‐acetyl‐l‐cysteine (NAC) were unequivocally identified, including the same product obtained upon ring‐opening of CBZ‐EP. The product profile is complex and consistent with radical‐mediated mechanisms. Importantly, side products and adducts compatible with this non‐enzymatic pathway were identified in liver extracts from CBZ‐treated Wistar rats. The reaction of CBZ with GSH and NAC is more extensive in the presence of oxygen. Taking into consideration that GSH conjugation is, in general, a detoxification pathway, these results suggest that under hyperoxia/oxidative stress conditions the bioavailability of the parent drug may be compromised. Additionally, this non‐enzymatic process can be anticipated to play, at least in part, a role in the onset of CBZ‐induced adverse reactions due to the concomitant generation of reactive oxygen species. Therefore, the search for causal relationships between the formation of non‐enzymatically‐driven CBZ products and the occurrence of CBZ‐induced adverse events in human patients merits further research, aiming the translation of basic mechanistic findings into a clinical context that may ultimately lead to a safer CBZ prescription. HighlightsCBZ reacts with biologically relevant thiols with no need for bioactivation.The product profile is complex and consistent with radical‐mediated mechanisms.The reaction is more extensive in the presence of oxygen.Products compatible with radical‐mediated mechanisms were identified in liver extracts from CBZ‐treated rats.The concomitant generation of reactive oxygen species can have a role on CBZ‐induced adverse reactions. Graphical abstract Figure. No caption available.

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches

ABSTRACT Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug‐induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk. Etravirine (ETR) was the first second‐generation Non‐Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV‐infected patients who developed resistance to the first‐generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post‐marketing reports of severe ETR‐induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent. In the current study, high resolution mass spectrometry‐based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co‐factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV‐infected patients on ETR therapy. We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N‐acetyl‐L‐cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR‐based regimens.

Quinoid derivatives of the nevirapine metabolites 2-hydroxy- and 3-hydroxy-nevirapine: activation pathway to amino acid adducts

Nevirapine (NVP) is the non-nucleoside HIV-1 reverse transcriptase inhibitor most commonly used in developing countries, both as a component of combined antiretroviral therapy and to prevent mother-to-child transmission of the virus; however, severe hepatotoxicity and serious adverse cutaneous effects raise concerns about its safety. NVP metabolism yields several phenolic derivatives conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid derivatives prone to react with bionucleophiles and initiate toxic responses. We investigated the ability of two phenolic NVP metabolites, 2-hydroxy-NVP and 3-hydroxy-NVP, to undergo oxidation and subsequent reaction with bionucleophiles. Both metabolites yielded the same ring-contraction product upon oxidation with Fremys salt in aqueous medium. This is consistent with the formation of a 2,3-NVP-quinone intermediate, which upon stabilization by reduction was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Additionally, we established that the oxidative activation of 2-hydroxy-NVP involved the transient formation of both the quinone and a quinone-imine, whereas 3-hydroxy-NVP was selectively converted into 2,3-NVP-quinone. The oxidations of 2-hydroxy-NVP and 3-hydroxy-NVP in the presence of the model amino acids ethyl valinate (to mimic the highly reactive N-terminal valine of hemoglobin) and N-acetylcysteine were also investigated. Ethyl valinate reacted with both 2,3-NVP-quinone and NVP-quinone-imine, yielding covalent adducts. By contrast, neither 2,3-NVP-quinone nor NVP-derived quinone-imine reacted with N-acetylcysteine. The product profile observed upon Fremys salt oxidation of 2-hydroxy-NVP in the presence of ethyl valinate was replicated with myeloperoxidase-mediated oxidation. Additionally, tyrosinase-mediated oxidations selectively yielded 2,3-NVP-quinone-derived products, while quinone-imine-derived products were obtained upon lactoperoxidase catalysis. These observations suggest that the metabolic conversion of phenolic NVP metabolites into quinoid electrophiles is biologically plausible. Moreover, the lack of reaction with sulfhydryl groups might hamper the in vivo detoxification of NVP-derived quinone and quinone-imine metabolites via glutathione conjugation. As a result, these metabolites could be available for reaction with nitrogen-based bionucleophiles (e.g., lysine residues of proteins) ultimately eliciting toxic events.