Thienopyrimidines are structural analogs of quinazolines, and the creation of new 2-alkyl derivatives of ethyl 4-aminothienopyrimidine-6-carboxylates for the study of their anti-proliferative properties is of great pharmacological interest. Some 2-alkyl-4-amino-thieno[2,3-d]pyrimidines 2-5 were synthesized, and their cyto- and phototoxicity against BALB 3T3 cells were established by an in vitro 3T3 NRU test. The obtained results indicate that the tested compounds are not cytotoxic or phototoxic, and that they are appropriate to be studied for their anti-proliferative and anti-tumor properties. The anti-proliferative potential of the compounds was investigated on MCF-7 and MDA-MB-231 cancer cells, as well as a MCF-10A cell line (normal human mammary epithelial cells). The most toxic to MCF-7 was thienopyrimidine 3 with IC50 13.42 μg/mL (IC50 0.045 μM), followed by compound 4 (IC50 28.89 μg/mL or IC50 0.11 μM). The thienopyrimidine 4 revealed higher selectivity to MCF-7 and lower activity (IC50 367 μg/mL i.e., 1.4 μM) than compound 3 with MCF-10A cells. With respect to MDA-MB-231 cells, ester 2 manifested the highest effect with IC50 52.56 μg/mL (IC50 0.16 μM), and 2-ethyl derivative 4 revealed IC50 62.86 μg/mL (IC50 0.24 μM). It was estimated that the effect of the substances on the cell cycle progression was due to cell cycle arrest in the G2 stage for MDA-MB-231, while arrest in G1 was detected for the estrogen (ER)-positive MCF-7 cell line. The tested compound's effects on the change of the zeta potential in the tumorigenic cells utilized in this study were determined. The calculation which we performed of the physicochemical properties and pharmacokinetic parameters influencing the biological activity suggested high intestinal absorption, as well as drug-likeness.
Keywords: 4-amino-thieno[2,3-d]pyrimidines; anti-proliferative activity; breast cancer cell lines MCF-10A, MCF-7, MDA-MB-231; cell cycle analysis; pharmacokinetics descriptors; synthesis; zeta potential.