Original article Synthesis of 2-amino-3-heteroaroylthiophenes and evaluation of their activity as potential allosteric enhancers at the human A 1 receptor Pier Giovanni Baraldi a, *, Maria Giovanna Pavani a , John C. Shryock b , Allan R. Moorman c , Valeria Iannotta d , Pier Andrea Borea d , Romeo Romagnoli a a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy b Department of Medicine, University of Florida, Gainesville, Florida, USA c King Pharmaceuticals Research and Development Inc., Cary, NC 27513, USA d Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara, 44100 Ferrara, Italy Received 26 March 2004; accepted 24 June 2004 Available online 12 August 2004 Abstract 2-Amino-3-benzoylthiophenes are allosteric enhancers of agonist binding to the adenosine A 1 receptor. New compounds bearing an heteroaroyl instead of the benzoyl moiety at the 3-position of the thiophene were synthesized. The phenyl ring was replaced with heterocycles that possess heteroatoms able to form hydrogen bonds (2-furanyl, 2-benzofuranyl, 2-pyridinyl in compounds 2–13) or with a thienyl moiety as isoster of the phenyl ring (2-thienyl, 3-thienyl and 5-halo-2-thienyl in compounds 14–29). The effect of several alkyl substituents at positions 4 and 5 of the thiophene ring to increase enhancer activity was determined. The ability of the new molecules to reduce the cAMP content in CHO cells expressing the human adenosine A 1 receptor was evaluated. Compounds 2–13 with hydrogen bond-forming heteroatoms did not show significant activity as allosteric enhancers. On the other hand, compounds 15–16 and 19–20 with an unsubstituted thienyl moiety as replacement for the phenyl ring were nearly as efficacious as PD 81,723, the prototypical A 1 allosteric enhancer. Alkyl substituents at positions 4 and 5 of the thiophene ring were tolerated while a substituted piperidine ring was not tolerated. We conclude that hydrogen bonds could not be formed in the domain of the receptor that accommodates the phenyl ring of 2-amino-3-benzoylthiophene derivatives, indicating that this domain is hydrophobic. © 2004 Elsevier SAS. All rights reserved. Keywords: Allosteric enhancer; Adenosine A 1 receptor; 2-Amino-3-heteroaroylthiophenes; Cyclic AMP 1. Introduction Adenosine is cardioprotective during periods of ischemia and a similar effect has been demonstrated in brain. These and other important physiological effects of adenosine on the cardiovascular and central nervous systems are mediated through the interaction with the A 1 receptor subtype [1]. The therapeutic potential of A 1 adenosine receptor agonists is undermined by a poor side effect profile due to the wides- pread distribution of adenosine receptors throughout the body. One way to avoid these side effects is offered by positive A 1 allosteric adenosine modulators whose action would be limited to times and locations at which significant release of adenosine occurs [2]. A series of 2-amino-3-benzoylthiophene derivatives has been shown to enhance the apparent binding of the radioli- gand agonist [ 3 H]CHA to A 1 adenosine receptors and to decrease the rate of dissociation of [ 3 H]CHA from the recep- tor. It has been suggested that these compounds act on an allosteric site, distinct from the orthosteric adenosine binding site, to stabilize the high affinity state of the receptor for agonist binding [3]. Among these compounds, PD 81,723 (1) has been pro- posed as a lead compound and chosen for pharmacological investigations. It increased agonist potencies 4- to 10-fold both in membrane and whole-cell function assays. As a drawback, this compound was also able to inhibit the binding * Corresponding author. Tel.: +39-0-532-291293; fax: +39-0-532-291296. E-mail address: baraldi@unife.it (P.G. Baraldi). European Journal of Medicinal Chemistry 39 (2004) 855–865 www.elsevier.com/locate/ejmech 0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.ejmech.2004.06.009