Studies on novel heterocyclic compounds having ...

2nd Indo-Italian Workshop on Chemistry and Biology of Antioxidants Organized by

Department of Human Physiology and Pharmacology “Vittorio Erspamer” Sapienza University of Rome

Institute for Biomolecular Chemistry of CNR

July 9-11, 2007

Location Aula Marconi CNR, P.le Aldo Moro 5, Rome

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PROGRAM July 9, 2007 14:00 Registration

14:30 Welcome Addresses Leonardo Gastaldi, Scientific Attaché, Embassy of Italy in New Delhi, India Fabrizio Eusebi, Director, Department of Human Physiology and Pharmacology “Vittorio Erspamer” Sapienza University of Rome Sesto Viticoli, Director, Department of Molecular Design of CNR, Italy Guido Cimino, Director, Institute for Biomolecular Chemistry of CNR

(A) Chemistry 15 :00

Session A1. Chairpersons: Subhash C. Jain and Armandodoriano Bianco

Virinder S. Parmar. Studies on novel heterocyclic compounds having anticancer, antimicrobial and antioxidant activities in the native and encapsulated forms. Paolo Bovicelli. New efficient synthesis of ubiquinones. Ashok K Prasad, Hanumantharao G. Raj and Virinder S. Parmar. Pyrazole derivatives as antioxidants. Mario C. Foti and Carmelo Daquino. The role of hydrogen bond in the antioxidant activity of phenols. Sunil Sharma. Polyphenols: antioxidants and beyond. 16 Discussion :45 17 Coffee Break :00 17 Session A2. Chairpersons: Gyan P. Garg and Virinder S. Parmar :15 Enzo Santaniello, Radmila Pavlovic and Giuseppe Meroni. Antioxidant properties of furane containing compounds. Subhash C Jain Mukesh K. Pandey, Shilpi Khurana and Leena Rohatgi. Bioactive natural products as leads for drug discovery. Giovanna Delogu, Davide Fabbri, Maria Antonietta Dettori. Synthetic strategies devoted to the preparation of hydroxylated natural-like biphenyls. Mazaahir Kidwai. Green chemistry and antioxidants. 19 Discussion :00

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July 10, 2007

(A) Chemistry 9: Session A3. Chairpersons: Ashok K. Prasad and Augusto Gambacorta 00 Anna Maria Serrilli and Armandodoriano Bianco. Biophenolic composition in callus cultures of Olea europaea. Brajendra Kumar Singh, Hanumantharao G. Raj, Balaram Ghosh, Erik Van der Eycken and Virinder S. Parmar. Antioxidant Activity of Some Selected Natural Polyphenolic Compounds, and Efficient Synthesis of Heterocyclic Compounds. Mauro Marchetti, Marianna Usai and Marzia Foddai. Composition and antioxidant activity of flavedo extracts from Sardinian citrus cultivar. Francesco Caruso, Miriam Rossi, Roberto Antonioletti, Justin Salciccioli and Alessandra Pompili. Crystal structure and DFT studies for scavenging properties of resveratrol derivatives including the resveratrol metabolite piceatannol. 10 Discussion :45 11 Coffee Break :00 11 Session A4. Chairpersons: Gino Lucente and Mauro Marchetti :15 Paolo La Colla, Roberta Loddo and Esther Marongiu. Development of new antiviral chemotherapeutic drugs. Enzo Santaniello, Radmila Pavlovic, Alessandro Repetto, Cristian E. M Bernardi and Pier Antonio Biondi. Synthesis of 4-hydroxy-2-hexenal (HHE) and development of analytical procedures for evaluation of cell and food damages by gas chromatography with electron capture detection (GC-ECD). Aidan Benson, Francesco Caruso and Miriam Rossi. Metal curcumin complexes. 12 Discussion :45 13 Lunch :00

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July 10, 2007

(B) Biology 14 Session B1. Chairpersons: Balaram Ghosh and Hanumantharao G. Raj :30 Monique Gardès-Albert and Daniel Jore. Steady-state radiolysis as a tool for caracterisation of antioxidant properties. Mauro Serafini, Monia Pecorari, Maria Francesca Testa and Debora Villano Valencia. Dietary antioxidants and oxidative stress prevention: the emerging role of the antioxidant network. Antonio Rescigno, Giuseppe Mocci, Paolo Zucca, Enrico Sanjust. (Auto)oxidation of catechols: implications towards antioxidant or prooxidant features. Jens Z. Pedersen, Sandra Incerpi, Ashok K. Prasad, Virinder S. Parmar, Luigi Iuliano and Luciano Saso. Organic antioxidants with catalytic activity: the surprising reaction mechanism of natural and synthetic polyphenolic compounds. Sandra Incerpi, Anna Maria Fiore, Paolo De Vito Ashok K. Prasad, Virinder S. Parmar and Jens. Z. Pedersen. Involvement of plasma membrane redox systems in hormone action. David Costantini. Minor antioxidant effects of carotenoids in birds. 16 Discussion :30 16 Coffee Break :45 17 Session B2. Chairpersons: Monique Gardès-Albert and Sandra Incerpi :00 Sarvesh Kumar, Ashok K. Prasad, Virinder S. Parmar and Balaram Ghosh. Antiinflammatory property of a novel cinnamic acid ester isolated from Piper longum. Petya Dimitrova and Nina Ivanovska. Antioxidant and anti-inflammatory effect of tyrphostin AG-490 on the development of zymosan-induced generalized inflammation. Nina Ivanovska and Petya Dimitrova.Vasoactive intestinal peptide (VIP) inhibits the production of NO and acute phase proteins in zymosan-induced generalized inflammation. Shvetambri Arora, Prabhjot Singh, Ajit Kumar, Yogesh K. Tyagi, Bilekere S. Dwarkanath, Jawahar S. Adhikari, Virinder S. Parmar and Hanumantharao G. Raj. The role of calreticulin transacetylase in the activation of nitrite reductase by polyphenolic per acetates in human platelets: a pathway for the enhancement of nitric oxide levels independent of nitric oxide synthase. Mimi Remichkova, Luciano Saso and Stefan Philipov. Inhibited NO release by stimulated peritoneal macrophages caused by some alkaloids. Tamara Pajpanova and Adriana Bocheva. Canavanine and dalargin as antioxidants. 19 Discussion :15

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July 11, 2007

(B) Biology 8: Session B3. Chairpersons: Liudmila Korkina and Silvia Bertuglia 30 Giuseppe Valacchi, Vihas Vasu, Wallace Yokoyama, Ana Corbacho, Anh Phung, Carroll Cross and Paul Davis. Vitamin E trafcking related genes are regulated by exogenous oxidants. Differences between young and old. Syed I. Rizvi and Pawan K. Maurya. Intracellular antioxidant systems in erythrocyte during aging in humans. Valentina Pallottini, Chiara Martini, Anna Trentalance. Prevention by caloric restriction of ROS-induced HMG-CoA reductase deregulation during aging. Fausta Natella and Cristina Scaccini. In vitro and ex vivo approaches to study dietary antioxidants. Gaetana A. Tonti, Francesca Benvenuti, Andrea Busetto, and Ferdinando Mannello. Antioxidant capacity and nutraceutical properties of crude bovine milk. Ferdinando Mannello, Gaetana A. Tonti, and Franco Canestrari. Prostaglandin concentrations and breast cancer risk using nipple aspirate fluids. 10 Discussion :30 10 Coffee Break :45 11 Session B4. Chairpersons: Jens Pedersen and Daniel Jore :00 Silvia Bertuglia. Hypoxic and hyperoxic preconditionings decrease oxidative stress in ischemia reperfusion injury. Mauro Todisco. Melatonin, platelets and endothelium. Chiara De Luca. The role of antioxidants in skin physiopathology: beneficial applications. Liudmila G. Korkina, Elena V. Mikhal’chik, Maxim V. Suprun, Roberto Dal Toso, and Saveria Pastore. Antioxidant, anti-inflammatory, and wound healing properties of naturally occurring and biotechnologically produced phenylpropanoids. Elisabetta Meucci, Andrea Silvestrini, Roberto Festa and Antonio Mancini. Role of antioxidants in male infertility. Cesare Mancuso. Role of the heme oxygenase/biliverdin reductase system in the central nervous system. Ashraf Virmani and Franco Gaetani. Role of coenzyme Q10 in Parkinson's disease therapy. Luciano Saso. Conclusions: pharmacological applications of antioxidants. 13 Discussion :45

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ABSTRACTS (A) CHEMISTRY ............................................................................................................................................ 7 STUDIES ON NOVEL HETEROCYCLIC COMPOUNDS HAVING ANTICANCER, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES IN THE NATIVE AND ENCAPSULATED FORMS.................................................................................................................. 7 NEW EFFICIENT SYNTHESIS OF UBIQUINONES ...................................................................................................................................... 8 PYRAZOLE DERIVATIVES AS ANTIOXIDANTS ........................................................................................................................................ 9 THE ROLE OF HYDROGEN BONDING ON THE ANTIOXIDANT ACTIVITY OF PHENOLS ................................................................ 10 POLYPHENOLS: ANTIOXIDANTS AND BEYOND ................................................................................................................................... 11 ANTIOXIDANT PROPERTIES OF FURANE CONTAINING COMPOUNDS............................................................................................. 12 BIOACTIVE NATURAL PRODUCTS AS LEADS FOR DRUG DISCOVERY ............................................................................................ 13 SYNTHETIC STRATEGIES DEVOTED TO THE PREPARATION OF HYDROXYLATED NATURAL-LIKE BIPHENYLS .................. 14 GREEN CHEMISTRY AND ANTIOXIDANTS ............................................................................................................................................. 15 BIOPHENOLIC COMPOSITION IN CALLUS CULTURES OF OLEA EUROPAEA .................................................................................. 16 ANTIOXIDANT ACTIVITY OF SOME SELECTED NATURAL POLYPHENOLIC COMPOUNDS, AND EFFICIENT SYNTHESIS OF HETEROCYCLIC COMPOUNDS.................................................................................................................................................................. 17 COMPOSITION AND ANTIOXIDANT ACTIVITY OF FLAVEDO EXTRACTS FROM SARDINIAN CITRUS CULTIVAR .................. 18 CRYSTAL STRUCTURE AND DFT STUDIES FOR SCAVENGING PROPERTIES OF RESVERATROL DERIVATIVES INCLUDING THE RESVERATROL METABOLITE PICEATANNOL .............................................................................................................................. 19 DEVELOPMENT OF NEW ANTIVIRAL CHEMOTHERAPEUTIC DRUGS .............................................................................................. 20 SYNTHESIS OF 4-HYDROXY-2-HEXENAL (HHE) AND DEVELOPMENT OF ANALYTICAL PROCEDURES FOR EVALUATION OF CELL AND FOOD DAMAGES BY GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION (GC-ECD) .............. 21 METAL CURCUMIN COMPLEXES ............................................................................................................................................................. 22

(B) BIOLOGY ................................................................................................................................................ 23 STEADY-STATE RADIOLYSIS AS A TOOL FOR CARACTERISATION OF ANTIOXIDANT PROPERTIES ....................................... 23 DIETARY ANTIOXIDANTS AND OXIDATIVE STRESS PREVENTION: THE EMERGING ROLE OF THE ANTIOXIDANT NETWORK ..................................................................................................................................................................................................... 24 (AUTO)OXIDATION OF CATECHOLS: IMPLICATIONS TOWARDS ANTIOXIDANT OR PROOXIDANT FEATURES ..................... 25 ORGANIC ANTIOXIDANTS WITH CATALYTIC ACTIVITY: THE SURPRISING REACTION MECHANISM OF NATURAL AND SYNTHETIC POLYPHENOLIC COMPOUNDS ........................................................................................................................................... 26 INVOLVEMENT OF PLASMA MEMBRANE REDOX SYSTEMS IN HORMONE ACTION .................................................................... 27 MINOR ANTIOXIDANT EFFECTS OF CAROTENOIDS IN BIRDS ........................................................................................................... 28 ANTI-INFLAMMATORY PROPERTY OF A NOVEL CINNAMIC ACID ESTER ISOLATED FROM PIPER LONGUM......................... 29 ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECT OF TYRPHOSTIN AG-490 ON THE DEVELOPMENT OF ZYMOSANINDUCED GENERALIZED INFLAMMATION ............................................................................................................................................ 30 VASOACTIVE INTESTINAL PEPTIDE (VIP) INHIBITS THE PRODUCTION OF NO AND ACUTE PHASE PROTEINS IN ZYMOSANINDUCED GENERALIZED INFLAMMATION ............................................................................................................................................ 31 THE ROLE OF CALRETICULIN TRANSACETYLASE IN THE ACTIVATION OF NITRITE REDUCTASE BY POLYPHENOLIC PER ACETATES IN HUMAN PLATELETS: A PATHWAY FOR THE ENHANCEMENT OF NITRIC OXIDE LEVELS INDEPENDENT OF NITRIC OXIDE SYNTHASE ......................................................................................................................................................................... 32 INHIBITED NO RELEASE BY STIMULATED PERITONEAL MACROPHAGES CAUSED BY SOME ALKALOIDS ........................... 33 CANAVANINE AND DALARGIN AS ANTIOXIDANTS ............................................................................................................................ 34 VITAMIN E TRAFCKING RELATED GENES ARE REGULATED BY EXOGENOUS OXIDANTS. DIFFERENCES BETWEEN YOUNG AND OLD......................................................................................................................................................................................... 35 INTRACELLULAR ANTIOXIDANT SYSTEMS IN ERYTHROCYTE DURING AGING IN HUMANS ................................................... 36 PREVENTION BY CALORIC RESTRICTION OF ROS-INDUCED HMG-COA REDUCTASE DEREGULATION DURING AGING .... 37 IN VITRO AND EX VIVO APPROACHES TO STUDY DIETARY ANTIOXIDANTS................................................................................ 38 ANTIOXIDANT CAPACITY AND NUTRACEUTICAL PROPERTIES OF CRUDE BOVINE MILK ........................................................ 39 PROSTAGLANDIN CONCENTRATIONS AND BREAST CANCER RISK USING NIPPLE ASPIRATE FLUIDS ................................... 40 HYPOXIC AND HYPEROXIC PRECONDITIONINGS DECREASE OXIDATIVE STRESS IN ISCHEMIA REPERFUSION INJURY ... 41 MELATONIN, PLATELETS AND ENDOTHELIUM.................................................................................................................................... 42 THE ROLE OF ANTIOXIDANTS IN SKIN PHYSIOPATHOLOGY: BENEFICIAL APPLICATIONS ....................................................... 43 ANTIOXIDANT, ANTI-INFLAMMATORY, AND WOUND HEALING PROPERTIES OF NATURALLY OCCURRING AND BIOTECHNOLOGICALLY PRODUCED PHENYLPROPANOIDS ............................................................................................................. 44 ROLE OF ANTIOXIDANTS IN MALE INFERTILITY ................................................................................................................................. 45 ROLE OF THE HEME OXYGENASE/BILIVERDIN REDUCTASE SYSTEM IN THE CENTRAL NERVOUS SYSTEM ........................ 46 ROLE OF COENZYME Q10 IN PARKINSON'S DISEASE THERAPY ........................................................................................................ 47 PHARMACOLOGICAL APPLICATIONS OF ANTIOXIDANT DRUGS ..................................................................................................... 48

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(A) CHEMISTRY

STUDIES ON NOVEL HETEROCYCLIC COMPOUNDS HAVING ANTICANCER, ANTIMICROBIAL AND ANTIOXIDANT ACTIVITIES IN THE NATIVE AND ENCAPSULATED FORMS. Virinder S. Parmar Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi – 110 007 (India) (E-mail: [email protected]) Heterocyclic compounds are widely distributed in nature and are essential to life; they play a vital role in the metabolism of all living cells. There are a vast number of pharmacologically active heterocylic compounds, many of which are in regular clinical use. There is no doubt that the chemistry of heterocylics will continue to grow for the creation of new drugs, agrochemicals, novel material, etc. In our laboratory, we have synthesized a large variety of heterocylic compounds which have exhibited interesting anti-inflammatory activity of importance in treating asthma and Alzheimer’s disease; antioxidant activity against the initiation of lipid peroxidation in rat liver microsomes; antiinvaise activity against human MCG-7/6 mammary carcinoma cells; antifungal activity against two deadly lung fungal infections; antiviral activity as nonnucleoside HIV-I reverse transcriptase inhibitors (NNRTIs) and antimycobacterial activity against Mycobacterium tuberculosis. Selected Publications 1.

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Raunak, V Kumar, S Mukherjee, Poonam, AK Prasad, CE Olsen, SJC Schaffer, SK Sharma, AC Watterson, W Errington and VS Parmar. Microwave mediated synthesis of spiro-(indolineisoxazolidines): mechanisitic study and biological activity evaluation. Tetrahedron 61, 2005, 56875697. S Kumar, BK Singh, N Kalra, V Kumar, A Kumar, AK Prasad, HG Raj, VS Parmar and B Ghosh. Novel thiocoumarins as inhibitors of TNF-α induced ICAM-1 expression on human umbilical vein endothelial cells (HUVECs) and microsomal lipid peroxidation. Bioogranic and Medicinal Chemistry 13, 2005, 1605-1613. BW Vanhoecke, HT Depypere, AD Beyter, SK Sharma, VS Parmar, DD Keukeleire and ME Bracke. New anti-invasive compounds: Results form the Indo-Belgian screening program. Pure and Applied Chemistry 77, 2005, 65-74. MC Foti, SK Sharma, G Shakya, AK Prasad, G Nicolosi, P Bovicelli, B Ghosh, HG Raj, RC Rastogi and VS Parmar, Biopolyphenolics as antioxidants: Studies under an Indo-Italian CSIR-CNR project. Pure and Applied Chemistry 77, 2005, 91-101. SV Slambrouck, VS Parmar, SK Sharma, BD Bondt, F Fore, P Coopman, BW Vanhoecke, T Boterberg, HT Depypere, G Leclercq and ME Bracke. Tangeretin inhibits extracellular-signalregulated kinase (ERK) phosphorylation. FEBS Letters 579, 2005, 1665-1669. S Kumar, P Arya, C Mukherjee, BK Singh, N Singh, VS Parmar, AK Prasad and B Ghosh. Novel aromatic ester from Piper longum and its analogues inhibit expression of cell adhesion on endothelial cells. Biochemistry 44, 2005, 15944-15952. AK Chhillar, P Arya, C Mukherjee, P Kumar, Y Yadav, AK Sharma, V Yadav, J Gupta, R Dabur, HN Jha, AC Watterson, VS Parmar, AK Prasad and GL Sharma. Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: Novel compounds against aspergillosis: Bioogranic and Medicinal Chemistry 14, 2006, 973-981. BK Singh, P Atppukktan, S Claerhout, VS Parmar and EV Eycken. Copper (II)-mediated crosscoupling of arylboronic acids and 2(1H)-pyrazinones facilitated by microwave irradiation with simultaneous cooling. Organic Letters 8, 2006, 1863-1866.

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NEW EFFICIENT SYNTHESIS OF UBIQUINONES Paolo Bovicelli Institute for Biomolecular Chemistry of CNR, Rome, Italy c/o Department of Chemistry "Sapienza University" P.le A. Moro, 5 – 00185 Rome, Italy (E-mail: [email protected]) Ubiquinones, also known as coenzyme Qn (n = number of isoprene units in the side chain) are lipophilic molecules constituted by a highly functionalised benzoquinone moiety and an all-trans polyisoprene hydrophobic chain, which assures their solubility in organic media. In a redox system, these compounds are present as p-benzoquinones (ubiquinones, CoQ) and phydroquinones (ubiquinonols, CoQH2). Ubiquinones are present in many natural systems in which electron transfer mechanisms are involved and they also function as antioxidants and scavengers of free radicals. Our approach to the synthesis of these compounds is based on a bromination/methanolysis procedure to prepare the phenol key intermadiates and on a acid mediated rearrangement of phenol-allyl ethers. Difficulties derived from lo yielding processes and not selective reactions were overlap by synthetising the suitable phenols. By these procedures, CoQ0-3 and CoQ9-10 were prepared in good yields. Selected Publications 1. Bovicelli, P.; Antonioletti, R.; Barontini, M.; Borioni, G.; Bernini, R.; Mincione, E. "New convenient synthesis of iridol. An approach to the synthesis of ubiquinones." Tetrah. Lett. 2005, 46(8), 1255-1257. 2. Foti, M. C.; Sharma, S. K.; Shakya, G.; Prasad, A. K.; Nicolosi, G.; Bovicelli, P.; Ghosh, B.; Raj, H. G.; Rastogi, Ramesh C.; Parmar, V. S. "Biopolyphenolics as antioxidants: studies under an Indo-Italian CSIR-CNR project" Pure App. Chem. 2005, 77(1), 91-101. 3. Bovicelli, P.; Antonioletti, R.; Onori, A.; Delogu, G.; Fabbri, D.; Dettori, M. A. "Regioselective halogenation of biphenyls for preparation of valuable polyhydroxylated biphenyls and diquinones" Tetrah. 2006, 62(4), 635-639. 4. Bovicelli, P.; Antonioletti, R.; Mancini, S.; Causio, S.; Borioni, G.; Ammendola, S.; Barontini, M. "Expeditious synthesis of hydroxytyrosol and its esters" Synth. Communic. 2007, 37(23), in press. 5. Bovicelli, P.; Antonioletti, R.; Borioni, G.; Fabbrini, D.; Barontini, M. "New efficient synthesis of ubiquionones" Synth. Communic. 2007, accepted. 6. Bovicelli, P.; D'Angelo, V.; Collalto, D.; Versino, A.; Lambusta, D.; Nicolosi, G. "Efficient synthesis of polyoxygenated flavones from naturally occurring flavanones" J. Pharm. Pharmac. 2007, submitted 7. Bovicelli, P. "Radical scavenging polyphenols: new strategies for their synthesis" J. Pharm. Pharmac. 2007, minireview, submitted.

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PYRAZOLE DERIVATIVES AS ANTIOXIDANTS Ashok K. Prasad1,* Hanumantha G. Raj2 and Virinder S. Parmar1 1

Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India; 2Department of Biochemistry, VP Chest Institute, University of Delhi, Delhi110 007, India (E-mail: [email protected]) Considerable progress has been made in recent years in relating aging to oxidation in biological cells. The reactive oxygen species (ROS), cause of oxidation in biological cells, are basically involved in detoxification of invading organisms and chemicals, but stray ROS also initiate lipid peroxidation in healthy cells leading to diverse pathologies such as Alzheimer’s disease, atherosclerosis, diabetes, Parkinson’s disease, etc. Thus, reduction of the rate of these life-limiting metabolic processes by use of chemicals has been a subject of current research. We have synthesized and evaluated a series pyrazoles and their derivatives against rat liver microsomal lipid peroxidation. Some of the pyrazole derivatives have exhibited very high radical scavenging and microsomal lipid peroxidation inhibition activity.

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THE ROLE OF HYDROGEN BONDING ON THE ANTIOXIDANT ACTIVITY OF PHENOLS Mario C. Foti and Carmelo Daquino Institute of Biomolecular Chemistry of the Italian CNR, Valverde (Catania), Italy (Email: [email protected]) Intermolecular and intramolecular hydrogen–bonding involving the phenolic hydroxyl influences profoundly the antioxidant properties of phenols (ArOH). Phenols function as antioxidants because they are able to quench the chain–carrying peroxyl radicals, ArOH + ROO•  ArO• + ROOH, responsible for the peroxidation process of organic matter. Polar solvents slow down this process because the hydrogen–bonded molecules ArOH•••S are not able to react with ROO•. Catechols are characterized by the presence of an intramolecular hydrogen–bond which reinforces their capability of transferring an H–atom to ROO•, the involved OH being the one free of hydrogen–bond. 2–Methoxyphenols show instead a decreased reactivity toward free–radicals. Selected Publications M. C. Foti; L. R. C. Barclay; K. U. Ingold J. Am. Chem. Soc. 2002, 124, 12881.

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POLYPHENOLS: ANTIOXIDANTS AND BEYOND Sunil K. Sharma Department of Chemistry, University of Delhi, Delhi 110 007, India (E-mail: [email protected]) Health, wellness, anti-aging - these are the growth areas of the 21st century. Never before have so many taken active measures to maintain good health as is the case today. Never before have they been as well informed as they are today and never before has there been as widespread a turning to preventative medicine based on natural substances. Plant foods, such as fruits, vegetables, and whole grains contain many components that are beneficial to human health. Research supports that some of these foods, as part of an overall healthful diet, have the potential to delay the onset of many age-related diseases. These observations have led to continuing research aimed at identifying specific bioactive components in foods, such as antioxidants, which may be responsible for improving and maintaining health. Antioxidants protect cells from damage caused by unstable molecules known as free radicals. Vitamins and secondary plant chemical substances such as polyphenols and particularly here the flavonoids are counted among the antioxidants. While the market potential may be largely exhausted for vitamins, the secondary plant chemical substances are about to experience a boom. Already today, due to their physiological effect, they are being called the “vitamins of the 21st century”. We have been working in the area of natural product chemistry for the last twenty years and have isolated / synthesized a variety of compounds e.g. flavonoids, coumarins, chromones, etc. and evaluated their antioxidant properties. Some of our compounds are found to possess highly efficient antioxidant properties. These results would be presented during the Workshop. Selected Publications 1. S Kumar, BK Singh, AK Panday, A Kumar, SK Sharma, HG Raj, AK Prasad, E Van der Eycken, VS Parmar and B Ghosh. A chromone analog inhibits TNFinduced expression of cell adhesion molecules on human endothelial cells via blocking TNF-2962 (2007). 2. A Kumar, BK Singh, NK Sharma, K Gyanda, SK Jain, YK Tyagi, AS Baghel, M Pandey, SK Sharma, AK Prasad, SC Jain, RC Rastogi, HG Raj, AC Watterson and VS Parmar. Specificities of acetoxy derivatives of coumarins, biscoumarins, flavones, isoflavones and xanthones for acetoxy drug: protein transacetylase. European Journal of Medicinal Chemistry 42, 2007, 447-455. 3. MC Foti, SK Sharma, G Shakya, AK Prasad, G Nicolosi, P Bovicelli, B Ghosh, HG Raj, RC Rastogi and VS Parmar. Biopolyphenolics as antioxidants: Studies under an Indo-Italian CSIR-CNR project. Pure and Applied Chemistry 77, 91-101 (2005).

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ANTIOXIDANT PROPERTIES OF FURANE CONTAINING COMPOUNDS Enzo Santaniello, Radmila Pavlovic and Giuseppe Meroni Department of Medicine, Surgery and Dentistry - School of Medicine c/o S. Paolo Hospital- University of Milan, Italy (E-mail: [email protected]) Many furan containing compounds are known to possess antioxidant activity, as, for instance, furan fatty acids that have shown to act as scavengers of hydroxyl radical .OH [1]. Among synthetic compounds, sorbityl furfural (SF) and the corresponding tetramethyl ether (MSF) have been proposed as antioxidant ingredients of some industrial preparations. SF is an excellent scavenger of .OH as shown by pulse-radiolysis [2] and more recent studies, that will be reported in this communication. MSF is less studied in this respect, but in vivo activity [3] has suggested properties similar to SF. We will refer about studies related to preparation of SF analogues and derivatives. Furane containing derivatives of glycerol [furfurylidene glycerol (FG) and glycerol furyl ether (GFE)] will be also described and preliminary account on their antioxidant activity illustrated. Selected Publications 1. Okada, Y. et al. Biol. Pharm. Bull. 19, 1607-1610 (1996) 2. Emmi, S. S. et al. J. Phys. Chem. A 106, 4598-4607 (2002) 3. Zanoli, P. et al. Agents Actions 12, 4-10 (1982)

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BIOACTIVE NATURAL PRODUCTS AS LEADS FOR DRUG DISCOVERY Subhash C. Jain, Mukesh K. Pandey, Shilpi Khurana and Leena Rohatgi Department of Chemistry, University of Delhi, Delhi 110 007; India (E-mail: [email protected] or [email protected])

The biologically active natural products, isolated from different sources serve as the lead compounds for the discovery of new drugs. This is why the importance of plants and marine sponges has been appreciated from time to time as natural resources in pharmaceutical research. Recently 3-substituted alkylpyridines from marine sponges and various anacardic acids from different plants have been isolated and were found to possess strong cytotoxic activity against P-388 marine leukemia cells and strong antimicrobial activities against many pathogens. 2-Alkyl/alkenyl benzopyranones (isoflavones) are known to possess strong antioxidant properties. A new strategy has been developed for the synthesis of these bioactive compounds using pyridyl, aryl and isoflavonyl sulfone as the potent intermediate. The sulfone unit was knocked off at the end of the synthesis to obtain the desired natural product. Using this methodology, we have synthesized 14 natural products for the first time. Taking leads from these compounds and using the above mentioned strategy we have also synthesized a large number of related compounds of biological importance in order to discover new drug candidate(s) for the future. Selected Publications Subhash C Jain; Rohtash Kumar; Rajeev Goswami; Mukesh K Pandey; Shilpi Khurana; Leena Rohatgi and Kapil Gyanda. Synthesis of novel non-isoprenoid phenolic acids and 3alkylpyridines. Pure and Applied Chemistry, 2005, 77(1), 185-193

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SYNTHETIC STRATEGIES DEVOTED TO THE PREPARATION OF HYDROXYLATED NATURAL-LIKE BIPHENYLS Giovanna Delogu, Davide Fabbri and Maria Antonietta Dettori CNR- National Research Council, Institute for Biomolecular Chemistry, Traversa La Crucca, 3 – I-07100 Sassari, ITALY (E-mail: [email protected]) Hydroxylated biphenyl unit is present in a large number of naturally occurring compounds such as vancomycin, biphenomycin, ellagitannins. There is an increasing interest toward use of the hydroxylated biphenyl unit as building block to prepare bioactive molecules involved in human pathologies because most of them manifest interesting pharmacological activities. One of the main features of these compounds is the antioxidant activity in virtue of fact that they can generate quinones under mild conditions. Recently, the concept that a drug with two or more mechanisms of action (eg. antioxidant activity) targeted at multiple etiologies of the same disease may offer great therapeutic benefit. As part of our continuing search among hydroxylated biphenyls as building block for the preparation of promising both pharmaceutics and new ligands, we have selected natural and natural-like 2-methoxy phenols and prenylated phenols of known antioxidant activity in order to prepare C2-symmetry dimers with interesting stereochemical and biological features. Selected Publications 1. ‘Eugenolo, bis-eugenol and synthetized related-dimer compounds produce antinociception in the acetic-induced-writhing responses’ Peana, A.; Chessa, G.; Carta, G.; Delogu, G.; Fabbri, D. Current Top. Phytochem. 2004, 6, 137-143. 2. ‘Synthesis and biocatalytic resolution of a new atropisomeric biphenyl: (2,2’,6,6’tetramethoxybiphenyl- 3,3’-diyl)dimethanethiol’ Sanfilippo, C.; Nicolosi, G.; Delogu, G.; Fabbri, D.; Dettori, M.A. Tetrahedron: Asymmetry 2005, 16, 10791084. 3. ‘Regioselective halogenation of biphenyls to prepare valuable polyhydroxylated biphenyls and diquinones’ P. Bovicelli, R. Antonioletti, A. Onori, G. Delogu, D. Fabbri, M. A. Dettori Tetrahedron: Lett. 2006, 62, 635-639. 4. ‘New Electroactive C2 symmetry assemblies based on the biphenyl scaffold and tetrathiafulvalene (TTF) units’ G. Delogu, D. Fabbri, M. A. Dettori, M. Sallé, F. Le Derf, M-J. Blesa, Magali A. J.Org. Chem. 2006, 71, 9096-9103. 5. ‘Antiproliferative and pro-apoptotic activity of eugenol-related biphenyls on malignant melanoma cells’ Pisano, M.; Pagnan, G.; Loi, M.; Mura, M.E.; Tilocca, M.G.; Palmieri, G.; Fabbri, D.; Dettori, M.A.; Delogu, G.; Ponzoni, M.; Rozzo, C. Molecular Cancer, 2007, 6 (8), 1-12. 6. ‘2,2’-Dihydroxy-3,3’-dimethoxy-5,5’-dimethyl-6,6’-dibromo-1,1’-biphenyl: preparation, resolution, structure and biological activity’ Fabbri, D.; Dettori, M.A.; Delogu, G.; Forni, A.; Casalone, G.; Palmieri, G.; Pisano, M.; Rozzo, C. Tetrahedron: Asymmetry 2007, 18,414-423.

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GREEN CHEMISTRY AND ANTIOXIDANTS Mazaahir Kidwai Department of Chemistry, [email protected])

University

of

Delhi,

India

(E-mail:

Chemical and biochemical studies under the domain of Green Chemistry is carried out and the term “ Green Chemistry “is the movement towards environmentally acceptable chemical process and products. In the context of green chemistry, Biocatalysis refers to the catalysis of various organic transformation using enzymes. Some of the important aspects of enzyme catalysis relevant of green chemistry, are high degree of selectivity, aqueous-phase reactions, non-toxic, low hazardous nature, possibility for asymmetric synthesis, energy-efficient reactions under moderate conditions, sequential one-pot synthesis etc. Enzymes, itself can act as antioxidant e.g. glutathione peroxidase and they can also be employed in the synthesis of antioxidants e.g. L-Ascorboyl fatty acid esters. Antioxidants are widely used in foods and cosmetics to prevent oxidation of lipids. The syntheses of these esters are highly challenging. New trends using green chemistry are employing now-a-days as it is used in the treatment or preventing sunburn damage to the skin and it is used as natural antioxidant. The synthesis using lipase (Novozyme 435) for the ester with L-Ascorbic acid and acyl donor is an illustrative example. O R O

OH HO

O

O

Lipase

+ HO

OH

HO

O

O

RCOOMe HO

OH

Selected Publications 1.

Kidwai, M.; K. Singhal, and S. Kukreja, 2007 One pot green synthesis for pyrimido[4,5d]pyrimidine derivatives Zeitschrift fuer Naturforschung B, (Germany) 62B,732-736 2. Kidwai, M. V. Bansal, A. Kumar, S. Mozumdar, 2007 The first Au-nanoparticles catalyzed green synthesis of propargylamines via a three-component coupling reaction of aldehyde, alkyne and amine Green Chemistry (RSC, London), 9, 1-5. 3. Kidwai, M. and K. Singhal, 2007 Aqua mediated one-pot synthesis and aromatization of pyrimido fused 1,4- dihydropyridine derivatives using ammonium salts. Candian. J. Chem., 85, 1-6. 4. Kidwai, M., V. Bansal, N. K. Mishra, A, Kumar, S. Mozumdar, 2007 Copper-NanoparticleCatalyzed A3 Coupling via C–H Activation. Synlett, 1, 1-4. M. 5. Kidwai, S. Saxena, M.K.R. Khan, S.S. Thukral, European Journal of Medicinal Chemistry, 2005, 42, 816-819. M. Kidwai, P. Mothsra, R. Mohan, S. Biswas, Bioorganic and Medicinal Chemistry Letters (U.K.), 2005, 15, 915-917. 6. M. Kidwai, S. Saxena, R. Mohan, J. Chem. Soc., Perkin Trans. 1, 2002, 16, 1845-1846. M. Kidwai, B. Dave, K.R. Bhushan, P. Misra, R.K. Saxena, R. Gupta, R. Gulati and M. Singh, Biocatalysis and Biotransformation, 2002, 20(5), 377-379. 7. M. Kidwai, P. Sapra, K.R. Bhushan, P. Misra, R.K. Saxena, R. Gupta and M. Singh, Biorganic Chemistry (USA), 2001, 29, 380-386. 8. M. Kidwai, R. Venkataramanan and B. Dave, Green Chemistry (USA), 2001, 3, 278-279. 9. M. Kidwai, P. Sapra, P. Misra, R.K. Saxena and M. Singh, Bioorganic and Medicinal Chemistry (UK), 2001, 9, 217-220. 10. M. Kidwai, K.R. Bhushan, P. Sapra, R.K. Saxena and R. Gupta, Bioorganic and Medicinal Chemistry (USA), 2000, 8, 69-72. 11. M. Kidwai, R. Kumar, A.K. Srivastava and H.P. Gupta, Bioorganic Chemistry (USA), 1998, 26, 289-294.

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BIOPHENOLIC COMPOSITION IN CALLUS CULTURES OF OLEA EUROPAEA Anna Maria Serrilli and Armandodoriano Bianco Institute for Biomolecular Chemistry of CNR and Department of Chemistry, Sapienza University of Rome, Italy (E-mail: [email protected]) Olea europaea L. subsp sativa, is a typical species of the Mediterranean area, always object of study. At the present, studies on in vitro systems of O. europaea concern mostly on the molecular aspects of the primary metabolism. One of goals of this research has been to verify if callus cells are able to producing secondary metabolites, in particular the phenolic compounds. An increasing interest in these compounds is due to their antioxidant and health enhancing properties. The amounts and types of these compounds in all parts of this plant are know. But the metabolic relationships between the various parts and phenolic content are poorly understood. Olive callus has been established from shoot explants. A moiety of the callus has been carried out on HPLC analysis, to search the phenolic component. The analysis showed the presence of oleuropein and this is a new data, not still present in the literature. Selected Publications 1. A.BIANCO, A.M.SERRILLI, C.MELCHIONI - Molecular composition and quality/taste of olive oil: monoterpenes and natural phenols, ARKIVOC, 2007, Part(vii), 146 2. A.BIANCO, A.RAMUNNO – The Chemistry of Olea europaea, in, Atta-urRahman Ed., Studies in Natural Products Chemistry, Elsevier, Vol 33 p.859-903 (2006). 3. A.BIANCO, M.A.CHIACCHIO, G.GRASSI, D.IANNAZZO, R.ROMEO Phenolic components of Olea europaea – Isolation of a new tyrosol and hydroxytyrosol derivatives – Food Chemistry, 2006, 95, 562-565. 4. A.BIANCO, C. MELCHIONI, A. RAMUNNO, G.ROMEO, N.UCCELLA Phenolic components of Olea europaea – Isolation of tyrosol derivatives – Natural Products Research, 2004, 18, 29-32. 5. A.BIANCO, F.BUIARELLI, G.CARTONI, F.COCCIOLI, I.MUZZALUPO, A.POLIDORI, N.UCCELLA - Analysis by HPLC/MS of biophenolic components in olives and oils - Analytical Letters, 2001, 34(6), 1033-1051.

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ANTIOXIDANT ACTIVITY OF SOME SELECTED NATURAL POLYPHENOLIC COMPOUNDS, AND EFFICIENT SYNTHESIS OF HETEROCYCLIC COMPOUNDS Brajendra Kumar Singh a,d H. G. Raj,b B. Ghoshc E. Van der Eyckend and V. S. Parmara a

Bioorganic Laboratory, Department of Chemistry, University of Delhi, India, Department of Biochemistry, V.P. Chest Institute, University of Delhi, India, cInstitute of Genomics and Integrative Biology, Delhi, India, dDepartment of Chemistry, University of Leuven, Leuven, Belgium (E-mail: [email protected]) b

The term antioxidant/antioxygen originally was used to refer specifically, to a chemical that prevents the consumption or slow down the oxidative damage to our body. They are believed to be a means of providing protection to the body by limiting damaging action of free radicals. Naturally occurring polyphenols, like xanthones, coumarins, flavones, chromones, etc. are well-known bioactive compounds. In addition to their other activities, results of their NADPH-catalyzed rat liver microsomal lipid peroxidation and TNF- induced ICAM-1 expression on endothelial cells will be discussed at the Workshop. Also results of our extensive work on efficient synthesis of heterocyclic compounds under microwaveirradiation conditions will be presented. Selected Publications 1. Sarvesh Kumar, Pragya Arya, Chandrani Mukherjee, Brajendra K. Singh, Naresh Singh, V. S. Parmar, A. K. Prasad, Balaram Ghosh. Biochemistry, 2005, 44, 1594415952. 2. Brajendra K. Singh, Prasad Appukkuttan, Stijn Claerhout, V. S. Parmar and Erik Van der Eycken. Organic Letters, 2006, 8, 1863-1866. 3. Nadya Kaval, Brajendra K. Singh, D. S. Ermolat’ev, Stijn Claerhout, V. S. Parmar, Johan Van der Eycken and Erik Van der Eycken. Journal of Combinatorial Chemistry, 2007, 9, 446-453. 4. Sarvesh Kumar, Brajendra K. Singh, A. K. Pandey, Ajit Kumar, S. K. Sharma, H. G. Raj, A. K. Prasad, Erik Van der Eycken, V. S. Parmar and Balaram Ghosh Bioorg. Med. Chem., 2007, 15, 2952-2962.

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COMPOSITION AND ANTIOXIDANT ACTIVITY OF FLAVEDO EXTRACTS FROM SARDINIAN CITRUS CULTIVAR Mauro Marchettia Marianna Usaib and Marzia Foddaic a

Institute of Biomolecular Chemistry, National Research Council, Trav. La Crucca 3, 07040 Li Punti – Sassari, Italy; bDepartment of Pharmaceutical Sciences, University of Sassari, Via Muroni, 23/A, 07100 Sassari, Italy; cDepartment of Biorganic Chemistry and Biopharmacy, University of Pisa, Via Bonanno Pisano 33, 56126 Pisa, Italy (E-mail: [email protected]) There is a strong need for effective antioxidants from natural sources as alternatives to prevent deterioration of conserved food and fats and oils, since synthetic antioxidants (BHT and BHA) are known to have toxic and carcinogenic effects on human health. The literature is replete with reports of extracts obtained from edible and non-edible plant materials, which possess antioxidant compounds. Citrus peel are a waste material, obtained after extraction of juice from citrus fruit. Methanolic extract of citrus peel is known to have different antioxidative compounds. A small number of studies have been carried out on antioxidant activity of citrus cultivars growing in Sardinia. The present study was undertaken to study the composition and the antioxidant activity of peel extracts of two citrus cultivars growing exclusively in Sardinia island: San Vito Orange and Pompia. The obtained experimental data showed that the citrus extracts of the two cultivars are effective in scavenging activity at the concentration of 0.1 mg/mL, and in the inhibition of Fenton reaction at the concentration of 0,75 g/mL Selected Publications 1. Bovill H., 1996, “Orange: sources of natural compounds”, Aromes Ingred. Addit., 7, 41-42. 2. Marini D.,Balestrieri F., 1995, “Multivariate analysis of flavanone glycosides in citrus juice” Ital. J. Food Sci., 3, 255-264. 3. Ooghe W.C., Detavernier C., 1997, “Detection of the addition of Citrus reticulata and Hybrids to Citrus sinensis by flavonoids” J. Agric. Food Chem., 45,1633-1637. 4. Joyeux M.,Lobstein A.,Anton R.,Mortier F.,1995: Planta Med.61,126. 5. Tadolini B., Cabrini L.,1988;Biochem.J:253,931. 6. Ito, N., Hiroze, M., Fukushima, G., Tauda, H., Shira, T., & Tatematsu, M. (1986). Studies on antioxidant: their carcinogenic and modifying effects on chemical carcinogensis. Food and Chemical Toxicology, 24, 1071–1081. 7. Alexander, P., Ritsuko, M., Miechal, S., Bat-Sheva, C., Fostik-Magyar, C., & Dubinsley, Z. (1998). Natural antioxidatnt activity in some microalgal spices. International Journal of Plant Science, 46, 169–176. 8. Alexandra, B., Marie Elisabeth, C., Hubert, R., & Clendette, B. (1998). Antioxidant activity and phenolic composition of citrus peel and seed extract. Journal of Agricultural and Food Chemistry, 46, 2123–2129. 9. Zia-ur-Rehman, Citrus peel extract – A natural source of antioxidant, Food Chemistry 99 (2006) 450–454 10. S. Palma, S. D'Aquino, M. Agabbio, S. Schirru. Changes in flavonoids, ascorbic acid, polyphenol content and antioxidant activity in cold-stored ´fortune´ mandarin. ISHS Acta Horticulturae 682: V International Postharvest Symposium

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CRYSTAL STRUCTURE AND DFT STUDIES FOR SCAVENGING PROPERTIES OF RESVERATROL DERIVATIVES INCLUDING THE RESVERATROL METABOLITE PICEATANNOL Francesco Caruso1, Miriam Rossi2, Roberto Antonioletti1, Justin Salciccioli2 and Alessandra Pompili1 1

Institute for Biomolecular Chemistry of CNR, Rome, Italy c/o Department of Chemistry "Sapienza University" P.le A. Moro, 5 – 00185 Rome, Italy; 1Vassar College, Department of Chemistry, Poughkeepsie, NY 12604, USA. 2CNR, Istituto di Chimica Biomolecolare, Roma, Italy (E-mail: [email protected]) The "French paradox" was coined years ago to explain why the French had lower mortality from coronary heart disease, compared to those in the USA, despite a cholesterol-rich diet. It was claimed that the paradox is due to the high consumption of wine, especially red wine [1]. Resveratrol is present in the grape skin, seeds and wine; other sources include Polygonium cuspidatum (Japanese knotweed) from where it is extracted and used as an ingredient in traditional Asian medicine (“Kojo-kon”) as a circulatory tonic. Resveratrol synthesis is triggered by plant stress conditions such as fungal infection, UV irradiation, etc. Besides its beneficial role against heart disease, resveratrol has demonstrated anti-tumor [2] and longevity properties [3,4] that are probably related to its antioxidant activity. To understand the chemical mechanisms behind these biological activities, we study these compounds using Density Functional Theory (DFT) and single crystal structure analysis. We conclude that piceatannol is more effective than resveratrol as a scavenger of free radicals. Selected Publications 1. Das, D. K.; Maulik, N. Wine, alcohol, and a healthy heart. Oxidative Stress Disease (2006) 21, 47-62. 2. O'Brian, C. A.; Stewart, J. R.; Chu, F. Cancer chemoprotective activity of stilbenes: resveratrol. Nutrition Cancer Prevention (2006) 369-383. 3. Valenzano, D. R.; Terzibasi, E.; Genade, T.; Cattaneo, A.; Domenici, L.; Cellerino, A. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived Vertebrate. Current Biology (2006) 16, 296-300. 4. Hirofumi, A. Drink red wine and enjoy longevity! Kagaku Seibutsu (2004) 42, 114115.

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DEVELOPMENT OF NEW ANTIVIRAL CHEMOTHERAPEUTIC DRUGS Paolo La Colla, Roberta Loddo and Esther Marongiu Department of Biomedical Sciences and Technologies - Section of General Microbiology and Virology & Microbial Biotechnologies - University of Cagliari (Italy) (E-mail: [email protected]) Viral diseases are an increasing health concern. Worldwide, infectious diseases account for approximately 50% of all deaths in tropical countries. In industrialized nations, epidemics due to drug resistant pathogens and the emergence of hitherto unknown disease-causing agents, pose enormous public health concerns. Infectious disease mortality rates are actually increasing also in developed countries. The existing drugs are prevalently synthetic, expensive and limitedly accessible to developing countries. Worldwide, less than 10% of individuals affected by the most severe infectious diseases has access to the available therapies. For this reason, and because there is no cure yet for some viral infections with high mortality rate, the development of new, safe, effective and low-cost drugs is one of the global priorities. This makes urgent to find solutions at sustainable costs also for the developing countries. The search for new antiviral agents is also driven by the development of resistance to existing compounds, and by the menacing presence of naturally resistant pathogens. Considering that approximately 80% of people living in developing countries rely almost completely on traditional medicine practices for their primary health care, substances derived from higher plants should be taken into consideration and should become part of a potential complementary strategy against viral diseases. Mention will be made of the major results obtained by our group in the field of antiviral chemotherapy. Selected Publications 1.

Jeannot F, Gosselin G, Standring D, Bryant M, Sommadossi JP, Loi A.G., La Colla P, Mathe C,. Syntesis and studies of 3’-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base. Bioorg Med Chem. 2002 Oct; 10 (10):3153-61. 2. Griffon JF, Dunkhan D, Pierra C, Benzaria S, Loi A.G., La Colla P, Sommadossi JP, Gosselin G,. 4’-C-methyl-beta-D-ribofuranosyl purine and pyrimidine nucleosides revisited. Nucleosides Nucleotides Nucleid Acids. 2003 May-Aug;22(5-8):707-9. 3. Pierra C, Benzaria S, Dunkhan D, Loi A.G., La Colla P, Bridges E, Mao J, Standring D, Sommadossi JP, Gosselin G. Syntesis,phicochemical and pharmacokinetic studies of potential prodrugs of beta-L-2’-deoxycytidine, a selective and specific anti- HBV agent. Antivir Chem Chemother. 2004 Sep;15(5):269-79 4. Dukan D, Leroy F, Peyrronet J, Bosc E, Chaves D, Durka M, Storer R, La Colla P, Seela F, Gosselin G. Syntesis of 5-aza-7-deazaguanine nucleoside derivatives as potential anti-flavivirus agents. Nucleosides Nucleotides Nucleic Acid 2005, 24 (5-7):671-4. 5. Seela F, Lin W, Kazimierczuk Z, Rosemeyer H, Glaron V, Peng X, He Y, Ming X, Andrzejewska M, Gorska A, Zhang X, Eickmeier H, La Colla P. L-nucleosides containing modified nucleobases. Nucleosides Nucleotides Nucleic Acids. 24 (5-7): 859-63. 6. Szymanska A, Szymczak M, Boryski J, Stawinski J, Kraszewski A, Collu G, Sanna G, Giliberti G, Loddo R, La Colla P. Aryl nucleoside H-phosphonates. Part 15: Synthesis, properties and anti-HIV activity of aryl nucleoside 5’-alpha-hydroxyphosphonates. Bioorg Med Chem. Mar 15; 14 (6): 192434. 7. Al- Masoudi NA, Al-Masoudi IA, Ali IA, Al-Soud YA, Saeed B, La Colla P. Amino acid derivatives. Part I. Synthesis, antiviral and antitumor evaluation of new alpha-amino acid esters bearing coumarin side chain. Acta Pharm. 2006 Jun; 56 (2): 175-88. 8. Carta A, Loriga M, Piras S, Paglietti G, La Colla P, Busonera B, Collu G, Loddo R. Synthesis of variously substituted 3-phenoxymethyl quinoxalin-2-ones and quinoxalines capable to potenziate in vitro the antiproliferative activity of anticancer drugs in multi-drug resistano cell lines. Med Chem. 2006 Mar; 2(2): 113-22. 9. Aly Y. L. and Pedersen E. B., La Colla P. and Loddo R. Synthesis and anti-HIV-1 activity of 1,3phenylene bis-uracil analogues of MKC-442. J. Heterocyclic Chem. 2006. 10. Carta A, Loriga G, Piras S, Paglietti G, Ferrone M, Fermeglia M, Pricl S, La Colla P, Secci B, Collu G, Loddo R,.Synthesis and In Vitro Evaluation of the Anti-Viral Activity of N-[4-(1H(2H)benzotriazol-1(2)-Yl)phenyl]alkylcarboxamides. Medicinal Chemistry 2006, 2, 577-589.

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SYNTHESIS OF 4-HYDROXY-2-HEXENAL (HHE) AND DEVELOPMENT OF ANALYTICAL PROCEDURES FOR EVALUATION OF CELL AND FOOD DAMAGES BY GAS CHROMATOGRAPHY WITH ELECTRON CAPTURE DETECTION (GC-ECD) Enzo Santaniello1, Radmila Pavlovic1, Alessandro Repetto1, Cristian E. M. Bernardi2 and Pier Antonio Biondi2 1

Department of Medicine, Surgery and Dentistry - School of Medicine c/o S. Paolo Hospital and 2Department of Veterinary Sciences and Technologies for Food Safety, Faculty of Veterinary Medicine- University of Milan, Italy (E-mail: [email protected]) 4-Hydroxy-2E-hexenal (HHE) is a cytotoxic aldehyde produced by peroxidation of ω-3 fatty acids [1], that are specially abundant in fishes. HHE can be, therefore, an important biomarker for assessment of quality and safety of fishery products [2]. Quantitative determination of HHE can rely on a wide array of analytical methods, among which we have selected gas chromatography with electron capture detection (GC-ECD), less expensive than GC-MS technique and nevertheless characterized by high sensitivity, as previously shown for malonaldehyde (MA) analysis in bovine plasma [3]. We have prepared HHE-dimethylacetal by modification of an established synthesis [4] and HHE derivatives with dichlorophenyl hydrazines or pentafluorobenzylhydroxylamine. With these derivatives in hands as standards, GC-ECD analytical protocols have been set up. Preliminary data on quantitative determination of HHE levels in fish meat will be presented and discussed. Selected Publications 1. Van Kuijk, F.J.M. et al. Biochim. Biophys. Acta 1043, 116-118 (1990) 2. Surh, J. And Kwon, H. Food Additives and Contaminants, 22, 701-708 (2005) 3. Sangalli, L. et al. J. Chromatogr. B., 796, 201-207 (2003) 4. Santaniello, E. et al. Redox Report, 12, 55-58 (2007)

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METAL CURCUMIN COMPLEXES Aidan Benson1, Francesco Caruso2 and Miriam Rossi1 1

Vassar College, Department of Chemistry, Poughkeepsie, NY 12604, USA. 2CNR, Istituto di Chimica Biomolecolare, Roma, Italy)Institute for Biomolecular Chemistry of CNR, Rome, Italy c/o Department of Chemistry "Sapienza University" P.le A. Moro, 5 – 00185 Rome, Italy; (E-mail: [email protected]) The naturally occurring polyphenolic antioxidant curcumin is considered the primary active ingredient of the curry spice turmeric. It has been used both in curries and in eastern medicine as an anti-inflammatory and wound healing agent among many other medicinal uses. Recent studies of curcuminoids and metal-curcuminoid complexes have shown them to have promising anti-tumor activity [1]; Cu-curcumin is also a good scavenger for Alzheimer's disease [2]. The Cu(II)acetate, Zn(II)acetate, and Sn(IV)Cl2-curcumin metal complexes have been synthesized and a new [p-cymene]Ru(II)Cl-curcumin complex has been synthesized and characterized using single crystal X-ray diffraction. The complexes are insoluble in most solvents though a combination of NMR, IR and UV studies have allowed confirmation of the products as a single curcumin molecule coordinating through the β-diketone fragment to the metal atom in a bidentate fashion. Elemental analysis suggests the Sn(IV) has octahedral coordination with two curcumin molecules and two chlorine atoms. X-ray crystal structures of curcumin have also shown that the predominant tautomeric form of the molecule is the enol. Density Functional Theory studies have been also performed. Selected Publications 1. Anti- tumour studies of metal chelates of synthetic curcuminoids. John, V. D.; Kuttan, G.; Krishnankutty, K. J. Experiment. Clinical Cancer Res. (2002) 21, 219224. 2. A theoretical study on Cu(II)-chelating properties of curcumin and its implications for curcumin as a multipotent agent to combat Alzheimer's disease. Shen, L.; Zhang, H-Y; Ji, H-F. Theochem (2005), 757, 199-202

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(B) BIOLOGY STEADY-STATE RADIOLYSIS AS A TOOL FOR CARACTERISATION OF ANTIOXIDANT PROPERTIES Monique Gardès-Albert and Daniel Jore Laboratory of Physical Chemistry, UMR 8601 CNRS, Biomedical Research Center, University Paris Descartes, 45 rue des Saints-Pères, 75270 Paris cedex 06 (E-mail: [email protected]) Reactive Oxygen Species (ROS) are formed as the result of absorption of ionizing radiations by water. Irradiations of aqueous solutions with gamma rays (from 137Cs or 60Co) give hydroxyl and superoxide free radicals, together with hydrogen peroxide, which are produced with constant rates (i.e. constant radiolytic yields). Water-soluble antioxidants can be attacked by the ROS formed in the aqueous medium, particularly by hydroxyl free radicals which are the main oxidizing species. It can be noted that the radiolysis method allows to selectively study one type of free radical as ●OH alone or O2●- alone, in order to precise the scavenging capacity of the antioxidant against each of these ROS. Bio-mimetic membrane models such as linoleate micelles, liposomes and low density lipoproteins, have been characterized by their radiation dose-dependent lipid peroxidation. They allow evaluating the ability of antioxidants to scavenge peroxyl free radicals from the lipidic phase, thus inhibiting the chain-mechanism peroxidation. Selected Publications 1. I Jedidi, P. Thérond, S. Zarev, C. Cosson, M. Couturier, C. Massot, D. Jore, M. Gardès-Albert, A. Legrand et D. Bonnefont-Rousselot. Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine. Biochemistry, 42, 11356-11365 (2003). 2. D. Bonnefont-Rousselot, V. Guilloz, S. Lepage, C. Bizard, P. Duriez, D. Lesieur, J. Delattre, D. Jore, M. Gardès-Albert. -carotene in LDL oxidized by oxygen free radicals in the presence of supraphysiological concentrations of melatonin. Redox Report, 8, 2, 95-104 (2003). 3. F. Collin, H. Khoury, D. Bonnefont-Rousselot, P. Thérond, A. Legrand, D. Jore et M. Gardès-Albert. Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end-products of metformin in aqueous solutions. J. Mass Spectrom., 39, 890-902 (2004). 4. H. Khoury, F. Colin, D. Bonnefont-Rousselot, P. Thérond, A. Legrand, D. Jore et M. Gardès-Albert. Radical-induced oxidation of metformin. Eur. J. Biochem. (FEBS 2004), 271, 4745-4752 (2004). 5. H. Vitrac, C. Hauville, F. Collin, M. Couturier, P. Thérond, M. Delaforge, S. Rémita, D. Jore et M. Gardès-Albert. Hydroxide characterisation as a signature of the micelle/monomer balance in radiation-induced peroxidation of arachidonate. Free Radical Research, 39 (5): 519-528 (2005). 6. J. Mekhloufi, D. Bonnefont-Rousselot, S. Yous, D. Lesieur, M. Couturier, P. Thérond, A. Legrand, D. Jore and M. Gardès-Albert. Antioxidant activity of melatonin and a pinoline derivative on linoleate model system. J. Pineal Res., 39 : 27-33 (2005). 7. J. Mekhloufi, H. Vitrac, S. Yous, P. Duriez, D . Jore, M. Gardès-Albert, D. BonnefontRousselot. Quantification of the water/lipid affinity of melatonin and a pinoline derivative in lipid models. J. Pineal Res. 42 : 330-337(2007). 23

DIETARY ANTIOXIDANTS AND OXIDATIVE STRESS PREVENTION: THE EMERGING ROLE OF THE ANTIOXIDANT NETWORK Mauro Serafini, Monia Pecorari, Maria Francesca Testa and Debora Villano Valencia Antioxidant Research Laboratory at the Unit of Human Nutrition, INRAN, Via Ardeatina 546, 00178, Rome, Italy (E-mail: [email protected]) Oxidative stress-related diseases have a complex etiology with multiple risk factors that involve the interplay between genetic and environmental influences. There is compelling evidence that dietary plant foods appear to be protective against degenerative diseases. Among a number of mechanistic hypotheses, diet-derived antioxidants have been proposed to contribute to explain these findings. However, contrasting results from intervention trials have raised strong concerns about the influence of antioxidants on human health. A vulnerable point of the research on antioxidants is the lack of information on the effect of the whole array of dietary antioxidants in disease prevention, as so far mainly single molecules have been investigated (1). Total Antioxidant Capacity (TAC) considers the single antioxidant activity as well as the synergistic interactions of all the redox molecules present in complex matrixes (such as foods and body fluids), thus giving an insight into the assessment of the antioxidant network (2). At epidemiological level, the development of a TAC database for food has provided the tool for assessing dietary TAC intake in population studies (3). Results from recent dietary intervention trials in humans and from the epidemiological application of TAC, furnishing new insights to the “antioxidant hypothesis,” will be highlighted and critically discussed. Selected Publications 1. Serafini, M. Valencia D. V. Spera, G. Pellegrini, N. (2006) Redox molecules and cancer prevention: the importance of understanding the role of the antioxidant network. Nutrition and Cancer 56(2) 232-240. 2. Serafini M, Bugianesi R, Maiani M, Valtueňa S, De Santis S & Crozier A. 3. Plasma antioxidants from chocolate. Nature 2003: 424, 1013. 4. Serafini M., Bellocco R., Wolk A., & Ekström AM. Total Antioxidant Potential of Fruit and Vegetables and risk of gastric cancer. Gastroenterology 2002:123:985991.

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(AUTO)OXIDATION OF CATECHOLS: IMPLICATIONS TOWARDS ANTIOXIDANT OR PROOXIDANT FEATURES Antonio Rescigno, Giuseppe Mocci, Paolo Zucca and Enrico Sanjust Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Chimica Biologica e Biotecnologie Biochimiche, Università di Cagliari, 09042 Monserrato (CA), Italy (Email: [email protected]) Catechols are widespread among living organisms, where they exert a number of functions. Catechols could be further classified in structural families, often strongly differing in chemical and biological properties. Many simple catechols are readily autoxidizable, especially under alkaline conditions and in the presence of redox active metal ions; the oxidation pathway implies the preliminary quinonization followed by a nucleophilic attack by water or hydroxide, and fast oxidative quinonization of the intermediate trihydroxy derivative leading to a hydroxyquinone. The (neuro)toxic nature of such hydroxyquinones has been in some cases well ascertained. By contrast, abietanoid catechols found in rosemary (Rosmarinus officinalis) follow an alternative oxidation pathway where the trihydroxy derivative is directly obtained starting from the catechol without passing through the o-quinone. Some implications of both the pathways are discussed in the light of the very high antioxidant properties of rosemary abietanoids. Selected Publications 1. A hydroxyquinone with amine oxidase activity: preparation and properties. E. Sanjust, A.C. Rinaldi, A. Rescigno, M.C. Porcu, G. Alberti, A. Rinaldi, A. FinazziAgrò. Biochem. Biophys. Res. Commun. 208: 825-834 (1995) 2. Autoxidation of 4-methylcatechol: a model for the study of the biosynthesis of copper amine oxidases quinonoid cofactor. A. C. Rinaldi, M.C. Porcu, N. Curreli, A. Rescigno, A. Finazzi-Agrò, Z. Pedersen, A. Rinaldi, E. Sanjust. Biochem. Biophys. Res Commun. 214: 559-567 (1995) 3. Biosynthesis of the topaquinone cofactor in copper amine oxidases - Evidence from model studies. A.C. Rinaldi, M.C. Porcu, S. Oliva, N. Curreli, A. Rescigno, F. Sollai, A. Rinaldi, A. Finazzi-Agrò, E. Sanjust. Eur. J. Biochem. 251: 91-97 (1998) 4. Some aspects of tyrosine secondary metabolism. A. Rescigno, A. C. Rinaldi, E. Sanjust. Biochem. Pharmacol. 56: 1089-1096 (1998)

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ORGANIC ANTIOXIDANTS WITH CATALYTIC ACTIVITY: THE SURPRISING REACTION MECHANISM OF NATURAL AND SYNTHETIC POLYPHENOLIC COMPOUNDS Jens Z. Pedersen1, Cristina Oliveira2, Sandra Incerpi3, Ashok K. Prasad4, Virinder S. Parmar4 and Luciano Saso2 1

Department of Biology, University of Rome “Tor Vergata”, Italy; 2Department of Human Physiology and Pharmacology “Vittorio Erspamer”, Sapienza-University of Rome, Italy; 3Department of Biology, University of Rome “Roma Tre”, Italy; 4 Department of Chemistry, University of Delhi, India; (E-mail: [email protected]) Classic dihydroxyphenyl antioxidants are believed to scavenge free radicals by two consecutive single-electron reductions, resulting in two radicals eliminated for each quinonetype oxidation product formed. During a study on hydroxycoumarin compounds we found a much higher antioxidant:radical reaction stoichiometry when very low levels of antioxidants were used, typically below 1 micromolar. We here report that both synthetic dihydroxy-4methylcoumarins and natural flavonoids (e.g. quercetin, morin, myricetin or luteolin) can react with free radicals by a catalytic mechanism, in which each antioxidant molecule may eliminate several hundred radical molecules. The catalytic effect could be demonstrated also in a biological system, using a standard experimental cell culture model. These results represent a unique example of small organic molecules acting as catalysts, and explain why flavonoids may have physiological effects even at concentrations as low as 10 nanomolar. Selected Publications 1. Pedersen, J. Z., De Maria, F., Turella, P., Federici, G., Mattei, M., Fabrini, R., Dawood, K. F., Massimi, M., Caccuri, A. M. and Ricci, G. (2007) Glutathione transferases sequester toxic dinitrosyl-iron complexes in cells: a protection mechanism against excess nitric oxide. J. Biol. Chem. 282, 6364-6371. 2. Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z. and Ricci, G. (2007) Electrostatic association of glutathione transferase to the nuclear membrane. J. Biol. Chem. 282, 6372-6379. 3. Filomeni, G., Cerchiaro, G., Ferreira, A. M. D. C., De Martino, A., Pedersen, J. Z., Rotilio, G. and Ciriolo, M. R. (2007) Pro-apoptotic activity of novel isatin-Schiff base copper(II) complexes depends on oxidative stress induction and organelleselective damage. J. Biol. Chem. 282, 12010-12021.

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INVOLVEMENT OF PLASMA MEMBRANE REDOX SYSTEMS IN HORMONE ACTION Sandra Incerpi, Anna Maria Fiore, Paolo De Vitoa, Ashok K. Prasadb, Virinder S. Parmarb and Jens Z. Pedersena Department of Biology, University of Rome “Roma Tre”, Viale Marconi 446, 00146 Roma, Italy; and aDepartment of Biology, University of Rome “Tor Vergata”, Viale della Ricerca Scientifica 1, 00133 Roma, Italy; and bDepartment of Chemistry, University of Delhi, India. (E-mail: [email protected]) Oxidative stress is an imbalance between oxidant production and the antioxidant capacity of the cell to prevent oxidative injury; this condition is involved in a large number of human diseases: atherosclerosis, cancer, neurodegenerative diseases and aging. ROS are not only injurious by-products of cellular metabolism but small amounts of ROS act as messenger molecules in cell signal transduction pathways. The plasma membrane of eukaryotic cells in particular contains a variety of different ROS-producing oxidases and reductases, of which the best characterized are the superoxide-producing NADPH oxidases. These membrane redox activities can be modulated by hormones and growth factors, but the molecular mechanisms involved and the physiological significance of this phenomenon have only recently begun to be unveiled. The apparent paradox of ROS being essential biomolecules in the regulation of cellular functions, but also toxic by-products of metabolism, may be important for the pharmacological application of natural and synthetic antioxidants. Selected Publications 1. Incerpi S., Scapin S., D’Arezzo S., Spagnuolo S., Leoni S. Short-term effect of thyroid hormone in prenatal development and cell differentiation. Steroids, 70: 434443, 2005 2. Incerpi S. Editorial.Thyroid hormones: rapid reply by surface delivery only. Endocrinology 146: 2561 2563, 2005 3. Pallottini V., Martini C., Pascolini A., Cavallini G., Gori Z., Bergamini E., Incerpi S., Trentalance A. 3-Hydroxy-3-Methyglutaryl Coenzyme A Reductase Deregulation and age related Hypercholesterolemia: A new role for ROS. Mech Ageing Dev. 126: 845-851, 2005 4. Farias N.R., Fiore A.M., Pedersen J.Z., Incerpi S. Nongenomic actions of thyroid hormones: Focus on membrane transport systems. Immunol Endocrine Metab. Agents in Med Chem 6: 241-254, 2006 5. Incerpi S. Editorial. The extranuclear life of the nuclear receptor hormone family: New therapeutic possibilities. Immunol Endocrine Metab. Agents in Med Chem 6: 233-234, 2006 6. Incerpi S., Fiore A.M., De Vito P., Pedersen J.Z. Involvement of plasma membrane redox systems in hormone action. J. Pharm. Pharmacol. (Submitted 2007) 7. Pedersen J.Z., Oliveira C., Incerpi S., Kumar V., Prasad A.K., Malhotra S.V., Parmar V. S., Saso L. Antioxidant activity of 4-methyl-coumarins. J. Pharm. Pharmacol. (Submitted 2007)

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MINOR ANTIOXIDANT EFFECTS OF CAROTENOIDS IN BIRDS David Costantini Department of Animal and Human Biology, University La Sapienza, Roma, Italy (Email: [email protected]) Carotenoids are fat-soluble natural pigments with anti-oxidant properties. Birds are incapable of synthesizing carotenoids de novo, and must obtain them through their diet. Carotenoids are often used by birds in visual displays through deposition in skin or feathers. Given these multiple uses that all require substantial amounts for normal functioning, carotenoids have been suggested to be in limited supply for reproduction, health-related functions, or the expression of sexual coloration. It is known that maternally transferred carotenoids are useful anti-oxidants in developing embryos or hatchlings. However, recent studies on nestling or adult birds challenge previous emphasis for these compounds as important anti-oxidants, suggesting that the capability to cope with oxidative stress is not limited by carotenoid availability. In conclusion, empirical evidence for carotenoids being important anti-oxidants in birds is very weak at best. Selected Publications 1. Costantini D., 2006. Carotenoids and oxidative stress in a life-history perspective: a study on the kestrel (Falco tinnunculus). Ph.D. Thesis in Animal Biology, University La Sapienza, Roma. 2. Casagrande S., Costantini D., Fanfani A., Tagliavini J. and Dell’Omo G., 2007. Patterns of serum carotenoid accumulation and skin colour variation in nestling kestrels in relation to breeding conditions and different terms of carotenoid supplementation. Journal of Comparative Physiology B, 177: 237-245. 3. Costantini D., Fanfani A. and Dell’Omo G., 2007. Carotenoid availability does not limit the capability of nestling kestrels (Falco tinnunculus) to cope with oxidative stress. The Journal of Experimental Biology, 210: 1238-1244. 4. Costantini D., Coluzza C., Fanfani A. and Dell’Omo G., 2007. Effects of carotenoid supplementation on colour expression, oxidative stress and body mass in rehabilitated captive adult kestrels (Falco tinnunculus). Journal of Comparative Physiology B, in press.

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ANTI-INFLAMMATORY PROPERTY OF A NOVEL CINNAMIC ACID ESTER ISOLATED FROM PIPER LONGUM Sarvesh Kumara, Ashok K.. Prasadb, Virinder S. Parmarb and Balaram Ghosha a

Molecular Immunogenetics Laboratory, Institute of Genomics and Integrative Biology, Mall Road, Delhi-110 007, India; and bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India (E-mail: [email protected]) We recently identified and characterized a novel cinnamic acid ester from Piper longum which was found to be effective in blocking cytokine induced expression of cell adhesion molecules. When the ester and its derivatives were tested for their efficacies in inhibiting cell adhesion molecule expression, a thio analogue of the ester was found to be more potent. Interestingly, the thio analogue was also found to be effective in alleviating inflammatory conditions in experimental allergic model of asthma in mice. Attempts have also been made to elucidate its mechanism of action. Selected Publications 1. Kumar, S., Arya, P., Mukherjee, C., Singh, BK., Singh, N., Parmar, VS., Prasad, AK., and Ghosh, B. (2005) Novel Aromatic Ester from Piper longum and Its Analogs Inhibit Expression of Cell Adhesion Molecules on Endothelial cells. Biochemistry 44(48):15944-52. 2. Singh, N, Sarvesh Kumar, S., Prabhjot Singh P., Raj HG, Prasad AK, Virinder S. Parmar, VS and Ghosh, B (2007) Piper longum Linn. Extract Inhibits TNF Induced Expression of Cell Adhesion Molecules by Inhibiting NF-B Activation and Microsomal Lipid Peroxidation. Phytomedicine (In press).

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ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECT OF TYRPHOSTIN AG490 ON THE DEVELOPMENT OF ZYMOSAN-INDUCED GENERALIZED INFLAMMATION Petya Dimitrova and Nina Ivanovska Department of Immunology, Institute of Microbiology 1113 Sofia, Bulgaria (E-mail: [email protected]) An excessive inflammatory response is the major cause for the development of multiple organ dysfunction (MODS) as reactive oxygen species, acute phase proteins and cytokines playing a central role. Tyrphostins AG-126 and AG-556 have been successfully applied in models of LPS shock, sepsis and multiple sclerosis. Their action is related to suppression of TNF-, iNOs and COX-2 release. Tyrphostin AG-490 was applied in zymosan-induced MODS in Balb/c and SCID mice. Dependently on the regimen of treatment the substance improved the exit of inflammation and attenuated the development of organ failure. Tyrphostin inhibited TNF- production by peritoneal macrophages and lowered the level of acute phase mediators MIP-1 and RANTES. We observed in vitro suppression of NO production induced by LPS. Its effect depends on the presence of functional T and B lymphocytes. Our results support the view that AG-490, may be useful in the treatment of acute and chronic inflammation. Selected Publications M. Hristova, M. Yordanov and N. Ivanovska. Effect of fangchinoline in murine models of multiple organ dysfunction syndrome and septic shock. Inflammation Research, 2003, .52, 1-7.

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VASOACTIVE INTESTINAL PEPTIDE (VIP) INHIBITS THE PRODUCTION OF NO AND ACUTE PHASE PROTEINS IN ZYMOSAN-INDUCED GENERALIZED INFLAMMATION Nina Ivanovska and Petya Dimitrova Department of Immunology, Institute of Microbiology 1113 Sofia, Bulgaria (E-mail: [email protected]) Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide, secreted by the nervous and immune cells. VIP functions through binding to three receptors, VPAC1 (type 1 VIP receptor), VPAC2 (type 2 VIP receptor) and PAC1 (pituitary adenylate cyclaseactivating polypeptide receptor). Neuropeptide suppresses the production of proinflammatory mediators TNF-, IL-6, IL-12, chemokines and NO and stimulates the production of antiinflammatory cytokines such as IL-10. In the present experiments we have administered VIP in a model of zymosan-induced generalized inflammation (ZIGI). Its beneficial action was associated with reduced TNF- and increased IL-10 production, diminished level of RANTES and MIP-1 in peritoneal exudate and circulation. The neuropeptide inhibited NO release from stimulated peritoneal macrophages. Decreased spleen, liver and kidney enlargement and less pathological changes in liver were observed. The effect of VIP was attenuated by pretreatment with VIP antagonist (anti-VIP) before the induction of shock. Selected Publications Ivanovska Nina, Kalfin Reni, Lazarova Maria, Dimitrova Petya. Exogenous VIP limits zymosan-induced generalized inflammation (ZIGI) in mice. Immunology Letters, 2007, 110, 126-132.

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THE ROLE OF CALRETICULIN TRANSACETYLASE IN THE ACTIVATION OF NITRITE REDUCTASE BY POLYPHENOLIC PER ACETATES IN HUMAN PLATELETS: A PATHWAY FOR THE ENHANCEMENT OF NITRIC OXIDE LEVELS INDEPENDENT OF NITRIC OXIDE SYNTHASE Shvetambri Arora1, Prabhjot Singh1, Ajit Kumar1, Yogesh K. Tyagi1, Bilekere S. Dwarkanath3, Jawahar S. Adhikari3, Virinder S. Parmar2 and Hanumantharao G. Raj1 1

Department of Biochemistry, V. P. Chest Institute, 2Department of Chemistry, Bioorganic laboratory, University of Delhi, Delhi-110007, India. 3Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur, Delhi, India (E-mail: [email protected]) Nitric oxide (NO) is principally produced by the action of Nitric Oxide Synthase (NOS) on L-Arginine. Previous work of our laboratory projected polyphenolic acetates (PA) as the remarkable activator of cytochrome P450 reductase (CPR) and NOS by 7,8-Diacetoxy-4methyl coumarin (DAMC), a model PA . Later we elaborated the role of Calreticulin transacetylase (CRTAase) in mediating the acetylation of NOS by DAMC resulting in the enhanced intracellular levels of NO. There are several reports on the existence of nitrite reductase in mammalian cells. Several proteins such as CPR and CP exhibit nitrite reductase activity. The incubation of DAMC with platelets caused substantial elevation of NO levels produced by nitrite. L-NAME had no effect on DAMC mediated elevation of NO levels in platelet due to nitrite. These results strongly implicate the role of CRTAase in the activation of platelet nitrite reductase by DAMC. Selected Publications 1. Pulkit Khurana, Ranju Kumari, Parag Vohra, Ajit Kumar, Seema, Garima Gupta, Hanumantharao G. Raj, Bilikere S. Dwarakanath, Virinder S. Parmar, Daman Saluja, Mridula Bose, Anjana Vij, Nabo K. Choudhary, Jawahar S. Adhikari, Yogesh K. Tyagi and Ekta Kohl Acetoxy drug: Protein transacetylase catalyzed activation of human platelet nitric oxide synthase by polyphenolic Peracetates. Bioorganic & Medicinal Chemistry 14 (2006) 575-583. 2. Seema, Ranju Kumari, Garima Gupta, Daman Saluja, Ajit Kumar, Sanjay Goel, Yogesh K. Tyagi, Ruchika Gulati, Anjali Vinocha, Kambadoor M. Muralidhar, Bilikere S. Dwarakanath, Ramesh C. Rastogi, Virinder S. Parmar, Samakant A. Patkar and Hanumantharao G. Raj,. Characterization of Protein Transacetylase from Human Placenta as a Signaling Molecule Calreticulin using Polyphenolic Peracetates as Acetyl Donors. Cell Biochemistry & Biophysics 47 (2007) 53-64. 3. Seema Bansal, Marco Gaspari, Hanumantharao G. Raj, Ajit Kumar, Giovanni Cuda, Elwin Verheij,Yogesh K. Tyagi, Ramesh C. Rastogi, Prija Ponnan, Virinder S. Parmar Calreticulin transacetylase mediates the acetylation of nitric oxide synthase by polyphenolic acetate. Appl. Biochem. Biotech. (Accepted)

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INHIBITED NO RELEASE BY STIMULATED PERITONEAL MACROPHAGES CAUSED BY SOME ALKALOIDS Mimi Remichkova1, Luciano Saso2 and Stephan Philipov3 1

Department of Immunology, Institute of Microbiology 1113 Sofia, Bulgaria; Department of Human Physiology and Pharmacology “Vittorio Erspamer”, University of Rome “La Sapienza”, P. le Aldo Moro 5, 00185 Rome, Italy; 3Institute of Orgaqnic Chemistry with Center of Phytochemistry, 1113 Sofia, Bulgaria (E-mail: [email protected]) 2

Nitric oxide is a key mediator of nonspecific immunity, which with other reactive nitrogen intermediates prevent the spread of infections. In response to an immune challenge, macrophages become activated and produce proinflammatory mediators, as their overproduction can result in tissue injury and cellular death. Toll-like receptor (TLR) activation of human macrophages up-regulated the release of reactive nitrogen intermediates. We have investigated the influence of reserpine, rescinamin, aspidospermine and of six glaucine derivatives, on the NO production by murine peritoneal macrophages. Dose response effect was determined against zymosan (TLR2 ligand), LPS (TLR4 ligand) and CpG (TLR9 ligand). Rescinamine, aspidospermine, 7-methyl-dehydroglaucine at a concentration of 50 g/ml completely inhibited LPS-induced NO production. Similar effect was established for oxoglaucine, 7-methyl-dehydroglaucine and 7-benzoyl-dehydroglaucine in respect to zymosan stimulated NO release. An understanding of the underlying molecular mechanisms responsible for the altered NO production should serve as a foundation for novel drug development aimed at modulating macrophage activity. Selected Publications 1. Philipov S., Ivanovska N., Nikolova P. Glaucine analogues as inhibitors of mouse splenocyte activity. Pharmazie, 1998, 53, 694-698. 2. Ivanovska N., Hristova M., Philipov S. Immunosuppression and recovery of drugimpaired host resistance against Candidda albicans infection by oxoglaucine. Pharmacol. Res., 2000, 41(1), 99-105.

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CANAVANINE AND DALARGIN AS ANTIOXIDANTS Tamara Pajpanova1 and Adriana Bocheva2 1

Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; Dept. of Pathophysiology, Medical University, 1431 Sofia, Bulgaria (E-mail: [email protected]) 2

Oxidative stress has been considered to be a major cause of cellular injuries in a variety of chronic health problems, such as arteriosclerosis, carcinogenesis and neurodegenerative disorders. The brain appears to be more susceptible to oxidative damage than other organs. Therefore, the existence of antioxidants may be essential in brain protective systems. The antioxidative and free radical scavenging effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain and other tyrosine amino-terminal peptides, has been well documented. In addition oxidative stress is believed to initiate and aggravate peptic ulcers and gastric carcinoma. It was observed an increase in rat gastric mucosal lipid peroxidation (LPO) and superoxide dismutase (SOD) and a decrease in catalase (CAT) levels in cold restraint stressinduced gastric ulceration while, in clinical peptic ulceration and gastric carcinoma patients, an increase in serum LPO and a tendency to decrease in SOD and CAT levels were observed. The result thus, indicated a positive correlation between free radical-induced oxidative stress both in gastric and duodenal ulcers and gastric carcinoma. Dalargin (Tyr-D-Ala-Gly-Phe-Leu-Arg) is a synthetic analogue of [D-Ala2]-Leuenkephalin (DALA), which molecule is elongated with arginine. Dalargin is the first opioid peptide with proven ability to stimulate tissue regeneration without producing any side effects. It offers a great potency as a therapeutic agent stimulating tissue regeneration and repair mechanisms in cases of gastric ulcers. On the other hand, L-canavanine, a structural analogue of L-arginine, has shown to be a selective inhibitor of iNOS in vivo and in vitro, ), with possible usage in the treatment of alcohol induced brain damage and certain other neurological disorders. It is interesting that the supplement of one only C-terminal amino acid (Arg) of the DALAmolecule the leads to a considerable change of the biological activity of the peptide. That's why the potential ability of dalargin as medicine and the structural analogy "argininecanavanine" stimulated us to derive ([D-Ala2,Cav6]-Leu-enkephalin (Cav6-dalargin )and investigate its effects. In the present study, dalargin, Cav6-dalargin and Cys(O2NH2)2-Leu-enkephalin on experimental model of gastric ulcers induced by water-immersion stress in rats has been studied with the aim of exploring a possible correlation between its antioxidant and antiulcer activities.

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VITAMIN E TRAFCKING RELATED GENES ARE REGULATED BY EXOGENOUS OXIDANTS. DIFFERENCES BETWEEN YOUNG AND OLD Giuseppe Valacchi1, Vihas Vasu2, Wallace Yokoyama2, Ana Corbacho2, Anh Phung2, Carroll Cross2 and Paul Davis2 1

Department of Physiology, University of Siena, Italy; 2Internal Medicine, Pulmonary Division, University of California Davis, Davis, CA, USA (E-mail: [email protected]) The molecular mechanisms involved in maintaining Alpha-tocopherol (a-Toc) cellular levels remain ill-defined. Plasma a-Toc, mainly transported as HDL lipoproteins, is regulated by hepatic a-Toc transfer protein (TTP). ATP-binding cassette transporter 1 (ABCA1), a member of the ABC family, mediates transfer of a-Toc from the cell into HDL, therefore modulating tocopherol content of the cell and tocopherol transport between tissues. Scavenger receptor B1 (SRB1) one of large family of scavenger receptors mediates a-Toc transfer from HDL via apo A-1 mediated binding to the cells. We have examined the effects of aging and the environmental pollutants O3 and CS on aToc trafficking related genes in lung tissues after 4 days of exposure. a-Toc concentrations in old animals was higher (+18%) in plasma and and lower (-15%) in lung. Older animals lung tissue showed declines in TTP and SRB1 mRNA. O3 or CS induced declines in young and aged animals lung TTP and SRB1 mRNA (ABCA1 ABCA3 levels were unchanged). These findings represent several novel observations: identification of TTP in lung; decrease levels of TTP and SRB1 by both aging and environmental stressors. Selected Publications 1. Lung vitamin e transport processes are affected by both age and environmental oxidants in mice by Valacchi G, Vasu V, Phung A, Lim Y, Aung H., Corbacho A, Cross CE, and Davis PA., Toxicology and Applied Pharmacology, in press. 2. In vivo ozone exposure induces antioxidant/stress-related responses in murine lung and skin”. Valacchi G, Pagnin E, Corbacho AM, Olano E, Davis PA, Packer L, Cross CE. Free Radic Biol Med. 2004 Mar 1;36(5):673-81. 3. Enhanced inflammatory responses in alpha-tocopherol transfer protein null mice Schock BC, Van der Vliet A, Corbacho AM, Leonard SW, Finkelstein E, Valacchi G, Obermueller-Jevic U, Cross CE, Traber MG. Arch Biochem Biophys. 2004 Mar 1;423(1):162-9.

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INTRACELLULAR ANTIOXIDANT SYSTEMS IN ERYTHROCYTE DURING AGING IN HUMANS Syed I. Rizvi and Pawan K. Maurya Department of Biochemistry, University of Allahabad, Allahabad 211002, India (Email: [email protected]) The oxidative stress theory of aging offers the best mechanistic elucidation of aging phenomenon and other age related diseases. The susceptibility of an individual depends on the antioxidant status of the body. In humans, antioxidant system includes a number of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and nonenzymatic antioxidants such as glutathione (GSH), protein –SH, ascorbic acid, uric acid and dietary antioxidants. Antioxidant enzymes form the first line of defense against reactive oxygen species. We have previously shown that the plasma antioxidant potential in humans decreases as a function of age and that there are compensatory mechanisms operating in the body which are induced to maintain the antioxidant capacity during aging. We report the relationship between human aging and the activity of antioxidant enzymes. Our results show a significantly higher plasma SOD and CAT activity in older individuals than in younger individuals. We also provide evidence for an age-dependent decline in the influx of Lcysteine in human erythrocytes. Since the biosynthesis of glutathione in erythrocytes is dependent primarily on the availability of L-cysteine, our results may help to explain the decreased level of erythrocyte glutathione during human aging. Selected Publications 1. Rizvi, S. I. and Maurya, P. K. Markers of oxidative stress in erythrocytes during aging in humans. Ann. N.Y. Acd. Sci. 2007, 1100, 373-382 2. Rizvi, S. I., Jha, R. and Maurya, P. K. Erythrocyte plasma membrane redox system in human aging. Rejuvenation Research 2006, 9, 470-474.

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PREVENTION BY CALORIC RESTRICTION OF ROS-INDUCED HMG-COA REDUCTASE DEREGULATION DURING AGING Valentina Pallottini, Chiara Martini and Anna Trentalance Department of Biology, University of Rome “ROMA TRE”, Viale Marconi 446 00146-ROMA (E-mail: [email protected]) During the ageing process in rats, hypercholesterolemia occurs in concert with full activation, lowered degradation rate and an unchanged level of the hepatic rate limiting cholesterol biosynthesis enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) (1,2). Moreover, it has been observed that the age-related total activation of the HMGCoAR is due to a lack of regulation by phosphorylation/dephosphorylation and that such fully activation has been related to ROS increase but the responsible mechanism is still unknown (3,4). Some of those alterations are partially reduced by caloric restriction (1,5). So, aim of this work has been to study the mechanism underling age and ROS-related HMG-CoAR full activation and the effect of caloric restriction on these processes. The data show that ROS induce, through p38 and PP2A activation, HMG-CoAR deregulation and that caloric restrictions are able to prevent enzyme full activation by reducing ROS levels. Selected Publications 1. Marino M, Pallottini V, D'Eramo C, Cavallini G, Bergamini E, Trentalance A. Mech Ageing Dev. 2002 123:1183-1189. 2. Pallottini V, Montanari L, Cavallini G, Bergamini E, Gori Z, Trentalance A. Mech Ageing Dev. 2004 125:633-639. 3. Pallottini V, Martini C, Pascolini A, Cavallini G, Gori Z, Bergamini E, Incerpi S, Trentalance A. Mech Ageing Dev. 2005 126:845-851. 4. Pallottini V, Martini C, Bassi AM, Romano P, Nanni G, Trentalance A. J Hepatol. 2006 44:368-374. 5. Martini C, Pallottini V, Cavallini G, Donati A, Bergamini E, Trentalance A. J Cell Biochem. 2007 101:235-243.

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IN VITRO AND EX VIVO APPROACHES TO STUDY DIETARY ANTIOXIDANTS Fausta Natella and Cristina Scaccini National Research Institute for Food and Nutrition (INRAN), Rome, Italy (E-mail: [email protected]) The inverse relationship observed between the intake of dietary antioxidants and the risk of degenerative diseases has increased the interest on studying the antioxidants present in food. To be defined a dietary antioxidants a compound must: 1) be an antioxidant, 2) be significantly present in food (i.e. in commonly consumed food), 3) decrease the adverse effects of free radicals in vivo. Thus, the first step into verifying the efficiency of a dietary antioxidant is to establish its antioxidant activity. For this purpose many in vitro methods are available, using different approaches and complexity levels. As for its in vivo activity, a dietary antioxidant can be active from the outside (acting in the gastrointestinal tract) or from inside (in plasma, tissues and organs). Obviously, to exert an internal action the antioxidant must be bioavailable. Thus to study the activity of a dietary antioxidant, besides the mere antioxidant functions, metabolic fate and biological action must be also investigated. Selected Publications 1. Natella F., Nardini M., Di Felice M. and Scaccini C.. Benzoic and cinnamic acid derivatives as antioxidants: structure-activity relation. J Agric Food Chem 47 (4):1453-1459 (1999) 2. Ghiselli A., Natella F., Guidi A., Montanari L., Fantozzi P., and Scaccini C. Beer increases plasma antioxidant capacity in humans. J. Nutr. Biochem. 11:76-80 (2000). 3. Nardini, M., Cirillo E., Natella F., Mencarelli, D., Comisso A. and Scaccini C. Detection of bound phenolic acids: prevention by ascorbic acid and ethylenediaminetetraacetic acid of degradation of phenolic acids during alkaline hydrolysis. Food Chem., 79 (1): 119-124 (2002). 4. Natella F, Nardini M, Giannetti I, Dattilo C & Scaccini C. Coffee drinking influences plasma antioxidant capacity in humans. J. Agr. Food Chem 50(22), (2002). 5. Nardini, M; Cirillo, E.; Natella, F. and Scaccini, C. Absorption of phenolic acids in humans after coffee consumption. J. Agr. Food Chem. 50:5735-5741 (2002). 6. Nardini M., Natella F., Scaccini C. and Ghiselli A. Phenolic acids from beer are absorbed and extensively metabolized in humans. J. Nutr. Biochem. 17:14-22 (2006).

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ANTIOXIDANT CAPACITY AND NUTRACEUTICAL PROPERTIES OF CRUDE BOVINE MILK Gaetana A. Tonti, Francesca Benvenuti, Andrea Busettoa, and Ferdinando Mannello Institute of Histology and Laboratory Analysis, Faculty of Sciences and Technologies, University “Carlo Bo”, Via O. Ubaldini 7, 61029 Urbino (PU), Italy ; aAzienda Agricola Vicari-Busetto, Montecalvo in Foglia (PU), Italy (E-mail: [email protected]) Heating is commonly used for processing milk in the diary industry, which produces conformational changes in peculiar components encrypted in milk proteins and limiting their peculiar health usefulness (1). Crude milk contains components that exert different biologically active effects through encrypted peptides that may be released as potential modulators of various regulatory processes in the body and functioning as nutraceuticals (1, 2). Total antioxidant capacity of bovine crude milk was significantly higher respect to heattreated commercially available bovine milk (p