Graphical abstract
Abbreviations: SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; KCB, Korea Chemical Bank; VOCs, variants of concern
Abstract
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.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus belonging to the betacoronavirus genus that exclusively infects mammalian species.1 Human coronaviruses, such as HCoV-NL63 and HCoV-HKU1, cause mild respiratory tract infection associated with symptoms of the ‘common cold’. By contrast, SARS-CoV-2 is pathogenic and infects the upper respiratory tract, causing life-threatening respiratory disease and lung damage.1, 2
The rapid spread of SARS-CoV-2 has had a significant impact on global health. COVID-19 continues to threaten human health and cause socioeconomic problems worldwide. SARS-CoV-2 spread rapidly across the globe, resulting in over 659 million confirmed cases and over 6.7 million deaths, as reported by the World Health Organization in 10 January 2023.3
Although COVID-19 vaccines are available, new variants of concern (VOCs), such as Delta or Omicron, could reduce vaccine effectiveness.4 In addition, vaccine booster shots must be taken periodically to maintain protection from SARS-CoV-2. Because of these limitations of vaccines, it is essential to develop antiviral drugs against SARS-CoV-2. Responding to an urgent need, Paxlovid™ (two tablets of nirmatrelvir and one tablet of ritonavir) and molnupiravir are authorized for emergency use by the USA Food and Drug Administration (FDA) as oral antiviral drugs.5 Although Paxlovid significantly reduces hospitalization and mortality resulting from SARS-CoV-2, drug-drug interaction must be taken into account because of potent CYP3A4 inhibition. Furthermore, the mutations in main protease (Mpro), which is the target protein of nirmatrelvir, could potentially raise drug resistance.6 Molnupiravir, which is an RNA-dependent RNA polymerase inhibitor, reduces the risk of hospitalization or death in unvaccinated outpatients with mild or moderate COVID-19.7 However, molnupiravir could induce genotoxicity arising from its mechanism of action.8 Another effective therapeutic agent should be developed to expand SARS-CoV-2 treatment options.
Small molecule library screening allows for the rapid introduction of drugs into clinical settings. This process is the quickest way to find bioactive compounds against SARS-CoV-2.9 Molnupiravir was originally developed for the treatment of influenza,10 and nirmatrelvir was developed by modification of PF-00835231 as a potent inhibitor of recombinant SARS-CoV-1 Mpro.11
We conducted high content screening of a library of 200,000 small molecule compounds from the Korea Chemical Bank (KCB). A primary hit from this screening was compound 1 with a quinolone structure,12 which showed an EC50 of 14.2 µM against SARS-CoV-2 in Vero cell (Fig. 1 ). Compound 1 contains an alkylamine and isopropyl ketone in the C-2 and C-3 position, respectively, and has been investigated as an agrochemical drug. Generally, the quinolone skeleton can be synthesized by a simple and flexible synthetic route as a preferred building block.13 Various biological activities, such as antibiotic, anxiolytic, anticancer, and antiviral activity were reported for quinolone skeleton-containing compounds.13, 14, 15 Fluoroquinolones, which are antimicrobial agents with broad spectrum and potent activity, show weak activities against SARS-CoV-2. For example, the fluoroquinolone, enoxacin, has an EC50 value of 126 µM in Vero cells.16 In addition, a molecular docking study shows that enoxacin binds to Mpro.17 Elvitegravir is another compound with a quinolone structure. It was approved by the FDA as an HIV integrase inhibitor for the treatment of AIDS in 2012. Thus, compounds with quinolone structures are valuable as antiviral agents. We designed a carboxylic acid instead of a ketone at the C-3 position of hit compound 1 (Fig. 2 ). Most of the currently approved quinolone-based drugs contain carboxylic acid, enabling the synthesis of more diverse structures through further modification, such as amide coupling. In addition, amino groups were introduced at the C-2 position on the quinolone acid core, and the C-6 or C-7 position on the ring was replaced with electron withdrawing groups or electron donating groups. Here, we report the synthesis of 2-amino-quinolone acid with a novel template and assess its antiviral activity, safety, and in vitro pharmacokinetics.
Fig. 1.
Structures of hit compound 1, enoxacin, and elvitegravir.
Fig. 2.
Design of novel 2-aminoquinolone derivatives.
A series of 2-aminoquinolone acid derivatives were prepared as shown in Scheme 1 . Aniline 2 and dithioacetals 3 were reacted with N-iodosuccimide through a one-pot reaction of Lim’s method.18 to obtain quinolone 4 by reflux in 1,2-dichlorobenzene. Oxidation of quinolone 4 with m-chloroperbenzoic acid yielded sulfoxide 5. Nucleophilic aromatic substitution of 5 with alkylamine and aniline derivatives led to compounds 6 and 8, respectively. Because aniline has low nucleophilicity, the reaction proceeded under high-temperature and high-pressure conditions in a sealed tube. We attempted hydrolysis of esters with acids or bases, but it failed to yield the desired carboxylic acid product.
Scheme 1.
Synthesis of 2-amino-4-quinolone acid derivatives. Reagent and conditions: (a) i) N-iodosuccinimide, THF, reflux, 24 h; ii) 1,2-dichlorobenzene, 190 °C, 30 min; (b) m-chloroperbenzoic acid, dichloromethane, rt, 4 h; (c) alkylamines, triethylamine, chloroform, 90 °C, 3 h; (d) anilines, 1,2-dichloroethane, 130 °C, 9 h; (e) iodotrimethylsilane, chloroform, 60 °C, 4 h; (f) 2 N HCl, THF, H2O, 100 °C, 9 h; (g) 2 M methylamine in EtOH, 50 °C, 5 h.
When the reaction temperature was raised under acidic or basic conditions, a decarboxylated compound 10 was formed. Demethylation of methyl ester 6 or 8 with iodotrimethylsilane as a mild and neutral reagent yielded 7 or 9, respectively. In addition, methyl ester 6 was converted to amide 11 using 2 M methylamine in ethanol.
The synthesized quinolone acid derivatives were assayed for their antiviral activities against SARS-CoV-2, using immunocytochemistry-based assessment of SARS-CoV-2 infection in Vero cells, as previously described.19 The cytotoxicity of the compounds in uninfected Vero cells was determined in parallel. Remdesivir, chloroquine and nirmatrevir were included as positive controls and reference compounds (Table 1 ). Because the prodrug remdesivir was discovered to be activated in Vero cells.20 it could be used as a control compound in vitro.
Table 1.
Inhibitory activities of quinolone derivatives against SARS-CoV-2.
| Compound | X | Y | R1 | R2 | EC50a(μM) | CC50b(μM) | SIc |
|---|---|---|---|---|---|---|---|
| 5a | H | H | -COOMe | SOMe | 13.4 | > 25 | 1.9 |
| 5b | H | Cl | -COOMe | SOMe | 9.4 | 12.0 | 1.3 |
| 7a | H | Cl | –COOH |  |
> 25 | > 25 | 1.0 |
| 7b | H | Cl | –COOH |  |
4.4 | > 25 | 5.7 |
| 9a | H | H | –COOH |  |
4.0 | > 25 | 6.2 |
| 10 | F | F | -H |  |
> 25 | > 25 | 1.0 |
| 11 | H | Cl | -CONHMe | |
7.9 | > 25 | 3.2 |
| Chloroquine | 8.2 | > 25 | 3.0 | ||||
| Remdesivir | 7.3 | > 25 | 3.4 | ||||
| Nirmatrevir | 14.1 | > 25 | 1.8 |
EC50 and CC50 values were derived from the results of at least two independent experiments conducted in Vero cells.
SI (selectivity index) = CC50/EC50 value for inhibiting SARS-CoV-2 infection.
Sulfoxide 5a and 5b showed moderate inhibitory activities with EC50 values of 13.4 µM and 9.4 µM, respectively. Compound 5b showed weak toxicity with a CC50 value of 12.0 µM, suggesting that its activity is due to cytotoxicity. The linear alkylamine 7a exhibited no activity, whereas the cyclohexylamine 7b displayed potent activity (EC50 = 4.4 µM). Additionally, compound 9a showed potent inhibitory activity against SARS-CoV-2 without causing cytotoxicity (EC50 = 4.0 µM). Decarboxylated compound 10 had no activity while 9f had antiviral activity, suggesting that the ketone at the C-3 position is important for its activity. The introduction of an amide group at the C-3 position of compound 11 slightly decreased its antiviral activity (EC50 = 7.9 µM) relative to compound 9a (EC50 = 4.0 µM).
We focused on changing the C-6 or C-7 of the quinolone core, which contains fluoroaniline at the C-2 position (Table 2 ). The activities of compound 9b-g, substituted with chloro or methoxy or fluoro at C-6 or C-7 on the quinolone core, were similar (EC50 = 1.5–3.5 µM). Chloro compounds 9b-d were slightly more active against SARS-CoV-2, than methoxy 9e and difluoro 9f and 9 g. In comparing 9c and 9d, antiviral activity of 3-fluoroaniline at the C-2 position was slightly better than that of 4-fluoroaniline.
Table 2.
The anti-SARS-CoV-2 activity of 2-aminoquinolone acid derivatives.
| Compound | X | Y | Z | EC50a (μM) | CC50b (μM) | SI c |
|---|---|---|---|---|---|---|
| 9b | H | Cl | 3-F | 1.5 | > 25 | 16 |
| 9c | Cl | H | 3-F | 1.5 | > 25 | 17 |
| 9d | Cl | H | 4-F | 2.8 | > 25 | 8.9 |
| 9e | OMe | H | 3-F | 3.3 | > 25 | 7.6 |
| 9f | F | F | 3-F | 3.3 | > 25 | 7.5 |
| 9g | F | F | 4-F | 3.5 | > 25 | 7.2 |
EC50 and CC50 values were derived from at least two independent experiments conducted in Vero cells.
SI (selectivity index) = CC50/EC50 value for inhibiting SARS-CoV-2 infection.
For the study of mode of action, we assessed the effects of 9b on viral entry using SARS-CoV-2 spike pseudo-type lentivirus system.21 Compound 9b inhibited the entry of the SARS-CoV-2 spike pseudo-typed virus in a concentration-dependent manner (EC50 = 0.69 µM), meaning that a series of 2-aminoquinolone acids derivatives act entry inhibitors (Fig. 3 ).22
Fig. 3.
Concentration-response inhibition curves of 9b against SARS-CoV-2 pseudovirus.
Compound 9b, which was found to have potential anti-SARS-CoV-2 activity, was evaluated for safety and in vitro pharmacokinetic profile (Table 3 ). Cytotoxicity was tested on four cell lines (HFL-1, L929, NIH 3 T3, and CHO-K1), using the EZ-Cytox cell viability assay kit. The CC50 values for all cell lines indicated that compound 9b is not generally cytotoxic. Compound 9b was subsequently assessed for cardiotoxicity through hERG fluorescence polarization assay. Results showed that no hERG binding was detected with compound 9b even at 100 µM. We next evaluated % inhibition of five subtypes of cytochrome P450 (CYP) at 10 µM. This method is a preliminary test using a mixture of five CYP isoenzymes. The inhibition levels for CYP1A2 and 2C9 were 72% and 59%, respectively, while the inhibition levels for CYP2C19, 2D6, and 3A4 were almost nonexistent with values of less than 10%. These results indicate that it may be necessary to pay attention to interactions of compound 9b with CYP1A2 or 2C9.
Table 3.
Safety and in vitro pharmacokinetic profile of compound 9b.
| Cytotoxicity (CC50, μM)a | hERG ligand binding assay (IC50, μM) | CYP inhibition at 10 µM (%) | Microsomal stability (%)b |
|---|---|---|---|
| HFL-1: 48 L929: > 100 NIH 3 T3: 67 CHO-K1: 55 |
> 100 | 1A2: 72 2C9: 59 2C19: 9 2D6: 3 3A4: <1 |
Mouse: 24 Rat: 14 Dog: 65 Human: 62 |
Cell information. HFL-1: human embryonic lung cell line, L929: mouse fibroblast cell line, NIH 3 T3: mouse embryonic fibroblast cell line, CHO-K1: Chinese hamster ovary cell line.
% Original compound remaining after 30 min incubation.
Furthermore, microsomal stability was determined by measuring how much compound 9b remained in the liver microsomes after 30 min of NADPH activation. This test is related to phase I metabolism. This is because liver microsomes activated by NADPH mainly carry out phase I metabolism. In vitro microsomal phase I metabolism assays revealed that compound 9b remained at 24% and 14% in mouse and rat, respectively, and 76% and 62% in dog and human, respectively. These results indicate that microsomal metabolism differs among species and is more stable in dogs and humans than in mouse and rat.
In summary, a series of derivatives of 2-aminoquinolone acids were found to be highly active entry inhibitors against SARS-CoV-2 without causing toxicity, while having satisfactory in vitro PK profiles. This study shows that 2-aminoquinolone acid 9b is a promising new template for the development of anti-SARS-CoV-2 agents.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The chemical library used in this study was kindly provided by Korea Chemical Bank (http://www.chembank.org/) of Korea Research Institute of Chemical Technology. This research was supported through the National Research Council of Science & Technology (NST) (No. CRC-16-01-KRICT) funded by the Ministry of Science and ICT (MSIP) and by the Korea Evaluation Institute of Industrial Technology (KEIT) grant funded by the Korea government (MOTIE) (No. RS-2022-00155902).
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmcl.2023.129214.
Appendix A. Supplementary data
The following are the Supplementary data to this article:
Data availability
Data will be made available on request.
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Supplementary Materials
Data Availability Statement
Data will be made available on request.