Synthesis and Cytotoxic Activity against K562 and MCF7 Cell Lines of Some N-(5-Arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2- ((4-oxo-3-phenyl-3,4-dihydroquinazoline-2-yl)thio) acetamide Compounds

Ethyl 2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetate (3) which was synthesized starting from anthranilic acid (1) via 2-thioxo-3-phenylquinazolin-4(3H)-one (2) reacted with hydrazine hydrate to afford 2-((4-oxo-3-phenyl-3,4-dihydroquinazolin2-yl)thio)acetohydrazide (4). Reaction of (4) with thiocarbonyl-bis-thioglycolic acid gave a new compound name N-(4-oxo-2thioxothiazolidin-3-yl)-2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamide (5). Knoevenagel condensation of (5) with appropriate aldehydes gave fourteen (Z)-N-(5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamide compounds (6a–o) with moderate yield. )e chemical structure of the compounds was elucidated on the basis of IR, H-NMR, C-NMR, and HR-MS spectral data. )e 5-arylidene-2-thioxothiazolidinone compounds exhibited mild-to-moderate cytotoxic activity against both K562 (chronic myelogenous leukemia) cells and MCF7 (breast cancer) cells.


Materials and Methods
All starting materials were purchased from Acros and used without purification.Melting points were measured in open capillary tubes on a Gallenkamp melting point apparatus.
e structure of all compounds was confirmed by their IR, 1 H-NMR, 13 C-NMR, and HR-MS spectral data.IR spectra (], cm − 1 ) were recorded on a FTIR-8400S-SHI-MADZU spectrometer using KBr pellets.
e NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR) using residual solvent DMSO-d 6 signals (δ H 2.50, δ C 39.52) as internal references.
e cytotoxic activity of 6a-o compounds was tested on K562 (chronic myelogenous leukemia) and MCF7 (breast cancer) cell lines using the MTT assay.

Experimental
e synthesis of the target compounds is carried out as outlined in Scheme 1.

Synthesis of 2-((4-Oxo
e reaction mixture was refluxed on a water bath for 10 h.After cooling, the solid separated was filtered and recrystallized from ethanol to give crystals of compound

2
Journal of Chemistry       3.6.Cell Viability Assay.e experimental procedure was followed by the steps presented in the literature [46,47].In brief, K562 cells and MCF7 cells were cultured in the DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin and maintained at 37 °C and 5% CO 2 with 95% humidity.Viable cells were counted and inoculated in 96-well plate with a density of 10 5 cells/100 μL/well for K562 and 5 × 10 4 cells/ 100 μL/well for MCF7.After 24 hours, the cells were treated with the compounds and doxorubicin (positive control) diluted in culture media at 100, 50, 25, 12.5, 6.25, 3.125, and 0 μg/mL concentration containing 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0% dimethyl sulfoxide (DMSO), respectively.DMSO in culture media was used as negative control.In addition, the culture medium without cells was used as blank.All experiments were done in triplicate.e plates were incubated in 5% CO 2 and 95% humidity at 37 °C for 72 hours.10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added into each well and incubated in 37 °C in 5% CO 2 for 3.5 hours.70 μL of detergent reagent (10% SDS) was added into each well, and the plate was maintained in 37 °C for 16 hours.e optical density of each well was read by using a scanning multiwall spectrophotometer (Sunrise) at a wavelength of 595 nm.Cell survival was measured as the percentage absorbance compared to the negative control (DMSO-treated cells).Cell death (% inhibition) was estimated by the following formula:
e reaction of a hydrazide compound with thiocarbonyl-bis-thioglycolic acid to form a 2-thioxothiazolidine-4-one compound was mentioned by some authors [36,[38][39][40][41][42][43].erefore, the hydrazide 4 was used in the reaction with thiocarbonyl-bis-thioglycolic acid in ethanol to obtain N-(4-oxo-2-thioxothiazolidin-3-yl)-2-Journal of Chemistry ((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)thio)acetamide (5).In the IR spectrum of compound 5, beside the absorption peak of the -NH-group at 3202 cm − 1 and the absorption peak of lactam and amide carbonyl groups at 1690 cm − 1 , the appearance of a new band at 1759 cm -1 indicated the presence of the C�O group in the 2-thioxothiazolidin-4-one ring.e mass spectrum of the product showed the [M + H] + ion peak at m/z 443.0367 in agreement with the molecular formula of C 19 H 15 N 4 O 3 S 3 ((M + H) � 443.0306).In the 1 H-NMR spectrum of the compound, besides the signal of the methylene group bonding with the quinazoline ring via the sulfur atom at δ 4.13, there was an appearance of a new signal at δ 4.41, which was imputed to the methylene group on the thiazolidinone ring.Similar to the 3-(4-methylcoumarin-7-yloxyacetylamino)-2thioxo-1,3-thiozolidin-4-one compound [36], the signals of the methylene groups in the molecule of compound 5 were not also singlets as expected.
ey were split by a non-first-order splitting effect.17 signals appeared in the 13  Compound 5 with the 2-thioxothiazolidin-4-one ring containing the active methylene group was then further reacted with appropriate aldehydes to give the raw of N-(5arylidene-4-oxo-2-thioxothiazolidin-3-yl)-2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)sulfanyl)acetamide compounds (6a-o) in the conditions of the Knoevenagel condensation reaction.In the IR spectra, the signal of the C�O group in the thioxothiazolidin-4-one ring of the 6a-o compounds in a comparison with that one of the compound 5 appeared at a lower frequency because of the conjugation of the carbonyl group with the benzylidene moiety.Mass spectra of the synthesized 6a-o compounds showed the molecular peaks in agreement with their molecular formula.In the 1 H-NMR spectra of 6a-o compounds, the signals of the methylene group outside the thiazolidinone ring (-SCH 2 CONH-) still appeared as multiplet peaks at around δ 4.16-4.19,but the signal of the methylene group on the ring was disappearance.Along with the additional signals of the aromatic protons in accordance with those ones of the initial aldehyde, a signal of the methylidene proton as a singlet at δ 7.79-7.93also appeared in the spectrum of each compound.
ese were evidence for the conversion of compound 5 to compounds 6a-h by Knoevenagel condensation.According to the previous reports [21,22,24,25,43,48], because of the interaction with the carbonyl group at the 4 position, the methylidene proton of the Z-isomer of 5-arylidene-2-thioxo-thiazolidin-4one compounds was more downfield (δ 7.9) than that of the E-isomer (δ 7.4).Comparing the vinylic proton shift in 1 H-NMR spectra of 6a-o with these chemical shifts indicated that the exocyclic double bond of the thiazolidinone 6a-o compounds exists in the Z-configuration.e formation of the Zisomers may be explained by the high degree of thermodynamic stability of these isomers [21,22,24].
All compounds were evaluated for their potential cytotoxicity against K562 and MCF7 tumor cell lines using doxorubicin as a positive control.e results were expressed in terms of the percentage growth inhibition (Table 1) and the IC 50 value of the compounds (Table 2).

Table 1 :
e selective cytotoxicity of compounds 5 and 6a-o against K562 and MCF7 cell lines

Table 2 :
IC 50 (μg/mL) on two cancer cell lines of the compounds.