A Proton Relay Process as the Mechanism of Activation of the Histamine H 3 -Receptor Determined by 1 H NMR and ab Initio Quantum Mechanical Calculations Jari T. Kovalainen,* ,² Johannes A. M. Christiaans, ²,‡ Risto Ropponen, ² Antti Poso, ² Mikael Pera1 kyla1 , § Jouko Vepsa1 la1 inen, § Reino Laatikainen, § and Jukka Gynther ², ⊥ Contribution from the Department of Pharmaceutical Chemistry, Department of Chemistry, UniVersity of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland ReceiVed September 13, 1999. ReVised Manuscript ReceiVed May 16, 2000 Abstract: This study proposes a new mechanism of activation of the histamine H 3 -receptor based on stabilization of the active state of the receptor protein by a proton relay process. A series of histamine H 3 -receptor agonists and one antagonist, all containing an imidazole and a side chain amino group, were studied using 1 H NMR and ab initio quantum mechanical calculations. A significant correlation (r )-0.87) between the formation energy of the active reaction intermediate and the agonistic activity is found. The calculated proton-transfer energies and pK a values of the reaction intermediates (at the MP2/6-31+G*//HF/6-31+G* level in the gas and aqueous phases), as well as, 1 H NMR conformational analysis, enable a qualitative and quantitative determination of intramolecular hydrogen bonding and its effect on proton release from the imidazole N(τ)-atom. The results indicate that the histamine H 3 -receptor is not activated by a proton release from imidazole N(τ)-atom but through adoption of a folded conformation of the ligand which stabilizes the active state of the receptor by an intramolecular hydrogen bond. Introduction The histamine H 3 -receptor 1 has been described as a presyn- aptically located auto- and heteroreceptor in histaminergic and nonhistaminergic neurones in the central and autonomic nervous system. Furthermore, the histamine H 3 -receptor is involved in the regulation of the release of several other neurotransmitters (i.e., acetylcholine, dopamine, γ-aminobutyric acid (GABA), glutamate, noradrenaline, and 5-hydroxytryptamine (5-HT)). The histamine H 3 -receptor is involved in control of sleep and wakefulness, food intake, and inhibition of seizures. Thus, many possible therapeutic targets for H 3 -receptor ligands have been suggested, i.e., asthma, migraine, septic shock, heart failure, acute myocardial infarction, learning and memory degenerative disorders (like Alzheimer’s disease), attention deficit hyperactive disorder (ADHD), epilepsy, and obesity. 2 The histamine H 3 -receptor belongs to the superfamily of G-protein coupled receptors. Recently the human histamine H 3 - receptor was cloned; however, the localization and topography of the binding sites were not resolved. 3 Therefore, approaches to study the molecular determinants for receptor binding and activation have to be focused on methods that utilize the information from molecular structures and structure activity relationships of known ligands. All known potent histamine H 3 -receptor agonists are small molecules. Common properties for these agonists are a 4(5)- substituted imidazole and an ionizable group in the side chain (Figure 1). The imidazole group plays a role in the activation mechanism of the histamine H 2 -receptor and 5-HT-receptor. 4-7 Because of the unique role of the imidazole group as a proton- transfer device, 8-10 its chemical and physical properties have widely been studied with many theoretical and experimental approaches. 11-15 * To whom correspondence should be addressed. E-mail: jari.kovalainen@ orionpharma.com. Present address: Orion Corporation ORION PHARMA, P.O. Box 1780, FIN-70701 Kuopio, Finland. ² Department of Pharmaceutical Chemistry. ‡ Present address: Byk Nederland bv, postbus 61, NL-1160 AB, Zwanenburg, The Netherlands. § Department of Chemistry, Kuopio. ⊥ Finncovery Ltd, Kuopio, Finland. (1) Arrang, J. M.; Garbarg, M.; Schwartz, J. C. Nature 1983, 302, 832- 837. (2) Leurs, R.; Blandina, P.; Tedford, C.; Timmerman, H. Trends Pharmacol. Sci. 1998, 19, 177-183. (3) Lovenberg, T. W.; Roland, B. L.; Wilson, S. J.; Jiang, X.; Pyati, J.; Huvar, A.; Jackson, M. R.; Erlander, M. G. Mol. Pharmacol. 1999, 55, 1101-1107. Figure 1. Histamine H3-receptor activating compounds: histamine 1, N R -methyl histamine 2, R(R),S()-dimethyl histamine 3a, S(R),R()- dimethyl histamine 3b, R(R)-methyl histamine 4a, S(R)-methyl hista- mine 4b, 4-(1H-imidazol-4(5)-ylmethyl)piperidine (immepip) 5, S-(2- (1H-imidazol-4(5)-yl)ethyl)isothiourea (imetit) 6, 2(S)-amino-3-(1H- imidazol-4(5)-yl)propyl 4-bromobenzyl ether dihydrochloride 7 (exhibits also H3-antagonistic activity 36 ), 2(S)-amino-3-(1H-imidazol-4(5)-yl)- propyl cyclohexylmethyl ether 8, 2(S)-amino-3-(1H-imidazol-4(5)-yl)- propyl 4-iodobenzyl ether 9 (exhibits only H3-antagonistic activity 36 ). 6989 J. Am. Chem. Soc. 2000, 122, 6989-6996 10.1021/ja993322f CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000