Synthesis and in Vitro Evaluation of CAPE Derivatives as Ferroptosis Inducers in Triple Negative Breast Cancer

Abstract

Herein, we describe the design, synthesis, and in vitro biological evaluation of HO-1 inducers endowed with cytotoxic effects mediated by ferroptosis activation. Using the natural HO-1 inducer caffeic acid phenethyl ester (CAPE) as a chemical scaffold, new derivatives were synthesized by performing modifications in the cathecol moiety and in the phenethyl ester aromatic ring. Biological assays aimed at evaluating an imbalanced activity of ferroptosis key players identified that 2-(1H-indol-3-yl)ethyl cinnamate (compound 24) possesses improved anticancer activity toward the MDA-MB 231 triple negative breast cancer cell line when compared to CAPE. Increased ROS and LOOH levels, reduced GSH levels, imbalanced mitochondrial activity, and restored cell viability after ferrostatin-1 treatment suggested a ferroptotic mechanism of action, which did not involve GPX4 inhibition. Compound 24 represents an intriguing hit compound useful for the identification of novel ferroptosis inducers.

Figure 1

Chemical structures of CAPE and VP961.

Figure 2

General structure and design strategy adopted for novel CAPE derivatives.

Scheme 1. Synthetic Strategy for the Synthesis of Compounds 11–13

Reagents and conditions: (a) NaOH, H₂O, r.t, acetic anhydride, 0 °C then r.t., 1.5 h. (b) LiAlH₄ in THF 1 M, dry THF, r.t., 4 h. (c) SOCl₂, dry CHCl₃, reflux, 2 h, then 5–7, dry pyridine, dry CH₂Cl₂, r.t., overnight. (d) Guanidinium carbonate, TEA, N₂, dry CH₂Cl₂/dry CH₃OH (1:2), r.t, 15–30 min.

Scheme 2. Synthetic Strategy for the Synthesis of Compounds 20–24

Reagents and conditions: (a) LiAlH₄ in THF 1M, dry THF, r.t., 4 h. (b) PBr₃, dry CH₂Cl₂, 0 °C, then r.t, 1.5 h. (c) CA or cinnamic acid, Na₂CO₃, KI, DMSO, r.t., 20 h.

Figure 3

Evaluation of the CAPE effect, alone or in combination with ferrostatin-1 (1 μM) and Z-VAD-FMK (20 μM), on cell viability after 48 h of treatment (A, B). Assessment of glutathione, ferrous iron, and IL-6 levels following CAPE exposure (C–E). Results are expressed as mean ± SEM (*compound vs CTRL; ^#vs CAPE; ***p < 0.0001; ^###p < 0.0001; ^##p < 0.001; ^#p < 0.01).

Figure 4

Evaluation of compounds 10–12, 21–25, VP961, and CAPE cytotoxic effects following 24 h of treatment. Results are expressed as mean ± SEM (^§CAPE vs CTRL; *compound vs CAPE 10 μM; ^#compound vs CAPE 50 μM; ^§p < 0.0001; ***p < 0.0001; ^###p < 0.0001; ^##p < 0.001; ^#p < 0.01).

Figure 5

Measurement of HO-1 expression (A) and enzymatic activity (B) following 18 h of treatment. Results are expressed as mean ± SEM (***p < 0.0001 vs CTRL).

Figure 6

Evaluation of a combination treatment (selected compounds/ferrostatin-1) effect on cell viability after 48 h (A). Measurement of lipid hydroperoxide (B), glutathione (D), and IL-6 (E) levels following treatment with the three selected compounds. Assessment of VP961, 11, and 24 GPX4 inhibitory capacity compared to positive control ML162 (C). Results are expressed as mean ± SEM (***p < 0.0001, **p < 0.001, *p < 0.01 vs CTRL; ^###p < 0.0001, ^#p < 0.01 vs reference compound).

Figure 7

Assessment of ROS profile following VP961, 11, and 24 treatments at different time points (1, 3, 6 h) by cytometric analysis (A) and relative quantification (B). Results are expressed as mean ± SEM (***p < 0.0001 vs CTRL).

Figure 8

Evaluation of treatment effects on mitochondrial membrane potential (A) and fluorescence images of JC-1 staining after 6 h of treatment (B). Results are expressed as mean ± SEM (***p < 0.0001 vs CTRL).