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COVID-19, caused by the SARS-CoV-2 virus, has become a global pandemic that is still present after more than two years. COVID-19 is mainly known as a respiratory disease that can cause long-term consequences referred to as long COVID. Molecular imaging of SARS-CoV-2 in COVID-19 patients would be a powerful tool for studying the pathological mechanisms and viral load in different organs, providing insights into the disease and the origin of long-term consequences and assessing the effectiveness of potential COVID-19 treatments. Current diagnostic methods used in the clinic do not allow direct imaging of SARS-CoV-2. In this work, a nanobody (NB) – a small, engineered protein derived from alpacas – and an Fc-fused NB which selectively target the SARS-CoV-2 Spike protein were developed as imaging agents for positron emission tomography (PET). We used the tetrazine ligation to 18F-label the NB under mild conditions once the NBs were successfully modified with trans-cyclooctenes (TCOs). We confirmed binding to the Spike protein by SDS-PAGE. Dynamic PET scans in rats showed excretion through the liver for both constructs. Future work will evaluate in vivo binding to the Spike protein with our radioligands.

Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure–activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD.

Almost all Iridium(III) complexes employed both as dopants in PhOLEDs and as pharmaceuticals and fluorescence bioprobes are racemic mixtures. In this study the single enantiomers of the most stable diastereomeric form fac-trans-N–N , bis[2-(4,6-difluorophenyl)pyridinato-C 2 , N ](picolinato)iridium(III) (FIrpic) were separated and analysed. The data obtained showed that the complex can be separated into stable optically active Λ and Δ isomers employing cellulose based chiral stationary phase both in normal and polar phase mode. Their chirality was confirmed and their absolute configuration assigned employing several methods (DFT and TDDFT calculations, CD and VCD). The CPL spectroscopy of the isolated enantiomers of FIrpic was also recorded due to its possible value in the OLEDs field. The chromatographic method was applied for a semipreparative purpose demonstrating that polar organic solvent chromatography (POSC) could be used to avoid the low-solubility issues associated with these Iridium(III) complexes. Finally, the chemical and stereochemical stability of the single isomers was evaluated under thermal stress by liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF) on both chiral and achiral columns. No racemization and/or isomerization was observed; however, the dissociation of the ancillary ligand was demonstrated employing LC-QTOF.

A “heart-cut” two-dimensional achiral-chiral liquid chromatography triple-quadrupole mass spectrometry method (LC–LC–MS/MS) was developed and coupled to in vivo cerebral microdialysis to evaluate the brain response to the chiral compound (±)-7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2 H -1,2,4-benzothiadiazine-1,1-dioxide ((±)- 1 ), a potent positive allosteric modulator (PAM) of AMPA receptor. The method was successfully employed to evaluate also its stereoselective metabolism and in vitro biological activity. In particular, the LC achiral method developed, employs a pentafluorinated silica based column (Discovery HS-F5) to separate dopamine, acetylcholine, serotonin, (±)- 1 and its two hepatic metabolites. In the “heart-cut” two-dimension achiral-chiral configuration, (±)- 1 and (±)- 1 - d 4 eluted from the achiral column (1st dimension), were transferred to a polysaccharide-based chiral column (2nd dimension, Chiralcel OD-RH) by using an automatic six-port valve. Single enantiomers of (±)- 1 were separated and detected using electrospray positive ionization mode and quantified in selected reaction monitoring mode. The method was validated and showed good performance in terms of linearity, accuracy and precision. The new method employed showed several possible applications in the evaluation of: (a) brain response to neuroactive compounds by measuring variations in the brain extracellular levels of selected neurotransmitters and other biomarkers; (b) blood brain barrier penetration of drug candidates by measuring the free concentration of the drug in selected brain areas; (c) the presence of drug metabolites in the brain extracellular fluid that could prove very useful during drug discovery; (d) a possible stereoselective metabolization or blood brain barrier stereoselective crossing of chiral drugs. Finally, compared to the methods reported in the literature, this technique avoids the necessity of euthanizing an animal at each time point to measure drug concentration in whole brain tissue and provides continuous monitoring of extracellular concentrations of single chiral drug enantiomers along with its metabolites in specific brain regions at each selected time point for a desired period by using a single animal.

5-Arylbenzothiadiazine type compounds acting as positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-PAMs) have received particular attention in the past decade for their nootropic activity and lack of the excitotoxic side effects of direct agonists. Recently, our research group has published the synthesis and biological activity of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (1), one of the most active benzothiadiazine-derived AMPA-PAMs in vitro to date. However, 1 exists as two stereolabile enantiomers, which rapidly racemize in physiological conditions, and only one isomer is responsible for the pharmacological activity. In the present work, experiments carried out with rat liver microsomes show that 1 is converted by hepatic cytochrome P450 to the corresponding unsaturated derivative 2 and to the corresponding pharmacologically inactive benzenesulfonamide 3. Surprisingly, patch-clamp experiments reveal that 2 displays an activity comparable to that of the parent compound. Molecular modeling studies were performed to rationalize these results. Furthermore, mice cerebral microdialysis studies suggest that 2 is able to cross the blood-brain barrier and increases acetylcholine and serotonin levels in the hippocampus. The experimental data disclose that the achiral hepatic metabolite 2 possesses the same pharmacological activity of its parent compound 1 but with an enhanced chemical and stereochemical stability, as well as an improved pharmacokinetic profile compared with 1.

A stopped-flow HPLC method was developed to evaluate configurational and chemical stability of pharmaceutical compounds employing immobilized artificial membranes (IAM) column to simulate conditions that pharmaceutical compounds will meet in vivo. The method was applied to recent developed chiral 5-arylbenzothiadiazine derivatives possessing high positive allosteric modulatory (PAM) activity on AMPA receptor. In particular the stopped-flow HPLC method developed used a chiral column to separate single enantiomer of the compounds that are forced into an IAM column where configurational and chemical stability was evaluated in simulated gastrointestinal fluids (pH 1.2 and 6.8 at 37.5 °C) to simulate in vivo conditions. The results were compared to those obtained by dynamic and off-column methods to evaluate the effects of stationary phases on kinetic constant of enantiomerization and hydrolysis. The results suggested that the phospholipids environment of IAM stationary phases, which mimes biological membrane, greatly influence the hydrolysis process increasing the chemical stability of tested compounds while no influence on enantiomerization kinetic was observed. Therefore it is possible to suppose that 5-arylbenzothiadiazine derivatives should not hydrolysed in vivo while they should rapidly racemized in aqueous solvents. The method could represents a rapid and value tool to predict chemical and configurational stability of new chemical entities to decrease the number of animal studies.