Reversible Covalent Inhibitors Research Articles

Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

The Design, Synthesis and Mechanism of Action of Paxlovid, a Protease Inhibitor Drug Combination for the Treatment of COVID-19.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has presented an enormous challenge to health care systems and medicine. As a result of global research efforts aimed at preventing and effectively treating SARS-CoV-2 infection, vaccines with fundamentally new mechanisms of action and some small-molecule antiviral drugs targeting key proteins in the viral cycle have been developed. The most effective small-molecule drug approved to date for the treatment of COVID-19 is PaxlovidTM, which is a combination of two protease inhibitors, nirmatrelvir and ritonavir. Nirmatrelvir is a reversible covalent peptidomimetic inhibitor of the main protease (Mpro) of SARS-CoV-2, which enzyme plays a crucial role in viral reproduction. In this combination, ritonavir serves as a pharmacokinetic enhancer, it irreversibly inhibits the cytochrome CYP3A4 enzyme responsible for the rapid metabolism of nirmatrelvir, thereby increasing the half-life and bioavailability of nirmatrelvir. In this tutorial review, we summarize the development and pharmaceutical chemistry aspects of Paxlovid, covering the evolution of protease inhibitors, the warhead design, synthesis and the mechanism of action of nirmatrelvir, as well as the synthesis of ritonavir and its CYP3A4 inhibition mechanism. The efficacy of Paxlovid to novel virus mutants is also overviewed.

Peptidyl nitroalkene inhibitors of main protease rationalized by computational and crystallographic investigations as antivirals against SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic continues to represent a global public health issue. The viral main protease (Mpro) represents one of the most attractive targets for the development of antiviral drugs. Herein we report peptidyl nitroalkenes exhibiting enzyme inhibitory activity against Mpro (Ki: 1–10 μM) good anti-SARS-CoV-2 infection activity in the low micromolar range (EC50: 1–12 μM) without significant toxicity. Additional kinetic studies of compounds FGA145, FGA146 and FGA147 show that all three compounds inhibit cathepsin L, denoting a possible multitarget effect of these compounds in the antiviral activity. Structural analysis shows the binding mode of FGA146 and FGA147 to the active site of the protein. Furthermore, our results illustrate that peptidyl nitroalkenes are effective covalent reversible inhibitors of the Mpro and cathepsin L, and that inhibitors FGA145, FGA146 and FGA147 prevent infection against SARS-CoV-2.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation

Thiophene-fused γ-lactams are reversible covalent inhibitors of the SARS-CoV-2 main protease, a nucleophilic cysteine enzyme. γ-Lactams can inhibit nucleophilic cysteine enzymes by S-acylation as well as nucleophilic serine enzymes by O-acylation.

Abstract B172: Potent anti-tumor activity of a selective and orally bioavailable reversible covalent CDK12 inhibitor

Abstract Cyclin-dependent kinase 12 (CDK12), in association with Cyclin K (CycK), regulates the elongation of transcription by modifying RNA polymerase II (RNAP II) through phosphorylation at Serine 2 (pS2) in the C-terminal domain, CDK12 plays an important role in maintaining genomic stability by modulating crucial cellular processes such as DNA damage response, splicing, and pre-mRNA processing. Overexpression of CDK12 has been observed in various tumor types, suggesting its potential oncogenic properties like other kinases involved in transcription. Given its significance in transcription and RNA processing, CDK12 has emerged as a promising therapeutic target for cancer treatment. We have identified a highly potent and selective reversible covalent inhibitor of CDK12, AU-22880, which exhibits significant anti-proliferative activity across various cancer cell lines. The covalent mode of action and on-target activity of AU-22880 were confirmed in a CDK12 target engagement assay and selective pS2 RNAPII inhibition respectively in a cellular context. Additionally, it has shown synergistic activity in combination with several standard-of-care agents in breast cancer, ovarian cancer, pancreatic cancer, and prostate cancer cell lines. AU-22880 shows desirable ADME-PK properties and demonstrates excellent anti-tumor activity in xenograft models. In the HCC-70 TNBC xenograft model, AU-22880 demonstrated anti-tumor efficacy at well-tolerated doses. A pharmacokinetic-pharmacodynamic (PK-PD) study revealed dose-dependent target engagement, as evidenced by the inhibition of pS2 RNAPII and DNA damage repair (DDR) genes. Furthermore, AU-22880 exhibited good tolerability in rodents, dog, and monkeys at exposure levels that demonstrated efficacy. Efficacy of AU-22880 in combination with approved SOCs targeting DDR pathway are currently being investigated. The findings reported here highlight the promising therapeutic potential of AU-22880, both as a monotherapy and in combination with existing treatments for the treatment of cancers characterized by DDR dysregulation. Citation Format: Leena Khare, Ramulu Poddutoori, Subhendu Mukherjee, Samiulla DS, Devaraja TS, Sivapriya Marappan, Shilpa Nayak, Sasirekha Sivakumar, Lakshmi Kaza, Suraj Tgore, Amit Dhudashiya, Raghavendra NR, Bhargav Gunnepalli, Aravind A B, Amith A, Charamanna KB, Thomas Antony, Kavitha Nellore, Girish Daginakatte, Shekar Chelur, Sanjeev Giri, Murali Ramachandra, Susanta Samajdar. Potent anti-tumor activity of a selective and orally bioavailable reversible covalent CDK12 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B172.

Expanding the Chemistry of Dihaloacetamides as Tunable Electrophiles for Reversible Covalent Targeting of Cysteines.

The choice of an appropriate electrophile is crucial in the design of targeted covalent inhibitors (TCIs). In this report, we systematically investigated the glutathione (GSH) reactivity of various haloacetamides and the aqueous stability of their thiol adducts. Our findings revealed that dihaloacetamides cover a broad range of GSH reactivity depending on the combination of the halogen atoms and the structure of the amine scaffold. Among the dihaloacetamides, dichloroacetamide (DCA) exhibited slightly lower GSH reactivity than chlorofluoroacetamide (CFA). The DCA-thiol adduct is readily hydrolyzed under aqueous conditions, but it can stably exist in the solvent-sequestered binding pocket of the protein. These reactivity profiles of DCA were successfully exploited in the design of TCIs targeting noncatalytic cysteines of KRASG12C and EGFRL858R/T790M. These inhibitors exhibited strong antiproliferative activities against cancer cells. Our findings provide valuable insights for designing dihaloacetamide-based reversible covalent inhibitors.

Covalent-reversible peptide-based protease inhibitors. Design, synthesis, and clinical success stories.

Dysregulated human peptidases are implicated in a large variety of diseases such as cancer, hypertension, and neurodegeneration. Viral proteases for their part are crucial for the pathogens' maturation and assembly. Several decades of research were devoted to exploring these precious therapeutic targets, often addressing them with synthetic substrate-based inhibitors to elucidate their biological roles and develop medications. The rational design of peptide-based inhibitors offered a rapid pathway to obtain a variety of research tools and drug candidates. Non-covalent modifiers were historically the first choice for protease inhibition due to their reversible enzyme binding mode and thus presumably safer profile. However, in recent years, covalent-irreversible inhibitors are having a resurgence with dramatic increase of their related publications, preclinical and clinical trials, and FDA-approved drugs. Depending on the context, covalent modifiers could provide more effective and selective drug candidates, hence requiring lower doses, thereby limiting off-target effects. Additionally, such molecules seem more suitable to tackle the crucial issue of cancer and viral drug resistances. At the frontier of reversible and irreversible based inhibitors, a new drug class, the covalent-reversible peptide-based inhibitors, has emerged with the FDA approval of Bortezomib in 2003, shortly followed by 4 other listings to date. The highlight in the field is the breathtakingly fast development of the first oral COVID-19 medication, Nirmatrelvir. Covalent-reversible inhibitors can hipothetically provide the safety of the reversible modifiers combined with the high potency and specificity of their irreversible counterparts. Herein, we will present the main groups of covalent-reversible peptide-based inhibitors, focusing on their design, synthesis, and successful drug development programs.

Structure-based computational design of novel covalent binders for the treatment of sickle cell disease

The quest in finding an everlasting panacea to the pernicious impact of sickle cell disease (SCD) in the society hit a turn of success since the recent discovery of a small molecule reversible covalent inhibitor, Voxelotor. A drug that primarily promotes the stability of oxygenated hemoglobin and inhibit the polymerization of HbS by enhancing hemoglobin's affinity for oxygen has opened a new frontier in drug discovery and development. Despite eminent efforts made to reproduce small molecules with better therapeutic targets, none has been successful. To this end, we employed the use of structure-based computational techniques with emphasis on the electrophilic warhead group of Voxelotor to harness novel covalent binders that could elicit better therapeutic response against HbS. The PubChem database and DataWarrior software were used to design random molecules using Voxelotor's electrophilic functionality. Following the compilation of these chemical entities, a high-throughput covalent docking-based virtual screening campaign was conducted which revealed three (Compound_166, Compound_2301, and Compound_2335) putative druglike candidates with higher baseline energy value compared to the standard drug. Subsequently, in silico ADMET profiling was carried out to evaluate their pharmacokinetics and pharmacodynamics properties, and their stability was evaluated for 1 μs (1 μs) using molecular dynamics simulation. Finally, to prioritize these compounds for further development in drug discovery, MM/PBSA calculations was employed to evaluate their molecular interactions and solvation energy within the HbS protein. Despite the admirable druglike and stability properties of these compounds, further experimental validations are required to establish their preclinical relevance for drug development.

An update on the discovery and development of reversible covalent inhibitors.

Small molecule drugs that covalently bind irreversibly to their target proteins have several advantages over conventional reversible inhibitors. They include increased duration of action, less-frequent drug dosing, reduced pharmacokinetic sensitivity, and the potential to target intractable shallow binding sites. Despite these advantages, the key challenges of irreversible covalent drugs are their potential for off-target toxicities and immunogenicity risks. Incorporating reversibility into covalent drugs would lead to less off-target toxicity by forming reversible adducts with off-target proteins and thus reducing the risk of idiosyncratic toxicities caused by the permanent modification of proteins, which leads to higher levels of potential haptens. Herein, we systematically review electrophilic warheads employed during the development of reversible covalent drugs. We hope the structural insights of electrophilic warheads would provide helpful information to medicinal chemists and aid in designing covalent drugs with better on-target selectivity and improved safety.

Rilzabrutinib, a reversible covalent Bruton's tyrosine kinase inhibitor: Absorption, metabolism, excretion and absolute bioavailability in healthy participants.

This single-center, open-label, non-randomized, two-part, phase I study was conducted 1) to evaluate the absolute oral bioavailability of rilzabrutinib 400 mg tablet following an IV microtracer dose of ~100 μg [14C]-rilzabrutinib (~1 μCi) and single oral dose of 400 mg rilzabrutinib tablet (Part 1), and 2) to characterize the absorption, metabolism, and excretion (AME) of 14C-radiolabeled rilzabrutinib following single oral dose (300 mg) of [14C]-rilzabrutinib (~1000 μCi) (administered as a liquid) in healthy male participants (Part 2). A total of 18 subjects were enrolled (n=8 in Part 1; n=10 in Part 2). The absolute bioavailability of 400 mg rilzabrutinib oral tablet was low (less than 5%). In Part 1, rilzabrutinib was absorbed rapidly after single oral dose of rilzabrutinib 400 mg tablet with a median (range) tmax value of 2.03 h (1.83-2.50). The geometric mean (CV%) t1/2 following the oral dose and IV microtracer dose of ~100 μg [14C]-rilzabrutinib, were 3.20 (51.0%) and 1.78 (37.6%) hours, respectively. In Part 2, rilzabrutinib was also absorbed rapidly following single oral dose of 300 mg [14C]-rilzabrutinib solution with a median (range) tmax value of 1.00 h (1.00-2.00). The majority of total radioactivity was in the feces for both non-bile collection subjects (92.9%) and bile collection subjects (87.6%), and approximately 5% of radioactivity was recovered in urine after oral administration. Urinary excretion of unchanged rilzabrutinib was low (3.02%). The results of this study advance the understanding of the absolute bioavailability and AME of rilzabrutinib and can help inform its further investigation.

Broad-spectrum coronavirus 3C-like protease peptidomimetic inhibitors effectively block SARS-CoV-2 replication in cells: Design, synthesis, biological evaluation, and X-ray structure determination

Despite the approval of vaccines, monoclonal antibodies and restrictions during the pandemic, the demand for new efficacious and safe antivirals is compelling to boost the therapeutic arsenal against the COVID-19. The viral 3-chymotrypsin-like protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants showing an almost unique specificity for Leu-Gln as P2–P1 residues, allowing the development of broad-spectrum inhibitors.The design, synthesis, biological activity, and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are herein described. The inhibitors display an aldehyde warhead, a Gln mimetic at P1 and modified P2–P3 residues. Particularly, functionalized proline residues were inserted at P2 to stabilize the β-turn like bioactive conformation, modulating the affinity. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited viral replication of three human CoVs, i.e. SARS-CoV-2, MERS-CoV, and HCoV 229 in different cell lines. Particularly, derivative 12 exhibited nM-low μM antiviral activity depending on the virus, and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro validating our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics.

Sulforaphane is a reversible covalent inhibitor of 3-chymotrypsin-like protease of SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a major public health threat worldwide and emphasizes an urgent need for effective therapeutics. Recently, Ordonez et al. identified sulforaphane (SFN) as a novel coronavirus inhibitor both in vitro and in mice, but the mechanism of action remains elusive. In this study, we independently discovered SFN for its inhibitory effect against SARS-CoV-2 using a target-based screening approach, identifying the viral 3-chymotrypsin-like protease (3CLpro ) as a target of SFN. Mechanistically, SFN inhibits 3CLpro in a reversible, mixed-type manner. Moreover, enzymatic kinetics studies reveal that SFN is a slow-binding inhibitor, following a two-step interaction. Initially, an encounter complex forms by specific binding of SFN to the active pocket of 3CLpro ; subsequently, the isothiocyanate group of SFN as "warhead" reacts covalently to the catalytic cysteine in a slower velocity, stabilizing the SFN-3CLpro complex. Our study has identified a new lead of the covalent 3CLpro inhibitors which has potential to be developed as a therapeutic agent to treat SARS-CoV-2 infection.

An approach combining deep learning and molecule docking for drug discovery of cathepsin L

ABSTRACT Objectives Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders and COVID-19. However, there are still no clinically available CTSL inhibitors. Our objective is to develop an approach for the discovery of potential reversible covalent CTSL inhibitors. Methods The authors combined Chemprop, a deep learning-based strategy, and the Schrödinger CovDock algorithm to identify potential CTSL inhibitors. First, they used Chemprop to train a deep learning model capable of predicting whether a molecule would inhibit the activity of CTSL and performed predictions on ZINC20 in-stock librarie (~9.2 million molecules). Then, they selected the top-200 predicted molecules and performed the Schrödinger covalent docking algorithm to explore the binding patterns to CTSL (PDB: 5MQY). The authors then calculated the binding energies using Prime MM/GBSA and examined the stability between the best two molecules and CTSL using 100ns molecular dynamics simulations. Results The authors found five molecules that showed better docking results than the well-known cathepsin inhibitor odanacatib. Notably, two of these molecules, ZINC-35287427 and ZINC-1857528743, showed better docking results with CTSL compared to other cathepsins. Conclusion Our approach enables drug discovery from large-scale databases with little computational consumption, which will save the cost and time required for drug discovery.

RGD Cyclopeptide Equipped with a Lysine-Engaging Salicylaldehyde Showing Enhanced Integrin Affinity and Cell Detachment Potency.

Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported.

Antiviral Pills against SARS-CoV-2 Virus to Combat Future Epidemic Threats in Pakistan

Antiviral pills are oral medications that treat infections through the inhibition of the viral growth and replication cycle. Paxlovid by Pfizer and Molnupiravir by Merck are the two pills effective for high-risk SARS-CoV-2 patients. Paxlovid works by inhibiting the replication cycle of SARS-CoV-2 using cysteine residues in-vitro. These cysteine residues inhibit the main protease of the virus by functioning as reversible covalent inhibitors. Molnupiravir works by introducing a high rate of mutations in the viral RNA causing the virus to become biologically unstable and non-functional. Both antiviral drugs can bridge the gap in the preparedness for viral outbreaks in low-income countries like Pakistan by mitigating the chances of fatality and inpatient treatment in high-risk, unvaccinated individuals. Pakistan has been plagued by various epidemics over the years however SARS-CoV-2 outbreak caused many deaths along with an economic crisis. The country lacks the resources to endure high inpatient treatment rates in case of SARS-CoV-2 infections, which is why the need for antiviral pills like Paxlovid and Molnupiravir is empirical to overcome epidemics and viral outbreaks. This work outlines the antiviral pills and their efficacy against SARS-CoV-2 with a focus on how these drugs can overcome significant gaps in epidemic preparedness and response in Pakistan. We aim to highlight how antiviral pills against SARS-CoV-2 can ensure resilience to future epidemic threats in Pakistan.

Discovery and Computational Studies of Potent Covalent Kinase Inhibitors with α-Substituent Electrophiles Targeting Cysteine.

Both reversible noncovalent inhibitors and irreversible covalent inhibitors targeting tyrosine kinases have their disadvantages. The reversible covalent inhibitors with electrophilic group cyanoacrylamide as warheads reacting with cysteine residues could solve the dilemmas. However, there are still several unresolved issues regarding the electrophilic groups. In this manuscript, a series of EGFR inhibitors with double electron-withdrawing substituents introduced into the Cα position on the olefin bond were designed and synthesized. The binding structures and characteristics of inhibitors with the kinase in both the first noncovalent binding phase and the second covalent binding step were explored and combined with molecular docking and molecular dynamics simulations. Then, the reverse β-elimination reactions of the thiol-Michael adducts were investigated by applying density functional theory calculations. In addition, the effects of different electrophilic substituents of Cα on the binding between the inhibitors and kinase were elucidated. The results suggested that the electrophilicity and size of the electron-withdrawing groups play an important role in the specific interactions during the reaction. The compounds with the electron-withdrawing groups that had medium electrostatic and steric complementarity to the kinase active site could cooperatively stabilize the complexes and showed relatively good potent activities in the kinase assay experiment. The mechanical and structural information in this study could enhance our understanding of the functioning of the electron-withdrawing groups in the covalent inhibitors. The results might help to design efficient cysteine targeting inhibitors in the future.

Mercapto-pyrimidines are reversible covalent inhibitors of the papain-like protease (PLpro) and inhibit SARS-CoV-2 (SCoV-2) replication.

The papain-like protease (PLpro) plays a critical role in SARS-CoV-2 (SCoV-2) pathogenesis and is essential for viral replication and for allowing the virus to evade the host immune response. Inhibitors of PLpro have great therapeutic potential, however, developing them has been challenging due to PLpro's restricted substrate binding pocket. In this report, we screened a 115 000-compound library for PLpro inhibitors and identified a new pharmacophore, based on a mercapto-pyrimidine fragment that is a reversible covalent inhibitor (RCI) of PLpro and inhibits viral replication in cells. Compound 5 had an IC50 of 5.1 μM for PLpro inhibition and hit optimization yielded a derivative with increased potency (IC50 0.85 μM, 6-fold higher). Activity based profiling of compound 5 demonstrated that it reacts with PLpro cysteines. We show here that compound 5 represents a new class of RCIs, which undergo an addition elimination reaction with cysteines in their target proteins. We further show that their reversibility is catalyzed by exogenous thiols and is dependent on the size of the incoming thiol. In contrast, traditional RCIs are all based upon the Michael addition reaction mechanism and their reversibility is base-catalyzed. We identify a new class of RCIs that introduces a more reactive warhead with a pronounced selectivity profile based on thiol ligand size. This could allow the expansion of RCI modality use towards a larger group of proteins important for human disease.

Multitarget inhibitors/probes that target LRRK2 and AURORA A kinases noncovalently and covalently.

Herein, we report a salicylaldehyde-based, reversible covalent inhibitor (A2) that possesses moderate cellular activity against AURKA with a prolonged residence time and shows significant non-covalent inhibition towards LRRK2. Our results indicated that this multitarget kinase inhibitor may be used as the starting point for future development of more potent, selective and dual-targeting covalent kinase inhibitors against AURKA and LRRK2 for mitophagy.

Discovery of DA‐7503 as a Novel Tau‐targeted Therapeutic Candidate for the Treatment of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is a late‐onset, progressive, age‐dependent neurodegenerative disorder. The load of neurofibrillary tangles correlates strongly with clinical progression of the disease. Although tauopathy has been extensively studied as a key hypothesis in AD, there are currently no clinical tau‐targeted drugs that produce a noticeable improvement. Therefore, we studied and developed novel tau‐targeted therapeutic drug candidates for the treatment of AD; in particular, tau aggregation inhibitors.MethodWe performed high‐content screening based on a Tau‐BiFC cell‐based assay platform and identified novel tau aggregation inhibitors. Lead optimization was subsequently performed to improve potency and ADME/Tox properties, resulting in the production of DA‐7503. In vivo efficacy of the candidate was validated by demonstration of memory impairment restoration in two transgenic animal models of P301L Tau‐BiFC and rTg4510. The non‐clinical GLP‐Tox studies of DA‐7503 is ongoing in order to proceed to clinical studies.ResultDA‐7503 exhibited excellent tau aggregation inhibitory activities (cell‐based IC50, 0.02 mM, in vitro IC50, 0.96 mM) with good cell viability (MTS IC50, 77.8 mM). DA‐7503 reduced tau oligomerization and protected against neuronal cell death. It restored memory impairment in P301L Tau‐BiFC and rTg4510 Tg mouse models (NOR, Open Field, and Morris Water Maze test) with reduction of tau pathology. The mode of action of DA‐7503 was confirmed by MALDI‐TOF Mass Spectrometric Analysis, 2D HSQC NMR and TR‐2 competition study to show reversible covalent inhibition of disulfide‐dependent oligomer formation.Conclusion DA‐7503, a highly potent inhibitor of tau aggregation, significantly improved memory impairment and reduced tau pathology in P301L Tau‐BiFC and rTg4510 Tg mouse models with good pharmacological properties. Currently, the GLP‐Tox safety profile of DA‐7503 as a non‐clinical candidate is being evaluated, and early results are promising enough to proceed to clinical studies.

Potent and biostable inhibitors of the main protease of SARS-CoV-2

Potent and biostable inhibitors of the main protease (Mpro) of SARS-CoV-2 were designed and synthesized based on an active hit compound 5h (2). Our strategy was based not only on the introduction of fluorine atoms into the inhibitor molecule for an increase of binding affinity for the pocket of Mpro and cell membrane permeability but also on the replacement of the digestible amide bond by a surrogate structure to increase the biostability of the compounds. Compound 3 is highly potent and blocks SARS-CoV-2 infection invitro without a viral breakthrough. The derivatives, which contain a thioamide surrogate in the P2-P1 amide bond of these compounds (2 and 3), showed remarkably preferable pharmacokinetics in mice compared with the corresponding parent compounds. These data show that compounds 3 and its biostable derivative 4 are potential drugs for treating COVID-19 and that replacement of the digestible amide bond by its thioamide surrogate structure is an effective method.