Synthesis, spectral characterization, and biological studies of 3,5-disubstituted-1,3,4-oxadiazole-2(3H)-thione derivatives

The reaction of 3,4-dichlorophenyl-1,3,4-oxadiazole-2( 3H )-thione with piperidine derivatives via Mannich reaction was used to generate eleven novel compounds in moderate to good yields. Synthesized molecules were characterized according to their structure with 1H NMR, 13C NMR and FT-IR spectral foundations, which were compatible with literature informations. Antimicrobial activity and cytotoxicity studies were done by disc diffusion and NCI-60 sulphordamine B assay methods. The antimicrobial test results revealed that synthesized compounds have better activity against gram-positive species than gram-negative ones. A total analysis of the antibacterial, antifungal, and antiyeast activity revealed that newly synthesized compounds were really active against Bacillus cereus , Bacillus ehimensis, and Bacillus thuringiensis species . For cytotoxicity, among three different cancer cell lines (HCT116, MCF7, HUH7) compounds 5c, 5d, 5e, 5f, 5g, 5i, 5j and 5k were seemed especially effective on HUH7 cancer cell line via moderate to good activity. More significantly, against liver carcinoma cell line (HUH7) most of the compounds of the series ( 5c-5g and 5i-5j ) have better IC50 values (IC50= 18.78 µM) than 5-Florouracil (5-FU) and also compound 5d possessed 10.1 µM value, which represents good druggable cytotoxic activity. Further, the molecules were also screened for in silico chemoinformatic and toxicity data to gather the predicted bioavailibity and safety measurements.

1,3,4-Oxadiazole is generally used entity for pharmacophore development and has been investigated because of its good metabolic profile and hydrogen-bonding capacity within the receptor site. Presence of azole group (N=C-O) also elevates lipophilicity feature of compound, which provides advantage for its transportation through cell membrane to reach the target site and show various biological activities [25]. These co-operative properties provide great benefits to obtain desired antimicrobial and anticancer activity within various proven in vitro and in vivo models. In 2013, Du and coworkers dealed with modeling 1,3,4-oxadiazole ring to obtain both of anticancer and antimicrobial effect by targeting thymidylate synthase (TS), which is an important enzyme for DNA synthesis. Newly synthesized compounds were identified as potent inhibitors against two kinds TS proteins with IC 50 values of 0.47-1.4 µM [26].
Nowadays, dual antimicrobial-anticancer activity of 1,3,4-oxadiazole core structure has been an important concern. Ahsan et al. dealed with disubstituted derivatives of 1,3,4-oxadiazole with antimicrobial-anticancer activity capacity ,and synthesized analogs showed moderate to severe potency for this binary physiological topics [27]. In another research, Savariz and coworkers studied with 3,5-disubstituted-1,3,4-oxadiazole pharmacophore group, which were derived with different functional moieties via Mannich reaction. Results showed that both anticancer and antimicrobial activity of synthesized series have intermediate to excellent effect, and especially one of the compounds, which has a heterocyclic ring from third position of core structure, improved antitumor activity, which was found to be 4.5 timefold compared to the precursor molecule [28]. Under the shadow of previous experiences, Selvaraj and coworkers synthesized fifteen 1,3,4-oxadiazole derivatives, and they were found to have moderate to severe inhibitor effect against different microbial and cancer cell lines in 2017 [29].
Based on the statements above, to develop new, potent antimicrobial and anticancer agents, we aimed to synthesize a new series of 3,5-disubstituted-1,3,4-oxadiazole-2(3H)-thione derivatives carrying different piperidine side chains. The compounds were evaluated for their antimicrobial and cytotoxicity profile to investigate the effect of molecular variations on activity against different bacteria, fungi, and yeasts. Further, compounds were tested in against various cancer cell lines for their cytotoxic activity.

Materials and measurements
Melting points of compounds were checked by Mettler Toledo FP62 capillary melting point apparatus (Mettler-Toledo, Greifensee, Switzerland) and are uncorrected. Infrared spectral data were obtained by the use of Perkin-Elmer Spectrum One series FT-IR apparatus (Version 5.0.1) (Perkin Elmer, Norwalk, CT, USA), with potassium bromide pellets and the frequencies were presented in cm -1 . The 1 H-NMR spectra were checked via Varian Mercury-400 FT-NMR spectrometer (Varian, Palo Alto, CA, USA) using tetramethylsilane as the internal reference, with dimethyl Sulfoxide (DMSO-d 6 ), as solvent, the chemical shifts were reported in parts per million (ppm), and coupling constants (J) were given in hertz (Hz). Elemental analyses were done by LECO 932 CHNS instrument (Leco-932, St. Joseph, MI, USA) and were within ± 0.4% of the theoretical values.

General procedure for the synthesis of 5-(3,4-dichlorophenyl)-1,3,4-oxadiazole-2(3H)-thione (4)
Solution of aroyl hydrazine (3.13 mmol) and carbon disulfide (6.27 mmol) in absolute ethanol (15 mL) were mixed in cold media (0 °C) and after the addition of potassium hydroxide (3.13 mmol) in one portion, the mixture was refluxed for 8 h. After the reaction was over, solvent was evaporated and the residue was acidified with 2M hydrochloric acid and extracted with ethyl acetate (2 × 20 mL). The organic layers were washed with water and dried with anhydrous sodium sulphate. Filtration and concentration in vacuo gave a solid, which was recrystallized from ethanol to give the compound [30].

. Disc diffusion method
Dimethylsulfoxide (DMSO) was used to dissolve and prepare the synthesized compounds with a concentration of 10 mg mL -1 . The lyophilized compounds sterilized by filtration via 0.45 mm millipore filters. Disc diffusion method was performed by using 100 mL of suspension containing 108 colony forming units (CFU) mL -1 of bacteria, 106 CFU mL -1 of yeast and 104 spore mL -1 of fungi spread on nutrient agar (NA), sabour dextrose agar (SDA), and potato dextrose agar (PDA) medium, in sequence. A total of 15 mL of each synthesized compounds (300 mg/disc) at the concentration of 10 mg mL -1 were impregnated to the discs (6 mm in diameter). DMSO impregnated discs were used for negative controls. The compounds and negative controls were located in the inoculated agar. In order to determine the sensitivity of one strain/ isolate standard, ofloxacin and nystatin were used as positive references for bacterial and fungus-yeast strains, respectively. The incubation at 37°C of inoculated plates took 24 h for bacterial strains, 48 h for yeast and 72 h for fungi isolates. The incubation of plant related microorganisms were held at 27°C, differently [31].

Cytotoxic activity
The human cancer cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM), with 10% fetal bovine serum (FBS) and 1% penicillin. They were incubated in 37 °C incubators containing 5% CO 2 and 95% air. Cancer cells (range of 2000 cells/well to 5000 cells/well) were inoculated into 96-well plates in 200 μL of media and incubated in 37 °C incubators containing 5% CO 2 and 95% air. After a 24 h incubation period, one plate for each cell line was fixed with 100 μL of 10% ice-cold trichloroacetic acid (TCA). This plate represents the behavior of the cells just prior to compound treatment and is accepted as the time-zero plate. The compounds to be tested were solubilized in dimethyl sulfoxide (DMSO) to a final concentration of 40 mM and stored at +4°C. While treating the cells with the compounds, the corresponding volume of the compound was applied to the cell to achieve the desired drug concentration and diluted through serial dilution (40, 20, 10, 5, 2.5 µM). After drug treatment, the cells were incubated in 37 °C incubators containing 5% CO 2 and 95% air for 72 h. Following the termination of the incubation period after drug treatment, the cells were fixed with 100 μL 10% ice-cold TCA and incubated in the dark at +4°C for 1 h. Then, the TCA was washed away with ddH 2 O five times and the plates were left to air dry. In the final step, the plates were stained with 100 μL of 0.4% SRB (cat.86183-5 g from Sigma) solution in 1% acetic acid solution. Following staining, the plates were incubated in dark for 10 min at room temperature. The unbound dye was washed away using 1% acetic acid and the plates were left to air dry. To measure the absorbance results, the bound stain was then solubilized using 200 μL of 10 mM Tris-Base. Camptothecin was the positive control and 5-Fluorouracil (5-FU) was standard drug for the cytotoxic effect. The OD values were obtained at 515nm [32].
The FT-IR spectrum of compounds displayed a strong band in range of 3080-2900 cm -1 which assigned to aromatic carbon-hydrogen sp 2 hybridizations in common for all compounds. Imine (C=N) and thione (C=S) groups in 1,3,4-oxadiazole-2(3H)-thione structure, generated two characteristic signals approximately at 1610 and 1330 cm -1 . Compounds 5c, 5g-5k had an extra sharp signal around 1740-1680 cm -1 , which corresponded to the carbonyl group, and compound 5d showed a spesific band at 2238 cm -1 , which was claimed as nitrile group. 1 H NMR spectra of compounds 4, 5a-5k demonstrated hydrogen signals of aromatic structures in the range of 8.00-7.00 ppm. Two proton integrationed and singlet coupled signal in the range of 5.13-5.01 ppm values was a strong evidence for methylene bridge protons between 1,3,4-oxadiazole and piperidine moieties which obtained via Mannich reaction procedure. Variable but compatible integrated signals between 3.60-1.30 ppm confirmed different piperidine protons for each compound. Compound 5c have an acetyl group and preserved a singlet signal in 1.84 ppm for alpha protons. Signals for compounds 5i-5k, which have different positioned ethyl ester groups on piperidine moiety emerged at 4.06-4.02 for methylene protons (-COOCH 2 -) and 1.18-1.15 ppm values for methyl protons (-COOCH 2 CH 3 ). Integration and multiplicity of signals for all compounds were compatible with literature data. 13 C NMR spectra of compounds 4, 5a-5k preserved two characteristic signals related to thione (C=S) and imine (C=N) groups at 179 and 158 ppm values originated from 1,3,4-oxadiazole-2(3H)-thione structure. Carbon signal of ketone carbonyl in compound 5b, carboxy and ester carbonyl in compounds 5g-5k were appeared at different ppm values due to different chemical environments of carbonyl functional groups. While ketone carbonyl carbon of 5c showed signal at 208 ppm, carboxy and ester carbonyl carbons of compound 5g-5k indicated their carbonyl carbon signals in the range of 179-174 ppm. Due to shielded-deshielded properties of carbon atoms in magnetic field of 13 C NMR, signal of ketone carbonyl in 5c occured in downfield region while carboxylic acid (5g, 5h) and ester (5i-5k) carbonyl carbons shifted through upfield part of the scale. In this direction, moderately shielded aromatic structures in compounds 4, 5a-k gave their spesific signals in the range of 138-122 ppm. 13 C NMR signals of remaining carbons associated with methylene and piperidine groups were observed at 70-65 and 69-32 ppm.

Biological evaluation 3.2.1. Antimicrobial activity
Antimicrobial activity was tested by measuring the zone of inhibition against test organisms with disc diffusion assay method, and results were summarized in Table 1 and Table 2 with positive control ofloxacin. Eleven compounds were screened for their antibacterial activity against three gram-negative (E. coli, P. aeruginosa, P. vulgaris) and sixteen grampositive bacterial strains. (Staphylococcus spp, Micrococcus spp, Bacillus spp). They were also evaluated for their antifungal potential against six fungal strains (Aspergillus spp, F. oxysporium, B. cinerea, Penicillium, Candida spp) and antiyeast activity against three yeast strains (K. marxianus, P. membranaefaciens, S. occidentalis). Ofloxacin and nystatin were used as positive controls. Antimicrobial data of compounds and reference drugs were given in Tables 1 and Table 2.
In vitro disc diffusion test was carried out to evaluate newly synthesized compounds (4, 5a-k) for their antibacterial activities towards pathogenic gram-positive and gram-negative bacteria, and ofloxacin was used as positive control under the same conditions. As shown in Table 1, compound series displayed serious inhibition of growth (mm) in certain bacterial strains. Especially compounds 5b, 5c, 5f-5k showed considerable antibacterial activity against gram-positive Bacillus spp. when compared to ofloxacin, while inhibition of growth values (mm) of other compounds in the series were either equal or lower than reference molecule. In vitro antibacterial screening results also revealed that, for all bacterial strains, lower inhibition values than reference material meaned as an indicator of antibacterial inability especially for compounds 5a, 5d and 5e (Table 1). None of compounds didn't show any antibacterial activity against E.coli and P. Aeruginosa but compounds 5b, 5f, 5g, 5h and 5j showed statistically significant antibacterial activity against B. ehimensis when they compared with ofloxacin.
The results obtained in parallel with this information was compound 5b, which had high level of antimicrobial property specifically against B. ehimensis due to its electron-donating hydroxyl moiety. In addition of these four compounds (5a-d), morpholine containing compound 5f was also found more active than reference drug on B. ehimensis strain. Heteroatoms in morpholine and their bonding capasities with active site of bacteria were serious indicators for biologic activity of 5f, which was an open point for further studies on heterocyclic structure substituted piperidine rings. Besides, substituent variation and their effects on biologic acitiy, structural design was modulated also to generate position effect on activity. So, on piperidine moiety, differently located carboxylic acid containing compounds 5g, 5h and differently positioned ethyl ester containing compounds 5i, 5j and 5k were added to series. Results clearly claimed that position differences of one substituent on piperidine did not cause a significant difference for their antimicrobial response (Table 1).
In vitro antimicrobial activity of compounds 4, 5a-k were further assessed in terms of antifungal and antiyeast activities relative to nystatin according to disc diffusion assay method. The results were presented in Table 2 and a review of data revealed that all compounds were possessed moderate activities against Candida spp and no inhibition against Aspergillus spp except compound 4. The best and comparable results were obtained against Penicillium sp. and F. oxysporium for compounds 4, 5g, and 5h. According to antiyeast activity profile, compounds showed weak to moderate activity against K. Marxianus wherease compounds 5b, 5g and 5h were equipotent against P. membranaefaciens and S. occidentalis when they were compared with nystatin. Also compound 5b, which has strong electron-donating hydroxyl group showed the best activity against all fungal and yeast strains according to other phenyl containing compounds 5a, 5c, and 5d. This unclear activity profile and lipophilicity relationship might be seen as described in previous study [39]. Due to the consistent results generated from antibacterial, antifungal and antiyeast activities, synthesized compounds were further analysed for their cytotoxic activities on certain cancer cell lines.

Cytotoxicity study
All synthesized target compounds 5a-5k were screened for their cytotoxic activity against three cancer cell lines: colon (HCT116), breast (MCF7), and liver (HUH7) with sulphorhodamine B (SRB) assay in triplicate aplication where 5-flourouracil (5-FU) was used as positive control. The IC 50 values obtained for these compounds were shown in Table 3.
The results of cytotoxicity studies revealed that activities of the compounds were not impressive against colon and breast cancer cells, all of the compounds showed cell viability with IC 50 values ranging from 13.9-80.4 µM concentrations. It was noteworthy that the cytotoxic effects were more pronounced against liver carcinoma cell line, HUH7. Most of the compounds of the series (5c-5g and 5i-5j) have better IC 50 values than 5-FLU (IC 50 = 18.78 µM) and also compound 5d possessed 10.1 µM value, which represents good druggable cytotoxic activity.
Our results indicate that the addition of phenyl and carbonyl group as substituents enhances the antimicrobial activity of the prepared compounds. It indicates that the structural differences is an important factor for the activity. It is notable, also, that phenyl and carbonyl substitutions in compounds 5c, 5d, 5f, 5g, 5i-5k lead to not only to an increase in the antimicrobial activity on certain bacterial and fungal strains, but also to a significant level of anticancer activity against liver carcinoma cell line (HUH7). According to the preliminary antimicrobial activity, compound 5b showed the best inhibitor activity against Bacillus spp strains, and compound 5d was evaluated to have strongest cytotoxic activity against liver carcinoma cells (HUH7). A total analysis of the antibacterial, antifungal, and antiyeast activity revealed that newly synthesized compounds were really active against Bacillus cereus, Bacillus ehimensis and Bacillus thuringiensis species.
In the situation of one deviation, corresponds to poor absorption of compounds. However, there are plenty of examples are available for RO5 violation amongst the existing drugs [41,42]. Furthermore, the polar surface area (PSA) of a molecule is defined as the surface sum over all polar atoms, primarily oxygen and nitrogen with their attached hydrogen atoms. The PSA value of a molecule reflects the ability to permeate cells, which is used for drug's optimization. Previous researches showed the standard value of PSA as <89 A 2 [43] in which this measure is supported by our newly synthesized compound 5a-5k serie. On the other hand, number of rotatable bond is a measurement for molecular flexibility and is significant in determining oral bioavailability of the compounds (rule of three-number of rotatable bonds ≤ 3), which explains oral usage of compound 5a-5k might be decrease bioavailibility (Table 4) [44]. Toxicity prediction is a tool is an useful method in the drug discovery due to many of the newly synthesized potential candidates had failed in clinical trial evaluation because of some pharmacokinetics and toxicity problems. In silico predictions are improved to overcome such scenario in this detailed process. Meanwhile, toxicity predictions were clearly revelead that all our novel compounds are seemed to have no predictable central nervous system side effects, carcinogenicity, and mutagenicity (Table 5).

Conclusion
In summary, we report the efficient synthesis, characterization, antimicrobial and cytotoxic activity evaluation of new compound series which contain different substituted piperidine bearing 1,3,4-oxadiazole-2(3H)-thione structures. According to biological consequences, phenyl and carbonyl group that substituted to piperidine ring seemed to have supportive property on antimicrobial activity of the novel compounds. Some compounds like 5c, 5d, 5f, 5g, 5i-5k that contain phenyl and carbonyl group revealed not only antimicrobial effect on certain bacterial and fungal strains but also significant level of anticancer activity against liver carcinoma cell line (HUH7). Especially compound 5b showed the best inhibitor activity against Bacillus spp whereas compound 5d represent valuable effect against liver carcinoma cells (HUH7). Besides, evaluating biological properties, compounds were predicted for the chemo-informatic and possible toxicity features within some software programmes. Synthesized molecules were calculated as to qualified the criteria to be a drug as per Lipinski Rule of Five and in silico predicted toxicology results were seemed as all of them have no mutagenic or carcinogenic profiles.

Supplementary Information
1 H and 13 C NMR spectra of all compounds, 5a-5k, are given as supplementary information at www.ias.ac.in/chemsci.