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.

A set of matrix metalloproteinases (MMPs) inhibitors, containing a bisphosphonate moiety (BP), has been evaluated for the inhibitory activity of carbonic anhydrases (CAs, EC 4.2.1.1). Human (h) isoforms hCA I, II, IX, XII and XIV were included in the study due to their involvement in crucial physiologic and pathologic processes. Some of these molecules selectively inhibited CA XII in the nanomolar range, showing an attractive dual mechanism (anti-MMP and anti-CA) of action as potential antitumor agents. The BP inhibitors investigated in this study are also excellent leads for obtaining even more effective compounds able to selectively target membrane-bound CA XII and having the potential to be used as tools for understanding physiologic processes regulated by this isoform.

The complexity of matrix metalloproteinase inhibitors (MMPIs) design derives from the difficulty in carefully addressing their inhibitory activity towards the MMP isoforms involved in many pathological conditions. In particular, specific metalloproteinases, such as MMP-2 and MMP-9, are key regulators of the 'vicious cycle' occurring between tumor metastases growth and bone remodeling. In an attempt to devise new approaches to selective inhibitor derivatives, we describe novel bisphosphonate bone seeking MMP inhibitors (BP-MMPIs), capable to be selectively targeted and to overcome undesired side effects of broad spectrum MMPIs. In vitro activity (IC50 values) for each inhibitor was determined against MMP-2, -8, -9 and -14, because of their relevant role in skeletal development and renewal. The results show that BP-MMPIs reached IC50 values of enzymatic inhibition in the low micromolar range. Computational studies, used to rationalize some trends in the observed inhibitory profiles, suggest a possible differential binding mode in MMP-2 that explains the selective inhibition of this isoform. In addition, survival assay was conducted on J774 cell line, a well known model system used to evaluate the structure-activity relationship of BPs for inhibiting bone resorption. The resulting data, confirming the specific activity of BP-MMPIs, and their additional proved propensity to bind hydroxyapatite powder in vitro, suggest a potential use of BP-MMPIs in skeletal malignancies.

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.

A number of matrix metalloproteinases (MMPs), proteins important in the balance of bone remodeling, play a critical role both in cancer metastasis and in bone matrix turnover associated with the presence of cancer cells in bone. Here, we report the synthesis and biological evaluation of a new class of MMP inhibitors characterized by a bisphosphonate function as the zinc binding group. Since the bisphosphonate group is also implicated in osteoclast inhibition and provides a preferential affinity to biological apatite, the new molecules can be regarded as bone-seeking medicinal agents. Docking experiments were performed to clarify the mode of binding of bisphosphonate inhibitors in the active site of MMP-2. The most promising of the studied bisphosphonates showed nanomolar inhibition against MMP-2 and resulted in potent inhibition of osteoclastic bone resorption in vitro.

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.

A set of bisphosphonate matrix metalloproteinase (MMP) inhibitors was investigated for inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes, some of which are overexpressed in hypoxic tumors. Some of the bisphosphonate revealed to be very potent inhibitors (in the low nanomolar range) of the cytosolic isoform CA II and the membrane-bound CA IX, XII and XIV isozymes, a feature useful for considering them as interesting compounds for bone resorption inhibition applications. We suggest here that it is possible to develop dual enzyme inhibitors bearing bisphosphonate moieties that may target both MMPs and CAs, two families of enzymes involved in tumor formation, growth, and metastasis.

Bisphosphonate drugs are well known to inhibit osteoclastic activity and have been proposed for the management of bone diseases, including periodontitis which is associated with alveolar bone destruction. In this study, we evaluated the effects of four arylsulfonamide bisphosphonates on growth of the periodontopathogenic bacterium Porphyromonas gingivalis as well as their capacity to reduce cytokine secretion by lipopolysaccharide (LPS)-stimulated oral epithelial cells. The growth of P. gingivalis was inhibited by (4'-Chloro-biphenyl-4-sulfonylamino)methyl-1,1-bisphosphonic acid while the three other arylsulfonamide bisphosphonates ((4-Methoxy-phenylsulfonylamino)methyl-1,1-bisphosphonic acid, (4-Nitro-phenylsulfonylamino)methyl-1,1-bisphosphonic acid, and (Biphenyl-4-sulfonylamino)methyl-1,1-bisphosphonic acid) had no effect. Growth inhibition was more pronounced under an iron-restricted condition. All four arylsulfonamide bisphosphonates decreased the production of the pro-inflammatory cytokines IL-6 and IL-8 by Aggregatibacter actinomycetemcomitans LPS-stimulated oral epithelial cells. In conclusion, we uncovered additional properties of bisphosphonates that may be beneficial for the treatment of periodontal diseases. In particular, (4'-Chloro-biphenyl-4-sulfonylamino)methyl-1,1-bisphosphonic acid combines the already disclosed antiresoptive activity with anti-inflammatory and antibacterial properties.

Effects of novel sintetic peroxisome proliferator-activation receptor  (PPARs) agonist on neuronal differentiation in the human neuroblastoma SH-SY5Y cell line MALLAMACI R1, LAGHEZZA A3,LOIODICE F3, VITIELLO F2, BUTTIGLIONE M2 1Dip.Pharmaceutical Biology, Faculty of Pharmacy, University of Bari 2Dip.Biomedical Sciences and Human Oncology, Faculty of Medicine, University of Bari 3Dip. Pharmaceutical Chemistry, Faculty of Pharmacy, University of Bari BACKGROUND: PPARs are a subfamily of the nuclear hormone receptor that heterodimerizes with the retinoid X receptor to act as a transcriptional regulator. Recently it  has been shown that the activation of PPARγ isoform promotes neuronal differentiation. AIM: In this study we have investigated the capability of synthetic compounds endowed with different activity profile on PPARα/γ subtypes to induce neuronal differentiation and neurite outgrowth. The experiments were carried out on cells of  the human neuroblastoma SH-SY5Y line. Retinoic acid (RA) was used as a  positive control for neurite outgrowth. METHODS: SH-SY5Y cultures were maintained in DMEM supplemented with 10% FCS. 10µM RA,  0,5 to 25µM PPAR agonist was added to the culture media in the experimental cultures. Cell viability was tested using the MTT assay. Changes in the expression and re-organization of specific markers involved in neuronal differentiation were investigated using immunofluorescence. RESULTS: Synthetic PPAR agonists promote cell differentiation and the outgrowth of cell processes in a concentration-dependent manner. The maximal effect was obtained at a concentration of 25µM. At this concentration we obtained a significant increase of the expression of neurofilament-200 (NF-H) and Gap-43; this agrees with a hypothesis of cell differentiation induced by these molecules.  CONCLUSION: Our results suggest that sintetic PPAR agonist promotes neuronal differentiation and neurite outgrowth in  SH-SY5Y human neuroblastoma cells. Further work on this subject is underway in our laboratories.

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.

In the last 20 years, a great variety of synthetic, low molecular weight MMP inhibitors (MMPIs) have been synthesized and tested, although none has reached clinical utility. Exploration of novel ZBGs and development of non-hydroxamate MMPI has become a focus in current research. It's well-known that polyphenols can produce beneficial effects on human health by their antioxidant properties as well as they have the ability to block gelatinase activity. In this work we tested a series of selected phenols as MMP inhibitors. The most interesting hit (B6) shows sub-micromolar activity against MMP-2 (IC(50) 0.59 +/- 0.05 microM, LE = 1.07) and a fairly good selectivity spectrum.

A new series of derivatives of the PPARα/γ dual agonist 1 allowed us to identify the ligand ( S)-6 as a potent partial agonist of both PPARα and γ subtypes. X-ray studies in PPARγ revealed two different binding modes of ( S)-6 to the canonical site. However, ( S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPARγ antagonist and supported from docking experiments. This compound did not activate the PPARγ-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, ( S)-6 inhibited the Cdk5-mediated phosphorylation of PPARγ at serine 273 that is currently considered the mechanism by which some PPARγ partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPARα/γ dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.

Background: PPARs are attractive targets of antidiabetic agents. However, PPAR ligands show side effects that hinder their clinical use. Results: LT175 improves insulin sensitivity and reduces body weight via selective gene activation in adipose tissue. Conclusion: LT175 shows an improved pharmacological profile linked to characteristic binding and differential coregulator recruitment. Significance: LT175 may be a scaffold molecule to design a safer generation of PPAR ligands.

Convenient synthetic routes to KN-93, N-(2-{[[(2E)-3-(4-chlorophenyl)prop-2-enyl](methyl)amino]methyl}phenyl)-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide, a well-known Ca(2+)/calmoduline-dependent protein kinase II (CaMKII) inhibitor, are described. The methods proposed start from easily available reagents and allow ready preparation of the final compound in moderate overall yields. Most of the synthetic steps proposed were microwave assisted.

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.

A series of saponins and sapogenins from Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome.

PPARs are nuclear receptors with a critical physiological role in lipid and glucose metabolism. As part of our effort to develop new and selective PPAR agonists containing stilbene and its bioisoster phenyldiazene, novel analogs were synthesized starting from tyrosine and evaluated as PPAR agonists. We tested the effects of phenyloxazole replacement of GW409544, a well-known PPARalpha/gamma dual agonist, with stilbene or phenyldiazene moiety, spaced by an ether bridge to tyrosine portion. These structural modifications provided potent and selective PPARgamma agonists. Molecular docking studies performed on these new compounds complemented the experimental results and allowed to gain some insights into the nature of binding of the ligands.

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.