Azepinone-Containing Tetrapeptide Analogues of Melanotropin Lead to Selective hMC4R Agonists and hMC5R Antagonist.

To address the need for highly potent, metabolically stable, and selective agonists, antagonists, and inverse agonists at the melanocortin receptor subtypes, conformationally constrained indolo- and benzazepinone residues were inserted into the α-MSH pharmacophore, His(6)-Phe(7)-Arg(8)-Trp(9)-domain. Replacement of His(6) by an aminoindoloazepinone (Aia) or aminobenzazepinone (Aba) moiety led to hMC4R and hMC5R selective agonist and antagonist ligands, respectively (tetrapeptides 1 to 3 and 4, respectively). In peptides 1 to 3 and depending on the para-substituent of the d-Phe residue in position 2, the activity goes from allosteric partial agonism (1, R = H) to allosteric full agonism (2, R = F) and finally allosteric partial agonism (3, R = Br).

Development and pharmacological characterization of conformationally constrained urotensin II-related peptide agonists.

Urotensin II (UII) and its paralog peptide, urotensin II-related peptide (URP), exert not only common but also divergent actions through the activation of UT, a specific membrane-bound receptor that belongs to the 1A G protein-coupled receptor subclass. In this study, we have designed and synthesized new URP analogues in which the intracyclic Trp residue was replaced with natural, unnatural, and constrained amino acids to determine important physicochemical features for receptor binding and activation. The biological data, highlighting the potent agonistic behavior of [Tiq(4)]URP and [Tpi(4)]URP, also suggest that the Trp residue, and more specifically the indole ring, is not critical for receptor interaction and could in fact be involved in the intramolecular stabilization of the bioactive conformation of URP. Finally, these analogues, which are intracyclic constrained URP-based agonists, could represent useful pharmacological tools for the study of the urotensinergic system.

Original ß,γ-diamino acid as an inducer of a γ-turn mimic in short peptides.

Original αγα tripeptides containing one ß,γ-diamino acid have been synthesized and their conformation determined by extensive NMR and molecular dynamic studies. These studies revealed the presence of a C(9) hydrogen bonded turn around the ß,γ-diamino acid which was stabilized by bulky side chains of the preceding residue. This turn can be considered as a mimic of the well-known γ-turn.

Local control of the cis-trans isomerization and backbone dihedral angles in peptides using trifluoromethylated pseudoprolines.

NMR studies and theoretical calculations have been performed on model peptides Ac-Ser(ΨPro)-NHMe, (S,S)Ac-Ser(Ψ(H,CF3)Pro)-NHMe, and (R,S)Ac-Ser(Ψ(CF3,H)Pro)-NHMe. Their thermodynamic and kinetic features have been analyzed in chloroform, DMSO, and water, allowing a precise description of their conformational properties. We found that trifluoromethyl C(δ)-substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. In CHCl(3), the configuration of the CF(3)-C(δ) entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn-like or PPI/PPII-like backbone conformations. Moreover, in water and DMSO, this C(δ)-configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. Theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the NMR observations.

Constrained α/γ-peptides: a new stable extended structure in solution without any hydrogen bond and characterized by a four-fold symmetry.

Small α/γ-peptides alternating α-aminoisobutyric acid and cyclic γ-amino acid residues are described. NMR studies together with restrained simulated annealing revealed that an extended backbone conformation largely dominates in solution for as short as 4-residues long oligomers. This new fold type is devoid of any hydrogen bond and characterized by a four-fold symmetry.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced µ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

p-Nitrobenzyl protection for cysteine and selenocysteine: a more stable alternative to the acetamidomethyl group.

This study evaluated the acidic lability of the acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), and the p-nitrobenzyl (pNB) as protecting groups for cysteine and selenocysteine (Sec) during the tert-butyloxycarbonyl (Boc)-chemistry solid-phase peptide synthesis of oxytocin (OT). Two novel Sec building blocks (Nalpha-tert-butyloxycarbonyl-Se(acetamidomethyl)-L-selenocysteine (Boc-L-Sec(Acm)-OH) and Nalpha-tert-butyloxycarbonyl-S(4-nitrobenzyl)-L-selenocysteine (Boc-L-Sec(pNB)-OH)) were developed for this study. Six partially protected thio- and seleno-OT analogues were synthesized, purified, and exposed to neat trifluoroacetic acid (TFA) at temperatures of 25, 40, 50, and 60 degrees C for 1 h, and HF treatment at 0 degrees C for 1 h. Significant losses were observed for the Acm and Tacm group in TFA at temperatures greater than 25 degrees C and during HF treatment at 0 degrees C, whereas the pNB group remained intact. Removal of the pNB was achieved via reduction to the p-aminobenzyl group either with zinc in acetic acid in solution or via tin chloride in hydrochloric acid on solid support, followed by oxidative cleavage with iodine yielding the corresponding disulfide or diselenide bond. No major side reactions were observed. This study confirms the occasionally described Acm instability and underpins the development of the pNB group as an alternative for cysteine and Sec protection.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds.

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced delta/mu antagonists, as determined by the functional [S(35)]GTPgammaS binding assay, the dimerization of potent Aia-based 'parent' ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds.

New tetracyclic tetrahydro-beta-carbolines as tryptophan-derived peptidomimetics.

The Pictet-Spengler reaction is known as a useful tool for the synthesis of constrained analogs of tryptophan. Herein, we present the further cyclization of 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid methyl esters with 1-(1'-aminoalkyl) side chain. These transformations lead to heterocyclic structures which can find useful applications in medicinal chemistry as peptide mimetics.

The replacement of His(4) in angiotensin IV by conformationally constrained residues provides highly potent and selective analogues.

The histidine residue in angiotensin IV was replaced by various conformationally constrained amino acids. The substitution of the His(4)-Pro(5) dipeptide sequence by the constrained Trp analogue Aia-Gly, in combination with beta(2)hVal substitution at the N-terminus, provided a new stable analogue H-(R)-beta(2)hVal-Tyr-Ile-Aia-Gly-Phe-OH (AL-40) that is a potent ligand for the Ang IV receptor IRAP and selective versus AP-N and the AT1 receptor.

A novel solid phase approach to Aia-containing peptides.

A strategy was developed to directly assemble 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]-azepin-3-ones on solid-phase-supported peptide sequences. Fmoc- and Boc-based strategies were investigated. The Fmoc-strategy approach strongly depends on the peptide sequence being synthesized while the Boc-based synthesis leads to excellent results for all the selected peptide analogs. The method was applied to prepare Aia-analogs of several bioactive peptides containing one or more Trp-residues which were shown to be important for biological recognition.

Highly potent 4-amino-indolo[2,3-c]azepin-3-one-containing somatostatin mimetics with a range of sst receptor selectivities.

The synthesis, biological evaluation, and conformational analysis of 4-amino-indolo[2,3-c]azepin-3-one (Aia)-containing SRIF mimetics are reported. Different subtype selectivities are observed depending on the N- and C-terminal substituents of the D-Aia-Lys dipeptide mimetic. An sst(5)-selective analogue with subnanomolar binding affinity was obtained that is the most potent agonist reported to date. A nonselective mimetic with high potency was also identified. This study allows a better definition of the bioactive conformation of the essential D-Trp side chain in the somatostatin pharmacophore.

Conformationally constrained opioid ligands: the Dmt-Aba and Dmt-Aia versus Dmt-Tic scaffold.

Replacement of the constrained phenylalanine analogue 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the opioid Dmt-Tic-Gly-NH-Bn scaffold by the 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]azepin-3-one (Aia) and 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffolds has led to the discovery of novel potent mu-selective agonists (Structures 5 and 12) as well as potent and selective delta-opioid receptor antagonists (Structures 9 and 15). Both stereochemistry and N-terminal N,N-dimethylation proved to be crucial factors for opioid receptor selectivity and functional bioactivity in the investigated small peptidomimetic templates. In addition to the in vitro pharmacological evaluation, automated docking models of Dmt-Tic and Dmt-Aba analogues were constructed in order to rationalize the observed structure-activity data.

Endomorphin-2 with a beta-turn backbone constraint retains the potent micro-opioid receptor agonist properties.

The constitutional similarity with different secondary structure preference between the Aba-Gly and the spiro-Aba-Gly scaffolds were exploited to design the novel endomorphin-2 analogs Tyr-spiro-( R/ S)-Aba-Gly-Phe-NH(2) ( 1 and 2) and Tyr-( R/ S)-Aba-Gly-Phe-NH(2) ( 3 and 4). The ( R)-spiro analog 1 was found to be a potent and selective micro-opioid agonist/partial agonist ( K (imicro) = 29.3 nM, IC(50) = 50 nM, K(e) = 0.57). NMR experiments and molecular modeling indicated that its backbone adopts mainly a beta-turn in aqueous solution.

New sst4/5-selective somatostatin peptidomimetics based on a constrained tryptophan scaffold.

The synthesis and biological evaluation of four peptidomimetic analogs of somatostatin based on a constrained Trp residue, 3-amino-indolo[2,3-c]azepin-2-one (Aia), are reported. It is shown that dipeptidomimetics with a D-Aia-Lys sequence, functionalized with N- and C-terminal aromatic substituents, display a good selectivity for both sst4 and sst5. This study allowed us to identify a new highly potent sst5 agonist with good selectivity over the other receptors, except versus sst4.