Dimeric Bis-Benzimidazole-Pyrroles DB2Py(n) - AT-Site-Specific Ligands: Synthesis, Physicochemical Analysis, and Biological Activity

Abstract

Its broad spectrum of biological activity makes benzimidazole a fundamental pharmacophore in pharmaceutics. The paper describes newly synthesized AT-specific fluorescent bis-benzimidazole molecules DB2Py(n) that contain a pyrrolcarboxamide fragment of the antibiotic drug netropsin. Physico-chemical methods using absorption, fluorescence, and circular dichroism spectra have shown the ability of bis-benzimidazole- pyrroles to form complexes with DNA. The new DB2Py(n) series have turned out to be more toxic to human tumor lines and less vulnerable to non-tumor cell lines. Bis-benzimidazole-pyrroles penetrated the cell nucleus, affected the cell-cycle synthesis (S) phase, and inhibited eukaryotic topoisomerase I in a cellfree model at low concentrations. A real-time tumor cell proliferation test confirmed the molecule's enhanced toxic properties upon dimerization. Preliminary cytotoxicity data for the bis-benzimidazole-pyrroles tested in a cell model with a MDR phenotype showed that monomeric compounds can overcome MDR, while dimerization weakens this ability to its intermediate values as compared to doxorubicin. In this respect, the newly synthesized cytotoxic structures seem promising for further, in-depth study of their properties and action mechanism in relation to human tumor cells, as well as for designing new AT-specific ligands.

Keywords: DNA narrow groove binding ligand; DNA-binding; bis-benzimidazole-pyrrole; cell cycle; cytotoxicity; multidrug resistance; topoisomerase I.

Fig. 1

Structural formulas of Hoechst 33258, netropsin, monomeric MB2Py, MB2Py(Ac), and dimeric compounds DB2Py(n). In MB2Py(Ac), the bis-benzimidazole fragment is highlighted in red; and the pyrrolecarboxamide fragment, in blue

Fig. 2

Synthesis scheme of monomeric (MB2Py, MB2Py(Ac)) and dimeric (DB2Py(4, 5)) compounds

Fig. 3

Absorption and fluorescence spectra of free DB2Py(n) and its complex with DNA. (A) – absorption spectra of DB2Py(4) in absence (1) and presence of DNA (2–8); [DB2Py(4)] 4.06 × 10-6 M; 0.001 M sodium cacodylate. [DNA] 1 – 0; 2 – 0.25; 3 – 0.49; 4 – 0.98; 5 – 1.48; 6 – 2.45; 7 – 14.67; 8 – 121.9 × 10-6 M b.p. respectively; optical path length, 10 mm. (B) – absorption spectra of DB2Py(5) in absence (1) and presence of DNA (2–8); [DB2Py(5)] 4.29 × 10-6 M; 0.001 M sodium cacodylate. [DNA] 1 – 0; 2 – 0.25; 3 – 0.49; 4 – 0.98; 5 – 1.48; 6 – 2.45; 7 – 14.67; 8 – 121.9 × 10-6 M b.p. respectively; optical path length, 10 mm. (C) – fluorescence spectra of DB2Py(4) in absence (1) and presence of DNA (2–6). [DB2Py(4)] 4.6 × 10-6 M; [DNA] 1 – 0; 2 – 3; 3 – 6; 4 – 18; 5 – 30; 6 – 54 × 10-6 M b.p. respectively. Buffer: 10 mM PBS (pH 7.4). Excitation wavelength, 320 nm; slot width, 5 nm; cell size 10 × 10 mm; 22°C. (D) – fluorescence spectra of DB2Py(5) in absence (1) and presence of DNA (2–6). [DB2Py(5)] 2.3 × 10-6 M; [DNA] 1 – 0; 2 – 3; 3 – 6; 4 – 18; 5 – 54; 6 – 78 × 10-6 M b.p. respectively. Buffer: 10 mM PBS (pH 7.4). Excitation wavelength, 320 nm; slot width, 5 nm; cell size 10 × 10 mm; 22°C

Fig. 4

(A) – CD spectra in absence (1) and presence of (2–9) of DB2Py(4); 0.3 M NaCl + 0.002 M Naphosphate buffer, pH 6.85; 170 mg/ml PEG-4000; [DNA] 4.545 × 10-5 M b.p.; [DB2Py(4)] 1 – 0; 2 – 0.41; 3 – 0.82; 4 – 1.63; 5 – 3.25; 6 – 4.87; 7 – 6.48; 8 – 8.08; 9 – 10.07 × 10-6 M respectively. Optical path length, 10 mm. (B) – CD spectra in absence (1) and presence of (2–8) DB2Py(5); 0.3 M NaCl + 0.002 M Naphosphate buffer, pH 6.85, 170 mg/ml PEG-4000; [DNA] 4.545 × 10-5 M b.p.; [DB2Py(5)] 1 – 0; 2 – 0.43; 3 – 0.86; 4 – 1.72; 5 – 3.44; 6 – 5.15; 7 – 6.85; 8 – 8.54 × 10-6 M respectively. Optical path length, 10 mm

Fig. 5

Cytotoxicity values (IC50) in micromoles (μM) of the monomeric and dimeric bis-benzimidazole- pyrroles compared to irinotecan in various human tumor cell lines. SD – standard deviation, SE – standard error, R2 – determination coefficient

Fig. 6

Cytotoxicity of the new bis-benzimidazole-pyrroles in MCF10A (human breast epithelial cells, normal cell line) and on MCF-7 (breast cancer). SD – standard deviation, SE – standard error, R2 – coefficient of determination, F-test – statistical criterion

Fig. 7

The effect of the bis-benzimidazoles MB2Py(Ac) and DB2Py(5) on the proliferation of osteosarcoma cells in real time. Growth curves were measured as the cell index over time. (A) – controls: puromycin 10 μg/ml (red line); growth of unexposed U2OS cell line (blue line); 0.5% DMSO (greenline). (B) – MB2Py exposure; (C) – DB2Py(5) exposure

Fig. 8

Fluorescent staining of Gbl13n glioblastoma cells incubated with monomeric and dimeric bis-benzimidazole-pyrroles at a concentration of 2 μM for 48 h. (A) – population of cells stained with the bis-benzimidazole- pyrrole DB2Py(5). On the left is a DAPI filter, on the right is a phase-contrast image. MB2Py(Ac) staining looks similar after 2 days; (B) – a picture of stained nuclei obtained using a confocal microscope

Fig. 9

Effect of bis-benzimidazole- pyrroles on the cell cycle in an HCT-116 cell line. (A) – after a 24 h of incubation; (B) – after a 48 h of incubation

Fig. 10

Calf-thymus Topo-I inhibition by MB2Py(Ac) and DB2Py(4). (A) – effect of the compounds on Topo-I activity at concentrations of 2.5 and 5 μM. (B) – at concentrations of 0.65 and 1.25 μM