Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method.

BACKGROUND: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. RESULTS: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. CONCLUSION: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

Elagolix Sodium Salt and Its Synthetic Intermediates: A Spectroscopic, Crystallographic, and Conformational Study.

Elagolix sodium salt is the first marketed orally active non-peptide gonadotropin-releasing hormone receptor antagonist (GnRHR-ant) for the management of hormone dependent diseases, such as endometriosis and uterine fibroids. Despite its presence on the market since 2018, a thorough NMR analysis of this drug, together with its synthetic intermediates, is still lacking. Hence, with the aim of filling this literature gap, we here performed a detailed NMR investigation, which allowed the complete assignment of the 1H, 13C, and 15N NMR signals. These data allowed, with the support of the conformational analysis, the determination of the stereochemical profile of the two atropisomers, detectable in solution. Moreover, these latter were also detected by means of cellulose-based chiral HPLC, starting from a sample prepared through an implemented synthetic procedure with respect to the reported ones. Overall, these results contribute to further understanding of the topic of atropisomerism in drug discovery and could be applied in the design of safe and stable analogs, endowed with improved target selectivity.

Heat-Treated Lysozyme Hydrochloride: A Study on Its Structural Modifications and Anti-SARS-CoV-2 Activity.

Lysozyme (E.C. 3.2.1.17), an about 14 kDa protein and pI 11, widely spread in nature, is present in humans mainly in milk, saliva, and intestinal mucus as a part of innate defense mechanisms. It is endowed with antimicrobial activity due to its action as an N-acetylmuramidase, cleaving the 1-4ß glycosidic linkage in the peptidoglycan layer of Gram-positive bacteria. This antimicrobial activity is exerted only against a limited number of Gram-negative bacteria. Different action mechanisms are proposed to explain its activity against Gram-negative bacteria, viruses, and fungi. The antiviral activity prompted the study of a possible application of lysozyme in the treatment of SARS-CoV-2 infections. Among the different sources of lysozyme, the chicken egg albumen was chosen, being the richest source of this protein (c-type lysozyme, 129 amino acids). Interestingly, the activity of lysozyme hydrochloride against SARS-CoV-2 was related to the heating (to about 100 °C) of this molecule. A chemical-physical characterization was required to investigate the possible modifications of native lysozyme hydrochloride by heat treatment. The FTIR analysis of the two preparations of lysozyme hydrochloride showed appreciable differences in the secondary structure of the two protein chains. HPLC and NMR analyses, as well as the enzymatic activity determination, did not show significant modifications.

Synthesis and automated fluorine-18 radiolabeling of new PSMA-617 derivatives with a CuAAC radiosynthetic approach.

In the last decade, the development of new radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research, especially focusing on the prostate-specific membrane antigen (PSMA), an antigen which is upregulated in prostate, as well as in other tumor cells. A large variety of PSMA ligands have been radiolabeled, to date. Among the various derivatives, PSMA-617 resulted to be one of the most interesting in terms of interaction with the antigen and clinical properties, and its lutetium-177 labeled version has recently been approved by regulatory agencies for therapeutic purposes. For this reasons, the radiolabeling with fluorine-18 of a PSMA-617 derivative might be of interest. Beside other methodologies to radiolabel macromolecules with fluorine-18, the "click-chemistry" approach resulted to be very useful, and the copper-catalyzed azide-alkyne cycloaddition (CuAAC) is considered one of most efficient and reliable. This paper proposes the synthesis of a suitable precursor for the radiolabeling with fluorine-18 of a new PSMA-617 derivative. The whole radiosynthetic procedure has been fully automated, and the final product, which proved to be stable in plasma, has been obtained with radiochemical yield and purity suitable for subsequent preclinical studies.

Applications of Lysozyme, an Innate Immune Defense Factor, as an Alternative Antibiotic.

Lysozyme is a ~14 kDa protein present in many mucosal secretions (tears, saliva, and mucus) and tissues of animals and plants, and plays an important role in the innate immunity, providing protection against bacteria, viruses, and fungi. Three main different types of lysozymes are known: the c-type (chicken or conventional type), the g-type (goose type), and the i-type (invertebrate type). It has long been the subject of several applications due to its antimicrobial properties. The problem of antibiotic resistance has stimulated the search for new molecules or new applications of known compounds. The use of lysozyme as an alternative antibiotic is the subject of this review, which covers the results published over the past two decades. This review is focused on the applications of lysozyme in medicine, (the treatment of infectious diseases, wound healing, and anti-biofilm), veterinary, feed, food preservation, and crop protection. It is available from a wide range of sources, in addition to the well-known chicken egg white, and its synergism with other compounds, endowed with antimicrobial activity, are also summarized. An overview of the modified lysozyme applications is provided in the form of tables.

An Outline of the Latest Crystallographic Studies on Inhibitor-Enzyme Complexes for the Design and Development of New Therapeutics against Tuberculosis.

The elucidation of the structure of enzymes and their complexes with ligands continues to provide invaluable insights for the development of drugs against many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. The structural elucidation of enzyme-ligand complexes is fundamental to identify hot-spots, define possible interaction sites, and elaborate strategies to develop optimized molecules with high affinity. This review offers a critical and comprehensive overview of the most recent structural information on traditional and emerging mycobacterial enzymatic targets. A selection of more than twenty enzymes is here discussed, with a special emphasis on the analysis of their binding sites, the definition of the structure-activity relationships (SARs) of their inhibitors, and the study of their main intermolecular interactions. This work corroborates the potential of structural studies, substantiating their relevance in future anti-mycobacterial drug discovery and development efforts.

Ovotransferrin Supplementation Improves the Iron Absorption: An In Vitro Gastro-Intestinal Model.

Transferrins constitute the most important iron regulation system in vertebrates and some invertebrates. Soluble transferrins, such as bovine lactoferrin and hen egg white ovotransferrin, are glycoproteins with a very similar structure with lobes that complex with iron. In this in vitro study, a comparison of bovine lactoferrin and ovotransferrin was undertaken to confirm the comparability of biological effects. An in vitro gastric barrier model using gastric epithelial cells GTL-16 and an in vitro intestinal barrier model using CaCo-2 cells was employed to evaluate iron absorption and barrier integrity. An analysis of the molecular pathways involving DMT-1 (divalent metal transporter 1), ferritin and ferroportin was also carried out. These in vitro data demonstrate the activity of both 15% saturated and 100% saturated ovotransferrin on the iron regulation system. Compared with the commercial bovine lactoferrin, both 15% saturated and 100% saturated ovotransferrin were found to act in a more physiological manner. Based on these data, it is possible to hypothesise that ovotransferrin may be an excellent candidate for iron supplementation in humans; in particular, 15% saturated ovotransferrin is the overall best performing product. In vivo studies should be performed to confirm this in vitro data.

Vecuronium bromide and its advanced intermediates: A crystallographic and spectroscopic study.

Vecuronium bromide (Piperidinium, 1-[(2ß,3α,5α,16ß,17ß)-3,17-bis(acetyloxy)-2-(1-piperidinyl)androstan-16-yl]-1-methyl-, bromide; Norcuron®) has been extensively used in anesthesiology practice as neuromuscular blocking agent since its launch on the market in 1982. However, a detailed crystallographic and NMR analysis of its advanced synthetic intermediates is still lacking. Hence, with the aim of filling this literature gap, vecuronium bromide was prepared starting from the commercially available 3ß-hydroxy-5α-androstan-17-one (epiandrosterone), implementing some modifications to a traditional synthetic procedure. A careful NMR study allowed the complete assignment of the 1H, 13C, and 15N NMR signals of vecuronium bromide and its synthetic intermediates. The structural and stereochemical characterization of 2ß,16ß-bispiperidino-5α-androstane-3α,17ß-diol, the first advanced synthetic intermediate carrying all the stereocenters in the final configuration, was described by means of single-crystal X-ray diffraction and Hirshfeld surface analysis, allowing a detailed conformational investigation.

Crystallographic and NMR Investigation of Ergometrine and Methylergometrine, Two Alkaloids from Claviceps purpurea.

Ergometrine and methylergometrine are two alkaloids that are used as maleate salts for the prevention and control of postpartum hemorrhage. Although the two molecules have been known for a long time, few and discordant crystallographic and NMR spectroscopic data are available in the literature. With the aim of providing more conclusive data, we performed a careful NMR study for the complete assignment of the 1H, 13C, and 15N NMR signals of ergometrine, methylergometrine, and their maleate salts. This information allowed for a better definition of their conformational equilibria. In addition, the stereochemistry and the intermolecular interactions in the solid state of the two maleate salts were deeply investigated by means of single-crystal X-ray diffraction, showing the capability of these derivatives to act as both hydrogen-bond donors and acceptors, and evidencing a correlation between the number of intermolecular interactions and their different solubility.

A New Chemoenzymatic Synthesis of the Chiral Key Intermediate of the Antiepileptic Brivaracetam.

Brivaracetam is a new anticonvulsant compound, recently approved as an antiepileptic drug. This drug substance presents a 4-substituted pyrrolidone structure: the (4R)-configuration of the stereocenter present on the heterocyclic ring is the main target of the synthesis. The described method allows to prepare the suitable optically pure 2-substituted primary alcohol by means of a Pseudomonas fluorescens lipase-catalyzed transesterification. The obtained (2R)-alcohol was easily transformed into the (3R)-3-propylbutyrolactone, an advanced intermediate of brivaracetam. The described synthetic pathway is completed with the chromatographic methods and the NMR analyses necessary to establish the chemical and the optical purity of the intermediates and of the final lactone.

Full spectroscopic characterization of two crystal pseudopolymorphic forms of the antiandrogen cortexolone 17α-propionate for topic application.

Cortexolone-17α-propionate (CP) is a topically active antiandrogen useful in the treatment of skin disorders. In the solid state, three anhydrous forms of this drug (CPI, CPII and CPIII) occur, together with one hydrated crystal (CPW). The single crystal structure of the monohydrated phase, CPW, compared with that of the anhydrous form CPIII, shows a markedly different solid state behavior. These latter pseudopolymorphic forms have also been fully characterized by spectroscopic methods.

Crystallographic and spectroscopic study on a known orally active progestin.

6,17α-Dimethyl-4,6-pregnadiene-3,20-dione (medrogestone, 2) is for a long time known steroid endowed with progestational activity. In order to study its crystallographic and NMR spectroscopic properties with the aim to fill the literature gap, we prepared medrogestone following a traditional procedure. A careful NMR study allowed the complete assignment of the (1)H and (13)C NMR signals not only of medrogestone but also of its synthetic intermediates. The structural and stereochemical characterizations of medrogestone together with its precursor 17α-methyl-3-ethoxy-pregna-3,5-dien-20-one were described by means of X-ray analysis, allowing a deepened conformational investigation.

Crystallographic, spectroscopic, and theoretical investigation of the efficiently separated 21R and 21S-diastereoisomers of argatroban.

Argatroban (I), a potent noncovalent reversible thrombin inhibitor, is used as an anticoagulant for the parenteral treatment of heparin-induced thrombocytopenia (HIT) patients. By virtue of its pharmacological properties and the well-balanced risks and benefits, argatroban is now emerging as a clinically relevant antithrombotic agent. The availability of this drug as a mixture of 21R and 21S-diastereoisomers, in a ratio of roughly 64:36, prompted us to design an efficient separation setup of the two epimers. We pursued our efforts on their detailed structural analysis with the aim of understanding their different activity and aqueous solubility. These investigations were accompanied by a modeling study of the two diastereoisomers, with particular attention on the easy interconverting half-chair of the tetrahydroquinoline system and its preferred conformation, which is determined by the configuration at C21. These results, together with the analysis of their physicochemical profiles, provide new useful information for the development of the individual diastereoisomers.

Evaluation, synthesis and characterization of tacrolimus impurities.

Tacrolimus is an immunosuppressant macrolactam of fermentative origin. By means of HPLC, LC-MS and NMR analyses, coupled with the reference standard synthesis, the main impurities of tacrolimus bulk drug samples were identified and their chemical-physical properties reported. Known ascomycin and tautomers I and II were detected. The correct relative retention time HPLC value of 39,40-dihydro tacrolimus was established. The not described 23,24-anhydro derivative was detected and completely characterized. A full characterization of ascomycin and 39,40-dihydro tacrolimus was also reported.

Corticosteroids 21-glucuronides: synthesis and complete characterization by (1)H and (13)C NMR.

Despite the important physiological role of the corticosteroids glucuronides, very poor NMR data for this class of compounds are reported. For this reason we prepared a set of corticosteroids and submitted them to a detailed NMR study. A complete assignment of (1)H and (13)C signals was accomplished arranging mono- and two-dimensional NMR techniques.

Anti-tumor-Promoting activity of tibolone and its metabolites.

The aim of this study was to evaluate the cancer chemopreventive potential of the widely prescribed drug tibolone (17alpha-ethynyl-7alpha-methyl-5(10)-estren-3-one, CAS 5630-53-5) and its main metabolites, 17alpha-ethynyl-7alpha-methyl-4-estren-3-one (CAS 1162-60-3), 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3alpha,17beta-diol (CAS 100239-44-9) and 17alpha-ethynyl-7alpha-methyl-5(10)-estrene-3beta,17beta-diol (CAS 100239-45-0), by studying their anti-tumor-promoting activity. To this aim the test compounds were submitted to the short term in vitro assay for the inhibition of Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) as a primary screening for anti-tumor promoters. All the compounds showed high inhibitory activity and low cytotoxicity as compared to literature data. To extend the study to an animal model, tibolone and its 3alpha-hydroxy metabolite (CAS 100239-44-9) were also assayed in the in vivo two-stage on mouse skin carcinogenesis test, exhibiting significant inhibitory effects on TPA promoted mouse skin papillomas formation. A comparison with literature data indicated them as more potent compounds than other steroids previously studied such as digitoxigenin, cortisone, hydrocortisone, and prednisolone.

Estimation and characterisation of budesonide tablets impurities.

Budesonide is the 16alpha,17alpha-acetal of 16alpha-hydroxyprednisolone with n-butyraldehyde, endowed with anti-inflammatory activity. In a sample of budesonide tablets, kept for 3 years at 25 degrees C and 60% RH unknown impurities, not reported in European Pharmacopoiea, were present. Their identification was achieved by means of chemical and spectroscopic methods.

Complete 1H and 13C assignments of (21R) and (21S) diastereomers of argatroban.

The complete 1H and 13C NMR assignments are reported for the antithrombotic (21R)- and (21S)-argatroban by 1D and 2D NMR experiments (HSQC, HMBC, NOESY and 1H--1H COSY). Some well-resolved signals could be used for an accurate measurement of the diastereomeric composition of argatroban.

Determination of aloesin and aloeresin A for the detection of aloe in beverages.

This work describes a sensitive high-performance liquid chromatography (HPLC) method for the quantification of aloesin and aloeresin A in alcoholic beverages containing aloe as a flavoring agent. The compounds were prepared from Aloe ferox juice. Sephadex LH20 and ion-exchange resin AG1X2 column chromatography were used for aloesin. Aloeresin A was obtained by Sephadex LH20 and silica gel column chromatography followed by purification on Discovery DSC-18 solid-phase extraction tubes. A 98 mg amount of aloesin (>99% purity) and 34 mg of aloeresin A (>98% purity) were recovered from 2.5 g of aloe juice. The HPLC method was validated, and intra- and interday performances were established. In-house validation was carried out by analyzing samples of beverages with and without aloe as a flavoring agent.

A full conformational characterization of 13-ethylprogestogens through theoretical calculations and nuclear magnetic resonance spectroscopy.

The conformational preferences of a group of 13-ethylsteroids and related estranes have been determined through theoretical calculations at the B3LYP/6-31G(*) level in order to ascertain differences and similarities in the conformational behavior which might, in principle, influence the activity. Attention was focussed on two geometrical features usually related to the progestational activity of this class of compounds, namely, the inversion of the A ring and the orientation of the 13-ethyl group. The present calculations show that the absence of the C10 methyl group, like in levonorgestrel, 3-ketodesogesterel, and gestodene, makes the inversion of the A ring easier than in norethisterone and ethisterone even if in any case the 1alpha,2beta-half-chair conformation remains preferred. The preference of the ethyl group for the trans orientation is maintained by all the 13-ethylprogestogens though in gestodene also the g(+)gauche conformation is significantly populated. This preference was experimentally supported through a high field NMR spectroscopy study of the ethylsteroids.