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Sono stati sintetizzati composti a struttura policiclica complessa. Tutti i composti sintetizzati sono stati saggiati dall'NCI (USA) su un panel di 60 linee cellulari tumorali umane. Sula base dei risultati ottenuti è stato possibile effettuare uno studio SAR che ha evidenziato come la presenza di gruppi idrofili è necessaria ai fini dell'attività antitumorale. E' stato inoltre studiato il meccanismo d'azione di queste molecole. E' stato notato un arresto delle cellule MDA-MB231 in fase G0/G1 correlato con la defosforilazione ed attivazione, indotta dai composti, della proteina pRb. Il meccanismo comunque varia arrestando il ciclo cellulare in fase G2/M quando per effetto della glicosilazione del derivato.

Isoxazolo[3,4-d]pyridazin-7(6H)-one derivatives endowed with antiproliferative Activity B. Maggio1, G. Cancemi1, D. Raffa1, M. V. Raimondi1, F. Plescia1, A. D’Anneo2,M. Lauricella3, G. Barone4, R. Bonsignore4, G. Daidone1 1. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Medicinal Chemistry and Pharmaceutical Technologies Section, University of Palermo, ViaArchirafi 32, 90123, Palermo, Italy 2. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Laboratory of Biochemistry, University of Palermo. 3.Department of Experimental Biomedicine and Clinical Neurosciences, Laboratory of Biochemistry, University of Palermo. 4. Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo. benedetta.maggio@unipa.it A screening carried out by NCI (Bethesda, USA) on compounds available in our laboratory,in order to ascertain their antiproliferative activity against a panel of 60 human tumor cell lines, allowed to discovery the 3,4-diphenylisoxazolo[3,4-d]pyridazin-7(6H)-one 1a [1] as an hit compound, often showing GI50values at sub-micromolar level. We synthesized some analogs of 1a, i.e.1b-gand other derivatives in which the NHgroup is variably alkylated, with the aim to obtain more active compounds as well as to perform structure-activity relationship(SAR) studies. We obtained a quite active antiproliferative compound, the 3,4-di-p-tolylisoxazolo[3,4-d]pyridazin-7(6H)- one 1d, and verified the importance for the antiproliferative activity of the aril, and not alkyl, groups linked to the isoxazolo-pyridazinone moiety. Studies performed on the cell cycle progression and on some cellular target (ATM, procaspase-2 proteins and H2AX histone) demonstrated that 1d produces an increase of the cell population in pre-G0/G1 and induces cellular death by apoptosis, damaging the DNA by double strand breaks. UV-vis titration and viscosity measurement showed that the compound is able to give an interaction with the B-DNA. 1. Renzi G., Dal Piaz V. (2004)Gazz. Chim. It.95: 1478–1491

Sintesi di due nuovi composti a struttura tetrazepinonica, analoghi della Temozolomide. I due nuovi composti hanno mostrato una interessante attività antiproliferativa nei confronti di linee cellulari tumorali esprimenti l'enzima MGMT

There is an urgent need of anti-virulence agents effective in the prevention or eradication of biofilms that are intrinsically resistant to conventional antibiotics. If we consider that the first step of staphylococcal pathogenesis and of biofilm formation is the bacterial adhesion, promoted by the surface exposed proteins at the cell wall, we believe that new anti-virulence agents could be developed by using as a target the Sortase A (SrtA), the enzyme responsible of linking surface exposed proteins to peptidoglycan. Therefore, SrtA inhibitors could act as anti-adhesion agents useful to prevent Gram positive virulence mechanisms as well as a virulence mechanism based on biofilm formation. Starting from these consideration and in continuing our research work on antibacterial/antibiofilm agents, we thought it was of interest to obtain novel SrtA inhibitors, showing a molecular diversity than the known ones.The esters and the acids were tested for their inhibitory activity on SrtA, utilizing the FRET technics (Fluorescenze Resonance Energy Transfer) [1]. Some of them showed IC50 values in the range 50-100 μM. For a preliminary assessment of antivirulence properties the most active SrtA inhibitors were tested for their ability to interfere with biofilm formation of S. aureus ATCC 29213, S. aureus ATCC 25923, S.aureus ATCC 6538 and S. epidermidis RP62A. We found that the above derivatives interfere with biofilm formation

Gram-positive bacteria are a significant cause of nosocomial and community-acquired infections associated with diseases of high morbidity and mortality. Moreover, antibiotic resistance of important Gram-positive pathogens, such as Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecalis is one of the major worldwide health problems. Over the last decade, many studies have focused on agents that target the virulence of important pathogens without killing or inhibiting their growth therefore imposing limited selective pressure to promote the development and spread of resistance mechanisms [1]enterococci and streptococci, sortase A plays a critical role in Gram-positive bacterial pathogenesis. It is thus considered a promising target for the development of new anti-infective drugs that aim to interfere with important Gram-positive virulence mechanisms, such as adhesion to host tissues, evasion of host defences, and biofilm formation. The additional properties of sortase A as an enzyme that is not required for Gram-positive bacterial growth or viability and is conveniently located on the cell membrane making it more accessible to inhibitor targeting, constitute additional reasons reinforcing the view that sortase A is an ideal target for anti-virulence drug development. Many inhibitors of sortase A have been identified to date using high-throughput or in silico screening of compound libraries (synthetic or natural. We synthesized two classes of molecules: curcumin analogues and N-phenyl-1H-pyrazole-4-carboxamides able to interfere with the bacterial adhesion, a fundamental step of Gram-positive pathogenesis [2].All derivatives obtained were tested for both the planktonic growth inhibition activity and the inhibition of biofilm formation against the following reference bacterial strains: S. aureus ATCC 6538, S. aureus ATCC 25923 and S. aureus ATCC 29213.

A new polycylic compound was synthesized and evaluated for its antiproliferative activity against the NCI panel of human tumoral cell lines showing IC50 values in the range 0.47-5.43 µM. The compound induced a decrease of the level of cyclin B1, whereas increased the level of the inhibitor p21 WAF1

The reaction of the diazonium salt 12 derived from N-(2-aminophenyl)-N,3-dimethyl-1-phenyl-1H-pyrazole-5-carboxamide with copper sulfate and sodium chloride in the presence of ascorbic acid afforded the unexpected products 3,5-dimethyl-1-phenyl-1,5-dihydro-4H-pyrazolo[4,3-c]¬quinolin-4-one (17) and N-methyl-2-(3-methyl-1-phenyl-1H-pyrazol-5-yl)aniline (19), accompanied by N-(2-chlorophenyl)-N,3-dimethyl-1-phenyl-1H-pyrazole-5-carboxamide (18). Products 17 and 19 are formed via a non-classical Pschorr reaction. The formation of 17 represents an alternative to the literature synthesis of this biologically active compound. The molecular structure of 18 was confirmed by single-crystal X-ray analysis.

Un nuovo derivato policiclico glicosilato è stato ottenuto partendo dal derivato policiclico iodurato, utilizzando la reazione di Sonogashira.Il composto glicosilato saggiato presso l’NCI è risultato attivo su tutte le 60 linee cellulari tumorali del panel, risultando più attivo degli altri composti policiclici precedentemente saggiati. Il composto induce l’arresto del ciclo cellulare in fase G2/M nella linea cellulare MDA-MB231, fa diminuire i livelli di ciclina B1e Cdc-2 mentre produce un aumento dell’inibitore p21WAF1, una chinasi ciclino-dipendente.

Staphylococcus aureus, due to its wide arsenal of virulence factors, is a very versatile pathogen responsible for a wide variety of infectious diseases. The virulence factors include the cell-wall associated proteins that have a direct role in the first stage of pathogenesis. The Sortase A (SrtA) transpeptidase is responsible for covalent anchoring to the cell wall of various surface proteins and it is considered a good target to design new antivirulence agents. In this study, we report the identification of an inhibitor of SrtA afforded from the random screening of a small molecular library of around 150 synthetic compounds, through a high throughput assay by using the standard Dabcyl-QALPETGEE-Edans fluorescence resonance energy transfer (FRET)-peptide substrate for measurement of enzyme activity. Such hit compound showed an IC50 value of 80µM. A group of derivatives of the hit compound has been obtained and evaluated for their activity as SrtA inhibitors. The efficacy of the most interesting SrtA inhibitors needs to be evaluated by in vivo models of infection, but we used a simple methodology in vitro, such as inhibition of biofilm formation, for a preliminary assessment of antivirulence properties of some of these novel inhibitors of SrtA. An interesting correlation between inhibition of SrtA and interference with the formation of staphylococcal biofilms has been observed in many cases especially at a concentration equal or more than IC50 determined as SrtA inhibitors.

By reacting methylaminopyrazoles with hexane-2,5-dione in 1,4-dioxane in the presence of p-toluensulfonic acid, new derivatives containing the policyclic sistem 5,7:7,13-dimethanopyrazolo[3,4-b]pyrazolo[3'.4':2,3]azepino[4,5-f]azocine were obtained. The new synthesized compounds were tested preliminarly at 10 microM against five human cancer cell lines showing a range of inhibition of 20-62% against the most susceptible cell lines K562 and HCT116.

Indazole nucleus represents a very attractive scaffol to obtain new molecole endowed with antineoplastic activity. On the basis of these literature data we have designed some indazole derivatives such as N-indazolylbenzamides and N-indazolyl-N’-phenylureas as potential CDK1 inhibitors. In fact the above compounds contain the structural feature, common to the majority of CDK inhibitors, requested to make hydrogen bonds with the molecular forks present in the hinge region of CDKs. The N-indazolylbenzamides 1 were obtained by reacting aminoindazoles and substituted benzoylchlorides. Among the synthesized compounds some derivatives 1 resulted to be CDK1 inhibitors showing IC50 values in the range 4.4-10.0 M

The cinnamoyl anthranilamides represent a class of biological active substances of great importance in medicinal chemistry. Moreover, despite their wide range of biological activities, a review of the literature revealed that no anticancer activity is described for this kind of substances. Starting from the 2-cinnamamido-5-iodobenzamide, resulted able to inhibit the leukemic cell line K-562 proliferation with a percent of inhibition of 74% at 10M concentration, we undertake the following structural modifications on cinnamamidobenzamide skeleton: the introduction of various substituents both on the benzamido and the cinnamamido moieties, the substitution of olefinic bond with the ethane, ethyne, cyclopropane and phenyl groups. Synthesized compounds caused growth inhibition against many tumor cell lines at low micromolar and submicromolar concentrations against every tumor cell line investigated The best activity was obtained when the 5 position of the benzamido moiety was substituted with an iodine moiety. COMPARE analysis, effects on tubulin polymerization in cells and with purified tubulin, and effects on cell cycle distribution (G2-M phase block), including induction of apoptosis, indicate that these new antiproliferative compounds act as antitubulin agents. Preliminary biological data on 2-(3-phenylpropiolamido)benzamides, bearing an ethyne bond, showed a different mechanism of action considering that they caused a G0-G1 block of the cell cycle on K526 cell line.

Recently our research group has reported the synthesis of some 3-amino-N-phenyl-1H-indazole-1-carboxamides able to inhibit at low micromolar concentrations the cell growth of many neoplastic cell lines. The above compounds are unsubstituted in the indazole nucleus and this gives the hope to obtain more active compounds if appropriate substituents are beared by the above nucleus. So, several new N-phenyl-1H-indazole-1-carboxamides 1c-h and 4l,m were prepared by reacting phenyl isocyanates 3a,b with 3-amino-1H-indazoles 2c,e,g, or 1H-indazole 2l respectively. Chemical trasformations of compounds 1a,b and 1g,h gave 3-acetamido-N-phenyl-1H-indazole-1-carboxamides 5a,b, and 3,5-diamino-N-phenyl-1H-indazole-1-carboxamides 4i,j respectively. Finally, 3,5-diacetamido-N-phenyl-1H-indazole-1-carboxamides 6a,b were prepared by acethylation of 4i,j. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against 60 human tumoral cell lines derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). Compound 1c, the most active of the series, was able to inhibit cell growth showing GI50 values in the 0.041-33.6 μM range, being very effective against colon and melanoma cell lines. Compound 1c induced an increase of cells in G0-G1 peak and a decrease of cells in S and G2-M phases when used at a concentration of or higher than 40µM which is the TGI calculated after 24 h of 1c exposure. This could be due to a block of G1-S transition as indicated by the ability of 1c to increase the hypophosphorylate pRb/ total pRb ratio.

Many infections such as otitis media, sinusitis, cholesteatoma, tonsillitis and adenoiditis are caused by biofilm forming mucosal pathogens (P. aeruginosa, S. aureus, S. peneomoniae, H. influenzae and M. catarrhalis). Moreover, the role of biofilms in the chronic otolaryngologic infections has been recognized for otitis media, tonsillitis and rhinosinusitis. Finally, bacterial biofilms of S. aureus, S. epidermis and E. faecalis are the leading cause of medical device-related infections. Pathogens growing as biofilms are intrinsically resistant to conventional antibiotics and therefore the discovery of new compounds able to act against biofilm aggregated micro organisms is an urgent task. Previously, we reported the study on the antibiofilm activity of 4-diazopyrazole derivatives of type 1 against S. aureus ATCC 29213, methycillin-resistant S. aureus ATCC 43866 and methycillin-resistant S. epidermis RP626 preformed biofilms. The compounds were active at the maximum tested concentration of 25 microg/ml. On the basis of these results, we have prepared a new series of 4-diazopyrazole derivatives 2a-i and 3 which take the phenylureido or phenylacetamido moiety at 5-position of the pyrazole nucleus. The compounds has been tested preliminarly against planktonic (single cells) strains of S. aureus (ATCC 25923), S. aureus (ATCC 29213), E. coli (ATCC 25922) and P. aeruginosa (ATCC 9027), showing MIC’s values in the range 50-0.097 microg/ml. Experimental work aimed to verify the effect of the above compounds on the corresponding microbial biofilms are in progress.

The pyrazolo[3,4-d]pyrimidine system shows a multitude of interesting pharmacological properties. Owing to the potential anti-inflammatory activity of 5-benzamido-pyrazolo[3,4-d]pyrimidin- 4-one derivatives and considering the easy synthesis of this class of compounds, a set of new 5- benzamido-1H-pyrazolo[3,4-d]pyrimidin-4-ones has been prepared in 42-80% yields by reacting 5- aminopyrazole-4(N-benzoyl)carbohydrazide derivatives and the opportune triethylorthoesters. Compounds 8a, b, 10a–d, and 11a, b revealed a superior inhibitory profile against COX-2, when compared to that of reference standards NS398 and indomethacin. Molecular modelling studies confirmed the obtained biological results.