Synthesis, Characterization, and Molecular Docking Study of Some Novel Imidazole Derivatives as Potential Antifungal Agents

Article abstract of DOI:10.1002/jhet.3388

The azole pharmacophore is still regarded as a viable lead structure for the synthesis of more effective antifungal agents. In this study, two novel series of imidazole derivatives containing dithiocarbamate (5a–5g) and (benz)azolethiol (6a–6n) side chains that are structurally related to the famous antifungal azole pharmacophore were synthesized, and the structures of them were characterized by spectral (IR, ¹H NMR, ¹³C NMR, and MS spectra) analyses. The synthesized compounds were screened in vitro antifungal activity against pathogenic strains fungi. Theoretical ADME (absorption, distribution, metabolism, and excretion) predictions were calculated for final compounds. A molecular docking study of the most active compound with target “lanosterol 14α-demethylase” (CYP51) was performed to unravel the mode of antifungal action. Compound 5e, which features imidazole and 4-methoxybenzyl piperazine scaffolds, showed the most promising antifungal activity with an MIC₅₀ value of 0.78?μg/mL against C. krusei. Effect of the compound 5e against ergosterol biosynthesis was observed by LC–MS–MS method, which is based on quantification of ergosterol level in C.?krusei.

Full text of DOI:10.1002/jhet.3388

Month 2018 Synthesis, Characterization, and Molecular Docking Study of Some Novel
Imidazole Derivatives as Potential Antifungal Agents
Ayşen Işık,a Ulviye Acar Çevik,a,b Begüm Nurpelin Sağlık,a,b and Yusuf Özkaya,b
*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
bDoping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir,
Turkey
*E-mail: yozkay@anadolu.edu.tr
Received May 23, 2018
DOI 10.1002/jhet.3388
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
The azole pharmacophore is still regarded as a viable lead structure for the synthesis of more effective an-
tifungal agents. In this study, two novel series of imidazole derivatives containing dithiocarbamate (5a–5g)
and (benz)azolethiol (6a–6n) side chains that are structurally related to the famous antifungal azole
1
pharmacophore were synthesized, and the structures of them were characterized by spectral (IR, H NMR,
13C NMR, and MS spectra) analyses. The synthesized compounds were screened in vitro antifungal activity
against pathogenic strains fungi. Theoretical ADME (absorption, distribution, metabolism, and excretion)
predictions were calculated for final compounds. A molecular docking study of the most active compound
with target “lanosterol 14α-demethylase” (CYP51) was performed to unravel the mode of antifungal action.
Compound 5e, which features imidazole and 4-methoxybenzyl piperazine scaffolds, showed the most prom-
ising antifungal activity with an MIC50 value of 0.78 μg/mL against C. krusei. Effect of the compound 5e
against ergosterol biosynthesis was observed by LC–MS–MS method, which is based on quantification of
ergosterol level in C. krusei.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
fluorocytosine) [7]. Among these agents, azole groups are
most widely used in antifungal therapy [8].
In recent years, the incidences of systemic fungal
infections are increasing dramatically as a consequence of
various factors, including the indiscriminate use of
antibiotics, the extensive practice of organ transplants,
and the rise in drug addiction and disease that suppress
the immune system [1–3]. Currently, the available
antifungal agents to treat fungal infections can be divided
into four categories based on their mode of action,
including the polyenes (e.g., amphotericin B and nystatin)
[4], echinocandins (e.g., caspofungin and micafungin)
[5], azoles (e.g., ketoconazole, fluconazole, voriconazole,
and itraconazole) [6], and antimetabolites (e.g., 5-
Azole antifungal drugs mainly act by inhibiting CYP51
or lanosterol-14α-demethylase, an enzyme necessary in
ergosterol biosynthesis, through a mechanism in which
the heterocyclic nitrogen of the azole binds (N-3 of
imidazole) as the sixth ligand to the heme iron present in
the enzyme, thereby altering the structure of the active
site and acting as non-competitive inhibitors. In addition
to the N-3 of imidazole, a second nitrogen is thought to
interact directly with the apoprotein of lanosterol
demethylase. It is thought that the position of this second
nitrogen in relation to the apoprotein may determine the
specificity of different azole drugs for the enzyme [9–11].
© 2018 Wiley Periodicals, Inc.

A. Işık, U. Acar Çevik, B. N. Sağlık, and Y. Özkay
Vol 000
The accumulation of 14-α-methylated sterols and depletion
of ergosterol alter membrane fluidity, thereby increasing its
permeability. The effectiveness of azoles as inhibitors of
the 14-α-demethylase has been supported through several
experiments [12–16].
In addition to the azole group, dithiocarbamates are a
well-known class of compounds that have been shown to
possess antifungal activity [17]. It has been reported that
thiocarbamate derivatives disturbed the cell wall
biosynthesis of the pathogen by inhibiting the ergosterol
biosynthesis [18]. In recent studies, the compounds in
which dithiocarbamate moiety was combined with
different heterocycles became promising candidates for
new antifungal agent investigation [19–24].
Clinically, representative antifungal drugs have certain
limitation such as narrow spectrum of activity, non-optimal
pharmacokinetics, and the emergence of drug resistance
(generally azoles). Therefore, there is an urgent need for
development of antifungal agents of new molecular
scaffolds with high efficiency, broad spectrum, and optimal
pharmacokinetics that are highly desirable [25,26].
In the current work, in order to take the advantage of the
antifungal properties of imidazoles, we synthesized new 2-
substituted-N-[4-(1H-imidazole-1-yl)phenyl]acetamide (5a–
5g, 6a–6n) derivatives and evaluated antifungal activities of
these compounds.
In the 1H-NMR spectra, piperazine was seen between 3.38
and 4.32 ppm as three singlet peaks. Conformational mobility
of cyclic compounds is greatly limited; therefore, less
rotational averaging of various chemical shift anisotropic
effects occurs. Thus, the chemical shift values of the
equatorial and axial hydrogens of the piperazine ring were
seen at different regions as different 2H, 2H, and 4H peaks
in agreement with the literature [27]. Methylene protons
between carbonyl and dithioate groups were recorded as a
singlet peak between 4.26 and 4.32 ppm. N–H proton of
amide group gave singlet at 10.45–10.50 ppm, and aromatic
protons were observed in the range of 6.89–8.40 ppm. The
protons of the methyl (–CH3) and methoxy (–OCH3)
substituents were observed as a singlet peak between 2.34
and 2.49 ppm and 3.82 and 3.89 ppm, respectively. In the
13C-NMR spectra, peaks about 195 and 165 ppm were
assigned to carbon atoms of C¼S and C¼O groups,
respectively. In the HRMS spectra, all measured mass and
isotope scores were compatible with calculated values for
the compounds (5a–g).
Antifungal activity.
The target compounds were
evaluated for their antifungal 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 [28]. Broth
microdiluation methods were used to determine the
minimum inhibitory concentrations (MICs) of the final
compounds in 96-well microtest plates. MIC50 values
were evaluated via fluorometric measurements, using
resazurin solution [29,30]. Ketoconazole and fluconazole
were used as reference drugs. The in vitro antifungal
activities of the synthesized compounds were listed in
Table 1.
In terms of chemical structure, synthesized compounds can
be separated in two main groups. The first group includes the
compounds 5a–5g, which carry dithiocarbamate side chain.
In the second group, the compounds 6a–6n bear (benz)
azolethiol substructure. In general, the compounds in the
first group displayed better antifungal activity than the
compounds in the second group. The compounds 5a–5g
indicated comparable anticandidal activity to reference
drugs ketoconazole and fluconazole. C. krusei (ATCC
6258) was the most sensitive fungal strain against these
compounds. Compound 5e showed the most potent activity
against C. krusei (ATCC 6258), with an MIC50 value of
0.78 μg/mL, while the MIC50 value of the reference drugs
was 1.56 μg/mL against same Candida strain.
RESULTS AND DISCUSSION
Chemistry.
In the synthetic procedure of the 2-
substituted-N-[4-(1H-imidazole-1-yl)phenyl]acetamide (5a–
g, 6a–n) derivatives, initially microwave supported
synthesis of 1-(4-nitrophenyl)-1H-imidazole (1) was
performed in DMF. In the next step, reduction of compound
1 by Zn/HCl in EtOH gave 1-(4-aminophenyl)-1H-
imidazole (2), which was then acetylated with chloroacetyl
chloride in conventional ways to afford 2-chloro-N-[4-(1H-
imidazole-1-yl)phenyl]acetamide (3). Compound 3 was
reacted with dithiocarbamate sodium salts (4a–4g) or
(benz)azolethiol derivatives in acetone to obtain target
compounds (5a–5g, 6a–6n). Synthetic route for the final
compounds was outlined in Scheme 1. Structure
elucidations of the final compounds (5a–g, 6a–n) were
performed by FT-IR, 1H-NMR, 13C-NMR, and MASS
spectroscopic methods. The stretching bands for C¼O
and C¼S were observed between 1622 and 1691 cmꢀ1
and 1200 and 1246 cmꢀ1
,
respectively. Stretching
In terms of short structure activity relationships, it is
observed that 4-benzylpiperazine moiety in the
dithiocarbamate side chain of compounds 5d and 5e
makes higher contribution to anticandidal effect when
compared with other moieties. Furthermore, 4-methoxy
substitution of benzyl fragment in compound 5e enhances
the anticandidal activity.
absorption of N–H groups was observed at 3041–
3305 cmꢀ1 as expected. The stretching absorption at
about 1419–1639 cmꢀ1 was recorded for C¼C and C¼N
double bonds in heteroaromatic rings. The stretching
absorption belonging to 1,4-disubstituted benzene was
determined at 813–843 cmꢀ1
.
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Scheme 1. Synthesis way for target compounds.
Inhibition of ergosterol biosynthesis. Infections caused
by eukaryotic organisms like fungus generally present
more difficult treatment problems than those of bacterial
infections. There are relatively few antifungal drugs that
can identify unique targets not shared with human hosts.
The fungal cell wall remains an underdeveloped
therapeutic target for selective antifungal agents because
of its chitin structure, which is absent in human cells
[16,31].
The present work is an attempt to understand the
mechanism of antifungal activity of newly synthesized
imidazole-dithiocarbamate hybrid compound 5e. Most
therapies, designed to treat fungal infections, target the
ergosterol biosynthesis pathway. It is the main sterol of
fungi cell membranes and is necessary for fluidity,
permeability, and protein function [32], and hence,
inhibition of ergosterol biosynthesis causes the end of life
functions.
and Owades [33]. Ergosterol standard (product no.
45480, Sigma-Aldrich, Germany) was used for quantifica-
tion of ergostrerol in both inhibitor-free (negative control)
and inhibitor including samples. The most active com-
pound 5e and reference drugs were used at 0.78-, 1.56-,
and 3.12-μg/mL concentrations. Ergosterol quantity in
negative control samples was regarded as 100%. All con-
centrations were analyzed in quadruplicate, and the results
were expressed as mean standard deviation (Fig. 1).
Ergosterol quantification studies revealed that
compound 5e and reference agents significantly decreased
the level of ergosterol at all tested concentrations. A
concentration-dependant decrease in the ergosterol level
was established for all tested agents. Hence, it can be
obviously suggested that compounds 5e have a role in the
ergosterol biosynthesis pathway.
Prediction of ADME parameters.
Most of new drug
candidates fail in clinical trials because of their poor
ADME (absorption, distribution, metabolism, and
excretion) properties. These late-stage failures contribute
significantly to the rapidly increasing cost of new drug
For quantitative determination of ergosterol level of
C. krusei, we used an LCMSMS method. Total
intracellular sterols were extracted as reported by Breivik
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Table 1
This program applies the Lipinski’s rule of five [36] and
Jorgensen’s rule of three [37], which evaluate the ADME
properties of drug like compounds, and is important for
the optimization of a biologically active compound. The
theoretical calculations of ADME parameters (molecular
weight, log P, polar surface area, number of hydrogen
donors, number of hydrogen acceptors, number of
rotatable bonds, and volume) are presented in Table 2
along with the violations of rules of three and five.
According to Lipinski’s rule of five, all compounds (5a–
g, 6a–n) abide to the rule by causing no more than one
violation. Also, these compounds are suited to
Jorgensen’s rule of three with no more than one
violation. Besides, it can be seen that all results of rules
of three and five are within recommended ranges. Thus, it
can be suggested that all synthesized compounds may
possess a good pharmacokinetic profile, increasing their
pharmacological importance.
MIC50 (μg/mL) values of compounds (5a–g, 6a–n).
Comp.
C. albicans C. glabrata C. krusei C. parapsilosis
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6.25
25
3.125
6.25
6.25
3.125
3.125
12.5
6.25
200
200
200
200
200
200
200
200
200
200
200
200
200
1.56
3.125
3.125
1.56
0.78
3.125
3.125
200
200
200
200
200
200
200
200
200
200
200
200
200
6.25
6.25
6.25
3.125
1.56
6.25
12.5
200
200
100
200
200
100
100
100
200
100
200
100
200
100
1.56
0.78
3.125
3.125
1.56
12.5
12.5
200
200
25
100
200
200
100
200
200
200
100
100
100
100
Molecular docking. Docking studies were performed
in order to gain more insight into the binding modes of
compound 5e to 14-α-sterol demethylase, which is a key
enzyme in ergosterol biosynthesis of fungi. Studies were
carried out by using the X-ray crystal structure of 14-α-
sterol demethylase from Mycobacterium tuberculosis in
complex with fluconazole (PDB ID: 1EA1) [38].
6m
6n
200
1.56
1.56
200
1.56
1.56
Ketoconazole 0.78
Fluconazole 0.78
development. The ability to detect problematic candidates
early can dramatically reduce the amount of wasted time
and resources and streamline the overall development
process. Hence, pharmacokinetic profiles of the new drug
candidates are very important and should be evaluated as
early as possible in the drug development process.
ADME prediction can be used to focus lead optimization
efforts to enhance the desired properties of a given
compound [34]. Thus, predictions of ADME properties of
the all synthesized compounds (5a–g, 6a–n) were
performed by QikProp 4.8 software [35].
According to the antifungal activity results, the
compound 5e shows significant antifungal activity against
C. krusei with an MIC50 value of 0.78 μg/mL. Thus, the
main purpose is to investigate the possible interaction of
this compound with cytochrome P450 14-α-sterol
demethylase from C. krusei. However, this enzyme is a
membrane-bound enzyme, and it is difficult to crystalize
for X-ray analysis and modeling studies. Moreover, there
are 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, origin
Figure 1. Relative ergosterol level (REL) of Candida krusei (ATCC 6258), after treatment with compound 5e, fluconazole, and ketoconazole. [Color
figure can be viewed at wileyonlinelibrary.com]
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Imidazole Derivatives as Potential Antifungal Agents
Table 2
Calculated ADME parameters (5a–g, 6a–n).
Comp.
MW
RB
V
DHB
AHB
QPlogPo/w
PSA
VRF
VRT
5a
5b
5c
5d
5e
5f
5 g
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
360.491
450.616
375.506
451.603
481.630
482.574
455.567
349.409
363.436
394.407
379.436
383.854
366.455
396.481
400.900
313.376
331.410
300.337
314.364
315.352
311.361
4
6
4
6
7
5
4
4
4
5
5
4
4
5
4
4
4
4
4
4
4
1.144.028
1.404.067
1.183.501
1.414.058
1.473.919
1.437.640
1.371.342
1.108.186
1.166.642
1.190.101
1.176.943
1.152.129
1.130.869
1.199.740
1.174.760
1.017.541
1.035.668
947.218
1
1
1
1
1
1
1
2
2
2
2
2
1
1
1
1
1
2
1
1
1
6
6
8
8
9
8
7
6
6
7
6
6
6
6
6
6
6
7
7
7
6
4.026
5.885
2.860
4.512
4.465
4.471
5.414
3.609
3.903
2.925
3.675
4.094
4.219
4.273
4.709
3.169
2.956
1.559
1.703
1.174
2.857
62.451
61.658
68.023
67.883
76.171
117.017
68.352
80.595
80.419
129.062
89.076
80.587
65.645
74.129
65.643
68.927
81.796
97.922
85.311
105.034
76.386
0
1
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
1
1
1
0
1
1
1
1
1
1
1
0
0
0
0
0
0
1.004.941
994.109
1.001.472
6m
6n
MW, molecular weight; RB, number of rotatable bonds (recommended value: 0–15); V, 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); QPlogPo/w, predicted octanol/water partition coefficient (recommended value: ꢀ2–6.5); PSA, Van
der Waals surface area of polar nitrogen and oxygen atoms and carbonyl carbon atoms (recommended value: 7–200); VRF, number of violations of
Lipinski’s rule of five [36]. The rules are mol_MW < 500, QPlogPo/w < 5, donorHB ≤ 5, and accptHB ≤ 10. Compounds that satisfy 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
[37]. The three rules are QPlogS > ꢀ5.7, QP PCaco > 22 nm/s, and # primary metabolites < 7. Compounds with fewer (and preferably no) violations of
these rules are more likely to be orally available (maximum is 3).
of Candida P450 and Mycobacterium P450. These
enzymes have high homology and high degree of
similarity between the hydrophobic cavities of the
catalytic site [39–42]. Among them, Mycobacterium
P450 have been presented with higher resolution.
Depending on these reasons, we choose the PDB ID:
1EA1 crystal structure from M. tuberculosis to obtain
clearer pose (Fig. 2).
Figure 2. The interacting mode of compound 5e in the active region of 14 alpha-sterol demethylase. The inhibitor is colored with purple and HEM with
red. [Color figure can be viewed at wileyonlinelibrary.com]
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The docking pose on 14-α-sterol demethylase reveals
that the interactions between the compound 5e and
HEM450 are very important in terms of binding to active
site of enzyme (Fig. 2). The HEM450 establishes five
metal coordination bonds with the 1st nitrogen of
piperazine, the carbonyl and amino groups of amide
function, and two nitrogens of imidazole ring. The 3rd
nitrogen of imidazole has a part in polar interactions
along with metal bond. There is a hydrogen bond
between this nitrogen and Thr264. Also, phenyl ring
creates two π–π interaction with the phenyl of Tyr76 and
Phe78. The methoxy group creates a hydrogen bond with
the amino group of Val434. This interaction emphasizes
why the compound 5e is more active than the other
synthesized derivatives. The methoxy group helps to
strengthen the polar interaction differently from other
substituents.
spectrometer. HRMS studies were performed on Shimadzu
LCMS-IT-TOF system (Shimadzu, Tokyo, Japan). LC–
MS–MS studies were performed on a Schimadzu, 8040
LC–MS–MS spectrophotometer (Shimadzu, Tokyo,
Japan). Chemical purities of the compounds were checked
by classical TLC applications performed on silica gel 60
F254 (Merck KGaA).
General procedure for 1-(4-nitrophenyl)-1H-imidazole
(1). 1-Iodo-4-nitrobenzene (9.96 g, 0.04 mol), K2CO3
(5.52 g, 0.04 mol), 1H-imidazole (2.72 g, 0.04 mol) in
DMF (10 mL) were put into a vial (30 mL) of microwave
synthesis reactor (Anton-Paar Monowave 300). The
reaction mixture was kept under the conditions of 200°C
and 10 bar for 15 min. After cooling, the mixture was
poured into iced-water; precipitated product was washed
with water, dried, and recrystallized from ethanol. Yield:
89%; m.p. 201–204°C. Literature m.p. 203°C [43].
General procedure for 1-(4-aminophenyl)-1H-imidazole
(2).
1-(4-Nitrophenyl)-1H-imidazole (1) (6.62 g,
CONCLUSIONS
0.035 mol) was dissolved in ethanol (50 mL), and 25%
HCl (50 mL) was added. Zinc powder (39.49 g,
0.35 mol) was divided into 10 equal portions, and each
portion was added to the stirring solution in 15-min
intervals. Once the addition of the zinc was completed,
reaction mixture was refluxed for 1 h. After cooling, the
mixture was poured into iced-water and then neutralized
by using 10% NaOH solution. The precipitate was
extracted with ethyl acetate in portions (3 × 100 mL).
Extracts were combined and dried with anhydrous
sodium sulfate. The solvent was evaporated, and the
residue was recrystallized from ethanol to give the 1-(4-
aminophenyl)-1H-imidazole (2). Yield: 71%; m.p. 142–
144°C. Literature m.p. 141–143°C [44].
In summary, we discovered a novel class of compounds
indicating important anticandidal effects. In addition to
good antifungal activity, all compounds in the series
exhibited a good predicted pharmacokinetics profile.
Furthermore, preliminary mechanism of action study
showed that the potent antifungal activity of compound
5e is related to inhibition of ergosterol biosynthesis in
C. krusei. Significant interactions were also observed
between compound 5e and 14-α-sterol demethylase. We
expect that in further studies, all these findings may have
an effect on medicinal chemists to discover more
promising anticandidal compounds.
General procedure for N-[4-(1H-imidazol-1-yl)phenyl]-2-
chloro-acetamide (3).
1-(4-Aminophenyl)-1H-imidazole
EXPERIMENTAL
(2) (3.18 g, 0.02 mol) and triethylamine (3.1 mL,
0.022 mol) in THF (100 mL) were allowed to stir on an
ice bath. Chloroacetylchloride (1.75 mL, 0.022 mol) in
THF (10 mL) was added drop by drop. After this stage,
the content was stirred for 1 h at room temperature. THF
was evaporated, and the product was recrystallized from
ethanol. Yield: 75%; m.p. 230–234°C.
Chemistry. All of the chemicals used in the study were
purchased from either Sigma-Aldrich Corp. (St. Louis,
MO) or Merck KGaA (Darmstadt, Germany) and used
without further chemical purifications. Microwave synthe-
ses were realized by using a Monowave 300 high-
performance microwave reactor (Anton-Paar, Austria).
Melting points of the synthesized compounds were deter-
mined by using a MP90 series automatic melting point
determination system (Mettler-Toledo, OH) and were pre-
General procedure for the synthesis of sodium
dithiocarbamate derivatives (4a–g).
Secondary amine
derivative (0.01 mol) and sodium hydroxide (0.4 g,
0.01 mol) in ethanol (10 mL) were cooled in an ice bath,
and CS2 (6 mL) was added drop by drop. The reaction
was allowed to stir for 1 h at room temperature. The
solvent and excess of CS2 were removed under reduced
pressure. The residue was washed with dry ether, and the
raw product was recrystallized from ethanol.
1
sented as uncorrected. H and 13C NMR spectra were re-
corded in DMSO-d6 by a Bruker digital FT-NMR
spectrometer (Bruker Bioscience, Billerica, MA) at 300,
75 or 500, and 125 MHz [splitting patterns in the NMR
spectra were designated as follows: s, singlet; d, doublet;
t, triplet; m, multiplet; coupling constants (J) were reported
in Hertz]. The IR spectra of the compounds were recorded
using an IRAffinity-1S Fourier transform IR (FTIR)
General procedure for the synthesis of 2-(substitute
ddithiocarbamoyl)-N-[4-(1H-imidazol-1-yl)phenyl]acetamide
derivatives (5a–g). Corresponding sodiumdithiocarbamate
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(4a–g) (0.001 mol) and N-[4-(1H-imidazol-1-yl)phenyl]-2-
chloro-acetamide (3) (0.24 g, 0.001 mol) in acetone were
refluxed for 2 h. After TLC control, the solvent was
evaporated; the residue was washed with water, dried,
and then recrystallized from ethanol to afford final
compounds (5a–g).
2-(Piperidine-1-yl-dithiocarbamoyl)-N-[4-(1H-imidazol-1-yl)
phenyl]acetamide (5a). Yield: 83%; m.p. 165.6–170.2°C.
2-(4-Benzylpiperazine-1-yl-dithiocarbamoyl)-N-[4-(1H-
imidazol-1-yl)phenyl]acetamide (5d).
Yield: 81%; m.p.
90.4–94.8°C. FTIR (ATR, cmꢀ1): 3116 (N–H), 1674
(C¼O), 1228 (C¼S), 833 (1,4-disubstituted benzene).
1H-NMR (300 MHz) (DMSO-d6) δ (ppm): 10.44 (1H, s,
N–H), 8.18 (1H, s, imidazole, C2-H), 7.70 (2H, d,
J = 8.8 Hz, 1,4-disubstituted benzene), 7.68 (1H, s,
imidazole-C5-H), 7.58 (2H, d, J = 8.8 Hz, 1,4-
disubstituted benzene), 7.26 (2H, d, J = 6.9 Hz, phenyl
H2, H6), 7.18–7.12 (3H, m, phenyl H3, H4, H5), 7.08
(1H, s, imidazole-C4-H), 4.26 (2H, s, –CH2–), 4.20 (2H,
br.s, piperazine –CH2–), 3.94 (2H, br.s, piperazine –CH2–),
3.35 (4H, br.s, piperazine –CH2–), 2.42 (2H, s, –CH2).
13C-NMR (75 MHz, DMSO-d6): δ (ppm): 41.70, 50.28,
51.59, 52.39, 61.73, 118.49, 120.50, 121.28, 127.60,
128.72, 129.43, 130.12, 132.67, 135.84, 137.96, 138.26,
165.96, 194.80. [M + H]+ calcd for C23H25N5OS2:
FTIR (ATR, cmꢀ1): 3041 (N–H), 1691 (C¼O), 1228
(C¼S), 842 (1,4-disubstituted benzene). 1H-NMR
(300 MHz) (DMSO-d6) δ (ppm): 10.45 (1H, s, N–H),
8.18 (1H, s, imidazole, C2-H), 7.70 (2H, d, J = 8.7 Hz,
1,4-disubstituted benzene), 7.68 (1H, s, imidazole-C5-H),
7.58 (2H, d, J = 8.7 Hz, 1,4-disubstituted benzene), 7.09
(1H, s, imidazole-C4-H), 4.26 (2H, s, –CH2–), 4.20 (2H,
s, piperidine –CH2–), 3.94 (2H, s, piperidine –CH2–),
1.60 (6H, br.s, piperidine –CH2–). 13C-NMR (75 MHz,
DMSO-d6): δ (ppm): 23.94, 25.66, 26.29, 41.75, 51.53,
53.04, 118.48, 120.50, 121.31, 130.14, 132.69, 135.83,
452.1573; found: 452.1565.
2-(4-(4-Methoxybenzyl)piperazine-1-yl-dithiocarbamoyl)-N-
[4-(1H-imidazol-1-yl)phenyl]acetamide (5e). Yield: 75%; m.
138.22, 166.06, 193.46. [M
+
H]+ calcd for
p. 184.5–187.9°C. FTIR (ATR, cmꢀ1): 3097 (N–H), 1683
(C¼O), 1224 (C¼S), 823 (1,4-disubstituted benzene). 1H-
NMR (300 MHz) (DMSO-d6) δ (ppm): 10.47 (1H, s, N–
H), 8.18 (1H, s, imidazole, C2-H), 7.71 (2H, d,
J = 8.8 Hz, 1,4-disubstituted benzene), 7.68 (1H, s,
imidazole-C5-H), 7.58 (2H, d, J = 8.8 Hz, 1,4-
disubstituted benzene), 7.22 (2H, d, J = 8.4 Hz, 1,4-
disubstituted benzene), 7.09 (1H, s, imidazole-C4-H),
6.89 (2H, d, J = 8.4 Hz, 1,4-disubstituted benzene), 4.27
(2H, s, –CH2–), 4.21 (2H, br.s, piperazine –CH2–), 3.95
(2H, br.s, piperazine –CH2–), 3.73 (3H, s, OCH3–), 3.40
(4H, br.s, piperazine –CH2), 2.45 (2H, s, –CH2). 13C-
NMR (75 MHz, DMSO-d6): δ (ppm): 41.71, 50.27,
51.59, 52.23, 55.48, 61.12, 114.08, 118.48, 120.50,
121.32, 129.65, 130.13, 130.72, 132.70, 135.83, 138.21,
C17H20N4OS2: 361.1151; found: 361.1146.
2-(4-Benzylpiperidine-1-yl-dithiocarbamoyl)-N-[4-(1H-imidazol-
1-yl)phenyl]acetamide (5b). Yield: 78%; m.p. 86.8–90.8°C.
FTIR (ATR, cmꢀ1): 3109 (N–H), 1681 (C¼O), 1232
(C¼S), 815 (1,4-disubstituted benzene). 1H-NMR
(300 MHz) (DMSO-d6) δ (ppm): 10.48 (1H, s, N–H), 8.19
(1H, s, imidazole, C2-H), 7.71 (2H, d, J = 8.7 Hz, 1,4-
disubstituted benzene), 7.68 (1H, s, imidazole-C5-H), 7.58
(2H, d, J = 8.7 Hz, 1,4-disubstituted benzene), 7.27 (2H, d,
J = 6.9 Hz, phenyl H2, H6), 7.19–7.17 (3H, m, phenyl H3,
H4, H5), 7.09 (1H, s, imidazole-C4-H), 4.26 (2H, s, –CH2–),
2.55–2.50 (4H, m, piperidine –CH2–, –CH2), 1.92 (1H, br.s,
piperidine –CH–), 1.70 (3H, br.s, piperidine –CH2–), 1.24–
1.17 (3H, m, piperidine, –CH2). 13C-NMR (75 MHz,
DMSO-d6): δ (ppm): 35.36, 37.35, 41.83, 41.95, 43.55,
50.56, 52.12, 118.49, 120.49, 121.31, 126.39, 128.68,
129.49, 130.11, 132.68, 135.83, 138.24, 140.33, 166.04,
193.60. [M + H]+ calcd for C24H26N4OS2: 451.1621; found:
158.88, 165.94, 194.73. [M
+
H]+ calcd for
C24H27N5O2S2: 482.1679; found: 482.1677.
2-(4-(4-Nitrophenyl)piperazine-1-yl-dithiocarbamoyl)-N-[4-
(1H-imidazol-1-yl)phenyl]acetamide (5f). Yield: 84%; m.p.
451.1621.
146.8–150.4°C. FTIR (ATR, cmꢀ1): 3109 (N–H), 1689
2-(4-Methylpiperazine-1-yl-dithiocarbamoyl)-N-[4-(1H-imidazol-
1-yl)phenyl]acetamide (5c). Yield: 86%; m.p. 131.3–134.9°C.
1
(C¼O), 1219 (C¼S), 821 (1,4-disubstituted benzene). H-
FTIR (ATR, cmꢀ1): 3118 (N–H), 1689 (C¼O), 1238 (C¼S),
815 (1,4-disubstituted benzene). 1H-NMR (300 MHz)
(DMSO-d6) δ (ppm): 10.76 (1H, s, N–H), 8.24 (1H, s,
imidazole, C2-H), 7.75 (1H, s, imidazole-C5-H), 7.72 (2H, d,
J = 8.7 Hz, 1,4-disubstituted benzene), 7.60 (2H, d,
J = 8.7 Hz, 1,4-disubstituted benzene), 7.11 (1H, s,
imidazole-C4-H), 4.26 (2H, s, –CH2–), 4.31 (2H, br.s,
piperazine –CH2–), 4.15 (2H, br.s, piperazine –CH2–), 3.40
(4H, br.s, piperazine –CH2–), 2.49 (3H, s, CH3). 13C-NMR
(75 MHz, DMSO-d6): δ (ppm): 13.94, 41.88, 43.85, 48.68,
53.14, 118.60, 120.54, 121.41, 129.85, 132.72, 135.83,
138.20, 165.52, 193.52. [M + H]+ calcd for C17H21N5OS2:
376.1260; found: 376.1252.
NMR (300 MHz) (DMSO-d6) δ (ppm): 10.50 (1H, s, N–
H), 8.19 (1H, s, imidazole, C2-H), 8.10 (2H, d, J = 9.3 Hz,
1,4-disubstituted benzene), 7.71 (2H, d, J = 8.7 Hz, 1,4-
disubstituted benzene), 7.68 (1H, s, imidazole-C5-H), 7.59
(2H, d, J = 8.7 Hz, 1,4-disubstituted benzene), 7.09 (1H, s,
imidazole-C4-H), 6.95 (2H, d, J = 9.3 Hz, 1,4-disubstituted
benzene), 4.32 (4H, s, –CH2–, piperazine –CH2–), 4.17
(2H, br.s, piperazine –CH2–), 3.73 (4H, br.s, piperazine
–CH2–). 13C-NMR (75 MHz, DMSO-d6): δ (ppm): 41.60,
42.77, 44.11, 45.16, 112.31, 113.87, 118.49, 121.33,
126.27, 130.11, 132.71, 135.83, 137.30, 138.21, 154.16,
165.87, 195.18. [M + H]+ calcd for C22H22N6O3S2:
483.1268; found: 483.1265.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Işık, U. Acar Çevik, B. N. Sağlık, and Y. Özkay
Vol 000
2-(4-(4-Fluorophenyl)piperazine-1-yl-dithiocarbamoyl)-N-
[4-(1H-imidazol-1-yl)phenyl]acetamide (5g).
(ppm): 21.2, 36.2, 109.1, 109.6, 118.5, 120.0, 121.2,
122.8, 123.2, 128.2, 130.2, 131.6, 131.9, 132.5, 135.2,
138.0, 166.4. LCMS (ESI) m/z: 182.60 (% 100.00),
364.15 (% 78.23), 365.15 (% 17.22), 366.30 (% 5.06).
2-(2-Mercapto-5-nitrobenzimidazole)-N-[4-(1H-imidazol-1-
Yield: 77%;
m.p. 187.2–190.2°C. FTIR (ATR, cmꢀ1): 3049 (N–H),
1670 (C¼O), 1217 (C¼S), 813 (1,4-disubstituted
benzene). 1H-NMR (300 MHz) (DMSO-d6) δ (ppm):
10.48 (1H, s, N–H), 8.18 (1H, s, imidazole, C2-H), 7.71
(2H, d, J = 8.8 Hz, 1,4-disubstituted benzene), 7.68 (1H,
s, imidazole-C5-H), 7.59 (2H, d, J = 8.8 Hz, 1,4-
disubstituted benzene), 7.08–7.05 (3H, m, 1,4-
disubstituted benzene, imidazole-C4-H), 7.00–6.99 (2H,
m, 1,4-disubstituted benzene), 4.31 (4H, s, –CH2–,
piperazine –CH2–), 4.13 (2H, br.s, piperazine –CH2–),
3.38 (4H, br.s, piperazine –CH2–). 13C-NMR (75 MHz,
DMSO-d6): δ (ppm): 41.69, 48.96, 50.01, 51.19, 115.91
(d, J = 21.8 Hz), 117.93 (d, J = 7.5 Hz), 118.49, 120.51,
121.33, 130.13, 132.72, 135.84, 138.20, 147.21, 156.78
(d, J = 234.75 Hz), 165.91, 195.09. [M + H]+ calcd for
C22H22N5OFS2: 456.1323; found: 456.1325.
yl)phenyl]acetamide (6c).
Yield: 73%; m.p. 202.5–
204.1°C. FTIR (ATR, cmꢀ1): 3211 (N–H), 1669 (C¼O),
1616–1453 (C¼C and C¼N), 819 (1,4-disubstituted
1
benzene). H-NMR (500 MHz) (DMSO-d6) δ (ppm): 4.39
(2H, s, CH2-S), 7.09 (1H, s, imidazole, C5-H), 7.59–7.62
(3H, m, benzimidazole, C7-H, phenyl C2,6-H), 7.68 (1H,s,
imidazole C4-H), 7.73 (2H, d, J = 9.0 Hz, phenyl, C3,5-H),
8.07 (1H, dd, J = 8.5 Hz, J = 2.5 Hz, benzimidazole, C6-
H), 8.18 (1H, s, imidazole, C2-H), 8.31 (1H, d,
J = 2.5 Hz, benzimidazole C4-H), 10.63 (1H, s, NH-CO),
13.39 (1H, s, benzimidazole N–H). 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 36.2, 110.5, 115.4, 117.5, 118.0,
120.1, 120.9, 129.7, 132.4, 135.4, 137.5, 138.4, 142.2,
145.8, 148.7, 165.8. LCMS (ESI) m/z: 198.05 (% 100.00),
General procedure for the synthesis of 2-(mercaptoheteroaryl)-
N-[4-(1H-imidazol-1-yl)phenyl]acetamide derivatives (6a–n).
350.15 (% 92.02), 351.15 (% 34.36), 352.10 (% 11.37).
2-(2-Mercapto-5-methoxybenzimidazole)-N-[4-(1H-imidazol-
An appropriate (benz)azole-thiol (0.001 mol), N-[4-(1H-
imidazol-1-yl)phenyl]-2-chloro-acetamide (3) (0.24 g,
0.001 mol), and K2CO3 (0.138 g, 0.001 mol) in acetone were
refluxed for 2 h. After TLC control, the solvent was evaporated;
the residue was washed with water, dried, and then
1-yl)phenyl]acetamide (6d).
Yield: 79%; m.p. 228.4–
230.8°C. FTIR (ATR, cmꢀ1): 3120 (N–H), 1674 (C¼O),
1620–1413 (C¼C and C¼N), 827 (1,4-disubstituted
benzene). 1H-NMR (500 MHz) (DMSO-d6) δ (ppm):
3.76 (3H, s, CH3), 4.24 (2H, s, CH2-S), 6.76 (1H, dd,
J = 8.5 Hz, J = 2.5 Hz, benzimidazole, C6-H), 6.92 (1H,
br.s, benzimidazole, C4-H), 7.08 (1H, t, J = 1.0 Hz,
imidazole, C5-H), 7.35 (1H, br.s, benzimidazole, C7-H),
7.60 (2H, d, J = 9.0 Hz, phenyl, C2,6-H), 7.67 (1H, t,
J = 1.0 Hz, imidazole C4-H), 7.71 (2H, d, J = 9.0 Hz,
phenyl, C3,5-H), 8.18 (1H, t, J = 1.0 Hz, imidazole C2-
H), 10.65 (1H, s, NH-CO), 12.51 (1H, s, benzimidazole-
NH). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 36.3,
55.5, 102.5, 118.0, 120.0, 120.9, 122.3, 123.1, 129.7,
130.4, 131.2, 132.4, 135.4, 137.6, 145.8, 155.7, 166.3.
LCMS (ESI) m/z: 190.60 (% 100.00), 380.10 (% 98.75),
recrystallized from ethanol to afford final compounds (6a–n).
2-(2-Mercaptobenzimidazole)-N-[4-(1H-imidazol-1-yl)
phenyl]acetamide (6a). Yield: 76%; m.p. 230.8–232.7°C.
FTIR (ATR, cmꢀ1): 3223 (N–H), 1641 (C¼O), 1612–
1440 (C¼C and C¼N), 835 (1,4-disubstituted benzene).
1H-NMR (500 MHz) (DMSO-d6) δ (ppm): 4.30 (2H, s,
CH2-S), 7.08 (1H, s, imidazole, C5-H), 7.11–7.15 (2H,
m, benzimidazole,
benzimidazole, C4,7-H), 7.60 (2H, d, J = 9.0 Hz, phenyl,
2,6-H), 7.68 (1H, s, imidazole, C4-H), 7.71 (2H, d,
C5,6-H), 7.40–7.49 (2H, m,
C
J = 9.0 Hz, phenyl, C3,5-H), 8.18 (1H, s, imidazole, C2-
H), 10.67 (1H, s, NH-CO), 12.67 (1H, s, benzimidazole-
NH). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 36.1,
114.6, 118.0, 120.0, 120.9, 129.7, 132.4, 135.4, 137.6,
139.3, 149.0, 149.8, 166.3. LCMS (ESI) m/z: 175.60 (%
100.00), 350.15 (% 92.02), 351.15 (% 34.36), 352.10 (%
381.15 (% 47.57), 382.10 (% 13.70).
2-(2-Mercapto-5-chlorobenzimidazole)-N-[4-(1H-imidazol-1-
yl)phenyl]acetamide (6e).
Yield: 62%; m.p. 232.5–
234.2°C. FTIR (ATR, cmꢀ1): 2985 (N–H), 1672
(C¼O), 1627–1523 (C¼C and C¼N), 839 (1,4-
disubstituted benzene). 1H-NMR (500 MHz) (DMSO-
d6) δ (ppm): 4.31 (2H, s, CH2-S), 7.08–7.14 (3H, m,
benzimidazole C-H), 7.16 (1H, s, imidazole, C5-H),
7.60 (1H, d, J = 8.5 Hz, phenyl, C2,6-H), 7.68 (1H, s,
imidazole, C4-H), 7.60 (2H, s, phenyl C3,5-H), 8.18
(1H, s, imidazole, C2-H), 10.62 (1H, s, NH-CO), 12.67
(1H, s, benzimidazole-NH). 13C-NMR (125 MHz,
DMSO-d6) δ (ppm): 36.2, 108.1, 109.1, 110.5, 118.1,
120.0, 120.9, 122.2, 123.6, 126.6, 128.3, 128.8, 130.9,
131.3, 133.2, 166.5. LCMS (ESI) m/z: 192.55 (%
100.00), 193.40 (% 45.00), 384.10 (% 87.00), 386.05
(% 34.86), 387.10 (% 7.16).
11.37).
2-(2-Mercapto-5-methylbenzimidazole)-N-[4-(1H-imidazol-1-
yl)phenyl]acetamide (6b).
Yield: 73%; m.p. 202.5–
204.1°C. FTIR (ATR, cmꢀ1): 3122 (N–H), 1672 (C¼O),
1618–1467 (C¼C and C¼N), 835 (1,4-disubstituted
benzene). 1H-NMR (500 MHz) (DMSO-d6) δ (ppm):
2.33 (3H, s, CH3), 4.27 (2H, s, CH2-S), 6.92–6.96 (3H,
m, imidazole, C5-H, benzimidazole, C4,6-H), 7.01 (1H, d,
J = 8.0 Hz, benzimidazole, C7-H), 7.62 (2H, d,
J = 9.0 Hz, phenyl, C2,6-H), 7.73 (2H, d, J = 9.0 Hz,
phenyl, C3,5-H), 7.78 (1H, s, imidazole, C4-H), 8.43 (1H,
s, imidazole, C5-H), 10.72 (1H, s, NH-CO), 12.41 (1H, s,
benzimidazole-NH). 13C-NMR (125 MHz, DMSO-d6) δ
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Imidazole Derivatives as Potential Antifungal Agents
2-(2-Mercaptobenzothiazole)-N-[4-(1H-imidazol-1-yl)phenyl]
acetamide (6f). Yield: 77%; m.p. 190.2–193.5°C. FTIR
1421 (C¼C and C¼N), 833 (1,4-disubstituted benzene).
1H-NMR (500 MHz) (DMSO-d6) δ (ppm): 3.61 (3H, s,
CH3), 3.89 (2H, s, CH2-S), 6.97 (1H, d, J = 1.0 Hz, N-
methylimidazole C4-H), 7.10 (1H, s, imidazole, C5-H),
7.26 (1H, d, J = 1.0 Hz, N-methylimidazole C5-H), 7.58
(2H, d, J = 9.0 Hz, phenyl C2,6-H), 7.67 (3H, d,
J = 9.0 Hz, phenyl C3,5-H, imidazole C4-H), 8.19 (1H, s,
imidazole C2-H), 10.52 (1H, s, NH-CO). 13C-NMR
(125 MHz, DMSO-d6) δ (ppm): 33.0, 38.3, 118.0, 120.0,
120.9, 123.6, 128.5, 129.6, 132.3, 135.4, 137.6, 139.5,
166.6. LCMS (ESI) m/z: 157.65 (% 100.00), 314.30 (%
(ATR, cmꢀ1): 3182 (N–H), 1665 (C¼O), 1608–1423
(C¼C and C¼N), 835 (1,4-disubstituted benzene). 1H-
NMR (500 MHz) (DMSO-d6) δ (ppm): 4.24 (2H, s, CH2-
S), 7.35 (1H, s, imidazole, C5-H), 7.36 (1H, t,
J = 7.0 Hz, benzothiazole, C5-H), 7.48 (1H, t, J = 7.0 Hz,
benzothiazole C6-H), 7.60 (2H, d, J = 9.0 Hz, phenyl
C
2,6-H), 7.68 (1H, s, imidazole, C4-H), 7.72 (2H, d,
J = 9.0 Hz, phenyl, C3,5-H), 7.83 (1H, d, J = 8.0 Hz,
benzothiazole C7-H), 8.03 (1H, d, 8.0 Hz,
J
=
88.16), 315.05 (% 23.16), 316.10 (% 9.78).
2-(2-Mercapto-5-methyl-1,3,4-thiadiazole)-N-[4-(1H-
benzothiazole C6-H), 8.19 (1H, s, imidazole C2-H), 10.61
(1H, s, NH-CO). 13C-NMR (125 MHz, DMSO-d6) δ
(ppm): 37.7, 118.0, 120.2, 120.9, 121.1, 121.9, 124.5,
126.4, 129.7, 132.5, 134.8, 135.4, 137.5, 152.5, 165.3,
166.0. LCMS (ESI) m/z: 184.55 (% 100.00), 367.10 (%
imidazol-1-yl)phenyl]acetamide (6j).
Yield: 72%; m.p.
225.6–228.9°C. FTIR (ATR, cmꢀ1): 3207 (N–H), 1686
(C¼O), 1622–1423 (C¼C and C¼N), 833 (1,4-
1
disubstituted benzene). H-NMR (500 MHz) (DMSO-d6)
89.12), 368.05 (% 35.96), 369.10 (% 6.10).
2-(2-Mercapto-5-methoxybenzothiazol)-N-[4-(1H-imidazol-
δ (ppm): 2.68 (3H, s, CH3), 4.29 (2H, s, CH2-S), 7.09
(1H, s, imidazole, C5-H), 7.60 (2H, d, J = 9.0 Hz, phenyl
C2,6-H), 7.68 (2H, d, J = 9.0 Hz, phenyl, C3,5-H), 7.71
(1H, s, imidazole C4-H), 8.19 (1H, s, imidazole C2-H),
10.54 (1H, s, NH-CO). 13C-NMR (125 MHz, DMSO-d6)
δ (ppm): 15.2, 38.1, 118.0, 120.1, 120.9, 129.7, 132.5,
135.4, 137.7, 164.3, 165.4, 165.7. LCMS (ESI) m/z:
167.65 (% 100.00), 332.30 (% 89.10), 333.05 (% 18.21),
1-yl)phenyl]acetamide (6g).
Yield: 65%; m.p. 146.7–
150.2°C. FTIR (ATR, cmꢀ1): 3244 (N–H), 1649 (C¼O),
1604–1452 (C¼C and C¼N), 833 (1,4-disubstituted
benzene). 1H-NMR (500 MHz) (DMSO-d6) δ (ppm):
3.82 (3H, s, OCH3), 4.40 (2H, s, CH2-S), 7.00 (1H, dd,
J = 8.5 Hz, J = 2.5 Hz, benzothiazole C6-H), 7.09 (1H, s,
imidazole, C5-H), 7.37 (1H, d, J = 2.5 Hz, benzothiazole
C3-H), 7.61 (2H, d, J = 9.0 Hz, phenyl, C3,5-H), 7.68
(1H, s, imidazole, C4-H), 7.72 (2H, d, J = 9.0 Hz,
phenyl, C2,6-H), 7.89 (1H, s, J = 8.5 Hz, benzothiazole
C7-H), 8.19 (1H, s, imidazole C2-H), 10.59 (1H, s, NH-
CO). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 37.6,
55.5, 104.6, 113.7, 118.0, 120.2, 120.9, 122.1, 126.3,
129.7, 132.5, 135.4, 137.5, 153.8, 158.7, 165.3 167.0.
LCMS (ESI) m/z: 199.55 (% 100.00), 397.10 (% 92.17),
334.10 (% 5.78).
2-(3-Mercapto-1,2,4-triazol)-N-[4-(1H-imidazol-1-yl)phenyl]
acetamide (6k). Yield: 71%; m.p. 169.2–173.8°C. FTIR
(ATR, cmꢀ1): 3275 (N–H), 1676 (C¼O), 1614–1419
(C¼C and C¼N), 840 (1,4-disubstituted benzene). 1H-
NMR (500 MHz) (DMSO-d6) δ (ppm): 4.07 (2H, s, CH2-
S), 7.09 (1H, t, J = 1.0 Hz, imidazole, C5-H), 7.59 (2H, d,
J = 9.0 Hz, phenyl C2,6-H), 7.68 (1H, t, J = 1.0 Hz,
imidazole C4-H), 7.70 (2H, d, J = 9.0 Hz, phenyl C3,5-H),
8.18 (1H, s, imidazole C2-H), 8.46 (1H, s, 1,2,4-triazole,
398.05 (% 33.06), 399.10 (% 6.80).
2-(2-Mercapto-5-chlorobenzothiazole)-N-[4-(1H-imidazol-1-
C
3,5-H), 10.45 (1H, s, NH-CO), 14.07 (1H, s, 1,2,4-
yl)phenyl]acetamide (6h).
Yield: 58%; m.p. 207.8–
triazole, N–H). 13C-NMR (125 MHz, DMSO-d6) δ (ppm):
36.3, 97.5, 118.0, 120.0, 120.9, 129.7, 132.3, 135.4,
137.7, 160.3, 166.4. LCMS (ESI) m/z: 151.45 (% 100.00),
210.3°C. FTIR (ATR, cmꢀ1): 2987 (N–H), 1680 (C¼O),
1612–1425 (C¼C and C¼N), 839 (1,4-disubstituted
1
benzene). H-NMR (500 MHz) (DMSO-d6) δ (ppm): 4.43
301.30 (% 94.62), 302.10 (% 19.89), 303.15 (% 5.66).
2-(3-Mercapto-4-methyl-4H-1,2,4-triazole)-N-[4-(1H-
(2H, s, CH2-S), 7.09 (1H, t, J = 1.0 Hz, imidazole C5-H),
7.42 (1H, dd, J = 8.5 Hz, J = 2.0 Hz, benzothiazole, C6-
H), 7.60 (2H, d, J = 9.0 Hz, phenyl C2,6-H), 7.68 (1H, t,
J = 1.0 Hz, imidazole C4-H), 7.73 (2H, d, J = 9.0 Hz,
phenyl C3,5-H), 7.89 (1H, d, J = 2.0 Hz, benzothiazole C4-
H), 8.06 (1H, d, J = 8.5 Hz, benzothiazole C7-H), 8.19
(1H, t, J = 1.0 Hz, imidazole C2-H), 10.62 (1H, s, NH-
CO). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 37.8,
118.0, 120.2, 120.5, 120.9, 123.3, 124.5, 129.7, 131.2,
132.5, 133.6, 135.4, 137.4, 153.4, 165.2, 169.0. LCMS
(ESI) m/z: 201.45 (% 100.00), 202.30 (% 42.07), 401.05
imidazol-1-yl)phenyl]acetamide (6l).
Yield: 81%; m.p.
230.5–234.8°C. FTIR (ATR, cmꢀ1): 2983 (N–H), 1681
(C¼O), 1620–1415 (C¼C and C¼N), 839 (1,4-
1
disubstituted benzene). H-NMR (500 MHz) (DMSO-d6)
δ (ppm): 3.61 (3H, s, CH3), 4.08 (2H, s, CH2-S), 7.09
(1H, t, J = 1.0 Hz, imidazole, C5-H), 7.60 (2H, d,
J = 9.0 Hz, phenyl C2,6-H), 7.66–7.68 (3H, m, phenyl,
C3,5-H, imidazole C4-H), 8.18 (1H, imidazole C2-H),
8.56 (1H, s, 1,2,4-triazole C5-H), 10.47 (1H, s, NH-CO).
13C-NMR (125 MHz, DMSO-d6) δ (ppm): 30.8, 37.7,
118.0, 120.1, 120.9, 129.7, 132.4, 135.4, 137.5, 146.2,
148.6, 165.9. LCMS (ESI) m/z: 158.35 (% 100.00),
315.20 (% 89.22), 316.10 (% 17.12), 317.15 (% 6.32).
(% 93.16), 402.10 (% 11.80), 403.05 (% 35.16).
2-(2-Mercapto-1-methylimidazole)-N-[4-(1H-imidazol-1-yl)
phenyl]acetamide (6i). Yield: 81%; m.p. 103.2–106.9°C.
FTIR (ATR, cmꢀ1): 3140 (N–H), 1697 (C¼O), 1620–
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. Işık, U. Acar Çevik, B. N. Sağlık, and Y. Özkay
Vol 000
2-(5-Mercapto-1-methyl-1H-tetrazole)-N-[4-(1H-imidazol-1-
yl)phenyl]acetamide (6m).
229.3°C. FTIR (ATR, cmꢀ1): 3265 (N–H), 1680 (C¼O),
1618–1425 (C¼C and C¼N), 831 (1,4-disubstituted
benzene). 1H-NMR (500 MHz) (DMSO-d6) δ (ppm):
3.99 (3H, s, CH3), 4.31 (2H, s, CH2-S), 7.09 (1H, s,
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.
Yield: 76%; m.p. 225.1–
imidazole, C5-H), 7.59 (2H, d, J = 9.0 Hz, phenyl C2,6
-
Quantification of ergosterol level.
Total intracellular
H), 7.67–7.69 (3H, m, phenyl, C3,5-H, imidazole C4-H),
8.18 (1H, s, imidazole C2-H), 10.55 (1H, s, NH-CO).
13C-NMR (125 MHz, DMSO-d6) δ (ppm): 33.7, 37.6,
118.0, 120.2, 120.9, 129.7, 132.5, 135.4, 137.3, 153.3,
165.1. LCMS (ESI) m/z: 158.35 (% 100.00), 316.20 (%
92.27), 317.15 (% 15.33), 318.05 (% 6.12).
2-(2-Mercaptopyrimidin)-N-[4-(1H-imidazol-1-yl)phenyl]
acetamide (6n). Yield: 73%; m.p. 147.4–150.6°C. FTIR
sterols were extracted as recorded by Breivik and
Owades [32] with slight modifications. In order to
inoculate 50 mL of Sabouraud dextrose broth (Difco)
containing 0, 0.78, 1.56, and 3.125 μg/mL of compound
5e, fluconazole and ketoconazole utilized a single
C. krusei 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
harvested 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 potas-
sium hydroxide solution was added to each pellet and vor-
tex mixed for 1 min. Cell suspensions were transferred to
sterile borosilicate glass screw-cap tubes and were incu-
bated in an 85°C water bath for 1 h. Following incubation,
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 to LCMSMS
system (Shimadzu LCMS 8040, Kyoto, Japan). The mass
spectrometric analysis was achieved by employing the
Nexera XR UFLC system coupled to an LCMS-8040 tan-
dem quadrupole mass spectrometer (Shimadzu, Kyoto,
Japan). Labsolutions LCMS software (Shimadzu) was used
to control the instruments and process the data. The Nexera
UFLC system used in the analysis consisted of two pumps
(LC-20ADxr), an autosampler (SIL-20ADxr), a column
heater (CTO-10ASvp), and a degasser (DGU-20A5R).
This instrument was equipped with ESI sources. Chro-
matographic separation was performed using a Shimadzu
Shimpack FC-ODS C18 column (150 mm × 2.0 mm,
3 μm) at a flow rate of 0.25 mL/min in ESI source. The
isocratic mobile phase consisted of acetonitrile-water
0.1% formic acid (50:50, v/v). The mass spectrometer op-
erating parameters were optimized as follows: nebulizer
gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation
line temperature, 250°C; and heat block temperature,
400°C in ESI source. Other parameters were tuned auto-
matically. MRM method was optimized by using ergos-
terol standard stock solution with concentration of
20 μg/mL. 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.
(ATR, cmꢀ1): 3304 (N–H), 1658 (C¼O), 1610–1425
(C¼C and C¼N), 831 (1,4-disubstituted benzene). 1H-
NMR (500 MHz) (DMSO-d6) δ (ppm): 4.15 (2H, s,
CH2-S), 7.23 (1H, t, J = 5.0 Hz, pyrimidin, C5-H),
7.34 (1H, s, imidazole, C5-H), 7.64 (2H, d, J = 9.0 Hz,
phenyl C2,6-H), 7.76 (2H, d, J = 9.0 Hz, phenyl C3,5
-
H), 7.85 (1H, s, imidazole C4-H), 8.64–8.66 (3H, m,
pyrimidin C4,6-H, imidazole C2-H), 10.60 (1H, s, NH-
CO). 13C-NMR (125 MHz, DMSO-d6) δ (ppm): 33.0,
117.4, 118.9, 120.0, 121.4, 126.9, 131.5, 135.0, 138.5,
157.8, 166.4, 170.3. LCMS (ESI) m/z: 156.55 (%
100.00), 312.20 (% 86.77), 313.15 (% 16.81), 314.05
(% 6.54).
Antifungal activity assays. Microbiological study was
performed according to EUCAST definitive method EDef
7.1 for Candida species [28]. 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
(ATCC 24433).
The yeasts were maintained in Roswell Park Memorial
Institute medium, after an overnight incubation at 37°C.
The inocula of test microorganisms adjusted to match the
turbidity of a Mac Farland 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 carried out for medium at pH = 7,
and twofold serial dilutions were applied. The last well
on the microplates, which was containing only the
inoculated broth, was kept as control, and the last well
with no growth of microorganism was recorded to
represent the 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
Roswell Park Memorial Institute medium. The completed
plates were incubated for 24 h, and at the end of the
incubation, resazurin (20 μg/mL) was added into each
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
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Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet