Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.

New bivalent thrombin inhibitors with N(alpha)(methyl)arginine at the P1-position.

A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.

Synthesis of different types of dipeptide building units containing N- or C-terminal arginine for the assembly of backbone cyclic peptides.

Different types of dipeptide building units containing N- or C-terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). For cyclization in the N-terminal sequence N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N-terminal arginine to the N-alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z-Arg(Z)2-OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C-terminal arginine was prepared to synthesize BK-analogues with backbone cyclization in the C-terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM technology was applied using the Boc-Arg(Alloc)2-OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc-protected amino aldehyde, derived from Boc-Phe-OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid-phase synthesis in the same manner as amino acid derivatives.

New thrombin inhibitors based on D-cha-Pro-derivatives.

A series of new analogs with modifications in the C-terminal residue were prepared based on the known thrombin inhibitor D-Phe-Pro-agmatine. These include several compounds alkylated at the N delta-, N omega- and N omega'-atoms of the guanidino group and a number of inhibitors derived from commercially available diamines. All analogs with alkylation of the guanidino group showed very poor activity. In contrast, the most potent and selective inhibitor with a cyclic and basic residue in the P1-position was found to be Ph-CH2-SO2-D-Cha-Pro-4-(amidomethyl) amidinopiperidine 11 with a Ki of 0.27 nM. In addition, a number of compounds were synthesized, in which the basic amidino group of the P1-residue was replaced by a hydroxyl group. Although the inhibition constants of these phenol derivatives showed still remarkable potency (16, Ki = 130 nM), their activity in clotting assays was strongly reduced.

A new type of bradykinin B2 receptor antagonists: bradykinin analogs with N-alkyl amino acids at position 2.

It is commonly assumed that bradykinin B2 receptor antagonists bind to a receptor site partially different from that for agonists. Thus, it is likely that there exists more than one key modification to convert bradykinin receptor agonists into antagonists. In this respect, [L-NMePhe2]-BK represents the basic structure of a new type of bradykinin B2 receptor antagonists without any replacement at position 7. This compound inhibits both in vitro bradykinin-induced contraction of the guinea pig lung strip and in vivo bradykinin-induced bronchoconstriction. Furthermore, this analog shows analgesic activity, blocks in a dose-dependent manner the bradykinin-induced Ca2+ release from macrophages and inhibits at a concentration of 10(-13) M the bradykinin-induced cytokine release from mononuclear cells. Combinations with structural modifications previously performed for other B2 receptor antagonists rather reduce than enhance the potency.

Highly selective bradykinin agonists and antagonists with replacement of proline residues by N-methyl-D- and L-phenylalanine.

For further studies on the structural and conformational requirements of positions 2,3, and 7 in the bradykinin sequence, we replaced the proline residues by the more hydrophobic and conformationally restricted N-methyl-L- and D-phenylalanine (NMF). The biological activities of the new analogs were evaluated on rat uterus, guinea pig ileum, and guinea pig lung strip. Receptor binding of the analogs was studied in membranes from rat uterus and guinea pig ileum. Influence of bradykinin analogs on the release of cytokines from mouse spleen cell cultures was also measured. Bradykinin analogs were synthesized by the solid phase method, using Boc strategy on PAM or Merrifield resins. The best results in the formation of the N-methylamide bond were obtained with the coupling reagent PyBrop. In position 7 the substitution of D-Phe by D-NMF, retaining the configuration of the amino acid, converts bradykinin antagonists into agonists. The bradykinin analogs with D-NMF at position 7 gave the highest known tissue selectivity for rat uterus among agonists. [L-NMF(2)]bradykinin has moderate agonist activity on rat uterus but antagonist activity on guinea pig lung strip. It represents a new antagonist for B(2) receptors without any replacement at position 7. The same analog completely inhibits bradykinin-evoked cytokine expression by mononuclear cells.

New cyclic bradykinin antagonists containing disulfide and lactam bridges at the N-terminal sequence.

Continuing our studies of the bioactive conformation of bradykinin (BK) antagonists, we synthesized a first series of analogues with side-chain cyclization in the N-terminal sequence. Through this conformational constraint it should be possible to gain insight into their three-dimensional structure. The cycles were proposed on the basis of existing ideas and hypotheses about the receptor bound conformation of BK and its antagonists. The reported peptides contain D-Phe at position 7 or D-Tic-Oic (D-Tetrahydroisoquinoline-3 -carboxyl-octahydroindole-2-carboxylic acid) at positions 7 and 8, respectively, and a disulfide or lactam bridge between positions 0 and 6. Syntheses, including cyclization reactions, were carried out on PAM resin. The biological activity of the lead compound [DPhe7]-BK, the linear precursors and the cyclic peptides, as estimated on isolated rat uterus, guinea pig ileum and lung strips, are in the same range. The conformational properties of the new cyclic analogues were studied through energy minimization on a model compound. The results of the calculations support the existence of low-energy structures containing a beta-turn. Therefore, such a turn in the N-terminal segment of the molecule can be proposed as an important structural feature of the bioactive conformation of BK antagonists.

Design, synthesis and characterization of bradykinin antagonists via cyclization of the modified backbone.

With the aim of synthesizing cyclic antagonists of the nonapeptide hormone bradykinin with minimal side chain modification, we performed backbone to backbone and backbone to side chain cyclization. To probe and compare different strategies for this new kind of cyclization, the branched peptide bonds were formed by both reductive alkylation on the solid phase and by using preformed building units. Lactam bridges between the modified amide groups were formed by the use of the phenylalanine derivatives N(CH2COOH)Phe and N(CH2CH2NH2)Phe. The best results in the formation of the N-alkylamide bond were obtained with the coupling reagent PyBrop. The coupling rate was monitored by estimation of the N-terminal Fmoc-group. The cyclization was performed on the solid support. Unexpected difficulties resulted from the instability of the N-alkylamide bond under strong acidic conditions, as used for deprotection and for removal from the resin. We synthesized peptides with backbone to backbone cyclization between positions 2 and 5, as well as backbone to side chain cyclizations between positions 0 and 5, and between 2 and 6. The relatively high biological activities of some of the cyclic analogues support the supposed receptor-bound conformation of bradykinin antagonists with a beta-turn in the N-terminal sequence.