Synthesis of Substituted Thieno[2,3-d]pyrimidin-4-ones and Their Testing for Evaluation of Cytotoxic Activity onMammalian Cell Models

1 S.Yu. Yunusov Institute of the Chemistry of Plant Substances AS RUz, Mirzo Ulugbek Street 77, Tashkent 100170, Uzbekistan Department for the Innovation in Biological, Agrofoods and Forests Systems (DIBAF), Tuscia University, Laboratory of Plant Cytology and Biotechnology, Largo dell Università, Blocco D, 01100 Viterbo, Italy Department of Biotechnology and Microbiology, National University of Uzbekistan, Tashkent 100174, Uzbekistan Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, China

ese �ndings clearly show the potential importance of such molecules as active principles of new pharmaceuticals and therefore the development of effective methods of synthesis and searching of biological activities among new synthesized compounds are a very important direction.Prompted by the various biological activities of thieno [2,3d]pyrimidin-4-ones and its substituted derivatives, we envisioned our approach towards the synthesis of a novel series of thieno [2,3-d]pyrimidin-4-ones derivatives and to evaluate their possible cytotoxic activity on mammalian cell models.

Chemicals and Reagents
. 1 H NMR spectra were recorded on Unity 400 + (400 MHz) in CDCl 3 .Chemical shis of 1 H were measured relative to HMDS as internal standard.IR spectra were recorded as KBr pellets on an IR Fury System 2000 (Perkin-Elmer).Melting points were measured on a Boetius (Germany) and MEL-TEMP (USA) apparatus and are not uncorrected.Reactions were monitored by TLC on Sorb�l (Russia) and �hatman UV-254 (Germany) (eluent: benzene-methanol, 2 : 1 (A) and benzene-methanol, 5 : 1 (B)), which were visualized under UV radiation.
2.1.1.Cytotoxic Activity.e evaluation of cytotoxic activity of the tested samples was carried out on HeLa (human epithelial cervical cancer, ATCC CCL-2) and P3X (murine myeloma, ATCC CRL-1580) cell models.For this purpose cells were cultured at 37 ∘ C, 90% of humidity and 5% CO 2 in 96-well plates (5 × 10 4 cells/well for HeLa and 1 × 10 5 for P3X cells) in DMEM medium culture (pH 7.4), containing 10% fetal bovine serum and 50 units/mL penicillin/streptomycin.Cells were treated for 24 hours with the individual substance (in concentrations of 500, 250, and 62.5 g/mL, resp.).Controls consisted of untreated cancer cells incubated for 24 hours.e obtained results were recalculated in percentage with respect to the control.All experiments were conducted in 3-time frequency.

MTT Assays.
Cytotoxic activity was evaluated by using the MTT test based on reducing [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) with the participation of cytoplasmatic dehydrogenases in insoluble formazan, the quantity of which correlates with metabolic activity of cells and a level of cell proliferation [21].e medium was replaced 4 hrs before the incubation period (100 L DMEM), containing 0.5 mg/mL MTT, and incubated for 3 hours at 37 ∘ C. At the end of the experiment the formed crystals of formazan were dissolved in DMSO and the absorbance was determined by a plate lecturer.Cytotoxic activity of substances was determined by percent of discolor in control samples at 595 nm.Cytotoxic activity is determined by the change in absorbance relative to the controls and has been expressed as cell viability rate (%).

Experimental Part
3.1.Sample Preparation.Compounds 1-4 were synthesized following to the procedure of Gewald et al., as already reported [17] with some minor modi�cations.To a suspension containing of 0.4 mole ketone, 0.4 mole ethyl cyanoacetate, and 0.44 g sulphur powder in 120 mL ethanol, 40 mL diethylamine was added dropwise and the temperature of the reaction mixture was kept at 45-50 ∘ C for 3 h; the reaction solution was allowed to stand overnight at +5 ∘ C (in Gewalds procedure [17], a reactionary mixture leave in a refrigerator at some times).700 mL Distilled water was added and was mixed for 3 h at room temperature.e formed crystals were �ltered off and washed with water and dried.e products 1-4 were puri�ed by recrystallization from corresponding solvent.Data on the prepared compounds 1-4 are given in Table 1.
Synthesis of compounds 5-16 was already has been carried out by the method changed by us [19,22].Addition to reagents of POCl 3 at cooling (0 ∘ C), instead of at room  temperature (20 ∘ C), the increasing in duration of reaction and processing of a reaction mixture by ice water (thus decomposition of reaction products decreases) have allowed to obtain main products 5-16 in high yields (82-96%) (Table 1).
Our results showed that the increase of methylene groups, that is, at transition from dihydropyrrolo-( 5), tetrahydropyrido-( 9) and tetrahydroazepino-( 13) derivatives, the inhibition activity on murine myeloma cells (P3X) of compounds in the line 5 > 9 > 13 (250 g/mL) decreased.e same behavior was observed for 5,6dimethyl-( 5), -trimethylene-( 6), -tetramethylene-( 7), and -pentamethylene-( 8) derivatives in the line of compounds 5 > 6 > 7 > 8.Among of them, compound 5 resulted as the most active and in this view, further investigations with derivatives of 2, ]pyrimidin-4-one (5) will be carried out in order to evaluate both the affecting level of single derivatives and their cellular targets.e 5-16 obtained compounds exhibited moderate cytotoxic activity on HeLa cells.  1 for HeLa cells and Figure 2 for P3X cells.All the tested substances showed weak antiproliferative activities against HeLa cells and as shown by our experiments many compounds enhance proliferation of such cancer cell model.ese compounds exhibited varying degrees of cytotoxic activity against P3X cell lines when tested of P3X murine myeloma cells.e substance 5 exhibited potent antiproliferative activity with IC50 values of 86,4 g/mL; substances 6, 7, 10, 11, 12 showed a moderate cytotoxic activity in P3X cells growth and the level of affection seemed dependent on the administered concentration.Substances 8, 9, 13-16 demonstrated weak inhibition of P3X cell proliferation, with IC50 values higher than 500 g/mL.However some compounds showed contrasting results by enhancing the proliferation of both the cell lines tested.

Conclusion
We synthesized a series of thieno [2,3-d]pyrimidin-4-ones in high yields; the synthesized 1-4 compounds were used for the synthesis of 5-16 compounds.e advantages of the obtained 5-16 compounds are low cost of the starting chemicals and simple experimental procedure of synthesis.e 5-16 obtained compounds exhibited moderate cytotoxic activity on HeLa cells, whereas more consistent antiproliferative activity was detected in P3X myeloma murine cells and in this view further studies will be addressed to investigate the cellular targets of compounds showing antiproliferative activity.In both cell models some of the tested compounds showed to enhance cell proliferation.