Capillary electrokinetic chromatography for chiral separation of potential SARS-CoV-2 3CL protease inhibitors.

In this work, a capillary electrophoresis method was developed as a quality control tool to determine the enantiomeric purity of a series of five chiral compounds evaluated as potential severe acute respiratory syndrome coronavirus 2 3CL protease inhibitors. The first cyclodextrin tested, that is, highly sulfated-ß-cyclodextrin, at 6% (m/v) in a 25 mM phosphate buffer, using a capillary dynamically coated with polyethylene oxide, at an applied voltage of 15 kV and a temperature of 25°C, was found to successfully separate the five derivatives. The limits of detection and quantification were calculated together with the greenness score of the method in order to evaluate the method in terms of analytical and environmental performance. In addition, it is noteworthy that simultaneously high-performance liquid chromatography separation of the enantiomers of the same compounds with two different columns, the amylose tris(3,5-dimethylphenylcarbamate)-coated and the cellulose tris(3,5-dichlorophenylcarbamate)-immobilized on silica stationary phases, was studied. Neither the former stationary phase nor the latter was able to separate all derivatives in a mobile phase consisting of n-heptane/propan-2-ol 80/20 (v/v).

Evaluation of greenness and analytical performances of separative methods for chiral separation of novel lactam-based P2RX7-antagonists.

In this work, a preparative supercritical fluid chromatography (SFC) method was first developed to separate a series of chiral compounds evaluated as lactam-based P2RX7 antagonists. Subsequently, high-performance liquid chromatography, SFC, and capillary electrophoresis (CE) were comparatively investigated as QC tools to determine the enantiomeric purity of the separated isomers, including analytical performance and greenness. The screening of the best conditions was carried out in liquid and SFC on the nine derivatives and the amylose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase was found to be highly efficient. The same screening was carried out in CE and very different conditions, either in acidic or basic background electrolyte and different cyclodextrins used as chiral selectors, allowed the separation of six of the nine derivatives. 1-((3,4-Dichlorophenyl)carbamoyl)-5-oxopyrrolidine-2-carboxylic acid (compound 1) was chosen as a probe, and its semi-preparative separation by SFC and enantiomeric verification using the three techniques are presented. Its limit of detection and limit of quantification are calculated for each method. Finally, the greenness of each quality control method was evaluated.

Development of Receptor for Advanced Glycation End Products (RAGE) ligands through target directed dynamic combinatorial chemistry: a novel class of possible antagonists.

RAGE is a transmembrane receptor of immunoglobulin family that can bind various endogenous and exogenous ligands, initiating the inflammatory downstream signaling pathways, including inflammaging. Therefore, RAGE represents an attractive drug target for age-related diseases. For the development of small-molecule RAGE antagonists, we employed protein-templated dynamic combinatorial chemistry (ptDCC) using RAGE's VC1 domain as a template, the first application of this approach in the context of RAGE. The affinities of DCC hits were validated using microscale thermophoresis. Subsequent screening against AGE2 (glyceraldehyde-modified AGE)-sRAGE (solubleRAGE) (AGE2-BSA/sRAGE) interaction using ELISA tests led to the identification of antagonists with micromolar potency. Our findings not only demonstrate the successful application of ptDCC on RAGE but also highlight its potential to address the pressing need for alternative strategies for the development of small-molecule RAGE antagonists, an area of research that has experienced a slowdown in recent years.

Electrophoretic separation of multiple chiral center analyte with a three cyclodextrins mixture.

A capillary electrokinetic chromatography method (CEKC) was developed for complete stereoisomeric separation of a neutral, hydrophobic, multiple chiral center dihydropyridone analogue, a drug candidate proposed in type 2 diabetes treatment. A background electrolyte comprising three cyclodextrins was found to successfully separate the eight isomers. First an anionic cyclodextrin, the SBE-ß-CD, was selected to allow the chiral separation of our neutral compound and partial resolutions of the eight isomers were obtained. Then, the effects of different parameters such as the nature and concentration of the other cyclodextrins added and pH of the buffer were examined. Finally, a triple CD-system consisted of 15 mM SBE-ß-CD plus 15 mM Î³-CD and 40 mM HP-γ-CD in a 50 mM borate background electrolyte at pH 10, was found to successfully separate the eight isomers. Last, the selectivity and limits of detection and quantification were evaluated for this optimized method.

γ-Lactam-Based Antifungal Compounds against the Wheat Pathogen Zymoseptoria tritici.

As new environmentally friendly and effective antifungal agents are deeply needed, efficient ecofriendly strategies were designed to access two series of compounds inspired from natural γ-lactams. Designed compounds were fully characterized and evaluated as antifungal candidates against Zymoseptoria tritici, the main pathogen on wheat crops. The targeted derivatives were prepared from natural resources using green solvents, simple procedures, and limited purification steps. These bio-inspired compounds revealed as good candidates for further development of efficient crop protection products. Indeed, the HIT compounds exhibited IC50 around 1 µg/mL and were more active than the references tebuconazole and bixafen towards some multidrug-resistant strains. Two dozen of derivatives have been obtained for each series and allowed to establish early structure-activity relationships useful for the development of next generation of γ-lactam derivatives with improved efficacy.

Design, synthesis and evaluation of hydrazine and acyl hydrazone derivatives of 5-pyrrolidin-2-one as antifungal agents.

Twenty-eight 5-pyrrolidine-2-ones decorated by hydrazine or acyl hydrazones groups have been designed, synthesized and evaluated as antifungal agents on a panel of twelve fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. Half of the target compounds exhibited good to high antifungal activities on at least one strain with MIC50 lower than the control antifungal agent - hymexazol or ketoconazole. 5-Arylhydrazino-pyrrolidin-2-ones were found active and the -NH-NH- linker proved to be essential to maintain the antifungal potential. Compound 2a is a broad-spectrum antifungal, active on 60% of the tested strains. Replacing the hydrazine linker by an acylhydrazone one narrowed the spectrum of activity but pyroglutamylaryl hydrazones, mainly aromatic ones, exhibited good activity, adequate "fungicide-like" properties and were devoted of cytotoxicity.

Development and validation of a reversed-phase HPLC method for the quantification of paclitaxel in different PLGA nanocarriers.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantification of paclitaxel encapsulated in biodegradable poly(lactic-co-glycolic) (PLGA) copolymer nanoparticles. This simple (isocratic mode, without additive) and rapid (retention time of the paclitaxel under 4 min) methodology permits the detection of low quantities of paclitaxel in nanoparticulate formulations and the determination of the encapsulation efficiency (EE). Analysis was achieved on an octadecyl stationary phase. The isocratic mobile phase consisted of acetonitrile:water 80:20 (v/v) (flow rate = 0.8 mL/min). Stability of free paclitaxel was preliminary studied in those chromatographic conditions. The calibration curve was linear in the concentration range of 2-10 µg/mL (R2  = 0.9994). The method was specific with valuable trueness, repeatability (intra-day precision) and intermediate precision (inter-day precision) based on relative standard deviation (RSD) values (less than 2%). The limits of detection (LOD) and quantification (LOQ) were 0.56 and 1.85 ng/mL, respectively. This developed method was successfully employed for quantifying paclitaxel in PLGA 50:50 co-polymer nanoparticles. The accurate knowledge of the encapsulated paclitaxel concentration is essential to define the quantities of PLGA nanoparticles necessary to achieve the in vitro cell viability study.

Antagonists of the P2X7 receptor: mechanism of enantioselective recognition using highly sulfated and sulfobutylether cyclodextrins by capillary electrokinetic chromatography.

This work concerns the successful enantiomeric separation of pyroglutamic acid derivatives, known to be P2X7 receptor antagonists, achieved by electrokinetic chromatography. After a broad screening, two negatively charged cyclodextrins, sulfobutylether-ß-cyclodextrin (SBE-ß-CD), and highly sulfated-γ-cyclodextrin (HS-γ-CD) were chosen as stereoselective agents to cooperate with the BGE for complexation. A fused silica capillary coated with polyethylene oxide, filled with a phosphate buffer (25 mM, pH 2.5) containing various concentrations of CD, was used. Assuming a 1:1 stoichiometry, calculations of the binding constants, employing the three different linearization plots, were performed from the corrected electrophoretic mobilities values of the enantiomers, at different concentrations of SBE-ß-CD and HS-γ-CD in the BGE. The highest complexation was found with the SBE-ß-CD. Among the three equations, results showed better linearity (R(2) > 0.99) using the y-reciprocal fit. This plotting method was then performed to determine the binding constants of each enantiomer at different temperature for compounds 1 and 2 with SBE-ß-CD and HS-γ-CD in order to access to the thermodynamic parameters of the eight complexes. The linearity of the Van't Hoff plot, in the range of 288-303 K leading to negative enthalpy values, showed that the complexation phenomenon is enthalpically controlled and thermodynamically favored.

Supercritical fluid chromatography for separation of chiral planar metallocenes.

Unsymmetrically disubstituted metallocene derivatives, characterized as the first sandwich structure, have found interest in asymmetrical synthesis and in medicinal chemistry as well. Besides, they present a particular case of chirality. Twenty original and six commercially available molecules presenting either i) a planar chirality or ii) an asymmetrical carbon containing group or iii) being symmetrically substituted were analyzed in supercritical fluid chromatography on eleven polysaccharide-based chiral stationary phases with carbon dioxide containing 30% of methanol or 2-propanol as a co-solvent mobile phase. A basic additive, either diethylamine, triethylamine or n-butylamine was also required at 1% to the co-solvent for elution. While some of the tested chiral stationary phases provided enantioseparation for the racemates, chlorinated cellulosic phases proved to be both highly retentive and highly enantioselective towards these particular species with the highest rate of success compared to their non-chlorinated counterparts. For instance, the resolution value was equal to 14.1 for one ferrocene derivative in one-hour analysis time on cellulose tris(3,5-dichlorophenylcarbamate) column with 30% 2-propanol/1% n-butylamine while a single peak was observed under the same conditions on cellulose tris(3,5-dimethylphenylcarbamate) column. Experimental parameters were arbitrarily set at 150 bar outlet pressure, 40 °C temperature and 3 mL/min flow-rate.

Separation of planar chiral ferrocenes by capillary electrokinetic chromatography and liquid chromatography.

Capillary electrokinetic chromatography (CEKC) and liquid chromatography (LC) methods were explored for the enantiomeric separation of six unsymmetrically disubstituted ferrocene derivatives. In normal-phase mode liquid chromatography, the use of n-heptane, ethanol or isopropanol with 1% n-butylamine as mobile phase on six polysaccharide-based columns, allowed to fully separate the enantiomers of three compounds out of the six (i.e 7-chloro-N-(2-((dimethylamino)methyl)ferrocenyl)quinolin-4-amine (ferroquine) (compound 1), 1-[(1S)-(1-Aminoethyl)]-2-(diphenylphosphino)ferrocene (compound 5) and 1-[(1R)-1-(Dicyclohexylphosphino)ethyl]-2-(diphenylphosphino)ferrocene (compound 6). Among the columns used, the Lux i-Cellulose-5 was the most effective. In capillary electrokinetic chromatography, a phosphate buffer of 25 mM concentration and pH equal to 2.5 was chosen as background electrolyte, leading to cationic ferrocene derivatives. The addition of neutral cyclodextrins was undertaken first and native ß- or γ-cyclodextrins were found to resolve the enantiomers of two derivatives. Then, 15 mM of anionic cyclodextrins were added to the background electrolyte. The use of SBE-ß-CD, S-ß-CD or S-γ-CD have allowed the separation of the enantiomers for most of the ferrocene derivatives studied with high resolution values in short migration time. For instance, for 1-(R)-2-(Diphenylphosphino)ethyldi-tert-butylphosphine ferrocene (compound 2), the migration times were less than 2 minutes and the resolution value was equal to 3.52 in short-end mode with 15 mM S-ß-CD, at 25 kV and 25°C. Finally, a dual cyclodextrins system was tested using 15 mM of S-ß-CD plus 15 mM HP-γ-CD in the phosphate buffer. This system allowed the improved separation of two ferrocene derivatives with an unusual resolution value equal to 41.5 in long-end mode. Overall, CEKC showed better enantioseparating power of the six chiral ferrocenes studied than liquid chromatography.

Sesamol-based terpenoids as promising bio-sourced crop protection compounds against the wheat pathogen Zymoseptoria tritici.

BACKGROUND: Research into environmentally friendly alternatives to conventional plant protection products, to promote sustainable agriculture and healthy food, is strongly encouraged. RESULTS: In this context, 20 naturally occurring terpenoids and phenolic compounds were selected and evaluated in vitro as crop protection compounds against Zymoseptoria tritici, the causal agent of Septoria tritici blotch of wheat. After selection of the most active compounds, some hemisynthetic modifications were conducted to modify their lipophilicity. These modifications led to the discovery of sesamol esters as promising antifungal agents, with IC50 around 10 µg/mL and a total absence of cytotoxicity against human cells. CONCLUSION: These sesamol-based derivatives should be selected for further evaluations in planta to validate their use as wheat crop protection agents. Moreover, the importance of a balanced hydrophily/lipophilicity ratio should be further studied. © 2021 Society of Chemical Industry.

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

Design, synthesis and antifungal activity of pterolactam-inspired amide Mannich bases.

Pterolactam (5-methoxypyrrolidin-2-one) is a heterocycle naturally occurring in plants. In an attempt to identify antifungal agents, a series of novel Mannich bases of amide derivated from Pterolactam have been designed, synthesized and their antifungal activities were evaluated on a panel of nine fungal strains and three non albicans candida yeasts species which have demonstrated reduced susceptibility to commonly used antifungal drugs. A third of the target compounds exhibited good to high antifungal activities on at least one strain with EC50 lower than the control antifungal agent. N,N'-aminals derivated from Pterolactam proved to be good candidates for the development of biosourced fungicides, with compound 3o being the most broader-spectrum agent, active against five strains and devoted of any cytotoxicity.

Pyroglutamide-Based P2X7 Receptor Antagonists Targeting Inflammatory Bowel Disease.

This report deals with the design, the synthesis, and the pharmacological evaluation of pyroglutamide-based P2X7 antagonists. A dozen were shown to possess improved properties, among which inhibition of YO-PRO-1/TO-PRO-3 uptake and IL1ß release upon BzATP activation of the receptor and dampening signs of DSS-induced colitis on mice, in comparison with reference antagonist GSK1370319A. Docking study and biological evaluation of synthesized compounds has highlighted new SAR, and low toxicity profiles of pyroglutamides herein described are clues for the finding of a usable h-P2X7 antagonist drug. Such a drug would raise the hope for a cure to many P2X7-dependent pathologies, including inflammatory, neurological, and immune diseases.

Ozonized low density lipoprotein (ozLDL) inhibits NF-kappaB and IRAK-1-associated signaling.

OBJECTIVE: Recent studies have provided strong evidence for the presence of ozone in atherosclerotic lesions. In addition, modification of LDL has been suggested to be involved in atherosclerosis. In the present study we wanted to investigate whether LDL exposed to ozone (ozLDL) is able to modulate the NF-kappaB system, as a paradigm for inflammatory signaling. METHODS AND RESULTS: We showed that activation of NF-kappaB by lipopolysaccharide (LPS), a prototypic inducer of innate immunity, was reversibly inhibited by ozLDL in monocytic THP-1 cells in a dose-dependent manner, whereas tumor necrosis factor (TNF) signaling was not affected. This was not attributable to a direct ozone effect or solely the presence of lipoprotein, and neither required direct contact to LPS nor was accompanied by a change in LPS binding. Comparable inhibitory effects of ozLDL were observed in human monocyte/macrophages and endothelial cells. The presence of ozLDL led to a decrease in LPS-induced IkappaB alpha proteolysis and a reduction of kappaB-dependent transcription/target-gene expression. Furthermore, ozLDL markedly lowered stimulus-induced IkappaB kinase (IKK) activity and phosphorylation/proteolysis of interleukin (IL)-1 receptor-associated kinase-1 (IRAK-1). Finally, cholesterol ozonization products were identified as effective ozLDL inhibitory compounds. CONCLUSIONS: Our study demonstrated that ozLDL inhibited NF-kappaB and IRAK-1-associated signaling which may impair immune function and promote apoptosis.

Antagonists of the adenosine A2A receptor based on a 2-arylbenzoxazole scaffold: Investigation of the C5- and C7-positions to enhance affinity.

We have recently reported a series of 2-furoyl-benzoxazoles as potential A2A adenosine receptor (A2AR) antagonists. Two hits were identified with interesting pharmacokinetic properties but were find to bind the hA2AR receptor in the micromolar-range. Herein, in order to enhance affinity toward the hA2AR, we explored the C5- and C7-position of hits 1 and 2 based on docking studies. These modifications led to compounds with nanomolar-range affinity (e.g. 6a, Ki = 40 nM) and high antagonist activity (e.g. 6a, IC50 = 70.6 nM). Selected compounds also exhibited interesting in vitro DMPK (Drug Metabolism and Pharmacokinetics) properties including high solubility and low cytotoxicity. Therefore, the benzoxazole ring appears as a highly effective scaffold for the design of new A2A antagonists.

Adipophilin increases triglyceride storage in human macrophages by stimulation of biosynthesis and inhibition of beta-oxidation.

Lipid accumulation alters macrophage biology and contributes to lipid retention within the vessel wall. In this study, we investigated the role of adipophilin on triglyceride accumulation and lipid-droplet formation in THP-1-derived macrophages (THP-1 macrophages). In the presence of acetylated low-density lipoprotein, macrophages infected with an adenovirus expressing human adipophilin showed a 31% increase in triglyceride content and a greater number of lipid droplets compared with control cells. Incubation of macrophages with very low-density lipoprotein (VLDL) dramatically increased cellular triglyceride content similarly in control and adipophilin-overexpressing cells. By itself, VLDL increased adipophilin expression, which explains the lack of effect of adipophilin overexpression on cellular triglyceride content in macrophages loaded with VLDL. The lipid-droplet content of macrophages was increased by overexpression of adipophilin and/or loading with VLDL. In contrast, inhibition of adipophilin expression using siRNA prevented lipid-droplet formation and significantly reduced intracellular triglyceride content. Using inhibitors of beta-oxidation and acyl-coenzyme A synthetase, results were obtained which suggest that adipophilin elevates cellular lipids by inhibition of beta-oxidation and stimulation of long-chain fatty acid incorporation into triglycerides. Adipophilin expression in THP-1 macrophages altered the cellular content of different lipids and enhanced the size of lipid droplets, consistent with a role for adipophilin in human foam cell formation.

Perilipin, a potential substitute for adipophilin in triglyceride storage in human macrophages.

Abnormal lipid deposition in human arteries leads to the formation of fatty streaks due to the accumulation of a large number of macrophage derived-foam cells. The formation and catabolism of intracellular lipid droplets is regulated by droplet-associated proteins. Among such proteins, the role of perilipin in human macrophages was unknown. In this study, we first showed that perilipin expression was increased during differentiation of human monocytes to macrophages. Interestingly, cellular perilipin content was unaffected by treatment of cells with OxLDL, AcLDL, VLDL or sterol esters. Moreover, its expression was not dependent on the presence of adipophilin, another lipid droplet-associated protein, since it was not affected by transfection of macrophages with siRNA-adipophilin. Perilipin overexpression in macrophages with an expression vector resulted in significant lipid droplet formation and TG accumulation and this was unaffected by decreasing adipophilin levels using siRNA. Consequently, perilipin, like adipophilin, might play an important role in the conversion of macrophages into foam cells and contribute to lesion formation. Therefore, inhibition of adipophilin might not be sufficient to prevent lesion formation as previously suggested, and perilipin inhibition might be additionally required.

Peroxisome proliferator-activated receptor-alpha activation as a mechanism of preventive neuroprotection induced by chronic fenofibrate treatment.

The treatment of ischemic strokes is limited to the prevention of cerebrovascular risk factors and to the modulation of the coagulation cascade during the acute phase. A new therapeutic strategy could be to preventively protect the brain against noxious biological reactions induced by cerebral ischemia such as oxidative stress and inflammation to minimize their neurological consequences. Here, we show that a peroxisome proliferator-activated receptor (PPAR-alpha) activator, fenofibrate, protects against cerebral injury by anti-oxidant and anti-inflammatory mechanisms. A 14 d preventive treatment with fenofibrate reduces susceptibility to stroke in apolipoprotein E-deficient mice as well as decreases cerebral infarct volume in C57BL/6 wild-type mice. The neuroprotective effect of fenofibrate is completely absent in PPAR-alpha-deficient mice, suggesting that PPAR-alpha activation is involved as a mechanism of the protection against cerebral injury. Furthermore, this neuroprotective effect appears independently of any improvement in plasma lipids or glycemia and is associated with (1) an improvement in middle cerebral artery sensitivity to endothelium-dependent relaxation unrelated to an increase in nitric oxide synthase (NOS) type III expression, (2) a decrease in cerebral oxidative stress depending on the increase in numerous antioxidant enzyme activities, and (3) the prevention of ischemia-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in cerebral vessels without any change in NOS II expression. These data demonstrate that PPAR-alpha could be a new pharmacological target to preventively reduce the deleterious neurological consequences of stroke in mice and suggest that PPAR-alpha activators could preventively decrease the severity of stroke in humans.

Adipophilin enhances lipid accumulation and prevents lipid efflux from THP-1 macrophages: potential role in atherogenesis.

OBJECTIVE: Uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors results in lipid droplets accumulation and foam cell formation. Excess lipid deposition in macrophages has been reported to modulate expression of several genes including adipophilin. In this study, we investigated the function of adipophilin in lipid accumulation and cholesterol efflux in THP-1 macrophages. METHODS AND RESULTS: Adipophilin mRNA expression was 3.5-fold higher in human atherosclerotic plaques compared with healthy areas of the same arteries. Moreover, in the presence of acetylated LDL (AcLDL), triglycerides and cholesteryl esters were increased in macrophages overexpressing adipophilin by 40% and 67%, respectively, whereas their accumulation was reduced when endogenous cellular adipophilin was depleted using siRNA approach. In addition, neither overexpression nor downregulation of adipophilin altered expression of genes involved in lipid efflux. However, the affinity and the number of AcLDL receptors were not affected. After 24-hour incubation of lipid-loaded macrophages with apolipoprotein A-I, cholesterol efflux was reduced by 47% in adipophilin transfected cells versus control cells. CONCLUSIONS: Our results showed that stimulation of adipophilin expression in macrophages by modified LDL promotes triglycerides and cholesterol storage and reduces cholesterol efflux. Therefore, adipophilin might contribute, in vivo, to lipid accumulation in the intima of the arterial wall.