Synthesis and anticandidal activity of new imidazole-chalcones

Article abstract of DOI:10.3390/molecules23040831

In the present work, 15 new 1-(4-(1H-imidazol-1-yl)phenyl)-3-(4-substituedphenyl)prop-2-en-1-one derivatives (3a–3o) were synthesized to evaluate their antifungal activity. Structures of newly synthesized imidazole derivatives (3a–3o) were characterized by IR,¹H-NMR,¹³C-NMR, and LCMSMS spectroscopic methods. The anticandidal activity of compounds (3a–3o) against C. albicans (ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and C. glabrata (ATCC 90030) was elucidated according to the EUCAST definitive (EDef 7.1) method. Consistent with the activity studies, 3a–3d were found to be more potent derivatives with their MIC₅₀ values (0.78 μg/mL–3.125 μg/mL) against Candida strains. Compound 3c indicated similar antifungal activity to ketoconazole against all Candida species and was evaluated as the most active derivative in the series. Effects of the most potent derivatives 3a–3d on ergosterol biosynthesis were observed by LC-MS-MS method, which is based on quantification of the ergosterol level in C. krusei. Moreover, these compounds were subjected to a cytotoxicity test for the preliminary toxicological profiles and were found as non-cytotoxic. Furthermore, docking studies for the most active derivative 3c were performed to evaluate its binding modes on lanosterol 14-α-demethylase. In addition to in vitro tests, docking studies also revealed that Compound 3c is a potential ergosterol biosynthesis inhibitor.

Full text of DOI:10.3390/molecules23040831

molecules
Article
Synthesis and Anticandidal Activity of
New Imidazole-Chalcones
Derya Osmaniye 1, Betul Kaya Cavusoglu1, Begum Nurpelin Saglik 1,2, Serkan Levent 1,2
,
Ulviye Acar Cevik 1,2, Ozlem Atli 3, Yusuf Ozkay 1,2,
*
and Zafer Asim Kaplancikli 1
ID
ID
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu Universty, 26470 Eskis¸ehir, Turkey;
dosmaniye@anadolu.edu.tr (D.O.); betulkaya@anadolu.edu.tr (B.K.Ç.); bnsaglik@anadolu.edu.tr (B.N.S.);
serkanlevent@anadolu.edu.tr (S.L.); uacar@anadolu.edu.tr (U.A.Ç.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu Universty,
26470 Eskis¸ehir, Turkey
2
3
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu Universty, 26470 Eskis¸ehir, Turkey;
oatli@anadolu.edu.tr
*
Correspondence: yozkay@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3603)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 March 2018; Accepted: 29 March 2018; Published: 4 April 2018
Abstract: In the present work, 15 new 1-(4-(1H-imidazol-1-yl)phenyl)-3-(4-substituedphenyl)prop-2-
en-1-one derivatives (3a
newly synthesized imidazole derivatives (3a
LCMSMS spectroscopic methods. The anticandidal activity of compounds (3a
–
3o) were synthesized to evaluate their antifungal activity. Structures of
3o) were characterized by IR, 1H-NMR, 13C-NMR, and
3o) against C. albicans
–
–
(ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and C. glabrata (ATCC 90030) was
elucidated according to the EUCAST definitive (EDef 7.1) method. Consistent with the activity studies,
3a–3d were found to be more potent derivatives with their MIC50 values (0.78 µg/mL–3.125 µg/mL)
against Candida strains. Compound 3c indicated similar antifungal activity to ketoconazole against
all Candida species and was evaluated as the most active derivative in the series. Effects of the most
potent derivatives 3a–3d on ergosterol biosynthesis were observed by LC-MS-MS method, which is
based on quantification of the ergosterol level in C. krusei. Moreover, these compounds were subjected
to a cytotoxicity test for the preliminary toxicological profiles and were found as non-cytotoxic.
Furthermore, docking studies for the most active derivative 3c were performed to evaluate its binding
modes on lanosterol 14-α-demethylase. In addition to in vitro tests, docking studies also revealed
that Compound 3c is a potential ergosterol biosynthesis inhibitor.
Keywords: imidazole; anticandidal activity; ergosterol inhibition; 14-alpha demethylase; docking study
1. Introduction
During the last few years, there has been an increased awareness of morbidity and mortality
related to invasive and systemic fungal disease due to resistant fungi and immunocompromised
infections such as AIDS. Candidiasis, aspergillosis, and cryptococcosis represent the three most
common invasive fungal infections and have the highest mortality rates [1–3]. Cryptococcus,
Trichosporon, Geotrichum, and Rhodotorula species also lead to diverse occurrence and restrict clinical
therapy owing to the fungus variety. However, Candida species have been the primary reason of fungal
infections [
in the USA [
tract and can exist in the oral cavity in 40–60% of the population without causing any problems [
If the environment of normal flora changes in a way that encourages fungal growth, Candida can
4
,
6
5
]. Candida spp. are presently the third-to-fourth leading reason of bloodstream infections
]. Candida is generally part of the normal flora of the mouth, vagina, skin, and intestinal
].
7,8
Molecules 2018, 23, 831; doi:10.3390/molecules23040831
www.mdpi.com/journal/molecules

Molecules 2018, 23, 831
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increase and cause infections. Currently, various antifungal drugs have been used clinically in an
attempt to reduce effects of fungal infections.
The “azoles” are class of antifungal agents whose molecules are based on a pharmacophore that
inhibits the activity of fungal cytochrome P45014DM (also known as lanosterol 14-
α-demethylase,
Erg11p, Cyp51p, and Erg16p). Azoles are administered against lanosterol 14- -demethylase in the
α
ergosterol pathway. Antifungal azoles bind with nitrogen atoms to iron atoms of the heme group in the
target protein and block fungal membrane ergosterol biosynthesis by inhibiting the demethylation of
lanosterol to ergosterol and altering the fungal membrane structure and function [9–11]. The variability
of azole nuclei is demonstrated in their present applications as pharmaceutical drugs used in the
cure of bacterial, viral, and fungal infections, worm infestations, acid reflux, cancer, inflammations,
and diabetes [12]. Imidazoles, the first group to be developed in azole antifungals also block the
accumulation of methylated sterols, and disrupt the ergosterol biosynthesis, which is an essential
component of the fungal cell wall. Moreover, some imidazole drugs, at high concentrations, could
display a direct inhibitory effect on membranes, without intervene in sterols and sterol esters [13
Ketoconazole, miconazole, and clotrimazole are some common drugs used for the treatment of patients
affected by different Candida species [16 18]. Chemical structures of these antifungal agents are
–15].
–
summarized in Figure 1 along with compounds targeted in the present study.
In other respects, it is well known that chalcones (1,3-diaryl-2-propen-1-one) plays an important
role for anticandidal activity [19–22]. Their anticandidal action has been mainly related to the reactive
enone moiety. As a Michael reaction acceptor, the enone unit binds thiol groups of certain proteins.
Hence, most chalcones inhibit biosynthesis of the fungal cell wall and thus clarify their antifungal
potential [23].
Knowledge of antifungal activities in both functional groups (azole and chalcone) has prompted
curiosity about the antifungal effects of compounds containing these two groups. Therefore, within
the scope of this study, a series of new imidazole–chalcone derivatives were synthesized and evaluated
for their antifungal activities.
Figure 1. Structures of some antifungal agents and synthesized compounds (3a–3o).
2. Results and Discussion
2.1. Chemistry
Compounds 3a
acetophenone ( ) was obtained under reflux by a reaction of 1-(4-fluorophenyl)ethan-1-one and
1H-imidazole. Secondly, 4-fluorobenzaldehyde and appropriate proton donoring group were reacted
–
3o were synthesized as summarized in Scheme 1. Initially, 40-(imidazol-1-yl)
1

Molecules 2018, 23, 831
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in order to obtained 4-substitutedbenzaldehydes (2a–2o). In the last step, the target compounds (3a–3o)
were synthesized by means of claisen schmidt condensation using 40-(imidazol-1-yl)acetophenone
1
(1
) and appropriate 4-substituted benzaldehydes (2a
–
2o). IR, H-NMR, 13C-NMR, and LCMSMS
spectroscopic methods were used to perform structure elucidations of the final compounds. In the IR
spectrum, C=O and C=N bonds were observed at 1601–1657 cm−1 and 1296–1389 cm−1, respectively.
1,4-Disubstituted benzene bands were observed at 808–814 cm−1
.
In the 1H-NMR spectrum,
1,4-disubstituted benzene protons had doublet peaks between 6.58 and 8.30 ppm. Three imidazole
hydrogens gave broad singlet or triplet peaks with small coupling constant values at 7.16–7.17 ppm,
7.90–7.91 ppm, and 8.44–8.47 ppm. Vinyl protons were observed as two doublet peaks with coupling
constant values, approximately 15 Hz. In the 13C-NMR spectrum, aromatic peaks were gained between
113 and 161 ppm. Carbonyl carbon gave a peak over 187 ppm. Aliphatic carbons belonging to
substituents were observed between 12 and 64 ppm. In the mass spectrum, all masses were matched
with the expected M + H values
Compounds
R
3a
3b
3c
3d
3e
3f
4-methylphenoxy
4-methylphenylthio
4-methoxyphenoxy
4-methoxyphenylthio
pyrrolidinyl
morpholinyl
3g
3h
3i
3j
3k
3l
3m
3n
3o
piperidinyl
3-methylpiperidinyl
4-methylpiperidinyl
3,5-dimethylpiperidinyl
4-benzylpiperidinyl
4-methylpiperazinyl
4-ethylpiperazinyl
4-(2-dimethylaminoethyl)piperazinyl
4-(3-dimethylaminopropyl)piperazinyl
Scheme 1. Synthesis way of the target compounds (3a–3o).
2.2. Antifungal Activity
Synthesized compounds (3a
(ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and C. glabrata (ATCC 90030). MIC50
values were determined via fluorometric measurements, using resazurin solution [24 25]. Ketoconazole
–
3o) were evaluated for anticandidal activity against C. albicans
,
and fluconazole were used as a standard drug in the activity test. Anticandidal activity results are
presented in Table 1.

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Consistent with the activity studies, Compounds 3a
with their MIC50 values (0.78–3.125
antifungal activity against C. krusei with an MIC50 value of 0.78
Furthermore, MIC50 value of 0.78 g/mL was recorded for Compounds 3c and 3d against C. glabrata.
–
3d, were found to be more potent derivatives
µg/mL) against Candida strains. Compounds 3a
–3c showed higher
µg/mL compared with standard drugs.
µ
Compound 3c indicated similar antifungal activity to ketoconazole and fluconazole against all Candida
species and was evaluated as the most active derivative in the series.
Antimicrobial activity results clearly indicated that variable groups at the 4th position of the
phenyl moiety have an essential impact on antifungal activity. It was observed that the presence of
more liphophilic phenoxy and phenylthio groups in Compounds 3a–3d have significantly enhanced
anticandidal activity when compared with the compounds that carry cyclic secondary amines. It is
known that lipophilicity is a key property that influences the ability of a drug to reach the target
by transmembrane diffusion and to have a major effect on the biological activity [26]. Besides,
augmented electron density may be another reason for better anticandidal activity due to the presence
of electronically rich aromatic rings in Compounds 3a–3d. Thus, it may be suggested that increased
lipophilic and/or electronic characters of Compounds 3a
–
3d caused an increase in anticandidal activity.
Table 1. MIC50 (µg/mL) values of Compounds 3a–3o.
Comp.
C. albicans
C. glabrata
C. krusei
C. parapsilosis
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3.125
3.125
1.56
1.56
50
50
50
50
50
50
50
50
50
50
50
0.78
0.78
3.125
3.125
0.78
0.78
0.78
0.78
1.56
12.50
12.50
25
25
12.50
25
0.78
0.78
0.78
0.78
3.125
12.50
12.50
12.50
12.50
12.50
25
12.50
12.50
25
12.50
12.50
12.50
12.50
12.50
12.50
25
12.50
12.50
12.50
12.50
1.56
25
25
12.50
12.50
12.50
1.56
1.56
12.50
12.50
1.56
Ketoconazole
Fluconazole
1.56
0.78
2.3. Quantification of the Ergosterol Level
Sterols are neutral lipids of eukaryotic cells, among which ergosterol is the main constituent of
fungal membranes. Ergosterol has biological functions such as membrane fluidity, regulation, activity
and distribution of integral membrane proteins, and control of the cell cycle [27
sterols in maintenance of cell membranes make ergosterol and its biosynthetic pathway essential for
fungal growth, and a primary target for antifungal drugs to treat fungal infections [ ]. From this point
of view, we performed an LCMSMS (Shimadzu LCMS 8040, Kyoto, Japan) method for quantitative
determination of ergosterol content of C. krusei as reported in our recent study [29]. Compounds 3a 3d
displaying the best anticandidal activity and low cytotoxic effect, and reference agents ketoconazole
and fluconazole were tested at concentrations of 0.78–3.12 g/mL. Ergosterol standard (Product No.:
45480, Sigma-Aldrich, Darmstadt, Germany) was used for the quantification of ergostrerol in samples,
which were treated with reference agents and Compounds 3a 3d. Ergosterol quantity of the negative
,28]. The critical role of
6
–
,
µ
–
control was considered as 100%. Quadruplicate analyses were performed for all concentrations and
the obtained data were expressed as mean ± standard deviation (SD) (Table 2).
According to obtained results, the decrease in ergosterol level after administration of Compounds
3a–3d are noticeable when compared to the reference agents. Compounds 3a–3d and reference

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agents significantly decreased the level of ergosterol at all tested concentrations. Therefore, it can be
interpreted that Compounds 3a–3d play a role in the ergosterol biosynthesis pathway.
2.4. Cytotoxicity Test
The main cause of failure in all stages of the new drug development process is toxicity. Early
identification of toxicities of drug candidates is very important in terms of drug development
studies [30]. Therefore, the MTT cell viability assay, which is suggested for cytotoxicity screening
of drug candidates by ISO (10993-5, 2009) was performed [31]. Cytotoxicity of selected compounds
(3a–3d), displaying strong anticandidal activity and good predicted pharmacokinetics, was determined
against NIH/3T3 mouse embryonic fibroblast cell lines (ATCC CRL1658).
The cytotoxicity results of the tested compounds are presented in Table 2. IC50 of compounds
against NIH/3T3 was much higher than their MIC50 values (0.78–3.125
µg/mL) against Candida strains.
This finding shows that the antifungal activity of Compounds 3a 3d is not due to general toxicity,
–
but can be ascribed to their selective action against Candida species. Thus, cytotoxicity test findings
enhanced the importance of Compounds 3a–3d as anticandidal drug candidates.
Table 2. Cytotoxic activity and ergosterol biosynthesis inhibition potency of Compounds 3a
NIH/3T3 cell line and C. krusei, respectively.
–3d against
Inhibition of Ergosterol Biosynthesis (%)
Compound
IC50 (µg/mL)
0.78 µg/mL
1.56 µg/mL
3.12 µg/mL
3a
3b
3c
>500
>500
66.19 ± 2.23
68.59 ± 1.98
71.14 ± 4.9
52.47 ± 1.83
60.99 ± 2.94
61.74 ± 1.70
79.45 ± 3.16
81.62 ± 4.07
78.16 ± 2.70
67.14 ± 2.70
73.12 ± 4.16
70.12 ± 3.22
86.47 ± 4.77
83.49 ± 3.18
84.28 ± 4.65
74.14 ± 2.21
84.56 ± 3.01
82.13 ± 4.45
436.04 ± 1.03
3d
387.64 ± 20.20
Ketoconazole
Fluconazole
-
-
2.5. Molecular Docking Studies
Docking studies were performed in order to gain more insight into the binding mode of
the most active compound 3c to lanosterol 14- -demethylase. Lanosterol 14- -demethylase from
Mycobacterium tuberculosis has high homology compared with lanosterol 14- -demethylase from
Candida species. It has been reported that these two enzymes have a high degree of similarity between
the hydrophobic cavities of the catalytic site [29 32]. Therefore, docking studies were carried out using
X-ray crystal structure of lanosterol 14- -demethylase from Mycobacterium tuberculosis in complex with
α
α
α
,
α
fluconazole (PDB ID: 1EA1) [33] obtained from the Protein Data Bank server (www.pdb.org).
As stated in the antifungal activity section, Compound 3c was found to be the most active
derivative against C. albicans, C. glabrata, C. krusei, and C. parapsilosis with 1.56, 0.78, 0.78, and
0.78
the possible interaction of this compound with lanosterol 14-α-demethylase enzyme.
The docking pose on lanesterol 14- -sterol demethylase reveals that the interactions between
Compound 3c and HEM450 are very important in terms of binding to the active site of the enzyme
(Figure 2). Imidazole and its neighboring phenyl established two interactions. The other two
interactions were formed by other phenyl rings of the structure. Phenyl of benzylidene was in
µg/mL MIC values, respectively. Thus, the main purpose of docking studies was to investigate
α
π–π
π–π
interaction with Arg96, while phenyl substituted with methoxy group created a π–π interaction with
the phenyl of Phe78. Additionally, the docking pose showed that there were three hydrogen bonds
providing polar interactions. Carbonyl of Compound 3c established a hydrogen bond with the amino
of Val395. Oxygen atoms of phenoxy created this interaction with the amino of Met79, whereas oxygen
atoms of the methoxy group formed with the amino of Ile323. These interactions explain the stronger
anticandidal activity of 3c. It could be that C-4 of phenyl is very important in terms of binding to the

Molecules 2018, 23, 831
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enzyme active site and anticandidal activity. As a result, this additional interaction could explain the
greater binding capability and stronger activity of Compound 3c compared with other compounds.
Figure 2. The interacting mode of Compound 3c in the active region of 14-alpha-sterol demethylase.
The inhibitor is colored with purple and HEM with grey.
3. Materials and Methods
3.1. Chemistry
All chemicals used in the syntheses were purchased either from Sigma-Aldrich Chemicals
(Sigma-Aldrich Corp., St. Louis, MO, USA) or Merck Chemicals (Merck KGaA, Darmstadt, Germany).
Melting points of the synthesized compounds were measured by MP90 digital melting point apparatus
1
(Mettler Toledo, Columbus, OH, USA) and were presented as uncorrected. H-NMR and 13C-NMR
spectra were recorded by a Bruker 300 MHz and 75 MHz digital FT-NMR spectrometer (Bruker
Bioscience, Billerica, MA, USA) in DMSO-d6, respectively. In the NMR spectra, splitting patterns were
designated as follows: s: singlet; d: doublet; t: triplet; m: multiplet. Coupling constants (J) were
reported as Hertz. The IR spectra of the compounds were recorded using an IRAffinity-1S Fourier
transform IR (FTIR) spectrometer (Shimadzu, Tokyo, Japan). LC-MS-MS studies were performed on a
Schimadzu, 8040 LCMSMS spectrophotometer (Shimadzu, Tokyo, Japan). The purities of compounds
were checked by TLC on silica gel 60 F254 (Merck KGaA, Darmstadt, Germany).
3.1.1. Synthesis of 40-(Imidazol-1-yl)acetophenone (1)
4-Fluoro acetofenone (4.976 mL, 0.041 mol), imidazole (2.789 g, 0.041 mol), and sodium hydride
(NaH) (1.080 g, 0.045 mol) in DMF were refluxed for 12 h. After cooling, the mixture was poured
into the ice water, and the precipitated product was washed with water, dried, and recrystallized
from ethanol.
3.1.2. Synthesis of Four Substituted Benzaldehydes (2a–2o)
A mixture of 4-fluoro benzaldehyde (0.259 mL, 0.002 mol), corresponding phenol, thiophenol,
or amine (0.002 mol), and a catalytic quantity of potassium carbonate (K2CO3) was refluxed in DMF
(20 mL) for 36 h. After completion of the reaction, the mixture was poured into ice water (50 mL),
and the precipitated product was filtered, washed with deionized water, dried, and recrystallized
from ethanol.

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3.1.3. General Procedure for the Synthesis of Target Compounds (3a–3o)
1-(4-(1H-imidazol-1-yl)phenyl)ethan-1-one (1) (0.316 g, 0.0017 mol) and appropriate 4-substituted
benzaldehydes (2a 2o) derivatives (0.0017 mol) in methanol were stirred for 10 h in the presence of
–
potassium hydroxide. The precipitated product was washed with water, dried, and recrystallized
from ethanol.
◦
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(p-tolyloxy)phenyl)prop-2-en-1-one (3a): Yield: 79%, M.P. = 146–148 C,
FTIR (ATR, cm−1): 3128 (C-H), 1653 (C=O), 1373 (C=N), 814. H-NMR (300 MHz, DMSO-d6): 2.32 (3H,
1
s, CH3), 7.01 (2H, d, J = 8.5 Hz, disubstituted CH), 7.02 (2H, d, J = 8.8 Hz, disubstituted CH), 7.17 (1H, s,
imidazole CH), 7.25 (2H, d, J = 8.2 Hz, methylphenyl CH), 7.76 (1H, d, J = 15.6 Hz, -HC=CH-), 7.87–7.94
(6H, m, disubstituted CH, methylphenyl CH, imidazole CH, -HC=CH-), 8.00 (1H, d, J = 15.6 Hz,
-HC=CH-), 8.30 (2H, d, J = 8.8 Hz, disubstituted CH), 8.47 (1H, s, imidazole CH). 13C-NMR (75 MHz,
DMSO-d6):
δ = 20.8, 118.2, 118.3, 120.1, 120.3, 121.0, 129.8, 130.9, 131.1, 131.5, 134.0, 136.1, 136.2, 140.7,
144.1, 153.6, 160.1, 188.2. ESI-MS [M + H]+: 381.25 (100%).
◦
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(p-tolylthio)phenyl)prop-2-en-1-one (3b): Yield: 84%, M.P. = 168–169 C,
FTIR (ATR, cm−1): 3120 (C-H), 1651 (C=O), 1337 (C=N), 812. H-NMR (300 MHz, DMSO-d6): 2.35 (3H,
1
s, CH3), 7.17 (1H, t, J = 1.1 Hz, imidazole CH), 7.21 (2H, d, J = 8.3 Hz, disubstituted CH), 7.29 (2H,
d, J = 7.9 Hz, methylphenyl CH), 7.40 (2H, d, J = 8.3 Hz, disubstituted CH), 7.72 (1H, d, J = 15.6 Hz,
-HC=CH-), 7.84–7.88 (3H, m, methylphenyl CH, imidazole CH), 7.89–7.98 (3H, m, disubstituted CH,
-HC=CH-), 8.00 (1H, d, J = 15.6 Hz, -HC=CH-), 8.29 (2H, d, J = 8.8 Hz, disubstituted CH), 8.47 (1H, s,
imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 21.2, 118.3, 120.3, 122.0, 128.4, 128.9, 130.3, 130.9,
130.9, 131.0, 132.9, 133.8, 135.9, 136.2, 139.1, 140.7, 141.1, 143.9, 188.2. ESI-MS [M + H]+: 397.20 (100%).
1-(4-(1H-◦Imidazol-1-yl)phenyl)-3-(4-(4-methoxyphenoxy)phenyl)prop-2-en-1-one (3c): Yield: 92%, M.P. =
168–170 C, FTIR (ATR, cm−1): 3144 (C-H), 1653 (C=O), 1342 (C=N), 814. 1H-NMR (300 MHz,
DMSO-d6): 3.78 (3H, s, OCH3), 6.98 (2H, d, J = 8.8 Hz, methoxyphenyl CH), 7.01 (2H, d, J = 9.2 Hz,
disubstituted CH), 7.09 (2H, d, J = 9.2 Hz, disubstituted CH), 7.17 (1H, t, J = 1.1 Hz, imidazole CH), 7.75
(1H, d, J = 15.5 Hz, -HC=CH-), 7.87–7.93 (6H, m, methoxyphenyl CH, disubstituted CH, imidazole CH,
-HC=CH-), 8.29 (2H, d, J = 8.8 Hz, disubstituted CH), 8.47 (1H, t, J = 1.1 Hz, imidazole CH). 13C-NMR
(75 MHz, DMSO-d6):
δ = 55.9, 115.7, 117.5, 118.3, 120.3, 120.8, 121.8, 129.5, 130.9, 131.3, 131.5, 136.1,
136.2, 140.6, 144.2, 148.9, 156.6, 160.8, 188.2. ESI-MS [M + H]+: 397.25 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-((4-methoxyphenyl)thio)phenyl)prop-2-en-1-one (3d): Yield: 80%,
1
M.P. = 177–179 ◦C, FTIR (ATR, cm−1): 3111 (C-H), 1657 (C=O), 1369 (C=N), 808. H-NMR (300 MHz,
DMSO-d6): 3.81 (3H, s, OCH3), 7.06 (2H, d, J = 8.8 Hz, methoxyphenyl CH), 7.12 (2H, d, J = 8.4 Hz,
disubstituted CH), 7.17 (1H, s, imidazole CH), 7.49 (2H, d, J = 8.8 Hz, methoxyphenyl CH), 7.70 (1H, d,
J = 15.6 Hz, -HC=CH-), 7.81 (2H, d, J = 8.4 Hz, disubstituted CH), 7.87 (2H, d, J = 8.8 Hz, disubstituted
CH), 7.91 (1H, t, J = 1.4 Hz, imidazole CH), 7.92 (1H, d, J = 15.6 Hz, -HC=CH-), 8.27 (2H, d, J = 8.8 Hz,
disubstituted CH), 8.46 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 55.9, 116.1, 118.3,
120.3, 121.7, 121.8, 127.2, 130.2, 130.9, 132.5, 136.0, 136.2, 136.6, 140.7, 142.6, 143.9, 160.7, 188.2. ESI-MS
[M + H]+: 413.20 (100%).
◦
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(pyrrolidin-1-yl)phenyl)prop-2-en-1-one (3e): Yield: 77%, M.P. = 186–188 C,
FTIR (ATR, cm−1): 3109 (C-H), 1655 (C=O), 1373 (C=N), 810. 1H-NMR (300 MHz, DMSO-d6):
δ
1.94–1.98 (4H, m, pyrrolidine CH2), 3.28–3.32 (4H, m, pyrrolidine CH2), 6.58 (2H, d, J = 8.8 Hz,
disubstituted CH), 7.16 (1H, s, imidazole CH), 7.63–7.73 (4H, m, disubstituted CH, -HC=CH-), 7.85
(2H, d, J = 8.7 Hz, disubstituted CH), 7.90 (1H, t, J = 1.2 Hz, imidazole CH), 8.25 (2H, d, J = 8.7 Hz,
disubstituted CH), 8.44 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 25.4, 47.8, 112.2,
115.6, 118.3, 120.2, 121.9, 130.5, 130.8, 131.6, 136.2, 136.9, 140.2, 146.2, 149.9, 187.6. ESI-MS [M + H]+:
344.25 (100%).

Molecules 2018, 23, 831
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◦
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-morpholinophenyl)prop-2-en-1-one (3f): Yield: 79%, M.P. = 214–215 C,
1
FTIR (ATR, cm−1): 3109 (C-H), 1653 (C=O), 1337 (C=N), 812. H-NMR (300 MHz, DMSO-d6):
δ 3.27
(4H, t, J = 4.9 Hz, morpholine CH2), 3.75 (4H, t, J = 4.9 Hz, morpholine CH2), 7.00 (2H, d, J = 8.8 Hz,
disubstituted CH), 7.16 (1H, t, J = 1.0 Hz, imidazole CH), 7.71 (2H, d, J = 15.4 Hz, -HC=CH-), 7.74–7.82
(3H, m, disubstituted CH, -HC=CH-), 7.86 (2H, d, J = 8.8 Hz, disubstituted CH), 7.90 (1H, t, J = 1.2 Hz,
imidazole CH), 8.25 (2H, d, J = 8.7 Hz, disubstituted CH), 8.44 (1H, s, imidazole CH). 13C-NMR
(75 MHz, DMSO-d6):
δ = 47.6, 63.4, 114.6, 117.9, 118.3, 120.2, 125.2, 130.7, 130.8, 131.1, 136.2, 136.6,
140.4, 145.3, 153.2, 187.9. ESI-MS [M + H]+: 360.25 (100%).
◦
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one (3g): Y Yield: 73%, M.P. = 198–199 C,
1
FTIR (ATR, cm−1): 3078 (C-H), 1657 (C=O), 1389 (C=N), 812. H-NMR (300 MHz, DMSO-d6):
δ 1.58
(6H, s, piperidine CH2), 3.32 (4H, s, piperidine CH2), 6.95 (2H, d, J = 8.9 Hz, disubstituted CH), 7.16
(1H, t, J = 1.0 Hz, imidazole CH), 7.71–7.82 (4H, m, disubstituted CH, -HC=CH-), 7.86 (2H, d, J = 8.7 Hz,
disubstituted CH), 7.91 (1H, t, J = 1.3 Hz, imidazole CH), 8.26 (2H, d, J = 8.7 Hz, disubstituted CH),
8.45 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 24.4, 25.4, 48.5, 114.6, 117.2, 118.3, 120.2,
123.9, 130.7, 130.8, 131.3, 136.2, 136.7, 140.3, 145.5, 153.2, 187.8. ESI-MS [M + 2H]2+: 179.70 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(3-methylpiperidin-1-yl)phenyl)prop-2-en-1-one (3h): Yield: 80%,
1
M.P. = 163–165 ◦C, FTIR (ATR, cm−1): 3107 (C-H), 1655 (C=O), 1339 (C=N), 812. H-NMR (300 MHz,
DMSO-d6): 0.92 (3H, d, J = 6.6 Hz, CH3), 1.04–1.17 (1H, m, piperidine CH), 1.43–1.80 (5H, m, piperidine
CH2), 2.73–2.82 (1H, m, piperidine CH2), 3.77–3.85 (2H, m, piperidine CH2), 6.96 (2H, d, J = 9.0 Hz,
disubstituted CH), 7.16 (1H, s, imidazole CH), 7.71–7.73 (4H, m, disubstituted CH, -HC=CH-), 7.86
(2H, d, J = 8.8 Hz, disubstituted CH), 7.91 (1H, t, J = 1.2 Hz, imidazole CH), 8.26 (2H, d, J = 8.8 Hz,
disubstituted CH), 8.45 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 19.6, 24.8, 30.6, 33.0,
47.9, 55.3, 114.6, 117.1, 118.3, 120.2, 123.8, 130.6, 130.8, 131.4, 136.2, 136.7, 140.3, 145.5, 152.9, 187.8.
ESI-MS [M + 2H]2+: 186.70 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-methylpiperidin-1-yl)phenyl)prop-2-en-1-one (3i): Yield: 77%,
1
M.P. = 189–191 ◦C, FTIR (ATR, cm−1): 3117 (C-H), 1657 (C=O), 1339 (C=N), 812. H-NMR (300 MHz,
DMSO-d6):
δ 0.92 (3H, d, J = 6.4 Hz, CH3), 1.10–1.24 (2H, m, piperidine CH2), 1.52–1.70 (3H, m,
piperidine CH2), 2.76–2.85 (2H, m, piperidine CH2), 3.87–3.91 (2H, m, piperidine CH2), 6.96 (2H, d,
J = 8.9 Hz, disubstituted CH), 7.16 (1H, t, J = 1.0 Hz, imidazole CH), 7.71–7.74 (4H, m, disubstituted CH,
-HC=CH-), 7.86 (2H, d, J = 8.7 Hz, disubstituted CH), 7.91 (1H, t, J = 1.3 Hz, imidazole CH), 8.26 (2H,
d, J = 8.7 Hz, disubstituted CH), 8.45 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 22.2,
30.8, 33.6, 47.9, 114.7, 117.2, 118.3, 120.2, 123.9, 130.7, 130.8, 131.3, 136.2, 136.7, 140.3, 145.5, 153.0, 187.8.
ESI-MS [M + 2H]2+: 186.75 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(3,5-dimethylpiperidin-1-yl)phenyl)prop-2-en-1-one (3j): Yield: 81%,
1
M.P. = 160–161 ◦C, FTIR (ATR, cm−1): 3035 (C-H), 1622 (C=O), 1335 (C=N), 815. H-NMR (300 MHz,
DMSO-d6):
δ 0.90 (6H, d, J = 6.5 Hz, CH3), 1.59–1.79 (4H, m, piperidine CH2), 2.28–2.36 (2H, m,
piperidine CH2), 3.85–3.90 (2H, m, piperidine CH2), 6.97 (2H, d, J = 9.0 Hz, disubstituted CH), 7.16 (1H,
t, J = 1.0 Hz, imidazole CH), 7.70–7.73 (4H, m, disubstituted CH, -HC=CH-), 7.86 (2H, d, J = 8.8 Hz,
disubstituted CH), 7.91 (1H, t, J = 1.3 Hz, imidazole CH), 8.23 (2H, d, J = 8.8 Hz, disubstituted CH), 8.45
(1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 19.5, 30.5, 42.4, 54.8, 114.5, 117.1, 118.3, 120.2,
123.7, 130.6, 130.8, 131.4, 136.2, 136.7, 140.3, 145.5, 152.7, 187.9. ESI-MS [M + 2H]2+: 193.70 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-benzylpiperidin-1-yl)phenyl)prop-2-en-1-one (3k): Yield: 83%,
1
M.P. = 178–180 ◦C, FTIR (ATR, cm−1): 3062 (C-H), 1601 (C=O), 1296 (C=N), 814. H-NMR (300 MHz,
DMSO-d6):
δ 1.17–1.31 (2H, m, piperidine CH2), 1.63–1.77 (3H, m, piperidine CH2), 2.54 (2H, d,
J = 7.1 Hz, CH2), 2.73–2.81 (2H, m, piperidine CH2), 3.88–3.92 (2H, m, piperidine CH2), 6.96 (2H,
d, J = 9.0 Hz, disubstituted CH), 7.16–7.21 (4H, m, monosubstituted benzene CH, imidazole CH),
7.27–7.29 (2H, m, monosubstituted benzene CH), 7.71–7.74 (4H, m, disubstituted CH, -HC=CH-), 7.86
(2H, d, J = 8.8 Hz, disubstituted CH), 7.91 (1H, t, J = 1.4 Hz, imidazole CH), 8.26 (2H, d, J = 8.8 Hz,

Molecules 2018, 23, 831
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disubstituted CH), 8.45 (1H, t, J = 1.0 Hz, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ
= 31.5, 37.8,
42.7, 47.8, 114.7, 117.2, 118.3, 120.2, 123.9, 126.3, 128.6, 129.5, 130.7, 130.8, 131.3, 136.2, 136.7, 140.3, 140.6,
145.5, 152.9, 187.9. ESI-MS [M + 2H]2+: 224.75 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-methylpiperazin-1-yl)phenyl)prop-2-en-1-one (3l): Yield: 82%,
1
M.P. = 205–207 ◦C, FTIR (ATR, cm−1): 3109 (C-H), 1655 (C=O), 1337 (C=N), 812. H-NMR (300 MHz,
DMSO-d6):
δ 2.22 (3H, s, CH3), 2.43 (4H, t, J = 5.0 Hz, piperazine CH2), 3.30 (4H, t, J = 5.0 Hz, piperazine
CH2), 6.98 (2H, d, J = 8.9 Hz, disubstituted CH), 7.16 (1H, t, J = 1.2 Hz, imidazole CH), 7.72–7.76 (4H,
m, disubstituted CH, -HC=CH-), 7.86 (2H, d, J = 8.8 Hz, disubstituted CH), 7.91 (1H, t, J = 1.3 Hz,
imidazole CH), 8.27 (2H, d, J = 8.8 Hz, disubstituted CH), 8.45 (1H, s, imidazole CH). 13C-NMR
(75 MHz, DMSO-d6):
δ = 46.2, 47.3, 54.8, 114.7, 117.7, 118.3, 120.2, 124.7, 130.7, 130.8, 131.2, 136.2, 136.6,
140.4, 145.3, 153.0, 187.9. ESI-MS [M + 2H]2+: 187.20 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-ethylpiperazin-1-yl)phenyl)prop-2-en-1-one (3m): Yield: 86%,
1
M.P. = 188–189 ◦C, FTIR (ATR, cm−1): 3125 (C-H), 1653 (C=O), 1337 (C=N), 814. H-NMR (300 MHz,
DMSO-d6):
δ 1.03 (3H, t, J = 7.2 Hz, CH3), 2.37 (2H, q, J = 7.2 Hz, CH2), 2.48 (4H, t, J = 5.0 Hz, piperazine
CH2), 3.30 (4H, t, J = 5.0 Hz, piperazine CH2), 6.99 (2H, d, J = 8.9 Hz, disubstituted CH), 7.17 (1H, s,
imidazole CH), 7.73–7.77 (4H, m, disubstituted CH, -HC=CH-), 7.87 (2H, d, J = 8.8 Hz, disubstituted
CH), 7.91 (1H, t, J = 1.3 Hz, imidazole CH), 8.28 (2H, d, J = 8.8 Hz, disubstituted CH), 8.46 (1H, s,
imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 12.4, 47.4, 52.1, 52.6, 114.6, 117.7, 118.3, 120.2, 124.7,
130.7, 130.8, 131.2, 136.2, 136.6, 140.4, 145.4, 153.1, 187.9. ESI-MS [M + 2H]2+: 194.20 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)phenyl)prop-2-en-1-one (3n):
1
Yield: 78%, M.P. = 146–149 ◦C, FTIR (ATR, cm−1): 3036 (C-H), 1655 (C=O), 1300 (C=N), 812. H-NMR
(300 MHz, DMSO-d6):
δ 2.14 (6H, s, CH3), 2.33–2.44 (4H, m, CH2), 2.52 (4H, t, J = 4.7 Hz, piperazine
CH2), 3.28 (4H, t, J = 4.7 Hz, piperazine CH2), 6.97 (2H, d, J = 8.9 Hz, disubstituted CH), 7.16 (1H, s,
imidazole CH), 7.72–7.76 (4H, m, disubstituted CH, -HC=CH-), 7.86 (2H, d, J = 8.7 Hz, disubstituted
CH), 7.91 (1H, t, J = 1.2 Hz, imidazole CH), 8.27 (2H, d, J = 8.7 Hz, disubstituted CH), 8.45 (1H, s,
imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ = 40.0, 47.4, 53.3, 56.3, 57.1, 114.6, 117.6, 118.3, 120.2,
124.7, 130.7, 130.8, 131.2, 136.2, 136.6, 140.4, 145.4, 153.0, 187.9. ESI-MS [M + H]+: 430.35 (100%).
1-(4-(1H-Imidazol-1-yl)phenyl)-3-(4-(4-(3-(dimethylamino)propyl)piperazin-1-yl)phenyl)prop-2-en-1-one (3o):
1
Yield: 79%, M.P. = 138–141 ◦C, FTIR (ATR, cm−1): 3086 (C-H), 1655 (C=O), 1339 (C=N), 812. H-NMR
(300 MHz, DMSO-d6):
δ 1.56 (2H, p, J = 7.3 Hz, CH2), 2.10 (6H, s, CH3), 2.20 (2H, t, J = 7.3 Hz, CH2), 2.31
(2H, t, J = 7.3 Hz, CH2), 2.47 (4H, t, J = 4.8 Hz, piperazine CH2), 3.28 (4H, t, J = 4.8 Hz, piperazine CH2),
6.97 (2H, d, J = 8.9 Hz, disubstituted CH), 7.16 (1H, s, imidazole CH), 7.70-7.76 (4H, m, disubstituted
CH, -HC=CH-), 7.86 (2H, d, J = 8.7 Hz, disubstituted CH), 7.91 (1H, t, J = 1.2 Hz, imidazole CH),
8.27 (2H, d, J = 8.7 Hz, disubstituted CH), 8.45 (1H, s, imidazole CH). 13C-NMR (75 MHz, DMSO-d6):
δ
= 24.9, 45.7, 47.3, 53.0, 56.5, 57.8, 113.7, 114.6, 118.2, 120.2, 124.6, 130.7, 130.8, 131.2, 136.2, 136.6, 140.4,
145.3, 153.0, 187.9. ESI-MS [M + H]+: 444.35 (100%).
3.2. Antifungal Activity
Microbiological studies were performed according to the EUCAST definitive (EDef 7.1)
method [34] for C. albicans (ATCC 24433), C. krusei (ATCC 6258), C. parapsilosis (ATCC 22019), and
C. glabrata (ATCC 90030). Fluconazole and ketoconazole were used as control drugs. Two MIC
readings were carried out for each chemical agent. The yeasts were maintained in RPMI after overnight
◦
incubation at 37 C. The inocula of test microorganisms was 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 the antifungal assay. Testing was carried out in RPMI at pH = 7, and
the two-fold serial dilutions technique was applied. The last well on the microplates containing
only inoculated broth was kept as a control, and the last well with no growth of microorganism was
recorded to represent the MIC50 expressed in µg/mL. For the antifungal assays, the compounds were

Molecules 2018, 23, 831
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dissolved in DMSO. Further dilutions of the compounds and standard drugs in the test medium were
prepared at the required quantities of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0.78 g/mL
concentrations RPMI. The completed plates were incubated for 24 h. At the end of the incubation,
resazurin (20 g/mL) was added into each well to control the growth in the wells. Completed plates
µ
µ
were incubated for 2 h. MIC50 values were determined using a microplate reader at 590 nm excitation
and 560 nm emission. Each experiment in the antimicrobial assays was replicated twice in order to
define the MIC50 values given in Table 1.
3.3. Quantification of Ergosterol Level
Extraction of total sterols from C. krusei was performed as recorded by Breivik and Owades [35].
Quantification of ergosterol level in this extract was carried out in accordance with our recently
described method [36].
3.4. Cytotoxicity Test
Cytotoxicity was tested using the NIH/3T3 mouse embryonic fibroblast cell line (ATCC®
CRL-1658
NIH/3T3 cells were seeded at 1
performed as previously described [37
™
, London, UK). NIH/3T3 cells were incubated according to the supplier’s recommendations.
104 cells into each well of the 96-well plates. The MTT assay was
38]. The compounds were tested between 800 and 0.78 g/mL
×
,
µ
concentrations. Inhibition % was calculated for each concentration according to the formula below,
and IC50 values were determined by plotting a dose–response curve of inhibition % versus compound
concentrations tested [39].
% inhibition = 100 − (mean sample × 100/mean solvent).
3.5. Molecular Docking Studies
A structure-based in silico procedure was applied to discover the binding modes of the most
active compound 3c to 14-alpha-sterol demethylase enzyme active sites. The crystal structure of the
enzyme (PDB ID: 1EA1) [33], which was crystallized with the reference drug (fluconazole) of antifungal
activity assay, was retrieved from the Protein Data Bank server (www.pdb.org).
The structure of the ligand was built using the Schrödinger Maestro [40] interface and was
then submitted to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016 Update 2 [41].
The ligands were prepared by LigPrep 3.8 [42] 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 Glide 7.1 [43], and docking runs were performed with the
standard precision docking mode (SP).
4. Conclusions
In the present study, 15 new imidazole compounds that incorporate chalcone pharmacophores as
antifungal agents were designed and synthesized. Activity studies revealed the potency of Compounds
3a–3d as antifungal agents. Furthermore, toxicological study enhanced the biological importance of
these compounds. Results of the ergosterol level quantification assay revealed that the mechanism
of action of the compounds is related to the inhibition of the biosynthesis of ergosterol, a vital sterol
regulating membrane fluidity, plasma membrane biogenesis, and function. The docking studies clearly
explained the molecular interacting mode of Compound 3c in the active region of 14-alpha-sterol
demethylase. Consequently, all of this information may pave the way for medicinal chemists to
synthesize similar compounds that have enhanced antifungal profiles.
Supplementary Materials: The following are available online.
Acknowledgments: This study was financially supported by Anadolu University Scientific Projects Fund, Project
No.: 1705S183.

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Author Contributions: Y.O. 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.C. performed activity tests; B.N.S. performed docking
studies; Ö.A. performed the toxicity tests; D.O., B.K.Ç., B.N.S., S.L., U.A.Ç., Ö.A., Y.O. and Z.A.K. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3a–3o are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).