Design, Synthesis, Conformational Analysis, and Biological Studies of Urotensin-II Lactam Analogues Paolo Grieco, a Alfonso Carotenuto, b Riccardo Patacchini, c Carlo A. Maggi, c Ettore Novellino a and Paolo Rovero b, * a Department of Pharmaceutical and Toxicological Chemistry, University of Naples ‘Federico II’, I-80131 Naples, Italy b Department of Pharmaceutical Sciences, University of Salerno, I-84084 Fisciano, Italy c Department of Pharmacology, Menarini Ricerche SpA, I-50131 Florence, Italy Received 3 May 2002; accepted 9 August 2002 Abstract—Human urotensin II (hU-II; H-Glu-Thr-Pro-Asp-cyclo[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) is a disulfide bridged unde- capeptide recently identified as the ligand of an orphan G protein-coupled receptor. hU-II has been described as the most potent vasoconstrictor compound identified to date. With the aim of replacing the disulfide bridge by a chemically more stable moiety, we have synthesized and tested a series of lactam analogues of hU-II minimum active fragment, that is hU-II(4–11). The contractile activity of the synthetic analogues on the rat isolated thoracic aorta was found to be dependent upon the dimension of the lactam bridge. The most active peptide, H-Asp-cyclo[Orn-Phe-Trp-Lys-Tyr-Asp]-Val-OH (3), is approximately 2 logs less potent than hU-II (pD 2 =6.3 vs 8.4). A conformational analysis in solution of the active peptide 3, one of the inactive analogues, and hU-II was performed, using NMR and molecular modelling techniques. A superposition of the calculated structures of hU-II and 3 clearly shows that three out of four key residues (i.e., Phe 6 , Lys 8 and Tyr 9 ) maintain the same side– chain orientation, while the fourth one, Trp 7 , cannot be superimposed. This observation could explain the reduced biological activity of the synthetic analogue. # 2002 Elsevier Science Ltd. All rights reserved. Introduction Urotensin II is a cyclic peptide originally isolated from the urophysis, the hormone storage-secretion organ of the caudal neurosecretory system of teleost fishes, and sequenced more than 20 years ago. 1 Several structural forms of U-II have subsequently been reported in dif- ferent species of fish and amphibians, with variation occurring in the five to seven N-terminal residues, fol- lowed by a C-terminal conserved, disulfide bridged cyc- lic hexapeptide (Fig. 1). These peptides showed general smooth muscle contracting activity in fish 2 and goby U-II additionally possesses vasoconstrictor activity in rats. 3 Recently, urotensin-II was cloned in several mammalian species, including humans. 4 Human U-II (hU-II) is an 11 amino acid peptide that retains the cyclic portion typical of fish U-II (Fig. 1). In 1999 Ames et al. 5 identified a new human G-protein coupled receptor homologous to the GPR14/SENR orphan receptor from rat. 6 The use of a ‘reverse molec- ular pharmacology’ approach 7 identified U-II as the ligand of this orphan receptor. Interestingly, three other independent groups reported similar results within 2 months. 8 As a result, there has been continued interest in U-II sequences and several reports have described hU-II to be a very potent constrictor of certain human isolated arteries and veins 9 as well as of several vessels from different mammalian species. 10 hU-II has been shown to be one to two orders of magnitude more potent than endothelin-1 in producing vasoconstriction in mammals and thus is one of the most effective vaso- constrictor compounds identified to date. 5,11 Moreover, hU-II produces contractions in a number of non-vas- cular smooth muscle tissues, such as primate airways 12 and human heart. 13 Interestingly, no differences were noted between the effects produced by human and rat isoforms of urotensin-II 14 indicating that the conserved cyclohexapeptide sequence is mainly responsible for bio- logical activity. Both hU-II immunoreactivity and recep- tors for hU-II have been identified in human cardiac tissues 5,9b and hU-II has been shown to produce very potent inotropic effects in human atrium and ventricle. 13 0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(02)00372-3 Bioorganic & Medicinal Chemistry 10 (2002) 3731–3739 *Corresponding author. Tel.: +39-089-962809; fax: +39-089-962828; e-mail: rovero@unisa.it