key: cord-0855851-s8iq7gnw authors: Ahmed, Tasmia; Rahman, S. M. Abdur; Asaduzzaman, Muhammad; Islam, Abul Bashar Mir Md. Khademul; Chowdhury, A. K. Azad title: Synthesis, in vitro bioassays, and computational study of heteroaryl nitazoxanide analogs date: 2021-06-04 journal: Pharmacol Res Perspect DOI: 10.1002/prp2.800 sha: 133f8fbdd064049f38eff3313b4248ee10afb22d doc_id: 855851 cord_uid: s8iq7gnw Antiprotozoal drug nitazoxanide (NTZ) has shown diverse pharmacological properties and has appeared in several clinical trials. Herein we present the synthesis, characterization, in vitro biological investigation, and in silico study of four hetero aryl amide analogs of NTZ. Among the synthesized molecules, compound 2 and compound 4 exhibited promising antibacterial activity against Escherichia coli (E. coli), superior to that displayed by the parent drug nitazoxanide as revealed from the in vitro antibacterial assay. Compound 2 displayed zone of inhibition of 20 mm, twice as large as the parent drug NTZ (10 mm) in their least concentration (12.5 µg/ml). Compound 1 also showed antibacterial effect similar to that of nitazoxanide. The analogs were also tested for in vitro cytotoxic activity by employing cell counting kit‐8 (CCK‐8) assay technique in HeLa cell line, and compound 2 was identified as a potential anticancer agent having IC(50) value of 172 µg which proves it to be more potent than nitazoxanide (IC(50) = 428 µg). Furthermore, the compounds were subjected to molecular docking study against various bacterial and cancer signaling proteins. The in vitro test results corroborated with the in silico docking study as compound 2 and compound 4 had comparatively stronger binding affinity against the proteins and showed a higher docking score than nitazoxanide toward human mitogen‐activated protein kinase (MAPK9) and fatty acid biosynthesis enzyme (FabH) of E. coli. Moreover, the docking study demonstrated dihydrofolate reductase (DHFR) and thymidylate synthase (TS) as probable new targets for nitazoxanide and its synthetic analogs. Overall, the study suggests that nitazoxanide and its analogs can be a potential lead compound in the drug development. Nitazoxanide (NTZ) or 2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide ( Figure 1 ) belongs to 5-nitrothiazole group of molecules which has already received ample attention in the field of drug discovery and drug development. 1,2 NTZ possessed broad spectrum of activity against protozoa, 3, 4 helminthes, 5, 6 and numerous Grampositive and Gram-negative anaerobic bacteria. [6] [7] [8] NTZ also exhibited potential antiviral properties 9,10 and, recently, it has been found to be effective against SARS-CoV-2 11 and a number of clinical trials are underway. 12, 13 Because of the potential biological properties, structurally modified NTZ-based analogs or structurally related molecules were synthesized and investigated for their biological activity. Some recently synthesized analogs of NTZ reportedly possess prominent antiprotozoal, [14] [15] [16] [17] antibacterial, and antimycobacterial 18, 19 and antiviral 20 activities even in some cases better than the parent drug NTZ itself. Hence, NTZ became the drug of interest for our research where we focused on synthesis of hetero-aryl amide analogs. Although, NTZ and some of its analogs were investigated for a wide range of biological activities, heteroaryl amide analogs were not screened for several bioactivities. Therefore, we planned to synthesize some heteroaryl amide analogs ( Figure 1 ) to evaluate their diverse biological activities such as antibacterial, antiviral, anticancer activity, in order to prove them to be a potential therapeutic choice. Based on the in vitro biological activities, we also conducted in silico molecular docking study to understand a mechanistic insight regarding the activity of NTZ and its synthetic analogs. So far, we have investigated in vitro antibacterial, cytotoxic and in vivo analgesic and anti-inflammatory effects of NTZ and four synthesized analogs. Although, the analogs did not produce significant analgesic and anti-inflammatory effects, we found some interesting results in antibacterial and cytotoxic screening which we are going to disclose in this paper. All the synthetic procedures were conducted in Chemical Biology and DNA Synthesis Laboratory, Faculty of Pharmacy, University of Dhaka. The solvents were dried and properly distilled. Progress of the reactions was monitored by Thin Layer Chromatography (TLC), and visualization was accomplished by using UV light at 254 nm. For column chromatography, silica gel 60 (0.06-0.2 mm, ROTH) was employed. 1 H NMR (400 MHz) was recorded on Ultra Shield Bruker 400 NMR instrument, using DMS0-d6, and the chemical shifts are reported as δ (ppm) with respect to tetramethylsilane (TMS) (ppm) as internal standard. Fourier-transform infrared (FT-IR) Spectra were recorded with FT-IR 8400S Shimadzu spectrophotometer in the range of 4000-400 cm −1 using KBr pressed pellet technique. All the reagents used in synthetic procedure were purchased from Sigma-Aldrich, Germany. Nitazoxanide, metronidazole, and nalidixic acid were procured from Incepta Pharmaceuticals Ltd. as dried powder. Triethylamine (TEA) (1.2 equiv.) was added to the solution of 2-amino-5-nitrothiazole (0.00689 mol) and dichloromethane (DCM). After the mixture was stirred for 15 min at 5℃, a solution of hetero aryl acid chloride (1.1 equiv.) in dichloromethane was added dropwise. The reaction mixture was stirred at room temperature for 24 h and after the completion of reaction indicated by TLC, the resulting residue was neutralized by saturated NaHCO 3 solution. The mixture was then extracted with ethyl acetate. The organic layer was washed with brine solution. The solvent was removed under reduced pressure and crude product was then subjected to column chromatography for purification. The structure of the synthetic analogs of nitazoxanide was elucidated spectroscopically by using IR and 1 H NMR and by comparing the data with that of the reported data. 17 NTZ and its four analogs were assayed for antibacterial activity against a gram-positive Staphylococcus aureus (S. aureus -coagulase (+) ve ATCC: 20121107-4) and a gram-negative (E. coli ATCC: 0157-CR3) strain by the standardized disc diffusion method. 22 All the test samples were dissolved in DMSO (0.1% v/v) and diluted to prepare four concentrations (100, 50, 25, and 12.5 µg/ml) for each test sample. Nalidixic acid was used as the standard drug. The zone of inhibition was compared with standard drug after 24 h of incubation at 37°C for antibacterial activity. HeLa, a human cervical carcinoma cell line, was used for the cytotoxicity assay. Here the assay was designed in two phases. Firstly, the quantity of cell viability was determined by Trypan blue dye exclusion technique. 23 Then MTT method was performed to determine the IC 50 value (50% growth inhibitory concentration) from the calculation of percent growth inhibition. Hela cell line was cultured in DMEM (Dulbecco's Modified Eagles' medium) containing 1% penicillin-streptomycin (1:1), 0.2% gentamycin, and 10% fetal bovine serum (FBS) and was incubated at 37°C with an atmosphere of 5% CO 2 . Cell Counting Kit-8 (CCK-8), a non-radioactive colorimetric cell proliferation and cytotoxic assay kit (Sigma-Aldrich), was employed for the in vitro cytotoxicity test. 24, 25 Cells were seeded onto 96-well plates at a concentration of (2 × 10 4 /100 µl) and incubated at 37°C with an atmosphere of 5% CO 2 for 24 h. Each sample measuring 25 µl (filtered) was added into each well in duplicate. Positive control (NTZ) and compounds 2 and 4 (500 and 100 μg/ml) were dissolved in DMSO (0.1% v/v). Cells were periodically checked for granularity, shrinkage, and swelling using trinocular microscope with camera (Optika) during the incubation period of 48 h. After incubation, 10 μl of CCK-8 (5 mg/ml) solution was added to each well followed by incubation at 37°C for 4 h for cytotoxicity. The viable cells were visualized by the presence of purple color formazan dye. As the amount of produced formazan dye is directly proportional to the number of living cells, the measurement of absorbance value will give the number of viable cells. 23, 24 The absorbance values were measured using a microplate reader at 570 nm wavelength where DMSO was used as blank. The percentage growth inhibition was calculated using the following where, A t = Mean absorbance value of test compound, A b = Mean absorbance value of blank, A c = Mean absorbance value of control. Percentage of inhibition is plotted against the drug concentration, and IC 50 value is determined. The current protocol followed 'rigid ligand-rigid receptor' dock- Nitazoxanide's accession no. in Drug Bank database, DB00507, 29 CASTp, 30 and DoGSiteScorer 31 were used to predict the active sites of the target proteins. The structures of the target proteins were converted into PDBQT format after performing necessary modifications in AutoDoc Tools (ADT) (version 1.5.6). 32 The ligands were also converted into PDBQT format with Open Bable tool. 33 Finally, the docking of the ligands with the target proteins was done by using the Autodock Vina (version 1.1.2). 34 Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 35 and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. 36 In the present work, 2-amino-5-nitrothiazole has been used as the starting material which was conjugated with various hetero arylchloride using well known Schotten Baumann 37,38 reaction in the presence of triethylamine (TEA) and dichloromethane (DCM) to produce the hetero aryl amide analogs of NTZ (Table 1) . During synthesis of different analogs, 5-nitro thiazole group was kept constant as drug activation largely depends on the redox potential of the 5-nitro group, because reduction of 5-nitro group by nitroreductases, including pyruvate ferredoxin oxidoreductase (PFOR), is responsible for NTZ's mechanistic activation and, likewise, spectrum of activity. 39 Moreover, heterocyclic rings (e.g. thiophene and furan) were incorporated replacing the benzene ring in the synthetic analogs to understand the role of hetero aryl residues. According to the synthetic method described above, four nitazoxanide analogs have been synthesized ( Table 1 ). The analogs designated as compound 1, 2, 3 and 4 were produced having a good yield 91%, 89%, 70%, and 53%, respectively. Structures of the synthesized compounds were elucidated by analyses of their high resolution 1 H-NMR and FT-IR spectroscopic data and were further confirmed by comparing their spectral data to that of the published values. 17, 21 The spectral features are in close agreement with the published data. In vitro antibacterial effect of the four NTZ analogs on E. coli and S. aureus was observed on the basis of differential concentration of the test samples. E. coli was found to be more sensitive toward the synthetic (Table 5) . For instance, in case of PFOR, all the compounds along with NTZ showed an excellent interaction (−7 to −9 kcal/mol) with Thr31, Glu64, Arg114, and Asp996, the active site of PFOR. NTZ demonstrates its bioactivity by blocking PFOR, an essential enzyme for energy metabolism in anaerobes, leading to bacterial and protozoal death. 45, 46 Among the synthetic compounds, 2 and 4 showed better interaction than the other compounds ( Figure 2 ). This computational finding is in consistence with a previous study by Scior, 15 where a notable binding affinity was reported by the author between their synthetic NTZ analogs and PFOR. Like PFOR, pyruvate dehydrogenase (PDH) also plays the same role in glucose consumption and energy metabolism via oxidation of pyruvate in aerobic organism (e.g. E. coli) and mammals. 17 NTZ had been reported to inhibit 35 to 80% growth of E. coli in a dose-dependent manner by inhibiting PDH. 46 This fact is well supported by our excellent docking score ranging from −7 to −9.1 kcal/mol, for NTZ and the analogs toward PDH (Table 5 , Figure 3A and compound 4 ( Figure 3 ) showed stronger affinity than NTZ toward MAPK9. Another two cancer signaling proteins, glutathione-S-transferase of the Pi class (GSTPI) 50 and mechanistic target of rapamycin complex 1 (mTORC1) 40, 43 have been reported to be a promising mamma- Interestingly, the synthesized analogs and NTZ also displayed a notable interaction (−6.4 to −7.7 kcal/mol) with DHFR in its NADPH binding site and with TS (−5.9 to −7.1 kcal/mol) in its active site Cys195 (Figure 3 ). These two enzymes are essential for cell proliferation and cell growth, 51 and inhibition of them causes disruption of DNA synthesis and cell death consequently. 52, 53 Therefore, the mentioned molecular affinity of the compounds toward DHFR and TS makes them a potential novel target for NTZ and its synthetic analogs in anticancer therapy. From the docking scores provided in Table 5 In most of the cases, compound 2 and 4 accounted for stronger binding affinity with the target proteins than do the other two compounds (Table 5 ). In fact, compound 2 and compound 4 showed better affinity than the parent drug NTZ against MAPK9 and FabH. We have synthesized four heteroaryl analogs of NTZ by condensing 2-amino-5-nitrothiazole with some acid chlorides. Among the four synthesized analogs of NTZ, compound 2 and compound 4 displayed better antibacterial activity than NTZ against E. coli. Additionally, cytotoxicity assay in HeLa cell line demonstrated greater cell growth inhibition for compound 2, which was proved to be more potent than NTZ. This is the very first report of cytotoxic as well as antibacterial activities of these synthesized compounds. The results of in vitro bioassays further corroborated with the molecular docking study that helped exploring probable mechanistic insights underlying their bioactivity. Moreover, the computational investigation identified DHFR and TS as potential novel targets for NTZ and its synthesized analogs. Although further detail experiments are warranted, these findings are unique and hence should draw attentions of structural, as well as chemical biologists, for future drug development considering nitazoxanide along with its analogs for repurposing it as a potential anticancer agent besides its conventional antibacterial and antiprotozoal uses. In addition, the analogs might be effective as antiviral agent against SARS-CoV-2 and, therefore, synthesis of new series of molecules followed by antibacterial, antiviral, and cytotoxicity assays will be focused in the future work. In addition, our future study will also focus on the in vivo and biochemical investigation of the synthesized analogs. No ethical permission required for this in vitro and in silico studies. Research (BCSIR) for providing facilities for 1 H NMR and Centre of Advanced Research in Sciences (CARS), University of Dhaka, for their support in antibacterial and cytotoxic assays. None. The data that support these results are available from the corresponding author upon reasonable request. S. M. Abdur Rahman https://orcid.org/0000-0002-9963-8885 Update on nitazoxanide: a multifunctional chemotherapeutic agent Structure-function relationship of thiazolides, a novel class of anti-parasitic drugs, investigated in intracellular and extracellular protozoan parasites and larval-stage cestodes Randomized clinical study of nitazoxanide compared to metronidazole in the treatment of symptomatic giardiasis in children from Northern Peru Efficacy of nitazoxanide to treat natural Giardia infections in dog Treatment of diarrhea caused by Cryptosporidium parvum: a prospective randomized, doubleblind, placebo-controlled study of nitazoxanide Nitazoxanide, a potential drug for eradication of Helicobacter pylori with no crossresistance to metronidazole In vitro and in vivo activities of nitazoxanide against Clostridium difficile Parish T nitazoxanide is active against mycobacterium leprae Effect of nitazoxanide for treatment of severe rotavirus diarrhoea: randomised doubleblind placebo-controlled trial Nitazoxanide: a first-in-class broad-sectrum antiviral agent Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Nitazoxanide/azithromycin combination for COVID-19: a suggested new protocol for early management Repurposing therapeutic agents against SARS-CoV-2 infection: most promising and neoteric progress Synthesis of benzologues of nitazoxanide and tizoxanide: a comparative study of their in vitro broad-spectrum antiprotozoal activity Antiprotozoal nitazoxanide derivatives: synthesis, bioassays and QSAR study combined with docking for mechanistic insight Synthesis and antiprotozoal activity of nitazoxanide-N-methylbenzimidazole hybrids Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide Nitazoxanide analogues as antimicrobial agents against nosocomial pathogens Development of 5-nitrothiazole derivatives: identification of leads against both replicative and latent Mycobacterium tuberculosis Second-generation nitazoxanide derivatives: thiazolides are effective inhibitors of the influenza A virus Synthesis and antimicrobial evaluation of nitazoxanide-based analogues: identification of selective and broad spectrum activity Antibiotic susceptibility testing by a standardized single disk method Trypan blue dye exclusion test of cell viability A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability A water-soluble tetrazolium salt useful for colorimetric cell viability assay Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays The protein data bank PubChem substance and compound databases Drug Bank 3.0: a comprehensive resource for 'omics' research on drugs CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues DoGSiteScorer: a web server for automatic binding site prediction, analysis and druggability assessment Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function Open Babel: an open chemical toolbox AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading The IUPHAR/BPS Guide to PHARMACOLOGY in 2019: updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY The Concise guide to pharmacology 2019/20: introduction and other protein targets Ueber die oxydation des piperidins Ueber eine einfache Methode der Darstellung von Benzoësäureäthern Enzymes associated with reductive activation and action of nitazoxanide, nitrofurans, and metronidazole in Helicobacter pylori Research perspective: potential role of nitazoxanide in ovarian cancer treatment. Old drug, new purpose? Three-dimensional cell culture-based screening identifies the anthelmintic drug nitazoxanide as a candidate for treatment of colorectal cancer Synergistic tumor inhibition of colon cancer cells by nitazoxanide and obeticholic acid, a farnesoid X receptor ligand. Cancer Gene Ther A functional perspective of nitazoxanide as a potential anticancer drug Comparing protein-ligand docking programs is difficult Towards a new therapeutic target: Helicobacter pylori flavodoxin Antiparasitic drug nitazoxanide inhibits the pyruvate oxidoreductases of Helicobacter pylori, selected anaerobic bacteria and parasites, and Campylobacter jejuni Synthesis, molecular docking and QSAR studies of 2, 4-disubstituted thiazoles as antimicrobial agents Design, synthesis and biological evaluation of metronidazole-thiazole derivatives as antibacterial inhibitors Computational prediction of the protein kinase that mediates the anti-HCV effect of nitazoxanide. Doctoral dissertation Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim axis and reveals glutathione-S-transferase P1 as Achilles' heel Structure, dynamics, and catalytic function of dihydrofolate reductase Antifolate drug selection results in duplication and rearrangement of chromosome 7 in Plasmodium chabaudi Additional 5-FU-LV significantly increases survival in gastrointestinal cancer Synthesis, in vitro bioassays, and computational study of heteroaryl nitazoxanide analogs