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Alfonso ZAMBON

Professore Associato
Dipartimento di Scienze Chimiche e Geologiche - Sede Dipartimento di Scienze Chimiche e Geologiche

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Pubblicazioni

2024 - New pan-ALK inhibitor-resistant EML4::ALK mutations detected by liquid biopsy in lung cancer patients [Articolo su rivista]
Villa, Matteo; Malighetti, Federica; Sala, Elisa; Sharma, Geeta G.; Arosio, Giulia; Gemelli, Maria; Manfroni, Chiara; Fontana, Diletta; Cordani, Nicoletta; Meneveri, Raffaella; Zambon, Alfonso; Piazza, Rocco; Pagni, Fabio; Cortinovis, Diego; Mologni, Luca
abstract

: ALK and ROS1 fusions are effectively targeted by tyrosine kinase inhibitors (TKIs), however patients inevitably relapse after an initial response, often due to kinase domain mutations. We investigated circulating DNA from TKI-relapsed NSCLC patients by deep-sequencing. New EML4::ALK substitutions, L1198R, C1237Y and L1196P, were identified in the plasma of NSCLC ALK patients and characterized in a Ba/F3 cell model. Variants C1237Y and L1196P demonstrated pan-inhibitor resistance across 5 clinical and 2 investigational TKIs.

2023 - Biostimulants derived from organic urban wastes and biomasses: An innovative approach [Articolo su rivista]
Giordana, Alessia; Malandrino, Mery; Zambon, Alfonso; Lusvardi, Gigliola; Operti, Lorenza; Cerrato, Giuseppina
abstract

We used humic and fulvic acids extracted from digestate to formulate nanohybrids with potential applications in agronomy. In order to obtain a synergic co-release of plant-beneficial agents, we functionalized with humic substances two inorganic matrixes: hydroxyapatite (Ca10(PO4)6(OH)2, HP) and silica (SiO2) nanoparticles (NPs). The former is a potential controlled-release fertilizer of P, and the latter has a beneficial effect on soil and plants. SiO2 NPs are obtained from rice husks by a reproducible and fast procedure, but their ability to absorb humic substances is very limited. HP NPs coated with fulvic acid are instead a very promising candidate, based on desorption and dilution studies. The different dissolutions observed for HP NPs coated with fulvic and humic acids could be related to the different interaction mechanisms, as suggested by the FT-IR study.

2023 - Ce-MBGs Loaded with Gentamicin: Characterization and In Vitro Evaluation [Articolo su rivista]
Fraulini, Francesca; Raimondi, Stefano; Candeliere, Francesco; Ranieri, Raffaella; Zambon, Alfonso; Lusvardi, Gigliola
abstract

Mesoporous Bioactive Glasses (MBGs) are biomaterials widely used in tissue engineering, particularly for hard tissue regeneration. One of the most frequent postoperative complications following a biomaterial surgical implant is a bacterial infection, which usually requires treatment by the systemic administration of drugs (e.g., antibiotics). In order to develop biomaterials with antibiotic properties, we investigated cerium-doped MBGs (Ce-MBGs) as in situ-controlled drug delivery systems (DDSs) of gentamicin (Gen), a wide spectrum antibiotic commonly employed against bacteria responsible of postoperative infections. Here we report the optimization of Gen loading on MBGs and the evaluation of the antibacterial properties and of retention of bioactivity and antioxidant properties of the resulting materials. The Gen loading (up to 7%) was found to be independent from cerium content, and the optimized Gen-loaded Ce-MBGs retain significant bioactivity and antioxidant properties. The antibacterial efficacy was verified up to 10 days of controlled release. These properties make Gen-loaded Ce-MBGs interesting candidates for simultaneous hard tissue regeneration and in situ antibiotic release.

2023 - Dual loaded Ce-MBGs with bioactivity, antioxidant and antibacterial properties [Articolo su rivista]
Zambon, Alfonso; Fraulini, Francesca; Raimondi, Stefano; Lusvardi, Gigliola
abstract

2022 - Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase [Articolo su rivista]
Mologni, Luca; Tardy, Sébastien; Zambon, Alfonso; Orsato, Alexandre; Bisson, William H.; Ceccon, Monica; Viltadi, Michela; D’Attoma, Joseph; Pannilunghi, Sara; Vece, Vito; Bertho, Jerome; Goekjian, Peter; Scapozza, Leonardo; Gambacorti-Passerini, Carlo
abstract

2022 - Erratum: Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase (ACS Omega (2022) 7:20 (17083-17097) DOI: 10.1021/acsomega.2c00507) [Articolo su rivista]
Mologni, L.; Tardy, S.; Zambon, A.; Orsato, A.; Schneider, C.; Bisson, W. H.; Ceccon, M.; Viltadi, M.; Goyard, D.; Garcia, P.; D'Attoma, J.; Pannilunghi, S.; Vece, V.; Bertho, J.; Gueyrard, D.; Goekjian, P.; Scapozza, L.; Gambacorti-Passerini, C.
abstract

(1) The following authors have been added: Ceìdric Schneider. Affiliations: Laboratoire Chimie Organique 2-Glycochimie, CNRS-UniversiteìClaude Bernard Lyon 1, Villeurbanne, France; Universiteì de Rouen, Mont-Saint- Aignan, France David Goyard. Affiliations: Laboratoire Chimie Organique 2-Glycochimie, CNRS-Universiteì Claude Bernard Lyon 1, Villeurbanne, France; Universiteì Grenoble Alpes, Grenoble, France Pierre Garcia. Affiliation: Laboratoire Chimie Organique 2- Glycochimie, CNRS-Universiteì Claude Bernard Lyon 1, Villeurbanne, France David Gueyrard. Affiliation: Laboratoire Chimie Organique 2-Glycochimie, CNRS-Universiteì Claude Bernard Lyon 1, Villeurbanne, France (2) In the third affiliation "Lyon, France"has been replaced by "Villeurbanne, France".These changes are reflected in the authorship of this Correction.

2022 - Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling [Articolo su rivista]
Destro, Lorenza; Van Melsen, Ross; Gobbi, Alex; Terzi, Andrea; Genitoni, Matteo; Zambon, Alfonso
abstract

2022 - Identification of non-ATP-competitive α-carboline inhibitors of the anaplastic lymphoma kinase [Articolo su rivista]
Mologni, Luca; Orsato, Alexandre; Zambon, Alfonso; Tardy, Sébastien; Bisson, William H.; Ceccon, Monica; Viltadi, Michela; D'Attoma, Joseph; Pannilunghi, Sara; Vece, Vito; Bertho, Jerome; Scapozza, Leonardo; Goekjian, Peter; Gambacorti-Passerini, Carlo
abstract

2022 - Investigation on the antimicrobial properties of cerium-doped bioactive glasses [Articolo su rivista]
Raimondi, S.; Zambon, A.; Ranieri, R.; Fraulini, F.; Amaretti, A.; Rossi, M.; Lusvardi, G.
abstract

Cerium-doped bioactive glasses (Ce-BGs) are implant materials that present high biocompatibility, modulate the levels of reactive oxygen species, and exert antimicrobial activity. The potential of BGs, 45S5, and K50S derived glasses doped with CeO2 (1.2, 3.6, and 5.3 mol%) to inhibit the growth of pathogen microbes was thoroughly investigated according to the ISO 22196:2011 method properly adapted. A significant reduction of the E. coli charge was detected in all glasses, including the BGs without cerium. The evolution of pH of the medium not inoculated following the immersion of the Ce-BGs was monitored. The presence of cerium did not affect markedly the pH trend, which increased rapidly for both compositions. The change of pH was strongly mitigated by the presence of 200 mM phosphate buffer pH 7.0 (PB) in the medium. In media buffered by PB, the growth of E. coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, and C. albicans was not affected by the presence of BGs doped or not with cerium, suggesting that the antibacterial activity of Ce-BGs is linked to the increase of environmental pH rather than to specific ion effects. However, Ce-BGs resulted promising biomaterials that associate low toxicity to normal cells to a considerable antimicrobial effect, albeit the latter is not directly associated with the presence of cerium.

2022 - Loading with Biomolecules Modulates the Antioxidant Activity of Cerium-Doped Bioactive Glasses [Articolo su rivista]
Lusvardi, Gigliola; Fraulini, Francesca; D'Addato, Sergio; Zambon, Alfonso
abstract

: In order to identify new bioactive glasses (BGs) with optimal antioxidant properties, we carried out an evaluation of a series of cerium-doped BGs [Ce-BGs─H, K, and mesoporous bioactive glasses (MBGs)] loaded with different biomolecules, namely, gallic acid, polyphenols (POLY), and anthocyanins. Quantification of loading at variable times highlighted POLY on MBGs as the system with the highest loading. The ability to dismutate hydrogen peroxide (catalase-like activity) of the BGs evaluated is strongly correlated with cerium doping, while it is marginally decreased compared to the parent BG upon loading with biomolecules. Conversely, unloaded Ce-BGs show only a marginal ability to dismutate the superoxide anion (SOD)-like activity, while upon loading with biomolecules, POLY in particular, the SOD-like activity is greatly enhanced for these materials. Doping with cerium and loading with biomolecules give complementary antioxidant properties to the BGs investigated; combined with the persistent bioactivity, this makes these materials prime candidates for upcoming studies on biological systems.

2022 - β-Glucuronidase Pattern Predicted From Gut Metagenomes Indicates Potentially Diversified Pharmacomicrobiomics [Articolo su rivista]
Candeliere, Francesco; Raimondi, Stefano; Ranieri, Raffaella; Musmeci, Eliana; Zambon, Alfonso; Amaretti, Alberto; Rossi, Maddalena
abstract

: β-glucuronidases (GUS) of intestinal bacteria remove glucuronic acid from glucoronides, reversing phase II metabolism of the liver and affecting the level of active deconjugated metabolites deriving from drugs or xenobiotics. Two hundred seventy-nine non-redundant GUS sequences are known in the gut microbiota, classified in seven structural categories (NL, L1, L2, mL1, mL2, mL1,2, and NC) with different biocatalytic properties. In the present study, the intestinal metagenome of 60 healthy subjects from five geographically different cohorts was assembled, binned, and mined to determine qualitative and quantitative differences in GUS profile, potentially affecting response to drugs and xenobiotics. Each metagenome harbored 4-70 different GUS, altogether accounting for 218. The amount of intestinal bacteria with at least one GUS gene was highly variable, from 0.7 to 82.2%, 25.7% on average. No significant difference among cohorts could be identified, except for the Ethiopia (ETH) cohort where GUS-encoding bacteria were significantly less abundant. The structural categories were differently distributed among the metagenomes, but without any statistical significance related to the cohorts. GUS profiles were generally dominated by the category NL, followed by mL1, L2, and L1. The GUS categories most involved in the hydrolysis of small molecules, including drugs, are L1 and mL1. Bacteria contributing to these categories belonged to Bacteroides ovatus, Bacteroides dorei, Bacteroides fragilis, Escherichia coli, Eubacterium eligens, Faecalibacterium prausnitzii, Parabacteroides merdae, and Ruminococcus gnavus. Bacteria harboring L1 GUS were generally scarcely abundant (<1.3%), except in three metagenomes, where they reached up to 24.3% for the contribution of E. coli and F. prausnitzii. Bacteria harboring mL1 GUS were significantly more abundant (mean = 4.6%), with Bacteroides representing a major contributor. Albeit mL1 enzymes are less active than L1 ones, Bacteroides likely plays a pivotal role in the deglucuronidation, due to its remarkable abundance in the microbiomes. The observed broad interindividual heterogeneity of GUS profiles, particularly of the L1 and mL1 categories, likely represent a major driver of pharmacomicrobiomics variability, affecting drug response and toxicity. Different geographical origins, genetic, nutritional, and lifestyle features of the hosts seemed not to be relevant in the definition of glucuronidase activity, albeit they influenced the richness of the GUS profile.

2021 - Boosting sunscreen stability: New hybrid materials from UV filters encapsulation [Articolo su rivista]
Fantini, Riccardo; Vezzalini, Giovanna; Zambon, Alfonso; Ferrari, Erika; Di Renzo, Francesco; Fabbiani, Marco; Arletti, Rossella
abstract

2021 - Cerium Containing Bioactive Glasses: A Review [Articolo su rivista]
Zambon, Alfonso; Malavasi, Gianluca; Pallini, Annalisa; Fraulini, Francesca; Lusvardi, Gigliola
abstract

Bioactive glasses (BGs) for biomedical applications are doped with therapeutic inorganic ions (TIIs) in order to improve their performance and reduce the side effects related to the surgical implant. Recent literature in the field shows a rekindled interest toward rare earth elements, in particular cerium, and their catalytic properties. Cerium-doped bioactive glasses (Ce-BGs) differ in compositions, synthetic methods, features, and in vitro assessment. This review provides an overview on the recent development of Ce-BGs for biomedical applications and on the evaluation of their bioactivity, cytocompatibility, antibacterial, antioxidant, and osteogenic and angiogenic properties as a function of their composition and physicochemical parameters.

2021 - Dual Kinase Targeting in Leukemia [Articolo su rivista]
Mologni, Luca; Marzaro, Giovanni; Redaelli, Sara; Zambon, Alfonso
abstract

2021 - The paradox-breaking panRAF plus SRC family kinase inhibitor, CCT3833, is effective in mutant KRAS-driven cancers [Articolo su rivista]
Saturno, G.; Lopes, F.; Niculescu-Duvaz, I.; Niculescu-Duvaz, D.; Zambon, A.; Davies, L.; Johnson, L.; Preece, N.; Lee, R.; Viros, A.; Holovanchuk, D.; Pedersen, M.; Mcleary, R.; Lorigan, P.; Dhomen, N.; Fisher, C.; Banerji, U.; Dean, E.; Krebs, M. G.; Gore, M.; Larkin, J.; Marais, R.; Springer, C.
abstract

2021 - Toward the Scale-Up of a Bicyclic Homopiperazine via Schmidt Rearrangement and Photochemical Oxaziridine Rearrangement in Continuous-Flow [Articolo su rivista]
Brown, Michael; Aljarah, Mohammed; Asiki, Hannah; Leung, Leo M. H.; Smithen, Deborah A.; Miller, Natalie; Nemeth, Gabor; Davies, Lawrence; Niculescu-Duvaz, Dan; Zambon, Alfonso; Springer, Caroline
abstract

2020 - Potential of wickerhamomyces anomalus in glycerol valorization [Articolo su rivista]
Amaretti, A.; Russo, B.; Raimondi, S.; Leonardi, A.; Foca, G.; Mucci, A.; Zambon, A.; Rossi, M.
abstract

Five-carbons polyalcohols, such as xylitol and arabitol, and microbial oils are important targets for biotechnological industries. Polyalcohols can find application as low-calories sweeteners and as building block in the synthesis of valuable compounds, while lipids are interesting for both biofuel and food industry. The osmophilic yeast Wickerhamomyces anomalus WC 1501 was preliminary known to produce arabitol from glycerol. Production kinetics were investigated in this study. Production was not growth-associated and occurred during a nitrogen-limited stationary phase, in presence of an excess of carbon source. Typical bioreactor batch cultures, carried out with 160 g/L glycerol, yielded 16.0 g/L arabitol in 160 h. A fed-batch process was developed, in which growth is carried out batchwise in a balanced medium containing 20 g/L glycerol, and arabitol production is induced at the entrance into the stationary phase with a pulse of concentrated glycerol to provide the remaining 140 g/L carbon source. At the end of the process 18.0 g/L arabitol were generated. Under these conditions, the yeast also accumulated intracellular triacylglycerols, with fatty acids of 16-18 carbons bearing 0 to 2 unsaturations, reaching up the 23% of biomass dry weight. Therefore, W. anomalus WC 1501 is a good candidate for the development of a fermentative process yielding arabitol and has potential also as oleaginous yeast for producing lipids, further improving the interest in this strain for glycerol biorefinery. The utilization of a fed-batch process allows to carry out distinct growth and production phases and thus allows the optimization of both phases separately, in order to achieve the highest concentration of catalytic biomass during growth and the maximum efficiency during production. This strain deserves further investigation to better exploit its biotechnological potential in the valorization of glycerol.

2020 - endo-1-Phenylborneol as a novel, alternative chiral auxiliary for the aza-Diels-Alder reaction [Articolo su rivista]
Bassani, M.; Scarso, A.; Drago, M.; Zambon, A.; Fabris, F.
abstract

We report the synthesis and preliminary evaluation of (1S,2S,4R)-7,7-dimethyl-1-phenylbicyclo[2.2.1]heptan-2-ol 1a, which was obtained in 3 steps from inexpensive starting materials, as a chiral auxiliary. The potential for asymmetric induction was investigated by carrying out aza-Diels-Alder reactions with cyclopentadiene and imine derivatives of 1a from (R)-(+)-1-phenylethylamine, (S)-(−)-1-phenylethylamine and benzylamine. The results showed marked exo selectivity and good diastereoisomeric excess when 1a was combined with (R)-(+)-1-phenylethylamine. These results are comparable with those reported using (−)-8-phenylmenthol, suggesting that 1a can represent an economically viable alternative to current chiral auxiliaries.

2019 - Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design and Structure-Activity Relationships [Articolo su rivista]
Leung, Leo; Niculescu-Duvaz, Dan; Smithen, Deborah; Lopes, Filipa; Callens, Cedric; Mcleary, Robert; Saturno, Grazia; Davies, Lawrence; Aljarah, Mohammed; Brown, Michael; Johnson, Louise; Zambon, Alfonso; Chambers, Tim; Ménard, Delphine; Bayliss, Natasha; Knight, Ruth; Fish, Laura; Lawrence, Rae; Challinor, Mairi; Tang, Haoran; Marais, Richard; Springer, Caroline
abstract

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that crosslinks collagens and elastin in the extracellular matrix (ECM) and is a critical mediator of tumor growth and metastatic spread. LOX is a target for cancer therapy and thus the search for therapeutic agents against LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High throughput screening (HTS) provided the initial hits. Structure-activity relationship (SAR) studies led to the discovery of AMT inhibitors with sub-micromolar half maximal inhibitory concentrations (IC50) in a LOX enzyme activity assay. Further SAR optimisation yielded the orally bioavailable LOX inhibitor CCT365623 with good anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy.

2019 - Cytocompatibility of potential bioactive cerium-doped glasses based on 45S5 [Articolo su rivista]
Malavasi, Gianluca; Salvatori, Roberta; Zambon, Alfonso; Lusvardi, Gigliola; Rigamonti, Luca; Chiarini, Luigi; Anesi, Alexandre
abstract

The cytocompatibility of potential bioactive cerium-containing (Ce 3+ /Ce 4+ ) glasses is here investigated by preparing three different glasses with increasing amount of doping CeO 2 (1.2, 3.6 and 5.3 mol% of CeO 2 , called BG_1.2, BG_3.6 and BG_5.3, respectively) based on 45S5 Bioglass®(called BG). These materials were characterized by Environmental Scanning Electron Microscopy (ESEM) and infrared spectroscopy (FTIR) after performing bioactivity tests in Dulbecco's Modified Eagle Medium (DMEM) solution, and the ions released in solution were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and Optical Emission Spectrometry (ICP-OES). The data obtained clearly show that the glass surfaces of BG, BG_1.2 and BG_3.6 were covered by hydroxyapatite (HA), while BG_5.3 favored the formation of a cerium phosphate crystal phase. The cytotoxicity tests were performed using both murine long bone osteocyte-like (MLO-Y4) and mouse embryonic fibroblast (NIH/3T3) cell lines. The cerium-containing bioactive glasses show an increment in cell viability with respect to BG, and at long times, no cell aggregation and deformation were observed. The proliferation of NIH/3T3 cells increased with the cerium content in the glasses; in particular, BG_3.6 and BG_5.3 showed a higher proliferation of cells than the negative control. These results highlight and enforce the proposal of cerium-doped bioactive glasses as a new class of biomaterials for hard-tissue applications.

2019 - Mesoporous bioactive glasses doped with cerium: Investigation over enzymatic-like mimetic activities and bioactivity [Articolo su rivista]
Nicolini, V.; Malavasi, G.; Lusvardi, G.; Zambon, A.; Benedetti, F.; Cerrato, G.; Valeri, S.; Luches, P.
abstract

In this work, we investigate the ability of mesoporous bioactive glasses (MBGs) modified with cerium ions (Ce3+/Ce4+) to act as catalase and superoxide dismutase (SOD) mimetic materials. We have previously reported that the catalytic properties of bioactive Ce-containing glasses based on 45S5 Bioglass® and synthesized via melting are influenced by: i) composition (presence/absence of P2O5); ii) Ce3+/Ce4+ molar ratio. The introduction of cerium species drastically decreased the bioactivity in terms of Hydroxyapatite formation during bioactivity tests in vitro. We thus decided to add cerium to MBGs, a class of glasses with improved bioactivity with respect to classical molten glasses. MBGs exhibit a high surface area and their reactivity is increased with respect to the molten glasses; they are able to induce the formation of Hydroxyapatite over the surface within shorter times with respect to the 45S5. The catalase and SOD mimetic activity tests revealed that the Ce-MGBs are able to act as mimetic materials for the two enzymes. Both Infrared Spectroscopy and X-ray diffraction analysis have confirmed the presence of Hydroxyapatite over both 80SiO2–15CaO–5P2O5, and 80SiO2–20CaO MBGs samples modified by 5.3% mol of CeO2; simultaneously the glasses maintain a good catalase activity. Moreover, the 80SiO2–15CaO–5P2O5 potential bioactive glasses showed SOD mimetic activity. These results highlight that it is possible to obtain a glass with both antioxidant and bioactivity properties.

2019 - NON CANONICAL CYCLIC NUCLEOTIDES MONOPHOSPHATES IN APHANIZOMENON FLOS-AQUAE: NUCLEAR MAGNETIC RESONANCE AND MASS SPECTROMETRY [Abstract in Atti di Convegno]
Righi, V.; Zambon, A.; Parenti, F.; Rossi, M. C.; Mucci, A.
abstract

Aphanizomenon os-aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a superfood", due to their complete nutritional prole that has proved to have health-enhancing properties. AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry, allows the detection of rather unusual phosphorylated metabolites in AFA. In this study we focused our attention on AFA phosphorylated metabolites giving 31P NMR signals at 20 ppm, a chemical shift that pointed to phosphonates. They instead revealed to be nucleoside 2',3'-cyclic monophosphates (cNMPs), that were characterized by multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry. Our data are fully consistent with the proposed structures and hence demonstrate the presence of cNMPs in AFA, for the rst time. The most studied of these biomolecules is cAMP that activates a protective mechanism in the case of brain tissue injury, whereas it inhibits mitophagy of damaged mitochondria in the kidney.

2019 - Non canonical Cyclic Nucleotides Monophosphates in Aphanizomenon flosaquae: nuclear magnetic resonance and mass spectrometry [Poster]
Righi, Valeria; Zambon, Alfonso; Parenti, Francesca; Rossi, Maria Cecilia; Libertini, Emanuela; Mucci, Adele
abstract

Aphanizomenon flos-aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a “superfood”, due to their complete nutritional profile that has proved to have health-enhancing properties. AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry, allows the detection of rather unusual phosphorylated metabolites in AFA. In this study we focused our attention on AFA phosphorylated metabolites giving 31P NMR signals at 20 ppm, a chemical shift that pointed to phosphonates. They instead revealed to be nucleoside 2’,3’-cyclic monophosphates (cNMPs), that were characterized by multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry. Our data are fully consistent with the proposed structures and hence demonstrate the presence of cNMPs in AFA, for the first time. The most studied of these biomolecules is cAMP that activates a protective mechanism in the case of brain tissue injury, whereas it inhibits mitophagy of damaged mitochondria in the kidney. The role of the other cNMPs there is much to be discovered.

2019 - Non-canonical Cyclic Nucleoside Monophosphates in Aphanizomenon flos-aquae: nuclear magnetic resonance and mass spectrometry [Poster]
Righi, V.; Zambon, A.; Parenti, F.; Rossi, M. C.; Mucci, A.
abstract

Aphanizomenon flos-aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a superfood", due to their complete nutritional prole that has proved to have health-enhancing properties. AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry, allows the detection of rather unusual phosphorylated metabolites in AFA [1,2]. In this study we focused our attention on AFA phosphorylated metabolites giving 31P NMR signals at 20 ppm, a chemical shift that pointed to phosphonates. They instead revealed to be nucleoside 2',3'-cyclic monophosphates (cNMPs), that were characterized by multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry. Our data are fully consistent with the proposed structures and hence demonstrate the presence of cNMPs in AFA, for the rst time. The most studied of these biomolecules is cAMP that activates a protective mechanism in the case of brain tissue injury, whereas it inhibits mitophagy of damaged mitochondria in the kidney [3]. The role of the other cNMPs there is much to be discovered.

2019 - Nucleoside 2’,3’-cyclic monophosphates in Aphanizomenon flos-aquae detected through nuclear magnetic resonance and mass spectrometry [Articolo su rivista]
Zambon, A.; Righi, V.; Parenti, F.; Libertini, E.; Rossi, M. C.; Mucci, A.
abstract

Aphanizomenon flos-aquae (AFA) cyanobacteria from Klamath Lake (Oregon) are considered a “superfood”, due to their broad nutritional profile that has proved to have health-enhancing properties. AFA metabolome is quite complex. Here, we present a study that, combining multinuclear 1H, 31P and 13C Nuclear Magnetic Resonance (NMR) spectroscopy and high-resolution mass spectrometry, led to the detection of uncommon phosphorylated metabolites in AFA. We focused our attention on 31P NMR signals at 20 ppm, a chemical shift that usually points to the presence of phosphonates. The molecules contributing to 20 ppm 31P NMR signals revealed, instead, to be nucleoside 2’,3’-cyclic monophosphates. These metabolites were fully characterized by multinuclear 1H, 31P and 13C NMR spectroscopy and high-resolution mass spectrometry.

2019 - Structure Model and Toxicity of the Product of Biodissolution of Chrysotile Asbestos in the Lungs [Articolo su rivista]
Gualtieri, A. F.; Lusvardi, G.; Pedone, A.; Di Giuseppe, D.; Zoboli, A.; Mucci, A.; Zambon, A.; Filaferro, M.; Vitale, G.; Benassi, M.; Avallone, R.; Pasquali, L.; Lassinantti Gualtieri, M.
abstract

Asbestos is a commercial term indicating six natural silicates with asbestiform crystal habit. Of these, five are double-chain silicates (amphibole) and one is a layer silicate (serpentine asbestos or chrysotile). Although all species are classified as human carcinogens, their degree of toxicity is still a matter of debate. Amphibole asbestos species are biopersistent in the human lungs and exert their chronic toxic action for decades, whereas chrysotile is not biopersistent and transforms into an amorphous silica structure prone to chemical/physical clearance when exposed to the acidic environment created by the alveolar macrophages. There is evidence in the literature of the toxicity of chrysotile, but its limited biopersistence is thought to explain the difference in toxicity with respect to amphibole asbestos. To date, no comprehensive model describing the toxic action of chrysotile in the lungs is available, as the structure and toxic action of the product formed by the biodissolution of chrysotile are unknown. This work is aimed at fulfilling this gap and explaining the toxic action in terms of structural, chemical, and physical properties. We show that chrysotile's fibrous structure induces cellular damage, mainly through physical interactions. Based on our previous work and novel findings, we propose the following toxicity model: inhaled chrysotile fibers exert their toxicity in the alveolar space by physical and biochemical action. The fibers are soon leached by the intracellular acid environment into a product with residual toxicity, and the dissolution process liberates toxic metals in the intracellular and extracellular environment.

2018 - Thiophene based A-D-A small molecules with a dithienosilole core: synthesis, theoretical calculations and optoelectronic properties [Abstract in Atti di Convegno]
Parenti, Francesca; Buffagni, Mirko; Caselli, Monica; Mucci, Adele; Pigani, Laura; Vanossi, Davide; Zambon, Alfonso
abstract

Polythiophenes are conjugated materials finding application in many optoelectronic devices.1 Recently, much attention has been devoted to oligothiophenes, the shorter analogous compounds of these polymers. These semiconductor materials are structurally well-defined, monodisperse, free of defects and often possess higher polarizability and charge mobility with respect to the corresponding polymers.2 Thiophene-based small molecules3 are well studied as photoactive materials in organic solar cells. In particular, molecules with A-π-D-π-A architecture, formed by a central electron donating building block (D) and two terminal electron accepting groups (A) linked by π-conjugated bridges, show a low energy absorption band making them suitable as active layers in organic solar cells.4,5 Little modifications of the backbone, i.e. by changing the length of the alkyl substituents or the type of the functional groups, will often result in remarkable modification of the band gap and other optical properties. Here, we present the theoretical calculations, the synthesis and the characterization of two A-π-D-π-A small molecules E1 and E2, where the central unit is a dithienosilole, the terminal units are methyldicyanovinyl functionalized thiophene rings and the π-bridges are alkyl or alkylsulfanyl functionalized thiophene rings, respectively. The aim of this research is to study the structural, electronic and optical properties of these molecules to better understand the role of the sulfur atom of the alkylsulfanyl chains and to gain insight on the properties of these materials in view of their application in optoelectronic and photovoltaic devices

2017 - 1 - ( 5 - TERT - BUTYL - 2 - ARYL - PYRAZOL - 3 - YL ) . 3 - 12 - FLUORO - 4 - [ ( 3 - OXO - 4H - PYRIDO [ 2 . 3 - BJPYRAZIN - 8 - YL ) OXY | PHENYLJUREA DERIVATIVES AS RAF INHIBITORS FOR THE TREATMENT OF CANCER [Brevetto]
Joy SPRINGER, Caroline; Marais, Richard; Girotti, Romina; - DUVAZ, Dan NICULESCU; - DUVAZ, Ion NICULESCU; Zambon, Alfonso
abstract

therapeutic compounds . More specifically the present invention pertains to certain 1 - ( 5 - tert - butyl - 2 - aryl - pyrazol - 3 - yl ) - 3 - [ 2 - fluoro - 4 - [ ( 3 - oxo 4H - pyrido [ 2 , 3 - b ] pyrazin - 8 - yl ) oxylphenyl ] urea compounds ( referred herein as “ TBAP compounds ” ) that , inter alia , inhibit RAF ( e . g . , BRAF , CRAF , etc . ) . The present invention also pertains to pharmaceutical com positions comprising such compounds , and the use of such compounds and compositions , both in vitro and in vivo , to inhibit RAF ( e . g . , BRAF , CRAF , etc . ) ; and to treat disorders including : proliferative disorders ; cancer ( including , e . g . , malignant melanoma , colorectal carcinoma , pancreatic adenocarcinoma ) ; inflammation ; immunological disorders ; viral infections ; fibrotic disorders ; disorders associated with a mutated form of RAF ( e . g . , BRAF , CRAF , etc . ) ; disorders ameliorated by the inhibition of RAF ( e . g . , BRAF , CRAF , etc . ) ; disorders ameliorated by the inhibition of mutant BRAF ; disorders ameliorated by the inhibition of BRAF and CRAF ; disorders associated with RAS mutations and / or MAPK pathway activation ; disorders ameliorated by the inhibition of SRC , p38 , FGFRA , VEGFR - 2 ( KDR ) , and / or LCK ; etc .

2017 - Erratum: Paradox-Breaking RAF Inhibitors that Also Target SRC Are Effective in Drug-Resistant BRAF Mutant Melanoma (Cancer Cell (2015) 27(1) (85–96) (S1535610814004577) (10.1016/j.ccell.2014.11.006)) [Articolo su rivista]
Girotti, M. R.; Lopes, F.; Preece, N.; Niculescu-Duvaz, D.; Zambon, A.; Davies, L.; Whittaker, S.; Saturno, G.; Viros, A.; Pedersen, M.; Suijkerbuijk, B. M. J. M.; Menard, D.; Mcleary, R.; Johnson, L.; Fish, L.; Ejiama, S.; Sanchez-Laorden, B.; Hohloch, J.; Carragher, N.; Macleod, K.; Ashton, G.; Marusiak, A. A.; Fusi, A.; Brognard, J.; Frame, M.; Lorigan, P.; Marais, R.; Springer, C.
abstract

(Cancer Cell 27, 85–96, January 12, 2015) The authors have noted an error in Figure 6A of the originally published version of this article. The image for pERK (top right panel) was incorrectly duplicated from Figure 4E (top right panel). The corrected Figure 6 is shown here. This error does not alter the original conclusions of the study, but the authors wish to apologize for this oversight and any confusion that may have resulted.

2017 - LYSYL OXIDASE INHIBITORS [Brevetto]
Marais, Richard; Springer, Caroline; NICULESCU-DUVAZ, Dan; Miller, Natalie; Aljarah, Mohammed; Zambon, Alfonso; Leung, Leo; Smithen, Deborah; Brown, Michael; Tang, Haoran
abstract

This invention relates to compounds useful as lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) family member (LOXL1, LOXL2, LOXL3, LOXL4) inhibitors. In addition there are contemplated pharmaceutical compositions comprising the compounds and the use of the compounds in the treatment of conditions mediated by LOX and LOXL, for example cancer. In particular a LOX inhibitor such as the present compounds may be for use in the treatment of a cancer associated with EGFR. The present invention also contemplates the identification of biomarkers that predict responsiveness to a LOX inhibitor.

2017 - Novel oligothiophenes with reduced HOMO-LUMO band gap for Optoelectonics [Abstract in Atti di Convegno]
Parenti, Francesca; Buffagni, Mirko; Zambon, Alfonso; Caselli, Monica; Vanossi, Davide; Mucci, Adele
abstract

Here we present the synthesis of A-π-D-π-A thiophene-based oligomers (Figure 1); in the design of these materials we incorporated both electron-donor and electron-acceptor groups by having a central donor dithienosilole, two terminal methyldicyanovinyl acceptor groups and two bithienyl units, functionalised with alkyl or alkylsulfanyl chains, as π-bridges.

2016 - A novel protocol for the one-pot borylation/Suzuki reaction provides easy access to hinge-binding groups for kinase inhibitors [Articolo su rivista]
Hooper, A.; Zambon, Alfonso; Springer, C. J.
abstract

The one-pot borylation/Suzuki reaction is a very efficient means of accessing cross-coupling products of two aryl-halide partners that generally requires the use of specific catalysts or ligands and/or relatively long reaction times. This new microwave-assisted method provides a quick one-pot borylation/Suzuki reaction protocol that we applied to the synthesis of various bi- or poly-aryl scaffolds, including a variety of aryl and heteroaryl ring systems and the core frameworks of kinase inhibitors vemurafenib and GDC-0879.

2015 - Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma [Articolo su rivista]
Girotti, Maria Romina; Lopes, Filipa; Preece, Natasha; Niculescu Duvaz, Dan; Zambon, Alfonso; Davies, Lawrence; Whittaker, Steven; Saturno, Grazia; Viros, Amaya; Pedersen, Malin; Suijkerbuijk, Bart M. J. M.; Menard, Delphine; Mcleary, Robert; Johnson, Louise; Fish, Laura; Ejiama, Sarah; Sanchez Laorden, Berta; Hohloch, Juliane; Carragher, Neil; Macleod, Kenneth; Ashton, Garry; Marusiak, Anna A.; Fusi, Alberto; Brognard, John; Frame, Margaret; Lorigan, Paul; Marais, Richard; Springer, Caroline
abstract

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutantmelanomas and second-line treatment for patients who develop resistance.

2014 - 1-(5-TERT-BUTYL-2-ARYL-PYRAZOL-3-YL)-3-2-FLUORO-4-(3-OXO-4H-PYRIDO2,3-BPYRAZIN-8-YL)OXYPHENYLUREA DERIVATIVES AS RAF INHIBITORS FOR THE TREATMENT OF CANCER [Brevetto]
Joy Springer, Caroline; Marais Romina Girotti, Richard; Niculescu-Duvaz, Dan; Niculescu-Duvaz, Ion; Zambon, Alfonso
abstract

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to certain 1-(5-tert-butyl-2-aryl-pyrazol-3-yl)- 3-2-fluoro-4-(3-oxo-4H-pyrido 2,3-bipyrazin-8-yl)oxy phenylurea compounds (referred herein as “TBAP compounds') that, inter alia, inhibit RAF (e.g., BRAF, CRAF, etc.). The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of Such compounds and compositions, both in vitro and in vivo, to inhibit RAF (e.g., BRAF, CRAF, etc.); and to treat disorders including proliferative disorders; cancer (including, e.g., malignant melanoma, colorectal carcinoma, pancreatic adenocarcinoma); inflammation; immunological disorders; viral infections; fibrotic disorders; disorders associated with a mutated form of RAF (e.g. BRAF, CRAF, etc.); disorders ameliorated by the inhibition of RAF (e.g., BRAF. CRAF, etc.); disorders ameliorated by the inhibition of mutant BRAF; disorders ameliorated by the inhibition of BRAF and CRAF; disorders associated with RAS mutations and/or MAPK pathway activation; disorders ameliorated by the inhibition of SRC, p38, FGFRA, VEGFR-2 (KDR), and/or LCK; etc.(I)

2014 - Abstract 3704: Novel panRAF inhibitors active in melanomas that are resistant to BRAF-selective, or BRAF-selective/MEK inhibitor combinations [Abstract in Rivista]
Girotti, Maria R.; Lopes, Filipa; Preece, Natasha; Niculescu Duvaz, Dan; Zambon, Alfonso; Davies, Lawrence; Whittaker, Steven; Saturno, Grazia; Viros, Amaya; Pedersen, Malin; Suijkerbuijk, Bart MJM; Menard, Delphine; Mcleary, Robert; Johnson, Louise; Fish, Laura; Ejiama, Sarah; Sanchez Laorden, Berta; Carragher, Neil; Macleod, Kenneth; Ashton, Garry; Marusiak, Anna; Fusi, Alberto; Brognard, John; Frame, Margaret; Lorigan, Paul; Springer, Caroline J.; Marais, Richard
abstract

The protein kinase BRAF is mutated ∼40% of human melanomas. BRAF is a component of the RAS/RAF/MEK/ERK pathway and BRAF or MEK inhibitors increase progression-free and overall survival in melanoma patients with BRAF mutations. However, most patients relapse with acquired resistance and ∼20% of patients present intrinsic resistance and do not respond to these drugs. We describe here two novel compounds that target mutant BRAF and wild-type CRAF. Our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors. ERK pathway reactivation is responsible for resistance to BRAF targeted therapies in ∼60% of the patients and in ∼25% of patients resistance is driven by acquisition of mutations in NRAS. We show that our compounds inhibited the growth of melanoma cells that were resistant to BRAF-selective inhibitors due to pathway reactivation mediated by different mechanisms. We show that the drugs were active against patient derived xenografts (PDXs) from patients with acquired or intrinsic resistance to BRAF-selective inhibitors and in whose tumors resistance was associated with ERK pathway reactivation. Further, our compounds are active in a PDX from a patient whose tumor developed acquired resistance to a combination of a BRAF-selective plus a MEK inhibitor and associated with acquisition of an NRAS mutation. Thus, our panRAF inhibitors can inhibit melanomas with different mechanisms of acquired or intrinsic resistance to BRAF-selective and BRAF-selective/MEK inhibitor combinations, potentially providing first-line treatment for naïve patients and second-line treatments for a range of relapsed patients.

2014 - BRAF inhibitors induce metastasis in RAS mutant or inhibitor-resistant melanoma cells by reactivating MEK and ERK signaling [Articolo su rivista]
Sanchez Laorden, Berta; Viros, Amaya; Girotti, Maria Romina; Pedersen, Malin; Saturno, Grazia; Zambon, Alfonso; Niculescu Duvaz, Dan; Turajlic, Samra; Hayes, Andrew; Gore, Martin; Larkin, James; Lorigan, Paul; Cook, Martin; Springer, Caroline; Marais, Richard
abstract

Melanoma is a highly metastatic and lethal form of skin cancer. The protein kinase BRAF is mutated in about 40% of melanomas, and BRAF inhibitors improve progression-free and overall survival in these patients. However, after a relatively short period of disease control, most patients develop resistance because of reactivation of the RAF-ERK (extracellular signal-regulated kinase) pathway, mediated in many cases by mutations in RAS. We found that BRAF inhibition induces invasion and metastasis in RAS mutant melanoma cells through a mechanism mediated by the reactivation of the MEK (mitogen-activated protein kinase kinase)-ERK pathway, increased expression and secretion of interleukin 8, and induction of protease-dependent invasion. These events were accompanied by a cell morphology switch from predominantly rounded to predominantly elongated cells. We also observed similar responses in BRAF inhibitor-resistant melanoma cells. These data show that BRAF inhibitors can induce melanoma cell invasion and metastasis in tumors that develop resistance to these drugs.

2014 - PROCESS FOR THE PREPARATION OF 8-(4-AMINOPHENOXY)-4H PYRIDO2,3-B PYRAZIN-3-ONE DERIVATIVES [Brevetto]
Zambon, Alfonso; Niculescu-Duvaz, Dan; Chubb, Richard; Springer, Caroline Joy
abstract

The present invention pertains generally to the field of organic chemical synthesis, and in particular to certain methods for the synthesis of 8-(4-aminophenyoxy)-4H pyrido2.3-bipyrazin-3-one and related compounds (de noted herein as (3)) from 4-(4-aminophenyoxy)pyridine-2, 3-diamine and related compounds (denoted herein as (1)), by reaction with glyoxylic acid (denoted herein as (2)). The compounds (3) are useful in the synthesis of known anti cancer agents, such as 1-(5-tert-butyl-2-(4-methyl-phenyl)- pyrazol-3-yl)-3-2-fluoro-4-(3-oxo-4H-pyrido 2,3-b pyrazin-8-yl)oxyphenylurea.

2013 - BRAF as a therapeutic target: A patent review (2006-2012) [Articolo su rivista]
Zambon, Alfonso; Niculescu Duvaz, Dan; Niculescu Duvaz, Ion; Marais, Richard; Springer, Caroline J.
abstract

Introduction: After its identification as an oncogene in 2002, mutant BRAF has become the target of a number of drug discovery programmes, primarily aimed at the treatment of late stage or unresectable melanoma. Some of the drugs thus developed, such as vemurafenib and dabrafenib, show impressive responses in melanoma patients harbouring a BRAF mutation. Areas covered: This review summarises the patent literature on BRAF from 2006 to 2012, focusing on the specific areas of inhibitors of mutant BRAF, drug combinations including BRAF inhibitors, diagnostic methods for use with mutant BRAF inhibitors & diagnosis and treatment of mutant BRAF cancers resistant to BRAF inhibitors. Expert opinion: Whilst these first-generation BRAF inhibitors initially mediate excellent responses in late stage or unresectable melanoma patients bearing the V600 mutation, resistance usually occurs and patients eventually relapse. The patent literature for new BRAF inhibitors and therapies reflects the desire to develop second-generation drugs able to overcome this resistance and combination treatments that increase the efficiency of current mutant BRAF inhibitors. © 2013 Informa UK, Ltd.

2013 - Erratum: Topical 5-fluorouracil elicits regressions of BRAF inhibitor-induced cutaneous squamous cell carcinoma (Journal of Investigative Dermatology (2013) 133 (274-276) DOI: 10.1038/jid.2012.268;0 [Articolo su rivista]
Viros, A.; Hayward, R.; Martin, M.; Yashar, S.; Yu, C. C.; Sanchez-Laorden, B.; Zambon, A.; Niculescu-Duvaz, D.; Springer, C.; Lo, R. S.; Marais, R.
abstract

2013 - Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma [Articolo su rivista]
Girotti, Maria R.; Pedersen, Malin; Sanchez Laorden, Berta; Viros, Amaya; Turajlic, Samra; Niculescu Duvaz, Dan; Zambon, Alfonso; Sinclair, John; Hayes, Andrew; Gore, Martin; Lorigan, Paul; Springer, Caroline; Larkin, James; Jorgensen, Claus; Marais, Richard
abstract

We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo, and monotherapy with the broad specifi city tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo. We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data show that BRAF inhibitor-mediated activation of EGFR-SFK-STAT3 signaling can mediate resistance in patients with BRAF-mutant melanoma. We describe 2 treatments that seem to overcome this resistance and could deliver therapeutic effi cacy in patients with drug-resistant BRAF-mutant melanoma. SIGNIFICANCE: Therapies that target the driver oncogenes in cancer can achieve remarkable responses if patients are stratifi ed for treatment. However, as with conventional therapies, patients often develop acquired resistance to targeted therapies, and a proportion of patients are intrinsically resistant and fail to respond despite the presence of an appropriate driver oncogene mutation. We found that the EGFR/SFK pathway mediated resistance to vemurafenib in BRAF -mutant melanoma and that BRAF and EGFR or SFK inhibition blocked proliferation and invasion of these resistant tumors, providing potentially effective therapeutic options for these patients. © 2012 American Association for Cancer Research.

2013 - Potent BRAF kinase inhibitors based on 2,4,5-trisubstituted imidazole with naphthyl and benzothiophene 4-substituents [Articolo su rivista]
Niculescu Duvaz, Dan; Niculescu Duvaz, Ion; Suijkerbuijk, Bart M. J. M.; Ménard, Delphine; Zambon, Alfonso; Davies, Lawrence; Pons, Jean Francois; Whittaker, Steven; Marais, Richard; Springer, Caroline J.
abstract

The RAS-RAF-MEK-ERK pathway is hyperactivated in 30% of human cancers. BRAF is a serine-threonine kinase, belonging to this pathway that is mutated with high frequency in human melanoma and other cancers thus BRAF is an important therapeutic target in melanoma. We have designed inhibitors of BRAF based on 2,4,5-trisubstituted imidazoles with naphthyl and benzothiophene-4-substituents. Two compounds were discovered to be potent BRAF inhibitors: 1-(6-2-[4-(2-dimethylamino-ethoxy)phenyl]-5-(pyridin-4-yl)-1H-imidazol-4-yl benzo[b]thiophen-3-yl)-2,2,2-trifluoroethanol (1i) with BRAF IC50 = 190 nM and with cellular GI50 = 2100 nM, and 6-2-[4-(2- dimethylamino-ethoxy)-phenyl]-5-pyridin-4-yl-3H-imidazol-4-yl-naphthalen-1-ol (1q) with IC50 = 9 nM and GI50 = 220 nM. © 2012 Elsevier Ltd. All rights reserved.

2013 - Preparation of 1-​(5-​tert-​butyl-​2-​aryl-​pyrazol-​3-​yl)​-​3-​[2-​fluoro-​4-​[(3-​oxo-​4H-​pyrido[2,​3-​b]​pyrazin-​8-​yl)​oxy]​phenyl]​urea derivatives as RAF inhibitors for treating cancer [Brevetto]
Springer, Caroline Joy; Marais, Richard; Girotti, Romina; Niculescu-Duvaz, Dan; Niculescu-Duvaz, Ion; Zambon, Alfonso
abstract

The present invention pertains generally to the field of therapeutic compounds. More specifically the present invention pertains to certain l-(5-tert-butyl-2-aryl-pyrazol-3-yl)- 3-[2-fluoro-4-[(3-oxo-4H-pyrido[2,3-b]pyrazin-8-yl)oxy]phenyl]urea compounds (referred herein as "TBAP compounds") that, inter alia, inhibit RAF (e.g., BRAF, CRAF, etc.). The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit RAF (e.g., BRAF, CRAF, etc.); and to treat disorders including: proliferative disorders; cancer (including, e.g., malignant melanoma, colorectal carcinoma, pancreatic adenocarcinoma); inflammation; immunological disorders; viral infections; fibrotic disorders; disorders associated with a mutated form of RAF (e.g., BRAF, CRAF, etc.); disorders ameliorated by the inhibition of RAF (e.g., BRAF, CRAF, etc.); disorders ameliorated by the inhibition of mutant BRAF; disorders ameliorated by the inhibition of BRAF and CRAF; disorders associated with RAS mutations and/or MAPK pathway activation; disorders ameliorated by the inhibition of SRC, p38, FGFRA, VEGFR-2 (KDR), and/or LCK; etc.

2013 - Topical 5-Fluorouracil Elicits Regressions of BRAF Inhibitor–Induced Cutaneous Squamous Cell Carcinoma [Articolo su rivista]
Viros, Amaya; Hayward, Robert; Martin, Matthew; Yashar, Sharona; Yu, Clarissa C.; Sanchez Laorden, Berta; Zambon, Alfonso; Niculescu Duvaz, Dan; Springer, Caroline; Lo, Roger S; Marais, Richard
abstract

We tested the efficacy of 5-FU on secondary SCC in two melanoma patients who were being treated with the BRAF inhibitor dabrafenib (GSK2118436). We show that 5-FU provides a safe, noninvasive, inexpensive, and effective alternative to surgical intervention in clinical cases in which multiple surgeries are difficult or in cases in which lesions are incipient.

2012 - Paradoxical activation of the MEK/ERK pathway promotes melanoma invasion and metastasis [Abstract in Rivista]
Sanchez Laorden, B.; Viros, A.; Girotti, R.; Pedersen, M.; Zambon, Alfonso; Niculescu Duvaz, D.; Springer, C; Marais, R.
abstract

Paradoxical activation of the MEK/ERK pathway promotes melanoma invasion and metastasis

2012 - RAS Mutations in Cutaneous Squamous-Cell Carcinomas in Patients Treated with BRAF Inhibitors [Articolo su rivista]
Su, F; Viros, A; Milagre, C; Trunzer, K; Bollag, G; Spleiss, O; Reis, Js; Kong, Xj; Koya, Rc; Flaherty, Kt; Chapman, Pb; Kim, Mj; Hayward, R; Martin, M; Yang, H; Wang, Qq; Hilton, H; Hang, Js; Noe, J; Lambros, M; Geyer, F; Dhomen, N; Niculescu Duvaz, I; Zambon, Alfonso; Niculescu Duvaz, D; Preece, N; Robert, L; Otte, Nj; Mok, S; Kee, D; Ma, Y; Zhang, C; Habets, G; Burton, Ea; Wong, B; Nguyen, H; Kockx, M; Andries, L; Lestini, B; Nolop, Kb; Lee, Rj; Joe, Ak; Troy, Jl; Gonzalez, R; Hutson, Te; Puzanov, I; Chmielowski, B; Springer, Cj; Mcarthur, Ga; Sosman, Ja; Lo, Rs; Ribas, A; Marais, R.
abstract

BACKGROUNDCutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.METHODSWe performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.RESULTSAmong 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.CONCLUSIONSMutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.)

2012 - Small molecule inhibitors of BRAF in clinical trials [Articolo su rivista]
Zambon, Alfonso; Niculescu Duvaz, Ion; Niculescu Duvaz, Dan; Marais, Richard; Springer, Caroline J.
abstract

Over the last few years, BRAF has emerged as a validated target in melanoma. This review summarises recent advances in the development of BRAF inhibitors, focussing on agents that have been assessed clinically. © 2011 Elsevier Ltd. All rights reserved.

2011 - 1-(5-tert-butyl-2-phenyl-2H-pyrazol-3-yl)-3-[2-fluoro-4-(1-methyl-2-oxo-2,3-dihydro-1H-imidazo[4,5-B]pyridin-7-yloxy)-phenyl]-urea and related compounds and their use in therapy [Brevetto]
Springer, Caroline; Niculescu-Duvaz, Ion; Marais, Richard; Niculescu-Duvaz, Dan; Zambon, Alfonso; Menard, Delphine
abstract

The present invention pertains generally to the field of therapeutic compds., and more specifically to certain compds. of the following formula (I) (for convenience, collectively referred to herein as "IP compds.")​, which, inter alia, are useful in the treatment of cancer, e.g., cancer characterized by (e.g., driven by) mutant RAS ("mutant RAS cancer")​. The present invention also pertains to pharmaceutical compns. comprising such compds., and the use of such compds. and compns. in the treatment of cancer, e.g., mutant RAS cancer.

2010 - A novel, selective, and efficacious nanomolar pyridopyrazinone inhibitor of V600EBRAF [Articolo su rivista]
Whittaker, Steven; Ménard, Delphine; Kirk, Ruth; Ogilvie, Lesley; Hedley, Douglas; Zambon, Alfonso; Lopes, Filipa; Preece, Natasha; Manne, Helen; Rana, Sareena; Lambros, Maryou; Reis Filho, Jorge S.; Marais, Richard; Springer, Caroline J.
abstract

Oncogenic BRAF is a critical driver of proliferation and survival and is thus a validated therapeutic target in cancer. We have developed a potent inhibitor, termed 1t (CCT239065), of the mutant protein kinase, V600EBRAF. 1t inhibits signaling downstream of V600EBRAF in cancer cells, blocking DNA synthesis, and inhibiting proliferation. Importantly, we show that 1t is considerably more selective for mutated BRAF cancer cell lines compared with wild-type BRAF lines. The inhibitor is well tolerated in mice and exhibits excellent oral bioavailability (F = 71%). Suppression of V600EBRAF-mediated signaling in human tumor xenografts was observed following oral administration of a single dose of 1t. As expected, the growth rate in vivo of a wild-type BRAF human tumor xenograft model is unaffected by inhibitor 1t. In contrast, 1t elicits significant therapeutic responses in mutant BRAF-driven human melanoma xenografts. ©2010 AACR.

2010 - BRAF inhibitors based on an imidazo[4,5]pyridin-2-one scaffold and a meta substituted middle ring [Articolo su rivista]
Nourry, Arnaud; Zambon, Alfonso; Davies, Lawrence; Niculescu Duvaz, Ion; Dijkstra, Harmen P.; Ménard, Delphine; Gaulon, Catherine; Niculescu Duvaz, Dan; Suijkerbuijk, Bart M. J. M.; Manne, Frank Friedlos Helen A.; Kirk, Ruth; Whittaker, Steven; Marais, Richard; Springer, Caroline J.
abstract

We recently reported on the development of a novel series of BRAF inhibitors based on a tripartite A-B-C system characterized by a para-substituted central aromatic core connected to an imidazo[4,5]pyridin-2-one scaffold and a substituted urea linker. Here, we present a new series of BRAF inhibitors in which the central phenyl ring connects to the hinge binder and substrate pocket of BRAF with a meta-substitution pattern. The optimization of this new scaffold led to the development of lownanomolar inhibitors that permits the use of a wider range of linkers and terminal C rings while enhancing the selectivity for the BRAF enzyme in comparison to the para series. © 2010 American Chemical Society.

2010 - Development of novel, highly potent inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF): Increasing cellular potency through optimization of a distal heteroaromatic group [Articolo su rivista]
Suijkerbuijk, Bart M. J. M.; Niculescu Duvaz, Ion; Gaulon, Catherine; Dijkstra, Harmen P.; Niculescu Duvaz, Dan; Ménard, Delphine; Zambon, Alfonso; Nourry, Arnaud; Davies, Lawrence; Manne, Helen A.; Friedlos, Frank; Ogilvie, Lesley M.; Hedley, Douglas; Lopes, Filipa; Preece, Natasha P. U.; Moreno Farre, Javier; Raynaud, Florence I.; Kirk, Ruth; Whittaker, Steven; Marais, Richard; Springer, Caroline J.
abstract

We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b] imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified V600EBRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated V600EBRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, V600EBRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo. © 2010 American Chemical Society.

2010 - Gatekeeper mutations mediate resistance to BRAF-targeted therapies [Articolo su rivista]
Whittaker, Steven; Kirk, Ruth; Hayward, Robert; Zambon, Alfonso; Viros, Amaya; Cantarino, Neus; Affolter, Annette; Nourry, Arnaud; Niculescu Duvaz, Dan; Springer, Caroline; Marais, Richard
abstract

BRAF is a serine-threonine - specific protein kinase that is mutated in 2% of human cancers. Oncogenic BRAF is a validated therapeutic target that constitutively activates mitogen-activated protein kinase kinase (MEK) - extracellular signal - regulated kinase (ERK) signaling, driving tumor cell proliferation and survival. Drugs designed to target BRAF have been developed, but it is difficult to prove that they mediate their antitumor effects by inhibiting BRAF rather than by working through off-target effects. We generated drug-resistant versions of oncogenic BRAF by mutating the gatekeeper residue. Signaling by the mutant proteins was resistant to the small-molecule inhibitor sorafenib, but sorafenib still inhibited the growth of tumors driven by the mutant protein. In contrast, both BRAF signaling and tumor growth were resistant to another RAF drug, PLX4720. These data provide unequivocal evidence that sorafenib mediates its antitumor effects in a manner that is independent of its ability to target oncogenic BRAF, whereas PLX4720 inhibits tumor growth by targeting oncogenic BRAF directly.

2010 - Novel hinge binder improves activity and pharmacokinetic properties of BRAF inhibitors [Articolo su rivista]
Zambon, Alfonso; Ménard, Delphine; Suijkerbuijk, Bart M. J. M.; Niculescu Duvaz, Ion; Whittaker, Steven; Niculescu Duvaz, Dan; Nourry, Arnaud; Davies, Lawrence; Manne, Helen A.; Lopes, Filipa; Preece, Natasha; Hedley, Douglas; Ogilvie, Lesley M.; Kirk, Ruth; Marais, Richard; Springer, Caroline J.
abstract

Mutated BRAF serine/threonine kinase is implicated in several types of cancer, with particularly high frequency in melanoma and colorectal carcinoma. We recently reported on the development of BRAF inhibitors based on a tripartite A?B?C system featuring an imidazo[4,5]pyridin-2-one group hinge binder. Here we present the design, synthesis, and optimization of a new series of inhibitors with a different A?B?C system that has been modified by the introduction of a range of novel hinge binders (A ring). The optimization of the hinge binding moiety has enabled the development of compounds with low nanomolar potencies in both BRAF inhibition and cellular assays. These compounds display optimal pharmacokinetic properties that warrant further in vivo investigations. © 2010 American Chemical Society.

2010 - Novel tricyclic pyrazole BRAF inhibitors with imidazole or furan central scaffolds [Articolo su rivista]
Niculescu Duvaz, Dan; Niculescu Duvaz, Ion; Suijkerbuijk, Bart M. J. M.; Ménard, Delphine; Zambon, Alfonso; Nourry, Arnaud; Davies, Lawrence; Manne, Helen A.; Friedlos, Frank; Ogilvie, Lesley; Hedley, Douglas; Takle, Andrew K.; Wilson, David M.; Pons, Jean Francois; Coulter, Tom; Kirk, Ruth; Cantarino, Neus; Whittaker, Steven; Marais, Richard; Springer, Caroline J.
abstract

V-RAF murine sarcoma viral oncogene homolog B1 (BRAF) is a serine/threonine-specific protein kinase that is mutated with high frequency in cutaneous melanoma, and many other cancers. Inhibition of mutant BRAF is an attractive therapeutic approach for the treatment of melanoma. A triarylimidazole BRAF inhibitor bearing a phenylpyrazole group (dimethyl-[2-(4-5-[4-(1H-pyrazol-3-yl)-phenyl]-4-pyridin-4-yl-1H-imidazol-2-yl -phenoxy)-ethyl]-amine, 1a) was identified as an active BRAF inhibitor. Based on this starting point, we synthesized a series of analogues leading to the discovery of 6-2-[4-(4-methyl-piperazin-1-yl)-phenyl]-5-pyridin-4-yl-3H- imidazol-4-yl-2,4-dihydro-indeno[1,2-c]pyrazole (1j), with nanomolar activity in three assays: inhibition of purified mutant BRAF activity in vitro; inhibition of oncogenic BRAF-driven extracellular regulated kinase (ERK) activation in BRAF mutant melanoma cell lines; and inhibition of proliferation in these cells. © 2010 Elsevier Ltd. All rights reserved.

2010 - Synthesis, structure-activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors [Articolo su rivista]
Mologni, Luca; Rostagno, Roberta; Brussolo, Stefania; Knowles, Phillip P.; Kjaer, Svend; Murray Rust, Judith; Rosso, Enrico; Zambon, Alfonso; Scapozza, Leonardo; Mcdonald, Neil Q.; Lucchini, Vittorio; Gambacorti Passerini, Carlo
abstract

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers. © 2010 Elsevier Ltd. All rights reserved.

2010 - The discovery of novel, highly potent inhibitors of BRAF [Abstract in Atti di Convegno]
Niculescu Duvaz, I.; Menard, D; Niculescu Duvaz, D.; Zambon, Alfonso; Davies, L; Preece, N.; Kirk, R.; Whittaker, S.; Marais, R.; Springer, C.
abstract

We describe the synthesis and optimisation of a series of new inhibitors of BRAF, a kinase whose mutant form (V600E) is implicated in several types of cancer, with particularly high frequency in melanoma. We designed and synthesised type II inhibitors interacting with the inactive conformation of the V600EBRAF.

2009 - Corrections to pyridoimidazolones as novel potent inhibitors of v-raf murine sarcoma viral oncogene homologue B1 (BRAF) (Journal of Medicinal Chemistry (2009) 52, (2255-2256) DOI: 10.1021/jm801509w) [Articolo su rivista]
Niculescu-Duvaz, D.; Gaulon, C.; Dijkstra, H. P.; Niculescu-Duvaz, I.; Zambon, A.; Menard, D.; Suijkerbuijk, B. M. J. M.; Nourry, A.; Davies, L.; Manne, H.; Friedlos, F.; Ogilvie, L.; Hedley, D.; Whittaker, S.; Kirk, R.; Gill, A.; Taylor, R. D.; Raynaud, F. I.; Moreno-Farre, J.; Marais, R.; Springer, C. J.
abstract

2009 - Novel Potent BRAF Inhibitors: Toward 1 nM Compounds through Optimization of the Central Phenyl Ring [Articolo su rivista]
Menard, Delphine; Niculescu Duvaz, Ion; Dijkstra, Harmen P; I. Niculescu Duvaz, Dan; Suijkerbuijk, Bart M. J. M.; Zambon, Alfonso; Nourry, Arnaud; Roman, Esteban; Davies, Lawrence; Marine, ; Friedlos, Frank; Helen A., And; Kirk, Ruth and; Gill, Adrian; Whittaker, Steven; Taylor, Richard D; Marais, Richard; Springer, Caroline
abstract

BRAF, a serine/threonine specific protein kinase that is part of the MAPK pathway and acts as a downstream effector of RAS, is a potential therapeutic target in melanoma. We have developed a series of small-molecule BRAF inhibitors based on a 1H-imidazo[4,5-b]pyridine-2(3H)-one scaffold (ring A) as the hinge binding moiety and a number of substituted phenyl rings C that interact with the allosteric binding site. The introduction of various groups on the central phenyl ring B combined with appropriate A- and C-ring modifications afford very potent compounds that inhibit V600EBRAF kinase activity in vitro and oncogenic BRAF signaling in melanoma cells. Substitution on the central phenyl ring of a 3-fluoro, a naphthyl, or a 3-thiomethyl group improves activity to yield compounds with an IC50 of 1 nM for purified V600EBRAF and nanomolar activity in cells.

2009 - Pyridoimidazolones as novel potent inhibitors of v-Raf murine sarcoma viral oncogene homologue B1 (BRAF) [Articolo su rivista]
Niculescu Duvaz, Dan; Gaulon, Catherine; Dijkstra, Harmen P.; Niculescu Duvaz, Ion; Zambon, Alfonso; Menard, Delphine; Suijkerbuijk, Bart M. J. M.; Nourry, Arnaud; Davies, Lawrence; Manne, Helen; Friedlos, Frank; Ogilvie, Lesley; Hedley, Douglas; Whittaker, Steven; Kirk, Ruth; Gill, Adrian; Taylor, Richard D.; Raynaud, Florence I.; Moreno Farre, Javier; Marais, Richard; Springer, Caroline J.
abstract

BRAF is a serine/threonine kinase that is mutated in a range of cancers, including 50-70% of melanomas, and has been validated as a therapeutic target. We have designed and synthesized mutant BRAF inhibitors containing pyridoimidazolone as a new hinge-binding scaffold. Compounds have been obtained which have low nanomolar potency for mutant BRAF (12 nM for compound 5i) and low micromolar cellular potency against a mutant BRAF melanoma cell line, WM266.4. The series benefits from very low metabolism, and pharmacokinetics (PK) that can be modulated by methylation of the NH groups of the imidazolone, resulting in compounds with fewer H-donors and a better PK profile. These compounds have great potential in the treatment of mutant BRAF melanomas. © 2009 American Chemical Society.

2009 - Qualitative and quantitative discrimination of fake and true alkene rotation processes in pd(η2-olefin) complexes. A new bimolecular mechanism [Articolo su rivista]
Lucchini, Vittorio; Borsato, Giuseppe; Canovese, Luciano; Santo, Claudio; Visentin, Fabiano; Zambon, Alfonso
abstract

The fluxional behaviour of [Pd(η2-fn)(N-SMe)] (2) (fn = fumaronitrile, N-SMe = 2-methylthiomethylpyridine) and of [Pd(η2-tmetc)(N-N′-4-anisyl)] (3) (tmetc = tetramethylethylenetetracarboxylate, N-N′-4-anisyl = 2-(4-methoxyphenyliminomethane)pyridine) were monitored by 1H NMR spectroscopy and quantitatively determined by line-shape analyses (for 2) and selective inversion recovery experiments (for 3). The coalescence of the AB multiplet of fn hydrogens of 2 is concentration dependent and presents a strongly negative ΔS≠, suggesting the intermediacy of a dimeric complex and ruling out the hypothesis of olefin rotation. The accurate evaluation of all spectral features also allows determination of the approaching mode of the monomeric units. The inversion transfer between the tmetc methyls of 3 reveals a true propeller-like olefin rotation. The presence of a nucleophilic electron pair at sulfur in 2 triggers the formation of the dimeric intermediate. © 2008 Elsevier B.V. All rights reserved.

2008 - ANTIPROLIFERATIVE COMPOUNDS AND THERAPEUTIC USES THEREOF [Brevetto]
Gambacorti Passerini, Carlo; Gunby, Rosalind Helen; Zambon, Alfonso; Scapozza, Leonardo; Ahmed, Shaheen; Goekjian, Peter G.; Gueyrard, David; Popowycz, Florence; Schneider, Cerric
abstract

Inhibitors of the oncogenic tyrosine kinase ALK and of the Bcr-Abl mutant T3 15I Bcr-Abl, pharmaceutical com positions comprising the same and the use thereof for the treatment of hyper-proliferative diseases.

2008 - ANTIPROLIFERATIVE COMPOUNDS AND THERAPEUTIC USES THEREOF [Brevetto]
Gambacorti Passerini, Carlo; Gunby, Rosalind Helen; Zambon, Alfonso; Scapozza, Leonardo; Ahmed, Shaheen; Goekjian, Peter G.; Gueyrard, David; Popowycz, Florence; Schneider, Cerric
abstract

nhibitors of the oncogenic tyrosine kinase ALK and of the Bcr-Abl mutant T3 15I Bcr-Abl, pharmaceutical com positions comprising the same and the use thereof for the treatment of hyper-proliferative diseases.

2008 - Characterization of compound 584, an Abl kinase inhibitor with lasting effects [Articolo su rivista]
Puttini, Miriam; Redaelli, Sara; Moretti, Loris; Brussolo, Stefania; Gunby, Rosalind H.; Mologni, Luca; Marchesi, Edoardo; Cleris, Loredana; Donella Deana, Arianna; Drueckes, Peter; Sala, Elisa; Lucchini, Vittorio; Kubbutat, Michael; Formelli, Franca; Zambon, Alfonso; Scapozza, Leonardo; Gambacorti Passerini, Carlo
abstract

Background: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosomepositive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotransplanted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former. ©2008 Ferrata Storti Foundation.

2008 - Efficient access to 5-substituted thiazoles by a novel metallotropic rearrangement [Articolo su rivista]
Zambon, Alfonso; Borsato, Giuseppe; Brussolo, Stefania; Frascella, Pietrogiulio; Lucchini, Vittorio
abstract

A novel rearrangement process involving the migration of trimethylstannanyl or trimethylsilanyl groups around the thiazole ring provides access to either 2- or 5-metallated thiazoles by tuning the reaction conditions. The proposed mechanism, based on experimental evidence, is characterized by the catalytic role of thiazole bisadducts as metal-transfer agents. © 2007 Elsevier Ltd. All rights reserved.

2007 - Chiral polycyclic ketones via desymmetrization of dihaloolefins [Articolo su rivista]
Borsato, Giuseppe; Linden, Anthony; De Lucchi, Ottorino; Lucchini, Vittorio; Wolstenholme, David; Zambon, Alfonso
abstract

(Chemical Equation Presented) 3-Chloronorbornenone (R)-1a (98% ee) was obtained from trichloronorbornene 5 in two steps by the in situ generation of dichloronorbornadiene 2a with t-BuOK and desymmetrization with (-)-ephedrine, followed by hydrolysis with PPTS. The generality of this desymmetrization with (-)-ephedrine was tested with dibromonorbornadiene 2c and other substituted dichloronorbornadienes. © 2007 American Chemical Society.

2007 - Synthesis and evaluation of antiparasitic activities of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives [Articolo su rivista]
Kuettel, Sabine; Zambon, Alfonso; Kaiser, Marcel; Brun, Reto; Scapozza, Leonardo; Perozzo, Remo
abstract

A series of new 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine derivatives, prepared by two synthetic routes, were in vitro assayed against three Trypanosoma strains, Leishmania donovani, and Plasmodium falciparum K1. Seven out of 17 compounds showed moderate to very good activity against blood stage T. b. rhodesiense, with 10 and 17 exhibiting highest potency (IC 50 of 1.0 and 1.1 μM. respectively). Interestingly, the β-diketone precursors 1-3 had good antitrypanosomal activity toward the insect stage, with IC50 values of 1.0-3.4 μM. Among different compounds with moderate activity against T. cruzi, compound 17 showed the lowest IC50 value of 9.5 μM; thus, the series seemed to act selectively toward the different Trypanosoma parasites. Eight compounds were moderately active against L. donovani, with 2, 3, and 12 being the most promising ones (IC50 values of 2.3-5.2 μM), whereas compound 14 was the only derivative with good activity against P. falciparum (IC50 of 3.7 μM). © 2007 American Chemical Society.

2006 - Rational design of inhibitors of the anaplastic lymphoma kinase [Abstract in Rivista]
Gunby, R; Ahmed, S; Zambon, Alfonso; Gasser, M.; Bertola, L; Porchia, F.; Tartari, C; Sottocornola, R.; Donella, A.; Goekjian, P; Scapozza, L; Gambacorti Passerini, C.
abstract

Rational design of inhibitors of the anaplastic lymphoma kinase

2006 - Synthetic approach to novel NMP/ALK inhibitors [Abstract in Rivista]
Zambon, Alfonso; Goekjian, P; Lucchini, V.
abstract

Synthetic approach to novel NMP/ALK inhibitors

2005 - "Hexacarboxytrindanes": Benzene rings with homotopic faces as scaffolds for the construction of D3 chiral architectures [Articolo su rivista]
Borsato, Giuseppe; Crisma, Marco; De Lucchi, Ottorino; Lucchini, Vittorio; Zambon, Alfonso
abstract

(Figure Presented) Thermodynamically driven epimerization at the trindane benzylic positions of "hexacarboxytrindanes" generates benzene rings with D3 symmetry and with R or S homotopic faces. Almost total resolution towards S trindanes was achieved by epimerization and transesterification with lithium (-)-mentholate. These structures are attractive cores for the construction of macromolecules in which asymmetry is induced by the center of the molecule. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.

2005 - Development and exploitation of CK2 inhibitors [Articolo su rivista]
Sarno, Stefania; Ruzzene, Maria; Frascella, Pietrogiulio; Pagano, Mario A.; Meggio, Flavio; Zambon, Alfonso; Mazzorana, Marco; Di Maira, Giovanni; Lucchini, Vittorio; Pinna, Lorenzo A.
abstract

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (Ki = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H- benzimidazole, DMAT (Ki = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (Ki = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (Ki = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs. © Springer Science + Business Media, Inc. 2005.

2005 - Study on the toxicity of phenolic and phenoxy herbicides using the submitochondrial particle assay [Articolo su rivista]
Argese, E; Bettiol, C.; Marchetto, D.; De Vettori, S.; Zambon, Alfonso; Miana, P.; Ghetti, P. F.
abstract

A simple and rapid in vitro toxicological assay, utilizing submitochondrial particles (SMP), has been used to evaluate the toxic effects of fifteen herbicides belonging to the phenol and phenoxyalkanoic acid chemical classes. The SMP assay allows the quantitative evaluation of the toxicity of compounds with different mechanisms of action: uncouplers, inhibitors of the enzyme complexes involved in reverse electron transfer and in oxidative phosphorylation and chemicals that alter the membrane structure. The two groups of herbicides showed different levels of toxicity. For phenol derivatives, EC50 values ranged from 0.16 μM (ioxynil) to 6.7 μM (2,4-dinitrophenol), whereas for phenoxy herbicides EC50 values ranged from 21 μM (2,4,5-trichlorophenoxyacetic acid, 2,4,5-T) to 110 μM (4-chloro-2- methylphenoxyacetic acid, MCPA). On the average, the toxicity of phenolic compounds is greater than that of phenoxyalkanoic acids by two orders of magnitude. Quantitative structure-activity relationships (QSAR) were developed between EC50 values and various molecular descriptors. The results suggest the existence of different mechanisms of action for the two classes of compounds. The findings obtained for phenolic herbicides are consistent with a protonophoric uncoupling mechanism, whereas for phenoxy herbicides a non-specific mode of action at membrane level can be hypothesized. © 2005 Elsevier Ltd. All rights reserved.

2005 - Tris-annelated benzenes selectively perfunctionalized on one side only: Hexachlorobenzotrinorbornadiene as a versatile scaffold for the construction of molecular domes [Articolo su rivista]
Borsato, Giuseppe; Brussolo, Stefania; Crisma, Marco; De Lucchi, Ottorino; Lucchini, Vittorio; Zambon, Alfonso
abstract

Well-defined, rigid molecular domes are obtained via substitution of the chlorine atoms of hexachlorobenzotrinorbornadiene syn-3, efficiently synthesized in 1:2 ratio along with its antiisomer by CuTC-promoted cyclotrimerization of 3-bromo-2-tri-methylstannylnorbornadiene 8. The three dichlorovinyl functions at the edge of syn-3 are displaced by sulfur nucleophiles and Grignard reagents with Ni(II)Cl2dppe as catalyst, gaining the persubstituted vinyl sulfides 9 and 10, and persubstituted methyl 12 and phenylethynyl 13.

2004 - Internal motions in a fulleropyrrolidine tertiary amide with axial chirality [Articolo su rivista]
Borsato, Giuseppe; Della Negra, Federico; Gasparrini, Francesco; Misiti, Domenico; Lucchini, Vittorio; Possamai, Giorgia; Villani, Claudio; Zambon, Alfonso
abstract

The kinetic parameters for topomerization around the N-CO bond and enantiomerization around the C-CO bond in N-1-naphthoyl fulleropyrrolidine 1 and N-1-naphthoyl pyrrolidine 2 have been determined by dynamic NMR (line shape simulation and selective inversion transfer). The ΔS ≠ values are negligible. The ΔH # value for topomerization of 1 is smaller with respect to that of 2 by 4.3 kcal mol -1 (explained by the electron-withdrawing effect of fullerene) and the value for enantiomerization is greater by 1.4 kcal mol -1 (explained by the greater rigidity of the fulleropyrrolidine ring, as confirmed by ab initio analyses).

2003 - Nucleophilic reactions at the ring carbons of thiiranium and thiirenium ions. An experimental and theoretical comparison of the S(N)2 and S(N)2-Vin mechanisms [Articolo su rivista]
Borsato, G; Lucchini, V; Modena, G; Pasquato, L; Zambon, Alfonso
abstract

Generally, the bimolecular nucleophilic substitution at the saturated carbon (S(N)2) and the mechanistically similar substitution at the vinyl carbon (S(N)2-Vin) cannot be quantitatively compared, because of the many interfering steric and electronic factors. The cis and trans rearrangements of trans c-2, t-3-di-tert-butyl-r-1-methylthiiranium ion 5a into thietanium ions 6a and 6b and that of 2,3-di-tert-butyl-1-methylthiirenium ion 7 into thietium ion 8 can be be compared both experimentally and computationally. They occur with intramolecular S(N)2 and S(N)2-Vin mechanisms respectively and are both almost exclusively governed by the nucleofugality of the sulfonium leaving group.

2003 - Synthesis and evaluation of new chiral diols based on the dicyclopentadiene skeleton [Articolo su rivista]
Borsato, G.; De Lucchi, O; Fabris, F.; Lucchini, V.; Frascella, P.; Zambon, Alfonso
abstract

The resolution by Lipase PS of rac-5 (from reduction of ketone 6, obtained from dicyclopentadiene with a new environment-friendly synthesis) gives (2S)-5, which was further reduced to the endo (2R)-1a alcohol. The endo (2S)-1b alcohol was obtained from camphor with a multistep synthesis. Pinacol couplings of 3a,b, carried out with Mg/Hg or Corey's general procedure respectively, afforded with high diastereoselectivity the C2 symmetry diols (2R,2′R)-2a and (2S,2′S)-2b, with endo oriented OH functions. The enantiogenic power of the endo alcohol (2R)-1a and (2S)-1b and of the diols (2R,2′R)-2a and (2S,2′S)-2b was tested towards the LiAlH4 reduction of acetophenone. The C2 symmetry appears to play a fundamental role.

2003 - Synthesis of the syn and anti isomer of 1,4,5,8,9,12-hexahydro-2,3,6,7,10,11-hexamethylidene-1,4 : 5,8 : 9,12-trimethanotriphenylene and Diels-Alder reactivity of the syn isomer [Articolo su rivista]
Borsato, G.; De, Lucchi; Fabris, O.; Lucchini, F.; Pasqualotti, V.; Zambon, Alfonso
abstract

The title compounds, new members in the class of tris-annelated benzenes with [2.2.1] bicyclic rings, were obtained in high yields by coupling of 2-bromo-3-trimethyltin-5,6-dimethylenenorborn-2-ene with copper(I) 2-thiophenecarboxylate (CuTC). The syn isomer was reacted with 3 equiv. of the dienophiles TCNE, DMAD, PTAD and norbornadiene to afford the corresponding cycloadducts.

2002 - A novel mechanism for the fluxional behaviour of [Pd(η2-tetramethylethylenetetracarboxylate)(2-methylthiomethylpyridine)] [Articolo su rivista]
Canovese, Luciano; Lucchini, Vittorio; Santo, Claudio; Visentin, Fabiano; Zambon, Alfonso
abstract

The fluxional behaviour of [Pd(η2-tmetc)(NSMe)] (tmetc=tetramethylethylenetetracarboxylate, NSMe=2-methylthiomethylpyridine) in CD2Cl2 is governed by two mechanisms: (i) the concentration independent inversion at sulfur which averages the methylenic signals and collapses the four methyl signals of tmetc to two; (ii) the concentration dependent mechanism occurring via a dimeric intermediate with CS symmetry which collapses the two residual tmetc signals into a singlet. The fluxional behaviour of [Pd(η2-tmetc)(NSMe)] (tmetc=tetramethylethylenetetracarboxylate, NSMe=2-methylthiomethylpyridine) in CD2Cl2 is governed by two mechanisms: (1) the concentration independent inversion at sulfur; and (2) the concentration dependent mechanism occurring via a dimeric intermediate with CS symmetry.

2002 - Efficient cyclotrimerization of bicyclic vic-bromostannylalkenes promoted by copper(I) thiophen-2-carboxylate [Articolo su rivista]
Borsato, Giuseppe; De Lucchi, Ottorino; Fabris, Fabrizio; Groppo, Luca; Lucchini, Vittorio; Zambon, Alfonso
abstract

Copper(I) thiophen-2-carboxylate was successfully employed in the trimerization of [2.2.1] bicyclic vicbromotrimethyltin olefins (in their racemic composition), bearing different functionalities, to invariably obtain almost quantitative yields of the syn and anti tris-annelated benzenes. The two isomers come in different ratios, smaller than or equal to the statistical 1:3 ratio, depending on the steric hindrance opposed by the functionalities. In the case of enantiopure (3-bromo-4,7,7-trimethylbicyclo[2.2.1]hept-2- en-2-yl)trimethylstannane, the 1:9 ratio found with Cu(NO3)2·3H2O increases to 1:6.

2002 - Substituted aniline interaction with submitochondrial particles and quantitative structure-activity relationships [Articolo su rivista]
Argese, Emanuele; Bettiol, Cinzia; Fasolo, Matteo; Zambon, Alfonso; Agnoli, Francesca
abstract

The toxic effects of eighteen substituted anilines were determined by means of a short-term in vitro assay, using submitochondrial particles (SMP) as biosensors. The assay allows for the quantification of the effects of toxicants that act specifically on mitochondrial respiratory functions, like uncouplers and inhibitors, or non-specifically, by disturbing the structure and functioning of the inner mitochondrial membrane. The obtained EC50 values range from 72.5 to 1910 μmol/l. The type and position of the substituents are of fundamental importance in determining the toxic potency. In general, the presence of electron-withdrawing substituents produces higher toxic effects, whereas electron-donating groups seem to reduce the toxicity. Quantitative structure-activity relationships (QSAR) showed that toxicity values were correlated with the Hammett σ constant and with hydrogen bonding capacity descriptors, such as ELUMO, EHOMO and Q+. The results indicate that toxicity increases with increasing the hydrogen bonding donor capacity of the NH2 group and support the hypothesis of a mechanism of action based on hydrogen bonding formation between the amino group of anilines and polar groups at the membrane/water interface. Such an interaction would cause a derangement of the membrane structure and, as a consequence, a disturbance of its functioning. © 2002 Elsevier Science B.V. All rights reserved.

2001 - Assessment of chloroaniline toxicity by the submitochondrial particle assay [Articolo su rivista]
Argese, E; Bettiol, C; Agnoli, F; Zambon, Alfonso; Mazzola, M; Ghirardini, Av
abstract

The effects on mitochondrial respiration of 15 chloroanilines were recorded by using the in vitro response of submitochondrial particles (SMP) from beef heart mitochondria. The bioassay procedure for SMP is based on the process of reverse electron transfer, which can be negatively affected by inhibitors of electron transport, by uncouplers, and by chemicals that impair membrane integrity. The EC50 values, determined for the tested chloroanilines, indicate a general tendency of increasing toxicity with increasing chlorine substitution. In order to validate the results obtained and to evaluate the capability of the SMP assay to reproduce the toxic effects of the examined compounds on different freshwater species, the EC50 values were compared with literature data from other biological assays regarding both in vitro systems and whole organisms. A good correlation was found in particular with two widely used testing systems, the Microtox((R)) and the Tetrahymena assays. In addition, quantitative structure-activity relationships (QSARs) were established between the EC50 values and various molecular descriptors for hydrophobic, steric, and electronic interactions. The results obtained were utilized to elucidate the mechanism of toxic action of chloroanilines, which are commonly reported to act by the polar narcosis mode of action. Moreover, they confirmed that the SMP assay can be a useful tool for studying the toxicity of chemicals that act nonspecifically by impairing membrane structure and functions.

2001 - Isolation and characterization of all eight bisadducts of fulleropyrrolidine derivatives [Articolo su rivista]
Kordatos, K.; Bosi, S.; Da Ros, T.; Zambon, Alfonso; Lucchini, V.; Prato, M.
abstract

We report the isolation and characterization of bisadducts of fulleropyrrolidine derivatives. The compounds were characterized by means of a variety of spectroscopic techniques, including ESMS, UV-vis, 1H NMR, and 13C NMR. The whole series of bisadducts was separated for the first time in the case of the bispyrrolidines, and the determination of their structure was obtained by NMR spectroscopy with the help of HMQC and HMBC techniques.

2000 - Surface acidity modifications induced by thermal treatments and acid leaching on microcrystalline H-BEA zeolite. A FTIR, XRD and MAS-NMR study [Articolo su rivista]
Trombetta, M.; Busca, ; Storaro, G.; Lenarda, L.; Casagrande, M.; Zambon, Alfonso
abstract

Samples of beta-zeolite thermally treated at di†erent temperatures and acid leached with diluted hydrochloric acid solution have been investigated by XRD, 27Al MAS-NMR spectroscopy and FTIR spectroscopy. The results have been used to interpret the behaviour of the samples as catalysts for the acylation of 2-methoxynaphthalene. It has been shown that the as prepared sample presents two types of extraframework species. These are identiÐed as Al hydroxo-ions highly dispersed in the internal zeolite channels and Al oxide nanoparticles. Calcination causes dealumination of the framework and progressive conversion of the Al hydroxo-ions into Al oxide nanoparticles that reduce the zeolite channels practicability, modifying the shape selectivity e†ect. The acid sites present in the sinusoidal channels cm BrÔnsted (l ~1) can be OH \ 3608 distinguished from those located in the larger ones cm (l ~1), due to the inability of the bulky OH \ 3620È3612 probe molecule pivalonitrile to enter the former. However, internal terminal silanols cm (l ~1) also OH \ 3735 apparently display a signiÐcant acidity, deÐnitely higher than the acidity of those absorbing at 3747 BrÔnsted cm~1, thought to be located at the external crystal surface. The strongest Lewis acidity is displayed by the aluminum hydroxo-ions, while that of alumina nanoparticles is a little weaker. Acid-leached beta-zeolite also displays a medium strength Lewis acidity, likely due to framework Al cations. Thus a partial reinterpretation of the real structure of beta-zeolite and a partial reassignment of the bands due to the surface hydroxy groups are proposed.