Vanadium complexes with hydrazone or thiosemicarbazone ligands as potential anti-mycobacterium tuberculosis agents

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Characterization of Primary Amine End-Functionalized Polystyrene and Poly(methyl methacrylate) Synthesized by Living Anionic Polymerization Techniques

The reaction of living anionic polymers with 2,2,5,5-tetramethyl-1-(3-bromopropyl)-1-aza-2,5- disilacyclopentane (1) was investigated using coupled thin layer chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of byproducts as well as the major product were determined. The anionic initiator having a protected primary amine functional group, 2,2,5,5-tetramethyl- 1-(3-lithiopropyl)-1-aza-2,5-disilacyclopentane (2), was synthesized using all-glass high-vacuum techniques, which allows the long-term stability of this initiator to be maintained. The use of 2 in the preparation of well-defined aliphatic primary amine R-end-functionalized polystyrene and poly(methyl methacrylate) was investigated. Primary amino R-end-functionalized poly(methyl methacrylate) can be obtained near-quantitatively by reacting 2 with 1,1-diphenylethylene in tetrahydrofuran at room temperature prior to polymerizing methyl methacrylate at -78 °C. When 2 is used to initiate styrene at room temperature in benzene, an additive such as N,N,N',N'- tetramethylethylenediamine is necessary to activate the polymerization. However, although the resulting polymers have narrow molecular weight distributions and well-controlled molecular weights, our mass spectra data suggest that the yield of primary amine α-end-functionalized polystyrene from these syntheses is very low. The majority of the products are methyl α-end-functionalized polystyrene.

I. Development of the In Situ Reductive Ozonolysis of Alkenes with Tertiary Amine N-Oxides. II. Progress toward the Asymmetric Synthesis of Peroxyplakoric Acid A3.

Ozone, first discovered in the mid 1800’s, is a triatomic allotrope of oxygen that is a powerful oxidant. For over a century, research has been conducted into the synthetic application and mechanism of reactions of ozone with organic compounds. One of the major areas of interest has been the ozonolysis of alkenes. The production of carbonyl compounds is the most common synthetic application of ozonolysis. The generally accepted mechanism developed by Rudolf Criegee for this reaction involves the 1,3-electrocyclic addition of ozone to the π bond of the alkene to form a 1,2,3-trioxolane or primary ozonide. The primary ozonide is unstable at temperatures above -100 °C and undergoes cycloreversion to produce the carbonyl oxide and carbonyl intermediates. These intermediates then recombine in another 1,3-electrocyclic addition step to form the 1,2,4-trioxolane or final ozonide. While the final ozonide is often isolable, most synthetic applications of ozonolysis require a subsequent reductive or oxidative step to form the desired carbonyl compound. During investigations into the nucleophilic trapping of the reactive carbonyl oxide, it was discovered that when amines were used as additives, an increased amount of reaction time was required in order to consume all of the starting material. Surprisingly, significant amounts of aldehydes and a suppression of ozonide formation also occurred which led to the discovery that amine N-oxides formed by the ozonation of the amine additives in the reaction were intercepting the carbonyl oxide. From the observed production of aldehydes, our proposed mechanism for the in situ reductive ozonolysis reaction with amine N-oxides involves the nucleophilic trapping of the carbonyl oxide intermediate to produce a zwitterionic adduct that fragments into 1O2, amine and the carbonyl thereby avoiding the formation of peroxidic intermediates. With the successful total syntheses of peroxyacarnoates A and D by Dr. Chunping Xu, the asymmetric total synthesis of peroxyplakorate A3 was investigated. The peroxyplakoric acids are cyclic peroxide natural products isolated from the Plakortis species of marine sponge that have been found to exhibit activity against malaria, cancer and fungi. Even though the peroxyplakorates differ from the peroxyacarnoates in the polyunsaturated tail and the head group, the lessons learned from the syntheses of the peroxyacarnoates have proven to be valuable in the asymmetric synthesis of peroxyplakorate A3. The challenges for the asymmetric synthesis of peroxyplakorate A3 include the stereospecific formation of the 3-methoxy-1,2-dioxane core with a propionate head group and the introduction of oxidation sensitive dienyl tail in the presence of a reduction sensitive 1,2-dioxane core. It was found that the stereochemistry of two of the chiral centers could be controlled by an anti-aldol reaction of a chiral propionate followed by the stereospecific intramolecular cyclization of a hydroperoxyacetal. The regioselective ozonolysis of a 1,2-disubstituted alkene in the presence of a terminal alkyne forms the required hydroperoxyacetal as a mixture of diastereomers. Finally, the dienyl tail is introduced by a hydrometallation/iodination of the alkyne to produce a vinyl iodide followed by a palladium catalyzed coupling reaction. While the coupling reaction was unsuccessful in these attempts, it is still believed that the intramolecular cyclization to introduce the 1,2-dioxane core could prove to be a general solution to many other cyclic peroxides natural products.

Capillary bioreactors based on human purine nucleoside phosphorylase: A new approach for ligands identification and characterization

The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The Km value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 +/- 29.2 mu M and 133 +/- 114.9 mu M, respectively). A new fourth-generation immucillin derivative (DI4G: IC50 = 40.6 +/- 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. (C) 2011 Elsevier B.V. All rights reserved.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

Advanced QSAR Studies on PPAR delta Ligands Related to Metabolic Diseases

PPAR delta is a nuclear receptor that, when activated, regulates the metabolism of carbohydrates and lipids and is related to metabolic syndrome and type 2 diabetes. To understand the main interactions between ligands and PPAR delta, we have constructed 2D and 3D QSAR models and compared them with HOMO, LUMO and electrostatic potential maps of the compounds studied, as well as docking results. All QSAR models showed good statistical parameters and prediction outcomes. The QSAR models were used to predict the biological activity of an external test set, and the predicted values are in good agreement with the experimental results. Furthermore, we employed all maps to evaluate the possible interactions between the ligands and PPAR delta. These predictive QSAR models, along with the HOMO, LUMO and MEP maps, can provide insights into the structural and chemical properties that are needed in the design of new PPAR delta ligands that have improved biological activity and can be employed to treat metabolic diseases.

Electrochromic Properties of a Metallo-supramolecular Polymer Derived from Tetra(2-pyridyl-1,4-pyrazine) Ligands Integrated in Thin Multilayer Films

The electrochromic behavior of iron complexes derived from tetra-2-pyridyl-1,4-pyrazine (TPPZ) and a hexacyanoferrate species in polyelectrolytic multilayer adsorbed films is described for the first time. This complex macromolecule was deposited onto indium-tin oxide (ITO) substrates via self-assembly, and the morphology of the modified electrodes was studied using atomic force microscopy (AFM), which indicated that the hybrid film containing the polyelectrolyte multilayer and the iron complex was highly homogeneous and was approximately 50 nm thick. The modified electrodes exhibited excellent electrochromic behavior with both intense and persistent coloration as well as a chromatic contrast of approximately 70%. In addition, this system achieved high electrochromic efficiency (over 70 cm(2) C-1 at 630 nm) and a response time that could be measured in milliseconds. The electrode was cycled more than 10(3) times, indicating excellent stability.

Preparation, characterization and structure of ruthenium phosphine complexes containing non-innocent ligands

Phosphine ruthenate complexes containing the non-innocent ligands 4-chloro-1,2-phenylenediamine (opda-CI) and 3,3',4,4'-tetraamminebiphenyl (diopda) were synthesized and characterized by means of X-ray diffraction, electrochemistry, P-31{H-1} NMR and electronic spectroscopies. Crystals of cis-[RuCl2 (dppb)(bqdi-CI)] complex were isolated as a mixture of two conformational isomers due to different positions of the chlorine atoms of the o-phenylene ligand in relation to the P1 atom of the phosphine moiety. (C) 2011 Elsevier Ltd. All rights reserved.

Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling

The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. (C) 2011 Elsevier Inc. All rights reserved.

Tailored norbornene-based copolymer with systematic variation of norbornadiene as a crosslinker obtained via ROMP with alternative amine Ru catalysts

Copolymers of norbornene (NBE) with norbomadiene (NBD) were obtained via ROMP with [RuCl2(PPh3)(2)(L)] type complexes as initiators (1 for L = piperidine and 2 for L = 3,5-Me(2)piperidine). The reactions were performed using a fixed quantity of NBE (5000 equivalents/[Ru]) for different concentrations of NBD (500, 1000, 1500 and 2000 equivalents/ [Ru]) in CHCl3, initiated with ethyl diazoacetate at room temperature. The presence of NBD in the NBE chains was characterized by H-1 and C-13 NMR. Whereas the copolymer microstructure was influenced neither by the NBD quantity nor by the initiator type, the N-n and PDI values were improved when increasing the NBD quantity in the medium. When raising the NBD amount, DMA results indicated increased cross-linking with increasing T-g and E ' storage modulus, as well as the fact that SEM micrographs indicated decreased pore sizes in the porous isolated copolymers. (C) 2011 Elsevier Ltd. All rights reserved.

Neutral Gold Complexes with Tridentate SNS Thiosemicarbazide Ligands

Na[AuCl4].2H(2)O reacts with tridentate thiosemicarbazide ligands, H(2)L1, derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chloride and thiosemicarbazides under formation of air-stable, green [AuCl(L1)] complexes. The organic ligands coordinate in a planar SNS coordination mode. Small amounts of gold(I) complexes of the composition [AuCl(L3)] are formed as side-products, where L3 is an S-bonded 5-diethylamino-3-phenyl-1-thiocarbamoyl-1,2,4-triazole. The formation of the triazole L3 can be explained by the oxidation of H(2)L1 to an intermediate thiatriazine L2 by Au3+, followed by a desulfurization reaction with ring contraction. The chloro ligands in the [AuCl(L1)] complexes can readily be replaced by other monoanionic ligands such as SCN- or CN- giving [Au(SCN)(L1)] or [Au(CN)(L1)] complexes. The complexes described in this paper represent the first examples of fully characterized neutral Gold(III) thiosemicarbazone complexes. All the [AuCl(L1)] compounds present a remarkable cell growth inhibition against human MCF-7 breast cancer cells. However, systematic variation of the alkyl groups in the N(4)-position of the thiosemicarbazone building blocks as well as the replacement of the chloride by thiocyanate ligands do not considerably influence the biological activity. On the other hand, the reduction of Au-III to Au-I leads to a considerable decrease of the cytotoxicity.

Organocatalytic asymmetric mannich-type reactions: an easy approach to optically active amine derivatives

The topics I came across during the period I spent as a Ph.D. student are mainly two. The first concerns new organocatalytic protocols for Mannich-type reactions mediated by Cinchona alkaloids derivatives (Scheme I, left); the second topic, instead, regards the study of a new approach towards the enantioselective total synthesis of Aspirochlorine, a potent gliotoxin that recent studies indicate as a highly selective and active agent against fungi (Scheme I, right). At the beginning of 2005 I had the chance to join the group of Prof. Alfredo Ricci at the Department of Organic Chemistry of the University of Bologna, starting my PhD studies. During the first period I started to study a new homogeneous organocatalytic aza-Henry reaction by means of Cinchona alkaloid derivatives as chiral base catalysts with good results. Soon after we introduced a new protocol which allowed the in situ synthesis of N-carbamoyl imines, scarcely stable, moisture sensitive compounds. For this purpose we used α-amido sulfones, bench stable white crystalline solids, as imine precursors (Scheme II). In particular we were able to obtain the aza-Henry adducts, by using chiral phase transfer catalysis, with a broad range of substituents as R-group and excellent results, unprecedented for Mannich-type transformations (Scheme II). With the optimised protocol in hand we have extended the methodology to the other Mannich-type reactions. We applied the new method to the Mannich, Strecker and Pudovik (hydrophosphonylation of imines) reactions with very good results in terms of enantioselections and yields, broadening the usefulness of this novel protocol. The Mannich reaction was certainly the most extensively studied work in this thesis (Scheme III). Initially we developed the reaction with α-amido sulfones as imine precursors and non-commercially available malonates with excellent results in terms of yields and enantioselections.3 In this particular case we recorded 1 mol% of catalyst loading, very low for organocatalytic processes. Then we thought to develop a new Mannich reaction by using simpler malonates, such as dimethyl malonate.4 With new optimised condition the reaction provided slightly lower enantioselections than the previous protocol, but the Mannich adducts were very versatile for the obtainment of β3-amino acids. Furthermore we performed the first addition of cyclic β-ketoester to α-amido sulfones obtaining the corresponding products in good yield with high level of diastereomeric and enantiomeric excess (Scheme III). Further studies were done about the Strecker reaction mediated by Cinchona alkaloid phase-transfer quaternary ammonium salt derivatives, using acetone cyanohydrin, a relatively harmless cyanide source (Scheme IV). The reaction proceeded very well providing the corresponding α-amino nitriles in good yields and enantiomeric excesses. Finally, we developed two new complementary methodologies for the hydrophosphonylation of imines (Scheme V). As a result of the low stability of the products derived from aromatic imines, we performed the reactions in mild homogeneous basic condition by using quinine as a chiral base catalyst giving the α-aryl-α-amido phosphonic acid esters as products (Scheme V, top).6 On the other hand, we performed the addition of dialkyl phosphite to aliphatic imines by using chiral Cinchona alkaloid phase transfer quaternary ammonium salt derivatives using our methodology based on α-amido sulfones (Scheme V, bottom). The results were good for both procedures covering a broad range of α-amino phosphonic acid ester. During the second year Ph.D. studies, I spent six months in the group of Prof. Steven V. Ley, at the Department of Chemistry of the University of Cambridge, in United Kingdom. During this fruitful period I have been involved in a project concerning the enantioselective synthesis of Aspirochlorine. We provided a new route for the synthesis of a key intermediate, reducing the number of steps and increasing the overall yield. Then we introduced a new enantioselective spirocyclisation for the synthesis of a chiral building block for the completion of the synthesis (Scheme VI).

Transformations of bridging ligands in diiron complexes: unconventional routes to new functionalized multisite bound organic frames

The main scope of this Ph.D. thesis has concerned the possible transformations of bridging ligands in diiron complexes, in order to explore unconventional routes to the synthesis of new functionalized multisite bound organic frames. The results achieved during the Ph.D. can be summarized in the following points: 1) We have extended the assembling between small unsaturated molecules and bridging carbyne ligands in diiron complexes to other species. In particular, we have investigated the coupling between olefins and thiocarbyne, leading to the synthesis of thioallylidene bridging diiron complexes. Then, we have extended the study to the coupling between olefins and aminocarbyne. This result shows that the coupling between activated olefins and heteroatom substituted bridging carbynes has a general character. 2) As we have shown, the coupling of bridging alkylidyne ligands with alkynes and alkenes provides excellent routes to the synthesis of bridging C3 hydrocarbyl ligands. As a possible extension of these results we have examined the synthesis of C4 bridging frames through the combination of bridging alkylidynes with allenes. Also in this case the reaction has a general character. 3) Diiron complexes bearing bridging functionalized C3 organic frames display the presence of donor atoms, such as N and S, potentially able to coordinate unsaturated metal fragments. Thus, we have studied the possibility for these systems to act as ‘organometallic ligands’, in particular towards Pd and Rh. 4) The possibility of releasing the organic frame from the bridging coordination appears particularly appealing in the direction of a metal-assisted organic synthesis. Within this field, we have investigated the possibility of involving the C3 bridging ligand in cycloaddition reactions with alkynes, with the aim of generating variously functionalized five-membered cycles. The [3+2] cyclization does not lead to the complete release of the organic fragment but rather it produces its transformation into a cyclopentadienyl ring, which remains coordinated to one Fe atom. This result introduces a new approach to the formation of polyfunctionalised ferrocenes. 5) Furthermore, I have spent a research period of about six months at the Department of Inorganic Chemistry of the Barcelona University, under the supervision of Prof. Concepción López, with the aim of studying the chemistry of polydentate ferrocenyl ligands and their use in organometallic synthesis.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.