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Preliminary studies and synthesis development for the preparation of a bicyclic homodetic peptide library have been carried out using orthogonal protection schemes. The best results have been obtained using two Fmoc/tBu-based strategies, in which the first cycle is carried out in the solid phase through side chain functional groups previously protected with Aloc/Al groups. The second cycle is performed either in the solid phase, which requires side chain anchoring of a trifunctional amino acid and Dmb protection for the C-terminus carboxyl group, or in solution, which requires the use of highly labile resins, such as the 2-chlorotrityl (Barlos) resin. Only when the cycles are formed in a ziplike manner, that is, first the small cycle and then the larger ring, is the desired final product obtained.

A series of analogues of desArg⁹-Lys-bradykinin (BK), Lys-Arg-X-Acnc-X-Ser-Pro-Phe, in which the spacer X-Acnc-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac6c, Ac7c, Ac8c, Ac9c, Ac12c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, βAla, γAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Acnc which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac7c-Gly-Ser-Pro-Phe and Lys-Arg-γAbu-Ac7c-γAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 Å for the Gly- and γAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH2)11-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.

Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg¹⁰-HOE 140 (H-d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-d-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg¹⁰-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-d-Arg-Arg-NH-(CH2)10-CO-Ser-d-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg¹⁰-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on C^α-tetrasubstituted α-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

We describe the in vitro and in vivo pharmacological properties of MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta), the first example of a polycyclic peptide tachykinin NK2 receptor antagonist. MEN 10,627 is endowed with high affinity for NK2 receptor expressed in various species with pKB values ranging between 10.1 (hamster trachea) and 8.1 (rabbit pulmonary artery). The antagonism is of competitive type in both functional and radioligand binding assays. A 100- to 10,000-fold selectivity was found vs. NK1 or NK3 receptors expressed in various species. As an NK2 receptor antagonist, MEN 10,627 is 10- to 100-fold more potent than the monocyclic peptide antagonist L 659,877 or cyclo(Met-Gln-Trp-Phe-Gly-Leu). At the hamster NK2 receptor, MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist SR 48,968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide], whereas the converse is true for the rabbit NK2 receptor. Furthermore, MEN 10,627 is, up to micromolar concentrations, devoid of antagonist properties toward a wide range of transmitters of both peptide and nonpeptide nature. In urethane-anesthetized rats in vivo, MEN 10,627 (10-100 nmol/kg i.v.) produced long-lasting inhibition of contraction of the urinary bladder and duodenum produced by i.v. administration of the NK2 receptor agonist [beta Ala8]NKA(4-10), without affecting the responses produced by i.v. administration of the NK1 receptor agonist [Sar9]SP sulfone or acetylcholine. In anesthetized rats, both MEN 10,627 and SR 48,968 blocked urinary bladder contraction induced by the NK2 receptor agonist after intravenous, intranasal or intraduodenal administration. Equieffective doses of MEN 10,627 producing about 50% inhibition of the response to [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo, were 0.01, 0.03 and 3 mumol/kg after intravenous, intranasal and intraduodenal administration, respectively. The corresponding doses of SR 48,968 were 0.03, 0.1 and 1 mumol/kg, after intravenous, intranasal and intraduodenal administration, respectively. In conclusion, MEN 10,627 is a potent and selective NK2 receptor antagonist, endowed with high potency and long duration of action in vivo, which is not restricted to parenteral administration.(ABSTRACT TRUNCATED AT 400 WORDS)

The pharmacological profile was studied of MEN 11420, or cyclo{[Asn(β‐D‐GlcNAc)‐Asp‐Trp‐Phe‐Dap‐Leu]cyclo(2β‐5β)}, a glycosylated derivative of the potent, selective, conformationally‐constrained tachykinin NK2receptor antagonist MEN 10627 (cyclo(Met‐Asp‐Trp‐Phe‐Dap‐Leu)cyclo(2β‐5β)).MEN 11420 competitively bound with high affinity to the human NK2receptor stably transfected in CHO cells, displacing radiolabelled [125I]‐neurokinin A and [3H]‐SR 48968 with Kivalues of 2.5±0.7 nM(n=6) and 2.6±0.4 nM(n=3), respectively.MEN 11420 showed negligible binding affinity (pIC50<6) at 50 different receptors (including tachykinin NK1and NK3receptors) and ion channels.In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2receptor‐mediated contractions (pKB=8.6±0.07, n=10, and 9.0±0.04, n=12; Schild plot slope=−1.06 (95% c.l.=−1.3; −0.8) and −1.17 (95% c.l.=−1.3; −1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pKBof 10.2±0.14, n= 9, and 9.8±0.15, n=9, was calculated in the hamster trachea and mouse urinary bladder, respectively.MEN 11420 showed low affinity (pKB<6) at guinea‐pig and rat tachykinin NK1(guinea‐pig ileum and rat urinary bladder) and NK3(guinea‐pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627.The in vivoantagonism of the contractions produced by [βAla8]neurokinin A(4–10) (1 nmol kg−1) was observed after intravenous (dose range: 1–10 nmol kg−1), intranasal (3–10 nmol kg−1), intrarectal (30–100 nmol kg−1) and intraduodenal (100–300 nmol kg−1) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability.A dose of MEN 11420 (100 nmol kg−1, i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2selective receptor agonist [βAla8]neurokinin A(4–10) (10–300 nmol kg−1), was without effect on the responses produced by the NK1receptor selective agonist [Sar9]substance P sulphone (1–10 nmol kg−1).These findings indicate that MEN 11420 is a potent and selective tachykinin NK2receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivopotency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2receptors in humans.

We have studied the effects of two newly developed tachykinin antagonists, MEN 10,207 and MEN 10,376, which are highly selective for NK-2 tachykinin receptors on tachykinin-induced contraction in the rat urinary bladder and guinea pig airways in vivo. MEN 10,207 exhibited antagonism only at doses which produced agonist effects. By contrast, MEN 10,376 was devoid of significant agonist activity at i.v. doses (1-3 mumol/kg) which selectively antagonized the effects of an NK-2 agonist [beta-Ala8]-neurokinin A(4-10) (bladder contraction in rats, bronchoconstriction in guinea pigs) without affecting the response to an NK-1 agonist [Sar9]-substance P sulfone (hypotension, salivation and bladder contraction in rats, bronchoconstriction in guinea pigs). At these NK-2-selective blocking doses, MEN 10,376: 1) did not affect urodynamic parameters at cystometry in normal rats but reduced amplitude of micturition contractions following induction of chemical (intravesical xylene) cystitis and 2) reduced by a maximum of 50% the noncholinergic bronchoconstrictor response to vagal nerve stimulation. These findings provide the first evidence for involvement of NK-2 receptors in physiological responses in the urinary and respiratory tracts.

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.