Novel Derivatives of Nitrobenzofurazan with Chromogenic and Fluorogenic Properties.

Five new derivatives were obtained utilizing 4-chloro-7-nitrobenzofurazan (NBD-chloride) in combination with furfurylamine, adamantylamine, aminohippuric acid, phenylalanine, and dehydroabietylamine. These derivatives were then subjected to a comparative analysis of their physical, chemical, and certain biological properties alongside two analogous and known compounds derived from the glycine and 4-amino-TEMPO free radical.

Insights into the Microbicidal, Antibiofilm, Antioxidant and Toxicity Profile of New O-Aryl-Carbamoyl-Oxymino-Fluorene Derivatives.

The unprecedented increase in microbial resistance rates to all current drugs raises an acute need for the design of more effective antimicrobial strategies. Moreover, the importance of oxidative stress due to chronic inflammation in infections with resistant bacteria represents a key factor for the development of new antibacterial agents with potential antioxidant effects. Thus, the purpose of this study was to bioevaluate new O-aryl-carbamoyl-oxymino-fluorene derivatives for their potential use against infectious diseases. With this aim, their antimicrobial effect was evaluated using quantitative assays (minimum inhibitory/bactericidal/biofilms inhibitory concentrations) (MIC/MBC/MBIC), the obtained values being 0.156-10/0.312-10/0.009-1.25 mg/mL), while some of the involved mechanisms (i.e., membrane depolarization) were investigated by flow cytometry. The antioxidant activity was evaluated by studying the scavenger capacity of DPPH and ABTS•+ radicals and the toxicity was tested in vitro on three cell lines and in vivo on the crustacean Artemia franciscana Kellog. The four compounds derived from 9H-fluoren-9-one oxime proved to exhibit promising antimicrobial features and particularly, a significant antibiofilm activity. The presence of chlorine induced an electron-withdrawing effect, favoring the anti-Staphylococcus aureus and that of the methyl group exhibited a +I effect of enhancing the anti-Candida albicans activity. The IC50 values calculated in the two toxicity assays revealed similar values and the potential of these compounds to inhibit the proliferation of tumoral cells. Taken together, all these data demonstrate the potential of the tested compounds to be further used for the development of novel antimicrobial and anticancer agents.

Biological Evaluation and Structural Analysis of Some Aminodiphenylamine Derivatives.

4-Aminodiphenylamino derivatives were investigated for their antioxidant and hydrophobicity character, together with other biological measurements, such as antimicrobial and antibiofilm activity. Among these nine compounds used, we obtained novel derivatives via reaction of the starting material with NBD-chloride. Therefore, we performed a full structural analysis for these compounds, i.e., elemental analysis, IR, UV-Vis, 1H- and 13C-NMR, ESI-MS, X-ray diffraction on single crystal, etc. The hydrophobicity of all the compounds was measured either experimentally using the RP-TLC technique, or via calculation using the fragments method. The other structural characteristics were analyzed, and a correlation between the experimental and computed properties was found. Moreover, the results of the biological evaluation showed that some of the synthesized compounds have antimicrobial and antibiofilm activity.

Synthesis and Anti-Melanoma Activity of L-Cysteine-Coated Iron Oxide Nanoparticles Loaded with Doxorubicin.

In this study, we report on the synthesis of L-Cysteine (L-Cys)-coated magnetic iron oxide nanoparticles (NPs) loaded with doxorubicin (Dox). The Fe3O4-L-Cys-Dox NPs were extensively characterized for their compositional and morpho-structural features using EDS, SAED, XRD, FTIR and TEM. XPS, MÓ§ssbauer spectroscopy and SQUID measurements were also performed to determine the electronic and magnetic properties of the Fe3O4-L-Cys-Dox nanoparticles. Moreover, by means of a FO-SPR sensor, we evidenced and confirmed the binding of Dox to L-Cys. Biological tests on mouse (B16F10) and human (A375) metastatic melanoma cells evidenced the internalization of magnetic nanoparticles delivering Dox. Half maximum inhibitory concentration IC50 values of Fe3O4-L-Cys-Dox were determined for both cell lines: 4.26 µg/mL for A375 and 2.74 µg/mL for B16F10, as compared to 60.74 and 98.75 µg/mL, respectively, for unloaded controls. Incubation of cells with Fe3O4-L-Cys-Dox modulated MAPK signaling pathway activity 3 h post-treatment and produced cell cycle arrest and increased apoptosis by 48 h. We show that within the first 2 h of incubation in physiological (pH = 7.4) media, ~10-15 µM Dox/h was released from a 200 µg/mL Fe3O4-L-Cys-Dox solution, as compared to double upon incubation in citrate solution (pH = 3), which resembles acidic environment conditions. Our results highlight the potential of Fe3O4-L-Cys-Dox NPs as efficient drug delivery vehicles in melanoma therapy.

Laccase-TEMPO as an Efficient System for Doxorubicin Removal from Wastewaters.

A large number of drugs are used to treat different diseases, and thus to improve the quality of life for humans. These represent a real ecological threat, as they end up in soil or ground waters in amounts that can affect the environment. Among these drugs, doxorubicin is a highly cytotoxic compound used as anticancer medicine. Doxorubicin can be efficiently removed from wastewater or polluted water using a simple enzymatic (biocatalytic) system, employing the oxidoreductase enzyme laccase and a stable organic nitroxide-free radical, TEMPO. Results presented in this work (as percentage of removal) were obtained at pH 5 and 7, after 2, 4, 6, and 24 h, using different ratios between doxorubicin, laccase, and TEMPO. It was shown that longer time, as well as an increased amount of catalyst, led to a higher percentage of removal, up to 100%. The influence of all these parameters is also discussed. In this way it was shown that the laccase-TEMPO biocatalytic system is highly efficient in the removal of the anticancer drug doxorubicin from wastewaters.

Chemical Degradation of Methylene Blue Dye Using TiO2/Au Nanoparticles.

Gold nanoparticles (~10 nm) were deposited on titanium dioxide nanoparticles (~21 nm) and the material obtained was characterized using IR, UV-Vis, N2 adsorption-desorption isotherm, DLS, EDS (EDX), TEM, XPS, and XRD techniques. It was found that the methylene blue dye is degraded in the presence of this material when using hydrogen peroxide as the oxidant. Tests were performed at 2, 4, 6, and 24 h, with hydrogen peroxide contents varying from 1 to 5 mg/mL. Longer exposure time and a higher content of oxidant led to the degradation of methylene blue dye at up to 90%. The material can be reused several times with no loss of activity.

Rational Functionalization Towards Redox-Active TEMPO Stable Free-Radical-Hydrochar Composites.

Although both stable free organic radicals and biomass-derived hydrochars have emerged as appealing, green, multifunctional materials, their association has not been explored. In this study, strength is found to lie in their union, which primarily leads to stable redox-active free-radical-hydrochar composites that can generate unexpected opportunities for the development of advanced metal-free sustainable materials. The composites are obtained by a straightforward green one-pot hydrothermal procedure. The loading of stable free radicals of nitroxide type and their localization is engineered by the nature of the carbohydrate and the reaction status; vigorous reaction parameters promote faster nucleation and growth kinetics of the hydrochar products, leading to a covalent immobilization of redox species on the surface of the carbonaceous microspherical aggregates. The nitroxide free-radical-hydrochar materials demonstrate enhancements in terms of both electrocatalytic activity and capacitive features.

The Chemistry of DPPH· Free Radical and Congeners.

Since the discovery in 1922 of 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl stable free radical (DPPH·), the chemistry of such open-shell compounds has developed continuously, allowing for both theoretical and practical advances in the free radical chemistry area. This review presents the important, general and modern aspects of the chemistry of hydrazyl free radicals and the science behind it.

Synthesis, Characterization, and Biologic Activity of New Acyl Hydrazides and 1,3,4-Oxadiazole Derivatives.

Starting from isoniazid and carboxylic acids as precursors, thirteen new hydrazides and 1,3,4-oxadiazoles of 2-(4-substituted-phenoxymethyl)-benzoic acids were synthesized and characterized by appropriate means. Their biological properties were evaluated in terms of apoptosis, cell cycle blocking, and drug metabolism gene expression on HCT-8 and HT-29 cell lines. In vitro antimicrobial tests were performed by the microplate Alamar Blue assay for the anti-mycobacterial activities and an adapted agar disk diffusion technique for other non-tubercular bacterial strains. The best antibacterial activity (anti-Mycobacterium tuberculosis effects) was proved by 9. Compounds 7, 8, and 9 determined blocking of G1 phase. Compound 7 proved to be toxic, inducing apoptosis in 54% of cells after 72 h, an effect that can be predicted by the increased expression of mRNA caspases 3 and 7 after 24 h. The influence of compounds on gene expression of enzymes implicated in drug metabolism indicates that synthesized compounds could be metabolized via other pathways than NAT2, spanning adverse effects of isoniazid. Compound 9 had the best antibacterial activity, being used as a disinfectant agent. Compounds 7, 8, and 9, seemed to have antitumor potential. Further studies on the action mechanism of these compounds on the cell cycle may bring new information regarding their biological activity.

Synthesis of Fluorescent Dansyl Derivatives of Methoxyamine and Diphenylhydrazine as Free Radical Precursors.

Starting from dansyl-chloride, in reaction with 1,1-diphenylhydrazine and methoxyamine, two new fluorescent derivatives 1 and 2 were obtained and characterized by NMR, IR, UV-Vis, HR-MS, and fluorescence spectroscopy. The single-crystal X-ray structure was obtained for compound 2. Both compounds generate free radicals by oxidation, as demonstrated by ESR spectroscopy. Compound 1 generates the corresponding hydrazyl-persistent free radical, evidenced directly by ESR spectroscopy, while compound 2 generates in the first instance the methoxyaminyl short-lived free radical, which decomposes rapidly with the formation of the methoxy radical, evidenced by the ESR spin-trapping technique. By oxidation of compounds 1 and 2, their fluorescence is quenched.

Capillary electrophoresis with dual detection UV/C4D for monitoring myrosinase-mediated hydrolysis of thiol glucosinolate designed for gold nanoparticle conjugation.

Capillary electrophoresis (CE) with dual UV and conductivity detection was used for the first time to monitor the functionalization of gold nanoparticles (AuNPs), a process catalyzed by an enzyme, myrosinase (Myr). A thiol glucosinolate (GL-SH) designed by our group was used as substrate. Hydrolysis of free and immobilized GL-SH was characterized using off-line and on-line CE-based enzymatic assays. The developed approaches were validated using sinigrin, a well-referenced substrate of Myr. Michaelis-Menten constant of the synthetized GL-SH was comparable to sinigrin, showing that they both have similar affinity towards Myr. It was demonstrated that transverse diffusion of laminar flow profiles was well adapted for in-capillary Mixing of nanoparticles (AuNPs) with proteins (Myr) provided that the incubation time is inferior to 20 min. Only low reaction volume (nL to few µL) and short analysis time (<5 min) were required. The electrophoretic conditions were optimized in order to evaluate and to confirm the AuNPs stability before and after functionalization by CE/UV based on surface plasmon resonance band red-shifting. The hydrolysis of the functionalized AuNPs was subsequently evaluated using the developed CE-C4D/UV approach. Repeatabilities of enzymatic assays, of electrophoretic analyses and of batch-to-batch functionalized AuNPs were excellent.

A novel profluorescent paramagnetic diaza-crown ether: synthesis, characterization and alkaline metal-ion complexation.

Starting from Kryptofix 22 two different branches were covalently attached through the nitrogen atoms, one containing a fluorescent moiety and the other the stable free radical TEMPO. The novel derivative exhibits fluorescence and paramagnetic properties, while the diaza-crown part ensures the affinity for alkaline metal-ions.

Lipoic Acid Gold Nanoparticles Functionalized with Organic Compounds as Bioactive Materials.

Water soluble gold nanoparticles protected by lipoic acid were obtained and further functionalized by standard coupling reaction with 1-naphtylamine, 4-aminoantipyrine, and 4'-aminobenzo-15-crown-5 ether. Derivatives of lipoic acid with 1-naphtylamine, 4-aminoantipyrine, and 4'-aminobenzo-15-crown-5 ether were also obtained and characterized. All these were tested for their antimicrobial activity, as well as for their influence on mammalian cell viability and cellular cycle. In all cases a decreased antimicrobial activity of the obtained bioactive nanoparticles was observed as compared with the organic compounds, proving that a possible inactivation of the bioactive groups could occur during functionalization. However, both the gold nanoparticles as well as the functionalized bioactive nanosystems proved to be biocompatible at concentrations lower than 50 µg/mL, as revealed by the cellular viability and cell cycle assay, demonstrating their potential for the development of novel antimicrobial agents.

Synthesis and biological activities of some new isonicotinic acid 2-(2-hydroxy-8-substituted-tricyclo[7.3.1.0(2.7)]tridec-13-ylidene)-hydrazides.

A series of several new isoniazid derivatives, isonicotinic acid 2-(2-hydroxy-8-substituted-tricyclo[7.3.1.0(2.7)]tridec-13-ylidene)-hydrazides, were synthesized and fully characterized. These new isoniazid derivatives were studied regarding their antibacterial activity and cytotoxicity, as well as their influences on some metabolizing enzymes. The best anti-mycobacterial activity was observed in the case of compounds containing alkyl side chains in the 8 position of tricyclo[7.3.1.0(2.7)]tridec-13-ylidene group. On contrary, the antimicrobial activity of these new compounds against various non-tuberculosis strains showed the best activity to be with the phenyl side chain of compound 6. It proved also to be the most toxic, inducing apoptosis and blocking the cell cycle in G0/G1 phase. The cell cycle was blocked in G0/G1 phase also by compound 3, but this compound did not show any toxicity. All compounds induced the expression of NAT1 and NAT2 genes in HT-29 cell line, and the expression of CYP1A1 in HT-29 and HCT-8 cell lines. The expression level of CYP3A4 was increased by compounds 1, 6 and 7 in HCT-8 cells. These results indicated that the activation of other metabolizing pathways, apart from those of isoniazid, take place. It might also point out the possibility of an increased isoniazid acetylation ratio by co-administration with new compounds in slow acetylators.

Synthesis and bioevaluation of some new isoniazid derivatives.

The aim of the study was to synthesize some new compounds with potential anti-tuberculosis activity, containing isoniazid and α,ß-unsaturated thiocinnamamide-like thioamides as precursors. The obtained derivatives were evaluated regarding their biological activity (antioxidant and antibacterial), as well as their influence on the eukaryotic cell cycle. The results suggested that the newly obtained derivatives of isoniazid exhibited different biological activities, depending on their structure; thus, the most active compound in terms of anti-oxidant and anti-Mycobacterium tuberculosis effects proved to be the isonicotinic acid N'-(1-amino-1-mercapto-3-phenyl-propen-1-yl)-hydrazide. This compound also increased the expression of NAT1 and NAT2 genes, which are implicated in the metabolism of the isoniazid, demonstrating that it could be rapidly metabolized, and thus well tolerated. The largest spectrum of antibacterial activity (excluding M. tuberculosis) was noticed for the isonicotinic acid N'-[1-amino-1-mercapto-3-(p-chloro-phenyl)-propen-1-yl]-hydrazide, which was also the most cytotoxic, especially at high concentrations, although not significantly affecting the cellular cycle phases. The obtained results showed that the new derivatives could represent potential candidates for the treatment of M. tuberculosis infections, but further research is needed in order to improve their pharmacological properties, by increasing their antimicrobial activity and reducing the risk of side-effects.

Lateral diffusion of thiol ligands on the surface of au nanoparticles: an electron paramagnetic resonance study.

The lateral mobility of the thiolate ligands on the surface of Au nanoparticles was probed by EPR spectroscopy. This was achieved by using bisnitroxide ligands, which contained a disulfide group (to ensure attachment to the Au surface) and a cleavable ester bridge connecting the two spin-labeled branches of the molecule. Upon adsorption of these ligands on the surface of Au nanoparticles, the two spin-labeled branches were held next to each other by the ester bridge as evidenced by the spin-spin interactions. Cleavage of the bridge removed the link that kept the branches together. CW and pulsed EPR (DEER) experiments showed that the average distance between the adjacent thiolate branches on the Au nanoparticle surface only marginally increased after cleaving the bridge and thermal treatment. This implies that the lateral diffusion of thiolate ligands on the nanoparticle surface is very slow at room temperature and takes hours even at elevated temperatures (90 degrees C). The changes in the distance distribution observed at high temperature are likely due to ligands hopping between the nanoparticles rather than diffusing on the particle surface.

Gold nanoparticle-initiated free radical oxidations and halogen abstractions.

We report on the use of EPR spectroscopy and spin trapping technique to detect free radical intermediates formed in the presence of gold nanoparticles. Phosphine- and amine-protected gold nanoparticles were found to initiate air oxidation of organic substrates containing active hydrogen atoms, such as amines and phosphine oxides. Nanoparticles protected by stronger bound ligands (e.g., thiols) were inactive in these reactions. We also found that gold nanoparticles are able to abstract a halogen atom from the halogenated compounds, presumably due to the high affinity of gold metal for halogens. Reaction of Au nanoparticles with chloroform showed an unusual inverse isotope effect. The trichloromethyl spin adduct was observed when Au nanoparticles were mixed with CDCl(3) but not with CHCl(3). This unexpected behaviour suggests that C-H bond breaking is not the rate-determining step in Au-initiated hydrogen abstraction.

Hydrazyl-nitrones, novel hybrid molecules in free radical research.

This work describes the synthesis and characterisation of some novel hybrid molecules which contains in the same molecule a free radical moiety of hydrazyl type and a spin-trap moiety of nitrone type. The new compounds synthesized have multiple and easy to follow spectroscopic properties, making them useful as sensors or probes in radical chemistry. The new class of hydrazyl-nitrone molecules can act, in a single step process, as both generator and spin-trap of short-lived radicals. The hybrid molecules can be also involved in acid-base or redox processes, and the chemical processes can be easily monitored by visible or electron paramagnetic resonance spectroscopy. The excellent generator and trap properties recommend them as valuable sensors and probes in radical chemistry.

Dipole-dipole interactions in spin-labeled au nanoparticles as a measure of interspin distances.

A series of Au nanoparticles modified with a nitroxide-functionalized ligand was prepared with a range of spin-label coverage. The X-band EPR spectra of frozen solutions of these nanoparticles showed coverage-dependent line-broadening due to dipole-dipole interactions between spin labels. We developed a methodology to analyze such spectra in terms of geometrical features of the nanoparticles (e.g., Au core size and the length of the spin-labeled ligand). Our method is based on the assumption that the spectral line shape is determined by the average distance between nearest-neighboring spin labels adsorbed on the Au particle. Geometrical and statistical analysis then relates this distance to the line shape parameter d1/d, which was calibrated using a model system. Application of this methodology to the experimental spectra provided information about the conformation of ligands on the Au surface. We found that, if the spin-labeled ligand is substantially longer than the surrounding protecting layer, it does not adopt a fully stretched conformation but wraps around the particle immediately above the layer of surrounding ligand. Our results also show that the ligands do not adsorb cooperatively on the Au surface.