Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products

doi:10.1016/j.chemosphere.2018.05.140

Chemosphere

Volume 207, September 2018, Pages 612-619

https://doi.org/10.1016/j.chemosphere.2018.05.140 Get rights and content

Highlights

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Chlorination products of 5-fluorouracil were characterized by NMR spectroscopy.
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Detailed reaction mechanism was calculated by DFT methods.
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Ecotoxicological profiles of degradation products differ from the parent compound.
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Duration of chlorination is an important parameter for EC50 values of byproducts.

Abstract

What happens to drugs in the chlorinating environment? Degradation products may vary in pharmacological profiles and in ecotoxicity potentials compared to the parent compound. This study combines synthesis, NMR spectroscopy, quantum chemical calculations, and toxicity experiments on Daphnia magna to investigate chemical fate of antineoplastic drug 5-fluorouracil (5-FU) in chlorinated environment, which is common in waste-water treatment procedures, but also endogenous in activated neutrophils. A reduction of toxicity (EC₅₀ after 48 h is 50% higher than for the parent 5-FU) was observed after the first chlorination step, in which a chlorohydrin 5-chloro-5-fluoro-6-hydroxy-5,6-dihydrouracil was formed. Further chlorination leads to N-chlorinated intermediate, that undergoes the pyrimidine ring opening reaction. The final product, 2-chloro-2-fluoro-3,3-dihydroxypropanoic acid was obtained after the loss of the chlorinated urea fragment. This is the most potent compound in the reaction sequence, with toxicity parameter EC₅₀, after 48 h, more than twice lower compared to the parent 5-FU. Clearly, the contact time between chlorinating species and degradation products provide different ecotoxicological properties of reaction mixtures. Interplay between experimental and theoretical procedures, to properly describe reaction pathways and provide more information on toxicity profiles, is a way forward in environmental science research.

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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View full text

Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

General procedures and equipments

The chlorination of 5-fluorouracil

Conclusions

Conflicts of interest

Acknowledgments

Water Res.

Water Res.

Trends Anal. Chem.

J. Chromatogr. A

Pharm. Biomed. Anal.

J. Hazard Mater.

Chemosphere

Chemosphere

Chem. Phys.

Chemosphere

5-Fluorouracil solutions: NMR study of acid–base equilibrium in water and DMSO

J. Phys. Org. Chem.

Microsolvation of uracil and its conjugate bases: a DFT study of the role of solvation on acidity

J. Phys. Chem.

Density-functional thermochemistry. III. The ole of exact exchange

J. Chem. Phys.

Nucleophilic reactivities of the anions of nucleobasees and their subunits

Chem. Eur J.

Results of toxic action of water pollutants on Daphnia magna straus tested by an improved standardized procedure

Wasser-Abwasser-Forsch

Aquatische ökotoxikologie ausgewahlter arzneimittel. Z. Umweltchem

Okotox.

Study of the factors that control the ratio of the products between 5-fluorouracil, uracil, and tetrahydrobenzoxazepine O,O-acetals bearing electron-withdrawing groups on the nitrogen atom

J. Org. Chem.

Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for Existing Substances. Part II; Environmental Risk Assessment

Gaussian 09, Revision D.01

A successful DFT calculation of carbon-13 NMR chemical shifts and carbon–fluorine spin–spin coupling constants in (η6-fluoroarene)tricarbonylchromium complexes

RSC Adv.

A Comparison of Aquatic Species Responses to Anticancer Drug Exposure