Gabriele Amari

Bronchodilator Activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a Potent, Long-Acting, and Selective Muscarinic M3 Receptor Antagonist

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t½ = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [3H]CHF5407 dissociated quickly from hM2 receptors (t½ = 31 min), whereas [3H]tiotropium dissociated slowly from both hM3 (t½ = 163 min) and hM2 receptor (t½ = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC50 = 9.0–9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED50 value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A–D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4+ T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.

Novel Class of Benzoic Acid Ester Derivatives as Potent PDE4 Inhibitors for Inhaled Administration in the Treatment of Respiratory Diseases

The first steps in the selection process of a new anti-inflammatory drug for the inhaled treatment of asthma and chronic obstructive pulmonary disease are herein described. A series of novel ester derivatives of 1-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl) ethanol have been synthesized and evaluated for inhibitory activity toward cAMP-specific phosphodiesterase-4 (PDE4). In particular, esters of variously substituted benzoic acids were extensively explored, and structural modification of the alcoholic and benzoic moieties were performed to maximize the inhibitory potency. Several compounds with high activity in cell-free and cell-based assays were obtained. Through the evaluation of opportune in vitro ADME properties, a potential candidate suitable for inhaled administration in respiratory diseases was identified and tested in an in vivo model of pulmonary inflammation, proving its efficacy.

The Molecular Structure and Crystal Organization Of Rac -terfenadine/β-cyclodextrin/tartaric Acid Multicomponent Inclusion Complex

The crystalline ternary inclusion complex terfenadine/ g -cyclodextrin/tartaric acid (TFN/ g CD/TA, 2:4:1) has been prepared from a aqueous solution (terfenadine, TFN, rac - f -[4-(1,1-dimethylethyl)phenyl]-4-(hydroxy-diphenylmethyl)-1-piperidine-butanol). The solubility of the multicomponent system in water is remarkably different from that of the single components. The crystal structure shows that the TFN guest adopts an extended conformation and that the diphenyl end of the molecule is docked in the cavity formed by the association of two independent g CD molecules through hydrogen bonds connecting their wide rims. The structure of the dimer is deformed with respect to uncomplexed g CDs, due to the shape of the guest. The two aromatic rings interact differently with the macrocycles forming the dimer, one being included perpendicular in the central cavity of one g CD, the other laying parallel to the interface between the two rims. The t-Bu- end of the guest is included in the cavity of a g CD belonging to a different dimer, entering from the side of the narrow rim. The central part of the guest is surrounded by water molecules and tartaric acid, which creates a hydrophilic microenvironment in the interstices among dimers. The enhanced solubility of the multicomponent system could be related to the hydrogen bonds between the tartaric acid and the oxygens belonging to the wide rims. The overall structural arrangement of the g CD units is driven by the shape of the TFN guest which needs a hydrophobic environment at both ends. The lipophilic interactions between TFN and g CD cavities are responsible for the relevant perturbation in the regularity of the packing of the hosts.

The Crystal Structure of the Inclusion Complex of the Sodium Salt of Piroxicam with β-cyclodextrin

Abstract The structural characterization by X-ray diffraction analysis of the title compound is reported. Crystallographic details are: C42H70O35°C15H12N3O4 SNa·8D2O, triclinic, space group P1, a = 14.767(3), b = 12.237(2), c = 11.537(2)A, α = 79.49(1), β = 110.91-(1), c = 104.61(1)°, d calc = 1.460 g/cm3, Z = 1, R = 0.045 for 6635 observed reflections. The crystal structure consists of head-to-tail β-cyclodextrin molecules (β-CD) linked together by piroxicam anions in infinite polymeric chains running along the c axis. In a chain each β-CD molecule takes up simultaneously two guest molecules including on the secondary hydroxyl end the benzene ring and on the primary hydroxyl end the pyridine ring of two adjacent piroxicam anions. The piroxicam anion is located with its central hydrophilic moiety at the interface between two adjacent β-CD molecules, forming with them hydrogen bonds through the enolate and amide oxygen atoms. Conformation of piroxicam is different from those observed for its neutral and zw...

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5, 6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HPLC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds.

Synthesis of glycose carbamides and evaluation of the induction of erythroid differentiation of human erythroleukemic K562 cells.

A series of carbamides derived from 1,2:5,6-di-O-isopropylidene-D-gluco- and D-allofuranose as well as their 5,6-O-deprotected analogues (2 and 4) and methyl 3,4-O-isopropylidene-alpha- and beta-D-galactopyranosides (5 and 6) have been prepared in order to evaluate their ability to induce erythroid differentiation of human erythroleukemic K562 cells. Twenty out of 51 carbamides tested exhibit an appreciable activity as inducers of erythroid differentiation and have been fully characterized and described.

Discovery and optimization of thiazolidinyl- and pyrrolidinyl- derivatives as inhaled PDE4 inhibitors for respiratory diseases

Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.

Structural characterization and solution rotational isomerism of delapril hydrochloride, a dipeptide angiotensin-converting enzyme inhibitor

Abstract The solid state structure of delapil hydrochloride was determined by single-crystal X-ray diffraction analysis (monoclinic, P21, a=16.098(5), b=10.712(3), c=7.856(2) A , β=97.85(2)° . The comparison between delapril and other ACE inhibitors of the same family is discussed with regard to the geometry of the phenomenological active site of the enzyme. In the solid state the amide bond conformation resulted in being trans, whereas, in solution, NMR spectra indicate that the molecule exists as a mixture of rotational isomers trans and cis (approximately 70:30). The free energy of activation for the hindered rotation about the amide bond was determined by line-shape analysis. The attempt to isolate possible conformational polymorphs failed, indicating that the trans conformation is favored when molecules pack together in the crystal.

Erythroid differentiation ability of butyric acid analogues: identification of basal chemical structures of new inducers of foetal haemoglobin.

Several investigations have demonstrated a mild clinical status in patients with β-globin disorders and congenital high persistence of foetal haemoglobin. This can be mimicked by a pharmacological increase of foetal γ-globin genes expression and foetal haemoglobin production. Our goal was to apply a multistep assay including few screening methods (benzidine staining, RT-PCR and HPLC analyses) and erythroid cellular model systems (the K562 cell line and erythroid precursors collected from peripheral blood) to select erythroid differentiation agents with foetal haemoglobin inducing potential. With this methodology, we have identified a butyric acid derivative, namely the 4174 cyclopropanecarboxylic acid compound, able to induce erythroid differentiation without antiproliferative effect in K562 cells and increase of γ-globin gene expression in erythroid precursor cells. The results are relevant for pharmacological treatments of haemoglobinopathies, including β-thalassaemia and sickle cell anaemia.