The spread of antibiotic-resistant bacteria poses a substantial threat with high morbidity and mortality worldwide. The discovery of new scaffolds derived by chemical synthesis could open the way to alternative classes of antimicrobial agents with new mechanism of action and activity against multidrug resistant (MDR) species of bacteria and fungi [1, 2]. Maleimide derivatives are known for their wide spectrum of biological activity [3,4,5,6]. The compounds of interest—3,4-bis(phenylthio)maleimides—are widely used by researchers for the purpose of incorporating fluorescent tags into proteins or other modifications of proteins and hormones, including binding them to a polymer [7,8,9,10].

Also bis-(alkylthio)maleimide 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 [11]. Bis(benzylthio)maleimide derivatives of teicoplanin pseudoaglycon showed good activity against vancomycin- and teicoplanin-resistant enterococci [12]. Antimicrobial activity of 3-(arylthio)maleimide derivatives was reported [13], but antimicrobial activity of 3,4-bis(arylthio)maleimides was not studied before. In the present work such compounds are shown to be rather active antimicrobial agents, capable of overcoming bacterial MDR.

The symmetrical 3,4-bis(arylthio)maleimides 3a–g were synthesized by the reaction of 3,4-dibromomaleimide with two equivalents of various thioaryl derivatives and triethylamine in THF (Scheme 1).

Scheme 1
scheme 1

Synthesis of 3,4-bis(arylthio)maleimides 3a-g

We failed to isolate monosubstituted products 2 even when 0.3 equivalents of thiophenol were used, carrying out the reaction at 0 °C. Changing the solvent to less polar (i.e. toluene) had no effect. We suggest that 3-bromo-4-(arylthio)maleimide intermediate 2 is much more reactive than dibromomaleimide 1, resulting in a mixture of disubstituted product 3 and unreacted 1.

All compounds 3a–g were tested against Gram-positive and Gram-negative bacteria and fungi. The minimum inhibitory concentrations (MIC) for Gram-positive and Gram-negative bacteria were determined by the microdilution method in a cation-adjusted Müller–Hinton medium in accordance with the requirements of the Institute of Clinical and Laboratory Standards (CLSI/NCCLS) [14]. The activity of the test compounds against various cultures of yeast and mycelial fungi was estimated using the recommendation by CLSI/NCCLS micromethod [15, 16] by twofold serial dilutions in the nutrient medium RPMI 1640 (liquid, with l-glutamine and without sodium bicarbonate). Observed MIC are presented in the following Tables 1 and 2.

Table 1 MIC (μg/ml) against bacterial strains
Table 2 MIC (μg/ml) against fungi strains

All compounds were active against Gram-positive S. aureus, S. epidermidis and S. haemoliticus. Compounds 3f and 3g, which both have an N-methyl group at the maleimide moiety, are generally less active against almost all strains, at the same time being more active against Gram-negative Escherichia coli and Pseudomonas aeruginosa. However, compounds 3d and 3e show an opposite pattern. All compounds turned out to be less active than the reference compound levofloxacin on all but three bacterial strains. In comparison to 3-(arylthio)maleimide derivatives [13] our compounds show mostly similar level of activity, but wider spectrum of action.

Some compounds were also tested of fungi strains (Table 2). Even the most active compound 3g is less active than the reference compound—amphotericin B.

All the reagents were obtained commercially and used without further purification. Melting points were determined by an open capillary method and are uncorrected. Purity of the compounds was checked by thin-layer chromatography using silica-gel 60 F254-coated Al plates (Merck) and spots were observed under UV light. 1H NMR and 13C NMR (in DMSO-d6) spectra were recorded on a Varian VXR-400 spectrometer at 400 MHz and 100 MHz, respectively; the chemical shift values are expressed in ppm (δ scale) using tetramethylsilane as an internal standard. The mass spectral measurements were carried out by ESI method on micrOTOF-QII (Brucker Daltonics GmbH).

General method for synthesizing 3,4-bis(arylthio)maleimides 3a-g: To the solution of dibromomaleimide 1a or N-methyldibromomaleimide 1b (1 mmol) in THF (20 ml) was added solution of the thiophenol (2.2 mmol) and triethylamine (2.2 mmol) in one portion. The resulting solution was stirred at room temperature for 1 h, then evaporated in vacuo and the residue was redissolved in ethyl acetate-water (20 + 20 ml) mixture. The organic layer was separated, washed with aq. NaHCO3, dried over anhydrous Na2SO4 and evaporated. The residue was purified by flash chromatography (ethyl acetate: petroleum ether 3:1).

3,4-Bis((4-fluorophenyl)thio)-1H-pyrrole-2,5-dione (3a) was obtained as a yellow solid, yield 64%. Mp 145–146 °C (diethyl ether). 1H NMR: δ 7.13 (4 H, t, J = 9.7 Hz), 7.29–7.32 (4 H, m), 11.37 (1 H, s, NH) 13C NMR: δ 115.95, 116.18, 124.68, 133.25, 133.33, 135.71, 160.77, 163.21, 167.57. ESI-HRMS: calculated for C16H9F2NO2S2 [M + H]+ 350.0116, found m/z 350.0107.

3,4-Bis((4-chlorophenyl)thio)-1H-pyrrole-2,5-dione (3b) was obtained as a yellow solid, yield 77%. Mp 165–166 °C (diethyl ether). 1H NMR: δ 7.26 (4 H, d, J = 8.5 Hz), 7.32 (4 H, d, J = 8.5 Hz), 11.43 (1 H, s, NH) 13C NMR: δ 128.20, 128.87, 132.32, 132.83, 135.80, 167.48. ESI-HRMS: calculated for C16H9Cl2NO2S2 [M + H]+ 381.9525, found m/z 381.9542.

3,4-Bis(o-tolylthio)-1H-pyrrole-2,5-dione (3c) was obtained as a yellow solid, yield 50%. Mp 149–151 °C. 1H NMR: δ 2.10 (6 H, s, CH3), 7.13 (10 H, m), 11.30 (1 H, s, NH). 13C NMR: δ 20.52, 126.88, 128.79, 129.04, 130.72, 132.48, 136.26, 139.32, 168.07. ESI-HRMS: calculated for C18H15NO2S2 [M + H]+ 342.0617, found m/z 342.0612.

3,4-Bis((4-methoxyphenyl)thio)-1-methyl-1H-pyrrole-2,5-dione (3d) was obtained as a red amorphous solid, yield 72%. 1H NMR: δ 2.82 (3 H, s, CH3), 3.73 (6 H, s, OCH3), 6.86 (4 H, d, J = 8.9 Hz), 7.26 (4 H, d, J = 8.9 Hz). 13C NMR δ 24.80, 39.49, 39.70, 39.91, 40.12, 40.33, 55.76, 115.16, 119.93, 134.10, 136.05, 160.08, 166.85. ESI-HRMS: calculated for C19H17NO4S2 [M + H]+ 388.0672, found m/z 388.0726.

3,4-Bis((4-methoxyphenyl)thio)-1H-pyrrole-2,5-dione (3e) was obtained as a red amorphous solid, yield 65%. 1H NMR: δ 3.74 (6 H, s, OCH3), 6.87 (4 H, d, J = 8.8 Hz), 7.26 (4 H, d, J = 8.8 Hz) 13C NMR: δ 55.30, 114.70, 119.64, 133.58, 136.13, 159.58, 167.51. ESI-HRMS: calculated for C20H19NO6S2 [M + H]+ 434.0727, found m/z 434.0697.

1-Methyl-3,4-bis(phenylthio)-1H-pyrrole-2,5-dione (3f) was obtained as a yellow solid, yield 80%. Mp 105–106 °C. 1H NMR: δ 2.90 (3 H, s, CH3), 7.21–7.31 (10 H, m) NMR 13C δ: 24.53, 127.92, 129.02, 130.71, 135.81, 166.50. ESI-HRMS: calculated for C21H21NO6S2 [M + H]+ 328.0460, found m/z 328.0486.

3,4-Bis((4-chlorophenyl)thio)-1-methyl-1H-pyrrole-2,5-dione (3g) was obtained as a yellow solid, yield 77%. Mp 120–121 °C. 1H NMR: δ 2.88 (3 H, s, CH3), 7.32 (4 H, d, J = 8.5 Hz), 7.26 (4 H, d, J = 8.5 Hz). NMR 13C δ: 25.01, 128.56, 129.35, 132.77, 133.36, 135.80, 166.87. ESI-HRMS: calculated for C17H11Cl2NO2S2 [M + H]+ 395.9681, found m/z 395.9662.