Novel SARS-CoV-2 entry inhibitors, 2-anilinoquinazolin-4(3H)-one derivatives, show potency as SARS-CoV-2 antivirals in a human ACE2 transgenic mouse model.

The ongoing COVID-19 has not only caused millions of deaths worldwide, but it has also led to economic recession and the collapse of public health systems. The vaccines and antivirals developed in response to the pandemic have improved the situation markedly; however, the pandemic is still not under control with recurring surges. Thus, it is still necessary to develop therapeutic agents. In our previous studies, we designed and synthesized a series of novel 2-anilinoquinazolin-4(3H)-one derivatives, and demonstrated inhibitory activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and MERS-CoV in vitro. We then conducted in vivo studies using modified compounds that are suitable for oral administration. These compounds demonstrated no toxicity in rats and inhibited viral entry. Here, we investigated the in vivo efficacy of these drug candidates against SARS-CoV-2. Three candidate drugs, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (1), N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-dichlorophenyl)acetamide (2), and N-(7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-N-(3,5-difluorophenyl)acetamide (3) were administered orally to hACE2 transgenic mice at a dose of 100 mg/kg. All three drugs improved survival rate and reduced the viral load in the lungs. These results show that the derivatives possess in vivo antiviral efficacy similar to that of molnupiravir, which is currently being used to treat COVID-19. Overall, our data suggest that 2-anilinoquinazolin-4(3H)-one derivatives are promising as potential oral antiviral drug candidates against SARS-CoV-2 infection.

Discovery of 2-aminoquinolone acid derivatives as potent inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to threaten human health and create socioeconomic problems worldwide. A library of 200,000 small molecules from the Korea Chemical Bank (KCB) were evaluated for their inhibitory activities against SARS-CoV-2 in a phenotypic-based screening assay to discover new therapeutics to combat COVID-19. A primary hit of this screen was the quinolone structure-containing compound 1. Based on the structure of compound 1 and enoxacin, which is a quinolone-based antibiotic previously reported to have weak activity against SARS-CoV-2, we designed and synthesized 2-aminoquinolone acid derivatives. Among them, compound 9b exhibited potent antiviral activity against SARS-CoV-2 (EC50 = 1.5 µM) without causing toxicity, while having satisfactory in vitro PK profiles. This study shows that 2-aminoquinolone acid 9b provides a promising new template for developing anti-SARS-CoV-2 entry inhibitors.

Optimization of 2-Aminoquinazolin-4-(3H)-one Derivatives as Potent Inhibitors of SARS-CoV-2: Improved Synthesis and Pharmacokinetic Properties.

We previously reported the potent antiviral effect of the 2-aminoquinazolin-4-(3H)-one 1, which shows significant activity (IC50 = 0.23 µM) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with no cytotoxicity. However, it is necessary to improve the in vivo pharmacokinetics of compound 1 because its area under the curve (AUC) and maximum plasma concentration are low. Here, we designed and synthesized N-substituted quinazolinone derivatives that had good pharmacokinetics and that retained their inhibitory activity against SARS-CoV-2. These compounds were conveniently prepared on a large scale through a one-pot reaction using Dimroth rearrangement as a key step. The synthesized compounds showed potent inhibitory activity, low binding to hERG channels, and good microsomal stability. In vivo pharmacokinetic studies showed that compound 2b had the highest exposure (AUC24h = 41.57 µg∙h/mL) of the synthesized compounds. An in vivo single-dose toxicity evaluation of compound 2b at 250 and 500 mg/kg in rats resulted in no deaths and an approximate lethal dose greater than 500 mg/kg. This study shows that N-acetyl 2-aminoquinazolin-4-(3H)-one 2b is a promising lead compound for developing anti-SARS-CoV-2 agents.

Anticoronaviral Activity of the Natural Phloroglucinols, Dryocrassin ABBA and Filixic Acid ABA from the Rhizome of Dryopteris crassirhizoma by Targeting the Main Protease of SARS-CoV-2.

The rhizome of Dryopteris crassirhizoma Nakai. (Dryopteridaceae) has been used in traditional medicine in East Asia and has recently been reported to have anticancer, anti-inflammation, and antibacterial activity as well as antiviral activity. Natural phloroglucinols from D. crassirhizoma, dryocrassin ABBA and filixic acid ABA were reported to inhibit influenza virus infection with an inhibitory activity on neuraminidase. In this study, we found that dryocrassin ABBA and filixic acid ABA have an inhibitory activity against the main protease of SARS-CoV-2. Therefore, dryocrassin ABBA and filixic acid ABA exhibited inhibitory activity against SARS-CoV-2 infection in Vero cells dose-dependently using the immunofluorescence-based antiviral assays. Moreover, these compounds inhibited SARS-CoV and MERS-CoV infection, suggesting their broad-spectrum anticoronaviral activity. In addition, a 5-day repeated-dose toxicity study of dryocrassin ABBA and filixic acid ABA suggested that an approximately lethal dose of these compounds in mice was >10 mg/kg. Pharmacokinetic studies of dryocrassin ABBA showed good microsomal stability, low hERG inhibition, and low CYP450 inhibition. In vivo pharmacokinetic properties of dryocrassin ABBA showed a long half-life (5.5-12.6 h) and high plasma exposure (AUC 19.3-65 µg·h/mL). Therefore, dryocrassin ABBA has therapeutic potential against emerging coronavirus infections, including COVID-19.

Synthesis and biological evaluation of 2-benzylaminoquinazolin-4(3H)-one derivatives as a potential treatment for SARS-CoV-2.

Despite the continuing global crisis caused by coronavirus disease 2019 (COVID-19), there is still no effective treatment. Therefore, we designed and synthesized a novel series of 2-benzylaminoquinazolin-4(3H)-one derivatives and demonstrated that they are effective against SARS-CoV-2. Among the synthesized derivatives, 7-chloro-2-(((4-chlorophenyl)(phenyl)methyl)amino)quinazolin-4(3H)-one (Compound 39) showed highest anti-SARS-CoV-2 activity, with a half-maximal inhibitory concentration value greater than that of remdesivir (IC50 = 4.2 µM vs. 7.6 µM, respectively), which gained urgent approval from the U.S. Food and Drug Administration. In addition, Compound 39 showed good results in various assays measuring metabolic stability, human ether a-go-go, Cytochromes P450 (CYPs) inhibition, and plasma protein binding (PPB), and showed better solubility and pharmacokinetics than our previous work.

Broad Spectrum Antiviral Properties of Cardiotonic Steroids Used as Potential Therapeutics for Emerging Coronavirus Infections.

Cardiotonic steroids are steroid-like natural compounds known to inhibit Na+/K+-ATPase pumps. To develop a broad-spectrum antiviral drug against the emerging coronavirus infection, this study assessed the antiviral properties of these compounds. The activity of seven types of cardiotonic steroids against the MERS-CoV, SARS-CoV, and SARS-CoV-2 coronavirus varieties was analyzed using immunofluorescence antiviral assay in virus-infected cells. Bufalin, cinobufagin, and telocinobufagin showed high anti-MERS-CoV activities (IC50, 0.017~0.027 µM); bufalin showed the most potent anti-SARS-CoV and SARS-CoV-2 activity (IC50, 0.016~0.019 µM); cinobufotalin and resibufogenin showed comparatively low anti-coronavirus activity (IC50, 0.231~1.612 µM). Differentially expressed genes in Calu3 cells treated with cinobufagin, telocinobufagin, or bufalin, which had high antiviral activity during MERS-CoV infection were analyzed using QuantSeq 3' mRNA-Seq analysis and data showed similar gene expression patterns. Furthermore, the intraperitoneal administration of 10 mg/kg/day bufalin, cinobufagin, or digitoxin induced 100% death after 1, 2, and 4 days in 5-day repeated dose toxicity studies and it indicated that bufalin had the strongest toxicity. Pharmacokinetic studies suggested that telocinobufagin, which had high anti-coronavirus activity and low toxicity, had better microsomal stability, lower CYP inhibition, and better oral bioavailability than cinobufagin. Therefore, telocinobufagin might be the most promising cardiotonic steroid as a therapeutic for emerging coronavirus infections, including COVID-19.

Design, synthesis and biological evaluation of 2-aminoquinazolin-4(3H)-one derivatives as potential SARS-CoV-2 and MERS-CoV treatments.

Despite the rising threat of fatal coronaviruses, there are no general proven effective antivirals to treat them. 2-Aminoquinazolin-4(3H)-one derivatives were newly designed, synthesized, and investigated to show the inhibitory effects on SARS-CoV-2 and MERS-CoV. Among the synthesized derivatives, 7-chloro-2-((3,5-dichlorophenyl)amino)quinazolin-4(3H)-one (9g) and 2-((3,5-dichlorophenyl)amino)-5-hydroxyquinazolin-4 (3H)-one (11e) showed the most potent anti-SARS-CoV-2 activities (IC50 < 0.25 µM) and anti-MERS-CoV activities (IC50 < 1.1 µM) with no cytotoxicity (CC50 > 25 µM). In addition, both compounds showed acceptable results in metabolic stabilities, hERG binding affinities, CYP inhibitions, and preliminary PK studies.

Lycorine, a non-nucleoside RNA dependent RNA polymerase inhibitor, as potential treatment for emerging coronavirus infections.

BACKGROUND: Highly effective novel treatments need to be developed to suppress emerging coronavirus (CoV) infections such as COVID-19. The RNA dependent RNA polymerase (RdRp) among the viral proteins is known as an effective antiviral target. Lycorine is a phenanthridine Amaryllidaceae alkaloid isolated from the bulbs of Lycoris radiata (L'Hér.) Herb. and has various pharmacological bioactivities including antiviral function. PURPOSE: We investigated the direct-inhibiting action of lycorine on CoV's RdRp, as potential treatment for emerging CoV infections. METHODS: We examined the inhibitory effect of lycorine on MERS-CoV, SARS-CoV, and SARS-CoV-2 infections, and then quantitatively measured the inhibitory effect of lycorine on MERS-CoV RdRp activity using a cell-based reporter assay. Finally, we performed the docking simulation with lycorine and SARS-CoV-2 RdRp. RESULTS: Lycorine efficiently inhibited these CoVs with IC50 values of 2.123 ± 0.053, 1.021 ± 0.025, and 0.878 ± 0.022 µM, respectively, comparable with anti-CoV effects of remdesivir. Lycorine directly inhibited MERS-CoV RdRp activity with an IC50 of 1.406 ± 0.260 µM, compared with remdesivir's IC50 value of 6.335 ± 0.731 µM. In addition, docking simulation showed that lycorine interacts with SARS-CoV-2 RdRp at the Asp623, Asn691, and Ser759 residues through hydrogen bonding, at which the binding affinities of lycorine (-6.2 kcal/mol) were higher than those of remdesivir (-4.7 kcal/mol). CONCLUSIONS: Lycorine is a potent non-nucleoside direct-acting antiviral against emerging coronavirus infections and acts by inhibiting viral RdRp activity; therefore, lycorine may be a candidate against the current COVID-19 pandemic.

Discovery of cyclic sulfonamide derivatives as potent inhibitors of SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) continues to spread worldwide, with 25 million confirmed cases and 800 thousand deaths. Effective treatments to target SARS-CoV-2 are urgently needed. In the present study, we have identified a class of cyclic sulfonamide derivatives as novel SARS-CoV-2 inhibitors. Compound 13c of the synthesized compounds exhibited robust inhibitory activity (IC50 = 0.88 µM) against SARS-CoV-2 without cytotoxicity (CC50 > 25 µM), with a selectivity index (SI) of 30.7. In addition, compound 13c exhibited high oral bioavailability (77%) and metabolic stability with good safety profiles in hERG and cytotoxicity studies. The present study identified that cyclic sulfonamide derivatives are a promising new template for the development of anti-SARS-CoV-2 agents.

Identification of 4-anilino-6-aminoquinazoline derivatives as potential MERS-CoV inhibitors.

New therapies for treating coronaviruses are urgently needed. A series of 4-anilino-6-aminoquinazoline derivatives were synthesized and evaluated to show high anti-MERS-CoV activities. N4-(3-Chloro-4-fluorophenyl)-N6-(3-methoxybenzyl)quinazoline-4,6-diamine (1) has been identified in a random screen as a hit compound for inhibiting MERS-CoV infection. Throughout optimization process, compound 20 was found to exhibit high inhibitory effect (IC50 = 0.157 µM, SI = 25) with no cytotoxicity and moderate in vivo PK properties.