Transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine by Salmonella typhimurium

The transport of 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP) by Salmonella typhimurium was studied using synthetic [methyl-³H₃]MAHMP. It was found that an active transport system existed for MAHMP, having K_m of 0.07 μM and V_max 45 nmol·min^?1·(g dry wt. cells)^?1, that required glucose as a source of energy and was pH and temperature dependent. Uptake was inhibited by cyanide, azide, N-ethylmaleimide, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Uptake was also weakly inhibited by oxythiamine, but not by thiamine, 2-methyl-4-amino-5-aminomethylpyrimidine, or 4-amino-5-hydroxymethylpyrimidine, indicating that the transport system is specific for MAHMP.     

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid in seeds of Lonchocarpus sericeus

3-[2-Amino-2-imidazolin-4(5)-yl]alanine (enduracididine) and 2-[2-amino-2-imidazolin-4(5)-yl] acetic acid have been isolated from seeds of Lonchocarpus sericeus. The concentration of each compound was ca 0.5 % of the fresh seed weight.     

Synthesis and activity of substituted heteroaromatics as positive allosteric modulators for α4β2α5 nicotinic acetylcholine receptors

The design and synthesis of a series of substituted heteroaromatic α4β2α5 positive allosteric modulators is reported. The optimization and development of the heteroaromatic series was carried out from NS9283, and several potent analogues, such as 3-(5-(pyridin-3-yl)-2H-tetrazol-2-yl)benzonitrile (5k) and 3,3′-(2H-tetrazole-2,5-diyl)dipyridine (12h) with good in vitro efficacy were discovered.     

Phosphatidylinositol (4, 5)‐bisphosphate targets double C2 domain protein B to the plasma membrane

Double C2 domain protein B (DOC2B) is a high‐affinity Ca²⁺ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)‐bisphosphate [PI(4, 5)P₂] and target SNAREs (t‐SNAREs). Here, we show that PI(4, 5)P₂ hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t‐SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P₂ in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P₂‐binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P₂ are required for the DOC2B–PM association and revealing multiple PI(4, 5)P₂–C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP‐independent and occurs in a diffusion‐like manner. Our data suggest that the Ca²⁺‐triggered translocation of DOC2B is diffusion‐driven and aimed at PI(4, 5)P₂‐containing membranes.      

Transport of thiamine and 4-methyl-5-hydroxyethylthiazole by Salmonella typhimurium

The transport of thiamine and 4-methyl-5-hydroxyethylthiazole (MHET), its thiazole moiety, was studied using whole cells of Salmonella typhimurium. It was found that the bacteria possessed an active transport system for thiamine that had K_m 0.21 μM and V_max 33 nmol·min^?1·(mg dry wt. cells)^?1. Transport of thiamine was glucose dependent, whereas MHET uptake was dependent on both glucose and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), the pyrimidine moiety of thiamine. Uptake of both thiamine and MHET was severely curtailed by cyanide, azide, N-ethylmaleimide and carbonyl cyanide m-chlorophenylhydrazone. Oxythiamine inhibited thiamine, but not MHET, uptake and thiamine slightly inhibited MHET uptake. 2-Methyl-4-amino-5-methoxymethylpyrimidine and 4-amino-5-hydroxymethylpyrimidine were unable to replace MAHMP as stimulators of MHET uptake, but 2-methyl-4-amino-5-aminomethylpyrimidine was marginally effective in this regard. Similar results were obtained with attempts to replace MAHMP as a growth requirement for a purD mutant of Salmonella typhimurium. MHET uptake showed saturation kinetics only in the presence of MAHMP, and is not otherwise actively transported.     

Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.     

5-Hydroxylevulinic acid,a new intermediate in the biosynthesis of protoanemonin

Administration of 5-hydroxy[1-¹⁴C]-and [4-¹⁴C]levulinic acid to Helleborus foetidus led to the isolation of [1-¹⁴C]- and [4-¹⁴C]protoanemonin, respectively. There was also incorporation of radioactivity into the four glucosides ranunculin, isoranunculin, ranuncoside and ranunculoside. Acid hydrolysis of radioactive ranuncoside gave labelled 5-hydroxylevulinic acid (HKV). A study of the incorporation of various ¹⁴C-labelled tracers into protoanemonin suggested that HKV is formed in higher plants by a new reduction of 2-ketoglutarate (2-KG) without free 4,5-dioxovalerate (DOVA) as an intermediate. A scheme for the biosynthesis of the antibiotic protoanemonin and its glucosidic precursors is proposed. It is shown that 5-(β-d-glucopyranosyloxy)levulinic acid could be the genuine precursor of all the compounds studied.     

Isolation of (2S,4R)-2-amino-4-methyl-hex-5-enoic acid,a nonprotein amino acid,as an allelochemical from the fruiting bodies of Boletus fraternus Peck.

The mushroom Boletus fraternus Peck. shows allelopathy and suppresses the growth of broad leaf plants in nature. According to a bioassay-guided fractionation of the fruiting body of the fungus, a rare nonprotein amino acid was isolated as a major allelochemical. The chemical structure of the compound was determined to be (2S,4R)-2-amino-4-methyl-hex-5-enoic acid (5-dehydrohomoleucine) by analysis of ¹H- and ¹³C-nuclear magnetic resonance spectra and comparison with data from the literature. The allelochemical caused 50% inhibition of lettuce seedling radicle growth at a concentration of 34 ppm (w/v). Further, since radicle growth was directed away from the filter paper to prevent contact with the allelochemical at concentrations higher than 300 ppm (w/v), the fungus may use the allelochemical to protect its immediate environment from contamination by other plants.     

(2S)-2-Amino-5-chloro-4-hydroxy-5-hexenoic acid,a new chloroamino acid,and related compounds fromAmanita gymnopus

Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of¹H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.     

Design,synthesis and biological evaluation of 4-aryl-5-aminoalkyl-thiazole-2-amines derivatives as ROCK II inhibitors

A series of 4-aryl-5-aminoalkyl-thiazole-2-amines were designed and synthesized, and their inhibitory activity on ROCK II was screened by enzyme-linked immunosorbent assay (ELISA). The results showed that 4-aryl-5-aminomethyl-thiazole-2-amines derivatives had certain ROCK II inhibitory activities. Compound 10l showed ROCK II inhibitory activity with IC₅₀ value of 20 nM.     

Probing the β-pocket of the active site of human liver glycogen phosphorylase with 3-(C-β-d-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors

Human liver glycogen phosphorylase (hlGP), a key enzyme in glycogen metabolism, is a valid pharmaceutical target for the development of new anti-hyperglycaemic agents for type 2 diabetes. Inhibitor discovery studies have focused on the active site and in particular on glucopyranose based compounds with a β-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the β-pocket. Recently, C-β-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the β-pocket by studying the inhibitory effect of six different groups at the para position of 3-(β-d-glucopyranosyl phenyl)-5-phenyl-, 1, 2, 4-triazoles in hlGP by kinetics and X-ray crystallography. The most bioactive compound was the one with an amine substituent to show a K_i value of 0.43 μM. Structural studies have revealed the physicochemical diversity of the β-pocket providing information for future rational inhibitor design studies.     

4-aminopyrimidine-5-carbaldehyde oximes as potent VEGFR-2 inhibitors. Part II

A series of 4-aminopyrimidine-5-carbaldehyde oxime was discovered to have potent VEGFR-2 inhibitory activity. Described here are the chemistry for analogue synthesis and SAR study results. The PK properties, kinase profiling, and in vivo efficacy study for compound 4b are also discussed.     

1, 2, 3, 4-Tetrahydro-2-Methyl-4, 6, 7-Isoquinolinetriol Depletes Catecholamines in Rat Brain

1, 2, 3, 4-Tetrahydro-2-methyl-4, 6, 7-isoquinolinetriol (TMIQ) was synthesised and tested for activity as a dopamine-depleting agent in rat brain. After intracerebroventricular infusion, TMIQ caused reductions in dopamine concentrations in substantia nigra, striatum, hypothalamus, and dorsal raphe, and reduction in noradrenaline concentrations in locus coeruleus. TMIQ also reduced 5-hydroxytryptamine concentrations in dorsal raphe and substantia nigra, although with a lower potency. Comparisons between TMIQ and MPTP showed that they were approximately equipotent in depleting dopamine in the substantia nigra, hypothalamus, and dorsal raphe. Pretreatment of animals with a combination of monoamine oxidase A and B inhibitors completely prevented the TMIQ-induced reductions in dopamine concentrations in substantia nigra and hypothalamus. Direct unilateral intrastriatal injections of TMIQ produced marked ipsilateral reductions in striatal dopamine, correlating with a behavioural response consisting of turning towards the side of injection. The results suggest that TMIQ should be evaluated further as a possible MPTP-like compound, which may derive from endogenous β-hydroxylated catecholamines.     

Association of a serotonin transporter gene (SLC6A4) 5-HTTLPR polymorphism with body mass index categories but not type 2 diabetes mellitus in Mexicans

The serotonergic system has been hypothesized to contribute to the biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI) categories. We investigate a possible association of 5-HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter gene (SLC6A4) with the development of T2DM and/or higher BMI by analyzing a sample of 138 individuals diagnosed with T2DM and 172 unrelated controls from the Mexican general population. In the total sample genotypes were distributed according to Hardy-Weinberg equilibrium, and S allele frequency was 0.58. There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Mexican population sample (p = 0.12). Nevertheless, logistic regression analysis of the L allele and increased BMI disclosed an association, after adjusting for age, sex and T2DM (p = 0.02, OR 1.74, 95% CI: 1.079–2.808).     

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.     

Non-covalent interaction of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein with proteins and its application

The interactions of 2', 4', 5', 7'-tetrabromo-4, 5, 6, 7-tetrachlorofluorescein (TBTCF) with BSA, ovalbumin (OVA) and poly-L-lysine (PLYS) at pH 3.70 have been investigated by combination of the spectral correction technique and the Langmuir isothermal adsorption. The active connection actions such as ion pairs, van der Waals' force, hydrogen bond, hydrophobic bond were proposed to explain the non-covalent interaction between TBTCF and BSA, OVA and PLYS. Effects of the electrolyte and high temperature indicated that union of the active connections between TBTCF and BSA and OVA was too firm to be destroyed. The relationship between the binding number of TBTCF and variety fraction of the amino acid residues was analyzed. The binding number of TBTCF depended on the number of positively charged amino acid residues. The other amino acid residues surrounded and seized TBTCF by hydrogen bonds and hydrophobic bonds when the electrostatic attraction pulled TBTCF to link protein. In addition, a novel method named the absorbance ratio difference was established for determination of protein in trace level and was applied with much higher sensitivity than the ordinary method.     

Discovery and pharmacological characterization of a novel small molecule inhibitor of phosphatidylinositol-5-phosphate 4-kinase,type II,beta

Phosphatidylinositol-5-phosphate 4-kinase, type II, beta (PIP5K2B) is linked to the pathogenesis of obesity, insulin resistance and diabetes. Here, we describe the identification of a novel pyrimidine-2,4-diamine PIP5K2B inhibitor, designated SAR088. The compound was identified by high-throughput screening and subsequently characterized in vitro and in vivo. SAR088 showed reasonable potency, selectivity and physicochemical properties in enzymatic and cellular assays. In vivo, SAR088 lowered blood glucose levels of obese and hyperglycemic male Zucker diabetic fatty rats treated for 3 weeks. Thus, SAR088 represents the first orally available and in vivo active PIP5K2B inhibitor and provides an excellent starting point for the development of potent and selective PIP5K2B inhibitors for the treatment of insulin resistance and diabetes.