Oxidative Cyclization of o-(1-Hydroxy-2-alkynyl)-N-tosylanilides for the Synthesis of 4-Quinolones.

The treatment of easily accessible o-(1-hydroxy-2-alkynyl)-N-tosylanilides 1 with excess manganese(IV) oxide in the presence of substoichiometric tetrabutylammonium iodide (TBAI) in chloroform (or in the absence of TBAI in dimethylformamide, DMF) promoted a sequential oxidation/intramolecular hydroamination to give 4-quinolones 3 and/or (Z)-2-alkylidene-3-oxindoles (Z)-4 in good yields. Possibly, MnO2 played dual roles as an oxidant and as a Lewis acidic activator of intermediary ynones 2. The product distributions between 3 and (Z)-4 could be controlled by the choice of solvents.

Reaction of para-hydroxybenzoic acid esters with singlet oxygen in the presence of glutathione produces glutathione conjugates of hydroquinone, potent inducers of oxidative stress.

The determination and toxicological characterization of products of the reaction between p-hydroxybenzoic acid esters (parabens) and singlet oxygen ((1)O(2)) are very important because of the frequent use of parabens in cosmetics and possible generation of (1)O(2) in the skin. We observed (1)O(2)-dependent production of mono-, di-, and tri-substituted glutathione (GSH) conjugates of hydroquinone (HQ) during visible light-irradiation of a mixture of methyl or ethyl paraben and GSH in the presence of rose bengal (RB). 1,4-Benzoquinone (BQ) and HQ were produced during the irradiation in the absence of GSH. While a mixture of BQ and GSH produced only mono-substituted conjugate, irradiation of the mixture with RB produced mono-, di-, and tri-substituted conjugates. These observations indicate that (1)O(2) is involved both in the production of BQ and HQ from parabens and in the formation of multi-substituted GSH conjugates from mono-substituted conjugate. Tri-substituted conjugate generated larger amounts of hydrogen peroxide in an aqueous solution than mono-substituted conjugates or HQ did. Detection of semiquinone radical suggests that the autoxidation of conjugates is related to the generation of hydrogen peroxide. The results obtained in this study indicate that parabens may induce oxidative stress in the skin after conversion to GSH conjugates of HQ by reacting with (1)O(2) and GSH.

Kinetic study on ESR signal decay of nitroxyl radicals, potent redox probes for in vivo ESR spectroscopy, caused by reactive oxygen species.

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants.

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (1O2) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of 1O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (*OH) adduct of DMPO (DMPO-OH) resulting from 1O2-dependent generation of *OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its *OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of *OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of *OH from 1O2, and that spin trap-mediated *OH generation hardly occurs with DEPMPO.

Singlet oxygen-mediated hydroxyl radical production in the presence of phenols: whether DMPO-*OH formation really indicates production of *OH?

The reaction of singlet oxygen (1O2) generated by ultraviolet-A (UVA)-visible light (lambda > 330 nm) irradiation of air-saturated solutions of hematoporphyrin with phenolic compounds in the presence of a spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), gave an electron spin resonance (ESR) spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-*OH). In contrast, the ESR signal of 5,5-dimethyl-2-pyrrolidone-N-oxyl, an oxidative product of DMPO, was observed in the absence of phenolic compounds. The ESR signal of DMPO-*OH decreased in the presence of either a *OH scavenger or a quencher of *O2 and under anaerobic conditions, whereas it increased depending on the concentration of DMPO. These results indicate both 1O2- and DMPO-mediated formation of free *OH during the reaction. When DMPO was replaced with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), no DEPMPO adduct of oxygen radical species was obtained. This suggests that 1O2, as an oxidizing agent, reacts little with DEPMPO, in which a strong electron-withdrawing phosphoryl group increases the oxidation potential of DEPMPO compared with DMPO. A linear correlation between the amounts of DMPO-*OH generated and the oxidation potentials of phenolic compounds was observed, suggesting that the electron-donating properties of phenolic compounds contribute to the appearance of *OH. These observations indicate that 1O2 reacts first with DMPO, and the resulting DMPO-1O2 intermediate is immediately decomposed/reduced to give *OH. Phenolic compounds would participate in this reaction as electron donors but would not contribute to the direct conversion of 1O2 to *OH. Furthermore, DEPMPO did not cause the spin-trapping agent-mediated generation of *OH like DMPO did.

Two reaction sites of a spin label, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), with hydroxyl radical.

The exposure of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) to hydroxyl radical ((.)OH) led to the appearance of a new triplet ESR signal along with a reduction in the intensity of the TEMPOL signal. The observed spectrum was in good agreement with the spectrum simulated with 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPOL, and the addition of sodium borohydride to the product solution led to the disappearance of the new signal. These results indicate the formation of TEMPON during the reaction. A comparison of the concentration of TEMPON formed with that of nitroxyl compounds that lost the electron spin resonance (ESR) signal showed that the rate of TEMPON formation is less than one-tenth that of the paramagnetism loss caused by (.)OH in the pH range 6.5-8.5. These results indicate that TEMPOL has two reaction sites with (.)OH; the nitroxyl group and the 4-position of the piperidine ring. The reaction at the 4-position should contribute to the (.)OH scavenging activity of TEMPOL together with that at the nitroxyl group, although the rate of the former reaction is relatively low.

Reactive oxygen species scavenging ability of a new compound derived from weathered coal.

The scavenging activity of three fulvic acids (named XWCS-1, XWCS-4, and XWCS-8 according to time taken for ozonolysis) obtained by ozonolysis of humic acid extracted from Xinjiang (China) weathered coal and a fulvic acid (named XWCFA) extracted from the same coal towards reactive oxygen species such as superoxide radical (O(2)(.)(-)) and hydroxyl radical ((.)OH) was investigated with an electron spin resonance (ESR)-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. O(2)(.)(-) was generated with a hypoxanthine-xanthine oxidase system. (.)OH was generated by three different methods; (i) FeSO(4)-hydrogen peroxide (H(2)O(2)) system, (ii) Cu(en)(2)-H(2)O(2) system, and (iii) UVB photolysis of H(2)O(2). At physiological pH, XWCS-1 had the greatest O(2)(.)(-) scavenging activity, followed by XWCS-4, XWCS-8 and XWCFA. XWCFA had the greatest ?OH scavenging activity among the four fulvic acids, whereas XWCS-1 and XWCS-4 enhanced the production of (.)OH from a metal-catalyzed hydroxyl radical generating system, suggesting that these molecules act as prooxidants in the presence of metal ion.

Acceleration of regeneration of mucosa in small intestine damaged by ionizing radiation using anabolic steroids.

Damage to intestine is a serious problem after accidental radiation exposure. To examine substances to ameliorate damage by postirradiation administration, we focused on the regeneration process after irradiation of the intestine. Using experimental systems, the effects of clinically used sex hormones on regeneration were compared. An anabolic steroid, nandrolone (19-nortestosterone), stimulated proliferation in IEC-6 epithelial cells. A single injection of 19-nortestosterone ester with prolonged action into mice 24 h after abdominal irradiation at a lethal dose of 15.7 Gy showed significant life-saving effects. Regeneration indicators such as microcolonies of BrdU-incorporated cells at day 5 and c-myb mRNA expression levels at day 4 were enhanced by 19-nortestosterone administration. In contrast, high concentrations of estradiol inhibited growth of IEC-6 cells. Treatment of abdominally irradiated mice with estradiol ester decreased levels of regeneration indicators and survival. These results suggest the effectiveness of the anabolic steroid as well as the importance of manipulation of steroid receptors in the recovery of mucosa damaged by radiation.

Circadian transitions in radiation dose-dependent augmentation of mRNA levels for DNA damage-induced genes elicited by accurate real-time RT-PCR quantification.

Molecular mechanisms of intracellular response after DNA-damage by exposure to ionizing radiation have been studied. In the case of cells isolated from living body of human and experimental animals, alteration of the responsiveness by physiological oscillation such as circadian rhythm must be considered. To examine the circadian variation in the response of p53-responsible genes p21, mdm2, bax, and puma, we established a method to quantitate their mRNA levels with high reproducibility and accuracy based on real-time RT-PCR and compared the levels of responsiveness in mouse hemocytes after diurnal irradiation to that after nocturnal irradiation. Augmentations of p21 and mdm2 mRNA levels with growth-arrest and of puma mRNA before apoptosis were confirmed by time-course experiment in RAW264.7, and dose-dependent increases in the peak levels of all the RNA were shown. Similarly, the relative RNA levels of p21, mdm2, bax, and puma per GAPDH also increased dose-dependently in peripheral blood and bone marrow cells isolated from whole-body-irradiated mice. Induction levels of all messages reduced by half after nighttime irradiation as compared with daytime irradiation in blood cells. In marrow cells, nighttime irradiation enhanced the p21 and mdm2 mRNA levels than daytime irradiation. No significant difference in bax or puma mRNA levels was observed between nighttime and daytime irradiation in marrow cells. This suggests that early-stage cellular responsiveness in DNA damage-induced genes is modulated between diurnal and nocturnal irradiation.

Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term.

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.

Efficient radical scavenging ability of artepillin C, a major component of Brazilian propolis, and the mechanism.

Hydrogen transfer from artepillin C to cumylperoxyl radical proceeds via one-step hydrogen atom transfer rather than via electron transfer, the rate constant of which is comparable to that of (+)-catechin, indicating that artepillin C can act as an efficient antioxidant.