Novel 2,4-Disubstituted-1,3-Thiazole Derivatives: Synthesis, Anti-Candida Activity Evaluation and Interaction with Bovine Serum Albumine.

Herein we report the synthesis of two novel series of 1,3-thiazole derivatives having a lipophilic C4-substituent on account of the increasing need for novel and versatile antifungal drugs for the treatment of resistant Candida sp.-based infections. Following their structural characterization, the anti-Candida activity was evaluated in vitro while using the broth microdilution method. Three compounds exhibited lower Minimum Inhibitory Concentration (MIC) values when compared to fluconazole, being used as the reference antifungal drug. An in silico molecular docking study was subsequently carried out in order to gain more insight into the antifungal mechanism of action, while using lanosterol-C14α-demethylase as the target enzyme. Fluorescence microscopy was employed to further investigate the cellular target of the most promising molecule, with the obtained results confirming its damaging effect towards the fungal cell membrane integrity. Finally, the distribution and the pharmacological potential in vivo of the novel thiazole derivatives was investigated through the study of their binding interaction with bovine serum albumin, while using fluorescence spectroscopy.


Introduction
Invasive candidiasis rank among the top four causes of nosocomial infectious diseases, being associated with an alarming mortality rate, especially in immunocompromised and critically ill patients [1]. terms of binding profile to plasmatic carrier proteins, such as human serum albumin (HSA), provides valuable insight into their pharmacological potential in vivo [18]. Generally, a weak binding interaction of a drug with serum albumin is associated with a short lifetime and poor distribution to the target site in vivo, while a strong binding interaction leads to low free drug plasma concentration, which is responsible for the therapeutic effect [19]. Bovine serum albumin (BSA) is widely used as a model protein for these experimental studies due to its high homology with HSA, having 75% identity and 87% similarity shared between amino acid sequences, and its relatively low cost [20]. Herein, fluorescence spectroscopy was employed for the analysis of the novel compounds-BSA molecular interactions, with this analytical method being preferred because of its sensitivity, rapidity, and simplicity [21].

Chemistry
The novel 4-substituted-1,3-thiazole derivatives (4a-d) were obtained according to the classical Hantzsch condensation protocol, as illustrated in Scheme 1. The synthesis of the intermediate thioamide 3 was previously reported by our group [14].
Molecules 2020, 25,1079 3 of 17 Protein-drug interaction studies of antifungals can provide a structural guideline and rational drug design, which is helpful in the synthesis of novel molecules with enhanced clinical efficiency and reduced toxicity [17]. The study of the pharmacokinetic profile of the newly synthesized compounds in terms of binding profile to plasmatic carrier proteins, such as human serum albumin (HSA), provides valuable insight into their pharmacological potential in vivo [18]. Generally, a weak binding interaction of a drug with serum albumin is associated with a short lifetime and poor distribution to the target site in vivo, while a strong binding interaction leads to low free drug plasma concentration, which is responsible for the therapeutic effect [19]. Bovine serum albumin (BSA) is widely used as a model protein for these experimental studies due to its high homology with HSA, having 75% identity and 87% similarity shared between amino acid sequences, and its relatively low cost [20]. Herein, fluorescence spectroscopy was employed for the analysis of the novel compounds-BSA molecular interactions, with this analytical method being preferred because of its sensitivity, rapidity, and simplicity [21].
The synthetic protocol that was employed for the chemical development of the compounds 7a-d is outlined in Scheme 2. The previously obtained thiosemicarbazone 6 [14] was subjected to a heterocyclization with the corresponding α-haloketones, yielding the targeted 2-hydrazinyl-4-substituted-1,3-thiazole derivatives. The synthetic protocol that was employed for the chemical development of the compounds 7a-d is outlined in Scheme 2.
The previously obtained thiosemicarbazone 6 [14] was subjected to a heterocyclization with the corresponding α-haloketones, yielding the targeted 2-hydrazinyl-4-substituted-1,3-thiazole derivatives. Protein-drug interaction studies of antifungals can provide a structural guideline and rational drug design, which is helpful in the synthesis of novel molecules with enhanced clinical efficiency and reduced toxicity [17]. The study of the pharmacokinetic profile of the newly synthesized compounds in terms of binding profile to plasmatic carrier proteins, such as human serum albumin (HSA), provides valuable insight into their pharmacological potential in vivo [18]. Generally, a weak binding interaction of a drug with serum albumin is associated with a short lifetime and poor distribution to the target site in vivo, while a strong binding interaction leads to low free drug plasma concentration, which is responsible for the therapeutic effect [19]. Bovine serum albumin (BSA) is widely used as a model protein for these experimental studies due to its high homology with HSA, having 75% identity and 87% similarity shared between amino acid sequences, and its relatively low cost [20]. Herein, fluorescence spectroscopy was employed for the analysis of the novel compounds-BSA molecular interactions, with this analytical method being preferred because of its sensitivity, rapidity, and simplicity [21].
Elemental analysis and spectral data ((Fourier transform infrared spectroscopy (FT-IR), electrospray ionization-mass spectrometry (ESI-MS), proton nuclear magnetic resonance ( 1 H-NMR), and carbon nuclear magnetic resonance ( 13 C-NMR)) were used for the structural confirmation of the newly synthesized compounds (see Supplementary Material, Figures S1-32). The results of the C, H, N, and S quantitative elemental analysis were consistent with the calculated values, within ± 0.4% of the theoretical values. The recorded molecular ion peaks (M + 1) were consistent with their molecular formulas.
In regard to the first series of compounds (4a-d), the spectral data confirmed the successful accomplishment of Hantzsch condensation through the appearance of specific thiazole-C5-H singlet at 7.97-8.31 ppm in the 1 H-NMR spectra, as well as the presence of a characteristic signal in the IR spectra at 3089-3106 cm −1 , which corresponded to the C5-H stretching vibration.
Concerning the 2-hydrazinyl-thiazole derivatives (7a-d), two specific signals appeared in their IR spectra, at 3318-3327 cm −1 and 3189-3200 cm −1 , corresponding to the N-H asymmetric and symmetric stretching vibrations. Moreover, the presence of a characteristic signal in the 13 C-NMR spectra at 143.2-144.1 ppm, belonging to the sp 2 carbon from hydrazone linker, along with the specific 1 H-NMR signals that were related to C5-H (7.15-7.50 ppm) and additional aromatic protons, confirmed the proposed structures.

In Vitro Anti-Candida Activity
The antifungal activity of the synthesized thiazole derivatives was evaluated in vitro against three human pathogenic Candida strains. The stock solutions (1 mg/mL) were prepared by dissolving the tested compounds and the reference antifungal drug, fluconazole, in sterile DMSO. The MIC and MFC values were determined through the broth microdilution method, and they are presented in Tables 1 and 2. The obtained MIC and MFC values revealed moderate to very good anti-Candida activities for the tested compounds.
2-Hydrazinyl-thiazole derivatives having a lipophilic (+π) para-substituent in the C4 position of the azole heterocycle (7a, 7b, and 7c) were the most promising, exhibiting MIC values (3.9 µg/mL) that were four times lower when compared to the reference drug fluconazole (15.62 µg/mL) against the pathogenic Candida albicans strain. The non-albicans species proved to be less susceptible to almost all of the tested molecules. This findings are in accordance with the results obtained in our previous study [14]. The replacement of the C4-para-substituted-phenyl ring with a naphthyl substituent, in the case of compound 7d, led to a decrease in the inhibitory activity on both albicans and non-albicans strains.
The obtained data emphasize that the antifungal activity is dependent on an optimal hydro-lipophilic balance. An excessive hydrophobicity and high aromaticity could bring forth an increased drug efflux mediated by the membrane-associated multidrug efflux pumps, and thus a lower biological activity, despite the higher permeability through the fungal cell membrane [22].
While analyzing the obtained results, it seems reasonable to assume that the presence of a hydrazine substituent in the C2 position of the azole heterocycle is correlated with an increased antifungal efficiency, since the MIC values that were obtained for the first series of 4-substituted-1,3-thiazole derivatives (4a-d) were significantly higher than those recorded for the second series of 4-substituted-2-hydrazinyl-1,3-thiazoles (7a-d).
The determination of MFC confirmed the previously obtained MIC values. The MFC/MIC ratio values for all of the tested compounds were equal to 2, which suggested that these molecules may exert a fungicidal effect [23].

Molecular Docking Study
Ergosterol biosynthesis is one of the most important cellular targets employed for the development of novel antifungals, being the main sterol responsible for the fungal cell membrane integrity and function, which is structurally different from the mammalian cholesterol [24,25].
We speculate that the newly synthesized compounds might act through altering the ergosterol biosynthetic pathway due to the promising MIC values obtained for the 2-hydrazinyl-1,3-thiazole derivatives 7a, 7b, and 7c, as compared with the reference drug fluconazole. Aiming to investigate this hypothesis, a molecular docking study was carried out on Candida albicans lanosterol-C 14 α-demethylase (CYP51), a validated target in the fungal cell membrane [26].
For each compound, the conformation with the best binding affinity, being expressed as the highest variation of Gibbs free energy (∆G) of the complex with the biomacromolecule, was predicted by a previously validated ligand-protein interaction protocol [27]. Table 3 presents the computed binding interaction energy (∆G) and the calculated inhibition constant (ki), together with the analysis of the cluster containing the energetically favorable binding conformations.
The obtained data indicated the existence of binding poses with favorable energies for all of the tested ligands. Concerning the second series of 2-hydrazinyl-4-substituted-1,3-thiazole derivatives (7a-d), the presence of a hydrazine substituent at the C2 position of the azole heterocycle and the supplementary phenyl ring translate into an improved interaction with the target enzyme in terms of binding energy and steric deviation. However, the increased molecular flexibility that is given out by the C 2 -hydrazine bridge is associated with a larger spatial dispersion of the predicted binding poses as compared with the first series of 4-substituted-1,3-thiazole compounds (4a-d), as outlined by the increased number of clusters containing residual conformations.
For both series of ligands, the differences in the predicted binding mode and ligand-protein affinity are linked to their electronic, steric, and hydrophobic properties. It can be observed that the binding affinity regularly increased with the lipophilicity of the C4-thiazole substituent. This might be explained by the hydrophobic interactions with the CYP51 lipophilic area that is located in the depth of the binding pocket (Figure 1), as previously reported by our research group [28]. Table 3. The predicted binding interaction energy (∆G, kcal/mol), the consequent inhibition constant (Ki, nM) and the cluster analysis containing the best conformation of each compound given by AutoDock. The obtained data indicated the existence of binding poses with favorable energies for all of the tested ligands. Concerning the second series of 2-hydrazinyl-4-substituted-1,3-thiazole derivatives (7a-d), the presence of a hydrazine substituent at the C2 position of the azole heterocycle and the supplementary phenyl ring translate into an improved interaction with the target enzyme in terms of binding energy and steric deviation. However, the increased molecular flexibility that is given out by the C 2 -hydrazine bridge is associated with a larger spatial dispersion of the predicted binding poses as compared with the first series of 4-substituted-1,3-thiazole compounds (4a-d), as outlined by the increased number of clusters containing residual conformations.
For both series of ligands, the differences in the predicted binding mode and ligand-protein affinity are linked to their electronic, steric, and hydrophobic properties. It can be observed that the binding affinity regularly increased with the lipophilicity of the C4-thiazole substituent. This might be explained by the hydrophobic interactions with the CYP51 lipophilic area that is located in the depth of the binding pocket (Figure 1), as previously reported by our research group [28]. An additional interaction with the polar Tyr132 amino acid sidechain, which is located at the hydrophilic domain of the access channel to the binding site, is due to the presence of the etheric oxygen, which acts as a hydrogen bond acceptor. Additionally, a π-cation interaction is allowed between the thymol fragment and the heme Fe 2+ , due to the parallel orientation of the first one to the catalytic site.
In regard to the 2-hydrazinyl-4-substituted-1,3 thiazole compounds (7a-d), a supplementary hydrogen bond is formed between the Tyr118 amino acid sidechain and the hydrazine bridge. An additional interaction with the polar Tyr132 amino acid sidechain, which is located at the hydrophilic domain of the access channel to the binding site, is due to the presence of the etheric oxygen, which acts as a hydrogen bond acceptor. Additionally, a π-cation interaction is allowed between the thymol fragment and the heme Fe 2+ , due to the parallel orientation of the first one to the catalytic site.
The results of the molecular docking study suggested that the tested compounds might act as non-competitive inhibitors of the fungal lanosterol C14α-demethylase, interfering with the access and the subsequent binding of the physiological substrate to the catalytic site. The newly synthesized thiazole derivatives might be associated with reduced toxicity and better activity against resistant strains since the mechanism of action is not related to the covalent coordination of the heme Fe 2+ , as in the case of classical antifungal azoles [29,30].

Propidium Iodide (PI) Dye Uptake Assay
The PI dye exclusion assay was performed to further support the cell membrane damaging effect of compound 7b, which exhibited the strongest anti-Candida activity in both in vitro and in silico studies. In fact, PI is a DNA-staining dye which upon binding to double stranded nucleic acid gives a red fluorescence when excited by a 480 nm laser [31]. This membrane-impermeant dye is generally excluded from viable cells, unless their membrane integrity is compromised [32].
Incubation of Candida albicans stained cells with MIC concentration of 7b resulted in a red fluorescence signal, as evidenced by fluorescence microscopy, as shown in Figure 2a. No fluorescence was observed in the control cells, being incubated with PI alone (Figure 2b).

Propidium Iodide (PI) Dye Uptake Assay
The PI dye exclusion assay was performed to further support the cell membrane damaging effect of compound 7b, which exhibited the strongest anti-Candida activity in both in vitro and in silico studies. In fact, PI is a DNA-staining dye which upon binding to double stranded nucleic acid gives a red fluorescence when excited by a 480 nm laser [31]. This membrane-impermeant dye is generally excluded from viable cells, unless their membrane integrity is compromised [32].
Incubation of Candida albicans stained cells with MIC concentration of 7b resulted in a red fluorescence signal, as evidenced by fluorescence microscopy, as shown in Figure 2a. No fluorescence was observed in the control cells, being incubated with PI alone (Figure 2b). The obtained results indicate the PI uptake in C. albicans cells exposed to the newly synthesized 2-hydrazinyl-1,3-thiazole derivative 7b, which suggest that this compound might act through the disruption of the fungal cell membrane structural integrity.

Protein Binding Study
BSA is a globular protein, which consists of a single polypeptide chain composed of 583 amino acid residues. At the physiological neutral pH, BSA presents a native form, its tertiary structure comprising three homologous domains (I, II, and III), each being divided into two subdomains (A and B) [20]. The intrinsic fluorescence properties of BSA are mainly due to the existence of two Trp residues. One is located on the surface of the macromolecule (Trp 134) and is more exposed to a hydrophilic environment. The other (Trp 213) is positioned in the hydrophobic pocket of the domain The obtained results indicate the PI uptake in C. albicans cells exposed to the newly synthesized 2-hydrazinyl-1,3-thiazole derivative 7b, which suggest that this compound might act through the disruption of the fungal cell membrane structural integrity.

Protein Binding Study
BSA is a globular protein, which consists of a single polypeptide chain composed of 583 amino acid residues. At the physiological neutral pH, BSA presents a native form, its tertiary structure comprising three homologous domains (I, II, and III), each being divided into two subdomains (A and B) [20]. The intrinsic fluorescence properties of BSA are mainly due to the existence of two Trp residues. One is located on the surface of the macromolecule (Trp 134) and is more exposed to a hydrophilic environment. The other (Trp 213) is positioned in the hydrophobic pocket of the domain II and it is highly sensitive to ligand binding-induced modifications in the microenvironment and to conformational changes [33].
The fluorescence spectra of BSA upon the addition of increasing concentrations (0.3 µM, 0.6 µM, 0.9 µM, 1.2 µM, 1.5 µM, and 1.8 µM) of the tested ligands were recorded in order to analyze the binding profile of compounds 4a-d and 7a-d to serum albumin, in terms of binding mechanism, binding constants and the number of binding sites. Spectral analysis was performed at room temperature under simulated physiological conditions (pH = 7.4), by the excitation of the probes at 290 nm and emission scans ranging between 300 to 450 nm.
The emission band that is centered at 341 nm (λ max ) originates from Trp residue. As a first observation, the fluorescent intensity of BSA regularly decreased with the increasing concentration of the tested molecules, which is in good agreement with the classical Stern-Volmer equation (1), which confirmed the binding interaction of the ligands with the biomacromolecule [34].
where F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher, respectively; K SV is the Stern-Volmer quenching constant; and, [Q] represents the concentration of the quencher. K q is the quenching rate constant of the biomolecule and г 0 is the average fluorescence lifetime of the biomolecule without the quencher, which is approximately 6 ns for BSA. Figure 3a illustrates the fluorescence spectra of BSA in the presence of increasing concentrations of 7b, being selected as the most active compound in both in vitro and in silico antifungal assays. The binding region should be in the vicinity of Trp residue, since a distant event cannot cause its fluorescence quenching [35]. No change of the maximum emission wavelength was observed in the case of the 4-phenyl-substituted-1,3 thiazole (4a-c) and 2-hydrazynil-1,3-thiazole (7a-d) derivatives. For compound 4d, having a naphthalene substituent at the C4 position of the thiazole ring, the fluorescence quenching was accompanied by a slight red shift of the λ max (from 341 nm to 345 nm), which suggested the tertiary structure modification of the protein and an increase in the polarity of the microenvironment around the Trp residue due to solvent exposure [21].
The drug binding-induced fluorescence quenching of serum albumin can occur due to either a dynamic or a static mechanism. Molecular collisions between the ligand and the fluorophore in excited state mediate dynamic fluorescence quenching, while the static quenching results from the formation of a ground state fluorophore-ligand complex, thus reducing the population of fluorescent motifs that are capable of excitation [36].
K SV and K q , according to Stern-Volmer equation, were determined by the slope and the intercept of the linear regression plot graph of the relative emission intensity (F0−F)/F versus г 0 × [Q], as illustrated in Figure 3b for compound 7b. Table 4 presents the calculated values for all of the tested compounds.
The calculated quenching constant (K q ) of the tested compounds is greater when compared to the value that was obtained for biological macromolecules quenching due to collision mechanism (2 × 10 10 M −1 × s −1 ), suggesting that a static quenching is involved, through the formation of a BSA-ligand complex [33].
The K sv value reflects the magnitude of quenching, which is dependent on the ligand molecule's availability to the fluorophore residue, as well as on the lipophilicity and electronic effects of the C4-thiazole substituent [18]. For compounds 4d and 7d, the increased hydrophobicity of the molecule as a whole, due to the presence of naphthalene substituent at C4 position of the thiazole heterocycle, might explain the strength of the interaction with BSA. The presence of two fused aromatic rings results in a higher ring planarity and greater conjugation (π-π interactions) to the hydrophobic pocket of domain II [37]. In the case of a static quenching mechanism, the number of binding sites (n) and binding constants (K b ) can be determined by the intercept and the slope of the regression curve, while using Equation (2) [38]. Table 5 presents the obtained results for all of the tested compounds.
(2) The K b values reflect the strength of the binding interaction. For the majority of the tested molecules, the K b values are in the range of 1−15 × 10 4 L/mol, which suggests the existence of a reversible and moderate interaction in BSA-ligand complex, which is associated with a faster diffusion rate in vivo to reach the target site [39]. The calculated n values are approximately 1, indicating the existence of a single binding site of the newly synthesized thiazole derivatives on BSA [40].

General Information
All of the chemicals (reagent grade) used for synthesis were obtained from commercial sources and used as supplied, without further purification. For the monitoring of the reaction progress and the purity of the newly synthesized compounds, an analytical thin layer chromatography (TLC) carried out on Merck precoated Silica Gel 60F254 sheets (Darmstadt, Germany) was employed. A mixture of ethyl acetate: n-heptane = 3:1 was used as an elution system and the visualization was made while using UV light (254 nm). The melting points were determined with an Electrothermal melting point meter through the open glass capillary method and they are presented uncorrected. The structures of the synthesized compounds were assigned through spectral data (mass spectrometry (MS), infrared spectroscopy (IR), and nuclear magnetic resonance (NMR)) and their purity was confirmed by elemental analysis. The IR spectra were recorded on a Jasco FT/IR 6100 spectrometer (Jasco, Easton, MD), while using anhydrous potassium bromide for sample preparation. The MS analyses were performed in positive ionization, while using an Agilent 1100 series and an Agilent Ion Trapp SL mass spectrometer (Agilent, Santa Clara, CA, USA). The 1 H-NMR spectra were recorded on a Bruker Advance NMR spectrometer (Karlsruhe, Germany), operating at 500 MHz, while using DMSO-d 6 as solvent and tetramethylsilane (TMS) as internal standard. 13 C-NMR analyses were performed on a Bruker Advance NMR spectrometer, operating at 125 MHz, in DMSO-d 6 , while using a Waltz-16 decoupling scheme, with TMS as the internal standard. Chemical shift (δ) values were reported in parts per million (ppm). Splitting patterns are given as s (singlet), d (doublet), t (triplet), and m (multiplet).

Chemistry
3.2.1. General Procedure for the Synthesis of 2-((2-isopropyl-5-methylphenoxy)methyl)-4-phenyl Thiazole Derivatives 4a-d Equimolar quantities (1 mmol) of carbothioamide 3 and corresponding α-haloketones were dissolved in dry acetone (3 mL) and stirred at room temperature for 6 h. The resulted precipitate was filtered under vacuum and then washed with a solution of NaHCO 3 10% until free of acid. The pure compounds were yielded through recrystallization from ethanol. Potato dextrose agar medium (Sifin, Germany) was used for the storage of the standardized cell cultures and Roswell Park Memorial Institute (RPMI) 1640 medium with L-glutamine, being adjusted to pH 7.0 with 3-(N-morpholino) propanesulfonic acid, was used for the susceptibility testing. Prior to antifungal susceptibility evaluation, each strain was inoculated on potato dextrose agar plates to ensure optical growth characteristics and purity. Subsequently, the yeast cells were suspended in saline and adjusted spectrophotometrically to RPMI 1640 medium. The initial density of Candida sp. was approximately 2 × 10 6 colony forming units (CFU)/mL. Inoculums (density of 0.5 in McFarland scale) were prepared in a sterile solution of 0.9% NaCl. Subsequently, the tested strains were suspended in nutrient broth and RPMI 1640 media to achieve a final density of 2 × 10 5 CFU/mL.
The stock solutions (1 mg/mL) were obtained by dissolving the newly synthesized compounds and the reference antifungal, fluconazole, in sterile DMSO, and they were kept at 4 • C. A series of double diluting solutions of the tested compounds were prepared in RPMI 1640 medium to achieve a final concentrations in the range of 500-0.015 µg/mL.
Media (100 µL) was placed into each 96 wells of the microplates for the determination of MIC/MFC according to broth microdilution method. Sample solutions (100 µL) at high concentration (500 µg/mL) were added into the first column of the microplates and two-fold dilutions of the compounds were obtained by the serial pipetting of the mixed solution (100 µL) into the remaining wells. Ten-microliter culture suspensions were inoculated into all of the wells and the sealed microplates were incubated at 37 • C for 18 h. Growth control, sterility control, and control of the antifungal compounds were used. The plates were incubated at 25 • C for 48 h. Next, minimum inhibitory concentration (MIC) values were spectrophotometrically determined, by recording the optical density test solutions at 600 nm, after the addition of 20 µL of resazurin solution (0.02%). The MIC was defined as the lowest concentration required arresting the growth of the fungi. 0.01 mL of the medium withdrawn from the culture tubes showing no macroscopic growth at the end of the 24 h was sub-cultured on potato dextrose agar plates to determine the number of vital organisms and then incubated further at 37 • C and 25 • C for 24 h and 48 h, respectively, for the determination of the minimum fungicidal concentration (MFC). The MFC was defined as the lowest concentration of the tested compounds at which no fungal colonies were observed. All the MIC and MFC measurements were made in triplicate.

Molecular Docking Study
The binding affinity of the newly synthesized thiazole derivatives (4a-d, 7a-d) to the catalytic site of the target fungal lanosterol C 14 α-demethylase (CYP51) was evaluated through a molecular docking study, while using AutoDock 4.2.6 [27].
The target enzyme was constructed through homology modeling that was based on the UniProt P10613 sequence from Candida albicans [42], while using SWISS-MODEL [43]. The homologous sequence that was used as template for the construction of the target macromolecule (PBD 5EQB) was chosen from Protein Data Bank based on the results of a BLAST search. The docking protocol was performed, as previously reported [14].
Visualization and analysis of the docking results were performed while using UCSF Chimera 1.10.2 [44].

Propidium Iodide (PI) Dye Uptake Assay
Fluorescence microscopy was used to evaluate the membrane integrity of yeast cells that were exposed to compound 7b. For this purpose, C. albicans cell suspension (1 × 10 7 cells/mL) that was prepared in sterile PBS was treated with MIC concentration of 7b and then incubated for two hours at 37 • C. Cells that were incubated in similar conditions without compound served as the negative control.
The suspensions were then centrifugated and the cell pellets were washed and resuspended in sterile PBS. PI solution was added to resuspended cells to achieve a final concentration of 1 µg/mL and the samples were incubated for another 30 minutes in the dark.
The cells were further analyzed with an inverted Zeiss Axio Observer Z1 microscope with a LD Plan Neofluar 20x objective (NA = 0.4, Zeiss). Concretely, the fluorescence images were collected using a Compact Light Source HXP 120 C mercury lamp, the light being reflected by a dichroic mirror while using an excitation filter BP 525/50. An AxioCam Icc digital camera was employed to capture the images, which were then processed while using the ZEN software.

Protein Binding Study
BSA fraction V was purchased from Merck (Darmstadt, Germany) and used as supplied, without further purification. The other used chemicals were of analytical grade purity.
All of the fluorescence emission spectra were recorded on a Jasco FP-6500 spectrofluorometer that was equipped with a DC-powered 150 W Xenon lamp. The preparation of the probes was done according to a previously reported protocol [14]. The BSA concentration was kept constant (1.5 µM) and the concentration of the newly synthesized thiazole derivatives was gradually increased from 0.3 µM to 1.8 µM. The fluorescence emission spectra of BSA-ligand solutions were recorded from 300 to 450 nm, with the excitation wavelength at 290 nm and slid widths of 3 nm. Quartz cells with 1.0 cm path length were employed for the spectral analysis.
It is worth mentioning that the tested compounds have no fluorescence at the emission wavelength (341 nm). Moreover, they have no absorption at the excitation and emission wavelengths, at the used concentrations, so their inner effect on BSA fluorescence intensity is negligible [19,21].

Conclusions
Altogether, two novel series of thiazole derivatives were synthesized according to a previously reported protocol, which is characterized by physicochemical properties and evaluated in vitro for their anti-Candida activity. Three compounds, 7a, 7b, and 7c, exhibited promising results in terms of MIC and MFC.
According to the data that were obtained from the performed molecular docking study, the newly synthesized compounds might interfere with the ergosterol biosynthetic pathway through a non-competitive inhibition of the fungal lanosterol-C14α-demethylase. Candida cells were incubated with the most active molecule, 7b, and then subjected to a fluorescence microscopy study to further support the proposed hypothesis. PI staining dye uptake, as shown by the captured fluorescence microscopy images, indicated that the tested compound damages the fungal cell membrane integrity.
Additionally, fluorescence spectroscopy studied the affinity of the novel synthesized thiazole derivatives towards carrier plasma proteins, with the obtained results suggesting a moderate and reversible binding interaction with bovine serum albumin, being mediated by the formation of a ground-state complex.
The present research provides comprehensive information on the development of novel antifungals that are endowed with improved therapeutic efficiency and pharmaco-toxicological profile.