Synthesis and biological evaluation of some new pyrimidine bearing 2,5-disubstituted 1,3,4-oxadiazole derivatives as cytotoxic agents Pirimidin içeren bazı yeni 2,5-disübstitüe 1,3,4-oksadiazol türevlerinin sentezi ve sitotoksik ajan olarak değerlendirilmesi

Objective: As a result of adverse effects including drug-resistance, toxicity and low bioavailability, there has been a crucial need for novel anticancer agents. In this present study, some novel 2,5-disubstituted 1,3,4-oxadiazole derivatives bearing pyridine moiety were synthesized and their potential cytotoxic activities were examined. Materials and methods: A series of seven new compounds of 2-[(5-(3-(pyrimidin-2-yl)thio)propyl)-1,3,4-oxa-diazol-2-yl)thio)]-1-(4-substituted)ethan-1-one derivatives were synthesized by reacting 5-[(3-(pyrimidin-2-yl)thio) propyl]-1,3,4-oxadiazole-2-thiol and 4-substituted phenacyl bromide derivatives in acetone with potassium carbonate. The structures of the obtained compounds were elucidated using FT-IR, 1 H-NMR and MS spectral data and elemental analyses. In vitro cytotoxic activity of the compounds was evaluated by MTT assay. Results: Among the tested compounds, compound 4a was found to be the most active cytotoxic agent against A549 cells, in compared with cisplatin as standard drug. Conclusions: It was determined that some of synthesized compounds had considerable anticancer activity against the A549 cell lines. Compound 4a including phenyl moiety was the most active compound against the A549 cell line and was identified as a lead moiety. Besides, compound 4c including 4-metoxy phenyl moiety exhibited cytotoxic activity against A549 cells. Consequently, compounds possess phenyl and 4-methoxy phenyl moieties have been determined to be important for cytotoxic activity of these compounds. known anticancer drug cisplatin.


Introduction
Cancer is described as a large group of disease that uncontrolled growth and expansion of abnormal cells included. Because of being responsible from 8.2 million deaths in 2012, cancer is one of the main causes of death in the world. It is presumed that within the next two decades the number of people with cancer will arise by about 70% [1]. The causes of cancer formation are generally using tobacco and alcohol, unhealthy diet, chemicals, radiation, immune conditions, hormones, infectious organisms [2]. Chemotherapy, radiation and surgery are leading treatments for cancer. Nitrogen mustards and antifolate drugs were used to treat cancer, primarily [3]. Although various chemotherapy drugs have been produced until today, most of them are not selective to neoplastic cells. Moreover, there are some adverse effects like low bioavailability, toxicity and drug-resistance [4]. That is why, new approaches for cancer therapy are required and become an emergence to develop new therapeutic drugs with less toxicity to the patients than is seen with currently used medicines. Anticancer drugs can be classified as cytotoxic agents, biological agents, bisphosphonates, hormones and others [5]. Among these drugs, the commonly used are cytotoxic drugs.
Due to severe side effects of substantial anticancer drugs, researchers concentrate on advancing more selective drugs. For this purpose, heterocyclic ring systems have become prominent by means of their similarity to biologically active compounds in our body. Oxadiazole is one of the significant heterocyclic ring systems with respect to what occurring in many drugs. Depending on the position of nitrogen and oxygen atoms in the ring, there are four different groups of oxadiazole rings, including 1,2,3-oxadiazole, 1,2,5-oxadiazole, 1,2,4-oxadiazole and 1,3,4-oxadiazole [6].
In the light of this information, we synthesized seven compounds (4a-g) possessing 1,3,4-oxadiazole and pyrimidine moieties and tested their antiproliferative activity against A549 lung cancer cells. The antiproliferative activity of the new compounds was evaluated by MTT method. The cytotoxic potency of compounds 4a-g was studied in comparison with the known anticancer drug cisplatin.

4-[(Pyrimidin-2-yl)thio]butano hydrazide (2) Ethyl
4-[(pyrimidin-2-yl)thio]butanoate (0.03 mol) was dissolved in ethanol (250 mL). Hydrazine hydrate (0.03 mol) dissolved in ethanol, was added gradually and the mixture stirred at room temperature. After completion of the reaction, the solvent was evaporated under reduced pressure then water was added to wash the resulting solid and the mixture was filtered, dried and recrystallized from ethanol to give compound 2.

5-[3-((Pyrimidin-2-yl)thio)propyl]-1,3,4-oxadiazole-2-thiol (3) 4-[(Pyrimidin-2-yl)thio]butano
hydrazide (0.02 mol) was dissolved in ethanol (250 mL). Potassium hydroxide (0.02 mol) was dissolved in ethanol (80 mL) with constant stirring. The second solution was added to first one. After carbon disulfide (0.02 mol) was added to this, the mixture was refluxed for 5 h. After completion of the reaction, dilute HCl was added to remove the salt form of the compound. Then water was added and the mixture was filtered, dried and recrystallized from ethanol to give compound 3. propyl]-1,3,4-oxadiazole-2-thiol (10 mmol) was dissolved in acetone (100 mL). Potassium carbonate (10 mmol) was added. After that, appropriate phenacyl bromide derivatives were added and stirred for 12 h at room temperature. After TLC screening, the solvent was evaporated under reduced pressure then water was added to wash the resulting solid and the mixture was filtered, dried and recrystallized from ethanol to give the final compounds 4a-g.

MTT assay
In order to examine cytotoxicity of the compounds against A549 (human lung adenocarcinoma cells) cell line, MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was applied in accordance with the reported data [37,38]. A549 Human lung adenocarcinoma cells were incubated in 90% RPMI supplemented with 10% fetal bovine serum (Gibco, Paisley, Scotland) and cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Then, growing cells were plated at 2 × 10 4 cells/mL into 96-well microtiter tissue culture plates (Nunc, Denmark) and incubated for 24 h. After this time, the synthesized compounds and cisplatin (reference drug) were dissolved in DMSO severally and added to give final concentration in the range 3.9-500 μg/mL and the cells were incubated for 24 h. At the end of this period, MTT was added to a final concentration of 0.5 mg/mL and the cells were incubated for 4 h at 37°C. Purple formazan crystals formed by MTT metabolism arised at the end of the process and crystals were dissolved in 200 mL DMSO. The absorbance was read at 540 nm with a microtiter plate spectrophotometer (Bio-Tek plate reader). The percentage of viable cells was calculated on the grounds that the medium control. IC 50 values were defined as the drug concentrations that diminished absorbance to 50% of control values.

Results and discussion
Target molecules (4a-g) were synthesized in four steps as shown in Figure 1

1-Phenyl-2-[(5-(3-((pyrimidin-2-yl) thio)propyl)-1,3,4-oxadiazol-2-yl)thio] ethan-1-one
Structures of the obtained compounds were elucidated by spectral data. In the IR spectra, significant stretching bands belonging to C=O were observed in between 1687 and 1672 cm − 1 , bands belonging to C=N and C=C were observed in between 1602 and 1448 cm − 1 and bands belonging to C-O were observed at about 1257-1085 cm − 1 . In the NMR spectra of the final compounds, a singlet peak at about 5.07 ppm was observed belonging to -CH 2 of the oxoethyl moiety. A triplet peak about 3.19 ppm was observed belonging to the -SCH 2 structure. Hydrogen atoms close to the nitrogen atom were observed at 8.63 ppm. The other hydrogen atom in pyrimidine was observed at 7.23 ppm in the NMR spectra. The other peaks belonging to aromatic and aliphatic protons were observed at the estimated areas.
Cytotoxical potencies of the compounds against tumor cells were measured by the colorimetric MTT assay. The MTT test is based on the cleavage of the yellow tetrazolium salt to form a soluble blue formazan product by mitochondrial enzymes. The amount of formazan produced Reactants, reagents and conditions; (i) K 2 CO 3 , acetone, reflux; (ii) NH 2 NH 2 ·H 2 O; (iii) KOH, CS 2 ; (iv) 2-bromo-1-phenylethan-1-one derivatives, K 2 CO 3 , acetone. is directly proportional to the number of living cells. The cytotoxic activities of the synthesized compounds (4a-g) were compared against positive controls by using A549 (human non-small cell lung cancer). Tested concentrations for compounds were in between 3.9 and 500 mg/ mL and for control (cisplatin) were in between 0.98 and 500 mg/mL. The corresponding IC 50 values are listed in Table 1.