Context: Translation of any chemical process from laboratory to pilot or commercial scale is by no means a simple linear process. This process is influenced by several parameters, such as temperature, agitation speed, the concentration of reactants, and their interactions. In addition, temperature and agitation speed are very related to heat and movement transfers, which are two critical unit operations that affect the scale-up process and reaction yield. A multicomponent reaction (MCR) is a process where more than three reagents react through two or more reactions, producing one biggest molecule. Its scale-up is complex considering the complex nature of the reaction pathway. Our project involves a 4-MCR for obtaining a 3,4-pyridone derivative, a very important product for obtaining a neuroprotective compound. Despite its importance in medicinal chemistry, no report about the scale-up of MCRs was found. Aims: To develop a practical way to scale up a multicomponent reaction with four components, from laboratory to pilot scales. Methods: A combination of the brute force method and dimensionless numbers was applied for the scale-up process based on the significant physical forces governing the process. Results: Experimentally, it was previously demonstrated that temperature and fluid agitation and their interaction are the most significant parameters affecting reaction yield. Its related unit operations, heat and movement transport, were selected as the critical physical forces for the scaling-up process. The dimensionless numbers related to those unit operations are the Nusselt and Reynold numbers. In addition to other criteria applied, those dimensionless numbers were used to calculate the agitation speed for the upper scale. Likewise, as the reaction product precipitated through the cooling process, the obstruction of the reactor discharge valve was observed. Thus, a critical agitation speed was determined at the laboratory scale by applying the Zwietering rule through direct observation of solid sedimentation in the reactor. Conclusions: The process showed high reproducibility at all scales, reaching good yields of around 73%. Product purity was higher than 99% for all batches. The method applied allows the successful scalability of the process to increase the scale to commercial.

This study evaluated the anti-hypernociceptive interaction between morphine and the neuroprotective compound 3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine (JM-20) in neuropathic rats. Single and repeated co-administration of both drugs was assessed using the chronic constriction injury (CCI) model, via subcutaneous and oral routes. Isobolographic analysis was performed to determine the nature of the pharmacological interaction. In addition, in silico pharmacokinetic and toxicological profiling was conducted, and JM-20's effects on human UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzyme systems were evaluated in vitro. Morphine, JM-20, and their 1:1 fixed-ratio combinations significantly attenuated mechanical hypersensitivity in a dose-dependent manner. The observed ED50 for the combination (2.15 ± 0.07 mg/kg) was significantly lower than the theoretical additive ED50 (8.05 ± 0.58 mg/kg), indicating a synergistic interaction. Repeated morphine administration led to tolerance, while the morphine:JM-20 combination maintained efficacy for 14 days and prevented morphine-induced facilitation of neuropathic hypersensitivity. JM-20 did not alter UGT activity, suggesting no interference with morphine glucuronidation or active metabolite formation. However, JM-20 strongly inhibited CYP3A4 and other major CYP isoforms (1A, 2C9, 2D6, 2A6), potentially relevant for interactions with co-administered drugs, though not with morphine. JM-20 may also inhibit P-glycoprotein, possibly enhancing morphine's central bioavailability. In silico and in vitro analyses indicated a potential risk of cardiotoxicity and hepatotoxicity. Overall, JM-20 enhances morphine analgesia in neuropathic pain, but further in vivo studies are required to evaluate the long-term safety of this combination.

JM-20 is a 1,5-benzodiazepine compound fused to a dihydropyridine fraction with different pharmacological properties. However, its potential toxic effects on blood cells have not yet been reported. Thus, the present study aimed to investigate, for the first time, the possible cytotoxicity of JM-20 through cell viability, cell cycle, morphology changes, reactive species (RS) to DCFH-DA, and lipid peroxidation in human leukocytes, its hemolytic effect on human erythrocytes, and its potential DNA genotoxicity using plasmid DNA in vitro. Furthermore, the compound's ability to reduce the DPPH radical was also measured. Human blood was obtained from healthy volunteers (30 +/- 10 years old), and the leukocytes or erythrocytes were immediately isolated and treated with different concentrations of JM-20. A cytoprotective effect was exhibited by 10 mu M JM-20 against 1 mM tert-butyl hydroperoxide (t-but-OOH) in the leukocytes. However, the highest tested concentrations of the compound (20 and 50 mu M) changed the morphology and caused a significant decrease in the cell viability of leukocytes (p < 0.05, in comparison with Control). All tested concentrations of JM-20 also resulted in a significant increase in intracellular RS as measured by DCFH-DA in these cells (p < 0.05, in comparison with Control). On the other hand, the results point out a potent antioxidant effect of JM-20, which was similar to the classical antioxidant alpha-tocopherol. The IC50 value of JM-20 against the lipid peroxidation induced by (FeII) was 1.051 mu M +/- 0.21, while the IC50 value of alpha-tocopherol in this parameter was 1.065 mu M +/- 0.34. Additionally, 50 and 100 mu M JM-20 reduced the DPPH radical in a statistically similar way to the 100 mu M alpha-tocopherol (p < 0.05, in comparison with the control). No significant hemolysis in erythrocytes, no cell cycle changes in leukocytes, and no genotoxic effects in plasmid DNA were induced by JM-20 at any tested concentration. The in silico pharmacokinetic and toxicological properties of JM-20, derivatives, and nifedipine were also studied. Here, our findings demonstrate that JM-20 and its putative metabolites exhibit similar characteristics to nifedipine, and the in vitro and in silico data support the low toxicity of JM-20 to mammals.

The present study examines the possible inhibitory effect of JM-20, a multi-target neuroprotective compound, on the development of morphine-induced hyperalgesia in Male Sprague-Dawley na & iuml;ve rats. Additionally, the impact of JM-20 on chronic constriction injury (CCI) rats under chronic morphine exposure was investigated, and its efficacy in reducing mechanical hypersensitivity and histopathological changes in the sciatic nerve was assessed. JM-20 (20 mg/kg, per os [p.o.]), administered 60 min before morphine (10 mg/kg, s.c. twice daily at 12 h intervals) for ten days, significantly inhibited the development of morphine-induced hyperalgesia assessed using an electronic pressure-meter paw test, hot-plate, and formalin test, as well as the appearance of spontaneous withdrawal somatic symptoms in rats. Furthermore, JM-20 decreases spinal pro-inflammatory interleukin-1 beta and restores glutathione to close physiological concentrations, biomarkers directly related to the intensity of mechanical hypernociception. After CCI and sham surgery, co-treatment with JM-20 (10 mg/kg, p.o.) for five days decreased morphine increased-mechanical hypersensitivity, even 12 days after its discontinuation. Continued morphine treatment imposed a neuroinflammatory challenge in CCI animals, further increasing cellularity (>75% immune cell infiltration) with lymphocytes and macrophages. However, JM-20 co-treatment still reduced the presence of cellular infiltrates (51-75%) with a predominance of lymphocytes. Even in the absence of nerve injury, JM-20 attenuated the peripheral neuroinflammatory response observed in morphine-treated sham-operated animals (0% vs. 1-25%). These findings suggest that JM-20 could prevent morphine-induced hyperalgesia by anti-inflammatory and antioxidant mechanisms.

Studies showed that JM-20, a benzodiazepine-dihydropyridine hybrid molecule, protects against rotenone and 6-hydroxydopamine neurotoxicity. However, its protective effects against cytotoxicity induced by endogenous neurotoxins involved in Parkinson's disease (PD) pathogenesis have never been investigated. In this study, we evaluated the ability of JM-20 to inhibit alpha-synuclein (aSyn) aggregation. We also evaluated the interactions of JM-20 with aSyn by molecular docking and molecular dynamics and assessed the protective effect of JM-20 against aminochrome cytotoxicity. We demonstrated that JM-20 induced the formation of heterogeneous amyloid fibrils, which were innocuous to primary cultures of mesencephalic cells. Moreover, JM-20 reduced the average size of aSyn positive inclusions in H4 cells transfected with SynT wild-type and synphilin-1-V5, but not in HEK cells transfected with synphilin-1-GFP. In silico studies showed the interaction between JM-20 and the aSyn-binding site. Additionally, we showed that JM-20 protects SH-SY5Y cells against aminochrome cytotoxicity. These results reinforce the potential of JM-20 as a neuroprotective compound for PD and suggest aSyn as a molecular target for JM-20.

Along with the discovery of new candidate molecules for pharmaceuticals, several studies have emerged showing different mechanisms of action and toxicological aspects. 3-ethoxycarbonyl-2-methyl-4- (2-nitrophenyl)4,11-dihydro-1 H-pyrido [2,3-b] [1,5] benzodiazepine (JM-20) is a hybrid molecule. It is derived from 1,5-benzodi-azepines and structurally differentiated by the addition of 1,4-dihydropyridine bonded to the benzodiazepine ring. This gives this molecule potential neuroprotective, antioxidant, and anxiolytic activity. As this is a prom-ising multi-target molecule, further studies are necessary to improve the knowledge about its mechanism of action. In our study, we used Caenorhabditis elegans (C. elegans) to investigate the effects of chronic treatment with JM-20. Nematodes from the wild-type strain (N2) were treated chronically at different concentrations of JM-20. Our results show that JM-20 does not cause mortality, but higher concentrations can delay the devel-opment of worms after 48 h exposure. We assessed basic behaviors in the worm, and our data demonstrate decreased defecation cycle. Our results suggest that JM-20 acts on the C. elegans GABAergic system because GABA neurotransmission is associated with the worm intestine. We also observed increased locomotor activity and decreased egg-laying after JM-20 treatment. When both behaviors were evaluated in mutants with have reduced levels of GABA (unc-25), this effect is no observed, suggesting the GABAergic modulation. Still, the JM -20 exert similar effect of Diazepam in basic behaviors observed. To reinforce neuromodulatory action, compu-tational analysis was performed, and results showed a JM-20 binding on allosteric sites of nematodes GABA receptors. Overall, this work provided a better understanding of the effects of JM-20 in C. elegans as well as showed the effects of this new molecule on the GABAergic system in this animal model.

Context: JM-20 is a hybrid synthetic molecule, which is based on a multimodal drug design paradigm for cerebrovascular disease. In addition to its neuroprotective effects, JM-20 also decreased sciatic nerve chronic constriction injury (CCI)-induced mechanical hypersensitivity in rats. JM-20 has a strong mitoprotective ability, and its effects could be in correspondence with the mitotoxicity hypothesis for paclitaxel-induced painful peripheral neuropathy. Aims: To evaluate the efficacy of the JM-20 to reduce neuropathic pain manifestations induced by the administration of paclitaxel in rats. Methods: In this study was implemented a rat model of painful peripheral neuropathy, produced by the chemotherapeutic agent paclitaxel, to determine whether JM-20 (10 mg/kg, p.o) could prevent the development of neuropathic pain during the exposure to paclitaxel. As well as to determine whether JM-20 (20 mg/kg, p.o) could reverse the established neuropathic pain. Mechanical behavioral assessment using von Frey filaments applied to the hind paws was applied before, during, and after treatments for 35 days. Results: Giving JM-20 during the exposure to paclitaxel significantly reduced the severity of mechanical allodynia and mechanical hyperalgesia. Moreover, JM-20 significantly reduced both established neuropathic pain manifestations. There was no evidence of tolerance to the effect during three days of dosing, and a long-term effect was observed after JM-20 discontinuation. Conclusions: JM-20 may be clinically relevant for both the prevention and treatment of paclitaxel-induced painful peripheral neuropathy.

Cerebral ischemia constitutes the most frequent type of cerebrovascular disease. The reduction of blood supply to the brain initiates the ischemic cascade starting from ionic imbalance to subsequent glutamate excitotoxicity, neuroinflammation and oxidative stress, eventually causing neuronal death. Previously, the authors have demonstrated the in vitro cytoprotective and antioxidant effects of a new arylidene malonate derivative, KM-34, against oxidizing agents like hydrogen peroxide, glutamate or Fe3+/ascorbate. Here, we examined for the first time the neuroprotective effect of KM-34 on ischemia/reperfusion models. In vitro, treatment with 10 and 50 μM KM-34 reduced the cellular death (propidium iodide incorporation) induced by oxygen glucose deprivation (OGD) in rat organotypic hippocampal slices cultures. In vivo, stroke was induced in male Wistar rats through middle cerebral artery occlusion (MCAO), followed by 23 h of reperfusion. KM-34 was orally administered 105 min after MCAO onset. We noticed that 1 mg/kg KM-34 reduced infarct volume and neurological score, and increased the latency to fall in the Hanging Wire test compared to vehicle-treated ischemic animals. While ischemic and sham-operated groups showed similar horizontal locomotor activity, vertical counts decreased after MCAO, suggesting that vertical movements are more sensitive to the ischemic injury. Treatment with KM-34 also alleviated the mitochondrial impairment (ROS generation, swelling and membrane potential dissipation) induced by transient MCAO but not significant alterations were found in oxidative stress parameters. Overall, the study provides preclinical evidences confirming the neuroprotective effects of a novel synthetic molecule and paved the way for future investigations regarding its therapeutic potential against brain ischemia/reperfusion injury.

We have previously shown that JM-20, a new chemical entity consisting of 1,5-benzodiazepine fused to a dihydropyridine moiety, protects against rotenone-induced neurotoxicity in an experimental model of Parkinson's disease (PD). The aim of this study was to investigate the effect of a novel hybrid molecule, named JM-20, in in vitro and in vivo models of PD induced by 6-hydroxydopamine (6-OHDA). PC-12 cells were exposed to 6-OHDA and treated with JM-20. Protection against mitochondrial damage induced by 6-OHDA was also investigated using isolated rat brain mitochondria. We found that JM-20 protected PC-12 cells against cytotoxicity induced by 6-OHDA and inhibited hydrogen peroxide generation, mitochondrial swelling and membrane potential dissipation. For in vivo experiments, adult male Wistar rats were lesioned in the substantia nigra pars compacta (SNpc) by 6-OHDA administration. JM-20 was orally administered (10, 20 or 40 mg/kg), intragastric via gavage, 24 h after surgery and daily for seven days. Treatment with JM-20 significantly reduced the percentage of motor asymmetry and increased vertical exploration. It improved the redox state of the SNpc and the striatal tissue of these animals. Also, JM-20 reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in SNpc. Altogether, these results demonstrate that JM-20 is a potential neuroprotective agent against 6-OHDA-induced damage in both in vitro and in vivo models. The mechanism underlying JM-20 neuroprotection against 6-OHDA appears to be associated with the control of oxidative injury and mitochondrial impairment.

The present study examines the possible effect of the novel hybrid molecule JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-411-dihydro-1H-pyrido[2,3-b] [1,5] benzodiazepine) on pain-related behaviours in a persistent pain model (5% formalin test) and in the neutrophil migration events during the inflammatory process. It further introduces JM-20 in a chronic constriction injury (CCI) model to clarify the possible subjacent mechanisms with its consequent clinical relevance. A single administration of JM-20 (20 or 40 mg/kg, per os [p.o.]) decreased licking/biting exclusively in the tonic phase of the formalin test in a GABA/benzodiazepine (BZD) receptor antagonist flumazenil-sensitive manner. JM-20 reduced in vivo neutrophil migration, rolling and adhesion to the endothelium induced by intraperitoneal administration of carrageenan in mice. In addition, plasma extravasation and tumour necrosis factor alpha production in the peritoneal fluid were decreased. Treatment with JM-20 (20 mg/kg, p.o.) for 7 days after CCI reduced mechanical hypersensitivity in a NG-monomethyl-l-arginine (L-NMMA)/methylene blue/glibenclamide-sensitive manner. Histopathological signs of Wallerian degeneration (WD) of the sciatic nerve were also attenuated, as well as interleukin-1 beta release in the spinal cord. The nitrate/nitrite concentration was increased centrally and did not show differences at the peripheral nerve level. The findings of this study suggest JM-20 can decrease persistent pain. A transient activity of its BDZ portion on nociceptive pathways mediated by GABA/BDZ receptors in association with its anti-inflammatory properties could be at least partially involved in this effect. JM-20 decreased CCI-induced mechanical hypersensitivity via the l-arginine/nitric oxide (NO)/cyclic GMP-sensitive ATP-sensitive potassium channel pathway. Its neuroprotective ability by preventing WD could be implicated in its anti-neuropathic mechanisms.

Context: Drug solubility is one of the most important physicochemical properties for the development of new pharmaceutical products, which, in our study, is a new synthetic molecule, which has been shown to have a potential neuroprotective effect in rat. Aims: To evaluate the equilibrium solubility of JM-20 within the physiological pH range and aqueous chemical stability. Methods: It was performed at pH 1.2, 4.5 and 6.8 at 37 ± 0.5°C for 27 h by the method of the saturated flasks. HPLC and FTIR were used for the study of aqueous stability and to evaluate the molecular structure of the non-ionized fraction by DSC/TGA. Results: Concentrations calculated at pH 1.2 were higher than at pH 4.5 and pH 6.8. A retention time greater than pH 4.5 and 6.8 after 24 h was detected in the aqueous stability study at pH 1.2, in addition to other peaks in these chromatograms. Analyses by FT-IR and DSC/TGA demonstrated the absence of polymorphism in the dissociated fraction of saturated solutions. Conclusions: The JM-20 presented at 6 hours its maximum steady state of solubility, being inferior to pH 4.5 and 6.8. It is corroborated by the qualitative analysis by HPLC, that the compound is chemically unstable from 24 h to pH 4.5 and 6.8. By FT-IR and DSC/TG it was elucidated that the fractions of the molecule not dissociated, did not undergo structural changes in it, and it is inferred that there is no polymorphism.

La solubilidad en farmacos es una de las propiedades fisicoquimicos mas importantes para el desarrollo de nuevos productos farmaceuticos que, en nuestro estudio, se trata de una nueva molecula sintetica que ha demostrado tener un efecto neuroprotector potencial en ratas. Objetivos: Evaluar la solubilidad de equilibrio de JM-20 dentro del rango de pH fisiologico y la estabilidad quimica acuosa. Metodos: Se realizo a pH 1,2; 4,5 y 6,8 a 37 ± 0,5°C por 27 h por el metodo de los matraces saturados. Para el estudio de la estabilidad acuosa se empleo HPLC y FTIR y para evaluar la estructura molecular de la fraccion no ionizada DSC/TGA. Resultados: Las concentraciones calculadas a pH 1,2 fueron mayores que a pH 4,5 y pH 6,8. En el estudio de estabilidad acuosa a pH 1.2 se detecto un tiempo de retencion mayor que a pH 4,5 y 6,8 despues de las 24 h, ademas de detectarse otros picos en dichos cromatogramas. Los analisis por FT-IR y DSC/TGA demostraron la ausencia de polimorfismo en las fracciones no disociadas de las soluciones saturadas. Conclusiones: El JM-20 presento a las 6 horas su maximo estado estacionario de solubilidad, siendo inferiores a pH 4,5 y 6,8. Se corroboro por el analisis cualitativo por HPLC, que la molecula es inestable quimicamente a partir de las 24 h a pH 4,5 y 6,8. Por FT-IR y DSC/TG se elucido que las fracciones de la molecula no disociada no sufrieron cambios estructurales en ella, y se infiere que no hay polimorfismo

The main function of AChE is the hydrolysis of the neurotransmitter acetylcholine (ACh) at the neuromuscular and in cholinergic brain synapses. In some pathologies, loss of cholinergic neurons may be associated with a deficiency of ACh in specific brain areas. Consequently, the study of new safe drugs that inhibit AChE is important, because they can increase ACh levels in the synaptic cleft without adverse effects. Here, we evaluated the effects of JM-20 (a benzodiazepine-dihydropyridine hybrid molecule) on cholinesterase (ChE) activities from distinct sources (AChE from Electrophorus electricus (EeAChE), human erythrocyte membranes (HsAChE (ghost)), total erythrocyte (HsAChE (erythrocyte)) and BChE from plasma (HsBChE) and purified enzyme from the horse ( EcBChE)). Kinetic parameters were determined in the presence of 0.05-1.6 mM of substrate concentration. The interactions ChEs with JM-20 were performed using molecular docking simulations. JM-20 inhibited all tested AChE but not BChE. The IC50 values were 123 nM +/- 0.2 (EeAChE), 158 nM +/- 0.1 (ghost HsAChE), and 172 nM +/- 0.2 (erythrocytic HsAChE). JM-20 caused a mixed type of inhibition (it altered Km and V-max of AChE). The molecular docking indicated the binding poses and the most plausible active isomer of JM-20. Besides giving important data for future drug design, our results help us understand the mode of action of JM-20 as a specific inhibitor of AChE enzymes. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Resumen Context: Drug solubility is one of the most important physicochemical properties for the development of new pharmaceutical products, which, in our study, is a new synthetic molecule, which has been shown to have a potential neuroprotective effect in rat. Aims: To evaluate the equilibrium solubility of JM-20 within the physiological pH range and aqueous chemical stability. Methods: It was performed at pH 1.2, 4.5 and 6.8 at 37 ± 0.5°C for 27 h by the method of the saturated flasks. HPLC and FTIR were used for the study of aqueous stability and to evaluate the molecular structure of the non-ionized fraction by DSC/TGA. Results: Concentrations calculated at pH 1.2 were higher than at pH 4.5 and pH 6.8. A retention time greater than pH 4.5 and 6.8 after 24 h was detected in the aqueous stability study at pH 1.2, in addition to other peaks in these chromatograms. Analyses by FT-IR and DSC/TGA demonstrated the absence of polymorphism in the dissociated fraction of saturated solutions. Conclusions: The JM-20 presented at 6 hours its maximum steady state of solubility, being inferior to pH 4.5 and 6.8. It is corroborated by the qualitative analysis by HPLC, that the compound is chemically unstable from 24 h to pH 4.5 and 6.8. By FT-IR and DSC/TG it was elucidated that the fractions of the molecule not dissociated, did not undergo structural changes in it, and it is inferred that there is no polymorphism. Contexto: La solubilidad en fármacos es una de las propiedades fisicoquímicos más importantes para el desarrollo de nuevos productos farmacéuticos que, en nuestro estudio, se trata de una nueva molécula sintética que ha demostrado tener un efecto neuroprotector potencial en ratas. Objetivos: Evaluar la solubilidad de equilibrio de JM-20 dentro del rango de pH fisiológico y la estabilidad química acuosa. Métodos: Se realizó a pH 1,2; 4,5 y 6,8 a 37 ± 0,5°C por 27 h por el método de los matraces saturados. Para el estudio de la estabilidad acuosa se empleó HPLC y FTIR y para evaluar la estructura molecular de la fracción no ionizada DSC/TGA. Resultados: Las concentraciones calculadas a pH 1,2 fueron mayores que a pH 4,5 y pH 6,8. En el estudio de estabilidad acuosa a pH 1.2 se detectó un tiempo de retención mayor que a pH 4,5 y 6,8 después de las 24 h, además de detectarse otros picos en dichos cromatogramas. Los análisis por FT-IR y DSC/TGA demostraron la ausencia de polimorfismo en las fracciones no disociadas de las soluciones saturadas. Conclusiones: El JM-20 presentó a las 6 horas su máximo estado estacionario de solubilidad, siendo inferiores a pH 4,5 y 6,8. Se corroboró por el análisis cualitativo por HPLC, que la molécula es inestable químicamente a partir de las 24 h a pH 4,5 y 6,8. Por FT-IR y DSC/TG se elucidó que las fracciones de la molécula no disociada no sufrieron cambios estructurales en ella, y se infiere que no hay polimorfismo.

Stroke is frequently associated with severe neurological decline and mortality, and its incidence is expected to increase due to aging population. The only available pharmacological treatment for cerebral ischemia is thrombolysis, with narrow therapeutic windows. Efforts aimed to identify new therapeutics are crucial. In this study, we look into plausible molecular and cellular targets for JM-20, a new hybrid molecule, against ischemic stroke in vivo. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) following 23 h of reperfusion. Animals treated with 8 mg/kg JM-20 (p.o., 1 h after reperfusion) showed minimal neurological impairment and lower GABA and IL-1β levels in CSF when compared to damaged rats that received vehicle. Immunocontent of pro-survival, phosphorylated Akt protein decreased in the cortex after 24 h as result of the ischemic insult, accompanied by decreased number of NeuN+ cells in the peri-infarct cortex, cornu ammonis 1 (CA1) and dentate gyrus (DG) areas. Widespread reactive astrogliosis in both cortex and hippocampus (CA1, CA3, and DG areas) was observed 24 h post-ischemia. JM-20 prevented the activated Akt reduction, neuronal death, and astrocytes reactivity throughout the brain. Overall, the results reinforce the pharmacological potential of JM-20 as neuroprotective agent and provide important evidences about its molecular and cellular targets in this model of cerebral ischemia.

The etiology of Parkinson's disease is not completely understood and is believed to be multifactorial. Neuronal disorders associated to oxidative stress and mitochondrial dysfunction are widely considered major consequences. The aim of this study was to investigate the effect of the synthetic arylidenmalonate derivative 5-(3,4-dihydroxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (KM-34), in oxidative stress and mitochondrial dysfunction induced by 6-hydroxydopamine (6-OHDA). Pretreatment (2 h) with KM-34 (1 and 10 μM) markedly attenuated 6-OHDA-induced PC12 cell death in a concentration-dependent manner. KM-34 also inhibited H2O2 generation, mitochondrial swelling, and membrane potential dissipation after 6-OHDA-induced mitochondrial damage. In vivo, KM-34 treatment (1 and 2 mg/Kg) reduced percentage of asymmetry (cylinder test) and increased the vertical exploration (open field) with respect to untreated injured animals; KM-34 also reduced glial fibrillary acidic protein overexpression and increased tyrosine hydroxylase-positive cell number, both in substantia nigra pars compacta. These results demonstrate that KM-34 present biological effects associated to mitoprotection and neuroprotection in vitro, moreover, glial response and neuroprotection in SNpc in vivo. We suggest that KM-34 could be a putative neuroprotective agent for inhibiting the progressive neurodegenerative disease associated to oxidative stress and mitochondrial dysfunction.

Several 1,4-dihydropyridine derivatives overcome the multidrug resistance in tumors, but their intrinsic cytotoxic mechanisms remain unclear. Here we addressed if mitochondria are involved in the cytotoxicity of the novel 1,4-dihydropyridine derivative VE-3N [ethyl 6-chloro-5-formyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate] towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In HepG2 cells, VE-3N induced mitochondrial membrane potential dissipation, ATP depletion, annexin V/propidium iodide double labeling, and Hoechst staining; events indicating apoptosis induction. In isolated rat liver mitochondria, VE-3N promoted mitochondrial uncoupling by exerting protonophoric actions and by increasing membrane fluidity. Mitochondrial uncoupling was evidenced by an increase in resting respiration, dissipation of mitochondrial membrane potential, inhibition of Ca2+ uptake, stimulation of Ca2+ release, decrease in ATP synthesis, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. Furthermore, uncoupling concentrations of VE-3N in the presence of Ca2+ plus ruthenium red induced the mitochondrial permeability transition process. These results indicate that mitochondrial uncoupling is potentially involved in the VE-3N cytotoxic actions towards HepG2 cells. Considering that hepatocellular carcinoma is the most common form of liver cancer, our findings may open a new avenue for the development of VE-3N-based cancer therapies, and help to unravel the cytotoxic mechanisms of 1,4-dihydropyridines towards cancer cells.

Ischemic stroke is a major cause of death and disability worldwide. Thrombolysis by tissue plasminogen activator is the only pharmacological treatment approved for clinical practice, but has a narrow therapeutic window and poor efficacy when the cell death cascade is activated. Numerous drugs that are thought to protect neurons against injury have previously failed in human trials despite showing efficacy in experimental models of stroke. Herein, we reviewed the main pre-clinical results of the neuroprotective effects of JM-20, a new hybrid molecule, against brain ischemia. JM-20 appears to protect the brain from ischemic damage by interfering with several elements of the ischemic cascade: antiexcitotoxic, anticalcic, antioxidant, antiapoptotic, and anti-inflammatory. Its ability to protect not only neurons but also glial cells together with its ability to target and preserve mitochondrial function makes JM-20 a promising molecule that may be able to shield the whole neurovascular unit. The multimodal and multi-cell action of JM-20 may explain the high degree of protection observed in a rat model of brain ischemia, as assayed through histological (hematoxylin-eosin, and luxol fast blue staining), neurochemical (glutamate and aspartate levels in cerebrospinal fluid), mitochondrial functionality and behavioural (neurological scale) analysis at doses of 4 and 8mg/kg. Furthermore, the wide therapeutic window of JM-20 of 8h also suggests that this molecule could be of potential interest in situations where brain perfusion is compromised.

This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.

Abstract Free radicals are important mediators in a number of neurodegenerative diseases and molecules capable of scavenging reactive oxygen species (ROS) may be a feasible strategy for protecting neuronal cells. In this sense, polyphenols have been studied for their antioxidant effects, KM-34 (5-(3, 4-dydroxyl-benzylidene)-2, 2-dimethyl-1, 3-dioxane-4, 6-Dione) is a novel synthetic catechol with potential neuroprotective and antioxidant properties. We have assessed the antioxidant (as scavenging and iron-chelating compound) and neuroprotectant in vitro (in PC12 cell injury induced by H2O2, glutamate or FeSO4/AA) of KM-34. KM-34 was found to be a potent antioxidant, as shown by (i) inhibition of iron induced-brain lipid peroxidation, (ii) inhibition of 2-deoxyribose degradation, (iii) inhibition of superoxide radicals generation (IC50=11.04 μM) and (iv) inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reduction (IC50=16.26 μM). The overall anti-oxidant action of KM-34 appears to be a combination of a direct reaction with free radicals and chelating the metal ions responsible for the production of ROS. Our work suggests that the antioxidant properties of KM-34 may provide future therapeutic approaches for neurodegenerative disorders.