Investigating the antiproliferative activities of new Cu^II complexes with pyridine hydrazone derivatives of nalidixic acid

Highlights

• New copper(II) complexes with nalidixic acid hidrazones were synthesized.

• The complexes were characterized by a set of chemical and spectroscopic techniques.

• Antiproliferative activity of the compounds over a panel of tumor cells is presented.

• Gel electrophoresis indicated potential artificial nuclease activities for the complexes.

• The interaction of the naphthyridyl groups may be related to the nuclease activity.

Abstract

To further explore the structural features and potential antibacterial and antitumor activities of polynuclear Cu^II coordination compounds with nalidixic acid (nx) derivatives, new complexes bearing nx hydrazones with N-pyridinyl moieties substituted at positions 2 and 3 (h2py and h3py) were synthesized. Complexes [Cu₃(C₁₈H₁₆N₅O₂)₂(C₁₈H₁₇N₅O₂)₂(H₂O)]4BF₄∙H₂O (1), and [Cu₃(C₁₈H₁₆N₅O₂)₂(C₁₈H₁₇N₅O₂)₂(H₂O)₃]4BF₄∙3H₂O (%) (2) were synthesized using h2py and h3py with Cu(BF₄)₂∙nH₂O as precursor, whereas the [Cu(C₁₈H₁₇N₅O₂)Cl₂]∙0.5H₂O complex (3) was synthesized with h2py and CuCl₂∙2H₂O. Crystallographic studies of complex 1, showed that coordination of hydrazones to Cu^II occurs by tridentate modes of type κ³(O,N,N′) as well as bidentate modes of type κ²(O′,N″). Complexes 1, 2 and 3 had their antiproliferative activities evaluated in vitro against a panel of tumor cells by the determination of GI₅₀ values. Complexes 1 and 2 were more active than complex 3, suggesting an effect of the complex charge on their activities. The interactions of such complexes towards bovine serum albumin (BSA) and DNA plasmid (pGEX-4 T1) were investigated using fluorescence spectroscopy and gel electrophoresis. All complexes were shown to interact with the DNA model as metallonucleases, but no interaction with BSA was observed. DNA molecular docking of complex 1 encompassing both its trinuclear (TN) form and a possible mononuclear (MN) derivative suggests that naphthyridyl ring performs π-stacking interactions with DNA. The TN species were also shown to be possible minor groove binders.

Introduction

Metal complexes are unique in their biological activities and have been explored for a multitude of applications such as antitumor, antiviral and antimicrobial agents [1,2]. Copper(II) complexes in particular, have been extensively explored as potential anticancer agents [3,4], especially due to potential DNA interactions and generation of reactive oxygen species, with emphasis to copper Casiopeinas® [[5], [6], [7], [8]].

In this context, Cu^II coordination compounds with nalidixic acid (nx) and its derivatives have been studied due to their promising antitumoral and antimicrobial activities [[9], [10], [11], [12], [13], [14], [15], [16], [17]]. Recently, we reported the synthesis, characterization and in vitro antiproliferative (GI₅₀) studies of highly active and selective polynuclear Cu^II complexes (SI = selectivity index) based on imidazolyl (2-imidazolyl and 4-imidazolyl, h2imi and h4imi, respectively), pirrolyl (hpyrr) and salicyl (hsali) hydrazone derivatives of nalidixic acid [9]. The Cu^II coordination compounds with hsali (C₁₉H₁₈N₄O₃) and hpyrr (C₁₇H₁₇N₅O₂) bearing BF₄⁻ as counter-ions, [Cu₃(C₁₉H₁₇N₄O₃)₄]∙2BF₄ and [Cu₃(C₁₇H₁₆N₅O₂)₂(C₁₇H₁₇N₅O₂)₂(H₂O)]4BF₄, presented higher antiproliferative activity and selectivity against the 786–0 renal tumor cell line (GI₅₀⁷⁸⁶⁻⁰ of 1.14 μmol∙L⁻¹ and 2.72 μmol∙L⁻¹ and SI_HaCat⁷⁸⁶⁻⁰ of 4.52 and 6.06, respectively) when compared to doxorubicin (GI₅₀⁷⁸⁶⁻⁰ = 85.0 μmol∙L⁻¹, SI_HaCat⁷⁸⁶⁻⁰= 5·10⁻³) [9]. Additionally, the complex synthesized from CuCl₂∙2H₂O and h2imi (C₁₆H₁₆N₆O₂), [Cu₂(C₁₆H₁₆N₆O₂)Cl₄], showed high selectivity indexes against U251 glioma tumor cells (GI₅₀^U251 = 2.53 μM, SI_HaCat^U251= 60.9) and NCI/ADR-RES resistant ovarian tumor cells (GI₅₀^{NCI/ADR−RES}= 1.69 μM, SI_HaCat^{NCI/ADR−RES}= 91.4), when compared to doxorubicin and cisplatin [9]. Human immortalized keratinocyte was used as non-tumor cell line (HaCat) for SI calculations [9].

Seeking to extend the comprehension of how the derivatization of nx into hydrazones affects the resultant structures of copper(II) complexes and their antitumor as well as antibacterial activities, a series of new Cu^II coordination compounds were synthesized bearing the 2-pyridinyl (h2py) and 3-pyridinyl (h3py) hydrazone derivatives of nx (Fig. 1) [18]. For the synthetic procedures of the complexes, CuCl₂∙2H₂O or Cu(BF₄)₂∙nH₂O were used as precursors as presented in this manuscript.

The synthesized complexes were characterized by elemental analysis, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis, electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS), electronic paramagnetic resonance spectroscopy (EPR) and Fourier-transformed infrared spectroscopies (FT-IR). The antibacterial activities of the Cu^II complexes were evaluated in vitro against Gram-positive bacteria Staphylococcus aureus, and Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, which were selected based on their hospital and community infections. Additionally, the antitumor activities of the complexes were screened in vitro against the human cancer cell lines U-251 (glioma), MCF-7 (breast), NCI-ADR/RES (multi-drug resistant ovarian), 786–0 (kidney), NCI-H460 (lung), PC3 (prostate), UACC62 (melanoma), OVCAR-3 (ovarian), HT29 (colon) and K562 (leukemia) as well as over the non-tumor cell line - HaCat (immortalized keratinocyte). Biophysical assays encompassing electrophoresis methods and fluorescence spectroscopy were carried out to evaluate possible interactions between the Cu^II complexes with (pGEX-4 T1) DNA or bovine-serum albumin (BSA). Molecular docking studies were then performed seeking to unveil possible interaction modes between the Cu^II complexes and DNA.