Novel 2,4,6-Trimethylbenzenesulfonyl Hydrazones with Antibacterial Activity: Synthesis and In Vitro Study

This research describes the synthesis and in vitro antimicrobial activity study of a series of 2,4,6-trimethylbenzenesulfonyl hydrazones. Twenty-five hydrazones (2–26) were synthesized on the basis of condensation reaction. The in vitro bioactivity study confirmed the potential application of obtained derivatives as antimicrobial agents. Among the tested compounds, the highest activity was discovered for derivative 24, which possessed minimal inhibitory concentration (MIC) ranging from 7.81 to 15.62 µg/mL against Gram-positive reference bacterial strains. Synthesized benzenesulfonyl hydrazones can be applied as potential ligands for the synthesis of bioactive metal complexes.


Introduction
In recent years, the problem of the emerging resistance of bacteria and fungi to antibiotics and chemotherapeutic agents has been observed [1]. Even 30-40 years ago, this problem was not so noticeable because from the 1940s to the 1980s, the pharmaceutical industry introduced many classes of medicines to the treatment [1][2][3]. In the fight against bacterial and fungal infections, antimicrobial agents are used, which can be divided into two main classes: obtained by isolating from naturally occurring organisms in the ecosystem and by chemical modification of them, namely, antibiotics, and obtained by chemical synthesis, namely, chemotherapeutic agents. Recent years have shown that antimicrobial agents that have been used for decades are not always effective in treating infections [1][2][3]. Due to these factors, scientists are seeking new molecules with antimicrobial properties. The discovery of new medicines with antimicrobial activity profile could eliminate infections that the current healthcare system cannot cope with, and it would also be possible to shorten hospitalization time and possible complications, as well as to reduce the number of therapeutic agents used [1][2][3].
Prompted by the above-mentioned fact, in this research, we designed, synthesized, and tested for in vitro antimicrobial activity a novel series of 2,4,6-trimethylbenzenesulfonyl hydrazones with the aim to obtain promising potential antimicrobial agents that can be used further as potential ligands for the synthesis of bioactive metal complexes.

Chemistry
All reagents and solvents used in this research were purchased from Sigma-Aldrich (Munich, Germany) and Merck Co. (Darmstadt, Germany) and used without further purification. Thin-layer chromatography (TLC) on plates covered with silica gel (aluminum oxide 60 F-254, Merck Co., Kenilworth, NJ, USA) was used to check the purity of the obtained compounds and to monitor the progress of the reaction. Chloroform-ethanol mixture in the 10:1 (v/v) ratio was used as the mobile phase. The spots were detected by irradiation with UV light at a wavelength of λ = 254 nm. 1 H NMR and 13 C NMR spectra were In the scientific literature findings, there is also some information that concerns the synthesis and activity of organometallic-based sulfonyl hydrazones [31]. Concha et al. synthesized series of organometallic tosylhydrazones containing cyrhetrenyl and cymantrenyl moieties and evaluated them for potential antitubercular and antitumor activity [31].
Prompted by the above-mentioned fact, in this research, we designed, synthesized, and tested for in vitro antimicrobial activity a novel series of 2,4,6-trimethylbenzenesulfonyl hydrazones with the aim to obtain promising potential antimicrobial agents that can be used further as potential ligands for the synthesis of bioactive metal complexes.

Chemistry
All reagents and solvents used in this research were purchased from Sigma-Aldrich (Munich, Germany) and Merck Co. (Darmstadt, Germany) and used without further purification. Thin-layer chromatography (TLC) on plates covered with silica gel (aluminum oxide 60 F-254, Merck Co., Kenilworth, NJ, USA) was used to check the purity of the obtained compounds and to monitor the progress of the reaction. Chloroform-ethanol mixture in the 10:1 (v/v) ratio was used as the mobile phase. The spots were detected by irradiation with UV light at a wavelength of λ = 254 nm. 1 H NMR and 13 C NMR spectra were recorded on the Bruker Avance 300 and 600 apparatus (Bruker BioSpin GmbH, Rheinstetten, Germany). The melting points of the obtained compounds were measured with a Fisher-Johns apparatus (Fisher Scientific, Schwerte, Germany) and presented without any correction. The elemental analysis was determined by the Perkin Elmer 2400 series II CHNS/O analyzer (Waltham, MA, USA), and the results were within ±0.4% of the theoretical value. The 2,4,6-trimethylbenzenesulfonyl hydrazones were synthesized with the use of the method described earlier by our group for the synthesis of hydrazide-hydrazones [32][33][34].
We dissolved 0.01 mole of 2,4,6-trimethylbenzenesulfonohydrazide (1) in ethanol (5 mL, 96%). Then, 0.011 mole of appropriate substituted benzaldehyde was added, and the mixture was heated under reflux for 3 h. The substituted benzaldehydes used in this research are presented in Scheme 1. After that, the solution was cooled to room temperature and placed in the refrigerator for 24 h. Subsequently the precipitate formed was filtered off and re-crystallized from ethanol (96%).   Figures S1-S8).

Microbiology
In Vitro Antimicrobial Activity Assay The examined compounds 2-26 were screened in vitro for antibacterial and antifungal activities according to the procedure described earlier by our group [35,36] with the use of the protocols of European Committee on Antimicrobial Susceptibility Testing (EUCAST) [37] and Clinical and Laboratory Standards Institute guidelines [38]. All the experiments were repeated three times, and representative data are presented. Detailed procedure for the in vitro antimicrobial activity assay is presented in the Supplementary Materials.
The chemical structure of synthesized 2,4,6-trimethylbenzenesulfonyl hydrazones (2-26) was established with the use of the analysis of 1 H NMR and 13 C NMR spectra. Compounds 2-26 on the 1 H NMR spectra possessed two characteristic singlet signals. First of them in the range of δ 7.83-8.98 ppm corresponded to proton in =CH group and confirmed the successful conduction of condensation reaction, whereas the other at δ 11.23-12.20 ppm corresponded to proton in NH group. In 13 C NMR spectra for compounds 2-26, we found peak for carbon atom of =CH group at δ 142.15-147.57 ppm. Other aliphatic and aromatic fragments of synthesized molecules in the 1 H and 13 C NMR spectra were found at the expected range of chemical shift.
The other compounds showed moderate or mild activity against Gram-positive bacteria with MIC from 250 to 1000 µg/mL, or had no activity.
In relation to the antibacterial activity of reference substances, it is worth underlining that the activity of compound 24 possessed 2-hydroxy-3,5-diiodophenyl substituent against M. luteus ATCC 10240 (MIC = 7.81 µg/mL), which was eight times greater that the activity of nitrofurantoin (MIC = 62.5 µg/mL). Towards B. subtilis ATCC 6633, this compound showed two times higher (MIC = 7.81 µg/mL) activity than cefuroxime (MIC = 15.62 µg/mL) and eight times higher activity than ampicillin (MIC = 62.5 µg/mL). Activity of this hydrazone towards B. cereus ATCC 10876 was equal to the activity of nitrofurantoin (MIC = 7.81 µg/mL) and four times greater than the activity of cefuroxime (MIC = 31.25 µg/mL). Towards Staphylococcus aureus ATCC 29213 and ATCC 25923, its activity was equal to the activity of nitrofurantoin (MIC = 15.62 µg/mL).  onyl hydrazones resulted in a decrease in activity in some cases. 2,4,6-Trimethylbenzenesulf onyl hydrazone 26 formed in the reaction of 2,4,6-trimethylbenzenesulfonohydrazide 1 with benzaldehyde was inactive towards all tested bacterial strains. However, the use of aldehydes that contained in the phenyl ring a strong electron-donating substituent, a hydroxy group, and a second electron-withdrawing halogen atom (23)(24)(25) increased the activity against Gram-positive bacteria. When analyzing the activity of hydrazide 1 and benzenesulfonyl hydrazones, we saw that the free amino group promoted the activity against Staphylococcus aureus ATCC 25923, but the introduction of two halogens into the hydrazone's phenyl ring caused an up to 16-fold increase in activity against this bacterial strain in the case of 2,4,6-trimethylbenzenesulfonyl hydrazone (24). In addition to this, the introduction of two electron-donating substituents in the phenyl ring caused a twofold increase in the activity of the compound 25 against this bacterial strain. The Bacillus subtilis ATCC 6633 was the most sensitive bacterial strain to tested 2,4,6-trimethylbenzenesylfonyl hydrazones. In its case, the free amino group was not conducive to activity.

Conclusions
In this research we designed, synthesized, and analyzed for potential antimicrobial activity a series of 2,4,6-trimethylbenzenesulfonyl hydrazones 2-26. Our antimicrobial activity assay results indicated that some of the newly obtained compounds 2-26 showed particular activity against Gram-positive bacteria. The highest antibacterial effect was indicated for compounds 7, 22, 23, 24, and 25. The bacteria from Staphylococcus spp., Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 10240, and Bacillus spp. were especially sensitive to compound 24. The minimal inhibitory concentration (MIC) values that inhibited growth of reference microorganisms for this hydrazone ranged from 7.81 to 15.62 µg/mL, indicating strong or very strong bactericidal effect of this molecule and its potential application as an antimicrobial agent. The antibacterial activity of the obtained compounds was connected both with the presence of sulfonyl hydrazone moiety in their molecules as well as with substitution with hydroxy and methoxy groups or chlorine and iodine atoms in the phenyl ring. The more substituents that were present in the aromatic ring of an aldehyde, which was used for the condensation reaction, the higher the observed activity of the resulting 2,4,6-trimethylbenzenesulfonyl hydrazones. The most potent compounds against tested bacterial strains will be applied as ligands for the synthesis of metal complexes.
Author Contributions: Ł.P. designed the study, participated in the synthesis of new 2,4,6-trimethylben zenesulfonyl hydrazones, performed the analysis of the spectral data of obtained compounds, wrote the first draft of the manuscript with the exception of the antimicrobial activity section, and critically revised the final version of the manuscript. S.S. participated in the synthesis of new 2,4,6-trimethylben zenesulfonyl hydrazones. A.B. performed the antimicrobial activity analysis of the obtained compounds and wrote the associated section of the manuscript. M.W. participated in the discussion section of the manuscript and critically revised the final version of manuscript. All authors have read and agreed to the published version of the manuscript.