Synthesis of Novel Benzenesulfonamide-Bearing Functionalized Imidazole Derivatives as Novel Candidates Targeting Multidrug-Resistant Mycobacterium abscessus Complex

Infections caused by drug-resistant (DR) Mycobacterium abscessus (M. abscessus) complex (MAC) are an important public health concern, particularly when affecting individuals with various immunodeficiencies or chronic pulmonary diseases. Rapidly growing antimicrobial resistance among MAC urges us to develop novel antimicrobial candidates for future optimization. Therefore, we have designed and synthesized benzenesulfonamide-bearing functionalized imidazole or S-alkylated derivatives and evaluated their antimicrobial activity using multidrug-resistant M. abscessus strains and compared their antimycobacterial activity using M. bovis BCG and M. tuberculosis H37Ra. Benzenesulfonamide-bearing imidazole-2-thiol compound 13, containing 4-CF3 substituent in benzene ring, showed strong antimicrobial activity against the tested mycobacterial strains and was more active than some antibiotics used as a reference. Furthermore, an imidazole-bearing 4-F substituent and S-methyl group demonstrated good antimicrobial activity against M. abscessus complex strains, as well as M. bovis BCG and M. tuberculosis H37Ra. In summary, these results demonstrated that novel benzenesulfonamide derivatives, bearing substituted imidazoles, could be further explored as potential candidates for the further hit-to-lead optimization of novel antimycobacterial compounds.


Introduction
Infections caused by nontuberculous mycobacteria (NTM) remain a challenging and emerging public health threat, particularly in individuals undergoing chemotherapy or patients with underlying lung conditions [1]. The incidence of infections caused by NTM is increasing globally, and it can be challenging to diagnose and treat due to the diverse range of NTM species and varying patterns of antibiotic susceptibility. In addition, some NTM developed resistance to multiple antibiotics, making the infections caused by NTM difficult to treat and worsening the treatment prognosis [2][3][4]. Therefore, it is critical to develop novel small molecule antimicrobial candidates targeting NTM in particularly multidrug-resistant (MDR) strains.
Amine 1 (1.72 g, 10 mmol) was dissolved in boiling water (40 mL). Then, the solution of corresponding α-haloketone (12 mmol) in 10 mL of 1,4-dioxane was added dropwise to the mixture. The reaction mixture was heated at reflux for 2 h, then it was cooled down and the precipitate was filtered off, washed with diethyl ether, and recrystallized from 1,4-dioxane to afford compounds 3 and 6.
Imidazole 9-15 (1.0 mmol) was dissolved in DMF (3 mL). Triethylamine (0.5 mL) and corresponding alkyl halide (1.5 mmol) were added dropwise, and the reaction mixture was stirred at room temperature for 2-3 h. Then, the reaction mixture was diluted with 20 mL of water. The precipitate was filtered off, washed with water and diethyl ether, dried, and recrystallized from propan-2-ol.

Preparation of Assay Microplates
The minimal inhibitory concentrations (MICs) of compounds 2-22a-c, as well as of clinically approved antibiotics (rifampin, isoniazid, amikacin, levofloxacin, and meropenem) were determined by microplate broth dilution method as described by Clinical Laboratory Standards Institute document M07-A8. The antimicrobials were selected to represent major antimicrobials used in clinical settings to treat MDR infections, as well as infections caused by rapidly growing Mycobacterium spp. The MICs for the compounds and comparator antibiotics were determined against the libraries of Gram-positive and Gram-negative pathogens, multidrug-resistant fungi, and mycobacteria.
Compounds and antibiotics that were used as a control were dissolved in molecular biology grade dimethyl sulfoxide (DMSO) to achieve a final concentration of 25-30 mg/mL. Compound dilutions were achieved in 1.5 mL polypropylene 96-well microplates to gener-ate 2× of concentrations of each drug (0.5-64 µg/mL). The 2× concentrates were then then transferred to flat bottom plates and used for inoculation or stored in argon purged sealed bags at −80 • C.

Antibacterial Activity Characterization Using Gram-Positive and Gram-Negative Pathogens
A microbial inoculum was prepared using the direct colony suspension method and densitometric analysis. The inoculum suspension of each test organism was prepared in 5 mL of sterile deionized water until densitometer reached 0.5 MFa and further diluted in sterile CAMBH media to achieve final concentrations of approximately 5 × 10 5 CFU/mL in each well after dispensing in microplates. The inoculum was transferred to the assay plates to achieve 1× assay concentration. A 10 µL of inoculum was plated on Sheep Blood agar plates to validate the purity and inoculum size. Inoculated microdilution plates were incubated at 35 • C for 16 to 20 h in an ambient-air incubator.

Antifungal Activity Characterization
The MIC of compounds 2-22a-c, as well as clinically approved antifungal drugs was determined by CLSI recommendations that were described in document M27-A3 [36,37]. Multidrug-resistant Candida spp. strains were sub-cultured on Sabouraud-Dextrose agar for 24 h at 35 • C. Drug-resistant Aspergillus fumigatus was cultured on Inhibitory mold agar slants for 5 days at 35 • C. The colonies of Candida isolates were suspended in sterile saline to reach approximately 5 × 10 6 CFU/mL. The conidia of A. fumigatus were collected by flooding the slants with saline containing 0.5% of Tween 80 and passing conidia through 75 µm cell strainer. The inoculums were quantified by using haematocytometer and then, the fungal suspension was diluted in RPMI/MOPS broth to reach 5 × 10 5 CFU/mL. The inoculum was then dispensed in assay microplates, and inoculated microdilution plates were incubated at 35 • C for 24 h in an ambient-air incubator within 15 min of the addition of the inoculum.

Antimycobacterial Activity Determination
Before the experiments, multidrug-resistant M. abscessus complex strains were cultured on Middlebrook 7H9 agar containing ODAC supplement for four days at 37 • C. M. bovis BCG and avirulent M. tuberculosis H37Ra strains were grown on Lowenstein-Jensen (LJ) media for 3 weeks.
The colonies of M. abscessus were scraped and suspended in tube with sterile saline to achieve approximately 5 × 10 6 CFU/mL. M. bovis BCG and M. tuberculosis H37Ra were scraped from the LJ media and transferred to the tube containing 4 mL of Middlebrook 7H9 broth and 3 borosilicate glass beads. The tube was vortexed on maximum speed for 2 min, and then, the bacterial suspension was adjusted to 5 × 10 6 CFU/mL. Prior to inoculation of the plates, the bacterial suspension was diluted 1:10 in Middlebrook 7H9 broth containing 20 µg/mL of resazurin, and microplates were inoculated by using multichannel pipette.
The plates were incubated at 37 • C in humidified incubator for 5 days (for M. abscessus complex) or two weeks (for M. bovis BCG and M. tuberculosis H37Ra) and the minimal inhibitory concentration was determined by visual evaluation.

Chemistry
Most of the compounds 2-15 (Scheme 1) were resynthesized according to our previous study [35] and were further investigated during this study. All the spectral data and reaction conditions can be found in previously mentioned research [35]. Moreover, to explore further on benzenesulfonamide-bearing 1H-imidazolethiol moieties, new compounds 3, 6, 10, and 13 were newly synthesized for this study (Scheme 1). 3-Aminobenzenesulfonamide (1) was treated with various α-halogenketones in water/1,4-dioxane solution to afford compound 3 and 6. These intermediate compounds were later cyclized with potassium thiocyanate in glacial acetic acid and in a presence of HCl as a catalyst into 1H-imidazole derivatives 10 and 13. The structures of compounds 3, 6, 10, and 13 have also been confirmed by the data of FT-IR, 1 H and 13 C NMR spectroscopy, as well as elemental analysis data. For instance, in a 1 H NMR spectrum for 10, the singlets assigned to the protons in the CH group at 8.18 ppm and in the SH group at 13.12 ppm have proven the presence of 1Himidazolethiol moiety in the molecule. One of the best-known properties of thioamides is the tautomerism [38]: thioamides can exist in their thione/thiol forms. However, the 13 C NMR spectral data showed that in DMSO-d 6 solvent, thiol tautomeric form is predominant for both compounds 10 and 13. The carbon attributed to the C-SH group resonated at 163.17 and 163.62 ppm, respectively.
Most of the compounds 2-15 (Scheme 1) were resynthesized according to our previous study [35] and were further investigated during this study. All the spectral data and reaction conditions can be found in previously mentioned research [35]. Moreover, to explore further on benzenesulfonamide-bearing 1H-imidazolethiol moieties, new compounds 3, 6, 10, and 13 were newly synthesized for this study (Scheme 1). 3-Aminobenzenesulfonamide (1) was treated with various α-halogenketones in water/1,4-dioxane solution to afford compound 3 and 6. These intermediate compounds were later cyclized with potassium thiocyanate in glacial acetic acid and in a presence of HCl as a catalyst into 1H-imidazole derivatives 10 and 13. The structures of compounds 3, 6, 10, and 13 have also been confirmed by the data of FT-IR, 1 H and 13 C NMR spectroscopy, as well as elemental analysis data. For instance, in a 1 H NMR spectrum for 10, the singlets assigned to the protons in the CH group at 8.18 ppm and in the SH group at 13.12 ppm have proven the presence of 1H-imidazolethiol moiety in the molecule. One of the best-known properties of thioamides is the tautomerism [38]: thioamides can exist in their thione/thiol forms. However, the 13 C NMR spectral data showed that in DMSO-d6 solvent, thiol tautomeric form is predominant for both compounds 10 and 13. The carbon attributed to the C-SH group resonated at 163.17 and 163.62 ppm, respectively. The main goal of this study was to further investigate 1H-imidazolethiol derivatives with various alkyl substituents. For this purpose, S-alkylation reactions with bromomethane, ethyl iodide, and n-propyl iodide in dimethyl formamide were carried out to obtain compounds 16-22a-c. Triethylamine was used as a base catalyst to increase the reaction rate. For example, in a 1 H NMR spectrum for 16a, the singlet assigned to the protons in the CH3 group at 2.63 ppm have proved the presence of methyl moiety in the molecule, while a triplet at 0.92 ppm, a sextet at 1.67 ppm, and a triplet at 3.12 ppm assigned to the protons in the CH3, CH2, and CH3, respectively, proved the presence of a propyl group in The main goal of this study was to further investigate 1H-imidazolethiol derivatives with various alkyl substituents. For this purpose, S-alkylation reactions with bromomethane, ethyl iodide, and n-propyl iodide in dimethyl formamide were carried out to obtain compounds 16-22a-c. Triethylamine was used as a base catalyst to increase the reaction rate. For example, in a 1 H NMR spectrum for 16a, the singlet assigned to the protons in the CH 3 group at 2.63 ppm have proved the presence of methyl moiety in the molecule, while a triplet at 0.92 ppm, a sextet at 1.67 ppm, and a triplet at 3.12 ppm assigned to the protons in the CH 3 , CH 2 , and CH 3 , respectively, proved the presence of a propyl group in compound 16c. Elemental analysis data of compounds 16-22a-c confirmed that all the molecules did not form hydroiodide or hydrobromide salts.

Conclusions
During this study, a series of imidazole-2-thiol bearing benzenesulfonamides was synthesized. To reach higher lipophilicity properties and potentially increase their membrane permeability through multidrug-resistant mycobacteria, various S-alkylation reactions were performed with alkyl halides.
Previous studies have explored the impact of alkyl substitution on the antimicrobial activity of various compounds against mycobacteria and other clinically important pathogens. Oh et al. [39] have reported the synthesis of a series of novel N-Alkyl-5-hydroxypyrimidinone carboxamides as potent inhibitors of M. tuberculosis decaprenylphosphoryl-β-d-ribose 2'-oxidase. Faria et al. [40] describes alkyl promising activity and the high reactivity of alkyl hydrazide derivatives of isoniazid, suggesting that the alkylation is an important modification leading to the in vitro and in silico activity. Yang Yong et al. [41] described the synthesis of novel 8-alkylberberine derivatives bearing aliphatic chains and evaluated their antimicrobial activity. The study showed that increasing the length of the aliphatic chain had a significant effect on the antibacterial activity of the compounds. However, antimicrobial activity started to decrease when alkyl chain consisted eight or more carbon atoms.
S-alkylation is widely employed strategy to increase the stability of biologically active compounds due to higher bond dissociation energy of the S-C bond compared to the N-C bond [42,43]. S-alkylated compounds are generally less susceptible to hydrolysis and more resistant to metabolic degradation compared to N-alkylated compounds, making S-alkylation an attractive strategy to enhance the biological activity of various compounds.
These results suggest that the S-alkylated benzenesulfonamide-bearing imidazole derivatives could be further explored as a scaffold for the development of novel, multidrugresistant M. abscesus complex-directed antimicrobials.