Exploring the Anti-Cancer Mechanism of Novel 3,4'-Substituted Diaryl Guanidinium Derivatives

Abstract

We previously identified a guanidinium-based lead compound that inhibited BRAF through a hypothetic type-III allosteric mechanism. Considering the pharmacophore identified in this lead compound (i.e., "lipophilic group", "di-substituted guanidine", "phenylguanidine polar end"), several modifications were investigated to improve its cytotoxicity in different cancer cell lines. Thus, several lipophilic groups were explored, the di-substituted guanidine was replaced by a secondary amine and the phenyl ring in the polar end was substituted by a pyridine. In a structure-based design approach, four representative derivatives were docked into an in-house model of an active triphosphate-containing BRAF protein, and the interactions established were analysed. Based on these computational studies, a variety of derivatives was synthesized, and their predicted drug-like properties calculated. Next, the effect on cell viability of these compounds was assessed in cell line models of promyelocytic leukaemia and breast, cervical and colorectal carcinomas. The potential of a selection of these compounds as apoptotic agents was assessed by screening in the promyelocytic leukaemia cell line HL-60. The toxicity against non-tumorigenic epithelial MCF10A cells was also investigated. These studies allowed for several structure-activity relationships to be derived. Investigations on the mechanism of action of representative compounds suggest a divergent effect on inhibition of the MAPK/ERK signalling pathway.

Keywords: 3,4′-bis-guanidino; 3-amino-4′-guanidino; BRAF; HL-60; apoptosis; cancer cell viability; diphenyl ether; intramolecular hydrogen bond; phenyl pyridyl ether.

Figure 1

Structural modifications proposed for the optimization of the compound 1.

Figure 2

Structures of the proposed new derivatives of 1 (compounds 2, 3 and 4), which docking to the in-house triphosphate (TP)-containing BRAF simplified model was studied.

Figure 3

Docking of derivative 2 in the TP-containing BRAF simplified model indicating the bifurcated (up left, bottom left and right) and single (up right) hydrogen bond (HB) interactions observed. Distances are expressed in Å.

Scheme 1

Preparation of mono-substituted 3,4’-bis-guanidinium diphenyl ether derivatives.

Scheme 2

Preparation of 3-amino,4’-guanidine diphenyl ether derivatives.

Scheme 3

Preparation of 3,4’-bis-guanidine pehylpyridyl ether derivatives.

Scheme 4

Preparation of 3-amino-4’-guanidine phenylpiridyl ether derivatives.

Scheme 5

Preparation of 3-amino-4’-isourea and 3-amino-4’-sulfamido diphenyl ether derivatives.

Figure 4

Structure Activity Relationship (SAR) deduced from the analysis of the HL-60 cytotoxicity results.

Figure 5

Annexin V-FITC vs. PI flow cytometry analysis of HL-60 cancer cells treated with compounds 3 (5 μM), 2 (5 μM) and 4 (4 μM) for 48 h. These figures are representative of three independent experiments. The viable cells, early apoptotic, necrotic and late apoptotic cells are represented by the lower left, lower right, upper left and upper right quadrants, respectively.

Figure 6

Western immunoblot of HL-60 cell extracts following incubation with compounds 2, 3, 4, 9, 52 and 1 (as a control). HL60 cells were seeded at 2 × 10⁵ cells/mL and were treated with either vehicle [0.5% EtOH (v/v)], compounds 52, 2, 9, 3 and 4 (5 µM) or compound 1 (5 and (*) 10 µM, as in reference [9]) for 16 h. Cells were lysed and equal amounts of protein were loaded and separated on 15% SDS-PAGE gels, transferred to PVDF membrane and probed with antibodies against total and phosphorylated ERK. Anti-GAPDH was used as a loading control. Results are representative of 2 independent experiments.