Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products
Graphical abstract
Introduction
5-Fluorouracil (5-FU) is a pyrimidine antimetabolite introduced in the clinic as an anticancer drug (Jordan, 2016). It is one of the most widely prescribed cytostatic pharmaceutical for the last 60 years. Up to 30% of its administered dose is excreted as the parent form that enters the environment (Kosjek and Heath, 2011). The measured environmental concentrations of 5-FU range from 5 to 100 ng/L in wastewater treatment plant influents (Kosjek et al., 2013), and may amount to more than 100 μg/L in hospital wastewaters (Mahnik et al., 2004). 5-FU is relatively stable in water, but undergoes different transformation reactions induced by chemical water treatments, such as ozonolysis or chlorination. The latter is the most widely used method for chemical treatment and disinfection of water (USEPA, 2004). Since 5-FU has been identified in wastewaters and surface waters (Mahnik et al., 2007) the potential exists for the formation of its disinfection byproducts during water chlorination.
The reaction between 5-FU and hypochlorous acid (HOCl) is the fundamental process which can occur in activated neutrophils in cancer patients (Winterbourn et al., 2016) or during chemical treatment of (hospital) wastewaters (Deborde and Gunten, 2008; Acero et al., 2010). Therefore, the chlorination of 5-FU is of utmost importance in medicinal and environmental chemistry. The elementary chemical reaction, the one with no enzyme assistance, is simple yet so intricate process. Mechanistic details underlying a HOCl-induced transformation of 5-FU have not been resolved. In addition, chlorinated products are unknown or only tentatively assigned, and their environmental effects have not been investigated. For this reason the chemical fate of 5-FU under chlorination conditions should be revisited.
We set to investigate the chlorination mechanism which gives rise to stable products. By using NMR spectroscopy coupled to high-level computational techniques, the relevant reaction profiles were described in details. The chlorinated products were isolated and their ecotoxicological effects were studied in acute immobilization assays with crustacean Daphnia magna.
In this work the interplay between experimental and theoretical methods has been shown as an efficient approach in solving some environmental problems. The results on the chlorination of 5-FU are relevant for a series of pyrimidines, and for nucleobase derivatives in particular.
Section snippets
General procedures and equipments
The syntheses were carried out in distilled water, and kinetic experiments in the phosphate buffer. Melting point was determined with a Büchi apparatus. For HPLC-MS analysis, ultra high-speed single quadropole mass spectrometer with ultra high-speed liquid chromatography, Prominence UFLC + LCMS-2020 from Shimadzu Corp. (Kyoto, Japan) was used. Separation was performed on a Zorbax SB C18 (150 × 2.1 mm 3.5 μm; Agilent Technologies Deutschland GmbH, Waldbronn, Germany) narrow bore LC column with
The chlorination of 5-fluorouracil
The initial site of chlorination of 5-FU is a conflicting issue. In the early report by Miyashita et al. (1982) the chlorination of 5-FU by molecular chlorine (Cl2) results in the formation of the C5-chlorinated product, whereas the recent study by Li and coworkers (Li et al., 2015) suggests that 5-FU is chlorinated at the C6-position. To resolve the controversy over the reaction regioselectivity, an experimental procedure for chlorination of 5-FU was repeated and product structures were
Conclusions
A detailed mechanism underlying the chlorination of 5-fluorouracil was explored by use of NMR analysis and quantum chemical calculations. In neutral aqueous solution the reactive species are N-anionic form of 5-FU and HOCl as a chlorinating agent. Both 1H and 19F NMR spectra of the reaction mixture confirm the formation of only one product, which corresponds to a chlorohydrin structure 3a. With the finding that the chlorination occurs at the C5-position, the old controversy on the site of the
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
We thank the Computing Centre SRCE of the University of Zagreb for allocating computer time on the Isabella cluster where a part of the calculations was performed.
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