Effects of MCI-186 upon neutrophil-derived active oxygens

Reactions of 3-methyl-1-phenyl-2-pyrazoline-5-one (MCI-186) with hypochlorous acid and superoxide were analysed by spectrophotometry and mass spectrometry. The results were applied to the neutrophil system to evaluate the scavenging activity of neutrophil-derived active oxygen species by MCI-186. MCI-186 reacted rapidly with hypochlorous acid (1 x 10(6) M(-1)s(-1)) to form a chlorinated intermediate, followed by a slow conversion to a new spectrum. MCI-186 consumed 3 moles of hypochlorous acid and did not react with superoxide. The newly synthesized fluorescence probes, 2-[6-(4'-amino)-phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) and 2-[6-(4'-hydroxy)phenoxy-3H-anthen-3-on-9-yl]benzoic acid (HPF) successfully detected neutrophil-derived active oxygens (Setsukinai K, Urano Y, Kakinuma K, Majima HJ, Nagano T. Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species. J Biol Chem 2003; 278: 3170-3175). The rate constants for the reaction of hypochlorous acid with MCI-186 and fluorescence probes was in the order of MCI-186 > APF > HPF. Fluorescence due to the oxidation of APF and HPF was observed with the stimulated neutrophils. The result that the intensity from APF oxidation was higher than that from HPF oxidation is compatible with reports that APF selectively reacts with hypochlorous acid. Fluorescence due to oxidation of both APF and HPF decreased when the reactions were carried out in the presence of a fluorescence probe and MCI-186 in a dose-dependent manner. These results indicate that MCI-186 effectively scavenges neutrophil-derived hypochlorous acid and other active oxygens.     

Evasion of Legionella pneumophila from the bactericidal system by reactive oxygen species (ROS) in macrophages

We demonstrated that the production of reactive oxygen species (ROS) by U937 macrophage-like cells was suppressed upon infection with a wild type Legionella pneumophila strain, whereas such suppression was not observed in the case of infection with intracellular growth-deficient mutants. This was supported not only by measuring ROS released into the supernatants of cell cultures by chemiluminescence assaying but also by detecting intracellular ROS with a fluorescent probe, 2-[6-(4'-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF), under a confocal laser scanning microscope. Furthermore, more than 60% of the phagosomes containing intracellular growth-deficient mutants were colocalized with p47(phox), which is the cytosolic subunit of NADPH oxidase, consistently throughout the observation period in an early stage of bacterial infection. In contrast, the colocalization of p47(phox) was suppressed after infection with the wild type strain. These results suggest that the interference with ROS production by U937 cells infected with wild type L. pneumophila is due to a failure of NADPH oxidase activation through inhibition of p47(phox) recruitment to phagosomes harboring bacteria. The results also highlighted the difference in the nature of phagosomes between ones harboring the wild type and ones the intracellular growth-deficient strains.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Reactive oxygen species, dietary restriction and neurotrophic factors in age-related loss of myenteric neurons

We have studied the mechanisms underlying nonpathological age-related neuronal cell death. Fifty per cent of neurons in the rat enteric nervous system are lost between 12 and 18 months of age in ad libitum (AL) fed rats. Caloric restriction (CR) protects almost entirely against this neuron loss. Using the ROS-sensitive dyes, dihydrorhodamine (DHR) and 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) in vitro, we show that the onset of cell death is linked with elevated intraneuronal levels of reactive oxygen species (ROS). Treatment with the neurotrophic factors NT3 and GDNF enhances neuronal antioxidant defence in CR rats at 12-15 months and 24 months but not in adult or aged AL-fed animals. To examine the link between elevated ROS and neuronal cell death, we assessed apoptotic cell death following in vitro treatment with the redox-cycling drug, menadione. Menadione fails to increase apoptosis in 6-month neurons. However, in 12-15mAL fed rats, when age-related cell death begins, menadione induces a 7- to 15-fold increase in the proportion of apoptotic neurons. CR protects age-matched neurons against ROS-induced apoptosis. Treatment with neurotrophic factors, in particular GDNF, rescues neurons from menadione-induced cell death, but only in 12-15mCR animals. We hypothesize that CR enhances antioxidant defence through neurotrophic factor signalling, thereby reducing age-related increases in neuronal ROS levels and in ROS-induced cell death.     

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.     

Isolation and identification of the intermediates during pyrazole formation of some carbohydrate hydrazone derivatives

Reaction of the oxidation product of L-ascorbic acid, dehydro-L-ascorbic acid, with o-phenylenediamine, followed by 2,4,6-trichlorophenylhydrazine (3) afforded 3-[1-(2,4,6-trichlorophenylhydrazono)-L-threo-2,3,4-trihydroxybut-1-yl]quinoxalin-2(1H)one (4), whose structure was deduced from studying its periodate oxidation, which gave the glyoxal derivative 3-[1-(2,4,6-trichlorophenylhydrazono)glyoxal-1-yl]quinoxalin-2(1H)one (5) that upon reduction afforded 3-[1-(2,4,6-trichlorophenylhydrazono)-2-hydroxyethy-1-yl]quinoxalin-2(1H)one (6). The reaction of 5 with 3 afforded the bishydrazone 3-[1,2-bis(2,4,6-trichlorophenylhydrazono)glyoxal-1-yl]quinoxalin-2(1H)one. The reaction of 5 with acetic anhydride in pyridine afforded the 2,3-dihydrofuro[2,3-b]quinoxaline derivative 2-acetoxy-3-[2-acetyl-2-(2,4,6-trichlorophenyl)hydrazono)]-2,3-dihydrofuro[2,3-b]quinoxaline. Acetylation of 4 with acetic anhydride in pyridine afforded the acyclic diacetate intermediate 3-[3,4-di-O-acetyl-2-deoxy-1-(2,4,6-trichlorophenylhydra-zono)but-2-en-1-yl]quinoxalin-2(1H)one (12), which was also obtained from the reaction of 4 with boiling acetic anhydride. Compound 12 rearranged under the reaction conditions to give the pyrazole derivatives 3-[5-(ace-toxymethyl)-1-(2,4,6-trichlorophenyl)pyrazol-3-yl]quinoxalin-2(1H)one (14) and 2-acetoxy-3-[5-(acetoxymethyl)-1-(2,4,6-trichlorophenyl)pyrazol-3-yl)]quinoxaline (15), as well as the 2,3-dihydrofuro[2,3-b]quinoxaline derivative 2-(2-acetoxyethen-2-yl)-3-[2-(2,4,6-trichlorophenyl)hydrazono]-2,3-dihydrofuro[2,3-b]quinoxaline. Acetylation of 3-[5-(hydroxymethyl)-l-(2,4,6-trichlorophenyl)pyrazol-3-yl]quinoxalin-2(1H)one (16) with acetic anhydride in pyridine or 12 with boiling acetic anhydride afforded 15 and 16, respectively. Treatment of 4 with diluted sodium hydroxide afforded the pyrazolo[2,3-b]quinoxaline (flavazole) derivative 1-(2,4,6-trichlorophenyl)-3-(L-threo-glycerol-1-yl)pyrazolo[2,3-b]quinoxaline whose acetylation afforded the acetyl derivative 3-(2,3,4-tri-O-acetyl-L-threo-glycerol-1-yl)-1-(2,4,6-trichlorophenyl)pyrazolo[2,3-b]quinoxaline. The assigned structures were based on spectral analysis. The activity of compound 4 against hepatitis B virus has been studied.     

Stereoselective synthesis and X-ray structure of 1-(4-O-benzyl-1,5-anhydro-2,3-dideoxy-D-arabino-hex-1-enitol-3-yl )-4-me thoxy-1H-pyrimidin-2-one

The intramolecular glycosylation of a 6-O-(4-methoxypyrimidin-2-yl) derivative of phenyl 2,3-didehydrohex-2-eno-1-thiopyranoside afforded 3-deoxy-3-(4-methoxypyrimidin-2-on-1-yl)glycal as the major product in a stereoselective manner. The isolated 3'-deoxyglycal nucleoside was characterized by X-ray and NMR spectral analyses.     

Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (DeltaPsim) and the production of reactive oxygen species. Fluorescent probes were used to assess DeltaPsim (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolyl-carbocyanine iodide), reactive oxygen species [DCF, 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester] and [Ca2+][Fluo-3, glycine N-[4-[6-[(acetyloxy)methoxy]-2,7-dichloro-3-oxo-3H-xanthen-9-yl]-2-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxyethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-N-[2-[(acetyloxy)methoxy]-2-oxyethyl]-(acetyloxy)methyl ester] in human kidney cells (HK-2 cells) and in a line of human small cell carcinoma cells (GLC4 cells), because these do not express cyclosporin A-sensitive P-glycoprotein. We used transfected GLC4 cells expressing P-glycoprotein as control for GLC4 cells. NIM811 (N-methyl-4-isoleucine-cyclosporin) and PSC833 (SDZ-PSC833) were applied as selective mitochondrial permeability transition pore and P-glycoprotein blockers, respectively. To study the effect of cyclosporin A on mitochondrial function, we isolated mitochondria from fresh pig livers. Cyclosporin A and PSC833 induced a more than two-fold increase in JC-1 fluorescence in HK-2 cells, whereas NIM811 had no effect. None of the three substances induced a significant increase in JC-1 fluorescence in GLC4 cells. Despite this, cyclosporin A, NIM811 and PSC833 induced a 1.5-fold increase in DCF fluorescence (P<0.05) and a two-fold increase in Fluo-3 fluorescence (P<0.05). Studies in isolated mitochondria showed that blockage of mitochondrial permeability transition pores by cyclosporin A affected neither DeltaPsim, ATP synthesis, nor respiration rate. The mitochondrial permeability transition pore blockers cyclosporin A and NIM811, but also the non-mitochondrial permeability transition pore blocker PSC833, induced comparable degrees of reactive oxygen species production and cytosolic [Ca2+]. Neither mitochondria, effects on P-glycoprotein nor inhibition of calcineurin therefore play a role in cyclosporin A-induced oxidative stress and disturbed Ca2+ homeostasis.     

Stereoselective Synthesis and X-Ray Structure of 1-(4-O-Benzyl-1,5-anhydro-2,3-dideoxy-D-arabino-hex-1-enitol-3-yl)-4-methoxy-1H-pyrimidin-2-one

Abstract

The intramolecular glycosylation of a 6-O-(4-methoxypyrimidin-2-yl) derivative of phenyl 2,3-didehydrohex-2-eno-1-thiopyranoside afforded 3-deoxy-3-(4-methoxypyrimidin-2-on-1-y1)glycal as the major product in a stereoselective manner. The isolated 3′-deoxyglycal nucleoside was characterized by X-ray and NMR spectral analyses.     

Chemiluminescence evaluation of oxidative damage to biomolecules induced by singlet oxygen and the protective effects of antioxidants

A chemiluminescence (CL) method was developed for the evaluation of oxidative damage to biomolecules induced by singlet oxygen ((1)O(2)) and for the evaluation of the protective effects of antioxidants. The (1)O(2) was generated from the reaction of H(2)O(2)+OCl(-). Results showed that the CL signal from the reaction of H(2)O(2)+OCl(-) was weak, however, it was enhanced dose-dependently with the addition of DNA and unsaturated fatty acid, respectively. Spectra analysis indicated that the enhanced CL could be ascribed to the decay of triplet-excited carbonyl compounds, which were generated from the reaction of (1)O(2) plus the biomolecules. On the other hand, the enhanced CL produced in the above systems could be effectively inhibited by lycopene, beta-carotene, VC, and VE, but could not be inhibited by mannitol, SOD, and NaN(3). The mechanism therein was discussed.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.

Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils

Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H(2)O(2) in Earl's solution (pH=7.2) at 37 degrees C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H(2)O(2) system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN(3)), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.     

The detection of hydroxyl radicals in vivo

Several indirect methods have been developed for the detection and quantification of highly reactive oxygen species (hROS), which may exist either as free hydroxyl radicals, bound “crypto” radicals or Fe(IV)-oxo species, in vivo. This review discusses the strengths and weaknesses associated with those most commonly used, which determine the hydroxylation of salicylate or phenylalanine. Chemical as well as biological arguments indicate that neither the hydroxylation of salicylate nor that of phenylalanine can guarantee an accurate hydroxyl radical quantitation in vivo. This is because not all hydroxylated product-species can be used for detection and the ratio of these species strongly depends on the chemical environment and on the reaction time. Furthermore, at least in the case of salicylate, the high concentrations of the chemical trap required (mM) are known to influence biological processes associated with oxidative stress.Two, newer, alternative methods described, the 4-hydroxy benzoic acid (4-HBA) and the terephthalate (TA) assays, do not have these drawbacks. In each case reaction with hROS leads to only one hydroxylated product. Thus, from a chemical viewpoint, they should provide a better hROS quantitation. Further work is needed to assess any possible biological effects of the required millimolar (4-HBA) and micromolar (TA) concentrations of the chemical traps.     

Generation of reactive oxygen species by a persulfide (BnSSH)

Hydropersulfides (RS(x)SH) have been implicated as important intermediates in the cell-killing action of the anticancer natural products leinamycin and varacin. It has been suggested that persulfides can mediate the conversion of molecular oxygen to reactive oxygen species (O2*-, H2O2, and HO*). Here, experiments with synthetic benzyl hydrodisulfide (BnSSH) provide direct evidence that persulfides readily decompose to produce reactive oxygen species under physiologically relevant conditions.     

Synthesis of enantiopure trans-N-Boc-3-aminobicyclo[2.2.2]octane-2-carboxylic acids and their bicyclic 1,3-amino alcohol derivatives via the [4+2] cycloaddition of 1,3-cyclohexadiene to a chiral β-nitroacrylate

The chiral β-nitroacrylate 2 derived from the (R)- or (S)-4-(3-hydroxy-4,4-dimethyl-2-oxopyrrolidin-1-yl) benzoic acid 1 acts as a reactive dienophile in a diastereoselective Diels-Alder reaction with 1,3-cyclohexadiene. The major cycloadducts have been isolated and transformed into enantiopure trans(2S,3S)- or (2R,3R)-N-Boc-3-aminobicyclic[2,2,2]octane-2-carboxylic acids 5. The trans-(2S,3S)- or (2R,3R)-N-Boc 3-(hydoxymethyl)-2-aminobicyclic[2,2,2]octane 6 derivatives were also obtained.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells

Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.     

The synthesis of some branched-chain-sugar nucleoside analogues.

1-(2,3-Epoxy-5-O-trityl-beta-D-lyxofuranosyl)uracil was treated with a number of carbon nucleophiles. Ethynyl lithium gave 3'-deoxy-3'-ethynyl-5'-O-trityl-ara-uridine, which was reduced to the corresponding 3'-ethenyl compound. Sodium cyanide gave 3'-cyano-3'-deoxy-5'-O-trityl-ara-uridine which upon alkaline hydrolysis gave the corresponding 3'-carboxamido compound. 1,3-Dithian-2-yl lithium gave 3'-deoxy-3'-(1,3-dithian-2-yl)-5'-O-trityl-ara-uridine. The trityl group was removed from each of these compounds by mild acidic hydrolysis. Treatment of 2 with 0.1M H2sO4 and mercury (II) acetate afforded 3'-acetyl-3'-deoxy-ara-uridine which upon reduction with NaBH4 gave 3'-deoxy-3'-(1-hydroxyethan-1-yl)-ara-uridine. Acetylation of 6 yielded 5'-O-acetyl-3'-acetyl-2',3'-didehydro-2',3'-dideoxyuridine which upon reduction with NaBH4 produced a mixture of 5'-O-acetyl-2',3'-didehydro-2',3'-dideoxy-3'-(1-hydroxyethan -1-yl)uridine and 1-(R)[5-(S)-acetoxymethyl-4-(1-hydroxyethan-1-yl)-tetrahydrofuran- 2-yl]- uracil. Reduction of 14 with Raney nickel followed by removal of the trityl group gave 3'-deoxy-3'-methyl-ara-uridine.     

Synthesis of some newer derivatives of 2-amino benzoic acid as potent anti-inflammatory and analgesic agents

Diazotization of N-benzylidene anthranilic acids 1a-1n at pH 9 yielded N-[alpha-(phenylazo) benzylidene] anthranilic acids 2a-2n and at pH 3 yielded N-benzylidene-5-(phenylazo) anthranilic acids 3a-3n. When compounds 3a-3n were treated with thioglycolic/thiolactic acid in the presence of anhydrous ZnCl(2), 2-(4-oxo-2-phenylthiazolidin-3-yl)-5-(phenylazo) benzoic acids 4a-4n were afforded. The newly synthesized compounds were screened for their anti-inflammatory and analgesic activities and were compared with standard drugs, aspirin and phenylbutazone. Out of the compounds studied, the most active compound 4n showed more potent activity than the standard drugs at all doses tested.