Hyaluronan derivatives bearing variable densities of ferulic acid residues.

A synthetic procedure has been developed to conjugate ferulic acid (FA) to an important natural polysaccharide derivative such as hyaluronic acid (HA). The activation of FA with 1,1'-carbonyldiimidazole (CDI) has been investigated. Two reactive intermediates, namely monoimidazolide 2 [i.e. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(1H-imidazol-1-yl)prop-2-en-1-one] and bisimidazolide 3 [i.e. (E)-4-(3-(1H-imidazol-1-yl)-3-oxoprop-1-enyl)-2-methoxyphenyl 1H-imidazole-1-carboxylate] were characterized from the point of view of their structure and reactivity. The ready isolation of bisimidazolide 3 and its reactivity support its potential usefulness in the feruloylation of molecular or macromolecular materials bearing hydroxyl moieties. Bisimidazolide derivative 3 has been found to be an effective reagent in the feruloylation of HA to give HAFA graft copolymers showing different grafting degrees (GD), which could be modulated by varying the reaction conditions. A series of HAFA derivatives showing different GD values has been prepared and submitted to an extensive macromolecular and rheological characterization in order to ascertain that the grafting of HA with FA does not degrade the polysaccharide backbone and to evaluate the role of GD in affecting solubility and rheological properties. The results suggested that relatively low GD values were sufficient to confer physical cross-linking capabilities resulting in the features of a strong gel of HAFA dispersions.

Evaluation of three quinoline-carboxamide derivatives as potential radioligands for the in vivo pet imaging of neurodegeneration.

The peripheral-type benzodiazepine receptors (PBRs) are only minimally expressed in normal brain parenchyma, where they are primarily localized in glial cells. Their basal expression rises in different neurodegenerative disorders, due to the presence of infiltrating inflammatory cells and activated microglia. [11C]PK11195, a selective PBR antagonist, has been used for the in vivo PET monitoring of neurodegeneration in clinical observations. We recently developed and labeled with carbon-11 three new carboxamide derivatives: [11C]VC193M, [11C]VC195 and [11C]VC198M. Aim of this study was to evaluate these ligands for the in vivo measuring of PBRs expression in neurodegenerations and compare their kinetic behavior with that of the reference tracer [11C]PK11195. Radioligands were evaluated in a preclinical model of Huntington's disease consisting in the monolateral striatal injection of quinolinic acid (QA). Activated microglia and astrocytic gliosis was present only within the affected striatum. A concomitant increase in radioactivity accumulation was observed for all the tracers examined (P<0.01). Among the new compounds, [11C]VC195 showed higher levels of lesioned/unlesioned striatum ratios (3.28+/-0.44), in comparison with [11C]VC193M and [11C]VC198M (2.69+/-0.53 and 1.52+/-0.36, respectively), but slightly inferior to that observed for [11C]PK11195 (3.76+/-1.41).In conclusion, the results of the study indicate that [11C]VC195 is a promising candidate for in vivo PET monitoring of neurodegenerative processes but its in vivo behavior overlap that of [11C]PK11195.

Determination of a novel angiotensin-AT1 antagonist CR 3210 in biological samples by HPLC.

A simple and sensitive method for the determination of a new angiotensin-AT(1) antagonist i.e. CR 3210, 4-[4-[(2-ethyl-5,7-dimethylimidazo[4,5-b]pyridin-3-yl)methyl]phenyl]-3-(2H-tetrazol-5-yl)quinoline, is described. The assay was utilised to describe the pharmacokinetic profile of the title compound after intravenous and intraperitoneal administration to Sprague Dawley rats. CR 3210 and the internal standard CR 1505 (loxiglumide, 4-[(3,4-dichlorobenzoyl)amino-5-[(3-methoxypropyl)pentylamino]-5-oxopentanoic acid) were isolated from rat plasma by solid-phase extraction. The sorbent extraction material along with the pH in the conditioning solution and the washing volume were considered pivotal parameters for the optimisation of the procedure. The separations were performed by reversed-phase high-performance liquid chromatography with ultraviolet detection. The samples were injected onto the analytical column (Tracer Extrasil ODS1) and detected at 238 nm, giving a retention time of 6.19 min for CR 3210 and 4.39 min for the internal standard, respectively. The selectivity of the method showed to be satisfactory. The mean recovery of CR 3210 from spiked rat plasma was 80.3 at 1 microg/ml and 79.9 at 2 microg/ml. The lower limit of detection (LOD) was taken as 0.014 microg/ml in plasma samples. The lower limit of quantification (LOQ) was taken as 0.02 microg/ml, the lowest calibration standard using 500 microg rat plasma. The procedures were validated according to international standards with a good reproducibility and linear response from 0.02 to 2 microg/ml. The sensitivity of the method allowed for its application to pharmacokinetic studies. The maximal concentration was detected 5' after the IV administration, whereas no significant absorption was evident after IP administration of CR 3210 to Sprague-Dawley rats. Our study suggests the absence of extensive bio-transformation of the drug in vivo, supported by the evidence that no metabolites were detected in plasma samples.

Mapping and fitting the peripheral benzodiazepine receptor binding site by carboxamide derivatives. Comparison of different approaches to quantitative ligand-receptor interaction modeling.

The synthetic-computational approach to the study of the binding site of peripheral benzodiazepine receptor (PBR) ligands related to 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195, 1) within their receptor (Cappelli et al. J. Med. Chem. 1997, 40, 2910-2921) has been extended. A series of carboxamide derivatives endowed with differently substituted planar aromatic or heteroaromatic systems was designed with the aim of getting further information on the topological requisites of the carbonyl and aromatic moieties for interaction with the PBR binding site. The synthesis of most of these compounds involves Weinreb amidation of the appropriate lactone as the key step. The most potent compound, among the newly synthesized ones, shows a nanomolar PBR affinity similar to that shown by 1 and the presence of a basic N-ethyl-N-benzylaminomethyl group in 3-position of the quinoline nucleus. Thus, it may be considered the first example of a new class of water soluble derivatives of 1. Several computational methods were used to furnish descriptors of the isolated ligands (indirect approaches) able to rationalize the variation in the binding affinity of the enlarged series of compounds. Sound QSAR models are obtained by size and shape descriptors (volume approach) which codify for the short-range contributions to ligand-receptor interactions. Molecular descriptors which explicitly account for the electrostatic contribution to the interaction (CoMFA, CoMSIA, and surface approaches) perform well, but they do not improve the quantitative models. Moreover, useful hints for the identification of the antagonist binding site in the three-dimensional modeling of the receptor (direct approach) were provided by the receptor hypothesis derived by the pharmacophoric approach. The ligand-receptor complexes obtained provided a detailed description of the modalities of the interaction and interesting suggestions for further experiments.

Labeling and evaluation of N-[11C]methylated quinoline-2-carboxamides as potential radioligands for visualization of peripheral benzodiazepine receptors.

The novel quinoline-2-carboxamide derivatives N-[methyl-11C]-3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide ([11C]4), (+/-)-N-[methyl-11C]-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide ([11C]5), and (+/-)-N-[methyl-11C]-3-methyl-4-(2-fluorophenyl)-N-(1-methylpropyl)quinoline-2-carboxamide ([11C]6) were labeled with carbon-11 (t1/2 = 20.4 min, beta+ = 99.8%) as potential radioligands for the noninvasive assessment of peripheral benzodiazepine type receptors (PBR) in vivo with positron emission tomography (PET). The radiosynthesis consisted of N-methylation of the desmethyl precursors 3-methyl-4-phenyl-N-(phenylmethyl)quinoline-2-carboxamide (4a), (+/-)-3-methyl-N-(1-methylpropyl)-4-phenylquinoline-2-carboxamide (5a), and (+/-)-4-(2-fluorophenyl)-3-methyl-N-(1-methylpropyl)quinoline-2-carboxamide (6a) with either [11C]methyl iodide or [11C]methyl triflate in the presence of tetrabutylammonium hydroxide or potassium hydroxide in dimethylformamide. The radioligands [11C]4, [11C]5, and [11C]6 were synthesized with over 99% radiochemical purity in 30 min, 30 +/- 5% radiochemical yield, calculated at the end of synthesis (EOS) non-decay-corrected, and 2.5 +/- 1.2 Ci/micromol of specific radioactivity. Inhibition studies in rats following intravenous pre-administration of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195, 1) showed high specific binding to PBR of [11C]4, [11C]5, and [11C]6 in heart, lung, kidney, adrenal gland, spleen, and brain. The biological data suggest that [11C]5, [11C]6, and particularly [11C]4 are promising radioligands for PBR imaging in vivo with PET.

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 2. Molecular basis of the intrinsic efficacy of arylpiperazine derivatives at the central 5-HT3 receptors.

Novel 5-HT3 receptor ligands were designed and synthesized with the aim of obtaining deeper insight into the molecular basis of the intrinsic efficacy of arylpiperazines interacting with the central 5-HT3 receptor. The newly synthesized compounds and some previously published compounds belonging to the same class of heteroarylpiperazines were tested for their potential ability to displace [3H]granisetron from rat cortical membranes. These 5-HT3 receptor binding studies revealed subnanomolar affinity in several of the compounds under study. The most active ligands were quipazine derivatives bearing a phenyl group in the 4-position and various oxygenated alkyl side chains in the 3-position of the quinoline nucleus. Qualitative and theoretical quantitative structure-affinity relationship studies were carried out, and the interaction model for the 5-HT3 ligands related to quipazine with their receptor, proposed in part 1 of the present work, was updated to incorporate the latest data. The potential 5-HT3 agonist/antagonist activity of 12 selected compounds was assessed in vitro on the 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Their intrinsic efficacy ranged from the 5-HT3 full agonist properties of compounds 7a and 8h, i to those of partial agonists 10a,d and antagonists 8b,d,e, and 9c, d,h,i. The comparison between these functional data and those relative to the previously described compounds suggested that in this class of 5-HT3 ligands the intrinsic efficacy is modulated in a rather subtle manner by the steric features of the heteroaryl moiety.

Novel potent and selective central 5-HT3 receptor ligands provided with different intrinsic efficacy. 1. Mapping the central 5-HT3 receptor binding site by arylpiperazine derivatives.

Synthesis and pharmacological evaluation of a series of condensed quinoline and pyridine derivatives bearing a N-methylpiperazine moiety attached to the 2-position of the quinoline or pyridine nucleus are described. 5-HT receptor binding studies revealed subnanomolar affinity for the 5-HT3 receptor subtype in some of the compounds under study. The most active compound (5b) displayed a Ki value about 1 order of magnitude higher than that of quipazine along with a higher selectivity. The potential 5-HT3 agonist/antagonist activity of four selected compounds was assessed in vitro on 5-HT3 receptor-dependent [14C]guanidinium uptake in NG 108-15 cells. Compound 5j acted as a 5-HT3 agonist in this assay with an EC50 value close to that reported for quipazine, while 5b was a partial agonist with an EC50 value of about 0.25 nM, and compound 5c possessed antagonist properties with an IC50 value (approximately 8 nM) in the same range as those of previously characterized 5-HT3 receptor antagonists. Qualitative and quantitative structure-affinity relationship studies carried out by making use of theoretical molecular descriptors allowed to elucidate the role of the main pharmacophoric components and to develop a model for the interaction of the 5-HT3 ligands related to quipazine with their receptor.

Novel and highly potent 5-HT3 receptor agonists based on a pyrroloquinoxaline structure.

The synthesis and the biological evaluation of a series of novel pyrroloquinoxaline derivatives are described. In binding studies several compounds proved to be potent and selective 5-HT3 receptor ligands. The most active pyrroloquinoxalines, 11d and 11e, showed a subnanomolar affinity for 5-HT3 receptor and were able to functionally discriminate the central and peripheral 5-HT3 receptor, being agonists and antagonists, respectively. In functional studies ([14C]-guanidinium accumulation test in NG 108-15 cells, in vitro) most of the synthesized compounds showed clear-cut 5-HT3 agonist properties. In in vivo studies on the von Bezold-Jarisch reflex test (a peripheral interaction model) the behavior of the tested compounds ranged from agonist to antagonist, while clear agonist properties were obtained with 12a on cortical acetylcholine release in freely moving rats. Pharmacokinetic studies with 11e and 12c indicate that the compounds easily cross the blood-brain barrier (BBB) after systemic administration with a brain/plasma ratio of 17.5 and 37.5, respectively. Thus compounds 11e and 12c represent the most potent central 5-HT3 agonists identified to date that are able to cross the blood-brain barrier.

Mapping the peripheral benzodiazepine receptor binding site by conformationally restrained derivatives of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinolinecarboxamide (PK11195).

A synthetic-computational approach to the study of the binding site of peripheral benzodiazepine receptor (PBR) ligands related to 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK11195, 1) within their receptor has been developed. A wide series of conformationally restrained derivatives of 1 has been designed with the aim of probing the PBR binding site systematically. The synthesis of these compounds involves palladium-catalyzed coupling and amidation as the key steps. Twenty-nine rigid and semirigid derivatives of 1 were tested in binding studies using [3H]-1, and most of these showed PBR affinities in the nanomolar range. The essential role of the carbonyl moiety as a primary pharmacophoric element in the recognition by and the binding to PBR has been confirmed, and the restricted range of the carbonyl orientations, which characterizes the most potent ligands, points to a specific hydrogen-bonding interaction, mainly directed by the geometrical factors, when the electronic ones are fulfilled. Moreover, the fundamental importance of the short-range dispersive interactions in the modulation of the binding affinity and, hence, in the stabilization of the ligand-receptor complex, emerged from the QSAR models reported.

Molecular basis of peripheral vs central benzodiazepine receptor selectivity in a new class of peripheral benzodiazepine receptor ligands related to alpidem.

Alpidem (1), the anxiolytic imidazopyridine, has nanomolar binding affinity for both the central benzodiazepine receptor (CBR) and the peripheral benzodiazepine receptor (PBR). A novel class of PBR ligands related to alpidem has been designed by comparing the interaction models of alpidem with PBR and CBR. Several compounds in this class have shown high selectivity for PBR vs CBR, and the selectivity has been discussed in terms of interaction models. The binding behavior of the three selected compounds was extensively studied by competition and saturation assays, and the results suggest that they are capable of recognizing two sites labeled by [3H]PK11195. The molecular structure of one of the most active compounds (4e) has been determined by X-ray diffraction and compared with that of alpidem. Molecular modeling studies suggest that the bioactive conformation of 4e is likely to be very similar to the conformation found in the crystal.

Molecular structure and dynamics of some potent 5-HT3 receptor antagonists. Insight into the interaction with the receptor.

The molecular structure and the dynamic behaviour of some potent 5-HT3 antagonists structurally related to quipazine have been investigated by NMR spectroscopy and by computational methods in order to gain insight into the structure-activity relationships at a molecular level. The role of the different dynamic behaviour of these compounds in the binding to 5-HT3 receptors is discussed. A model of ligand-receptor interaction has been developed on the basis of molecular orbital calculations and on the reference ligands quipazine, ondansetron and LY278584. The interaction model proposed herein rationalizes the observed agonist-antagonist shift between quipazine and investigated compounds with the assumption of different but overlapping binding domains for antagonists and agonists at the 5-HT3 receptor.

Synthesis, biological evaluation, and quantitative receptor docking simulations of 2-[(acylamino)ethyl]-1,4-benzodiazepines as novel tifluadom-like ligands with high affinity and selectivity for kappa-opioid receptors.

The synthesis and biological evaluation of a series of 2-substituted 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a kappa-opioid agonist and a cholecystokinin-A (CCK-A) antagonist, are reported. The radioligand binding models used in these studies were [(125)I](BH)-CCK-8 in rat pancreas (CCK-A), [(3)H]-(MENLE(28,31))-cck-8 in guinea pig cerebral cortex (CCK-B), and [(3)H]U-69593 (kappa(1)), [(3)H]DAMGO (mu), and [(3)H]DADLE (delta) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for kappa-opioid receptors. In particular, the 2-thienyl derivative 7A(X = H) with a K(i) = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig kappa (kappa(1))-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure-affinity relationships in this class of receptors.

Characterization and differentiation of heterocyclic isomers. Part 2. Mass spectrometry and molecular orbital calculations on pyrrolo[1,2-a][1,4]benzodiazepin-4-one, -6-one, and -4,6-dione.

Pyrrolo[1,2-a][1,4]benzodiazepin-4-one (1), -6-one (2), and -4,6-dione (3), which are starting materials for the synthesis of pharmacologically interesting compounds that are active as neurotropic agents, have been characterized in the gas phase. The application of different mass spectrometric techniques, such as electron ionization, high-resolution, and tandem mass spectrometry, has allowed the structural characterization and differentiation of their molecular ions and most abundant fragment ions formed in the source. In particular, the two positional isomers 1 and 2 produce quite different mass spectra, and their molecular and the most intense fragment ions yield different metastable mass-analyzed ion kinetic energy spectra. Furthermore, high-resolution mass spectrometry and accurate mass measurements have revealed different elemental compositions and abundances for isobaric fragment ions produced by isomers 1 and 2. From these data and from the comparison with those relevant to compound 3, it has been possible to evaluate the influence of the position of the carbonyl group on the fragmentation pathways.Semiempirical molecular orbital calculations carried out by both the modified neglect of differential overlap and Austin 1 methods have provided useful information on the characterization of the neutrals as well as the molecular ions of compounds 1-3.

Novel, potent, and selective 5-HT3 receptor antagonists based on the arylpiperazine skeleton: synthesis, structure, biological activity, and comparative molecular field analysis studies.

Synthesis and pharmacological evaluation of a series of condensed quinoline derivatives bearing a basic nitrogen on piperazine or [(dimethylamino)ethyl]thio moieties attached at the 2-position of the quinoline nucleus are described. 5-HT receptor binding studies revealed, for most of the compounds studied, nanomolar affinity for the 5-HT3 receptor subtype. The most active compound, benzopyrano[3,4-c]quinoline derivative 5f, displayed a Ki value very similar to that reported for quipazine along with an improved selectivity. Functional and in vivo testing carried out on three selected compounds showed that 5f,j,n are potent 5-HT3 receptor antagonists with potencies in the same range as the best known 5-HT3 receptor antagonists ondansetron, tropisetron, and zacopride. The crystal and molecular structures of compounds 5f,j,n were determined by single-crystal X-ray diffraction and used as starting structures for molecular modeling studies. Comparative molecular field analysis (CoMFA) was applied to binding constants of compounds 5a-p and 6a-h. The cross-validated r2, derived from partial least-squares calculations, indicated a good predictive capacity for affinity values in the series of compounds investigated. Evidence for the prediction capacity is provided in the form of plots of actual vs predicted pKi values. The steric and electrostatic features of the CoMFA-derived model are presented as standard coefficient contour maps of steric and electrostatic fields.

Synthesis and receptor binding studies of 2-functionalized 1,4-benzodiazepine derivatives as potential metaclazepam-like antianxiety agents.

The synthesis of 7-chloro-2,3-dihydro-2-(2-methoxyethyl)-1-methyl-5-phenyl- 1H-1,4-benzodiazepine (2) is described. While the metaclazepam-like compound 2 showed an affinity for benzodiazepine receptor central type (CBZRs) comparable to that of metaclazepam (1a), its intermediates bearing the exocyclic double bond resulted more active.

Synthesis and benzodiazepine receptors affinity of 2,3-dihydro-9-phenyl-1H-pyrrolo[3,4-b]quinolin-1-one and 3-carbethoxy-4-phenylquinoline derivatives.

A series of 2,3-dihydro-9-phenyl-1H-pyrrolo[3,4-b]quinolin-1-one derivatives and related compounds were tested for their ability to bind benzodiazepine receptors (BZRs). Most of the synthesized compounds showed micromolar affinity for BZR peripheral type, but not for the central one, with the exception of compound 4m which displayed an IC50 = 0.407 microM, only 2 fold higher than IC50 for chlordiazepoxide.

Synthesis and 5-HT receptors binding studies of some 3-substituted-2-(4-methyl-1-piperazinyl)-4-phenylquinolines.

The syntheses of some 3-substituted-2-(4-methyl-1-piperazinyl)-4-phenylquinolines are reported. The title compounds were tested for their potential activities on 5-HT receptor subtypes and 5-HT uptake site; compounds 4b-d showed micromolar affinity for 5-HT3 and 5-HT uptake site.

5-HT and benzodiazepine receptor ligands. III. Synthesis and receptor affinities of 1,2,4-triazolo[4',3':1,6]pyridazino[4,5-b]quinoline and 2,3-dihydro-9-phenyl-1H-pyrrolo[3,4-b]quinoline-1-one derivatives.

The syntheses as well as 5-HT and Benzodiazepine receptor binding studies of some 1,2,4-Triazolo[4',3':1,6]piridazino[4,5-b]quinoline and 2,3-Dihydro-9-phenyl-1H-pyrrolo[3,4-b]quinoline-1-one derivatives are reported. While the triazole-containing heterocycles are devoid of any biological activity, 2-benzyl-2,3-dihydro-9-phenyl-1H-pyrrolo[3,4-b]quinoline-1-one shows some affinity for the central type of benzodiazepine receptors.

Synthesis and 5HT-receptors affinity of some 4-phenylquinoline derivatives.

The synthesis of a series of 4-phenylquinolines with substituents in 2,3 and 6-position of the quinoline nucleus is described. Serotoninergic activity towards 5HT1A, 5HT1B, 5HT2, 5HT1A + B receptors and the 5-HT-uptake site was also studied.

[Substances inhibiting amine oxidases. III. Synthesis and amine oxidase inhibition of imidazoquinoline derivatives]. / Ricerche su sostanze inibitrici delle aminossidasi. Nota III. Sintesi ed attività inibitrice sulle aminossidasi di derivati delle imidazochinoline.

The activity of copper and FAD dependent amine oxidases was tested with some derivatives of 3H-imidazo[4,5-h]quinoline and its isomers 3H-imidazo[4,5-f]quinoline, the chemistry of which is described in the literature (1), and Ki calculated. The methyl derivative of 3H-imidazo[4,5-f]quinoline was found to activate the copper bovine serum enzyme, but inhibits the FAD mitochondrial enzyme.