European Journal of Medicinal Chemistry 261 (2023) 115819 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: www.elsevier.com/locate/ejmech Research paper Synthesis and evaluation of aromatic BDSF bioisosteres on biofilm formation and colistin sensitivity in pathogenic bacteria Andromeda-Celeste Gómez a, b, 1, Conor Horgan c, 1, Daniel Yero a, b, Marc Bravo a, b, Xavier Daura a, d, e, Michelle O’Driscoll c, f, g, Isidre Gibert a, b, **, Timothy P. O’Sullivan c, f, g, * 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 Chemistry, University College Cork, Cork, Ireland d Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain e CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Cerdanyola de Vallès, Spain f School of Pharmacy, University College Cork, Cork, Ireland g 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: The diffusible signal factor family (DSF) of molecules play an important role in regulating intercellular Diffusible signal factor communication, or quorum sensing, in several disease-causing bacteria. These messenger molecules, which are Quorum sensing comprised of cis-unsaturated fatty acids, are involved in the regulation of biofilm formation, antibiotic tolerance, Biofilms virulence and the control of bacterial resistance. We have previously demonstrated how olefinic N-acyl sulfon­ Resistance amide bioisosteric analogues of diffusible signal factor can reduce biofilm formation or enhance antibiotic Acinetobacter baumannii Escherichia coli sensitivity in a number of bacterial strains. This work describes the design and synthesis of a second generation of Burkholderia multivorans aromatic N-acyl sulfonamide bioisosteres. The impact of these compounds on biofilm production in Acinetobacter Burkholderia cepacia baumannii, Escherichia coli, Burkholderia multivorans, Burkholderia cepacia, Burkholderia cenocepacia, Pseudomonas Burkholderia cenocepacia aeruginosa and Stenotrophomonas maltophilia is evaluated, in addition to their effects on antibiotic tolerance. The Pseudomonas aeruginosa and Stenotrophomonas ability of these molecules to increase survival rates on co-administration with colistin is also investigated using maltophilia the Galleria infection model. 1. Introduction its biosynthesis. The rpfF gene likely encodes an enoyl-coenzyme A (CoA) hydratase while the rpfB gene probably encodes a long chain fatty Diffusible signal factor (DSF) is one of an important family of quorum acyl CoA ligase. The structure of DSF was eventually confirmed to be the sensing (QS) signalling molecules found in Gram negative bacteria [1]. α,β-unsaturated fatty acid, cis-11-methyl-2-dodecenoic acid (1) [5]. DSF (1) was first identified in Xanthomonas campestris pv campestris Subsequently, a homologue of DSF from B. cenocepacia was isolated (Xcc), the bacterium responsible for black rot, a major disease of by Boon and colleagues, called BDSF (2) [6]. B. cenocepacia is a key cruciferous plants (Fig. 1) [2]. Xcc contains a cluster of genes, known as bacterium in the Burkholderia cepacia complex (BCC) and is an oppor­ regulation of pathogenicity factors (rpf), which are essential for the tunistic pathogen commonly found in immunocompromised in­ synthesis of enzymes and extracellular polysaccharides [3]. Mutation of dividuals, including those with cystic fibrosis (CF) [7]. The key one of these genes results in activity failure in related enzymes. Daniels structural difference between the two molecules is the absence of the and co-workers demonstrated that the phenotype of an rpfF mutant methyl group at C-11 in BDSF. It was later established that BDSF can also could be corrected when cultivated in proximity to its wild-type parental inhibit the ability of Candida albicans to switch between yeast and hy­ strain [4]. It was postulated that a signalling molecule was responsible phal forms [6,8]. In recent years, several additional species of bacteria for this restoration and that the rpfB and rpfF genes played some part in have been found to employ members of the DSF family in their quorum * Corresponding author. School of Chemistry, 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.2023.115819 Received 14 August 2023; Received in revised form 11 September 2023; Accepted 12 September 2023 Available online 15 September 2023 0223-5234/© 2023 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 261 (2023) 115819 of BDSF retain twelve carbons in their backbone. This article describes the preparation of a library of aromatic N-acyl sulfonamide derivatives of BDSF. The individual sulfonamides are evaluated for their inhibitory effects on biofilm production as well as their antimicrobial properties in nosocomial pathogens including Aci­ netobacter baumannii, E. coli, B. multivorans, B. cepacia, B. cenocepacia, P. aeruginosa and S. maltophilia. Additionally, the enhancing effects of the novel bioisosteres when co-administered with colistin are also reported. Fig. 1. Structures of DSF and BDSF signalling molecules. 2. Results and discussion 2.1. Chemistry sensing systems including Pseudomonas sp. [9], Stenotrophomonas mal­ tophilia (Sm) [10], Xanthamonas oryzae [11] and Xylella fastidiosa [12]. The preparation of the library of aromatic N-acyl sulfonamides Wang and co-workers investigated the effects of a library of DSF revolved around carboxylic acid 7 (Fig. 3). As a common intermediate, 7 derivatives on Xcc virulence in order to determine the structural features could be coupled with various sulfonamides to create a library of bio­ that were important for biological activity [5]. A key finding was that isosteric analogues. Starting from 2-iodobenzoic acid (3), methyl 2-iodo­ the location and configuration of the double bond was important for benzoate (4) was synthesised using standard Fisher esterification activity. For example, the saturated fatty acid 11-methyldodecanoic acid conditions by heating 3 and catalytic sulfuric acid in methanol to reflux was 20,000-fold less active in the induction of virulence gene expression to afford the methyl ester in 91% yield [24]. The next step was to than DSF. Additionally, cis-unsaturated DSF was 200-fold more active introduce the alkyl chain via Pd/Cu-mediated Sonogashira coupling. than its trans isomer (i.e. trans-11-methyl-2-dodecenoic acid). The cis-­ Coupling of 4 and 1-nonyne using bis(triphenylphosphine)palladium(II) configuration is likewise important in other DSF-sensitive pathogens dichloride and copper (I) iodide as the catalyst system at room tem­ including BCC, Pseudomonas aeruginosa, C. albicans, Escherichia coli, perature did furnish the target 5, albeit in a low yield of 22%. Increasing Klebsiella pneumoniae, Proteus mirabilis, Streptococcus pyogenes, Bacillus the temperature to reflux saw a modest improvement in yield to 27% subtilis, and Streptococcus mutans [1,13,14]. A similar pattern was after 24 h. The use of microwave irradiation in metal-catalysed reactions observed for BDSF – trans-BDSF, or trans-2-dodecenoic acid, was 33-fold is often associated with higher yields and reduced reaction times less active than BDSF in Xcc while saturated dodecanoic acid was 333 [25–27]. When the reaction mixture was heated to 100 ◦ C under mi­ times less effective than BDSF. Shortening or increasing the fatty acid crowave irradiation in anhydrous acetonitrile, alkyne 5 was recovered chain length was also found to be detrimental. in a much improved yield of 66% yield. The copper acetylide species In our search for novel quorum sensing inhibitors of DSF and BDSF, generated in situ can render Sonogashira reactions sensitive to air, with we previously synthesised a series of bioisosteres where the original Glaser homocoupling of the terminal alkyne being the main undesired carboxylic acid was replaced with an N-acyl sulfonamide group [15,16]. side reaction [28,29]. Repeating the reaction in degassed solvent saw There is ample literature precedent to support the use of sulfonamides as full consumption of 4 after 60 min and elimination of the unwanted bioisosteric equivalents of carboxylic acids [17–19]. Each of these homocoupled by-product. Thereafter, target alkyne 5 could be consis­ ‘olefinic sulfonamides’ retained the cis-unsaturated double bond char­ tently recovered in 88% yield under these optimised conditions. Alkyne acteristic of the DSF family (Fig. 2). These molecules were found to 5 was reduced to corresponding alkane 6 via hydrogenation at atmo­ possess anti-biofilm and anti-quorum sensing activity in S. maltophilia, spheric pressure after 16 h with a 10 mol% loading of palladium on BCC and P. aeruginosa. DSF production in S. maltophilia was also carbon. Evidence for this transformation was provided by the shift of inhibited. The olefinic N-acyl sulfonamides not only reduced the mini­ two signals at 79.2 ppm and 96.1 ppm to 34.5 ppm and 31.8 ppm mal inhibitory concentration (MIC) of colistin in Sm-resistant isolates respectively in the 13C NMR spectrum of 6. Halving the loading of the but also increased the larval survival of infected Galleria mellonella when catalyst resulted in incomplete consumption of 5, even with extended co-administered with colistin. reaction times. By contrast, full conversion of 5 was again observed on One of the problems encountered in the synthesis of our olefinic N- replacing Pd/C with Adam’s catalyst at the lower 5 mol% loading, with acyl sulfonamide analogues was the tendency for the cis-alkene to iso­ 6 obtained in quantitative yield. Methyl ester 6 was hydrolysed with merise to the thermodynamically favoured trans-isomer [20]. This lithium hydroxide in THF/methanol/water when heated to reflux over behaviour is not uncommon, and many cis-unsaturated fatty acids 16 h. Reacidification and concentration in vacuo provided 2-nonylben­ readily isomerise due to the relatively low energy barrier between the zoic acid (7) as a pale-yellow oil in 94% yield without need of further cis- and trans-isomers [21]. We considered modifications to the structure purification. Formation of the carboxylic acid was confirmed by the that would allow us to essentially lock the double bond into the cis-­ appearance of a broad O–H stretch centred on 2923 cm− 1 in the infra-red configuration and thus prevent isomerisation. The incorporation of an spectrum of 7. aromatic ring at the C-2 position was proposed as a possible stable de­ The next step was the preparation of the N-acyl sulfonamide de­ rivative of BDSF (Fig. 2). A similar approach has been exploited for rivatives for subsequent evaluation. A range of alkyl- and aryl- lipoxin and resolvin analogues where a triene motif is replaced with a substituted sulfonamides were selected for coupling with 7 to generate stable aromatic ring [22,23]. These ‘aromatic sulfonamide’ derivatives a library with sufficient structural diversity. Taking N-(meth­ anesulfonyl)-2-nonylbenzamide (8) as a typical example, meth­ anesulfonamide was coupled with 2-nonyl benzoic acid (7) in the presence of 1.1 equivalents of DCC and DMAP in dichloromethane (Table 1, entry 1). Following work up and purification by silica gel column chromatography, N-acyl sulfonamide 8 was obtained as an off- white solid in 82% yield. A broad 1H singlet at 8.24 ppm representing the N–H signal was apparent in the 1H NMR spectrum of 8. A similar approach was employed for the remaining sulfonamides (entries 2–16) to create a library of 16 bioisosteres. The yields ranged from 31% (entry Fig. 2. Previous vs current work. 16) to 96% (entry 13) with an average yield of 66%. The lowest yields 2 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 3. Synthetic route to carboxylic acid intermediate 7. complete the library, electron rich analogues 4-tert-butylphenyl sulfon­ Table 1 amide 21 (entry 14), 4-methoxyphenyl sulfonamide 22 (entry 15) and Preparation of aromatic N-acyl sulfonamides. electron poor analogue 4-nitrophenyl sulfonamide 23 (entry 16) were also prepared. 2.2. Biological evaluation 2.2.1. Bacterial growth Sixteen aromatic N-acyl sulfonamide analogues were tested against clinically relevant isolates of seven important nosocomial bacteria, namely A. baumannii, E. coli, B. multivorans, B. cepacia, B. cenocepacia, P. aeruginosa and S. maltophilia. BDSF (1) was also included in the assays Entry R Solvent Product Yield as a reference compound. The N-acyl sulfonamides were tested for their 1 Me DCM 8 82% intrinsic antimicrobial activity at 50 μM concentration (Fig. 4). None of 2 Cyclopropyl DCM 9 58% the candidates displayed a significant growth inhibitory effect in 3 tBu DCM 10 73% A. baumannii, E. coli and P. aeruginosa (Supplementary Figure A1, A2 and 4 CF3(CH2)2 DCM/DMF 11 36% A6). In S. maltophilia, both meta-bromophenylsulfonamide 14 and para- 5 Ph DCM 12 61% iodophenylsulfonamide 20 exhibited significant inhibitory activity, 6 2-BrC6H4 DCM 13 67% 7 3-BrC6H4 DCM 14 86% although the effect was much less pronounced in the case of 14 (Sup­ 8 4-BrC6H4 DCM 15 72% plementary Figure A7). In Burkholderia, several of the compounds, in 9 2-FC6H4 DCM 16 66% particular para-iodophenylsulfonamide 20, were highly inhibitory with 10 3-FC6H4 DCM 17 73% B. multivorans proving especially susceptible (Supplementary 11 4-FC6H4 DCM 18 57% 12 4-ClC6H4 DCM 19 64% Figures A3, A4 and A5). These initial experiments suggest that any 13 4-IC6H4 DCM 20 96% subsequent biological effects (e.g. reduced biofilm formation) observed 14 4-tBuC6H4 DCM 21 69% in A. baumannii and E. coli in the presence of these molecules are likely 15 4-MeOC6H4 DCM 22 57% due to disruption of intracellular communication rather than inhibition 16 4-NO2C6H4 DCM/DMF 23 31% of bacterial growth. were recorded for 3,3,3-trifluoropropanesulfonamide 11 (36%, entry 4) 2.3. Biofilm inhibition and 4-nitrobenzenesulfonamide 23 (31%, entry 16). For both reactions, the starting sulfonamides were not fully soluble in dichloromethane and The inhibitory effect of aromatic BDSF bioisosteres on biofilm for­ required the addition of DMF as a co-solvent to improve solubility. In mation in the seven bacterial strains was investigated on a polystyrene terms of structural diversity, the library could be split into alkyl- (entries surface at 50 μM concentrations. A concentration of 50 μM was chosen 1–4) and aryl-substituted (entries 5–12) sulfonamides. Methyl analogue as it is lies within the range at which natural BDSF or DSF typically 8 (entry 1) was the simplest alkyl derivative. The cyclopropyl- exhibit physiological effects [32,33]. Additionally, this concentration containing sulfonamide (entry 2) is a privileged motif in medicinal allows for comparison with our prior work on olefinic BDSF analogues chemistry which frequently appears in preclinical and clinical drug [15] or, more broadly, with halofuranone inhibitors of autoinducer-2 candidates, and thus warranted inclusion [30]. tert-Butyl analogue 10 [34]. Bacterial biofilm formation in the presence of the compounds (entry 3) provided more steric bulk while the 3,3,3-trifluoropropyl was compared with a control containing neat DMSO, which was group (entry 4) appears in several pharmaceuticals and agrochemicals benchmarked as 100% biofilm formation. Results were expressed as the [31]. In addition to phenyl sulfonamide 12 (entry 5), a series of ortho-, average and standard deviation of duplicates of at least two independent meta- and para-brominated aryl sulfonamides (entries 6–8) was pre­ experiments. Statistical significance was analysed by one-way ANOVA. pared. In our previous study, ortho-brominated analogues proved espe­ The impact on biofilm growth in A. baumannii was first investigated. cially active. For comparsion, a number of similarly substituted A. baumannii is a leading nosocomial pathogen which exhibits high fluorinated analogues (9-11) were included, along with para-substituted levels of virulence and is intrinsically resistant to many antimicrobials chlorine- (entry 12) and iodine-containing (entry 13) derivatives. To [35]. This biofilm-producing species of bacteria poses a significant threat, particularly in intensive care units [36,37]. The biofilms 3 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 4. Intrinsic inhibitory properties of aromatic sulfonamides. Heat map of mean growth inhibition values from at least two independent experiments for A. baumannii ATCC 15308 (ABA), E. coli ATCC 9637 (ECO), B. multivorans B10 (BMU), B. cepacia R6193 (BCE), B. cenocepacia 289 (BCN), P. aeruginosa PAO1 (PAO) and S. maltophilia K279a (SMA). The scale displays 0%–100% growth inhibition at 50 μM concentration of each sulfonamide. Control contains the same volume of neat DMSO. produced by A. baumannii are maintained for the production of adhesins 9637, a strain deemed safe for laboratory purposes, was selected as our and capsular polysaccharides [38]. The strain used in this study was test candidate [42]. E. coli ATCC 9637 proved quite susceptible to our ATCC 15308. Most of our compounds inhibited biofilm growth to a aromatic analogues with the majority of compounds, apart from meth­ statistically significant level with an average 26% inhibition recorded ylsulfonamide 8, reducing biofilm formation to some extent (Fig. 7). By (Fig. 5). The most potent inhibitor was electron poor para-­ far the most potent inhibitors were halogenated analogues 14, 15, 19 nitrophenylsulfonamide 23 which reduced biofilm formation by 50% and 20 each of which reduced biofilm formation by over 59% - a more (Fig. 6). Interestingly, the next most effective compound was electron pronounced effect than that observed in A. baumannii. These results rich para-methoxy analogue 22 which caused a 41% reduction. The suggest that a para- or meta-halogenated aryl sulfonamide could be an substitution pattern was also found to be important. For example, important structural feature for inhibition of biofilm growth in this ortho-bromophenylsulfonamide 13 (30% inhibition) was noticeably species. The most active compound was para-iodophenylsulfonamide 20 more active than meta-bromophenylsulfonamide 14 (1.5% inhibition) or which exhibited 68% biofilm inhibition. When the iodine atom was para-bromophenylsulfonamide 15 (8% inhibition). The activity of replaced by a bromine or chlorine, a comparable effect was recorded (i. ortho-, meta- and para-fluorophenylsulfonamides 16–18 was more e. 63% for 14 and 59% for 19). Contrastingly, when the iodine was consistent with inhibition values of 27%, 27% and 25% respectively, substituted with a fluorine atom, the inhibitory effect was appreciably similar to 13. Notably, the signalling molecule BDSF itself inhibited reduced with 22% inhibition observed for 18. meta-­ biofilm formation in the same strain by 38%. This behaviour has not Bromophenylsulfonamide 14 caused a 63% reduction in biofilm for­ been previously described in the literature. However, unsaturated fatty mation whereas its ortho-brominated analogue 13 resulted in a 43% acids such as palmitoleic and myristoleic acid are reported to decrease decrease, highlighting the sensitivity to substituent location. The biofilm formation in A. baumannii at 20 μg/mL concentrations [39]. structure activity relationships between our compound library and the E. coli is a Gram-negative pathogen which is responsible for roughly different bacterial species proved to be highly variable. As a case in three quarters of all urinary tract infections [40]. The elevated ability of point, para-halogenated analogues 15, 19 and 20, which were among E. coli to form biofilms enhances its persistence making it difficult to the most effective analogues for E. coli (Fig. 7), comprised the least treat. In hospital settings, these biofilms are frequently found on medical effective inhibitors for A. baumannii (Fig. 6). devices such as urethral and intravascular catheters, prosthetic joints, BCC is a group of closely related Gram-negative bacteria species that shunts and grafts [41]. BDSF is not known to affect biofilm formation in are involved in nosocomial infections and exhibit a high degree of E. coli but it has been reported that the closely related cis-2-decenoic acid antimicrobial resistance [43–45]. This complex includes not less than 20 can induce dispersion of biofilms formed by E. coli [9]. E. coli ATCC bacterial species, including B. multivorans, B. cepacia and B. cenocepacia 4 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 5. Percentage biofilm inhibition by aromatic sulfonamides. Heat map of mean biofilm inhibition values from at least two independent experiments for A. baumannii ATCC 15308 (ABA), E. coli ATCC 9637 (ECO), B. multivorans B10 (BMU), B. cepacia R6193 (BCE), B. cenocepacia 289 (BCN), P. aeruginosa PAO1 (PAO) and S. maltophilia K279a (SMA). The scale displays 0%–100% biofilm inhibition at 50 μM concentration of each sulfonamide. Control contains the same volume of neat DMSO. [46]. Members of BCC are known to form biofilms on biotic and abiotic biofilm production in B. cepacia R6193 (Fig. 9) while acting as biofilm surfaces, including medical devices [47]. As human pathogens, they are inhibitors in B. multivorans B10 (Fig. 7). often associated with respiratory tract infections and are prevalent in P. aeruginosa is one of the most common biofilm-producing patho­ patients with cystic fibrosis [48]. Bacterial behaviour, including viru­ gens found in healthcare settings [52]. It is responsible for 10% of all lence and biofilm formation, are regulated in BCC via BDSF-mediated nosocomial infections [36,37]. Often characterised by its high degree of quorum sensing [49]. In recent times, B. multivorans has overtaken virulence, P. aeruginosa is known to exhibit elevated levels of antimi­ B. cenocepacia as the most common BCC isolate in patients with CF in crobial resistance (AMR) [35]. Although P. aeruginosa does not produce United States and the United Kingdom [50]. Accordingly, one of the BDSF, it is capable of sensing the signalling molecule, resulting in the clinical isolates investigated in this study was B. multivorans B10 [51]. suppression of biofilm formation and virulence factor production [53]. Most of the 16 sulfonamides inhibited biofilm production in The strain of P. aeruginosa employed in this study was PAO1 [54]. Bio­ B. multivorans B10 (Fig. 8). This species of bacteria was most sensitive to film formation in P. aeruginosa was not affected by any of the compounds para-chlorophenylsulfonamide 19 which reduced biofilm growth by in this library (Fig. 11). Instead, the N-acyl sulfonamides appeared to 47% (Fig. 8). This was better than the corresponding para-­ enhance biofilm growth with >100% normalised biofilm observed for bromophenylsulfonamide 15 (31% inhibition), para-fluor­ all compounds. ophenylsulfonamide 18 (27% inhibition), or S. maltophilia is a Gram-negative, nosocomial pathogen associated para-iodophenylsulfonamide 20 (35% inhibition). The second most with pneumonia, bacteremia, UTIs and septic arthritis in immunocom­ active molecule was meta-bromophenylsulfonamide 14 with 41% inhi­ promised patients [44,55]. Its prominent biofilm producing capabilities bition. meta-Bromophenylsulfonamide 14 outperformed its corre­ make S. maltophilia highly resistant to antimicrobials [43]. DSF is the sponding ortho- and para-brominated analogues 13 and 15 (38% and main QS signalling molecule used by this pathogen to regulate biofilm 31% inhibition respectively). Non-halogenated sulfonamides 10 and 21 formation and virulence [56]. The clinical isolate S. maltophilia K279a were also highly active. Both structures contained a tert-butyl motif and (belonging to the rpf-1 subpopulation) was used in the study. None of inhibited biofilm formation by more than 35%. our aromatic sulfonamides inhibited biofilm formation and apparently By contrast, biofilm growth in the clinical strains B. cepacia R6193 caused increased biofilm growth compared to the DMSO control (Fig. 9) and B. cenocepacia 289 (Fig. 10) was not significantly inhibited (Fig. 12). This result was unsurprising as we had previously observed a by the library of aromatic sulfonamides, highlighting the species-specific similar outcome with our olefinic sulfonamides [15]. activity of the compounds tested. Indeed, meta-bromophenylsulfona­ As a general trend, aromatic N-acyl sulfonamides which contained a mide 14 and para-bromophenylsulfonamide 15 appeared to promote halogen group were found to be among the most effective inhibitors of 5 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 6. Effect of sulfonamides on biofilm formation of A. baumannii ATCC15308 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Fig. 7. Effect of sulfonamides on biofilm formation of E. coli ATCC9637 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). biofilm growth across several bacterial species. This outcome matched A. baumannii and E. coli, both BDSF and the sulfonamide analogues were similar findings with our earlier olefinic N-acyl sulfonamides, where a 2- observed to inhibit biofilm formation. By contrast, BDSF promoted bromobenzenesulfonamide was the standout compound. The superior biofilm growth in B. cepacia, P. aeruginosa and S. maltophilia which activity of halogen-containing analogues may be in part due to increased matched the effect of the sulfonamides. The main exception was in lipophilicity (e.g. cLogP of 5.15 for 12 vs. cLogP of 6.04 for 13) and B. cenocepacia where biofilm production was inhibited by BDSF but the improved permeation of these molecules through membranes or existing sulfonamides were inactive. bacterial biofilms [57,58]. It is also interesting to note that the aromatic N-acyl sulfonamides broadly behaved in a similar manner to the parent signalling molecule BDSF for most of the species under investigation. In 6 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 8. Effect of sulfonamides on biofilm formation of B. multivorans B10 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Fig. 9. Effect of sulfonamides on biofilm formation of B. cepacia R61993 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). 2.4. Colistin enhancing effects S. maltophilia K279a. Resistance to colistin has been identified in all five genera [60–63]. The strongest colistin enhancing effects were observed Colistin is a last-resort antibiotic which is used to treat hospital- in S. maltophilia despite these compounds not inhibiting biofilm forma­ acquired infections caused by multi-drug resistant (MDR) Gram- tion in this bacterium (Fig. 5). The MIC values were at least 2-fold lower negative pathogens [59]. The colistin enhancing effect of our aromatic than the DMSO control (2 μg/mL) in the presence of a majority of our N-acyl sulfonamide analogues was next investigated. Compounds were compounds. Seven compounds produced a 2-4-fold improvement administered at a fixed dose of 50 μM concentration to the seven strains compared to the DMSO control, namely methylsulfonamide 8 (entry 4), A. baumannii ATCC 15308, E. coli ATCC 9637, B. multivorans B10, tert-butylsulfonamide 10 (entry 6), 3,3,3-trifluoropropylsulfonamide 11 B. cepacia R6193 and B. cenocepacia 289, P. aeruginosa PAO1 and (entry 7), phenylsulfonamide 12 (entry 8), 7 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 10. Effect of sulfonamides on biofilm formation of B. cenocepacia 289 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Fig. 11. Effect of sulfonamides on biofilm formation of P. aeruginosa PAO1 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ortho-bromophenylsulfonamide 13 (entry 9), meta-fluor­ A. baumannii (Fig. 5), this did not directly translate to increased colistin ophenylsulfonamide 17 (entry 13) and para-fluorophenylsulfonamide sensitivity. Only two compounds exhibited an enhancing effect with a 2- 18 (entry 14). This is well below the threshold required for use in colistin fold reduction in MIC noted for methylsulfonamide 8 (entry 4) and inhalation therapy [63]. Cyclopropylsulfonamide 9 (entry 5), ortho-­ cyclopropylsulfonamide 9 (entry 5). No reduction in MIC values was fluorophenylsulfonamide 16 (entry 12), para-­ observed in E. coli even though biofilm production was also disrupted in methoxyphenylsulfonamide 22 (entry 18) and this species. para-nitrophenylsulfonamide 23 (entry 19) were also found to reduce Neither B. multivorans B10, B. cepacia R6493 or B. cenocepacia 289 the MIC to 1 μg/mL which represented a 2-fold improvement compared responded to any colistin/analogue combination and MIC values to colistin alone. remained unchanged. Additionally, none of our aromatic N-acyl sul­ Although most sulfonamides inhibited biofilm formation in fonamides displayed an antibiotic enhancing effect in P. aeruginosa. 8 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Fig. 12. Effect of sulfonamides on biofilm formation of S. maltophilia K279a (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Surprisingly, halogenated sulfonamides 14 (entry 10), 15 (entry 11) colistin compared to untreated larvae or larvae treated with colistin and 20 (entry 16) increased resistance levels to colistin in all strains alone (Fig. 13). Although sulfonamide 10 was not associated with a except Burkholderia. The MIC values were between 2- and 8-fold higher decrease in biofilm formation in S. maltophilia, co-administration with than the DMSO control suggesting these analogues were protecting the colistin did result in a 2–4 fold reduction in MIC (Table 2, entry 6). These bacterial cells from the effects of colistin. results, which mirror similar trends in the olefinic sulfonamides [15], Galleria mellonella has been widely used as an effective model to suggest that tert-butylsulfonamide 10 is a good enhancer of colistin ac­ evaluate the efficacy of antimicrobial compounds [64]. Based on the tivity although the exact mechanism involved in the survival improve­ reduction in MIC observed in S. maltophilia, we decided to investigate ment remains to be elucidated. the in vivo effects of sulfonamides 8, 10, 14 and 20 on G. mellonella. G. mellonella is increasingly popular both as a means of measuring 3. Conclusion chemical toxicity [65] and as a model for bacterial lung infections [66]. When the selected sulfonamides were initially screened for toxicity at The DSF family of compounds are all characterised by the presence of 50 μM concentration, larval survival reached 100% for all compounds a cis-alkene which lies in conjugation with a carboxylic acid. Several tested (data not shown). Given their apparent non-toxicity in vivo, studies have highlighted the importance of the configuration of the G. mellonella larvae were infected with S. maltophilia K279a and treated double bond, with the corresponding trans-isomers possessing little or with colistin alone or in combination with the sulfonamides. Of the four no biological activity. We have previously demonstrated that replacing sulfonamides examined, tert-butylsulfonamide 10 was the most effective the carboxylic acid in BDSF with an N-acyl sulfonamide affords com­ and showed an improvement in larval survival when combined with pounds with the ability to inhibit biofilm production and enhance colistin sensitivity in BDSF-sensitive bacteria. However, the preparation of these compounds is problematic due to tendency of the cis-alkene to isomerise. In this work, we have created a second generation library of com­ pounds where the cis-alkene is replaced with a benzene ring. Despite this structural modification, the resulting aromatic N-acyl sulfonamides retained biological activity, with several molecules significantly inhib­ iting biofilm production in A. baumannii, E. coli and B. multivorans. The strongest effects were observed in E. coli with halogenated analogues 14, 15, 19 and 20 proving most potent. There was a degree of overlap in B. multivorans with the same halogen-containing molecules among the most effective biofilm inhibitors. Additionally, in the case of A. baumannii and E. coli, the sulfonamide library did not affect cell growth, suggesting likely inhibition via interference with QS-regulated biofilm production. The inhibition of biofilm growth did not directly result in decreased antibiotic tolerance, however, with only a 2-fold reduction in colistin MIC recorded for A. baumannii and no reduction Fig. 13. Kaplan-Meier survival curves of Galleria mellonella infected with observed in E. coli. By contrast, several aromatic N-acyl sulfonamides S. maltophilia K279a. G. mellonella was treated with colistin (CST) at MIC caused a reduction in colistin MIC in S. maltophilia, despite not affecting 0.5 μg/mL or in combination with sulfonamide 10 at 50 μM in PBS. DMSO at 0.5% in PBS was used as a vehicle control for untreated larvae. biofilm growth in this species, matching our earlier findings with 9 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 Table 2 Minimal inhibitory concentrations (MIC) to colistin of A. baumannii ATCC 15308 (ABA), E. coli ATCC 9637 (ECO), B. multivorans B10 (BMU), B. cepacia R6193 (BCE), B. cenocepacia 289 (BCN), P. aeruginosa (PAO) and S. maltophilia K279a (SMA) isolates in the presence of sulfonamides at a fixed dose of 50 μM. Entry Compound Colisin MIC (μg/ml) ABA ECO BMU BCE BCN PAO SMA 1 w/o 0.25–0.5 0.125–0.25 >128 >256 >256 1–2 2–8 2 DMSO 0.25 0.0625–0.125 >128 >256 >256 1 2 3 BDSF 0.125–0.25 0.0625–0.125 >128 >256 >256 1 0.5 4 08 0.125–0.25 0.125 >128 >256 >256 1 0.5–1 5 09 0.125–0.25 0.125 >128 >256 >256 1 1 6 10 0.25–0.5 0.5 >128 >256 >256 1 0.5–1 7 11 0.25–0.5 0.25–0.5 >128 >256 >256 1 0.5–1 8 12 0.25–0.5 0.25–0.5 >128 >256 >256 1 0.5–1 9 13 0.5 0.5 >128 >256 >256 1 0.5–1 10 14 1 >1 >128 >256 >256 4–8 4 11 15 2 >1 >128 >256 >256 8 2–4 12 16 0.25–0.5 0.25–0.5 >128 >256 >256 1 1 13 17 0.25–0.5 0.25 >128 >256 >256 1 0.5–1 14 18 0.25–0.5 0.5 >128 >256 >256 1 0.5–1 15 19 1 1 >128 >256 >256 4 2 16 20 2 >1 >128 >256 >256 8 4 17 21 0.5 0.5 >128 >256 >256 1 4–8 18 22 0.25–0.5 0.25 >128 >256 >256 1 1 19 23 0.5–1 0.5 >128 >256 >256 2 1 olefinic sulfonamides. Importantly, the levels achieved are below the solvents removed in vacuo. The crude mixture was purified by column threshold required for colistin inhalation therapy. These in vitro results chromatography on silica gel using hexane-diethyl ether (100:0–98:2) to were reflected in vivo with increased Galleria larval survival observed on yield 5 as a pale yellow oil (270 mg, 1.048 mmol, 88%). 1 co-administration of colistin with tert-butylsulfonamide 10 compared to H NMR (400 MHz, CDCl3) δ 0.89 (t, 3H, J = 6.7 Hz, C9′), 1.24–1.39 colistin alone. Taken together, these findings demonstrate the potential (m, 6H, C6′-C8′), 1.47 (tt, 2H, J = 7.6 Hz, C5′), 1.63 (tt, 2H, J = 7.5 Hz, of aromatic N-acyl sulfonamide erivatives of BDSF as biofilm inhibitors C4′), 2.47 (t, 2H, J = 7.1 Hz, C3′), 3.91 (s, 3H, C8), 7.30 (ddd, 1H, and disruptors of antimicrobial resistance in pathogenic bacteria. J = 7.6, 1.3 Hz, C5), 7.42 (ddd, 1H, J = 7.6, 1.3 Hz, C4), 7.51 (dd, 1H, J = 7.8, 1.2 Hz, C3), 7.88 (dd, 1H, J = 7.9, 1.2 Hz, C6). 13 4. Experimental section C NMR (100 MHz, CDCl3) δ 14.1 (C9′, CH3), 19.8 (C3′, CH2), 22.7 (C8′, CH2), 28.7 (C4′, CH2), 28.9 (C5′, CH2), 29.0 (C6′, CH2), 31.8 (C7′, 4.1. Chemistry CH2), 52.1 (C8, CH3), 79.2 (C1′), 96.1 (C2’), 124.5 (C2), 127.1 (C5, CH), 130.1 (C6, CH), 131.5 (C4, CH), 131.9 (C1), 134.2 (C3, CH), 167.0 (C7). Acetonitrile, benzenesulfonamide, bis(triphenylphosphine)palla­ IR (ATR) υmax cm− 1 2928, 2856, 2232, 1734, 1485, 1432, 1447, dium(II) dichloride, copper ioidide, dichloromethane, diethyl ether, 1292, 1249, 1129, 1083, 757, 702 dimethyl formamide, hexane, lithium hydroxide, methanol, platinum (IV) oxide and triethylamine were obtained from Sigma-Aldrich. 2-Bro­ 4.1.2. Synthesis of methyl 2-nonylbenzoate (6) mobenzenesulfonamide, 3-bromobenzenesulfonamide, 4-bromobenze­ Platinum(IV) oxide hydrate (30 mg, 0.123 mmol, 5 mol%) was added nesulfonamide, 4-tert-butylbenzenesulfonamide, tert-butylsulfonamide, to an oven-dried round bottom flask before 5 (636 mg, 2.465 mmol, 1.00 4-chlorobenzenesulfonamide, cyclopropylsulfonamide, DMAP, DCC, 2- eq.) in methanol (8 mL) was added. The flask was evacuated and back fluorobenzenesulfonamide, 3-fluorobenzenesulfonamide, 4-fluoroben­ filled with hydrogen gas. The suspension was stirred for 24 h under an zenesulfonamide, 4-iodobenzenesulfonamide, methanesulfonamide, 4- atmosphere of hydrogen. The reaction mixture was filtered through a methoxybenzenesulfonamide, 4-nitrobenzenesulfonamide and 3,3,3-tri­ pad of celite and the filtrate was concentrated under reduced pressure to fluoropropanesulfonamide were obtained from Fluorochem Ltd. 1-Non­ yield 6 as a pale yellow oil (644 mg, 2.461 mmol, 100%). 1 yne was obtained from TCI Chemicals (Europe). Unless otherwise noted, H NMR (400 MHz, CDCl3) δ 0.80 (t, 3H, J = 6.8 Hz, C9′), 1.11–1.39 all the purchased materials and solvents were used without further pu­ (m, 12H, C3′-C8′), 1.57 (tt, 2H, J = 7.5 Hz, C2′), 2.86 (t, 2H, J = 7.8 Hz, rification. Compounds were purified by silica gel (Kieselgel 60, C1′), 3.82 (s, 3H, C8), 7.12–7.24 (m, 2H, C3, C5). 7.33 (ddd, 1H, J = 7.6, 0.040–0.063 mm, Merck) column chromatography. 1H NMR and 13C 1.1 Hz, C4), 7.77 (dd, 1H, J = 8.0, 0.9 Hz, C6). 13 NMR spectra were recorded on Bruker Avance 300 (300/75 MHz) or C NMR (100 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.4 Bruker Avance 400 (400/100 MHz) NMR spectrometers respectively. (CH2), 29.6 (CH2), 29.60 (CH2), 29.8 (C3′, CH2), 31.8 (C2′, CH2), 31.9 cLogP values were calculated using SwissADME [67]. (C7′, CH2), 34.5 (C1’, CH2), 51.9 (C8, CH3), 125.6 (C5, CH), 129.5 (C1), 130.5 (C6, CH), 130.9 (C3, CH), 131.8 (C4, CH), 144.8 (C2), 168.3 (C7). 4.1.1. Synthesis of methyl 2-(non-1-yn-1-yl)benzoate (5) [24] IR (ATR) υmax cm− 1 3483, 2924, 2854, 1725, 1433, 1253, 1102, Bis(triphenylphosphine)palladium(II) dichloride (25 mg, 1078, 750, 710 0.035 mmol, 3 mol%) and copper iodide (11 mg, 0.059 mmol, 5 mol%) HRMS (ESI) m/z: [M+H] Calcd for C17H26O2 263.2005; Found were added to an oven dried microwave tube. Freshly distilled trie­ 263.2016. thylamine (1.60 mL, 11.501 mmol, 10.00 eq.) and 4 (312 mg, 1.191 mmol, 1.00 eq.) in dry degassed acetonitrile (6 mL) were then 4.1.3. Synthesis of 2-nonylbenzoic acid (7) [68] added to the vial before 1-nonyne (0.230 mL, 1.428 mmol, 1.20 eq.) was Methyl 2-nonylbenzoate (6) (670 mg, 2.557 mmol, 1.00 eq.) and added. The mixture was stirred under microwave irradiation at 100 ◦ C lithium hydroxide (336 mg, 14.000 mmol, 5.50 eq.) were dissolved in (120 W) for 1 h. The reaction mixture was filtered through a pad of THF/methanol/water 3:1:1 (20 mL) and refluxed overnight in a round celite, washed with water and extracted with diethyl ether (3 × 30 mL). bottom flask. The reaction mixture was cooled to room temperature The combined organic layers were washed with water (20 mL) and brine before the solvent was removed under reduced pressure. Water (15 m L) (20 mL) and dried with MgSO4. The solution was filtered and the was added and the mixture was acidified to pH 2 using 2 M aqueous HCl 10 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 (15 mL) before extracting with diethyl ether (5 × 30 mL). The combined 3.09–3.18 (m, 1H, C1″), 7.21–7.33 (m, 2H, C3, C5), 7.39–7.48 (m, 2H, organic extracts were washed with brine, dried with MgSO4, filtered, C4, C6), 8.17 (s, 1H, N–H). 13 and evaporated in vacuo to yield 7 as a pale yellow oil (596 mg, C NMR (75 MHz, CDCl3) δ 6.3 (C2″, C3″, 2 × CH2), 14.1 (C9′, CH3), 2.403 mmol, 94%). 22.7 (C8′, CH2), 29.3 (CH2), 29.5 (CH2), 29.5 (CH2), 29.6 (CH2), 31.3 1 H NMR (400 MHz, CDCl3) δ 0.86 (t, 3H, J = 6.7 Hz, C9′), 1.16–1.46 (C1”, CH), 31.8 (C2′, CH2), 31.9 (C7′, CH2), 33.4 (C1’, CH2), 126.1 (C5, (m, 12H, C3′-8′), 1.62 (tt, 2H, J = 7.5 Hz, C2′), 3.02 (t, 2H, J = 7.8 Hz, CH), 127.1 (C6, CH), 131.0 (C3, CH), 131.8 (C4, CH), 132.3 (C1), 142.7 C1′), 7.20–7.33 (m, 2H, C3, C5), 7.46 (ddd, 1H, J = 7.41 Hz, C4), (C2), 167.2 (C7). 7.97–8.09 (m, 1H, C6). IR (ATR) υmax cm− 1 3232, 2925, 2854, 1698, 1455, 1427, 1344, 158, 13 C NMR (100 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.4 1059, 1042, 885, 847, 750, 574 (C6′, CH2), 29.5 (C5′, CH2), 29.6 (C4′, CH2), 29.8 (C3′, CH2), 31.8 (C2′, HRMS (ESI) m/z: [M+H] Calcd for C19H29NO3S 352.1941; Found CH2), 31.9 (C7′, CH2), 34.6 (C1’, CH2), 125.8 (C5, CH), 128.1 (C1), 352.1943. 131.2 (C3, CH), 131.6 (C6, CH), 132.8 (C4, CH), 146.0 (C2), 173.2 (C7). IR (ATR) υmax cm− 1 2923, 2853, 1688, 1456, 1403, 1297, 1264, 907, 4.1.7. N-(tert-Butylsulfonyl)-2-nonylbenzamide (10) 733, 656, 561 Prepared following the general procedure using 2-nonylbenzoic acid HRMS (ESI) m/z: [M+Na] Calcd for C22H37NO3S 271.1668; Found (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 271.1672. eq.), tert-butylsulfonamide (26 mg, 0.192 mmol, 0.95 eq.) and DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture was 4.1.4. General procedure for the preparation of N-(sulfonyl)-2- purified by column chromatography on silica gel using hexane-diethyl nonylbenzamides ether (70:30) to yield 10 as a yellow solid (51 mg, 0.139 mmol, 73%), A solution of 2-nonylbenzoic acid (50 mg, 0.202 mmol, 1.00 eq.) and mp 52–53 ◦ C. 1 DMAP (27 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL) was cooled to 0 ◦ C H NMR (500 MHz, CDCl3) δ 0.87 (t, 3H, J = 6.9 Hz, C9′), 1.19–1.38 before the appropriate sulfonamide (0.192 mmol, 0.95 eq.) was added. (m, 12H, C3′-C8′), 1.56 (s, 9H, C2″-C4″), 1.57–1.67 (m, 2H, C2′), 2.80 (t, After stirring for 15 min under a nitrogen atmosphere, DCC (45 mg, 2H, J = 7.9 Hz, C1′), 7.22–7.31 (m, 2H, C3, C4), 7.38–7.47 (m, 2H, C5, 0.221 mmol, 1.10 eq.) was added and the mixture was stirred for 16 h at C6), 7.78 (s, 1H, N–H). 13 room temperature. The reaction mixture was filtered through a thin pad C NMR (125 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 24.5 of celite to remove the insoluble urea and the solvent was removed in (C2″-C4″, 3 × CH3), 29.3 (CH2), 29.5 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 vacuo. The resulting residue was redissolved in diethyl ether (10 mL), (C7′, CH2), 32.0 (C2′, CH2), 33.4 (C1′, CH2), 62.5 (C1″), 126.0 (C5, CH), poured onto 2 M aqueous HCl (20 mL) and extracted with diethyl ether 127.0 (C6, CH), 130.8 (C3, CH), 131.5 (C4, CH), 133.2 (C1), 142.4 (C2), (3 × 30 mL). The combined organic extracts were washed with brine 166.8 (C7). (20 mL), dried over MgSO4 and filtered. The solvent was removed in IR (ATR) υmax cm− 1 3236, 2925, 2854, 1714, 1448, 1429, 1332, vacuo and the resulting residue was purified by silica gel column chro­ 1259, 1139, 1056, 843, 801, 649, 567, 511 matography using hexane-diethyl ether (70:30) to afford the target HRMS (ESI) m/z: [M+H] Calcd for C20H33NO3S 368.2254; Found sulfonamide. 368.2251. 4.1.5. N-(Methanesulfonyl)-2-nonylbenzamide (8) 4.1.8. 2-Nonyl-N-(3,3,3-trifluoropropanesulfonyl)benzamide (11) Prepared following the general procedure using 2-nonylbenzoic acid Prepared following the general procedure using 2-nonylbenzoic acid (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 eq.), methanesulfonamide (18 mg, 0.192 mmol, 0.95 eq.) and DCC eq.), 3,3,3-trifluoropropanesulfonamide (34 mg, 0.192 mmol, 0.95 eq.) (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture was and DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude purified by column chromatography on silica gel using hexane-diethyl mixture was purified by column chromatography on silica gel using ether (70:30) to yield 8 as an off-white solid (51 mg, 0.157 mmol, hexane-diethyl ether (90:10) to yield 11 as a yellow waxy solid (27 mg, 82%), mp 57–59 ◦ C. 0.066 mmol, 36%). 1 1 H NMR (400 MHz, CDCl3) δ 0.87 (t, 3H, J = 6.8 Hz, C9′), 1.16–1.40 H NMR (400 MHz, CDCl3) δ 0.87 (t,3H, J = 6.9 Hz, C9′), 1.21–1.38 (m, 12H, C3′-C8′), 1.53–1.67 (m, 2H, C2′), 2.82 (t, 2H, J = 7.9 Hz, C1′), (m,12H, C3′-C8′), 1.54–1.64 (m, 2H, C2′), 2.66–2.78 (m,2H, C2″), 2.81 3.43 (s, 3H, C8), 7.22–7.34 (m, 2H, C3, C5), 7.40–7.50 (m, 2H, C4, C6), (t, 2H, J = 7.9 Hz, C1′), 3.77–3.85 (m, 2H, C1″), 7.24–7.34 (m,2H, C3, 8.24 (s, 1H, N–H). C5), 7.43–7.50 (m, 2H, C4, C6). 13 13 C NMR (100 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 C NMR (100 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 28.8 (CH2), 29.4 (CH2), 29.5 (C4′, CH2), 29.6 (C3′, CH2), 31.9 (C2′, C7′, (d, 2JC-F = 31.6, C2″, CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 2 × CH2), 33.4 (C1’, CH2), 41.6 (C8, CH3), 126.1 (C5, CH), 127.2 (C6, (CH2), 31.9 (C14, CH2), 32.0 (C9, CH2), 33.4 (C1’, CH2), 46.9 (d, 3JC- 1 CH), 131.2 (C3, CH), 131.8 (C1), 132.1 (C4, CH), 143.1 (C2), 167.4 (C7). F = 3.3 Hz, C1”, CH2), 125.2 (d, JC-F = 276.8 Hz, CF3), 126.2 (C3, CH), IR (ATR) υmax cm− 1 3232, 2925, 2854, 1698, 1435, 1403, 1341, 127.3 (C4, CH), 131.2 (C1), 131.3 (C5, CH), 132.4 (C6, CH), 143.5 (C2), 1164, 972, 893, 750, 520 167.3 (C7). 19 HRMS (ESI) m/z: [M+H] Calcd for C17H27NO3S 326.1784; Found F NMR (376 MHz, CDCl3) δ − 65.8 (s, CF3). 326.1780. IR (ATR) υmax cm− 1 2961, 2917, 2849, 1746, 1630, 1462, 1260, 1089, 1017, 797, 415 4.1.6. N-(Cyclopropanesulfonyl)-2-nonylbenzamide (9) HRMS (ESI) m/z: [M+H] Calcd for C19H28F3NO3S 408.1814; Found Prepared following the general procedure using 2-nonylbenzoic acid 408.1813. (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 eq.), cyclopropylsulfonamide (23 mg, 0.192 mmol, 0.95 eq.) and DCC 4.1.9. N-(Benzenesulfonyl)-2-nonylbenzamide (12) (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture was Prepared following the general procedure using 2-nonylbenzoic acid purified by column chromatography on silica gel using hexane-diethyl (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 ether (70:30) to yield 9 as an off-white solid (39 mg, 0.111 mmol, eq.), benzenesulfonamide (30 mg, 0.192 mmol, 0.95 eq.) and DCC 58%), mp 50–52 ◦ C. (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture was 1 H NMR (300 MHz, CDCl3) δ 0.87 (t, 3H, J = 6.8 Hz, C9′), 1.12–1.20 purified by column chromatography on silica gel using hexane-diethyl (m, 2H, C2″, C3″), 1.20–1.38 (m, 12H, C3′-C8′), 1.40–1.47 (m, 2H, C2″, ether (80:20) to yield 12 as a yellow solid (45 mg, 0.116 mmol, 61%), C3″), 1.61 (tt, 2H, J = 7.5 Hz, C2′), 2.82 (t, 2H, J = 7.8 Hz, C1′), mp 42–43 ◦ C. 11 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 1 H NMR (400 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.9 Hz, C9′), 1.04–1.39 eq.), 4-bromobenzenesulfonamide (45 mg, 0.192 mmol, 0.95 eq.) and (m, 14H, C2′-C8′), 2.59 (t, 2H, J = 7.8 Hz, C1′), 7.15–7.24 (m, 2H, C3, DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture C4), 7.33–7.42 (m, 2H, C5, C6), 7.57 (dd, 2H, J = 7.5 Hz, C3″, C5″), 7.67 was purified by column chromatography on silica gel using hexane- (dd, 1H, J = 7.5 Hz, C4″), 8.14 (d, 2H, J = 7.5 Hz, C2″, C6″), 8.51 (s, 1H, diethyl ether (85:15) to yield 15 as an off-white solid (64 mg, N–H). 0.144 mmol, 72%), mp 94–96 ◦ C. 13 1 C NMR (100 MHz, CDCl3) δ 14.2 (C9′, CH3), 22.7 (C8′, CH2), 29.3 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, C9′), 1.07–1.41 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.7 (C2′, CH2), 31.9 (C7′, (m, 14H, C2′-C8′), 2.61 (t, 2H, J = 7.8 Hz, C1′), 7.16–7.27 (m, 2H, C3, CH2), 33.1 (C1’, CH2), 126.0 (C5, CH), 127.1 (C6, CH), 128.5 (C2″, C6″, C5), 7.32–7.42 (m, 2H, C4, C6), 7.70 (app. dt, 2H, J = 8.7, 2.2 Hz, C3″, 2 × CH), 129.0 (C3″, C5″, 2 × CH), 130.9 (C3, CH), 131.7 (C4, CH), C5″), 8.00 (app. dt, 2H, J = 8.7, 2.2 Hz, C2″, C6″), 8.37 (s, 1H, N–H). 13 132.2 (C1), 134.1 (C4″, CH), 138.4 (C1”), 142.6 (C2), 166.4 (C7). C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 IR (ATR) υmax cm− 1 3238, 2925, 2854, 1705, 1450, 1428, 1349, (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (C3′, CH2), 31.7 (C2′, CH2), 31.9 1172, 1089, 1059, 751, 686, 584, 566 (C7′, CH2), 33.2 (C1’, CH2), 126.0 (C5, CH), 127.1 (C6, CH), 129.4 (C4″), HRMS (ESI) m/z: [M+H] Calcd for C22H29NO3S 388.1941; Found 130.1 (C2″, C6″, 2 × CH), 130.9 (C3, CH), 131.8 (C4, CH), 132.0 (C1), 388.1947. 132.3 (C3″, C5″, 2 × CH), 137.4 (C1”), 142.7 (C2), 166.3 (C7). IR (ATR) υmax cm− 1 3234, 2923, 2853, 1686, 1574, 1428, 1351, 4.1.10. N-(2-Bromobenzenesulfonyl)-2-nonylbenzamide (13) 1172, 1068, 738, 602, 567 Prepared following the general procedure using 2-nonylbenzoic acid HRMS (ESI) m/z: Calcd for C22H28BrNO3S 466.1046; Found (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 466.1042. eq.), 2-bromobenzenesulfonamide (45 mg, 0.192 mmol, 0.95 eq.) and DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture 4.1.13. Synthesis of N-(2-Fluorobenzenesulfonyl)-2-nonylbenzamide (16) was purified by column chromatography on silica gel using hexane- Prepared following the general procedure using 2-nonylbenzoic acid diethyl ether (85:15) to yield 13 as an off-white solid (59 mg, (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 0.126 mmol, 67%), mp 116–118 ◦ C. eq.), 2-fluorobenzenesulfonamide (33 mg, 0.192 mmol, 0.95 eq.) and 1 H NMR (400 MHz, CDCl3) δ 0.81 (t, 3H, J = 6.8 Hz, C9′), 1.03–1.28 DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture (m, 12H, C3′-8′), 1.28–1.42 (m, 2H, C2′), 2.59 (t, 2H, J = 7.8 Hz, C1′), was purified by column chromatography on silica gel using hexane- 7.21–7.29 (m, 2H, C3, C5), 7.41 (dt, 1H, J = 7.6, 1.3 Hz, C4), 7.46–7.54 diethyl ether (85:15) to yield 16 as a white solid (51 mg, 0.126 mmol, (m, 2H, C6, C4″), 7.58 (ddd, 1H, J = 7.7, 1.2 Hz, C5″), 7.75 (dd, 1H, 66%), mp 53–54 ◦ C. 1 J = 7.7, 1.2 Hz, C3″), 8.42 (dd, 1H, J = 7.9, 1.8 Hz, C6″), 8.67 (s, 1H, H NMR (400 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.9 Hz, C9′), 1.10–1.35 N–H). (m, 12H, C3′-8′). 1.35–1.48 (m, 2H, C2′), 2.65 (t, 2H, J = 7.8 Hz, C1′), 13 C NMR (100 MHz, CDCl3) δ 14.2 (C9′, CH3), 22.8 (C8′, CH2), 29.3 7.18–7.30 (m, 3H, C3, C5, C3″), 7.33–7.48 (m, 3H, C4, C6, C5″), (2 × CH2), 29.5 (CH2), 29.6 (CH2), 31.7 (C2′, CH2), 31.9 (C7′, CH2), 33.1 7.62–7.73 (m, 1H, C4″), 8.18 (ddd, 1H, J = 7.6, 1.4 Hz, C6″), 8.64 (s, 1H, (C1’, CH2), 120.0 (C2″), 126.0 (C5, CH), 127.2 (C6, CH), 128.0 (C5″, N–H). 13 CH), 131.0 (C3, CH), 131.8 (C1), 131.9 (C4, CH), 133.8 (C6″, CH), 134.9 C NMR (100 MHz, CDCl3) δ 14.2 (C9′, CH3), 22.7 (C8′, CH2), 29.3 (C4″, CH), 135.1 (C3″, CH), 137.6 (C1”), 143.1 (C2), 166.1 (C7). (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.6 (C2′, CH2), 31.9 (C7′, IR (ATR) υmax cm− 1 3247, 2923, 2852, 1707, 1575, 1416, 1345, CH2), 33.1 (C1’, CH2), 116.9 (d, 2JC-F = 20.7 Hz, C3″, CH), 124.7 (d, 4JC- 2 1177, 1051, 1024, 581 F = 3.8 Hz, C5″, CH), 126.1 (C5, CH), 126.5 (d, JC-F = 12.5 Hz, C1″), HRMS (ESI) m/z: Calcd for C22H28BrNO3S 466.1046; Found 127.2 (C6, CH), 131.0 (C3, CH), 131.8 (C1, CH), 131.9 (C4, CH), 132.4 466.1044. (C6″, CH), 136.4 (d, 3JC-F = 8.8 Hz, C4″, CH), 143.0 (C2), 159.0 (d, 1JC- F = 256.5 Hz, C2”, C–F), 166.3 (C7). 19 4.1.11. N-(3-Bromobenzenesulfonyl)-2-nonylbenzamide (14) F NMR (376 MHz, CDCl3) δ − 109.8 (s, C–F). Prepared following the general procedure using 2-nonylbenzoic acid IR (ATR) υmax cm− 1 3247, 2925, 2854, 1708, 1599, 1477, 1413, (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 1352, 1176, 1054, 761, 587 eq.), 3-bromobenzenesulfonamide (45 mg, 0.192 mmol, 0.95 eq.) and HRMS (ESI) m/z: [M+H] Calcd for C22H28NO3SF 406.1846; Found DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture 406.1849. was purified by column chromatography on silica gel using hexane- diethyl ether (85:15) to yield 14 as an off-white solid (76 mg, 4.1.14. N-(3-Fluorobenzenesulfonyl)-2-nonylbenzamide (17) 0.163 mmol, 86%), mp 68–70 ◦ C. Prepared following the general procedure using 2-nonylbenzoic acid 1 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, C9′), 1.09–1.42 (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 (m, 14H, C2′-C8′), 2.62 (t, 2H, J = 7.8 Hz, C1′), 7.18–7.27 (m, 2H, C3, eq.), 3-fluorobenzenesulfonamide (33 mg, 0.192 mmol, 0.95 eq.) and C5), 7.34–7.42 (m, 2H, C4, C6), 7.45 (dd, 1H, J = 8.0 Hz, C5″), 7.79 DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture (ddd, 1H, J = 8.0, 1.8, 1.0 Hz, C4″), 8.11 (ddd, 1H, J = 8.0, 1.8, 1.0 Hz, was purified by column chromatography on silica gel using hexane- C6″), 8.26 (dd, 1H, J = 1.8 Hz, C2″), 8.35 (s, 1H, N–H). diethyl ether (85:15) to yield 17 as a white solid (56 mg, 0.138 mmol, 13 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 73%), mp 75–77 ◦ C. 1 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.7 (C2′, CH2), 31.9 (C7′, H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, C9′), 1.09–1.43 CH2), 33.2 (C1’, CH2), 122.8 (C3″), 126.1 (C5, CH), 127.0 (C6, CH), (m, 14H, C2′-C8′), 2.62 (t, 2H, J = 7.8 Hz, C1′), 7.16–7.28 (m, 2H, C3, 127.3 (C6″, CH), 130.4 (C5″, CH), 131.0 (C3, CH), 131.2 (C2″, CH), 131.8 C5), 73.1–7.45 (m, 3H, C4, C6, C4″), 7.56 (app dt., 1H, J = 8.1, 5.2 Hz, (C4, CH), 131.9 (C1), 137.1 (C4″, CH), 140.3 (C1”), 142.8 (C2), 166.2 C5″), 7.85 (app. dt, 1H, J = 8.0, 2.1 Hz, C2″), 7.91–8.01 (m, 1H, C6″), (C7). 8.34 (s, 1H, N–H). IR (ATR) υmax cm− 1 3233, 2924, 2853, 1693, 1428, 1350, 1173, 13 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 1066, 892, 784, 677, 588 (CH2), 29.4 (CH2), 29.5 (CH2), 29.5 (CH2), 31.7 (C2′, CH2), 31.9 (C7′, HRMS (ESI) m/z: Calcd for C22H28BrNO3S 466.1046; Found CH2), 33.2 (C1’, CH2), 115.9 (d, 2JC-F = 25.0 Hz, C2″, CH), 121.3 (d, 2JC- 4 466.1043. F = 21.3 Hz, C4″, CH), 124.4 (d, JC-F = 3.5 Hz, C6″, CH), 126.1 (C5, CH), 3 127.0 (C6, CH), 130.7 (d, JC-F = 7.7 Hz, C5″, CH), 131.0 (C3, CH), 131.8 4.1.12. N-(4-Bromobenzene)sulfonyl)-2-nonylbenzamide (15) (C4, CH), 132.0 (C1), 140.4 (d, 3JC-F = 7.2 Hz, C1″), 142.8 (C2), 162.2 (d, 1 Prepared following the general procedure using 2-nonylbenzoic acid JC-F = 252.1 Hz, C3”), 166.2 (C7). 19 (7) (50 mg, 0.400 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 F NMR (282 MHz, CDCl3) δ − 109.3 (s, C–F). 12 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 IR (ATR) υmax cm− 1 3243, 2925, 2854, 1693, 1598, 1436, 1350, 129.9 (C2″, C6″, 2 × CH), 131.0 (C3, CH), 131.8 (C4, CH), 132.0 (C1), 1228, 1168, 1059, 902, 677, 599, 580 138.1 (C1″), 138.3 (C3″, C5”, 2 × CH), 142.7 (C2), 166.2 (C7). HRMS (ESI) m/z: [M+H] Calcd for C22H28NO3SF 406.1846; Found IR (ATR) υmax cm− 1 3232, 2924, 2853, 1688, 1569, 1429, 1350, 406.1846. 1172, 1054, 1007, 730, 598, 567 HRMS (ESI) m/z: [M+H] Calcd for C22H28INO3S 514.0907; Found 4.1.15. N-(4-Fluorobenzenesulfonyl)-2-nonylbenzamide (18) 514.0914. Prepared following the general procedure using 2-nonylbenzoic acid (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 4.1.18. N-(4-tert-Butylbenzenesulfonyl)-2-nonylbenzamide (21) eq.), 4-fluorobenzenesulfonamide (33 mg, 0.192 mmol, 0.95 eq.) and Prepared following the general procedure using 2-nonylbenzoic acid DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 was purified by column chromatography on silica gel using hexane- eq.), 4-tert-butylbenzenesulfonamide (42 mg, 0.192 mmol, 0.95 eq.) and diethyl ether (85:15) to yield 18 as a white solid (44 mg, 0.108 mmol, DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture 57%), mp 112–115 ◦ C. was purified by column chromatography on silica gel using hexane- 1 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.7 Hz, C9′), 1.08–1.41 diethyl ether (70:30) to yield 21 as an off-white solid (58 mg, (m, 14H, C2′-C8′), 2.61 (t, 2H, J = 7.8 Hz, C1′), 7.15–7.30 (m, 4H, C3, C5, 0.131 mmol, 69%), mp 108–109 ◦ C. 1 C3″, C5″), 7.32–7.43 (m, 2H, C4, C6), 8.10–8.22 (m, 2H, C2″, C6″), 8.50 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, C9′), 1.09–1.45 (s, 1H, N–H). (m, 14H, overlapped, C2′-C8′), 1.35 (s, 9H, C9–C11), 2.62 (t, 2H, 13 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 J = 7.7 Hz, C1′), 7.15–7.28 (m, 2H, C3, C5), 7.31–7.43 (m, 2H, C4, C6), (CH2), 29.4 (CH2), 29.5 (2 × CH2), 31.7 (C2′, CH2), 31.9 (C7′, CH2), 33.2 7.57 (app. dt, 2H, J = 8.7, 2.1 Hz, C3″, C5″), 8.06 (app. dt, 2H, J = 8.7, (C1’, CH2), 116.2 (d, 2JC-F = 22.8 Hz, C3″, C5″), 126.0 (C5, CH), 127.1 2.1 Hz, C2″, C6″), 8.18 (s, 1H, N–H). (C6, CH), 130.9 (C3, CH), 131.6 (d, 3JC-F = 9.9 Hz, C2″, C6″), 131.7 (C4, 13 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 CH), 132.1 (C1), 134.4 (d, 4JC-F = 3.1 Hz, C1″), 142.7 (C2), 166.0 (d, 1JC- (CH2), 29.4 (CH2), 29.4 (CH2), 29.5 (CH2), 31.0 (C9–C11, 3 × CH3), 31.5 F = 256.8 Hz, C4”), 166.4 (C7). (C2′, CH2), 31.9 (C7′, CH2), 33.1 (C1’, CH2), 35.3 (C8), 126.0 (C5, C3″, 19 F NMR (282 MHz, CDCl3) δ − 102.6 (s, C–F). C5″, 3 × CH), 126.9 (C6, CH), 128.4 (C2″, C6″, 2 × CH), 130.8 (C3, CH), IR (ATR) υmax cm− 1 3234, 2925, 2854, 1682, 1591, 1493, 1432, 131.5 (C4, CH), 132.5 (C1), 135.4 (C1″), 142.4 (C2), 158.1 (C4”), 166.3 1352, 1235, 1176, 1156, 838, 570, 546 (C7). HRMS (ESI) m/z: [M+H] Calcd for C22H28NO3SF 406.1846; Found IR (ATR) υmax cm− 1 3237, 2958, 2926, 2854, 1704, 1428, 1348, 406.1852. 1172, 1112, 846, 624, 579 HRMS (ESI) m/z: [M+H] Calcd for C26H37NO3S 444.2566; Found 4.1.16. N-(4-Chlorobenzenesulfonyl)-2-nonylbenzamide (19) 444.2569. Prepared following the general procedure using 2-nonylbenzoic acid (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 4.1.19. N-(4-Methoxybenzenesulfonyl)-2-nonylbenzamide (22) eq.), 4-chlorobenzenesulfonamide (36 mg, 0.192 mmol, 0.95 eq.) and Prepared following the general procedure using 2-nonylbenzoic acid DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 was purified by column chromatography on silica gel using hexane- eq.), 4-methoxybenzenesulfonamide (35 mg, 0.192 mmol, 0.95 eq.) and diethyl ether (80:20) to yield 19 as a white solid (52 mg, 0.123 mmol, DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture 64%), mp 99–101 ◦ C. was purified by column chromatography on silica gel using hexane- 1 H NMR (400 MHz, CDCl3) δ 0.89 (t, 3H, J = 7.0 Hz, C9′), 1.09–1.41 diethyl ether (60:40) to yield 22 as a white solid (45 mg, 0.108 mmol, (m, 14H, C2′-C8′), 2.62 (t, 2H, J = 7.8 Hz, C1′), 7.17–7.32 (m, 2H, C3, 57%), mp 90–92 ◦ C. 1 C5), 7.33–7.47 (m, 2H, C4, C6), 7.49–7.64 (m, 2H, C3″, C5″), 8.04–8.17 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.8 Hz, C9′), 1.06–1.45 (m, 2H, C2″, C6″), 8.27 (s, 1H, N–H). (m, 14H, C2′-C8′), 2.61 (t, 2H, J = 7.8 Hz, C1′), 3.88 (s, 3H, C8), 7.01 13 C NMR (100 MHz, CDCl3) δ 14.2 (C9′, CH3), 22.7 (C8′, CH2), 29.3 (app. dt, 2H, J = 9.0, 2.5 Hz, C3″, C5″), 7.11–7.29 (m, 2H, C3, C5), (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.7 (C2′, CH2), 31.9 (C7′, 7.29–7.43 (m, 2H, C4, C6), 8.07 (app. dt, 2H, J = 9.0, 2.5 Hz, C2″, C6″), CH2), 33.2 (C1’, CH2), 126.1 (C5, CH), 127.0 (C6, CH), 129.3 (C3″, C5″, 8.23 (s, 1H, N–H). 13 2 × CH), 130.1 (C2″, C6″, 2 × CH), 131.0 (C3, CH), 131.9 (C4, CH), C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 132.0 (C1), 136.8 (C1″), 140.9 (C4”), 142.7 (C2), 166.2 (C7). (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.6 (C2′, CH2), 31.9 (C7′, IR (ATR) υmax cm− 1 3235, 2923, 2853, 1687, 1427, 1348, 1169, CH2), 33.1 (C1’, CH2), 55.7 (C8, CH3), 114.1 (C3″, C5″, 2 × CH), 126.0 1085, 827, 753, 615, 568, 483 (C5, CH), 127.0 (C6, CH), 129.9 (C1″), 130.8 (C3, CH), 130.9 (C2″, C6″, HRMS (ESI) m/z: [M+H] Calcd for C22H28ClNO3S 422.1551; Found 2 × CH), 131.5 (C4, CH), 132.5 (C1), 142.4 (C2), 164.0 (C4”), 166.3 422.1541. (C7). IR (ATR) υmax cm− 1 3239, 2925, 2854, 1702, 1596, 1498, 1428, 4.1.17. N-(4-Iodobenzenesulfonyl)-2-nonylbenzamide (20) 1345, 1262, 1162, 834, 574, 557 Prepared following the general procedure using 2-nonylbenzoic acid HRMS (ESI) m/z: [M+H] Calcd for C23H31NO4S 418.2046; Found (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 418.2054. eq.), 4-iodobenzenesulfonamide (54 mg, 0.192 mmol, 0.95 eq.) and DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL). The crude mixture was 4.1.20. N-(4-Nitrobenzenesulfonyl)-2-nonylbenzamide (23) purified by column chromatography on silica gel using hexane-diethyl Prepared following the general procedure using 2-nonylbenzoic acid ether (70:30) to yield 20 as an off-white solid (94 mg, 0.183 mmol, (7) (50 mg, 0.202 mmol, 1.00 eq.), DMAP (27 mg, 0.221 mmol, 1.10 96%), mp 66–68 ◦ C. eq.), 4-nitrobenzenesulfonamide (38 mg, 0.192 mmol, 0.95 eq.) and 1 H NMR (300 MHz, CDCl3) δ 0.89 (t, 3H, J = 6.8 Hz, C9′), 1.08–1.42 DCC (45 mg, 0.221 mmol, 1.10 eq.) in DCM (5 mL) and DMF (1 mL). The (m, 14H, C2′-C8′), 2.62 (t, 2H, J = 7.8 Hz, C1′), 7.18–7.27 (m, 2H, C3, crude mixture was purified by column chromatography on silica gel C5), 7.32–7.43 (m, 2H, C4, C6), 7.85 (app. dt, 2H, J = 8.7, 2.0 Hz, C2″, using hexane-diethyl ether (60:40) to yield 23 as an off-white solid C6″), 7.94 (app. dt, 2H, J = 8.7, 2.0 Hz, C3″, C5″), 8.27 (s, 1H, N–H). (25 mg, 0.058 mmol, 31%), mp 120–122 ◦ C. 13 1 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 H NMR (300 MHz, CDCl3) δ 0.88 (t, 3H, J = 6.9 Hz, C9′), 1.08–1.45 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (C3′, CH2), 31.7 (C2′, CH2), 31.9 (m, 14H, C2′-C8′), 2.63 (t, 2H, J = 7.7 Hz, C1′), 7.19–7.29 (m, 2H, C3, (C7′, CH2), 33.2 (C1’, CH2), 102.1 (C4″), 126.1 (C5, CH), 127.0 (C6, CH), C5), 7.34–7.48 (m, 2H, C4, C6), 8.33–8.46 (m, 4H, C2″, C3″, C5″, C6″) 13 A.-C. Gómez et al. European Journal of Medicinal Chemistry 261 (2023) 115819 13 C NMR (75 MHz, CDCl3) δ 14.1 (C9′, CH3), 22.7 (C8′, CH2), 29.3 (cMHB) to a final concentration of 5 × 105 cfu/mL. Aliquots of 50 μl (CH2), 29.4 (CH2), 29.5 (CH2), 29.5 (CH2), 31.8 (C2′, CH2), 31.9 (C7′, were added to each well of a 96-well plate containing 50 μl of 2-fold CH2), 33.2 (C1’, CH2), 124.1 (C3″, C5″, 2 × CH), 126.2 (C5, CH), 127.1 serial dilutions of colistin in duplicate and 100 μl of sulfonamides at (C6, CH), 130.1 (C2″, C6″, 2 × CH), 131.2 (C3, CH), 131.5 (C1), 132.2 0.1 mM (from a stock solution at 10 mM dissolved in DMSO) to obtain a (C4, CH), 143.0 (C2), 144.0 (C1″), 151.0 (C4”), 166.1 (C7). final concentration of 0.05 mM. Sterility (no bacteria) and growth (no IR (ATR) υmax cm− 1 3110, 2926, 2853, 1678, 1529, 1432, 1350, antibiotic) controls were also prepared. Plates were incubated at 37 ◦ C 1185, 1174, 845, 606 for 20 h. The optical density, was determined at 550 nm (OD550nm) using HRMS (ESI) m/z: [M+H] Calcd for C22H28N2O5S 433.1791; Found a multilabel microtitre plate reader (Victor 3, PerkinElmer LAS, Wal­ 433.1787. tham, MA, USA). MICs was determined as the antibiotic concentration that reduced ≥80% of bacterial growth compared with the positive 4.2. Biological evaluation control. At least two replicates per strain and condition were tested. 4.2.1. Bacterial strains 4.2.5. In vivo assays A. baumannii ATCC 15308, E. coli ATCC 9637, B. multivorans B10, Galleria mellonella larvae were used to evaluate the effect of sulfon­ B. cepacia R6193, B. cenocepacia 289, P. aeruginosa PAO1 and amides after S. maltophilia infection as was previously described [34]. S. maltophilia K279a, were the strains used in this study. Bacterial strains Briefly, At least 15 larvae with a weight of 300–400 mg and no signs of were obtained from our own collection. All the strains were grown in LB melanisation were infected with S. maltophilia K279a. Log-phase culture medium at 37 ◦ C. in LB medium was washed and adjusted with PBS to obtain 1 × 104 cfu per larva. Infective dose was confirmed by viable counts on LB agar 4.2.2. Intrinsic cell growth inhibition plates. Larvae were infected injecting 5 μl of K7279a in the last proleg To evaluate the growth inhibition of sulfonamides, the broth using a 50 μl- Hamilton® Microliter™ syringe. Infected larvae were microdilution method was used, as previously described [69,70]. incubated at 37 ◦ C for 60 min. After incubation, larvae were incubated Briefly, bacterial strains were grown overnight in cation-adjusted Mul­ on ice for 15 min to anesthetise. 5 μl of PBS solution containing either ler-Hinton Broth (cMHB) and then subcultured to new cultures with DMSO (untreated group), colistin at final concentration of 0,5 μg/mL or 1:100 dilution. 100 μL of fresh log phase cultures, adjusted to a final sulfonamides at final concentration of 50 μM were injected in the last concentration of 1 × 105 cfu/mL, were added per well to 96-well sterile right proleg. The amounts of sulfonamides, colistin and DMSO injected microtiter plates containing 100 μL of sulfonamides at 0.1 mM (from a were calculated as was previously described [64]. Larvae were incu­ stock solution at 10 mM dissolved in DMSO) to obtain a final concen­ bated at 37 ◦ C and the survival was recorded every 24 h. At least two tration of 0.05 mM. Plates were incubated at 37 ◦ C for 20 h and then the replicates were made with different batches of larvae. optical density, determined at 550 nm (OD550nm), was measured using a multilabel microtitre plate reader (Victor 3, PerkinElmer LAS, Waltham, Declaration of competing interest MA, USA). Before readout, plates were shaken for homogenization. DMSO alone was included as control at final concentration of 0,5%. The authors declare that they have no known competing financial Bacterial growth was expressed in percentage on the basis of growth in interests or personal relationships that could have appeared to influence control wells. The screening was done at least two times. The average of the work reported in this paper. OD550nm values of each sample was used to calculate the percentage of inhibition. Data availability 4.2.3. Biofilm inhibition assay Data available in supplementary information For biofilm formation, the crystal violet method was used as previ­ ously described [71]. Briefly, 100 μl of fresh log phase bacteria at a final Acknowledgements concentration of 1 × 105 cfu/mL, were added to 96-well non-treated flat-bottom sterile microtiter plates (Deltalab S.L., Spain) containing Conor Horgan is grateful for funding by way of a Government of 100 μL of sufonamides at 0.1 mM (0.05 mM as a final concentration). Ireland Postgraduate Research Scholarship (GOIPG/2017/1111) pro­ Plates were incubated for 24 h at 37 ◦ C. The OD620nm was determined vided by the Irish Research Council. Michelle O’Driscoll thanks the Irish using the microtitre plate reader (Victor 3, PerkinElmer LAS, Waltham, Research Council for a Government of Ireland Postgraduate Research MA, USA) to measure bacterial biomass. Supernatants were then aspi­ Scholarhsip (GOIPG/2021/227). Xavier Daura acknowledges funding rated and the plates were washed three times with distilled water. In from the Spanish MICINN (PID2019-111364RB-I00). Isidre Gibert ac­ order to fix the biofilm, plates were incubated at 65 ◦ C for 15 min. The knowledges support from the Catalan AGAUR (2021 SGR00092). biofilm was stained by adding 200 μL of 0.1% crystal violet and the plates were incubated for 15 min at RT. The crystal violet was discarded Appendix A. Supplementary data and the plates were washed three times with distilled water. The plates were allowed to dry, incubating at 37 ◦ C for at least 30 min. To dissolve Supplementary data to this article can be found online at https://doi. the crystal violet, 250 μl of 30% acetic acid was added. Plates were org/10.1016/j.ejmech.2023.115819. incubated for 15 min at room temperature and 150 μL were transferred to a new plate and the OD550nm was measured. At least two replicates per Abbreviations strain and condition were tested. Growth control wells containing bac­ teria in LB medium and control wells containing only sterile medium ABA A. baumannii ATCC 15308 were included. BCC Burkholderia cepacia complex BCE B. cepacia R6193 4.2.4. MIC determination BCN B. cenocepacia 289 Minimal inhibitory concentrations (MIC) of colistin, was tested in BDSF Burkholderia diffusible signal factor accordance with Clinical and Laboratory Standards Institute and The BMU B. multivorans B10 European Committee on Antimicrobial Susceptibility Testing recom­ CoA Coenzyme A mendations with some modifications [70,72]. 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