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Synthesis and Bioactivity of Thiazolethioacetamides As Potential Metallo-Β-Lactamase Inhibitors

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Synthesis and Bioactivity of Thiazolethioacetamides As Potential Metallo-Β-Lactamase Inhibitors antibiotics Communication Synthesis and Bioactivity of Thiazolethioacetamides as Potential Metallo-β-Lactamase Inhibitors Yi-Lin Zhang 1,*, Yong Yan 1, Xue-Jun Wang 1 and Ke-Wu Yang 2 1 College of Biology Pharmacy and Food Engineering, Shangluo University, Shangluo 726000, China; [email protected] (Y.Y.); [email protected] (X.W.) 2 Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127, China; [email protected] * Correspondence: [email protected] Received: 2 February 2020; Accepted: 23 February 2020; Published: 26 February 2020 Abstract: Metallo-β-lactamase (MβLs) mediated antibiotic resistance seriously threatens the treatment of bacterial diseases. Recently, we found that thioacetamides can be a potential MβL inhibitor skeleton. In order to improve the information of the skeleton, twelve new thiazolethioacetamides were designed by modifying the aromatic substituent. Biological activity assays identify the thiazolethioacetamides can inhibit ImiS with IC50 values of 0.17 to 0.70 µM. For two of them, the IC50 values against VIM-2 were 2.2 and 19.2 µM, which is lower than in our previous report. Eight of the thiazolethioacetamides are able to restore antibacterial activity of cefazolin against E.coli-ImiS by 2–4 fold. An analysis of the structure–activity relation and molecule docking show that the style and position of electron withdrawing groups in aromatic substituents play a crucial role in the inhibitory activity of thiazolethioacetamides. These results indicate that thiazolethioacetamides can serve as a potential skeleton of MβL inhibitors. Keywords: thiazolethioacetamides; metallo-β-lactamase; inhibitor 1. Introduction Antibacterial resistance has now reached alarming levels worldwide. As classical antibiotics, β-Lactam antibiotics account for over 65% in clinical settings because of their effective antibacterial activity and selectivity [1]. Unfortunately, like other antibacterial drugs, the overuse and misuse of β-lactam antibiotics accelerates the emergence of drug-resistant strains [2]. The main mechanisms of β-lactam antibiotic resistance are due to the production of β-lactamases by bacteria, the resistant strains of which can rapidly spread on a global scale [3]. The β-lactamases can hydrolyze the β-lactam ring, which leads to a loss of antibacterial activity in antibiotics including penicillin, cephalosporins and carbapenems [4]. Based on amino acid sequence homologies, β-lactamases have been categorized into serine β-lactamases (SβLs) and metallo-β-lactamases (MβLs) [5]. SβLs use serine as an active site, existing in successful clinical inhibitors such as sulbactam, tazobactam and clavulanic acid [6]. MβLs can be further categorized into subclasses B1-B3, which utilize either one or two equivalents of Zn(II) as the active site [7]. MβLs currently threaten almost all β-lactamase antibiotics in treating bacterial infection. The rapid diffusion of NDM-1-producing K.pneumoniae isolates is a striking example [8]. Because of variations in MβL structures, clinical MβL inhibitors are still lacking. Over the past several years, the literature has provided various approaches for overcoming antibiotic resistance and a number of MβL inhibitors have been designed including sulfonamides, dicarboxylate, β-lactams, cyclic boronates and multivalent chelators [1,9–11]. Sulfur-containing compounds occupy an important position in the Antibiotics 2020, 9, 99; doi:10.3390/antibiotics9030099 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 99 2 of 7 Antibiotics 2020, 9, 99 2 of 7 design of M βLL inhibitors inhibitors because because the sulf sulfurur atom can reduce the M βLsLs activity activity by by binding binding to the zinc zinc ionsions which which are are enzyme active center and replacing the bridging waterwater moleculesmolecules [[12,13].12,13]. Recently, our group has reported that thioacetamid thioacetamidee derivatives exhibit biological activity which which may inhibitinhibit M MβLsβLs [14 [14–17]–17]. In. addition,In addition, some some of the of thioacetamides the thioacetamides showed broad-spectrumshowed broad-spectrum inhibitory inhibitoryactivity against activity all threeagainst subclasses all three ofsubclasses MβLs. In of order MβLs. to developIn order the to structure–activitydevelop the structure–activity relationships, relationships,twelve new thiazolethioacetamides twelve new thiazolethioacetamides were synthesized andwere characterized. synthesized Theand inhibitory characterized. activity wasThe inhibitoryevaluated againstactivity MwasβLs evaluated VIM-2, ImiS, against and M L1,βLs which VIM-2, are ImiS, representatives and L1, which of the are B1, representatives B2 and B3 subclasses of the B1,of MB2β Ls,and respectively.B3 subclasses Theof M abilityβLs, respectively. of these thiazolethioacetamides The ability of these thiazolethioacetamides to defend against the to resistant defend againstbacterial the strain resistant was evaluatedbacterial strain by a minimumwas evaluate inhibitoryd by a minimum concentrations inhibitory (MICs) concentrations assay. Furthermore, (MICs) assay.molecular Furthermore, docking was molecular employed docking when studying was empl theoyed possible when interactions studying betweenthe possible the inhibitors interactions and betweenthe corresponding the inhibitors Mβ Ls.and the corresponding MβLs. 2. Results Results To acquire eeffectiveffective MMβL inhibitors, twelve diaryl-substituteddiaryl-substituted thiazolethioacetamides were synthesized as shown in the Supporting Information and characterized by NMR and MS. The yields of the compounds ranged from from 56 56.9%.9% to 87.4% and the structures of these compounds are shown in Figure 11.. Cl Cl O O O OH OH OH S S S S S S N N Cl N Cl H H H N N N N N N 12 3 Cl Cl Cl NO OH O O 2 OH OH O S S S S S S N N NH Cl H H N N N N NO2 N N 45 6 F Cl NO2 OCF3 O OH O OH O OH S S S S S S N N N H H H N N N N N N 78 9 F F OH O OH O OH O S S S S S S N F NH N H H N N N N N N F 10 11 12 Figure 1. StructuresStructures of of the the synthesi synthesizedzed thiazolethioacetamides. thiazolethioacetamides. β β To testtest thethe inhibitory inhibitory activity activity of compoundsof compounds 1–12 1–12 against against M Ls, M threeβLs, three representative representative M Ls, M VIM-2βLs, (B1), ImiS (B2), and L1 (B3), were chosen for evaluation. The IC50 values of the compounds against VIM-2 (B1), ImiS (B2), and L1 (B3), were chosen for evaluation. The IC50 values of the compounds β againstM Ls with MβLs cefazolin with cefazolin as the substrate as the substr are listedate are in listed Table 1in. TheTable inhibition 1. The inhibition studies indicated studies indicated that the thatthiazolethioacetamides the thiazolethioacetamides had specific had inhibitoryspecific inhibito activityry againstactivity ImiSagainst and ImiS VIM-2, and thoughVIM-2, nonethough of none them ofshowed them showed any activity any againstactivity L1against until L1 the un inhibitortil the inhibitor concentration concentration reached reached 1 mM. 1 mM. Table 1. IC50 values of thiazolethioacetamides against MβLs ImiS and VIM-2. Table 1. IC50 values of thiazolethioacetamides against MβLs ImiS and VIM-2. IC50(µM) IC50(µM) Compds IC50(μM) Compds IC50(μM) Compds ImiS VIM-2Compds ImiS VIM-2 ImiS VIM-2 ImiS VIM-2 1 0.58 - 7 0.42 - 1 20.58 0.53 - - 87 0.310.42 2.2 - 2 30.53 0.69 - - 98 0.460.31 - 2.2 3 40.69 0.58 - - 109 0.610.46 - - 4 50.58 0.17 - - 1110 0.680.61 - - 6 0.36 - 12 0.70 19.2 5 0.17 - 11 0.68 - 6 0.36 - 12 0.70 19.2 Antibiotics 2020, 9, 99 3 of 7 It can be observed that compounds 1–12 exhibited an ability to inhibit ImiS with an IC50 value range of 0.17–0.70 µM, while compounds 8 and 12 also showed inhibitory activity against VIM-2 with an IC50 value 2.2 and 19.2 µM. When the substituent was at the p-position of the benzene ring, compound 8 with trifluoromethoxy had a lower IC50 value of 0.31 µM for ImiS than compound 1 containing chlorine (0.58 µM) and compound 9 containing fluorine (0.46 µM). The number and position of chlorine atoms had little effect on the inhibitory activity of compounds 1–4 which had almost the same IC50 value. However, with compounds 9–12, fluorine in the p-position improved the ability of the compounds to inhibit ImiS. In addition, compounds 5–7 with chlorine and nitro inhibited ImiS with a better IC50 value range of 0.17–0.42 µM, whilst compound 5 gave the lowest IC50 value of 0.17 µM among all the compounds. The nitro group may bind selectively with the active site of the enzyme. The capacity of thiazolethioacetamides to restore the antibacterial activity of cefazolin against E.coli BL21 (DE3) cells expressing ImiS and VIM-2 was investigated by determining the minimum inhibitory concentrations (MIC). No compounds had synergistic bacteriostatic effect on E.coli and E.coli-VIM-2 with cefazolin, and the results to inhibit E.coli-ImiS are shown in Table2. Compounds 5–12 resulted in a 2–4 fold reduction of MIC value for E.coli-ImiS in vivo. Inhibitors 1–4 did not change the MIC value relative to the blank control. Table 2. Minimum inhibitory concentrations (MIC)(µg/mL) value of cefazolin against E. coli-ImiS in the presence of thiazolethioacetamides. Compds E.coli-ImiS Compds E.coli-ImiS Compds E.coli-ImiS Blank 20 5 10 10 5 1 20 6 10 11 10 2 20 7 10 12 10 3 20 8 5 4 20 9 5 In order to explore how the inhibitors bind to MβLs, compounds 8 and 12 were docked into the active pocket of VIM-2 (PDB code 4NQ2), whilst 5 and 8 were docked into CphA (PDB code 2QDS).
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