Improving the Stability of EGFR Inhibitor Cobalt(III) Prodrugs

Abstract

Although tyrosine kinase inhibitors (TKIs) have revolutionized cancer therapy in the past two decades, severe drawbacks such as strong adverse effects and drug resistance limit their clinical application. Prodrugs represent a valuable approach to overcoming these disadvantages by administration of an inactive drug with tumor-specific activation. We have recently shown that hypoxic prodrug activation is a promising strategy for a cobalt(III) complex bearing a TKI of the epidermal growth factor receptor (EGFR). The aim of this study was the optimization of the physicochemical properties and enhancement of the stability of this compound class. Therefore, we synthesized a series of novel derivatives to investigate the influence of the electron-donating properties of methyl substituents at the metal-chelating moiety of the EGFR inhibitor and/or the ancillary acetylacetonate (acac) ligand. To understand the effect of the different methylations on the redox properties, the newly synthesized complexes were analyzed by cyclic voltammetry and their behavior was studied in the presence of natural low-molecular weight reducing agents. Furthermore, it was proven that reduction to cobalt(II) resulted in a lower stability of the complexes and subsequent release of the coordinated TKI ligand. Moreover, the stability of the cobalt(III) prodrugs was investigated in blood serum as well as in cell culture by diverse cell and molecular biological methods. These analyses revealed that the complexes bearing the methylated acac ligand are characterized by distinctly enhanced stability. Finally, the cytotoxic activity of all new compounds was tested in cell culture under normoxic and various hypoxic conditions, and their prodrug nature could be correlated convincingly with the stability data. In summary, the performed chemical modifications resulted in new cobalt(III) prodrugs with strongly improved stabilities together with retained hypoxia-activatable properties.

Figure 1

Proposed mechanism of the hypoxia-activated cobalt(III) prodrug system. In healthy tissue (left), the cobalt(III) complex is too bulky to fit into the ATP-binding pocket of the EGFR and is therefore biologically inactive. In the hypoxic environment of the tumor (right) an irreversible reduction takes place. This results in the release of the TKI ligand with formation of cobalt(II) species {[Co(H₂O)₆]²⁺ and mixed acac/H₂O complexes} and subsequent inhibition of EGFR-downstream signaling.

Scheme 1. Chemical Structures of EGFR Inhibitor Ligands L and MeL as well as Cobalt(III) Complexes Co(acac)₂L⁺, Co(Meacac)₂L⁺, Co(acac)₂MeL⁺, and Co(Meacac)₂MeL⁺

Figure 2

(A) 3D full excitation–emission landscape of MeL (Rayleigh scattering of first and second order appears as diagonal ridges). (B) Fluorescence emission spectra at a λ_ex of 365 nm of MeL, Co(acac)₂MeL⁺, and Co(Meacac)₂MeL⁺ (the peaks at 420 nm are caused by Raman scattering). All measurements were performed in PBS at pH 7.40 (30 μM ligand, 30 μM complex, and 25.0 °C).

Figure 3

Cyclic voltammograms of Co(acac)₂L⁺, Co(Meacac)₂L⁺, Co(acac)₂MeL⁺, and Co(Meacac)₂MeL⁺ in 10 mM phosphate buffer (pH 7.40) (1.5 mM complex, I = 0.10 M KCl, scan rate of 30 mV/s, 25.0 °C). Potentials are referenced to the NHE.

Figure 4

Cobalt(III) model complexes synthesized to investigate the effect of the methyl and phenyl substitution at the “en” and/or acac moiety. E_c is the cathodic peak potential vs NHE of the cobalt complexes measured at a scan rate of 30 mV/s in 10 mM phosphate buffer (pH 7.4).

Figure 5

Concentration distribution diagram for the 1:2:1 cobalt(II)–acac–PhEn system. A = acac; B = PhEn [1 mM cobalt(II); I = 0.10 M (KCl); 25.0 °C].

Figure 6

Fluorescence emission spectra of Co(Meacac)₂L⁺ in the presence of 10 equiv of GSH followed for 24 h. The dashed spectrum corresponds to the emission spectrum of free EGFR inhibitor L [c_complex = 15 μM; c_free ligand = 15 μM; λ_EX = 350 nm; pH 7.40 (10 mM phosphate buffer and 0.1 M KCl); 25.0 °C].

Figure 7

Time-dependent stability of (A) Co(acac)₂L⁺ and (B) Co(Meacac)₂L⁺ incubated in FCS at 37 °C (pH 7.4, 150 mM phosphate buffer) and analyzed by HPLC–mass spectrometry (depicted are the extracted ion mass chromatograms). Due to the different ionization properties, the intensities of the free ligand (m/z 358.1) and cobalt(III) complexes (m/z 614.0 or 642.1) cannot be directly compared.

Figure 8

Stability measurements of Co(acac)₂L⁺, Co(Meacac)₂L⁺, Co(acac)₂MeL⁺, and Co(Meacac)₂MeL⁺ incubated in FCS at 37 °C (pH 7.4, 150 mM phosphate buffer) and analyzed by mass spectrometry over a period of 26 h. The y-axis shows the relative ratio of the integrated peak areas of the intact complex over time (in percent) compared to the area at the starting point (0 h).

Figure 9

Fluorescence microscopic measurements indicating the release of the ligand from the different cobalt(III) complexes. Release of (A) L and (B) MeL from the different cobalt(III) complexes under normoxic cell culture conditions (37 °C, 21% O₂, and 5% CO₂) using UV fluorescence microscopy. A431 cells were incubated with 10 μM drugs for 6 or 24 h. Images are overlays of representative fluorescence and differential interference contrast microscopies (10× objective) of the different treatments processed by ImageJ software.

Figure 10

Release of (A) L or (B) MeL from the indicated cobalt(III) complexes under normoxic cell culture conditions (37 °C, 21% O₂, and 5% CO₂) by flow cytometry. A431 cells were incubated with 10 μM drugs for 6 or 24 h, and the fold change in fluorescence intensity (left, after normalization with fluorescence intensity of the cells) and the percent of fluorescence-positive cells (right) were evaluated using Diva Software and GraphPad Prism. Statistical significance was calculated via two-way analysis of variance with a multiple-comparison test and Bonferroni correction with p < 0.001 (***).

Figure 11

Impact of new cobalt(III) complexes on the EGFR signaling cascade (pEGFR, pERK 1/2) under normoxic conditions. A431 cells were grown in medium with or without FCS and treated with the indicated drug for 2 h. After EGFR stimulation with 50 ng/mL EGF for 10 min, cells were harvested, lysated, and further analyzed by Western blotting. The ratios of pEGFR or pERK 1/2 levels of the treated samples (after normalization to the loading control β-actin) to the levels of the control (−FCS and +EGF) are given below the respective bands.

Figure 12

Cytotoxic activity of the indicated compounds against A431 cancer cells. The incubation time of the compounds on the cells was 72 h under normoxic and three different hypoxic conditions (5%, 1%, or 0.1% O₂). Values are given as means ± the standard deviation of one representative experiment performed in triplicate.