RESUMEN

The design of compounds able to combine the selective inhibition of cyclooxygenase-2 (COX-2) with the release of nitric oxide (NO) is a promising strategy to achieve potent anti-inflammatory agents endowed with an overall safer profile and reduced toxicity upon gastrointestinal and cardiovascular systems. With the aim of generating novel and selective COX-2 inhibiting NO-donors (CINOD) and encouraged by the promising results obtained with our nitrooxy- and hydroxyethyl ethers 11 and 12 reported in previous works, we shifted our attention on the synthesis of isosteric thioanalogs nitrooxy- and hydroxy ethyl sulfides 13a-c and 14a-c, respectively, along with their oxidation products nitrooxy- and hydroxyethyl sulfoxides 15a-c and 16a-c, respectively, also referred to as thio-CINOD. Preliminary data and metabolic analysis highlighted how the isosteric substitution of the ethereal oxygen atom of 11a-c with sulfur in compounds 13a-c, independently from the presence and the number of fluorine atoms in N1-phenyl ring, leads to new selective and highly potent COX-2 inhibitors, capable to induce vasorelaxant responses in vivo. The same behavior is observed with their oxidized counterparts nitrooxyethyl sulfoxides 15a-c, in which the oxidation state of the sulfur atom and the presence of the additional oxygen atom play a substantial role in enhancing compounds activity and vasorelaxation. In addition, the screened compounds proved significantly efficacious in mouse models of inflammation and nociception at the dose of 20 mg/kg.

Asunto(s)

Inhibidores de la Ciclooxigenasa 2 , Donantes de Óxido Nítrico , Analgésicos/farmacología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios no Esteroideos/farmacología , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Éteres , Ratones , Donantes de Óxido Nítrico/farmacología , Oxígeno , Pirroles/farmacología , Sulfuros , Sulfóxidos , Azufre , Vasodilatadores

RESUMEN

HBDI-like chromophores represent a novel set of biomimetic switches mimicking the fluorophore of the green fluorescent protein that are currently studied with the hope to expand the molecular switch/motor toolbox. However, until now members capable of absorbing visible light in their neutral (i. e. non-anionic) form have not been reported. In this contribution we report the preparation of an HBDI-like chromophore based on a 3-phenylbenzofulvene scaffold capable of absorbing blue light and photoisomerizing on the picosecond timescale. More specifically, we show that double-bond photoisomerization occurs in both the E-to-Z and Z-to-E directions and that these can be controlled by irradiating with blue and UV light, respectively. Finally, as a preliminary applicative result, we report the incorporation of the chromophore in an amphiphilic molecule and demonstrate the formation of a visible-light-sensitive nanoaggregated state in water.

Asunto(s)

Luz , Proteínas Fluorescentes Verdes/química

RESUMEN

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

Asunto(s)

Receptores de Ácido Retinoico/metabolismo , Rodopsina/metabolismo , Química Clic , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Receptores de Ácido Retinoico/química , Rodopsina/química , Estereoisomerismo

RESUMEN

The synthesis of new bis-deoxy-coelenterazine (1) derivatives bearing ester protective groups (acetate, propionate and butyrate esters) was accomplished. Moreover, their hydrolytic stability at room temperature was evaluated in dimethylsulfoxide (DMSO) as solvent, using the nuclear magnetic resonance (NMR) spectra of the key products at different time intervals. The results showed an increasing hydrolysis rate according to longest aliphatic chain, with a half-life of 24 days of the more stable acetate derivative (4a). Furthermore, the analysis of the experimental data revealed the greater stability of the enol tautomer in this aprotic polar solvent. This result was confirmed by theoretical calculations using the density functional theory (DFT) approach, which gave us the opportunity to propose a detailed decomposition mechanism. Additionally, the derivatives obtained were tested by bioluminescence luciferase assays to evaluate their potential use as extracellular pH-sensitive reporter substrates of luciferase. The biological data support the idea that further structural modifications of these molecules may open promising perspectives in this field of research.

Asunto(s)

Concentración de Iones de Hidrógeno , Imidazoles/química , Sondas Moleculares/química , Pirazinas/química , Semivida , Hidrólisis , Cinética , Espectroscopía de Resonancia Magnética/métodos , Termodinámica

RESUMEN

A series of 4-phenyl-6H-imidazo[1,5-a]thieno[3,2-f][1,4]diazepine-7-carboxylate esters were synthesized and tested as central benzodiazepine receptor (CBR) ligands by the ability to displace [3H]flumazenil from rat cortical membranes. All the compounds showed high affinity with IC50 values ranging from 5.19 to 16.22 nM. In particular, compounds 12b (IC50 = 8.66 nM) and 12d (IC50 = 5.19 nM) appeared as the most effective ligands being their affinity values significantly lower than that of diazepam (IC50 = 18.52 nM). Compounds 12a-f were examined in vivo for their pharmacological effects in mice and five potential benzodiazepine (BDZ) actions were thus taken into consideration: anxiolytic, anticonvulsant, anti-amnesic, hypnotic, and locomotor activities. All the new synthesized compounds were able to induce a significant antianxiety effect and, among them, compound 12f protected pentylenetetrazole (PTZ)-induced convulsions in a dose-dependent manner reaching a 40% effect at 30 mg/kg. In addition, all the compounds were able to significantly prevent the memory impairment evoked by scopolamine, while none of them was able to interfere with pentobarbital-evoked sleep and influence motor coordination. Moreover, title compounds did not affect locomotor and exploratory activity at the same time and doses at which the anti-anxiety effect was observed. Finally, molecular docking simulations were carried out in order to assess the binding mode for compounds 12a-f. The obtained results demonstrated that these compounds bind the BDZ binding site in a similar fashion to flumazenil.

Asunto(s)

Ansiolíticos/síntesis química , Benzodiazepinas/química , Diseño de Fármacos , Animales , Ansiolíticos/farmacología , Anticonvulsivantes , Benzodiazepinas/metabolismo , Sitios de Unión , Locomoción/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/prevención & control , Ratones , Simulación del Acoplamiento Molecular , Ratas , Receptores de GABA-A/metabolismo

RESUMEN

In this paper we report about the preparation, physicochemical and biological characterization of a magneto responsive nanostructured material based on magnetite nanoparticles (NP) coated with hyaluronic acid (HA). A synthetic approach, based on a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition "click" reaction between azido-functionalized magnetite NP and a derivative of hyaluronic acid bearing propargylated ferulic acid groups (HA-FA-Pg), was developed to link covalently the polymer layer to the magnetite NP. The functionalization steps of the magnetite NP and their coating with the HA-FA-Pg layer were monitored by Fourier Transform Infrared (FTIR) spectroscopy and Thermal Gravimetric Analysis (TGA) while Dynamic Light Scattering (DLS) and ζ-potential measurements were performed to characterize the aqueous dispersions of the HA-coated magnetite NP. Aggregation and sedimentation processes were investigated also by UV-visible spectroscopy and the dispersions of HA-coated magnetite NP were found significantly more stable than those of bare NP. Magnetization and zero field cooled/field cooled curves revealed that both bare and HA-coated magnetite NP are superparamagnetic at room temperature. Moreover, cytotoxicity studies showed that the coating with HA-FA-Pg significantly reduces the cytotoxicity of the magnetite NP providing the rational basis for the application of the HA-coated magnetite NP as healthcare material.

Asunto(s)

Ácido Hialurónico/química , Nanopartículas de Magnetita/química , Animales , Supervivencia Celular/efectos de los fármacos , Química Clic , Coloides/química , Ácidos Cumáricos/química , Nanopartículas de Magnetita/toxicidad , Ratones , Células 3T3 NIH , Polímeros/química

RESUMEN

In order to evaluate the potential of a technology platform based on hyaluronan copolymers grafted with propargylated ferulate fluorophores (HA-FA-Pg) in the development of drug delivery systems, the propargyl groups of HA-FA-Pg derivatives were employed with oleic acid (OA) or stearic acid (SA) residues across a biocompatible hexa(ethylene glycol) (HEG) spacer. The designed materials (i.e., HA-FA-HEG-OA or HA-FA-HEG-SA) showed clear-cut aggregation features in an aqueous environment, as confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), generating nanoaggregate systems. In fact, HA-FA-HEG-OA and HA-FA-HEG-SA derivatives showed the property to create self-assembled cytocompatible nanostructured aggregates in water, thanks to the simultaneous presence of hydrophilic portions in the polymeric backbone, such as hyaluronic acid, and hydrophobic portions in the side chains. Furthermore, the designed materials interact with living cells showing a high degree of cytocompatibility. The potential ability of nanosystems to load pharmacologically active molecules was assessed by the physical entrapment of olanzapine into both polymeric systems. The drug loading evaluation demonstrated that the nanoparticles are able to incorporate a good quantity of olanzapine, as well as improve drug solubility, release profile, and cytocompatibility.

RESUMEN

Cyclooxygenase-2 (COX-2) is involved in the inflammatory response, and its recurrent overexpression in cancers as well as in neurodegenerative disorders has made it an important target for therapy. For this reason, noninvasive imaging of COX-2 expression may represent an important diagnostic tool. In this work, a COX-2 inhibitor analogue, VA426 [1-(4-fluorophenyl)-3-(2-methoxyethyl)-2-methyl-5-(4-(methylsulfonil)phenyl)-1H-pyrrole], was synthesized and radiolabelled with the 11C radioisotope. The ex vivo biodistribution profile of 11C-VA426 was evaluated in the brain and periphery of healthy rats and mice and in brain and periphery of inflammation models, based on the administration of LPS. 11C-VA426 synthesis with the tBuOK base showed optimal radiochemical yield (15 ± 2%) based on triflate activity, molar activity (range 37-148 GBq/µmol), and radiochemical purity (>95%). Ex vivo biodistribution studies showed a fast uptake of radioactivity but a rapid washout, except in regions expressing COX-2 (lungs, liver, and kidney) both in rats and in mice, with maximum values at 30 and 10 minutes p.i., respectively. LPS administration did not show significant effect on radioactivity accumulation. Celecoxib competition experiments performed in rats and mice treated with LPS produced a general target unrelated reduction of radioactivity concentration in all peripheral tissues and brain areas examined. Finally, in agreement with the negative results obtained from biodistribution experiments, radiometabolites analysis revealed that 11C-VA426 is highly unstable in vivo. This study indicates that the compound 11C-VA426 is not currently suitable to be used as radiopharmaceutical for PET imaging. This family of compounds needs further implementation in order to improve in vivo stability.

Asunto(s)

Radioisótopos de Carbono , Ciclooxigenasa 2/análisis , Inhibidores de la Ciclooxigenasa , Marcaje Isotópico/métodos , Radiofármacos/síntesis química , Animales , Biotransformación , Celecoxib/farmacología , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa/síntesis química , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacocinética , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Inflamación/inducido químicamente , Inflamación/diagnóstico por imagen , Ligandos , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Masculino , Ratones , Especificidad de Órganos , Tomografía de Emisión de Positrones , Radiofármacos/química , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Distribución Tisular

RESUMEN

A new polymer brush was synthesized by spontaneous polymerization of benzofulvene macromonomer 6-MOEG-9-T-BF3k bearing a nona(ethylene glycol) side chain linked to the 3-phenylindene scaffold by means of a triazole heterocycle. The polymer structure was studied by SEC-MALS, NMR spectroscopy, and MALDI-TOF MS techniques, and the results supported the role of oligomeric initiatory species in the spontaneous polymerization of polybenzofulvene derivatives. The aggregation features of high molecular weight poly-6-MOEG-9-T-BF3k-FE were investigated by pyrene fluorescence analysis, dynamic light scattering studies, and transmission electron microscopy, which suggested a tendency towards the formation of spherical objects showing dimensions in the range of 20-200 nm. Moreover, poly-6-MOEG-9-T-BF3k-FE showed an interesting cytocompatibility in the whole concentration range tested that, besides its aggregation features, makes this polybenzofulvene brush a good polymer candidate for nanoencapsulation and delivery of drug molecules. Finally, the photo-physical features of poly-6-MOEG-9-T-BF3k-FE could allow the biodistribution of the resulting drug delivery systems to be monitored by fluorescence microscopy techniques.

RESUMEN

A novel series of 1,5-diarylpyrrol-3-sulfur derivatives (10-12) was synthesized and characterized by NMR and mass spectroscopy and x-ray diffraction. The biological activity of these compounds was evaluated in in vitro and in vivo tests to assess their COX-2 inhibitory activity along with anti-inflammatory and antinociceptive effect. Results showed that the bioisosteric transformation of previously reported alkoxyethyl ethers (9a-c) into the corresponding alkyl thioethers (10a-c) still leads to selective and active compounds being the COX-2 inhibitory activity for most of them in the low nanomolar range. The oxidation products of 10a,b were also investigated and both couple of sulfoxides (11a,b) and sulfones (12a,b) showed an appreciable COX-2 inhibitory activity. Molecular modeling studies were performed to investigate the binding mode of the representative compounds 10b, 11b, and 12b into COX-2 enzyme and to explore the potential site of metabolism of 10a and 10b due to the different in vivo efficacy. Among the developed compounds, compound 10b showed a significant in vivo anti-inflammatory and antinociceptive activity paving the way to develop novel anti-inflammatory drugs.

Asunto(s)

Inhibidores de la Ciclooxigenasa 2/uso terapéutico , Pirroles/uso terapéutico , Sulfuros/uso terapéutico , Sulfonas/uso terapéutico , Sulfóxidos/uso terapéutico , Analgésicos/síntesis química , Analgésicos/metabolismo , Analgésicos/uso terapéutico , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/metabolismo , Antiinflamatorios/uso terapéutico , Carragenina , Línea Celular , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/síntesis química , Inhibidores de la Ciclooxigenasa 2/metabolismo , Diseño de Fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Masculino , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Pirroles/síntesis química , Pirroles/metabolismo , Ratas Sprague-Dawley , Ratas Wistar , Relación Estructura-Actividad , Sulfuros/síntesis química , Sulfuros/metabolismo , Sulfonas/síntesis química , Sulfonas/metabolismo , Sulfóxidos/síntesis química , Sulfóxidos/metabolismo

RESUMEN

The technique of grafting side chains onto a linear polymeric backbone is commonly used to confer to the new polymeric material with desired properties, such as tunable solubility, ionic charge, biocompatibility, or specific interactions with biological systems. In this paper, two new polybenzofulvene backbones were assembled by spontaneous polymerization of the appropriate benzofulvene monomers (4,6-PO-BF3k and 4',6-PO-BF3k) bearing two clickable propargyloxy groups in different positions of the 3-phenylindene scaffold. Poly-4,6-PO-BF3k and poly-4',6-PO-BF3k were grafted with monomethyl oligo(ethylene glycol) (MOEG) to prepare two new polybenzofulvene brushes (i.e., poly-4,6-MOEG-9-TM-BF3k and poly-4',6-MOEG-9-TM-BF3k) by means of a "grafting onto" approach, that were characterized from the point of view of their macromolecular features, aggregation liability, and in a preliminary evaluation of biocompatibility. The obtained results make these PEGylated polybenzofulvene brushes (PPBFB) derivatives potentially useful as nanocarriers for nanoencapsulation and delivery of drug molecules.

RESUMEN

Two small series of quinoline derivatives were designed starting from previously published quinoline derivatives 7a and b in order to obtain information about their interaction with the 5-HT4R binding site. Initially, the structure of 7a and b was modified by replacing their basic moiety with that of partial agonist 4 (ML10302) or with that of reference ligand 6 (RS-67-333). Then, the aromatic moieties of 4-quinolinecarboxylates 7a, d-f, and h-k or 4-quinolinecarboxamides 7b, c, and g were modified into those of 2-quinolinecarboxamides 9a-e. Very interestingly, this structure-affinity relationship study led to the discovery of 7h-j as novel 5-HT4R ligands showing K i values in the subnanomolar range. The structures of all these compounds contain the N-butyl-4-piperidinylmethyl substituent, which appear to behave as an optimized basic moiety in the interaction of these 4-quinolinecarboxylates with the 5-HT4R binding site. However, this basic moiety was ineffective in providing 5-HT4R affinity in the corresponding 4-quinolinecarboxamide 7g, but it did in 2-quinolinecarboxamide ligands 9c-e. Thus, a subtle interrelationship of several structural parameters appeared to play a major role in the interaction of the ligands with the 5-HT4R binding site. They include the kind of basic moiety, the position of the carbonyl linking group with respect to the aromatic moiety and its orientation, which could be affected by the presence of the intramolecular H-bond as in compounds 9c-e.

RESUMEN

In order to develop a technology platform based on two natural compounds from biorenewable resources, a short series of hyaluronan (HA) copolymers grafted with propargylated ferulic acid (HA-FA-Pg) were designed and synthesized to show different grafting degree values and their optical properties were characterized in comparison with reference compounds containing the same ferulate fluorophore. Interestingly, these studies revealed that the ferulate fluorophore was quite sensitive to the restriction of intramolecular motion and its introduction into the rigid HA backbone, as in HA-FA-Pg graft copolymers, led to higher photoluminescence quantum yield values than those obtained with the isolated fluorophore. Thus, the propargyl groups of HA-FA-Pg derivatives were exploited in the coupling with oleic acid through a biocompatible nona(ethylene glycol) spacer as an example of the possible applications of this technology platform. The resulting HA-FA-NEG-OA materials showed self-assembling capabilities in aqueous environment. Furthermore, HA-FA-NEG-OA derivatives have been shown to interact with phospholipid bilayers both in liposomes and living cells, retaining their fluorogenic properties and showing a high degree of cytocompatibility and for this reason they were proposed as potential biocompatible self-assembled aggregates forming new materials for biomedical applications.

RESUMEN

A small series of Morita-Baylis-Hillman adduct (MBHA) derivatives was synthesized and made to react with imidazole, N-acetylhistidine, and N-acetylhexahistidine as models of poly-histidine derivatives. Intriguingly, the reaction of MBHA derivatives 1a and b with imidazole in acetonitrile-phosphate buffered saline (PBS) gave the imidazolium salt biadducts 3a and b as the main reaction products. These results were confirmed by experiments performed with N-acetylhistidine and 1b and suggested the possible occurrence of these structures in the products of poly-histidine labeling with MBHA derivatives 1a and b. These compounds were then transformed into the corresponding water-soluble derivatives 1c-e by introducing oligo(ethylene glycol) chains and their reactivity was evaluated in preliminary experiments with imidazole and then with N-acetylhexahistidine in PBS. The structure of polymeric materials Ac-His-6-MBHA-1d and Ac-His-6-MBHA-1e obtained using ten-fold excesses of compounds 1d and e was investigated using mass spectrometry, NMR spectroscopy, and photophysical studies, which suggested the presence of biadduct residues in both polymeric materials. These results provide the basis for the preparation of fishbone-like polymer brushes, the characterization of their properties, and the exploration of their potential applications in different fields of science such as in vivo fluorogenic labeling, fluorescence microscopy, protein PEGylation, up to the production of smart materials and biosensors.

RESUMEN

The electrochemical behavior of some polybenzofulvene derivatives bearing bithiophene (BT) or terthiophene (TT) side chains was investigated by cyclic voltammetry. Very interestingly, the presence of unsubstituted terminal thiophene moieties allowed poly-6-BT-BF3k and poly-6-TT-BF3k to be cross-linked by electrochemical procedures. Conductive films were obtained by electrodeposition from solutions of these polymers onto electrode surfaces through the formation of covalent cross-linking due to dimerization (i.e. electrochemical oxidation) of the BT or TT side chains. The films showed electrochromic features and switched from yellow-orange (neutral) to green (positively charged) by switching the potential, and were stable to tenths of cycles, without degradation in the wet state in the electrolyte solution. Finally, the thin film obtained by electrodeposition of poly-6-TT-BF3k on a indium tin oxide (ITO) glass substrate showed in the neutral state a significantly red-shifted photoluminescence (PL) emission (∼40 nm red-shifted with respect to that of the corresponding film obtained by casting procedures), which was consistent with the presence of more conjugated moieties produced by the oxidative dimerization of the TT side chains. The innovative architecture and the easy preparation could lead to a broad range of applications in optoelectronics and bioelectronics for these cross-linked hybrid materials based on π-stacked polybenzofulvene backbones bearing oligothiophene side chains.

RESUMEN

Spontaneous polymerization is an intriguing phenomenon in which pure monomers begin their polymerization without initiators or catalysts. Previously, 3-phenylbenzofulvene monomers were found to polymerize spontaneously after solvent removal. Here, eight new 3-substituted benzofulvene monomers 1a⁻h were synthesized in order to investigate the effects of differently substituted aromatic rings in position 3 of the benzofulvene scaffold on spontaneous polymerization. The newly synthesized monomers maintained the tendency toward spontaneous polymerization. However, monomer 1a, bearing an ortho-methoxy substituted phenyl, polymerized hardly, thus producing low polymerization yields, inhomogeneous structure, and low molecular weight of the obtained polymeric material. This result suggested the importance of the presence of hydrogen atoms in the 2'-position to achieve productive interactions among the monomers in the recognition step preluding the spontaneous polymerization and among the monomeric units in the polybenzofulvene backbones. Moreover, this study paves the way to modify the pendant rings in position 3 of the indene scaffold to synthesize new polybenzofulvene derivatives variously decorated.

RESUMEN

In order to obtain new fluorophores potentially useful in imidazole labeling and subsequent conjugation, a small series of Morita-Baylis-Hillman acetates (3a-c) was designed, synthesized, and reacted with imidazole. The optical properties of the corresponding imidazole derivatives 4a-c were analyzed both in solution and in the solid state. Although the solutions display a very weak emission, the powders show a blue emission, particularly enhanced in the case of compound 4c possessing two methoxy groups in the cinnamic scaffold. The photophysical study confirmed the hypothesis that the molecular rigidity of the solid state enhances the emission properties of these compounds by triggering the restriction of intramolecular motions, paving the way for their applications in fluorogenic labeling.