ABSTRACT BOOK
Savigliano (CN), 29-31 Maggio 2013
Comitato organizzatore
Università di Torino
Roberta Fruttero (Presidente - TO)
Massimo Bertinaria
Clara Cena
Antonella Di Stilo
Università del Piemonte Orientale
Giovanni Sorba (Presidente - PMN)
Marco Arlorio
Giorgio Grosa
Gian Cesare Tron
Sito Web Alberto Massarotti - Paolo Tosco
Comitato scientifico
Girolamo Cirrincione, Presidente, Università di Palermo
Stefano Alcaro, Università della Magna Grecia di Catanzaro
Pietro Campiglia, Università di Salerno
Federico Da Settimo, Università di Pisa
Roberta Fruttero, Università di Torino
Elias Maccioni, Università di Cagliari
Maurizio Recanatini, Università di Bologna
Giovanni Sorba, Università del Piemonte Orientale
Giampiero Spalluto, Università di Trieste
Patrocinio
Sponsor
Comune di Savigliano
Programma scientifico
Programma scientifico
29 Maggio 2013
15.30 - 17.30
Registrazione
Crosà Neira
Opening
18.00 - 18.30
Apertura lavori
18.30 - 19.30
Plenary lecture
Multitarget drugs: focus on the NO-donor hybrids
Alberto Gasco (Università di Torino)
19.30 - 20.15
Intermezzo musicale (Classico Terzetto Italiano)
20.30
Welcome buffet
30 Maggio 2013
Sala A
Antitumor agents
08.40 - 09.10
Main lecture
Polymer bioconjugates for selective tumour targeting
Paolo Caliceti (Università di Padova)
Short lectures
09.10 - 09.30
Design, synthesis and SARs of azolylmethyl-pyrroloquinolines as non steroidal aromatase
inhibitors
Davide Carta (Università di Padova)
09.30 - 09.50
Toward the design of new DNA G-quadruplex ligands through rational analysis of
polymorphism and binding data
Giosuè Costa (Università di Catanzaro)
09.50 - 10.10
Synthesis and antiproliferative activity of novel 13-arylalkyl berberine derivatives
Gaetano Fiorillo (Naxospharma srl)
10.10 - 10.30
Beyond the Pt(II)-based chemotherapy: Pt(IV) complexes as oral anticancer prodrugs
Elisabetta Gabano (Università del Piemonte Orientale)
10.30 - 10.50
Identification of novel pseudopeptidic inhibitors of human sirtuins 1-3 with antiproliferative
effect in cancer cells
Paolo Mellini (Università di Roma Sapienza, University of Eastern Finland)
Programma scientifico
10.50 - 11.20
Coffee break
Short lectures
11.20 - 11.40
Synthesis of a new water-soluble Re(I)-porphyrin conjugate as a future prospect for nuclear
medicine applications
Giuliana Mion (Università di Trieste)
11.40 - 12.00
Pt-based drugs and DNA adduct formation: the spotlight will be on proteins
Odra Pinato (Università di Padova)
12.00 - 12.20
2-Substituted-[1,3]thiazolo[4,5-e]isoindoles as kinases inhibiting compounds
Virginia Spanò (Università di Palermo)
12.20 - 12.40
Bone seeking MMP inhibitors for the treatment of tumor induced bone metastases
Marilena Tauro (Università di Bari)
12.40 - 13.00
Ni , Cu and Zn Salphen-like complexes: selective G-quadruplex stabilizers
Alessio Terenzi (Università di Palermo)
Sala B
Metabolic and inflammatory disease agents
09.40 - 10.10
Main lecture
An introduction to the initiation, propagation and resolution of inflammation
Adriano Rossi (University of Edinburgh)
II
II
II
Short lectures
10.10 - 10.30
New biphenylic derivatives as CB2 receptor selective ligands
Simone Bertini (Università di Pisa)
10.30 - 10.50
Identification of new potent TRPV1 ligands: design, synthesis and structure-activity
relationships of amides exhibiting an agonist pharmacological profile
Antonella Brizzi (Università di Siena)
10.50 - 11.20
Coffee break
Short lectures
11.20 - 11.40
Development of a novel class of derivatives as selective COX-2 inhibiting nitric oxide donors:
improving the solubility and the biological profile
Sara Consalvi (Università di Roma Sapienza)
11.40 - 12.00
Drug design, synthesis and biological evaluation of 3-substituted-1,5-diaryl-2-alkylpyrroles as
COX-2/CINODs endowed with anti-inflammatory and antiproliferative activity
Angela Di Capua (Università di Siena)
12.00 - 12.20
Ligand-based NMR screening methods applied to discovery of GRK2 Inhibitors
Antonio Limatola (Università di Napoli Federico II)
12.20 - 12.40
Design and synthesis of new multitarget drugs for autoimmune inflammatory diseases
Matteo Massa (Università di Genova)
12.40 - 13.00
Synthetic analogues of natural polyphenolic compounds as novel agents for the treatment of
atherosclerosis
Stefania Sartini (Università di Pisa)
13.00 - 15.00
Pranzo a buffet & Sessione poster
Programma scientifico
Sala A
Neurodegenerative disease agents
15.20 - 15.50
Main lecture
Advances in Alzheimer's disease drug discovery
Andrea Cavalli (Università di Bologna, IIT - Genova)
Short lectures
15.50 - 16.10
Exploring a new scaffold of 2-/3-/4-acetylpyridine derivatives as potent and selective human
MAO-B inhibitors
Simone Carradori (Università di Roma Sapienza)
16.10 - 16.30
Advantageous tools potentially useful to reduce withdrawal syndrome and associated
psychopathologies
Fabio Del Bello (Università di Camerino)
16.30 - 16.50
Drug design and synthesis of GSK-3β inhibitors
Valeria Famiglini (Università di Roma Sapienza)
16.50 - 17.20
Coffee break
Short lectures
17.20 - 17.40
The multi-target drug approach in Alzheimer's disease therapy
Antonella Federico (Università di Torino)
17.40 - 18.00
Novel fluorescent hA3 adenosine receptor antagonists
Stephanie Federico (Università di Trieste)
18.00 - 18.20
Design, synthesis and pharmacological properties of alkylbisammonio bitopic ligands of
muscarinic acetylcholine receptors
Carlo Matera (Università di Milano)
18.20 - 18.40
Discovery of a new, chemically stable class of 3-HAO inhibitors, endowed with
neuroprotective activity in vivo
Gian Paolo Vallerini (Università di Parma)
Sala B
Analytical methods in medicinal chemistry
15.00 - 15.30
Main lecture
Analytical challenges in drug discovery
Vincenza Andrisano (Università di Bologna)
Short lectures
15.30 - 15.50
Development of capillary bioreactors based on purine nucleoside phosphorylase from
Aeromonas hydrophila for biocatalytic applications
Giulia Cattaneo (Università di Pavia)
15.50 - 16.10
LC-ESI-MSn analyses in the identification of chemically reactive metabolites
Erika Del Grosso (Università del Piemonte Orientale)
16.10 - 16.30
Hydroxypyrazolo[1,5-a]pyridine as a fluorescent carboxylic acid isostere: probing first
MedChem bioisosteric applications
Alex Ducime (Università di Torino)
16.30 - 16.50
Monitoring GSM peptide phosphorylation by Glycogen Synthase Kinase-3β using
Electrospray Ionization Ion Trap Mass Spectrometry (ESI-IT-MS)
Annalisa D'Urzo (Università di Bologna)
16.50 - 17.20
Coffee break
Short lectures
Programma scientifico
17.20 - 17.40
Drug binding to albumins by high performance affinity chromatography and circular
dichroism
Cecilia Fortugno (Università di Bologna)
17.40 - 18.00
Mass spectrometric strategies for studying albumin covalent modifications induced by
xenobiotics and endogenous electrophilic cytotoxic compounds
Davide Garzon (Università di Milano)
18.00 - 18.20
Evaporative Light Scattering Detector C-650 - Advantages in flash chromatography
Simone Giordano (Büchi Italia srl)
18.20 - 18.40
Nexera method scouting: maximing efficiency for method development
Stefano Maria Lucini (Shimadzu Italia srl)
20.00
Cena sociale
31 Maggio 2013
Sala A
Nutraceutics
08.40 - 09.10
Main lecture
New perspectives in nutraceutics and functional foods
Vincenzo Fogliano (Università di Napoli Federico II)
Short lectures
09.10 - 09.30
Extraction, synthesis and biological activity evaluation of some new semisynthetic
polyphenols
Anna Baldisserotto (Università di Ferrara)
09.30 - 09.50
Quantification of "cholesterol lowering" soyasaponins in legumes and assessment of their
bioaccessibility
Giovanni Caprioli (Università di Camerino)
09.50 - 10.10
Nutraceutical properties of methyl 3-O-methyl gallate
Valeria Curti (Università di Pavia)
10.10 - 10.30
Phenolic compounds as inhibitors of the amyloid aggregation of Hen Egg White Lysozyme: a
multidisciplinary approach
Camilla Giuliani (Università di Firenze)
10.30 - 10.50
In-Cell Western Assay: a tool to study the hypocholesterolemic effects of potentially bioactive
food components at HepG2 cell line
Carmen Lammi (Università di Milano)
10.50 - 11.20
Coffee break
Short lectures
11.20 - 11.40
Clovamide: a new interesting antioxidant compound with potential nutraceutical properties
Monica Locatelli (Università del Piemonte Orientale)
11.40 - 12.00
Nutraceutics with potential preventive action against oral cavity pathologies
Dora Mascherpa (Università di Pavia)
12.00 - 12.20
Nutraceuticals and functional foods from turmeric: a useful tool in intestinal motility alteration
in mice with DSS induced colitis
Matteo Micucci (Università di Bologna)
12.20 - 12.40
In vitro cardioprotective potential of polyphenols from red grape juice (Vitis vinifera L. cv.
Aglianico N.): nutraceutical perspectives
Gian Carlo Tenore (Università di Napoli Federico II)
12.40 - 13.00
Functional foods for human health benefits: focus on lupin
Chiara Zanoni (Università di Milano)
Programma scientifico
Sala B
Antimicrobial agents
09.00 - 09.30
Main lecture
The quest for new antimicrobial agents: a global emergency
Stefano Biondi (Allecra Anti Infectives - Saint Louis, France)
Short lectures
09.30 - 09.50
New dual inhibitors of RT associated functions: investigation on their mechanism and mode
of action
Giulia Bianco (Università di Cagliari)
09.50 - 10.10
Design and synthesis of new inhibitors of human helicase DDX3 as antiviral agents
Annalaura Brai (Università di Siena)
10.10 - 10.30
Praziquantel analogues containing NO-donor furoxans and related furazans as agents active
against Schistosoma mansoni
Daniela Cortese (Università di Torino)
10.30 - 10.50
Development of a new scaffold of 1,3-thiazolidin-4-one derivatives as promising antifungal
agents with low cytotoxicity
Celeste De Monte (Università di Roma Sapienza)
10.50 - 11.20
Coffee break
Short lectures
11.20 - 11.40
Structural investigation of small molecules targeting influenza A virus polymerase
Jenny Desantis (Università di Perugia)
11.40 - 12.00
Development of novel rhodesain inhibitors characterized by the presence of the 3-bromo
isoxazoline moiety as the warhead
Roberta Ettari (Università di Milano)
12.00 - 12.20
Sulfonamidomethaneboronic acids as β-lactamase inhibitors: from fragment-based drug
design to in vivo activity
Chiara Romagnoli (Università di Modena e Reggio Emilia)
12.20 - 12.40
2-Phenylquinazolinone fragment imparts anti-CDKs and anti-HIV activities
Luca Sancineto (Università di Perugia)
12.40 - 13.00
Identification of HIV-1 nucleocapsid protein inhibitors by FRET technological platform
Marta Serena (Università di Padova)
13.00 - 13.30
Chiusura lavori
13.30 - 15.30
Pranzo a buffet
16.00 - 18.00
Visita ai luoghi storici della Città di Savigliano (su prenotazione)
1 Giugno 2013
09.30 - 15.00
Escursione a Monforte d'Alba con visita alla cantina Conterno Fantino
(su prenotazione)
Poster
Poster
Antitumor agents
P1.1
Design and synthesis of high affinity compounds for the Hsp60 expression control in carcinogenic
processes
1
1
1
2
1
1
1
Almerico A. M., Martorana A., Giacalone V., Mingoia F., Barone G., Terenzi A., Lauria A.
1
2
( Università di Palermo; CNR, Palermo)
P1.2
VEGFR-2 inhibitors. New perspectives from molecular modeling
1,2
1
1
2
2
2
1 1
Ballante F., Pirolli A., Patsilinakos A., Gadais C., Hesse S., Kirsch G., Ragno R. ( Università
2
di Roma Sapienza; Université de Lorraine, Metz, France)
P1.3
Unprecedented one-pot synthesis of pharmaceutical interest derivatives via in situ condensation of
heterocyclic iminium salts with nitrogen nucleophiles
Caneva C., Alfei S., De Maria M., Debenedetti S., Spallarossa A., Ranise A. (Università di Genova)
P1.4
Small-molecule enhancers of CARM1 catalyzed arginine methylation: identification of uracandolates
1
1
2
1
1
1
1
Cianciulli A., Castellano S., Spannhoff A., Milite C., Dal Piaz F., Cheng D., Tosco A., Viviano
1
3
1
4
4
5
6
2
M., Yamani A., Sala M., Cura V., Cavarelli J., Novellino E., Mai A., Bedford M.T., Sbardella
1 1
2
3
4
G. ( Università di Salerno; University of Texas, USA; Celon Pharma, Poland; Institut de
5
Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France; Università di Napoli Federico II;
6
Università di Roma Sapienza)
P1.5
Polymer functionalization with selected peptides aimed to nanoparticles preparation for tumour
targeting
1
2
2
1
1
1 1
Colzani B., Biagiotti M., Speranza G., Dorati R., Conti B., Genta I. ( Università di Pavia;
2
Università di Milano)
P1.6
Phenylpyrazolo[1,5-a]quinazolin-5(4H)-one: a suitable scaffold for the development of noncamptothecin Topoisomerase I (top1) inhibitors.
Pugliesi I.,1 Barresi E.,1 Taliani S.,1 Salerno S.,1 Marchand C.,2 Agama K.,2 Simorini F.,1 La Motta
1
1
2
3
1 1
2
C., Marini A. M., Pommier Y., Di Santo R., Da Settimo F. ( Università di Pisa; NCI, Bethesda,
3
Maryland, USA; Università di Roma Sapienza)
P1.7
Use of Microscale Thermophoresis (MST) for studying binding interactions of PRSet-7/SETD8 with
small molecule specific inhibitors EPI-9 and EPI-23
1
1
1
1
2
1
1 1
Feoli A., Castellano S., Milite C., Viviano M., Imhof A., Tosco A., Sbardella G. ( Università di
2
Salerno; Ludwig-Maximilians University of Munich, Germany)
P1.8
New N-alkanol-N-cyclohexanolamine aryl ester derivatives as reverting agents of Multidrug
Resistance (MDR)
1
1
1
1
1
2
1
Floriddia E., Orlandi F., Bellucci C., Manetti D., Romanelli M. N., Salerno M., Scapecchi S.,
1
1 1
2
Teodori E., Dei S. ( Università di Firenze; Université Paris 13, France)
P1.9
Synthesis and in vitro evaluation of tetrasubstituted pyridines as potential PI3Ks inhibitors
1
2
2
1
1 1
Galli U., Ciraolo E., Hirsch E., Sorba G., Tron G. C. ( Università del Piemonte Orientale;
2
Università di Torino)
P1.10
Biological evaluation of novel tamoxifen analogues
García Argáez A. N.,1 Christodoulou M. S.,2,3 Passarella D.,3 Iannoli M.,1 Gia O. M.,1 Dalla Via L.1
(1Università di Padova; 2University of Athens, Greece; 3Università di Milano)
P1.11
Human lactate dehydrogenase 5 (hLDH5) inhibitors based on N-hydroxyindole and malonic
scaffolds: a comparison of their anti-cancer activities
1
1
2
1
1
1
Granchi C., Paterni I., Calvaresi E. C., Tuccinardi T., Macchia M., Martinelli A., Hergenrother P.
2
1 1
2
J., Minutolo F. ( Università di Pisa; University of Illinois, Illinois, USA)
P1.12
Structural analysis of G-rich oncogenic promoter sequences
Greco M. L.,1 Folini M.,2 Sissi C.1 (1Università di Padova; 2IRCCS Istituto Nazionale dei Tumori,
Milano)
Poster
Antitumor agents
P1.13
New potent and selective P-gp inhibitors
1
1
2
2
2
1
1
Guglielmo S., Bonomo S., Colabufo N. A., Contino M., Perrone M. G., Fruttero R., Gasco A.
1
2
( Università di Torino; Università di Bari)
P1.14
1,2,4-Triazole derivatives as ATP competitive tyrosine kinase RET inhibitors: novel drug candidates
for the treatment of thyroid carcinoma
1
1
2
2
2
2
1
Coviello V., Sartini S., La Pietra V., Marinelli L., Cosconati S., Di Maio D., Marini A. M.,
1
1
1
1
2
1 1
Simorini F., Taliani S., Salerno S., Da Settimo F., Novellino E., La Motta C. ( Università di Pisa;
2
Università di Napoli Federico II)
P1.15
Design, synthesis and biological activity evaluation of c-myc proto-oncogene binders as potential
antitumor agents
1
1
2
2
1
1
3
3
Meleddu R., Distinto S., Sissi C., Musetti C., Arridu A., Bianco G., Parrotta L., Ortuso F.,
3
1 1
2
3
Alcaro S., Maccioni E. ( Università di Cagliari; Università di Padova; Università di Catanzaro)
P1.16
Synthesis, structure-activity relationships and stereochemical investigations on new tricyclic
pyridazinone derivatives as potential STAT3 inhibitors
1
1
1
1
1
1
1
Meneghetti F., Masciocchi D., Gelain A., Pedretti A., Celentano G., Barlocco D., Porta F.,
2
2
3
4
1 1
2
Legnani L., Toma L., Kwon B.-M., Asai A., Villa S. ( Università di Milano; Università di Pavia;
3
University of Science and Technology, Daejeon, Republic of Korea; 4University of Shizuoka, Japan)
P1.17
From anacardic acid to LoCAMs: development of a chemical tool for tuning histone acetylation
Milite C.,1 Castellano S.,1 Tosco A.,1 Feoli A.,1 Mai A.,2 Sbardella G.1 (1Università di Salerno;
2
Università di Roma Sapienza)
P1.18
Synthesis of pyrazolo[3,4-d]pyrimidines active on Bcr-Abl T315I mutation
Musumeci F.,1 Desogus A.,1 Brullo C.,1 Tintori C.,2 Fallacara A.,2 Maga G.,3 Crespan E.,3 Radi M.,4
Schenone S.1 (1Università di Genova; 2Università di Siena; 3IGM-CNR, Pavia; 4Università di Parma)
P1.19
Preparation and characterization of liposomes containing a new anthracycline derivative
Pedrini I., Gazzano E., Arpicco S., Riganti C., Rolando B., Chegaev K., Fruttero R., Gasco A.
(Università di Torino)
P1.20
Highly potent cancer agents by modulating the C-2 group of the arylthioindole class of tubulin
polymerization inhibitors
1
2
2
2
2
3
4
Pelliccia S., La Regina G., Coluccia A., Famiglini V., Reggio A., Brancale A., Hamel E.,
1
2 1
2
3
Novellino E., Silvestri R. ( Università di Napoli Federico II; Università di Roma Sapienza; Cardiff
University, UK; 4NCI, Frederick, USA)
P1.21
Targeting drug resistance in ovarian cancer: design and development of peptide based inhibitor
Santucci M.,1 Pelà M.,2 Saxena P.,1 Luciani R.,1 Ferrari S.,1 Marverti G.,1 Marraccini C.,1 Martello A.,1
D'Arca D.,1 Ponterini G.,1 Guerrini R.,2 Costi M. P.1 (1Università di Modena e Reggio-Emilia;
2
Università di Ferrara)
P1.22
Indole-based modulators of p53/MDM2 interaction as antitumoral agents
Barresi E.,1 Pugliesi I.,1 Taliani S.,1 Da Settimo F.,1 La Pietra V.,2 Marinelli L.,2 Novellino E.,2 Daniele
1
1
1
1 1
2
S., Da Pozzo E., Costa B., Martini C. ( Università di Pisa; Università di Napoli Federico II)
P1.23
Synthesis and biological evaluation of Δ2-isoxazoline derivatives as DNA methyl-transferase 1
(DNMT1) inhibitors
1
1
2
2
3
4
5
Viviano M., Castellano S., Conti P., Tamborini L., Kuck D., Novellino E., Medina-Franco J. L.,
3
1 1
2
3
Lyko F., Sbardella G. ( Università di Salerno; Università di Milano; Deutsches
4
5
Krebsforschungzentrum, Germany; Università di Napoli Federico II; Torrey Pines Institute for
Molecular Studies, Florida, USA)
P1.24
Identification of a new series of non-covalent proteasome inhibitors with selectivity for β5-subunits
Scarbaci K.,1 Troiano V.,1 Micale N.,1 Ettari R.,2 Lavecchia A.,3 Di Giovanni C.,3 Grasso S.,1
Novellino E.,3 Schirmeister T.,4 Zappalà M.1 (1Università di Messina; 2Università di Milano;
3
Università di Napoli Federico II; 4University of Mainz, Germany)
P1.25
Synthesis and structure-activity relationships of bis-3-chloropiperidines as bifunctional DNA
alkylating agents
Zuravka I.,1 Göttlich R.,1 Gatto B.2 (1University of Giessen, Germany; 2Università di Padova)
Poster
Metabolic and inflammatory disease agents
P2.1
N-alkyl dien- and trienamides with binding affinity for opioid and cannabinoid receptors
Anzani N.,1 Ruiu S.,2 Orrù A.,2 Floris C.,1 Caboni P.,1 Cottiglia F.1 (1Università di Cagliari; 2Istituto di
Farmacologia Traslazionale, Cagliari)
P2.2
Synthesis of 3β,7α-Dihydroxy-Δ -cholenoic acid derivatives: 'atypical' bile acid metabolites and
useful biomarkers to detect 3β-HSDH deficiency
1
1
2
1 1
2
Gioiello A., Cerra B., Setchell K. D., Pellicciari R. ( Università di Perugia; Cincinnati Children's
Hospital Medical Center, Ohio, USA)
P2.3
Synthesis and cycloxygenase inhibitory activity of new naphthalene-methylsulfonamido and
naphthalene-methylsulfonyl compounds
Nencetti S., Ciccone L., Rossello A., Nuti E., Orlandini E. (Università di Pisa)
P2.4
Novel tools for the elucidation of physio-pathological role of H2S
Corvino A., Severino B., Vellecco V., Bucci M. R., Cirino G., Santagada V., Caliendo G., Frecentese
F. (Università di Napoli Federico II)
P2.5
[1,2,4]Triazolo[1,5-c]pyrimidines as new potent human A3 adenosine receptor antagonists
1
1
2
3
4
2 1
Spalluto G., Federico S., Ciancetta A., Cacciari B., Klotz K. N., Moro S. ( Università di Trieste;
2
3
4
Università di Padova; Università di Ferrara; University of Würzburg, Germany)
P2.6
Synthesis and preliminary evaluation of model compounds targeting the NLRP3 inflammasome
pathways
Garella D., Cocco M., Di Stilo A., Miglio G., Collino M., Fantozzi R., Bertinaria M. (Università di
Torino)
P2.7
Structure-activity relationship, biological and conformational studies of derivatives of the pepducin
S1P3 agonist KRX-725
1
1
1
1
2
1
1
Incisivo G. M., Perissutti E., Bertolino A., Di Vaio P., Benfenati E., Santagada V., Caliendo G.,
1
1 1
2
Roviezzo F., Severino B. ( Università di Napoli Federico II; Istituto di Ricerche Farmacologiche
Mario Negri, Milano)
P2.8
Dimeric analogs of the dual PPARα/γ agonist LT-175: design, synthesis and biological evaluation
Laghezza A., Piemontese L., Sblano S., Carrieri A., Fracchiolla G., Carbonara G., Tortorella P.,
Loiodice F. (Università di Bari)
P2.9
Solid state interconversion of drug-triacylglycerol systems
Foglio Bonda A., Mannina P., Giovannelli L., Segale L., Pattarino F. (Università del Piemonte
Orientale)
P2.10
Molecular docking of biaryl tetrazolyl ureas as inhibitors of endocannabinoid metabolism
Pirolli A., Ballante F., Patsilinakos A., Ortar G., Morera E., Ragno R. (Università di Roma Sapienza)
P2.11
H2S-releasing properties of new thioamide derivatives
Pugliesi I., Barresi E., Taliani S., Da Settimo F., Martelli A., Testai L., Calderone V. (Università di
Pisa)
P2.12
From agonist to antagonist profile in LP1 analogues through the introduction of different Nsubstituents in benzomorphan-based ligands
Ronsisvalle S., Turnaturi R., Prezzavento O., Arena E., Calò G., Camarda V., Ronsisvalle G.,
Pasquinucci L. (Università di Catania)
5
Poster
Neurodegenerative disease agents
P3.1
Radiosynthesis of the TSPO 18 kDa PET radiotracer [18F](R)-PK14105
1,2
1
2
2
2
1 1
Barresi E., Siméon F. G., Taliani S., Pugliesi I., Da Settimo F., Pike V. W. ( NIH, Bethesda,
2
Maryland, USA; Università di Pisa)
P3.2
New PDE4D selective inhibitors able to restore memory function
Brullo C., Rotolo C., Rocca M., Massa M., Bruno O. (Università di Genova)
P3.3
Hydroxypyrazolo[1,5-a]pyridine as a fluorescent carboxylic acid isostere: introducing GABAA
receptor system affinities in the scaffold
1
2
1
2
1 1
2
Ducime A., Nielsen B., Boschi D., Frølund B., Lolli M. L. ( Università di Torino; University of
Copenhagen)
P3.4
A new series of 5-HT1A, 5-HT2A and 5-HT2C receptor ligands containing an isonicotinic nucleus
1
1
1
2
1
1
1
Magli E., Perissutti E., Ciano A., Massarelli P., Capasso R., Santagada V., Caliendo G.,
1 1
2
Fiorino F. ( Università di Napoli Federico II; Università di Siena)
P3.5
Hydroxypyrazolol[1,5-a]pyridine as a fluorescent carboxylic acid isostere: development of new
fluorescent 4-PHP analogues
1
2
1
2
1 1
2
Sainas S., Nielsen B., Boschi D., Frølund B., Lolli M. L. ( Università di Torino; University of
Copenhagen)
P3.6
New heterocyclic derivatives of benzylpiperidin-4-yl methyl and benzyl(methyl)aminobutyl sequences
as sigma ligands
Zampieri D., Laurini E., Sveronis A., Vio L., Mamolo M. G. (1Università di Trieste)
Analytical methods in medicinal chemistry
P4.1
Assessment of a HPLC assay for monoacylglycerol lipase activity
Del Carlo S.,1 Arena C.,1 Chicca A.,2 Bertini S.,1 Chidini S.,1 Manera C.,1 Saccomanni G.,1 Gertsch
J.,2 Macchia M.1 (1Università di Pisa; 2University of Bern)
P4.2
HPTLC-AMD and HPLC Characterization of the saccharides fraction of the novel food Adansonia
digitata (Baobab) fruit pulp
1
1
1,2
1,2 1
2
De Lucia D., Bernardi T., Vertuani S. Manfredini S. ( Università di Ferrara; Ambrosialab,
Ferrara)
P4.3
Determination of the pyruvic acid content of three italian onion varieties
Scorzoni S., Marinozzi M., Ianni F., Sardella R., Natalini B. (1Università di Perugia)
P4.4
New biotinilated spacers for studying the interaction of ELAV mimicking peptides with the target
mRNA
Tripodo G., Maggi L., Amadio M. L., Pascale A., Marra A., Rossi D., Collina, S. (Università di Pavia)
Nutraceutics
P5.1
An approach towards the total synthesis of the Yunnaneic acids
Botta L., Griffith D. R., Snyder S. A., (Columbia University, New York, USA)
P5.2
Extracts of cocoa and chocolate a comparative valuation of functional efficacy
Vertuani S.,1,2 Scalambra E.,1 Trotta V.,1 Malisardi G.,1 Baldisserotto A.,1 Manfredini S.,1,2
(1Università di Ferrara; 2Ambrosialab, Ferrara)
P5.3
Novel nutraceuticals from etnopharmacy: Nigella sativa seeds essential oils against recurrent
bladder infections
1
1,4
2
1,4
4
2
3 1
Haloci E., Manfredini S., Toska V., Vertuani S., Ziosi P., Topi I., Kolani H. ( Università di
2
3
4
Ferrara; Tirana University, Albania; QSUT Nene Tereza, Tirana, Albania; Ambrosialab, Ferrara)
Poster
Antimicrobial agents
P6.1
Computer-aided identification of fragments as CDK9 inhibitors with anti-HIV-1 Tat-mediated
transcription activity
Iraci N., Sancineto L., Barreca M. L., Massari S., Sabatini S., Manfroni G., Cecchetti V., Tabarrini O.
(1Università di Perugia)
P6.2
Development of novel anti-HIV agents acting as non-nucleoside reverse trascriptase inhibitors
1
1
1
2
1
1 1
Lo Surdo G., Ferro S., De Luca L., Pannecouque C., Chimirri A., Monforte A. M. ( Università di
2
Messina; University of Leuven, Belgium)
P6.3
Synthesis of new Plasmodium Falciparum dihydroorotate dehydrogenase inhibitors through the
application of an isosteric portfolio
1
1
1
2
3
2
2
Pippione A. C., Federico A., Boschi D., Nilsson U. J., Fritzson I., Sundin A. P., Al-Karadaghi S.
1 1
2
3
Lolli M. L. ( Università di Torino; University of Lund, Sweden; Active Biotech AB, Lund, Sweden)
P6.4
Enzymatic PEGylation of oligonucleotides
Sosic A., Pasqualin M., Pasut G., Gatto B. (Università di Padova)
P6.5
Structure-Activity Relationship of zosteric acid analogues as inhibitors of biofilm formation by
Escherichia coli
Villa S., Dell'Orto S., Gelain A., Villa F., Cattò C., Cappitelli F. (Università di Milano)
ABSTRACT DELLE
O RAL COMMUNICATION
Plenary lecture
Multi-Target Drugs: Focus on NO-Donor Hybrids
Alberto Gasco
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino
Via Pietro Giuria 9, 10125, Torino, Italy
[email protected]
Multi-target (or multifunctional) drugs are single compounds which are able to modulate
simultaneously more than one receptor. They are an alternative to the use of mixtures
of single-target drugs in the treatment of complex diseases. The nitric oxide(NO)-donor
hybrids are an important class of such products. They are designed in order to combine
therapeutic properties of established drugs with NO-dependent physiological actions.
These hybrids are generally obtained by joining or partly overlapping a lead drug, or
crucial part of it, with/to appropriate NO-donors including nitrooxy and nitrosothiol
derivatives, diazeniumdiolates and furoxan (1,2,5-oxadiazole 2-oxide) systems bearing
various substituents The latter systems represent a particularly flexible tool to obtain a
finely-tuned “balance” of the final hybrids. NO-aspirins (NO-ASA) are an example of
NO-hybrids which have been object of particular attention. Examples of such products
taken from literature and from the author’s own laboratory are analysed and discussed.
Antitumor agents
Main lecture M1
Polymer bioconjugates for selective tumour targeting
Paolo Caliceti
Department of Pharmaceutical and Pharmacological Sciences, University of Padua –
Via F. Marzolo 5 – 35131 Padova, Italy
[email protected]
Over the past years, multifunctional nanotechnology has emerged as a novel approach
to create new systems with optimized therapeutic performance. As a result, last
generation delivery systems are capable of complex functions, which enable sequential
overcoming of multiple biobarriers following a certain time/site determined “logic” of
events. These nanocarriers provide longer drug circulation times, higher tolerability,
and site specific delivery; factors that result in better patient outcomes. Cancer
represents the field of medicine application to which multifunctional nanotechnology
made the most prominent contributions.
Natural and synthetic polymers are landmark materials for production of novel smart
nanomedicines. Multivalent, amphiphilic and stimuli responsive polymers have been
exploited to produce drug bioconjugates and self-assembling colloidal systems or to
bestow peculiar physicochemical and biological properties on inert colloidal scaffolds.
Polysaccharides have been investigated for drug bioconjugation as the therapeutic
properties of anticancer drugs can be combined with the biological properties of these
polymers. Tumour targeting and selective anti-cancer drug delivery can in fact be
accomplished by receptor-mediated uptake or passive fluid-phase endocytosis of
polysaccharide prodrugs while polysaccharides may be exploited for tumour cell
targeting or to inhibit tumor angiogenesis and metastasis.
Bioconjugates obtained by pullulan derivatization with doxorubicin via hydrazone bond
have been found to possess suitable physicochemical properties for passive tumor
targeting via EPR, prolonged body exposure and pH dependent drug release. The
introduction of folic acid was found to bestow active targeting properties. A comparative
study performed using folated and unfolated doxorubicin loaded liposmes and pullulan
showed that both folated systems were rapidly and selectively internalizated into FR(+)
human cervical cancer HeLa cells and inhibited the proliferation of FR(+) KB human
nasopharyngeal carcinoma cells and of human umbilical vein endothelial cells
(HUVEC). Treatment of KB-bearing mice with both delivery systems showed simiilar
anticancer activity of PLD and PLD-FA, both equally efficient in reducing the tumor
size, while the Pull-PEG-FA-Dox conjugate displayed enhanced activity compared to
the non-targeted Pull-PEG-Dox. This study highlighted the effect of the structural
composition and architecture of the colloidal systems.
A different approach of tumour targeting involves the exploitation of the environmental
features of the cancer tissue. Stimuli sensitive polyacrylates represent unique
functional modules for drug delivery as they can be used to produce assemblies,
namely micelles and polymersomes, for selective drug release or to trigger the surface
properties of inert surfaces, namely gold nanoparticles.
Gold nanoparticles decorated with stimuli sensitive polymers have been found to gain
switchable properties: particle aggregation and cell interaction and internalisation. The
combination of the polymer decorated nanoparticles with targeting agents has been
found to bestow switchable recognition properties on the colloidal system that can be
exploited for surface recognition or cell targeting and may be used for theragnostic
applications.
Antitumor agents
Short lecture S1.1
Design, synthesis and SARs of azolylmethyl-pyrroloquinolines as non steroidal
aromatase inhibitors.
D. Carta, M. G. Ferlin†, R. Bortolozzi‡, R. Ghodsi¥, A. Chimento±, V. Pezzi±, S. Moro†,
G. Basso‡, G. Viola‡
†
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy
Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad/Iran
±
Department of Pharmaco-Biology, University of Calabria, Arcavata, Italy
‡
Department of Woman and Child health, Laboratory of Oncohematology University of Padova,
Padova, Italy
¥
[email protected]
Nowadays, aromatase CYP19 competitive inhibitors (AI) are the first choice as
adjuvant therapeutics for postmenopausal breast cancer patients. However, aimed to
overcome the little specificity and the resistance drawback of the clinically used
inhibitors such as Letrozole, the search for potent and selective AIs still remains an
attractive subject. Our design strategy consisted in combining the angular tricyclic
pyrroloquinoline core from our antimitotic phenyl-pyrroloquinolinone derivatives
(PPyQs) and in particular from 2-PPyQs endowed with a certain aromatase inhibitory
activity1, with a determinant structural element derived from non-steroidal CYP19
inhibitors of the third generation: the azole ring. 2-PPyQs were chemically modified by
introducing an imidazolylmethyl or triazolylmethyl group at 4 position (Figure 1). By this
way, the proposed compounds are endowed with an aza-ring for a strong interaction
with the heme iron atom of CYP192 and the bulky tricycle structure having a geometry
similar to that of the natural substrate (Figure 1). Their inhibitory activity was evaluated
both by an enzymatic HTS kit and the tritiated water release assay in H295R cells. The
results showed that, among the synthesized compounds, imidazol derivatives (11, 13,
14 and 21) and the triazolylmethyl derivative 22 exhibited an inhibitory potency against
aromatase comparable to Letrozole chosen as reference compound. The molecular
modelling study along with the predict pharmacokinetics profiles of compounds
demonstrated that the pyrroloquinoline scaffold represents a starting point for the
development of new pyrroloquinoline-based aromatase inhibitors.
X
O
A
C
O
D
N
N
A
B
C
HN D
B
N
R
Figure 1. Structural similarities between the aromatase substrate androstenedione and the
proposed inhibitors 4-azolylmethyl-PQs.
References
1. Ferlin, M. G.; Chiarelotto, G.; Gasparotto, V.; Dalla Via, L.; Pezzi, V.; Barzon, L.; Palu, G.;
Castagliuolo, I. Synthesis and in vitro and in vivo antitumor activity of 2phenylpyrroloquinolin-4-ones. J. Med. Chem. 2005, 48, 3417-3427.
2. Chumsri, S.; Howes, T.; Bao, T.; Sabnis, G.; Brodie, A. Aromatase, aromatase inhibitors,
and breast cancer. J. Steroid Biochem. Mol. Biol. 2011, 125, 13-22.
Antitumor agents
Short lecture S1.2
Toward the design of new DNA G-quadruplex ligands through rational analysis
of polymorphism and binding data
Artese A.a, Costa G.a, Distinto S.b, Moraca F.a, Ortuso F.a, Parrotta L.a and Alcaro S.a
a
Dipartimento di Scienze della Salute, Università degli Studi “Magna Græcia”, Campus “S.
Venuta”, Viale Europa, Germaneto, 88100, Catanzaro, Italy.
b
Dipartimento di Scienze della Vita e dell'Ambiente, Università degli Studi di Cagliari, Via
Ospedale 72, 09124, Cagliari, Italy.
[email protected]
Human telomeres play a key role in protecting chromosomal ends from fusion events;
they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form
G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and
are involved in several biological processes. The central cation has a crucial role in
maintaining the stability of the structure. Based on its nature, it can be associated with
different topological telomeric quadruplexes, which depend also on the orientation of
the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism,
confirmed by the different structures deposited in the Protein Data Bank (PDB)1,
prompted us to apply a computational protocol in order to investigate the
conformational properties of a set of known G-quadruplex ligands and their molecular
recognition against six different experimental models of the human telomeric sequence
d[AG3(T2AG3)3].
chemically diverse
G-quadruplex
ligands
from nano to micro M
TRAP data pIC50
Experimental
Theoretical
Docking
Free Energy evaluation
G-quadruplex
d[AG3(T2AG3)3]
PDB models
NMR, X-ray and hybrid folds
Comparison
Figure 1. Summary of the experimental vs theoretical flow chart adopted in the selection of
ligand, target and computational models
A good linear correlation between theoretical and experimental data was observed, in
particular with the parallel fold. Among the studied binders, a trisubstituted acridine and
a dibenzophenanthroline derivative were well recognized by the parallel and the mixed
G-quadruplex structures, allowing the identification of specific key contacts with DNA
and the further design of more potent or target specific G-quadruplex ligands.
References
1. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G. ; Bhat, T.N.; Weissig, H.; Shindyalov, I.
N.; Bourne, P. E. The Protein Data Bank. Nucleic acids research 2000, 28, 235-242.
Antitumor agents
Short lecture S1.3
SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY OF NOVEL 13-ARYLALKYL
BERBERINE DERIVATIVES
Gaetano Fiorillo, Fabrizio Orzi, Franco Buzzetti, Paolo Lombardi
Naxospharma srl, via G. Di Vittorio 70, I-20026 Novate Milanese
[email protected]
Berberine is an isoquinoline quaternary alkaloid (or a 5,6-dihydrodibenzo[a,g]
quinolizinium derivative) isolated from many kinds of medicinal plants belonging to the
genus Anonaceae, Berbiridaceae, Papaveraceae, Ranunculaceae and others [1]. The
alkaloid, in use in the Ayurvedic and Chinese medicines since hundreds of years, has
drawn extensive attention as a therapeutic against a number of diseases including
hyperlipidemia, diabetes, metabolic syndrome, polycystic ovary syndrome, obesity,
fatty liver disease, coronary artery disease [2], as supported by scientific and patent
literature, and 13 ongoing clinical trials (www.clinicaltrials.gov). The diverse
pharmacological properties exhibited by berberine indicate that the alkaloid has a
definite potential as drug in a wide spectrum of clinical applications [3]. Consequently,
the structure of berberine represents a biologically important skeleton and an attractive
natural lead compound for the introduction of chemical modifications to search for more
selective and specific medical indications.
Anticancer properties of berberine have also been reported [4]. In this respect we
disclosed novel 13-arylakyl berberine derivatives with improved anticancer properties
[5,6]. This class of compounds can be prepared starting from 7,8-dihydroberberine
precursors by using various synthetic methodologies, including an uncommon
aldehyde-enamine condensation. Several of the instant new berberine derivatives
show in vitro remarkable antiproliferative effects on a variety of human mesothelioma
cell lines, including the environmental and occupational asbestos exposure malignant
mesothelioma. Although the precise molecular basis of the biological activities of
berberine is still debated, at least for the anticancer activity, we present new
informations and data regarding a putative major effective biological target of this class
of compounds which is exploitable for clinical applications.
References
1. Imanshahidi M, Hosseinzadeh H. Pharmacological and therapeutic effects of Berberis vulgaris and its active
constituent, berberine. Phytother Res 2008;22:999–1012.
2. Cordell GA, Quinn-Beattle ML, Farnsworth NR. The potential of alkaloids in drug discovery. Phytother Res
2001;15:183–205.
3. Tillhon M, Guamàn Ortiz LM, Lombardi P, Scovassi AI. Berberine: new perspective for old remedies. Biochem
Pharmacol 2012; accepted online, in press.
4. Sun Y, Xun K, Wang Y, Chen X. Systematic review of the anticancer properties of berberine, a natural product
from Chinese herbs, Anti-Cancer Drugs, 2009, 20, 757-769.
5. Lombardi P, Buzzetti F, Arcamone AG. Benzoquinolizinium salt derivatives as anticancer agents. US Patent
8,188,109B2 to Naxospharma srl.
6. Bhowmik D, Hossain M, Buzzetti F, D’Auria R, Lombardi P, Kumar GS. Biophysical studies on the effect of the 13
position substitution of the anticancer alkaloid berberine on its DNA binding. J. Phys. Chem. B 2012, 116,
2314−2324.
Acknowledgements: Research supported by Regione Lombardia, Italy (Bando
ATP2009, Project: Plant Cell, grant No. 13810040.
Antitumor agents
Short lecture S1.4
Beyond the Pt(II)-based chemotherapy: Pt(IV) complexes as oral anticancer prodrugs
Gabano E., Ravera M., Alessio M., Bianco S., Zanellato I., Bonarrigo I. and Osella D.
Università del Piemonte Orientale, Dipartimento di Scienze e Innovazione Tecnologica, Viale T.
Michel 11, 15121 Alessandria, Italy
[email protected]
The current clinical chemotherapeutic protocol for the treatment of several solid
tumours consists of cisplatin (or its analogues carboplatin and oxaliplatin) combined
with drugs acting with a different mechanism (possibly) in a synergistic way.
Pharmacological properties of Pt(II) drugs might be improved with the use of Pt(IV)
complexes. Such compounds are believed to act as pro-drugs being activated in vivo in
the hypoxic, reducing and acidic environment of the tumour tissue, that converts the
inert octahedral Pt(IV) compounds into the active square planar Pt(II) metabolites via a
two electron reductive elimination (Figure 1). Pt(IV) complexes are more inert to
substitution than their Pt(II) counterparts and therefore are involved in fewer side
reactions with biological nucleophiles. Moreover, they can cross the gastrointestinal
tract and therefore be administered per os instead of i.v.
The choice of the axial ligands is
O
essential to modulate the water
solubility, the lipophilicity (and
the related cellular uptake) and
R
O
the
redox properties
(the
H3N
Cl
+ 2 eH3N
Cl
+ 2 RCOOH
Pt
Pt
reduction
peak
potential
should
H3N
Cl
H3N
Cl
+ 2 H+
fall into the biological window) of
R
O
these complexes.1
The axial ligands may have their
O
own biological role as active
targeting and delivery vectors or
Figure 1. Scheme of activation of cisplatin-based Pt(IV)
as co-adjuvant drugs. 2
pro-drugs with two carboxylato ligands
The quantitative structure-property relationship (QSPR) approach3 can be applied to
Pt(IV) complexes to rationalize their key features as pro-drugs with the final aim of
conceiving the design and synthesis of new effective drug candidates.
References
1. Hall, M.D.; Hambley, T.W. Platinum(IV) antitumour compounds: their bioinorganic
chemistry. Coord. Chem. Rev. 2002, 232, 49-67.
2. Graf, N.; Lippard, S.J. Redox activation of metal-based prodrugs as a strategy for drug
delivery. Adv. Drug. Deliv. Rev. 2012, 64, 993-1004.
3. Gramatica, P.; Papa, E.; Luini, M.; Monti, E.; Gariboldi, M.B.; Ravera, M.; Gabano, E.;
Gaviglio, L.; Osella, D. Antiproliferative Pt(IV) complexes: synthesis, biological activity, and
quantitative structure-activity relationship modelling. J. Biol. Inorg. Chem. 2010, 15, 11571169.
Antitumor agents
Short lecture S1.5
Identification of Novel Pseudopeptidic Inhibitors of Human Sirtuins 1-3 with
Antiproliferative Effect in Cancer Cells
Paolo Mellini,a ,b Elina M. Jarho,b Maija Lahtela-Kakkonen,b Tarja Kokkola,b Tiina
Suuronen,c Heikki Salo,b Laura Tolvanen,b and Antonello Mai a
a
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del
Farmaco, Università degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Rome, Italy
b
School of Pharmacy and cDepartment of Neurology, Institute of Clinical Medicine, University of
Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland
[email protected]
1. Introduction
The class III histone deacetylases, also called sirtuins (SIRT1-7) have gained
growing attention for their involvement in many biological processes such as cellular
metabolism regulation, neuroprotection, apoptosis, inflammation, telomere
maintenance etc.1 SIRT1/2 have been found to be involved in tumorigenesis through
their inhibitory effect on p53 associated with tumor suppression. Lately has the
hypothesis that targeting SIRT1/2 may represent an intriguing anticancer therapeutic
option made its way2. Herein we focused our efforts on the preparation and biological
evaluation of a novel fragment based library of pseudopeptidic inhibitors generated
considering our previous binding hypothesis3 developed using SIRT3, in which a
potential sirtuin inhibitor should create an H-bond network with Val292, Gly295,
Glu296, Glu325 and aromatic interactions (Figure1).
Figure 1. A scheme of the interaction pattern reported for a pseudopeptidic inhibitor in SIRT3.
2. Results and Discussion
A library of 30 novel thioacetylated pseudopeptidic compounds was synthesized and
screened against human SIRT1-3. Three sirtuin inhibitors were selected and subjected
to IC50 profile and cellular studies. All of them showed increase in acetylation of Lys382
of p53 after DNA damage. Furthermore, two of the compounds were able to inhibit both
A549 lung carcinoma and MCF-7 breast carcinoma cell growth in micromolar
concentration with the ability to arrest cancer cell cycle in the G1 phase.
3. References
1. Haigis, M.C. ; Sinclair, D.A. Mammalian Sirtuins: Biological Insights and Disease Relevance.
Annu Rev Pathol-Mech 2010, 5, 253–295.
2. Peck, B.; Chen, C. Y. SIRT inhibitors induce cell death and p53 acetylation through targeting
both SIRT1 and SIRT2. Mol Cancer Ther 2010, 9, 844-855.
3. Huhtiniemi, T.; Salo, H.S. Structure-Based Design of Pseudopeptidic Inhibitors for SIRT1
and SIRT2. J Med Chem 2011, 54, 6456–6468.
Antitumor agents
Short lecture S1.6
Synthesis of a new water-soluble Re(I)-porphyrin conjugate as a future prospect
for nuclear medicine applications
Mion G., Gianferrara T., Alessio E.
Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.le Europa 1,
34127 Trieste
[email protected]
Water soluble synthetic porphyrins and metalloporphyrins have attracted considerable
interest for their potential applications in many biological fields, and in photodynamic
therapy (PDT) in particular. Another interesting field of investigation concerns the
preparation of novel porphyrin conjugates with Re/99mTc complexes. Indeed, the
labeling of biologically active molecules with 99mTc moieties is a field of intense
research, as 99mTc is one of the most widely employed isotopes for imaging in nuclear
medicine (SPECT) and the biomolecules are meant to provide targeting efficacy. In this
respect, porphyrins are appealing as they typically show preferential uptake and
retention by tumor tissues and might serve as carrier ligands for the active transport of
metal compounds into cancer cells. In addition, their fluorescent emission can be
exploited for imaging purposes. The characterization of the 99mTc(I) conjugates is
performed with the corresponding non-radioactive Re(I) compounds.
Recently our research group synthesized two new water-soluble 99mTc(I)/Re(I)porphyrin conjugates bearing in peripheral position either one fac-{Re(CO)3}+ or one
fac-{99mTc(CO)3}+ fragment. This matched pair of conjugates are multimodal agents that
combine PDT activity with both fluorescence and radio-imaging functionalities for in
vitro and in vivo applications.1
Herein we report the preparation of the novel extended-arms porphyrin 1 and of its
Re(I) conjugate 2 (Figure 1). The precursor porphyrin bears four hydrophilic 2,2'(ethylendioxy)diethylamine chains in each meso position of the macrocycle. The
terminal amino groups of each flexible spacers were coupled with a tridentate ligand, a
functionalized 1,4,7-triazacyclononane (tacn), to obtain porphyrin 1 (Figure 1). As this
fragment is suitable for facial coordination, we performed the coordination of 1 with 4
eq of theRe(I) compound, [Re(CO)3(dmso)3](OTf), to obtain the conjugate 2. The water
soluble porphyrin 1 will be investigated for coordination with fac-{99mTc(CO)3}+ in future
studies for nuclear medicine applications.
NH N
N HN
+
H
N
=
O
O
O
O
N
H
N H NH
N
=
H
N
O
O
O
CO
O OC Re CO
NH
N
N
N
H
H
CF3SO3-
1
2
Figure 1. Structure of porphyrin 1 and of its Re(I)-conjugate 2.
1
Spagnul, C.; Alberto, R.; Gasser, G.; Ferrari, S.; Pierroz, V.; Bergamo, A.; Gianferrara, T.;
Alessio, E. J. Inorg. Biochem. 2013, 122, 57–65.
Antitumor agents
Short lecture S1.7
Pt-based drugs and DNA adduct formation: the spotlight will be on proteins
Pinato O.a, Benetazzo A. a, Farrell N.P. b, and Sissi C a.
a
Dipartimento di Scienze del Farmaco, v. F. Marzolo 5, 35131 Padova
Department of Chemistry, Virginia Commonwealth University, Richmond, VA, USA
b
[email protected]
Platinum-complexes represent one of the most successful group of clinically used
anticancer drugs. The mechanism of action of Pt-based drugs is based on the
formation of stable DNA adducts occurring at the nitrogen in position 7 of guanine (N7)
and involving one or two spatially closed residues. The formation of stable DNA
adducts is recognized as a DNA damage event and can ultimately drive to cell death.
Nevertheless, nucleobases are not the only reliable target of these drugs. Indeed,
sulphur containing biomolecules can be potential targets since platinum can efficiently
interact with this S-donor groups1. This is the case of proteins containing cysteine or
methionine residues and, actually, formation of DNA-protein cross-linking has been
further take into account in defining cisplatin mechanism of action. From a clinical point
of view, the possible role of protein as reaction intermediates or reservoir systems in
platinum drugs metabolism must be considered. This feature prompt us to deeper
investigate the potential role of Pt-protein adducts in modulating the drug action and
metabolism. In this study, we compared the reactivity profile of structurally different
platinum-complexes, including cisplatin, transplatin and trans planar platinum ammines
(TPAs) were considered. This finding helps to rationalize the promising
pharmacological profile exerted by TPA which show cytotoxic activity comparable to
cisplatin while displaying negligible cross-resistance2. We characterized the reaction of
these Pt-complexes with proteins and nucleic acids in terms of efficiency, kinetic and
nature of products formation. Our results emphasize how the reactivity towards
proteins and nucleic acids may be differently affected by the nature of the leaving
groups as well as by the ligands. Furthermore, during our research activity we set up
some experimental models, including transfer and competition reactions, to further
elucidate a potential direct role of proteins in DNA platination event. Our results
confirmed that while interaction with proteins are kinetically promoted, the reaction at
guanine-N7 is thermodynamically preferred. Moreover, Pt-complexes can efficiently be
transferred from platinated protein to DNA, which turned out to represent the final
platinum acceptor3. All these outcomes clearly claimed a role of proteins as reactive
intermediates for DNA platination and it define them as relevant to describe the clinical
potential of this class of Pt-complexes.
References
1. Reedijk, J. Why does Cisplatin reach guanine-N7 with competing S-donor ligands available
in the cell?. Chem. Rev. 1999, 99, 2499-2510.
2. Benedetti, B.T.; Quintal. S.; Farrell. N.P. Modulation of drug activation profiles through
carboxylate ligand modification in citotoxic trans-platinum planar amine compounds. Dalton
Trans. 2011, 40, 10983-10988.
3. Pinato, O.; Musetti, C; Farrell. N.P; Sissi. C. Platinum-based drugs and proteins: Reactivity
and relevance to DNA adduct formation. J. Inorg. Biochem. 2013, 122, 27-37.
Antitumor agents
Short lecture S1.8
2-Substituted-[1,3]thiazolo[4,5-e]isoindoles as kinases inhibiting compounds
Spanò Virginia, Diana Patrizia, Montalbano Alessandra, Carbone Anna, Parrino
Barbara, Cirrincione Girolamo, Barraja Paola
Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche
Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo (Italy).
[email protected]
Condensed thiazoles have been widely investigated as potential drugs. Among these,
benzothiazoles in the past two decades have been largely studied for their remarkable
anticancer activity.1 Tricyclic compounds incorporating the thiazole nucleus such as
thiazolyl-dihydro-indazoles 1 are described as kinases inhibiting compounds. This class
of compounds has received great attention in the last few years for the potent inhibitory
effect toward kinases, and many compounds have been covered by patents.2,3 We
have previously reported the synthesis of heterocycles in which a six membered ring, in
particular a pyridine,4 a pyrane,5 or a pyrimidine6 moiety is annelated to the isoindole
system with interesting antitumor properties. In the light of the potent activity showed
by thiazole compounds, we thought to synthetize a new class of tricyclic dihydro-4H[1,3]thiazolo[4,5-e]isoindoles of type 2 to evaluate their antitumor properties and their
inhibitory activity toward kinases. Our synthetic approach consisted in the annelation of
the thiazole core on the isoindole ring, using α-halogenated ketones. Evaluation of the
antiproliferative activity of the new compounds was performed at the NCI of Bethesda
on a panel of about 60 tumor cell lines. Six compounds showed antiproliferative activity
in the micromolar-submicromolar range, making this class of compounds very
promising. Results will be discussed.
R1
R1
N N
N1
2
S
S
3
R
N
1
Figure 1. General structures of compounds 1,2.
R2
8
NR
4
7
5
6
2
References
1. Smirnova, N. G.; Zavarzin, I. V.; Krayushkin M. M. Chemistry of Heterocyclic Compounds
2006, 42, 144.
2. Alexander, R. P.; Aujla, P. S.; Brown, J. A.; De Candole, B. C.; Trevitt, G. P. PCT Int. Appl.
WO 2009071890. Chem. Abstr. 2009, 151, 33574.
3. Van der Veen, L.; McConnell, D.; Schneider, S.; Grauert, M.; Schoop, A.; Wunberg, T. PCT
Int. Appl., WO 2010122091. Chem. Abstr. 2010, 153, 555154.
4. Barraja, P.; Diana, P.; Montalbano, A.; Carbone, A.; Viola, G.; Basso, G.; Salvador, A.;
Vedaldi, D.; Dall’Acqua, F.; Cirrincione, G. Bioorg. Med. Chem. 2011, 19, 2326.
5. Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G.; Vedaldi, D.; Salvador, A.;
Viola, G.; Dall'Acqua, F. Bioorg. Med. Chem. Lett. 2009, 19, 1711.
6. Barraja, P.; Spanò, V.; Diana, P.; Carbone, A.; Cirrincione, G. Tetrahedron Lett. 2009, 50,
5389.
Antitumor agents
Short lecture S1.9
BONE SEEKING MMP INHIBITORS FOR THE TREATMENT OF TUMOR INDUCED BONE
METASTASES
Tauro M.,(1) Shay G., (2) Laghezza A., (1) Loiodice F., (1) Lynch C.C. (2) and Tortorella P. (1)
(1) Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi Aldo Moro di Bari, Via Orabona 4,
70125 Bari (Italy)
(2) Tumor Biology Department, H. Lee Moffitt Cancer Center and Research Institute
SRB-3, 12902 Magnolia Dr., Tampa, FL, 33612
[email protected]
Matrix metalloproteinases (MMPs) can process all components of the extracellular matrix and have
been implicated in the progression of several cancer types including bone metastases. Because of
the relevant therapeutic potential, a wide variety of synthetic broad spectrum MMP inhibitors
(MMPIs) have been synthesized and tested over the past 20 years, although none reached clinical
utility as anticancer drug. In hindsight, this failure was due to non-specific side effects, largely due
to the systemic delivery of the inhibitors, a failure to reach the targeted cancers at sufficiently high
enough doses to inhibit tumor associated MMPs and a poor understanding of individual MMP
function.
Recently, we have identified that individual MMPs can impact tumor growth and tumor induced
osteolysis in skeletal malignancies such as metastasis and multiple myeloma that greatly affect the
patient’s quality of life [1]. At the bone, these cancers manipulate osteoblasts and osteoclasts to
induce bone formation or destruction respectively. This increase in bone turnover results in the
release of growth factors that feeds the cancer cells and initiates a “vicious cycle” of tumor-bone
interaction. We have demonstrated that MMPs, in particular
O
MMP-2, are important regulators of this cycle. Therefore, we
OH
propose that the generation of specific bone seeking MMPIs
P
OH
O
O
(BMMPIs) and target the bone would be of benefit for the
S
treatment of skeletal malignancies.
N
P OH
We have synthesized new potent MMPIs characterized by a
H
bisphosphonic structure (Fig. 1), ML104, ML111, ML115 with
Ki
O OH
R
values against MMP-2 in the nM range [2]. Using cell lines and
animal models of multiple myeloma, prostate and breast Fig. 1. MMPIs structure.
cancer to bone metastases, we confirmed BMMPIs efficacy in
preventing the growth of cancer cells and osteoclasts in vitro while in vivo reducing the size of
osteolytic lesions and extending overall survival in the multiple myeloma (MM) model.
Treatment of the 5TGM1 mouse MM cell line with low micromolar concentrations of the BMMPIs
significantly reduced cell viability through inhibition of cell growth (p<0.05). In vivo, 5TGMI tumorbearing mice receiving BMMP inhibitors three times a week, showed significant increase in overall
survival over 80 days (p<0.05). This was associated with a reduction in tumor burden and
protection from tumor-associated bone loss compared to vehicle treated mice as measured by xray and histomorphometry. These data suggest that given the roles for MMPs in tumor progression
in bone, that our novel BMMPIs may be effective in the treatment of MM.
We predict that MMP-2 specific BMMPIs may be more effective than current clinically used
bisphosphonates for the treatment of tumor induced bone metastases and may eliminate
undesirable side effects of broad-spectrum MMP inhibitors due to their high selectivity and bone
seeking nature. We are currently continuing to characterize these BMMPIs both as single agents,
and in combination with existing MM treatments (eg. Bortezomib) for the treatment and eradication
of MM.
In conclusion, we anticipate that the BMMPIs will provide new treatment options patients suffering
with these tumor induced incurable lesions.
References
[1] Lynch, C. C. Matrix metalloproteinases as master regulators of the vicious cycle of bone metastasis.
Bone 2011, 48(1), 44-53.
[2] Tortorella, P. et al. Biphenyl sulfonylamino methyl bisphosphonic acids as inhibitors of matrix
metalloproteinases and bone resorption. ChemMedChem 2011, 6(7), 1258-1268.
Antitumor agents
Short lecture S1.10
NiII, CuII and ZnII Salphen-like complexes: selective G-quadruplex stabilizers
Alessio Terenzi, Annamaria Martorana, Riccardo Bonsignore, Carla Gentile,
Giampaolo Barone, Antonino Lauria, Anna Maria Almerico
1
Università degli Studi di Palermo, Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e
Farmaceutiche (STEBICEF), Via Archirafi, 32 - 90123 Palermo, Italy
[email protected]
In efforts to find agents with improved biological activity against cancer cells, recent
years have seen an increased interest in the study of small molecules able to bind the
deoxyribonucleic acid (DNA) when it assumes secondary structures known as Gquadruplexes (G4) in preference over its B form.1
As Schiff base complexes derived from N,N′-bridged tetradentate ligands involving an
N2O2 donor atoms present very favourable features to act as G4 binders, a series of
square-planar and square pyramidal metal complexes, ML2+ (M = Ni, Cu, and Zn), have
been synthesized as perchlorate salts and characterized. Their affinity for wild-type
hTelo and c-myc G-quadruplexes DNA and for ct-DNA was investigated by UV
absorption spectroscopy, circular dichroism and viscosimetry. The data collectively
suggest that the complexes bind effectively to G-quadruplexes by direct end-stacking,
stabilizing the oligonucleotides secondary structure in its antiparallel form. The three
complexes are also able to behave as typical intercalators towards B-DNA.
Remarkably, the association constants between ML2+ and the G4s structures are about
10 fold higher than those for B-DNA, with a promising selectivity. The values of the
binding constants, Kb, within the range 105-106 M-1, present the following decreasing
order of interaction strength: NiL2+ > CuL2+ > ZnL2+.
Figure 1. Schematic representations of ML2+ stacked on the face of a G-quadruplex DNA (left)
and intercalated into B-DNA (right).
The biological activity of the three complexes was tested against MCF7 and HeLa
cancer cell lines. The antiproliferative activity follows the same trend of the complexes
binding capability.
References
1. Balasubramanian, S.; Hurley, L. H.; Neidle, S. Targeting G-quadruplexes in gene
promoters: a novel anticancer strategy? Nat. Rev. Drug Discov. 2011, 10, 261–275.
Metabolic and inflammatory disease agents
Main lecture M2
An introduction to the initiation, propagation and resolution of inflammation:
Targeting the resolution of inflammation by pharmacological intervention
Adriano G. Rossi
MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of
Edinburgh Medical School, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK.
[email protected]
White blood cells such as neutrophils and macrophages are vital for host defence in
the fight against invading organisms and are responsible, in part, for the cellular and
tissue damage associated with chronic inflammatory diseases (e.g., rheumatoid
arthritis, atherosclerosis, inflammatory bowel disease, bronchitis, etc). The processes
regulating to the initiation, propagation and resolution of inflammation are highly
regulated and importantly can be manipulated by pharmacological intervention1,2.
Specific recent attention has focussed on the resolution of inflammation with apoptosis
and non-phlogistic clearance of apoptotic cells by phagocytes such as macrophages
(sometimes called efferocytosis) identified as important cellular processes involved in
terminating inflammation3,4. In this presentation, I will attempt to convince you that
deliberately targeting these resolution processes by pharmacological means is a
realistic approach for reducing and resolving established inflammation and therefore a
novel approach for the development of new drugs for the treatment of acute and
chronic inflammatory diseases5.
References
1. Gilroy, D.W.; Lawrence, T.; Perretti, M.; Rossi, A.G. Inflammatory resolution: new
opportunities for drug discovery. Nat. Rev. Drug Discov. 2004, 3(5), 401-416.
2. Serhan, C.N.; Brain, S.D.; Buckley, C.D.; Gilroy, D.W.; Haslett, C.; O'Neill, L.A.; Perretti, M.;
Rossi, A.G.; Wallace, J.L. Resolution of inflammation: state of the art, definitions and terms.
FASEB J. 2007, 21(2), 325-332.
3. Fox, S.; Leitch, A.E.; Duffin, R,; Haslett, C,; Rossi, A.G. Neutrophil apoptosis: relevance to
the innate immune response and inflammatory disease. J. Innate Immun. 2010, 2(3), 216227.
4. Duffin, R.; Leitch, A.E.; Fox, S.; Haslett, C.; Rossi, A.G. Targeting granulocyte apoptosis:
mechanisms, models, and therapies. Immunol. Rev. 2010, 236, 28-40.
5. Hallett, J.M.; Leitch, A.E.; Riley, N.A.; Duffin R.; Haslett C.; Rossi AG. Novel pharmacological
strategies for driving inflammatory cell apoptosis and enhancing the resolution of
inflammation. Trends Pharmacol. Sci. 2008, 29(5), 250-257.
Metabolic and inflammatory disease agents
Short lecture S2.1
New biphenylic derivatives as CB2 receptor selective ligands
Bertini S.,a Arena C.,a Del Carlo S.,a Ligresti A.,b Saccomanni G.,a Saguto S.,a
Parkkari T.,c, d Savinainen J. R.,d Di Marzo V.,b Macchia M.,a Manera C.a
a
Dipartimento di Farmacia, Via Bonanno 6, 56126 Pisa, Italy; bEndocannabinoid Research
Group, Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei
c
34, 80078 Pozzuoli, Napoli, Italy. University of Eastern Finland, Faculty of Health Sciences,
d
School of Pharmacy, POBox 1627, 70211 Kuopio, Finland; University of Eastern Finland,
Faculty of Health Sciences, Institute of Biomedicine, School of Medicine, POBox 1627, 70211
Kuopio, Finland
[email protected]
The CB2 receptor (CB2R) was initially considered as an exclusively peripheral
receptor, restricted to cells of the immune system. It is now widely accepted that CB2R
is present in some central and peripheral neurons. Recent studies have shown the
increasing importance of CB2R as a therapeutic target for several diseases, such as
neuroinflammation, neurodegeneration and cancer.1 Therefore, great efforts have been
devoted to the development of CB2R selective ligands, in order to identify novel
therapeutic approaches, avoiding the undesirable (CB1R-mediated) central effects, and
to study more in detail the role of this receptor in physiopathological conditions. We
recently reported 1,2-dihydro-2-oxo-pyridine-3-carboxamide derivatives (A, Figure 1)
showing interesting CB2R affinity and selectivity.2 Subsequently, we introduced
substituents at position 5 of the central nucleus (B, Figure 1), thus determining in
general a further increase in the CB2R binding properties. On these basis we have
then developed a new class of compounds bearing a biphenylic structure (C, Figure 1),
derived from compounds of type B, in which the 1,2-dihydro-2-oxo-pyridine nucleus is
replaced with a phenylic ring and the "pattern" of substitution is substantially retained.
R3
O
NH
N
O
R3
O
R2
NH
N
O
R2
NH
R2
OMe
O
R1
R1
R1
A
B
C
Figure 1. Biphenylic compounds derived from 5-substituted-1,2-dihydro-2-oxo-pyridine-3carboxamides.
The new biphenylic derivatives showed, in some cases, good Ki values for CB2R (in
the nM range) and high levels of CB2R-selectivity. The compounds that gave the best
results in terms of binding properties were submitted to functional assays: preliminary
results showed that some of them seems to have an interesting pharmacological
character of CB2R-neutral antagonist.
References
1. Pertwee, R. G. Emerging strategies for exploiting cannabinoid receptor agonists as
medicines. Br. J. Pharmacol. 2009, 156, 397-411.
2. Manera, C.; Saccomanni, G.; Malfitano, A. M.; Bertini, S.; Castelli, F.; Laezza, C.; Ligresti,
A.; Lucchesi, V.; Tuccinardi, T.; Rizzolio, F.; Bifulco, M.; Di Marzo, V.; Giordano, A.;
Macchia, M.; Martinelli, A. Rational design, synthesis and anti-proliferative properties of
new CB2 selective cannabinoid receptor ligands: An investigation of the 1,8-naphthyridin2(1H)-one scaffold. Eur. J. Med. Chem. 2012, 52, 284-294.
Metabolic and inflammatory disease agents
Short lecture S2.2
Identification of new potent TRPV1 ligands: design, synthesis and structure-activity
relationships of amides exhibiting an agonist pharmacological profile.
A. Brizzi1, F. Aiello2, A. Artese4, G. Costa4, A. Ligresti3, F. Grande2, S. Alcaro4, F. Corelli1,
L. De Petrocellis3, A. Garofalo2, V. Di Marzo3
1
Dipartimento di Biotecnologie, Chimica e Farmacia Università degli Studi di Siena 53100 Siena,
Dipartimento di Farmacia e Scienze della Salute e della Nutrizione Università della Calabria 87036
3
Arcavacata di Rende Cosenza, Endocannabinoid Research Group Istituto di Chimica Biomolecolare
4
Consiglio Nazionale delle Ricerche 80078 Pozzuoli Napoli, Dipartimento Scienze della Salute Università
degli Studi “Magna Grecia” 88100 Catanzaro
2
[email protected]
The capsaicin receptor TRPV1 is a ligand-gated ion channel, activated by endogenous arachidonic
acid-derived lipids, called “Endovanilloids”, and expressed in sensory neurons [1] but also in higher
brain structures [2]. Experimental studies highlight the important role of this receptor in several
human diseases, such as inflammatory, visceral, cancer and neuropathic pain [3] as well as
pancreatitis [4] and migraine [5]. Nowadays, there is a strong evidence that the combination of
direct and indirect mechanisms finely tune the TRPV1 activity [6]. Accordingly, both agonists and
antagonists of this non-selective cation channel have been evaluated as potential analgesics; in
fact, its agonists lead to receptor desensitization, silencing previously excited neurons, while its
antagonists relieve pain behaviors in animal models of inflammation and cancer.
Inside the very extensive chemical diversity of agonist and antagonist compounds, we have
identified new TRPV1 ligands, chemically characterized by a 4-(thiophen-2-yl)butanoic acid amide
moiety and endowed with agonist activity. With the aim to better understand the structure-activity
relationships and deeply investigate how the chemical decoration of the thiophene-ring and/or the
electronic/steric properties of amide can affect the interaction with the vannilloid receptor and
modulate the TRPV1 activity, we have designed and synthesized a new set of derivatives,
employing modifications both on the acidic moiety and the aromatic amidic head.
Moreover a molecular modeling analysis has been used to better rationalize the different activities
of the TRPV1 derivatives. In particular the pharmacophoric and the conformational properties of
the molecules have been investigated by ligand-based 3D-QSAR techniques using a comparative
approach, allowing to define the common features crucial for the activity.
References
1. Sanchez, J.F.; Krause, J.E.; Cortright, D.N. The distribution and regulation of vanilloid receptor VR1 and
VR1 5' splice variant RNA expression in rat. Neuroscience 2001, 107, 373-381.
2. Szallasi, A.; Di Marzo, V. New perspectives on enigmatic vanilloid receptors. Trends Neurosci. 2000, 23,
491-497.
3. Immke, D.C.; Gavva, N.R. The TRPV1 receptor and nociception. Seminars in Cell & Developmental
Biology 2006, 17, 582-591.
4. Liddle, R.A.; Nathan, J.D. Neurogenic inflammation and pancreatitis. Pancreatology 2004, 4, 551-559.
5. Goadsby, P.J. Post-triptan era for the treatment of acute migraine. Curr. Pain Headache Rep. 2004, 8,
393-398.
6. Nagy, I.; Santha, P; Jancsò, G.; Urban, L. The role of the vanilloid (capsaicin) receptor (TRPV1) in
physiology and phatology. Eur. J. Pharmacol. 2004, 500, 351-369.
Metabolic and inflammatory disease agents
Short lecture S2.3
Development of a Novel Class of Derivatives as Selective COX-2 Inhibiting Nitric
Oxide Donors: Improving the Solubility and the Biological Profile
Consalvi S.a, Poce G.a, Battilocchio C.a, Alfonso S.a, Calderone V.b, Martelli A.b , Testai
L.b, Sautebin L.c , Rossi A.c, Ghelardini C.d, Di Cesare Mannelli L.d, Giordani Ae.,
Patrignani P.f, Anzini M.g, Biava M.a
a
Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma “Sapienza”, P.le A.
b
Moro, 5, 00185 Roma. Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, I-56126
c
Pisa. Dipartimento di Farmacologia, Università di Firenze, viale G. Pieraccini 6, I-50139
d
Firenze. Dipartimento. di Farmacia, Università di Napoli “Federico II”, via D. Montesano 49,
80131 Napoli.eRottapharm S.p.A., via Valosa di Sopra 7, I-20052 Monza. fDip. Medicina e
Centro di Eccellenza per l’Invecchiamento, Università degli Studi di Chieti “G. d’Annunzio”
g
University e CeSI, Via dei Vestini 31, 66013 Chieti. Dipartimento Farmaco Chimico
Tecnologico, Università degli Studi di Siena, Via Alcide de Gasperi 2, I-53100 Siena
[email protected]
The family of cyclooxygenase-2 (COX-2) selective inhibitors (COXIBs) is a class of
compounds developed to overcome gastrointestinal toxicity caused by use of traditional
non steroidal anti-inflammatory drugs (t-NSAIDs)[1]. Moreover, it is well known that the
cardiovascular side effects of COXIBs are due to the selective COX-2 inhibition[2];
therefore, in the last years research attention focused on developing pharmacodynamic
hybrids that conjugate the COX-2 selective skeleton with different Nitric oxide (NO)releasing chains, to make use of NO-mediated vasorelaxing effects[3]. In the past years
we synthesized a first class of COXIBs characterized by a 1,5-diphenyl-pyrrole scaffold
and a NO releasing moiety[4-5]; these derivatives showed good biological profiles,
despite their low water solubility. On these grounds and on the basis of literature data,
the study progressed by means of modifying the central core in order to improve the
solubility of this class of derivatives. Following the previous adopted protocol, selected
compounds have been tested: i) in vitro: to assess their cyclooxygenase-1 (COX-1)
and COX-2 inhibition activities; ii) ex vivo: to investigate their NO-releasing properties;
iii) in vivo: to evaluate their efficacy in analgesia, by means of the abdominal writhing
test.
Furthermore, since COX-inhibitors play important opposite roles in carcinogenesis and
they are also involved in reducing the Histone Deacetylase Inhibitors (HDACi) side
effects of upregulating COX-1 and COX-2[6], in our future studies, we aim to investigate
antitumor activity of these compounds, also in association with HDACi.
References
1. Richy,F., Bruyere, O. et al. Time dependent risk of gastrointestinal complications induced by
non-steroidal anti-inflammatory drug use: a consensus statement using a meta-analytic
approach, Ann Rheum Dis, 2004, 63, 759-766.
2. Bresalier, R.S. Sandler, R.S. et al. Adenomatous Polyp Prevention on Vioxx (APPROVe),
Trial Investigators, Cardiovascular events associated with rofecoxib in a colorectal adenoma
chemoprevention trial, New Eng J Med, 2005, 352, 1092-1102.
3. Ignarro, L.J. Nitric oxide as a unique signaling molecule in the vascular system: a historical
overview, J Physiol Pharmacol, 2002, 53, 503-514.
4. Biava, M., Porretta G.C. et al. Novel analgesic/anti-inflammatory agents: diarylpyrrole acetic
esters endowed with nitric oxide releasing properties, J Med Chem, 2011, 54, 7759-7771.
5. Biava, M., Battilocchio, C. et al., Improving the solubility of a new class of antiinflammatory
pharmacodynamic hybrids, that release nitric oxide and inhibit cycloxygenase-2 isoenzyme,
Eur J Med Chem, 2012, 58, 287-298.
6. Wang, X., Li, G. et al., Combined histone deacetylase and cyclooxygenase inhibition
achieves enhanced antiangiogenic effects in lung cancer cells. Mol Carcinog, 2013, 52,
218-228.
Metabolic and inflammatory disease agents
Short lecture S2.4
Drug design, synthesis and biological evaluation of 3-substituted-1,5-diaryl-2alkylpyrroles as COX-2/CINODs endowed with anti-inflammatory and
antiproliferative activity
Di Capua A.,1 Valenti S.,1 Cappelli A.,1 Ghelardini C., Di Cesare Mannelli L.,2 Calderone
V.,3 Martelli A.,3 Testai L.,3 Sautebin L.,4 Rossi A.,4 Patrignani P.,5 Sticozzi C.,6 Valacchi
G.,6 Giordani A.,7 Biava M.,8 and Anzini M.1
1
Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, Via A. Moro, 2 - 53100
2
Siena, Italy; Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino
(NEUROFARBA) - Sez. Farmacologia e Tossicologia, Università di Firenze, Viale G. Pieraccini,
3
6 - 50139 Firenze, Italy; Dipartimento di Psichiatria, Neurobiologia, Farmacologia e
4
Biotecnologie, Università degli Studi di Pisa, Via Bonanno 6, I-56126 Pisa, Italy; Dipartimento
di Farmacia, Università degli Studi di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli,
5
Italy; Dipartimento di Neuroscienze ed Imaging, Università degli Studi di Chieti, “G. d’Annunzio”
6
and CeSI, Via dei Vestini 31, I-66100 Chieti, Italy; Dipartimento di Scienze della Vita e
Biotecnologie, Università degli Studi di Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy;
7
8
Rottapharm Madaus , Via Valosa di Sopra 7, I-20052 Monza, Italy;. Dipartimento di Studi di
Chimica e Tecnologie del Farmaco, Università degli Studi di Roma "La Sapienza”, Piazzale
Aldo Moro 5, I-00185 Roma, Italy.
[email protected]
Long-term therapy with tNSAIDs generates adverse gastrointestinal complications
ranging from stomach erosions and silent intestinal ulcerations to life-threatening
complications. Selective inhibitors of COX-2 have overcome this problem.
Nevertheless, they depress prostacyclin (PGI2), an atheroprotective and vasorelaxant
agent, but not COX-1-derived thromboxane A2 (TXA2), a pro-aggregatory and
vasoconstrictor mediator. This might predispose patients to heart attack and stroke.
The discovery that Nitric Oxide (NO) produces many of the same effects of PGI2 at the
cardiovascular level has led to the disclosure of a new class of drugs, COX-2 Inhibitor
NO Donors (CINODs). In addition to its similar properties to PGI2 within the
cardiovascular system, evidence is accumulating for the role of NO as a new
oncopreventive agent and as a novel therapeutic tool to overcome cell resistance. We
reported the synthesis and the biological evaluation of a series of 1,5-diarylpyrrole
nitro-oxyalkyl congeners [1-3] in which different linkers between the 1,5-diarylpyrrole
scaffold and the NO releasing moiety were introduced. In this way, novel compounds
are prepared and evaluated in vitro and in vivo in order to disclose new chemical
entities potentially useful in the long-term treatment of osteoarthritis (OA) with improved
cardiovascular safety and in order to evaluate their antiproliferative activity in different
types of cancer cell lines.
References
1. Giordani, A.; Biava, M.; Anzini, M.; Calderone, V.; Rovati, L. 1,5-Diaryl-2-alkyl-pyrrole-3Substituted nitro esters selective COX-2 inhibitors and nitric oxide donors. WO2012/032479
A1.
2. Biava, M. et al.Novel Analgesic/Anti-inflammatory Agents: Diarylpyrrole Acetic Esters
Endowed with Nitric Oxide Releasing Properties. J. Med. Chem. 2011, 54, 7759-7771.
3. Anzini M.; Di Capua A. et al. Novel Analgesic/Anti-inflammatory Agents: 1,5-Diarylpyrrole
Nitro-oxyalkyl Ethers and Related Compounds as Cyclooxygenase-2 Inhibiting Nitric Oxide
Donors J. Med. Chem. submitted.
Metabolic and inflammatory disease agents
Short lecture S2.5
Ligand-based NMR Screening Methods applied to discovery of GRK2 Inhibitors
A. Limatola1, E. Vernieri2, I. M. Gomez-Monterrey1, M. Sala2, G. Iaccarino3,
P. Grieco1, E. Novellino1, P. Campiglia2, A. Carotenuto1
1
Department of Pharmacy, University of Naples “Federico II”, 80131– Napoli.
Department of Pharmaceutical and Biomedical Science, University of Salerno , 84084–
Salerno.
3
Department of Clinical Medicine Cardiovascular and Immunological Sciences, University of
Naples“Federico II”, 80131– Napoli.
2
[email protected]
G protein-coupled receptor kinase 2 (GRK2) regulates cell signaling by promoting
agonist-specific desensitization of several metabolism-related GPCRs, including the βadrenergic receptors, endothelin, and glucose-dependent insulinotropic polypeptides.
The up-regulation of GRK2 and corresponding desensitization of these metabolismrelated GPCRs seem play an important role in the onset or progression of diseases
such as heart failure, myocardial ischemia, hypertension, Type 2 diabetes and in cell
cycle progression. Recent studies suggest that GRK2 participates in the regulatory
network controlling cell cycle arrest and survival in such conditions.1 Understanding of
the molecular mechanisms leading to altered GRK2 levels, as well as the identification
of GRK2 inhibitors is a very active field of research.
We report a simple approach for ligand binding studies by NMR spectroscopy. In
particular, ligand-based methods such as saturation transfer difference (STD) and
water ligand observed gradient spectroscopy (waterLOGSY) are widely used for ligand
screening. These experiments allows to detect transient binding of small molecule
ligands to macromolecular receptors.2 We performed competition-based NMR
experiments, using GRK2 as target.
We report the preliminary results obtained with known small-molecule inhibitors and a
library of short peptides. This peptide library was developed by two precursors which
show a positive effect on glucose metabolism in animal models of Type 2 diabetes,
increasing insuline sensitivity and improving glucose homeostasis and emerge as a
valuable starting point for the development of a novel class of GRK2 inhibitors.3
References
1. Penela P, Rivas V, Salcedo A et al. Cell Biology, 2010, 17, 1118-1123.
2. Pellecchia M, Bertini I, Cowburn D, Dalvit C et al. Nat Rev Drug Discovery, 2008, 9,738-45
3. Y. Anis, O. Leshem, H. Reuveni et al., Diabetologia, 2004, 47, 1232-1244.
Metabolic and inflammatory disease agents
Short lecture S2.6
Design and synthesis of new multitarget drugs for autoimmune inflammatory diseases
Brullo C., Massa M., Rotolo C. and Bruno O.
Department of Pharmacy; University of Genoa, Viale Benedetto XV, 3-16132, Genoa-Italy
[email protected]
In several non-infectious human diseases, such as ulcerous colitis, rheumatoid arthritis, chronic
obstructive pulmonary disease (COPD), the extravasal recruitment of neutrophils (chemotaxis)
plays a crucial role in the development of tissue damage, which, when persistent, can lead to the
irreversible organ dysfunction. The neutrophil activation is controlled by a number of intracellular
pathways, particularly by a cAMP dependent protein-Kinase A (PKA) which also acts on
phosphodiesterase 4 (PDE4) gene stimulating the synthesis of this enzyme, able to transform
cAMP to inactive AMP. PDE4 inhibitors enhance intracellular cAMP and decrease inflammatory
cell-activation. Since 2010 the PDE4B/4D inhibitor Roflumilast (Daxas®) has been approved for
COPD treatment.
R
O
In the last ten years, we published several pyrazolyl-ureas R
1
N
and two series of imidazo-pyrazoles inhibiting fMLP- and
N
N
H
N
H
IL8-induced chemotaxis at very low concentration (in the
OH R = H, COOEt, CONH2, CONR2
R1 = C6H5, C6H4X,
sub-nanomolar range) (compounds A and B) 1,2,3,4.
C6H4R3, CH2C6H5
A
Moreover, we synthesized several cathecolic derivatives
(structurally related to Roflumilast and Rolipram as well)
O
N
(compounds (C))5,6, which were able to reduce the
N
R4
O
superoxide anion production in neutrophil α-TNF
HN
N R6
stimulated and increased the cAMP levels by inhibiting
O
C
B
PDE4 enzymes.
R4 = OH, NH2, NR5
R6 = cycloamines,
five member heterocycles
Taking in account the new drug discovery paradigm which considers the development of “multiple
targeted drugs” more profitable than the classic “one target-one disease” approach, we projected
new molecules able to inhibit simultaneously PDE4 enzymes and neutrophil regulation. They
could be used in the sympthomatic treatment of inflammatory events of various diseases that
affect respiratory system (bronchial asthma, COPD, cystic fibrosis).
To obtain new molecules with dual activity, we
O
OH
N
bounded the pyrazole or the imidazo-pyrazole
O
R7 = NH2,
N
N
N NH
scaffolds of the compounds A and B with the
R7
O
cathecolic moiety of the compounds C, obtaining
D
compounds D and E.
O
N
They have been tested on different PDE4 isoforms
O
N
(PDE4A4, PDE4D3 and PDE4B2) and they showed
N NH
HN
O
a good inhibitory activity with IC50 in the micromolar
range. Further tests are in progress to state its ability
E
to interfere with neutrophil activation and
recruitment.
References
1. Bruno, O.; Brullo, C.; et al. J. Med. Chem. 2007, 50, 3618-3626.
2. Bruno, O.; Brullo, C.; et al. Bioorg. Med. Chem. 2009, 17, 3379-3387
3. Bruno, O.; Brullo, C.; et al. Bioorg. Med. Chem. Lett. 2007, 17, 3696-3701.
4. Brullo, C.; Spisani, S.; et al. Eur. J. Med. Chem. 2012, 47, 573-579.
5. Bruno, O.; Brullo, C.; et al. Farmaco 2004, 59, 223-235
6. Bruno, O.; Romussi. A.; et al. J. Med. Chem. 2009, 52, 6546-6557.
Metabolic and inflammatory disease agents
Short lecture S2.7
Synthetic Analogues of Natural Polyphenolic Compounds as
Novel Agents for the treatment of Atherosclerosis
S. Sartini,1 V. Coviello,1 A.M. Marini,1 F. Simorini,1 S. Taliani,1 S. Salerno,1
S. Del Turco,2 C. Sentieri,2 G. Basta,2 F. Da Settimo,1 and C. La Motta1
1
Dipartimento di Farmacia, Università di Pisa, via Bonanno, 6, 56126, Pisa
2
Dipartimento di Fisiologia Clinica, CNR, via Moruzzi, 1, 56124, Pisa
[email protected]
Atherosclerosis is a complex disease in which an artery wall thickens accumulating
fatty materials. Illnesses related to the phenomenon of atherosclerosis are the most
common cause of death. The atherosclerotic lesion mainly affects the inner arteries of
large and medium caliber, especially abdominal aorta, renal arteries and femoral
arteries, coronary arteries and the brain.1 To date, patients affected by atherosclerosis
are faced with different treatment opportunities, represented by drug therapy, coronary
artery by-pass graft surgery (CABG) and percutaneous coronary intervention (PCI).
Drug therapy represents a reasonable option for the prevention of short term cardiac
mortality in patients with stable angina. Intermediate- and high-risk patients, however,
exhibit longer survival when subjected to CABG or PCI. Among these latter, PCI is
currently the most frequently exploited treatment around the world. Different techniques
and devices have been developed to either remodel or remove causes of acute and
chronic artery obstruction, increasing luminal cross-sectional area and improving blood
flow. However, despite improvement in equipments, limitations still remains as patients
may experience abrupt vessel closure at the treated site, due to coronary artery spasm,
localized thrombus, coronary dissection and neointimal hyperplasia. Coronary
implantation of bare metal stents allowed a great advance in the treatment of CAD.
Actually, behaving like a mechanical scaffold, they reduce restenosis, hampering
elastic recoil and negative remodelling. However, although currently used, they pose
the risk of an additional issue, represented by in-stent restenosis (ISR), mainly
sustained by inflammatory response to metallic stent. Failures of suitable drug
treatment for ISR, both orally administered and locally delivered, led to the
development of drug-eluting stent (DES), which may reasonably be considered the first
example of intravascular drug delivery device.
Actually, the ideal therapeutic agent for injuried vessel wall should combine an antirestenotic effect with a pro-healing action. This is why the search of novel and more
effective compounds is still a challenging issue. In this sense, development of multifunctional agents, targeting different molecular pathways at the same time, represents
a fully original strategy to address the site of endovascular intervention.2
Pursuing our interest in the development of multi-functional drug candidates, we
recently disclosed a novel class of 2,3-diphenylpyrido[1,2-a]pyrimidin-4-one derivatives
which, tested in vitro both on human aortic smooth muscle cells (HAoSMCs) and on
human umbilical vein endothelial cells (HUVECs), proved to combine an anti-restenotic
effect with a pro-healing action, inhibiting endothelial cells dysfunction. Moreover, they
also showed a remarkable anti-aggregating effects on platelets. In this presentation,
synthesis and biological activity of suitably substituted derivatives acting on injured
vessels will be discussed.
References
1. Hansson, G. K. New Engl. J. Med. 2005, 352, 1685-1696.
2. Zimmermann, G. R.; Lehar, J.; Keith, C. T. Drug Discov. Today, 2007, 12, 34-42.
Neurodegenerative disease agents
Main lecture M3
Advances in Alzheimer’s disease drug discovery
Andrea Cavalli
Dept. of Pharmacy and Biotechnology, University of Bologna
Dept. of Drug Discovery and Development, IIT Genova
Alzheimer's disease (AD) is the major unmet medical need in neurology.1 The currently
available drugs for the treatment of AD are three inhibitors of the acetylcholinesterase
enzyme (i.e. donepezil, rivastigmine, and galantamine), and memantine, a noncompetitive NMDA receptor antagonist. The discovery of these drugs had been based
on the observation that the cholinergic and the glutamatergic systems are
compromised in AD.2 However, these drugs can only treat some AD symptoms for a
limited period of time. More recently, based on the observation that amyloid plaques
and neurofibrillary tangles are the two major hallmarks of AD, two hypotheses (i.e.
amyloid and tau hypotheses3,4) have been pursued in AD drug discovery, aimed at
obtaining innovative disease-modifying drugs. The amyloid hypothesis has generated
several drug candidates, mainly targeting the enzymes beta- and gamma-secretases.
Unfortunately, none of these molecules has so far reached the market. The most
recent failure has been semagagestat, a gamma-secretase inhibitor, whose
development was stopped in phase III clinical trials because of lack of efficacy and
dangerous side effects (i.e. skin cancer). The tau hypothesis has been aimed at
discovering molecules able to tackle the hyperphosphorylation of tau and the
subsequent formation of the neurofibrillary tangles. Also this hypothesis has not so far
generated valuable clinical candidates for AD. Recently, other aspects of AD have
been investigated: i) the neuroinflammation,5 which is a major component of the
neurodegeneration associated with AD, and ii) the oxidative stress,6 which is greatly
increased in the disease. Based on these pathways, some small organic molecules
have recently been discovered and developed as drug candidates for AD.
In this lecture, the major pathological pathways in AD will be presented, and discussed
from a drug discovery standpoint. Several drug candidates will be presented along with
their major benefits and drawbacks.
References
1.
2.
3.
4.
5.
6.
Citron, M. Nat. Rev. Drug Discovery 2010, 9, 387−398.
Palmer, A. M., Gershon, S. FASEB J. 1990, 4, 2745−2752.
Hardy J., Selkoe D.J. Science, 2002, 297, 353-356.
Ghoshal N., et al. Exp. Neurol. 2002, 177, 475-493.
Heneka M.T., et al. Nature, 2013, 493, 674-678.
Bishop N.A., et al. Nature, 2010, 464, 529-535.
Neurodegenerative disease agents
Short lecture S3.1
Exploring a new scaffold of 2-/3-/4-acetylpyridine derivatives as potent and
selective human MAO-B inhibitors
Simone Carradoria, Melissa D’Ascenzioa, Daniela Seccia, Adriana Bolascoa, Paola
Chimentia, Matilde Yáñezb
a
Dip. di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5,
b
00185 Rome, Italy; Departamento de Farmacología, Universidad de Santiago de Compostela,
E-15782 Santiago de Compostela (La Coruña), Spain.
[email protected]
Human monoamine oxidases (hMAO-A and -B) metabolize monoamine
neurotransmitters and exogenous amines by oxidative deamination, playing an
important role in regulating emotional (anxiety and depression) and other brain
functions. In addition, patients suffering from Parkinson and Alzheimer diseases might
take advantage of selective hMAO-B inhibition to reduce the oxidative stress involved
in the neurodegenerative processes. We previously reported on the synthesis, hMAO
inhibitory activity, and 3D-QSAR studies of a large number of substituted (thiazol-2yl)hydrazines.1-3 On the basis of these results, we designed a new series of (thiazol-2yl)hydrazine derivatives, keeping constant the pyridine ring on the N1-hydrazine at C2
of the thiazole nucleus, with the aim to explore the C4 position of the thiazole ring
influence to display monoamine oxidase A and B inhibitory activity. The MAO inhibitory
properties of these pyridine derivatives were investigated using commercially available
microsomes from insect cells containing recombinant human MAO-A and MAO-B with
kynuramine as the substrate. These compounds displayed selective and reversible
inhibition of hMAO-B isoform in the nanomolar range.
Figure 1. General structure of selective hMAO-B inhibitors object of this work
References
1. Carradori, S.; D’Ascenzio, M.; De Monte, C.; Secci, D.; Yáñez, M. Synthesis and selective
human MAO-B inhibition of heterocyclic hybrids based on hydrazine and thiazole scaffolds.
Arch. Pharm., 2013, 346, 17-22.
2. Secci, D.; Bolasco, A.; Carradori, S.; D’Ascenzio, M.; Nescatelli, R.; Yáñez, M. Recent
advances in the development of selective human MAO-B inhibitors: (Hetero)arylidene-(4substituted-thiazol-2-yl)hydrazines. Eur. J. Med. Chem., 2012, 58, 405-417.
3. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Carradori, S.; D’Ascenzio,
M.; Yáñez, M.; Orallo, F. Synthesis and selective inhibition of human monoamine oxidases
of a large scaffold of (4,5-substituted-thiazol-2-yl)hydrazones. Med. Chem. Commun., 2010,
1, 61-72.
Neurodegenerative disease agents
Short lecture S3.2
Advantageous tools potentially useful to reduce withdrawal syndrome
and associated psycopatologies
Del Bello F., Bonifazi A., Diamanti E., Giannella M., Mammoli V.,
Mattioli L., Piergentili A., Quaglia W., Titomanlio F., Pigini M.
School of Pharmacy, University of Camerino
[email protected]
Addiction to opioids is a complex syndrome involving tolerance, drug-seeking and
physical dependence with withdrawal avoidance behaviors. α2-Adrenoreceptors (α2ARs) have been demonstrated to be extremely sensitive to opioid exposure. The nonsubtype selective α2-AR agonist clonidine is clinically used for relief of withdrawal
symptoms, but its α2A-AR subtype activation is responsible for sedation and
hypotension side effects. Selective α2C-AR agonists, alone or in combination with
opioids, might afford an improvement over current therapies with clonidine-like drugs.
Strong association between protracted abstinence and depressive disorders,
contributing to relapse, emerges from several studies. Since prolonged abstinence
remains a major challenge, strategies addressed to discover multifunctional agents
beneficial to withdrawal symptoms and depressive disorders should be explored. Our
recent allyphenyline (1) and cyclomethyline (2), devoid of sedative side effect, at the
same low dose (0.05 mg/Kg), were able to significantly decrease the naloxoneprecipitated withdrawal syndrome and to exert a potent antidepressant-like effect. Such
advantageous behaviour appeared consequent to their α2C-AR agonism/α2A-AR
antagonism/5-HT1A agonism.1 We have previously demonstrated that in the structures
sharing the common pharmacophore reported in figure, the -OCH(CH3)- bridge (X) of
1-6, suitable for the α2-AR interaction, negatively affected the I2-IBS affinity.
Interestingly, the -OCH2- bridge proved to be compatible with both systems.2
I2-IBS are also involved in depression, modulation of morphine analgesia as well as
opioid addiction. Aims of the present study were (i) to confirm our previous
observations; (ii) to discover other multitarget ligands potentially useful in managing
opioid addiction and associated disorders. For this latter purpose the designed
compounds 7-12, bearing the same aromatic area of their corresponding leads 1-6
(effective α2C-AR agonists/α2A-AR antagonists),3 were characterized by the -OCH2bridge which, as aforementioned, proved compatible also with I2-IBS interaction.
Compounds 3 and 7 were selected for the in vivo studies.
References
1. Del Bello, F. et al. ACS Med. Chem. Lett. 2012, 3, 535-539 and references therein.
2. Cardinaletti, C. et al. J. Med. Chem. 2009, 52, 7319-7322 and references therein.
3. Diamanti, E. et al. Bioorg. Med. Chem.. 2012, 20, 2082-2090.
Neurodegenerative disease agents
Short lecture S3.3
DRUG DESIGN AND SYNTHESIS OF GSK-3β INHIBITORS
Valeria Famiglini,a Valeria Lapietra,b Giuseppe La Regina,a Antonio Coluccia,a Sveva
Pelliccia,b Batya Plotkin,c Hagit Eldar-Finkelman,c Andrea Brancale,d Luciana Marinelli,b
Ettore Novellino,b and Romano Silvestria
a
Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma.
Dipartimento di Farmacia, Università di Napoli Federico II. cDepartment of Human Molecular
Genetics and Biochemistry,
Sackler Medical School, Tel Aviv University. dWelsh School of
Pharmacy, Cardiff University, UK.
b
[email protected]
Alzheimer’s disease (AD) is a leading cause of death worldwide. The development
of AD is accompanied by neuronal loss, presence of intracellular neurofibrillary tangles
and the formation of extracellular senile plaques.
Recent evidence suggests that glycogen synthase kinase 3 (GSK3) proteins play
key roles in many fundamental processes during neurodevelopment1. Over the past
decades have been reported a number of diverse GSK-3β inhibitors. We observed that
compound 1 synthesized previously by our group2 shared some chemical features
common with known GSK-3β inhibitors. We selected compound 1 as a useful hit
compound for the development of new GSK-3β inhibitors (Chart 1). Thus, we have
carried out SAR studies and synthesized a number of new derivatives that showed
potent GSK-3β inhibitory activity in the low micromolar range of concentration.
Chart 1. New GSK-3β inhibitors and their binding mode
References
[1] Castano, Z.; Gordon-Weeks, P. R. et al. J. Neurochem. 2010, 113,117–130.
[2] Artico, M.; Massa, S.; Stefancich, G. et al. J. Heterocyclic Chem. 1989, 26, 503-507.
Neurodegenerative disease agents
Short lecture S3.4
The Multi-Target Drug Approach in Alzheimer Disease Therapy
Federico A.,1 Chegaev K.,1 Lazzarato L.,1 Marini E.,1 Fruttero R.,1 Gasco A.,1
Ripoli C.,2 Grassi C.,2 Bastone A.,3 Morbin M.,3 Rossi G3
1
Dipartimento di Scienza e Tecnologia Del Farmaco, Via P. Giuria 9 Torino
Istituto di Fisiologia Umana dell'Università Cattolica, Largo F. Vito 1 Roma
3
Istituto Neurologico Carlo Besta, Via Celoria 11 Milano
2
[email protected]
Today the term Alzheimer’s disease (AD) indicates a progressive neurodegenerative
disorder caused by various processes such as cognitive decline, irreversible memory loss,
disorientation, language impairment.1
The difficulty for developing satisfactory therapy of AD lies in the complex pathophysiology
of disease which involves numerous pathways. These include deficiency in cholinergic
neurotransmission, defective β-amyloid protein metabolism, accumulation of aggregated
Tau proteins within neuronal cells and involvement of inflammatory, oxidative and hormonal
pathway.2 Recent findings indicate that AD is also accompanied by neurovascular
dysfunction which involves the reduction of physiological levels of an important
neuroprotective agent, nitric oxide (NO.).3 Considering the complexity of the disease, the
project was focused on the use of the multi-target drug approach in order to obtain new
molecular hybrids potentially active in AD therapy. The defective β-amyloid protein
metabolism, the accumulation of aggregate Tau–proteins and the endothelial dysfunction
were potential targets chosen. On this basis new multi-target drugs were designed in which
an inhibitor of both β-amyloid and Tau protein aggregation were combined with NO-donors.
Recent studies have identified several β-amyloid and Tau protein aggregation inhibitors;
among these, the class of thiacarbocyanines was one of the most potent.4 For this reason
our choice focused on this class of compounds. As NO-donor moieties, nitrate esters and
furoxan heterocycle system were chosen for building the molecular hybrids. Successively,
molecules synthesized were characterized by the following biological assays: activities on
β-amyloid fibrillization inhibition, cytotoxicity, NO-release, and the effect on tau aggregation.
Preliminary results showed two molecular hybrids (Figure 1) were able to inhibit β-amyloid
aggregation, to reduce the fibrillogenic ability of Tau to form fibrils and to release NO
simultaneously.
O
O
S
N
S
+
N O
S
O
N
I
N
S
+
O
N
I
N
O
N
+
O
Figure 1. Most promising Cyanine-NO donors.
For these two promising compounds studies of long lasting synaptic plasticity (LTP) are in
progress. If results will confirm the expected values, they might be developed either as new
potential AD drugs or as new pharmacological tools to explore this complex disease.
References
1. Hamley, W.; Author, B. The Amyloid Beta Peptide: A Chemist’s Perspective. Role in
Alzheimer’s and Fibrillization. Chem. Rev., 2012, 112 (10), 5147–5192
2. Kung, F. The β-Amyloid Hypothesis in Alzheimer’s Disease: Seeing Is Believing Med. Lett.
3, 2012, 265-267
3. Thatcher, G. Nitrate and NO Release: Contemporary Aspect in Biological and Medicinal
Chemistry. J. Free Rad .Bio. Med. 2004, 37, pages 1122-1143
4. Chang, E. Structure-Activity Relationship of Cyanine Tau Aggregation Inhibitors. J. Med.
Chem. 2009, 52, pages 3539-3547
Neurodegenerative disease agents
Short lecture S3.5
Novel fluorescent hA3 adenosine receptor antagonists
Federico S.,a Kozma E.,b Kumar T.S.,b Jacobson K.A.,b Moro S.c and Spalluto G.a
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Piazzale Europa 1,
b
I-34127 Trieste, Italy; Molecular Recognition Section, Laboratory of Bioorganic Chemistry,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
c
Bethesda, MD 20892-0810, USA; Molecular Modeling Section (MMS), Dipartimento di Scienze
Farmaceutiche, Università di Padova, via Marzolo 5, I-35131 Padova, Italy
[email protected]
Activation of the Gi protein-coupled A3 adenosine receptor (AR) is associated with
anticancer, antiischemic and anti-inflammatory effects. hA3 AR antagonists are being
examined as promising agents for the treatment of glaucoma.1
Fluorescent probes are useful tools to investigate specific subcellular components in
cells, tissues and organisms. In high throughput screening, fluorescent ligands
represents a safer, more powerful and more versatile alternative to radioligands.2
We have recently reported a series of pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine
(PTP) based fluorophore-conjugated hA3 AR antagonists.3 Unfortunately, none of them
reached affinities in the nM range. Here we report a novel series of PTP based
fluorophore-conjugated hA3 AR antagonist with an additional pharmacophoric group at
the 5-position, in order to obtain more potent hA3 AR antagonists.
Figure 1. Previous vs. novel strategy to obtain hA3 AR fluorescent antagonists.
References
1. Müller, C.E.; Jacobson, K.A. Recent developments in adenosine receptor ligands and their
potential as novel drugs. Biochim. Biophys. Acta 2011, 1808, 1290-1308.
2. Kuder,K.; Kiec-Kononowicz, K. Fluorescent GPCR ligands as new tools in pharmacology.
Curr. Med. Chem. 2008, 15, 2132–2143.
3. Kozma, E.; Kumar, T.S.; Federico, S.; Phan, K.; Balasubramanian, R.; Gao, Z.G.; Paoletta,
S.; Moro, S.; Spalluto, G.; Jacobson, K.A. Novel fluorescent antagonist as a molecular
probe in A3 adenosine receptor binding assays using flow cytometry. Biochem. Pharmacol.
2012, 83, 1552-1561.
Neurodegenerative disease agents
Short lecture S3.6
Design, synthesis and pharmacological properties of alkylbisammonio bitopic
ligands of muscarinic acetylcholine receptors
Matera C., Dallanoce C., De Amici M.
Dipartimento di Scienze Farmaceutiche - Sezione di Chimica Farmaceutica “Pietro Pratesi”
Università degli Studi di Milano - Via L. Mangiagalli, 25 - 20133 - Milan (Italy)
[email protected]
Over the past two decades, novel opportunities for drug discovery have risen from a
greater understanding of the complexity of GPCR signaling. A striking example of this
is the appreciation that GPCRs possess functional allosteric binding sites.1 The five
muscarinic acetylcholine receptor (mAChR) subtypes bind their physiological
transmitter in the highly conserved orthosteric site within the transmembrane domain of
the receptors. Orthosteric muscarinic agonists have negligible binding selectivity and
poor signaling specificity. A less conserved allosteric site has been also characterized
at the extracellular entrance of the binding pocket of mAChRs. Our interest in the
research field on mAChR ligands has been recently extended to the study of
derivatives designed to simultaneously interact with the orthosteric and the allosteric
site (bitopic ligands) of these receptors, thus exploiting the complementary
characteristics of the two different binding sites by a single ligand molecule.2,3 This
approach is a variation of the more traditional bivalent ligand concept and shares some
of the same challenges, including the choice and role of the linker between the two
pharmacophores and the validation of mechanism of action.4 The most interesting
bitopic compounds which emerged from our investigation (represented by the general
molecular skeleton A in Figure 1 are alkylbisammonio derivatives incorporating a)
iperoxo, an oxotremorine-related unselective muscarinic superagonist, b) a polymethylene spacer chain, and c) a heteroaromatic fragment targeting the allosteric site.
Figure 1. General structure of mAChR bitopic ligands and the orthosteric superagonist Iperoxo.
The synthetic approach of target compounds and the most relevant results of their
pharmacological investigation will be presented. In particular, the analgesic effects
observed for a group of the compounds under study will be illustrated in detail.
References
1. Wang, L.; Martin, B.; Brenneman, R.; Luttrell, L. M.; Maudsley, S. Allosteric Modulators of G
Protein-Coupled Receptors: Future Therapeutics for Complex Physiological Disorders. J.
Pharmacol. Exp. Ther. 2009, 331, 340-348
2. Antony, J.; Kellershohn, K.; Disingrini, T.; Dallanoce, C.; Tränkle, C.; Christopoulos, A.;
Barocelli, E.; De Amici, M.; Holzgrabe, U.; Mohr, K. et al. Dualsteric GPCR targeting: a
novel route to binding and signaling pathway selectivity. FASEB J. 2009, 23, 442-450
3. Bock, A.; Merten, N.; Schrage, R.; Dallanoce, C.; Matera, C.; Hoffmann, C.; De Amici, M.;
Holzgrabe, U.; Kostenis, E.; Mohr, K. et al. The allosteric vestibule of a seven
transmembrane helical receptor controls G-protein coupling. Nat. Commun. 2012, 3, 1044
4. Valant, C.; Lane, J. R.; Sexton, P. M.; Christopoulos, A. The Best of Both Worlds? Bitopic
Orthosteric/Allosteric Ligands of G Protein-Coupled Receptors. Annu. Rev. Pharmacol.
Toxicol. 2012, 52, 153-178
Neurodegenerative disease agents
Short lecture S3.7
Discovery of a new, chemically stable class of 3-HAO inhibitors, endowed with
neuroprotective activity in vivo
Vallerini G. P.*, Amori L.*, Schwarcz R.§, Costantino G.*
*
Università degli Studi di Parma - Dipartimento di Farmacia, Parco Area delle Scienze, 27/A 43124 - Parma - Italy
§
Maryland Psychiatric Research Center - University of Maryland School of Medicine, Baltimore Maryland - 21228 - USA
[email protected]
The kynurenine pathway of tryptophan metabolism (KP) attracted a great interest from
neuroscientists since the observations that several intermediates are endowed with
biological activities in the central nervous system and that an impaired balance
between neuroprotective and neurotoxic metabolites is associated to a number of
neurodegenerative conditions.1 In this framework, the search for inhibitors of 3hydroxyanthranilic acid 3,4-dioxygenase (3-HAO), the enzyme responsible for the
conversion of 3-hydroxyanthranilic acid (3-HANA) into the neurotoxin quinolinic acid
(QUIN) is still a rather unexplored but very promising approach.
Over the last 35 years, only one class of halogenated substrate analogues has been
described as 3-HAO inhibitors.2 This class of compounds is characterized by high
activity, but also by intrinsic chemical instability, due to the propensity of the common
o-aminophenol core to auto-oxidize and generate reactive radical species.
With the aim of developing novel, chemically stable 3-HAO inhibitors, we proposed the
2-aminopyridine 1-oxide scaffold as a bioisosteric replacement of o-aminophenol.
Thus, we designed and synthesized a library of 2-aminonicotinic acid 1-oxide
analogues. The biological results showed interesting 3-HAO inhibitory activity in vitro
for some of the synthesized compounds, in both rat and human brain homogenate, with
IC50 values inferior to 100 µM and maximum activity registered for 2-amino-6methylnicotinic acid 1-oxide (IC50 = 0.6 µM in human brain homogenate). In addition,
HPLC/UV-VIS analysis showed complete chemical stability for these compounds, in
the same conditions in which previously reported inhibitors underwent moderate to full
decomposition. These findings confirmed our initial hypothesis of the bioisosterism
between 3-hydroxyanthranilate and 2-aminonicotinate 1-oxide and, along with data
deriving from inactive derivatives, allowed us to draw a preliminary SAR profile for this
class of novel 3-HAO inhibitors.
The most interesting compound was also assayed for its ability to prevent the
production of QUIN in an in vivo animal model of neurodegenerative condition, the
QUIN-induced brain lesion of the rat striatum. Intrastriatal administration of 2-amino-6methylnicotinic acid 1-oxide was able to strongly decrease the production of QUIN at
the lesion site, also affecting, at a minor extent, that of the synergistic neurotoxin 3hydroxykynurenine (3-HK). Surprisingly, an increase in the production of the
neuroprotective agent, kynurenic acid (KYNA), was also observed. These results show
that localized administration of our 3-HAO inhibitor is able to switch the balance
between neurotoxic and neuroprotective KP metabolites towards the latter, in an in vivo
animal model of excitotoxic stress.
References
1. Schwarcz, R.; Bruno, J.; Muchowski, P.; Wu, H.-Q. Kynurenines in the mammalian brain:
when physiology meets pathology. Nature Reviews. Neuroscience 2012, 13, 465–477
2. Linderberg, M.; Hellberg, S.; Björk, S.; Gotthammar, B.; Högberg, T.; Persson, K.;
Schwarcz, R.; Luthman, J.; Johansson, R. Synthesis and QSAR of substituted 3hydroxyanthranilic acid derivatives as inhibitors of 3-hydroxyanthranilic acid dioxygenase (3HAO). European Journal of Medicinal Chemistry 1999, 34, 729–744
Analytical methods in medicinal chemistry
Main lecture M4
Analytical Challenges in Drug Discovery
Vincenza Andrisano
Department for the Quality of Life Studies, Rimini Campus, University of Bologna
[email protected]
The development of new lead compounds in drug discovery has been of continuous
importance. However, high attrition rates and the decreasing number of new drug
approvals in the past few years have increased the necessity for new development
tools. Lately, “pharmaceutical analysis” has been also focusing on the field of drug
discovery, benefiting strongly from different technological improvements in separation
sciences, modern soft impact mass spectrometry methods coupled with liquid
chromatography, and the use of bioanalytical tools for molecular recognition such as
pure immobilized recombinant enzymes, receptor and transport proteins.
Over the years, many different high throughput screening technologies and
subsequently follow-up methodologies have been developed. All of these can be
categorized, for example according to measurement of analyte classes, assay
mechanisms, readout principles, or screening of drug target classes. When categorized
according to drug target class, assay formats can be subdivided into early hit stage
assays (usually ligand-binding based) that are often straightforward and robust up to
analysis of final cellular effects exerted by ligands. The latter do not only provide higher
content data but also represent anticipated effects in the body more closely. These
assay formats, however, are often elaborate, non-robust, and very time consuming to
conduct but become pivotal when going from the hit-to-lead discovery stage.
In this presentation, an overview of recent developments in the field of protein affinity
selection methods and to the hit-to-lead process is provided. Methods such as
immobilized or dynamic protein/enzyme affinity selection and surface plasmon
resonance (SPR) are reported for the screening of bioactives (ligands) and for the
subtle definition of their binding mode.
Finally, an example of affinity selection methods utilizing mass spectrometry (MS) is
reported among the more recently developed methods that can be informative on hitto-lead cellular effects, overcoming intrinsic problems related to traditional drug
discovery techniques.
Analytical methods in medicinal chemistry
Short lecture S4.1
Development of capillary bioreactors based on purine nucleoside phosphorylase
from Aeromonas hydrophila for biocatalytic applications
Cattaneo G. a, Ubiali D. a, Serra I. a, Rabuffetti M. b, Temporini C. a, Calleri E. a and
Massolini G. a
a
University of Pavia, Department of Drug Sciences, Viale Taramelli 12, 27100 Pavia, Italy
b
University of Milan, Department of Chemistry, Via Golgi 19, 20133 Milano, Italy
[email protected]
Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze the reversible cleavage of the
glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate
(Pi) to generate the nucleobase and α-D-(deoxy)ribose-1-phosphate (R-1-P) (Eq. 1).1
If a second nucleobase is added to the reaction medium the formation of a new
nucleoside can result (transglycosylation). Thus, NPs can be used for chemoenzymatic
synthesis of both natural and unnatural nucleosides, as an alternative to conventional
chemical methods which are generally plagued by low stereoselectivity, multi-step
procedures and modest yields.
We have cloned and over-expressed a purine nucleoside phosphorylase (PNP) from A.
hydrophila (encoded by deoD gene), tested its substrate specificity, and used it for few
synthetic applications, also as immobilized biocatalyst.2,3
The aim of the present work is the development of a biochromatographic system based
on this PNP as a tool to speed up the screening of new nucleoside libraries through a
medium-high throughput approach and to characterize the catalytic efficiency of the
biocatalyst.
To this end, immobilization trials of PNP on different chromatographic supports (e.g.
monolithic and/or Open Tubular Capillary) will be perfomed.
The prepared bioreactors will be typified by the determination of kinetic constants (kM
and Vmax) using the Michaelis-Menten kinetic model for a natural substrate (e.g.
inosine) in phosphorolysis reaction and comparing the results with kinetic constants
calculated for the in batch free enzyme in the same operative conditions.
Experimentally, the characterization will be realized through a bidimensional
chromatographic system with the sample on-line transfer, from the bioreactor to the
analytical column, for the separation and quantification of the substrate and product.4
The most promising bioreactor will be used to verify the system synthetic potential
towards modified nucleosides.
(deoxy)ribonucleoside + Pi
NP
nucleobase + R-1-P
Eq. 1
References
1. Pugmire, M. J.; Ealick, S. E. Structural analyses reveal two distinct families of nucleoside
phosphorylases. Biochem. J. 2002, 361, 1–25.
2. Ubiali, D.; Serra, C. D. et al. Product characterization and synthetic application of a purine
nucleoside phosphorylase from Aeromonas hydrophila. Adv. Synth. Cat. 2012, 354, 96–
104.
3. Serra, I.; Ubiali, D. et al. Developing a collection of immobilized nucleoside phosphorylases
for the preparation of nucleoside analogues: enzymatic synthesis of arabinosyladenine and
2′,3′-dideoxyinosine. ChemPlusChem 2013, 78, 157–165.
4. Moraes, M. C.; Ducati, R. G. et al. Capillary bioreactors based on human purine nucleoside
phosphorylase: a new approach for ligands identification and characterization. J. Chrom. A
2012, 1232, 110–115.
Analytical methods in medicinal chemistry
Short lecture S4.2
LC-ESI-MSn ANALYSES IN THE IDENTIFICATION
of CHEMICALLY REACTIVE METABOLITES
Del Grosso Erika, Aprile Silvio and Grosa Giorgio
Dipartimento di Scienze del Farmaco (DSF), Università del Piemonte Orientale “A. Avogadro”,
Largo Donegani 2, 28100 Novara
[email protected]
Metabolic activation of drugs leading to chemically reactive metabolites (CRMs) able to
covalently interact with proteins is considered an initial step that may lead to druginduced organ toxicity. Hence the characterization of CRMs is critical to design new
drug candidates with an improved safety profile. From the analytical point of view, high
performance liquid chromatography coupled with mass spectrometry predominates
over all analytical tools used for the screening and characterization of CRMs in drug
metabolism studies.1,2 Since the introduction of electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI), LC-MS has been the preeminent
analytical tool used in small molecule drug metabolism investigation. This is primarily
due to the high sensitivity and selectivity of this hyphenated technique and its ability to
separate, detect and structurally identify drug metabolites.2,3
In this work we focused our attention on the structure determination of different classes
of CRMs arising from the metabolism of new candidate antitumoral drugs (e.g.
combretastatin A-1)4,5,6. The presence and the structural characterization of the CRMs
were highlighted by LC-ESI-MSn analyses, also studying the formation of their stable
adducts with nucleophilic trapping agents such as glutathione (GSH), semicarbazide
and 2-mercaptoethanol.
References
[1] Kevin Park B. et al. Nat. Rev. Drug Discov. Managing the challenge of chemically reactive
metabolites in drug development 2011, 10, 292-306.
[2] Shuguang M. Subramanian R. J. Mass Spectrom. Detecting and characterizing reactive
metabolites by liquid chromatography/tandem mass spectrometry2006, 41, 1121-1139.
[3] Kostiainen R. et al. J. Mass Spectrom. Liquid chromatography/atmospheric pressure
ionization–mass spectrometry in drug metabolism studies, 2003, 38, 357.
[4] Saussi MA. et al. ChemMedChem. The Metabolic Fate of isoCombretastatin A-4 in Human
Liver Microsomes: Identification, Synthesis and Biological Evaluation of Metabolites 2011,
6, 1781-1788
[5] Theeramunkong S. et al. J. Med. Chem. Regioselective Suzuki Coupling of
Dihaloheteroaromatic Compounds as a Rapid Strategy To Synthesize Potent Rigid
Combretastatin Analogues, 2011, 54, 4977-4986
[6] Aprile S. et al. J. Pharm.Biomed. Anal. Metabolic fate of combretastatin A-1: LC-DADMS/MS investigation and biological evaluation of its reactive metabolites, 2013, 78-79, 233242
Analytical methods in medicinal chemistry
Short lecture S4.3
Hydroxypyrazolo[1,5-a]pyridine as a fluorescent carboxylic acid isostere:
probing first MedChem bioisosteric applications
Alex Ducime
DSTF – Department of Drug Science and Technology, University of Turin,
Via Pietro Giuria, 9 – 10125 Torino, Italy.
[email protected]
Bioisosteric replacement is a widely used strategy in Medicinal Chemistry for the
rational design of new drugs. Using this approach, the accurate modulation of lead
compounds allow to improve properties such as bioavailability, selectivity and potency.1
A number of bioisosteric relationships have been established for a series of functional
groups including the carboxylic acid.2
Since 2006, at DSTF is investigated the application of hydroxylated pentaatomic
heterocyclic systems (1) as possible bioisosters of acidic moieties, such as carboxyl
groups as well as phenols and salicylamides.3 At the moment, we are focusing our
attention on the hydroxypyrazolo[1,5-a]pyridine scaffold (2), that besides owning an
acidity comparable to the carboxyl group (pKa around 5) shows also interesting
fluorescent properties. A preliminary analysis revealed that this fluorophore possesses
a very impressive quantum yield (around 0.7), suggesting that this structure could be
also used to provide tracking properties. In this sense this moiety can be seen as a first
example of fluorescent isostere of acidic moieties.
Modulation in terms of
electronic (pKa) and
other properties
Taking advantage of these characteristics, we are already investigating first
applications of this new tool. Studies are directed towards the development of possible
fluorescent analogues with applications in Malaria and Neurotransmission fields. In this
latter, hydroxypyrazolo[1,5-a]pyridine-based GABA analogues should afford fluorescent
ligands for the orthosteric binding site in the GABAA receptor.
References
1. Burger, A. Isosterism and Bioisosterism in Drug Design. Progress in Drug Research, 1991,
37, 337.
2. Meanwell Synopsis of some recent tactical application of bioisosteres in drug design.J.
Med. Chem. 2011, 54, 2529.
3. Lolli, M. L.; Hansen, S. L.; Rolando, B.; Nielsen, B.; Wellendorph, P.; Madsen, K.; Larsen,
O. M.; Kristiansen, U.; Fruttero, R.; Gasco, A.; Johansen, T. N. Hydroxy-1,2,5-oxadiazolyl
Moiety as Bioisoster of the Carboxy Function. Synthesis, Ionization Constants, and
Pharmacological Characterization of gamma-Aminobutyric Acid (GABA) Related
Compounds. J. Med. Chem. 2006, 49(14), 4442-4446.
Analytical methods in medicinal chemistry
Short lecture S4.4
Monitoring GSM peptide phosphorylation by Glycogen Synthase Kinase-3β
using Electrospray Ionization Ion Trap Mass Spectrometry (ESI-IT-MS)
D’Urzo A.1, Fiori J.2, De Simone A.3 and Andrisano V.1
1
Dipartimento di Scienze per la Qualità della Vita, 2Dipartimento di Farmacia e Biotecnologie,
3
Università di Bologna, IIT, Fondazione Istituto Italiano di Tecnologia, Genova
[email protected]
Glycogen synthase kinase-3β (GSK-3β) is involved in the phosphorylation of previously
phosphorylated (primed) substrates. Altered GSK-3β activity, normally involving
hyperphosphorylation of its main target tau protein, can contribute to a number of
pathological processes including Alzheimer’s disease (AD). Therefore GSK-3β
represents a very important target for the development of new therapeutic agents for
AD [1].
At present, there are a wide variety of kinase assays that can be used to measure
inhibition [2,3]. ATP-dependent assay as bioluminescent read-outs is an indirect
method where another enzyme (luciferase) is used to detect the degree of
phosphorylation and also is not possible to discriminate between substrate and
product. Radioassay is another approach to assess hyperphosphorylation of primed
substrate by Gsk-3β, but it is not scalable to high-throughput screening of large
compound collections and has significant issues related to the use and disposal of
radioactive materials [2].
On the basis of these considerations, we focused our attention on the development of a
selective methodology suited to specifically monitoring the product of the enzymatic
reaction catalyzed by GSK 3 beta, in view of a more detailed description of the
enzymatic reaction for highlighting the mechanism of action of potential inhibitors.
Therefore, we are validating an Electrospray-Ionization Ion Trap mass spectrometry
(ESI-IT-MS) study to allow for the direct detection of the phosphorylated product which
is formed in a solution reaction involving a primed peptide substrate (GSM peptide) and
GSK-3β. Thus, we will use this method to screen potential inhibitors. Selected hits will
be further characterized by defining their competitive mode of action with the substrate
rather than with the ATP cofactor in view of the discovery of compounds endowed of an
increased selectivity over other protein-kinases.
References
1. C. A. Grimes, R. S. Jope. The multifaceted roles of glycogen synthase kinase 3beta in
cellular signaling. Prog.Neurob. 2001, 65, 391–426
2. Partserniak I, Werstuck G, Capretta A, Brennan JD. An ESI-MS/MS method for screening of
small-molecule mixtures against glycogen synthase kinase-3beta (GSK-3beta).
Chembiochem. 2008 May 5;9(7):1065-73.
3. Andrea Baki, Attila Bielik, László Molnár, Györgyi Szendrei, and György M. Keserü.A. High
Throughput Luminescent Assay for Glycogen Synthase Kinase-3 β Inhibitors. ASSAY and
Drug Development Technologies, 2007 Volume 5, Number 1.75-83
Analytical methods in medicinal chemistry
Short lecture S4.5
Drug binding to albumins by high performance affinity chromatography and
circular dichroism
1
C.Fortugno, 2M.Muraglia, 2F.Corbo, 2C.Franchini, 1C.Bertucci
1. Department of Pharmacy and Biotechnology, University of Bologna,
via Belmeloro 6, 40126 Bologna, Italy
2. Department of Pharmaceutical Chemistry, University of Bari,
Via E. Orabona n.4, 70125 Bari, Italy
[email protected]
The early determination of ADMET (absorbtion, distribution, metabolism, elimination
and toxicity) parameters is nowadays considered a crucial issue of drug discovery and
development. In fact, about half of all drugs in development fail to make it to the market
because of ADME deficiencies. The study of drug binding to serum proteins is
becoming essential in the field of drug discovery to characterize the drug distribution in
human body. Human serum albumin (HSA) is the most abundant protein in plasma and
plays a fundamental role in the transport of drugs, metabolites and endogenous
factors, through the formation of non covalent complexes at specific binding sites; so
the study of the binding mechanism to HSA is essential to the early characterization of
the pharmacokinetic profile of new potential lead1. In this study a series of synthesized
tocainide analogues were characterized for their HSA binding, using two different
analytical techniques: high-performance liquid affinity chromatography (HPLAC) and
circular dichroism (CD). In the HPLAC study HSA was previously covalently
immobilized to the silica matrix of the HPLC column, allowing the binding properties of
the protein to be maintained. With this HSA column it was possible to rank the HSA
binding level by measuring the degree of binding at only one concentration value. This
technique was also used to characterize the high affinity binding sites of these
tocainide analogues to the protein. For this purpose displacement experiments were
carried out utilizing increasing concentrations of competitors known to bind selectively
to the main binding sites of the HSA. To confirm the results obtained by HPALC, the
same drug-protein system was investigated in solution by CD2,3. The same study was
made with rat serum albumin (RSA) to underline the major differences between these
two systems (drug-HSA and drug-RSA) and to evaluate the risk in the extrapolation to
the human of the distribution data obtained during the in-vivo phase of drug discovery.
Comparing the collected data came out that, despite the high similarity of the two
proteins, the slight differences in the aminoacidic sequence could lead to significant
differences in their tridimensional structure that will influence their binding capacity and
their stereoselectivity. Therefore, in the pharmacokinetic studies it’s crucial to consider
the significant differences of various species utilized; and it’s clear also the great
advantage of being able to know this parameter at an early stage of drug discovery.
References
1. C. Bertucci et al. Curr Med Chem. 2002, 9, 1463-1481
2. GA. Ascoli et al. Chirality 2006, 18, 667-679
3. M. Pistolozzi et al. J. Pharmaceut. Biomed. Anal. 2010, 53, 179-185
Analytical methods in medicinal chemistry
Short lecture S4.6
Mass spectrometric strategies for studying albumin covalent modifications
induced by xenobiotics and endogenous electrophilic cytotoxic compounds
Garzon D., Colzani M., Cannizzaro L., Carini M. and Aldini G.
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli
25,20133, Milano, Italia.
[email protected]
Protein covalent modification is involved in the toxic mechanisms of electrophilic
xenobiotics as well as of endogenous cytotoxic compounds such as reactive carbonyl
species generated by oxidative stress. Protein covalent modification is irreversible and
unrepairable and besides inducing a loss of protein conformation and of functional
integrity, it can make the adducted proteins immunogenic and pro-inflammatory.
Identification and characterization of covalent protein adducts represent an important
aspect of pharmaceutical analysis, not only to predict idiosyncratic reactions but also to
elucidate cytotoxic mechanism at cellular levels and to find novel biomarkers of
electrophilic/oxidative stress.
Human serum albumin (HSA) is considered the main blood protein target of circulating
electrophilic compounds and this is due not only to its relevant plasma concentration (≈
0.6 mM) but also because the presence of several accessible nucleophilic sites such
as Cys34, Lys199 and 525. For this reason, HSA protein adducts are searched by
using different analytical techniques and among these, mass spectrometry has attained
a central role because of the wealth of structural and molecular information that can be
obtained. MS approach not only reveals the identity of the adducted protein but also
clarifies the stoichiometry of reaction, the aminoacid site undergoing biotransformation,
the reaction products and hence the mechanism of reaction. Such an approach has
been applied in our laboratory to fully elucidate the HSA binding by amoxicillin (AX) and
by 4-hydroxy-trans-2-nonenal (HNE) which is an endogenous electrophilic RCS
generated by lipid-peroxidation.
The binding of HSA with amoxicillin was investigated since it is considered a key
process for the allergic response of the drug. HSA binding by AX was firstly studied by
a top-down MS approach and then the identification of the adducted HSA sites was
carried out by a novel bottom-up approach based on a precursor ion approach. At the
lowest AX concentration, we detected one main AX-HSA adduct involving residues Lys
190, 199 or 541, whereas higher AX concentrations elicited a more extensive
modification. These findings provide novel tools and insight for the study of protein
haptenation and the mechanisms involved in AX-elicited allergic reactions.
HNE is one of most abundant and toxic lipid-peroxidation derived compound, which
reacts with HSA. In a previous study we fully identified the protein adduct in terms of
stoichiometry, reaction kinetic and sites of adduction [1]. We now set-up a LC-ESIMS/MS approach in order to quantitate the protein adduct. The method consists to
digest the protein and quantitate the adducted peptides by using a triple quadrupole
MS analyzer working in multiple reaction monitoring mode. The isotope dilution
technique was used for quantitative analysis and using deuterium labelled HNE
adducted peptides. The method was firstly validated and it is now applied to measure
HNE-adducted albumin in the sera of patients as a novel biomarker of oxidative stress.
References
1. Aldini G et al.Albumin is the main nucleophilic target of human plasma: a protective role
against pro-atherogenic electrophilic reactive carbonyl species? Chem Res Toxicol. 2008,
21,824-35.
Analytical methods in medicinal chemistry
Short lecture S4.7
Evaporative Light Scattering Detector C-650 – Advantages in flash
chromatography
Daniel Meier, Aleksandra Szpak
Buchi Labortechnik AG, Flawil, Switzerland
[email protected]
Flash chromatography is the method of choice for separation and purification of
reaction mixtures and natural product compositions. Conventionally, compounds are
detected using a UV/VIS detector or differential refractometer - these detectors
have inherent limitations. Here, a novel detector, the Evaporative Light Scattering
Detector (ELSD) C-650 is presented, allowing detection of UV/VIS and non-UV/VIS
absorbing molecules. The separation of a mixture of cholesterol and 4-aminobenzoic
acid with ELS-detection of the pure compounds is reported.
Introduction
Commonly used UV/VIS detectors and differential rifractometer for the detection of
compounds separated by flash column chromatography have significant drawbacks.
The UV/VIS detection principle relies on the molecular interaction with light in the
UV/VIS range. But, only specific molecules, bearing functional groups, e.g. aromatic
moieties, allow this interaction – other molecules cannot be measured. Furthermore,
when eluting with a solvent gradient, the use of refracting index detectors is limited due
to gradient dependent refraction properties of the liquid. It is shown how these
limitations can be avoided by employing an ELS-detector instead. Inside the ELSD the
mobile phase is finely dispersed by means of a nebulizer (Figure 1). The formed
droplets are heated in order to evaporate the mobile phase. Remaining solid particles
of less volatile substances are then detected by a light scattering detector. In addition,
low-temperature evaporation at 30-40 °C, facilitates the detection of thermo-labile and
semi-volatile compounds. Here, the ELS detector was connected to the BUCHI
Sepacore X-50 System. Before detection, two compounds, UV-light absorbing 4aminobenzoeic acid, and non UV-light absorbing cholesterol, were separated using a
hexane/ethyl acetate gradient.
Figure 1. Splitter (left) and nebulizer (right) of the C-650 ELSD
Analytical methods in medicinal chemistry
Short lecture S4.8
Nexera Method Scouting: Maximizing Efficiency for Method Development
Stefano Maria Lucini
Shimadzu Italia S.r.l., Via Giovanni Battista Cassinis, 20139 Milano
[email protected]
The Nexera Method Scouting System design is based on the Nexera next-generation
ultra high performance liquid chromatograph. It uses special Method Scouting Solution
control software and a robust, high-pressure resistant column switching system to
switch mobile phases and columns automatically, achieving a quick and accurate
method scouting workflow. As a result, this system provides strong support for efficient
HPLC method development.
In a typical method scouting procedure, data is collected using a number of mobile
phase and column combinations. Analyzers switching manually between these
combinations are limited by the number of work hours in a single day, making it
impossible to perform method scouting efficiently. The Nexera Method Scouting
System is capable of automatically investigating up to 96 combinations of mobile
phases and columns, without such time restrictions, thereby significantly improving
method development productivity.
If the transfer program is used, the ultra-high speed conditions obtained with the
Nexera Method Scouting System can be easily transferred to conventional conditions.
Furthermore, this software can be used to investigate the robustness of methods under
conventional conditions. The Nexera Method Scouting System is an all-round LC that
enables the collection of ultra-high speed data and conventional data using a single
system.
Method scouting results are important data for determining analysis conditions, so
obtaining highly reliable data is essential. Method Scouting Solution provides the data
required for analysis via a graphical user interface, as well as automated controls
conforming to laboratory operations configured in the system settings, thereby
providing data reliably.
Mobile phase purge conditions and equilibration when automatically switching mobile
phases and columns are key issues in method scouting. With Method Scouting
Solution, scouting reflects laboratory operations, thanks to automatic control of mobile
phase purging and equilibration based on preset system configurations.
The Nexera Method Scouting System is based on the Nexera UHPLC, which has an
established reputation for ultra-high speed analysis. As a result, highly reliable method
scouting results are provided using ultra-high speed analysis featuring stable solvent
delivery at ultra-high pressures, excellent injection repeatability from low injection
quantities, and ultra-low carryover.
The Nexera Method Scouting System achieves stable data acquisition by adopting the
newly developed FCV-34AH (100 MPa pressure resistance) high-pressure-resistant
column switching valve and manifold. In addition, with auto-purging, mobile phase
equilibration times are reduced by purging the mobile phase inside the autosampler,
which means a faster method scouting workflow.
Nutraceutics
Main lecture M5
New perspectives in nutraceutics and functional foods
Vincenzo Fogliano
Department of Agricultural and Food Science University of Naples “Federico II” Portici, Italy
The discussion on functional foods among scientific communities and EC commissions
is mainly focused on definitions and on legislative issues related to the presence on the
market of this kind of products. Although many new products continuously appear, the
functional food market is characterized by a high rate of failure. To realize a successful
functional food, experts having different background should work together following a
detailed workplan. In this paper, the problems related to planning and development of
functional foods are considered using a step-by-step approach. The strategies for
invention and development, formulation and validation of nutritional claims are
regarded also illustrating practical examples of functional food development. The
differences between functional foods and nutraceuticals will be also highlighted.
Nutraceutics
Short lecture S5.1
EXTRACTION, SYNTHESIS AND BIOLOGICAL ACTIVITY EVALUATION OF SOME
NEW SEMISYNTHETIC POLYPHENOLS.
A. Baldisserotto, G. Malisardi, C.B. Vicentini, I. Lampronti, R. Gambari,
S. Vertuani, S. Manfredini
Ferrara University, Department of Life Science and Biotechnology, Ferrara, Italy
[email protected]
Polyphenols are molecules present in vegetable foods with marked antioxidant
properties in vitro. Besides the activity reducing which has so far been attributed to
their biological activity, experimental data show that polyphenols also lead to a
strengthening of endogenous antioxidant defenses and that this enhancement is
achieved through the activation of antioxidant response element (antioxidant
responsive elements, ARE), involved in the induction of antioxidant enzymes and
detoxifying. The molecules that have been selected for this study are Verbascoside
and phloridzna. The Verbascoside is a phenylpropanoid glycoside of plant origin
present in plants of Syringa vulgaris and Buddleja davidii belonging to the Oleaceae
and Scrophulariaceae family respectively, and better known by the common name of
Lilac and Lilac summer. The biological properties of verbascoside, also known as
acteoside, have been described in the literature and include a wide spectrum of
activities including the antioxidant, anti-inflammatory, photoprotective and chelating.
Phloridzin is the main phenolic glucoside from the apple tree and is present in roots,
shoots and leaves, while being also in the skin and in small amounts in the pulp of the
"fruit". Phloridzin plays an important physiological role for the plant, as it acts as a
regulator for the growth and development of the tree and at the same time performs an
action fungicide. Phloridzin may be useful in treating inflammatory states resulting from
oxidative stress, to protect the cardiovascular system from damage by free radicals, in
check osteopenia due to menopause. In order to improve the stability and the
framework of applicability in topical formulations containing these polyphenols, have
been developed the two analogs semisynthesis, named VPP and ES2 able to allow the
use of the molecule in the functional lipid formulations and with cutaneous pH range.
The active ingredients and the two derivatives were then investigated to evaluate the
antioxidant activity (PCL method), antifungal activity (on a selection of dermatophytes),
the inhibitory capacity against the transcription factor NF-kB (Nuclear Factor-kappa B)
and the anti-inflammatory activity, assessing the modulation of IL-8 (Interleukin-8) in
IB3-1 cells fibrocystic.
This work was supported by: the Italian Ministry of Education and Research (PRIN,
Grant 20082L3NFT_003) and Ambrosialab.
Nutraceutics
Short lecture S5.2
QUANTIFICATION OF “CHOLESTEROL LOWERING” SOYASAPONINS IN
LEGUMES AND ASSESSMENT OF THEIR BIOACCESSIBILITY
Caprioli G.1, Sagratini G. 1, Maggi F.1, Font G.2, Mañes J.2, Meca G.2, Ricciutelli M.1,
Sirocchi V.1, Torregiani E.1, Vila-Donat P.1, S. Vittori1
1
School of Pharmacy, University of Camerino, via S. Agostino 1, 62032 Camerino, Italy,
Faculty of Pharmacy, University of Valencia, Avenue Vicent Andres Estelles s/n, 46100
Burjassot, Valencia, Spain
2
[email protected]
Legumes include lentils, beans, peas, chickpeas, lupins, fava beans, soybeans, and
others. Legumes, particularly lentils, are a primary dietary source of food saponins,
which are bioactive compounds that have been demonstrated to possess multiple
health-promoting properties, such as reduction of cholesterol levels, anticarcinogenic
and antihepatotoxic properties, and antireplicative effects against HIV.1 There is
evidence that dietary saponins (including soyasaponins) can lower plasma cholesterol
values; this happens directly by inhibiting absorption of cholesterol from the small
intestine or, indirectly by inhibiting the reabsorption of bile acids. Reduced entry of
cholesterol or bile acids into the enterohepatic circulation results in the stimulation of
cholesterol synthesis mainly by the liver.2 Legumes mainly contain soyasaponin I
(soyasaponin βb) and soyasaponin βg (also called soyasaponin VI), both belonging to
the B group soyasaponins. The aim of this work was the quantification of SS I and SS
βg in raw and cooked legumes of difference provenience and variety (60 samples) by a
SPE-HPLC-MS method developed in our lab.3 Results of this study showed that the 60
raw legume samples analyzed had a content of soyasaponin I that ranged from 636 to
907 mg kg-1, while that of soyasaponin βg ranged from 647 to 1807 mg kg-1. Soybean
contains the highest level of soyasaponins, and also pea and chickpea could be
considered a great source of “cholesterol lowering” soyasaponins. Additionally, it is
important also to understand absorption and metabolic process of soyasaponins in the
body. So, six legume samples were chosen for cooking studies for the investigation of
the amount of soyasaponins that persists after cooking. Changes in the soyasaponins
content were investigated for the first time in chickpea, lentil, bean, pea and chuckling
after the seeds were soaked and cooked in distilled water. Cooking did not modify the
total soyasaponins content and soyasaponin βg was partially degraded during cooking
to soyasaponin I. Moreover, a small amount of soyasaponins leached into the cooking
solution, and the percentage was depending on the legumes analyzed. Moreover, a
study on the bioaccessibility of SS I and βg contained in lentils was determined by
using an in vitro digestion model in such a way to understand the amount that can
reach the duodenum and colon and that can explicate the cholesterol lowering activity.
To our knowledge, no studies have been reported in the literature concerning the
bioaccessibility of soyasaponins in food. This study could be very useful for indicating
the amount of soyasaponins that really reach the duodenal and intestinal tract, which
might be beneficial for inhibiting cholesterol absorption.
References
1. Hu, J.; Lee, S. O.; Hendrich, S.; Murphy, P. A. Quantification of the group B soyasaponins
by high-performance liquid chromatography. J. Agric. Food Chem. 2002, 50, 2587−2594.
2. Ruiz, R. G.; Price, K. R.; Arthur, A. E.; Rose, M. E.; Rhodes, M. J. C.; Fenwick, R. G. Effect
of soaking and cooking on the saponin content and composition of chickpeas (Cicer
arietinum) and lentils (Lens culinaris). J. Agric. Food Chem. 1996, 44, 1526–1530.
3. Sagratini, G.; Zuo, Y.; Caprioli, G.; Cristalli, G.; Giardinà, D.; Maggi, F.; Molin, L.; Ricciutelli,
M.; Traldi, P.; Vittori, S. Quantification of soyasaponins I and βg in Italian lentil seeds by
solid phase extraction (SPE) and high performance liquid chromatography-mass
spectrometry HPLC-MS). J. Agric. Food Chem. 2009, 57, 11226−11233.
Nutraceutics
Short lecture S5.3
Nutraceutical Properties of Methyl 3-O-Methyl Gallate
Curti V.1,2, Capelli E.2, Nabavi SF.3,4, Habtemariam S.5, Nabavi SM.3,4, Daglia M.1
1
Department of Drug Sciences, University of Pavia, Via Taramelli 12, 27100 Pavia, Italy.
2
Department of Earth and Environmental Sciences, Via Taramelli 24, 27100 Pavia,
3
Italy. Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences,
4
5
Tehran,Iran. National Elites Foundation of Iran, Tehran, Iran. Pharmacognosy Research
Laboratories, Medway School of Science, University of Greenwich, Central Avenue, ChathamMaritime, Kent ME4 4TB, United Kingdom.
[email protected]
Over the course of the last 20 years, polyphenols have been studied for their potential
involvement in the prevention of chronic diseases, such as cardiovascular diseases,
cancer, osteoporosis, diabetes mellitus, and neurodegenerative diseases. Their
protective activity has been attributed initially to their antioxidant, free radical
scavenger, and metal chelator properties, then to the capability of inhibiting or reducing
different enzymes, such as telomerase, cycloxygenase, or lipoxygenase, and in more
recent years, to the interaction with signal transduction pathways and cell receptors.1
As regard to chemical structure, they comprise a wide variety of molecules with
polyphenol structure and are generally divided into flavonoids and nonflavonoids.
Among nonflavonoids there are phenolic acids, such as gallic acid, a benzoic acid
derivative. Gallic acid is a natural polyphenolic acid found in gall nuts, sumac, oak bark,
tea leaves, grapes and wine, with potent antioxidant and antiapoptotic activity.2 Methyl3-O-methyl gallate (M3OMG) is a rare natural product, occurring in small amounts in
few species of plants, such as Peltiphyllum peltatum, a medicinal and food plant widely
distributed throughout the world, that can be also synthetized from methyl gallate. It
showed in vitro antioxidant activity and in vivo cardiovascular and neuroprotective
effects.3 Our previous results showed that synthetic origin M3OMG mitigates the NaFinduced cardiotoxicity by modification of abnormality in the oxidant-antioxidant status
measured by the changes in the level of thiobarbituric acid reactive substances
(TBARS), reduced glutathione (GSH) and the activities of the antioxidant enzymes
superoxide dismutase (SOD) and catalase (CAT) in cardiac tissues of rats.
The aim of the present study was to investigate the molecular mechanisms underlying
the antioxidant effects of M3OMG on mononucleated cells obtained from peripheral
blood of healty subjects. To this purpose the variation of the expression of mir-17*
(microRNA) that regulates the expression of antioxidant enzymes, such as manganese
superoxide dismutase (MnSOD), glutathione peroxidase-2 (GPX2) and thioredoxin
reductase-2 (TrxR2), was considered.
The cells were cultured in the presence of M3OMG lasting 1 cell cycle. Survival and
mitotic index were assessed in order to select the useful concentrations. On this basis,
the M3OMG concentrations allowing between 70% and 100% of cell survival have
been selected. Exosomes were recovered from the culture medium and the miRNAs
were isolated. After the reverse transcription reaction, during the which we obtained
the cDNA, we performed a qRT-PCR to investigate the expression of the miR-17* after
the treatment with M3OMG.
References
1. Daglia M. Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 2011, 23, 174-81.
2. Nabavi SM., Habtemariam S., Nabavi SF., Sureda A., Daglia M., Moghaddam AH., Amani
MA. Protective effect of gallic acid isolated from Peltiphyllum peltatum against sodium
fluoride-induced oxidative stress in rat's kidney. Mol Cell Biochem. 2013, 372, 233-9.
3. Nabavi SF., Nabavi SM., Habtemariam S., Moghaddam AH., Daglia M., Abolhasani F.
Cardioprotective effect of methyl 3-O-methyl gallate against NaF-induced oxidative stress
in rats. Fluoride, 2012, 45, 262–268.
Nutraceutics
Short lecture S5.4
Phenolic compounds as inhibitors of the amyloid aggregation of Hen Egg White
Lysozyme: a multidisciplinary approach.
Camilla Giuliani; Marzia Innocenti; Fabrizio Melani; Valentina Noccioli;
Donatella degl’Innocenti; Nadia Mulinacci
University of Florence, Department of NEUROFARBA, Via Ugo Schiff 6, Sesto Fiorentino,
Firenze, Italy
[email protected]
An increasing number of diseases has been linked to proteins amyloid aggregation.
This process consists in tissue deposition, inside or outside the cells, of β sheets
protein structures; these evolve until the formation of mature fibrils in which the β
filaments are aligned perpendicularly to the main axis, from which the name cross β1.
Thus formed amyloidosis were divided into three main classes: neurodegenerative,
localized non-neuropathic and systemic non-neuropathic. To study this phenomena
different proteins for each class are used as in vitro model systems.
This work was focused to study the interaction between natural phenols and hen egg
white lysozyme (HEWL) used as in vitro model of systemic non-neuropathic
amyloidosis2.
The natural polyphenols have been described to be effective in interfering with the
amyloidosis process in some model systems3,4. However the study of the interaction of
these compounds with the protein target was faced with methodological approaches
which show several criticality5.
In our experimental work we tried to define a suitable methodological procedure able to
provide congruent and reproducible qualitative/quantitative results by the integrated
use of different techniques.
The rosmarinic acid and a commercial standardized extract, Vineatrol® (containing tresveratrol and trans-ε-viniferin) were selected to assess their anti-aggregating activity
on HEWL. Cutoff filter, CD and UV spectra, HPLC/DAD analyses and MALDI spectra
were mainly used in this integrated approach.
The analyses carried out both on samples containing HEWL alone and incubated with
the phenols, pointed out anti-aggregation properties for the rosmarinic acid, whereas
the Vineatrol® didn’t show any activity in our model.
To investigate on the possible mechanism of action of rosmarinic acid molecular
dynamics simulations were also performed and the obtained theoretical findings
showed good coherence with the experimental results.
In conclusion this work describes a multidisciplinary approach suitable to be applied for
evaluating the anti-amyloidosis activity of different phenolic compounds in the HEWL in
vitro model.
References
1. Sipe, JD; Cohen, AS. Review: history of the amyloid fibril. Journal of Structure Biology 2000, 130:8898
2. Frare, E; Polverino de Laureto, P; Zurdo, j; Dobson, CM; Fontana, A. A highly amyloidogenic region of
Hen Egg White Lysozyme. Journal of Molecular Biology 2004, 340,5:1153-1165
3. Rivière, C; Papastamoulis, Y; Fortin, PY; Delchier, N; Andriamanarivo, S; Waffo-Teguo, P; Kapche,
WF; Amira-Guebalia, H; Delaunay, JP; Mérillon, JM; Richard, T; Monti, JP.; New stilbene dimers
against amyloid fibril formation. Bioorganic & Medicinal Chemistry Letters 2010, 20:3441–3443
4. Feng, s; Song, XH; Zeng, CM. Inhibition of amyloid fibrillation of lysozyme by phenolic compounds
involves quinoprotein formation. Febs Letters 2012, 586:3951–3955
5. Hudson, SA; Ecroyd, H; Kee, TW; Carver, JA; The thioflavin T fluorescence assay for amyloid fibril
detection can be biased by presence of exogenous compound, The Febs Journal 2009
Nutraceutics
Short lecture S5.5
In-Cell Western Assay: a tool to study the hypocholesterolemic effects of
potentially bioactive food components at HepG2 cell line.
Carmen Lammi, Chiara Zanoni, Anna Arnoldi
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli, 25 –
20133 Milan (ITALY)
[email protected]
The low density lipoprotein (LDL) receptor (LDLR) plays a pivotal role in the binding
and endocytosis of circulating LDL increasing its plasma clearance1. The aim of the
present work is the development of the In-Cell Western (ICW), a quantitative
colorimetric cell-based technique, in order to screen and to evaluate the effects of
potentially active compounds at the LDLR-SREBP2 pathway in a throughput way
(Figure 1). The ICW was optimized and validated at HepG2 cell line using mevinolin as
reference compound. Experiments were performed using fixed cells in order to
establish the best conditions, in which mevinolin is able to properly stimulate the LDLR
protein level. Subsequently, the ICW was used to investigate the activity of peptide
mixtures obtained by partial hydrolysis of lupin protein with pepsin and trypsin. The
ICW optimization and validation provided a robust and reproducible assay useful for
characterizing the cholesterol-lowering properties of bioactive food components.
Figure 1. Assay Principle
References
1. Goldstein, J. L., Brown, M. S.; The LDL receptor. Arterioscler Thromb Vasc Biol 2009, 29,
431-438
Nutraceutics
Short lecture S5.6
Clovamide: a new interesting antioxidant compound with potential nutraceutical
properties
Locatelli M., Coïsson J.D., Travaglia F., *Brunelleschi S.,
*Prat M. and Arlorio M.
Dipartimento di Scienze del Farmaco and DFB Center, Largo Donegani 2, 28100 Novara
*Dipartimento di Scienze della Salute, via Solaroli 17, 28100 Novara
[email protected]
Clovamide is a polyphenolic amide, belonging to the class of N-phenylpropenoyl amino
acids. It is the isosteric amidic analogue of rosmarinic acid and it was firstly identified in
the red clover; successively, clovamide was also described in cocoa (cocoa liquor,
which is the liquid - or paste - produced during chocolate manufacturing, after cocoa
beans are roasted and finely ground) and was suggested as a natural antioxidant,
having elicited interesting properties against lipid peroxidation.1
Since the clovamide molecule is not commercially available, we synthesized it from L3,4-dihydroxyphenylalanine (previously protected as methylester) and caffeic acid, via
DCC (dicyclohexylcarbodiimide)-mediated amidation. The crude clovamide methylester
was then hydrolyzed, and the residue was purified on silica gel column. The natural-like
clovamide was used as a chemical standard for the quantification in cocoa and to test
its potential biological properties.
Clovamide content in cocoa ranged from 1.36 to 2.64 mg/kg in unroasted fermented
beans (dry weight), varying depending on their geographic origin, and was drastically
reduced after roasting process (up to 59% in Arriba cocoa from Ecuador). Also cocoa
hulls, obtained as by-product of pre-roasting of whole beans, contained interesting
amounts of clovamide (about 1.82 mg/kg), suggesting them as an interesting and lowcost source.
Clovamide was then evaluated for their antioxidant (antiradical activity, inhibition of lipid
peroxidation) and anti-inflammatory properties, using both in vitro chemical systems
and in vitro/ex vivo biological models. Preliminary studies on in vitro chemical models
(DPPH• method) revealed that clovamide presents radical scavenging properties
comparable to those showed by rosmarinic and caffeic acid, resulting more effective
than other well known antioxidant, such as epicatechin, trolox, kaempferol and
butylated hydroxyanisole (BHA). Clovamide was also effective in the inhibition of
oxidative stress-induced apoptosis in rat cardiomyoblasts (H9c2 cell line) at nanomicro-molar concentrations, and presented interesting anti-inflammatory effects on
human monocytes, resulting more active than rosmarinic acid concerninig the inhibition
of i) the respiratory burst, ii) the NF-kB activation and iii) the pro-inflammatory cytokine
release.
Finally clovamide was added to phosphatidylcholine liposomes and, under accelerated
oxidation standardized conditions, a significant protective effect against lipid
peroxidation was observed. Moreover, taking into account that more than 50% of
clovamide was incorporated in the liposomes, their use as a structured vehicle for
clovamide could be suggested.
Concluding, although clovamide is present in low amounts in cocoa, its nutraceutical
value and its possible beneficial effects for the treatment of inflammatory disorders was
demonstrated, suggesting its use in functional foods or nutraceuticals design.
References
1. Sanbongi, C.; Osakabe, N.; Natsume, M.; Takizawa, T.; Gomi, S.; Osawa, T.J. Antioxidative
polyphenols isolated from Theobroma cacao. Journal of Agricultural and Food Chemistry,
1998, 46, 454–457
Nutraceutics
Short lecture S5.7
Nutraceutics with potential preventive action against oral cavity pathologies
Papetti A.1, Mascherpa D.1, Carazzone C.1, Stauder M.2 David A. Spratt3, Michael
Wilson3, Jonathan Pratten3, Peter Lingström4, Egija Zaura5, Itzak Ofek6, Caterina
Signoretto7, Carla Pruzzo2, Gabriella Gazzani1
1
Dept. Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
2
DIPTERIS, University of Genoa, Corso Europa 26, 16132 Genoa, Italy
Dept. Microbial Diseases, UCL, 256 Gray’s Inn Road, London, WC1X 8LD, UK
4
Dept. Cariology, Inst. Odontology, University of Gothenburg, Götegborg, Sweden
5
Dept. Cariology, ACTA, University of Amsterdam and Free University Amsterdam, Gustav
Mahlerlaan 3004, 1081 LA Amsterdam, the Netherlands.
6
Dept. Prosthodontics, Hebrew University-Hadassah, 91120 Jerusalem, Israel
7
Dip. Patologia e Diagnostica-Sezione di Microbiologia, Università di Verona, Strada Le Grazie
8 37134 Verona, Italy
3
[email protected]
Caries and Gingivitis are among the most prevalent infectious diseases in humans.
Diet can positively or negatively influence their development, so it could be useful to
individuate foods that can contribute to promote oral health, and identify natural
compounds able to inhibit virulence-linked properties of oral pathogens, such as
Streptococcus mutans, Actinomyces naeslundii and Prevotella intermedia1-6.
To this purpose, a number of foods and beverages were investigated. Among them
Cichorium intybus var. silvestre (red chicory) and Lentinus edodes (shiitake mushroom)
gave extracts active in inhibiting virulence-linked properties of oral pathogens; in fact,
they were able to interfere with the ability of growth of microorganisms, with their
capability of adhesion to hydroxyapatite and KB cells and biofilm production.
Furthermore the extracts induced the disruption of the preformed biofilm.
The fractionation of the extracts showed that in both vegetables a number of active
components able to interfere with growth and/or some virulence traits of the considered
oral pathogens occur. Using HPLC-DAD-ESI-MS/MS, oxalic, succinic, shikimic and
quinic acids were identified in the most active subfractions of both red chicory and
shiitake mushroom. Inosine, adenine, adenosine, uridine and deoxyguanosine were
also found In mushroom. The biological tests on the standard compounds showed that
all the acidic compounds, and above all quinic acid can contribute to the extracts
activity. Among the nucleobases, adenosine, uridine and inosine showed to be active in
at least one biological test. Since these compounds were active in vitro, we can
hypothesise that, in vivo they might interfere with plaque formation that is a prerequisite
for both caries and gingivitis development.
The research leading to these results has received funding from the European Union’s
Sixth Framework Programme (FP6) under the contract FOOD-CT-2006-036210
(Project NUTRIDENT).
References
1. Shmuely, H. Ofek, I., Weiss, E.I., Rones, Z., Houri-Haddad Y. Cranberry components for
the therapy of infectious disease. Curr. Opin. Biotechnol.2012, 23, 148-152.
2. Signoretto, C., Canepari, P., Stauder, M., Vezzulli, L., Pruzzo, C. Functional
foodsbandstrategies contrasting bacterial adhesion. Curr. Opin. Biotechnol.2012, 23, 160167.
3. Loesche, W.J., Grossman, N.S. Periodontal disease as a specific, albeit chronic, infection:
diagnosis and treatment. Clin. Microbiol. Rev. 2001, 14, 727–752.
4. Deng, D.M. van Loveren, C., ten Cate, J.M. Caries-preventive agents induce
remineralization of dentin in a biofilm model. Caries Res.2005, 39, 216–223.
Nutraceutics
Short lecture S5.8
Nutraceuticals and Functional Foods from turmeric: a useful tool in intestinal
motility alteration in mice with DSS induced colitis.
Micucci M.,1 Aldini R.,1 Cevenini M.,2 Roda G.,2 Chiarini A.,1
Mazzella G.,2 and Budriesi R.1
1
Dipartimento di Scienze Farmaceutiche e Biotecnologia, via Belmeloro, 6, 40126, Bologna
2
Dipartimento di Medicina e Chirurgia, Via Massarenti, 9, 40126, Bologna
[email protected]
Ulcerative colitis (UC) is a chronic inflammatory condition of the rectum and colon.
The conventional medical treatment of inflammatory bowel disease (IBD) involves antiinflammatory, immunosuppressive drugs and antibiotics.1 Recently, considerable
attention has been given to the identification of naturally occurring substances,
particularly those present in dietary and medicinal plants as a useful tool in the
prevention and treatment of many pathological conditions. Turmeric (Curcuma longa
L.) is extensively used in Ayurvedic medicine as a treatment for several inflammatory
and functional conditions of the gastrointestinal tract such as diarrhea. Since intestinal
motility has been reported to be impaired in IBD patients, our investigation was aimed
at evaluating whether turmeric, besides its anti-inflammatory properties, exerts also a
myorelaxant effect on the intestine.
Acute and chronic colitis were induced in Balb/c mice by Dextran Sulphate Sodium
(DSS) administration (5% and 2.5%,w/v, respectively) and either Curcuma extract (200
mg/kg/day) or placebo was thereafter administered for 7 and 21 days respectively.
Similarly Curcuma extract was administered to healthy mice and in vitro evaluated in
healthy intestine. The biological activity of Curcuma extract was evaluated towards
Carbachol induced contraction in isolated ileum and colon in healthy animals, acute
and chronic colitis animals and after administration of Curcuma.3
Curcuma extract reduced the spontaneous contractions in the ileum and colon both in
the healthy intestine and in the pathological intestinal strips. In addition, it inhibited the
maximal response to Carbachol in a non-competitive and reversible manner. Similar
results were observed in ileum and colon from Curcuma fed mice. The administration
of DSS reduced the motility, mainly in the colon; Curcuma almost restored both the
spontaneous contractions and the response to Carbachol after 14 days assumption,
compared to standard diet; however a prolonged administration of Curcuma, once the
mucosal damage of the intestine was repaired, reduced the spontaneous and
Carbachol-induced contractions.3
Curcuma extract has been shown to exert a direct and indirect myorelaxant effect on
mouse ileum and colon, independent of the anti-inflammatory effect. The effect is
reversible and non-competitive and it is present also in the normal intestine. These
results suggest the use of curcuma extract as a spasmolytic agent 3 not only in IBD, but
also as a useful tool in the management of functional disorders
References
1. Bossa, F.; Colombo, E.; Andriulli, A.; Annese, V. Treatment of steroid-naive ulcerative
colitis. Expert Opin Pharmacother, 2009, 10, 1449-1460.
2. Jurenka, J.S. Anti-inflammatory properties of curcumin, a major constituent of Curcuma
longa: a review of preclinical and clinical research. Altern Med Rev, 2009, 14, 141-153.
3. Aldini, R.; Budriesi, R.; Roda, G.; Micucci, M.; Ioan, P.; D’Errico-Grigioni, A.; Sartini, A.;
Guidetti, E.; Marocchi, M.; Cevenini, M.; Rosini, F.; Montagnani, M.; Chiarini, A.; Mazzella,
G. Effect of Curcuma longa extract on the intestinal motility in acute and chronic DSS
induced colitis in mice. Plose One, 2012, 7, e44650.
Nutraceutics
Short lecture S5.9
In vitro cardioprotective potential of polyphenols from red grape juice (Vitis
vinifera L. cv. Aglianico N.): nutraceutical perspectives
Tenore G. C.a, Stiuso P.b, Coppola L.c, Campiglia P.d and Novellino E.a
a
Department of Pharmacy, University of Naples Federico II, via D. Montesano 49, 80131 Napoli,
Italy
b
Department of Biochemistry and Biophysics, Second University of Naples, via Costantinopoli
16, 80138 Italy
c
Coppola S.p.a., via S. Maria la Carità, Vicinale De Risi, Scafati (SA), Italy
d
Department of Pharmacy, University of Salerno, via Ponte Don Melillo, 84084, Fisciano (SA),
Italy
[email protected]
Several works have reported on the pharmaceutical usefulness of grape
phytochemicals. Nevertheless, scientific literature needs further studies to consider
grape extracts as useful dietary supplements. The aim of the present work was to
hypothesize for the first time the use of whole commercial red grape juices as food
supplements potentially useful against both physiological and induced cardiac oxidative
stress. First of all, our results indicated a good antioxidant stability of the juice sample
to lyophilisation that may be reasonably regarded as a suitable process for the
formulation of food supplements. Then, the processed sample (lioRGJ) was tested on
cardiac derived H9C2 myocytes in order to ascertain its effects on reactive oxygen
species (ROS) generation and caspase-3 activity incubating cardiomyocytes with
lioRGJ at increasing doses (0.01-1 µg). Experiments showed an appreciable direct
radical scavenging activity at a maximum sample dose of 0.01 µg that made the
caspase-3 activity decrease by about 47% (P < 0.001). Cardiac cells were exposed to
1 μM doxorubicin and its combination with different doses of lioRGJ. Maximum sample
aliquot of 0.01 µg seemed to effectively contrast the induced oxidant injury decreasing
the ROS levels by about 31% and depressing the caspase-3 activity by about 60% (P <
0.001). In both assays, pro-oxidant effects at higher sample concentrations were
detected as indicated by the increase in both ROS generation and apoptotic activity.
Our data suggested for the juice sample the possibility to be employed as a food
supplement with prospective cardioprotective benefits although further studies are
needed to optimize its dosages in order to avoid harmful pro-oxidant effects.
Nutraceutics
Short lecture S5.10
Functional foods for human health benefits: focus on lupin
Chiara Zanoni, Carmen Lammi, Anna Arnoldi
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano,
Via Mangiagalli, 25 – 20133 Milan (ITALY)
[email protected]
In the last decades, numerous studies have reported the beneficial effects of soy
proteins and in 1999, the America Food and Drug Administration has approved the
health claim that soy protein is useful to reduce the risk of cardiovascular disease,
since it is able to control the serum cholesterol level. Subsequently, nutritionists have
now a main interest in soy and other legume proteins not only for their nutritional role,
but also for the positive effects that they may exert on the human body, including
hypotensive, hypoglycemic, hypolipidemic, anti-carcinogenic and anti-obesity activities.
Numerous teams around the world have started to investigate the health benefits
deriving from the consumption of different legumes, such as lupin, chickpea, common
bean, faba bean, pea, lentils, etc.
In this scenario, lupin is a very special case, since its seed contain up to 35-40%
protein as in the case of soybean, but are completely devoid of isoflavones1.
Nowadays, four Mediterranean species of lupin are cultivated, i.e. Lupinus albus, L.
angustifolius, L. luteus and L. mutabilis.
Numerous teams have studied the biological activities of lupin proteins and
investigated the possible health benefits derived from their consumption. In particular,
studies have evaluated either in animal model (rats) or in clinical study the
hypocholesterolemic, hypoglycemic and anti-hypertensive effect of lupin.2-4 Another
study has shown, in a rabbit model, a protective activity of lupin protein against
atherosclerosis progression.5 In parallel, the mechanism of action at molecular level is
under investigation6.
References
1. Sirtori, C. R.; Galli, C.; Anderson, J. W.; Sirtori, E.; Arnoldi, A. Functional foods for
dyslipidaemia and cardiovascular risk prevention. Nutrition Research Reviews 2009, 22,
244-261.
2. Sirtori, C. R.; Lovati, M. R.; Manzoni, C.; Castiglioni, S.; Duranti, M.; Magni, C.; Morandi, S.;
D'Agostina, A.; Arnoldi, A. Proteins of white lupin seed, a naturally isoflavone-poor legume,
reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. Journal of
Nutrition 2004, 134, 18-23.
3. Lovati, M. R.; Manzoni, C.; Castiglioni, S.; Parolari, A.; Magni, C.; Duranti, M. Lupin seed γconglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption
of HepG2 cells. Br J Nutr 2012, 107, 67-73.
4. Pilvi, T. K.; Jauhiainen, T.; Cheng, Z. J.; Mervaala, E. M.; Vapaatalo, H.; Korpela, R. Lupin
protein attenuates the development of hypertension and normalises the vascular function of
NaCl-loaded Goto-Kakizaki rats. J Physiol Pharmacol 2006, 57, 167-76.
5. Marchesi, M.; Parolini, C.; Diani, E.; Rigamonti, E.; Cornelli, L.; Arnoldi, A.; Sirtori, C. R.;
Chiesa, G. Hypolipidaemic and anti-atherosclerotic effects of lupin proteins in a rabbit
model. British Journal of Nutrition 2008, 100, 707-710.
6. Lammi,C.; Zanoni, C.; Arnoldi, A.; In-Cell Western Assay: a tool to study the
hypocholesterolemic effects of potentially bioactive food components at HepG2 cell line.
Molecular Nutrition and Food Research submitted
Antimicrobial agents
Main lecture M6
The quest for new antimicrobial agents: a global emergency
Stefano Biondi
Allecra Anti Infectives - Saint Louis, France
[email protected]
The considerable therapeutic arsenal constituted by available antibiotics generated a
false sense of confidence, inducing researchers to think that available drugs against
bacterial infections were adequate and sufficient, thus diverting investments from this
therapeutic area.
Conversely, bacteria kept evolving, creating an ever-growing number of resistant
strains, which have a potential to develop untreatable, life-threatening infections.
Therefore, research is currently living its second childhood, and efforts are being made
to recover lost ground and regain control of the situation.
During this talk the main problems connected with multidrug-resistant bacterial
infections will be illustrated, and the ongoing lines of research aimed at fighting
resistant strains will be presented.
Antimicrobial agents
Short lecture S6.1
New dual inhibitors of RT associated functions: investigation on their
mechanism and mode of action.
Distinto S.,1 Bianco G.,1 Meleddu R.,1 Cannas C.,1 Del Vecchio C.,2 Cottiglia F.,1 Alcaro
S.,3 Parolin C.,4 Tramontano E.,1 and Maccioni E.1
Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy;
Department of Molecular Medicine, University of Padua, 35121 Padua, Italy;
Department of Health Sciences, University of Catanzaro, 88100 Catanzaro, Italy;
Department of Biology, University of Padua, 35121 Padua, Italy.
[email protected]
The design of multiple-acting ligands has become a fascinating challenge for the
therapy of diseases with complex pathological mechanism such HIV/AIDS.1 The aim of
this approach is targeting with one drug two or more receptors/enzymes or even two
separate functions of a single enzyme. In this regard the development of compounds
inhibiting both RT associate RNA-dependent DNA polymerase (RDDP) and RNase H
activities (Figure 1) would have several advantages, leading to a complete block of RT
functions, new favourable drug resistance profiles, the reduction of combining more
drugs, and, hence, their toxic side effects. In a previous study we have identified novel
inhibitors of both HIV-1 RT-associated activities in the low micromolar range.2 Pursuing
this aim we have designed and synthesized a new class of molecules. The compounds
were assayed for RT enzyme activities and exhibited dual inhibition properties in the
low micromolar range. Interestingly, single amino acids mutations in the NonNucleoside Inhibitors binding pocket strongly affected the RNase H inhibition, without
reducing their DNA polymerase inhibition ability. In order to understand the possible
mechanism of action biochemical and computational approaches were applied.
Figure 1. Structure of HIV-1 RT. The polymerase catalytic triad is highlighted in yellow and the
RNase H DEDD motif, located on the other side of the p66 subunit, in orange.
References
1. Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging drug discovery paradigm.
J. Med. Chem. 2005, 48, 6523-6543.
2. Distinto, S.; Esposito, F.; Kirchmair, J.; Cardia, M. C.; Gaspari, M.; Maccioni, E.; Alcaro, S.;
Markt, P.; Wolber, G.; Zinzula, L.; Tramontano, E. Identification of HIV-1 reverse
transcriptase dual inhibitors by a combined shape-, 2D-fingerprint- and pharmacophorebased virtual screening approach. Eur. J. Med. Chem. 2012, 50, 216-229.
Antimicrobial agents
Short lecture S6.2
Design and synthesis of new inhibitors
of human helicase DDX3 as antiviral agents
A. Brai,a M.Gallo,a R. Fazi,a C. Tintori,a J. Esté,b A. Garbelli,c G. Maga,c and M. Botta a
a
Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena,
via Aldo Moro, 53100 Siena, Italy.
b
Retrovirology Laboratory Irsi Caixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de
Barcelona, 08916 Barcelona, Spain.
c
Istituto di Genetica Molecolare, IGM-CNR, Via Abbiategrasso 207, 127100 Pavia, Italy.
[email protected]
Compounds currently used for the treatment of viral infections are targeted to viral proteins.
However, the high intrinsic mutation and replication rates, often lead to the emergence of drug
resistant strains and consequent therapeutic failure. On this basis, the main argument in favor of
targeting a host factor instead of a viral protein is the predicted low drug resistance level. One
protein that has recently attracted much attention is DDX3. DEAD box polypeptide 3 (DDX3) is a
DEAD box RNA helicase whose endogenous function is involved in mRNA splicing, export,
transcriptional and translational regulation, and ribosome biogenesis. Interestingly, DDX3 appears
to be a prime target for viral manipulation: at least four different viruses, namely Hepatitis C virus
(HCV), Hepatitis B virus (HBV), Human Immunodeficiency Virus (HIV) and poxviruses, encode
proteins that interact with DDX3 and modulate its function. HIV and HCV seem to co-opt DDX3 and
require it for their replication. It has therefore been suggested that DDX3 could be a novel target for
the development of drugs against these two viruses. DDX3 has multiple enzymatic activities
(ATPase and RNA helicase) and functional domains that may be targeted by potential inhibitors.
Several compounds identified through computational studies, have been synthesized and sent to
the biological assays1, among them some compounds showed submicromolar activity towards the
helicase binding site of DDX3.
References
1. Radi, M.; Falchi, F.; Garbelli , A.; Samuele, A.; Bernardo, V.; Paolucci, S.; Baldanti, F.; Schenone, S.;
Manetti, F.; Maga, G.; Botta, M. Discovery of the first small molecule inhibitor of
human DDX3 specifically designed to target the RNA binding site: Towards the next generation HIV-1
inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 2094-2098.
Antimicrobial agents
Short lecture S6.3
Praziquantel analogues containing no-donor furoxans and related furazans
as agents active against Schistosoma mansoni.
Cortese D.1,2, Guglielmo S.1, Vottero F.1, Kommer V. P. 2, Williams D. L.2*, Fruttero R.1*, Gasco A.1
1
Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Via P. Giuria 9, 10125 Torino, Italy.
2
Department of Immunology/Microbiology, Rush University Medical Center, 1735 West Harrison Street,
Chicago, IL 60612 USA
[email protected]
Today around 200 million people worldwide are parasitized by several species of
Schistosoma and in particular S. mansoni, and more than 200 000 people die every year from this
neglected disease. Praziquantel (PZQ) is the main drug currently used in infection by schistosome.
The mechanism of action of this drug has not been elucidated; however, inhibition of calcium
channels and inhibition of adenosine uptake have been suggested to be involved in PZQ activity.
Since PZQ-resistant worms have been identified, the development of new anti-schistosomiasis
drugs is urgently needed.1 Recently, several furoxans (1,2,5-oxadiazole 2-oxides) (Figure 1 A)
have been shown to be endowed with good activity against S. mansoni. The proposed mechanism
of action consists in the inhibition of thioredoxin glutathione reductase (TGR), an essential enzyme
for parasite redox balance, through nitrosylation of cysteine and/or selenocysteine residues of the
protein. This reaction takes place as a consequence of the interaction of the compounds with the
enzyme and subsequent NO release.2 On this basis we decided to conjugate PZQ and furoxan
pharmacophores in a single molecule in order to obtain dual drugs exploiting antischistosomal
properties through two different mechanisms of action. Through slight modifications of inexpensive
and straightforward synthesis,3,4 two different series of novel PZQ derivatives were prepared. In the
first group of products, the furoxan substructures were substituted for the cyclohexyl group of PZQ
(Figure 1 C) whereas in the second group of hybrids they were linked through appropriate bridges
to the 10-position of PZQ (Figure 1 D). For both these groups we prepared the related products
bearing the corresponding furazan (1,2,5-oxadiazole) (Figure 1 B) moieties devoid of NO-donor
properties. All the compounds have been subjected to structural and pharmacological
characterization for their activity against adult ex vivo worms and their capability to inhibit TGR
activity.
N
N
O
PZQ
O
4
N
5
N
3
O
+
O
N (O )
n
2
Furoxan/furazan
moieties
O
O
N
N
1
A n=1 Furoxan
B n=0 Furazan
N
Furoxan/furazan
moieties
C
O
D
Figure 1. General structure of NO-donor PZQ hybrids and of the related des-NO furazans.
References
1. Doemling, A.; Khoury, K. Praziquantel and schistosomiasis. ChemMedChem 2010, 5, 1420-1434.
2. Simeonov, A.; Jadhav, A.; Quantitative high-throughput screen identifies inhibitors of the Schistosoma
mansoni redox cascade. PLoS. Negl. Trop. Dis. 2008, 2 (1), e127.; Sayed, A. A.; Simeonov, A.;
Identification of oxadiazoles as new drug leads for the control of schistosomiasis. Nat. Med. 2008, 14,
407-412.; Rai, G.; Sayed, A. A.; Structure mechanism insights and the role of nitric oxide donation guide
the development of oxadiazole-2-oxides as therapeutic agents against schistosomiasis. J. Med. Chem.
2009, 52, 6474-6483.
3. Formation of pyrazinoisoquinoline ring system by the tandem amidoalkylation and N-acyliminium ion
cyclization: an efficient synthesis of praziquantel. Tetrahedron 1998, 54, 7395-7400.
4. Cao, H.; Liu, H.; Doemling, A. Efficient multicomponent reaction synthesis of the schistosomiasis drug
praziquantel. Chem. Eur. J. 2010, 16, 12296-12298.
Antimicrobial agents
Short lecture S6.4
Development of a new scaffold of 1,3-thiazolidin-4-one derivatives as promising
antifungal agents with low cytotoxicity
Celeste De Montea, Simone Carradoria, Melissa D’Ascenzioa, Bruna Bizzarria, Daniela
Seccia, Daniela Rivanerab, Alessanda Zicaric, Emanuela Maric
a
c
Dip. di Chimica e Tecnologie del Farmaco, bDip. di Sanità Pubblica e Malattie Infettive, and
Dip. di Medicina Sperimentale,Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
[email protected]
Candida species are the most common cause of opportunistic mycoses in humans,
ranging from superficial mucocutaneous infections to invasive systemic diseases,
especially in immunocompromised patients and in those receiving broad spectrum
antibiotic therapy, aggressive cancer treatment, or transplantation therapy. Due to all
these risks factors and to the increasing development of drug resistance, there is an
emergent demand for the design of new antifungal agents.
On the basis of the recent reports on the therapeutic importance of thiazolidin-4-one
nucleus with regard to its antifungal activity,1 we developed a new scaffold of promising
anti-Candida derivatives by combining the thiazolidin-4-one nucleus and
(cyclo)aliphatic or (hetero)aryl substituted hydrazines (compounds 1A-59A).
Functionalization of the lactamic nitrogen with a benzyl group (compounds 1B-59B)
was performed in order to evaluate the influence of steric hindrance and lipophilicity of
this part of the molecule on the pharmacological effect (Figure 1). Compounds 1A-59A
and 1B-59B were tested against twenty-two clinical Candida spp. strains (C. albicans,
C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, and C. sakè), and their minimum
inhibitory concentration (MIC) was determined according to the established guidelines
of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee
for Antimicrobial Susceptibility Testing (EUCAST).2 The antifungal activity of tested
compounds was similar or even better than topical and systemic reference drugs
(clotrimazole, fluconazole, ketoconazole, miconazole, tioconazole, amphotericin B, and
griseofulvin). Finally, the most active derivatives were also tested for cell toxicity (EC50)
against Hep2 cells showing less toxicity than clotrimazole.
Figure 1. Chemical modifications of the thiazolidin-4-one scaffold
Aliphatic chains
(C3-C8)
Unsubstituted or alkyl-cycloaliphatic rings
(C3-C8)
(Hetero)aryl or bicyclic rings
furan, thiophene, pyridine, (substituted)aryl,
pyrazine, indole, benzofuran, benzodioxole,
benzodioxane, chromene, naphthalene,
phenantrene, ferrocene
R
N
N
N
S
O
R = H (1A-59A),
Bz (1B-59B)
2-substituted-1,3-thiazolidin-4-ones
References
1. Jain, A. K.; Vaidya, A.; Ravichandran, V.; Kashaw, S. K.; Agrawal, R. K. Recent
developments and biological activities of thiazolidinone derivatives: A review. Bioorg. Med.
Chem. 2012, 20, 3378-3395.
2. National Committee for Clinical Laboratory Standard. Reference method for broth dilution
antifungal susceptibility testing of yeasts; approved standard 3rd edition. DM27-A3 vol 28 n
14 National Committee for Clinical Laboratory Standard, Wayne, Pa.
Antimicrobial agents
Short lecture S6.5
Structural investigation of Small Molecules Targeting
Influenza A Virus Polymerase
Jenny Desantis,a Giulio Nannetti,b Serena Massari,a Giulia Muratore,b Stefano
Sabatini,a Luca Sancineto,a Giuseppe Manfroni, a Giorgio Palù,b
Arianna Loregian,b Oriana Tabarrinia
a
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, 06123 Perugia;
b
Dipartimento di Medicina Molecolare, Università di Padova, 35121, Padova
[email protected]
Influenza (Flu) viruses are human respiratory pathogens, responsible for both seasonal
epidemics and global pandemics. The currently available anti-Flu drugs, i.e., M2
blockers and neuraminidase inhibitors, are considered still inadequate and may
become ineffective when resistant viral strains emerge.1 New antiviral strategies
against Flu, with an innovative mechanism of action, are urgently needed.
The viral RNA-dependent RNA polymerase (RdRP), and in particular its correct
assembly to a heterotrimer formed by the subunits PB1, PB2, and PA, provides an
underexploited drug target.
Using the crystal structure of a truncated form of PA bound to a PB1-derived peptide,2
an in-silico screening of small molecule libraries recently identified some promising
compounds able to specifically interfere with the PA-PB1 interaction.3,4 The inhibitory
activity translated in the ability to block virus growth in cell cultures at non-cytotoxic
concentrations, providing a proof-of-principle for this antiviral strategy that could
present several advantages compared to the existing drugs.
Starting from one of the most interesting hits, a series of analogues have been
designed and synthesized with the aim to increase the PA/PB1 inhibitory activity and to
achieve a potent anti-Flu activity also encompassing clinical isolates and drug-resistant
strains.
In this work, the structural optimization cycle that led to identify improved anti-Flu
derivatives along with preliminary SAR information, will be presented.
References
1. De Clercq, E. Antiviral agents active against influenza A viruses. Nat. Rev. Drug. Discov.
2006, 5, 1015-25.
2. Obayashi, E.; Yoshida, H.; Kawai, F.; Shibayama, N.; Kawaguchi, A.; Nagata, K.; Tame,
JR.; Park, SY. The structural basis for an essential subunit interaction in influenza virus
RNA polymerase. Nature. 2008, 454,1127-31.
3. Muratore, G.; Goracci, L.; Mercorelli, B.; Foeglein, Á.; Digard, P.; Cruciani, G.; Palù, G.;
Loregian, A. Small molecule inhibitors of influenza A and B viruses that act by disrupting
subunit interactions of the viral polymerase. Proc. Natl. Acad. Sci. USA 2012, 109, 6247-52.
4. Loregian, A.; Palù, G.; Muratore, G.; Cruciani, G.; Tabarrini, O. Nuovi composti contro i
virus dell’influenza A e B che interferiscono con interazioni tra subunita’ della polimerasi
virale. PCT Application PCT/EP2012/052914. 2. 21. 2012.
Antimicrobial agents
Short lecture S6.6
Development of Novel Rhodesain Inhibitors characterized by the presence of the
3-Bromo Isoxazoline Moiety as the Warhead
R. Ettari,a L. Tamborini,a A. Pinto,a I. C. Angelo,b S. Grasso,b Tanja Schirmeister,c C. De
Micheli,a P. Contia
a
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Mangiagalli 25,
b
20133 Milano, Italy; Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Università
degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy; cInstitute of Pharmacy and
Biochemistry, University of Mainz, Staudinger Weg 5, D-55099 Mainz, Germany.
[email protected]
Human African Trypanosomiasis (HAT) is a parasitic disease transmitted by tse-tse flies
that afflicts millions of people in 36 countries in sub-Saharan Africa, with an estimated
30,000 cases/year. The parasites responsible of the human disease are Trypanosoma
brucei gambiese and T. b. rhodesiense..Current chemotherapy of HAT relies on the
use of the following four drugs: suramin, melarsoprol, pentamidine and eflornithine.
These drugs possess a sizable toxicity, are difficult to administer and produce an
acquired drug resistance. For all these reasons there is an urgent need to identify new
potential molecular targets.
Rhodesain, a cathepsin L-like cysteine protease of T. brucei rhodesiense, is needed to
the parasite to cross the blood-brain barrier leading to the second stage of the
pathology, the sleeping sickness. It takes also part to the degradation of host
immunoglobulines and it is involved in the turnover of variant surface glycoproteins
(VSG) of T. brucei coat, preventing the host immune response. For these reasons,
rhodesain is considered a relevant target for HAT treatment.1
In the recent past, our research group has been actively involved in the development of
novel rhodesain inhibitors characterized by the presence of a 1,4-benzodiazepine
scaffold as peptidomimetic moiety which bears different warheads.2 We now report a
series of peptidomimetic rhodesain inhibitors bearing the 3-bromo isoxazoline nucleus
(i.e. 1, Figure 1), as an innovative electrophilic moiety capable to react with Cys25
present in the catalytic site of the enzyme. In a second series of derivatives such a
moiety was incorporated into a bicyclic structure (i.e. 2, Figure 1). The results of this
investigation will be reported and discussed.
Figure 1. General structure of novel peptidomimetic rhodesain inhibitors
References
1. Steverding, D.; Sexton, D. W.; Wang, X.; Gehrke, S. S.; Wagner, G. K.; Caffrey, C. R.
Trypanosoma brucei: Chemical evidence that cathepsin L is essential for survival and a
relevant drug target. Int. J. Parasitol. 2012, 42, 481–488.
2. (a) Ettari, R.; Zappalà, M.; Micale, N.; Schirmeister, T.; Gelhaus, C.; Leippe, M.; Evers, A.;
Grasso, S. Synthesis of novel peptidomimetics as inhibitors of protozoan cysteine proteases
falcipain-2 and rhodesain. Eur. J. Med. Chem. 2010, 7, 3228–3233; (b) Bova, F.; Ettari, R.;
Micale, N.; Carnovale, C.; Schirmeister, T.; Gelhaus, C.; Leippe, M.; Grasso, S.; Zappalà,
M. Constrained peptidomimetics as antiplasmodial falcipain-2 inhibitors. Bioorg. Med.
Chem. 2010, 18, 4928–4938.
“With the financial support of Ministero degli Affari Esteri, Direzione Generale per la Promozione del Sistema Paese”.
Antimicrobial agents
Short lecture S6.7
Sulfonamidomethaneboronic acids as β-lactamase inhibitors:
from fragment-based drug design to in vivo activity
Romagnoli C., Eidam O., Dalmasso G., Bonnet R., Shoichet B. K. and Prati F.
Università degli Studi di Modena e Reggio Emilia, Dipartimento di Scienze della Vita, Via Campi
287, 41125 Modena
[email protected]
Target. Antibiotic resistance has been identified by the World Health Organization as
one of the three most important problems for human health. Particularly, β-lactamases
are the largest spread and most efficient method of resistance to β-lactam antibiotics,
such as penicillins and cephalosporins. One strategy for restoring β-lactams activity is
the development of novel β-lactamase inhibitors. Recently we developed
sulfonamidomethaneboronic acids with Ki in the nanomolar range.1 To rationally guide
their optimization we turned towards the recent developed fragment-based drug
discovery technique.
H
H
Design. Our idea was to derivatize our
(TMS) N
N
N
S
S
O O B
O O B
B
previous inhibitors adding anionic groups,
O
O
O
O
OH
HO
COOH
COOMe
such as a carboxylate (I) or its bioisostere
I
K vs AmpC 1.3 nM
tetrazole (II) (Figure 1), that are known to bind
N N
the distal site of AmpC in non boronic NC
N N
N
N
2,3
H
N
N
X
N
H
H
fragments.
H
H
S
N
X
X
N
S
S
R O O B
O
O
O
O
Synthesis. Sulfonamidomethaneboronic acids
R
B
O O B
R
O
O
HO
OH
I-II were obtained through sulfonylation of the
II
K vs AmpC 0.05-3.0 nM
intermediate
aminomethaneboronate.
Cycloaddition of a cyano group with Figure 1. Synthetic scheme for anionic
trimethylsilylazide allowed to obtain tetrazole sulfonamidomethaneboronic acids.
derivatives II.
Enzimology. All the new compounds exhibit Ki values against AmpC, a class C βlactamase, in the singlet digit and subnanomolar range, with activity improved as much
as 4000-fold in comparison to previous compounds that don’t target the distal site.
Structural analysis. X-ray crystal structures of the inhibitors in complex with AmpC
confirm that the inhibitors interact both with the catalytic and the distal sites, validating
the design procedure.
In vivo test. In a mouse systemic infectious model, cefotaxime allowed only 15%
survival at 120h post infection. Interestingly, the combination of one selected
compound II with cefotaxime rescued 65% of animals. For what we know, this is the
first proof of in vivo efficacy of a boronic acid as β-lactamase inhibitor.
2
i
i
References
1. Eidam, O.; Romagnoli, C.; Caselli, E.; Babaoglu, K.; Teotico Pohlhaus, D.; Karpiak, J.;
Bonnet, R.; Shoichet, B. K.; Prati, F. Design, Synthesis, Crystal Structures, and Antimicrobial
Activity of Sulfonamide Boronic Acids as β-Lactamase Inhibitors, Journal of Medicinal
Chemistry 2010, 53 (21), 7852-7863.
2. Babaoglu, K.; Shoichet, B. K. Deconstructing fragment-based inhibitor discovery. Nat. Chem.
Biol. 2006, 2, 720-3.
3. Teotico, D. G.; Babaoglu, K.; Rocklin, G. J.; Ferreira, R. S.; Giannetti, A. M.; Shoichet, B. K.
Docking for fragment inhibitors of AmpC beta-lactamase. Proc. Natl. Acad. Sci. USA 2009,
106, 7455-60.
Antimicrobial agents
Short lecture S6.8
2-Phenylquinazolinone fragment imparts anti-CDKs and anti-HIV activities
Luca Sancineto, Nunzio Iraci, Serena Massari, Stefano Sabatini, Giuseppe Manfroni,
Violetta Cecchetti, Oriana Tabarrini.
Dipartimento di Chimica e Tecnologia del Farmaco, Universita’ di Perugia, Via del Liceo,1
06123
[email protected]
The emergence of multidrug resistant viral strains along with the inability of the
current drug regimen to completely eradicate the virus in the HIV infected individuals,
demands new drugs capable of interfering with alternative targets or steps of the viral
replicative cycle. An appealing strategy could be the interference with host factors
involved in the Tat-mediated transcription.1 Among them, CDK9, the catalytic subunit of
the Positive Transcriptional Elongation Factor b (P-TEFb), plays a pivotal role in
sustaining high levels of HIV transcription representing a promising target for antiviral
therapy.2
Taking advantage of the crystallographic data of CDK9 in complex with the best
characterized inhibitor, flavopiridol,3 we have performed a structure-based drug design
that followed by a structural optimization cycle, led to identify and characterize a new
class of nontoxic anti-CDK9 chemotypes based on the 2-phenylquinazolinone scaffold.
Inhibition of CDK9 translated into the ability to interfere selectively with Tat-mediated
transactivation of the viral promoter and in the inhibition of HIV-1 reactivation from
latently infected cells at low micromolar concentrations.
The appeal of the reported 2-phenylquinazolinones is strenghtened by the lack of
toxicity in several cell lines. In addition, being still fragments,4 there is plenty of room for
optimization to increase both potency and selectivity
The whole drug discovery workflow will be the object of the presentation.
References
1. Massari, S.; Sabatini, S.;Tabarrini, O. Blocking HIV-1 Replication by Targeting the TatHijacked Transcriptional Machinery. Curr Pharm Des. 2013,19, 1860-79.
2. Flores, O.; Lee, G.; Kessler, J.; Miller, M.; Schlief, W.; Tomassini, J.; Hazuda, D. Host-cell
positive transcription elongation factor b kinase activity is essential and limiting for HIV type
1 replication. Proc Natl Acad Sci U S A 1999, 96, 7208-7213.
3. .Baumli, S.; Lolli, G.; Lowe, E. D.; Troiani, S.; Rusconi, L.; Bullock, A. N.; Debreczeni, J. E.;
Knapp, S.; Johnson, L. N. The structure of P-TEFb (CDK9/cyclin T1), its complex with
flavopiridol and regulation by phosphorylation. Embo J 2008, 27, 1907-18.
4. Sancineto, L.; Iraci, N.; Massari, S.; Tabarrini, O. From Small to Powerful: The Fragments
Universe and its "Chem-Appeal". Curr. Med. Chem. 2013, 20, 1355-1381.
Antimicrobial agents
Short lecture S6.9
Identification of HIV-1 nucleocapsid protein inhibitors by FRET technological
platform
M. Serena1, L. Sinigaglia1, A. Sosic 1, B. Gatto 1
1
Dipartimento di Scienze del Farmaco, via Marzolo 5, 35131 Padova
[email protected]
HIV-1 Nucleocapsid protein (NCp7) is a short, basic, nucleic-acid binding protein,
which has a crucial role at different stages of HIV replicative cycle: it acts as nucleic
acid chaperone at several points in the replication cycle of retrovirus, in particular at
several strand transfer steps that occur subsequent to the initiation of reverse
transcription. NC binds to the trans-activation response element TAR and its
complementary DNA sequence cTAR, destabilizes their highly stable secondary
structures thus allowing correct strand transfer reactions to proceed during reverse
transcription1. Therefore the NC-viral nucleic acids complex could represent an
attractive target for the development of new antiviral agents. Indeed, the potential
interference induced by small molecules towards NC would result in a reduction of the
reverse transcription of the viral genome as a consequence of a compromised NC
activity. This approach would ultimately bring new insights into the development of
successful anti-HIV agents.
In our work we developed a simple, fast and reliable FRET based assay2 to test the
inhibitory activity of a large number of small molecules on recombinant full-length NC.
The protein possesses several functions during viral replicative cycle in addition to the
chaperone activity. Using the same technological platform, we characterized NC
biological activity on both TAR and cTAR structures and determined the best
experimental conditions to avoid its aggregation ability for better performance of the
assay.
We screened our in-house chemical library constituted by more than 100 molecules,
representing a wide range of structures, using the same FRET based technological
platform. We tested the ability of these compounds to inhibit NC chaperone activity on
TAR and cTAR and the IC50 value was calculated for each screened molecule. Their
interactions with viral nucleic acids was also evaluated through melting assay, to
correlate the inhibition of NC chaperone activity with the ability of all compounds to
interact and stabilize these sequences.
The obtained results show that the highly active compounds strongly stabilize the
tested stem-loop structures; however other active compounds did not affect the stability
of the viral sequences, suggesting a different mechanism of action which probably
involves a direct interaction with NC protein. This work could be considered a reliable
starting point for further studies and for the design and synthesis of more active
derivatives.
References
1. Judith G. Levin, Mithun Mitra, Anjali Mascarenhas and Karin Musier-Forsyth. Role of HIV-1
nucleocapsid protein in HIV-1 reverse transcription. RNA Biology 2010, 7, 754-774
2. Volodymyr Shvadchak, Sarah Sanglier, Sandrine Rocle, Pascal Villa, Jacques Haiech,
Marcel Hibert, Alain Van Dorsselaer, Yves Mély, Hugues de Rocquigny. Identification by
high throughput screening of small compounds inhibiting the nucleic acid destabilization
activity of the HIV-1 nucleocapsid protein. Biochimie 2009, 7, 916–923.
3.
4.
5.
6.
ABSTRACT DEI
P OSTER
Antitumor agents
Poster P1.1
Design and synthesis of high affinity compounds for the Hsp60 expression
control in carcinogenic processes
Anna Maria Almerico,1 Annamaria Martorana,1 Valentina Giacalone,1 Francesco
Mingoia,2 Giampaolo Barone,1 Alessio Terenzi,1 Antonino Lauria1
1
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF),
2
Università di Palermo, Via Archirafi, 32 - 90123 Palermo (I); Istituto per lo Studio dei Materiali
Nanostrutturati, Consiglio Nazionale delle Ricerche (CNR) Via La Malfa 153, 90146 Palermo (I)
[email protected]
First observed in cells exposed to high temperatures, Heat shock proteins (Hsps) are
nowadays considered the most important cell “chaperone” complexes over-expressed
in response to a number of cell stress stimuli.1 The chaperone activity is the main
function of the eukaryotic Heat shock protein 60 kDa (Hsp60), involved in the capture
and refold of unfolded or misfolded proteins. Additional roles in signal transduction,2
senescence activation3 and apoptosis4 have been ascribed to cytosolic Hsp60. During
the carcinogenIc process, in vivo studies demonstrated increased levels of human
Hsp60 in several organs, such as uterine exocervix,5 large bowel,6 and prostate.6
In this context, our study aims to elucidate the structural details of the interaction
between Hsp60 and novel designed antagonists able to specifically block this
chaperonine. A preliminary virtual screening of 24 million molecules, available in the
Zinc database, was carried out on the ATP-binding site of a bacterial Hsp60 monomer,
the coordinates of which were taken from Protein Data Bank (ID: 1WE3), figure 1.
Compounds with high affinity were further refined by other in silico protocols previously
and successfully applied by us in the study of several biological targets.7
Figure 1. Bacterial Hsp60 monomer (left), selected benzofuran core structure (right).
The analysis of virtual screening results highlights the N-{5-[1H-imidazol-4-yl-methyl)amino]-benzofuran-3-yl}-benzamides of type 1 as interesting series for the inhibition of
Hsp60 ATP-binding site. Selected compounds were prepared in excellent yields,
following appropriate synthetic pathways. All compounds are currently tested in order
to proof they potential anticancer activity as modulator of Hsp60 function in tumor cells.
References
1. Haak J., Kriegel, K.C. Nov. Found Symp., 2008, 291, 3.
2. Ellis R.J. Sem. Cell. Biol., 1990, 1, 1.
3. Di Felice V. et al. Anat. Rec., 2005, 284A, 446.
4. Chandra D.et al. J. Biol. Chem., 2007, 282, 31289.
5. Cappello F. et al. Pathobiology, 2002, 70, 83.
6. Cappello F. et al. Anticancer Res., 2003, 23, 1325.
7. Lauria A. et al. Eur. J. Med. Chem., 2011, 46, 4274.
Antitumor agents
Poster P1.2
VEGFR-2 Inhibitors. New perspectives from molecular modeling
Flavio Ballante,†,‡ Adele Pirolli,† Alexandros Patsilinakos,† Charlène Gadais,‡
Stéphanie Hesse,‡ Gilbert Kirsch,‡ Rino Ragno†
†
Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco,
Università degli Studi di Roma “La Sapienza”, P. le A. Moro 5, 00185 Roma, Italy.
‡
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Institut Jean Barriol, FR
CNRS 2843, Université de Lorraine, 1 Boulevard Arago, 57070 Metz, France.
[email protected]
Angiogenesis is the process of new blood vessels growth, creating new capillaries from
existing vasculature. Angiogenic dys-regulation may be involved in various diseases
development and progression as tumor growth1 and metastasis.2 The vascular
endothelial growth factor (VEGF) pathway provides several opportunities by which
small molecules can act as inhibitors of endothelial proliferation and migration and thus
as anticancer agents. Among VEGF receptors, VEGFR-2 seems to play a key role in
tumor angiogenesis. Structure-based (SB) 3-D QSAR studies characterized a
multidisciplinary approach combination3 to detect new thienopyrimidines as new class
of VEGFR-2 inhibitors. Binding mode analysis led to the design of a new series of
thienopyrimidines: among the newly synthesized, compound 2f, showed the highest
activity, as predicted by the 3-D QSAR approach.4 Further biological assays on
endothelial cell tube formation proved compound 2f as a new anti-angiogenic lead
compound that showed to be more efficient in inhibiting endothelial cell tube formation
induced by VEGF compared with Sunitinib. Therefore, molecular modeling based on
Three-Dimensional Quantitative Structure-Activity Relationships (3-D QSAR)5 provided
crucial information about the structure of potent inhibitors revealing to be an effective
tool to predict compounds’ activities. Details and methodologies will be reported.
References
1. Folkman, J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 1972,
175, 409-416.
2. Liotta, L. A.; Steeg, P. S.; Stetler-Stevenson, W. G. Cancer metastasis and angiogenesis:
an imbalance of positive and negative regulation. Cell 1991, 64, 327-336.
3. Perspicace, E.; Jouan-Hureaux, V.; Ragno, R.; Ballante, F.; Sartini, S.; La Motta, C.; Da
Settimo, F.; Chen, B.; Kirsch, G.; Schneider, S.; Faivre, B.; Hesse, S. Design, synthesis and
biological evaluation of new classes of thieno[3,2-d]pyrimidinone and thieno[1,2,3]triazine
as inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2). Eur J Med Chem
2013, 63C, 765-781.
4. Ballante, F.; Ragno, R. 3-D QSAutogrid/R: an alternative procedure to build 3-D QSAR
models. Methodologies and applications. J Chem Inf Model 2012, 52, 1674-1685.
5. Ragno, R. In VEGFR-2 Inhibitors. Ligand-Based, Structure-Based and 3-D QSAR Studies
as Tools to Design New Small Molecules, XXIII Congresso Nazionale della Società Chimica
Italiana, Sorrento, Italy, 2009; Sorrento, Italy, 2009.
Antitumor agents
Poster P1.3
Unprecedented one-pot synthesis of pharmaceutical interest derivatives via in
situ condensation of heterocyclic iminium salts with nitrogen nucleophiles
Chiara Caneva, Silvana Alfei, Monica De Maria, Simone Debenedetti, Andrea
Spallarossa, Angelo Ranise
Dipartimento di Farmacia, Università di Genova, Viale Benedetto XV 3, 16132, Genova
[email protected]
Unprecedented, highly convergent, high yielding, one-pot synthesis of
phenylhydrazones 7, acylhydrazones 8 and thiosemicarbazones 9 (most of them
obtained as Z-isomers) was carried out starting from 1,2,3 by in situ condensing
isolable key intermediate iminium chlorides 4,5,1a 61b with nitrogen nucleophiles (RNHNH2) in the presence of N,N-dimethylaniline (Scheme 1). The reaction of 4,5,6 with
hydrazine gave azines instead of hydrazones, while with primary aromatic amines
(e.g., aniline) the reaction afforded the corresponding benzamides, rather than imines.
Scheme 1. General synthesis of derivatives 7, 8 and 9.
G
H
1, 2, 3
CH3
H
C6H5COCl
+
N
DMF
G
Cl
-
CH3
G
4, 5, 6
H2N
NH
H
R
N,N-dimethylaniline
Cpds
R
NH
7, 8, 9
N
* HN
N
*
N
H
N
G
HN
*
S
S
1, 4, 7
*
H5C6
2, 5, 8
O
*
S
3, 6, 9
*
NH2
O
*
CH 2 CN
(Et)Ar
R
At present, synthetic procedures for thioureidic derivatives 7 and 8 are unknown, while
indole products 9 are obtainable from the corresponding aldehyde.
Recently, metal ion chelation therapy has attracted much attention. In this context,
hydrazones, acylhydrazones, and thiosemicarbazones show a variety of relevant
biological activity, especially as bioavailable metal (iron, copper, zinc) chelators,
potentially useful in cancer2. and neurodegenerative3. diseases. Derivatives 7, 8, and 9
are currently under biological screening.
References
1. (a) Ranise, A.; Cesarini, S.; Spallarossa A.; Sancassan F.; Bondavalli F.; Bruno O.;
Schenone S.; Menozzi G.; Fossa P.; Mosti L. Unprecedented One-Pot Stereoselective
Synthesis of Knoevenagel-Type Derivatives via in situ Condensation of N-Methyleniminium
Salts of Ethylenethiourea and Ethyleneurea with Active Methylene Reagents. Synthesis
2007, 16, 2495-2502. (b) Manuscript submitted toTetrahedron.
2. Bernhardt, P. V.; Sharpe, P. C.; Islam, M.; Lovejoy, D. B.; Kalinowski, D. S.; Richardson, D.
R. Iron Chelators of the Dipyridylketone Thiosemicarbazone Class: Precomplexation and
Transmetalation Effects on Anticancer Activity. J. Med. Chem. 2009, 52, 407–415.
3. Gaeta, A.; Hider, R. C. The crucial role of metals ions in neurodegeneration: the basis for a
promising therapeutic strategy. Br. J. Clin. Pharmacol. 2005, 146, 1041-1059.
Antitumor agents
Poster P1.4
Small-molecule enhancers of CARM1 catalyzed Arginine methylation:
Identification of Uracandolates
Cianciulli A.,a Castellano S.,a Spannhoff A.,b Milite C.,a Dal Piaz F.,a Cheng D.,b
Tosco A.,a Viviano M.,a Yamani A.,c Sala M.,a Cura V.,d Cavarelli J., d
Novellino E.,e Mai A.,f Bedford M.T.,b and Sbardella G.a
a
Dipartimento di Farmacia, Epigenetic Med Chem Lab, Università degli Studi di Salerno;
University of Texas M.D. Anderson Cancer Center, Science Park-Research Division; cCelon
Pharma Sp. Z o.o., Laboratory of Medicinal Chemistry; dDépartement de Biologie Structurale
e
Intégrative, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire); Dipartimento di
f
Chimica Farmaceutica e Tossicologica, Università di Napoli “Federico II”; Istituto Pasteur –
Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza
Università di Roma
b
[email protected]
Arginine methylation is a common post-translational modification that is crucial in
modulating gene expression at multiple critical levels. The arginine methyltransferases
(PRMTs)1,2 are envisaged as promising druggable targets, but their role in
physiological and pathological pathways is far from
being clear due to the limited number of modulators reported to date. In this effort,
enzyme activators can be invaluable tools useful as gain-of-function reagents to
interrogate the biological
roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely
pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates
(dubbed “uracandolates”)3, able to increase the methylation of histone (H3) or
nonhistone (polyadenylate-binding protein 1, PABP1) (fig.1) substrates induced by
coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and
cellular settings. To the best of our knowledge, this is the first report of compounds
acting as CARM1 activators.
Figure 1. General structure of Uracandolates activators of CARM1.
References:
1. Bedford, M. T., et al. Arginine Methylation: An Emerging Regulator of protein Function.
Molecular Cell 2005, 18, 263-272.
2. Castellano, S., et al. Design,Synthesis and Biological Avaluation of Carboxy Analogues of
Arginine Methyltransferase Inhibitor1 (AMI-1).ChemMedChem 2010, 5, 398-414.
3. Castellano, S., et al. Identification of Small-Molecule Enhancers of Arginine Methylation
Catalyzed by Coactivator-Associated Arginine Methyltransferase 1. J. Med. Chem 2012, 55,
9875-9890.
Antitumor agents
Poster P1.5
Polymer functionalization with selected peptides aimed to nanoparticles
preparation for tumour targeting
a
Colzani B., b Biagiotti M., bSperanza G., aDorati R., aConti B., and a,Genta I.
a
University of Pavia, Dept. Drug Sciences, V.le Taramelli,12 – 27100 Pavia (Italy)
b
University of Milan, Dept. Chemistry, Via Golgi, 19 – 20133 Milano (Italy)
[email protected]
Hypotesis and Purpose
1
To exploit the agent YHWYGYTPQNVI (GE11) , a dodecapeptide that has been demonstrated
to selectively recognise the epidermal growth factor receptor (EGFR), in order to realize
selective nanosized drug delivery systems by combining passive and active cell targeting.
To set-up chemical and technological protocols to obtain a GE11-poly-lactide-co-glicolide
conjugate (GE11-PLGA) as “smart” nanoparticulate platform for drug delivery.
Methods
A model tetrapeptide (FQPV, Mw 489.57 g/mol) and GE11 were synthesized by standard
fluorenyl-9-methoxy-carbonyl (Fmoc) protocol using a Biotage Initiator SP Wave synthesizer.
They were purified by semi-preparative HPLC using an AKTA Basic100 instrument and its purity
characterized by analytical RP-HPLC. The peptides identity and molecular weight were
confirmed by MALDI TOF mass spectrometry (Bruker Microflex LT Spectrometer).
FQPV was used to set up the best protocol for chemical conjugation to PLGA (7525 DLG 3A,
Mw 35,000 Da, Lakeshore Biomaterials, USA) (FQPV-PLGA), using carbodiimmide chemistry.
The FQPV peptide was labelled with rhodamine B (Merck) in order to quantify, by fluorescence
spectrometry, the amount of peptide covalently bound to the polymer.
FQPV-PLGA and rhodamine-labelled-FQPV-PLGA nanoparticles were prepared by suitably set
2
up nanoprecipitation technique . Rhodamine-labelled-FQPV loaded PLGA nanoparticles were
also prepared by adsorption or encapsulation protocols in order to compare different techniques
to load peptide to the polymer. The nanoparticles were recovered by high speed
ultracentrifugation.
The nanoparticles were characterized for their morphology (TEM), particle size and Z-potential
(NICOMP 380 ZLS apparatus) and peptide loading (fluorescence spectrometry).
Results
GE11 peptide was obtained in 35% yield and its purity was shown to be > 95%. NMR spectra
showed covalent bond of FQPV peptide to PLGA, as further demonstrated by fluorescence
spectrometry.
Placebo PLGA nanoparticles showed homogeneous size distribution and suitable dimensions
(330.0±77.200nm, PI 0.055). Zeta potential was -4.27 mV. FQPV-PLGA nanoparticles didn’t
show significant differences in terms of dimensions and surface charge. TEM images confirmed
nanoparticle size distribution and revealed regular spherical shape.
References
1. Li, Z.; Zhao, R.; Wu, X. et al.. Identification and characterization of a novel peptide ligand of
epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 2005,19,
1978-85.
2. Yung-Chu Chen; Wen- Yuan Hsieh; Wen-Fu Lee; Ding-Tai Zeng. Effects of surface
modification of PLGAPEG-PLGA nanoparticles on loperamide delivery efficiency across the
blood–brain barrier. Journal of Biomaterials Applications 2011 0(0) 1–14.
Antitumor agents
Poster P1.6
Phenylpyrazolo[1,5-a]quinazolin-5(4H)-one: a suitable scaffold for the development
of non-camptothecin Topoisomerase I (top1) inhibitors
Pugliesi, I.;1 Barresi, E.;1 Taliani, S.;1 Salerno, S.;1 Marchand, C.;2 Agama, K.;2 Simorini, F.;1 La
Motta, C.;1 Marini, A.M.;1 Pommier, Y.;2 Di Santo, R.;3 Da Settimo, F.1
1
Dipartimento di Farmacia, Università di Pisa, via Bonanno, 6, 56126 Pisa. 2Laboratory of Molecular
3
Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255.
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco,
“Sapienza” Università di Roma, P.le Aldo Moro 5, I-00185, Rome, Italy
[email protected]
DNA topoisomerases are essential enzymes inducing topological modification in the DNA double
helix that are required during important cellular processes such as replication, transcription, repair,
and chromosome condensation.1 Topoisomerase type 1 (top1) relaxes supercoiled DNA by
forming DNA single-strand breaks which are produced when top1 forms a covalent bond between
its tyrosine active site and a 3´-DNA phosphate. These top1 linked breaks allow controlled rotation
of the free 5'-end of the broken DNA around the intact strand; after the DNA is relaxed, top1
spontaneously religates the breaks and rapidly restores intact duplex DNA.2 At this stage, the
enzyme is particularly vulnerable to a group of anticancer agents that reversibly trap the complex
by intercalating between DNA base pairs and the cleavage site, thereby inhibiting religation.
Collision of the replication machinery with the trapped complex leads to irreversible DNA strand
breaks, resulting in activation of apoptotic and cell cycle arrest pathways. Camptothecin, a natural
product, was the first small molecule to be identified as a top1 inhibitor. Efforts to improve its water
solubility, toxicity profile, and pharmacokinetics led to the development of the two clinically useful
water-soluble CPT derivatives topotecan and irinotecan.3 However, the CPTs are not ideal drugs
because they are compromised by a number of limitations. In order to overcome the main
drawbacks of CPTs, have been developed non-CPT Top1 poisons. Several chemical classes
emerged as promising antitumor drugs, that share similar specificity and potency with respect to
CPT, including the indolocarbazoles, indenoisoquinolines, and the phenanthridines.4
In the present research, we focussed our attention on the pyrazolo[1,5-a]quinazoline nucleus,
structurally related to the indenoisoquinoline system, with the aim to develop novel potential
anticancer agents acting as Top1 inhibitors. Various decorations have been introduced on the
central core scaffold, including a substituted phenyl ring at 2- or 3-positions, and a protonable side
chain featuring linkers different in nature (-CH2-, -O-, -NH-), length (2 or 3 methylene units), and
terminal nitrogen-containing group (dimethylamino, diethylamino, imidazole nucleus) at 5- or 6positions.
O
C
A
B
D
N
O
Indenoisoquinolines
A
D
N
NC
B
N
X (CH2)n N
2(3)-Phenyl-pyrazolo[1,5-a]quinazolines
References
1. Wang, J. C. Nat. Rev. Mol. Cell. Biol. 2002, 3, 430-40. 2. Wall, M. E. W., M. C.; Cook, C. E.; Palmer K.
H.; McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888-3890. 3. Garcia-Carbonero, R.; Supko,
J. G. Clin Cancer Res 2002, 8, 641-61. 4. Pommier, Y. Nat Rev Cancer 2006, 6, 789-802.
Antitumor agents
Poster P1.7
Use of Microscale Thermophoresis (MST) for studying binding interactions of
PRSet-7/SETD8 with small molecule specific inhibitors EPI-9 and EPI-23
Feoli A.1, Castellano S.1, Milite C.1, Viviano M.1, Imhof A.2, Tosco A.1, Sbardella G.1
1
Dipartimento di Farmacia, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084
2
Fisciano (SA), Italy, Adolf-Butenandt Institute and Munich Center of Integrated Protein Science
(CIPS), Ludwig-Maximilians University of Munich, Schillerstrasse 44, 80336 Munich, Germany.
[email protected]
Histone methylation plays a key role in establishing and maintaining stable gene
expression patterns during cellular differentiation and embryonic development.
Considering that a number of small molecules identified as modulators of
methyltransferases by high-throughput screening were dyes or derivatives, a small
focused library of dye-like compounds was prepared and the small molecules
synthesized were pre-screened for potential inhibition of histone lysine
methyltransferases (HKMT) using in vitro HMT assays. Two compounds, EPI-9 and
EPI-23, showed low IC50 values against nucleosomal HKMT PR-Set7. Prompted by our
interest in the study of small molecule modulators of these epigenetic targets, we
applied MST (Microscale Thermophoresis) to the investigation of the interaction of
PRset7 with small molecule ligands EPI-9 and EPI-23. Microscale thermophoresis
(MST) is a new method that enables the quantitative analysis of molecular interactions
in solution. MST is the directed movement of particles in a microscopic temperature
gradient: any change of the hydration shell of biomolecules due to changes in their
structure/conformation results in a relative change of movement along the temperature
gradient and is used to determine binding affinities, binding kinetics and activity
kinetics. Events such as the binding of small molecules to a target can be monitored by
this tecnique.
Figure 1. MST assays carried out using mouse the PRSet7 with EPI-9 and EPI-23.
Antitumor agents
Poster P1.8
New N-alkanol-N-cyclohexanolamine aryl ester derivatives as reverting agents of
Multidrug Resistance (MDR)
Floriddia E.,a Orlandi F.,a Bellucci C.,a Manetti D.,a Romanelli M.N.,a Salerno M.,b
Scapecchi S.,a Teodori E.a and Dei S.a
a
Dipartimento NEUROFARBA, Sezione di Farmaceutica e Nutraceutica, Università degli Studi
b
di Firenze, Via U. Schiff 6, 50019 Sesto Fiorentino (FI), Italy. Laboratoire BioMoCeTi UMR
7033, Université Paris 13, 74 rue Marcel Cachin, 93017 Bobigny, France
[email protected]
Multidrug resistance (MDR) is an acquired drug resistance of cancer cells and
microorganisms to a variety of chemotherapeutic drugs. It is due to overexpression of
proteins such as ABCB1 (Pgp) and ABCC1 (MRP1) that act as extrusion pumps
causing a lower cell concentration of various anticancer drugs that usually are
structurally and mechanistically unrelated.1 Inhibition of the functions of Pgp and
related proteins, is considered a suitable approach to circumvent MDR. This is the
main reason prompting the design and synthesis of Pgp inhibitors to co-administrate
with cytotoxic substrates of Pgp.2
Recently, we have described two new families of MDR reverters, N,N-bis(alkanol)amine
aryl esters and N,N-bis(cyclohexanol)amine aryl esters, endowed with fairly good
potency.3,4 Now we report an extension of structure-activity relationships and of
pharmacological studies in this series of compounds. The new obtained derivatives are
hybrid compounds characterized by the presence of two different linkers: a methylene
chain of different length and a cyclohexylic scaffold.
Figure 1. General structure of N-alkanol-N-cyclohexanolamine aryl esters
CH3
N
O
Ar
(CH2)n
Ar1
O
O
Ar, Ar1 =
O
H3CO
H3CO
OCH3
H3CO
n = 3, 4, 5
OCH3
H3CO
The new hybrid compounds show a good potency and efficacy in the preliminary
pharmacological tests. The pharmacological profile of the new molecules will be
reported and discussed.
References:
1. Mitcher, L.A.; Pillai, S.P.; Gentry, E.J.; Shankel, D.M. Multiple Drug Resistance. Med. Res.
Rev. 1999, 19, 477-496.
2. Teodori, E.; Dei, S.; Martelli, C.; Scapecchi, S.; Gualtieri, F. The functions and structure of
ABC transporters: implications for the design of new inhibitors of Pgp and MRP1 to control
multidrug resistance (MDR). Curr. Drug Targets 2006, 7, 893-909.
3. Martelli, C.; Coronnello, M.; Dei, S. et al. Structure-activity relationships studies in a series
of N,N-bis(alkanol)amine aryl esters as P-glicoprotein (Pgp) dependent multidrug resistance
(MDR) inhibitors. J. Med. Chem. 2010, 53, 1755-1762.
4. Orlandi, F.; Coronnello, M.; Bellucci, C. et al. Structure-activity relationships studies in a
series of N,N-bis(cyclohexanol)amine aryl esters as P-glycoprotein-mediated Multidrug
Resistance (MDR) inhibitors. Bioorg. Med. Chem. 2013, 21, 456-465.
Antitumor agents
Poster P1.9
Synthesis and in vitro evaluation of tetrasubstituted pyridines as potential PI3Ks inhibitors
Galli U.,a Ciraolo E.,b Hirsch E.,b Sorba G.,a Tron G.C.a
a
Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale,
Largo Donegani 2 - 28100 Novara
b
Dipartimento di Genetica, Biologia e Biochimica, Molecular Biotechnology Center,
Università degli Studi di Torino, Via Nizza 52 - 10126 Torino,
[email protected]
Phosphatidylinositol-3-kinases (PI3Ks) are a family of lipid kinases mediating numerous cell
process such as proliferation, survival, motility, metabolism and differentiation.1
The PI3Ks are divided into classes IA, IB, II and III according to their structural characteristics and
substrate specificity. Class IA consists of three catalytic isoforms: p110α, p110β and p110δ.
Whereas the expression of p110δ is restricted to the immune system, p110α and p110β are
ubiquitously expressed. Class IB consists of a single p110γ catalytic isoform, which is mainly
expressed in leukocytes.
The PI3K pathway is often de-regulated in cancer through PI3K p110α overexpression, gene
amplification, mutations and PTEN phosphatase deletion.2
PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment and
other proliferative diseases.3
The pyridofuropyrimidine PI-103 1 (Fig.1) is a potent, ATP-competitive PI3K p110α inhibitor with
IC50 of 2 nM and shows anti-proliferative activity at submicromolar concentrations in various human
tumor cell lines, including multi-drug resistant cancer cells.4
PI-103 is also a potent inhibitor of p110β (IC50 3 nM) and displays excellent selectivity over p110γ
(IC50 0.25 µM). Unfortunately, the compound has poor pharmacokinetic profile (low solubility, high
metabolic instability).4
In the course of our efforts toward the identification of isoform-specific PI3K inhibitors, we modified
the structure of 1 in order to generate structurally simpler analogues 2 (Fig. 1), which can be
prepared using an improved Guareschi pyridine synthesis.5
Figure 1. Structure of PI-103 1 and proposed new scaffold 2.
In this contribution we describe the synthesis and biological evaluation of a new series of 2-(3hydroxyphenyl)-3-cyano-4-substituted-6-morpholino pyridines as potential PI3K inhibitors, showing
low micromolar activity against classes IA and IB of PI3Ks in the biochemical assays.
References
1. Katso, R.; Okkenhaug, K.; et al. Annu. Rev. Cell Dev. Biol. 2001, 17, 615
2. Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Nat. Rev. Drug Disc. 2009, 8, 627
3. Workman, P.; Clarke, P. A.; Raynaud, F. I.; et al. Cancer Res. 2010, 70, 2146
4. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; et al. Bioorg. Med. Chem. Lett. 2007, 17, 2438
5. Bobbitt, J.M.; Scola, D.A. J. Org. Chem. 1960, 25, 560
Antitumor agents
Poster P1.10
Biological evaluation of novel tamoxifen analogues
Aída Nelly García Argáeza, Michael S. Christodouloub,c, Daniele Passarellac, Michela
Iannolia, Ornella Maria Giaa, Lisa Dalla Viaa
a
Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via F. Marzolo 5,
b
35131 Padova – Italy; Chemistry Laboratory, Agricultural University of Athens, Iera odos 75,
c
Athens 11855, Greece; Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19,
20133 Milano – Italy.
[email protected]
Tamoxifen (TAM) and its active metabolite, 4-hydroxytamoxifen, are non steroidal
selective estrogen receptor (ER) modulators that compete with estrogens for binding to
ER and are widely used in the treatment of breast cancer. Nevertheless the use of
TAM has also been associated with some undesirable side effects.1 Therefore, the
attainment of new anticancer agents, which retain all the beneficial effects but are
devoid of toxicity, has to be considered an intriguing and current goal.
In the present study 14 new TAM analogues, E and Z isomers, were synthesized and
their effect on human tumor cell lines, (MCF-7, HeLa and MSTO-211H) was
determined. These novel derivatives maintain the triarylethylene skeleton, bearing OH
groups in position 4 of the phenyl moieties and substitute the side chain of TAM with an
alkyl or aryl amide side chain. The investigation on antiproliferative activity evidenced a
significant cytoxicity highlighting for the compounds carrying a branched group, or the
piperidine moiety an antiproliferative effect comparable or even higher with respect to
that of TAM. In addition Z isomers seem generally slightly more active toward MCF-7
cells. In an attempt to establish if the cell death occurs through the apoptotic pathway,
the apoptosis induction and the decrease of mitochondrial membrane potential of
selected compounds were determined by cytofluorimetric assays. The results showed
that these derivatives induced intrinsic apoptosis.
The primary mechanism of action of TAM is believed to involve the inhibition of ER,
nevertheless, research over the years has indicated that additional non ER-mediate
mechanisms exist.1 Indeed it has been suggested that TAM has DNA intercalative
properties and inhibits topoisomerases.2 In order to verify the occurrence of an
interaction of these compounds with DNA, flow linear dichroism experiments were
performed. The spectra showed only a strong negative signal at 260 nm typical of the
macromolecule then, it has to be concluded that these compounds are unable to form a
complex with DNA.
In order to assess the effects of some representative compounds on topoisomerase
activities the supercoiled pBR322 plasmid was utilized. The partial inhibitory effect on
topoisomerase II relaxation of the compounds at 50 µM was observed while by
increasing the concentration up to 100 µM the complete inhibition was reached. These
data could suggest the contribution of the inhibitory effect on topoisomerase II to the
cytotoxicity. Otherwise, experiments performed on topoisomerase I highlighted the
inability of the derivatives to affect its enzymatic activity.
References
1. Mandlekar, S.; Kong, AN. Mechanisms of tamoxifen-induced apoptosis. Apoptosis 2001,
6,469-477
2. Larosche, I.; Lettéron, P.; Fromenty, B.; Vadrot, N.; Abbey-Toby, A.; Feldmann, G.;
Pessayre, D.; Mansouri, A. Tamoxifen Inhibits Topoisomerases, Depletes Mitochondrial
DNA, and Triggers Steatosis in Mouse Liver. J. Pharmacol. Exp. Ther. 2007, 321, 526-535.
Antitumor agents
Poster P1.11
Human lactate dehydrogenase 5 (hLDH5) inhibitors based on N-hydroxyindole
and malonic scaffolds: a comparison of their anti-cancer activities
Granchi C.,1 Paterni I.,1 Calvaresi E.C.,2 Tuccinardi T.,1 Macchia M.,1 Martinelli A.,1
Hergenrother P.J.,2,3 Minutolo F.1
1
Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy, 2 Department
3
of Biochemistry and Department of Chemistry, University of Illinois, Urbana, Illinois 61801
[email protected]
Invasive cancer cells are characterized by a metabolic switch from oxidative
phosphorylation to aerobic glycolysis, with higher glucose intake and elevated lactate
production (Warburg effect). Isoform 5 of human lactate dehydrogenase (hLDH5) is the
enzyme accounting for the transformation of pyruvate into lactate and it is a critical
enzyme in the metabolism of tumours, allowing their survival. hLDH5 is currently being
considered as a strategic target for cancer treatment, since hLDH5 inhibition should
block energetic production and lead to cell death.1 Inhibition of this enzyme has
represented a very challenging goal over the past few years. We have developed a
new class of hLDH5 inhibitors based on a N-hydroxyindole-2-carboxylate scaffold (NHI,
Figure 1).2 These compounds showed Ki values in the low micromolar range on the
isolated enzyme and proved to be competitive versus both the substrate (pyruvate) and
the cofactor (NADH). Moreover, they displayed antiproliferative activity and provoked a
significant reduction of cellular lactate production in vitro. Recently, AstraZeneca UK
discovered a new class of hLDH5 inhibitors based on a malonic scaffold (Mal, Figure
1). These derivatives showed inhibition potencies in the sub-micromolar range, but they
had the drawback of poor cellular penetration.3 A head-to-head study of representative
examples of N-hydroxyindole and malonic derivatives was conducted, by comparing
enzyme inhibition potency, cellular uptake, reduction of lactate production in cancer
cells and anti-proliferative activity. Among the compounds tested, a methyl ester
belonging to the NHI class displays optimal properties in the cell-based assays, proving
to be superior to the malonic derivatives as an anti-glycolytic agent against cancer
cells.
Figure 1. Structures of N-hydroxyinole and malonic derivatives.
References
1. Fantin, V.R.; St-Pierre, J.; Leder, P. Attenuation of LDH-A expression uncovers a link
between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 2006, 9,
2. Granchi, C.; Roy, S.; Giacomelli, C.; et al. Discovery of N-hydroxyindole-based inhibitors of
human lactate dehydrogenase isoform A (LDH-A) as starvation agents against cancer cells.
J. Med. Chem. 2011, 54, 1599-1612.
3. Ward, R.A.; Brassington, C.; Breeze, A.L.; Design and synthesis of novel lactate
dehydrogenase A inhibitors by fragment-based lead generation. J. Med. Chem., 2012, 55,
3285-3306.
Antitumor agents
Poster P1.12
Structural analysis of G-rich oncogenic promoter sequences
Greco M.L.a, Folini M.b, and Sissi C.a
a
BMCS, Dept. of Pharmaceutical and Pharmacological Sciences,
v. Marzolo 5, 35131 Padova, Italy.
b
Dept. of Experimental Oncology and Molecular Medicine; IRCCS Istituto Nazionale dei Tumori;
v.Giacomo Venezian, 1, 20133 Milan, Italy.
[email protected]
The conformational switch of promoter sequences is known to represent a mechanism
of modulation of gene transcription, since it is a tool to displace/recruit the proteins
involved in the transcription processes at the starting site. A bioinformatic genome
analysis showed that many oncogene promoters are characterized by a G-rich
signature. Accordingly, these sequences can potentially assume non canonical
secondary structures called G-quadruplexes. They are promoted by monovalent
cations such as K+, Na+, which are physiologically present in the cellular
environment1,2,3. Moreover, literature data confirmed that also small molecules able to
stabilize the G4 forming within MYC, TERT, KIT and KRAS promoters induce a specific
decrease of gene expression at both mRNA and protein levels. Thus, a functional role
of G4 structures as a switch-off element for gene transcription has been envisaged. In
this study, we focused our attention on two well known oncogenes, EGFR (Epidermal
Growth Factor Receptor) and BRAF, whose products play essential roles in the
development and progression of several cancers. Moreover, activating mutations in the
BRAF oncogene (BRAFV600E) and the overexpression of EGFR are correlated to
different types of cancer thus suggesting that the inhibition of their transcription could
be a powerful strategy for anticancer therapy. By an in silico prediction tool we
identified and selected three sequences potentially able to assume a G4 conformation
within EGFR and BRAF promoters. Starting from these assumptions, we worked to
answer some questions: can the oncogenic sequences fold in a G-quadruplex
structure? Which is the predominant conformation in physiological condition? Can
small molecules selectively bind/stabilize the G-rich motif? Can the complementary Crich strand be involved in other conformations? Herein we will report the result of our
spectroscopic and electrophoretic analysis which confirmed that the G-rich EGFR and
BRAF promoter regions are able to fold into a G-quadruplex conformation. Moreover,
the binding process of anthraquinones and naphthalene diimides derivatives to these
sequences in different conformational arrangements will be discussed.
References
1. Parkinson, G. N.; Lee, M. P. H.; Neidle, S., Crystal structure of parallel quadruplexes from
human telomeric DNA. Nature 2002, 417, 876-880.
2. Wang, Y.; Patel, D. J., Solution structure of the human telomeric repeat d[AG3(T2AG3)3]
G-tetraplex. Structure 1993, 1, 263-282.
3. Dai J.; Punchihewa, C.; Ambrus, A.; Chen, D.; Jones, R. A.; Yang D., Structure of the
intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple
formation. Nucleic Acids Res., 2007, 35, 2440-2450.
Antitumor agents
Poster P1.13
New potent and selective P-gp inhibitors
Guglielmo S.,a Bonomo S.,a Colabufo N. A.,b Contino M.,b Perrone M. G.,
b
Fruttero R.,a and Gasco A.a
a
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino,
Via Pietro Giuria 9, 10125 Torino, Italy
b
Dipartimento Farmacia, Università degli Studi di Bari ALDO MORO, Via Orabona 4, 70125 Bari, Italy
[email protected]
Expression of ATP-binding cassette (ABC) transporters such as ABCB1 (P-glycoprotein, Pgp), ABCC1 (MRP1), or ABCG2 (breast cancer resistance protein, BCRP) by cancer cells is
associated with multidrug resistance (MDR) which is a major cause of chemotherapeutic
treatment failure in cancer therapy.1
Moreover, P-gp is expressed in several physiological barriers, thus exerting a protective
function in several tissues. In particular a dysfunction of P-gp activity at the blood-brain
barrier is considered one of the causes of the onset of Parkinson’s Disease (PD) and
Alzheimer’s Disease (AD) since a correlation between aging and the decreased function of
the pump in the BBB has been established in vivo.2
On these basis the development of P-gp inhibitors can be of primary importance in oncology
field, where they should contribute to increase the concentration of the chemotherapeutic
agents in cancer cells; moreover these compounds are potential novel tools in the diagnosis
of CNS diseases by sophisticated imaging techniques such as PET and SPECT: in
particular, PD and AD patients can be monitored in an early stage and during disease
progression with radiolabeled inhibitors that bind to the pump in the high affinity state.
In an effort to discover novel potent and selective P-gp inhibitors, we synthesized a series of
compounds based on a previously developed inhibitor (13, fig. 1). The compounds were
obtained modulating the phenol group of 1 with alkyl and hydroxyalkyl chains (general
structure A, fig. 1); they showed moderate to very good and selective inhibitory activity
against P-gp over-expressed in MDCK-MDR1 cells, some of them being effective in the submicromolar range.
O
O
N
N
O
O
1 (MC70, EC50 = 690 nM4)
OH
A, R = alkyl/hydroxyalkyl chain
(R = n-Butyl, EC50 = 5.2 nM)
OR
Figure 1. Structure of lead compound 1 and general structure A of the synthesized novel P-gp
inhibitors.
References
1. Gottesman, M. M. et al. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat.
Rev. Cancer 2002, 1, 48−58.
2. Bartels, A. L. et al. Blood-brain barrier P-glycoprotein function decreases in specific brain regions
with aging: a possible role in progressive neurodegeneration. Neurobiol. Aging 2009, 30, 1818–
1824.
3. Colabufo, N. A. et al. 4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline
derivatives as potent P-gp modulators. Bioorg. Med. Chem. 2008, 7, 3732-3743.
4. Colabufo, N. A. et al. Multi drug resistance reverting agents: 2-aryloxazole and 2-arylthiazole
derivatives as potent BCRP or MRP1 inhibitors. ChemMedChem 2009, 4, 188–195.
Antitumor agents
Poster P1.14
1,2,4-Triazole Derivatives as
ATP Competitive Tyrosine Kinase RET Inhibitors:
Novel Drug Candidates for the Treatment of Thyroid Carcinoma
V. Coviello,1 S. Sartini,1 V. La Pietra,2 L. Marinelli,2 S. Cosconati,2 D. Di Maio,2
A.M. Marini,1 F. Simorini,1 S. Taliani,1 S. Salerno,1
F. Da Settimo,1 E. Novellino,2 and C. La Motta1
1
Dipartimento di Farmacia, Università di Pisa, via Bonanno, 6 56126, Pisa.
Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli “Federico II”,
via D. Montesano, 49, 80131, Napoli.
2
[email protected]
Thyroid carcinoma (TC) is the most common endocrine tumour, accounting for up to
1% of all human malignancies. Its worldwide incidence rate has been increasing
sharply since the mid 1990s, thus becoming the fastest increasing cancer in both men
and women in western countries, including Italy. Its treatment of choice is represented
by medical surgery, radioiodine and hormone suppressive therapy. However, disease
can persist or recur, with local and distant metastases which are often fatal. Recent
advances in the knowledge of TC development identified receptor tyrosine kinase RET
as a viable and promising target. Actually, gain of function mutations and overexpression of RET are causally linked to tumour growth and aggressiveness.
Accordingly, a number of ATP competitive RET inhibitors are current involved in clinical
trials, showing a promising degree of efficacy.1,2
Exploiting a receptor-based virtual screening campaign, a novel 1,2,4-triazole hit has
been disclosed, DP01920, which proved to inhibit effectively both wild-type and V804L
mutant RET. Moreover, tested on a panel of both receptor and cytoplasmic kinases,
DP01920 showed good inhibitory properties against VEGFR-1, VEGFR-2 ;VEGFR-3
and PDGFRβ, thus standing out as a novel and promising multi-effective kinase
inhibitor. Moving from this exciting result, a small library of 1,2,4-triazole derivatives has
been developed, to analyze thoroughly structure-activity relationships of this class of
compounds and get to more effective analogues.
Here we present the synthesis and the functional evaluation of the novel compounds.
H
N N
S
Cl
N
Cl
DP01920
References
1. Wong, K. P.; Lang, B. H. J. Thyroid Research, 2012, 818204.
2. Lodish, M. A. J. Clin. Endocrinol. Metab. 2013, doi: 10.1210/jc.2012-4085.
Antitumor agents
Poster P1.15
Design, synthesis and biological activity evaluation of c-myc proto-oncogene
binders as potential antitumor agents
Meleddu R.,a Distinto S.,a Sissi C.,b Musetti C.,b Arridu A.,a Bianco G.,a Parrotta L.,c
Ortuso F.,c Alcaro S.,c and Maccioni E.a
a
University of Cagliari, Department of Life and Environmental Sciences, via Ospedale n 72
b
09124 Cagliari Italy, University of Padua, Department of Pharmaceutical Sciences, Via
Marzolo 5, 35131 Padova, Italy. c University of Magna Graecia Catanzaro, Department of
Healt Sciences, Campus “Salvatore Venuta”, Viale Europa, 88100 Catanzaro, Italy.
[email protected]
G-quadruplex structures are nucleic acid arrangements assumed by guanine rich
sequences and stabilised by the planar pairing of four guanines through eight
Hoogsteen hydrogen bonds. Recently, a method to quantitatively visualise DNA Gquadruplex structures in human cells was published. This study corroborates the
application of stabilizing ligands in a cellular contests to target G-quadruplex and
interfere with their function.1. G-quadruplexes regions have been found in crucial
position of the genoma, such as telomeric ends, ribosomal DNA (rDNA), RNA or gene
promoter region (for es. c-myc bcl-2 or c-kit).2.
Due to its strategic presence in gene promoter regions, G-quadruplexes are viewed
as emerging therapeutic targets in oncology: their stabilization could lead to a
transcriptional repression of oncogenes.
In fact the overexpression of mutated protoncogene is often associate with a
significant number of human malignancies.3.
Our research is focused on the design and synthesis of new compounds able to
stabilise the c-myc oncogene promoter region. In the present study we describe the
design and the synthetic route devised for the synthesis of a new series of fluorenone
derivatives (Figure 1).
O
O
CH3
Y
n
X
NH
n
O
NH
n
X
CH3
Y
n
X= N, C
Y= N, O, C, NCH3
n= 1, 2, 3
Figure 1. General structure of 9-oxo-9H-fluorene derivatives
References
2. Biffi, G.; Tannahill, D.; McCafferty, J.; Balasubramanian, S. Quantitative Visualization of
DNA G-Quadruplex Structures in Human Cells.) Nature Chemistry 2013, 5, 182-186
3. Huppert, J. L. Hunting G-Quadruplexes. Biochimie 2008, 90, 1140-1148
4. Balasubramanian, S.; Hurley, L. H.; Neidle, S. Targeting G-quadruplexes in gene
promoters: a novel anticancer strategy? Nat Rev Drug Discov. 2011,10, 261–275
Antitumor agents
Poster P1.16
Synthesis, structure-activity relationships and stereochemical investigations on
new tricyclic pyridazinone derivatives as potential STAT3 inhibitors
Fiorella Meneghetti[a], Daniela Masciocchi[a], Arianna Gelain[a], Alessandro Pedretti[a],
Giuseppe Celentano[a], Daniela Barlocco[a], Federica Porta[a], Laura Legnani[b], Lucio
Toma[b], Byoung-Mog Kwon[c], Akira Asai[d], Stefania Villa[a]
[a]
Dipartimento di Scienze Farmaceutiche,Università degli Studi di Milano, Via Mangiagalli 25,
[b]
Milano (Italy), Dipartimento di Chimica, Università degli Studi di Pavia, Via Taramelli 12, Pavia
[c]
(Italy), KRIBB, University of Science and Technology, Eoun-Dong, Yuseong-gu, Daejeon
[d]
(Republic of Korea), Center for Drug Discovery, Graduate School of Pharmaceutical
Sciences,University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka (Japan)
[email protected]
Signal Transducer and Activator of Transcription 3 (STAT3) directly relates signals
from the cytoplasmic membrane to the nucleus and regulate transcription of target
genes. While the inhibition of STAT3 signaling leads tumor cells to apoptosis, it has
only minimal effects in normal cells. On this basis, the design of new molecules as
STAT3 inhibitors is crucial for the development of new anticancer drugs endowed with
interesting pharmacodynamic properties and reduced side effects.1 During our
ongoing researches,2 we screened our internal library through a cell-based biological
assay and the benzocinnolinone derivative (±)-1 was identified as promising STAT3
inhibitor. Since SAR studies on a series of compounds structurally related to (±)-1
have shown that the latter had the most significant inhibitory activity, we more deeply
investigated its essential structural features. In particular, enantiomeric separation
was performed, and the absolute configuration of the stereoisomers was assigned by
theoretical and crystallographic studies. The biological evaluation highlighted that (S)(-)-1 was twice as potent as (R)-(+)-1.
(S)-(-)-1
R = ester, carboxylic acid,
amide, amine, alkyl, Phe residue,
tetrazole derivatives
R1,R2,R3 = H, OCH3,Cl
n = 1,2,3
Figure 1. Structures of the potential STAT3 inhibitors presented in this work
References
1. Masciocchi, D.; Gelain, A.; Villa, S.; Meneghetti, F.; Barlocco, D.; Future Med. Chem. 2011,
3(5), 367-397.
2. Masciocchi, D.; Villa, S.; Meneghetti, F.; Pedretti, A.; Barlocco, D.; Legnani, L.; Toma, L.;
Kwon, B-M.; Nakano, S.; Asai, A.; Gelain, A. Med. Chem. Commun., 2012, 3, 592-599.
Antitumor agents
Poster P1.17
From Anacardic acid to LoCAMs: Development of a chemical tool for tuning
histone acetylation
Milite C.1, Castellano S.1, Tosco A.1, Feoli A.1, Mai A.2, Sbardella G.1
1
Dipartimento di Farmacia, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084
2
Fisciano (SA), Italy, Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Chimica e
Tecnologie del Farmaco, "Sapienza" Università di Roma, P.le A. Moro 5, I-00185 Rome, Italy
[email protected]
Lysine acetylation is among the prominent posttranslational modifications in eukaryotic
cells. Protein acetylation level is a consequence of the balance between the opposite
activities of protein acetyltransferases (KATs) and deacetylases (KDACs), and its
deregulation has been linked to several diseases, including cancer, inflammation and,
probably, neurodegenerative diseases. At present, only a small number of KATs
modulators have been reported and just a few of them show selectivity between KATs
isoforms. Among these, anacardic acid is a potent inhibitor of histone
acetyltransferases p300 and PCAF1. Prompted by our interest in the discovery of small
molecule modulators of epigenetic target, we focused our attention on this compound.
Here we report the design, synthesis and the biological evaluation of novel LoCAMs
(Long Chain AlkylidenMalonates) derivatives obtained by molecular pruning and lead
optimization of anacardic acid2,3.
Figure 1. From anacardic acid to selective modulators of KATs.
References
1. Kundu T., Swaminathan, V. Ranganathan, A. et al. J. Biol. Chem. 2003, 278, 19134.3
2. Sbardella G., Castellano S., Mai A. et al. Bioorg. Med. Chem. Lett., 2008, 9, 2788.
3. Milite C., Castellano S., Sbardella G. et al., Bioorg. Med. Chem., 2011, 19, 3690
Antitumor agents
Poster P1.18
Synthesis of pyrazolo[3,4-d]pyrimidines active on Bcr-Abl T315I mutation
Musumeci F.a, Desogus A.a, Brullo C.a, Tintori C.b, Fallacara A.b, Maga G.c, Crespan
E.c, Radi M.d, Schenone S.a
a
Dipartimento di Farmacia, Università degli Studi di Genova
Viale Benedetto XV, 3, 16132 Genova, Italy
b
Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena
Via Aldo Moro 2, 53100 Siena, Italy
c
Istituto di Genetica Molecolare, IGM-CNR
Via Abbiategrasso 207, 27100 Pavia, Italy
d
Dipartimento di Farmacia, Università degli Studi di Parma
Viale delle Scienze, 27/A, 43124 Parma, Italy
[email protected]
The discovery of compounds targeting the Bcr-Abl T315 mutation represents a primary
challenge for chronic myeloid leukemia therapy. Indeed, even if several drugs inhibiting
wild-type Bcr-Abl have been approved for this diseases, only few drugs acting on T315I
mutation are being studied in clinical trials to date.
In this context, our research group has deeply investigated a class of pyrazolo[3,4d]pyrimidine derivatives acting as Src and/or Abl kinase inhibitors. These compounds
showed potent antiproliferative and proapoptotic activity towards different cancer cell
lines depending on the nature and position of substituents on the heterocyclic core.1 In
particular compounds 1a,b showed to be active also against the T315I mutation.2
Following the suggestion of a molecular modeling study that highlighted the importance
of a bromine atom in the para position of the N1 side chain phenyl ring for the activity
against the T315I mutant, we synthesized the series of derivatives 2 (Figure 1).
Among these, the compound bearing an anilino group in C4 was identified as the most
promising derivative showing a good balance of different ADME properties, and activity
in vitro and in vivo. Experimental procedures and biological results will be presented in
the poster session.
NH
R1
R
N
N
N
R2
N
N
N
Cl
1a R1 = H, R2 = H
1b R1 = F, R2 = Cl
N
N
Br
Cl
2
R = amino groups
Figure 1. Pyrazolo[3,4-d]pyrimidines 2 synthesized by our group on the basis of previous
results and molecular modelling studies.
References
1. Radi, M.; Dreassi, E.; Brullo, C.; Crespan, E.; Tintori, C.; Bernardo, V.; Valoti, M.;
Zamperini, C.; Daigl, H.; Musumeci, F.; Carraro, F.; Naldini, A.; Filippi, I.; Maga, G.;
Schenone, S.; Botta, M. Design, synthesis, biological activity, and ADME properties of
pyrazolo[3,4-d]pyrimidines active in hypoxic human leukemia cells: a lead optimization
study. J. Med. Chem. 2011, 54, 2610-2626.
2. Santucci, M. A.; Mancini, M.; Corradi, V.; Iacobucci, I.; Martinelli, G.; Botta, M.; Schenone,
S. New SRC/ABL inhibitors for chronic myeloid leukemia therapy show selectivity for T315I
+
ABL mutant CD34 cells. Invest. New Drugs 2010, 28, 876-878.
Antitumor agents
Poster P1.19
Preparation and characterization of liposomes containing
a new anthracycline derivative
Isabella Pedrini1, Elena Gazzano2, Silvia Arpicco1, Chiara Riganti2, Barbara Rolando1,
Konstantin Chegaev1, Roberta Fruttero1, Alberto Gasco1
1
Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino,
Via Giuria 9, 10125, Torino, Italy
2
Dipartimento di Oncologia, Università di Torino, Via Santena 5/bis, 10126, Torino, Italy
E-mail: [email protected]
Purpose: We have previously described a new multi-target drug named nitrooxydoxorubicin (Nit-DOX) that exhibits a novel and unexpected mechanism of action and
high anticancer activity. In this work we show the results obtained with the preparation
of liposomal formulation of the compound in order to improve its delivery and its
targetability.
Methods: Liposomal Nit-DOX was prepared by thin lipid film hydration and extrusion
method and characterized in terms of size and surface charge. Drug release into
phosphate buffer pH 7.4 and serum was evaluated. In vitro cytotoxicity, intracellular
doxorubicin accumulation and nitric oxide release were evaluated in human colon
cancer chemosensitive HT29 cells, in their resistant counterpart HT29-dx (which
express high levels of drug efflux transporters, such as P-glycoprotein and multidrug
resistance related protein 1), and in rat cardiomyocytes H9c2.
Results: Two liposomal Nit-DOX formulation containing different amounts of
cholesterol (15 and 30%) were prepared. They showed similar size and Z potential
values, while the amount of Nit-DOX encapsulated increased with the decreasing of the
rate of cholesterol. The formulation were stable for more than 28 days in buffer at 4°C,
retained 90% of their initial content and no drug precipitation or liposomes aggregation
were observed. The drug leakage was higher in buffer and serum at 37°C with the
liposomes containing 15% of cholesterol. The biological data showed that the in vitro
efficacy of Nit-DOX was further increased when the drug was encapsulated in
liposomes..
Conclusion: We were able to obtain liposomal formulation that permitted to increase
the stability and the cytotoxicity of Nit-DOX. Studies are in progress to further
investigate the molecular mechanisms by which this system overcomes drug
resistance.
Antitumor agents
Poster P1.20
HIGHLY POTENT CANCER AGENTS BY MODULATING THE C-2 GROUP OF THE
ARYLTHIOINDOLE CLASS OF TUBULIN POLYMERIZATION INHIBITORS
Sveva Pelliccia,a Giuseppe La Regina,b Antonio Coluccia,b Valeria Famiglini,b Alessia
Reggio,b Andrea Brancale,c Ernest Hamel,d Ettore Novellino,a and Romano Silvestrib
a
b
Dipartimento di Farmacia, Università di Napoli Federico II. Dipartimento di Chimica e
c
Tecnologie del Farmaco, Sapienza Università di Roma. Welsh School of Pharmacy, Cardiff
d
University, UK. Frederick National Laboratory for Cancer Research, National Cancer Institute,
Frederick, USA
[email protected]
Microtubules (MTs) are highly dynamic cylindrical structures, composed of α,β-tubulin
heterodimers. MTs are required for many essential functions, including the
maintenance of cell shape, cell motility, intracellular transport and cell division. Our
previous study showed that arylthioindoles (ATIs) bearing an alkoxycarbonyl group at
position 2 of the indole, were potent tubulin assembly inhibitors. In this study we
included the ester function in a heterocyclic nucleus. New arylthioindole derivatives (273) were potent tubulin assembly inhibitors that bound to the colchicine site.
Furthermore, they showed metabolic stability superior to the previously synthesized
ATIs (e.g., 1).1-3 Compound 13 was exceptionally potent as an inhibitor of cell growth
and it was uniformly active in the whole panel of cancer cells and superior to colchicine
and combretastatin A-4. It showed water solubility and high metabolic stability in
human liver microsomes.
Figure 1. General Structure of ATIs and binding mode into the Colchicine site of tubulin.
References
1. La Regina, G.; Sarkar, T.; Bai, R. et al. J. Med. Chem. 2009, 52, 7512−7527.
2. La Regina, G.; Bai, R.; Rensen, W. et al. J. Med. Chem. 2011, 54, 8394−8406.
3. La Regina, G.; Bai, R.; Pelliccia, S. et al. J. Med. Chem. 2013, 56, 123-149.
Antitumor agents
Poster P1.21
Targeting drug resistance in ovarian cancer:
design and development of peptide based inhibitor
Matteo Santucci1, Michela Pelà2, Puneet Saxena1, Rosaria Luciani1, Stefania Ferrari1,
Gaetano Marverti1, Chiara Marraccini1, Andrea Martello1, Domenico D’Arca1, Glauco
Ponterini1, Remo Guerrini2, Maria Paola Costi1
1
University of Modena and Reggio-Emilia, Department of Life Sciences, via Giuseppe Campi,
2
287 - 41125 Modena, Italy; University of Ferrara, Department of Pharmaceutical Sciences, via
Fossato di Mortara 17-19, 44100 Ferrara, Italy.
[email protected]
The gradual accretion in the number of ovarian cancer cases has sought serious
attention at the worldwide level. It is becoming the leading cause of deaths in the
western world [1], effecting about 22,240 women in US by the end of year 2013 (a
study performed by The American Cancer Society). Since, the surfacing of the disease,
several laboratories have started slogging their significant hours for the development of
drugs that could completely eradicate the lethal disease and overcome the problem of
ovarian cancer drug resistance. Thymidylate synthase is one such enzyme that plays
important role in the biosynthesis of DNA base Thymine. As per its functioning, the
enzyme catalyzes the reductive methylation of the uracil moiety of 2’-deoxyuridine5‘monophosphate (dUMP), turning it into dTMP. This characteristic mechanism has
been well demonstrated in humans. And due to its involvement in the DNA pathway,
the enzyme has become an important target for treating cancer disease, especially
ovarian cancer. In an attempt to find suitable drug-like candidates, we have discovered
peptide-based inhibitor that binds at the dimeric interface of the protein and provides
hindrance in the functioning of the enzyme. Thus, this inhibitor, namely LR-peptide, is a
protein-protein interaction modulator and its different mechanism of action could help to
overcome the drug cross-resistance development, which is one of the major drawbacks
in the ovarian cancer’s treatment, to keep into consideration when the conventionalclassic platinum-based drugs are used [2]. Based on these findings, we have
performed structure-activity relationship study of the LR-peptide, in order to understand
the structural features responsible for inducing activity. We have also performed tests
for the various derived compounds on some ovarian cancer cell lines. In particular, the
D-scanning approach was adopted, where we observed that mutating Gln4 to its Disoform, enhanced the enzyme inhibition percentage value from 60% of LR to 80% at
100uM [3]. Our previous CD experiments have shown a relationship between the
activity and the conformation of the peptide: active peptides (LN, LR, CG and C8)
adopt secondary structures while inactive peptides (QE, SY and VQ) were found to be
unordered. Based on this, we also performed CD experiments to examine whether the
same holds true for the D-scanning results. To analyze the reason behind structural
variability and its effect on the activity of the peptide, Molecular dynamics technique
was implemented. In the present communication major results will be presented.
References
1. Ozols, R. F; Daly, M. B.; et al., Specific Keynote: Chemoprevention of Ovarian Cancer: The
Journey Begins. Gynecologic Oncology, 2003, 88, S59–S66.
2. Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R. et al., Protein-protein interface-binding
peptides inhibit the cancer therapy target human Thymidylate synthase. PNAS, 2011, 108
(34), 542-549.
3. Pela, M.; Saxena P.; Luciani, R.; et al., Design and synthesis of optimized peptides
inhibitors of human Thymidylate synthase and platinum resistant ovarian cancer cell
growths. In preparation.
Antitumor agents
Poster P1.22
Indole-based modulators of p53/MDM2 interaction as antitumoral agents
Barresi E.,1 Pugliesi I.,1 Taliani S.,1 Da Settimo F.,1 La Pietra V.,2 Marinelli L.,2 Novellino E.,2 Daniele
S.,1 Da Pozzo E.,1 Costa B.,1 Martini C.1
1
Dipartimento di Farmacia, via Bonanno, 6, Università di Pisa, 56126 Pisa. 2 Dipartimento di Farmacia,
Università di Napoli “Federico II”, via D. Montesano, 49, 80131 Napoli.
[email protected]
The tumor suppressor protein p53, also known as the guardian of genoma, is a transcription factor
that controls cellular response to stress which may be caused by hypoxia and DNA damage. To
maintain genomic integrity, p53 can: (i) activate DNA repair proteins when DNA has sustained
damage; (ii) induce cell cycle arrest by holding the cell cycle at the G1/S regulation point by the
expression of the cyclin-dependent kinase inhibitor p21; (iii) initiate apoptosis, if DNA damage
proves to be irreparable. Under normal conditions, p53 is maintained at a low steady-state level
through proteasome-mediated degradation, while in human cancer its level is frequently
altered.(1,2) The inactivation of p53 is often due to the overexpression of its mean negative
regulator that is the ubiquitine E3 Ligase Murine Double Minute 2 protein, MDM2. The aminoacids
implicated in the interaction between p53 and MDM2 are three: Phe19, Trp23, and Leu26 which
are inserted into a deep hydrophobic pocket on the surface of MDM2.(3) The first reported potent
and selective small molecule MDM2 antagonists were a class of cis-imidazolines, the nutlins. Other
small molecules have been developed, such as calchones, benzodiazepinones and spirooxindoles.(4)
In this research, we have developed novel small molecules able to activate the p53 through the
inhibition of the MDM2-p53 interaction. In the rational design of these novel inhibitors, we have
considered indole-based scaffolds as indole is frequently used as “ privileged structure” in biology
and medicine due to its favorable physico-chemical properties.(5) Functionalization of the central
scaffold was driven by molecular modeling studies in order to mimic the contacts and the
orientations of the key amino acid side chains implied in the protein-protein binding. In particular,
we developed a number of 2-phenylindol-3-ylglyoxyl derivatives featuring dipeptide moieties on the
amide side chain (series I). Design, synthesis and biological evaluation of compounds I will be
discussed.
R1
NH
O
O
COOC2H5
N
H
R2
O
X
N
H
Y
I
References
1. Meulmeester, E.; Jochemsen, A. Curr. Cancer Drug Targets 2008, 8, 87-97.
2. Lakin, N. D.; Jackson, S. P. Oncogene 1999, 18, 7644-7655.
3. Kussie, P. H.; Gorina, S.; Marechal, V. Science 1996, 274, 948-953.
4. Vassilaev, L. T.; Vu, B. T.; Graves, B. Science 2004, 303, 844-848. (5) Welsch, M.; Snyder, S. A.;
Stockwell B. R. Curr. Opin. Chem. Biol. 2010, 14, 347-361.
Antitumor agents
Poster P1.23
SYNTHESIS AND BIOLOGICAL EVALUATION OF ∆2-ISOXAZOLINE DERIVATIVES
AS DNA METHYL-TRANSFERASE 1 (DNMT1) INHIBITORS
Viviano, M.1, Castellano, S.1, Conti, P.2, Tamborini, L.2, Kuck, D.3, Novellino, E.4,
Medina-Franco, J. L.5, Lyko, F.3 and Sbardella, G.1
1
Dipartimento di Farmacia, Università degli Studi di Salerno,Italy; 2Dipartimento di Scienze
3
Farmaceutiche ‘Pietro Pratesi’, Università degli Studi di Milano, Italy; Division of Epigenetics,
4
Deutsches Krebsforschungzentrum, Germany; Dipartimento di Chimica Farmaceutica e
Tossicologica, Università di Napoli “Federico II” Via D. Montesano 49, 80131 Napoli, Italy;
5
Torrey Pines Institute for Molecular Studies, FL 34987, USA.
[email protected]
The inactivation of tumor suppressor genes, which often results from epigenetic
silencing associated with DNA hypermethylation, plays a pivotal role in the
development of most forms of human cancer. Functional knockdown of DNA
methyltransferase 1 (DNMT1) is reported to reduce the genomic methylation patterns
by approximately 93%.1 Moreover, there are several reports demonstrating a strictly
link between DNMT1 disregulation and oncogenensis.
Nucleoside analogues of cytosine (e.g., 5-azacytidine) effectively inhibit the activity of
DNA methyltransferases, but their high cytotoxicity and the low therapeutic index make
the development of novel non-nucleoside inhibitors highly desirable. Procaine and
procainamide, already used as a local anesthetic and an antiarrhythmic drug,
respectively, exhibit a weak DNA demethylating activity and are “repositionable” as
non-nucleoside inhibitors.
We recently identified a ∆2-isoxazoline constrained analogue of procaine/procainamide
as a lead compound for the development of non-nucleoside DNMT inhibitors.2-4 Herein
we report the synthesis and the biological evaluation of new, longer compounds,
obtained by the combination of this SAM-competitive scaffold with “warheads” targeting
the nucleotide binding site, as bisubstrate inhibitors of DNMT1 (Figure 1).
Figure 1. General structure of DNMT1 inhibitors object of this work
References
1. Rhee, I. Bachman, K. E. Park, B. H. et al. Nature, 2002, 416, 552-556.
2. S. Castellano, D. Kuck, M. Sala et al., J. Med. Chem., 2008, 51, 2321-2325.
3. S. Castellano, L. Tamborini, M. Viviano et al., J. Org. Chem., 2010, 75, 7439-7442.
4. S. Castellano, D. Kuck, M. Viviano et al., J. Med. Chem., 2011, 54, 7663-7677.
Antitumor agents
Poster P1.24
Identification of a new series of non-covalent proteasome inhibitors with
selectivity for β5-subunits
Scarbaci K.,a Troiano V.,a Micale N.,a Ettari R.,b Lavecchia A.,c Di Giovanni C.,c
Grasso S.,a Novellino E.,c Schirmeister T.,d Zappalà M.a
a
Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Università degli Studi di Messina
b
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano
c
Dipartimento di Farmacia, Università degli Studi di Napoli Federico II
d
Institute of Pharmacy and Biochemistry, University of Mainz
[email protected]
Proteasome is a multisubunit, multicatalytic threonine protease complex with caspaselike (β1), trypsin-like (β2), and chymotrypsin-like (β5). It is responsible for the turnover
of cellular proteins including those involved in signal transduction, cell cycle control,
apoptosis and inflammation. Defects in the proteasome activity can lead to anarchic
cell proliferation and tumors development. For these reasons, proteasome is a target of
great interest in drug discovery for anticancer therapy.1
The majority of 20S inhibitors developed nowadays are peptide-based compounds
endowed with a C-terminal electrophilic warhead that forms covalent adducts with the
active site Thr1Oγ. However, very often this mechanism of action may lead to off-target
interactions due to high reactivity of compounds. Non-covalent inhibitors provide an
alternative mechanism for proteasome inhibition and, due to the rapid dissociation from
the 20S subunits, may be devoid of all inherent drawbacks related to the covalent
inhibition. In this context, we report a series of amide derivatives (1-2, Fig. 1) built on a
constrained motif suitable for proteasomal β5 inhibition.2-3 Preliminary screening
demonstrated that several derivatives are good inhibitors the proteasome β5 subunit
with Ki values in the micromolar range. Docking models confirmed the expected noncovalent interaction with the target for this new class of amide derivatives.
Figure 1. General structure of proteasome inhibitors synthesized
References
1. Goldberg, A. L. Functions of the proteasome: from protein degradation and immune
surveillance to cancer therapy. Biochem. Soc. Trans. 2007, 35, 12-17.
2. Ettari, R.; Bonaccorso, C.; Micale, N.; Schirmeister, T.; Calabrò, M. L.; Grasso, S.; Zappalà,
M. Development of novel peptidomometics containing a vinyl sulfone moiety as proteasome
inhibotors. ChemMedChem 2011, 6, 1228-1237.
3. Micale N., Ettari R., Lavecchia A., Di Giovanni C., Scarbaci K., Troiano V., Grasso S.,
Novellino E., Schirmeister T., Zappalà M. Development of peptidomimetic boronates as
proteasome inhibitors. Eur. J. Med. Chem. 2013, in press.
Antitumor agents
Poster P1.25
Synthesis and Structure-Activity Relationships of Bis-3-chloropiperidines
as Bifunctional DNA Alkylating Agents
Zuravka, I.a, Göttlich, R.a and Gatto, B.b
a
b
Department of Organic Chemistry, Heinrich-Buff-Ring 58, 35392 Giessen, Germany
Dept. of Pharmaceutical and Pharmacological Sciences, v. Marzolo 5, 35131 Padova, Italy
[email protected]
Bifunctional alkylating agents such as chlorambucil and melphalan represent an
important class of clinical cancer chemotherapeutics. The antitumor antibiotics
azinomycin A and B have the ability to alkylate and cross-link DNA. However, their
poor chemical stability suggests that these natural products are unlikely to progress as
therapeutic candidates. Yet they can act as lead structures from which to develop
potentially useful new molecules.1 Due to our interest in developing more effective
bifunctional alkylating agents, we designed novel bis-3-chloropiperidines based upon
the azinomycins backbone (fig.1).
Figure 1. Bis-3-chloropiperidines: structurally simplified analogues of the azinomycins.
The nitrogen mustard moiety is more stable and the alkylating functions generated via
intermediate aziridinium ions are positioned in a similar distance as has been observed
for the azinomycins.2,3 Accordingly, we synthesised a series of derivatives and
evaluated the alkylating properties towards nucleic acids to assess their molecular
mechanism of action and elaborate SAR useful to direct the synthesis of new
molecules. The biochemical results as well as the synthetic approach to bis-3chloropiperidines will be presented.
References
1. Watanabe, C. M. H. et al. Mode of action and biosynthesis of the azabicycle-containing
natural products azinomycin and ficellomycin. Nat. Prod. Rep. 2011, 28, 693-704.
2. Shipman, M. et al. Delineating noncovalent interactions between the azinomycins and
double-stranded DNA: Importance of the naphthalene substitution pattern on interstrand
cross-linking efficiency. Organic Letters 2004, 6, 3505-3507.
3. Searcey M. et al. Design and synthesis of a DNA-crosslinking azinomycin analogue. Org.
Biomol. Chem. 2005, 3, 3585-3589.
Metabolic and inflammatory disease agents
Poster P2.1
N-alkyl dien- and trienamides with binding affinity for opioid
and cannabinoid receptors
1
Anzani N., 2Ruiu S., 2Orrù A., 3Floris C., 1Caboni P. and 1Cottiglia F.
1
Dipartimento di Scienze della Vita e dell’Ambiente, University of Cagliari, via Ospedale 72,
2
09124 Cagliari, Italy. Institute of Translational Pharmacology, UOS of Cagliari, National
3
Research Council, Parco Scientifico e Tecnologico - Pula (Cagliari), Italy. Dipartimento di
Scienze Chimiche, University of Cagliari, Complesso di Monserrato, Monserrato, 09042, Italy
[email protected]
Echinacea purpurea and Echinacea angustifolia alcoholic extracts are widely used for
the prevention and treatment of common cold, bronchitis and coughs.1 These extracts
are popularly believed to act as immunomodulators. Despite the popularity and the
clinical effectiveness of Echinacea preparations, the molecular mechanism of action of
these herbs has been clarified only recently and various studies indicated certain fatty
acid N-isobutylamides (alkylamides) as the active principles of Echinacea extracts. In
particular, dodeca-2E,4E-dienoic acid isobuthylamide (1) and dodeca-2E,4E,8Z,10Ztetraenoic acid isobuthylamide (2) (Figure 1) were identified as full agonists at the
cannabinoid type 2 receptor (CB2) at nanomolar level showing a higher selectivity
versus CB2 receptors.2 Otanthus maritimus (L.) is an aromatic herb belonging to
Asteraceae that grows on sandy beaches along the Mediterranean coasts. In traditional
medicine O. maritimus is used to treat asthmatic bronchitis, dysentery and
inflammation of the urinary bladder. Taking into account of the importance of
alkylamides as CB2 selective natural ligands and of the very unusual
thienylheptatrienamides contained in O. maritimus roots,3 we decided to isolate
Otanthus amides and evaluate their binding affinity for the CB1 and CB2 as well as for
the µ and δ opioid receptors. Two new thienylheptatrienamides and one new neolignan, together with eight known amides were isolated from the roots of Otanthus
maritimus. The structures of the new compounds were elucidated on the basis of
extensive 1D and 2D NMR experiments as well as high resolution mass spectrometry.
Some alkylamides showed moderately high binding affinity for CB2 receptors at
nanomolar level. In addition, all the compounds showed low binding affinity versus
opioid receptors.
NH
1
O
NH
2
O
Figure 1. Alkylamides from Echinacea that act as full agonists at CB2 receptors
References
1. Goel, V.; Lovlin, R.; Barton, R.; et al. Efficacy of standardized Echinacea preparation
TM
(Echinilin ) for the treatment of common cold: A randomized, double-blind, placebocontrolled trial. J. Clin. Pharmacol. Ther. 2004, 29,75-83.
2. Raduner, S.; Majewska, A.; Chen, J-Z.; et al. Alkylamides from Echinacea are a new class
of cannabinomimetics. J. Biol. Chem. 2006, 281, 14192-14206.
3. Bohlmann, F.; Zdero, C.; Suwita, A. Weitere amide aus der tribus Anthemideae. Chem. Ber.
1974, 107, 1038-1043.
Metabolic and inflammatory disease agents
Poster P2.2
Synthesis of 3β,7α-Dihydroxy-Δ5-cholenoic Acid Derivatives: ‘Atypical’ Bile Acid
Metabolites and Useful Biomarkers to Detect 3β-HSDH Deficiency
Antimo Gioiello,a Bruno Cerra,a Kenneth D. Setchell,b Roberto Pellicciari.a
a
Dipartimento di Chimica e Tecnologia del Farmaco, Via del Liceo 1, 06123, Perugia, Italy;
Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center,
3333 Burnet Avenue, Cincinnati, OH 45229.
b
[email protected]
Inborn errors responsible for impaired bile acid (BA) biosynthesis and metabolism are
demonstrated to be potential cause of severe gastrointestinal diseases including
neonatal cholestasis, neurological disorders, as well as lipids and fat-soluble vitamins
malabsorption.1 If not diagnosed and treated, progressive liver and intestinal disorders
may develop leading, in some circumstances, to serious morbidity or mortality.2-4 These
defects which derive from the failed action of key enzymes, block the production of
'normal' BAs whereas favour the accumulation of 'atypical' BAs and intermediary
metabolites.2 In this framework, the presence and/or the variation of the concentration
related to specific BAs and their metabolic intermediates in plasma, serum, bile or urine
are considered important biomarkers for diagnostic purposes.
Based on these premises, the need to identify and confirm the biochemical diagnosis of
these defects has required the use of sophisticated analytical techniques, sensitive and
validated analytical protocols, as well as the development of efficient synthetic methods
for the preparation of suitable reference standard compounds.
As a continuation of our interest in the field of BAs, herein we report the synthesis of
3β,7α-dihydroxy-5-cholen-24-oic acid derivatives, which represent the signature
metabolites of the 3β-hydroxy-C27-steroid oxidoreductase (3β-HSDH) deficiency.
According to the published literature,5 clinical manifestations of this metabolic disorder
include jaundice, elevated serum γ-glutamyl transpeptidase, fat-soluble vitamine
malabsorption and conjugated bilirubinemia.
References
1. Mayatepek, E. Inborn errors of bile acid metabolism and their diagnostic confirmation by
means of mass spectrometry. J. Pediatric Sciences 2011,3, e68.
2. Heubi, J. E.; Setchell, K. D. R.; Bove, K. E. Inborn errors of bile acid metabolism. Seminars
Liver Dis. 2007, 27, 282-294.
3. Setchell, K. D. R.; O’ Connell N. C. Inborn errors of bile acid metabolism. Liver Disease in
Children 1994, 835-851.
4. Setchell, K. D. R. Disorders of bile acid synthesis and metabolism. Pediatric Gastrointestinal
Disease: pathophysiology, diagnosis, management 1996, 1205-1233.
5. Mizuochi, T.; et al. Molecular genetic and bile acid profiles in two Japanese patients with 3βhydroxy-∆5-C27-steroid dehydrogenase/isomerase deficiency. Ped. Res. 2010, 68, 258-263.
Metabolic and inflammatory disease agents
Poster P2.3
Synthesis and cycloxygenase inhibitory activity of new naphthalenemethylsulfonamido and naphthalene-methylsulfonyl compounds
Nencetti S., Ciccone L., Rossello A., Nuti E. and Orlandini E.
Dipartimento di Farmacia, Università Pisa Via Bonanno 6 56126 Pisa
[email protected]
Non-steroidal anti-inflammatory drugs (NSAIDs) are competitive inhibitors of the
ciclooxygenase (COX) enzyme. Their use is associate with side effects such as
gastrointestinal and renal toxicity. The anti-inflammatory therapeutic action of NSAIDs
is due to the inhibition of COX-2, while the undesired side effects arise from the
inhibition of COX-1 activity. This enzyme exists in two isoforms: COX-1, which is
constitutive, and COX-2 which is inducible. COX-1 is expressed in the most of tissues
and is involved in physiological production of PGs -responsible of the gastric
cytoprotection-, COX-2 has lower expression levels in tissues in normal conditions, but
its expression is increased during inflammatory responses. The expression of COX-2
is induced by cytokines, mitogens and endotoxins thus it plays the major role in
prostaglandin biosynthesis during inflammation. The up-regulation of COX-2 was
observed in premalignant and malignant conditions of various cancers such as breast,
prostate and lung ones which metastasize in the bones.1 Particular attention has been
recently focused on the role that COX enzyme could play in Alzheimer's diseases
(AD).2,3
After the removal from the market of some selective COX-2 inhibitors (COXIBs), due
to their cardiovascular side effects, has focused the research towards the evaluation of
alternative chemical structures able to maintain the COX inhibitory activity with reduced
side effects.
With this aim, we synthesized a series of new naphthalene derivatives: 6-MNA- and
naproxen-like (1-5), and indomethacin-like (6a-c), figure 1. In these compounds the
carboxylic function, typical of the classical NSAIDs, was replaced by a
methylsulfonamido or methylsulfonyl group present in some selective COX-2
inhibitors. The compounds 1-6 were tested “in vitro” to assess the activity towards COX
enzymes.
Figure 1. General structure of new naphthalene compounds 1-6
References
1. Suraneni, M.V.; Reddy, G.V. and Pallu, R. NSAIDs, COXIBs, CLOXIBs and cancer pain.
Horizons in Cancer Research 2012, 47, 253-270
2. Townsed, K.P. and Patricò D. Novel therapeutic opportunities for Alheimer’s disease: focus
on nonsteroidal anti-inflammatory drugs. The FASEB Journal 2005, 19, 1592-1061
3. Nivsarkar, M.; Banerjee, A. and Padh, H. Cyclooxygenase inhibitor: a novel direction for
Alzheimer’s management. Pharmacological Reports 2008, 60, 692-698
Metabolic and inflammatory disease agents
Poster P2.4
Novel Tools for the Elucidation of Physio-Pathological Role of H2S
A. Corvino, B. Severino, V. Vellecco, M.R. Bucci, G. Cirino,
V. Santagada, G. Caliendo, F. Frecentese
Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”
email: [email protected]
Hydrogen sulphide is a colourless, flammable, watersoluble gas with the characteristic smell
of rotten eggs. It is emerging as an important endogenous modulator, which is responsible
for the beneficial effects associated with Allium species intaking, such as garlic, onion, leek,
which are rich in biologically active sulphur containing compounds, such as S-allylcysteine,
allicin, diallyl sulfide (DADS) and diallyl trisulfide (DATS). H2S is endogenously synthesized in
various mammalian tissues by two pyridoxal-5′-phosphate-dependent enzymes involved in Lcysteine metabolism: cystathionine-ß-sythase (CBS)
and cystathionine-γ-lyase (CSE) [1].
H2S behaves as a scavenger of reactive oxygen
species and several evidences suggest that it
triggers other important effects, mainly mediated by
the activation of ATP-sensitive potassium channels
(KATP). This mechanism accounts for the
vasorelaxing and cardioprotective effects of H2S.
Furthermore,
H2S
inhibits
smooth
muscle
proliferation and platelet aggregation and regulates
central nervous system functions, as well as
respiratory, gastroenteric and endocrine systems
[1]. Retrospective analysis of the literature on
biological effects of H2S suggests two different
approaches for investigating physio-pathological
conditions in which it is involved and to develop novel therapeutic approaches based on its
activities: i) synthesis and evaluation of "sulfur-precursors" or "prodrugs", able to release
H2S directly or via specific enzyme interaction or synthesis and ii) evaluation of CBS/CSE
inhibitors aiming to endogenous production of H2S inhibition. As already known,
pharmacological inhibitors of H2S biosynthesis include DL-propargylglycine (PAG) and betacyanoalanine (BCA), which are not specific and are likely to interact with other pyridoxal
phosphate dependent enzymes. Considering the lack of a pharmacophoric model as a lead
for rational design of targeted enzyme inhibitors, we selected and preliminarly tested
commercially available cysteine surrogates such as L-2-amino-3-(carbamoylthio) propanoic
acid, L-2-oxotiazolidin-4-carboxylic acid, L-2-amino-3-(2-aminoetilthio) propanoic acid, Lthiazolidine-4-carboxylic acid, D-penicillamine and DL-penicillamine. Catalytic profile of
recombinant CBS and CSE was assessed in the presence of selected compounds. The
interesting modulatory effect showed by tested compounds prompted us to design and
synthesize propargyl amide derivatives, combining structural features of natural substrates
and chemical residues supported onto known enzymatic inhibitors. Furthermore, following
crystallographic structural data available on CSE and considering the presence of
hydrophobic aminoacids near to catalytic site, we designed and synthesized butyl amide
derivatives characterized by enhanced lipophilic profile [2]. Finally, structural variations were
also conducted on an aromatic ring able to impair π stacking interactions between Tyr114
and the PLP. Synthesized compounds (Figure) were tested on rat aortic rings. Butyl
derivatives showed vasorelaxant activity acting as enzymatic substrates, while propargyl
amide derivatives evidenced an interesting inhibitory effect, reverting vasorelaxant action
induced by L-cysteine. This result will be further assessed on recombinant CBS and CSE by
direct H2S determination.
References
[1] Caliendo, G.; Cirino, G..; Santagada, V.; Wallace, J. J. Med. Chem 2010, 53, 6275-6286.
[2] Huang, S; Chua, J.H.; Yew, W.S.; Sivaraman, J.; Moore, P.K.; Tan C.H., Deng, L.W. J. Mol. Biol.
2010, 396, 708-718.
Metabolic and inflammatory disease agents
Poster P2.5
[1,2,4]Triazolo[1,5-c]pyrimidines as New Potent
Human A3 Adenosine Receptor Antagonists
Spalluto G.,a Federico S.,a Ciancetta A.,b Cacciari B.,c Klotz K.N.,d Moro S.b
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Piazzale Europa 1,
b
I-34127 Trieste, Italy; Molecular Modeling Section (MMS), Dipartimento di Scienze
c
Farmaceutiche, Università di Padova, via Marzolo 5, I-35131 Padova, Italy; Dipartimento di
Scienze Farmaceutiche, Università degli Studi di Ferrara, via Fossato di Mortara 17-19, I-44100
d
Ferrara, Italy; Institut für Pharmakologie, Universität of Würzburg, D-97078 Würzburg,
Germany.
[email protected]
[1,2,4]Triazolo[1,5-c]pyrimidine derivatives are reported in literature as A2A adenosine
receptor (AR) antagonists useful for the treatment of Parkinson’s disease (PD), senile
dementia and depression (1).1 We decided to explore the [1,2,4]triazolo[1,5-c]
pyrimidine scaffold in order to obtain antagonists selective towards hA3 AR. Inactivation
of the A3 AR is being studied in glaucoma but also in inflammatory diseases such as
asthma.2 In a previous work, we have found that, in order to obtain good hA3 AR
antagonists, the better substitutions at the 5 and 8 positions are a little alkylamino
(methyl or ethylamino) and an ethylester moieties, respectively. Here we report a novel
series of [1,2,4]triazolo[1,5-c]pyrimidines where we have introduced different alkyl,
cycloalkyl, aryl and heteroaryl moieties at the 2 position (2). Surprisingly, the
introduction of substituted-phenyl rings led to subnanomolar affinities at the hA3 AR.
The best compound of this series is represented by the ethyl 2-(4-methoxyphenyl)-5(methylamino)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate (3) which shows a Ki of
0.47 nM towards hA3 AR and a 4200- and 8400-fold selectivity over hA1 and hA2A ARs,
respectively.
Figure 1. [1,2,4]Triazolo[1,5-c]pyrimidine derivatives as adenosine receptor antagonists.
References
1. Shimada, J.; Imma, H.; Osakada, N.; Shiozaki, S.; Kanda, T.; Kuwana, Y.
[1,2,4]Triazolo[1,5-c]pyrimidine derivatives. 2003, US6545000.
2. Cheong, S.L; Federico, S.; Venkatesan, G.; Mandel, A.L.; Shao, Y.M.; Moro, S.; Spalluto,
G.; Pastorin, G. The A3 adenosine receptor as multifaceted therapeutic target:
pharmacology, medicinal chemistry and in silico approaches. Med. Res. Rev. 2013, 33,
235-335.
Metabolic and inflammatory disease agents
Poster P2.6
Synthesis and preliminary evaluation of model compounds targeting the NLRP3
inflammasome pathways
Davide Garella, Mattia Cocco, Antonella Di Stilo, Gianluca Miglio, Massimo Collino,
Roberto Fantozzi, Massimo Bertinaria
Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via P.
Giuria 9, I-10125 Torino, Italy
[email protected]
The inflammasomes have recently emerged as key mediators of inflammation and
immunity. Four inflammasome complexes have been described to date. The most
intensely studied is the NLRP3 inflammasome formed by the NLR (nucleotide-binding
domain leucine-rich repeat) family member NLRP3 and the adapter protein ASC, its
assembling is triggered by a diverse series of endogenous and exogenous stimuli.1
NLRP3 assembly leads to caspase-1 activation which, in turn, causes the maturation
and secretion of the pro-inflammatory cytokine IL-1β and IL-18 (figure 1). The onset of
chronic inflammation due to NLRP3 inflammasome overactivation has been recently
linked to a wide range of metabolic disorders such as type 2 diabetes, atherosclerosis,
intestinal inflammation leading to colorectal cancer, inflammatory bowel disease, liver
injury and Alzheimer’s disease.2 A series of autosomal and dominant or de novo
mutation of NLRP3 lead to auto-inflammatory syndromes known as CAPS (cryopyrin–
associated periodic syndromes) which are characterized by high levels of IL-1β release
and associated sterile systemic inflammation.
Nowadays clinical application of this knowledge has been limited by a lack of selective
drug-like inhibitors of the NLRP3 inflammasome-related pathways. At the present the
therapeutic approaches are based on the use of recombinant IL-1 receptor antagonists
(Anakinra), anti-IL1 monoclonal antibody (Canakinumab) or IL-1 trapping agents
(Rilonacept). Few small molecules acting upstream or downstream of NLRP3 activation
or directly on the NLRP3 assembly have been reported to date.3 In this communication
we describe the synthesis and preliminary evaluation of some model compounds
designed to inhibit the NLRP3 inflammasome-related pathways.
Figure 1. NLRP3 inflammasome activation in macrophages and general structure of designed
compounds
R4
R1
R3
X
n
R2
References
1. De Nardo, D.; Latz, E. NLRP3 inflammasomes link inflammation and metabolic disease.
Trends Immunol. 2011, 32(8), 373-379
2. Davis B. K.; Wen H., Ting, J. P.-Y. The inflammasome NLRs in immunity, inflammation and
associated diseases. Annu. Rev. Immunol. 2011, 29, 707-735
3. Lopez-Castejon, G.; Pelegrin, P. Current status of inflammasome blockers as antiinflammatory drugs. Expert Opin. Investig. Drugs 2012, 21(7), 995-1007.
Metabolic and inflammatory disease agents
Poster P2.7
Structure-Activity Relationship, Biological and Conformational Studies of Derivatives of the
Pepducin S1P3 Agonist KRX-725
Giuseppina Maria Incisivo1, Elisa Perissutti1, Antonio Bertolino1, Paola Di Vaio1, Emilio Benfenati2,
Vincenzo Santagada1, Giuseppe Caliendo1, Fiorentina Roviezzo1, Beatrice Severino1
1
Dipartimento di Farmacia, Università di Napoli «Federico II» Via D. Montesano, 49, 80131, Napoli, Italy.
2
Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156, Milano, Italy.
[email protected]
Sphingosine-1-phosphate (S1P) is a bioactive lipid with key functions in the immune,inflammatory,
and cardiovascular systems. S1P exerts its action through the interaction with a family of five
known G protein-coupled receptors (GPCRs), named S1P1-5.[1] Among them, S1P3 has been
implicated in the pathological processes of a number of diseases, including sepsis and cancer.[2,3]
KRX-725 (compound 1, Myr-GMRPYDANKR-NH2) is a pepducin that mimics the effects of S1P by
triggering specifically S1P3.[4] Here, to address the contribution of the side chains of each amino
acid residue to the peptide function, an alanine scanning analysis was carried out. Then deleted
peptides both from the C- and N-terminus were prepared in order to determine the minimal
sequence for activity and to identify the structural requirements for agonistic and, possibly,
antagonistic behavior.
Figure 1: A. Pretreatment of mouse aortic rings with compound 16 significantly inhibits in a concentration
dependent manner compound 1-induced vasorelaxation.. B. Pretreatment of mouse fibroblasts with
compound 16 abrogates compound 1-induced effect on cell proliferation.
The pharmacological results of the Alascan derived
compounds (2-10) suggested a high tolerance of the
pepducin 1 to amino acid substitutions. Importantly, the
deleted peptide 16 (Myr-GRPYDAN-NH2) has the ability to
inhibit, in a dose-dependent manner, both pepducin 1
induced vasorelaxation and fibroblast proliferation (Figure
1).
Finally a computational analysis was performed on the
prepared compounds showing that supposed antagonists
16 and 17 (Myr-GRPYDA-NH2), appeared to be aligned
each other, but not with the others (Figure 2). These results
suggested a correlation between specific conformations
and activities.
Figure 2: Superposition of compounds
16 (magenta) and 17 (yellow).
References
1. Rosen, H.; Gonzalez-Cabrera, P. J.; Sanna, M. G.; Brown, S.. Annu. Rev. Biochem. 2009, 78, 743–768.
2. Keul, P., Lucke, S.; von Wnuck Lipinski, K.; Bode, C.; Graler, M.; Heusch, G.; Levkau, B. Circ. Res.
2011, 108, 314–323.
3. Hsu, A.; Zhang, W.; Lee, J. F.; An, J.; Ekambaram, P.; Liu, J.; Honn, K. V.; Klinge, C. M.; Lee, M. J. Int.
J. Oncol. 2012, 40, 1619–1626.
4. Licht, T.; Tsirulnikov, L.; Reuveni, H.; Yarnitzky, T.; Ben-Sasson, S. A. Blood 2003, 102, 2099–2107.
Metabolic and inflammatory disease agents
Poster P2.8
Dimeric analogs of the dual PPARα/γ agonist LT-175:
design, synthesis and biological evaluation
Antonio Laghezza, Luca Piemontese, Sabina Sblano, Antonio Carrieri, Giuseppe
Fracchiolla, Giuseppe Carbonara, Paolo Tortorella and Fulvio Loiodice
Dipartimento di Farmacia-Scienze del Farmaco,
Università degli Studi di Bari “Aldo Moro”, via Orabona 4, 70125 Bari
[email protected]
Peroxisome Proliferator-Activated Receptors (PPARs) are members of the nuclear
receptor superfamily playing a crucial role in the regulation of metabolic homeostasis.
As such, the three PPAR isoforms designated α, γ and δ, certainly represent attractive
targets for molecules that could be used in diabetes and dyslipidemia [1]. Following the
recent indications available in the literature, research is currently addressed to the
preparation and characterization of new molecules able to simultaneously activate
more PPAR subtypes (PPAR dual agonists and/or pan-agonists), and/or to selectively
modulate them (SPPARMs). Recently, we reported the synthesis and biological activity
of some chiral carboxylic acid derivatives showing an interesting dual activity towards
PPARα and PPARγ with the stereochemistry playing a crucial role in the receptor
activation [2,3]. In particular, one of these newly identified PPAR ligands (LT-175,
Figure 1) has been shown to occupy a branch of the PPARγ ligand binding domain
(LBD), named “diphenyl pocket”, exhibiting the typical profile of partial agonists [2]. The
corresponding R enantiomer, instead, is less active fitting another part of the receptor.
With the aim to evaluate the effects resulting from both interaction modes, we
synthesized the dimeric analogs shown in Figure 1 and tested the corresponding
stereoisomers. Preliminary results show very interesting activity profiles with marked
differences depending on the configuration of the two stereogenic centers.
Figure 1. Structure of LT-175 and dimeric analogs.
References
1. Pinelli, A. et al. J. Med. Chem., 2005, 48, 5509-5519.
2. Pochetti, G. et al. J. Med. Chem., 2008, 51, 7768-7776.
3. Laghezza, A. et al. J. Med. Chem., 2013, 56, 60-72.
Metabolic and inflammatory disease agents
Poster P2.9
Solid state interconversion of drug-triacylglycerol systems
Foglio Bonda A., Mannina P., Giovannelli L., Segale L., Pattarino F.
Dipartimento di Scienze del Farmaco, Università del Piemonte Orientale, Largo Donegani, 2 28100 Novara
[email protected]
The relevant number of research works on lipid pharmaceutical dosage forms has
stimulated the researchers to study in depth the intimate structure-function relationship
in lipid materials1-2. The characteristics of lipid dosage forms depend on the choice of
lipids, the selected processing technique and also on the chemical and physicochemical properties of the drug. Solid systems can be produced by a variety of different
methods, most of which involve the melting of lipids which can crystallize during resolidification as unstable polymorphs. Therefore, the processing of dosage forms is
often accompanied by undesired solid-state changes. In addition, during storage the
polymorphic form of lipids might undergo a change to another form, often referred to as
“aging” in the literature. These changes can lead to alteration of the dissolution
characteristics of the drug and of its bioavailability in human body. At present, there is a
lack of understanding of the underlying principles of such transformations during the
manufacturing of pharmaceutical dosage forms. Therefore, for reliable and
reproducible dosage forms, an accurate study of the physico-chemical behavior of lipidbased drug dosage forms is mandatory to prevent potential stability problems.
In the present work binary and ternary mixtures constituted of tristearoylglycerol (TSG),
caprylic/capric triglyceride (Labrafac®-CCT) and one of three model drugs were
investigated using differential scanning calorimetry (DSC) and X-ray powder diffraction
(XRPD) in order to acquire information about the effects of composition, manufacturing
process and storage on the crystal structure and thermodynamic behavior (phase
transition behavior) of these lipid materials. An anthelmintic (FM213) and two antiinflammatory drugs (ketoprofen and ibuprofen) were used as model drugs.
These systems were prepared using the same protocol: solid dispersions obtained comelting drug and excipient(s) were heated to 100 °C and held for 5 min at this
temperature (to erase crystal memory), then rapidly cooled down to 25 °C. Samples of
each system were stored at 25 °C over the examined period of time. The XRPD pattern
and DSC profile of blends were obtained immediately after preparation (t=0) and at
scheduled times within 90 days.
DSC profiles and XRPD spectra indicated the presence of different crystalline
arrangements of blends; solid-solid conversion from unstable to stable crystalline forms
occurred during storage and the rate and pathway of interconversion changed over
time, primarily induced by the drug incorporated in the lipid vehicle.
References
1. Himawan, C.; Starov, V.M.; Stapley, A.G.F. Thermodynamic and kinetic aspects of fat
crystallization. Adv. Colloid. Interface Sci. 2006, 122, 3-33.
2. Sato, K. Crystallization behaviour of fats and lipids: a review. Chem. Eng. Sci. 2001, 56,
2255-2265.
Metabolic and inflammatory disease agents
Poster P2.10
MOLECULAR DOCKING OF BIARYL TETRAZOLYL UREAS
AS INHIBITORS OF ENDOCANNABINOID METABOLISM
Pirolli Adele¥§, Ballante Flavio¥§, Patsilinakos Alexandros¥§, Giorgio Ortar¥,
Enrico Morera¥, Rino Ragno¥§
§
Rome Center for Molecular Design
Dipartimento di Chimica e Tecnologie del farmaco, Sapienza Universita` di Roma,
Piazzale Aldo Moro 5, 00185 Roma, Italy
¥
[email protected]
Endogenous ligands of cannabinoid receptors (endocannabinoids) are involved
as lipid messengers in a wide array of physiological and pathological conditions, and
the regulation of their levels appear to be of remarkable therapeutic interest in a variety
of human disorder, including appetite, pain-sensation, mood and memory.1
In the present study, we have further extended the structure–activity
relationships for the tetrazolyl ureas class of compounds as potential FAAH and/or
MAGL inhibitors, by replacing the dimethylamino group of the parent compounds 1 and
2 with bulkier groups or by introducing on the distal phenyl ring of 1 and 2 a selected
set of substituents. Some of the new compounds inhibited FAAH potently (IC50 = 3.0–
9.7 nM) and selectively (39- to more than 141-fold) over MAGL.2 Molecular docking
studies were therefore conducted on a series of 32 tetrazolic compounds with
AutoDock Vina docking program. AutoDock Vina usage was assessed through an
extensive re-docking and cross-docking previously reported protocol and using the
available experimental three-dimensional structures of FAAH co-crystallized with
different inhibitors. Covalent docking studies on FAAH indicated that the binding modes
of tetrazoles did not display a unique pattern, and were determined by the regioisomery
of compounds, the nature of nitrogen substituents of the carbamylating moiety, and the
nature and position of the distal phenyl ring substituents. The ability of tetrazoles to act
as TRPV1 and TRPA1 modulators was also investigated.
References
1. Battista, N.; Di Tommaso, M.; Bari, M.; Maccarrone, M. Frontiers in behavioral neuroscience
2012, 6, 9.
2. Ortar, G.; Morera, E.; De Petrocellis, L.; Ligresti, A.; Schiano Moriello, A.; Morera, L.; Nalli,
M.; Ragno, R.; Pirolli, A.; Di Marzo, V. European journal of medicinal chemistry 2013, 63C,
118.
Metabolic and inflammatory disease agents
Poster P2.11
H2S-releasing properties of new thioamide derivatives
I. Pugliesi, E. Barresi, S. Taliani, F. Da Settimo, A. Martelli, L. Testai, V. Calderone
Dipartimento di Farmacia, Università di Pisa, via Bonanno, 6. 56126 Pisa Italy.
[email protected]
Hydrogen sulphide (H2S) is emerging as an important endogenous modulator, which
exhibits the beneficial effects of nitric oxide (NO) on the cardiovascular (CV) system,
without producing toxic metabolites.[1] H2S is biosynthesized in mammalian tissues by
cystathionine-β-synthase and cystathionine-γ-lyase. H2S exhibits the antioxidant
properties of inorganic and organic sulphides, behaving as a scavenger of reactive
oxygen species. H2S trigger other important effects and the activation of ATP-sensitive
potassium channel (KATP) accounts for its vasorelaxing and cardioprotective effects.[2]
Furthermore, H2S inhibits smooth muscle proliferation and platelet aggregation. In nonCV systems, H2S regulates the functions of the central nervous system, as well as
respiratory, gastro enteric, and endocrine systems. Conversely, H2S deficiency
contributes to the pathogenesis of hypertension.[1] Likewise, impairment of H2S
biosynthesis is involved in CV complications associated with diabetes mellitus.[1] There
is also evidence of a cross-talk between the H2S and the endothelial NO pathways. In
particular, recent observations indicate a possible pathogenic link between deficiencies
of H2S activity and the progress of endothelial dysfunction. These biological aspects of
endogenous H2S have led several authors to look at this mediator as ‘‘the new NO’’
that has given attractive opportunities to develop innovative classes of drugs.[2]
In this study, we report the synthesis and the pharmacological evaluation of a series of
new thioamide derivatives of general formula I and II as H2S donor.
S
NH2
NH2
R
I
R1
S
N
II
References
1. Martelli A, Testai L, Breschi MC, Blandizzi C, Virdis A, Taddei S, Calderone V. Med Res
Rev 2012; 32, 1093-1130.
2. Martelli A, Testai L, Breschi M.C, Da Settimo F, Calderone V. Curr. Med. Chem. 2012,
19(20), 3325-3336.
Metabolic and inflammatory disease agents
Poster P2.12
From agonist to antagonist profile in LP1 analogues through the introduction of
different N-substituents in benzomorphan-based ligands
Ronsisvalle S, Turnaturi R, Prezzavento O, Arena E, Girolamo Calò, Valeria Camarda,
Ronsisvalle G, Pasquinucci L
Department of Drug Sciences, Medicinal Chemistry section, University of Catania, Viale A.
Doria 6, 95125 Catania, Italy
[email protected]
The design and discovery of new analgesics with diminished adverse side effects are a
major challenge for medicinal chemists1. An emerging approach is the use of multitarget
opioid ligands, e.g. mixed MOR/DOR compounds2. The benzomorphan-based compound
LP1, a multitarget opioid ligand with a MOR agonist/DOR antagonist profile, produced
centrally-mediated antinociception with a low tolerance-inducing capability3. Moreover, a
significant antiallodynic and antihyperalgesic of LP1 in animal models of neuropathic pain
and inflammatory pain determined effect was detected4. These results emphasized LP1 as
analgesic candidate for chronic pain treatment. The nature of the N-substituent conferred to
LP1 affinity and intrinsic activity versus MOR and DOR. In this study, we examined the
results obtained by a structure-activity relationships performed by the introduction of psubstituted-phenyl, naphtyl, diphenyl, indolinyl and tetrahydroquinolyl rings in the Nsubstituent. Thus, nine analogues of LP1 were synthesized and tested for the calcium
mobilization in CHO cells stably expressing human MOR or KOR coupled to the G〈qi5
chimeric protein or DOR coupled to the G〈qG66Di5 chimeric protein in the fluorometric imaging
plate reader FlexStation II. The data showed that the increase of N-substituent size led to a
MOR antagonist profile. An antagonist activity at all three opioid receptors was detected for
derivatives with 1-Nph and 2-Nph as substituent. Interestingly, the 1-Nph-substuted
compound inhibited the effects of Dermorphin, DPDPE and Dynorphin A agonists showing
higher potency at MOR (pKB 7.90) than DOR (pKB 6.78) and KOR (pKB 5.86). Moreover, a
MOR antagonist profile was detected for derivatives bearing in the N-substituent the
diphenyl or the indolinyl rings. The insertion of tetrahydroquinolinyl ring led to a shift of the
KOR profile from antagonist to agonist maintaining the MOR antagonist activity. In
comparison with LP1, the p-substituted-phenyl compounds showed a lower potency on
MOR and DOR and a switched functional profile. Briefly, the bulkier size of the substituent
modified the DOR and KOR functional profile from agonist to antagonist with retention of
the MOR agonist activity. The p-methoxy derivative did not follow this trend and just the pF-phenyl analogue shared with LP1 the same functional profile. Collectively, these data
suggest that the benzomorphan-N-phenylpropanamide-based structure may represent a
tool to achieve a specific opioid functional profile going from a pure agonist to a pure
antagonist via mixed agonist/antagonist ligands, simply by increasing or decreasing the
aromatic group size.
References
1. Morphy, R.; Rankovic, Z. Designing multiple ligands - medicinal chemistry strategies and
challenges.Curr. Pharm. Des. 2009, 15, 587-600.
2. Dietis, N.; Guerrini, R.; Calò, G. et al. Simultaneous targeting of multiple opioid receptors: a
strategy to improve side-effect profile. Br. J. Anaesth. 2009, 103, 38-49.
3. Pasquinucci, L.; Prezzavento, O.; Marrazzo, A. et al. Evaluation of N-substitution in 6,7benzomorphan compounds.Bioorg. Med. Chem. 2010, 18, 4975-82.
4. Parenti, C., Turnaturi, R., Aricò, G. et al. The multitarget opioid ligand LP1’s effects in persistent
pain and in primary cell neuronal cultures. Neuropharmacology, in press.
Neurodegenerative disease agents
Poster P3.1
Radiosynthesis of the TSPO 18 kDa PET Radiotracer [18F](R)-PK14105
Barresi E.,1,2 Siméon F.G.,1 Taliani S.,2 Pugliesi I.,2 Da Settimo F.2 and Pike V.W.1
1
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health,
Bethesda, MD 20892, USA.
2
Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
[email protected]
The isoquinoline carboxamide, PK14105, has previously been labeled with fluorine-18 (t1/2 =
109.7 min)1 and evaluated in rat as a prospective radiotracer for imaging human TSPO 18
kDa, a marker of neuroinflammation2, with positron emission tomography (PET). This
radiotracer initially appeared promising, but extensive evaluation was hindered by its rather
difficult radiosynthesis based on substitution of a chloro-substituent with cyclotron–produced
[18F]fluoride ion. Recently, we have shown that simple p-nitroaryl sulfoxides react readily
with [18F]fluoride ion to produce the corresponding [18F]p-nitrofluoroarenes in good
radiochemical yields.3 We considered that this method might be applied to enhance the
radiosynthesis of [18F]PK14105 as its higher affinity single R-enantiomer, and so permit
detailed evaluation of its radiotracer properties in monkey with PET. (R)-PK14105 was
treated with benzenethiol to produce the p-nitroaryl sulfide which was then converted into the
p-nitroaryl sulfoxide. Treatment of this sulfoxide with [18F]fluoride ion produced [18F](R)PK14105, which will now be characterized with PET as a TSPO 18 kDa radiotracer in
monkey. Further optimization of the radiosynthesis is in progress.
O
H
N
N
18F
NO2
Figure 1. [18F](R)-PK14105.
This work was supported by the Intramural Research Program of the National Institutes of
Health (NIMH).
References
1. Pascali, C.; Luthra, S. K.; Pike, V. W.; Price, G. W.; Ahier, R. G.; Hume, S. P.; Myers, R.; Manjil,
18
L.; Cremer, J. E. The radiosynthesis of [ F]PK 14105 as an alternative radioligand for peripheral
type benzodiazepine binding sites. Appl. Radiat. Isot. 1990, 41, 477−482.
2. Price, G. W.; Ahier, R. G.; Hume, S. P.; Myers, R.; Manjil, L.; Cremer, J. E.; Luthra, S. K.;
Pascali,C.; Pike, V.; Frackowiak, R. S. J. In vivo binding to peripheral benzodiazepine binding
3
18
sites in lesioned rat brain: comparison between [ H]PK11195 and [ F]PK14105 as markers for
neuronal damage. J. Neurochem. 1990, 55, 175−185.
18
3. Chun, J.-H.; Morse, C. L.; Chin, F. T.; Pike, V. W. No-carrier-added [ F]fluoroarenes from the
radiofluorination of diaryl sulfoxides. Chem. Commun. 2013, 49, 2151-2153.
Neurodegenerative disease agents
Poster P3.2
New PDE4D selective inhibitors able to restore memory function
Brullo C., Rotolo C., Rocca M., Massa M., Bruno O.
Department of Pharmacy, University of Genoa, Viale Benedetto XV, 3-16132, Genoa-Italy
[email protected]
Our recent research on PDE4 inhibitors (PDE4Is) gave a series of small molecules
bearing a catecholic moiety (typical of rolipram related PDE4Is) and an amino function
linked to the aromatic portion by an imino-ether chain. SAR studies on those
compounds evidenced a pivotal role for the chain length and its spatial direction,
particularly in determining the selectivity towards the different PDE4 isoforms 1. Two
compounds (namely 4a and 7b, Figure 1) showed a good selective inhibition of PDE4D
isoforms in respect to other PDE4 isoforms (PDE4A, PDE4B and PDE4C). From the
chemical point of view the two compounds differ in the nature of the linker, which in one
case is a propylic alcoholic chain (compound 4a), while in compound 7b is a shorter
amide 1.
Compound 7b, being the most active, was selected for additional studies in vitro and in
vivo to assess its ability to restore memory function in mice and rats and to evidence
possible lacking in side effects 2.
CH3
O
H3C
O
N O
H
linker
N
O
4a linker = CH2CHOHCH2
7b linker = CH2CO
CH3
Figure 1. General structure of our leads 4a and 7b.
In that study we demonstrated that the selective inhibition of PDE4D can increase
spatial and object recognition in rats and mice without causing emesis, the typical side
effect of non selective PDE4Is. These results prompted us to design new selective
PDE4DIs in order to obtain a possible drug candidate for patent application and
preclinical development.
Starting from the structures of our lead compounds 4a and 7b we planned the
synthesis of a new generation of small molecules bearing the same
dimethylmorpholino group linked to the catecholic portion by a modified chains.
In detail, we planned to:
1) modify the length of the linker chain.
2) improve the pharmacokinetic properties of our leads 4a and 7b, particularly to
increase the blood-brain-barrier (BBE) crossing.
The biological results gave us interesting information about the chemical modification
useful to improve selective PDE4D inhibition. Synthesis of new derivatives and
pharmacological results will be reported in the poster session.
References
1. Bruno O.; Romussi A. et al. New selective phosphodiesterase 4D inhibitors differently acting
on long, short, and supershort isoforms. J. Med. Chem. 2009, 52, 6546.
2. Bruno O.; Fedele E.; Prickaerts J. et al. GEBR-7b, a novel PDE4D selective inhibitor that
improves memory in rodents at non-emetic doses. Br .J. Pharm, 2011, 164, 2054.
Neurodegenerative disease agents
Poster P3.3
Hydroxypyrazolo[1,5-a]pyridine as a fluorescent carboxylic acid isostere:
introducing GABAA receptor system affinities in the scaffold
Alex Ducime,1 Birgitte Nielsen,2 Donatella Boschi,1 Bente Frølund,2 Marco L. Lolli1
1
DSTF - Department of Drug Science and Technology, University of Turin,
Via Pietro Giuria, 9 – 10125 Torino, Italy.
2
PHARMA - Department of Drug Design and Pharmacology, The Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
[email protected]
Bioisosteric replacement is a widely used strategy in Medicinal Chemistry for the
rational design of new drugs. Using this approach, the accurate modulation of lead
compounds allow to improve properties such as bioavailability, selectivity and potency.1
A number of bioisosteric relationships have been established for a series of functional
groups including the carboxylic acid.2
Medicinal chemistry programs have provided an extensive variety of bioisosteric
replacements for the carboxylic acid in GABA, the major inhibitory neurotransmitter in
the mammalian central nervous system.3,4 In this study, we firstly introduce the bicyclic
hydroxypyrazolo[1,5-a]pyridine scaffold (1) as a possible fluorescent isostere of the
carboxylic group. Besides being a strong emitter of blue light, the acidic conformational
locked hydroxypyrazolo[1,5-a]pyridine moiety offer additional positions for introducing
substituents in fixed directions. Taking advantage of this option, we have investigated
the effect of introducing the amino containing substituents in different positions, in order
to develop potential ligands for the orthosteric binding site in the GABAA receptor.
Designed GABA analogues (2) based on 1 should permit the investigation of the
requirement for the mutual position of the functional groups and exposing access to
cavities/channels associated to the orthosteric binding site, reaching out for subtypeselectivity.
The series of hydroxypyrazolo[1,5-a]pyridine (2) derivatives synthesized were
pharmacologically characterized in a [3H]-muscimol displacement assay at native
GABAA receptors and electrophysiological assays at relevant GABAA receptor
subtypes. The synthesis and pharmacological properties are reported and discussed in
terms of the structural knowledge available for the GABAA receptor.
References
1. Burger, A. Isosterism and Bioisosterism in Drug Design. Progress in Drug Research, 1991,
37, 337.
2. Meanwell Synopsis of some recent tactical application of bioisosteres in drug design.J.
Med. Chem. 2011, 54, 2529.
3. Frølund, B., Ebert, B., Kristiansen, U., Liljefors, T., Krogsgaard-Larsen, P. GABAA receptor
ligands and their therapeutic potentials. Curr, Topics Med. Chem. 2002, 2, 817–832.
4. Lolli, M. L.; Hansen, S. L.; Rolando, B.; Nielsen, B.; Wellendorph, P.; Madsen, K.; Larsen,
O. M.; Kristiansen, U.; Fruttero, R.; Gasco, A.; Johansen, T. N. Hydroxy-1,2,5-oxadiazolyl
Moiety as Bioisoster of the Carboxy Function. Synthesis, Ionization Constants, and
Pharmacological Characterization of gamma-Aminobutyric Acid (GABA) Related
Compounds. J. Med. Chem. 2006, 49(14), 4442-4446.
Neurodegenerative disease agents
Poster P3.4
A New Series of 5-HT1A, 5-HT2A and 5-HT2C Receptor Ligands
containing an Isonicotinic Nucleus
E. Magli1, E. Perissutti1, A.Ciano1, P. Massarelli2, R. Capasso1,
V. Santagada1, G. Caliendo1, F. Fiorino1
1
Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”.2Dipartimento di Farmacologia
“G. Segre”, Università degli Studi di Siena
[email protected]
Serotonin (5-hydroxytryptamine, 5-HT), one of the most important neurotransmitter in the
central and peripheral nervous systems (CNS and PNS, respectively), is implicated in
numerous physiological and physiopathological processes. Serotonin receptors may be
involved in the regulation of impulsivity and alcoholism, in the different phases of sleep,
sexual behavior, appetite control, thermoregulation, cardiovascular function and recently it
has been found to show growth-promoting activity and to be functionally related to
oncogenes. In particular, the 5-HT1A receptor, is a major target for research and drug
development due to its implication in many physiological processes. Instead, 5-HT2A receptor
is known to play a key role as a therapeutic target for the treatment of schizophrenia and
hypertension.
Moreover,
5-HT2A
O
receptors
are
widely
expressed
in the
N
H
gastrointestinal tract, where serotonin
causes contraction of longitudinal and
circular smooth muscle cells. Finally,
5-HT2C
receptor
subtype
is
considered to be an attractive target
X=
HC
HC
CH
for the design of novel drugs for
treatment of CNS-related diseases
such
as
obesity,
obsessive
compulsive disorders and sexual
dysfunction. Several chemical classes of agents are already known for their high affinity
toward these receptors (aminotetralines, ergolines, arylpiperazine, indolylalkylamines,
indoles, aporphines, arylalkylpiperidines, aryloxyalkylamines) and one of the most studied
group is the long-chain arylpiperazine (LCAPs) one, that have provided interesting drugs
acting on CNS (Buspirone, Aripiprazol) and compounds with a potential therapeutic profile
(Flesinoxan, Bifeprunox). Their diversified receptor binding profiles and intrinsic activities,
depending on either the kind of substituent attached to the N-4 atom of the piperazine moiety
or the nature of an amide or imide terminal fragment, open possibility for discovery of new
potent therapeutic agents. In continuation of our research program, the main goal of this
work was also to better clarify the structural features needed in order to make the LCAPs
able to interact with 5-HT1A and 5-HT2A receptors. Based on this considerations we have
analyzed a new set of derivatives where the piperazine-N-alkyl moiety has been linked, via
two or three methylene spacing units, to an isonicotinic fragment (Figure), that represents a
molecular simplification of N’-cyanoisonicotinamidine nucleus already reported, as
arylpiperazine derivatives.1 Moreover, the multireceptor profiles of promising new isonicotinic
derivatives were also evaluated in terms of binding affinities for D1, D2 and α1, α2 receptors.
Binding data presented in this study have shed additional light on the influence of the LCAPs
on the 5-HT receptors affinity and selectivity. In particularly, we have disclosed interesting
compounds as 5-HT1A, 5-HT2A and mixed 5-HT1A/5-HT2C ligands. Successively, the most
interesting derivatives have also been evaluated on rat ileus disclosing some interesting
compounds showing an inhibitory effect on serotonin-inducted contraction and evidencing
the role of these receptors in gastrointestinal tract, with a potential therapeutical profile as
spasmolytic agents.
(CH2)n
N
N
N
X
n = 2,3
OCH2CH3
OCH3
Cl
OCH3
Cl
Cl
Cl
3
3
3
Cl
N
Reference
[1] Fiorino, F.; Severino, B.; De Angelis, F.; Perissutti, E.; Magli, E.; Frecentese, F.; Esposito, A.; Massarelli, P.;
Nencini, C.; Santagada, V.; Caliendo, G. Bioorganic & Medicinal Chemistry Letters 2010, 20(9), 2978-2982.
Neurodegenerative disease agents
Poster P3.5
Hydroxypyrazolol[1,5-a]pyridine as a fluorescent carboxylic acid isostere:
development of new fluorescent 4-PHP analogues
Stefano Sainas,*1 Birgitte Nielsen,2 Donatella Boschi,1 Bente Frølund2 and
Marco L. Lolli1
1
DSTF - Science and Drug Technology Department, University of Turin,
Via Pietro Giuria, 9 – 10125 Torino, Italy.
2
PHARMA - Department of Drug Design and Pharmacology, The Faculty of Health and Medical Sciences,
University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
[email protected]
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system
(CNS). Disfunctions of the GABAergic transmission, expecially associated to the GABAA suptype
are involved in several CNS disorders which makes it a relevant drug target.1 To date the 3-D
structure of the GABAA receptor is still not available. Instead, during the last couple of years
pharmacophore models and homology models of the orthosteric binding site of the GABAA
receptor have been developed.2 Previous structure-activity relationships studies of the GABAA
receptor, using the 1-hydroxypyrazole moiety of 4-(piperidine-4-yl)-1H-pyrazol-1-ol (4-PHP, 1) as a
bioisostere to the carboxyl group of GABA, have shown the presence of two large hydrophobic
pockets near the GABA binding site.3
At DSTF the acidic bicyclic hydroxypyrazolol[1,5-a]pyridine scaffold is currently under investigation
in bioisosteric applications at the carboxylic group. Beside its acidic characters, its strongly
fluorescent properties bring it a quite attractive tool for introducing new probe opportunities. In this
occasion, this scaffold was used to develop a first example of GABA fluorescent 4-PHP analogue.
Beside introducing fluorescent properties in the scaffold, the quite lipophilic character of the
structure should offer possible binding opportunities with the accessible lipophilic cavities inside the
GABA binding area.
As first example, the derivative 1 was synthesized and pharmacologically characterized in a [3H]muscimol deplacements assay at native GABAA receptors and electrophysiological assays at
relevant GABAA receptor subtypes. The synthesis and pharmacological properties are reported
and discussed in terms of the structural knowledge available for the GABAA receptor.
References
1. Graham A.R. Johnston, GABAA Receptor Channel Pharmacology. Current Pharmaceutical Design 2005,
11, 1867-1885, 18
2. .Frølund, B.; Jørgensen, A. T.; Liljefors, T.; Mortensen, M.; Krogsgaard-Larsen, P. Characterization of
the GABAA Receptor Recognition Site Through Ligand Design and Pharmacophore Modeling. Molecular
Neuropharmacology 2004, 1, 113-127.
3. Møller, H. A.; Sander, T.; Kristensen, J. L.; Nielsen, B.; Krall, J.; Bergmann, M. L.; Christensen, B.; Balle,
T.; Jensen, A. A.; Frølund, B. Novel 4-(Piperidin-4-yl)-1-hydroxypyrazoles as γ-Aminobutyric Acid
receptor Ligands: Synthesis, Pharmacology, and Structure-Activity Relationships. J. Med. Chem. 2010,
53, 3417-3421.
Neurodegenerative disease agents
Poster P3.6
New heterocyclic derivatives of benzylpiperidin-4-yl methyl and
benzyl(methyl)aminobutyl sequences as sigma ligands
D. Zampieria, E. Laurinib, A. Sveronisa, L. Vioa and M. G. Mamoloa
a
Department of Chemical and Pharmaceutical Sciences, University of Trieste,
P.le Europa,1, 34127 Trieste, Italy
b
Department of Chemical, Environmental and Raw Materials Engineering,
Piazzale Europa 1, 34127, University of Trieste, Italy
[email protected]
Sigma (σ) receptors are involved in several functions such as modulation and
biosynthesis of several neurotransmitters, motor control, cell growth and proliferation.
Several classes of structurally unrelated compounds interact with σ receptors, but only
few σ ligands are gifted with affinity and selectivity against a specific receptor subtypes.
The interest in σ ligands stems from the possibility to develop clinical agents for the
treatment of several CNS diseases, for neuroprotection, tumor treatment and
diagnosis. Therefore σ1 receptor ligands could be involved in treatment for
schizophrenia, depression, lack of memorization skill, difficulty of learning and increase
of analgesic action.
On the basis of some benzo[d]oxazol-2(3H)-one derivatives previously developed by
us and gifted with excellent σ1 affinity and selectivity1,2, we have synthesized a new
series of compounds 1 and 2, changing the benzoxazolone moiety with similar groups
to evaluate their effects toward the sigma affinity and selectivity.
Figure 1. σ1-docking of derivative 1a:
(3-((1-benzylpiperidin-4-yl)methyl)-4phenyloxazolidin-2-one).
The preliminary docking study (Fig.1), confirm the interactions between the structure of
our new derivative and the features of the σ1 3D-receptor model.
References
1. D. Zampieri, M.G. Mamolo, E. Laurini, et al., Substituted benzo[d]oxazol-2(3H)-one
derivatives with preference for the σ1 binding site. Eur. J. Med. Chem., 2009, 44, 124-130.
2. D. Zampieri, M.G. Mamolo, E. Laurini, et al., Synthesis, biological evaluation, and threedimensional in silico pharmacophore model for σ1 receptor ligands based on a series of
substituted benzo[d]oxazol-2(3H)-one derivatives. J. Med. Chem., 2009, 52, 5380-5393.
Analytical methods in medicinal chemistry
Poster P4.1
Assessment of a HPLC Assay for Monoacylglycerol Lipase Activity
Del Carlo S.,a Arena C.,a Chicca A.,b Bertini S.,a Chidini S.,a
Manera C.,a Saccomanni G.,a Gertsch J.,b Macchia M.a
b
a
Dipartimento di Farmacia, Via Bonanno 6, 56126 Pisa, Italy; Institute of Biochemistry and
Molecular Medicine, National Center of Competence in Research TransCure, University of Bern,
CH 3012 Bern, Switzerland
[email protected]
Endocannabinoids are lipid transmitters acting as endogenous ligands towards the
cannabinoid receptors CB1 and CB2. The two main endocannabinoid ligands are 2arachidonylglycerol (2-AG) and anandamide (AEA) which play important roles in a wide
range of physiological and pathological processes. 2-AG is the most abundant
endocannabinoid in the brain, and it acts as full agonist at both cannabinoid receptors
unlike anandamide. 2-AG is involved in a wide range of beneficial properties, among
which analgesic, anti-inflammatory, immunomodulating, neuroprotective and
hypotensive effects, as well as inhibition of prostate and breast cancer cells growth.
Monoacylglycerol lipase (MAGL) is the major enzyme responsible for 2-AG hydrolysis.
Inhibition of MAGL leads to an increase of 2-AG levels, therefore it may be an attractive
therapeutic approach in the treatment of various disorders.1,2
The main assay used to screen compounds capability to inhibit MAGL action involves
the use of tritium substrate ([3H]-2-OG).3 4-nitrofenilacetate has been identified as good
and safe substrate for MAGL.4 The interaction of this compound with the enzyme leads
to the production of p-nitrophenol (PNP) as metabolite (Fig.).
Figure. MAGL hydrolysis of 4-NPA leading to PNP
Employing 4-nitrofenilacetate as substrate, a simple, fast and sensitive HPLC-UV
based assay for screening of potential MAGL inhibitors has been developed. MAGL
activity was related to PNP concentration obtained after incubation of the enzyme and
4-NPA. Initial velocity of the enzymatic hydrolysis of 4-NPA (to PNP) was evaluated
and the assay substrate concentration was selected in relation to the assessed Km and
Vmax. Some well known MAGL inhibitors such as JZL184, CAY 10499 and JW 642
were tested in order to validate the method, which results sensible, repeatable and
efficacious.
References:
1. Savinainen J. R., Saario S. M et al. The serine hydrolases MAGL, ABHD6 and ABHD12 as
guardians of 2-arachidonoylglycerol signalling through cannabinoid receptors. Acta Physiol.
2012, 204, 267-276.
2. Ortega-Gutierrez S., Viso A. et al. The Medicinal Chemistry of Agents Targeting
Monoacylglycerol Lipase. Curr. Top. in Med. Chem. 2008, 8, 231-246.
3. Ulloa N. M.; and Deutch D. G. Assessment of a Spectrophotometric Assay for
Monoacylglycerol Lipase Activity. AAPS J. 2010, 12(2), 197-201.
4. Muccioli G. G., Labar G. et al. CAY10499, a novel monoglyceride lipase inhibitor evidenced
by an expeditious MGL assay. Chembiochem 2008, 9(16), 2704-2710.
Analytical methods in medicinal chemistry
Poster P4.2
HPTLC-AMD and HPLC Characterization of the saccharides fraction of the novel
food Adansonia digitata (Baobab) fruit pulp
De Lucia D.1;Bernardi T.2; Vertuani S.1,3; Manfredini S.1,3
1. Department of Life Science and Biotechnology, via L. Borsari 46, Section of Medicinal and
Health Products, Via Fossato di Mortara 17-19, University of Ferrara,
2. Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via L. Borsari46
3. Ambrosialab, via Mortara 171, 44121 Ferrara, Italy
[email protected]
Soluble fibers are and important component of diet. De Caluwé et al. reported that
increasing concentrations of baobab dry pulp, led to an increase in the population of
bifidobacteria and lactic bacteria because the gut microflora is able to use complex
sugars as fructooligosaccharides, xilooligosaccharides etc..as carbon source.
Continuing our studies on Adansonia digitata and in the lack of any analytical
characterization, we undertook qualitative-quantitative analysis of the latter
oligosaccharides fraction, potentially responsible of the known prebiotic activity. The
quality control is a compelling issue in the food industry, frequently the lack of rapid and
efficient methods does not consent adequate characterization of foods. In this study we
continued our previous investigations on high performance thin layer chromatography
(HPTLC), applying it to a novel food, very interesting for its nutritional properties, the
Adansonia digitata fruit pulp. The objective was to evaluate the oligosaccharides
content indicated as responsible of the known prebiotic activity of the fruit. A novel high
performance thin layer chromatography (HPTLC) based method was developed using
also high performance liquid chromatography (HPLC) to confirm results before and
after hydrolytic treatment. Samples were prepared following and adapting widely
accepted extracting procedures for fibers. HPTLC confirmed to be a simple and reliable
analytical technique for oligosaccharides detection in different matrix. However, despite
what claimed, unattended results were obtained for this dried fruit, even with several
different extractions only sucrose, glucose and fructose were recovered. This confirms
the need of rapid methods for composition assessment of fiber rich vegetables and
fruits.
This work was supported by: the Italian Ministry of Education and Research (PRIN,
Grant 20082L3NFT_003) and Ambrosialab
Analytical methods in medicinal chemistry
Poster P4.3
Determination of the Pyruvic Acid Content of Three Italian Onion Varieties
Stefania Scorzoni, Maura Marinozzi, Federica Ianni, Roccaldo Sardella and
Benedetto Natalini
Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Perugia,
Via del Liceo, 1-06123 Perugia (Italy)
[email protected]
Onion (Allium cepa L.), a member of the Liliaceae family, is a species of great
economic importance, widely cultivated all over the world, especially in Asia and
Europe. It is mainly used as a spice to enhance the taste and flavor of numerous
dishes and in many countries it is also used as a fresh, cooked, and dehydrated
vegetable. Since ancient times, onion has been used in folk medicine as a remedy
against common cold, cough as well as local treatment for insect bites. A variety of
recent studies suggests a positive effect of onions in the prevention and treatment of
cancer, cardiovascular diseases, asthma, infections and hyperlipidemia. Most of these
biological effects have been ascribed to the presence of sulfur-containing compounds.
The precursors of this class of compounds are odorless, non-proteinogenic sulfur
amino acids, namely, S-alk(en)yl cysteine sulfoxides (ACSO) which, after tissue
disruption and mixture of the vacuole and cytoplasmic contents, are cleaved by the
enzyme alliinase into pyruvate, ammonia and the corresponding sulfenic acid (Figure
1). The latter compounds are highly unstable and condense to form thiosulfinates (TS),
intermediates in the formation of sulphur volatiles and thus responsible for the onion’s
typical odor and flavor.1 Because pyruvic acid and TSs are produced in the same
pathway, pyruvate content analysis is commonly used as an estimation of the total TS
content and an indicator of the pungency.2
In the heart of the Umbra Valley, a small town, Cannara, is well-known for the onion
production. The onion growing in this area has very ancient origins and has developed
thanks to the water abundance and the geo-pedological characteristics of the soil.
Traditionally, in the Cannara area three onion varieties are cultivated: Rossa di
Toscana, which has purple tunics, and is round and globular, Borettana di Rovato, with
a flat, pale yellow bulb, and Dorata di Parma, characterized by a golden bulb similar to
a spinning. With the aim to promote and defend the characteristics of this production
and considering the shortage of literature about the Italian onion cultivars, we directed
our attention to the characterization of the chemical and nutraceutical properties of
these three typical onion varieties. In particular, the determination of pyruvic acid
content of the three cultivars by both colorimetric and HPLC methods will be reported.
Figure 1. Alliinase-mediated hydrolysis of S-alk(en)yl cysteine sulfoxides (ACSO).
References
1. Lanzottti, V. The analysis of onion and garlic. J. Chromatogr. A. 2006, 1112, 3–22.
2. Galdon B. R.; Rodriguez C. T.; Rodriguez E. R.; Romero C. D. Organic acid contents in
onion cultivars (Allium cepa L.). J. Agric. Food Chem. 2008, 56, 6512-6519.
Analytical methods in medicinal chemistry
Poster P4.4
New biotinilated spacers for studying the interaction of ELAV mimicking
peptides with the target mRNA
Giuseppe Tripodo,a Lauretta Maggi,a Maria Laura Amadio,b Alessia Pascale,b
Annamaria Marra,a Daniela Rossi,a Simona Collinaa
Department of Drug Sciences, viale Taramelli, 12 27100 Pavia
a
Section of Medicinal Chemistry and Technology
b
Section of Pharmacology, University of Pavia, Italy
[email protected]
Embryonic Lethal Abnormal Vision (ELAV) proteins are RNA-binding proteins that bind
specific Adenine and Uridine-Rich Elements mainly located in the 3'-untranslated
region of target mRNAs, preventing their otherwise rapid degradation and thus
increasing gene expression. Four peptides corresponding to highly conserved motives
within the first two RNA Recognition Motif-type domains of ELAV proteins were
prepared and their effect on the stability of NOVA-1 and VEGF ELAV-target mRNAs
was evaluated. The stabilizing effect on the VEGF transcript (mRNA VEGF) exerted by
each peptide, tested individually, was initially evaluated and no effects were
evidenced.1 The biological effects of all peptides couples were also investigated.
Interestingly, in accordance with preliminary molecular dynamics results, only one of all
possible peptide couples resulted highly effective in stabilizing mRNA VEGF. Two
peptides were identified as valuable starting point for designing the first class of small
molecules endowed with ELAV-like stabilizing effects, which could represent a highly
innovative therapeutic tool potentially useful in the treatment of neurodegenerative
diseases and other pathologies characterized by ELAV impairment.2 Our current efforts
are directed toward a deeper understanding of the interaction of peptides with the
target transcript, using the ALPHA (Amplified Luminescent Proximity Homogeneous
Assay) Technology.
In this communication we focus on the set up of a synthetic strategy to prepare
versatile tools based on biotinilated spacers (figure 1) suitable for derivatizing our
peptides. In this way we will obtain labelled peptides for the ALPHA investigation.
O
HN
NH
H
N
S
O
O
n=3
n
NH2
n=8
Figure 1. General structure of biotinilated spacers object of this work
Such a study will provide useful information for performing further studies able to lead
to the next rational design of new chemical entities with ELAV mimicking properties.
References
1. Rossi D.; Amadio M.; Carnevale Baraglia A.; Azzolina O.; Ratti A.; Govoni S.;
Pascale; Collina S. Discovery of small peptides derived from embryonic lethal abnormal
vision proteins structure showing RNA-stabilizing properties. J. Med. Chem. 2009, 52, 50175019.
2. Amadio M.; Pascale A.; Govoni S.; Laurini E.; Pricl S.; Gaggeri R.; Rossi D.; Collina S.
Identification of peptides with ELAV-like mRNA stabilizing effect: an integrated in vitro/in
silico approach. Chem. Biol. Drug Des. 2013, DOI: 10.1111/cbdd.12117.
Nutraceutics
Poster P5.1
An approach towards the Total Synthesis of the Yunnaneic Acids
Lorenzo Botta, Daniel R. Griffith, Scott A. Snyder*
Dept. of Chemistry, Columbia University, Havemeyer Hall, 3000 Broadway
New York, NY 10027 (USA)
[email protected]
Yunnaneic acids A, B, C, and D (1-4, Figure 1) were isolated from the roots of Salvia
yunnanensis, a plant found in southern China that has been used in traditional folk
medicine.1 These interesting structures are hexamers (1 and 2) or trimers (3 and 4) of
caffeic acid. Despite their intricate architectural features, no reports on the synthesis of
these molecules have appeared to date. In this poster, a model system study to
determine a synthetic route to fully functionalized molecules is described. The pathway
developed consists of a cascade-based approach to fashion the [2.2.2]-core in addition
to a number of different approaches to effect dimerization leading to the spiroketal
moiety linking together the two halves of both monomers into the cores of 1 and 2.
These efforts are quite unique relative to those we have used towards related targets.2
Figure 1. Structures of the Yunnaneic Acids A-D (1-4) and of the model system monomers (7,8)
and dimers (5,6).
References
1. Takashi, T.; Akiko, N.; Isao, K.; Gen-ichiro, N.; Tsuong-Jen, Y. Isolation and
Characterization of Yunnaneic Acids A-D, Four Novel Caffeic Acid Metabolites from Salvia
yunnanensis. J. Nat. Prod. 1996, 59, 843–849.
2. Snyder, S.A.; Kontes, F. Explorations into Neolignan Biosynthesis: Concise Total Syntheses
of Helicterin B, Helisorin, and Helisterculin A from a Common Intermediate.
J.A.C.S. 2009, 5, 1745–1752
Nutraceutics
Poster P5.2
Extracts of cocoa and chocolate a comparative valuation of functional efficacy
Silvia Vertuani,1,2 Emanuela Scalambra,1 Vittorio Trotta,1 Gemma Malisardi,1
Anna Baldisserotto1 and Stefano Manfredini1,2
1. Department of Life Science and Biotechnology, via L. Borsari 64, Section of Medicinal and
Health Products, Via Fossato di Mortara 17-19, University of Ferrara,
2. Ambrosialab, via Mortara 171, 44121 Ferrara, Italy
[email protected]
Chocolate antioxidant properties are often claimed, however they are frequently
different from the parent natural sources due to the industry or artisan transformation.
In this study we report the preliminary results of antioxidant capacity and total phenolic
content of cocoa nibs, cocoa masses and corresponding chocolate bars with different
percentages of cocoa from different origin. The antioxidant capacity of the different
samples was measured by two different assays (DPPH and FRAP tests). Folin–
Ciocalteu reagent was used to assess the total phenolics content. The masses showed
a higher antioxidant power than the nibs, and this has been attributed to the fact that in
the nibs is still present the lipid part which will form the cocoa butter. Ours results
showed that the extra dark cocoa bar 100% cocoa chocolate is the best in terms of
total polyphenols content and in terms of antioxidant capacity according to the DPPH
and FRAP tests. Also the bars of Organic dark chocolate 80%, dark Tanzania 80% and
Trinidad 80% products are good in all respects.
This work was supported by: the Italian Ministry of Education and Research (PRIN,
Grant 20082L3NFT_003) and Ambrosialab.
Nutraceutics
Poster P5.3
NOVEL NUTRACEUTICALS FROM ETNOPHARMACY: NIGELLA SATIVA SEEDS
ESSENTIAL OILS AGAINST RECURRENT BLADDER INFECTIONS
E. Haloci1, S.Manfredini1,5, V.Toska2, S.Vertuani1,5, P.Ziosi5, I.Topi3, H.Kolani4
1. Department of Life Science and Biotechnology, via L. Borsari 46, Section of Medicinal
and Health Products, Via Fossato di Mortara 17-19, University of Ferrara
2. Tirana University, Medicine Faculty, Pharmaceutical Department, Rr.Dibres, Tirane, Albania
3. Tirana University , National Laboratory of Drug Control
4. QSUT Nene Tereza, Klinika nr 1, Pavioni i Kirurgjise,Tirane ,Albania
5. Ambrosialab, via Mortara 171, 44121 Ferrara, Italy
[email protected]
Seeds of Nigella sativa L.(Ranunculaceae), known commonly as “black cumin” have
been employed for thousands years in traditional use as a spice and food preservative.
Recently, many biological activities of Nigella sativa L. seeds have been reported,
including: antioxidant, anti-inflammatory, anticancer and antimicrobial and antifungal
ones. Several effects have been attributed to active principles of Nigella sativa L. which
includes thymoquinone, thymohydroquinone, dithymoquinone, thymol,carvacrol,
nigellicine, nigellimine-x-oxide, nigellidine and alpha-hedrin. Nigella sativa L. seed
extract inhibits fungal strains. In the present study we started a systemathic evaluation
on fungi and pathogenic bacteria of ether and methanolic extracts of oils from Nigella
Sativa seeds. The oils were found to have important antifungal and antibacterial
activities as compared to tetracycline, cefuroxime and ciprofloxacin positive controls.
The ether esxtracts showed the stonger activity. These interesting findings has
prompted us to propose Nigella sativa seeds and their extracts as possible treantment
in the prevention of recurrent infection (i.e. bladder, gut and kidney) in which othe
natural treatments has already shown interesting results especially in association. A
novel food supplement, based on Nigella sativa, Cranberry and Mannose, is proposed
for the treatment of urinary and digestive tracts recurrent infection.
Ciprofluoxacin
Cefuroxime
Tetracyclin
M2
M1
This work was supported by: the Italian Ministry of Education and Research (PRIN,
Grant 20082L3NFT_003) and Ambrosialab
Figure 1. General structure of thymoquinone and a graphic showing activity of
essential oils from Nigella sativa seeds on P. Vulgaris.
Antimicrobial agents
Poster P6.1
Computer-Aided Identification of Fragments as CDK9 Inhibitors with Anti-HIV-1
Tat-Mediated Transcription Activity
Nunzio Iraci, Luca Sancineto, M. Letizia Barreca, Serena Massari, Stefano Sabatini,
Giuseppe Manfroni, Violetta Cecchetti, Oriana Tabarrini
Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia
[email protected]
Although remarkable progress has been made in the treatment of HIV infection, the
current therapy, beside suffering from the emergence of drug-resistant viral strains,
results unable to completely eradicate the virus from infected individuals, thus
prompting for innovative ad more effective treatment options.1
Among the many host factors involved in the modulation of the post integration latency,
the Positive Transcriptional Elongation Factor b (P-TEFb) was shown by several
experimental evidences to be druggable.2 In particular, the inhibition of kinase subunit
CDK9 could be a good strategy to obtain indirect antiviral drugs that should be active
against mutant viruses eventually escaping current therapies besides controlling the
reactivation from viral latency.
Using as target the crystallographic structure of P-TEFb in complex with flavopiridol,3 a
docking-based virtual screening was performed on vendor and in-house libraries, and
on a fragment-based database we built specifically for this task. The selected hits were
submitted to biologically testing, confirming for some of them the ability to inhibit the
activity of CDK9. The optimization of two fragment-based “real” hits was carried on
taking advantage of several in silico and experimental techniques, and led to the
identification of non toxic 2-phenylquinazolinone derivatives able to inhibit Tat-mediated
transactivation as well as HIV-1 reactivation from latency in latently infected cells.
Of note, a derivative showed an anti-HIV potency in the latently infected cell model
superior to what could be predicted looking just at its anti-CDK9 activity, thus leading
us to speculate about an additional target. Indeed, CDK2, which plays a supportive role
within the transcriptional machinery and shares structural features with CDK9, was also
inhibited by the derivative at comparable concentrations.
An insight into the double activity of the compound has been achieved studying the
docking-predicted binding mode into both enzymes followed by its comparison to
several published crystallographic structures and to literature data that show the
chemical features needed to obtain selectivity.
Thus, the identified 2-phenylquinazolinones, which conforming to the rule of 3 can be
categorized as fragments,4 represent a good template for the development of anti-HIV
agents optimized in both potency and selectivity.
References
1. Colin, L.; Van Lint, C.. Molecular Control of HIV-1 Postintegration Latency: Implications for
the Development of New Therapeutic Strategies. Retrovirology 2009, 6, 111
2. Schang, L. M. Cyclin-Dependent Kinases as Cellular Targets for Antiviral Drugs. J.
Antimicrob. Chemother. 2002, 50, 779-792.
3. Baumli, S.; Lolli, G.; Lowe, E. D.; Troiani, S.; Rusconi, L.; Bullock, A. N.; Debreczeni, J. E.;
Knapp, S.; Johnson, L. N. The Structure of P-TEFb (CDK9/cyclin T1), its Complex with
Flavopiridol and Regulation by Phosphorylation. EMBO J. 2008, 27, 1907-1918.
4. Sancineto, L.; Massari S; Iraci, N.; Tabarrini, O. From Small to Powerful: The Fragments
Universe and its "Chem-Appeal". Curr. Med. Chem. 2013, 20, 1355-1381.
Antimicrobial agents
Poster P6.2
DEVELOPMENT OF NOVEL ANTI-HIV AGENTS ACTING AS NON-NUCLEOSIDE
REVERSE TRASCRIPTASE INHIBITORS
Lo Surdo G.,a Ferro S.,a De Luca L.,a Pannecouque C.,b Chimirri A.,a Monforte A.M.a
a
Dipartimento di Scienze del Farmaco e Prodotti per la Salute, Università degli studi di
Messina, Viale SS. Annunziata, 98168 Messina, Italy.
b
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10,
B-3000 Leuven, Belgium.
[email protected]
Reverse transcriptase (RT) is a crucial enzyme in the life cycle of HIV-1 by performing
the retrotranscription of the viral RNA into viral DNA, and thereby has been the prime
drug target for antiretroviral therapy.
Non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are a mainstay of
combination therapies for the treatment of AIDS. However, the effectiveness of these
drugs is hampered by rapid emergence of drug-resistant viruses and severe side
effects upon long-term use. Therefore, there is an urgent need to develop novel, highly
potent NNRTIs with improved resistance profiles and pharmacokinetic properties.
We previously reported a series of N1-substituted 1,3-dihydro-2H-benzimidazol-2-ones
and their sulfones which proved to effectively inhibit HIV-1 replication at nanomolar
concentration with low citotoxicity1,2,3.
With the aim of clarifying the structural characteristics able to improve the biological
profile of these promising molecules, new
R'
benzimidazole derivatives were designed and
different structural modifications have been
introduced. Docking studies were also performed to
X
rationalize the interesting biological results obtained
for most of these novel RT inhibitors showing potent
R
N
anti-HIV activity and very high selectivity index.
R''
N
S
Figure 1. General structure
References
1. Monforte, A.M.; Rao, A.; Logoteta, P.; Ferro, S.; De
Luca, L.; Barreca, M.L.; Iraci, N.; Maga, G.; De Clercq, E.; Pannecouque, C.; Chimirri A.
Bioorg. Med. Chem., 2008, 16(15), 7429-7435.
2. Monforte, A.M.; Logoteta, P.; Ferro, S.; De Luca, L.; Iraci, N.; Maga, G.; De Clercq, E.;
Pannecouque, C.; Chimirri, A. Bioorg. Med. Chem., 2009, 17, 5962-5967.
3. Monforte, A.M.; Logoteta, P.; De Luca, L.; Iraci, N.; Ferro, S.; Maga, G.; De Clercq, E.;
Pannecouque, C.; Chimirri, A. Bioorg. Med. Chem., 2010, 18, 1702-1710.
Research supported by Fondo Ateneo di Ricerca (2008/2009, Messina, Italy).
Antimicrobial agents
Poster P6.3
Synthesis of new Plasmodium Falciparum dihydroorotate dehydrogenase
inhibitors through the application of an isosteric portfolio
Agnese Chiara Pippione,1 Antonella Federico,1 Donatella Boschi,1 Ulf J. Nilsson,2
Ingela Fritzson,3 Anders P. Sundin2 and Salam Al-Karadaghi4 and Marco L. Lolli1
1
2
DSTF - Science and Drug Technologies Department, University of Turin, Torino, Italy
Centre for Analysis and Synthesis, Department of Chemistry, Lund University, Lund, Sweden
3
Active Biotech AB, Lund, Sweden
4
Center for Molecular Protein Science, Department of Chemistry, Lund University, Lund,
Sweden
[email protected]; [email protected]
Malaria causes great suffering, with annual mortalities of over 800.000 people,
principally in Africa and Asia. To fight the resistance to current drug treatments, several
strategies have been employed. The de novo pyrimidine biosynthesis enzyme
dihydroorotate dehydrogenase (DHODH) is an emerging drug target for the treatment
of malaria. In this context, the followed key strategy is to selectively inhibit the
Plasmodium falciparum DHODH isoform (PfDHODH) over its human homologue
(HsDHODH). Recently, we presented PfDHODH inhibitors based on N-substituted
salicylamides scaffold.1 Compound 1, see figure, showed low micromolar range activity
over PfDHODH together with a quite interesting selectivity over human DHODH.1 Since
2006, the DSTF Med Chem group investigate hydroxylated pentatomic heterocyclic
systems as iso/bioisoster mimic of carboxylic group2 as well as other acidic systems.3
Here, the salicylamide moiety present in 1 is identified as a possible target for a
bioisosteric modulation. We present here the synthesis, dissociation constant and
preliminary PfDHODH in vitro inhibition assays of a small library of compounds of
general structures 2. This allowed us to suggest important considerations about
inhibition of PfDHODH.
Figure 1. General structure of PfDHODH inhibitor and iso/bioisosteric modulation.
References
1. Fritzson, I.; Bedingfield P.T.P. et al. N-Substituted salicylamides as selective malaria
parasite dihydroorotate dehydrogenase inhibitors. MedChemComm 2011, 2, 895-898.
2. Lolli M.L.; Giordano C. et al. 4-hydroxy-1,2,5-oxadiazol-3-yl moiety as bioisoster of the
carboxy function. Synthesis, ionization constants, and molecular pharmacological
characterization at ionotropic glutamate receptors of compounds related to glutamate and
its homologues. J. Med. Chem. 2010, 53, 4110-4118.
3. Lolli M.L.; Giorgis M. et al. New inhibitors of dihydroorotate dehydrogenase (DHODH) based
on the 4-hydroxy-1,2,5-oxadiazol-3-yl (hydroxyfurazanyl) scaffold. Eur. J. Med. Chem. 2012,
49, 102-109.
Antimicrobial agents
Poster P6.4
Enzymatic PEGylation of oligonucleotides
Sosic A., Pasqualin M., Pasut G. and Gatto B.
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F.
Marzolo 5, 35131 Padua, Italy
[email protected]
The gene and RNAi therapies as well as therapeutic aptamers attracted a great
interest owing to the potential high selectivity and the theoretical possibility to treat a
wide range of diseases, even those that have no other therapies. Unfortunately, this
promise was partially reconsidered in face of the many difficulties related to the in vivo
application of these molecules and the issues associated with their stability and
delivery. Unmodified DNAs are rapidly degraded by 3’ exonucleases [1] and,
furthermore, they suffer a fast kidney clearance and scarce cell internalization.
To address these limitations, two main approaches of nucleic acids delivery have
been proposed i.e. viral vectors and lipidic or polymeric non-viral vectors. PEGylation,
the covalent attachment of polyethylene glycol (PEG) [2], has been proposed for
prolonging the pharmacokinetic profile of nucleic acid materials: PEGylated
oligonucleotides and siRNAs have demonstrated an improved cell internalization and
stability with respect to the free nucleic acids [3, 4].
A method for the direct selective conjugation of PEG to nucleic acid would be
desired to overcome the constrains of the nucleic acids PEGylation based on the
covalent attachment of a properly activated PEG to modified DNA or RNA
oligonucleotides. In the field of protein PEGylation, the attention has been recently
directed towards the development of enzymatic methods of conjugation [2, 5]. These
new perspectives in protein PEGylation inspired us to develop a method consisting of
an enzymatic PEGylation of oligonucleotides.
T4 DNA ligase enzyme was used to ligate double stranded oligonucleotides to a
20kDa PEG bearing a short oligonucleotide sequence. The chemical synthesis of the
oligo-PEG conjugate was characterized and standardized. Hence, the enzymatic
ligation of the oligo-PEG conjugate to a dsDNA was performed using a set of
appositely designed oligonucleotides as model system. The ligation protocol was
optimized allowing the complete achievement of a stable PEGylated dsDNA system.
This early study on enzymatic PEGylation of oligonucleotides can potentially be applied
to every DNA or RNA sequence and can stimulate new researches in this field.
References
1. Eder PS, DeVine RJ, Dagle JM, Walder JA. Substrate specificity and kinetics of degradation
of antisense oligonucleotides by a 3' exonuclease in plasma. Antisense Res Dev.
1991;1:141-151.
2. Pasut G, Veronese FM. State of the art in PEGylation: the great versatility achieved after
forty years of research. J Control Release. 2012;161:461-472.
3. Rapozzi V, Cogoi S, Spessotto P, Risso A, Bonora GM, Quadrifoglio F, Xodo LE. Antigene
effect in K562 cells of a PEG-conjugated triplex-forming oligonucleotide targeted to the
bcr/abl oncogene. Biochemistry. 2002;41:502-510.
4. Jung S, Lee SH, Mok H, Chung HJ, Park TG. Gene silencing efficiency of siRNA-PEG
conjugates: effect of PEGylation site and PEG molecular weight. J Control Release.
2010;144:306-313.
5. Mero A, Schiavon M, Veronese FM, Pasut G. A new method to increase selectivity of
transglutaminase mediated PEGylation of salmon calcitonin and human growth hormone. J
Control Release. 2011;154:27-34
Antimicrobial agents
Poster P6.5
Structure-Activity Relationship of zosteric acid analogues as inhibitors of biofilm
formation by Escherichia coli
Stefania Villa [a], Silvia Dell’Orto[a], Arianna Gelain[a],
Federica Villa[b], Cristina Cattò[b], Francesca Cappitelli[b]
[a]
Dipartimento di Scienze Farmaceutiche,Università degli Studi di Milano, Via Mangiagalli 25,
[b]
Milano (Italy), Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente, Università
degli Studi di Milano, Via Mangiagalli 25, Milano (Italy)
[email protected]
Biofilm is defined as a complex microbial community characterized by adhesion to a
solid surface and by production of a matrix, which surrounds the bacterial cells and
includes extracellular polysaccharides (EPS), proteins and DNA. Biofilm plays a pivotal
role in healthcare-associated infections, especially those related to the implant of
medical devices, such as vascular and urinary catheters and orthopedic implants.
Attempt to use conventional antibiotics to retard the formation of bacterial films on
implanted medical devices suffer from the problem of the emergence and increasing
prevalence of microbial strains in form of biofilm that are resistant to available
antimicrobial-based therapies. [1] The new approach consists in using sublethal doses
of bio-inspired molecules to interfere with the key-steps that orchestrate biofilm
formation (like surface sensing process and/or cell-to-cell signalling pathways), thus
infection cascade might be hampered. In addition, as these approaches do not directly
kill cells or prevent their growth, there is presumably less evolutionary pressure for the
development of resistance than with traditional antimicrobial agents. In this contest,
zosteric acid or p-(sulfo-oxy) cinnamic acid (Figure 1), a secondary metabolite
produced by the seagrass Zostera marina, might be suitable for implementation as a
preventive or integrative approach against device-related infections. In this project
supported by Cariplo Fundation a series of zosteric acid derivatives was synthesized
and tested as inhibitors of biofilm formation by Escherichia coli. The results will be
reported in the poster.
Figure 1. Structural formula of zosteric acid.
References
1. Sintim, H.O., et al., Paradigm shift in discovering next-generation anti-infective agents:
targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small
molecules. Future Medicinal Chemistry, 2010, 2(6), 1005-1035.
2. Villa, F., et al., Hindering biofilm formation with zosteric acid. Biofouling, 2010, 26(6), 739752.
3. Villa F., et al., Altered expression level of Escherichia coli proteins in response to treatment
with the antifouling agent zosteric acid sodium salt. Environmental Microbiology 2012,
14(7), 1753-1761.