key: cord-0927784-g7v2vaiz
authors: Lal, Jhajan; Kaul, Grace; Akhir, Abdul; Ansari, Shabina B.; Chopra, Sidharth; Reddy, Damodara N.
title: Bio-evaluation of fluoro and trifluoromethyl-substituted salicylanilides against multidrug-resistant S. aureus
date: 2021-10-27
journal: Med Chem Res
DOI: 10.1007/s00044-021-02808-4
sha: 392b4e56e2ba4dc1180838809c3c30b59b0d3be3
doc_id: 927784
cord_uid: g7v2vaiz

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA) are primary causes of skin and soft tissue infections worldwide. To address the emergency caused due to increasing multidrug-resistant (MDR) bacterial infections, a series of novel fluoro and trifluoromethyl-substituted salicylanilide derivatives were synthesized and their antimicrobial activity was investigated. MIC data reveal that the compounds inhibited S. aureus specifically (MIC 0.25–64 µg/mL). The in vitro cytotoxicity of compounds with MIC < 1 µg/mL against Vero cells led to identification of four compounds (20, 22, 24 and 25) with selectivity index above 10. These four compounds were tested against MDR S. aureus panel. Remarkably, 5-chloro-N-(4’-bromo-3’-trifluoromethylphenyl)-2-hydroxybenzamide (22) demonstrated excellent activity against nine MRSA and three VRSA strains with MIC 0.031–0.062 µg/mL, which is significantly better than the control drugs methicillin and vancomycin. The comparative time–kill kinetic experiment revealed that the effect of bacterial killing of 22 is comparable with vancomycin. Compound 22 did not synergize with or antagonize any FDA-approved antibiotic and reduced pre-formed S. aureus biofilm better than vancomycin. Overall, study suggested that 22 could be further developed as a potent anti-staphylococcal therapeutic. [Image: see text]

The unceasing emergence of antimicrobial resistance in bacterial pathogens is a significant threat to global human health [1] . Staphylococcus aureus, a gram-positive, priority II pathogen, is a significant human pathogen that is responsible for causing life-threatening infections, consequently increasing mortality rates in the absence of effective containment and therapeutic solutions [2] [3] [4] . S. aureus biofilm formation and persister cells are associated with recurring and difficult to treat infections such as endocarditis, intravenous catheter-related bacteremia, and osteomyelitis [5] [6] [7] . The biofilm's extracellular matrix blocks entry of antibiotics and persister cells are dormant, thus making both physiological states as highly antibiotic tolerant [7] .

Methicillin-resistant staphylococcus aureus (MRSA) is one of the primary cause of skin and soft tissue infections [8] . Despite the availability of number of antibiotics, effective treatments are very limited for MRSA [9] and include fusidic acid, mupirocin, retapamulin, clindamycin, and vancomycin with other newer drugs such as ceftobiprole, nafithromycin, XF-73, TNP-2092, and ATx201 that are in clinical trials [10] . The situation is getting more worrisome, particularly due to emergence of vancomycinresistant S. aureus (VRSA) [2] [3] [4] . For successful treatment of infections caused due to multidrug-resistant (MDR) S. aureus, discovery and development of new antibiotic molecules with a novel mode of action is an urgent and unmet medical need [4, 11] .

In search of developing potential anti-infective agents, salicylanilides biological activity profile is well explored in parasitic [12] , microbial [13] [14] [15] [16] , rheumatology [17] , and cancer [18] . These compounds also demonstrate potency against various viral pathogens such as SARS-CoV, MERS-CoV, SARS-CoV-2, ZIKV, HCV, and human adenovirus [19, 20] . Salicylanilide family of anthelmintic drugs, e.g., closantel, niclosamide, oxyclozanide, and kinase inhibitor IMD0354, was recently identified as a potent antistaphylococcals [21] [22] [23] [24] . Niclosamide is currently in phase-II clinical trial for the treatment of S. aureus-infected patients (NCT03429595) [10] . However, Niclosamide is known to possess limited aqueous solubility, poor intestinal tract absorption, and low metabolic stability [24, 25] . These features represent significant drawbacks in using Niclosamide for the treatment of systemic bacterial infections. O-Alkylamino-tethered niclosamide derivatives have been designed to improve water solubility, but demonstrated poor activity (MIC of ≥15 μg/mL) against colistin-resistant Enterobacteriaceae [26] . These drawbacks motivated us to investigate the impact of fluorine and trifluoromethyl functional group in salicylanilides as potent antistaphylococcals.

The C-F bond has been used as a bioisostere for a number of functional groups, including C-H, C-OH, C=O, and CN [27] . The high dissociation energy (105.4 kcal/mol) of C-F bond is difficult to break and therefore less prone for metabolic transformation [28] . The incorporation of fluorine atoms or fluorinated functional group has become a growing trend in drug development, as it can be used to improve activity, improve bioavailability, and slow metabolic degradation. In recent years, FDA has approved~20% of drugs containing fluorine atom or a fluorinated functional group (e.g., trifluoromethyl, CF 3 ) [29] . The fluorine containing antibiotics ciprofloxacin, norfloxacin, levofloxacin, linezolid, and eravacycline is currently under clinical utilization ( Fig. 1 ) [10] . However, a recent study has demonstrated maximum gain of fluoroquinolone resistance against MRSA [30] and highlighted a series of side effects such as central nervous system toxicity, cartilage damage, and spermatogenesis impairment [31, 32] . Because of insurgence of linezolid-resistant strains, the usage of linezolid is also limited to the hospitalized patients infected with superbugs [33, 34] . Due to increasing resistance of these Considering the importance of fluorine in medicinal chemistry and growing demand of fluorine containing drugs, we report herein a series of systematically incorporated fluoro and trifluoromethyl salicylanilide derivatives that were synthesized and investigated their activity against MDR S. aureus to identify a potential therapeutic option. Structure-activity relationship (SAR) of fluoro and trifluoromethyl salicylanilide derivatives was examined to identify a new hit molecule for the treatment of MDR S. aureus infections with increased antimicrobial potency and low toxicity.

The fluoro and trifluoromethyl-substituted salicylanilides and other derivatives synthesis is shown in Scheme 1 and synthesis was accessed in one-pot condensation reaction. Substituted salicylic or benzoic acid (1a-d) was treated with substituted anilines (2a-k) in the presence of phosphorous trichloride in mixture of xylenes at 120°C for 3 h. No side products were observed during the reaction and all compounds were isolated without column chromatography. Filtration and washing with hot water (70°C) and 10% ethanol in water gave the desired salicylanilides 3-25 in high purity (Table 1 ). All synthesized compounds were characterized by NMR, HRMS, and ESI. The purity of synthesized compounds was determined by analytical highperformance liquid chromatography (HPLC).

The initial antimicrobial screening was performed against panel of five clinically relevant bacterial strains namely: (1) Escherichia coli ATCC 25922, (2) Klebsiella pneumoniae BAA 1705, (3) Pseudomonas aeruginosa ATCC 27853, (4) Acinetobacter baumannii BAA 1605, and (5) S. aureus ATCC 29213. The MIC data are shown in Table 2 and reveal that the synthesized salicylanilide derivatives inhibited S. aureus ATCC 29213 specifically (MIC 0.25-64 µg/mL) and did not exhibit any gram-negative bacterial coverage (Table 2) .

As it can be seen in Table 2 , benzanilide (3) and 5-chlorobenzanilide (4) demonstrated no growth inhibition, while salicylanilide (5) and 5-chloro salicylanilide (6) inhibited S. aureus ATCC 29213 at 64 and 8 µg/mL, respectively. These preliminary results are comparable with the reported activity of benzanilide derivatives [13] [14] [15] [16] . Fluorine substitution at 2ʹ or 4ʹ position in salicylanilides 7 and 9 exhibited improved potency (32 µg/mL). More interestingly, ten-fold increased activity observed in 5-chloro-N-(2ʹ/4ʹ-fluorophenyl)-salicylanilides 8 and 10 (2 and 4 µg/mL, respectively). As expected with the reported results [13, 22] , the activity improved on substitution of chlorine at 5-position in salicylanilides and same trend was observed in entire series (Table 2) .

Based on these preliminary results, we fixed chlorine at the fifth position in salicylic acid and varied the substitution on aniline ring. The activity was further improved eight-fold in 11 and 12 (0.44 µg/mL), in which additional fluorine and chlorine was incorporated at 3ʹ-position in compound 8, indicating that substitution at 3ʹ-position improves activity. The activity diminished in 2ʹ-nitro-4ʹ-fluoro substituted compounds 15 and 16 (MIC 32 and 3.88 µg/mL, respectively). However, activity improved four-fold in 17 and 18 (MIC 2 and 1 µg/mL, respectively) on exchanging nitro and fluoro substituent positions in 15 and 16. More interestingly, 3ʹ-trifluoromethylsubstituted salicylanilides 19-23 (MIC 0.25-0.5 µg/mL) showed improved activity. In the absence of 3ʹ-trifluoromethyl substitution, reduction in activity was observed in compounds 24 and 25. Overall eight compounds inhibited S. aureus ATCC 29213 with MIC below 1 µg/mL.

Next, we examined in vitro cytotoxicity of active compounds (MIC < 1 µg/mL) against Vero cells (ATCC CCL-81) using MTT assay to determine the selectivity index (SI) of active compounds. The SI of at least >10 is considered to be essential to take the compound forward for pre-clinical development. As summarized in Table 2 , four compounds 20, 22, 24, and 25 demonstrated SI > 10, thus indicating the specificity of these compounds toward bacterial rather than host cells.

As the next step, 20, 22, 24, and 25 were taken further to test activity against clinical drug-resistant S. aureus isolates along with levofloxacin, meropenem, and vancomycin as comparators. The MIC against clinical drugresistant S. aureus isolates is listed in Table 3 . Since MDR S. aureus are a global threat to human health, it is an urgent unmet need to develop potential drug candidates for the treatment of MDR S. aureus infections. As can be seen in Table 3 , compounds were tested against nine MRSA strains (NRS 10100, 10119, 10129, 10186, 10191-10194, and 10198) and three VRSA (VRS 1, 4, and 12). As shown in Table 3 , 20, 24, and 25 inhibited MRSA and VRSA with MICs 0.25-0.5 µg/mL. As expected, 22 found to be the most potent molecule inhibiting all MDR S. aureus isolates with MIC 0.031-0.062 µg/mL, which is significantly better active than that of vancomycin (Table 3) .

Finally, comparative time-kill kinetics was assessed for 22 along with vancomycin as a comparator to determine whether it exhibits bactericidal activity (Fig. 2) . The experiment was conducted for 24 h by adding 1× and 10× MIC of 22 and vancomycin, aliquots of cultures were removed, plated at 1, 6, and 24 h and the cfu are plotted in Fig. 2 . As can be seen at 24 h, 10× MIC of 22 lead to~8 log 10 cfu/mL reduction, whereas 10× MIC of vancomycin reduced~9 log 10 cfu/mL with no regrowth (Fig. 2) . Thus, 22 exhibits concentration-dependent bactericidal activity, which is comparable to vancomycin.

The formation of biofilm is a self-protection phenotype of bacteria, which often leads to prolonged therapeutic intervention and increasing drug resistance. The effect of 22 on in vitro pre-formed biofilm was tested [21] . Figure 3 represents the bacterial biofilm inhibition activity of 22 and comparator drugs. As can be seen, treatment with 10× MIC of 22 leads to a more significant reduction (P < 0.0005) in pre-formed biofilm as compared to either of comparators (P < 0.05). Thus, 22 is able to exert its effect against bacteria in different growth phases, whereas vancomycin is not.

Treatment of MDR infections typically requires a combination of drugs to achieve therapeutic clearance; thus, it is imperative to determine activity of drugs in combination with other approved FDA antibiotics. In this context, activity of 22 was determined in the presence of a panel of antibiotics that are clinically utilized for the treatment of staphylococcal infections. As shown in Table 4 , 22 did not synergize with or antagonize any of FDA-approved antibiotic; thus, it can be utilized as a therapeutic combination.

The improved activity exhibited by salicylanilide than that of benzanilide indicated the importance of 2-hydroxyl group in benzoic acid (Fig. 4) . 

A series of novel fluoro and trifluoromethyl-substituted salicylanilide derivatives were synthesized and investigated for their MDR S. aureus inhibition activity. The in vitro cytotoxicity assay of compounds with MIC < 1 µg/mL identified four compounds (20, 22, 24 , and 25) with SI > 10. Remarkably, 22 bearing trifluoromethyl group demonstrates excellent 

Reactions were performed in oven-dried glassware apparatus and magnetic stir bars were used to make homogeneous solution. 5-Chlorosalicylic acid and aniline derivatives were procured from Sigma-Aldrich. Thin layer chromatography (TLC) was used to monitor reaction progress, which was carried out on Merck silica-gel plates (0.25 mm thick, 60F254), and visualized by using UV (254 nm) or ninhydrin. 1 H, 13 C, 19 F, COSY, HSQC, and HMBC NMR spectra were measured on a Bruker 400 MHz NMR instrument and processed using TopSpin 3.6.1. Chemical shifts (δ) are represented in parts per million (ppm) from the residual of nondeuterated solvents that is used as an internal standard ( 1 H NMR: TMS δ = 0.00 ppm, CDCl 3 δ = 7.26 ppm, DMSO-d6 δ = 2.50 ppm, 13 C NMR: TMS δ = 0.00 ppm, CDCl 3 δ = 77.16 ppm, DMSO-d 6 δ = 39.52 ppm) and coupling constants (J) are given in hertz (Hz). The multiplicities were expressed using the following abbreviations: s = singlet; d = doublet; t = triplet; q = quartet; p = pentet; [35, 36] . The panel was further expanded to include drugresistant clinical S. aureus and Enterococci strains including those resistant to vancomycin and other clinically utilized antibiotics. These strains were procured from Biodefense and Emerging Infections Research Resources Repository/Network on Antimicrobial Resistance in Staphylococcus aureus/ American Type Culture Collection (BEI/NARSA/ATCC, USA) and routinely cultivated on MHA and MHBII (Difco). Before starting the experiment, single colony was picked from MHA plate, inoculated in Muller-Hinton broth (MHB, Difco) and incubated at 37°C for overnight to get the starter culture.

Antibiotic susceptibility testing was carried out by broth microdilution assay. Test compounds were prepared in DMSO as stock solutions (10 mg/mL) [37] . Bacterial cultures were inoculated in MHB. Optical density (OD) of the cultures was measured at the wavelength of 600 nm followed by dilution to achieve~10 6 CFU/ml. The concentrations of test compounds used in the study ranged from 64 to 0.5 mg/L in serially diluted fashion in DMSO from stock solutions and 2.5 μL of each concentration was added to each well of a 96-well microtiter plate (Polypropylene, Corning Inc., Corning, USA). Later, 97.5 μL of bacterial suspension in MHB medium was added to each well containing the test compound. Two controls were also included, i.e., cells alone and media alone (without compound + cells) and plates were incubated at 37°C for 16-18 h. MIC values were observed by the absence or presence of visible growth. For each compound, MIC determinations were carried independently three times using duplicate samples.

Cell toxicity was performed against Vero cells using the MTT assay [38] . In all,~10 3 cells/well were seeded in 96well plate and incubated at 37°C in an 5% CO 2 atmosphere. After 24 h, compound was added ranging from 100 to 12.5 µg/mL concentration and incubated for 72 h. After the incubation was over, MTT was added in each well, incubated at 37°C for further 4 h, residual medium was discarded, 0.1 mL of DMSO was added to solubilize the formazan crystals, and OD was taken at 540 nm for the calculation of CC 50 . CC 50 is defined as the lowest concentration of compound that leads to a 50% reduction in cell viability. Doxorubicin was used as positive control and each experiment was repeated in triplicate.

Interaction of 22 with FDA-approved drugs was tested by checkerboard method [39] . Serial two-fold dilutions of each drug were freshly prepared prior to testing. 20 was two-fold diluted along abscissa, while the antibiotics were serially diluted along ordinate in 96-well microtiter plate. Then, 95 µL of~10 5 CFU/mL was added to each well and plates were incubated at 37°C for 24 h. After the incubation, the ΣFICs (fractional inhibitory concentrations) were calculated as follows: ΣFIC = FIC A + FIC B, where FIC A is the MIC of drug A in the combination/ MIC of drug A alone and FIC B is the MIC of drug B in the combination/MIC of drug B alone. The combination is considered synergistic when the ∑FIC is ≤0.5, indifferent when the ∑FIC is >0.5-4, and antagonistic when the ∑FIC is >4.

The bactericidal activity was assessed by time-kill method [35] . Briefly, S. aureus ATCC 29213 was diluted to~10 6 cfu/mL, cells were treated with individual inhibitors along with appropriate controls at 1× and 10× MIC followed by incubation at 37°C for 24 h. A 0.1 mL sample was removed at various time points, serial ten-fold dilutions in 0.9 mL of PBS and 0.1 mL of the respective dilution were spread on an MHA. The plates will be incubated at 37°C for 24 h and colonies enumerated. Kill curves were constructed by counting the colonies from plates and plotting the cfu/mL of surviving bacteria at each time point in the presence and absence of compounds.

The determination of 22 anti-biofilm activity was performed as described earlier [35] . Briefly, S. aureus ATCC 29213 were grown overnight in 1% TSB with shaking (180 RPM) at 37°C. The overnight culture was diluted in fresh TSB broth (1:100) and 0.2 mL of freshly diluted culture was transferred into 96-well polystyrene flat bottom plate, covered with adhesive foil lid for maintaining low oxygen, and incubated in static condition for 48 h at 37°C. After incubation, media was decanted and plate was rinsed gently three times with the 1X PBS (pH 7.4) to remove the planktonic bacteria. Plates were refilled with TSB with different drug concentration and incubated for 24 h at 37°C. After drug treatment, the media was decanted, washed three times with 1X PBS (pH 7.4), and biofilm was fixed by incubating the plate at 60°C for 1 h. After fixing, the biofilm is stained by 0.06% crystal violet for 10 min, rinsed with PBS, and dried at room temperature. For quantification of biofilm, the bound crystal violet was eluted by 30% acetic acid (0.2 mL). Absorbance was taken on microtiter plate reader at 600 nm for biofilm quantification.

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