Improving Activity of New Arylurea Agents against Multidrug-Resistant and Biofilm-Producing Staphylococcus epidermidis

Multidrug-resistant (MDR) strains of Staphylococcus epidermidis (S. epidermidis), prevalent in hospital environments, contribute to increased morbidity and mortality, especially among newborns, posing a critical concern for neonatal sepsis. In response to the pressing demand for novel antibacterial therapies, we present findings from synthetic chemistry and structure–activity relationship studies focused on arylsulfonamide/arylurea derivatives of aryloxy[1-(thien-2-yl)propyl]piperidines. Through bioisosteric replacement of the sulfonamide fragment with a urea moiety, compound 25 was identified, demonstrating potent bacteriostatic activity against clinical multidrug-resistant S. epidermidis strains (MIC50 and MIC90 = 1.6 and 3.125 μg/mL). Importantly, it showed activity against linezolid-resistant strains and exhibited selectivity over mammalian cells. Compound 25 displayed antibiofilm-forming properties against clinical S. epidermidis strains and demonstrated the capacity to eliminate existing biofilm layers. Additionally, it induced complete depolarization of the bacterial membrane in clinical S. epidermidis strains. In light of these findings, targeting bacterial cell membranes with compound 25 emerges as a promising strategy in the fight against multidrug-resistant S. epidermidis strains.

T he spread of multidrug resistant (MDR) bacteria, stemming from the excessive and inappropriate use of antimicrobials, has emerged as a significant impediment to the effective treatment of infectious diseases. 1The European Centre for Disease Prevention and Control (ECDC) reported that around 33 000 deaths in Europe can be directly linked to infections caused by MDR bacteria. 2he widespread presence of MDR S. epidermidis strains in hospital environments stands as the primary factor behind the escalating incidence of nosocomial infections.Up to 90% of S. epidermidis strains within healthcare settings commonly exhibit resistance to multiple drugs, including methicillin, or demonstrate cross-resistance to macrolides, lincosamides, and type B streptogramins (referred to as MRSE and MLS B S. epidermidis, respectively).Notably, infections associated with S. epidermidis contribute to elevated morbidity and mortality rates, especially among immunocompromised patients including neonates. 3,4−7 LOS typically manifests after 72 h of life, particularly in very low birth weight (VLBW) infants, often originating from the surrounding nosocomial/hospital environment.Late infections in VLBW neonates, prolonged hospital stays, or invasive medical procedures amplify mortality rates among neonates. 8Furthermore, the ability of S. epidermidis to form biofilms on medical devices such as central venous and urinary tract catheters and prosthetic implants intensifies the risk of nosocomial infection.−11 Despite numerous efforts to ensure sterility and encourage the prudent use of currently available antibiotics, morbidity rates persist at high levels.These findings underscore the pressing need for the development of a novel class of potential antimicrobial agents targeting multidrug-resistant and biofilmforming S. epidermidis.To address this challenge, screening our in-house library of arylsulfonamide derivatives of aryloxy(1phenylpropyl)piperidines identified compound I (Figure 1), exhibiting promising antibacterial activity against reference susceptible and multidrug-resistant S. epidermidis and S. aureus strains (MIC ranged from 3.125 and 6.25 μg/mL).Of note, compound I does not inhibit the growth of the tested Gramnegative bacteria (MIC ≥ 25 μg/mL).Herein we present the discovery of new aryloxy[1-(thien-2-yl)propyl]piperidines with improved antibacterial activity compared to compound I, evaluated against a spectrum of clinical S. epidermidis isolates (81 strains), including multidrug-resistant and biofilm-forming strains.We also determined their bacteriostatic/bactericidal properties and conducted a real-time analysis of bacterial growth.Finally, we evaluated the ability of the most promising compound, 25, in terms of its activity and safety over mammalian cell lines, to disrupt the staphylococcal cell membranes of clinical S. epidermidis.
The synthesis of the designed arylsulfonamide/arylurea derivatives of aryloxy(1-phenylpropyl) and [1-(thien-2-yl)propyl]piperidines I and 12−27 followed a previously established multistep procedure (Scheme 1). 12In the first step, chloropropiophenone 1 or a commercially unavailable 3bromo-1-(thiophen-2-yl)propan-1-one 3, derived from the Friedel−Craft acylation of thiophene with 3-bromopropionyl chloride, underwent a reaction with 4-N-Boc-aminopiperidine under basic conditions, yielding intermediates 4 and 5 (with isolated yields of 76% and 65%, respectively).A highly effective reduction (yield up to 90%) of the ketone function was achieved by adding a 2.5 M solution of lithium aluminum hydride (LiAlH 4 ) in THF at 0 °C for 1 h, resulting in formation of secondary alcohols 6 and 7. Subsequent Mitsunobu coupling between these intermediates and the appropriate 3-CF 3 -phenol or 4-CF 3 -phenol, in the presence of triphenylphosphine and diethylazodicarboxylate (DEAD), produced O-arylated derivatives 8−11 in good yields (ranging from 31% to 52%).The deprotection of the Boc moiety present in intermediates 8 and 9 was carried out under classical conditions in an acidic medium, utilizing a mixture of TFA/ DCM (20/80 v/v).In contrast, due to the instability of the thiophene moiety under strongly acidic conditions, the removal of the Boc function from compounds 10 and 11 was performed in a basic environment using sodium tertbutoxylate in DMSO.The final arylsulfonamide/arylurea derivatives I and 12−27 were obtained in good yields (55− 85%) through the acylation of the primary amines with the appropriate aryl sulfonyl chloride or aryl isocyanates in the presence of triethylamine.
The antimicrobial activity of the newly synthesized compounds 12−27 was preliminarily assessed based on MIC values determined by the broth microdilution method 13 against three reference strains (Table 1 and Table S1).These strains included susceptible, multidrug-resistant, and biofilm-producing strains of S. epidermidis and S. aureus (MSSE, MDR/BPSE, and MDR/BPSA, respectively).The cornerstone antibiotic linezolid served as a positive control (Table 1).The impact of structural modifications at the aryloxy fragment and the aromatic moiety bound to the propyl linker on the inhibitory activity was initially investigated.The shifting of the trifluoromethyl group in the aryloxy fragment from meta-to para-position increased the activity of compound 12 against the MDR/BPSE strain up to 8-fold when compared to the hit I.Of note, this modification significantly reduced potency against the MDR/BPSA strain (MIC = 50 μg/mL).
Compounds featuring the thienyl moiety in position 1 of the propyl linker (13 and 14) exhibited higher anti-MSSE and anti-MRD/BPSE properties compared to their phenyl analogs, with 14 emerging as the most potent derivative (MIC = 1.6 and 0.8 μg/mL for MSSE and MRD/BPSE, respectively).The antibiotic concentration inhibiting the growth of 50% clinical isolates.b The antibiotic concentration inhibiting the growth of 90% of clinical isolates.c Mean of MIC values against clinical MDR-SE isolates reported in Table 2-SI.d Results were obtained after treating cardiomyocytes H9c2 (ATCC-CRL-1446) or skin BJ (ATCC-CRL-2522) fibroblasts for 24 h using MTT test; n = 3. e Hemolytic activity was detected after treating red blood cells of horse with compounds at the concentration of 200 μM for 1 h; the positive control Triton X-100 produced 100% lysis; n = 3; NT = not tested.
Further optimization focused on evaluating the impact of the type of substituent and its position on the arylsulfonamide fragment's activity.Except for 3-fluorobenzenesulfonamide 17, all tested compounds bearing an electron-donating or electronwithdrawing substituent in the meta-position displayed significant antibacterial activity (MIC ranged from 0.1 to 0.4 μg/mL).They demonstrated higher potency than that of linezolid (MIC = 1.6 μg/mL) against the MDR/BPSE strain.Nevertheless, they exhibited weaker antibacterial properties than the chlorine analog 14 against the MSSE strain and MDR/BPSA.Compounds with a fluorine or chlorine atom in the para position were less potent in inhibiting the growth of susceptible S. epidermidis while maintaining high antibacterial activity toward multidrug-resistant and biofilm-producing S. epidermidis strains (MIC = 1.6 and 0.2 μg/mL for 22 and 23, respectively).
In the next move, arylsulfonamide group was bioisosterically replaced with the arylurea yielding compounds 24−27. 14mong the synthesized compounds, only 25 demonstrated potency against both susceptible and multidrug-resistant strains (MIC = 0.8 and 1.6 μg/mL), exhibiting antibacterial efficacy comparable to that of the last-resort antibiotic linezolid.These results confirmed the importance of the presence of the chlorine atom in the meta rather than para position at the arylurea fragment for antibacterial activity.
Subsequently selected compounds (12, 14, 18, 21, 25) with favorable antimicrobial properties against the reference MSSE (MIC ≤ 6.25 μg/mL) and higher anti-MDR/BPSE activity than linezolid (MIC ≤ 0.8 μg/mL) were evaluated against 81 clinical MDR strains of S. epidermidis (Table 2, Table S2) including LRSE (linezolid-resistant S. epidermidis) pathogens.The 3-chlorobenzenesulfonamide derivatives 12 and 14 featuring the phenyl or the 2-thienyl moiety at the propyl linker exhibited moderate-to-low antibacterial activity (MIC 50 and MIC 90 ranging from 6.25 to 50 μg/mL) against clinical S. epidermidis isolates.Likewise, compounds 18 and 21, demonstrating promising anti-MDR/BPSE activity against the reference strain (MIC ≤ 0.4 μg/mL), did not inhibit the growth of all tested strains.In contrast, the urea-containing analogue 25 displayed potency against the majority of clinical multidrug-resistant SE bacteria showing antibacterial activity comparable to that of the reference linezolid (Table 2).Remarkably, compound 25 demonstrated the ability to overcome resistance to the linezolid associated with clinically important LRSE strains.It was also observed that compound 25 exhibited selective antimicrobial activity against Grampositive bacteria as MIC values exceeded 50 μg/mL for reference strains of Gram-negative bacteria such as Acinetobacter baumannii ATCC 19606, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 (Table S3).
The selectivity toward mammalian cells is one of the major concerns in the development of new antibacterial agents for clinical applications.Therefore, the cytotoxicity of the selected compounds 12, 14, and 25 was determined against cardiomyocytes (H9c2) and skin fibroblasts (BJ) using the MTT assay (Table 2, Figure S1, Figure S2).The anthracycline chemotherapeutic doxorubicin served as a positive control (IC 50 = 0.39 μg/mL for both cell lines).The tested compounds displayed IC 50 values ranging from 14 to 28 μg/ mL against H9c2 cells and exhibited 2-fold higher toxicity against skin BJ fibroblasts.Linezolid produced a cardiotoxic effect against H9c2 with an IC 50 value of 16.9 μg/mL.Compound 25 showed relatively low toxicity toward the tested mammalian cells within the MIC values against most of the clinical S. epidermidis strains.Next, the hemolytic activity of the selected compounds (12, 14, and 25) was assessed on horse red blood cells.None of them caused lysis of erythrocytes at concentrations up to 200 μM.The most potent anti-SE agent 25, with the highest safety margin, was also found to be metabolically stable (Cl in = 26.7 mg/μL/min) after 60 min of incubation using rat liver microsomes (RLM). 15,16Furthermore, thiophene-containing compounds may undergo cytochrome P450-dependent bioactivation into reactive electrophilic epoxides or S-oxides species which cause idiosyncratic adverse drug reactions. 17,18Specific structural alerts are conditional, depending on, among other things, the type of incorporation of thiophene in molecules (e.g., mono/ disubstituted ring, fused ring) and the reactivity of metabolites.This prompted us to predict bioactivation pathways for compound 25 and selected reference thiophene-containing drugs (i.e., duloxetine, eprosartan, rotigotine, suprofen, tienilic acid) using built-in cytochrome P450 homology models incorporated in MetaSite software. 19The 3-chlorophenylurea fragment of compound 25 was the most likely susceptible to hydroxylation of the phenyl ring, suggesting a low propensity of 25 to generate thiophene-associated reactive metabolites (<25% of relative scores for CYP1A2, CYP2D6, and CYP3A4 isoforms, Figure S3).Demonstrated results are consistent with those assessed for neither bioactivated nor toxic thiophenebased drugs, i.e., duloxetine, eprosartan, and rotigotine (Figure S3).
Simultaneously, we evaluated the antibiofilm activity of compounds 12, 14, and 25 to determine whether these compounds inhibit biofilm formation, expressed by minimal biofilm inhibition concentration (MBIC) or eliminate persistent biofilm, i.e., release cells back to a planktonic state, expressed by minimal biofilm elimination concentration (MBEC).Of note, biofilm-forming MDR S. epidermidis strains which colonize hospital environments contribute to an increased risk of nosocomial infection. 20,21This heightening risk results, in part, from an increased likelihood of transmitting strains from the environment or medical devices to patients.Among the selected compounds, arylsulfonamides 12 and 14 were devoid of antibiofilm activity (MBIC 90 and MBEC 90 ≥ 125 μg/mL), while arylurea derivative 25 emerged as a promising antibiofilm agent.Compound 25 exhibited promising inhibitory properties against biofilm formation as well as the ability to eliminate existing biofilm layers (Figure 2).Notably, 25 exhibits enhanced antibiofilm activity compared to the last-resort drug, linezolid (MBIC 90 and MBEC 90 amounted to 31.25 μg/mL for comp.25 vs 50 μg/mL for linezolid (Table S4)).
Additionally, through a real-time bacterial growth analysis (Figure S4) and an examination of the MBC/MIC ratio, we observed that, similarly to linezolid compound 25, it demonstrated bacteriostatic properties.The MBC/MIC ratio for compound 25 is 16 (Table S5).
Finally, we investigated the impact of the most active compound 25, on cell membrane permeability against MSSE, S. epidermidis clinical strain no.23, and S. aureus Newman (reference strain without known antibiotic resistance determinants).Bacterial strains were stained with 3,3′-diethyloxacarbocyanine iodide (DiOC 2 ) and then treated with the tested compound 25, with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a positive control.Daptomycin was used as a reference compound that causes depolarization of bacterial cell membranes. 22At a concentration equal to 4 × MIC, compound 25 induced depolarization in nearly all bacterial cells within 15 min (Figure 3).The effect of compound 25 against the cell membrane of S. epidermidis was comparable to that produced by CCCP.
An expanded phenotypic testing approach, employed to evaluate the antibacterial activity of compound 25, allowed us to juxtapose the MIC data with the clinical MIC breakpoints recommended by EUCAST for currently available antibiotics.In line with EUCAST guidelines, compounds with low MIC values -below clinical breakpoints for resistant strains are regarded as conceptual advances and may serve as complementary therapeutic alternatives.Importantly, the activity of compound 25 against all reference and clinical MDR S. epidermidis strains was within the susceptible category according to the EUCAST clinical breakpoints for linezolid (Table 1, Table S2, Figure S5). 23he hospital environment is a reservoir for multidrugresistant and biofilm-forming S. epidermidis strains that causes numerous nosocomial infections.The increasing antibiotic resistance among S. epidermidis, especially with the emergence of strains that are simultaneously linezolid-resistant and biofilm-producing, poses a therapeutic challenge.Given the looming shortage of effective therapeutic options against these bacteria, a series of arylsulfonamide/arylurea derivatives of aryloxy[1-(thien-2-yl)propyl]piperidines has been designed and synthesized.We identified arylurea derivative 25 as an effective bacteriostatic agent (MBC/MIC ratio = 16), demonstrating significant antibacterial activity against clinical multidrug-resistant and biofilm-producing S. epidermidis strains (MIC 50 and MIC 90 equaling 1.6 μg/mL and 3.125 μg/mL, respectively).Compound 25 also exhibited activity against S. epidermidis strains resistant to linezolid, the last-resort drug used in the treatment of challenging infections caused by multidrug-resistant biofilm-producing strains.The favorable selectivity of compound 25 over mammalian cells (cardiomyocytes and skin fibroblasts) and no cytotoxic effects on horse red blood cells further confirmed its potential.Compound 25 inhibited biofilm formation and disrupted persistent biofilm, thus preventing the widespread dissemination of MDR strains and eliminating persistent biofilms on medical devices.Mechanistic studies of compound 25 conclusively revealed that membrane-targeting antibacterial agents should be considered a promising strategy for eradicating MDR S. epidermidis strains.

* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00536.Synthetic procedures and characterization data for all intermediates and final compounds, biological assay protocols, UPLC/MS, 1 H and 13 C NMR spectra of representative compounds, supporting tables and figures (PDF)

Figure 2 .
Figure 2. Impact of compound 25 on the biofilm structure of the clinical strain Staphylococcus epidermidis no. 25, visualized using a confocal microscope and the fluorescent dye FilmTracer SYPROTM Ruby Biofilm Matrix Stain (ThermoFisher Scientific).Scale = 100 μm.Sectors A and B depict the compact biofilm structure before addition and incubation with compound 25 (control).Sector C shows extracellular mucus on the biofilm surface.Sectors D and E illustrate the disintegration of the biofilm structure after 24 h of incubation with compound 25 (MBIC = 7.8 μg/ mL).(F) Magnification of the stained biofilm fragment.

Figure 3 .
Figure 3. Flow cytometry dot plots showing alterations in the cell membrane potential of the clinical Staphylococcus epidermidis no.23 strain under the influence of various concentrations of the tested compound 25.Red, polarized cells; green, depolarized cells.(A) Negative control�cells stained by DiOC 2 ; (B) positive control�cells treated with CCCP; (C) cells treated with daptomycin (positive control) at a concentration of 0.4 μg/mL; (D) cells treated with the tested compound 25 at a concentration of 0.8 μg/mL; (E) cells treated with the tested compound 25 at a concentration of 1.6 μg/mL; (F) cells treated with the tested compound 25 at a concentration of 3.125 μg/mL There was no statistically significant relationship (p > 0.05) between the average fluorescence value for the tested strains and compound 25 and CCCP.