Effects of tethered ligands and of metal oxidation state on the interactions of cobalt complexes with the 26S proteasome

Abstract

In this paper we report on the synthesis and characterization of three cobalt complexes described as [Co(II)(L(1))(2)] (1), [Co(II)(L(2))] (2), and [Co(III)(L(1))(2)]ClO(4)(3). These complexes contain the deprotonated forms of the [NN'O] tridentate ligand HL(1) and its newly synthesized [N(2)N'(2)O(2)] hexadentate counterpart H(2)L(2), namely, 2,4-diiodo-6-((pyridine-2-ylmethylamino)methyl)phenol and 6,6'-((ethane-1,2-diylbis((pyridin-2-ylmethyl) azanediyl))bis(methylene))bis(2,4-diiodophenol). Characterizations for 1-3 include electrospray ionization (ESI) spectrometry, infrared, and UV-visible spectroscopies, and elemental analyses. A detailed (1)H-NMR study was conducted for 3 and X-ray structural data was obtained for 2. The viability of this series as potential agents for proteasome inhibition and cell apoptotic induction involving PC-3 cancer cells is presented comparing the behavior of the untethered [NN'O](2) six-coordinate 1 and 3 and the tethered counterpart 2 with a 1:1 metal-to-ligand ratio. It is observed that the tethering in 2 decreases inhibition activity. When 1 and 3 are compared, the most inert, but redox-active, cobalt(III) species shows the highest chymotrypsin-like activity inhibition on purified proteasome and PC-3 cancer cells. A hypothesis based on the role of oxidation states for proteasome inhibition is offered.

Fig. 1

Isotopic distribution for complex 2 (left) and 3 (right). The bars indicate the experimental results and the continuous spectra indicate the simulated results.

Fig. 2

ORTEP diagram at 50% probability level for 2. Selected bond lengths include Co(1)-O(1) = 2.003(3), Co(1)-N(1) = 2.139(3), Co(1)-N(2) = 2.234(3) Å. Selected angles include N(1)-Co(1)-N(2) = 76.57(12), O(1)-Co(1)-N(1) = 90.32(12), O(1)-Co(1)-N(2) = 89.65(11). Goodness of fit is given by R(F) (%) = 3.13.

Fig. 3

NMR spectroscopic measurements for 3; (a) ¹H-NMR spectrum and (b) HMQC spectra.

Fig. 4

UV–visible spectra of complexes 1–3 in N,N-dimethylformamide, 1.0×10⁻⁴ M.

Fig. 5

MTT, PC-3 cells after 18 h treatment. Control is DMSO, for each concentration from 10 to 50 μM, compound 1 is indicated in the left column and 3 is indicated in the right column.

Fig. 6

Chymotrypsin-activity inhibition in human purified 20S proteasome; DMSO and Co^II(ClO₄)₂ are controls. Top: comparison between 1 (left column) and 2 (right column); bottom comparison between 1 (left column) and 3 (right column).

Fig. 7

Chymotrypsin-activity inhibition in PC-3 cell lysates after 18 h treatment. Compound 1 was measured at 40 and 50 μM and is shown in the left column. Compound 3 is shown as a single column for 1–30 μM and in the right column for 40 and 50 μM. DMSO is the control.

Fig. 8

Comparison between 1 (left column) and 3 (right column) for Caspase-3 (apoptosis) Induction in PC-3 cell lysates after 18 h treatment. DMSO is the control.

Fig. 9

Western blot for PC-3 cell lysates after 18 h treatment.

Scheme 1

Cobalt complexes.

Scheme 2

Synthesis of the H₂L² ligand.