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Assignment of the absolute configuration at the sulfur atom of thioridazine metabolites by the analysis of their chiroptical properties: The case of thioridazine 2-sulfoxide

https://doi.org/10.1016/j.jpba.2010.01.047Get rights and content

Abstract

Thioridazine (THD) is a commonly prescribed phenotiazine neuroleptic drug, which is extensively biotransformed in the organism producing as main metabolites sulfoxides and a sulfone by sulfur oxidation. Significant differences have been observed in the activity of the THD enantiomers as well as for its main metabolites, and enantioselectivity phenomena have been proved in the metabolic pathway. Here the assignment of the absolute configuration at the sulfur atom of enantiomeric THD-2-sulfoxide (THD-2-SO) has been carried out by circular dichroism (CD) spectroscopy. The stereoisomers were separated by HPLC on Chiralpak AS column, recording the CD spectra for the two collected enantiomeric fractions. The theoretical electronic CD spectrum has been obtained by the TDDFT/B3LYP/6-31G*, as Boltzmann averaging of the contributions calculated for the most stable conformations of the drug. The comparison of the simulated and experimental spectra allowed the absolute configuration at the sulfur atom of the four THD-2-SO stereoisomers to be assigned. The developed method should be useful for a reliable correlation between stereochemistry and activity and/or toxicity.

Introduction

Thioridazine (THD), a commonly prescribed phenotiazine neuroleptic drug, used for the treatment of schizophrenia and other psychiatric disorders, is extensively biotransformed in the organism producing as main metabolites the sulfoxides at positions 2 and 5, and the sulfone derivate at position 2 [1], [2].

THD is currently administered as a racemate, although significant differences have been observed for the activity of this drug depending on the stereochemistry; the antipsychotic effect of racemic THD is mainly associated with (R)-THD [2]. In the metabolic process a new stereocenter is formed at the sulfur atom in thioridazine-2-sulfoxide (THD-2-SO) and thioridazine-5-sulfoxide (THD-5-SO) metabolites (Fig. 1). So for each of these two compounds two pairs of enantiomers are possible: (Rc, Rs) (Sc, Ss) and (Rc, Ss) (Sc, Rs), the first and second pairs are diastereoisomers. The (R) and (S) configurations of THD and their metabolites are related to the chiral carbon at position 2 in the piperidyl ring (c) and to the sulfur atom of the sulfoxide metabolites (s).

Considering that THD-2-SO and THD-2-SO2 metabolites are also therapeutically active [3], [4], [5], but with undesirable side effects that can be attributed to specific stereoisomers, the assignment of the absolute configuration to each of these streoisomeric forms of the drug and of its metabolites should allow reliable information on the relationship structure/activity in clinical and/or toxicological studies. In addition, THD-5-SO metabolite contributes to the cardio toxicity of the drug [6], [7].

We report here the determination of the absolute configuration at the sulfur atom of THD-2-SO. The diastereoisomers were separated by non-chiral HPLC and referred as fast-eluting (FE) THD-2-SO and slow-eluting (SE) THD-2-SO, based on their chromatographic behaviour [1], [2]. The enantiomers of the fast-eluted couple (FE) have been separated by HPLC on Chiralpack AS, recording the circular dichroism (CD) spectra for the two collected enantiomeric fractions.

To simulate the experimental electronic CD spectrum of THD 2-SO, we arbitrarily assumed S absolute configuration for the sulfur atom and removed the chain linked to the N atom to reduce the numbers of conformers, without neglecting significant chromophores. Two stable conformers have been found in the gas phase: using the above two conformers as input geometries and the TDDFT/B3LYP/6-31G* method [8], [9], [10] the theoretical CD spectrum (after Boltzmann averaging) was obtained, with the simulated curve (S) AC of the sulfur atom that corresponds to the first eluted peak.

Section snippets

Chemicals

rac-Thioridazine, thioridazine-2-sulfone and thioridazine-2-sulfoxide, were kindly supplied by Novartis Pharma AG (Basel, Switzerland). The THD-2-SO standard sample consisted mainly of the racemic mixture of the enantiomers of the fast-eluted (FE) diastereoisomer, according to the separation procedure reported in the literature [1]. Stock standard solutions were prepared in methanol at concentrations of 200 and 400 μg mL−1 and were stored at −20 °C in the absence of light. HPLC-grade hexane,

Stereochemical characterization of THD and of its metabolites by enantioselective HPLC and circular dichroism

Enantioselective HPLC was successfully applied for the stereoisomeric resolution of THD and of its main metabolites. In particular, enantiomeric fractions of THD, THD-2SO2 and of both the diastereoisomers of THD-2SO were obtained at preparative level, in order to carrying out the CD spectra of each fraction, and then to establish the relationship between sign of the CD spectra and absolute configuration. Thus different chiral stationary phases were used in order to have the enantioselectivity

Conclusions

The Sc, Ss and Rc, Rs absolute configuration was assigned to the first and second eluted fractions of THD-2SO, respectively. The satisfactory simulation of the (Sc, Ss)-THD-2SO CD spectrum by the calculated CD spectrum of (Ss)-1, demonstrates that the CD spectrum depends primarily on the absolute configuration at the sulfur atom. This result, which concerns one of the main metabolites of such an important drug, is really relevant for these studies because this information allows a deeper

Acknowledgments

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support and for the grant of research fellowships. Thanks are also due to Università di Bologna (C.B.) and to Università della Basilicata (G.M., C.R.) for financial support.

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