Synthesis, antimicrobial properties and in silico studies of aryloxyacetic acid derivatives with hydrazone or thiazolidine-4-one scaffold

Abstract In this work, twenty hydrazide-hydrazone and 4-thiazolidinone derivatives were synthesized starting from m-cresol. Antimicrobial evaluation was carried out by microdilution method against Enterococcus faecalis and Staphylococcus aureus as Gram-positive bacteria and Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria, and three pathogenic fungi Candida albicans, Candida parapsilosis and Candida krusei. Some compounds possessed considerable antimicrobial properties against the tested microorganisms, particularly against E. coli. 4-Thiazolidinones containing 3-methoxyphenyl and 3,5-dichlorophenyl moieties (4h and 4i) were found to be the most active derivatives with MICs of 2 μg/mL against E. coli. N'-[(3,5-dichlorophenyl)methylidene]-2-(3-methylphenoxy)acetohydrazide (3i) also displayed antifungal activity against Candida krusei that was comparable to fluconazole. Calculated drug-likeness and ADMET parameters of the most active compounds confirmed their potential as antimicrobial drug candidates. Molecular docking investigations were carried out in the thiamine diphosphate-binding site of pyruvate dehydrogenase multienzyme complex E1 component (PDHc-E1) to clarify the potential antibacterial mechanism against E. coli. The results showed the potential and importance of developing new hydrazones and 4-thiazolidinones that would be effective against microbial strains. Communicated by Ramaswamy H. Sarma


Introduction
Antimicrobial resistance (AMR)-that occurs when microorganisms become resistant to the effects of antimicrobialsis recognized as one of the prominent global public health threats in the 21st century. According to The Lancet's most comprehensive analysis on bacterial AMR to date, while 4.95 million people died in 2019 because of antibiotic-resistant bacterial infections, 1.27 million people died directly as a result of AMR (Murray et al., 2022). Antimicrobials, notably antibiotics, have been the keystone in modern medicine. The discovery of antibiotics and their usage in the treatment of infectious diseases worldwide eventually have been regarded as a revolution (Gajd acs & Albericio, 2019). Although all beneficial outcomes of antimicrobials in the process of dealing with bacterial infections, over the years their effectiveness has started to decrease due to the evolution of bacterial resistance in these pathogens, making the need for the new generation of antimicrobials more urgent to reduce the spread of antibiotic resistance (de la Fuente-Nunez et al., 2017;Laxminarayan et al., 2016). For instance, Escherichia coli is a typical commensal of the vaginal and skin microbiota, as well as the stomach (Sharma et al., 2022). Due to the development of resistance to the majority of mostly used antimicrobial drugs (e.g., amoxicillin, cefixime, and ciprofloxacin), the management of E. coli infections has grown more challenging (Laxminarayan et al., 2013). It is therefore also necessary to develop new agents with excellent antibacterial effects against drug-resistant clinical pathogens such as E. coli, S. aureus and K. pneumonia (Mccarthy et al., 2020).
Hydrazones are a significant class of organic compounds because of not only their biological activities but also azomethine group (-NH-N ¼ CH-) attached to the carbonyl functionality, which allows applying various synthetic routes to reach various heterocyclic scaffolds, such as 4-thiazolidinones (Rollas & K€ uç€ ukg€ uzel, 2007). Hydrazone moiety is also present in the chemical structure of medicines with antimicrobial activity (K€ uç€ ukg€ uzel et al., 2002, 2003), such as nitrofurazone, furazolidone, or nitrofurantoin ( Figure 1).
In medicinal chemistry, heterocyclic scaffolds are known for their great importance. It has been proved that more than 85% of all chemical entities which have biological activity include at least one heterocycle (Jampilek, 2019). 4-Thiazolidinones are one of the most important five-membered heterocyclic rings, which attracted special interest over the years with their chemical structure containing one nitrogen and one sulfur atom, as well as a C ¼ O group at the 4-position. It has been reported that while substituents in the other positions may be varied, the difference in the properties is mainly provided by the group attached to the carbon in the 2-position (Verma & Saraf, 2008). They are associated with their broad biological activities, such as antitumoral (Cikla et al., 2013;Han et al., 2021;Koç et al., 2022;Şenkardeş & Kucukguzel, 2016), antidiabetic (Datar & Aher, 2016), antiinflammatory (Shawky et al., 2020), antiviral (Kaushik-Basu et al., 2008), and antitubercular (K€ uç€ ukg€ uzel et al., 2006). The following thiazolidinone-based compounds are used as registered drugs: ralitoline (anticonvulsant), piprozolin (choleretic), etozolin (antihypertensive, diuretic), spiclomazine (psychotropic) (Figure 2). In addition, the researchers have determined by molecular modeling that some structures containing hydrazone and thiazolidone inhibit potential target enzymes of SARS-CoV-2 (S¸ahin et al., 2021).
The valuable biological activities of hydrazone and 4-thiazolidinone based compounds prompted us to synthesize new derivatives. In continuation of our work on these moieties, we have evaluated the antimicrobial activities of a series of hydrazones and 4-thiazolidinones derived from m-cresol and supported the obtained biological results by computational techniques.

Chemistry
All melting points are recorded on digital Electrothermal Thermo Scientific IA9300 instrument and are uncorrected. The IR spectra (cm À1 ) was characterized on a Fourier transform infrared spectrometer (FTIR, 8400S, Shimadzu, Japan). 1 H and 13 C-NMR spectra were recorded in CDCl 3 and/or DMSO-d 6 on BRUKER NMR spectrometer and are reported relative to deuterated solvent signals. Elemental microanalysis was carried out on a CHNS-932 (LECO) analyzer.
A mixture of hydrazone derivatives (3a-j) (0.001 mol) and thioglicolic acid (0.0012 mol) in toluene (80-100 ml) was refluxed using Dean-Stark apparatus for 10-12 h. After distillation of toluene, the separated product was crystallized from ethanol-water to give compounds 4a-j.   The fungal and bacterial isolates were subcultured onto Sabouraud dextrose agar and Mueller Hinton agar, respectively prior to testing. For fungi, broth microdilution was performed using RPMI 1640 broth (ICN-Flow, with glutamine, without bicarbonate and with pH indicator) buffered to pH 7.0 with 3-N-morpholinopropanesulfonic acid (MOPS). For bacteria, microdilution test was conducted using Mueller Hinton broth (MHB, Difco Laboratories, USA) buffered to pH 7.0 with 3-N-morpholinopropanesulfonic acid (MOPS, Sigma, USA). The final test concentration of fungi and bacteria was 0.5 to 2.5 Â 10 3 cfu/mL and 5 Â 10 5 cfu/mL, respectively. After dissolving the compounds in DMSO, their final two fold concentrations (1024 to 1 mg/mL) were prepared in the wells of the microtiter plates. The plates for bacteria and fungi were incubated at 35 C for 18-24 h and 48 h, respectively. MIC values were read as the lowest concentration (mg/mL) of test compound that fully inhibited visual microorganism growth.

Drug likeness and ADMET analysis
The chemical structures of the selected molecules were sketched, SMILES codes were generated, and the descriptors indicating the compounds' drug-likeness were computed in SwissADME (Daina et al., 2017). ADMET properties such as water solubility, Caco-2 permeability, intestinal absorption, blood brain barrier (BBB) permeability along with metabolism and toxicity parameters were predicted using pkCSM web server (Pires et al., 2015). AMES: assay of the ability of a chemical compound to induce mutations in DNA; BBB: blood-brain barrier (log BB > 0.3 (cross BBB), log BB < -1 (poorly distributed brain); (http://biosig.unimelb.edu.au/pkcsm/prediction).

Molecular docking
The crystal structure of pyruvate dehydrogenase multienzyme complex E1 component (PDHc-E1) from E. coli was obtained from Protein Data Bank (PDB ID: 1L8A, Resolution: 1.85 Å) (Arjunan et al., 2002). The chemical structures of 3h, 4d, and m-cresol were sketched and energy minimized using the MMFF94 force field in LigandScout 4.4 (Wolber & Langer, 2005). The compounds were subsequently docked into the binding site of the cofactor thiamin diphosphate (ThDP) using AutoDock 4.2 (Morris et al., 2009), implemented in LigandScout, with default parameters. The most plausible binding modes were selected upon the visual analysis of the obtained docking poses in LigandScout. 3D and 2D representations of the interactions were prepared using Maestro (Schr€ odinger Release 2019-1: Maestro, 2019).

Chemistry
The title products 3a-j and 4a-j were synthesized using the reactions depicted in Scheme 1. Compounds 1 and 2 were obtained by previously described procedures (Kulabaş et al., 2016;Şenkardeş et al., 2021). Condensation of 2 with the various aldehydes in ethanol yielded the hydrazide-hydrazones 3a-j. The reaction of 3a-j with thioglycolic acid in toluene yielded 4-thiazolidinones. Among the newly synthesized compounds, compounds 3d and 3 g have got only CAS numbers (CAS No: 351871-47-1 and 2486106-08-3, respectively) with no spectroscopic data. So, all newly synthesized compounds were checked for purity using elemental microanalysis and fully characterized by their spectral data and melting points. The structures of the molecules were confirmed by elemental analysis, FTIR spectra and NMR analyses. In the IR spectra of 3a-j, C¼ O bands were observed in the 1674-1680 cm À1 regions. The IR spectra of 4a-j exhibited another C ¼ O band (1712-1716 cm À1 ) which indicated the presence of a thiazolidinone ring.
According to literature, the hydrazone moieties may present as E/Z isomers about C ¼ N bonds and cis/trans CO-NH conformers. Literature survey reveals that both cis and trans conformers are observed in polar aprotic solvents (Koc¸et al., 2022;Palla et al., 1986). When we analyse 1 H-NMR spectra of hydrazone derivatives in DMSO-d 6 as an aprotic solvent; we observed two singlets for each of the methylene, azomethine and amide protons corresponding to E and Z forms. As indicated in our previous article (S¸enkardeş et al., 2021), aromatic methyl protons of 3a-j appeared at 2.25-2.29 ppm as a double singlet. The disappearance of the azomethine signal and the appearance of a signal for the thiazolidinone ring CH displayed the ring closure. Multiplets or double doublet peaks at d values between 3.52 and 4.04 ppm suggest the presence of CH 2 protons of the ring in the 4a-j derivatives. Finally, peaks for phenyl and thienyl protons in the compounds appeared between 6.59-8.33 ppm.

Biological evaluation
Antibacterial and antifungal activities of the compounds 3a-j and 4a-j were tested using microbroth dilution method. Tested organism strains were; S. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), E. faecalis (ATCC 29212), and E. coli (ATCC 25922) as bacteria and C. albicans (ATCC 90028), C. parapsilosis (ATCC 90018) and C. krusei (ATCC 6258) as fungal strains. The observed data on the activity of the products are shown in Table 1.
P. aeruginosa causes severe acute and chronic infections in the host body, including the skin, urinary tract, and respiratory system (Mansuri et al., 2022). Compounds 3b, 3i Scheme 1. Syntetic route of m-cresol derivatives (3a-j and 4a-j). and 4h have shown same potency (MIC¼ 16 mg/mL) against P. aeruginosa. Furthermore, compounds 3h, 3i, 4a, 4b, 4d, 4h and 4i showed low MICs in the range of 2-8 mg/mL against E. coli. Compound 4i with 3,5-dichlorophenyl substituent also showed the highest effect against S. aureus, which represents Gram positive bacteria with MIC of 8 mg/ mL. This study showed that these m-cresol derivatives were particularly effective against E. coli.
On the other hand, from the data shown in Table 1, it is clear that N'-[(3,5-dichlorophenyl)methylidene]-2-(3-methylphenoxy)acetohydrazide (3i) exhibited the lowest MIC values in the range of 16-32 mg/mL against pathogenic fungal strains, particularly as effective as standard Fluconazole (MIC value¼ 16 mg/mL) against C. krusei. These data clearly demonstrated that the 3,5-dichlorophenyl substituent plays a major role in antimicrobial activity of these derivatives.

In silico prediction of drug-likeness and ADMET profiles
When turning bioactive compounds into drug molecules, unsuitable physicochemical properties can stand as a significant impediment. Hence, we computed drug-likeness parameters of the selected compounds. We picked up the molecules exhibiting antibacterial activity against E. coli with MIC value 4 mg/mL (3h, 4a, 4d, 4h, and 4i) and also 3i as the most active antifungal derivative. Initially, we calculated the physicochemical properties constituting Lipinski's rule-offive. This rule of thumb states that an orally active drug should not break more than one of these criteria: molecular weight less than 500 Da, the octanol-water partition coefficient (Log P) not greater than 5, no more than 5 hydrogen bond donors, and no more than 10 hydrogen bond acceptors (Lipinski et al., 2001). Furthermore, we computed topological polar surface area and the number of rotatable bonds which are also considered critical parameters for the oral bioavailability prediction of novel compounds (Veber et al., 2002). The calculated parameters of the most active molecules are reported in Table 2. Based on these results, all selected compounds adhered to Lipinski's rules. Among them, lipophilicity is particularly regarded as an important parameter for drug design and development process (Arnott & Planey, 2012). This molecular descriptor plays a central role not only in the transport of drugs through biological systems but also in the interactions of drugs with their biological targets (Testa et al., 2000). Lipophilicity is considered one of the main structural properties of antimicrobial agents correlating with their biological effects (Testa et al., 2000;Ullah et al., 2018). Therefore, one of the factors determining the antimicrobial activity of the selected compounds is likely to be their lipophilic characters. Rotatable bonds count is a considerable parameter indicating the number of bonds that rotate freely around themselves and should be <10. Our selected compounds obeyed this rule with 10 rotatable bonds. Since most therapeutics have topological polar surface area less than 140-150 Å 2 , our compounds also stay in the favorable range with their values varying from 50.69 to 93.17 Å 2 (Ertl et al., 2000).  Organic molecules' solubility is another important parameter. Poor aqueous solubility of drug candidates is one of the most significant barriers to drug discovery. From the pkCSM results shown in Table 3, it was observed that the compounds tested are moderately soluble. (Insoluble -10 poorly soluble < -6 < moderately < -4 < soluble < -2 very soluble < 0 highly soluble) (Amin et al., 2022).
The predictive model of pkCSM shows that these molecules with predicted values >0.9 have high Caco2 cell permeability. According to Chander et al. (2017), a compound is considered to have good absorption if its absorption value is greater than 80%, and it is considered to have poor absorption if it is less than 30%. Table 3 shows that the six compounds have good absorption. These derivatives have log values of K p <-2.5, meaning that these compounds have good permeability on the skin (Pires et al., 2015). Also, these compounds cannot penetrate the blood-brain barrier because they do not have log BB values of >0.3 (Pires et al., 2015).
The cytochrome P450 (CYP) family catalyzes drug metabolism and thus is relevant to drug development. The subtype of cytochrome P450 CYP2D6 shows that compounds could not be substrates or inhibitors of this main subtype, which can reduce the possibility of drug-drug interactions. According to data, compounds 4a, 4d, 4h and 4i are CYP2C9 and CYP3A4 inhibitors, while CYP1A2 was not inhibited by these compounds in silico. Additionally, all the studied compounds were found to be a substrate of CYP3A4 and an inhibitor of CYP2C19.
To predict the compounds' excretion process, the total clearance, a combination of hepatic clearance and renal clearance, was calculated. A drug with a high total clearance value will be excreted quickly. Compounds present total clearance values ranging from -0.219 to 0.261. Also, they present no AMES toxicity and skin sensibilization, only compound 4h can be hepatotoxic.
Consequently, the studied molecules possess suitable physicochemical and ADMET properties that make them antimicrobial drug candidates.

Molecular docking
The synthesized compounds were particularly effective against E. coli. Therefore, we aimed to rationalize the obtained biological data and find out the potential antibacterial activity mechanism through molecular docking studies.
The pyruvate dehydrogenase complex (PDHc) consisting of three enzymes catalyzes the conversion of pyruvate into acetyl coenzyme A (acetyl-CoA) initiated by the oxidative decarboxylation of pyruvate. Due to this function, PDHc plays a central role in the cellular metabolism of organisms (Patel & Korotchkina, 2003).  . ThDP is represented as orange stick and balls, amino acid residues as gray sticks, protein backbone as white cartoons, binding interactions as color dashes (purple for hydrogen bond, cyan for p-p), and the magnesium ion as faded pink sphere.
The first member of this complex is pyruvate dehydrogenase (E1) which is responsible for the initial step of this multistep process employing thiamine diphosphate (ThDP) and magnesium ion (Mg 2þ ) as cofactors (Patel et al., 2014). Hence, inhibiting PDHc-E1 is considered an effective strategy to block PDHc activity. As ThDP is one of the essential cofactors that regulate PDHc-E1 activity, blocking its active site with an inhibitor is a rational approach to inactivate the enzyme.
The researchers gave much effort to design and synthesize new PDHc-E1 inhibitors. ThDP analogs carrying core thiazole-based moieties were often developed to target the binding site of ThDP. Despite their high binding affinities, they exhibited low bioavailability and no potential utility because of their highly charged phosphate groups (Arjunan et al., 2006;Nemeria et al., 2001). Subsequently, other chemical groups of compounds were considered to block PDHc-E1 activity. Among them, hydrazone functionality was one of the most common pharmacophores present in the structures of newly designed inhibitors (Figure 3) (He et al., 2017;Zhou et al., 2021).
Prompted by these considerations and the pharmacophore similarity of our compounds with the previously reported inhibitors, we aimed to support the high antibacterial activity against E. coli by molecular docking studies into the cofactor (ThDP) binding site of E. coli PDHc-E1. We picked up the most active compound from each series: 3h from hydrazone derivatives and 4d from thiazolidinones. We additionally docked m-cresol to determine how the modifications on this core moiety affect the activities of our compounds on E. coli PDHc-E1.
We initially examined the interactions of thiamine diphosphate (ThDP) within the cofactor binding site of E. coli PDHc-E1 (Figure 4).
Diphosphate group of ThDP interacts with the enzyme in two ways; through forming hydrogen bonds to Gly231 and Asn260, and through interacting with Mg 2þ ion. Furthermore, ThDP adopts a "V" conformation allowing thiazolium ring mainly interact with Hid142. Aminopyrimidine moiety participates in binding to the active site of E. coli PDHc-E1 through hydrogen bonds with Val192 and Val194. p-p stacking between aminopyrimidine ring and Phe602 is also observed.
Subsequently, we compared the binding interactions of 3h and 4d in the ThDP binding site of E. coli PDHc-E1 with the co-crystallized ligand ThDP.
Based on the obtained interactions shown in Figure 5, 3h occupied ThDP binding site in a "V" conformation similar to ThDP with the estimated binding energy of -10.42 kcal/mol. 3-Methylphenyl ring was responsible for hydrophobic contacts, particularly to Tyr599, Phe602, and Met194. On the other side of the molecule, 2-(trifluoromethoxy)phenyl moiety formed additional hydrophobic interactions. 2-OCF 3 substituent on this phenyl ring oriented towards the Mg ion and formed two key hydrogen bonds to Ser109 and Asn260, in the same way as ThDP. The low MIC value of 3h against E. coli can be explained due to the similar binding mode of 3 h to ThDP. When the interactions of 4d in the same binding site (binding energy: -8.86 kcal/mol) were examined, it was observed that 3-methylphenoxy moiety occupied the same lipophilic pocket providing hydrophobic interactions with the enzyme. N-H group of acetamide functionality formed a hydrogen bond to Asp521 to stabilize the ligand in the active pocket. It is noteworthy that two phenyl rings on the other side of the molecule formed p-cation interactions with Mg 2þ , the other co-factor of the enzyme.  (3h) and green (4d) balls and sticks, some key amino acid residues participating in ligand binding as gray sticks, protein backbone as white cartoon, hydrogen bonds as purple dashes, and the magnesium ion as faded pink sphere.
We also docked our starting compound m-cresol in the same binding pocket. Due to its less bulky structure compared to our final compounds, m-cresol could only occupy the phosphate binding site of the pocket. Therefore, as expected, its estimated binding energy is higher (-4.70 kcal/ mol) than our final compounds. This situation shows that the modifications on m-cresol leading to hydrazone or thiazolidine-4-one moieties are beneficial to the binding of the studied molecules to the cofactor (ThDP) binding site of E. coli PDHc-E1.
As a result, molecular docking studies point out the inhibition of the cofactor binding site of PDHc-E1 as the potential antibacterial mechanism of our compounds against E. coli. It is also highlighted that the substituent type on the phenyl ring is significant for the antibacterial activity trend of these molecules against E. coli.

Conclusion
The hydrazones and 4-thiazolidinones of m-cresol were readily prepared for evaluation of their antimicrobial activity. According to the activity results, some of the synthesized compounds demonstrated remarkable antibacterial effects on E. coli that causes serious infections by consuming contaminated food and water or by contacting infected people. Among them, compounds 4d and 4i were the most promising derivatives against E. coli with MIC value of 2 lg/mL. Additionally, compound 3i exhibited comparable antifungal activity to fluconazole against Candida krusei is well known as a fungal nosocomial pathogen. Calculated physicochemical descriptors of the most active compounds demonstrated that they can be considered drug candidates. The inhibition of the cofactor binding site of E. coli PDHc-E1 was determined as the potential activity mechanism of the compounds against E. coli through molecular docking studies. Altogether, our results may be useful for further improvements in development of new antimicrobial drugs.