Cytotoxicity of Pyrazine-Based Cyclometalated (C^Npz^C)Au(III) Carbene Complexes: Impact of the Nature of the Ancillary Ligand on the Biological Properties

Abstract

The synthesis of a series of cyclometalated gold(III) complexes supported by pyrazine-based (C^N^C)-type pincer ligands is reported, including the crystal structure of a cationic example. The compounds provide a new platform for the study of antiproliferative properties of gold(III) complexes. Seven complexes were tested: the neutral series (C^N^pz^C)AuX [X = Cl (1), 6-thioguanine (4), C≡CPh (5), SPh (6)] and an ionic series that included the N-methyl complex [(C^N^pzMe^C)AuCl]BF₄ (7) and the N-heterocyclic carbene complexes [(C^N^pz^C)AuL]⁺ with L = 1,3-dimethylbenzimidazol-2-ylidene (2) or 1,3,7,9-tetramethylxanthin-8-ylidene (3). Tests against human leukemia cells identified 1, 2, 3, and 4 as particularly promising, whereas protecting the noncoordinated N atom on the pyrazine ring by methylation (as in 7) reduced the cytotoxicity. Complex 2 proved to be the most effective of the entire series against the HL60 leukemia, MCF-7 breast cancer, and A549 lung cancer cell lines, with IC₅₀ values down to submicromolar levels, associated with a lower toxicity toward healthy human lung fibroblast cells. The benzimidazolylidene complex 2 accumulated more effectively in human lung cancer cells than its caffeine-based analogue 3 and the gold(III) chloride 1. Compound 2 proved to be unaffected by glutathione under physiological conditions for periods of up to 6 days and stabilizes the DNA G-quadruplex and i-motif structures; the latter is the first such report for gold compounds. We also show the first evidence of inhibition of MDM2-p53 protein-protein interactions by a gold-based compound and identified the binding mode of the compound with MDM2 using saturation transfer difference NMR spectroscopy combined with docking calculations.

Figure 1

Examples of cyclometalated (C^N^C)Au(III) complexes based on pyridine (A, B)^, and pyrazine (C) ligands^,

Scheme 1. Synthesis of Pyrazine-Based (C^N^pz^C)Au(III) Complexes 2–4 and Structures of the Previously Reported Complexes 5–7; The Structure of 1 Shows the Numbering System Used for NMR Assignments

Figure 2

(left) Molecular structure of the cation in 2·toluene. The atomic numbering scheme is also shown. Selected bond distances [Å] and angles [deg]: Au1–N1 1.991(9), Au1–C25 1.995(10), Au1–C6 2.099(10), Au1–C16 2.096(11), C6–Au1–C25 97.8(4), C16–Au1–C25 100.9(4), torsion angle N1–Au1–C25–N3 125.73. (right) Crystal packing and hydrogen-bonding interactions involving the pyrazine moiety. Color coding: C (gray), Au (yellow), N (blue), P (orange); F (light green); toluene (dark green).

Figure 3

Inhibition of HL60 cell proliferation by (C^N^pz^C)Au(III) complexes 1–7; data represent the average ± standard error of three experiments.

Figure 4

Cell uptake of compounds 1–3 by A549 cells after 6 h of treatment with compounds at 10 μM in DMSO. The significance of the results was analyzed by the t test: *, p < 0.05.

Figure 5

Stabilization of different DNA structures (0.2 μM) by pyrazine-based (C^N^pz^C)Au complexes 1–7 at 10 μM measured by FRET DNA-melting assay. Data represent the average and standard deviation of three experiments.

Figure 6

Interaction of 2 with MDM2 in solution studied by STD NMR (13.5 μM MDM2, 500 μM 2, 1% DMSO-d₆, 800 MHz, 25 °C). (top) ¹H NMR reference spectrum. The inset shows the group epitope mapping of 2 from STD NMR experiments derived from STD NMR intensities at very low saturation time (0.5 s). Ligand hydrogens showing high % values have close contacts with the surface of the binding pocket of MDM2. (bottom) STD NMR spectrum (4 s saturation time) showing signals of 2 resulting from the binding in solution (intense signals around 3.5–3.7 ppm in the reference spectrum belong to glycerol, which does not bind to MDM2.

Figure 7

Representation of 2 (gray) docked in MDM2 (PDB entry 1T4E, green). On the left, the side chains of residues surrounding 2 (closer than 4 Å) are displayed. On the right, the surface of MDM2 and relevant pockets occupied by p53 residues are indicated.