Effects of Modifications of Residues in Position 3 of Dynorphin A(1-11)-NH 2 on K Receptor Selectivity and Potency Feng-Di T. Lung, † J.-P. Meyer, † Bih-Show Lou, † Li Xiang, † Guigen Li, † Peg Davis, ‡ Irene A. De Leon, ‡ Henry I. Yamamura, ‡ Frank Porreca, ‡ and Victor J. Hruby* ,† Departments of Chemistry and Pharmacology, University of Arizona, Tucson, Arizona, 85721 Received September 1, 1995 X Tyrosine 1 and phenylalanine 4 in dynorphin A (Dyn A) have been reported to be important residues for opioid agonist activity and for potency at κ receptors. The glycine residues in the 2 and 3 positions of dynorphin A may affect the relative orientation of the aromatic rings in positions 1 and 4, but their flexibility precludes careful analysis. To examine these effects on dynorphin A, we previously have synthesized the linear analogues [D-Ala 3 ]Dyn A(1-11)-NH 2 (2) and [Ala 3 ]Dyn A(1-11)-NH 2 (3) and reported their biological activities. Analogues 2 and 3 displayed affinities for the central κ opioid receptor (IC 50 ) 0.76 and 1.1 nM, respectively) similar to that of Dyn A(1-11)-NH 2 (1) (IC 50 ) 0.58 nM) and greatly enhanced selectivities for κ vs µ and κ vs δ receptors (IC 50 ratios of 350 and 1300 for 2, and 190 and 660 for 3, respectively). These results suggest that the structure and lipophilicity of the amino acid present in position 3 of Dyn A(1-11)-NH 2 as well as the conformational changes they induce in the message sequence of dynorphin have important effects on potency and selectivity for κ opioid receptors. To further investigate structure-activity relationships involving the residue at the 3 position of Dyn A(1-11)-NH 2 , a series of Dyn A analogues with aromatic, charged, and aliphatic side chain substitutions at the 3 position was designed, synthesized, and evaluated for their affinities for κ, µ, and δ opioid receptors. It was found that analogues with lipophilic amino acids at the 3 position of Dyn A(1-11)-NH 2 generally displayed higher affinity but similar selectivities for the κ receptor than analogues with charged residues at the same position. It is suggested that the structural, configurational, and steric/lipophilic effects of amino acids at position 3 of Dyn A(1-11)-NH 2 may play an important role in potency and selectivity for the κ receptor. Introduction Dynorphin A (Dyn A(1-17)), a heptadecapeptide, H-Tyr-Gly-Gly-Phe-Leu 5 -Arg-Arg-Ile-Arg-Pro 10 -Lys-Leu- Lys-Trp-Asp 15 -Asn-Gln-OH, was first isolated from por- cine pituitary and recognized as a potent opioid agonist. 2-4 It is now well established that there are at least three types of opioid receptors, namely µ (mu), δ (delta), and κ (kappa). 5-7 However, the physiological and pharmacological roles of these receptors still need clarification, thus requiring the design and synthesis of highly potent and selective ligands. Research in this area has expanded rapidly in the past decade, with considerable effort devoted to the development of δ and µ opioid receptor selective peptide ligands primarily based on enkephalin. 8-11 The potential of targeting the κ opioid receptor as an effector for analgesia has yet to be explored in detail. 12 Previous structure-activity studies have shown that the truncated derivative, dynorphin A(1-11)-NH 2 , retains the high binding po- tency of dynorphin A at the κ receptor. Thus we and others have primarily used Dyn A(1-11)-NH 2 as a template to examine the structure-activity relation- ships of dynorphin. 13,14 Since Tyr 1 and Phe 4 are re- ported to be important for opioid agonist activity, the effects of the glycine residues in positions 2 and 3 of Dyn A on the relative orientation of the two aromatic rings may be biologically important. 13 To assess these possible effects, we previously synthesized [D-Ala 3 ]Dyn A(1-11)-NH 2 and [Ala 3 ]Dyn A(1-11)-NH 2 and evalu- ated for their bioactivities. Interestingly, both ana- logues were found to be very potent for κ receptors and very selective for κ vs µ and κ vs δ receptors. 15 These results suggest that substitution of lipophilic residues and/or certain D-amino acids at position 3 of Dyn A(1- 11)-NH 2 may lead to novel Dyn A analogues that exhibit enhanced selectivities for κ receptors, while retaining strong affinities. Therefore, we designed and synthe- sized a series of linear Dyn A(1-11)-NH 2 analogues with various substitutions at the 3 position and evaluated their binding affinity, selectivity, and bioactivity for κ receptors. Results and Discussion Opioid Receptor Binding Affinities and Selec- tivities in the Guinea Pig Brain (GPB). The peptide analogues of dynorphin A(1-11)-NH 2 (Chart 1) were evaluated for their receptor binding affinities at κ, δ, and µ receptors by measuring the inhibition of binding of [ 3 H]U-69,593 (N-methyl-N-[7-(1-pyrrolidinyl)-1-oxa- spiro[4.5]dec-8-yl]benzo[b]furan-4-acetamide) (κ), [ 3 H]- cyclo[D-Pen 2 , p-Cl-Phe 4 , D-Pen 5 ]enkephalin (δ), and [ 3 H]DAMGO ([D-Ala 2 , MePhe 4 , Glyol 5 ]enkephalin) (µ) to opioid receptors in guinea pig brain (GPB) homogenates (Table 1). 16 In the GPB binding assay, substitution of Gly 3 with D- and L-alanine leads to analogues 2 and 3 that displayed affinities at the central κ opioid receptor (IC 50 ) 0.76 and 1.1 nM, respectively), similar to that of Dyn A(1-11)-NH 2 1 (IC 50 ) 0.58 nM), and greatly enhanced selectivities for κ vs µ and κ vs δ receptors (IC 50 ratios of 350 and 1300 for 2, and 190 and 660 for 3 respec- tively, compared to 17 and 44 for 1), due to poor * Author to whom reprint request and correspondence should be addressed at the Department of Chemistry. X Abstract published in Advance ACS Abstracts, June 1, 1996. 2456 J. Med. Chem. 1996, 39, 2456-2460 S0022-2623(95)00655-8 CCC: $12.00 © 1996 American Chemical Society