Oxidative dealkylation and reductive denitrosation of nitrosomethylaniline in vitro

N-Nitrosomethylaniline (NMA) was incubated with liver microsomes from female mice and rats. Both formaldehyde and nitrite formation were determined in the same incubation mixture under various experimental conditions. The animals were pretreated with phenobarbital (PB) or butylhydroxytoluene (BHT) in order to modify microsomal monooxygenase activities. Furthermore, various possibilities were tried to supply the microsomal system with reducing equivalents (NADPH-regenerating system, NADPH-regenerating system plus NADH or NADH alone). It can be deduced from these experiments that both enzymatic activities--oxidative demethylation and reductive denitrosation of NMA--do not proceed in a parallel manner. Thus both reactions are different from each other. They represent two separate pathways in nitrosamine metabolism.     

Synthesis of derivatives of phosphonic glutathione analogs

Five analogs of S-t-butyl glutathione containing phosphonic analogs of glycine and glutamic acid were obtained by standard procedures of MA activation in solution. Simultaneous deprotection of phosphonic, carboxylic and amino groups was achieved in the silylation reaction.     

Cyclobutane analogs of GABA

Bothcis-andtrans-3-aminocyclobutane-1-carboxylic acid have been synthesized as conformationally restricted analogs of GABA. The cis isomer displayed weak to moderate GABA-like activity with respect to (1) inhibition of GABA uptake in rat brain minislices, (2) inhibition of sodium-independent binding of GABA to rat brain membranes, (3) activity as a substrate for GABA aminotransferase, and (4) depression of the firing rate of cat spinal neurons in vivo. The trans isomer was less effective on all four assays. The result has been interpreted in terms of the conformational pinning back of the polar groups by the cyclobutane ring in the trans GABA analog so that unfavorable steric interactions would occur between one of the methylene groups and a region of steric hindrance at the active sites for particular GABA processes.     

Incorporation of phosphonic acid diesters into lipid model membranes. Part II. X-ray and neutron diffraction studies.

Mixtures of egg phosphatidylcholine and phosphonic acid diethyl or dibutyl esters of the general type RP(O)(OR')2 with R = hexane or dodecane were studied at room temperature in the fluid lamellar state by X-ray and by neutron diffraction. Generally a molar ratio of lipid and ester of 1:0.5 was used. Additionally an equimolar lipid/ester mixture of hexane phosphonic acid diethyl ester was studied. Depending on the ester used and its concentration a single L alpha-phase was observed above a certain water content which changes to an L alpha + water two phase system at high water concentration. Despite the large amounts of the amphiphilic ester molecules incorporated in the membrane and their high molecular asymmetry, the mixtures qualitatively show the typical hydration and swelling behaviour of non-charged lipid membranes. However, the incorporation of the esters induces a higher hydration capacity, a lateral extension and a decrease in membrane thickness. The position of the ester molecules and their orientation in the membrane were determined by neutron diffraction using partially deuterated esters. The esters were found to be located with their phosphonic moiety near or in the lipid/water interface. The lamellar structure contradicts this location of the cone-shaped ester molecules which should increase the tendency to form hexagonal structures. However, the experimental findings can be understood if one considers a partial interdigitation of the last hydrocarbon groups of the lipid chains accompanied by a larger disorder in the hydrophobic centre of the membrane. In the case of hexane phosphonic acid dibutyl ester, a vertical translocation of the ester takes place below a certain water content where it is distributed between two locations at the lipid water interface and the centre of the membrane.     

Phosphinic,phosphonic and seleninic acid bioisosteres of isonipecotic acid as novel and selective GABA(C) receptor antagonists

A number of amino acids bioisosterically derived from the specific GABA_A agonist, isonipecotic acid, were electrophysiologically characterized as antagonists at GABA_C ρ₁ receptors expressed in Xenopus oocytes. The phosphinic acid analogue of isonipecotic acid, piperidin-4-ylphosphinic acid (2), was comparable with the standard GABA_C antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), in terms of potency and GABA_C versus GABA_A receptor selectivity. Whereas the phosphonic acid analogue, piperidin-4-ylphosphonic acid (4), was at least an order of magnitude weaker than piperidin-4-ylphosphinic acid as a GABA_C antagonist, the seleninic acid analogue, piperidin-4-ylseleninic acid (SEPI, 6), was the most potent and selective GABA_C antagonist within the group of isonipecotic acid derived amino acids studied.     

Hydrolysis of novel diacylglycerol analogs and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [3H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, over crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-14C]palmitoyl-2-acetyl-sn-glycerol and [1-14C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-times the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[3H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

Exploring the UDP pocket of LpxC through amino acid analogs

Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.     

Boronic acid mono- and diesters of the aldopentoses

The four aldopentoses ribose, arabinose, xylose, and lyxose were evaluated to test their suitability as linear linkers for the formation of intrinsically chiral covalent organic boronic ester networks. Based on X-ray crystal structures of the reaction products with phenylboronic acid, arabinose and xylose formed boronic acid diesters. Lyxose and ribose formed monoesters under the conditions employed. NMR shielding constants were calculated by DFT methods. The results are highly correlated with the experimentally observed NMR shift values.     

Covalent molecular imprinting of bisphenol A using its diesters followed by the reductive cleavage with LiAlH(4)

Bisphenol A (BPA) recognition materials were synthesized by a covalent imprinting technique using BPA-dimethacrylate or BPA-diacrylate as the template monomer. Binding sites in the polymers consisted of two hydroxyl groups that are generated by reducing the ester bonds of the template monomer with lithium aluminum hydride. The polymers strongly adsorbed BPA and structurally related compounds, however, other endocrine disruptors were hardly adsorbed. The BPA-dimethacrylate-based polymer interacted with the samples more strongly than the BPA-diacrylate-based polymer.     

Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1

The sodium- and chloride-dependent electrogenic gamma-aminobutyric acid (GABA) transporter GAT-1, which transports two sodium ions together with GABA, is essential for synaptic transmission by this neurotransmitter. Although lithium by itself does not support GABA transport, it has been proposed that lithium can replace sodium at one of the binding sites but not at the other. To identify putative lithium selectivity determinants, we have mutated the five GAT-1 residues corresponding to those whose side chains participate in the sodium binding sites Na1 and Na2 of the bacterial leucine-transporting homologue LeuT(Aa). In GAT-1 and in most other neurotransmitter transporter family members, four of these residues are conserved, but aspartate 395 replaces the Na2 residue threonine 354. At varying extracellular sodium, lithium stimulated sodium-dependent transport currents as well as [3H]GABA uptake in wild type GAT-1. The extent of this stimulation was dependent on the GABA concentration. In mutants in which aspartate 395 was replaced by threonine or serine, the stimulation of transport by lithium was abolished. Moreover, these mutants were unable to mediate the lithium leak currents. This phenotype was not observed in mutants at the four other positions, although their transport properties were severely impacted. Thus at saturating GABA, the site corresponding to Na2 behaves as a low affinity sodium binding site where lithium can replace sodium. We propose that GABA participates in the other sodium binding site, just like leucine does in the Na1 site, and that at limiting GABA, this site determines the apparent sodium affinity of GABA transport.     

Synthesis of phosphonic analogs of enkephalins. Pentapeptides with COOH replaced by PO3H2 group

Eight analogs of enkephalins containing the phosphonic analogs of Gly, Leu, Met, Phe and Pro have been synthesized by standard procedures in solution. The key intermediates in the synthesis were pure diastereomers of dialkyl N-L-phenylalanyl/aminoalkanephosphonates obtained from racemic dialkyl aminoalkanephosphonates by coupling with phenylalanine and resolution of the resulting mixture of L, D and L, L dipeptides by means of column chromatography.     

Lipase-catalyzed preparation of mono- and diesters of ferulic acid

Lipophilic and stable derivatives of ferulic acid are required to improve its efficacy in fatty foods and to optimize its use in cosmetic and pharmaceutical preparations. We report an improved synthesis of ferulic acid monoesters (ethyl ferulate and lauryl ferulate) using immobilized lipase from Candida antarctica B (CALB) in diisopropyl ether (DIPE). Maximum yields were 89% and 85% in 200 h for ethyl and lauryl ferulate, respectively. Ethyl ferulate was further acylated with vinyl esters to form ferulate diesters. 4-Acetoxy-ethyl ferulate was obtained with the immobilized lipase from Alcaligenes sp. (QLG) with 59% yield in 72 h, whereas 4-dodecanoyloxy-ethyl ferulate (a new compound) was synthesized with 52% yield in 72 h using CALB. DIPE was the best solvent for the transesterifications. Finally, the anti-inflammatory activity of the synthesized derivatives was evaluated in vitro; the compounds bearing a dodecyl chain showed improved anti-inflammatory activity compared with short-chain esters.     

Novel phosphinic and phosphonic acid analogues of the anticonvulsant valproic acid

1-Propylbutylphosphinic acid 2, (1-propylbutyl)methylphosphinic acid 3 and 1-propylbutylphosphonic acid 4 have been synthesized as bioisosteres of the corresponding carboxylic acid valproate 1, which is a potent anticonvulsant. The novel phosphinic and phosphonic acids were tested for their anticonvulsant activity and were found to be inactive.     

Phosphonic acid-containing analogues of mycophenolic acid as inhibitors of IMPDH

The design, synthesis, and IMPDH inhibitory activity of a series of phosphonic acid-containing analogues of mycophenolic acid are described.     

Lipoic acid analogs with enhanced pharmacological activity

Lipoic acid (1,2-dithiolane-3-pentanoic acid) is a pharmacophore with unique antioxidant and cytoprotective properties. We synthesized a library based upon the condensation of natural and unnatural amino acids with the carboxylic acid moiety of lipoic acid. SAR studies were conducted using a cardiac ischemia-reperfusion animal model. Cytoprotective efficacy was associated with the R-enantiomer of the dithiolane. Potency of library compounds was dictated by the acidic strength of the adduct. α-N-[(R)-1,2-dithiolane-3-pentanoyl]-l-glutamyl-l-alanine, designated CMX-2043, was chosen for further pharmacologic evaluation.     

Synthesis of phosphonic analogues of carnitine and gamma-amino-beta-hydroxybutyric acid

The involvement of carnitine and gamma-amino-beta-hydroxybutyric acid in the biology of mammalian cells, the physiology of the human body, and some important aspects of medicinal treatment has induced many research groups to develop their pharmacologically potent analogues. Among them are the very important phosphonic analogues: phosphocarnitine and gamma-amino-beta-hydroxypropylphosphonic acid. This mini-review describes the various methodologies used for the synthesis of these compounds.     

Neuroexcitatory amino acids: phosphonic analogue of kainic acid

Summary The enantioselective synthesis of phosphonic analogue of kainic acid is described.     

The oxidative dealkylation of insecticidal phosphoric acid triesters by mammalian liver enzymes

1. The dealkylation of the insecticidal phosphoric acid triester, 2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate, proceeds in mammalian liver slices via an oxidative mechanism and not by hydrolysis. 2. The enzyme that catalyses the reaction is located in the microsomal fraction of liver homogenate and is dependent for activity on molecular oxygen and NADPH. 3. There are large species differences between rat, mouse, rabbit and dog in the activity of the enzymes, the relative rates of dealkylation being 1, 8, 24 and 88 respectively in liver slices. 4. Dimethyl and di-isopropyl phosphate triesters are also dealkylated by rabbit liver microsomal preparations. 5. The mechanism of dealkylation involves hydroxylation at the alpha-carbon atom of an alkyl group, which is removed as the corresponding aldehyde, and is thus analogous to that of similar reactions catalysed by the microsomal mixed-function oxidases. 6. The relevance of these findings in the toxicology of phosphoric acid triesters is discussed.