11th National Meeting of Organic Chemistry and 4th Meeting of Therapeutic Chemistry

For the first time under the auspices of Sociedade Portuguesa de Química, the competences of two important fields of Chemistry are brought together into a single event, the 11st National Organic Chemistry Meeting and the the 4th National Medicinal Chemistry Meeting, to highlight complementarities and to promote new synergies. Abstracts of plenary lectures, oral communications, and posters presented during the meeting are collected in this report.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's and Parkinson's diseases, or rare neurodegenerative diseases like Familial Amyloid Polyneuropathy (FAP). FAP is an amyloid disease caused by mutations in the protein transthyretin (TTR) and characterized by progressive peripheral and autonomic polyneuropathy. It manifests by early impairment of pain and heat sensations in the lower limbs and progresses to a general lowering of the state of health and severe autonomic dysfunction with fatal consequences in many patients, if untreated, 10-15 years after the onset of the first symptoms.
TTR is a homotetrameric protein mainly synthesized in the liver, the choroid plexus, and the retina, forming independent TTR pools in the plasma, cerebrospinal fluid and the eye, respectively. Liver transplantation (LT) has been the standard treatment option for FAP for nearly two decades. LT halts progression of clinical symptoms by replacing the main organ producing the disease-associated mutant TTR. More recently, tafamidis meglumine (brand name Vyndaqel) reached the European and Japanese drug markets as the first drug therapy directed to the treatment of FAP. Tafamidis has shown that stabilization of the native tetrameric form of TTR by molecules endowed with chaperone-like activity is a viable approach to prevent (or at least stall) the formation of amyloid aggregates and fibrils, thus delaying disease progression. However, tafamidis demonstrated improvement of symptoms in only approximately 60% of the FAP patients.
Nevirapine (NVP, 1) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type 1. NVP's clinical efficacy, along with low cost, favourable lipid profile, and suitability for use during pregnancy largely account for the widespread use of the drug (Lockman, S., et al. N. Engl. J. Med. 2007, 356, 135). Despite its benefits, NVP is associated with immune-mediated hepatotoxicity and skin rash, which can be fatal and are major causes of drug discontinuation. In addition, although direct correlation between NVP-based therapies and human cancer has yet to be demonstrated, long-term administration of the drug to rodent models resulted in increased incidences of hepatocellular adenomas and carcinomas (PDR staff, VIRAMUNE ® (nevirapine), In Physicians' Desk Reference, 63rd ed., Physicians' Desk Reference, Inc., Montvale, NJ, USA, 2009;873); moreover, epidemiological data indicated an association between the chronic use of NNRTIs and the occurrence of non-AIDS-defining cancers in HIV-positive patients (Powles, T., et al. J. Clin. Oncol. 2009, 27, 884).
While the exact mechanisms underlying NVP-induced toxic events are still not fully understood, a considerable amount of work on the development of mass spectrometry-based tools to assess covalent NVP-protein and NVP-DNA adducts as biomarkers of metabolic activation has been The best compounds in each series display in vitro inhibitory activity unmatched by any known inhibitor of transthyretin-related amyloid. Some of the compounds are almost insensitive to TTR mutations and display very high TTR stabilization activity ex vivo (in plasma of V30M-TTR carriers). Additionally, several compounds in each series display very favorable solubility and toxicity profiles.
Nevirapine (NVP, 1) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type 1. NVP's clinical efficacy, along with low cost, favourable lipid profile, and suitability for use during pregnancy largely account for the widespread use of the drug (Lockman, S., et al. N. Engl. J. Med. 2007, 356, 135). Despite its benefits, NVP is associated with immune-mediated hepatotoxicity and skin rash, which can be fatal and are major causes of drug discontinuation. In addition, although direct correlation between NVP-based therapies and human cancer has yet to be demonstrated, long-term administration of the drug to rodent models resulted in increased incidences of hepatocellular adenomas and carcinomas (PDR staff, VIRAMUNE ® (nevirapine), In Physicians' Desk Reference, 63rd ed., Physicians' Desk Reference, Inc., Montvale, NJ, USA, 2009;873); moreover, epidemiological data indicated an association between the chronic use of NNRTIs and the occurrence of non-AIDS-defining cancers in HIV-positive patients (Powles, T., et al. J. Clin. Oncol. 2009, 27, 884). Pharmaceuticals 2016, 9, 15 discovery and optimization was carried out in silico followed by experimental in vitro, ex vivo and in vivo validation. Three novel lead compound series (AT09, AT40 and AT50) have been identified, characterized and expanded.
The best compounds in each series display in vitro inhibitory activity unmatched by any known inhibitor of transthyretin-related amyloid. Some of the compounds are almost insensitive to TTR mutations and display very high TTR stabilization activity ex vivo (in plasma of V30M-TTR carriers). Additionally, several compounds in each series display very favorable solubility and toxicity profiles.
Nevirapine (NVP, 1) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in combined antiretroviral therapy and to prevent mother-to-child transmission of the human immunodeficiency virus type 1. NVP's clinical efficacy, along with low cost, favourable lipid profile, and suitability for use during pregnancy largely account for the widespread use of the drug (Lockman, S., et al. N. Engl. J. Med. 2007, 356, 135). Despite its benefits, NVP is associated with immune-mediated hepatotoxicity and skin rash, which can be fatal and are major causes of drug discontinuation. In addition, although direct correlation between NVP-based therapies and human cancer has yet to be demonstrated, long-term administration of the drug to rodent models resulted in increased incidences of hepatocellular adenomas and carcinomas (PDR staff, VIRAMUNE ® (nevirapine), In Physicians' Desk Reference, 63rd ed., Physicians' Desk Reference, Inc., Montvale, NJ, USA, 2009;873); moreover, epidemiological data indicated an association between the chronic use of NNRTIs and the occurrence of non-AIDS-defining cancers in HIV-positive patients (Powles, T., et al. J. Clin. Oncol. 2009, 27, 884).
While the exact mechanisms underlying NVP-induced toxic events are still not fully understood, a considerable amount of work on the development of mass spectrometry-based tools to assess covalent NVP-protein and NVP-DNA adducts as biomarkers of metabolic activation has been While the exact mechanisms underlying NVP-induced toxic events are still not fully understood, a considerable amount of work on the development of mass spectrometry-based tools to assess covalent Pharmaceuticals 2016, 9, 15 7 of 56 NVP-protein and NVP-DNA adducts as biomarkers of metabolic activation has been conducted in recent years. The rationale for using these biomarkers is based on evidence that NVP is bioactivated to reactive metabolites able to modify biomacromolecules by forming covalent adducts that may trigger carcinogenic and immune-mediated processes (Marinho, A.T., et al. J. Antimicrob. Chemother. 2014, 69, 476).
The current presentation addresses our efforts to assess the contribution of benzylic (at C12) versus aromatic (at C2/C3) hydroxylation of NVP to the generation of electrophilic metabolites prone to bind biomacromolecules in vitro and in vivo. The ensuing implications to NVP's toxicity are discussed.
Acknowledgments: Thanks are due to the Portuguese NMR and MS Networks (IST-UL nodes) for providing access to the facilities. This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through grants UID/QUI/00100/2013, RECI/QEQ-QIN/0189/2012 and RECI/QEQ-MED/0330/2012. Some of us have previously identified TXA1 as a hit thioxanthone with antitumor potential (Palmeira, A., et al. Biochem. Pharmacol. 2012, 83, 57-68). The present study aimed to investigate its mechanism of action in vitro and in human non-small cell lung cancer (NSCLC) cells xenografted in nude mice. TXA1 presented antitumor activity associated with the induction of autophagy and apoptosis, in melanoma and NSCLC cell lines. Interestingly, this molecule (soluble salt) affected lipid biosynthesis and resulted in an abnormal cellular cholesterol localization in NSCLC cells. The soluble salt of TXA1 was not toxic to nude mice, significantly reducing the growth of human NSCLC cells xenografts. Overall this study provides new insights into the mechanism of action of a novel small molecule, which may be relevant for the development of anticancer strategies.

Oral Presentations
Tumor suppressor p53 is a transcription factor widely regarded as the "guardian of the genome" that plays an important role in the regulation of several biological processes. So, it is not surprising that the p53-signaling pathway is inactivated in all types of cancers and that restoring p53 function in cancer cells represents a valuable anticancer approach (Hoe, K.K., et al. Nat. Rev. Drug Discov. 2014, 13, 217). In tumors that retain wild-type p53 but have defects in p53 regulatory pathways, the main goal is to inhibit the function of its negative regulators, such as MDM2. Generally, p53-MDM2 interaction inhibitors contain three lipophilic groups attached to a rigid heterocyclic scaffold to mimic the three most important p53 amino acids (Phe19, Trp23 and Leu26) that interact with MDM2. Furthermore, all the interactions are primarily hydrophobic, with potency increasing essentially by introduction of halide-substituted aromatic groups. Based on this information, we developed several novel chemical scaffolds with potential anticancer activity (Ribeiro, C.J. A., et al. Bioorg. Med. Chem. 2014, 22, 577-584;Soares, J., et al. Eur. J. Pharm. Sci. 2015, 66, 138-147;Soares, J., et al. Pharmacol. Res. 2015, 95-96, 42-52;Monteiro, A., et al. Eur. J. Med. Chem. 2014, 79, 266-272). Herein, we report the synthesis of a library of spirooxadiazoline oxindoles and the biological evaluation as p53-MDM2 interaction inhibitors. The most active compound showed a GI50 value of 1.7 µM in HCT 116 p53 (+/+) cell line, representing a 15.4-fold increase in potency when compared to the most active spiroisoxazoline oxindole obtained previously (Ribeiro, C.J. A., et al. Bioorg. Med. Chem. 2014, 22, 577-584). Together, our results indicate that spirooxadiazoline oxindoles reduce the p53 inhibition by MDM2, subsequently increasing the expression levels of p53 target genes, representing a promising scaffold for the development of novel anticancer agents (Ribeiro, C.J. A., et al. MedChemComm. 2016, doi:10.1039/C5MD00450K).

Surpassing Multidrug Resistance in Cancer: A Study on Jolkinol D Derivatives (MC4)
Mariana A. Reis 1 , Omar B. Ahmed 2 , Gabriella Spengler 3 , Hermann Lage 2 and Maria-José U. Ferreira 1 Synthesis of Ionic Liquids from Active Principle Ingredients (API-ILs) has been the main focus of our group for the last years. The combination of APIs as anions or cations with appropriate organic counter ions can be an innovative solution to the polymorphism behavior of several drugs as well as to improve their water solubility, permeability and corresponding bioavailability and biological activity (Ferraz, R., et al. ChemMedChem 2011, 6, 975-985;Marrucho, I.M., et al. Annu. Rev. Chem. Biomol. Eng. 2014, 5, 527-546). Within this context, novel ionic liquids with anti-cancer properties and decreased toxicity have recently been investigated.
In this communication we present the anti-proliferative effect against diverse tumor cell lines of novel Ionic Liquids based on anionic Ampicillin (1) and Bisphosphonates (2) combined with appropriate biocompatible organic cations, e.g., choline, cetylpyridinium and alkylimidazolium. This approach has conferred antitumor activity against five different human cancer cell lines with IC50 values in the low micromolar/nanomolar ranges to Ampicillin while showing enhanced antibacterial properties against sensitive and Gram-negative resistant bacteria (Ferraz, R., et al. ChemMedChem 2015, 10, 1480-1483Ferraz, R., et al. RSC Adv. 2014, 4, 4301-4307;Florindo, C., et al. Int. J. Pharm. 2013, 456, 553-559;Ferraz, R., et al. Med. Chem. Commun. 2012, 3, 494-497). On the other hand, by taking advantage of the high affinity of bisphosphonates towards bone tissue (Ben-Aharon, I., et al. PLoS ONE 2013, 8, e70044), the new API-ILs based on these compounds have also been tested against related cancer cell lines. The discussion will be complemented with the study of different physicochemical properties. Glioma is a type of primary brain tumour that arises from glial cells. The most common of malignant gliomas, glioblastoma multiforme, has a median survival of approximately 14 months after diagnosis (Sathornsumetee, S., et al. Ann. N.Y. Acad. Sci. 2000, 1142.

Hybrid
Temozolomide is a triazene alkylating agent used for the treatment of gliomas. However, its therapeutic effectiveness is often disappointing, largely in consequence of the lack of selectivity for tumor cells, insufficient drug concentration in the tumor and notorious resistance (Agarwala, S.S., et al. Oncologist 2000, 5, 144-151).
Valproic acid is an anticonvulsant used in the treatment of epilepsy. Recently, it was shown to inhibit a subset of histone deacetylases (HDAC), that consequently leads to the inhibition of DNA repair, thereby potentiating cytotoxic treatments such as chemotherapy or radiation therapy (Kazantsev, A.G., et al. Nat. Rev. Drug Discov. 2008, 7, 854-68).
Our team has been involved in the design of novel hybrid compounds with two units, a triazene as an alkylating agent and a carboxylic acid with known HDAC inhibitory activity. Preliminary results towards glioma cell line (GL261), demonstrated that the first hybrid compound tested has improved efficacy compared to Temozolomide. In this research work we report the evaluation of hybrid compounds. The stability of the compounds in phosphate buffer pH 7.4 and human plasma (80% v/v) were evaluated by HPLC. Our first results demonstrated a high stability in physiological conditions. The lipophilicity of the hybrid compounds is described. The screening for their inhibitory activity against HDAC showed improved activity comparing to the parental HDAC inhibitor, however, the exact mechanism of action is yet to be fully determined. β-secretase (BACE-1) inhibitors are potential useful drugs for the management of Alzheimer's disease (AD), but their incapacity to cross the blood-brain barrier and reach the Central Nervous System (CNS) is still a major reason for failure (Butini, S., et al. Curr. Top. Med. Chem. 2013, 13, 1787-1807. In this work we have tested the hypothesis of whether the conjugation of a peptidomimetic inhibitor, OM00-3, with a β-amyloid peptide sequence, Aβ18-23, facilitates its delivery into the brain.
Inhibitors were synthesized by Solid Phase Peptide Synthesis. Their potency against BACE-1/2, cytotoxicity in Caco-2 cells, metabolization in serum and mice brain were determined. A pharmacokinetic assay was performed in mice.
HVR-3 was found to be as potent as OM00-3 but 4-fold more selective toward BACE-1 in relation to BACE-2 and also 3-fold more stable against in vitro metabolization in Human serum. Intravenous administration to mice generated an active metabolite recovered from the rodent's brain. The success of this conjugation strategy to target the CNS corroborates the potential of HVR-3 as new anti-AD drug (Vila-Real, H., et al. J. Med. Chem. 2015, 58, 5408-5418).
In recent years, marine macroinvertebrates gained great importance by their fatty acid composition. Sea hares of Aplysia genus are known to be consumed, in oriental countries (Titcomb, M., et al. Pac. Sci. 1978, 32, 325-386); however, their nutritional composition and potential health effects are nearly unknown. In the present study we intended to characterize the fatty acids composition and evaluate the anti-inflammatory potential of lipophilic extracts of two sea hares, Aplysia fasciata Poiret and Aplysia punctata Cuvier.
Twenty-five fatty acids were identified, nine of them not yet reported in these species. Both extracts revealed similar anti-inflammatory properties in the culture medium of LPS-stimulated macrophages, as proved by the decreased ‚ NO levels. A similar decrease was also observed in L-citrulline levels, indicating a possible modulation of inducible nitric oxide synthase (iNOS) by the action of the compounds found in the extracts (A). Regarding lipoxygenase (LOX) inhibition, A. punctata extract was more effective (B), probably because it contains more polyunsaturated fatty acids (PUFA) that can compete with linoleic acid for the active site. Pharmaceuticals 2016, 9, 15 Effect in • NO and L-citrulline levels of RAW 264.7 cells pre-treated with Aplysia fasciata extract (A) and inhibition of soybean lipoxygenase by extracts of Aplysia spp. (B).
Overall, the results indicate that, in addition to their direct ingestion, A. fasciata and A. punctata may be good sources of nutraceuticals providing beneficial health effects, by reducing the levels of inflammatory mediators involved in the genesis of several diseases.
Overall, the results indicate that, in addition to their direct ingestion, A. fasciata and A. punctata may be good sources of nutraceuticals providing beneficial health effects, by reducing the levels of inflammatory mediators involved in the genesis of several diseases. Aromatic Schiff bases represent versatile pharmacophores with potential antimicrobial properties. As part of a program aimed at identifying new drug candidates with multi-target antibacterial activity, we have been exploring several classes of bis-hydrazone compounds and their iron complexes.
The antimicrobial activities of the synthesized compounds against the Gram-positive S. aureus Newman and the Gram-negative P. aeruginosa 477 were assessed using the Disk Diffusion Test. The Minimum Inhibition Concentrations (MICs) were also determined. Although the results are still preliminary, moderate activities were observed for compounds 1 and 7. The results obtained will be discussed on the basis of structure-activity relationships.
Effect in • NO and L-citrulline levels of RAW 264.7 cells pre-treated with Aplysia fasciata extract (A) and inhibition of soybean lipoxygenase by extracts of Aplysia spp. (B).
Overall, the results indicate that, in addition to their direct ingestion, A. fasciata and A. punctata may be good sources of nutraceuticals providing beneficial health effects, by reducing the levels of inflammatory mediators involved in the genesis of several diseases.

Design, Synthesis and Biological Evaluation of Novel Anti-Bacterial Agents (MC15)
Filipa Aromatic Schiff bases represent versatile pharmacophores with potential antimicrobial properties. As part of a program aimed at identifying new drug candidates with multi-target antibacterial activity, we have been exploring several classes of bis-hydrazone compounds and their iron complexes.
The antimicrobial activities of the synthesized compounds against the Gram-positive S. aureus Newman and the Gram-negative P. aeruginosa 477 were assessed using the Disk Diffusion Test. The Minimum Inhibition Concentrations (MICs) were also determined. Although the results are still preliminary, moderate activities were observed for compounds 1 and 7. The results obtained will be discussed on the basis of structure-activity relationships.
The use of herbal drugs for improving health and for treating several disorders, including depression, is becoming increasingly popular in western societies. The enzyme monoamine oxidase A (MAO-A) is one of the targets of the antidepressant drugs available in the market. Since almost 30% of the patients do not respond to the current treatments it is urgent to find new antidepressants ( The use of herbal drugs for improving health and for treating several disorders, including depression, is becoming increasingly popular in western societies. The enzyme monoamine oxidase A (MAO-A) is one of the targets of the antidepressant drugs available in the market. Since almost Pharmaceuticals 2016, 9, 15 13 of 56 30% of the patients do not respond to the current treatments it is urgent to find new antidepressants (Caraci, F., et al. Eur. J. Pharmacol. 2010, 626, 64-71). Herbal teas prepared from several plant species with claimed antidepressant properties, as well as from other species non-related with depression treatment, have been screened for the first time against MAO-A by our group, with promising results. The following IC 50 values were obtained: 699.8 µg/mL for Jasminum officinalis L. (Ferreres, F., et al. J. Pharm. Biomed. Anal. 2014, 88, 157-161), 19.3 µg/mL for Annona muricata L., 40.5 µg/mL for Hyssopus officinalis L., 428.1 µg/mL for Cereus grandiflorum L. (Grosso, C., et al. Microchem. J. 2015, 119, 176-182), 38.5 µg/mL for Cochlospermum angolensis Welw. (Ferreres, F., et al. Phytochem. Anal. 2013, 24, 534-540), 17.4 µg/mL for Jacaranda caroba (Vell.) A. DC. (Ferreres, F., et al. Food Chem. Toxicol. 2013, 57, 91-98) and 99.5 µg/mL for Grindelia robusta Nutt. (Ferreres, F., et al. J. Pharm. Biomed. Anal. 2014, 94, 163-172).
The herbal teas were also analysed by HPLC-DAD-ESI-MS n and HPLC-DAD, revealing the presence of different hydroxybenzoic acids, hydroxycinnamic acids and flavonoids.
Since A. muricata displayed a strong anti-MAO-A activity and is traditionally used against depression, its herbal tea was selected to be further incorporated in liposomes functionalized with ApoE. From the chemical point of view, the extract is composed by 5-Ocaffeoylquinic acid, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside.
Further studies will include the assessment of the effect of these herbal teas on serotonin and/or noradrenaline reuptake transporters, which are also extremely important targets for antidepressant drug design.
Acknowledgments: This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e a Tecnologia, FCT) through the project UID/QUI/50006/2013. C. Grosso thanks FCT for the FCT Investigator (IF/01332/2014). The p53 tumor suppressor is a transcription factor inactivated in all human cancers due to mutations in the p53 protein or to the overexpression of endogenous negative regulators of wild-type (wt) p53. The high prevalence, and the often observed increased drug resistance of mutant p53-expressing tumors, make mutant p53 a highly appealing target for novel anticancer therapies (Bykov, V.J.N., et al. FEBS Lett. 2014, 588, 2622-2627. In the present work, yeast assays consisting of Saccharomyces cerevisiae cells expressing human wt p53 or the most prevalent human mutant p53 forms were used to screen for reactivators of these inactive forms of p53. Using this approach, a chemical library of enantiopure tryptophanol-derived oxazoloisoindolinones was tested, and SLMP53-1 was selected as a potential activator of wt p53 and reactivator of mutant p53R280K (Soares, J., et al. 2014, WO2014/207688A1).
(wt) p53. The high prevalence, and the often observed increased drug resistance of mutant p53expressing tumors, make mutant p53 a highly appealing target for novel anticancer therapies (Bykov, V.J.N., et al. FEBS Lett. 2014, 588, 2622-2627. In the present work, yeast assays consisting of Saccharomyces cerevisiae cells expressing human wt p53 or the most prevalent human mutant p53 forms were used to screen for reactivators of these inactive forms of p53. Using this approach, a chemical library of enantiopure tryptophanol-derived oxazoloisoindolinones was tested, and SLMP53-1 was selected as a potential activator of wt p53 and reactivator of mutant p53R280K (Soares, J., et al. 2014, WO2014/207688A1).
Collectively, besides its potential as anticancer drug, SLMP53-1 belongs to a new chemical family, and its scaffold is a starting point for the development of effective drugs targeting mutant p53 forms. The molecular mechanism of SLMP53-1 was further validated in human colon carcinoma tumor cells with wt p53 (HCT116 p53 +/+ ) and in its p53-null isogenic derivative (HCT116 p53´{´), as well as in breast adenocarcinoma MDA-MB-231 cells expressing the mutant p53R280K. In these cells, SLMP53-1 exhibited a p53-dependent growth inhibitory effect associated with G1-phase cell cycle arrest (in HCT116 p53 +/+ cells) and apoptosis (in HCT116 p53 +/+ and MDA-MB-231 cells), and increased the expression levels of several p53 target genes in HCT116 p53 +/+ and MDA-MB-231 cells, but not in HCT116 p53´{´cells. In MDA-MB-231 cells, SLMP53-1 reestablished the wt-like DNA binding ability to mutant p53R280K. Additionally, SLMP53-1 potently triggered a mitochondrial apoptotic pathway in HCT116 p53 +/+ and MDA-MB-231 cells, involving Bax and wt/mutant p53 translocation to mitochondria. Besides this, SLMP53-1 sensitized HCT116 p53 +/+ and MDA-MB-231 cells to the effects of conventional chemotherapeutic agents and inhibited cell migration. Contrary to the majority of known p53 activators, no genotoxicity and in vivo toxicity were observed with SLMP53-1. Finally, the p53-dependent antitumor activity of SLMP53-1 were validated in vivo using xenograft mouse models (Soares, J., et al. Oncotarget 2015, 7, 4326-4343).

Antimalarial Activity of s-Triazine Based Hybrids in Both
Collectively, besides its potential as anticancer drug, SLMP53-1 belongs to a new chemical family, and its scaffold is a starting point for the development of effective drugs targeting mutant p53 forms. Malaria is a deadly disease that, despite being preventable and curable, is threatening the world wide health. Due to the fast resistance acquired by the Plasmodium parasite to the new developed drugs, an efficient molecule in both liver and blood stages was not yet established. (Dechy-Cabaret, O., et al. J. Med. Chem. 2012, 55, 10328-10344). The combination of active structures that act by different mechanisms in one single molecule (Meunier, B., Acc. Chem. Res. 2008, 41, 69-77) is a commonly used strategy to circumvent this inefficiency drawback. s-Triazine is a versatile core widely applied in the synthesis of hybrids with antimalarial activity, namely 4-aminoquinoline-s-triazine (Kumar, A., et al. Eur. J. Med. Chem. 2011, 46, 676-690).
Primaquine presents the highest activity in liver stage and is also the only registered drug for radical cure of blood and liver stages malaria caused by P. vivax and P.ovale infection (Vale, N., et al. Eur. J. Med. Chem. 2009, 44, 937-953).
Herein is presented the study of s-triazine hybrids with the liver stage active primaquine, aiming to find a molecule active in both liver and blood stage of malaria disease. In vitro tests in blood stage against P. Falciparum W2 strain have shown encouraging results, with IC 50 ranging from 0.2 to 8.3 microM. One primaquine-s-triazine hybrid showed very promising results in in vitro human hepatoma Huh-7 cells infected with a P. berghei line at a 1 microM dose. mechanisms in one single molecule (Meunier, B., Acc. Chem. Res. 2008, 41, 69-77) is a commonly used strategy to circumvent this inefficiency drawback. s-Triazine is a versatile core widely applied in the synthesis of hybrids with antimalarial activity, namely 4-aminoquinoline-s-triazine (Kumar, A., et al. Eur. J. Med. Chem. 2011, 46, 676-690).
Primaquine presents the highest activity in liver stage and is also the only registered drug for radical cure of blood and liver stages malaria caused by P. vivax and P.ovale infection (Vale, N., et al. Eur. J. Med. Chem. 2009, 44, 937-953).
Herein is presented the study of s-triazine hybrids with the liver stage active primaquine, aiming to find a molecule active in both liver and blood stage of malaria disease. In vitro tests in blood stage against P. Falciparum W2 strain have shown encouraging results, with IC50 ranging from 0.2 to 8.3 microM. One primaquine-s-triazine hybrid showed very promising results in in vitro human hepatoma Huh-7 cells infected with a P. berghei line at a 1 microM dose. The increasing number of chemotherapy failures reported worldwide identifies multidrug resistance (MDR) to anticancer drugs as a serious health concern. Since the over-expression of ABC transporters in cancer cell lines is one of the most reported MDR mechanisms, the inhibition of MDRrelated efflux pumps as P-glycoprotein (Pgp) remains a promising approach for overcoming MDR (Eckford, P.D. W., et al. Chem. Rev. 2009, 109, 2989.
Macrocyclic diterpenes isolated from Euphorbia species have been characterized as potent Pgp efflux modulators. However, their potency may be further improved by molecular manipulation of the diterpenic core. Thus, in silico approaches can be valuable tools for the identification of the most suitable modifications for MDR modulation (Ferreira, M.J. U., et al. Phytochem. Rev. 2014, 13, 915 The increasing number of chemotherapy failures reported worldwide identifies multidrug resistance (MDR) to anticancer drugs as a serious health concern. Since the over-expression of ABC transporters in cancer cell lines is one of the most reported MDR mechanisms, the inhibition of MDR-related efflux pumps as P-glycoprotein (Pgp) remains a promising approach for overcoming MDR (Eckford, P.D. W., et al. Chem. Rev. 2009, 109, 2989. Macrocyclic diterpenes isolated from Euphorbia species have been characterized as potent Pgp efflux modulators. However, their potency may be further improved by molecular manipulation of the diterpenic core. Thus, in silico approaches can be valuable tools for the identification of the most suitable modifications for MDR modulation (Ferreira, M.J.U., et al. Phytochem. Rev. 2014, 13, 915). Several computational approaches were applied to a small diterpenic library (n = 25) obtained by chemical derivatization of compounds isolated from Euphorbia boetica (Matos, A.M., et al. J. Nat. Prod. 2015, 78, 2215. A virtual screening procedure involving pharmacophoric identification followed by molecular docking was used to select and predict experimentally active MDR-reversal molecules (Ferreira, R.J., et al. J. Chem. Theory Comput. 2012, 8, 1853Ferreira, R.J., et al. J. Chem. Inf. Model. 2013, 53, 1747, while ligand-based drug discovery techniques as quantitative structure-activity relationship (QSAR, Weka software) and pharmacophore modeling (calculated from molecular interaction fields with Open3DQSAR) were used to characterize the relationship between chemical modifications and the respective modulation capabilities (Baptista R., et al., Future Med. Chem. 2015, submitted). From these procedures, a thorough characterization of the groups involved in the MDR-reversal activity was obtained, which can be further used to guide chemical derivatization, hopefully avoiding the synthesis of low-activity compounds. Rilpivirine (RPV) is a 2nd-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) that was added to the available therapeutic options with the aim to overcome the most common adverse effects of the 1st-generation NNRTIs as well as their viral cross-resistance. However, post-marketing reports of RPV-associated depressive disorders are a cause for concern when contemplating chronic administration regimens. Therefore the development of reliable prognostic tools for pharmacovigilance procedures is urgent.
With the ultimate goals of disclosing the mechanisms underlying RPV-induced neurotoxicity and developing suitable biomarkers of toxicity, the reactivity of RPV towards amino acids (N-acetyl-lysine and N-acetyl-cysteine) and model proteins such as Human Serum Albumin (HSA) was investigated by liquid chromatography-mass spectrometry (LC-MS) methodologies. The results obtained suggest the key role of RPV-derived covalent adduct formation in the onset of the adverse effects induced by this 2nd generation NNRTI.
Xanthones are an important group of oxygenated heterocyclic compounds, which are known to exhibit interesting pharmacological properties, such as anti-tumoral activity. Numerous studies have revealed that DNA is a primary intracellular target of anticancer drugs, due to the interaction of small molecules with DNA. Hence, characterization of the interaction of xanthones with DNA can be an important contribution for the development of a new class of anti-cancer agents.
In this communication we report the study of the interaction of xanthone with double stranded DNA, using UV-vis spectroscopy, including UV melting experiments, and viscosity measurements. The denaturation temperature and the thermodynamic parameters of DNA thermal denaturation were obtained from the curves of melted base pairs as a function of temperature. The binding constant of the xanthone-DNA complex, at 293 K, was calculated from the UV spectra.
The results indicate a strong binding affinity of xanthone with DNA, affecting the stability of the double helix, and suggest the binding of xanthone to DNA mainly by intercalation. The biological properties of olive oil polyphenols in vivo depend on the extent to which they are absorbed and metabolized. In a recent work, the metabolites hydroxytyrosol (3,4dihydroxyphenylethanol) sulfate and hydroxytyrosol acetate sulfate were found to be the most useful metabolites for monitoring the intake compliance of extra virgin olive oil (Rubió, L., et al. Food Res. Int. 2014, 65, 59-68). Pharmaceuticals 2016, 9, 15 The growing interest in the bioactivity of natural polyphenols requires their metabolites to be used in bioassays and as standards in research protocols. Therefore, we report here the synthesis of several polyphenol sulfates namely hydroxytyrosol, hydroxytyrosol acetate, homovanillyl alcohol, homovanillyl alcohol acetate, homovanillilic acid, ferulic acid, and 3,4-dihydroxyphenylethanoic acid sulfates. A relatively fast and cheap synthetic solution based on avoidance of high temperature conditions during the synthesis and of low pressure conditions during purification has been established. Compounds were efficiently synthesized in 1-2 steps in a good yield (>75%). Flavonoids have been associated with various health benefits, in which their anti-inflammatory effects play an important role. These properties associated with their ubiquitous distribution in nature, and their presence in the great majority of foods, as part of our daily diet, confer flavonoids great value-added molecules. Taking into account the potential anti-inflammatory properties, flavonoids started to be considered a valuable alternative to modulate and prevent inflammatory processes; and moreover, to be the base for the synthesis of more potent and efficient antiinflammatory drugs (Ribeiro, D., et al. Med. Res. Rev. 2015, 35, 877). The work herein developed intended to extend and rationalize the current knowledge on the alleged anti-inflammatory

Flavonoids Effects in Proinflammatory Signaling
The growing interest in the bioactivity of natural polyphenols requires their metabolites to be used in bioassays and as standards in research protocols. Therefore, we report here the synthesis of several polyphenol sulfates namely hydroxytyrosol, hydroxytyrosol acetate, homovanillyl alcohol, homovanillyl alcohol acetate, homovanillilic acid, ferulic acid, and 3,4-dihydroxyphenylethanoic acid sulfates. A relatively fast and cheap synthetic solution based on avoidance of high temperature conditions during the synthesis and of low pressure conditions during purification has been established. Compounds were efficiently synthesized in 1-2 steps in a good yield (>75%). Flavonoids have been associated with various health benefits, in which their anti-inflammatory effects play an important role. These properties associated with their ubiquitous distribution in nature, and their presence in the great majority of foods, as part of our daily diet, confer flavonoids great value-added molecules. Taking into account the potential anti-inflammatory properties, flavonoids started to be considered a valuable alternative to modulate and prevent inflammatory processes; and moreover, to be the base for the synthesis of more potent and efficient anti-inflammatory drugs (Ribeiro, D., et al. Med. Res. Rev. 2015, 35, 877). The work herein developed intended to extend and rationalize the current knowledge on the alleged anti-inflammatory properties of flavonoids by elucidating the mechanism of action related with their structure (structure-activity relationship). For this purpose, a group of 24 flavonoids were selected, belonging to three flavonoid classes, flavones, flavanones and flavonols. Flavonoids have been associated with various health benefits, in which their anti-inflammatory effects play an important role. These properties associated with their ubiquitous distribution in nature, and their presence in the great majority of foods, as part of our daily diet, confer flavonoids great value-added molecules. Taking into account the potential anti-inflammatory properties, flavonoids started to be considered a valuable alternative to modulate and prevent inflammatory processes; and moreover, to be the base for the synthesis of more potent and efficient antiinflammatory drugs (Ribeiro, D., et al. Med. Res. Rev. 2015, 35, 877). The work herein developed intended to extend and rationalize the current knowledge on the alleged anti-inflammatory properties of flavonoids by elucidating the mechanism of action related with their structure (structure-activity relationship). For this purpose, a group of 24 flavonoids were selected, belonging to three flavonoid classes, flavones, flavanones and flavonols.

Flavonoids Effects in Proinflammatory Signaling
These flavonoids' ability to modulate various proinflammatory signaling systems was assessed using various approaches: modulation of human neutrophils' oxidative burst; inhibition of leukotriene B4, via 5-lipoxygenase, and prostaglandin E2, via cyclooxygenases -1 and -2, production; These flavonoids' ability to modulate various proinflammatory signaling systems was assessed using various approaches: modulation of human neutrophils' oxidative burst; inhibition of leukotriene B 4 , via 5-lipoxygenase, and prostaglandin E2 , via cyclooxygenases -1 and -2, production; and apoptosis induction. The experimental studies performed in the scope of this work allowed the conclusion that among the tested flavonoids, the ones with a catechol group in B-ring revealed overall best anti-inflammatory activities. Indeed, flavonoids 3 1 ,4 1 -dihydroxyflavone, 5,3 1 ,4 1 -trihydroxyflavone, 7,3 1 ,4 1 -trihydroxyflavone, and luteolin are undoubtedly good modulators of all the proinflammatory mediators evaluated, constituting promising alternatives for the resolution of inflammatory processes.

Acknowledgments:
We would like to thank FCT (Fundação para a Ciência e a Tecnologia) for the financial support to UCIBIO-REQUIMTE (project: UID/Multi/04378/2013), trough National and European Union Funds, and to thank University of Aveiro and FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement. Daniela Ribeiro and Marisa Freitas acknowledge FCT for the financial support to their Ph.D. grant (SFRH/BD/72966/2010) and Pos-doc grant (SFRH/BPD/76909/2011), respectively, in the ambit of "QREN-POPH-Tipologia 4.1-Formação Avançada", co-sponsored by FSE and by national funds of MCTES. The oxygen supply to the cells need meet the metabolic demand, and have a critical effect in energy requirement in the cardiorespiratory system, this offer can be seriously affected in the case a person suffering a injury because of a surgery trauma or car accident causing blood loss, in the cases it is normal doctors recommend the transfusion of concentrates of red blood cells to help O 2 supply maintenance in the human organism. The red blood cells (CH) have a complex system (Razouk, F.H., et al. Rev. Bras. Hematol. Hemoter. 2004, 26, 126-134) having three different stages of Pharmaceuticals 2016, 9, 15 19 of 56 C = 3 substances (plasma, proteins and oxygen) being multiphase P + 3 (solid, liquid and gas). For -CH be stored is necessary that it be carried out at temperatures 2˝C to 6˝C, depending on how this occurs can lead to formation of ice crystals, causing damage to the red blood cells. Depending on the severity of the damage caused during the cryopreservation process can lead to cell death. To reduce effect caused because formation of ice crystals in the blood component, in the storage process is added cryoprotectant solutions such as mannitol (C 6 H 14 O 6 ), in the bags blood derived bags (Drozdov, A. D., Mech. Res. Commun. 1996, 23, 543-548). Besides the natural toxicity caused by the use of cryoprotectants agents in the cryopreservation process, also observed the formation of reactive oxygen species (ROS), and in the case of mannitol (C 6 H 14 O 6 ) subject to the following reaction:

Analysis of Characteristics
The use of mannitol (C 6 H 14 O 6 ), leads to the consumption of oxygen released by molecules of hemoglobin contained in the concentrated bags of red cell-CH, leading to decreased efficacy of oxygen transport, since only 50% of Hemogloina-Hb to P50 is covers carry molecules of O 2 , also taking into account that the human body at room temperature is covers to extract only 30% of O 2 Hb, and this forms an efficiency of only 15% extraction of the total of all a bag CH hemacia-concentrate 3,4 . Thus the addition of mannitol (C 6 H 14 O 6 ) as cryoprotectants ultimately further reduce the effectiveness of blood transfusion and its blood products.  [268][269][270][271][272][273][274][275][276][277][278][279][280][281][282], although C2-derivatives of azoles has shown to be associated with some relevant biological properties of azoles. Therefore, the reactivity of 2-methyl-imidazole derived salts was studied, in the presence of imines and aromatic aldehydes.

Recent Developments in the Synthesis of Novel Xanthone-1,2,3-triazole Dyads (OC2)
Hélio Albuquerque 1, *, Clementina Santos 1,2 , José Cavaleiro 1 and Artur Silva 1 The development of multi-target drugs for treating complex multifactorial diseases constitutes an active research field. This kind of drugs has gained much importance as alternative strategy to combination therapy ("cocktail drugs") (Nepali, K., et al. Eur. J. Med. Chem. 2014, 77, 422-487). A common way to design them brings together two different pharmacophores in one single molecule (so-called dyads). Following this idea and being aware that xanthones (Pinto, M. M., et al. Curr. Med. Chem.. 2005, 12, 2517-2538 and 1,2,3-triazoles (Lauria, A., et al. Eur. J. Org. Chem. 2014, 3289-3306) possess important pharmacological properties, we combined these two heterocycles in one molecule to create new dyads with improved therapeutic potential. In this work, new xanthone-1,2,3-triazole dyads were prepared from novel (E)-2-(4-arylbut-1-en-3-yn-1-yl)chromones by two different approaches to evaluate their efficiency and sustainability. Both methodologies involved Diels-Alder reactions to build the xanthone core, which were optimized using microwave irradiation as alternative heating method, and 1,3-dipolar cycloadditions to insert the 1,2,3-triazole moiety ( Chalcones and dihydropyrimidine-2(1H)-thiones are biologically active compounds that have shown rather diverse pharmacological properties, such as antiviral, antiparasitic and anticancer activities (Kappe, C.O., Eur. J. Med. Chem. 2000, 35, 1043-1052. Beyond the classic Biginelli reaction, within the last decade, there are very few examples in the literature describing the synthesis of 3,4dihydropyrimidin-2(1H)-ones or thiones carrying no substituents at the 5 or 6 positions of the heterocycle. Organic chemists have been looking for more sustainable methodologies and ways to improve de greenness of organic synthesis. Exclude solvent from the reaction medium, generally the main source of waste in a synthetic process, is probably the most efficient way to attain this objective. Mechanical activation (MA), which is normally carried out in the absence of, or with minimal use of solvents, could be and alternative to improve the sustainabiliaty of organic synthesis (Friscic, T., Chem. Soc. Rev. 2012, 41, 3493-3510;James, S.L., et al Chem. Soc. Rev. 2012, 41, 413-447). Herein we present the synthesis of chalcones and 4,6-diaryldihydropyrimidine-2(1H)-thiones in short reaction times and high yields using automatized mechanical action. The improvement of the sustainability of this method, compared with the use of conventional methodologies, was assessed by E-factor values under 10 and EcoScale values above 70. heterocycle. Organic chemists have been looking for more sustainable methodologies and ways to improve de greenness of organic synthesis. Exclude solvent from the reaction medium, generally the main source of waste in a synthetic process, is probably the most efficient way to attain this objective. Mechanical activation (MA), which is normally carried out in the absence of, or with minimal use of solvents, could be and alternative to improve the sustainabiliaty of organic synthesis (Friscic, T., Chem. Soc. Rev. 2012, 41, 3493-3510;James, S.L., et al Chem. Soc. Rev. 2012, 41, 413-447). Herein we present the synthesis of chalcones and 4,6-diaryldihydropyrimidine-2(1H)-thiones in short reaction times and high yields using automatized mechanical action. The improvement of the sustainability of this method, compared with the use of conventional methodologies, was assessed by E-factor values under 10 and EcoScale values above 70. In this communication, we describe the synthesis of novel tetrazol-5-yl-allenes 2 and their reactivity towards aziridines 3 leading to the synthesis of functionalized methylenepyrrolidines 4 and pyrroles 5. The Wittig reaction between the ylide, formed from the phosphonium chloride 1, and ketenes gave the target allenes in high yields.  , et al. Eur. J. Org. Chem. 2014, 24, 5159-5165).
In this communication, we describe the synthesis of novel tetrazol-5-yl-allenes 2 and their reactivity towards aziridines 3 leading to the synthesis of functionalized methylenepyrrolidines 4 and pyrroles 5. The Wittig reaction between the ylide, formed from the phosphonium chloride 1, and ketenes gave the target allenes in high yields. The biological importance of glycostructures made them popular targets in synthetic chemistry, in particular those incorporating N-acetyl-D-glucosamine (NAG) units. The urgent need of these compounds in pure form and in significant amount has implied vast synthetic efforts. Usually oligosaccharides are constructed through sugar monomers manipulation, which implies timeconsuming protection/deprotection steps and wild glycosylations. Several approaches have been developed to attain complex glycostructures (Boltje, T.J., et al. Nat. Chem. 2009, 611-622). However, it has been demonstrated that glycosylation using NAG derivatives as glycosyl donors is still a difficult task (Zhu, X., et al. Angew. Chem. Int. Ed. 2009, 48, 1900-1934. Our group has been involved in the synthesis of glycans containing NAG, specifically on the assembly of small fragments of bacterial peptidoglycan ( The biological importance of glycostructures made them popular targets in synthetic chemistry, in particular those incorporating N-acetyl-D-glucosamine (NAG) units. The urgent need of these compounds in pure form and in significant amount has implied vast synthetic efforts. Usually oligosaccharides are constructed through sugar monomers manipulation, which implies time-consuming protection/deprotection steps and wild glycosylations. Several approaches have been developed to attain complex glycostructures (Boltje, T.J., et al. Nat. Chem. 2009, 611-622). However, it has been demonstrated that glycosylation using NAG derivatives as glycosyl donors is still a difficult task (Zhu, X., et al. Angew. Chem. Int. Ed. 2009, 48, 1900-1934. Our group has been involved in the synthesis of glycans containing NAG, specifically on the assembly of small fragments of bacterial peptidoglycan (Enugala, R., et al. Synlett 2010, 18, 2711-2716Enugala, R., et al. Chem. Asian J. 2012, 7, 2482-2501Enugala, R., et al. Carbohydr. Res. 2014, 384, 112-118). Due to our interest in glycostructures involved in important biological mechanisms, it was envisaged the direct modification of chitobiose to attain the desired compounds. Clozapine, a molecule incorporating the N-methyl piperazine nucleus, is a gold standard among antipsychotic medications for schizophrenia. Its therapeutic use is restricted by agranulocytosis, a fatal blood disorder associated with the use of the drug (Lieberman, J. A., et al. J. Clin. Psychiatry 1988, 49, 271-277). It is crucial to develop new molecules inspired in the clozapine scaffold, with potential antipsychotic activity and reduced side effects, capable of interacting with different receptors associated to this type of disorders, namely the 5-HT serotonine receptors (Ye, N., et al. Chem. Rev. 2013, 113, 123-178).

Strategies towards the Synthesis of New (E)-2-Aryl-3-styryl-4H-chromen-4-ones and (E)-2-Aryl-1methyl-3-styrylquinolin-4(1H)-ones (OC9)
The furan and cyclopropane rings can be found in a variety of biologically active synthetic and natural compounds (Luo, Y., et al. Bioorg. Med. Chem. Lett. 2015, 25, 2421-2424Sampson, P.B., et al. J. Med. Chem. 2015, 58, 130-146). Due to their importance as potential pharmaceutical agents, the synthesis of novel furan-and cyclopropane-containing compounds is a highly active research field. For this purpose, 3-bromochromones arise as desirable and versatile starting materials (Sousa, J.L. C., et al. Synlett 2015C., et al. Synlett , 26, 2724C., et al. Synlett -2729. Exploring the singular chemical features of such chromone derivatives, herein we present the synthesis of two different families of oxygen-containing heterocycles-furan-based polyphenolics and fused chromanone-cyclopropanes-starting from the same 3-bromochromones and using one-pot base-catalyzed reactions. Specifically, our methodologies relay on tandem reactions of 3-bromochromones 1 with 1,3-dicarbonyl compounds 2 to afford a series of furan-based polyphenolic derivatives 3, and ketone compounds 4 to prepare a library of fused chromanone-cyclopropane derivatives 5 in the presence of organic and inorganic bases, respectively. et al. Synlett 2015Synlett , 26, 2724Synlett -2729. Exploring the singular chemical features of such chromone derivatives, herein we present the synthesis of two different families of oxygen-containing heterocycles-furan-based polyphenolics and fused chromanone-cyclopropanes-starting from the same 3-bromochromones and using one-pot base-catalyzed reactions. Specifically, our methodologies relay on tandem reactions of 3-bromochromones 1 with 1,3-dicarbonyl compounds 2 to afford a series of furan-based polyphenolic derivatives 3, and ketone compounds 4 to prepare a library of fused chromanone-cyclopropane derivatives 5 in the presence of organic and inorganic bases, respectively.

Synthesis of New Bis(indolyl)methanes With Anti-Cancer Activity (OC16)
Carla Grosso  We have recently disclosed an approach to novel bis(indolyl)methane oximes (BIM Oximes) via Hetero-Diels-Alder reactions of nitrosoalkenes with indoles. This class of compounds showed very interesting anti-cancer activity, in particular against leukaemia and lymphoma cell lines (Pinho e Melo, T., et al. Eur. J. Med. 2015, 93, 9). In this communication, the tuning of the structure of scaffold 1 which led to the preparation of a range of new BIM oximes will be described.
Moreover, the one-pot synthetic strategy to BIMs was extended to novel bis(indolyl)methane hydrazones via bis-hetero-Diels-Alder reaction of azoalkenes with indoles. The biological evaluation of these new BIMs as anti-cancer agents will also be disclosed. We have recently disclosed an approach to novel bis(indolyl)methane oximes (BIM Oximes) via Hetero-Diels-Alder reactions of nitrosoalkenes with indoles. This class of compounds showed very interesting anti-cancer activity, in particular against leukaemia and lymphoma cell lines (Pinho e Melo, T., et al. Eur. J. Med. 2015, 93, 9). In this communication, the tuning of the structure of scaffold 1 which led to the preparation of a range of new BIM oximes will be described.
Moreover, the one-pot synthetic strategy to BIMs was extended to novel bis(indolyl)methane hydrazones via bis-hetero-Diels-Alder reaction of azoalkenes with indoles. The biological evaluation of these new BIMs as anti-cancer agents will also be disclosed. "Krokodil" is a homemade substitute to heroin, which psychoactive substance is believed to be desomorphine 1, an opioid ten times more potent than morphine. The users of this drug can develop severe skin ulcerations, and scale-like skin that resembles a crocodile, hence its street name. "Krokodil" is prepared at home with easily available materials and therefore its chemical composition is complex and still poorly understood (Alves, E. A., et al. Forensic Sci. Int. 2015, 249, 207-213). Since its consume is spreading in Europe, it is important to elucidate and characterize "Krokodil" composition.
With the aim of studying the chemical composition of "Krokodil", its street synthesis was mimicked resorting to the same materials and conditions used by street manufacturers. The chemical profile of "Krokodil" was outlined by chromatographic (HPLC and GC-MS) and spectroscopic techniques (IR, 1 H-NMR, MS). With these data it was possible to establish the typical chromatographic and spectroscopic profiles of "Krokodil".
The analysis of phytoprostanes in natural matrices is extremely challenging, requiring highly sensitive and specific tools for their profiling and characterization. Moreover, the great diversity granted by the presence of racemic mixtures of phytoprostanes increases the complexity of those analyses (Barbosa, M., et al. J. Agric. Food Chem. 2015, 63, 6466-6474).
Our work aimed at determining naturally occurring classes of free phytoprostanes in 24 macroalgae species belonging to Chlorophyta, Phaeophyta and Rhodophyta, collected along the western coast of Portugal and from integrated multi-trophic aquaculture (IMTA) systems. For this, a fast, selective, and robust ultrahigh-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) method was employed. Three classes of phytoprostanes were identified and quantitated for the first time in the analyzed species.
Our work aimed at determining naturally occurring classes of free phytoprostanes in 24 macroalgae species belonging to Chlorophyta, Phaeophyta and Rhodophyta, collected along the western coast of Portugal and from integrated multi-trophic aquaculture (IMTA) systems. For this, a fast, selective, and robust ultrahigh-performance liquid chromatography coupled to triplequadrupole mass spectrometry (UHPLC-QqQ-MS/MS) method was employed. Three classes of phytoprostanes were identified and quantitated for the first time in the analyzed species.
Currently, the interest in phytoprostanes comprises two general areas: as biomarkers of oxidative stress in plant-derived foodstuffs and as bioactive mediators with potential benefits in human health. Therefore, the determination of phytoprostane levels in macroalgae is of extreme importance, encouraging the exploitation and characterization of new natural dietary sources of these compounds.  that daily consumption of caffeine causes improvements in memory capacity, however, ha indication of adverse consequences (Diukova, A, et al. NeuroImage 2012, 62, 239-249), such as increased anxiety, among various other cognitive effects in humans and animals (Smit, H.J., et al. Psychopharmacology 2000, 152, 167-173). After the analysis in two databases-Science Library Online (Scielo) and PubMed-using the criteria search "free full text" and "last five years", we obtained the following data from the terms: Although caffeine is consumed daily by a significant portion of the world's population, yet there are few clinical studies that demonstrate the actual effects of it in humans and animals, which indicates a great need for experimental research in humans and animals based on consumption caffeine.  Ethnopharmacol. 2006, 103, 1-24), which may be supported by the presence of bioactive abietane diterpenes (Burmistrova, O., et al. Phytomedicine 2013, 22, 1009-1016. The intrinsic instability of these and their unfavorable partition coefficient, affects their bioavailability and therefore the bioactivity. New drug delivery systems as the Phytosome ® , have proven to be effective in surpassing those limitations (Semalty, A., et al. Fitoterapia 2010, 81, 306-314).
The most potent antibacterial extract (PM 4) was encapsulated into phytosomes microencapsulated with chitosan/TPP. The average size of the microspheres was 1082 ± 363 nm showing a low polydispersitivity index (0.224), zeta potential of 20.59 ± 12.02 mV and encapsulation efficiency of 57.70% ± 6.00%. Preliminary studies showed short-term stability, sustained release and antibacterial activity of the formulation. The results confirm the presence of bioactive compounds in Plectranthus extracts and usefulness of the encapsulation technology applied to improve the stability The most potent antibacterial extract (PM 4) was encapsulated into phytosomes microencapsulated with chitosan/TPP. The average size of the microspheres was 1082˘363 nm showing a low polydispersitivity index (0.224), zeta potential of 20.59˘12.02 mV and encapsulation efficiency of 57.70%˘6.00%. Preliminary studies showed short-term stability, sustained release and antibacterial activity of the formulation. The results confirm the presence of bioactive compounds in Plectranthus extracts and usefulness of the encapsulation technology applied to improve the stability of bioactive extracts of PM. The endoplasmic reticulum (ER) is an organelle comprising a network of branching tubules and sacs that is present in all eukaryotic cells. ER stress has been identified as a hallmark, and sometimes trigger, of several pathologies, notably cancer, inflammation and neurodegenerative diseases such as Alzheimer's and Parkinson's.

ER Stress and
One of the factors that can trigger ER stress is the presence of unfolded or misfolded proteins, which can be a consequence of the inhibition of the proteasome, a eukaryotic protein complex that is involved in proteolysis of undesired proteins.
Among the molecules described in literature known to affect ER and proteasome function, the majority are natural products, suggesting that natural molecules may constitute a significant arsenal of chemical entities for modulating this cellular target.
In this presentation, we will briefly outline current knowledge of ER biology and the hallmarks of ER stress, thus paving the way for presenting the natural products that have been described as being ER modulators, either stress inducers or ER protectors.
The chemistry, distribution and mechanism of action of these compounds will be presented and discussed, including examples both from the literature and from our laboratory.

Poster Presentations
4.1. CotA-Laccase as Biocatalyst in the "Green" Synthesis of Phenazine and Acridine Cores (P1) Phenazines and acridines are an important class of benzoheterocycle compounds which exhibit a broad spectrum of biological activities and a number of derivatives are widely used as antibacterial, antifungal, antiviral and anticancer drugs (Laursen, J.B., et al. Chem. Rev. 2004, 104, 1663-1686Lian, Y., et al. J. Am. Chem. Soc. 2013, 135, 12548-12551). There are several chemical methods for the synthesis of these important cores starting from substituted o-aromatic amines that are also good substrates for laccases. Based on this property and seeking for a cleaner synthetic method, we present a "green" approach for the formation of symmetric and asymmetric phenazines and acridines using CotA-laccase, a multicopper oxidase, as biocatalyst. Laccase promote the oxidation of the precursor molecules to the O-quinone-diimine intermediates which undergo a Michael addition leading to the final heterocycle cores (Sousa, A.C., et al. Green Chem. 2014, 16, 4127-4136). Reactions were performed in aqueous media and with mild reaction conditions of pH and temperature. a broad spectrum of biological activities and a number of derivatives are widely used as antibacterial, antifungal, antiviral and anticancer drugs (Laursen, J.B., et al. Chem. Rev. 2004, 104, 1663-1686Lian, Y., et al. J. Am. Chem. Soc. 2013, 135, 12548-12551). There are several chemical methods for the synthesis of these important cores starting from substituted o-aromatic amines that are also good substrates for laccases. Based on this property and seeking for a cleaner synthetic method, we present a "green" approach for the formation of symmetric and asymmetric phenazines and acridines using CotA-laccase, a multicopper oxidase, as biocatalyst. Laccase promote the oxidation of the precursor molecules to the O-quinone-diimine intermediates which undergo a Michael addition leading to the final heterocycle cores (Sousa, A.C., et al. Green Chem. 2014, 16, 4127-4136). Reactions were performed in aqueous media and with mild reaction conditions of pH and temperature.

Impact of Citrus Pectin on Oenin Copigmentation Mechanisms (P2)
Ana Fernandes 1, *, Natércia Brás 2 , Nuno Mateus 1 and Victor de Freitas 1 1  Copigmentation is regarded as one mechanism for anthocyanin's color stabilization (Boulton, R. Am. J. Enol. Vitic. 2001, 52, 67-87). Also, interaction with polysaccharides is an important topic to be considered on anthocyanin's stabilization, as anthocyanins are synthetized in a polysaccharide environment where extensive contact may occur (Padayachee, A., et al. Food Chem. 2012, 134, 155-161). These associations may affect pigment's stability and color. This work aimed to evaluate the impact of pectin on anthocyanin's copigmentation mechanisms. Oenin copigmentation was observed in the presence of (+)-catechin, as indicated by an increase in the absorbance. However, the copigmentation binding constants (K CP ) determined for the interaction of catechin with oenin in the presence of a pectic polysaccharide showed that this polysaccharide induced a decrease on the copigmentation binding constants. These results probably suggest the occurrence of competition equilibrium in which the presence of this pectic polysaccharide limited the association between catechin and oenin. Pharmaceuticals 2016, 9, 15 considered on anthocyanin's stabilization, as anthocyanins are synthetized in a polysaccharide environment where extensive contact may occur (Padayachee, A., et al. Food Chem. 2012, 134, 155-161). These associations may affect pigment's stability and color. This work aimed to evaluate the impact of pectin on anthocyanin's copigmentation mechanisms. Oenin copigmentation was observed in the presence of (+)-catechin, as indicated by an increase in the absorbance. However, the copigmentation binding constants (KCP) determined for the interaction of catechin with oenin in the presence of a pectic polysaccharide showed that this polysaccharide induced a decrease on the copigmentation binding constants. These results probably suggest the occurrence of competition equilibrium in which the presence of this pectic polysaccharide limited the association between catechin and oenin. Here we present the biological activity of bark and leaves extracts from M. faya and some compounds isolated from these extracts.
The bark acetone extract presented a strong anti-acetylcholinesterase activity, comparable to the commercial drug galanthamine. Moreover, the inhibition was also reversible, which means that it will be less toxic than an irreversible inhibitor. Both acetone extracts presented comparable inhibition values against xanthine oxidase, with only approximately 4 to 5-fold the IC 50 for standard drug allopurinol. Considering that these extracts were reversible inhibitors, it means that they have the potential to be less toxic than allopurinol, which is approved for medical purposes.
From the most active extracts were isolated fatty alcohol (1)(2), pentacyclic triterpenes with lupane and oleanane skeleton (3)(4)(5)(6), cyclic diarylheptanoid (7) and a phthalate (8). All these compounds are reported in M. faya for the first time and belong to organic families well known by the broad spectrum of biological activities exhibited by its members. These results show the potential of M. faya as medicinal plant and source of pharmacologically active compounds. Pharmaceuticals 2016, 9, 15 flavonoids can be highlighted. In this work, the ability of Grindelia robusta Nutt aqueous extract and of some of its flavonoids, to reduce nitric oxide (NO) levels in RAW 264.7 cells was assessed. Results revealed that the extract of G. robusta reduced cells' viability. The recurrent use of anti-inflammatory drugs and their side effects led to a growing demand for viable and safer alternatives. In this context, natural products arise, playing an important role in the treatment of this pathology. Amongst natural compounds with anti-inflammatory properties, flavonoids can be highlighted. In this work, the ability of Grindelia robusta Nutt aqueous extract and of some of its flavonoids, to reduce nitric oxide (NO) levels in RAW 264.7 cells was assessed. Results revealed that the extract of G. robusta reduced cells' viability.
Cell viability of RAW 264.7 cells pre-treated for 2 h with G. robusta aqueous extract, followed by 22h co-treatment with LPS (1 µg/mL) with LPS or vehicle (culture medium). Results represent the mean ± standard deviation of four independent experiments performed in triplicate.
A tendency to reduce NO levels, in a dose-dependent way, was also observed. All flavonoids were able to decrease NO levels in a concentration-dependent manner, quercetin being the most effective one (IC50 values of 7.47 µM). The presence of quercetin, apigenin and luteolin derivatives in the extract of G. robusta can partially explain its capacity to decrease NO levels. In a general way, aglycones revealed to be more active than the respective glycosides. Furthermore, the catechol group on ring B and the hydroxyl group in C3 seem to be essential for the anti-inflammatory activity of these compounds.
Cell viability of RAW 264.7 cells pre-treated for 2 h with G. robusta aqueous extract, followed by 22h co-treatment with LPS (1 µg/mL) with LPS or vehicle (culture medium). Results represent the mean˘standard deviation of four independent experiments performed in triplicate.
A tendency to reduce NO levels, in a dose-dependent way, was also observed. All flavonoids were able to decrease NO levels in a concentration-dependent manner, quercetin being the most effective one (IC 50 values of 7.47 µM). The presence of quercetin, apigenin and luteolin derivatives in the extract of G. robusta can partially explain its capacity to decrease NO levels. In a general way, aglycones revealed to be more active than the respective glycosides. Furthermore, the catechol group on ring B and the hydroxyl group in C3 seem to be essential for the anti-inflammatory activity of these compounds. None of the extracts was active against A549 cell line or against any cell line in log phase up to 200 µg/mL. In lag phase, the dichloromethane extract from Z. verticillatum presented the best activity against MCF-7 cells and was the only extract active against HeLa cells (with selectivity Index of 1.94 and 4.64, respectively).The results obtained show the potential of M. squamiger, B. neritina and Z. verticillatum as sources of antitumor agents.

Acknowledgments:
FRC/Azores ASMAS-M2. Antibiotic residues have been detected in various environmental matrices, such as surface and drinking waters. Although present at low levels (µg/L, ng/L), many antibiotics are bioaccumulative, pseudo-persistent and can promote resistance/alterations in bacterial populations (Doorslaer, V.X., et al. Sci. Total Environ. 2014, 500, 250-269). In this work we present the biosorption of three widely used fluoroquinolones (FQ)-ofloxacin (OFL), norfloxacin (NOR) and ciprofloxacin (CPF)-to activated sludge (AS) and aerobic granular sludge (AGS). A HPLC-FD method was validated and optimized to follow the biosorption of the targeted FQ (Maia, A.S., et al. J. Chrom. A 2014, 1333, 87-98). The validated method demonstrated good selectivity, linearity (r 2 > 0.999), intra-day and inter-day precisions (RSD < 3%) and accuracy. LOD and LOQ were, respectively, 0.7 ng/mL and 1 ng/mL for the three FQ. Several parameters that can affect biossorption kinetics, namely, contact time, pH, and biosortion mass were also studied. At pH 7 AS showed better performance to biosorb OFL, NOR and CPF. The equilibrium data for AS showed a better fit to the Langmuir model, while AGS showed a better fit to the Freundlich model. The FQ could be desorbed from AGS at pH 3, 8 and 9, whereas at pH 4 the biosorption process was promoted. Flavonoids are chemically based on a fifteen-carbon skeleton consisting of two benzene rings (ring A and ring B) joined by a linear three carbon chain (C 6 -C 3 -C 6 ) forming an oxygenated heterocycle pyran ring (ring C). 2-styrylchromones (2-SC), a small group of compounds characterized by the attachment of a styryl group to the 2-position of the chromone skeleton, have structural similarities with flavonoids, particularly those belonging to the class of flavones. Flavonoids possess many biological activities, from which the antioxidant properties are the best described. Considering the structural similarities of 2-SC and the fact that their styryl moiety may greatly contribute to their molecular stabilization under redox challenges, some of its biological activities are likely to be similar or even enhanced in comparison to flavonoids, although it needs to be experimentally confirmed. Thus, the purpose of the present study was to evaluate and compare the putative scavenging of reactive oxygen (ROS) and nitrogen (RNS) species by synthetic 3-hydroxyflavones and 3-hydroxy-2-styrylchromones, using in vitro non-cellular systems. The obtained results show that both groups of compounds have high capacity to scavenge ROS and RNS. Interestingly, the efficacy of 3-hydroxy-2-styrylchromones and 3-hydroxyflavones vary among the tested reactive species, constituting a good option as antioxidant agents.
with flavonoids, particularly those belonging to the class of flavones. Flavonoids possess many biological activities, from which the antioxidant properties are the best described. Considering the structural similarities of 2-SC and the fact that their styryl moiety may greatly contribute to their molecular stabilization under redox challenges, some of its biological activities are likely to be similar or even enhanced in comparison to flavonoids, although it needs to be experimentally confirmed. Thus, the purpose of the present study was to evaluate and compare the putative scavenging of reactive oxygen (ROS) and nitrogen (RNS) species by synthetic 3-hydroxyflavones and 3-hydroxy-2styrylchromones, using in vitro non-cellular systems. The obtained results show that both groups of compounds have high capacity to scavenge ROS and RNS. Interestingly, the efficacy of 3-hydroxy-2styrylchromones and 3-hydroxyflavones vary among the tested reactive species, constituting a good option as antioxidant agents. 4 For the last several years, searching of new xanthone derivatives (XD) with potential pharmacological properties has remained in the area of interest of our group. In addition, the assessment of the drug-likeness of the bioactive XD synthesized is crucial in an early stage of the drug discovery pipeline considering that problems related to physicochemical properties are one of the main reasons of failure during pre-clinical trials.
Lipophilicity, commonly expressed by the logarithm of the partition coefficient (log P), is one of the most important physicochemical properties having a great impact both in pharmacokinetics (absorption, distribution, metabolism, excretion, and toxicity-ADMET) and pharmacodynamic processes.
In this work, we describe the lipophilicity of a series of XD, previously synthesized in our group (Fernandes, C., et al. Bioorgan. Med. Chem. 2014, 22, 1049-1062, using two different methods: RP-HPLC (Ayouni, L., et al. Chromatographia 2005, 62, 251-255) and vortex-assisted liquid-liquid microextraction coupled with liquid chromatography (VALLME-HPLC) (Roman, I.P. et al. J Chromatogr. A 2014, 1330. Both methods were validated accordingly with OCDE guidelines. In the RP-HPLC method retention factors (log k) were determined and correlated to log P. The analyses were carried out under optimized isocratic conditions, using two different hydrophobic silica-based stationary phases (C8 and C18) and different water/methanol ratios as mobile phases. Linear correlations were found between log k values and the volume fraction of methanol in the mobile phase (R 2 higher than 0.99) for both stationary phases. The log k values were extrapolated for pure aqueous eluent to correlate with the values obtained using VALLME method. Low sample consumption, low sensitivity to impurities, good accuracy and excellent reproducibility were observed.
The VALLME-HPLC method demonstrated to be a successful technique for log P evaluation. Linear correlations were obtained, with R 2 >0.99 in all cases and, in most cases, low variation coefficients (%) were observed for all the XD tested.
HPLC technique was crucial for the determination of log P values, showing its importance in the evaluation of physicochemical parameters that can be useful for pharmacokinetics prevision.
Acknowledgments: This research was partially supported by the Strategic Funding UID/Multi/ 04423/2013 through national funds provided by FCT-Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. In order to find new agents to combat bacteria resistance it is important to understand the details of the mechanism of action of glycopeptides antibiotics as vancomycin (Vorpagel, E., et al. J. Am. Chem. Soc. 2008, 130, 13013-13022). Those antibiotics prevent the formation of peptidoglycan (PGN), the major component of the bacterial cell wall which is constituted by a glycan chain of alternating β(1 Ñ 4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide bridges (Höltje, J.V., et al. Microbiol. Mol. Biol. Rev. 1998, 62, 181-203).

Investigation of the Interaction of Vancomycin with Synthetic Bacterial Muropeptides (P11)
It is well established that the replacement of the last amino acid in the peptide chain linked to the MurNAc moiety changes the interaction with the glycopeptides antibiotics. However the interactions studies have been limited to the use of N-protected dipeptides and tripeptides (Kim, C., et al. Bmb Rep. 2008, 41, 93-101;Jimenez-Barbero, J., et al. Chem. Eur. 2014, 20, 7363-7372). In order to clarify how the different compositions of the bacterial fragments affect the recognition by vancomycin we have developed a study involving synthesis and screening of small cell wall bacterial fragments.
Herein we present our preliminary studies on the interaction between vancomycin and the synthesized compounds.  Metab. Agents 2003, 3, 349-360). Currently, there is no fully effective treatment for TTR amyloidosis and several therapeutic strategies targeting different steps of the fibril formation pathway are under development (Ueda, M., et al. Transl. Neurodegener. 2014, 3, 19-29). The disruption of amyloid aggregates and fibrils is one of those strategies. To properly select compounds for this purpose a specific screening protocol that can identify and characterize the interaction of the compounds with the fibrils must be developed. With this purpose in mind three wild type-TTR fibril formation protocols were studied by dynamic light scattering, circular dichroism, turbidimetry, transmission electron microscopy and fluorescence spectroscopy to select and characterize the most appropriate fibril formation protocol. The results show that the heat-induced fibril formation protocol at neutral pH has advantages over other fibril formation protocols but requires further characterization. Additionally, saturation transfer difference nuclear magnetic resonance was used to study the interaction of doxycycline with WT-TTR fibrils, and a DLS assay was developed to characterize the effect of this compound on fibril disruption.  , et al. Biochem. Pharmacol. 2013, 85, 1234-1245.

Identification and Characterization of Small Molecule Interactions with Transthyretin Amyloid
Based on the substitution pattern of xanthone LEM2, discovered as a potent antitumor agent, and on amine moiety of p53-MDM2 inhibitors, new potential disruptors with drug-like properties were obtained. By reductive amination, eleven LEM2-aminated derivatives were successfully synthesized. Their binding affinity was predicted by docking studies towards MDM2. Using yeastbased assays, their inhibitory activity on p53-MDM2 was investigated. These studies will provide the most favorable requirements for the construction of new antitumor agents. A wide spectrum of biological activities including antitumor, anti-inflammatory and antimicrobial are displayed by the abietane dehydroabietic acid (DHA) (González, M.A., et al. Eur. J. Med. Chem. 2010, 45, 811-816). Recently, DHA was shown to prevent Staphylococcus aureus bacterial colonization and also inhibiting biofilm formation (Fallarero, A., et al. Int. J. Mol. Sci. 2013, 14, 12054-72). Considering these promising results, the aim of this study was to investigate the efficacy of DHA for inhibiting the biofilm formation as well as its efficiency against standard and isolate strains. Minimum Inhibitory Concentration (MIC) and Minimum Biofilm Inhibitory Concentration (MBIC) were determined by two-fold serial broth microdilution assay, as well as crystal violet staining methods (Stepanović, S., et al. Apmis 2007, 115, 891-899). DHA showed MIC values between 15.63-500 µg/mL and a range of 32%-90% of biofilm inhibition, therefore demonstrating efficacy against Based on the substitution pattern of xanthone LEM2, discovered as a potent antitumor agent, and on amine moiety of p53-MDM2 inhibitors, new potential disruptors with drug-like properties were obtained. By reductive amination, eleven LEM2-aminated derivatives were successfully synthesized. Their binding affinity was predicted by docking studies towards MDM2. Using yeast-based assays, their inhibitory activity on p53-MDM2 was investigated. These studies will provide the most favorable requirements for the construction of new antitumor agents. A wide spectrum of biological activities including antitumor, anti-inflammatory and antimicrobial are displayed by the abietane dehydroabietic acid (DHA) (González, M.A., et al. Eur. J. Med. Chem. 2010, 45, 811-816). Recently, DHA was shown to prevent Staphylococcus aureus bacterial colonization and also inhibiting biofilm formation (Fallarero, A., et al. Int. J. Mol. Sci. 2013, 14, 12054-72). Considering these promising results, the aim of this study was to investigate the efficacy of DHA for inhibiting the biofilm formation as well as its efficiency against standard and isolate strains. Minimum Inhibitory Concentration (MIC) and Minimum Biofilm Inhibitory Concentration (MBIC) were determined by two-fold serial broth microdilution assay, as well as crystal violet staining methods (Stepanović, S., et al. Apmis 2007, 115, 891-899). DHA showed MIC values between 15.63-500 µg/mL and a range of 32%-90% of biofilm inhibition, therefore demonstrating efficacy against resistant bacteria and their biofilms.

Antimicrobial Activity and Biofilm Formation Inhibition of Alginate Microspheres of Dehydroabietic
Future studies will focus on a synergistic effect between an antimicrobial synthetic molecule and an encapsulation polymer, namely a new abietane cationic amphiphile (ACA) derived from dehydroabietic acid. The aim of this work was to perform a human crossover trial in which volunteers ingested a blackberry fruit juice as an acute dose, containing mainly cyanidin-3-glucoside (Cy3glc) with or without 12% ethanol. A through screening analysis for anthocyanins and/or metabolites was performed in plasma and urine samples by mass spectrometry. Cy3glc in its parent form was detected in plasma and urine samples. The identification and quantification of anthocyanin conjugates was also performed yielding several sulphate, glucuronyl and methyl conjugates of Cy3glc and Cy.

Identification and Quantification of Blackberry Anthocyanin Metabolites in Human
Anthocyanins can be further metabolized by colon microbiota and excreted in the urine, yielding several phenolic acid and their metabolized forms. Some protocatechuic, vanilic, benzoic and hippuric acid conjugates were identified in urine samples, mainly with sulfate groups.
So far, it has been quite difficult to clearly assess both native and metabolized forms and also catabolites in vivo and to distinguish their different biological roles.

Synthesis of 3-Oxo-β-Sultam Activity-based Probes for Biomarker Discovery in Chronic Obstructive
* Correspondence: luis.carvalho@ff.ulisboa.pt Chronic Obstructive Pulmonary Disease (COPD) refers to a condition of inflammation with progressive weakening of the structure of the lung and irreversible narrowing of the airways (Lucas, S.D. et al. Med. Res. Rev. 2013, 33 (Suppl. S1), e73-e101). Novel biomarkers for this disease are urgently needed. COPD induced inflammation modulates proteolytic enzymes in the lungs, like Human Neutrophil Elastase (HNE), which are clinically relevant for the diagnosis of COPD. Adequately targeted probes can be designed to quantify the active catalytic state of these enzymes (Carvalho, L.A.R., et al. MedChemComm 2015, 6, 536). Following the extensive work of our group with HNE inhibitor synthesis (Mulchande, J., et al. J. Med. Chem. 2008, 51, 1783 we developed a library of HNE inhibitors and activity-based probes (ABPs) based on the 3-oxo-β-sultam scaffold (Tsang, W.Y., et al. Org. Biomol. Chem. 2007, 5, 3993).
These ABPs will be used to covalently tag the active enzyme, providing protein activity quantification by proteomics-based analysis. We envisage the study and validation of HNE as a potential biomarker in COPD.

Chemical Acylation vs. Enzymatic Esterification of Malvidin-3-Glucoside-Rich Wine Extract with Oleic Acid (P24)
Luis Cruz *, Marta Guimarães, Victor de Freitas and Nuno Mateus REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal * Correspondence: luis.cruz@fc.up.pt These ABPs will be used to covalently tag the active enzyme, providing protein activity quantification by proteomics-based analysis. We envisage the study and validation of HNE as a potential biomarker in COPD. The structural modification of anthocyanins (water soluble pigments) into more lipophilic compounds is very important to expand their application in the food, medicinal and cosmetic industries (Cruz, L., et al. Food Chem. 2015, 174, 480-486;Ardhaoui, M., et al. Biocatal. Biotransform. 2004, 22, 253-259). In this work, the synthesis of anthocyanin oleic acid ester derivatives was achieved by chemical and enzymatic approaches. Pharmaceuticals 2016, 9, 15 The structural modification of anthocyanins (water soluble pigments) into more lipophilic compounds is very important to expand their application in the food, medicinal and cosmetic industries (Cruz, L., et al. Food Chem. 2015, 174, 480-486;Ardhaoui, M., et al. Biocatal. Biotransform. 2004, 22, 253-259). In this work, the synthesis of anthocyanin oleic acid ester derivatives was achieved by chemical and enzymatic approaches.

Acknowledgments
Enzymatic approach allowed to synthesize mv3glc-oleic acid ester which was structurally characterized by mass spectrometry and for the first time by NMR spectroscopy. Enzymatic reaction revealed to be more efficient and regioselective in the conversion of native anthocyanins into their ester product rather than the chemical reaction. Antioxidant features of the obtained products by means of DPPH and FRAP assays confirms that their antioxidant potential was not compromised, which is an important insight for future technological applications.

Synthesis of Innovative Anticoagulant Hybrids (P26)
Marta Correia-da-Silva 1,2, *, Catarina Carvalho 1 , Emília Sousa 1,2 and Madalena Pinto 1,2  Enzymatic approach allowed to synthesize mv3glc-oleic acid ester which was structurally characterized by mass spectrometry and for the first time by NMR spectroscopy. Enzymatic reaction revealed to be more efficient and regioselective in the conversion of native anthocyanins into their ester product rather than the chemical reaction. Antioxidant features of the obtained products by means of DPPH and FRAP assays confirms that their antioxidant potential was not compromised, which is an important insight for future technological applications.

Synthesis of Innovative Anticoagulant Hybrids (P26)
Marta Correia-da-Silva 1,2, *, Catarina Carvalho 1 , Emília Sousa 1,2  In this work, a hybridization strategy was planned in order to develop new anticoagulants joining a coumarin scaffold with a heparin-like sugar sulfated moiety. With this approach we expected to mimetize the sulfated polysaccharide anticoagulants, while adding some hydrophobic character to the resulting molecule. Due to the evidence that 6-substituted coumarins with heteroaromatic rings can act as FXa inhibitors (Amin, K.M., et al. Bioorg. Chem. 2014, 52, 31-43;Frédérick, R., et al. J. Med. Chem. 2005, 48, 7592-7603), the 6-hydroxycoumarin (1) was selected for molecular modification and triazole was used as a linker between the coumarin scaffold and the glucosidic moiety (compounds 5-7). For structure-activity relationships purposes, the syntheses of 7-triazole-linked coumarin glucosides (compounds 13, 14, and 15) as well as of sulfates of non-triazole linked 6-and 7-coumarin glucosides (compounds 10, 18, and 19) were performed. Pharmaceuticals 2016, 9, 15 In conclusion, thirteen new coumarin derivatives were obtained, i.e., two propynyl, three acetyl glucosides, three glucosides, and five sulfated derivatives, and the structure elucidation of the synthesized compounds was stablished by IR, NMR, and HRMS for the first time. This small library of compounds will allow the study of the effect of the presence of the triazole moiety on the anticoagulant activity and mode of action of these new anticoagulant hybrids.
In conclusion, thirteen new coumarin derivatives were obtained, i.e., two propynyl, three acetyl glucosides, three glucosides, and five sulfated derivatives, and the structure elucidation of the synthesized compounds was stablished by IR, NMR, and HRMS for the first time. This small library of compounds will allow the study of the effect of the presence of the triazole moiety on the anticoagulant activity and mode of action of these new anticoagulant hybrids. Astringency is an important factor in food quality. Different theories about astringency mechanism have been attributed but the most established process is the interaction between food tannins and human salivary proteins (Kallithraka, S., et al. J. Sens. Stud. 1998, 13, 29-43). Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested and the tannin/protein ratio (Soares, S., et al. Food Res. Int. 2012, 49, 807-813). Highlight the astringency mechanism through the study of tannin-protein interactions became relevant. The goal of this study aims to study the effect of different salivas (A, B and C) and different tannin concentration (0.5 and 1 mg/mL) in the interaction process. Human salivary proteins are divided into different groups mainly histatins (His), statherins (Stah), proline rich proteins (PRPs) and cystatins (Huq, N.L., et al. Int. J. Pep. Res. Ther. 2007, 13, 547-564). This study is is focused in the identification of new procyanidin B3-salivary protein complexes complexes created between a common food tannin, the procyanidin B3 (B3), and the Stath, His and PRPs originating from saliva with different protein profiles.  Biol. 2010, 1, 108). Nevertheless, little is known about the chemical composition of C. arvensis and even less regarding the different subspecies of C. suffruticosa. Therefore, the present study aims the elucidation of these plants' chemical composition and to compare and identify differences and/or similarities among them.
To accomplish this, one sample of each taxon, C arvensis, C. officinalis, and C suffruticosa subsp. lusitanica, and two samples, from two different populations, of C. suffruticosa subsp. algarbiensis were collected in the field, washed with running water, and dried in a woven at 40˝C until stabilization of weight was reached. The hexane extract of each taxon was obtained from dried and powdered plant and completely characterized by GC-MS after silylation, which allowed the identification and quantification of their constituents.
The achieved data showed the presence of mono-and disaccharides, terpenoids, fatty acids, sterols, alkanes, long chain alcohols and some amino acids. It was found that the monosaccharides and fatty acids are the most abundant families in C. officinalis being the palmitic acid and α-linoleic acid the most abundant compounds. The last one was also found in higher quantities in C. arvensis. Fatty acids like α-linoleic acid, palmitic acid and linoleic acid are also the most abundant in C. suffruticosa subsp. lusitanica. Lastly, the two samples of C. suffruticosa subsp. algarbiensis showed in major quantities a branched alkane and one compound from the ursano family. Some carbohydrates as well as lignoceric acid and linoleic acid were described for the first time in the Calendula L. genus.
Through the accomplished findings, including a preliminary Principal Component Analysis (PCA), a taxonomic differentiation among the taxa can be made. Irrelevant variations were also found in the two samples of the subsp. algarbiensis. The compounds detected for the first time improved our knowledge of the chemical profile of this genus. Additionally, some of the reported compounds have a major importance on a nutritional level. Cancer persists as one of the major global public health concerns due to its large incidence and mortality. Searching for novel anticancer agents with higher potency and lower toxicity, our work aims the development of monastrol analogues. For this, forty five dihydropyrimidin(thi)ones were synthesized via the Biginelli reaction by condensation of an aldehyde, a β-ketoester and urea/thiourea. The screening of the antiproliferative effects of the compounds was evaluated at 30 µM on MCF-7, T47D, LNCaP, HepaRG, Caco-2 and NHDF cell lines by the MTT assay. The concentration inducing 50% inhibition of cell growth (IC 50 ) was assessed for the most toxic compounds using different concentrations (0.01, 0.1, 1, 10, 50 and 100 µM). The results revealed that the compounds did not show significant toxicity neither in normal dermal cells (NHDF) nor in prostatic and breast (T47D) cancer cell lines; however, the chloro-containing compounds of the urea series showed selective toxicity for HepaRG cells (5.28 µM ď IC 50 ď 15.9 µM) whereas their thiourea analogs evidenced lower selectivity, being significantly toxic for hepatic, colon and breast (MCF-7) cancer cell lines (0.749 µM ď IC 50 ď 31.9 µM for HepaRG; 5.51 µM ď IC 50 ď 13.7 µM for Caco-2; and 2.95 µM ď IC 50 ď 10.9 µM for MCF-7). Thus, it was found that the molecules containing chloro atoms in their structure, particularly those belonging to urea series, demonstrated selective toxicity for hepatic cancer cells. Additional studies are ongoing to understand what mechanisms of action are involved in the toxicity of these molecules as well as the existence of differences between the two series. The enantioselective alkylation of aromatic aldehydes with diethylzinc in the presence of chiral ligands is a valuable method for synthesizing chiral secondary alcohols. Chiral 1,3-thiazolidine-4-carboxylates derived from L-cysteine have been sparingly used as catalysts in this reaction, giving secondary alcohols with good to excellent enantiomeric excesses (Meng, Q., et al. Tetrahedron Asymmetry 2000, 11, 4255-4261;Jin, M.J., et al. Bull. Korean Chem. Soc. 2002, 23, 509-510).
To the best of our knowledge, structurally identical D-penicillamine-derived thiazolidines 2 have not been used in these reactions. These two types of thiazolidines, easily obtained by a simple synthetic process could bring singular advantages to many catalytic processes. Because L-cysteine has an (S) chiral center, while D-penicillamine's chiral center is (R), the alkylation of several aromatic aldehydes which we carried out using 1 and 2 as catalysts, gave (S) and (R) alcohols, respectively, leading to a myriad of useful chiral products with opposite absolute configurations in excellent yields and ee up to >99%. Hydrolysis by β-lactamases (BL) is one of the major mechanisms that drive resistance to β-lactam antibiotics. The major strategy to overcome their activity is the use of inhibitors that have little to none antibiotic activity, but bind with greater affinity to BLs, allowing an effective antibiotic therapy.
The study of inhibitors for MBLs has highlighted some classes of compounds potentially useful for the design of a successful inhibitor in the future. However, none have yet entered further development stages (Klingler, F.M., et al. J. Med. Chem. 2015, 58, 3626-3630;Fast, W., et al. Biochim. Biophys. Acta 2013, 1834, 1648-1659. In this study, we have developed a structure-based pharmacophore model to screen large compound databases (NCI, ZINC and DrugBank) for candidate ligands to IMP-1 MBL, followed by docking of the successful results. Hydrolysis by β-lactamases (BL) is one of the major mechanisms that drive resistance to β-lactam antibiotics. The major strategy to overcome their activity is the use of inhibitors that have little to none antibiotic activity, but bind with greater affinity to BLs, allowing an effective antibiotic therapy.
The study of inhibitors for MBLs has highlighted some classes of compounds potentially useful for the design of a successful inhibitor in the future. However, none have yet entered further development stages (Klingler, F.M., et al. J. Med. Chem. 2015, 58, 3626-3630;Fast, W., et al. Biochim. Biophys. Acta 2013, 1834, 1648-1659. In this study, we have developed a structure-based pharmacophore model to screen large compound databases (NCI, ZINC and DrugBank) for candidate ligands to IMP-1 MBL, followed by docking of the successful results.
which raises some questions about the automation of the methodology due to the complexity of sample preparation. Methodologies based on liquid chromatography-mass spectrometry analysis of protein digests provide better alternatives, involving conditions that result in low thermal input and mild ionization. Moreover, these methodologies offer a wider applicability, enabling the analysis of covalent adducts formed with polar electrophiles. Nonetheless, levels of protein adducts are small compared to the unmodified proteins, and therefore they are difficult to detect and identify without prior enrichment. To increase analytical sensitivity for detecting less-abundant protein adducts, we report herein a methodology to enrich GA adducts of Human Serum Albumin (HSA) prior to nano-electrospray ionization mass spectrometry analysis. Following trypsin digestion of HSA modified in vitro with GA, the GA-modified peptides were enriched through solid-phase extraction and then analyzed by nano liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (Q-ToF) using a data-dependent auto-MS/MS method. This analytical method provides a straightforward approach for detecting GA-protein adducts and, therefore, stimulating the common use of HSA adducts as biomarkers of acrylamide exposure. The development of new and better molecular probes for the early diagnosis of amyloid diseases such as Alzheimer's, Parkinson's and type II diabetes are of critical essence (Buell, A.K., et al, Phys. Chem. Chem. Phys. 2011, 13, 20044-20052). We employed molecular dynamics (MD) simulations to characterize the interaction of the protein β-2 microglobulin (β2m) with the well-known amyloid probe thioflavin-T (ThT). The results obtained are in agreement with reports found in the literature, where residues Q8 and Y10 of β2m were shown to be crucial for the interaction with ThT (Wolfe, L.S., et al. Proc. Natl. Acad. Sci. USA 2010, 107, 16863-16868). To better characterize the topological constraints imposed by these residues, we have constructed in silico the mutants Q8A, Y10A and Y10F, and performed several MD simulations. The results show that the aromatic ring of Y10 is crucial for the interaction of ThT with β2m. Additionally, the energetic contribution of these residues to the stability of the β2m-ThT complex was quantified using alchemical free energy calculations (AFEC) which also confirmed the importance of this interaction.

Activation of Cytotoxic Responses of NK Cells (P41)
Pedro F. Pinheiro *, Gonçalo C. Justino and M. Matilde Marques Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal * Correspondence: pedro.pinheiro@tecnico.ulisboa.pt Natural killer (NK) cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cells provide rapid responses to viral-infected cells, and play a role in tumor immunosurveillance by directly inducing the death of tumor cells. Instead of acting via antigen-specific receptors, lysis of tumor cells by NK cells is mediated by alternative receptors, including NKG2D, NKp44, NKp46 and NKp30 (Terunuma, H., et al. Int. Rev. Immunol. 2008, 27, 93-110).
In recent developments, B7-H6, a surface protein present on a broad panel of tumor cells including lymphoma, melanoma, and carcinoma, was identified as a ligand for the NKp30 receptor. The structure of the NKp30-B7H6 complex has also been resolved (Li, Y., et al. J. Exp. Med. 2011, 208, 703-714). The comparison between the 3D structures of unbound and B7-H6-bound NKp30 demonstrated marked conformational changes that may be a key-factor for the NK-response activation role of B7-H6.
Our current work aims at designing a family of small organic molecules (SOMs) capable of mimicking the effect of B7-H6 on the NKp30 receptor. A combination of computational docking and molecular dynamics tools was extensively used to scan several ligand libraries, yielding core-structures as possible ligands for the receptor. These were further optimized to generate lead structures that will be screened as NKp30 ligands through mass spectrometry tools.
The main goal is to obtain an SOM capable of inducing the activation of an NK response, through binding of the NKp30 receptor, and structurally amenable to derivatization with specific tumor-targeting molecular units to produce a specific immune response against cancer cells.  Cellulose is considered one of the most promising polymeric resource as alternative for petroleum-based polymers (Pang, J., et al. Materials 2013, 6, 1270-1284. It has many advantages such as low cost, biocompatibility and biodegradability (Fink, D.K., et al. Angew. Chem. Int. 2005, 44, 3358-3393). Carboxymethyl cellulose (CMC) is the most important ionic cellulose ether widely used due to its thickening, suspending and film forming properties (Ramos, L. A., et al. Carbohydr. Polym. 2005, 60, 259-267). With the aim of producing cellulose filters able to adsorb positive proteins at low pH, we have produced surface carboxymethylated cellulose filters using commercial filters has starting material.

Production of Surface Carboxymethylated Cellulose Filters in
Commercial filters were carboxymethylated using sodium hydroxide (NaOH) and monochloroacetic acid (MCA) in aqueous medium under heterogeneous conditions. The produced CMCs were characterized by FTIR and protein adsorption capacity was evaluated by static method using lysozyme. The CMC surface modified filters had a DS between 0.139 and 0.179 and an adsorption capacity between 2.5 to 10.4 mg of lysozyme per gram of cellulose at pH 5. Hence, it is possible to produce water insoluble carboxymethylated cellulose filters for the adsorption of positive proteins at low pH in aqueous medium. [5,4d] Cellulose is considered one of the most promising polymeric resource as alternative for petroleum-based polymers (Pang, J., et al. Materials 2013, 6, 1270-1284. It has many advantages such as low cost, biocompatibility and biodegradability (Fink, D.K., et al. Angew. Chem. Int. 2005, 44, 3358-3393). Carboxymethyl cellulose (CMC) is the most important ionic cellulose ether widely used due to its thickening, suspending and film forming properties (Ramos, L. A., et al. Carbohydr. Polym. 2005, 60, 259-267). With the aim of producing cellulose filters able to adsorb positive proteins at low pH, we have produced surface carboxymethylated cellulose filters using commercial filters has starting material.

Synthesis and in Vitro Activity in HCT116 Human Colon Cancer Cells of New Phenol-Pyrimido
Commercial filters were carboxymethylated using sodium hydroxide (NaOH) and monochloroacetic acid (MCA) in aqueous medium under heterogeneous conditions. The produced CMCs were characterized by FTIR and protein adsorption capacity was evaluated by static method using lysozyme. The CMC surface modified filters had a DS between 0.139 and 0.179 and an adsorption capacity between 2.5 to 10.4 mg of lysozyme per gram of cellulose at pH 5. Hence, it is possible to produce water insoluble carboxymethylated cellulose filters for the adsorption of positive proteins at low pH in aqueous medium. [5,4-d] Colon cancer is the third cause of death worldwide and it is more frequent in industrialized countries. In the USA it is the second cause of death and in Europe approximately 25,000 cases are reported every year. The existent treatments are based in the combination of 5-Fluorouracil (5-FU) and LV or 5-FU alone. However, these treatments are not effective in all patients due to multidrug resistance developed by tumour cells and the lack of selectivity (Levin, B., et al. J. Gastroenterology 2008, 134, 1570-1595Labianca, R., et al. J. Crit. Rev. Oncol. Hematol. 2010, 74, 106-133).
Recently, we synthesized new derivatives of pyrimido [5,4-d]pyrimidines, 2, combining pyrimido [5,4-d]pyrimidine core with phenolic aldehydes. The in vitro activity of the new compounds was assessed in HCT116 human cancer cells. The new compounds showed high activity that depends on the R 2 group. The synthetic approach and the biological results will be presented. Bile acids have been widely applied in synthetic chemistry mainly due to their enantiomeric purity, high stability of the steroid nucleus, reactivity of the side chain groups, low cost and ready availability, as well as anticancer properties (Davis, A., Molecules 2007, 12, 2106-2122. This work aims to develop potential new antitumor agents by combining the bile acid core with different polyamines.
Of these, the compound 1 evidenced a relevant cytotoxic effect on hormone-dependent cells, and flow cytometry studies suggest that it can promote cytotoxicity with 13% of cells death at 50 µM. The compound 2 showed an antiproliferative effect only on LNCaP cells.
In conclusion, it was possible to synthetize novel aminated steroids some of which show promising biological activity and can constitute interesting hit compounds for the development of potential new anticancer agents.
Recently, we synthesized new derivatives of pyrimido [5,4-d]pyrimidines, 2, combining pyrimido [5,4-d]pyrimidine core with phenolic aldehydes. The in vitro activity of the new compounds was assessed in HCT116 human cancer cells. The new compounds showed high activity that depends on the R 2 group. The synthetic approach and the biological results will be presented. Bile acids have been widely applied in synthetic chemistry mainly due to their enantiomeric purity, high stability of the steroid nucleus, reactivity of the side chain groups, low cost and ready availability, as well as anticancer properties (Davis, A., Molecules 2007Molecules , 12, 2106Molecules -2122. This work aims to develop potential new antitumor agents by combining the bile acid core with different polyamines.
Of these, the compound 1 evidenced a relevant cytotoxic effect on hormone-dependent cells, and flow cytometry studies suggest that it can promote cytotoxicity with 13% of cells death at 50 µM. The compound 2 showed an antiproliferative effect only on LNCaP cells.
In conclusion, it was possible to synthetize novel aminated steroids some of which show promising biological activity and can constitute interesting hit compounds for the development of potential new anticancer agents.  Cashew nut shell liquid (CNSL) is obtained in the cashew nut processing industries as a byproduct of little commercial value but with high technological potential due to its phenolic constitution and its various biological properties such as antimicrobial, anti-inflammatory, antitumor, antioxidant, anti-acetylcholinesterase, etc (Kubo, I., et al. Food Chem. 2006, 99, 555-562;Kubo, I., et al. J. Agric. Food Chem. 1993, 41, 1012-1015Oliveira, M.S.C., et al. Acta Trop. 2010, 117, 165-170). The anacardic acid, the major constituent of CNSL, is a mixture of three derivatives of salicylic acid with side chains of fifteen carbon atoms presenting different degree of unsaturation. In this study, the extraction of natural CNSL from cashew shell was made with hexane by maceration for obtaining anacardic acid (Paramashivappa, P., et al. J. Agric. Food Chem. 2001, 49, 2548-2551. The major components of anacardic acid (15:1, 15:2 and 15:3) were isolated by means of column chromatography impregnated with silver nitrate, its structures were characterized by nuclear magnetic resonance, making a complete assignment of proton and carbon atoms in the spectra. It was evaluated the effect of unsaturation of the side chain in the biological activities such as antioxidant, anticholinesterasic and toxicity against Artemia salina. Antioxidant activity was assessed by inhibition of the free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl): monoene anacardic acid presents an IC50 = 2.06 ± 0.28 mg/mL, similar to the diene with IC50 = 1.78 ± 0.01 mg/mL, the triene was more active with IC50 = 0.13 ± 0.81 mg/mL, and the standard BHT showed an IC50 = 0.266 ± 0.005 mg/mL. The cytotoxicity against the A. salina was higher for anacardic acid triene with IC50 of 109.71 mg/mL, and even lower, a cytotoxic effect was also observed for other constituents which present LC50 <1000 g/mL (Meyer, B.N. et al. Planta Med. 1982, 45, 31-34). Regarding antiacetilcolinesterase activity, the triene anacardic acid showed the highest inhibition zone (1.0 cm), when compared to standard physostigmine (0.9 cm). Thus, among anacardic acids, triene showed better biological activities, followed by the diene and  Cashew nut shell liquid (CNSL) is obtained in the cashew nut processing industries as a by-product of little commercial value but with high technological potential due to its phenolic constitution and its various biological properties such as antimicrobial, anti-inflammatory, anti-tumor, antioxidant, anti-acetylcholinesterase, etc (Kubo, I., et al. Food Chem. 2006, 99, 555-562;Kubo, I., et al. J. Agric. Food Chem. 1993, 41, 1012-1015Oliveira, M.S.C., et al. Acta Trop. 2010, 117, 165-170). The anacardic acid, the major constituent of CNSL, is a mixture of three derivatives of salicylic acid with side chains of fifteen carbon atoms presenting different degree of unsaturation. In this study, the extraction of natural CNSL from cashew shell was made with hexane by maceration for obtaining anacardic acid (Paramashivappa, P., et al. J. Agric. Food Chem. 2001, 49, 2548-2551. The major components of anacardic acid (15:1, 15:2 and 15:3) were isolated by means of column chromatography impregnated with silver nitrate, its structures were characterized by nuclear magnetic resonance, making a complete assignment of proton and carbon atoms in the spectra. It was evaluated the effect of unsaturation of the side chain in the biological activities such as antioxidant, anticholinesterasic and toxicity against Artemia salina. Antioxidant activity was assessed by inhibition of the free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl): monoene anacardic acid presents an IC 50 = 2.06˘0.28 mg/mL, similar to the diene with IC 50 = 1.78˘0.01 mg/mL, the triene was more active with IC 50 = 0.13˘0.81 mg/mL, and the standard BHT showed an IC 50 = 0.266˘0.005 mg/mL. The cytotoxicity against the A. salina was higher for anacardic acid triene with IC 50 of 109.71 mg/mL, and even lower, a cytotoxic effect was also observed for other constituents which present LC 50 <1000 g/mL (Meyer, B.N. et al. Planta Med. 1982, 45, 31-34). Regarding antiacetilcolinesterase activity, the triene anacardic acid showed the highest inhibition zone (1.0 cm), when compared to standard physostigmine (0.9 cm). Thus, among anacardic acids, triene showed better biological activities, followed by the diene and monoene. Therefore the greater the number of unsaturations higher will be its action against free radicals, enzymes and organisms tested. Pharmaceuticals 2016, 9, 15 monoene. Therefore the greater the number of unsaturations higher will be its action against free radicals, enzymes and organisms tested.
Efavirenz (EFV, 1) is a non-nucleoside reverse transcriptase inhibitor administered as first-line treatment against HIV and prescribed as part of combination therapy. EFV is extensively metabolized by cytochrome P450, undergoing primary oxidation on the aromatic ring to the phenolic products 7-OH-EFV (minor, 2) and 8-OH-EFV (major, 3), and secondary oxidation on the cyclopropane ring (at C14) to 8,14-diOH-EFV (4). Despite the drug's efficacy, clinically restrictive neurotoxic and hepatotoxic events are a major limitation of EFV administration. Bioactivation to reactive electrophiles (e.g., to catechol metabolites and their quinoid derivatives) capable of reacting with biomacromolecules is likely to be involved.
We obtained and fully characterized a new catechol (6) from direct oxidation of 8-OH-EFV with Frémy´s salt; its quinone-imine precursor (5) was also detected. Incubation of EFV and its monohydroxy metabolites 2 and 3 with rat and human liver microsomes, followed by LC-ESI-MS characterization, led to the first-time evidence for the formation of catechol 6 in metabolically competent systems. By contrast, direct oxidation of 2 afforded a stable quinoline derivative (7)  Efavirenz (EFV, 1) is a non-nucleoside reverse transcriptase inhibitor administered as first-line treatment against HIV and prescribed as part of combination therapy. EFV is extensively metabolized by cytochrome P450, undergoing primary oxidation on the aromatic ring to the phenolic products 7-OH-EFV (minor, 2) and 8-OH-EFV (major, 3), and secondary oxidation on the cyclopropane ring (at C14) to 8,14-diOH-EFV (4). Despite the drug's efficacy, clinically restrictive neurotoxic and hepatotoxic events are a major limitation of EFV administration. Bioactivation to reactive electrophiles (e.g., to catechol metabolites and their quinoid derivatives) capable of reacting with biomacromolecules is likely to be involved. Pharmaceuticals 2016, 9, 15 monoene. Therefore the greater the number of unsaturations higher will be its action against free radicals, enzymes and organisms tested.
We obtained and fully characterized a new catechol (6) from direct oxidation of 8-OH-EFV with Frémy´s salt; its quinone-imine precursor (5) was also detected. Incubation of EFV and its monohydroxy metabolites 2 and 3 with rat and human liver microsomes, followed by LC-ESI-MS characterization, led to the first-time evidence for the formation of catechol 6 in metabolically Procyanidins (PC) are polyphenols (polymeric flavan-3-ols) that enter the human diet through fruits and vegetable-based beverages like red wine. These compounds have the ability to interact with salivary proteins. This interaction is related to astringency sensation defined as dryness, tightening and puckering sensations perceived in the oral cavity during the ingestion of food/beverages.
Most of the works done in astringency research do not characterize this interaction at molecular level. So, herein was intended to study the interaction between mucin (salivary) protein and PC by fluorescence quenching and Saturation Transference Difference-NMR. Thus, we focused in synthesize/isolate PC commonly present in red wine (dimers B3/B4, tetramer and oligomeric PC) and characterize their interaction with mucin.
The results showed that the values of binding constants obtained by fluorescence extinction increase with PC polymerization degree. Hydrophobic and hydrogen bonds were identified as the major driving forces for the interaction. The discovery of new and better photosensitizers for alternative therapies, such as Photodynamic Therapy (PDT), has aroused the interest of the scientific community. The squarylium cyanines dyes possess some of the essential properties for a good PDT photosensitizer, including a strong absorption in the Visible (Vis) or NIR region and the significant production of reactive oxygen species (Santos, P., et al. J. Photochem. Photobiol. A 2003, 160, 159-161). However, some modifications in their Pharmaceuticals 2016, 9, 15 50 of 56 structure may improve these properties enhancing its biological application. Previous studies suggest that the replacement of the oxygen atoms bonded at central ring by sulfur atoms increase the singlet oxygen production (Webster, S., et al., J. Phys. Chem. Lett. 2010, 1, 2354-2360. In this study a new symmetric dithiosquarylium cyanine dye 4 was synthesized with a moderated yield and its structural characterization was elucidated by, melting point, IR, Vis, 1 H-NMR, 13 C-NMR and HRMS-ESI-TOF. The citotoxicity in dark and under by LEDs irradiation, with an appropriate wavelength, was also evaluated in the cell lines HepG2 and Caco-2. The continuous study and development of organic chemistry has enabled the discovery of compounds with different applications. Among them, the discovery of new synthetic dyes, such as squarylium cyanine dyes which have shown great interest in many areas and applications (Chang, J., et al. J. Power Sources 2013, 240, 779-785). The squarylium cyanine dyes are a class of compounds, discovered in 1965 by Treibs (Treibs, A., et al. Angew. Chem. Int. Ed. 1965, 4, 694), whose photochemical and photophysical properties showed ideal behaviour for many biotechnological applications, such as photodynamic therapy (Avirah, R. R., et al. Org. Biomol. Chem. 2012, 10, 911-920).
In this work a new squarylium cyanine dye 7 was synthesized and fully characterized by melting point, IR, Vis, 1 H-NMR, 13 C-NMR and HRMS-ESI-TOF spectra. This dye was obtained, according to methods adapted from literature (Tatarets, A., et al. Dyes Pigment. 2005, 64, 125-134), with moderate yield. Later, its cytotoxic effect was evaluated on Caco-2 and HepG2 cells, in the dark and after LEDs irradiation with an appropriate wavelength. The continuous study and development of organic chemistry has enabled the discovery of compounds with different applications. Among them, the discovery of new synthetic dyes, such as squarylium cyanine dyes which have shown great interest in many areas and applications (Chang, J., et al. J. Power Sources 2013, 240, 779-785). The squarylium cyanine dyes are a class of compounds, discovered in 1965 by Treibs (Treibs, A., et al. Angew. Chem. Int. Ed. 1965, 4, 694), whose photochemical and photophysical properties showed ideal behaviour for many biotechnological applications, such as photodynamic therapy (Avirah, R. R., et al. Org. Biomol. Chem. 2012, 10, 911-920).