Synthesis and antibacterial evaluation of some teicoplanin pseudoaglycon derivatives containing alkyl- and arylthiosubstituted maleimides

Abstract

Bis-alkylthio maleimido derivatives have been prepared from teicoplanin pseudoaglycon by reaction of its primary amino group with N-ethoxycarbonyl bis-alkylthiomaleimides. Some of the new derivatives displayed excellent antibacterial activity against resistant bacteria.

Introduction

Glycopeptide antibiotics exert their antibacterial activity by inhibiting two sequential enzymatic reactions—transglycosylation and transpeptidation—in the bacterial cell-wall biosynthesis. The antibiotics recognize and tightly bind to the L-Lys-D-Ala-D-Ala termini of peptidoglycan precursors at the external side of the developing bacterial membrane. In this way transglycosylation and transpeptidation are physically prevented, arresting cell-wall elongation and cross-linking and leading to cell lysis.¹ Due to the lack of cross-resistance to other antibacterial drugs, the glycopeptide antibiotics have become first-line drugs for the treatment of life-threatening multi-drug resistant infections by Gram-positive bacteria.²

The emergence and spread of glycopeptide-resistant enterococci and glycopeptide intermediate-resistant Staphylococcus aureus, as well as teicoplanin-resistant Staphylococcus haemolyticus³ present a serious global challenge and have led to renewed interest in the development of novel, effective and safe antibacterials including new derivatives of glycopeptide antibiotics.^{4, 5, 6}

Inspired by the high activity of the semisynthetic lipoglycopeptide antibiotics telavancin,⁷ dalbavancin⁸ and oritavancin⁹ against vancomycin-resistant bacteria, we have started a program to produce new antibiotics by introducing lipophilic subtituents to the primary amino function of ristocetin aglycon and of teicoplanin pseudoaglycon. Applying various approaches including squaric acid conjugation method, azide-alkyne cycloaddition reaction or three-component isoindole formation, we have prepared a large set of new derivatives exhibiting high antibacterial^{10, 11, 12, 13} and, in some cases, robust anti-influenza virus activity.^{14, 15, 16, 17}

Recently, Caddick, Baker and coworkers^{18, 19, 20, 21} reported on applications of 3,4-dibromomaleimides for site-specific protein modification and bioconjugation. The method is based on addition–elimination reaction of thiols to the bromomaleimides leading to regeneration of the double bond resulting in thiomaleimide products (Scheme 1). Last year the group of Caddick and Baker published a simple method for the synthesis of N-functionalised bromo- and thiomaleimides through the corresponding N-ethoxycarbonyl maleimide derivatives.²² Applying these recent results of maleimide chemistry we describe here derivatisation of teicoplanin pseudoaglycon with thiomaleimide substituents carrying two lipophilic alkyl or aryl sulfide side chains.

Results and Discussion

Dibromomaleimide (1) that can be obtained by simple bromination of maleimide²³ has been allowed to react with a range of thiols including the 6-thio-D-galactose derivative 2a, thiophenol 2b, phenylmethanethiol 2c, dodecanethiol 2d, octanethiol 2e, propanethiol 2f and t-butyl mercaptane 2h, representing a series of substituents of different lipophilicity.

The obtained sulfides 3a–g have been then ethoxycarbonylated with ethyl chloroformate in the presence of potassium carbonate to provide 5a–g, ready for a reaction with a primary amino group (Scheme 2). Direct methoxycarbonylation²² of dibromomaleimide offers an alternative route for the synthesis of the targeted N-functionalized dithiomaleimide as it is illustrated by the synthesis of 6g. We tested this route with several thiols such as 2d–2g, however, the sulfide formation showed low efficacy in all cases.

Next, teicoplanin pseudoaglycon 7¹⁶ has been reacted with N-ethoxycarbonyl maleimides 5a–g and 6g in the presence of triethylamine (Table 1). In these reactions bis-alkyl- or arylthiomaleimide 8a–f were formed in moderate yields, together with the N-alkoxycarbonyl derivatives of the teicoplanin pseudoaglycon (9 and 10). The formation of 9 and 10 can be explained by the steric hindrance of the amino function of 7. In the case of 5g and 6g, the undesired carbamate derivatives 9 and 10 were dominantly formed, probably due to the presence of bulky t-butyl substituents of the reagents.

Table 1 Synthesis and structure of teicoplanin pseudoaglycon-maleimide conjugates

Antibacterial activity of maleimido-teicoplanin-pseudoaglycons was evaluated on a panel of Gram-positive bacteria (Table 2). The D-galactose-containing 8a, the bis-phenylthio derivative 8b and the bis-benzylthio derivative 8c displayed similar activities than teicoplanin pseudoaglycon 7 with one exception: the maleimido compounds 8a–c were active against Enterococcus faecalis 15 376 having vanA resistance gene while teicoplanin and 7 were completely inactive against this bacterium strain.

Table 2 Antibacterial activity of compounds 7–10

The detected antibacterial activities of 8d, 8e and 8f were related to the length of the alkyl chain substituents of their maleimide residues. The bis-dodecyl derivative 8d was inactive, the bis-octyl derivative 8e was a weak antibacterial and the bis-propylthio compound 8f displayed very high activity. It can be supposed that a correlation exists between lipophilicity of the maleimide substituents and antibacterial activity, and the high lipophilicity erodes the activity. To test this hypothesis, logP (logarithm of partition coefficient between n-octanol and water) values were calculated for N-methyl maleimide derivatives 11a–f and the calculated logP values corroborate our postulation (Table 3).

Table 3 Calculated logP for N-methyl maleimide derivatives 11a–f

In conclusion we have utilized, for the first time, bis-sulfide derivatives of N-alkoxycarbonyl maleimide for versatile derivatisation of teicoplanin pseudoaglycon. It turned out that lipophilicity of substituents of the maleimide ring has strong influence on the antibacterial activity of these derivatives. Further synthetic tuning of these chemical structures hopefully will result in even more effective antibacterials.

Experimental procedure

General information

Maleimide and thiols 2b–2g were purchased from Sigma-Aldrich Chemical (St Louis, MO, USA). 2,3-Dibromomaleimide 1, 1,2:3,4-di-O-isopropylidene-6-deoxy-6-thio-α-D-galactopyranose 2a and teicoplanin pseudoaglycon 7 were prepared according to literature procedures. TLC analysis was performed on Kieselgel 60 F₂₅₄ (Merck, Darmstadt, Germany) silica gel plates with visualization by immersing in ammonium-molibdate solution followed by heating or Pauly-reagent in the case of teicoplanin derivatives. Column chromatography was performed on silica gel 60 (Merck 0.063–0.200 mm), flash column chromatography was performed on silica gel 60 (Merck 0.040–0 0.063 mm). Organic solutions were dried over MgSO₄ and concentrated under vacuum. The ¹H (400 and 500 MHz) and ¹³C NMR (100.28, 125.76 MHz) spectra were recorded with Bruker DRX-400 and Bruker Avance II 500 spectrometers. Chemical shifts are referenced to Me₄Si or DSS (2,2-Dimethyl-2-silapentane-5-sulfonate sodium salt) (0.00 p.p.m. for ¹H) and to solvent signals (CDCl₃: 77.00 p.p.m., DMSO-d₆: 39.51 p.p.m. for ¹³C). MALDI-TOF MS analyses for the compounds 8b, 8c, 8e, 9 and 10 were carried out in positive reflectron mode using a BIFLEX III mass spectrometer (Bruker, Bremen, Germany) equipped with delayed-ion extraction. In the case of 8a, 8d and 8f, Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) MS spectra were recorded by a Voyager-DE STR MALDI-TOF Biospectrometry Workstation (Applied Biosystems, Budapest, Hungary). 2,5-Dihydroxybenzoic acid was used as matrix and CF₃COONa as cationising agent in DMF. Elemental analysis (C, H, S) was performed on an Elementar Vario MicroCube instrument. The antibacterial activity of 8a–f, 9 and 10 was tested against a panel of Gram-positive bacteria using broth microdilution method as described earlier.²⁴

General method A for preparation maleimide bis-sulfides (3a–3g)

To a stirred solution of 2,3-dibromomaleimide²³ (1.0 mmol) in CH₂Cl₂ (20 ml) Et₃N (2.0 mmol) and thiol (2.1 mmol) were added under argon atmosphere and stirred for 3 h at room temperature. The reaction mixture was evaporated, and the crude product was purified by flash chromatography to give the desired compound.

General method B for preparation N-ethoxycarbonyl maleimide bis-sulfides (5a–5g)

To a stirred solution of maleimide bis-sulfide (1.0 mmol) in dry acetone (20 ml) K₂CO₃ (1.2 mmol) and ethyl chloroformate (1.2 mmol) were added under argon atmosphere and stirred for 3 h at room temperature. The reaction mixture was diluted with CH₂Cl₂, filtered through a pad of Celite and evaporated. The crude product was used for further step without purification.

General method C for the synthesis of teicoplanin pseudoaglycon derivatives (8a–8f)

To a stirred solution of teicoplanin pseudoaglycon¹⁶ (0.1 mmol) in dry DMF (5 ml) N-ethoxycarbonyl maleimide bis-sulfides (0.14 mmol) and Et₃N (0.1 mmol) were added under argon atmosphere and stirred for overnight at room temperature. The reaction mixture was evaporated, and the crude product was purified by flash chromatography to give the desired compound.

Compound 3a

2,3-Dibromomaleimide (255 mg, 1.0 mmol) was reacted with thiol 2a²⁵ (580.4 mg, 2.1 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:acetone=8:2, to give 3a (550 mg, 85%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.58 (1H, s, NH), 5.51 (2H, d, J_1,2=0.3 Hz, 2 × H-1), 4.62 (2H, d, J_2,3=8.0 Hz, 2 × H-2), 4.32–4.30 (4H, m, 2 × H-3, 2 × H-4), 3.98–3.95 (2H, m, 2 × H-5), 3.57–3.36 (4H, m, 2 × H-6a,b), 1.48, 1.44, 1.33, 1.32 (24H, 4 × s, 8 × CH₃-ip); ¹³C NMR (100 MHz, CDCl₃) δ 165.8 (2C, 2 × C=O), 137.2, 136.9 (2C, C=C), 109.5, 108.7 (4C, 4 × C_q-ip), 96.5 (2C, 2 × C-1), 71.5, 70.9, 70.4, 67.9 (8C, skeleton carbons), 31.6 (2C, 2 × C-6), 25.9, 24.9, 24.4 (8C, 8 × CH₃); analysis calculated for C₂₈H₃₉NO₁₂S₂ C 52.08, H 6.09, N 2.17, O 29.73, S 9.93. Found: C 51.99, H 6.08, S 9.90.

Compound 3b

2,3-Dibromomaleimide (255 mg, 1.0 mmol) was reacted with thiophenol 2b (215 μl, 2.1 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:acetone=8:2, to give 3b (310 mg, 98%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (1H, s, NH), 7.29–7.17 (10H, m, arom); ¹³C NMR (100 MHz, CDCl₃) δ 166.5 (2C, 2 × C=O), 136.8 (2C, C=C), 131.9, 129.1, 128.6 (10C, arom), 128.9 (2C, C_q arom); analysis calculated for C₁₆H₁₁NO₂S₂ C 61.32, H 3.54, N 4.47, O 10.21, S 20.46. Found: C 61.15, H 3.53, S 20.39.

Compound 3c

2,3-Dibromomaleimide (510 mg, 2.0 mmol) was reacted with benzyl mercaptan 2c (490 μl, 4.2 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:acetone=8:2, to give 3c (460 mg, 67%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (1H, s, NH), 7.29–7.26 (10H, m, arom), 4.42 (4H, s, 2 × SCH₂); ¹³C NMR (100 MHz, CDCl₃) δ 175.3, 166.3 (2C, 2 × C=O), 136.5 (2C, C=C), 128.9, 128.8, 128.7, 127.7 (10C, arom), 36.2 (2C, 2 × SCH₂); analysis calculated for C₁₈H₁₅NO₂S₂ C 63.32, H 4.43, N 4.10, O 9.37, S 18.78. Found: C 63.19, H 4.45, S 18.69.

Compound 3d

2,3-Dibromomaleimide (510 mg, 2.0 mmol) was reacted with dodecyl mercaptan 2d (950 μl, 4.2 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:ethyl acetate=9:1, to give 3d (670 mg, 67%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.55 (1H, s, NH), 3.29–3.25 (4H, m, 2 × SCH₂), 1.64–1.25 (40H, m, 20 × CH₂), 0.89–0.86 (6H, m, 2 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 165.8 (2C, 2 × C=O), 136.4 (2C, C=C), 31.5, 31.4, 30.2, 29.3, 29.1, 28.9, 28.7, 28.1 (20C, 20 × CH2), 22.3 (2C, 2 × SCH₂), 13.7 (2C, 2 × CH₃). Analysis calculated for C₂₈H₅₁NO₂S₂ C 67.55, H 10.33, N 2.81, O 6.43, S 12.88. Found: C 66.59, H 10.23, S 12.03.

Compound 3e

2,3-Dibromomaleimide (255 mg, 1.0 mmol) was reacted with octyl mercaptan 2e (364 μl, 2.1 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:acetone=8:2, to give 3e (317 mg, 82%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (1H, s, NH), 3.28 (4H, t, J=7.5 Hz, 2 × SCH₂), 1.69–1.60 (8H, m, 4 × CH₂), 1.43–1.27 (20H, m, 10 × CH₂), 0.88 (6H, t, J=6.8 Hz, 2 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 166.3 (2C, 2 × C=O), 136.7 (2C, C=C), 31.8, 30.5, 29.0, 28.5 (12C, 12 × CH₂), 22.6 (2C, 2 × SCH₂), 14.0 (2C, 2 × CH₃); analysis calculated for C₂₀H₃₅NO₂S₂ C 62.29, H 9.15, N 3.63, O 8.30, S 16.63. Found: C 61.03, H 9.08, S 16.08.

Compound 3f

2,3-Dibromomaleimide (510 mg, 2.0 mmol) was reacted with propyl mercaptane 2f (380 μl, 4.2 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:ethyl acetate=9:1, to give 3f (430 mg, 87%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (1H, s, NH), 3.28–3.25 (4H, m, 2 × SCH₂), 1.73–1.66 (4H, m, 2 × CH₂), 1.06–1.02 (6H, m, 2 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 166.3 (2C, 2 × C=O), 137.2 (2C, C=C), 33.6 (2C, 2 × CH₂), 23.8 (2C, 2 × SCH₂), 13.1 (2C, 2 × CH₃); analysis calculated for C₁₀H₁₅NO₂S₂ C 48.95, H 6.16, N 5.71, O 13.04, S 26.14. Found: C 48.18, H 5.70, S 26.01.

Compound 3g

2,3-Dibromomaleimide (510 mg, 2.0 mmol) was reacted with t-butyl mercaptane 2g (473 μl, 4.2 mmol) according to general method A. The crude product was purified by silica gel chromatography in n-hexane:ethyl acetate=9:1, to give 3g (432 mg, 80%) as a yellow sirup. ¹H NMR (400 MHz, CDCl₃) δ 8.09 (1H, s, NH), 1.54 (18H, s, 6 × CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 166.9 (2C, 2 × C=O), 145.3 (2C, C=C), 51.9 (2C, 2 × SC_q), 32.2 (6C, 6 × CH₃); analysis calculated for C₁₂H₁₉NO₂S₂ C 52.71, H 7.00, N 5.12, O 11.70, S 23.46. Found: C 51.66, H 6.93, S 22.89.

Compound 6g

To a stirred solution of 2,3-dibromomaleimide (0.255 g, 1.0 mmol) in tetrahydrofuran (4 ml) N-methylmorpholine (76 μl, 1.1 mmol) and methyl chloroformate (85 μl, 1.1 mmol) were added at 0 °C. When TLC (n-hexane:acetone=8:2) showed complete conversion of the starting material (3 h), the reaction mixture was diluted with CH₂Cl₂, filtered through a pad of Celite and evaporated. The obtained crude 4 (0.308 g) was reacted, without purification, with t-butyl mercaptan 2g (237 μl, 2.1 mmol) according to general method A to give compound 6g (0.150 g). The crude product was used for further step without purification.

Compound 8a

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5a (100 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=8:2, to give 8a (30 mg, 15%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=2051.39 m/z. Calcd for C₉₄H₉₄Cl₂N₈O₃₅S₂Na 2051.45 m/z.

Compound 8b

Teicoplanin pseudoaglycon (140 mg 0.1 mmol) was reacted with compound 5b (58 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=8:2, to give 8a (35 mg, 21%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1719.41 m/z. Calcd for C₈₂H₆₆Cl₂N₈O₂₅S₂Na 1719.29 m/z.

Compound 8c

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5c (41 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=7:3, to give 8c (27 mg, 16%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1747.47 m/z. Calcd for C₈₄H₇₀Cl₂N₈O₂₅S₂Na 1747.32 m/z.

Compound 8d

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5d (74 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=9:1, to give 8d (85 mg, 44%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1903.66 m/z. Calcd for C₉₄H₁₀₆Cl₂N₈O₂₅S₂Na 1903.60 m/z.

Compound 8e

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5e (69 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=8:2, to give 8d (38 mg, 22%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1791.64 m/z. Calcd for C₈₆H₉₀Cl₂N₈O₂₅S₂Na 1791.47 m/z.

Compound 8f

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5f (40 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=9:1, to give 8d (110 mg, 66%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1651.02 m/z. Calcd for C₇₆H₇₀Cl₂N₈O₂₅S₂Na 1651.32 m/z.

Compound 9

Teicoplanin pseudoaglycon (140 mg, 0.1 mmol) was reacted with compound 5g (49 mg, 0.14 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=9:1, to give 9 (87 mg, 59%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1495.34 m/z. Calcd for C₆₉H₆₂Cl₂N₈O₂₅Na 1495.31 m/z.

Compound 10

Teicoplanin pseudoaglycon (210 mg, 0.15 mmol) was reacted with compound 6g (70 mg, 0.21 mmol) according to general method C. The crude product was purified by silica gel chromatography in toluene:methanol=8:2, to give 10 (120 mg, 48%) as a yellow powder. MALDI-TOF MS: [M+Na]⁺=1481.51 m/z. Calcd for C₆₈H₆₀Cl₂N₈O₂₅Na 1481.29 m/z.

NMR analysis

The ¹H and ¹³C NMR data of the teicoplanin derivatives 8a–f, 9 and 10 are collected in Tables 4 and 5. The spectra were recorded at 500.13/125.76 MHz frequencies, respectively, at 300 K, using DMSO-d₆, as solvent. Numbering atoms in teicoplanin derivatives are given in Figure 1. Signal assignments were aided by 2D HSQC, TOCSY (15 and 60 ms mixing times) and HMBC (60 ms mixing time) experiments.

Table 4 ¹H and ¹³C NMR data for compounds 8a, 8b, 8c and 8d (chemical shifts in ppm)

Table 5 ¹H and ¹³C NMR data for compounds 8e, 8f, 9 and 10 (chemical shifts in p.p.m.)

Figure 1

Structure and numbering for compounds 8a–f, 9 and 10. A full color version of this figure is available at The Journal of Antibiotics journal online.

Reaction of thiols with 3,4-dibromomaleimide.

Synthesis of N-alkoxycarbonylated di-alkyl/arylthio-maleimide derivatives.