The Scientific World JournalVolume 2012 (2012), Article ID 290628, 7 pagesdoi:10.1100/2012/290628

Research Article

In Vitro Antibacterial and Antifungal Activity of Salicylanilide Benzoates

Martin Krátký,¹ Jarmila Vinšová,¹ and Vladimír Buchta2,3

¹Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic²Department of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University, Sokolská 581, 500 12 Hradec Králové, Czech Republic³Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic

Received 14 October 2011; Accepted 10 January 2012

Academic Editor: Adam Shih-Yuan Lee

Copyright © 2012 Martin Krátký et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The resistance to antimicrobial agents brings a need of novel antimicrobial agents. We have synthesized and found the in vitro antibacterial activity of salicylanilide esters with benzoic acid (2-(phenylcarbamoyl)phenyl benzoates) in micromolar range. They were evaluated in vitro for the activity against eight fungal and eight bacterial species. All derivatives showed a significant antibacterial activity against Gram-positive strains with minimum inhibitory concentrations ≥0.98 μmol/L including methicillin-resistant Staphylococcus aureus strain. The most active compounds were 5-chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl benzoate and 4-chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl benzoate. The antifungal activity is significantly lower.

1. Introduction

The worldwide epidemic of antibiotic resistance is in danger of ending the “golden age” of antibiotic therapy and therefore is touching all people [1]. Major current problems arise from the spread of nosocomial antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum -lactamases-producing (ESBL) Escherichia coli or Klebsiella spp., multiresistant Pseudomonas, or Acinetobacter sp. as well as Clostridium difficile [2].

The situation in the fungal kingdom is a bit different. Over the past decades there has been a growing number of immunocompromised patients (e.g., patients with AIDS or after transplantations) who can develop opportunistic mycoses caused by expanding spectrum of fungal pathogens, including those with problematic susceptibility to current antifungal drugs. Pathogenic fungi can use different mechanisms of resistance to diverse drugs with unrelated modes of action [3].

The searching for potential antimicrobial agents is still challenging and new groups of compounds are desired [4].

Various salicylanilide (2-hydroxy-N-phenylbenzamide) esters have displayed good antibacterial and antifungal activities, especially against Gram-positive strains [5–7]. Recently salicylanilides were described besides an excellent antibacterial acting against both drug-sensitive and methicillin-resistant S. aureus inhibition activity towards bacterial transglycosylase, an enzyme necessary for the formation of the cell wall [8].

Benzoic acid alone is known as a nonspecific antimicrobial agent with the wide spectrum of the activities against human pathogenic fungi and bacteria with different minimum inhibitory concentration (MIC) values [9–14]; moreover it was being evaluated as an inhibitor of -carbonic anhydrase, a new molecular target occurring in C. albicans and Cryptococcus neoformans [15]. The review of the benzoic acid as preservative agent, the mechanisms of action, and resistance were published [16].

Based on these facts, we designed and evaluated new salicylanilide benzoates as potential antibacterial and antifungal agents.

2. Material and Methods

2.1. Chemistry

Salicylanilides were prepared by the procedure described previously [7]. The esters were prepared from salicylanilides by using benzoic acid and N,N′-dicyclohexylcarbodiimide as dehydrating and condensation agent (e.g., [7]). The general structure is presented in the head of Table 1.

Table 1: Antibacterial activity of benzoates 1-18.

MIC/IC₈₀ [μmol/L]

R¹
R²
Staphylococcus aureus
MRSA S. aureus
Staphylococcus epidermidis
Enterococcus sp.
Pseudomonas aeruginosa

24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h

(1)
4-Cl
3-Cl
0.98
3.9
0.98
3.9
1.95
7.81
31.25
62.5
3.9
7.81

(2)
5-Cl
3-Cl
3.9
3.9
3.9
3.9
3.9
62.5
>125
>125
>125
>125

(3)
4-Cl
4-Cl
3.9
3.9
3.9
3.9
>125
>125
>125
>125
>125
>125

(4)
5-Cl
4-Cl
15.62
31.25
15.62
31.25
31.25
31.25
>125
>125
>125
>125

(5)
4-Cl
3,4-diCl
1.95
3.9
1.95
7.81
1.95
1.95
1.95
1.95
>125
>125

(6)
5-Cl
3,4-diCl
1.95
1.95
1.95
1.95
1.95
1.95
1.95
1.95
>125
>125

(7)
4-Cl
3-Br
3.9
7.81
3.9
7.81
1.95
3.9
7.81
>125
>125
>125

(8)
5-Cl
3-Br
1.95
3.9
3.9
7.81
1.95
3.9
7.81
>125
>125
>125

(9)
4-Cl
4-Br
1.95
7.81
7.81
15.62
3.9
15.62
125
>125
>125
>125

(10)
5-Cl
4-Br
62.5
>125
125
>125
15.62
>125
>125
>125
>125
>125

(11)
4-Cl
3-F
3.9
7.81
3.9
7.81
1.95
7.81
31.25
>125
>125
>125

(12)
5-Cl
3-F
3.9
7.81
3.9
15.62
1.95
7.81
7.81
>125
>125
>125

(13)
4-Cl
4-F
7.81
7.81
15.62
15.62
1.95
3.9
62.5
>125
>125
>125

(14)
5-Cl
4-F
3.9
7.81
7.81
15.62
1.95
3.9
>125
>125
>125
>125

(15)
4-Cl
4-CF₃
1.95
1.95
1.95
1.95
1.95
1.95
1.95
1.95
>125
>125

(16)
5-Cl
4-CF₃
3.9
3.9
3.9
3.9
3.9
3.9
>125
>125
>125
>125

(17)
4-Cl
3-CF₃
1.95
3.9
1.95
7.81
1.95
3.9
7.81
>125
>125
>125

(18)
4-Br
4-CF₃
1.95
1.95
1.95
1.95
1.95
1.95
1.95
>125
>125
>125

PNC
—
—
0.98
0.98
62.5
125
250
250
7.81
15.62
>500
>500

BA
—
—
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500

PNC: penicillin G; BA: benzoic acid. The lowest MIC value(s) for each strain are bolded.

All used chemicals were purchased from commercial sources (Sigma-Aldrich) and they were used without a further purification. Reactions were monitored by thin-layer chromatography plates coated with 0.2 mm silica gel 60 F₂₅₄ (Merck) visualized by UV irradiation (254 nm). All synthesized compounds were characterized. Elemental analysis (C, H, N) was performed on an automatic microanalyser CHNS-O CE instrument (FISONS EA 1110, Italy). Melting points were determined on a Melting Point machine B-540 (Büchi) apparatus using open capillaries and they are uncorrected. Infrared spectra (ATR) were recorded on FT-IR spectrometer Nicolet 6700 FT-IR in the range of 400–4000 cm⁻¹. The NMR spectra were recorded on a Varian VNMR S500 (500 MHz for ¹H and 125 MHz for ¹³C; Varian, Inc., Palo Alto, USA) at ambient temperature using deuterated dimethyl sulfoxide (DMSO-d6) solutions of the samples. The chemical shifts are given in ppm, with respect to tetramethylsilane as an internal standard. The coupling constants () are reported in Hz.

2.2. Biology

2.2.1. Antibacterial Evaluation

The in vitro antibacterial activity was assayed against next Gram-positive and Gram-negative strains: Staphylococcus aureus CCM 4516/08, methicillin-resistant Staphylococcus aureus H 5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08, Escherichia coli CCM4517, Klebsiella pneumoniae D 11750/08, ESBL-positive Klebsiella pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961.

The microdilution broth method modified according to standard M07-A07 [17] in Mueller-Hinton broth (HiMedia Laboratories, India) was adjusted to pH 7.4 (±0.2). The investigated compounds were dissolved in DMSO to the final concentrations ranging from 500 to 0.49 μmol/L. Penicillin G (benzylpenicillin) and benzoic acid were used as comparative standard drugs. Bacterial inoculum in sterile water was prepared to match 0.5 McFarland scale (1.5 × 10⁸ CFU/mL). The minimum inhibitory concentrations (MICs) were assayed as 80% (IC₈₀) or higher reduction of growth in comparison to the control. The determination of results was performed visually and spectrophotometrically (at 540 nm). The values of MICs were determined after 24 and 48 h of incubation in the darkness at 35°C (±0.1) in a humid atmosphere.

2.2.2. Antifungal Evaluation

The inhibitory activity was determined in vitro against four yeast strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, and Candida glabrata 20/I) and four moulds (Trichosporon asahii 1188, Aspergillus fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445).

The method used was microdilution broth method in the format of the CLSI M27-A3 and M38 A2 guidelines for yeasts and moulds [18, 19] in RPMI 1640 with glutamine (KlinLab, the Czech Republic) buffered to pH 7.0 with 0.165 M of 3-morpholino-propane-1-sulphonic acid (Sigma-Aldrich, Germany). DMSO served as a diluent for all compounds. Fungal inoculum was prepared to give a final concentration of 5 × 10³ ± 0.2 CFU/mL. Fluconazole was used as a reference drug. Other conditions were the same as for antibacterial assay; only for T. mentagrophytes the final MIC were determined after 72 and 120 h of incubation. MICs were determined twice and in duplicate.

3. Results and Discussion

3.1. Chemistry

Eighteen new salicylanilide benzoates were synthesized. The yields ranged from 44 to 88%.

4-Chloro-2-(3-chlorophenylcarbamoyl)phenyl Benzoate (1)White solid; yield 82%; mp 146.5–149°C. IR (ATR): 3325 (NH amide; m), 3081, 2932, 2853, 1716 (CO ester; s), 1672 (CO amide; s), 1590, 1525, 1483, 1451, 1424, 1309, 1286, 1267, 1251, 1208, 1181, 1104, 1085, 1067, 1023, 902, 873, 786, 735, 703, 681. ¹H NMR (500 MHz, DMSO): 10.69 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.83 (1H, d,  Hz, H3), 7.76–7.69 (3H, m, H5, H6, H2′), 7.56 (1H, t,  Hz, H4′′), 7.53–7.49 (3H, m, H6′, H3′′, H5′′), 7.31 (1H, t,  Hz, H5′), 7.11 (1H, dd,  Hz,  Hz, H4′). ¹³C NMR (125 MHz, DMSO): 164.3, 163.0, 147.0, 140.3, 134.4, 133.1, 131.8, 131.2, 130.6, 130.4, 130.0, 129.1, 128.6, 128.0, 125.7, 123.8, 119.4, 118.4. Anal. Calcd. for C₂₀H₁₃Cl₂NO₃ (386.23): C, 62.19; H, 3.39; N, 3.63. Found: C, 61.89; H, 3.50; N, 3.87.

5-Chloro-2-(3-chlorophenylcarbamoyl)phenyl Benzoate (2)White solid; yield 68%; mp 166–168°C. IR (ATR): 3282 (NH amide; m), 3072, 1739 (CO ester; s), 1647 (CO amide; s), 1600, 1589, 1548, 1481, 1450, 1410, 1320, 1255, 1241, 1192, 1075, 1051, 1021, 915, 896, 873, 854, 829, 782, 702, 676, 660. ¹H NMR (500 MHz, DMSO): 10.65 (1H, bs, NH), 8.08 (2H, d,  Hz, H2′′, H6′′), 7.79 (1H, d,  Hz, H3), 7.76–7.67 (3H, m, H4, H6, H2′), 7.58–7.48 (4H, m, H6′, H3′′, H4′′, H5′′) 7.30 (1H, t,  Hz, H5′), 7.10 (1H, dd,  Hz,  Hz, H4′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.5, 148.9, 140.3, 135.8, 134.4, 133.1, 131.0, 130.6, 130.1, 129.1, 128.6, 128.5, 126.5, 124.0, 123.7, 119.4, 118.4. Anal. Calcd. for C₂₀H₁₃Cl₂NO₃ (386.23): C, 62.19; H, 3.39; N, 3.63. Found: C, 62.34; H, 3.22; N, 3.79.

4-Chloro-2-(4-chlorophenylcarbamoyl)phenyl Benzoate (3)White solid; yield 80%; mp 185–187°C. IR (ATR): 3309 (NH amide; m), 3072, 2928, 2850, 1741 (CO ester; s), 1649 (CO amide; s), 1593, 1543, 1537, 1490, 1451, 1405, 1314, 1257, 1245, 1197, 1099, 1053, 1023, 875, 836, 814, 724, 706, 669. ¹H NMR (500 MHz, DMSO): 10.65 (1H, bs, NH), 8.06 (2H, d,  Hz, H2′′, H6′′), 7.82 (1H, d,  Hz, H3), 7.73–7.68 (2H, m, H5, H6), 7.63 (2H, d,  Hz, H2′, H6′), 7.55 (1H, t,  Hz, H4′′), 7.50 (2H, t,  Hz, H3′′, H5′′), 7.33 (2H, d,  Hz, H3′, H5′). ¹³C NMR (125 MHz, DMSO): 164.3, 162.8, 146.9, 137.8, 134.3, 131.6, 131.3, 130.4, 130.0, 129.1, 128.7, 128.0, 127.7, 127.1, 125.7, 121.5. Anal. Calcd. for C₂₀H₁₃Cl₂NO₃ (386.23): C, 62.19; H, 3.39; N, 3.63. Found: C, 62.00; H, 3.45; N, 3.87.

5-Chloro-2-(4-chlorophenylcarbamoyl)phenyl Benzoate (4)White solid; yield 81%; mp 192–194°C. IR (ATR): 3344 (NH amide; m), 3070, 1746 (CO ester; s), 1650 (CO amide; s), 1592, 1533, 1491, 1453, 1401, 1307, 1259, 1243, 1191, 1176, 1077, 1052, 1021, 915, 893, 827, 762, 703. ¹H NMR (500 MHz, DMSO): 10.61 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.78 (1H, d,  Hz, H3), 7.73–7.67 (2H, m, H4, H6), 7.63 (2H, d,  Hz, H2′, H6′), 7.58–7.53 (3H, m, H3′′, H4′′, H5′′), 7.32 (2H, d,  Hz, H3′, H5′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.3, 148.9, 137.9, 135.7, 134.4, 130.9, 130.1, 129.1, 128.8, 128.7, 128.6, 127.6, 126.5, 123.9, 121.4. Anal. Calcd. for C₂₀H₁₃Cl₂NO₃ (386.23): C, 62.19; H, 3.39; N, 3.63. Found: C, 61.87; H, 3.54; N, 3.90.

4-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl Benzoate (5)White solid; yield 52%; mp 169.5–172°C. IR (ATR): 3304 (NH amide; m), 3074, 2928, 2850, 1713 (CO ester; s), 1668 (CO amide; s), 1578, 1516, 1478, 1469, 1449, 1384, 1298, 1275, 1247, 1208, 1101, 1087, 1066, 1026, 889, 866, 704. ¹H NMR (500 MHz, DMSO): 10.79 (1H, bs, NH), 8.06 (2H, d,  Hz, H2′′, H6′′), 7.92 (1H, s, H2′), 7.84 (1H, d,  Hz, H3), 7.74–7.69 (2H, m, H5, H6), 7.58–7.50 (5H, m, H5′, H6′, H3′′, H4′′, H5′′). ¹³C NMR (125 MHz, DMSO): 164.3, 163.0, 147.0, 138.9, 134.4, 131.9, 131.1, 130.9, 130.4, 130.0, 129.1, 128.6, 128.0, 127.9, 127.0, 125.7, 121.1, 120.0. Anal. Calcd. for C₂₀H₁₂Cl₃NO₃ (420.67): C, 57.10; H, 2.88; N, 3.33. Found: C, 57.40; H, 2.99; N, 3.41.

5-Chloro-2-(3,4-dichlorophenylcarbamoyl)phenyl Benzoate (6)White solid; yield 82%; mp 155–157°C. IR (ATR): 3412 (NH amide; m), 3093, 2930, 2851, 1754 (CO ester; s), 1682 (CO amide; s), 1593, 1527, 1475, 1450, 1400, 1375, 1303, 1244, 1180, 1135, 1042, 1020, 914, 881, 823, 759, 700. ¹H NMR (500 MHz, DMSO): 10.75 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.92 (1H, s, H2′), 7.79 (1H, d,  Hz, H3), 7.74–7.69 (2H, m, H4, H6), 7.59–7.53 (5H, m, H5′, H6′, H3′′, H4′′, H5′′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.6, 148.9, 139.0, 136.0, 134.4, 131.1, 130.9, 130.1, 129.1, 128.8, 128.0, 127.1, 126.5, 125.6, 124.0, 121.1, 119.9. Anal. Calcd. for C₂₀H₁₂Cl₃NO₃ (420.67): C, 57.10; H, 2.88; N, 3.33. Found: C, 56.95; H, 2.82; N, 3.59.

2-(3-Bromophenylcarbamoyl)-4-chlorophenyl Benzoate (7)White solid; yield 84%; mp 144–146°C. IR (ATR): 3325 (NH amide; m), 3079, 2930, 2852, 1716 (CO ester; s), 1671 (CO amide; s), 1586, 1520, 1479, 1450, 1419, 1305, 1284, 1266, 1249, 1208, 1103, 1084, 1065, 1023, 893, 872, 783, 733, 702, 684, 672. ¹H NMR (500 MHz, DMSO): 10.67 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.89 (1H, s, H2′), 7.83 (1H, d,  Hz, H3), 7.73–7.69 (2H, m, H5, H6), 7.58–7.50 (3H, m, H3′′, H4′′, H5′′), 7.45 (1H, d,  Hz, H6′), 7.36 (1H, t,  Hz, H5′), 7.24 (1H, d,  Hz, H4′). ¹³C NMR (125 MHz, DMSO): 164.3, 163.0, 147.0, 140.4, 134.4, 131.7, 130.9, 130.4, 130.0, 129.1, 128.6, 128.0, 127.1, 126.7, 125.7, 122.3, 121.6, 118.7. Anal. Calcd. for C₂₀H₁₃BrClNO₃ (430.68): C, 55.78; H, 3.04; N, 3.25. Found: C, 55.49; H, 3.20; N, 3.48.

2-(3-Bromophenylcarbamoyl)-5-chlorophenyl Benzoate (8)White solid; yield 73%; mp 153.5–156°C. IR (ATR): 3279 (NH amide; m), 1739 (CO ester; s), 1647 (CO amide; s), 1599, 1585, 1541, 1476, 1452, 1407, 1320, 1254, 1241, 1190, 1075, 1050, 1020, 914, 894, 854, 781, 702, 659. ¹H NMR (500 MHz, DMSO): 10.63 (1H, bs, NH), 8.08 (2H, d,  Hz, H2′′, H6′′), 7.89 (1H, s, H2′), 7.79 (1H, d,  Hz, H3), 7.73–7.68 (2H, m, H4, H6), 7.58–7.53 (3H, m, H3′′, H4′′, H5′′), 7.45 (1H, d,  Hz, H6′), 7.36 (1H, t,  Hz, H5′), 7.23 (1H, d,  Hz, H4′). ¹³C NMR (125 MHz, DMSO): 164.1, 163.5, 148.9, 140.5, 135.8, 134.4, 130.9, 130.1, 129.1, 128.6, 128.0, 127.1, 126.6, 126.5, 124.0, 122.2, 121.6, 118.7. Anal. Calcd. for C₂₀H₁₃BrClNO₃ (430.68): C, 55.78; H, 3.04; N, 3.25. Found: C, 55.87; H, 3.31; N, 3.46.

2-(4-Bromophenylcarbamoyl)-4-chlorophenyl Benzoate (9)White solid; yield 79%; mp 199–201°C. IR (ATR): 3308 (NH amide; m), 2932, 1739 (CO ester; s), 1668 (CO amide; s), 1597, 1541, 1487, 1449, 1403, 1314, 1258, 1246, 1197, 1099, 1053, 1023, 875, 833, 813, 706. ¹H NMR (500 MHz, DMSO): 10.64 (1H, bs, NH), 8.06 (2H, d,  Hz, H2′′, H6′′), 7.82 (1H, d,  Hz, H3), 7.73–7.68 (2H, m, H5, H6), 7.59–7.45 (7H, m, H2′, H3′, H5′, H6′, H3′′, H4′′, H5′′). ¹³C NMR (125 MHz, DMSO): 164.3, 162.8, 147.0, 138.3, 134.4, 131.8, 131.7, 131.3, 130.4, 130.0, 129.1, 129.0, 128.7, 125.6, 121.9, 115.7. Anal. Calcd. for C₂₀H₁₃BrClNO₃ (430.68): C, 55.78; H, 3.04; N, 3.25. Found: C, 55.55; H, 3.01; N, 3.51.

2-(4-Bromophenylcarbamoyl)-5-chlorophenyl Benzoate (10)White solid; yield 73%; mp 201–203°C. IR (ATR): 3326 (NH amide; m), 2929, 1745 (CO ester; s), 1651 (CO amide; s), 1601, 1587, 1531, 1487, 1450, 1397, 1260, 1241, 1189, 1176, 1073, 1051, 1020, 914, 893, 823, 810, 762, 703. ¹H NMR (500 MHz, DMSO): 10.61 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.78 (1H, d,  Hz, H3), 7.73–7.67 (2H, m, H4, H6), 7.59–7.53 (5H, m, H2′, H6′, H3′′, H4′′, H5′′), 7.45 (2H, d,  Hz, H3′, H5′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.3, 148.9, 138.3, 135.7, 134.4, 131.7, 130.9, 130.0, 129.1, 128.7, 128.6, 126.5, 123.9, 121.8, 115.7. Anal. Calcd. for C₂₀H₁₃BrClNO₃ (430.68): C, 55.78; H, 3.04; N, 3.25. Found: C, 55.67; H, 2.93; N, 3.29.

4-Chloro-2-(3-fluorophenylcarbamoyl)phenyl Benzoate (11)White solid; yield 61%; mp 143–144.5°C. IR (ATR): 3324 (NH amide; m), 2930, 2852, 1716 (CO ester; s), 1673 (CO amide; s), 1601, 1531, 1485, 1452, 1437, 1316, 1268, 1208, 1176, 1104, 1086, 1067, 1022, 965, 858, 784, 733, 704, 681. ¹H NMR (500 MHz, DMSO): 10.71 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.83 (1H, d,  Hz, H3), 7.73–7.69 (2H, m, H5, H6), 7.57–7.46 (4H, m, H2′, H3′′, H4′′, H5′′), 7.40–7.29 (2H, m, H5′, H6′), 6.92–6.86 (1H, m, H4′). ¹³C NMR (125 MHz, DMSO): 164.3, 162.9, 163.1 and 161.2 ( Hz), 147.0, 140.6 and 140.5 ( Hz), 131.7, 131.2, 130.6 and 130.5 ( Hz), 130.4, 130.0, 129.1, 128.6, 128.0, 127.1, 125.7, 115.7 and 115.7 ( Hz), 110.6 and 110.5 ( Hz), 106.8 and 106.6 ( Hz). Anal. Calcd. for C₂₀H₁₃ClFNO₃ (369.77): C, 64.96; H, 3.54; N, 3.79. Found: C, 64.80; H, 3.29; N, 3.58.

5-Chloro-2-(3-fluorophenylcarbamoyl)phenyl Benzoate (12)White solid; yield 78%; mp 149–151°C. IR (ATR): 3295 (NH amide; m), 3073, 2931, 1739 (CO ester; s), 1650 (CO amide; s), 1596, 1550, 1489, 1450, 1423, 1324, 1256, 1241, 1196, 1171, 1149, 1075, 1053, 1022, 911, 845, 779, 704, 662. ¹H NMR (500 MHz, DMSO): 10.68 (1H, bs, NH), 8.08 (2H, d,  Hz, H2′′, H6′′), 7.79 (1H, d,  Hz, H3), 7.73–7.68 (2H, m, H4, H6), 7.58–7.51 (4H, m, H2′, H3′′, H4′′, H5′′), 7.39–7.27 (2H, m, H5′, H6′), 6.90–6.85 (1H, m, H4′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.5, 163.1 and 161.2 ( Hz), 148.9, 140.7 and 140.6 ( Hz), 135.8, 134.4, 130.9, 130.6 and 130.5 ( Hz), 130.1, 129.1, 128.6, 128.5, 126.5, 124.0, 115.7 and 115.6 ( Hz), 110.5 and 110.4 ( Hz), 106.7 and 106.5 ( Hz). Anal. Calcd. for C₂₀H₁₃ClFNO₃ (369.77): C, 64.96; H, 3.54; N, 3.79. Found: C, 64.90; H, 3.24; N, 3.99.

4-Chloro-2-(4-fluorophenylcarbamoyl)phenyl Benzoate (13)White solid; yield 88%; mp 149–151°C. IR (ATR): 3318 (NH amide; m), 3076, 2929, 2852, 1715 (CO ester; s), 1665 (CO amide; s), 1622, 1571, 1527, 1505, 1479, 1450, 1406, 1310, 1275, 1250, 1207, 1177, 1151, 1098, 1086, 1064, 1023, 892, 822, 778, 733, 697. ¹H NMR (500 MHz, DMSO): 10.56 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.81 (1H, d,  Hz, H3), 7.73–7.68 (2H, m, H5, H6), 7.63–7.59 (2H, m, H2′, H6′), 7.55 (1H, t,  Hz, H4′′), 7.51–7.48 (2H, m, H3′′, H5′′), 7.14–7.09 (2H, m, H3′, H5′). ¹³C NMR (125 MHz, DMSO): 164.3, 162.6, 159.5 and 157.5 ( Hz), 147.0, 135.2 and 135.2 ( Hz), 134.3, 131.5, 130.4, 130.0, 129.1, 128.7, 128.0, 127.1, 125.6, 121.8 and 121.8 ( Hz), 115.5 and 115.4 ( Hz). Anal. Calcd. for C₂₀H₁₃ClFNO₃ (369.77): C, 64.96; H, 3.54; N, 3.79. Found: C, 65.15; H, 3.47; N, 3.70.

5-Chloro-2-(4-fluorophenylcarbamoyl)phenyl Benzoate (14)White solid; yield 84%; mp 142–144°C. IR (ATR): 3295 (NH amide; m), 2930, 2852, 1739 (CO ester; s), 1647 (CO amide; s), 1601, 1547, 1505, 1452, 1411, 1317, 1258, 1245, 1194, 1177, 1155, 1072, 1054, 1023, 893, 835, 826, 706. ¹H NMR (500 MHz, DMSO): 10.52 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.78 (1H, d,  Hz, H3), 7.73–7.66 (2H, m, H4, H6), 7.63–7.59 (2H, m, H2′, H6′), 7.57–7.53 (3H, m, H3′′, H4′′, H5′′), 7.13–7.08 (2H, m, H3′, H5′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.1, 159.4 and 157.5 ( Hz), 148.9, 135.6, 135.3 and 135.3 ( Hz), 134.4, 130.9, 130.0, 129.1, 128.6, 128.0, 126.4, 123.9, 121.8 and 121.7 ( Hz), 115.5 and 115.3 ( Hz). Anal. Calcd. for C₂₀H₁₃ClFNO₃ (369.77): C, 64.96; H, 3.54; N, 3.79. Found: C, 64.74; H, 3.61; N, 3.94.

4-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl Benzoate (15)White solid; yield 44%; mp 179.5–181.5°C. IR (ATR): 3310 (NH amide; m), 1715 (CO ester; s), 1672 (CO amide; s), 1601, 1526, 1476, 1452, 1407, 1317, 1278, 1253, 1207, 1168, 1110, 1086, 1063, 1015, 891, 867, 844, 820, 775, 703. ¹H NMR (500 MHz, DMSO): 10.87 (1H, bs, NH), 8.06 (2H, d,  Hz, H2′′, H6′′), 7.86 (1H, d,  Hz, H3), 7.82 (2H, d,  Hz, H2′, H6′), 7.75–7.68 (3H, m, H5, H6), 7.65 (2H, d,  Hz, H3′, H5′), 7.57–7.51 (3H, m, H3′′, H4′′, H5′′). ¹³C NMR (125 MHz, DMSO): 164.3, 163.2, 147.0, 142.5, 134.4, 131.9, 131.1, 130.4, 130.0, 129.2, 129.1, 128.6, 126.8 (q,  Hz), 126.2 (d,  Hz), 125.7, 124.4 (q,  Hz), 119.9. Anal. Calcd. for C₂₁H₁₃ClF₃NO₃ (419.78): C, 60.08; H, 3.12; N, 3.34. Found: C, 60.31; H, 2.87; N, 3.09.

5-Chloro-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl Benzoate (16)White solid; yield 80%; mp 177.5–179.5°C. IR (ATR): 3325 (NH amide; m), 2927, 1746 (CO ester; s), 1654 (CO amide; s), 1598, 1534, 1481, 1450, 1408, 1323, 1243, 1192, 1160, 1104, 1067, 1048, 1020, 916, 894, 845, 825, 763, 702. ¹H NMR (500 MHz, DMSO): 10.84 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 7.83–7.79 (3H, m, H3, H2′, H6′), 7.71–7.67 (2H, m, H4, H6), 7.64 (2H, d,  Hz, H3′, H5′), 7.60–7.54 (3H, m, H3′′, H4′′, H5′′). ¹³C NMR (125 MHz, DMSO): 164.2, 163.7, 149.0, 142.5, 135.9, 134.4, 131.0, 130.0, 129.1, 128.5, 128.5, 126.5, 126.2 (q,  Hz), 124.6 (q,  Hz), 124.0, 123.9 (q,  Hz), 119.8. Anal. Calcd. for C₂₁H₁₃ClF₃NO₃ (419.78): C, 60.08; H, 3.12; N, 3.34. Found: C, 60.22; H, 3.21; N, 3.43.

4-Chloro-2-(3-(trifluoromethyl)phenylcarbamoyl)phenyl Benzoate (17)White solid; yield 72%; mp 159.5–162°C. IR (ATR): 3314 (NH amide; m), 2931, 2853, 1714 (CO ester; s), 1670 (CO amide; s), 1597, 1540, 1443, 1326, 1274, 1206, 1166, 1119, 1097, 1084, 1064, 1023, 906, 800, 735, 695. ¹H NMR (500 MHz, DMSO): 10.83 (1H, bs, NH), 8.07 (2H, d,  Hz, H2′′, H6′′), 8.00 (1H, s, H2′), 7.87 (1H, d,  Hz, H3), 7.74–7.68 (2H, m, H5, H6), 7.56–7.50 (4H, m, H6′, H3′′, H4′′, H5′′), 7.46–7.35 (2H, m, H4′, H5′). ¹³C NMR (125 MHz, DMSO): 164.3, 163.1, 147.0, 139.6, 134.4, 131.8, 131.1, 130.4, 130.2, 130.0, 129.6 (q,  Hz), 129.1, 128.6, 128.0, 125.7, 124.2 (q,  Hz), 123.6, 120.4 (q,  Hz), 116.1 (q,  Hz). Anal. Calcd. for C₂₁H₁₃ClF₃NO₃ (419.78): C, 60.08; H, 3.12; N, 3.34. Found: C, 60.19; H, 3.35; N, 2.99.

4-Bromo-2-(4-(trifluoromethyl)phenylcarbamoyl)phenyl Benzoate (18)White solid; yield 79%; mp 177.5–180°C. IR (ATR): 3309 (NH amide; m), 1714 (CO ester; s), 1672 (CO amide; s), 1600, 1526, 1474, 1451, 1406, 1317, 1280, 1252, 1208, 1169, 1111, 1086, 1064, 1041, 862, 843, 818, 720, 703. ¹H NMR (500 MHz, DMSO): 10.87 (1H, bs, NH), 8.06 (2H, d,  Hz, H2′′, H6′′), 7.97 (1H, d,  Hz, H3), 7.85 (1H, dd,  Hz,  Hz, H5), 7.82 (2H, d,  Hz, H2′, H6′), 7.70 (1H, t,  Hz, H4′′), 7.65 (2H, d,  Hz, H3′, H5′), 7.54 (2H, t,  Hz, H3′′, H5′′), 7.45 (1H, d,  Hz, H6). ¹³C NMR (125 MHz, DMSO): 164.2, 163.1, 147.5, 142.5, 134.8, 134.4, 132.0, 131.4, 130.0, 129.1, 128.6, 126.7 (q,  Hz), 126.2 (d,  Hz), 126.0, 124.4 (q,  Hz), 119.9, 118.5. Anal. Calcd. for C₂₁H₁₃BrF₃NO₃ (464.23): C, 54.33; H, 2.82; N, 3.02. Found: C, 54.16; H, 3.03; N, 3.35.

3.2. Biology

3.2.1. Antibacterial Evaluation

Salicylanilide benzoates were assayed for their in vitro antibacterial activity towards eight strains. Benzoic acid expressed no activity up to 500 μmol/L. Table 1 summarizes the results with respect to structure of the esters.

Almost all benzoates exhibited a very good activity against both strains of S. aureus with MIC from 0.98 to 31.25 μmol/L (10 being an exception). Importantly, MIC for drug-sensitive and MRSA strain did not differ practically, which indicates none cross resistance. Also S. epidermidis was affected by the majority of evaluated compounds (without 3) at only slightly higher concentrations from 1.95 μmol/L. Enterococcus was the most insensitive Gram-positive strain, although it was inhibited by four esters (5, 6, 15, 18) at 1.95 μmol/L; six esters (2, 3, 4, 10, 14, 16) were inactive at 125 μmol/L. Molecules 6 and 15 were found having the highest in vitro inhibitory potency. MIC values against three Staphylococci support the hypothesis that these derivatives act as bactericidal agents.

Some salicylanilide benzoates MIC values are comparable to benzylpenicillin towards S. aureus, but almost all esters are favorable against MRSA and S. epidermidis. The situation for Enterococcus is quite more complex; some benzoates exhibited better MIC value (5, 6, 15, 18), some comparable after 24 h (7, 8, 12, 17) and other worse than benzylpenicillin.

When concentrated on the structure-activity relationships, the position of the substituents on the salicylic ring is ambiguous—in some cases are superior 4-chloro derivatives (e.g., 1 versus 2, 9 versus 10, 15 versus 16), in other 5-chloro ones (5 versus 6 or 7 versus 8). For the substitution of the aniline ring, 3,4-dichloro (5, 6) and CF₃- (15–18) moieties improved the antibacterial activity the most significantly. In general, it seems that 3-substituted anilines produced a higher antibacterial activity than 4-substituted anilines.

Gram-negative species (E. coli, two strains of Klebsiella pneumoniae, P. aeruginosa) were almost completely resistant to benzoates up to 125 μmol/L at the testing conditions with one notable exception; P. aeruginosa was inhibited by 1 at low concentration of 3.9/7.81 μmol/L.

3.2.2. Antifungal Evaluation

Synthesized esters were tested for their in vitro activity against eight human pathogenic fungi. MIC values are presented in Table 2. Benzoic acid alone was completely inactive at the concentration of 500 μmol/L at both neutral and slightly acidic (pH ~ 5) environment.

Table 2: In vitro antifungal activity of salicylanilide benzoates.

MIC/IC₈₀ [μmol/L]

Candida albicans
Candida krusei
Trichosporon asahii
Absidia corymbifera
Trichophyton mentagrophytes

24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
72 h
120 h

(1)
250
>500
125
>500
>500
>500
500
>500
250
>500

(6)
125
>125
15.62
>125
3.9
15.62
125
125
31.25
31.25

(8)
>250
>250
>250
>250
>250
>250
250
>250
>250
>250

(9)
>125
>125
>125
>125
>125
>125
125
125
62.5
62.5

(13)
>125
>125
>125
>125
>125
>125
62.5
>125
125
125

(18)
>125
>125
>125
>125
>125
>125
>125
>125
62.5
125

BA
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500

FLU
1.00
2.00
>50.0
>50.0
4.00
9.00
>50.0
>50.0
17.0
26.0

FLU: fluconazole; BA: benzoic acid. The best MIC value for each strain is bolded.

Unforeseen, in contrast to the antibacterial activity, salicylanilide benzoates expressed only mild antifungal potency. From eight strains, C. tropicalis, C. glabrata, and A. fumigatus showed a complete insensitivity to all tested derivatives at the value of 125 μmol/L. When concentrated on the esters, benzoates 2 and 11 did not affect the growth of any fungal species at the concentration of 500 μmol/L and lower, derivatives 12, 14, and 17 up to 250 μmol/L and 3, 4, 5, 7, 10, 15, 16 expressed activity levels >125 μmol/L. Only six derivatives (1, 6, 8, 9, 13, 18) displayed certain in vitro efficacy. The most active compound was assayed trichlorinated ester 6 (MIC ≥ 3.9 μmol/L), which surpassed standard fluconazole against C. krusei. In general, Candida is more resistant to salicylanilide esters than moulds and moreover, it seems that the mechanism of the action is only fungistatic. On the other side, the growth of T. mentagrophytes was affected by the highest number of the derivatives; based on the MIC values, activity against filamentous fungi is probably fungicidal.

Because pH effect on the efficiency of benzoic acid derivatives (generally of weak organic acids) was described (e.g., [16]), we measured MIC values of the esters 6 and 15 not only at approximately neutral environment, but additionally at slightly acidic pH (~5; without buffering of the testing medium). Unfortunately, the change of pH did not result in the improvement of activity, even it led to a bit worse values.

Although we expected that the introduction of benzoyl fragment into salicylanilide molecules resulted in the significantly increased antifungal potency, this modification failed in this point.

4. Conclusion

In sum, we have designed and synthesized new esters of halogenated salicylanilides with benzoic acid. This series of compounds was evaluated to be a new group with promising in vitro antibacterial (against Gram-positive strains) activity. Unfortunately these derivatives disappointed expectation about them as potential antifungal agents.

Acknowledgments

This work was financially supported by GAUK 27610/2010, the Research Project MSM 0021620822, IGA NS 10367-3, and SVV 2012-265-001.

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