N-(pyrimidin-4-yl) and N-(pyridin-2-yl) phenylalanine derivatives as VLA-4 integrin antagonists

The SAR studies to optimise both potency and rate of clearance in the rat for a series of pyrimidine and pyridine based VLA-4 antagonists are described.     

Photoaffinity labeling of rat liver ribosomes by N-(2-nitro-4-azidobenzoyl)puromycin

N-(2-Nitro-4-azidobenzoyl)-[3H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes.     

Synthesis and anti-Candidal activity of N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide

Eighteen N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide derivatives were synthesized, evaluated against ten clinical isolates of Candida spp. and compared with itraconazole. Introduction of p-chloro (2c), p-iodo (2q), m-chloro (2l) and o-nitro (2r) substitution at phenyl ring of thiosemicarbazide enhanced the anti-Candida activity. Compound (2c) bearing p-cholorophenyl ring was found to be the most effective against Candida albicans ATCC 66027, Candida spp. 12810 (blood) and Candida spp. 178 (HVS) with MIC value of 0.09–0.78 μg/mL, whereas itraconazole exhibits the inhibitory activity with MIC value of 0.04–1.56 μg/mL against all tested strains. There is a correlation between anti-Candidal activity and p-chloro substitution at phenyl ring of thiosemicarbazide. All synthesized compounds were investigated for their potential cytotoxicity against non cancer cell line MCF-10A. The active compounds 2c, 2r and 2a were further investigated for their cytotoxic effects on three cancer cell lines; HT1080 (skin), HepG2 (liver) and A549 (lung). The active compounds showed minimal cytotoxic activity against non cancer cell line and all three cancer cell lines. Moreover, compound 2c displaying better activity against C. albicans ATCC66027 and Candida spp. [blood] compared to reference drug (itraconazole), represents a good lead for the development of newer, potent and broad spectrum anti-Candidal agents.     

Synthesis and antibacterial activity of N-(5-benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-yl)piperazinyl quinolone derivatives

A series of N-(5-benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-yl) derivatives of piperazinyl quinolones was synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative microorganisms. Some of these derivatives exhibit high activity against Gram-positive bacteria; Staphylococcus aureus and Staphylococcus epidermidis, comparable or more potent than their parent N-piperazinyl quinolones norfloxacin and ciprofloxacin as reference drugs. The SAR of this series indicates that both the structure of the benzyl unit and the S or SO(2) linker dramatically impact antibacterial activity.     

Chronic ethanol ingestion modifies liver microsomal phosphatidylserine inducing resistance to hydrolysis by exogenous phospholipase A2.

Chronic ethanol ingestion leads to the acquisition of a tolerance to membrane lipid disordering, a lowered partition coefficient to hydrophobic compounds and a resistance to the hydrolysis of the phospholipids by exogenous phospholipase A2. Anionic phospholipids have been implicated as being responsible for the resistance to lipid disordering and a number of modifications to these phospholipids are known to occur as a result of chronic ethanol-ingestion. In this study the basis of the resistance to phospholipase A2 in hepatic microsomes was investigated. It was found that chronic ethanol-induced modifications to each of the major phospholipid classes was responsible to some extent for the resistance to phospholipase A2, however, PS was particularly potent considering it is a compositionally minor constituent. The effect was interpreted as a reduced ability to activate the phospholipase A2 since PS acts as an essential activator of phospholipase A2 (along with PI). Fatty acid analysis revealed that the chronic ethanol-treatment resulted in a elevated level of docosahexaenoate with a parallel reduction in arachidonate in phosphatidylserine. Lipid packing and organization is important in the regulating the level of exogenous phospholipase A2 activity but the activity was not found to correlate with lipid order of different phosphatidylserine species. It is concluded that subtle differences in the molecular species arrangement or disposition around the enzyme may be responsible for the altered phospholipase A2 interaction with the membrane induced by chronic ethanol-treatment. One implication of this study is that other anionic phospholipid dependent membrane proteins, of which there are many known examples, may also be modified as a result of chronic ethanol-ingestion.     

Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: a fluorescence approach

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. NBD-labeled lipids have previously been used to monitor the organization and dynamics of molecular assemblies such as membranes, micelles and reverse micelles utilizing the wavelength-selective fluorescence approach. In this paper, we have characterized the organization and dynamics of various NBD-labeled lipids using red edge excitation shift (REES) and other fluorescence approaches which include analysis of membrane penetration depths of the NBD group using the parallax method. We show here that the environment and location experienced by the NBD group of the NBD-labeled lipids could depend on the ionization state of the lipid. This could have potentially important implications in future studies involving NBD-labeled lipids as tracers in a cellular context.     

Amino acids analysis using a monolithic silica column after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.     

Novel application of 4-nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole to visualize lysosomes in live cells

4-Nitro-7-(1-piperazinyl)-2,1,3-benzoxadiazole (NBD-PZ) reacts with carboxylic acids in the presence of condensing agents and is utilized for the fluorescence detection of the generated derivatives in high performance liquid chromatography or capillary electro-phoresis. Although the fluorescence intensity of derivatives of NBD-PZ-CH2CH2NH2 with carboxylic acids is elevated at low pH, the pH-dependent fluorescence of NBD-PZ itself has not yet been investigated. In this study, we determined the fluorescence spectra of NBD-PZ at various pH and found that the fluorescence intensity of NBD-PZ was elevated dramatically at pH 相似文献   

Antidiabetic activity of N-(6-substituted-1,3-benzothiazol-2-yl)benzenesulfonamides

N-(6-Substituted-1,3-benzothiazol-2-yl)benzenesulfonamide derivatives 1–8 were synthesized and evaluated for their in vivo antidiabetic activity in a non-insulin-dependent diabetes mellitus rat model. Several compounds synthesized showed significant lowering of plasma glucose level in this model. As a possible mode of action, the compounds were in vitro evaluated as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. The most active compounds (3 and 4) were docked into the crystal structure of 11β-HSD1. Docking results indicate potential hydrogen bond interactions with catalytic amino acid residues.     

Cytosolic phospholipase A(2) activity during the ongoing cell cycle

Cytosolic phospholipase A(2) (cPLA(2)) is of special interest because it selectively releases arachidonic acid from membrane phospholipids. Arachidonic acid has been implicated to play an important role in various cellular responses. Recently arachidonic acid release and prostaglandin synthesis have been shown to be cell cycle dependent and therefore the activity of cPLA(2) during the ongoing cell cycle was investigated, using the mitotic shake off method for cell synchronisation. cPLA(2) activity was high in mitotic cells and decreased rapidly in the early G1 phase. A strong increase in activity was measured following the G1/S transition in both neuroblastoma and Chinese hamster ovary cells. The changes in activity were not due to a difference in cPLA(2) expression but due to phosphorylation of cPLA(2). Phosphorylation of cPLA(2) occurs through MAPK since the use of a specific MAPK kinase inhibitor and serum depletion of synchronised cells inhibited cPLA(2) activity.     

Enhancement of mast cell survival: a novel function of some secretory phospholipase A(2) isotypes

This study tested the hypothesis that certain secretory phospholipase A(2) (sPLA(2)) isotypes act in a cytokine-like fashion through cell surface receptors to influence mast cell survival. Initial experiments revealed that sPLA(2) activity and sPLA(2) receptor expression are increased, and mast cells lost their capacity to maintain membrane asymmetry upon cytokine depletion. Groups IB and III, but not group IIA PLA(2), prevented the loss of membrane asymmetry. Similarly, group IB prevented nucleosomal DNA fragmentation in mast cells. Providing putative products of sPLA(2) hydrolysis to cytokine-depleted mast cells did not influence survival. Furthermore, catalytic inactivation of sPLA(2) did not alter its capacity to prevent apoptosis. Inhibition of protein synthesis using cycloheximide or actinomycin reversed the antiapoptotic effect of sPLA(2). Additionally, both wild-type and catalytically inactive group IB PLA(2) induced IL-3 synthesis in mast cells. However, adding IL-3-neutralizing Ab did not change Annexin V(FITC) binding and only partially inhibited thymidine incorporation in sPLA(2)-supplemented mast cells. In contrast, IL-3-neutralizing Ab inhibited both Annexin V(FITC) binding and thymidine incorporation in mast cells maintained with IL-3. sPLA(2) enhanced phosphoinositide 3'-kinase activity, and a specific inhibitor of phosphoinositide 3'-kinase reversed the antiapoptotic effects of sPLA(2). Likewise, sPLA(2) increased the degradation of I-kappaBalpha, and specific inhibitors of nuclear factor kappa activation (NF-kappaB) reversed the antiapoptotic effects of sPLA(2). Together, these experiments reveal that certain isotypes of sPLA(2) enhance the survival of mast cells in a cytokine-like fashion by activating antiapoptotic signaling pathways independent of IL-3 and probably via sPLA(2) receptors rather than sPLA(2) catalytic products.     

Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids in model membranes

The spectroscopic and ionization properties of various lipids labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group have been studied in model membranes using fluorescence, absorbance and electrophoretic mobility measurements. Electrophoretic measurements show that the NBD group is uncharged at neutral pH. However, at high pH, hydroxyl addition or deprotonation occurs with a pKa, depending upon conditions, of 11.5-11.8 for the NBD group of headgroup-labeled phosphatidylethanolamine (NBD-PE) and 11.1-11.5 for NBD labels placed at the end of one fatty acyl chain of a phosphatidylcholine (6-NBD-PC and 12-NBD-PC). This type of behavior is not observed in the case of a methylated NBD label placed in the flexible 'tail' of cholesterol (NBD-cholesterol). The similarity in pKa for NBD-PE and NBD-PCs suggests that in these cases the NBD group is at a similar depth in the membrane. This was examined further by comparison of the fluorescence emission maximum of the NBD group in model membranes with that in solvents of varying polarity. The apparent polarity experienced by NBD groups in model membranes indicates that for NBD-PE and 12-NBD-PC they are located at the polar region whereas the NBD group of NBD-cholesterol is deeply buried in a nonpolar region of the membrane. This conclusion is supported further by fluorescence quenching experiments measuring NBD exposure to the aqueous quencher Co2+. The results of this study confirm the tentative conclusions of our previous fluorescence quenching studies on the location of NBD groups in model membranes.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Synthesis and CYP24A1 inhibitory activity of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides

A series of N-(2-(1H-imidazol-1-yl)-2-phenylethyl)arylamides were prepared, using an efficient three- to five-step synthesis, and evaluated for their inhibitory activity against human cytochrome P450C24A1 (CYP24A1) hydroxylase. Inhibition ranged from IC₅₀ 0.3–72 μM compared with the standard ketoconazole IC₅₀ 0.52 μM, with the styryl derivative (11c) displaying enhanced activity (IC₅₀ = 0.3 μM) compared with the standard, providing a useful preliminary lead for drug development.     

New antiprotozoal agents: Synthesis and biological evaluation of different 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives

In an endeavor to develop efficacious antiprotozoal agents 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives (5–14) were synthesized, characterized and biologically evaluated for antiprotozoal activity. The compounds were screened in vitro against the HM1: IMSS strain of Entamoeba histolytica and NF54 chloroquine-sensitive strain of Plasmodium falciparum. Among the synthesized compounds six exhibited promising antiamoebic activity with IC₅₀ values (0.14–1.26 μM) lower than the standard drug metronidazole (IC₅₀ 1.80 μM). All nine compounds exhibited antimalarial activity (IC₅₀ range: 1.42–19.62 μM), while maintaining a favorable safety profile to host red blood cells. All the compounds were less effective as an antimalarial and more toxic (IC₅₀ range: 14.67–81.24 μM) than quinine (IC₅₀: 275.6 ± 16.46 μM) against the human kidney epithelial cells. None of the compounds exhibited any inhibitory effect on the viability of Anopheles arabiensis mosquito larvae.     

Synthesis and preliminary pharmacological evaluation of N-2-(4-(4-(2-substitutedthiazol-4-yl) piperazin-1-yl)-2-oxoethyl)acetamides as novel atypical antipsychotic agents

A series of N-2-(4-(4-(2-substitutedthiazol-4-yl) piperazin-1-yl)-2-oxoethyl)acetamides were synthesized in an effort to prepare novel atypical antipsychotic agents. The compounds were synthesized by either microwave irradiation technique or by conventional synthesis and were characterized by spectral data (IR, (1)H NMR, and MS) and the purity was ascertained by microanalysis. All the synthesized compounds were screened for their in vivo pharmacological activity in Swiss albino mice. D(2) antagonism studies were performed using climbing mouse assay model and 5-HT(2A) antagonism studies were performed using quipazine induced head twitches in mice. It was observed that none of the new chemical entities exhibited catalepsy. AG 3 was found to be the most active compound.     

Synthesis and antiplasmodial activity of new N-[3-(4-{3-[(7-chloroquinolin-4-yl)amino]propyl}piperazin-1-yl)propyl]carboxamides

The parallel acylation of N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-7-chloroquinolin-4-amine with polymer-bound carboxylic acids opened straightforward access to novel aminoquinolines with activity against Plasmodium falciparum strains in vitro. Using this amino scaffold prepared in solution and polymer-bound carboxylic, we have synthesized a series of 29 new compounds in good to excellent yield and purity. Biological evaluation included determination of the activity against a chloroquine (CQ) sensitive strain and a CQ resistant mutant. Most of the novel structures presented here displayed activity against both strains in the lower nanomolar range, four compounds showed an at least fourfold increase in the ratio of inhibition of CQ resistant to sensitive strains over CQ itself. These results suggest that this derivatization technique is a useful method to speed up structure-activity relationship studies on aminoquinolines toward improved activity versus CQ resistant strains of P. falciparum in vitro.     

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.     

Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4-fluor-7-nitro-2,1,3-benzoxadiazole derivatization and fluorescence detection

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 x 10(-8) M for spermine to 2.1 x 10(-8) M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.     

Internalization and degradation of type IIA phospholipase A(2) in mast cells

Whereas exogenous types IB and X secretory phospholipase A(2) (sPLA(2)) elicited prostaglandin D(2) (PGD(2)) production in mouse bone marrow-derived mast cells (BMMC), sPLA(2)-IIA was unable to do so. In search of a mechanism underlying this cellular refractoriness to exogenous sPLA(2)-IIA, we now report that this isozyme is promptly associated with cell surfaces, internalized, and then degraded in BMMC. Adsorption of sPLA(2)-IIA to BMMC was prevented by addition of heparin to the medium. Moreover, a heparin-nonbinding sPLA(2)-IIA mutant did not bind to BMMC. These results indicate that this sPLA(2)-IIA inactivation process depends on its rapid binding to heparan sulfate proteoglycan (HSPG) on BMMC surfaces. Thus, the present observations represent a particular situation in which cell surface HSPG exhibit a negative regulatory effect on cellular function of sPLA(2)-IIA, and argue that HSPG does not always act as a functional adapter for heparin-binding sPLA(2)s in mammalian cells as has been demonstrated before.