Synthesis and antioxidant activity evaluation of new hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones obtained by Biginelli reaction in two steps.

New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-carboxylates, previously prepared by Biginelli reaction using appropriate aldehyde, urea derivatives and ethyl acetoacetate. Their antioxidant properties were evaluated by two methods: scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and scavenging effect on hydroxyl radicals. The results show that the compounds containing thiourea moiety have better activity.

Immobilization of arginase and its application in an enzymatic chromatographic column: thermodynamic studies of nor-NOHA/arginase binding and role of the reactive histidine residue.

A biochromatographic approach is developed to measure for the first time changes in enthalpy, heat capacity change and protonation for the binding of nor-NOHA to arginase in a wide temperature range. For this, the arginase enzyme was immobilized on a chromatographic support. It was established that this novel arginase column was stable during an extended period of time. The affinity of nor-NOHA to arginase is high and changes slightly with the pH, because the number of protons linked to binding is low. The determination of the enthalpy change at different pH values suggested that the protonated group in the nor-NOHA-arginase complex exhibits a heat protonation of approximately -33 kJ/mol. This value agrees with the protonation of an imidazole group. Our result confirmed that active-site residue Hist 141 is protonated as imidazolium cation. Hist 141 can function as a general acid to protonate the leaving amino group of L-ornithine during catalysis. The thermodynamic data showed that nor-NOHA-arginase binding, for low temperature (<15 degrees C), is enthalpically unfavourable and being dominated by a positive entropy change. This result suggests that dehydration at the binding interface and charge-charge interactions contribute to the nor-NOHA-arginase complex formation. The temperature dependence of the free energy of binding is weak because of the enthalpy-entropy compensation caused by a large heat capacity change, DeltaC(p)=-2.43 kJ/mol/K, of arginase. Above 15 degrees C, the thermodynamic data DeltaH and DeltaS became negative due to van der Waals interactions and hydrogen bonding which are engaged at the complex interface confirming strong enzyme-inhibitor hydrogen bond networks. As well, by the use of these thermodynamic data and known correlations it was clearly demonstrated that the binding of nor-NOHA to arginase produces slight conformational changes in the vicinity of the active site. Our work indicated that our biochromatographic approach could soon become very attractive for studying other enzyme-ligand binding.

Effect of metals on herbicides-alpha-synuclein association: a possible factor in neurodegenerative disease studied by capillary electrophoresis.

The aggregation of alpha-synuclein in the dopaminergic neurons of the substantia nigra is a critical step in the Parkinson's disease (PD). The etiology of the disease is unknown but recent epidemiological and experimental studies have renewed interest in the hypothesis that environmental factors, especially herbicides and metals, have a role on the pathogenesis of PD. For the first time, the association constants of alpha-synuclein with five herbicides have been calculated using a capillary electrophoresis (CE) method. In addition, the effect of a number of metals on this binding has been investigated. It appears that the herbicides preferentially bind to a partially folded intermediate conformation of alpha-synuclein induced by manganese, aluminium, cadmium, copper and zinc. Then, metal increases the synuclein-herbicide association. However, this study shows contrasting actions with the antibiotic rifampicin and magnesium addition leading to a decrease of the alpha-synuclein-herbicide interaction even if other metals are present in the bulk solvent. Considering epidemiological studies, all these results suggest an underlying molecular basis for PD and related body diseases.

Reanalysis of the testosterone displacement from its HSA binding site by DHEA using competitive Langmuir isotherm.

In a previous manuscript [C. Andre, A. Berthelot, J.F. Robert, M. Thomassin, Y.C. Guillaume, J. Pharm. Bio. Med., in press], the Mg(2+) effect on the testosterone displacement equilibrium from its human serum albumin (HSA) binding site by DHEA was investigated using a thermodynamic approach. In this paper, a novel concept based on the competitive Langmuir distribution isotherms was proposed to calculate the association constant of the HSA-hormone binding and to confirm the Mg(2+) role on the testosterone displacement equilibrium from its HSA binding site by DHEA. Thus, both the HSA-hormone binding and the displacement equilibrium processes were reanalysed. The results obtained confirmed that:

Synthesis of 4-hydroxycoumarin and 2,4-quinolinediol derivatives and evaluation of their effects on the viability of HepG2 cells and human hepatocytes culture.

We report here the synthesis of aromatic coumarins and aromatic alpha-quinolones which were evaluated in vitro for their protective potentialities against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage on human liver cell death, i.e., human hepatoma HepG2 cell line and human hepatocytes in primary culture. We found that the presence of a benzylidene at the 3-position or a heterocycle with N and S heteroatoms on the benzopyrone or quinolone system was essential for the protective effect of these compounds against t-BHP-induced decrease in viability of cells. We found also that a methoxy group on the aromatic ring systems decreased this potential. t-BHP-induced cytotoxicity in primary cultures of human hepatocytes could be therefore prevented by these compounds suggesting that they could display hepatoprotective effects in humans.

Testimony of the correlation between DHEA and bioavailable testosterone using a biochromatographic concept: effect of two salts.

In a previous paper (C. André et al., submitted to J. Chromatogr. B) a mathematical model based on the Langmuir theory was developed to visualize the competition effect between testosterone and deshydroepiandrosterone (DHEA) for their identical human serum albumin (HSA) binding cavity. In this work, the thermodynamic mechanisms of (i) the binding of two hormones, DHEA and testosterone to HSA and (ii) the testosterone displacement of its HSA binding cavity by DHEA was studied by biochromatography. The Na+ cation effect used as physico-chemical marker of these binding processes was clearly described. The Gibbs free energy value (DeltaGo ) of the displacement equilibrium was always negative demonstrating that DHEA well displaced testosterone of its HSA binding cavity. The thermodynamic data also showed that this displacement equilibrium was enthalpically controlled. Moreover, the effect of (Mg2+) concentration (x') on the two binding mechanisms was analyzed. It appeared that for old men with a deficit of testosterone, Mg(2+) supplementation during treatment with DHEA can increased the free testosterone concentration and its biological effect. All these results must be confirmed by in vivo test.

Concentration of sodium ion as the determining factor for the association of dansyl amino acids with the teicoplanin molecule in reversed-phase liquid chromatography.

The mechanism of the binding of D,L dansyl amino acids to teicoplanin was investigated. Na+ was used as an indicator of the interactions between the solutes and teicoplanin. The number (n) of sodium ions, Na+, excluded from the solute-teicoplanin interface when analyte transfer occurred was determined. A thermodynamic study and enthalpy-entropy compensation were performed to further explore the interaction mechanism. From these results, it was shown that teicoplanin was balanced between 2 conformational states characterized by distinct enantioselective properties. This approach indicates that liquid chromatography (LC) is a useful tool to extract physicochemical and molecular information from retention data. Thus, LC can be used as a complementary technique with the conventional techniques of molecular interaction analysis.