The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented an enormous challenge to health care systems and medicine. As a result of global research efforts aimed at preventing and effectively treating SARS-CoV-2 infection, vaccines with fundamentally new mechanisms of action and some small-molecule antiviral drugs targeting key proteins in the viral cycle have been developed. The most effective small-molecule drug approved to date for the treatment of COVID-19 is PaxlovidTM, which is a combination of two protease inhibitors, nirmatrelvir and ritonavir. Nirmatrelvir is a reversible covalent peptidomimetic inhibitor of the main protease (Mpro) of SARS-CoV-2, which enzyme plays a crucial role in viral reproduction. In this combination, ritonavir serves as a pharmacokinetic enhancer, it irreversibly inhibits the cytochrome CYP3A4 enzyme responsible for the rapid metabolism of nirmatrelvir, thereby increasing the half-life and bioavailability of nirmatrelvir. In this tutorial review, we summarize the development and pharmaceutical chemistry aspects of Paxlovid, covering the evolution of protease inhibitors, the warhead design, synthesis and the mechanism of action of nirmatrelvir, as well as the synthesis of ritonavir and its CYP3A4 inhibition mechanism. The efficacy of Paxlovid to novel virus mutants is also overviewed.

In vitro and in vivo antiplasmodial evaluation of sugar-modified nucleoside analogues.

Drug-resistant Plasmodium falciparum (Pf) infections are a major burden on the population and the healthcare system. The establishment of Pf resistance to most existing antimalarial therapies has complicated the problem, and the emergence of resistance to artemisinin derivatives is even more concerning. It is increasingly difficult to cure malaria patients due to the limited availability of effective antimalarial drugs, resulting in an urgent need for more efficacious and affordable treatments to eradicate this disease. Herein, new nucleoside analogues including morpholino-nucleoside hybrids and thio-substituted nucleoside derivatives were prepared and evaluated for in vitro and in vivo antiparasitic activity that led a few hits especially nucleoside-thiopyranoside conjugates, which are highly effective against Pf3D7 and PfRKL-9 strains in submicromolar concentration. One adenosine derivative and four pyrimidine nucleoside analogues significantly reduced the parasite burden in mouse models infected with Plasmodium berghei ANKA. Importantly, no significant hemolysis and cytotoxicity towards human cell line (RAW) was observed for the hits, suggesting their safety profile. Preliminary research suggested that these thiosugar-nucleoside conjugates could be used to accelerate the antimalarial drug development pipeline and thus deserve further investigation.

Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Triaza-tricyclanos - synthesis of a new class of tricyclic nucleoside analogues by stereoselective cascade cyclocondensation.

Herein, we report a stereoselective synthesis of a novel type of conformationally constrained nucleoside analogue in which the sugar part is replaced by a new symmetrical tricycle consisting of a morpholine ring condensed with two imidazolidines. 1,5-Dialdehydes obtained from trityl- and dimethoxytrityl-protected uridine, ribothymidine, inosine, cytidine, adenosine and guanosine by metaperiodate oxidation were reacted with N1,N3-dibenzyl-1,2,3-triaminopropane; the latter reactant was produced using a new method that avoids explosive intermediates. Reactions of dialdehydes with propane-triamine via cascade tricyclization resulted in the corresponding triaza-tricyclic derivatives bearing three new stereogenic centers in high yields. Out of the eight possible diastereoisomers, one stereoisomer was formed in each case due to the chiral control of the starting nucleoside-dialdehydes and the steric constraint of the condensed ring system. The absolute configuration of the new stereotriad was determined by X-ray diffraction and NMR experiments. A mechanistic study performed under reductive conditions to trap the presumed bicyclic intermediate showed that the triamine reactant first attacks the 2'-aldehyde group, followed by a rapid bicyclization to form the imidazolidino-morpholine unit.

N-Fluoroalkylated Morpholinos - a New Class of Nucleoside Analogues.

The first concise and efficient synthesis of some fluorine-containing morpholino nucleosides has been developed. One synthetic strategy was based on the oxidative ring cleavage of the vicinal diol unit of uridine, cytidine adenosine and guanosine derivatives, followed by cyclisation of the dialdehyde intermediates by double reductive amination with fluorinated primary amines to obtain various N-fluoroalkylated morpholinos. Another approach involved cyclisation of the diformyl intermediates with ammonia source, followed by dithiocarbamate formation and desulfurization-fluorination with diethylaminosulfur trifluoride yielding the corresponding morpholine-based nucleoside analogues with a N-CF3 element in their structure.

Synthesis and Anticancer and Antiviral Activities of C-2'-Branched Arabinonucleosides.

d-Arabinofuranosyl-pyrimidine and -purine nucleoside analogues containing alkylthio-, acetylthio- or 1-thiosugar substituents at the C2' position were prepared from the corresponding 3',5'-O-silylene acetal-protected nucleoside 2'-exomethylenes by photoinitiated, radical-mediated hydrothiolation reactions. Although the stereochemical outcome of the hydrothiolation depended on the structure of both the thiol and the furanoside aglycone, in general, high d-arabino selectivity was obtained. The cytotoxic effect of the arabinonucleosides was studied on tumorous SCC (mouse squamous cell) and immortalized control HaCaT (human keratinocyte) cell lines by MTT assay. Three pyrimidine nucleosides containing C2'-butylsulfanylmethyl or -acetylthiomethyl groups showed promising cytotoxicity at low micromolar concentrations with good selectivity towards tumor cells. SAR analysis using a methyl ß-d-arabinofuranoside reference compound showed that the silyl-protecting group, the nucleobase and the corresponding C2' substituent are crucial for the cell growth inhibitory activity. The effects of the three most active nucleoside analogues on parameters indicative of cytotoxicity, such as cell size, division time and cell generation time, were investigated by near-infrared live cell imaging, which showed that the 2'-acetylthiomethyluridine derivative induced the most significant functional and morphological changes. Some nucleoside analogues also exerted anti-SARS-CoV-2 and/or anti-HCoV-229E activity with low micromolar EC50 values; however, the antiviral activity was always accompanied by significant cytotoxicity.

The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides.

Nucleic acids play a central role in human biology, making them suitable and attractive tools for therapeutic applications. While conventional drugs generally target proteins and induce transient therapeutic effects, nucleic acid medicines can achieve long-lasting or curative effects by targeting the genetic bases of diseases. However, native oligonucleotides are characterized by low in vivo stability due to nuclease sensitivity and unfavourable physicochemical properties due to their polyanionic nature, which are obstacles to their therapeutic use. A myriad of synthetic oligonucleotides have been prepared in the last few decades and it has been shown that proper chemical modifications to either the nucleobase, the ribofuranose unit or the phosphate backbone can protect the nucleic acids from degradation, enable efficient cellular uptake and target localization ensuring the efficiency of the oligonucleotide-based therapy. In this review, we present a summary of structure and properties of artificial nucleic acids containing nucleobase, sugar or backbone modifications, and provide an overview of the structure and mechanism of action of approved oligonucleotide drugs including gene silencing agents, aptamers and mRNA vaccines.

The Very First Modification of Pleuromutilin and Lefamulin by Photoinitiated Radical Addition Reactions-Synthesis and Antibacterial Studies.

Pleuromutilin is a fungal diterpene natural product with antimicrobial properties, semisynthetic derivatives of which are used in veterinary and human medicine. The development of bacterial resistance to pleuromutilins is known to be very slow, which makes the tricyclic diterpene skeleton of pleuromutilin a very attractive starting structure for the development of new antibiotic derivatives that are unlikely to induce resistance. Here, we report the very first synthetic modifications of pleuromutilin and lefamulin at alkene position C19-C20, by two different photoinduced addition reactions, the radical thiol-ene coupling reaction, and the atom transfer radical additions (ATRAs) of perfluoroalkyl iodides. Pleuromutilin were modified with the addition of several alkyl- and aryl-thiols, thiol-containing amino acids and nucleoside and carbohydrate thiols, as well as perfluoroalkylated side chains. The antibacterial properties of the novel semisynthetic pleuromutilin derivatives were investigated on a panel of bacterial strains, including susceptible and multiresistant pathogens and normal flora members. We have identified some novel semisynthetic pleuromutilin and lefamulin derivatives with promising antimicrobial properties.

Tightly linked morpholino-nucleoside chimeras: new, compact cationic oligonucleotide analogues.

The polyanionic phosphodiester backbone of nucleic acids contributes to high nuclease sensitivity and low cellular uptake and is therefore a major obstacle to the biological application of native oligonucleotides. Backbone modifications, particularly charge alterations is a proven strategy to provide artificial oligonucleotides with improved properties. Here, we describe the synthesis of a new type of oligonucleotide analogues consisting of a morpholino and a ribo- or deoxyribonucleoside in which the 5'-amino group of the nucleoside unit provides the nitrogen of the morpholine ring. The synthetic protocol is compatible with trityl and dimethoxytrityl protecting groups and azido functionality, and was extended to the synthesis of higher oligomers. The chimeras are positively charged in aqueous medium, due to the N-alkylated tertiary amine structure of the morpholino unit.

MTT Test and Time-lapse Microscopy to Evaluate the Antitumor Potential of Nucleoside Analogues.

BACKGROUND/AIM: Conventional viability tests, help to screen the cellular effects of candidate molecules, but the endpoint of these measurements lacks sufficient information regarding the molecular aspects. A non-invasive, easy-to-setup live-cell microscopic method served to in-depth analysis of mechanisms of potential anticancer drugs. MATERIALS AND METHODS: The proposed method combining the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test with time-lapse scanning microscopy (TLS), provided additional data related to the cell-cycle and the dynamic properties of cell morphology. Apoptotic and necrotic events became detectable with these methods. RESULTS: Quantification of the results was assisted by image analysis of the acquired image sequences. After demonstrating the potential of the TLS method, a series of experiments compared the in vitro effect of a known and a newly synthesized nucleoside analogue. CONCLUSION: The proposed approach provided a more in-depth insight into the cellular processes that can be affected by known chemotherapeutic agents including nucleoside analogues rather than applying repeated individual treatments.

Synthesis and oligomerization of cysteinyl nucleosides.

Nucleoside and nucleic acid analogues are known to possess a considerable therapeutic potential. In this work, by coupling cysteine to nucleosides, we successfully synthesized compounds that may not only have interesting biological properties in their monomeric form, but can be used beyond that, for oligomerization, in order to produce new types of synthetic nucleic acids. We elaborated different strategies for the synthesis of cysteinyl nucleosides as monomers of cysteinyl nucleic acids using nucleophilic substitution or thiol-ene coupling as a synthetic tool, and utilised on two complementary nucleosides, uridine and adenosine. Dipeptidyl dinucleosides and pentameric cysteinyl uridine were prepared from the monomeric building blocks, which are the first members of a new class of peptide nucleic acids containing the entire ribofuranosyl nucleoside units bound to the peptide backbone.

Stereoselective Thioconjugation by Photoinduced Thiol-ene Coupling Reactions of Hexo- and Pentopyranosyl d- and l-Glycals at Low-Temperature-Reactivity and Stereoselectivity Study.

A comprehensive optimization and mechanistic study on the photoinduced hydrothiolation of different d- and l- hexo- and pentoglycals with various thiols was performed, at the temperature range of RT to -120 °C. Addition of thiols onto 2-substituted hexoglycals proceeded with complete 1,2-cis-α-stereoselectivity in all cases. Hydrothiolation of 2-substituted pentoglycals resulted in mixtures of 1,2-cis-α- and -ß-thioglycosides of varying ratio depending on the configuration of the reactants. Hydrothiolation of unsubstituted glycals at -80 °C proceeded with excellent yields and, except for galactal, provided the axially C2-S-linked isomers with high selectivity. Cooling was always beneficial to the efficacy, increased the yields and in most cases significantly raised the stereoselectivity. The suggested mechanism explains the different conformational preferences of the intermediate carbon-centered radicals, which is a crucial factor in the stereoselectivity of the reactions.

Synthesis and Cytostatic Effect of 3'-deoxy-3'-C-Sulfanylmethyl Nucleoside Derivatives with d-xylo Configuration.

A small library of 3'-deoxy-C3'-substituted xylofuranosyl-pyrimidine nucleoside analogues were prepared by photoinduced thiol-ene addition of various thiols, including normal and branched alkyl-, 2-hydroxyethyl, benzyl-, and sugar thiols, to 3'-exomethylene derivatives of 2',5'-di-O-tert-butyldimethylsilyl-protected ribothymidine and uridine. The bioactivity of these derivatives was studied on tumorous SCC (mouse squamous carcinoma cell) and immortalized control HaCaT (human keratinocyte) cell lines. Several alkyl-substituted analogues elicited promising cytostatic activity in low micromolar concentrations with a slight selectivity toward tumor cells. Near-infrared live-cell imaging revealed SCC tumor cell-specific mitotic blockade via genotoxicity of analogue 10, bearing an n-butyl side chain. This analogue essentially affects the chromatin structure of SCC tumor cells, inducing a condensed nuclear material and micronuclei as also supported by fluorescent microscopy. The results highlight that thiol-ene chemistry represents an efficient strategy to discover novel nucleoside analogues with non-natural sugar structures as anticancer agents.

Promotion of a Reaction by Cooling: Stereoselective 1,2-cis-α-Thioglycoconjugation by Thiol-Ene Coupling at -80 °C.

The photoinitiated thiol-ene coupling reactions of 2-substituted glycals were studied as a generally applicable strategy for stereoselective 1,2-cis-α-thioconjugation. Although all glycals reacted with full α-selectivity, the efficacy of the reactions varied in a broad range depending on their configuration and glycals bearing axial acetoxy substituents reacted with very low efficacy at room temperature. The study revealed that the reaction progress could be promoted by cooling and inhibited by heating. At -80 °C, the equilibrium of the rapidly reversible addition of the thiyl radical to alkenes is shifted almost completely toward products, leading to efficient addition reactions. By exploiting this unique temperature effect a series of α-thio-l-fucosides, -d-galactosides, and d-GlcNAc derivatives were prepared with high efficacy and complete stereoselectivity.

A low-temperature, photoinduced thiol-ene click reaction: a mild and efficient method for the synthesis of sugar-modified nucleosides.

Sugar-modified nucleosides are prime synthetic targets in anticancer and antiviral drug development. Radical mediated thiol-ene coupling was applied for the first time on nucleoside enofuranoside derivatives to produce a broad range of thio-substituted d-ribo, -arabino, -xylo and l-lyxo configured pyrimidine nucleosides. In contrast to the analogous reactions of simple sugar exomethylenes, surprisingly, hydrothiolation of nucleoside alkenes under the standard conditions of various initiation methods showed low to moderate yields and very low stereoselectivity. Optimizing the reaction conditions, we have found that cooling the reaction mixture has a significant beneficial effect on both the conversion and the stereoselectivity, and UV-light initiated hydrothiolation of C2'-, C3'- and C4'-exomethylene derivatives of nucleosides at -80 °C proceeded in good to high yields, and, in most cases, in excellent diastereoselectivity. Beyond the temperature, the solvent, the protecting groups on nucleosides and, in some cases, the configuration of the thiols also affected the stereochemical outcome of the additions. The anomalous l-lyxo diastereoselectivity observed upon the addition of 1-thio-ß-d-gluco- and galactopyranose derivatives onto C4',5'-unsaturated uridines is attributed to steric mismatch between the d-ribo C4'-radical intermediates and the ß-configured 1-thiosugars.

A three-component reagent system for rapid and mild removal of O-, N- and S-trityl protecting groups.

A new reagent system consisting of a Lewis acid such as BF3·Et2O or Cu(OTf)2, the mild protic acid hexafluoroisopropanol and the reducing quenching agent triethylsilane was elaborated for O-, N- and S-detritylation of nucleoside, carbohydrate and amino acid derivatives. The method is compatible with acetyl, silyl, acetal and Fmoc groups.