2H-1,4-Benzoxazine amidine derivatives are drugs acting as modulators of the skeletal muscle and pancreatic beta cell ATP-sensitive-K+ (KATP) channels. With the aim of evaluating the influence of absolute configuration on the biological activity of these drugs, we herein report the optimization of a synthetic route to obtain both enantiomers of some of these compounds with improved chemical yield and high enantiomeric excess.

In this study we report the behavior of two PPAR enantiomeric ligands (R-1 and S-1). Cell-based reporter assays indicate that both enantiomers are dual PPAR/ ligands, being R-1 a full agonist of PPAR and and S-1 a partial agonist of PPAR. 3D-structure analysis of the PPAR ligand binding domain in the complex with the two ligands shows that the suboptimal conformation of helix 12 in the PPAR/S-1 complex is the consequence of a steric hindrance between the ethyl group of the ligand and the Q286 in helix 3. Site-directed mutagenesis confirms that Q286 is a key residue in determining the activity of different ligands. By using FRET assays, we found that the coactivators SRC-1, PGC-1, RIP140, TIF-2 are recruited by R-1, S-1 and rosiglitazone with similar EC50, whereas CBP affinity is higher in the presence of rosiglitazone. Conversely, only S-1 allows the association of the corepressor N-CoR to PPAR as opposed to rosiglitazone and R-1, providing a functional explanation to the partial agonist behavior of S-1. ChIP assays confirmed that S-1 enhances the association of N-CoR to PPAR target promoters in the native chromatin context, whereas rosiglitazone and R-1 recruit less N-CoR. Altogether, our results provide a rationale for the distinct behavior of different PPAR ligands.

Improved experimental conditions were carried out for the preparation in high yields of some 3-phenyl-1-benzofuran-2-carboxylic acids, potent inhibitors of C1C-K chloride channels. A one-pot condensation-cyclization was set up starting from different 2-hydroxybenzophenones whose reactivity was significantly affected from the electronic properties of their substituents.

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARalpha/gamma dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARalpha and fine-tuned moderate activity on PPARgamma. For the most interesting compound (S)-3, docking studies in PPARalpha and PPARgamma provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARalpha/gamma activity which probably involves a synergistic effect on both receptor subtypes.

In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC–MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC50 values reported in literature. The optimized PPARγ-OT capillary was validated by determining the Kd values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.

A series of previously synthesized chiral derivatives of clofibric and phenylacetic acids, acting as dual agonists towards the peroxisome proliferator-activated receptors (PPARs) alpha and gamma, was taken into account, and the efficacy of these compounds was analyzed by means of 2D-, 3D-QSAR and docking studies with the goal to gain deeper insights into the three-dimensional determinants governing ligands selectivity for PPARs. By multiregressional analysis a correlation between the lipophilicity and PPARalpha activity was found, whereas for PPARgamma the correlation was achieved once efficacy was related to the presence of polar groups on agonists scaffold. Docking of these compounds further corroborated this hypothesis, and then provided a valid support for subsequent chemometric analysis and pharmacophore models development for both receptors subtypes. Computational results suggested site directed mutagenesis experiments which confirmed the importance of amino acid residues in PPAR activity, allowing the identification of critical hotspots most likely taking over PPARs selectivity.

The preparation of a series of 2-(aryloxy)-3-phenylpropanoic acids, resulting from the introduction of different substituents into the biphenyl system of the previously reported peroxisome proliferator-activated receptor α/γ (PPARα/γ) dual agonist 1, allowed the identification of new ligands with higher potency on PPARα and fine-tuned moderate PPARγ activity. For the most promising stereoisomer (S)-16, X-ray and calorimetric studies in PPARγ revealed, at high ligand concentration, the presence of two molecules simultaneously bound to the receptor. On the basis of these results and docking experiments in both receptor subtypes, a molecular explanation was provided for its different behavior as a full and partial agonist of PPARα and PPARγ, respectively. The effects of (S)-16 on mitochondrial acylcarnitine carrier and carnitine-palmitoyl-transferase 1 gene expression, two key components of the carnitine shuttle system, were also investigated, allowing the hypothesis of a more beneficial pharmacological profile of this compound compared to the less potent PPARα agonist fibrates currently used in therapy

Molecular dynamics simulations were performed on two ureidofibrate-like enantiomers to gain insight into their different potency and efficacy against PPARgamma. The partial agonism of the S enantiomer seems to be due to its capability to stabilize different regions of the receptor allowing the interaction with both coactivators and corepressors as shown by fluorescence resonance energy transfer (FRET) assays. The recruitment of the corepressor N-CoR1 by the S enantiomer on two different responsive elements of PPARgamma regulated promoters was confirmed by chromatin immunoprecipitation assays. Cell-based transcription assays show that PPARgamma coactivator 1alpha (PGC-1alpha) and cAMP response element binding protein-binding protein (CBP) enhance the basal and ligand-stimulated receptor activity acting as coactivators of PPARgamma, whereas the receptor interacting protein 140 (RIP140) and the nuclear corepressor 1 (N-CoR1) repress the transcriptional activity of PPARgamma. We also tested the importance of the residue Q286 on the transcriptional activity of the receptor by site-directed mutagenesis and confirmed its key role in the stabilization of helix 12. Molecular modeling studies were performed to provide a molecular explanation for the different behavior of the mutants.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic beta-cell ATP-sensitive-K(+)(KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic beta-cell KATP channel and the Kir6.2C36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by i.p. GTT and on fasted glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP(IC50=10.1x10(-9)M) and Kir6.2C36(IC50=9.6x10(-9)M) channels which induced depolarization. In contrast, the 2-phenyl analog was a potent opener(DE50=0.04x10(-9)M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl>2-benzyl>2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg kg(-1)) and 2-phenyl analogs (2-30 mg kg(-1)) reduced and enhanced the glucose AUC curves, respectively, following the glucose loading in mice. These compounds did not affect the fasted glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which respectively confer the KATP channel blocking action with glucose lowering effects and the opening action with increased glucose levels. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel anti-diabetic drugs.

PPARs are transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new agonists for these receptors. Given that the introduction of fluorine generally has a profound effect on the physical and/or biological properties of the target molecule, we synthesized a series of fluorinated analogs of the previously reported compound 2, some of which turned out to be remarkable PPARα and PPARγ dual agonists. Docking experiments were also carried out to gain insight into the interactions of the most active derivatives with both receptors.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARgamma-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARgamma target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARalpha target genes indicating that their in vivo effects stemmed from an activation of both PPARalpha and gamma. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Some aminophenyl benzyl sulfides or benzyl pyridyl sulfides were asymmetrically oxidized with tert-butyl hydroperoxide in the presence of a complex between titanium i-propoxide and (S, S)-hydrobenzoin, an oxidation system that works particularly well with a vast set of aryl benzyl sulfides. Notwithstanding the presence of nitrogen-containing moieties that, in principle, could interfere with the correct co-ordination of the sulfide to the metal center, satisfactory levels of enantioselectivity (up to 78%) were measured for this oxidation process.