Design and Optimization of Novel Competitive, Non-peptidic, SARS-CoV-2 Mpro Inhibitors.

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles.

Development of a Dihydroquinoline-Pyrazoline GluN2C/2D-Selective Negative Allosteric Modulator of the N-Methyl-d-aspartate Receptor.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 µM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 µM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Discovery of 5'-Substituted 5-Fluoro-2'-deoxyuridine Monophosphate Analogs: A Novel Class of Thymidylate Synthase Inhibitors.

5-Fluorouracil and 5-fluorouracil-based prodrugs have been used clinically for decades to treat cancer. Their anticancer effects are most prominently ascribed to inhibition of thymidylate synthase (TS) by metabolite 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). However, 5-fluorouracil and FdUMP are subject to numerous unfavorable metabolic events that can drive undesired systemic toxicity. Our previous research on antiviral nucleotides suggested that substitution at the nucleoside 5'-carbon imposes conformational restrictions on the corresponding nucleoside monophosphates, rendering them poor substrates for productive intracellular conversion to viral polymerase-inhibiting triphosphate metabolites. Accordingly, we hypothesized that 5'-substituted analogs of FdUMP, which is uniquely active at the monophosphate stage, would inhibit TS while preventing undesirable metabolism. Free energy perturbation-derived relative binding energy calculations suggested that 5'(R)-CH3 and 5'(S)-CF3 FdUMP analogs would maintain TS potency. Herein, we report our computational design strategy, synthesis of 5'-substituted FdUMP analogs, and pharmacological assessment of TS inhibitory activity.

Fluorous-Phase Approach to α-Hydroxytropolone Synthesis.

α-Hydroxytropolones (αHTs) are troponoids that demonstrate inhibition against an array of therapeutically significant targets, making them potential drug leads for several human diseases. We have utilized a recently discovered one-pot three-component oxidopyrylium cycloaddition in a solid-supported synthesis of αHTs. Though the procedure is time efficient and generates assay-ready molecules, the system suffers from low yields and an inability to perform reaction modifications on resin-bound intermediates. In order to combat these issues with the solid-phase platform, we incorporated fluorous tags into our synthetic route. Through the implementation of fluorous phase chemistry, we demonstrate a substantial increase in the overall yield of αHTs, as well as an ability to execute metal-catalyzed cross coupling and amide coupling on fluorous tagged intermediates. We also show that tagged molecules can be separated from nonfluorous impurities, and vice versa, by utilizing fluorous liquid-liquid and solid-phase extractions. Hence, these proof-of-principle investigations describe the viability of a fluorous phase approach to αHT synthesis and its potential to serve as a combinatorial technique to produce structurally diverse substrates.

Free Energy-Based Virtual Screening and Optimization of RNase H Inhibitors of HIV-1 Reverse Transcriptase.

We report the results of a binding free energy-based virtual screening campaign of a library of 77 α-hydroxytropolone derivatives against the challenging RNase H active site of the reverse transcriptase (RT) enzyme of human immunodeficiency virus-1. Multiple protonation states, rotamer states, and binding modalities of each compound were individually evaluated. The work involved more than 300 individual absolute alchemical binding free energy parallel molecular dynamics calculations and over 1 million CPU hours on national computing clusters and a local campus computational grid. The thermodynamic and structural measures obtained in this work rationalize a series of characteristics of this system useful for guiding future synthetic and biochemical efforts. The free energy model identified key ligand-dependent entropic and conformational reorganization processes difficult to capture using standard docking and scoring approaches. Binding free energy-based optimization of the lead compounds emerging from the virtual screen has yielded four compounds with very favorable binding properties, which will be the subject of further experimental investigations. This work is one of the few reported applications of advanced-binding free energy models to large-scale virtual screening and optimization projects. It further demonstrates that, with suitable algorithms and automation, advanced-binding free energy models can have a useful role in early-stage drug-discovery programs.

Discovery and Development of a Three-Component Oxidopyrylium [5 + 2] Cycloaddition.

α-Hydroxy-γ-pyrone-based oxidopyrylium cycloaddition reactions are useful methods for accessing a highly diverse range of oxabicyclo[3.2.1]octane products. Intermolecular variants of the reaction require the formation of a methyl triflate-based pre-ylide salt that upon treatment with base in the presence of alkenes or alkynes leads to α-methoxyenone-containing bicyclic products. Herein, we describe our discovery that the use of ethanol-stabilized chloroform as solvent leads to the generation of α-ethoxyenone-containing bicyclic byproducts. This three-component process was further optimized by gently heating a mixture of a purified version of the oxidopyrylium dimer in the presence of an alcohol prior to addition of a dipolarophile. Using this convenient procedure, several new oxidopyrylium cycloaddition products can be generated in moderate yields. We also highlight the method in a tandem ring-opening/debenzylation method for the generation of α-hydroxytropolones.

Characterization of the C-Terminal Nuclease Domain of Herpes Simplex Virus pUL15 as a Target of Nucleotidyltransferase Inhibitors.

The natural product α-hydroxytropolones manicol and ß-thujaplicinol inhibit replication of herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, respectively) at nontoxic concentrations. Because these were originally developed as divalent metal-sequestering inhibitors of the ribonuclease H activity of HIV-1 reverse transcriptase, α-hydroxytropolones likely target related HSV proteins of the nucleotidyltransferase (NTase) superfamily, which share an "RNase H-like" fold. One potential candidate is pUL15, a component of the viral terminase molecular motor complex, whose C-terminal nuclease domain, pUL15C, has recently been crystallized. Crystallography also provided a working model for DNA occupancy of the nuclease active site, suggesting potential protein-nucleic acid contacts over a region of ∼ 14 bp. In this work, we extend crystallographic analysis by examining pUL15C-mediated hydrolysis of short, closely related DNA duplexes. In addition to defining a minimal substrate length, this strategy facilitated construction of a dual-probe fluorescence assay for rapid kinetic analysis of wild-type and mutant nucleases. On the basis of its proposed role in binding the phosphate backbone, studies with pUL15C variant Lys700Ala showed that this mutation affected neither binding of duplex DNA nor binding of small molecule to the active site but caused a 17-fold reduction in the turnover rate (kcat), possibly by slowing conversion of the enzyme-substrate complex to the enzyme-product complex and/or inhibiting dissociation from the hydrolysis product. Finally, with a view of pUL15-associated nuclease activity as an antiviral target, the dual-probe fluorescence assay, in combination with differential scanning fluorimetry, was used to demonstrate inhibition by several classes of small molecules that target divalent metal at the active site.

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses.

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as ß-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 µM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 µM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 µM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.

Traceless Solid-Phase α-Hydroxytropolone Synthesis.

α-Hydroxytropolones are established inhibitors of several therapeutically relevant binuclear metalloenzymes, and thus lead drug targets for various human diseases. We have leveraged a recently-disclosed three-component oxidopyrylium cycloaddition in the first solid-phase synthesis of α-hydroxytropolones. We also showed that, while minor impurities exist after cleavage and aqueous wash, the semi-crude products display activity in HIV RT-associated RNaseH enzymatic and cell-based assays consistent with pure molecules made in solution phase. These proof-of-principle studies demonstrate the feasibility of solid-phase α-hydroxytropolone synthesis and its potential to serve as a powerful platform for α-hydroxytropolone-based drug discovery and development.

Synthetic α-Hydroxytropolones as Inhibitors of HIV Reverse Transcriptase Ribonuclease H Activity.

HIV Reverse Transcriptase-associated ribonuclease H activity is a promising enzymatic target for drug development that has not been successfully targeted in the clinic. While the α-hydroxytropolone-containing natural products ß-thujaplicinol and manicol have emerged as some of the most potent leads described to date, structure-function studies have been limited to the natural products and semi-synthetic derivatives of manicol. Thus, a library of α-hydroxytropolones synthesized through a convenient oxidopyrylium cycloaddition/ring-opening sequence have been tested in in vitro and cell-based assays, and have been analyzed using computational support. These studies reveal new synthetic α-hydroxytropolones that, unlike the natural product leads they are derived from, demonstrate protective antiviral activity in cellular assays.

Hydroxylated tropolones inhibit hepatitis B virus replication by blocking viral ribonuclease H activity.

Hepatitis B virus (HBV) remains a major human pathogen despite the development of both antiviral drugs and a vaccine, in part because the current therapies do not suppress HBV replication far enough to eradicate the virus. Here, we screened 51 troponoid compounds for their ability to suppress HBV RNaseH activity and HBV replication based on the activities of α-hydroxytropolones against HIV RNaseH, with the goal of determining whether the tropolone pharmacophore may be a promising scaffold for anti-HBV drug development. Thirteen compounds inhibited HBV RNaseH, with the best 50% inhibitory concentration (IC50) being 2.3 µM. Similar inhibition patterns were observed against HBV genotype D and C RNaseHs, implying limited genotype specificity. Six of 10 compounds tested against HBV replication in culture suppressed replication via blocking of viral RNaseH activity, with the best 50% effective concentration (EC50) being 0.34 µM. Eighteen compounds inhibited recombinant human RNaseH1, and moderate cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50]=25 to 79 µM). Therapeutic indexes ranged from 3.8 to 94. Efficient inhibition required an intact α-hydroxytropolone moiety plus one or more short appendages on the tropolone ring, but a wide variety of constituents were permissible. These data indicate that troponoids and specifically α-hydroxytropolones are promising lead candidates for development as anti-HBV drugs, providing that toxicity can be minimized. Potential anti-RNaseH drugs are envisioned to be employed in combination with the existing nucleos(t)ide analogs to suppress HBV replication far enough to block genomic maintenance, with the goal of eradicating infection.

Inhibition of the ANT(2")-Ia resistance enzyme and rescue of aminoglycoside antibiotic activity by synthetic α-hydroxytropolones.

Aminoglycoside-2"-O-nucleotidyltransferase ANT(2")-Ia is an aminoglycoside resistance enzyme prevalent among Gram-negative bacteria, and is one of the most common determinants of enzyme-dependant aminoglycoside-resistance. The following report outlines the use of our recently described oxidopyrylium cycloaddition/ring-opening strategy in the synthesis and profiling of a library of synthetic α-hydroxytropolones against ANT(2")-Ia. In addition, we show that two of these synthetic constructs are capable of rescuing gentamicin activity against ANT-(2")-Ia-expressing bacteria.

The biology and synthesis of α-hydroxytropolones.

α-Hydroxytropolones are a subclass of the troponoid family of natural products that are of high interest due to their broad biological activity and potential as treatment options for several diseases. Despite this promise, there have been scarce synthetic chemistry-driven optimization studies on the molecules. The following review highlights key developments in the biological studies conducted on α-hydroxytropolones to date, including the few synthetic chemistry-driven optimization studies. In addition, we provide an overview of the methods currently available to access these molecules. This review is intended to serve as a resource for those interested in biological activity of α-hydroxytropolones, and inspire the development of new synthetic methods and strategies that could aid in this pursuit.

7,9-Diaryl-1,6,8-trioxaspiro[4.5]dec-3-en-2-ones: readily accessible and highly potent anticancer compounds.

7,9-Diaryl-1,6,8-trioxaspiro[4.5]dec-3-en-2-ones are a recently described group of spirocyclic butenolides that can be generated rapidly and as a single diastereomer through a cascade process between γ-hydroxybutenolides and aromatic aldehydes. The following outlines our findings that these spirocycles are potently cytotoxic and have a dramatic structure-function profile that provides excellent insight into the structural features required for this potency.