2 PERKIN DOI: 10.1039/b002357o J. Chem. Soc., Perkin Trans. 2, 2000, 1781–1787 1781 This journal is © The Royal Society of Chemistry 2000 Empirical and molecular modeling study of the pyridinium species RHPP  , an abundant and potentially neurotoxic metabolite of haloperidol Frédéric Ooms, a * Sébastien Delvosal, a Johan Wouters, a François Durant, a Gisella Dockendolf, d Clinton Van’t Land, b Thomas Glass, b Neal Castagnoli, Jr. b and Cornelis J. Van der Schyf b,c,d a Laboratoire de Chimie Moléculaire Structurale, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium b Department of Chemistry and the Harvey W. Peters Center for the Study of Parkinson’s Disease and Disorders of the Central Nervous System, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA c Department of Biomedical Sciences and Pathobiology, VA-MD Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA d Department of Pharmaceutical Chemistry, Potchefstroom University for Christian Higher Education, Potchefstroom 2520, South Africa Received (in Cambridge, UK) 24th March 2000, Accepted 6th June 2000 Published on the Web 11th August 2000 The “reduced” haloperidol pyridinium metabolite (RHPP + ) is found in the brain, plasma and urine of patients treated with the neuroleptic drug haloperidol (HP). RHPP + is suspected to be neurotoxic through a mechanism that entails interference with the mitochondrial electron transport chain. We have studied the conformation of this flexible molecule in solution (using NMR) and in the solid state by single crystal X-ray analysis. Using the solid state structure as initial input, molecular dynamics runs indicated that the molecule preferably exists in an unfolded, rather than a folded conformation. We propose that the interaction of RHPP + with complexes in the mitochondrial respiratory chain is stabilized primarily by an ionic bond involving the cationic nitrogen and secondarily by hydrogen-bond anchoring originating from the hydroxy group. A comparison with HPP + , the “unreduced” pyridinium metabolite of HP, suggests that this latter interaction may—among other considerations such as lipophilicity—account for differences in the in vitro toxicological profiles of RHPP + and HPP + , which carries a ketone group in lieu of the hydroxy. Introduction Haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxypiperidino]-4'- fluorobutyrophenone, (HP, 1, Scheme 1) is a potent neuroleptic agent that, like other members of this pharmacological class known as typical neuroleptics, causes severe extrapyramidal side effects including parkinsonism and a condition known as tardive dyskinesia (TD) 1 that, in many cases, is irreversible. 2 The irreversibility of TD suggests an underlying pathology 3 that may be the result of neuroleptic treatment. HP is a 4-piperidinol derivative that resembles the parkinsonian- inducing pro-neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetra- hydropyridine (MPTP, 2, Scheme 1) in that it bears an aryl group at C-4 of the azacycle. 4 MPTP is transformed through brain monoamine oxidase B (MAO-B) 5 and subsequent oxidation to the N-methyl-4-pyridinium (MPP + ) species, its ultimate neurotoxic form. 6 Simple dehydration of HP, a reaction which is reported to occur in microsomal incubations, 7 gives rise to the corresponding 1,2,3,6-tetra- hydropyridine derivative HPTP (12, Scheme 1), an even closer analog of MPTP. Similar to MPTP, the structural features present in HP also predispose this compound to an oxidative pathway leading to pyridinium metabolites. 8 Ring α-carbon oxidation of the piperidine derivatives (1, 7) bearing a leaving group (OH) at C-4 generates the corresponding iminium metabolites 3 and 8 that, via the enamines 4 and 9, will undergo spontaneous conversion to the dihydropyridinium intermediates 5 and 10 (Scheme 1). Dihydropyridinium compounds 5 and 10 undergo spontaneous autoxidation to form the pyridinium species HPP + (6) 8 and RHPP + (11). 9,10 HP (1) and its principal circulating metabolite in humans, (S)-(-) “reduced” HP (RHP, 7), 11 are both candidates for such reaction sequences (1→3→4→5→6, in the case of HP, and 7→8→9→10→11, in the case of RHP). Alternatively, simple dehydration of 1 and 7 could generate the corresponding tetrahydropyridine derivatives HPTP (12) 12 and RHPTP (13) respectively which, following ring α-carbon oxidation, would yield the same dihydropyridinium inter- mediates 5 and 10. 13 The metabolic fate of HP (1) has been examined in rodents 12,19 and baboons 13 treated with either HP or HPTP (12) and in humans treated with HP 9,21 employing LC-MS/MS and LC-fluorescence techniques designed to detect pyridinium metabolites. The results of the studies in rodents and baboons have established that both HP and HPTP are biotransformed to the HP pyridinium metabolite HPP + (6), as is HP in humans. Unlike the corresponding conversion of MPTP to the neuro- toxic pyridinium species MPP + , the oxidative biotrans- formation of HP to HPP + is not mediated by MAO-B. 8 Neither HP nor HPTP are substrates for MAO-B, although both are biotransformed to the pyridinium product by rat and human liver microsomal preparations, 8 but the conversion of HPTP to HPP + , presumably via the dihydropyridinium intermediate 5, is catalyzed by MAO-A. 22 Recent findings 10,14–16 have led to the