European Journal of Medicinal Chemistry 242 (2022) 114678 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: www.elsevier.com/locate/ejmech Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens Andromeda-Celeste Gómez a, b, 1, Thérèse Lyons c, 1, Uwe Mamat d, 1, Daniel Yero a, b, Marc Bravo a, b, Xavier Daura a, e, f, Osama Elshafee g, Sascha Brunke g, Cormac G.M. Gahan c, h, i, Michelle O’Driscoll c, j, k, Isidre Gibert a, b, **, Timothy P. O’Sullivan c, j, k, * a Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain b Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain c School of Pharmacy, University College Cork, Cork, Ireland d Cellular Microbiology, Research Center Borstel, Leibniz Lung Center, Parkallee 4a, 23845 Borstel, Germany e Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain f Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Barcelona, Spain g Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology – Hans Knoell Institute, Jena, Germany h School of Microbiology, University College Cork, Cork, Ireland i APC Microbiome Ireland, University College Cork, Cork, Ireland j School of Chemistry, University College Cork, Cork, Ireland k Analytical and Biological Chemistry Research Facility, University College Cork, Cork, Ireland A R T I C L E I N F O A B S T R A C T Keywords: Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat Furanones to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for Biofilms example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from Quorum sensing marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report Salmonella enterica Staphylococcus aureus the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several Pseudomonas aeruginosa opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. Escherichia coli A number of the most potent compounds were subjected to further analysis by confocal laser-scanning micro­ Stenotrophomonas maltophilia scopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial Candida albicans biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model. intervening decades, so too has the problem of antimicrobial resistance 1. Introduction (AMR). The World Bank has calculated that AMR could lead to ten million deaths each year by 2050, accompanied by an economic impact The discovery and development of antibiotics represents one of the comparable to the 2008 financial collapse [6]. Over the next 35 years, major advances in human health [1,2]. Bacterial infections, which were 300 million are predicted to die prematurely as a result of AMR [7]. The previously potentially fatal, were suddenly made readily treatable [3,4]. six leading pathogens for deaths associated with resistance are Escher­ However, even at the very early stages of the so-called ‘golden age’ of ichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus antibiotics, some warning signs were noted. In 1947, the first bacterial pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, in strain resistant to penicillin was identified and within a year of its that order [8]. They constitute important opportunistic pathogens and clinical introduction, resistant strains of Staphylococci were isolated in are a frequent cause of nosocomial infections. patient samples [5]. This challenge has been made all the greater by problems with the As the use, overuse and misuse of antibiotics has increased over the antibiotics pipeline where few new classes of antimicrobials have come * Corresponding author. School of Pharmacy, University College Cork, Cork, Ireland. ** Corresponding author. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain. E-mail addresses: Isidre.Gibert@uab.cat (I. Gibert), tim.osullivan@ucc.ie (T.P. O’Sullivan). 1 These authors contributed equally. https://doi.org/10.1016/j.ejmech.2022.114678 Received 7 May 2022; Received in revised form 8 August 2022; Accepted 10 August 2022 Available online 20 August 2022 0223-5234/© 2022 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Abbreviations PAR P. aeruginosa PAR7244 PBS Phosphate-buffered saline AHL Acyl Homoserine Lactone PCR Polymerase chain reaction AI-2 Autoinducer-2 QS Quorum sensing AMR Antimicrobial Resistance RFP Red fluorescence protein CAL C. albicans M2396 RBF Round bottomed flask CLSM Confocal laser-scanning microscopy SAU S. aureus Newman DME Dimethoxyethane SEN S. enterica sv. Typhimurium DMSO Dimethyl sulfoxide SMA S. maltophilia K279a ECO E. coli ATCC 9637 SOB Super Optimal Broth LB Lysogeny broth SPhos 2-Dicyclohexylphosphino-2′ ,6′ -dimethoxybiphenyl NOESY Nuclear Overhauser Effect Spectroscopy TSB Tryptic Soy Broth PAO P. aeruginosa PAO1 YPD Yeast Extract–Peptone–Dextrose to market in recent years. Only three new classes of antibiotics have and Staphylococcus epidermis [23]. We have previously reviewed the been launched since 1990, and for one of these, the oxazolidiones, structure-activity relationships of these bioactive furanones, although resistance was identified soon after their introduction [9]. the impact of structure on activity does vary considerably depending on Taking these factors into account, it is clear that new strategies are the microbe under investigation [24]. It has been suggested that these required. One such strategy involves targetting the resistance mecha­ compounds may constitute potential QS inhibitors. There is strong evi­ nisms of pathogenic bacteria, such as the production of biofilms. dence that halogenated furanones, such as 2 and 3, interfere with the Biofilm-producing bacteria can be over 1000-fold more resistant to an­ AI-2 (4) signalling system present in several species [16,25]. For timicrobials than their planktonic equivalents [10]. Additionally, the US example, Zang et al. demonstrated that certain furanones deactivate the National Institute of Health has noted that 80% of persistent infections LuxS enzyme which plays a key role in the production of AI-2 [26]. Some in patients are associated with bacterial biofilms [11]. As biofilms are evidence also exists that these compounds can inhibit AHL-dependent often polymicrobial in nature, the innate resistance of one population phenotypes [27,28]. can reduce the susceptibility of another population in the biofilm lead­ Herein, we describe the synthesis of a range of structurally diverse ing to “herd-protection” [12]. Such polymicrobial infections are closely furanones using a variety of palladium-cataysed coupling reactions. associated with diseases such as cystic fibrosis, ear and urinary in­ These compounds are evaluated for their inhibitory effects on biofilm fections, respiratory tract infections, diabetic ulcers, wounds, as well as production in opportunistic human pathogens such as S. enterica, with certain medical devices [13]. S. aureus, P. aeruginosa, E. coli and S. maltophilia, as well as the fungal Biofilms are regulated by quorum sensing (QS), an intercellular pathogen C. albicans. The impact of furanone structure on biological communication system, which influences microbial virulence, second­ activity is also discussed. ary metabolites production and DNA uptake [14,15]. QS is coordinated by small signalling molecules known as autoinducers (AI). Autoinducers 2. Results and Discussion are biosynthesised by bacteria until their extracellular concentration reaches a particular threshold, which then triggers a change in gene 2.1. Chemistry expression [16]. One of the main autoinducers in Gram-negative bac­ teria are the N-acylated-L-homoserine lactones (AHLs). Autoinducer-2 We have previously described the preparation of gem-dibromofur­ (AI-2) is an intra- and interspecies communication molecule which has anone 3 from maleic anhydride via a modified Ramirez olefination [29]. been identified in both Gram-negative and -positive bacteria. The Subsequent palladium-mediated coupling of 3, in the presence of the development of new antimicrobial agents that interfere with bacterial SPhos ligand, was attempted with a variety of boronic acids (Table 1). In quorum sensing, thereby inhibiting biofilm formation and increasing general, monoarylated furanones resulted when 1.1 equivalents of the antibiotic sensitivity, has been the focus of significant research [17]. boronic acid were employed and the reaction conducted at room tem­ De Nys et al. have previously reported the isolation of halogenated perature (entries 1, 4, 6, 8, 10). These couplings proceeded with high furanones from the marine alga Delisea pulchra [18]. A number of these stereoselectivity, with formation of the Z-isomers favoured. Similar molecules were subsequently found to inhibit colonisation of the algal selectivity has been previously noted with comparable gem-di­ surface and were identified as potential anti-fouling agents, with bro­ bromoolefin substrates [30–32]. The preparation of diarylated fur­ mofuranone 1 being recognised as one of the more promising candidates anones was achieved by increasing the equivalents of boronic acid from (Fig. 1) [19]. This discovery prompted further research into this class of 1.1 to 3, and by raising the reaction temperature to 100 ◦ C (entries 2, 5, compounds in the intervening years. A large number of halogenated 7, 9, 11, 15). The introduction of a m- or p-nitro group to the arylboronic furanones, and related synthetic analogues, were later discovered to acid was associated with significantly reduced reactivity, and couplings inhibit biofilm formation in a variety of bacterial pathogens including failed to proceed at room temperature. When these reactions were Salmonella enterica sv. Typhimurium [20], P. aeruginosa [21], E. coli [22] repeated at 100 ◦ C, a mixture of mono- and diarylated furanones were recovered (entries 12, 13). Attempted coupling of p-hydrox­ yphenylboronic acid under these conditions was not achieved and pro­ todeboronation instead afforded phenol as the major by-product. By adapting the conditions developed by Noteberg and colleagues [33], where Pd(OAc)2/SPhos was replaced with Pd(PPh3)2Cl2 and DME/e­ thanol used in place of toluene, bis(phenol)-substituted furanone 21 was produced in 58% yield under microwave irradiation (entry 14). Dibromoolefination of methylsuccinic anhydride (23) afforded a mixture of regioisomeric products (Scheme 1). 4-Methyl-substituted Fig. 1. Comparison of AI-2 with furanone-based quorum sensing inhibitors. gem-dibromofuranone 24 was isolated in 46% yield along with minor 2 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Table 1 Synthesis of arylated furanones via Suzuki coupling. Entry R1 R1B(OH)2 Temp. (◦ C) Conditions Product R2 Yield (eq.) 1 1.1 r.t. i 5 Br 32% 2 3 100 ii 6 82% 3 1.1 50 ii 7 Br 59% 8 7% 4 1.1 r.t. i 9 Br 67% 5 3 100 ii 10 75% 6 1.1 r.t. i 11 Br 29% 7 3 100 ii 12 83% 8 1 r.t. i 13 Br 15% 9 3 100 ii 14 86% 10 1 10 i 15 Br 26% 11 3 100 ii 16 84% 12 3 100 ii 17 Br 24% 18 11% 13 3 100 ii 19 Br 20% 20 8% (continued on next page) 3 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Table 1 (continued ) 14 3 120 iii 21 58% 15 3 100 ii 22 25% i. Pd(OAc)2 (3 mol%), SPhos (2 mol%), K3PO4 (2 eq.), PhMe, 48 h; ii. Pd(OAc)2 (1 mol%), SPhos (2 mol%), K3PO4 (3 eq.), PhMe, 3 h; iii. Pd(PPh3)2Cl2 (8 mol%), Na2CO3 (3 eq.), m.w., DME/EtOH 4:1, 40 min. Table 2 Introduction of bromine onto the furanone ring. Scheme 1. Preparation of methylated gem-dibromofuranone 24. product 25 in 31% yield. 25 has been previously investigated as a QS Entry R Solvent Product Yield inhibitor and was not evaluated in this study [22]. 1 Et2O 29 81% The addition of an allylic halide to a furanone ring has been linked to improved QS activity [20,22,34]. With that in mind, a modified Ramirez olefination of anhydride 26, prepared using a procedure described by 2 Et2O 30 36% Reynauld et al. [35], was attempted (Scheme 2). Treating 26 with a combination of carbon tetrabromide and triisopropyl phosphite fur­ nished dibromoolefin 27 in 36% yield as the major product. Known furanone 28 was also identified as a minor product but proved impos­ Et2O 31 34% sible to isolate as a pure compound. 27 could be distinguished from regioisomer 28 by comparison of the chemical shift of H-3 at 6.58 ppm in 27 which was significantly upfield of the equivalent signal in 28 [35]. Several groups have investigated ring-brominated furanones as po­ 3 CHCl3 32 76% tential QS inhibitors [20,22,23,34,36]. Accordingly, a number of dia­ rylated furanones were initially treated with bromine at reflux, followed by addition of triethylamine at 0 ◦ C, to produce the corresponding ring-brominated furanones in high overall yields (Table 2). Bromination was found to proceed with high regioselectivity (entries 1–3), with the boronic acid proceeded more readily, with 36 recovered in 76% yield halogen introduced into the 3-position exclusively as confirmed by again as its Z-isomer. gem-Dichlorofuranone 34 proved to be a less correlations between H-4 and the aromatic protons in 2D-NOESY reactive substrate than gem-dibromofuranone 3, as reflected by the spectra. The electron-donating effect of a 4-methoxy group led to the complete absence of diarylated equivalents of 35 or 36 after 48 h. formation of two products, with concomitant ortho-bromination of the Furanones bearing alkyne substituents have exhibited promising QS activated aromatic rings being observed (entry 2). Bromination of 4-tri­ inhibition against P. aeruginosa [36]. Sonogashira coupling of dibro­ fluoromethylphenyl-substituted furanones in diethyl ether proceeded moolefin 3 with 2.2 equivalents of 4-ethynylanisole at 80 ◦ C furnished sluggishly with significant amounts of unreacted substrate recovered. 37 exclusively in 34% yield under solvent-free conditions (Scheme 4). Gratifyingly, switching to higher boiling point chloroform saw complete Attempted isolation of the corresponding monoalkynyl-substituted consumption of starting materials with a yield of 76% recorded (entry analogue using 1.1 equivalents of 4-ethynylanisole was ultimately un­ 3). successful, as the product was found to co-elute with trace amounts of Chlorine- and iodine-containing furanones have received far less 37. attention to date than their brominated analogues [24]. For example, gem-dichlorofuranone 33, prepared by addition of trichloroacetate to 2.2. Biofilm inhibition activity of furanone library succinic anhydride at reflux and subsequent acidification with concen­ trated sulfuric acid [37,38], has never been investigated for its QS ac­ Our library of novel furanones was next evaluated for their effects on tivity (Scheme 3). A subsequent microwave-mediated Finkelstein different microorganisms of public health significance. We first tested reaction transformed 33 into gem-chloroiodofuranone 34 in 43% yield. their impact on microbial growth. Most of the compounds assessed did Alternatively, Suzuki coupling of 33 with phenyl boronic acid furnished not impair growth significantly (Fig. 2). The largest effects were 35 solely as the Z-isomer in 50% yield. Coupling with 4-methoxyphenyl observed in the fungal pathogen C. albicans, with control furanones 2 and 3 inhibiting growth to the greatest extent. By contrast, both strains of P. aeruginosa proved largely insensitive to our library. These findings are of interest, as an ideal QS disruptor should minimally impact mi­ crobial growth, thus avoiding increased evolutionary pressure on the target. S. enterica is a foodborne bacterium that colonises the gastrointes­ tinal tract of infected individuals [39]. Tribromofuranone 27, which incorporates an allylic bromide in addition to the common dibromoo­ Scheme 2. Preparation of allylic bromide 27. lefin motif, was the most active compound and significantly reduced 4 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Scheme 3. Synthesis of chlorine- and iodine-containing furanones. Scheme 4. Synthesis of 37 via Sonogashira coupling. Fig. 3. Percentage biofilm inhibition by individual furanones. Heat map of mean biofilm inhibition values from at least three independent experiments for S. enterica sv. Typhimurium (SEN), S. aureus Newman (SAU), E. coli ATCC 9637 (ECO), S. maltophilia K279a (SMA), P. aeruginosa PAO1 (PAO), P. aeruginosa PAR7244 (PAR) and C. albicans M2396 (CA). For C. albicans M2396, inhibition of biofilm formation was analysed by sustained (CAS) and developmental (CAD) inhibition assays. The scale displays 0%–100% biofilm inhibition at 50 μM concentration of each furanone. Controls contained the same volume of neat DMSO. furanones have traditionally been underexplored for their effects on bacterial biofilms. Interestingly, gem-chloroiodofuranone 34 was highly active with 59% inhibition recorded. Activity in S. enterica was quite structure specific, with most of our library having little or no impact on biofilm formation. Control 3 was found to reduce bacterial growth, along with 27 and 34 to a lesser extent (Fig. 2 & Supplementary Figure A1). Fig. 2. Percentage growth inhibition by individual furanones. Heat map of S. aureus is a Gram-positive opportunist bacterium that colonises the mean growth inhibition values from at least three independent experiments for skin and mucosae of approximately 30% of the human population [40]. S. enterica sv. Typhimurium (SEN), S. aureus Newman (SAU), E. coli ATCC 9637 S. aureus causes a wide range of illnesses including impetigo, endo­ (ECO), S. maltophilia K279a (SMA), P. aeruginosa PAO1 (PAO), P. aeruginosa carditis, osteoarticular infection, pneumonia and toxic shock syndrome, PAR7244 (PAR) and C. albicans M2396 (CAL). The scale displays 0%–100% as well as prosthetic device and catheter infections [41]. growth inhibition at 50 μM concentration of each furanone. Controls contain Methicillin-resistant S. aureus (MRSA) is currently responsible for 10 the same volume of neat DMSO. times more infections than all other multi-drug resistant Gram-negative pathogens combined [42]. MRSA poses a major challenge as current biofilm formation in S. enterica sv. Typhimurium by 72% at 50 μM antimicrobial therapies are often associated with poor outcomes. As concentration (Fig. 3 & Supplementary Figure B1). Known gem-di­ with S. enterica, tribromofuranone 27 was the most potent molecule, bromofuranone 3, which was used as a control in this study, displayed significantly inhibiting S. aureus Newman biofilm production by 71% comparable activity with 67% inhibition. Chlorine-substituted (Fig. 3 & Supplementary Figure B2). Of the remaining candidates, 5 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 bis-4-hydroxyphenyl-substituted furanone 21 was the next most effica­ inhibitor in PAO1, had no impact on biofilm formation in PAR7244. As cious compound with 52% inhibition recorded. The structurally similar with PAO1, bacterial growth in P. aeruginosa PAR7244 was mostly un­ 4-methoxyphenyl-substituted analogue 12 displayed a comparable de­ affected by our library (Supplementary Figure A6). gree of inhibition, and reduced biofilm growth by 44%. Neither of these While C. albicans is prevalent in the gastrointestinal and genitouri­ compounds contains a halogen atom, which is often considered a pre­ nary tracts of healthy individuals, it may become pathogenic when the quisite for biofilm-inihibiting furanones [24]. Transposing the methoxy host’s microbiota or immunity are disrupted [49,50]. C. albicans bio­ group to either the 2- or 3-position saw a fall-off in activity with values films can be 1000 times more resistant to antimicrobials than their of 7% recorded for 8 and 30% for 10. 4-Methoxyphenylacetylene-substi­ planktonic equivalents [51]. The presence of biofilm increases resis­ tuted furanone 37, a close analogue of 12 but incorporating an acetylene tance to both antifungal agents and the host’s immune system. Such spacer, also exhibited a moderate effect (36% inhibition). Several of the biofilms pose a significant clinical challenge as they frequently form on biofilm inhibitors identified above (e.g. 12 and 21) had minimal impact implanted medical devices such as pacemakers, catheters and prosthetic on bacterial growth (Supplementary Figure A2). joints [52]. Although a small number of brominated furanones have The Gram-negative bacterium E. coli is a common cause of several been investigated as fungal growth inhibitors of C. albicans [53], their debilitating infections, including enteritis, urinary tract infections, sep­ impact on Candida biofilm formation remains unexplored. ticaemia and neonatal meningitis [43]. The effect on biofilm production Several furanones as analysed by sustained inhibition assays dis­ in E. coli ATCC 9637 was reduced relative to the other species under rupted biofilm formation in C. albicans M2396 (Fig. 3). At 50 μM con­ investigation (Fig. 3). Molecules lacking a dihaloolefin motif were centration, controls 2 and 3 completely inhibited biofilm production, generally more potent. Bis-4-methoxyphenylacetylene-substituted 37 with a strong inhibitory effect (92% inhibition) also observed for was the most active compound, inhibiting biofilm growth by 31% chloroiodofuranone 34 (Supplementary Figure B7). There was a (Supplementary Figure B3). The ring-brominated furanone 31 was noticeable overlap between compounds that inhibited biofilm formation moderately active with a 15% reduction noted. Neither of the two with those furanones that also retarded cell growth (Supplementary brominated controls 2 or 3 affected biofilm production. Several of these Figure A7). By contrast, the next most active candidate, namely 4-tri­ biofilm inhibitors moderately reduced bacterial growth in the order of fluoromethylphenyl-substituted 15, reduced biofilm production by 8% for 37 (Supplementary Figure A3). The largest effect was recorded 75% but had a much smaller effect on cell growth. Tribromofuranone with chloroiodofuranone 34 (28% reduction) which was not itself a 27, which was the most potent inhibitor in several bacterial species, was biofilm inhibitor. less active against C. albicans biofilm with an inhibition value of 51%. S. maltophilia is also an important nosocomial pathogen that has been The replacement of the two bromines in 3 with chlorines saw a fall-off in linked to respiratory tract and blood infections, and is particularly sig­ activity for 33 (21% inhibition). Other structural changes, such as the nificant in immunocompromised lung cancer patients [44]. None of our introduction of a methyl group onto the C4-position of the furanone furanones inhibited growth (Supplementary Figure A4) or biofilm for­ ring, were similarly detrimental with 24 reducing biofilm formation by mation in S. maltophilia K279a (Fig. 3 & Supplementary Figure B4). The only 38%. As previously observed with bacterial biofilms, the presence latter result is, perhaps, unsurprising as neither AI-2 nor AHL play sig­ of a vinylic halogen was not a prequisite for activity. Bis-4- nificant roles in S. maltophilia QS [45]. methoxyphenyl-substituted furanone 12 and the closely related bis-3- P. aeruginosa is a significant driver of nosocomial infections, mainly methoxyphenyl-substituted furanone 10 reduced fungal biofilm in intensive unit care, where it is associated with ventilator-associated growth by 61% and 53% respectively. pneumonia, urinary tract infections, wound infections and various We also examined the effect of our library on biofilm growth in the sepsis syndromes [46]. This pathogen poses a particular threat to developmental phase of C. albicans M2396 where a different pattern immunocompromised patients [47]. In comparison to other bacteria, emerged. In particular, the correlation of inhibitory effects on biofilm P. aeruginosa PAO1 biofilms proved sensitive to a wider range of fur­ growth with cell growth was less pronounced. Once again, dibromo­ anone structures (Fig. 3). The most active compound was found to be furanone 3 completely inhibited Candida biofilm, but mono­ control monobromofuranone 2 which inhibited biofilm growth by 75% bromofuranone 2 proved much less effective (Supplementary (Supplementary Figure B5). Methyl-containing dibromofuranone 24 Figure B8). 4-Trifluoromethylphenyl-substituted 15 was more active in was almost equipotent with 70% inhibition observed. Its tribominated the developmental phase with 85% inhibition recorded. Non- analogue 27, which was the most active inhibitor of S. enterica and halogenated bis-4-methoxyphenyl-substituted furanone 12 and bis-3- S. aureus biofilms, was slightly less effective against P. aeruginosa (60% methoxyphenyl-substituted furanone 10 reduced fungal biofilm inhibition). Although bromine-containing compounds were generally growth by 75% and 71% respectively. A reduced inhibitory effect was more potent, both dichlorofuranone 33 and chloroiodofuranone 34 observed with chloroiodofuranone 34 (52% inhibition) in the develop­ inhibited biofilm production in P. aeruginosa PAO1 by 52% and 51% mental phase. respectively. However, as with S. enterica, the presence of a halogen atom was not necessarily required for activity. Indeed, diarylated fur­ 2.3. Disruption of mixed bacterial-fungal biofilms by selected furanones anones 8 and 10, which bear methoxy substituents at the 2- and 3-posi­ tions respectively, were found to be more active than their In both natural and clinical environments, biofilms are often poly­ monoarylated equivalents 7 and 9, and reduced biofilm growth by 41% microbial in composition. Inter-kingdom (e.g. bacteria/fungi) biofilms, and 44% respectively. None of the furanones inhibited the growth of such P. aeruginosa/C. albicans, have been identified in infections of the P. aeruginosa PAO1 by more than 5% except for bromofuranone 24 lungs, skin and in medical devices [13]. When these microorganisms are which reduced bacterial growth by just 11% (Supplementary Figure A5). co-isolated, their interaction may be simultaneously competitive, syn­ This is noteworthy as it suggests that these compounds reduce bacterial ergistic and/or antagonistic [54]. We wondered if our furanones with biofilm production without causing cell death, highlighting their po­ significant anti-biofilm activity were also capable of disrupting mixed tential as QS inhibitors. fungal-bacterial biofilms. The chosen furanones were initially subjected P. aeruginosa PAR7244 is a recent clinical isolate identified in pa­ to further analysis for their effect on single-species biofilms of tients with tracheobronchitis and is classified as a high biofilm former P. aeruginosa and C. albicans using confocal laser-scanning microscopy [48]. In contrast to PAO1, biofilm production in PAR7244 was less (CLSM). Under these conditions, some of the compounds assayed dis­ sensitive overall (Fig. 3). Methyl-substituted dibromofuranone 24 was rupted biofilm formation in both target pathogens (Fig. 4). These results the most potent inhibitor, reducing biofilm growth significantly by 44% were in accordance with those obtained by the crystal violet staining compared to a 70% reduction in PAO1 (Supplementary Figure B6). method (Supplementary Figure B5) and the XTT reduction assay (Sup­ Surprisingly, control bromofuranone 2, which was the most effective plementary Figure B7) using microtiter plates. For C. albicans, the most 6 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Fig. 4. Furanones with significant anti-biofilm activity as analysed by confocal laser-scanning microscopy (CLSM). (A) CLSM images of C. albicans M2396 in the presence of 2, 3, 7, 10, 12, 15, 27, 29 and 34. (B) Representative CLSM images of biofilm formed by P. aeruginosa PAO1 in the presence of 2, 3, 10, 24, 33 and 34. In both panels, control (Ctrl) represents biofilms treated with neat DMSO. pronounced effects were observed with bromofuranones 2, 3, tri­ for its reduced toxicity. The other compounds tested, namely 2, 24, 27 bromofuranone 27 and chloroiodofuranone 34 (Fig. 4A). Chlor­ and 33, proved toxic to human kidney cells which could prevent their oiodofuranone 34 was also one of the most effective compounds against use in humans, at least in their current formulation (data not shown). By P. aeruginosa PAO1 (Fig. 4B). contrast, in vivo toxicity screening of the same compounds at double Based on these results, we studied the effect of bromofuranones 2, 3 concentration (100 μM) showed that none was toxic to the larvae as no and chloroiodofuranone 34 on the formation of mixed biofilms animal died or showed signs of melanization (Fig. 6B). Toxicity testing composed of both species. Fig. 5 shows CLSM images of dual-species in G. mellonella larvae has recently emerged as a reliable predictor of biofilms formed by C. albicans M2396 with P. aeruginosa PAO1. Under chemical toxicity [55]. the growth conditions used, C. albicans showed less adherence to Since 10 was non-toxic in human cells, we investigated its in vivo μ-Slides than P. aeruginosa, probably due to the inhibitory effect of antimicrobial activity in the G. mellonella acute model following infec­ P. aeruginosa exoproducts on the fungus [13]. Bromofuranones 2, 3 and tion with P. aeruginosa. The strain PAO1 is known to be highly virulent chloroiodofuranone 34 were all found to decrease the biomass of both for Galleria larvae [56]. Despite the low concentration tested (100 cfu microorganisms within the mixed biofilms (Fig. 5). per larva), we recorded 100% mortality after 24 h (data not shown). We instead infected the larvae with the clinical isolate PAR7244 which has been shown to be less virulent for Galleria [48]. Treatment with 10 2.4. In vitro and in vivo toxicity and efficacy of candidate compounds resulted in a significant increase in larvae survival (72%) compared with non-treated larvae at 48% survival (Fig. 7). Furanone 10 was not asso­ We next evaluated the toxicity of our most promising furanones in ciated with significant biofilm inhibition in P. aeruginosa PAR7244 vitro and in vivo, using HK-2 human kidney cells and G. mellonella larvae (Supplementary Figure B6), so the basis of increased larval survival respectively. We first examined the in vitro effect of 2, 10, 24, 27 and 33 remains to be elucidated. at 50 μM on the HK-2 human cell line. Bis-3-methoxyphenyl-substituted furanone 10 showed no indication of significant toxicity (Fig. 6A). 10 was the sole non-halogenated furanone in this group which may account 7 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Fig. 5. Confocal laser-scaning microscopy (CLSM) images of dual-species biofilms formed by C. albicans M2396 (red) and P. aeruginosa PAO1 (yellow) in the presence of furnanones 2, 3 and 34. The control (Ctrl) represents a mixed biofilm treated with neat DMSO. 3. Conclusion trifluoromethylphenyl-substituted 15 being identified as a promising candidate. These effects were confirmed by confocal laser-scaning mi­ We have described the synthesis of a variety of bromine-, chlorine- croscopy. While compound cytotoxicity was identified as a potential and iodine-containing furanones which were subjected to further issue, this was not replicated in toxicity testing with G. mellonella. modification via Pd-catalysed coupling reactions. Several compounds in Interestingly, diarylated furanone 10 was not only found to be non-toxic this diverse library were found to strongly inhibit bacterial biofilm but also increased the survival rates of G. mellonella infected with formation in S. enterica, S. aureus, P. aeruginosa and to a lesser extent in P. aeruginosa PAR7244. E. coli. In particular, tribromofuranone 27 exhibited equal or better The discovery of antibiotics remains one of the crowning achieve­ inhibitory activity against S. enterica, S. aureus, P. aeruginosa compared ments of the 20th century. However, since their introduction, the to known dibromofuranones 2 or 3. Compounds which inhibited biofilm problem of antimicrobial resistance has grown exponentially, with ever formation did not generally impact on bacterial growth, highlighting more cases of resistant infections being reported in the literature. Dis­ their potential as QS inhibitors. A number of compounds were also found eases caused by resistant bacteria now constitute the second most to impede biofilm growth in the fungal pathogen C. albicans with 4- common cause of death globally [57]. It is clear that a multipronged 8 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 approach to tackling AMR is required. The use of small molecule in­ hibitors of QS has long been touted as one promising strategy. The re­ sults reported here support this approach for the treatment of common nocosomial infections. 4. Experimental section 4.1. Chemistry Known compounds 2 [58], 3 [29,59] & 33 [29,37] were prepared via previously reported procedures. Analytical data are provided as Sup­ plementary data. 4.1.1. General procedure for the preparation of monoarylated furanones To an oven-dried RBF was added 3 (202 mg, 0.787 mmol, 1 eq.), the appropriate boronic acid (0.866 mmol, 1.1 eq.), tripotassium phosphate (334 mg, 1.574 mmol, 2 eq.), palladium(II) acetate (5 mg, 3 mol%), SPhos (7 mg, 2 mol%) and toluene (4 mL) under a flow of nitrogen. The reaction mixture was stirred at room temperature for 48 h and then diluted with dichloromethane (30 mL). The solution was filtered through a layer of Celite and the solvents were evaporated under reduced pressure. The crude product was purified via column chroma­ tography with 4%–8% ethyl acetate in hexane to afford the mono­ arylated furanone product. 4.1.1.1. (Z)-5-(Bromo(phenyl)methylene)furan-2(5H)-one (5) [32]. Yield: 32% 1 H NMR (CDCl3, 400 MHz): 6.32 (1H, d, J = 5.5 Hz, H-3), 7.42–7.48 (6H, m, H-4, H-2′ , H-3′ , H-4′ , H-5′ , H-6′ ); 13C NMR (CDCl3, 100 MHz): 110.98 (CBr), 120.81 (C3), 128.78 and 130.12 (C2′ , C6′ , C3′ , C5′ ), 130.27 (C4′ ), 135.36 (C1’), 140.96 (C4), 149.08 (C5), 168.53 (C2); IR (ATR, cm− 1): 2922, 1776, 1759, 1624, 1547, 1487, 1444, 1230, 1107, 1069, 1033, 955, 925, 902, 861, 809, 755, 711. 693, 627; HRMS (ESI+): Exact mass calculated for C11H8BrO+ 2 [M+H] = 250.9708; Found = + 250.9704. Fig. 6. In vitro and in vivo toxicity of selected furanones. A) Cytotoxicity in 4.1.1.2. (Z)-5-(Bromo(2-methoxyphenyl)methylene)furan-2(5H)-one human kidney cells. MTT-based assay with furanone 10 at a concentration of (7). Yield: 59% 50 μM on HK-2 cells. The viability percentage was calculated using non-treated 1 H NMR (CDCl3, 400 MHz): 3.86 (3H, s, OCH3), 6.23 (1H, d, J = 5.5 cells (Control). Vehicle is DMSO with a concentration of 0.5%. DMSO 5% was Hz, H-3), 6.96–7.02 (2H, m, H-3′ , H-5′ ), 7.17 (1H, d, J = 5.5 Hz H-4), used as a cytotoxicity control. B) Toxicity in G. mellonella of 2, 10, 24, 27 and 7.25–7.28 (1H, m, H-6′ ), 7.42 (1H, td, J = 7.8 Hz, 1.31 Hz, H-4′ ); 13C 33 at 100 μM. Vehicle is DMSO with a concentration of 1%. The surviving percentage was calculated using larvae injected with the vehicle. NMR (CDCl3, 100 MHz): 55.79 (OCH3), 106.13 (CBr), 111.59 (C3′ ), 120.11 (C3), 120.57 (C5′ ), 123.63 (C1′ ), 131.87 (C4′ ), 132.03 (C6’), 141.11 (C4), 149.92 (C5), 157.47 (COCH3), 168.82 (C2); IR (ATR, cm− 1): 3071, 2932, 2839, 1776, 1762, 1595, 1578, 1488, 1462, 1250, 1108, 1068, 1048, 956, 862, 810, 776, 754, 704, 624,; HRMS (ESI+): Exact mass calculated for C12H10BrO+ 3 [M+H] = 280.9813; Found = + 280.9806. 4.1.1.3. (Z)-5-(Bromo(3-methoxyphenyl)methylene)furan-2(5H)-one (9). Yield: 67% 1 H NMR (CDCl3, 400 MHz): 3.85 (3H, s, OCH3), 6.31 (1H, d, J = 5.5 Hz, H-3), 6.97–7.03 (3H, m, H-2′ , H-4′ , H-6′ ), 7.32 (1H, t, J = 7.8 Hz, H- 5′ ), 7.46 (1H, d, J = 5.5 Hz, H-4); 13C NMR (CDCl3, 100 MHz): 55.48 (OCH3), 110.65 (CBr), 115.72 (C2′ ), 115.88 (C4′ ), 120.75 (C3), 122.43 (C6′ ), 129.77 (C5′ ), 136.54 (C1′ ), 141.04 (C4), 149.15 (C5), 159.62 (C3’), 168.51 (C2); IR (ATR, cm− 1): 3139, 2937, 2843, 1782, 1758, Fig. 7. Kaplan-Meier survival curves of G. mellonella larvae infected with 1596, 1576, 1548, 1486, 1427, 1287, 1251, 1202, 1108, 1049, 1033, P. aeruginosa PAR7244. The larvae were treated with furanone 10 at a con­ 976, 883, 812, 774, 711, 690; HRMS (ESI+): Exact mass calculated for centration of 50 μM in PBS (difference significant by log-rank test, P < 0.001). C12H10BrO+ 3 [M+H] = 280.9813; Found = 280.9806. + DMSO at concentrations of 0.5% in PBS was used as vehicle control for un­ treated animals. 4.1.1.4. (Z)-5-(Bromo(4-methoxyphenyl)methylene)furan-2(5H)-one (11). Yield: 29% 1 H NMR (CDCl3, 300 MHz): 3.86 (3H, s, OCH3), 6.29 (1H, d, J = 5.5 Hz, H-3), 6.95 (2H, dt, J = 8.9 Hz, 3.0 Hz, H-3′ , H-5′ ), 7.40–7.44 (3H, m, 9 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 H-4, H-2′ , H-6′ ); 13C NMR (CDCl3, 75.5 MHz): 55.53 (OCH3), 111.43 4.1.2.1. 5-(Diphenylmethylene)furan-2(5H)-one (6) [32]. Yield: 82% 1 (CBr), 114.18 (C3′ , C5′ ), 120.31 (C3), 127.64 (C1′ ), 131.63 (C2′ , C6′ ), H NMR (CDCl3, 400 MHz): 6.21 (1H, d, J = 5.2 Hz, H-3), 7.26–7.50 140.91 (C4), 148.43 (C5), 161.19 (C4’), 168.71 (C2); IR (ATR, cm− 1): (11H, m, H-4, H-2′ , H-2′′ , H-3′ , H-3′′ , H-4′ , H-4′′ , H-5′ , H-5′′ , H-6′ , H-6′′ ); 13 3138, 3109, 2840, 1759, 1776, 1602, 1508, 1545, 1253, 1237, 1175, C NMR (CDCl3, 100 MHz): 118.60 (C3), 128.24 and 128.49 (C2′ , C6′ , 1110, 1031, 904, 864, 809, 703, 579; HRMS (ESI+): Exact mass calcu­ C2′′ , C6′′ ), 128.75 (C6), 128.90 (C4′ ), 129.08 (C4′′ ), 131.23 and 131.25 lated for C12H10BrO+ 3 [M+H] = 280.9813; Found = 280.9803. + (C3′ , C5′ , C3′′ , C5′′ ), 136.55 (C1′ ), 137.24 (C1′′ ), 143.93 (C4), 147.13 (C5), 170.50 (C2); IR (ATR, cm− 1): 3056, 1768, 1746, 1545, 1491, 1444, 4.1.1.5. (Z)-5-(Bromo(4-fluorophenyl)methylene)furan-2(5H)-one (13). 1230, 1110, 1069, 1031, 999, 931, 877, 772, 757, 695, 659; HRMS Yield: 15% (ESI+): Exact mass calculated for C17H13O+ 2 [M+H] + = 249.0916; 1 Found = 249.0910. H NMR (CDCl3, 400 MHz): 6.34 (1H, d, J = 5.5 Hz, H-3), 7.14 (2H, t, J = 8.6 Hz, H-3′ , H-5′ ), 7.39 (1H, d, J = 5.5 Hz, H-4), 7.45–7.48 (2H, m, H-2′ , H-6′ ); 13C NMR (CDCl3, 100 MHz): 109.55 (CBr), 116.02 (d, J = 4.1.2.2. 5-(Bis(2-methoxyphenyl)methylene)furan-2(5H)-one (8). Yield: 22.2 Hz, C3′ , C5′ ), 121.08 (C3), 131.47 (d, J = 3.4 Hz, C1′ ), 132.07 (d, J 7% 1 = 8.7 Hz, C2′ , C6′ ), 140.58 (C4), 149.14 (C5), 163.65 (d, J = 252.2 Hz, H NMR (CDCl3, 400 MHz): 3.65 (3H, s, OCH3’), 3.74 (3H, s, OCH3′′ ), C4’), 168.37 (C2); IR (ATR, cm− 1): 3072, 2955, 2853, 2922, 1782, 1765, 6.15 (1H, d, J = 5.8 Hz, H-3), 6.89–6.98 (4H, m, H-5′ , H-5′′ , H-3′ , H-3′′ ), 1601, 1549, 1505, 1236, 1106, 866, 807, 567, 535; HRMS (ESI+): Exact 7.19 (2H, td, J = 8.3 Hz, 1.56 Hz, H-4′ , H-4′′ ), 7.26–7.35 (2H, m, H-6′ , H- mass calculated for C11H7BrFO+ 2 [M+H] + = 268.9613; Found = 6′′ ), 7.37 (1H, d, J = 5.8 Hz, H-4); 13C NMR (CDCl3, 100 MHz): 55.74 268.9607. (OCH3’), 55.86 (OCH3′′ ), 111.55 and 111.62 (C3′ , C3′′ ), 118.98 (C3), 120.21 and 120.30 (C5′ , C5′′ ), 122.73 and 126.31 (C1′ , C1′′ ), 126.50 4.1.1.6. (Z)-5-(Bromo(4-(trifluoromethyl)phenyl)methylene)furan-2(5H)- (C6), 129.79 and 130.01 (C6′ , C6′′ ), 131.06 and 132.38 (C4′ , C4′′ ), one (15). Yield: 26% 143.12 (C4), 148.09 (C5), 157.20 and 157.75 (C2′ and C2′′ ), 170.51 1 H NMR (CDCl3, 400 MHz): 6.39 (1H, d, J = 5.5 Hz, H-3), 7.41 (1H, (C2); IR (ATR, cm− 1): 3069, 3000, 2935, 2836, 1771, 1744, 1595, 1488, d, J = 5.5 Hz, H-4), 7.60 (2H, d, J = 8.1 Hz, H-2′ , H-6′ ), 7.72 (2H, d, J = 1246, 1228, 1179, 1162, 1111, 1023, 961, 937, 881, 751; HRMS (ESI+): 8.1 Hz, H-3′ , H-5′ ); 13C NMR (CDCl3, 75.5 MHz): 108.42 (CBr), 121.75 Exact mass calculated for C19H17O+ 4 [M+H] = 309.1127; Found = + (C3), 123.37 (q, J = 272.5 Hz, CF3), 125.81 (q, J = 3.8 Hz, C3′ , C5′ ), 309.1117. 130.50 (C2′ , C6′ ), 132.04 (q, J = 32.9 Hz, C4′ ), 138.87 (C1’), 140.35 (C4), 149.86 (C5), 168.01 (C2); IR (ATR, cm− 1): 2957, 2923, 2853, 4.1.2.3. 5-(Bis(3-methoxyphenyl)methylene)furan-2(5H)-one (10). 1782, 1765, 1615, 1551, 1408, 1320, 1166, 1122, 1104, 1065, 1018, Yield: 75% 1 957, 844, 809, 765, 710, 600,; HRMS (ESI+): Exact mass calculated for H NMR (CDCl3, 400 MHz): 3.79 (3H, s, OCH3’), 3.80 (3H, s, OCH3′′ ), C12H7BrF3O+ 2 [M+H] = 318.9582; Found = 318.9574. + 6.20 (1H, d, J = 5.5 Hz, H-3), 6.80 (1H, dd, J = 2.4 Hz, 1.67 Hz, H-2′ ), 6.86–6.91 (2H, m, H-4′ , H-6′ ), 6.96 (1H, ddd, J = 8.5 Hz, 2.7 Hz, 1.0 Hz, 4.1.1.7. (Z)-5-(Bromo(3-nitrophenyl)methylene)furan-2(5H)-one (17). H-4′′ ), 7.05–7.11 (2H, m, H-2′′ , H-6′′ ), 7.26–7.35 (2H, m, H-5′ , H-5′′ ), Yield: 24% 7.43 (1H, d, J = 5.5 Hz, H-4); 13C NMR (CDCl3, 100 MHz): 55.33 1 (OCH3’), 55.36 (OCH3′′ ), 114.32 (C4′′ ), 114.71 (C4′ ), 116.73 (C2′′ ), H NMR (CDCl3, 300 MHz): 6.43 (1H, d, J = 5.6 Hz, H-3), 7.41 (1H, d, J = 5.6 Hz, H-4), 7.67 (1H, td, J = 8.0 Hz, 0.64 Hz, H-5′ ), 7.82 (1H, 116.82 (C2′ ), 118.63 (C3), 123.68 (C6′ ), 123.82 (C6′′ ), 128.22 (C6), ddd, J = 8.0 Hz, 1.6 Hz, 1.2 Hz, H-6′ ), 8.30–8.34 (2H, m, H-2′ , H-4′ ); 13C 129.15 (C5′ ), 129.45 (C5′′ ), 137.61 (C1′ ), 138.41 (C1′′ ), 143.99 (C4), NMR (CDCl3, 75.5 MHz): 106.88 (CBr), 122.30 (C3), 124.75 (C2′ ), 147.22 (C5), 159.28 (C3′ ), 159.49 (C3′′ ), 170.37 (C2); IR (ATR, cm− 1): 124.84 (C4′ ), 130.05 (C5′ ), 135.77 (C6′ ), 137.09 (C1′ ), 139.92 (C4), 2959, 2935, 2835, 1779, 1750, 1594, 1574, 1486, 1429, 1286, 1247, 148.31 (C5), 150.22 (C3’), 167.68 (C2); IR (ATR, cm− 1): 3081, 2904, 1207, 1109, 1047, 983, 889, 809, 792, 771, 736,; HRMS (ESI+): Exact 2854, 1781, 1762, 1612, 1550, 1528, 1474, 1348, 1290, 1107, 984, 882, mass calculated for C19H17O+ 4 [M+H] = 309.1127; Found = 309.1129. + 812, 737, 676; HRMS (ESI+): Exact mass calculated for C11H7BrNO+ 4 [M+H]+ = 295.9558; Found = 295.9551. 4.1.2.4. 5-(Bis(4-methoxyphenyl)methylene)furan-2(5H)-one (12). Yield: 83% 1 4.1.1.8. (Z)-5-(Bromo(4-nitrophenyl)methylene)furan-2(5H)-one (19). H NMR (CDCl3, 400 MHz): 3.84 (3H, s, OCH3’), 3.87 (3H, s, OCH3′′ ), Yield: 20% 6.14 (1H, d, J = 5.3 Hz, H-3), 6.88 (2H, d, J = 8.9 Hz, H-3′′ , H-5′′ ), 6.94 1 (2H, d, J = 8.7 Hz, H-3′ , H-5′ ), 7.19 (2H, d, J = 8.7 Hz, H-2′ , H-6′ ), 7.40 H NMR (CDCl3, 400 MHz): 6.44 (1H, d, J = 5.6 Hz, H-3), 7.41 (1H, d, J = 5.6 Hz, H-4), 7.67 (2H, d, J = 8.7 Hz, H-2′ , H-6′ ), 8.32 (2H, d, J = (1H, d, J = 5.4 Hz, H-4), 7.47 (2H,d, J = 9.0 Hz, H-2′′ , H-6′′ ); 13C NMR 8.7 Hz, H-3′ , H-5′ ); 13C NMR (CDCl3, 100 MHz): 107.18 (CBr), 122.36 (CDCl3, 100 MHz): 55.37 (OCH3’), 55.41 (OCH3′′ ), 113.68 (C3′′ , C5′′ ), (C3), 123.99 (C3′ , C5′ ), 131.10 (C2′ , C6′ ), 140.02 (C4), 141.55 (C1′ ), 113.86 (C3′ , C5′ ), 117.13 (C3), 128.72 (C6), 129.27 (C1′′ ), 129.61 (C1′ ), 148.46 (C4’), 150.32 (C5), 167.67 (C2); IR (ATR, cm− 1): 3141, 3108, 132.60 (C2′ , C6′ ), 133.06 (C2′′ , C6′′ ), 143.93 (C4), 146.07 (C5), 160.21 3053, 1770, 1767, 1595, 1551, 1517, 1346, 1232, 1108, 1033, 957, 859, (C4′ ), 160.28 (C4′′ ), 170.98 (C2); IR (ATR, cm− 1): 3004, 2956, 2838, 767; HRMS (ESI+): Exact mass calculated for C11H7BrNO+ 4 [M+H] = + 1770, 1744, 1602, 1540, 1509, 1251, 1175, 1031, 883, 835, 562; HRMS 295.9558; Found = 295.9548. (ESI+): Exact mass calculated for C19H17O+ 4 [M+H] + = 309.1127; Found = 309.1118. 4.1.2. General procedure for the preparation of diarylated furanones To an oven-dried RBF was added 3 (200 mg, 0.787 mmol, 1 eq.), the 4.1.2.5. 5-(Bis(4-fluorophenyl)methylene)furan-2(5H)-one (14). Yield: appropriate boronic acid (2.361 mmol, 3 eq.), tripotassium phosphate 86% 1 (501 mg, 2.361 mmol, 3 eq.), palladium(II) acetate (3 mg, 1 mol%), H NMR (CDCl3, 400 MHz): 6.23 (1H, d, J = 5.5 Hz, H-3), 7.06 (2H, t, SPhos (7 mg, 2 mol%) and toluene (5 mL). The reaction mixture was J = 8.7 Hz, H-3′′ , H-5′′ ), 7.14 (2H, t, J = 8.8 Hz, H-3′ , H-5′ ), 7.23–7.26 heated to 100 ◦ C for 3 h under a flow of nitrogen. The reaction was (2H, m, H-2′ , H-6′ ), 7.38 (1H, d, J = 5.5 Hz, H-4), 7.46–7.50 (2H, m, H- cooled to room temperature and dichloromethane (30 mL) was added to 2′′ , H-6′′ ); 13C NMR (CDCl3, 100 MHz): 115.47 (d, J = 21.7 Hz, C3′′ , the reaction flask. The solution was filtered through a layer of Celite and C5′′ ), 115.81 (d, J = 21.9 Hz, C3′ , C5′ ), 118.88 (C3), 126.36 (C6), 132.48 the solvents were evaporated under reduced pressure. The crude prod­ (d, J = 3.47 Hz, C1′ ), 132.98 (d, J = 3.3 Hz, C1′′ ), 132.8 (d, J = 8.3 Hz, uct was purified via column chromatography with 5% ethyl acetate and C2′ , C6′ ), 133.18 (d, J = 8.5 Hz, C2′′ , C6′′ ), 143.54 (C4), 146.97 (C5), hexane to afford the diarylated furanone product. 162.99 (d, J = 251.0, C4′ ), 163.15 (d, J = 250.0 Hz, C4′′ ), 170.21 (C2); 10 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 IR (ATR, cm− 1): 2923, 1770, 1746, 1596, 1546, 1504, 1343, 1233, sodium carbonate (251 mg, 2.368 mmol), bis(triphenylphosphine) 1158, 1107, 951, 879, 838, 807, 801, 570, 592, 524; HRMS (ESI+): Exact palladium(II) dichloride (44 mg, 0.062 mmol), DME (2 mL) and ethanol mass calculated for C17H11F2O+ 2 [M+H] + = 285.0721; Found = (0.5 mL). The vial was flushed with nitrogen and heated at 120 ◦ C in a 285.0722. microwave reactor for 40 min. The crude reaction mixture was filtered over a layer of Celite and the solvent was evaporated under reduced 4.1.2.6. 5-(Bis(4-(trifluoromethyl)phenyl)methylene)furan-2(5H)-one pressure. The crude product was purified via column chromatography (16). Yield: 84% with 10%–20% ethyl acetate in hexane to afford 21. 1 Yield: 58% H NMR (CDCl3, 400 MHz): 6.34 (1H, d, J = 5.50 Hz, H-3), 7.41–7.43 1 (3H, m, H-4, H-2′ , H-6′ ), 7.57 (2H, d, J = 8.5 Hz, H-2′′ , H-6′′ ), 7.63 (2H, H NMR (CDCl3, 300 MHz): 6.09 (1H, d, J = 5.4 Hz, H-3), 6.84 (2H, d, J = 8.51 Hz, H-3′′ , H-5′′ ), 7.73 (2H, d, J = 8.1 Hz, H-3′ , H-5′ ); 13C NMR d, J = 8.7 Hz, H-3′′ , H-5′′ ), 6.89 (2H, d, J = 8.5 Hz, H-3′ , H-5′ ), 7.09 (2H, (CDCl3, 75.5 MHz): 120.56 (C3), 122.53 (C6), 123.77 (q, J = 272.2 Hz, d, J = 8.5 Hz, H-2′ , H-6′ ), 7.38–7.41 (3H, m, H-4, H-2′′ , H-6′′ ); 13C NMR CF3′′ ), 123.80 (q, J = 272.2 Hz, CF3’), 125.33 (q, J = 3.6 Hz, C3′′ , C5′′ ), (DMSO‑d6), 75.5 MHz): 115.62 (C3′′ , C5′′ ),115.81 (C3′ , C5′ ), 117.19 125.76 (q, J = 3.6 Hz, C3′ , C5′ ) 130.86 (q, J = 32.7 Hz, C4′′ ), 131.32 (q, (C6), 127.91 (C3), 128.07 (C1′′ ), 129.23 (C1′ ), 132.92 (C2′ , C6′ ), 133.18 J = 32.7 Hz, C4′ ), 131.24 (C2′′ , C6′′ ), 131.49 (C2′ , C6′ ), 139.35 (C1′′ ), (C2′′ , C6′′ ), 144.85 (C4), 145.52 (C5), 158.73 (C4′′ ), 158.87 (C4’), 140.20 (C1), 143.11 (C4), 148.39 (C5), 169.36 (C2); IR (ATR, cm− 1): 170.80 (C2); IR (ATR, cm− 1): 3210, 2924, 2853, 1774, 1742, 1605, 3144, 1776, 1752, 1616, 1573, 1320, 1235, 1165, 1110, 1067, 1017, 1536, 1512, 1275, 1234, 1171, 1046, 1024, 954, 842, 810; HRMS 966, 957, 923, 877, 769, 752, 731; HRMS (ESI+): Exact mass calculated (ESI+): Exact mass calculated for C17H13O+ 4 [M+H] + = 281.0814; for C19H11F6O+ 2 [M+H] = 385.0663; Found = 385.0656. + Found = 281.0808. 4.1.2.7. 5-(Bis(3-nitrophenyl)methylene)furan-2(5H)-one (18). Yield: 4.1.4. Synthesis of 5-(Dibromomethylene)-4-methylfuran-2(5H)-one (24) 11% [29] 1 H NMR (CDCl3, 300 MHz): 6.42 (1H, d, J = 5.6 Hz, H-3), 7.44 (1H, To a solution of methylsuccinic anhydride (1.606 mL, 17.844 mmol) d, J = 5.6 Hz, H-4), 7.61–7.66 (2H, m, H-5′′ , H-6′ ), 7.72 (1H, t, J = 8.0 in dichloromethane (40 mL) was slowly added a solution of carbon Hz, H-5′ ), 7.89 (1H, ddd, J = 7.9 Hz, 1.7 Hz, 1.1 Hz, H-6′′ ), 8.17–8.20 tetrabromide (8.876 g, 26.766 mmol) in dichloromethane (20 mL). The (2H, m, H-2′ , H-2′′ ), 8.24 (1H, ddd, J = 8.2 Hz, 2.3 Hz, 1.1 Hz, H-4′′ ), reaction mixture was cooled to 0 ◦ C and triisopropylphosphite (8.804 8.36 (1H, ddd, J = 8.0 Hz, 2.3 Hz, 1.2 Hz, H-4′ ); 13C NMR (CDCl3, 75.5 mL, 35.684 mmol) in dichloromethane (10 mL) was added over 1 h. The MHz): 121.83 (C3), 122.97 (C6), 124.27 (C4′′ ), 124.60 (C4′ ), 125.62 mixture was stirred at room temperature for 2 h under nitrogen. The (C2′′ ), 125.95 (C2′ ), 130.00 (C5′′ ), 130.55 (C5′ ), 136.88 (C6′′ ), 137.12 reaction was quenched with sodium bicarbonate (30 mL) and the (C6′ ), 137.53 (C1′′ ), 137.97 (C1′ ), 142.88 (C4), 148.63 (C3′′ ), 148.84 mixture extracted with dichloromethane (3 x 30 mL). The combined (C3’), 149.58 (C5), 169.05 (C2); IR (ATR, cm− 1): 3082, 3924, 2854, organic layers were washed with brine (20 mL), dried with magnesium 1781, 1754, 1526, 1345, 1235, 1201, 1075, 994, 879, 815, 762, 709, sulfate and the solvent was removed in vacuo. The crude product was 662; HRMS (ESI+): Exact mass calculated for C17H11N2O+ 6 [M+H] = + purified by column chromatography with 2%–5% ethyl acetate in hex­ 339.0617; Found = 339.0607. ane to afford 24. Yield: 46% 4.1.2.8. 5-(Bis(4-nitrophenyl)methylene)furan-2(5H)-one (20). Yield: H NMR (CDCl3, 400 MHz): 2.48 (3H, d, J = 1.4 Hz, CH3), 6.20 (1H, q, 8% J = 1.4 Hz, H-3); 13C NMR (CDCl3, 100 MHz): 17.04 (CH3), 80.68 (CBr2), 1 H NMR (CDCl3, 300 MHz): 6.43 (1H, d, J = 5.5 Hz, H-3), 7.42 (1H, 121.04 (C3), 150.08 (C5), 153.87 (C4), 165.87 (C2); IR (ATR, cm− 1): d, J = 5.5 Hz, H-4), 7.48 (2H, d, J = 8.5 Hz, H-2′ , H-6′ ), 7.63 (2H, d, J = 3114, 2922, 1770, 1759, 1603, 1383, 1325, 1254, 1235, 1056, 1033, 8.7 Hz, H-2′′ , H-6′′ ), 8.25 (2H, d, J = 8.7 Hz, H-3′′ , H-5′′ ), 8.35 (2H, d, J 949, 905, 848, 820, 711, 651; HRMS (ESI+): Exact mass calculated for = 8.5 Hz, H-3′ , H-5′ ); 13C NMR (CDCl3, 75.5 MHz): 121.77 (C3), 123.05 C6H5Br2O+ 2 [M+H] = 266.8656; Found = 266.8648. + (C6), 123.70 (C3′′ , C5′′ ), 124.18 (C3′ , C5′ ), 131.73 (C2′′ , C6′′ ), 132.06 (C2′ , C6′ ), 141.67 (C1′ , C1′′ ), 142.63 (C4), 147.67 (C4′ ), 148.31 (C4′′ ), 4.1.5. Synthesis of 4-(Bromomethyl)-5-(dibromomethylene)furan-2(5H)- 149.21 (C5), 168.63 (C2); IR (ATR, cm− 1): 3109, 3077, 1778, 1754, one (27) 1552, 1515, 1406, 1344, 1191, 1107, 1041, 857, 845, 752, 734, 625; To a solution of 3-(bromomethyl)furan-2,5-dione (100 mg, 0.523 HRMS (ESI+): Exact mass calculated for C17H11N2O+ mmol) in dichloromethane (2 mL) was slowly added a solution of carbon 6 [M+H] + = 339.0617; Found = 339.0606. tetrabromide (277 mg, 0.835 mmol) in dichloromethane (1 mL). The reaction mixture was cooled to 0 ◦ C and triisopropylphosphite (0.258 mL, 1.047 mmol) in dichloromethane (0.5 mL) was added over 2 h. The 4.1.2.9. 5-(Di(thiophen-2-yl)methylene)furan-2(5H)-one (22) [32]. Yield: 25% mixture was stirred at room temperature for 4 h under nitrogen. The 1 reaction was quenched with sodium bicarbonate and the mixture H NMR (CDCl3, 300 MHz): 6.19 (1H, d, J = 5.7 Hz, H-3), 7.08 (1H, dd, J = 5.2 Hz, 3.9 Hz, H-4′′ ), 7.13 (1H, dd, J = 5.2 Hz, 3.5 Hz, H-4′ ), extracted with dichloromethane (3 x 10 mL). The combined organic layers were washed with brine, dried with magnesium sulfate and the 7.21 (1H, dd, J = 3.5 Hz, 1.3 Hz, H-3′ ), 7.42 (1H, dd, J = 3.9 Hz, 1.3 Hz, H-3′′ ), 7.49 (1H, d, J = 5.7 Hz, H-4), 7.51 (1H, dd, J = 5.2 Hz, 1.3 Hz, H- solvent was removed in vacuo. The crude product was purified by careful column chromatography with 2%–10% ethyl acetate in hexane to 5′ ), 7.55 (1H, dd, J = 5.2 Hz, 1.3 Hz, H-5′′ ); 13C NMR (CDCl3, 75.5 MHz): 115.01 (C6), 118.11 (C3), 127.03 (C4′ ), 127.50 (C4′′ ), 128.08 (C3′′ ), furnish 27. Yield: 36% 130.79 (C3′ ), 131.00 (C5′′ ), 132.08 (C5′ ), 136.31 (C2′ ), 139.55 (C2′′ ), 1 143.04 (C4), 146.23 (C5), 169.55 (C2); IR (ATR, cm− 1): 3106, 2921, H NMR (CDCl3, 400 MHz): 4.57 (2H, d, J = 1.3 Hz, CH2Br), 6.58 (1H, t, J = 1.3 Hz, H-3); 13C NMR (CDCl3, 100 MHz): 23.60 (CH2Br), 1780, 1761, 1579, 1539, 1412, 1238, 1105, 1066, 892, 806, 706; HRMS (ESI+): Exact mass calculated for C13H9O2S+ 81.45 (C6), 123.59 (C3), 147.64 (C5), 152.18 (C4), 164.36 (C2); IR 2 [M+H] = 261.0044; + (ATR, cm− 1): 3117, 2926, 1762, 1730, 1600, 1404, 1367, 1313, 1205, Found = 261.0036. 1165, 1065, 1044, 956, 898, 864, 852, 832, 754, 736, 720, 679, 636, 4.1.3. Synthesis of 5-(Bis(4-hydroxyphenyl)methylene)furan-2(5H)-one 536, 426; HRMS (ESI+): Exact mass calculated for C6H4Br3O+ 2 [M+H] + (21) = 344.7761; Found 344.7766. To an oven dried microwave reaction vial was added 3 (200 mg, 0.787 mmol), 4-hydroxyphenylboronic acid (326 mg, 2.363 mmol), 4.1.6. General ring-bromination procedure To a stirred solution of the appropriate diarylated furanone (0.253 11 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 mmol, 1 eq.) in diethyl ether (0.4 mL) was slowly added a solution of for C19H10BrF6O+ 2 [M+H] = 462.9768; Found = 462.9758. + bromine (0.01 mL, 0.251 mmol, 1 eq.) in diethyl ether or chloroform (0.4 mL). The reaction mixture was heated to reflux for 4 h, after which 4.1.7. Synthesis of (Z)-5-(chloroiodomethylene)furan-2(5H)-one (34) time the mixture was sparged with nitrogen for 10 min to remove excess [29] bromine. The resulting solution was then cooled to 0 ◦ C and a solution of gem-Dichlorofuranone 33 (50 mg, 0.303 mmol), sodium iodide (454 triethylamine (0.07 mL, 0.503 mmol, 2 eq.) in diethyl ether (0.2 mL) was mg, 3.030 mmol) and anhydrous DMF (1.8 mL) were placed in an oven- added dropwise over 10 min. The reaction was stirred for an additional dried microwave reaction vial, flushed with nitrogen and sealed. The hour at 0 ◦ C before the reaction was allowed to reach room temperature. reaction mixture was heated to 160 ◦ C for 3 min in a microwave reactor. The reaction mixture was washed with water (3 x 5 mL) and brine (5 The reaction mixture was then cooled to room temperature, poured onto mL), dried over anhydrous magnesium sulfate and solvents were ice water and extracted into diethyl ether (3 x 15 mL). The combined removed under reduced pressure. The crude reaction mixture was pu­ organic extracts were washed with water (15 mL) and brine (15 mL), rified via column chromatography with 5%–10% ethyl acetate in hexane dried over magnesium sulfate and the solvent was evaporated under to afford the ring-brominated product. reduced pressure. Purification of the crude reaction mixture via column chromatography with 4% ethyl acetate in hexane afforded 34. 4.1.6.1. 3-Bromo-5-(diphenylmethylene)furan-2(5H)-one (29). Yield: Yield: 43% 1 81% H NMR (CDCl3, 400 MHz): 6.43 (1H, d, J = 5.5 Hz, H-3), 7.75 (1H, 1 H NMR (CDCl3, 300 MHz): 7.26–7.47 (10H, m, H-2′ , H-2′′ , H-3′ , H- d, J = 5.5 Hz, H-4); 13C NMR (CDCl3, 100 MHz): 63.28 (CICl), 122.28 3′′ , H-4′ , H-4′′ , H-5′ , H-5′′ , H-6′ , H-6′′ ), 7.52 (1H, s, H-4); 13C NMR (C3), 137.86 (C4), 154.15 (C5), 167.42 (C– – O); IR (ATR, cm− 1): 3129, (CDCl3, 75.5 MHz): 111.80 (C6), 128.35 and 128.65 (C2′ , C6′ , C2′′ , C6′′ ), 3103, 3063, 1750, 1737, 1633, 1546, 1337, 1248, 1217, 1124, 1079, 129.25 and 129.45 (C4′ , C4′′ ), 129.69 (C5), 131.13 and 131.29 (C3′ , C5′ , 971, 889, 844, 812, 711, 538; HRMS (ESI+): Exact mass calculated for C3′′ , C5′′ ), 136.05 and 136.90 (C1′ , C1′′ ), 141.87 (C4), 145.38 (C5), C5H3ClIO+ 2 [M+H] = 256.8866; Found = 256.8856. + 165.97 (C2); IR (ATR, cm− 1): 3057, 1761, 1545, 1492, 1444, 1228, 1109, 980, 924, 772, 741, 696, 659, 640; HRMS (ESI+): Exact mass 4.1.7.1. (Z)-5-(Chloro(phenyl)methylene)furan-2(5H)-one (35) [29]. calculated for C14H12BrO+ 2 [M+H] = 327.0021; Found = 327.0009. + Prepared using the general procedure for monoarylated furanones from gem-dichlorofuranone 33. 4.1.6.2. 5-(Bis(4-methoxyphenyl)methylene)-3-bromofuran-2(5H)-one Yield: 50% 1 (30). Yield: 36% H NMR (CDCl3, 300 MHz): 6.28 (1H, d, J = 5.5 Hz, H-3), 7.47–7.51 1 H NMR (CDCl3, 400 MHz): 3.84 (3H, s, OCH3′′ ), 3.88 (3H, s, OCH3’), (6H, m, H-4, H-2′ , H-3′ , H-4′ , H-5′ , H-6′ ); 13C NMR (CDCl3, 100 MHz): 6.89 (2H, d, J = 8.7 Hz, H-3′′ , H-5′′ ), 6.95 (2H, d, J = 8.5 Hz, H-3′ , H-5′ ), 120.42 (C3), 120.48 (CCl), 128.85 and 129.60 (C2′ , C6′ , C3′ , C5′ ), 7.19 (2H, d, J = 8.5 Hz, H-2′ , H-6′ ), 7.44 (2H, d, J = 8.7 Hz, H-2′′ , H-6′′ ), 130.43 (C4′ ), 134.00 (C1’), 141.44 (C4), 147.32 (C5), 168.46 (C2); IR 7.49 (1H, s, H-4); 13C NMR (CDCl3, 75.5 MHz): 55.40 (OCH3′′ ), 55.45 (ATR, cm− 1): 3112, 3064, 1769, 1742, 1627, 1547, 1445, 1230, 1108, (OCH3’), 109.89 (C6), 113.81 (C3′′ , C5′′ ), 114.03 (C3′ , C5′ ), 128.74 1072, 961, 939, 869, 809, 759, 713, 695, 632, 543; HRMS (ESI+) Exact (C3), 129.23 (C1′ ), 129.73 (C1′′ ), 132.56 (C2′ , C6′ ), 133.18 (C2′′ , C6′′ ), mass calculated for C11H8ClO+ 2 [M+H] + = 207.0213; Found = 141.98 (C4), 144.28 (C5), 160.49 (C4′ ), 160.59 (C4′′ ), 166.36 (C2); IR 207.0207. (ATR, cm− 1): 2917, 2848, 1768, 1594, 1496, 1287, 1266, 1054, 1016, 988, 816, 747, 668; HRMS (ESI+): Exact mass calculated for C19H16BrO+ 4 4.1.7.2. (Z)-5-(Chloro(4-methoxyphenyl)methylene)furan-2(5H)-one [M+H]+ = 387.0232; Found = 387.0231. (36). Prepared using the general procedure for monoarylated furanones from gem-dichlorofuranone 33. 4.1.6.3. 5-(Bis(3-bromo-4-methoxyphenyl)methylene)-3-bromofuran-2 Yield: 76% 1 (5H)-one (31). Yield: 34% H NMR (CDCl3, 300 MHz): 3.87 (3H, s, OCH3), 6.25 (1H, d, J = 5.6 1 Hz, H-3), 6.96–6.98 (2H, m, H-3′ , H-5′ ), 7.43–7.45 (2H, m, H-2′ , H-6′ ), H NMR (CDCl3, 400 MHz): 3.94 (3H, s, OCH3′′ ), 3.98 (3H, s, OCH3’), 6.91 (1H, d, J = 8.7 Hz, H-5′′ ), 6.97 (1H, d, J = 8.4 Hz, H-5′ ), 7.18 (1H, 7.50 (1H, d, J = 5.6 Hz, H-4); 13C NMR (CDCl3, 75.5 MHz): 55.51 dd, J = 8.4 Hz, 2.18 Hz, H-6′ ), 7.44 (1H, d, J = 2.2 Hz, H-2′ ), 7.47 (1H, s, (OCH3), 114.28 (C3′ , C5′ ), 119.84 (C3), 120.72 (CCl), 126.27 (C1′ ), H-4), 7.48 (1H, dd, J = 8.7 Hz, 2.2 Hz, H-6′′ ), 7.57 (1H, d, J = 2.2 Hz, H- 131.09 (C2′ , C6′ ), 141.37 (C5), 146.63 (C4), 161.37 (C4’), 168.59 (C2); 2′′ ); 13C NMR (CDCl3, 100 MHz): 56.40 (OCH3′′ ), 56.45 (OCH3’), 111.47 IR (ATR, cm− 1): 3025, 2922, 2848, 1770, 1746, 1604, 1544, 1509, (C5′′ ), 111.62 (C6), 111.78 (C5′ ), 111.84 (C3′′ ), 112.17 (C3′ ), 126.17 1413, 1301, 1254, 1237, 1176, 1110, 1029, 959, 872, 836, 808, 699, (C3), 129.59 (C1′′ ), 129.94 (C1), 131.39 (C6′ ), 132.97 (C6′′ ), 135.52 682, 548; HRMS (ESI+) Exact mass calculated for C12H10ClO+ 3 [M+H] + (C2′ ), 135.76 (C2′′ ), 141.32 (C4), 145.10 (C5), 156.81 (C4′ ), 156.88 = 237.0318; Found = 237.0312. (C4′′ ), 165.67 (C2); IR (ATR, cm− 1): 2917, 2848, 1768, 1594, 1496, 1461, 1439, 1287, 1266, 1054, 1016, 988, 816, 747, 668, 623; HRMS 4.1.8. Synthesis of 5-(1,5-dis(4-methoxyphenyl)penta-1,4-diyn-3-ylidene) (ESI+): Exact mass calculated for C19H14Br3O+ 4 [M+H] = 542.8436; + furan-2(5H)-one (37) Found = 542.8435. gem-Dibromofuranone 3 (300 mg, 1.181 mmol), triphenylphosphine (62 mg, 20 mol%), bis(triphenylphosphine)palladium(II) dichloride (83 4.1.6.4. 5-(Bis(4-(trifluoromethyl)phenyl)methylene)-3-bromofuran-2 mg, 10 mol%) and copper iodide (45 mg, 20 mol%) were added to an (5H)-one (32). Yield: 76% oven-dried RBF under a flow of nitrogen. Diisopropylamine (5 mL) and 1 H NMR (CDCl3, 400 MHz): 7.42 (2H, d, J = 8.1 Hz, H-2′ , H-6′ ), 7.51 4-ethynylanisole (0.33 mL, 2.599 mmol) were subsequently added to the (1H, s, H-4), 7.55 (2H, d, J = 8.3 Hz, H-2′′ , H-6′′ ), 7.64 (2H, d, J = 8.3 Hz, reaction flask. The mixture was stirred at 80 ◦ C for 24 h after which time H-3′′ , H-5′′ ), 7.74 (2H, d, J = 8.1 Hz, H-3′ , H-5′ ); 13C NMR (CDCl3, 100 the reaction was quenched with saturated aqueous ammonium chloride MHz): 114.38 (C6) 123.68 (q, J = 272.0 Hz, CF3’), 123.79 (q, J = 271.7 solution. The mixture was extracted with dichloromethane (3 x 30 mL) Hz, CF3′′ ), 125.47 (q, J = 3.7 Hz, C3′′ , C5′′ ), 125.64 (C3), 125.95 (q, J = and the combined organic layers were washed with water (3 x 30 mL) 3.6 Hz, C3′ , C5′ ), 131.03 (q, J = 32.62 Hz, C4′ ), 131.26 (C2′′ , C6′′ ), and brine (3 x 30 mL), dried with anhydrous magnesium sulfate and 131.37 (C2′ , C6′ ), 131.61 (q, J = 33.0 Hz, C4′′ ), 138.86 (C1′ ), 139.87 concentrated under reduced pressure. The crude product was purified by (C1′′ ), 140.87 (C4), 146.73 (C5), 164.96 (C2); IR (ATR, cm− 1): 2923, column chromatography with 5%–10% ethyl acetate in hexane to afford 1852, 1772, 1617, 1552, 1410, 1321, 1235, 1160, 1130, 1117, 1109, 37. 1069, 986, 855, 845, 730, 665, 610; HRMS (ESI+): Exact mass calculated Yield: 34% 12 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 1 H NMR (CDCl3, 400 MHz): 3.84–3.85 (6H, m, OCH3’, OCH3′′ ), 6.29 strain C. albicans SC5314 [67] with an RFP-cassette obtained from (1H, d, J = 5.3 Hz, H-3), 6.88–6.91 (4H, m, H-3′ , H-5′ , H-3′′ , H-5′′ ), 7.49 plasmid pADH1R1 [68]. A single copy of the ADH1 gene was replaced by (2H, d, J = 8.8 Hz, H-2′ , H-6′ ), 7.54 (2H, d, J = 8.9 Hz, H-2′′ , H-6′′ ), 7.86 the RFP-cassette yielding the RFP-labelled strain. Positive transformants (1H, d, J = 5.3 Hz, H-4); 13C NMR (CDCl3, 100 MHz) 55.38 and 55.40 were selected on YPD agar plates containing 200 μg/mL nourseothricin. (OCH3’, OCH3′′ ), 81.95 (C6), 82.61 and 92.76 (C’≡C′ , C’‘≡C′′ ), 95.28 The RFP signal was detected using the red channel (excitation/emission and 100.65 (C’≡C′ , C’‘≡C′′ ), 113.86 and 114.06 (C1′ , C1′′ ), 114.14 and 555/584 – FLUO-N3) of a Leica microscope (Leica DM5500 B). The 144.22 (C3′ , C5′ , C3′′ , C5′′ ), 119.80 (C3), 133.41 (C2′ , C6′ ), 133.82 (C2′′ , correct genomic integration of the RFP-cassette was confirmed by PCR C6′′ ), 140.32 (C4), 158.38 (C5), 160.50 and 160.65 (C4′ , C4′′ ), 168.63 and Southern blot analyses (data not shown). (C2); IR (ATR, cm− 1): 2936, 2841, 2187, 1774, 1748, 1603, 1509, 1411, 1291, 1250, 1173, 1119, 1105, 1031, 875, 831, 806, 740, 565; HRMS 4.2.3. Intrinsic cell growth inhibition (ESI+): Exact mass calculated for NaC23H16O+ 4 [M+Na] = 379.0936; + To evaluate the growth inhibition potential of furanone compounds, Found = 379.0946. a variant of the broth microdilution method was used. Bacterial strains were grown overnight in the appropriated medium and then subcultured 4.2. Biological evaluation to new cultures with 1:100 dilution. 100 μL of fresh log phase cultures, adjusted to a final concentration of 1 × 105 cfu/mL, were added per well 4.2.1. Microbial strains and growth conditions to 96-well sterile microtiter plates containing 100 μL of furanone at 0.1 Bacterial strains were obtained from our own collection. S. enterica mM (from a stock solution at 10 mM dissolved in DMSO) to obtain a final sv. Typhimurium was grown in tryptic soy broth 20 times diluted (1/20 concentration of 0.05 mM. Plates were incubated at 37 ◦ C for 20 h and TSB). S. aureus Newman (ATCC 25904) was grown in TSB 0.5 × TSB then the optical density, determined at 550 nm (OD550nm), was supplemented with 1% glucose. P. aeruginosa strains PAO1 and measured using a multilabel microtitre plate reader (Victor 3, Perki­ PAR7244, E. coli ATCC 9637 and S. maltophilia K279a were grown in LB nElmer LAS, Waltham, MA, USA). Before readout, plates were shaken for broth (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl). All cultures homogenization. DMSO alone was included as control at final concen­ were incubated at 37 ◦ C and maintained on LB agar plates. If not stated tration of 0.5%. Bacterial growth was expressed in percentage on the otherwise, C. albicans M2396 [adh1::(P_ADH1-RFP CaSAT1)] was grown basis of growth in control wells. The screening was done at least three aerobically with shaking (180 rpm) at either 30 ◦ C or 37 ◦ C in YPD times. The average of OD550nm values of each sample was used to media (10 g/L yeast extract, 20 g/L peptone and 20 g/L D-(+)-glucose) calculate the percentage of inhibition. supplemented with 200 μg/mL nourseothricin. To assess the growth of C. albicans M2396 in the presence of fur­ anones at 50 μM each, a fresh overnight culture of the yeast grown 4.2.2. Fluorescently labelled strains aerobically (180 rpm) at 30 ◦ C in YPD medium was diluted to an OD600 The P. aeruginosa strain PAO1 was fluorescently labelled with of 0.05, followed by transfer of 199 μL of the diluted cell suspension to enhanced yellow fluorescent protein (eYFP), using the mini-Tn7 de­ each well of a Corning 96 flat bottom transparent polystyrol microplate livery plasmid pUC18T-mini-Tn7T-Gm-eyfp (Addgene #65031) essen­ containing 1 μl of the compound stock solution (10 mM in DMSO). tially as described previously [60,61]. For the construction of Corresponding controls consisting of 199 μL of the diluted cell suspen­ P. aeruginosa PAO1:eYFP, the plasmid pUC18T-mini-Tn7T-Gm-eyfp was sion and 1 μL of DMSO were used as a reference to calculate the average transferred to the P. aeruginosa PAO1 wild-type by four-parental mating, percentage of growth inhibition by each individual furanone from four involving E. coli DH5α/pRK2013 [62–64] and E. coli SM10 independent experiments. The microplates were incubated in a humid­ λpir/pUX-BF13 [63,65] as helper strains, E. coli DH5α/pUC18T-mi­ ified chamber at 30 ◦ C with shaking (100 rpm) for 20 h, followed by ni-Tn7T-Gm-eyfp as the donor, and P. aeruginosa PAO1 as the recipient measurement of the OD600 values of the cell suspensions using a strain. The strains were grown in LB media, containing kanamycin (30 NanoQuant Infinite 200 Microplate Reader (Tecan Group, Gröding, μg/mL) for E. coli DH5α/pRK2013, 100 μg/mL of ampicillin for E. coli Austria). SM10 λpir/pUX-BF13, and ampicillin (100 μg/mL) and gentamycin (15 μg/mL) for E. coli DH5α/pUC18T-mini-Tn7T-Gm-eyfp. All strains were 4.2.4. Biofilm inhibition assay grown aerobically with shaking (220 rpm) at 37 ◦ C in LB media, except For biofilm formation, the crystal violet method was used as previ­ E. coli SM10 λpir/pUX-BF13 was routinely cultured at 30 ◦ C. After ously described, with some modifications [69]. Briefly, 100 μl of fresh overnight growth, the cells from 1 mL of each E. coli culture and 330 μL log phase bacteria at a final concentration of 1 × 105 cfu/mL, were of P. aeruginosa PAO1 were harvested by centrifugation. To prepare a added to 96-well non-treated flat-bottom sterile microtiter plates (Del­ mating mixture, the cell pellets of all strains were pooled in 1 mL of LB talab S.L., Spain) containing 100 μL of furanones at 0.1 mM (0.05 mM as medium, sedimented by centrifugation, resuspended in 100 μL of super a final concentration). Plates were incubated for 24 h at 37 ◦ C. The optimal broth (SOB) (5 g/L yeast extract, 20 g/L tryptone, 10 mM NaCl, OD620nm was determined using the microtitre plate reader (Victor 3) to 2.5 mM KCl, 20 mM MgSO4) and then spotted onto an SOB agar plate as measure bacterial biomass. Supernatants were then aspirated and the described elsewhere for triparental mating experiments [66]. Following plates were washed three times with distilled water. In order to fix the overnight incubation of the SOB agar plate at 30 ◦ C, the bacterial spot biofilm, plates were incubated at 65 ◦ C for 15 min. The biofilm was was scraped and resuspended in 1 mL of PBS by rigorous vortexing to stained by adding 200 μL of 0.1% crystal violet and the plates were interrupt mating pairs. The P. aeruginosa PAO1 transconjugants were incubated for 15 min at room temperature. The crystal violet was dis­ selected at 37 ◦ C on Vogel-Bonner minimal medium (VBBM) (3 g/L carded and the plates were washed three times with distilled water. The trisodium citrate, 2 g/L citric acid, 10 g/L K2HPO4, 3.5 g/L NaNH4 plates were allowed to dry, incubating at 37 ◦ C for at least 30 min. To PO4⋅4H2O, 1 mM MgSO4, 100 μM CaCl2, pH 7.0) agar plates containing dissolve the crystal violet, 250 μl of 30% acetic acid was added. The 30 μg/ml of gentamycin [61]. The chromosomal insertion of the min­ plates were incubated for 15 min at room temperature and 150 μL was i-Tn7 element downstream of the glmS gene for glutamine–­ transferred to a new plate and the OD550nm was measured. At least three fructose-6-phosphate aminotransferase was verified by PCR, using the replicates per strain and condition were tested. Growth control wells primer pairs PTn7R [61] and PAO1-glmS-Ctrl1 (GCTGAAGCTCAAG­ containing bacteria in the corresponding medium and control wells GAAATTTCC), and PTn7L [61] and PAO1-PA5548-Ctr (TTACCTGC­ containing only sterile medium were included. GACGTTATCTGAAGC) to amplify the flanking mini-Tn7 regions, Biofilm formation of C. albicans M2396 upon exposure to furanones respectively. at 50 μM each was investigated by sustained and developmental inhi­ The RFP-expressing C. albicans strain M2396 expressing the red bition assays according to published protocols [70]. For the sustained fluorescence protein (RFP) was generated by transforming the wild-type inhibition assay, the furanones were present in both a 90-min adherence 13 ­ A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 step and a 24-h growth period, whereas the developmental inhibition and incubated for 24 and 48 h. The culture medium was then removed. assay consisted of a 90-min adherence step in absence and a 24-h biofilm Next, 100 μL of fresh medium and 20 μL of CellTiter 96 aqueous reagent formation period in presence of the compounds. All other steps were were added to each well. The absorbance was read after 4 h of incuba­ identical for both assays. The OD600 of a fresh overnight culture of tion. A microplate reader (PerkinElmer Victor 3) was used with a C. albicans M2396 grown aerobically (180 rpm) at 30 ◦ C in YPD medium wavelength of 450 nm and 620 nm. The results are expressed as viability was adjusted to a value of 0.5. For the sustained inhibition assay, 199 μL percentage using cells unexposed as a control. of the cell suspension was added to each well of a Corning 96 flat bottom transparent polystyrol microplate along with 1 μL of the compound 4.2.7. In vivo efficacy in the Galleria mellonella infection model stock solution (10 mM in DMSO), whereas the wells of the develop­ The larval stage of greater wax moth G. mellonella was used to mental inhibition assay contained instead 1 μL of DMSO. The micro­ evaluate the effect of furanones following bacterial infection. Larvae plates were then incubated without shaking at 37 ◦ C for 90 min. The were obtained from our own hatchery. At least 15 larvae with no signs of media were aspirated and the wells were washed with 200 μL of PBS. A melanization and a weight of 300–400 mg were used to infect total volume of 200 μL of YPD medium with the appropriate compound P. aeruginosa PAR7244. The inoculum was obtained from a log-phase was added to the corresponding well, and after an incubation of the culture in appropriate media. Log-phase cultures were washed and microplates in a humidified chamber at 37 ◦ C with shaking (100 rpm) adjusted in PBS to the desired doses, based on pilot studies (data not for another 24 h, the medium was aspirated from each well. Following a shown). The infective dose for P. aeruginosa PAR7244 was 1.6–3.2 × 102 washing step of the biofilm with 200 μL of PBS, cell viability was cfu per larva. The cell number of the inoculum was confirmed by viable examined with the colorimetric 2,3-bis-(2-methoxy-4-ni­ counts on LB agar plates. Larvae were infected injecting 5 μL of bacteria tro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction in the last left proleg using a 50-μL Hamilton® Microliter™ syringe. assay as described [70]. The OD492 values obtained in quadruplicate for Larvae were incubated at 30 ◦ C for 60 min. After incubation, 5 μL of a each condition were averaged and normalized to averaged OD492 values PBS solution containing either DMSO (untreated group) or furanone at a of control biofilms formed in YPD medium with 0.5% of DMSO in both final concentration of 50 μM was injected in the last right proleg. The the adherence step and the growth period. amount of furanones or DMSO solution injected was obtained according to the larvae weight as was previously described [71]. Larvae were 4.2.5. Confocal laser-scanning microscopy of mono- and dual-species incubated at 30 ◦ C in darkness and the survival was recorded every 24 h. biofilms Larvae were considered dead when they were unresponsive to touch and Confocal laser-scanning microscopy (CLSM) was used to investigate turned dark brown or black in colour. At least two replicates of the ex­ the effect of selected furanones at 50 μM each on the formation of mono- periments were made with different batches of larvae. Kaplan-Meier and dual-species biofilms. To prepare the inoculum for biofilm forma­ survival curves were plotted using GraphPad Prism and statistical sig­ tion, the cell numbers of exponentially growing cultures of P. aeruginosa nificance was determined using the log rank (Mantel Cox) test. PAO1:eYFP and C. albicans M2396 were adjusted to 6.67 × 105 cfu/mL in LB media. Each well of a μ-Slide 8-Well ibiTreat slide (ibidi GmbH, Declaration of competing interest Gräfelfing, Germany) was then seeded with a total volume of 300 μL, containing 1 × 105 cfu of each microorganism and 1.5 μL of the com­ The authors declare that they have no known competing financial pound stock solution (10 mM in DMSO). For mono-species biofilms, 150 interests or personal relationships that could have appeared to influence μL of the diluted cell suspension was mixed with 150 μL of LB medium, the work reported in this paper. whereas the inoculum for dual-species biofilm formation consisted of 150 μL of each microorganism with a cell number of 6.67 × 105 cfu/ml. Data availability The corresponding control biofilms were grown in LB media containing 0.5% of DMSO. After incubation of the μ-Slides at 37 ◦ C without shaking Data is available in the Supplementary Data. in a humidified chamber for 24 h, the culture supernatants were aspi­ rated and the wells were washed with 300 μL of PBS. The biofilms were Acknowledgements then fixed in the dark with 300 μL of a 2% paraformaldehyde solution in PBS at room temperature for 20 min. Finally, the biofilms were washed TL was supported by the School of Pharmacy University College Cork with 300 μL of PBS and overlayed with 150 μL of VECTASHIELD Anti­ and Future University Egypt with a PhD Scholarship. CGMG acknowl­ fade Mounting Medium (Biozol GmbH, Eching, Germany). edges support from Science Foundation Ireland in the form of a center CLSM of the biofilms was performed with a TCS SP5 inverse confocal grant (APC Microbiome Ireland grant SFI/12/RC/2273_P2). ACG, DY, laser-scanning microscope (Leica Microsystems, Mannheim, Germany) MB, XD and IG acknowledge financial support from the Spanish Ministry and analysed with LAS AF software (version 2.73). The microscope was for Science, Innovation and Universities (grant PID2019-111364RB- equipped with a Leica 63x/NA 1.40 HCX Plan Apochromat CS oil im­ I00). UM and SB are grateful to the Leibniz Association for financial mersion objective. RFP of C. albicans M2396 was excited with 561 nm support (grant SAS-2021-1-FZB of the Leibniz Research Alliance IN­ laser light and fluorescence emission was detected between 566 and 600 FECTIONS in an Urbanizing World - Humans, Animals, Environments). We nm, whereas eYFP of P. aeruginosa PAO1:eYFP was excited with 514 nm thank Manuel Hein, Dörte Grella (RCB) for expert technical assistance laser light and fluorescence emission was detected between 520 and 560 and the Fluorescence Cytometry Core Facility of the RCB for continuous nm. support. We thank Herbert P. Schweizer (Colorado State University, Fort Collins, USA) for the plasmid pUC18T-mini-Tn7T-Gm-eyfp (Addgene # 4.2.6. Cytotoxicity assay 65031) and Holger Lößner (Paul-Ehrlich-Institute, Federal Institute for To determinate the cytotoxic effect of furanones on HK-2 tubular Vaccines and Biomedicines, Langen, Germany) for E. coli SM10 λpir/ cells, the kit CellTiter 96 Aqueous solution (Promega) was used pUX-BF13. following the manufacturer’s instructions. HK-2 cells were seeded at a concentration of 3.5 × 103 cells per well in 96-well tissue culture plates Appendix A. Supplementary data with clear bottoms (Nunc™ MicroWell™ 96-Well, Nunclon Delta- Treated®), using DMEM/F-12 GlutaMAX™ (Gibco™) supplemented Supplementary data to this article can be found online at https://doi. with 10% Fetal Bovine Serum and 1 × Insulin-Transferrin-Selenium org/10.1016/j.ejmech.2022.114678. (Gibco™). The plates were incubated for 24 h (37 ◦ C in 5% CO2 atmo­ sphere). Furanones were diluted to 50 μM in 200 μl of DMEM per well 14 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 Supplementary data [24] T. Lyons, C.G.M. Gahan, T.P. O’Sullivan, Structure–activity relationships of furanones, dihydropyrrolones and thiophenones as potential quorum sensing inhibitors, Future Med. Chem. 12 (2020) 1925–1943, https://doi.org/10.4155/ Supplementary data associated with this article can be found in the fmc-2020-0244. online version, at XXXXX. [25] C.S. Pereira, J.A. Thompson, K.B. Xavier, AI-2-mediated signalling in bacteria, FEMS Microbiol. Rev. 37 (2013) 156–181, https://doi.org/10.1111/j.1574- 6976.2012.00345.x. References [26] T. Zang, B.W.K. Lee, L.M. Cannon, K.A. Ritter, S. Dai, D. Ren, T.K. Wood, Z.S. Zhou, A naturally occurring brominated furanone covalently modifies and inactivates [1] L. Zaffiri, J. Gardner, L.-H. Toledo-Pereyra, History of antibiotics. From Salvarsan LuxS, Bioorg. Med. Chem. Lett 19 (2009) 6200–6204, https://doi.org/10.1016/j. to Cephalosporins, J. Invest. Surg. 25 (2012) 67–77, https://doi.org/10.3109/ bmcl.2009.08.095. 08941939.2012.664099. [27] M. Manefield, R. de Nys, N. Kumar, R. Read, M. Givskov, P. Steinberg, [2] K.C. Nicolaou, S. Rigol, A brief history of antibiotics and select advances in their S. Kjelleberg, Evidence that halogenated furanones from Delisea pulchra inhibit synthesis, J. Antibiot. 71 (2018) 153–184, https://doi.org/10.1038/ja.2017.62. acylated homoserine lactone (AHL)-mediated gene expression by displacing the [3] E.B. Chain, Academic and industrial contributions to drug research, Nature 200 AHL signal from its receptor protein, Microbiol. 145 (Pt 2) (1999) 283–291, (1963) 441–451, https://doi.org/10.1038/200441a0. https://doi.org/10.1099/13500872-145-2-283. [4] T.C. Porco, T. Gebre, B. Ayele, J. House, J. Keenan, Z. Zhou, K.C. Hong, N. Stoller, [28] H. Wu, Z. Song, M. Hentzer, J.B. Andersen, S. Molin, M. Givskov, N. Hoiby, K.J. Ray, P. Emerson, B.D. Gaynor, T.M. Lietman, Effect of mass distribution of Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in azithromycin for trachoma control on overall mortality in Ethiopian children: a Pseudomonas aeruginosa lung infection in mice, J. Antimicrob. Chemother. 53 randomized trial, JAMA 302 (2009) 962–968, https://doi.org/10.1001/ (2004) 1054–1061, https://doi.org/10.1093/jac/dkh223. jama.2009.1266. [29] T. Lyons, C.G.M. Gahan, T.P. O’Sullivan, Synthesis and reactivity of [5] C.P. Miller, Development of bacterial resistance to antibiotics, JAMA 135 (1947) dihalofuranones, Lett. Org. Chem. 19 (2022) 662–667, https://doi.org/10.2174/ 749–751, https://doi.org/10.1001/jama.1947.02890120003002. 1570178618666211027103633. [6] O.B. Jonas, A. Irwin, F.C.J. Berthe, F.G. Le Gall, P.V. Marquez, Drug-resistant [30] S. Couty, M. Barbazanges, C. Meyer, J. Cossy, Palladium-catalyzed Suzuki-Miyaura Infections : a Threat to Our Economic Future, The World Bank, 2017. coupling reactions involving β,β-dihaloenamides: application to the synthesis of [7] J. O’Neill, Review on Antimicrobial Resistance, Antimicrobial Resistance - Tackling disubstituted Ynamides, Synlett 2005 (2005) 905–910, https://doi.org/10.1055/s- a Crisis for the Health and Wealth of Nations, 2014. 2005-864821. [8] Antimicrobial Resistance Collaborators, Global burden of bacterial antimicrobial [31] L. Jeanne-Julien, G. Masson, E. Astier, G. Genta-Jouve, V. Servajean, J.-M. Beau, resistance in 2019: a systematic analysis, Lancet 399 (2022) 629–655, https://doi. S. Norsikian, E. Roulland, Study of the construction of the Tiacumicin B aglycone, org/10.1016/s0140-6736(21)02724-0. J. Org. Chem. 83 (2018) 921–929, https://doi.org/10.1021/acs.joc.7b02909. [9] S.J. Baker, D.J. Payne, R. Rappuoli, E. De Gregoriob, Technologies to address [32] E. Marchal, P. Uriac, Y. Brunel, S. Poigny, Methylene Furanone Derivatives and Use antimicrobial resistance, Proc. Natl. Acad. Sci. USA 115 (2018) 12887–12895, of Said Derivatives as a Photoprotecting or Antioxidant or Depigmentation Agent https://doi.org/10.1073/pnas.1717160115. in Cosmetic or Dermatological Compositions, US Patent US8563548B2, 2013. [10] M.E. Olson, H. Ceri, D.W. Morck, A.G. Buret, R.R. Read, Biofilm bacteria: formation [33] D. Nöteberg, W. Schaal, E. Hamelink, L. Vrang, M. Larhed, High-speed and comparative susceptibility to antibiotics,, Can. J. Vet. Res. 66 (2002) 86–92. optimization of inhibitors of the malarial proteases plasmepsin I and II, J. Comb. PMCID: PMC226988. Chem. 5 (2003) 456–464, https://doi.org/10.1021/cc0301014. [11] D. Sharma, L. Misba, A.U. Khan, Antibiotics versus biofilm: an emerging [34] H.P. Steenackers, J. Levin, J.C. Janssens, A. De Weerdt, J. Balzarini, battleground in microbial communities, Antimicrob. Resist. Infect. Control 8 J. Vanderleyden, D.E. De Vos, S.C. De Keersmaecker, Structure-activity (2019) 1–10, https://doi.org/10.1186/s13756-019-0533-3. relationship of brominated 3-alkyl-5-methylene-2(5H)-furanones and alkylmaleic [12] I.S. Sharafutdinov, E.Y. Trizna, D.R. Baidamshina, M.N. Ryzhikova, R. anhydrides as inhibitors of Salmonella biofilm formation and quorum sensing R. Sibgatullina, A.M. Khabibrakhmanova, L.Z. Latypova, A.R. Kurbangalieva, E. regulated bioluminescence in Vibrio harveyi, Bioorg. Med. Chem. 18 (2010) V. Rozhina, M. Klinger-Strobel, R.F. Fakhrullin, M.W. Pletz, M.I. Bogachev, A. 5224–5233, https://doi.org/10.1016/j.bmc.2010.05.055. R. Kayumov, O. Makarewicz, Antimicrobial effects of sulfonyl derivative of 2(5H)- [35] C. Reynaud, H. Doucet, M. Santelli, Synthesis of Spherical Polyols from Itaconic Furanone against planktonic and biofilm associated methicillin-resistant and Acid, Synthesis 2010 (2010) 1787–1792, https://doi.org/10.1055/s-0029- -susceptible Staphylococcus aureus, Front. Microbiol. 8 (2017), https://doi.org/ 1218764. 10.3389/fmicb.2017.02246. [36] N.N. Biswas, G.M. Iskander, M. Mielczarek, T.T. Yu, D.S. Black, N. Kumar, Alkyne- [13] T. Grainha, P. Jorge, D. Alves, S.P. Lopes, M.O. Pereira, Unraveling Pseudomonas substituted fimbrolide analogues as novel bacterial quorum-sensing inhibitors, aeruginosa and Candida albicans communication in coinfection scenarios: insights Aust. J. Chem. 71 (2018) 708–715, https://doi.org/10.1071/CH18194. through network analysis, Front. Cell. Infect. Microbiol. 10 (2020), https://doi. [37] R. Franzén, L. Kronberg, Synthetic approaches to chlorinated 5-hydroxy-5-methyl- org/10.3389/fcimb.2020.550505. 2-furanones, Tetrahedron 49 (1993) 10945–10958, https://doi.org/10.1016/ [14] M.B. Miller, B.L. Bassler, Quorum sensing in bacteria, Annu. Rev. Microbiol. 55 S0040-4020(01)80248-9. (2001) 165–199, https://doi.org/10.1146/annurev.micro.55.1.165. [38] R. Franzén, L. Kronberg, Synthesis of chlorinated 5-hydroxy 4-methyl-2(5H)- [15] B.L. Bassler, S. Mukherjee, Bacterial quorum sensing in complex and dynamically furanones and mucochloric acid, Tetrahedron Lett. 36 (1995) 3905–3908, https:// changing environments, Nat. Rev. Microbiol. 17 (2019) 371–382, https://doi.org/ doi.org/10.1016/0040-4039(95)00638-S. 10.1038/s41579-019-0186-5. [39] S.M. Jajere, A review of Salmonella enterica with particular focus on the [16] W.R.J.D. Galloway, J.T. Hodgkinson, S.D. Bowden, M. Welch, D.R. Spring, Quorum pathogenicity and virulence factors, host specificity and antimicrobial resistance sensing in gram-negative bacteria: small-molecule modulation of AHL and AI-2 including multidrug resistance, Vet. World 12 (2019) 504–521, https://doi.org/ quorum sensing pathways, Chem. Rev. 111 (2011) 28–67, https://doi.org/ 10.14202/vetworld.2019.504-521. 10.1021/cr100109t. [40] K.M. Craft, J.M. Nguyen, L.J. Berg, S.D. Townsend, Methicillin-resistant [17] L. Zhou, Y. Zhang, Y. Ge, X. Zhu, J. Pan, Regulatory mechanisms and promising Staphylococcus aureus (MRSA): antibiotic-resistance and the biofilm phenotype, applications of quorum sensing-inhibiting agents in control of bacterial biofilm Med. Chem. Comm. 10 (2019) 1231–1241, https://doi.org/10.1039/c9md00044e. formation, Front. Microbiol. 11 (2020), https://doi.org/10.3389/ [41] C.B. Creech, D.N. Al-Zubeidi, S.A. Fritz, Prevention of recurrent staphylococcal fmicb.2020.589640. skin infections, Infect. Dis. Clin. 29 (2015) 429–464, https://doi.org/10.1016/j. [18] R. de Nys, A.D. Wright, G.M. König, O. Sticher, New halogenated furanones from idc.2015.05.007. the marine alga delisea pulchra (cf. fimbriata), Tetrahedron 49 (1993) [42] S.Y. Tong, J.S. Davis, E. Eichenberger, T.L. Holland, V.G. Fowler Jr., Staphylococcus 11213–11220, https://doi.org/10.1016/S0040-4020(01)81808-1. aureus infections: epidemiology, pathophysiology, clinical manifestations, and [19] S. Kjelleberg, P. Steinberg, M. Givskov, L. Gram, M. Manefield, R. de Nys, Do management, Clin. Microbiol. Rev. 28 (2015) 603–661, https://doi.org/10.1128/ marine natural products interfere with prokaryotic AHL regulatory systems? Aquat. cmr.00134-14. Microb. Ecol. 13 (1997) 85–93, https://doi.org/10.3354/ame013085. [43] N. Allocati, M. Masulli, M.F. Alexeyev, C. Di Ilio, Escherichia coli in Europe: an [20] J.C.A. Janssens, H. Steenackers, S. Robijns, E. Gellens, J. Levin, H. Zhao, overview, Int. J. Environ. Res. Publ. Health 10 (2013) 6235–6254, https://doi.org/ K. Hermans, D. De Coster, T.L. Verhoeven, K. Marchal, J. Vanderleyden, D.E. De 10.3390/ijerph10126235. Vos, S.C.J. De Keersmaecker, Brominated furanones inhibit biofilm formation by [44] J.S. Brooke, Stenotrophomonas maltophilia: an emerging global opportunistic Salmonella enterica Serovar Typhimurium,, Appl. Environ. Microbiol. 74 (2008) pathogen, Clin. Microbiol. Rev. 25 (2012) 2–41, https://doi.org/10.1128/ 6639–6648, https://doi.org/10.1128/AEM.01262-08. cmr.00019-11. [21] M. Hentzer, M. Givskov, Pharmacological inhibition of quorum sensing for the [45] P. Huedo, X. Coves, X. Daura, I. Gibert, D. Yero, Quorum sensing signaling and treatment of chronic bacterial infections, J. Clin. Investig. 112 (2003) 1300–1307, quenching in the multidrug-resistant pathogen Stenotrophomonas maltophilia, Front. https://doi.org/10.1172/JCI200320074. Cell. Infect. Microbiol. 8 (2018) 1–8, https://doi.org/10.3389/fcimb.2018.00122. [22] Y. Han, S. Hou, K.A. Simon, D. Ren, Y.-Y. Luk, Identifying the important structural [46] A. Litwin, S. Rojek, W. Gozdzik, W. Duszynska, Pseudomonas aeruginosa device elements of brominated furanones for inhibiting biofilm formation by Escherichia associated - healthcare associated infections and its multidrug resistance at coli,, Bioorg. Med. Chem. Lett. 18 (2008) 1006–1010, https://doi.org/10.1016/j. intensive care unit of University Hospital: polish, 8.5-year, prospective, single- bmcl.2007.12.032. centre study, BMC Infect. Dis. 21 (2021) 1–8, https://doi.org/10.1186/s12879- [23] J. Lonn-Stensrud, M.A. Landin, T. Benneche, F.C. Petersen, A.A. Scheie, Furanones, 021-05883-5. potential agents for preventing Staphylococcus epidermidis biofilm infections? [47] G.P. Bodey, R. Bolivar, V. Fainstein, L. Jadeja, Infections caused by Pseudomonas J. Antimicrob. Chemother. 63 (2009) 309–316, https://doi.org/10.1093/jac/ aeruginosa, Rev. Infect. Dis. 5 (1983) 279–313, https://doi.org/10.1093/clinids/ dkn501. 5.2.279. 15 A.-C. Gómez et al. European Journal of Medicinal Chemistry 242 (2022) 114678 [48] M. Gomes-Fernandes, A.-C. Gomez, M. Bravo, P. Huedo, X. Coves, C. Prat- [61] K.-H. Choi, H.P. Schweizer, mini-Tn7 insertion in bacteria with single attTn7 sites: Aymerich, I. Gibert, A. Lacoma, D. Yero, Strain-specific interspecies interactions example Pseudomonas aeruginosa, Nat. Protoc. 1 (2006) 153–161, https://doi.org/ between co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa 10.1038/nprot.2006.24. from patients with tracheobronchitis or bronchial colonization, Sci. Rep. 12 (2022) [62] D.H. Figurski, D.R. Helinski, Replication of an origin-containing derivative of 1–15, https://doi.org/10.1038/s41598-022-07018-5. plasmid RK2 dependent on a plasmid function provided in trans, Proc. Natl. Acad. [49] F.L. Mayer, D. Wilson, B. Hube, Candida albicans pathogenicity mechanisms, Sci. USA 76 (1979) 1648–1652, https://doi.org/10.1073/pnas.76.4.1648. Virulence 4 (2013) 119–128, https://doi.org/10.4161/viru.22913. [63] V.L. Miller, J.J. Mekalanos, A novel suicide vector and its use in construction of [50] R. Pereira, R.O. Dos Santos Fontenelle, E.H.S. De Brito, S.M. De Morais, Biofilm of insertion mutations: osmoregulation of outer membrane proteins and virulence Candida albicans: formation, regulation and resistance, J. Appl. Microbiol. 131 determinants in Vibrio cholerae requires toxR, J. Bacteriol. 170 (1988) 2575–2583, (2020) 11–22, https://doi.org/10.1111/jam.14949. https://doi.org/10.1128/jb.170.6.2575-2583.1988. [51] P. Uppuluri, J. Nett, J. Heitman, D. Andes, Synergistic effect of calcineurin [64] R. Simon, U. Priefer, A. Pühler, A broad host range mobilization system for in vivo inhibitors and fluconazole against Candida albicans biofilms, Antimicrob. Agents genetic engineering: transposon mutagenesis in Gram negative bacteria, Chemother. 52 (2008) 1127–1132, https://doi.org/10.1128/aac.01397-07. Biotechnology 1 (1983) 784–791, https://doi.org/10.1038/nbt1183-784. [52] M. Gulati, C.J. Nobile, Candida albicans biofilms: development, regulation, and [65] Y. Bao, D.P. Lies, H. Fu, G.P. Roberts, An improved Tn7-based system for the single- molecular mechanisms, Microb. Infect. 18 (2016) 310–321, https://doi.org/ copy insertion of cloned genes into chromosomes of Gram-negative bacteria, Gene 10.1016/j.micinf.2016.01.002. 109 (1991) 167–168, https://doi.org/10.1016/0378-1119(91)90604-a. [53] M. Duo, M. Zhang, Y.-Y. Luk, D. Ren, Inhibition of Candida albicans growth by [66] D.F. Aubert, M.A. Hamad, M.A. Valvano, A markerless deletion method for genetic brominated furanones, Appl. Microbiol. Biotechnol. 85 (2010) 1551–1563, https:// manipulation of Burkholderia cenocepacia and other multidrug-resistant gram- doi.org/10.1007/s00253-009-2174-6. negative bacteria, in: A. Vergunst, D. O’Callaghan (Eds.), Host-Bacteria [54] M.E. Shirtliff, B.M. Peters, M.A. Jabra-Rizk, Cross-kingdom interactions: Candida Interactions, Humana Press, New York, 2014, pp. 311–327. albicans and bacteria, FEMS Microbiol. Lett. 299 (2009) 1–8, https://doi.org/ [67] A.M. Gillum, E.Y. Tsay, D.R. Kirsch, Isolation of the Candida albicans gene for 10.1111/j.1574-6968.2009.01668.x. orotidine-5’-phosphate decarboxylase by complementation of S. cerevisiae ura3 and [55] E. Allegra, R.W. Titball, J. Carter, O.L. Champion, Galleria mellonella larvae allow E. coli pyrF mutations, Mol. Gen. Genet. 198 (1984) 179–182, https://doi.org/ the discrimination of toxic and non-toxic chemicals, Chemosphere 198 (2018) 10.1007/bf00328721. 469–472, https://doi.org/10.1016/j.chemosphere.2018.01.175. [68] C. Sasse, N. Dunkel, T. Schäfer, S. Schneider, F. Dierolf, K. Ohlsen, [56] L. Hill, N. Veli, P.J. Coote, Evaluation of Galleria mellonella larvae for measuring the J. Morschhäuser, The stepwise acquisition of fluconazole resistance mutations efficacy and pharmacokinetics of antibiotic therapies against Pseudomonas causes a gradual loss of fitness in Candida albicans, Mol. Microbiol. 86 (2012) aeruginosa infection, Int. J. Antimicrob. Agents 43 (2014) 254–261, https://doi. 539–556, https://doi.org/10.1111/j.1365-2958.2012.08210.x. org/10.1016/j.ijantimicag.2013.11.001. [69] P. Huedo, D. Yero, S. Martínez-Servat, I. Estibariz, R. Planell, P. Martínez, A. Ruyra, [57] WHO, Antimicrobial Resistance, Global Report on Surveillance, WHO Reports, N. Roher, I. Roca, J. Vila, X. Daura, I. Gibert, Two different rpf clusters distributed World Health Organization, Geneva, Switzerland, 2014. among a population of Stenotrophomonas maltophilia clinical strains display [58] A. Sorg, K. Siegel, R. Brukner, Stereoselective syntheses of dihydroxerulin and differential diffusible signal factor production and virulence regulation, xerulinic acid, anti-hypocholesterolemic dyes from the fungus Xerula melanotricha, J. Bacteriol. 196 (2014) 2431–2442, https://doi.org/10.1128/jb.01540-14. Chem. Eur J. 11 (2005) 1610–1624, https://doi.org/10.1002/chem.200400913. [70] M. Gulati, M.B. Lohse, C.L. Ennis, R.E. Gonzalez, A.M. Perry, P. Bapat, A. [59] A.J. Manny, S. Kjelleberg, N. Kumar, R. de Nys, R.W. Read, P. Steinberg, V. Arevalo, D.L. Rodriguez, C.J. Nobile, In vitro culturing and screening of Candida Reinvestigation of the sulfuric acid-catalysed cyclisation of brominated 2-alkylle­ albicans biofilms, Curr. Protoc. Microbiol. 50 (2018) e60, https://doi.org/10.1002/ vulinic acids to 3-alkyl-5-methylene-2(5H)-furanones, Tetrahedron 53 (1997) cpmc.60. 15813–15826, https://doi.org/10.1016/S0040-4020(97)10034-5. [71] A. Andrea, K.A. Krogfelt, H. Jenssen, Methods and challenges of using the greater [60] K.-H. Choi, J.B. Gaynor, K.G. White, C. Lopez, C.M. Bosio, R.R. Karkhoff-Schweizer, wax moth (Galleria mellonella) as a model organism in antimicrobial compound H.P. Schweizer, A Tn7-based broad-range bacterial cloning and expression system, discovery, Microorganisms 7 (2019) 1–9, https://doi.org/10.3390/ Nat. Methods 2 (2005) 443–448, https://doi.org/10.1038/nmeth765. microorganisms7030085. 16