Discovery of Di- and Trihaloacetamides as Covalent SARS-CoV-2 Main Protease Inhibitors with High Target Specificity.

The main protease (Mpro) is a validated antiviral drug target of SARS-CoV-2. A number of Mpro inhibitors have now advanced to animal model study and human clinical trials. However, one issue yet to be addressed is the target selectivity over host proteases such as cathepsin L. In this study we describe the rational design of covalent SARS-CoV-2 Mpro inhibitors with novel cysteine reactive warheads including dichloroacetamide, dibromoacetamide, tribromoacetamide, 2-bromo-2,2-dichloroacetamide, and 2-chloro-2,2-dibromoacetamide. The promising lead candidates Jun9-62-2R (dichloroacetamide) and Jun9-88-6R (tribromoacetamide) had not only potent enzymatic inhibition and antiviral activity but also significantly improved target specificity over caplain and cathepsins. Compared to GC-376, these new compounds did not inhibit the host cysteine proteases including calpain I, cathepsin B, cathepsin K, cathepsin L, and caspase-3. To the best of our knowledge, they are among the most selective covalent Mpro inhibitors reported thus far. The cocrystal structures of SARS-CoV-2 Mpro with Jun9-62-2R and Jun9-57-3R reaffirmed our design hypothesis, showing that both compounds form a covalent adduct with the catalytic C145. Overall, these novel compounds represent valuable chemical probes for target validation and drug candidates for further development as SARS-CoV-2 antivirals.

Discovery of SARS-CoV-2 Papain-like Protease Inhibitors through a Combination of High-Throughput Screening and a FlipGFP-Based Reporter Assay.

The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. Through a fluorescence resonance energy transfer-based high-throughput screening and subsequent lead optimization, we identified several PLpro inhibitors including Jun9-72-2 and Jun9-75-4 with improved enzymatic inhibition and antiviral activity compared to GRL0617, which was reported as a SARS-CoV PLpro inhibitor. Significantly, we developed a cell-based FlipGFP assay that can be applied to predict the cellular antiviral activity of PLpro inhibitors in the BSL-2 setting. X-ray crystal structure of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to a more closed conformation. Molecular dynamics simulations showed that Jun9-72-2 and Jun9-75-4 engaged in more extensive interactions than GRL0617. Overall, the PLpro inhibitors identified in this study represent promising candidates for further development as SARS-CoV-2 antivirals, and the FlipGFP-PLpro assay is a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

Rational Design of Hybrid SARS-CoV-2 Main Protease Inhibitors Guided by the Superimposed Cocrystal Structures with the Peptidomimetic Inhibitors GC-376, Telaprevir, and Boceprevir.

SARS-CoV-2 main protease (Mpro) is a cysteine protease that mediates the cleavage of viral polyproteins and is a validated antiviral drug target. Mpro is highly conserved among all seven human coronaviruses, with certain Mpro inhibitors having broad-spectrum antiviral activity. In this study, we designed two hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 based on the superimposed X-ray crystal structures of SARS-CoV-2 Mpro with GC-376, telaprevir, and boceprevir. Both UAWJ9-36-1 and UAWJ9-36-3 showed potent binding and enzymatic inhibition against the Mpro's from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-229E, and HCoV-HKU1. Cell-based Flip-GFP Mpro assay results show that UAWJ9-36-1 and UAWJ9-36-3 inhibited the intracellular protease activity of SARS-CoV-2 Mpro. In addition, UAWJ9-36-1 and UAWJ9-36-3 had potent antiviral activity against SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E, with UAWJ9-36-3 being more potent than GC-376 in inhibiting SARS-CoV-2. Selectivity profiling revealed that UAWJ9-36-1 and UAWJ9-36-3 had an improved selectivity index over that of GC-376 against host cysteine proteases calpain I and cathepsin L, but not cathepsin K. The X-ray crystal structures of SARS-CoV-2 Mpro with UAWJ9-36-1 and UAWJ9-36-3 were both solved at 1.9 Å, which validated our design hypothesis. Overall, hybrid inhibitors UAWJ9-36-1 and UAWJ9-36-3 are promising candidates to be further developed as broad-spectrum coronavirus antivirals.

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay.

The papain-like protease (PL pro ) of SARS-CoV-2 is a validated antiviral drug target. PL pro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PL pro inhibitors with IC 50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC 50 values ranging from 0.56 to 0.90 µM. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PL pro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PL pro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC 50 values of 8.89 and 8.32 µM, respectively, which were 3-fold more potent than GRL0617 (EC 50 = 25.1 µM). The X-ray crystal structures of PL pro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PL pro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PL pro assay might be a suitable surrogate for screening PL pro inhibitors in the BSL-2 setting.

Chiral phosphoric acid-catalyzed asymmetric dearomatization reactions.

We summarize in this review the recent development of chiral phosphoric acid (CPA)-catalyzed asymmetric dearomatization reactions. A wide array of electron-rich arenes (indoles, phenols, naphthols, benzothiophenes, benzofurans, etc.) and electron-poor arenes (pyridines, quinolines, isoquinolines, etc.) has been proved reactive towards various reaction partners in the presence of a CPA catalyst, enabling asymmetric dearomatization reactions that lead to structurally-diverse polycyclic molecules. The reactions are grouped according to the roles of the arenes in the reactions (as nucleophiles or electrophiles) and the types of reaction partners. This review closes with a personal perspective on the dynamic research area of asymmetric dearomatization reactions by CPAs.

Potent HIV-1 Protease Inhibitors Containing Carboxylic and Boronic Acids: Effect on Enzyme Inhibition and Antiviral Activity and Protein-Ligand X-ray Structural Studies.

We report the synthesis and biological evaluation of phenylcarboxylic acid and phenylboronic acid containing HIV-1 protease inhibitors and their functional effect on enzyme inhibition and antiviral activity in MT-2 cell lines. Inhibitors bearing bis-THF ligand as P2 ligand and phenylcarboxylic acids and carboxamide as the P2' ligands, showed very potent HIV-1 protease inhibitory activity. However, carboxylic acid containing inhibitors showed very poor antiviral activity relative to carboxamide-derived inhibitors which showed good antiviral IC50 value. Boronic acid derived inhibitor with bis-THF as the P2 ligand showed very potent enzyme inhibitory activity, but it showed lower antiviral activity than darunavir in the same assay. Boronic acid containing inhibitor with a P2-Crn-THF ligand also showed potent enzyme Ki but significantly decreased antiviral activity. We have evaluated antiviral activity against a panel of highly drug-resistant HIV-1 variants. One of the inhibitors maintained good antiviral activity against HIVDRVRP20 and HIVDRVRP30 viruses. We have determined high resolution X-ray structures of two synthetic inhibitors bound to HIV-1 protease and obtained molecular insight into the ligand-binding site interactions.

Chiral phosphoric acid catalyzed aminative dearomatization of α-naphthols/Michael addition sequence.

Asymmetric dearomatization reactions have recently emerged as a powerful tool for the rapid build-up of the molecular complexity. Chiral three-dimensional polycyclic molecules bearing contiguous stereogenic centers can be synthesized from readily available planar aromatic feedstocks. Here we report that an intermolecular asymmetric dearomatization reaction of α-naphthols bearing a tethered nucleophile at the C4 position of the naphthol ring is achieved by a chiral phosphoric acid. The reaction proceeds via a highly chemo- and regioselective aminative dearomatization/Michael addition sequence, affording a wide array of functionalized cyclic ketones in good yields (up to 93%) with excellent enantioselectivity (up to >99% ee). The catalyst loading can be reduced to 0.1 mol%. Preliminary mechanistic investigations identify that the enantioselectivity is established in the dearomatization step, while the Michael addition is the rate-limiting step. A working model accounting for the origin of the stereochemistry is proposed based on DFT calculations.

Manipulation of Spiroindolenine Intermediates for Enantioselective Synthesis of 3-(Indol-3-yl)-Pyrrolidines.

By manipulating the reactivity of spiroindolenine species, a sequential Michael/retro-Mannich/Mannich reaction of ω-indol-3-yl α,ß-unsaturated ketones was developed. In the presence of 10 mol % of a chiral phosphoric acid as the catalyst, a series of 3-(indol-3-yl)-pyrrolidines were synthesized in high yields (up to 91 %) with excellent stereoselectivities (up to 92 % ee, >19:1 d.r.). The products obtained here undergo diverse functional-group transformations. The mechanistic proposal of this reaction is supported by DFT calculations.

Catalytic Asymmetric Dearomatization of Indolyl Dihydropyridines through an Enamine Isomerization/Spirocyclization/Transfer Hydrogenation Sequence.

A highly efficient synthesis of enantioenriched spiroindolines by catalytic asymmetric dearomatization of indolyl dihydropyridines through a chiral phosphoric acid catalyzed enamine isomerization/spirocyclization/transfer hydrogenation sequence has been developed. This reaction proceeds under mild reaction conditions, affording novel spiroindolines in good yields (up to 88 %) with excellent enantioselectivity (up to 97 % ee). DFT calculations provide insights into the reaction mechanism as well as the origin of stereochemistry.

Facile access to 2,2-disubstituted indolin-3-ones via a cascade Fischer indolization/Claisen rearrangement reaction.

An efficient approach for the synthesis of 2,2-disubstituted indolin-3-ones is described. From readily accessible aryl hydrazines and allyloxyketones, 2,2-disubstituted indolin-3-ones could be obtained in good to excellent yields under mild reaction conditions via a cascade Fischer indolization/Claisen rearrangement process. This protocol provides a facile entry to 2,2-disubstituted indolin-3-ones, which have been applied in the construction of the benzofuroindoline framework related to Phalarine.

Construction of Chiral Tetrahydro-ß-Carbolines: Asymmetric Pictet-Spengler Reaction of Indolyl Dihydropyridines.

A highly efficient synthesis of the enantioenriched tetrahydro-ß-carbolines was developed by using a chiral phosphoric acid catalyzed Pictet-Spengler reaction of indolyl dihydropyridines. The reaction proceeds under mild reaction conditions to afford the desired chiral tetrahydro-ß-carbolines in good to excellent yields (up to 96 %) and high enantioselectivities (up to 99 % ee). With this method, a formal synthesis of tangutorine and a total synthesis of deplancheine were achieved in a highly efficient manner.

Chiral Phosphoric Acid Catalyzed Intramolecular Dearomative Michael Addition of Indoles to Enones.

An enantioselective intramolecular dearomative Michael addition of indolyl enones is presented. In the presence of catalytic amount of chiral phosphoric acid, various enantioenriched spiro-indolenines bearing a quaternary stereogenic center were obtained with good yields and enantioselectivity (up to 97% ee) under mild reaction conditions.

Enantioselective Bromo-oxycyclization of Silanol.

Relying on the nucleophilicity of silanol for building up silicon-incorporated scaffold with an enantiopure tetrasubstituted carbon center remains elusive. In this report, asymmetric bromo-oxycyclization of olefinic silanol by using chiral anionic phase-transfer catalyst is described. This protocol provided a facile entry to a wide arrangement of enantiopure benzoxasilole in moderate to excellent enantioselectivities depending on the unique reactivity of bromine/N-benzyl-DABCO complex.

Total Synthesis of (-)-Conolutinine.

The first enantioselective synthesis of (-)-conolutinine was achieved in 10 steps. The synthesis featured a catalytic asymmetric bromocyclization of tryptamine to forge the tricycle intermediate. Hydration of an alkene catalyzed by Co(acac)2 was also employed as a key step to diastereoselectively introduce the tertiary alcohol moiety. The absolute configuration of (-)-conolutinine was established to be (2S,5aS,8aS,13aR) based on this asymmetric total synthesis.

Synthesis of naked amino-pyrroloindoline via direct aminocyclization of tryptamine.

The first direct access to unprotected amino-pyrroloindoline via aminocyclization of tryptamine and tryptophan has been described. A variety of structurally diverse amino-pyrroloindolines are furnished by the use of O-(2,4-dinitrophenyl)hydroxylamine (DPH) as the nitrogen source in the presence of catalytic Rh2(esp)2.