Boronic acids as partners in multicomponent reactions

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014In the last three decades, the boronic acid notoriety and usefulness changed from almost neglected to a highly used class of synthetic reactant. The mild Lewis acidity, together with their stability, easy handling and environmental friendly impact attracted the attention of the synthetic community towards these peculiar compounds. They found use in classical organic chemistry as synthetic reagents as well as in the medicinal chemistry as potent inhibitors. The purpose of this work was to explore a relatively new field of application, in which the boronic acid functionality acts as a template to promote the assembly of simple building blocks into more complex molecules, and explore this strategy as a tool to easily synthesize small collections of active compounds. The biological activity of such collections was tested against eligible targets (HNE; PHA) in order to select the most promising compounds and use them as a new benchmark for a new round of interactive optimization aiming to amplify the percentage of success in the discovery of new lead compounds. reaction design to mimic the natural architecture using boronic acids as tethers for building blocks: Synthesis of the fused tricycle-boronate heterocycles. The B ability to act as a tether was used to simply assemble heterocycles through covalent bond formation with two oxygen and a dative bond with the N. A highly efficient one-pot four component reaction is the protocol exploited to assemble 23 tricycle boronated heterocycles in yields and diastereoselectivities up to 95% and 97% respectively. DFT calculation were performed to clarify the reaction mechanism. Synthesis of fused bicycle-boronate heterocycles and their enzymatic evaluation as new human neutrophil elastase (HNE)inhibitors Herein we demonstrate that boron tether strategy may synthetize collection of molecules to discover new hit compounds. Through a one pot three components reaction 17 new compounds were synthesize in excellent yields up to 98%, high diastereoselectivities up to de100%. The compounds were tested as novel enzyme inhibitors for HNE and reaching the IC50 up to 1.10 μM. Docking studies were performed to enlighten the enzymatic interaction between the molecules and the active site of the HNE. Design of phenylalanine hydroxylase (PAH) inhibitors based on fused bicycle-boronate heterocycles The bicycle boronate heterocycles were also tested against the wild type PHA compound 27 seems able to stabilize the protein affording a new scaffold for further optimization. Synthesis of diazaborines and their enzymatic evaluation as new HNE inhibitors Herein we disclose the value of the diazaborines as inhibitor of the HNE. The collection was synthetize through optimization of the Dewar’s procedure that enabled the synthesis of the diazaborine in yield up to 89%. and IC50 2.5μM potency against HNE.No decorrer das últimas 3 décadas, os ácidos borónicos evoluíram de quase curiosidades químicas até ao reconhecimento como reagentes de grande utilidade preparativa. A acidez de Lewis moderada, acompanhada de uma boa estabilidade, facilidade de manuseamento e baixo impacto ambiental, chamaram a atenção da comunidade sintética para estes compostos peculiares. Encontraram aplicação em química orgânica clássica como reagentes sintéticos, bem como em química medicinal como poderosos inibidores enzimáticos. O objecto deste trabalho foi explorar um campo de aplicação emergente, no qual o grupo funcional ácido borónico actua como molde para promover a associação de blocos constitutivos simples em moléculas mais complexas, e aplicar esta estratégia como ferramenta para a síntese de pequenas bibliotecas de compostos activos. A eficácia destas bibliotecas foi ensaiada em alvos selecionados (HNE; PHA) por forma a eleger os compostos mais promissores, por sua vez utilizados como padrões para um novo ciclo de optimização estrutural. Procurou-se assim maximizar a percentagem de sucesso no processo de descoberta de novas moléculas promissoras. Ensaiaram-se os seguintes conceitos estruturais por forma a mimetizar arquitecturas encontradas em produtos naturais: Síntese de arquiteturas bora-tricíclicas fundidas: A capacidade do boro para actuar como elo de ligação foi aproveitada para a construção de estruturas heterocíclicas via ligação covalente com dois átomos de oxigénio e uma ligação dativa com um átomo de azoto. Através de uma reacção de uma só etapa envolvendo quatro componentes, construíram-se 23 espécies tricíclicas boradas em rendimentos e diastereoselectividades de até, respectivamente, 95 e 97%. Cálculos de DFT permitiram obter informação sobre o mecanismo da reacção. Síntese de arquiteturas bora-bicíclicas fundidas e respectiva avaliação como inibidores da elastase neutrofílica humana (HNE): Demonstrou-se que a estratégia de utilizar o boro como elo permite a síntese rápida de um conjunto de moléculas, acelerando a descoberta de novas moléculas activas. A reacção de três componentes numa só etapa permitiu a síntese de 17 novos compostos em excelentes rendimentos de até 98%, e diastereoselectividades de até 100%. O ensaio XXIV dos compostos como inibidores enzimáticos da HNE evidenciou IC50’s de até 1,10 μM. Efectuaram-se estudos de docking no sentido de elucidar a interacção entre as moléculas e o centro activo da HNE. Desenvolvimento de inibidores da fenilalanina hidroxilase (PAH) baseados na arquitetura bora-bicíclica fundida: Os referidos bora-bicíclicos foram também ensaiados perante a PAH de tipo selvagem. O composto 27 aparenta ser capaz de estabilizar a proteína, servindo de ponto de partida para posterior optimização. Síntese de diazaborinas e respectiva avaliação como novos inibidores da HNE: Demonstrou-se o valor de certas diazaborinas como inibidores da HNE. Esta biblioteca foi sintetizada por meio da optimização do procedimento de Dewar, tendo-se obtido os compostos pretendidos com rendimentos de até 89%, os quais evidenciaram IC50’s de até 2,5μm contra a HNE

Gastro-Resistant Microparticles Produced by Spray-Drying as Controlled Release Systems for Liposoluble Vitamins

In the present study, gastro-resistant microparticles (MPs) were produced using the spray-drying technique as controlled-release systems for some model liposoluble vitamins, including retinyl-palmitate, retinyl-acetate, beta-carotene, cholecalciferol and alpha-tocopherol. The gastroprotective action of three different gastro-resistant excipients, the anionic methacrylic copolymer (Eudraguard (R) Biotic, E1207), the cellulose acetate phthalate (CAP) and whey proteins (WPs), was compared. The latter was used to produce a novel delivery system manufactured with only food-derived components, such as milk, and showed several improvements over the two synthetic gastro-resistant agents. Scanning electron microscopy (SEM) images showed a quite homogeneous spherical shape of all microparticle batches, with an average diameter between 7 and 15 mu m. FTIR analysis was used to evaluate the effective incorporation of vitamins within the microparticles and the absence of any degradation to the components of the formulation. The comparison graphs of differential scanning calorimetry (DSC) confirmed that the spray drying technique generates a solid in which the physical interactions between the excipients and the vitamins are very strong. Release studies showed a prominent pH-controlled release and partially a delayed-release profile. Ex vivo permeation studies of retinyl palmitate, retinyl acetate and alpha-tocopherol revealed greater transmucosal permeation capacity for microparticles produced with the WPs and milk

Sudden vestibular and hearing loss in mediterranean spotted fever: The first case report

Mediterranean spotted fever is an infection disease caused by Rickettsiae and transmitted by tick puncture; the diffusion of such disease is wide in countryside of the Mediterranean regions. The disease may be complicated by several organ failure due to a vasculitis, and also the inner ear can be involved although rarely. We report the first documented case of total involvement of inner ear with sensorineural hearing loss and acute vestibular impairment. The patient was treated with medical therapy and resolution of the infection, but the inner ear damage was not reversible with persistent loss of all functions, documented by instrumental vestibular analysis

Mille general practice governance (MilleGPG): an interactive tool to address an effective quality of care through the Italian general practice network.

The General Practitioner (GP) is the "gate-keeper" in patients' treatment and management. Herein, the use of Electronic Medical Records (EMR) could represent an effective support for GPs. Software capable of managing EMRs are available and they can be functional in adopting treatment guidelines by means of computerized prompts and reminders systems. These tools can be also programmed to include clinical algorithms with which to measure the quality of care to make possible the identification of clinical issues, and to take actions for addressing them. Given that similar tools were not available in Italy, we developed MilleGPG, an interactive tool aimed to evaluate, and subsequently improve the quality of care among patients with comorbidities

Comparative genomic hybridization on microarray (a-CGH) in constitutional and acquired mosaicism may detect as low as 8% abnormal cells

<p>Abstract</p> <p>Background</p> <p>The results of cytogenetic investigations on unbalanced chromosome anomalies, both constitutional and acquired, were largely improved by comparative genomic hybridization on microarray (a-CGH), but in mosaicism the ability of a-CGH to reliably detect imbalances is not yet well established. This problem of sensitivity is even more relevant in acquired mosaicism in neoplastic diseases, where cells carrying acquired imbalances coexist with normal cells, in particular when the proportion of abnormal cells may be low.</p> <p>We constructed a synthetic mosaicism by mixing the DNA of three patients carrying altogether seven chromosome imbalances with normal sex-matched DNA. Dilutions were prepared mimicking 5%, 6%, 7%, 8%, 10% and 15% levels of mosaicism. Oligomer-based a-CGH (244 K whole-genome system) was applied on the patients' DNA and customized slides designed around the regions of imbalance were used for the synthetic mosaics.</p> <p>Results and conclusions</p> <p>The a-CGH on the synthetic mosaics proved to be able to detect as low as 8% abnormal cells in the tissue examined. Although in our experiment some regions of imbalances escaped to be revealed at this level, and were detected only at 10-15% level, it should be remarked that these ones were the smallest analyzed, and that the imbalances recurrent as clonal anomalies in cancer and leukaemia are similar in size to those revealed at 8% level.</p

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal-oxide-semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast

Anionic Methacrylate Copolymer Microparticles for the Delivery of Myo-Inositol Produced by Spray-Drying: In Vitro and In Vivo Bioavailability

: In this study, a new micro delivery system based on an anionic methacrylate copolymer, able to improve the biological response of myo-inositol by daily oral administration, was manufactured by spray-drying. It has an ideal dose form for oral administration, with an experimental drug loading (DL)% of 14% and a regulated particle size of less than 15 μm. The new formulation features an improvement on traditional formulations used as a chronic therapy for the treatment of polycystic ovary syndrome. The microparticles' release profile was studied and ex vivo porcine intestinal mucosa permeation experiments were performed to predict potential improvements in oral absorption. Batch n. 3, with the higher Eudragit/MI weight ratio (ratio = 6), showed the best-modified release profiles of the active ingredient, ensuring the lowest myo-inositol loss in an acidic environment. The in vivo evaluation of the myo-inositol micro delivery system was carried out in a rat animal model to demonstrate that the bioavailability of myo-inositol was increased when compared to the administration of the same dosage of the pure active ingredient. The AUC and Cmax of the loaded active molecule in the micro delivery system was improved by a minimum of 1.5 times when compared with the pure substance, administered with same dosage and route. Finally, the increase of myo-inositol levels in the ovary follicles was assessed to confirm that a daily administration of the new formulation improves myo-inositol concentration at the site of action, resulting in an improvement of about 1.25 times for the single administration and 1.66 times after 7 days of repeated administration when compared to pure MI

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value

Chromosome anomalies in bone marrow as primarycause of aplastic or hypoplastic conditions andperipheral cytopenia: disorders due to secondaryimpairment of RUNX1 and MPL genes

Background Chromosome changes in the bone marrow (BM) of patients with persistent cytopenia are often considered diagnostic for a myelodysplastic syndrome (MDS). Comprehensive cytogenetic evaluations may give evidence of the real pathogenetic role of these changes in cases with cytopenia without morphological signs of MDS. Results Chromosome anomalies were found in the BM of three patients, without any morphological evidence of MDS: 1) an acquired complex rearrangement of chromosome 21 in a boy with severe aplastic anaemia (SAA); the rearrangement caused the loss of exons 2-8 of the RUNX1 gene with subsequent hypoexpression. 2) a constitutional complex rearrangement of chromosome 21 in a girl with congenital thrombocytopenia; the rearrangement led to RUNX1 disruption and hypoexpression. 3) an acquired paracentric inversion of chromosome 1, in which two regions at the breakpoints were shown to be lost, in a boy with aplastic anaemia; the MPL gene, localized in chromosome 1 short arms was not mutated neither disrupted, but its expression was severely reduced: we postulate that the aplastic anaemia was due to position effects acting both in cis and in trans, and causing Congenital Amegakaryocytic Thrombocytopenia (CAMT). Conclusions A clonal anomaly in BM does not imply per se a diagnosis of MDS: a subgroup of BM hypoplastic disorders is directly due to chromosome structural anomalies with effects on specific genes, as was the case of RUNX1 and MPL in the patients here reported with diagnosis of SAA, thrombocytopenia, and CAMT. The anomaly may be either acquired or constitutional, and it may act by deletion/disruption of the gene, or by position effects. Full cytogenetic investigations, including a-CGH, should always be part of the diagnostic evaluation of patients with BM aplasia/hypoplasia and peripheral cytopenias

Improvement of a low-cost buoy for the measurement of the sea wave weather

This article presents the result of an experimental meteorological buoy designed by the Department of Engineering of the University of Palermo, characterized by low costs and great versatility, and equipped with industrially certified measuring and detection instruments. This buoy can measure the parameters of the water column and continuously transmit the data to a remote acquisition centre. Maintenance requirements are very limited