Cis-trans isomerization of nitrofuran derivatives by xanthine oxidase

Enzymatic cis-trans isomerization of nitrofuran derivatives was 3-(5-Nitro-2-furyl)-2-(2-furyl)-demonstrated with milk xanthine oxidase. acrylamide (AF-2) and 3-(5-nitro-2-furyl)-2-(5-bromo-2-furyl)acrylamide (NFBFA) were mainly converted from the cis to the trans form by this enzyme supplemented with an electron donor. This enzymatic reaction was further characterized with respect to its cofactor requirements. Finally, a new cis-trans isomerization mechanism, which is based on transfer of a single electron by a nitroreductase such as xanthine oxidase to a nitrofuran derivative to give the anion free radical, was proposed.     

Mutagenic activity of carcinogenic and noncarcinogenic nitrofurans and of urine of rats fed these compounds

The nitrofurans, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), nitrofurantoin, 5-nitro-2-furoic acid, 5-nitro-2-furamidoxime, 5-nitrofurfurylidene diacetate and the urine of rats fed these compounds, were assayed for mutagenic activity in Salmonella typhimurium strains TA100 and TA100FR1. All the nitrofurans were mutagenic in the order: AF-2 and FANFT > nitrofurantoin > 5-nitro-2-furamidoxime > 5-nitrofurfurylidene diacetate > 5-nitro-2-furoic acid. Strain TA100 was more sensitive than TA100FR1 to the mutagenic influence of these nitrofurans. Only the urine of rats fed AF-2, FANFT and nitrofurantoin had mutagenic activity. Again, TA100 was more sensitive than TA100FR1. The mutagenicity of the urine was not increased by treatment with β-glucuronidase. AF-2, 2-amino-4-(5-nitro-2-furyl)thiazole (deformylated product of FANFT) and nitrofurantoin were excreted in the urine of rats fed these compounds; whereas the other nitrofurans were not excreted.     

Chromosome breakage and neoplastic transformation of Syrian golden hamster embryonic cells in tissue culture by transplacental application of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Hamster embryos were treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) in vivo (in the mother) by transplacental application. The fetuses were isolated 24 h after administration of the AF-2 and cultured. Within the first 24h of primary culture, some parts of the cells were treated with colcemide for 3 h so that mitotic cells could be observed in the first cell cycle in vitro. Cultured embryonic fibroblasts in metaphase plates showed a marked dose-dependence in chromosomal aberrations. Transplacental application of AF-2 also caused slightly dose-dependent morphological transformation. When some transformed colonies were cloned and transferred to the hamster cheek pouch, these cells produced tumors in the host animals. This new in vivo--in vitro combination assay system is considered to be useful for detection of environmental potential carcinogens.     

Mutagenesis in cultured human diploid cells. III. Induction of 8-azaguanine-resistant mutations by furylfuramide.

Trans-2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furylfuramide: FF or AF2) was tested for ability to induce 8-azaguanine (8AG) resistant mutations in cultured human diploid cells. FF had a relatively severe cytotoxic effect on the cells. From the concentration-survival curve, the D0 value for 2-h treatment with FF was estimated to be 11 mug/ml. When cells were treated with FF at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected in medium containing 8AG at 30 mug/ml, the induced mutation frequency increased gradually with increase in concentration of FF. When cells were treated with FF at 10 mug/ml for 2 h, cultured in normal medium for various periods of mutation expression time, and selected with 8AG at 30 mug/ml, the highest induced mutation frequency was obtained with 48 h of mutation expression time. Microscopic examination of the numbers of cells in colonies indicated that the total number of cells increased by half during this mutation expression time of 48 h.     

Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues

The deacylations of N-[4-(5-nitro-2-furyl)-2-thiazolyl] fonnamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl] acetarnide (NFTA) and formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl] hydrazide (FNT) by liver, kidney, small intestines and stomach of mouse, rat, hamster and guinea pig were investigated. FANFT was deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). FANFT formamidase activity was higher in the liver and small intestines of mouse, hamster and guinea pig, and small intestines and stomach of rat. There was no detectable FANFT formamidase activity in the stomach of the mouse and hamster. Neither NFTA nor FNT was deacylated by the rodent tissue homogenates studied. It is suggested that (1)4 ANFT is a metabolite of FANFT but not NFTA; (2) 2-hydrazino-4-(5-nitro-2-furyl)thiazole (HNFT) may not be a metabolite of FNT; and (3) the induction of tumors by FANFT, NFTA and FNT may not be due to a common carcinogenic metabolite, although these chemicals demonstrate similar organ specificities in some of these rodents.     

A cis-trans isomerising activity of Escherichia coli. Isomerization from 2-(2-furyl)-3-cis-(5-nitro-2-furyl) acrylamide (furylfuramide) to its trans isomer.

The soluble enzyme fraction derived from Escherichia coli K-12 JE2100 cells was found to exhibit, in addition to Nadh- and NADPH-dependent reductase activities, NADH-dependent cis-trans isomerising activity toward 2-(2-furyl-3-(5-nitro-2-furyl)acrylamide leading to a specific change in geometrical configuration of the vinyl group at the 2-position from cis to trans but not in the reverse direction. This furylfuramide-isomerising action of bacteria was dicoumarol insensitive, and did not require glutathione for full activity. The particulate enzyme fraction derived from JE2100 cells, although it showed little reductase activity toward furylfuramide in the presence of either NADH or NADPH, revealed an isomerising activity in the presence of NADH.     

2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) is a weak in vivo clastogen as revealed by the micronucleus assay

The in vivo clastogenicity of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) was examined in the micronucleus test using peripheral blood from three mouse strains (ICR, CD-1, and MS/Ae) and bone marrow from one rat strain (Sprague-Dawley). Doses up to the maximum tolerated were tested. The chemical was given once, twice, thrice, or four times via either the i.p. or p.o. route. Under some conditions, ICR and CD-1 mice showed an increased frequency of micronucleated reticulocytes, but definite conclusions were difficult to draw because the increases were very slight. MS/Ae mice showed a markedly elevated micronucleated reticulocyte frequency after the double and triple ip treatments. Rats showed a slightly but statistically significantly increased frequency of micronucleated polychromatic erythrocytes after double i.p. treatments. These results indicate that AF-2 is a weak in vivo clastogen.     

Metabolism and mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine in Chinese hamster ovary cells

The mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine (THFMP) have been studied in Chinese hamster ovary (CHO) cells in tissue culture. THFMP is relatively unstable in physiological buffers, being facilely converted to 6-mercaptopurine (6-MP) even in the absence of cells. Consequently, THFMP undergoes metabolic conversions characteristic of 6-MP, namely formation of 6-thioIMP and incorporation into DNA as 6-thioguanine (6-TG) nucleotide. A number of purines are capable of preventing the toxicity of THFMP in wild-type cells in a manner similar to that of 6-MP. However, exogenous purines and pyrimidines did not prevent the toxicity of THFMP to cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase). Cells lacking HGPRTase were 20–40-fold resistant to 6-TG and 6-MP but were only 2–4-fold resistant to THFMP. Furthermore, the time-course for killing CHO cells deficient in HGPRTase was different from that in wild-type cells containing the enzyme. There was no apparent effect of THFMP on the utilization of precursors for DNA, RNA or protein synthesis in the enzyme-deficient mutant cell line. The results suggest that THFMP is converted non-enzymatically to 6-MP and shares its mechanisms of action in wild-type cells containing HGPRTase, i.e., inhibition of de novo purine biosynthesis and incorporation into DNA as 6-TG nucleotide. However, the mechanism of action of THFMP in cells lacking HGPRTase is probably unique and is presently unknown.     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Mutagenicity of urine of various species fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide or 2-amino-4-(5-nitro-2-furyl)thiazole

Rats, mice and hamsters, which are susceptible to the bladder carcinogenesis by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), and guinea pigs, which are not, were fed a diet containing 0.188% FANFT or 0.188% 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) for 1 week and their urine was then examined for mutagenicity for S. typhimurium TA100. The mutagenicities of the urine of these species fed FANFT were approximately equal. Similarly, that of the urine of these species fed ANFT were also approximately equal. However, the urine from FANFT-fed animals was approximately 10 times as mutagenic as that from ANFT-fed animals. ANFT was detected only in the urine of rats, mice or hamsters fed FANFT. A positive correlation between the susceptibility toward bladder carcinogenesis by FANFT and urinary ANFT excretion was demonstrated, although the correlation between this susceptibility and urine mutagenicity was lacking.     

Synthesis and in vitro leishmanicidal activity of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles

A series of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles (5a-d and 6a-j) were synthesized and evaluated against Leishmania major promastigotes using (3)H-thymidine incorporation. Most of the compounds showed activity better than the reference drug sodium stibogluconate (Pentostam). The most active compound was 6c (IC(50)=0.1 microM).     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.     

Inhibitors of HCV NS5B polymerase. Part 1: Evaluation of the southern region of (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid

A novel series of nonnucleoside HCV NS5B polymerase inhibitors were prepared from (2Z)-2-(benzoylamino)-3-(5-phenyl-2-furyl)acrylic acid, a high throughput screening lead. SAR studies combined with structure based drug design focusing on the southern heterobiaryl region of the template led to the synthesis of several potent and orally bioavailable lead compounds. X-ray crystallography studies were also performed to understand the interaction of these inhibitors with HCV NS5B polymerase.     

Substrate inhibition of Pseudomonas aeruginosa elastase by 3-(2-furyl)acryloyl-glycyl-L-phenylalanyl-L-phenylalanine

The kinetics of hydrolysis by Pseudomonas aeruginosa elastase at 37 degrees C and pH 7.3 of 3-(2-furyl)acryloyl-glycyl-L-phenylalanyl-L-phenylalanine is compatible with nonproductive substrate inhibition, i.e., v = V.[S]/(Km + [S] + [S]2/K1), and the values of Km, Ki, and kappa cat are 1.4 mM, 5.0 mM, and 240 s-1, respectively. Product inhibition experiments are in agreement with an ordered release of product, with L-phenylalanyl-L-phenylalanine, the amino-containing product, being released first from the elastase.product complex. The values of Ki for L-phenylalanyl-L-phenylalanine and 3-(2-furyl)acryloyl-glycine are 1.5 and 4.0 mM, respectively. Kinetic experiments indicate that the second molecule of substrate combines with elastase.substrate to form a dead-end elastase . (substrate)2 complex.     

Microbial synthesis of S (+)-1-(2-furyl)-3-pentanol: A pheromone intermediate of bark beetle

Summary The synthesis of S (+)-1-(2-furyl)-3-pentanol (3), a key intermediate in the synthesis of chalcogran (1) is described via microbial reduction of 1-(2-furyl)-1-pentene-3-one (2) using fungusRhizopus arrhizus.     

1-(Benzofuran-2-yl)-2-(3,3,3-trifluoropropyl)aminopentane HCl, 3-F-BPAP,antagonizes the enhancer effect of (-)-BPAP in the shuttle box and leaves the effect of (-)-deprenyl unchanged

The subcutaneous administration of 1 mg/kg tetrabenazine, once daily for 5 days, which depletes the catecholamine stores in the brain, significantly inhibits in rats the acquisition of a two-way conditioned avoidance reflex in the shuttle box. Enhancer substances, the tryptamine-derived selective and highly potent enhancer, R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane HCl [(-)-BPAP] (0.05-10 mg/kg), the beta-phenylethylamine (PEA)-derived enhancer, (-)-deprenyl (1-5 mg/kg) and the (-)-deprenyl analogue, free of MAO-B inhibitory potency, (-)-1-phenyl-2-propylaminopentane HCl [(-)-PPAP], (1-5 mg/kg), antagonize in a dose-dependent manner the inhibition of learning caused by tetrabenazine. 1-(Benzofuran 2 yl)-2-(3,3,3-trifluoropropyl)aminopentane HCl [3 F BPAP], a newly synthetized analogue of (-)-BPAP with low specific activity, significantly antagonized the enhancer effect of (-)-BPAP but left the effect of (-)-deprenyl and (-)-PPAP unchanged. This is the first proof for a difference in the mechanism of action between a PEA-derived enhancer substance and its tryptamine-derived peer.     

柯拉斯那沉香中2-(2-苯乙基)色酮类化学成分研究

为研究柯拉斯那(Aquilaria crassna Pierre ex Lecomte)沉香的化学成分。实验采用多种柱色谱方法从该沉香中分离得到9个2-(2-苯乙基)色酮类化合物,通过现代波谱学技术分别鉴定为6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(1)、5-羟基-6-甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(2)、tetrahydrochromone F(3)、6-甲氧基-2-[2-(3′-甲氧基-4′-羟基苯基)乙基]色酮(4)、6-甲氧基-7-羟基-2-[2-(4′-甲氧基苯基)乙基]色酮(5)、6,7-二甲氧基-2-[2-(3′-羟基-4′-甲氧基苯基)乙基]色酮(6)、6,7-二甲氧基-2-[2-(4′-甲氧基苯基)乙基]色酮(7)、6-羟基-2-[2-(4′-羟基苯基)乙基]色酮(8)、5-羟基-2-[2-(2′-羟基苯基)乙基]色酮(9)。化合物2、3和5～9均为首次从柯拉斯那所得沉香中分离得到。采用MTT法对单体化合物的细胞毒活性进行测试,测试结果表明,化合物1,2和4具有微弱的细胞毒活性。     

Product profiles in enzymic and non-enzymic oxidations of the lignin model compound erythro-1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol

The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H₂O₂, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.     

Reduction of nitrofuran derivatives by xanthine oxidase and microsomes: Isolation and identification of reduction products

An investigation was carried out to identify the reduction products of nitrofurazone and AF-2 (2-furyl)-3-(5-nitro-2-furyl)acrylamide by milk xanthine oxidase, rat liver xanthine oxidase, and rat liver microsomes. Data obtained from mass spectrometry and other methods indicated that the ethyl acetate-extractable major product of each nitrofuran derivative should be the corresponding amine derivative or the equivalent compound. This conclusion was further confirmed by an examination of stoichiometry. The reduced nitrofurazone was finally identified as 5-amino-2-furfural semicarbazone by comparative studies with the authentic specimen. The reduced AF-2 was tentatively identified as 2-(2-furyl)-3-(5-oxo-2-pyrrolin-2-yl)acrylamide. A reduction pathway for this conversion is postulated.