Comparison of metabolic pathways of different α-N-heterocyclic thiosemicarbazones.

Clinical failure of novel drugs is often related to their rapid metabolism and excretion. This highlights the importance of elucidation of their pharmacokinetic profile already at the preclinical stage of drug development. Triapine, the most prominent representative of α-N-heterocyclic thiosemicarbazones, was investigated in more than 30 clinical phase I/II trials, but the results against solid tumors were disappointing. Recent investigations from our group suggested that this is, at least partially, based on the fast metabolism and excretion. In order to establish more detailed structure/activity/metabolism relationships, herein a panel of 10 different Triapine derivatives was investigated for their metabolic pathways. From the biological point of view, the panel consists of terminally dimethylated thiosemicarbazones with nanomolar IC50 values, derivatives with micromolar cytotoxicities comparable to Triapine and a completely inactive representative. To study the oxidative metabolism, a purely instrumental approach based on electrochemistry/mass spectrometry was applied and the results were compared to the data obtained from microsomal incubations. Overall, the investigated thiosemicarbazones underwent the phase I metabolic reactions dehydrogenation, hydroxylation, oxidative desulfuration (to semicarbazone and amidrazone) and demethylation. Notably, dehydrogenation resulted in a ring-closure reaction with formation of thiadiazoles. Although strong differences between the metabolic pathways of the different thiosemicarbazones were observed, they could not be directly correlated to their cytotoxicities. Finally, the metabolic pathways for the most cytotoxic compound were elucidated also in tissues collected from drug-treated mice, confirming the data obtained by electrochemical oxidation and microsomes. In addition, the in vivo experiments revealed a very fast metabolism and excretion of the compound. Graphical abstract Structure/activity/metabolisation relationships for 10 anticancer thiosemicarbazones were established using electrochemical oxidation coupled to mass spectrometry (EC-MS) and human liver microsomes analyzed by LC-MS.

Understanding the metabolism of the anticancer drug Triapine: electrochemical oxidation, microsomal incubation and in vivo analysis using LC-HRMS.

α-N-Heterocyclic thiosemicarbazones are among the most promising ribonucleotide reductase inhibitors identified so far. Triapine, the most prominent representative of this class of substances, has been investigated in multiple phase I and II clinical trials. With regard to clinical practice, Triapine showed activity against hematological diseases, but ineffectiveness against a variety of solid tumors. However, the reasons are still vague and the amount of ADME (absorption, distribution, metabolism and excretion) data for Triapine available in the literature is very limited. Therefore, different analytical tools were used to investigate the metabolism of Triapine including electrochemical oxidations, liver microsomes and in vivo samples from mice. The main metabolic reactions, observed by all three methods, were dehydrogenation and hydroxylations, confirming that electrochemistry, as a purely instrumental approach, can be applied for the simulation of metabolic pathways. The dehydrogenated metabolite M1 was identified as a thiadiazole ring-closed oxidation product of Triapine. From a biological point of view, M1, as a key metabolite, is of interest since the crucial chemical property of α-N-heterocyclic thiosemicarbazones to bind metal ions is lost and cytotoxicity studies showed no anticancer activity of M1. The in vivo data of the urine samples revealed very high levels of the metabolites and Triapine itself already 15 min after treatment. This clearly indicates that Triapine is rapidly metabolised and excreted, which represents an important step forward to understand the possible reason for the inefficiency of Triapine against solid tumors.

Antioxidant Properties and Oxidative Transformation of Different Chromone Derivatives.

Nowadays, there is an increase in the application of natural products for the prevention of different disorders or adjuvant substances next to pharmacological treatment. Phytochemicals include different chromone derivatives, which possess a wide spectrum of biological activity. The aim of the present study was the investigation of the antioxidant activity, cytotoxicity and oxidative transformation of nine chromone derivatives. First, we investigated the radical scavenging activity (ABTS), the oxygen radical absorption capacity (ORAC) and the ferric reducing antioxidant power (FRAP) of the investigated molecules. The cytotoxic effects of the compounds were tested on H9c2 cell cultures by the MTT assay. Each compound showed a significant ORAC value compared to the reference. However, the compound 865 possess significantly higher FRAP and ABTS activity in comparison with the reference and other tested molecules, respectively. Based on these assays, the compound 865 was selected for further analysis. In these experiments, we investigated the oxidative metabolism of the compound in vitro. The molecule was oxidized by the Fenton reaction, artificial porphyrin and electrochemistry; then, the formed products were identified by mass spectrometry. Four possible metabolites were detected. The results revealed the compound 865 to possess good antioxidant properties and to be stable metabolically; hence, it is worth investigating its effects in vivo.

Dual reductive/oxidative electrochemistry/liquid chromatography/mass spectrometry: Towards peptide and protein modification, separation and identification.

A new hyphenated technique based on on-line dual (oxidative and reductive) electrochemistry coupled to liquid chromatography and high resolution electrospray mass spectrometry is presented. Two liquid streams are combined, with one containing a disulfide, which is reduced to the respective thiol in an electrochemical cell based on a titanium working electrode. The other stream contains phenol, which is electrochemically activated to benzoquinone on a boron-doped diamond working electrode. Upon combination of the two streams, a Michael addition takes places, leading to the covalent coupling of thiol to quinone. In continuous flow, the reaction mixture is transferred into an injection valve and the products are separated by reversed phase liquid chromatography and detected by electrospray-high resolution mass spectrometry. Proof of concept is demonstrated for low molecular mass disulfides and peptides, but further optimization will be required in future work to achieve efficient protein labelling.

Liquid chromatography/mass spectrometry to study oxidative degradation of environmentally relevant pharmaceuticals by electrochemistry and ozonation.

In this work, the potential of electrochemical oxidation as a tool for the rapid prediction of transformation products in water appearing after ozonation is investigated. These two approaches were compared by choosing the two environmentally relevant model compounds diclofenac and metoprolol and comparison of their transformation products after electrochemical oxidation and treatment with ozone. Within these two approaches, certain similarities were observed in the resulting chromatograms: Six transformation products of the electrochemical oxidation of metoprolol were also detected in the ozone samples. For diclofenac two transformation products matched. Additionally, five of the electrochemically generated oxidation products were reported in literature to occur after water treatment processes. The application of a boron-doped diamond working electrode for electrochemical oxidation allowed the generation of hydroxyl radicals, which was shown by spin trapping experiments with p-chlorobenzoic acid. This allowed the generation of certain transformation products previously not obtained by electrochemical oxidation. Concluding, the hyphenation of electrochemistry with liquid chromatography and mass spectrometry offers a useful tool in transformation studies.