molecules Article Thiazole Analogues of the Marine Alkaloid Nortopsentin as Inhibitors of Bacterial Biofilm Formation Anna Carbone †,‡, Stella Cascioferro † , Barbara Parrino, Daniela Carbone , Camilla Pecoraro, Domenico Schillaci , Maria Grazia Cusimano, Girolamo Cirrincione and Patrizia Diana * Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Università degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy; [email protected] (A.C.); [email protected] (S.C.); [email protected] (B.P.); [email protected] (D.C.); [email protected] (C.P.); [email protected] (D.S.); [email protected] (M.G.C.); [email protected] (G.C.) * Correspondence: [email protected] † Present address: Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy. ‡ These authors contributed equally to this work. Abstract: Anti-virulence strategy is currently considered a promising approach to overcome the global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm formation is recognized as one of the most relevant. Considering the high and growing percentage of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of counteracting the formation of biofilms are urgently required. In this scenario, a new series of 18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923 and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably, Citation: Carbone, A.; Cascioferro, S.; five compounds exhibited BIC50 values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 µM. Parrino, B.; Carbone, D.; Pecoraro, C.; The new compounds, affecting the biofilm formation without any interference on microbial growth, Schillaci, D.; Cusimano, M.G.; can be considered promising lead compounds for the development of a new class of anti-virulence Cirrincione, G.; Diana, P. Thiazole Analogues of the Marine Alkaloid agents. Nortopsentin as Inhibitors of Bacterial Biofilm Formation. Molecules Keywords: antibiofilm agents; antibiotic resistance; thiazole derivatives; marine alkaloids ana- 2021, 26, 81. https://dx.doi.org/ logues; nortopsentin 10.3390/molecules26010081 Academic Editor: Manuel Simões Received: 30 October 2020 1. Introduction Accepted: 23 December 2020 The development of synthetic small molecules able to counteract antibiotic resistance Published: 27 December 2020 (AMR) mechanisms is urgently needed [1]. In fact, most antibiotics used to date to treat the most common infections are becoming ineffective. Many bacteria, including the well- Publisher’s Note: MDPI stays neu- known ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, tral with regard to jurisdictional claims Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) evolved in highly in published maps and institutional affiliations. resistant forms through different mechanisms that include the inactivation of the antibiotic, chemical modification of the antibiotic target, alteration of cell permeability, and biofilm formation [2]. In particular, bacterial biofilm is currently considered one of the most relevant virulence factors, which is capable of making pathogens up to 1000 times more Copyright: © 2020 by the authors. Li- resistant than their planktonic form [3]. It was estimated that more than 80% of chronic censee MDPI, Basel, Switzerland. This infections are caused by biofilm formation on indwelling medical devices or host tissues [4]. article is an open access article distributed Biofilm is a complex multicellular structure in which bacterial cells are embedded in a under the terms and conditions of the matrix constituted of extracellular polymeric substance (EPS), which is mainly formed by Creative Commons Attribution (CC BY) polysaccharides, proteins, lipids, extracellular DNA (e-DNA), and molecules originating license (https://creativecommons.org/ from the host including mucus and DNA [5]. In the past decade, many efforts have licenses/by/4.0/). Molecules 2021, 26, 81. https://dx.doi.org/10.3390/molecules26010081 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 16 Molecules 2021, 26, 81 2 of 16 nating from the host including mucus and DNA [5]. In the past decade, many efforts have been made for identifying new therapeutic strategy able to eradicate bio- beenfilm-associated made for identifying infections, [6] new and, therapeutic despite numerous strategy able compounds to eradicate being biofilm-associated described as po- infections,tent anti-biofilm [6] and, agents, despite no numerous new derivative compounds has reached being describedthe clinic. asThe potent lack anti-biofilmof approved agents,anti-biofilm no new drugs derivative together has with reached the theincrease clinic. in The the lack spread of approved of chronic anti-biofilm biofilm-related drugs togethernosocomial with infections the increase make in the the research spread of in chronic this field biofilm-related particularly relevant. nosocomial infections makeAmong the research the bioactive in this field scaffolds particularly recently relevant. described for their interesting anti-biofilm properties,Among thiazole the bioactive derivatives scaffolds are recentlyconsidered described among forthe theirmost interestingpromising anti-biofilmcompounds properties,[7]. thiazole derivatives are considered among the most promising compounds [7]. Sulfur-containingSulfur-containing heterocycles heterocycles are are often often involved involved in attractivein attractive nonbonding nonbonding interactions interac- thattions play that an play important an important role in role the in control the control of molecular of molecular conformation. conformation. In comparison In comparison with otherwith other five-membered five-membered heterocycles, heterocycles, the thiazole the thiazole nucleus nucleus has uniquehas unique features features due due to the to presencethe presence of the of low-lying the low-lying C−S σ C*− orbitals.S σ* orbitals. The small The regionssmall region of lows electronof low electron density presentdensity onpresent the sulfur on the atom, sulfur known atom, as knownσ-holes, as areσ-holes, often involvedare often ininvolved drug–target in drug–target interactions, interac- thus improvingtions, thus theimproving affinity towardthe affinity the biologicaltoward the receptor biological [8]. receptor [8]. ManyMany thiazolethiazole compoundscompounds werewere reportedreported inin thethe lastlast decadedecade asas potentpotent anti-biofilmanti-biofilm agents.agents. TheThe 4-(4-(oo-methoxyphenyl)-2-aminothiazoles-methoxyphenyl)-2-aminothiazoles1a,b 1a,b(Figure (Figure1) were1) were found found to beto ablebe able to significantlyto significantly inhibit inhibitP. aeruginosa P. aeruginosabiofilm biofilm formation formation at concentrations at concentrations in the low in the micromolar low mi- range,cromolar interfering range, interfering with the quorum with the sensing quorum (QS) sensing system (QS) [9]. Thesystem thiazole [9]. The derivatives thiazole2a,b de- (Figurerivatives1) showed2a,b (Figure potent 1) anti-biofilm showed potent activity anti-bio againstfilm eight activity methicillin-resistant against eight methicil- (MRSE) andlin-resistant two reference (MRSE) (ATCC and 12228,two reference ATCC 35984) (ATCC strains 12228, of StaphylococcusATCC 35984) epidermidisstrains of Staphylo-eliciting BICcoccus50 values epidermidis ranging eliciting from BIC 0.3550 to values 7.32 µ rangingg/mL [ 10from]. 0.35 to 7.32 µg/mL [10]. OCH3 HO R N S N N R S N N Cl S H H 1a, R=5-Br 2a, R=Cl 1b, R=2-NH 2b, R=F R S N N N N R1 R2 3 FigureFigure 1.1. ChemicalChemical structuresstructures ofof thethe thiazolethiazole compoundscompounds 1a,b1a,b;; 2a,b2a,b;; andand3 3.. OnOn thethe basis basis of of the the interesting interesting anti-biofilm anti-biofilm properties properties described described for theforthiazole the thiazole scaf- foldscaffold and and continuing continuing our searchour search for new for new nortopsentin nortopsentin alkaloid alkaloid analogues analogues with with promising prom- biologicalising biological activity activity [11–13 ],[11–13], we recently we recently reported reported the synthesis the synthesis and the and anti-biofilm the anti-biofilm activity ofactivity the new of nortopsentinthe new nortopsentin analogues analogues of type 3, inof whichtype 3 the, in imidazolewhich the nucleus imidazole of the nucleus natural of compoundthe natural wascompound replaced was by replaced the thiazole by the ring, thia andzole the ring, indole and the moiety indole in positionmoiety in 4 posi- was replacedtion 4 was by replaced 7-azaindole by 7-azaindole (Figure1)[ (Figure14]. The 1) thiazole[14]. The derivativesthiazole derivatives3 were tested 3 were against tested S. aureus ATCC 25923, S. aureus ATCC 6538, and P. aeuruginosa ATCC 15442 in order to against S. aureus ATCC 25923, S. aureus ATCC 6538, and P. aeuruginosa ATCC 15442 in evaluate their ability to inhibit biofilm formation and microbial growth. Most of the new order to evaluate their ability to inhibit biofilm formation and microbial growth. Most of thiazole nortopsentin analogues proved to be