1-Methyl-3-nitropyridine: an efficient oxidant of NADH in non-enzymatic and enzyme-mediated processes

It is shown that NADH can be effectively oxidized by 1-methyl-3-nitropyridine in non-enzymatic and enzyme-mediated processes. Mechanistic issues of these reactions are discussed. These processes seem to contribute to the observed cytotoxicity of 1-methyl-3-nitropyridine. A key role of 1-methyl-3-nitropyridinyl radical formed in the enzyme-mediated processes is emphasized.     

Microbial transformation of 2-amino-4-methyl-3-nitropyridine

Biotransformation of the highly substituted pyridine derivative 2-amino-4-methyl-3-nitropyridine by Cunninghamella elegans ATCC 26269 yielded three products each with a molecular weight of 169?Da which were identified as 2-amino-5-hydroxy-4-methyl-3-nitropyridine, 2-amino-4-hydroxymethyl-3-nitropyridine, and 2-amino-4-methyl-3-nitropyridine-1-oxide. Biotransformation by Streptomyces antibioticus ATCC 14890 gave two different products each with a molecular weight of 169?Da; one was acid labile and converted to the other stable product under acidic conditions. The structure of the stable product was established as 2-amino-4-methyl-3-nitro-6(1H)-pyridinone, and that of the less stable product was assigned as its tautomer 2-amino-6-hydroxy-4-methyl-3-nitropyridine. Four of the five biotransformation products are new compounds. Several strains of Aspergillus also converted the same substrate to the lactam 2-amino-4-methyl-3-nitro-6(1H)-pyridinone. Microbial hydroxylation by C. elegans was found to be inhibited by sulfate ion. In order to improve the yield and productivity of the 5-hydroxylation reaction by C. elegans, critical process parameters were determined and Design of Experiments (DOE) analyses were performed. Biotransformation by C. elegans was scaled up to 15-l fermentors providing 2-amino-5-hydroxy-4-methyl-3-nitropyridine at ca. 13?% yield in multi-gram levels. A simple isolation process not requiring chromatography was developed to provide purified 2-amino-5-hydroxy-4-methyl-3-nitropyridine of excellent quality.     

Photoacoustic and luminescence characterization of nitrogen heterocyclic aromatic UV-MALDI matrices in solution

A group of nitrogen heterocyclic aromatic compounds used, among others, as UV-MALDI matrices was studied. By using spectroscopic, luminescence and photoacoustic techniques, as well as time resolved phosphorescence for singlet oxygen production determination, the behaviour of 9-aminoacridine (9AA), 3-aminoquinoline (3AQ), 2-(2-aminoethylamino)-5-nitropyridine (AAN) and 3,4-dihydro-7-methoxy-1-methyl-9H-pyrido[3,4-b] indole (harmaline, HLA) in acetonitrile solutions is described. The results show that for these compounds radiationless processes that release prompt heat to the media are a quite important deactivation mechanism.     

Mononuclear copper(II) nitrato complexes with methyl-substituted 4-nitropyridine N-oxide. Physicochemical and cytotoxic characteristics

Three new complexes, products of the interaction of Cu(NO₃)₂ and methyl-substituted 4-nitropyridine N-oxides were synthesized and characterized by elemental analysis, magnetic, spectroscopic (IR, FIR and EPR), thermal and X-ray methods. The complexes (magnetic moments 1.70-1.81 BM at 300 K) of general formula [Cu(H₂O)(NO₃)₂L₂], L = 2-methyl-4-nitropyridine N-oxide and [Cu(NO₃)₂ L′₂], where L′ = 2,6-dimethyl- and 2,3,6-trimethyl-4-nitropyridine N-oxide were obtained. The compounds were unstable upon dissolution. The X-ray single crystal structure of Cu(II) complex with 2,6-dimethyl-4-nitropyridine N-oxide was determined and analysed. The compounds and free ligands were tested in vitro on the cytotoxic activity against MCF-7 and SW-707 human cancer cell lines. The complexes with 4-nitropyridine N-oxide (a reference) and 2-methyl-4-nitropyridine N-oxide show a significant anti-proliferative activity against studied cell lines. A reciprocal relationship between the activity and the number of methyl groups was observed. Both ligands and complexes are cytotoxic active but to the different cell lines.     

Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for CRF1 receptors

Synthesis and evaluation of [O-methyl-11C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [11C]SN003 ([11C]6), as a PET imaging agent for CRF1 receptors, in baboons is described. 4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5), the precursor molecule for the radiolabeling, was synthesized from 2,4-dichloro-6-methyl-3-nitropyridine in seven steps with 20% overall yield. The total time required for the synthesis of [11C]SN003 is 30 min from EOB using [11C]methyl triflate in the presence of NaOH in acetone. The yield of the synthesis is 22% (EOS) with >99% chemical and radiochemical purities and a specific activity of >2000 Ci/mmol. PET studies in baboon show that [11C]6 penetrates the BBB and accumulates in brain. No detectable specific binding was observed, likely due to the rapid metabolism or low density of CRF1 receptors in primate brain.     

Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P₄-R) agonists

High affinity and selective small molecule agonists of the S1P(4) receptor (S1P(4)-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P(4)-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P(4)-R agonists exquisitely selective over the remaining S1P(1-3,5)-Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P(4)-R.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents.

1. O-Iodosobenzoate and 2,2'-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol. 2. The inhibition of the Ca2+-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2'-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide. 3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher Km for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Nitroreductase activity of heart lipoamide dehydrogenase.

A novel reaction catalysed by lipoamide dehydrogenase is described. In the presence of NADH, lipoamide dehydrogenase reduces the nitro group of 4-nitropyridine and 4-nitropyridine N-oxide. The elution profiles from a DEAE-cellulose column for the dehydrogenase and nitroreductase activities are identical. Chemical modifications of critical amino acid residues suggest that the two activities share a common catalytic domain. Nitro reduction catalysed by lipoamide dehydrogenase was monitored spectrophotometrically and chromatographically. The major product from the enzymic reduction of 4-nitropyridine was isolated and characterized structurally as NN-bis(pyridinyl)hydroxylamine, which is formed presumably via 4-hydroxyaminopyridine in a four-electron redox reaction.     

Systematic coordination chemistry and cytotoxicity of copper(II) complexes with methyl substituted 4-nitropyridine N-oxides

Three new nitrato copper(II) complexes of dimethyl substituted 4-nitropyridine N-oxide were synthesized and characterized by elemental analysis, magnetic, spectroscopic, thermal and X-ray methods, respectively. They were isolated as trans isomers, mononuclear (μ = 1.70-1.88 BM), five (1-2) and four (3) coordinate species of general formula [Cu(NO₃)₂(H₂O)L₂] where L = 2,3-dimethyl-, 2,5-dimethyl-4-nitropyridine N-oxide and [Cu (NO₃)₂L₂], L = 3,5-dimethyl-4-nitropyridine N-oxide, respectively. The X-ray crystal structure of (1) (L = 2,3-dimethyl-4-nitropyridine N-oxide) was determined. The organic ligands, the complexes and copper hexaqua ion as a reference were tested in vitro on the cytotoxic activity against human cancer cell lines: MCF-7 (breast), SW-707 (colon) and P-388 (murine leukemia). The complexes are relatively strong cytotoxic agents towards P-388 cell line. Comparative analysis was performed for all known copper(II) complexes containing methyl derivatives of the 4-nitropyridine N-oxide on the basis of their composition, structure and cytotoxic activities. To obtain the typical structure for these species (i.e., 4-coordinate mononuclear of the type trans-[Cu(inorganic anion)₂L₂]), two methyl groups must be situated on both sides of nitrogen atom(s) (i.e., NO and NO₂) in the ligand. The biological activity was found to be strongly dependent upon the number of the methyl groups and the type of cell line. The best cytotoxic results were found for the complexes without substituents or with one methyl group. Generally, for all cell lines, the complexation increased cytotoxicity when compared with the free ligands.     

Sulphydryl groups in photosynthetic energy conservation. IV. Inhibition of the ATPase of chloroplast coupling factor 1 by sulphydryl reagents

1. o-Iodosobenzoate and 2,2′-dithio bis-(5-nitropyridine) inhibited by about fifty per cent the ATPase activity of heat-activated chloroplast coupling factor 1 only when present during the heating but were without effect when added before or after the activation. Reversion of this inhibition was only obtained by a second heat treatment with 10 mM dithioerythritol.2. The inhibition of the Ca²⁺-ATPase of coupling factor 1 by o-iodosobenzoate or 2,2′-dithio bis-(5-nitropyridine) was not additive with similar inhibitions obtained with the alkylating reagents iodoacetamide and N-ethylmaleimide.3. The heat-activated ATPase of o-iodosobenzoate-treated coupling factor 1 had a higher K_m for ATP, without modification of V. The modified enzyme was desensitized against the allosteric inhibitor ADP.     

CRF ligands via Suzuki and Negishi couplings of 3-pyridyl boronic acids or halides with 2-benzyloxy-4-chloro-3-nitropyridine

A series of imidazo[4,5-b]pyridines with a 7-(3-pyridyl) substituent is described as high affinity CRF receptor ligands. Individual analogues were synthesized from key intermediates obtained via palladium-catalyzed coupling of 3-pyridyl zinc or boronic acid organometallic intermediates with 2-benzyloxy-4-chloro-3-nitropyridine 12.     

The Mode of Action of Certain 3- and 5-Nitropyridines and Pyrimidines. IV. Development of Drug-Resistance by Trichomonas vaginalis.

SYNOPSIS. Certain 5-nitropyridines and pyrimidines are growth inhibitory to Trichomonas vaginalis in vitro. Populations were made resistant either to 2-amino-5-nitropyrimidine, or 2-amino-5-nitropyridine, or 2-hydroxy-5-nitropyridine, by growth in gradually increasing concentrations of one of these inhibitors. After resistance was maximal, clonal lines were established. The development of resistance to any one of these inhibitors was accompanied by resistance to the other two. Since cross-resistance was complete, these inhibitors act at the same locus. This in vitro resistance persisted in vivo.
Resistance was accompanied by an increase in the generation time and a decrease in the maximal obtainable population. These growth parameters were unique for each resistant clone. The growth of the clone made resistant to 2-amino-5-nitropyridine was the closest to the normal clone. Its metabolic capacity, as measured by conventional Warburg techniques, was less than half of the parent strain and anaerobically was the same as had been previously found for non-resistant cells which had been grown for 3 hr with inhibitor. The mechanisms of killing by the inhibitor and the development of drug resistance are the same. The inhibitor kills the cells by holding them in an early metabolic sequence. Some of them succeed in growing by using this metabolic sequence and reinforcing it with an alternate aerobic route.     

Neurotoxicity studies with the monoamine oxidase B substrate 1-methyl-3-phenyl-3-pyrroline

The neurotoxic properties of the parkinsonian inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are dependent on its metabolic activation in a reaction catalyzed by centrally located monoamine oxidase B (MAO-B). This reaction ultimately leads to the permanently charged 1-methyl-4-phenylpyridinium species MPP(+), a 4-electron oxidation product of MPTP and a potent mitochondrial toxin. The corresponding 5-membered analogue, 1-methyl-3-phenyl-3-pyrroline, is also a selective MAO-B substrate. Unlike MPTP, the MAO-B-catalyzed oxidation of 1-methyl-3-phenyl-3-pyrroline is a 2-electron process that leads to the neutral 1-methyl-3-phenylpyrrole. MPP(+) is thought to exert its toxic effects only after accumulating in the mitochondria, a process driven by the transmembrane electrochemical gradient. Since this energy-dependent accumulation of MPP(+) relies upon its permanent charge, 1-methyl-3-phenyl-3-pyrrolines and their pyrrolyl oxidation products should not be neurotoxic. We have tested this hypothesis by examining the neurotoxic potential of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-chlorophenyl)-3-pyrroline in the C57BL/6 mouse model. These pyrrolines did not deplete striatal dopamine while analogous treatment with MPTP resulted in 65-73% depletion. Kinetic studies revealed that both 1-methyl-3-phenyl-3-pyrroline and its pyrrolyl oxidation product were present in the brain in relatively high concentrations. Unlike MPP(+), however, 1-methyl-3-phenylpyrrole was cleared from the brain quickly. These results suggest that the brain MAO-B-catalyzed oxidation of xenobiotic amines is not, in itself, sufficient to account for the neurodegenerative properties of a compound like MPTP. The rapid clearance of 1-methyl-3-phenylpyrroles from the brain may contribute to their lack of neurotoxicity.     

The identification of pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum

Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1). that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.     

Mechanisms in microbial evolution

Molecular genetic studies with prokaryotic microorganisms reveal that many different molecular processes contribute to the formation of spontaneous mutations. Besides infidelities in DNA replication and the consequences of environmental mutagens, enzyme-mediated DNA rearrangements bring about important, evolutionarily relevant alterations in the genetic information. Particular attention is given in this article to site-specific recombination at secondary crossover sites and to the transposition of mobile genetic elements with relaxed target specificity. Besides these diverse processes of genomic mutation the acquisition of genetic information from other organisms plays an uncontested role in microbial evolution. Enzymes and organelles mediating any of these mutational processes can be looked at as biological functions acting at the level of populations for the needs of biological evolution, rather than to fulfill the needs of individual living organisms.     

Pentanol isomer synthesis in engineered microorganisms

Pentanol isomers such as 2-methyl-1-butanol and 3-methyl-1-butanol are a useful class of chemicals with a potential application as biofuels. They are found as natural by-products of microbial fermentations from amino acid substrates. However, the production titer and yield of the natural processes are too low to be considered for practical applications. Through metabolic engineering, microbial strains for the production of these isomers have been developed, as well as that for 1-pentanol and pentenol. Although the current production levels are still too low for immediate industrial applications, the approach holds significant promise for major breakthroughs in production efficiency.     

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.     

Generation of genetic diversity by DNA rearrangements in resting bacteria

Abstract Transpositional DNA rearrangements importantly contribute to the genomic plasticity of bacteria, their viruses and plasmids. Interestingly, enzymatically mediated transposition is not limited to propagating bacteria, but it also occurs in prolonged periods of rest. As revealed with RFLP analysis, genetic polymorphism increases steadily upon storage of bacteria in the resting phase. These results are discussed here in the wider context of accumulating knowledge on molecular mechanisms contributing to overall spontaneous mutagenesis, which is the result of a multitude of specific, often enzyme-mediated processes of variation generation.     

Formycin Analogs II. Antiviral and Cytotoxic s-Triazolo [4, 3-a] - and [1, 5-a] Pyridine Derivatives

Abstract

The novel N-bridgehead formycin analog 3- β-D-ribofuranosyl-8-amino-s-triazolo [4, 3-a] pyridine (8a-aza-4, 6-dideaza formycin) has been prepared from 5- [2, 3, 5-tri-O-benzoyl- β-D-ribofuranosyl] - (2H)-tetrazole and 2-chloro-3-nitropyridine. The synthetic route used an initial condensation followed by deprotection and subsequent hydrogenation to afford 2a. 2-Hydroxyethoxymethyl group, an acyclic group, that mimics the ribofuranose unit was also introduced. These compounds were tested against type 1 herpes and poliovirus in tissue culture and their effect on cellular RNA and DNA synthesis was determined. All derivatives possess considerable cytotoxic effect which is expressed more with ribofuranosyl derivatives.