Synthesis of four stereoisomers of 4-amino-2-(hydroxymethyl)tetrahydrofuran-4-carboxylic acid

The 5-benzyl ether, 15, of a 1,2,4,5-pentanetetrol of known 2S configuration was made by a multistep synthesis from d-ribose. Ring-closure of the 1-O-tosyl derivative, 17, with retention of configuration, followed by oxidation, gave the 2S enantiomer, 22, of 2-benzyloxymethyl-4-oxotetrahydrofuran. The latter was converted by a hydantion synthesis into the 4-amino-4-carboxylic acid (mixture of 2S,4R and 2S,4S isomers, 28 and 29). Spontaneous lactonization of the 2S,4R diastereomer proved it to have the “cis” configuration. The remaining, 2S,4S diastereomer then must be “trans” it is identical with a natural compound recently isolated from an acid hydrolyzate of diabetic urine. In a parallel synthesis, the 4-O-mesyl derivative (de-O-isopropylidenated 19) was cyclized, with inversion at ring-position 2, leading after oxidation to the 2R enantiomer, 25, of the 4-oxotetrahydrofuran. The hydantoin synthesis this time yielded a mixture of the 2R,4R and 2R,4S amino-acids. Spontaneous lactonization of the latter showed it to have the “cis” configuration. Absolute configurations were assigned to the four optically active products, based on the known absolute configuration of d-ribose and the known mechanisms of the synthetic reactions.     

New 4H-chromen-4-one and 2H-chromene derivatives as anti-picornavirus capsid-binders

Substituted (E)-3-styryl-4H-chromen-4-ones 1a–d, 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-4H-chromen-4-ones 2a–d, (E)-3-styryl-2H-chromenes 3a–d and 3-[(1E,3E)-4-phenylbuta-1,3-dienyl]-2H-chromenes 4a–d were designed and synthesized to improve the anti-picornavirus activity of previously tested analogues. The new compounds were evaluated in vitro against human rhinovirus (HRV) serotypes 1B and 14 and enterovirus (EV) 71. All the compounds interfered with the replication of picornaviruses, although considerable differences were observed in the sensitivity of viruses to each compound. Generally, both HRVs were more susceptible than EV71 and their sensitivity was dependent upon the linker chain length as well as upon the oxidation state of the heterocyclic ring. (E)-3-Styryl-2H-chromene (3a) emerged as the most effective inhibitor of both HRVs showing IC₅₀ values of 0.20 μM and 1.38 μM towards serotype 1B and 14, respectively. The potent activity was also coupled with low cytotoxicity resulting in high therapeutic indexes (250 and 36, respectively). Mechanism of action studies indicated that 3a, like structurally related compounds, behaves as a capsid binder interfering with the early stages of rhinovirus infection, probably at the adsorption and/or uncoating level.     

Synthesis and HMG-CoA reductase inhibition of 2-cyclopropyl-4-thiophenyl-quinoline mevalonolactones

A novel series of 2-cyclopropyl-4-thiophenyl quinoline-based mevalonolactones were synthesized from the substituted anilines by several reactions. Among them, (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(4-fluoro-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1d), (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1f) and (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4,7-di(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1q) showed potent HMG-CoA reductase inhibitory activity comparable with pitavastatin.     

Microbial enantioselective reduction of ethyl-2-oxo-4-phenyl-butanoate

Different microorganisms (MOs) were used to carry out the enantioselective reduction of ethyl-2-oxo-4-phenylbutanoate to (S)-(+)-2-hydroxy-4-phenylbutanoate or (R)-(+)-2-hydroxy-4-phenylbutanoate. Commercially available Saccharomyces cerevisiae and Dekera sp. led to over 92% ee of (S)-(+)-2-hydroxy-4-phenylbutanoate. Kluyveromyces marxianus gave the opposite isomer with 32% ee (R). All reactions, except those with Hansenula sp., proceeded to greater than 90% conversion. This the first report on the use of Dekera sp., Hansenula sp. and K. marxianus in the reduction of α-ketoesters.     

Functional dissection of XDppa2/4 structural domains in Xenopus development

The maintenance of pluripotency in mammalian embryonic stem cells depends upon the expression of regulatory genes like Oct3/4 and Sox2. While homologues of these genes are also characterized in non-mammalian vertebrates, like birds, amphibians and fish, existence and function of developmental pluripotency associated genes (Dppa) in lower vertebrates have not yet been reported. Here we describe a Dppa2/4-like gene, XDppa2/4, in Xenopus. The protein contains a SAP domain and a conserved C-terminal region. Overexpression of XDppa2/4, murine Dppa2 or Dppa4 produces similar phenotypes (defects in blastopore closure), while injection of XDppa2/4 morpholino generates a loss of blastopore closure and neural fold formation. Embryos die up to tailbud stage. mDppa2 (but not mDppa4) rescues blastopore closure and neurulation defects caused by XDppaMO, but does not prevent subsequent death of embryos. Although XDppa2/4 exhibits a Dppa-like expression pattern and is indispensable for embryogenesis, analyses of various marker genes make its role as a pluripotency factor rather unlikely. Both the gain and loss of function effects until the end of neurulation are caused by the conserved C-terminal region but not by the SAP domain. The SAP domain is required for association of XDppa2/4 to chromatin and for embryonic survival at later stages of development suggesting epigenetic programming events.     

Modifications at C-3 and C-4 of 2-amino-2-deoxy-d-glucose

Modifications at C-3 and C-4 of 2-amino-2-deoxy-d-glucose have been developed. A 3,4-double bond was introduced into benzyl 2-acetamido-2-deoxy-3,4-di-O-Methylsulfonyl-α-d-glucopyranoside by treatment with NaI and Zn. Epoxidation of the double bond with m-chloroperoxybenzoic acid gave an exo-epoxide exclusively. The stereochemistry of the epoxidation product has been confirmed by an alternative synthesis. An analysis of the ¹H-n.m.r. spectra indicates that both the 3,4-unsaturated derivatives and the epoxide exist in the °H₁ (d) conformation. Nucleophilic reagents (F^?, I^?) opened the 3,4-epoxide to give 4-substituted derivatives having the d-gulo configuration. Thus, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-iodo-α-d-gulopyranose and 2-acetamido-1,3,6-tri-O-acetyl-3,4-dideoxy-4-fluoro-α-d-gulopyranose have been synthesized. Reduction of the double bond in the key intermediate and deprotection gave 2-acetamido-2,3,4-trideoxy-d-glucopyranose. Some of the derivatives were active as inhibitors of growth of mouse, mammary adenocarcinoma cells in culture.     

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.     

Synthesis of 2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes via LiAlH4-induced reductive cyclization of 2-(4-chloro-2-cyano-2-phenylbutyl)aziridines and evaluation of their antimalarial activity

2-(4-Chloro-2-cyano-2-phenylbutyl)aziridines were employed for the one-step stereoselective construction of both endo- and exo-2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes as new azaheterobicyclic scaffolds via a double LiAlH₄-induced reductive cyclization protocol. Antiplasmodial assessment of these 1-azabicyclo[2.2.1]heptanes revealed moderate to good activities in the micromolar range, with the exo-isomers being the most promising structures. Furthermore, the proposed mode of action was supported by ligand docking studies, pointing to a strong binding interaction with the enzyme plasmepsin II.     

Carbamazepine derivatives with P2X4 receptor-blocking activity

Antagonists for the P2 receptor subtype P2X4, an ATP-activated cation channel receptor, have potential as novel drugs for the treatment of neuropathic pain and other inflammatory diseases. In the present study, a series of 47 carbamazepine derivatives including 32 novel compounds were designed, synthesized, and evaluated as P2X4 receptor antagonists. Their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor was determined. Additionally, species selectivity (human, rat, mouse) and receptor subtype selectivity (P2X4 vs P2X1, 2, 3, 7) were investigated for selected derivatives. The most potent compound of the present series, which exhibited an allosteric mechanism of P2X4 inhibition, was N,N-diisopropyl-5H-dibenz[b,f]azepine-5-carboxamide (34, IC₅₀ of 3.44 μM). The present study extends the so far very limited knowledge on structure–activity relationships of P2X4 receptor antagonists.     

Synthesis of copper(II) complexes with 3,5-bis(4,6-dimethylpyrimidin-2-yl)-4H-1,2,4-triazol-4-amine (L). Molecular and crystal structures of L and [Cu2L2Cl4]·2MeCN

A series of copper(II) complexes, i.e. Cu₂LCl₄, CuLCl₂·H₂O and [Cu₂L₂Cl₄]·2MeCN (8), based on a new potentially polytopic ligand, 3,5-bis(4,6-dimethylpyrimidin-2-yl)-4H-1,2,4-triazol-4-amine (3b, L), have been synthesized. The crystal structures of L and [Cu₂L₂Cl₄]·2MeCN were studied by X-ray single crystal analysis. The dinuclear compound [Cu₂L₂Cl₄]·2MeCN represents the first example of structurally characterized metal complexes with 3,5-di(pyrimidin-2-yl)-4H-1,2,4-triazol-4-amines. Both copper atoms have distorted tetragonal-pyramidal 3N + 2Cl environment. Surprisingly, in contrast to the complexes based on 3,5-di(pyridin-2-yl)-4H-1,2,4-triazol-4-amine (pyridinyl analog of L), the compound [Cu₂L₂Cl₄]·2MeCN adopts a dinuclear trans-(N′,N¹,N²)₂ double bridging binding mode which is due to tridentate coordination of two L molecules linking two copper atoms through N¹,N²-triazole and N′-pyrimidine atoms. It seems to be reasonable that it is methyl groups in pyrimidinyl moiety that obstruct the expected dinuclear (N′,N¹,N²,N″)₂ double bridging coordination being one of the most common for 4-substituted 3,5-di(pyridin-2-yl)-4H-1,2,4-triazoles and 3,5-di(pyridin-2-yl)-1,2,4-triazolates. Due to π-π stacking interactions, molecules of Cu₂L₂Cl₄ in the structure of [Cu₂L₂Cl₄]·2MeCN form 1D chains.     

Design,synthesis and antimicrobial evaluation of novel 1-benzyl 2-butyl-4-chloroimidazole embodied 4-azafluorenones via molecular hybridization approach

A series of novel 1-benzyl-2-butyl-4-chloroimidazole embodied 4-azafluorenone hybrids, designed via molecular hybridization approach, were synthesized in very good yields using one pot condensation of 1-benzyl-2-butyl-4-chloroimidazole-5-carboxaldehyde, 1,3-indanedione, aryl/heteroaryl methyl ketones and ammonium acetate. All the synthetic derivatives were fully characterized by spectral data and evaluated for antimicrobial activity by disc diffusion method against selected bacteria and fungal strains. Among the 15 new compounds screened, 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(furan-2-yl)-5H-indeno[1,2-b]pyridin-5-one(10k) has pronounced activity with higher zone of inhibition (ZoI) against Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Aspergillus flavus and Candida albicans. Also 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(dibenzo[b,d]thiophen-2-yl)-5H-indeno [1,2-b]pyridin-5-one (10n) and 4-(1-benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-2-(3-tosyl-3H-inden-1-yl)-5H-indeno[1,2-b]pyridin-5-one (10o) showed selective higher inhibitory activity against Aspergillus flavus and Candida albicans. The results demonstrated potential importance of molecular hybridization in the development of 10k as potential antimicrobial agent.     

Expression of the Blood-Group-Related Gene B4galnt2 Alters Susceptibility to Salmonella Infection

Glycans play important roles in host-microbe interactions. Tissue-specific expression patterns of the blood group glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) are variable in wild mouse populations, and loss of B4galnt2 expression is associated with altered intestinal microbiota. We hypothesized that variation in B4galnt2 expression alters susceptibility to intestinal pathogens. To test this, we challenged mice genetically engineered to express different B4galnt2 tissue-specific patterns with a Salmonella Typhimurium infection model. We found B4galnt2 intestinal expression was strongly associated with bacterial community composition and increased Salmonella susceptibility as evidenced by increased intestinal inflammatory cytokines and infiltrating immune cells. Fecal transfer experiments demonstrated a crucial role of the B4galnt2-dependent microbiota in conferring susceptibility to intestinal inflammation, while epithelial B4galnt2 expression facilitated epithelial invasion of S. Typhimurium. These data support a critical role for B4galnt2 in gastrointestinal infections. We speculate that B4galnt2-specific differences in host susceptibility to intestinal pathogens underlie the strong signatures of balancing selection observed at the B4galnt2 locus in wild mouse populations.     

Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation

Pear accessions and species show a broad response to tissue culture media due to the wide genetic diversity that exists in the available pear germplasm. An initial study of mineral nutrition using a systematic response surface approach with five Murashige and Skoog medium mineral stock solutions indicated that the mesos factor (CaCl₂, MgSO₄, and KH₂PO₄) affected most plant responses and genotypes, suggesting that additional studies were needed to further optimize these three mesos components for a wide range of genotypes. Short stature, leaf spots, edge necrosis, and red or yellow coloration were the main symptoms of poor nutrition in shoot cultures of 10 diverse pear genotypes from six species. A surface response experimental design was used to model the optimal factor and factor levels for responses that included overall quality, leaf character, shoot multiplication, and shoot height. The growth morphology, shoot length, and multiplication of these pear shoots could be manipulated by adjusting the mesos components. The highest quality for the majority of genotypes, including five P. communis cultivars, P. koehnei, P. dimorphophylla, and P. pyrifolia ‘Sion Szu Mi’, required higher concentrations (>1.2× to 2.5×) of all the components than are present in Murashige and Skoog medium. ‘Capital’ (P. calleryana) required high CaCl₂ and MgSO₄ with low KH₂PO₄; for ‘Hang Pa Li’ (P. ussuriensis), low CaCl₂ and moderate to low MgSO₄ and KH₂PO₄ produced high-quality shoots. Suitable combinations of the meso nutrients produced both optimum shoot number and shoot length in addition to general good plant quality.     

4-Carboxy-4-hydroxy-2-aminoadipic acid and other acidic amino acids in Caylusea abyssinica

Two diastereoisomers of 4-carboxy-4-hydroxy-2-aminoadipic acid have been isolated from leaves and inflorescences of Caylusea abyssinica. Green parts of the plant also contain appreciable amounts of the two diastereoisomers of 4-hydroxy-4-methylglutamic acid, 3-(3-carboxyphenyl)alanine, (3-carboxyphenyl)glycine, 3-(3-carboxy-4-hydroxyphenyl)alanine, (3-carboxy-4-hydroxyphenyl)glycine and in low concentration 2-aminoadipic acid, saccharopine [(2S, 2′S)-N⁶-(2-glutaryl)lysine] and some γ-glutamyl peptides. The acidic amino acids were separated from other amino acids on an Ecteola ion exchange column with M pyridine as eluant.     

Pou4f1 and Pou4f2 Are Dispensable for the Long-Term Survival of Adult Retinal Ganglion Cells in Mice

Purpose

To investigate the role of Pou4f1 and Pou4f2 in the survival of adult retinal ganglion cells (RGCs).

Methods

Conditional alleles of Pou4f1 and Pou4f2 were generated (Pou4f1^loxP and Pou4f2^loxP respectively) for the removal of Pou4f1 and Pou4f2 in adult retinas. A tamoxifen-inducible Cre was used to delete Pou4f1 and Pou4f2 in adult mice and retinal sections and flat mounts were subjected to immunohistochemistry to confirm the deletion of both alleles and to quantify the changes in the number of RGCs and other retinal neurons. To determine the effect of loss of Pou4f1 and Pou4f2 on RGC survival after axonal injury, controlled optic nerve crush (CONC) was performed and RGC death was assessed.

Results

Pou4f1 and Pou4f2 were ablated two weeks after tamoxifen treatment. Retinal interneurons and Müller glial cells are not affected by the ablation of Pou4f1 or Pou4f2 or both. Although the deletion of both Pou4f1 and Pou4f2 slightly delays the death of RGCs at 3 days post-CONC in adult mice, it does not affect the cell death progress afterwards. Moreoever, deletion of Pou4f1 or Pou4f2 or both has no impact on the long-term viability of RGCs at up to 6 months post-tamoxifen treatment.

Conclusion

Pou4f1 and Pou4f2 are involved in the acute response to damage to RGCs but are dispensable for the long-term survival of adult RGC in mice.     

Preparation,structure and properties of dicyano silver complexes of the M(en)3Ag2(CN)4 and M(en)2Ag2(CN)4 type

Complexes of the M(en)₃Ag₂(CN)₄ (M = Ni, Zn, Cd) and M(en)₂Ag₂(CN)₄ (M = Ni, Cu, Zn, Cd) type were prepared and identified by elemental analysis, infrared spectroscopy, measurement of magnetic susceptibility, and X-ray powder diffractometry. The crystal structures of Ni(en)₃Ag₂(CN)₄ (I) and Zn(en)₂Ag₂(CN)₄ (II) were determined by the method of monocrystal structure analysis. Complex I crystallizes in the space group C2/c, a = 1.2639(5), b = 1.3739(4), c = 1.2494(4) nm, β = 113.25(4)°, D_m = 1.86(1), D_c = 1.86 gcm⁻³ Z = 4, R = 0.0429. The crystal structure of I consists of complex cations [Ni(en)₃]²⁺ and complex anions [Ag(CN)₂]⁻. Complex II crystallizes in the space group I2/m, a = 0.9150(3), b = 1.3308(4), c = 0.6442(2) nm, β = 95.80(3)°, D_m = 2.14(1), D_c = 2.15 gcm⁻³, Z = 2, R = 0.0334. Its crystal structure consists of infinite, positively charged chains of the [-NCAgCNZn- (en)₂]_nⁿ⁺ type and isolated [Ag(CN)₂]⁻ anions. The atoms of Ag are positioned parallely to the z axis and the AgAg distance is equal to 0.3221(2) nm.