Dihydropyrimidine-2-thione derivatives as SARS-CoV-2 main protease inhibitors: synthesis, SAR and in vitro profiling

Abstract

Despite the passage of approximately five years since the outbreak, an efficacious remedy for SARS-CoV-2 remains elusive, highlighting the urgent imperative for developing SARS-CoV-2 potent inhibitors. In our current study, we have unmasked the hitherto unrealized potential of dihydropyrimidine-2-thiones against the Main Protease (M^pro) of SARS-CoV-2. Employing a predictive docking tool, we identified promising lead compounds and optimized them via comprehensive Structural Activity Relationship (SAR) studies. Key design elements included proton donor/acceptor groups, six-membered rings, and fluorinated moieties to enhance interactions. These leads underwent in vitro inhibition assays to enhance their interaction with key M^pro amino acid residues. Our findings indicated that all synthesized compounds exhibited significant inhibition of the M^pro. Compounds 12j (IC₅₀ = 0.063 μM), and 12l (IC₅₀ = 0.054 μM) displayed exceptional in vitro binding affinities. In addition to their string inhibitory activity, CC₅₀ values were assessed, confirming acceptable cytotoxicity profiles for potent compounds. Molecular dynamic simulation substantiated the binding mechanism revealing that compound 12l maintains robust stability with the target protein. Furthermore, compounds predicted to have minimal oral toxicity and high intestinal absorption make them promising candidates for drug development. These findings paved the way for the potent clinical application of these dihydropyrimidine-2-thiones as efficient SARS-CoV-2 therapeutics.

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Introduction

COVID-19 is caused by a deadly Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) that targets the human respiratory system. Coronaviruses such as NL63, OC43, 229E, and HKU1, have generally caused mild respiratory infections like the common cold in humans.^1–5 However, highly pathogenic strains like SARS-CoV-1 (2003), MERS-CoV (2012), and SARS-CoV-2 (2019) have emerged in the past 20 years, posing severe threats to public health. The first SARS-CoV-2 outbreak, reported in Wuhan, China, in 2019, signified the beginning of a global pandemic. SARS-CoV-2, a positive-sense, single-stranded RNA virus within the mammalian beta-coronavirus genus causes respiratory and lung damage leading to potentially fatal illnesses.⁶ On March 11, 2020, the World Health Organization (WHO) declared COVID-19, a global pandemic, due to the rapid spread of the virus and the rising mortality rate.^7,8 As of November 25, 2024, WHO reported 776.41 million confirmed cases, 7.075 million deaths, and over 13.64 billion vaccinations globally. Notably, SARS-CoV-2 is among the most infectious virus in the coronavirus family impacting both human and animal populations.^8,9

SARS-CoV-2 encodes 16 non-structural proteins (NSPs), key structural proteins, membrane (M), nucleocapsid (N), and spike (S) and a Main Protease (M^pro) or 3C-like protease (3CL^pro), Papain Like Protease (PL^pro), and RNA-dependent RNA polymerase (RdRp).^10–13 The viral genome, approximately 30 000 nucleotides long, contains a 5′-cap structure, a 3′-poly (A) tail, and multiple open reading frames (ORFs). NSPs possess PL^pro and M^pro which play a crucial role in viral replication. M^pro, comprised of three domains (I–III) and a unique Cys–His dyad active site, is highly conserved across coronaviruses, highlighting its potential as a target for antivirals.^10,14–18 With S1, S1′, S2, and S3 forming the active binding pocket for substrate engagement, M^pro facilitates viral protein maturation through peptide bond cleavage, a process validated by mixed quantum mechanics/molecular mechanics (QM/MM) simulations.^19,20 Due to its essential function in the viral life cycle and lack of homologs in humans, M^pro is an ideal candidate for therapeutic intervention and drug design.^21–23 Fig. 1 illustrates the M^pro's role in viral replication, making protease inhibition a prime target for antiviral drug development.

Fig. 1 SARS-CoV-2 M^pro inhibition and mechanism of action.

Currently, FDA-approved COVID-19 inhibitors (Fig. 2) are limited. However, PF-07321332 (nirmatrelvir, K_i = 0.003 μM) (1a), demonstrates safety, selectivity, and high antiviral activity (EC₅₀ of 0.074 μM).^4,24 To counteract its rapid metabolism by CYP3A, the HIV protease inhibitor ritonavir (1b) was added, forming Paxlovid, which received FDA approval in 2021 for treating mild to moderate COVID-19. Past studies have identified effective M^pro inhibitors, such as PF-00835231 (1c) during the SARS-CoV-1 outbreak in 2003.²⁵ In 2021, Pfizer developed an oral SARS-CoV-2 M^pro inhibitor. Japan approved ensitrelvir (S-217622) (1d) in 2022, which showed potent M^pro inhibition with IC₅₀ = 0.013 μM and EC₅₀ = 0.37 μM.^26,27 Several other M^pro inhibitors (Fig. 3), including lufotrelvir (2a), ebselen (2b), and masitinib (2c) are in clinical trials while repurposed drugs like boceprevir (IC₅₀ = 4.13 μM) also exhibit notable inhibitory effects.^28–32

Fig. 2 FDA-approved medication for the treatment of SARS-CoV-2 (1a–d).

Fig. 3 Medication in clinical trials for the treatment of SARS-CoV-2 (2a–c).

This work seeks to identify novel dihydropyrimidine-2-thione-based inhibitors with favourable pharmacokinetic properties to combat SARS-CoV-2 effectively by focusing on the design of dihydropyrimidine-2-thione-based derivatives targeting SARS-CoV-2 M^pro, aiming to maximize scaffold inhibitory potency. Structure-Based Drug Design (SBDD) techniques were used to optimize and evaluate these scaffolds.³³ The sulphur-rich dihydropyrimidine-2-thione has shown antiviral, antibacterial, and potential anticancer activities. It disrupts viral replication and hosts immune responses.^34–36 Notably, dihydropyrimidine derivatives such as batzelladine A & B exhibit antiviral properties, while monastrol is known for anticancer activity.^37,38 The antihypertensive drug terazosin exemplified its diverse biological activities.³⁹

Materials and methods

General

Solvents and reagents used for current work were purchased from commercial sources and were used without purification. Compounds include substituted aromatic aldehydes (6a–c), thiourea, ethyl 4,4,4-trifluoro-3-oxobutanoate, diverse primary amines (L-glutamic acid (7a), L-aspartic acid (7b), sulfanilic acid (7c), L-histidine (7d), L-tyrosine (7e)), bromoacetyl bromide and diverse secondary amines (11a–c) were purchased from Sigma-Aldrich. 400 MHz/100 MHz Bruker NMR spectrometer was used to record ¹H and ¹³C NMR spectra respectively in the DMSO solvent. The solvent was used as an internal reference in NMR analysis. Chemical shifts in NMR analysis are recorded in δ scale part per million (ppm). Thin Layer Chromatography (TLC) was used to monitor the progress of all reactions for current research work on 2 × 5 cm precoated aluminum sheets with silica gel (60-F254), the coating layer thickness specification was 0.25 mm (Merck). Liquid chromatography-mass spectrometry (LC-MS) was performed using Agilent Technologies 1200 series high-performance liquid chromatography system with C18 reversed-phase column (particle size: 3.5 μm, length: 100 mm, internal diameter: 4.6 mm, vendor: Agilent Technologies). Elemental analyses were conducted using an Elemental Vario EI III CHN analyzer. Elemental analysis (±0.4% of the calculated values) was performed for all the tested compounds. Final products were checked for their purity on a HPLC system using a C18 RP column (particle size: 5 μm, length: 150 mm, internal diameter: 4.6 mm, vendor: Shimadzu) and an isocratic solvent system (mentioned in the experimental part) at room temperature. Biologically screened compounds are >95% pure as determined by HPLC.

General method for the synthesis of compounds (6a–c)

Dihydropyrimidine-2-thiones (6a–c) core was synthesized by 10 mmol aldehydes (4-(trifluoromethyl)benzaldehyde (3a), 4-nitrobenzaldehyde (3b) and methyl 4-formyl benzoate (3c)) reaction with thiourea (4) (10 mmol) and ethyl 4,4,4-trifluoro-3-oxobutanoate (5) (12 mmol) through classical multicomponent Biginelli approach using acetonitrile solvent under reflux for 6 h's in the presence of SnCl₂·2H₂O. Reaction was monitored by TLC, after completion of the reaction the reaction mixture was poured into ice-cold distilled water and allowed to stir for 5 minutes. Precipitates formed, filtered off, dried, washed with cold ethanol, and recrystallized with ethanol to afford pure dihydropyrimidine-2-thiones (6a–c).

General method for the synthesis of compounds (8a–h)

Compounds (8a–h) are synthesized by the reaction of the substituted dihydropyrimidine-2-thiones (20 mmol, 6a–c) with diverse primary amines (25 mmol, 7a–e) in the presence of K₂CO₃ base (1.2 equivalent) in a solvent dimethylformamide (DMF) on heating. The reaction was monitored by TLC, after completion, the reaction mixture was poured into ice water. The precipitates were filtered and washed with cold water and purified with the help of column chromatography (hexane : ethyl acetate 9 : 1 system) to afford pure compounds (8a–h). Characterization data of intermediate compounds is presented in the ESI† file.

General method for the synthesis of compounds (10a–h)

To the stirred solution of (5 mmol) synthesized dihydropyrimidine-thione amide derivatives (8a–h) in 10 mL acetone solvent, at room temperature, and K₂CO₃ as base and bromoacetyl bromide (9) (7.5 mmol) was added dropwise with continuous stirring. The reaction was monitored by TLC and stirring continued for 4 hours. At the end of the reaction precipitated solid was obtained which was further recrystallized by dioxane : ethanol (1 : 1) to furnish S-acetylated product/compounds (10a–h). Characterization data of intermediate compounds is presented in the ESI† file.

General method for the synthesis of compounds (12a–l)

Diverse secondary amines (5 mmol) (11a–c) along with K₂CO₃ base (1.5 eq.) were taken in DMF (10 mL). To this stirred solution previously synthesized S-bromoacylated products (10a–h) (7.5 mmol) were added. The reaction was monitored by TLC. Stirring continued till the completion of the reaction. Water was added after completion of the reaction to afford precipitation of the products (12a–l). In some products, precipitation was not observed. If precipitates were not formed product was not extracted in the organic layer. Later on, all products were purified through column chromatography.

(6-(Trifluoromethyl)-2-((2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]-pyrazin-7(8H)-yl)acetyl)thio)-4-(4 (trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-glutamic acid (12a)

Light yellow solid, yield = 48%, m.p. 199–201 °C; R_f = 0.47; (n-hexane/EA; 3 : 1); HPLC purity = 97.5% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 12.1 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.39 (s, 1H, COOH), 11.75 (s, 1H, COOH), 9.38 (s, 1H, py-NH), 8.41 (d, J = 4.72 Hz, –NH), 7.81 (d, J = 8.52 Hz, –2H, ArH), 7.27 (d, J = 8.52 Hz, –2H, ArH), 5.46 (s, 1H, –CH), 4.49–4.45 (m, 1H, glu), 4.26 (t, J = 5.92 Hz, 2H, pip-CH₂), 3.83 (s, 2H, CH₂), 3.28 (s, 2H, pip-CH₂), 3.11 (t, J = 5.92 Hz, 2H, pip-CH₂), 2.27–2.19 (m, 2H, glu), 2.03–1.93 (m, 2H, glu). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.5, 174.1, 173.6, 162.6, 147.6, 139.7 (q, J = 36.0 Hz, –C–CF₃), 133.8 (q, J = 32.49 Hz, –C–CF₃), 131.7 (q, J = 32.12 Hz, –C–CF₃), 128.1 (2C), 126.9, 125.9, 124.2, 123.4, 122.4, 122.2 (q, J = 269 Hz, –CF₃), 119.9, 116.2, 110.7, 64.8, 59.6, 57.7, 48.4, 41.5, 32.2, 23.4. Analysis calculated for C₂₆H₂₂F₉N₇O₆S; C, 42.69; H, 3.03; F, 23.37; N, 13.40; O, 13.12; S, 4.38; found C, 42.56; H, 3.04; N, 13.42; LCMS: m/z = 732.1 [M + H]⁺.

(6-(Trifluoromethyl)-2-((2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]-pyrazin-7(8H)-yl)acetyl)thio)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-aspartic acid (12b)

White solid, yield = 65%, m.p. 204–206 °C; R_f = 0.48; (n-hexane/EA; 3 : 1); HPLC purity = 98.6% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 11.9 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.46 (s, 1H, COOH), 11.81 (s, 1H, COOH), 9.36 (s, 1H, py-NH), 8.40 (d, J = 6.08 Hz, –NH), 7.81 (d, J = 8.36 Hz, –2H, ArH), 7.27 (d, J = 8.36 Hz, –2H, ArH), 5.46 (s, 1H, –CH), 4.44–4.39 (m, 1H, glu), 4.22 (t, J = 5.8 Hz, 2H, pip-CH₂), 3.83 (s, 2H, CH₂), 3.53–3.45 (m, 2H, pip-CH₂), 3.28 (s, 2H, pip-CH₂), 3.10 (t, J = 5.8 Hz, 2H, glu). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.7, 173.5, 172.9, 164.1, 148.8, 144.9, 142.4, 140.5, 140.2, 132.7, 132.9 (q, J = 36.7 Hz, –C–CF₃), 132.8 (q, J = 32 Hz, –C–CF₃), 131.6 (q, J = 36.5 Hz, –C–CF₃), 127.5, 125.7, 125.2, 124.3, 123.6, 122.4 (q, J = 274 Hz, –CF₃), 117.7, 115.6, 90.7, 61.3, 57.2, 52.8, 50.5, 49.8, 42.7, 36.2. Analysis calculated for C₂₅H₂₀F₉N₇O₆S; C, 41.85; H, 2.81; F, 23.83; N, 13.66; O, 13.38; S, 4.47; found C, 41.70; H, 2.82; N, 13.72; LCMS: m/z = 718.1 [M + H]⁺.

4-(6-(Trifluoromethyl)-2-((2-(3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]-pyrazin-7(8H)-yl)acetyl)thio)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carboxamide)benzenesulfonic acid (12c)

Off white solid, yield = 52%, m.p. 182–184 °C; R_f = 0.51; (n-hexane/EA; 3 : 1); HPLC purity = 97.3% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 14.4 min. ¹H NMR (400 MHz, DMSO-d₆) δ 10.38 (s, 1H, OH, sulpha), 9.36 (s, 1H, py-NH), 8.69 (s, –NH, sulpha), 7.82 (d, J = 8.32 Hz, –2H, ArH), 7.71 (d, J = 8.56 Hz, –2H, ArH), 7.41 (d, J = 8.56 Hz, –2H, ArH, sulpha), 7.27 (d, J = 8.36 Hz, –2H, ArH), 5.46 (s, 1H, –CH), 4.26 (t, J = 5.84 Hz, 2H, pip-CH₂), 3.83 (s, 2H, CH₂), 3.28 (s, 2H, pip-CH₂), 3.10 (t, J = 5.80 Hz, 2H, pip-CH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.7, 166.1, 147.5, 144.9, 142.4, 140.7, 139.4, 139.1, 136.3, 133.1, 132.8 (q, J = 36.3 Hz, –C–CF₃), 132.6 (q, J = 32.4 Hz, –C–CF₃), 131.3 (q, J = 36.6 Hz, –C–CF₃), 128.0, 127.5, 125.7, 125.2, 124.7, 123.2, 122.1 (q, J = 271 Hz, –CF₃), 121.3, 119.3, 117.2, 100.1, 61.6, 57.2, 51.2, 50.2, 42.8. Analysis calculated for C₂₇H₂₀F₉N₇O₅S₂; C, 42.81; H, 2.66; F, 22.57; N, 12.94; O, 10.56; S, 8.46; found C, 42.94; H, 2.67; F, 22.57; N, 13.00; O, 10.56; S, 8.46; LCMS: m/z = 758.0 [M + H]⁺.

(6-(Trifluoromethyl)-2-((2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)acetyl)thio)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)histidine (12d)

Off white solid, yield = 51%, m.p. 226–228 °C; R_f = 0.45; (n-hexane/EA; 3 : 1); HPLC purity = 97.3% (C18 RP, acetonitrile/H2O-80 : 20), TR = 13.7 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.16 (brs, 1H, COOH, His), 9.36 (brs, 1H, py-NH), 8.86 (s, 1H, imidazole, NH), 8.49 (d, J = 6.6 Hz, 1H, CONH, His), 8.03 (s, 1H, imidazole, ArH), 7.82 (d, J = 8.4 Hz, –2H, ArH), 7.26 (d, J = 8.4 Hz, –2H, ArH), 7.07 (s, 1H, imidazole, ArH), 5.46 (s, 1H, –CH), 4.36–4.31 (m, 1H, CHCH₂, His), 4.25 (t, J = 576 Hz, 2H, pip-CH₂), 3.83 (s, 2H, CH₂), 3.53–3.45 (m, 2H, CHCH₂, His), 3.28 (s, 2H, pip-CH₂), 3.10 (t, J = 5.72 Hz, 2H, pip-CH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.7, 175.3, 164.5, 148.8, 144.9, 142.4, 140.5, 140.2, 138.8, 135.2, 133.1 (q, J = 36.3 Hz, –C–CF₃), 132.7 (q, J = 36.7 Hz, –C–CF₃), 131.4 (q, J = 36.4 Hz, –C–CF₃), 130.4, 127.5, 125.7, 125.2, 124.9, 123.0, 122.1 (q, J = 272.4 Hz, –CF₃) 118.8, 117.7, 115.6, 90.7, 61.3, 57.2, 54.0, 52.8, 49.8, 42.7, 29.6. Analysis calculated for C₂₇H₂₂F₉N₉O₄S; C, 43.85; H, 3.00; F, 23.12; N, 17.05; O, 8.65; S, 4.33; found C, 43.73; H, 3.04; F, 23.12; N, 17.23; O, 8.65; S, 4.33; LCMS: m/z = 740.5 [M + H]⁺.

(6-(Trifluoromethyl)-2-((2-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]-pyrazin-7(8H)-yl)acetyl)thio)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-tyrosine (12e)

Cream yellow solid, yield = 54%, m.p. 191–193 °C; R_f = 0.47; (n-hexane/EA; 3 : 1); HPLC purity = 97.6% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 14.3 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.36 (s, 1H, COOH, Tyr), 9.37 (s, 1H, py-NH), 9.08 (s, 1H, Tyr–OH), 8.53 (d, J = 5.28 Hz, 1H, CO–NH), 7.82 (d, J = 8.2 Hz, –2H, ArH), 7.26 (d, J = 8.16 Hz, –2H, ArH), 7.03 (d, J = 7.52 Hz, 2H, Tyr–ArH), 6.76 (d, J = 7.56 Hz, 2H), 5.46 (s, 1H, –CH), 4.98–4.88 (m, 1H, CHCH₂ of Tyr), 4.26 (t, J = 5.8 Hz, 2H, pip-CH₂), 3.83 (s, 2H, CH₂), 3.71 (dd, J = 12.44 Hz, J = 9.68 Hz, 1H, CH₂ of Tyr), 3.49 (dd, J = 12.16 Hz, J = 5.16 Hz, 1H, CH₂ of Tyr), 3.28 (s, 2H, pip-CH₂), 3.10 (t, J = 5.8 Hz, 2H, pip-CH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.4, 177.9, 166.4, 164.5, 157.8, 148.3, 143.8, 141.7, 138.3, 132.3, 131.4, 130.4 (2C), 128.5, 127.6, 125.7 (2C), 125.5 (2C), 124.5, 122.1, 117.3 (2C), 91.5, 61.5, 55.1, 49.3, 48.6, 41.9, 34.1. Analysis calculated for C₃₀H₂₄F₉N₇O₅S; C, 47.06; H, 3.16; F, 22.33; N, 12.81; O, 10.45; S, 4.19; found C, 46.88; H, 3.17; F, 22.33; N, 12.85; O, 10.45; S, 4.19; LCMS: m/z = 766.1 [M + H]⁺.

(2-((2-(3-Aminopiperidin-1-yl)acetyl)thio)-6-(trifluoromethyl)-4-(4-(trifluoromethyl)-phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-aspartic acid (12f)

Light yellow solid, yield = 61%, m.p. 188–190 °C; R_f = 0.45; (DCM/MeOH; 5 : 1); HPLC purity = 97.8% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 7.9 min. ¹H NMR (400 MHz, CDCl₃) δ 12.43 (s, COOH, 1H), 11.80 (s, COOH, 1H), 9.36 (s, py-NH, 1H), 8.40 (d, J = 6.16 Hz, –NH), 7.81 (d, J = 8.4 Hz, 2H, ArH), 7.28 (d, J = 8.4 Hz, 2H, ArH), 5.46 (s, 1H, –CH), 4.44–4.38 (m, 1H, glu), 3.50–3.45 (m, 4H, –CH), 3.31 (s, 2H, CH₂), 3.16–3.12 (m, 1H, CH), 2.67 (d, J = 3.92 Hz, 2H, NH₂), 2.48 (dd, J = 17.28 Hz, J = 5.16 Hz, 6.2 Hz, 1H, CH), 1.60–1.49 (m, 2H, glu), 1.41–1.38 (m, 2H, –CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 188.6, 173.6, 172.4, 166.2, 144.9, 143.1, 132.3, 130.6, 127.5 (2C), 125.3 (2C), 124.3, 123.8, 91.5, 63.1, 59.6, 57.5, 53.6, 49.8, 47.6, 35.8, 34.1, 21.7. Analysis calculated for C₂₄H₂₅F₆N₅O₆S; C, 46.08; H, 4.03; F, 18.22; N, 11.20; O, 15.35; S, 5.13; found C, 45.89; H, 4.05; F, 18.22; N, 11.23; O, 15.35; S, 5.13; LCMS: m/z = 626.1 [M + H]⁺.

(2-((2-(3-Aminopiperidin-1-yl)acetyl)thio)-6-(trifluoromethyl)-4-(4-(trifluoromethyl)-phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-tyrosine (12g)

Cream yellow solid, yield = 51%, m.p. 199–201 °C; R_f = 0.48; (n-hexane/EA; 3 : 1); HPLC purity = 97.6% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 13.5 min. ¹H NMR (400 MHz, CDCl₃) δ 12.36 (s, 1H, COOH, Tyr), 9.36 (s, 1H, py-NH), 9.09 (s, 1H, Tyr–OH), 8.54 (d, J = 5.36 Hz, 1H, CO–NH), 7.81 (d, J = 8.2 Hz, 2H, ArH), 7.28 (d, J = 8.2 Hz, 2H, ArH), 7.03 (d, J = 7.56 Hz, 2H, Tyr–ArH), 6.75 (d, J = 7.56 Hz, 2H, Tyr–ArH), 5.45 (s, 1H, –CH), 4.44–4.38 (m, 1H, CHCH₂ of Tyr), 3.30 (s, 2H, CH₂ of Tyr), 3.18–3.14 (m, 3H, –CH), 3.08 (t, J = 5.28 Hz, 2H, CH₂), 2.67 (d, J = 4.4 Hz, 2H, NH₂), 2.47 (dd, J = 17.12 Hz, J = 5.08 Hz, 1H, CH), 1.65–1.50 (m, 2H, –CH₂), 1.44–1.38 (m, 2H, –CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 188.7, 177.7, 166.0, 157.7, 145.9, 143.0, 134.2 (q, J = 32.2 Hz), 132.2, (q, J = 32.4 Hz), 130.9, 129.7 (2C), 126.9 (2C), 125.8 (q, J = 280 Hz, CF₃), 125.4, (2C) 121.6, 116.2 (2C), 91.9, 63.9, 61.7, 59.4, 56.6, 53.6, 47.7, 38.9, 34.4, 21.6. Analysis calculated for C₂₉H₂₉F₆N₅O₅S; C, 51.71; H, 4.34; F, 16.92; N, 10.40; O, 11.88; S, 4.76; found C, 51.91; H, 4.32; F, 16.92; N, 10.37; O, 11.88; S, 4.76; LCMS: m/z = 674.1 [M + H]⁺.

(2-((2-(2-(Methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)acetyl)thio)-6-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)histidine (12h)

Light brown solid, yield = 48%, m.p. 231–233 °C; R_f = 0.46; (n-hexane/EA; 3 : 1); HPLC purity = 97.6% (C18 RP, acetonitrile/H₂O-80 : 20), TR = 13.3 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.01 (s, 1H, COOH, His), 9.46 (s, 1H, py-NH), 8.68 (s, 1H, imidazole, NH), 8.53 (s, 1H, CONH, His), 7.94 (s, 1H, pyrazol–ArH), 7.79 (s, 1H, imidazole, ArH), 7.75 (d, J = 8.16 Hz, –2H, ArH), 7.24 (d, J = 8.16 Hz, –2H, ArH), 7.10 (s, 1H, imidazole, ArH), 5.28 (s, 1H, –CH), 4.34–4.29 (m, 1H, CHCH₂, His), 4.19–4.14 (m, 4H, 2× CH₂), 3.48–3.42 (m, 2H, CHCH₂, His), 3.10 (s, 3H, SO₂–CH₃), 3.01 (s, 2H, CH₂). ¹³C NMR (100 MHz, CDCl₃) δ 189.8, 175.3, 164.5, 150.8, 144.9, 142.4, 138.8, 135.2, 133.5 (q, J = 36.6 Hz, –C–CF₃), 131.2 (q, J = 36.1 Hz, –C–CF₃), 130.6, 130.4, 127.5, 126.1, 125.7, 125.2 (q, J = 272.8 Hz, –CF₃), 124.9, 124.4, 123.0, 122.8, 118.8, 90.7, 63.8, 57.2, 55.4, 54.6, 54.0, 41.0, 29.6. Analysis calculated for C₂₇H₂₄F₆N₈O₆S₂; C, 44.14; H, 3.29; F, 15.52; N, 15.25; O, 13.07; S, 8.73; found C, 44.11; H, 3.31; F, 15.52; N, 15.22; O, 13.07; S, 8.73; LCMS: m/z = 735.12 [M + H]⁺.

(2-((2-(2-(Methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)acetyl)thio)-6-(trifluoromethyl)-4-(4-(trifluoromethyl)phenyl)-1,4-dihydropyrimidine-5-carbonyl)-L-tyrosine (12i)

Off white solid, yield = 49%, m.p. 221–223 °C; R_f = 0.47; (n-hexane/EA; 3 : 1); HPLC purity = 97.7% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 13.4 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.28 (s, 1H, COOH, Tyr), 9.45 (s, 1H, py-NH), 9.24 (s, 1H, Tyr–OH), 8.50 (d, J = 2.64 Hz, 1H, CO–NH), 7.83 (s, 1H, pyrazol–ArH), 7.76 (d, J = 7.88 Hz, –2H, ArH), 7.24 (d, J = 8.72 Hz, –2H, ArH), 7.04 (d, J = 7.6 Hz, 2H, Tyr–ArH), 6.75 (d, J = 7.48 Hz, 2H), 5.29 (s, 1H, –CH), 4.94–4.89 (m, 1H, CHCH₂ of Tyr), 4.20–4.13 (m, 4H, 2× CH₂), 3.66 (dd, 1H, J = 12.12 Hz, J = 9.64 Hz, CH₂ of Tyr), 3.48 (dd, 1H, J = 11.92 Hz, J = 5.04 Hz, CH₂ of Tyr), 3.12 (s, 3H, CH₂), 2.98 (s, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-d₆) δ 189.8, 174.9, 164.5, 156.4, 150.8, 144.9, 142.4, 132.3, 132.0, 132.4 (q, J = 36.9 Hz, –C–CF₃), 131.8 (q, J = 36.3 Hz, –C–CF₃), 129.6, 127.5, 126.1, 125.7, 125.2, 124.9, 124.4, 123.6, 122.5 (q, J = 273 Hz, –CF₃), 115.8, 90.7, 63.8, 57.2, 55.4, 55.0, 54.6, 41.0, 37.2. Analysis calculated for C₃₀H₂₆F₆N₆O₇S₂; C, 47.37; H, 3.45; F, 14.99; N, 11.05; O, 14.72; S, 8.43; found C, 47.19; H, 3.46; F, 14.99; N, 11.02; O, 14.72; S, 8.43; LCMS: m/z = 761.1 [M + H]⁺.

(2-((2-(2-(Methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)acetyl)thio)-4-(4-nitrophenyl)-6-(trifluoromethyl)-1,4-dihydro pyrimidine-5-carbonyl)-L-glutamic acid (12j)

Yellow solid, yield = 56%, m.p. 234–236 °C; R_f = 0.41; (DCM/MeOH; 5 : 1); HPLC purity = 97.8% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 9.1 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.36 (s, 1H, COOH), 11.70 (s, 1H, COOH), 9.36 (s, 1H, py-NH), 8.44 (d, J = 4.4 Hz, –NH), 8.26 (d, J = 8.7 Hz, 2H, ArH), 7.95 (d, J = 8.7 Hz, 1H, pyrazol–ArH), 7.76 (s, 2H, ArH), 5.47 (s, 1H, –CH), 4.52–4.42 (m, 1H, glu), 4.24–4.22 (m, 4H, 2× CH₂), 3.12 (s, 3H, SO₂–CH₃), 2.97 (s, 2H, CH₂), 2.31 (t, J = 7.24 Hz, 2H, glu), 2.29–1.92 (m, 2H, glu). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.3, 177.0, 176.5, 151.7, 146.4, 145.7, 131.8 (q, J = 32.5 Hz, C–CF₃), 129.0, 126.4 (q, J = 273.1 Hz, CF₃), 124.5, 124.0, 123.7, 121.2, 91.7, 64.9, 56.0, 55.1, 48.9, 41.9, 32.6, 23.8. Analysis calculated for C₂₅H₂₄F₃N₇O₁₀S₂; C, 42.68; H, 3.44; F, 8.10; N, 13.93; O, 22.74; S, 9.11; found C, 42.53; H, 3.45; F, 8.10; N, 13.97; O, 22.74; S, 9.11; LCMS: m/z = 704.1 [M + H]⁺.

4-(2-((2-(2-(Methylsulfonyl)-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)acetyl)thio)-4-(4-nitrophenyl)-6-(trifluoromethyl)-1,4-dihydropyrimidine-5-carboxamido)benzene-sulfonic acid (12k)

Brownish solid, yield = 64%, m.p. 254–256 °C; R_f = 0.43; (n-hexane/EA; 3 : 1); HPLC purity = 97.6% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 12.5 min. ¹H NMR (400 MHz, CDCl₃) δ 10.15 (s, 1H, SO₂–OH), 9.42 (s, 1H, py-NH), 8.49 (s, 1H, CO–NH), 8.32 (d, J = 8.9 Hz, 2H, ArH), 8.23 (d, J = 8.4 Hz, 2H, ArH), 7.89 (d, J = 8.1 Hz, 2H, ArH), 7.87 (s, 1H, pyrazol–ArH), 7.86 (d, J = 8.4 Hz, 2H, ArH), 5.41 (s, 1H, –CH), 4.17–4.17 (m, 4H, 2× CH₂), 3.11 (s, 2H, CH₂), 3.09 (s, 3H, SO₂–CH₃). ¹³C NMR (100 MHz, CDCl₃) δ 189.8, 166.1, 150.8, 147.3, 144.9, 140.7, 136.3, 133.1, 132.8 (q, J = 32.8 Hz, –C–CF₃), 129.6, 128.0, 124.7, 123.1, 122.5, 121.3, 119.8, 100.1, 64.2, 57.4, 56.0, 55.8, 41.0. Analysis calculated for C₂₆H₂₂F₃N₇O₉S₃; C, 42.80; H, 3.04; F, 7.81; N, 13.44; O, 19.73; S, 13.18; found C, 42.93; H, 3.05; F, 7.81; N, 13.48; O, 19.73; S, 13.18, LCMS: m/z = 730.0 [M + H]⁺.

(4-(4-(Methoxycarbonyl)phenyl)-6-(trifluoromethyl)-2-((2-(3-(tri fluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl) acetyl)thio)-1,4-dihydropyrimidine-5-carbonyl)-L-histidine (12l)

White solid, yield = 57%, m.p. 195–197 °C; R_f = 0.44; (DCM/MeOH; 5 : 1); HPLC purity = 98.6% (C18 RP, acetonitrile/H₂O-80 : 20), T_R = 8.8 min. ¹H NMR (400 MHz, DMSO-d₆) δ 12.01 (s, 1H, COOH, His), 9.53 (s, 1H, py-NH), 8.69 (s, 1H, imidazole, NH), 8.53 (s, 1H, CONH, His), 7.94 (s, 1H, imidazole, ArH), 7.92 (d, J = 8.6 Hz, 2H, ArH), 7.44 (d, J = 8.7 Hz, 2H, ArH), 7.10 (s, 1H, imidazole, ArH), 5.28 (s, 1H, –CH), 4.35–4.30 (m, 1H, CHCH₂, His), 4.35 (t, 2H, pip-CH₂), 3.92 (s, 3H, –OCH₃), 3.79 (s, 2H, CH₂), 3.41–3.48 (m, 2H, CHCH₂, His), 3.31 (s, 2H, pip-CH₂), 3.26 (t, 2H, pip-CH₂), 2.33 (t, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ 188.7, 175.3, 168.0, 164.5, 148.8, 145.4, 144.9, 140.5, 140.2, 138.8, 135.2, 132.8, 132.6 (q, J = 32.4 Hz, –C–CF₃), 132.5 (q, J = 36.0 Hz, –C–CF₃), 129.6, 124.9, 123.6, 122.8, 118.8, 117.7, 115.6, 90.7, 61.3, 56.9, 54.0, 52.8, 52.1, 49.8, 42.7, 29.6. Analysis calculated for C₂₈H₂₅F₆N₉O₆S; C, 46.09; H, 3.45; F, 15.62; N, 17.28; O, 13.16; S, 4.39; found C, 45.91; H, 3.46; F, 15.62; N, 17.33; O, 13.16; S, 4.39; LCMS: m/z = 730.1 [M + H]⁺.

Docking studies

Docking studies were conducted by AutoDock4 version (v4.2.6). The crystal structure of SARS-CoV-2 M^pro, along with its native ligand, was retrieved from the Protein Data Bank (PDB) with accession code 6XHM, which offers the target's highest resolution. The docking protocol was adapted from established procedures. The chemical structures of the designed intermediates and inhibitors were sketched using ChemDraw software, followed by geometry optimization, and energy minimization with the Avogadro tool using the MMFF94 force field. Optimized structures were saved in PDB format and converted to PDBQT format using AutoDock Tools (ADTs) with appropriate torsion settings and flexible bonds. Upon ligand preparation, the protein was downloaded from PDB and underwent pre-docking optimization, which included the removal of water molecules, addition of hydrogen atoms, and assigning of Kollman charges. The protein was first saved in PDB format followed by conversion to PDBQT format via ADTs. The active site of protein (6XHM) was identified based on coordinates (X: 10.541898, Y: 14.409504, Z: 27.098183), obtained using Discovery Studio Visualizer v24.1.0.23298. A grid box with the blind dimensions was set to encompass the entire ligand, ensuring sufficient coverage for binding conformations sampling. The file is saved in .gpf format. Docking parameters were defined using genetic algorithm configurations with 10 runs and 2 500 000 energy evaluations per run. AutoDock4 version (v4.2.6) was employed for docking simulations, which predicted binding energies. The resulting .dpf file was generated and subsequently used to produce .glg and .dlg files.^40,41 A more favorable pose is associated with a lower score and the scoring function used kcal per mole as the unit. After docking, protein–ligand interactions, including H-bonding, π–π stacking, π–sigma stacking, etc., were analyzed using 2D and 3D visualization through Discovery Studio Visualizer v24.1.0.23298.⁴²

Methodology of SARS-CoV-2 M^pro assay

The in vitro enzymatic inhibition assay was conducted using the SARS-CoV-2 M^pro Inhibitor Screening Assay Kit supplied by BPS Biosciences. This assay provides a reliable and user-friendly platform for evaluating potential inhibitors of M^pro. The assay followed the manufacturer's recommended protocols and was performed using a 96-well plate format. Inhibitor solutions were prepared by diluting the synthesized compound into the M^pro assay buffer. The assay utilized a fluorogenic substrate containing a cleavage site between nsp4 and nsp5, which, under normal conditions, is cleaved by M^pro. The cleavage released the EDANS fluorophores for the Dabcyl quencher, producing a measurable fluorescent signal. Fluorescence intensity was measured using a PerkinElmer 2030 Victor X Multilabel Plate Reader with excitation and emission wavelengths set to 355 nm and 535 nm, respectively. GC376, a known M^pro inhibitor, was included as a standard reference drug. In the 1st step, 5 ng of MBP-tagged M^pro enzyme was combined with 30 μL of assay buffer containing 1 mM dithiothreitol (DTT). Synthesized compounds dissolved in DMSO (10 μL) were added to the mixture and pre-incubated for 1 h at rt. Subsequently, 10 μL of the fluorescent substrate was added to initiate the enzymatic reaction, bringing the final reaction volume to 50 μL. The final concentration of the substrate and inhibitor in the reaction mixture was maintained at 50 μL. The incubation was carried out at rt for 12–17 hours. The assay tested a range of inhibitor concentrations (0.005–50 μL) to determine IC₅₀ values. Positive control of well-containing enzymes, 1% DMSO, and substrate showed no inhibition of enzymatic activity. As reference control GC376 inhibitor was tested at 100, 10 and 0.1 μM. The IC₅₀ values of the synthesized compounds were calculated using the nonlinear regression (curve fit) function in the GraphPad Prism 8.0 software.⁴³

M^pro cytotoxic assay

The cytotoxic activity of the synthesized compounds was assessed in Vero-E6 cells using the MTT assay with minor modifications. Stock solutions of the compounds were initially dissolved in 10% aqueous DMSO solution, followed by further dilution in DMEM to prepare working solutions. Vero-E6 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and incubated for 24 hours at 37 °C in the five percent CO₂ atmosphere. Various concentrations of the synthesized compound were then applied to the cells, and they were incubated for 48 hours under the same conditions. Cytotoxicity was determined by adding 10 μL of MTT reagent (5 mg mL⁻¹ in PBS) to each well, followed by additional incubation for 4 hours to allow formazan crystal formation. The resulting crystals were dissolved in 100 μL of DMSO, and absorbance was measured at 570 nm using a microplate reader.⁴⁴ The percentage of cell viability was plotted against the compound concentration, and the 50% cytotoxic concentration (CC₅₀) was subsequently calculated using the non-linear regression function in GraphPad Prism 8.0.^43,45,46

Molecular dynamic simulation

After docking studies, the best and most potent COVID-19 inhibitor and conformation were selected for the molecular dynamic simulation (MDS) to evaluate their interaction at the molecular level and in real body conditions. GROMACS software was used to perform molecular dynamic simulation. Charmm GUI server was used to generate input files for MD simulation. The protein–ligand complex was enclosed in a cubic box and solved using the TIP3P water model. The system was equilibrated with a human body temperature at 37 °C and 1 atm pressure. The system was minimized using 50 000 steps before performing MD simulation.

After MD simulation, root means square deviation (RMSD) analysis of protein and complex backbone has been performed to evaluate the structural changes of protein before and after ligand inhibition. RMSD gives a clear picture of overall protein dynamics (such as folding and unfolding) and conformational changes during the MD simulation. On the other hand, the Root Mean Square Fluctuation (RMSF) calculation analyzes the movement/fluctuation of protein regions and residue during the simulation. Another key parameter to understand the ligand inhibition potential and protein–ligand complex at the molecular level is the number of hydrogen bonding interactions throughout simulations.

Results and discussion

Design rational

We have conducted a comprehensive literature review on the surface topology of the M^pro active site and existing M^pro inhibitors (repurposed and rationalized). DHPM-based inhibitors have demonstrated anti-HIV,^47,48 anti-SARS-CoV-1,^49–51 and anti-SARS-CoV-2 (ref. 49, 50 and 52–54) properties. Due to the notable pharmacological relevance, we focused on DHPM's derivatives featuring EWGs and EDGs to design M^pro inhibitors. Our laboratory has previously explored the DHPM scaffold and reported its diverse activity profiles targeting various biological assays.^55–61 We designed and docked DHPM derivatives against the best resolution X-ray crystallographic structure of the M^pro (PDB id: 6XHM), identified three promising DHPM-2-thiones (6a–c) with the lowest binding energies as a hit for guiding our lead identification process. To refine these hits, SAR analysis was employed to delineate critical structural elements that enhance biological activity. Subsequently, lead compounds (8a–h) were docked, and their IC₅₀ values were determined (Fig. 5). This systematic approach enables precise lead optimization. The M^pro comprises four pockets S1, S1′, S2, and S3 pockets. Notable inhibitors such as N3, 13b, 11a, 11b, X77, boceprevir, and dipyridamole include five- and six-membered rings, showcasing the S1 pocket's capacity to accommodate bulky substituents.^62,63 The S1′ pocket is the hydrated region within the active site that forms a water bridge with Thr26, and requires proton donors and acceptors, essential to maintain the water bridge in S1′ and H-bond within S1, inhibitors lacking these groups often exhibit reduced activity. The NGSC motif (Asn142–Gly143–Ser144–Cys145) in S1′ is crucial for forming H-bonds with inhibitors.^64–66 The S2 pocket of M^pro is deeply embedded in nature, favoring six-membered ring and halogenated moieties enhancing bindings affinities e.g. nirmatrelvir, 13b and 11b.⁶³ The hydrophobic S3 pocket supports cyclic structures and branched alkyl chains (e.g. X77, 11a, PF-00835231, boceprevir, and 13b),⁶³ with full occupancy observed for six-membered ring.⁶⁷ In line with these findings, we synthesized DHPM derivatives with proton donor/acceptor groups, aromatic bicyclic moieties, aliphatic moieties, and six-membered rings to optimize interactions across M^pro. Additionally, we strategically incorporated fluorine into our inhibitors, a modification known to increase the inhibitory potency of various medications such as anti-HIV (e.g. efavirenz) and antivirals (e.g. raltegravir, emtricitabine, ledipasvir, and tenofovir). Its presence is particularly relevant to the S1, S2, and S3 pockets where its electronegativity enhances the H-bonding and hydrophobic interactions, improving binding affinity.⁶⁸ The synthesis of these potent compounds was carried out. To validate in silico findings, in vitro analysis was conducted confirming biological activity safety & efficacy. These results provided quantitative data for further optimization in drug development (Fig. 4).

Fig. 4 Design strategy for SARS-CoV-2 M^pro inhibitors.

Chemistry

In the present study, we have synthesized a series of DHPM-based SARS-CoV-2 M^pro inhibitors. Initially, a range of aromatic aldehydes, thiourea and ethyl 4,4,4-trifluoro-3-oxobutanoate underwent a multi-component, one-pot classical Biginelli reaction in the presence of tin(II) chloride catalyst, leading to the formation of a variety of substituted DHPM-thiones (6a–c) (Scheme 1).

Scheme 1 DHPM-thiones (6a–c) synthesis through Biginelli reaction.

Subsequently, DHPM-thione derivatives reacted with various primary amines via a substitution reaction, resulting in the synthesis of amide derivatives of DHPM-thiones (8a–h) (Scheme 2).

Scheme 2 Synthesis of DHPM-thiones amide derivatives (8a–h) by reaction of DHPM's & 1° amines.

In the next step, bromoacetylated DHPM-thione (10a–h) (Scheme 3) were prepared through the reaction of amide derivatives of DHPM-thione with bromoacetyl bromide.

Scheme 3 Bromoacetylated DHPM-thiones (10a–h) synthesis by reaction of (8a–h) with (9).

Finally, potent SARS-CoV-2 inhibitors (12a–l) based on DHPM-thiones scaffold were synthesized by reacting the bromoacetylated DHPM-thiones with diverse secondary amines (Scheme 4).

Scheme 4 Reaction of bromoacetylated products (10a–h) with diverse 2° amines.

In vitro pharmacology [inhibitory concentrations (IC₅₀) and cytotoxicity concentration (CC₅₀)]

The M^pro of SARS-CoV-2 plays a critical role in the virus replication by cleaving viral polyproteins, making it a pivotal target for the therapeutic intervention. Numerous covalent and non-covalent inhibitors such as carmofur, PX-12, and GC376 have been developed to combat SARS-CoV-2. These inhibitors feature N-heterocycles, that mimic the glutamine amino acid at P1 active site. Covalent inhibitors target the catalytic Cys145 residue leading to potent antiviral activity through both cellular and enzymatic inhibition. Noteworthy, ML188.47, a non-covalent M^pro inhibitor, has demonstrated significant efficacy against M^pro.^69–71

In this study we identified lead compounds through comprehensive Structure–Activity Relationship (SAR) analysis, followed by their optimization. Various amino acids were conjugated on the western side of the DHPM-thione scaffold using different synthetic strategies. The half-minimal inhibitory concentration (IC₅₀) values of these intermediates were determined using M^pro assay kit. The in vitro inhibition results of the intermediates are summarized in Fig. 5. SAR analysis revealed that intermediate 8a containing a trifluoromethyl benzene moiety on the northern side and glutamic acid at the western side of scaffold DHPM-thione, exhibited potent inhibitory activity with an IC₅₀ of 23.2 μM ± 0.92. Substituting glutamic acid with aspartic acid in 8b reduced inhibitory activity (IC₅₀ = 44.7 ± 1.36 μM), indicating that the carbon chain length influences inhibition. Conjugation with sulfanilic acid in 8c enhanced inhibitory potency (IC₅₀ = 28.2 μM ± 1.71). Based on these findings, we hypothesized that cyclic moieties could enhance activity. Replacing sulfanilic acid with histidine in 8d resulted in a marginally altered IC₅₀ of 30.7 μM ± 1.43 while substituting with tyrosine in 8e yielded a significant inhibitory effect (IC₅₀ = 25.3 μM ± 1.02). Modifying the northern side with nitrobenzene, while retaining glutamic acid in 8f and sulfanilic acid in 8g on the western side, resulted in reduced potency, with IC₅₀ values of 36.8 μM ± 1.36 and 55.5 μM ± 1.43 respectively. Further SAR exploration incorporating histidine (8h) at the western side, paired with methyl benzoate at the northern side resulted in IC₅₀ values of 34.9 μM ± 1.042 μM. The histidine derivative 8h demonstrated superior inhibitory activity. In conclusion, the SAR analysis highlights the critical role of cyclic moieties on the western side of the DHPM-thione scaffold, along with strategic substitution on the northern side, in influencing the inhibitory efficacy against the SARS-CoV-2 M^pro. This SAR investigation underscores the potential of strategic substitution to enhance the potency of M^pro inhibitors (Fig. 5).

Fig. 5 Intermediates (8a–h) synthesized during the SARS-CoV-M^pro inhibitor development and standard drug GC376.

In our study, we synthesized a series of dihydropyrimidine-2-thione-based compounds (12a–l) based on SAR insights and evaluated in vitro inhibitory potential against SARS-CoV-2 M^pro. GC376 (a well-known SARS-CoV-2 inhibitor) was used as a benchmark to compare the in vitro results. Several exhibited notable inhibitory activities at the sub-micromolar range against SARS-CoV-2 M^pro, ranging from moderate to excellent.

Notably, compound 12l demonstrated remarkable potency, with an IC₅₀ value of 0.054 μM ± 0.001, significantly surpassing the benchmark inhibitor GC376 (IC₅₀ = 0.175 μM ± 0.004). The incorporation of histidine on the western side of 12l provided a key structure–activity relationship (SAR) insight essential for M^pro inhibition. However, contrary to expectations, (12d) and (12h), which also features histidine, exhibited lower inhibitory activity (IC₅₀ = 14.38 μM ± 1.05 and 16.27 μM ± 0.81, respectively), suggesting that additional structural elements critically modulate efficacy.

The methyl benzoate moiety at the northern side of 12l further contributed to its enhanced activity, underscoring its importance in M^pro inhibition. Additionally, halogenated substituents, particularly fluorine, on the northern side influenced inhibitory potential across (12a–i). While most fluorinated compounds, including (12b), (12c), (12d), (12f), and (12h), displayed limited activity, compounds (12a), (12e), (12g), and (12h) exhibited improved inhibition (IC₅₀ = 0.15 μM ± 0.01, 0.73 ± 0.04, 0.21 ± 0.01, and 16.27 μM ± 0.81, respectively) relative to GC376. The IC₅₀ values for (12a–l) are summarized in Fig. 6.

Fig. 6 In vitro M^pro inhibition results of synthesized compounds 12a–l.

Furthermore, (12a) and (12e) display superior efficacy when combined with additional functional groups, such as glutamic acid and tyrosine, on the western side. Further lead optimization revealed that 12j (IC₅₀ = 0.063 μM ± 0.001) and 12k (IC₅₀ = 0.146 μM ± 0.001) exhibited excellent inhibitory activity. These derivatives leveraged glutamic and sulfanilic acid moieties on the western side and nitrogen- and sulfur-containing groups on the northern and eastern sides, respectively.

The exceptional potency of compound 12l was attributed to the fluorinated triazolo-pyrazine group on its eastern side, a feature also observed in the potent 12a and 12e. Moreover, the piperidinyl amine group in (12g) and the sulfonyl pyrrolo-pyrazole moiety in 12i, 12j, and 12k further enhanced inhibition. These findings underscore the critical role of diverse substituents including histidine, halogens (fluorine), and functional groups containing nitrogen, oxygen, and sulfur in optimizing the efficacy of M^pro inhibitors.

Notably, compounds 12a, 12j, 12k, and 12l outperformed GC376 in M^pro inhibition, while others displayed moderate to good activity. Among the most potent inhibitors, 12j and 12l each achieved over 50% inhibition against M^pro. Dose–response experiments for 12j, 12k, and 12l confirmed their IC₅₀ values, as depicted in Fig. 7. Compound 12l emerged as a highly promising candidate, demonstrating exceptional inhibitory potency against SARS-CoV-2 M^pro, making it a viable candidate for further drug development.

Fig. 7 Dose–response curves for IC₅₀ values of compounds 12a, 12j, 12k, 12l, and standard drug GC376 ± SEM; n = 3.

The cytotoxic potential of the synthesized DHPM-2-thione derivatives were evaluated to determine their half-maximal cytotoxic concentration (CC₅₀) against SARS-CoV-2. The MTT assay revealed a wide spectrum of cytotoxic responses among the compounds, ranging from low to high. By employing non-linear regression analysis, the data were converted into the percent cell viability, enabling the precise calculation of CC₅₀ values. Furthermore, these values were utilized to compute the selectivity index (SI) as the ratio of CC₅₀ to IC₅₀, providing insight into the therapeutic safety margins of the compounds.

The IC₅₀ and CC₅₀ graphs for the compounds are summarized in Fig. 7 and 8 respectively. Among these compounds, (12c) exhibited the highest CC₅₀ of 247 μM, (lowest cytotoxicity) followed by 12k (CC₅₀ = 239 μM), 12i (CC₅₀ = 243 μM), 12j (CC₅₀ = 211 μM) and 12l (CC₅₀ = 198 μM). Particularly, structural optimization contributed significantly to improving CC₅₀ values for selected derivatives, highlighting the critical role of the chemical modifications in enhancing their safety profiles. The CC₅₀ value for these compounds, as observed on Vero E6 cells, offers a detailed comparison of their cytotoxic profile.

Fig. 8 Half-maximal cytotoxicity concentration (CC₅₀) of final compounds 12c, 12i, 12j, 12k and 12l on Vero E6 cell ± SEM; n = 3.

Docking studies

Docking studies were conducted to thoroughly analyze the spatial orientation and binding configurations of ligand–enzyme complexes. The crystal structure of the M^pro, co-crystallized with native ligands N-[(2S)-1-({(2S,3S)-3,4-dihydroxy-1-[(3S)-2-oxopyrrolidin-3-yl]butan-2-yl}amino)-4-methyl-1-oxopentan-2-yl]-4-methoxy-1H-indole-2-carboxamide and EDO (1,2-ethanediol), was retrieved from the Protein Data Bank (PDB) under accession code 6XHM, featuring the best resolution. To confirm the reliability of the docking protocol, a re-docking of the native ligand with the M^pro was performed. The observed root-mean-square deviation (RMSD) values were <2.0 Å, indicating a high degree of consistency between the experimental and modelled configurations.

Subsequently, synthesized DHPM-thione derivatives (12a–l) were subjected to docking analysis against the binding sites of SARS-CoV-2 M^pro, in alignment with SAR study guidelines. Extensive literature reports that M^pro/3CL^pro comprises four binding pockets: a bulky S1 pocket that accommodates six-member rings and facilitates H-bond formation, a hydrated S1′ pocket, a narrow and hydrophobic S2 pocket and the S3 pocket capable of binding both aliphatic and aromatic moieties. Key amino acid residues involved in the drug design for each pocket include: Phel40, Leul41, Asnl42, Hisl63, Glul66, Hisl72 (S1 pocket); Thr24, Thr25–26, Leu27, His41, Met49, Asnl42, Glyl43, Serl44, Cysl45 (S1′ pocket); His41, Met49, Tyr54, Glnl89, His164, Asp187, Arg188 (S2 pocket); and Metl65, Leul67, Prol68, Glnl89, Thrl90, Alal91, Glnl92, Leu167, Gly170 (S3 pocket).^64,72–80

To optimize the lead compounds, a detailed SAR analysis was performed against the binding pockets of the M^pro using an advanced computational docking approach to assess binding interactions and to identify potent intermediate for further inhibitor development. Intermediates (8a–h) were docked into M^pro binding pockets, with four intermediates (8a, 8c, 8e, 8h) showing promising potential. To further enhance the binding affinity of the intermediates, the introduction of additional functional groups is required, which could improve the overall potency of inhibitors.

The 2D interaction plot of intermediate 8a (Fig. 9(a)) revealed six conventional H-bonds, three halogens (fluorine) interactions, two π–alkyl interactions, and a π–sulfur interaction. Specifically, intermediate 8a interacted with all four M^pro pockets: H-bonds formed with His163, halogen (fluorine) interactions with Phe140 and Leu141, and a π–alkyl interaction with His172 in the S1 pocket. The S1′ pocket exhibited H-bonds and a π–sulfur interaction Ser144 and Cys145 respectively. The S2 pocket showed halogen (fluorine) interactions and H-bonds interactions with His164 and His41, while the S3 pocket displayed H-bond interactions with Gln189 and Thr190 along with a π–alkyl interaction with Met165. The estimated free binding energy value of 8a in the binding pocket of M^pro was −6.9 kcal mol⁻¹, indicating moderate binding affinity and promising potential for further development as an inhibitor.

Fig. 9 Two-dimensional (2D) interaction plot of intermediate (a) 8a (b) 8c.

Intermediate 8c (Fig. 9(b)) exhibited a similar interaction profile with six conventional H-bonds, three halogens (fluorine) interactions, a π–sulfur and a π–alkyl interaction. H-bond formed with Asn142 and Glu166 in the S1 pocket and with Gly143 in the S1′ pocket. In S2 pocket halogen (fluorine) and π–alkyl interactions were observed with His41, while halogen (fluorine) and π–sulphur interactions with Asp187 and Met49.

The S3 pocket demonstrated H-bond interactions with Glnl89, Thrl90, and Glnl92, along with π–sulphur and π–alkyl interactions with Met49 and Met165 respectively. The estimated free binding energy of the intermediate 8c was −7.4 kcal mol⁻¹, indicating moderate to strong binding affinity, suggesting its potential for optimization and further investigation as an inhibitor.

The 2D interaction plot for intermediate 8e (Fig. 10(a)) demonstrated six H-bonds, three halogens (fluorine) interactions, and two π–sulfur interactions. The H-bonds were formed with His163, Asn142, and Glu166 in the S1 pocket, while halogen (fluorine) interactions were observed with Leu141. The S1′ pocket H-bonds were formed with Cys145 and Ser144 accompanied by a π–sulfur interaction with Met49. The S2 pocket exhibited halogen (fluorine) interactions with His164 and His41 along with a π–sulfur interaction. The S3 pocket showed halogen (fluorine) interactions with Met165 and an H-bond with Gln189. The estimated free binding energy of the 8e was −7.3 kcal mol⁻¹ indicating moderate to strong binding affinity, suggesting its potential for optimization as an inhibitor.

Fig. 10 Two-dimensional (2D) interaction plot of intermediate (a) 8e (b) 8h.

The 2D interaction plot of intermediate 8h (Fig. 10(b)) indicated diverse interactions, including four H-bonds, a halogen (fluorine) interaction, one π–sulfur interaction, one sulphur–X interaction, a π–π T shaped interaction, and one π–alkyl interaction. H-bonds were observed with Glu166, HIS163, and LEU141 in S1 pocket. In the S1′ a π–sulfur interaction was noted with Cys145; however, this pocket is not addressed properly as the NGSC motif is not fully engaged. Additionally, the S3 pocket is not occupied comprehensively, suggesting further modifications and indicating the need for further optimization to enhance binding interactions in the pocket. The S2 pocket exhibited a π–π T-shaped interaction with His42, a π–alkyl interaction with Met49 and H-bond with Asp187. The S3 pocket demonstrated halogen (fluorine) interaction with Gln189 and a π–alkyl interaction with Met165. The estimated free binding energy of 8h was −7.7 kcal mol⁻¹ demonstrating highly favorable binding interaction and promising potential for further development as an inhibitor.

The SAR analysis of intermediates 8a, 8c, 8e, and 8h confirmed their potential as effective M^pro inhibitors. These intermediate display robust binding interactions across all four defined binding pockets of SARS-CoV-2 M^pro, including H-bonds, halogen (fluorine), π–S, S–X, π–π T-shaped, and π–alkyl, contributing to their high binding affinities. The estimated free binding energies of intermediates ranged from −6.9 to −7.7 kcal mol⁻¹ with RMSD values suggesting stable docked conformation. These findings underscore the promising therapeutic potential of these intermediates, warranting further optimization for M^pro inhibitors.

The two-dimensional (2D) interaction plot for compound (12a), presented in Fig. 11(a) revealed strong binding interactions with M^pro. Compound (12a) demonstrates versatile interactions with all four defined pockets of M^pro, including four halogen–fluorine interactions, six hydrogen bonds (H-bonds), and three π–alkyl interactions. The compound's proton donor and acceptor functionalities enable it to engage key residues across M^pro's pocket. Specifically, in the S1 pocket, (12a) forms halogen (F) interactions & π–alkyl interactions with Leu141 and halogen (F) interactions Phe140, H-bonds interaction with Glu166 & His163. In the S1′ pocket, H-bonds were observed with NGSC motif Ser144, and H-bond & π–alkyl interactions were observed with Cys145. In the S2 hydrophobic pocket, halogen (F) interactions & H-bond observed with His164. Furthermore, in the large S3 pocket, which accommodates both cyclic and aliphatic motifs, π–alkyl interactions with Pro168, H-bond observed with Gln189, and halogen (F) interactions were observed with Met165 & Thr190 and the chloro thiophene group. The estimated free binding energy of compound 12a was calculated to be −10.2 kcal mol⁻¹, indicating a potent inhibitor against M^pro. In contrast, compound 12j, as shown in Fig. 11(b), demonstrated an even more extensive set of interactions with the M^pro. This compound formed seven H-bonds, three halogens (F) interactions, and a π–alkyl interaction. The proton donor and acceptor moieties of compound 12j facilitated its engagement with key residues across all M^pro pockets. In the S1 pocket, 12j formed H-bonds with Asn142 & Glu166, halogen (fluorine) interaction with Leu141 & Phe140, and H-bonds and π–alkyl interaction with His163.

Fig. 11 Two-dimensional (2D) interaction plot of synthesized compound (a) (12a) (b) (12j).

In the S1′ pocket, H-bonds were observed with NGSC residues Ser144, Cys145 & Thr26 involving both carbonyl and fluorine groups. In the S2 pocket H bonding is observed with His164, and in the S3 pocket, Met165 halogen (fluorine) interaction was observed with (trifluoromethyl)benzene. The estimated free binding energy value of 12j was −11.5 kcal mol⁻¹, reflecting its exceptional binding affinity as an M^pro inhibitor. Visual inspection of the docking results revealed that while all synthesized compounds exhibited significant interactions with M^pro, compound 12l displayed the most favorable interactions. It effectively engaged all the key pocket residues and demonstrated superior binding affinity compared to the standard drug against M^pro. The synthesized compound (12l) demonstrated excellent interactions with the SARS-CoV-2 M^pro. As shown in Fig. 12(a), a two-dimensional (2D) interaction plot reveals that compound 12l forms eight conventional H-bonds, six halogen (F) bonds, one π–sulphur interactions, two π–alkyl bonds, and a π–lone pair interaction. The compound comprehensively engages all key residues across all four pockets of M^pro. The presence of proton-withdrawing and proton-donating groups in 12l facilitates H-bonding interactions via the carbonyl group and (trifluoromethyl)benzene with key amino acid residues Asnl42, Hisl63. Additionally, halogen (F) bond interactions were observed with residues Glu166 and Leu141 of the S1 pocket, via (trifluoromethyl)-triazole and (trifluoromethyl)benzene moieties. In the S1′ pocket, Cys145 forms π–alkyl and conventional H-bond interactions with the carbonyl group and trifluoro moiety. The important residue Thr26 engages via H-bonding via the imidazole moiety. Furthermore, a π–sulphur interaction with Met49 and H-bonding with Glyl43 and Serl44, facilitated by carbonyl and trifluoro moieties, were also observed. All the NGSC motifs of the S1′ pocket of M^pro were effectively addressed by the compound 12l. In the hydrophobic S2 pocket, π–alkyl interactions with His41, and Met49 were facilitated by the benzene ring, while a π–lone pair interaction with Gln189 was noted. Halogen (F) bond interaction was observed with His164 & Arg188 via the trifluoromethyl-triazole moiety. In the S3 pocket Metl65, and Leul67 exhibit halogen (F) interaction through fluorine moiety of DHPM and triazole. Additionally, π–alkyl interaction with Pro168, halogen (F) interaction with Thr190, and H-bonding interaction with Gln192 was observed via the trifluoromethyl-triazole moiety in the S3 pocket of the M^pro target. The estimated free binding energy for 12l in the binding pocket of SARS-CoV-2 M^pro was −11.9 kcal mol⁻¹, demonstrating a superior binding affinity against M^pro making it a highly promising inhibitor. For comparison GC376 (the standard drug), as shown in Fig. 12(b), exhibited diverse but fewer interactions with M^pro. The interactions include five H-hydrogen bond interactions, three π–alkyl bond interactions, a π–sulphur interaction, and one metal acceptor interaction with M^pro. GC376 exhibited H-bond interactions with Phel40, Hisl63, and Glul66 of the S1 pocket residues. In the S1′ pocket, hydrophobic π–alkyl interaction was observed with Leu27 and Cysl45, while H-bonding was observed with Serl44. With the S2 pocket residues Met49 the π–sulphur interaction was observed and with residue Glnl89 the H-bond interactions were observed. In the S3 pocket residue Metl65, hydrophobic interaction π–alkyl was observed while with residue Thrl90 metal acceptor interaction was observed.

Fig. 12 Two-dimensional (2D) interaction plot (a) synthesized compound (12l) (b) referenced drug GC376.

Comparative molecular modeling and surface analysis of the intermediate (8h) and final compound (12l)

Our molecular modeling studies revealed that intermediate 8h, while capable of engaging some key residues, does not comprehensively interact with all four binding pockets of M^pro. It fails to fully engage the NGSC motifs in the S1′ pocket and does not extend into the S3 pocket (Fig. 13).

Fig. 13 Interaction of 8h with the M^pro binding sites: the S3 pocket is not occupied by 8h.

In contrast, the final product 12l incorporates a trifluoromethyl-triazole moiety that facilitates additional interactions across the S1, S1′, S2, and S3 pockets as shown in Fig. 14. This modification enhances both hydrogen bonding and halogen interactions with residues across all pockets, resulting in the optimal alignment of compound 12l within the M^pro binding clefts. This improvement is reflected in its high inhibitory potency, with an IC₅₀ of 0.054 μM, when compared to the intermediate compound 8h. Overall, our docking analysis via surface diagram confirms that the structural modification introduced in 12l is critical for achieving comprehensive engagement of M^pro's binding pockets and, consequently, for its enhanced antiviral activity.

Fig. 14 Comprehensive engagement of 12l with the M^pro binding sites.

Molecular dynamic simulations (MDS)

Molecular dynamic simulations of protein and protein–ligand complex were performed to understand the inhibition of our compound at the molecular level and verify the docking results in possible body conditions. Compound inhibition was analyzed in terms of how it changes or controls the protein overall and regional dynamics as well as their conformation changes. The RMSD graph (Fig. 15) shows that target protein M^pro (PDB Id = 6XHM) shows considerable stability in the first 20 ns and after 20 ns there are so many conformational changes during the simulation which indicates their active conformation changes. The frequency of conformation changes has been reduced in the last 20 ns. The overall RMSD of protein M^pro is more than 4 Å. On the other hand, after the binding of the ligand, the protein–ligand complex shows complete stability with RMSD below 2.5 nm throughout the simulation.

Fig. 15 The root means square deviation (RMSD) graph of protein (blue) and protein–ligand (12l) complex (brown).

The root means square fluctuations (RMSF) (Fig. 16) of target protein 6XHM and protein–ligand complex are almost similar, with few exceptions in the complex RMSF plot.

Fig. 16 The root means square fluctuations (RMSF) graph of protein (6XHM) (blue) and protein–ligand (12l) complex (brown).

The RMSF graph for both is between 0 and 0.6 nm, mostly below 0.2 nm, except for four peaks, which show the flexibility of protein in the ranges of 1–10, 49–55, 190–200, and 290–300. The protein–ligand complex shows more fluctuations at 49–55 and 190–200. Hydrogen bonding analysis was performed to analyze the interaction between the target protein (6XHM) and ligand (12l).

The histogram (Fig. 17) shows that the minimum number of hydrogen bonds in every frame throughout the simulation is three and the maximum is five, which is evident in the strong inhibition of our ligand.

Fig. 17 The H-bonding interactions histogram of protein (6XHM) and ligand (12l).

Pharmacokinetic predictions

Pharmacokinetic properties of the most potent derivatives (12a, 12j, 12k, 12l) were evaluated using in silico tools to predict their potential for Blood–Brain Barrier (BBB) penetration, Human Intestinal Absorption (HIA), and AMES toxicity.

The SMILES strings of these synthesized compounds were submitted to the online admetSAR server (https://lmmd.ecust.edu.cn/admetsar2/) for prediction.

As summarized in Table 1, the results indicate that all tested compounds are likely to penetrate the BBB, exhibit significant absorption in the intestine, and are predicted to be non-AMES toxic. These predictions suggest favourable pharmacokinetic profiles, underscoring their potential for further development as therapeutic agents against SARS-CoV-2 M^pro.⁸¹

Table 1 Pharmacokinetic properties of the most potent derivatives (12a, 12j, 12k, 12l)

Comp. no.	BBB	HIA	AMES toxicity
12a	−(0.9399)	+(0.8739)	Non-toxic (0.5949)
12j	−(0.9205)	+(0.6844)	Non-toxic (0.5488)
12k	−(0.8224)	+(0.5774)	Non-toxic (0.5496)
12l	−(0.9665)	+(0.8197)	Non-toxic (0.6035)

---

Conclusions

The study harnessed the dihydropyrimidine-2-thiones as a scaffold due to its extensive applications in various medications and the availability of an in-house library within our research group. Among diverse derivatized dihydropyridines-2-thiones, 6a, 6b, and 6c DHPM's based intermediate emerged as potent leads subsequently optimized through Structure Activity Relationship (SAR) and computational docking interaction analysis through AutoDock4 version (v4.2.6). These intermediates were also docked and subjected to in vitro analysis. Conclusively the study identifies fifteen potent dihydropyrimidine-2-thione-based compounds (12a–l) as potential inhibitors of SARS-CoV-2 M^pro. These compounds underwent biological assay against the M^pro confirming significant inhibitory activity. Notably, compounds (12a), 12j, 12k, and 12l exhibited superior inhibition compared to the standard drug GC376. Conversely, compounds (12b), (12c), (12e), (12f), (12g), and (12i) demonstrated less inhibitory efficacy. Molecular docking and Molecular Dynamic (MD) simulation revealed significant coherence with biological assay and confirmed the ligand (12l)–protein complex (6XHM) stability respectively. The cytotoxic profile of compounds 12c, 12i, 12j, 12k, and 12l were evaluated, with CC₅₀ values confirming an acceptable safety margin for human consumption. Furthermore, the oral toxicity of compounds 12j, and 12l were predicted and these were found inactive to the oral toxicities. Collectively designating these compounds as promising non-covalent inhibitors for M^pro, compound 12l exhibited exceptional potency against the M^pro of SARS-CoV-2.

Data availability

The data supporting this article have been included as part of the ESI.†

Author contributions

Umer Rashid conceived, designed, and supervised this study. He was involved in all the phases (from synthesis to pharmacological evaluation and manuscript writing/editing) that led to the manuscript's completion. Anees Saeed synthesized the compounds. In vitro experiments were performed by Anees Saeed, Fahad Hussain, and Abdul Sadiq. Docking studies were performed by Anees Saeed. Docking results were analyzed and written by Ayesha Tahir and UR. MD simulations were performed by Muhammad Shah. The manuscript was drafted by Anees Saeed. Umer Rashid reviewed and edited the drafts. All the authors have read the manuscript and approved it for publication.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The research is financially supported by a Project Grant from the Higher Education Commission Pakistan to Umer Rashid (PI) under the National Research Program for Universities (NRPU) (20-14513/NRPU/R&D/HEC/2021 2021).

References

1. A. R. Fehr and S. Perlman, Coronaviruses: Methods and Protocols, 2015, pp. 1–23.
2. W. S. T. Consortium, N. Engl. J. Med., 2021, 384, 497–511.
3. L. Riva, S. Yuan, X. Yin, L. Martin-Sancho, N. Matsunaga, L. Pache, S. Burgstaller-Muehlbacher, P. D. De Jesus, P. Teriete and M. V. Hull, Nature, 2020, 586, 113–119.
4. D. R. Owen, C. M. Allerton, A. S. Anderson, L. Aschenbrenner, M. Avery, S. Berritt, B. Boras, R. D. Cardin, A. Carlo and K. J. Coffman, Science, 2021, 374, 1586–1593.
5. P. V’kovski, A. Kratzel, S. Steiner, H. Stalder and V. Thiel, Nat. Rev. Microbiol., 2021, 19, 155–170.
6. M. M. Lamers and B. L. Haagmans, Nat. Rev. Microbiol., 2022, 20, 270–284.
7. D. Cucinotta and M. Vanelli, Acta Bio Med. Atenei Parmensis, 2020, 91, 157.
8. E. Mahase, BMJ, 2020, 368, m1036.
9. D. S. Hui, E. I. Azhar, T. A. Madani, F. Ntoumi, R. Kock, O. Dar, G. Ippolito, T. D. Mchugh, Z. A. Memish and C. Drosten, Int. J. Infect. Dis., 2020, 91, 264–266.
10. F. G. Hayden, R. B. Turner, J. M. Gwaltney, K. Chi-Burris, M. Gersten, P. Hsyu, A. K. Patick, G. J. Smith III and L. S. Zalman, Antimicrob. Agents Chemother., 2003, 47, 3907–3916.
11. Y. Kim, H. Liu, A. C. Galasiti Kankanamalage, S. Weerasekara, D. H. Hua, W. C. Groutas, K.-O. Chang and N. C. Pedersen, PLoS Biol., 2016, 12, e1005531.
12. H. Yang, W. Xie, X. Xue, K. Yang, J. Ma, W. Liang, Q. Zhao, Z. Zhou, D. Pei and J. Ziebuhr, PLoS Biol., 2005, 3, e324.
13. W. Dai, B. Zhang, X.-M. Jiang, H. Su, J. Li, Y. Zhao, X. Xie, Z. Jin, J. Peng and F. Liu, Science, 2020, 368, 1331–1335.
14. Y. Chen, Q. Liu and D. Guo, J. Med. Virol., 2020, 92, 418–423.
15. S. Hussain, J. a. Pan, Y. Chen, Y. Yang, J. Xu, Y. Peng, Y. Wu, Z. Li, Y. Zhu and P. Tien, J. Virol., 2005, 79, 5288–5295.
16. R. Ramajayam, K.-P. Tan and P.-H. Liang, Biochem. Soc. Trans., 2011, 39, 1371–1375.
17. K. Anand, G. J. Palm, J. R. Mesters, S. G. Siddell, J. Ziebuhr and R. Hilgenfeld, EMBO J., 2002, 21, 3213–3224.
18. H. Yang, M. Yang, Y. Ding, Y. Liu, Z. Lou, Z. Zhou, L. Sun, L. Mo, S. Ye and H. Pang, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 13190–13195.
19. S. Zhang, M. Krumberger, M. A. Morris, C. M. T. Parrocha, A. G. Kreutzer and J. S. Nowick, Eur. J. Med. Chem., 2021, 218, 113390.
20. K. Świderek and V. Moliner, Chem. Sci., 2020, 11, 10626–10630.
21. M. Bartlam, H. Yang and Z. Rao, Curr. Opin. Struct. Biol., 2005, 15, 664–672.
22. D. Stern and B. Sefton, J. Virol., 1982, 44, 804–812.
23. M. A. Marra, S. J. Jones, C. R. Astell, R. A. Holt, A. Brooks-Wilson, Y. S. Butterfield, J. Khattra, J. K. Asano, S. A. Barber and S. Y. Chan, Science, 2003, 300, 1399–1404.
24. D. F. Veber, S. R. Johnson, H.-Y. Cheng, B. R. Smith, K. W. Ward and K. D. Kopple, J. Med. Chem., 2002, 45, 2615–2623.
25. R. L. Hoffman, R. S. Kania, M. A. Brothers, J. F. Davies, R. A. Ferre, K. S. Gajiwala, M. He, R. J. Hogan, K. Kozminski and L. Y. Li, J. Med. Chem., 2020, 63, 12725–12747.
26. Y. Unoh, S. Uehara, K. Nakahara, H. Nobori, Y. Yamatsu, S. Yamamoto, Y. Maruyama, Y. Taoda, K. Kasamatsu and T. Suto, J. Med. Chem., 2022, 65, 6499–6512.
27. J. D. Tyndall, J. Med. Chem., 2022, 65, 6496–6498.
28. S. G. V. Rosa and W. C. Santos, Rev. Panam. Salud Públic, 2020, 44, e40.
29. N. Drayman, J. K. DeMarco, K. A. Jones, S.-A. Azizi, H. M. Froggatt, K. Tan, N. I. Maltseva, S. Chen, V. Nicolaescu and S. Dvorkin, Science, 2021, 373, 931–936.
30. Z. Jin, X. Du, Y. Xu, Y. Deng, M. Liu, Y. Zhao, B. Zhang, X. Li, L. Zhang and C. Peng, Nature, 2020, 582, 289–293.
31. C. Ma, M. D. Sacco, B. Hurst, J. A. Townsend, Y. Hu, T. Szeto, X. Zhang, B. Tarbet, M. T. Marty and Y. Chen, Cell Res., 2020, 30, 678–692.
32. L. Fu, F. Ye, Y. Feng, F. Yu, Q. Wang, Y. Wu, C. Zhao, H. Sun, B. Huang and P. Niu, Nat. Commun., 2020, 11, 1–8.
33. P. Aparoy, K. Kumar Reddy and P. Reddanna, Curr. Med. Chem., 2012, 19, 3763–3778.
34. A. Saeed, S. A. Ejaz, A. Khalid, P. A. Channar, M. Aziz, Q. Abbas, T. A. Wani, N. A. Alsaif, M. M. Alanazi and A. M. Al-Hossaini, Int. J. Mol. Sci., 2022, 23, 13164.
35. A. Elmaghraby, I. Mousa, A. Harb and M. Mahgoub, Int. Scholarly Res. Not., 2013, 2013, 706437.
36. F. Sánchez-Sancho, M. Escolano, D. Gaviña, A. G. Csáky, M. Sánchez-Roselló, S. Díaz-Oltra and C. Del Pozo, Pharmaceuticals, 2022, 15, 948.
37. I. S. Zorkun, S. Saraç, S. Çelebi and K. Erol, Bioorg. Med. Chem., 2006, 14, 8582–8589.
38. R. Kaur, S. Chaudhary, K. Kumar, M. K. Gupta and R. K. Rawal, Eur. J. Med. Chem., 2017, 132, 108–134.
39. G. C. Rovnyak, K. S. Atwal, A. Hedberg, S. D. Kimball, S. Moreland, J. Z. Gougoutas, B. C. O'Reilly, J. Schwartz and M. F. Malley, J. Med. Chem., 1992, 35, 3254–3263.
40. S. M. D. Rizvi, S. Shakil and M. Haneef, EXCLI Journal, 2013, 12, 831.
41. R. Quiroga and M. A. Villarreal, PLoS One, 2016, 11, e0155183.
42. A. A. Jabbar, R. A. Mothana, M. A. Abdulla, F. O. Abdullah, K. A.-A. Ahmed, R. R. Hussen, M. F. Hawwal, O. I. Fantoukh and S. Hasson, Saudi Pharm. J., 2023, 31, 101850.
43. M. A. Abu-Zaied, G. H. Elgemeie and N. M. Mahmoud, ACS Omega, 2021, 6, 16890–16904.
44. N. P. Marques, C. S. Lopes, N. C. T. Marques, L. Cosme-Silva, T. M. Oliveira, C. Duque, V. T. Sakai and J. A. C. Hanemann, Laser Med. Sci., 2019, 34, 465–471.
45. C. Shivanika, D. Kumar, V. Ragunathan, P. Tiwari and A. Sumitha, J. Biomol. Struct. Dyn., 2022, 40, 585–611.
46. E. Radchenko, A. Dyabina, V. Palyulin and N. Zefirov, Russ. Chem. Bull., 2016, 65, 576–580.
47. B. H. Sarvaiya, P. I. Vaja, N. A. Paghdar and S. M. Ghelani, J. Heterocycl. Chem., 2024, 61, 1325–1348.
48. Z. Wang, H. Zhang, Z. Gao, Z. Sang, E. De Clercq, C. Pannecouque, D. Kang, P. Zhan and X. Liu, Acta Pharm. Sin. B, 2024, 14, 1257–1282.
49. C. Gege, F. Hahn, C. Wangen, S. Häge, A. Herrmann, N. Uhlig, V. Eberlein, L. Issmail, R. Klopfleisch and T. Grunwald, ChemMedChem, 2024, e202400292.
50. A. M. Elshamsy, T. F. Ali, M. Osman and N. A. El-Koussi, Journal of Advanced Biomedical and Pharmaceutical Sciences, 2023, 6, 114–123.
51. D. S. E. Sayed and E.-S. M. Abdelrehim, BMC Chem., 2022, 16, 82.
52. D. C. Schultz, R. M. Johnson, K. Ayyanathan, J. Miller, K. Whig, B. Kamalia, M. Dittmar, S. Weston, H. L. Hammond and C. Dillen, Nature, 2022, 604, 134–140.
53. E. Mansour, A. M. Sayed and S. I. Elewa, Polycyclic Aromat. Compd., 2024, 1–26.
54. R. Xiong, L. Zhang, S. Li, Y. Sun, M. Ding, Y. Wang, Y. Zhao, Y. Wu, W. Shang and X. Jiang, Protein Cell, 2020, 11, 723–739.
55. U. Rashid, S. F. Hassan, S. Nazir, A. Wadood, M. Waseem and F. L. Ansari, Med. Chem. Res., 2015, 24, 304–315.
56. U. Rashid, R. Sultana, N. Shaheen, S. F. Hassan, F. Yaqoob, M. J. Ahmad, F. Iftikhar, N. Sultana, S. Asghar and M. Yasinzai, Eur. J. Med. Chem., 2016, 115, 230–244.
57. G. Ahmad, N. Rasool, K. Rizwan, A. A. Altaf, U. Rashid, M. Z. Hussein, T. Mahmood and K. Ayub, Molecules, 2019, 24, 2609.
58. M. J. Ahmad, S. F. Hassan, R. U. Nisa, K. Ayub, M. S. Nadeem, S. Nazir, F. L. Ansari, N. A. Qureshi and U. Rashid, Med. Chem. Res., 2016, 25, 1877–1894.
59. F. Iftikhar, F. Yaqoob, N. Tabassum, M. S. Jan, A. Sadiq, S. Tahir, T. Batool, B. Niaz, F. L. Ansari and M. I. Choudhary, Bioorg. Chem., 2018, 80, 99–111.
60. M. Bibi, N. A. Qureshi, A. Sadiq, U. Farooq, A. Hassan, N. Shaheen, I. Asghar, D. Umer, A. Ullah and F. A. Khan, Eur. J. Med. Chem., 2021, 210, 112986.
61. M. A. Javed, N. Ashraf, M. Saeed Jan, M. H. Mahnashi, Y. S. Alqahtani, B. A. Alyami, A. O. Alqarni, Y. I. Asiri, M. Ikram and A. Sadiq, ACS Chem. Neurosci., 2021, 12, 4123–4143.
62. L. Zhang, D. Lin, X. Sun, U. Curth, C. Drosten, L. Sauerhering, S. Becker, K. Rox and R. Hilgenfeld, Science, 2020, 368, 409–412.
63. C.-H. Zhang, E. A. Stone, M. Deshmukh, J. A. Ippolito, M. M. Ghahremanpour, J. Tirado-Rives, K. A. Spasov, S. Zhang, Y. Takeo and S. N. Kudalkar, ACS Cent. Sci., 2021, 7, 467–475.
64. A. Thakur, G. Sharma, V. N. Badavath, V. Jayaprakash, K. M. Merz Jr, G. Blum and O. Acevedo, J. Phys. Chem. Lett., 2022, 13, 5776–5786.
65. S. Tomar, M. L. Johnston, S. E. S. John, H. L. Osswald, P. R. Nyalapatla, L. N. Paul, A. K. Ghosh, M. R. Denison and A. D. Mesecar, J. Biol. Chem., 2015, 290, 19403–19422.
66. J. Mondal, P. Tiwary and B. Berne, J. Am. Chem. Soc., 2016, 138, 4608–4615.
67. H. H. Chan, M. A. Moesser, R. K. Walters, T. R. Malla, R. M. Twidale, T. John, H. M. Deeks, T. Johnston-Wood, V. Mikhailov and R. B. Sessions, Chem. Sci., 2021, 12, 13686–13703.
68. C. Zhang, ACS omega, 2022, 7, 18206–18212.
69. C. Ma, Y. Hu, J. A. Townsend, P. I. Lagarias, M. T. Marty, A. Kolocouris and J. Wang, ACS Pharmacol. Transl. Sci., 2020, 3, 1265–1277.
70. W. Vuong, M. B. Khan, C. Fischer, E. Arutyunova, T. Lamer, J. Shields, H. A. Saffran, R. T. McKay, M. J. van Belkum and M. A. Joyce, Nat. Commun., 2020, 11, 4282.
71. A. K. Ghosh, M. Brindisi, D. Shahabi, M. E. Chapman and A. D. Mesecar, ChemMedChem, 2020, 15, 907–932.
72. I. Y. Akbayrak, S. I. Caglayan, L. Kurgan, V. N. Uversky and O. Coskuner-Weber, Curr. Res. Struct. Biol., 2022, 4, 349–355.
73. A. Chandra Manivannan, A. Malaisamy, M. Eswaran, A. Meyyazhagan, V. A. Arumugam, K. R. Rengasamy, B. Balasubramanian and W.-C. Liu, Front. Mol. Biosci., 2022, 9, 918101.
74. A. M. Shaqra, S. N. Zvornicanin, Q. Y. J. Huang, G. J. Lockbaum, M. Knapp, L. Tandeske, D. T. Bakan, J. Flynn, D. N. Bolon and S. Moquin, Nat. Commun., 2022, 13, 3556.
75. M. Macchiagodena, M. Pagliai and P. Procacci, Chem. Phys. Lett., 2020, 750, 137489.
76. C. Lin, H. Jiang, W. Li, P. Zeng, X. Zhou, J. Zhang and J. Li, Structure, 2023, 31, 1016–1024.e3.
77. H. P. Shao, T. H. Wang, H. L. Zhai, K. X. Bi and B. Q. Zhao, Chem.-Biol. Interact., 2023, 371, 110352.
78. L. Alzyoud, M. A. Ghattas and N. Atatreh, Drug Des., Dev. Ther., 2022, 16, 2463.
79. Q. Hu, Y. Xiong, G. H. Zhu, Y. N. Zhang, Y. W. Zhang, P. Huang and G. B. Ge, MedComm, 2022, 3, e151.
80. M. D. Sacco, C. Ma, P. Lagarias, A. Gao, J. A. Townsend, X. Meng, P. Dube, X. Zhang, Y. Hu and N. Kitamura, Sci. Adv., 2020, 6, eabe0751.
81. J. Shen, F. Cheng, Y. Xu, W. Li and Y. Tang, J. Chem. Inf. Model., 2010, 50, 1034–1041.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra08449g

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