A Conformationally Restricted Gold(III) Complex Elicits Antiproliferative Activity in Cancer Cells

Abstract

Diamine ligands are effective structural scaffolds for tuning the reactivity of transition-metal complexes for catalytic, materials, and phosphorescent applications and have been leveraged for biological use. In this work, we report the synthesis and characterization of a novel class of cyclometalated [C^N] Au(III) complexes bearing secondary diamines including a norbornane backbone, (2R,3S)-N²,N³-dibenzylbicyclo[2.2.1]heptane-2,3-diamine, or a cyclohexane backbone, (1R,2R)-N¹,N²-dibenzylcyclohexane-1,2-diamine. X-ray crystallography confirms the square-planar geometry and chirality at nitrogen. The electronic character of the conformationally restricted norbornane backbone influences the electrochemical behavior with redox potentials of -0.8 to -1.1 V, atypical for Au(III) complexes. These compounds demonstrate promising anticancer activity, particularly, complex 1, which bears a benzylpyridine organogold framework, and supported by the bicyclic conformationally restricted diaminonorbornane, shows good potency in A2780 cells. We further show that a cellular response to 1 evokes reactive oxygen species (ROS) production and does not induce mitochondrial dysfunction. This class of complexes provides significant stability and reactivity for different applications in protein modification, catalysis, and therapeutics.

Figure 1.

UV-Vis absorption spectrum of complexes 1–4 in biologically relevant media RPMI at room temperature, final concentration of the complexes was 50 μM.

Figure 2.

Cyclic voltammogram recorded at a platinum electrode in DMSO solution of 10 mM 2 or 3 with 0.10 M N(Bu)₄PF₆ supporting electrolyte at a scan rate of 0.1 V/s using Ag/AgCl reference electrode at room temperature.

Figure 3.

X-ray crystal structure of complex 1, 2, and 3, ellipsoids drawn at 50%. Hydrogen atoms and solvents were omitted for clarity.

Figure 4.

Stability studies. A. HPLC trace (A₂₆₀ nm) following the incubation of 1 with RPMI medium for 20 h. At respective time points reaction was aliquoted (100 μL) and diluted with methanol (100 μL) prior to HPLC runs. Blank run of RPMI-methanol is included to account for the baseline. B. Formation of 1-GSH by the chemical reaction of 1 and L-GSH as observed by LC-MS and monitored over 24 h period. C. HPLC trace (A₂₆₀ nm) following the incubation of 1 in methanol and BSA in PBS for 24 h.

Figure 5.

Cellular uptake. Intracellular accumulation of 1–4 in A2780 cancer cells. Cells were treated with 5 μM (left) or 10 μM (right) for 18 h.

Figure 6.

Dual staining flow cytometry experiment showing apoptosis at 5 μM of 1 in A2780 cells for 24 h. Propidium iodide and FITC-labeled Annexin V was used to detect apoptotic populations (a) control and (b) complex 1 treated. (c) Bar chart representation of the apoptotic populations extrapolated from a and b. Data analyzed by two-tailed unpaired Student’s t test (*p < 0.05). (d) Expression levels of apoptotic proteins in A2780 following a 24 h treatment of 1 at indicated concentrations.

Figure 7.

(a) Total intracellular ROS levels induced by 1 using DCFDA flow cytometry assay. (b) Bar chart representation of data extrapolated from a. (c) mitochondrial ROS measured by MitoSOX flow cytometry (PE channel) assay. (d) Bar chart representation of data extrapolated from c. Cells were exposed to complex 1 for 2 h prior to assay. Data are plotted as the mean ± SD. (n =3). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test and two-tailed unpaired Student’s t test (** p < 0.01, * p < 0.05, n.s. = not significant).

Figure 8.

(a) OCR from cell mito stress study using Seahorse XF96. A2780 cancer cells were pretreated with 1 (12 h) and various inhibitors of ETC were added at indicated time points. (b) ECAR from cell mito stress study using Seahorse XF96. A2780 cancer cells were pretreated with 1 (12 h) and various inhibitors of ETC were added at indicated time points. (c) Mitochondria membrane potential measured by flow cytometry analysis of TMRE fluorescence intensity. (d) Bar chart of MMP extrapolated from (c). Data are plotted as the mean ± SD. (n = 3). Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparison test (**** p < 0.0001, n.s. = not significant).

Scheme 1:

Synthetic procedure for complexes 1–4.