First Advisor

Robert M. Strongin

Term of Graduation

Winter 2022

Date of Publication


Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry







Physical Description

1 online resource (xxiii, 148 pages)


Type B hepatotoxicity has been reported for thiopurines, and this has limited their clinical uses. Although a lot of research has been done on thiopurines, the precise mechanism of their hepatotoxicity is still unclear. The results of the in vitro and in vivo experiments aimed at assessing the products of their CYP450-mediated bioactivation have been contradictory. Although it has been theorized that the hepatic toxicity of this class of drug is caused by their bioactivation to electrophilic reactive metabolite (RM) and concomitant protein binding to form immunogenic adducts, resulting in direct cellular dysfunction or an immune response via the formation of hapten, this has not been substantiated.

Electrochemistry coupled to mass spectrometry (EC-MS) has the potential to evaluate the toxicity of drugs, as it allows for the detection of the highly electrophilic intermediates that would otherwise not have been in the highly reducing physiological environment. This thesis describes the application of EC-MS to correlate bioactivation of thiopurine drugs with their hepatoxicity which could lead to better understanding and improved treatment outcomes for thiopurine drugs.

In this study, EC-MS was used as a biomimetic tool to study the formation of RMs as a causative factor in Type B hepatotoxicity of thiopurine drugs. Their oxidative metabolism was mimicked through the electrochemical generation and spectroscopic characterization of oxidation intermediates and products. Electrochemical oxidation was performed under controlled conditions to generate the oxidation intermediates and products, which were characterized using MS. Trapping of the formed RMs was attempted, and any conjugate formed was characterized with MS. UV-Vis spectrophotometry, and quantum chemical calculation (TD-DFT) were used to confirm the structure of the adduct.

Our results suggest two competing reaction pathways for the electrooxidation of thiopurine drugs; First, a reaction initiated through an electron abstraction leads to a thiyl radical that terminates in a disulfide. Second, a two-electron pathway that forms sulfenic acid (this is the rate-determining step for this pathway) through to sulfonic acid. While the former dominated in 6-thioguanine (6-TG), the latter was found to dominate in 6-mercaptopurine (6-MP). Electrooxidation of azathioprine (AZA) proceeded through the formation of 6-MP. When a freshly prepared oxidized sample was incubated with N-acetylcysteine (NAC), an adduct was formed only with 6-MP intermediate (sulfenic acid). The abundance and stability of the adduct formed depended on the relative amount of the NAC. The formation of the adduct strongly indicates that intermediate (sulfenic acid) is electrophilic and reactive. The 6-MP/NAC adduct molecule was were subjected to TD-DFT to obtain their theoretical UV-Vis spectra. Theoretical and experimental UV-Vis spectra were compared to verify the adduct's proposed structure, and there was a good agreement between experimental and quantum chemical calculation results. The lack of adducts from 6-TG confirms the dominancy of the thiyl radical in the EC oxidation of 6-TG.

The reaction intermediates and products were detected and were used to explore the mechanisms of the oxidation reaction. The formation of RM in the oxidation of 6-MP has been hypothesized before but has never been isolated. Moreover, our results show that very electrophilic and hepatotoxic sulfenic acid (S-oxide) is produced in the oxidation of 6-MP. The toxicity of 6-MP may be attributed to its bioactivation to RMs. EC-MS techniques are quite informative in the evaluation of toxicities from drugs.

Also, chemical oxidation was used to investigate the kinetics and mechanism of oxidation of the 6-MP and 6-TG with the biologically relevant hypoiodous acid (HOI) and iodine (I2). Kinetic data obtained from double mixing stopped-flow spectrophotometer and UV-vis spectrophotometer were used to deduce the dependence of the S-oxidation reaction on oxidant concentration, reductant concentration, and pH. Also, the products from the chemical oxidations were analyzed with MS and NMR and were compared to the electrochemically generated metabolites; though there were similarities, some differences were evident, probably due to reaction conditions. Lower limit bimolecular rate constants for the oxidation reactions were calculated, and the overall scheme of the oxidation reaction was fully explained using 19 composite reactions.


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