Uracil derivatives/ursolic acid hybrids - naturally derived compounds as anticancer agents

Abstract

A series of new uracil derivatives/ursolic acid hybrids were designed and synthesised as potential cytotoxic agents. The uracil, thymine, 6-methyluracil and 2-thiouracil moieties were linked to ursolic acid (1) through alkyl chains of different lengths (either four or six -CH₂- units). The cytotoxic activity of the synthesised conjugates was determined using the hormone-dependent breast cancer cell line MCF-7, the triple-negative breast cancer (TNBC) cell line MDA-MB-231 and normal cell lines: human skin fibroblasts (CCD-25Sk) and human bronchial epithelium (BEAS-2B). The five compounds, 4a, 5a, 6a, 7a and 9a, exhibited a significant reduction in the cell viability of human BC cell lines. Analog 6a, which demonstrated high cytotoxic activity against MCF-7 and MDA-MB-231 cell lines with IC₅₀ values of 14.00 µM and 5.83 µM, respectively, was also antitumorigenic in all biochemical assays. It increased p53 and Bax levels in MDA-MB-231 cells as well as significantly decreased Akt kinase levels in the tested cells, and effectively inhibited collagen biosynthesis. Finally, for the selected compounds, we computationally predicted their ADME properties and performed molecular docking to Akt protein kinase. The results of computational docking indicated that the preferred binding mode for all of them was the inactive form of Akt kinase, as in the case with known Akt allosteric inhibitors.

Keywords: ADME; Breast cancer; Cytotoxic activity; Molecular docking; Uracil derivatives; Ursolic acid.

Fig. 1

Examples of natural compounds having thymine and uracil in their structure.

Fig. 2

Examples of ursolic acid hybrids with anticancer activity against selected breast cancer lines: FZU3010, UA-5a, 4c, 15.

Fig. 3

Schematic diagram of the designed group of compounds.

Fig. 4

Synthesis of new ursolic acid derivatives. Conditions: a) K₂CO₃, 1,4-dibromobutane or 1,6-dibromohexane, DMF, rt.

Fig. 5

Synthesis of new uracil derivatives/ursolic acid hybrids. Reaction conditions:   K₂CO₃, DMF, 50°C.

Fig. 6

Selectivity index (SI) relative to skin fibroblasts and BEAS-2B.

Fig. 7

Scheme showing proline and collagen biosynthesis pathways from glutamine and ornithine. Pyrroline-5-carboxylate reductase (PYCR), ^Δ1-pyrroline-5-carboxylate (P5C), glutaminase (GLS), ornithine aminotransferase (OAT).

Fig. 8

Collagen biosynthesis (incorporation of 5-[³H]-proline) in MDA-MB-231 breast cancer cells after 24 h incubation with tested compounds and reference drug (UA) at 10–100 µM.

Fig. 9

Molecular mechanisms induced apoptosis via the activation of DNA damage proteins such as p53.

Fig. 10

Concentration of p53 tumour suppressor in MDA-MB-231 cells after 24 h incubation with tested compounds and reference drug (UA) at 20 µM. The data are presented as the mean ± SD from three independent experiments (n = 3) conducted in duplicate. *P < 0.05 vs. control group.

Fig. 11

The concentration of proapoptotic Bax in the MDA-MB-231 cells after 24 h incubation with tested compounds and reference drug (UA) at 20 µM. The data are presented as the mean ± SD from three independent experiments (n = 3) conducted in duplicate. *P < 0.05 vs. control group.

Fig. 12

Phosphorylated Bax protects cells from apoptosis. Akt phosphorylates (P) Bax at position S184 in cells with increased Akt signaling. Phosphorylation prevents Bax from entering the outer mitochondrial membrane, allowing binding of the activator BH3, and making cells resistant.

Fig. 13

The concentration of Akt protein in MDA-MB-231 after 24 h incubation with the tested compounds and reference drug (UA) at 20 µM. The data are presented as the mean ± SD from three independent experiments (n = 3) conducted in duplicate. *P < 0.05 vs. control group.

Fig. 14

(a) The predicted (cyan) and experimental (green) binding pose of miransertib in the 5KCV crystal structure; (b) The predicted binding pose of the ursolic acid (1) in the 5KCV crystal structure; (c) The predicted binding pose of 4a in the 5KCV crystal structure; (d) The predicted binding site of derivative 5a’ in the inactive form of Akt (4GV1 crystal structure). Figure 14 has been created using The PyMOL Molecular Graphics System ver. 1.3 (https://pymol.org).

Fig. 15

Hybrid 6a exerts anticancer activity by inducing cell apoptosis and inhibiting cell migration.