Novel 2,4-disubstituted-1,3-thiazole derivatives: Synthesis, anti-Candida activity evaluation and interaction with bovine serum albumine

Article abstract of DOI:10.3390/molecules25051079

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. Follo

Full text of DOI:10.3390/molecules25051079

Article
Novel 2,4-Disubstituted-1,3-Thiazole Derivatives:
Synthesis, Anti-Candida Activity Evaluation and
Interaction with Bovine Serum Albumine
1
2,
1,
1
3
Andreea-Iulia Pricopie , Monica Focșan *, Ioana Ionuț *, Gabriel Marc , Laurian Vlase ,
4
5
5
5
6
Luiza-Ioana Găină , Dan C. Vodnar , Simon Elemer , Gabriel Barta , Adrian Pîrnău
1
and Ovidiu Oniga
1
Department of Pharmaceutical Chemistry, “Iuliu Hațieganu” University of Medicine and Pharmacy, 41
Victor Babeș Street, 400012 Cluj-Napoca, Romania; pricopie.andreea@umfcluj.ro (A.I.P.);
marc.gabriel@umfcluj.ro (G.M.); onigao65@yahoo.com (O.O.)
Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on
Bio-Nano-Sciences, Babes-Bolyai University, Treboniu Laurean No. 42, 400271 Cluj-Napoca, Romania
Department of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Hațieganu” University of
Medicine and Pharmacy, 41 Victor Babeș Street, 400012 Cluj-Napoca, Romania;
laurian.vlase@umfcluj.ro (L.V.)
2
3
4
5
6
Research Center on Fundamental and Applied Heterochemistry, Faculty of Chemistry and Chemical
Engineering, “Babeş-Bolyai” University, 11 Arany Janos Street, 400028 Cluj-Napoca, Romania;
gaina.ioana.luiza@gmail.com (L.G.)
Department of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine,
3-5 Mănăștur Street, 400372 Cluj-Napoca, Romania; dan.vodnar@usamvcluj.ro (D.V.);
simon.elemer@usamvcluj.ro (S.E.); gabriel.barta@usamvcluj.ro (G.B.)
National Institute for Research and Development of Isotopic and Molecular Technologies,
67-103 Donath Street, 400293 Cluj-Napoca, Romania; adrian.pirnau@itim-cj.ro (A.P)
* Correspondence: monica.iosin@phys.ubbcluj.ro (M.F.); ionut.ioana@umfcluj.ro (I.I.);
Tel.: +0264454554 (int. 117) (M.F.); +40747-507-629 (I.I.);
Received: 14 February 2020; Accepted: 26 February 2020; Published: 28 February 2020
Abstract: 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.
Keywords: 1,3-thiazole; anti-Candida; cell membrane integrity; fluorescence microscopy; bovine
serum albumin
Molecules 2020, 25, 1079; doi:10.3390/molecules25051079
www.mdpi.com/journal/molecules

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1. 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].
Candida albicans is the predominant pathogenic strain that is involved in candidaemia.
However, the increased antifungal selective pressure, which is mainly mediated by the overuse of
fluconazole and other azole drugs, led to a progressive aethiological shift to non-albicans Candida sp.,
such as C. glabrata or C. auris, together with an increasing resistance to the marketed antifungals [2].
Among the virulence traits of C. albicans, is its ability to form polymicrobial biofilms is noteworthy,
which enables the development of mixed bacterial and fungal infections that are highly resistant to
antimicrobial agents [3,4]. C. albicans acts synergistically with some bacterial species, such as
methicillin resistant Staphylococcus aureus (MRSA), providing protection against the bactericidal
effect of antibiotics. Moreover, the fungal biofilm supplies the growth and development of anaerobic
strains, such as Clostridium difficile and Bacterioides fragilis, in external aerobic conditions by creating a
protective hypoxic microenvironment [5].
Resistance to antifungal treatment is a serious concern on account of both the limited number of
available chemotherapeutic agents and the phylogenetic relatedness between fungal and human
eukaryotic cells, which hinder the development of novel active drugs with an acceptable
pharmaco-toxicological profile [6]. However, fungi also possess distinct cytoplasmic organelles and
biosynthetic pathways [7]. The enzymes that are involved in the biosynthesis of ergosterol are
important targets for several clinically approved antifungals. Among these, the most widely used
are the azole compounds, which inhibit the CYP51 enzyme-mediated demethylation of lanosterol
into ergosterol, which is a major component of the fungal cytoplasmic membrane, being absent in
the mammalian cells, and it is also a bioregulator of membrane integrity and proper function of
membrane-bound enzymes [8,9]. The resulted disruption of fungal cell membrane morphology and
functional integrity, with the subsequent loss of intracellular constituents and cell lysis, is
responsible for their fungistatic effect [7]. However, drawbacks, such as narrow antifungal spectrum,
low bioavailability, systemic toxicity, and increasing drug tolerance, require the development of
novel molecules with improved pharmacokinetics and therapeutic efficiency [10].
In the field of antifungal drugs discovery, significant efforts have been made towards the
chemical development of molecules containing azole heterocyclic structures as key pharmacophores
[11]. 1,3-thiazole and 2-hydrazynil-1,3-thiazole derivatives having a lipophilic C4-substituent are
endowed with potent anti-Candida activity against clinically relevant fungal strains, as reported in
the literature [12,13] and supported by the results that were obtained in our previous research [14].
Thus, we decided to synthesize and evaluate the antifungal potential of two novel series of
compounds, keeping the key pharmacophores constant, 1,3-thiazole and 2-hydrazinyl-1,3-thiazole,
and introducing different moieties at the C4-position of the thiazole heterocycle.
The inhibitory activity of the newly synthesized compounds against pathogenic Candida sp.
strains was evaluated in vitro and the results obtained, being expressed in terms of Minimum
Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC), were compared to
those of the reference drug fluconazole.
A molecular docking study was performed while using fungal lanosterol-C14α-demethylase
(CYP51) as a target enzyme in an attempt to gain more insight into the mechanism of action of the
novel thiazole derivatives. Molecular docking is widely used for the approximate prediction of the
binding affinity of a molecule towards a biological target using scoring functions, owing to the
progress in structural analysis techniques and the consequential accessibility to experimentally
derived ligand-receptor complexes [15].
The most active molecule in both in vitro and in silico antifungal assay was subjected to a
fluorescence microscopy study, while using the membrane-impermeable DNA-binding fluorescent
dye propidium iodide (PI) as a tracer, in order to confirm the disruptive effect on the integrity of the
fungal cell membrane [16].

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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].
2. Results and Discussion
2.1. 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].
Scheme 1. Synthetic protocol of the 4-phenyl-1,3-thiazole derivatives 4a–d.
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.
Scheme 2. Synthetic protocol of the 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives 7a–d.

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Elemental analysis and spectral data ((Fourier transform infrared spectroscopy (FT-IR),
1
electrospray ionization-mass spectrometry (ESI-MS), proton nuclear magnetic resonance ( H-NMR),
13
and carbon nuclear magnetic resonance ( C-NMR)) were used for the structural confirmation of the
newly synthesized compounds (see Supplementary Material, Figures 1–32). 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
1
at 7.97–8.31 ppm in the H-NMR spectra, as well as the presence of a characteristic signal in the IR
−1
spectra at 3089–3106 cm , which corresponded to the C5-H stretching vibration.
Concerning the 2-hydrazinyl-thiazole derivatives (7a–d), two specific signals appeared in their
−1
−1
IR spectra, at 3318-3327 cm and 3189–3200 cm , corresponding to the N-H asymmetric and
13
symmetric stretching vibrations. Moreover, the presence of a characteristic signal in the C-NMR
spectra at 143.2–144.1 ppm, belonging to the sp carbon from hydrazone linker, along with the
specific H-NMR signals that were related to C5-H (7.15–7.50 ppm) and additional aromatic
2
1
protons, confirmed the proposed structures.
2.2. 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.
Table 1. The minimum inhibitory concentration values (MIC, μg/mL) of the newly synthesized
4-phenyl-1,3-thiazole (4a–d) and 2-hydrazinyl-4-phenyl-1,3-thiazole (7a–d) derivatives.
C. albicans
ATCC 10231
62.5
C. parapsilosis
ATCC 22019
62.5
C. zeylanoides
ATCC 201082
Compound
4a
4b
125
125
62.5
62.5
4c
4d
62.5
62.5
62.5
62.5
125
62.5
7a
7b
7c
3.9
3.9
3.9
62.5
15.62
15.62
15.62
62.5
15.62
15.62
15.62
125
7d
Fluconazole
Broth control
15.62
7.81
No growth
15.62

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Table 2. The minimum fungicidal concentration values (MFC, μg/mL) of the newly synthesized
4-phenyl-1,3-thiazole (4a–d) and 2-hydrazinyl-4-phenyl-1,3-thiazole (7a–d) derivatives.
C. albicans
ATCC 10231
C. parapsilosis
ATCC 22019
125
C. zeylanoides
ATCC 201082
Compound
4a
4b
4c
4d
7a
7b
7c
7d
125
125
125
125
7.8
7.8
7.8
125
250
250
250
125
31.24
31.24
31.24
250
125
125
125
31.24
31.24
31.24
125
Fluconazole
Broth control
31.24
15.62
No growth
31.24
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].
2.3. 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
14
lanosterol-C 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.

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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.
Best Binding
Conformation
The 2 Å Cluster Containing the
Top Binding Conformation
Number of Distinct
Clusters
Compound
Standard Deviation
Average
G
G
Ki
NoC1
G
(kcal/mol)
0.49
Cartesian
Coordinates
0.59
(kcal/mol)
(nM)
Total
Multi-Member
(kcal/mol)
−10.24
−10.61
−10.48
−11.38
−11.90
−11.95
−12.54
−13.05
4a
4b
4c
4d
7a
7b
7c
−10.87
−11.17
−11.45
−12.36
−12.63
−13.16
−12.46
−13.61
10.77
6.49
4.05
0.87
0.55
0.74
0.23
0.11
30
24
29
17
10
10
4
20
22
19
27
48
54
49
47
14
13
11
16
19
23
15
16
0.56
0.52
0.57
0.46
0.50
0.54
0.45
0.67
0.65
0.90
0.61
0.45
0.44
0.64
7d
9
1 = number of 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
2
by the C -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].
Figure 1. The top binding conformation of compound 7b. Carbon atoms of the docked compound to
the active site of fungal lanosterol C14α-demethylase are depicted in magenta. Unnecessary protein
fragments in the background and foreground were removed for clarity.
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
2+
between the thymol fragment and the heme Fe , 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.

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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
2+
the heme Fe , as in the case of classical antifungal azoles [29,30].
2.4. 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).
Figure 2. Representative bright-field, fluorescence and merged images of Candida albicans cells
stained with Propidium Iodide (PI): (a) cells exposed to MIC concentration of 7b; and, (b) control
cells.
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.
2.5. 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

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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 (λmax) 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].
F0/F = 1 + KSV × [Q] = 1+K
q
× г0
× [Q] or (F0 − F)/F = KSV × [Q] = K
q
× г
0
× [Q]
(1)
where F0 and F are the fluorescence intensities of BSA in the absence and presence of the quencher,
respectively; KSV 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 is the average fluorescence
0
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.
Figure 3. (a) Fluorescence spectra of bovine serum albumin (BSA) in the presence of compound 7b.
(b) The Stern–Volmer curve for the quenching of BSA (1.5 μM) by increasing concentrations (0.3 μM,
0.6 μM, 0.9 μM, 1.2 μM, 1.5 μM, and 1.8 μM ) of 7b (λex = 289 nm, T = 298K).
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 λmax (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],

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as illustrated in Figure 3b for compound 7b. Table 4 presents the calculated values for all of the
tested compounds.
Table 4. Stern–Volmer equation constants for the interaction of BSA with the compounds 4a–d and
7a–d.
Compound
Kq × 1012 (L/mol·s)
8.59 ± 0.0004
4.94 ± 0.0001
3.41 ± 0.0001
17.77 ± 0.0008
9.36 ± 0.0003
22.21 ± 0.0012
2.35 ± 0.0001
12.75 ± 0.0007
a KSV × 104 (L/mol)
5.15± 0.0034
b R2
4a
4b
4c
4d
7a
7b
7c
0.9838
0.9986
0.9877
0.9899
0.9918
0.9847
0.9872
0.9834
2.94 ± 0.0008
2.04 ± 0.0011
10.66 ± 0.0059
5.58 ± 0.0026
13.32 ± 0.0086
1.41 ± 0.0011
7.65 ± 0.0051
7d
a KSV = Kq× г0, г0 ≈ 6 ns; b R2 is the correlation coefficient.
The calculated quenching constant (Kq) of the tested compounds is greater when compared to
the value that was obtained for biological macromolecules quenching due to collision mechanism (2
10
−1
−1
× 10
M
× s ), suggesting that a static quenching is involved, through the formation of a
BSA-ligand complex [33].
The Ksv 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 (Kb) 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.
log[(F0 − F)/F] = log Kb + n log[Q]
(2)
Table 5. Binding constant values and number of binding sites for the compounds 4a–d and 7a–d into
BSA.
4
−1
2
Compound
n
logKb
Kb × 10 (M )
6.60
R
4a
4b
4c
4d
7a
7b
7c
0.773 ± 0.0327
0380 ± 0.0276
0.796 ± 0.0260
1.305 ± 0.0690
0.987 ± 0.0360
0.642 ± 0.0445
0.696 ± 0.0575
0.912 ± 0.0623
−1.1801 ± 0.0087
−1.1559 ± 0.0073
−1.5819 ± 0.0070
−1.0696 ± 0.0186
−1.2482 ± 0.0096
−0.7441 ± 0.0122
−1.7314 ± 0.0158
−1.0390 ± 0.0160
0.9911
0.9740
0.9944
0.9859
0.9933
0.9810
0.9732
0.9770
6.98
2.61
8.51
5.64
18.02
1.85
9.14
7d
The Kb values reflect the strength of the binding interaction. For the majority of the tested
4
molecules, the Kb values are in the range of 1−15 × 10 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].

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3. Materials and Methods
3.1. 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
1
series and an Agilent Ion Trapp SL mass spectrometer (Agilent, Santa Clara, CA, USA). The H-NMR
spectra were recorded on a Bruker Advance NMR spectrometer (Karlsruhe, Germany), operating at
500 MHz, while using DMSO-d6 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-d6, 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).
3.2. 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 NaHCO3 10% until free of acid. The pure
compounds were yielded through recrystallization from ethanol.
2-((2-isopropyl-5-methylphenoxy)methyl)-4-(4-methoxyphenyl)thiazole (4a): 0.33 g, yield 63%; m.p.
−1
186–187 °C; FT-IR (KBr) νmax cm : 3089 (C5–H thiazole str), 3048 (C–Har str), 2921 (C-Halif str), 1612
1
(C=N str), 1257 (C–O–C asym str), 1050 (C–O–C sym str); H NMR (500 MHz, DMSO-d6, δ/ppm): 7.97
(s, 1H, thiazole-C5H), 7.90 (d, J = 9.0 Hz, 2H, Ar-H), 7.11 (d, J = 7.5 Hz, 1H, Ar-H), 7.01 (d, J = 9.0 Hz,
2H, Ar-H), 6.95 (s, 1H, Ar-H), 6.78 (d, J = 7.5 Hz, 1H, Ar-H), 5.46 (s, 2H, O-CH2), 3.80 (s, 3H, Ar-CH3),
13
3.33–3.27 (m, 1H, Ar-CH-(CH3)2)), 2.27 (s, 3H, Ar-CH3), 1.20 (d, J = 7.0 Hz, 6H, Ar-CH-(CH3)2);
C
NMR (125 MHz, DMSO-d6, δ/ppm): 167.5 (C), 159.7 (C), 155.0 (C), 154.5 (C), 136.5 (C), 133.7 (C), 127.8
(2CH), 127.3 (C), 126.3 (CH), 122.4 (CH), 114.6 (2CH), 113.5 (CH), 113.1 (CH), 67.5 (CH2), 55.6 (CH3),
+
26.8 (CH), 23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: calculated for C21H23NO2S [M+H] 354.1, found
354.3; Anal. calculated for C21H23NO2S (%): C, 71.36; H, 6.56; N, 3.96; S, 9.09; found (%): C, 71.49; H,
6.53; N, 3.94; S, 9.07.
4-(4-chlorophenyl)-2-((2-isopropyl-5-methylphenoxy)methyl)thiazole (4b): 0.31 g, yield 58%; m.p. 182–183
−1
°C; FT-IR (KBr) νmax cm : 3106 (C5–H thiazole str), 3050 (C–Har str), 2920 (C-Halif str), 1613 (C=N str),
1
1256 (C–O–C asym str), 1049 (C–O–C sym str), 745 (C–Cl str); H NMR (500 MHz, DMSO-d6, δ/ppm):
8.23 (s, 1H, thiazole-C5H), 8.00 (d, J = 9.0 Hz, 2H, Ar-H), 7.53 (d, J = 9.0 Hz, 2H, Ar-H), 7.12 (d, J= 7.5
Hz, 1H, Ar-H), 6.95 (s, 1H, Ar-H), 6.79 (d, J = 7.5 Hz, 1H, Ar-H), 5.48 (s, 2H, O-CH2), 3.33–3.28 (m, 1H,
13
Ar-CH-(CH3)2)), 2.27 (s, 3H, Ar-CH3), 1.20 (d, J = 7.0 Hz, 6H, Ar-CH-(CH3)2); C NMR (125 MHz,
DMSO-d6, δ/ppm): 168.2 (C), 154.9 (C), 153.3 (C), 136.5 (2C), 133.7 (C), 133.3 (C), 129.3 (2CH), 128.1
(2CH), 126.3 (CH), 122.4 (CH), 115.9 (CH), 113.5 (CH), 67.4 (CH2), 26.8 (CH), 23.1 (2CH3), 21.4 (CH3);

Molecules 2020, 25, 1079
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+
MS (ESI) m/z: calculated for C20H20ClNOS [M+H] 358.1, found 358.5; Anal. calculated for
C20H20ClNOS (%): C, 67.12; H, 5.63; N, 3.91; S, 8.96; Cl, 9.91; found (%): C, 66.98; H, 5.65; N, 3.90; S,
8.99; Cl, 9.88.
4-(4-bromophenyl)-2-((2-isopropyl-5-methylphenoxy)methyl)thiazole (4c): 0.41 g, yield 69%; m.p. 198–199
−1
°C; FT-IR (KBr) νmax cm : 3101 (C5–H thiazole str), 3046 (C–Har str), 2919 (C-Halif str), 1614 (C=N str),
1
1255 (C–O–C asym str), 1049 (C–O–C sym str), 676 (C–Br str); H NMR (500 MHz, DMSO-d6, δ/ppm):
8.24 (s, 1H, thiazole-C5H), 7.94 (d, J = 9.0 Hz, 2H, Ar-H), 7.67 (d, J = 9.0 Hz, 2H, Ar-H), 7.12 (d, J = 7.5
Hz, 1H, Ar-H), 6.95 (s, 1H, Ar-H), 6.79 (d, J = 7.5 Hz, 1H, Ar-H), 5.48 (s, 2H, O-CH2), 3.33–3.28 (m, 1H,
13
Ar-CH-(CH3)2)), 2.27 (s, 3H, Ar-CH3), 1.20 (d, J = 7.0 Hz, 6H, Ar-CH-(CH3)2); C NMR (125 MHz,
DMSO-d6, δ/ppm): 168.2 (C), 154.9 (C), 153.3 (C), 136.5 (2C), 133.7 (C), 132.29 (2CH), 128.4 (2CH),
126.3 (CH), 122.4 (CH), 121.7 (C), 116.0 (CH), 113.5 (CH), 67.4 (CH2), 26.8 (CH), 23.1 (2CH3), 21.4
+
(CH3); MS (ESI) m/z: calculated for C20H20BrNOS [M+H] 402.0, found 402.8; Anal. calculated for
C20H20BrNOS (%): C, 59.70; H, 5.01; N, 3.48; S, 7.97; Br, 19.86; found (%): C, 59.88; H, 4.98; N, 3.50; S,
7.94; Br, 19.91.
2-((2-isopropyl-5-methylphenoxy)methyl)-4-(naphthalen-2-yl)thiazole (4d): 0.39 g, yield 71%; m.p. 179–180
−1
°C; FT-IR (KBr) νmax cm : 3103 (C5–H thiazole str), 3049 (C–Har str), 2921 (C-Halif str), 1610 (C=N str),
1
1257 (C–O–C asym str), 1039 (C–O–C sym str); H NMR (500 MHz, DMSO-d6, δ/ppm): 8.55 (s, 1H,
Ar-H), 8.31 (s, 1H, thiazole-C5H), 8.13 (d, J = 8.5 Hz, 1H, Ar-H), 8.00 (d, J = 8.5 Hz, 2H, Ar-H), 7.95 (d, J
= 8.5 Hz, 1H, Ar-H), 7.55 (t, 2H, Ar-H), 7.13 (d, J = 7.5 Hz, 1H, Ar-H), 6.98 (s, 1H, Ar-H), 6.80 (d, J = 7.5
Hz, 1H, Ar-H), 5.53 (s, 2H, O-CH2), 3.36–3.31 (m, 1H, Ar-CH-(CH3)2)), 2.29 (s, 3H, Ar-CH3), 1.22 (d, J =
13
7.5 Hz, 6H, Ar-CH-(CH3)2); C NMR (125 MHz, DMSO-d6, δ/ppm): 168.1 (C), 155.0 (C), 154.5 (C),
136.6 (2C), 133.7 (C), 133.1 (C), 131.8 (C), 128.9 (CH), 128.7 (CH), 128.1 (CH), 127.0 (CH), 126.8 (CH),
126.3 (CH), 125.1 (CH), 124.6 (CH), 122.4 (CH), 115.7 (CH), 113.5 (CH), 67.5 (CH2), 26.8 (CH), 23.1
+
(2CH3), 21.4 (CH3); MS (ESI) m/z: calculated for C24H23NOS [M+H] 374.1, found 374.2; anal.
calculated for C24H23NOS (%): C, 77.18; H, 6.21; N, 3.75; S, 8.58; found (%): C, 77.31; H, 6.19; N, 3.76; S,
8.55.
3.2.2. General Procedure for the Synthesis of 2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenyl
ethylidene)hydrazineyl)-4-phenylthiazole Derivatives 7a–d
To a solution of hydrazinyl-1-carbothioamide 6 (1 mmol) that was dissolved in dry acetone
(3mL), equimolar quantities of corresponding α-haloketones were added and the mixture was
stirred at room temperature for 6h. The resulted precipitate was filtered under vacuum and then
washed with a solution of NaHCO3 10% until free of acid. The pure compounds were yielded
through recrystallization from ethanol.
2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)-4-(4-methoxyphenyl)thiazole (7a):
−1
0.50 g, yield 72%; m.p. 195–196 °C; FT-IR (KBr) νmax cm : 3327 (N–H asym str), 3194 (N–H sym str),
3106 (C5–H thiazole str), 3050 (C–Har str), 2917 (C-Halif str), 1608 (C=N str), 1253 (C–O–C asym str),
1
1062 (C–O–C sym str); H NMR (500 MHz, DMSO-d6, δ/ppm): 11.44 (br, 1H, NH), 7.79 (d, J = 8.8 Hz,
2H, Ar-H), 7.76 (d, J = 8.0 Hz, 2H, Ar-H), 7.42 (t, J = 7.0 Hz, 2H, Ar–H), 7.37 (t, J = 7.0 Hz, 1H, Ar–H),
7.15 (s, 1H, thiazole-C5H), 7.05 (d, J = 7.5 Hz, 1H, Ar-H), 6.98 (d, J = 8.8 Hz, 2H, Ar-H), 6.92 (s, 1H,
Ar-H), 6.75 (d, J = 7.5 Hz, 1H, Ar-H), 5.30 (s, 2H, O-CH2), 3.79 (s, 3H, Ar-CH3), 3.05–3.00 (m, 1H,
13
Ar-CH-(CH3)2)), 2.28 (s, 3H, Ar-CH3), 1.00 (d, J = 7.0 Hz, 6H, Ar-CH-(CH3)2); C NMR (125 MHz,
DMSO-d6, δ/ppm): 170.1 (C), 159.3 (C), 155.2 (C), 143.2 (C), 136.6 (C), 136.2 (2C), 133.6 (2C), 129.3
(CH), 128.7 (2CH), 127.3 (2CH), 126.7 (2CH), 126.1 (CH), 121.8 (CH), 114.5 (2CH), 113.1 (CH), 112.6
(CH), 61.1 (CH2), 55.6 (CH3) 26.3 (CH), 23.1 (2CH3), 21.5 (CH3); MS (ESI) m/z: calculated for
+
C28H29N3O2S [M+H] 472.2, found 472.7; Anal. calculated for C28H29N3O2S (%): C, 71.31; H, 6.20; N,
8.91; S, 6.80; found (%): C, 71.52; H, 6.18; N, 8.89; S, 6.82.
4-(4-chlorophenyl)-2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)thiazole
0.54 g, yield 76%; m.p. 190–191 °C; FT-IR (KBr) νmax cm : 3318 (N–H asym str), 3189 (N–H sym str),
(7b):
−1

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3108 (C5–H thiazole str), 3053 (C–Har str), 2920 (C-Halif str), 1606 (C=N str), 1246 (C–O–C asym str),
1
1060 (C–O–C sym str), 750 (C–Cl str); H NMR (500 MHz, DMSO-d6, δ/ppm): 12.21 (br , 1H, NH), 7.88
(d, J = 8.5 Hz, 2H, Ar-H), 7.75 (d, J = 7.0 Hz, 2H, Ar-H), 7.56–7.51 (m, 1H, Ar-H), 7.48 (d, J = 8.5 Hz, 1H,
Ar-H), 7.44 (s, 1H, thiazole-C5H), 7.42 (t, J = 7.0 Hz, 2H, Ar–H), 7.37 (t, J = 7.0 Hz, 1H, Ar–H), 7.04 (d, J
= 7.5 Hz, 1H, Ar-H), 6.91 (s, 1H, Ar-H), 6.74 (d, J = 7.5 Hz, 1H, Ar-H), 5.28 (s, 2H, O-CH2), 3.03–2.97
13
(m, 1H, Ar-CH-(CH3)2)), 2.28 (s, 3H, Ar-CH3), 0.99 (d, J = 6.5 Hz, 6H, Ar-CH-(CH3)2); C NMR (125
MHz, DMSO-d6, δ/ppm): 168.8 (C), 155.2 (C), 151.2 (C), 143.6 (C), 136.5 (C), 136.2 (C), 133.5 (C), 132.5
(C), 130.4 (C), 129.1 (2CH), 128.7 (2CH), 127.6 (2CH), 126.7 (2CH), 126.1 (CH), 121.8 (CH), 113.1 (CH),
113.0 (CH), 61.1 (CH2), 26.3 (CH), 23.1 (2CH3), 21.5 (CH3); MS (ESI) m/z: calculated for C27H26ClN3OS
+
[M+H] 476.1, found 476.5; Anal. calculated for C27H26ClN3OS (%): C, 68.12; H, 5.51; N, 8.83; S, 6.73;
Cl, 7.45; found (%): C, 68.34; H, 5.52; N, 8.81; S, 6.75; Cl, 7.46.
4-(4-bromophenyl)-2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)thiazole (7c): 0.52
−1
g, yield 67%; m.p. 187–188 °C; FT-IR (KBr) νmax cm : 3318 (N–H asym str), 3192 (N–H sym str), 3113
(C5–H thiazole str), 3053 (C–Har str), 2919 (C-Halif str), 1604 (C=N str), 1245 (C–O–C asym str), 1071
1
(C–O–C sym str), 685 (C–Br str); H NMR (500 MHz, DMSO-d6, δ/ppm): 11.99 (br, 1H, NH), 7.82 (d, J
= 8.5 Hz, 2H, Ar-H), 7.76 (d, J = 6.5 Hz, 2H, Ar-H), 7.62 (d, J = 8.5 Hz, 2H, Ar-H), 7.46 (s, 1H,
thiazole-C5H), 7.42 (t, J = 7.0 Hz, 2H, Ar–H), 7.38 (t, J = 7.0 Hz, 1H, Ar–H), 7.05 (d, J = 7.5 Hz, 1H,
Ar-H), 6.91 (s, 1H, Ar-H), 6.75 (d, J = 7.5 Hz, 1H, Ar-H), 5.28 (s, 2H, O-CH2), 3.04–2.98 (m, 1H,
13
Ar-CH-(CH3)2)), 2.28 (s, 3H, Ar-CH3), 0.99 (d, J = 7.0 Hz, 6H, Ar-CH-(CH3)2); C NMR (125 MHz,
DMSO-d6, δ/ppm): 169.2 (C), 155.2 (C), 151.7 (C), 143.6(C), 136.5 (C), 136.2 (C), 133.6 (C), 133.5 (C),
132.08 (2CH), 129.2 (CH), 128.8 (2CH), 128.0 (2CH), 126.7 (2CH), 126.1 (CH), 121.8 (CH), 121.1 (C),
113.0 (CH), 111.9 (CH), 61.1 (CH2), 26.3 (CH), 23.1 (2CH3), 21.5 (CH3); MS (ESI) m/z: calculated for
+
C27H26BrN3OS [M+H] 520.1, found 521.1; Anal. calculated for C27H26BrN3OS (%): C, 62.31; H, 5.04; N,
8.07; S, 6.16; Br, 15.35; found (%): C, 62.19; H, 5.05; N, 8.10; S, 6.14; Br 15.31.
2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)-4-(naphthalen-2-yl)thiazole
0.45 g, yield 61%; m.p. 183–184 °C; FT-IR (KBr) νmax cm : 3326 (N–H asym str), 3200 (N–H sym str),
(7d):
−1
3089 (C5–H thiazole str), 3050 (C–Har str), 2922 (C-Halif str), 1605 (C=N str), 1256 (C–O–C asym str),
1
1091 (C–O–C sym str); H NMR (500 MHz, DMSO-d6, δ/ppm): 12.02 (br, 1H, NH), 8.41 (s, 1H, Ar-H),
8.02 (d, J = 9.0 Hz, 1H, Ar-H), 7.95 (d, J = 9.0 Hz, 1H, Ar-H), 7.92 (d, J = 9.0 Hz, 2H, Ar-H), 7.77 (d, J =
7.0 Hz, 2H, Ar-H), 7.52 (t, 2H, Ar-H), 7.50 (s, 1H, thiazole-C5H), 7.43 (t, J = 7.0 Hz, 2H, Ar–H), 7.39 (t, J
= 7.0 Hz, 1H, Ar–H), 7.06 (d, J = 7.5 Hz, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 6.76 (d, J = 7.5 Hz, 1H, Ar-H),
5.32 (s, 2H, O-CH2), 3.05–3.00 (m, 1H, Ar-CH-(CH3)2)), 2.30 (s, 3H, Ar-CH3), 1.01 (d, J = 6.5 Hz, 6H,
13
Ar-CH-(CH3)2); C NMR (125 MHz, DMSO-d6, δ/ppm): 168.5 (C), 155.2 (C), 154.6 (C), 144.1 (C), 136.6
(2C), 136.2 (C), 133.5 (2C), 132.9 (C), 129.2 (CH), 128.8 (2CH), 128.6 (2CH), 128.0 (CH), 126.9 (CH),
126.7 (2CH), 126.5 (CH), 126.1 (CH), 124.5 (CH), 124.3 (CH), 121.8 (CH), 113.1 (CH), 111.8 (CH), 61.1
+
(CH2), 26.3 (CH), 23.1 (2CH3), 21.5 (CH3); MS (ESI) m/z: calculated for C31H29N3OS [M+H] 492.2,
found 492.4; Anal. calculated for C31H29N3OS (%): C, 75.73; H, 5.95; N, 8.55; S, 6.52; found (%): C,
75.92; H, 5.93; N, 8.58; S, 6.50.
3.3. In Vitro Anti-Candida Activity
The in vitro anti-Candida screening was completed according to the guidelines of Clinical
Laboratory Standards Institute (CLSI) [41], with the broth microdilution method being employed for
the determination of MIC and MFC values. All of the used fungal strains were obtained from the
Food Biotechnology Laboratory, Life Sciences Institute, University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca, Romania.
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

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suspended in saline and adjusted spectrophotometrically to RPMI 1640 medium. The initial density
6
of Candida sp. was approximately 2 × 10 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
5
were suspended in nutrient broth and RPMI 1640 media to achieve a final density of 2 × 10 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.
3.4. Molecular Docking Study
The binding affinity of the newly synthesized thiazole derivatives (4a–d, 7a–d) to the catalytic
14
site of the target fungal lanosterol C 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].
The inhibition constant (Ki) values were calculated based on the in silico predicted binding
(∆G), while using the following formula: Ki = e^((∆G × 1000)/(R × T)) (R represents the Regnault
constant = 198,719 kcal/(K × mol) and T = 298.15 K).
Visualization and analysis of the docking results were performed while using UCSF Chimera
1.10.2 [44].
3.5. Propidium Iodide (PI) Dye Uptake Assay
Fluorescence microscopy was used to evaluate the membrane integrity of yeast cells that were
7
exposed to compound 7b. For this purpose, C. albicans cell suspension (1 × 10 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

Molecules 2020, 25, 1079
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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.
3.6. 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].
4. 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.
Supplementary Materials: The following are available online, Figure 1–32, containing structural information
related to compounds 4a–d and 7a–d
Author Contributions: Conceptualization, O.O. and A.I.P.; investigation, A.-I.P., G.M., D.C.V., S.E., G.B., L.V.,
A.P., L.I.G. and M.F.; software, G.M.; writing—Original draft preparation, A.-I.P.; writing—review and editing,
L.I.G., I.I. and M.F.; supervision, O.O.; project administration, O.O.; funding acquisition, A.I.P. and I.I. All
authors have read and agreed to the published version of the manuscript.
Funding: This paper was published under the frame of European Social Found, Human Capital Operational
Programme 2014-2020, project no. POCU/380/6/13/125171. Andreea-Iulia Pricopie would like to acknowledge
the financial support provided by “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca,
Romania, through doctoral research project number 1300/55/13.01.2017, 3066/45/01.02.2018, 1531/7/18.01.2019
and 2461/62/17.01.2020, and the Romanian Ministry of Research and Innovation, through the Core Program, PN
19 35 02 01.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 4a–d and 7a–d are available from the authors.
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