Carboxylic Acid Isostere Derivatives of Hydroxypyridinones as Core Scaffolds for Influenza Endonuclease Inhibitors.

pubs.acs.org/acsmedchemlett Letter Carboxylic Acid Isostere Derivatives of Hydroxypyridinones as Core Scaffolds for Influenza Endonuclease Inhibitors Ryjul W. Stokes,‡ Alysia J. Kohlbrand,‡ Hyeonglim Seo, Banumathi Sankaran, Johannes Karges, and Seth M. Cohen* Cite This: ACS Med. Chem. Lett. 2023, 14, 75−82 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Among the most important influenza virus targets is the RNAdependent RNA polymerase acidic N-terminal (PAN) endonuclease, which is a critical component of the viral replication machinery. To inhibit the activity of this metalloenzyme, small-molecule inhibitors employ metal-binding pharmacophores (MBPs) that coordinate to the dinuclear Mn2+ active site. In this study, several metalbinding isosteres (MBIs) were examined where the carboxylic acid moiety of a hydroxypyridinone MBP is replaced with other groups to modulate the physicochemical properties of the compound. MBIs were evaluated for their ability to inhibit PAN using a FRET-based enzymatic assay, and their mode of binding in PAN was determined using X-ray crystallography. KEYWORDS: drug discovery, metal-binding pharmacophore, isosteres, influenza endonuclease, medicinal inorganic chemistry C translated into viral proteins.16 The PA subunit, which enables endonucleolytic cleavage, is composed of a C-terminal domain that is mostly structural and an N-terminal domain that is catalytically active. The N-terminal domain contains a dinuclear Mg 2+ or Mn 2+ active site that is highly conserved.17−19 Some efforts have used metalloenzymefocused fragment-based drug discovery (FBDD) to identify potent inhibitors of PAN.20−23 These FBDD campaigns have identified metal-binding pharmacophores (MBPs) that utilize a triad of oxygen donors, including a carboxylic acid, to bind the metal ions, making these fragments very polar and non-ideal starting points for the development of novel therapeutics. To this end, (bio)isosteric replacement is a strategy that can mitigate pharmacological liabilities.24 Herein, the design and experimental evaluation of novel metal-binding isosteres (MBIs) are reported (Figure 1). The MBIs were found to possess good inhibition of PAN endonuclease, with many displaying half-maximal inhibitory concentration (IC50) values in the low nanomolar range, comparable to the parent carboxylic acid MBP (compound 1, Figure 1). Using X-ray crystallography, the binding of these compounds to PAN was urrent estimates suggest that annual influenza epidemics are responsible for up to 650,000 deaths globally.1 One recent study concluded that during the 2009 H1N1 pandemic there were ∼61 million cases in the United States alone, resulting in ∼275,000 hospitalizations and 12,000 deaths.2 Vaccines are available; however, efficacy depends on the ability to predict antigenic changes, and requires semi-annual reformulation.3 To address acute cases of infection, smallmolecule therapeutics have been approved by the U.S. Food and Drug Administration (FDA). Adamantane-based structures that inhibited the matrix protein 2 (M2) ion channel have been used,4 but are now largely not prescribed due to resistance.5,6 Newer neuraminidase inhibitors are now used,7 and resistance to neuraminidase inhibitors remains low; however, emergence of resistance remains a threat to public health.5,8 Other small-molecule strategies to address influenza include targeting the hemagglutinin protein9 and the RNAdependent RNA polymerase acidic N-terminal (PA N) endonuclease.10 PAN represents an especially attractive target due to its role in viral replication, its high conservation, and its lack of a human analog.11 FDA approval of baloxavir marboxil, a first-in-class PAN inhibitor, has validated this approach,12 but despite its clinical success, resistance against baloxavir has also begun to emerge.13 PAN is part of a heterotrimeric RNA-dependent RNA polymerase complex, which is composed of PA, PB1, and PB2 subunits.14 Together, they facilitate replication and transcription of the viral genome.15 A “cap-snatching” mechanism enables the synthesis of viral mRNA, which can later be © 2022 The Authors. Published by American Chemical Society Received: October 1, 2022 Accepted: December 2, 2022 Published: December 9, 2022 75 https://doi.org/10.1021/acsmedchemlett.2c00434 ACS Med. Chem. Lett. 2023, 14, 75−82 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Figure 1. Chemical structures of metal-binding isosteres (MBIs) investigated in this study. ■ COMPOUND SYNTHESIS All bromine-containing compounds (2−11) were synthesized from precursor 12, 3-hydroxypicolinonitrile (Scheme 1). Commercially available 12 can be dibrominated selectively in the 4- and 6-positions upon treatment with elemental bromine to afford 13 in high yield. An oxygen atom that will later participate as a Lewis base to bind the Mn2+ center can be introduced via SNAr chemistry. This nucleophilic addition results in the selective introduction of benzyl alcohol in the 4position to afford intermediate 14 in good yield. Compound 14 serves as a crucial intermediate for the compounds evaluated in this study. Hydrolysis of 14 under basic conditions yields the benzyl-protected compounds 2a and 3a, which can be deprotected under acidic conditions to afford compounds 2 and 3, the acid- and amide-functionalized molecules, respectively. Compound 2 can be further functionalized to incorporate an N-methoxycarboxamide group (5) by conversion to the acid chloride and subsequent treatment with methoxyamine hydrochloride. A click reaction between compound 14 and sodium azide results in the formation of tetrazole 4a, a commonly employed carboxylic acid isostere, which can be deprotected to afford the tridentate MBI 4. Treatment of compound 14 with zinc chloride and ethanolamine or ethylenediamine results in the partially saturated heterocycles 10a and 11a. Saturated heterocycles can be advantageous as they tend to have higher lipophilicity, solubility, and three-dimensionality than their unsaturated counterparts.30 Deprotection of these compounds with HCl results in the formation of the partially saturated imidazoline (10) and oxazoline (11). The nitrile group in compound 14 can also be transformed into an N-hydroxyamidine functional group when treated with hydroxylamine hydrochloride and triethylamine, followed by debenzylation to afford compound 6. Finally, simple deprotection of compound 14 under acidic conditions results in the nitrile-containing MBI 9. In some cases, protection of the phenol in compound 14 was beneficial. Treatment of 14 with benzyl bromide and potassium carbonate resulted in the formation of 15 in good yield. The protected Nhydroxyamide intermediate was synthesized using conditions similar to those used for 6a, though longer reaction times were elucidated. While many compounds bound as expected (based on prior findings),21,22 some isosteres exhibited somewh (...truncated)