Synthesis of oxadiazole-thiadiazole hybrids and their anticandidal activity

Article abstract of DOI:10.3390/molecules22112004

In the field of infection management, it is a major challenge to discover a potent and safe antifungal agent due to the emergence of resistant strains. Hence, the goal of this paper is to design and synthesize novel oxadiazole-thiadiazole hybrid compounds

Full text of DOI:10.3390/molecules22112004

molecules
Article
Synthesis of Oxadiazole-Thiadiazole Hybrids and
Their Anticandidal Activity
Serkan Levent 1 , Betül Kaya Çavu s¸ o g˘ lu , Begüm Nurpelin Sa g˘ lık , Derya Osmaniye 1,2,
,2
1
1,2
Ulviye Acar Çevik 1 , Özlem Atlı , Yusuf Özkay 1,2,* and Zafer Asım Kaplancıklı 1
,2
3
ID
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eski s¸ ehir, Turkey;
serkanlevent@anadolu.edu.tr (S.L.); betulkaya@anadolu.edu.tr (B.K.Ç.); bnsaglik@anadolu.edu.tr (B.N.S.);
dosmaniye@anadolu.edu.tr (D.O.); uacar@anadolu.edu.tr (U.A.Ç.); zakaplan@anadolu.edu.tr (Z.A.K.)
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University,
2
3
2
6470 Eski s¸ ehir, Turkey
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eski s¸ ehir,
Turkey; oatli@anadolu.edu.tr
*
Correspondence: yozkay@anadolu.edu.tr; Tel.: +90-222-335-0580 (ext. 3603)
Received: 8 November 2017; Accepted: 15 November 2017; Published: 18 November 2017
Abstract: In the field of infection management, it is a major challenge to discover a potent and
safe antifungal agent due to the emergence of resistant strains. Hence, the goal of this paper is to
design and synthesize novel oxadiazole-thiadiazole hybrid compounds (6a
–6s) and evaluate their
antifungal activity. The structures of synthesized compounds were elucidated by various methods
1
13
including FT-IR, H-NMR, C-NMR and HR-MS spectral data. Compounds were tested against
four Candida species by broth microdilution assay. Compounds 6e, 6k and 6r, bearing a nitro group,
showed significant antifungal activity against all fungi with minimum inhibitory concentration (MIC)
in the range of 0.78–3.12 µg/mL. These compounds were also screened for their in vitro cytotoxic
effects by MTT assay and detected as nontoxic at their active concentrations against Candida strains.
To examine the effects of these compounds on ergosterol biosynthesis, the LC-MS-MS method,
which is based on quantification of ergosterol level in C. krusei, was carried out. Finally, the most
active molecule (6e) was docked in the active site of the lanosterol 14α-demethylase enzyme, and it
was determined that there is a strong interaction between the compound and enzyme.
Keywords: 1,3,4-oxadiazole; 1,3,4-thiadiazole; antifungal activity; ergosterol; cytotoxicity; docking
1
. Introduction
Fungal infections represent a challenging problem worldwide. High prevalence of antimicrobial
resistance of pathogenic micro-organisms to conventional drugs reduces the efficacy of treatment,
requiring the development of alternative agents [ ]. Among many drugs in clinic, azole antifungals
are commonly employed in the therapy of fungal diseases. Their mechanism of action is to inhibit
the lanosterol 14 -demethylase (CYP51) in yeast and fungi [ ]. CYP51 is a key enzyme in fungal
1
α
2
membrane ergosterol biosynthesis, and azoles, thereby, cause depletion of ergosterol and resulting in
inhibition of fungal growth [3].
It is well-known that azole derivatives represent a series of synthetic compounds of remarkable
medicinal importance. Azole antifungal agents are of ample interest due to their broad spectrum-
activity, high therapeutic index and low toxicity [
,3,4-oxadiazole compounds were reported to possess various biological activities including antifungal
activity [ ]. The –N=C–O– linkage in oxadiazoles can react with the nucleophilic centers of
4]. Among a large variety of azole derivatives,
1
5–9
microbial cells and is attributed with its biological activity [10]. Its electronic and charge-transporting
properties ensure connection with targets through non-covalent interactions. Oxadiazoles have been
Molecules 2017, 22, 2004; doi:10.3390/molecules22112004
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2004
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described as bioisosteres for carboxylic acids, esters and carboxamides [11]. These characteristics of
the 1,3,4-oxadiazole ring resulted in the diverse pharmaceutical applications of these molecules in the
field of medicinal chemistry.
1,3,4-Thiadiazole, containing hydrogen-binding domain, sulfur atom, and two-electron donor
nitrogen system, is an important class of the five-membered heterocyclic family. The wide range
of therapeutic values of this ring systems encourage researchers to perform an extensive research
on the synthesis of new potent antifungal agents that contain the 1,3,4-thiadiazole ring in their
structures. Due to the bioisosterism between oxygen and sulphur atoms, 1,3,4-thiadiazoles possess
similar physical and chemical properties to 1,3,4-oxadiazoles. The extensive literature survey revealed
that 1,3,4-thiadiazole derivatives exhibit antifungal activity [12–16].
The combination of two or more pharmacophores in one molecule is a strategy in the search
for new antifungal agents, due to a broader antimicrobial spectrum and the potential to slow the
development of drug resistance [17]. Thus, we planned to combine both pharmacophores into
a compact system to obtain a new class of antifungal agents. The hybrid 1,3,4-oxadiazole-1,3,4-
thiadiazole compounds were linked via a thioethyl chain and substituted with different aliphatic and
aromatic rings in order to investigate the effects of structural variation on biological activity.
2
. Results and Discussion
2
.1. Chemistry
Title compounds (6a
The thiosemicarbazide derivative (
and hydrazine hydrate. The ring closure reaction of compound
presence of sodium hydroxide gave 5-(cyclohexylamino)-1,3,4-thiadiazole-2-thiol (
reacted with ethyl bromopropionate to obtain ethyl 3-[[5-(cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]
propanoate ). 3-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]propanehydrazide was
synthesized via the treatment of ester ( ) with hydrazine hydrate. The second ring closure reaction of the
corresponding hydrazide ( ) with carbon disulfide resulted in the formation of 5-[2-[[5-(cyclohexylamino)-
,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazole-2-thiol ( ). Finally, compound was reacted
with appropriate 4-substituted phenacyl bromides, 4-substituted anilines or 6-substituted-
-aminobenzothiazoles to afford the target compounds (6a–6s).
The formation of synthesized compounds (6a 6s) was confirmed by IR, mass spectra, H-NMR
and 13C-NMR. In the IR spectra of all synthesized compounds, bands at ~3340 cm , ~3070 cm
–
6s) were synthesized according to the pathway as outlined in Scheme 1.
) was obtained through the reaction of cyclohexyl isothiocyanate
with carbon disulfide in the
) which then was
1
1
2
(3
(4)
3
4
1
5
5
2
1
–
−1
−1
,
~
1580 cm 1 and ~1480 cm were attributed to the stretching of the N-H, aromatic C-H, C=N and C=C
− −1
−
1
groups. The carbonyl band belonging to ketone group was observed between 1660–1701 cm
.
−
1
−1
The compounds 6e
,
6k and 6r showed two bands around ~1520 cm and ~1330 cm due to
asymmetric and symmetric stretching of the NO group. The H-NMR of all synthesized compounds
1
2
showed peaks at ~3.25 ppm (SCH CH ) and ~3.41 ppm (-SCH CH -) due to the methylene protons of
2
2
2
2
thioethyl moiety. The signals of oxoethyl moiety (COCH ) were resonated at ~5.09 ppm, ~4.27 ppm
2
and ~4.41 ppm as singlets for compounds 6a
the NH proton bonded to cyclohexyl ring appeared at ~7.79 ppm as a doublet peak. A singlet peak
belonging to the NH proton of acetamide moiety (-NHCOCH ) was designated at ~10.38 ppm in
–6f, 6g–6l and 6m–6s, respectively. For all compounds,
2
1
3
compounds 6g
due to the carbonyl group appeared at ~192.50 ppm for compounds 6a
compounds 6g 6s. The spectra of all compounds showed peaks in the range of ~105–169 ppm due to
the presence of aromatic carbons. The mass spectrum of all the compounds were in full accordance
–
6l and ~12.76 ppm in compounds 6m
–
6s. In the C-NMR of compounds, the signal
–
6f and at ~169.20 ppm for
–
1
13
with their molecular weights. The IR, H-NMR, C-NMR, and HRMS spectra of all compounds are
presented in supplementary file.

Molecules 2017, 22, 2004
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R/Ar
Compound
R’
Compound
R’
Compound
R’
6
a
H
Cl
H
Cl
H
6b
6e
6h
6k
6n
6r
CH3
NO2
CH3
NO2
CH3
NO2
6c
6f
6i
6l
6o
6s
OCH3
6
6
d
g
F
OCH3
F
OCH3
F
6
j
6
6
m
p
Cl
Scheme 1. The synthesis of the compounds (6a–6s). Reagents and conditions: (i) BrCH CH COOEt, K CO ,
2 2 2 3
acetone, reflux, 9 h; (ii) NH NH2
pH: 4–5; (iv) 4-substitutedphenacyl bromides (6a
·
H O, ethanol, rt, 4 h; (iii) (1) CS /NaOH, ethanol, reflux, 10 h; (2) HCl,
2
2
2
–6f), 2-chloro-N-(4-substitutedphenyl)acetamides (6g–6l),
or 2-chloro-N-(6-substitutedbenzothiazol-2-yl)acetamides (6m–6s), K CO , acetone, rt.
2
3
2
.2. Antifungal Activity Assay
The results of anticandidal activity of the synthesized compounds 6a
are presented with minimum inhibitory concentration (MIC ; g/mL) values in Table 1. Minimum
6s that inhibited the growth of yeast strains ranged from 0.78 to
g/mL. According to antifungal screening, compounds 6e 6k and 6r were found to be active
–
6s and reference drugs
µ
0
5
concentrations of compounds 6a
00
–
1
µ
,
against all tested fungi. Compound 6e indicated similar antifungal activity to ketoconazole against all
Candida strains and was evaluated as the most active derivative in the series. This compound showed
higher anticandidal activity than ketoconazole against C. krusei and C. parapsilopsis with an MIC50
value of 0.78 µg/mL. Compounds 6e and 6k showed equal levels of activity as ketoconazole against
C. glabrata. Also these compounds showed half as much activity as ketoconazole against C. albicans.
Compound 6r exhibited equal level of activity with ketoconazole against C. krusei and C. parapsilopsis
with an MIC50 value of 1.56 µg/mL. The most sensitive yeast to all compounds was C. parapsilopsis.
In compounds 6a
–
6f, the introduction of a chloro atom in the 4 position of phenyl ring improved
6l, the effects of
activity against C. krusei, compared to 4-methoxy derivative. In compounds 6g
–

Molecules 2017, 22, 2004
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4-methoxy and 4-methyl groups on the compound’s activity were the same against all strains.
A common structural feature of the most active compounds 6e, 6k, and 6r is the presence of a nitro
substituent. Hence, it can be assumed that the introduction of a nitro group is highly beneficial
for antifungal activity [18 20]. It is possible to note a correlation between antifungal activity with
–
an electron withdrawing nitro group because the substition of this group affected pKa, hydrophobic
interactions and also lipophilicity, which are citical to enhanced biological activity.
Table 1. Minimum inhibitory concentration (MIC ; µg/mL) values of compounds on four strains of
5
0
Candida (6a–6s).
Compound
C. albicans
C. glabrata
C. krusei
C. parapsilopsis
6
a
25
50
100
100
1.56
50
50
50
25
25
1.56
50
50
100
100
50
0.78
25
50
50
50
50
0.78
50
6
b
6
c
6
d
6
6
e
f
6
g
50
25
25
25
50
50
50
50
50
50
6
h
100
100
50
100
100
50
6
6
i
j
6
6
k
l
1.56
100
50
1.56
50
50
1.56
50
50
1.56
25
25
6
m
6
n
100
100
50
25
25
25
50
25
25
25
50
50
6o
p
6
6
r
3.12
50
0.78
3.12
6.25
1.56
1.56
50
1.56
1.56
50
1.56
6
s
Ketoconazole
2
.3. Effect of Compounds 6e, 6k, and 6r on Ergosterol Content of C. krusei
Compounds 6e 6k and 6r were selected for the second step of the study due to their potent activity
against tested Candida strains. In this step, these compounds were tested for their effect on the ergosterol
level of C. krusei. Inhibition of ergosterol biosynthesis was measured at their 0.78 g/mL, 1.56 g/mL,
.12 g/mL concentrations and expressed as (%) inhibition. The results are respresented in Table 2.
,
µ
µ
3
µ
All tested compounds inhibited ergosterol biosynthesis, concentration-dependently. The results were
comparable to the reference drug ketoconazole. At all tested concentrations, the (%) inhibition of
compound 6e was higher than that of the reference. These results provide further support for the
hypothesis that compounds 6e, 6k and 6r exhibited their antifungal activity through the inhibition of
ergosterol biosynthesis.
Table 2. Cytotoxic activity and ergosterol biosynthesis potency of the compounds 6e, 6k and 6r against
NIH/3T3 cell line and C. krusei, respectively.
Inhibition of Ergosterol Biosynthesis (%)
Comp.
IC50 (µg/mL)
0
.78 µg/mL
1.56 µg/mL
3.12 µg/mL
6
e
>500
>500
76.45 ± 3.67
58.72 ± 2.41
61.43 ± 1.88
60.99 ± 2.94
82.94 ± 4.72
74.65 ± 3.26
70.59 ± 4.23
73.12 ± 4.16
88.56 ± 4.52
81.29 ± 4.19
80.21 ± 3.70
84.56 ± 3.01
6
k
6
r
406.066 ± 9.36
Ketoconazole
-

Molecules 2017, 22, 2004
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2
.4. Cytotoxicity Test
In drug discovery, cytotoxicity is an important and essential criteria for a drug candidate.
MTT assay was carried out for screening the cytotoxic effects of the compounds [21]. The IC50
values of the compounds are indicated in Table 2. The IC50 values of compounds 6e and 6k were
recorded above 500
µg/mL and that of compound 6r was found to be 406.066 µg/mL. It can thus be
suggested that the tested compounds are nontoxic at their active concentrations against Candida strains.
2
.5. Molecular Docking Studies
4- -Sterol demethylase is a key enzyme in the membrane ergosterol biosynthesis of fungi.
In docking studies, the binding modes of compound 6e to 14- -sterol demethylase was clarified.
Studies were performed with the X-ray crystal structure of 14- -sterol demethylase from
1
α
α
α
Mycobacterium tuberculosis in complex with fluconazole (Protein Data Bank (PDB) ID: 1EA1) [22].
The docking pose on 14-α-sterol demethylase reveals that the interactions between the compound 6e
and amino acid residues are very important in terms of binding to the active site of enzyme (Figure 1).
It is seen that there are two π-π interactions. The first π-π interaction is between oxadiazole ring and
Arg96, while the second is between phenyl and phenyl of Phe78. The amino of the structure, between
cyclohexyl and the thiadiazole ring establishes a hydrogen with the carbonyl of Ala256. Also, there is
another hydrogen bond between the carbonyl group and the amino of Hid259. An additional
interaction is related to the nitro substituent. The nitro group, at C-4 of phenyl, is in interaction
with the amino of Ile323 by hydrogen bonding. These interactions explain the reason for the stronger
anticandidal activity of 6e. It can be suggested that electron withdrawing groups such as the nitro at
C-4 of phenyl is very important in terms of binding to enzyme active site and anticandidal activity.
As a result, it is considered that this additional interaction could explain the higher binding capability
and stronger activity of compound 6e, as compared with the other compounds.
Figure 1. The interacting mode of compound 6e in the active region of 14 alpha-sterol demethylase.
The inhibitor is colored with dark green and HEM is colored with pink.

Molecules 2017, 22, 2004
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3
. Materials and Methods
3
.1. Chemistry
All chemicals were purchased from Sigma-Aldrich Chemical Co (Sigma-Aldrich Corp., St. Louis,
MO, USA) and Merck Chemicals (Merck KGaA, Darmstadt, Germany). All melting points (m.p.)
were determined by Electrothermal 9100 digital melting point apparatus (Electrothermal, Essex, UK)
and are uncorrected. All the reactions were monitored by thin-layer chromatography (TLC) using
Silica Gel 60 F254 TLC plates (Merck KGaA, Darmstadt, Germany). Spectroscopic data were recorded
with the following instruments: IR, Shimadzu 8400S spectrophotometer (Shimadzu, Tokyo, Japan);
1
H-NMR, Bruker DPX 300 NMR spectrometer (Bruker Bioscience, Billerica, MA, USA), 13C-NMR,
Bruker DPX 75 NMR spectrometer (Bruker Bioscience, Billerica, MA, USA) in DMSO-d , using TMS as
6
the internal standard; M + 1 peaks were determined by Shimadzu LC/MS ITTOF system (Shimadzu,
Tokyo, Japan).
3
.1.1. Synthesis of N-Cyclohexylhydrazinecarbothioamide (1)
Cyclohexyl isothiocyanate (0.1 mol, 15 mL) and hydrazine hydrate (0.2 mol) were stirred in ethanol
(
150 mL) at room temperature. After the completion of the reaction, the precipitated product (1) was
filtered and recrystallized from ethanol [23].
3
.1.2. Synthesis of 5-(Cyclohexylamino)-1,3,4-thiadiazole-2-thiol (2)
Compound (0.09 mol) was dissolved in ethanol (150 mL) and a solution of NaOH (0.1 mol) in
1
ethanol was added. Carbon disulfide (0.1 mol, 6 mL) was added and the mixture was refluxed for 8 h.
After this period, the solution was cooled and acidified to pH4–5 with hydrochloric acid solution and
crystallized from ethanol [23].
3
.1.3. Synthesis of Ethyl 3-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]propanoate (3)
A mixture of compound (0.08 mol), ethyl bromopropionate (0.08 mol) and potassium carbonate
0.09 mol) was stirred under reflux for 9 h in acetone (150 mL). The solvent was evaporated under
reduced pressure and the product was washed with water, dried and crystallized from ethanol [23].
2
(
3
.1.4. Synthesis of 3-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl)thio]propanehydrazide (4)
Compound (0.06 mol) and hydrazine hydrate (0.06 mol) were stirred in ethanol (150 mL) at
room temperature for 4 h and filtered [23].
3
3
.1.5. Synthesis of 5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazole-2-thiol (
5)
Compound (0.05 mol) was added to a solution of NaOH (0.06 mol) in ethanol (100 mL). After the
4
addition of carbon disulfide (0.06 mol), the mixture was heated under reflux for 10 h. The solution was
cooled and acidified to pH4–5 with hydrochloric acid solution and recrystallized from ethanol [24].
3
.1.6. Synthesis of 2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]
thio]-1-substituted ethan-1-one (6a–6s)
Compound (2 mmol) and appropriate 4-substituted phenacyl bromide (2 mmol), 4-substituted
5
aniline (2 mmol) or 6-substituted 2-amino benzothiazole (2 mmol) were stirred in acetone (40 mL) at
room temperature in the presence of potassium carbonate (2.4 mmol). Acetone was evaporated under
reduced pressure and the product was washed with water, dried and crystallized from ethanol.
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-phenylethan-1-one (6a):
◦
−1
Yield 67%, m.p. 141 C. IR νmax (cm ): 3340 (N-H) 3064 (aromatic C-H), 2933 (aliphatic C-H),
1
1
681 (C=O ketone), 1587–1487 (C=N, C=C), 1197–1085 (C-N, C-O). H-NMR (300 MHz, DMSO-d , ppm)
6

Molecules 2017, 22, 2004
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δ
1.14–1.30 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H),
1
.91–1.95 (2H, m, cyclohexyl-H), 3.25 (2H, t, J = 6.6 Hz, SCH CH2), 3.41 (2H, t, J = 7.4 Hz, SCH2CH2),
2
3
.46–3.52 (1H, m, cyclohexyl-H), 5.09 (2H, s, COCH ), 7.58 (2H, t, J = 7.8 Hz, Ar-H), 7.71 (1H, t,
2
13
J = 7.3 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 8.04 (2H, d, J = 8.5 Hz, Ar-H). C-NMR (75 MHz,
DMSO-d , ppm)
δ
24.67, 25.65, 26.08, 31.04, 32.46, 41.04, 54.00, 128.91, 129.36, 134.45, 166.34, 169.19,
6
+
1
92.99. HR-MS (m/z): [M + H] calcd for C H N O S : 462.1087; found: 462.1089.
20
23
5
2 3
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-methylphenyl)
◦
−1
ethan-1-one (6b): Yield 70%, m.p. 136 C. IR νmax (cm ): 3327 (N-H) 3055 (aromatic C–H),
2
1
926 (aliphatic C–H), 1678 (C=O ketone), 1589–1481 (C=N, C=C), 1298–1089 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.30 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H),
6
1
.65–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 2.40 (3H, s, CH ), 3.25 (2H, t,
3
J = 6.5 Hz, SCH CH ), 3.41 (2H, t, J = 7.0 Hz, SCH CH ), 3.45–3.51 (1H, m, cyclohexyl-H), 5.05 (2H, s,
2
2
2
2
COCH ), 7.38 (2H, d, J = 8.0 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 7.94 (2H, d, J = 8.3 Hz, Ar-H).
2
13
C-NMR (75 MHz, DMSO-d , ppm)
δ
21.71, 24.67, 25.65, 26.08, 31.05, 32.47, 41.00, 54.00, 129.03,
6
+
1
29.89, 132.99, 145.03, 148.52, 163.53, 166.31, 169.18, 192.48 (C=O). HR-MS (m/z): [M + H] calcd for
C H N O S : 476.1243; found: 476.1241.
21
25
5
2 3
2
1
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-methoxyphenyl)ethan-
◦ −1
-one (6c): Yield 75%, m.p. 120 C. IR νmax (cm ): 3338 (N-H) 3076 (aromatic C–H), 2927 (aliphatic C–H), 1660
1
(
C=O ketone), 1593–1483 (C=N, C=C), 1259–1095 (C-N, C-O). H-NMR (300 MHz, DMSO-d , ppm) δ
6
1
1
.16–1.30 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.65–1.70 (2H, m, cyclohexyl-H),
.91–1.95 (2H, m, cyclohexyl-H), 3.25 (2H, t, J = 6.5 Hz, SCH CH2), 3.39–3.48 (3H, m, SCH2CH2,
2
cyclohexyl-H), 3.86 (3H, s, OCH ), 5.03 (2H, s, COCH ), 7.09 (2H, d, J = 8.9 Hz, Ar-H), 7.79 (1H, d,
3
2
13
J = 7.3 Hz, NH), 8.02 (2H, d, J = 8.9 Hz, Ar-H). C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.65,
6
2
6.08, 31.05, 32.46, 54.00, 56.12, 114.58, 128.32, 131.35, 148.52, 163.60, 164.21, 166.29, 169.18, 191.28.
+
HR-MS (m/z): [M + H] calcd for C H N O S : 492.1187; found: 492.1192.
21
25
5
3 3
2
1
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-chlorophenyl)ethan-
◦ −1
-one (6d): Yield 74%, m.p. 167 C. IR νmax (cm ): 3336 (N-H) 3057 (aromatic C–H), 2929 (aliphatic C–H),
1
1
680 (C=O ketone), 1587–1481 (C=N, C=C), 1197–1087 (C-N, C-O). H-NMR (300 MHz, DMSO-d , ppm)
6
δ
1.10–1.35 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H),
1
.91–1.95 (2H, m, cyclohexyl-H), 3.25 (2H, t, J = 6.9 Hz, SCH CH2), 3.39–3.52 (3H, m, SCH2CH2,
2
cyclohexyl-H), 5.09 (2H, s, COCH ), 7.65 (2H, d, J = 8.6 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 8.05 (2H,
2
d, J = 8.7 Hz, Ar-H). 13C-NMR (75 MHz, DMSO-d , ppm)
1
δ
24.67, 25.65, 26.07, 31.02, 32.46, 40.92, 54.01,
29.49, 130.84, 134.20, 139.37, 148.51, 163.38, 166.39, 169.18, 192.16. HR-MS (m/z): [M + H]+ calcd for
C H ClN O S : 496.0697; found: 496.0679.
6
20
22
5
2 3
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-nitrophenyl)ethan-
◦
−1
1-one (6e): Yield 77%, m.p. 144 C. IR νmax (cm ): 3336 (N-H) 3068 (aromatic C–H), 2929 (aliphatic
1
C–H), 1701 (C=O ketone), 1583–1481 (C=N, C=C), 1180–1051 (C-N, C-O). H-NMR (300 MHz,
DMSO-d , ppm)
δ 1.11–1.35 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m,
6
cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2), 3.35–3.49 (3H, m,
2
SCH2CH , cyclohexyl-H), 5.15 (2H, s, COCH ), 7.79 (1H, d, J = 7.3 Hz, NH), 8.27 (2H, d, J = 9.0 Hz,
2
2
13
Ar-H), 8.39 (2H, d, J = 8.9 Hz, Ar-H). C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.65, 26.05, 31.00,
6
3
+
2.46, 41.16, 54.01, 124.42, 130.37, 140.16, 148.51, 150.74, 163.23, 166.48, 169.18, 192.50. HRMS (m/z): [M
H] calcd for C H N O S : 507.0937; found: 507.0924.
20 22 6 4 3
+
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-fluorophenyl)ethan-
◦
−1
1-one (6f): Yield 64%, m.p. 131 C. IR νmax (cm ): 3342 (N-H) 3061 (aromatic C–H), 2929 (aliphatic
1
C–H), 1680 (C=O ketone), 1587–1485 (C=N, C=C), 1232–1049 (C-N, C-O). H-NMR (300 MHz,

Molecules 2017, 22, 2004
8 of 13
DMSO-d , ppm)
δ
1.14–1.35 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m,
6
cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.25 (2H, t, J = 6.3 Hz, SCH CH2), 3.39-3.51 (3H, m,
2
SCH2CH , cyclohexyl-H), 5.08 (2H, s, COCH ), 7.41 (2H, t, J = 8.9 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz,
2
2
13
NH), 8.11–8.16 (2H, m, Ar-H). C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.65, 26.07, 31.03, 32.46,
6
4
0.91, 54.01, 116.45 (d, J = 21.8 Hz), 132.04 (d, J = 9.8 Hz), 132. 28 (d, J = 3.5 Hz), 148.51, 163.43, 165.50
+
(d, J = 250.0 Hz), 167.57, 169.18, 191.68. HRMS (m/z): [M + H] calcd for C H FN O S : 480.0992;
20 22 5 2 3
found: 480.0987.
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-phenyl acetamide (6g):
◦
−1
Yield 72%, m.p. 177 C. IR νmax (cm ): 3342 (N-H) 3057 (aromatic C–H), 2931 (aliphatic C–H), 1672 (C=O
ketone), 1598–1483 (C=N, C=C), 1170–1093 (C-N, C-O). H-NMR (300 MHz, DMSO-d , ppm)
1
δ
1.10–1.36
6
(5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.95
(
2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH ), 3.39–3.53 (3H, m, SCH CH , cyclohexyl-H),
2
2
2
2
4
.27 (2H, s, COCH ), 7.07 (1H, t, J = 7.4 Hz, Ar-H), 7.32 (2H, t, J = 7.6 Hz, Ar-H), 7.56 (2H, d, J = 7.6 Hz,
2
13
Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.38 (1H, s, NH). C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67,
6
2
5.66, 26.10, 31.01, 32.47, 37.27, 54.02, 119.60, 124.12, 129.31, 139.10, 148.50, 163.58, 165.22, 166.38, 169.20.
+
HR-MS (m/z): [M + H] calcd for C H N O S : 477.1196; found: 477.1191.
20
24
6
2 3
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-methyl phenyl)
◦
−1
acetamide (6h): Yield 79%, m.p. 173 C. IR νmax (cm ): 3336 (N-H) 3068 (aromatic C–H),
2
1
916 (aliphatic C–H), 1668 (C=O ketone), 1521–1483 (C=N, C=C), 1240–1085 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.96 (2H, m, cyclohexyl-H), 2.25 (3H, s, CH ), 3.26 (2H, t,
3
J = 6.5 Hz, SCH CH2), 3.39–3.50 (3H, m, SCH2CH , cyclohexyl-H), 4.25 (2H, s, COCH ), 7.12 (2H,
2
2
2
d, J = 8.3 Hz, Ar-H), 7.45 (2H, d, J = 8.4 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.29 (1H, s, NH).
1
3
C-NMR (75 MHz, DMSO-d , ppm)
δ
20.91, 24.68, 25.66, 26.10, 31.01, 32.47, 37.25, 54.02, 119.61, 129.67,
6
+
1
33.08, 136.60, 148.51, 163.60, 164.95, 166.36, 169.20. HR-MS (m/z): [M + H] calcd for C H N O S :
21
26
6
2 3
4
91.1352; found: 491.1343.
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-methoxy
◦
−1
phenyl)acetamide (6i): Yield 71%, m.p. 147 C. IR νmax (cm ): 3336 (N-H) 3064 (aromatic C–H),
2
1
920 (aliphatic C–H), 1662 (C=O ketone), 1514–1421 (C=N, C=C), 1286–1035 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
3
.39–3.52 (3H, m, SCH2CH , cyclohexyl-H), 3.72 (3H, s, OCH ), 4.23 (2H, s, COCH ), 6.89 (2H, d,
2 3 2
J = 9.1 Hz, Ar-H), 7.47 (2H, d, J = 9.1 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.24 (1H, s, NH).
1
3
C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.66, 26.11, 31.02, 32.47, 37.17, 54.02, 55.61 (OCH ),
6
3
+
1
14.41, 121.17, 132.22, 148.51, 155.93, 163.61, 164.68, 166.36, 169.20. HR-MS (m/z): [M + H] calcd for
C H N O S : 507.1301; found: 507.1291.
21
26
6
3 3
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-chloro phenyl)
◦
−1
acetamide (6j): Yield 69%, m.p. 131 C. IR νmax (cm ): 3338 (N-H) 3066 (aromatic C–H),
1
2933 (aliphatic C–H), 1668 (C=O ketone), 1583–1483 (C=N, C=C), 1238–1083 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.35 (5H, m, cyclohexyl-H), 1.52–1.57 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
3
.39–3.52 (3H, m, SCH2CH , cyclohexyl-H), 4.27 (2H, s, COCH ), 7.38 (2H, d, J = 8.9 Hz, Ar-H), 7.60 (2H,
2 2
13
d, J = 8.9 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.53 (1H, s, NH). C-NMR (75 MHz, DMSO-d6,
ppm)
δ
24.68, 25.66, 26.10, 31.00, 32.47, 37.19, 54.03, 60.71, 121.17, 127.69, 129.24, 138.04, 148.50, 163.51,
+
1
65.44, 166.41, 169.20. HR-MS (m/z): [M + H] calcd for C H ClN O S : 511.0806; found: 511.0788.
20
23
6
2 3

Molecules 2017, 22, 2004
9 of 13
2
(
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-nitrophenyl)acetamide
◦ −1
6k): Yield 67%, m.p. 149 C. IR νmax (cm ): 3340 (N-H) 3049 (aromatic C–H), 2939 (aliphatic C–H),
1
1
697 (C=O ketone), 1616–1490 (C=N, C=C), 1521 and 1328 (NO ), 1159–1072 (C-N, C-O). H-NMR
2
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.36 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
3
8
2
.39–3.51 (3H, m, SCH2CH , cyclohexyl-H), 4.33 (2H, s, COCH ), 7.78–7.84 (3H, m, NH, Ar-H),
2 2
13
.24 (2H, d, J = 9.2 Hz, Ar-H), 11.00 (1H, s, NH). C-NMR (75 MHz, DMSO-d , ppm) δ 24.67, 25.66,
6
6.08, 30.98, 32.46, 37.28, 54.02, 119.40, 125.57, 142.96, 145.16, 148.50, 163.41, 166.38, 166.47, 169.18.
+
HRMS (m/z): [M + H] calcd for C H N O S : 522.1046; found: 522.1035.
20
23
7
4 3
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-fluoro phenyl)
◦
−1
acetamide (6l): Yield 73%, m.p. 160 C. IR νmax (cm ): 3342 (N-H) 3032 (aromatic C–H),
2
1
854 (aliphatic C–H), 1670 (C=O ketone), 1583–1481 (C=N, C=C), 1168–1083 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.35 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
3
.39–3.53 (3H, m, SCH2CH , cyclohexyl-H), 4.25 (2H, s, COCH ), 7.16 (2H, t, J = 8.9 Hz, Ar-H), 7.56–7.60
2 2
13
(2H, m, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.45 (1H, s, NH). C-NMR (75 MHz, DMSO-d , ppm)
6
δ
24.67, 25.66, 26.10, 31.00, 32.47, 37.13, 54.02, 115.91 (d, J = 22.5 Hz), 121.42 (d, J = 8.3 Hz), 135.49 (d,
J = 3.0 Hz), 148.50, 157.05, 161.89 (d, J = 248.0 Hz), 165.18, 166.39, 169.19. HR-MS (m/z): [M + H]+ calcd
for C H FN O S : 495.1101; found: 495.1087.
20
23
6
2 3
2
2
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(benzothiazol-
◦
−1
-yl)acetamide (6m): Yield 74%, m.p. 214 C. IR νmax (cm ): 3338 (N-H) 3062 (aromatic C–H),
1
931 (aliphatic C–H), 1678 (C=O ketone), 1554–1492 (C=N, C=C), 1257–1085 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.31 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.4 Hz, SCH CH2),
2
3
7
.39–3.48 (3H, m, SCH2CH , cyclohexyl-H), 4.42 (2H, s, COCH ), 7.32 (1H, t, J = 7.2 Hz, Ar-H),
2 2
.45 (1H, t, J = 7.1 Hz, Ar-H), 7.76–7.80 (2H, m, Ar-H, NH), 7.98 (1H, d, J = 7.9 Hz, Ar-H), 12.76 (1H, s,
13
NH). C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.66, 26.11, 30.99, 32.46, 36.09, 54.03, 121.16, 122.26,
6
+
1
24.22, 126.69, 131.90, 148.49, 158.11, 163.23, 166.58, 166.82, 169.20. HR-MS (m/z): [M + H] calcd for
C H N O S : 534.0869; found: 534.0866.
21
23
7
2 4
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-methyl
◦
−1
benzothiazol-2-yl)acetamide (6n): Yield 77%, m.p. 209 C. IR νmax (cm ): 3336 (N-H) 3026 (aromatic
C–H), 2920 (aliphatic C–H), 1674 (C=O ketone), 1585–1467 (C=N, C=C), 1257–1087 (C-N, C-O). H-NMR
1
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 2.41 (3H, s, CH ), 3.26 (2H, t,
3
J = 6.5 Hz, SCH CH2), 3.39–3.48 (3H, m, SCH2CH , cyclohexyl-H), 4.40 (2H, s, COCH ), 7.26 (1H, dd,
2
2
2
J = 1.4 Hz, J = 9.0 Hz, Ar-H), 7.65 (1H, d, J = 8.3 Hz, Ar-H), 7.77–7.80 (2H, m, Ar-H, NH), 12.68 (1H, s,
1
2
13
NH). C-NMR (75 MHz, DMSO-d , ppm)
δ
21.45, 24.67, 25.66, 26.10, 30.99, 32.46, 36.05, 54.02, 120.82,
6
+
1
21.83, 128.02, 132.05, 133.71, 148.49, 157.19, 163.24, 166.57, 169.20. HR-MS (m/z): [M + H] calcd for
C H N O S : 548.1025; found: 548.1007.
22
25
7
2 4
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-methoxy
◦
−1
benzothiazol-2-yl)acetamide (6o): Yield 75%, m.p. 198 C. IR νmax (cm ): 3334 (N-H) 3072 (aromatic C–H),
2
1
933 (aliphatic C–H), 1681 (C=O ketone), 1614–1467 (C=N, C=C), 1257–1033 (C-N, C-O). H-NMR
(
300 MHz, DMSO-d , ppm)
δ
1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
3
.39–3.48 (3H, m, SCH2CH , cyclohexyl-H), 3.80 (3H, s, OCH ), 4.39 (2H, s, COCH ), 7.04 (1H, dd,
2 3 2
J1 = 2.6 Hz, J = 9.0 Hz, Ar-H), 7.57 (1H, d, J = 2.5 Hz, Ar-H), 7.66 (1H, d, J = 8.1 Hz, Ar-H), 7.79 (1H, d,
2
13
J = 7.3 Hz, NH), 12.62 (1H, s, NH). C-NMR (75 MHz, DMSO-d , ppm)
δ 24.67, 25.66, 26.11, 30.99,
6

Molecules 2017, 22, 2004
10 of 13
3
2.47, 36.02, 54.03, 56.10, 105.20, 115.53, 121.79, 133.25, 143.03, 148.49, 155.98, 156.72, 163.25, 166.48,
+
1
66.56, 169.20. HR-MS (m/z): [M + H] calcd for C H N O S : 564.0974; found: 564.0965.
22 25 7 3 4
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-chloro
◦
−1
benzothiazol-2-yl)acetamide (6p): Yield 74%, m.p. 213 C. IR νmax (cm ): 3334 (N-H) 3088 (aromatic
C–H), 2931 (aliphatic C–H), 1681 (C=O ketone), 1612–1467 (C=N, C=C), 1261–1093 (C-N, C-O). H-NMR
1
(
300 MHz, DMSO-d , ppm)
δ
1.10–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H),
6
1
3
.65–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH CH2),
2
.39–3.49 (3H, m, SCH2CH , cyclohexyl-H), 4.41 (2H, s, COCH ), 7.47 (1H, dd, J = 2.3 Hz, J = 9.0 Hz
,
2
2
1
2
1
3
Ar-H), 7.75–7.80 (2H, m, Ar-H, NH), 8.13 (1H, d, J = 2.1 Hz, Ar-H), 12.85 (1H, s, NH). C-NMR
(
1
75 MHz, DMSO-d , ppm)
28.27, 133.61, 147.82, 148.49, 158.96, 163.19, 166.60, 167.05, 169.19. HR-MS (m/z): [M + H] calcd for
δ
24.67, 25.66, 26.10, 30.98, 32.46, 36.03, 54.03, 121.99, 122.39, 127.05,
6
+
C H ClN O S : 568.0479; found: 568.0469.
21
22
7
2 4
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-nitro
◦
−1
benzothiazol-2-yl)acetamide (6r): Yield 68%, m.p. 205 C. IR νmax (cm ): 3338 (N-H) 3051 (aromatic
C–H), 2933 (aliphatic C–H), 1687 (C=O ketone), 1614–1446 (C=N, C=C), 1514 and 1334 (NO ),
2
1
1
1
3
7
9
2
288–1093 (C-N, C-O). H-NMR (300 MHz, DMSO-d , ppm)
δ
1.13–1.34 (5H, m, cyclohexyl-H),
6
.52–1.56 (1H, m, cyclohexyl-H), 1.65–1.69 (2H, m, cyclohexyl-H), 1.91–1.94 (2H, m, cyclohexyl-H),
.26 (2H, t, J = 6.5 Hz, SCH CH2), 3.39–3.48 (3H, m, SCH2CH , cyclohexyl-H), 4.45 (2H, s, COCH ),
2
2
2
.78 (1H, d, J = 7.3 Hz, NH), 7.92 (1H, d, J = 9.0 Hz, Ar-H), 8.29 (1H, dd, J = 2.4 Hz, J = 8.9 Hz, Ar-H),
1
2
.06 (1H, d, J = 2.3 Hz, Ar-H), 13.17 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d , ppm)
δ 24.67, 25.66,
6
6.09, 30.97, 32.45, 36.09, 54.02, 119.61, 121.28, 122.30, 132.67, 143.59, 148.48, 153.82, 163.14, 163.65,
+
1
66.64, 167.65, 169.17. HR-MS (m/z): [M + H] calcd for C H N O S : 579.0720; found: 579.0697.
21
22
8
4 4
2
-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-fluoro
◦
−1
benzothiazol-2-yl)acetamide (6s): Yield 67%, m.p. 218 C. IR νmax (cm ): 3338 (N-H) 3072 (aromatic
C–H), 2927 (aliphatic C–H), 1674 (C=O ketone), 1562–1458 (C = N, C = C), 1147–1049 (C-N,
1
C-O). H-NMR (300 MHz, DMSO-d , ppm)
δ 1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m,
6
cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz
,
SCH CH2), 3.39–3.52 (3H, m, SCH2CH , cyclohexyl-H), 4.41 (2H, s, COCH ), 7.26–7.33 (1H, m,
2
2
2
Ar-H), 7.76–7.80 (2H, m, Ar-H, NH), 7.91 (1H, dd, J = 2.7 Hz, J = 9.0 Hz, Ar-H), 12.78 (1H, s,
1
2
NH). 13C-NMR (75 MHz, DMSO-d , ppm)
δ
24.67, 25.66, 26.10, 30.98, 32.46, 36.01, 54.03, 108.72 (d,
J = 27.0 Hz), 114.83 (d, J = 24.0 Hz), 122.32 (d, J = 9.8 Hz), 133.17 (d, J = 11.3 Hz), 145.66, 148.49,
58.10 (d, J = 2.2 Hz), 159.20 (d, J = 239.3 Hz), 163.21, 166.59, 166.92, 169.19. HR-MS (m/z): [M + H]+
calcd for C H FN O S : 552.0775; found: 552.0751.
6
1
21
22
7
2 4
3
.2. Antifungal Activity Assay
The anticandidal effect of compounds 6a–6s were screened in accord with the EUCAST definitive
(
EDef 7.1) method [25]. Ketoconazole was employed as a reference drug. The in vitro growth inhibitory
activity of the compounds were tested against C. albicans (ATCC 24433), C. glabrata (ATCC 90030),
C. krusei (ATCC 6258) and C. parapsilosis (ATCC 22019). The results were obtained as MIC values and
◦
MIC readings were accomplished twice for each chemical agent. After incubating overnight at 37 C,
the fungal strains were sustained in Roswell Park Memorial Institute (RPMI) medium.
Adjustment of the inoculum density to a 0.5 McFarland turbidity standard was carried out by
5
5
using a spectrophotometer. The resulting inoculum suspension contained 0.5
×
10 –2.5
×
10 cfu/mL
for fungi. The two-fold serial dilutions technique were applied and the test was carried out in
Mueller–Hinton broth and RPMI at pH7 and the test was performed for medium at pH7. The last well
on the microplates containing only inoculated broth and the last well with no growth of microorganism
represent controls and the MIC50 in µg/mL, respectively. DMSO was used as solvent. Further dilutions

Molecules 2017, 22, 2004
11 of 13
of the compounds and standard drugs in the RPMI medium were prepared in the required quantities
at contcentrations of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78 g/mL. After a 24 h
incubation of the completed plates, resazurin (20 g/mL) was added into each well in order to observe
µ
µ
the fungal growth. At the end of a 2 h incubation period of the final plates including microorganism
strains, MIC50 values were determined with a microplate reader at 590 nm excitation and 560 nm
emission. Each experiment in antifungal assay was performed twice.
3
.3. Effect of Compounds 6e, 6k, and 6s on Ergosterol Content of C. krusei
The ergosterol level was determined using the method described by Breivik and Owades [26
]
with adjustments according to [
2]. C. krusei was inoculated with Sabouraud dextrose broth (Difco)
supplemented with compounds 6e
,
6k 6r and the positive control, ketoconazole. The cultures
,
were incubated, and the stationary-phase cells were harvested. After the addition of 25% alcoholic
potassium hydroxide solution, cell suspensions were separated and incubated. Sterols were extracted
by sterile distilled water and chloroform, and then the extract was injected into the LC-MS-MS system
(Shimadzu LCMS 8040, Kyoto, Japan). The ergosterol quantity in the negative control samples was
considered as 100%. All concentrations were analysed in quadruplicate, and the results were presented
as mean ± standard deviation (SD).
3
.4. Cytotoxicity Test
The MTT assay was carried out to determine the cytotoxicity of the synthesized compounds
as previously reported in [21]. NIH/3T3 mouse embryonic fibroblast cells (ATCC® CRL-1658
™,
4
London, UK) were incubated according to the supplier’s references and seeded at 1
each well of 96-well plates. After this process, cells were treated with the compounds at concentrations
ranging from 800 g/mL to 0.78 g/mL. By means of the following formula, percent inhibition was
×
10 cells into
µ
µ
evaluated for each concentration. The IC50 values were determined by using dose-response curves
plotted against compound concentrations applied [27].
%
Inhibition = 100 − (Mean Sample × 100/Mean Solvent)
3
.5. Molecular Docking Studies
The crystal structures of 14 alpha-sterol demethylase enzyme (PDB ID: 1EA1) [22] that was
crystallized with fluconazole, was retrieved from the Protein Data Bank server (www.pdb.org).
The structure of ligand was built using the Schrödinger Maestro [28] interface and then was submitted
to the Protein Preparation Wizard protocol of the Schrödinger Suite 2016, Update 2 [29]. The ligand
was prepared by the LigPrep 3.8 [30] to correctly assign the protonation states at pH 7.4
±
1.0 and the
atom types. Bond orders were designated and hydrogen atoms were added to the structures. The grid
generation was formed using the Glide 7.1 [31] program and docking runs were performed with single
precision docking mode (SP).
4
. Conclusions
In conclusion, the eighteen newly synthesized 2-[[5-[2-[[5-(cyclohexylamino)-1,3,4-thiadiazol-2-yl]
thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-substituted ethan-1-one derivatives (6a 6s) were tested for
their inhibitory activity against the growth of four Candida species, and three of them (6e 6k and 6r
–
,
)
exhibited comparable antifungal activity to that of reference drug ketoconazole. After preliminary
studies, ergosterol level quantification assay and docking studies provided insight into the mechanism
of their antifungal activity. It is therefore likely that such connections exist between their mechanism
of action and the inhibition of ergosterol biosynthesis. Consequently, all experiments indicated that
this kind of structures may help to improve and/or design new antifungals due to their high activity
and nontoxic nature.

Molecules 2017, 22, 2004
12 of 13
Supplementary Materials: The spectra of all compounds are available online.
Acknowledgments: This study was financially supported by Anadolu University Scientific Projects Fund, Project
No: 1705S312.
Author Contributions: Y.Ö. and Z.A.K. conceived and designed the experiments; B.K.Ç. and D.O. performed the
synthesis; S.L. performed analysis studies; Ö.A. performed the cytotoxicity test; B.N.S. and U.A.Ç. performed
the microbiological studies; B.N.S. performed docking studies; 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.
References
1
.
Pfaller, M.A. Antifungal drug resistance: Mechanisms, epidemiology, and consequences for treatment.
Am. J. Med. 2012, 125, 3–13. [CrossRef] [PubMed]
2
.
Karaca Gençer, H.; Acar Çevik, U.; Levent, S.; Sa g˘ lık, B.N.; Korkut, B.; Özkay, Y.; Ilgın, S.; Öztürk, Y. New
benzimidazole-1,2,4-triazole hybrid compounds: synthesis, anticandidal activity and cytotoxicity evaluation.
Molecules 2017, 22, 507. [CrossRef] [PubMed]
3
.
Sadeghpour, H.; Khabnadideh, S.; Zomorodian, K.; Pakshir, K.; Hoseinpour, K.; Javid, N.; Faghih-Mirzaei, E.;
Rezaei, Z. Design, synthesis, and biological activity of new triazole and nitro-triazole derivatives as antifungal
agents. Molecules 2017, 22, 1150. [CrossRef] [PubMed]
4
.
.
Karyotakis, N.C.; Anaissie, E.J. The new antifungal azoles: Fluconazole and itraconazole. Curr. Opin. Infect. Dis.
1
994, 7, 658–666. [CrossRef]
Sangshetti, J.N.; Chabukswar, A.R.; Shinde, D.B. Microwave assisted one pot synthesis of some novel
,5-disubstituted 1,3,4-oxadiazoles as antifungal agents. Bioorg. Med. Chem. Lett. 2011, 21, 444–448. [CrossRef]
PubMed]
5
2
[
6
.
Xu, W.; He, J.; He, M.; Han, F.; Chen, X.; Pan, Z.; Wang, J.; Tong, M. Synthesis and antifungal activity of
novel sulfone derivatives containing 1,3,4-oxadiazole moieties. Molecules 2011, 16, 9129–9141. [CrossRef]
[PubMed]
7
8
9
1
1
1
.
.
.
Maslat, A.O.; Abussaud, M.; Tashtoush, H.; Al-Talib, M. Synthesis, antibacterial, antifungal and genotoxic
activity of bis-1,3,4-oxadiazole derivatives. Pol. J. Pharmacol. 2002, 54, 55–59. [PubMed]
Subhashinia, N.J.P.; Bhadraiaha, B.; Janaki, P. Synthesis and biological evaluation of 1,3,4-oxadiazole fused
pyridine derivatives as antibacterial and antifungal agents. Russ. J. Gen. Chem. 2017, 87, 550–553. [CrossRef]
Wang, F.; Qin, Z.; Huang, Q. Synthesis and fungicidal activity of 1,3,4-oxadiazole substituted acylthioureas.
Front. Chem. China 2006, 1, 112–114. [CrossRef]
0. Yurtta s¸ , L.; Bülbül, E.F.; Tekinkoca, S.; Demirayak, S. Antimicrobial activity evaluation of new 1,3,4-oxadiazole
derivatives. Acta Pharm. Sci. 2017, 55, 45–54. [CrossRef]
1. Boström, J.; Hogner, A.; Llinàs, A.; Wellner, E.; Plowright, A.T. Oxadiazoles in medicinal chemistry.
J. Med. Chem. 2012, 55, 1817–1830. [CrossRef] [PubMed]
2. Salgin-Göksen, U.; Gökhan-Kelekçi, N.; Göktas, O.; Köysal, Y.; Kiliç, E.; Isik, S.; Aktay, G.; Ozalp, M.
1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones, 1,3,4-thiadiazoles and hydrazones containing
5-methyl-2-benzoxazolinones: Synthesis, analgesic-anti-inflammatory and antimicrobial activities.
Bioorg. Med. Chem. 2007, 15, 5738–5751. [CrossRef] [PubMed]
1
3. Chen, C.J.; Song, B.A.; Yang, S.; Xu, G.F.; Bhadury, P.S.; Jin, L.H.; Hu, D.Y.; Li, Q.Z.; Liu, F.; Xue, W.; et al.
Synthesis and antifungal activities of 5-(3,4,5-trimethoxyphenyl)-2-sulfonyl-1,3,4-thiadiazole and 5-(3,4,5-
trimethoxyphenyl)-2-sulfonyl-1,3,4-oxadiazole derivatives. Bioorg. Med. Chem. 2007, 15, 3981–3989.
[
CrossRef] [PubMed]
4. Abdel-Wahab, B.F.; Abdel-Aziz, H.A.; Ahmed, E.M. Synthesis and antimicrobial evaluation of some
,3-thiazole, 1,3,4-thiadiazole, 1,2,4-triazole, and 1,2,4-triazolo[3,4-b] [1,3,4]-thiadiazine derivatives including
1
1
a 5-(benzofuran-2-yl)-1-phenylpyrazole moiety. Monatsh. Chem. 2009, 140, 601–605. [CrossRef]
5. Matysiak, J.; Malinski, Z. 2-(2,4-Dihydroxyphenyl)-1,3,4 thiadiazole analogues: Antifungal activity in vitro
against Candida species. Russ. J. Bioorg. Chem. 2007, 33, 594–601. [CrossRef]
6. Klip, N.T.; Capan, G.; Gursoy, A.; Uzun, M.; Satana, D. Synthesis, structure, and antifungal evaluation of some
novel 1,2,4-triazolylmercaptoacetylthiosemicarbazide and 1,2,4-triazolylmercaptomethyl-1,3,4-thiadiazole
analogs. J. Enzyme Inhib. Med. Chem. 2010, 25, 126–131. [CrossRef] [PubMed]
1
1

Molecules 2017, 22, 2004
13 of 13
1
7. Demirci, S.; Mermer, A.; Ak, G.; Aksakal, F.; Colak, N.; Demirbas, A.; Ayaz, F.A.; Demirbas, N. Conventional
and microwave-assisted total synthesis, antioxidant capacity, biological activity, and molecular docking
studies of new hybrid compounds. J. Heterocycl. Chem. 2017, 54, 1785–1805. [CrossRef]
1
8. Janakiramudu, D.B.; Subba Rao, D.; Srikanth, C.; Madhusudhana, S.; Murthy, P.S.; Nagalakshmidevamma, M.;
Chalapathi, P.V.; Naga Raju, C. Sulfonamides and carbamates of 3-fluoro-4-morpholinoaniline (linezolid
intermediate): Synthesis, antimicrobial activity and molecular docking study. Res. Chem. Intermed. 2017
CrossRef]
.
[
1
2
9. Sowmya, D.V.; Basha, S.S.; Padmaja, A.; Padmavathi, V.; Devi, P.U.M.; Lavanyalatha, Y. Synthesis,
antimicrobial, and anti-inflammatory activities of acetamido pyrrolyl azoles. Med. Chem. Res. 2017, 26,
1
010–1021. [CrossRef]
0. Malhotra, M.; Rawal, R.K.; Malhotra, D.; Dhingra, R.; Deep, A.; Sharma, P.C. Synthesis, characterization and
pharmacological evaluation of (Z)-2-(5-(biphenyl-4-yl)-3-(1-(imino)ethyl)-2,3-dihydro-1,3,4-oxadiazol-2-yl)
phenol derivatives as potent antimicrobial and antioxidant agents. Arab. J. Chem. 2017, 10, S1022–S1031.
[CrossRef]
2
2
1. Sa g˘ lık, B.N.; Ilgın, S.; Özkay, Y. Synthesis of new donepezil analogues and investigation of their effects on
cholinesterase enzymes. Eur. J. Med. Chem. 2016, 124, 1026–1040. [CrossRef] [PubMed]
2. Podust, L.M.; Poulos, T.L.; Waterman, M.R. Crystal structure of cytochrome P450 14a-sterol demethylase
(CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors. Proc. Natl. Acad. Sci. USA
2
001, 98, 3068–3073. [CrossRef] [PubMed]
2
3. Altıntop, M.D.; Sever, B.; Özdemir, A.; Küçüko g˘ lu, K.; Onem, H.; Nadaro g˘ lu, H.; Kaplancıklı, Z.A. Potential
inhibitors of human carbonic anhydrase isozymes I and II: Design, synthesis and docking studies of new
1
,3,4-thiadiazole derivatives. Bioorg. Med. Chem. 2017, 25, 3547–3554. [CrossRef] [PubMed]
4. Kaplancıklı, Z.A. Synthesis of some oxadiazole derivatives as new anticandidal agents. Molecules 2011, 16,
662–7671. [CrossRef] [PubMed]
5. Rodriguez-Tudela, J.L.; Arendrup, M.C.; Barchiesi, F.; Bille, J.; Chryssanthou, E.; Cuenca-Estrella, M.;
Dannaoui, E.; Denning, D.W.; Donnelly, J.P.; Dromer, F.; et al. EUCAST Definitive document EDef 7.1: Method
for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee
on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial
Susceptibility Testing (EUCAST). Clin. Microbiol. Infect. 2008, 14, 398–405.
2
2
7
2
6. Breivik, O.N.; Owades, J.L. Spectrophotometric semi-micro determination of ergosterol in yeast. Agric. Food Chem.
1
957, 5, 360–363. [CrossRef]
2
7. Patel, S.; Gheewala, N.; Suthar, A.; Shah, A. In-vitro cytotoxicity activity of Solanum nigrum extract against
Hela cell line and Vero cell line. Int. J. Pharm. Pharm. Sci. 2009, 1, 38–46.
2
2
3
3
8. Maestro, version 10.6; Schrödinger Maestro LLC: New York, NY, USA, 2016.
9. Schrödinger, version 2016-2; Schrödinger Suite Update 2; Schrödinger LLC: New York, NY, USA, 2016.
0. LigPrep, version 3.8; Schrödinger LLC: New York, NY, USA, 2016.
1. Glide, version 7.1; Schrödinger LLC: New York, NY, USA, 2016.
Sample Availability: Samples of the compounds 6a–6s from the authors.
©
2017 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/).