Synthesis and anticandidal activity evaluation of new benzimidazole-thiazole derivatives

Article abstract of DOI:10.3390/molecules22122051

Azole-based antifungal agents constitute one of the important classes of antifungal drugs. Hence, in the present work, 12 new benzimidazole-thiazole derivatives 3a-3l were synthesized to evaluate their anticandidal activity against C. albicans, C. glabrata, C. krusei, and C. parapsilopsis. The structures of the newly synthesized compounds 3a-3l were confirmed by IR, H-NMR, C-NMR, and ESI-MS spectroscopic methods. ADME parameters of synthesized compounds 3a-3l were predicted by an in-slico study and it was determined that all synthesized compounds may have a good pharmacokinetic profile. In the anticandidal activity studies, compounds 3c and 3d were found to be the most active compounds against all Candida species. In addition, cytoxicity studies showed that these compounds are nontoxic with a IC50 value higher than 500 μg/mL. The effect of compounds 3c and 3d on the ergosterol level of C. albicans was determined by an LC-MS-MS method. It was observed that both compounds cause a decrease in the ergosterol level. A molecular docking study including binding modes of 3c to lanosterol 14α-demethylase (CYP51), a key enzyme in ergosterol biosynthesis, was performed to elucidate the mechanism of the antifungal action. The docking studies revealed that there is a strong interaction between CYP51 and the most active compound 3c.

Full text of DOI:10.3390/molecules22122051

molecules
Article
Synthesis and Anticandidal Activity Evaluation
of New Benzimidazole-Thiazole Derivatives
Zafer Asım Kaplancıklı 1,
* ,
ID , Serkan Levent 1,2, Derya Osmaniye 1,2, Begüm Nurpelin Sag˘lık 1,2
Ulviye Acar Çevik 1,2, Betül Kaya Çavus¸og˘lu 1, Yusuf Özkay 1,2 and Sinem Ilgın 3
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu Universty, 26470 Eskisehir, Turkey;
serkanlevent@anadolu.edu.tr (S.L.); dosmaniye@anadolu.edu.tr (D.O.); bnsaglik@anadolu.edu.tr (B.N.S.);
uacar@anadolu.edu.tr (U.A.C.); betulkaya@anadolu.edu.tr (B.K.C.); yozkay@anadolu.edu.tr (Y.O.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu Universty,
26470 Eskisehir, Turkey
2
3
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu Universty, 26470 Eskisehir, Turkey;
silgin@anadolu.edu.tr
*
Correspondence: zakaplan@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 1180)
Received: 31 October 2017; Accepted: 21 November 2017; Published: 23 November 2017
Abstract: Azole-based antifungal agents constitute one of the important classes of antifungal drugs.
Hence, in the present work, 12 new benzimidazole-thiazole derivatives 3a
to evaluate their anticandidal activity against C. albicans, C. glabrata, C. krusei, and C. parapsilopsis.
The structures of the newly synthesized compounds 3a
3l were confirmed by IR, 1H-NMR, 13C-NMR,
and ESI-MS spectroscopic methods. ADME parameters of synthesized compounds 3a 3l were
–3l were synthesized
–
–
predicted by an in-slico study and it was determined that all synthesized compounds may have a
good pharmacokinetic profile. In the anticandidal activity studies, compounds 3c and 3d were found
to be the most active compounds against all Candida species. In addition, cytoxicity studies showed
that these compounds are nontoxic with a IC50 value higher than 500 µg/mL. The effect of compounds
3c and 3d on the ergosterol level of C. albicans was determined by an LC-MS-MS method. It was
observed that both compounds cause a decrease in the ergosterol level. A molecular docking study
including binding modes of 3c to lanosterol 14α-demethylase (CYP51), a key enzyme in ergosterol
biosynthesis, was performed to elucidate the mechanism of the antifungal action. The docking studies
revealed that there is a strong interaction between CYP51 and the most active compound 3c.
Keywords: benzimidazole; thiazole; anticandidal; docking studies; CYP51; cytotoxicity; ergosterol
1. Introduction
Fungal infections represent a serious and presently unresolved health problem, particularly in
developed countries. Fungal infections represent 17% of all intensive care unit infections in Europe and
the statistics for the United States are similar. Treatment, especially of systemic infections, is accompanied
not only by moderate success rates but also by high costs. Furthermore, emerging resistance to
commercially offered antifungals has been reported. Additionally, common non-life threatening by
aggressive superficial infections such as recurrent vulvovaginal candidiasis, cause important restrictions
to patients, resulting in reduced quality of life [1]. Invasive fungal infections and dermatomycoses are
the other type of fungal infections, caused by fungal organisms in people with increased vulnerability
such as burn patients, neonates, organ transplant patients, and cancer patients receiving chemotherapy.
Other risk factors include antibiotic and steroid uses, diabetes, lesions of the dermis and epidermis,
neutropenia, malnutrition, and surgery. In recent years, the frequency and severity of fungal infections
have increased, mainly in patients with impaired immunity. Besides, an increasing number of fungal
cases involved in sepsis is an observed regular trend. Fungal infections in humans can be classified into
Molecules 2017, 22, 2051; doi:10.3390/molecules22122051
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three groups: (a) infections (mycoses); (b) toxic reactions to toxins present in certain fungi; and (c) allergic
reactions to fungal proteins [2,3].
Like their human hosts, fungi are eukaryotic, which limits the number of molecular targets that
can be selectively used for drug development without risk of cross-target toxicity. The current portfolio
of antifungal compounds approved for the treatment of invasive infections comprises only four classes
of antifungal agents active against a limited number of cellular processes. Of perhaps greater concern
is the existence of few discovery programs devoted to the development of new antifungal therapeutics
because of the probability of limited financial return for the pharmaceutical industry [4].
Presently, azoles, polyenes, pyrimidine analogs, and echinocandins constitute the four classes of
antifungal agents used for the treatment of fungal infections [5]. There is also a fifth class, the
allylamines, but compounds of this class are only used for treating superficial dermathophytic
infections. Current antifungals have many disadvantages such as narrow spectra of activity, toxicity,
safety issues, and poor pharmacokinetic properties. In addition, the emergence of strains resistant
to the current antifungal agents has highlighted the urgency of developing new drugs with different
mechanisms of action that target the biosynthesis of fungal proteins, lipids, and cell walls [6–8].
Most antifungal agents, especially the azoles, target the biosynthesis of ergosterol, a main
component of the fungal cell wall. This scarcity of fungus-specific targets is a difficult problem
because of the frequency of cross-resistance to all drugs with a common target. Resistance to all major
current antifungals has been reported in both laboratory and clinic, and continues to be a growing
problem in the medical community [9,10]. The growing emergence of azole-resistant yeast and fungal
strains is related to the prophylactic use of azole drugs, long treatment programs in the clinic, and usage
of agricultural azole fungicides in crop protection. Thus, there is a need for development of new azole
antifungal compounds with augmented selectivity for the fungal sterol 14α-demethylase enzyme
(CYP51), alternative antifungal approaches, and treatment regimens against drug-resistant strains.
Currently, new azole and nonazole CYP51 inhibitors are being developed as the next generation of
antifungal drugs [11
of nucleophilic nitrogen of the azole heterocyclic ring to the sixth ligand of the heme ferric ion,
and interactions of the azole drug side chains with the CYP51 polypeptide structure [13 14]. CYP51 is
,12]. The mechanism of action of azole antifungals involves the direct coordination
,
a member of the cytochrome P450 superfamily and is necessary for ergosterol biosynthesis in fungi
and cholesterol biosynthesis in mammals. Therefore, fungal CYP51 is the main target for therapeutic
azole antifungal agents and agricultural azole fungicides [15]. The similar biosynthesis pathways of
ergosterol and cholesterol have led researchers to develop azole inhibitors that are selective for the
fungal CYP51 enzyme, and are usually used to treat fungal infections caused by Candida albicans and
Aspergillus fumigatus [16,17].
Several triazole-based azole antimycotic agents have been incessantly optimized and presented
in clinical practice [18]. However, increased triazole resistance, reproductive toxicity, and hepatic
toxicity cases are associated with long-term use of triazole compounds. The capability of triazoles
to inhibit CYP-reliant enzymes raises alarms about triazole effects on hormone synthesis and drug
metabolism [19–22]. Therefore, some research group efforts include compound collections of the
more encouraging benzimidazole type for antifungal discovery [1,3, , ,
14 23 24]. For instance, in our
recent study [25], we reported 2-((5-(4-(6-fluoro-1H-benzimidazol-2-yl)phenyl)-4 methyl-4H-1,2,4-
triazol-3-yl)thio)-1-(4-fluorophenyl)ethan-1-one (BT-23, Figure 1), a new benzimidazole-triazole hybrid
compound with a significant MIC50 (0.78 µg/mL) against Candida strains. We also determined that
BT-23 acts as an ergosterol biosynthesis inhibitor at 0.78–3.12 µg/mL concentrations.
In addition to classical activity methods, high throughput screening-compatible assays have been
adapted in the presence of fungal pathogens to classify novel and selective antifungal lead compounds.
In these screenings, a lot of heterocyclic scaffold-based small molecules have been evaluated and several
benzimidazole compounds with potential antifungal activity have been identified [
1,23,26]. A novel
benzimidazole derivative EMC120B12 ((S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole,

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Figure 1), displaying high antifungal activities against the major Candida species, is an example
identified in such screenings [1,26].
In addition to benzimidazoles, new thiazolyl-triazole Schiff bases (TTSBs), which have
MICs close to those of reference agents ketoconazole and fluconazole, have been reported as
potential anticandidal agents [27]. Besides, the newly synthesized thiazolin-4-one derivative,
5-(2,4-dichlorobenzylidene)-2-(naphthalen-1-ylamino)thiazol-4(5H)-one (DCBNAT), has been found as
highly effective (MIC = 0.015 µg/mL) against a series of pathogenic fungi [28] (Figure 1).
Figure 1. Structures of BT-23, EMC120B12, TTSBs and DCBNAT.
As described above, we have reported the anticandidal potential of benzimidazole-triazole
hybrid compounds in our recent work [25]. However, there was no in-silico study, displaying
interactions between potent compound (BT-23) and an enzyme like CYP51. Hence, with the purpose of
developing new lead antifungal agents, we initially designed novel compounds depending on a similar
hybridization strategy as used in our recent work. Benzimidazole and thiazole pharmacophore groups,
which have a significance in terms of anticandidal activity, were combined on the same chemical
structure and then investigated for their anticandidal effect, cytotoxicity and ability to inhibit CYP51.
2. Results and Discussion
2.1. Chemistry
The compounds 3a
yl)benzaldehyde ( ) was prepared by reacting 1H-benzimidazole and 4-fluorobenzaldehyde under
microwave irradiation. Secondly, 4-(1H-benzimidazol-1-yl)benzaldehyde ( ) and hydrazine-carbothioamide
were reacted to obtain 2-(4-(1H-benzimidazol-1-yl)benzylidene)hydrazine-1-carbothioamide ( ). Finally,
reaction of compound and an appropriate 2-bromoacetophenone afforded the target compounds 3a
–3l were synthesized as outlined in Scheme 1. Firstly, 4-(1H-benzimidazol-1-
1
1
2
2
–
3l
.
−1
−1
In the IR spectra (Supplementary Materials Figures S1 and S5), stretching absorptions at 3097–3218 cm
indicated the presence of hydrazone group N-H bonds. The stretching absorption at about 1585–1610 cm
was attributed to C=N double bonds. The stretching absorption belonging to the C–N single bond was
determined at 1105–1141 cm−1. Stretching absorptions for the out of plane bending of a 1,4-disubstituted
1
benzene moiety was observed at 825–846 cm−1. In the H-NMR spectra (Supplementary Materials
Figures S3 and S7), the aromatic protons of benzene, benzimidazole, thiazole and –CH=N– groups were
recorded between 6.97 ppm and 9.27 ppm. Methyl protons in compounds 3b and 3j were recorded
between 2.30 ppm and 2.42 ppm as a singlet. Moreover, the benzimidazole H2 and –CH=N– group protons
were seen as a singlet between 8.13 ppm and 9.27 ppm. However, the proton of the thiazole ring was

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recorded as a multiplet (compounds 3a
,
3c
–
3f
,
3h
–
3k) or a singlet (3b
,
3g
,
3l) at 6.19–7.67 ppm. The N–H
proton on the hydrazide moiety showed a signal over 12 ppm. In the 13C-NMR (Supplementary Materials
Figures S4 and S8), all aromatic carbons gave peaks from 102.16 ppm to 168.75 ppm. In fluorinated
derivatives (compounds 3f, 3k and 3i), carbon fluorine coupling was observed. In the MS spectra
(Supplementary Materials Figures S2 and S6), all masses matched well with the expected [M + H]+ or
[M + 2H]2+ values.
Compounds
R1
R2
3a
3b
3c
3d
3e
3f
3g
3h
3i
–H
–H
–H
–H
–H
–H
–H
–H
–H
–CH3
–F
–H
–CH3
–NO2
–CN
–OCH3
–F
–Cl
–Br
–CF3
–CH3
–F
3j
3k
3l
–Cl
–Cl
Scheme 1. Synthesis of the target compounds 3a–3l.
2.2. Antifungal Activity Assay
The obtained compounds 3a
–3l were evaluated for anticandidal activity against C. albicans
(ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019) and C. glabrata (ATCC 90030)
according to the protocol of the EUCAST [29]. The minimum inhibitory concentrations (MICs) of the
final compounds were recorded by fluorometric measurements [30,31]. Ketoconazole and fluconazole
were used as reference drugs in the activity tests. The antifungal activity results are presented in Table 1.
As regards to the chemical structure of the compounds, the substituents on the phenyl ring
are different. According to the antifungal screening results, the most active compound 3c indicated
similar antifungal activity to ketoconazole and fluconazole against all Candida strains with a MIC50
value of 1.56
exhibited antifungal activity against C. glabrata and C. krusei with a MIC50 value of 1.56
displayed a MIC50 value of 3.12 g/mL against C. albicans and C. parapsilopsis. The only difference
µg/mL. Moreover, compound 3d displayed remarkable activity too. This compound
µg/mL, while it
µ
between these two compounds is the substitution at C-4 of the phenyl ring. Compound 3c bears
a nitro group, whereas compound 3d has a nitrile group at this position. In terms of electronic features,

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both nitro and nitrile substituents act as strong electron withdrawing groups, so it can be assumed that
higher antifungal activity of compounds 3c and 3d than other derivatives is related to the presence of
electron withdrawing groups, which enhances the antifungal activity.
Table 1. MIC50 (µg/mL) values of compounds (3a–3l).
Compounds
C. albicans
C. glabrata
C. krusei
C. parapsilopsis
3a
3b
3c
3d
3e
3f
3g
3h
100
100
1.56
3.12
6.25
6.25
6.25
100
100
50
25
25
100
100
1.56
1.56
100
6.25
6.25
100
100
100
12.5
6.25
1.56
1.56
100
100
1.56
3.12
100
6.25
6.25
100
100
100
6.25
6.25
1.56
0.78
1.56
1.56
100
25
12.5
12.5
100
100
6.25
12.5
1.56
1.56
3i
3j
3k
6.25
6.25
0.78
0.78
3l
Ketoconazole
Fluconazole
2.3. Quantification of Ergosterol Level
Due to the fact both humans and fungi are eukaryotic, there are comparatively limited antifungal
agents able to attack unique fungal targets not shared with human hosts. The fungal cell wall is a
however a significant target for selective antifungal drugs owing to the chitin structure that is lacking
in the human cells [32,33]. Most of the therapies for fungal infections target the ergosterol biosynthesis
pathway, as it is a vital membrane sterol for the normal fungal cell cycle [34].
From this point of view, in the current work newly synthesized benzimidazole-thiazole compounds
3c and 3d were selected to investigate their probable mechanism of action. Thus, a LC-MS-MS-based
method that quantifies the ergosterol level was applied [25]. Compound 3c, ketoconazole, and fluconazole
were used at 0.78 µg/mL, 1.56 µg/mL, and 3.12 µg/mL concentrations. Ergosterol quantity in negative
control samples was regarded as 100%. All concentrations were analyzed in quadruplicate, and the results
were expressed as mean ± standard deviation (SD, Table 2).
Ergosterol quantification studies indicated that compounds 3c, 3d, and the reference agents
significantly reduced the level of ergosterol of C. albicans at all tested concentrations. It was determined
that tested compounds and reference agent caused a concentration dependent decrease in the ergosterol
level. As a result, it can be proposed that compounds 3c and 3d have an impact on the biosynthesis
pathway of ergosterol.
Table 2. Inhibition potency (%) of compounds 3c and 3d on ergosterol biosynthesis of C. albicans.
Concentrations (µg/mL)
Compounds
0.78
1.56
3.12
3c
3d
56.83 ± 2.96
48.25 ± 3.17
60.99 ± 2.94
61.74 ± 1.70
65.81 ± 3.88
58.77 ± 4.03
73.12 ± 4.16
70.12 ± 3.22
79.14 ± 4.29
66.58 ± 3.27
84.56 ± 3.01
82.13 ± 4.45
Ketoconazole
Fluconazole
2.4. Cytotoxicity Test
The toxicity of compounds 3c and 3d was examined using the MTT assay, which is based upon
the reduction of yellow MTT dye by metabolically active eukaryotic and prokaryotic cells to form a

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purple formazan product. The MTT assay was carried out using healthy NIH/3T3 mouse embryonic
fibroblast cell lines (ATCC CRL1658), which are recommended for cytotoxicity screening by ISO
(10993-5, 2009) [35]. The IC50 values of the compounds are presented in Table 3. The IC50 values
of compounds 3c and 3d were recorded above 500 µg/mL. Consequently, it can be stated that the
obtained compounds are nontoxic at their active concentrations against Candida species.
Table 3. Cytotoxic activity of the compounds 3c and 3d against NIH/3T3 Cell Line.
Compounds
IC50 (µg/mL)
R2
3c
3d
>500
>500
0.9754
0.9839
2.5. Prediction of ADME Parameters
Most new drug candidates fail in clinical trials due to their poor absorption, distribution,
metabolism, and excretion (ADME) properties. Late-stage failures cause significant costs in new
drug development. The ability to ientiify problematic issues early can significantly decrease the
amount of lost time and costs, and rationalize the complete development progression. Therefore, the
pharmacokinetic properties of new drug candidates are very vital and should be evaluated as early
as possible in the drug development process [36]. Therefore, predictions of ADME parameters of
synthesized compounds 3a–3l were performed by using the QikProp 4.8 software [37].
This program applies the Lipinski’s rule of five [38] and Jorgensen’s rule of three [39], which evaluate
the ADME properties of drug-like compounds, and are important for the optimization of a biologically
active compound. The theoretical calculations of ADME parameters (molecular weight, log P, polar surface
area (PSA), number of hydrogen donors, number of hydrogen acceptors, number of rotatable bonds and
volume) are presented in Table 4, along with the violations of the rules of three and five.
Table 4. Calculated ADME parameters of compounds 3a–3l.
Compounds
MW
RB
MV
DHB AHB PSA
log P
VRT VRF
3a
3b
3c
3d
3e
3f
3g
3h
3i
395.48
409.51
440.48
420.49
425.51
413.47
429.93
474.38
463.48
423.53
431.46
464.37
4
4
5
5
5
4
4
4
4
4
4
4
5
6
1263.60
1322.54
1346.00
1330.30
1331.38
1279.71
1307.72
1316.63
1360.43
1346.88
1289.37
1316.53
1511.39
883.50
1
1
1
1
1
1
1
1
1
1
1
1
0
1
5.5
5.5
6.5
7
51.98
51.98
100.64
77.77
60.46
51.98
51.98
51.98
51.98
47.09
50.54
48.57
55.75
72.55
5.33
5.63
4.62
4.56
5.37
5.56
5.82
5.90
6.31
5.80
5.72
6.00
4.91
3.55
1
1
0
0
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
6.25
5.5
5.5
5.5
5.5
5.5
5.5
5.5
6.75
6.75
3j
3k
3l
Ketoconazole 530.45
Fluconazole 306.27
MW: Molecular weight; RB: Number of rotatable bonds (recommended value: 0–15); MV: Total solvent-accessible
volume in cubic angstroms using a probe with a 1.4 Å radius (recommended value: 500–2000); DHB: Estimated
number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution
(recommended value: 0–6); AHB: Estimated number of hydrogen bonds that would be accepted by the solute
from water molecules in an aqueous solution (recommended value: 2–20); PSA: Van der Waals surface area of
polar nitrogen and oxygen atoms and carbonyl carbon atoms (recommended value: 7–200); log P: Predicted
octanol/water partition coefficient (recommended value: −2–6.5); VRF: Number of violations of Lipinski’s rule of
five [38]. The rules are: mol_MW < 500, QPlogPo/w < 5, donorHB
these rules are considered druglike. (The “five” refers to the limits, which are multiples of 5.) (maximum is 4); VRT:
Number of violations of Jorgensen’s rule of three [39]. The three rules are: QPlogS > 5.7, QP PCaco > 22 nm/s, #
Primary Metabolites < 7. Compounds with fewer (and preferably no) violations of these rules are more likely to be
orally available (maximum is 3).
≤
5, accptHB
≤
10. Compounds that satisfy
−

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According to Lipinski’s rule of five, all compounds 3a
–3l abide by the rules by causing no more
than one violation. Furthermore, these compounds fulfil Jorgensen’s rule of three with no more than
one violation. Also, it can be seen that all rule of three and five results are within the desired ranges.
Thus, it can be stated that all synthesized compounds may have a good pharmacokinetic profile,
increasing their pharmacological significance.
2.6. Molecular Docking Studies
Docking studies were performed in order to gain more insight into the binding modes of compound
3c to 14-α-sterol demethylase, which is a key enzyme in ergosterol biosynthesis in fungi. Studies were
performed with the X-ray crystal structure of 14-α-sterol demethylase from Mycobacterium tuberculosis
in complex with our reference agent fluconazole (PDB ID: 1EA1) [40].
According to the antifungal activity results, the compound 3c shows significant antifungal
activity against all Candida species with a MIC50 value of 1.56
µg/mL. Thus, the main purpose is
to investigate the possible interaction of this compound with cytochrome P450 14-α-sterol demethylase
from Candida species. However, this enzyme is a membrane-bound enzyme and it is difficult to
crystallize for X-ray analysis and modelling studies. Moreover, there is no experimental data or
crystal structure of this enzyme in Protein Data Bank server. On the other hand, in the database there
are two-analogous enzymes from Candida P450 and Mycobacterium P450. These enzymes have high
homology and a high degree of similarity between the hydrophobic cavities of the catalytic site [41–44].
Among them Mycobacterium P450 has been resolved with higher resolution. For these reasons, we choose
the PDB ID: 1EA1 crystal structure from Mycobacterium tuberculosis to obtain a clearer pose.
In docking studies the crystal structure of 14-α-sterol demethylase, which has been reported in a
complex with our reference agent fluconazole [40], was used to compare the binding modes of 3c and
fluconazole. It has been reported that the HEM molecule has an interaction with the triazole ring of
fluconazole. Two different π-π interactions were also assigned between the 2,4-difluorophenyl moiety
and amino acids of Phe83 and Phe255. In Figure 2, the docking pose of the same enzyme reveals that
the interactions between compound 3c and amino acid residues are very important in terms of binding
to the active site.
Figure 2. The interacting mode of compound 3c in the active region of 14 alpha-sterol demethylase.
The inhibitor is colored with green and HEM with turquoise.

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Compound 3c has some similar interactions to those of fluconazole. The HEM molecule creates
a π–π interaction with the phenyl ring, near the benzimidazole ring. It is seen that there are three
hydrogen bonds. The nitrogen atom of benzimidazole establishes a hydrogen bond with the amino
group of Gly257. The amino of the hydrazone is in an interaction with carbonyl of Thr260. The last
hydrogen bond is formed between the nitro group at the C-4 position of the phenyl ring, and the amino
group of Arg326. These interactions help explain the stronger anticandidal activity of 3c. It can be
suggested that electron withdrawing groups such as nitro at C-4 of the phenyl ring are very important
in terms of binding to the enzyme active site and anticandidal activity. As a result, it is considered that
these interactions could explain the better binding capability and stronger activity of compound 3c
than other compounds in the series.
3. Materials and Methods
3.1. Chemistry
All chemicals were obtained either from Sigma-Aldrich Corp. (St. Louis, MO, USA) or Merck
KGaA (Darmstadt, Germany), and used without further chemical purification. Melting points of
the compounds were measured by using an automatic melting point determination instrument
1
(MP90, Mettler-Toledo, Columbus, OH, USA) and were presented as uncorrected. H- and 13C-NMR
spectra were recorded in DMSO-d6 by a Bruker digital FT-NMR spectrometer (Bruker Bioscience,
Billerica, MA, USA) at 300 MHz and 75 MHz, respectively. The IR spectra of the compounds were
recorded using an IRAffinity-1S Fourier transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan).
MS studies were performed on an LCMS-8040 tandem mass system (Shimadzu, Tokyo, Japan). Chemical
purities of the compounds were checked by classical TLC applications performed on silica gel 60 F254
(Merck KGaA).
3.1.1. Synthesis of 4-(1H-Benzimidazol-1-yl)benzaldehyde (1)
4-Fluorobenzaldehyde (1.607 mL, 0.015 mol), benzimidazole (1.770 g, 0.015 mol), and sodium
hydride (NaH) (0.396 g, 0.016 mol) in DMF (10 mL) were put into a 30 mL microwave synthesis reactor
vial (Monowave 300, Anton-Paar, Graz, Austria). The reaction mixture was kept under the conditions
◦
of 170 C and 10 bar for 25 min. After cooling, the mixture was poured into ice-water, the precipitated
product was washed with water, dried, and recrystallized from ethanol.
3.1.2. Synthesis of 2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazine-1-carbothioamide (2)
A mixture of 4-(1H-benzimidazol-1-yl)benzaldehyde (1, 2.887 g, 0.013 mol) and thiosemicarbazide
(1.183 g, 0.013 mol) were refluxed in EtOH (50 mL) for 3 h. After completion of the reaction the mixture
was cooled in an ice-bath, the precipitated product was filtered, dried, and recrystallized from EtOH.
3.1.3. General Procedure for the Synthesis of the Target Compounds 3a–3l
2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazine-1-carbothioamide (2, 0.3 g, 0.001 mol) and
appropriate 2-bromoacetophenone derivative (0.001 mol) in EtOH (20 mL) were refluxed for 4 h.
The mixture was cooled in an ice-bath, the precipitated product was filtered, dried, and recrystallized
from EtOH.
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-phenylthiazole (3a). Yield: 88%, m.p. = 270–271 ◦C,
FTIR (ATR, cm−1): 1610 (C=N), 1141 (C–N), 738, 696. 1H-NMR (DMSO-d6):
δ = 7.29–7.36 (3H, m,
benzimidazole CH, thiazole CH), 7.42 (2H, t, J = 7.20 Hz, phenyl CH), 7.69–7.72 (1H, m, phenyl CH),
7.77 (2H, d, J = 8.58 Hz, phenyl CH), 7.80–7.82 (1H, m, benzimidazole CH), 7.88 (2H, d, J = 7.17 Hz, phenyl
CH), 7.91 (2H, d, J = 8.73 Hz, phenyl CH), 8.15 (1H, s, –CH=N–), 8.64 (1H, s, benzimidazole CH), 12.32 (1H,
s, –NH). 13C-NMR (DMSO-d6):
δ = 104.32, 111.34, 120.41, 123.15, 124.13, 124.34, 126.00, 128.04, 128.19,

Molecules 2017, 22, 2051
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129.09, 133.30, 134.21, 135.11, 136.75, 140.49, 143.61, 144.15, 151.11, 168.61. ESI-MS (m/z): [M + 2H]2+:
198 (100%).
◦
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(p-tolyl)thiazole (3b). Yield: 83%, m.p. = 266–267 C,
FTIR (ATR, cm−1): 3082 (N–H), 1604 (C=N), 1139 (C–N), 837. 1H-NMR (DMSO-d6):
δ = 2.33 (3H, s, –CH3),
7.22 (2H, d, J = 8.07 Hz, phenyl CH), 7.27 (1H, s, thiazole), 7.33–7.37 (2H, m, benzimidazole CH), 7.68–7.71
(1H, m, benzimidazole CH), 7.75–7.78 (4H, m, phenyl CH), 7.81–7.85 (1H, m, benzimidazole CH), 7.91 (2H,
d, J = 8.61 Hz, phenyl CH), 8.14 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole CH), 12.28 (1H, s, –NH).
13C-NMR (DMSO-d6):
δ = 21.27, 103.39, 111.30, 120.50, 123.08, 124.07, 124.30, 125.95, 128.17, 129.65, 132.49,
133.34, 134.19, 136.78, 137.30, 140.41, 143.63, 144.37, 151.16, 168.51. ESI-MS (m/z): [M + 2H]2+: 205 (100%).
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4-nitrophenyl)thiazole (3c). Yield: 89%, m.p. =
1
274–275 ◦C, FTIR (ATR, cm−1): 3163 (N–H), 1598 (C=N), 1105 (C–N), 846. H-NMR (DMSO-d6):
δ =
7.49–7.53 (2H, m, benzimidazole CH), 7.76–7.80 (2H, m, benzimidazole CH, thiazole CH), 7.84 (2H,
d, J = 8.52 Hz, phenyl CH), 7.88–7.91 (1H, m, benzimidazole CH), 7.96 (2H, d, J = 8.55 Hz, phenyl),
8.12 (2H, d, J = 8.91 Hz, phenyl CH), 8.19 (1H, s, –CH=N–), 8.28 (2H, d, J = 8.97 Hz, phenyl CH),
9.27 (1H, s, benzimidazole CH), 12.48 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ = 109.30, 112.38, 118.30,
124.15, 124.57, 125.08, 125.60, 126.82, 128.26, 129.21, 132.41, 135.15, 135.60, 140.77, 141.04, 143.02, 146.69,
149.06, 168.97. ESI-MS (m/z): [M + H]+: 441 (100%).
4-(2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)thiazol-4-yl)benzonitrile (3d). Yield: 85%, m.p.
1
= 283–284 ◦C, FTIR (ATR, cm−1): 3196 (N–H), 1610 (C=N), 1139 (C–N), 837. H-NMR (DMSO-d6):
δ
= 7.33–7.38 (2H, m, benzimidazole CH), 7.67 (1H, s, thiazole CH), 7.68–7.71 (1H, m, benzimidazole
CH), 7.77 (2H, d, J = 8.58 Hz, phenyl CH), 7.79–7.83 (1H, m, benzimidazole CH), 7.88 (2H, d, J = 8.52
Hz, phenyl CH), 7.91 (2H, d, J = 8.64 Hz, phenyl CH), 8.06 (2H, d, J = 8.43 Hz, phenyl CH), 8.17 (1H, s,
–CH=N–), 8.62 (1H, s, benzimidazole CH), 12.39 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ = 108.20, 110.10,
111.30, 119.45, 120.50, 123.09, 124.08, 124.28, 125.15, 126.59, 128.27, 133.16, 134.02, 136.90, 139.20, 140.96,
143.62, 144.35, 149.41, 168.95. ESI-MS (m/z): [M + H]+: 421 (100%).
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4-methoxyphenyl)thiazole (3e). Yield: 81%, m.p.
1
= 253–254 ◦C, FTIR (ATR, cm-1): 3105 (N–H), 1600 (C=N), 1138 (C–N), 831. H-NMR (DMSO-d6):
δ
= 3.77 (3H, s, –OCH3), 6.97 (2H, d, J = 8.91 Hz, phenyl CH), 7.17 (1H, s, thiazole CH) 7.32–7.37 (2H,
m, benzimidazole CH), 7.67–7.71 (1H, m, benzimidazole CH), 7.76 (2H, d, J = 8.61 Hz, phenyl CH),
7.77–7.78 (1H, m, benzimidazole CH), 7.80 (2H, d, J = 8.85 Hz, phenyl CH), 7.90 (2H, d, J = 8.64 Hz,
phenyl CH), 8.13 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole CH), 12.27 (1H, s, –NH). 13C-NMR
(DMSO-d6):
δ = 55.61, 102.16, 111.31, 114.45, 120.48, 123.09, 124.09, 124.31, 127.33, 128.00, 128.16, 133.33,
134.22, 136.75, 140.36, 143.62, 144.31, 150.87, 159.29, 168.50. ESI-MS (m/z): [M + 2H]2+: 213 (100%).
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4-fluorophenyl)thiazole (3f). Yield: 80%, m.p. =
1
260–261 ◦C, FTIR (ATR, cm−1): 3091 (N-H), 1606 (C=N), 1138 (C–N), 825. H-NMR (DMSO-d6):
δ =
7.24 (2H, t, J = 8.91 Hz, phenyl CH), 7.32–7.37 (3H, m, benzimidazole CH, thiazole CH), 7.68–7.71 (1H,
m, benzimidazole CH), 7.75-7.81 (3H, m, benzimidazole CH, phenyl CH), 7.88–7.93 (4H, m, phenyl
CH), 8.14 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole CH), 12.30 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ
= 104.12, 111.30, 115.92 (J2 = 21.41 Hz), 120.50, 123.08, 124.08, 124.31, 127.98 (J3 = 8.06 Hz), 128.21,
131.77 (J4 = 2.55 Hz), 133.33, 134.13, 136.83, 140.58, 143.64, 144.37, 150.14, 162.09 (J1 = 242.71 Hz), 168.70.
ESI-MS (m/z): [M + 2H]2+: 215 (100%).
2-(2-(4-(1H◦-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4-chlorophenyl)thiazole (3g). Yield: 84%, m.p.
1
= 279–280 C, FTIR (ATR, cm−1): 3213 (N–H), 1585 (C=N), 1141 (C–N), 831. H-NMR (DMSO-d6):
δ
= 7.33–7.38 (2H, m, benzimidazole CH), 7.43 (1H, s, thiazole CH), 7.48 (2H, d, J = 8.58 Hz, phenyl
CH), 7.68–7.71 (1H, m, benzimidazole CH), 7.77 (2H, d, J = 8.67 Hz, phenyl CH), 7.80–7.82 (1H, m,
benzimidazole CH), 7.88–7.93 (4H, m, phenyl CH), 8.15 (1H, s, –CH=N–), 8.64 (1H, s, benzimidazole
CH), 12.33 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ = 105.16, 111.35, 120.40, 123.17, 124.15, 124.34, 127.71,

Molecules 2017, 22, 2051
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128.22, 129.11, 132.43, 133.29, 133.98, 134.15, 136.79, 140.68, 143.61, 144.08, 149.87, 168.75. ESI-MS (m/z):
[M + 2H]2+: 207 (100%).
2-(2-(4-(1H-benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4-bromophenyl)thiazole (3h). Yield: 85%, m.p. =
1
274–275 ◦C, FTIR (ATR, cm−1): 3188 (N–H), 1608 (C=N), 1141 (C–N), 829. H-NMR (DMSO-d6):
δ =
7.33–7.37 (2H, m, benzimidazole CH), 7.44 (1H, s, thiazole CH), 7.61 (2H, d, J = 8.55 Hz, phenyl CH),
7.68–7.71 (1H, m, benzimidazole CH), 7.76–7.79 (3H, m, benzimidazole CH, Phenyl CH), 7.83 (2H,
d, J = 8.49 Hz, phenyl CH), 7.91 (2H, d, J = 8.55 Hz, phenyl CH), 8.15 (1H, s, –CH=N–), 8.62 (1H, s,
benzimidazole CH), 12.32 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ = 105.25, 111.31, 120.49, 121.02, 123.10,
124.09, 124.32, 128.02, 128.23, 132.02, 133.32, 134.10, 134.32, 136.84, 140.69, 143.63, 144.33, 153.18, 168.75.
ESI-MS (m/z): [M + 2H]2+: 238 (100%).
2-(2-(4-(1H-benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(4 (trifluoromethyl)phenyl)thiazole (3i). Yield: 83%,
◦
1
m.p. = 279–280 C, FTIR (ATR, cm−1): 3097 (N–H), 1606 (C=N), 1139 (C–N), 837. H-NMR (DMSO-d6):
δ
= 7.32–7.37 (2H, m, benzimidazole CH), 7.60 (1H, s, thiazole CH), 7.68–7.71 (1H, m, benzimidazole
CH), 7.77 (4H, m, phenyl CH), 7.80–7.81 (1H, m, benzimidazole CH), 7.91 (2H, d, J = 8.58 Hz, phenyl
CH), 8.08 (2H, d, J = 8.04 Hz, phenyl CH), 8.15 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole CH),
12.37 (1H, s, –NH). 13C-NMR (DMSO-d6):
δ = 107.16, 111.30, 120.50, 123.08, 124.08, 124.31, 124.82 (q,
J = 270.2 Hz), 126.08 (q, J = 3.5 Hz), 126.54, 128.26, 126.08 (q, J = 37.4 Hz), 133.32, 134.05, 136.86, 138.80,
140.86, 143.63, 144.37, 149.56, 168.91. ESI-MS (m/z): [M + H]+: 464 (100%).
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(2,4-dimethylphenyl)thiazole (3j). Yield: 87%, m.p.
◦
1
= 215–216 C, FTIR (ATR, cm−1): 3097 (N–H), 1606 (C=N), 1126 (C–N), 831, 875. H-NMR (DMSO-d6):
δ
= 2.30 (3H, s, –CH3), 2.42 (3H, s, –CH3), 6.91 (1H, s, thiazole CH), 7.04-7.08 (2H, m, phenyl CH),
7.48–7.51 (3H, m, benzimidazole CH, phenyl CH), 7.75–7.79 (1H, m, benzimidazole CH), 7.82 (2H, d,
J = 8.61 Hz, phenyl CH), 7.86–7.90 (1H, m, benzimidazole CH), 7.94 (2H, d, J = 8.70 Hz, phenyl CH),
8.15 (1H, s, –CH=N–), 9.20 (1H, s, benzimidazole CH). 13C-NMR (DMSO-d6):
δ = 21.14, 21.53, 106.51,
112.29, 118.57, 124.83, 125.06, 125.44, 126.86, 128.15, 129.63, 131.89, 132.33, 132.57, 135.28, 135.56, 135.59,
137.22, 139.12, 140.18, 143.11, 150.98, 167.57. ESI-MS (m/z): [M + H]+: 424 (100%).
2-(2-(4-(1H-Benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(2,4-difluorophenyl)thiazole (3k). Yield: 81%, m.p.
◦
1
= 274–276 C, FTIR (ATR, cm−1): 3147 (N–H), 1608 (C=N), 1141 (C–N), 831, 840. H-NMR (DMSO-d6):
δ
= 7.19 (1H, td, J1 = 2.49 Hz, J2 = 8.91 Hz, phenyl CH), 7.25 (1H, d, J = 2.58 Hz, phenyl CH), 7.30–7.39
(3H, m, benzimidazole CH, thiazole CH), 7.68–7.71 (1H, m, benzimidazole CH), 7.77 (2H, d, J = 8.70 Hz,
phenyl CH), 7.80-7.86 (1H, m, benzimidazole CH), 7.91 (2H, d, J = 8.61 Hz, phenyl CH), 8.00–8.08 (1H,
m, phenyl CH), 8.15 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole CH), 12.33 (1H, s, –NH). 13C-NMR
(DMSO-d6):
δ
= 105.03 (t, J = 26.1 Hz), 108.48 (d, J = 13.5 Hz), 111.30, 112.31 (dd, J = 21.0 Hz 3.1 Hz),
−
120.50, 123.08, 124.32, 128.24, 130.85 (dd, J = 9.1 Hz–4.6 Hz), 133.33, 134.08, 136.88, 140.68, 140.77, 143.64,
144.00, 144.36, 144.38, 160.01 (dd, J = 248.2 Hz–12.6 Hz), 161.70 (dd, J = 246.2 Hz–11.3 Hz), 168.11.
ESI-MS (m/z): [M + 2H]2+: 216 (100%).
2-(2-(4-(1H-benzimidazol-1-yl)benzylidene)hydrazineyl)-4-(2,4-dichlorophenyl)thiazole (3l). Yield: 84%, m.p.
= 271–271 ◦C, FTIR (ATR, cm−1): 3061 (N–H), 1585 (C=N), 1139 (C–N), 825, 854. 1H-NMR (DMSO-d6):
δ
= 7.33–7.37 (2H, m, benzimidazole CH), 7.44 (1H, s, thiazole CH), 7.52 (1H, dd, J1 = 2.16 Hz,
J2 = 8.49 Hz, phenyl CH), 7.68–7.72 (2H, m, benzimidazole CH, phenyl CH), 7.76–7.79 (3H, m, phenyl
CH, benzimidazole CH), 7.91 (3H, m, phenyl CH), 8.14 (1H, s, –CH=N–), 8.61 (1H, s, benzimidazole
CH). 13C-NMR (DMSO-d6):
δ = 109.90, 111.30, 120.50, 123.08, 124.32, 127.98, 128.24, 129.34, 130.24, 132.06,
132.99, 133.33, 134.09, 136.87, 140.75, 141.47, 143.64, 144.37, 167.86. ESI-MS (m/z): [M + 2H]2+: 232 (100%).
3.2. Antifungal Activity Assays
Microbiological study was performed according to EUCAST definitive method EDef 7.1 for
Candida species [29]. Synthesized compounds were tested for their in vitro growth inhibitory activity
against C. glabrata (ATCC 90030), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019) and C. albicans

Molecules 2017, 22, 2051
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(ATCC 24433). The yeasts were sustained in Roswell Park Memorial Institute (RPMI) medium, after
an overnight incubation at 37 ◦C. The inocula of test microorganisms adjusted to match the turbidity
of a MacFarland 0.5 standard tube as determined with a spectrophotometer and the final inoculum
size was 0.5–2.5
×
105 cfu/mL for antifungal assay. The test was performed for medium at pH = 7
and two-fold serial dilutions were applied. 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 µg/mL. For the antifungal
assays, the test compounds and reference drugs were firstly dissolved in DMSO, and further dilutions
were performed to the desired concentrations of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and
0.78
µg/mL using RPMI medium. The completed plates were incubated for 24 h, and at the end of
the incubation, resazurin (20
µg/mL) was added into each well to control the growth in the wells.
Final plates including microorganism strains were incubated for 2 h. MIC50 values were determined
using microplate reader at 590 nm excitation and 560 nm emission wavelengths; MIC50 readings were
performed twice for entire compounds. Ketoconazole and fluconazole were used as reference drugs.
3.3. Quantification of Ergosterol Level
Extraction of total sterols from C. krusei was performed as recorded by Breivik and Owades [45].
Quantification of ergosterol level in this extract was carried out in accordance with our recently
described method [25].
3.4. Cytotoxicity Test
Cytotoxicity was experienced using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC®
CRL-1658
they were seeded at 1
reported data [46 48]. Treated with the compounds at concentrations ranging from 800
0.78 g/mL. Percent inhibition was evaluated for each concentration in accordance with the following
™
, London, UK). NIH/3T3 cells were incubated according to the supplier’s references and
104 cells into each well of 96-well plates. MTT assay was carried out as
g/mL to
×
–
µ
µ
formula. Moreover, dose-response curves were plotted against compound concentrations to determine
IC50 values [49].
3.5. Prediction of ADME Parameters
Physicochemical parameters of compounds (3a–3l) were analyzed by using QikProp 4.8 [37].
3.6. Molecular Docking Studies
A structure-based in silico procedure was applied to discover the binding modes of the most
active compound 3c to 14 α-sterol demethylase enzyme active sites. The crystal structures of enzyme
(PDB ID: 1EA1) [40] in complex with our reference agent fluconazole, was retrieved from the Protein
Data Bank server (www.pdb.org).
The structure of ligand was built using the Schrödinger Maestro [50] interface and then was
submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [51].
The ligand was prepared by the LigPrep 3.8 [52] to assign the protonation states at pH 7.4
±
1.0 and the
atom types, correctly. Bond orders were assigned and hydrogen atoms were added to the structures.
The grid generation was formed using the Glide 7.1 [53] program and docking runs were performed
with single precision docking mode (SP).
4. Conclusions
In summary, we have synthesized novel benzimidazole-thiazole derivatives showing significant
anticandidal effects. Additionally, antifungal activity studies, quantification of ergosterol level, preliminary
toxicological screening, ADME prediction and docking evaluations of obtained compounds were
undertaken in the current study. Activity studies showed that the compound 3c was the most active

Molecules 2017, 22, 2051
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compound in the series, with a MIC50 value of 1.56
µg/mL against Candida strains. Toxicological and
ADME studies enhanced the biological importance of this compound. Docking evaluations demonstrated
the binding modes of this compound to enzyme active site. We presume that in further studies all these
findings may help medicinal chemists to discover more promising anticandidal compounds.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This study was financially supported by Anadolu University Scientific Projects Fund, Project
No.: 1705S183.
Author Contributions: Y.Ö. and Z.A.K. conceived and designed the experiments; D.O. and B.K.Ç. performed the
synthesis; S.L. performed analysis studies; B.N.S. and U.A.Ç. performed activity tests; B.N.S. performed docking
studies; S.I. performed the toxicity tests; Z.A.K., Y.Ö., U.A.Ç., B.N.S., S.L., B.K.Ç. and D.O. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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