Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p < 0.001) in MR signal intensity or a significant decrease (p < 0.001) in T₁ relaxation, measured as %T₁ change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p < 0.0001) in T₁ relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p < 0.001) and 3-nitrotyrosine (3-NT) (p < 0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.     

Inhibition of Fe2+- and Fe3+- induced hydroxyl radical production by the iron-chelating drug deferiprone

Deferiprone (L1) is an effective iron-chelating drug that is widely used for the treatment of iron-overload diseases. It is known that in aqueous solutions Fe²⁺ and Fe³⁺ ions can produce hydroxyl radicals via Fenton and photo-Fenton reactions. Although previous studies with Fe²⁺ have reported ferroxidase activity by L1 followed by the formation of Fe³⁺ chelate complexes and potential inhibition of Fenton reaction, no detailed data are available on the molecular antioxidant mechanisms involved. Similarly, in vitro studies have also shown that L1–Fe³⁺ complexes exhibit intense absorption bands up to 800 nm and might be potential sources of phototoxicity. In this study we have applied an EPR spin trapping technique to answer two questions: (1) does L1 inhibit the Fenton reaction catalyzed by Fe²⁺ and Fe³⁺ ions and (2) does UV–Vis irradiation of the L1–Fe³⁺ complex result in the formation of reactive oxygen species. PBN and TMIO spin traps were used for detection of oxygen free radicals, and TEMP was used to trap singlet oxygen if it was formed via energy transfer from L1 in the triplet excited state. It was demonstrated that irradiation of Fe³⁺ aqua complexes by UV and visible light in the presence of spin traps results in the appearance of an EPR signal of the OH spin adduct (TMIO–OH, a(N)=14.15 G, a(H)=16.25 G; PBN–OH, a(N)=16.0 G, a(H)=2.7 G). The presence of L1 completely inhibited the OH radical production. The mechanism of OH spin adduct formation was confirmed by the detection of methyl radicals in the presence of dimethyl sulfoxide. No formation of singlet oxygen was detected under irradiation of L1 or its iron complexes. Furthermore, the interaction of L1 with Fe²⁺ ions completely inhibited hydroxyl radical production in the presence of hydrogen peroxide. These findings confirm an antioxidant targeting potential of L1 in diseases related to oxidative damage.     

Efficient repair of protein radicals by ascorbate

Protein radicals were selectively generated by reaction with azide radicals on Trp and Tyr residues in insulin, β-lactoglobulin, pepsin, chymotrypsin, and bovine serum albumin at rate constants in the range (2.9–19) × 10⁸ M^? 1 s^? 1. Monohydrogen ascorbate reduced tryptophanyl radicals in chymotrypsin and pepsin with rate constants in the narrow range of (1.6–1.8) × 10⁸ M^? 1 s^? 1, whereas β-lactoglobulin tryptophanyl radicals reacted almost 10 times slower. The corresponding values for the protein tyrosyl radicals were about an order of magnitude smaller. Comparison of the rate constants of reactions of free and protein-bound tryptophanyl and tyrosyl radicals showed that, in most cases, the location of the radicals in the protein chain did not constitute a major barrier to the reaction with monohydrogen ascorbate. The results suggest that, under physiological concentrations of dioxygen, monohydrogen ascorbate is likely to be a significant target of protein radicals. It seems likely, therefore, that reaction with protein radicals may be responsible for much of the well-documented loss of ascorbate in living organisms subjected to oxidative stress.     

Design,synthesis and anti-Alzheimer properties of dimethylaminomethyl-substituted curcumin derivatives

Eight dimethylaminomethyl-substituted curcumin derivatives were designed and synthesized. The antioxidant test revealed that the synthesized compounds had higher free radical scavenging activity towards both 2,2-diphenyl-1-picrylhydrazyl free radicals (DPPH) (IC₅₀ 1.5–29.9 μM) and galvinoxyl radicals (IC₅₀ 4.9–41.1 μM) than the lead compound curcumin. Besides, compound 3a could effectively inhibit the Aβ self-aggregation in vitro. Investigated in phosphate-buffered solutions (pH = 7.4) in the presence or absence of 0.1% FBS 3a showed a good stability while curcumin did not. Furthermore, 3a showed a good lipophilicity (log P = 3.48), suggesting a potential ability to penetrate the blood–brain-barrier. The aqueous solubility of the hydrochloride salt of 3a (16.7 mg/mL) has also been significantly improved as compared with curcumin (<0.1 mg/mL).     

Isoorientin-6″-O-glucoside,a water-soluble antioxidant isolated from Gentiana arisanensis

The antioxidant activities of isoorientin-6″-O-glucoside were studied using various models. Isoorientin-6″-O-glucoside was more potent than Trolox, probucol and butylated hydroxytoluene (BHT) in reducing the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). It also scavenged superoxide anion, peroxyl and hydroxyl radicals that were generated by xanthine/xanthine oxidase, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and Fe³⁺–ascorbate–EDTA–H₂O₂ system, respectively. The IC₅₀ value, stoichiometry factor and second-order rate constant were 9.0 ± 0.8 μM, 1.8 ± 0.1 and 2.6 × 10¹⁰ M⁻¹ s⁻¹ for superoxide generation, peroxyl and hydroxyl radicals. However, isoorientin-6″-O-glucoside did not inhibit xanthine oxidase activity or scavenge hydrogen peroxide (H₂O₂), carbon radical or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN)-derived peroxyl radical in hexane. Isoorientin-6″-O-glucoside inhibited Cu²⁺-induced oxidation of human low-density lipoprotein (LDL) as measured by fluorescence intensity, thiobarbituric acid-reactive substance formation and electrophoretic mobility. Since isoorientin-6″-O-glucoside did not possess pro-oxidant activity, it may be an effective water-soluble antioxidant that can prevent LDL against oxidation.     

Synthesis,crystal structure and magnetic properties of the spin crossover system [Fe(pq)3]2+

Three new compounds formulated (ClO₄)₂[Fe(pq)₃] (1), (BF₄)₂[Fe(pq)₃] · EtOH (2) and {(ClO₄)[MnCr(C₂O₄)₃][Fe(pq)₂(H₂O)₂]} (3), where pq is 2,2′-pyridylquinoline, have been synthesised and characterised. Despite the different crystal packing exhibited by 1 and 2, the cationic species [Fe(pq)₃]²⁺ are structurally quite similar. At 293 K, the Fe–N bond lengths are characteristic of the iron(II) in the high-spin state. In contrast to 1, 2 undergoes a continuous spin transition. Indeed, at 95 K its structure experiences a noticeable change in the Fe–N bonds and angles, i.e. the Fe–N bonds shorten by 0.194 Å on the average. The magnetic behaviour confirms that 1 is fully high-spin in the 4–300 K temperature range while 2 shows a spin transition centred at T_1/2 = 150 K. The corresponding enthalpy, entropy and interaction parameter are ΔH = 7.49 kJ mol^?1, ΔS = 50 J K^?1 mol^?1and Γ = 1.35 kJ mol^?1. Compound 3 has been obtained as a microcrystalline powder. The magnetic properties of 3 point at the occurrence of ferromagnetic coupling below 100 K and the onset of a ferromagnetic ordering below 10 K (Weiss constant equal to 6.8 K). The Mössbauer spectra of 3 show the occurrence of a magnetic order at T ? 4.2 K.     

Isostructural M(RL)2(hfac)2 complexes with RL = 5-(4-[N-tert-butyl-N-aminoxyl]phenyl)pyrimidine

5-(4-(N-tert-Butyl-N-aminoxylphenyl))pyrimidine (RL, 4PPN) forms crystallographically isostructural and isomorphic pseudo-octahedral M(RL)₂(hfac)₂ complexes with M(hfac)₂, M = Zn, Cu, Ni, Co, and Mn. Multiple close contacts occur between sites of significant spin density of the organic radical units. Magnetic behavior of the Zn, Cu, Ni, Co complexes appears to involve multiple exchange pathways, with multiple close crystallographic contacts between sites that EPR (of 4PPN) indicates to have observable spin density. Powder EPR spectra at room temperature and low temperature are reported for each complex. Near room temperature, the magnetic moments of the complexes are roughly equal to those expected by a sum of non-interacting moments (two radicals plus ion). As temperature decreases, AFM exchange interactions become evident in all of the complexes. The closest fits to the magnetic data were found for a 1-D Heisenberg AFM chain model in the Zn(II) complex (J/k = (?)7 K), and for three-spin RL—M—RL exchange in the other complexes (J/k = (?)26 K, (?)3 K, (?)6 K, for Cu(II), Ni(II), and Co(II) complexes, respectively).     

Electromyographic,but not mechanomyographic amplitude–force relationships,distinguished differences in voluntary activation capabilities between individuals

The purpose of the present study was to examine the influence of activation capabilities on the electromyography (EMG_RMS) and mechanomyography amplitude (MMG_RMS)–force relationships of the vastus lateralis (VL) and rectus femoris (RF). Thirteen men (mean ± SD; age = 22 ± 3 year) performed nine submaximal contractions (10–90% maximal voluntary contraction [MVC]) with the interpolated twitch technique performed during a separate contraction at 90% MVC to calculate percent voluntary activation (%VA). Nine participants with >90% VA were categorized into the high-activated group with the remaining categorized into the moderate-activated group. Slopes (b terms) were calculated from the log-transformed EMG_RMS and MMG_RMS–force relationships. The b terms (collapsed across the VL and RF) for the EMG_RMS–force relationships were greater for the high- (1.29 ± 0.31) than the moderate-activated (1.10 ± 0.20) group. In contrast, there were no differences in the b terms for the MMG_RMS–force relationships between the high- and moderate-activated groups. For the EMG_RMS and MMG_RMS–force relationships, the b terms were greater for the RF (1.38 ± 0.30, 0.81 ± 0.20) than the VL (1.08 ± 0.19, 0.60 ± 0.13) collapsed across groups. The b terms from the EMG_RMS–force relationships, but not the MMG_RMS–force relationships, reflected differences in %VA.     

Zinc protects Ceratophyllum demersum L. (free-floating hydrophyte) against reactive oxygen species induced by cadmium

Evidence for Zn protection against Cd-induced reactive oxygen species in the free-floating hydrophyte Ceratophyllum demersum L. is presented in this paper. Metal treatments of 10 μmol/L Cd, 10 Cd μmol/L supplemented with Zn (10, 50, 100 and 200 μmol/L) and Zn-alone treatments of the same concentrations were used. Using 5,5 dimethyl pyrroline-N-oxide as the spin-probe, electron spin resonance spectra indicated a drastic increase in hydroxyl radicals (OH) in Cd-10 μmol/L treatments, which was closely correlating with the enhanced formation of hydrogen peroxide (H₂O₂) and generation of superoxide radical (O₂^?) triggered by the oxidation of NADPH. The supplementation of adding Zn (10–200 μmol/L) to the Cd-10 μmol/L treatments significantly decreased the production of free radicals especially by eliminating the precursors of OH through inhibition of NADPH oxidation. Cd-enhanced ROS production which substantially increased the oxidative products of proteins measured as carbonyls was effectively inhibited by Zn supplementation.     

Metal–organic chemical vapor deposition of Bi2Mn4O10 films on SrTiO3 〈1 0 0〉

Bi₂Mn₄O₁₀ films were deposited on SrTiO₃ (1 0 0) substrates via metal–organic chemical vapor deposition (MOCVD) from the Bi(phenyl)₃ and Mn(tmhd)₃ (Htmhd = 2,2,6,6-tetramethyl-3,5-heptanedione) precursors. The films were deposited in the temperature range of 600–800 °C. The X-ray diffraction (XRD) characterization indicates that the Bi₂Mn₄O₁₀ phase is stable within the investigated range, but the temperature plays a crucial role in determining the out-of-plane orientation of the films. The SEM shows very homogeneous surfaces with a fiber texture morphology at the highest deposition temperature. The AFM data indicate a textured surface with a root mean square roughness of 77.67 nm for films deposited at 800 °C.     

Angiotensin-(1–7) abolishes AGE-induced cellular hypertrophy and myofibroblast transformation via inhibition of ERK1/2

Angiotensin-(1–7) (Ang-(1–7))/AT₇-Mas receptor axis is an alternative pathway within the renin–angiotensin system (RAS) that generally opposes the actions of Ang II/AT₁ receptor pathway. Advanced glycated end product (AGEs) including glucose- and methylglyoxal-modified albumin (MGA) may contribute to the development and progression of diabetic nephropathy in part through activation of the Ang II/AT₁ receptor system; however, the influence of AGE on the Ang-(1–7) arm of the RAS within the kidney is unclear. The present study assessed the impact of AGE on the Ang-(1–7) axis in NRK-52E renal epithelial cells. MGA exposure for 48 h significantly reduced the intracellular levels of Ang-(1–7) approximately 50%; however, Ang I or Ang II expression was not altered. The reduced cellular content of Ang-(1–7) was associated with increased metabolism of the peptide to the inactive metabolite Ang-(1–4) [MGA: 175 ± 9 vs. Control: 115 ± 11 fmol/min/mg protein, p < 0.05, n = 3] but no change in the processing of Ang I to Ang-(1–7). Treatment with Ang-(1–7) reversed MGA-induced cellular hypertrophy and myofibroblast transition evidenced by reduced immunostaining and protein expression of α-smooth muscle actin (α-SMA) [0.4 ± 0.1 vs. 1.0 ± 0.1, respectively, n = 3, p < 0.05]. Ang-(1–7) abolished AGE-induced activation of the MAP kinase ERK1/2 to a similar extent as the TGF-β receptor kinase inhibitor SB58059; however, Ang-(1–7) did not attenuate the MGA-stimulated release of TGF-β. The AT₇-Mas receptor antagonist D-Ala⁷-Ang-(1–7) abolished the inhibitory actions of Ang-(1–7). In contrast, AT₁ receptor antagonist losartan did not attenuate the MGA-induced effects. We conclude that Ang-(1–7) may provide an additional therapeutic approach to the conventional RAS blockade regimen to attenuate AGE-dependent renal injury.     

Magnetic properties of six-coordinated high-spin cobalt(II) complexes: Theoretical background and its application

In this contribution we study and analyse the influence of the different parameters involved in the magnetic susceptibility of six-coordinated high-spin Co(II) complexes. We propose an empirical expression to fit the magnetic susceptibility of polycrystalline samples of mononuclear Co(II) complexes with an axial distortion, the variable parameters being Δ (axial distortion), α (orbital reduction factor) and λ (spin–orbit coupling). This expression avoids solving the 12 × 12 matrix associated to the distortion of the ⁴T_1g term. In order to take into account the magnetic coupling (J) in the polynuclear Co(II) complexes, a perturbational approach is proposed to describe their magnetic susceptibility in the whole temperature range (2–300 K) as a function of J, Δ, α and λ. This approach is valid in the limit of the weak magnetic coupling as compared to the spin–orbit coupling, |J/λ| < 0.1. The model allows the treatment of each cobalt(II) ion in axial symmetry as an effective spin S_eff = 1/2. That causes a drastic reduction of the matrix size of the polynuclear compounds from 12ⁿ × 12ⁿ to 2ⁿ × 2ⁿ, n being the number of Co(II) ions in the complex. The main advantage of the model is to make possible the fit of the magnetic susceptibility data of those polynuclear Co(II) complexes whose high nuclearity involved intractable matrices.     

In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes

Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively.The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC₅₀ values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0–14.4 μg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC₅₀ values in the range of 17.9–33 μg/ml, and aromatase (Cyp19) activity with IC₅₀ values in the range of 1.2–5.6 μg/ml.     

Real evidence about zeolite as microorganisms immobilizer in anaerobic fluidized bed reactors

Using the scanning electronic microscopy, it was observed that natural zeolite possesses excellent physical characteristics as a support medium in anaerobic fluidized bed reactors (AFBR). Samples for biomass analysis were taken from two identical laboratory-scale AFBR (R-1 and R-2), which were operated with 25% of fluidization. These reactors treated distillery wastewaters (vinasses) at mesophilic temperature (30 ± 2 °C). The experiments were carried out with 0.25–0.50 and 0.50–0.80 mm zeolite particle diameter in reactors R-1 and R-2, respectively. The biomass concentration attached to zeolite in both reactors was found to be in the range of 40–45 g volatile solids/l. COD removal efficiencies as high as 90% were achieved at organic loading rate (OLRs) of up to 20 g COD/l day. The volatile fatty acid (VFA) levels were always lower that the suggested limits for digester failure. The yield coefficient of methane production was 0.29 l CH₄(at STP)/g COD consumed and was virtually independent of the OLR applied. A hybridization technique (fluorescence in situ hybridization, FISH) helped determine the predominant anaerobic microorganisms that colonized the natural zeolite, which were found to be Methanosaeta and Methanosarcinaceae, observing a reduced number of sulphate reducing bacteria. The results obtained for reactors R-1 and R-2 were very similar, showing that the particle size did not significantly influence the microbial community immobilized on zeolite.     

Hydrogen sulphide removal from air by biotrickling filter using open-pore polyurethane foam as a carrier

A study was conducted on H₂S removal in a biotrickling filter packed with open-pore polyurethane foam. Thiobacillus denitrificans was used as inoculum and a mixed culture population was developed during the process. The inhibitory effect of sulphate concentration (1.8–16.8 g L⁻¹), pH (6.9–8.6), trickling liquid velocity (TLV, 9.1–22.8 m h⁻¹), H₂S inlet concentration (20–157 ppmv) and the empty bed residence time (EBRT, 9–57 s) on the H₂S removal efficiency (RE) were thoroughly investigated. An increase in pH from 6.9 to 8.5 led to a corresponding increase in H₂S removal. In addition, an inhibitory effect of sulphate concentration was observed from 16.8 g L⁻¹ and the maximum elimination capacity was found to be 22 gS m⁻³ h⁻¹ (RE 98%). The RE was constant (98.8 ± 0.30%) for EBRT ≥ 16 s, but a decrease in the EBRT from 16 to 9 s led to a corresponding decrease in RE from 98.2 to 89.6% for a TLV of 9.1 m h⁻¹ and from 97.9 to 94.9% for a TLV of 22.8 m h⁻¹ (inlet load of 11.0 ± 0.2 gS m⁻³ h⁻¹). The sulphur oxidation capacity in the biotrickling filter was not diminished by the presence of other bacteria.     

Phosphorus retention capacity of agricultural headwater ditch sediments under alkaline condition in purple soils area,China

Phosphorus (P) retention by headwater ditch sediments adsorption plays a pivotal ecological role in P buffering in freshwater ecosystems. Previous studies focused on headwater ditch sediment adsorption and its P retention capacity in acid conditions, but little information is available for headwater ditches under alkaline condition. In this study, adsorption behavior of phosphorus was investigated in headwater ditch sediments under alkaline condition using a batch equilibrium technique, thus determining phosphorus retention capacity of headwater ditch sediments collected at 11 sites at base-flow on 2 March 2006 in purple soils area of China. Results showed that headwater ditch sediments had elevated phosphorus sorption maximum (S_max) values (122.72–293.23 mg P kg^?1) and P binding energy (K) values (1.64–8.65 L mg^?1), while they had low equilibrium phosphorus concentration (EPC₀) (0.001–0.108 mg L^?1) and degree of phosphorus saturation (DSP) (1.93–10.19%). Analysis of EPC₀ and soluble P concentration indicated that sediments acted as a sink for P across all headwater ditches. Therefore, there were high intrinsic P retention capacities of headwater ditch sediments. Positive correlations of both K and S_max with oxalate-extractable Fe (r of 0.93 and 0.81, p < 0.05) and total carbon (TC) (r of 0.89 and 0.74, p < 0.05) were found, thus suggesting that organic matter and amorphous or poorly crystalline Fe would play dominant roles in P adsorption in the headwater ditch sediments under alkaline condition. Since neither S_max nor K were correlated with CCE (CaCO₃) (r of 0.15 and ?0.06, p > 0.05), the high-energy sorptive surfaces of Fe oxides were more important than CaCO₃ in P sorption of sediment under alkaline condition. At the same time, these poor correlations between CCE and K and S_max imply a non-linear relationship between P retention and the content of carbonate. The negative correlations of both K and S_max with pH (r of–0.73, and–0.58, p < 0.05) revealed that an increase in pH would not improve sediment retention capacity under alkaline conditions.     

Using short columns to speed up LC–MS quantification in MS binding assays

The present study describes the use of short columns to speed up LC–MS quantification in MS binding assays. The concept of MS binding assays follows closely the principle of traditional radioligand binding but uses MS for the quantification of bound marker thus eliminating the need for a radiolabelled ligand. The general strategy of increasing the throughput of this type of binding assay by the use of short columns is exemplified for NO 711 binding addressing GAT1, the most prevalent GABA transporter in the CNS. Employing short RP-18 columns with the dimension of 20 mm × 2 mm and 10 mm × 2 mm at flow rates up to 1000 μL/min in an isocratic mode retention times of 8–9 s and chromatographic cycle times of 18 s could be achieved. Based on the internal standard [²H₁₀]NO 711 fast chromatography methods were developed for four different columns that enabled quantification of NO 711 in a range from 50 pM up to 5 nM directly out of reconstituted matrix samples without further sample preparation. A validation of the established methods with respect to linearity, intra- and inter-batch accuracy and precision showed that the requirements according to the FDA guideline for bioanalytical methods are met. Furthermore the established short column methods were applied to the quantification of NO 711 in saturation experiments. The results obtained (i.e., K_d- and B_max-values) were almost identical as compared to those determined employing standard column dimension (55 mm × 2 mm).     

Synthesis and biological evaluation of compounds which contain pyrazole,thiazole and naphthalene ring as antitumor agents

A series of compounds which contain pyrazole, thiazole and naphthalene ring (1a–7a, 1b–7b, 1c–7c, 1d–7d) were firstly synthesized and their anti-proliferative activity, EGFR inhibitory activity, cytotoxicity and inhibition to Hela cell migration were evaluated. Compound 2-(3-(3,4-dimethylphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)thiazol-4(5H)-one (7d) displayed the most potent inhibitory activity (IC₅₀ = 0.86 μM for Hela and IC₅₀ = 0.12 μM for EGFR). Structure–activity relationship (SAR) analysis showed that the anti-proliferative activity was affected by A-ring-substituent (–OCH₃ > –CH₃ > –H > –Br > –Cl > –F). Docking simulation of compound 7d into EGFR active site showed that naphthalene ring of 7d with LYS721 formed two p–π bonds, which enhanced antitumor activity. Therefore, compound 7d may be developed as a potential antitumor agent.     

New heteroaryl nitrones with spin trap properties: Identification of a 4-furoxanyl derivative with excellent properties to be used in biological systems

A new series of heteroaryl nitrones, 1–7, bearing furoxanyl and thiadiazolyl moieties, were evaluated for their free radical-trapping properties. The physicochemical characterization by electron paramagnetic resonance (EPR) demonstrated its capability to trap and stabilize oxygen-, carbon-, sulfur-, and nitrogen-centered free radicals. The 4-furoxanyl nitrone 3 (FxBN), α(Z)-(3-methylfuroxan-4-yl)-N-tert-butylnitrone, showed appropriate solubility in aqueous solution and taking into account that this physicochemical property is very important for biological applications, we studied it deeply in terms of its trapping and kinetic behaviors. For this, kinetic studies of the hydroxyl adduct decay gave rate constants k_ST of 1.22 × 10¹⁰ dm³ mol^?1 s^?1 and half-live up to 7200 s at physiological pH, without any artifactual signals. The ability of FxBN to directly traps and stabilizes superoxide free radical, with a half-life of 1620 s at physiological pH, was also demonstrated. Besides, FxBN-hydroxyl and -superoxide adducts exhibited distinct and characteristic EPR spectral patterns. Finally, we confirmed the ability of FxBN to act as spin trap in a specific biological system, that is, in the free radical production of experimental anti-trypanosomatid drugs using Trypanosoma cruzi microsomes as biological system. Moreover, previous observations of low FxBN toxicity transform it in a good candidate for in vivo spin trapping.     

Determination of mildronate by LC–MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of mildronate in human plasma. Following a simple protein precipitation with methanol, the analyte was separated on a C₁₈ column by isocratic elution with methanol and 10 mM ammonium acetate (55:45; v/v), and then analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 0.01–20 μg/mL. The intra- and inter-batch precisions (as RSD, %) were less than 7.1%. The average extraction recovery was 87.5%. The method described above has been used, for the first time, to reveal the pharmacokinetics of mildronate injection in healthy subjects. After single intravenously administration of 250, 500 and 1000 mg mildronate, the elimination half-life (t_1/2) were (5.56 ± 1.55), (6.46 ± 1.07) and (6.55 ± 1.17) h, respectively. The Student–Newman–Keuls test results showed that peak plasma concentration (C_max) and the area under the plasma concentration versus time curve from time 0 to 24 h (AUC_0–24) were both linearly related to dose. The pharmacokinetics of mildronate fitted the linear dynamic feature over the dose range studied. The essential pharmacokinetic parameters of multidoses administration intravenously (500 mg, b.i.d) were as follows: t_1/2 was (15.34 ± 3.14) h; C_max was (25.50 ± 3.63) μg/mL; AUC_0–24 was (58.56 ± 5.57) mg h/L. The t_1/2 and AUC of multidoses administration intravenously were different from those of single-dose administration significantly. These findings suggested that accumulation of mildronate in plasma occurred.