Laura Legnani

Mechanism of falcipain-2 inhibition by α,β-unsaturated benzo[1,4]diazepin-2-one methyl ester.

Falcipain-2 (FP-2) is a papain-family cysteine protease of Plasmodium falciparum whose primary function is to degrade the host red cell hemoglobin, within the food vacuole, in order to provide free amino acids for parasite protein synthesis. Additionally it promotes host cell rupture by cleaving the skeletal proteins of the erythrocyte membrane. Therefore, the inhibition of FP-2 represents a promising target in the search of novel anti-malarial drugs. A potent FP-2 inhibitor, characterized by the presence in its structure of the 1,4-benzodiazepine scaffold and an α,β-unsaturated methyl ester moiety capable to react with the Cys42 thiol group located in the active site of FP-2, has been recently reported in literature. In order to study in depth the inhibition mechanism triggered by this interesting compound, we carried out, through ONIOM hybrid calculations, a computational investigation of the processes occurring when the inhibitor targets the enzyme and eventually leads to an irreversible covalent Michael adduct. Each step of the reaction mechanism has been accurately characterized and a detailed description of each possible intermediate and transition state along the pathway has been reported.

Stable GM3 lactone mimetic raises antibodies specific for the antigens expressed on melanoma cells.

Immunotherapy of tumors and of melanoma in particular has a long history, and recently this therapeutic approach found a reliable scientific rationale. This biological therapy aims to teach the patients immune system to recognize the antigens expressed on tumor cells and destroy them, leaving normal cells intact. The success of this therapy highly depends on the selection of target antigens that are essential for tumors growth and progression. The overexpression of GM(3) ganglioside 1 and especially the expression of its metabolite GM(3) lactone 2 characterize murine and human melanomas, playing an important role in tumor progression and making such self-antigens potential targets for the immunotherapy of these neoplasms. Although more immunogenic than its precursor, GM(3) lactone 2 is unsuitable to be used in immunotherapy as a melanoma-associated antigen (MAA) because it is unstable under physiological conditions. We designed and synthesized the hydrolytically stable mimetic 3, which is remarkably simpler than the native lactone 2; after conjugation of 3 to the protein carrier keyhole-limpet hemocyanin (KLH), the obtained glycoprotein 5 was used as the immunogen in vivo to successfully elicit specific antimelanoma antibodies. In fact, no appreciable binding to GM(1) was observed. Capitalizing on the stability and on the reduced structural complexity of mimetic 3, the immunostimulant 5 we report represents a new promising synthetic glycoconjugate for the immunotherapy of melanoma.

Synthesis, Conformational Studies, Binding Assessment and Liposome Insertion of a Thioether-Bridged Mimetic of the Antigen GM3-Ganglioside Lactone.

Oncogenic transformations are often associated with abnormal glycosylation in human tumours. In recent years a variety of monoclonal antibodies have been developed to specifically recognize carbohydrate epitopes that result from these faulty glycosylation processes. These epitopes, known as tumourassociated antigens (TAAs), have been successfully used as markers of tumour progression, and include catbohyrates expressed on normal tissues, but which are accumulated in high density on the surface of tumour cells. GM3 Ganglioside, a glycosphingolipid found in essentially all types of cells and tissues, is over expressed in melanoma cells with metastatic potential. The corresponding GM3 lactone (1a, Scheme 1) has also been found in melanoma as a minor component; its formation is likely promoted by the lower pH environment of tumour cells and, possibly, by a different conformation of GM3 ganglioside induced on the tumour cell surface as a result of localised high density. In the development of a biological therapy for tumours, GM3 ganglioside and GM3 lactone 1a have been extensively investigated as potential vaccines against cancer. Immunization experiments have indicated that GM3 lactone is more immunogenic than GM3 itself ; however, under physiological conditions, the equilibrium amount of lactone is below the recognition threshold. In addition, GM3 lactone 1a and its analogue 1b are both potent inducers of antimelanoma cytotoxic T cell (CTL) response. These data suggest that the antigenic ACHTUNGTRENNUNGdeterminant of 1a might not include the acetamido function, and that the folded shape, which is characteristic of a lactone ring-containing structure, might be responsible for the enhanced immunogenicity of 1a compared to GM3 ganglioACHTUNGTRENNUNGside. A few years ago two hydrolytically stable analogues of GM3 lactone, namely a GM3 lactam and a GM3 ether, were prepared but they have to date not been used for anticancer therapy. The development of mimetics of 1a that are resistant to hydrolysis remains a challenging target in the immunotherapy domain. Here, we report the conformational analysis and synthesis of the spiro glycoside 2 (Scheme 1), a thioether-bridged Figure 1. Three-dimensional plot of the preferred conformations of compounds 1a and 2 separately and superimposed; the hydrogen atoms are omitted in the superposition for clarity.

Modeling of synthetic phosphono and carba analogues of N-acetyl-α-D-mannosamine 1-phosphate, the repeating unit of the capsular polysaccharide from Neisseria meningitidis serovar A

The conformational behavior of methyl (2-acetamido-2-deoxy-alpha-d-mannopyranosyl)phosphate 1, and its analogues, methyl C-(2-acetamido-2-deoxy-alpha-d-mannopyranosyl)methanephosphonate 2 and methyl O-(2-acetamido-2-deoxy-5a-carba-alpha-d-mannopyranosyl)phosphate , where a methylene group replaces, respectively, the anomeric and the pyranose oxygen atom, was investigated at the B3LYP/6-311+G(d,p) level [6-311+G(2df,p) for the phosphorus atom]. The energy of the optimized structures was recalculated using the continuum solvent model C-PCM choosing water as the solvent. The compounds exhibited several populated conformations, but they all showed a marked preference for the (4)C(1) geometry of the pyranose ring; this preference was almost complete for 1, very large for the phosphono analogue 2, and large for the carba analogue 3. To give experimental support to these results, compounds 2 and 3 were synthesized and characterized by NMR spectroscopy. The comparison of the theoretical and experimental vicinal coupling constants confirmed the marked preference for the (4)C(1) geometry in the case of 2 and suggested that the same holds true for compound 3.

Computational Mechanistic Study of Thionation of Carbonyl Compounds with Lawesson’s Reagent

The thionation reaction of carbonyl compounds with Lawessons reagent (LR) has been studied using density functional theory methods and topological analyses. After dissociation of LR, the reaction takes place through a two-step mechanism involving (i) a concerted cycloaddition between one monomer and the carbonyl compound to form a four-membered intermediate and (ii) a cycloreversion leading to the thiocarbonyl derivative and phenyl(thioxo)phosphine oxide. Topological analyses confirmed the concertedness and asynchronicity of the process. The second step is the rate-limiting one, and the whole process resembles the currently accepted mechanism for the lithium salt-free Wittig reaction. No zwitterionic intermediates are formed during the reaction, although stabilizing electrostatic interactions are present in initial stages. Phenyl(thioxo)phosphine oxide formed in the thionation reaction is capable of performing a second thionation, although with energy barriers higher than the first one. The driving force of the thionation reactions is the formation of trimers from the resulting monomers. In agreement with experimental observations, the amides are the most reactive when compared with esters, aldehydes, and ketones and the reaction is slightly influenced by the polarity of the solvent. Whereas for amides and esters substituents have little effect, aldehydes and ketones are influenced by both steric and electronic effects.

Enantioselective Synthesis and Olfactory Evaluation of Bicyclic α- and γ-Ionone Derivatives: The 3D Arrangement of Key Molecular Features Relevant to the Violet Odor of Ionones

Violet smelling ionones 1-3, occurring in the headspace of different flowers, are well-known perfumery raw materials. With the goal to recognize the still ill-defined spatial arrangement of structural features relevant to the binding of ionones to olfactory G-protein coupled receptors, through B3LYP/6-31G(d) modeling studies we identified bicyclic compounds 7-9 as conformationally constrained 13-alkyl-substituted analogues of monocyclic alpha- and gamma-ionones. They were thus synthesized to evaluate the olfactory properties. The enantioselective syntheses of 7-9 entailed two common key steps: (i) a Diels-Alder reaction to construct the octalinic core and (ii) a Julia-Lythgoe olefination to install the alpha,beta-enone side chain. The odor thresholds of synthetic 7 and 9 were significantly lower than the corresponding parent ionones, and 9 showed the lowest threshold value among violet-smelling odorants examined so far. Modeling studies suggested a nearly identical spatial orientation of key hydrophobic and polar moieties of compounds 1, 3, and 4-9. Presumably, interaction of these moieties with ionone olfactory receptors (ORs) triggers a similar receptor code that is ultimately interpreted by the human brain as a pleasant woody-violet smell. These results open the way to studies aimed at identifying and modeling complementary binding sites on alpha-helical domains of ionone receptor proteins.

Synthesis, structure–activity relationships and stereochemical investigations of new tricyclic pyridazinone derivatives as potential STAT3 inhibitors

Through a cell-based biological screening, the benzocinnolinone derivative (±)-2c was identified as a promising STAT3 inhibitor. Since SAR studies on a series of compounds structurally related to (±)-2c (1c, 2a–p, 3c, 4c, 6) showed that the latter had the most significant inhibitory activity, we investigated in depth its essential structural features. In particular, enantiomeric separation was performed, and the absolute configuration of the stereoisomers was assigned by theoretical and crystallographic studies. The biological evaluation highlighted that (S)-(−)-2c is twice as potent as (R)-(+)-2c.

Synthesis of 3,6-diazabicyclo[3.1.1]heptanes as novel ligands for neuronal nicotinic acetylcholine receptors.

Alpha series of novel 3,6-diazabicyclo[3.1.1]heptane derivatives 4a-f was synthesized and their affinity and selectivity towards alpha4beta2 and alpha7 nAChR subtypes were evaluated. The results of the current study revealed a number of compounds (4a, 4b and 4c) having a very high affinity for alpha4beta2 (K(i) at alpha4beta2 ranging from 0.023 to 0.056 nM) versus alpha7 nAChR subtypes; among these compounds, the 3-(6-bromopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptane 4c was found to be the most alpha7alpha4beta2 selective term in receptor binding assays (alpha7alpha4beta2=1295). Moreover, compound 4d also had high affinity for the alpha4beta2 nAChR subtype (K(i)=1.2 nM) with considerably high selectivity (alpha7/alpha4beta2=23300).

Computational Mechanistic Study of the Julia–Kocieński Reaction

This paper describes the first detailed computational mechanistic study of the Julia-Kocieński olefination between acetaldehyde (1) and ethyl 1-phenyl-1H-tetrazol-5-yl sulfone (2), considered a paradigmatic example of the reaction between unsubstituted alkyl PT sulfones and linear aliphatic aldehydes. The theoretical study was performed within the density functional approach through calculations at the B3LYP/6-311+G(d,p) level for all atoms except sulfur for which the 6-311+G(2df,p) basis set was used. All the different intermediates and transition states encountered along the reaction pathways leading to final E and Z olefins have been located and the relative energies calculated, both for the reactions with potassium- and lithium-metalated sulfones, in THF and toluene, respectively. We have essentially confirmed the complex multistep mechanistic manifold proposed by others; however, the formation of a spirocyclic intermediate in the Smiles rearrangement was excluded. Instead, we found that this step involves a concerted, though asynchronous, mechanism. Moreover, our calculations nicely fit with the diastereoselectivities observed experimentally for potassium- and lithium-metalated sulfones, in THF and toluene, respectively.

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold.

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.