Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: synthesis, biological characterization, and behavioral studies.

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

Discovery of potent inhibitors of human and mouse fatty acid amide hydrolases.

Fatty acid amide hydrolase (FAAH, EC 3.5.1.99) is the main enzyme catabolizing endocannabinoid fatty acid amides. FAAH inactivation promotes beneficial effects upon pain and anxiety without the side effects accompanying agonists of type-1 cannabinoid receptors. Aiming at discovering new selective FAAH inhibitors, we developed a series of compounds (5a-u) characterized by a functionalized heteroaromatic scaffold. Particularly, 5c and 5d were identified as extremely potent, noncompetitive, and reversible FAAH inhibitors endowed with a remarkable selectivity profile and lacking interaction with the hERG channels. In vivo antinociceptive activity was demonstrated for 5c, 5d, and 5n at a dose much lower than that able to induce either striatal and limbic stereotypies or anxiolytic activity, thus outlining their potential to turn into optimum preclinical candidates. Aiming at improving pharmacokinetic properties and metabolic stability of 5d, we developed a subset of nanomolar dialyzable FAAH inhibitors (5v-z), functionalized by specific polyethereal lateral chains and fluorinated aromatic rings.

C5-DNA methyltransferase inhibitors: from screening to effects on zebrafish embryo development.

DNA methylation is involved in the regulation of gene expression and plays an important role in normal developmental processes and diseases, such as cancer. DNA methyltransferases are the enzymes responsible for DNA methylation on the position 5 of cytidine in a CpG context. In order to identify and characterize novel inhibitors of these enzymes, we developed a fluorescence-based throughput screening by using a short DNA duplex immobilized on 96-well plates. We have screened 114 flavones and flavanones for the inhibition of the murine catalytic Dnmt3a/3L complex and found 36 hits with IC(50) values in the lower micromolar and high nanomolar ranges. The assay, together with inhibition tests on two other methyltransferases, structure-activity relationships and docking studies, gave insights on the mechanism of inhibition. Finally, two derivatives effected zebrafish embryo development, and induced a global demethylation of the genome, at doses lower than the control drug, 5-azacytidine.

Specific targeting of peripheral serotonin 5-HT(3) receptors. Synthesis, biological investigation, and structure-activity relationships.

The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT(3) receptor (5-HT(3)R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated cardiac parameters by direct interaction with myocardial 5-HT(3)Rs. The potent 5-HT(3)R ligands 4h and 4n modulate chronotropy (right atrium) but not inotropy (left atrium) at the cardiac level, being antagonist and partial agonist, respectively. Preliminary pharmacokinetic studies indicate that (S)-4n and 4a, representatives of the novel 5-HT(3)R ligands, possess poor blood-brain barrier permeability, being the prototypes of peripherally acting 5-HT(3)R modulators endowed with a clear-cut pharmacological activity at the cardiac level. The unique properties of 4h and 4n, compared to their previously described centrally active N-methyl analogue 5a, are mainly due to the hydrophilic groups at the distal piperazine nitrogen. These analogues represent novel pharmacological tools for investigating the role of peripheral 5-HT(3)R in the modulation of cardiac parameters.

Discovery of a new class of potential multifunctional atypical antipsychotic agents targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and effects on behavior.

Dopamine D(3) antagonism combined with serotonin 5-HT(1A) and 5-HT(2A) receptor occupancy may represent a novel paradigm for developing innovative antipsychotics. The unique pharmacological features of 5i are a high affinity for dopamine D(3), serotonin 5-HT(1A) and 5-HT(2A) receptors, together with a low affinity for dopamine D(2) receptors (to minimize extrapyramidal side effects), serotonin 5-HT(2C) receptors (to reduce the risk of obesity under chronic treatment), and for hERG channels (to reduce incidence of torsade des pointes). Pharmacological and biochemical data, including specific c-fos expression in mesocorticolimbic areas, confirmed an atypical antipsychotic profile of 5i in vivo, characterized by the absence of catalepsy at antipsychotic dose.

An efficient approach to chiral C8/C9-piperazino-substituted 1,4-benzodiazepin-2-ones as peptidomimetic scaffolds.

A promising way to interfere with biological processes is through the modulation of protein-protein interactions by means of small molecules acting as peptidomimetics. The 1,4-benzodiazepine scaffold has been widely reported as a peptide-mimicking, pharmacogenic system. While several synthetic pathways to C6-8 substituted benzodiazepines have been disclosed, few 1,4-benzodiazepines substituted at C9 have been reported. Herein, we describe a versatile approach to introduce cyclic, protonatable functionality at C8/C9. Introduction of the piperazine system at C8 and C9 gave access to a unique functionalization of the versatile benzodiazepine skeleton, broadening tailoring options on the benzofused side of the molecule, and the possibility of discovering novel peptidomimetics potentially able to modulate protein-protein interactions. Coupling of activated amino acids with poorly reactive anilines under mild conditions, while avoiding racemization, gave easy access to these compounds. Efficient amino acid activation was obtained by exploiting the rapid formation of acid chlorides under low temperature and acid/base free conditions, using triphenylphosphine and hexachloroacetone. This procedure successfully resulted in high reaction yields, did not produce racemization (ee > 98%, as demonstrated by using chiral solvating agents), and was compatible with the acid sensitive protecting groups present in the substrates.

Exploiting protein fluctuations at the active-site gorge of human cholinesterases: further optimization of the design strategy to develop extremely potent inhibitors.

Protein conformational fluctuations are critical for biological functions, although the relationship between protein motion and function has yet to be fully explored. By a thorough bioinformatics analysis of cholinesterases (ChEs), we identified specific hot spots, responsible for protein fluctuations and functions, and those active-site residues that play a role in modulating the cooperative network among the key substructures. This drew the optimization of our design strategy to discover potent and reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase (hAChE and hBuChE) that selectively interact with specific protein substructures. Accordingly, two tricyclic moieties differently spaced by functionalized linkers were investigated as molecular yardsticks to probe the finest interactions with specific hot spots in the hChE gorge. A number of SAR trends were identified, and the multisite inhibitors 3a and 3d were found to be the most potent inhibitors of hBuChE and hAChE known to date.

Design, synthesis, and structure-activity relationship studies of 4-quinolinyl- and 9-acrydinylhydrazones as potent antimalarial agents.

Malaria is a major health problem in poverty-stricken regions where new antiparasitic drugs are urgently required at an affordable price. We report herein the design, synthesis, and biological investigation of novel antimalarial agents with low potential to develop resistance and structurally based on a highly conjugated scaffold. Starting from a new hit, the designed modifications were performed hypothesizing a specific interaction with free heme and generation of radical intermediates. This approach provided antimalarials with improved potency against chloroquine-resistant plasmodia over known drugs. A number of structure-activity relationship (SAR) trends were identified and among the analogues synthesized, the pyrrolidinylmethylarylidene and the imidazole derivatives 5r, 5t, and 8b were found as the most potent antimalarial agents of the new series. The mechanism of action of the novel compounds was investigated and their in vivo activity was assessed.