Synthesis of new 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino)thiazolidin-4-one derivatives as anti-MRSA and anti-H. pylori agents

In this work, we have synthesized twenty five new 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino)thiazolidin-4-one derivatives bearing an aryl or heteroaryl methylene group on position 5 of thiazolidinone and evaluated their antimicrobial activity against Gram-positive and -negative bacteria as well as three metronidazole resistant Helicobacter pylori strains. Most of the compounds were very potent towards tested Gram-positive bacteria and showed an antibacterial efficacy substantially greater than ampicillin as the reference drug. However, no effectiveness was observed for the Gram-negative microorganisms. The compounds 9, 20 and 29 exhibited strong antimicrobial activity against Helicobacter pylori strains (inhibition zone > 30 mm) in 100 μg/disc and (inhibition zone > 20 mm) in 50 μg/disc. Taking these findings together, it seems that these potent antibacterial derivatives could be considered as promising agents for developing new anti-infectious drugs against microorganisms resistant to currently available antibiotics. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13065-022-00829-7.


Introduction
Treatment of infectious diseases remains one of the most important and challenging areas in global public health. Infections that are caused by microbes are a paramount cause of death worldwide, specifically in low-income countries. The World Health Organization (WHO) has mentioned three infectious diseases: lower Tabei et al. BMC Chemistry (2022) 16:38 respiratory infections, diarrheal diseases, and tuberculosis in the list of top ten causes of death worldwide in 2016 [1]. Although various antimicrobial and antifungal agents have been discovered in the last decades, the substantial need for finding new potent antimicrobials still remains of great concern owing to the rapid growth in microbial resistance and emergence of multi-drug resistant pathogens [2]. Designing new agents that utilize different targets and mechanisms of action can be a useful approach to deal with microbial resistance and particularly cross-resistance with conventional antimicrobial therapeutics.
Another interesting heterocyclic core is 5-nitrofuran that is used in several antibacterial agents already available in the market like nitrofurantoin, nifuroxazide, furazolidone, nitrofural, nifurtoinol, furazidin, difurazone, and nifurquinazol [23,24]. Different nitroreductase enzymes, available in both aerobic and anaerobic bacteria, easily reduce 5-nitrofuran derivatives and form various nitro radical anions and cyano derivatives. Although, the exact toxic intermediate species as well as their cellular targets are still undiscovered, but it is supposed that these reduced reactive intermediates can interfere with diverse critical bacterial pathways through damaging both DNA and proteins [23,25].
The structures of newly synthesized compounds 7-31 were characterized by 1 H-NMR, FT-IR, and mass spectra. In the 1 H-NMR spectral data, the signal for thiazolidinone NH group was observed as a broad singlet at δ 12.84-13.25 ppm, and the singlet signal for = CH in the arylidene appeared at δ 7.63-7.83 ppm. The signals for two hydrogens of furan ring were found as two broad singlets at δ 7.73-8.07 and 7.53-7.61 ppm. In the FT-IR spectra, the characteristic signals for carbonyl moiety appeared at 1694-1743 cm −1 . Additionally, the absorption bands of the NH group at 3072-3262 cm −1 and asymmetric and symmetric bands of NO 2 group at 1518-1581 and 1347-1362 cm −1 confirmed the structures.

Biological activity
The antibacterial activity of the final compounds 7-31 was evaluated by determining their minimal inhibitory concentration (MIC) against the following strains: S . These compounds were also tested against three clinically isolate metronidazole resistant strains of H. pylori [26].
As shown in Table 1, most of the compounds showed significant antibacterial activity that was better than ampicillin as the reference drug against the rest of Grampositive bacteria; however all the Gram-negative bacteria including E. coli, K. pneumonia, P. aeruginosa, and S. typhimurium Most tested derivatives showed respectable activity toward E. faecalis; however they were less active than ampicillin as the reference drug, with the exception of 17 (MIC = 0.78 µg/ml), that was 2.5 times more potent than ampicillin (MIC = 2 µg/ml). Also, only compound 28 (MIC = 0.097 µg/ml) was more potent than the reference drug (MIC = 0.125 µg/ml) in terms of activity toward B. subitilis. Besides, although M. luteus was not resistant to the rest of the compounds, none of the target compounds was more potent than the reference drug against M. luteus. Unlike our previous study that had shown compounds bearing 2-, 3-pyridyl moiety on thiazolidinone ring were the most active compounds against S. aureus, S. epidermidis, B. cereus and B. subtilis, 3-pyridyl containing derivative 26 was only more potent than ampicillin against B. cereus.
Considering proper anti-H. pylori activity of similar derivatives in previous studies as well as good antibacterial activity of the title compounds against various Gram-positive bacteria, the anti-H. pylori activities of our target compounds were evaluated [3,27]. The activity of compounds 7-31 was assessed by in vitro paper disk diffusion bioassay and measuring Inhibition Zone Diameters (IZDs) of compounds at 100, 50, 25, and 12.5 µg/disk concentrations against three metronidazole resistant strains of H. pylori. The inhibition zone diameters (IZDs) were recorded and the antimicrobial activity was expressed as mean ± SD IZDs (mm) produced by selected compound against three clinical H. pylori isolates. H. pylori strains were considered as susceptible when exhibited growth inhibition zones of ≥ 15 mm for each compound. Metronidazole was used as reference drug and no inhibition zone was found in all recruited doses.
In order to investigate the safety and cytotoxicity of the target compounds, the in silico toxicity evaluation was performed by means of osiris property explorer (OPE) (https:// www. organ ic-chemi stry. org/ prog/ peo/) which shows the probable mutagenic, tumorgenic, irritant and reproductive effects of compounds and vNN web server was used to study their cytotoxicity [28]. The results were shown in Additional file 1: Table S1. According to the results the mutagenic property of 28 and 29, the tumurgenic activity of 14 and 29 and reproductive effect of 20 are possible. The medium irritant and reproductive effects of 24 were predicted by OPE. The cytotoxicity results of vNN indicate that none of the compounds 7-31 were cytotoxic. The MTT test showed that the IC 50 of compounds 8 and 18 was more than 50 µg/ml and the the IC 50 of compounds 17 and 29 was 19.45 ± 1.09 and 32.93 ± 1.05 respectively. The comparison of their antibacterial concentrations with the related cytotoxic results depicted their acceptable selectivity index.

Structure-Activity Relationship (SAR)
As Table 1 shows, among five different aryl and heteroaryles introduced to position 5 of thizolidinone ring, attachment of 2-furyl ring 28 led to excellent activity against S. aureus, MRSA, S. epidermidis, B. subtilis and B. cereus. Replacement of 2-furyl with unsubstituted phenyl 7 and 2-thienyl 30 moieties resulted in a slight decrease in the activity, but the compounds were still very active. However, unlike the results from our previous study [3], the insertion of 3-pyridyl 26 moiety had a deteriorative effect on antibacterial activity. The substitution of 4-Me group on phenyl ring improved activity, while 4-OMe and 4-SMe decreased the activity of 9 and 10. In addition, the introduction of the more polar electron-donating OH group on meta and para positions of phenyl ring, reduced antibacterial activity.
Introduction of non-polar electron-withdrawing halogens on the para position of phenyl ring affected the antimicrobial activity in the following order: Also, the effect of positioning each halogen at the ortho, meta, and para sites of phenyl ring was as follows: 4-Cl > 3-Cl > 2-Cl and 2-Br > 3-Br > 4-Br. 2,6-dichloro substitution on the phenyl ring resulted in high antimicrobial activity in compound 17. Yet again, the introduction of polar electron withdrawing nitro substituent on both aryl and heteroaryl ring systems led to significant diminishing in antibacterial activity. Bulky substituents at meta and para positions of 24 and 25 seem unfavorable for antibacterial activity.
With these findings in mind, it seems that the presence of bulky polar substituents on the para position of the phenyl ring has a negative impact on antibacterial activity and 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino) thiazolidin-4-one derivatives bearing small aryl or heteroaryl groups with non-bulky and non-polar substituents, are favorable for antibacterial activity against Gram-positive bacteria.
Anti-H. pylori assay data, as shown in Table 2, revealed that the unsubstituted phenyl ring led to better anti-H. pylori activity in comparison to other unsubstituted heteroaryles. The sequence of their activity was in the following order: phenyl > 2-thienyl > 2-furyl > 2-indolyl > 3-pyridyl. Introduction of electron-donating groups on the para position of the phenyl ring, improved activity and compounds 8, 9 and 20 bearing 4-Me, 4-OMe and 4-OH showed strong anti-H. pylori activity [MIC (12.5 µg/disk)]. However, the displacement of the hydroxyl group to meta position reduced activity. Generally, it seems that presence of both polar and nonpolar electron-withdrawing groups on the phenyl ring reduces the efficacy of compounds on H. pylori. The only exceptions were compound 17, having 2,6-dichloro substitution on the phenyl ring, and compound 29, bearing 5-nitro-2-furyl moiety, which showed stronger activities.
These results indicate that small electron-donating substituent on the para position of aryl ring can lead to better anti-H. pylori activity.

Conclusions
In summary, new 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino)thiazolidin-4-one derivatives bearing an aryl or heteroaryl methylene group on position 5 of thiazolidinone were synthesized and examined for their antimicrobial and anti-H. pylori activity. The in vitro Biological data illustrated that many of these derivatives were potent active growth inhibitors against Gram-positive bacteria as well as H. pylori whereas Gram-negative microorganisms were not susceptible to them.
The MIC determination results exhibited that most of the compounds showed better activity than ampicillin as the reference drug versus MRSA, S. epidermidis and B. cereus and Compounds 17 and 28 were the most active compounds. The anti-H. pylori assay showed that compounds 8, 9, 20 and 29 had strong growth inhibitory activity at 12.5 µg/disk concentrations against three metronidazole resistant strains. Based on these results, it seems that 2-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-ylimino)thiazolidin-4-one derivatives bearing small aryl or heteroaryl groups with non-bulky non-polar substituents, are favorable for antibacterial activity against Gram-positive bacteria. On the other hand, the small polar substituents on the para position of aryl or heteroaryl methylene group can lead to better anti-H. pylori activity. .

Experimental section
General procedure for the synthesis of compounds 7-31 To a well-stirred solution of 6 (0.2 g, 0.64 mmol) in glacial acetic acid (35 ml) buffered with anhydrous sodium acetate (1.83 mmol), the respective aryl aldehyde (1.28 mmol) was added. The solution was refluxed for 24-72 h and then poured into ice-cold water. The precipitate was filtered and recrystallized from ethanol to give compounds 7-31 (All the compounds were recrystallized from ethanol except compounds 11, 12, 13, 18, 19 and 20 that were recrystallized in ethyl acetate).