Ultrapotent influenza hemagglutinin fusion inhibitors developed through SuFEx-enabled high-throughput medicinal chemistry.

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.

SuFEx-based chemical diversification for the systematic discovery of CRBN molecular glues.

Molecular glues are small molecules that stabilize protein-protein interactions, enabling new molecular pharmacologies, such as targeted protein degradation. They offer advantages over proteolysis targeting chimeras (PROTACs), which present challenges associated with the size and properties of heterobifunctional constructions, but glues lack the rational design principles analogous to PROTACs. One notable exception is the ability to alter the structure of Cereblon (CRBN)-based molecular glues and redirect their activity toward new neo-substrate proteins. We took a focused approach toward modifying the CRBN ligand, 5'-amino lenalidomide, to alter its neo-substrate specificity using high-throughput chemical diversification by parallelized sulfur(VI)-fluoride exchange (SuFEx) transformations. We synthesized over 3,000 analogs of 5'-amino lenalidomide using this approach and screened the crude products using a phenotypic screen for cell viability, identifying dozens of analogs with differentiated activity. We characterized four compounds that degrade G-to-S phase transition 1 (GSPT1) protein, providing a proof-of-concept model for SuFEx-based discovery of CRBN molecular glues.

Synthesis of thiodisaccharides related to 4-thiolactose. Specific structural modifications increase the inhibitory activity against E. coli ß-galactosidase.

In the search for new glycosidase inhibitors, a set of benzyl ß-D-Gal-S-(1→4)-3-deoxy-4-thio-α-D-hexopyranosides was synthesized. Diverse configurations were installed at C-2 and C-4 of the glucose residue. The benzyl glycosidic group was kept intact or substituted by an electron-donating or electron-withdrawing group that could also participate in hydrogen bonding. All thiodisaccharides were found to be inhibitors of E. coli ß-galactosidase. In general, benzyl thiodisaccharides were better inhibitors than those substituted (NO2 or NH2) on the benzyl ring. Thiodisaccharides containing a hexopyranoside, instead of a pentopyranoside, showed a weaker inhibitory activity, except for those having the α-D-xylo configuration, which exhibited inhibition constants of the same order of magnitude. These and previous results indicated that the inhibition process by thiodisaccharides is strongly dependent on the configuration of the 3-deoxy-4-thiopyranoside, as well as its substitution pattern (such as the presence of a benzyl glycoside). The enzyme-inhibitor interaction during the hydrolysis process involves a conformational selection resulting from rotation around the thioglycosidic bond and the flexibility of the terminal six-membered ring. Thus, the mentioned structural features of the inhibitor could give rise to favorable ground state conformations for the interaction with the enzyme, similar to those found for selected thiodisaccharides in the bound state. These studies demonstrated that the performance of thiodisaccharides as enzyme inhibitors could be increased by selecting the appropriate configuration and substitution of the hexopyranoside replacing the glucose moiety of 4-thiolactose.

Synthesis of sugar enones and their use as powerful synthetic precursors of thiodisaccharides.

Monosaccharide derivatives having a double bond conjugated to a carbonyl (sugar enones or enuloses) are relevant synthetic tools. They are also suitable starting materials, or versatile intermediates, for the synthesis of a wide variety of natural or synthetic compounds with a broad spectrum of biological and pharmacological activities. The preparation of enones is mainly focused on the search for more efficient and diastereoselective synthetic methodologies. The usefulness of enuloses relies on the diverse reaction possibilities offered by alkene and carbonyl double bonds, which are prone to undergo varied reactions such as halogenation, nitration, epoxidation, reduction, addition, etc. The addition of thiol groups that led to sulfur glycomimetics, such as thiooligosaccharides, is particularly relevant. Therefore, the synthesis of enuloses and the Michael addition of sulfur nucleophiles to give thiosugars or thiodisaccharides are discussed here. Chemical modifications of the conjugate addition products to afford biologically active compounds are also reported.

Synthesis of enantiomerically pure enones (2-benzyloxypyran-3-ones) derived from pentoses.

The useful synthons sugar enones (2-benzyloxypyran-3-ones) derived from pentoses have been prepared starting from 2-acetoxyglycals or benzyl pentopyranosides. The glycals were glycosylated with benzyl alcohol in the presence of a Lewis acid (SnCl4 or InCl3) to give enantioenriched enones (ee = 80-90%). Under catalysis with InCl3, benzyl 2-enopyranosides gave also the enones (ee = 87%). On the other hand, enantiomerically pure enones were synthesized via an improved straightforward and high yielding sequence (70% overall) from benzyl pentopyranosides. However, the yields of both, the glycosylation of glycals as well as some specific reactions of the sequence from glycosides, were lowered when a p-nitro substituent was introduced into the benzyl group. These routes became impractical in the case of p-acetamidobenzyl derivatives, because of the large extent of decomposition. Therefore, alternative sequences have been developed for the synthesis of 2-(p-acetamidobenzyloxy)pyran-3-ones.

Design and Synthesis of 2-Acetamido-2,3-dideoxythiodisaccharides via Diastereoselective Conjugate Addition to Sugar Enone O-Acetyl Oximes. Galactosidase Inhibition Studies.

The key step in a new synthesis of 2-acetamido-2,3-dideoxy-(1→4)-thiodisaccharides was the conjugate addition of a 1-thiogalactose derivative to E and Z acetyl oximes derived from sugar enones. This reaction was shown to be completely diastereoselective for both the formation of the thioglycosidic linkage and the configuration of acetyl oxime. The thiodisaccharides have been designed as inhibitors of the ß-galactosidase from E. coli, and they have been shown to successfully meet such requirements.