Novel Cyclic Analogs of Angiotensin II with Cyclization between Positions 5 and 7: Conformational and Biological Implications Wei-Jun Zhang, † Gregory V. Nikiforovich,* ,‡ Jacqueline Pe ´rodin, § Darren E. Richard, § Emanuel Escher, § and Garland R. Marshall †,‡ Department of Molecular Biology and Pharmacology and Center for Molecular Design, Washington University, St. Louis, Missouri 63130, and Faculte ´ de me ´ decine c , Universite ´ de Sherbrooke, Canada J1H 5N4 Received October 16, 1995 X To study the conformational features of molecular recognition of angiotensin II (Asp 1 -Arg 2 - Val 3 -Tyr 4 -Val/Ile 5 -His 6 -Pro 7 -Phe 8 , AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Cys 5 -His 6 -Pen 7 )-Phe 8 (3), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Asp 5 -His 6 - Apt 7 )-Phe 8 (4), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Glu 5 -His 6 -Apt 7 )-Phe 8 (5), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo- (Cys 5 -His 6 -Mpt 7 )-Phe 8 (6), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Cys 5 -His 6 -Mpc 7 )-Phe 8 (7), Sar 1 -Arg 2 -Val 3 - Tyr 4 -cyclo(Hcy 5 -His 6 -Mpt 7 )-Phe 8 (8), and Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Hcy 5 -His 6 -Mpc 7 )-Phe 8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis- prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a K D ) 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, RE, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His 6 and Phe 8 residues. Introduction During the past two decades, extensive structure- activity studies of angiotensin II (Asp 1 -Arg 2 -Val 3 -Tyr 4 - Val/Ile 5 -His 6 -Pro 7 -Phe 8 , AII, AT, angiotensin, 1), the known pressor and myotropic agent, have been per- formed to establish a realistic model for the biologically active conformation of AII, i.e., for the conformation it adopts when binding specific receptors (the “receptor- bound” conformation). Conformational studies of AII have been performed by a variety of approaches (e.g., see Duncia et al. 1 and references therein) including physicochemical measurements, energy calculations, and the synthesis and biological testing of conforma- tionally constrained analogs (see Nikiforovich 2 ). Several cyclic analogs of AII, mostly stabilized by either lactam 3-5 or disulfide bonds 6 between different backbone positions, were synthesized from 1975 until 1994. Unfortunately, all of these compounds proved to be virtually inactive. A breakthrough in the study of cyclic AII analogs came with the synthesis of compounds containing a Hcy 3 -Hcy 5 cycle 7,8 which showed practically the same binding potency at AT-1 receptors as AII itself. The cycle in question allows the peptide backbone to adopt a -turn in the region of the Tyr 4 and Val 5 residues, which had been suggested earlier by independent mo- lecular modeling studies. 9,10 Further development of this model resulted in new potent cyclic analogs, namely cyclo[Sar 1 , Cys/Hcy 3 , Mpt 5 ]AII. 11 However, both energy calculations and NMR data favored an “open turn” model for the cycles of the two Mpt-containing analogs, with φ ≈ -130°, ψ ≈ 60° for the Tyr 4 residue. 12 This discrepancy suggested that the biologically active con- formation(s) of AII and its analogs should not be considered solely in terms of backbone structures, i.e., different kinds of turns, peptide chain reversals, etc., and an attempt was, therefore, made to find a common spatial arrangement of important functional groups for receptor interaction in low-energy conformers of AII and its active analogs. Using the assumption that the aromatic moieties of Tyr 4 , His 6 , and Phe 8 residues and C-terminal carboxyl were important for biological activ- ity (see Regoli et al. 13 ), a model was developed for the receptor-bound conformation of AII 14 which was the basis for the design of a new analog, [Sar 1 , D-Tyr 4 , Pro 5 ]- AII (2). The compound is an agonist with a relatively good affinity toward AT-1 receptors (K D ) 58 nM) and with an EC 50 of 94 nM (see Tables 1 and 2). Despite this success of this new model for the biologi- cally active conformation of AII, 14 its verification by experimental methods is still far from complete. Whereas conformations of the AII 3-5 fragment, which bears the crucial Tyr aromatic ring, were studied quite thoroughly by synthesis and biological testing of cyclic analogs (see above) as well as by NMR of these conformationally restricted analogs, 12 fragment AII 5-7 which includes the His imidazole moiety has not been thoroughly investigated with conformational constraints. There- fore, the present study describes the synthesis, biological testing, and molecular modeling studies of several cyclic analogs of AII cyclized between positions 5 and 7, namely, Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Cys 5 -His 6 -Pen 7 )-Phe 8 (3), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Asp 5 -His 6 -Apt 7 )-Phe 8 (4), Sar 1 -Arg 2 -Val 3 -Tyr 4 -cyclo(Glu 5 -His 6 -Apt 7 )-Phe 8 (5), Sar 1 - Arg 2 -Val 3 -Tyr 4 -cyclo(Cys 5 -His 6 -Mpt 7 )-Phe 8 (6), Sar 1 - Arg 2 -Val 3 -Tyr 4 -cyclo(Cys 5 -His 6 -Mpc 7 )-Phe 8 (7), Sar 1 - Arg 2 -Val 3 -Tyr 4 -cyclo(Hcy 5 -His 6 -Mpt 7 )-Phe 8 (8), and Sar 1 - Arg 2 -Val 3 -Tyr 4 -cyclo(Hcy 5 -His 6 -Mpc 7 )-Phe 8 (9), where Apt stands for 4-amino-trans-proline and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. † Department of Molecular Biology and Pharmacology, Washington University. ‡ Universite ´ de Sherbrooke. § Center for Molecular Design, Washington University. X Abstract published in Advance ACS Abstracts, June 1, 1996. 2738 J. Med. Chem. 1996, 39, 2738-2744 S0022-2623(95)00774-6 CCC: $12.00 © 1996 American Chemical Society