Thiazolidin-4-Ones as a Promising Scaffold in the Development of Antibiofilm Agents—A Review

Thiazolidin-4-ones have a broad range of medical and clinical implementation, which is important for pharmaceutical and medicinal chemistry. This heterocyclic core has been reported to possess a diversity of bioactivities, including antimicrobial and antibiofilm-forming potential. The resistance of biofilms to antibiotics or disinfectants is a serious medical problem. Therefore, there is a natural need to discover new effective structures with properties that inhibit biofilm formation. This review aims to analyze the antibiofilm features of thiazolidin-4-ones described in the literature over the last two decades. The information gathered in this review could benefit the rational design of new effective antibiofilm small molecules with thiazolidin-4-one cores.


Introduction
The effectiveness of antimicrobial therapy faces a significant challenge in the form of resistance development, observed in both bacteria and fungi.This resistance is a strategic adaptation for self-survival.It is now known that more than 99% of bacteria in the natural environment exist in the form of biofilm and not as single planktonic cells.The resistance of bacterial biofilms to antibiotics or commonly used disinfectants is a serious problem in modern medicine and industrial practice.Currently, it is estimated that biofilmmediated infections account for approximately 80% of all infections affecting animals and humans [1,2].Microorganisms that adhere to the surface form durable, thin films called biological membranes or biofilms.Currently, biofilms are defined as complex multicellular structures of bacteria surrounded by a layer of organic and inorganic substances produced by these microorganisms, showing adhesion to both biological and abiotic surfaces.Microbial cells, which are part of the biofilm, are nearly 1000 times more resistant to the effects of toxic substances (disinfectants, antibiotics, and surfactants) than those remaining in suspension [3,4].Living in clusters, microorganisms develop multi-level defense mechanisms against the degrading effects of chemotherapy drugs on cells.
The role of biofilms in antimicrobial resistance is highly complex and may significantly drive resistance.As an illustration, consider a study that investigated the antibiotic resistance of Staphylococcus epidermidis within biofilms.In the planktonic state, all isolates demonstrated susceptibility to the antibiotic vancomycin, reaching 100%.However, when tested within a biofilm, almost 75% of these isolates exhibited complete resistance to the same antibiotic [5].
Bacteria commonly employ antibiotic resistance mechanisms such as point mutations, enzymes, and efflux pumps [6][7][8].However, these mechanisms are less likely to account for the resistance observed in biofilm organisms.Within a biofilm, various components collaboratively act to diminish or completely impede antibiotic effectiveness, actively contributing to the reinforcement of resistance.When these mechanisms operate in concert, they enable the survival of organisms within the biofilm even in the presence of elevated antibiotic concentrations, a phenomenon referred to as resistance.
One of the directions to combat biofilm is the use of new small molecules with confirmed biological potency, especially antimicrobial and/or antibiofilm properties.Such a group of derivatives may be compounds with a thiazolidin-4-one core.Thiazolidin-4-one derivatives exhibit a broad spectrum of therapeutic applications and hold clinical significance in the field of medicinal chemistry [9][10][11][12][13].For example, thiazolidine-2,4-diones are a well-known group of antidiabetic drugs, glitazones, that act by activating PPARγ.Representants of this group, pioglitazones and rosiglitazones, are still present in the pharmaceutical market in the EU and the US.Another thiazolidine-4-one derivative, the drug ponesimod, is used for the treatment of multiple sclerosis.It was approved for medical use in the US and EU in 2021.It is a sphingosine-1-phosphate receptor modulator [14,15].Moreover, the compounds with a thiazolidin-4-one core have been documented to display diverse biological activities such as antibacterial [16][17][18], antifungal [19], antitubercular [20], anticancer [21,22], antiviral [23], and antiprotozoal activities [24].
Previous information about the antibiofilm activity of thiazolidin-4-ones was unsystematic and generally found in reviews about the biological potential of thiazolidin-4ones [25].Nevertheless, over the last two decades, various thiazolidin-4-one derivatives have been tested for their antibiofilm properties, and some of them have demonstrated promising results.
The main assumption of this study is to collect, systemize, and analyze information about the antibiofilm potential of thiazolidin-4-one derivatives reported in the scientific literature between the years 2004 and 2023.This review describes thiazolidin-4-ones, which have antibiofilm activity against different bacteria and fungi by inhibiting their formation or interfering with their construction, leading to the removal of existing biofilm.

Biofilm Life-Cycle
The formation of a biofilm is a multi-stage process that depends on the structure and properties of the colonized surface, as well as on the properties of the microorganisms.In the first stage, the bacteria settle in planktonic form and attach themselves to the substrate.Cells bind to the medium by nonspecific, reversible interactions.In the second step, a specific reaction takes place between the bacterial adhesins and the substrate.The close adherence of the microbial cell to the substrate for a sufficiently long time results in an irreversible connection.In this phase, microorganisms produce an extracellular polymeric substance (EPS), which becomes the extracellular matrix.In the next stage, the multiplication and differentiation of microorganisms are observed.In the final stage of biofilm formation, bacterial cells detach from the formed structure and, because of moving to new surfaces, give rise to a new biofilm (Figure 1) [26].

Strategies to Control Harmful Biofilms
Current strategies to control harmful biofilms may be classified into three categories: (i) changing abiotic surface characteristics to prevent biofilm formation; (ii) regulating the signaling pathways to inhibit biofilm formation and stimulate biofilm dispersal; (iii)

Strategies to Control Harmful Biofilms
Current strategies to control harmful biofilms may be classified into three categories: (i) changing abiotic surface characteristics to prevent biofilm formation; (ii) regulating the signaling pathways to inhibit biofilm formation and stimulate biofilm dispersal; (iii) applying external forces to eradicate the biofilm [27].The first category is changing abiotic surfaces including by treating abiotic surfaces and coating surfaces.Biofilm development is dependent on the physicochemical features of adsorptive surfaces.Consequently, altering the properties of material surfaces, including smoothness, wettability, or hydrophilicity, holds the potential to put a stop to the formation of biofilm.In turn, the coating of small molecules can also change the adhesive properties of surface materials.For example, coating tissue implants and medical devices can decrease microbial adhesion to the material surface to prevent biofilm formation and reduce bacterial infection.
The next category is the regulation of signaling pathways, such as quorum sensing (QS), leading to the inhibition of biofilm development.QS is a phenomenon of chemical "communication" of microorganisms consisting of the production and secretion of signal molecules (autoinducers) into the environment, which are used in various physiological processes, including in the formation of biofilm.The increase in the concentration of autoinductors (AIs) is a function of the number of cells.QS systems typically comprise an enzyme responsible for synthesizing the signaling molecules (e.g., acyl-homoserine lactones (AHL) or cyclic peptides) and a receptor that binds these signals, triggering the reprogramming of gene expression.This process includes the activation of genes, including those encoding the enzyme responsible for signal production, establishing a positive feedback loop.In bacterial pathogens, most QS-controlled genes codify diverse virulence factors, such as proteases, toxins, and adhesins.Shortly, QS regulates the metabolic activity of planktonic cells, and it can induce microbial biofilm formation and increased virulence [28].
Mechanisms of the QS inhibiting agents in controlling bacterial biofilm formation may include the next important points: (i) inhibition of AI synthesis; (ii) degradation or inactivation of AIs by AHL-lactonases, -oxidoreductases, and antibodies; (iii) interference in the signal receptors using AI antagonists; (iv) interference in the response regulators, thus disturbing signaling cascade; (v) reducing the extracellular AI accumulation by inhibiting AI efflux, thus inhibited cell-to-cell signaling [29].
Microorganisms' communication can occur between cells of one species or of different species.The chemical nature of the signals, their mechanisms of action, and the genes that control quorum sensing vary from case to case.Regulation of biofilm formation affords a novel potential target to control biofilms [26].Other biofilm inhibitors are based on nucleotide second messenger molecules.These molecules play an important role in regulating the various physiological functions of bacteria [30].The last category is the use of external forces for biofilm eradication, such as physical methods (ultrasound and magnetic fields) and biochemical methods (application of phage lysins and degradative enzymes).
reduction for 1h, 1m, and 1n was above 50% (61.34%,62.69%, and 56.74%, respe recorded after application of concentrations equal to their MIC (125.4 µM, 162.1 µ 157.9 µM, respectively), indicating good biofilm-inhibiting potential.It is worth that compounds with good antibiofilm potential had 3-F, 3-Br, or 2,6-diCl substit the benzylidene fragment of the molecule.The introduction at the fifth position of thiazolidine ring pyrrolemethylidene (blue color in Figure 3) allowed the obtaining of the effective compound 2 against S (MIC = 0.5 µg/mL).Therefore, this compound was tested on its antibiofilm acti shown in the results, compound 2 reduced S. aureus biofilm by > 11% at 10xM result was compared to standard drugs such as levofloxacin and vancomycin [32  The introduction at the fifth position of thiazolidine ring pyrrolemethylidene moiety (blue color in Figure 3) allowed the obtaining of the effective compound 2 against S. aureus (MIC = 0.5 µg/mL).Therefore, this compound was tested on its antibiofilm activity.As shown in the results, compound 2 reduced S. aureus biofilm by >11% at 10× MIC.This result was compared to standard drugs such as levofloxacin and vancomycin [32].
157.9 µM, respectively), indicating good biofilm-inhibiting potential.It is worth n that compounds with good antibiofilm potential had 3-F, 3-Br, or 2,6-diCl substit the benzylidene fragment of the molecule.The introduction at the fifth position of thiazolidine ring pyrrolemethylidene (blue color in Figure 3) allowed the obtaining of the effective compound 2 against S (MIC = 0.5 µg/mL).Therefore, this compound was tested on its antibiofilm acti shown in the results, compound 2 reduced S. aureus biofilm by > 11% at 10xM result was compared to standard drugs such as levofloxacin and vancomycin [32]   Gullapelli and Maroju [33] synthesized a series of iminothiazolidin-4-ones and evaluated their antibacterial and antibiofilm potential.Compounds 3a and 3b showed good antibacterial potential against four strains including two resistants with MIC in the range of 3.62 to 7.14 µg/mL for compound 3a and with MIC in the range of 2.95 to 4.63 µg/mL for compound 3b.The antibiofilm activity of compounds 3a and 3b was very promising (Figure 4).Compound 3a showed antibiofilm activity against resistant strains MRSA and VRE with biofilm inhibitory concentrations (BICs) of 8.23 µg/mL and 7.56 µg/mL, respectively.Moreover, derivative 3a exhibited activity against K. pneumoniae and E. coli biofilms with BICs of 6.25 and 6.62 µg/mL, respectively.Significant activity against biofilm formation of the above strains showed also in compound 3b.Their BICs were 2.22 µg/mL against MRSA and 3.05 µg/mL against VRE strains.In addition, 3b was found to be significant against K. pneumoniae and E. coli biofilms with BICs of 3.25 and 2.03 µg/mL, respectively.µg/mL, respectively.Moreover, derivative 3a exhibited activity against K. pneumoniae and E. coli biofilms with BICs of 6.25 and 6.62 µg/mL, respectively.Significant activity against biofilm formation of the above strains showed also in compound 3b.Their BICs were 2.22 µg/mL against MRSA and 3.05 µg/mL against VRE strains.In addition, 3b was found to be significant against K. pneumoniae and E. coli biofilms with BICs of 3.25 and 2.03 µg/mL, respectively.The Hemeda research group [34] synthesized two series of 2-iminothiazolidin-4-one derivatives with benzothiazole substituent (blue color in Figure 5) in position 3 of the thiazolidine ring (5-arylidene derivatives (4a-4l) and Mannich bases (5a, 5b, 6a, 6b, 7a, and 7b)).These compounds were evaluated for their C. albicans antibiofilm activity.Only 10 compounds (4a-4d, 4h, 4j, 5b, 6b, 7a, and 7b) that showed MICs in the range of 25 to 100 µg/mL in antimicrobial screening were studied for their antibiofilm activity.Most of the tested antifungal agents against two pathogenic Candida isolates, CA1 and CA2, demonstrated significant antibiofilm activity compared to the reference, fluconazole, but were not more effective.Compound 4j with the para-methoxy group in the benzylidene substituent showed the most antibiofilm activity against both CA1 and CA2 isolates (OD570 mm = 0.297 and 0.218, respectively).The Mannich base 7b with a dimethylamino substituent in position 2 of the thiazolidine ring showed almost similar OD (OD570 mm = 0.266) to compound 4j (MIC = 25 µg/mL) against only the CA2 pathogen, although its MIC value was 100 µg/mL.On the other hand, a lack of a substituent (compounds 4a and 4b) or 4-dimethylamino group (compound 4h) in the benzylidene fragment allows decreasing antibiofilm activity.Their OD570 mm was in the range of 0.779 to 1.269.The remaining compounds (4c, 4d, 5b, 6b, and 7a) showed good to moderate antibiofilm activity against both isolates (OD570 mm were in the range 0.307 to 0.530) comparing to fluconazole (OD570 mm = 0.088 and 0.105 for CA1 and CA2, respectively).The Hemeda research group [34] synthesized two series of 2-iminothiazolidin-4-one derivatives with benzothiazole substituent (blue color in Figure 5) in position 3 of the thiazolidine ring (5-arylidene derivatives (4a-4l) and Mannich bases (5a, 5b, 6a, 6b, 7a, and 7b)).These compounds were evaluated for their C. albicans antibiofilm activity.Only 10 compounds (4a-4d, 4h, 4j, 5b, 6b, 7a, and 7b) that showed MICs in the range of 25 to 100 µg/mL in antimicrobial screening were studied for their antibiofilm activity.Most of the tested antifungal agents against two pathogenic Candida isolates, CA1 and CA2, demonstrated significant antibiofilm activity compared to the reference, fluconazole, but were not more effective.Compound 4j with the para-methoxy group in the benzylidene substituent showed the most antibiofilm activity against both CA1 and CA2 isolates (OD 570mm = 0.297 and 0.218, respectively).The Mannich base 7b with a dimethylamino substituent in position 2 of the thiazolidine ring showed almost similar OD (OD 570mm = 0.266) to compound 4j (MIC = 25 µg/mL) against only the CA2 pathogen, although its MIC value was 100 µg/mL.On the other hand, a lack of a substituent (compounds 4a and 4b) or 4-dimethylamino group (compound 4h) in the benzylidene fragment allows decreasing antibiofilm activity.Their OD 570mm was in the range of 0.779 to 1.269.The remaining compounds (4c, 4d, 5b, 6b, and 7a) showed good to moderate antibiofilm activity against both isolates (OD 570mm were in the range 0.307 to 0.530) comparing to fluconazole (OD 570mm = 0.088 and 0.105 for CA1 and CA2, respectively).Pan et al. [35] designed and synthesized a series of 2-arylimino-3-arylthiazolidinones (8a-11) (blue color in Figure 6).All 25 compounds of this series were evaluated fo their antibacterial and antibiofilm activity against S. epidermidis RP62A using a standar tube-dilution assay.Recently, some thiazolidin-4-one derivatives were documented wi moderate antibiofilm activity against S. epidermidis RP62A, which caused their effe through inhibition of the YycG histidine kinase [36].YycG plays an essential role in ce viability and related cell wall metabolism, biofilm formation, virulence, and antibiot resistance.For these reasons, YycG histidine kinase may be considered as a potenti target for this series of compounds.Pan et al. [35] designed and synthesized a series of 2-arylimino-3-arylthiazolidin-4ones (8a-11) (blue color in Figure 6).All 25 compounds of this series were evaluated for their antibacterial and antibiofilm activity against S. epidermidis RP62A using a standard tube-dilution assay.Recently, some thiazolidin-4-one derivatives were documented with moderate antibiofilm activity against S. epidermidis RP62A, which caused their effect through inhibition of the YycG histidine kinase [36].YycG plays an essential role in cell viability and related cell wall metabolism, biofilm formation, virulence, and antibiotic resistance.For these reasons, YycG histidine kinase may be considered as a potential target for this series of compounds.Pan et al. [35] designed and synthesized a series of 2-arylimino-3-arylthiazolidin-4ones (8a-11) (blue color in Figure 6).All 25 compounds of this series were evaluated for their antibacterial and antibiofilm activity against S. epidermidis RP62A using a standard tube-dilution assay.Recently, some thiazolidin-4-one derivatives were documented with moderate antibiofilm activity against S. epidermidis RP62A, which caused their effect through inhibition of the YycG histidine kinase [36].YycG plays an essential role in cell viability and related cell wall metabolism, biofilm formation, virulence, and antibiotic resistance.For these reasons, YycG histidine kinase may be considered as a potential target for this series of compounds.The eighteen derivatives of this series showed weak antibacterial effects and antibiofilm activity.Their MIC values were >200 µM, and antibiofilm concentration was more than 200 µM.Compounds 10a, 10b, and 11 exhibited a little better antibacterial and antibiofilm activity.MIC and antibiofilm concentrations were 50 and 100 µM, respectively.The most active group of compounds in this series were derivatives 8a-8d with MIC values in the range of 6.25 to 12.5 µM and antibiofilm concentration in the range of 6.25 to The eighteen derivatives of this series showed weak antibacterial effects and antibiofilm activity.Their MIC values were >200 µM, and antibiofilm concentration was more than 200 µM.Compounds 10a, 10b, and 11 exhibited a little better antibacterial and antibiofilm activity.MIC and antibiofilm concentrations were 50 and 100 µM, respectively.The most active group of compounds in this series were derivatives 8a-8d with MIC values in the range of 6.25 to 12.5 µM and antibiofilm concentration in the range of 6.25 to 25 µM.Compound 8d with the 4-methoxy group in the benzene ring showed the best activity in the series with MIC and an antibiofilm concentration of 6.25 µM.It is worth noticing that replacement of the carboxylic group in the 8a-8d derivatives by the methoxycarbonyl group (9a-9d) led to abolished antibacterial (MIC > 200 µM) and antibiofilm activity (antibiofilm concentration > 200 µM).
Continuing their research, Pan et al. [37] focused on optimizing the structure with documented antibiofilm activity (compound 13) by different pathways of modification: functional group modification, ring opening, chain shortening, and pharmacophore overlapping (Figure 7).The antibacterial and antibiofilm activity was determined using the S. epidermidis ATCC 35984 strain.The structure-activity relationship analysis was carried out.Compound 13 is a pioneering antibiofilm agent that has moderate inhibition of S. epidermidis biofilm growth (antibiofilm concentration was 25 µM).Its antibiofilm effect is associated with the inhibition of the YycG histidine kinase [36].The modification of functional groups did not improve antibacterial and antibiofilm activities.Only the para-methoxyphenyl derivative (12) displayed antibiofilm and antibacterial activities comparable to compound 13.In contrast, pharmacophore overlapping modification (compounds 16 and 17) is a less potent agent than compound 13 with an antibiofilm concentration of 50 µM and MIC = 25 µM.This confirms that the furanylbenzoic acid fragment is more significant than the 2-phenoxyacetic acid substituent (blue and red colors in Figure 7, respectively).Interest-ingly, that ring opening led to compound 18, which was more active against biofilm growth than derivative 16.Unexpectedly, modification by chain shortening (8a, 8c, 8d, and 14 derivatives) led to the improvement of antibacterial and antibiofilm activities.Compound 8d showed eightfold better antibiofilm activity than their 2,3-di(4-methoxyphenyl)methyl analogue (15), whereas compound 14 was fourfold more active relative to biofilm formation than derivative 13 (antibiofilm concentration value was 6.25 µM).
concentration of 50 µM and MIC = 25 µM.This confirms that the furanylbenzoic acid fragment is more significant than the 2-phenoxyacetic acid substituent (blue and red colors in Figure 7, respectively).Interestingly, that ring opening led to compound 18, which was more active against biofilm growth than derivative 16.Unexpectedly, modification by chain shortening (8a, 8c, 8d, and 14 derivatives) led to the improvement of antibacterial and antibiofilm activities.Compound 8d showed eightfold better antibiofilm activity than their 2,3-di(4-methoxyphenyl)methyl analogue (15), whereas compound 14 was fourfold more active relative to biofilm formation than derivative 13 (antibiofilm concentration value was 6.25 µM).
These results showed that the presence of a 4-(5-ylidenemethylfuran-2-yl)benzoic acid fragment and substituted phenyl moiety, which directly connected with the N atom of the thiazolidine ring, might be two decisive factors that advantageously influenced the antibiofilm activity.These results showed that the presence of a 4-(5-ylidenemethylfuran-2-yl)benzoic acid fragment and substituted phenyl moiety, which directly connected with the N atom of the thiazolidine ring, might be two decisive factors that advantageously influenced the antibiofilm activity.
The next step for the continuation of research on the antibiofilm potential of 2iminothiazolidin-4-one derivatives was a synthesis of halogenphenyl analogues (19)(20)(21)(22)(23)(24) of compounds 8a-8d, 10a, and 14 (green color in Figure 8).This paper evaluated the inhibition effects of new derivatives on the autophosphorylation activity of the purified recombinant YycG's activity on S. epidermidis biofilm, as well as their antibacterial efficacy in a rabbit subcutaneous S. epidermidis biofilm infection model [39].The IC 50 values of derivatives 19-24 toward YycG's protein were in the range of 24.2 to 71.2 µM.The most active was compound 24 (IC 50 = 24.2µM), which demonstrated a twofold lower concentration than precursor 10b (IC 50 = 47.9 µM).Furthermore, compound 24 was most effective against S. epidermidis biofilms.Their MBEC was 6.3 µM.Other derivatives (20-23), except 19 (MBEC = 50 µM), showed antibiofilm activity with MBEC = 12.5 µM, which was eight-fold better than precursor 10b (MBEC = 100 µM).It is worth noticing that the incorporation of a furan (20-22) and thiophene (23 and 24) ring and the introduction of halogen into the appropriate position of phenyl substituents may improve antibacterial and antibiofilm activities.
Additionally, two compounds, 19 and 24, were tested in vivo using a rabbit subcutaneous S. epidermidis biofilm infection model.Bacterial viability after being treated by 19 and 24 was significantly reduced compared to controls (DMSO and vancomycin 5.3 log10CFU/cm 2 and 5.21 log10CFU/cm 2 , respectively).CFUs in biofilms treated with 19 were reduced to 2.91 log10CFU/cm 2 and with 24 to 2.18 log10CFU/cm 2 .This is in accordance with antibiofilm activity in vitro.Additionally, two compounds, 19 and 24, were tested in vivo using a rabbit subcutaneous S. epidermidis biofilm infection model.Bacterial viability after being treated by 19 and 24 was significantly reduced compared to controls (DMSO and vancomycin 5.3 log 10 CFU/cm 2 and 5.21 log 10 CFU/cm 2 , respectively).CFUs in biofilms treated with 19 were reduced to 2.91 log 10 CFU/cm 2 and with 24 to 2.18 log 10 CFU/cm 2 .This is in accordance with antibiofilm activity in vitro.
Compounds 20 and 24 with antibiofilm activity against S. epidermidis ATCC 35984 (MBEC values were 12.5 µM and 6.3 µM, respectively) were evaluated on their antibiofilm potential against clinical staphylococcal strains [40].Confocal microscopy with live/dead staining exhibited that at a concentration of 4× MIC (6.25 µM) of compounds 20 and 24 had bactericidal activity against six strong biofilm-forming clinical isolates (MRSA 1000234, MRSA 916054, MSSA 1815, MRSE 1020081, MRSE 915296, and MSSE 914111).The percentages of viable cells in the biofilms of six strains were 3.5% to 16.2% after compound 20 was treated.In the case of compound 24, the percentage of viable cells in the biofilms was 1.6% to 11.6%.For the controls vancomycin and DMSO, 75.4-80.8%and 88.5-93.6% of the cells were viable in the biofilm, respectively.
Hammad and co-workers carried out the synthesis and antimicrobial evaluation of two series of halogenated thiazolidin-4-ones against clinically relevant bacterial strains.The most effective, compound 25 (MIC = 32 µg/mL against methicillin-resistant Staphylococcus aureus NRS384 (MRSA USA300)), was tested for its antibiofilm activity against the abovementioned staphylococcal strain (Figure 9).The test evaluated the percentage of eradication of MRSA USA300 mature biofilm in relation to DMSO.Compound 25, at 2× MIC, disrupted about 17% of the MRSA USA300 biofilm mass, which was better in biofilm eradication with comparison to the reference, vancomycin (which disrupted about 12% of the biofilm mass) [41].
Staphylococcus aureus NRS384 (MRSA USA300)), was tested for its antibiofilm activity against the above-mentioned staphylococcal strain (Figure 9).The test evaluated the percentage of eradication of MRSA USA300 mature biofilm in relation to DMSO.Compound 25, at 2 x MIC, disrupted about 17% of the MRSA USA300 biofilm mass, which was better in biofilm eradication with comparison to the reference, vancomycin (which disrupted about 12% of the biofilm mass) [41].These results [39][40][41][42] confirm the importance of the halogenphenyl substituent (green color in Figures 8 and 9) in the thiazolidine ring for the antibiofilm activity among 2iminothiazolidin-4-ones.
Staphylococcus aureus NRS384 (MRSA USA300)), was tested for its antibiofilm activity against the above-mentioned staphylococcal strain (Figure 9).The test evaluated the percentage of eradication of MRSA USA300 mature biofilm in relation to DMSO.Compound 25, at 2 x MIC, disrupted about 17% of the MRSA USA300 biofilm mass, which was better in biofilm eradication with comparison to the reference, vancomycin (which disrupted about 12% of the biofilm mass) [41].These results [39][40][41][42] confirm the importance of the halogenphenyl substituent (green color in Figures 8 and 9) in the thiazolidine ring for the antibiofilm activity among 2iminothiazolidin-4-ones.
Another 2-iminothiazolidin-4-one derivative ( 26) conjugated with coumarin moiety (red color in Figure 10) showed high activity against Gram-negative bacteria Acinetobacter baumannii (MIC = 0.25 µg/mL).Viability of A. baumannii biofilm was decreased to 45% at a concentration of 2 µg/mL (8xMIC) [43].El-Hossary et al. synthesized a series of 2-(benzylidenehydrazono)thiazolidin-4-ones with a sulfonamide group in the third position of the thiazolidine ring (blue color in Figure 11).Among this series of derivatives, only two compounds (27 and 28) showed 70% and 80% inhibition of the biofilm formation in S. epidermidis at a concentration of 40 µM, respectively (Figure 11).Analyzing the substitution at the thiazolidine ring, the authors concluded that the presence of an ethylcarboxylate fragment at position 5 was not beneficial for antibacterial and biofilm inhibition activity [44].El-Hossary et al. synthesized a series of 2-(benzylidenehydrazono)thiazolidin-4-ones with a sulfonamide group in the third position of the thiazolidine ring (blue color in Figure 11).Among this series of derivatives, only two compounds (27 and 28) showed 70% and 80% inhibition of the biofilm formation in S. epidermidis at a concentration of 40 µM, respectively (Figure 11).Analyzing the substitution at the thiazolidine ring, the authors concluded that the presence of an ethylcarboxylate fragment at position 5 was not beneficial for antibacterial and biofilm inhibition activity [44].
In the case of P. aeruginosa, compounds 29b and 29c displayed no activity.It is worth noticing that compound 29f was the most active among other derivatives, and its MIC (7.81 µg/mL) was comparable to the reference, vancomycin.Other analogues exhibited moderate activity against P. aeruginosa with MIC in the range of 31.25 to 62.5 µg/mL.New series of indazole-thiazolidin-4-one hybrids were obtained and evaluated for their antibacterial and antibiofilm activity (blue color in Figure 12) [46].Klebsiela planticola MTCC 530 is an important nosocomial pathogen causing urinary tract infections.For this reason, compounds 30a-30c with promising bactericidal activity against K. planticola (MIC New series of indazole-thiazolidin-4-one hybrids were obtained and evaluated for their antibacterial and antibiofilm activity (blue color in Figure 12) [46].Klebsiela planticola MTCC 530 is an important nosocomial pathogen causing urinary tract infections.For this reason, compounds 30a-30c with promising bactericidal activity against K. planticola (MIC = 3.9 µg/mL and MBC = 15.6 µg/mL) were tested for specific K. planticola antibiofilm activity.These hybrids showed promising antibiofilm activity with IC 50 values in the range of 20.28 to 20.79 µg/mL [46].
A series of 2-aryl-3-aminothiazolidin-4-one derivatives with 1,2,4-triazole moiety (31a-31r) (blue color in Figure 13) were synthesized and studied for antibacterial and antibiofilm activity.Screening results showed that derivatives inhibited the S. aureus biofilm formation with IC 50 in the range of 25 to 100 µg/mL.There is a mild to moderate effect.Hybrid 31n with 4-nitrophenyl substituent in the 1,2,4-triazole ring and 4-fluorophenyl in position 2 of the thiazolidine ring was most effective in this series.Its IC 50 was 12.5 µg/mL, which was a higher potential than the standard ciprofloxacin (IC 50 = 25 µg/mL) [47].
biofilm formation with IC50 in the range of 25 to 100 µg/mL.There is a mild to m effect.Hybrid 31n with 4-nitrophenyl substituent in the 1,2,4-triazole ring fluorophenyl in position 2 of the thiazolidine ring was most effective in this series was 12.5 µg/mL, which was a higher potential than the standard ciprofloxacin ( µg/mL) [47].Another series of 2-aryl-3-aminothiazolidin-4-one derivatives wit dibromopyrrole substituent (red color in Figure 14) (32a-32h, and 33) show promising antibiofilm potential (Figure 14).The compounds of these series e inhibition of S. aureus biofilm at a concentration in the range of 0.78 to 6.25 µg epidermidis biofilm with MIC = 3.125-12.5µg/mL and E. faecalis biofilm was inhib concentration in the range of 6.25-12.5 µg/mL.The compounds 32b-32h exhibited S. epidermidis antibiofilm activity comparable to reference vancomycin (MIC µg/mL).It is worth noticing that compounds 32b and 32c showed antibiofilm against S. aureus with MIC = 0.78 µg/mL, and compounds 32d, 32f, 32g, and 32h w = 1.56 µg/mL, which speaks to their potential in the development of newer ant agents [48].In the context of SAR analysis, it is also worth noting that the replacement imino group of compounds 4a-4l (with benzothiazole substituent at the third po  Another series of 2-aryl-3-aminothiazolidin-4-one derivatives wit dibromopyrrole substituent (red color in Figure 14) (32a-32h, and 33) show promising antibiofilm potential (Figure 14).The compounds of these series ex inhibition of S. aureus biofilm at a concentration in the range of 0.78 to 6.25 µg epidermidis biofilm with MIC = 3.125-12.5µg/mL and E. faecalis biofilm was inhib concentration in the range of 6.25-12.5 µg/mL.The compounds 32b-32h exhibited S. epidermidis antibiofilm activity comparable to reference vancomycin (MIC µg/mL).It is worth noticing that compounds 32b and 32c showed antibiofilm against S. aureus with MIC = 0.78 µg/mL, and compounds 32d, 32f, 32g, and 32h w = 1.56 µg/mL, which speaks to their potential in the development of newer ant agents [48].In the context of SAR analysis, it is also worth noting that the replacement o imino group of compounds 4a-4l (with benzothiazole substituent at the third po In the context of SAR analysis, it is also worth noting that the replacement of the 2-imino group of compounds 4a-4l (with benzothiazole substituent at the third position of the thiazolidine ring (blue color in Figure 5)) by 2-aryl substituent completely abolished antibiofilm activity [49].Compound 34 demonstrated a similar potential of biofilm inhibition to rifampicin against S. epidermidis (ATCC 35984) biofilm at a concentration of 20 µg/mL (3.9 ± 0.083 vs. 3.48 ± 0.087 log 10 cfu/mL).Moreover, 34 showed similar levels of activity at a concentration of 20 µg/mL toward three S. epidermidis clinical isolates: 4 ± 0.239 (DSM 3269), 3.8 ± 0.055 (40004), and 3.4 ± 0.032 (48155) log 10 cfu/mL [50].As a continuation of these studies, a series of phenyl-furanyl-rhodanines and their analogues as inhibitors of RNA polymerase were synthesized and evaluated for their antibacterial and antibiofilm activity.Among the tested compounds, five derivatives displayed good activities on S. epidermidis biofilm at a concentration of eightfold the MIC (MIC = 25 µg/mL for 36-39 and 12.5 µg/mL for 35) and a decreased amount of bacteria by 2-3 log 10 cfu/well.It is worth noticing that compound 36 (homologue of 34) demonstrated similar activities on the three S. epidermidis strains (RP62A, 40004, and 48155) at fourfold and eightfold the MIC.These results highlighted the potential role of distancing the carboxylic group from the phenyl ring concerning biofilms.The most active against three S. epidermidis strains was compound 37 at a concentration of eight-fold MIC, with a decreased amount of bacteria by 2.8 log 10 cfu/well (RP62A), 2.3 log 10 cfu/well (40004), and 1.8 log 10 cfu/well (48155) [52].

40004, S. epidermidis 48155).
Compound 34 demonstrated a similar potential of inhibition to rifampicin against S. epidermidis (ATCC 35984) biofilm at a concentr 20 µg/mL (3.9 ± 0.083 vs. 3.48 ± 0.087 log10cfu/mL).Moreover, 34 showed similar activity at a concentration of 20 µg/mL toward three S. epidermidis clinical isolates: (DSM 3269), 3.8 ± 0.055 (40004), and 3.4 ± 0.032 (48155) log10cfu/mL [50].As a cont of these studies, a series of phenyl-furanyl-rhodanines and their analogues as in of RNA polymerase were synthesized and evaluated for their antibacterial and ant activity.Among the tested compounds, five derivatives displayed good activiti epidermidis biofilm at a concentration of eightfold the MIC (MIC = 25 µg/mL for 36 12.5 µg/mL for 35) and a decreased amount of bacteria by 2-3 log10cfu/well.It noticing that compound 36 (homologue of 34) demonstrated similar activities on t S. epidermidis strains (RP62A, 40004, and 48155) at fourfold and eightfold the MIC results highlighted the potential role of distancing the carboxylic group from the ring concerning biofilms.The most active against three S. epidermidis stra compound 37 at a concentration of eight-fold MIC, with a decreased amount of by 2.8 log10cfu/well (RP62A), 2.3 log10cfu/well (40004), and 1.8 log10cfu/well (48155 The next research concerned antibiofilm activities of the 3-arylrhodanines (blue color in Figure 16).The evaluation of antibiofilm activities was carried out to all tested rhodanine derivatives (MBIC ≥ 100 µM).None of the compounds 40-43 showed antibiofilm activity toward Gram-negative E. coli ATCC 25922 and P. aeruginosa PAO1 strains (MBIC ≥ 100 µM).
Additionally, the mechanism of action studies revealed that 3-arylrhodanine derivatives specifically inhibit the early stages of biofilm formation by preventing the adhesion of the bacteria to surfaces.Rhodanine derivative 44 at four-and eight-fold its MIC (4 µM) significantly decreased S. epidermidis biofilm mass by more than 35% and 45%, respectively.In contrast, even at high concentrations, neither linezolid nor vancomycin significantly reduced biofilm formation [54].
The rhodanine derivative (Les-3166) with benzothiazole moiety (blue color in Figure 17) induced significant degradation of the P. aeruginosa biofilm after 24-h exposure to all examined concentrations.The most pronounced antibacterial effects were observed at concentrations of 10, 50, and 100 µM, resulting in reductions of 37%, 34%, and 58%, respectively, compared to the control.In turn, the 5-(indol-3-yl)methylidenerhodanine derivative (Les-6009) exhibited the ability to inhibit the formation of P. aeruginosa biofilm at concentrations of 10, 50, and 100 µM, leading to decreases in biofilm biomass of 28%, In turn, only one Gram-positive strain in the panel of E. faecium F1181 was resistant to all tested rhodanine derivatives (MBIC ≥ 100 µM).None of the compounds 40-43 showed antibiofilm activity toward Gram-negative E. coli ATCC 25922 and P. aeruginosa PAO1 strains (MBIC ≥ 100 µM).
Additionally, the mechanism of action studies revealed that 3-arylrhodanine derivatives specifically inhibit the early stages of biofilm formation by preventing the adhesion of the bacteria to surfaces.
Rhodanine derivative 44 at four-and eight-fold its MIC (4 µM) significantly decreased S. epidermidis biofilm mass by more than 35% and 45%, respectively.In contrast, even at high concentrations, neither linezolid nor vancomycin significantly reduced biofilm formation [54].
The rhodanine derivative (Les-3166) with benzothiazole moiety (blue color in Figure 17) induced significant degradation of the P. aeruginosa biofilm after 24-h exposure to all examined concentrations.The most pronounced antibacterial effects were observed at concentrations of 10, 50, and 100 µM, resulting in reductions of 37%, 34%, and 58%, respectively, compared to the control.In turn, the 5-(indol-3-yl)methylidenerhodanine derivative (Les-6009) exhibited the ability to inhibit the formation of P. aeruginosa biofilm at concentrations of 10, 50, and 100 µM, leading to decreases in biofilm biomass of 28%, 29%, and 45%, respectively, compared to the control.Notably, the replacement sulfanyl group at the second position of the thiazolidine ring by a 4-hydroxyphenylamine moiety (Les-6166) (marked red in Figure 17) did not induce any changes in biofilm eradication at any tested concentration [55].
29%, and 45%, respectively, compared to the control.Notably, the replacement sulfanyl group at the second position of the thiazolidine ring by a 4-hydroxyphenylamine moiety (Les-6166) (marked red in Figure 17) did not induce any changes in biofilm eradication at any tested concentration [55].
29%, and 45%, respectively, compared to the control.Notably, the replacement sulfanyl group at the second position of the thiazolidine ring by a 4-hydroxyphenylamine moiety (Les-6166) (marked red in Figure 17) did not induce any changes in biofilm eradication at any tested concentration [55].
In the next studies, TZD-8 was tested on antibiofilm activity against co-species C. albicans-Streptococcus mutans [58].No change in biofilm formation was observed when using 4 µg/mL of TZD-8.However, increasing concentrations of TZD-8 to 8 and 16 µg/mL revealed biofilm inhibition by 42% and 59%, respectively.Additionally, TZD-8 substantially reduces EPS production in a dose-dependent manner.Furthermore, TZD-8 induces a complex alteration in the symbiotic relationship between these species.The expression of Streptococcal genes associated with quorum sensing (QS) (comDE and luxS), EPS production (gtfBCD and gbpB), and genes related to protection against oxidative stress (nox and sodA) are notably upregulated by TZD-8.In contrast, fungal genes related to hyphae formation (hwp1), adhesion (als3), hydrophobicity (csh1), and oxidative stress response (sod1, sod2, and cat1) are downregulated in the presence of TZD-8.These results suggest that TZD-8 disrupts the symbiotic balance between C. albicans and S. mutans within the dual-species biofilm [58].
A sustained-release membrane (SRM) of TZD-8, which has the properties of an anti-quorum-sensing agent, displayed good potential against oral fungal (C.albicans) biofilms [60].Moreover, SRM-TZD-8 revealed an advantageous effect on the formation of Candida/Streptococcus mixed biofilm on hydroxyapatite in a continuous flow model [59].The utilization of a locally applied sustained-release drug delivery system for TZD-8 has the potential to influence dental polymicrobial biofilm, leading to observable clinical improvements and enhanced patient compliance.Additionally, it was reported that local sustained-release delivery systems with antibiofilm agent TZD-8 were used for the prevention of catheter-associated urinary tract infections.As a matrix that influences the release rate of an antibiofilm agent, ethylcellulose was used [61].
In addition to its antibiofilm activity against C. albicans, TZD-8 also showed activity against other microorganisms that form biofilms.
Lidor et al. reported that TZD-8 displayed an anti-quorum-sensing effect on P. aeruginosa biofilm potentially through the inhibition of LasI and the associated gene regulation.The Pseudomonas quorum-sensing signaling in the PAO1 strain exhibited a dose-responsive manner upon exposure to the TZD-8 molecule.The reduction became noticeable with the initial exposure to 2 µM and 20 µM concentrations of TZD-8.The inhibitor targeting P. aeruginosa quorum-sensing, particularly the TZD-8 compound, holds significant promise (confirmed by in vitro and in silico tests).Its effectiveness not only showcases potential therapeutic benefits but also validates the mechanistic approach employed in the discovery of inhibitors targeting LuxI-type acyl-homoserine lactone synthases [62].
Furthermore, the potential of TZD-8 as an antibiofilm agent has also been confirmed against Cryptococcal biofilm.The compound was tested by XTT reduction assay for biofilm metabolic activity.TZD-8 demonstrated reductions in biofilm metabolic activity of two Cryptococcal strains when treated with sub-inhibitory concentrations (1/4 and 1/16 MIC).The metabolic activity of Cryptococcal neoformans H-99 biofilm was reduced by 51.6% and 47.8% at concentrations of 3.125 and 0.78 µg/mL, respectively.On the other hand, the metabolic activity of Cryptococcal gattii R-272 biofilm was reduced to a lower extent by 34.8% and 26.5% at concentrations of 6.25 and 1.56 µg/mL, respectively [63].
In addition, TZD-8 and TZD-10 (Figure 15) showed activity against Propionibacterium acnes biofilms as quorum-sensing inhibitors [64].Another homologue of 5-octanylidenethiazolidine-2,4-dione, TZD-6, displayed activity against Pectobacterium carotovorum biofilm.Based on TZD-6, an anti-biofilm polymer designed to coat the surfaces of corrugated cardboard was developed, effectively moderating bacterial biofilm formation on those surfaces.The incorporation of a novel thiazolidinedione derivative into acrylic emulsion polymers was evaluated through energy-dispersive X-ray spectrometry analysis, while surface topography on corrugated cardboard surfaces was observed and quantified.Biofilm growth was assessed using q-PCR, targeting the gene responsible for encoding 16s rRNA.The thorough analysis confirmed the consistent integration of the thiazolidinedione derivative TZD-6.Through q-PCR analysis, it was established that TZD-6 reduced biofilm growth by approximately 80% on the surfaces under examination [65].
34.6 µM against Vibrio harveyi BB170 and V. harveyi MM32, respectively.They inhibited the AI-2-mediated bioluminescence of V. harveyi BB170 and V. harveyi MM32 [66].The most effective QS inhibitor among them was TZD-10 with EC50 values of 2.1 µM and 9.8 µM in Vibrio harveyi BB170 and V. harveyi MM32, respectively.These outcomes demonstrated that the length of alkylidene chain at the fifth position of the thiazolidine ring affected QS inhibitory activity.TZD might act on the transcriptional regulatory LuxR of an AI-2regulated QS system to interfere with the binding between LuxR and DNA.The saturated analogue of TZD-10, compound 46, exhibited QS inhibitory activity with EC50 = 21.9 µM (for V. harveyi BB170) and EC50 = 37.9 µM (for V. harveyi MM32) [67].
34.6 µM against Vibrio harveyi BB170 and V. harveyi MM32, respectively.They inhibited the AI-2-mediated bioluminescence of V. harveyi BB170 and V. harveyi MM32 [66].The most effective QS inhibitor among them was TZD-10 with EC50 values of 2.1 µM and 9.8 µM in Vibrio harveyi BB170 and V. harveyi MM32, respectively.These outcomes demonstrated that the length of alkylidene chain at the fifth position of the thiazolidine ring affected QS inhibitory activity.TZD might act on the transcriptional regulatory LuxR of an AI-2regulated QS system to interfere with the binding between LuxR and DNA.The saturated analogue of TZD-10, compound 46, exhibited QS inhibitory activity with EC50 = 21.9 µM (for V. harveyi BB170) and EC50 = 37.9 µM (for V. harveyi MM32) [67].
dioxothiazolidin-5-ylidene)acetic acid, featuring a double bond in the fifth positio TZD ring.Notably, these compounds predominantly incorporated a rhodanine r their structure, as observed in compounds 54-56.
Interestingly, the substituent located in the second position of the phenyl ring appear to exert a significant influence on the anti-adhesive properties.Furtherm isomerization within the phenyl ring, as evidenced by the meta isomer compo seemed to have a negligible impact on these properties.This structural analysis p insights into the specific features associated with the compounds that contribute heightened anti-adhesive effects in the context of biofilm formation.
The ability of compounds to affect microbial adherence as the first and key biofilm formation may be an important criterion for the design of the substan improved antibiofilm activity.As reported by Shakour et al., TZD-imidazole hybrids (57a-57c) (blue color i 22) showed a biofilm reduction of 87.94% (57a) for the P. aeruginosa PAO1 strain as of 30.53% (57b) and 44.65% (57c) for the P. aeruginosa 1074 and P. aeruginosa 1707 The anti-adhesive properties of tested compounds were assessed after 1 h incubation in the conditions and by a method such as a biofilm.The assay was carried out according to the biofilm detection procedure.After 1 h of incubation, the cultures from microplates were removed; the wells were rinsed and, after drying, stained with 0.1% crystal violet for 10 min.After washing off the excess dye, the microwells were poured for 15 min with ethanol.Absorbance values were obtained using a 570 nm wavelength spectrometer (OD570) [70].
Among the compounds examined, namely 53-56, those exhibiting the most pronounced anti-adhesive properties were characterized as derivatives of (2,4-dioxothiazolidin-5-ylidene)acetic acid, featuring a double bond in the fifth position of the TZD ring.Notably, these compounds predominantly incorporated a rhodanine ring into their structure, as observed in compounds 54-56.
Interestingly, the substituent located in the second position of the phenyl ring did not appear to exert a significant influence on the anti-adhesive properties.Furthermore, the isomerization within the phenyl ring, as evidenced by the meta isomer compound 56, seemed to have a negligible impact on these properties.This structural analysis provides insights into the specific features associated with the compounds that contribute to their heightened anti-adhesive effects in the context of biofilm formation.
The ability of compounds to affect microbial adherence as the first and key stage of biofilm formation may be an important criterion for the design of the substances with improved antibiofilm activity.
respectively.It is worth noticing that tested compounds did not show any toxicity against human dermal fibroblasts and 4T1 cells (viability higher than 90%) [71].

Key Structural Features of Thiazolidine-4-Ones Responsible for Antibiofilm Activity
Based on the description provided in this review and on a thorough critical analysis of the scientific information presented herein, the pivotal structural features influencing the antibiofilm activity of thiazolidin-4-one derivatives can be identified.The important structural elements are included in Figure 23.The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.
On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.

Key Structural Features of Thiazolidine-4-Ones Responsible for Antibiofilm Activity
Based on the description provided in this review and on a thorough critical analysis of the scientific information presented herein, the pivotal structural features influencing the antibiofilm activity of thiazolidin-4-one derivatives can be identified.The important structural elements are included in Figure 23.respectively.It is worth noticing that tested compounds did not show any toxicity against human dermal fibroblasts and 4T1 cells (viability higher than 90%) [71].

Key Structural Features of Thiazolidine-4-Ones Responsible for Antibiofilm Activity
Based on the description provided in this review and on a thorough critical analysis of the scientific information presented herein, the pivotal structural features influencing the antibiofilm activity of thiazolidin-4-one derivatives can be identified.The important structural elements are included in Figure 23.The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.
On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.
Introduction of the 2-aryl 3-(2,3-dibromopyrrole) moiety provides effective compounds against S. epidermidis and S. aureus biofilms.The presence of 3-aryl 2-thiazole The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.
On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.
Compounds bearing thiazole in position 2 of thiazolidine showed good antibiofilm potential against P. aeruginosa.

Challenges and Future Perspective
The rise of severe biofilm infections and their resistance to antimicrobial treatment pose significant challenges in the medical field.Over the last two decades, there has been rapid progress in the study of microbial biofilms, revealing the intricate nature of this phenomenon.Despite this progress, the persistence of infections linked to biofilms continues to pose a significant health crisis.Therefore, concerted efforts are essential to deepen our comprehension of the genetics, physiology, and dynamics of bacterial biofilms, particularly concerning chronic infections.
Further investigations should aim to pinpoint the genes responsible for each stage of biofilm development, including those critical for the initial transition from individual cells to aggregate forms.Additionally, understanding the mechanisms through which biofilms acquire antimicrobial resistance is crucial.Simultaneously, the quest for innovative antibiofilm agents targeting biofilm-specific bacterial components is imperative.Thiazolidin-4-ones completely fit into this assumption.
The future directions of small molecule development as antibiofilm agents include thiazolidin-4-ones and involve using innovative multipronged antibiofilm therapeutic strategies, incorporating nanoengineering (antibiofilm nanoparticles), antibiofilm surface coating, or antibiofilm microneedles.The latter can effectively penetrate the EPS barrier of biofilms, offering an efficient drug delivery system.

Conclusions
In conclusion, this paper provides an overview of the antibiofilm properties of various thiazolidin-4-ones, which have been discovered during the last two decades.There are tests against biofilm formation, as well as more in-depth research related to finding the mechanisms of action of thiazolidin-4-ones.Some of them have promising anti-adhesive properties, reduce EPS production, or inhibit quorum-sensing signaling.Compound 3b showed significant antibiofilm activity against the MRSA and VRE strains (BIC values of 2.22 µg/mL and 3.05 µg/mL, respectively), as well as significant activity against K. pneumoniae and E. coli biofilms with BIC values of 3.25 µg/mL and 2.03 µg/mL, respectively.Compounds 20 and 24 exhibited bactericidal activity against six strong biofilm-forming staphylococcal clinical isolates.Thiazolidine-4-ones (32a-32h) demonstrated equal S. epidermidis antibiofilm activity compared to the standard vancomycin (MIC = 3.125 µg/mL).Moreover, 32b and 32c exhibited high effective inhibition of S. aureus biofilm at a concentration of 0.78 µg/mL.
In turn, some thiazolidine-4-ones showed antibiofilm activity against S. epidermidis biofilm as a YycG histidine kinase inhibitor.The compound TZD-8 demonstrated broadspectrum antibiofilm activity against C. albicans, S. epidermidis, and P. aeruginosa, as well as mixed Candida/Bacteria biofilm formation.Furthermore, TZD-8 can be used in clinical aspects for the prevention of catheter-associated urinary tract infections or can affect dental polymicrobial biofilm.Its homologue (TZD-10) was the most effective QS inhibitor in V. harveyi BB170 at a micromolar concentration (EC 50 = 2.1 µM).
Analyzing the results, it can be concluded that the group of thiazolidin-4-one derivatives have great potential as antibiofilm agents and can act as inhibitors of QS signaling, reduce EPS production, or display anti-adhesive properties.Therefore, this review may be useful for the further development of new molecules based on a thiazolidin-4-one core as potential bioactive agents to combat biofilm formation.

Figure 15 .
Figure 15.The structures of 3-allylrhodanines (blue color).The next research concerned antibiofilm activities of the 3-arylrhodanines (40-43) (blue color in Figure 16).The evaluation of antibiofilm activities was carried out using a panel of Gram-positive (S. aureus, S. epidermidis, E. faecalis, Enterococcus faecium, and Enterococcus gallinarum) and Gram-negative strains (E. coli and P. aeruginosa) including resistant strains.The results of this study demonstrated that the 3-arylrhodanines 40-43 have potent antibiofilm activity against a wide range of Gram-positive bacteria.Compounds 40-43 exhibited good antibiofilm activity toward Staphylococcus spp. in the range of MBIC = 2.6-100 µM.Most active among them were compounds 40 and 43 with MBIC 2.6 µM against S. aureus ATCC 35556 and derivatives 40, 41, and 43 that inhibited biofilm growth at a concentration of 8.8 µM against methicillin-resistant S. aureus MRSA-47263.

Figure 16 .
Figure 16.The structures of 3-arylrhodanines.However, taking into account all panels of Gram-positive strains, compound 42 showed the best antibiofilm potential.Its MBICs against S. aureus strains were in the range of 10.5 to 31.5 µM, against S. epidermidis strains from 12.5 to 59.5 µM, against E. faecalis from 8.8 to 21 µM, against E. faecium from 15.7 to 21 µM, and toward E. gallinarum 2392 MBIC = 17.7 µM[53].In turn, only one Gram-positive strain in the panel of E. faecium F1181 was resistant to all tested rhodanine derivatives (MBIC ≥ 100 µM).None of the compounds 40-43 showed antibiofilm activity toward Gram-negative E. coli ATCC 25922 and P. aeruginosa PAO1 strains (MBIC ≥ 100 µM).Additionally, the mechanism of action studies revealed that 3-arylrhodanine derivatives specifically inhibit the early stages of biofilm formation by preventing the adhesion of the bacteria to surfaces.Rhodanine derivative 44 at four-and eight-fold its MIC (4 µM) significantly decreased S. epidermidis biofilm mass by more than 35% and 45%, respectively.In contrast, even at high concentrations, neither linezolid nor vancomycin significantly reduced biofilm formation[54].The rhodanine derivative (Les-3166) with benzothiazole moiety (blue color in Figure17) induced significant degradation of the P. aeruginosa biofilm after 24-h exposure to all examined concentrations.The most pronounced antibacterial effects were observed at concentrations of 10, 50, and 100 µM, resulting in reductions of 37%, 34%, and 58%, respectively, compared to the control.In turn, the 5-(indol-3-yl)methylidenerhodanine derivative (Les-6009) exhibited the ability to inhibit the formation of P. aeruginosa biofilm at concentrations of 10, 50, and 100 µM, leading to decreases in biofilm biomass of 28%, 29%, and 45%, respectively, compared to the control.Notably, the replacement sulfanyl group at the second position of the thiazolidine ring by a 4-hydroxyphenylamine moiety (Les-6166) (marked red in Figure17) did not induce any changes in biofilm eradication at any tested concentration[55].

Figure 17 .
Figure 17.The structures of rhodanine derivatives with benzothiazole and indole moieties.

Figure 18 .
Figure 18.The structures of 5-alkylidene-TZD and some of their saturated analogues.

Figure 17 .
Figure 17.The structures of rhodanine derivatives with benzothiazole and indole moieties.

Figure 17 .
Figure 17.The structures of rhodanine derivatives with benzothiazole and indole moieties.

Figure 18 .
Figure 18.The structures of 5-alkylidene-TZD and some of their saturated analogues.

Figure 18 .
Figure 18.The structures of 5-alkylidene-TZD and some of their saturated analogues.

Figure 23 .
Figure 23.Key structural features for thiazolidine-4-ones as antibiofilm agents.Yellow colorsubstitution at the position 2; green color-substitutions at the position 3; beige color-substitutions at the position 5; pink color-substitutions at the positions 2 and 3; cyan color-substitutions at the position 3 and 5; blue color-substitutions at the positions 2, 3 and 5 of thiazolidine ring.Introduction of the 5-alkylidene chain to the TZD ring provides compounds with good antibiofilm activity against C. albicans, S. epidermidis, S. aureus, P. aeruginosa, and Candida/Streptococcus mixed biofilm.Moreover, some of them inhibited QS signals against V. harveyi, S. epidermidis, S. aureus, and P. aeruginosa.The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.Introduction of the 2-aryl 3-(2,3-dibromopyrrole) moiety provides effective compounds against S. epidermidis and S. aureus biofilms.The presence of 3-aryl 2-thiazole

Figure 23 .
Figure 23.Key structural features for thiazolidine-4-ones as antibiofilm agents.Yellow colorsubstitution at the position 2; green color-substitutions at the position 3; beige color-substitutions at the position 5; pink color-substitutions at the positions 2 and 3; cyan color-substitutions at the position 3 and 5; blue color-substitutions at the positions 2, 3 and 5 of thiazolidine ring.Introduction of the 5-alkylidene chain to the TZD ring provides compounds with good antibiofilm activity against C. albicans, S. epidermidis, S. aureus, P. aeruginosa, and Candida/Streptococcus mixed biofilm.Moreover, some of them inhibited QS signals against V. harveyi, S. epidermidis, S. aureus, and P. aeruginosa.The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.Introduction of the 2-aryl 3-(2,3-dibromopyrrole) moiety provides effective compounds against S. epidermidis and S. aureus biofilms.The presence of 3-aryl 2-thiazole

Figure 23 .
Figure23.Key structural features for thiazolidine-4-ones as antibiofilm agents.Yellow colorsubstitution at the position 2; green color-substitutions at the position 3; beige color-substitutions at the position 5; pink color-substitutions at the positions 2 and 3; cyan color-substitutions at the position 3 and 5; blue color-substitutions at the positions 2, 3 and 5 of thiazolidine ring.Introduction of the 5-alkylidene chain to the TZD ring provides compounds with good antibiofilm activity against C. albicans, S. epidermidis, S. aureus, P. aeruginosa, and Candida/Streptococcus mixed biofilm.Moreover, some of them inhibited QS signals against V. harveyi, S. epidermidis, S. aureus, and P. aeruginosa.The presence of arylidene and norfloxacin moieties in positions 5 and 3 of the thiazolidine ring, respectively, was effective against bacterial biofilms but showed a weak effect on fungal biofilm formation.On the other hand, the presence of the 1,3,5-triazine moiety in the third position in thiazolidine provides moderate antibiofilm activity against S. aureus and P. aeruginosa.Introduction of the 2-aryl 3-(2,3-dibromopyrrole) moiety provides effective compounds against S. epidermidis and S. aureus biofilms.The presence of 3-aryl 2-thiazole substituents significantly increased antibiofilm activity against MRSA, VRE, K. neumoniae, and E. coli.Compounds bearing thiazole in position 2 of thiazolidine showed good antibiofilm potential against P. aeruginosa.