Nonpeptide analogues of dynorphin A(1-8): design, synthesis, and pharmacological evaluation of kappa-selective agonists.

Two novel series of kappa opioid receptor agonist analogues of MPCB-GRRI and MPCB-RRI, hybrid ligands of MPCB ((-)-cis-N-(2-phenyl-2-carbomethoxy)cyclopropylmethyl-N-normetazocine ) and of the C-terminal fragments of dynorphin A(1-8), have been synthesized. The critical functional groups of the peptide fragments of hybrid compounds were maintained, and the binding affinities and selectivities for compounds 1-40 to mu, delta, and kappa opioid receptors were analyzed. Compounds 15 and 16, MPCB-Gly-Leu-NH-(CH(2))(n)()-NH-C(=NH)-C(4)H(9) (n = 5, 6), displayed high affinity and selectivity for kappa opioid receptors (K(i)(kappa) = 6.7 and 5.3 nM, K(i)(mu)/K(i)(kappa) = 375 and 408, and K(i)(delta)/K(i)(kappa) = 408 and 424, respectively). Since kappa agonists may also cause psychotomimetic effects by interaction with sigma sites, binding assays to sigma(1) sites were performed where compounds 15 and 16 showed negligible affinity (K(i) > 10 000). Compounds 15 and 16 were further characterized in vivo and showed potent antinociceptive activity in mouse abdominal constriction tests (ED(50) = 0.88 and 1.1 mg/kg, respectively), fully prevented by nor-BNI. Thus, these novel analogues open an exciting avenue for the design of peptidomimetics of dynorphin A(1-8).

Substituted 1-phenyl-2-cyclopropylmethylamines with high affinity and selectivity for sigma sites.

A series of 1-phenyl-2-cyclopropylmethylamines structurally related to (+)- and (-)-MPCB were synthesized and their binding affinities for sigma1, sigma2, opioid and dopamine (D2) receptors were evaluated. Substitution of the cis-N-normetazocine with different aminic moieties provided compounds with high affinity and selectivity for sigma binding sites with respect to opioid and dopamine (D2) receptors. The observed increase in sigma2 affinity as compared to the parent (+)-MPCB, supports the idea that the particular stereochemistry of (+)-cis-N-normetazocine affects sigma1 selectivity but does not affect sigma1 affinity. The (+/-)-cis isomers of methyl 2-[(1-adamantylamino)methyl]-1-phenylcyclopropane-1-carboxyl ate (18) displayed a higher affinity and selectivity for the sigma1 and sigma2 receptor subtypes compared to the (+/-)-trans 19. Interestingly, the enantiomer (-)-cis 18 displayed a preference for sigma1 receptor subtype whereas the (+)-cis 18 did for sigma2. These results prompt us to synthesize compounds with modification of nitrogen and carboxyl groups. The compounds obtained showed high affinities and selectivity for sigma sites. Moreover, modifications of carboxyl groups provided compounds with the highest affinities in the series. In particular, compound 25 with reverse-type ester showed a Ki of 0.6 and 4.05 nM for sigma1 and sigma2 binding sites, respectively.

Characterization of a putative calcitonin receptor in IMR 32 human neuroblastoma cells.

In this study we characterized calcitonin (CT) receptors in human neuroblastoma IMR 32 cells. Saturation binding assays indicated that [125I]-human CT bound with high affinity to IMR 32 cell membranes (K(d) = 253.6 pM; Bmax = 3.84 fmol/ mg protein). In competition binding studies, human adrenomedullin displayed high affinity for these sites (IC50 = 30 nM) whereas human alpha calcitonin-gene related peptide (alphaCGRP; IC50 = 145 nM) and human amylin (IC50 = 415 nM) showed lower affinity. These peptides increased cAMP levels in viable cells; the relative potencies were: human CT > human adrenomedullin > human cCGRP > or = human amylin. The expression of mRNA coding for the published sequences of the human calcitonin receptor and of the human calcitonin receptor-like receptorwas evaluated by reverse transcriptase-polymerase chain reaction. Electrophoretic analysis did not confirm the occurrence of mRNA coding for the above mentioned receptors in these cells. This study suggests the presence of a novel, putative CT receptor in IMR 32 cells.