Optimization of Pyrazole Compounds as Antibiotic Adjuvants Active against Colistin- and Carbapenem-Resistant Acinetobacter baumannii

Abstract

The diffusion of antibiotic-resistant, Gram-negative, opportunistic pathogens, an increasingly important global public health issue, causes a significant socioeconomic burden. Acinetobacter baumannii isolates, despite causing a lower number of infections than Enterobacterales, often show multidrug-resistant phenotypes. Carbapenem resistance is also rather common, prompting the WHO to include carbapenem-resistant A. baumannii as a "critical priority" for the discovery and development of new antibacterial agents. In a previous work, we identified several series of compounds showing either direct-acting or synergistic activity against relevant Gram-negative species, including A. baumannii. Among these, two pyrazole compounds, despite being devoid of any direct-acting activity, showed remarkable synergistic activity in the presence of a subinhibitory concentration of colistin on K. pneumoniae and A. baumannii and served as a starting point for the synthesis of new analogues. In this work, a new series of 47 pyrazole compounds was synthesized. Some compounds showed significant direct-acting antibacterial activity on Gram-positive organisms. Furthermore, an evaluation of their activity as potential antibiotic adjuvants allowed for the identification of two highly active compounds on MDR Acinetobacter baumannii, including colistin-resistant isolates. This work confirms the interest in pyrazole amides as a starting point for the optimization of synergistic antibacterial compounds active on antibiotic-resistant, Gram-negative pathogens.

Keywords: Acinetobacter baumannii; ESKAPE bacteria; antibacterials; antibiotic adjuvant; antibiotic potentiation; colistin.

Figure 1

Chemical structure of hit compounds identified during a previous antibacterial screening campaign of an unfocused library [12].

Figure 2

Exploration of the chemical space around pyrazole-4-carboxamides.

Figure 3

Exploration of the chemical space around pyrazole-3-carboxamides.

Scheme 1

Synthesis of pyrazole-4-carboxamide derivatives 3–14. Reagents and conditions: (a) diethyl ethoxymethylenemalonate, EtOH, reflux; (b) 2,5-dimethoxytetrahydrofuran, glacial acetic acid, 120 °C; (c) p-tolylboronic acid, Pd(OAc)₂, PPh₃, 1 M Na₂CO₃, dioxane, reflux, N₂ atmosphere, 4 h; (d) NaOH, H₂O, reflux; (e) appropriate amine, HOBt, EDC, DCM, RT.

Scheme 2

Synthesis of pyrazole-4-carboxamide derivatives 15–19. Reagents and conditions: (a) LiOH, H₂O/THF/MeOH, RT, 4 h; (b) (R)-alanine ethyl ester hydrochloride or (R)-valine ethyl ester hydrochloride, HOBt, EDC, DCM, RT.

Scheme 3

Synthesis of pyrazole-4-carboxamide derivatives 20–22. Reagents and conditions: (a) Boc₂O, DCM, RT, 2–4 h; (b) KOH, EtOH/THF, 55 °C, 1 h; (c) appropriate amine, HOBt, EDC, DCM, RT.

Scheme 4

Synthesis of pyrazole-3-carboxamide derivatives 23–40. Reagents and conditions: (a) appropriate amine, HOBt, EDC, DCM, RT; (b) NaOH, MeOH, H₂O, reflux, 3 h.

Scheme 5

Synthesis of pyrazole-3-carboxamide derivatives 41–49. Reagents and conditions: (a) appropriate aniline, HOBt, EDC, DCM, RT; (b) NaOH, MeOH, H₂O, reflux, 3 h; (c) 4-aminophenol, PyBOP, HOBt, dry DMF, RT, 4 h; (d) (COCl)₂, DCM, RT, 1 h, and then sulfanilamide, TEA, dry THF, RT, 2 h; (e) methyl chloroformate, TEA, DCM, RT, 1 h; (f) PhI(OAc)₂, NH₄OH, MeOH, RT, 2 h.

Figure 4

Direct antibacterial activity of compounds 3–40, 42, 44–47, and 49 (agar diffusion method) on a panel of eight representative bacterial species (type strains), including four Gram-negative and four Gram-positive organisms. (a) Pyrazole-4-carboxamide analogues (data for parent compound 1 are shown for comparison); (b) pyrazole-3-carboxamide analogues (data for parent compound 2 are shown for comparison) [12].

Figure 5

Isobolograms showing the strong synergistic activity of compounds 8 (top) and 9 (bottom) with colistin on A. baumannii reference strain (ATCC 17978) (left) and an MDR, colistin-resistant, and carbapenem-resistant A. baumannii clinical isolate (N50) (right).

Figure 6

Time-dependent kill-curve analysis of the MDR, colistin-resistant A. baumannii clinical isolate (N50) in the presence 2 µg/mL of colistin (squares), 2 µg/mL of colistin, and 8 µg/mL of 9 (circles). The growth control is shown as triangles (similar results were obtained in the presence of 8 µg/mL of 9, as this compound alone does not exhibit antibacterial activity).