A novel arylpiperazine derivative (LQFM181) protects against neurotoxicity induced by 3- nitropropionic acid in in vitro and in vivo models.

In the pursuit of novel antioxidant therapies for the prevention and treatment of neurodegenerative diseases, three new arylpiperazine derivatives (LQFM181, LQFM276, and LQFM277) were synthesized through a molecular hybridization approach involving piribedil and butylated hydroxytoluene lead compounds. To evaluate the antioxidant and neuroprotective activities of the arylpiperazine derivatives, we employed an integrated approach using both in vitro (SH-SY5Y cells) and in vivo (neurotoxicity induced by 3-nitropropionic acid in Swiss mice) models. In the in vitro tests, LQFM181 showed the most promising antioxidant activity at the neuronal membrane and cytoplasmic levels, and significant neuroprotective activity against the neurotoxicity induced by 3-nitropropionic acid. Hence, this compound was further subjected to in vivo evaluation, which demonstrated remarkable antioxidant capacity such as reduction of MDA and carbonyl protein levels, increased activities of succinate dehydrogenase, catalase, and superoxide dismutase. Interestingly, using the same in vivo model, LQFM181 also reduced locomotor behavior and memory dysfunction through its ability to decrease cholinesterase activity. Consequently, LQFM181 emerges as a promising candidate for further investigation into its neuroprotective potential, positioning it as a new therapeutic agent for neuroprotection.

Overdiagnosis of vaccine allergy: Skin testing and challenge at a public specialized unit (CRIE) in Rio de Janeiro, Brazil.

Background: Vaccination is an extremely safe public health intervention, but rare IgE-mediated adverse events must be identified to avoid the risk of anaphylaxis in the event of reexposure. However, using only clinical history to diagnose previous allergic reactions may lead to overdiagnosis of vaccine allergy and even to the use of medical exemptions as a subterfuge to mandatory vaccination. Methods: We conducted a retrospective study to describe the outcomes of patients with a history of vaccine or vaccine component allergy who were evaluated at our unit from 2011 to 2017. Data on allergy history, skin test results, vaccines prescribed, and adverse events were retrieved from the medical records at the Centro de Referência para Imunobiológicos Especiais (Reference Center of Special Immunobiologicals)-Fiocruz, in Rio de Janeiro, Brazil. Results: Of 34 adults with history of allergy to vaccine or vaccine components, 32 (94.1%) were successfully vaccinated without serious adverse events after our evaluation. In 12 patients (35%), the time elapsed between the allergy symptoms and evaluation in the Centro de Referência para Imunobiológicos Especiais-Fiocruz was more than 10 years. Conclusion: Specialized care and use of skin tests allowed safe vaccination of the majority of patients. An objective, systematic evaluation of a history of vaccine allergy can prevent its improper use to avoid mandatory vaccination and reduce missed opportunities for immunization.

Preclinical pharmacokinetics of a promising antineoplastic prototype piperazine-containing compound (LQFM018) in rats by a new LC-MS/MS bioanalytical method.

LQFM018 is a novel antineoplastic prototype, showing an expressive drug-triggered K562 leukemic cells death mechanism, through necroptotic signaling. Due to its promising effect, this study aimed to evaluate the pharmacokinetics of LQFM018 in rats, using a new validated bioanalytical LC-MS/MS-based method. Chromatographic column was an ACE® C18 (100 mm × 4.6 mm, 5 µm) eluted by a mobile phase composed of ammonium acetate 2 mM and formic acid 0.025%:methanol (50:50, v/v), under flow of 1.2 mL/min and injection volume of 3.0 µL. LQFM018 was extracted from rat plasma by a simple liquid-liquid method, using MTBE solvent. Rats were administered intraperitoneally at LQFM018 100 mg/kg dose and blood samples were collect at times of 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 h. Bioanalytical-LC-MS/MS-based method was rapid, high throughput and sensitive with a good linearity ranging from 10 (LLOQ) to 15000 ng/mL, besides precise and accurate, ranging of 0.8-7.3% and 96.8-107.6%, respectively. The prototype LQFM018 was rapid and well absorbed, and highly distributed, apparently due to its high lipid solubility. These features are primordial for an anticancer agent in the treatment of deep tumors, such as bone marrow neoplasms, in which the drug might permeate easily tissue barriers. Also, LQFM018 has demonstrated a high clearance, according to a low t1/2in rats, indicating a relative fast elimination phase related to a possible intense hepatic biotransformation. These information support further studies to establish new understands on pharmacokinetics of promising antineoplastic prototype LQFM018 from preclinical and clinical evaluations.

Anti-inflammatory, antinociceptive, and vasorelaxant effects of a new pyrazole compound 5-(1-(2-fluorophenyl)-1H-pyrazol-4-yl)-1H-tetrazole: role of NO/cGMP pathway and calcium channels.

Molecular modification of compounds remains important strategy towards the discovery of new drugs. In this sense, this study presents a new pyrazole derivative 5-(1-(2-fluorophenyl)-1H-pyrazol-4-yl)-1H-tetrazole (LQFM039) and evaluated the anti-inflammatory, analgesic, and vasorelaxant effects of this compound as well the mechanisms of action involved in the pharmacological effects. For this, mice were orally treated with LQFM039 (17.5, 35, or 70 mg/kg) prior acetic acid-induced abdominal writhing, formalin, tail flick, and carrageenan-induced paw edema protocols. In addition, vascular reactivity protocols were made with aortic rings contraction with phenylephrine and stimulated with graded concentrations of LQFM039. Abdominal writhing and licking time in both neurogenic and inflammatory phases of formalin were reduced with LQFM039 without altering latency to nociceptive response in the tail flick test. Carrageenan-induced paw edema showed that LQFM039 reduces edema and cell migration. In addition, the mechanism of action of LQFM039 involves NO/cGMP pathway and calcium channels, since this new pyrazole derivate elicited concentration-dependent relaxation attenuated by Nω-nitro-l-arginine methyl ester and 1H-[1,2,4] oxadiazolo [4,3-alpha]quinoxalin-1-one, and blockade of CaCl2-induced contraction. Altogether, our finding suggests anti-inflammatory, antinociceptive, and vasorelaxant effect of this new pyrazole derivative with involvement of NO/cGMP pathway and calcium channels.

L-proline transporter inhibitor (LQFM215) promotes neuroprotection in ischemic stroke.

BACKGROUND: L-proline transporter (PROT/SLC6A7) is closely associated with glutamatergic neurotransmission, where L-proline modulates the NMDA receptor (NMDAR) function. NMDAR-mediated excitotoxicity is a primary cause of neuronal death following stroke, which is triggered by the uncontrolled release of glutamate during the ischemic process. After ischemic stroke, L-proline levels show a reduction in the plasma, but high circulating levels of this molecule indicate good functional recovery. This work aimed to produce new PROT inhibitors and explore their effects on ischemic stroke. METHODS: Initially, we built a three-dimensional model of the PROT protein and run a molecular docking with the newly designed compounds (LQFM215, LQFM216, and LQFM217). Then, we synthesized new PROT inhibitors by molecular hybridization, and proline uptake was measured in ex vivo and in vivo models. The behavioral characterization of the treated mice was performed by the open-field test, elevated plus-maze, Y-maze, and forced swimming test. We used the permanent middle cerebral artery occlusion (MCAO) model to study the ischemic stroke damage and analyzed the motor impairment with limb clasping or cylinder tests. RESULTS: LQFM215 inhibited proline uptake in hippocampal synaptosomes, and the LQFM215 treatment reduced proline levels in the mouse hippocampus. LQFM215 reduced the locomotor and exploratory activity in mice and did not show any anxiety-related or working memory impairments. In the MCAO model, LQFM215 pre-treatment and treatment reduced the infarcted area and reduced motor impairments in the cylinder test and limb clasping. CONCLUSIONS: This dataset suggests that the new compounds inhibit cerebral L-proline uptake and that LQFM215 promotes neuroprotection and neuro-repair in the acute ischemic stroke model.

Telemedicine in the National Immunization Program (Brazil): A promising tool.

The coronavirus disease 2019 pandemic abruptly changed the dynamics of basic health care, with the consequent need for adjustments in essential services. The objective of this study was to evaluate the acceptance and impact of telemedicine at a Reference Center for Special Immunobiologicals (CRIE). Methods: Patients aged 18 years or older who had a medical referral to CRIE and agreed to have a telemedicine consultation were included. After the medical appointments, participants answered a satisfaction survey. Results: From April 2021 to February 2022, 702 telemedicine consultation were conducted. Over 3,380 vaccines were prescribed via telemedicine. Of all the participants who answered the satisfaction questionnaire, 99.8% stated that they would recommend the service to other people. Conclusions: Telemedicine proved to be promising tool for healthcare at CRIE and had good acceptance by users, potentially improving access and extending the reach of the National Immunization Program.

Structure-activity relationship of three new piperazine derivates with anxiolytic-like and antidepressant-like effects.

Anxiety and depression are common mental disorders affecting millions of people worldwide. Unsatisfactory clinical outcomes with the use of the available pharmacological interventions among some patients demand newer drugs with proven efficacy, safety, and tolerability profile. In this study, the LQFM211, LQFM213, and LQFM214 were designed from the piperazine scaffold and administered orally in mice. These mice were later evaluated in the open field, elevated plus maze, and forced swimming tests to assess the exploratory, anxiolytic, and antidepressant-like activities, respectively. The mechanism of action of these new derivatives was evaluated using flumazenil (benzodiazepine antagonist) and WAY100635 (5-HT1A receptor antagonist). Unlike LQFM214, the LQFM211 and LQFM213 elicited anxiolytic and antidepressant-like effects. The blockade of the effect of LQFM213 by WAY100635 suggests the involvement of the serotonergic pathway.

Anxiolytic- and antidepressant-like effects of new phenylpiperazine derivative LQFM005 and its hydroxylated metabolite in mice.

The current treatments available for anxiety and depression are only palliative. Full remission has remained elusive, characterizing unmet medical needs. In the scope of an academic drug discovery program, we describe here the design, synthesis, in vitro metabolism prediction and pharmacological characterization of a new piperazine compound, 1-(4-methoxyphenyl)-4-((1-phenyl-1H-pyrazol-4-yl)methyl)piperazine (LQFM005), and of its main putative metabolite, 4-(4-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)- 1H-pyrazol-1-yl)phenol (LQFM235). The production of the metabolite was initially performed by in vitro biotransformation of LQFM005 using Aspergillus candidus and then by chemical synthesis. Oral administration of either 12 or 24 µmol/kg LQFM005 to mice did not affect spontaneous locomotor activity but increased the time spent in the center of the open field. Both LQFM005 and LQFM235 (24 µmol/kg) increased the time spent by the mice in the open arms of the elevated plus maze (EPM), a good indication of anxiolytic-like effect, and decreased the immobility time in the forced swimming test (FST), suggesting an antidepressant-like effect. The previous administration of WAY-100635 (a 5-HT1A antagonist) abolished the effects of LQFM005 in both EPM and FST. Binding experiments showed that LQFM005 and its metabolite bind to the 5-HT1A receptor with a moderate affinity (Ki around 5-9 µM). The two compounds are relatively safe, as indicated by cytotoxic assessment using the 3T3 fibroblast cell line and estimated LD50 around 600 mg/kg. In conclusion, oral administration of the newly synthesized phenylpiperazines produced anxiolytic- and antidepressant-like effects in behavioral tests, putatively in part through the activation of 5-HT1A receptors.

Neuropharmacological Activity of the New Piperazine Derivative 2-(4-((1- Phenyl-1H-Pyrazol-4-yl)Methyl)Piperazin-1-yl)Ethyl Acetate is Modulated by Serotonergic and GABAergic Pathways.

BACKGROUND: Pharmacological treatments for mental disorders, such as anxiety and depression, present several limitations and adverse effects. Therefore, new pharmacotherapy with anxiolytic and antidepressant potential is necessary, and the study of compounds capable of interacting with more than one pharmacological target may provide new therapeutic options. OBJECTIVES: In this study, we proposed the design, synthesis of a new compound, 2-(4-((1- phenyl-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethyl acetate (LQFM192), pharmacological evaluation of its anxiolytic-like and antidepressant-like activities, as well as the possible mechanisms of action involved. METHODS: Administration of LQFM192 was carried out prior to the exposure of male Swiss mice to behavioral tests, such as the elevated plus-maze and forced swimming test. The involvement of the serotonergic system was studied by pretreatment with WAY-100635 or p-chlorophenylalanine (PCPA) and the involvement of the benzodiazepine site of the GABAA receptor by pretreatment with flumazenil. RESULTS: The treatment with LQFM192 at doses of 54 and 162 µmol/kg demonstrated anxiolyticlike activity that was blocked by WAY-100635, PCPA, and flumazenil pretreatments. The potential antidepressant-like activity was visualized at the same doses and blocked by WAY-100635 and PCPA. CONCLUSION: In summary, the anxiolytic-like activity of LQFM192 is mediated by the serotonergic system and the benzodiazepine site of the GABAA receptor, and the antidepressant-like activity through the serotonergic system.

Comparison of Conventional and Microwave Synthesis of Phenyl-1H-pyrazoles and Phenyl-1H-pyrazoles-4-carboxylic Acid Derivatives.

BACKGROUND: Privileged scaffolds are of high importance for molecules containing the pyrazole subunit due to their broad spectrum of pharmacological activities. For this reason, a method that is more efficient needs to be developed for the preparation of pyrazole derivatives. OBJECTIVE: The purpose of this study was the optimisation of the conventional synthesis of the pyrazole ring and the oxidation of phenyl-1H-pyrazole-4-carbaldehyde to phenyl-1H-pyrazole-4-carboxylic acid through Microwave- Assisted Organic Synthesis (MAOS). METHODS: We performed a comparison between conventional synthesis and conventional synthesis with microwave heating using the synthesis method of pyrazole ring described by Finar and Godfrey and for the oxidation of phenyl-1H-pyrazole-4-carbaldehyde, the method described by Shriner and Kleiderer was used. RESULTS: MAOS reduces the reaction time to obtain all compounds compared to conventional heating. At a temperature of 60°C, 5 minutes of reaction time, and power of 50 W, the yield of phenyl-1H-pyrazoles (3a-m) compounds was in the range of 91 - 98% using MAOS, which is better than conventional heating (72 - 90%, 75ºC, 2 hours). An improvement in the yield for the oxidation reaction was also achieved with MAOS. The compounds (5a-m) were obtained with yields ranging from 62 - 92% (80ºC, 2 minutes, 150 W), while the yields with conventional heating were in the range of 48 - 85% (80ºC, 1 hour). The 26 compounds were achieved through an easy work-up procedure with no chromatographic separation. The pure products were characterised by the spectral data obtained from IR, MS, 1H and 13C NMR or HSQC/HMBC techniques. CONCLUSION: The advantages of MAOS include short reaction time and increased yield, due to which it is an attractive option for pyrazole compounds synthesis.

Neuropharmacological assessment in mice and molecular docking of piperazine derivative LQFM212.

Piperazine derivatives are an attractive class of chemical compounds for the treatment of various mental illness. Herein, we demonstrated the synthesis of LQFM212, a piperazine derivative, behavioral evaluation in mice and computational studies. In neuropharmacological assessment, LQFM212 treatment at doses of 18, 54 or 162 µmol/kg increased the sleep duration in sodium pentobarbital-induced sleep test. LQFM212 at dose of 162 µmol/kg increased climbing time in the chimney test and decreased the number of squares crossed in the open field test, suggesting that LQFM212 in high doses reduces spontaneous movement. However, LQFM212 treatment at the doses of 18 or 54 µmol/kg increased the preference for the center of field which could be indicative of anxiolytic-like effects. In elevated plus maze and light-dark box tests, LQFM212 treatment altered all parameters observed that demonstrate anxiolytic-like activity. These effects were reversed by flumazenil, mecamylamine, WAY-100635 and PCPA, but not with ketanserin, showing that anxiolytic-like activity involve benzodiazepine site of GABAA receptor, nicotinic and serotonergic pathways. Molecular docking of LQFM212 showed that the ligand has more interactions with GABAA receptor than with 5-HT1A receptor. Despite the involvement of benzodiazepine site on anxiolytic-like effect of LQFM212, treatment with this compound did not alter cognitive function in the step-down avoidance test. In this sense, this piperazine derivative is a good prototype for treating anxiety disorders with putative mechanism of action.

Design, synthesis and pharmacological assessment of new pyrazole compounds.

AIMS: This study investigated the antinociceptive and anti-inflammatory effects of new pyrazole compounds LQFM011(5), LQFM043(6) and LQFM044(7) as well as the mechanisms of action and acute in vitro toxicity. MAIN METHODS: The antinociceptive activity was evaluated using the acetic acid-induced abdominal writhing test, formalin-induced pain test and the Randall-Selitto test. The anti-inflammatory activity was evaluated using models of paw oedema and pleurisy induced by carrageenan; cell migration, the levels of tumour necrosis factor α (TNF-α) and myeloperoxidase (MPO) enzyme activity were evaluated. In addition, the ability to inhibit phospholipase A2 (PLA2) in vitro and docking in PLA2 were used. Acute oral systemic toxicity in mice was evaluated through the neutral red uptake assay. KEY FINDINGS: The synthesised compounds (5-7), delivered via gavage (p.o.) at 70, 140 or 280 µmol/kg, decreased the number of writhings induced by acetic acid; the three compounds (280 µmol/kg p.o.) reduced the paw licking time in the first and second phase of the formalin test and decreased the nociceptive threshold variation in the Randall-Selitto test. Furthermore, this dose reduced oedema formation, leucocyte migration (specifically through reduction in polymorphonuclear cell movement) and increased mononuclear cells. MPO activity and the levels of pro-inflammatory cytokines TNF-α were decreased. Evaluation of PLA2 inhibition via the docking simulation revealed more interactions of LQFM043R(6) and LQFM044(7), data that corroborated the half-maximal inhibitory concentration (IC50) of PLA2 inhibition in vitro. Therefore, LQFM011(5), LQFM043(6) and LQFM044(7) were classified with the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) as category 4.

Antiangiogenic and antitumoral activity of LQFM126 prototype against B16F10 melanoma cells.

Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candidates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10 melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cytoplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle retention in G0/G1 phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126 was classified as UN GHS category 4 (LD50 > 300-2000 mg/kg), suggesting it has low acute systemic toxicity. LQFM126 can be a promising prototype for anticancer therapy.

Correction to: Invited Discussion on: Skin Reducing Mastectomy and Prepectoral Breast Reconstruction in Large Ptotic Breasts.

Aesth Plast Surg.

Invited Discussion on: Skin Reducing Mastectomy and Prepectoral Breast Reconstruction in Large Ptotic Breasts.

The applicability of acellular dermal matrix (ADM) for breast reconstruction is a consolidated reality, as skin and nipple sparing techniques became standard mastectomy approaches. ADM is a soft connective tissue graft generated via a decellularization process that preserves intact the extracellular skin matrix. ADM not only provides tissue reinforcement, but also better pocket control, and shape without the compressive effects of total sub-muscular coverage. Our preference is using one "Strattice®" ADM in pocket's format to cover the implant's inferior pole, protecting the totality of the implant in its inferior pole by the ADM. This technique besides its versatility is cheaper than other techniques presented. The success of ADM prepectoral breast reconstruction depends on three pillars: careful patient selection, flap perfusion and postoperative management. The challenge in large and/or ptotic breasts under the risk of large badly perfused flaps as well as of nipple, due to the nipple-furcules distance which can be handled with well-selected criteria as mentioned and safe management of nipple ascension as well as the nipple graft. Traditionally, they need to be associated with reducing mammoplasty techniques to achieve the expected aesthetic results in a single stage. Therefore, the plastic surgeon must be prepared for new reconstructive approaches postmastectomy, having the ADM as an excellent option for that.

Small-molecule MDM2 inhibitor LQFM030-induced apoptosis in p53-null K562 chronic myeloid leukemia cells.

Our group designed and synthesized the N-phenyl-piperazine LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone], a small molecule derived from molecular simplification of the Nutlin-1, an inhibitor of the human homologue of murine double minute 2 (MDM2) protein that is expressed in several types of cancer. To better investigate the effects of LQFM030 regarding the p53 mutation status, this study investigated the antiproliferative activity of LQFM030 against the p53-null K562 leukemia cells as well as the cell death pathways involved. In addition, the effects of LQFM030 on the levels of the p53/MDM2 complex were also carried out using 3T3 cells as a p53 wild-type model. Our data suggest that LQFM030 triggered apoptosis in K562 cells via different mechanisms including cell cycle arrest, caspase activation, reduction of mitochondrial activity, decrease in MDM2 expression, and transcriptional modulation of MDMX, p73, MYC, and NF-ĸB. Additionally, it promoted effects in p53/MDM2 binding in p53 wild-type 3T3 cells. Therefore, LQFM030 has antiproliferative effects in cancer cells by a p53 mutation status-independent manner with different signaling pathways. These findings open new perspectives to the treatment of leukemic cells considering the resistance development associated with cancer treatment with conventional cytotoxic drugs.

Antinociceptive effects of new pyrazoles compounds mediated by the ASIC-1α channel, TRPV-1 and µMOR receptors.

Pyrazoles are potent medicinal scaffolds and exhibit a wide spectrum of biological activities, such as analgesic, anti-inflammatory and antipyretic. In this paper we report on research we have performed with the aim of continuing the biological evaluation of the regio-isomeric pyrazole compounds, LQFM-020 (fluorine, para position), LQFM-021 (fluorine, meta position), and LQFM-039 (fluorine, ortho position) in models of pain induced by acidified saline, capsaicin, and formalin. We also investigated the mechanisms of action of these compounds via electrophysiological analyses using the two-electrode voltage-clamp technique and heterologous expression in Xenopus laevis oocytes. This enabled us to study different potassium channel subtypes: the ASIC-1α channel, TRPV-1, and µMOR receptors. Our results indicate that LQFM-020, LQFM-021, and LQFM-039 (15, 30 or 60 mg.kg-1) compounds inhibited the nociceptive response induced by acidified saline in a dose-dependent manner. The dose of 30 mg.kg-1 inhibited the nociceptive response induced by capsaicin by 53.3%, 51.4%, and 52.1%, respectively. In addition, we found that naloxone reverses the antinociceptive effect produced by the compounds in both phases of the formalin test. In electrophysiological analyses, we observed that the LQFM-020, LQFM-021, and LQFM-039 compounds did not modulate voltage-gated K + channel subtypes. In contrast, all the compounds tested inhibited the ASIC-1α channel at pH 4.5, with IC50-values of 96.1, 91.6, and 235.2 µM, respectively. All compounds also inhibited the TRPV-1 channel with IC50-values of 139.1, 212.5, and 159.1 µM, respectively. In contrast to the ASIC-1α and TRPV-1 targets, all compounds showed agonist activity on the µMOR receptor with an EC50-value of 117.4, 98.9, and 86.3 µM, respectively. We thus conclude that the ASIC-1α, TRPV-1, and µMOR channels are targets that are directly involved in the antinociceptive effect of LQFM-020, LQFM-021, and LQFM-039. Furthermore, the modifications of the fluorine positions in the phenyl analogs do not change the analgesic effect. However, LQFM-039 showed lower interaction with ASIC-1α channel.

Toxico-pharmacological evaluations of the small-molecule LQFM166: Inducer of apoptosis and MDM2 antagonist.

Inhibition of p53-MDM2 complex has been emerging as a strategy for antitumoral drug development considering the pro-apoptotic role of functional p53 in tumor cells. In our study, the prototype LQFM166 (2), designed through molecular simplification strategy inspired in the Nutlins compounds, was synthetized, characterized and the mechanisms of cell death were investigated. In addition, we estimated the starting doses for acute oral systemic toxicity tests according to the OECD Guidance Document No.129 - 3T3 NRU. The cytotoxic profile of LQFM166 (2) was determined in K-562 cells, a p53-null cell line, since previous studies also showed activity of LQFM166 (2) on this cells. After 24, 48 or 72 h of compound treatment, using MTT reduction assay, the IC50 values found were 100.1 µM, 56.76 µM and 45.11 µM, respectively. LQFM166 (2) was cytotoxic for leukemia cells in a concentration-time-dependent manner. Cell death mechanisms studies of LQFM166 on K-562 cells, revealed that the compound induced cell cycle arrest, increased the expression of caspase 3/7, 8 and 9, cytochrome c, Bax, p21 and p27. Additionally, a decrease in the expression of the Bcl-2 and cyclin-B1 was observed. The apoptotic inducer profile of the compound was confirmed by phosphatidylserine externalization. Investigation of complexation of p53/MDM2 was carried out by ELISA assay using 3T3 cell, showing a decrease in the p53-MDM2 complex induced by the compound. Furthermore, the cytotoxicity in basal fibroblasts 3T3 was determined to estimate LD50. LQFM166 (2) reduced 3T3 cells viability with the IC50 of 185.3 µM and estimated LD50 of 706.7 mg/kg (category 4 of GHS). The rationally designed of the prototype LQFM166 (2) induced cell death by apoptotic mechanisms in leukemic cells and showed MDM2 complexation antagonism in 3T3 cells.

Tert-butyl 4-((1-phenyl-1H-pyrazol-4-yl) methyl) piperazine-1-carboxylate (LQFM104)- New piperazine derivative with antianxiety and antidepressant-like effects: Putative role of serotonergic system.

The piperazine derivatives correspond to an extensive chemical class of compounds with numerous neuropharmacological activities, including antidepressant (e.g., nefazodone, trazodone) and anxiolytic (e.g., buspirone) properties. Therefore, aiming to identify a new antidepressant and antianxiety lead-compound, our group designed, synthesized, and investigated the effects of a new piperazine compound, namely, LQFM104, on the behavior of mice. Male albino Swiss mice were treated with LQFM104 prior to predictive behavioral tests as open field (OFT), elevated plus maze (EPM), forced swimming (FST), and tail suspension tests (TST). The participation of the serotonergic system was evaluated by pretreatment with a 5-HT1A antagonist receptor (WAY100635) and serotonin (5-HT) synthesis inhibitor (p-chlorphenylalanine, pCPA) before oral administration of LQFM104 and behavioral tests. The treatment with LQFM104 did not interfere with locomotor activity but revealed suggestive data of anxiolytic-like effects by the increase in the time spent in the center of the OFT. This activity was confirmed by the results obtained in the EPM, and it was abolished after pretreatment with WAY100635 and pCPA. The immobility time decreased in both the FST and TST. The antidepressant-like activity was completely abolished after WAY100635 pretreatment. Altogether, these data revealed that LQFM104 possesses anxiolytic and antidepressant-like properties in behavioral tests on mice, and these activities are possibly mediated, directly and/or indirectly, by serotonergic pathways.

The novel piperazine-containing compound LQFM018: Necroptosis cell death mechanisms, dopamine D4 receptor binding and toxicological assessment.

Piperazine is a promising scaffold for drug development due to its broad spectrum of biological activities. Based on this, the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate] was synthetized and some biological activities investigated. In this work, we described its ability to bind aminergic receptors, antiproliferative effects as well as the LQFM018 (2)-triggered cell death mechanisms, in K562 leukemic cells, by flow cytometric analyses. Furthermore, acute oral systemic toxicity and potential myelotoxicity assessments of LQFM018 (2) were carried out. LQFM018 (2) was originally obtained by molecular simplification from LASSBio579 (1), an analogue compound of clozapine, with 33% of global yield. Binding profile assay to aminergic receptors showed that LQFM018 (2) has affinity for the dopamine D4 receptor (Ki = 0.26 µM). Moreover, it showed cytotoxicity in K562 cells, in a concentration and time-dependent manner; IC50 values obtained were 399, 242 and 119 µM for trypan blue assay and 427, 259 and 50 µM for MTT method at 24, 48 or 72 h, respectively. This compound (427 µM) also promoted increase in LDH release and cell cycle arrest in G2/M phase. Furthermore, it triggered necrotic morphologies in K562 cells associated with intense cell membrane rupture as confirmed by Annexin V/propidium iodide double-staining. LQFM018 (2) also triggered mitochondrial disturb through loss of ΔΨm associated with increase of ROS production. No significant accumulation of cytosolic cytochrome c was verified in treated cells. Furthermore, it was verified an increase of expression of TNF-R1 and mRNA levels of CYLD with no involviment in caspase-3 and -8 activation and NF-κB in K562 cells. LQFM018 (2) showed in vitro myelotoxicity potential, but it was orally well tolerated and classified as UN GHS category 5 (LD50 > 2000-5000 mg/Kg). Thus, LQFM018 (2) seems to have a non-selective action considering hematopoietic cells. In conclusion, it is suggested LQFM018 (2) promotes cell death in K562 cells via necroptotic signaling, probably with involvement of dopamine D4 receptor. These findings open new perspectives in cancer therapy by use of necroptosis inducing agents as a strategy of reverse cancer cell chemoresistance.