**1. Introduction**

It was recently reported that worldwide, an estimated 19.3 million new cancer cases and almost 10.0 million cancer deaths occurred in 2020 [1]. It is therefore apparent that the search for new anticancer drugs is still extremely topical. The discovery of *cis*- {[PtCl2(NH3)2]}, cisplatin, a platinum-based drug, was a major breakthrough in cancer treatment strategies [2]. However, the toxicity and drug resistance associated with this compound steered drug discovery research toward the rational development of metalcontaining agents with more specific activity and less toxicity, and a mode of action different from *cis*-{[PtCl2(NH3)2]} and its derivatives. Metallodrugs have been used for centuries, but only now are methods and techniques becoming available to characterize such drugs more precisely, to identify their target sites, and to elucidate their often unique mechanisms of action [3]. It is also noteworthy that a better understanding of the roles played by metal compounds at a mechanistic level will help in the implementation of new metal-based therapies by providing an alternative, targeted, and rational approach to supplement non-targeted screening of novel chemical entities for biological activity [4]. In the search for new coordination compounds with promising biological properties, the choice of the metal ion is crucial [3,4]. Amongst metal ions, copper plays an important role thanks to

**Citation:** Pelosi, G.; Pinelli, S.; Bisceglie, F. DNA and BSA Interaction Studies and Antileukemic Evaluation of Polyaromatic Thiosemicarbazones and Their Copper Complexes. *Compounds* **2022**, *2*, 144–162. https://doi.org/10.3390/ compounds2020011

Academic Editor: Juan Mejuto

Received: 11 April 2022 Accepted: 17 May 2022 Published: 23 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

its distinct properties [5,6]. Copper is a bioessential element in biology with truly unique chemical characteristics in its two biologically relevant oxidation states, i.e., +1 and +2. Its most notable features are its almost exclusive function in the metabolism of O2 or N/O compounds (NO2<sup>−</sup>, N2O) and its frequent association with the oxidation/generation of organic and inorganic radicals such as tyrosyl, semiquinones, superoxide, or nitrosyl [7]. Many ligands can be chosen to bind copper and create valuable coordination complexes. Thiosemicarbazones are particularly interesting ligands because they present at least a couple of N, S donor atoms that can modulate the hard and soft character. Moreover, they can be suitably modified to increase the denticity of the ligand or the number of donor atoms, or to adjust parameters such as solubility and the partition coefficient [8–11], thereby modulating the biological activity of the compound in question. Thiosemicarbazones are a class of compounds which are known to exert different biological properties, e.g., catalysis [12] antibacterial [13,14], antifungal [15–21], antiparasitic [22], antiviral [23,24] and anticancer [25–32]. The antitumor activity provided by thiosemicarbazones is usually enhanced upon complexation. Many mechanisms of action have been proposed, including ribonucleotide reductase and topoisomerase II inhibitors, ROS generators (which, it is assumed, interact with DNA) and others which are attributed, for example, to their strong iron chelating ability [8,29]. With these hypotheses, we decided to synthesize new thiosemicarbazones and their copper complexes and perform interaction studies with in vitro biological systems in order to preliminarily evaluate their antitumor activity. As mentioned, DNA is a major target for both anticancer therapy and metal based drugs, and it is also known that polycyclic aromatic hydrocarbons tend to intercalate into DNA nitrogenous bases stackings [33]. Based on these hypotheses, analogues of naphthaldehyde and anthraldehyde thiosemicarbazone derivatives were synthesized and characterized, because naphthaldehyde [34–41] and anthraldehyde [37,42–48] have already shown interesting and promising chemical and biological properties. Structural modifications on the thiosemicarbazide terminal nitrogen, which seems to play a relevant role in its biological activity, have been investigated. Unexpectedly, reaction of the ligands with Cu(II) salts produced ligand oxidation products and the isolation of Cu(I) metal complexes. The nature of these compounds, formed upon reduction of copper, was assessed by means of X-ray crystallography. The ligand and its Cu(I) complex were subjected to biological tests (UV-Vis and CD titration) and showed important interaction with DNA which was not ascribable to intercalation. The same compounds also showed affinity toward BSA, as established by FT-IR experiments. Preliminary in vitro biological tests against a histiocytic lymphoma cell line resulted in a very low IC50 value, i.e., 5.46 μM, for the Cu(I) complex, highlighting the interesting behavior of this compound.

#### **2. Materials and Methods**

1H NMR were recorded on a Bruker Anova spectrometer at 300 MHz, with chemical shift reported in δ units (ppm). NMR spectra were referenced relative to residual NMR solvent peaks. Coupling constants (J) are reported in hertz (Hz). The solvent used in the acquisitions of spectra was DMSO-d6.

The FT-IR measurements were recorded on Perkin Elmer's Spectrum Two in the 4000–400 cm<sup>−</sup><sup>1</sup> range, equipped with the ATR accessory. The shapes and signal intensities are reported as w (weak), m (medium), s (strong), sh (sharp), b (broad).

Elemental analyses were performed using Flashsmart CHNS Elemental Analyzer (Thermofisher Scientific, Waltham, MA, USA).

Mass analyses were carried out using a Waters Acquity Ultraperformance ESI-MS spectrometer with Single Quadrupole Detector (Mode used: Flow Injection; Source temperature (◦C) 150; Desolvation Temperature (◦C) 300; Cone Gas Flow (L/Hr) 100; Desolvation Gas Flow (L/Hr) 480; Solvent Flow (mL/min) 0.2; Capillary voltage (kV) 3, Cone voltage (V) 20/50/80). The compounds were dissolved in MeOH.

Melting points were determined using a SPM3 apparatus (Stuart Scientific, Nicosia, Cyprus).

Circular dichroism spectra were recorded with a Jasco J-715 spectropolarimeter.

UV-Vis spectra were collected using Thermofisher Scientific's Evolution 260 Bio Spectrophotometer in a quartz cuvette.

The crystallographic data of compounds L1, L2, L3, L5 and [CuI2(SO4)(L2)5] (**2**) were collected with a SMART APEX2 diffractometer using Mo-Kα radiation and a graphite crystal monochromator [λ(Mo-Kα) 0.71073 Å]. Intensities data for compounds L4 and [CuI(L1)2](HSO4) (**1**) were collected on a Siemens AED diffractometer using Cu-Kα radiation [λ(Cu-Kα) 1.54178 Å]. For the data collected on the SMART APEX2 diffractometer, the SAINT [49] software was used for integrating reflection intensities and scaling, and SADABS [50] for absorption correction. The structures were solved by direct methods using SHELXS [51]] and refined by full-matrix least-squares on all F2 using SHELXL97 [52] implemented in the OLEX package [53]. The structure drawings were obtained with the ORTEPIII [54] and Mercury [55] programs.

#### *2.1. Synthetic Procedures*

#### 2.1.1. General Information

The following compounds were used: 4-methyl-3-thiosemicarbazide, 97% (Aldrich, St. Louis, MO, USA), 4,4-dimethyl-3-thiosemicarbazide, 98% (TCI Europe N.V., Zwijndrecht, Belgium), 2-naphthaldehyde (Aldrich), 10-chloro-9-anthraldehyde, 97% (Aldrich), Cu(SO4)2·5H2O (Aldrich), disodium salt of calf thymus DNA (CT DNA) (Serva), bovine serum albumin (BSA) (Aldrich).

#### 2.1.2. Synthesis of the Ligands

L1, 2-naphthaldehyde 4,4-dimethyl-3-thiosemicarbazone was synthesized as follows. First, 2-naphthaldehyde (0.0826 g, 0.529 mmol) was placed in a round bottomed flask with 25 mL of absolute ethanol. Subsequently, a slightly larger amount of 4,4-dimethyl-3- thiosemicarbazide (0.0815 g, 0.684 mmol) was added to the reaction flask. A representation of the synthesis is presented in Scheme 1. The mixture was gently heated until dissolution of both reagents, and then the flask was placed in an ice bath to limit the formation of by-products for one day under magnetic stirring. The solution took on a more intense yellow color over time, until a yellow suspension formed which was filtered on Buchner and then analyzed. The analyses highlighted the purity of the product, which was then recrystallized from acetonitrile, yielding straw yellow, needle-like crystals which were subjected to X-ray diffractometric analysis (Figure 1). Crystal data details are reported in the Supplementary Materials.

**Scheme 1.** Representation of the syntheses of ligands L1 and L2.

**Figure 1.** X-ray structure of L1 with ellipsoid probability at 50%.

Yield: 52%.

1H NMR (300 MHz, ppm, DMSO d6): 11.05 (s, 1H, N–NH–C=S), 8.36 (s, 1H, CH=N), 7.97 (m, 5H, aromatic), 7.55 (q, 2H, aromatic), 3.33 (s, 6H, N–(CH3)2).

FT–IR: 3139 cm<sup>−</sup>1, m, sh, ν N–H; 3008 cm<sup>−</sup>1, w, broad, ν sp<sup>2</sup> C–H; 2980 cm<sup>−</sup>1, w, broad, ν CH3; 1550 cm<sup>−</sup>1, s, ν C=C; 1520 cm<sup>−</sup>1, s, ν C=N; 1282 cm<sup>−</sup>1, s, ν C–N; 1121 cm<sup>−</sup>1, m, ν C=S.

L2, 2-naphthaldehyde 4-methyl-3-thiosemicarbazone was synthesized as follows.

First, 2-naphthaldehyde (0.0947 g, 0.606 mmol) was placed in a round bottomed flask with 25 mL of ethanol and a slight excess amount of 4-methyl-3-thiosemicarbazide (0.0769 g, 0.727 mmol) was added. A representation of the synthesis is reported in Scheme 1. The mixture was then gently heated to dissolve both reagents, and then the reaction flask was placed in an ice bath under magnetic stirring for three days, monitoring the reaction by means of TLC. The product was then extracted from the solution by evaporation of the solvent under vacuum and analyzed. Due to the presence of reagen<sup>t</sup> impurities, the product was then subjected to a purification silica column (ethyl acetate/cyclohexane 1/5 as mobile phase). The central fraction was then dried and recrystallized from acetonitrile, yielding white crystals in the shape of rice grains, which were suitable for diffractometric analysis (Figure 2). Crystal data details are reported in the Supplementary Materials.

**Figure 2.** X-ray structure of L2 with ellipsoid probability at 50%.
