Synthesis and Development of N-2,5-Dimethylphenylthioureido Acid Derivatives as Scaffolds for New Antimicrobial Candidates Targeting Multidrug-Resistant Gram-Positive Pathogens

The growing antimicrobial resistance to last-line antimicrobials among Gram-positive pathogens remains a major healthcare emergency worldwide. Therefore, the search for new small molecules targeting multidrug-resistant pathogens remains of great importance. In this paper, we report the synthesis and in vitro antimicrobial activity characterisation of novel thiazole derivatives using representative Gram-negative and Gram-positive strains, including tedizolid/linezolid-resistant S. aureus, as well as emerging fungal pathogens. The 4-substituted thiazoles 3h, and 3j with naphthoquinone-fused thiazole derivative 7 with excellent activity against methicillin and tedizolid/linezolid-resistant S. aureus. Moreover, compounds 3h, 3j and 7 showed favourable activity against vancomycin-resistant E. faecium. Compounds 9f and 14f showed broad-spectrum antifungal activity against drug-resistant Candida strains, while ester 8f showed good activity against Candida auris which was greater than fluconazole. Collectively, these data demonstrate that N-2,5-dimethylphenylthioureido acid derivatives could be further explored as novel scaffolds for the development of antimicrobial candidates targeting Gram-positive bacteria and drug-resistant pathogenic fungi.


Introduction
Infections caused by Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) remain among one of the most common infectious agents worldwide. Infections caused by MRSA and VRE are responsible for increased mortality rates among hospitalised patients with chronic illness [1,2]. Numerous virulence factors are harboured by MRSA and VRE as well as biofilm production often leading to infections in surgically implanted catheters, especially in patients receiving cancer chemotherapy, hematopoietic stem-cell transplantation, and solid-organ transplantation. The profound antimicrobial resistance among MRSA and VRSA shortens the available treatment options, resulting in the systemic manifestation of the pathogen and death. Therefore, it is important to develop and investigate novel compounds with antimicrobial activity directed to MRSA and VRE.
The ability of pathogens to form biofilms on indwelling catheters has previously been associated with greater morbidity [3,4]. Moreover, the formation of interkingdom biofilms, consisting of bacterial pathogens and Candida species worsens the clinical prognosis. Despite being the most common fungal pathogen in clinical settings responsible for great morbidity worldwide, Candida spp. can form synergistic relationships with S. aureus leading to bacterial protection from antimicrobial treatment. Moreover, there is evidence that S. aureus provides a niche for C. albicans to evade antifungal drugs and thus survive the treatment [5,6]. Moreover, the recent emergence of the highly resistant C. auris with an instinctive resistance to azoles remains an increasing hardcore threat. Therefore, it is critical to apply novel concepts and develop multifunctional compounds with the ability to evade pre-existing antimicrobial activity in Gram-positive pathogens and clinically important fungi [7][8][9].
The development of new antimicrobials is focused on aspects of enhancing antimicrobial properties as well as evading antimicrobial resistance of bacterial and fungal pathogens or restoring the susceptibility of the pathogens to clinically approved antimicrobials [10][11][12][13][14][15]. In addition, compounds should remain minimally toxic to the host and show good pharmacological properties. However, the main aspect of developing effective drugs is their structural characteristics and the rate of activity. Lipophilicity is a significant physicochemical parameter that influences the membrane's transport and the binding's ability to act [16][17][18][19]. The study of lipophilicity-related parameters of thiazole derivatives showed these compounds to be promising drug candidates [20].
The 2,5-dimethylphenyl scaffold is a common structural feature in many antimicrobial compounds, particularly in the class of compounds known as phenylpropanoids [44]. These compounds have been found to have antimicrobial activity against a wide range of microorganisms, including bacteria, fungi and viruses [45][46][47]. They have been the subject of extensive research for the development of new antimicrobial agents, particularly in the fight against antibiotic-resistant infections [48]. Some examples of drugs that have been developed from the 2,5-dimethylphenyl scaffold include antifungal echinocandins and antibacterial agents such as linezolid. Therefore, novel compounds bearing 2,5-dimethylphenyl substituents may pose antimicrobial activity against Gram-positive and Gram-negative pathogens with novel or emerging resistance mechanisms. To explore aminothiazole derivatives as novel candidates targeting clinically important and multidrugresistant WHO priority pathogens, we generated a series of aminothiazole derivatives bearing N-2,5-dimethylphenyl and β-alanine and characterised their antimicrobial activity using pathogens with defined resistance mechanisms. In addition to that, we characterised anticancer activity using A549 and Caco-2 cell culture models. A SAR investigation of aminothiazole derivatives revealed the influence of modification to a carboxylic acid moiety on the antimicrobial activity of the synthesized compounds [49]. In this paper, we describe the synthesis and in vitro characterisation of antimicrobial and anticancer properties of a series of novel thiazole derivatives bearing 4-quinolone, quinoxaline, naphthoquinone, hydrazone, benzimidazole, and benzenesulphonamide moieties. It is known that thiazoles can be synthesized from α-bromoketone and thiourea via Hantzsch thiazole synthesis in high yields.

Synthesis
This research work is a continuation of projects on the synthesis and biological evaluation of various thiazole derivatives. 3-(1-(2,5-Dimethylphenyl)thioureido)propanoic acid (1) as the starting compound was synthesized by multistep reactions according to the method described in [50]. It is known that thiazoles can be synthesised from α-haloketone and a thioamide via Hantzsch thiazole synthesis in high yields [51]. To synthesize thiazolone 2 (Scheme 1), thioureido acid 1 was reacted with monochloroacetic acid in an aqueous 10% potassium carbonate solution at room temperature followed by the acidification with acetic acid to pH 6. In this reaction, the conventional reaction conditions at reflux were not suitable due to the formation of a large number of impurities. A singlet at 3.91 ppm of CH 2 group protons in the 1 H NMR spectrum and resonance lines at 183.3 ppm (C=O) and 187.0 ppm (C=N) in the 13 C NMR spectrum of compound 2 confirmed the formation of the thiazolone ring. 2-Amino-1,3-thiazole derivative 3a was obtained from thioureido acid 1 and a chloroacetaldehyde 50% aqueous solution. The reaction was performed by refluxing acetone for 12 h. Product 3a was obtained in the form of water-soluble hydrochloride salt [52]. The convenience of the method is the crystallisation of the product from the reaction mixture already during the process. The data of the 1 H and 13 C NMR spectra of synthesized compounds 1, 2 3a are included in the Supplementary Material (Figures S1-S6).

Synthesis
This research work is a continuation of projects on the synthesis and biological evaluation of various thiazole derivatives. 3-(1-(2,5-Dimethylphenyl)thioureido)propanoic acid (1) as the starting compound was synthesized by multistep reactions according to the method described in [50]. It is known that thiazoles can be synthesised from α-haloketone and a thioamide via Hantzsch thiazole synthesis in high yields [51]. To synthesize thiazolone 2 (Scheme 1), thioureido acid 1 was reacted with monochloroacetic acid in an aqueous 10% potassium carbonate solution at room temperature followed by the acidification with acetic acid to pH 6. In this reaction, the conventional reaction conditions at reflux were not suitable due to the formation of a large number of impurities. A singlet at 3.91 ppm of CH2 group protons in the 1 H NMR spectrum and resonance lines at 183.3 ppm (C=O) and 187.0 ppm (C=N) in the 13 C NMR spectrum of compound 2 confirmed the formation of the thiazolone ring. 2-Amino-1,3-thiazole derivative 3a was obtained from thioureido acid 1 and a chloroacetaldehyde 50% aqueous solution. The reaction was performed by refluxing acetone for 12 h. Product 3a was obtained in the form of water-soluble hydrochloride salt [52]. The convenience of the method is the crystallisation of the product from the reaction mixture already during the process. The data of the 1 H and 13 C NMR spectra of synthesized compounds 1, 2 3a are included in the Supplementary Material ( Figures S1-S6).
Next, the 4-methyl substituted thiazole 3b was synthesized as shown in Scheme 1. Previously, in [53], 4-methyl-2-aminothiazole was prepared in acetone, but for 3b, the interaction of thioureido acid 1 with chloroacetone proceeded better in water than in acetone. The reaction at room temperature for 24 h and the following addition of sodium acetate to the reaction mixture gave the desired 4-methyl-1,3-thiazole derivative 3b, the structure of which was easily confirmed by the NMR spectral data (Supplementary Material, Figures S7 and S8).
According to the publication [54], 4-substituted thiazoles and especially those with chromen-3-yl and naphthalen-2-yl moieties demonstrate convincing antibacterial properties. Based on this, we have prepared a library of 4-substituted thiazoles 3c-k. The wellknown Hantzsch thiazole synthesis method was applied and the generation of the target products 3c-k was performed by condensation of thioureido acid 1 with a series of αbromoacetophenones without the use of a base. The isolated hydrobromide salts were transformed to free-based by dissolving them in 10% aqueous sodium carbonate and acidifying the solutions with acetic acid to pH 6. The structures of the obtained compounds 3c-k were confirmed by the data of the 1 H and 13 C NMR spectra (Supplementary Material, Figures S9-S26).
The activity of broad-spectrum quinolone-based antibiotics against both Gram-positive and Gram-negative bacteria, including mycobacteria and anaerobes, promotes the synthesis and development of new quinolone-type compounds which are relevant to the development of knowledge on this topic [55]. Heating of N-aryl-β-alanines with strong dehydrating agents such as Eaton's reagent [56], polyphosphoric acid [57,58] or phosphorus pentoxide [59] causes the formation of compounds containing 2,3-dihydroquinolin-4(1H)one moiety. To obtain quinolone-type compounds 4i-k, thiazoles 3i-k were treated with polyphosphoric acid at 120 • C. The intramolecular cyclisation occurred over 2-3 h and 2,3-dihydroquinolin-4(1H)-ones 4i-k were obtained in 71-88% yield. The data of the 1 H and 13 C NMR spectra of synthesized compounds 4i-k are included in the Supplementary Material ( Figures S27-S32).
Compounds bearing a 5-acetyl-4-methylthiazole structure were found to exhibit high cytotoxicity against the MCF-7 cell line [60] as well as demonstrate up-and-coming antimicrobial properties [61,62]. For that purpose, we have included the synthesis of a compound containing this fragment into the goals of our study, and 5-acetyl-4-methylthiazole 5 was prepared by the same technique as for 3c-k. The product was separated in 75% yield and its structure was confirmed by the methods of IR, NMR spectroscopy and the data of elemental analysis (NMR data are included in the Supplementary Material, Figures S33 and S34).
The carboxyl functional group can be an important constituent of a pharmacophore [68]. Its functionalisation can greatly expand the library of biological properties of compounds [69,70]. To expand the library of thiazole derivatives and to evaluate the influence of substituents on their biological properties, some transformations of the carboxyl group were performed (Scheme 2). Firstly, acid 3f was esterified with methanol to obtain ester 8f. No additional catalyst was required for this reaction with hydrobromide analogue. Afterwards, ester 8f was refluxed in 1,4-dioxane with hydrazine monohydrate and the resulting acid hydrazide 9f was then condensed with aldehydes and ketones. The structure of 9f was confirmed by the NMR techniques and microanalysis data. The presence Antibiotics 2023, 12, 220 5 of 27 of the CONHNH 2 group protons were indicated by the singlets at 9.11 (NH) and 4.07 (NH 2 ) ppm in the 1 H NMR spectrum of 9f, whereas the spectral line of the carbon of the C=O group resonated at the characteristic area of the 13  of substituents on their biological properties, some transformations of the carboxyl group were performed (Scheme 2). Firstly, acid 3f was esterified with methanol to obtain ester 8f. No additional catalyst was required for this reaction with hydrobromide analogue. Afterwards, ester 8f was refluxed in 1,4-dioxane with hydrazine monohydrate and the resulting acid hydrazide 9f was then condensed with aldehydes and ketones. The structure of 9f was confirmed by the NMR techniques and microanalysis data. The presence of the CONHNH2 group protons were indicated by the singlets at 9.11 (NH) and 4.07 (NH2) ppm in the 1 H NMR spectrum of 9f, whereas the spectral line of the carbon of the C=O group resonated at the characteristic area of the 13  As previously reported in [71], 4-aryl substituted thiazoles bearing hydrazonyl fragments appeared to be an eligible scaffold for the future development of antifungal and antibacterial agents targeting highly resistant pathogenic microorganisms. Further, a series of various hydrazones were synthesized and investigated for their bioactivity. To obtain hydrazones 10-15f, hydrazide 9f was condensed with heterocyclic and aliphatic ketones. The products were isolated in a 75-85% yield. The NMR spectra of the synthesized hydrazones 10-15f showed that in the DMSO-d6 solution, they exist as a mixture of E/Z conformers due to the presence of a CO-NH fragment in the molecule and the restricted rotation around it; in the studies [57,72], the Z-form predominate are described NMR data are included in the Supplementary Material, Figures S43-S54).
One of the methods for the synthesis of a benzimidazole heterosystem involves to condensation of carboxylic acids with 1,2-diaminobenzenes. The target compounds 16c, f, h were synthesized by Phillip's method (heating of both reagents in 4 M hydrochloric acid). The reflux for 72 h gave benzimidazoles 16c, f, h in good yields (NMR data are in the Supplementary Material, Figures S45-S60).
Finally, amides 17c, f, h were prepared by direct coupling of acids 3 with the sulphanilamide using HBTU as the coupling reagent and triethylamine as the base (Scheme 2).
The reactions were performed in dimethylformamide at room temperature in an inert argon atmosphere. The products were isolated by the dilution of the reaction mixture with an aqueous 10% potassium carbonate and were characterised using NMR and IR As previously reported in [71], 4-aryl substituted thiazoles bearing hydrazonyl fragments appeared to be an eligible scaffold for the future development of antifungal and antibacterial agents targeting highly resistant pathogenic microorganisms. Further, a series of various hydrazones were synthesized and investigated for their bioactivity. To obtain hydrazones 10-15f, hydrazide 9f was condensed with heterocyclic and aliphatic ketones. The products were isolated in a 75-85% yield. The NMR spectra of the synthesized hydrazones 10-15f showed that in the DMSO-d 6 solution, they exist as a mixture of E/Z conformers due to the presence of a CO-NH fragment in the molecule and the restricted rotation around it; in the studies [57,72], the Z-form predominate are described NMR data are included in the Supplementary Material, Figures S43-S54).
One of the methods for the synthesis of a benzimidazole heterosystem involves to condensation of carboxylic acids with 1,2-diaminobenzenes. The target compounds 16c, f, h were synthesized by Phillip's method (heating of both reagents in 4 M hydrochloric acid). The reflux for 72 h gave benzimidazoles 16c, f, h in good yields (NMR data are in the Supplementary Material, Figures S45-S60).
Finally, amides 17c, f, h were prepared by direct coupling of acids 3 with the sulphanilamide using HBTU as the coupling reagent and triethylamine as the base (Scheme 2).
The reactions were performed in dimethylformamide at room temperature in an inert argon atmosphere. The products were isolated by the dilution of the reaction mixture with an aqueous 10% potassium carbonate and were characterised using NMR and IR spectroscopy and elemental analysis (NMR data are included in the Supplementary Material, Figures S61-S66).
It is worth noting that the analysis of NMR spectra revealed that the CH 2 N protons are often observed as two broad singlets. The characteristic splitting of resonances was observed for all compounds except 4i-k. The literature in [64] suggests that such splitting of the proton resonances of the compounds possessing methyl group in the 2nd position of the benzene ring was caused by the restricted rotation of benzene and other N-substituents around the C(1 )-N(1) bond and the possibility of diastereomers.

Antibacterial Activity of Compounds 1-17 against Multidrug-Resistant Pathogens
In order to understand the structure-dependent antibacterial activity of novel thiazole derivatives 1-17, we first used a representative collection of multidrug-resistant Grampositive and Gram-negative bacterial pathogens with genetically defined antimicrobial resistance profiles [73]. The bacterial strains were selected to represent WHO ESKAPE group pathogens with emerging and challenging antimicrobial resistance mechanisms. To do so, the compounds were screened by using clinically approved minimal inhibitory concentration (MIC) determination by using the Clinical Laboratory Standard Institute (CLSI) recommendations.
The replacement of the thiazole ring with the ring-fused structure strongly enhanced the antibacterial activity against S. aureus TCH 1516 strain (MIC 1 µg/mL), while the antibacterial activity against E. faecium AR-0783 increased two-fold (MIC 8 µg/mL) if compared to 3h ( Table 1). The antibacterial activity of compound 7 against S. aureus was similar to daptomycin (MIC 1 µg/mL) and twice stronger than vancomycin (MIC 2 µg/mL) and excellent if compared to ampicillin (MIC > 64 µg/mL).
The emerging resistance to oxazolidinones (linezolid and tedizolid) which are last-line antimicrobial pharmaceuticals used to treat infections caused by Staphylococcus remains one of the major healthcare threats worldwide. Therefore, we further evaluated if the most promising thiazole derivatives 3h, 3j and 7 will be active against linezolid/tedizolidresistant S. aureus strains ( Table 2). The compounds 3h, 3j and 7 showed promising activity against linezolid/tedizolid-resistant S. aureus strains with MIC ranging from 1 to 32 µg/mL ( Table 2). Compound 3j showed the highest activity against tested strains (MIC 1-2 µg/mL), and its antimicrobial activity was greater than linezolid (8-32 µg/mL).  Collectively, these results demonstrated that substituted thiazoles exert in vitro antibacterial activity against Gram-positive bacterial pathogens harbouring emerging antibacterial resistance profiles. Moreover, 3,4-diCl-C 6 H 3 and naphthalen-2-yl substitutions, as well as naphthothiazoledione moiety, are important for the potent in vitro antibacterial activity against S. aureus with challenging resistance mechanisms.

Antifungal Activity of Thiazoles 1-17 against Drug-Resistant Candida Species
Antifungal drugs of the azole group are one of the most widely used for the treatment of invasive and systemic fungal infections [74]. Candida species are responsible for the majority of fungal infections, as rapidly rising resistance to azole antifungals among Candida species, in particular C. albicans is responsible for increased morbidity and mortality worldwide [75]. We, therefore, evaluated novel thiazoles 1-17 for their in vitro antifungal activity against multidrug-resistant Candida species by exposing the fungal strains with clinically relevant concentrations of each compound or control antifungal drug (Table 3).   Interestingly, only thiazoles 8f, 9f and 14f showed in vitro antifungal activity against Candida strains, expressing the multidrug-resistance phenotype to azoles ( Table 3). The transformation of thiazole 3f (MIC > 64 µg/mL) to ester 8f was observed to result in striking changes in antifungal activity. (Table 3). The derivative 8f showed antifungal activity against all tested strains (1-8 µg/mL) including Candida auris AR-0383 strains to harbour instinctive resistance to clinically approved antifungal drugs. The ester 8f transformation to hydrazide 9f greatly affected the antifungal activity. The hydrazide 9f was no longer active against C. auris (MIC > 64 µg/mL) and had decreased activity against C. albicans (MIC 8 µg/mL) in comparison to ester 8f (MIC 1 µg/mL). Interestingly, despite the loss of activity against C. auris, hydrazide 9f showed increased activity against C. parapsilosis AR-0335 (MIC 4 µg/mL). Numerous hydrazones were previously reported to show antifungal activity [76][77][78]; therefore, we further transformed hydrazide 9f to the corresponding hydrazones 10-15f and evaluated their structure-dependent antifungal activity in vitro. Hydrazone 14f bearing the butan-2-ylidene substitution exhibited antifungal activity against the majority of tested fungi (MIC 8-32 µg/mL) with the exception of C. auris (MIC > 64 µg/mL). The substitution of this moiety with a longer octan-2-ylidene carbon chain (15f) results in a complete loss of antifungal activity against all tested strains.
Thus, these results demonstrate novel thiazole derivatives 9f, 14f and, especially, 8f with a moderately functionalised carboxyl group exert the most promising antifungal activity against highly multidrug-resistant fungal pathogens. On the other hand, the functionalization of the carboxyl group is important for the broad-spectrum antifungal activity since bulky substitutions result in decreased antifungal activity spectrum or potency in these novel compounds.

Anticancer Activity of Compounds 1-17
The thiazole scaffold of often explored by medicinal chemists as an attractive pharmacophore for the development of novel antineoplastic drug candidates [79][80][81]. Therefore, we used two well-described pulmonary (A549) [82] and corectal adenocarcinoma (Caco-2) [83] models to evaluate the anticancer activity of compounds 1-17. We exposed the cells to 100 µM of each compound or cisplatin (CP) that was used as a clinically approved drug for the neoplastic disease treatment. The treatment was initiated for 24 h and after that, the cellular viability was measured by using MTT assay.
5-Acetyl-4-methylthazole 5 did not significantly reduce the viability of A549 or Caco-2, although the compound was able to decrease the viability to 63 and 55%, respectively. Thiazole fusion with quinoxaline (compound 6) did not result in significant viability reduction, although fusion with naphthoquinone lead to compound 7 with significantly high anticancer activity. Compound 7 exhibited broad-spectrum anticancer activity and was able to reduce A549 and Caco-2 viability to 14.0 and 13.1% compared to UC (p < 0.001) ( Figure 1A,B).
The carboxyl group transformation to ester 8f in compound 3f slightly enhanced the anticancer activity if compared to parent acid 3f and showed similar anticancer efficacy against A549 and Caco-2 by reducing the viability to 48.1 and 56.4%, respectively. Interestingly subsequent ester 8f transformation to hydrazide 9f greatly enhanced the anticancer activity against Caco-2 cells (20.6%) while ameliorating compound-mediated cytotoxic activity against A549 cells ( Figure 1A,B).
The carboxyl group transformation to ester 8f in compound 3f slightly enhanced the anticancer activity if compared to parent acid 3f and showed similar anticancer efficacy against A549 and Caco-2 by reducing the viability to 48.1 and 56.4%, respectively. Interestingly subsequent ester 8f transformation to hydrazide 9f greatly enhanced the anticancer activity against Caco-2 cells (20.6%) while ameliorating compound-mediated cytotoxic activity against A549 cells ( Figure 1A,B).
On the other hand, hydrazones 10-15f showed highly structure-dependent activity. Hydrazones 10f and 11f bearing the 5-nitrothien-2-yl and 5-nitrofuran-2-yl substitutions showed no anticancer activity against both tested cancer cell lines. Compound 12f containing indol-3-yl substitution demonstrated greater anticancer activity against A549 (35.0%) than Caco-2 (53.1%). The incorporation of cyclopentyl substitution resulted in compound 13f with a complete loss of anticancer activity against both cell lines, while the incorporation of ethyl radical (14f) significantly enhanced the anticancer activity against Caco-2 cells, but not A549. On the other hand, the incorporation of the hexyl chain (15f) resulted in a complete loss of anticancer activity against Caco-2, suggesting the importance of ethyl radical in novel thiazole structures ( Figure 1A,B).
These results demonstrate that novel thiazole derivatives possess strong anticancer activity against different adenocarcinoma cell lines. The structure-activity relation studies showed that naphthoquinone-fusion and 2,4-diCl-C 6 H 3 substitution are required for broadspectrum anticancer activity.

Bacterial Strains and Culture Conditions
The multidrug-resistant and genetically defined isolates were obtained from the AR isolate bank at the Centre for Disease Control (CDC, United States). S. aureus TCH 1516 (USA300) was obtained from the American Type Culture Collection. Prior to the study, all strains were maintained in commercial cryopreservation systems at −80 • C. Bacterial strains were subcultured on Columbia Sheep Blood agar or Tryptic-Soy agar (Becton Dickenson, Franklin Lakes, NJ, USA). Fungal strains were cultured on Saburoud-Dextrose agar. Unless otherwise specified, all antimicrobial susceptibility studies with bacterial pathogens were performed in Cation-Adjusted Mueller-Hinton broth (CAMBH) for liquid cultures (Liofilchem, Italy). Antifungal studies were conducted using RPMI/MOPS broth.

Antibacterial Activity Characterization
The minimal inhibitory concentrations (MICs) of compounds 1-17 as well as various antibiotics were determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The MICs for the compounds and comparator antibiotics were determined according to the testing standard broth microdilution methods described in CLSI document M07-A8 against the libraries of Gram-positive and Gramnegative pathogens. Compounds and antibiotics that were used as a control were dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 15-30 mg/mL. A series of compound dilutions were prepared in deep, polypropylene 96-well microplates to achieve 2× of concentrations (0.5-64 µg/mL) and were then transferred to the assay single-use, flat bottom plates. The prepared microplates were stored at −80 • C until the day of the experiment.
A standardized bacterial inoculum was prepared using the colony suspension method. The inoculum suspension was diluted in sterile CAMBH to achieve final concentrations of approximately 5 × 10 5 CFU/mL (range, 2 × 10 5 to 8 × 10 5 CFU/mL) in each well. The inoculum was transferred to the assay plates to achieve 1× assay concentration. Inoculated microdilution plates were incubated at 35 • C for 16 to 20 h in an ambient-air incubator within 15 min of the addition of the inoculum.

Antifungal Activity Characterization
The MIC of compounds 1-17 as well as clinically approved antifungal drugs was determined by CLSI recommendations, described in document M27-A3 [84,85]. Briefly, before the experiments, multidrug-resistant Candida spp., strains were sub-cultured on Saburoud-Dextrose agar for 24 h at 35 • C. The colonies were suspended in sterile saline to reach approximately 5 × 10 6 CFU/mL. Then, the fungal suspension is diluted in RPMI/MOPS broth to reach 5 × 10 5 CFU/mL and microplates containing test compounds, prepared as described above are inoculated using a multichannel pipette. Inoculated microdilution plates were incubated at 35 • C for 16 to 20 h in an ambient-air incubator within 15 min of the addition of the inoculum.

In Vitro Cytotoxic Activity Determination
The viability of A549 and Caco-2 cells after the treatment with compounds or cisplatin that served as cytotoxicity control was evaluated by using commercial MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (ThermoFisher Scientific, United States). Briefly, cells were plated in the flat-bottomed 96-well microplates (1 × 10 4 cells/well) and incubated overnight to facilitate the attachment. The test compounds were dissolved in hybridoma-grade DMSO (Sigma-Aldrich, St. Louis, MO, USA) and then further serially diluted in cell culture media containing 0.25 % DMSO to achieve 100 µM of each compound.
Subsequently, the media from the cells was aspirated and the compounds were added to the microplates. The cells were incubated at 37 • C, 5% CO 2 for 24 h. After incubation, a 10 µL of Vybrant ® MTT Cell Proliferation Reagent (ThermoFisher Scientific) was added, and cells were further incubated for 4 h. After incubation, the media was aspirated, and the resulting formazan was solubilized by the addition of 100 µL of DMSO. The absorbance was then measured at 570 nm by using a microplate reader (Multiscan, Ther-moFisher Scientific). The following formula was used to calculate the % of A549 viability: ([AE-AB]/[AC-AB]) × 100%. AE, AC, and AB were defined as the absorbance of experimental samples, untreated samples, and blank controls, respectively. The experiments were performed in triplicates.

Statistical Analysis
The results are expressed as mean ± standard deviation (SD). Statistical analyses were performed with Prism (GraphPad Software, San Diego, CA, USA), using Kruskal-Wallis test and two-way ANOVA. p < 0.05 was accepted as significant.

Conclusions
In the present study, a series of 2-aminothiazole derivatives containing N-2,5-dimethyl phenyl and β-alanine moieties in the molecules were synthesized and evaluated for their in vitro antimicrobial activity using a panel of multidrug-resistant bacterial and fungal pathogens with emerging and genetically defined resistance mechanisms. In addition, we characterized the cytotoxic and anticancer properties using A549 and Caco-2 pulmonary and corectal adenocarcinoma models.
The results revealed that thiazoles with the 4-aryl substituted showed profound and selective antimicrobial activity against Gram-positive pathogens. Compound 3j possessed the most potent activity against tested strains multidrug-resistant S. aureus and E. faecium (MIC 1-2 µg/mL). Strikingly, compounds 3h, 3j and 7 showed antibacterial activity against especially clinically challenging tedizolid/linezolid-resistant S. aureus strains.
On the other hand, the modified carboxyl group in thiazoles 8f, 9f and 14f provided promising antifungal properties against drug-resistant Candida strains, including C. glabrata, C. parapsilosis and C. hemulonii. Moreover, ester 8f was found to have broader antifungal activity on multiple Candida species (1-8 µg/mL) including the emerging fungal pathogen C. auris.
During the anticancer activity study, a structure-dependent anticancer activity against A549 and Caco-2 cells was observed. Structure-activity relation analysis revealed naphthoquinone-fused thiazole 7 structure and 2,4-diCl-C 6 H 3 moiety 3j to provide broad-spectrum anticancer against both A549 and Caco-2 cells.
Compounds based on 2-aminothiazole derivatives containing N-2,5-dimethyl phenyl and β-alanine moieties could be further explored as novel scaffolds for the development of novel and highly active compounds targeting multidrug-resistant pathogens, including tedizolid/linezolid-resistant S. aureus as well as vancomycin-resistant Enterococcus. Furthermore, a series of novel compounds based on 8f could be further explored as novel antifungals targeting challenging and multidrug-resistant Candida auris. Further studies are needed to better understand the safety, pharmacological properties as well as cellular targets of 2-aminothiazole derivatives 1-17.