Inhibitory effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1 H-pyrazole-1-carbothioamide on Haemophilus spp. planktonic or biofilm-forming cells

Abstract

During this study, we have investigated in vitro activity of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide derivatives with N-ethyl, N-(4-metoxyphenyl) and N-cyclohexyl substituents against Gram-negative Haemophilus influenzae and H. parainfluenzae bacteria. A spectrophotometric assay was used in order to determine the bacterial growth and biofilm formation using a microtiter plate to estimate minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC). Among the tested N-substituted pyrazole derivatives, only N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide showed a significant in vitro activity against both planktonic cells of H. parainfluenzae (MIC = 0.49-31.25 μg ml^-1) and H. influenzae (MIC = 0.24-31.25 μg ml^-1) as well as biofilm-forming cells of H. parainfluenzae (MBIC = 0.24-31.25 μg ml^-1) and H. influenzae (MBIC = 0.49 to ≥31.25 μg ml^-1). The pyrazole compound exerted higher inhibitory effect both on the growth of planktonic cells and biofilm formation by penicillinase-positive and penicillinase-negative isolates of H. parainfluenzae than the activity of commonly used antibiotics such as ampicillin. No cytotoxicity of the tested compound in vitro at concentrations used was found. The tested pyrazole N-ethyl derivative could be considered as a compound for the design of agents active against both pathogenic H. influenzae and opportunistic H. parainfluenzae, showing also anti-biofilm activity. This appears important because biofilms are determinants of bacterial persistence in long-term and recurrent infections recalcitrant to standard therapy.

Keywords: Anti-biofilm activity; Antibacterial activity; Cytotoxicity; Haemophilus spp.; Pyrazole derivatives.

Fig. 1

The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide compound on biofilms formation by Haemophilus spp. on the basis of MBIC/MIC ratio

Fig. 2

The effect of N-ethyl-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide compound and ampicillin on the penicillinase-negative (filled diamond S85Pen−) and penicillinase-positive (filled square S86Pen+) Haemophilus parainfluenzae planktonic or biofilm-forming cells (broth without bacteria: OD₅₇₀ = 0.09–0.11)

Scheme 1

Synthesis and structure of N-substituted-3-amino-5-oxo-4-phenyl-2,5-dihydro-1H-pyrazole-1-carbothioamide