A Classic Photochemical Approach Inducing an Unexpected Rearrangement: Exploring the Photoreactivity of Pentacyclic Triterpenic Acids.

The discovery of new bioactivities is closely related to the generation of novel scaffolds, and in the past few years different strategies have been proposed to obtain unknown architectures from the manipulation of known compounds. In the present study, we exploited a vintage photochemical approach for the discovery of an unexpected pathway of reactivity related to Δ1-3-oxo-pentacyclic triterpenic acids gaining access to a new class of natural-unnatural 5(10→1)abeo-pentacyclic triterpenic acids.

Functionalization of ß-cyclodextrin with a urea-based PSMA ligand and preliminary studies on targeting prostate cancer cells.

Targeted delivery of drugs into specific cancer cells is an effective way to enhance the efficacy and minimize the side effects of therapy. Prostate malignant cells overexpress the prostate-specific membrane antigen (PSMA), a membrane protein that may be a valid target for selective drug administration. To target prostate cancer cells, a ß-cyclodextrin perfunctionalised with dipeptide-like urea arms, a well-established mimic of a selective ligand against PSMA, is herein reported, to develop a multivalent drug delivery and targeting system. Firstly, fluorescein was used to validate the system on cells that express high levels of PSMA (prostate tumoral cells, LNCap) or very low levels of PSMA (non-tumoral cells, Hek293T). Then, the antineoplastic agent doxorubicin complexed with ß-cyclodextrin functionalized with PSMA-like ligand takes less time to induce cytotoxicity on LNCap cells compared to doxorubicin alone. This might represent a promising drug-delivery approach to selectively target prostate cancer cells.

Synthesis, equilibrium, and biological study of a C-7 glucose boronic acid derivative as a potential candidate for boron neutron capture therapy.

The synthesis of d-glucoheptose derivative containing a boronic moiety is described herein. Starting from benzyl 6,7-dideoxy-2,3,4-tri-O-benzyl-ß-d-gluco-ept-6-enopyranoside, the introduction of the boronic acid was performed through a metathesis reaction by using MIDA vinyl boronic acid and the 2nd generation Grubbs catalyst. Hydrogenation led to the final product in only two reaction steps. This new sugar-containing boronic acid in the skeleton could mimic carbohydrate behavior and follow the glucose uptake in living cells. The in vitro toxicity tests performed in fibroblasts and glioma tumor cell lines showed minimal toxicity. Boron uptake measured using ICP-MS was minimal in fibroblasts, while in glioma cells showed a value of 6 ng of total boron accumulation per mg of cells, implying that compound 1a is able to accumulate selectively in the tumor tissues compared to normal.

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD+ salvage pathway in the nucleus.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Aryl Azides as Forgotten Electrophiles in the Van Leusen Reaction: A Multicomponent Transformation Affording 4-Tosyl-1-arylimidazoles.

Considering aryl azides as electrophilic partners for the TosMIC mediated Van Leusen reaction, a novel multicomponent synthesis of 4-tosyl-1-arylimidazoles is reported. In this transformation, two molecules of TosMIC participate in the reaction mechanism in two different ways, with the second molecule undergoing a novel type of fragmentation resulting in the incorporation of a C-H into the final product.

Exploiting the Nucleophilicity of the Nitrogen Atom of Imidazoles: One-Pot Three-Component Synthesis of Imidazo-Pyrazines.

A novel one-pot multicomponent reaction to synthesize substituted imidazopyrazines is described. In brief, 1H-(imidazol-5-yl)-N-substituted methanamines react with aldehydes and isocyanides in methanol at room temperature to give imidazopyrazine derivatives in excellent yields. The imidazole nitrogen atom was able to intercept the nascent nitrilium ion, channeling the reaction toward to the sole formation of imidazopyrazines, suppressing the competitive formation of other possible side products deriving from the reaction with the high-energy nitrilium ion. The number of examples and the variability of the nature of isocyanides, aldehydes, and amine components herein employed, witness the robustness of this novel methodology.

Anomeric sugar boronic acid analogues as potential agents for boron neutron capture therapy.

After the development of accelerators as neutron source, the access to new suitable agents for boron neutron capture therapy (BNCT) became a major need. Among many others, sugar boronic acids have recently attracted attention as boron carriers. Herein we report the synthesis and preliminary biological studies of two new sugar analogues containing a boronic acid at the anomeric position. The analogues were obtained by hydroboration of proper open-chain terminal alkenes that, after quenching with water, spontaneously afforded cyclic boronic acids with hemiacetal-like structures.

New insights in the metabolism of oxybutynin: evidence of N-oxidation of propargylamine moiety and rearrangement to enaminoketone.

1. Oxybutynin hydrochloride is an antimuscarinic agent prescribed to patients with an overactive bladder (OAB) and symptoms of urinary urge incontinence. Oxybutynin undergoes pre-systemic metabolism, and the N-desethyloxybutynin (Oxy-DE), is reported to have similar anticholinergic effects. 2. We revisited the oxidative metabolic fate of oxybutynin by liquid chromatography-tandem mass spectrometry analysis of incubations with rat and human liver fractions, and by measuring plasma and urine samples collected after oral administration of oxybutynin in rats. This investigation highlighted that not only N-deethylation but also N-oxidation participates in the clearance of oxybutynin after oral administration. 3. A new metabolic scheme for oxybutynin was delineated, identifying three distinct oxidative metabolic pathways: N-deethylation (Oxy-DE) followed by the oxidation of the secondary amine function to form the hydroxylamine (Oxy-HA), N-oxidation (Oxy-NO) followed by rearrangement of the tertiary propargylamine N-oxide moiety (Oxy-EK), and hydroxylation on the cyclohexyl ring. 4. The functional activity of Oxy-EK was investigated on the muscarinic receptors (M1-3) demonstrating its lack of antimuscarinic activity. 5. Despite the presence of the α,ß-unsaturated function, Oxy-EK does not react with glutathione indicating that in the clearance of oxybutynin no reactive and potentially toxic metabolites were formed.

Quantification of the Chemical Chaperone 4-Phenylbutyric Acid (4-PBA) in Cell Culture Media via LC-HRMS: Applications in Fields of Neurodegeneration and Cancer.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.

Biochemical and structural analysis of a cytosolic sulfotransferase of the malaria vector Anopheles gambiae overexpressed in the reproductive tissues.

The temporary or permanent chemical modification of biomolecules is a crucial aspect in the physiology of all living species. However, while some modules are well characterised also in insects, others did not receive the same attention. This holds true for sulfo-conjugation that is catalysed by cytosolic sulfotransferases (SULT), a central component of the metabolism of endogenous low molecular weight molecules and xenobiotics. In particular, limited information is available about the functional roles of the mosquito predicted enzymes annotated as SULTs in genomic databases. The herein described research is the first example of a biochemical and structural study of a SULT of a mosquito species, in general, and of the malaria vector Anopheles gambiae in particular. We confirmed that the AGAP001425 transcript displays a peculiar expression pattern that is suggestive of a possible involvement in modulating the mosquito reproductive tissues physiology, a fact that could raise attention on the enzyme as a potential target for insect-containment strategies. The crystal structures of the enzyme in alternative ligand-bound states revealed elements distinguishing AgSULT-001425 from other characterized SULTs, including a peculiar conformational plasticity of a discrete region that shields the catalytic cleft and that could play a main role in the dynamics of the reaction and in the substrate selectivity of the enzyme. Along with further in vitro biochemical studies, our structural investigations could provide a framework for the discovery of small-molecule inhibitors to assess the effect of interfering with AgSULT-001425-mediated catalysis at the organismal level.

Organotrifluoroborate Sugar Conjugates for a Guided Boron Neutron Capture Therapy: From Synthesis to Positron Emission Tomography.

Sugars are a versatile tool for targeting malignant cells and have been extensively used for drug delivery and imaging techniques. Their prototype, fluorodeoxyglucose ([18F]FDG), is currently used for positron emission tomography. Boron neutron capture therapy (BNCT) is a cancer treatment that relies on irradiation with thermal neutrons of cancer cells previously loaded with [10B]-containing compounds. The recent introduction of accelerators as a neutron source for clinical use prompts the planning of delivery compounds enriched with boron able to be traced in real time. This work describes the first synthesis of a new class of sugar derivatives conjugated to a trifluoroborate moiety as potential theranostic agents. Stability and cytotoxicity studies are reported for all compounds, together with [18F] radiolabeling optimization and in vivo preliminary positron emission tomography (PET) experiments on a selected compound.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

In vitro metabolism study of 2-isopropyl-9H-thioxanthen-9-one (2-ITX) in rat and human: evidence for the formation of an epoxide metabolite.

1. 2-Isopropyl-9H-thioxanthen-9-one (2-ITX) is one of the most extensively used photoinitiators in inks found in paper and/or packaging materials for foodstuffs. Recently, traces of 2-ITX as a contaminant were discovered in baby milk and other foodstuffs at a level sufficient to pose a risk to human health. However, little is known about the toxicological profile of 2-ITX. 2. The high lipophilicity of this substance would suggest that it could be a good substrate for hepatic metabolizing enzymes and that these metabolites could have a role in the toxicological properties of 2-ITX. 3. The metabolism of 2-ITX, using both rat and human subcellular preparations, was studied and has resulted in the formation of eight polar metabolites (M1-M8) as revealed in the liquid chromatograpy/ultraviolet (LC/UV) and liquid chromatograpy/mass spectrometry (LC/MS) analyses. Their structures highlight a marked regioselectivity in the metabolism of 2-ITX; it is directed mainly toward the isopropyl moiety and the sulfur atom. 4. The unsaturated metabolite 6 causes the formation of a reactive epoxide metabolite 7. This finding was supported by identification in microsomal incubations of 1,2-diol metabolite 8 arising from the epoxide by hydrolysis and it was validated by incubating in the same conditions the synthetic epoxide 7: the formation of metabolite 8 was again observed. 5. On the basis of these data, we propose that the metabolite 6 could be included in toxicological studies of 2-ITX.

Tritylamine as an Ammonia Surrogate in the Ugi Reaction Provides Access to Unprecedented 5-Sulfamido Oxazoles Using Burgess-type Reagents.

Starting from a wide range of α-acylamino amide substructures synthesized using tritylamine as an ammonia surrogate in the Ugi reaction, Burgess-type reagents enable cyclodehydration and afford unprecedented oxazole scaffolds with four points of diversity, including a sulfamide moiety in the 5-position. The synthetic procedure employs readily available starting materials and proceeds smoothly under mild reaction conditions with good tolerance for a variety of functional groups, coming to fill a gap in the field of oxazole compounds.

Identification of the human UDP-glucuronosyltransferases involved in the glucuronidation of combretastatin A-4.

The stilbenic compound (Z)-combretastatin A-4 (CA-4) has been described as a potent tubulin polymerization inhibitor. In vivo, CA-4 binds to tubulin and inhibits microtubule depolymerization, which results in morphological changes in proliferating endothelial cells. Combretastatin A-4 prodrug phosphate is a leading vascular disrupting agent and is currently being evaluated in multiple clinical trials as a treatment for solid tumors. The aim of this study was to identify and characterize the UDP-glucuronosyltransferase (UGT) isoforms involved in CA-4 glucuronidation by incubation with human liver microsomes and a panel of nine liver-expressed recombinant UGT Supersomes (1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, 2B15, and 2B17). As we observed, the high rate of formation of CA-4 glucuronide (V(max) = 12.78 +/- 0.29 nmol/min/mg protein) and the low K(m) (6.98 +/- 0.65 microM) denoted that UGT1A9 was primarily responsible for the in vitro glucuronidation of CA-4. UGT1A6 was also a significant contributor to CA-4 glucuronidation (V(max) = 3.95 +/- 0.13 nmol/min/mg protein and S(50) = 44.80 +/- 3.54 microM). Furthermore, we demonstrated that the kinetics of CA-4 glucuronidation with liver microsomes but also with a panel of recombinant UGTs is atypical as it fits two different models: the substrate inhibition and also the sigmoidal kinetic model. Finally, experiments conducted to inhibit the glucuronosyltransferase activity in the human liver microsomes assay showed that phenylbutazone, trifluoperazine, propofol, and 1-naphthol effectively inhibited CA-4 glucuronidation.

Data on metabolic stability, aqueous solubility and CYP inhibition of novel triazole-based nicotinamide phosphoribosyltransferase (NAMPT) inhibitors.

In the related research article, entitled "Identification of novel triazole-based nicotinamide phosphoribosyltransferase (NAMPT) inhibitors endowed with antiproliferative and antiinflammatory activity" [1], we reported the in vitro hepatic metabolism data for compounds 30c, 48b, and 31b (here named as E5, A6, and T1), in comparison with the reference compounds GPP78 and FK866 [1-3]. In this article, we retrieved the available data about the hepatic microsomal stability and metabolites structural characterization of the entire library of triazole-based NAMPT inhibitors, also implementing the given information with data regarding aqueous solubility and CYP inhibition. Compounds are divided in subclasses based on the hydrolytic resistant groups replacing the amide function of GPP78 [1, 2].

Study of Anopheles gambiae 3-hydroxykynurenine transaminase activity and inhibition by LC-MS/MS method.

In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-hydroxykynurenine is removed by a specific transaminase (3-hydroxykynurenine transaminase, 3-HKT) which converts the compound into the more stable xanthurenic acid. Interfering with 3-hydroxykynurenine metabolism in A. gambiae is a potential approach for the development of transmission-blocking drugs and insecticides. Hence, the aims of this work were to develop and validate a new LC-MS/MS method for the evaluation of A. gambiae 3-hydroxykynurenine transaminase (Ag-HKT) activity and the determination of the potency of inhibitors of the enzyme. We set up a LC-MS/MS based enzymatic assay for the determination of kinetic constants values of the recombinant Ag-HKT enzyme and for the evaluation of Ag-HKT inhibition by a known protein inhibitor used as reference and a newly synthesized compound. The chromatographic separation was performed in a gradient mode on a Phenomenex Synergi Polar-RP (150 mm × 2.0, 4 µm) with methanol and water containing both 0.2% formic acid. Mass spectrometric detection was achieved with an ion trap equipped with an ESI source, in positive ionization scan, operating in SRM mode. The LC-MS/MS method was validated in terms of selectivity, linearity, precision and accuracy.

Solution-phase parallel synthesis and biological evaluation of combretatriazoles.

Combretastatin A-4 is an antitumoral and antitubulin agent that is active only in its cis configuration. In the present manuscript, we have synthesized cis-locked combretastatins containing a triazole ring (combretatriazoles). To achieve this, we have developed a column chromatography-free parallel solution-phase synthesis that exploits the reaction between azides and alpha-keto phosphorus ylids, which is known to regioselectively generate the 1,5-disubstituted triazoles. The prepared compounds were screened as antitubulinic agents, allowing us to identify three new compounds with high potency, two of which show a new mechanism of action that induces cells to appear multinucleated and display a high number of mitotic spindles.

Troxerutin, a mixture of O-hydroxyethyl derivatives of the natural flavonoid rutin: Chemical stability and analytical aspects.

Troxerutin (TRX) is a mixture of semisynthetic hydroxyethylrutosides (Hers) arising from hydroxyethylation of rutin, a natural occurring flavonoid. TRX is commonly used for its anti-oxidant and anti-inflammatory properties in chronic venous insufficiency and other vascular disorders. In recent studies, the protective effects of TRX in Alzheimer's disease, colon carcinogenesis and hepatocellular carcinoma are emerged. However, the chemical stability of TRX has never been studied. Hence, the aims of the work were to study the TRX chemical stability through a forced degradation study and to develop and validate a new stability indicating LC-UV method for determination of TRX. In order to perform the study, TRX stability was tested in various stress conditions analysing the degradation samples by LC-MS. Three degradation products (DPs; D1, D2 and D3, 3',4',7-Tri-O-(ß-hydroxyethyl)quercetin, 3',4',5,7-Tetra-O-(ß-hydroxyethyl)quercetin and 3',4'-Di-O-(ß-hydroxyethyl)quercetin respectively) arising from degradation in acidic conditions were identified and synthesized: among them, D1 resulted the stability indicator for hydrolytic degradation. Furthermore, a stability-indicating LC-UV method for simultaneous determination of triHer (3',4',7-Tri-O-(ß-hydroxyethyl)rutin, the principal component of the mixture) and D1 was developed and validated. The LC-UV method consisted in a gradient elution on a Phenomenex Kinetex EVO C18 (150 × 3 mm, 5 µm) with acetonitrile and ammonium bicarbonate buffer (10 mM, pH 9.2). The method was linear for triHer (20-60 µg mL-1) and D1 (5.1-35 µg mL-1). The intraday and interday precision were determined and expressed as RSDs: all the values were ≤ 2% for both triHer and D1. The method demonstrated also to be accurate and robust and the average recoveries were 98.8 and 97.9% for triHer and D1, respectively. Moreover, the method resulted selective and specific for all of the components present in the degradation pattern of TRX (diHer (3',4'-Di-O-(ß-hydroxyethyl)rutin), triHer, tetraHer (3',4',5,7-Tetra-O-(ß-hydroxyethyl)rutin), D3, D1 and D2) and it was successfully applied for the stability studies of both drug substances and drug products.