Design, Synthesis and Pharmacological Activity of New Pyrrolo[1,2-A] Pyrazine Translocator Protein (TSPO) Ligands.

BACKGROUND: Translocator protein 18 kDa (TSPO) is a promising target for the creation of effective and safe neuropsychotropic drugs. The ligands of TSPO exhibit anxiolytic, antidepressant, neuroprotective and other activities without the side effects of benzodiazepines. METHODS: New TSPO ligands in the series of N,1-diphenylpyrrolo[1,2-a]pyrazine-3-carboxamides derivatives were designed using calculated pharmacophore model and molecular docking analysis. The synthesis of new compounds was carried out by two schemes using [3+3]-cycloaddition reaction of 2-azidoacrylic acid derivatives with pyrrolphenylketone as a key stage. The anxiolytic activity of new substances has been established using open field test with flash. RESULTS: Several synthesized N,1-diphenylpyrrolo[1,2-a]pyrazine-3-carboxamides derivatives significantly increased the total motor activity of Balb/c mice compared to the control. The structureactivity relationship was investigated. The most effective compound was found to be GML-11 (Nbenzyl- N,1-diphenylpyrrolo[1,2-a]pyrazine-3-carboxamide), which had anxiolytic action in the dose range from 0.001 to 0.100 mg/kg (Balb/c, i.p.). This compound is two orders of magnitude higher in dose activity than all other pyrrolo[1,2-a]pyrazine TSPO ligands. CONCLUSION: Molecular modelling methods allowed us to create new TSPO ligands in the series of N,1-diphenylpyrrolo[1,2-a]pyrazine-3-carboxamides with high anxiolytic activity.

The New Dipeptide TSPO Ligands: Design, Synthesis and Structure-Anxiolytic Activity Relationship.

The translocator protein (TSPO, 18 kDa) plays an important role in the synthesis of neurosteroids by promoting the transport of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step in neurosteroidogenesis. Stimulation of TSPO by appropriate ligands increases the level of neurosteroids. The present study describes the design, synthesis and investigation of anxiolytic-like effects of a series of N-acyl-tryptophanyl-containing dipeptides. These novel dipeptide TSPO ligands were designed with the original drug-based peptide design strategy using alpidem as non-peptide prototype. The anxiolytic activities were investigated in Balb/C mice using the illuminated open-field and elevated plus-maze tests in outbred laboratory mice ICR (CD-1). Dipeptide GD-102 (N-phenylpropionyl-l-tryptophanyl-l-leucine amide) in the dose range of 0.01-0.5 mg/kg intraperitoneally (i.p.) has a pronounced anxiolytic activity. The anxiolytic effect of GD-102 was abolished by PK11195, a specific TSPO antagonist. The structure-activity relationship study made it possible to identify a pharmacophore fragment for the dipeptide TSPO ligand. It was shown that l,d-diastereomer of GD-102 has no activity, and the d,l-isomer has less pronounced activity. The anxiolytic activity also disappears by replacing the C-amide group with the methyl ester, a free carboxyl group or methylamide. Consecutive replacement of each amino acid residue with glycine showed the importance of each of the amino acid residues in the structure of the ligand. The most active and technologically available compound GD-102, was selected for evaluation as a potential anxiolytic drug.

Design, Synthesis and Anxiolytic Activity Evaluation of N-Acyltryptophanyl- Containing Dipeptides, Potential TSPO Ligands.

BACKGROUND: The 18 kDa translocator protein (TSPO), previously known as the peripheral- type benzodiazepine receptor, plays a key role for the synthesis of neurosteroids by promoting transport of cholesterol from the outer to the inner mitochondrial membrane, which is the ratelimiting step in neurosteroid biosynthesis. Neurosteroids interact with nonbenzodiazepine site of GABAa receptor causing an anxiolytic effect without the side effects. METHODS: Using the original peptide drug-based design strategy, the first putative dipeptide ligand of the TSPO N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23) was obtained. Molecular docking of GD-23 in the active pocket of the TSPO receptor using Glide software was carried out. The lead compounds GD-23 and its analogues were synthesized using activated succinimide esters coupling method. The anxiolytic activity of GD-23 and its analogues was investigated in vivo, using two validated behavioral tests, illuminated open field and elevated plus-maze. RESULTS: The in vivo studies revealed that the following parameters are necessary for the manifestation of anxiolytic activity of new compounds: the L-configuration of tryptophan, the presence of an amide group at the C-terminus, the specific size of the N-acyl substituent at the Nterminus. Compound GD-23 (N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide) demonstrated a high anxiolytic-like effect in the doses of 0.05-1.0 mg/kg i.p. comparable with that of diazepam. Compound GD-23 was also active in the open field test when was administered orally in the doses of 0.1-5.0 mg/kg. The involvement of TSPO receptor in the mechanism of anxiolytic-like activity of new compounds was proved by the antagonism of compound GD-23 with TSPO selective inhibitor PK11195 as well as with inhibitors of enzymes which are involved in the biosynthesis of neurosteroids, trilostane and finasteride. CONCLUSION: A series of N-acyl-tryptophanyl-containing dipeptides were designed and synthesized as 18 kDa translocator protein (TSPO) ligands. Using a drug-based peptide design method a series of the first dipeptide TSPO ligands have been designed and synthesized and their anxiolytic activity has been evaluated. In general, some of the compounds displayed a high level of anxiolytic efficacy comparable with that of diazepam. The involvement of TSPO receptor in the mechanism of anxiolytic activity of new compounds was proved using two methods. On this basis, the N-acyl-Ltryptophanyl- isoleucine amides could potentially be a novel class of TSPO ligands with anxiolytic activity.

In vivo effects of afobazole (2-mercaptobenzimidazole derivative) on the 7,12-dimethylbenz [alpha]anthracene-induced oncogene and suppressor gene expression.

BACKGROUND: Afobazole, a new 2-mercapto-benzimidazole derivative, exhibited antimutagenic activity in chromosome aberration tests and antioxidant properties. The aim of this study was to demonstrate the potential chemopreventive effect of afobazole on the level of early biological effects by analysing changes in oncogene and tumor suppressor gene expression. MATERIALS AND METHODS: Single intraperitoneal (i.p.) treatment with 7,12-dimethylbenz[alpha]anthracene (DMBA) combined with afobazole was administered to CBA/Ca (sensitive H-2K haplotype) female mice. The expression of Ha-ras and p53 was determined in the vital organs (liver, spleen, lung, kidney, thymus, lymph nodes and bone marrow) 24, 48 and 72 hours later. RESULTS: Coadministration of afobazole and DMBA resulted in a decrease of DMBA-induced overexpression of Ha-ras and p53. Reduction of the DMBA-induced gene expression was most striking when afobazole was given in parallel with DMBA. DISCUSSION: Our results strengthen the previous assumption, which was based on in vitro results, that afobazole has a chemopreventive effect in vivo.