Synthesis and evaluation of new 1,3,4-thiadiazole derivatives as potent antifungal agents

Article abstract of DOI:10.3390/molecules23123129

With the goal of obtaining a novel bioactive compound with significant antifungal activity, a series of 1,3,4-thiadiazole derivatives (3a–3l) were synthesized and characterized. Due to thione-thiol tautomerism in the intermediate compound 2, type of subst

Full text of DOI:10.3390/molecules23123129

molecules
Article
Synthesis and Evaluation of New 1,3,4-Thiadiazole
Derivatives as Potent Antifungal Agents
Ahmet Çag˘rı Karaburun 1, Ulviye Acar Çevik 1,2,*, Derya Osmaniye 1,2
,
Begüm Nurpelin Sag˘lık 1,2, Betül Kaya Çavus¸og˘lu 1, Serkan Levent 1,2 , Yusuf Özkay 1,2
,
Ali Savas¸ Koparal 3, Mustafa Behçet 4 and Zafer Asım Kaplancıklı 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eskis¸ehir 26470,
Turkey; ackarabu@anadolu.edu.tr (A.C.K.); dosmaniye@anadolu.edu.tr (D.O.);
bnsaglik@anadolu.edu.tr (B.N.S.); betulkaya@anadolu.edu.tr (B.K.Ç.); serkanlevent@anadolu.edu.tr (S.L.);
yozkay@anadolu.edu.tr (Y.Ö.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
Eskis¸ehir 26470, Turkey
2
3
4
Open Education Faculty, Anadolu University, Eskis¸ehir 26470, Turkey; askopara@anadolu.edu.tr
˙
Department of Medical Microbiology, Faculty of Medicine, Abant Izzet Baysal University,
Bolu 14280, Turkey; mustafabehcet@ibu.edu.tr
*
Correspondence: uacar@anadolu.edu.tr; Tel.: +90-222-335-0580/3774
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 5 November 2018; Accepted: 27 November 2018; Published: 29 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: With the goal of obtaining a novel bioactive compound with significant antifungal
activity, a series of 1,3,4-thiadiazole derivatives (3a 3l) were synthesized and characterized. Due to
thione-thiol tautomerism in the intermediate compound , type of substitution reaction in the final
–
2
step was determined by two-dimensional (2D) NMR. In vitro antifungal activity of the synthesized
compounds was evaluated against eight Candida species. The active compounds 3k and 3l displayed
very notable antifungal effects. The probable mechanisms of action of active compounds were
investigated using an ergosterol quantification assay. Docking studies on 14-α-sterol demethylase
enzyme were also performed to investigate the inhibition potency of compounds on ergosterol
biosynthesis. Theoretical absorption, distribution, metabolism, and excretion (ADME) predictions
were calculated to seek their drug likeness of final compounds. The results of the antifungal
activity test, ergosterol biosynthesis assay, docking study, and ADME predictions indicated that
the synthesized compounds are potential antifungal agents, which inhibit ergosterol biosynthesis
probably interacting with the fungal 14-α-sterol demethylase.
Keywords: 1,3,4-thiadiazole; thione-thiol tautomerism; docking; antifungal; 14-α-sterol demethylase;
ADME
1. Introduction
Invasive fungal infections due to Candida species are being increasingly shown as a main reason
of morbidity and mortality in the healthcare environment [ ]. These infections typically occur due to
1,2
the effect of five most general pathogens C. glabrata, C. albicans, C. krusei, C. parapsilosis, and C. tropicalis.
Each of these organisms has unique virulence potency, antifungal susceptibility and epidemiology;
however, when considered as a whole, important organism-related infections are often referred to as
invasive candidiasis. The high prevalence of antifungal resistance of pathogenic Candida species to
existing drugs reduces the effectiveness of treatment; thus, the development of alternative antifungal
agents is required [3,4].
Due to the low incidence of fungal infections, the rate of development of new antifungal agents is
lower than that of antibacterial agents. In addition, fungi are eukaryotic (as are their human hosts)
Molecules 2018, 23, 3129; doi:10.3390/molecules23123129
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3129
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and, thus, it is difficult to investigate antifungal targets. However, detailed information on the nature
of fungal cells contributed to the understanding of the mechanisms of action of many antifungal
agents in addition to a variety of fungal infections [5,6]. Efforts are expected to reduce toxicity,
increase bioavailability, improve the antifungal spectrum and combat resistance, and enhance the
efficacy of existing antifungals. In fact, elucidation of the mechanism of action of a potential antifungal
compound can reduce the time period from lead compound to drug candidate agent [7].
Currently, the available antifungal agents to treat Candida infections can be divided into
four categories based on their mode of action, including azoles, polyenes, echinocandins,
and antimetabolites [8,9]. Among these agents, azole groups are most widely used in antifungal
therapy [10].
The 1,3,4-thiadiazole compound is a five-membered heterocyclic scaffold including diverse
physicochemical properties. It is a mesoionic system associated with the discrete regions of
positive and negative charges leading to
σ and π electrons and highly polarizable derivatives [11].
This distinguishing feature allows mesoionic compounds to effectively cross cellular membranes
and interact with biological molecules in unique ways, which highlights the high potential of this
ring system in medicinal chemistry [12]. Furthermore, it was reported that the toxophoric –N–C–S
moiety of the 1,3,4-thiadiazoles is probably responsible for a broad range of biological activity [13].
For instance, 1,3,4-thiadiazole derivatives were extensively used as pesticides with a wide range
of biocidal properties, among which antifungal activities attracted particular attention as part of a
comprehensive project for the development of agricultural fungicides [14–18]. The good liposolubility
of the sulfur atom in the thiadiazole compounds might also have a positive effect on the biological
activity [19–21].
Thiadiazole is a bioisoster of triazole and imidazole, which are members of the azole group
with antifungal importance (Figure 1). Imidazole- and triazole-based synthetic antifungal drugs
prevent the conversion of lanosterol to ergosterol and inhibit the enzyme cytochrome P450
14-α-demethylase enzyme [13,22–27]. Based on the bioisosterism and antifungal potency of azole-type
compounds, researchers reported several studies including thiadiazole derivatives and their antifungal
activity [28–30]. From this point of view, in our previous paper, we reported the synthesis and
antifungal activity evaluation of some oxadiazole–thiadiazole derivatives, which target the P450
14-α-demethylase enzyme [28]. Results of our study encouraged us to synthesize more compounds
including thiadiazole nuclei. Thus, we prepared a new series of thiadiazole derivatives, and tested
their antifungal activities with in vitro and in silico studies.

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Figure 1. Chemical structures of targeted compounds and some antifungal drugs which are in clinical use.
2. Results and Discussion
2.1. Chemistry
The target compounds (3a
–
3l) were prepared via three steps, starting from 1-chloro-4-
isothiocyanatobenzene. In the first step, in an ice bath, 4-chlorophenyl isothiocyanate reacted
with hydrazine hydrate to give N-(4-chlorophenyl)hydrazinecarbothioamide ( ). Then, compound
reacted with CS2 under alkaline conditions, such as NaOH. The synthesized compound
(5-((4-chlorophenyl)amino)-1,3,4-thiadiazole-2-thiol) was then converted to the target compounds
3l upon reacting with 2-bromoacetophenone derivatives in acetone. The synthetic method
employed to synthesize these compounds is given in Scheme 1.
Compounds 3a
3l were confirmed by infrared (IR), 1H-NMR, 13C-NMR, and LC–MS
1
1
2
3a
–
–
(see Supplementary Materials). The IR spectra of compounds 3a–3l showed N–H absorption
bands in the range of 3244 to 3265 cm−1. The stretching bands for C=O were observed between
1637 and 1689 cm−1 as expected. The stretching bands for 1,4-disubstituted benzene were determined
at 810–852 cm−1
For the 1H-NMR spectra of compounds 3a
.
–
3l, the signal of the N–H proton was seen in the region
of 10.51–10.55 ppm as a singlet. The signal of S–CH2 protons was observed at 4.69–5.01 ppm as a
singlet peak. The hydrogens of two phenyl rings and other aliphatic hydrogens were observed in the
expected areas.
The 13C-NMR spectra exhibited different types of signals at various ppm which were assigned
for the different types of carbons of all the target compounds. All compounds (3a–3l) gave rise to the
molecular-ion peak [M + H]+ in the LC–MS-8040 tandem mass system, which confirmed the calculated
mass data.
In addition to structure elucidations using routine spectroscopic methods, two-dimensional (2D)
NMR heteronuclear multiple bond correlation (HMBC) analysis was also performed for a selected final
compound (3l). As seen in Scheme 1, compound
2 has two tautomeric forms. Thus, it can be suspected
whether the substitution reaction in the final step is N-substitution or S-substitution. In fact it is known
that –SH proton is more acidic than –NH proton and, therefore, the –SH group is more susceptible

Molecules 2018, 23, 3129
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to nucleophilic substitution than the –NH group in alkaline conditions. Nevertheless, we carried out
the 2D NMR analysis of compound 3l to determine whether the substitution pathway was a sulfur
atom or a nitrogen atom. At first glance, in the 13 C-NMR spectrum of compound 3l, 165-ppm and
194-ppm peaks may be considered as belonging to carbonyl and thione groups, respectively. In this
case, the presence of the thione group indicates that the reaction occurred as N-alkylation. However,
in the HMBC plot (Figure 2) there is an interaction between the carbon peak at 194 ppm and the
hydrogen peak at 7.9 ppm, belonging to the sixth position of 2,4-dichlorobenzene. Furthermore, it is
clearly observed that hydrogens of methylene (–CH2) at 4.8 ppm correlate with the carbons at 194 ppm
and 152 ppm, and do not show any interaction with the carbon at 165 ppm. Therefore, it can easily be
suggested that the peak at 194 ppm belongs to the carbonyl group instead of the thione group, and the
peaks at 152 ppm and 165 ppm belong to carbons at the second and fifth position of the thiadiazole
ring. As a result, HMBC analysis reveals that the reaction in the final step occurs as an S-substitution
as expected.
Compound
R1
R2
3a
3b
3c
3d
3e
3f
3g
3h
3i
–H
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–F
–H
–CH3
–OCH3
–CN
–NO2
–F
–Cl
–Br
–Phenyl
–CH3
–F
3j
3k
3l
–Cl
–Cl
Scheme 1. The synthetic route of the compounds (3a–3l).

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Figure 2. HMBC plot of compound 3l.
2.2. Biological Activity Screening
2.2.1. Antifungal Activity
The in vitro antifungal activity results of the title compounds against C. albicans (ATCC 90028),
C. crusei (ATCC 6258), C. parapsilosis (ATCC 22019), C. tropicalis (ATCC 13803), C. albicans (ATCC 10231),
˙
C. glabrata (ATCC 2001), C. famata (Abant Izzet Baysal University Medical Faculty Hospital, clinical
˙
isolate), and C. lusitaniae (Abant Izzet Baysal University Medical Faculty Hospital, clinical isolate) are
listed in Table 1, where fluconazole was used as a control.
Compounds 3e, 3i, and 3j were the least active among this series as antifungal agents against
all tested fungus strains. Compound 3a exhibited same activity when compared with the standard
drug fluconazole against four tested fungus strains. Compounds 3f and 3g displayed very similar
antifungal activity against the same Candida strains. The compounds 3b, 3c, 3d, and 3h also exhibited
similar anticandidal activity to fluconazole against most of the tested fungus strains.
Compound 3l bearing 2,4-dichlorophenyl was the most active compound in the series against
C. albicans ATCC 10231 with a minimum inhibitory concentration (MIC) value of 5
compound 3l had significant antifungal activity against C. crusei ATCC 6258, C. glabrata ATCC 2001,
and C. famata with an MIC value of 10 g/mL. Compound 3k bearing 2,4-difluorophenyl also possessed
good activity against C. albicans ATCC 10231, C. crusei ATCC 6528, and C. famata with an MIC value of
10 g/mL. Compounds 3k and 3l also exhibited comparable antifungal activity to the standard drug
µg/mL. Moreover,
µ
µ
fluconazole against the other Candida strains. However, compounds 3f and 3g with chloro and fluoro
substituents at the fourth position of the phenyl ring showed lower activity than compounds 3k and
3l. These findings indicated that the antifungal activity of the compounds is enhanced when halogens
such as chloro or fluoro are added to the second position of the phenyl ring.

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Table 1. Minimum inhibitory concentration (MIC50; µg/mL) values of compounds on eight strains of
Candida (3a–3l).
Comp.
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
40
40
40
80
80
40
20
20
40
40
10
10
10
40
20
20
20
80
20
20
20
40
80
10
10
20
20
20
40
20
80
20
20
10
40
20
10
10
10
20
20
40
20
80
20
20
20
40
20
20
20
40
80
20
20
20
40
40
10
5
20
20
20
20
80
20
20
20
40
40
20
10
20
20
20
40
80
80
20
20
40
40
40
10
10
20
80
10
20
40
80
20
20
20
40
40
10
10
10
3j
3k
3l
>160
20
20
Fluconazole
20
20
1. Candida albicans ATCC 90028, 2. Candida crusei ATCC 6258,
ATCC 13803, 5. Candida albicans ATCC 10231, 6. Candida glabrata ATCC 2001, 7. Candida famata (Abant Izzet Baysal
3. Candida parapsilosis ATCC 22019, 4. Candida tropicalis
˙
˙
University Medical Faculty Hospital, clinic isolate),
Faculty Hospital, clinic isolate).
8. Candida lusitaniae (Abant Izzet Baysal University Medical
2.2.2. Quantification of Ergosterol Level
Ergosterol, a major sterol of the fungal cell membrane, is essential for membrane integrity
and fluidity, nutrient transport, and chitin synthesis. Furthermore, trace amounts of ergosterol
are required as a “sparking” function to allow progression through the cell cycle. The difference in
composition of sterols between mammalian cells, which primarily feature cholesterol, and fungal cells,
primarily ergosterol, is what allows for many of the popular antifungal treatments in use today to be
effective [31,32].
The present study was undertaken to investigate the probable mechanism of action of the
antifungal activity of newly synthesized thiadiazole compounds 3k and 3l. It is known that current
therapies of fungal infections mostly target the ergosterol biosynthesis pathway. The reason for this
medicinal strategy is the significance of ergosterol, which causes end-of-life functions as a result of the
inhibition of its biosynthesis.
For quantitative determination of the ergosterol level of C. albicans ATCC 10231, we used an
LC–MS/MS method. Total intracellular sterols were extracted as reported by Breivik and Owades [33].
Ergosterol standard (Product No: 45480, Sigma-Aldrich, Steinheim, Germany) was used to quantify
the ergosterol level in both inhibitor-free (negative control) and inhibitor-including samples. The most
active compounds 3k and 3l and the reference drug were tested in the concentration range of 2.5
µg/mL
to 40 g/mL. The level of ergosterol in the negative control was considered as 100%. Both compounds
µ
were analyzed four times for all concentrations. Then, the results were presented as means
deviation (SD) (Table 2).
±
standard
Table 2. Percentage ergosterol level of C. albicans (ATCC 10231) after treatment with compounds 3k
and 3l and fluconazole.
Compound
2.5 µg/mL
5 µg/mL
10 µg/mL
20 µg/mL
40 µg/mL
3k
3l
45.38 ± 1.75
39.21 ± 1.24
77.82 ± 1.43
38.17 ± 1.21
33.24 ± 1.18
58.02 ± 1.12
36.24 ± 1.17
26.55 ± 0.97
44.82 ± 1.24
28.33 ± 1.12
21.74 ± 0.82
30.65 ± 1.11
20.97 ± 0.88
17.29 ± 0.64
18.27 ± 0.72
Fluconazole
The studies to determine ergosterol level indicated that compounds 3k and 3l and the reference
agent possess an important effect on ergosterol level. Thus, the results show that the level of ergestrol

Molecules 2018, 23, 3129
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decreases with increasing compound concentration. As a result, it can be obviously suggested that
compounds 3k and 3l may have a role in the biosynthesis of ergosterol.
2.2.3. Molecular Docking Studies
Compounds 3k and 3l were found as the most potent antifungal derivatives in the series, and an
in vitro ergosterol quantification assay was carried out for them. In order to evaluate the in silico
features of these compounds, docking studies were performed on the 14-
which is a key enzyme for ergosterol biosynthesis in fungi.
α-sterol demethylase enzyme,
It can be understood by looking at the anticandidal activity results that compounds 3k and 3l
were the most active derivatives, especially against C. albicans, with MIC50 values of 5 and 10 g/mL.
µ
Therefore, in silico docking studies were carried out using the X-ray crystal structure originating from
C. albicans (Protein Data Bank identifier (PDB ID): 5FSA) [34], which was obtained from the Protein
Data Bank server (www.pdb.org).
Figure 3 presents the superimposition of all compounds in the active region of 14-α-sterol
demethylase. It was determined that compounds 3k and 3l settle down in the active region in
different conformations, which may be the reason for more interactions than other compounds.
Figure 3. The superimposition of all compounds (3a–3l) in the active region of 14-α-sterol demethylase
(Protein Data Bank (PDB) code: 5FSA). The heme molecule is colored orange, while compounds 3k
and 3l are colored purple and pink, respectively.
The docking poses of compounds 3k and 3l are presented in Figures 4–7. It can be seen that there
are two common interactions for these compounds. The first of them is related to the thiadiazole
ring. The nitrogen atom at the third position of the thiadiazole ring forms a hydrogen bond with
the hydroxyl group of Tyr132 for compound 3k, whereas this hydrogen bond is observed with the
hydroxyl group of Tyr118 for compound 3l. The other common interaction is created between the
amino group in the structure and the hydroxyl group of Tyr118 for compound 3k. This interaction is
observed with the carbonyl of Met508 in compound 3l.

Molecules 2018, 23, 3129
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Figure 4. Active compound 3k in the active region of 14-α-sterol demethylase (PDB code: 5FSA).
The inhibitor is colored purple and the heme molecule is colored orange.
Figure 5. The interacting mode of compound 3k in the active region of 14-α-sterol demethylase
(PDB code: 5FSA). The inhibitor is colored purple, and the heme molecule is colored orange.

Molecules 2018, 23, 3129
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Figure 6. Active compound 3l in the active region of 14-α-sterol demethylase (PDB code: 5FSA).
The inhibitor is colored pink and the heme molecule is colored orange.
Figure 7. The interacting mode of compound 3l in the active region of 14-α-sterol demethylase
(PDB code: 5FSA). The inhibitor is colored pink and the heme molecule is colored orange.
The main structural difference of these compounds is the presence of halogen atoms at the C2
and C4 positions of the phenyl ring. It can be seen that this disubstituted phenyl ring is in a π–π
interaction with the heme molecule. While compound 3k carries fluorine atoms at the C2 and C4
positions, compound 3l has chlorine atoms. It can be suggested based on anticandidal activity and
molecular docking studies that the presence of halogen atoms, especially at the C2 position of the

Molecules 2018, 23, 3129
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phenyl ring, strengthens the antifungal activity and ergosterol biosynthesis. As can be seen from the
docking poses (Figures 5 and 7), the ortho fluorine and chlorine atoms establish a halogen bond with
the hydrogen of the amino group of Gly308 in compounds 3k and 3l, respectively.
Furthermore, by looking at Figure 7, it can be seen that compound 3l has two additional
interactions compared to compound 3k
para-chlorophenyl ring next to the amino group in the structure. The para-chlorophenyl ring is
in interaction with the imidazole ring of Hid377 via a interaction. The other interaction is related
. These additional two interactions are formed by the
π–π
to the chlorine atom at the C4 position of the phenyl ring. The chlorine atom creates a halogen bond
with the hydrogen of the hydroxyl group of Tyr64. It was theorized that compound 3l can show these
additional interactions compared to compound 3k owing to its different conformation caused by the
volume of chlorine atoms at the C2 and C4 positions of the phenyl ring.
2.2.4. Theoretical Determination of ADME Properties
ADME refers to the absorption, distribution, metabolism, and excretion of a molecule in an
organism. All these characteristics are very important for any drug. Having favorable ADME
characteristics is one of the most daunting hurdles for drug development. Thus, early optimization is
very essential in this process. A waste of time and money in the later stages of drug development can be
prevented thanks to early optimization. The identification and elimination of unfavorable compounds
makes the research process more cost-effective and efficient [35]. For this reason, the prediction of
the pharmacokinetic properties of new drug candidates as early as possible in the drug development
process is very important.
ADME parameters of synthesized compounds (3a–3l) were calculated with the help of QikProp 4.8
software [36]. QikProp also allows providing acceptable ranges for comparing the predicted
properties of compounds with those of 95% of known drugs, allowing the estimation of drug-likeness
properties. The drug-likeness of a compounds was assessed according to Lipinski’s Rule of Five [37],
which considers molecular weight (<500 Da), number of hydrogen-bond acceptors (
5), and octanol/water partition coefficient ( 5), and Jorgensen’s rule of three [38], which regards
logS (> 5.7), PCaco (>22 nm/s), and primary metabolites (PM) (<7). The violations of these rules are
≤
10) and donors
(
≤
≤
−
essential for the optimization of biologically active compounds and should not be more than 1.
Table 3 presents predicted ADME properties of all compounds, and this table contains the
following parameters: molecular weight (MW), number of rotatable bonds (RB), dipole moment
(DM), molecular volume (MV), number of hydrogen donors (DHB), number of hydrogen acceptors
(AHB), polar surface area (PSA), octanol/water partition coefficient (log P), aqueous solubility
(log S), apparent Caco-2 cell permeability (PCaco), number of likely primer metabolic reactions
(PM), percentage of human oral absorption (%HOA), and the violations of rules of three (VRT) and
five (VRF).
The theoretical calculations of ADME parameters (molecular weight, log P, topological polar
surface area (tPSA), number of hydrogen donors (nON), number of hydrogen acceptors (nOHNH),
and molecular volume (MV)) are presented in Table 3 along with the violations of Lipinski’s and
Jorgensen’s rules. According to these data, all compounds (3a–3l) follow both rules, causing no more
than one violation. Thus, it can be suggested that the synthesized compounds may possess a good
pharmacokinetic profile, increasing their pharmacological importance.
Consequently, according to predictions of the ADME properties, it can be suggested that the final
compounds may have a good pharmacokinetic profile.

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Table 3. Calculated absorption, distribution, metabolism, and excretion (ADME) parameters of compounds 3a–3l.
Compound
MW
RB
DM
MV
DHB
AHB
PSA
LogP
LogS
PCaco
PM
%HOA
VRF
VRT
3a
3b
3c
3d
3e
3f
3g
3h
3i
361.86
375.89
391.89
386.87
406.86
379.85
396.31
440.76
437.96
389.92
397.84
430.75
6
6
7
7
7
6
6
6
7
6
6
6
5.741
6.021
6.627
6.187
8.43
5.193
5.161
5.241
8.053
4.88
1067.2
1126.1
1133.6
1133.9
1149.6
1083.3
1111.3
1120.2
1305.8
1175.2
1072.2
1158.7
1
1
1
1
1
1
1
1
1
1
1
1
4.5
4.5
5.25
6
5.5
4.5
4.5
4.5
4.5
4.5
4.5
4.5
68.51
68.51
4.233
4.537
4.247
3.48
3.538
4.469
4.727
4.804
5.862
4.78
−5.731
−6.293
−5.796
−6.702
−5.987
−6.096
−6.472
−6.587
−7.79
797.651
797.651
794.837
164.955
81.982
797.555
797.588
797.605
742.617
813.51
2
3
3
2
3
2
2
2
2
4
2
2
100
100
100
87.011
81.911
100
100
100
100
100
0
0
0
0
0
0
0
0
1
0
0
1
1
1
1
1
1
1
1
1
1
1
0
1
78.875
94.305
117.18
68.514
68.513
68.513
70.177
66.349
69.187
64.786
3j
3k
3l
−6.611
−5.216
−6.907
5.396
5.548
4.371
5.318
765.672
1112.803
100
100
MW: molecular weight; RB: number of rotatable bonds; DM: computed dipole moment; MV: total solvent-accessible volume; DHB: estimated number of hydrogen-bond donors;
AHB: estimated number of hydrogen-bond acceptors; PSA: van der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms; LogP: predicted octanol/water
partition coefficient; LogS: predicted aqueous solubility; PCaco: predicted apparent Caco-2 cell permeability; PM: number of likely metabolic reactions; %HOA: predicted human oral
absorption percentage; VRF: number of violations of Lipinski’s rule of five (the rules are as follows: MW < 500, logP < 5, DHB
violations of Jorgensen’s rule of three (the rules are as follows: logS > −5.7, PCaco > 22 nm/s, PM < 7).
≤
5, AHB
≤
10, positive PSA value); VRT: number of

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3. Materials and Methods
3.1. Chemistry
All chemicals used in the synthesis studies were obtained from Merck Chemicals (Merck KGaA,
Darmstadt, Germany) or Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, Mo., USA).
The MP90 digital melting point apparatus (Mettler Toledo, OH, USA) was used to determine the
melting points (MPs) of the resulting compounds, which were presented uncorrected. Bruker 300-MHz
and 75-MHz digital Fourier transform (FT) NMR spectrometers (Bruker Bioscience, Billerica, MA, USA)
in dimethyl sulfoxide (DMSO-d6) were used to record the 1H NMR and 13C NMR spectra, respectively.
In the NMR spectra, splitting patterns were determined as follows: s, singlet; d, doublet; t, triplet;
dd, double doublet; m, multiplet. Coupling constants (J) are reported in units of Hertz (Hz).
An IRAffinity-1S Fourier transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan) was used to
record the IR spectra of the compounds. Mass spectra were recorded on an LCMS-8040 tandem mass
spectrometer (Shimadzu, Kyoto, Japan) by means of electrospray ionization (ESI). Silica gel 60 F254
thin-layer chromatography (TLC; Merck KGaA, Darmstadt, Germany) was used to control the purity
of the obtained compounds.
3.1.1. Synthesis of N-(4-Chlorophenyl)hydrazinecarbothioamide (1)
An excess of hydrazine hydrate was added to the mixture of 4-chlorophenyl isothiocyanate
(0.22 mol, 3.73 g) in ethanol (EtOH; 40 mL) and stirred in an ice bath for 2h. After the reaction was
complete, the precipitated product was filtered and washed with cold EtOH. Then, the residue was
dried and recrystallized from EtOH [39].
3.1.2. Synthesis of 5-((4-Chlorophenyl)amino)-1,3,4-thiadiazole-2-thiol (2)
A mixture of N-(4-chlorophenyl)hydrazinecarbothioamide (0.02 mol, 4.03 g) and carbon disulfide
(0.024 mol, 1.44 mL) was added to EtOH (40 mL) containing NaOH (0.024 mol, 0.96 g) and refluxed
for 4 h. The reaction mixture was poured into iced water and acidified by 20% HCl down to pH 4–5,
after completion of the reaction. The precipitated product was filtered and washed with water. Then,
the residue was dried and recrystallized from EtOH [39].
3.1.3. General Procedure for the Synthesis of Target Compounds (3a–3l)
Firstly, 5-((4-chlorophenyl)amino)-1,3,4-thiadiazole-2-thiol (2) (0.83 mmol), appropriate
2-bromoacetophenone (0.83 mmol), and potassium carbonate (0.83 mmol, 0.11 g) as a catalyst were
stirred for 4 h in acetone (20 mL). The acetone was evaporated under reduced pressure after completion
of the reaction. The precipitated product was washed with water in order to remove potassium
carbonate, before being dried and recrystallized from EtOH [39].
2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-phenylethan-1-one
MP = 208–210 ◦C, FTIR (ATR, cm−1): 3265 (N–H), 2953 (C–H), 1689 (C=O), 684, 748. 1H-NMR
(300 MHz, DMSO-d6): = 4.97 (2H, s, –CH2–), 7.37 (2H, d, J = 8.9 Hz, C–H aromatic), 7.58–7.73 (5H,
m, C–H aromatic), 8.05 (2H, d, J = 8.5 Hz, C–H aromatic), 10.52 (1H, s, –NH). 13C-NMR (75 MHz,
(
3a).
Yield:
85%,
δ
DMSO-d6):
δ = 41.81, 119.33, 125.80, 128.96, 129.33, 129.39, 134.29, 135.67, 139.68, 153.14, 164.91, 193.62.
ESI-MS (m/z): [M + H]+: 362.1 (100%).
2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(p-tolyl)ethan-1-one (3b).
Yield:
88%,
1
MP = 200–201 ◦C, FTIR (ATR, cm−1): 3248 (N–H), 2891 (C–H), 1687 (C=O), 835. H-NMR (300 MHz,
DMSO-d6):
2H, d, J = 8.9 Hz, C–H aromatic), 7.94 (2H, d, J = 8.2 Hz, C–H aromatic), 10.51 (1H, s, –NH). 13C-NMR
(75 MHz, DMSO-d6): = 21.69, 41.77, 119.32, 125.79, 129.09, 129.86, 130.53, 133.17, 139.68, 144.83, 164.90,
193.14. ESI-MS (m/z): [M + H]+: 376.1 (100%).
δ = 2.40 (3H, s, –CH3), 4.93 (2H, s, –CH2–), 7.37 (4H, d, J = 8.9 Hz, C–H aromatic), 7.59
(
δ

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2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(4-methoxyphenyl)ethan-1-one (3c). Yield: 82%,
MP = 209–211 ◦C, FTIR (ATR, cm−1): 3263 (N–H), 2966 (C–H), 1681 (C=O), 833. 1H-NMR
(300 MHz, DMSO-d6):
δ = 3.86 (3H, s, –OCH3), 4.90 (2H, s, –CH2–), 7.09 (2H, d, J = 8.9 Hz,
C–H aromatic), 7.38 (2H, d, J = 8.9 Hz, C–H aromatic), 7.59 (2H, d, J = 8.9 Hz, C–H aromatic), 8.02 (2H,
d, J = 8.9 Hz C–H aromatic,), 10.51 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 41.64, 56.13, 114.53,
119.32, 125.79, 128.51, 129.40, 131.39, 139.69, 153.31, 164.07, 164.91, 191.97. ESI-MS (m/z): [M + H]+:
392.1 (100%).
4-(2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)acetyl)benzonitrile
MP = 182–184 ◦C, FTIR (ATR, cm−1): 3244 (N–H), 2906 (C–H), 2229 (C
1H-NMR (300 MHz, DMSO-d6):
= 4.90 (2H, s, –CH2–), 7.36 (2H, d, J = 8.9 Hz, C–H aromatic), 7.58
(2H, d, J = 8.9 Hz, C–H aromatic), 8.06 (2H, d, J = 8.6 Hz, C–H aromatic), 8.18 (2H, d, J = 8.6 Hz, C–H
(
3d).
Yield:
86%,
≡
N), 1681 (C=O), 833.
δ
aromatic), 10.52 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 41.77, 116.06, 118.57, 119.34, 125.84,
129.39, 129.55, 133.34, 138.93, 139.64, 152.69, 165.03, 193.23. ESI-MS (m/z): [M + H]+: 387.1 (100%).
2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(4-nitrophenyl)ethan-1-one (3e). Yield: 81%, MP
◦
1
= 197–199 C, FTIR (ATR, cm−1): 3251 (N–H), 2931 (C–H), 1685 (C=O), 1517, 1344 (NO2), 852. H-NMR
(300 MHz, DMSO-d6):
δ = 5.01 (2H, s, –CH2–), 7.37 (2H, d, J = 8.9 Hz, C–H aromatic), 7.58 (2H, d, J =
8.9 Hz, C–H aromatic), 8.27 (2H, d, J = 8.9 Hz, C–H aromatic), 8.38 (2H, d, J = 8.9 Hz, C–H aromatic),
10.52 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 41.93, 119.34, 124.39, 125.84, 129.39, 130.38,
139.63, 140.40, 150.63, 152.65, 165.04, 193.08. ESI-MS (m/z): [M + H]+: 407.1 (100%).
2-((5-((4-Ch◦lorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(4-fluorophenyl)ethan-1-one (3f). Yield: 79%, MP
= 205–207 C, FTIR (ATR, cm−1): 3251 (N–H), 2899 (C–H), 1676 (C=O), 821. 1H-NMR (300 MHz,
DMSO-d6):
aromatic), 8.14 (2H, dd, J1 = 5.5 Hz, J2 = 8.9 Hz, C–H aromatic), 10.51 (1H, s, –NH). 13C-NMR
(75 MHz, DMSO-d6): = 41.71, 116.39 (J2 = 21.9 Hz), 119.33, 125.81, 129.39, 132.06 (J3 = 9.6 Hz), 132.44
J4 = 2.6 Hz), 139.67, 153.03, 164.95, 165.80 (J1 = 251.1 Hz), 192.30. ESI-MS (m/z): [M + H]+: 380.1
(100%).
δ = 4.95 (2H, s, –CH2–), 7.35–7.44 (4H, m, C–H aromatic), 7.59 (2H, d, J = 8.9 Hz, C–H
δ
(
1-(4-Chlorophenyl)-2-((5-((4-chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one (3g). Yield: 75%,
1
MP = 198–200 ◦C, FTIR (ATR, cm−1): 3246 (N–H), 3037 (C–H), 1676 (C=O), 813. H-NMR (300 MHz,
DMSO-d6):
δ = 4.95 (2H, s, –CH2–), 7.37 (2H, d, J = 8.9 Hz, C–H aromatic), 7.58 (2H, d, J = 8.9 Hz,
C–H aromatic), 7.65 (2H, d, J = 8.6 Hz, C–H aromatic), 8.06 (2H, d, J = 8.6 Hz, C–H aromatic), 10.52
(1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 41.69, 119.33, 125.81, 129.40, 129.45, 130.89, 134.38,
139.19, 139.66, 153.05, 164.97, 192.78. ESI-MS (m/z): [M + H]+: 396.1 (100%).
1-(4-Bromophenyl)-2-((5-((4-chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one (3h). Yield: 84%,
1
MP = 185–187 ◦C, FTIR (ATR, cm−1): 3250 (N–H), 2927 (C–H), 1676 (C=O), 810. H-NMR (300 MHz,
DMSO-d6):
δ = 4.94 (2H, s, –CH2–), 7.37 (2H, d, J = 8.9 Hz, C–H aromatic), 7.58 (2H, d, J = 8.9 Hz,
C–H aromatic), 7.79 (2H, d, J = 8.6 Hz, C–H aromatic), 7.97 (2H, d, J = 8.6 Hz, C–H aromatic), 10.52
(1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
δ = 41.66, 119.33, 125.81, 128.44, 129.40, 130.96, 132.40,
134.70, 139.66, 152.93, 164.96, 193.00. ESI-MS (m/z): [M + H]+: 440.0 (100%).
1-([1,10-Biphenyl]-4-yl)-2-((5-((4-chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)ethan-1-one (3i). Yield: 89%,
MP = 221–223 ◦C, FTIR (ATR, cm−1): 3263 (N–H), 2968 (C–H), 1687 (C=O), 833. 1H-NMR
(300 MHz, DMSO-d6):
δ = 4.99 (2H, s, –CH2–), 7.37 (2H, d, J = 8.9 Hz, C–H aromatic), 7.44–7.47
(
1H, m, C–H aromatic), 7.52 (2H, t, J = 7.5 Hz, C–H aromatic), 7.59 (2H, d, J = 8.9 Hz, C–H aromatic),
7.77 (2H, d, J = 7.0 Hz, C–H aromatic), 7.87 (2H, d, J = 8.5 Hz, C–H aromatic), 8.13 (2H, d, J = 8.5 Hz,
C–H aromatic), 10.53 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
= 41.82, 119.33, 125.81, 127.47,
δ
127.53, 129.02, 129.40, 129.60, 129.73, 134.47, 139.21, 139.68, 145.56, 153.14, 164.94, 193.21. ESI-MS (m/z):
[M + H]+: 438.1 (100%).

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2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(2,4-dimethylphenyl)ethan-1-one (3j). Yield: 77%,
MP = 172–174 ◦C, FTIR (ATR, cm−1): 3259 (N–H), 2914 (C–H), 1683 (C=O), 835. 1H-NMR
(300 MHz, DMSO-d6):
2H, m, C–H aromatic), 7.38 (2H, d, J = 8.9 Hz, C–H aromatic), 7.59 (2H, d, J = 8.9 Hz, C–H aromatic),
7.84 (1H, d, J = 7.7 Hz, C–H aromatic), 10.55 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
= 21.40,
δ = 2.33 (3H, s, –CH3), 2.38 (3H, s, –CH3), 4.82 (2H, s, –CH2–), 7.13–7.18
(
δ
21.41, 43.78, 119.32, 125.76, 126.90, 129.39, 130.25, 132.97, 133.41, 138.73, 139.74, 142.75, 153.21, 164.87,
196.28. ESI-MS (m/z): [M + H]+: 390.1 (100%).
2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(2,4-difluorophenyl)ethan-1-one (3k). Yield: 80%,
1
MP = 203–204 ◦C, FTIR (ATR, cm−1): 3248 (N–H), 2914 (C–H), 1680 (C=O), 829. H-NMR (300 MHz,
DMSO-d6):
aromatic), 7.46–7.54 (1H, m, C–H aromatic), 7.59 (2H, d, J = 8.9 Hz, C–H aromatic), 7.97–8.05 (1H, m,
C–H aromatic), 10.52 (1H, s, –NH). 13C-NMR (75 MHz, DMSO-d6):
= 44.92 (d, J = 7.3 Hz), 105.83
t, J = 26.6 Hz), 113.07 (dd, J1 = 3.0 Hz, J2 = 21.6 Hz), 119.33, 121.22 (dd, J1 = 3.3 Hz, J2 = 12.1 Hz), 125.82,
δ = 4.82 (2H, s, –CH2–), 7.25–7.32 (1H, m, C–H aromatic), 7.37 (2H, d, J = 8.9 Hz, C–H
δ
(
129.39, 133.38 (dd, J1 = 3.5 Hz, J2 = 10.9 Hz), 139.65, 152.83, 162.54 (d, J1 = 13.2 Hz, J2 = 256.4 Hz),
164.91, 165.94 (dd, J1 = 12.9 Hz, J2 = 253.5 Hz), 190.18 (d, J = 4.25 Hz). ESI-MS (m/z): [M + H]+: 398.1
(100%).
2-((5-((4-Chlorophenyl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-(2,4-dichlorophenyl)ethan-1-one (3l). Yield: 78%,
MP = 176–178 ◦C, FTIR (ATR, cm−1): 3248 (N–H), 2914 (C–H), 1660 (C=O), 831. 1H-NMR
(300 MHz, DMSO-d6): δ = 4.80 (2H, s, –CH2–), 7.38 (2H, d, J = 8.9 Hz, C–H aromatic), 7.59 (2H,
d, J = 8.9 Hz, C–H aromatic), 7.62 (1H, dd, J1 = 2.0 Hz, J2 = 8.3 Hz, C–H aromatic) 7.78 (1H, d, J = 2.0
Hz, C–H aromatic), 7.87 (1H, d, J = 8.3 Hz, C–H aromatic), 10.53 (1H, s, –NH). 13C-NMR (75 MHz,
DMSO-d6):
δ = 43.85, 119.35, 125.85, 128.07, 129.42, 130.67, 131.91, 132.19, 135.62, 137.32, 139.64, 152.58,
165.02, 194.77. ESI-MS (m/z): [M + H]+: 430.0 (100%).
3.2. Antifungal Assay
The anticandidal effects of compounds 3a–3l were screened according to the method of EUCAST
determinant (EDef 7.1) [40]. The antifungal assays were performed as previously described [28].
Fluconazole was used as a reference drug. The in vitro growth inhibitory activity of the compounds
was tested against Candida albicans (ATCC 90028), Candida crusei (ATCC 6258), Candida parapsilosis
(ATCC 22019), Candida tropicalis (ATCC 13803), Candida albicans (ATCC 10231), Candida glabrata
˙
(ATCC 2001), Candida famata (Abant Izzet Baysal University Medical Faculty Hospital, clinical isolate),
˙
and Candida lusitaniae (Abant Izzet Baysal University Medical Faculty Hospital, clinical isolate).
Results for MIC values were determined in duplicate for each compound. Fungal strains incubated
overnight at 37 ◦C were maintained in Roswell Park Memorial Institute (RPMI) medium. Adjustment
of the inoculum density to a 0.5 McFarland turbidity standard was performed by means of a
spectrophotometer. The obtained inoculum suspension included 0.5
×
105 to 2.5
×
105 colony-forming
units (cfu)/mL for fungi. The test, in which the two-fold serial dilution technique was applied,
was performed in RPMI at pH 7. The last well on the microplates, which contained only the inoculated
broth, was kept as control, and the last well with no growth of microorganism was recorded to
represent the minimum inhibitory concentration (MIC50) in
Further dilutions of the standard drug and obtained compounds in the RPMI medium were carried
out in the required doses at concentrations of 160–0.312 g/mL. After a 24-h incubation of the final
µg/mL. DMSO was used as a solvent.
µ
plates containing microorganism strains, MIC50 values were determined using a microplate reader at
490 nm. Each experiment was performed twice in the antifungal assay.
3.3. Quantification of Ergosterol Level
Total sterols were extracted from C. albicans ATCC 10231, which was licensed by Breivik and
Owades to the literature [33]. Quantification of ergosterol levels in this extract was performed as
previously described [41]. In order to inoculate, 50 mL of Sabouraud dextrose broth (Difco) including

Molecules 2018, 23, 3129
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2.5, 5, 10, 20, and 40
µg/mL of compounds 3k
,
3l and fluconazole utilized a single C. albicans colony
from an overnight Sabouraud dextrose agar plate culture. The cultures were incubated for 16 h
with shaking at 35 ◦C. The stationary-phase cells were taken by centrifugation at 2700 rpm (Hettich,
Rotina 380 R, Germany) for 5 min and washed once with sterile distilled water. Three milliliters of
25% alcoholic potassium hydroxide solution was added to each pellet and vortex mixed for 1 min.
Cell suspensions were carried to sterile borosilicate glass screw-cap tubes and were incubated in an
◦
85 C water bath for 1 h. After this stage, tubes were allowed to cool to room temperature. Sterols were
then extracted by addition of a mixture of 1 mL of sterile distilled water and 3 mL of chloroform
followed by vigorous vortex mixing for 3 min. The chloroform layer was transferred to a clean
borosilicate glass screw-cap tube and 1 µL of sterol extract was injected into the LC–MS/MS system
(Shimadzu LCMS 8040, Kyoto, Japan). The ergosterol quantity in the negative control samples was
regarded as 100%. All concentrations were analyzed in quadruplicate, and the results were expressed
as means ± standard deviation (SD).
3.4. Molecular Docking Studies
Molecular docking studies were performed using an in silico procedure to define the binding
modes of compounds 3k and 3l in the 14-α-sterol demethylase enzyme active region. X-ray crystal
structures of 14- -sterol demethylase (PDB code: 5FSA) [34] were retrieved from the Protein Data
α
Bank server (www.pdb.org). The docking procedure was applied using the Schrödinger Maestro [42
interface as in previously described by our research group [43].
]
The structure of the ligand was built using the Schrödinger Maestro [42] interface and was then
submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [44].
The ligands were prepared using LigPrep 3.8 [45] to correctly assign the protonation states at pH
7.4 ± 1.0, as well as the atom types. Bond orders were assigned and hydrogen atoms were added to
the structures. The grid generation was formed using the Glide 7.1 [46] program and docking runs
were performed with standard precision docking mode (SP).
3.5. Theoretical Determination of ADME Properties
Physicochemical parameters of obtained compounds (3a
4.8 [36].
–3l) were evaluated using QikProp
4. Conclusions
In this study, we synthesized a series of new 1,3,4-thiadiazole derivatives to evaluate their possible
antifungal properties against different fungal strains. The preliminary results indicated that final
compounds 3a–3l showed antifungal activities to different extents. In the series, compounds 3k and 3l
displayed excellent antifungal activities against all of the tested fungi. In particular, compound 3l was
the most effective derivative against C. albicans ATCC 10231. Compounds 3k and 3l possess significant
activities due to the fluoro and chloro groups at the second position of the phenyl moiety. In addition to
their good antifungal activity, all compounds in the series exhibited a good predicted pharmacokinetics
profile. Furthermore, a primary mechanism-of-action study showed that the antifungal activity of
compounds 3k and 3l was related to the inhibition of ergosterol biosynthesis in C. albicans. Moreover,
in the docking study, significant interactions were observed between compounds 3k and 3l and
14-α-sterol demethylase, which is a key enzyme in ergosterol biosynthesis. Consequently, the present
study introduced new 1,3,4-thiadiazole derivatives as potent antifungal candidates, which may assist
scientists in the synthesis of more active compounds with a similar structure.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Z.A.K and. Y.Ö. conceived and designed the experiments; U.A.Ç. and D.O. performed the
synthesis; S.L. and B.K.Ç. performed and analyzed the spectral data; M.B. performed the microbiological studies;
B.N.S. performed the docking and ADME studies; A.Ç.K. and A.S.K. wrote the paper.

Molecules 2018, 23, 3129
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Funding: This study was financially supported by the Anadolu University Scientific Research Projects
Commission, Project no. 1803S050.
Conflicts of Interest: The authors declare no conflicts of interest.
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