Design and synthesis of novel 1,3-thiazole and 2-hydrazinyl-1,3-thiazole derivatives as anti-Candida agents: In vitro antifungal screening, molecular docking study, and spectroscopic investigation of their binding interaction with bovine serum albumin

Article abstract of DOI:10.3390/molecules24193435

In the context of there being a limited number of clinically approved drugs for the treatment of Candida sp.-based infections, along with the rapid development of resistance to the existing antifungals, two novel series of 4-phenyl-1,3-thiazole and 2-hydr

Full text of DOI:10.3390/molecules24193435

molecules
Article
Design and Synthesis of Novel 1,3-Thiazole
and 2-Hydrazinyl-1,3-Thiazole Derivatives
as Anti-Candida Agents: In Vitro Antifungal
Screening, Molecular Docking Study,
and Spectroscopic Investigation of their Binding
Interaction with Bovine Serum Albumin
Andreea-Iulia Pricopie 1, Ioana Ionut 1,*, Gabriel Marc 1,
* ,
, Anca-Maria Arseniu 1,2
,
Laurian Vlase 3 , Adriana Grozav 4, Luiza Ioana Găină 5, Dan C. Vodnar 6 , Adrian Pîrnău 7,
Brîndusa Tiperciuc 1 and Ovidiu Oniga 1
,
1
Department of Pharmaceutical Chemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy,
,
41 Victor Babes Street, 400012 Cluj-Napoca, Romania; pricopie.andreea@umfcluj.ro (A.-I.P.);
,
borcea.anca@umfcluj.ro (A.-M.A.); brandu32@yahoo.com (B.T.); onigao65@yahoo.com (O.O.)
Preclinic Department, Pharmacy Specialization, Faculty of Medicine, Lucian Blaga University of Sibiu,
2
2A Lucian Blaga Street, 550169 Sibiu, Romania
Department of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Hat, ieganu” University of Medicine
3
and Pharmacy, 41 Victor Babes Street, 400012 Cluj-Napoca, Romania; laurian.vlase@umfcluj.ro
,
4
5
Department of Organic Chemistry, “Iuliu Hatieganu” University of Medicine and Pharmacy,
,
41 Victor Babes Street, 400012 Cluj-Napoca, Romania; adriana.ignat@umfcluj.ro
,
Research Center on Fundamental and Applied Heterochemistry, Faculty of Chemistry and Chemical
Engineering, “Babes¸-Bolyai” University, 11 Arany Janos Street, 400028 Cluj-Napoca, Romania;
lusi@chem.ubbcluj.ro
6
7
Department of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine,
3-5 Mănăstur Street, 400372 Cluj-Napoca, Romania; dan.vodnar@usamvcluj.ro
,
National Institute for Research and Development of Isotopic and Molecular Technologies,
67-103 Donath Street, 400293 Cluj-Napoca, Romania; adrian.pirnau@itim-cj.ro
*
Correspondence: ionut.ioana@umfcluj.ro (I.I.); marc.gabriel@umfcluj.ro (G.M.); Tel.: +40747-507-629 (I.I.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 8 August 2019; Accepted: 20 September 2019; Published: 21 September 2019
Abstract: In the context of there being a limited number of clinically approved drugs for the treatment of
Candida sp.-based infections, along with the rapid development of resistance to the existing antifungals,
two novel series of 4-phenyl-1,3-thiazole and 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives were
synthesized and tested in vitro for their anti-Candida potential. Two compounds (7a and 7e) showed
promising inhibitory activity against the pathogenic C. albicans strain, exhibiting substantially lower
MIC values (7.81
(15.62 g/mL). Their anti-Candida activity is also supported by molecular docking studies, using the
fungal lanosterol C14 -demethylase as the target enzyme. The interaction of the most biologically
µg/mL and 3.9 µg/mL, respectively) as compared with the reference drug fluconazole
µ
α
active synthesized compound 7e with bovine serum albumin was investigated through fluorescence
spectroscopy, and the obtained data suggested that this molecule might efficiently bind carrier
proteins in vivo in order to reach the target site.
Keywords: 1,3-thiazole; anti-Candida; molecular docking; bovine serum albumin; fluorescence quenching
Molecules 2019, 24, 3435; doi:10.3390/molecules24193435
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3435
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1. Introduction
The increasing incidence of Candida sp.-based infections poses a direct threat to the healthcare
system, being associated with high mortality rates and expensive medical costs for governments and
hospitalized patients [
of superficial and systemic fungal infections. The most widely used, the azole compounds, are directed
against ergosterol biosynthesis through the inhibition of lanosterol-C14 -demethylase (CYP51).
1]. Currently, only five classes of antifungal drugs are available for the treatment
α
The resulted ergosterol depletion and the methylated sterols accumulation lead to alterations in the
integrity and the functioning of the fungal cell membrane, thus inhibiting fungal cell replication
and enhancing susceptibility to host defense mechanisms [2]. The effectiveness of these marketed
drugs is significantly reduced by the development of robust resistance mechanisms in medically
important fungal strains, such as drug target overexpression, the up-regulation of drug efflux pumps,
and activation of the stress response [
agents, with a distinct mechanism of action, is hindered by the phylogenetic relatedness between fungi
and humans, and is responsible for the reduced number of differential targets to be exploited [ ]. In this
setting, the development of novel chemical entities that are endowed with lanosterol C14 -demethylase
3]. Furthermore, the development of novel chemotherapeutic
4
α
enzyme inhibitory potential and are less susceptible to the above-mentioned fungal adaptation
mechanisms is a priority.
The identification of drug-like chemical features and theoretical ligand structures as biologically
active hits or leads is easily achievable nowadays due to the availability of high resolution structural
analysis techniques such as X-ray crystallography and in silico drug design tools. Both structure-based
molecular docking programs and ligand-based molecular modeling studies can be employed for this
purpose [5].
Nitrogen-containing heterocycles have received particular interest in recently reported research
studies due to their valuable biological activities [6]. In particular, 1,3-thiazole scaffold is a versatile
building block for lead generation, and is readily accessible for subsequent lead optimization through
chemical derivatization. The antifungal activity against clinically relevant Candida sp. strains was
described for a large number of small molecules bearing thiazole scaffolds [7,8].
The main structural features identified in the family of 1,3-thiazole derivatives with antifungal
activity by means of pharmacophore-based approaches consist of: An aromatic nitrogen fragment
with an accessible lone pair, functioning as a hydrogen bond acceptor, a hydrophobic area represented
by a phenyl ring with a para-lipophilic substituent, along with a second aromatic ring and a hydrogen
bond donor motif [9,10].
Prompted by this validated hypothesis, and maintaining consistency with our research group
interest for thiazole-based compounds, we decided to synthesize a novel series of molecules containing
this chemical feature in order to investigate their antifungal potential. On account of the recently
reported data regarding the importance of a hydrazine substituent at the C2 position of the thiazole ring
in terms of improved anti-Candida activity [11,12], we were encouraged to work toward the chemical
development of a second series of substituted-2-hydrazinyl-1,3-thiazole derivatives (Figure 1).
The inhibitory activity of the newly synthesized compounds 4a–e and 7a–e was evaluated
in vitro against opportunistic Candida sp. strains and the results obtained, which were expressed
in terms of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC),
were compared to those of fluconazole, which was used as a reference drug. In order to confirm
their lanosterol-C14α-demethylase inhibitory potential, both series were subjected to a molecular
docking study. Additionally, their drug-like properties were assessed through an in silico absorption,
distribution, metabolism, excretion and toxicity (ADMET) prediction. It has been shown that the
pharmacokinetic properties, as well as the stability and toxicity of a drug, are actively influenced
by drug–protein interactions in the blood stream [13]. Human serum albumin (HSA) is an abundant
plasma protein that binds a wide range of drugs and metabolites, so the investigation of binding
interactions between the biologically active small molecules and serum albumin is an important
step for the assessment of their distribution and pharmacological activity. Bovine serum albumin

Molecules 2019, 24, 3435
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(BSA) and HSA are homologous proteins, and their interactions with different drugs have been
extensively studied by using both experimental and theoretical calculation methods. Due to its low
cost and highly structural homology with HSA, BSA represents the serum albumin that is preferred
in laboratory practice for the above-mentioned type of pharmacokinetic studies [14]. Fluorescence
spectroscopy was employed for the analysis of the binding profile of the most promising of the newly
synthesized compounds with BSA in terms of binding mechanism, binding constants, and the number
of binding sites.
Figure 1. Rational design of the titled 4-substituted thiazole derivatives (HF–hydrophobic feature;
HBA–hydrogen bond acceptor; HBD–hydrogen bond donor).
2. Results and Discussion
2.1. Chemistry
Thesyntheticprotocolemployedforthechemicaldevelopmentofthefirstseriesof4-phenyl-1,3-thiazole
compounds (4a–e) is outlined in Scheme 1.
The O-alkylation reaction of the phenolic hydroxyl group of thymol (1) with 2-iodoacetamide
yielded the amide compound . Next, a thionation process using Lawesson’s reagent (LR) in refluxing
2
toluene, followed by the Hantzsch condensation of the intermediate thioamide
3 with the appropriate
α-haloketones afforded the targeted 4-phenyl-1,3-thiazole derivatives.
The second series of compounds was synthesized according to the route described in Scheme 2.
By reacting thymol with 2-bromoacetophenone in alkaline media, we obtained the ketone derivative
The desired 2-hydrazinyl-4-phenyl-1,3-thiazoles (7a ) were synthesized in a two-step process,
5.
–e
by classical condensation of the carbonyl group with thiosemicarbazide in refluxing ethanol,
in the presence of glacial acetic acid as catalyst, followed by heterocyclization with the corresponding
α-halocarbonyl compounds.

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Scheme 1. Synthetic protocol of the 4-phenyl-1,3-thiazole derivatives 4a–e
Scheme 2. Synthetic protocol of the 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives 7a–e.
The reaction progress was periodically monitored by thin-layer chromatography (TLC).
The structures of the newly synthesized compounds were assigned by elemental analysis and spectral
data: Fourier transform infrared spectroscopy (FT-IR), electrospray ionization-mass spectrometry
(ESI-MS), hydrogen nuclear magnetic resonance (1H-NMR) and carbon nuclear magnetic resonance
(13C-NMR). The results of the C, H, N, and S quantitative elemental analysis of the titled thiazole
derivatives were consistent with the calculated values, within
The recorded molecular ion peaks were consistent with their molecular formulas. Concerning
compounds 4a , the thionation of the intermediate amide was confirmed by the FT-IR spectral
±
0.4% of the theoretical values.
–
e
2
data through the disappearance of the high-intensity signal corresponding to the carbonyl group
stretching vibration at 1698 cm−1. Subsequent cyclization to the desired 4-phenyl-1,3-thiazoles is
supported by the appearance in their IR spectra of a sharp medium intensity signal at 3180–3100 cm−1
corresponding to the thiazole-methine group stretching vibration, as well as by MS and NMR analysis.
Spectroscopic data related to the methine located at the C5 position of the thiazole ring are highly
specific for this class of compounds [15]. Regarding the newly synthesized 4-phenyl-1,3-thiazole
,

Molecules 2019, 24, 3435
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derivatives, the presence of a singlet at around 7.62–8.55 ppm in the 1H-NMR spectra, along with the
13C-NMR corresponding signal at about 110–115 ppm, confirmed their proposed structure.
The structure of the thiosemicarbazone intermediate
6 was confirmed by the characteristic
signal in the 13C-NMR spectra, at 145.3 ppm, belonging to the tertiary carbon atom involved in the
hydrazone bridge (C=N), as well as the two broad signals in the 1H-NMR spectra, at 10.80 ppm and
8.07 ppm, respectively, corresponding to the –NH and –NH2 protons. The successful accomplishment
of Hantzsch condensation is also supported by 1H-NMR through the disappearance of the NH2-related
signals together with the appearance of thiazole-methine proton specific singlet, at 8.19–8.54 ppm,
and additional aromatic protons, according to the specific structure of 7a–e derivatives.
2.2. Anti-Candida Activity Assay
The antifungal potential of the newly synthesized compounds was evaluated in vitro against
three human pathogenic Candida strains. The broth microdilution method was employed
for the determination of MIC and MFC values. Stock solutions (1 mg/mL) were prepared by dissolving
the tested compounds and the reference antifungal drug, fluconazole, in sterile DMSO. The obtained
values 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–e) and 2-hydrazinyl-4-phenyl-1,3-thiazole (7a–e) derivatives.
C. albicans
ATCC 10231
C. parapsilosis
ATCC 22019
C. zeylanoides
ATCC 201082
Compound
4a
4b
4c
4d
4e
7a
7b
7c
62.5
62.5
62.5
62.5
62.5
7.81
62.5
62.5
62.5
3.9
62.5
62.5
62.5
62.5
62.5
15.62
62.5
62.5
62.5
15.62
7.81
125
125
125
125
125
62.5
62.5
125
125
15.62
15.62
7d
7e
Fluconazole
15.62
Table 2. The minimum fungicidal concentration values (MFC,
µg/mL) of the newly synthesized
4-phenyl-1,3-thiazole (4a–e) and 2-hydrazinyl-4-phenyl-1,3-thiazole (7a–e) derivatives.
C. albicans
ATCC 10231
C. parapsilosis
ATCC 22019
C. zeylanoides
ATCC 201082
Compound
4a
4b
4c
4d
4e
7a
7b
7c
125
125
125
125
125
15.62
125
125
125
7.8
125
125
125
125
125
31.24
125
125
125
31.24
15.62
250
250
250
250
250
125
125
250
250
7d
7e
31.24
31.24
Fluconazole
31.24
The newly synthesized compounds exhibited moderate to good anti-Candida activity, according
to the obtained MIC and MFC values. The best inhibitory activity was exerted against C. albicans strain
ATCC 10231, the non-albicans species being less susceptible to almost all of the tested molecules.

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The 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives (7a
–e) proved to be superior to those lacking
the C2-hydrazone linkage (4a–e) in terms of antifungal potency. This superiority may be explained
either by the increased lipophilicity of the former, which is supported by the calculated logP values, and
correlates with an increased ability to penetrate the fungal cell membrane. Compounds 7a 7e were the
most promising, exhibiting substantially lower MIC values (7.81 µg/mL and 3.9 µg/mL, respectively)
as compared with the reference drug fluconazole (15.62 µg/mL).
Some structure-dependent differences in activity could be noticed in the second series
of compounds (7a ), which was related to the para-substitution of the C4-phenyl ring. It seems
–
e
that the presence of a lipophilic, electron-donating substituent (–CH3) is correlated with an increased
inhibitory activity on both albicans and non-albicans Candida strains, as compared with hydrophilic,
polar substituents (–CN, –NO2, –OH). These findings are in accordance with the data reported in the
literature regarding the antifungal potential of hydrazinyl-thiazoles with hydrophobic features and are
also supported by the results obtained from the in silico antifungal screening [9,11].
The determination of MFC confirmed the previously obtained MIC values. The MFC/MIC ratio
values for all the tested compounds were equal to two, suggesting that they may exert a fungicidal
effect [16].
2.3. Molecular Docking
Docking simulation is a widely used structure-based drug design (SBDD) method because of its
ability to predict the affinity of the ligands toward the active site of the biological targets through the
prediction of the interaction energy associated with each of the potential binding conformations [17].
The rational design and development of antifungals is particularly challenging, as approximately 80%
of the antifungal targets reported in the literature turned out to be false positive, with little potential
to develop target-based inhibitors. Another aspect worth mentioning is the potential lead-like molecules’
lack of selectivity toward fungal enzymatic machinery. Lanosterol-C14α-demethylase is a validated
target in the fungal cell membrane that is not present in the host cell membrane, which explains the
growing interest for the discovery of novel compounds that are capable of inhibiting it [2].
In order to evaluate the binding affinity of the newly synthesized compounds toward the
catalytic site of the target enzyme, a molecular docking study was carried out. For each compound,
the conformation with the best binding affinity, expressed as the highest variation of Gibbs-free
energy (∆G) of the complex with the target CYP51, and the computed inhibition constant (Ki) are
presented in Table 3, together with the conformational analysis of the cluster containing the top
binding conformations.
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 AutoDs.
Best Binding Conformation
The 2 Å Cluster Containing the Top Binding Conformation
Number of Distinct
Clusters
Standard Deviation
Compound
∆G
Average ∆G
(kcal/mol)
Ki
NoC 1
∆G
Cartesian
(kcal/mol)
(nM)
Total
Multi-Member
(kcal/mol)
Coordinates
4a
4b
4c
4d
4e
7a
7b
7c
−10.51
−11.49
−10.93
−10.83
−11.00
−12.52
−13.21
−12.20
−12.39
−12.71
19.77
3.78
9.73
11.52
8.65
0.66
0.21
1.14
0.83
0.48
19
41
32
6
27
3
14
1
1
−9.89
−10.60
−9.95
−10.52
−10.63
−11.35
−12.51
–
0.54
0.46
0.56
0.27
0.51
1.20
0.74
–
0.73
0.36
0.51
0.76
0.47
1.04
0.50
–
26
21
23
38
23
50
42
44
64
48
15
15
12
19
16
18
16
18
17
19
7d
7e
–
–
0.70
–
0.70
10
−12.63
1
= number of conformations.

Molecules 2019, 24, 3435
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The results of the molecular docking found poses with favorable binding energies toward C.
albicans lanosterol C14 -demethylase for all the tested molecules. The differences observed, in terms
α
of interaction energy (∆G), might be correlated with the C2-substitution of the thiazole ring.
According to the obtained data, the presence of the hydrazone bridge and the additional aromatic
phenyl in the second series of 2-hydrazinyl-4-phenyl-1,3-thiazoles (7a
for the target fungal enzyme, as compared with the first series of 4-phenyl-1,3-thiazole derivatives (4a
An aspect worth mentioning here is the higher flexibility of the compounds 7a , as compared to 4a–e,
–e) improved their affinity
–e).
–e
which lack the C2-substituent, as outlined by the increased number of clustered conformations with
improved binding affinity to the target enzyme. Moreover, the hydrogen-bonding capacity of N1-hydrazine
nitrogen allows additional interaction with the target lanosterol-C14α-demethylase.
For the compounds 4a–e, some important structural rigid elements, such as aromatic nuclei or the
sp2 hybrid imine carbon, are responsible for the heterogeneity of their binding mode. These molecules
have an important number of clusters containing residual conformations, which don’t match the main
cluster. The influence of structural flexibility on ∆G is also supported by the larger dispersion of Gibbs
free energy values and the large spatial dispersion of the predicted poses in the case of the second
series of compounds (7a–e).
The observed differences in terms of ∆G for compounds from both series could be attributed to the
lipophilicity and mesomeric effects of the para-substituent of C4-phenyl ring. It could be noticed that
the presence of a lipophilic, electron-donating substituent (–CH3) in compounds 4e and 7e is associated
with improved binding energy, as compared with the hydrophilic, electron withdrawing functional
groups (–CN, –NO2), given the hydrophobic environment (Phe233, Val510 amino acid residues) of the
phenyl-thiazole fragment. The two distinct areas with different polarity identified in the binding
regions from the access channel to the catalytic site of the lanosterol C14
described in a previous study reported by our research group [18].
α-demethylase were thoroughly
A comparative analysis of the linking mode of the most active compound 7e with its structural
analog 4e was made. In Figure 2, significant differences can be observed between the two.
Both compounds 4e and 7e enter the depth of the enzyme’s catalytic pocket along the access channel,
with the thymol fragment parallel to the active site, favoring a п-cation interaction. The 2-phenyl-thiazole
fragment of both compounds 4e and 7e fits well between the hydrophobic Leu376 and Phe233. On the
other side, the Leu121, Phe233, and Thr122 amino acid residues create another lipophilic subpocket into
the nonpolar zone of the binding pocket, which fits the supplementary phenyl ring of the compound 7e
.
Moreover, for the compound 7e, the presence of the hydrazine bridge allows additional interactions
with the polar area of the access channel. The electron lone pair of the N1-hydrazine nitrogen allows
the interaction of the compound with the polar Tyr118 residues through a hydrogen bond. For both
compounds 4e and 7e, a second polar interaction with the Tyr132 residue is endowed by the etheric
oxygen, which also acts as a hydrogen bond acceptor.
According to the data obtained from the molecular docking study, the newly synthesized
compounds might act as noncompetitive inhibitors of the fungal lanosterol C14α-demethylase.
The induced enzyme inhibition is not related to the covalent coordination of the heme Fe2+, as in the
case of classical antifungal azoles, but they compete with the physiological substrate of the enzyme
for access to the active site. This mechanism of action has been reported in the literature as being
associated with a reduced toxicity and resistance of fungal pathogens to the clinically approved
azole drugs [19,20]. Further studies on the fungal sterols’ biosynthetic pathway, involving ergosterol
extraction and quantitation assay, are required in order to confirm the inhibitory effect of the tested
thiazole derivatives against the target enzyme.

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Figure 2. The top binding conformation of compounds 4e (subsection (
C atoms of the docked compounds to the active site of fungal lanosterol C14
a
α
)) and 7e (subsection (
-demethylase are depicted
b)).
in magenta. Unnecessary protein fragments in the background and foreground were removed for clarity.
2.4. ADMET Profiling
In silico ADMET prediction studies are a central component of pharmaceutical research and
drug design, as it provides helpful guidance in the early evaluation of the in vivo efficiency and
safety of a drug. The magnitude of the biological activity of an active molecule, in terms of specific
interactions with the molecular target, along with the drug-related side effects, are strongly influenced
by its pharmacokinetic properties [21]. Integrated ADMET prediction platforms allow an early research
stage determination of the molecular descriptors, which predicts the suitability of a small molecule for
oral administration, thus reducing the number of synthesis–evaluation cycles and the more expensive
late-stage failures [22].
Drug-likeness descriptors of the newly synthesized compounds, according to the Lipinski Rule
of Five [23], are presented in Table 4.
Table 4. Computationally predicted drug-like descriptors of compounds 4a–e and 7a–e.
Compound MW 1 (Da)
RoB 2
HBA 3
HBD 4
tPSA 5
mLogP 6
Lipinski Violations
Veber Violations
4a
4b
4c
4d
4e
7a
7b
7c
323.45
348.46
368.45
382.48
337.48
441.59
466.60
486.59
500.61
455.61
5
5
6
6
5
8
8
9
9
8
2
3
4
4
2
3
4
5
5
3
0
0
0
2
0
1
1
1
3
1
50.36
74.15
96.18
113.68
50.36
74.75
98.54
120.57
138.07
74.75
3.54
2.81
2.39
2.07
3.76
4.49
3.78
3.46
3.11
4.68
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
7d
7e
1
Molecular weight; 2 Number of rotatable bonds; 3 Number of hydrogen bond acceptors; 4 Number of hydrogen
bond donors; 5 Topological polar surface area; 6 Moriguchi logarithm of the compound partition coefficient between
n-octanol and water.
According to the Lipinski Rule of Five, a newly synthesized small molecule is more likely to be an
orally active drug if it has no more than one violation of the following criteria: a molecular weight under
500 Da, an octanol–water partition coefficient (the Moriguchi logarithm of the compound partition
coefficient, or mLogP) lower than five, less than 10 nitrogen and oxygen atoms in the molecule, and no
more than five potential hydrogen donors [24]. As it can be observed in Table 4, the newly synthesized
4-phenyl-1,3-thiazole 4a–e and 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives 7a–e meet the criteria
for drug-likeness. It is worth mentioning that all the tested compounds comply with Veber’s rule
regarding the influence of polar surface area and molecular flexibility on oral bioavailability [25].

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The predicted molecular properties correlated with the pharmacokinetic profile and central
nervous system (CNS) activity of the titled compounds are presented in Table 5.
Table 5. Computationally predicted pharmacokinetic profile of compounds 4a–e and 7a–e.
CYP450 Inhibition
%GI Abs 1
BBBP 2
PGP 3 Substrate
Compound
CYP3A4
CYP2C9
4a
4b
4c
4d
4e
7a
7b
7c
7d
7e
1
91.62
83.41
75.81
69.78
91.62
83.21
75.00
67.40
61.36
83.21
Yes
No
No
No
No
No
No
No
No
No
Yes
Yes
No
No
Yes
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
No
gastrointestinal absorption (%); 2 blood–brain barrier permeability; 3 P-glycoprotein.
Permeability through biological cell membranes, including the gastrointestinal tract barrier, proved
to be predicted by a series of molecular properties, such as the partition coefficient (logP), topological
polar surface area (tPSA), intrinsic water solubility, carrier proteins-mediated efflux, and molecular
flexibility [26]. The cell membrane partitioning of a drug is considered a two-step process. In the first
step, drug incorporation into the phospholipid bilayer is strongly dependent on its lipophilicity. In the
second step, by contrast, drug delivery through the interior of the phospholipid bilayer is dependent
on its hydrogen-bonding capacity and polarity.
The dependence of gastrointestinal absorption on tPSA is assessed by Equation (1) [27]:
%GI Abs = 109 − (0.345 × tPSA)
(1)
Drugs that are almost completely absorbed (>90%) after oral administration have a tPSA < 60 Å,
while a tPSA > 140 Å results in unacceptably low (<10%) oral absorption [28].
As is can be observed in Table 4, all the newly synthesized compounds have tPSA values
under the threshold of 140 Å, which might suggest a good oral bioavailability. The lower values,
which are associated with a higher level of gastrointestinal absorption (%GI Abs = 69.78–91.62%),
were recorded for the 4-phenyl-1,3-thiazole derivatives (4a–e). The slightly reduced oral bioavailability
(%GI Abs = 61.36–83.21%) of the 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives (7a–e) may be explained
by their higher polarity and increased molecular flexibility, owing to the superior number of rotatable
bonds [25,28].
Classification of a drug as CNS active or inactive is based on evidence related to its blood–brain
barrier (BBB) permeability, which can be evaluated based on the predicted molecular weight (MW),
mLogP, HBA, HBD, and tPSA [16]. In particular, a tPSA value under 60–70 Å tends to identify CNS
active compounds [23]. The vast majority of the tested compounds have no ability to penetrate
the blood–brain barrier, thus presenting a low risk for CNS side effects. Only one compound, 7a
,
exhibits BBB penetration, which may be explained by its proper lipophilicity and lower molecular
weight [29,30].
Lanosterol C14α-demethylase (CYP51) belongs to the cytochrome P450 superfamily, which is
a large class of hemoproteins involved in the metabolism of a wide variety of endogenic metabolism
products and xenobiotics. Due to the high homology in the binding pocket of the CYP450 isoforms,
the development of selective inhibitors represents an important challenge in the field of drug design [31].
The clinically approved CYP51 inhibitor fluconazole is known to be a moderate inhibitor of CYP2C9
and CYP3A4 isoenzymes and is recommended in regulatory guidance as a prototype inhibitor to assess
the potential for drug–drug interactions mediated through CYP2C9 inhibition [32].

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The ligand-induced CYP450 cross-inhibition proved to be influenced by a series of molecular
descriptors, such as the molecular size and shape, hydrogen-bonding capacity, mLogP, and molecular
weight [33].
In respect of the newly synthesized compounds, the obtained results showed that the majority
of 2-hydrazinyl-4-phenyl-1,3-thiazole derivatives (7a, 7c–e) exhibit no cross-inhibition of the human
CYP2C9 isoenzyme, thus being less susceptible for drug–drug interactions-mediated side effects.
It may be explained by their large molecular size, compared with CYP2C9 substrates, and their higher
hydrogen-bonding capacity. The lower hydrophobicity and the presence of more hydrogen bond
donors and acceptors as compared with the substrates may also explain the higher degree of CYP3A4
cross-inhibition potential for both series of tested molecules [32].
2.5. Protein-Binding Study
The antimicrobial potency of heterocyclic compounds may be hindered by their restricted access
to the target site in vivo, because of the hydrophobicity of these molecules [34]. The absorption of a drug
into the blood stream and its distribution to the molecular target depend largely on its interactions
with carrier proteins, such as serum albumin or α1-acidic glycoprotein (α1-AGP) [35]. Human serum
albumin (HSA) is the most abundant carrier protein in plasma, having a high affinity for a wide range
of exogenous and endogenous ligands. HSA increases the apparent plasma solubility of hydrophobic
drugs and modulates their pharmacokinetic properties, free active concentration, and toxicity [36].
The nature and magnitude of small molecules’ binding interactions with HSA represent an important
predictor of their efficacy and delivery rate to the specific cellular target in vivo [37]. Therefore,
the investigation of the main factors involved in this process may be useful for the rational design and
development of novel derivatives with improved biological activity [38]. Given the high structural
homology with HSA and its relatively low cost, bovine serum albumin (BSA) is often used in laboratory
practice for the establishment of the protein-binding profile of various drugs [14].
On account of the intrinsic fluorescence of HSA, originating from tryptophan (Trp) residues,
the measurement of fluorescence quenching is a suitable analytical method to study drug–protein
interactions [39]. The fluorescent emission of serum albumin is sensitive to changes in the local
environment of the Trp-134 and Trp-212 amino acid residues, which is induced by the binding
of a small molecule [34]. The ligand binding induced decrease in intensity of the Trp fluorescence
may occur through a static or/and dynamic quenching mechanism, and is mediated by different
types of molecular interactions, such as ground state complex formation, excited state reactions,
energy transfer, and collision quenching [37]. In the case of static quenching, the ligand binding to the
macromolecule takes place in the ground state, and the resulting complex is nonfluorescent in nature.
By contrast, in the case of dynamic quenching, the fluorophore residues interact with the quencher
during the excitation [40].
In order to analyze the binding profile of the most biologically active molecule of the newly
synthesized thiazole derivatives, the fluorescence spectra of BSA upon the addition of an increasing
concentration of compound 7e (0.3
µM; 0.6
µM; 0.9
µM; 1.2
µM; 1.5
µM 1.8
µM) were recorded.
Spectral analysis was performed at room temperature by excitation of the probes at 289 nm (
λex)
and an emission scan from 300 to 500 nm. On excitation at 289 nm, the emission spectrum of BSA
showed an intense band at 341 nm. The obtained data demonstrated a linear decrease of BSA fluorescent
emission on increasing ligand concentration, as illustrated in Figure 3. A slight hypsochromic shift
of the BSA maximum emission wavelength was also observed from 341 nm to 339 nm, these suggesting
a static quenching mechanism [36,41].
This blue shift indicates that the ligand binding and formation of a ground-state complex with
BSA expose the fluorophore Trp residues to a more hydrophobic environment, thus changing their
emission properties [41].

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Figure 3. Fluorescence spectra of bovine serum albumin (BSA) (1.5
µM; λmax = 341 nm) in the presence
of compound 7e (0.3 µM; 0.6 µM; 0.9 µM; 1.2 µM; 1.5 µM 1.8 µM; λmax = 339 nm).
The magnitude of fluorescent quenching of the protein induced by the tested compound 7e and
the mechanism involved in the process were investigated using the Stern–Volmer Equation (2) [14]:
F0/F = 1 + KSV × [Q] = 1 + Kq × τ0 × [Q] or (F0 − F)/F = KSV × [Q] = Kq × τ0 × [Q]
(2)
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. Kq is the quenching rate constant of the biomolecule, and τ0 is the average fluorescence
lifetime of the biomolecule without the quencher, which is about 6 ns for BSA.
KSV and Kq were determined by the slope and the intercept of the linear regression plot graph
of the relative emission intensity (F0
for the compound 7e are presented in Table 6.
− F)/F versus τ0 × [Q], as shown in Figure 4. The calculated values
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
5
10
[Q]*τ0 (mol*L-1*s)
Figure 4. The Stern–Volmer curves for the quenching of BSA (1.5
1.2 µM; 1.5 µM 1.8 µM) (λex = 289 nm, T = 298K).
µM) by 7e (0.3 µM; 0.6 µM; 0.9 µM;
Table 6. Stern–Volmer equation constants for the interaction of BSA with the compound 7e at room
temperature (λmax BSA = 341 nm).
a
Kq × 1013 (L/mol·s)
3.29 ± 0.0017
KSV × 105 (L/mol)
1.97± 0.0124
b
R2
Compound
7e
0.9856
KSV = Kq × τ0, τ0 ≈ 6 ns; b R2 is the correlation coefficient.
a

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Kq values greater than that of the maximum scatter collision quenching constant with biopolymers
1010 mol−1 −1) confirm the static quenching of BSA induced by the newly synthesized compounds
through the formation of a ground-state complex [39].
(2
×
L
·
·
s
For the static quenching interaction, the number of binding sites (n) and the binding constants
(Kb) can be determined by the intercept and the slope of the regression curve, using Equation (3) [42].
The obtained results are presented in Table 7.
Table 7. Binding constant and binding sites for compound 7e binding into BSA.
Kb × 105 (M−1
)
Compound
7e
n
logKb
R2
0.873 ± 0.0355
−0.6448 ± 0.0094
2.26
0.9917
log[(F0 − F)/F] = log Kb + n log[Q]
(3)
The strength of the binding interaction is assessed based on the values of Kb: the higher the value
of Kb, the stronger the binding between the protein and the ligand [40].
The obtained n value was about 1, indicating the existence of a single binding site of compound 7e
on BSA. The kb values are in the order of 105, which support a strong binding interaction and a high
affinity of the macromolecule for the tested ligand [43].
As reported for clinically approved azole antifungals, an improved uptake of drugs into the
fungal cells and a more rapid inhibition of cellular replication may be associated with a higher affinity
and binding interaction with the serum albumin [44]. In this setting, the binding profile of the tested
compounds suggests that HSA may act as a drug delivery system, improving the targeting and
decreasing their plasma-free concentration and toxicity.
3. Materials and Methods
3.1. General Information
All the chemicals and reagents used for synthesis were obtained from Alfa Aesar (Karlsruhe, Germany)
and Merck (Darmstadt, Germany), and were used as supplied, without further purification.
Analytical thin-layer chromatography (TLC) carried out on Merck precoated Silica Gel 60F254 sheets
(Darmstadt, Germany) was employed for the monitoring of the reaction progress and confirming the purity
of the newly synthesized compounds, using a mixture of ethyl acetate:n-hexane = 3:1 as the elution system
and UV light (254 nm) for visualization. Melting points were determined with an electrothermal melting
point meter through the open glass capillary method and are presented uncorrected. Spectral analytical
methods (mass spectrometry [MS], infrared spectroscopy [IR], and nuclear magnetic resonance [NMR])
were used to confirm the structures of the synthesized compounds. IR spectra were recorded on a Jasco
FT-IR 6100 spectrometer (Jasco, Easton, MD, USA), using anhydrous potassium bromide for sample
preparation. MS analyses were performed in positive ionization at 70 eV, using an Agilent 1100 series
and an Agilent Ion Trapp SL mass spectrometer (Agilent, Santa Clara, CA, USA). 1H-NMR spectra
were recorded on a Bruker Advance NMR spectrometer (Karlsruhe, Germany), operating at 500 MHz,
using DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal standard. 13C-NMR analyses
were performed on a Bruker Advance NMR spectrometer operating at 125 MHz, in DMSO-d6 and using
a Waltz-16 decoupling scheme, with TMS as the internal standard. Chemical shift (δ) values were reported
in parts per million (ppm). Spin multiplets are given as s (singlet), d (doublet), t (triplet), and m (multiplet).
For the elemental analysis, a Vario El CHNS instrument (Hanau, Germany) was employed.

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3.2. Chemistry
3.2.1. Synthesis of 2-(2-isopropyl-5-methylphenoxy)acetamide 2
A solution of thymol (10 mmol) and potassium hydroxide (KOH) (11 mmol) in dry acetone was
cooled at 0 ◦C in an ice–salt bath. Then, equimolar quantities of 2-iodoacetamide were slowly added,
and the reaction mixture was further stirred at room temperature for 3 h. The obtained white solid
mass was filtered under vacuum, washed thoroughly with a mixture of ethanol:water = 1:3, and dried.
◦
Yield 87%; m.p. 99.5–100.5 C; FT-IR (KBr)
2932 (C-Halif str), 1698 (C=O amide str), 1613 (C=N str), 1257 (C–O–C asym str), 1064 (C–O–C sym str);
1H-NMR (500 MHz, DMSO-d6,
/ppm): 7.19 (s, 2H, –NH), 7.10 (d, J = 7.50, 1H, Ar–H),
6.78 (d, J = 7.50, 1H, Ar–H), 6.66 (s, 1H, Ar–H), 4.38 (s, 2H, O–CH2), 2.50-2.51 (m, 1H, Ar–CH–(CH3)2),
2.19 (s, 3H, Ar–CH3), 1.09 (d, J = 6.84, 6H, Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
/ppm):
ν
cm−1: 3424-3283 (N–H str), 3055 (C-Har str),
max
δ
δ
166.3 (C=O), 155.1 (C), 136.3 (C), 132.6 (C), 126.1 (CH), 122.5 (CH), 113.6 (CH), 65.3 (CH2), 25.9 (CH),
23.3 (2CH3), 21.3 (CH3); MS (ESI) m/z: [M + H]+ 208.1; anal. calculated for C12H17NO2 (%): C, 69.54;
H, 8.27; N, 6.76; found (%): C, 69.77; H, 8.38; N, 6.71.
3.2.2. Synthesis of 2-(2-isopropyl-5-methylphenoxy)ethanethioamide 3
To a solution containing 10 mmol of compound
2 in 30 mL of anhydrous toluene, 12 mmol
of Lawesson’s reagent (LR) were slowly added, under vigorous stirring. The reaction mixture was
further refluxed at 150 ◦C, under continuous stirring, until the TLC (ethyl acetate:n-hexane = 3:1 v/v)
confirmed the completion of the chemical process (3 h). The solid mass was filtered off in order to get
rid of the excessive LR and insoluble impurities, and the filtrate was concentrated under reduced
pressure, using a rotary evaporator. The cooled residue in a mixture of ethanol:diethylether = 1:1 was
filtered under vacuum to yield the desired carbothioamide 3.
Yield 51%; m.p. 125 ◦C; FT-IR (KBr)
ν
max
cm−1
:
3407–3229 (N–H str), 3052 (C-Har str),
2925 (C-Halif str), 1617 (C=N str), 1254 (C-O-C asym str), 1117 (C=S str), 1049 (C–O–C sym str);
1H-NMR (500 MHz, DMSO-d6, δ/ppm): 9.99 (s, 1H, –NH), 9.09 (s, 1H, –NH), 7.10 (d, J = 7.50, 1H, Ar–H),
6.78 (d, J = 7.50, 1H, Ar–H), 6.66 (s, 1H, Ar–H), 4.73 (s, 2H, O–CH2), 2.50-2.51 (m, 1H, Ar–CH–(CH3)2),
2.24 (s, 3H, Ar–CH3), 1.15 (d, J = 6.85, 6H, Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm):
201.2 (C=S), 154.9 (C), 136.3 (C), 132.6 (C), 126.2 (CH), 122.5 (CH), 113.6 (CH), 74.4 (CH2), 25.9 (CH),
23.3 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M + H]+ 224.0; anal. calculated for C12H17NOS (%): C, 64.54;
H, 7.67; N, 6.27; S, 14.36; found (%): C, 64.76; H, 7.60; N, 6.35; S, 14.27.
3.2.3. General Procedure for the Synthesis of 2-((2-isopropyl-5-methylphenoxy)methyl)-4-phenyl
thiazole Derivatives 4a–e
Equimolar quantities (1 mmol) of carbothioamide 3 and the 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 washed with a solution of Na2CO3 until it was free of acid. The pure
compounds were yielded through recrystallization from ethanol.
2-((2-isopropyl-5-methylphenoxy)methyl)-4-phenylthiazole (4a): Yield 76%; m.p. 149–150 ◦C; FT-IR (KBr)
ν
max
cm−1: 3109 (C5–H thiazole str), 3049 (C-Har str), 2920 (C-Halif str), 1615 (C=N str), 1259 (C–O–C asym str),
1046 (C–O–C sym str); 1H-NMR (500 MHz, DMSO-d6,
δ/ppm): 8.15 (s, 1H, thiazole-C5H),
7.98 (dd, J = 7.05; 1.25, 2H, Ar–H), 7.47 (t, 2H, Ar–H), 7.37 (t, 1H, Ar–H), 7.12 (d, J = 7.50, 1H),
6.97 (s, 1H, Ar–H), 6.79 (d, J = 7.50, 1H, Ar–H), 5.49 (s, 2H, O–CH2), 2.50–2.51 (m, 1H, Ar–CH–(CH3)2),
2.28 (s, 3H, Ar–CH3), 1.21 (d, J = 6.85, 6H, Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm):
167.8 (C), 155.0 (C), 154.6 (C), 138.7 (C), 136.5 (C), 133.7 (C), 129.3 (2CH), 128.6 (CH), 126.4 (2CH), 126.3 (CH),
122.4 (CH), 115.2 (CH), 113.6 (CH), 67.5 (CH2), 26.8 (CH), 23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M + H]+

Molecules 2019, 24, 3435
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324.3; anal. calculated for C20H21NOS (%): C, 74.27; H, 6.54; N, 4.33; S, 9.91; found (%): C, 74.40; H, 6.49;
N, 4.26; S, 9.85.
4-(2-((2-isopropyl-5-methylphenoxy)methyl)thiazol-4-yl)benzonitrile(4b): Yield68%; m.p. 126–127◦C;FT-IR(KBr)
ν
cm−1: 3121 (C5-H thiazole str), 3031 (C-Har str), 2921 (C-Halif str), 2221 (C≡N str),
max
1616 (C=N str), 1256 (C–O–C asym str), 1048 (C–O–C sym str); 1H-NMR (500 MHz, DMSO-d6,
δ/ppm):
8.43 (s, 1H, thiazole-C5H), 8.17 (d, J = 8.55 Hz, 2H, Ar–H), 7.93 (d, J = 8.55 Hz, 2H, Ar–H),
7.12 (d, J = 7.50, 1H, Ar–H), 6.95 (s, 1H, Ar–H), 6.79 (d, J = 7.50, 1H, Ar–H), 5.50 (s, 2H, O–CH2),
2.50–2.51 (m, 1H, Ar–CH– (CH3)2), 2.27 (s, 3H, Ar–CH3), 1.21 (d, J = 7.1 Hz, 6H, Ar–CH–(CH3)2);
13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 168.6 (C), 154.9 (C), 152.7 (C), 138.5 (C), 136.6 (C), 133.7 (C),
133.4 (2CH), 127.1 (2CH), 126.3 (CH), 122.5 (CH), 119.3 (C N), 118.6 (CH), 113.5 (CH), 110.8 (C), 67.4 (CH2),
≡
26.8 (CH), 23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M+H]+ 349.3; anal. calculated for C21H20N2OS (%):
C, 72.38; H, 5.79; N, 8.04; S, 9.20; found (%): C, 72.55; H, 5.71; N, 8.10; S, 9.29.
◦
2-((2-isopropyl-5-methylphenoxy)methyl)-4-(4-nitrophenyl)thiazole (4c): Yield 71%; m.p. 155–156 C;
FT-IR (KBr)
1521 (N–O asym str), 1342 (N–O sym str), 1257 (C–O–C asym str), 1062 (C–O–C sym str);
1H-NMR (500 MHz, DMSO-d6,
/ppm): 8.51 (s, 1H, thiazole-C5H), 8.67 (d, J = 8.5 Hz, 2H, Ar–H),
ν
max cm−1: 3119 (C5-H thiazole str), 3042 (C-Har str), 2923 (C-Halif str), 1617 (C=N str),
δ
8.11 (d, J = 8.5 Hz, 2H, Ar–H), 7.12 (d, J = 7.50, 1H, Ar–H), 6.95 (s, 1H, Ar–H), 6.78 (d, J = 7.50, 1H,
Ar–H), 5.51 (s, 2H, O–CH2), 2.50-2.51 (m, 1H, Ar–CH–(CH3)2), 2.27 (s, 3H, Ar–CH3), 1.20 (d, J = 6.50 Hz,
6H, Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 168.6 (C), 154.9 (C), 152.8 (C), 145.2 (C),
138.5 (C), 137.0 (C), 134.0 (C), 133.3 (2CH), 127.3 (2CH), 126.3 (CH), 122.5 (CH), 118.7 (CH), 113.6 (CH),
67.4 (CH2), 26.8 (CH), 23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M+H]+ 369. 2; anal. calculated for
C20H20N2O3S (%): C, 65.20; H, 5.47; N, 7.60; S, 8.70; found (%): C, 65.32; H, 5.54; N, 7.53; S, 8.61.
2-hydroxy-5-(2-((2-isopropyl-5-methylphenoxy)methyl)thiazol-4-yl)benzamide (4d): Yield 77%; m.p. 197–198 ◦C;
FT-IR (KBr)
2920 (C-Halif str), 1671 (C=O str), 1617 (C=N str), 1255 (C-O-C asym str), 1097 (C–O–C sym str);
1H-NMR (500 MHz, DMSO-d6,
/ppm): 10.03 (s, 1H, –OH), 9.63 (s, 1H, –NH2), 8.55 (s, 1H, –NH2),
ν
cm−1: 3415 (O–H str), 3264 (N–H str), 3180 (C5-H thiazole str), 3064 (C-Har str),
max
δ
8.47 (d, J = 1.95 Hz, 1H, Ar–H), 8.02 (dd, J = 8.55, 1.22 Hz, 1H, Ar–H), 7.98 (s, 1H, C5H thiazole),
7.12 (d, J = 7.50, 1H, Ar–H), 6.98 (d, J = 8.55 Hz, 2H, Ar–H), 6.94 (s, 1H, Ar–H), 6.78 (d, J = 7.50, 1H, Ar–H),
5.43 (s, 2H, O-CH2), 2.50–2.51 (m, 1H, Ar–CH–(CH3)2), 2.27 (s, 3H, Ar–CH3), 1.20 (d, J = 7.05 Hz, 6H,
Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 172.2 (C), 167.7 (C), 161.2 (C), 155.0 (C), 154.1 (C),
136.5 (C), 133.7 (C), 132.2 (CH), 126.3 (2CH), 125.5 (C), 122.4 (CH), 118.2 (CH), 115.2 (C), 113.5 (CH),
113.3 (CH), 67.5 (CH2), 26.8 (CH), 23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M+H]+ 383.4; anal. calculated
for C21H22N2O3S (%): C, 65.95; H, 5.80; N, 7.32; S, 8.38; found (%): C, 66.07; H, 5.71; N, 7.29; S, 8.47.
2-((2-isopropyl-5-methylphenoxy)methyl)-4-(p-tolyl)thiazole (4e): Yield 78%; m.p. 194–195 ◦C; FT-IR (KBr)
ν
cm−1: 3117 (C5-H thiazole str), 3071 (C-Har str), 2923 (C-Halif str), 1616 (C=N str),
max
1257 (C–O–C asym str), 1097 (C–O–C sym str); 1H-NMR (500 MHz, DMSO-d6,
8.07 (s, 1H, thiazole-C5H), 7.98 (d, J = 8.5 Hz, 2H, Ar–H), 7.31 (d, J = 8.5 Hz, 2H, Ar–H), 7.13 (d, J = 7.50
δ/ppm):
,
1H, Ar–H), 6.94 (s, 1H, Ar–H), 6.80 (d, J = 7.50, 1H, Ar–H), 5.49 (s, 2H, O–CH2), 2.50–2.51 (m, 1H,
Ar–CH–(CH3)2), 2.37 (s, 3H, Ar–CH3), 2.29 (s, 3H, Ar–CH3), 1.20 (d, J = 6.5 Hz, 6H, Ar–CH–(CH3)2);
13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 168.8 (C), 155.0 (C), 153.1 (C), 138.1 (C), 137.1 (C), 136.8 (C),
131.2 (C), 133.8 (2CH), 127.4 (2CH), 127.1 (CH), 123.1 (CH), 115.9 (CH), 113.8 (CH), 67.4 (CH2), 26.8 (CH),
23.1 (2CH3), 21.4 (CH3), 21.1 (CH3); MS (ESI) m/z: [M + H]+ 338.4; anal. calculated for C21H23NOS (%):
C, 74.74; H, 6.87; N, 4.15; S, 9.50; found (%): C, 74.91; H, 6.72; N, 4.23; S, 9.46.
3.2.4. Synthesis of 2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazine-1-carbo-thioamide
6
To a solution containing thymol (10 mmol) and potassium hydroxide (KOH) (11 mmol) in dry
acetone (10 mL) cooled at 0 ◦C in an ice–salt bath, equimolar quantities of 2-Br-acetophenone (10 mmol

Molecules 2019, 24, 3435
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dissolved in 2 mL of dry acetone) was added dropwise under stirring, over a period of 1h. The reaction
mixture was further refluxed on a steam bath, at 55–60 ◦C, for 8 h, until TLC indicated the completion
of the chemical process. The solvent was removed under reduced pressure, using a rotary evaporator,
thus obtaining the intermediate ketone 5. Then, equimolar quantities of thiosemicarbazide were
added, and the reaction mixture was refluxed in absolute ethanol (15 mL) for 12 h, in the presence
of catalytic amounts of glacial acetic acid (2 drops). After cooling at room temperature overnight,
the resulting precipitate was filtered under vacuum, dried, and recrystallized from ethanol to yield the
pure intermediate 6.
Yield 67%; m.p. 163.5 ◦C; FT-IR (KBr)
1613 (C=N str), 1249 (C–O–C asym str), 1161 (C=S str), 1063 (C–O–C sym str); H-NMR (500 MHz,
ν
max cm−1: 3237, 3154 (N–H str), 3057 (C-Har str), 2922 (C-Halif str),
1
DMSO-d6, δ/ppm): 10.80 (s, 1H, NH), 8.45 (s, 1H, NH2), 8.07 (s, 1H, NH2), 7.93 (d, J = 2 Hz, 1H,
Ar–H), 7.92 (t, 1H, Ar–H), 7.39 (d, J = 2 Hz, 1H, Ar–H), 7.38 (t, 2H, Ar–H), 7.05 (d, J = 7.50, 1H, Ar–H),
6.88 (s, 1H, Ar–H), 6.76 (d, J = 7.50, 1H, Ar–H), 5.30 (s, 2H, O–CH2), 2.50-2.51 (m, 1H, Ar–CH–(CH3)2),
2.28 (s, 3H, Ar–CH3), 1.01 (d, J = 7 Hz, 6H, Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm):
179.6 (C=S), 145.3 (C=N), 136.2 (C), 136.1 (C), 133.7 (C), 129.6 (CH), 128.0 (2CH), 127.6 (CH), 126.1 (2CH),
122.1 (CH), 113.2 (CH), 61.7 (CH2), 26.2 (CH), 22.9 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M + H]+ 342.2;
anal. calculated for C19H23N3OS (%): C, 66.83; H, 6.79; N, 12.31; S, 9.39; found (%): C, 66.98; H, 6.68;
N, 12.27; S, 9.31.
3.2.5. General Procedure for the Synthesis of 2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenyl
ethylidene)hydrazineyl)-4-phenylthiazole Derivatives 7a–e
To a solution of hydrazinyl-1-carbothioamide
6 (1 mmol) dissolved in dry acetone (3 mL),
equimolar quantities of the corresponding -haloketones were added, and the mixture was stirred
α
at room temperature for 6 h. The resulted precipitate was filtered under vacuum and washed with
a solution of Na2CO3 until free of acid. The pure compounds were yielded through recrystallization
from ethanol.
2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)-4-phenylthiazole (7a): Yield 76%;
m.p. 174–175 ◦C; FT-IR (KBr)
ν
cm−1: 3230 (N–H str), 3122 (C5–H thiazole str), 3031 (C-Har str),
max
2927 (C-Halif str), 1630 (C=N str), 1248 (C–O–C asym str), 1068 (C–O–C sym str); 1H-NMR (500 MHz,
DMSO-d6, /ppm): 8.29 (s, 1H, NH), 7.99 (d, J = 6.00 Hz, 2H, Ar–H), 7.81 (d, J = 7.2 Hz, 2H, Ar–H),
δ
7.71 (s, 1H, thiazole-C5H), 7.48 (t, 2H, Ar–H), 7.36 (t, 1H, Ar–H), 7.31 (t, 2H, Ar–H), 7.25 (t, 1H, Ar–H),
7.03 (d, J = 7.50, 1H, Ar–H), 6.93 (s, 1H, Ar–H), 6.77 (d, J = 7.50, 1H, Ar–H), 5.28 (s, 2H, O–CH2),
2.50-2.51 (m, 1H, Ar–CH–(CH3)2), 2.28 (s, 3H, Ar–CH3), 1.01 (d, J = 7.1 Hz, 6H, Ar–CH–(CH3)2);
13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 163.2 (C), 155.0 (C), 146.1 (C=N), 144.0 (C), 137.0 (C),
136.8 (C), 134.2 (C), 133.7 (C), 129.8 (2CH), 129.2 (CH), 128.3 (2CH), 127.0 (2CH), 127.2 (CH), 127.4 (CH),
125.5 (2CH), 123.1 (CH), 113.9 (CH), 110.3 (CH), 65.8 (CH2), 26.3 (CH), 23.1 (2CH3), 21.5 (CH3); MS (ESI)
m/z: [M + H]+ 442.6; anal. calculated for C27H27N3OS (%): C, 73.44; H, 6.16; N, 9.52; S, 7.26; found (%):
C, 73.78; H, 6.09; N, 9.61; S, 7.35.
4-(2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)thiazol-4-yl)benzonitrile (7b): Yield 78%;
◦
m.p. 192–194 C; FT-IR (KBr)
2921 (C-Halif str), 2310 (C
N str), 1607 (C=N str), 1241 (C–O–C asym str), 1078 (C–O–C sym str); 1H-NMR
(500 MHz, DMSO-d6, /ppm): 8.34 (s, 1H, NH), 8.05 (d, J = 8.3 Hz, 2H, Ar–H), 7.87 (d, J = 8.3 Hz
ν
cm−1: 3246 (N-H str), 3130 (C5-H thiazole str), 3052 (C-Har str),
max
≡
δ
,
2H, Ar–H), 7.76 (d, J = 8.3 Hz, 2H, Ar–H), 7.67 (s, 1H, thiazole-C5H), 7.42 (t, 1H, Ar–H), 7.37 (t, 2H,
Ar–H), 7.05 (d, J = 7.50, 1H, Ar–H), 6.92 (s, 1H, Ar–H), 6.75 (d, J = 7.50, 1H, Ar–H), 5.29 (s, 2H, O–CH2),
2.50–2.51 (m, 1H, Ar–CH–(CH3)2), 2.28 (s, 3H, Ar–CH3), 1.00 (d, J = 7.1 Hz, 6H, Ar–CH–(CH3)2); 13C-NMR
(125 MHz, DMSO-d6,
133.6 (C), 133.1 (C), 129.1 (2CH), 128.7 (CH), 128.7 (2CH), 126.7 (2CH), 126.5 (2CH), 126.1 (CH), 121.8 (CH),
119.4 (C
N), 113.1 (CH), 110.0 (CH), 61.2 (CH2), 26.3 (CH), 23.0 (2CH3), 21.5 (CH3); MS (ESI) m/z: [M + H]+
δ/ppm): 162.8 (C), 155.1 (C), 148.2 (C=N), 143.5 (C), 136.6 (C), 136.2 (C), 136.1 (C),
≡

Molecules 2019, 24, 3435
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467.5; anal. calculated for C28H26N4OS (%): C, 72.08; H, 5.62; N, 12.01; S, 6.87; found (%): C, 72.21; H, 5.65;
N, 12.04; S, 6.93.
2-(2-(2-(2-isopro◦pyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)-4-(4-nitrophenyl)thiazole (7c): Yield 75%;
cm−1: 3243 (N–H str), 3100 (C5-H thiazole str), 3042 (C-Har str),
max
m.p. 168–169 C; FT-IR (KBr)
ν
2921 (C-Halif str), 1617 (C=N str), 1510 (N–O asym str), 1341 (N–O sym str), 1242 (C–O–C asym str),
1
1036 (C–O–C sym str); H-NMR (500 MHz, DMSO-d6,
δ/ppm): 8.42 (s, 1H, NH), 8.21 (d, J = 7.5 Hz,
2H, Ar–H), 7.92 (d, J = 7.5 Hz, 2H, Ar–H), 7.58 (d, J = 7.10 Hz, 2H, Ar–H), 7.62 (s, 1H, thiazole-C5H),
7.38 (t, 1H, Ar–H), 7.33 (t, 2H, Ar–H), 7.12 (d, J = 7.50, 1H, Ar–H), 6.94 (s, 1H, Ar–H), 6.77 (d, J = 7.50, 1H,
Ar–H), 5.27 (s, 2H, O–CH2), 2.50-2.51 (m, 1H, Ar–CH–(CH3)2), 2.29 (s, 3H, Ar–CH3), 1.20 (d, J = 7.2 Hz, 6H,
Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 163.7 (C), 156.2 (C), 149.2 (C–NO2), 147.5 (C=N),
143.8 (C), 138.0 (C), 137.1 (C), 136.5 (C), 133.6 (C), 129.2 (CH), 128.5 (2CH), 127.5 (CH), 127.1 (2CH),
126.0 (2CH), 125.6 (2CH), 123.0 (CH), 114.7 (CH), 110.4 (CH), 62.7 (CH2), 26.4 (CH), 22.9 (2CH3), 21.3 (CH3);
MS (ESI) m/z: [M + H]+ 487.4; anal. calculated for C27H26N4O3S (%): C, 66.65; H, 5.39; N, 11.51; S, 6.59;
found (%): C, 66.82; H, 5.27; N, 11.58; S, 6.64.
2-hydroxy-5-(2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)thiazol-4-yl)benzamide
◦
(
7d): Yield 80%; m.p. 197–198 C; FT-IR (KBr)
3123 (C5–H thiazole str), 3042 (C-Har str), 2918 (C-Halif str), 1654 (C=O str), 1616 (C=N str),
1246 (C–O–C asym str), 1091 (C–O–C sym str); 1H-NMR (500 MHz, DMSO-d6,
/ppm):
ν
cm−1: 3474 (O–H str), 3409, 3232 (N–H str),
max
δ
13.30 (s, 1H, –OH), 8.54 (s, 1H, NH), 8.43 (s, 1H, NH2), 7.94 (s, 1H, NH2), 8.47 (d, J = 2.50 Hz, 1H,
Ar–H), 8.02 (dd, J = 8.55, 2.22 Hz, 1H, Ar–H), 7.76 (s, 1H, thiazole–C5H), 7.77 (d, J = 1.5 Hz, 1H, Ar–H),
7.48 (d, J = 1.5 Hz, 1H, Ar–H), 7.41 (t, 1H, Ar–H), 7.37 (t, 2H, Ar–H), 7.12 (d, J = 8.00, 1H, Ar–H),
6.98 (d, J = 8.5 Hz, 2H, Ar–H), 6.95 (s, 1H, Ar–H), 6.79 (d, J = 8.00, 1H, Ar–H), 5.47 (s, 2H, O–CH2),
2.50–2.51 (m, 1H, Ar–CH–(CH3)2), 2.27 (s, 3H, Ar–CH3), 1.20 (d, J = 6.85 Hz, 6H, Ar–CH–(CH3)2);
13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 172.2 (C=O), 167.7 (C), 161.2 (C), 155.2 (C), 145.8 (C=N),
141.3 (C), 136.2 (C), 136.6 (C), 133.2 (C), 133.7 (C), 131.7 (CH), 129.1 (CH), 128.7 (2CH), 126.7 (CH),
126.0 (CH), 125.5 (2CH), 121.8 (CH), 117.9 (CH), 115.2 (CH), 113.4 (C), 113.1 (CH), 67.5 (CH2), 26.8 (CH),
23.1 (2CH3), 21.4 (CH3); MS (ESI) m/z: [M + H]+ 501.4; anal. calculated for C28H28N4O3S (%): C, 67.18;
H, 5.64; N, 11.19; S, 6.40; found (%): C, 67.35; H, 5.59; N, 11.25; S, 6.51.
2-(2-(2-(2-isopropyl-5-methylphenoxy)-1-phenylethylidene)hydrazineyl)-4-(p-tolyl)thiazole (7e): Yield 78%;
m.p. 158.5–159.5 ◦C; FT-IR (KBr)
ν
cm−1: 3236 (N–H str), 3144 (C5–H thiazole str), 3030 (C-Har str),
max
2922 (C-Halif str), 1616 (C=N str), 1242 (C–O–C asym str), 1078 (C–O–C sym str); 1H-NMR (500 MHz,
DMSO-d6, /ppm): 8.19 (s, 1H, NH), 7.98 (s, 1H, thiazole–C5H), 7.91 (d, J = 9.00 Hz, 2H, Ar–H),
δ
7.82 (d, J = 7.5 Hz, 2H, Ar–H), 7.45 (d, J = 7.5 Hz, 2H, Ar–H), 7.39 (t, 2H, Ar–H), 7.35 (t, 1H, Ar–H),
7.12 (d, J = 7.50, 1H, Ar–H), 6.93 (s, 1H, Ar–H), 6.81 (d, J = 7.50, 1H, Ar–H), 5.35 (s, 2H, O–CH2),
2.50–2.51 (m, 1H, Ar–CH–(CH3)2), 2.36 (s, 3H, Ar–CH3), 2.29 (s, 3H, Ar–CH3), 1.20 (d, J = 7.1 Hz, 6H,
Ar–CH–(CH3)2); 13C-NMR (125 MHz, DMSO-d6,
δ/ppm): 163.1 (C), 155.1 (C), 145.5 (C=N), 143.8 (C),
138.5 (C), 137.1 (C), 136.9 (C), 135.0 (C), 133.7 (2CH), 133.6 (C), 129.0 (CH), 128.4 (2CH), 127.5 (2CH),
127.1 (CH), 125.9 (2CH), 123.0 (CH), 114.0 (CH), 110.3 (CH), 67.1 (CH2), 26.7 (CH), 23.0 (2CH3),
21.4 (CH3), 21.1 (CH3); MS (ESI) m/z: [M + H]+ 456.5; anal. calculated for C28H29N3OS (%): C, 73.81;
H, 6.42; N, 9.22; S, 7.04; found (%): C, 73.65; H, 6.35; N, 9.31; S, 7.12.
3.3. Anti-Candida Activity Assay
The in vitro anti-Candida screening was done according to the guidelines of the Clinical Laboratory
Standards Institute (CLSI) [45]. The broth microdilution method was employed for the determination
of MIC and MFC, as previously reported [18]. All the tested fungal strains were obtained from the Food
Biotechnology Laboratory, Life Sciences Institute, University of Agricultural Sciences and Veterinary
Medicine Cluj-Napoca, Romania.

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3.4. Molecular Docking
The binding affinity of the newly synthesized compounds (4a
–e
,
7a–
e) to the catalytic site of the
target fungal lanosterol C14
using AutoDock 4.2.6 [46].
α-demethylase (CYP51) was evaluated through a molecular docking study,
The docking protocol in terms of search space size, grid spacing, center Cartesian coordinates,
and 2Å cluster analysis was applied as previously reported [47 48], with the exception of the number
,
of generated conformations for each ligand. This parameter was increased to 100 in order to assess
a higher reproducibility of the results obtained from the molecular docking study [18].
The inhibition constant (Ki) values were calculated based on the in silico predicted binding
(
∆
G), using the following formula: Ki = eˆ((
∆
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 using UCSF Chimera 1.10.2 [49].
3.5. ADMET Predictions
A SwissADME web tool [50] was employed for the in silico assignment of drug-likeness
for the newly synthesized compounds.
The pharmacokinetic profile (ADME) and drug-likeness of the tested compounds was assessed
through the analysis of a series of physicochemical properties, according to the Lipinski Rule of Five [23]:
molecular weight (MW), hydrogen bond acceptors (HBA), hydrogen bond donors (HBD), and Moriguchi
LogP (mLogP). Additional molecular property-related filters for oral bioavailability, as topological
polar surface area (tPSA) and the number of rotatable bonds (RoB), were also applied [51].
Oral bioavailability in terms of human gastrointestinal absorption and blood–brain barrier (BBB)
permeation of the target compounds were evaluated based on tPSA calculated values and their
predicted ability to act as glycoprotein P (Pgp) substrates. The percentage of gastrointestinal absorption
(%GI Abs) was calculated using the formula: %GI Abs = 109 − (0.345 × tPSA) [27].
The predicted potential for the cross-inhibition of human CYP450 isoenzymes was used to assess
the susceptibility for pharmacokinetic drug–drug interactions of the newly synthesized molecules.
3.6. Protein-Binding Study
BSA fraction V was purchased from Merck (Darmstadt, Germany) and used as supplied, without
further purification. All the other chemicals were of analytical grade purity.
All the fluorescence emission spectra were recorded on a Jasco FP-6500 spectrofluorometer
equipped with a DC-powered 150 W Xenon lamp and a 1.0-cm quartz cell. Spectral analysis was
performed at room temperature under simulated physiological conditions (pH = 7.4) by excitation
of the probes at 289 nm and an emission scan ranging from 300 to 500 nm. Excitation and emission slit
width was fixed at 3 nm.
It is worthwhile to highlight that the BSA fluorescence intensity is reduced by the UV-VIS
absorption of the ligands at the excitation and emission wavelengths [14]. For the tested compounds,
the inner-filter effect of fluorescence was ignored, as they have no absorbance at the above-mentioned
parameters, at the used concentrations.
BSA stock solution was prepared in a Tris-HCl buffer solution (consisted of Tris 0.05 M and adjusted
◦
to pH = 7.4 using HCl 36%) containing NaCl 0.05 M, and was kept in the dark at 4 C. The stock
solutions of the tested compounds (15 mM) were prepared in DMSO and accordingly diluted to obtain
a final concentration ranging from 0.6 to 1.8 µM.
The probes were prepared by mixing a fixed concentration of BSA solution (1.5
µM) with
appropriate amounts of the tested compound 7e in a 10-mL volumetric flask and diluting with Tris
buffer solution to obtain final ligand concentrations of 0.3, 0.6, 0.9, 1.2, 1.5 and 1.8 µM.

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4. Conclusions
Two series of novel 1,3-thiazole derivatives were designed as potential anti-Candida agents and
synthesized through a Hantzsch condensation protocol. The chemical structures were confirmed
by spectral analysis.
In order to evaluate the drug-likeness and pharmacokinetic profile of the newly synthesized
compounds, an in silico ADMET screening was performed. The obtained results support their suitability
for further development as orally active drugs. Additionally, the distribution profile of the most
biologically active compound (7e), which influences the access to the target site in vivo, was investigated
through a serum albumin-binding study, using fluorescence spectroscopy. The experiment showed
a strong binding interaction of the tested compound with the target carrier protein, mediated by the
formation of a ground-state nonfluorescent complex.
The antifungal potential of the novel 1,3-thiazoles, in terms of MIC and MFC, was evaluated
in vitro against three pathogenic Candida strains. The results of the anti-Candida assay pointed to some
structure-dependent differences in the inhibitory activity, which were related to the C2-substitution
of the thiazole ring and para-phenyl substituent properties. Compounds 7a and 7e are the most
promising ones, exhibiting substantially lower MIC values (7.81
µg/mL and 3.9
µg/mL) as compared
with the reference drug fluconazole (15.62 µg/mL).
A molecular docking study was performed toward fungal lanosterol-C14
α
-demethylase (CYP51),
aiming to investigate the molecular mechanism of action of the newly synthesized compounds.
All the tested molecules demonstrated a good binding affinity toward the target enzyme. The induced
enzyme inhibition is not related to the covalent coordination of the heme Fe2+, as in the case of classical
antifungal azoles, but they compete with the physiological substrate of the CYP51 for the access channel
to the active site.
The above-mentioned data represent a helpful support for the rational design and development
of novel antifungal drugs with improved activity and pharmacokinetic properties, and lower toxicity.
Author Contributions: Conceptualization, O.O. and A.-I.P.; investigation, A.I.P., G.M., D.C.V., L.V., A.P., L.I.G.
and A.G.; software, G.M.; Writing—Original draft preparation, A.-I.P.; Writing—Review and editing, I.I. and A.-M.A.;
supervision, O.O. and B.T.; project administration, O.O.; funding acquisition, A.-I.P., I.I., G.M. and A.P.
Funding: This research was funded by “Iuliu Hat, ieganu” University of Medicine and Pharmacy, Cluj-Napoca,
Romania, through doctoral research project number 1531/7/18.01.2019, and by the Romanian Ministry of Research
and Innovation, through the Core Program, PN 19 35 02 01.
Acknowledgments: We gratefully thank Nanostructured Materials and Bio-Nano-Interfaces Centre, Institute for
Interdisciplinary Research on Bio-Nano-Sciences, Treboniu Laurian 42, RO–400271 Cluj-Napoca, Romania,
and Monica Focsan for their help with the fluorescence spectroscopy analysis.
,
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4a–e and 7a–e are available from the authors.
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