Introduction

In recent years, the emergence of widespread antibiotic resistance has been getting increasing publicity, while there is also a worldwide attempt to provide financial support for the research and development of new antibiotics.1, 2 Also, numerous events are organized to raise awareness to this global health-care crisis. Although the discovery of effective antibiotics against resistant Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, etc. are of main interest, many resistant Gram-positive bacteria (for example, Clostridium difficile, multidrug-resistant Staphylococcus aureus, vancomycin-resistant enterococci) are also found on the list of the most dangerous organisms causing life-threatening infections,3, 4 therefore constant research in this area should not be neglected.

An enormous collection of derivatives of glycopeptide antibiotics has been synthesized by numerous research groups throughout the years5, 6, 7, 8, 9, 10, 11, 12 to yield compounds that are active against resistant Gram-positive bacteria, unlike the original molecules. In several cases, the chemical modifications did not only improve the antibacterial spectrum or activity but also many of them came out to be superior to the parent compounds. The successful development and approval of telavancin,13 dalbavancin14 and oritavancin15 proves that semisynthetic work, especially lipophilic derivatization, is undoubtedly useful for preparing compounds with favorable biological characteristics.

Besides others, we have been working on the chemical transformations of glycopeptide antibiotics for a long time. Our synthetic work has essentially been focused on derivatives of the acid hydrolysis products of glycopeptides; that is, aglycons or teicoplanin pseudoaglycon. In order to obtain new derivatives with improved antibacterial activity, in the synthetic work we have carried out in this field, we introduced a large series of more or less lipophilic groups at the N-terminal position of aglycons or pseudoaglycons prepared from ristocetin, teicoplanin or dechloroteicoplanin.16, 17, 18, 19 Compounds obtained in this way form nanosized aggregates in water.20, 21 This phenomenon; that is, formation of multivalent clusters, can be one of the explanations of their improved antibacterial activity. In the sense of antibacterial activity, our best results were mostly achieved by starting from the N-acetylglucosaminyl teicoplanin aglycon. The antibacterial activity of these compounds has been evaluated on a standard panel of Gram-positive bacteria, including one vancomycin and one teicoplanin-resistant Enterococcus strain possessing resistance genes vanB or vanA accordingly, but none of the compounds have ever been tested against multiple clinical strains of vancomycin-resistant Enterococcus (VRE).

Although, as mentioned, several large systematic series of teicoplanin derivatives are found in the literature, the panel of bacteria for the measurement of bioactivity rarely includes more than a few glycopeptides-resistant Enterococcus strains and only a limited number of derivatives (mainly candidates for further development) are tested against larger collections of VRE. Here we wish to report the antibacterial activity of nine of our most active teicoplanin pseudoaglycon derivatives from recent years’ work against a collection of clinical isolates of VRE.

Materials and methods

General information

Optical rotations were measured at room temperature with a Perkin-Elmer 241 automatic polarimeter (Perkin-Elmer, Waltham, MA, USA). TLC was performed on Kieselgel 60 F254 (Merck, Darmstadt, Germany) with detection by immersing into 5% ethanolic sulfuric acid/ammonium molybdate solution followed by heating, or in the case of the teicoplanin derivatives, Pauly’s reagent was used for detection. Flash column chromatography was performed on Silica gel 60 (Merck 0.040–0.063 mm). Organic solutions were dried over Na2SO4 and concentrated in vacuum. The 1H NMR (360, 400 and 500 MHz) and 13C NMR (91, 101 and 125.76 MHz) spectra were recorded with Bruker DRX-360, DRX-400 and Bruker Avance II 500 spectrometers at 25 °C. Chemical shifts are referenced to Me4Si (0.00 p.p.m. for 1H) and to the solvent signals (dimethylsulfoxide (DMSO)-d6: 2.50 p.p.m. for 1H and CDCl3: 77.16 p.p.m., DMSO-d6: 39.52 p.p.m. for 13C). Matrix assisted laser desorption/ionization–time of flight MS (MALDI-TOF-MS) analyses of the compounds were carried out in the positive reflectron mode using a BIFLEX III mass spectrometer (Bruker, Bremen, Germany) equipped with delayed-ion extraction. 2,5-Dihydroxybenzoic acid was used as matrix and CF3COONa as cationizing agent in dimethylformamide (DMF). ESI-TOF MS spectra were recorded by a microTOF-Q type QqTOFMS mass spectrometer (Bruker) in the positive ion mode using MeOH as the solvent. Elemental analysis was performed on an Elementar Vario MicroCube instrument. The logP values were calculated using the logP calculation plugin of Marvin Sketch (version 16.5.2) from ChemAxon (Budapest, Hungary) using the Consensus Method with electrolyte concentrations of 0.1 M.

Bacterial strains

The Department of Medical Microbiology, Semmelweis University, Budapest, Hungary cultured and characterized 44 VRE strains. All the strains were isolated from routine laboratory samples of patients suffering from wound, urinary tract or blood stream infections. Clinical samples were cultured on COS agar (Columbia agar+5% sheep blood, Biomérieux, Budapest, Hungary), and vancomycin-resistant strains were isolated on VRE chromogenic medium (Biomérieux). Species of strains were identified by MALDI-TOF-MS. Minimum inhibitory concentrations (MICs; mg l−1) were determined for vancomycin and teicoplanin on Mueller Hinton E agar (Biomérieux using MIC strip tests (Liofilchem, Roseto degli Abruzzi, Italy), according to the manufacturer’s instructions, and by using a direct colony suspension equivalent to a McFarland standard of 0.5. Testing conditions also included incubation at 35.5 °C for 24 h. All results were interpreted by using breakpoints for susceptibility and resistance according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)22 Concerning vancomycin/teicoplanin MIC breakpoints, strains with an MIC>4 or 2 mg l−1 were considered to be vancomycin- or teicoplanin-resistant, respectively.

MIC of lipophilic teicoplanin pseudoaglycon derivatives

The efficacy of the prepared compounds was determined with the broth micro-dilution method according to the EUCAST guideline. Bacterial strains were grown on COS agar at 35.5 °C overnight. Appropriate numbers of colonies were suspended in physiological saline in order to reach the density of 0.5 McFarland for inoculation.

Stock solutions containing the substances were prepared in distilled water and DMSO (1:1). These were two-fold serially diluted from 20 to 0.044 mg l−1 in cation-adjusted Mueller–Hinton broth (Biolab, Budapest, Hungary) and 100 μl of each dilution was transferred into microplate holes. Inoculation was carried out with 10 μl of each bacterial suspension. Incubation was performed at 35 °C for 24 h and determination of MIC was made with the naked eyes on a black background.

Determination of vanA and vanB genes

Enterococcus strains were retrieved from storage at −80 °C on Cryobank breads (Mast Diagnostica, Reinfeld, Germany), cultured on COS agar and incubated overnight at 35.5 °C. A turbid suspension of the subculture was made in PCR water and DNA was isolated with the GeneAll Ribospin Extraction Kit (Geneall Biotechnology, Seoul, Korea).

The presence of glycopeptides-resistance genes was investigated by PCR using primers for the identification of vanA or vanB sequence described by Dutka-Malen et al.23 The full reaction mixture (25 μl) included 12.5 μl master mix (ImmoMix Red, BIOLINE, London, UK), 0.5 μl forward primer, 0.5 μl reverse primer, 6.5 μl PCR water and 5 μl DNA template. PCR thermal profile was 94 °C 2 min, (92 °C 1 min, 54 °C 1 min, 72 °C 1 min) 30 cycles, 72 °C 10 min. PCR products were detected by Gelgreen nucleic acid gel stain (BIOLINE) in 2% agarose under UV light.

Compound 1

Teicoplanin pseudoaglycon 10 (200 mg, 0.14 mmol) was dissolved in dry DMF (3 ml) and Et3N (20 μl) and compound 1124 (57.6 mg, 0.19 mmol) was added. After 3 h, TLC showed complete disappearance of starting material 10. The reaction mixture was concentrated and the residue was purified by flash column chromatography (toluene:MeOH 4:6+1% acetic acid) to give 1 (153 mg, 68%) as an off-white powder. MALDI-TOF-MS m/z 1611.35 (M+Na)+; calcd. 1611.32 for C74H70Cl2N8NaO24S2.

Compound 2

Teicoplanin pseudoaglycon 10 (200 mg, 0.14 mmol) was dissolved in dry DMF (3 ml) and Et3N (20 μl, 0.14 mmol) and compound 1224 (77.2 mg, 0.19 mmol) was added. After 3 h, TLC showed complete disappearance of starting material 10. The reaction mixture was concentrated and the residue was purified by flash column chromatography (toluene:MeOH 1:1+1% acetic acid) to give 2 (166 mg, 68%) as an off-white powder. MALDI-TOF-MS m/z 1724.45 (M+Na)+; calcd. 1724.41 for C80H81Cl2N9NaO25S2.

Methyl 2,3-di-O-n-butyl-4,6-O-p-methoxybenzylidene-α-D-glucopyranoside (14)

To the mixture of NaH (50% in mineral oil, 922 mg, 19.2 mmol) washed with hexane and dry DMF (30 ml), methyl 4,6-O-p-methoxybenzylidene-α-D-glucopyranoside 1325 (1.5 g, 4.8 mmol) was added, and the suspension was stirred for 45 min under argon. Then n-butyl bromide (1.55 ml, 14.4 mmol) was added in four portions in an hour. After two days, MeOH (2 ml) and distilled water (50 ml) was added carefully, and the mixture was stirred for half an hour. The precipitated solids were filtered off and purified by flash chromatography (n-hexane:acetone 9:1) to give 14 (1.30 g, 64%) as a white powder. [α]D25 +46.7 (c 0.08, CHCl3) MALDI-TOF-MS m/z 447.36 (M+Na)+; calcd. 447.24 for C23H36NaO7. 1H NMR (400 MHz, CDCl3) δ (p.p.m.): 7.43 (d, J=8.7 Hz, 2H, aromatic), 6.90 (d, 2H, J=8.7 Hz aromatic), 5.51 (s, 1H, H acetalic), 4.81 (d, J=3.6 Hz, 1H, H-1), 4.27 (dd, J=9.8, 4.5 Hz, 1H, CH2), 3.85–3.60 (m, 10H), 3.50 (t, J=9.3 Hz, 1H), 3.45 (s, 3H, OMe), 3.37 (dd, J=9.3, 3.7 Hz, 1H, H-2), 1.63–1.52 (m, 4H, butyl CH2), 1.46–1.33 (m, 4H, butyl CH2), 0.94 (t, J=7.4Hz, 3H, butyl CH3), 0.89 (t, J=7.4Hz, 3H, butyl CH3). 13C NMR (101 MHz, CDCl3) δ (p.p.m.): 160.3, 130.0, 127.3, 113.5 (6C, aromatic), 101.2 (1C, acetalic), 99.1 (1C, C-1), 82.0, 80.4, 78.3 (3C, skeleton), 73.4, 72.0 (2C, butyl CH2), 69.0 (1C, C-6), 62.4 (1C, C-5), 55.2 (2C, 2 OMe), 32.3, 32.1, 19.2, 19.1 (4C, butyl CH2), 13.8 (2C, butyl CH3).

Methyl 2,3-di-O-n-butyl-4-O-p-methoxybenzyl-α-D-glucopyranoside (15)

p-Methoxybenzylidene derivative 14 (1.20 g, 2.24 mmol) was dissolved in a mixture of dry CH2Cl2 (20 ml) and dry Et2O (7 ml) under an argon atmosphere. The stirred mixture was cooled to 0 °C, and LiAlH4 (382 mg, 10.1 mmol) was added in four portions. Dry Et2O (7 ml) was cooled to 0 °C by the use of drying tube filled with CaCl2, and AlCl3 (447 mg, 3.4 mmol) was added. The solution of AlCl3 was stirred for 5 min, then it was added to the previously prepared mixture containing the p-methoxybenzylidene derivative and it was stirred at 0 °C under an argon atmosphere. After 1 h, ethyl acetate (45 ml) was added to the reaction mixture and it was stirred for 10 min, then water (10 ml) was added, and the mixture was stirred for further 10 min. The mixture was filtered through a Celite pad (Sigma Aldrich, Budapest, Hungary), and the solid part was washed with ethyl acetate. The filtrate was diluted with ethyl acetate (100 ml) and washed twice with water (2 × 40 ml). The organic solution was dried and concentrated. The residue was purified by flash column chromatography (n-hexane:acetone 8:2) to yield 15 (902 mg, 75%) as a colorless syrup. [α]D25 +78.43 (c 0.07, CHCl3); MALDI-TOF-MS m/z 449.36 (M+Na)+; calcd. 449.25 for C23H38NaO7. 1H NMR (400 MHz, CDCl3) δ (p.p.m.): 7.29 (d, J=8.8 Hz, 2H, aromatic), 6.90 (d, J=8.6 Hz, 2H, aromatic), 4.85 (d, J=10.7 Hz, 1H, benzyl CH2), 4.78 (d, J=3.5 Hz, 1H, H-1), 4.60 (d, J=10.7 Hz, 1H, benzyl CH2), 3.94–3.86 (m, 1H); 3.82 (s, 3H, OMe), 3.81–3.57 (m, 4H), 3.46–3.44 (m, 1H), 3.40 (s, 3H, OMe), 3.30 (dd, J=9.6, 3.6 Hz, 1H, H-2), 1.67–1.57 (m, 4H, butyl CH2), 1.48–1.36 (m, 4H, butyl CH2), 0.95 (t, 6H, J=7.4 Hz, butyl CH3). 13C NMR (101 MHz, CDCl3) δ (p.p.m.): 159.3, 130.4, 129.8, 113.9 (6C, aromatic), 98.0 (1C, C-1), 81.6, 80.9, 77.3 (3C, skeleton), 74.6 (1C, benzyl CH2), 73.4, 71.4 (2C, butyl CH2), 70.6 (1C, C-5), 62.0 (1C, C-6), 55.2, 55.1 (2C, OMe), 32.7, 32.1, 19.4, 19.1 (4C, butyl CH2), 14.1, 14.0 (2C, butyl CH3).

Methyl 2,3-di-O-n-butyl-4-O-p-methoxybenzyl-6-O-(2,5,8,11,14-pentaoxaheptadec-16-ynyl)-α-D-glucopyranoside (16)

To the solution of sugar derivative 15 (500 mg, 1.17 mmol) in dry DMF (20 ml), NaH was added (50% in mineral oil, 112 mg, 2.34 mmol), and the mixture was stirred for 20 min under an argon atmosphere. The solution of triethylene glycol 2-bromoethyl propargyl ether26 (414 mg, 1.40 mmol) in dry DMF (2 ml) was added to the mixture and it was stirred overnight. Additional NaH (50%, 50 mg) and triethylene glycol 2-bromoethyl propargyl ether (50 mg) were added to the reaction mixture, and it was stirred at 60 °C for 2 days. The reaction was quenched by the addition of MeOH (2 ml) and water (2 ml) and the mixture was stirred for 10 min. After removing the solvents under vacuum, the residue was purified by flash chromatography (n-hexane:acetone 95:5) to yield 16 (360 mg, 48%) as a colorless syrup. [α]D25 +63.40 (c 1.4, CHCl3) ESI-TOF-MS m/z 663.3728 (M+Na)+; calcd. 663.3715 for C34H56NaO11. 1H NMR (400 MHz, CDCl3) δ (p.p.m.) 7.31–7.23 (m, 2H, aromatic); 6.90–6.84 (m, 2H, aromatic); 4.83-4.75 (m, 2H, benzyl CH2 and H-1); 4.57 (d, J=10.5 Hz, 1H, benzyl CH2); 4.19 (d, J=2.3 Hz, 2H, propargyl CH2); 3.89-3.81 (m, 1H); 3.80 (s, 3H, OMe); 3.76-3.53 (m, 23H); 3.49 (t, J=9.2 Hz, 1H); 3.38 (s, 3H, OMe); 3.30 (dd, J=9.7, 3.5 Hz, 1H); 2.43 (t, J=2.3 Hz, 1H, propargyl CH); 1.67-1.52 (m, 4H, butyl CH2); 1.46-1.32 (m, 4H, butyl CH2); 0.92-0.83 (m, 6H, butyl CH3). 13C NMR (101 MHz, CDCl3) δ (p.p.m.) 159.2, 130.7, 129.5, 113.7 (6C, aromatic), 98.0 (1C, C-1), 81.6, 80.7, 77.3, 70.0 (4C, skeleton), 74.6, 74.4, 73.3, 71.2, 70.8, 70.7, 70.6, 70.5, 70.4, 70.0, 69.8 (11C, OCH2 tetraethyleneglycol, OCH2 butyl, OCH2 PMB (p-methoxybenzyl)), 58.3 (1C, C-6), 55.2, 55.0 (2C, OMe), 32.63, 32.02, 19.33, 19.05, (4C, CH2 butyl), 14.0, 13.8 (2C, CH3 butyl).

Methyl 2,3-di-O-n-butyl-6-O-(2,5,8,11,14-pentaoxaheptadec-16-ynyl)-α-D-glucopyranoside (17)

Compound 16 (225 mg, 0.35 mmol) and DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone; 120 mg, 0.53 mmol) were dissolved in CH2Cl2:water 9:1 (25 ml), and the mixture was stirred vigorously. After 1 h, the reaction mixture was diluted with CH2Cl2 (300 ml), washed with saturated aq NaHCO3 (2 × 50 ml) and with water (50 ml). The organic phase was dried and concentrated. The residue was purified by flash column chromatography to yield 17 (153 mg, 84%) as a colorless syrup. [α]D25 +48.26 (c 0.09, CHCl3); MALDI-TOF-MS m/z 655.45 (M+Na)+; calcd. 655.44 for C34H64NaO10. 1H NMR (360 MHz, CDCl3) δ (p.p.m.): 4.79 (d, J=3.5 Hz, 1H, H-1), 4.21 (d, J=2.4 Hz, 2H, propargyl CH2), 3.91-3.82 (m, 1H), 3.80-3.48 (m, 25H); 3.41 (s, 3H, OMe), 3.29 (dd, J=9.1, 3.5 Hz, 1H, H-2), 2.45 (t, J=2.4 Hz, 1H, propargyl CH), 1.62-1.51 (m, 4H, butyl CH2), 1.43-1.32 (m, 8H, butyl CH2), 0.91 (t, J=7.4 Hz, 6H, CH3). 13C NMR (91 MHz, CDCl3) δ (p.p.m.): 98.0 (1C, C-1), 80.9, 80.3, 70.0 (3C, skeleton) 74.4, 73.1, 70.9, 70.7, 70.3, 70.0 (OCH2), 68.9 (1C, C-6), 58.2, 55.0 (1C, OMe), 32.3, 31.9, 19.1, 19.0 (4C, CH2 butyl), 13.8, 13.7 (2C, CH3 butyl).

Compound 4

To the solution of azido teicoplanin pseudoaglycon 1820 (150 mg, 0.11 mmol) and the propargyl derivative 16 (81 mg, 0.126 mmol) in dry DMF (5 ml), CuI (2 mg, 0.011 mmol) and Et3N (0.11 mmol, 15 μl) were added under an argon atmosphere and the mixture was stirred for 20 h. The mixture was concentrated and the residue was purified by flash column chromatography (toluene:MeOH 6:4+0.1% acetic acid) to yield 4 (84 mg, 39%) as a white powder. MALDI-TOF-MS m/z 2089.66 (M+Na)+; calcd. 2089.66 for C100H112Cl2N10NaO34.

Compound 5

To the solution of azido teicoplanin pseudoaglycon 18 (150 mg, 0.11 mmol) and the propargyl derivative 17 (66 mg, 0.126 mmol) in dry DMF (5 ml), CuI (2 mg, 0.01 mmol) and Et3N (0.11 mmol, 15 μl) were added under an argon atmosphere and the mixture was stirred for 2 days and at 60 °C for a day. The mixture was concentrated and the residue was purified by flash chromatography (toluene:MeOH 6:4+0.1% acetic acid) to yield 5 (98 mg, 48%) as a white powder. MALDI-TOF-MS m/z 1969.60 (M+Na)+; calcd. 1969.60 for C92H104Cl2N10NaO33.

Compound 20

To a stirred solution of compound 1927 (107 mg, 0.15 mmol) in CH2Cl2 (30 ml), propargyl amine (12 μl, 0.19 mmol) and Et3N (26 μl, 0.19 mmol) were added under an argon atmosphere and stirred overnight at room temperature. The reaction mixture was diluted with CH2Cl2 (100 ml), washed with cc. aq NH4Cl (20 ml) and water (2 × 20 ml), dried and concentrated. The crude product was purified by flash column chromatography (n-hexane:acetone 8:2) to give 20 (102 mg, 99%) as a yellow syrup. [α]D25=−87.5 (c 0.11, CHCl3); 1H NMR (360 MHz, CDCl3) δ (p.p.m.): 5.52 (d, J=5.0 Hz, 2H, 2xH-1), 4.70–4.52 (m, 2H), 4.44–4.13 (m, 4H), 4.06–3.85 (m, 2H), 3.62-3.56 (m, 2H), 3.46–3.30 (m, 2H), 2.17 (m, 1H), 1.63–1.51 (m, 2H), 1.46 (s, 6H), 1.44 (s, 6H), 1.33 (s, 6H), 1.32 (s, 6H). 13C NMR (91 MHz, CDCl3) δ 165.3 (2 × C=O), 127.6 (C=C), 113.8 (4 C, Cacetalic), 96.6 (2 C-1), 71.6, 71.0, 70.6, 68.0 (2 × C-2,3,4,5), 31.7 (2 × C-6), 26.0, 26.0, 24.9, 24.5 (8 C, CH3,ip).

Compound 6

To the solution of azido teicoplanin pseudoaglycon 18 (105 mg, 0.075 mmol) and the propargyl derivative 20 (68 mg, 0.09 mmol) in dry DMF (5 ml), CuI (1.4 mg, 0.008 mmol) and Et3N (10 μl, 0.075 mmol) were added under an argon atmosphere and the mixture was stirred overnight. The reaction mixture was concentrated, and the crude product was purified by flash chromatography (toluene:MeOH 1:1+1% acetic acid) and gel filtration by Sephadex-LH 20 gel in methanol to give 6 (61 mg, 41%) as a yellow powder. MALDI-TOF-MS m/z 2132.62 (M+Na)+; calcd. 2132.49 for C97H97Cl2N11NaO35S2.

Results and Discussion

On the basis of the routine antibacterial screening (Supplementary information, Supplementary Table S1), teicoplanin pseudoaglycon derivatives 1–9 (Table 1) have been selected to the present antibacterial study. Synthesis of 327 and 7–9 has been published recently.28

Table 1 Structure and lipophilicity of teicoplanin pseudoaglycon derivatives 1–9
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Lipoic acid derivatives 1 and 2 were prepared from N-acetylglucosaminyl teicoplanin aglycon29 10 by simple acylation reactions with hydroxysuccinimide active esters 11 and 1224 (Scheme 1).

In the framework of a systematic structure–anti-influenza virus activity study,30 compounds 4 and 5 have been prepared by derivatization of azido teicoplanin pseudoaglycon. The synthesis of the substituents 16 and 17 started from methyl 4,6-O-p-methoxybenzylidene-α-D-glucopyranoside 13.25 Di-O-n-butylation of 13 followed by reductive opening of the 4,6-acetal ring31, 32 of the obtained 14 gave 15. Subsequent alkylation of the liberated 6-hydroxyl group with a propargyl tetraethyleneglycol derivative produced 16, conjugation of which to azido teicoplanin pseudoaglycon 1820 by the copper catalyzed azide–alkyne dipolar cycloaddition click reaction33, 34 furnished compound 4. DDQ-mediated oxidative PMB deprotection of 16 resulted in 17. Then compound 5 was obtained using click reaction of 17 and 18 (Scheme 2).

In a search for antibacterial and antiviral teicoplanin pseudoaglycon derivatives, we have synthesized compound 6 by the copper catalyzed azide–alkyne click reaction of azide 18 with alkyne 20 obtained from N-ethoxycarbonyl maleimide derivative 1927 (Scheme 3).

Our derivatives were characterized using calculated logP values (Table 1). According to these calculations, eight of the nine compounds displayed logP values >3 at their isoelectric point, showing their high lipophilicity.

Evaluation of antibacterial activity on a standard panel of Gram-positives showed that compounds 1–6 are very active against the two Enterococcus strains possessing resistance genes vanA or vanB. Compounds 7–9 that we have published earlier also displayed high activity against resistant enterococci (Supplementary Table S1).

In order to obtain structure–activity relationship information in this collection of teicoplanin pseudoaglycon derivatives, we evaluated the antibacterial activity of compounds 1–9 against a collection of 44 vancomycin-resistant Enterococcus faecalis and Enterococcus faecium strains isolated from routine laboratory samples of patients suffering from wound, urinary tract or blood stream infections. All the strains were characterized using PCR using primers for the identification of vanA- or vanB-resistance gene sequences. Eight of the strains proved to have vanB, 32 strains had vanA and interestingly 3 of them possessed both genes.35 The prevalence of VREs carrying the vanA gene is higher owing to the enhanced mobility of the mentioned gene cluster.36

Against the first group of enterococci (the VanB group, Table 2, lines 1–8), all of our nine compounds exhibited low MIC values, mostly lower than that of teicoplanin. In the evaluation of antibacterial activities against the vanA-positive group of E. faecalis and E. faecium (Table 3, lines 9–41) and for the group of three strains possessing both resistance genes (Table 4, lines 42–44), the same compounds displayed diverse results. Four of them (3, 4, 7 and 8) proved to be active against about one-third of the Enterococcus strains; the other compounds had moderate or low activity. Analyzing the data further in Tables 3 and 4, it can be noticed that some of the strains are completely resistant to all of our teicoplanin pseudoaglycon derivatives, and some of them are more or less sensitive (see Table 5 for details). This phenomenon might be related to the different degrees of expression of the resistance genes in the different strains.37

Table 2 MIC of compounds 1–9 against VanB enterococci in μg ml−1
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Table 3 MIC of compounds 1–9 against VanA enterococci in μg ml−1
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Table 4 MIC of compounds 1–9 against VanA and VanB enterococci in μg ml−1
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Table 5 Number of vancomycin- and teicoplanin-resistant Enterococcus strains susceptible to teicoplanin pseudoaglycon derivatives 1–9
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The logP calculations for the whole collection of compounds revealed that all of them are lipophilic compounds with logP values close to or >3. Based on the results presented here and in our previous articles,16, 17, 18, 19, 20, 21, 27, 28 it seems that there is no linear correlation between the lipophilicity of glycopeptide antibiotics and their antibacterial activity against VRE strains, but the presence of a hydrophobic moiety on the antibiotic is critical in most cases.

It has been shown that other attributes than lipophilicity can also substantially influence the activity of glycopeptide derivatives; for example, the overall charge38 and the capability of the compounds to dimerize39 in the presence of their target, D-Ala-D-Ala, and obviously the structure of the sidechain itself. As all molecules presented here are of acidic character, they are uniformly negatively charged at the pH of the antibacterial tests (close to neutral pH). As the differences in activity owing to ionization characteristics can be ruled out and the dimerization of teicoplanin, its aglycon or pseudoaglycons was also shown to be insignificant,40 we can conclude that among these derivatives the structure of the substituents is the most important factor in their activity against VRE. Considering these and some of our other results (Szucs Z, unpublished data) relatively simple aromatic substituents like phenyl, biphenyl, naphthyl, etc. seem to be very beneficial for high in vitro antibacterial activity.

In summary, evaluation of antibacterial activities of nine teicoplanin pseudoaglycon derivatives against a series of resistant enterococci demonstrated that introduction of hydrophobic side chains into the antibiotic molecule, in spite of the possible side effects of this structure manipulation, is still a good strategy in fighting against bacterial resistance. Compounds 4, 7 and 8 could be good lead derivatives for obtaining new highly active antibiotics against multiresistant enterococci.

scheme 1

Conjugation of lipoic acid derivatives 11 and 12 to the N-terminal part of teicoplanin pseudoaglycon.

scheme 2

Derivatization of azido teicoplanin pseudoaglycon with sugar-based lipophilic moieties by CuAAC.

scheme 3

Coupling of bis-(diisopropylidene-α-D-Galp-6-thio)-maleimide 20–18 by CuAAC.