key: cord-1014342-kbk365v4 authors: Al-Wahaibi, Lamya H.; Mostafa, Ahmed; Mostafa, Yaser A.; Abou-Ghadir, Ola F.; Abdelazeem, Ahmed H.; Gouda, Ahmed M.; Kutkat, Omnia; Abo Shama, Noura M.; Shehata, Mahmoud; Gomaa, Hesham A.M.; Abdelrahman, Mostafa H.; Mohamed, Fatma A.M.; Gu, Xuyuan; Ali, Mohamed A.; Trembleau, Laurent; Youssif, Bahaa G.M. title: Discovery of novel oxazole-based macrocycles as anti-coronaviral agents targeting SARS-CoV-2 main protease date: 2021-09-17 journal: Bioorg Chem DOI: 10.1016/j.bioorg.2021.105363 sha: 0a75ce01acaadef5bb7dc1b5382a0339edd1408a doc_id: 1014342 cord_uid: kbk365v4 We have discovered a family of synthetic oxazole-based macrocycles to be active against SARS-CoV-2. The synthesis, pharmacological properties, and docking studies of the compounds are reported in this study. The structure of the new macrocycles was confirmed by NMR spectroscopy and mass spectrometry. Compounds 13, 14, and 15a-c were evaluated for their anti-SARS-CoV-2 activity on SARS-COV-2 (NRC-03-nhCoV) virus in Vero-E6 cells. Isopropyl triester 13 and triacid 14 demonstrated superior inhibitory activities against SARS-CoV-2 compared to carboxamides 15a-c. MTT cytotoxicity assays showed that the CC(50) (50% cytotoxicity concentration) of 13, 14, and 15a-c ranged from 159.1 to 741.8 μM and their safety indices ranged from 2.50 to 39.1. Study of the viral inhibition via different mechanisms of action (viral adsorption, replication, or virucidal property) showed that 14 had mild virucidal (60%) and inhibitory effects on virus adsorption (66%) at 20 μM concentrations. Compound 13 displayed several inhibitory effects at three levels, but the potency of its action is primarily virucidal. The inhibitory activity of compounds 13, 14, and 15a-c against the enzyme SARS-CoV-2 M(pro) was evaluated. Isopropyl triester 13 had a significant inhibition activity against SARS-CoV-2 M(pro) with an IC(50) of 2.58 µM. Large substituents on the macrocyclic template significantly reduced the inhibitory effects of the compounds. Study of the docking of the compounds in the SARS CoV-2-M(pro) active site showed that the most potent macrocycles 13 and 14 exhibited the best fit and highest affinity for the active site binding pocket. Taken together, the present study shows that the new macrocyclic compounds constitute a new family of SARS CoV-2-M(pro) inhibitors that are worth being further optimized and developed. The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) virus was first detected in China's Wuhan province at the end of 2019 and has greatly affected human societies due to the global pandemic. The new virus has now infected over 120 million people around the world and resulted in over 2.6 million deaths [1] [2] [3] [4] [5] . SARS-CoV-2, like SARS-CoV and MERS-CoV, belongs to the genus Beta coronavirus and is responsible for the infection of the lower respiratory tract in particular, resulting in the disease COVID-19 [1, 6] . SARS-CoV-2 is a 30,000-base pair single stranded RNA virus, which possesses an envelope containing spike (S) proteins on its surface which binds with high affinity to the human angiotensin-converting enzyme 2 (ACE2) embedded in the membrane of cells found in the lungs, heart, kidneys, intestines, and arteries. This results in the infection of these cells by the virus [1, 7] . The incubation time can vary from 2 days up to 24 days and is asymptomatic in many cases, which facilitated the virus transmission [6] . Several efficient and safe vaccines have rapidly been developed by several pharmaceutical companies [7] . The production of sufficient doses for the vaccination of the world population has proved challenging with the concomitant threat of the emergence of vaccine-resistant variants of the virus. Very few known drugs have proved effective in reducing the gravity of the disease in severely affect patients with COVID-19 [2, 6] . COVID-19-related research has been able to elucidate many druggable SARS-CoV-2 targets, including the spike (S) protein, the 3-chymotrypsin-like protease/main protease (3CL pro /M pro ), the papain-like protease (PL pro ), and the RNA-dependent polymerase [3, 7] . Of these, viral proteases PL pro and M pro constitute the most promising targets for the development of effective drugs against SARS-CoV-2. Viral proteases in coronaviruses are responsible for the development of non-structural proteins (nsps) by processing viral RNA translated polyproteins [3] [4] 7] . Thus, M pro and PL pro have acknowledged a great deal of publicity for their significant role in the enzymatic operation leading to their post-translational treatment of replicate polyproteins that are essential to the life cycle of the corona virus [8] [9] [10] [11] [12] [13] [14] [15] [16] . In the last year, several peptide-like derivatives were introduced as COVID-19 M pro inhibitors I-IX as shown in Fig. 1 [17] [18] [19] [20] [21] . Virtual screening and docking studies have played a pivotal role in the discovery of these kinds of inhibitors. On the other hand, more than 40 cyclic peptides are used clinically in treatment of different diseases [22] . These peptides came from natural and synthetic origin. Takagi et al. have reported a series of cyclic peptide as NS2B-NS3 protease inhibitor [23] . The design of these peptides was done to mimic the substrate of NS2B-NS3 protease. Among this series, the cyclic peptide X (Fig. 2 ) exhibited inhibitory activity of NS2B-NS3 protease of dengue virus at IC 50 of 0.95 μM. Aspergilli peptide D XI is a natural product isolated from Aspergillus sp and exhibited inhibitory activity against herpes simplex virus type 1 at IC 50 value of 9.5 μM [24] . The eight-residue cyclic D,L-α-peptide XII exhibited antiviral activity against adenovirus infection without an apparent adverse effect on cell viability [25] . The plausible mechanism of action of this compound could be mediated by targeting endosomal compartments in pHdependent viral infections. Liang et al. have reported a series of cyclic peptide XIII with significant antifungal and obvious antiviral activity against herpes simplex virus [26] . Among these derivatives, Simplicilliumtide J XIV exhibited significant antiviral activities against herpes simplex virus type 1 (IC 50 = 14.0 μM) [26] . The cyclic peptide ICU-1 (XI) was designed as potential anti-COVID-19 activity [27] . The design was performed using SARS coronavirus (SARS-CoV) main protease bound to a peptide substrate, which was modified to give a cyclic peptide inhibitor VI. A molecular docking study was used to illustrate the binding interactions of the designed peptide with main protease of both SARS-CoV and SARS-CoV-2. Macrocyclic peptides are notoriously promising privilege structures in drug discovery due to their improved selectivity and stability against proteolytic enzymes in addition to their higher membrane permeability compared to their linear counterparts [28, 29] . Motivated by the aforementioned data and over the course of the screening of in-house collections of molecules we discovered that a family of oxazole-based macrocycles, previously developed for other applications [30] [31] [32] possessed significant activity against SARS-CoV M pro . The large macrocyclic structures were made following the work of Rebek et al who developed this type of nature-inspired molecules for applications in supramolecular chemistry [30] and tackle the inhibition of protein-protein interactions [31, 32] (Fig. 3 We report herein the inhibitory activity of the new macrocyclic compounds 13, 14, and 15a-c ( Fig. 3) against the SARS-COV-2 main protease, their pharmacological properties in infected Very-E6 cells, and results of docking studies in the SARS-COV-2 main protease active site. The synthesis of the macrocyclic compounds is largely inspired from the synthesis of Rebek triacid platform and Galmic [30] [31] [32] . However, we prepared the required oxazole building blocks having the opposite absolute configuration to that required for the synthesis of Galmic, leading to a slightly simplified synthesis of the macrocycles. Enantiomerically pure amino acid 6 was prepared in five steps from commercially available amino diester 1 (Scheme 1). Boc-protection of this compound followed by -methylation, and kinetic resolution using pig liver esterase gave enantiomerically pure Boc-protected amino acid 4. This compound was then esterified as an isopropyl ester and the methyl ester was hydrolyzed to afford the required compound 6 in good overall yield. A colorimetric MTT assay [33] was used to assess the cytotoxicity of 13, 14, and 15a-c on Vero-E6 cells. The results showed that their CC 50 ranged from 159.1 to 741.8 μM and their safety indices ranged from 2.50 to 39.1 ( Table 1 The inhibitory effect of compounds 13, 14, and 15a-c on SARS-COV-2 (NRC-03-nhCoV) virus in Vero-E6 cells were evaluated as previously described [34] [35] [36] [37] . Results from Table 1 showing that most of the compounds tested significantly inhibited the replication of the NRC-03-nhCoV virus in a dose-dependent profile. Triester macrocycle 13 and triacid 14 demonstrated superior inhibitory activity compared to carboxamides 15a-c ( Table 2 , Fig. 4b ). The strongest anti-SARS-COV-2 compounds are 13, 14 and 15a, which showed IC 50s of 18. The percent inhibition of different mechanisms of action (viral adsorption, replication, and virucidal) for the most active compounds 13, 14 and 15a is shown in Table 2 [36] . Compound 15a has a virucidal effect of up to 90%, meaning that it acts directly on the virion and inactivates it. Furthermore, it inhibited viral replication by up to 50% at a concentration of 20 µM with a marginal effect on viral adsorption. Compound 14 had a mild viral replication effect with a 60% inhibitory effect and a 65% inhibitory effect on virus adsorption. Finally, Compound 13 displayed several inhibitory effects at three levels, but the potency of its action is primarily a virucidal influence. * The mechanism of action of the three compounds were done at concentrations higher than the halfmaximal inhibitory effect "IC 50 " to better resolve the mechanism of action. Several potential target proteins in coronavirus (COVID-19) have been isolated and are now available through protein data bank. Protease is one of these important proteins that has been targeted by a panel of inhibitors with different chemical scaffolds and were suggested as potential drugs to be developed against this challenging virus [38, 39] . Interestingly, there is no similarity in cleavage specificity of this enzyme and the corresponding human proteases which in turn makes it an ideal antiviral target with high margins of safety and selectivity [4] . Inspired by these findings, it was of interest to investigate the potential protease inhibitory activities of compounds 13, 14, and 15a-c against COVID-19. The inhibitory activity of compounds 13, 14, and 15a-c against SARS-CoV-2 M pro were evaluated following the previously published procedure [40] . Results recorded in Table 1 demonstrated that the isopropyl triester 13 (R = isopropyl) had a promising inhibition activity Rebek triacid platform as well as ester and amide derivatives are known to adopt a quasiplanar structure stabilized by six bifurcated hydrogen bonds provided by the alternating oxazole (hydrogen-bond acceptor) and trans amide (hydrogen-bond donor) groups of the macrocycle [46, 47] . The structures of compounds 13, 14 and 15a-c were prepared in Discovery Studio Visualizer using the MMFF94 force field [48] and submitted to docking via ADT on SARS-CoV-2 M pro (PDB ID: 6LU7) with the ligand N3 and water molecules being removed. Docking was initially performed using triester 13, which was found to dock in cavities of SARS-CoV-2 M pro , in particular the enzyme active site, site 2 and site 3 [ Fig. 6] . However, the docking scores for the poses docked in the active site were significantly better. This also proved true for triacid and triamides 15a-c. We therefore focused on the binding mode of these compounds in the enzyme active site. Fig. 7C and D) . Discovery studio visualizer suggests the presence of a significant anion- interaction between glutamate-166 and one of the oxazole rings of the ligand (Fig. 7E) . The docking score for this pose was -7.5 kcal/mol. Glu-189 and Asp-144 side chains also appear to lock the molecule in place. In fact, re-docking of the molecule with the enzyme where these side chains were rotated to offer additional hydrogen-bonding interactions with the ligand yielded a higher docking score of -7.8 kcal/mol (Fig. 7F) . Additional hydrophobic interactions are also seen in this case with Pro-168. Following the docking of triester 13, which has the best anti-COVID activity among the macrocycles tested, we studied the possible interactions that the enzyme could make with the other bioactive compounds triacid 14 and amide 15a (Fig. 8) . The authors declare no conflict of interest Abdulrahman University through the Fast-track Research Funding Program. General details: See Appendix A To a stirred suspension of dimethyl amino malonate hydrochloride (11.9 g, 64.26 mmol, 1eq) in CHCl 3 (50mL), NaHCO 3 (6.4g, 1.2 eq) dissolved in H 2 O (10mL), NaCl (4.54 g, 1.2 eq), and A mixture of 2 (18 g, 72. In three-liter beaker with magnetic stirrer, K 2 HPO 4 (31.23 g, 3.5eq) and KH 2 To a stirred solution of 4 (6 g, 24.27 mmol, 1 eq) in DCM (100 mL), isopropyl alcohol (3 mL, To a solution of 5 ( To a stirred solution of 6 (5 g, 18 To a solution of 8 (3.6 g, 7.74 mmol, 1 eq) in DCM (70 mL), Dess-Martin periodinane (4.26 g, 1.3 eq) was added with stirring at 0 o C for 30 mins then the temperature was warmed to rt for 4 h. Saturated solution of Na 2 S 2 O 3 (20 mL) and saturated solution of NaHCO 3 (20 mL) were added to reaction mixture with stirring at rt for 15 mins. The organic layer was separated, dried over Na 2 SO 4, and evaporated under reduced pressure to yield a crude product which was used for next step without further purification. To a solution of 9 (2.48 g, 5.34 mmol, 1 eq) in DCM (100 ml), triethylamine (TEA, 2.96 ml, 4 eq), and DMAP (0.064 g, 0.1 eq) were added with stirring for 1 h at rt. Triphenylphosphine (PPh 3 , 1.68 g, 1.2 eq) was added to reaction mixture followed by portion wise addition of iodine (1.63 g, 1.2 eq) at 0 o C. The cooling bath was removed after 30 mins and stirring was continued at rt for 5 h. The solvent was removed under reduced pressure. The residue was diluted with Et 2 O to enhance the precipitation of triphenylphosphine oxide (OPPh 3 ) which was filtered out. Pure oxazole was obtained after flash chromatography of crude product using a mixture of ethyl acetate, hexanes (1:4) as an eluent to give 2 g (84%) of 10 as a colourless oil. ν max (neat, To a vigorous stirred solution of oxazole 10 (1. A mixture of triester macrocycle 13 (0.2 g, 0.32 mmol) and 5% NaOH (8 ml A mixture of the triacid platform 14 (0. 05 g, 0.085 mmol, 1 eq), BOP (0.14 g, 3.6 eq.), and DIPEA (0.1 mL, 6 eq.) in DCM (10 mL) was stirred for 10 mins at rt before addition of an appropriate amine (3.4 eq) e.g., phenethylamine, 4-(piperidin-1-yl)phenylethylamine, 4-(N,Ndimethylamine)phenylethylamine and the resulting reaction mixture was stirred overnight at rt. After removing of the solvent in vacuo, the residue was extracted with EtOAc, washed with 5% HCl, saturated NaHCO 3 solution, brine, dried over MgSO 4 , and evaporated under reduced pressure to give a crude product which was purified by flash chromatography on silica gel using EtOAc/ hexane (1:2) as an eluent to afford the corresponding carboxamides. The crystallography coordinates of the complexes were downloaded as pdb files. The files were loaded in AutoDockTools, the ligand(s) and water molecules were removed, hydrogens and charges were added, and the structures were saved as pdbqt files. The ligands were drawn in Discovery Studio Visualizer, energy minimized by molecular mechanics (MMFF force field) and saved as mol2 files. These ligand files were loaded in AutoDockTools, hydrogen atoms and charges were added, all the relevant bonds were made rotatable, and the files were saved as pdbqt files. (Fig. 4a) and IC 50 (50% inhibitory concentration) against NRC-03-nhCoV virus in Vero-E6 cells (4b). The CC 50 and IC 50 were plotted for each tested compound using Graph Pad Prism and were calculated from the non-linear regression curve-fit analysis, relative to the virus and cell controls. Abbreviations: "CC 50 " half maximal cytotoxic concentration; "IC 50 " half maximal inhibitory concentration; "SI" Safety index * The mechanism of action of the three compounds were done at concentrations higher than the halfmaximal inhibitory effect "IC50" to better resolve the mechanism of action. Highlights  A small set of macrocyclic peptidomimetics as SARS CoV-2 main protease inhibitors was designed and synthesized.  The new targets were tested for their inhibitory effects on SARS-COV-2 (NRC-03-nhCoV) virus in Vero-E6 cells.  The inhibitory effects of the most active compounds against SARS CoV-2 main protease have been reported.  A docking study was conducted to investigate potential binding patterns and crucial interactions inside the active site of COVID-19 main protease. 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