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. 2022 Dec 9;14(1):75–82. doi: 10.1021/acsmedchemlett.2c00434

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 †, Seth M Cohen †,*
PMCID: PMC9841593  PMID: 36655124

Abstract

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Among the most important influenza virus targets is the RNA-dependent 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 metal-binding 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

Current 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 re-formulation.3 To address acute cases of infection, small-molecule 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 RNA-dependent RNA polymerase acidic N-terminal (PAN) 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 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 Mg2+ or Mn2+ active site that is highly conserved.17−19 Some efforts have used metalloenzyme-focused 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 elucidated. While many compounds bound as expected (based on prior findings),21,22 some isosteres exhibited somewhat unexpected binding interactions. A preliminary analysis of the physical properties, such as calculated log partition coefficient (clogP) and pKa, shows that the isosteres have tunable properties, potentially circumventing the limitations of the carboxylic acid scaffold.

Figure 1.

Figure 1

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Chemical structures of metal-binding isosteres (MBIs) investigated in this study.

Previous work has focused on the identification of MBP fragments and their evolution into lead-like molecules by FBDD.20−22 A carboxylic acid-modified hydroxypyridinone MBP was reported to achieve IC50 values as low as ∼17 nM against PAN.21 Hydroxypyridinones have been utilized in drug discovery campaigns, including for their specific ability to coordinate metal ions.25 Despite impressive activity, this MBP (and elaborated inhibitors derived from it) was found to be only moderately active in influenza-infected cells. This disparity between antiviral activity and enzymatic inhibition is likely due to low membrane permeability originating from the high polarity of these molecules.19 Additionally, it is well-known that carboxylic acid moieties are associated with pharmacological liabilities, which can limit their use in drug development.26−28 The carboxylic acid functional group can be replaced by a different moiety with similar biochemical interactions, but different physiochemical properties, via isosteric replacement to try to address possible shortcomings of the carboxylic acid-containing MBP 1 (Figure 1).24,29

Nine MBIs (3–11) of hydroxypyridinone MBP 1 were synthesized, with isosteric replacements for the carboxylic acid moiety installed (Figure 1). These MBIs also possess a bromine atom in the 6-position of the ring to aid in crystallography (i.e., via heavy-atom substitution and anomalous scattering) and to serve as a synthetic handle for future compound elaboration.21 To account for the electronic and lipophilic effects of adding this bromine substituent, a direct analog of 1 was synthesized that contains a bromine atom (2, Figure 1). Table S1 lists the calculated clogP values of these fragments compared to analogues substituted with -H, -CH3, or -Ph groups.

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 4-position 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 N-hydroxyamide intermediate was synthesized using conditions similar to those used for 6a, though longer reaction times were employed. Compound 16 can be acylated upon the addition of acetic anhydride to afford compound 17, which when exposed to heat and a mixture of hydrochloric acid, acetic acid, and trifluoroacetic acid undergoes both debenzylation and an acid-promoted cyclization to afford oxadiazole 8 in a single step. Additionally, treatment of compound 16 with carbonyldiimidazole (CDI) results in the formation of the oxadiazolone-functionalized molecule 7a, which can be deprotected to afford compound 7.

Scheme 1. Synthesis of MBIs.

Scheme 1

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Reagents and conditions: (a) NaOAc, H2O:MeOH, Br2, 0 °C to 25 °C; (b) NaH, BnOH, DMSO, 60 °C, 18 h; (c) BnBr, K2CO3, DMF, 45 °C, 19 h; (d) HONH2·HCl, NEt3, EtOH, 50 °C, 15 h; (e) CDI, NEt3, DMF, 80 °C, 8 h; (f) i. SOCl2, DCM, reflux, 12 h. ii. Methoxyamine hydrochloride, NEt3, DCM, −78 °C to 40 °C, 48 h; (g) NaOH, EtOH, 100 °C, 20 h; (h) KOH, EtOH:H2O, 100 °C, 16 h; (i) NaN3, NH4Cl, DMF, 115 °C, 4 h; (j) ethane-1,2-diamine or 2-aminoethan-1-ol, ZnCl2, toluene, 90 °C to 130 °C, 16–20 h; (k) HONH2·HCl, NEt3, EtOH, 50 °C 1 h; (l) HCl:AcOH:TFA (5:5:1), 50 °C, 48 h; (m) Ac2O, DBU, THF, 0 °C to 80 °C, 8 h; (n) HCl, 25 °C to 100 °C, 12–48 h.

Previous studies have correlated the acidity of the phenolic proton (pKa) of the hydroxypyridinone fragment with the ability to inhibit the activity of PAN.22 In addition, changes in pKa can be telling indicators of changes in the electronic structure of the heterocycle. Accordingly, pKa values of the MBIs were calculated to probe the electronic differences introduced by the isostere groups. The pKa value of the phenolic proton was estimated using density functional theory (DFT) calculations. More precisely, the energetic states within a thermodynamic cycle of the proton-transfer reaction were calculated, and the resulting free energy was correlated to the pKa values as has been previously described (see Supporting Information for details).31 Compound 2 was predicted to have a pKa value of 9.4. Using a pH-metric titration, the measured pKa value of compound 2 was measured at 9.79 ± 0.01, showing reasonable agreement with the DFT calculation. The isosteric replacement of this functional group was identified as an influencing factor of the acidity of the phenolic proton (a full list of predicted and several additional measured pKa values is provided in Table S2). The isostere modification of the carbonyl group gave calculated pKa values ranging from 8.1 to 12.6 (Table S2), indicating that changes in the 2-position of the hydroxypyridinone ring can have large effects on the electronics of the ring system, and is a useful means to tune the electronic properties of the ring.

To determine the binding mode of the MBIs in the PAN active site, several compounds were crystallized with the enzyme for structure determination. Tables of collection and refinement statistics are provided in the Supporting Information (Tables S3, S4). In native PAN, the Mn2+ cations are coordinated by His41, Glu80, Asp108, Glu 119, and Ile120 and five water molecules, producing an octahedral coordination geometry at each metal center. As anticipated, the addition of a bromine atom to the MBIs produced no apparent change in the binding to the active site metal ions (compare structures of 1 and 2, Figure 2) and aided in ligand placement within the electron density map. Both compounds 1 and 2 coordinated by bridging the metal ions through the phenolate donor. This provides a triad of oxygen donor atoms (including oxygen donor atoms from the ketone and carboxylic acid groups), creating a 5-membered chelate ring at Mn1 and a 6-membered chelate ring at Mn2. Compounds 1 and 2 also displayed hydrogen bonding between the ketone donor from the hydroxypyridinone ring and Lys134.32

Figure 2.

Figure 2

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Co-crystal structures of PAN endonuclease with compounds 1–5 and 8−10. The large panel depicts MBI 5 coordinating Mn1 through the exocyclic carbonyl oxygen atom, Mn2 through the carboxylate group, and the hydroxyl donor bridging between Mn1 and Mn2. The protein backbone is shown as a gray cartoon. Mn2+ ions and water molecules are shown as purple and red spheres, respectively. The structure with compound 8 is shown from two perspectives. Residues coordinating the Mn2+ ions are shown in sticks. Atom colors are as follow: carbon (green MBI, gray protein), oxygen (red), nitrogen (blue), bromine (dark red). The electron density of each MBI and the Mn2+ ions is displayed as a blue mesh. Mesh is 2Fo – Fc contoured at 2σ.

Previous crystallographic studies have focused on compounds containing a triad of chelating oxygen atoms that bind in a tridentate fashion, displacing three of the coordinating water molecules.21,22 Compounds 1, 2, 3, and 5 present the same triad and display similar binding poses, coordinating Mn1 through the ketone and Mn2 through the carboxylate, with the hydroxyl group bridging Mn1 and Mn2 (Figure 2).21 The binding and inhibitory activity (vide infra) of these compounds are comparable to those of the parent MBP 1. The average distance from Mn2 to the carboxylic acid isosteric group in each of these structures is 2.2 Å. A list of metal–donor atom distances is provided in Table S5. Interestingly, several nitrogen-containing isosteres were found to have a bridging water molecule between the nitrogen of the hydroxypyridinone core and a nitrogen from the isosteric groups (see compounds 3, 4, 5, and 9, Figure 2). This interaction is reminiscent of the carbamoylpyridine bicycle in Baloxavir,19 but creates a cyclized, rigid structure by hydrogen bonding with water, rather than a synthetic, covalent ring structure. This pseudo-five-membered ring may be a useful in future inhibitor design, as it provides conformational stabilization without the necessity to form a bicycle, affording additional synthetic opportunities to develop interactions in the pocket toward Tyr24 and α-helix 2.

Among structures, compounds 4, 8, 9, and 10 break with the chelating oxygen triad motif by replacing the carboxylic acid oxygen with a nitrogen atom. As expected, Mn1 is bound between the phenol and the ketone of the hydroxypyridinone core, but unlike previous MBIs with a third oxygen donor, rotations of varying degrees were observed with the heterocyclic MBIs when bound to Mn2. Interestingly, the oxadiazolone ring of compound 8 is noticeably rotated out of the plane of the hydroxypyridinone core. A possible explanation for the rotation is the driving force to maintain an octahedral coordination geometry at both metal centers. To preserve an acceptable angle/distance between the metal and the donor atom at both metal sites, a rotation of the heterocyclic isostere may occur, the metal–ligand distances may be adjusted, or a combination thereof. To further examine this hypothesis, octahedral distortion parameters ζ and Σ were calculated for compound 8 (relative to Mn2) and compared with the distortion parameters of compound 3 and the ligand-free structure, which presented the most ideal octahedral geometries. To calculate and visualize changes in octahedral geometry, Octadist,33 an octahedral distortion analysis software, was used (Figure S1, Table S6). The ζ and Σ values are general octahedral distortion parameters used to characterize deviations from ideal octahedral geometry and are used here to evaluate the isostere binding geometry to Mn2.33 The value of ζ is the average of the sum of the deviation of 6 unique metal–ligand bond lengths around the central metal atom (di) from the average value (dmean).34 The value of Σ is the sum of the deviation of 12 unique cis ligand–metal–ligand angles (ϕi) from 90°.35,36 These distortion values were calculated for compound 8, although it should be noted that these values do not take into account the resolution of the crystal structure, and should be considered accordingly. The value for ζ is very small, 0.574 Å, suggesting only very small changes to ligand–metal distances for each of the six donor atoms. The Σ value is also rather small, 90.81°, with the largest deviations from 90° resultant from contributions of the more rigid angles associated with Glu80 and Asp108. When these distortion parameters are calculated for a structure of compound 8 that is forced into an idealized co-planar ligand geometry, the ζ value increases to 0.606 Å and Σ increases to 98.347°, suggesting that the planar MBI structure enforces an inferior octahedral geometry at the metal centers compared to the experimental structure. Comparing both the rotated and planar structures of compound 8 to those of compound 3 (0.357 Å, 46.669°) and the ligand-free structure (bound water, 0.685 Å, 44.571°), both conformations of compound 8 exhibit poorer octahedral character.

The tetrazole in compound 4 displays a slight twist, perhaps due to the same reason as compound 8, though the rotation appears less pronounced. Interestingly, instead of incorporating a more distinct twist, this structure adopts different metal–ligand binding distances. The distance between Mn1 and the ketone functional group is 2.3 Å, while the distance between the tetrazole and Mn2 is 1.8 Å, both the longest and shortest metal–donor distances observed among these inhibitors, respectively. The average distance from the metal cation in each nitrogen-donating structure is 2.2 Å, matching the donor–metal distance in oxygen triad MBIs, furthering the argument that the rotations of compounds 4 and 8 result from attempting to optimize metal-binding interactions.

Overall, MBIs following the same donor oxygen triad displayed metal-binding activity and binding poses similar to those of previously reported structures. MBIs presenting a nitrogen donor atom instead of the carboxylate oxygen have more variability in metal-binding behavior and pose. Heterocyclic nitrogen MBIs were found to have varying degrees of internal ligand rotation, most notably with compound 8, which is most likely explained by the need to optimize the octahedral coordination geometry at the Mn2 metal center.

To determine the activity of these molecules against PAN, MBIs were evaluated using a fluorescence-quenching-based nuclease activity assay (see Supporting Information for details).37 Using this assay, dose–response curves were obtained to determine the half-maximal inhibitory concentration (IC50) values of these compounds. Importantly, IC50 values are strongly dependent on the buffer, pH, enzyme concentration, and substrate used, and as such may vary somewhat with assay conditions.38 Here, a protein concentration of 25 nM was employed.17 As previously identified hydroxypyridinones have been shown to be tight binders of PAN,21 it is expected that molecules with good metal-binding motifs will demonstrate low IC50 values under these assay conditions.39 The results of the dose–response experiment are shown in Table 1.

Table 1. Half-Maximal Inhibitory Concentration (IC50), 95% Confidence Interval (95% CI), log IC50 (pIC50), and Ligand Efficiency (LE) of Compounds 1–11 against PAN Endonuclease.

graphic file with name ml2c00434_0004.jpg

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Compound 2 was used as a control to determine whether the addition of a bromine atom negatively affected the metal-binding ability of the MBP. Compounds 1 and 2 showed similar inhibition activity (Table 1), indicating that the bromide did not impact metal-binding relative to the parent compound. Both compounds display high binding energy per atom (ligand efficiency, LE) (Table 1). Despite conversion from a potentially anionic carboxylate to a neutral carboxamide, compound 3 showed little difference in activity when compared with compounds 1 or 2. Compound 4 showed activity comparable to that of the parent structure, possibly due to shorter metal-ligand bond distances (Table S5). This is perhaps unsurprising, as tetrazoles have been widely investigated as isosteres for carboxylic acids.40 Compound 5 demonstrated moderate inhibition, suggesting somewhat poorer metal-binding ability, perhaps due to the electron-withdrawing nature of the methoxy group. Compound 7 demonstrated the most potent IC50 value of the evaluated compounds; however, a crystal structure was not obtained. Compound 8, functionalized with a methyl-oxadiazole, showed good inhibition, indicating that the rotation of the heterocycle may contribute to improved metal-binding via optimizing the metal–ligand geometry. While compound 8 does bind favorably, it demonstrates higher distortion parameters than compound 3, possibly accounting for the decreased activity of 8 compared to 3. Compound 9 showed relatively poor inhibition, due to the loss of a strong interaction with Mn2, which cannot be provided by the nitrile isostere in this context. Previous structures have shown bidentate hydroxypyridinones coordinating to Mn1, with the ketone bridging the Mn1 and Mn2 metals with the hydroxyl group bound to only Mn1.17,21 Consistent with every structure reported in this study, compound 9 bridges the metal ions through the anionic phenolic group, while the ketone group interacts with Mn2 and Lys134. This result indicates that even with poorer-binding isosteres like 9, the introduction of a substituent in the 2-position of the hydroxypyridine ring drives the MBI to coordinate in such a way that bridges the metals with the phenolic group, which is a stronger donor than the ketone and should give better overall affinity. Finally, the saturated heterocycle isosteres 10 and 11 showed markedly poorer inhibition than the parent MBI, possibly due to the softer nature of the heterocyclic donor nitrogen atoms, resulting in poorer electron donation to Mn2.

In summary, MBIs of the highly active hydroxypyridinone scaffold were designed to replace the carboxylic acid group and retain inhibition of PAN endonuclease. A variety of fragments with isosteric replacements were synthesized via a common intermediate and evaluated in a FRET-based enzymatic assay. Many of these molecules retain inhibitory activity comparable to that of the parent carboxylic acid fragment. These molecules were further evaluated crystallographically to improve our understanding of how the structural and metal-binding properties are affected by replacement of the carboxylic acid group. This study shows that MBIs may be useful in the development of small-molecule therapeutics of influenza PAN and other related nucleic-acid-processing viral metalloenzymes.41,42

Acknowledgments

This work was supported by grants from the National Institutes of Health (R01 AI149444) and a National Science Foundation Graduate Research Fellowship Program (DGE-1650112 to R.W.S.). The computational studies were supported in part by the W. M. Keck Foundation through computing resources at the W. M. Keck Laboratory for Integrated Biology. The work used beamlines 5.0.3 and 8.2.1 of the Advanced Light Source, a U.S. DOE Office of Science User Facility, supported in part under Contract No. DE-AC02-05CH11231, and is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, Grant P30 GM124169-01. The authors acknowledge Yongxuan Su (U.C. San Diego, Molecular Mass Spectrometry Facility) for aid with MS and HR-MS analysis. The authors also acknowledge Dr. Carlo Ballatore, Bobby Lucero, and Karol Francisco for aid with pKa determination and preparatory HPLC.

Glossary

Abbreviations

clogP

calculated log partition coefficient

CDI

carbonyldiimidazole

DBU

1,8-diazabicyclo(5.4.0)undec-7-ene

DCM

dichloromethane

DFT

density functional theory

DMF

dimethylformamide

EtOAc

ethyl acetate

FDA

U.S. Food and Drug Administration

IC50

half-maximal inhibitory concentration

HAC

heavy atom count

Hex

hexanes

LE

ligand efficiency

M2

matrix protein 2

MBI

metal-binding isostere

MeOH

methanol

MBP

metal-binding pharmacophore

PAN

N-terminal domain of the RNA-dependent RNA polymerase PA subunit

SNAr

nucleophilic aromatic substitution

SAR

structure–activity relationship

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00434.

  • Computational prediction of pKa, comparative predictions of clogP, predicted phenolic pKa values, protein expression and purification, protein crystallography; protein crystallography tables, metal-binding distances, octahedral distortion parameters, octahedral distortion, representative HPLC traces, and lipophilic ligand efficiencies (LLE) (PDF)

  • SMILES strings formula (CSV)

Author Contributions

‡ R.W.S. and A.J.K. contributed equally to this work.

The authors declare the following competing financial interest(s): S.M.C. is a co-founder, has an equity interest, and receives income as member of the Scientific Advisory Board for Forge Therapeutics; is a co-founder, has an equity interest, and is a member of the Scientific Advisory Board for Blacksmith Medicines; and is a co-founder and has an equity interest Cleave Therapeutics (formerly Cleave Biosciences). These companies may potentially benefit from the research results of certain projects in the laboratory of S.M.C. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

Supplementary Material

ml2c00434_si_001.pdf (834.1KB, pdf)
ml2c00434_si_002.xlsx (9.1KB, xlsx)

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