Synthesis and Antimicrobial Activity of 4-Substituted 1,2,3-Triazole-Coumarin Derivatives

A new series of coumarin-1,2,3-triazole conjugates with varied alkyl, phenyl and heterocycle moieties at C-4 of the triazole nucleus were synthesized using a copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of corresponding O-propargylated coumarin (3) or N-propargylated coumarin (6) with alkyl or aryl azides. Based on their minimal inhibitory concentrations (MICs) against selected microorganisms, six out of twenty-six compounds showed significant antibacterial activity towards Enterococcus faecalis (MIC = 12.5–50 µg/mL). Moreover, the synthesized triazoles show relatively low toxicity against human erythrocytes.


Introduction
Antimicrobial resistance has been listed by the World Health Organization (WHO) as one of the biggest threats to global health today [1]. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements [2][3][4][5][6]. Over the past decade, it has become apparent that several highly resistant bacterial pathogens have acquired clever mechanisms to negate the effectiveness of numerous therapeutic agents [7]. Staphylococcus aureus is one bacterial pathogen that has emerged as a significant concern to healthcare professionals worldwide. In this sense, isolated strains of S. aureus have exhibited resistance to several classes of antibacterial drugs, including β-lactam antibiotics [8], macrolides [9], fluoroquinolones [10][11][12], glycopeptides [13] and oxazolidinones [14]. Enterococci were previously considered commensal organisms of little clinical importance but have emerged as serious nosocomial pathogens responsible for e.g. endocarditis and infections of the urinary tract, bloodstream, meninges, wounds and the biliary tract [15]. Recent surveillance data indicate that Enterococcus is the third most commonly isolated nosocomial pathogen (12% of all hospital infections), only behind coagulase-negative Staphylococcus and Staphylococcus aureus [16]. The clinical importance of the genus Enterococcus is directly related to its antibiotic resistance, which contributes to the risk of colonization and infection. Enterococci are intrinsically resistant to many commonly used antimicrobial agents (penicillins, ampicillins, cephalosporins, clindamycin) and exhibit native resistance to clinically achievable concentrations of aminoglycosides. Although E. faecalis is naturally resistant to quinupristin-dalfopristin, this combination is highly active against E. faecium strains that lack specific resistance determinants. Enterococci are tolerant to the (normally) bactericidal activity of cell-wall active agents, such as β-lactam antibiotics and vancomycin. Tolerance implies that the The 4-substituted 1,2,3-triazole-coumarin derivatives were synthesized using a copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction [46] of the corresponding O-propargylated coumarin (3) or N-propargylated coumarin (6) with alkyl or aryl azides (Scheme 2). (3) and N-propargylated coumarin (6).
As can be seen, two isosteric series of coumarin derivatives were obtained (X=O, X=NH). Each series presents different substituents at the triazole moiety in order to evaluate their influence on the antimicrobial activity. Thus, coumarin derivatives with an aromatic ring having electron-donating groups or electron-withdrawing groups were prepared (8a-8h, 9a-9h). Coumarin-triazole derivatives with alkyl moieties (8k, 9k, 8l, 9l) and coumarin-indole hybrids (8i, 9i) were synthesized Following the reaction shown in Scheme 2 and using azides 7a-7m, compounds 8a-8m and 9a-9m were obtained as illustrated in Figure 1. studies. All of them showed the characteristic proton of the triazol ring in the 1 H-NMR spectral region between δ 7.63 and 9.31. The hydrogen of the coumarin nucleus was detected as a singlet at δ 5.18-6.22 and the methylene hydrogens in the oxygenated series appeared as a singlet at δ 5.35-5.59 and as a doublet at δ 4.66-4.46 (J = 5.6 Hz) in the nitrogenated series.
The best yields were obtained from the N-propargylated coumarin (6) and from the aliphatic azides (7k, 7l and 7m).
The in vitro antimicrobial activity of the novel coumarin-1,2,3-triazole conjugates was tested against the yeast Candida albicans, Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis and Gram-negative bacteria Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. The minimum inhibitory concentrations (MICs) were determined and given in Table 3. As can be seen, most of the coumarin-triazole hybrids did not exhibit considerable activity against the tested microorganisms. The best results were obtained with conjugates 8a, 8b, 8f, 9h and 9k, which displayed promising activity against Enterococcus faecalis at MICs ranging from 12.5 to 50.0 µg/mL. Compound 8b having a 2-OMe-Ph group attached at the triazol nucleus and an -OCH2linker was the best of the series, while the corresponding isoster 9b (-NHCH2-) turned out to be 64-fold less active than 8b. The position of the OMe group in the phenyl ring also plays an important role in the activity, since compounds 8c (3-OMe-Ph) and 8d (4-OMe-Ph) showed an 8-and 16-fold lower As can be seen, two isosteric series of coumarin derivatives were obtained (X=O, X=NH). Each series presents different substituents at the triazole moiety in order to evaluate their influence on the antimicrobial activity. Thus, coumarin derivatives with an aromatic ring having electron-donating groups or electron-withdrawing groups were prepared (8a-8h, 9a-9h). Coumarin-triazole derivatives with alkyl moieties (8k, 9k, 8l, 9l) and coumarin-indole hybrids (8i, 9i) were synthesized as well. Moderate yields were obtained with aromatic azides while the use of the more stable aliphatic azides (7k, 7l and 7m) led to high yields, in agreement with the more favourable HOMO of the dipole in the 1,3-dipolar cycloaddition. The structures of all adducts were determined by spectroscopic studies. All of them showed the characteristic proton of the triazol ring in the 1 H-NMR spectral region between δ 7.63 and 9.31. The hydrogen of the coumarin nucleus was detected as a singlet at δ 5.18-6.22 and the methylene hydrogens in the oxygenated series appeared as a singlet at δ 5.35-5.59 and as a doublet at δ 4.66-4.46 (J = 5.6 Hz) in the nitrogenated series.
The best yields were obtained from the N-propargylated coumarin (6) and from the aliphatic azides (7k, 7l and 7m).
The in vitro antimicrobial activity of the novel coumarin-1,2,3-triazole conjugates was tested against the yeast Candida albicans, Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis and Gram-negative bacteria Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa. The minimum inhibitory concentrations (MICs) were determined and given in Table 3. As can be seen, most of the coumarin-triazole hybrids did not exhibit considerable activity against the tested microorganisms. The best results were obtained with conjugates 8a, 8b, 8f, 9h and 9k, which displayed promising activity against Enterococcus faecalis at MICs ranging from 12.5 to 50.0 µg/mL. Compound 8b having a 2-OMe-Ph group attached at the triazol nucleus and an -OCH 2 -linker was the best of the series, while the corresponding isoster 9b (-NHCH 2 -) turned out to be 64-fold less active than 8b. The position of the OMe group in the phenyl ring also plays an important role in the activity, since compounds 8c (3-OMe-Ph) and 8d (4-OMe-Ph) showed an 8-and 16-fold lower antibacterial activity, respectively, than 8b. In the nitrogenated series, compounds 9h (3-NO 2 -Ph) and 9k having an undecyl chain showed the best activities. In other studies, menthyl 1,4-disubstituted 1,2,3-triazole derivatives of hydroxybenzaldehydes, phenols and bile acids showed a strong inhibitory effect against E. faecium with the minimum inhibitory concentration (MIC) values in the range of 1-3 µM [52]. Kant and co-workers reported that 1,2,3-triazole linked chalcone and flavone hybrids showed activity against Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Shigella boydii, Klebsiella pneumoniae) with MIC values in the range of 6.25-100 µg/mL [53]. In turn, 1,2,4-triazolo [3,4a]phthalazine derivatives showed inhibitory activity against Staphylococcus aureus (MIC 16-128 µg/mL) [54].
In order to verify if the newly synthesized triazoles could be considered as potential antimicrobial therapeutics, the most active compounds, namely 8a, 8b, 8f, 9h and 9k were examined in terms of their haemolytic activity against human erythrocytes. The results are shown in Figure 2. In order to verify if the newly synthesized triazoles could be considered as potential antimicrobial therapeutics, the most active compounds, namely 8a, 8b, 8f, 9h and 9k were examined in terms of their haemolytic activity against human erythrocytes. The results are shown in Figure 2. Compounds 8b, 8f and 9h exhibit minimal toxicity towards human blood cells (1.6-3.1% of lysed cells) in MIC. Although compound 8b appears to be the most active antimicrobial agent, simultaneously it moderately affects the erythrocytes (6.9% of lysed cells) in MIC. The presence of an undecyl chain in the triazole ring (9k) results in a drastic increase in the haemolytic activity (94% of lysed cells) in MIC.

General Experimental Procedures
IR spectra were obtained using a Fourier Transform Infrared spectrometer. NMR spectra were recorded in CDCl3 or DMSO at 500 or 600 MHz for 1 H NMR and 125 or 150 MHz for 13 C-NMR. Chemical shifts are given in (δ) parts per million and coupling constants (J) in hertz (Hz). 1 H-and 13 Cspectra were referenced using the solvent signal as an internal standard. Melting points were taken on a capillary melting point apparatus and are uncorrected. Microwave reactions were conducted in sealed glass vessels (capacity 5 mL) using a CEM Discover microwave reactor. HREIMS were recorded using a high-resolution magnetic trisector (EBE) mass analyser. The analytical thin-layer Compounds 8b, 8f and 9h exhibit minimal toxicity towards human blood cells (1.6-3.1% of lysed cells) in MIC. Although compound 8b appears to be the most active antimicrobial agent, simultaneously it moderately affects the erythrocytes (6.9% of lysed cells) in MIC. The presence of an undecyl chain in the triazole ring (9k) results in a drastic increase in the haemolytic activity (94% of lysed cells) in MIC.

Haemolytic Assay
Haemolytic properties of the selected compounds were determined according to the method described previously [45]. The human blood samples were centrifuged at 500× g for 10 min at 4 • C and the supernatant was discarded. Next, the erythrocytes were resuspended with PBS buffer (10 mM phosphate, pH 7.5; 150 mM NaCl) and centrifuged as previously. The washing procedure was repeated until a transparent supernatant was obtained. The washed erythrocytes were finally resuspended in PBS buffer to a final concentration of 2%. Simultaneously, appropriate concentrations (5, 10, 25, 50, 100 and 500 µg/mL for 8a, 8f, 9h and 9k, or 2, 5, 12.5, 50, 125 and 250 µg/mL for 8b) of the examined compounds were prepared in a final volume of 50 mL DMSO. The compounds prepared in this way were mixed with 450 mL of 2% erythrocyte suspension and incubated for 1 h at 37 • C. Then, the samples were centrifuged at 5000× g for 10 min and absorbance at wavelength 415 nm was measured.