Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Comparison of different protein precipitation and solid-phase extraction protocols for the study of the catabolism of peptide drugs by LC-HRMS.

LC-HRMS-based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent-related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid-phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP-2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (HR ) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed-mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP-2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the HR and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.

Recognition mechanism of aromatic derivatives resolved by argentation chromatography: The driving role played by substituent groups.

Argentation chromatography is widely used nowadays as a powerful tool to separate complex mixtures of analytes containing unsaturated and/or aromatic fragments. Here we present the results of chromatographic and computational studies on a silver-thiolate stationary phase, in which the silver metal is covalently bonded to mercaptopropyl silica particles. The exceptionally high selectivity displayed by this organometallic moiety prompted us to deeply investigate its molecular recognition properties. The interactions of the silver atom with a series of benzene derivatives was investigated to gain information on the mechanism by which the different ring substituents modulate retention factors and selectivity. The experimental trend was fully rationalized by means of quantum-mechanical Density Functional Theory (DFT) calculations, which allowed us to elucidate the chromatographic results in the light of unusual and unexpected substituent effects.

Analysis of imidazoles and triazoles in biological samples after MicroExtraction by packed sorbent.

This paper reports the MEPS-HPLC-DAD method for the simultaneous determination of 12 azole drugs (bifonazole, butoconazole, clotrimazole, econazole, itraconazole, ketoconazole, miconazole, posaconazole, ravuconazole, terconazole, tioconazole and voriconazole) administered to treat different systemic and topical fungal infections, in biological samples. Azole drugs separation was performed in 36 min. The analytical method was validated in the ranges as follows: 0.02-5 µg mL-1 for ravuconazole; 0.2-5 µg mL-1 for terconazole; 0.05-5 µg mL-1 for the other compounds. Human plasma and urine were used as biological samples during the analysis, while benzyl-4-hydroxybenzoate was used as an internal standard. The precision (RSD%) and trueness (Bias%) values fulfill with International Guidelines requirements. To the best of our knowledge, this is the first HPLC-DAD procedure coupled to MEPS, which provides the simultaneous analysis of 12 azole drugs, available in the market, in human plasma and urine. Moreover, the method was successfully applied for the quantitative determination of two model drugs (itraconazole and miconazole) after oral administration in real samples.

High-Affinity "Click" RGD Peptidomimetics as Radiolabeled Probes for Imaging αv ß3 Integrin.

Nonpeptidic Arg-Gly-Asp (RGD)-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αv ß3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g., tritium and fluorine-18, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD mimics to αv ß3 or αIIb ß3 receptors was investigated by molecular modeling simulations. Lead compound 12 was successfully radiofluorinated and used for in vivo positron emission tomography/computed tomography (PET/CT) studies in U87 tumor models, which showed only modest tumor uptake and retention, owing to rapid excretion. These results demonstrate that the novel click RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αv ß3 integrin, whereas further optimization of their pharmacokinetic and dynamic profiles is necessary for successful use in in vivo imaging.

3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole: A fascinating molecular framework to study the enantioseparation ability of the amylose (3,5-dimethylphenylcarbamate) chiral stationary phase. Part II. Solvophobic effects in enantiorecognition process.

The enantiomers of five chiral compounds incorporating the 3-(phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole scaffold and differing only in the linear alkyl chain (varying in length from 1 to 5 carbon atoms) linked to the oxygen atom were directly separated on the amylose (3,5-dimethylphenylcarbamate) based Chiralpak AD-3 chiral stationary phase. The effects of the mobile phase composition, the structure of the analytes and temperature on the retention and enantioselectivity were investigated. It was found that the enantiomeric separations were in all cases enthalpy-driven and disfavored by entropic term. U-shape curves obtained by plotting the chromatographic data versus the alcoholic percentage in n-pentane-methanol and n-hexane-ethanol mobile phases highlighted that, at higher alcohol concentrations, solvophobic interactions were operative in the retention mechanism. The unusual trend of such curves was linked to the nature of alkyl chain of the pyrazolines and it was indicative of the solvophobic contribution to the achievement of a high degree of enantioseparation.

3-(Phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole: A fascinating molecular framework to study the enantioseparation ability of the amylose (3,5-dimethylphenylcarbamate) chiral stationary phase. Part I. Structure-enantioselectivity relationships.

Chiral stationary phases (CSPs) based on amylose (3,5-dimethylphenylcarbamate) (ADMPC) exhibit a wide-range of enantioselectivity in high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Although this class of CSPs has been extensively used, chiral discriminations at receptorial level, which are useful to develop predictive molecular models, have been rarely reported in the literature. Herein, we describe the results obtained in the enantioselective HPLC of a set of six C5-chiral 4,5-dihydro-(1H)-pyrazole derivatives on the ADMPC-based Chiralpak AD-3 CSP (CSP) under normal-phase and polar organic conditions. Using pure methanol as a mobile phase the exceptional enantioseparation factor value of 50 at 25°C was found for one of the investigated analytes. To the best of our knowledge, the enantiomeric bias represents the most outstanding enantioseparation ever recorded on ADMPC-based CSPs. Systematic variations in chemical groups in specific positions of the 3-(phenyl-4-oxy)-5-phenyl-4,5-dihydro-(1H)-pyrazole molecular framework resulted in peculiar changes in retention and enantioselectivity. A careful analysis of the chromatographic data permitted to advance some hypotheses concerning the role played by the individual chemical groups in determining the exceptional enantioseparation. In particular, under methanol-rich mode, the prenyl moiety of the second eluted enantiomer of the better resolved analyte was recognized as a critical structural element to establish direct and favorable solvophobic interactions with apolar portions of selector.

Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.

The Anancomeric Character of the Pharmacophore 1,3,4-Thiadiazoline Framework in Chiral Spiro-Cyclohexyl Derivatives: Effects on Stereochemistry and Spiro-Junction Lability. Thermodynamic Aspects.

Three new and easily accessible chiral compounds, containing the pharmacophore 1,3,4-thiadiazoline nucleus joined by a spiro center to a monoalkyl (methyl or t-butyl) substituted cyclohexyl fragment, have been synthesized and fully characterized from the structural and stereochemical point of view. The formation of a spiro-cyclohexyl-thiadiazoline system (sCT) offered the rare opportunity to generate at room temperature both anancomeric structures, displaying alkyl groups bound to the cyclohexyl ring in equatorial position, and other quite stable stereoisomers in which the same alkyl moieties are, instead, inserted in axial position, even for the extreme case represented by the really bulky t-butyl group. DFT calculations led to a clear rationalization of such stereochemical behaviors, pointing out that in all cases they arise from the unexpected strong anancomeric character possessed by the sCT framework in its 4-acetyl substituted version. In consideration of the large number of substances in which the 1,3,4-thiadiazoline heterocycle has been found as the active pharmacophore, the results discussed in this work may provide solid bases to allow a rational design of new chiral bioactive spiro-thiadiazolines characterized by well-defined stereochemical structures and single anancomeric geometries.

Determination of the Enantiomerization Barrier of the Residual Enantiomers of C3 -Symmetric Tris[3-(1-Methyl-2-Alkyl)Indolyl]Phosphane Oxides: Case Study of a Multitasking HPLC Investigation Based on an Immobilized Polysaccharide Stationary Phase.

The residual enantiomers of three tris-(3-indolyl)-phosphane oxides bearing different alkyl groups (methyl, ethyl or i-propyl) in position 2 of the indole rings constituting the blades were separated on the immobilized type Chiralpak IC column in polar organic and reversed-phase modes. The good enantioselectivity and versatility of the IC CSP allowed easy isolation of the enantiomerically highly enriched samples suitable for configurational stability studies. The enantiomerization barriers of residual phosphane oxides were evaluated both by off-column techniques (CD signal and enantiomeric purity decay kinetics) and by dynamic enantioselective high-performance liquid chromatography (HPLC).

Stereolability of chiral ruthenium catalysts with frozen NHC ligand conformations investigated by dynamic-HPLC.

The stereolability of chiral Hoveyda-Grubbs II type ruthenium complexes bearing N-heterocyclic carbene (NHC) ligands with Syn-phenyl groups on the backbone and Syn- or Anti-oriented o-tolyl N-substituents was studied by resorting to dynamic high-performance liquid chromatography (D-HPLC). A complete chromatographic picture of the involved stereoisomers (four for Anti- and two for Syn-complexes) was achieved at very low temperatures (-53°C and -40°C respectively), at which the NHC-Ru bond rotations were frozen out. Inspection of the chromatographic profiles recorded at higher temperatures revealed the presence of plateau zones between the couples of either Syn or Anti stereoisomers, attesting to the active interconversion between the eluted species. Such dynamic chromatograms were successfully simulated through procedures based on both theoretical plate and classical stochastic models. The good superimposition achieved between experimental and simulated chromatographic profiles allowed determination of the related isomerization energy barriers (ΔGisom (#) ), all derived by rotation around the NHC-Ru bond. The obtained diastereomerization barriers between the Anti isomers were found in very good agreement with those previously measured by experimental nuclear magnetic resonance (NMR) and assessed through Density Functional Theory (DFT) calculations. With the same approach, for the first time we also determined the enantiomerization barrier of the Syn isomer. Focused changes to the structure of complex Syn, studied by a molecular modeling approach, were found suitable to strongly reduce the stereolability arising from rotation around the NHC-Ru bond.

Hydrolytic inhibition of α-chymotrypsin by 2,8,14,20-tetrakis(D-leucyl-D-valinamido)resorc[4]arenecarboxylic acid: a spectroscopic NMR and computational combined approach.

The stereochemical features of 2,8,14,20-tetrakis(D-leucyl-D-valinamido)resorc[4]arenecarboxylic acid and the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-4-nitroanilide polypeptide substrate were investigated by nuclear magnetic resonance spectroscopy. Proton selective relaxation parameters gave the basis for the inhibitory activity of resorcin[4]arene in the hydrolysis of the polypeptide substrate by α-chymotrypsin. Results showed that an interaction between the resorcin[4]arene and α-chymotrypsin does occur, and involves the hydrophobic moiety of the macrocycle. This interaction is further reinforced by polar groups located on the side chains of the resorcin[4]arene, whereas the macrocycle-polypeptide substrate interaction is negligible. Conformational analysis and interaction studies carried out by molecular modeling are in good agreement with the NMR data, thus providing an additional support to the rationalization of the inhibitory potential of resorcin[4]arenes on the α-chymotrypsin activity.

Influence of the nature of alkyl substituents on the high-performance liquid chromatography enantioseparation and retention of new atropisomeric 1,1'-bibenzimidazole derivatives on amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase.

Six new atropisomeric heteroarenes were synthesized by connecting two 2-alkylbenzimidazole fragments via N-N junction. They differ by the substituent nature (methyl, ethyl, propyl, butyl, pentyl and hexyl) of the aliphatic function. The novel atropisomeric compounds were used as chiral probes to study the chromatographic behavior of the amylose tris(3,5-dimethylphenyl carbamate) (Chiralpak AD-3) chiral stationary phase (CSP) under normal phase mode. The pivotal role of the length and flexibility of the 2,2'-alkyl groups on retention, enantioselectivity and enantiomer elution order was demonstrated by enantioselective HPLC analysis. Additional information on the chiral recognition mechanism was obtained from the evaluation of the correlated thermodynamic data.

Exploring the role of 2-chloro-6-fluoro substitution in 2-alkylthio-6-benzyl-5-alkylpyrimidin-4(3H)-ones: effects in HIV-1-infected cells and in HIV-1 reverse transcriptase enzymes.

A comparison of the effects of the 6-(2-chloro-6-fluorobenzyl)-2-(alkylthio)pyrimidin-4(3H)-ones (2-Cl-6-F-S-DABOs) 7-12 and the related 6-(2,6-difluorobenzyl) counterparts 13-15 in HIV-1 infected cells and in the HIV-1 reverse transcriptase (RT) assays is here described. The new 2-Cl-6-F-S-DABOs showed up to picomolar activity against wt HIV-1. Against clinically relevant HIV-1 mutants and in enzyme assays, the simultaneous C5(methyl)/C6(methyl/ethyl) substitution in the 2-Cl-6-F- and 2,6-F2-benzyl series furnished compounds with the highest, wide-spectrum inhibitory activity against HIV-1. Three representative 2-Cl-6-F-S-DABOs carrying two (9c, 10c) or one (10a) stereogenic centers were resolved into their individual stereoisomers and showed a significant diastereo- and enantioselectivity in HIV-1 inhibition, the highest antiviral activity well correlating with the R absolute configuration to the stereogenic center of the C6-benzylic position in both cellular and enzymatic tests. Application of previously reported COMBINEr protocol on 9c and 10c confirmed the influence of the stereogenic centers on their binding modes in the HIV-1 RT.

A chromatographic and computational study on the driving force operating in the exceptionally large enantioseparation of N-thiocarbamoyl-3-(4'-biphenyl)-5-phenyl-4,5-dihydro-(1H) pyrazole on a 4-methylbenzoate cellulose-based chiral stationary phase.

This paper describes the results obtained in the HPLC enantioseparation of N-thiocarbamoyl-3-(4'-biphenyl)-5-phenyl-4,5-dihydro-(1H) pyrazole on a cellulose tris(4-methylbenzoate) chiral stationary phase (OJ-3 CSP) using normal-phase and polar organic conditions. The enantioseparation factor (α=207) observed using the mixture n-hexane-2-propanol 70:30 as a mobile phase is among the highest values ever reported in enantioselective HPLC. The enantioseparation process was investigated by means of molecular modelling techniques. Chromatographic and theoretical investigations addressed the extreme affinity of the most CSP retained (S)-enantiomer to the intermolecular H bond network between the ligand thioamide group and the stationary phase O atoms.

3-methylcyclohexanone thiosemicarbazone: determination of E/Z isomerization barrier by dynamic high-performance liquid chromatography, configuration assignment and theoretical study of the mechanisms involved by the spontaneous, acid and base catalyzed processes.

Here, we report on the simultaneous direct HPLC diastereo- and enantioseparation of 3-methylcyclohexanone thiosemicarbazone (3-MCET) on a polysaccharide-based chiral stationary phase under normal-phase conditions. The optimized chromatographic system was employed in dynamic HPLC experiments (DHPLC), as well as detection technique in a batch wise approach to determine the rate constants and the corresponding free energy activation barriers of the spontaneous, base- and acid-promoted E/Z diastereomerization of 3-MCET. The stereochemical characterization of four stereoisomers of 3-MCET was fully accomplished by integrating the results obtained by chemical correlation method with those derived by theoretical calculations and experimental investigations of circular dichroism (CD). As a final goal, a deepened analysis of the perturbing effect exercised by the stationary phase on rate constant values measured through DHPLC determinations as a function of the chromatographic separation factor α of the interconverting species was successfully accomplished. This revealed quite small deviations from the equivalent kinetic values obtained by off-column batch wise procedure, and suggested a possible effective correction of rate constants measured by DHPLC approach.