Gonadotropin-releasing hormone (GnRH) is the main regulator of the reproductive system, acting on gonadotropic cells by binding to the GnRH1 receptor (GnRH1R). Traditionally, therapies targeting this receptor have relied on peptide modulators, which required subcutaneous or intramuscular injections. Due to the limitations of the parenteral administrations, there is a growing interest in developing oral small molecule modulators of GnRH1R as more convenient therapeutic alternatives. In this study, we examined the potential of chemically modifying elagolix, the first approved non-peptide, orally active GnRH1R antagonist, to increase its atropisomeric properties by introducing new moieties. We designed and synthesized the thio-uracil (1) 1 ) and cytosine (2) 2 ) derivatives of elagolix, both demonstrating GnRH1R antagonistic activities, with EC50 50 values of 39 and 110 nM, respectively. The atropisomers of 1 and 2 were efficiently separated using silica gel chromatography, and extensive NMR investigation, supported by Density Functional Theory (DFT) calculations, allowed us to define their conformations and rotational barriers. Docking and Molecular Dynamics (MD) studies revealed that 1 and 2 bind to GnRH1R with Delta G values comparable to elagolix, but through distinct binding modes. These results highlight the potential of non-peptide modulators to effectively modulate GnRH1R activity and pave the way for developing novel modulators.

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 1-4β 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.

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.

Vecuronium bromide (Piperidinium, 1-[(2β,3α,5α,16β,17β)-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 3β-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 2β,16β-bispiperidino-5α-androstane-3α,17β-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.

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.

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.

A new chemoenzymatic method has been developed for the synthesis of (S)- and (R)-N-(6-hydroxy-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl) acetamide, two key synthons for the preparation of (S)-pramipexole, an anti-Parkinson drug, and its enantiomer dexpramipexole, which is currently under investigation for the treatment of eosinophil-associated disorders. These two building blocks have been obtained in good yields and high enantiomeric excess (30% and >98% ee for the R-enantiomer, and 31% and >99% ee for the S- one) through a careful optimization of the reaction conditions, starting from the corresponding racemic mixture and using two consecutive irreversible transesterifications, catalyzed by Candida antarctica lipase type A. Single crystal X-ray analysis has been carried out to unambiguously define the stereochemistry of the two enantiomers, and to explore in depth their three-dimensional features.

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.

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.

Fluoropyrimidines 5-FluorouracilInterest in the fluoropyrimidines stemmed from studies of the metabolism of uracil in rat hepatoma cells.The observation that these cells utilize uracil more avidly than normal rat intestinal mucosa prompted the preparation of fluorinated pyrimidines in order to improve disruption of tumor DNA biosynthesis. 3n 1957 5-fluorouracil (5-FU) 1 was synthesized by Heidelberger et al., 4 with the aim of blocking metabolism in malignant cells.The replacement of a hydrogen atom at C-5 by the fluorine atom modifies the interaction with the active sites of enzymes involved in metabolism.This antimetabolite, although toxic, is still one of the most widely used agents against solid tumors.Its action is due to two different mechanisms: 5 after penetration into the cell, 5-FU 1 is transformed into the 5-fluorouridine triphosphate that mimics UTP, is recognized by RNA polymerase and consequently incorporated into RNA.The most significant action, however, is due to the 5-FU conversion into 5-fluoro-2 0 -deoxyuridine (FdUMP) 2, a known inhibitor of thymidylate synthetase (TS), a key enzyme in the DNA synthesis. 6,7TS, in the presence of methylene tetrahydrofolate and deoxyuridine monophosphate (dUMP) 3 forms a ternary complex that catalyzes the substitution of 5-H uracil with a methyl group, affording thymine.If FdUMP 2 is present, the above ternary complex is not able to carry out this reaction, due to the presence of fluorine in the 5-position: the formation of TMP 4 (2 0 -deoxythymidine 5 0 -monophosphate), the only nucleotide precursor specific to DNA, is, therefore, blocked (Scheme 1), decreasing the availability of TTP (2 0 -deoxythymidine 5 0 -triphosphate) for DNA synthesis.

Capecitabine is a 5'-deoxynucleoside endowed with antitumoral activity. We planned a new approach to its synthesis: a cross linked enzyme aggregate subtilisin (Alcalase CLEA)-catalyzed alcoholysis allowed the selective deprotection of primary acetyl ester of the N-1-(2',3',5'-tri-O-acetyl-beta-o-ribofuranosyl)-5-fluoro-N-4-(n-pentyloxycarbonyl)cytosine affording the corresponding 5'-hydroxyderivative; the 5'-alcohol was transformed into the methyl group of capecitabine after a careful investigation about the best reducing agent and reaction conditions. (C) 2015 Elsevier Ltd. All rights reserved.

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.

L'invention concerne un procede de preparation de la forme amorphe du regadenoson de formule avec de nouvelles formes polymorphes cristallines E, F et G, et leurs procedes de preparation. Une forme amorphe du regadenoson peut etre preparee dans des conditions de reaction douces a une haute purete chimique (>99,6%) et une haute stabilite a la chaleur. Une forme cristalline anhydre particulierement stable d'un point de vue thermodynamique du regadenoson (forme G) est egalement decrite, et presente une haute stabilite lorsqu'elle n'est pas exposee a 90 % RH a 25 °C pendant 96 heures, ni a la chaleur jusqu'a 200 °C.

A biocatalyzed reduction of a prochiral bicyclic ketone afforded enantiomerically pure (R)-2-acetylamino-6-hydroxy-4,5,6,7-tetrahydrobenzothiazole, a synthon of the anti-Parkinson (S)-pramipexole and its (R)-isomer, which is currently under investigation for the treatment of amyotrophic lateral sclerosis (ALS).

The synthetic antithrombotic argatroban is a dipeptide between the nonproteogenic (2R,4R)-4-methyl-2-piperidine carboxylic acid and l-arginine, in turn bonded to a 3-methyltetrahydroquinoline sulfonyl group; the drug is usually prepared and administered as a mixture of C-21-diastereoisomers. By means of a biocatalytic transformation enantiomerically pure (R)- and (S)-synthons, suitable for the synthesis of separate (21R)- and (21S)- argatroban, were obtained.