Synthesis and SARS-CoV-2 3CL Protease Inhibitory Effects of Oxazolidinone Derivatives

Abstract

The 3-chymotrypsin-like protease (3CL^pro) is an attractive target for the development of anti-SARS (severe acute respiratory syndrome) drugs. In this work, a series of oxazolidinone derivatives 3a-3v were synthesized and their inhibitory activities against SARS coronavirus 2 (SARS-CoV-2) 3CL^pro were evaluated by the fluorescence resonance energy transfer (FRET)-based enzymatic assay. Among synthesized compounds, 3g displayed the best inhibitory activity, with a half maximal inhibitory concentration (IC₅₀) value of 14.47 μM. Also, docking studies implied that compound 3g was fitted into the active pocket of 3CL^pro, forming a hydrogen bond with Glu166.

Keywords:
oxazolidinones; SARS-CoV-2; 3CL protease; inhibitory activity

Introduction

Since the isolation of the first human coronavirus in 1965,¹,² seven family members have been identified.³ Three of them, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have caused very severe respiratory diseases in humans over the past 20 years.²-⁵ The coronavirus disease 2019 (COVID-19) outbreak has taken a large number of human deaths and severe economic losses globally.⁶,⁷ This virus is most likely to coexist with humans for a long time,⁸ so there is an urgent demand to develop preventive and therapeutic measures. From what has been reported so far,⁹ the vaccine may not be effective enough due to the mutation of the virus. In the meantime, no specific antiviral drugs are currently available for the prevention and treatment of COVID-19 infections.¹⁰

After coronavirus infection, its genetic material RNA (ribonucleic acid) first encodes into two polymeric precursors pp1a and pp1ab. Under the action of 3-chymotrypsin-like protease (3CL^pro) and papain-like protease (PL^pro), the polymeric precursors undergo intramolecular cleavage to produce multiple non-structural proteins. Because the 3CL protease is responsible for cutting at least 11 sites and is extremely important for virus replication, it is also called the main protease (M^pro), and this protease has no homologous protein in the human body.¹¹,¹² Gene sequence analysis showed that this protease is relatively conserved, and the 3CL proteases of SARS-CoV and SARS-CoV-2 are highly similar on the gene sequences and active site.¹³ Therefore, 3CL^pro is an ideal target for the development of anti-coronavirus drug.

The coronavirus 3CL^pro inhibitors reported in the literature are mainly divided into peptide inhibitors¹²-¹⁸ and non-peptide inhibitors.¹⁹-²² Commonly non-peptide inhibitors belong to reversible inhibitors, which compete for the active site of protease with natural substrates. The oxazolidinone derivatives have shown antibacterial,²³ anticoagulant,²⁴ and cholesteryl ester transfer protein inhibiting activity.²⁵ Linezolid, tedizolid, rivaroxaban, anacetrapib, and other oxazolidinone derivatives have been on the market for many years, as the safety profile has been proved. The sulfonamide hybrided oxazolidinones displayed excellent activities toward human immunodeficiency virus-1 (HIV-1) protease and significant antiviral activities.²⁶ Further, the oxazolidinone ring was designed as a novel design element for the binding interaction with active sites of norovirus 3CL^pro.²⁷ On the other hand, the aryl sulfones and sulfonamides showed the inhibitory activities against SARS-CoV 3CL^pro.²⁸ Hence, target compounds 3a-3v (Figure 1) were designed as possible inhibitors of 3CL^pro and docking analysis demonstrated that these compounds had a moderate binding affinity with 3CL^pro. Herein, we report the identification of 3g as a novel inhibitor of SARS-CoV-2 3CL protease.

Figure 1
Design of target compounds 3a-3v.

Experimental

Chemistry

Materials and methods

All chemicals were analytically pure and purchased from Shanghai Baishun Biotechnology Co., Ltd. (Shanghai, China). All melting points were determined on a WRS-1B Digital Melting Point Apparatus (uncorrected, Shanghai Precision Instruments Co., Ltd., Shanghai, China). All new compounds were characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high-resolution mass spectrometry (HRMS). NMR spectra were recorded in dimethyl sulfoxide (DMSO-d₆) on a Bruker Avance Neo 500 MHz instrument (Fällanden, Switzerland). HRMS spectra were obtained on an Agilent 1290II+6545 mass spectrometer (Waldbronn, Germany).

General procedure for the synthesis of target compounds 3a 3v

To a solution of sulfonyl chloride (1 mmol) and amine (1 mmol) in dichloromethane (12 mL) was added triethylamine (1.2 mmol, 0.1214 g). The mixture was stirred at room temperature for 5 h, and monitored by thin layer chromatography (TLC, ethyl acetate:n-hexane 2:1). After completion of the reaction, the mixture was washed three times with water, dried over anhydrous sodium sulfate, and purified by column chromatography (ethyl acetate:n-hexane 2:1) to obtain the products 3a-3v.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)benzenesulfonamide (3a)

White solid, yield: 92%; mp 157.8-159.3 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.15 (t, J 7.5 Hz, 1H), 7.84-7.82 (m, 2H), 7.68-7.64 (m, 3H), 7.49 (dd, J 2.5, 15.0 Hz, 1H), 7.16 (dd, J 2.1, 15.0 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.73 4.69 (m, 1H), 4.08 (t, J 10.0 Hz, 1H), 3.78-3.73 (m, 5H), 3.16-3.06 (m, 2H), 2.97-2.95 (m, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.9, 140.3, 135.6 (d, J 8.8 Hz), 133.4 (d, J 10.0 Hz), 132.6, 129.3, 126.5, 119.3 (d, J 3.8 Hz), 114.1 (d, J 3.8 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.2; HRMS (electrospray ionization (ESI)) m/z, calculated for C₂₀H₂₃FN₃O₅S [M + H]⁺: 436.1337, found: 436.1342; ∆ 1.1 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-4-methylbenzenesulfonamide (3b)

Off-white solid, yield: 78%; mp 149.4-150.0 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.05 (t, J 7.5 Hz, 1H), 7.70 (d, J 10.0 Hz, 2H), 7.48 (dt, J 1.1, 15.0 Hz, 1H), 7.40 (d, J 10.0 Hz, 2H), 7.16 (dd, J 5.0, 10.0 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.72-4.67 (m, 1H), 4.08-4.01 (m, 1H), 3.75-3.73 (m, 5H), 3.13-3.03 (m, 2H), 2.96 (t, J 5.0 Hz, 4H), 2.38 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.9, 142.9, 137.5, 135.6 (d, J 8.8 Hz), 133.4 (d, J 10.0 Hz), 129.7, 126.5, 119.3 (d, J 3.8 Hz), 114.1 (d, J 2.5 Hz), 106.6 (d, J 25.0 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.1, 21.0; HRMS (ESI) m/z, calculated for C₂₁H₂₅FN₃O₅S [M + H]⁺: 450.1493, found: 450.1497; ∆ 0.9 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-4-methoxybenzenesulfonamide (3c)

White solid, yield: 93%; mp 174.2-175.7 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.97 (t, J 5.0 Hz, 1H), 7.77-7.74 (m, 2H), 7.48 (dd, J 2.5, 15.0 Hz, 1H), 7.17-7.05 (m, 4H), 4.73-4.68 (m, 1H), 4.07 (t, J 10.0 Hz, 1H), 3.84 (s, 3H), 3.77-3.73 (m, 5H), 3.12-3.02 (m, 2H), 2.96 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 162.3, 154.6 (d, J 242.5 Hz), 154.0, 135.6 (d, J 8.8 Hz), 133.4 (d, J 10.0 Hz), 132.0, 128.7, 119.3 (d, J 3.8 Hz), 114.4, 114.1 (d, J 2.5 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 55.7, 50.7 (d, J 3.8 Hz), 46.8, 45.1; HRMS (ESI) m/z, calculated for C₂₁H₂₅FN₃O₆S [M + H]⁺: 466.1443, found: 466.1448; ∆ 1.1 ppm.

(R)-4-Fluoro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxo­oxazolidin-5-yl)methyl)benzenesulfonamide (3d)

White solid, yield: 79%; mp 168.0-168.8 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.18 (t, J 5.0 Hz, 1H), 7.90-7.87 (m, 2H), 7.50-7.42 (m, 3H), 7.16 (dd, J 5.0, 10.0 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.74-4.68 (m, 1H), 4.08 (t, J 10.0 Hz, 1H), 3.76-3.73 (m, 5H), 3.18-3.07 (m, 2H), 2.97 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 164.2 (d, J 248.8 Hz), 154.6 (d, J 242.5 Hz), 153.9, 136.8 (d, J 3.8 Hz), 135.6 (d, J 8.8 Hz), 133.3 (d, J 11.3 Hz), 129.5 (d, J 8.8 Hz), 119.3 (d, J 3.8 Hz), 116.4 (d, J 22.5 Hz), 114.1 (d, J 2.5 Hz), 106.6 (d, J 25.0 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₂F₂N₃O₅S [M + H]⁺: 454.1243, found: 454.1248; ∆ 1.1 ppm.

(R)-4-Chloro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxo­oxazolidin-5-yl)methyl)benzenesulfonamide (3e)

White solid, yield: 88%; mp 181.2-182.9 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.25 (t, J 5.0 Hz, 1H), 7.84-7.81 (m, 2H), 7.69-7.66 (m, 2H), 7.48 (dd, J 5.0, 15.0 Hz, 1H), 7.15 (dd, J 2.2, 8.8 Hz, 1H), 7.07 (t, J 7.5 Hz, 1H), 4.71-4.68 (m, 1H), 4.08 (t, J 10.0 Hz, 1H), 3.75-3.72 (m, 5H), 3.20-3.09 (m, 2H), 2.96 (t, J 4.6 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.9, 139.3, 137.5, 135.6 (d, J 8.8 Hz), 133.3 (d, J 11.3 Hz), 129.5, 128.5, 119.3 (d, J 3.8 Hz), 114.1 (d, J 3.8 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 3.8 Hz), 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₂ClFN₃O₅S [M + H]⁺: 470.0947, found: 470.0951; ∆ 0.9 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-4-(trifluoromethyl)benzenesulfonamide (3f)

White solid, yield: 91%; mp 179.7-181.6 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.42 (t, J 5.0 Hz, 1H), 8.01 (dd, J 7.5, 22.5 Hz, 4H), 7.49-7.46 (m, 1H), 7.15 (dd, J 5.0, 10.0 Hz, 1H), 7.07 (t, J 7.5 Hz, 1H), 4.74-4.69 (m, 1H), 4.10-4.01 (m, 1H), 3.75-3.72 (m, 5H), 3.24-3.13 (m, 2H), 2.96 (t, J 2.5 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.8, 144.4, 135.6 (d, J 8.8 Hz), 133.3 (d, J 11.3 Hz), 132.3 (q, J 32.5 Hz), 127.5, 126.5 (q, J 3.8 Hz), 123.5 (q, J 267.5 Hz), 119.3 (d, J 5.0 Hz), 114.0 (d, J 2.5 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.8, 45.2; HRMS (ESI) m/z, calculated for C₂₁H₂₂F₄N₃O₅S [M + H]⁺: 504.1211, found: 504.1216; ∆ 1.0 ppm.

(R)-3-Fluoro-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxo­oxazolidin-5-yl)methyl)benzenesulfonamide (3g)

White solid, yield: 94%; mp 166.4-166.8 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.27 (t, J 7.5 Hz, 1H), 7.70-7.61 (m, 3H), 7.56-7.51 (m, 1H), 7.48 (dd, J 5.0, 15.0 Hz, 1H), 7.16 (dd, J 5.0, 10.0 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.74-4.69 (m, 1H), 4.08 (t, J 10.0 Hz, 1H), 3.76-3.73 (m, 5H), 3.22-3.11 (m, 2H), 2.97 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 161.8 (d, J 246.3 Hz), 154.6 (d, J 242.5 Hz), 153.9, 142.5 (d, J 6.3 Hz), 135.6 (d, J 8.8 Hz), 133.3 (d, J 11.3 Hz), 131.7 (d, J 7.5 Hz), 122.7 (d, J 3.8 Hz), 119.8 (d, J 20.0 Hz), 119.3 (d, J 3.8 Hz), 114.1 (d, J 2.5 Hz), 113.6 (d, J 23.8 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₂F₂N₃O₅S [M + H]⁺: 454.1243, found: 454.1247; ∆ 0.9 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-3-(trifluoromethyl)benzenesulfonamide (3h)

White solid, yield: 76%; mp 137.8-138.7 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.38 (t, J 7.5 Hz, 1H), 8.13 (t, J 10.0 Hz, 2H), 8.06 (d, J 5.0 Hz, 1H), 7.87 (t, J 7.5 Hz, 1H), 7.48 (dd, J 2.5, 15.0 Hz, 1H), 7.15 (dd, J 2.2, 8.8 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.74-4.69 (m, 1H), 4.08 (t, J 7.5 Hz, 1H), 3.75-3.72 (m, 5H), 3.24-3.13 (m, 2H), 2.96 (t, J 4.6 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.9, 141.7, 135.6 (d, J 8.8 Hz), 133.3 (d, J 11.3 Hz), 131.0, 130.6, 130.0 (q, J 32.5 Hz), 129.4 (q, J 15.0 Hz), 123.5 (q, J 271.3 Hz), 123.1 (q, J 3.8 Hz), 119.3 (d, J 3.8 Hz), 114.1 (d, J 2.5 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.2; HRMS (ESI) m/z, for C₂₁H₂₂F₄N₃O₅S [M + H]⁺: 504.1211, found: 504.1215; ∆ 0.8 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-3-nitrobenzenesulfonamide (3i)

Yellow solid, yield: 89%; mp 179.3-179.7 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.56-8.46 (m, 3H), 8.24-8.22 (m, 1H), 7.90 (t, J 7.5 Hz, 1H), 7.45 (dd, J 5.0, 15.0 Hz, 1H), 7.13 (dd, J 5.0, 10.0 Hz, 1H), 7.05 (t, J 7.5 Hz, 1H), 4.74-4.69 (m, 1H), 4.07 (t, J 7.5 Hz, 1H), 3.75-3.70 (m, 5H), 3.28-3.17 (m, 2H), 2.96 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.8, 148.0, 142.1, 135.6 (d, J 8.8 Hz), 133.3 (d, J 10.0 Hz), 132.5, 131.4, 127.2, 121.4, 119.3 (d, J 5.0 Hz), 114.0 (d, J 3.8 Hz), 106.6 (d, J 26.3 Hz), 71.1, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₂FN₄O₇S [M + H]⁺: 481.1188, found: 481.1193; ∆ 1.0 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)-2-(trifluoromethyl)benzenesulfonamide (3j)

Off-white solid, yield: 87%; mp 102.5-104.1 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.54 (t, J 7.5 Hz, 1H), 8.11 (d, J 5.0 Hz, 1H), 7.98 (dd, J 0.8, 7.7 Hz, 1H), 7.91-7.88 (m, 1H), 7.84 (t, J 7.5 Hz, 1H), 7.47 (dd, J 5.0, 15.0 Hz, 1H), 7.15 (dd, J 5.0, 7.5 Hz, 1H), 7.07 (t, J 10.0 Hz, 1H), 4.76-4.71 (m, 1H), 4.09 (t, J 10.0 Hz, 1H), 3.79-3.73 (m, 5H), 3.34-3.24 (m, 2H), 2.97 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.6 (d, J 242.5 Hz), 153.9, 139.5 (d, J 1.3 Hz), 135.6 (d, J 8.8 Hz), 133.4, 133.36 (d, J 11.3 Hz), 133.0, 129.8, 128.5 (q, J 6.3 Hz), 126.1 (q, J 32.5 Hz), 122.9 (q, J 272.5 Hz), 119.3 (d, J 3.8 Hz), 114.1 (d, J 3.8 Hz), 106.6 (d, J 26.3 Hz), 71.2, 66.2, 50.7 (d, J 2.5 Hz), 46.9, 45.5; HRMS (ESI) m/z, calculated for C₂₁H₂₂F₄N₃O₅S [M + H]⁺: 504.1211, found: 504.1214; ∆ 0.6 ppm.

(R)-N-((3-(3-Fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl)methyl)quinoline-8-sulfonamide (3k)

White solid, yield: 96%; mp 100.2-100.8 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 9.04-9.03 (m, 1H), 8.52-8.50 (m, 1H), 8.34-8.27 (m, 2H), 7.77-7.65 (m, 3H), 7.35-7.32 (m, 1H), 7.04-7.00 (m, 2H), 4.71-4.66 (m, 1H), 3.98 (t, J 7.5 Hz, 1H), 3.76-3.73 (m, 5H), 3.34-3.25 (m, 2H), 2.96 (t, J 5.0 Hz, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 154.5 (d, J 241.3 Hz), 153.7, 151.3, 142.6, 137.0, 136.6, 135.5 (d, J 8.8 Hz), 133.7, 133.3 (d, J 10.0 Hz), 130.5, 128.6, 125.7, 122.5, 119.2 (d, J 5.0 Hz), 113.9 (d, J 3.8 Hz), 106.5 (d, J 25.0 Hz), 71.3, 66.2, 50.7 (d, J 2.5 Hz), 46.8, 45.5; HRMS (ESI) m/z, calculated for C₂₃H₂₄FN₄O₅S [M + H]⁺: 487.1446, found: 487.1451; ∆ 1.0 ppm.

(R)-N-((2-Oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5 yl)methyl)benzenesulfonamide (3l)

White solid, yield: 90%; mp 158.5-159.3 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.16 (t, J 7.5 Hz, 1H), 7.83 (d, J 10.0 Hz, 2H), 7.68-7.54 (m, 5H), 7.43-7.40 (m, 2H), 4.75-4.70 (m, 1H), 4.20 (s, 2H), 4.12 (t, J 7.5 Hz, 1H), 3.98-3.96 (m, 2H), 3.83-3.79 (m, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.18-3.08 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 154.0, 140.4, 137.1, 136.5, 132.6, 129.3, 126.5, 126.0, 118.3, 71.2, 67.8, 63.5, 49.1, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₂N₃O₆S [M + H]⁺: 432.1224, found: 432.1231; ∆ 1.6 ppm.

(R)-4-Methyl-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3m)

White solid, yield: 87%; mp 173.1-174.2 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.06 (t, J 7.5 Hz, 1H), 7.71 (d, J 5.0 Hz, 2H), 7.56-7.53 (m, 2H), 7.43-7.39 (m, 4H), 4.74-4.69 (m, 1H), 4.20 (s, 2H), 4.11 (t, J 10.0 Hz, 1H), 3.98-3.96 (m, 2H), 3.81-3.78 (m, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.15-3.04 (m, 2H), 2.38 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 154.0, 142.9, 137.5, 137.1, 136.5, 129.7, 126.5, 126.0, 118.3, 71.2, 67.8, 63.5, 49.1, 46.9, 45.2, 21.0; HRMS (ESI) m/z, calculated for C₂₁H₂₄N₃O₆S [M + H]⁺: 446.1380, found: 446.1385; ∆ 1.1 ppm.

(R)-4-Methoxy-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3n)

Off-yellow solid, yield: 90%; mp 142.2-143.4 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 7.98 (t, J 5.0 Hz, 1H), 7.77-7.74 (m, 2H), 7.57-7.54 (m, 2H), 7.43-7.40 (m, 2H), 7.14-7.11 (m, 2H), 4.74-4.70 (m, 1H), 4.20 (s, 2H), 4.12 (t, J 10.0 Hz, 1H), 3.98-3.96 (m, 2H), 3.84 (s, 3H), 3.82-3.79 (m, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.14-3.03 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 162.3, 154.0, 137.1, 136.5, 132.0, 128.7, 126.0, 118.3, 114.4, 71.2, 67.8, 63.5, 55.7, 49.0, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₁H₂₄N₃O₇S [M + H]⁺: 462.1329, found: 462.1336; ∆ 1.5 ppm.

(R)-4-Fluoro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3o)

White solid, yield: 86%; mp 176.2-177.6 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.20 (t, J 7.5 Hz, 1H), 7.89 (t, J 5.0 Hz, 2H), 7.57-7.54 (m, 2H), 7.47-7.40 (m, 4H), 4.75-4.70 (m, 1H), 4.20 (s, 2H), 4.13 (t, J 10.0 Hz, 1H), 3.98-3.96 (m, 2H), 3.82-3.80 (m, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.20-3.09 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 164.2 (d, J 248.8 Hz), 154.0, 137.1, 136.8 (d, J 3.8 Hz), 136.5, 129.5 (d, J 10.0 Hz), 126.0, 118.3, 116.5 (d, J 22.5 Hz), 71.2, 67.8, 63.5, 49.1, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₁FN₃O₆S [M + H]⁺: 450.1130, found: 450.1135; ∆ 1.1 ppm.

(R)-4-Chloro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3p)

White solid, yield: 91%; mp 187.4-188.9 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.19 (t, J 7.5 Hz, 1H), 7.77-7.74 (m, 2H), 7.62-7.60 (m, 2H), 7.49-7.46 (m, 2H), 7.35-7.33 (m, 2H), 4.68-4.63 (m, 1H), 4.12 (s, 2H), 4.05 (t, J 7.5 Hz, 1H), 3.90 (t, J 5.0 Hz, 2H), 3.72 (dd, J 5.0, 10.0 Hz, 1H), 3.64 (t, J 5.0 Hz, 2H), 3.13-3.02 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 154.0, 139.3, 137.5, 137.1, 136.5, 129.5, 128.5, 126.0, 118.3, 71.2, 67.8, 63.5, 49.1, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₁C_lN₃O₆S [M + H]⁺: 466.0834, found: 466.0839; ∆ 1.1 ppm.

(R)-N-((2-Oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)-4-(trifluoromethyl)benzenesulfonamide (3q)

White solid, yield: 73%; mp 171.3-172.1 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.43 (t, J 10.0 Hz, 1H), 8.02 (q, J 10.0 Hz, 4H), 7.55 (d, J 10.0 Hz, 2H), 7.42 (d, J 5.0 Hz, 2H), 4.76-4.71 (m, 1H), 4.20 (s, 2H), 4.13 (t, J 7.5 Hz, 1H), 3.98 (t, J 5.0 Hz, 2H), 3.81-3.78 (m, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.26-3.14 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 153.9, 144.4, 137.1, 136.5, 132.3 (q, J 32.1 Hz), 127.5, 126.6 (q, J 3.8 Hz), 126.0, 123.5 (q, J 271.3 Hz), 118.3, 71.2, 67.8, 63.5, 49.1, 46.8, 45.2; HRMS (ESI) m/z, calculated for C₂₁H₂₁F₃N₃O₆S [M + H]⁺: 500.1098, found: 500.1103; ∆ 1.0 ppm.

(R)-3-Fluoro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3r)

White solid, yield: 83%; mp 172.9-173.6 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.29 (t, J 5.0 Hz, 1H), 7.70-7.61 (m, 3H), 7.56-7.50 (m, 3H), 7.43-7.40 (m, 2H), 4.75-4.71 (m, 1H), 4.20 (s, 2H), 4.13 (t, J 10.0 Hz, 1H), 3.98-3.96 (m, 2H), 3.80 (dd, J 5.0, 10.0 Hz, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.24-3.12 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 161.8 (d, J 246.3 Hz), 154.0, 142.5 (d, J 6.3 Hz), 137.1, 136.5, 131.7 (d, J 7.5 Hz), 126.0, 122.7 (d, J 10.0 Hz), 119.8 (d, J 20.0 Hz), 118.3, 113.6 (d, J 23.8 Hz), 71.2, 67.8, 63.5, 49.1, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₁FN₃O₆S [M + H]⁺: 450.1130, found: 450.1134; ∆ 0.9 ppm.

(R)-N-((2-Oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)-3-(trifluoromethyl)benzenesulfonamide (3s)

Off-yellow solid, yield: 80%; mp 158.4-159.5 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.40 (t, J 5.0 Hz, 1H), 8.14 (d, J 5.0 Hz, 2H), 8.06 (d, J 5.0 Hz, 1H), 7.87 (t, J 7.5 Hz, 1H), 7.56-7.53 (m, 2H), 7.43-7.40 (m, 2H), 4.76-4.72 (m, 1H), 4.20 (s, 2H), 4.14 (t, J 10.0 Hz, 1H), 3.99-3.97 (m, 2H), 3.80 (dd, J 5.0, 10.0 Hz, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.26-3.15 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 154.0, 141.7, 137.1, 136.5, 131.0, 130.6, 130.0 (q, J 32.5 Hz), 129.4 (q, J 3.8 Hz), 126.0, 123.5 (q, J 271.3 Hz), 123.1 (q, J 3.8 Hz), 118.3, 71.2, 67.8, 63.5, 49.1, 46.9, 45.2; HRMS (ESI) m/z, calculated for C₂₁H₂₁F₃N₃O₆S [M + H]⁺: 500.1098, found: 500.1103; ∆ 1.0 ppm.

(R)-3-Nitro-N-((2-oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)benzenesulfonamide (3t)

Off-yellow solid, yield: 82%; mp 137.7-138.8 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.49 (t, J 1.9 Hz, 1H), 8.45 (t, J 5.0 Hz, 1H), 8.41-8.39 (m, 1H), 8.17-8.15 (m, 1H), 7.83 (t, J 7.5 Hz, 1H), 7.47-7.44 (m, 2H), 7.35-7.32 (m, 2H), 4.68-4.63 (m, 1H), 4.13 (s, 2H), 4.05 (t, J 7.5 Hz, 1H), 3.90 (t, J 5.0 Hz, 2H), 3.70 (dd, J 5.0, 10.0 Hz, 1H), 3.65 (t, J 5.0 Hz, 2H), 3.22-3.10 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 153.9, 148.0, 142.1, 137.1, 136.4, 132.5, 131.4, 127.2, 126.0, 121.4, 118.2, 71.1, 67.8, 63.5, 49.1, 46.8, 45.2; HRMS (ESI) m/z, calculated for C₂₀H₂₁N₄O₈S [M + H]⁺: 477.1075, found: 477.1080; ∆ 1.0 ppm.

(R)-N-((2-Oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5-yl)methyl)-2-(trifluoromethyl)benzenesulfonamide (3u)

White solid, yield: 93%; mp 127.6-128.9 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (t, J 7.5 Hz, 1H), 8.11 (d, J 5.0 Hz, 1H), 7.98 (dd, J 2.5, 7.5 Hz, 1H), 7.91-7.88 (m, 1H), 7.84 (t, J 7.5 Hz, 1H), 7.56-7.53 (m, 2H), 7.43-7.40 (m, 2H), 4.78-4.73 (m, 1H), 4.20 (s, 2H), 4.14 (t, J 10.0 Hz, 1H), 3.99-3.97 (m, 2H), 3.83 (dd, J 5.0, 10.0 Hz, 1H), 3.72 (t, J 5.0 Hz, 2H), 3.34-3.26 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 153.9, 139.5, 137.1, 136.5, 133.4, 133.0, 129.8, 128.5 (q, J 6.3 Hz), 126.1 (q, J 32.5 Hz), 126.0, 122.9 (q, J 272.5 Hz), 118.3, 71.2, 67.8, 63.5, 49.1, 46.9, 45.5; HRMS (ESI) m/z, calculated for C₂₁H₂₁F₃N₃O₆S [M + H]⁺: 500.1098, found: 500.1101; ∆ 0.6 ppm.

(R)-N-((2-Oxo-3-(4-(3-oxomorpholino)phenyl)oxazolidin-5 yl)methyl)quinoline-8-sulfonamide (3v)

White solid, yield: 75%; mp 222.4-224.1 °C; ¹H NMR (500 MHz, DMSO-d₆) δ 8.96 (dd, J 1.7, 4.2 Hz, 1H), 8.43 (dd, J 1.7, 8.4 Hz, 1H), 8.26 (dd, J 1.2, 7.3 Hz, 1H), 8.20 (dd, J 1.2, 8.3 Hz, 1H), 7.69-7.64 (m, 2H), 7.59 (q, J 4.2 Hz, 1H), 7.36-7.28 (m, 4H), 4.65-4.60 (m, 1H), 4.13 (s, 2H), 3.95 (t, J 10.0 Hz, 1H), 3.91-3.89 (m, 2H), 3.72 (dd, J 5.0, 10.0 Hz, 1H), 3.64 (t, J 5.0 Hz, 2H), 3.26-3.18 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.0, 153.8, 151.3, 142.6, 137.1, 137.0, 136.6, 136.4, 133.7, 130.5, 128.6, 125.9, 125.7, 122.5, 118.1, 71.3, 67.8, 63.5, 49.1, 46.8, 45.5; HRMS (ESI) m/z, calculated for C₂₃H₂₃N₄O₆S [M + H]⁺: 483.1333, found: 483.1337; ∆ 0.8 ppm.

Biological assays

Materials

The fluorescent intensity was monitored by envision multi-mode reader (PerkinElmer, Llantrisant, United Kingdom). SARS-COV-2 3CL^pro inhibitor screening kit was purchased from Xiamen Lablead Biotechnology Co., Ltd. (Xiamen, China). All chemicals were analytically pure and purchased from commercial sources (Shanghai Baishun Biotechnology Co., Ltd., Shanghai, China).

3CL^pro inhibition rates

The 3CL^pro enzyme assay was conducted in 384-well black microplates (PerkinElmer, Boston, USA) with a total volume of 41 μL. In a 384-well plate format, 20 μL enzyme in reaction buffer was added into each well, followed by the addition of 1 μL target compound (different concentrations dissolved in DMSO). SARS-COV-2 3CL^pro and compound were mixed and pre-incubated at 30 °C for 10 min. Fluorescent intensity was measured at different time points on a plate reader with λ_Ex = 320 nm and λ_Em = 405 nm after the addition of 20 μL substrate with the cleavage site of M^pro (indicated by the arrow, MCA-AVLQ↓SGFR-K(Dnp)K). The experiment was conducted at room temperature (RT).

First, 0.2 μM (final concentration) SARS-CoV-2 3CL^pro and different concentrations of substrates (2.5 100 μM) were mixed and then the analysis was started. The fluorescent intensity was monitored by envision multi-mode reader. The kinetic parameters Michaelis constant (K_m) and catalytic constant (K_cat) were calculated by a double reciprocal diagram. Then all compounds with a concentration of 50 μM were screened. When different compounds were added to the mixture, the change of initial rate was calculated to evaluate the inhibitory effect of the compounds. The specific methods are as follows: (i) 20 μL enzyme was incubated with 1 μL compound (2 mM) at 30 °C for 10 min, and the substrate was added rapidly, the final concentration of substrate was 20 μM; (ii) the absorbance was measured by excitation at 320 nm and emission at 405 nm for 10 min; (iii) plot of the growth value of time and excitation light relative to 0 time, slope calculation, and comparison with the slope without compound, which is the inhibition rate of a drug on enzyme activity.

Half maximal inhibitory concentration (IC₅₀) of 3CL^pro inhibition

For the determination of the IC₅₀, 0.1 mM SARS CoV-2 3CL^pro was incubated with compounds at various concentrations (0.048-50 μM) in an assay buffer at 30 °C for 10 min. Afterward, the reaction was initiated by adding the substrate at a 20 μM final concentration (final well volume: 41 μL). The IC₅₀ value for 3g was determined using the GraphPad Prism software.²⁹

Docking analysis

Schrödinger software (version 2017-1, Maestro 9.3)³⁰ was employed in our molecular docking study. The crystal structure of the SARS-COV-2 3CL^pro complex (PDB ID: 6LU7) was retrieved from the Protein Data Bank.³¹ The protein 3CL^pro was prepared with the Protein Preparation Wizard panel.³² The glide grid center was set according to the geometrical center of native ligand N3 and the grid size was 10 × 10 × 10 ų. The native ligand and the active compound 3g were prepared using the LigPrep suite.³⁰ Then, they were docked into the active site of 3CL^pro using the induced-fit docking protocol in Schrodinger Maestro 9.3.³⁰ All of docking results were processed by Maestro 9.3³⁰ or PyMOL software.³³ For the native ligand N3, the root-mean-square deviation (RMSD) value of the docked pose referred to crystal pose was 1.9374 Å, which implicated this dock process was enough reliable.

Results and Discussion

Chemistry

Target compounds 3a-3v were prepared by condensation of sulfonyl chlorides with linezolid/rivaroxaban amines (Scheme 1). Although some of the starting materials are dissolved in DMSO rather than methylene chloride, the yield in DMSO is much lower than in methylene chloride, not more than 40% in general, therefore the condensation was carried out in methylene chloride. The structures of target compounds 3a-3v were characterized by ¹H NMR, ¹³C NMR, and HRMS. When linezolid amine was used as the starting material (R₂ = F), the carbon atom of 3-CH₂ of morpholine ring appears as a doublet at around 50.7 ppm (J 2.5 Hz), this signal splitting is due to the through-space coupling with the space adjacent fluorine atom.³⁴

Scheme 1
Synthesis of target compounds 3a-3v.

3CL^pro inhibitory activities

In this study, it was evaluated the inhibition activities of synthesized compounds against SARS-CoV-2 3CL^pro using a procedure mentioned earlier.²⁹ The inhibition rates of target compounds 3a-3v at 50 μM against SARS-CoV-2 3CL^pro activity are shown in Table 1. All synthesized compounds at 50 μM exhibited potent inhibition on 3CL^pro activity. Especially, compounds 3d, 3g, and 3h at 50 μM displayed remarkable inhibitory activity of SARS CoV 2 3CL protease, with inhibition rates of 92.2, 99.5, and 89.3%, respectively. According to these data in Table 1, we attempted to establish the preliminary SARs of these novel oxazolidinone derivatives. In general, compounds 3a-3k with R₂ = F and X = CH₂ displayed more inhibitory effect than compounds 3l-3v with R₂ = H and X = CO. In the target compounds 3a-3j which contain mono-substituted phenyl ring (R₁), the type and substituted position of substitutes displayed an important relationship with the inhibitory activity. When the moiety R₁ was the para-mono-substituted phenyl, the introduction of a halogen atom caused a great increase in 3CL^pro inhibition. For example, both 3d (R₁ = 4-fluorophenyl) and 3e (R₁ = 4-chlorine phenyl) showed better inhibitory activity than 3a (R₁ = phenyl), while compounds 3b (R₁ = 4-methylphenyl) and 3c (R₁ = 4-methoxyphenyl) exhibited a similar inhibition activity with 3a (R₁ = phenyl), suggesting that electron donating groups substituted at phenyl ring could not improve the 3CL^pro inhibition. Additionally, 3g (R₁ = 3-fluorophenyl) had a better inhibitor activity than 3d (R₁ = 4-fluorophenyl), and 3h (R₁ = 3-(trifluoromethyl)phenyl) had a better inhibitor activity than 3f (R₁ = 4-(trifluoromethyl)phenyl) and 3j (R₁ = 2-(trifluoromethyl)phenyl), which implied that meta substitution was helpful for inhibitory activity compared with the ortho- and para- substitutions. Finally, compound 3k which had a quinolyl group at R₁ retained moderate inhibitory activity.

Table 1
Inhibition rates of target compounds 3a-3v at 50 μM on 3CL^pro activity

ID	Structure	Inhibition rate		ID	Structure	Inhibition rate
3a		48.5		3l		58.4
3b		43.9		3m		50.8
3c		55.3		3n		60.2
3d		92.2		3o		51.6
3e		78.2		3p		66.3
3f		58.1		3q		72.3
3g		99.5		3r		71.7
3h		89.3		3s		70.2
3i		59.8		3t		61.0
3j		76.4		3u		65.9
3k		62.5		3v		57.9

At the concentration of 50 μM, compound 3g was found to be the most potent SARS-CoV-2 3CL^pro inhibitor among all synthesized compounds. So, the 3CL^pro inhibitory activities of 3g at variable concentrations were further evaluated. As shown in Figure 2a, the inhibition of SARS CoV-2 3CL^pro by 3g slowly increased in the concentration range from 0 to 50 M of compound 3g. It suggested that 3g could enter into the active pocket of enzyme SARS-CoV-2 3CL^pro and inhibit its activity in a concentration dependent manner. Also, the IC₅₀ value of compound 3g in inhibiting the catalytic activity of SARS CoV-2 3CL protease was calculated by the dose dependent inhibitory curves of 3g. Compound 3g was confirmed to have a prominent inhibitory activity, with an IC₅₀ value of 14.47 μM.

Figure 2
(a) Enzyme activities were measured using fluorogenic substrate (20 μM) in the presence of compound 3g (0-50 μM). Over the entire 600 s time window, the inhibited enzyme with a different concentration of inhibitor showed a time-dependent reduction of activity; (b) inhibitory activity profiles of compound 3g against SARS-CoV-2 3CL^pro.

Docking analysis of compound 3g with 3CL^pro

To predict the possible binding mode of compound 3g to the active site of SARS-CoV-2 3CL^pro, docking analysis was performed using the induced-fit docking protocol in Schrodinger Maestro 9.3.³⁰ The docking study revealed that compound 3g exhibited a moderate binding affinity with SARS-CoV-2 3CL^pro, with a docking score of -6.317 kcal mol^-1 for the first top-scored docking mode shown in Figure 3. As shown in Figure 3a, compound 3g was well-fitted into the active pocket of SARS CoV 2 3CL^pro. The morpholine ring of 3g was inside the drug binding pocket of 3CL^pro. The five membered ring of 3g located near the solvent exposure area made close contact with SARS CoV-2 3CL^pro. Moreover, the hydrogen atom of sulfonamide of 3g formed a hydrogen bond (2.14 Å) with Glu166 (Figure 3b). These molecular docking results further supported that 3g was a potential SARS CoV 2 3CL^pro inhibitor.

Figure 3
(a) Surface presentation of the docking mode of 3CL^pro complex with 3g (PDB ID: 6LU7); (b) 2D binding model of 3g with 3CL^pro. Hydrogen bond is indicated with purple solid arrows, color lines around 3g stand for the binding pocket and the residues in colors nearby established the pocket. The green denotes the hydrophobic nature of amino acids, the red denotes the acid amino acids, the purple denotes the alkalinity of amino acids, the cyan denotes the polar amino acids, and the grey points of ligand atoms denote the solvent accessibility.

Conclusions

In summary, a total of 22 oxazolidinone derivatives 3a 3v were synthesized and their inhibitory activities against SARS-CoV-2 3CL^pro were screened. Compound 3g displayed the most potent inhibitory activity with an IC₅₀ value of 14.47 μmol L^-1. Moreover, molecular docking analysis showed the modes of action of compound 3g with 3CL^pro, indicating that the hydrogen bond interaction plays a key role in stabilizing 3g in the binding pocket. Taken together, compound 3g may be a valuable lead compound for further development of the effective SARS-CoV-2 3CL^pro inhibitors.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This research was funded by the Natural Science Foundation of Ningbo (grant No. 202003N4160), Key Research Projects on Emergency Prevention and Control of New Coronavirus Infected Pneumonia from Jinhua Science and Technology Bureau (2020XG-11) and sponsored by K. C. Wong Magna Fund in Ningbo University.

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Edited by

Editor handled this article: Brenno A. D. Neto (Associate)

Publication Dates

• Publication in this collection
  26 Aug 2022
• Date of issue
  Sept 2022

History

• Received
  13 Dec 2021
• Published
  21 Feb 2022