Perspectives on Synthetic Adducts (Salts) of Nitroxoline^TM and 2-Aminoquinolin-8-ol as Promising Antibacterial Agents ^†

Abstract

The threatening phenomenon of antibiotic failure in the future determines the intensive research of antibacterial active compounds, which are promising candidates as antibiotics. Quinolines, with only the representative in clinical practice being Nitroxoline^TM, are, in addition to being effective beta-lactams, macrolides, tetracyclines, and other antibiotic categories, forgotten antibiotics. The antibacterial efficiency of Nitroxoline^TM and 2-aminoquinolin-8-ol on eight selected highly resistant bacterial species that are the most problematic (Klebsiella ssp., Enterococcus ssp., Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus) could lead to higher solubility and thus bioavailability and increased antibacterial effects. In the first phase, the basic salts of Nitroxoline^TM, with sodium hydroxide, benzylamine, 4-(aminomethyl)pyridine, and other primary amines, were synthesized. In the second phase, the corresponding acidic salts of 2-aminoquinolin-8-ol were synthesized with the following acids: oxalic acid, pyrazine-2,3-dicarboxylic acid, chelidonic acid, quinaldic acid, 3,5-dinitrosalycilic acid, quinoline-2-carboxylic acid, quinoline-3-carboxylic acid, kynurenic acid, and xanthurenic acid. Nitroxoline^TM and 2-aminoquinolin-8-ol both demonstrated moderate antibacterial effects, with the average value for the eight mentioned bacterial strains being 16 mg/L (84 μM) and 50 mg/L (301 μM), respectively. The synthetized salts of both quinolinols demonstrated significantly higher solubility and slightly increased antibacterial activity. The identity and purity of the prepared products were determined by NMR and IR spectroscopy. The MW values of both quinolinols are relatively low and offer better use of the largest molecule limit, defined by Lipinski’s rule of five at 500 g/M. The options of amines and acids offer the achievement of quaternary salts with improved antibacterial activity.

1. Introduction

8-hydroxyquinolines (8OHQs) are the one of the subjects of intensive research. Its antibacterial activity has been well known for several centuries because it has been part of the formulation of disinfectants in army medicine. 8OHQs are known to be produced by the roots of several plants, such as Broussonetia zeylanica or Allium stipitatum, grown in the Mediterranean region of Europe [1]. 8OHQs have been the subject of intensive research over the last four decades up to now, as documented by several patent inventions [2,3] as well as original, scientific papers [4,5,6,7]. The only clinically approved and launched antibiotic of this structural group is Nitroxoline^TM (NQ), prescribed for the therapy of urinary system infections [8,9,10]. Numerous collections of 8OHQ derivatives (n ≥ 100) were screened from the eight most problematic clinical isolates with clearly defined mechanisms of resistance from our previous research (data not yet published). Surprisingly, moderate antibacterial effect was observed with our “alert collection” of selected bacterial strains. The moderate antibacterial effect, with no known mechanism (except for the ion chelating effect in bacterial cells, which has been published), is the main motivation for the applications of our current research and this paper. The published category [2,11] of NQ or 8OHQ salts seems to be from at least two points of view—antibacterial activity and increasing solubility in polar solvents and water.

2. Methods

All chemicals/Nitroxoline^TM, sodium hydroxide, benzylamine, 4-(aminomethyl)pyridine, 2-amino-8-quinoplinol, oxalic acid, pyrazine-2,3-dicarboxylic acid, chelidonic acid, quinaldic acid, 3,5-dinitrosalycilic acid, quinoline-2-carboxylic acid, quinoline-3-carboxylic acid, xanthurenic acid, and kynurenic acid, as well as solvents for synthesis 2-propanol and tetrahydrofuran, were purchased from Meck/Sigma-Aldrich (St. Louis, MO, USA), and were of the highest purity grade.

All syntheses were carried out through the mixing (10 mL) of 100 mM solutions of both reactants, dissolution in THF (2-propanol eventually) at room temperature for 24 h in darkness, followed by filtration, washing, and drying of the synthetized crystals. The identity and purity of the prepared products were determined by ¹H NMR spectroscopy in denatured methanol (with Me₄Si as an internal standard) and IR spectroscopy using KBr.

The solubility of the prepared salts and parent compounds (Nitroxoline^TM and 2A8OHQ) was determined gravimetrically from saturated solutions at 25 °C, after filtration and evaporation.

The antibacterial activity of the prepared salts was screened using eight bacterial strains: Klebsiella pneumonae of two different strains, No. 4498 and 4352; Klebsiella oxytoca, strain No. 3541; Klebsiella aerogenes, strain No. 500; Pseudomonas aeruginosa, strain No. 3396; Acinetobacter baumannii, strain No. 3333; vancomycin-resistant Enterococcus, strain No. 636; and Staphylococcus aureus, strain No. 1942, in accordance with the CLSI standard.

3. Results

In the first phase, three basic salts of Nitroxoline^TM with sodium hydroxide, benzylamine, 4-(aminomethyl)pyridine, and other primary amines were synthesized. In the second phase, nine corresponding acidic salts of 2-aminoquinolin-8-ol were synthesized with the following acids: oxalic acid, pyrazine-2,3-dicarboxylic acid, chelidonic acid, quinaldic acid, 3,5-dinitrosalycilic acid, quinoline-2-carboxylic acid, quinoline-3-carboxylic acid, xanthurenic acid, and finally kynurenic acid (

Table 1. Structures of NQ, 2A8OHQ, and synthesized salts.

Code	Compound	Structure
NQ	NitroxolineTM
NQs1	NitroxolineTM, natrium salt
NQs2	NitroxolineTM salt with 4-benzylamine
NQs3	NitroxolineTM salt with, 4-(aminomethyl)pyridine
2A8OHQ	2-Amino-8-hydroxyquinoline
2A8OHQs1	2-Amino-8-hydroxyquinoline salt with oxalic acid
2A8OHQs2	2-Amino-8-hydroxyquinoline salt with 2,3-pyrazinedicarboxylic acid
2A8OHQs3	2-Amino-8-hydroxyquinoline salt with chelidonic acid
2A8OHQs4	2-Amino-8-hydroxyquinoline salt with quinalid acid
2A8OHQs5	2-Amino-8-hydroxyquinoline salt with 3,5-dinitrosalicylic acid
2A8OHQs6	2-Amino-8-hydroxyquinoline salt with quinoline-2-carboxylic acid
2A8OHQs7	2-Amino-8-hydroxyquinoline salt with quinoline-3-carboxylic acid
2A8OHQs8	2-Amino-8-hydroxyquinoline salt with xanthurenic acid
2A8OHQs9	2-Amino-8-hydroxyquinoline salt with kynurenic acid

).

Mainly, monocarboxylic aromatic acids with higher pKa values were selected for random synthesis of the salts of 2-aminoquinolin-8-ol. The products were brown or yellow crystals with moderate yield (50–75%) of sufficient purity (over 95%). The solubility of the prepared salts was within a narrow range of values and has evidently increased compared to the “parent” molecule; the solubility of Nitroxoline^TM vs. Nitroxoline^TM salts was 0.03 g/L vs. 0.5–1 g/L. Similarly, the solubility was 0.2 g/L for 2A8OHQ vs. about 2 g/L for its salts.

All compounds of sufficient amount and purity were subjected to evaluation of antibacterial activity with eight selected bacterial strains that cause nosocomial, problematic infections. The results of the antibacterial activity of the standards NQ and 2A8OHQ, 12 prepared salts, and 2 parent compounds are in

Table 2. Antibacterial activity of NQ, 2A8OHQ and prepared salts.

Strain No./ Compound Code	MIC (mg/L)
	4498	3541	500	3396	3333	636	1942	4352
NQ	32	16	16	16	8	16	8	32
NQs1	8	8	12	32	4	8	4	12
NQs2	16	16	8	32	8	8	4	8
NQs3	32	8	8	32	8	8	4	8
2A8OHQ	64	64	32	64	32	16	64	64
2A8OHQs1	64	32	32	64	32	16	32	64
2A8OHQs2	32	32	32	64	32	16	32	64
2A8OHQs3	64	32	32	64	32	8	32	64
2A8OHQs4	32	32	32	64	32	8	32	64
2A8OHQs5	32	32	32	32	32	16	32	64
2A8OHQs6	64	32	32	64	32	16	32	64
2A8OHQs7	64	32	32	64	32	4	16	64
2A8OHQs8	32	32	32	64	32	16	32	64
2A8OHQs9	64	32	32	64	32	4	16	64

.

The results presented in Table 2 show that all the prepared salts are comparable or have lower MIC values, which means higher antibacterial activity. From the achieved results, it is evident that there are moderate effects from both the compounds and the prepared salts from their “parent” compounds.

4. Discussion

This short paper revealed the simple synthesis of the prospective salts of “parent” molecules/compounds, particularly Nitroxoline^TM and 2-aminoquinolin-8-ol. Whereas Nitroxoline^TM is well known and frequently covered in publications, a somewhat-documented, clinically applied antibiotic, 2-aminoquinolin-8-ol, is a less potent yet moderately effective antibacterial compound, holding promise for more effective derivatives or salts. This study represents only a brief, preliminary screening of three salts of Nitroxoline and nine salts of 2A8OHQ to demonstrate a representation of this direction. The significant increase in water solubility (at least ten times) and conservation or improvement in antibacterial potency is strongly evident.