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metodologia per la sintesi di derivati delle benzotieno[2, 3-b]piridine e dmiostrazione della loro attività come inibitori della ß-secretasi

The patent describes new peptidomimetic inhibitors of retroviral proteases, in particular of human immunodeficiency virus (HIV) protease. These inhibitors comprise as the core structure a new diaminodiol isostere of the dipeptide Phe-Pro having four stereogenic centres. The inhibitors of the invention have been shown to inhibit HIV protease and can therefore be usefully employed as antivirals for post-exposure prophylaxis and as a therapy for viral infections by a retrovirus, in particular HIV. The syntheses processes of the isosteres and inhibitors are also described.

A practical and general method for the Biginelli cyclocondensation of guanidine with aldehydes and β-dicarbonyl compounds is described and illustrated with the synthesis of a set of 26 functionalized 2-amino-3,4-dihydropyrimidines. The simple protocol involves the ­microwave-mediated reaction of a twofold excess of guanidine hydrochloride with the required reaction partners in an alcohol at 120 °C. Yields are generally good, with short reaction times and a simple workup. The scope is considerably wider than that of similar reactions ­carried out under conventional heating.

Microgels can be considered soft, porous and deformable particles with an internal gel structure swollen by a solvent and an average size between 100 and 1000 nm. Due to their biocompatibility, colloidal stability, their unique dynamicity and the permeability of their architecture, they are emerging as important candidates for drug delivery systems, sensing and biocatalysis. In clinical applications, the research on responsive microgels is aimed at the development of “smart” delivery systems that undergo a critical change in conformation and size in reaction to a change in environmental conditions (temperature, magnetic fields, pH, concentration gradient). Recent achievements in biodegradable polymer fabrication have resulted in new appealing strategies, including the combination of synthetic and natural-origin polymers with inorganic nanoparticles, as well as the possibility of controlling drug release remotely. In this review, we provide a literature review on the use of dual and multi-responsive chitosan-grafted-poly-(N-vinylcaprolactam) (CP) microgels in drug delivery and oncological applications.

A simple laboratory procedure for the isolation of pure cafestol and 16-O-methylcafestol together with β-sitosterol from coffee is disclosed. Cafestol and 16-O-methylcafestol have been exhaustively characterized through 1D and 2D 1H, 13C NMR, CD and X-ray diffraction. For the first time, the molecular structure of cafestol is reported and the assignment of the absolute configuration is unequivocally given by exploiting anomalous scattering of a brominated derivative.

The activation of signals in fluorescent nanosensors upon interaction with their targets is highly desirable. To this aim, several molecularly imprinted nanogels have been synthetized for the recognition of tyrosol, hydroxytyrosol and oleuropein in aqueous extracts using the non-covalent approach. Two of them contain fluorescein derivatives as co-monomers, and their fluorescence emission is switched on upon binding of the target phenols. The selection of functional monomers was previously done by analyzing the interactions by nuclear magnetic resonance (NMR) in deuterated dimethylsulfoxide (DMSO-d6) of the monomers with tyrosol and hydroxytyrosol. Polymers were synthetized under high dilution conditions to obtain micro- and nano-particles, as verified by transmission electron microscopy (TEM). 1,4-Divinylbenzene (DVB) was used in the fluorescent polymers in order to enhance the interactions with the aromatic ring of the templates tyrosol and hydroxytyrosol by &pi, &pi, stacking. The results were fully satisfactory as to rebinding: DVB-crosslinked molecularly imprinted polymers (MIPs) gave over 50 nmol/mg rebinding. The sensitivity of the fluorescent MIPs was excellent, with LODs in the pM range. The sensing polymers were tested on real olive leaves extracts, with very good performance and negligible matrix effects.

New heteroaryl HIV-protease inhibitors bearing a carbamoyl spacer were synthesized in few steps and high yield, from commercially available homochiral epoxides. Different substitution patterns were introduced onto a given isopropanoyl-sulfonamide core that can have either H or benzyl group. The in vitro inhibition activity against recombinant protease showed a general beneficial effect of both carbamoyl moiety and the benzyl group, ranging the IC50 values between 11 and 0.6 nM. In particular, benzofuryl and indolyl derivatives showed IC50 values among the best for such structurally simple inhibitors. Docking analysis allowed to identify the favorable situation of such derivatives in terms of number of interactions in the active site, supporting the experimental results. The inhibition activity was also confirmed in HEK293 mammalian cells and was maintained against protease mutants. Furthermore, the metabolic stability was comparable with that of the commercially available inhibitors.

Estrogens play a key role in cellular proliferation of estrogen-receptor-positive (ER+) breast cancers (BCs). Suppression of estrogen production by competitive inhibitors of the enzyme aromatase (AIs) is currently one of the most effective therapies against ER + BC. Yet, the development of acquired resistance, after prolonged treatments with AIs, represents a clinical major concern. Serendipitous findings indicate that aromatase may be non-competitively inhibited by clinically employed drugs and/or industrial chemicals. Here, by performing in silico screening on two putative allosteric sites, molecular dynamics and free energy simulations, supported by enzymatic and cell-based assays, we identified five leads inhibiting the enzyme via a non-active site-directed mechanism. This study provides new compelling evidences for the existence of an allosteric regulation of aromatase and for the possibility of exploiting it to modulate estrogens biosynthesis. Such modulation can aptly reduce side effects caused by the complete estrogen deprivation therapy, and, possibly, delay/avoid the onset of resistance. (C) 2019 Elsevier Masson SAS. All rights reserved.

Quantitative analyses of mono-p-coumaroylquinic acids (pCoQAs) and total chlorogenic acids (CGAs) in green coffee commercial lots of C. arabica, C. canephora and C. liberica from different geographical origins and eight wild Coffea species were carried out. Among the commercial lots, pCoQAs average content of C. arabica (0.67 mg/g) is higher than that of C. canephora (0.40 mg/g) being C. liberica intermediate (0.58 mg/g). As far as the analyzed wild Coffea species is concerned, C. pseudozanguebariae is characterized by the lower pCoQAs content (0.12 mg/g) whereas C. sessiliflora is by far the richest source of pCoQAs (2.18 mg/g). Effect of the roasting process on the mono-p-coumaroylquinic acids profile was evaluated for the economically exploited species C. arabica and C. canephora. For the first time distribution of mono-p-coumaroylquinic acid isomers in wild coffee species by fast and accurate UHPLC-DAD analyses using authentic standards previously synthetized, is reported.

Peptidomimetic inhibitors of HIV-1 PR are still a key resource in the fight against AIDS. Here we describe the synthesis and biological activity of HIV-1 PR inhibitors based on four novel dihydroxyethylene isosteres of the Phe-Pro and Pro- Pro dipeptides. The isosteres, containing four stereogenic centers, were synthesized in high yield and excellent stereoselectivity via the cyclization of epoxy amines derived from α-amino acids. The inhibitors were assembled by coupling the isosteres with suitable flanking groups and were screened against recombinant HIV PR showing activities in the subnanomolar to micromolar range. Two Phe-Pro-based inhibitors active at the nanomolar level were further investigated: both inhibitors combine the ability to suppress HIV-1 replication in infected MT-2 cells with low cytotoxicity against the same cells, thereby displaying a high therapeutic index. These results demonstrate the potential of the new Phe-Pro dihydroxyethylene isostere as a core unit of powerful HIV-1 PR inhibitors.

Ostreopsis cf. ovata, a benthic dinoflagellate often blooming along the Mediterranean coasts, has been associated with toxic events ranging from dyspnea to mild dermatitis. In late September 2009, an Ostreopsis cf. ovata bloom occurred in the Gulf of Trieste (Northern Adriatic Sea; Italy), causing pruritus and mild dermatitis in beachgoers. An integrated study was initiated to characterize Ostreopsis cells by light and confocal microscopy, PCR techniques, immunocytochemistry, and high resolution liquid chromatography-mass spectrometry (HR LC-MS). The presence of Ostreopsis cf. ovata of the Atlantic/Mediterranean clade was unambiguously established by morphological and genetic analyses in field samples. Several palytoxin-like compounds (ovatoxin-a,-b,-c,-d,-e) were identified by HR LC-MS, ovatoxin-a being the most abundant (45-64 pg/cell). Surprisingly, no palytoxin was detected. For the first time, monoclonal and polyclonal antipalytoxin antibodies revealed the intracellular cytoplasmic localization of ovatoxins, suggesting their cross-reactivity with these antibodies. Since harmful dinoflagellates do not always produce toxins, the immunocytochemical localization of ovatoxins, although qualitative, can provide an early warning for toxic Ostreopsis cells before their massive diffusion and/or concentration in seafood.

A series of new thienyl ring containing analogues of nelfinavir and saquinavir with different substitution patterns were synthesized from suitable enantiopure diols. Their inhibitory activity against wild type recombinant HIV-1 protease was evaluated. In general thienyl groups spaced from the core by a methylene group gave products showing IC(50) in the nanomolar range, irrespective of the type and the substitution pattern of the heterocycle. The range of activity of the two most active compounds is substantially maintained or even increased against two commonly selected mutants, under drug pressure, such as V32I and V82A.

The anthranilic acid diamides represent the more recent class of nonpeptide CCK(1) receptor antagonists. This class is characterized by the presence of anthranilic acid, used as a molecular scaffold, and two pharmacophores selected from the C-terminal tetrapeptide of CCK. The lead compound coded VL-0395, endowed with sub-micromolar affinity towards CCK(1) receptors, was characterized by the presence of Phe and 2-indole moiety at the C- and N-termini of anthranilic acid, respectively. Herein we describe the first step of the anthranilic acid C-terminal optimization using, instead of Phe, aminoacids belonging to the primary structure of CCK-8 and other not coded residues. Thus we demonstrate that the CCK(1) receptor affinity depends on the nature of the aminoacidic side chain as well as that the free carboxy group of the alpha-aminoacids is crucial for the binding. The R enantiomers of the most active compounds represent the eutomers of this class of antagonists confirming thus the stereo preference of the receptor. Moreover this SAR study demonstrates that the receptor binding pocket, that host the aminoacidic side chain, results much more tolerant respect to that accommodating the indole ring. As a result, an appropriate variation of the aminoacidic side chain could provide a better CCK(1) receptor affinity diorthosis.

We exploit a recently developed computational approach [I. Gladich et al, J. Chem. Phys. B, DOI: 10.1021/acs.jpcb.5b06227 (2015)] to design cyclic peptides capable to recognize chlorogenic acid and related phenolic compound. A peptide designed by this procedure was synthesized and characterized by circular dichroism and fluores-cence spectroscopy, cyclic voltammetry and differential pulse voltammetry. We found that the peptide is selective for chlorogenic acid against other ortho-diphenols such as caffeic acid, and mono-phenols as ferulic and coumaric acid. Indeed, when chlorogenic or caffeic acid are bound to the cyclic peptide, the ortho-diphenol moiety capable to undergo oxidation is not available to electrode surface due to diffusion limitation and steric hindrance. This phenomenon did not occur for cumaric and ferulic acid possibly because of limited complex formation with the cyclic peptide. In an electrochemical sensing system the peptide can therefore discriminate ortho-diphenols in a mixture of phenols.

An assay for routine therapeutic drug monitoring of anti-HIV HAART drugs in clinical use is highly desirable, in order to rapidly measure the pharmacokinetic parameters on single patients. We have started a project to develop a panel of enzyme-linked immunosorbent assays (ELISA) for the whole set of HAART drugs, and the development, performance and evaluation of the assay for amprenavir is described here. A diazo conjugate of amprenavir has been used in order to raise polyclonal anti-amprenavir antibodies in rabbits. Antisera have been used to set up a quantitative and rapid competitive assay. Plasma samples are simply diluted in the assay buffer after thermal inactivation, before running the assay. The assay allows the detection of amprenavir in the quantification range 400-5000 ng/ml, in a diluted plasma sample. The assay has been compared with an HPLC reference technique, on 27 samples from treated patients. Within the quantification range, the ELISA data are well correlated with the HPLC results by a regression line close to the identity, and a Bland-Altman analysis shows the agreement between the two methods.

Cafestol and 16-O-methylcafestol are diterpenes present in coffee, but whilst cafestol is found in both Coffea canephora and Coffea arabica, 16-O-methylcafestol (16-OMC) was reported to be specific of only C. canephora. The interactions of such compounds, with serum albumins, have been studied. Three albumins have been considered, namely human serum albumin (HSA), fatty acid free HSA (ffHSA) and bovine serum albumin (BSA). The proteins interact with the diterpenes at the interface between Sudlow site I and the fatty acid binding site 6 in a very peculiar way, leading to a significant change in the secondary structure. The diterpenes do not displace reference binding drugs of site 2, but rather they enhance the affinity of the site for the drugs. They, therefore, may alter the pharmacokinetic profile of albumin - bound drugs.

The aspartic protease of HIV-1 represents a valid therapeutic target of antiviral agents suitable for the treatment of AIDS. We have designed peptidomimetic inhibitors for this enzyme with a hydroxyethylenediamine core, based on a molecular modeling approach that predicts the effectiveness of the designed compounds in terms of computed enzyme-inhibitor complexation Gibbs free energies. This structurebased molecular design was then combined with a synthetic strategy that couples stereochemical control with full flexibility in the choice of the central core side chains and of the flanking residues. A series of peptidomimetic inhibitors was thus assembled from readily available amino acids and carboxylic acids and -Phe-ω[CH2-(r/s)CHOH]-Phe- cores. The IC50 values for these compounds ranged from 3 nM to 80 μM, allowing a QSAR analysis and identification of factors that determine the inhibition potency of the compounds. Predicted ADME-related properties of the inhibitor candidates span a range of pharmacokinetics profiles, which allows selection of a potent and bioavailable lead compound for further development.

Density functional theory calculations with the B3LYP hybrid functional have been performed to determine the equilibrium structures of a representative series of thiono-, oxo,thiono-, dioxo-, and dithiono-derivatives of sparteine. All these molecules except 2,13-dioxo-sparteine adopt one most stable conformation or exhibit a marked preference for one of the two lowest-energy conformations of the parent sparteine (with each functionalized ring distorted as a sofa or half-chair). 2,13-Dioxo-sparteine is instead predicted to exist as a nearly 1:1 mixture of “ring C chair” and “ring C boat” conformers in solution. The theoretical structural models are consistent with the available X-ray experimental results. The electronic structure of these bis-quinolizidine alkaloids has been studied by measuring and calculating important features of their NMR and photoelectron spectra. In particular, a representative set of NMR chemical shifts and nuclear spin–spin coupling constants, calculated by means of DFT formalisms, compare favourably with experiment. Notably, the repercussion of stereoelectronic hyperconjugative effects on Δ δ (H eq /H ax ) and Δ 1 J (CH eq /CH ax ) of the N–CO(S)– groups is correctly accounted for by the DFT results. Based on ab initio outer valence Green’s function calculations, a reliable interpretation of the uppermost bands in the photoelectron spectra has been advanced. The theoretical results indicate a complex interaction of the n (N), n (O), n (S), π (CO), and π (CS) chromophores within the functionalized sparteine framework. The theoretical lowest-energy conformer of all compounds but 2,13-dioxo-sparteine allows a consistent interpretation of the photoelectron spectrum. The conformation adopted by 2,13-dioxo-sparteine likely changes on passing from the crystalline form at room temperature (“ring C boat”) to the gas phase at high temperature (“ring C chair”).

Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo.

Screening of a large number of clones produced in a fusion is often the bottleneck in the isolation of catalytic antibodies. The usual approach requires two steps: clones are first selected for their high affinity to the antigen, and then the good binders are tested for their catalytic activity. To simplify this selection process, a competitive enzyme-linked immunosorbent assay (ELISA) has been developed that allows direct screening of the antibodies on the basis of their catalytic activity. In this assay, the product of the catalyzed reaction binds to an immobilized anti-product antibody in competition with a peroxidase-product conjugate. The screening assay has been developed for the antibody-catalyzed hydrolysis of esters of p-aminophenylacetic acid and has been tested on the porcine liver esterase (PLE)-catalyzed hydrolysis of the same substrates. This test allows the detection of product formation at the nanomolar level, while, in a typical assay, the catalytic activity of PLE can be traced down to 200 fmol of enzyme. Under standard conditions for the screening of hybridomas obtained from a fusion, the competitive ELISA allows detection of catalytic species with values of kcator = 5 x 10(-7) mol l-1 s-1 and kcat/kuncator = 50. While the assay has been designed for the selection of catalytic antibodies, other potential applications of this methodology are in the screening of libraries of engineered and designed enzymes and, in general, in the quantitative measurement of enzyme activity.

A computational investigation of the specific optical rotation and of the electronic circular dichroism spectra of two chiral 1,4-dihydropyridazines was performed and compared with existing experimental data to verify a previous assignment of their absolute configuration based on a well-accepted mechanism of catalysis of the organocatalyst used in their synthesis. Both the optical rotation and circular dichroism calculations indicate that the absolute configuration is opposite to the one assigned on the basis of the mechanism originally assumed. An alternative reaction mechanism is therefore suggested.

We have identified a 101-amino-acid polypeptide derived from the sequence surrounding the IIA binding site of human albumin. The polypeptide contains residues that make contact with ligands as warfarin in the parent protein, and eight cysteine residues to form disulfide bridges, which stabilize the polypeptide structure. Seventy-four amino acids are located in six [alpha]-helical regions, with the remaining amino acids forming six connecting coil/loop regions. Codon usage optimization was used to express a GST fusion protein in E. coli in yields as high as 4 mg/l. This fusion protein retains its structural integrity and aldolase activity, the ability to direct the stereochemical outcome of a diketone reduction, and its binding capacity to warfarin and efavirenz. Notably, this newly cloned polypeptide represents a valuable starting point for the construction of libraries of binders and catalysts with improved proficiency.

Synthesis and biological evaluation of a new series of potential HIV-1 protease inhibitors incorporating different heterocycles are described. The variation of heteroatom in such molecules has displayed totally different biological activities and a benzothiophene containing inhibitor has shown high potency against wild type HIV-1 protease with IC50 = 60 nM, thanks to the lower desolvation penalty to be payed by such hydrophobic moiety.

A method for the determination of zeranol and its metabolite beta-zearalanol in bovine urine is described. It has been applied to samples from calves given multiple subcutaneous doses of zeranol. Samples were extracted with immunoaffinity columns containing antibodies raised against zeranol and were analysed by gas chromatography-mass spectrometry. The immunoaffinity columns were prepared by coupling immunoglobulin G fractions obtained from rabbit antisera with a Sepharose matrix. The immunizing agent was carboxybutylzeranol coupled to bovine serum albumin. Gas chromatography-mass spectrometry was performed in the negative-ion chemical ionization mode, after derivatization of the compounds to their pentafluorobenzyl ethers, and allowed detection of analytes with a sensitivity of 0.01 ppb in spiked urine. The derivatization method and the gas chromatographic determination were also applied to the similar compounds zearalanone, zearalenone and beta-zearalenol. A synthesis of dideuterated zeranol and beta-zearalanol by isotopic exchange is described. These deuterated analogues had an isotopic purity of more than 99% and were used for quantitation of zeranol and beta-zearalanol by isotope dilution mass spectrometry. The recoveries of zeranol and beta-zearalanol, using the immunoaffinity columns, were determined after extraction from spiked urine and were 84 and 64%, respectively. The urines of treated calves were collected for several days after treatments and were analysed after hydrolysis with beta-glucuronidase and arylsulphatase. The samples showed variable but generally decreasing concentrations of zeranol and beta-zearalanol. The levels of beta-zearalanol ranged from less than 0.01 to 98 ppb and were 1.2-3.2 times higher than those of zeranol.

A stereoselective synthesis of hydroxyethylene dipeptide isosteres based on the 1,4-diamino-2-hydroxybutane structure is described. Horner-Emmons olefination of phosphonates derived from alpha-amino acids, stereoselective reduction of the resulting enones to allylic alcohols, and syn epoxidation of the latter lead to enantiomerically pure 1-amino-2-hydroxy-3,4-epoxybutanes, key intermediates in the synthesis. Reductive cleavage of the epoxy alcohols with Red-Al proceeds in a highly regioselective way, giving 1-amino-2,4-dihydroxybutanes, from which diamino alcohol hydroxyethylene isosteres are obtained by selective protection of the secondary 2-hydroxy group, via cyclization to 1,3-oxazolidinone, and further elaboration of the 4-hydroxy. Both C-2 epimers of 1,4-diamino-2-hydroxybutanes are accessible by appropriate choice of the conditions for cyclization. The approach is demonstrated by the synthesis of a series of six hydroxyethylene dipeptide isosteres, including the diamino alcohol core of potent HIV-protease inhibitor ritonavir 18 and its C-2 epimer 11a.

A series of new N-substituted anthranilic acid dimer derivatives having a C-terminal Phe residue was synthesized and evaluated for their affinity for CCK receptors. These compounds resulted from a blended approach based firstly on the use of an alternative substructure embedded within asperlicin and secondly on the derivatization of this template with substituents chosen considering the C-terminal primary structure of the endogenous ligand. Although these compounds exhibited a regnylogical-type organization similar to that of CCK-4, they are characterized by about 1000-fold greater affinity for CCK-A receptor than the C-terminal tetrapeptide.

Bovine and human serum albumins catalyze the aldol reaction of aromatic aldehyedes and acetone, with saturation kinetics and moderate and opposite enantioselectivity. The reaction occurs at the binding site in domain IIa, and is inhibited by warfarin. Kinetic data are consistent with an enamine mechanism. The activity is conserved in a 103 aminoacid peptide derived from the albumin sequence.

The hydrolysis of N-p-toluoylindole and its 3-formyl derivative, at pH 8.0, are accelerated by a factor of nearly 103 by an antibody obtained against a KLH conjugate of N-p-toluensulfonylindole.

Esterase antibody 522c2, the first example of a catalytic antibody specifically programmed to control the reactivity of functional groups on the side chain of tyrosine, accelerates the hydrolysis of benzoate esters of L-tyrosine and tyrosine-containing dipeptides by a factor of 104 and is moderately active against other benzoate esters.

Two peptidomimetic inhibitors based on a novel epoxyalcohol core were designed to target the epoxide ring at the catalytic aspartates of HIV-protease for irreversible inhibition of the enzyme. The inhibitors were synthesized with a multi-step approach which includes Horner-Emmons olefination of a phenylalanine-derived phosphono ketone, stereoselective reduction of the resulting trans-enones to allylic alcohols and syn-epoxidation of the latters. The epoxyalcohols thus obtained were assayed for their ability to inhibit HIV-PR and were shown to inhibit the protease with IC50 values of 39 and 150 µM, respectively. This confirms that the designed epoxides are recognised with fairly good affinity by the enzyme’s active site, a pre-requisite for selective irreversible inhibition.