Design and synthesis of Diphenyl-1H-imidazole analogs targeting MPro/3CLpro enzyme of SARS-CoV-2

Design and synthesis of Diphenyl-1H-imidazole analogs targeting MPro/3CLpro enzyme of SARS-CoV-2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Design and synthesis of Diphenyl-1H-imidazole analogs targeting MPro/3CLpro enzyme of SARS-CoV-2 Amisha Vora, Ashish Kanhed, Ami Thakkar, Rudramurthy Renukaiah Gudepalya, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3975613/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract The prevailing COVID-19 pandemic, triggered by the novel coronavirus SARS-CoV-2, stands as the predominant global health crisis of the decade, claiming millions of lives and causing profound disruptions to society. Despite the rapid development of vaccines against COVID-19, the situation remains challenging, necessitating the exploration of new antiviral drugs. In this study, we present the design and synthesis of Diphenyl-1H-imidazole derivatives as a potential lead series for inhibiting the SARS-CoV-2 3CLpro enzyme. The synthesized molecules underwent screening for inhibiting the SARS-CoV-2 3CLpro enzyme at a concentration of 20µM. Compounds 6-14 exhibited inhibition ranging from 88% to 99%. Further assessments were conducted to evaluate the anti-SARS-CoV-2 activity of these compounds against both the ancestral Wuhan strain and the Delta variant in virus-infected cells. Compounds such as 4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)- 1H -imidazole (9) , 4-(2,4-dichlorophenyl)-2-(3,4-dimethoxyphenyl)- 1H -imidazole (10) , and 4-(4-(2,4-dichlorophenyl)- 1H -imidazol-2-yl)benzene-1,2-diol (14) exhibited promising activity against both the Wuhan strain (with IC50 values of 7.7 µM, 12.6 µM, and 11.8 µM, respectively) and the Delta variant (with IC50 values of 7.4 µM, 13.8 µM, and 12.1 µM, respectively). Moreover, the 3CLpro inhibition IC50 values for these compounds correlated well with the observed antiviral activity, measuring at 5.1 µM (9) , 10.9 µM (10) , and 7.3 µM (14) . These findings underscore the efficacy of diphenyl- 1H -imidazole derivatives as promising candidates for further development and optimization in the fight against COVID-19. SARS-CoV-2 3CLpro inhibition Diphenyl-1H-imidazole Wuhan variant Delta variant Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction COVID-19 caused by novel corona virus (SARS-CoV-2) is the major pandemic of the decade claiming approximately over 6.6 million lives, causing severe socio-economic implications globally [ 1 ]. Vaccination has helped to get considerable control on the spread and fatality of the condition; however, complete treatment is not available [ 2 – 4 ]. Certain repurposed drugs have found benefits; however, they have their own side effects and limitations. FDA has approved some anti-viral drugs to treat mild to moderate COVID-19 condition, if detected in early stages. The NIH has suggested few treatment combinations for healthcare workers, which includes Nirmatrelvir with Ritonavir, Remdesivir, Molnupiravir for different age groups. These antiviral drugs have many side effects and may show drug-drug interactions [ 5 ]. Despite substantial efforts, there no precise cure for COVID-19 and discovery of new molecules with promising antiviral activity is required. Viral proteases are the potentially validated target for anti-viral drug discovery over many years. In addition to this, in case of SARS-CoV-2 virus, many variants (Wuhan, Delta, Omicron) are observed with different virulence capacity and morbidity. In all these variants, the virulence capacity varies mainly because of mutations in spike protein. However, major mutations in active site of in 3CLpro (main protease / Mpro) are not observed. Thus, the antiviral activity of 3CLpro inhibitors is expected to remain unaffected by any of the mutations of spike protein, generally observed in different SARS-CoV-2 variants [ 3 , 6 ]. Therefore, 3CLpro is a promising target for the treatment of COVID-19. In the current study, we have identified 2,4-diphenyl- 1H -imidazole as a promising scaffold and explored its interactions with the active site of the SARS-CoV-2 3CLpro enzyme. Following the assessment of its enzyme inhibition potential in vitro and its antiviral activity in virus-infected cells, we proceeded to synthesize derivatives and subjected them to screening for both SARS-CoV-2 3CLpro enzyme inhibition and anti-SARS-CoV-2 activity (against both the ancestral Wuhan and Delta variants) in virus-infected cells. Dose-response curves were generated for the top three molecules to ascertain their IC50 values. Additionally, an in-silico analysis was conducted to investigate their interactions with the active site of the SARS-CoV-2 3CLpro enzyme. Detailed discussion of all these findings is provided in the subsequent sections. 2. Results & Discussion 2.1 Designing aspect: Majority of the antiviral drugs contain heterocyclic moieties with diverse substitutions. Mainly because of its active site interaction abilities, good synthetic feasibility for derivatization and amphoteric nature, imidazole is one of the important heterocycles in the field of anti-viral drug discovery [7]. Various anti-viral agents like ledipasvir, daclatasvir (anti-hepatitis C virus), capravirine (anti-HIV), enviroxime (anti- rhinovirus and polio virus) contains imidazole scaffold. Various imidazole alkaloids (naamidine, pyronaamidine, leucettaamine) isolated from the marine sponge Pericharax heteroraphis were found effective on H1N1 influenza virus [8-10]. Apart from these, various research groups are working on imidazole-based drug discovery for viral infections like COVID-19, Dengue, Zika virus. Considering imidazole as a promising heterocycle, here we initiated imidazole-based lead identification. In the present report our focus is on substituted 2,4-diphenyl imidazole. The 3CLpro is cysteine protease enzyme made up of 306 amino acids having three major domains. Domain 1 is 8 – 101 amino acid residue region, domain 2 is comprised of amino acids from 102 - 184, and the 3 rd domain is from 201 - 203 amino acids. The loop region with amino acids from 185 – 200 connects domain 2 with domain 3. The active site/binding site region is present between domain 1 and 2 with a Cys145-His41 catalytic dyad. This active site region is mainly comprised of S1, S1’, S2 and S4 sub-regions. The S1 region is made up of Phe140, Leu141, Asn142, His163, Glu166 and His172 amino acids. The S1’ small region is made up from S1 subsite by intrusion of Asn142 and it consist of Thr25, Thr26 and Leu27. The S2 subsite is hydrophobic and is made up of His41, Met49, Tyr54, Met165 and Asp187. The final S4 region comprises Met165, Leu167, Phe185, Gln189 and Gln192 amino acids [11,12] (Fig. 1). In virtual interaction studies, the 2-phenyl moiety of 2,4-diphenyl imidazole was observed in S2 binding subsite surrounded by Met49, Tyr54, Arg188 and Gln189. Whereas the 4-phenyl of same scaffold observed in S1 sub site surrounded by Phe140, Ser144 and His163. The imidazole ring was observed located centrally with free N-H facing towards S4 subsite (Fig. 2). To understand the enzyme inhibition potency of basic scaffold 6 , it was tested using FRET based SARS-CoV-2 3CLpro enzyme inhibition assay and at 20 µM concentration, 89.96% enzyme inhibition was observed. Further, this compound was tested against ancestral Wuhan and Delta variants of SARS-CoV-2 infected Vero E6 cells at the same concentration and showed 14.1% and 6.9% antiviral activity respectively. This considerably good enzyme inhibition and low to moderate antiviral activity could be because of poor cell permeability. This suggested the possibility of the scaffold for further development. Thus, we decided to make some hydrophobic and hydrophilic modifications in the scaffold to extemporize the lead activity. 2.2 S ynthesis of 2, 4-diphenyl-1 H -imidazole derivatives Compounds 6-14 were synthesized as depicted in scheme 1 . The starting material 2-Bromo-1-(substitutedphenyl)ethanone ( 2a – 2d ) were synthesized using various acetophenone ( 1a-1d ). Second starting material analogs ( 5a, 5b ) were synthesized from benzaldehyde / 3,4-dimethoxybenzaldehyde ( 3a, 3b ) via benzonitrile / 3,4-dimethoxybenzonitrile ( 4a, 4b ). The benzimidamide ( 5a/5b ) were treated with different bromoketone ( 2a-2d ) to get final compounds 4-(Substitutedphenyl)-2-(3,4-dimethoxyphenyl)-1 H -imidazole ( 6-10 ). To improve the polar properties of compounds 6-10 , O -demethylation was carried to obtain 4-(4-substitutedphenyl-1 H -imidazol-2-yl)benzene-1,2-diol ( 11-14 ). 2.3 Antiviral activity of 2,4-diphenyl-1 H -imidazole derivatives The synthesized compounds were evaluated for 3CLpro enzyme inhibition by quenched fluorescence resonance energy transfer (FRET) assay and antiviral activity against SARS-CoV-2 (ancestral Wuhan and the Delta) by plaque assay. The FRET assay showed promising activity by all the synthesized compounds ( 6-14 ) against the 3CLpro enzyme with 88% to 99% inhibition at 20 µM concentration. Considering the observed 3CLpro enzyme inhibition activity, all the synthesized molecules were tested against SARS-CoV-2 (ancestral Wuhan and the Delta variant) to understand viral inhibition potency. The initial screening of compounds at 20 µM against SARS-CoV-2 by plaque assay identified 3 hit molecules such as compounds 9 , 10 , and 14 (Table 1). Further, the IC50 of the hit molecules 9 , 10 , and 14 was found to be 7.7 µM, 12.6 µM, and 11.08 µM respectively against the ancestral Wuhan strain, while the IC50 against Delta variant was found to be 7.4 µM, 13.8 µM, and 12.1 µM respectively. The positive control remdesivir used in the antiviral assay showed an IC50 value of 0.8 µM and 0.7 µM respectively against ancestral Wuhan and Delta variants ( Fig. 3 ). To correlate the antiviral activity and 3CLpro enzyme inhibition the IC50 of hit compounds 9 , 10 , and 14 was determined against the 3CLpro enzyme and it was found to be 5.1 µM, 10.9 µM, and 7.3 µM respectively. A good correlation was observed between enzyme inhibition assay and antiviral activity with hit molecules 9 , 10 , and 14 . The details of enzyme inhibition and antiviral activity is mentioned in Table 1 . Table 1: 3CLpro enzyme inhibition (by FRET assay) and ancestral Wuhan & Delta variant inhibition (by plaque assay) by compounds 6-14 . The IC50 value of active compounds ( 9 , 10 , and 14 against 3CLpro and SARS-CoV-2 were determined with a starting concentration of 20 µM. Compound code % Inhibition IC50 (µM) 3CLpro (FRET) at 20 µM SARS-CoV-2 (Plaque assay) at 20 µM SARS-CoV-2 3CLpro Wuhan Delta Wuhan Delta 5a 92.95±0.2 4.7 3.4 nd nd nd 6 97.81±0.0 14.1 6.9 nd nd nd 7 99.61±0.4 40.6 34.5 nd nd nd 8 88.02±0.1 54.7 51.7 nd nd nd 9 99.63±0.5 98.31 100 7.7 7.4 5.1 10 95.64±0.7 96.61 96.49 12.6 13.8 10.9 11 94.84±0.4 6.3 0 nd nd nd 12 90.26±0.7 1.6 3.4 nd nd nd 13 91.97±0.6 28.1 24.1 nd nd nd 14 94.24±0.3 100 98.25 11.08 12.1 7.3 GC-376 100.00 - - - - Remdesivir (Positive Control) - 100 100 0.8 0.7 nd *nd= Not determined 2.4 Structure Activity Relationship: All the synthesized lead compounds ( 6-14 ) expressed promising enzyme inhibition activity from 84% to 99% in FRET assay. When these compounds were tested against ancestral Wuhan and Delta strain, the results did not correlate with the enzyme activity. The basic amidine intermediate 5a showed 4.7% and 3.4% antiviral activity against ancestral Wuhan and delta strains, respectively. Cyclization of this amidine to diaryl imidazole ( 6 ) showed improvement in the antiviral activities against both the strains (ancestral Wuhan: 14.1% & Delta: 6.9%). Introducing 3,4-dimethoxy substitution on B ring of diarylimidazole scaffold as in compound 7 showed drastic improvement in the antiviral activities. This modification improved the electron density of ring B , reduced the percentage of unsaturation of the overall molecule as well as has helped in improvement of lipophilicity of the molecule. The corresponding demethylated compound 11, however was found to have reduced antiviral activity against ancestral Wuhan strain (6.3%) and inactive against delta strain. Considerable enzyme inhibition in FRET assay, but poor activity in cell-based assay could be due to increased polarity and consequently reduced cell permeability of the molecule. This observation was also supported when its activity is compared with the basic diarylimidazole scaffold (compound 6 ) (Fig. 4) Introducing methyl substitution on ring A and retaining the dimethoxy substitution on ring B as in compound 8 showed further improvement in the antiviral activity against both the strains (ancestral Wuhan: 54.7% & Delta: 51.7%). The corresponding dihydroxy derivative (compound 12 ) showed drastically reduced antiviral activity (ancestral Wuhan: 1.6% & Delta: 3.4%). Replacement of methyl group (compound 8 ) with chloro group (compound 9, 10 ) showed better antiviral activity (compound 9 : IC 50 values of 7.7 μM and 7.4 μM; Compound 10 : 12.6 μM, 13.8 μM against Wuhan and delta strain, respectively). Demethylation of compound 9 leads to compound 13 , which showed improved enzyme inhibition, but considerably reduced viral inhibition (ancestral Wuhan: 28.1% & Delta: 24.1%). Compound 14 is demethylated compound 10 , having dichloro substitution on ring A , showed very good viral inhibition activity (ancestral Wuhan: 11.8 μM & Delta: 12.1 μM). The presence of extra chloro on ring A of compound 14 , as compared to compound 13 , could be the reason and compensated the additional lipophilicity even after demethylation of compound 14 and supported enhanced activity. Considering molecules 9 , 10 and 14 as promising lead molecules, various substitutions, and modifications at free N-H in all three leads are under progress in our lab and planned for separate communications. 2.5 Molecular Docking: To understand the interactions of synthesized molecules with 3CLpro enzyme (PDB code: 6LU7), molecular docking studies were carried out using ‘flexible docking’ protocol within BIOVIA Discovery Studio. The protocol uses a combination of components from other protocols available within BIOVIA Discovery Studio such as CDOCKER to perform the docking and is based on methods within CHARMm to sample sidechain and ligand conformations. Here the molecular interactions of identified potential hits ( 9 , 10 and 14 ) are discussed (Fig. 5). Unlike compound 6 , in all three cases, the 2-phenyl ring was observed to be stabilized in S1 subsite of receptor active site. Whereas the 4-phenyl ring of all three leads was stabilizing receptor-ligand complex by interacting with S4 subsite. The free N-H group is facing the S2 subsite. Considering the linear small molecular nature of synthesized ligands and substantially larger active site cavity, the orientation of ligands within active site can vary while having very stable interactions. The substitutions at N-H of imidazole will decide the final orientation of the ligands in the receptor active site. 3. Conclusion In summary, 2,4-Diphenyl- 1H -imidazole is a promising lead to inhibit SARS-CoV-2 3CLpro, which is a validated target for developing treatments for COVID-19. Substitution of the phenyl rings with methoxy and chloro groups increases the antiviral potency, probably via increasing the overall lipophilicity and subsequently the cell-permeability. Further these substituents are found to stabilize the molecular interactions with the active sites on SARS-CoV-2 3CLpro in virtual studies. The free NH of the imidazole ring provides additional site for further modifications in order to achieve maximum potency. 4. Experimental General: All the chemicals were acquired from S. D. fine chemicals, Spectrochem, Sigma-Aldrich, or Avra chemicals. Pre-coated silica gel TLC plates were used for reaction monitoring. All the mentioned yields are from un-optimized processes. Melting points were determined either using melting point apparatus or by differential scanning calorimetry (DSC) and are uncorrected. Using Perkin-Elmer FT-IR / Bruker spectrophotometer all IR spectra were recorded. The 1 H-NMR spectra of final lead molecules were recorded on a Bruker Advance-II 500 MHz spectrometer using DMSO- d 6 solvents and corresponding chemical shifts (δ) are expressed in parts per million (ppm). LC-MS-, Shimadzu (EI) was used to record the mass of compounds. 4.1 Chemistry: 2-Bromo-1-(substitutedphenyl)ethanone (2a – 2d) Bromine was added drop-wise to a stirred solution of an acetophenone (1 mM) ( 1a – 1d ) in ethanol (30 ml) and the solution was stirred at room temperature for 1 h and then poured into water to form a precipitate. This was re-crystallized from ethanol to give pure bromoacetophenone derivatives ( 2a – 2d ) with an 85-95% yield. 2-bromo-1-phenylethan-1-one (2a): yield 87%; m.p. 53-56 ˚C; IR (KBr, cm -1 ): 3098, 3072, 2939, 1686, 1599, 1572, 748, 687. 2-bromo-1-(p-tolyl)ethan-1-one (2b): yield 92%; m.p. 51-52 ˚C; IR (KBr, cm -1 ): 3085, 3038, 2999, 2955, 1691, 1591, 725, 665. 2-bromo-1-(4-chlorophenyl)ethan-1-one (2c): yield 95%; m.p. 94-97 ˚C; IR (KBr, cm -1 ): 3083, 3019, 2966, 2940, 1676, 1592, 798, 758. 2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (2d): yield 85%; m.p. 32-34 ˚C; IR (KBr, cm -1 ): 3082, 3010, 2965, 2940, 1681, 1585, 868, 797, 769. 3,4-Dimethoxybenzonitrile (4a, 4b) Added a solution of benzaldehyde / 3,4-dimethoxybenzaldehyde ( 3a, 3b ) (1 equiv.) in 10 mL dimethylsulfoxide (DMSO) along with hydroxylamine hydrochloride (1.2 equiv.). The reaction mixture was stirred at 110 °C for 10 hours. After the reaction completion, the reaction mixture was poured into the ice-cooled water. Filtered the obtained precipitate, washed it with water and dried it under vacuum to get 4a, 4b . Benzonitrile (4a): yield 83%; b.p. 188-190 ˚C; IR (KBr, cm -1 ): 3078, 3016, 2922, 2223, 1618, 1604, 1488, 812. 3,4-Dimethoxybenzonitrile (4b): yield 88%; m.p. 68-70 ˚C; IR (KBr, cm -1 ): 3122, 3085, 2962, 2840, 2223, 1596, 1582, 1466, 818. 3,4-Dimethoxybenzimidamide (5a, 5b) To a solution of benzonitrile / 3,4-dimethoxybenzonitrile (1 equiv.) in ethanol, dry HCl gas was purged to saturation. The resulted solution was stirred for 10 hrs. Excess HCl gas was removed and ammonium carbonate (3 equivalent) was added to it. The resulted solution was stirred further for 10 hrs and concentrated on rotary evaporator to obtain the product 5a, 5b . Benzimidamide (5a): yield 87%; m.p. 78-80 ˚C; IR (KBr, cm -1 ): 3333 (broad), 3053, 2968, 2903, 2842, 1615, 1598, 852, 808. 3,4-Dimethoxybenzimidamide (5b): yield 85%; m.p. 110-112 ˚C; IR (KBr, cm -1 ): 3305 (broad), 3210, 3089, 2996, 2835, 1644, 1606, 1591, 814. 4-(Substitutedphenyl)-2-(3,4-dimethoxyphenyl)-1 H -imidazole (6-10) A solution of benzimidamide ( 5a ) / 3,4-dimethoxybenzimidamide ( 5b ) (3 mmol), potassium bicarbonate (12 mmol) in THF (16 ml) and water (4 ml) was heated vigorously at reflux. Various bromoketone ( 2a-2d ) (3 mmol) in THF (4 ml) was added over period of 30 mins and reflux further maintained for 2 hrs. THF was then recovered, and product was washed with water and recrystallized with ethanol to get pure products 6-10 respectively. 2,4-diphenyl- 1H -imidazole (6): yield 67%; m.p. 168-170 ˚C; IR (KBr, cm -1 ): 3065, 3032, 1607, 1583, 1459, 714; 1 H NMR (DMSO- d 6 ): δ 12.63 (s, 1H, -N H ), 8.02-8.01 (d, 2H, Ar H ), 7.86-7.85 (d, 2H, Ar H ), 7.72 (s, 1H, Ar H ), 7.49-7.46 (t, 2H, -SC H 2 ), 7.40-7.35 (m, 3H, Ar H ), 7.24-7.21 (t, 1H, Ar H ); MS (m/z): 221.20 (M+H) + . 2-(3,4-dimethoxyphenyl)-4-phenyl- 1H -imidazole (7): yield 71%; m.p. 120-122 ˚C; IR (KBr, cm -1 ): 3455, 3078, 3002, 2938, 2840, 1606, 1504, 765; 1 H NMR (DMSO- d 6 ): δ 12.48 (s, 1H, -N H ), 7.86-7.85 (d, 2H, Ar H ), 7.72 (s, 1H, Ar H ), 7.61 (s, 1H, Ar H ), 7.56-7.54 (d, 1H, Ar H ), 7.38-7.35 (t, 2H, Ar H ), 7.21-7.18 (t, 1H, Ar H ), 7.06-7.04 (d, 1H, Ar H ), 3.86 (s, 3H, -OC H 3 ), 3.81 (s, 3H, -OC H 3 ); MS (m/z): 281.40 (M+H) + . 2-(3,4-dimethoxyphenyl)-4-(p-tolyl)- 1H -imidazole (8): yield 68%; m.p. 105-108 ˚C; IR (KBr, cm -1 ): 3460, 3060, 3002, 2915, 2835, 1606, 1505, 764; 1 H NMR (DMSO- d 6 ): δ 12.42 (s, 1H, -N H ), 7.73 (br, 2H, Ar H ), 7.61 (s, 1H, Ar H ), 7.57-7.56 (d, 2H, Ar H ), 7.20-7.19 (d, 2H, Ar H ), 7.05-7.04 (d, 1H, Ar H ), 3.85 (s, 3H, -OC H 3 ), 3.80 (s, 3H, -OC H 3 ), 2.31 (s, 3H, -C H 3 ); MS (m/z): 295.20 (M+H) + . 4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)- 1H -imidazole (9): yield 70%; m.p. 107-110 ˚C; IR (KBr, cm -1 ): 3457, 3000, 2920, 2835, 1590, 1495, 765; 1 H NMR (DMSO- d 6 ): δ 12.53 (s, 1H, -N H ), 7.87-7.86 (d, 2H, Ar H ), 7.77 (s, 1H, Ar H ), 7.60 (s, 1H, Ar H ), 7.56-7.54 (d, 1H, Ar H ), 7.43-7.42 (d, 2H, Ar H ), 7.06-7.04 (d, 1H, Ar H ), 3.85 (s, 3H, -OC H 3 ), 3.81 (s, 3H, -OC H 3 ); MS (m/z): 315.20 (M+H) + , 317.20 (M+H+2) + . 4-(2,4-dichlorophenyl)-2-(3,4-dimethoxyphenyl)-1H-imidazole (10): yield 73%; m.p. 189-190 ˚C; IR (KBr, cm -1 ): 3459, 3035, 3002, 2957, 2836, 1590, 1498, 765; 1 H NMR (DMSO- d 6 ): δ 12.71 (s, 1H, -N H ), 8.27-8.25 (d, 1H, Ar H ), 7.87 (s, 1H, Ar H ), 7.62 (s, 2H, Ar H ), 7.59-7.57 (d, 1H, Ar H ), 7.49-7.47 (d, 1H, Ar H ), 7.07-7.06 (d, 1H, Ar H ), 3.85 (s, 3H, -OC H 3 ), 3.81 (s, 3H, -OC H 3 ); MS (m/z): 349.25 (M) + , 351.20 (M+2) + , 353.25 (M+4) + . 4-(4-substitutedphenyl-1 H -imidazol-2-yl)benzene-1,2-diol (11-14) To obtain products 11-14 , A solution of compound 6-10 respectively (1 equiv.) in dry DCM (36 mL) at 0 °C under N 2 was treated drop wise with BBr 3 (1M in DCM, 4 equiv.). The resulting mixture was allowed to attain the RT and stirred overnight, then it was drop wise poured to a stirring ice water (50 mL). The mixture was stirred for 30 min at RT then filtered and dried to obtain the products 11-14 as a yellow solid. 4-(4-phenyl- 1H -imidazol-2-yl)benzene-1,2-diol (11): yield 73%; m.p. 98-100 ˚C; IR (KBr, cm -1 ): 3426, 3369, 3035, 2799, 1643, 1607, 1518, 686; 1 H NMR (DMSO- d 6 ): δ 14.33 (s, 1H, -N H ), 10.13 (s, 1H, broad, variable –O H proton peaks), 9.53 (s, 1H, broad, variable –O H proton peaks), 8.20-8.18 (s, 1H, Ar H ), 7.91-7.90 (d, 2H, Ar H ), 7.58-7.39 (m, 5H, Ar H ), 7.02-7.00 (d, 1H, Ar H ); MS (m/z): 253.25 (M+H) + . 4-(4-(p-tolyl)- 1H -imidazol-2-yl)benzene-1,2-diol (12): yield 69%; m.p. 118-120 ˚C; IR (KBr, cm -1 ): 3366, 3167, 2967, 1642, 1601, 1520, 715; 1 H NMR (DMSO- d 6 ): δ 14.15 (s, 1H, -N H ), 10.12 (s, 1H, broad, variable –O H proton peaks), 9.51 (s, 1H, broad, variable –O H proton peaks), 8.13 (s, 1H, Ar H ), 7.80-7.78 (d, 2H, Ar H ), 7.48 (s, 1H, Ar H ), 7.44-7.42 (d, 1H, Ar H ), 7.36-7.35 (d, 2H, Ar H ), 7.01-6.99 (d, 1H, Ar H ), 2.37 (s, 3H, -C H 3 ); MS (m/z): 267.25 (M+H) + . 4-(4-(4-chlorophenyl)- 1H -imidazol-2-yl)benzene-1,2-diol (13): yield 72%; m.p. 305-307 ˚C; IR (KBr, cm -1 ): 3301, 3276, 3135, 2972, 2888, 1639, 1601, 1510, 808, 704, 647; 1 H NMR (DMSO- d 6 ): δ 14.35 (s, 1H, -N H ), 10.13 (s, 1H, broad, variable –O H proton peaks), 9.53 (s, 1H, broad, variable –O H proton peaks), 8.23 (s, 1H, Ar H ), 7.95-7.93 (d, 2H, Ar H ), 7.65-7.63 (d, 2H, Ar H ), 7.49-7.48 (d, 1H, Ar H ), 7.45-7.43 (d, 1H, Ar H ), 7.01-6.99 (d, 1H, Ar H ); MS (m/z): 287.20 (M+H) + , 289.20 (M+H+2) + . 4-(4-(2,4-dichlorophenyl)- 1H -imidazol-2-yl)benzene-1,2-diol (14): yield 70%; m.p. 269-271 ˚C; IR (KBr, cm -1 ): 3377, 3140, 2987, 1635, 1603, 849, 808, 705; 1 H NMR (DMSO- d 6 ): δ 14.55 (s, 1H, -N H ), 10.07 (s, 1H, broad, variable –O H proton peaks), 9.51 (s, 1H, broad, variable –O H proton peaks), 8.04 (s, 1H, Ar H ), 7.89-7.88 (d, 1H, Ar H ), 7.85-7.83 (d, 1H, Ar H ), 7.69-7.67 ((d)d, 1H, Ar H ), 7.46-7.45 (d, 1H, Ar H ), 7.41-7.39 ((d)d, 1H, Ar H ), 6.99-6.97 (s, 1H, Ar H ); MS (m/z): 321.15 (M) + , 323.15 (M+2) + , 325.15 (M+4) + . 4.2. Enzyme inhibition (3CLpro) assay The synthesized compounds were screened as inhibitors of SARS-CoV-2 3CL Pro at 20 µM using an in vitro quenched fluorescence resonance energy transfer (FRET) assay using a fluorogenic substrate to measure the residual activity. For the study, the MBP-tagged 3CL Protease (SARS-CoV-2) Assay Kit (BPS Bioscience, San Diego, CA, USA) was used according to the manufacturer’s instructions [13,14]. For determination of the 3CL Pro activity, 10 µL of the compounds was pre-incubated with 30µl of the 3CL Pro for 30 min. Subsequently, the fluorogenic substrate was added to a final concentration of 50 µM and the reaction was incubated for 4 h in the dark in the presence of 1 mM 1,4-dithio-D, L-threitol (DTT). The fluorescence intensity was recorded at 460 nm / 360 nm. A positive control was included to measure the maximum activity of the protease in the absence of potential inhibitors. Moreover, an inhibition control GC 376 at 20 µM was included in the study. All the compounds were tested initially at 20 µM to establish the enzyme inhibition and antiviral activity. The hit molecules identified from the antiviral screening against SARS-CoV-2 (ancestral Wuhan and Delta) were subjected to IC50 determination against SARS-CoV-2 variants by plaque assay and 3CLpro inhibition by FRET assay. 4.3 Cell lines and viruses The African green monkey kidney epithelial cell (Vero E6) was cultured in a humidified CO 2 (5%) incubator at 37 0 C, in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 µg/mL) and Amphotericin B (0.25 µg/mL). SARS-CoV-2 isolate USA-WA1/2020 (ancestral Wuhan strain) and B.1.617.2 (Delta) were obtained from bei Resources, USA. The virus stocks were prepared by propagating in Vero E6 cells by following the standard protocol [15,16]. The virus stocks were quantified by the gold standard plaque assay (Case, et. al., 2020). The SARS-CoV-2 infection study was carried out in high containment (BSL-3) facility. 4.4 Screening and determination of IC 50 value through dose response curve generation The compounds were solubilized in dimethyl sulfoxide (DMSO) and screened against SARS-CoV-2 (ancestral Wuhan and Delta) by plaque assay. For the initial antiviral screening, Vero E6 cells were seeded at a density of ~30000 cells per well, in 96 well flat bottom tissue culture plate in 200 µL of complete DMEM. The plate was incubated for 18-24 h, at 37 0 C in a humidified CO 2 (5%) incubator. Next day, the medium was removed from the wells and the test compounds were added in duplicate to respective wells at a final concentration of 20 µM, followed by infection with SARS-CoV-2 isolates at approximately 30 plaque forming units per well. The plate was incubated at 37 0 C for 1 h, in a humidified CO 2 (5%) incubator for adsorption of virus. The DMEM supplemented with 2.5% FBS (infection medium) was used to dilute the test compounds and virus. The final volume of infection medium containing test compounds and virus was maintained at 40µL per well to maximize the virus adsorption. After 1 h of incubation, the infection medium was removed from the wells and overlayed with DMEM-CMC and incubated for 72 h and then the plates were processed to score the plaques. The controls including virus only wells (with infection and without test compound) and cell only wells (without infection and test compound) were maintained as positive and negative controls, respectively. The percentage reduction of virus in test compound treated wells were calculated in comparison to positive control (virus only well). The hit molecules identified in the initial screen were subjected to six-point dose response curve (DRC) generation (20 µM, 10 µM, 5 µM, 2.5 µM, 1.25 µM, and 0.625 µM) and IC 50 determination in Vero E6 cells. The IC 50 of the test compounds was calculated by non-regression analysis using GraphPad prism version 9.2.0. 4.5 Molecular Docking: The interactions of molecules under study with 3CLpro enzyme (PDB Code: 6LU7) were analyzed using ‘flexible docking’ protocol within BIOVIA Discovery Studio software [17]. To perform the flexible docking, residues (THR25, LEU27, HIS41, VAL42, CYS44, SER46, MET49, LEU50, TYR54, PHE140, LEU141, ASN142, SER144, CYS145, HIS163, HIS164, MET165, GLU166, LEU167, HIS172, ALA173, PHE181, VAL186, ASP187, ARG188, GLN189, THR190 and GLN192) were considered flexible in study. In the study, these residues were considered in creating flexible protein conformations using ChiFlex and side-chain refinement in the presence of the ligand using ChiRotor. In the process, generating protein confirmation was kept true with maximum number to 100. Ligand conformation generation was allowed using BEST method with maximum 255 conformations and energy threshold value to 20 for each ligand under study. Docking protocol was run with 100 number of hotspots and docking refinement was carried using simulated annealing with 2000 heating and 5000 cooling steps. The x,y,z coordinates for docking was set as -11.859595 13.885757 69.446622 respectively. Declarations Acknowledgement: Authors (AMK & AV) are thankful to SVKM’s NMIMS University for providing financial support under ‘University Seed Grant’ scheme to carry out this work. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281. The following reagent was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta Variant), NR-55611, con-tributed by Dr. Richard Webby and Dr. Anami Patel. Author contributions: AMK & AV: Designing of project, in-silico studies, synthesis of the compounds and drafting and editing the manuscript; AT: Preliminary screening of compounds; GRR, RKS, MY, RH, MS, SN: MPro inhibition assay and anti-SARS-CoV-2 activity, drafting and editing the manuscript. Statements and Declarations: Authors (AMK and AV) received financial support was received SVKM’s NMIMS under ‘University Seed Grant’ scheme for this work. References https://www.who.int/publications/m/item/covid-19-weekly-epidemiological-update---21-december-2022 (cited on 02-Jan-2023) Pelly S, Liotta D (2021) Potent SARS-CoV-2 Direct-acting antivirals provide an important complement to COVID-19 vaccines. ACS Cent. Sci 7:396−399 Unoh Y, Uehara S, Nakahara K, Nobori H, Yamatsu Y, Yamamoto S et al (2022) Discovery of S‑217622, a Noncovalent Oral SARS-CoV‑2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J. Med Chem 65:6499−6512 Fan H, Lou F, Fan J, Li M, Tong Y (2022) The emergence of powerful oral anti-COVID-19 drugs in the post-vaccine era. The lancet 3:e91 https://www.cdc.gov/coronavirus/2019-ncov/your-health/treatments-for-severe-illness.html (cited on 02-Jan-2023) Shahanshah MFH, Jain S, Sharma B, Grewall A, Swami S (2022) Comparative Analysis of B.1.617.2 (Delta) Variant of SARS-CoV-2. JMID 12(1):38-51 DeSimone RW, Currie KS, Mitchell SA, Darrow JW, Pippin DA (2004) Privileged structures: applications in drug discovery. Comb Chem High Throughput Screen 7(5):473–493 Fei F, Zhou Z (2013) New substituted benzimidazole derivatives: a patent review (2010−2012). Expert Opin Ther Pat 23(9):1157–1179 Ingle RG, Magar DD (2011) Heterocyclic chemistry of benzimidazoles and potential activities of derivatives. Int J Drug Res Technol 1:26–32 Gong KK, Tang XL, Liu YS, Li PL, Li GQ (2016) Imidazole alkaloids from the South China Sea sponge Pericharax heteroraphis and their cytotoxic and antiviral activities. Molecules 21(2):150 Kanhed AM, Patel DV, Teli DM, Patel NR, Chhabria MT, Yadav MR (2021) Identification of potential Mpro inhibitors for the treatment of COVID-19 by using systematic virtual screening approach. MolDiv 25:383–401 Patel DV, Teli DM, Kanhed AM, Patel NR et al (2021) Identification of potential Mpro inhibitors for the treatment of COVID-19 by targeted covalent inhibition: An in silico approach. IJQSPR 6(2):58-77 Morse JS, Lalonde T, Xu S, Ray Liu W (2020) Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV. ChemBioChem 21(5): 730-738 Zhang L, Lin D, Sun X, Curth U et al (2020) Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science 368(6489):409-412 Case JB, Bailey AL, Kim AS, Chen RE, Diamond MS (2020) Growth, Detection, Quantification, and Inactivation of SARS-CoV-2. Virology 548:39–48 Jureka AS, Silvas JA, Basler CF (2020) Propagation, Inactivation, and Safety Testing of SARS-CoV-2. Viruses 12(6):622 Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2022, San Diego: Dassault Systèmes, 2022 Scheme Scheme 1 is available in the Supplementary Files section. Supplementary Files Graphicalabstract.png ReactionScheme1.png Reaction Scheme 1: Syntheses of 2,4-diphenyl-1 H -imidazole derivatives. Reagents and conditions: (i) Bromine, ethanol; (ii) Hydroxylamine HCl, DMSO; (iii) (a) dry HCl, ethanol, (b) Ammonium carbonate; (iv) Potassium carbonate, THF, water; (v) BBr 3 (1M in DCM), 0 °C to RT. Supplementarydata.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Minor Revisions 09 Apr, 2024 Reviewers invited by journal 13 Mar, 2024 Reviewers agreed at journal 08 Mar, 2024 Editor assigned by journal 21 Feb, 2024 First submitted to journal 19 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3975613","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277569121,"identity":"70d9822a-0338-449a-b653-cb3934e651b3","order_by":0,"name":"Amisha Vora","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYFACxgZmGOsBA4MEmCVBrBZmAyK1AJVCaTa4Srxa+Kcdbv5cUMOQ2C99+Fl1YY5FtHwD88HbPHi0SNxObJOecYwhcWZfmtntmdskcjccYEu2xqeFAaiFmYeNwdjgDIPZbV6QFgYeM2l8WuRvJzZ/5vkH0sL+rRikZX4D/ze8WgxuJzZI87YxyBmc4TFjBmlpOMDDhleLIcgvvH0ScpI9PMXSYIcdZjO2nINHi9zt9Mefeb7Z8PDzsG/8zLutLnd+e/PDG2/weR8CkCOCGaeqUTAKRsEoGAXEAgB6kEMyVzygEgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6229-1574","institution":"Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management: NMIMS Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management","correspondingAuthor":true,"prefix":"","firstName":"Amisha","middleName":"","lastName":"Vora","suffix":""},{"id":277569122,"identity":"a201c19d-853e-40d0-a39b-1058a2379915","order_by":1,"name":"Ashish Kanhed","email":"","orcid":"","institution":"Shobhaben Pratapbhai Patel School of Pharmacy and Technology 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13:24:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3975613/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3975613/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52512226,"identity":"f527d7eb-32f5-4df2-9c48-318cd2270e6e","added_by":"auto","created_at":"2024-03-12 12:14:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":413462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003ective site of enzyme 3CLpro comprising of S1, S1’, S2 and S4 sub-regions\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/c104fea5e32342b69a0143e0.png"},{"id":52512230,"identity":"92480754-3946-4b3f-8cc3-5223c19d91d8","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77397,"visible":true,"origin":"","legend":"\u003cp\u003eInteractions of 2,4-diphenyl imidazole \u003cstrong\u003e(6)\u003c/strong\u003e with 3CLpro active site.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/d7d0f1c339212f1c6582bc3e.png"},{"id":52512229,"identity":"d4c434b7-be8a-4867-bce9-1d877911de12","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84896,"visible":true,"origin":"","legend":"\u003cp\u003eDose Response Curve (DRC) and determination of IC\u003csub\u003e50\u003c/sub\u003e against 3CLpro enzyme and SARS-CoV-2 (ancestral Wuhan strain and delta variant).\u0026nbsp; \u003cstrong\u003eA;\u003c/strong\u003e Remdesivir; \u003cstrong\u003eB;\u003c/strong\u003e Compound \u003cstrong\u003e9\u003c/strong\u003e; \u003cstrong\u003eC;\u003c/strong\u003e Compound \u003cstrong\u003e10\u003c/strong\u003e; \u003cstrong\u003eD;\u003c/strong\u003e Compound \u003cstrong\u003e14\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/1617a0dd1e5034408d0fc8e6.png"},{"id":52512231,"identity":"77d20f54-7723-4c33-ab86-024dda96107b","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79553,"visible":true,"origin":"","legend":"\u003cp\u003eSAR representation of synthesized lead molecules (\u003cstrong\u003e6-14\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/82156a40b82b52a72d2fed87.png"},{"id":52512232,"identity":"7769d829-4fdb-4744-ab66-79798851024a","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":450557,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking interactions of: (\u003cstrong\u003ea\u003c/strong\u003e) compound \u003cstrong\u003e9\u003c/strong\u003e; (\u003cstrong\u003eb\u003c/strong\u003e) compound \u003cstrong\u003e10\u003c/strong\u003e; (\u003cstrong\u003ec\u003c/strong\u003e) compound \u003cstrong\u003e14\u003c/strong\u003e with 3CLpro (PDB code: 6LU7)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/253ea16cbae706f6e8dabf85.png"},{"id":52513698,"identity":"06b8727a-b883-4dfe-b52d-143927ed6c79","added_by":"auto","created_at":"2024-03-12 12:22:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1655860,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/898602e4-c595-43c2-8c90-3d549351f65a.pdf"},{"id":52512228,"identity":"59b46c2a-c572-41d4-a2ee-855fd8ac80d5","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":350469,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/c711055c7401ec35be76afcc.png"},{"id":52512227,"identity":"01407a8e-6979-4d0f-ac34-a087eaec06a5","added_by":"auto","created_at":"2024-03-12 12:14:36","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReaction Scheme 1: \u003c/strong\u003eSyntheses of 2,4-diphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazole derivatives. \u003cstrong\u003eReagents and conditions\u003c/strong\u003e: \u003cstrong\u003e(i)\u003c/strong\u003e Bromine, ethanol; \u003cstrong\u003e(ii)\u003c/strong\u003e Hydroxylamine HCl, DMSO; \u003cstrong\u003e(iii)\u003c/strong\u003e (a) dry HCl, ethanol, (b) Ammonium carbonate; \u003cstrong\u003e(iv)\u003c/strong\u003e Potassium carbonate, THF, water; \u003cstrong\u003e(v)\u003c/strong\u003e BBr\u003csub\u003e3 \u003c/sub\u003e(1M in DCM), 0 °C to RT.\u003c/p\u003e","description":"","filename":"ReactionScheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/cef5a7313c6b1bbb90a5e628.png"},{"id":52512233,"identity":"01b33629-0d74-48bf-ab36-f1419efad143","added_by":"auto","created_at":"2024-03-12 12:14:37","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2127508,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-3975613/v1/45475bb6e61db7d9fa0a9781.docx"}],"financialInterests":"","formattedTitle":"Design and synthesis of Diphenyl-1H-imidazole analogs targeting MPro/3CLpro enzyme of SARS-CoV-2","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCOVID-19 caused by novel corona virus (SARS-CoV-2) is the major pandemic of the decade claiming approximately over 6.6\u0026nbsp;million lives, causing severe socio-economic implications globally [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Vaccination has helped to get considerable control on the spread and fatality of the condition; however, complete treatment is not available [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Certain repurposed drugs have found benefits; however, they have their own side effects and limitations. FDA has approved some anti-viral drugs to treat mild to moderate COVID-19 condition, if detected in early stages. The NIH has suggested few treatment combinations for healthcare workers, which includes Nirmatrelvir with Ritonavir, Remdesivir, Molnupiravir for different age groups. These antiviral drugs have many side effects and may show drug-drug interactions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Despite substantial efforts, there no precise cure for COVID-19 and discovery of new molecules with promising antiviral activity is required.\u003c/p\u003e \u003cp\u003eViral proteases are the potentially validated target for anti-viral drug discovery over many years. In addition to this, in case of SARS-CoV-2 virus, many variants (Wuhan, Delta, Omicron) are observed with different virulence capacity and morbidity. In all these variants, the virulence capacity varies mainly because of mutations in spike protein. However, major mutations in active site of in 3CLpro (main protease / Mpro) are not observed. Thus, the antiviral activity of 3CLpro inhibitors is expected to remain unaffected by any of the mutations of spike protein, generally observed in different SARS-CoV-2 variants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, 3CLpro is a promising target for the treatment of COVID-19.\u003c/p\u003e \u003cp\u003eIn the current study, we have identified 2,4-diphenyl-\u003cem\u003e1H\u003c/em\u003e-imidazole as a promising scaffold and explored its interactions with the active site of the SARS-CoV-2 3CLpro enzyme. Following the assessment of its enzyme inhibition potential in vitro and its antiviral activity in virus-infected cells, we proceeded to synthesize derivatives and subjected them to screening for both SARS-CoV-2 3CLpro enzyme inhibition and anti-SARS-CoV-2 activity (against both the ancestral Wuhan and Delta variants) in virus-infected cells. Dose-response curves were generated for the top three molecules to ascertain their IC50 values. Additionally, an in-silico analysis was conducted to investigate their interactions with the active site of the SARS-CoV-2 3CLpro enzyme. Detailed discussion of all these findings is provided in the subsequent sections.\u003c/p\u003e"},{"header":"2. Results \u0026 Discussion","content":"\u003cp\u003e\u003cstrong\u003e2.1 Designing aspect: \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMajority of the antiviral drugs contain heterocyclic moieties with diverse substitutions. Mainly because of its active site interaction abilities, good synthetic feasibility for derivatization and amphoteric nature, imidazole is one of the important heterocycles in the field of anti-viral drug discovery [7]. Various anti-viral agents like ledipasvir, daclatasvir (anti-hepatitis C virus), capravirine (anti-HIV), enviroxime (anti- rhinovirus and polio virus) contains imidazole scaffold. Various imidazole alkaloids (naamidine, pyronaamidine, leucettaamine) isolated from the marine sponge \u003cem\u003ePericharax heteroraphis\u0026nbsp;\u003c/em\u003ewere found effective on H1N1 influenza virus [8-10]. Apart from these, various research groups are working on imidazole-based drug discovery for viral infections like COVID-19, Dengue, Zika virus. Considering imidazole as a promising heterocycle, here we initiated imidazole-based lead identification. In the present report our focus is on substituted 2,4-diphenyl imidazole.\u003c/p\u003e\n\u003cp\u003eThe 3CLpro is cysteine protease enzyme made up of 306 amino acids having three major domains. Domain 1 is 8 \u0026ndash; 101 amino acid residue region, domain 2 is comprised of amino acids from 102 - 184, and the 3\u003csup\u003erd\u003c/sup\u003e domain is from 201 - 203 amino acids. The loop region with amino acids from 185 \u0026ndash; 200 connects domain 2 with domain 3. The active site/binding site region is present between domain 1 and 2 with a Cys145-His41 catalytic dyad. This active site region is mainly comprised of S1, S1\u0026rsquo;, S2 and S4 sub-regions. The S1 region is made up of Phe140, Leu141, Asn142, His163, Glu166 and His172 amino acids. The S1\u0026rsquo; small region is made up from S1 subsite by intrusion of Asn142 and it consist of Thr25, Thr26 and Leu27. The S2 subsite is hydrophobic and is made up of His41, Met49, Tyr54, Met165 and Asp187. The final S4 region comprises Met165, Leu167, Phe185, Gln189 and Gln192 amino acids [11,12] (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn virtual interaction studies, the 2-phenyl moiety of 2,4-diphenyl imidazole was observed in S2 binding subsite surrounded by Met49, Tyr54, Arg188 and Gln189. Whereas the 4-phenyl of same scaffold observed in S1 sub site surrounded by Phe140, Ser144 and His163. The imidazole ring was observed located centrally with free N-H facing towards S4 subsite (Fig. 2).\u003c/p\u003e\n\u003cp\u003eTo understand the enzyme inhibition potency of basic scaffold \u003cstrong\u003e6\u003c/strong\u003e, it was tested using FRET based\u0026nbsp;SARS-CoV-2 3CLpro\u0026nbsp;enzyme inhibition assay and at 20 \u0026micro;M concentration, 89.96% enzyme inhibition was observed. Further, this compound was tested against\u0026nbsp;ancestral\u0026nbsp;Wuhan and Delta variants of SARS-CoV-2 infected Vero E6 cells at the same concentration and showed 14.1% and 6.9% antiviral activity respectively. This considerably good enzyme inhibition and low to moderate antiviral activity could be because of poor cell permeability. This suggested the possibility of the scaffold for further development. Thus, we decided to make some hydrophobic and hydrophilic modifications in the scaffold to extemporize the lead activity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003eynthesis of 2, 4-diphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazole derivatives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompounds \u003cstrong\u003e6-14\u003c/strong\u003e were synthesized as depicted in \u003cstrong\u003escheme 1\u003c/strong\u003e. The starting material\u0026nbsp;2-Bromo-1-(substitutedphenyl)ethanone (\u003cstrong\u003e2a \u0026ndash; 2d\u003c/strong\u003e)\u0026nbsp;were synthesized using various acetophenone (\u003cstrong\u003e1a-1d\u003c/strong\u003e). Second starting material analogs (\u003cstrong\u003e5a, 5b\u003c/strong\u003e) were synthesized from\u0026nbsp;benzaldehyde / 3,4-dimethoxybenzaldehyde (\u003cstrong\u003e3a, 3b\u003c/strong\u003e) via benzonitrile / 3,4-dimethoxybenzonitrile (\u003cstrong\u003e4a, 4b\u003c/strong\u003e). The benzimidamide (\u003cstrong\u003e5a/5b\u003c/strong\u003e) were treated with different bromoketone (\u003cstrong\u003e2a-2d\u003c/strong\u003e) to get final compounds 4-(Substitutedphenyl)-2-(3,4-dimethoxyphenyl)-1\u003cem\u003eH\u003c/em\u003e-imidazole (\u003cstrong\u003e6-10\u003c/strong\u003e). To improve the polar properties of compounds \u003cstrong\u003e6-10\u003c/strong\u003e, \u003cem\u003eO\u003c/em\u003e-demethylation was carried to obtain 4-(4-substitutedphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (\u003cstrong\u003e11-14\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Antiviral activity of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2,4-diphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazole derivatives\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe synthesized compounds were evaluated for 3CLpro enzyme inhibition by quenched fluorescence resonance energy transfer (FRET) assay and antiviral activity against SARS-CoV-2 (ancestral\u0026nbsp;Wuhan and the Delta) by plaque assay. The FRET assay showed promising activity\u0026nbsp;by all the synthesized compounds (\u003cstrong\u003e6-14\u003c/strong\u003e) against the 3CLpro enzyme with 88% to 99% inhibition at 20 \u0026micro;M concentration. Considering the observed 3CLpro enzyme inhibition activity, all the synthesized molecules were tested against SARS-CoV-2 (ancestral Wuhan and the Delta variant) to understand viral inhibition potency. The initial screening of compounds at 20 \u0026micro;M against SARS-CoV-2 by plaque assay identified 3 hit molecules such as compounds \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e, and \u003cstrong\u003e14\u003c/strong\u003e (Table 1). Further, the IC50 of the hit molecules \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e, and \u003cstrong\u003e14\u003c/strong\u003e was found to be 7.7 \u0026micro;M, 12.6 \u0026micro;M, and 11.08 \u0026micro;M respectively against the ancestral Wuhan strain, while the IC50 against Delta variant was found to be 7.4 \u0026micro;M, 13.8 \u0026micro;M, and 12.1 \u0026micro;M respectively. The positive control remdesivir used in the antiviral assay showed an IC50 value of 0.8 \u0026micro;M and 0.7 \u0026micro;M respectively against ancestral Wuhan and Delta variants (\u003cstrong\u003eFig. 3\u003c/strong\u003e). To correlate the antiviral activity and 3CLpro enzyme inhibition the IC50 of hit compounds \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e, and \u003cstrong\u003e14\u003c/strong\u003e was determined against the 3CLpro enzyme and it was found to be 5.1 \u0026micro;M, 10.9 \u0026micro;M, and 7.3 \u0026micro;M respectively. A good correlation was observed between enzyme inhibition assay and antiviral activity with hit molecules \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e, and \u003cstrong\u003e14\u003c/strong\u003e. The details of enzyme inhibition and antiviral activity is mentioned in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e 3CLpro enzyme inhibition (by FRET assay) and ancestral Wuhan \u0026amp; Delta variant inhibition (by plaque assay) by \u003cstrong\u003ecompounds 6-14\u003c/strong\u003e.\u0026nbsp;The IC50 value of active compounds (\u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e, and \u003cstrong\u003e14\u003c/strong\u003e against 3CLpro and SARS-CoV-2 were determined with a starting concentration of 20 \u0026micro;M.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Inhibition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC50 (\u0026micro;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"30.56092843326886%\"\u003e\n \u003cp\u003e3CLpro (FRET) at 20 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"31.528046421663444%\" colspan=\"2\"\u003e\n \u003cp\u003e\u0026nbsp;SARS-CoV-2 (Plaque assay) at 20 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.63056092843327%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSARS-CoV-2\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.28046421663443%\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e3CLpro\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eWuhan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDelta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eWuhan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eDelta\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e5a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e92.95\u0026plusmn;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026nbsp;97.81\u0026plusmn;0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e14.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.61\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e40.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e34.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e88.02\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e54.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e51.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e99.63\u0026plusmn;0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e7.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e95.64\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e96.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e12.6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e13.8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e10.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94.84\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e90.26\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e91.97\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e24.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e94.24\u0026plusmn;0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e98.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e11.08\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e12.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eGC-376\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eRemdesivir (Positive Control)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*nd= Not determined\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Structure Activity Relationship:\u003c/strong\u003e All the synthesized lead compounds (\u003cstrong\u003e6-14\u003c/strong\u003e) expressed promising enzyme inhibition activity from 84% to 99% in FRET assay. When these compounds were tested against ancestral Wuhan and Delta strain, the results did not correlate with the enzyme activity. The basic amidine intermediate \u003cstrong\u003e5a\u003c/strong\u003e showed 4.7% and 3.4% antiviral activity against ancestral Wuhan and delta strains, respectively. Cyclization of this amidine to diaryl imidazole (\u003cstrong\u003e6\u003c/strong\u003e) showed improvement in the antiviral activities against both the strains (ancestral Wuhan: 14.1% \u0026amp; Delta: 6.9%). Introducing 3,4-dimethoxy substitution on B ring of diarylimidazole scaffold as in compound \u003cstrong\u003e7\u003c/strong\u003e showed drastic improvement in the antiviral activities. This modification improved the electron density of ring \u003cstrong\u003eB\u003c/strong\u003e, reduced the percentage of unsaturation of the overall molecule as well as has helped in improvement of lipophilicity of the molecule. The corresponding demethylated compound \u003cstrong\u003e11,\u0026nbsp;\u003c/strong\u003ehowever was found to have reduced antiviral activity against ancestral Wuhan strain (6.3%) and inactive against delta strain. Considerable enzyme inhibition in FRET assay, but poor activity in cell-based assay could be due to increased polarity and consequently reduced cell permeability of the molecule. This observation was also supported when its activity is compared with the basic diarylimidazole scaffold (compound \u003cstrong\u003e6\u003c/strong\u003e) (Fig. 4)\u003c/p\u003e\n\u003cp\u003eIntroducing methyl substitution on ring \u003cstrong\u003eA\u003c/strong\u003e and retaining the dimethoxy substitution on ring \u003cstrong\u003eB\u003c/strong\u003e as in compound \u003cstrong\u003e8\u003c/strong\u003e showed further improvement in the antiviral activity against both the strains (ancestral Wuhan: 54.7% \u0026amp; Delta: 51.7%). The corresponding dihydroxy derivative (compound \u003cstrong\u003e12\u003c/strong\u003e) showed drastically reduced antiviral activity (ancestral Wuhan: 1.6% \u0026amp; Delta: 3.4%). Replacement of methyl group (compound \u003cstrong\u003e8\u003c/strong\u003e) with chloro group (compound \u003cstrong\u003e9, 10\u003c/strong\u003e) showed better antiviral activity (compound \u003cstrong\u003e9\u003c/strong\u003e: IC\u003csub\u003e50\u003c/sub\u003e values of 7.7 \u0026mu;M and 7.4 \u0026mu;M; Compound \u003cstrong\u003e10\u003c/strong\u003e: 12.6 \u0026mu;M, 13.8 \u0026mu;M against Wuhan and delta strain, respectively).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDemethylation of compound \u003cstrong\u003e9\u003c/strong\u003e leads to compound \u003cstrong\u003e13\u003c/strong\u003e, which showed improved enzyme inhibition, but considerably reduced viral inhibition (ancestral Wuhan: 28.1% \u0026amp; Delta: 24.1%). Compound \u003cstrong\u003e14\u003c/strong\u003e is demethylated compound \u003cstrong\u003e10\u003c/strong\u003e, having dichloro substitution on ring \u003cstrong\u003eA\u003c/strong\u003e, showed very good viral inhibition activity (ancestral Wuhan: 11.8 \u0026mu;M \u0026amp; Delta: 12.1 \u0026mu;M). The presence of extra chloro on ring \u003cstrong\u003eA\u003c/strong\u003e of compound \u003cstrong\u003e14\u003c/strong\u003e, as compared to compound \u003cstrong\u003e13\u003c/strong\u003e, could be the reason and compensated the additional lipophilicity even after demethylation of compound \u003cstrong\u003e14\u003c/strong\u003e and supported enhanced activity. Considering molecules \u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e and \u003cstrong\u003e14\u003c/strong\u003e as promising lead molecules, various substitutions, and modifications at free N-H in all three leads are under progress in our lab and planned for separate communications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Molecular Docking:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the interactions of synthesized molecules with 3CLpro enzyme (PDB code: 6LU7), molecular docking studies were carried out using \u0026lsquo;flexible docking\u0026rsquo; protocol within BIOVIA Discovery Studio. The protocol uses a combination of components from other protocols available within BIOVIA Discovery Studio such as CDOCKER to perform the docking and is based on methods within CHARMm to sample sidechain and ligand conformations. Here the molecular interactions of identified potential hits (\u003cstrong\u003e9\u003c/strong\u003e, \u003cstrong\u003e10\u003c/strong\u003e and \u003cstrong\u003e14\u003c/strong\u003e) are discussed (Fig. 5). Unlike compound \u003cstrong\u003e6\u003c/strong\u003e, in all three cases, the 2-phenyl ring was observed to be stabilized in S1 subsite of receptor active site. Whereas the 4-phenyl ring of all three leads was stabilizing receptor-ligand complex by interacting with S4 subsite. The free N-H group is facing the S2 subsite. Considering the linear small molecular nature of synthesized ligands and substantially larger active site cavity, the orientation of ligands within active site can vary while having very stable interactions. The substitutions at N-H of imidazole will decide the final orientation of the ligands in the receptor active site.\u003c/p\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eIn summary, 2,4-Diphenyl-\u003cem\u003e1H\u003c/em\u003e-imidazole is a promising lead to inhibit SARS-CoV-2 3CLpro, which is a validated target for developing treatments for COVID-19. Substitution of the phenyl rings with methoxy and chloro groups increases the antiviral potency, probably via increasing the overall lipophilicity and subsequently the cell-permeability. Further these substituents are found to stabilize the molecular interactions with the active sites on SARS-CoV-2 3CLpro in virtual studies. The free NH of the imidazole ring provides additional site for further modifications in order to achieve maximum potency.\u003c/p\u003e"},{"header":"4. Experimental","content":"\u003cp\u003e\u003cstrong\u003eGeneral:\u0026nbsp;\u003c/strong\u003eAll the chemicals were acquired from S. D. fine chemicals, Spectrochem, Sigma-Aldrich, or Avra chemicals. Pre-coated silica gel TLC plates were used for reaction monitoring. All the mentioned yields are from un-optimized processes. Melting points were determined either using melting point apparatus or by differential scanning calorimetry (DSC) and are uncorrected. Using Perkin-Elmer FT-IR / Bruker spectrophotometer all IR spectra were recorded. The \u003csup\u003e1\u003c/sup\u003eH-NMR spectra of final lead molecules were recorded on a Bruker Advance-II 500 MHz spectrometer using DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e solvents and corresponding chemical shifts (\u0026delta;) are expressed in parts per million (ppm).\u0026nbsp;LC-MS-, Shimadzu\u0026nbsp;(EI) was used to record the mass of compounds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1 Chemistry:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-Bromo-1-(substitutedphenyl)ethanone (2a \u0026ndash; 2d)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBromine was added drop-wise to a stirred solution of an acetophenone (1 mM) (\u003cstrong\u003e1a \u0026ndash; 1d\u003c/strong\u003e) in ethanol (30 ml) and the solution was stirred at room temperature for 1 h and then poured into water to form a precipitate. This was re-crystallized from ethanol to give pure bromoacetophenone derivatives (\u003cstrong\u003e2a \u0026ndash; 2d\u003c/strong\u003e) with an 85-95% yield.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-bromo-1-phenylethan-1-one (2a):\u0026nbsp;\u003c/strong\u003eyield 87%; m.p. 53-56 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3098, 3072, 2939, 1686, 1599, 1572, 748, 687. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-bromo-1-(p-tolyl)ethan-1-one (2b):\u0026nbsp;\u003c/strong\u003eyield 92%; m.p. 51-52 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3085, 3038, 2999, 2955, 1691, 1591, 725, 665.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-bromo-1-(4-chlorophenyl)ethan-1-one (2c):\u0026nbsp;\u003c/strong\u003eyield 95%; m.p. 94-97 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3083, 3019, 2966, 2940, 1676, 1592, 798, 758.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (2d):\u0026nbsp;\u003c/strong\u003eyield 85%; m.p. 32-34 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3082, 3010, 2965, 2940, 1681, 1585, 868, 797, 769.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3,4-Dimethoxybenzonitrile (4a, 4b)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdded a solution of benzaldehyde / 3,4-dimethoxybenzaldehyde (\u003cstrong\u003e3a, 3b\u003c/strong\u003e) (1 equiv.) in 10 mL dimethylsulfoxide (DMSO) along with hydroxylamine hydrochloride (1.2 equiv.). The reaction mixture was stirred at 110 \u0026deg;C for 10 hours. After the reaction completion, the reaction mixture was poured into the ice-cooled water. Filtered the obtained precipitate, washed it with water and dried it under vacuum to get\u0026nbsp;\u003cstrong\u003e4a, 4b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBenzonitrile (4a):\u0026nbsp;\u003c/strong\u003eyield 83%; b.p. 188-190 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3078, 3016, 2922, 2223, 1618, 1604, 1488, 812.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3,4-Dimethoxybenzonitrile (4b):\u0026nbsp;\u003c/strong\u003eyield 88%; m.p. 68-70 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3122, 3085, 2962, 2840, 2223, 1596, 1582, 1466, 818.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3,4-Dimethoxybenzimidamide (5a, 5b)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo a solution of benzonitrile / 3,4-dimethoxybenzonitrile (1 equiv.) in ethanol, dry HCl gas was purged to saturation. The resulted solution was stirred for 10 hrs. Excess HCl gas was removed and ammonium carbonate (3 equivalent) was added to it. The resulted solution was stirred further for 10 hrs and concentrated on rotary evaporator to obtain the product \u003cstrong\u003e5a, 5b\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBenzimidamide (5a):\u0026nbsp;\u003c/strong\u003eyield 87%; m.p. 78-80 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3333 (broad), 3053, 2968, 2903, 2842, 1615, 1598, 852, 808.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3,4-Dimethoxybenzimidamide (5b):\u0026nbsp;\u003c/strong\u003eyield 85%; m.p. 110-112 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3305 (broad), 3210, 3089, 2996, 2835, 1644, 1606, 1591, 814.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(Substitutedphenyl)-2-(3,4-dimethoxyphenyl)-1\u003cem\u003eH\u003c/em\u003e-imidazole (6-10)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA solution of benzimidamide (\u003cstrong\u003e5a\u003c/strong\u003e) / 3,4-dimethoxybenzimidamide (\u003cstrong\u003e5b\u003c/strong\u003e) (3 mmol), potassium bicarbonate (12 mmol) in THF (16 ml) and water (4 ml) was heated vigorously at reflux. Various bromoketone (\u003cstrong\u003e2a-2d\u003c/strong\u003e) (3 mmol) in THF (4 ml) was added over period of 30 mins and reflux further maintained for 2 hrs. THF was then recovered, and product was washed with water and recrystallized with ethanol to get pure products \u003cstrong\u003e6-10\u003c/strong\u003e respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2,4-diphenyl-\u003cem\u003e1H\u003c/em\u003e-imidazole (6):\u003c/strong\u003e yield 67%; m.p. 168-170 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3065, 3032, 1607, 1583, 1459, 714; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 12.63 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 8.02-8.01 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.86-7.85 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.72 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.49-7.46 (t, 2H, -SC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e), 7.40-7.35 (m, 3H, Ar\u003cem\u003eH\u003c/em\u003e), 7.24-7.21 (t, 1H, Ar\u003cem\u003eH\u003c/em\u003e); MS (m/z): 221.20 (M+H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-(3,4-dimethoxyphenyl)-4-phenyl-\u003cem\u003e1H\u003c/em\u003e-imidazole (7):\u0026nbsp;\u003c/strong\u003eyield 71%; m.p. 120-122 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e):\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e3455, 3078, 3002, 2938, 2840, 1606, 1504, 765; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 12.48 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 7.86-7.85 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.72 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.61 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.56-7.54 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.38-7.35 (t, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.21-7.18 (t, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.06-7.04 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 3.86 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 3.81 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e); MS (m/z): 281.40 (M+H)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-(3,4-dimethoxyphenyl)-4-(p-tolyl)-\u003cem\u003e1H\u003c/em\u003e-imidazole (8):\u0026nbsp;\u003c/strong\u003eyield 68%; m.p. 105-108 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3460, 3060, 3002, 2915, 2835, 1606, 1505, 764; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 12.42 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 7.73 (br, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.61 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.57-7.56 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.20-7.19 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.05-7.04 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 3.85 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 3.80 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 2.31 (s, 3H, -C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e); MS (m/z): 295.20 \u0026nbsp;(M+H)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazole (9):\u0026nbsp;\u003c/strong\u003eyield 70%; m.p. 107-110 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3457, 3000, 2920, 2835, 1590, 1495, 765; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 12.53 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 7.87-7.86 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.77 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.60 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.56-7.54 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.43-7.42 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.06-7.04 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 3.85 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 3.81 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e); MS (m/z): 315.20 (M+H)\u003csup\u003e+\u003c/sup\u003e, 317.20 (M+H+2)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(2,4-dichlorophenyl)-2-(3,4-dimethoxyphenyl)-1H-imidazole (10):\u0026nbsp;\u003c/strong\u003eyield 73%; m.p. 189-190 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3459, 3035, 3002, 2957, 2836, 1590, 1498, 765; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 12.71 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 8.27-8.25 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.87 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.62 (s, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.59-7.57 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.49-7.47 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.07-7.06 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 3.85 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e), 3.81 (s, 3H, -OC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e); MS (m/z): 349.25 (M)\u003csup\u003e+\u003c/sup\u003e, 351.20 (M+2)\u003csup\u003e+\u003c/sup\u003e, 353.25 (M+4)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-substitutedphenyl-1\u003cem\u003eH\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (11-14)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain products \u003cstrong\u003e11-14\u003c/strong\u003e, A solution of compound \u003cstrong\u003e6-10\u003c/strong\u003e respectively (1 equiv.) in dry DCM (36 mL) at\u0026nbsp;0 \u0026deg;C\u0026nbsp;under N\u003csub\u003e2\u003c/sub\u003e was treated drop wise with BBr\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(1M in DCM, 4 equiv.). The resulting mixture was allowed to attain the RT and stirred overnight, then it was drop wise poured to a stirring ice water (50 mL). The mixture was stirred for 30 min at RT then filtered and dried to obtain the products \u003cstrong\u003e11-14\u003c/strong\u003e as a yellow solid.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-phenyl-\u003cem\u003e1H\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (11):\u0026nbsp;\u003c/strong\u003eyield 73%; m.p. 98-100 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3426, 3369, 3035, 2799, 1643, 1607, 1518, 686; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 14.33 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 10.13 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 9.53 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 8.20-8.18 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.91-7.90 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.58-7.39 (m, 5H, Ar\u003cem\u003eH\u003c/em\u003e), 7.02-7.00 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e); MS (m/z): 253.25 \u0026nbsp;(M+H)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-(p-tolyl)-\u003cem\u003e1H\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (12):\u0026nbsp;\u003c/strong\u003eyield 69%; m.p. 118-120 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3366, 3167, 2967, 1642, 1601, 1520, 715; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 14.15 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 10.12 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 9.51 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 8.13 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.80-7.78 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.48 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.44-7.42 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.36-7.35 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.01-6.99 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 2.37 (s, 3H, -C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e3\u003c/sub\u003e); MS (m/z): 267.25 \u0026nbsp;(M+H)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-(4-chlorophenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (13):\u0026nbsp;\u003c/strong\u003eyield 72%; m.p. 305-307 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3301, 3276, 3135, 2972, 2888, 1639, 1601, 1510, 808, 704, 647; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 14.35 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 10.13 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 9.53 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 8.23 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.95-7.93 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.65-7.63 (d, 2H, Ar\u003cem\u003eH\u003c/em\u003e), 7.49-7.48 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.45-7.43 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.01-6.99 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e); MS (m/z): 287.20 (M+H)\u003csup\u003e+\u003c/sup\u003e, 289.20 (M+H+2)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4-(4-(2,4-dichlorophenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol (14):\u0026nbsp;\u003c/strong\u003eyield 70%; m.p. 269-271 ˚C; IR (KBr, cm\u003csup\u003e-1\u003c/sup\u003e): 3377, 3140, 2987, 1635, 1603, 849, 808, 705; \u003csup\u003e1\u003c/sup\u003eH NMR (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): \u0026delta; 14.55 (s, 1H, -N\u003cem\u003eH\u003c/em\u003e), 10.07 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 9.51 (s, 1H, broad, variable \u0026ndash;O\u003cem\u003eH\u003c/em\u003e proton peaks), 8.04 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.89-7.88 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.85-7.83 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.69-7.67 ((d)d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.46-7.45 (d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 7.41-7.39 ((d)d, 1H, Ar\u003cem\u003eH\u003c/em\u003e), 6.99-6.97 (s, 1H, Ar\u003cem\u003eH\u003c/em\u003e); MS (m/z): 321.15 (M)\u003csup\u003e+\u003c/sup\u003e, 323.15 (M+2)\u003csup\u003e+\u003c/sup\u003e, 325.15 (M+4)\u003csup\u003e+\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Enzyme inhibition (3CLpro) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesized compounds were screened as inhibitors of SARS-CoV-2 3CL\u003csup\u003ePro\u003c/sup\u003e at 20 \u0026micro;M using an \u003cem\u003ein vitro\u003c/em\u003e quenched fluorescence resonance energy transfer (FRET) assay using a fluorogenic substrate to measure the residual activity. For the study, the MBP-tagged 3CL Protease (SARS-CoV-2) Assay Kit (BPS Bioscience, San Diego, CA, USA) was used according to the manufacturer\u0026rsquo;s instructions [13,14]. For determination of the 3CL\u003csup\u003ePro\u003c/sup\u003e activity, 10 \u0026micro;L of the compounds was pre-incubated with 30\u0026micro;l of the 3CL\u003csup\u003ePro\u003c/sup\u003e for 30 min. Subsequently, the fluorogenic substrate was added to a final concentration of 50 \u0026micro;M and the reaction was incubated for 4 h in the dark in the presence of 1 mM 1,4-dithio-D, L-threitol (DTT). The fluorescence intensity was recorded at 460 nm / 360 nm. A positive control was included to measure the maximum activity of the protease in the absence of potential inhibitors. Moreover, an inhibition control GC 376 at 20 \u0026micro;M was included in the study. All the compounds were tested initially at 20 \u0026micro;M to establish the enzyme inhibition and antiviral activity. The hit molecules identified from the antiviral screening against SARS-CoV-2 (ancestral Wuhan and Delta) were subjected to IC50 determination against SARS-CoV-2 variants by plaque assay and 3CLpro inhibition by FRET assay. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Cell lines and viruses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe African green monkey kidney epithelial cell (Vero E6) was cultured in a humidified CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(5%) incubator at 37\u003csup\u003e0\u003c/sup\u003eC, in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 \u0026micro;g/mL) and Amphotericin B (0.25 \u0026micro;g/mL).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSARS-CoV-2 isolate USA-WA1/2020 (ancestral Wuhan strain) and B.1.617.2 (Delta) were obtained from bei Resources, USA. The virus stocks were prepared by propagating in Vero E6 cells by following the standard protocol [15,16]. \u0026nbsp;The virus stocks were quantified by the gold standard plaque assay (Case, et. al., 2020). The SARS-CoV-2 infection study was carried out in high containment (BSL-3) facility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Screening and determination of IC\u003csub\u003e50\u003c/sub\u003e value through dose response curve generation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe compounds were solubilized in dimethyl sulfoxide (DMSO) and screened against SARS-CoV-2 (ancestral Wuhan and Delta) by plaque assay. For the initial antiviral screening, Vero E6 cells were seeded at a density of ~30000 cells per well, in 96 well flat bottom tissue culture plate in 200 \u0026micro;L of complete DMEM. The plate was incubated for 18-24 h, at 37\u003csup\u003e0\u003c/sup\u003e C in a humidified CO\u003csub\u003e2\u003c/sub\u003e (5%) incubator. Next day, the medium was removed from the wells and the test compounds were added in duplicate to respective wells at a final concentration of 20 \u0026micro;M, followed by infection with SARS-CoV-2 isolates at approximately 30 plaque forming units per well. The plate was incubated at 37\u003csup\u003e0\u003c/sup\u003e C for 1 h, in a humidified CO\u003csub\u003e2\u003c/sub\u003e (5%) incubator for adsorption of virus. The DMEM supplemented with 2.5% FBS (infection medium) was used to dilute the test compounds and virus. The final volume of infection medium containing test compounds and virus was maintained at 40\u0026micro;L per well to maximize the virus adsorption. After 1 h of incubation, the infection medium was removed from the wells and overlayed with DMEM-CMC and incubated for 72 h and then the plates were processed to score the plaques. The controls including virus only wells (with infection and without test compound) and cell only wells (without infection and test compound) were maintained as positive and negative controls, respectively. The percentage reduction of virus in test compound treated wells were calculated in comparison to positive control (virus only well).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe hit molecules identified in the initial screen were subjected to six-point dose response curve (DRC) generation (20 \u0026micro;M, 10 \u0026micro;M, 5 \u0026micro;M, 2.5 \u0026micro;M, 1.25 \u0026micro;M, and 0.625 \u0026micro;M) and IC\u003csub\u003e50\u003c/sub\u003e determination in Vero E6 cells. The IC\u003csub\u003e50\u003c/sub\u003e of the test compounds was calculated by non-regression analysis using GraphPad prism version 9.2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5 Molecular Docking:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interactions of molecules under study with 3CLpro enzyme (PDB Code: 6LU7) were analyzed using \u0026lsquo;flexible docking\u0026rsquo; protocol within BIOVIA Discovery Studio software [17]. To perform the flexible docking, residues (THR25, LEU27, HIS41, VAL42, CYS44, SER46, MET49, LEU50, TYR54, PHE140, LEU141, ASN142, SER144, CYS145, HIS163, HIS164, MET165, GLU166, LEU167, HIS172, ALA173, PHE181, VAL186, ASP187, ARG188, GLN189, THR190 and GLN192) were considered flexible in study. In the study, these residues were considered in creating flexible protein conformations using ChiFlex and side-chain refinement in the presence of the ligand using ChiRotor. In the process, generating protein confirmation was kept true with maximum number to 100. Ligand conformation generation was allowed using BEST method with maximum 255 conformations and energy threshold value to 20 for each ligand under study. Docking protocol was run with 100 number of hotspots and docking refinement was carried using simulated annealing with 2000 heating and 5000 cooling steps. The x,y,z coordinates for docking was set as -11.859595 13.885757 69.446622 respectively.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors (AMK \u0026amp; AV) are thankful to SVKM\u0026rsquo;s NMIMS University for providing financial support under \u0026lsquo;University Seed Grant\u0026rsquo; scheme to carry out this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe following reagent was obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2; Delta Variant), NR-55611, con-tributed by Dr. Richard Webby and Dr. Anami Patel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMK \u0026amp; AV: Designing of project, \u003cem\u003ein-silico\u003c/em\u003e studies, synthesis of the compounds and drafting and editing the manuscript; AT: Preliminary screening of compounds; GRR, RKS, MY, RH, MS, SN: MPro inhibition assay and anti-SARS-CoV-2 activity, drafting and editing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors (AMK and AV) received financial support was received SVKM\u0026rsquo;s NMIMS under \u0026lsquo;University Seed Grant\u0026rsquo; scheme for this work.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ehttps://www.who.int/publications/m/item/covid-19-weekly-epidemiological-update---21-december-2022 (cited on 02-Jan-2023)\u003c/li\u003e\n \u003cli\u003ePelly S, Liotta D (2021) Potent SARS-CoV-2 Direct-acting antivirals provide an important complement to COVID-19 vaccines. ACS Cent. Sci 7:396\u0026minus;399\u003c/li\u003e\n \u003cli\u003eUnoh Y, Uehara S, Nakahara K, Nobori H, Yamatsu Y, Yamamoto S et al (2022) Discovery of S‑217622, a Noncovalent Oral SARS-CoV‑2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19. J. Med Chem 65:6499\u0026minus;6512\u003c/li\u003e\n \u003cli\u003eFan H, Lou F, Fan J, Li M, Tong Y (2022) The emergence of powerful oral anti-COVID-19 drugs in the post-vaccine era. The lancet 3:e91\u003c/li\u003e\n \u003cli\u003ehttps://www.cdc.gov/coronavirus/2019-ncov/your-health/treatments-for-severe-illness.html (cited on 02-Jan-2023)\u003c/li\u003e\n \u003cli\u003eShahanshah MFH, Jain S, Sharma B, Grewall A, Swami S (2022) Comparative Analysis of B.1.617.2 (Delta) Variant of SARS-CoV-2. JMID 12(1):38-51\u003c/li\u003e\n \u003cli\u003eDeSimone RW, Currie KS, Mitchell SA, Darrow JW, Pippin DA (2004) Privileged structures: applications in drug discovery. Comb Chem High Throughput Screen 7(5):473\u0026ndash;493\u003c/li\u003e\n \u003cli\u003eFei F, Zhou Z (2013) New substituted benzimidazole derivatives: a patent review (2010\u0026minus;2012). Expert Opin Ther Pat 23(9):1157\u0026ndash;1179\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Ingle RG, Magar DD (2011) Heterocyclic chemistry of benzimidazoles and potential activities of derivatives. Int J Drug Res Technol 1:26\u0026ndash;32\u003c/li\u003e\n \u003cli\u003eGong KK, Tang XL, Liu YS, Li PL, Li GQ (2016) Imidazole alkaloids from the South China Sea sponge \u003cem\u003ePericharax heteroraphis\u003c/em\u003e and their cytotoxic and antiviral activities. Molecules 21(2):150\u003c/li\u003e\n \u003cli\u003eKanhed AM, Patel DV, Teli DM, Patel NR, Chhabria MT, Yadav MR (2021) Identification of potential Mpro inhibitors for the treatment of COVID-19 by using systematic virtual screening approach. MolDiv 25:383\u0026ndash;401\u003c/li\u003e\n \u003cli\u003ePatel DV, Teli DM, Kanhed AM, Patel NR et al (2021) Identification of potential Mpro inhibitors for the treatment of COVID-19 by targeted covalent inhibition: An in silico approach. IJQSPR 6(2):58-77\u003c/li\u003e\n \u003cli\u003eMorse JS, Lalonde T, Xu S, Ray Liu W (2020) Learning from the Past: Possible Urgent Prevention and Treatment Options for Severe Acute Respiratory Infections Caused by 2019-nCoV. ChemBioChem 21(5): 730-738\u003c/li\u003e\n \u003cli\u003eZhang L, Lin D, Sun X, Curth U et al (2020) Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved \u0026alpha;-ketoamide inhibitors. Science 368(6489):409-412\u003c/li\u003e\n \u003cli\u003eCase JB, Bailey AL, Kim AS, Chen RE, Diamond MS (2020) Growth, Detection, Quantification, and Inactivation of SARS-CoV-2. Virology 548:39\u0026ndash;48\u003c/li\u003e\n \u003cli\u003eJureka\u0026nbsp;AS, Silvas JA, Basler CF (2020) Propagation, Inactivation, and Safety Testing of SARS-CoV-2. Viruses 12(6):622\u003c/li\u003e\n \u003cli\u003eDassault Syst\u0026egrave;mes BIOVIA, Discovery Studio Modeling Environment, Release 2022, San Diego: Dassault Syst\u0026egrave;mes, 2022\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SARS-CoV-2, 3CLpro inhibition, Diphenyl-1H-imidazole, Wuhan variant, Delta variant","lastPublishedDoi":"10.21203/rs.3.rs-3975613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3975613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe prevailing COVID-19 pandemic, triggered by the novel coronavirus SARS-CoV-2, stands as the predominant global health crisis of the decade, claiming millions of lives and causing profound disruptions to society. Despite the rapid development of vaccines against COVID-19, the situation remains challenging, necessitating the exploration of new antiviral drugs. In this study, we present the design and synthesis of Diphenyl-1H-imidazole derivatives as a potential lead series for inhibiting the SARS-CoV-2 3CLpro enzyme. The synthesized molecules underwent screening for inhibiting the SARS-CoV-2 3CLpro enzyme at a concentration of 20µM. Compounds 6-14 exhibited inhibition ranging from 88% to 99%. Further assessments were conducted to evaluate the anti-SARS-CoV-2 activity of these compounds against both the ancestral Wuhan strain and the Delta variant in virus-infected cells. Compounds such as 4-(4-chlorophenyl)-2-(3,4-dimethoxyphenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazole \u003cstrong\u003e(9)\u003c/strong\u003e, 4-(2,4-dichlorophenyl)-2-(3,4-dimethoxyphenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazole \u003cstrong\u003e(10)\u003c/strong\u003e, and 4-(4-(2,4-dichlorophenyl)-\u003cem\u003e1H\u003c/em\u003e-imidazol-2-yl)benzene-1,2-diol \u003cstrong\u003e(14)\u003c/strong\u003eexhibited promising activity against both the Wuhan strain (with IC50 values of 7.7 µM, 12.6 µM, and 11.8 µM, respectively) and the Delta variant (with IC50 values of 7.4 µM, 13.8 µM, and 12.1 µM, respectively). Moreover, the 3CLpro inhibition IC50 values for these compounds correlated well with the observed antiviral activity, measuring at 5.1 µM \u003cstrong\u003e(9)\u003c/strong\u003e, 10.9 µM \u003cstrong\u003e(10)\u003c/strong\u003e, and 7.3 µM \u003cstrong\u003e(14)\u003c/strong\u003e. These findings underscore the efficacy of diphenyl-\u003cem\u003e1H\u003c/em\u003e-imidazole derivatives as promising candidates for further development and optimization in the fight against COVID-19.\u003c/p\u003e","manuscriptTitle":"Design and synthesis of Diphenyl-1H-imidazole analogs targeting MPro/3CLpro enzyme of SARS-CoV-2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-12 12:14:32","doi":"10.21203/rs.3.rs-3975613/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revisions","date":"2024-04-09T11:57:08+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-13T13:41:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-09T02:29:55+00:00","index":0,"fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-21T09:49:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medicinal Chemistry Research","date":"2024-02-20T04:01:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medicinal-chemistry-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcre","sideBox":"Learn more about [Medicinal Chemistry Research](https://www.springer.com/journal/44)","snPcode":"44","submissionUrl":"https://submission.nature.com/new-submission/44/3","title":"Medicinal Chemistry Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"771be3b1-b630-4cc2-8f78-f09ce3b5edd8","owner":[],"postedDate":"March 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-06T13:37:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-12 12:14:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3975613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3975613","identity":"rs-3975613","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}