Multitarget Anticancer Agents Based on Histone Deacetylase and Protein Kinase CK2 inhibitors

Abstract

The design of multitarget drugs (MTDs) has become an innovative approach for the search of effective treatments in complex diseases such as cancer. In this work, we communicate our efforts in the design of multi-targeting histone deacetylase (HDAC) and protein kinase CK2 inhibitors as a novel therapeutic strategy against cancer. Using tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole (DMAT) as scaffolds for CK2 inhibition, and a hydroxamate to coordinate the zinc atom present in the active site of HDAC (zinc binding group, ZBG), new multitarget inhibitors have been designed and synthesized. According to the in vitro assays, N-Hydroxy-6-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)hexanamide (11b) is the most interesting compound, with IC₅₀ values of 0.66; 1.46 and 3.67 µM. for HDAC6; HDAC1 and CK2; respectively. Cellular assays on different cancer cell lines rendered promising results for N-Hydroxy-8-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)octanamide (11d). This inhibitor presented the highest cytotoxic activity, proapoptotic capability, and the best mitochondria-targeting and multidrug-circumventing properties, thus being the most promising drug candidate for further in vivo studies.

Keywords: CK2; CuAAC; HDAC; cytotoxic activity; docking; molecular dynamics; multi-target inhibitors.

Figure 1

Representative CK2 inhibitors.

Figure 2

FDA-approved histone deacetylase (HDAC) inhibitors [50].

Scheme 1

General approach for the synthesis of dual HDAC/CK2 based on TBB.

Figure 3

Examples of previously described CK2/HDAC1 dual inhibitors [30].

Scheme 2

Synthetic route for compounds 7a–d. Reaction conditions: (a) K₂CO₃, acetone, MW, 110 °C, 60 min; (b) HNMe₂, MeOH, 110 °C, 48 h; (c) CuSO₄, sodium ascorbate, TBTA, RT, overnight; (d) AcCl, MeOH, 0 °C, 15 min.

Scheme 3

Synthetic route for compounds 11a–d. Reactions conditions: (a) K₂CO₃, acetone, reflux, 72 h; (b) LiOH, THF/H₂O, RT, 2h; (c) H₂N-OTHP, EDCI, HOBt, NMM, DMF, RT, overnight; (d) AcCl, MeOH, 0 °C, 15 min.

Figure 4

Phenylhydroxamate-based HDAC-6 inhibitors.

Scheme 4

Synthetic route for compounds 13–19. Reactions conditions: (a) methyl 4-(bromomethyl)benzoate, K₂CO₃, acetone, 150 °C, MW; (b) NaOH, THF, RT; (c) H₂N-OTHP, EDCI, HOBt, NMM, DMF, RT, 24 h; (d) AcCl, MeOH, 0 °C, 15 min.

Figure 5

PyMOL stick and cartoon representation of the proposed binding modes of compound 11b to CK2 (magenta), HDAC1 (cyan) and HDAC6 (light green). For the sake of clarity, only heavy atoms are shown.

Figure 6

Comparison of cytotoxic activity of selected CK2-HDAC dual inhibitors (7c, 11b, 11c, 11d, 15a and 19) towards human leukemia (top left), carcinoma (top right and down left,) and pseudonormal (down right) cell lines. Trypan blue assay, 24 h incubation. Means and SD of at least three experiments in triplicate are shown.

Figure 7

Cell cycle arrest and cell death induction by dual CK2-HDAC inhibitors. (A) the impact of the indicated compound doses on the distribution of Jurkat T-leukemia cells in the different phases of the cell cycle was determined by FACS of propidium iodide-stained cells after 24 h of continuous exposure. (B) evaluation of the impact of selected dual CK2-HDAC inhibitors (10 µM and 30 µM, 24 h) on phosphatidylserine externalization in Jurkat T-leukemia cells was determined by FACS, using FITC-labeled annexin V and PI staining. One of three experiments delivering comparable data is shown.

Figure 8

Evaluation of cytotoxic activity of selected CK2-HDAC dual inhibitors towards human leukemia cells of the HL-60 line and its drug-resistant sublines HL-60/adr (MRP-1 overexpression) and HL-60/vinc (P-glycoprotein overexpression). Trypan blue assay, 24 h incubation. Means and SD of at least three experiments in triplicate are shown.

Figure 9

Depolarization of mitochondria in HL-60/wt cells under the action of selected CK2-HDAC dual inhibitors (10 µM and 30 µM, 24 h) was determined by FACS using the Mitochondrial Membrane Potential Assay JC-1. One of three experiments delivering comparable data is shown.