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Article

Synthesis of Oxadiazole-Thiadiazole Hybrids and Their Anticandidal Activity

by
Serkan Levent
1,2,
Betül Kaya Çavuşoğlu
1,
Begüm Nurpelin Sağlık
1,2,
Derya Osmaniye
1,2,
Ulviye Acar Çevik
1,2,
Özlem Atlı
3,
Yusuf Özkay
1,2,* and
Zafer Asım Kaplancıklı
1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
2
Doping and Narcotic Compounds Analysis Laboratory, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
3
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, 26470 Eskişehir, Turkey
*
Author to whom correspondence should be addressed.
Molecules 2017, 22(11), 2004; https://doi.org/10.3390/molecules22112004
Submission received: 8 November 2017 / Revised: 13 November 2017 / Accepted: 15 November 2017 / Published: 18 November 2017
(This article belongs to the Special Issue Emerging Drug Discovery Approaches against Infectious Diseases)
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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 including FT-IR, 1H-NMR, 13C-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.

Graphical Abstract

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 [1]. 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 14α-demethylase (CYP51) in yeast and fungi [2]. CYP51 is a key enzyme in fungal 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 [4]. Among a large variety of azole derivatives, 1,3,4-oxadiazole compounds were reported to possess various biological activities including antifungal activity [5,6,7,8,9]. The –N=C–O– linkage in oxadiazoles can react with the nucleophilic centers of 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 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 [12,13,14,15,16].
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–6s) were synthesized according to the pathway as outlined in Scheme 1. The thiosemicarbazide derivative (1) was obtained through the reaction of cyclohexyl isothiocyanate and hydrazine hydrate. The ring closure reaction of compound 1 with carbon disulfide in the presence of sodium hydroxide gave 5-(cyclohexylamino)-1,3,4-thiadiazole-2-thiol (2) which then was reacted with ethyl bromopropionate to obtain ethyl 3-[[5-(cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]propanoate (3). 3-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]propanehydrazide (4) was synthesized via the treatment of ester (3) with hydrazine hydrate. The second ring closure reaction of the corresponding hydrazide (4) with carbon disulfide resulted in the formation of 5-[2-[[5-(cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazole-2-thiol (5). Finally, compound 5 was reacted with appropriate 4-substituted phenacyl bromides, 4-substituted anilines or 6-substituted-2-aminobenzothiazoles to afford the target compounds (6a–6s).
The formation of synthesized compounds (6a–6s) was confirmed by IR, mass spectra, 1H-NMR and 13C-NMR. In the IR spectra of all synthesized compounds, bands at ~3340 cm−1, ~3070 cm−1, ~1580 cm−1 and ~1480 cm−1 were attributed to the stretching of the N-H, aromatic C-H, C=N and C=C groups. The carbonyl band belonging to ketone group was observed between 1660–1701 cm−1. The compounds 6e, 6k and 6r showed two bands around ~1520 cm−1 and ~1330 cm−1 due to asymmetric and symmetric stretching of the NO2 group. The 1H-NMR of all synthesized compounds showed peaks at ~3.25 ppm (SCH2CH2) and ~3.41 ppm (-SCH2CH2-) due to the methylene protons of thioethyl moiety. The signals of oxoethyl moiety (COCH2) were resonated at ~5.09 ppm, ~4.27 ppm and ~4.41 ppm as singlets for compounds 6a–6f, 6g–6l and 6m–6s, respectively. For all compounds, 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 (-NHCOCH2) was designated at ~10.38 ppm in compounds 6g–6l and ~12.76 ppm in compounds 6m–6s. In the 13C-NMR of compounds, the signal due to the carbonyl group appeared at ~192.50 ppm for compounds 6a–6f and at ~169.20 ppm for 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 with their molecular weights. The IR, 1H-NMR, 13C-NMR, and HRMS spectra of all compounds are presented in supplementary file.

2.2. Antifungal Activity Assay

The results of anticandidal activity of the synthesized compounds 6a–6s and reference drugs are presented with minimum inhibitory concentration (MIC50; µg/mL) values in Table 1. Minimum concentrations of compounds 6a–6s that inhibited the growth of yeast strains ranged from 0.78 to 100 µg/mL. According to antifungal screening, compounds 6e, 6k and 6r were found to be active 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 activity against C. krusei, compared to 4-methoxy derivative. In compounds 6g–6l, the effects of 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 [18,19,20]. 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.

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 µg/mL, 1.56 µg/mL, 3.12 µg/mL concentrations and expressed as (%) inhibition. The results are respresented in Table 2. 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.

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

14-α-Sterol demethylase is a key enzyme in the membrane ergosterol biosynthesis of fungi. In docking studies, the binding modes of compound 6e to 14-α-sterol demethylase was clarified. Studies were performed with the X-ray crystal structure of 14-α-sterol demethylase from 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.

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); 1H-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-d6, using TMS as 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 1 (0.09 mol) was dissolved in ethanol (150 mL) and a solution of NaOH (0.1 mol) in 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 2 (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].

3.1.4. Synthesis of 3-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl)thio]propanehydrazide (4)

Compound 3 (0.06 mol) and hydrazine hydrate (0.06 mol) were stirred in ethanol (150 mL) at room temperature for 4 h and filtered [23].

3.1.5. Synthesis of 5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazole-2-thiol (5)

Compound 4 (0.05 mol) was added to a solution of NaOH (0.06 mol) in ethanol (100 mL). After the 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 5 (2 mmol) and appropriate 4-substituted phenacyl bromide (2 mmol), 4-substituted 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): Yield 67%, m.p. 141 °C. IR νmax (cm−1): 3340 (N-H) 3064 (aromatic C-H), 2933 (aliphatic C-H), 1681 (C=O ketone), 1587–1487 (C=N, C=C), 1197–1085 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) 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, SCH2CH2), 3.41 (2H, t, J = 7.4 Hz, SCH2CH2), 3.46–3.52 (1H, m, cyclohexyl-H), 5.09 (2H, s, COCH2), 7.58 (2H, t, J = 7.8 Hz, Ar-H), 7.71 (1H, t, 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). 13C-NMR (75 MHz, DMSO-d6, 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, 192.99. HR-MS (m/z): [M + H]+ calcd for C20H23N5O2S3: 462.1087; found: 462.1089.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-1-(4-methyl phenyl)ethan-1-one (6b): Yield 70%, m.p. 136 °C. IR νmax (cm−1): 3327 (N-H) 3055 (aromatic C–H), 2926 (aliphatic C–H), 1678 (C=O ketone), 1589–1481 (C=N, C=C), 1298–1089 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.30 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.65–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 2.40 (3H, s, CH3), 3.25 (2H, t, J = 6.5 Hz, SCH2CH2), 3.41 (2H, t, J = 7.0 Hz, SCH2CH2), 3.45–3.51 (1H, m, cyclohexyl-H), 5.05 (2H, s, COCH2), 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). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 21.71, 24.67, 25.65, 26.08, 31.05, 32.47, 41.00, 54.00, 129.03, 129.89, 132.99, 145.03, 148.52, 163.53, 166.31, 169.18, 192.48 (C=O). HR-MS (m/z): [M + H]+ calcd for C21H25N5O2S3: 476.1243; found: 476.1241.
2-[[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−1): 3338 (N-H) 3076 (aromatic C–H), 2927 (aliphatic C–H), 1660 (C=O ketone), 1593–1483 (C=N, C=C), 1259–1095 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.16–1.30 (5H, m, cyclohexyl-H), 1.53–1.56 (1H, m, cyclohexyl-H), 1.65–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.25 (2H, t, J = 6.5 Hz, SCH2CH2), 3.39–3.48 (3H, m, SCH2CH2, cyclohexyl-H), 3.86 (3H, s, OCH3), 5.03 (2H, s, COCH2), 7.09 (2H, d, J = 8.9 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 8.02 (2H, d, J = 8.9 Hz, Ar-H). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.65, 26.08, 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 C21H25N5O3S3: 492.1187; found: 492.1192.
2-[[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−1): 3336 (N-H) 3057 (aromatic C–H), 2929 (aliphatic C–H), 1680 (C=O ketone), 1587–1481 (C=N, C=C), 1197–1087 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) 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, SCH2CH2), 3.39–3.52 (3H, m, SCH2CH2, cyclohexyl-H), 5.09 (2H, s, COCH2), 7.65 (2H, d, J = 8.6 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 8.05 (2H, d, J = 8.7 Hz, Ar-H). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.65, 26.07, 31.02, 32.46, 40.92, 54.01, 129.49, 130.84, 134.20, 139.37, 148.51, 163.38, 166.39, 169.18, 192.16. HR-MS (m/z): [M + H]+ calcd for C20H22ClN5O2S3: 496.0697; found: 496.0679.
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-one (6e): Yield 77%, m.p. 144 °C. IR νmax (cm−1): 3336 (N-H) 3068 (aromatic C–H), 2929 (aliphatic C–H), 1701 (C=O ketone), 1583–1481 (C=N, C=C), 1180–1051 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.11–1.35 (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), 3.26 (2H, t, J = 6.5 Hz, SCH2CH2), 3.35–3.49 (3H, m, SCH2CH2, cyclohexyl-H), 5.15 (2H, s, COCH2), 7.79 (1H, d, J = 7.3 Hz, NH), 8.27 (2H, d, J = 9.0 Hz, Ar-H), 8.39 (2H, d, J = 8.9 Hz, Ar-H). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.65, 26.05, 31.00, 32.46, 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 C20H22N6O4S3: 507.0937; found: 507.0924.
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-one (6f): Yield 64%, m.p. 131 °C. IR νmax (cm−1): 3342 (N-H) 3061 (aromatic C–H), 2929 (aliphatic C–H), 1680 (C=O ketone), 1587–1485 (C=N, C=C), 1232–1049 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.35 (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 = 6.3 Hz, SCH2CH2), 3.39-3.51 (3H, m, SCH2CH2, cyclohexyl-H), 5.08 (2H, s, COCH2), 7.41 (2H, t, J = 8.9 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 8.11–8.16 (2H, m, Ar-H). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.65, 26.07, 31.03, 32.46, 40.91, 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 C20H22FN5O2S3: 480.0992; 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): Yield 72%, m.p. 177 °C. IR νmax (cm−1): 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). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.10–1.36 (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, SCH2CH2), 3.39–3.53 (3H, m, SCH2CH2, cyclohexyl-H), 4.27 (2H, s, COCH2), 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, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.38 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 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 C20H24N6O2S3: 477.1196; found: 477.1191.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-methyl phenyl)acetamide (6h): Yield 79%, m.p. 173 °C. IR νmax (cm−1): 3336 (N-H) 3068 (aromatic C–H), 2916 (aliphatic C–H), 1668 (C=O ketone), 1521–1483 (C=N, C=C), 1240–1085 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H), 1.92–1.96 (2H, m, cyclohexyl-H), 2.25 (3H, s, CH3), 3.26 (2H, t, J = 6.5 Hz, SCH2CH2), 3.39–3.50 (3H, m, SCH2CH2, cyclohexyl-H), 4.25 (2H, s, COCH2), 7.12 (2H, 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). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 20.91, 24.68, 25.66, 26.10, 31.01, 32.47, 37.25, 54.02, 119.61, 129.67, 133.08, 136.60, 148.51, 163.60, 164.95, 166.36, 169.20. HR-MS (m/z): [M + H]+ calcd for C21H26N6O2S3: 491.1352; 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 phenyl)acetamide (6i): Yield 71%, m.p. 147 °C. IR νmax (cm−1): 3336 (N-H) 3064 (aromatic C–H), 2920 (aliphatic C–H), 1662 (C=O ketone), 1514–1421 (C=N, C=C), 1286–1035 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (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, SCH2CH2), 3.39–3.52 (3H, m, SCH2CH2, cyclohexyl-H), 3.72 (3H, s, OCH3), 4.23 (2H, s, COCH2), 6.89 (2H, d, 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). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.11, 31.02, 32.47, 37.17, 54.02, 55.61 (OCH3), 114.41, 121.17, 132.22, 148.51, 155.93, 163.61, 164.68, 166.36, 169.20. HR-MS (m/z): [M + H]+ calcd for C21H26N6O3S3: 507.1301; found: 507.1291.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-chloro phenyl)acetamide (6j): Yield 69%, m.p. 131 °C. IR νmax (cm−1): 3338 (N-H) 3066 (aromatic C–H), 2933 (aliphatic C–H), 1668 (C=O ketone), 1583–1483 (C=N, C=C), 1238–1083 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.35 (5H, m, cyclohexyl-H), 1.52–1.57 (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, SCH2CH2), 3.39–3.52 (3H, m, SCH2CH2, cyclohexyl-H), 4.27 (2H, s, COCH2), 7.38 (2H, d, J = 8.9 Hz, Ar-H), 7.60 (2H, d, J = 8.9 Hz, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.53 (1H, s, NH). 13C-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, 165.44, 166.41, 169.20. HR-MS (m/z): [M + H]+ calcd for C20H23ClN6O2S3: 511.0806; found: 511.0788.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-nitrophenyl)acetamide (6k): Yield 67%, m.p. 149 °C. IR νmax (cm−1): 3340 (N-H) 3049 (aromatic C–H), 2939 (aliphatic C–H), 1697 (C=O ketone), 1616–1490 (C=N, C=C), 1521 and 1328 (NO2), 1159–1072 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (5H, m, cyclohexyl-H), 1.53–1.56 (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, SCH2CH2), 3.39–3.51 (3H, m, SCH2CH2, cyclohexyl-H), 4.33 (2H, s, COCH2), 7.78–7.84 (3H, m, NH, Ar-H), 8.24 (2H, d, J = 9.2 Hz, Ar-H), 11.00 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.08, 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 C20H23N7O4S3: 522.1046; found: 522.1035.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(4-fluoro phenyl)acetamide (6l): Yield 73%, m.p. 160 °C. IR νmax (cm−1): 3342 (N-H) 3032 (aromatic C–H), 2854 (aliphatic C–H), 1670 (C=O ketone), 1583–1481 (C=N, C=C), 1168–1083 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.35 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, 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, SCH2CH2), 3.39–3.53 (3H, m, SCH2CH2, cyclohexyl-H), 4.25 (2H, s, COCH2), 7.16 (2H, t, J = 8.9 Hz, Ar-H), 7.56–7.60 (2H, m, Ar-H), 7.79 (1H, d, J = 7.3 Hz, NH), 10.45 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) 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 C20H23FN6O2S3: 495.1101; found: 495.1087.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(benzothiazol-2-yl)acetamide (6m): Yield 74%, m.p. 214 °C. IR νmax (cm−1): 3338 (N-H) 3062 (aromatic C–H), 2931 (aliphatic C–H), 1678 (C=O ketone), 1554–1492 (C=N, C=C), 1257–1085 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.31 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.4 Hz, SCH2CH2), 3.39–3.48 (3H, m, SCH2CH2, cyclohexyl-H), 4.42 (2H, s, COCH2), 7.32 (1H, t, J = 7.2 Hz, Ar-H), 7.45 (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, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.11, 30.99, 32.46, 36.09, 54.03, 121.16, 122.26, 124.22, 126.69, 131.90, 148.49, 158.11, 163.23, 166.58, 166.82, 169.20. HR-MS (m/z): [M + H]+ calcd for C21H23N7O2S4: 534.0869; found: 534.0866.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-methyl benzothiazol-2-yl)acetamide (6n): Yield 77%, m.p. 209 °C. IR νmax (cm−1): 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). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.66–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 2.41 (3H, s, CH3), 3.26 (2H, t, J = 6.5 Hz, SCH2CH2), 3.39–3.48 (3H, m, SCH2CH2, cyclohexyl-H), 4.40 (2H, s, COCH2), 7.26 (1H, dd, J1 = 1.4 Hz, J2 = 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, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 21.45, 24.67, 25.66, 26.10, 30.99, 32.46, 36.05, 54.02, 120.82, 121.83, 128.02, 132.05, 133.71, 148.49, 157.19, 163.24, 166.57, 169.20. HR-MS (m/z): [M + H]+ calcd for C22H25N7O2S4: 548.1025; found: 548.1007.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-methoxy benzothiazol-2-yl)acetamide (6o): Yield 75%, m.p. 198 °C. IR νmax (cm−1): 3334 (N-H) 3072 (aromatic C–H), 2933 (aliphatic C–H), 1681 (C=O ketone), 1614–1467 (C=N, C=C), 1257–1033 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (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, SCH2CH2), 3.39–3.48 (3H, m, SCH2CH2, cyclohexyl-H), 3.80 (3H, s, OCH3), 4.39 (2H, s, COCH2), 7.04 (1H, dd, J1 = 2.6 Hz, J2 = 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, J = 7.3 Hz, NH), 12.62 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.11, 30.99, 32.47, 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, 166.56, 169.20. HR-MS (m/z): [M + H]+ calcd for C22H25N7O3S4: 564.0974; found: 564.0965.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-chloro benzothiazol-2-yl)acetamide (6p): Yield 74%, m.p. 213 °C. IR νmax (cm−1): 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). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.10–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.65–1.70 (2H, m, cyclohexyl-H), 1.91–1.95 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH2CH2), 3.39–3.49 (3H, m, SCH2CH2, cyclohexyl-H), 4.41 (2H, s, COCH2), 7.47 (1H, dd, J1 = 2.3 Hz, J2 = 9.0 Hz, 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). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.10, 30.98, 32.46, 36.03, 54.03, 121.99, 122.39, 127.05, 128.27, 133.61, 147.82, 148.49, 158.96, 163.19, 166.60, 167.05, 169.19. HR-MS (m/z): [M + H]+ calcd for C21H22ClN7O2S4: 568.0479; found: 568.0469.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-nitro benzothiazol-2-yl)acetamide (6r): Yield 68%, m.p. 205 °C. IR νmax (cm−1): 3338 (N-H) 3051 (aromatic C–H), 2933 (aliphatic C–H), 1687 (C=O ketone), 1614–1446 (C=N, C=C), 1514 and 1334 (NO2), 1288–1093 (C-N, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.13–1.34 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, cyclohexyl-H), 1.65–1.69 (2H, m, cyclohexyl-H), 1.91–1.94 (2H, m, cyclohexyl-H), 3.26 (2H, t, J = 6.5 Hz, SCH2CH2), 3.39–3.48 (3H, m, SCH2CH2, cyclohexyl-H), 4.45 (2H, s, COCH2), 7.78 (1H, d, J = 7.3 Hz, NH), 7.92 (1H, d, J = 9.0 Hz, Ar-H), 8.29 (1H, dd, J1 = 2.4 Hz, J2 = 8.9 Hz, Ar-H), 9.06 (1H, d, J = 2.3 Hz, Ar-H), 13.17 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, ppm) δ 24.67, 25.66, 26.09, 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, 166.64, 167.65, 169.17. HR-MS (m/z): [M + H]+ calcd for C21H22N8O4S4: 579.0720; found: 579.0697.
2-[[5-[2-[[5-(Cyclohexylamino)-1,3,4-thiadiazol-2-yl]thio]ethyl]-1,3,4-oxadiazol-2-yl]thio]-N-(6-fluoro benzothiazol-2-yl)acetamide (6s): Yield 67%, m.p. 218 °C. IR νmax (cm−1): 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, C-O). 1H-NMR (300 MHz, DMSO-d6, ppm) δ 1.14–1.36 (5H, m, cyclohexyl-H), 1.52–1.56 (1H, m, 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, SCH2CH2), 3.39–3.52 (3H, m, SCH2CH2, cyclohexyl-H), 4.41 (2H, s, COCH2), 7.26–7.33 (1H, m, Ar-H), 7.76–7.80 (2H, m, Ar-H, NH), 7.91 (1H, dd, J1 = 2.7 Hz, J2 = 9.0 Hz, Ar-H), 12.78 (1H, s, NH). 13C-NMR (75 MHz, DMSO-d6, 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, 158.10 (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 C21H22FN7O2S4: 552.0775; found: 552.0751.

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 using a spectrophotometer. The resulting inoculum suspension contained 0.5 × 105–2.5 × 105 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 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 µg/mL. After a 24 h incubation of the completed plates, resazurin (20 µg/mL) was added into each well in order to observe 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™, London, UK) were incubated according to the supplier’s references and seeded at 1 × 104 cells into each well of 96-well plates. After this process, cells were treated with the compounds at concentrations ranging from 800 µg/mL to 0.78 µg/mL. By means of the following formula, percent inhibition was 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.

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. [Google Scholar] [CrossRef] [PubMed]
  2. Karaca Gençer, H.; Acar Çevik, U.; Levent, S.; Sağlık, 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. [Google Scholar] [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. [Google Scholar] [CrossRef] [PubMed]
  4. Karyotakis, N.C.; Anaissie, E.J. The new antifungal azoles: Fluconazole and itraconazole. Curr. Opin. Infect. Dis. 1994, 7, 658–666. [Google Scholar] [CrossRef]
  5. Sangshetti, J.N.; Chabukswar, A.R.; Shinde, D.B. Microwave assisted one pot synthesis of some novel 2,5-disubstituted 1,3,4-oxadiazoles as antifungal agents. Bioorg. Med. Chem. Lett. 2011, 21, 444–448. [Google Scholar] [CrossRef] [PubMed]
  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. [Google Scholar] [CrossRef] [PubMed]
  7. 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. [Google Scholar] [PubMed]
  8. 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. [Google Scholar] [CrossRef]
  9. Wang, F.; Qin, Z.; Huang, Q. Synthesis and fungicidal activity of 1,3,4-oxadiazole substituted acylthioureas. Front. Chem. China 2006, 1, 112–114. [Google Scholar] [CrossRef]
  10. Yurttaş, 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. [Google Scholar] [CrossRef]
  11. Boström, J.; Hogner, A.; Llinàs, A.; Wellner, E.; Plowright, A.T. Oxadiazoles in medicinal chemistry. J. Med. Chem. 2012, 55, 1817–1830. [Google Scholar] [CrossRef] [PubMed]
  12. 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. [Google Scholar] [CrossRef] [PubMed]
  13. 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. [Google Scholar] [CrossRef] [PubMed]
  14. Abdel-Wahab, B.F.; Abdel-Aziz, H.A.; Ahmed, E.M. Synthesis and antimicrobial evaluation of some 1,3-thiazole, 1,3,4-thiadiazole, 1,2,4-triazole, and 1,2,4-triazolo[3,4-b] [1,3,4]-thiadiazine derivatives including a 5-(benzofuran-2-yl)-1-phenylpyrazole moiety. Monatsh. Chem. 2009, 140, 601–605. [Google Scholar] [CrossRef]
  15. 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. [Google Scholar] [CrossRef]
  16. 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. [Google Scholar] [CrossRef] [PubMed]
  17. 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. [Google Scholar] [CrossRef]
  18. 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. [Google Scholar] [CrossRef]
  19. 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, 1010–1021. [Google Scholar] [CrossRef]
  20. 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. [Google Scholar] [CrossRef]
  21. Sağlık, 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. [Google Scholar] [CrossRef] [PubMed]
  22. 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 2001, 98, 3068–3073. [Google Scholar] [CrossRef] [PubMed]
  23. Altıntop, M.D.; Sever, B.; Özdemir, A.; Küçükoğlu, K.; Onem, H.; Nadaroğlu, 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. [Google Scholar] [CrossRef] [PubMed]
  24. Kaplancıklı, Z.A. Synthesis of some oxadiazole derivatives as new anticandidal agents. Molecules 2011, 16, 7662–7671. [Google Scholar] [CrossRef] [PubMed]
  25. 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. [Google Scholar]
  26. Breivik, O.N.; Owades, J.L. Spectrophotometric semi-micro determination of ergosterol in yeast. Agric. Food Chem. 1957, 5, 360–363. [Google Scholar] [CrossRef]
  27. 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. [Google Scholar]
  28. Maestro, version 10.6; Schrödinger Maestro LLC: New York, NY, USA, 2016.
  29. Schrödinger, version 2016-2; Schrödinger Suite Update 2; Schrödinger LLC: New York, NY, USA, 2016.
  30. LigPrep, version 3.8; Schrödinger LLC: New York, NY, USA, 2016.
  31. Glide, version 7.1; Schrödinger LLC: New York, NY, USA, 2016.
Sample Availability: Samples of the compounds 6a–6s from the authors.
Molecules 22 02004 sch001 550
Scheme 1. The synthesis of the compounds (6a–6s). Reagents and conditions: (i) BrCH2CH2COOEt, K2CO3, acetone, reflux, 9 h; (ii) NH2NH2·H2O, ethanol, rt, 4 h; (iii) (1) CS2/NaOH, ethanol, reflux, 10 h; (2) HCl, pH: 4–5; (iv) 4-substitutedphenacyl bromides (6a–6f), 2-chloro-N-(4-substitutedphenyl)acetamides (6g–6l), or 2-chloro-N-(6-substitutedbenzothiazol-2-yl)acetamides (6m–6s), K2CO3, acetone, rt.
Scheme 1. The synthesis of the compounds (6a–6s). Reagents and conditions: (i) BrCH2CH2COOEt, K2CO3, acetone, reflux, 9 h; (ii) NH2NH2·H2O, ethanol, rt, 4 h; (iii) (1) CS2/NaOH, ethanol, reflux, 10 h; (2) HCl, pH: 4–5; (iv) 4-substitutedphenacyl bromides (6a–6f), 2-chloro-N-(4-substitutedphenyl)acetamides (6g–6l), or 2-chloro-N-(6-substitutedbenzothiazol-2-yl)acetamides (6m–6s), K2CO3, acetone, rt.
Molecules 22 02004 sch001

R/ArCompoundR’CompoundR’CompoundR’
Molecules 22 02004 i0016aH6bCH36cOCH3
6dCl6eNO26fF
Molecules 22 02004 i0026gH6hCH36iOCH3
6jCl6kNO26lF
Molecules 22 02004 i0036mH6nCH36oOCH3
6pCl6rNO26sF
Molecules 22 02004 g001 550
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.
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 22 02004 g001
Table
Table 1. Minimum inhibitory concentration (MIC50; µg/mL) values of compounds on four strains of Candida (6a–6s).
Table 1. Minimum inhibitory concentration (MIC50; µg/mL) values of compounds on four strains of Candida (6a–6s).
CompoundC. albicansC. glabrataC. kruseiC. parapsilopsis
6a25505050
6b505010050
6c1002510050
6d100255050
6e1.561.560.780.78
6f50502550
6g50505050
6h2510010050
6i2510010050
6j25505050
6k1.561.561.561.56
6l100505025
6m50505025
6n100255025
6o100252550
6p50252550
6r3.123.121.561.56
6s506.255050
Ketoconazole0.781.561.561.56
Table
Table 2. Cytotoxic activity and ergosterol biosynthesis potency of the compounds 6e, 6k and 6r against NIH/3T3 cell line and C. krusei, respectively.
Table 2. Cytotoxic activity and ergosterol biosynthesis potency of the compounds 6e, 6k and 6r against NIH/3T3 cell line and C. krusei, respectively.
Comp.IC50 (µg/mL)Inhibition of Ergosterol Biosynthesis (%)
0.78 µg/mL1.56 µg/mL3.12 µg/mL
6e>50076.45 ± 3.6782.94 ± 4.7288.56 ± 4.52
6k>50058.72 ± 2.4174.65 ± 3.2681.29 ± 4.19
6r406.066 ± 9.3661.43 ± 1.8870.59 ± 4.2380.21 ± 3.70
Ketoconazole-60.99 ± 2.9473.12 ± 4.1684.56 ± 3.01

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Levent, S.; Kaya Çavuşoğlu, B.; Sağlık, B.N.; Osmaniye, D.; Acar Çevik, U.; Atlı, Ö.; Özkay, Y.; Kaplancıklı, Z.A. Synthesis of Oxadiazole-Thiadiazole Hybrids and Their Anticandidal Activity. Molecules 2017, 22, 2004. https://doi.org/10.3390/molecules22112004

AMA Style

Levent S, Kaya Çavuşoğlu B, Sağlık BN, Osmaniye D, Acar Çevik U, Atlı Ö, Özkay Y, Kaplancıklı ZA. Synthesis of Oxadiazole-Thiadiazole Hybrids and Their Anticandidal Activity. Molecules. 2017; 22(11):2004. https://doi.org/10.3390/molecules22112004

Chicago/Turabian Style

Levent, Serkan, Betül Kaya Çavuşoğlu, Begüm Nurpelin Sağlık, Derya Osmaniye, Ulviye Acar Çevik, Özlem Atlı, Yusuf Özkay, and Zafer Asım Kaplancıklı. 2017. "Synthesis of Oxadiazole-Thiadiazole Hybrids and Their Anticandidal Activity" Molecules 22, no. 11: 2004. https://doi.org/10.3390/molecules22112004

APA Style

Levent, S., Kaya Çavuşoğlu, B., Sağlık, B. N., Osmaniye, D., Acar Çevik, U., Atlı, Ö., Özkay, Y., & Kaplancıklı, Z. A. (2017). Synthesis of Oxadiazole-Thiadiazole Hybrids and Their Anticandidal Activity. Molecules, 22(11), 2004. https://doi.org/10.3390/molecules22112004

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