Synthesis of Novel Halogenated Heterocycles Based on o-Phenylenediamine and Their Interactions with the Catalytic Subunit of Protein Kinase CK2

Abstract

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay.

Keywords: cell toxicity; differential scanning fluorimetry; halogenated heterocycles; hydrophobic contribution; inhibitory activity; kinase CK2; ligand binding; molecular modeling.

Figure 1

Structures of potent inhibitors of kinase CK2. Bolded fragments represent the common o-phenylenediamine substructure.

Scheme 1

Synthesis of compounds 2–7. Conditions: (a) SOCl₂, Et₃N, DCM, 40 °C; (b) TFA, HCl reflux; (c) HOCH₂COOH, HCl, H₂O, reflux; (d) CDI, DMF, RT; (e) 40% glyoxal solution, EtOH, reflux; (f) oxalic acid, DMF, 120 °C.

Figure 2

Effect of substitution at the two distant positions of the benzene ring with a halogen atom on HPLC-derived hydrophobicity index (log(τ)) (A). The same values are represented relative to the unsubstituted compound of the same scaffold (B).

Figure 3

Correlation between HPLC-derived hydrophobicity index (log(τ)) and the ΔT_m determined using nanoDSF.

Figure 4

Correlation between cell viability, IC₅₀, and hydrophobicity of tested compounds, log(τ).

Figure 5

Correlation between the calculated Gibbs free energy of binding of synthesized compounds to CK2 obtained from molecular modeling (ΔG_bind) and the ΔT_m, estimated with the aid of nanoDSF.