# Noncovalent Inhibitors of SARS-CoV-2 Main Protease: A Rescaffolding Attempt

Tim Krischuns, Sylvain Paisant, Kuang-Yu Chen, Laura N Thirion, Agnès Zettor, Jeanne Chiaravalli, Yves Jacob, Marco Bellinzoni, Nadia Naffakh, Yves L Janin

► To cite this version:

Tim Krischuns, Sylvain Paisant, Kuang-Yu Chen, Laura N Thirion, Agnès Zettor, et al. Noncovalent Inhibitors of SARS-CoV-2 Main Protease: A Rescaffolding Attempt. Synthesis: Journal of Synthetic Organic Chemistry, 2025, 57 (09), pp.1627-1634. 10.1055/a-2519-9876. pasteur-05081440

HAL Id: pasteur-05081440

https://pasteur.hal.science/pasteur-05081440v1

Submitted on 5 Nov 2025

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Distributed under a Creative Commons Attribution 4.0 International License
Non covalent inhibitors of SARS-CoV-2 main protease, a rescaffolding attempt

Tim Krischuns<sup>a</sup>, Sylvain Paisant<sup>a</sup>, Kuang-Yu Chen<sup>a</sup>, Laura N. Thirion<sup>b</sup>, Agnès Zettor<sup>c</sup>, Jeanne Chiaravalli<sup>c</sup>, Yves Jacob<sup>d</sup>, Marco Bellinzoni<sup>e</sup>, Nadia Naffakh<sup>a</sup>, Yves L. Janin<sup>b*</sup>

Early version before submission to *Synthesis*.

a : RNA Biology and Influenza Virus Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 25-28 rue du Dr. Roux, F-75015 Paris, France

b : Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France.

c : Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, Paris, France.

d : Molecular Genetics of RNA Viruses Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 25-28 rue du Dr. Roux, F-75015 Paris, France

e : Structural Microbiology Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3528, 25-28 rue du Dr. Roux, F-75015 Paris, France

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**Abstract**

Out of the results of the sole large-scale screening for inhibitors of SARS-CoV-1 main protease reported in 2013, attempts to improve the 3-pyridyl-bearing hits found have been conducted in research laboratories, either on this enzyme or more recently on the closely related SARS-CoV-2 main protease. From the resulting structural information reported, we sought to design analogues featuring some of the components providing an affinity for the active site of these proteases along with a different scaffold which would allow for further structure-activity relationship studies and/or pharmacological improvements. We describe here the introduction of a bridging component aiming at the stabilization of the ligand conformation adopted when bound to these proteases. Accordingly, this led us to prepare 3,3-disubstituted piperazin-2-ones from an array of ketones, via either a Bargellini reaction or a multistage condensation/cyclization/hydrolysis involving ethylene diamine and potassium cyanide. However, even the most elaborate biphenyl-bearing analog made, displayed only a weak effect in a bioluminescence-based SARS-CoV-2 main protease inhibition assay.
**Introduction**

As reviewed recently, SARS-CoV-2 main protease (SARS-CoV-2 M^pro^) has become a central target for the design of drug treatments against COVID-19 infection and this protease became a validated target of several drugs which alleviate the outcome of a COVID-19 infection in fragile patients.¹ Aside from these remarkable successes, research also focused on the results of the sole large scale screening on of SARS-CoV-1 M^pro^ reported in 2013.² Out of these screenings, compounds **1a** or the corresponding enantiomer ML188 (**1b**) were reported and this 3-pyridyl-bearing class of inhibitors was also studied for its inhibition properties on related viral proteases such as the porcine epidemic diarrhea virus protease.³ This compound turned out to also inhibit⁴ the SARS-CoV-2 M^pro^ and thus became the focus of a lot more research. Structure-wise, compound **1b** had been co-crystallized with SARS-CoV-1 M^pro^ (see PDB 3V3M)²b and was then co-crystallized with SARS-CoV-2 M^pro^ (see PDB 7L0D).⁴ As accounted in more details,¹ these structures drove many X ray-based studies of 3-pyridyl-bearing analogues⁵ as well as structure-activity relationship iterations,⁴,⁶ illustrated by the improved inhibitors **2-8**. It appears that nitrile-bearing MAT-POS-e194df51-1 (**5**), one of the many fruits of the remarkable COVID Moonshot initiative,⁶a was selected for preclinical development and the hydantoin **8b** is also featuring a rather favorable profile.⁶e
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Structures of selected pyridyl or isoquinolyl-bearing inhibitors of SARS-CoV-2 M^pro^.</figcaption></figure>

Concerning the nature of the interaction between these inhibitors and their target, the reported⁶c X-ray structure of compound **2a** co-crystallized with SARS-CoV-2 M^pro^ (see PDB 7KX5) or the more recently deposited compound **4a** (see PDB 7S4B) are providing many insights. As depicted in figure 2, compound **2a** binds to the catalytic site of the SARS-CoV-2 M^pro^ via a pyridyl nitrogen’s interaction with the hydrogen of the imidazole ring of histidine-163, an interaction between its amide moiety and the backbone NH of glutamine-166 and an interaction between its carboxyfuranne part and the backbone NH of Gly-143. As seen on the 3D structure, the conformation adopted by compound **2a** leads to the CH and NH bonds depicted in red to be almost on the same plane. It is this structural information which led us to envisage the synthesis of analogues **9** which would feature a bridge hopefully locking the pyridyl-amide diad into its active conformation. It is in fact a similar reasoning which led to the design of the bridge-bearing analogue **6** described above.⁷ Moreover, the side chain (R) of compound **9** could plausibly be able to replace some of the lipophilic character featured by the tertiary amide component of analogue **3**. Finally, when comparing the binding modes of compound **2a** and **4a**, as well as the series of benzotriazole-bearing inhibitors (see PDB 7LMF for instance),⁸ quite a few changes are seen in the protease binding pocket to accommodate the fairly different conformations and/or decoration of these
compounds. This observation was in a way encouraging to undertake the preparation of analogues 9 with a potential for a large variation of the binding properties provided by its R group.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Structures of compound **2a** and analogues **9**; binding mode of **2a** to SARS-CoV-2 M^pro^ (from PDB 7KX5; generated using the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC)</figcaption></figure>

## Results and discussion

As described in scheme 1, an initial synthesis of a diaminoethyl-bearing analogues 9, was achieved with a three components Bargellini reaction⁹ between 3-acetylpyridine (10), 1,2-diaminoethane and chloroform. This led in one step to the very water-soluble 3,3-disubstituted piperazin-2-one 11 which was isolated in a 49% yield. Then, probably because of a steric hindrance, we could not protect the amine function of compound 11 with a Boc group under standard conditions.¹⁰ However, the regioselective N-alkylation of the amide function of compounds 11 with (1-chloroethyl)benzene (12) turned out to be possible in DMF using a strong base to give the methyl-bearing analogue 13 as a mixture of diastereoisomers. In order to reach more elaborate aryl-bearing homologues, such as compounds **16a-b**, the starting phenyl or bisphenyl ketones **14a-b** were prepared by α-arylation of 3-acetylpyridine (10) using a previously reported palladium-catalyzed method.¹¹ However, in the next stage, when using ketones **14a-b** extensive decompositions were the sole result of Bargellini reactions. Amongst many unidentified reaction products, benzoic acid was actually detected when starting from **14a**. We then focused on a previously patented¹² preparation of piperazin-2-ones from aldehydes or ketones, ethylene diamine and hydrogen cyanide which was later used in very few instances.¹³ Following some optimization of the procedure (condition ii in scheme 1) compounds **15a-b** were obtained in respectively 29 and 42% yields. Finally, the phenethyl-bearing ketone **17**, was prepared in three steps from ethyl nicotinate, as described in the experimental part. From this ketone, the phenethyl-bearing piperazin-2-one **18** was then obtained using the Bargellini reaction in a 43% yield. This last result actually hints at the influence of the ketone acidity on the proper proceeding of a Bargellini reaction but we have yet to investigate this in more details. In any case, the ensuing N-alkylation of the piperazin-
2-ones **15ab** and **18** with (1-chloroethyl)benzene (**12**) gave the target bridged analogues **16a-b** and **19** as mixtures of diastereoisomers.

<figure><img src="image_5.png" /><figcaption>**Scheme 1.** i: 50% NaOH, CHCl<sub>3</sub>, H<sub>2</sub>N(CH<sub>2</sub>)<sub>2</sub>NH<sub>2</sub>, BnNEt<sub>3</sub>Cl, CH<sub>2</sub>Cl<sub>2</sub>, 0°C. ii: a) H<sub>2</sub>N(CH<sub>2</sub>)<sub>2</sub>NH<sub>2</sub>, KCN, AcOH, EtOH, 20-140°C; b) H<sub>2</sub>O, 150°C. iii: PhCHClMe (**12**), NaH, DMF, 20°C. iv: ArBr, Pd(OAc)<sub>2</sub>, XantPhos, K<sub>3</sub>PO<sub>4</sub>, dioxane, THF, toluene, 100°C.</figcaption></figure>

The SARS-CoV-2 M^pro^ inhibition properties of the analogues described in Scheme 1 were then determined, using a gain-of-signal, highly sensitive cell-based luciferase assay we recently developed<sup>14</sup>. Briefly, HEK-293T cells are co-transfected with an M^pro^ expression plasmid and a Nanoluciferase-based reporter plasmid which includes the M^pro^ cleavage site VRLQ/S. At 6 hours post-transfection, the transfected cells are harvested and dispensed into 96- or 384-well plates containing the compounds to be tested, at various concentrations. The assay is performed in parallel on a wild-type and a catalytically inactive (C145A) M^pro^, which improves the selectivity of the assay. Indeed, it allows to identify compounds capable of enhancing or reducing a luciferase activity in both conditions in order to discard them as, respectively, false-positives or false-negatives/toxics. Here each compound was tested at a final concentration of 50, 10, 2 or 0.4 µM. The results are provided in figure 1 of the supplementary information section. The previously reported<sup>15</sup> and large spectrum 3C and 3C-like proteases inhibitor GC376 was used as a positive control and a dose-dependent increase of the luciferase signal (RLU) was observed in the presence of the wild-type Mpro, but not in the presence of its inactive counterpart (C145A mutant). Concerning the compounds described here, a moderate but specific activity was observed for the biphenyl-bearing compound **16b** at 25 µM, but it also showed some degree of cytotoxicity at the higher concentration of 50 µM. In one last approach, we also prepared analogues of the N-3-pyridine or N-1-isoquinoline derivatives **4-8** as well as even more recently reported inhibitors of SARS-CoV-2 M^pro^.<sup>6f</sup> Accordingly, we “turned around” the central pyrazinone scaffold of the compounds described in scheme 1 and prepared the N-pyridyl derivative **22** and **25** which can arguably be considered as analogues of the hydantoins **7** and **8**. As depicted in scheme 2 these compounds were prepared in only two steps from ketones **20** and **23**. The construction of the pyrazinones **21** and **24** made good use of the method described above and their palladium-catalyzed N-arylation using 3-bromopyridine was
achieved by adapting a reported method.¹⁶ Unfortunately and again, no tangible inhibition of SARS-CoV-2 M^pro^ was observed with compounds **22** or **25**.

<figure><img src="image_6.png" /><figcaption>**Scheme 2.** i: a) H₂N(CH₂)₂NH₂, KCN, AcOH, EtOH, 140°C; b) H₂O, 150 °C. ii: Pd(OAc)₂, XantPhos, Cs₂CO₃, 3-bromopyridine, 1,4-dioxane, 110°C.</figcaption></figure>

## Conclusion

In an attempt to strengthen the active conformation of an inhibitor of SARS-CoV-2 M^pro^, the synthesis of analogues **13-16a-b** and **19** featuring a bridging component was achieved. In a second approach, we also prepared the “turned around” N-pyridyl analogues **22** or **25**. However, only compounds **16b** showed some activity at 25 µM in our SARS-CoV-2 M^pro^ luciferase-based assay.¹⁴ On the chemistry point of view, the extension of the Bargellini reaction to prepare the 3,3-disubstituted piperazin-2-ones **11** and **18**, or, when this failed, the recourse to a rather simple alternative to reach the 3,3-disubstituted piperazin-2-ones **15a-b** as well as **21** and **25** should be of interest to generate original chemical libraries.¹⁷ Indeed, 3,3-disubstituted piperazin-2-ones appears to be a rather underexplored class of compounds as a data base search came out with only 168 compounds featuring this component which were described in 84 patents and 28 articles. This search result was obtained when retaining both NH as well as at least one hydrogen on carbon 5 and 6 of the 3,3-disubstituted piperazin-2-one in order to avoid the much more numerous piperazinediones or benzo derivatives reported.

## Funding sources

This work was supported by the «URGENCE COVID-19» fundraising campaign of the Institut Pasteur. KYC and TK were funded by the Agence Nationale de la Recherche (grants ANR-18-CE18-0026 and ANR-18-CE18-0028).

## Experimental part

**Chemistry.** ¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz and 100 MHz, respectively. Shifts (δ) are given in ppm with respect to the TMS signal and cross-coupling constants (J) are given in Hertz. Column chromatography were performed either on Merck silica gel 60 (0.035 - 0.070 mm) or neutral alumina containing 1.5% of added water using a solvent pump and an automated collecting
system driven by a UV detector set to 254 nm unless required otherwise. Sample deposition was carried out by
absorption of the mixture to be purified on a small amount of the solid phase followed by its deposition of the top
of the column. The low-resolution mass spectra were obtained on an Agilent 1200 series LC/MSD system using
an Agilent Jet-Stream atmospheric electrospray ionization system and the high-resolution mass spectra (HRMS)
were obtained using a Thermo Fisher Q Exactive Orbitrap.

More recent HRMS measurements were carried out, by the plateau technique de Spectrométrie de Masse Bio-
organique, using an ESI-Q-TOF MS (compact, Bruker, France) by directly injecting 1µl of the diluted sample
solution. The composition of the mobile phase was 1:1 containing ACN (with 0.08 % formic acid) and milli-Q
water (containing 0.1 % formic acid). The flow rate was maintained at 100µl/min. Mass spectra were recorded in
the positive ionization mode in the m/z range between 50 and 1200. Calibration of the mass spectrometer was
carried out with sodium-formate clusters formed in the gas phase. When specified, the anhydrous solvents used
were purchased. Experiments under inert atmosphere were carried out by purging the glassware with a stream of
dry argon. Then, an argon balloon, fitted with a needle, was used to insure a positive pressure of inert gas during
the reaction. Unless stated otherwise, a purity of at least 95% was obtained for all the compounds by means of
chromatography or recrystallization and this level of purity was established by TLC, LC/MS and NMR
spectroscopy.

Synthesis of 3-methyl-3-(pyridin-3-yl)piperazin-2-one (11) via a Bargellini reaction: The procedure followed is essentially the one described on page 15 of a patent.⁹c A solution of sodium hydroxide 50% (6.10 g, 82.5 mmol) was slowly added, while stirring, to an ice-cold mixture of ethylene diamine (1.19 g, 19.8 mmol) and benzyltriethylammonium chloride (0.19 g, 0.82 mmol) dissolved in dichloromethane (8 mL). A solution of 3-acetylpyridine (2.01 g, 16.5 mmol) and chloroform (2.60 g, 21.5 mmol) dissolved in dichloromethane (8 mL) was then added drop-wise while stirring rapidly the ice-cold solution described above. This was stirred overnight while allowing the bath to warm back to room temperature. The resulting suspension was dispersed in water and ethyl acetate and made slightly acid with acetic acid. The organic phase was removed and the aqueous phase subjected to an extraction in boiling ethyl acetate overnight using a liquid/liquid extractor. The resulting solution was concentrated to dryness and purified by a chromatography over silica gel (dichloromethane/ethanol 90/10) to give compound 11 as a slowly crystallizing pale yellow solid (1.57 g, 49%). ¹H NMR (400 MHz, CDCl₃): δ = 8.88 (m, 1H), 8.52 (dd, 1H, J = 1.6 and 4.7 Hz), 7.98 (m, 1H), 7.28 (ddd, 1H, J = 0.7, 4.7 and 8.0 Hz), 6.74 (s(br), 1H), 3.49 (m, 1H), 3.28 (m, 1H), 3.01 (m, 1H), 2.83 (m, 1H), 2.24 (s(br), 1H), 1.68 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ = 172.8, 148.5, 148.0, 138.9, 134.3, 123.1, 61.4, 43.9, 38.3, 28.3. HRMS (m/z): [M+H]⁺ calcd for C₁₀H₁₄N₃O: 192.1131; found: 192.1128. Note: a similar yield was obtained when using tetrabutyl ammonium bromide as a phase transfer catalyst, but this ammonium could not be separated from the reaction product.

Alternative synthesis of 3-methyl-3-(pyridin-3-yl)piperazin-2-one (11) using ethylene diamine and potassium cyanide. In a 20 mL Biotage microwave oven tube, 3-acetylpyridine (1.05 g, 8.66 mmol), ethylene diamine (1.56 g, 26.00 mmol) and potassium cyanide (0.68 g, 10.04 mmol) were dispersed in ethanol (12 mL, dried over 4Å molecular sieves). Under a well-ventilated hood, acetic acid (1.2 mL, 20.80 mmol) was added and the tube was sealed. This was stirred for two hours at room temperature and the resulting solution was heated at 140 °C for 3 hours in a Biotage microwave oven. The resulting solution was concentrated to dryness, dissolved in water (15
mL) and heated at 150 °C for two hours in a Biotage microwave oven. This solution was subjected to an extraction in boiling ethyl acetate overnight using a liquid/liquid extractor, the organic solution was concentrated to dryness and purified by a chromatography over silica gel (dichloromethane/ethanol 90/10) to give compound **11** as a slowly crystallizing solid (0.67 g). Note: with this method (a) highly polar and low molecular mass impurity(ies) (¹H NMR: δ = 8.12 (m), 6.95 (s(br), 3.36 (m), 3.32 (m) and 1.93 (s)) could not be removed. However, this could be used in the next step without any problems.

**Synthesis of 3-methyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (13):** Compound **11** (0.23 g, 1.20 mmol) was dissolved in dry dimethylformamide (3 mL, dried over 4Å molecular sieves). Sodium hydride 60% in oil (0.05 g, 1.44 mmol) was added followed, at the end of the hydrogen evolution, by (1-chloroethyl)benzene (0.17 g, 1.20 mmol). This was stirred for three hours, diluted in water and ethyl acetate, the organic phase was washed with water, brine, dried over magnesium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol 95/5) to give compound **13** (0.08 g, 22%) as an oil. ¹H NMR (400 MHz, CDCl₃, 1/1 mixture of two diastereoisomers): δ = 8.90 (m, 1H), 8.53 (m, 1H), 7.99 (m, 1H), 7.40-7.24 (m, 6H), 6.19 (m, 1H), 3.31 (m, 0.5H), 3.08-2.72 (m, 3.5H), 2.15 (s(br), 1H), 1.74 (s, 1.5H), 1.73 (s, 1.5H), 1.58 (d, 1.5H, J = 7.00 Hz), 1.51 (d, 1.5H, J = 7.00 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 170.5, 170.2, 148.3, 148.2, 148.0, 147.9, 140.1, 139.8, 134.4, 134.3, 128.5, 127.6, 127.5, 127.2, 123.2, 62.0, 61.9, 50.4, 50.3, 42.3, 42.0, 39.0, 38.8, 29.0, 28.7, 15.5, 15.3. HRMS (m/z): [M+H]⁺ calcd for C₁₈H₂₂N₃O: 296.1757; found: 296.1738.

**Synthesis of 2-phenyl-1-(pyridin-3-yl)ethan-1-one (14a):** The following is an adaptation of the reported procedure.¹¹ In a 60 mL tube featuring a Teflon-coated screw cap, 3-acetylpyridine (3.02 g, 24.92 mmol), bromobenzene (1.95 g, 12.46 mmol), potassium phosphate tribasic (7.40 g, 34.90 mmol), palladium acetate (0.11 g, 0.498 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.58 g, 0.997 mmol) were weighted. This tube was flushed with a slow stream of argon and dry dioxane (30 mL), dry toluene (5 mL) and dry tetrahydrofuran (5 mL) were injected under while retaining the inert atmosphere. The tube was tightly closed and stirred and heated at 100 °C for 23 hours. The resulting black suspension was dispersed in water and ethyl acetate, filtered, the aqueous phase was extracted with ethyl acetate (3 x 25 mL), the organic phase was washed with water (20 mL), brine (20 mL), dried over magnesium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (cyclohexane/ethyl acetate 2/1) to give compound **14a** as a solid (7.71 g, 69%) with spectral data identical with the reported one.¹⁸

**Synthesis of 3-benzyl-3-(pyridin-3-yl)piperazin-2-one (15a):** In a 20 mL Biotage microwave oven tube, compound **14a** (1.01 g, 5.12 mmol), ethylene diamine (0.92 g, 15.36 mmol) and potassium cyanide (0.40 g, 6.14 mmol) were dispersed in ethanol (10 mL, dried over 4Å molecular sieves). Under a well-ventilated hood, acetic acid (0.70 mL, 12.28 mmol) was added and the tube was sealed. This was stirred for two hours at room temperature and the resulting solution was heated at 140 °C for three hours in a Biotage microwave oven. The resulting solution was partially concentrated, dispersed in a 2/1 mixture of water and ethanol (15 mL) and heated at 150 °C for two hours in a Biotage microwave oven. The resulting solution was diluted in water extracted with ethyl acetate (3 x 25 mL), the organic layer was washed with brine (20 mL) dried over magnesium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol
95/5) to give some unreacted ketone (0.25 g, 24%) and then compound **15a** as a solid (0.40 g, 29%). ¹H NMR (400 MHz, CDCl₃): δ = 9.00 (m, 1H), 8.56 (m, 1H), 8.17 (m, 1H), 7.33-7.26 (m, 4H), 7.21 (m, 2H), 6.23 (s(br), 1H), 3.71 (d, 1H, J = 13.5 Hz), 3.25-3.12 (m, 2H), 2.93 (m, 1H), 2.90 (d, 1H, J = 13.5 Hz), 2.82 (m, 1H), 2.35 (s(br), 1H). ¹³C NMR (100 MHz, CDCl₃): δ = 171.2, 148.2 (two signals), 137.9, 135.6, 135.2, 130.6, 128.5, 127.3, 123.1, 65.1, 47.2, 44.0, 38.1. HRMS (*m/z*): [M+H]⁺ calcd for C₁₆H₁₈N₃O: 268.1444; found: 268.1147.

Synthesis of 3-benzyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (**16a**): Compound **15a** (0.19 g, 0.71 mmol) was dissolved in dry dimethylformamide (4 mL, dried over 4Å molecular sieves). Sodium hydride 60% in oil (0.034 g, 0.85 mmol) was added followed, at the end of the hydrogen evolution, by (1-chloroethyl)benzene (0.1 g, 0.71 mmol). This was stirred for 10 hours, diluted in water and ethyl acetate, the organic phase was washed with water, brine, dried over magnesium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol 97/3) to give compound **16a** as a glassy mixture of diastereoisomers (0.14 g, 53%). ¹H NMR (400 MHz, CDCl₃, 1/1 mixture of diastereoisomers, traces of solvents): δ = 9.06 (m, 1H), 8.57 (m, 1H), 8.23 (m, 1H), 7.35-7.18 (m, 10H), 7.06 (m, 1H), 6.24 (q, 0.5 H, J = 7.1 Hz), 6.17 (q, 0.5 H, J = 7.1 Hz), 3.85 (d, 0.5 H, J = 13.1 Hz), 3.84 (q, 0.5 H, J = 13.1 Hz), 2.96-2.48 (m, 5H), 2.18 (s(br), 1H), 1.46 (d, 1.5H, J = 7.2 Hz), 1.40 (d, 1.5H, J = 7.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 169.0, 168.8, 148.5, 148.2, 148.1, 140.0, 139.3, 139.0, 138.9, 136.1, 136.0, 135.3, 135.2, 130.9, 130.8, 128.6, 128.5, 128.4, 128.3, 127.5, 127.4, 127.3, 127.2, 127.1, 123.2, 65.9, 65.7, 50.3, 50.2, 47.9, 47.6, 42.4, 42.3, 38.5, 15.4, 15.3. HRMS (*m/z*): [M+H]⁺ calcd for C₂₄H₂₆N₃O: 372.2070; found: 372.2063.

Synthesis of 2-([1,1'-biphenyl]-4-yl)-1-(pyridin-3-yl)ethan-1-one (**14b**): The procedure described above for the preparation of compound **14a** was used and the biphenyl derivative **14b** (1.31 g, 57%) was obtained as a white solid following a chromatography over silica gel (cyclohexane/ethyl acetate 7/3) and a recrystallization in cyclohexane. ¹H NMR (400 MHz, CDCl₃): δ = 9.29 (m, 1H), 8.80 (dd, 1H, J = 1.7 and 4.9 Hz), 8.34 (m, 1H), 7.59 (m, 4H), 7.49-7.43 (m, 3H), 7.36 (m, 3H), 4.37 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 196.1, 153.0, 149.6, 140.6, 140.3, 136.4, 132.4, 132.0, 129.8, 128.8, 127.6, 127.3, 127.0, 123.9, 45.5. HRMS (*m/z*): [M+H]⁺ calcd for C₁₉H₁₇NO: 274.1226; found: 274.1221.

Synthesis of 3-([1,1'-biphenyl]-4-ylmethyl)-3-(pyridin-3-yl)piperazin-2-one (**15b**): In a 20 mL Biotage microwave oven tube, compound **14b** (0.82 g, 2.99 mmol), ethylene diamine (0.54 g, 8.99 mmol) and potassium cyanide (0.23 g, 3.59 mmol) were dispersed in ethanol (10 mL, dried over 4Å molecular sieves). Under a well-ventilated hood, acetic acid (0.41 mL, 7.20 mmol) was added and the tube was sealed. This suspension was stirred for two hours at 50 °C and the resulting solution was then heated at 140 °C for three hours in a Biotage microwave oven. Water (8 mL) was then added to the resulting solution and this was heated at 150 °C for two hours in a Biotage microwave oven. The resulting suspension was diluted in water and ethyl acetate, the aqueous phase was extracted with ethyl acetate (3 x 25 mL), the organic phase was washed with brine (20 mL), dried over magnesium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol 97/3 to 95/5) to give some unreacted ketone (0.24 g, 29%) and then compound **15b** as a solid (0.44 g, 42%, traces of ethanol). ¹H NMR (400 MHz, CDCl₃): δ = 9.04 (m, 1H), 8.57 (dd, 1H, J = 1.6 and 4.7 Hz), 8.19 (m, 1H), 7.60 (m, 2H), 7.54 (m, 2H), 7.45 (m, 2H), 7.38-7.26 (m, 4H), 6.29 (s(br), 1H), 3.76 (d, 1H, J = 13.3 Hz), 3.30-3.23 (m, 1H), 3.20-3.14 (m, 1H), 2.97 (m, 1H), 2.95 (d, 1H, J = 13.3 Hz), 2.89-2.81 (m,
1H), 2.30 (s(br), 1H). ¹³C NMR (100 MHz, CDCl₃): δ = 171.4, 148.3, 148.2, 140.6, 140.1, 137.9, 135.1, 134.6, 135.1, 134.6, 131.1, 128.8, 127.3, 127.2, 126.9, 123.2, 65.1, 46.8, 44.0, 38.1. HRMS (*m/z*): [M+H]⁺ calcd for C₂₂H₂₂N₃O: 344.1757; found: 344.1761.

**Synthesis of 3-([1,1'-biphenyl]-4-ylmethyl)-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (16b):** By following the procedure described above for the preparation of compound **16a** and after a chromatography over silica gel (dichloromethane/ethanol 97/3) and prolonged heating at 70 °C under vacuum to remove some pesky DMF, compound **16b** was obtained as a glassy mixture of diastereoisomers (0.12 g, 38%). ¹H NMR (400 MHz, CDCl₃, 1/1 mixture of diastereoisomers): δ = 9.10 (m, 1H), 8.60 (m, 1H), 8.25 (m, 1H), 7.63 (m, 2H), 7.58 (m, 2H), 7.48 (m, 2H), 7.42-7.18 (m, 8H), 7.07 (m, 1H), 6.26 (q, 0.5 H, *J* = 7.1 Hz), 6.18 (q, 0.5 H, *J* = 7.0 Hz), 3.89 (d, 0.5 H, *J* = 13.1 Hz), 3.86 (q, 0.5 H, *J* = 13.0 Hz), 3.02-2.51 (m, 5H), 2.15 (s(br), 1H), 1.47 (d, 1.5H, *J* = 7.2 Hz), 1.43 (d, 1.5H, *J* = 7.3 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 169.0, 168.8, 148.5, 148.3, 148.2, 140.7, 140.6, 140.1, 140.0, 139.3, 139.0, 138.9, 135.3, 135.2, 135.1, 131.4, 131.2, 128.9, 128.8, 128.5, 128.4, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0, 126.9, 123.2, 66.0, 65.8, 50.4, 50.3, 47.6, 47.3, 42.4, 42.3, 38.6, 38.5, 15.4, 15.3. HRMS (*m/z*): [M+H]⁺ calcd for C₃₀H₃₀N₃O: 448.2383; found: 448.2376.

**Synthesis of 3-phenyl-1-(pyridin-3-yl)propan-1-one (17):** This compound was prepared in three steps. Step 1: ethyl nicotinate (6.57 g, 43.4 mmol) and dry ethyl acetate (8.52 g, 86.9 mmol, dried over 4Å molecular sieves) were dissolved in dry toluene (40 mL, dried over 4Å molecular sieves). Sodium hydride 60% in oil (3.47 g, 86.9 mmol) was added and, at the end of the slight hydrogen evolution, this was heated at 90 °C under a calcium chloride guard for 20 hours. The resulting precipitate was dissolved in water and ethyl acetate and neutralized with solid ammonium chloride (4.64 g, 86.9 mmol). The organic phase was washed with water, brine, dried over magnesium sulfate and concentrated to dryness to give the crude ethyl 3-oxo-3-(pyridin-3-yl)propanoate (8.91 g) as an oil. Step 2: this oil was dissolved in dry dimethylformamide (50 mL, dried over 4Å molecular sieves), sodium hydride 60% in oil (1.56 g, 39.0 mmol) was added and, at the end of the slight hydrogen evolution, benzyl bromide (6.7 g, 39.0 mmol). This was stirred at room temperature under a calcium chloride guard for 16 hours, diluted in water and ethyl acetate, the organic phase was washed with water (three time), brine, dried over magnesium sulfate and concentrated to dryness to give the crude ethyl 2-benzyl-3-oxo-3-(pyridin-3-yl)propanoate (9.87 g) as an oil. Step 3: this oil was dissolved in acetic acid (10 mL) and hydrochloric acid (5 mL) and heated at 100 °C for four hours. This was cooled, dissolved in water and ethyl acetate and cautiously made basic with solid sodium carbonate. The organic phase was washed with water, brine, dried over magnesium sulfate and concentrated to dryness and the residue purified by a chromatography over silica gel (cyclohexane/ethyl acetate 3/1) to give compound **17** (3.57 g, 37% from ethyl nicotinate) as a clear oil with analytical characteristic similar to the reported one. ¹⁹ ¹H NMR (400 MHz, CDCl₃): δ = 9.18 (d(br), 1H, *J* = 2.0 Hz), 8.79 (dd, 1H, *J* = 1.7 and 4.8 Hz), 8.26 (m, 1H), 7.45 (m, 1H), 7.35-7.22 (m 5H), 3.35 (m, 2H), 3.11 (t, 2H, *J* = 7.5 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 198.0, 153.3, 149.4, 140.7, 135.6, 132.1, 128.6, 128.4, 126.3, 123.7, 40.7, 29.8. HRMS (*m/z*): [M+H]⁺ calcd for C₁₄H₁₄NO: 212.1067; found: 212.1070.

**Synthesis of 3-phenethyl-3-(pyridin-3-yl)piperazin-2-one (18):** The procedure followed is essentially the one described on page 15 of a patent.⁹c A solution of sodium hydroxide 50% (4.5 g, 56.3 mmol) was slowly added, while stirring, to an ice-cold mixture of ethylene diamine (0.81 g, 13.5 mmol) and benzyltriethylammonium
chloride (0.13 g, 0.56 mmol) dissolved in dichloromethane (8 mL). A solution of 3-phenyl-1-(pyridin-3-yl)propan-1-one (17) (2.38 g, 11.2 mmol) and chloroform (1.75 g, 14.6 mmol) dissolved in dichloromethane (8 mL) was then added drop-wise while stirring rapidly the ice-cold solution described above. This was stirred overnight while allowing the bath to warm back to room temperature. The resulting suspension was dispersed in water and ethyl acetate and made slightly acid with acetic acid. This was extracted with ethyl acetate (3 x 25 mL), the organic phase was washed with water (20 mL), brine (20 mL), dried over magnesium sulfate and concentrated to dryness and the residue purified by a chromatography over silica gel (dichloromethane/ethanol 95/5 to 94/6) to give some unreacted ketone (0.43 g, 18%) and then compound 18 as a glass (1.36 g, 43%). ¹H NMR (400 MHz, CDCl₃, traces of EtOH): δ = 8.96 (m, 1H), 8.54 (dd, 1H, J = 1.7 and 4.8 Hz), 8.08 (m, 1H), 7.31-7.24 (m, 3H), 7.18-7.14 (m, 3H), 6.54 (s(br), 1H), 3.53 (m, 1H), 3.27 (m, 1H), 3.06 (m, 1H), 2.90 (m, 1H), 2.70 (m, 2H), 2.24-2.13 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 171.8, 148.3, 148.0, 141.4, 137.7, 134.8, 128.5, 128.4, 126.0, 123.2, 64.7, 43.8, 43.4, 38.4, 30.6. HRMS (*m/z*): [M+H]⁺ calcd for C₁₇H₂₀N₃O: 282.16009; found: 282.1585.

**Synthesis of 3-phenethyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (19):** By using the protocol described for the preparation of 3-methyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (13) and stirring overnight, compound 19 was obtained as an oil (0.15 g, 40%) after a chromatography over silica gel (cyclohexane/ethyl acetate 1/1 to 1/2; note: compound 19 is very weakly visible under normal setting at 280 nm).

¹H NMR (400 MHz, CDCl₃, 1/1 mixture of two diastereoisomers): δ = 8.96 (m, 1H), 8.53 (m, 1H), 8.08 (m, 1H), 7.37 (m, 2H), 7.33-7.16 (m, 8H), 6.25 (m, 1H), 3.35 (m, 0.5H), 3.03-2.86 (m, 3.5H), 2.82-2.64 (m, 3H), 2.53 (m, 1H), 2.21 (m, 2H), 1.61 (d, 1.5H, J = 7.00 Hz), 1.51 (d, 1.5H, J = 7.00 Hz). ¹³C NMR (100 MHz, CDCl₃): δ = 169.5, 169.3, 148.3, 148.2, 148.1, 148.0, 141.6, 140.1, 139.7, 138.7, 138.6, 134.9, 134.8, 128.6, 128.5, 128.4, 127.6, 127.5, 127.2, 126.0, 123.2, 65.3, 65.1, 50.5, 50.4, 44.2, 43.8, 42.3, 42.1, 40.0, 38.9, 30.9, 30.8, 15.6, 15.4. HRMS (*m/z*): [M+H]⁺ calcd for C₂₅H₂₈N₃O: 386.22269; found: 386.2219.

**Synthesis of 3-(3-chlorobenzyl)-3-methylpiperazin-2-one (21):** In a 20 mL Biotage microwave oven tube, ketone 20 (0.98 g, 5.81 mmol), ethylene diamine (1.05 g, 17.32 mmol) and potassium cyanide (0.38 g, 5.83 mmol) were dispersed in ethanol (12 mL, dried over 4Å molecular sieves). Under a well-ventilated hood, acetic acid (0.8 mL, 13.97 mmol) was added and the tube was sealed. This was heated at 140 °C for three hours in a Biotage microwave oven. To the resulting gel water (4 mL) was added and the vial was heated at 150 °C for two hours in a Biotage microwave oven. The resulting solution was diluted in water extracted with ethyl acetate (3 x 25 mL), the organic layer was washed with brine (20 mL) dried over sodium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol 97/3) to give compound 21 as a slowly solidifying solid (0.55 g, 40%). ¹H NMR (400 MHz, CDCl₃): δ = 7.27-7.21 (m, 3H), 7.15 (m, 1H), 3.34 (d, 1H, J = 13.5 Hz), 3.28 (3, 1H), 3.19 (m, 1H), 3.05 (m, 1H), 2.96 (m, 1H), 2.73 (d, 1H, J = 13.5 Hz), 1.39 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ = 174.8, 138.9, 134.2, 130.6, 129.6, 128.9, 127.1, 60.0, 44.0, 43.9, 38.3, 25.7. HRMS (*m/z*): [M+H]⁺ calculated for C₁₂H₁₅N₂OCl: 239.0946; found: 239.0941.

**3-(3-chlorophenyl)-3-ethylpiperazin-2-one (24).** In a 5 mL Biotage microwave oven tube, ketone 23 (0.49 g, 2.90 mmol), ethylene diamine (0.88 g, 14.64 mmol) and potassium cyanide (0.57 g, 8.75 mmol) were dispersed in ethanol (2.5 mL, dried over 4Å molecular sieves). Under a well-ventilated hood, acetic acid (0.6 mL, 10.48 mmol)
was added and the tube was sealed. This was heated at 140 °C for three hours using a Biotage microwave oven. To the resulting gel water (1 mL) was added and the vial was heated at 150 °C for two hours. The resulting solution was diluted in water extracted with ethyl acetate (3 x 25 mL), the organic layer was washed with brine (20 mL) dried over sodium sulfate and concentrated to dryness. The residue was purified by a chromatography over silica gel (dichloromethane/ethanol 98/2) to give compound **21** as white powder (0.36 g, 52 %). ¹H NMR (400 MHz, CDCl₃): $\delta$ = 7.69 (m, 1H), 7.57 (m, 1H), 7.25 (m, 1H), 6.53 (s (br), 1H), 3.48 (m, 1H), 3.23 (m, 1H), 3.02 (m, 1H), 2.90 (m, 1H), 2.12 (m, 1H), 1.85 (m, 1H), 0.91 (t, 3H, J = 7.43 Hz). ¹³C NMR (100 MHz, CDCl₃): $\delta$ = 172.68, 144.66, 134.42, 129.62, 127.41, 126.75, 124.77, 66.1, 43.88, 38.37, 34.47, 8.71. HRMS (*m/z*): [M+H]⁺ calculated for C₁₂H₁₅N₂OCl: 239.0946; found: 239.0948.

3-(3-chlorobenzyl)-3-methyl-1-(pyridin-3-yl)piperazin-2-one (**22**). This procedure was adapted from the one described in page 40 of a patent.¹⁶ In a 5 mL Biotage microwave oven tube, 3-(3-chlorobenzyl)-3-methylpiperazin-2-one (**21**) (0.4 g, 1.67 mmol), 3-bromo-pyridine (0.52 g, 3.35 mmol), Pd(OAc)₂ (0.037 g, 0.16 mmol), XantPhos (0.19 g, 0.33 mmol) and Cs₂CO₃ (1.64 g, 5.03 mmol) were dispersed in anhydrous 1,4-dioxane (3 mL), and degassed under argon for 1 hour. The tube was sealed and the reaction mixture was heated at 110°C for 4 hours in a microwave oven. The resulting solution was diluted in water extracted with ethyl acetate (3 x 25 mL), the organic layer was washed with brine (20 mL) dried over sodium sulfate and concentrated to dryness. This residue was purified by a column chromatography over neutral alumina containing 2% of water (dichloromethane/ethanol 99/1) and the relevant fraction was further purified by a chromatography over silica gel (dichloromethane/ethanol 98/2 to 96/4) to yield compound **22** a yellow oil (0.31 g, 59%). ¹H NMR (400 MHz, CDCl₃): $\delta$ = 8.48 (dd, 1H, J = 4.75 Hz, J = 1.5 Hz), 8.44 (d, 1H, J = 2.4 Hz), 7.54 (m, 1H), 7.32 (m, 2H), 7.26 (m, 2H), 7.16 (m, 1H), 3.5 (m, 2H), 3.43 (d, 1H, J = 13.24 Hz), 3.24 (m, 1H), 3.07 (m, 1H), 2.75 (d, 1H, J = 13.28 Hz), 1.52 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): $\delta$ = 172.8, 147.81, 147.3, 139.24, 138.86, 134.13, 133.74, 130.55, 129.53, 128.92, 127.17, 123.65, 61.04, 52.62, 46.07, 38.83, 26.37. HRMS (*m/z*): [M+H]⁺ calculated for C₁₇H₁₉ClN₂O: 316,1211; found: 316,1211.

3-(3-chlorophenyl)-3-ethyl-1-(pyridin-3-yl)piperazin-2-one (**25**). By using the procedure described above for the preparation of compound **22** and starting with 3-(3-chlorophenyl)-3-ethylpiperazin-2-one (**24**) (0.2 g, 0.9 mmol). This product was obtained as a yellow solid (0.13 g, 46%) following a purification by column chromatography over neutral alumina containing 2% of water (dichloromethane). ¹H NMR (400 MHz, CDCl₃): $\delta$ = 8.57 (m, 1H), 8.50 (dd, 1H, J = 4.79 Hz, J = 1.5 Hz), 7.71 (m, 1H), 7.57 (ddd, 1H, J = 8.16 Hz, J = 2.53 Hz, J = 1.54 Hz), 7.59 (dt, 1H, J = 7.28 Hz, J = 1.77 Hz), 7.3 (m, 3H), 3.91 (m, 1H), 3.5 (m, 1H), 3.19 (m, 2H), 2.2 (m, 1H), 1.94 (m, 1H), 0.94 (t, 3H, J = 7.4 Hz). ¹³C NMR (100 MHz, CDCl₃): $\delta$ = 171.22, 147.77, 147.18, 144.45, 138.37, 134.61, 133.72, 129.80, 127.56, 126.60, 124.69, 123.68, 66.65, 51.91, 39.01, 35.05, 8.79. HRMS (*m/z*): [M+H]⁺ calculated for C₁₇H₁₉ClN₂O: 316,1211; found: 316,1218.

**Bioluminescence-based SARS-CoV-2 protease inhibition assay.** HEK-293T cells (purchased from ATCC, # CRL-11268). The Mpro expression plasmids (pcDNA3.1-nsp4-5-6 WT and pcDNA-3.1-nsp4-5-6 C145A) and Nanoluciferase reporter plasmid (Rev-Nluc-CoV) used were previously described.¹⁴ HEK-293T cells were transfected in 50 cm² dishes with 1 µg of the Rev-Nluc-CoV plasmid and 15 µg of the nsp4-5-6 WT or nsp4-5-
6 C145A expression plasmids. Increasing amounts of the tested compounds, corresponding to final concentrations of 0.4 to 50 µM with a constant DMSO concentration of 0.5%, were distributed in 96-well white opaque plates (Greiner Bio-One, 10 µL per well). Transfected HEK-293T cells were trypsinized at 6 hours post-transfected and distributed in the 96-well plates (105 cells per well in 40 µL). Nanoluciferase activity was measured at 24 hours post-transfection using the Nano-Glo® Luciferase Assay System (#1120, Promega) and a Berthold Centro XS3 luminometer (integration time 1s/well using the MikroWin® software).

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Non covalent inhibitors of SARS-CoV-2 main protease, a rescaffolding attempt

Tim Krischuns<sup>a</sup>, Sylvain Paisant<sup>a</sup>, Kuang-Yu Chen<sup>a</sup>, Laura N. Thirion<sup>b</sup>, Agnès Zettor<sup>c</sup>, Jeanne Chiaravalli<sup>c</sup>, Yves Jacob<sup>d</sup>, Marco Bellinzoni<sup>e</sup>, Nadia Naffakh<sup>a</sup>, Yves L. Janin<sup>b,e</sup>

a : RNA Biology and Influenza Virus Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 25-28 rue du Dr. Roux, F-75015 Paris, France

b : Structure et Instabilité des Génomes (StrInG), Muséum National d'Histoire Naturelle, INSERM, CNRS, Alliance Sorbonne Université, 75005 Paris, France.

c : Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, Paris, France.

d : Molecular Genetics of RNA Viruses Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3569, 25-28 rue du Dr. Roux, F-75015 Paris, France

e : Structural Microbiology Unit, Institut Pasteur, Université Paris Cité, CNRS UMR 3528, 25-28 rue du Dr. Roux, F-75015 Paris, France

Supporting information

This document contains

- The ¹H NMR and ¹³C NMR spectra of all the compounds described in this manuscript. These were recorded on a Bruker Avance 400 spectrometer at 400 MHz and 100 MHz, respectively.

- A graph obtained using the bioluminescence-based SARS-CoV-2 main protease inhibition assays
<figure><img src="image_7.png" /><figcaption>3-methyl-3-(pyridin-3-yl)piperazin-2-one (11)</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>3-methyl-3-(pyridin-3-yl)piperazin-2-one (11)</figcaption></figure>

<figure><img src="image_9.png" /></figure>
$$ \chemfig{C(=[phenyl])N(-[6]CH_2-CH=N-CH(-[2]CH_3)([9]CH_3)-C(=[pyridin])N} $$

3-methyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (13)

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>3-methyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (13)</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>2-phenyl-1-(pyridin-3-yl)ethan-1-one (14a)</figcaption></figure>
<figure><img src="image_13.png" /><figcaption>2-phenyl-1-(pyridin-3-yl)ethan-1-one (14a)</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>2-([1,1'-biphenyl]-4-yl)-1-(pyridin-3-yl)ethan-1-one (14b)</figcaption></figure>
<figure><img src="image_15.png" /></figure>

$$2-([1, 1'-biphenyl]-4-yl)-1-(pyridin-3-yl)ethan-1-one (14b)$$
<figure><img src="image_16.png" /><figcaption>3-benzyl-3-(pyridin-3-yl)piperazin-2-one (15a)</figcaption></figure>
$$3-\text{benzyl}-3-(\text{pyridin-3-yl})\text{piperazin-2-one (15a)}$$

<figure><img src="image_17.png" /></figure>
<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>3-([1,1'-biphenyl]-4-ylmethyl)-3-(pyridin-3-yl)piperazin-2-one (15b)</figcaption></figure>

<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /><figcaption>3-[[1,1'-biphenyl]-4-ylmethyl]-3-(pyridin-3-yl)piperazin-2-one (15b)</figcaption></figure>
<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /><figcaption>3-benzyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (16a)</figcaption></figure>
<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /><figcaption>3-benzyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (16a)</figcaption></figure>
<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /><figcaption>3-[(1,1'-biphenyl]-4-ylmethyl)-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (16b)</figcaption></figure>
<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

3-([1,1'-biphenyl]-4-ylmethyl)-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (16b)
$$ \chemfig{*6(=(-N=C(-[2]CH_2CH(CH_3)_2)-CH_2-)=)} $$

<figure><img src="image_30.png" /><figcaption>3-phenyl-1-(pyridin-3-yl)propan-1-one (17)</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>3-phenyl-1-(pyridin-3-yl)propan-1-one (17)</figcaption></figure>
<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /><figcaption>3-phenethyl-3-(pyridin-3-yl)piperazin-2-one (18)</figcaption></figure>
<figure><img src="image_34.png" /><figcaption>3-phenethyl-3-(pyridin-3-yl)piperazin-2-one (18)</figcaption></figure>
<figure><img src="image_35.png" /></figure>

$$3-phenethyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (19)$$
<figure><img src="image_36.png" /><figcaption>3-phenethyl-1-(1-phenylethyl)-3-(pyridin-3-yl)piperazin-2-one (19)</figcaption></figure>
<figure><img src="image_37.png" /><figcaption>3-(3-chlorobenzyl)-3-methylpiperazin-2-one (21)</figcaption></figure>
<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /><figcaption>3-(3-chlorobenzyl)-3-methylpiperazin-2-one (21)</figcaption></figure>
<figure><img src="image_40.png" /><figcaption>3-(3-chlorobenzyl)-3-methyl-1-(pyridin-3-yl)piperazin-2-one (22)</figcaption></figure>
<figure><img src="image_41.png" /><figcaption>3-(3-chlorobenzyl)-3-methyl-1-(pyridin-3-yl)piperazin-2-one (22)</figcaption></figure>
<figure><img src="image_42.png" /></figure>

<figure><img src="image_43.png" /><figcaption>3-(3-chlorophenyl)-3-ethylpiperazin-2-one (24)</figcaption></figure>
<figure><img src="image_44.png" /><figcaption>3-(3-chlorophenyl)-3-ethylpiperazin-2-one (24)</figcaption></figure>
<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /><figcaption>3-(3-chlorophenyl)-3-ethyl-1-(pyridin-3-yl)piperazin-2-one (25)</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>3-(3-chlorophenyl)-3-ethyl-1-(pyridin-3-yl)piperazin-2-one (25)</figcaption></figure>
Bioluminescence-based SARS-CoV-2 protease inhibition assay
<figure><img src="image_48.png" /></figure>

○ Wild-type Mpro

• C145A mutant Mpro

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

<figure><img src="image_53.png" /></figure>

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /></figure>

<figure><img src="image_56.png" /></figure>