Synthesis and properties of vinyl carbamate epoxide, a possible ultimate electrophilic and carcinogenic metabolite of vinyl carbamate and ethyl carbamate

Vinyl carbamate reacted with dimethyldioxirane in dry acetone to give a high yield of pure crystalline vinyl carbamate epoxide. This epoxide was characterized by its NMR and MS spectra and elementary analysis. It is unstable at room temperature and has a half-life in water solution of approximately 32 minutes. It reacts with adenosine to form 1,N6-ethenoadenosine and more of this etheno nucleoside was found in hydrolysates of hepatic RNA of male mice injected i.p. with the epoxide than with vinyl carbamate. Tests with Salmonella typhimurium TA1535 showed that this epoxide is a strong direct mutagen. It is also more toxic in the mouse than vinyl carbamate. Studies on the carcinogenicity of this epoxide are in progress.     

The effect of vinyl acetate in acetoclastic methanogenesis

The influence of vinyl acetate (VA) in the methanogenesis was evaluated, by using an upflow anaerobic sludge blanket reactor of 1.5L. The reactor was operated at 33.5 g/L volatile suspended solids to 30±2 °C, a hydraulic residence time of 1 day, an organic loading rate of 1 kgCOD/m3/d of two different mixtures of VA and glucose. The VA was methanized to 81% when its proportion was of 10% into reactor loading rate, when VA proportion increased to 25%, the methane production rate decreased to 62% and the acetate production rate increased almost 8 times. These results indicated that VA was only hydrolyzed and glucose was not used as a co-substrate. The effect of glucose on VA methanogenic degradation was evaluated through batch reactors of 60 mL, concluding that the glucose supported the methanogenesis without favoring the VA elimination. On the other hand, the results of the sludge from the reactor in the presence of VA demonstrated that VA caused an irreversibly inhibition of acetoclastic methanogenesis when the anaerobic sludge was exposed to this compound.     

Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins

Twenty-six epoxide and corresponding pyrazole derivatives, of the structurally related chalcones and combretastatin A-4 (CA-4), were synthesized and tested for in vitro cytotoxicity. These molecules were synthesized by epoxidation of the relevant chalcones, followed by reaction with hydrazine. The structures of epoxides 3 and 7, and pyrazole 17, were confirmed by X-ray diffraction studies. The relatively coplanar conformation of a 3',3',4',4',5',5'-hexamethoxypyrazole 17 was in good agreement with the shape for 3',3',4',4',5'-pentamethoxypyrazole 16, which was determined from molecular mechanics optimization. In vitro cytotoxicity of each class of compounds was obtained using a 72 h continuous exposure MTT assay against two murine cancer cell lines; B16 melanoma and L1210 leukemia. The effect of substitution in the A-ring is addressed: three methoxy groups versus two, generally increased cytotoxicity across both cell lines. In the majority of cases, the pyrazoles are generally more active than the epoxides, with the most active, 5-(3'-amino-4'-methoxyphenyl)-3-(3',4',5'-trimethoxyphenyl)pyrazole 21, possessing an IC(50) value of 5 and 2.4 microM (B16 and L1210, respectively). Due to their planar conformations, the pyrazoles are typically less active than the corresponding chalcones, which adopt angular conformations similar to CA-4. B-ring modifications confirmed that in general the amino compounds are more active than the corresponding nitro compounds. Varying the number and orientation of methoxy groups on the A-ring did not produce any significant differences in toxicity in the cell lines studied.     

Degradation of vinyl acetate by soil, sewage, sludge, and the newly isolated aerobic bacterium V2

Vinyl acetate is subject to microbial degradation in the environment and by pure cultures. It was hydrolyzed by samples of soil, sludge, and sewage at rates of up to 6.38 and 1 mmol/h per g (dry weight) under aerobic and anaerobic conditions, respectively. Four yeasts and thirteen bacteria that feed aerobically on vinyl acetate were isolated. The pathway of vinyl acetate degradation was studied in bacterium V2. Vinyl acetate was degraded to acetate as follows: vinyl acetate + NAD(P)+----2 acetate + NAD(P)H + H+. The acetate was then converted to acetyl coenzyme A and oxidized through the tricarboxylic acid cycle and the glyoxylate bypass. The key enzyme of the pathway is vinyl acetate esterase, which hydrolyzed the ester to acetate and vinyl alcohol. The latter isomerized spontaneously to acetaldehyde and was then converted to acetate. The acetaldehyde was disproportionated into ethanol and acetate. The enzymes involved in the metabolism of vinyl acetate were studied in extracts. Vinyl acetate esterase (Km = 6.13 mM) was also active with indoxyl acetate (Km = 0.98 mM), providing the basis for a convenient spectrophotometric test. Substrates of aldehyde dehydrogenase were formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde. The enzyme was equally active with NAD+ or NADP+. Alcohol dehydrogenase was active with ethanol (Km = 0.24 mM), 1-propanol (Km = 0.34 mM), and 1-butanol (Km = 0.16 mM) and was linked to NAD+. The molecular sizes of aldehyde dehydrogenase and alcohol dehydrogenase were 145 and 215 kilodaltons, respectively.     

Distribution of the coenzyme M pathway of epoxide metabolism among ethene- and vinyl chloride-degrading Mycobacterium strains

An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of Mycobacterium strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from Mycobacterium strain JS60 and the propene oxidizers Xanthobacter strain Py2 and Rhodococcus strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The Mycobacterium EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of Rhodococcus strain B-276 than that of Xanthobacter strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the Mycobacterium strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.     

Degradation of vinyl acetate by soil, sewage, sludge, and the newly isolated aerobic bacterium V2.

Vinyl acetate is subject to microbial degradation in the environment and by pure cultures. It was hydrolyzed by samples of soil, sludge, and sewage at rates of up to 6.38 and 1 mmol/h per g (dry weight) under aerobic and anaerobic conditions, respectively. Four yeasts and thirteen bacteria that feed aerobically on vinyl acetate were isolated. The pathway of vinyl acetate degradation was studied in bacterium V2. Vinyl acetate was degraded to acetate as follows: vinyl acetate + NAD(P)+----2 acetate + NAD(P)H + H+. The acetate was then converted to acetyl coenzyme A and oxidized through the tricarboxylic acid cycle and the glyoxylate bypass. The key enzyme of the pathway is vinyl acetate esterase, which hydrolyzed the ester to acetate and vinyl alcohol. The latter isomerized spontaneously to acetaldehyde and was then converted to acetate. The acetaldehyde was disproportionated into ethanol and acetate. The enzymes involved in the metabolism of vinyl acetate were studied in extracts. Vinyl acetate esterase (Km = 6.13 mM) was also active with indoxyl acetate (Km = 0.98 mM), providing the basis for a convenient spectrophotometric test. Substrates of aldehyde dehydrogenase were formaldehyde, acetaldehyde, propionaldehyde, and butyraldehyde. The enzyme was equally active with NAD+ or NADP+. Alcohol dehydrogenase was active with ethanol (Km = 0.24 mM), 1-propanol (Km = 0.34 mM), and 1-butanol (Km = 0.16 mM) and was linked to NAD+. The molecular sizes of aldehyde dehydrogenase and alcohol dehydrogenase were 145 and 215 kilodaltons, respectively.     

Lipase catalyzed synthesis of benzyl acetate in solvent-free medium using vinyl acetate as acyl donor

Use of vinyl acetate as acyl donor in transesterification of benzyl alcohol catalyzed by a commercially available lipase (Lipozyme RM IM) gave 100% conversion in 10 min. The excess acyl donor and the enzyme could be recovered and reused. Unlike the chemical catalytic processes, it produced no undesirable side product.     

Synthesis, X-ray crystal structural study, antiviral and cytostatic evaluations of the novel unsaturated acyclic and epoxide nucleoside analogues

A series of the novel purine and pyrimidine nucleoside analogues were synthesised in which the sugar moiety was replaced by the 4-amino-2-butenyl (2-6 and 10-18) and oxiranyl (8 and 20) spacer. The Z- (2-6) and E-isomers (10-18) of unsaturated acyclic nucleoside analogues were synthesized by condensation of 2- and 6-substituted purine and 5-substituted uracil bases with Z- (1) or E-phthalimide (9) precursors. The oxiranyl nucleoside analogues (8 and 20) were obtained by epoxidation of 1 and 9 with m-chloroperoxybenzoic acid and subsequent coupling with adenine. The new compounds were evaluated for their antiviral and antitumor cell activities. Among the olefinic nucleoside analogues, Z-isomer of adenine containing 4-amino-2-butenyl side chain (6) exhibited the best cytostatic activities, particularly against colon carcinoma (SW 620, IC50 = 26 microM). Its E-isomer 15 did not show any antiproliferative activity against malignant tumor cell lines, except for a slight inhibition of colon carcinoma (SW 620, IC50 = 56.5 microM) cells. In general, Z-isomers showed better cytostatic activities than the corresponding E-isomers. (Z)-4-Amino-2-butenyl-adenine nucleoside analogue 6 showed albeit modest but selective activity against HIV-1 (EC50 = 4.83 microg mL(-1)).     

Synthesis and SAR of conformationally restricted inhibitors of soluble epoxide hydrolase

A series of conformationally restricted inhibitors of human soluble epoxide hydrolase (sEH) has been developed. Inhibition potency of the described compounds ranges from 4.2 microM to 1.1 nM against recombinant sEH. N-(1-Acetylpiperidin-4-yl)-N'-(adamant-1-yl) urea (5a) was found to be a potent inhibitor (IC(50) = 7.0 nM) that was also orally bioavailable in canines.     

Synthesis, antiproliferative activity and genotoxicity of novel anthracene-containing aminophosphonates and a new anthracene-derived Schiff base

A new Schiff base, 9-anthrylidene-furfurylamine and three novel anthracene-containing α-aminophosphonates, [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine, [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were synthesized. The compounds have been characterized by elemental analysis, TLC, IR, NMR and fluorescent spectra. The aminophosphonates and their synthetic precursors were tested for in vitro antitumor activity on a panel of seven human epithelial cancer cell lines. Safety testing was performed both in vitro (3T3 NRU test) and in vivo on ICR mice for genotoxicity and antiproliferative activity. 9-Anthrylidene-furfurylamine and [N-methyl(diethoxyphosphonyl)-1-(9-anthryl)]furfurylamine were most potent cytotoxic agents towards colon carcinoma cell line HT-29. The latter compound exhibited also antiproliferative activity to HBL-100, MDA-MB-231 and 647-V cells. The aminophosphonate [N-methyl(dimethoxyphosphonyl)-1-(9-anthryl)]-p-toluidine and its synthetic precursor 9-anthrylidene-p-toluidine were found to be cytotoxic to HBL-100 and HT-29 tumor cell lines, respectively. Moderate genotoxic and antiproliferative activity in vivo and low toxicity to Balb/c 3T3 (clone 31) mouse embryo cells were observed for all tested compounds. The subcellular distribution of two tested compounds in a tumor cell culture system was also studied.     

Synthesis of epoxide and vicinal diol regioisomers from docosahexaenoate methyl esters

Docosahexaenoic acid (22:6(n-3)) was recently shown to be metabolized by liver microsomes to vicinal diol regioisomers. To identify the diols, and to compare their biological actions with those of epoxide precursors, we developed a chemical method to synthesize microgram to milligram amounts of epoxides and corresponding diols. In brief, methylated docosahexaenoate was reacted for 15 min with 0.1 eq m-chloroperoxybenzoic acid. After normal- and reverse-phase high performance liquid chromatography, six products were isolated. The underivatized or hydrogenated products were characterized and identified using capillary gas-liquid chromatography and mass spectrometry. The products were identified as 19,20-, 16,17-, 13,14-, 10,11-, 7,8-, and 4,5-epoxy-docosapentaenoate. Per incubation, the total epoxide yield from 22:6(n-3) was 8.6%. By reincubating unused substrate 10-20 times (cycling), the total epoxide could be increased to 55-70%. As found for epoxides of arachidonic and eicosapentaenoic acids, the yield of individual regioisomers increased as the distance between the targeted double bond and carbomethoxy group increased. Each epoxide regioisomer was hydrolyzed to its corresponding vicinal diol. The gas-liquid chromatographic retention times and mass spectra of the diol products were found to match those of metabolites produced by cytochrome P-450 monooxygenases.     

Effects of vinyl acetate and acetaldehyde on sperm morphology and meiotic micronuclei in mice

The testicular genotoxic effects of vinylacetate (VA) and its hydrolysis product, acetaldehyde (AA), were studied in mice by analyzing the induction of morphologically abnormal sperm and meiotic micronuclei. VA significantly increased the frequency of sperm abnormalities at 500 mg/kg/day while lower doses were ineffective. AA did not induce abnormal sperm. Neither of the compounds influenced the frequency of meiotic micronuclei. VA, but not AA, caused a dose-dependent decrease in sperm production and a reduction of testicular weight at 500 and 125 mg/kg/day.     

Synthesis and characterization of camphorsulfonyl acetate of cellulose

Novel cellulose derivatives were prepared from reacting (1R)-(+)-camphor-10-sulfonic chloride (CSC) with cellulose acetate (CA) in acetone and triethylamine. The reaction conditions, including reaction time and reactant molar ratios, were optimized. The structure of the products was confirmed by means of 1H NMR, 13C NMR, FT-IR and elementary analysis. The techniques were also used to determine the degree of the substitution of camphorsulfonyl groups (DSCS). The data calculated from 1H NMR, 13C NMR, percent grafting (G %) and elementary analysis coincided with those from chemical analysis. Compared to cellulose acetate, the cellulose derivatives exhibited decreased thermal stability, improved solubility in organic solvents and enhanced enantioselectivity towards tyrosine isomers. The solubility and enantioselectivity increased with increasing degrees of camphorsulfonyl substitution.     

In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA-protein cross-links

Lead is present in the natural and occupational environment and is reported to interact with DNA, but the mechanism of this interaction is not fully understood. Using the alkaline comet assay we showed that lead acetate at 1-100 microM induced DNA damage in isolated human lymphocytes measured the change in the comet tail length. At 1 and 10 microM we observed an increase in the tail length, whereas at 100 microM a decrease was seen. The former effect could follow from the induction of DNA strand breaks and/or alkali-labile sites (ALS), the latter from the formation of DNA-DNA and/or DNA-protein cross-links. No difference was observed between tail length for the alkaline and pH 12.1 versions of the assay, which indicates that strand breaks and not ALS are responsible for the tail length increase induced by lead. The neutral version of the test revealed that lead acetate induced DNA double-strand breaks at all concentrations tested. The presence of spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) did not influence the level of DNA damage induced by lead. Post-treatment of the lead-damaged DNA (at 100 microM treatment concentration) by endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing oxidized DNA bases, as well as 3-methyladenine-DNA glycosylase II, an enzyme recognizing alkylated bases, gave rise to a significant increase in the extent of DNA damage. Proteinase K caused an increase in comet tail length, suggesting that lead acetate might cross-link DNA with nuclear proteins. Vitamin A, E, C, calcium chloride and zinc chloride acted synergistically on DNA damage evoked by lead. The results obtained suggest that lead acetate may induce single-strand breaks (SSB) and double-strand breaks (DSB) in DNA as well as DNA-protein cross-links. The participation of free radicals in DNA-damaging potential of lead is not important and it concerns other reactive species than could be trapped by DMPO or PBN.     

DNA cross-links in human leucocytes treated with vinyl acetate and acetaldehyde in vitro

Human leucocytes were incubated in the presence of vinyl acetate or acetaldehyde (10-20 mM) for 4 h at 37 degrees C in vitro. DNA damage was analysed by alkaline elution. None of the compounds induced a detectable increase in the frequency of DNA strand breaks. Cells exposed to 5 Gy of X-ray immediately after treatment and before alkaline elution showed a clear, dose-dependent retardation of the elution rate in comparison with X-irradiated control cells. These results demonstrate that both vinyl acetate and acetaldehyde induce DNA cross-links in human cells.