Keto-enol tautomerism of hydroxynaphthoquinoneoxime ligands: Copper complexes and topoisomerase inhibition activity

Highlights

• Keto-enol tautomers of hydroxynaphthoquinoneoximes characterized by NMR.

• Chemical shifts of tautomer are assigned and kinetics studied by NMR.

• Copper(II) complexes synthesized and characterized by EPR and cyclic voltammetry.

• Coordination of the ligand in keto and enol form in Cu-4 confirmed by X-ray structure.

• Topoisomerase inhibition activity of complexes evaluated.

Abstract

Six copper(II) complexes of 3‑hydroxy-4-(hydroxylamine)naphthalene-1(4H)-one; (1) (in Cu-1; [Cu(1)₂] and Cu-2;(Py⁺)[Cu(1)₂], 3‑hydroxy-4-(hydroxylamine)-2-methylnaphthalen-1(4H)-one; (2) (in Cu-3; [Cu(2)₂] and Cu-4; (Py⁺)₂[Cu₄(2)₈]) and 2‑chloro-3‑hydroxy-4-(hydroxylamine)naphthalene-1(4H)-one(3) (in Cu-5; [Cu(3)₂] and Cu-6;[Cu(3)₂] (Py) and their ligands are targeted as topoisomerase II inhibitors. The NMR technique revealed tautomers of ligands 1 to 3 and their concentration. The tautomer's ¹H and ¹³C chemical shifts are assigned from CD₃OD-d₄ solvent from ¹H, ¹³C, DEPT, gDQCOSY, gHSQCAD NMR experiments. Complexes Cu-1, Cu-3, and Cu-5 are complexes of ligands 1, 2, and 3, respectively, while in complexes Cu-2, Cu-4, and Cu-6 contain either pyridine as a cation or in lattice. Cu-2 crystallizes in the monoclinic P2_1/C space group, one of the naphthoquinoneoxime ligands coordinates to Cu(II) ion in the dianionic form. Copper-Copper distance is ∼3.25 Å in the stacked dimer. A tetrameric complex Cu-4 crystallizes in the triclinic P-1 space group, X-ray structure of Cu-4 confirms the coordination of ligand 2 in both keto and enol form to Cu(II). Topoisomerase II inhibition, DNA Cleavage assay, and growth inhibition assay were performed on all six copper complexes.

Introduction

Topoisomerases (TOPO) are DNA enzymes that play an essential role in cellular processes like DNA replication, transcription, chromosome condensation, and most importantly, in the mitosis to decatenate the daughter chromosomes. During DNA replication and transcription processes, a large number of positive and negative supercoils are formed ahead of the replication. These supercoils lead to stalling replication machinery [1] wherein the TOPO enzyme fine-tunes and modulates the topology of DNA supercoiling and helps them relax to facilitate protein interactions to further replication [2]. Therefore, these enzymes are considered magicians of the DNA world to solve topological problems of DNA [1]. Based on their activity, these DNA cleaving enzymes are classified into two types [2] types I (TOPO I) that cleave only one of the strands and relax the DNA through the strand passage mechanism, and type II(TOPO II) that cleaves both the strands and do the same by swivel mechanism. These enzymes are subdivided into type IA and type IB based on protein attachment to the phosphate (5′ and 3′) and type IIA and type IIB on the structural considerations [3].

Several antitumor drugs inhibit the DNA function by either directly binding to the DNA (intercalating agents or groove binding agents) or indirectly inhibiting the function of DNA enzymes like TOPO [4]. Some compounds act directly on the DNA and damage it by modifying or cleaving the same, altering the cell's biochemical activities [5]. Understanding TOPO's role in many disease conditions, researchers studied and characterized these enzymes using topoisomerase inhibitors (TOPO-In) in different experiments. TOPO-In can also be classified as catalytic inhibitors and TOPO poisons. TOPO poisons act on the drug stabilized cleavable complexes to the extreme level that cells can no longer tolerate [6]. Catalytic inhibitors interact with the TOPO and do not bind to the DNA by inhibiting the enzyme activity. TOPO poisons act differently where these molecules bind to the TOPO only after the DNA is cleaved by TOPO to stabilize these cleaved complexes and lead to cell death [7].

Tumor cells are highly proliferative, and elevated TOPO expression is reported in these cells compared to normal cells [8]. Therefore, drugs that can bind and damage DNA have been developed and have an excellent chemotherapy scope. Camptothecin (CPT) and etoposide (VP-16) act as TOPO poisons to the TOPO I and TOPO II family. Despite several TOPO-In being available in size, potency, and selectivity, several parameters can be optimized. To date, the TOPO-In developed has a polycyclic scaffold as its preferred structure that can bind to the catalytic site of DNA and enzyme interface. Such chemical structures are not feasible to synthesize and make the molecule very rigid; moreover, their cellular uptake is too low.

On the other hand, metal ions are potent and can cleave DNA but are very non-selective. Few studies also show metal complexes as potential inhibitors of TOPO [9,10]. However, some reports suggest increasing TOPO inhibition activity upon complexation with copper ion [11,12]. A strategy would be to develop smaller scaffold structures with functional groups that can complex with a metal and form homo-oligomers, showing synergistic activity upon cellular uptake. Therefore, in the present work, a panel of small molecules was synthesized that were complexed with copper.

Several nonmetallic synthetic topoisomerase inhibitors were proposed in recent years, including benzoxanthone derivatives, acridines, trisubstituted pyridines, and thiosemicarbazones benzophenanthridines, nitrofurans, purine analogs, anilinothiazoloquinolines, etc. [13]. Metal complexes of Cu(II) [14], [15], [16] Ru(II) [17], [18], [19], [20], [21], [22], Pt(II) [23], Pd(II) [24], Au(III) [25], Mn(II) [26], Zn(II) [27,28] and Ru(III) [18] are also known as topoisomerase inhibitors; however, their studies are scarce as compared to nonmetallic synthetic topoisomerase inhibitors. Osimertinib, a third generation inhibitor used in lung cancer treatment. Multilayer films are prepared by Xu et al. for controlled release of drug release [29], [30], [31].

An enormous number of naphthoquinones exhibit cytotoxic and anticancer activity towards a large number of cancer cell lines. It has been proved that naphthoquinone possesses anticancer activity through inhibition of the topoisomerase-II enzyme [32,33]. The compounds containing quinone scaffold as topoisomerase inhibitors include HU331, Cpd-9 naphthothiophenedione, TU100 naphthoquinone, and natural molecules products α-lapachone, eleutherin, SH-7, plumbagin, and clinically used include doxorubicin mitoxantrone. To the best of our knowledge, there is only one report in the literature of quinone metal complex as a topoisomerase II inhibitor [34]. This investigation targets naphthoquinoneoxime-based Cu(II) complexes as topoisomerase inhibitors. Copper complexes of the deprotonated ligands 3‑hydroxy-4-(hydroxylamine)naphthalene-1(4H)-one; 1(Cu-1; [Cu(1)₂], Cu-2; (Py⁺)[Cu(1)₂]), 3‑hydroxy-4-(hydroxylamine)−2-methylnaphthalen-1(4H)-one; 2 (Cu-3; [Cu(2)₂], Cu-4; (Py⁺)₂[Cu₄(2)₈], 2‑chloro-3‑hydroxy-4-(hydroxylamine)naphthalene-1(4H)-one; 3 (Cu-5; [Cu(3)₂], Cu-6; [Cu(3)₂](Py) are synthesized and characterized. Complex Cu-4, a tetramer, is crystallized in triclinic space group P-1. Coordination of ligand 2 in this complex is as ‘keto’ and ‘enol’ tautomeric forms. Hence the existence of tautomeric forms of the ligands 1 to 3 in polar solvents are extensively studied by NMR spectroscopy, and ¹H, ¹³C, gDQCOSY, gHSQCAD methods are used to assign the proton and carbon chemical shift of tautomers in this investigation.

Moreover, tautomers are separated by preparative HPLC. Time-dependent NMR experiments in CD₃OD-d₄ solvent monitored their tautomer interconversion of the isolated tautomers. Anticancer activity of Cu-1 to Cu-6 complexes has been evaluated against cancer cell lines THP 1 and COLO205. The potential topoisomerase II inhibitor activity, DNA cleavage activity of the ligands 1, 2, and 3, their precursors of all copper(II) complexes were evaluated.