Synthesis and Biological Evaluation of Piperazine Hybridized Coumarin Indolylcyanoenones with Antibacterial Potential

Abstract

A class of piperazine hybridized coumarin indolylcyanoenones was exploited as new structural antibacterial frameworks to combat intractable bacterial resistance. Bioactive assessment discovered that 4-chlorobenzyl derivative 11f showed a prominent inhibition on Pseudomonas aeruginosa ATCC 27853 with a low MIC of 1 μg/mL, which was four-fold more effective than norfloxacin. Importantly, the highly active 11f with inconspicuous hemolysis towards human red blood cells displayed quite low proneness to trigger bacterial resistance. Preliminary explorations on its antibacterial behavior disclosed that 11f possessed the ability to destroy bacterial cell membrane, leading to increased permeability of inner and outer membranes, the depolarization and fracture of membrane, and the effusion of intracellular components. Furthermore, bacterial oxidative stress and metabolic turbulence aroused by 11f also accelerated bacterial apoptosis. In particular, 11f could not only effectively inset into DNA, but also bind with DNA gyrase through forming supramolecular complex, thereby affecting the biological function of DNA. The above findings of new piperazine hybridized coumarin indolylcyanoenones provided an inspired possibility for the treatment of resistant bacterial infections.

Keywords: antibacterial; coumarin; membrane; piperazine; resistance.

Figure 1

Structures of some cyanoenone and indole derivatives.

Figure 2

Design of piperazine hybridized coumarin indolylcyanoenones as new structural antibacterial agents.

Scheme 1

Synthesis of N-unsubstituted piperazine hybridized coumarin indolylcyanoenones 5a–g. Reagents and conditions: (i) EtOH, piperazine, rt; (ii) DMF, cyanoacetic acid, HBTU, DIPEA, rt; (iii) EtOH, piperidine, 4a–g, 80 °C.

Scheme 2

Synthesis of alkyl and unsaturated hydrocarbyl piperazine hybridized coumarin indolylcyanoenones 7a–f, cycloalkyl piperazine hybridized coumarin indolylcyanoenones 9a–c, benzyl piperazine hybridized coumarin indolylcyanoenones 11a–i, and hydrophilic potential piperazine hybridized coumarin indolylcyanoenones 13a–c. Reagents and conditions: (iv) EtOH, piperidine, 6a–f, 80 °C; (v) EtOH, piperidine, 8a–c, 80 °C; (vi) EtOH, piperidine, 10a–i, 80 °C; (vii) EtOH, piperidine, 12a–c, 80 °C.

Figure 3

Resistance development of P. aeruginosa 27,853 towards molecule 11f and norfloxacin in 20 days.

Figure 4

Hemolytic ratio of human red blood cells caused by indolylcyanoenone 11f.

Figure 5

Outer membrane permeability of P. aeruginosa 27,853 treated by coumarin 11f.

Figure 6

Inner membrane permeability of P. aeruginosa 27,853 caused by compound 11f.

Figure 7

The depolarization assay of P. aeruginosa 27,853 in the presence of varying concentrations of derivative 11f.

Figure 8

The SEM images of P. aeruginosa 27,853 without treatment (A) and treated with 4-chlorobenzyl 11f at 2 μg/mL for 3 h (B), 6 h (C), and 12 h (D).

Figure 8

The SEM images of P. aeruginosa 27,853 without treatment (A) and treated with 4-chlorobenzyl 11f at 2 μg/mL for 3 h (B), 6 h (C), and 12 h (D).

Figure 9

Intracellular nucleic acids (A) and proteins (B) leakage following treatment with the 4-chlorobenzyl derivative 11f.

Figure 10

The cellular ROS production (A) and fluorescent images (B) of P. aeruginosa 27,853 exposed to different concentrations of compound 11f. Scale bar for all fluorescent images was 100 μm.

Figure 11

The metabolic activity of P. aeruginosa 27,853 influenced by molecule 11f.

Figure 12

The absorption spectra of DNA in the presence of coumarin indolylcyanoenone 11f. c(DNA) = 7.1 × 10⁻⁵ mol·L⁻¹, c(11f)/(10⁻⁵ mol·L⁻¹), a–h: 0–1.333 (increment: 0.167). Inset: Absorption comparison at 260 nm between the DNA–11f complex and the simple addition of DNA alone alone 11f alone.

Figure 13

Fluorescence variation of DNA–AO system with increased coumarin 11f. c(DNA) = 7.1 × 10⁻⁵ mol·L⁻¹, c(11f)/(10⁻⁵ mol·L⁻¹), a–h: 0–1.333 (increment: 0.167).

Figure 14

3D (left) and 2D (right) conformational images of DNA gyrase (PDB: 2XCS) docked with derivative 11f.