Design, synthesis and characterization of novel chromone based-copper(ii) antitumor agents with N, N-donor ligands: comparative DNA/RNA binding profile and cytotoxicity

Abstract

A series of new chromone based-Cu(ii) complexes 1-3 derived from bioactive pharmacophore, 3-formylchromone and N,N-donor ligands viz., 1,10-phenanthroline, 2,2'-bipyridine and 1R,2R-DACH were synthesized as potential antitumor agents and thoroughly characterized by UV-vis, FT-IR, EPR, ESI-MS and elemental analysis. Single X-crystal diffraction studies of complex 2 revealed triclinic P1̄ space group with square pyramidal geometry around the Cu(ii) center. Comparative in vitro binding studies with ct-DNA and tRNA were carried out using absorption and emission titration experiments which revealed intercalative mode of binding with higher binding propensity of complexes 1-3 towards tRNA as compared to ct-DNA. Additionally, complex 1 exhibited high binding affinity among all the three complexes due to the involvement of phen co-ligands via π-stacking interactions in between nucleic acid base pairs. Furthermore, Hirshfeld surface analysis was carried out for complex 2 to investigate various intra and intermolecular non-covalent interactions (H-bonding, C-H⋯π etc.) accountable for stabilization of crystal lattice. The cleavage activity of complex 1 was performed by gel electrophoretic assay with pBR322 DNA and tRNA which revealed efficient DNA/tRNA cleaving ability of complex, suggesting tRNA cleavage both concentration and time dependent. Furthermore, in vitro cytotoxic activity of complexes 1-3 on a selected panel of human cancer cell lines was performed which revealed that all three complexes exhibited remarkably good cytotoxic activity with GI₅₀ value < 10 μg mL^-1 (<20 μM).

Scheme 1. Synthetic route of complexes 1, 2 and 3.

Scheme 2. Mechanism of in situ nucleophilic substitution of methoxide anion at 2-position of 3-formylchromone.

Fig. 1. (a) ORTEP view of complex 2 with partial numbering. Solid thermal ellipsoids are reported at the 50% probability level. (b) Packing diagram of complex 2 showing π–π stacking interaction. Hydrogen atoms have been omitted for clarity.

Fig. 2. Absorption spectra of complexes 1 (a), 2 (b) and 3 (c) in absence and presence of increasing concentrations of tRNA in Tris–HCl buffer (pH 7.2). Inset: plots of [RNA]/(ε_a − ε_f) (M² cm) vs. [RNA] for the titration with complexes 1–3. The arrows specify the intensity change with increasing [tRNA].

Fig. 3. Emission spectra of complexes 1 (a), 2 (b) and 3 (c) (2 × 10⁻⁴ M) in Tris–HCl buffer at pH 7.2 [RNA] = (0–4.00 × 10⁻⁵ M). Arrows depicts the intensity change upon increasing concentration of tRNA.

Fig. 4. Emission spectra of EB–tRNA in the absence and presence of complexes 1 (a), 2 (b) and 3 (c) in Tris–HCl buffer at pH 7.2. [Complexes 1–3] = [EB] = [RNA] = 1.11 × 10⁻⁴ M. Arrow indicates change in intensity with increasing concentration of complexes 1–3.

Fig. 5. X-band EPR spectra of 0.3 mM complex 1 (blue), 0.3 mM complex 1 + 1.4 mg mL⁻¹ tRNA (black) and 0.3 mM complex 1 + 1.4 mg mL⁻¹ ct-DNA (red). Experimental conditions: T = 298 K; microwave frequency = 9.46 GHz; microwave power = 20 mW; 10 G field modulation amplitude; time constant 81.92 ms; conversion time 81.92 ms; 3 accumulations.

Fig. 6. (A) Hirshfeld surface of complex 2 mapped with d norm (a), shape index (b) and curvedness (c). (B) The 2D fingerprint plots of interatomic interactions of complex 2 showing the percentage of contacts contributed to the total Hirshfeld surface area of the molecules.

Fig. 7. Agarose gel electrophoretic pattern depicting the cleavage of pBR322 plasmid DNA (300 ng) by complex under different conditions for an incubation time of 45 min at 37 °C. (a) Lane 1, DNA control; lane 2, DNA + 1 (5 μM); lane 3, DNA + 1 (10 μM); lane 4, DNA + 1 (15 μM); lane 5, DNA + 1 (20 μM); lane 6, DNA + 1 (25 μM); lane 7 DNA + 1 (30 μM); (b) lane 1, DNA control, lane 2, DNA + 1 (30 μM) + Asc (0.4 mM), lane 3, DNA + 1 (30 μM) + GSH (0.4 mM), lane 4, DNA + 1 (30 μM) + H₂O₂ (0.4 mM), lane 5, DNA + 1 (30 μM) + MPA (0.4 mM), lane 6, DNA + 1 (30 μM) + DMSO (5 μL); lane 7, DNA + 1 (30 μM) + EtOH (5 μL); lane 8, DNA + 1 (30 μM) + NaN₃ (20 mM); lane 9, DNA + 1 (30 μM) + SOD (5 units).

Fig. 8. Agarose gel electrophoretic pattern showing RNA cleavage in buffer after 8 h of incubation time at 37 °C with increasing concentration of complex 1 in a Tris–borate–EDTA (TBE) buffer 40 mM, pH 8.0 buffer, 30 mL of tRNA solution (3 × 10⁻³ M). Lane 1: tRNA control; lane 2: 6.25 μM of 1 + tRNA; lane 3: 9.25 μM of 1 + tRNA; lane 4: 12.37 μM of 1 + tRNA; lane 5: 15.87 μM of 1 + tRNA; lane 6: 19.37 μM of 1 + tRNA.

Fig. 9. Growth curve showing % control growth vs. drug concentration (μg mL⁻¹) against four different human carcinoma cell lines: (a) MIA-Pa-Ca-2 (pancreas), (b) HeLa (cervix), (c) A-498 (kidney) and (d) MCF-7 (breast) and (e) Hep-G2 (human hepatoma).

Fig. 10. Morphological changes observed in cancer cell lines on treating with (a) MIA-Pa-Ca-2 control, (b) complexes 1, (c) 2 and (d) 3; (a′) MCF-7 control, (b′) complexes, 1 (c′) 2 and (d′) 3.

Fig. 11. Molecular docked model of complex 2 with (a) ct-DNA (PDB ID: 1BNA) and (b) tRNA (PDB ID: 6TNA).