Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA)

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Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) Iveta Zadrazilova a,b,c,⇑, Martina Masarikova b,c, Sarka Pospisilova b, Ales Imramovsky d, Juana Monreal Ferriz e, Jarmila Vinsova e, Alois Cizek b,c, Josef Jampilek a,⇑ a

Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic c CEITEC VFU, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic d Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentska 95, 532 10 Pardubice, Czech Republic e Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic b

a r t i c l e

i n f o

Article history: Received 7 April 2015 Received in revised form 4 June 2015 Accepted 12 June 2015 Available online xxxx Keywords: MRSA Salicylanilides Alkylcarbamates Antibacterial activity Time-kill assay Structure–activity relationships

a b s t r a c t A series of twenty one salicylanilide N-alkylcarbamates was assessed for novel antibacterial characteristics against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum inhibitory concentration was determined by the broth dilution micro-method with subsequent subcultivation of aliquots to assess minimum bactericidal concentration. The bactericidal kinetics was established by time-kill assay. Ampicillin, ciprofloxacin and vancomycin were used as reference antibacterial drugs. All the tested compounds exhibited highly potent anti-MRSA activity (60.008–4 lg/mL) comparable or up to 250 higher than that of vancomycin, the standard in the treatment of serious MRSA infections. 4-Chloro-2-(3,4-dic hlorophenylcarbamoyl)phenyl butylcarbamate and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl ethylcarbamate were the most active compounds. In most cases, compounds provided reliable bacteriostatic activity, except for 4-chloro-2-(4-chlorophenylcarbamoyl)phenyl decylcarbamate exhibiting bactericidal effect at 8 h (for clinical isolate of MRSA 63718) and at 24 h (for clinical isolates of MRSA SA 630 and MRSA SA 3202) at 4 MIC. Structure–activity relationships are discussed. Ó 2015 Published by Elsevier B.V.

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1. Introduction Recent studies have shown that, despite antibacterial therapy, methicillin-resistant Staphylococcus aureus (MRSA) infections are still associated with serious clinical consequences, especially treatment failure, higher morbidity and mortality (up to 40%) (Kaku et al., 2014), prolonged hospitalization (4.5-fold longer length of stay compared to the general hospitalized population) (Peres et al., 2011), increased health care costs (from $3767 to $42,286, approaching 10 times the cost of susceptible strains) (Thampi et al., in press), etc. Activity against MRSA is of great importance in the new generation of antibacterial agents because of the

⇑ Corresponding authors at: Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic (I. Zadrazilova). E-mail addresses: [email protected] (I. Zadrazilova), [email protected] (J. Jampilek).

worldwide increasing prevalence of this pathogen (Stefani et al., 2012), more frequent antibiotic resistance to available anti-MRSA drugs, their toxicity and general lack of oral agents (Wilcox, 2011). Since vancomycin, the first line therapic agent of MRSA infections for many years (Clemens et al., 2011), appears to be getting ineffective (CDCP, 2002; Clemens et al., 2011; Sakoulas et al., 2004), limited possibilities to handle this problem make the discovery of new molecular scaffolds a priority, and the current situation even necessitates the re-engineering and repositioning of some old drug families to achieve effective control of these bacteria (Wilcox, 2011). The discovery of salicylanilides (SALs) dates back to early phenol applications and research for new antiseptics. Step by step, to improve disinfectant properties, a large number of substituted compounds were prepared culminating in the synthesis of 20 ,5-di chloro-40 -nitrosalicylanilide (niclosamide) as a potent molluscicide and taeniacide agent in 1955 (Anand and Sharma, 1997). Nowadays, SALs are well-known organic compounds exhibiting a broad spectrum of interesting biological activities, such as

http://dx.doi.org/10.1016/j.ejps.2015.06.009 0928-0987/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: Zadrazilova, I., et al. Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.009

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anthelmintic (Bartram et al., 2012), antibacterial (Cheng et al., 2010; Mollaghan et al., 2011; Pauk et al., 2013), antifungal (Vinsova et al., 2014), antimycobacterial (Pauk et al., 2013) and antiviral (Liu et al., 2008; Wu et al., 2004), among others. SALs have been described to affect a wide range of targets (Brown et al., 2008; Cheng et al., 2010; Hlasta et al., 1998; Kratky et al., 2012; Liechti et al., 2004; Liu et al., 2008; Macielag et al., 1998; Wu et al., 2004), although the appropriate mechanism of action responsible for overall biological activities of these compounds has not been proposed so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria (Hlasta et al., 1998; Macielag et al., 1998). They serve as inhibitors of protein kinase epidermal growth factor receptor (EGFR PTK) and are generally designed to compete with ATP for binding in the catalytic domain of tyrosin kinase (Liechti et al., 2004). The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion and control of cellular response to tuberculosis (Brown et al., 2008). Furthermore, SALs have been determined as inhibitors of some bacterial enzymes, such as transglycosylases from S. aureus (but not from Mycobacterium tuberculosis) in the cell wall biosynthesis (Cheng et al., 2010). Inhibition of isocitrate lyase and methionine aminopeptidase has been described to be involved in the antimycobacterial activity of SAL derivatives (Kratky et al., 2012). Thus, SALs seem to be promising candidates of antibacterial agents, which could be a solution to the resistance challenges. To assure higher antibacterial and especially antimycobacterial activity, SALs and their derivatives were chemically modified in the recent years (Brown et al., 2008; Cheng et al., 2010; Ferriz et al., 2010; Imramovsky et al., 2009, 2011a; Kratky et al., 2012; Liechti et al., 2004; Liu et al., 2008; Mollaghan et al., 2011; Pauk et al., 2013; Vinsova et al., 2007, 2014; Waisser et al., 2003). The synthesis of a series of novel SAL N-alkylcarbamates (see Table 1 for the general structure) was described previously (Ferriz et al., 2010), and their antimycobacterial (Ferriz et al., 2010), photosynthesis-inhibiting (Imramovsky et al., 2011b) and acetyl cholinesterase-inhibiting (Imramovsky et al., 2012) activities were reported recently. The aim of the current study was to evaluate the antibacterial activities of these previously described twenty-one SAL N-alkylcarbamates, which were the most active of the prepared salicylanilide-like compounds, against the three clinical isolates of MRSA (vancomycin-susceptible with increased vancomycin MICs within the susceptible range) and S. aureus ATCC 29213 (vancomycin-susceptible, methicillin-susceptible) as the reference and quality control strain. To the best of our knowledge, this is the first time when SAL N-alkylcarbamates have been screened as anti-MRSA agents; moreover, time-kill kinetics of these agents against MRSA has not been analyzed so far.

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2. Materials and methods

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2.1. Synthesis of compounds

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All the series of novel SAL N-alkylcarbamates were synthesized previously, and the synthetic pathway of these compounds was described recently (Ferriz et al., 2010). The structures of the compounds (see Table 1) were confirmed by IR, NMR and HR-MS spectrometry, and the purity was checked by HPLC and CHN analyses. The stability in the range of pH from 3 to 8 was determined by HPLC (Ferriz et al., 2010).

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2.2. Lipophilicity and other molecular descriptors

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Determination of the lipophilicity of the discussed compounds by HPLC with subsequent calculation of the capacity factor k and

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log k was described in Imramovsky et al. (2011b). Log P values (i.e. the logarithm of the partition coefficient for octanol/water), molar volume (MV (cm3)) and surface tension (ST (dyne/cm)) were predicted by means of ACD/Percepta (ACD/Labs, ver. 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012). All the results are shown in Table 1.

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2.3. Culture media and antibiotics

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All media were prepared from dehydrated powders (Oxoid, Basingstoke, UK) according to the manufacturer’s instructions. Ampicillin (AMP), ciprofloxacin (CPX) and vancomycin (VAN) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Stock solutions were prepared by dissolving the antibiotic in sterile deionized water (CLSI, 2012).

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2.4. Bacterial strains

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The in vitro antibacterial activity of the synthesized compounds was evaluated against representatives of multidrug-resistant bacteria, three clinical isolates of MRSA: clinical isolate of animal origin MRSA 63718 (Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic) carrying mecA gene; MRSA SA 630 (Nubel et al., 2010) and MRSA SA 3202 (Nubel et al., 2010) (National Institute of Public Health, Prague, Czech Republic), both of human origin. These three clinical isolates were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 lg/mL (VA2-MRSA) within the susceptible range for MRSA 63718), methicillin-resistant S. aureus (VS-MRSA) (Nubel et al., 2010). For the MICs of vancomycin see Table 1. S. aureus ATCC 29213 (vancomycin-susceptible, methicillin-susceptible S. aureus, VS-MSSA), obtained from the American Type Culture Collection, was used as the reference and quality control strain. The bacteria were stored at 80 °C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments.

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2.5. Determination of minimum inhibitory concentrations (MICs)

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The MICs (i.e. the lowest concentrations of the antimicrobials that will inhibit the visible growth of a microorganism) were determined by the broth dilution micro-method according to the CLSI, 2012 guidelines in sterile fresh Mueller–Hinton broth (MHB) with some modifications. Bacterial inoculum in sterile phosphate buffered saline (pH 7.2–7.3 to simulate physiological pH) was prepared to match 0.5 McFarland scale and then diluted 1:20 in MHB. The evaluated compounds were dissolved in dimethylsulfoxide (DMSO) (Sigma–Aldrich, Germany) to the concentration of 10 000 lg/mL, and twofold serial dilution of the stock solution was performed in a 96-wells plate with bacterial suspension described above to obtain the final concentrations of the tested compounds ranging from 256 to 0.008 lg/mL. The final concentration of bacterial inoculum was approximately 7.5  106 colony forming units (CFU)/mL in the final volume of 100 lL. The concentration of DMSO in the MHB did not exceed 2.5% of the total solution composition. AMP, CPX and VAN were used as the reference antibacterial drugs. Drug-free controls, sterility controls and controls consisting of MHB with DMSO were included. The determination of results was performed visually after 24 h of static incubation in the darkness at 37 °C in an aerobic atmosphere. The MIC was defined as the lowest concentration of the compound at which no visible bacterial growth was observed (Sato et al., 2004). To ensure reproducibility, each MIC assay was performed in at least triplicate on separate occasions.

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Table 1 Chemical structures of tested compounds and in vitro MIC and MBC (lg/mL) values (bactericidal effect of individual compounds against particular strains is marked in bold).

R1

R2

log k

log P

MV (cm3)

ST (dyne/ cm)

MIC (lg/mL (lmol/L)) MRSA 63718

MRSA SA 630

MRSA SA 3202

S.a.

MBC (lg/mL (lmol/L)) MRSA 63718

MRSA SA 630

MRSA SA 3202

S.a.

32 (80.96) 16 (39.09) 8 (18.29) 8 (16.69)

0.5 (1.27) 2 (4.89)

64 (161.91) 32 (78.18)

4 (9.15) 2 (4.17)

32 (73.17) 32 (66.74)

64 (161.91) 8 (19.55) 4 (9.15) 8 (16.69) 4 (11.33) 2 (5.25)

1

3-Cl

C5H11

0.773

5.54

306.93

49.28

0.5 (1.27)

0.5 (1.27)

0.25 (0.63)

0.25 (0.63)

2

3-Cl

C6H13

0.776

6.01

323.10

48.37

0.5 (1.22)

0.25 (0.61)

0.5 (1.22)

0.5 (1.22)

3 4

3-Cl 3-Cl

C8H17 C11H23

0.783 0.790

7.20 9.07

355.39 403.76

46.84 45.05

0.5 (1.14) 1 (2.09)

0.5 (1.14) 1 (2.09)

0.5 (1.14) 1 (2.09)

0.25 (0.57) 0.5 (1.04)

5

4-Cl

C2H5

0.770

2.72

258.37

52.83

0.25 (0.71)

4 (11.33)

C4H9

0.776

4.38

290.75

50.31

4 (10.49)

4 (10.49)

8 (20.98)

7

4-Cl

C5H11

0.784

5.45

306.93

49.28

0.063 (0.17) 0.5 (1.26)

4 (10.12)

4 (10.12)

4 (10.12)

8 9 10 11

4-Cl 4-Cl 4-Cl 4-Cl

C6H13 C7H15 C8H17 C9H19

0.786 0.787 0.790 0.792

5.78 6.51 6.88 7.73

323.10 339.25 355.39 371.53

48.37 47.56 46.84 46.18

0.5 (1.22) 0.5 (1.18) 1 (2.29) 0.5 (1.11)

0.5 (1.22) 0.5 (1.18) 1 (2.29) 1 (2.22)

0.063 (0.18) 0.032 (0.08) 0.125 (0.32) 0.25 (0.61) 0.5 (1.18) 0.25 (0.57) 0.5 (1.11)

2 (5.66)

4-Cl

0.125 (0.35) 0.25 (0.66)

2 (5.66)

6

0.125 (0.35) 0.125 (0.33) 0.125 (0.32) 0.25 (0.61) 0.5 (1.18) 1 (2.29) 1 (2.22)

8 4 4 4

4 2 2 2

8 8 4 8

12 13

4-Cl 4-Cl

C10H21 C11H23

0.794 0.795

8.17 8.61

387.65 403.76

45.59 45.05

2 (4.30) 1 (2.09)

4 (8.59) 1 (2.09)

1 (2.15) 0.5 (1.04)

8 (17.19) 8 (16.69)

2 (4.30) 2 (4.17)

8 (17.19) 8 (16.69)

14

C2H5

1.023

4.56

269.24

53.91

4 (10.32)

16 (41.27)

1.046

5.38

301.61

51.33

4 (9.62)

4 (9.62)

8 (19.24)

C5H11

1.060

6.31

317.77

50.28

4 (9.31)

2 (4.65)

8 (18.62)

C7H15

1.067

7.36

350.07

48.5

8 (17.48)

1 (2.18)

8 (17.48)

C8H17

1.085

7.94

366.20

47.74

4 (8.48)

2 (4.24)

8 (16.96)

8 (18.62) 8 (17.48) 4 (8.48)

C9H19

1.088

8.71

382.33

47.06

4 (8.23)

4 (8.23)

16 (32.93)

2 (4.12)

C10H21

1.089

9.24

398.44

46.45

4 (8.00)

4 (8.00)

8 (16.00)

4 (8.00)

C11H23

1.090

9.58

414.55

45.88

60.008 (60.02) 0.016 (0.04) 0.032 (0.07) 0.063 (0.14) 0.125 (0.26) 0.125 (0.26) 0.125 (0.25) 0.063 (0.12) 0.5 (1.43)

2 (5.16)

C4H9

4 (7.78)

4 (7.78)

–

–

V

–

–

0.25 (0.49) >16 (>45.79) >16 (>48.29) 1 (0.69)

4 (7.78)

C

60.008 (60.02) 60.008 (60.02) 0.063 (0.15) 0.063 (0.14) 0.125 (0.26) 0.125 (0.26) 0.063 (0.13) 0.063 (0.12) >16 (>45.79) >16 (>48.29) 1 (0.69)

0.125 (0.32) 0.032 (0.08) 1 (2.33)

A

3,4Cl 3,4Cl 3,4Cl 3,4Cl 3,4Cl 3,4Cl 3,4Cl 3,4Cl –

4 (8.59) 0.063 (0.13) 60.008 (60.02) 60.008 (60.02) 0.032 (0.07) 0.063 (0.14) 0.125 (0.26) 0.125 (0.26) 0.063 (0.13) 0.125 (0.24) >16 (>45.79) >16 (>48.29) 2 (1.38)

>16 (>45.79) >16 (>48.29) 1 (0.69)

0.5 (1.43) 0.5 (1.51) 1 (0.69)

15 16 17 18 19 20 21

–

0.5 (1.26)

0.25 (0.55) 0.5 (1.06) 2 (4.12) 1 (2.00) 1 (1.95) >16 (>45.79) >16 (>48.29) 1 (0.69)

0.5 (1.51) 1 (0.69)

(19.55) (9.45) (9.15) (8.86)

>16 (>45.79) >16 (>48.29) 2 (1.38)

(9.77) (4.72) (4.57) (4.43)

(19.55) (18.90) (9.15) (17.72)

4 (10.12) 4 (9.77) 4 (9.45) 4 (9.15) 0.5 (1.11) 2 (4.30) 0.5 (1.04) 4 (10.32) 4 (9.62)

A = ampicillin, C = ciprofloxacin, V = vancomycin; MIC breakpoints for S. aureus ATCC 29213 (lg/mL): A > 2, C > 1, V > 2 (CLSI, 2006, 2014).

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2.6. Determination of minimum bactericidal concentrations (MBCs)

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The MBCs (i.e. the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 lL) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller–Hinton agar (MHA) plates. The plates were incubated aerobically at 37 °C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced P99.9% killing after 24 h of incubation as compared to the colony count of the starting inoculum (Schwalbe et al., 2007). To ensure reproducibility, each MBC assay was performed in at least triplicate on separate occasions.

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2.7. Time-kill assays

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Time-kill assays were performed by the broth macrodilution method according to the previously described methodology

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(Schwalbe et al., 2007) with some modifications. Briefly, flasks containing sterile fresh MHB with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5  106 CFU/mL (actual inoculum concentrations ranged from 2.6  105 to 2.2  106 CFU/mL) and a final concentration of the antibiotic equal to 1, 2 and 4 MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8 and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 lL) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6–60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 °C for 24 –48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate

Please cite this article in press as: Zadrazilova, I., et al. Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.009

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decylcarbamate (12) expressed strong anti-MRSA activity as well with the MICs ranging from 1 to 4 lg/mL for all the tested strains. However, the MICs of compound 12 for all tested strains were identical (S. aureus ATCC 29213) or within 1 (MRSA 63718, MRSA SA 630) or 2 (MRSA SA 3202) dilutions to those of vancomycin. The susceptibility of the S. aureus reference strain and the clinical isolates of MRSA to all the tested compounds was comparable. As for the MICs, the MBC values did not exceed the highest drug concentration tested. The MBCs of all the tested compounds ranged from 0.5 to 64 lg/mL. In most cases, there were comparable MBC values for the S. aureus reference strain and the clinical isolates of MRSA.

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occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log10 CFU per milliliter versus time (over 24 h), and the change in bacterial concentration was determined. The results were analyzed by evaluating the numbers of strains that yielded D(log10 CFU/mL) values of 1 (corresponding to 90% killing), 2 (99% killing), and 3 (99.9% killing) at 4, 6, 8 and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of 99.9% (P3 log10) of the total count of CFU/mL in the original inoculum.

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2.8. In vitro antiproliferative assay

3.2. Type of antibacterial effect

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245

Bactericidal activity was defined as a ratio of MBC to MIC of 64 (Cha et al., 2011). Comparison of the MIC and MBC values of the discussed compounds for each isolate indicates that the activity of the derivatives of SAL carbamates was predominantly bacteriostatic. Compounds 10–13 and 21 possessed bactericidal activity against at least 2 tested strains. Interestingly, compound 12, the least active substance from all the series, seems to be an exception as it was the only compound, the activity of which was bactericidal against all 4 strains. In Table 1 bactericidal activity is shown in bold.

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3.3. Time-kill assays

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3. Results

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3.1. Antibacterial activity

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The studied compounds were tested for in vitro antibacterial activity against representatives of multidrug-resistant bacteria, three clinical isolates of MRSA and S. aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. The antibacterial activities of twenty-one SAL N-alkylcarbamates, expressed as MICs and MBCs (all values are expressed both in lg/mL and lmol/L), are shown in Table 1 along with the activity of the reference antibacterial drugs, ampicillin, ciprofloxacin and vancomycin. Quality control limits for these drugs and the reference strain S. aureus ATCC 29213 are 0.5–2.0 lg/mL (ampicillin), 0.12–0.5 lg/mL (ciprofloxacin) and 0.5–2.0 lg/mL (vancomycin) (CLSI, 2006, 2014). As indicated in Table 1, the MICs of these compounds were within the acceptable limits in this study. DMSO as the solvent did not affect the bacterial growth. All the tested compounds (except compound 12) showed excellent anti-Staphylococcus efficiency with MIC values ranging from 60.008 to 1 lg/mL against all four strains. The MICs of all these substances were 16–4000 lower than those of the reference antibacterial drugs; in the case of vancomycin the MICs of all the compounds were equal to those of vancomycin or up to 250 lower. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl butylcarbamate (15) and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl ethylcarbamate (14) were the most potent antimicrobials against all the tested strains with the MICs 6 0.008 lg/mL for the two MRSA strains. 4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl

Compound 12 was tested in time-kill studies at 1, 2 and 4 MIC against all the MRSA isolates and the S. aureus reference strain. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log10 CFU/mL in viable cell count at the different times after incubation. A summary of these data is presented in Table 2. No bactericidal activity (i.e. P3 log10 CFU/mL decrease) was observed at 4 and 6 h after incubation for any concentration tested. Compound 12 was bactericidal only when tested at 4 MIC as follows: at 8 h against one (MRSA 63718 with the highest vancomycin MIC value among all tested strains) of the four strains and at 24 h against two (MRSA SA 630 and MRSA SA 3202) of the four strains. The findings of time-kill studies for each of the 4 staphylococci strains during exposure to compound 12 are summarized in Table 3. Bactericidal activity (i.e. P3 log10 CFU/mL decrease) is expressed in bold. For the clinical isolate of MRSA 63718, compound 12 displayed concentration-dependent antibacterial effect. The highest efficiency was reached at 4 MIC with a reduction in bacterial count ranging from 2.77 to 3.55 log10 CFU/mL. A reduction consistent with bacteriostatic effect (0.71–2.42 log10 CFU/mL) was observed at 2 MIC over time for this isolate. For the remaining strains, the clinical isolates of MRSA SA 630 and MRSA SA 3202, concentration-independent bactericidal effect was recorded. Incubation time was the predictive factor influencing killing activity, as bactericidal effect was observed only at 24 h after incubation with a reduction in bacterial count of 4.76 and 5.94 log10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 24 h after incubation at 4 MIC. No bactericidal effect was observed for the S. aureus reference strain; compound 12 demonstrated a pattern of bacteriostatic activity against this strain with a reduction in bacterial count of 0.78 log10 CFU/mL at 24 h at 4 MIC. In other cases, a slight increase in bacterial counts (i.e. overgrowth) in comparison with the starting inoculum was observed with the values ranging from 0.05 to 1.08 log10 CFU/mL for this reference strain. For illustrative purpose, representative time-kill curves for compound 12 were plotted for all the clinical isolates of MRSA and the S. aureus reference strain. Results are shown in Fig. 1.

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Human monocytic leukemia THP-1 cells were used for in vitro antiproliferative assay. Cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2% L-glutamine, 1% penicillin and streptomycin at 37 °C with 5% CO2. Cells were passaged at approximately one week intervals. Antiproliferative activity of the compounds was determined using a WST-1 assay kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The tested compounds were dissolved in DMSO and added in five increasing concentrations (0.37, 1.1, 3.3, 10 and 20 lM) to the cell suspension in the culture RPMI 1640 medium. The maximum concentration of DMSO in the assays never exceeded 0.1%. Subsequently, the cells were incubated for 24 h at 37 °C with 5% CO2. For WST-1 assays, cells were seeded into 96-well plates (5  104 cells/well in 100 lL culture medium) in triplicate in serum-free RPMI 1640 medium, and measurements were taken 24 h after the treatment with the compounds. The median inhibition concentration values, IC50, were deduced through the production of a dose–response curve. All data were evaluated using GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA).

232 233 234 235 236 237 238 239 240 241 242

246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264

269 270 271 272 273

274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292

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I. Zadrazilova et al. / European Journal of Pharmaceutical Sciences xxx (2015) xxx–xxx Table 2 Extent of bacterial killing exerted by compound 12 over time against MRSA 63718, MRSA SA 630, MRSA SA 3202, S. aureus ATCC 29213. Drug and concentration (multiplicity of MIC)

No. of strains showing following log10 CFU/mL decreasea at designated incubation time 4h

Comp. 12 4 MIC 2 MIC 1 MIC

6h

8h

24 h

1

2

3

1

2

3

1

2

3

1

2

3

1 0 0

1 0 0

0 0 0

1 1 0

1 0 0

0 0 0

1 1 0

1 1 0

1 0 0

3 2 0

3 1 0

2 0 0

CFU = colony-forming units. a D(log10 CFU/mL) values of 1, 2, and 3 log10 CFU/mL correspond to 90% (bacteriostatic), 99% (bacteriostatic), and 99.9% (bactericidal) of killing, respectively.

Table 3 Change in viable counts (log10 CFU/mL) of MRSA and S. aureus strains following incubation for 24 h with compound 12 (bactericidal effect is expressed in bold). Strain

MRSA 63718

MRSA SA 630

MRSA SA 3202

S.a.

Compound 12 MIC/MBC

Log10 difference in CFU/mL from inoculum Conc.

4h

4/8

1 MIC 2 MIC 4 MIC 1 MIC 2 MIC 4 MIC 1 MIC 2 MIC 4 MIC 1 MIC 2 MIC 4 MIC

0.01a

2/2

4/8

1/2

6h

8h

24 h

0.00

0.22

0.02

0.71

1.97

2.42

0.78

2.90

2.88

3.55b

2.77

0.02

0.04

0.07

0.04

0.13

0.20

0.33

1.31

0.23

0.51

0.67

4.76b

0.07

0.02

0.01

0.07

0.02

0.00

0.07

2.01

0.07

0.02

0.07

5.94b

0.99

0.93

0.92

1.08

0.37

0.40

0.44

0.31

0.11

0.05

0.19

0.78

CFU = colony-forming units; Conc., concentration (multiplicity of MIC). a <3 log10 reduction in CFU implies bacteriostatic effect. b P3 log10 reduction in CFU implies bactericidal effect.

355

3.4. In vitro antiproliferative activity

356

367

The preliminary in vitro screening of the antiproliferative activity of compound 12 (in comparison with vancomycin) was performed using WST-1 assay kit (ROCHE, 2011) and the human monocytic leukemia THP-1 cell line by means of the method described recently (Kos et al., 2015). The antiproliferative activity was evaluated as the IC50 value (compound concentration causing 50% inhibition of cell population proliferation). The treatment with 20 lmol/L of vancomycin did not lead to significant antiproliferative effect on THP-1 cells. Compound 12 demonstrated antiproliferative activity IC50 = 4.8 ± 0.4 lmol/L against THP-1. For comparison, e.g., IC50 of camptothecin, assessed in this line formerly, was 0.16 ± 0.07 lmol/L, which is much lower value.

368

4. Discussion

369

Based on the obtained results, twenty-one tested SAL N-alkylcarbamates seem to be promising candidates of antibacterial agents with highly potent anti-MRSA activity. All the discussed compounds exhibited MIC values ranging from 60.008 to 4 lg/mL and MBC values ranging from 0.5 to 64 lg/mL. The activity of the

357 358 359 360 361 362 363 364 365 366

370 371 372 373

discussed compounds against all the tested strains was higher than that of the reference antibacterial drugs used in this study, ampicillin and ciprofloxacin. While the reference strain S. aureus ATCC 29213 was susceptible to AMP and CPX according to CLSI (2006, 2014) MIC breakpoints, these antibacterial drugs were inactive against MRSA (MIC > 16 lg/mL for both antibacterial drugs). Compared with vancomycin, the treatment standard for serious MRSA infections (Clemens et al., 2011), the MICs of all the compounds were similar to those of vancomycin or up to 250 lower. Since the MRSA strains tested in this study had higher MIC values for vancomycin, and increased vancomycin MIC levels within the susceptible range were previously reported to be associated with treatment failure (Sakoulas et al., 2004), SAL N-alkylcarbamates could present alternative antibacterial agents against these strains superior even to vancomycin. A study evaluating the in vitro antibacterial activity of ceftaroline and various comparable anti-MRSA clinically used drugs against clinical isolates of MRSA and MSSA by the broth microdilution method was published recently (Sader et al., 2013). The MIC values of these agents are listed in Table 4. It can be stated that the anti-staphylococcal activity of all the SAL N-alkylcarbamates is comparable to or, in the case of compounds 14 and 15, higher than that of antibacterial agents used in clinical practice, see Table 4. This fact is a very significant finding which makes the tested compounds promising candidates of anti-MRSA agents for the future. High antimycobacterial activity of SAL N-alkylcarbamates against multidrug-resistant tuberculosis strains was reported previously (Ferriz et al., 2010). To allow a meaningful comparison, the compounds were initially tested at molar equivalent concentrations. Comparing the MICs of SAL N-alkylcarbamates for M. tuberculosis (MICs from 0.5 to 4 lmol/L) and MRSA MICs calculated as molar concentrations (data not shown), there is even superior antibacterial activity against MRSA (MICs from 60.02 to 8 lmol/L). However, based on the fact that the susceptibility of the S. aureus reference strain, the clinical isolates of MRSA and the susceptible and multidrug-resistant tuberculosis strains is similar, it can be assumed that the mechanism of action of SAL N-alkylcarbamates affects a common element of all these microorganisms. Concerning antibacterial effect, generally it is not important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient’s immune system is capable to cope with the infection then (Lullmann et al., 2004). However, bactericidal therapy could produce a better treatment result by rapid reduction of bacterial load. Resistant mutants cannot arise in the case of complete sterilisation (Gould, 2007). Moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, AIDS patients, etc.) bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis or osteomyelitis in recent years, it will be necessary to achieve bacterial killing and broaden

Please cite this article in press as: Zadrazilova, I., et al. Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.009

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8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

B 9.0 log (CFU/mL)

log (CFU/mL)

A 9.0

Bactericidal level

0

6

4

8

24

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

Bactericidal level

0

4

Time [h] 1× MIC

2× MIC

4× MIC

Growth control

DMSO control

C 9.0

1× MIC

8

24

2× MIC

4× MIC

Growth control

DMSO control

D 9.0

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

log (CFU/mL)

log (CFU/mL)

6 Time [h]

Bactericidal level

0

4

6

8

24

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0

Bactericidal level

0

4

1× MIC

2× MIC

4× MIC

6

8

24

Growth control

DMSO control

Time [h]

Time [h] Growth control

DMSO control

1× MIC

2× MIC

4× MIC

Fig. 1. In vitro time-kill kinetics performed in fresh MHB at 1, 2 and 4 MIC of compound 12 against MRSA 63718 (A), MRSA SA 630 (B), MRSA SA 3202 (C) and S. aureus ATCC 29213 (D). (CFU = colony-forming units).

Table 4 Activity of ceftaroline and other antimicrobial agents clinically used in the treatment of serious MRSA infections in comparison with the most active compounds from the present study. Antibacterial agent

Ceftaroline Vancomycin Tigecycline Daptomycin Linezolid Comp. 14a Comp. 15a

MIC (lg/mL (lmol/L)) MRSA

MSSA

2 (2.69) 1 (0.69) 0.25 (0.43) 0.5 (0.31) 2 (5.93) 60.008 (60.02) 60.008 (60.02)

1 (1.34) 1 (0.69) 0.25 (0.43) 0.5 (0.31) 2 (5.93) 60.008 (60.02) 0.016 (0.04)

Refs.

Sader et al. (2013) Sader et al. (2013) Sader et al. (2013) Sader et al. (2013) Sader et al. (2013) Present study Present study

a

The most active compounds from the present study; MRSA = methicillin-resistant S. aureus; MSSA = methicillin-susceptible S. aureus.

427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445

the spectrum of antimicrobial agents with bactericidal active compounds (Schwalbe et al., 2007). The potential bactericidal activity of SAL N-alkylcarbamates was assessed using MBC assay (bactericidal activity is defined as a ratio of MBC to MIC of 64 (Cha et al., 2011)) and time-kill assay (bactericidal activity is defined as a reduction of 99.9% (P3 log10) of the total count of CFU/mL in the original inoculum (Schwalbe et al., 2007)). According to the MBC assay, the activity of the derivatives of SAL carbamates was predominantly bacteriostatic. However, the least active substance from all series, 4-chloro-2-(4-chlorophenyl carbamoyl)phenyl decylcarbamate (12, R1 = 3-Cl, R2 = C10H21), exhibited bactericidal effect against all 4 strains. This compound was chosen for subsequent time-kill analysis to study bactericidal kinetics over 24 h. By time-kill assay, concentration-dependent bactericidal effect was recorded for clinical isolate of MRSA 63718 with the highest efficiency at 4 MIC. For the remaining clinical isolates, MRSA SA 630 and MRSA SA 3202, antibacterial effect was dependent on time after incubation; bactericidal activity was observed only at 24 h at

4 MIC with a reduction in bacterial count of 4.76 and 5.94 log10 CFU/mL, respectively. Compound 12 demonstrated a pattern of bacteriostatic activity against S. aureus reference strain ATCC 29213 at 24 h at 4 MIC with slight overgrowth in comparison with the starting inoculum in other cases; no bactericidal effect was observed. The antibacterial effect of DMSO (Basch and Gadebusch, 1968) was excluded in this assay as time-kill curves of the solvent were identical or very similar to those of growth control. There is a discrepancy between the bactericidal results of the MBC assay and the results of the time-kill assay. Although time-kill assays are more labor intensive and time consuming than MBC assays, they are recognized to provide a greater degree of characterization of the cell eradication potential of antibacterial agents (NCCLS, 1999). Moreover, this difference could be caused by comparing microtiter (MBC assay) to macrobroth (time-kill assay) dilutions (Lin et al., 2012). It is of note that the clinical isolates of MRSA were effectively killed by compound 12 while the reference strain remained unaffected, although all these strains had similar MICs for compound 12. All of the compounds selected for testing exhibited very promising anti-MRSA activity in the range of the concentrations tested; however, the activity varied depending on the chemical structure and physicochemical properties. It can be stated that the anti-staphylococci activity of SAL N-alkylcarbamates is affected by both variable structural components R1 (substitution of the aniline part) and R2 (the length of the alkyl chain). Nevertheless differences can be observed within structure–activity relationships of MIC values of the discussed compounds and their bactericidal effect. Within investigation of structure–activity relationships (SAR) various parameters describing physicochemical properties are used. In the current investigation the logarithm of experimentally determined capacity factor (log k) and log P values were used as lipophilicity parameters. The results obtained with all the compounds show that the experimentally-determined lipophilicity (log k) values of

Please cite this article in press as: Zadrazilova, I., et al. Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.009

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545

logð1=MICÞ ¼ 53:708ð9:767Þ  64:099ð12:39Þ log k

504

the discussed compounds are in accordance with the calculated values log P of 3-Cl (compounds 1–4, correlation coefficient r = 0.9929), 4-Cl (compounds 5–13, correlation coefficient r = 0.9873) and 3,4-Cl (compounds 14–21, correlation coefficient r = 0.9694) substituted series. Due to the length of the alkyl chain as R2 substituent the discussed compounds can be also considered as potential non-ionic surfactants, i.e. their biological effect is also influenced by the length of this hydrocarbon tail. This parameter is reflected in bulkiness/molar volume (MV (cm3)) of the compounds. The surface tension (ST (dyne/cm)) of the compound is also connected with the length of the hydrocarbon chain. The calculated values of surface tension increase (i.e. surface activity decreases) with decreasing bulkiness expressed as molar volume (reflecting a decrease in the length of the hydrocarbon chain). The log P values, molar volume and surface tension of all the compounds (see Table 1) were calculated by ACD/Percepta. Fig. 2 shows the dependence of the antibacterial activity against S. aureus ATCC 29213 expressed as log(1/MIC (mol/L)) of all the tested compounds on lipophilicity expressed as log k. The dependences of the antibacterial activity on bulkiness and on surface tension are not illustrated, because they are very similar to that of log k. It can be concluded that lipophilicity is the most important for high anti-Staphylococcus activity. High lipophilicity is caused by disubstitution of chlorine in C0ð3;4Þ of the anilide ring.

505

Monosubstitution of the C0ð3Þ or C0ð4Þ position of the anilide ring

logð1=MICÞ ¼ 2:672ð1:275Þ þ 0:136ð0:026Þ ST

506

caused an activity decrease in comparison with disubstituted derivatives, whereas the activity of 4-Cl substituted compounds seems to be slightly higher than that of 3-Cl derivatives (e.g., compare 5/14, 1/7/16), see Table 1. The activity within the series of 3-Cl substituted compounds seems to be practically constant independently on any physicochemical parameter or very slightly decreases with increasing lipophilicity, molar volume or surface activity (see Table 1), nevertheless this fact can be connected with the small number of investigated compounds (mainly missing compounds with shorter alkyl chains C2H5 and C4H9 for which lower activity could be assumed), therefore SAR is not discussed for this series. For C0ð4Þ substituted compounds a biphasic course

481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535

536

of the dependence of the activity on all the physicochemical parameters can be found. Within the series of investigated compounds the activity increases to the optimum log k = 0.776, the optimum bulkiness MV  290 cm3 and surface tension ST  50 dyne/cm (compound 6, R2 = C4H9) and decreases linearly with subsequently increasing lipophilicity (prolongation of the hydrocarbon chain), see Fig. 2, and increasing values of surface tension. The activity within C0ð3;4Þ disubstituted series decreases with an increase of lipophilicity and molar volume and increases with increasing surface tension (i.e. with a decrease of surface activity). The biphasic course of the dependences log(1/MIC) on log k, MV or ST was reflected also in the results of statistical analysis of corresponding regression equations, by which they were evaluated for all nine compounds 5–13 of 4-Cl series (Eqs. (1)–(3)) in comparison with those related to eight compounds 6–13 showing a linear increase with increasing log k and MV or decreasing ST (Eqs. (4)–(6)). It is evident that the results of statistical analysis for Eqs. (4)–(6) are better.

logð1=MICÞ ¼ 39:959ð8:027Þ  46:693ð10:212Þ log k 538

r ¼ 0:866; s ¼ 0:241; F ¼ 20:90; n ¼ 9

ð1Þ

539

logð1=MICÞ ¼ 59:492ð6:655Þ  79:737ð0:002Þ MV 541

r ¼ 0:839; s ¼ 0:262; F ¼ 16:63; n ¼ 9

ð2Þ

542

logð1=MICÞ ¼ 3:993ð1:796Þ þ 0:151ð0:037Þ ST 544

r ¼ 0:837; s ¼ 0:264; F ¼ 16:32; n ¼ 9

ð3Þ

r ¼ 0:904; s ¼ 0:204; F ¼ 26:74; n ¼ 8

ð4Þ

547 548

logð1=MICÞ ¼ 63:743ð0:907Þ  91:458ð0:003Þ MV r ¼ 0:821; s ¼ 0:272; F ¼ 12:41; n ¼ 8

ð5Þ

550 551

logð1=MICÞ ¼ 6:622ð2:347Þ þ 0:207ð0:049Þ ST r ¼ 0:863; s ¼ 0:240; F ¼ 17:52; n ¼ 8

ð6Þ

553

The above mentioned relationships are also supported by the correlations between log(1/MIC) and log k, MV, ST that could be expressed for all eight 3,4-Cl disubstituted compounds 14–21 by the following Eqs. (7)–(9), or by regression Eqs. (10)–(12) for seven 3,4-Cl disubstituted compounds 14–20. These equations reflect a linear increase with increasing log k and MV or decreasing ST (the results of statistical analysis for Eqs. (10)–(12) are better).

554 555 556 557 558 559 560

561

logð1=MICÞ ¼ 20:974ð2:469Þ  15:092ð2:310Þ log k r ¼ 0:942; s ¼ 0:413; F ¼ 47:43; n ¼ 8

ð7Þ

563 564

logð1=MICÞ ¼ 6:447ð0:603Þ  0:007ð0:002Þ MV r ¼ 0:861; s ¼ 0:227; F ¼ 17:14; n ¼ 8

ð8Þ

566 567

r ¼ 0:905; s ¼ 0:189; F ¼ 27:24; n ¼ 8

ð9Þ

569 570

logð1=MICÞ ¼ 22:991ð1:267Þ  17:841ð1:189Þ log k r ¼ 0:989; s ¼ 0:072; F ¼ 225:24; n ¼ 7

ð10Þ

572 573

logð1=MICÞ ¼ 7:168ð0:325Þ  0:009ð0:009Þ MV r ¼ 0:975; s ¼ 0:107; F ¼ 97:69; n ¼ 7

ð11Þ

575 576

logð1=MICÞ ¼ 4:141ð0:642Þ þ 0:165ð0:013Þ ST r ¼ 0:985; s ¼ 0:189; F ¼ 160:43:24; n ¼ 7

ð12Þ

578

Dependences of the antibacterial activities of the discussed compounds against the three MRSA strains can be considered as similar. In general it can be stated that the activity of the compounds within individual series decreases with increasing lipophilicity, bulkiness and surface activity (i.e. with a decrease of surface tension), see Table 1, similarly as illustrated in Fig. 2, therefore these dependences are not illustrated. Within individual series the most potent derivatives are those with the shortest alkyl chain (C5 among 3-Cl and C2/C4 among 4-Cl, 3,4-Cl substituted compounds). The above mentioned observations underline the importance of the suitable lipophilicity of compounds shown by the chlorine substituents in the molecule together with a short alkyl chain as R2 substituent. There is a discrepancy between the optimal lengths of the alkyl chain. The most potent anti-MRSA/SA agents are substituted by C4 (compound 15) or C2 (compound 14) alkyl chain contrary to the most potent antimycobacterial agents where C6, C5 and C7 alkyl chains as R2 substituents are preferable (Ferriz et al., 2010). This fact could be connected with structure/composition differences of the mycobacterial and the gram-positive bacterial cell walls. The mycobacterial cell wall is composed of various highly lipophilic mycolic acids, and thus more lipophilic compounds are needed to permeate through this wall to a mycobacterial cell (Chatterjee, 1997; Dedieu et al., 2013; Dolezal et al., 2012; Imramovsky et al., 2007; Minnikin et al., 2002). As mentioned above the discussed compounds can act as non-ionic surfactants, i.e. their antimycobacterial effect is also connected with the increasing surface activity (i.e. a longer tail as R2

579

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log (1/MIC [mol/L])

5.0 4.5 4.0 3.5 3.0 2.5 2.0 0.76

0.77

0.78

0.79

0.80

1.02

1.04

1.06

1.08

1.10

log k 3-Cl

4-Cl

3,4-Cl

Fig. 2. Dependence of antibacterial activity against S. aureus ATCC 29213 expressed as log(1/MIC (mol/L)) of tested compounds on lipophilicity expressed as log k.

609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645

646

on lipophilicity (expressed as log k) and surface tension are illustrated in Fig. 3. It is evident that the activity increases almost linearly with increasing lipophilicity (i.e. length of the chain) and surface activity within the series of 4-Cl substituted compounds. The above mentioned statements are supported by the correlations between log(1/MBC) of 4-Cl substituted compounds 9–13 and lipophilicity (log k, log P), molar volume (MV) and surface tension (ST) that could be expressed by the following regression Eqs. (13)–(16):

logð1=MBCÞ ¼ 0:2333ð0:7352Þ þ 6:4689ð0:9288Þ log k 648

r ¼ 0:970; s ¼ 0:0058; F ¼ 48:51; n ¼ 5

ð13Þ

649

logð1=MBCÞ ¼ 5:1782ð0:0212Þ þ 0:0232ð0:0028Þ log P 651

r ¼ 0:9790; s ¼ 0:0049; F ¼ 69:25; n ¼ 5

ð14Þ

652

logð1=MBCÞ ¼ 5:0545ð0:02312Þ þ 0:0008ð0:0006Þ MV 654

r ¼ 0:9912; s ¼ 0:0032; F ¼ 168:44; n ¼ 5

ð15Þ

5.39

log (1/MBC [mol/L])

608

substituent is preferable). These compounds can easier disturb lipophilic mycobacterial cell wall and thus penetrate through it to a cell (Tengler et al., 2013). On the contrary, too high lipophilicity associated, inter alia, with a long-alkyl chain (i.e. limited water solubility) prevents penetration and permeation of compounds through the peptidoglycan layer of the cell wall of gram-positive bacteria (Abou-Rahma et al., 2009; He et al., 2003; Lode, 2001, 2008; Mori et al., 2001; Sriram et al., 2005; Takacs-Novak et al., 1992; Wube et al., 2011). Thus the lipophilicity of chloro-salicylanilide N-butyl-/ethyl-carbamates (compounds 6/5, 15/14) seems to be favorable for good permeation of the compounds through the gram-positive bacterial cell walls. As the mechanism of action of SAL in S. aureus strains is manifold, molecules with a small flexible chain, as competitive antagonists with high affinity, can more easily target various binding sites (e.g., transglycosylases and other enzymatic systems) concerned with their bacteriostatic effect (Cheng et al., 2010; Hilliard et al., 1999). Different dependences were observed for bactericidal activity, see bolded values in Table 1. Based on these data, SAR can be described, in spite of relatively limited number of compounds that demonstrated bactericidal effect. The bactericidal activity against all four tested Staphylococcus strains was shown especially by compounds with medium or moderate activity (higher MIC values). Based on the structure of the compounds with bactericidal effect it can be stated that this type of activity is especially connected with chlorine substitution of the anilide ring at position 4 and more likely with a longer alkyl chain (C8–C11) as R2 substituent (see compounds 10–13). It is not possible to analyse SAR for 3-Cl and 3,4-Cl, but the dependences of the bactericidal activity against MRSA SA 630 strain expressed as log(1/MBC (mol/L)) of the C0ð4Þ substituted compounds

5.38

A r = 0.9841

5.37 5.36 5.35 5.34 5.33 5.32 0.786

0.788

0.790

0.792

0.794

0.796

log k 5.39

log (1/MBC [mol/L])

607

5.38

B

5.37 r = -0.9992

5.36 5.35 5.34 5.33 5.32 44.5

45.0

45.5

46.0

46.5

47.0

47.5

48.0

ST [dyne/cm] Fig. 3. Dependence of antibacterial activity against methicillin-resistant S. aureus SA 630 strain expressed as log(1/MBC (mol/L)) of 4-Cl substituted compounds on lipophilicity expressed as log k (A) and on surface tension (ST (dyne/cm)) (B).

655

logð1=MBCÞ ¼ 6:3057ð0:0944Þ  0:0206ð0:0020Þ ST r ¼ 0:9912; s ¼ 0:0032; F ¼ 168:44; n ¼ 5

ð16Þ

657

Based on the obtained results it is evident that the activity of studied compounds is connected with the presence of a longer alkyl chain in the molecule. The variation in the length of the long hydrocarbon tail influencing the extent of the antimicrobial activity of surfactants was published previously by Birnie et al. (2000). The decrease in biological activity at higher chain lengths is known as a cut-off effect (Balgavy and Devinsky, 1996). The effect of these compounds is based on membrane activity. The long alkyl chain that penetrates to the membrane and disturbs its architecture is necessary for the biological effect of this type of compounds. For this primary mechanism of action (the first attack of the molecule to bacteria) the rest of the molecule plays only a secondary role (only modifies lipophilicity and solubility). Generally, surfactants are mostly believed to be membrane perturbants, with hydrocarbon tail integrated with the lipid bilayer of membranes (see also

658

Please cite this article in press as: Zadrazilova, I., et al. Salicylanilide carbamates: Promising antibacterial agents with high in vitro activity against methicillin-resistant Staphylococcus aureus (MRSA). Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.06.009

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in Jampilek and Brychtova (2012), Kralova and Sersen (2012)). This integration causes a disruption in the membrane followed by, in the case of cell/bacteria, the leakage of the intracellular contents. The length of the alkyl chain of the surfactants is thought to contribute to the extent of membrane disruption, because longer chains may be incorporated into the lipid bilayers of the plasma membrane. The decreased activity of compounds with a longer alkyl chain (cut-off effect) can be associated with their limited solubility. As the alkyl chain increases, lipid solubility increases at a faster rate than that of partition coefficient change (lipid/aqueous). For these longer alkyl chains, partitioning is limited, making the concentration at the site of action insufficient to have a significant effect on the membrane of the cell wall (Birnie et al., 2000). However, the decrease of activity with the alkyl chain reaching a certain length can also be explained as follows: the terminal methyl group of a compound may localize in the area of the terminal groups of the hydrocarbon chains of lipids that form the opposite monolayer, where surfactants are not incorporated (Przestalski et al., 2000). The occurrence of such ‘‘sewing’’ of both monolayers (Sarapuk and Kubica, 1998) and the resulting structure may exhibit an increased stability and diminished potential biological activity of an amphiphile. This phenomenon is commonly called interdigitation and has been observed for some amphiphilic compounds, alcohols, acetone, drugs, anesthetics and other substances (Lobbecke and Cevc, 1995). Based on the results presented in Table 1 it can be summarized that the optimal bactericidal activity is associated with the C10 alkyl chain (compound 12). The MBC values of other compounds are similar to the MBC values of compound 12, i.e. the activity of other compounds is primarily bacteriostatic, but these compounds possess bactericidal effect at higher concentrations (i.e. higher MBC values). Compound 12 is the least potent from the discussed compounds, i.e. it shows higher MIC values and this is the reason why this compound demonstrates primary bactericidal efficacy in comparison with the rest of the compounds (following the rule for a ratio of MBC to MIC of 64 for bactericidal activity (Cha et al., 2011)). This apparent discrepancy can be explained as follows: The studied compounds possess an alkyl chain that is able to form the ‘‘input channel’’ for the ‘‘active compound’’ (i.e. some channel or pore enabling the entry into bacteria) and at suitable structure of the rest part of molecule, that is active against bacteria, destruction of bacteria can occur. This theory is supported by similarity with the effect of pore-forming toxins (e.g., Genestier et al., 2005; Boyle-Vavra and Daum, 2007; Takano et al., 2007). Since the studied compounds have a relatively labile carbamate group that can tend to hydrolyze in time and thus releasing of 5-chloro-N-(4-chlorophenyl)-2-hydroxybenzamide, it can be supposed that bactericidal effect is caused especially by the unsubstituted phenolic moiety in position C(2) of salicylanilide and the 4-chlorophenyl group (that occurs also in many pesticides, herbicides and disinfectants). It can be stated that compound 12 with higher MIC values and lower MBC values acts as a biocide, and the cell of Staphylococcus is able to exhaust this compound by efflux pumps norA up to the MIC concentration (Schindler et al., 2015). Higher concentration of the compound than the MIC value causes the bactericidal effect. Therefore it can be summarized that the long alkyl chain (C10H23) in the molecule of 12 can result in its weak bacteriostatic effect, however, after formation of pores and permeation of the active fragment of the molecule into the cell, bactericidal effect occurs by means of binding to various SAL-sensitive enzymatic systems as mentioned above. This hypothesis also corresponds with the finding of antiproliferative activity of compound 12 (IC50 = 4.8 ± 0.4 lmol/L) against THP-1. These results indicate a different mechanism of action of compound 12 in comparison with vancomycin, because compound

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12 inhibits mitochondrial dehydrogenases (the principle of WST-1 assay kit), which activity directly correlates with the number of metabolically active cells in the culture. Vancomycin inhibits cell wall synthesis in gram-positive bacteria, therefore it does not have any influence on proliferation of mammalian cells. Based on the various modes of actions of SAL derivatives mentioned in Section 1 it can be hypothesized that compound 12 interacts with enzymatic systems affecting proliferative cell functions. Generally, there is a choice of effective anti-MRSA agents with reliable in vitro activity, including vancomycin, linezolid, daptomycin or teicoplanin (Wilcox, 2011). Their side effects and general lack of oral agents, however, usually makes the therapy less comfortable for the patient. Considering the pharmacokinetic properties of SAL N-alkylcarbamates, the presence of the carbamate residue in the salicylanilide molecule seems to be very advantageous for protection of the phenolic hydroxyl (Thorberg et al., 1987), which can lower the susceptibility of the molecule toward the first-pass effect after oral administration. Based on the results of this study, SAL N-alkylcarbamates seem to be promising candidates of potential anti-MRSA agents. Moreover, compound 12 could serve as bactericidal active agent interacting with enzymatic systems affecting proliferative cell functions.

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A series of twenty-one salicylanilide N-alkylcarbamates was evaluated on their antibacterial activity against three clinical isolates of methicillin-resistant S. aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. These compounds seem to be promising candidates of antibacterial agents with high anti-MRSA activity with MIC values ranging from 60.008 to 4 lg/mL. 4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl butylcarbamate (15) and 4-chloro-2-(3,4-dichlorophenylcarbamoyl)phe nyl ethylcarbamate (14) were the most potent anti-MRSA agents. In most cases, these compounds provided reliable bacteriostatic activity against the clinically important pathogen. 4-Chloro-2-(4-c hlorophenylcarbamoyl)phenyl decylcarbamate (12) exhibited bactericidal effect at 8 h (for clinical isolate of MRSA 63718) and at 24 h (for clinical isolates of MRSA SA 630 and MRSA SA 3202) at 4 MIC. The bacteriostatic activity is influenced especially by the lipophilicity of the compounds, and the bactericidal effect of these membrane-disrupting agents shows good correlation with their surface activity. It can be concluded that the hydrophobic tail is important for causing membrane damage, suggesting ‘‘a detergent-like mode of action’’, that is combined, after permeation of the compound through bacterial cell, with binding to proliferative enzyme systems characteristic for SAL derivatives, as described. However, further investigation is necessary, e.g., to ascertain real in vivo activity and the exact mechanism of action.

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Acknowledgements

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The authors would like to thank Marie Slavikova and Jana Hofirkova for their help and excellent laboratory cooperation. Thanks go to Helena Zemlickova from the National Institute of Public Health, Prague, Czech Republic, for providing clinical isolates of MRSA. This study was financially supported by the IGA VFU Brno, Projects No. 65/2012/FVL, 52/2014/FaF and 304/2015/FaF and by the project ‘‘CEITEC – Central European Institute of Technology’’ (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund. The authors also wish to acknowledge the institutional support of the Faculty of Chemical Technology, University of Pardubice funded by the Ministry of Education, Youth and Sports of the Czech Republic.

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References

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Abou-Rahma, G.E.-D.A.A., Sarhan, H.A., Gad, G.F.M., 2009. Design, synthesis, antibacterial activity and physicochemical parameters of novel N-4piperazinyl derivatives of norfloxacin. Bioorg. Med. Chem. 17, 3879–3886. Anand, N., Sharma, S., 1997. Salicylanilides. In: Approaches to Design and Synthesis of Antiparasitic Drugs. Book series Pharmacochemistry Library 25, pp. 239–257. Balgavy, P., Devinsky, F., 1996. Cut-off effects in biological activities of surfactants. Adv. Colloid Interface Sci. 66, 23–63. Bartram, D.J., Leathwick, D.M., Taylor, M.A., Geurden, T., Maeder, S.J., 2012. The role of combination anthelmintic formulations in the sustainable control of sheep nematodes. Vet. Parasitol. 186, 151–158. Basch, H., Gadebusch, H.H., 1968. In vitro antimicrobial activity of dimethylsulfoxide. Appl. Environ. Microbiol. 16, 1953–1954. Birnie, C.R., Malamud, D., Schnaare, R.L., 2000. Antimicrobial evaluation of N-alkyl betaines and N-alkyl-N, N-dimethylamine oxides with variations in chain length. Antimicrob. Agents Chemother. 44, 2514–2517. Boyle-Vavra, S., Daum, R.S., 2007. Community-acquired methicillin-resistant Staphylococcus aureus: the role of Panton-Valentine leukocidin. Lab. Invest. 87, 3–9. Brown, M.E., Fitzner, J.N., Stevens, T., Chin, W., Wright, C.D., Boyce, J.P., 2008. Salicylanilides: selective inhibitors of interleukin-12p40 production. Bioorg. Med. Chem. 16, 8760–8764. Centers for Disease Control and Prevention, 2002. Staphylococcus aureus resistant to vancomycin – United States. Morb. Mortal. Wkly. Rep. 51, pp. 565–567. (cited 20.12.14). Cha, J.O., Park, Y.K., Lee, Y.S., Chung, G.T., 2011. In vitro biofilm formation and bactericidal activities of methicillin-resistant Staphylococcus aureus clones prevalent in Korea. Diagn. Microbiol. Infect. Dis. 70, 112–118. Chatterjee, D., 1997. The mycobacterial cell wall: structure, biosynthesis and sites of drug action. Curr. Opin. Chem. Biol. 1, 579–588. Cheng, T.-J.R., Wu, Y.-T., Yang, S.-T., Lo, K.-H., Chen, S.-K., Chen, Y.-H., Huang, W.-I., Yuan, C.-H., Guo, C.-W., Huang, L.-Y., Chen, K.-T., Shih, H.-W., Cheng, Y.-S.E., Cheng, W.-C., Wong, C.-H., 2010. High-throughput identification of antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase. Bioorg. Med. Chem. 18, 8512–8529. Clemens, E.C., Chan, J.D., Lynch, J.B., Dellit, T.H., 2011. Relationships between vancomycin minimum inhibitory concentration, dosing strategies, and outcomes in methicillin-resistant Staphylococcus aureus bacteremia. Diagn. Microbiol. Infect. Dis. 71, 408–414. Clinical and Laboratory Standards Institute, 2006. Performance Standards for Antimicrobial Susceptibility Testing; Sixteenth Informational Supplement. CLSI document M100-S16, CLSI, Wayne, Pa, USA. Clinical and Laboratory Standards Institute, 2012. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; approved standard-ninth edition. CLSI document M07-A9 PK, CLSI, Wayne, Pa, USA. Clinical and Laboratory Standards Institute, 2014. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fourth Informational Supplement. CLSI document M100-S24, CLSI, Wayne, Pa, USA. Dedieu, L., Serveau-Avesque, C., Kremer, L., Canaan, S., 2013. Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research. Biochimie 95, 66–73. Dolezal, M., Zitko, J., Jampilek, J., 2012. Pyrazinecarboxylic acid derivatives with antimycobacterial activity. In: Cardona, P.J. (Ed.), Understanding Tuberculosis – New Approaches to Fighting Against Drug Resistance. InTech, Rijeka, pp. 233– 262. Ferriz, J.M., Vavrova, K., Kunc, F., Imramovsky, A., Stolarikova, J., Vavrikova, E., Vinsova, J., 2010. Salicylanilide carbamates: antitubercular agents active against multidrug-resistant Mycobacterium tuberculosis strains. Bioorg. Med. Chem. 18, 1054–1061. Genestier, A.L., Michallet, M.C., Prevost, G., Bellot, G., Chalabreysse, L., Peyrol, S., Thivolet, F., Etienne, J., Lina, G., Vallette, F.M., Vandenesch, F., Genestier, L., 2005. Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J. Clin. Invest. 115, 3117–3127. Gould, I.M., 2007. MRSA bacteraemia. Int. J. Antimicrob. Agents 30, 66–70. He, X., Sugawara, M., Kobayashi, M., Takekuma, Y., Miyazaki, K., 2003. An in vitro system for prediction of oral absorption of relatively water-soluble drugs and ester prodrugs. Int. J. Pharm. 263, 35–44. Hilliard, J.J., Goldschmidt, R.M., Licata, L., Baum, E.Z., Bush, K., 1999. Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems. Antimicrob. Agents Chemother. 43, 1693–1699. Hlasta, D.J., Demers, J.P., Foleno, B.D., Fraga-Spano, S.A., Guan, J., Hilliard, J.J., Macielag, M.J., Ohemeng, K.A., Sheppard, C.M., Sui, Z., Webb, G.C., WeidnerWells, M.A., Werblood, H., Barrett, J.F., 1998. Novel inhibitors of bacterial twocomponent systems with gram positive antibacterial activity: pharmacophore identification based on the screening hit closantel. Bioorg. Med. Chem. Lett. 8, 1923–1928. Imramovsky, A., Polanc, S., Vinsova, J., Kocevar, M., Jampilek, J., Reckova, Z., Kaustova, J., 2007. A new modification of anti-tubercular active molecules. Bioorg. Med. Chem. 15, 2551–2559. Imramovsky, A., Vinsova, J., Ferriz, J.M., Dolezal, R., Jampilek, J., Kaustova, J., Kunc, F., 2009. New antituberculotics originated from salicylanilides with promising in vitro activity against atypical mycobacterial strains. Bioorg. Med. Chem. 17, 3572–3579.

Imramovsky, A., Pauk, K., Pejchal, V., Hanusek, J., 2011a. Salicylanilides and their derivates as perspective anti-tuberculosis drugs: synthetic routes and biological evaluations. Mini-Rev. Org. Chem. 8, 211–220. Imramovsky, A., Pesko, M., Ferriz, J.M., Kralova, K., Vinsova, J., Jampilek, J., 2011b. Photosynthesis-inhibiting efficiency of 4-chloro-2(chlorophenylcarbamoyl)phenyl alkylcarbamates. Bioorg. Med. Chem. Lett. 21, 4564–4567. Imramovsky, A., Stepankova, S., Vanco, J., Pauk, K., Ferriz, J.M., Vinsova, J., Jampilek, J., 2012. Acetylcholinesterase-inhibiting activity of salicylanilide Nalkylcarbamates and their molecular docking. Molecules 17, 10142–10158. Jampilek, J., Brychtova, K., 2012. Azone analogues: classification, design, and transdermal penetration principles. Med. Res. Rev. 32, 907–947. Kaku, N., Yanagihara, K., Morinaga, Y., Yamada, K., Harada, Y., Migiyama, Y., Nagaoka, K., Matsuda, J., Uno, N., Hasegawa, H., Miyazaki, T., Izumikawa, K., Kakeya, H., Yamamoto, Y., Kohno, S., 2014. Influence of antimicrobial regimen on decreased in-hospital mortality of patients with MRSA bacteremia. J. Infect. Chemother. 20, 350–355. Kos, J., Nevin, E., Soral, M., Kushkevych, I., Gonec, T., Bobal, P., Kollar, P., Coffey, A., O’Mahony, J., Liptaj, T., Kralova, K., Jampilek, J., 2015. Synthesis and antimycobacterial properties of ring-substituted 6-hydroxynaphthalene-2carboxanilides. Bioorg. Med. Chem. 23, 2035–2043. Kralova, K., Sersen, F., 2012. Effects of bioactive natural and synthetic compounds with different alkyl chain length on photosynthetic apparatus. In: Najafpour, M. (Ed.), Applied Photosynthesis. InTech, Rijeka, pp. 165–190. Kratky, M., Vinsova, J., Novotna, E., Mandikova, J., Wsol, V., Trejtnar, F., Ulmann, V., Stolarikova, J., Fernandes, S., Bhat, S., Liu, J.O., 2012. Salicylanilide derivatives block Mycobacterium tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase. Tuberculosis 92, 434–439. Liechti, C., Sequin, U., Bold, G., Furet, P., Meyer, T., Traxler, P., 2004. Salicylanilides as inhibitors of the protein tyrosine kinase epidermal growth factor receptor. Eur. J. Med. Chem. 39, 11–26. Lin, G., Pankuch, G.A., Appelbaum, P.C., Kosowska-Shick, K., 2012. Activity of telavancin compared to other agents against coagulase-negative staphylococci with different resistotypes by time kill. Diagn. Microbiol. Infect. Dis. 73, 287– 289. Liu, Y., Donner, P.L., Pratt, J.K., Jiang, W.W., Ng, T., Gracias, V., Baumeister, S., Wiedeman, P.E., Traphagen, L., Warrior, U., Maring, C., Kati, W.M., Djuric, S.W., Molla, A., 2008. Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase. Bioorg. Med. Chem. Lett. 18, 3173–3177. Lobbecke, L., Cevc, G., 1995. Effects of short-chain alcohols on phase behaviour and interdigitation of phosphatidylcholine bilayer membranes. Biochim. Biophys. Acta 1237, 59–69. Lode, H., 2001. Role of sultamicillin and ampicillin/sulbactam in the treatment of upper and lower bacterial respiratory tract infections. Int. J. Antimicrob. Agents 18, 199–209. Lode, H.M., 2008. Rational antibiotic therapy and the position of ampicillin/sulbactam. Int. J. Antimicrob. Agents 32, 10–28. Lullmann, H., Mohr, K., Wehling, M., 2004. Pharmacology and Toxicology. Grada, Prague. Macielag, M.J., Demers, J.P., Fraga-Spano, S.A., Hlasta, D.J., Johnson, S.G., Kanojia, R.M., Russell, R.K., Sui, Z., Weidner-Wells, M.A., Werblood, H., Foleno, B.D., Goldschmidt, R.M., Loeloff, M.J., Webb, G.C., Barrett, J.F., 1998. Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria. J. Med. Chem. 41, 2939–2945. Minnikin, D.E., Kremer, L., Dover, L.G., Besra, G.S., 2002. The methyl-branched fortifications of Mycobacterium tuberculosis. Chem. Biol. 9, 545–553. Mollaghan, A., Vinsova, J., Imramovsky, A., Cotter, L., Lucey, B., O´Mahony, J., Costelloe, A., Coffey, A., 2011. Antistaphylococcal activity of novel salicylanilide derivatives. Curr. Drug Discov. Technol. 8, 39–47. Mori, N., Kodama, T., Sakai, A., Suzuki, T., Sugihara, T., Yamaguchi, S., Nishijima, T., Aoki, A., Toriya, M., Kasai, M., Hatano, S., Kitagawa, M., Yoshimi, A., Nishimura, K., 2001. AS-924, a novel, orally active, bifunctional prodrug of ceftizoxime: physicochemical properties, oral absorption in animals, and antibacterial activity. Int. J. Antimicrob. Agents 18, 451–461. National Committee for Clinical Laboratory Standards, 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents, Approved Guideline; Document M26-A. NCCLS, Wayne, PA, USA. Nubel, U., Dordel, J., Kurt, K., Strommenger, B., Westh, H., Shukla, S.K., Zemlickova, H., Leblois, R., Wirth, T., Jombart, T., Balloux, F., Witte, W., 2010. A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS Pathog. 6, e1000855. Pauk, K., Zadrazilova, I., Imramovsky, A., Vinsova, J., Pokorna, M., Masarikova, M., Cizek, A., Jampilek, J., 2013. New derivatives of salicylamides: preparation and antimicrobial activity against various bacterial species. Bioorg. Med. Chem. 21, 6574–6581. Peres, D., Pina, E., Cardoso, M.F., 2011. Methicillin-resistant Staphylococcus aureus (MRSA) in a Portuguese hospital and its risk perception by health care professionals. Rev. Port. Saúde Públ. 29, 132–139. Przestalski, S., Sarapuk, J., Kleszczynska, H., Gabrielska, J., Hladyszowski, J., Trela, Z., Kuczera, J., 2000. Influence of amphiphilic compounds on membranes. Acta Biochim. Pol. 47, 627–638. ROCHE, 2011. Cell proliferation reagent WST-1. Roche Diagnostics GmbH, Mannheim, Germany. (cited 27.05.15).

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Sader, H.S., Flamm, R.K., Jones, R.N., 2013. Antimicrobial activity of ceftaroline and comparator agents tested against bacterial isolates causing skin and soft tissue infections and community-acquired respiratory tract infections isolated from the Asia-Pacific region and South Africa (2010). Diagn. Microbiol. Infect. Dis. 76, 61–68. Sakoulas, G., Moise-Broder, P.A., Schentag, J., Forrest, A., Moellering Jr., R.C., Eliopoulos, G.M., 2004. Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant Staphylococcus aureus bacteremia. J. Clin. Microbiol. 42, 2398–2402. Sarapuk, J., Kubica, K., 1998. Cut-off phenomenon. Cell. Mol. Biol. Lett. 3, 261–269. Sato, M., Tanaka, H., Yamaguchi, R., Kato, K., Etoh, H., 2004. Synergistic effects of mupirocin and an isoflavanone isolated from Erythrina variegata on growth and recovery of methicillin-resistant Staphylococcus aureus. Int. J. Antimicrob. Agents 24, 241–246. Schindler, B.D., Jacinto, P.L., Buensalido, J.A.L., Seo, S.M., Kaatz, G.W., 2015. Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus. Int. J. Antimicrob. Agents 45, 464–470. Schwalbe, R., Steele-Moore, L., Goodwin, A.C., 2007. Antimicrobial Susceptibility Testing Protocols. CRC Press, Boca Raton. Sriram, D., Yogeeswari, P., Basha, J.S., Radha, D.R., Nagaraja, V., 2005. Synthesis and antimycobacterial evaluation of various 7-substituted ciprofloxacin derivatives. Bioorg. Med. Chem. 13, 5774–5778. Stefani, S., Chung, D.R., Lindsay, J.A., Friedrich, A.W., Kearns, A.M., Westh, H., Mackenzie, F.M., 2012. Methicillin-resistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods. Int. J. Antimicrob. Agents 39, 273–282. Takacs-Novak, K., Jozan, M., Hermecz, I., Szasz, G., 1992. Lipophilicity of antibacterial fluoroquinolones. Int. J. Pharm. 79, 89–96. Takano, T., Saito, K., Teng, L.-J., Yamamoto, T., 2007. Spread of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) in hospitals in Taipei, Taiwan in 2005, and comparison of its drug resistance with previous hospitalacquired MRSA. Microbiol. Immunol. 51, 627–632.

11

Tengler, J., Kapustikova, I., Pesko, M., Govender, M., Keltosova, S., Mokry, P., Kollar, P., O’Mahony, J., Coffey, A., Kralova, K., Jampilek, J., 2013. Synthesis and biological evaluation of 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4[(alkoxycarbonyl)amino]benzoates. Sci. World J. 2013, 1–13. Thampi, N., Showler, A., Burry, L., Bai, A.D., Steinberg, M., Ricciuto, D.R., Bell, C.M., Morris, A.M., 2015. Multicenter study of health care cost of patients admitted to hospital with Staphylococcus aureus bacteremia: impact of length of stay and intensity of care. Am. J. Infect. Control (in press), http://dx.doi.org/10.1016/j. ajic.2015.01.031. Thorberg, S.O., Berg, S., Lundstrom, J., Pettersson, B., Wijkstrom, A., Sanchez, D., Lindberg, P., Nilsson, J.L., 1987. Carbamate ester derivatives as potential prodrugs of the presynaptic dopamine autoreceptor agonist (-)-3-(3hydroxyphenyl)-N-propylpiperidine. J. Med. Chem. 30, 2008–2012. Vinsova, J., Imramovsky, A., Buchta, V., Ceckova, M., Dolezal, M., Staud, F., Jampilek, J., Kaustova, J., 2007. Salicylanilide acetates: synthesis and antibacterial evaluation. Molecules 12, 1–12. Vinsova, J., Kozic, J., Kratky, M., Stolarikova, J., Mandikova, J., Trejtnar, F., Buchta, V., 2014. Salicylanilide diethyl phosphates: synthesis, antimicrobial activity and cytotoxicity. Bioorg. Med. Chem. 22, 728–737. Waisser, K., Pesina, M., Holy, P., Pour, M., Bures, O., Kunes, J., Klimesova, V., Buchta, V., Kubanova, P., Kaustova, J., 2003. Antimycobacterial and antifungal isosters of salicylamides. Arch. Pharm. 336, 322–335. Wilcox, M.H., 2011. MRSA new treatments on the horizon: current status. Injury 42, S42–S44. Wu, C.J., Jan, J.T., Chen, C.M., Hsieh, H.P., Hwang, D.R., Liu, H.W., Liu, C.Y., Huang, H.W., Chen, S.C., Hong, C.F., Lin, R.K., Chao, Y.S., Hsu, J.T., 2004. Inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide. Antimicrob. Agents Chemother. 48, 2693–2696. Wube, A.A., Hufner, A., Thomaschitz, C., Blunder, M., Kollroser, M., Bauer, R., Bucar, F., 2011. Design, synthesis and antimycobacterial activities of 1-methyl-2alkenyl-4(1H)-quinolones. Bioorg. Med. Chem. 19, 567–579.

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