key: cord-330660-tx20im6w authors: Mahmoud, Huda K.; Asghar, Basim H.; Harras, Marwa F.; Farghaly, Thoraya A. title: Nano-sized Formazan analogues: Synthesis, structure elucidation, antimicrobial activity and Docking study for COVID-19 date: 2020-10-07 journal: Bioorg Chem DOI: 10.1016/j.bioorg.2020.104354 sha: doc_id: 330660 cord_uid: tx20im6w Three series of nanosized-formazan analogues were synthesized from the reaction of dithiazone with various types of α-haloketones (ester and acetyl substituted hydrazonoyl chlorides and phenacyl bromides) in sodium ethoxide solution. The structure and the crystal size of the new synthesized derivatives were assured based on the spectral analyses, XRD and SEM data. The antibacterial and antifungal activities were evaluated by agar diffusion technique. The results showed mild to moderate antibacterial activities and moderate to potent antifungal activities. Significant antifungal activities were observed for four derivatives 3a, 3d, 5a and 5g on the pathogenic fungal strains; Aspergillus flavus and Candida albicans with inhibition zone ranging from 16 to 20 mm. Molecular docking simulations of the synthesized compounds into leucyl-tRNA synthetase editing domain of Candida albicans suggested that most formazan analogues can fit deeply forming stable complexes in the active site. Furthermore, we utilized the docking approach to examine the potential of these compounds to inhibit SARS-CoV-2 3CLpro. The results were very promising verifying these formazan analogues as a hopeful antiviral agents. According to World Health Organization (WHO) reports, the misuse of the antibiotics have led to development of Multidrug Resistance among various strains of microorganisms [1] . Accordingly, new antimicrobial agents acting on novel targets have to be developed to overwhelm the increased incidence of microbial resistance to antibiotic remedy. From the best validated antimicrobial targets are the aminoacyl-tRNA synthetase enzymes which are key enzymes in the protein translation, producing the charged tRNAs required for proper assembly of peptide chains. From these enzymes, LeuRS have been considered as a drug target in fungi and bacteria, it is reported to be inhibited in the editing site by the potent antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690) which is in clinical phases [2] [3] [4] . Recently, the outbreak of COVID-19, a new coronavirus pneumonia, caused by a new coronavirus (SARS-CoV-2) has emerged as a pandemic according to WHO in March 2020 [5] . To date, millions of infections and thousands of deaths have been recorded all over the world. In this scenario, there is a crucial need for developing antiviral agents interfering with the life cycle of the virus or with the virus replication or membrane fusion [6] . Many researchers have considered the SARS-CoV-2 major protease (Mpro), in addition called chymotrypsin-like protease (3CLpr-2) as a potential target to anti-COVID-19 drugs [7] [8] [9] . Literature survey demonstrates that a lot of formazans have been described to possess broad spectrum of biological activities and pharmacological applications [10] such as antimicrobial [11] [12] [13] [14] , antiviral [15] [16] [17] , anticancer [18] , and anti-inflammatory [19] [20] . As for instance, the formazan derivative I was reported to display 100% inhibition of the Ranikhet diseases virus [21] . Additionally, the two formazans II and III synthesized by Misra and Dhar [22] showed 87% and 83% protections against the Ranikhet disease virus, respectively. Furthermore, many formazans IV were reported by Lakshmi et al. [23] to have significant antibacterial and antifungal activities. Also, Uraz et al. [24] synthesized some formazan derivatives V, VI with different substituents and evaluated their antibacterial and antifungal activities against some selected microorganism species. The results revealed high activity against C. tropicalis, C. kefir, S. cerevisiae and C. neofarmans (Figure 1) . Recently, the synthesized drugs in the nanoscale have demonstrated a superior ability to penetrate the DNA of various diseases, which have increased their biological activity and their ability to inhibit the diseases [25, 26] . Inspired by the promising previously reported antimicrobial and antiviral activities of formazans and the urgent need of new effective antimicrobial agent and antiviral drugs to act mainly against COVID-19 and in continuation of our research work in synthesis of bioactive compounds [27] [28] [29] [30] [31] [32] , we synthesized herein a new series of nano-sized formazan analogues to study their potential as antibacterial and antifungal agents in vitro. Additionally, molecular docking simulations were done to propose their mode of binding in the editing domain of LeuRS and to show the possibility of these compounds to act against SARS-CoV-2 main protease enzyme. The first series of formazan analogues 3a-f were synthesized in sodium ethoxide solution at The XRD diffraction is a good tool to predict the size of the solid sample and the degree crystal regularity. Five sample 3d, 3f, 5g, 5h and 7b of the synthesized formazan analogues were screened over 10˚<2θ <80˚ range to determined their crystallographic features (Figures 6a-e). All investigated formazan analogues revealed sharp peaks which indicated the crystalline feature of them. The size of the crystals of the five samples was calculated according to the reported Debye-Scherrer equation [33] and the calculated size was tabulated in Table 1 . The results referred to the formazan analogues moieties synthesized in the nanometer-scale. In addition, SEM is another tool useful to give excellent insight about the crystallinity as well as surface topography for the tested solid samples. Figure 7 contains two SEM images for two formazan analogues 5h and 7b as examples for the prepared series. The two images indicated that the crystals of the two derivatives are found in the nanometer-scale. Antimicrobial activities were carried out at the Regional Center for Mycology and biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Target compounds 3a-e, 5a-h, 7b were evaluated for their in vitro antibacterial, and antifungal activities, by inhibition zone method against two gram-positive bacteria: Staphylococcus aureus (RCMB 010010) and Bacillus subtilis (RCMB 015), two gram-negative bacteria: Escherichia coli (RCMB 010052) and Proteus vulgaris (RCMB 004) and two fungi: Aspergillus flavus (RCMB 002002) and Candida albicans (RCMB 005003) using gentamycin and ketoconazole as reference antibacterial and antifungal drugs, respectively (Table 2&3 ). Concerning the antibacterial activity against gram-positive bacteria, most compounds showed mild to moderate activities. The best activities were seen for the acetyl derivatives 5a, 5b, 5c, 5g and 5h with inhibition zones values ranging from 15 to 18 mm, compared to gentamycin with IZ = 24 and 26 mm for S. aureus and B. subtilis, respectively. Regarding gram-negative bacteria, they were resistant to 3a and 3b, while 3d having 4-Br group revealed moderate activity against P. vulgaris (IZ=17 mm) compared to gentamycin (IZ=25 mm). On the other hand, compounds 5a, 5b, 5g and 5h displayed reasonable antibacterial activities against P. vulgaris with IZ values 16, 18, 17 and 18, respectively. Additionally, most compounds revealed mild antibacterial activities against the gram negative E. coli. Most of the tested formazan analogues exhibited promising antifungal activities with the exception of 3e, 5f and 7b that were inactive against used fungal strains. Focusing on the antifungal activities of compounds 3a-e, compound 3a elicited comparable antifungal activity (IZ= 17 and 18 mm against A. flavus and C. Albicans, respectively) to ketoconazole (IZ= 16 and 20 mm against A. flavus and C. Albicans, respectively). Also, significant activities were observed for compounds 3b and 3c (IZ=13-16 mm). In addition, compound 3d demonstrated superior antifungal activity against A. flavus (Z= 20 mm) that was more potent than ketoconazole. Furthermore, by studying the antifungal activities of 5a-h, moderate to potent activities were displayed. Among these compounds, compound 5g with 3-Cl group showed IZ= 20 mm against A. flavus and 18 mm against C. Albicans that were higher than those of ketoconazole. Additionally, compound 5a showed comparble activity (IZ= 16 and 19 mm) to ketoconazole. These antifungal activities results were highly appreciated since A. fumigatus is the second main cause of invasive aspergillosis and is the first leading cause of cutaneous aspergillosis [34] . In addition to the increased incidence of infections by C. albicans, the most common cause of candidiasis, and the increased resistance of C. albicans to antifungal drugs [35, 36] . Leucyl-tRNA synthetase (LeuRS) belongs to the family aminoacyl tRNA synthetases (aaRSs), group of central enzymes that play a crucial role in protein synthesis, which is vital for survival of micro-organism and hence its inhibition presented a novel and attractive target for developing antimicrobials [37] . Many recent reviews have discussed the importance of aaRSs in the discovery and development of antibacterial and antifungal agents [38] [39] [40] [41] . In this study, molecular docking study of the newly synthesized formazan analogues 3a-f, 5a-h and 7a,b have been performed onto the active site of Candida albicans editing domain of cytosolic leucyl-tRNA synthetase to demonstrate their binding affinity and orientation. Results of the docking simulation were displayed in Table 4 showing the docking scores and the different formed interactions such as hydrogen bond, pi-H and non-polar pi-cation interactions. For majority of compounds, Ala315, Lys407, and Lys483 were recognized as key amino acid residues responsible for hydrogen bonds generation and Thr316, Leu317, Arg318, Asp421and Tyr487 were identified for minor interaction. Whereas, for pi-H and pi-cation interactions, Lys407, Ser419, Lys483 and Tyr487 were the only observed residues. Focusing on the binding mode of the most active analogues as antifungals, 3a, 3d, 5a and 5g (Figure 8) , it was observed that they are well stabilized into the active site through strong it an attractive target for developing anti-coronavirus drug [42] . The protein structure of 3CLpro-2 consists of 9 α-helices and 13 β-strands making together three distinctive domains: Domain I (residues 8-101), Domain II (residues 102-184) and Domain III (residues 201-306) connected to Domain II by an extended loop (residues 185-200). 3CLpro-2 contains a catalytic dyad that is composed of conserved residues His41 and Cys145 and the key substrate-binding site is formed as a split between Domain I and Domain II [43] . To estimate the binding affinity of derivatives 3a-f, 5a-h and 7a,b with COVID-19 3CLpro (PDB code: 6LU7), molecular docking study was done. As shown in Table 5 and Gln189 indicating the importance of these hydrophobic moieties. Molecular modeling study revealed that these compounds could bind properly to C. albicans leucyl-tRNA synthetase editing domain. Additionally, docking simulation into the active site of COVID-19 3CL protease showed superior fitting into the active site with binding scores from -5.6064 to -8.0555 Kcal/mol. The melting points of all new formazan analogues were recorded using a SMP3 melting All the microbial strains that used in the current study have been supplied from Al-Azhar University in Cairo, Egypt from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB). The method used for recording the antimicrobial activity according to the literature method [44] . Molecular docking was analyzed through using MOE-Dock 2014 software [45] . Chemical structures of 3a-f, 5a-h and 7a,b were drawn by the MOE builder followed by minimization using the force field MMFF94x in this program. Hydrogen atoms were then added and unwanted water molecules were cancelled. Docking was then performed using London dG for rescoring 1 and GBVI/WSA dG for rescoring 2. At the same time, refinement was done through forcefield. "Ligand Interactions" was utilized for the 2D visualization o the proteinligand interactions. The best pose was then selected depending on the binding energy and the interactions found in the active site. cm -1 . 1 H NMR (DMSO-d 6 ) 1.60 (s, 3H Anal. Calcd. for C 22 H 19 ClN 6 OS (450.94) Calcd: C, 58 -Nitrophenyl)-2-oxopropanehydrazonic-N',2-diphenyldiazenecarbohydrazonic thioanhydride (5e) IR ύ: 3479 (br. 2NH), 1662 (CO), 1596 (C=N) IR ύ: 3436 (br 2NH), 1653 (CO), 1594 (C=N) -Chlorophenyl)-2-oxopropanehydrazonic-N',2-diphenyldiazenecarbohydrazonic thioanhydride (5g) Orange solid, yield (0.38g, 85%), mp 160-162°С (ethanol /dioxane), IR ύ: 3437 (br 2NH), 1659 (CO), 1593 (C=N) M/z (%) 452 (M + +2, 0.16), 451 (M + +1, 0.11), 450 (M + , 0.38) Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site Crystal structures of the human and fungal cytosolic Leucyl-Trna synthetase editing domains: a structural basis for the rational design of antifungal benzoxaboroles Molecular Investigation of SARS-CoV-2 Proteins and Their Interactions with Antiviral Drugs Putative Inhibitors of SARS-CoV-2 Main Protease from A Library of Marine Natural Products: A Virtual Screening and Molecular Modeling Study Molecular Docking Reveals the Potential of Aliskiren, Dipyridamole, Mopidamol, Rosuvastatin, Rolitetracycline and Metamizole to Inhibit COVID-19 Virus Main Protease TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Functionalized formazans: A review on recent progress in their pharmacological activities Synthesis, evaluation and docking studies of novel formazan derivatives as an enoyl-ACP reductase inhibitors Synthesis and evaluation of antimicrobial activity of some new Schiff bases and formazans Synthesis, characeterization and in vitro antibacterial activity of novel 3-(4-methoxyphenyl)-1-isonicotinoyl-5-(substituted phenyl)-formazans Synthesis and biological studies of some novel formazans Synthesis of some new formazans as potential antiviral agents Synthesis and antiviral activity of some new formazans Synthesis of 1-(20aryl-40-oxo-3Hquinazolyl)-3-aryl-5-phenyl-formazans as potential anti-viral agents Synthesis of some new formazans and their biological activity Novel formazans as potent anti-inflammatory and analgesic agents Antiinflammatory activity of quinazolinoformazans Synthesis of some newer formazans and tetrazolium salts as antiviral agents Synthesis of some newer formazans and tetrazolium salts and their effect on Ranikhet disease virus and the vaccinia virus Synthesis and their possible biological activities of few formazans of 3-amino-2-sulphanyl-2 Discovery of thiazole-based-chalcones and 4-hetarylthiazoles as potent anticancer agents: Synthesis, docking study and anticancer activity New azoloazine derivatives as antimicrobial agents: Synthesis under microwave irradiations, structure elucidation, and antimicrobial activity New series of thiazole derivatives: Synthesis, structural elucidation, antimicrobial activity, molecular modeling and MOE docking ZnO Nanoparticles catalyst in Synthesis of Bioactive Fused Pyrimidines as Anti-breast Cancer Agents Targeting VEGFR-2 Antitumor activity of pyrrolizines and their Cu(II) complexes: Design, synthesis and cytotoxic screening with potential apoptosis-inducing activity New and efficient approach for synthesis of novel bioactive [1,3,4]thiadiazoles incorporated with 1,3-thiazole moiety Elements of X-ray Diffraction Aspergillus flavus: human pathogen, allergen and mycotoxin producer Mechanisms of Pathogenic Candida Species to Evade the Host Complement Attack Pathogenicity and drug resistance in Candida albicans and other yeast species Hybrid phenylthiazole and 1,3,5-triazine target cytosolic leucyl-tRNA synthetase for antifungal action as revealed by molecular docking studies Aminoacyl-tRNA synthetases and their inhibitors as a novel family of antibiotics Aminoacyl-tRNA synthetases: essential and still promising targets for new anti-infective agents Aminoacyl-tRNA synthetase inhibitors as potential antibiotics Aminoacyl-tRNA synthetase inhibitors as potent antibacterials Molecular Investigation of SARS-CoV-2 Proteins and Their Interactions with Antiviral Drugs Anti-HIV drug repurposing against SARS-CoV-2 Molecular Operating Environment (MOE) 2014.09, Chemical Computing Group Inc., 1010 Sherbrooke Street West, Suite 910 ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: _______________________________________________________________________________ ______ Nano-sized Formazan analogues: Synthesis, structure elucidation, antimicrobial activity and Docking study for Harras and Thoraya A. 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