Neurotoxicity Assessment of 1-[(2,3-Dihydro-1-Benzofuran-2-yl)Methyl]Piperazine (LINS01 Series) Derivatives and their Protective Effect on Cocaine-Induced Neurotoxicity Model in SH-SY5Y Cell Culture.

Excessive levels of dopamine in the synaptic cleft, induced by cocaine for example, activates dopaminergic receptors, mainly D1R, D2R, and D3R subtypes, contributing to neurotoxic effects. New synthetic 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazine derivatives (the LINS01 compounds), designed as histaminergic receptor (H3R) ligands, are also dopaminergic receptor ligands, mainly D2R and D3R. This study aims to evaluate the neurotoxicity of these new synthetic LINS01 compounds (LINS01003, LINS01004, LINS01011, and LINS01018), as well as to investigate their protective potential on a cocaine model of dopamine-induced neurotoxicity using SH-SY5Y cell line culture. Neurotoxicity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and automated cell counting with fluorescent dyes (acridyl orange and propidium iodide) assays. Concentration-response curves (CRCs) were performed for all LINS compounds and cocaine using MTT assay. The results show that LINS series did not decrease cell viability after 48h of exposure-except for 100 µM LINS01018, which was discontinued from the study. Likewise, MTT, LDH, and fluorescent dyes staining showed no difference is cell viability for LINS compounds at 10 µM. When incubated with 2.5 mM cocaine (lethal concentration 50) for 48h, 10 µM of each LINS compound, metoclopramide (D2R antagonist) and haloperidol (D2R/D3R antagonist), ameliorated cocaine-induced neurotoxicity. However, only metoclopramide, haloperidol, and LINS01011 compound significantly decreased LDH released in the culture medium, suggesting that this new synthetic compound presents a more robust effect. This preliminary in vitro neurotoxicity study suggests that LINS01 compounds are not neurotoxic, and that they play a promising role in preventing cocaine-induced neurotoxicity.

Assessment of the Physicochemical Properties and Stability for Pharmacokinetic Prediction of Pyrazinoic Acid Derivatives.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which still has high prevalence worldwide. In addition, cases of drug resistance are frequently observed. In the search for new anti-TB drugs, compounds with antimycobacterial activity have been developed, such as derivatives of pyrazinoic acid, which is the main pyrazinamide metabolite. In a previous study, the compounds were evaluated and showed moderate antimycobacterial activity and no important cytotoxic profile; however, information about their pharmacokinetic profile is lacking. OBJECTIVE: The aim of this work was to perform physicochemical, permeability, and metabolic properties of four pyrazinoic acid esters. METHOD: The compounds were analyzed for their chemical stability, n-octanol:water partition coefficient (logP) and apparent permeability (Papp) in monolayer of Caco-2 cells. The stability of the compounds in rat and human microsomes and in rat plasma was also evaluated. RESULTS: The compounds I, II and IV were found to be hydrophilic, while compound III was the most lipophilic (logP 1.59) compound. All compounds showed stability at the three evaluated pHs (1.2, 7.4 and 8.8). The apparent permeability measured suggests good intestinal absorption of the compounds. Additionally, the compounds showed metabolic stability under action of human and rat microsomal enzymes and stability in rat plasma for at least 6 hours. CONCLUSION: The results bring favorable perspectives for the future development of the evaluated compounds and other pyrazinoic acid derivatives.

Differential contribution of H3R antagonism by LINS01 compounds on memory, anxiety-like behaviour and spontaneous locomotor activity in healthy rats.

Histamine H3 receptors (H3R) have attracted interest of research groups as drug target to several CNS disorders. Data suggests that H3R antagonists exert neuroprotective, cognitive enhancement and antidepressant effects in rodents. The LINS01 compounds were reported as selective H3R antagonists, but their effects on memory, anxiety-like behaviour and spontaneous locomotor activity were not evaluated to date. Therefore, this study employed the plus-maze discriminative avoidance task (PM-DAT) to assess concomitantly the effects of LINS01 compounds on short- and long-term memory, anxiety-like behaviour and spontaneous locomotor activity. Thirty-eight adult male Wistar rats were divided into five groups (n = 7-8 per group) according to the treatment. The animals were treated with donepezil (1 mg/kg) and clobenpropit (3 mg/kg) (reference compounds), and with two LINS01 compounds at doses of 5 mg/kg (LINS01003 and LINS01004), and then submitted to the PM-DAT protocol. Saline (vehicle) was used as control group. The behavioural data showed that anxiety-like behaviour, spontaneous locomotor activity and memory effects (short- and long-term) were not affected by the treatment with LINS01004 or clobenpropit. Conversely, treatment with LINS01003 and donepezil impaired the maintenance of discriminative avoidance long-term memory, a hippocampal-dependent memory. Donepezil-treated rats also showed decreased spontaneous locomotor activity and anxiolytic-like effects. In summary, considering that hippocampal damage and memory impairment are associated with Alzheimer's disease (AD), this work brought important findings regarding the contribution of the histamine system to the effects of LINS01 compounds on memory, anxiety and motility, and suggests that H3R antagonism had no effects on anxiety-like behaviour and do not impair discriminative avoidance memory. Furthermore, the findings herein raise new questions about donepezil's function in an "impaired" system such as AD, since it prevented the long-term memory formation in healthy rats.

Pharmacological and SAR analysis of the LINS01 compounds at the human histamine H1 , H2 , and H3 receptors.

Histamine is a transmitter that activates the four receptors H1 R to H4 R. The H3 R is found in the nervous system as an autoreceptor and heteroreceptor, and controls the release of neurotransmitters, making it a potential drug target for neuropsychiatric conditions. We have previously reported that the 1-(2,3-dihydro-1-benzofuran-2-yl)methylpiperazines (LINS01 compounds) have the selectivity for the H3 R over the H4 R. Here, we describe their pharmacological properties at the human H1 R and H2 R in parallel with the H3 R, thus providing a full analysis of these compounds as histamine receptor ligands through reporter gene assays. Eight of the nine LINS01 compounds inhibited H3 R-induced histamine responses, but no inhibition of H2 R-induced responses was seen. Three compounds were weakly able to inhibit H1 R-induced responses. No agonist responses were seen to any of the compounds at any receptor. SAR analysis shows that the N-methyl group improves H3 R affinity while the N-phenyl group is detrimental. The methoxy derivative, LINS01009, had the highest affinity.

QSAR Modeling of Histamine H3R Antagonists/inverse Agonists as Future Drugs for Neurodegenerative Diseases.

BACKGROUND: Histamine H3 receptor (H3R) is associated with several neuropsychological diseases, and thus it is an important target involved in several CNS disorders, such as narcolepsy, attention deficit hyperactivity disorder and schizophrenia. Since QSAR modeling is a feasible approach to explain the role of the molecular substituents in the biological activity, it can help in improving the design of better H3R ligands for these conditions. METHODS: This article reviews papers previously published in literature to show the current status of the contribution from QSAR modeling to reach H3R antagonists/inverse agonists. RESULTS: Classical and 3D-QSAR models were retrieved, showing that the steric and hydrophobic properties of the H3R ligands are most important to reach good affinity. CONCLUSION: Although QSAR methods are valuable to design better H3R antagonists/inverse agonists, pharmacokinetics should also be considered in future models to ensure good CNS penetration.

1-[(2,3-Dihydro-1-benzofuran-2-yl) methyl]piperazines as novel anti-inflammatory compounds: Synthesis and evaluation on H3 R/H4 R.

The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H3 R and H4 R have been explored as targets for drug discovery, including in the search for dual-acting H3 R/H4 R ligands. The H4 R, the most recent histamine receptor, is a promising target for novel anti-inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines were synthesized and evaluated in competitive binding assays as H3 R/H4 R ligands herein. The results showed the compounds presented affinity (Ki ) for H3 R/H4 R in micromolar range, and they are more selective to H3 R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl-substituted compound LINS01005 has shown the higher affinity of the set for H4 R, but no considerable selectivity toward this receptor over H3 R. LINS01005 showed interesting anti-inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX-2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti-inflammatory agents.

1-[(2,3-Dihydro-1-benzofuran-2-yl) methyl]piperazines as novel anti-inflammatory compounds: synthesis and evaluation on H3R/H4R

The histamine receptors (HRs) are members of G-protein-coupled receptor superfamily and traditional targets of huge therapeutic interests. Recently, H3R and H4R have been explored as targets for drug discovery, including in the search for dual-acting H3R/H4R ligands. The H4R, the most recent histamine receptor, is a promising target for novel anti-inflammatory agents in several conditions such as asthma and other chronic inflammatory diseases. Due to similarity with previously reported ligands of HRs, a set of 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazines were synthesized and evaluated in competitive binding assays as H3R/H4R ligands herein. The results showed the compounds presented affinity (K-i) for H3R/H4R in micromolar range, and they are more selective to H3R. All the compounds showed no important cytotoxicity to mammalian cells. The phenyl-substituted compound LINS01005 has shown the higher affinity of the set for H4R, but no considerable selectivity toward this receptor over H3R. LINS01005 showed interesting anti-inflammatory activity in murine asthma model, reducing the eosinophil counts in bronchoalveolar lavage fluid, as well as the COX-2 expression. The presented compounds are valuable prototypes for further improvements to achieve better anti-inflammatory agents.

Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile.

Pyrazinamide and Pyrazinoic Acid Derivatives Directed to Mycobacterial Enzymes Against Tuberculosis.

Tuberculosis (TB) is an infectious diseases responsible for thousands of deaths worldwide. Due to the use of antimycobacterial drugs, TB prevalence seemed to be controlled, but with the appearance of resistant tuberculosis cases, the concern about the disease had become significant again, as well as the need for new alternatives to TB treatment. Since pyrazinamide (PZA) is part of the firstline agents in TB treatment, several derivatives of this drug were described, besides pyrazinoic acid (POA) derivatives, the active form of PZA. POA has been used mainly to design prodrugs to be activated by mycobacterial esterases, while PZA derivatives should be activated specifically by the nicotinamidase/ pyrazinamidase (PZAse), or other PZAse-independent pathways. The intention of this paper is to discuss the state of art of PZA and POA derivatives and their activity against Mycobacterium tuberculosis and other mycobacteria, besides the therapeutic potential. Focus was given in prodrugs and derivatives directed to mycobacterial enzymes involved in its activation or mechanism of action.

Histamine H4 receptor ligands: future applications and state of art.

Histamine is a chemical transmitter found practically in whole organism and exerts its effects through the interaction with H1 to H4 histaminergic receptors. Specifically, H4 receptors are found mainly in immune cells and blood-forming tissues, thus are involved in inflammatory and immune processes, as well as some actions in central nervous system. Therefore, H4 receptor ligands can have applications in the treatment of chronic inflammatory and immune diseases and may be novel therapeutic option in these conditions. Several H4 receptor ligands have been described from early 2000's until nowadays, being imidazole, indolecarboxamide, 2-aminopyrimidine, quinazoline, and quinoxaline scaffolds the most explored and discussed in this review. Moreover, several studies of molecular modeling using homology models of H4 receptor and QSAR data of the ligands are summarized. The increasing and promising therapeutic applications are leading these compounds to clinical trials, which probably will be part of the next generation of blockbuster drugs.