In Vitro and in Vivo Characterization of 3-{2-[6-(2-tert-Butoxyethoxy)pyridin-3-yl]- 1H-imidazol-4-yl}benzonitrile Hydrochloride Salt, a Potent and Selective NPY5 Receptor Antagonist Richard L. Elliott,* ,† Robert M. Oliver, † Marlys Hammond, † Terrell A. Patterson, † Li She, † Diane M. Hargrove, † Kelly A. Martin, † Tristan S. Maurer, † J. Cory Kalvass, † Bradley P. Morgan, † Paul A. DaSilva-Jardine, † Ralph W. Stevenson, † Christine M. Mack, ‡ and James V. Cassella ‡ Cardiovascular and Metabolic Diseases, PGRD, Pfizer Inc., Groton, Connecticut 06340, and Neurogen Corporation, Branford, Connecticut 06405 Received September 13, 2002 Abstract: To investigate the anorectic potential of NPY5 receptor antagonists, we have profiled the in vitro and in vivo properties of 3-{2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]-1H-imi- dazol-4-yl}benzonitrile hydrochloride salt (1). This compound was found to have excellent NPY5 receptor affinity and selectivity, potent functional antagonism, and good peripheral and central nervous system exposure in rats. This compound attenuated bovine pancreatic polypeptide induced food intake in rats but failed to demonstrate anorectic activity in rodent natural feeding models. Introduction. Neuropeptide Y (NPY) is a 36 amino acid peptide with broad distribution within the central nervous system (CNS). On the basis of the potent orexigenic effects of NPY in vivo, many researchers have investigated the utility of NPY antagonists as potential anorectic agents. The pharmacological activity of NPY is mediated by at least six receptor subtypes: Y1-Y5 and y6 (in mouse and rabbit, a pseudogene in humans). Two of these NPY receptor subtypes, NPY1 and NPY5, have been implicated in mediating the orexigenic effects of NPY. Evidence supporting the role of the NPY5 receptor subtype in regulating food intake and body weight includes the finding that the orexigenic effects of peptide analogues of NPY correlate with affinity for the NPY5 receptor, 1 the anatomical location and physi- ological regulation of NPY5 receptors in areas of the CNS relevant to feeding, 2 and the determination that NPY5 antisense oligodeoxynulceotides have anorectic effects in rodents. 3 In addition, a number of papers have been published claiming anorectic effects with NPY5 receptor antagonists. 4 However, the relative importance and physiological roles of NPY1 and NPY5 receptors in mediating feeding behavior are still not fully defined, 5 and differences have been observed in the anorectic effects of NPY5 receptor antagonists on food intake induced by exogenous NPY5 agonists versus the effect observed in natural feeding models (vide infra). We originally identified a series of 2,4-diarylimida- zoles 6 as NPY5 antagonists from a high-throughput screen and subsequently optimized the in vitro potency and PK properties of this series. We now report on our in vitro and in vivo findings with a key compound in this series, 3-{2-[6-(2-tert-butoxyethoxy)pyridin-3-yl]- 1H-imidazol-4-yl}benzonitrile hydrochloride salt (1). 7 Chemistry. Compound 1 was synthesized as outlined in Scheme 1. Condensation of the anion of 2-tert- butoxyethanol, generated using lithium bistrimethylsi- lylamide as the base, with 6-chloronicotinonitrile af- forded the 2-alkoxy-5-cyanopyridine 2 in 87% yield. Construction of the imidazole ring of 1 relied on the condensation of the amidine, generated from the nitrile moiety of 2, with an R-bromo ketone. To circumvent difficulties in isolating and purifying the polar and often water-soluble amidines, we devel- oped a convenient one-pot method to convert aryl- and heteroarylnitriles to amidines in situ, followed by further condensation with an R-bromoketone to obtain the corresponding aryl- or heteroarylimidazole. By use of this methodology, treatment of 2 with lithium bist- rimethylsilylamide followed by addition of 3-bromoacetyl- benzonitrile and conversion to the HCl salt afforded imidazole 1 in 23% yield from 2. Results and Discussion. As shown in Table 1, compound 1 has high affinity for the human and rat NPY5 receptors (human K i ) 1.2 nM; rat K i ) 1.7 nM) and low affinity (IC 50 . 1 µM) for the NPY1 and NPY2 receptors. Compound 1 also potently antagonized NPY- induced Ca 2+ mobilization in a stably transfected mela- noma cell line expressing the human NPY5 receptor. In addition, compound 1 did not have any significant * To whom correspondence should be addressed. Phone: 860-715- 5076. Fax: 860-715-8069. E-mail: richard_l_elliot@groton.pfizer.com. † Pfizer Inc. ‡ Neurogen Corporation. Scheme 1 a a Reagents: (i) lithium bistrimethylsilyamide/THF; (ii) H2O/ NaHCO3/K2CO3, then 3-bromoacetylbenzonitrile; (iii) HCl. Table 1. In Vitro Potency of Compound 1 at NPY Receptors rat NPY5, Ki, a,e nM hu NPY5 Ki, b,e nM NPY1 IC50, c,e µM NPY2 IC50, c,e µM NPY5 Ca 2+ mobil. IC50, d,e nM 1.7 ( 0.3 (n ) 4) 1.2 ( 0.4 (n ) 4) .1 (n ) 1) .1 (n ) 1) 0.4 ( 0.3 (n ) 2) a [ 125 I][Leu31,Pro34]PYY binding in the whole rat brain in the presence of 1 µM BIBP3226 to block the NPY-1 receptors. b Cloned human chimeric receptors, using [ 125 I]PYY as the radioligand. c Cerep data (Cerep, Paris, France; values are from one experi- ment, performed in duplicate). d Ca 2+ mobilization in NPY5 trans- fected human Bowes melanoma (HMCB) cell line. e All values are mean values ( SEM. 670 J. Med. Chem. 2003, 46, 670-673 10.1021/jm025584p CCC: $25.00 © 2003 American Chemical Society Published on Web 02/04/2003