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. 2025 Aug 27;10(36):41608–41619. doi: 10.1021/acsomega.5c05168

SARS-CoV‑2 Main Protease Inhibitors Containing 5‑Substituted Benzothiazole-2-carbonyl Moieties at the P1′ Site and Their Derivatives

Kohei Tsuji †, Takuya Kobayakawa †, Nobuyo Higashi-Kuwata ‡, Takahiro Ishii †, Kouki Shinohara †, Ryusei Yamamoto †, Yutaro Miura †, Naoya Wada †, Hiroaki Mitsuya ‡,§,∥, Hirokazu Tamamura †,*
PMCID: PMC12444507  PMID: 40978346

Abstract

Development of anti-SARS-CoV-2 agents is still required because of the appearance of drug-resistant strains, and targeting the main protease (Mpro) of SARS-CoV-2 is a powerful way to develop therapeutics against COVID-19. To date, we have developed several Mpro inhibitors including compounds 3, 4, TKB245 (6), and TKB248 (7), which have a 4-fluorobezothiazole ketone moiety as a warhead structure. Further studies led to the development of a more potent Mpro inhibitor, TKB272 (8), which has a 5-fluorobenzothiazole ketone moiety. The slight difference in the introduction position of a fluorine atom enhanced its antiviral activity approximately 3-fold for VeroE6 cells and 15-fold for HeLahACE2‑TMPRSS2 cells when TKB272 (8) was compared to TKB245 (6). Herein, we report the synthesis and structure–activity relationship (SAR) studies of TKB272 (8). TKB272 (8) is a promising drug candidate for COVID-19 therapy, and the present data of the SAR studies are useful for the further development of Mpro inhibitors.

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Introduction

Five years have already passed since a positive-sense single-stranded RNA virus, the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), caused the pandemic of the novel corona virus disease 2019 (COVID-19). − To date, various vaccines covering mutant strains have been developed and used clinically to suppress the infection of SARS-CoV-2 and aggravation of the symptoms. − However, the spike proteins on the virion surface easily mutate, and its mutation tends to decrease the efficacy of the vaccines. , In addition, there are many people who decline to take the vaccines for various reasons. Accordingly, anti-COVID-19 drugs are required. A repositioning inhibitor of RNA-dependent RNA polymerase (RdRp), remdesivir, which had been developed as a drug for Ebola hemorrhagic fever, was initially authorized by FDA as an anti-COVID-19 drug. , Subsequently, a SARS-CoV-2 RdRp inhibitor, molnupiravir (LAGEVRIO), was developed by Merck & Co. − Two SARS-CoV-2 main protease (Mpro) inhibitors were also produced by Pfizer Inc. (nirmatrelvir 1, PAXLOVID, nirmatrelvir, and ritonavir tablets, approved by FDA in 2021), and Shionogi & Co., Ltd. (ensitrelvir fumaric acid, Xocova, approved by the Ministry of Health, Labour, and Welfare (MHLW) in Japan in an emergency regulatory approval in 2022).

In cells infected with SARS-CoV-2, ORF1a and ORF1b in its genome are translated into two nonstructural polyproteins, pp1a and pp1ab. These proteins are processed by two viral proteases, Mpro and a papain-like protease (PLpro), to produce nonstructural proteins 1–16 (nsp1–16), which are essential for viral replication. − Therefore, Mpro is a key enzyme for SARS-CoV-2 replication. Because of the high conservation of its amino acid sequence and no overlap with human proteases, which recognize and process homologous substrates of Mpro, this enzyme is an attractive target for drug discovery to treat COVID-19. Since the beginning of the emergence of SARS-CoV-2, many Mpro inhibitors have been developed by various research groups. − Several studies found the drug-resistant SARS-CoV-2 variants against the first generation inhibitor, nirmatrelvir (1, Figure ). − In addition, the nirmatrelvir-resistant strains were found from a patient and naturally occurring mutants. , Therefore, novel drugs that combat such strains are urgently needed. To meet this challenge, we have developed SARS-CoV-2 Mpro inhibitors based on optimization of a tripeptide mimetic, YH-53/5h (2), which was initially developed as a SARS-CoV Mpro inhibitor by Hayashi et al. in 2013, − and then identified as a SARS-CoV-2 Mpro inhibitor by Mitsuya et al. (Figure ). In order to occupy effectively the S1′ pocket, appropriate introduction of fluorine atoms into the lead compound (2) afforded compound (3), which have potent antiviral activity, and the additional replacement of a metabolically digestible P1–P2 amide in 3 with the corresponding thioamide unit produced compound (4) with an improved PK profile. − Subsequently, we developed TKB245 (6) and TKB248 (7) with orally administrable PK profiles, which have higher potency than those of 3 and 4. , These Mpro inhibitors have a 4-fluorobenzothiazole ketone moiety as a common characteristic. Although the reason why compound (5) having a 5-fluorobenzothiazole ketone moiety showed decreased potency compared to compound (3) having a 4-fluorobenzothiazole ketone moiety remains unclear, further study led to the finding of a more potent Mpro inhibitor, TKB272 (8), possessing a 5-fluorobenzothiazole ketone moiety. The slight difference in the position of the fluorine atom between TKB272 (8, 5-F) and TKB245 (6, 4-F) dramatically improved the potency especially for the use of HeLahACE2‑TMPRSS2 cells expressing human angiotensin converting enzyme 2 (hACE2, a SARS-CoV-2 receptor) and transmembrane protease serine 2 (TMPRSS2, an activator of virus entry). Although nirmatrelvir needs combination with a CYP3A inhibitor, ritonavir, TKB272 (8) potently blocked SARS-CoV-2 without ritonavir in mice. In addition, TKB272 (8) showed substantial antiviral activity against nirmatrelvir-resistant E166 V-carrying SARS-CoV-2E166V.

1.

1

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Structures of SARS-CoV-2 Mpro inhibitors, nirmatrelvir (1), YH-53/5h (2), 3, 4, 5, TKB245 (6), TKB248 (7), and TKB272 (8).

In this study, we report the synthesis and structure–activity relationship (SAR) studies of TKB272 (8).

Results and Discussion

Concept of Compound Design

Our previous fluorine scanning of the lead compound, YH-53/5h (2), disclosed that the combination of 4-methoxy-7-fluoro-indole and 4-fluorobenzothiazole ketone moieties (compound (3)) produced a highly potent compound in a cell-based anti-SARS-CoV-2 assay. Compound (5) having 4-methoxy-7-fluoro-indole and 5-fluorobenzothiazole ketone moieties showed lower potency than that of compound (3) and comparable potency with that of YH-53/5h (2). Therefore, we decided to adopt a 4-fluorobenzothiazole ketone moiety at the P1′ site as a warhead structure for further optimization, and found that the hybrid molecule of nirmatrelvir (1) and (3), which has a trifluoroacetyl-l-tert-Leu residue at the P3 site and a (1R,2S,5S)-6,6-dimethyl-3-azabicyclo­[3.1.0]­hexane-2-carboxy residue at the P2 site derived from nirmatrelvir (1), and an (αS,3S)-α-amino-2-oxo-3-pyrrolidinepropanoyl residue at the P1 site and a 4-fluorobenzothiazole ketone at the P1′ site derived from (3), TKB245 (6), showed greatly improved anti-SARS-CoV-2 potency compared to the parent compounds nirmatrelvir (1) and (3), and showed satisfied PK profiles (Figure ). , Recently, we conducted the fluorine scanning again based on TKB245 (6) and found that the 5-fluorobenzothiazole ketone derivative, TKB272 (8), was the most potent compound among the monofluorobenzothiazole derivatives TKB245 (6), and TKB272 (8)–TKB252 (10). According to the solved cocrystal structures of TKB245 (6)-Mpro (PDB: 8DOX) and TKB272 (8)-Mpro (PDB: 8UH5), these compounds showed almost the same interaction mode with Mpro except for the fluorine atom on the benzothiazole ring (Figure a–c). , In the interaction of TKB272 with Mpro, the fluorine atom on the 5-position of the benzothiazole ring gains new fluorine-based interactions with the side chain oxygen atom of Thr25 and the mainchain nitrogen atom of Ser46, suggesting that these interactions produced a more potent Mpro inhibitor, TKB272 (8), from TKB245 (6, Figure e). Therefore, we initially designed and synthesized (11) having a 4,7-difluorobenzothiazole ketone moiety with an increased number of fluorine atoms and possible new interaction modes with the Mpro. In the conversion of compound (3) and TKB245 (6) to compound (4) and TKB248 (7), respectively, the replacement of the P1–P2 amide with the corresponding thioamide unit improved their PK profiles in mice with a maintenance or a slight attenuation of anti-SARS-CoV-2 activity in cell-based assays. Therefore, we synthesized a TKB272 (8) thioamide derivative (12). We previously reported that the replacement of the P2 unit in TKB245 (6), a (1R,2S,5S)-6,6-dimethyl-3-azabicyclo­[3.1.0]­hexane-2-carboxy residue, which had been used in the hepatitis C viral (HCV) enzyme NS3/4A serine protease inhibitor, boceprevir, by a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue, which had been used in an HCV NS3/4A serine protease inhibitor, telaprevir, brought tolerable potency attenuation. Therefore, we designed and synthesized (13) and (14) having a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue as the P2 unit. According to the results of the fluorine scanning, introduction of a fluorine atom at the 5-position in the benzothiazole ketone moiety is superior to the introduction at the other positions. Hence, we focused on TKB272 (8) derivatives with 5-substituted benzothiazoles, and 16 derivatives (15)–(30) were designed and synthesized.

2.

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X-ray cocrystal structures of SARS-CoV-2 Mpro with TKB245 (6) and with TKB272 (8). (a) Binding mode of TKB245 (6, magenta sticks) to Mpro (white sticks and ribbon, PDB: 8DOX). Fluorine, nitrogen, oxygen, and sulfur atoms are shown in light green, blue, red, and yellow, respectively. (b) Superimposed structures of TKB245 (6, magenta sticks) bound to Mpro (white sticks and ribbon, PDB: 8DOX) and TKB272 (8, cyan sticks) bound to Mpro (dark gray sticks and ribbon, PDB: 8UH5). (c) The binding mode of TKB272 (8, cyan sticks) to Mpro (dark gray sticks and ribbon; PDB: 8UH5). (d) Mpro are shown in white surface presentations, and TKB245 (6) is shown with magenta sticks. (e) The fluorine-based interactions of TKB272 (8) with Mpro are shown. The fluorine atom on the 5-position of the benzothiazole ring interacts with the hydrogen atom of the side chain hydroxy group of Thr25 and the hydrogen atom of the amino group of Ser46. (f) Mpro are shown in dark gray surface presentations, and TKB272 (8) is shown with cyan sticks. All structural figures were produced with MOE (ver. 2020.0901).

Chemistry

The purity of the final synthesized compounds was determined to be >95% by analytical RP-HPLC or NMR analysis. Experimental procedures of the synthesis of all of the compounds including characterization data are provided in the Supporting Information. The synthetic routes of TKB272 (8) shown in Schemes and are similar to those of our previously reported synthetic routes for TKB245 (6). , The thioamide derivative (12) was synthesized as a similar fashion to the synthesis of TKB248 (7, Scheme ).

1. Scheme for the Synthesis of TKB272 (8)­ .

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a dox = 1,4-dioxane, TFAA = trifluoroacetic anhydride, DIPEA = N,N-diisopropylethylamine, COMU = 1-[(1-(cyano-2-ethoxy-2-oxoethyl-ideneaminooxy)­dimethylaminomorpholino)]­uronium hexafluorophosphate.

2. Alternative Scheme for the Synthesis of TKB272 (8)­ .

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a T3P = propylphosphonic acid anhydride (cyclic trimer).

3. Scheme for the Synthesis of Compound (12).

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In the first synthetic route of TKB272 (8) shown in Scheme , the Boc-protected methyl ester (31) was saponified using LiOH, and the resulting Boc-protected amine (32) was deprotected by the treatment of HCl. The obtained amine hydrochloride salt (33) was trifluoroacetylated by trifluoroacetic anhydride (TFAA) to yield carboxylic acid (34). Coupling of (34) with the amine tetrafluoroboric acid (HBF4) salt (36), which was achieved by deprotection of the Boc group in (35) using a tetrafluoroboric acid diethyl ether complex (HBF4·OEt2), using 1-[(1-(cyano-2-ethoxy-2-oxoethylideneamino-oxy)-dimethylaminomorpholino)]­uronium hexafluorophosphate (COMU) in the presence of N,N-diisopropylethylamine (DIPEA) provided TKB272 (8).

In the second synthetic route of TKB272 (8) shown in Scheme , methyl (S)-2-(Boc-amino)-3-((S)-2-oxopyrrolidin-3-yl)­propanoate (37) was deprotected using HCl, and the resulting amine hydrochloride salt (38) was coupled with the carboxylic acid (34) using propylphosphonic acid anhydride (cyclic trimer, T3P) in the presence of pyridine to afford the methyl ester (39). The methyl ester (39) was then treated with n-BuLi-pretreated 5-fluorobenzothiazole to yield TKB272 (8). As in the case of TKB245 (6), the synthetic route of TKB272 shown in Scheme demonstrated a higher yield and easier preparation of the P1′ derivatives than that shown in Scheme . All of the reported compounds were synthesized according to these two synthetic schemes except for (12).

In the synthesis of (12) shown in Scheme , coupling of (1R,2S,5S)-3-Boc-6,6-dimethyl-3-azabicyclo­[3.1.0]­hexane-2-carboxylic acid (40) with 4-nitro-1,2-phenylenediamine by a mixed anhydride reaction using isobutylchloroformate and N-methylmorpholine provided the Boc-protected amide (41). Treatment of compound (41) with phosphorus pentasulfide (P4S10) in the presence of Na2CO3 gave the thioamide (42), which was followed by treatment with sodium nitrite in the presence of aqueous acetic acid to yield the triazole (43). Conjugation of the amine HBF4 salt (36) and the triazole (43) in the presence of DIPEA produced thioamide (44). The N α-Boc group of the obtained thioamide (44) was deprotected by HBF4·OEt2 followed by coupling of the resulting amine HBF4 salt (45) with the carboxylic acid (46) using COMU in the presence of DIPEA to afford (12).

Structure–Activity Relationship Studies

We have demonstrated that a fluorobenzothiazole ketone structure is a suitable warhead structure for SARS-CoV-2 Mpro inhibitors, which can reversibly form a covalent bond with the catalytic Cys145 residue of Mpro. ,− , The EC50 and CC50 values of the synthesized compounds were determined with RNA-qPCR and WST-8 assays, respectively, using VeroE6 cells (see the Experimental Section). In consistent with the recent report, TKB272 (8) showed the most potent anti-SARS-CoV-2 activity among the mono- or difluorobenzothiazole ketone derivatives TKB245 (6) and TKB272 (8)–(11) (Figure , the EC50 and CC50 values of compounds in parentheses are from the reported paper). The X-ray structural analyses of TKB245 (6) or TKB272 (8) complexed with Mpro (PDB: 8DOX or 8UH5, respectively) suggested that the 4- or 5-fluorobenzothiazole moiety effectively occupies the inhibitor-binding pocket of Mpro, and that substitution of fluorine atom(s) at the 6- or 7-position (TKB273 (9), TKB252 (10), or (11)) might be sterically unfavored (Figure a,d). Although the 4-position of the benzothiazole ring is exposed to the solvent, the binding of TKB245 (6) to Mpro might be more preferable than those of the other three derivatives (TKB273 (9), TKB252 (10), and (11)) (Figure a,d). Furthermore, TKB272 (8) with the 5-fluorine-substituted benzothiazole gains additional interactions of the fluorine atom on the 5-position of the benzothiazole ring with the Thr25 side chain hydroxyl group and the Ser46 mainchain amino group (Figure b,c,e,f). Accordingly, TKB272 (8) has a more potent antiviral activity than the parent compound, TKB245 (6).

3.

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Structures of Mpro inhibitors possessing a fluorinated benzothiazole ketone moiety as a warhead structure. EC50 and CC50 values were determined with RNA-qPCR and WST-8 assays, respectively, using VeroE6 cells. The numbers represent the average EC50 value ± SD (μM) and CC50 value (μM) from at least two independent experiments.

Next, we evaluated anti-SARS-CoV-2 potencies of a TKB272 (8) derivative with the replacement of the P1–P2 amide by thioamide, (12), and TKB272 (8) and (11) derivatives with the replacement at the P2 site from a (1R,2S,5S)-6,6-dimethyl-3-azabicyclo­[3.1.0]­hexane-2-carboxy residue, derived from boceprevir, into a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue, derived from telaprevir, (13) and (14), respectively (Figure ). As a result, a thioamide-type derivative (12) showed one order lower potency compared to TKB272 (8), and (13) and (14) having a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue at the P2 site also showed remarkably lower potencies. This tendency was consistent with that of the corresponding TKB245 (6) derivatives. Although a thioamide-type derivative, TKB248 (7), showed great potency with preferable PK profiles, an amide-type inhibitor, TKB245 (6), is more accessible in synthesis. , In consideration of the drug development, the amide-type inhibitors such as TKB245 (6) and TKB272 (8) would be more suitable than the corresponding thioamide derivatives, TKB248 (7) and (12), which might accompany difficulties and/or problems in manufacturing. Taken together, a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue derived from telaprevir would be an alternative P2 unit; however, TKB272 (8) is still an optimal SARS-CoV-2 inhibitor among the tested compounds.

4.

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Structures of TKB272 (8) derivatives modified at the P1–P2 amide bond or the P2 site.

The effects of the 5-position substitutions on the benzothiazole moiety were then investigated (Table ). The bis-halogenated (4-bromo, 5-fluoro) derivative (15) showed approximately 3-fold lower potency than the monohalogenated (5-fluoro) compound, TKB272 (8), and the other bis-halogenated (5,7-fluoro or 5,7-bromo) derivatives, (16) or (17), respectively, also significantly decreased their potencies compared to TKB272 (8). These results suggested that the 5-fluorobenzothiazole moiety is the most suitable among the tested halogenated benzothiazole moieties. In the comparison of TKB272 (8) and (18)–(22), only the 5-Me derivative (20) showed a tolerable potency decrease but significant cytotoxicity (CC50 = 68 ± 2.0 μM), and other derivatives (18), (19), (21), and (22) showed dramatically decreased potencies. It suggests that lacks of the interaction of TKB272 (8) with Thr25 and Ser46 might attenuate the potency in addition of steric hindrance by the substituent groups at the 5-position (Figure e). To obtain the interaction with Thr25 and/or Ser46 as observed in TKB272 (8), an oxygen or nitrogen atom was introduced at the 5-position of the benzothiazole ring. Compound (25) having the 5-OMe substitution on the benzothiazole ring showed moderate potency, whereas compound (24) having the 5-OH substitution showed a remarkably decreased potency. However, (25) showed significant cytotoxicity (CC50 = 65 ± 2.5 μM). According to the cocrystal structure of TKB272 (8) and Mpro (Figure ), the introduction of a hydrogen bond acceptor on the 5-position of the benzothiazole ring would be more suitable than that of a hydrogen bond donor. The other oxygen-introduced derivatives (23), (26), and (27) showed decreased potencies. This suggests that these substituted groups are too bulky to fit the S1′ site in the binding pocket. In the nitrogen-introduced derivatives (28), (29), and (30), which showed decreased potencies, the introduction of hydrogen bond donors or their protected forms at the 5-position of the benzothiazole ring seemed to be unsuitable due to difficulties in achieving the desired interactions and/or these steric hindrances.

1. Antiviral Activities of Derivatives with 5-Substituted Benzothiazole Moieties (15)–(30) Against the Ancestral Wuhan Strain (WK-521),

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compound structure R = EC50 (μM) compound structure R = EC50 (μM)
TKB272 (8) 5-F 0.0070 ± 0.0010 23 5-OTBS 1.2 ± 0.14
15 4-Br, 5-F 0.022 ± 0.0020 24 5-OH 0.72 ± 0.013
16 5,7-F 0.20 ± 0.0050 25 5-OMe 0.069 ± 0.0030
17 5,7-Br 1.4 ± 0.050 26 5-OEt 0.25 ± 0.025
18 5-CN 0.15 ± 0.0030 27 5-OCF3 0.25 ± 0.070
19 5-CH2OH 0.39 ± 0.011 28 5-NHBoc 0.33 ± 0.049
20 5-Me 0.062 ± 0.0020 29 5-NH2 0.10 ± 0.015
21 5-Ph 0.38 ± 0.035 30 5-NHAc 4.1 ± 0.27
22 5-C5H11 0.38 ± 0.036 nirmatrelvir (1)   0.77 ± 0.18
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a

EC50 values were determined with RNA-qPCR using VeroE6 cells..

b

Numbers represent the average EC50 value ± SD (μM) from at least two independent experiments.

c

EC50 values against the ancestral Wuhan strain (WK-521) are from Figure or ref .

d

CC50 > 100 μM.

e

CC50 = 68 ± 2.0 μM.

f

CC50 = 65 ± 2.5 μM. The CC50 values were determined with WST-8 assays using VeroE6 cells. Numbers represent the average CC50 value ± SD (μM) from at least two independent experiments.

Furthermore, immunostaining experiments were performed to confirm that the anti-SARS-CoV-2 activity of the compounds is not brought due to nonspecific cytotoxicity, as described previously (Figure S1). ,

Conclusions

Since the emergence of SARS-CoV-2, this virus produces various mutants with enhanced infectivity and escapes from the human immune system. Therefore, the development of novel anti-COVID-19 drugs is still highly required. Herein, we performed SAR studies based on our recently developed highly potent and orally administrable SARS-CoV-2 Mpro inhibitor, TKB272 (8). TKB272 (8) has some positive features including no need of combination with a CYP3A inhibitor, ritonavir, which nirmatrelvir needs, and substantial antiviral activity against nirmatrelvir-resistant SARS-CoV-2E166V. Initially, we synthesized and evaluated Mpro inhibitors that have mono- or difluorination at different positions of the benzothiazole moiety and found that fluorine-substitution at the 5-position is much preferable than those at the other positions (Figure ).

Next, a thioamide derivative of TKB272 (8), (12), and P2 unit derivatives of TKB272 (8) and (11), (13) and (14), respectively, were investigated (Figure ). The results of antiviral assays revealed that (12) showed potency attenuated from TKB272 (8), and that (13) and (14) showed decreased potencies from the corresponding parent compounds TKB272 (8) and (11), respectively. These tendency is consistent with that of TKB245 (6) derivatives. Therefore, a (1S,3aR,6aS)-octahydrocyclopenta­[c]­pyrrole-1-carboxy residue, which is contained in an HCV NS3/4A serine protease inhibitor, telaprevir, would not be an alternative P2 unit of the (1R,2S,5S)-6,6-dimethyl-3-azabicyclo­[3.1.0]­hexane-2-carboxy residue, which is contained in boceprevir, nirmatrelvir (1), TKB245 (6), TKB248 (7), and TKB272 (8) as the P2 unit.

As a result of the SAR studies of TKB272 (8), none of the tested TKB272 (8) derivatives with 5-substituted benzothiazole ketone moieties improved their potencies in cell-based assays (Table ). This suggested that the 5-fluorobenzothiazole ketone moiety is extremely suitable as a P1′ unit of SARS-CoV-2 Mpro inhibitors.

Immunostaining experiments and the micro-Ames bacterial mutation tests demonstrated that TKB272 (8) has no significant cytotoxicity (Figure S1) and no revertant mutagenicity, respectively.

TKB272 (8) can be synthesized by an efficient synthetic route, and exhibits potent antiviral activity, high enzymatic inhibitory activity, favorable pharmacokinetics, enough membrane permeability and effectiveness against nirmatrelvir-resistant Mpro mutants without coadministration with ritonavir. These results underscore the exceptional profile of TKB272 as a highly optimized SARS-CoV-2 Mpro inhibitor. These data strongly supported that the Mpro inhibitor, TKB272 (8), can become a drug candidate for the treatment of COVID-19, and further investigation for drug development based on this compound is in progress. The present data are useful for the design of Mpro inhibitors, which are valid for nirmatrelvir-resistant strains and would be effective against new corona virus that might appear in the future.

Experimental Section

Compound Synthesis

The synthetic methods for representative compounds TKB272 (8) and (12) are described in Schemes – . The purity of all of the final compounds, measured by analytical RP-HPLC or NMR is >95%. Experimental procedures including characterization data of all of the synthesized compounds are provided in the Supporting information.

Cells, Viruses, and Test Compounds

VeroE6 cells were obtained from the American Type Culture Collection (ATCC) (CRL-1586) (Manassas, VA) and were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL of streptomycin. The SARS-CoV-2 strain JPN/TY/WK-521 (SARS-CoV-2WK‑521, ancestral Wuhan strain, GISAID Accession ID; EPI_ISL_408667) and the SARS-CoV-2 strain hCoV-19/Japan/TY41-796/2022 (SARS-CoV-2TY41‑796, omicron BQ.1.1, GISAID Accession ID; EPI_ISL_15579783) were obtained from the National Institute of Infectious Diseases (Tokyo, Japan). An antiviral agent Nirmatrelvir (1) (PF-07321332) was purchased from MedChemExpress (Monmouth Junction, NJ). Each compound was dissolved in DMSO at a concentration of 20 mM as a stock solution.

Anti-SARS-CoV-2 and Cytotoxicity Assays

For antiviral assays, cells were seeded in a 96-well plate (2 × 104 cells/well) and incubated. After 1 day, the virus was inoculated into cells at each multiplicity of infection (MOI): SARS-CoV-2WK‑521 (80). After an additional 3 days, cell culture supernatants were harvested, viral RNA was extracted using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany), and quantitative RT-PCR (RT-qPCR) was then performed using One Step PrimeScript III RT-qPCR mix (TaKaRa Bio, Shiga, Japan) following the instructions of the manufacturers. The primers and probe used for detecting SARS-CoV-2 nucleocapsid were 5′-AAATTTTGGGGACCAGGAAC-3′ (forward), 5′-TGGCAGCTGTGTAGGTCAAC-3′ (reverse), and 5′-FAM-ATGTCGCGCATTGGCATGGA-black hole quencher 1 (BHQ1)-3′ (probe). To determine the cytotoxicity of each compound, cells were seeded in a 96-well plate (2 × 104 cells/well). One day later, various concentrations of each compound were added, and the cells were then incubated for an additional 3 days. Values of the 50% cytotoxic concentration (CC50) were determined based on the WST-8 assays using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan).

Supplementary Material

ao5c05168_si_001.pdf (5.4MB, pdf)
ao5c05168_si_002.csv (3.5KB, csv)

Acknowledgments

This work was supported in part by Research Projects on COVID-19, Japan Agency for Medical Research and Development (AMED) 20fk0108510 and 21fk0108480 (H.M. and H.T.); JSPS KAKENHI Grant Numbers 24K02144 (H.T.), 22K15243 (K.T.), and 23K14318 (T.K.); AMED under Grant Number JP24ama121043 (Platform Project for Supporting Drug Discovery and Life Science Research, BINDS) (H.T.); JPMJFS2109 (MEXT, the establishment of university fellowships toward the creation of science technology innovation) (T.I.); and JST SPRING, Grant Number JPMJSP2180 (K.S. and Y.M.). This research is based on the Cooperative Research Project of Research Center for Biomedical Engineering.

Glossary

Abbreviations

ACE2

angiotensin converting enzyme 2

BSA

bovine serum albumin

CC50

half-maximum cytotoxicity concentration

COMU

1-[(1-(cyano-2-ethoxy-2-oxoethylideneamino-oxy)-dimethylaminomorpholino)]­uronium hexafluorophosphate

DIPEA

N,N-diisopropylethylamine

D-MEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

HATU

1-[bis­(dimethylamino)­methylene]-1H-1,2,3-triazolo­[4,5-b]­pyridinium 3-oxide hexafluorophosphate

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOAt

1-hydroxy-7-azabenzotriazole

Mpro

main protease

PLpro

papain-like protease

qPCR

quantitative polymerase chain reaction

RP

reversed-phase

RT-qPCR

reverse transcription-qPCR

SD

standard deviation

TFAA

trifluoroacetic anhydride

TMPRSS2

transmembrane protease, serine 2

T3P

propylphosphonic acid anhydride (cyclic trimer)

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05168.

  • Experimental procedures of compound synthesis including characterization data (1H NMR, 13C NMR, and analytical RP-HPLC charts), and immunostaining experimental data (Figure S1) (PDF)

  • Molecular formula strings of the synthesized compounds (CSV)

⊥.

K.T. and T.K. contributed equally to the study. Conceptualization, H.M. and H.T.; methodology, K.T., T.K., N.H.-K., T.I., K.S., R.Y., Y.M., N.W.; drafted and finalized the manuscript, H.M. and H.T. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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