New Arylpiperazine Derivatives as Antagonists of the Human Cloned 5-HT 4 Receptor Isoforms Sophie Curtet, § Jean-Louis Soulier, § Ivan Zahradnik, † Mireille Giner, § Isabelle Berque-Bestel, § Jeanne Mialet, ‡ Frank Lezoualc’h, ‡ Patrick Donzeau-Gouge, # Sames Sicsic, § Rodolphe Fischmeister, ‡ and Michel Langlois* ,§ CNRS-BIOCIS (UPRES A 8076) and Laboratoire de Cardiologie Cellulaire et Mole ´ culaire, INSERM U-446, Institut de Signalisation et Innovation The ´ rapeutique (IFR-ISIT), Faculte ´ de Pharmacie, Universite ´ de Paris-Sud, 92296 Cha ˆ tenay-Malabry, France Received June 19, 2000 New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT 4 receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5- chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human 5-HT 4(e) isoform stably expressed in C6 glial cells with [ 3 H]GR 113808 as the radioligand. The affinity values (K i ) depended upon the substituent on the aromatic ring. A chlorine atom produced a marked drop in activity (K i > 100 nM), while a m-methoxy group gave a compound with nanomolar affinity (K i ) 3 nM). The most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, respectively). K i values for 9a and 9r were determined for the 5-HT 4(a) , 5-HT 4(b) , 5-HT 4(c) , and 5-HT 4(d) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT 4(e) receptor and shifted the 5-HT concentration-effect curve on adenylyl cyclase activity with pK D values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT on the L-type calcium current (I Ca ) with a K D value of 0.7 nM. Introduction The 5-HT 4 receptor is a member of the seven trans- membrane-spanning G protein-coupled family of receptors. 1-3 Initially, it was characterized by Dumuis 4 in neuronal cells and shown to be linked positively to adenylyl cyclase. This neuronal receptor was similar to that discovered by Clarke 5 in the gastrointestinal (GI) system where it was shown to be responsible for stimulating motility. Considerable interest has devel- oped in this receptor because it provided a mechanism of action for the gastric prokinetic drugs, a number of which were found to be 5-HT 4 receptor agonists. 6 Many of these compounds are members of the generic benza- mide family, derived from metoclopramide. 7 They are amides or esters of 4-amino-5-chloro-2-methoxybenzoic acid derivatives such as renzapride (2), 8a SC-53116 (3), 8b ML 10302 8c (1), cisapride (4), 8c prucalopride (5), 8d and mosapride (6) (Chart 1). 8e The pharmacological charac- terization of this receptor was facilitated by the syn- thesis of potent and selective antagonists such as GR 113808 (7) 9a and SB 207710 (8). 9b 5-HT 4 receptors are expressed in a wide variety of tissues: brain, heart, bladder, intestine, and kidney 10,11 and have been implicated in a number of pathological disorders: memory deficits, 12a irritable bowel syndrome, gastroparesis, urinary incontinence, 11 and cardiac atrial arrhythmias. 12b Recently, several groups reported the cloning of the receptors from different species. 1-3 They are characterized by several splice variants which differ in the length and sequence of their C termini. We described 2 the cloning and pharmacological character- ization of four splice variants of the human 5-HT 4 receptor: 5-HT 4(a) , 5-HT 4(b), 5-HT 4(c) , and 5-HT 4(d). More recently another isoform (5-HT 4(e) ) has been character- ized. 13 Other 5-HT receptor subtypes also have splice variants, in particular the 5-HT 7 receptor which has four isoforms 14 produced by alternative splicing in the car- boxyl terminus. The expression of 5-HT 4 receptor iso- forms depends on the tissue: three or four variants were expressed in heart atrium, brain, and intestine, while only one was found in kidney and bladder. 2 On the other hand, the 5-HT 4(d) variant was only present in the intestine. 2 The availability of the cloned 5-HT 4 receptors stimu- lated more interest in the search for new ligands. In particular the potential use of 5-HT 4 receptor antago- nists in the treatment of the human atrial arrhythmias has become an emerging concept 12b,15,16 since the 5-HT 4 receptor stimulates two potentially arrhythmogenic ion channel currents in human atrial tissue: the L-type calcium current (I Ca ) 17 and the pacemaker f channel current (I f ). 15 A very recent study has demonstrated the potential therapeutic value of a selective 5-HT 4 receptor antagonist (RS-100302) as an antiarrhythmic drug. 16b Therefore, there is much interest in the clinical evalu- * To whom correspondence should be addressed. Tel: 33 1 46 83 57 36. Fax: 33 1 46 83 57 40. E-mail: michel.langlois@cep.u-psud.fr. § CNRS-BIOCIS (UPRES A 8076). ‡ Laboratoire de Cardiologie Cellulaire et Mole ´culaire, INSERM U-446. † On leave from: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia. # Permanent address: Institut Hospitalier Jacques Cartier, Service de Chirurgie Cardiaque, Avenue du Noyer Lambert, F-91349 Massy Cedex, France. 3761 J. Med. Chem. 2000, 43, 3761-3769 10.1021/jm0009538 CCC: $19.00 © 2000 American Chemical Society Published on Web 09/19/2000