Enzymatic Synthesis of a Novel Coumarin Aminophosphonates: Antibacterial Effects and Oxidative Stress Modulation on Selected E. coli Strains

Abstract

The objective of the present study was to evaluate the synergistic effect of two important pharmacophores, coumarin and α-amino dimethyl phosphonate moieties, on antimicrobial activity toward selected LPS-varied E. coli strains. Studied antimicrobial agents were prepared via a Kabachnik-Fields reaction promoted by lipases. The products were provided with an excellent yield (up to 92%) under mild, solvent- and metal-free conditions. A preliminary exploration of coumarin α-amino dimethyl phosphonate analogs as novel antimicrobial agents was carried out to determine the basic features of the structure responsible for the observed biological activity. The structure-activity relationship revealed that an inhibitory activity of the synthesized compounds is strongly related to the type of the substituents located in the phenyl ring. The collected data demonstrated that coumarin-based α-aminophosphonates can be potential antimicrobial drug candidates, which is particularly crucial due to the constantly increasing resistance of bacteria to commonly used antibiotics.

Keywords: E. coli cells; Kabachnik-Fields reaction; MIC; aminophosphonates; antimicrobial activity; coumarins; enzymatic catalytic promiscuity; lipases; multicomponent reaction.

Figure 1

Biologically active antimicrobial coumarin and α-aminophosphonate derivatives.

Scheme 1

Enzyme-catalyzed synthesis of coumarin aminophosphonates 1–13.

Figure 2

Reuse of the Novozym 435 catalyst.

Figure 3

The synthesis of various coumarin α-aminophoshonates 1-13 under optimized conditions. Reaction conditions: aldehyde (1 mmol), 7-amino-4-(trifluoromethyl)coumarin (1 mmol), dimethyl phosphite (1 mmol), and Novozym 435 (80 mg) in neat for 18 h at 30 °C, 200 rpm. ^a 2-Me-THF (2 mL). Yields in brackets provided for isolated products 1–13.

Scheme 2

Plausible mechanism of CaLB lipase-catalyzed Kabachnik-Fields reaction.

Figure 4

Minimum inhibitory concentration (MIC) of the coumarin derivatives 1–14 in the model bacterial strains. The x-axis features compounds 1–14. The y-axis shows the MIC value in µg/mL. Investigated strains of E. coli K12 as control (blue), R2 strains (orange), R3 strain (grey), and R4 strain (yellow), Fpg overexpression strain (red bars). The order in which the compounds were applied to the plate are shown in Supplementary Materials Figure S1.

Figure 5

Minimum bactericidal concentration (MBC) of the coumarin derivatives. The x-axis features compounds 1–14. The y-axis shows the MIC value in µg/mL. Investigated strains of E. coli K12 as control (blue), R2 strains (orange), R3 strain (grey), and R4 strain (yellow), Fpg overexpression strain (red bars). The order in which the compounds were applied to the plate are shown in Supplementary Materials Figure S1.

Figure 6

The ratio of MBC/MIC of the coumarin derivatives. The x-axis features compounds 1–14. The y-axis shows the MBC/MIC value in µg/mL. Investigated strains of E. coli K12 as control (blue), R2 strains (orange), R3 strain (grey), and R4 strain (yellow), Fpg overexpression strain (red bars). The order in which the compounds were applied to the plate are shown in Supplementary Materials Figure S1.

Figure 7

Percentage of plasmid DNA recognized by Fpg enzyme (y-axis) with model bacterial, K12, and R2–R4 strains (x-axis). All analyzed compounds numbered were statistically significant at <0.05 (see Table 2). The data are shown in Supplementary Materials Figure S2.

Figure 8

Examples of MIC with model bacterial strains K12, R2, R3, and R4 for studying antibiotics ciprofloxacin (cipro), bleomycin (bleo), and cloxacillin (clox). The x-axis features antibiotics used sequentially. The y-axis features the MIC value in µg/mL. The order in which the compounds were applied to the plate are shown in Supplementary Materials Figure S1.

Figure 9

Percentage of bacterial DNA recognized by Fpg enzyme in model bacterial strains after ciprofloxacin, bleomycin, and cloxacillin treatment. The compounds were statistically significant at p < 0.05.—(Supplementary Materials Figure S3).