Antitumor Activity of New Methylpyrazolotriazine and Pyrazolobenzotriazine Derivatives Tested on MCF-7, MDA-MB-231, and BT-474 Breast Cancer Cell Lines

Abstract

Introduction. Breast cancer (BC) remains a leading cause of cancer incidence and mortality among women worldwide. The high heterogeneity of tumor cells and their morphological and functional variability often complicate the selection of effective treatment regimens. A primary challenge in BC therapy is the development of drug resistance, underscoring the need for novel therapeutic agents.

Aim. To evaluate the potential of two new methylpyrazolotriazine derivatives (MPTA 1, MPTA 2) and two new pyrazolobenzotriazine derivatives (PBTA 1, PBTA 2) as anticancer agents, including their application in BC chemotherapy.

Materials and Methods. The study assessed the cytotoxic (CTA) and cytostatic (CSA) activity of these derivatives at concentrations ranging from 0.25 to 10.0 µM. The effects were tested on BC cell lines MCF-7, MDA-MB-231, and BT-474, and on non-tumorigenic human breast epithelial cells MCF-10A. CTA was determined using the methyl tetrazolium (MTT) assay, with the half-maximal cytotoxic concentration (IC₅₀T) calculated. CSA was evaluated after longer cultivation time (72 hours), with the IC₅₀S defined as the concentration that inhibited cell growth by 50 %. Statistical analysis employed nonparametric tests (Kruskal–Wallis and Mann–Whitney) with a significance level of p < 0.05.

Results. The highest CTA against MCF-7 cells was observed for derivative MPTA 1 (IC₅₀T 8.15 µM), against MDA-MB-231 cells for PBTA1 (IC₅₀T 3.79 µM), and against BT-474 cells for MPTA 1 (IC₅₀T 4.48 µM) and MPTA 2 (IC₅₀T 6.15 µM). All tested compounds exhibited similar CSA against MCF-7 cells, averaging 1.20–1.45 times higher than the reference drug temozolomide (minimum cell viability 55 %). In MDA-MB-231 and BT-474 cultures, MPTA 1 and MPTA 2 exhibited superior CSA compared to PBTA 1, PBTA 2, and temozolomide, with a 1.16–1.50-fold increase. All the investigated derivatives of methylpyrazolotriazine and pyrazolobenzotriazine and the reference drug temozolomide showed low CSA against non-cancerous epithelial MCF-10A cells, with IC₅₀S values exceeding the tested concentration range (> 10.0 µM). Ranking the new methylpyrazolotriazine and pyrazolobenzotriazine derivatives by the combined potency of CTA and CSA yields the following order: MPTA 2, PBTA 1 < temozolomide < PBTA 2 < MPTA 1.

Conclusion. Derivative MPTA 1 (3-(3'-Phenyl-4'-methoxycarbonyl-isoxazolyl)-7-methyl-pyrazolo[5,1-c]triazine) demonstrated the highest CTA and CSA among the tested derivatives and is recommended for further preclinical studies.