Effects of environmental factors on the fleroxacin photodegradation with the identification of reaction pathways.

The abuse of fluoroquinolones (FQs) antibiotics leads to bacterial resistance and environmental pollution, so it is of great significance to verify the decomposition mechanism for eliminating antibiotic efficiently and conveniently. The effects of various environmental factors and the fleroxacin (FLE) photodegradation mechanisms were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS), UV-Vis absorption spectroscopy, fluorescence spectroscopy and quantum chemical calculation. Six possible photodegradation reaction paths on T1 (excited triplet state) were proposed and simulated. The departure of the piperazine ring and the substitution of F atom at C-6 position by OH group were determined as the main reactions based on the reaction rates and energy barriers of each path. The multi-pathway reactions resulted in the fastest photodegradation rates of FLE at pH 6-7 than other pH conditions. NaN3 would promote FLE photodegradation by inhibiting the reverse reaction of the separation process of F atom at C-8 and the generation of biphenyl molecules, which was a novel and distinctive phenomenon in this report. ·OH would rapidly combine with the free radicals generated in photolysis processes and made a great contribution to FLE photodegradation. Ca2+, Mg2+ and Ba2+ could stabilize the carboxyl group to impede the photo-competitive process of the decarboxylation reaction, while NO3- could generate reactive oxygen species to promote photodegradation.

Collection and Separation of Fleroxacin and Ciprofloxacin in Ultrasound-Assisted Ionic Liquid Salting-Out Microextraction System.

An ultrasound-assisted ionic liquid (IL) salting-out microextraction system was developed and applied for the extraction of quinolone antibiotics from urine. A precipitate was formed from the salt and IL, and it acted as the sorbent for the analytes. The precipitate containing the analyte was separated by filtration, redissolved, and the solution then was evaporated. The resulting extract was redissolved for high-performance liquid chromatographic analysis. Several parameters, including type and volume of IL, the type and amount of salts, sample pH, temperature and extraction time were optimized. Under the optimal experimental conditions, the limits of detection for fleroxacin and ciprofloxacin were 3.12 and 4.97 µg L-1, respectively. When the present method was applied to real urine sample analysis, the analyte recoveries ranged from 82.3 to 106.8%. This ultrasound-assisted IL salting-out microextraction system had the characteristics of high recoveries, shorter separation time and easy-to-perform collection procedure, which yielded the method to have potential for wide application.

A highly sensitive and selective "turn-on" fluorescent probe for detection of fleroxacin in human serum and urine based on a lanthanide functionalized metal-organic framework.

Cation exchange, a facile and non-destructive post-synthetic modification method, is applied to [(Me)4N]2[Pb6K6(m-BDC)9(OH)2]·H2O (1) (1,3-H2BDC = 1,3-benzenedicarboxylic acid) to prepare a series of lanthanide functionalized metal-organic frameworks. The fluorescence properties of Ln3+@1 (Ln = Eu, Tb, Sm and Dy) are investigated. The results demonstrate that the framework of 1 is capable of sensitizing both Eu3+ and Tb3+ ions effectively. Remarkably, the rapid and stable fluorescence sensitization of Eu3+@1 can be observed in the presence of fleroxacin in aqueous solution, indicating that the hybrid system can be designed as a highly sensitive and selective probe for fleroxacin. As a novel "turn-on" fluorescent probe, Eu3+@1 is regarded as a promising candidate for applications in clinical diagnosis, due to its merits of high antidisturbance, chemical stability and a low detection limit (43.91 ng mL-1). In this paper, the practical application of luminescent Eu3+@1 is highlighted, and its possible sensing mechanism is also described.

Investigation of binding between fluoroquinolones and pepsin by fluorescence spectroscopy and molecular simulation.

In this paper, the interactions of pepsin with fluoroquinolones, including norfloxacin (NFX) or ofloxacin (OFX), were investigated using fluorescence spectroscopy. The effects of NFX or OFX on pepsin showed that the molecular conformation of pepsin and the microenvironment of tryptophan residues were changed under mimicked physiological conditions. Static quenching was suggested as a factor. Quenching constants and binding constants were determined and thermodynamic parameters were calculated at three temperatures (25°C, 31°C and 37°C). Molecular interaction distances (binding distance r) were obtained. Binding was enthalpy driven and the process was spontaneous. Synchronous fluorescence, three-dimensional fluorescence spectroscopy and molecular simulation were used for analysis. Interactions were further tested using molecular modelling. Quenching and binding constants of NFX with pepsin were the highest when testing NFX/OFX/fleroxacin/gatifloxacin with pepsin combinations. NFX was the strongest quencher, and affinity of NFX for pepsin was higher than that of OFX/fleroxacin/gatifloxacin.

Development of photoprotective, antiphototoxic, and antiphotogenotoxic formulations of ocular drugs with fluoroquinolones.

The development of innovative solutions in photosafety of photolabile pharmaceutical products may help to reduce the adverse effects of these products, caused by light exposure. Providing new data in this area of study is particularly important in case of drugs applied topically on sensitive organs such as eyes. The main goal of this research is to investigate whether two potential excipients, namely: p-coumaric acid and benzophenone-4, affect the photodegradation, phototoxicity and photogenotoxicity of water solutions of four fluoroquinolones: ciprofloxacin, lomefloxacin, fleroxacin and clinafloxacin. We conducted a set of bioassays combined with the application of high-performance liquid chromatography and mass spectrometry techniques. The significant reduction of phototoxic and photogenotoxic abilities was evaluated in mixtures with ciprofloxacin and p-coumaric acid by using the umu test with Salmonella typhimurium TA1535/pSK1002, the methylthiazol tetrazolium reduction assay, and the micronucleus assay with the V79 cell line. In the bacterial assay the opposite effect was observed for the formulation with lomefloxacin and p-coumaric acid. This may be explained by the significant differences in the profile of the lomefloxacin photodegradation products. Further, the photoprotective and antiphotomutagenic abilities of ciprofloxacin mixed with benzophenone-4 were assessed. Promising results obtained in compositions with ciprofloxacin may be a basis for further research. Nevertheless, the increase in the DNA damage potential in mixtures with p-coumaric acid and two other antibiotics shows the importance of the safety evaluation of such innovative combinations.

Spectroscopic and Molecular Modeling Studies on Binding of Fleroxacin with Human Serum Albumin.

Fleroxacin (FLRX) is a new member of the class of fluoroquinolones, its effects on human serum albumin (HSA) and the mechanism of action are poorly understood, Especially, the secondary structural alterations of HSA induced by FLRX and the inner filter effect, which resulted in a spurious decrease in the observed fluorescence intensity and affected the binding parameters calculated from it are not considered. In this paper, binding of FLRX to HSA has been studied using multi-spectroscopy and molecular modeling methods. Fluorescence spectra revealed that the observed fluorescence quenching of HSA by FLRX was due to a 1∶1 complex formation by a static quenching process with a constant of 105 L·mol-1. The thermodynamic parameters (ΔH and ΔS) were calculated to be -107.99 kJ·mol-1 and -240.99 J·mol-1·K-1 via the Van't Hoff equation, which indicated that hydrogen bond and van der Waals force were the dominant intermolecular force. From the synchronous fluorescence, FT-IR and three dimensional fluorescence spectra, it was evident that the binding of FLRX to HSA induced a conformational change in the protein, and the alterations of secondary structure were quantitatively calculated by the evidence from FTIR spectra with reductions of α-helices of about 18.3%, decreases of ß-sheet structures of about 9.6%, and increases of ß-turn structures of about 18.0%. Site marker competitive experiments showed that phenylbutazone and FLRX shared a common binding site Ⅰ corresponding to the subdomain Ⅱ A of HSA. The binding details between FLRX and HSA were further confirmed by molecular docking studies, which revealed that FLRX was bound at subdomain Ⅱ A through multiple interactions, such as hydrogen bond, hydrophobic and van der Waals, etc. The accurate and full basic data in the work is beneficial to clarify the binding mechanism of FLRX with HSA and is helpful for understanding its effect on protein function during the blood transportation process.

Synthesis, structure, and biological evaluation of a copper(ii) complex with fleroxacin and 1,10-phenanthroline.

A novel mixed-ligand Cu(ii) complex combined with the quinolone drug fleroxacin and 1,10-phenanthroline was synthesized in this work. The crystal structure of the complex was characterized via X-ray crystallography, which was the first reported single crystal complex of fleroxacin. Results showed that Cu(ii) was coordinated through pyridone oxygen and one carboxylate oxygen atom of fleroxacin, as well as two nitrogen atoms from 1,10-phenanthroline. Various characterization methods, including Fourier transform infrared, elementary analysis, thermogravimetry, and X-ray powder diffraction, were applied. The Cu(ii)-quinolone complex exhibited favorable biological activities, and was proved to be capable of transforming supercoiled PUC19 DNA into nicked form under hydrolytic conditions. The obtained pseudo-Michaelis-Menten kinetic parameter was 12.64 h(-1), which corresponded to a million-fold rate enhancement in DNA cleavage. In addition, the interaction capacity of the complex with human serum albumin (HSA) was investigated. The results demonstrated a moderately intense combination between HSA and the complex. The complex evidently quenched the fluorescence of HSA. Approximately 19.2% of the quenching was attributed to Förster resonance energy transfer (FRET), whereas the rest was caused by ground-state complex formation (molar ratio of HSA : complex = 1 : 2). The energy of the complex was excited during FRET, which increased the fluorescence of the complex by approximately 18%.

Intramolecular charge transfer effects on the diradical character and second hyperpolarizabilities of open-shell singlet X-π-X (X = donor/acceptor) systems.

We investigate the effect of the quadrupole-type intramolecular charge transfer (ICT) in open-shell singlet donor-π-donor (D-π-D) molecules on the singlet open-shell (diradical) character and the longitudinal second hyperpolarizabilities γ (the third-order nonlinear optical (NLO) properties at the molecular scale). For this investigation we used the para-quinodimethane (PQM) with point charges (pc's) model calculated with the unrestricted coupled cluster method including single and double excitations with a perturbative treatment of the triple excitations (UCCSD(T)). In this model, the diradical character y and the amount of the ICT, that is, the D-π-D nature, can be varied primarily by changing the exocyclic carbon-carbon bond (C-C) lengths and the external pc's Q, respectively. It turns out that the increase in the D-π-D nature decreases the y values, moves the y values (ymax) giving the maximum γ (γmax) to the large y region, and enhances the γmax values, for example, the γmax of the singlet diradical PQM with Q = -2.8 au reaches twice that of the singlet diradical PQM without any pc's. This result indicates that open-shell singlet D-π-D systems with ICT are promising candidates for a new class of third-order NLO molecules, whose γ values are more enhanced than those of conventional closed-shell D-π-D systems and of symmetric open-shell singlet systems without the ICT. To confirm this tendency, we examine the boron-disubstituted PQM dianion model, which is found to exhibit further enhancement of γ as compared to the PQM model with intermediate diradical character due to the synergy effects of the intermediate open-shell singlet nature and the strong field-induced ICT nature in the dianionic state of the D-π-D system. Further investigation of the acceptor-π-acceptor (A-π-A) type ICT effect in the PQM-pc model shows that both D-π-D and A-π-A type symmetric ICTs give similar effects on the relationship between y and γ, though there are some differences originating in the orbital contraction and extension induced by the pc's. The present results contribute to understanding the third-order NLO properties of open-shell symmetric ICT systems and thus to constructing new design guidelines for highly efficient third-order NLO systems.

Pressure-dependent relaxation in the photoexcited mott insulator ET-F2TCNQ: influence of hopping and correlations on quasiparticle recombination rates.

We measure the ultrafast recombination of photoexcited quasiparticles (holon-doublon pairs) in the one dimensional Mott insulator ET-F(2)TCNQ as a function of external pressure, which is used to tune the electronic structure. At each pressure value, we first fit the static optical properties and extract the electronic bandwidth t and the intersite correlation energy V. We then measure the recombination times as a function of pressure, and we correlate them with the corresponding microscopic parameters. We find that the recombination times scale differently than for metals and semiconductors. A fit to our data based on the time-dependent extended Hubbard Hamiltonian suggests that the competition between local recombination and delocalization of the Mott-Hubbard exciton dictates the efficiency of the recombination.

Tetrathiafulvalene-based mixed-valence acceptor-donor-acceptor triads: a joint theoretical and experimental approach.

This work presents a joint theoretical and experimental characterisation of the structural and electronic properties of two tetrathiafulvalene (TTF)-based acceptor-donor-acceptor triads (BQ-TTF-BQ and BTCNQ-TTF-BTCNQ; BQ is naphthoquinone and BTCNQ is benzotetracyano-p-quinodimethane) in their neutral and reduced states. The study is performed with the use of electrochemical, electron paramagnetic resonance (EPR), and UV/Vis/NIR spectroelectrochemical techniques guided by quantum-chemical calculations. Emphasis is placed on the mixed-valence properties of both triads in their radical anion states. The electrochemical and EPR results reveal that both BQ-TTF-BQ and BTCNQ-TTF-BTCNQ triads in their radical anion states behave as class-II mixed-valence compounds with significant electronic communication between the acceptor moieties. Density functional theory calculations (BLYP35/cc-pVTZ), taking into account the solvent effects, predict charge-localised species (BQ(.-)-TTF-BQ and BTCNQ(.-)-TTF-BTCNQ) as the most stable structures for the radical anion states of both triads. A stronger localisation is found both experimentally and theoretically for the BTCNQ-TTF-BTCNQ anion, in accordance with the more electron-withdrawing character of the BTCNQ acceptor. CASSCF/CASPT2 calculations suggest that the low-energy, broad absorption bands observed experimentally for the BQ-TTF-BQ and BTCNQ-TTF-BTCNQ radical anions are associated with the intervalence charge transfer (IV-CT) electronic transition and two nearby donor-to-acceptor CT excitations. The study highlights the molecular efficiency of the electron-donor TTF unit as a molecular wire connecting two acceptor redox centres.

Photodegradation of fleroxacin injection: II. Kinetics and toxicological evaluation.

Photodegradation kinetics of fleroxacin were investigated in different injections. Five commercial formulations were analyzed before and after irradiation by determining residual volumes of fleroxacin with high-pressure liquid chromatography (HPLC), and different decomposition functions and models were obtained. Concentration levels of fleroxacin in injections caused the differences in photodegradation kinetics instead of ingredients. Influences of different pH values and presence of NaCl on photodegradation of fleroxacin were observed. Low pH value decreased the efficacy of photolysis and enhanced photostability of fleroxacin injections. Tentative structure of a new degradation product afforded was proposed. An acute toxicity assay using the bioluminescent bacterium Q67 was performed for fleroxacin injections after exposure to light. The research proved that fleroxacin was more photolabile in dilute injection, and acute toxicity of dilute injection increased more rapidly than that of concentrated injection during irradiation.

Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin.

The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non-radiation energy transfer. The quenching constants KSV , binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non-radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX-pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study.

Mass spectrometic study of speciation in aluminium-fluoroquinolone solutions.

Fluoroquinolones (FQLs) are synthetic antibacterial agents containing a 4-oxo-1,4-dihydroquinoline skeleton. When concomintantly administered with other drugs which may contain metal ions, particularly Al(3+) (antacids, phosphate binders, vaccines etc) they may form metal-drug complexes. Pharmacokinetic studies showed that aluminium-quinolone interactions lead to reduced bio- availability and altered activity of the drug with possible development of the toxic effects of aluminum ion. Reliable speciation in Al(3+) - quinolone systems at micromolar concentration level is needed to better understand pharmaco- and toxicokinetics of the FQLs in the presence of Al. In this work, the speciation in solutions containing Al(3+) and FQL family members (fleroxacin, moxifloxacin and ciprofloxacin) was studied by electrospray mass spectrometry (ESI-MS), ESI-MS/MS, and laser desorption ionization (LDI) MS. The dominating species identified in all the three Al(3+)-FQL solutions, at ca 30-50 µmol L(-1) total Al concentration and 2:1 to 1:3 metal-to-ligand ratio in the pH range 3.0- 6.0, were the ions related to the complexes AlL(2+), AlL(2)(+) and AlL(3)(0) (L = ligand in the monodeprotonated form). Mixed protonated and hydroxo complexes were also formed at lower and higher pH values respectively and, as expected, dimeric and polymeric species were not observed in ESI spectra. LDI measurements confirmed the existence of the mononuclear complexes found by ESI, and indicated the formation of polymeric species. The ion [2Al(3+) +5(-)](+) was identified with all three FQLs. This ionic species most probably arises from Al(2)L(2) by clustering with free ligand anions. Comparison of literature potentiometric data with mass spectral data indicated good agreement between speciation schemes. The obtained results suggest the presence of strong interaction between FQLs and Al(3+) which may be important in affecting absorption of these drugs in the gastrointestinal tract.

Air- and heat-stable planar tri-p-quinodimethane with distinct biradical characteristics.

A heptacyclic carbocycle possessing three p-quinodimethane units conjugated in one plane has been synthesized and shown to exhibit distinct biradical characteristics. The molecule has a HOMO/LUMO band gap of ca. 1 eV and a S(0)-T(1) energy gap of 2.12 kcal/mol, and it absorbs and emits near-IR light at room temperature. It is air-stable under ambient light for several months and thermally stable up to 160 °C under nitrogen, and it undergoes reversible two-electron oxidation and reduction. The synthetic approach is such that a smaller and larger oligo-p-quinodimethane can be synthesized.

Photodegradation of fleroxacin injection: different products with different concentration levels.

Photodegradation of fleroxacin is investigated in different injections and solutions. After UV irradiation, fleroxacin was degraded to afford two major products in large-volume injection (specification, 200 mg:100 ml), while degraded to afford another major product in small-volume injection (specification, 200 mg:2 ml). The photodegradation products were detected and isolated by reversed-phase HPLC. Based on the spectral data (FT-IR, MS(n), TOF-MS, (1)H/(13)C, DEPT, and 2D NMR), the structures of these products were: 8-fluoro-9-(4-methyl-piperazin-1-yl)-6-oxo-2,3-dihydro-6H-1-oxa-3a-aza-phenalene-5-carboxylic acid (impurity-I); 6-fluoro-1-(2-fluoro-ethyl)-7-(2-methylamino-ethylamino)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (impurity-II); and 6,8-difluoro-1-(2-fluoro-ethyl)-7-(2-methylamino-ethylamino)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (impurity-III), respectively. Different photodegradation pathways of fleroxacin were proposed, which led to the different stability characteristics of fleroxacin in the injections. The fluorine atom at C8 is more photolabile in dilute injection, so defluorination and cyclization reactions are prone to take place, whereas photo irradiation only cause ring-opening oxidation reaction of piperazine side chain in concentrated injection.

Inter- and intramolecular photochemical reactions of fleroxacin.

In the cation formed by photoinduced C-F bond cleavage in fleroxacin, intramolecular reaction with the N-ethyl chain is prevented by the electron-withdrawing effect of fluorine and intermolecular attack by nucleophiles is facilitated.

[Study on the ternary complex of FLRX, zinc(II) and BSA by the method of fluorescence].

The influences of fleroxacin (FLRX) on the fluorescence of bovine serum albumin(BSA), zinc(II) on that of bovine serum albumin, and zinc(II) on the of fleroxacin and bovine serum albumin were studied under imitated the physiological condition. It was shown that both fleroxacin and zinc(II) have a powerful ability to quench the BSA fluorescence via a nonradiative energy transfer mechanism. But the fluorescence quenching action of fleroxacin on BSA was much stronger in the presence of zinc (II). The fluorescence quenching data were analyzed according to Stern-Volmer equation and double-reciprocal equation, and the binding constant(K) and the binding sites(n) were obtained. In the system of binary complex of FLRX and BSA, K = 5.44 x 10(4) and n = 1.05, while in the system of binary complex of zinc(II) and BSA, K = 2.19 x 10(9) and n = 2.

1H and 19F NMR relaxation studies of fleroxacin with Micrococcus luteus.

In order to investigate and characterize interaction processes between the fluoroquinolone fleroxacin and bacterial cells we used non-selective (all resonances are excited), selective (observed resonance is excited) spin-lattice relaxation rates and spin-spin relaxation measurements. The signals of three hydrogens at different moieties of the fleroxacin molecule were considered to get an insight in the complexation behavior. The enhancement of selective relaxation rates was observed with increasing fleroxacin concentrations and keeping the bacterial mass constant. The obtained relaxation rates of the affected hydrogens were analyzed via a Lineweaver-Burk-plot to determine the KD values. Furthermore, 19F NMR spectra were recorded and spin-spin relaxation rates (R2) were determined by a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. Because of the dependency of the line width of NMR peaks on transversal relaxation time T2, we compared the line width at half-height at different fleroxacin concentrations in order to investigate the involvement of fluorine atoms in different positions in the complexation. All findings point to core quinolone moiety to be involved in the interaction with bacterial cells.

[Determination of fleroxacin by photochemical fluorescence of zinc-fleroxacin complex].

Based on the complex formed by Zn2+ which can strengthen the relative fluorescence intensity of fleroxacin evidently, a novel photochemical fluorescence method was developed. The effects of the acidity, the concentration ratio of Zn2+ to fleroxacin, and the time for illumination were studied. Under the optimum experiment conditions, the linear range of the determination was 5.00 x 10(-8) - 5.00 x 10(-6) mol x L(-1). The detection limit was 4.2 x 10(-8) mol x L(-1). The relative standard deviation of the determination of fleroxacin (5.0 x 10(-7) mol x L(-1)) was 1.7% (n = 20). The method was successfully applied to the determination of fleroxacin in specimens, and the recoveries were in the range of 95.0%-105%. The mechanism of this system is proposed.

Synthesis and antibacterial activity of 1-(2-fluorovinyl)-7-substituted-4-quinolone-3-carboxylic acid derivatives, conformationally restricted analogues of fleroxacin.

The novel 1-(2-fluorovinyl)-4-quinolone-3-carboxylic acid derivatives Z-15a-c, E-15a-c, Z-16a-c, and E-16a-c, conformationally restricted analogues of fleroxacin (5), were synthesized, and their in vitro antibacterial activity was evaluated. A dehydrosulfenylation of a 2-fluoro-2-[(4-methoxyphenyl)sulfinyl]ethyl group was employed as a key step for the construction of a 2-fluorovinyl group at the N-1 position. It appeared evident that the Z-isomers Z-15a-c and Z-16a-c exhibited 2- to 32-fold more potent in vitro antibacterial activity than the corresponding E-isomers E-15a-c and E-16a-c. Furthermore, since Z-15b showed in vitro antibacterial activity and DNA gyrase inhibition comparable to that of 5, it was hypothesized that the conformation of Z-15b would be equivalent to the active conformer of 5. The results revealed that the antibacterial Z-1-(2-fluorovinyl)quinolone derivatives carry the novel N-1 substituent of the fluoroquinolones.