1-Heteroaryl-6-(3,4-dichlorophenyl)-3-azabicyclo[4.1.0]heptane: further insights into a class of triple re-uptake inhibitors.

Further exploration around the recently disclosed potent triple re-uptake inhibitor 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptane led to the identification of a new series of potent triple re-uptake inhibitors endowed with good developability characteristics. The insertion of a further aryl moiety into the template allowed the 'titration' of the SERT/NET/DAT ratio leading to the identification of further tools in this important area.

Tumor-cell-targeted methionine-enkephalin analogues containing unnatural amino acids: design, synthesis, and in vitro antitumor activity.

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing C(alpha alpha)-dialkylated glycine (Aaa1, 1) or C(alpha)-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

The first ferrocene analogues of muramyldipeptide.

The two structurally interesting bioorganometallic analogues of muramyldipeptide (MDP) with potential immunomodulatory activity were synthesized starting from the O-protected N-acetylmuramic acid (MurNAc), L- or D-Ala and 1'-aminoferrocene-1-carboxylic acid (Fca). They were fully characterized by IR, (1)H NMR, (13)C NMR and CD spectroscopy as well as by FD mass spectrometry.

Glycosylation of lysine-containing pentapeptides by glucuronic acid: new insights into the Maillard reaction.

The formation of glycosylation products in model systems consisting of d-glucuronic acid (GlcA) and lysine-containing peptides, such as Lys-Gly-Gly-Phe-Leu (1), Gly-Lys-Gly-Phe-Leu (4) and Ac-Gly-Lys-Gly-Phe-Leu (6), was examined to evaluate the site specificity as well as the extent and nature of the modification. Peptides were reacted with GlcA either in solution or under dry-heating conditions. From the incubations performed in solution (MeOH), the corresponding (1-deoxy-D-fructofuranos-1-yluronic acid)-peptide derivatives (Amadori compounds) were isolated. Whereas reaction of 1 resulted in the formation of mono-glycosylated Amadori compound 2 with the sugar moiety attached to the N(epsilon)-amino group of the Lys residue and its di-glycosylated analogue 3, exposure of 4 to GlcA afforded only di-glycosylated peptide 5. From the incubation of GlcA with Ac-Gly-Lys-Gly-Phe-Leu (6) performed under mild dry-heating conditions (50 degrees C) in an environment of 75% relative humidity, besides Amadori compound 7, two new Maillard reaction products were isolated that contained 3-hydroxypyridinium (8) and 3-hydroxy-picolinic acid moiety (9). The mechanism for the formation of pyridinium products is discussed.

Novel spirotetracyclic zwitterionic dual H(1)/5-HT(2A) receptor antagonists for the treatment of sleep disorders.

Histamine H(1) and serotonin 5-HT(2A) receptors mediate two different mechanisms involved in sleep regulation: H(1) antagonists are sleep inducers, while 5-HT(2A) antagonists are sleep maintainers. Starting from 9'a, a novel spirotetracyclic compound endowed with good H(1)/5-HT(2A) potency but poor selectivity, very high Cli, and a poor P450 profile, a specific optimization strategy was set up. In particular, we investigated the possibility of introducing appropriate amino acid moieties to optimize the developability profile of the series. Following this zwitterionic approach, we were able to identify several advanced leads (51, 65, and 73) with potent dual H(1)/5-HT(2A) activity and appropriate developability profiles. These compounds exhibited efficacy as hypnotic agents in a rat telemetric sleep model with minimal effective doses in the range 3-10 mg/kg po.

6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptane: a new potent and selective triple reuptake inhibitor.

A pharmacophore model for triple reuptake inhibitors and the new class of 1-(aryl)-6-[alkoxyalkyl]-3-azabicyclo[3.1.0]hexanes were recently reported. Further investigation in this area led to the identification of a new series of potent and selective triple reuptake inhibitors endowed with good developability characteristics. Excellent bioavailability and brain penetration are associated with this series of 6-(3,4-dichlorophenyl)-1-[(methyloxy)methyl]-3-azabicyclo[4.1.0]heptanes together with high in vitro potency and selectivity at SERT, NET, and DAT. In vivo microdialysis experiments in different animal models and receptor occupancy studies in rat confirmed that derivative 17 showed an appropriate profile to guarantee further progression of the compound.

Novel side-chain glucosylated and adamantylated [Asp(2)/Glu(2)]enkephalin analogs: synthesis and in vitro growth inhibition of human tumor cells.

A series of new backbone-modified Leu- and Met-enkephalin analogs (13-20 a and b) were synthesized. Backbone manipulations involved the replacement of the Gly(2) residue in Tyr-Gly-Gly-Phe-Leu/Met with side-chain glucosylated or adamantylated D/L-aspartic or -glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met-enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20-50% inhibition at millimolar concentrations). Among the tested compounds, Tyr-D-Glu(AdNH)-Gly-Phe-Met (20b) showed significant antiproliferative activity, somewhat more pronounced on MCF-7 (breast carcinoma) and MOLT-4 (lymphoblastic leukemia) cells.

Formation pathways and opioid activity data for 3-hydroxypyridinium compounds derived from glucuronic acid and opioid peptides by Maillard processes.

The kinetics of formation and identity of the reaction products of the glucuronic acid with three representative opioid peptides were investigated in vitro. Peptides were conjugated with glucuronic acid either in solution or under dry-heating conditions. From the incubations performed in solution N-(1-deoxy-D-fructofuranos-1-yluronic acid)-peptide derivatives (Amadori compounds) were isolated, whereas from the dry-heated reactions products containing the 3-hydroxypyridinium moiety at the N-terminal of the peptide chain were obtained. Experiments performed under mild dry-heating conditions (40 degrees C) in model systems based on Leu-enkephalin and glucuronic acid, and in environment of either 40% or 75% relative humidity, revealed that the higher level of humidity promoted a process that enhanced 3-hydroxypyridinium compound generation. The mechanism of 3-hydroxypyridinium formation is discussed. In comparison with their respective parent peptides, the N-(1-deoxy-D-fructofuranosyl-uronic acid) derivatives of the opioid peptides showed three- to 11-fold lower mu- and delta-receptor-binding affinities and agonist potencies in the functional assays, likely as a consequence of the steric bulk introduced at the N-terminal amino group. The further decrease in opioid activity observed with the 3-hydroxypyridinium-containing peptides may be due to the lower pK(a) of the 3-hydroxypyridinium moiety and to delocalization of the positive charge in the pyridinium ring system.

Transformations of bioactive peptides in the presence of sugars--characterization and stability studies of the adducts generated via the Maillard reaction.

Glycation of biomolecules, such as proteins, peptide hormones, nucleic acids, and lipids, may be a major contributor to the pathological manifestations of aging and diabetes mellitus. These nonenzymatic reactions, also termed the Maillard reaction, alter the biological and chemical properties of biomolecules. In order to investigate the effect of various reducing sugars on the products formed from small bioactive peptides (Tyr-Gly-Gly-Phe-Leu, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OMe, Tyr-Gly-Gly-Phe, and Tyr-Gly-Gly), model systems were prepared with glucose, mannose or galactose. Peptide-sugar mixtures were incubated at 37 or 50 degrees C in phosphate-buffered saline, pH 7.4, or in methanol. The extent of glycation was determined periodically by RP HPLC. All sugar-peptide mixtures generated two different types of glycation products: N-(1-deoxy-ketos-1-yl)-peptide (Amadori compound) and the imidazolidinone compound substituted by sugar pentitol and peptide residue. The amount and distribution of peptide glycation products depended on the structure of the reactants, and increased in both concentration- and time-dependent manner in relation to exposure to sugar. Additionally, the rate of hydrolysis of glucose-derived imidazolidinone compounds, obtained either from leucine-enkephalin (1) or its shorter N-terminal fragments 2 and 3, was determined by incubation at 37 degrees C in human serum. These results revealed that imidazolidinones obtained from glucose and small peptides are almost completely protected from the action of enzymes in serum, the predominant route of degradation being spontaneous hydrolysis to initial sugar and peptide compound.

Novel carbohydrate-induced modification of peptides: crystal structure and NMR analysis of ester-bridged bicyclic galactose-enkephalin adduct containing imidazolidinone moiety.

Comparative studies based on x-ray crystallography and NMR spectroscopy were used for structural characterization of the novel minor, imidazolidinone moiety containing, product 2b of the Maillard reaction obtained in vitro by using the galactose-modified endogenous opioid pentapeptide leucine-enkephalin (Tyr-Gly-Gly-Phe-Leu) 1. The x-ray analysis uniquely defined the molecular structure as cyclo-(N-(12-[-4)-D-galacto-pentitol-1-yl]-4-(4-hydroxybenzyl)-5-oxoimidazolidin-1-yl-(1 --> O]acetyl]glycyl-L-phenylalanyl-L-leucyl-] (3), having an 18-membered ring with an ester bond between the secondary (C4') hydroxyl group of a D-galacto-pentitolyl residue and the C-terminal carboxy group of leucine-enkephalin. The absolute configuration of the new chiral centre at the imidazolidinone moiety was established as C2(S), indicating a cis arrangement of C2 and C4 substituents at the 5-membered heterocyclic ring. The NMR analysis of compound 2b carried out in CH3CN-d3 and DMSO-d6, indicated the existence of two isomers in solution, differing only in the position of the ester group in the molecule. NMR data for the minor isomer (13%-16%) are in agreement with structure 3. The migratory tendency of the peptidyl group from the primary (2b) to the secondary hydroxyl group (3) of a D-galacto-pentitolyl residue in methanol/water solution was confirmed by RP HPLC analysis.