Polyoxazolines based mixed micelles as PEG free formulations for an effective quercetin antioxidant topical delivery.

This study aims to prove the value of the polyoxazolines polymer family as surfactant in formulations for topical application and as an alternative to PEG overuse. The amphiphilic polyoxazolines (POx) were demonstrated to have less impact on cell viability of mice fibroblasts (NIH3T3) than their PEG counterparts. Mixed micelles, made of POx and phosphatidylcholine, were manufactured using thin film and high pressure homogenizer process. The mixed micelles were optimized to produce nanosized vesicles of about 20 nm with a spherical shape and stable over 28 days. The natural lipophilic antioxidant, quercetin, was successfully encapsulated (encapsulation efficiency 94 ±â€¯4% and drug loading 3.6 ±â€¯0.2%) in the mixed micelles with no morphological variation. Once loaded in the formulation, the quercetin impact on cell viability of NIH3T3 was decreased while its antioxidant activity remained unchanged. This work highlights the capacity of amphiphilic POx to create, in association with phospholipids, stable nanoformulations which show promise for topical delivery of antioxidant and ensure skin protection against oxidative stress.

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.

CsF-Catalyzed Transannulation Reaction of Oxazolones: Diastereoselective Synthesis of Diversified trans- N-(6-Oxo-1,4,5,6-tetrahydropyrimidin-5-yl)benzamides with Arylidene Azlactones and Amidines.

A versatile and straightforward synthetic strategy for the construction of new tetrasubstituted 1,3-diazinones is described. The procedure is based on CsF-catalyzed, microwave-assisted, ring transformation reaction of arylidene azlactones with amidines. Moreover, this technique provides diversified trans- N-(6-oxo-1,4,5,6-tetrahydropyrimidin-5-yl)benzamides with a good antimicrobial activity.

Partially Hydrolyzed Poly(n-propyl-2-oxazoline): Synthesis, Aqueous Solution Properties, and Preparation of Gene Delivery Systems.

Random copolymers of n-propyl-2-oxazoline and ethylenimine (PPrOx-PEI) were prepared by partial acidic hydrolysis of poly(n-propyl-2-oxazoline) (PPrOx). Dynamic and electrophoretic light scattering and diffusion-ordered NMR spectroscopy were utilized to investigate aqueous solution properties of the copolymers. Above a specific cloud point temperature, well-defined nanoparticles were formed. The latter consisted of a core composed predominantly of PPrOx and a thin positively charged shell from PEI moieties that mediated formation of polyplexes with DNA. The polyplexes were prepared at 65 °C at varying N/P (amine-to-phosphate groups) ratios. They underwent structural changes upon temperature variations 65-25-37 °C depending on N/P. At N/P < 2, the polyplex particles underwent minor changes because of formation of a surface layer of DNA that acted as a barrier and prevented swelling and disintegration of the initial particles. Dramatic rearrangements at N/P ≥ 2 resulting in large swollen microgel particles were overcome by coating of the polyplex particles with a cross-linked polymeric shell. The shell retained the colloidal stability and preserved the physicochemical parameters of the initial polyplex particles while it reduced the high surface potential values. Progressive loss of cytotoxicity upon complexation with DNA and coating of polyplex particles was displayed.

Dihydrooxazolones and dihydroimidazolones derived from acylglycines: syntheses, molecular structures and supramolecular assembly.

Syntheses and structures are described for some alkylidene-substituted dihydrooxazolones and dihydroimidazoles derived from simple acylglycines. A second, triclinic, polymorph of 4-benzylidene-2-(4-methylphenyl)-1,3-oxazol-5(4H)-one, C17H13NO2, (I), has been identified and the structure of 2-methyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one, C9H7NO2S, (II), has been rerefined taking into account the orientational disorder of the thienyl group in each of the two independent molecules. The reactions of phenylhydrazine with 2-phenyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one or 2-(4-methylphenyl)-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one yield, respectively, 3-anilino-2-phenyl-5-[(thiophen-2-yl)methylidene]-3,5-dihydro-4H-imidazol-4-one, C10H15N3OS, (III), and 3-anilino-2-(4-methylphenyl)-5-[(thiophen-2-yl)methylidene]-3,5-dihydro-4H-imidazol-4-one, C21H17N3OS, (IV), which both exhibit orientational disorder in their thienyl groups. The reactions of 2-phenyl-4-[(thiophen-2-yl)methylidene]-1,3-oxazol-5(4H)-one with hydrazine hydrate or with water yield, respectively, N-[3-hydrazinyl-3-oxo-1-(thiophen-2-yl)prop-1-en-2-yl]benzamide and 2-(benzoylamino)-3-(thiophen-2-yl)prop-2-enoic acid, which in turn react, respectively, with thiophene-2-carbaldehyde to form 2-phenyl-5-[(thiophen-2-yl)methylidene]-3-{[(E)-(thiophen-2-yl)methylidene]amino}-3,5-dihydro-4H-imidazol-4-one, C19H13N3OS2, (V), which exhibits orientational disorder in only one of its thienyl groups, and with methanol to give methyl (2Z)-2-(benzoylamino)-3-(thiophen-2-yl)prop-2-enoate, C15H13NO3S, (VI). There are no direction-specific intermolecular interactions in the crystal structure of the triclinic polymorph of (I), but the molecules of (II) are linked by two independent C-H···O hydrogen bonds to form C2(2)(14) chains. Compounds (III) and (IV) both form centrosymmetric R2(2)(10) dimers built from N-H···O hydrogen bonds, while compound (V) forms a centrosymmetric R2(2)(10) dimer built from C-H···O hydrogen bonds. In the structure of compound (VI), a combination of N-H···O and C-H···π(arene) hydrogen bonds links the molecules into sheets. Comparisons are made with some similar compounds.

The discovery of oxazolones-grafted spirooxindoles via three-component diversity oriented synthesis and their preliminary biological evaluation.

A facile method via 1,3-dipolar cycloaddition of substituted benzylidene-2-phenyloxazolone under mild conditions with azomethine ylides, which were generated in situ by a decarboxylative route from a common set of diverse isatins and amino acid derivatives was developed for a 15-membered library of regio- and stereoselective oxazolones-grafted spirooxindole-pyrrolidine, pyrrolizidines and pyrrolothiazoles. After screening their cytotoxic activities against a spectrum of cell-lines, compound 4h was identified as potent antitumor agent and inducing apoptosis. The present study has provided an effective entry to rapidly construct a chemical library of oxazolones-grafted spirooxindoles and developed a good lead compound for subsequent optimization.

In vitro reconstitution of indolmycin biosynthesis reveals the molecular basis of oxazolinone assembly.

The bacterial tryptophanyl-tRNA synthetase inhibitor indolmycin features a unique oxazolinone heterocycle whose biogenetic origins have remained obscure for over 50 years. Here we identify and characterize the indolmycin biosynthetic pathway, using systematic in vivo gene inactivation, in vitro biochemical assays, and total enzymatic synthesis. Our work reveals that a phenylacetate-CoA ligase-like enzyme Ind3 catalyzes an unusual ATP-dependent condensation of indolmycenic acid and dehydroarginine, driving oxazolinone ring assembly. We find that Ind6, which also has chaperone-like properties, acts as a gatekeeper to direct the outcome of this reaction. With Ind6 present, the normal pathway ensues. Without Ind6, the pathway derails to an unusual shunt product. Our work reveals the complete pathway for indolmycin formation and sets the stage for using genetic and chemoenzymatic methods to generate indolmycin derivatives as potential therapeutic agents.

Formation of a(1) ions directly from oxazolone b(2) ions: an energy-resolved and computational study.

It is well-known that oxazolone b2 ions fragment extensively by elimination of CO to form a2 ions, which often fragment further to form a1 ions. Less well-known is that some oxazolone b2 ions may fragment directly to form a1 ions. The present study uses energy-resolved collision-induced dissociation experiments to explore the occurrence of the direct b2→a1 fragmentation reaction. The experimental results show that the direct b2→a1 reaction is generally observed when Gly is the C-terminal residue of the oxazolone. When the C-terminal residue is more complex, it is able to provide increased stability of the a2 product in the b2→a2 fragmentation pathway. Our computational studies of the relative critical reaction energies for the b2→a2 reaction compared with those for the b2→a1 reaction provide support that the critical reaction energies are similar for the two pathways when the C-terminal residue of the oxazolone is Gly. By contrast, when the nitrogen of the oxazolone ring in the b2 ion does not bear a hydrogen, as in the Ala-Sar and Tyr-Sar (Sar = N-methylglycine) oxazolone b2 ions, a1 ions are not formed but rather neutral imine elimination from the N-terminus of the b2 ion becomes a dominant fragmentation reaction. The M06-2X/6-31+G(d,p) density functional theory calculations are in general agreement with the experimental data for both types of reaction. In contrast, the B3LYP/6-31+G(d,p) model systematically underestimates the barriers of these SN2-like b2→a1 reaction. The difference between the two methods of barrier calculation are highly significant (P < 0.001) for the b2→a1 reaction, but only marginally significant (P = 0.05) for the b2→a2 reaction. The computations provide further evidence of the limitations of the B3LYP functional when describing SN2-like reactions.

Lead optimization and modulation of hERG activity in a series of aminooxazoline xanthene ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

The optimization of a series of aminooxazoline xanthene inhibitors of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aß lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2' side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aß reduction in a rat pharmacodynamic model (78% Aß reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.

Death-associated protein kinase 3 mediates vascular structural remodelling via stimulating smooth muscle cell proliferation and migration.

Death-associated protein kinase 3 (DAPK3) also known as zipper-interacting kinase is a serine/threonine kinase that mainly regulates cell death and smooth muscle contraction. We have previously found that protein expression of DAPK3 increases in the mesenteric artery from spontaneously hypertensive rats (SHRs) and that DAPK3 mediates the development of hypertension in SHRs partly through promoting reactive oxygen species-dependent vascular inflammation. However, it remains to be clarified how DAPK3 controls smooth muscle cell (SMC) proliferation and migration, which are also important processes for hypertension development. We, therefore, sought to investigate whether DAPK3 affects SMC proliferation and migration. siRNA against DAPK3 significantly inhibited platelet-derived growth factor (PDGF)-BB-induced SMC proliferation and migration as determined by bromodeoxyuridine (BrdU) incorporation and a cell counting assay as well as a Boyden chamber assay respectively. DAPK3 siRNA or a pharmacological inhibitor of DAPK3 inhibited PDGF-BB-induced lamellipodia formation as determined by rhodamine-phalloidin staining. DAPK3 siRNA or the DAPK inhibitor significantly reduced PDGF-BB-induced activation of p38 and heat-shock protein 27 (HSP27) as determined by Western blotting. In ex vivo studies, PDGF-BB-induced SMC out-growth was significantly inhibited by the DAPK inhibitor. In vivo, the DAPK inhibitor significantly prevented carotid neointimal hyperplasia in a mouse ligation model. The present results, for the first time, revealed that DAPK3 mediates PDGF-BB-induced SMC proliferation and migration through activation of p38/HSP27 signals, which may lead to vascular structural remodelling including neointimal hyperplasia. The present study suggests DAPK3 as a novel pharmaceutical target for the prevention of hypertensive cardiovascular diseases.

GANRA-5 protects both cultured cells and mice from various radiation types by functioning as a free radical scavenger.

The radio-protective effects of the oxazolone derivative chemical compound 4-(4-methoxy-3-methoxyphenyl-methyl)-2-phenyl- 5(4H)-oxazolone (GANRA-5) against different types of radiation including X-rays, carbon ion beams, microwaves and ultraviolet light (UV) were studied. Cell proliferation/cytotoxicity assay and colony-forming assay were conducted to evaluate the toxicity of GANRA-5. To test its influence on the induction of double-stranded break (DSB) formation and genomic instability, γH2AX focus-forming assay as well as cytokinesis-block micronucleus assay was utilized. Our results indicate that GANRA-5 exhibits low toxicity, while providing high radio-protective effects for MRC-5 cells against different types of radiation. We also found that GANRA-5 acts as a free radical scavenger. Our animal studies provided evidence that GANRA-5 significantly increases the survival rate of mice after X-ray irradiation. Analyses of hemogram, visceral index and detection of superoxide dismutase (SOD) and malondialdehyde (MDA) in the viscera indicate both low toxicity of GANRA-5, combined with its ability to shield radiation risk. In conclusion, our results suggest that GANRA-5 has the potential to be used as a safe and efficient radio-protectant.

Spectral characteristics of 4-(p-N,N-dimethyl-aminophenylmethylene)-2-phenyl-5-oxazolone (DPO) in different media.

The absorption and fluorescence characteristics of 4-(p-N,N-dimethyl-aminophenylmethylene)-2-phenyl-5-oxazolone (DPO) have been investigated in different solvents. DPO dye exhibits a large red shift in both absorption and emission spectra as solvent polarity increases, indicating a large change in the dipole moment of dye molecules upon excitation due to an intramolecular charge transfer interaction. The fluorescence quantum yield depends strongly on the properties of the solvents. Crystalline solids of DPO gave strong red emission at 605nm due to the excitation of molecular aggregates. The absorption and fluorescence emission spectral properties of DPO have also been investigated in organized media of aqueous micellar and ß-cyclodextrin (ß-CD) solutions. The critical micelle concentrations (CMCs) of SDS and CTAB as well as the binding constants of DPO in micellar solution and ß-cyclodextrin have been also determined.

A novel ring oxidation of 4- or 5-substituted 2H-oxazole to corresponding 2-oxazolone catalyzed by cytosolic aldehyde oxidase.

The ring oxidation of 2H-oxazole, or C2-unsubstituted oxazole, to 2-oxazolone, a cyclic carbamate, was observed on various 4- or 5-substituted oxazoles. Using 5-(3-bromophenyl)oxazole as a model compound, its 2-oxazolone metabolite M1 was fully characterized by liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance. The reaction mainly occurred in the liver cytosolic fraction without the requirement of cytochrome P450 enzymes and cofactor NADPH. Investigations into the mechanism of formation of 2-oxazolone using various chemical inhibitors indicated that the reaction was primarily catalyzed by aldehyde oxidase and not by xanthine oxidase. In addition, cytosol incubation of 5-(3-bromophenyl)oxazole in the medium containing H2¹8O led to the ¹8O incorporation into M1, substantiating the reaction mechanism of a typical molybdenum hydroxylase. The rank order of liver cytosols for the 2-oxazolone formation was mouse > monkey ≫ rat and human liver cytosol, whereas M1 was not formed in dog liver cytosol. Because the reaction was observed with a number of 4- or 5-substituted 2H-oxazoles in mouse liver cytosols, 2H-oxazoles represent a new substrate chemotype for ring oxidation catalyzed by aldehyde oxidase.

Immunization induces activation of bone marrow eosinophils required for plasma cell survival.

Eosinophils not only have multiple functions as effector cells of the innate immune system but also as modulators of immune responses. As producers of cytokines required for plasma cell survival, they are essential for the long-term maintenance of plasma cells in the BM. Here we show that the activation of eosinophils both in vitro and in vivo enhances the expression of the plasma cell survival factors APRIL, IL-6, IL-4, IL-10 and TNF-α. The in vivo activation of eosinophils was independent of the type of adjuvant used for primary immunization. Although eosinophils were activated by adjuvant itself, a stable activation and a constant increase in BM eosinophils were observed only in the presence of antigen. Thus, the numbers and the quality of eosinophils were dependent on priming the adaptive immune system. With secondary immunization and re-activation of antigen-dependent memory cells, the ability of eosinophils to promote plasma cell survival was further increased. These findings suggest that in T-cell-dependent immune responses eosinophils are conditioned to support the long-term survival of plasma cells in the BM, and furthermore imply that through accelerated numbers of eosinophils, stable plasma cell survival niches are established and the long-term survival of plasma cells is ensured.

Novel immunomodulators based on an oxazolin-2-one-4-carboxamide scaffold.

A series of oxazolidin-2-one-4-carboxylic amide compounds (1a-f) were designed and synthesized as the non-phosphate S1P(1) receptor agonists. The single crystal of 1e was prepared and solved to elucidate the structure of 1a-f. EC(50) of 1a-d were about 1.1-3.6 µM in S1P(1) Redistribution® assay, and their cytotoxicity was 8-40-fold lower than FTY720. Though its S1P(1) agonist activities in vitro were about 1000-folds weaker than (S)-FTY720-P, at a dose of 10mg/Kg, the immunosuppressive effects of 1a were comparable to FTY720. So oxazolidin-2-one-4-carboxylic amide derivatives were found as potential immunomodulator, compound 1a could be considered as a lead compound, rational modifications of 1a are anticipated using medicinal chemistry techniques and molecular modeling to obtain analogs with higher affinity and better clinical therapeutic properties.

Is there a typical germinal center? A large-scale immunohistological study on the cellular composition of germinal centers during the hapten-carrier-driven primary immune response in mice.

Germinal centers (GCs) are complex, multicell-type, transient structures that form in secondary lymphatic tissues in response to T cell-dependent stimulation. This process is crucial to the adaptive immune response because it is the source of affinity maturation and long-lived B cell memory. Our previous studies showed that the growth of murine splenic GCs is nonsynchronized, involving broad-volume distributions of individual GCs at any time. This raises the question whether such a thing as a typical GC exists. To address this matter, we acquired large-scale confocal data on GCs throughout the course of the 2-phenyl-5-oxazolone chicken serum albumin-driven primary immune response in BALB/c mice. Semiautomated image analysis of 3457 GC sections revealed that, although there is no typical GC in terms of size, GCs have a typical cellular composition in that the cell ratios of resident T cells, macrophages, proliferating cells, and apoptotic nuclei are maintained during the established phase of the response. Moreover, our data provide evidence that the dark zone (DZ) and light zone (LZ) compartments of GCs are about the same size and led us to estimate that the minimal cell loss rate in GCs is 3% per hour. Furthermore, we found that the population of GC macrophages is larger and more heterogeneous than previously thought, and that despite enrichment of T cells in the LZ, the DZ of murine splenic GCs is not poor in T cells. DZ and LZ differ in the T cell-to-macrophage ratio rather than in the density of T cells.

Poly(3-alkylthiophenes): new sorption materials for solid phase microextraction of drugs isolated from human plasma.

A novel sorbent in solid phase microextraction (SPME) method based on poly(3-alkylthiophenes) was used in the isolation of linezolid from human plasma samples following liquid chromatography determination. The effect of extraction time on the sorption capacity of the SPME process was studied and pointed at 10 min both for adsorption and desorption. Poly(3-methylthiophene) and poly(3-nonylthiophene) were applied for the extraction of linezolid from water solutions. In plasma samples, four coatings including polythiophene and poly(3-penthylthiophene) were investigated. With these measurements, correlation coefficients were calculated in the range from 0.9820 to 0.9995, and the relative standard deviations were below 15%. That allowed claiming that the synthesized and described materials can be successfully applied in the analysis of linezolid also from other matrices such as urine or blood.

Regioselective 1,3-dipolar cycloaddition of phenanthrolinium N-ylides to substituted arylidene oxazolones.

The 1,3-dipolar cycloaddition reactions of phenanthrolinium ylides have been investigated. Thus 15 novel aryl substituted spiro[oxazole-4,10'-pyrrolo[1,2-a][1,10] phenanthrolin]-5-ones were prepared in a regioselective manner, by the treatment of phenanthrolinium ylides with (Z)-arylidene oxazolones.

IL-33 activates B1 cells and exacerbates contact sensitivity.

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33-activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33-activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2(-/-) mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.