Prévia do material em texto

Journal of Molecular Structure 1294 (2023) 136460
Available online 21 August 2023
0022-2860/© 2023 Elsevier B.V. All rights reserved.
Palladium (II) complexes as inhibitors of cathepsin B and topoisomerase I 
beta: Synthesis, characterization, and cytotoxicity 
Amos O. Akinyemi a,c, George B.S. Pereira a, Gabriela P. Oliveira a, Mauro A. Lima a, 
Josias S. Rocha a, Vinicius A. Costa a, Dario B. Fortaleza a, Tamara Teixeira a, Karine Zanotti a, 
Moacir Rossi Forim a, João H. Araujo-Neto b, Javier Ellena b, Fillipe Vieira Rocha a,* 
a Department of Chemistry, Federal University of São Carlos, São Carlos, São Paulo, CEP 13.565-905, Brazil 
b São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, CEP 13566-590, Brazil 
c Department of Toxicology and Cancer Biology, University of Kentucky, USA 
A R T I C L E I N F O 
Keywords: 
Palladium (II) 
Cathepsin B 
Topoisomerase 
Cytotoxicity 
DNA interactions 
A B S T R A C T 
This research presents the synthesis, characterization, anticancer activity, and investigation of biological targets 
of three new palladium (II) complexes. The compounds were characterized by spectroscopies techniques (UV-Vis, 
NMR, and IR), elemental analysis, mass spectrometry, and one for monocrystal X-ray diffraction. The cytotoxic 
activity of the complexes against two cell lines, A2780cis (cisplatin resistance ovarian tumor cell line) and MRC-5 
(non-tumor lung cell), was evaluated. The in vitro cytotoxicity assays using the MTT method revealed a signif-
icant cytotoxic activity of the palladium complexes against the evaluated cell lines, highlighting the resistance 
tumor cell line, A2780cis. Also, DNA interaction studies suggested that the compounds either do not interact, or 
interact weakly with DNA via groove and/or electrostatically. The interaction assay demonstrated that the 
compounds can weakly to moderately bind to the HSA biomolecule through electrostatic or van der Waals forces. 
In addition, agarose gel electrophoresis assays indicated that the complexes could not inhibit the action of the 
enzyme, topoisomerase II alpha. Nonetheless, the complexes demonstrated a promising target against topo-
isomerase I beta by promoting its inhibition. Finally, fluorescence studies revealed the capacity of irreversible 
inhibition of the complexes against the enzyme, cathepsin B. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) antioxidant 
assay did not show significant scavenging of DPPH radical. The results showed that the proposed structural 
features generate compounds of high cytotoxicity that can inhibit the action of cathepsin B and topoisomerase I 
beta. 
1. Introduction 
Cathepsin B, originally named CatB1, is a lysosomal cysteine prote-
ase that plays a role in several physiological and pathological processes 
[1]. It is predominantly found in subcellular endosomal and lysosomal 
compartments, and it is expressed in most cell lines. This enzyme de-
grades proteins and regulates their typical body turnover (proteolysis). 
It is also produced as an enzyme called myokine, which is essential in 
muscle–brain communication and dendritic remodeling in neurons [1]. 
Cathepsin B is significant because it is implicated in various diseases and 
oncogenic processes [2]. It has been associated with the degeneration of 
neurons and may play a role as a neuronal death mediator in a variety of 
neurodegenerative illnesses [3]. A recent study shows that the leakage of 
cathepsin B from lysosomes contributes to mitochondrial stress, 
inflammation activation, and nuclear senescence without an acidic 
environment [1]. According to the literature, the translocation of the 
lysosomal cysteine protease, cathepsin B, into the nucleus is a critical 
molecular event in human pancreatic cancer cells that promotes 
organelle-specific activation of ferroptosis [4]. It has also been revealed 
that cathepsin B is highly expressed in lung adenocarcinoma tissues 
following SARS-COV-2 infection. A condition correlated with immune 
cell infiltration and proinflammatory cytokine expression suggests that 
cathepsin B could be a therapeutic target for lung adenocarcinoma pa-
tients with coronavirus disease [5]. These conditions indicate the 
importance of developing compounds that are capable of inhibiting the 
proliferation of tumor cells. 
Several metallodrugs have been shown to target cysteine proteases, 
including cathepsin [6,7]. For instance, inorganic gold (I) and 
* Corresponding author. 
E-mail address: fillipe@ufscar.br (F.V. Rocha). 
Contents lists available at ScienceDirect 
Journal of Molecular Structure 
journal homepage: www.elsevier.com/locate/molstr 
https://doi.org/10.1016/j.molstruc.2023.136460 
Received 29 March 2023; Received in revised form 15 August 2023; Accepted 20 August 2023 
mailto:fillipe@ufscar.br
www.sciencedirect.com/science/journal/00222860
https://www.elsevier.com/locate/molstr
https://doi.org/10.1016/j.molstruc.2023.136460
https://doi.org/10.1016/j.molstruc.2023.136460
https://doi.org/10.1016/j.molstruc.2023.136460
http://crossmark.crossref.org/dialog/?doi=10.1016/j.molstruc.2023.136460&domain=pdf
Journal of Molecular Structure 1294 (2023) 136460
2
organometallic gold (III) complexes are effective cathepsin B inhibitors 
due to the aurophilicity of sulfur. The inhibitory effect of gold (I) com-
plexes is thought to be reversible because adding more cysteine restores 
cathepsin B activity [8,9]. However, despite this research, no structural 
data about cathepsin B metalloinhibitors is known, although it is 
assumed that they work by binding directly to the Cys29 residue in the 
enzyme’s active site, near the nitroxoline binding site [10]. An 
active-site titration and protection from inactivation assays, as well as 
docking simulations, were used to suggest the binding mode of metal-
loinhibitors [10,11]. 
Other reversible inhibitors of cathepsin B contain different metal 
centers such as Ru (II) and Pd (II) [12–14]. Among these, palladium has 
aroused significant scientific interest due to its similarity to Pt (II) metal 
complexes [15,16]. However, Pd (II) complexes are less stable, and can 
rapidly hydrolyze before reaching their target, resulting in minimal 
antitumor efficacy and toxicity. Furthermore, their penchant for 
generating trans isomers may encourage cis–trans isomerization, 
resulting in inactive species, at least at first [17]. Therefore, the inclu-
sion of thiosemicarbazone ligands is an efficient structural strategy to 
obtain more stable complexes due to the chelate effect, allowing their 
accumulation in pharmacological targets [18,19]. Additionally, ligands 
that contribute to structural conservation are bulky phosphines such as 
triphenylphosphine, which makes it challenging to replace ligands, thus 
increasing the stability and lipophilicity of the compounds [20–22]. 
DNA topoisomerases are enzymes considered necessary for DNA 
replication through the temporarily induced breakup of the DNA strand 
to catalyze the transformation of the DNA topology [23]. This enzyme 
plays a pivotal role in DNA, and is an essential source of cancer drugs 
[24]. Hence, DNA topoisomerases are widely divided into two main 
groups, called type I and type II DNA topoisomerases, depending on 
whether they form single or two-strand breaches of DNA during catal-
ysis, respectively [25]. Topoisomerase stability and inhibition of DNA 
break are the two most important strategies used to treat cancer. 
Intriguingly, HPyCT4BrPh and its copper (II) complex, [Cu(PyCT4BrPh) 
Cl], were investigated by Vutey and his colleagues for their capacity to 
inhibit human topoisomerase I beta [26]. Thiosemicarbazone has 
become a potent inhibitor of topoisomerase I beta after complexing with 
copper [27]. It was also reported that topoisomerase I beta is inhibited 
by stable novel Pd (II) complexes with thiosemicarbazonate ligands 
derivatized with pyrene. Investigations into the mechanism of action 
reveal that topoisomerase I betahttp://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0024
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0025
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0025
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0026
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0026
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0027
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0027
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0027
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0028
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0028
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0028
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0029
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0029
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0029
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0030
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0030
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0030
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0031
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0031
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0031
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0032
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0032
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0032
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0033
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0033
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0033
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0034
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0034
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0035
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0035
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0035
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0035
Journal of Molecular Structure 1294 (2023) 136460
14
[36] J.G. Da Silva, et al., Chalcone-derived thiosemicarbazones and their zinc (II) and 
gallium (III) complexes: spectral studies and antimicrobial activity, J. Coord. 
Chem. 66 (3) (2013) 385–401. 
[37] T. Diao, et al., Characterization of DMSO coordination to palladium (II) in solution 
and insights into the aerobic oxidation catalyst, Pd (DMSO) 2 (TFA) 2, Inorg. 
Chem. 51 (21) (2012) 11898–11909. 
[38] S. Bhattacharya, et al., Bioinspired oxidation of oximes to nitric oxide with 
dioxygen by a nonheme iron (II) complex, JBIC J. Biol. Inorg. Chem. 25 (2020) 
3–11. 
[39] N. Bandyopadhyay, et al., Synthesis, structure, DFT calculations, electrochemistry, 
fluorescence, DNA binding and molecular docking aspects of a novel oxime based 
ligand and its palladium (II) complex, J. Photochem. Photobiol. B Biol. 160 (2016) 
336–346. 
[40] V.Y. Almeida, et al., Cytotoxicity and antibacterial activity of silver complexes 
bearing semicarbazones and triphenylphosphine, ChemistrySelect 5 (46) (2020) 
14559–14563. 
[41] G. Faraglia, S. Sitran, The behaviour of dithiocarbamic ester palladium (II) and 
platinum (II) complexes in dimethyl sulfoxide, Inorg. Chim. Acta 176 (1) (1990) 
67–73. 
[42] H. Repich, et al., Mono-and binuclear Pd (II) complexes with 2-(5, 6-dimethyl-4- 
oxo-3, 4-dihydrothieno [2, 3-d] pyrimidin-2-yl)-N- 
phenylhydrazinecarbothioamide: Synthesis, crystal structure and spectroscopic 
characterization, J. Mol. Struct. 1102 (2015) 161–169. 
[43] J. Baruah, et al., A thiosemicarbazone–palladium (II)–imidazole complex as an 
efficient pre-catalyst for Suzuki–Miyaura cross-coupling reactions at room 
temperature in aqueous media, Transit. Met. Chem. 42 (2017) 683–692. 
[44] S.S. Batsanov, Van der Waals radii of elements, Inorg. Mater. 37 (9) (2001) 
871–885. 
[45] T. Steiner, The hydrogen bond in the solid state, Angew. Chem. Int. Ed. 41 (1) 
(2002) 48–76. 
[46] M.R. Khan, et al., Pd (II)-based heteroleptic complexes with N-(acyl)-N′, N′- 
(disubstituted) thioureas and phosphine ligands: synthesis, characterization and 
cytotoxic studies against lung squamous, breast adenocarcinoma and Leishmania 
tropica, Inorg. Chim. Acta 479 (2018) 189–196. 
[47] E.A. Nyawade, et al., Synthesis, characterization and anticancer activity of new 2- 
acetyl-5-methyl thiophene and cinnamaldehyde thiosemicarbazones and their 
palladium (II) complexes, Inorg. Chim. Acta 515 (2021), 120036. 
[48] M.A. Spackman, D. Jayatilaka, Hirshfeld surface analysis, CrystEngComm 11 (1) 
(2009) 19–32. 
[49] A. Kellett, et al., Molecular methods for assessment of non-covalent 
metallodrug–DNA interactions, Chem. Soc. Rev. 48 (4) (2019) 971–988. 
[50] J.S. Rocha, et al., Synthesis and characterization of silver (I) complexes bearing 
phenanthroline derivatives as ligands: cytotoxicity and DNA interaction 
evaluation, Inorg. Chem. Commun. 131 (2021), 108757. 
[51] N. Biswas, et al., Example of two novel thiocyanato bridged copper (II) complexes 
derived from substituted thiosemicarbazone ligand: structural elucidation, DNA/ 
albumin binding, biological profile analysis, and molecular docking study, 
J. Biomol. Struct. Dyn. 37 (11) (2019) 2801–2822. 
[52] C.V. Barra, et al., DNA binding, topoisomerase inhibition and cytotoxicity of 
palladium (II) complexes with 1, 10-phenanthroline and thioureas, Inorg. Chim. 
Acta 446 (2016) 54–60. 
[53] W. Villarreal, et al., Copper (I)–phosphine polypyridyl complexes: synthesis, 
characterization, DNA/HSA binding study, and antiproliferative activity, Inorg. 
Chem. 56 (7) (2017) 3781–3793. 
[54] J. Kypr, et al., Circular dichroism and conformational polymorphism of DNA, 
Nucleic Acids Res. 37 (6) (2009) 1713–1725. 
[55] J.J. Champoux, DNA topoisomerases: structure, function, and mechanism, Annu. 
Rev. Biochem. 70 (1) (2001) 369–413. 
[56] C.V. Barra, A.V. Netto, Antitumour complexes and DNA interactions and their tools 
of analysis: an approach to metalointercalators, Rev. Virtual Quim. (2015) 
1998–2016. 
[57] I.J. Bruno, et al., New software for searching the Cambridge structural database 
and visualizing crystal structures, Acta. Crystallogr. B. Struct. Sci. Cryst. Eng. 
Mater. 58 (3) (2002) 389–397. 
[58] Y.Y. Li, J. Fang, G.Z. Ao, B Cathepsin, L inhibitors: a patent review (2010-present), 
Expert Opin. Ther. Pat. 27 (6) (2017) 643–656. 
[59] H.H. Otto, T. Schirmeister, Cysteine proteases and their inhibitors, Chem. Rev. 97 
(1) (1997) 133–172. 
[60] J. Spencer, et al., Excellent correlation between cathepsin B inhibition and 
cytotoxicity for a series of palladacycles, Dalton Trans. (48) (2009) 10731–10735. 
[61] K. Mishra, H. Ojha, N.K. Chaudhury, Estimation of antiradical properties of 
antioxidants using DPPH assay: a critical review and results, Food Chem. 130 (4) 
(2012) 1036–1043. 
[62] B.N. Cunha, et al., Selective coordination mode of acylthiourea ligands in half- 
sandwich Ru (II) complexes and their cytotoxic evaluation, Inorg. Chem. 59 (7) 
(2020) 5072–5085. 
[63] A.P.M. Guedes, et al., Heterobimetallic Ru (ii)/Fe (ii) complexes as potent 
anticancer agents against breast cancer cells, inducing apoptosis through multiple 
targets, Metallomics 12 (4) (2020) 547–561. 
[64] O.V. Dolomanov, et al., OLEX2: a complete structure solution, refinement and 
analysis program, J. Appl. Crystallogr. 42 (2) (2009) 339–341. 
[65] M.A. Lima, et al., Palladium (II) complexes bearing thiosemicarbazone and 
phosphines as inhibitors of DNA-Topoisomerase II enzyme: synthesis, 
characterizations and biological studies, Inorg. Chem. Commun. 112 (2020), 
107708. 
[66] M. Fandzloch, et al., New organometallic ruthenium (II) complexes with purine 
analogs–a wide perspective on their biological application, Dalton Trans. 50 (16) 
(2021) 5557–5573. 
[67] C.P. Popolin, et al., Cytotoxicity and anti-tumor effects of new ruthenium 
complexes on triple negative breast cancer cells, PLoS One 12 (9) (2017), 
e0183275. 
[68] H.A. Benesi, J. Hildebrand, A spectrophotometric investigation of the interaction of 
iodine with aromatic hydrocarbons, J. Am. Chem. Soc 71 (8) (1949) 2703–2707.[69] A. Banerjee, et al., Synthesis, structure and characterization of new 
dithiocarbazate-based mixed ligand oxidovanadium (IV) complexes: DNA/HSA 
interaction, cytotoxic activity and DFT studies, New J. Chem. 44 (26) (2020) 
10946–10963. 
A.O. Akinyemi et al. 
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0036
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0036
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0036
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0037
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0037
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0037
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0038
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0038
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0038
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0039
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0039
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0039
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0039
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0040
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0040
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0040
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0041
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0041
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0041
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0042
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0042
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0042
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0042
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0043
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0043
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0043
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0044
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0044
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0045
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0045
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0046
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0046
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0046
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0046
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0047
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0047
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0047
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0048
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0048
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0049
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0049
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0050
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0050
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0050
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0051
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0051
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0051
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0051
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0052
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0052
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0052
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0053
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0053
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0053
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0054
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0054
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0055
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0055
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0056
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0056
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0056
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0057
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0057
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0057
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0058
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0058
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0059
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0059
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0060
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0060
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0061
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0061
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0061
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0062
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0062
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0062
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0063
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0063
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0063
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0064
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0064
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0065
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0065
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0065
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0065
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0066
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0066
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0066
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0067
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0067
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0067
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0068
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0068
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0069
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0069
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0069
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0069
	Palladium (II) complexes as inhibitors of cathepsin B and topoisomerase I beta: Synthesis, characterization, and cytotoxicity
	1 Introduction
	2 Results and discussion
	2.1 Synthesis of complexes
	2.2 Spectroscopic study
	2.3 Single crystal X-ray analysis
	2.4 Biomolecules assays
	2.5 Cytotoxic assays
	3 Conclusions
	4 Materials and methods
	4.1 General methods
	4.2 X-ray diffraction
	4.3 Synthesis of complexes
	4.3.1 Ligand TSC
	4.3.2 Precursor cis-[Pd(CH3CN)2Cl2]
	4.3.3 Synthesis of [PdCl(TSC)(PPh3)] (1)
	4.3.4 [Pd(SCN)(TSC)(PPh3)] (2)
	4.3.5 [Pd(TSC)(DMSO)(PPh3)] (3)
	4.4 Lipophilicity (Partition coefficient logP)
	4.5 UV-Vis Stability
	4.6 Cytotoxic assay
	4.7 Spectrophotometer titration with CT-DNA
	4.8 Circular dichroism
	4.9 Topoisomerase I beta and II alpha interaction
	5 Cathepsin B
	5.1 DPPH antioxidant assay
	5.2 Human serum albumin (HSA) binding studies
	Author contributions
	Funding
	Supplementary materials
	Declaration of Competing Interest
	Data availability
	Supplementary materials
	Referencesis a likely target for [PdCl(PPh3) 
(PrCh)]. Interestingly, the substance blocks the reconnection reaction 
without blocking DNA cleavage [27]. 
Considering these enzymes as prospective therapeutic targets for 
palladium compounds, three Pd(II) complexes of the type [PdCl(TSC) 
(PPh3)] (1), [Pd(SCN)(TSC)(PPh3)] (2), [Pd(TSC)(DMSO)(PPh3)] (3), 
PPh3 = Triphenylphosphine, TSC = 1-methyl- 3-phenylprop-2-en- 1- 
ylidene hydrazine carbothioamide were synthesized. The compounds 
were fully characterized by spectroscopic techniques and evaluated as 
cytotoxic agents. 
2. Results and discussion 
2.1. Synthesis of complexes 
In this work, we synthesized three palladium complexes of the type 
[Pd(TSC)(X)PPh3], where X= Chloride(Cl− )(1), Thiocyanate (SCN− )(2), 
and DMSO (Dimethyl sulfoxide)(3), Fig. S1. The ligands were synthe-
sized according to the literature [28]. The precursor 
cis-[PdCl2(CH3CN)2] was first synthesized [29]. As a result, the pre-
cursor complex reacted with an equimolar amount of thiosemicarbazone 
and the triphenylphosphine ligand to generate the compounds. Com-
plexes 1–3 were air stable powders, which were yellow to deep orange, 
and Scheme 1 represents the synthetic pathway for complex 1. The 
monodentate ligand on complex 1 would lead to and be replaced by 
SCN− and DMSO to form new complexes 2 and 3, respectively. The 
higher affinity of palladium íon for sulfur atoms allows for the change of 
ligands that occurs by an associative mechanism in square-planar 
complexes. 
2.2. Spectroscopic study 
The characterization techniques, NMR, IR, UV-Vis, and mass spec-
trometry, painstakingly confirmed the structures of the complexes (1-3). 
In the IR spectra of the compounds (Fig. S2), bands were observed in the 
2800 cm− 1, referring to the aliphatic C-H stretches. When comparing the 
IR spectra of the free ligand (TSC) and the complexes, some changes 
were observed, indicating the coordination of the ligand to the metallic 
center. The attribution of the stretches allowed us to infer that the li-
gands coordinate to the metallic center in the bidentate form via N and S 
atoms. This coordination is indicated by the displacement of the C=S 
stretch from 694 cm− 1 in the ligand to 690 cm− 1 in the complexes, 
Scheme 1. Representation of the synthetic steps to obtain complexes 1–3. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
3
characterizing a shift to a less energetic region. Furthermore, this change 
indicates a donation of the electron density of sulfur from thio-
semicarbazone to the metal. The absence of the N-H(13) band in the 
3230 cm− 1 in complexes 2 and 3 reveals the ligand’s deprotonation, 
which generates a delocalization of the electron density. 
Consequently, the C=S bond weakens since there is a decrease in the 
π character of this bond. In contrast, the electron density of the C-N bond 
increases. This behavior is evidenced by the significant displacement of 
the C-N stretch to a more energetic region in the complexes (Table S1) 
[30–33]. The presence or absence of the N-H(13) band in complex 1 is 
hampered by overlapping bands that constitute an extended band, 
which probably results from hydrogen interactions, thus requiring more 
characterization techniques. 
Furthermore, from the analysis of the infrared spectra, it was 
possible to suggest the N-terminal coordination for the thiocyanate 
ligand in complex 2 due to an intense band at 2125 cm− 1. Such behavior 
is reinforced by the absence of a band around 862 cm− 1 corresponding 
to the C-S stretch of the NCS ligand and an intense band at 2110 cm− 1 
attributed to the C-N stretch [19,31,34]. This coordination mode is 
suggested according to the literature since the author’s evidence that the 
M-SCN bond, because of the angular structure of M-S-C, has a more 
significant steric requirement when compared to the linear form of 
M-NCS. Thus, bulky ligands such as triphenylphosphine could generate 
an M-SCN bond tension and promote the M-NCS coordination mode, 
driven by the steric factor [34]. Additionally, the trans position between 
the phosphorous atom and sulfur could create a back-bonding compe-
tition metal to P or S, weakening both bonds. Furthermore, the presence 
of a characteristic band at 1090 cm− 1 was highlighted, referring to the 
angular demormation (τ) between the carbon and phosphorus atoms of 
triphenylphosphine, which further indicate the presence of the ligand in 
the molecular framework [32]. 
Table S2 and Figs. S3–S9 show the NMR signal and their attributions. 
From the 1H NMR analysis of the compounds, it was possible to observe 
the presence of two doublets in the regions of 8.15–8.67 ppm and 
7.01–7.20 ppm, referring to the hydrogens, H6 and H7. The value of the 
coupling constant of 16 Hz indicates that the hydrogens are in a trans 
position [35]. Also, in the spectra of the complexes, the presence of 
multiplets was observed in the region of 7.08–7.80 ppm, attributable to 
the aromatic hydrogens of both the phenyl groups of triphenylphosphine 
and the phenyl group belonging to the organic portion of the molecule 
[31]. The anionic coordination of the ligands was also confirmed by 1H 
NMR due to the absence of the singlet at 13 ppm in the spectra of 
complexes 2 and 3, which corresponds to H(13), indicating deprotona-
tion of the ligand [36]. Except for complex 1 (Fig. S5), it was possible to 
verify the presence of the signal at 12.91 ppm, indicating the ligand’s 
coordination in the neutral form. When comparing the spectra of the 
ligand and the complexes, it is possible to infer that after complexation, 
there was a shift concerning the signal from 2.08 ppm in the ligand to 
2.4–2.6 ppm for the hydrogens of the CH3(8) methyl group. This shift to 
a less shielded field is due to the removal of electron density for the 
metal, indicating coordination via azomethine atom (C=N). The ethyl 
group hydrogens also shifted after coordination; hence, the CH2(11) 
hydrogens moved from 3.74 ppm in the ligand to 3.22–3.29 ppm in the 
complexes. Furthermore, the CH3(12) hydrogens shifted from 1.31 ppm 
in the ligand to 1.13–1.21 ppm in the complexes. Both underwent 
displacement to a more unshielded field due to the removal of electron 
density through the coordination of the S atom in the C=S thioamide 
bond [32,33]. 
For complex 3 (Fig. S7), the singlet at 2.61 ppm was attributable to 
the hydrogens of the coordinated DMSO. The literature reports that the 
signal in the region between 3.03 ppm and 2.59 ppm corresponds to the 
coordination of DMSO through the oxygen atom [37]. The 31P {1H} 
NMR spectra obtained for complexes 1 and 3 indicated that a single 
product was obtained from the presence of a signal around 20–40 ppm, 
ruling out the formation of by-products (Fig. S9). This analysis can 
confirm the presence of coordinated triphenylphosphine, since no 
signals referring to free phosphine were observed in the negative spec-
trum region around -7.3 ppm [36] (Fig. S8). In the case of complex 2, 
two signals were kept in the 31P NMR, the most pronounced signal at 
27.23 ppm referring to the product of interest and the signal at 29.55 
ppm coming from the phosphine oxide [31,38]. 
The mass spectrum of the compounds (Fig. S14 [complex 1], S15 
[complex 2] and S16 [complex 3]) showed high confluency between the 
theoretical structural proposal and the experimental result. The peaks 
referred to the formulas [PdX(TSC)(PPh3)]+, and Table 1 presents the 
data obtained, describing the error (ppm) for each formula mentioned 
above. The calculated error did not exceed 5 ppm, which corroborates 
the structural elucidation and strongly indicates that the proposed 
structure was synthesized. 
In UV-Vis spectra, bands less energetic than 300 nm are attributedto 
intraligand transitions. In the ligand spectrum, a band close to 328 nm 
was observed to be referring to the transitions of the π-π* and n-π* type 
from the chromophore groups of the molecule: NH2, C=C, C=N, and 
C=S, Fig. S9 [37]. In the spectra of the complexes, this band underwent a 
significant bathochromic shift to the region between 290 nm and 390 
nm, reinforcing the coordination of ligands to the metallic center [39] 
(Fig. S10). This red shift comes from a reorganization of electron density 
from the deprotonation of the ligand that occurs due to coordination. 
This region is related to charge transfer (MLCT or LMCT) to palladium 
complexes, since the flat quadratic conformation stabilizes the orbitals 
at the center of the metal ion, making them energetically more distant 
from the ligand orbitals. 
Consequently, the charge transition becomes more energetic. Iden-
tifying bands corresponding to d–d electronic transitions is rugged due 
to the widening of the charge transfer band [32,39]. For instance, from 
the superposition of the UV-Vis spectra of the complexes, it is possible to 
verify a similar behavior between them (Fig. S11). Wavelengths, molar 
absorptivity, and band assignments are shown in Table S3. 
Studying the lipophilicity of compounds is vital because it impacts 
their ability to cross biological barriers and affects drug distribution and 
metabolism. Thus, the lipophilicity of the compounds was determined, 
and solvents were employed in this study to mimic both the extracellular 
(aqueous) and intracellular (n-octanol) environments. The log P values 
for all the complexes are presented in (Fig. 12). The results indicate that 
these complexes exhibit lipophilic properties with positive logP values 
clustered closely together in approximately ~0.36–0.52 (Fig. S12). 
Furthermore, the observed logP values suggest that the complexes have 
the potential to permeate the cell membrane due to their lipophilic 
nature. This finding highlights the significance of structural design, 
particularly the incorporation of the co-ligand, phosphine, which en-
hances the lipophilicity of the complexes [40]. 
The complexes demonstrated remarkable stability when subjected to 
analysis against DMSO over a period ranging from 0 to 48 h (Figs. 1a and 
S13). For instance, by observing their behavior in the culture medium 
(Figs. 1b and S14), minimal changes were noted, and two isosbestic 
points were observed at 369 nm and 416 nm. These findings may indi-
cate the presence of an equilibrium between the species in the culture 
medium and the complexes. Beside, the high concentrations of inorganic 
salts, amino acids, and vitamins in the DMEM medium could have 
contributed to this equilibrium, as leaving groups such as Cl− , NCS− , and 
DMSO were present. However, it was not anticipated that the other 
portion of the complexes containing the chelating ligand and phosphine 
would easily substitute these species. 
Table 1 
Theoretical masses and mass obtained by mass spectrometry for Pd(II) 
complexes. 
Compounds Theorical (m/z) Experimental (m/z) Error (ppm) 
[M+H]+ (1) 650.0776 650.0765 1.69 
[M+Na]+ (2) 695.0655 695.0689 4.80 
[M-DMSO]+ (3) 614.1011 614.1041 4.80 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
4
2.3. Single crystal X-ray analysis 
The single crystals of complex (1) were obtained by the slow evap-
oration of the supernatant of the reaction solution (acetonitrile: chlo-
roform [1:1]) at room temperature. The ORTEP representation with the 
atom-numbering scheme is illustrated in Fig. 2. 
The Pd(II) compounds adopted a distorted square-planar geometry, 
with interatomic bond angles of 89.61◦ between the P1-Pd1-Cl1 atoms 
(Table S4). According to the literature, the bond lengths and the angles 
were within the expected length [41]. TSC served as a neutral bidentate 
ligand that leads to cis coordination through S1 and N1 atoms to form a 
five-membered ring. The remaining binding sites were occupied by a 
chloride ion that was coordinated trans to the S1 atom and with a 
phosphine ligand coordinated trans to the N1 atom [42]. The intra-
molecular interactions observed for complex 1 were C(6)—H(6)⋅⋅⋅Cl(1) 
forming S(6)-type arrangements. These interaction had bond lengths of 
2.693 Å, which are close to the values described in the literature for 
H⋅⋅⋅Cl hydrogen bonds [43]. These interactions presented bond lengths 
smaller than the sum of the van der Waals radii of the atoms involved, 
which is characterized as 3.0 Å [44]. The X-ray diffraction technique 
enabled the verification of the presence of chloride as a counter-ion. This 
atom established intermolecular interactions, N(2)—H(2)⋅⋅⋅Cl(2) and N 
(3)—H(3)⋅⋅⋅Cl(2), forming an arrangement of type S(6) in that chlorine 
acts as a bifurcated acceptor atom [43,45]. It was also observed that the 
counter-ion participates in non-classical bonds such as C(132)—H 
(132)⋅⋅⋅Cl(2), whose hydrogen atoms belong to the phenyl groups of 
triphenylphosphine [30]. In addition, the intermolecular interaction, C 
(134)—H(134)⋅⋅⋅Cl(2), was observed through the Hirshfeld surface 
(Fig. 4a) [46]. The non-classical interactions, C(125)—H(125)⋅⋅⋅C(134), 
exhibited bond lengths of 2.895 Å, which is close to the literature value 
of 2.776 Å [47]. The intermolecular interactions, C(132)—H(132)⋅⋅⋅Cl 
(2), are illustrated in Fig. 3 and described in Table 2, while data resulting 
from the X-Ray diffraction technique are described in Table S5. 
The non-covalent intermolecular interactions present in the crystal-
line lattice were explored using the Hirshfeld surface (HS) using the 
Cristal Explorer 21.5 program. From the qualitative analysis of the HS 
mapping, it was possible to observe the presence of strong contacts 
represented in red, referring to the regions whose contact distance is 
smaller than the van der Waals radius of the atoms involved. Meanwhile, 
weak interactions are represented by white and blue areas [48]. 
Therefore, the regions in red indicate interactions between the atoms of 
the H‧‧‧Cl type (Fig. 4a). The projection of the HS in a two-dimensional 
graph as a function of the parameters of (y-axis) and di (x-axis) 
allowed for quantification of the contributions of the interatomic con-
tacts to the crystalline packing [48]. Thus, the contacts that make up the 
most notable contributions were H—H (56.2%), C—H (22.1%), and 
H—Cl (11.1%) (Fig. 4b). The other contacts with the lowest contribution 
to crystalline cohesion were N-H (2.9%), S-H (2.6%), and H-Pd (0.7%) 
(Fig. S12). 
2.4. Biomolecules assays 
DNA is widely regarded as a biological target against various anti-
neoplastic agents, which may interact with DNA in a covalent or non- 
covalent manner. When a covalent interaction occurs, the labile ligand 
of the complexes is replaced by a nitrogenous DNA base, while non- 
covalent interactions include intercalation, interaction with grooves, 
and electrostatic attraction [49]. The UV-Vis titration assay with DNA 
provided evidence to suggest that the complexes do not interact with 
Fig. 1. (a) Complex (1) analyzed at intervals 0–48 h in DMSO. (b) Complex (1) analyzed in fractions of DMSO:DMEM (1:99%). 1 × 10− 5 M. 
Fig. 2. ORTEP representation of the asymmetric unit of complex 1. Thermal 
ellipsoids are presented with a probability of 30%. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
5
DNA, or that they tend to interact through weak interactions such as 
electrostatics and/or via the groove, as no significant changes are 
observedin the spectra of the complexes after successive additions of 
DNA (Fig. 5) [50]. No bathochromic shift and high hypochromism were 
observed in the spectra, which characterizes the intercalative interac-
tion, ruling out this possibility [51]. Since a covalent bond or interca-
lation leads to drastic changes in the UV-vis spectrum profile, there was 
only one hypochromic effect, 1 and 2, which was less pronounced in 
complex 3 (Fig. S13). This hypothesis is reinforced by the values of the 
intrinsic binding constant (Kb) obtained in the order of 104 (Table 3) 
from the Benesi–Hildebran equation: [DNA]/(εa – εf) = [DNA]/(εb – εf) 
+ 1/[Kb (εb – εf)]. The compounds of interest showed lower Kb values 
than the classical Ethide Bromide intercalator (Kb=105 to 107 M− 1), and 
are close to the values described for interactions via grooves, which 
range from 103 to 104 M− 1 [50,52]. 
Circular dichroism (CD) is a commonly used technique for investi-
gating the interaction between compounds and DNA with diverse 
conformational properties [2]. This study examined the interaction of 
ct-DNA with the synthesized compounds. CD spectra revealed a promi-
nent positive band at approximately 260–280 nm and a negative band 
around 248 nm, corresponding to the helical conformation of the 
ct-DNA double helix and the stacking of nitrogenous bases, respectively 
[53]. 
However, interpreting the band at 248 nm was challenging due to 
interferences from solvent and compound bands in this region. In 
contrast, the band at 260–280 nm slightly fluctuated in its maximum 
during the interaction with DMSO and NCS complexes. Compound 1 
showed no changes in the bands compared to the control ct-DNA alone 
(Fig. 6). These findings indicate that there is minimal or no significant 
interaction between the synthesized compounds and ct-DNA [54]. 
Topoisomerases can change the topology of DNA, allowing it to relax 
the supercoiled form. Since these enzymes function in essential pro-
cesses such as transcription and DNA replication, they have emerged as 
an attractive biological target for cytotoxic agents [55]. Thus, the 
influence of complexes 1–3 in inhibiting the relaxation on the structure 
of the supercoiled DNA caused by topo enzymes was determined by their 
ability to modify the mobility of the pBR322 plasmid in a gel electro-
phoresis assay. Furthermore, it is well known that relaxed or cleavage 
plasmid structures produce different plasmid forms with a slower 
migration [56]. 
Complexes 1–3 were incubated with pBR322 plasmid and top-
oisomerases I beta and II alpha (Figs. 7 and 8, respectively) at 1.00, 10.0, 
and 100 μM concentrations. Fig. 7 shows that the complexes interfered 
with the action of topoisomerase I beta in all the tested concentrations, 
following the same pattern as negative control (only supercoiled 
plasmid). However, a decrease in the intensity of the band related to 
supercoiled plasmid was observed in the positive control (TOPO +
plasmid). In contrast, in Fig. 8, compounds 1–3 did not affect the 
mobility or intensity of the bands referring to the DNA structure, 
keeping the identical profile of the positive control. These data indicate 
that compounds 1–3 did not interfere with the action of topoisomerase II 
alpha. In previous works carried out by our research group, it was 
demonstrated that a similar compound [PdCl(PPh3)TSC-CC] was able to 
inhibit the total action of topoisomerase II at a concentration of 12.5 
µmol⋅L− 1[57]. Thus, from the present work, it is possible to suggest that 
the group at the N4 position of the thiosemicarbazones can influence the 
interaction with the enzyme. In this way, the steric volume of the ethyl 
group—when compared to the methyl group—may hinder the interac-
tion in the active site of the enzyme (Fig. 9). Hence, there are no in-
dications that the complexes stop the relaxation of DNA provided by 
topoisomerase II alpha, which is promising and selective against topo-
isomerase I beta. 
The importance of cathepsin B in cell degradation and its involve-
ment in tumor invasion, cancer, osteoporosis, autoimmune disorders, 
and metastases has led to its investigation as a potential target for the 
complexes of interest [58,59]. For instance, compounds were evaluated 
for their ability to inhibit enzyme action by fluorescence assay. The three 
complexes were assayed against cathepsin B at seven different concen-
trations (25.0–300 µL). The data obtained from fluorescence were 
further analyzed using the slope of the line for all test inhibitor samples 
[S] and the enzyme control (EC), where Slope = (RFU2-RFU1)/ T2-T1 or 
ΔRFU/ΔT. The relative activity of the three compounds was shown 
versus their concentrations, with IC50 values ranging from 60.3 to 101 
µM (Table 4). The results showed that the inhibition capacity of the 
compounds changes depending on the labile group. 
Complex 2 with SCN- as ligand showed the highest IC50 value 
probably due to its strong interaction with the palladium ion. As such, 
the SCN- could hinder the metal’s interaction with the free sulfur atom 
of the enzyme and its subsequent reduction of inhibition through irre-
versible bonds [19]. The most accepted hypothesis that the thiolate 
Fig. 3. Intermolecular interactions C(132)—H(132)⋅⋅⋅Cl(2) for complex 1 along the crystallographic axis a. 
Table 2 
Data referring to inter- and intramolecular hydrogen interactions (*) identified 
in the crystal structure of complex 1. 
D—H⋅⋅⋅A d(D—H), Å d(H⋅⋅⋅A), Å d(D⋅⋅⋅A), Åsuggesting 
an interaction between the compounds and the protein (Fig. S20). 
A quantitative analysis of the fluorescence suppression process was 
performed and the values for the Stern–Volmer interaction constant 
(Ksv) as well as the bimolecular suppression velocity (kq) were obtained 
according to the following equation: 
F0
F
= 1 + KSV [Q] = 1 + kqt0[Q]
Where F0 is the fluorescence intensity of HSA without the complex, F 
is the fluorescence intensity in the presence of the complexes, t0 is the 
average lifetime of HSA without quencher (approximately 10− 8s), [Q] is 
the complex concentration, kq is the bimolecular constant, and Ksv is the 
Fig. 4. (a) View of the d-norm mapped on the Hirshfeld surface of complex 1. The red spots represent the intermolecular contacts between the atoms. (b) Fingerprint 
plots of contacts of higher H—H, C—H, and H—Cl contributions to crystalline packing. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
7
Stern–Volmer constant. 
The values in Table 5 indicate that the Stern–Volmer constant (Ksv) 
decreases with increasing temperature, suggesting a static quenching 
mechanism for the interaction between the complexes and HSA. Addi-
tionally, the calculated values of the bimolecular quenching rate con-
stant (kq) are larger than the maximum possible value for a dynamic 
quenching mechanism (2 × 1010 M− 1 s1)[ref]. Further, these data sup-
port the idea that the suppression of HSA fluorescence occurs through 
the formation of an intermediate species. 
In addition, the binding constant (Kb) was determined from the 
following equation: log[(F0-F)/F] = logKb + nlog[Q], from the linear 
Fig. 5. Interaction of Complex 1 with CT-DNA by Ultraviolet-Visible Spectroscopic Titration. 
Table 3 
Values of the intrinsic binding constant (Kb) and percentage of hypochromism 
for the complexes. 
Compound Kb (M− 1) x 104 H (%) 
1 8.73 30 
2 8.20 18 
3 5.57 08 
Fig. 6. Circular dichroism (CD) spectra of ct-DNA (80.0 μmol‧L− 1) incubated with complex 1 (Cl− ), 2 (SCN− ), and 3 (DMSO) at different [complex]/DNA ratios at 
37 ◦C within 24 h. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
8
coefficient of the straight line constructed using the graph of log(F0-F)/F 
versus log[Q]. According to the obtained data in Table 5, the complexes 
presented Kb values in the order of 103 to 104 M− 1, indicating a weak to 
moderate interaction with HSA. 
Thermodynamic parameters were determined based on the following 
equations: 
ln
Kb3
Kb1
=
[
1
T1
−
1
T3
]
ΔH∘
R 
ΔG∘ = − RTlnKb = ΔH∘ − TΔS∘ 
Where R = constant of gases (8.314 J mol− 1K− 1) and K is the 
Fig. 7. Inhibitory capacity of TOPO Iβ by compounds 1– 3. Lane 1 (C+): plasmid (DNA and TOPO Iβ). Lane 2 (C-): (plasmid DNA only). Lane 3: 1 (1.00 µmolL− 1), 
Lane 4: 1 (10.0 µmolL− 1), Lane 5: 1 (100 µmolL− 1). Lane 6: 2 (1.00 µmolL− 1), Lane 7: 2 (10.0 µmolL− 1), Lane 8: 2 (100 µmolL− 1). Lane 9: 3 (1.00 µmolL− 1), Lane 10: 3 
(10.0 µmolL− 1), Lane 11: 3 (100 µmol⋅L− 1). 
Fig. 8. Inhibitory capacity of TOPO IIα by compounds 1–3. Lane 1(C+): plasmid (DNA and TOPO IIα). Lane 2 (C-): (DNA plasmid only). Lane 3: 1 (1.00 µmolL− 1), 
Lane 4: 1 (10.0 µmolL− 1), Lane 5: 1 (100 µmolL− 1). Lane 6: 2 (1.00 µmolL− 1), Lane 7: 2 (10.0 µmolL− 1), Lane 8: 2 (100 µmolL− 1). Lane 9: 3 (1.00 µmolL− 1), Lane 10: 3 
(10.0 µmolL− 1), Lane 11: 3 (100 µmolL− 1). 
Fig. 9. Comparison of the inhibition capacity of topoisomerase II between compounds (a) synthesized in the present work and (b) those synthesized previously by the 
research group. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
9
interaction constant with HSA (Kb). 
Based on the data, it can be inferred that the interaction of the 
complexes with HSA is spontaneous, as evidenced by the negative values 
of ΔGo. Additionally, the thermodynamic parameters, ΔH (enthalpy 
change) and ΔS (entropy change), provide insight into the nature of the 
interactions. 
Complexes 1 and 3 exhibited negative values for ΔH and positive 
values for ΔS, which indicates that the binding process involves an 
Table 4 
IC50 values of the complexes referring to the ability to inhibit 
the enzyme, cathepsin B. 
Complexes IC50 (μM) 
PdCl(TSC)(PPh3) (1) 86.4 
Pd(SCN)(TSC)(PPh3) (2) 101 
Pd(TSC)(DMSO)(PPh3) (3) 60.3 
Fig. 10. Comparison of the inhibition capacity of the complexes (1–3) with the enzymatic control (cathepsin B) (violet) and enzymatic inhibitor (blue). 
Fig. 11. Scavenging of DPPH radical by compounds 1–3 and the control (ascorbic acid) in different concentrations. 
Table 5 
Values of Ksv, kq, Kb, n, and the thermodynamic parameters for the complexes. 
Compounds Temperature KSV ± SD Kq Kb ± SD n ΔH ΔS ΔG ± SD 
(K) 104 (L•mol− 1) 1013 (L•mol− 1•s− 1) 104 (L•mol− 1) (kJ•mol− 1) (J•mol− 1) (kJ•mol− 1) 
293 6.76 ± 0.36 1.13 3.33 ± 0.00 0.93 -25.38 ± 5.17 
1 (Cl) 298 6.25 ± 0.54 1.04 2.72 ± 0.00 0.92 -17.99 24.99 -25.31 ± 5.10 
303 6.03 ± 0.50 1.00 2.61 ± 0.00 0.92 -25.64 ± 5.38 
293 6.13 ± 0.22 1.02 8.21 ± 0.00 1.03 -27.58 ± 4.98 
2 (SCN) 298 5.73 ± 0.15 0.96 7.85 ± 0.00 1.03 -33.79 -20.66 -27.94 ± 5.05 
303 5.55 ± 0.20 0.92 5.18 ± 0.00 1.00 -27.36 ± 5.34 
293 3.81 ± 0.15 0.64 0.58 ± 0.00 0.81 -21.137 ± 5.56 
3 (DMSO) 298 3.50 ± 0.23 0.58 0.50 ± 0.00 0.81 -9.70 38.77 -21.101 ± 5.39 
303 3.55 ± 0.25 0.59 0.51 ± 0.00 0.81 -21.530 ± 5.31 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
10
electrostatic interaction with HSA. A negative ΔH suggests that the 
interaction is exothermic, i.e., it releases heat during binding. The pos-
itive ΔS implies an increase in the disorder or freedom of movement of 
the system, which is typical for electrostatic interactions. 
In contrast, Complex 2 showed negative values for both ΔH and ΔS, 
suggesting the occurrence of van der Waals forces in the binding process. 
The negative ΔH indicates an exothermic interaction, while the negative 
ΔS implies a reduction in system disorder upon binding. These findings 
shed light on the interactions between the complexes and HSA, with 
Complexes 1 and 3 likely engaging in electrostatic interactions. At the 
same time, Complex 2 is more likely to interact through van der Waals 
forces. 
2.5. Cytotoxic assays 
The cytotoxic assay was performed in vitro using the MTT colori-
metric method [57]. The cytotoxic activity of the TSC ligand, complexes 
(1–3) and cisplatin was determined for two cell lines: A2780cis (ovarian 
tumor cell) and MRC-5 (non-tumor lung cell), and the corresponding 
IC50 values were compared to the standard drug, cisplatin (Table 6). 
The cytotoxicity assays revealed that the free ligand is inactive 
against the tested cell lines in concentrations below 50 μM. Conversely, 
the three complexes showed outstanding activity. The activity of the 
compounds was more prominent against the ovarian tumor cell line 
(A2780cis), showing considerably low IC50 values and surpassing 
cisplatin values between 93 and 1470 folds. Furthermore, the selectivity 
index showed a higher preference for ovarian cancercells. An SI ratio of 
72:668 was found between the compounds and cisplatin. These results 
indicate that these structural features are crucial build blocks for 
achieving high cytotoxic activity against ovarian cancer cells. 
Cell morphology is an important parameter to be monitored, since it 
indicates different relationship types between the study compounds and 
the target cell. For example, reducing the number of viable cells without 
changing their morphology suggests an antiproliferative action. In 
contrast, a morphological change followed by a decrease in the number 
of cells may indicate cell death. Thus, the capacity of complex 3 to 
change the morphology of A280cis cells was tested at different con-
centrations (¼ × IC50, IC50, and 2 × IC50) between 0 and 24 h. 
By analyzing the images (Fig. 12), one can observe an increase in the 
number of cells as well as the maintenance of morphology in the control 
image and for the ¼ × IC50 concentration after 24 h of incubation. 
However, cell morphology at IC50 and 2 × IC50 concentrations was 
entirely altered in the spherical shape, and did not adhere to the surface 
of the culture plate after the incubation period, indicating cell death [62, 
63]. 
3. Conclusions 
In this study, three novel palladium (II) compounds were successfully 
synthesized and thoroughly characterized using various techniques, 
including UV-vis, NMR, infrared, elemental analysis, and mass spec-
trometry. The cytotoxic activity of these complexes was evaluated 
against different cell lines, and they demonstrated superior potency to 
that of the well-known anticancer drug, cisplatin. For instance, Complex 
3 exhibited a highly selective index, being 314 times more sensitive to 
tumor cells than non-tumor cells. The investigation of potential phar-
macological targets using UV-visible spectroscopy titration and circular 
dichroism indicated that the complexes likely do not interact with DNA. 
However, further testing of their inhibitory actions on topoisomerase II 
alpha did not yield any evidence of their action on this enzyme. 
Nevertheless, partial inhibition of topoisomerase I beta was observed for 
the three compounds. Furthermore, the assessment of the inhibitory 
capacity of the complexes against the enzyme, cathepsin B, provided 
valuable insights into possible multiple modes of their action. All three 
compounds exhibited irreversible inhibition. Complex 3 displayed a 
significantly stronger inhibitory effect with a low relative intensity (%) 
value. HAS interaction assay showed that the compounds can bind to the 
biomolecule via electrostatic or van der Waals interaction in a weak to 
moderate manner. 
Finally, this research demonstrates that the proposed structural 
features can lead to the development of highly cytotoxic compounds that 
effectively inhibit the activity of cathepsin B and topoisomerase I beta. 
Understanding the activity mode against cathepsin B may pave a way for 
proposing improvements in the molecular framework of these 
compounds. 
4. Materials and methods 
4.1. General methods 
The reagents and the solvents were purchased as reagent grades from 
Acros, Sigma–Aldrich, and Fisher Scientific, and they were used without 
any further purification. The nuclear magnetic resonance spectra of 1H, 
13C{1H}, and 31P{1H} (NMR) were recorded when using the Fourier 
transform tool with a BRUKER DRX 400, 100.6, and 161.98 MHz to 1H, 
13C, and 31P nucleus, respectively, chloroform at 298 K. The 31P NMR 
spectrum was taken just after the 1H resonance, and the measurement 
was carried out in the same deuterated solution. The electronic spectra 
of UV-Vis were obtained on a SHIMADZU-1650PC spectrophotometer. 
The IR spectra were recorded on an FT-IR SHIMADZU IRTracer-100 
spectrometer in the range 4000–400 cm− 1 using KBr pellets. The com-
pounds were analyzed using a high-resolution mass spectrometer with 
electron spray ionization (HRMS-ESI) in the positive mode using an 
Agilent LC-6545 Q-TOF-MS mass spectrometer (Agilent Technologies, 
Santa Clara, CA, EUA). 
4.2. X-ray diffraction 
A single crystal of compound 1 that is suitable for X-ray diffraction 
was obtained. Single-crystal X-ray diffraction measurements were per-
formed on a Rigaku XtaLAB mini diffractometer with graphite- 
monochromated Mo Kα radiation (λ = 0.71073 Å) at 298 K. The struc-
ture was solved through direct phase retrieval methods using SHELXT, 
and the refinement by full-matrix least-square using SHELXL [57,64], 
both hosted in an OLEX2 software [64]. Non-hydrogen atoms were 
refined anisotropically, and the hydrogen atoms were fixed at calculated 
positions and refined using the riding mode. The artwork and structure 
analysis were prepared using Mercury software [57], and Table S4 
shows the crystallographic data and refinement conditions. 
4.3. Synthesis of complexes 
4.3.1. Ligand TSC 
Based on the literature [28,29], the 1-methyl- 3-phenyl-
prop-2-en-1-ylidene hydrazine carbothioamide (TSC) ligand was syn-
thesized with minor modifications. First, equal amounts of 
4-methyl-3-thiosemicarbazide (1.90 mmol) and 0.280 g of benzalace-
tone (1.90 mmol) were solubilized in 50 mL of ethanol. Then 5 drops of 
concentrated HCl were added to act as a catalyst. The condensation 
reaction was refluxed for 24 h, after which the solution was concen-
trated in a rota evaporator, cooled in a bath of ice, and filtered. 
Table 6 
IC50 (µM) values for Pd(II) complexes (1–3) obtained by the in vitro MTT method. 
Complexes A2780cis MRC-5 *SI 
TSC > 50 μM > 50 μM - 
(1) 0.268 ± 0.015 μM 9.28 ± 0.3 μM 34.63 
(2) 0.201 ± 0.013 μM 9.5 ± 0.082 μM 47.26 
(3) 0.017 ± 0.004 μM 5.346 ± 0.002 μM 314.47 
Cisplatin 25.087 ± 0.891 μM 11.84 ± 1.19 μM 0.47 
* SI =[MRC-5]/[A2780 Cis]. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
11
Afterward, ice-cold ethanol was used to wash the product, and the solid 
was later dried under a desiccator (0.23 g of dry mass was obtained, 53% 
yield). IR (KBr; ν/cm− 1): 1625 n(C=N); 694 n(C=S); 3105 nN-H(13); 
3320 nN-H(10). UV-vis in chloroform λ[nm](log(εM− 1cm− 1): 203 
(3.68); 328 (3.98). 1H NMR (400 MHz, Chloroform-d) δ 8.59 (s, 1H, 
H13), 7.56 (d, J = = 6.3 Hz, 1H, H10), 7.5–7.3 (m, 5H, phenyl), 6.91 (s, 
1H, H7), 6.86 (s, 1H, H6), 3.74 (s, 2H, H11), 2.08 (s, 3H, H8), 1.31 (s, 
3H, H12). 
4.3.2. Precursor cis-[Pd(CH3CN)2Cl2] 
In a 125 mL Erlenmeyer flask with 50 mL of acetonitrile previously 
heated to 80 ◦C, 500 mg of palladium (II) chloride was slowly added. 
After the addition of the salt, the formation of a yellow solution was 
observed. After 4 h of reaction, the volume of the solution was reduced, 
and the yellow solid formed was filtered off (0.585 g of [PdCl2(CH3CN)2] 
was obtained, 80% yield) [65]. 
4.3.3. Synthesis of [PdCl(TSC)(PPh3)] (1) 
The synthesis was adapted from literature [25,63] with minor ad-
aptations. In a 25 mL beaker, 15 mL of acetonitrile was added, 50.0 mg 
(0.190 mmol) of cis-[PdCl2(CH3CN)2] and 47.0 mg (0.190 mmol) of 
ligand TSC-CC was solubilized in the acetonitrile. After 24 h, 51.0 mg (0, 
190 mmol) of PPh3 was added to the reaction and stood for more than 
24 h. The solid form was filtered and dry (0.25 g of dry mass was ob-
tained, 54% yield). IR (KBr; ν/cm− 1): 1591 n(C=N); 689 n(C=S); 1093 n 
(P-C). UV-Vis in chloroform λ[nm](log(εM− 1cm− 1): 243 (4.18); 351 
(4.10). 1H NMR (400 MHz, Chloroform-d) δ 12.91 (s, 1H, H13), 10.42 (s, 
1H, H10), 8.50 (d, J = = 16.1 Hz, 1H, H7), 7.87–7.71 (m, 6H), 7.68 (dd, 
J == 7.2, 2.3 Hz, 2H), 7.61–7.53 (m, 3H), 7.49 (td, J = 7.7, 2.7 Hz, 6H), 
7.41–7.31 (m, 3H), 7.18 (d, J = 16.1 Hz, 1H,H6), 3.22 (s, 2H,H11), 2.63 
(s, 3H,H8), 1.21 (s, 3H, H12). 31P NMR (162 MHz, CDCl3) δ30.89. 
4.3.4. [Pd(SCN)(TSC)(PPh3)] (2) 
50.0 mg (0.0770 mMol) ofpalladium complex [PdCl(TSC)(PPh3)] 
was weighed out and solubilized in methanol. An addition of 15.00 mg 
of KSCN followed, dissolved in 1.00 mL of water in the reaction (Note: 
color changes took place from light orange to deep orange). Finally, the 
solid form was filtered and stored. (Obtained 0.230 g of dry mass, 53% 
yield) [5]. IR (KBr; ν/cm− 1): 1557 n(C=N); 692 n(C=S); 1097 n(P-C); 
3410 nN-H(10); 2125 n(SCN).UV-Vis in chloroform 
λ[nm](log(εM− 1cm− 1): 242 (3.87); 348 (3.76). 1H NMR (400 MHz, 
Chloroform-d) δ 8.17 (d, J = 16.1 Hz, 1H, H7), 7.77–7.70 (m, 6H), 7.67 
(d, J = 7.7 Hz, 2H), 7.54 (dd, J = 9.0, 6.7 Hz, 3H), 7.47 (td, J = 7.8, 2.8 
Hz, 6H), 7.38 (t, J = 7.5 Hz, 2H), 7.32–7.28 (m, 1H), 7.10 (d, J = 16.0 
Hz, 1H, H6), 4.46 (s, 1H, H10), 3.29 (s, 2H, H11), 2.39 (s, 3H, H8), 1.14 
(s, 3H, H12). 31P NMR (162 MHz, CDCl3) δ27.23. 
4.3.5. [Pd(TSC)(DMSO)(PPh3)] (3) 
At first, 50.0 mg of palladium complex [PdCl(TSC)(PPh3)] was dis-
solved in 20.0 mL of dichloromethane (CH2Cl2). However, 8.20 μL of 
DMSO was further added. The reaction was refluxed and stirred over-
night. Then, the solution was allowed to cool down, after which the 
synthesis was kept in the fridge for 3 days to allow for evaporation. 
However much, 98% of KPF6 (potassium hexafluorophosphate) was 
added and warmed. The precipitate formed was further filtered and 
weighed (0.310 g of dry mass was obtained, 56% yield). IR (KBr; 
ν/cm− 1): 1625 n(C=N); 690 n(C=S); 1097 n(P-C) 3412 nN-H(10). UV- 
Vis in chloroform λ[nm](log(εM− 1cm− 1): 243 (4.17); 353 (5.06). 1H NMR 
(400 MHz, Chloroform-d) δ 8.65 (d, J = 16.1 Hz, 1H, H7), 7.79 (ddd, J =
12.0, 7.0, 1.3 Hz, 6H), 7.68 (d, J = 7.6 Hz, 2H), 7.53–7.44 (m, 3H), 
7.46–7.38 (m, 6H), 7.35 (t, J = 7.5 Hz, 2H), 7.31–7.27 (m, 1H), 6.99 (d, 
J = 16.1 Hz, 1H, H6), 4.43 (s, 1H, H10), 3.28 (s, 2H, H11), 2.36 (s, 3H, 
H8), 1.13 (s, 3H, H12).31P NMR (162 MHz, CDCl3) δ28.01. 
4.4. Lipophilicity (Partition coefficient logP) 
The lipophilicity or partition coefficient (log P) of the complexes was 
determined using the shake-flash method in n-octanol/water (MilliQ™) 
[66,67]. Equal volumes of water (750 μL) and n-octanol (750 μL) con-
taining 6% DMSO were mixed, and the complexes were added to this 
mixture. The solution was continuously shaken at 310 K for 24 h at 200 
rpm using a Thermo-Shaker Agmaxx, AG-100 model. After the incuba-
tion period, the solutions (in triplicate) were centrifuged at 1000 rpm for 
5 min. Measurement of organic layers was spectrophotometrically car-
ried out, and the concentration of the complexes was determined by 
UV-Visible calibration curves in n-octanol. Finally, the value of logP was 
determined using the equation below [66] 
logP = log([nOc])/([H2O]
4.5. UV-Vis Stability 
The UV-Vis stability of the compounds was assessed in two different 
media: pure DMSO and a mixture of DMSO:DMEM (1:99%). The 
monitoring times for stability were set at 0, 24, and 48 h, which corre-
sponded to the same incubation times used in the cytotoxic assays. 
4.6. Cytotoxic assay 
The MRC-5 cells (non-tumor lung cells) were maintained in Dul-
becco’s Modified Eagle’s Medium (DMEM) supplemented with 10% 
heat-inactivated FBS and 80.0 μgmL–1 gentamicin, which was defined as 
the complete medium. In contrast, the A2780cis (ovarian cancer cells 
Fig. 12. Effect of complex 3 on the A2780cis cell line morphology in 24 h at ¼ × IC50, IC50, and 2 × IC50 concentrations. 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
12
resistant to cisplatin) were maintained in RPMI 1640 supplemented with 
10% heat-inactivated FBS. Then the cells were placed in plastic flasks 
(Corning) at 37 ◦C in a humidified 5% CO2 atmosphere. The number of 
cells was determined using the Trypan Blue dye exclusion method. 
Samples (150 μL) containing the cancer cells (1.5 × 104 cell mL–1) 
were added to each well of a 96-well culture plate, then they were pre- 
incubated in the absence of the compounds for 24 h to allow for the cells 
to adhere before the addition of the test agents. Subsequently, 0.750 μL 
of a solution of the compounds was added to the wells, with concen-
trations of DMSO in the range of 0.800–100 or 0.200–25.0 μmolL-1 also 
being added. The maximum concentration of DMSO in each well was 
0.5%; the same volume of complete medium culture, plus 0.5% of DMSO 
was added to be used as cell viability control. The effects of the com-
plexes on the cells were determined 48 h after the culture incubation 
when 50.0 μL of a 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium 
bromide solution (MTT; 1 mgL− 1) was added to the wells, and the plates 
were incubated for another 4 h. Their absorbances were measured in a 
microplate spectrophotometer reader (BioTek™, Epoch™) at a wave-
length of 540 nm. The IC50 values were determined graphically (the % 
cell viability was plotted against the drug concentrations [μmolL− 1] 
when using the logarithmic scale) using GraphPad Prism 6.01 software 
(GraphPad Software, San Diego, CA, USA)[50, 57, 65]. The data were 
obtained from three independent experiments. 
4.7. Spectrophotometer titration with CT-DNA 
The coordination complex concentration (2.0 × 10− 5 molL− 1) was 
kept constant throughout the absorbance titration tests, while CT-DNA 
concentrations were steadily increased in a quartz cell. The absor-
bance ratio between 260 and 280 nm of the CT-DNA spectrum was 1.83, 
showing that CT-DNA was sufficiently free of protein. After each 10-µL 
addition of DNA, the electronic absorption spectra were recorded, and 
the bands were monitored at 294–305 nm. An equal amount of DNA was 
added to both the complex and reference solutions to minimize free DNA 
absorbance before analyzing the absorption spectra. The measurements 
were performed in a Tris-HCl buffer containing 2% DMSO (5 mmolL− 1 
Tris-HCl and 50.0 mmolL− 1 NaCl pH 7.4). The intrinsic binding con-
stants, Kb, for the complex’s binding to DNA was estimated using the 
Banesi–Hildebrand method [68]. 
Equation :
[DNA]
εa − εf
=
[DNA]
εb − εf
+
1
Kb
(
εb − εf
)
Where: εa was the extinction coefficient that was observed for the 
charge-transfer absorption at a given DNA concentration; εf was the 
extinction coefficient of the complex when free in solution; εb was the 
extinction coefficient of the complex when fully bound to the DNA; Kb 
was the equilibrium binding constant; [DNA] was the concentration in 
the nucleotides. 
4.8. Circular dichroism 
The circular dichroism (CD) experiments were conducted to study 
the interaction of the complexes with ct-DNA. The CD spectra were 
recorded on a JASCO J-815 spectropolarimeter in the wavelength range 
of 235–320 nm using a continuous scanning mode at a rate of 200 nm‧ 
min-1 with 5 accumulations, all at room temperature [53]. An optical 
quartz cell with a path length of 0.5 cm was used for the measurements. 
The buffer solution used was Tris-HCl (pH = 7.4) containing 40% DMSO. 
The samples were incubated at 310 K for 24 h before recording the CD 
spectra. The fixed concentration of ct-DNA used was 80.0 μmol⋅L-1, and 
different concentrations of the complexes (ranging from 0 to 24.0 
μmol⋅L-1) were added to the ct-DNA solution. 
4.9. Topoisomerase I beta and II alpha interaction 
The DNA-topoisomerase II alpha inhibition assay was performed 
using the DNA relaxation kit supplied by Inspiralis Limited. In the assay 
performed, 0.5 µL (500 mg) of super-folded pBR322 DNA, 3.00 µL of 
assay buffer (Tris.HCl (10.0 mmolL− 1), NaCl (50.0 mmolL− 1), KCl (50.0 
mmol⋅L− 1), MgCl2 (5.00 mmolL− 1), Na2H2EDTA (0.100 mmolL− 1), BSA 
(15.0 mgmL− 1) with pH 7.9), 1.00 µL (1, 00 mmolL− 1) of ATP, 1.00 µL of 
the compound of interest at different concentrations, and finally, water 
and 1.00 µL (4.00 nmolL− 1) of TOPO II were addedto reach a final 
volume of 30.0 µL. The reaction mixture was kept in incubation at 37 ◦C 
for 40 min, after which 3 µL of sodium dodecyl sulfate (SDS) was added 
to the solution and a thermal shock was carried out at 60 ◦C for 2 min to 
interrupt the enzymatic process. After stopping the enzymatic action, 15 
µL of STEB (40% (w/v) of sucrose, 100 mmol⋅L− 1 of Tris.HCl, pH = 7.5, 
1.00 mmolL− 1 of EDTA, 0.500 mgmL− 1 of bromophenol blue) and 60.0 
µL of a mixture of chloroform: isoamyl alcohol (24:1 v/v). The samples 
were centrifuged at 5000 rpm for 5 min, then the aqueous phase was 
added to the 1% (w/v) agarose gel in a 1x TBE buffer solution (Tris/ 
Borate/EDTA), pH = 8.2. The same procedure was applied in the 
interaction with the supercoiled plasmid, the gel run, and its develop-
ment. The DNA-topoisomerase I & II inhibition assay was performed 
with the kit provided by Inspiralis Limited, using plasmid pBR322 
(supercoiled DNA), which was incubated with all complexes individu-
ally at two different concentrations, 5 and 12.5 µmolL− 1. The result 
showed the best inhibition of the enzyme was associated with com-
pounds 1, 2, and 3. 
5. Cathepsin B 
The cathepsin B assay was done using an inhibitor screening kit from 
Abcam (ab185438). A microplate white opaque was used, adding 48.0 
µL of buffer reaction, 1.00 µL of the substrate (Ac-RR-AFC), and 1.00 µL 
of cathepsin B. The plate was mixed, and finally, different concentra-
tions (12.5, 25.0, 50.0, 100, 150, 200, 250, and 300 µmolL− 1) of com-
plexes 1, 2, 3 and a commercial inhibitor of cathepsin B (F-F-FMK) were 
added in the wells to a total volume was 60.0 µL. The microplate was 
incubated on an angular shaker for 15 min at room temperature. Then, 
the fluorescence was read using a microplate reader Synergy H1 (Agi-
lent) that utilizes the fluorescence parameters of catalytic product AFC 
(Coumarin 151): Ex/Em 400/505 nm, employing the kinetic mode of the 
equipment ready at 30 and 60 min after the initial assay. The reading 
temperature was 37 ◦C. 
After the measure, it was a plot of the time versus intensity, the 
relative inhibition (I%) was calculated using the slope of the curve, S0- 
cathepsin B full action, and S - sample, through this equation: I% = 100 
× (S0-S)/S0. 
5.1. DPPH antioxidant assay 
A DPPH⋅ solution was prepared in 0.100 mM of methanol and stocks 
of solutions of complexes and ascorbic acid (positive control) in DMSO. 
The experiment was performed in 96 wells of the microplate in 100, 
50.0, 25.0, 12.5, 6.25, 3.12, 1.56, and 0 µM of the pallidium (II) com-
pounds and methanolic DPPH⋅ solution. The mix was performed in 
triplicate, and the mixtures were kept at room temperature for 30 min in 
the dark. Spectrophotometric measurements were done at 515 nm using 
a microplate spectrophotometer reader (BioTek™, Epoch™). The scav-
enging effects of the DPPH· was calculated using the following equation 
[57]: 
DPPH⋅ scavengingeffects (%) = [(A − A0) /A0] × 100 
5.2. Human serum albumin (HSA) binding studies 
To investigate the interaction of complexes 1–3 with human serum 
A.O. Akinyemi et al. 
Journal of Molecular Structure 1294 (2023) 136460
13
albumin (HSA), various concentrations of the compounds (ranging from 
0 to 30 µM) were added to a solution containing 2.5 µM of HSA in 
Trisma-HCl buffer at pH = 7.4. The concentration of HSA was deter-
mined using the molar absorption coefficient at 280 nm. Subsequently, 
200 µL of the solutions were transferred into an opaque 96-well plate for 
analysis [69]. The measurements were carried out using a Synergy H1 
(BioTek) fluorimeter at three different temperatures: 293, 298, and 303 
K. The experiment started with excitation at 270 nm, and the emission 
was recorded at 305 nm. The compounds did not show any emission 
spectra in the wavelength range of 305–500 nm. 
Author contributions 
Amos O. Akinyemi and Vinicius A. Costa worked on the synthesis and 
characterization of the compounds; Karine Zanotti and Moacir Rossi 
Forim, spectrometry and spectroscopy characterizations; Josias S. Rocha 
and George performed the biomolecules interactions assays; Gabriela P. 
Oliveira, Mauro A. Lima, and Dario B. Fortaleza carried out the MTT 
assay; Tamara Teixeira carried out the HSA and circular dichroism 
assay; João H. Araujo-Neto diffracted and solved the crystal structure, 
further analyzed by Eduardo E. Castellano (refinement of the crystal 
structure); Amos O. A. wrote the manuscript with the support of F. V. 
Rocha, who supervised the project. All authors discussed the results and 
contributed to the final manuscript. 
Funding 
Please add: We thank the Brazilian funding agencies: Conselho 
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant 
#146769/2018-0, Coordenação de Aperfeiçoamento de Pessoal de Nível 
Superior (CAPES) Finance Code 001, and Fundação de Amparo à Pes-
quisa do Estado de São Paulo (FAPESP 2019/11242-1, 2022/02876- 
0,17/15840-0 and 21/04876-4) for financial support. 
Supplementary materials 
The attached document contains crucial data that support the main 
text, including all spectral figures and attributions (vibrational, elec-
tronic, 1H NMR, 31P NMR spectroscopies, and mass spectrometry). In 
addition, the description of solid structure analysis by x-ray diffraction 
incorporates the checkcif of Complex 1 and the Hirshfeld surface. 
Declaration of Competing Interest 
The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper. 
Data availability 
Data will be made available on request. 
Supplementary materials 
Supplementary material associated with this article can be found, in 
the online version, at doi:10.1016/j.molstruc.2023.136460. 
References 
[1] J. Ni, et al., Extralysosomal cathepsin B in central nervous system: Mechanisms and 
therapeutic implications, Brain Pathol. 32 (5) (2022) e13071. 
[2] A. Sharma, et al., Cathepsin B-A neuronal death mediator in Alzheimer’s disease 
leading to neurodegeneration, Mini Rev. Med. Chem. 22 (15) (2022) 2012–2023. 
[3] M. Fonović, B. Turk, Cysteine cathepsins and extracellular matrix degradation, 
Biochim. Biophys. Acta 1840 (8) (2014) 2560–2570. 
[4] F. Kuang, et al., Cathepsin B is a mediator of organelle-specific initiation of 
ferroptosis, Biochem. Biophys. Res. Commun. 533 (4) (2020) 1464–1469. 
[5] X. Ding, et al., Cathepsin B is a potential therapeutic target for coronavirus disease 
2019 patients with lung adenocarcinoma, Chem. Biol. Interact. 353 (2022), 
109796. 
[6] S.P. Fricker, Cysteine proteases as targets for metal-based drugs, Metallomics 2 (6) 
(2010) 366–377. 
[7] A.O. Akinyemi, G.B.S. Pereira, F.V. Rocha, Role of cathepsin B in cancer 
progression: a potential target for coordination compounds, Mini Rev. Med. Chem. 
21 (13) (2021) 1612–1624. 
[8] Y. Zhu, et al., Inhibition of the cathepsin cysteine proteases B and K by square- 
planar cycloaurated gold(III) compounds and investigation of their anti-cancer 
activity, J. Inorg. Biochem. 105 (5) (2011) 754–762. 
[9] S.S. Gunatilleke, A.M. Barrios, Tuning the Au(I)-mediated inhibition of cathepsin B 
through ligand substitutions, J. Inorg. Biochem. 102 (3) (2008) 555–563. 
[10] A. Casini, et al., Emerging protein targets for anticancer metallodrugs: inhibition of 
thioredoxin reductase and cathepsin B by antitumor ruthenium (II)− arene 
compounds, J. Med. Chem. 51 (21) (2008) 6773–6781. 
[11] R. Mosi, et al., Rhenium inhibitors of cathepsin B (ReO(SYS)X (where Y = S, py; X 
= Cl, Br, SPhOMe-p)): Synthesis and mechanism of inhibition, J. Med. Chem. 49 
(17) (2006) 5262–5272. 
[12] A. Mitrović, et al., Clioquinol-ruthenium complex impairs tumour cell invasion by 
inhibitingcathepsin B activity, Dalton Trans. 45 (42) (2016) 16913–16921. 
[13] A. Mitrović, et al., Organoruthenated nitroxoline derivatives impair tumor cell 
invasion through inhibition of cathepsin B activity, Inorg. Chem. 58 (18) (2019) 
12334–12347. 
[14] L. Oehninger, et al., Evaluation of arene ruthenium(II) N-heterocyclic carbene 
complexes as organometallics interacting with thiol and selenol containing 
biomolecules, Dalton Trans. 42 (5) (2013) 1657–1666. 
[15] M.K. Amir, et al., Anticancer activity, DNA-binding and DNA-denaturing aptitude 
of palladium (II) dithiocarbamates, Inorg. Chim. Acta 451 (2016) 31–40. 
[16] T. Zou, et al., Anticancer metal-N-heterocyclic carbene complexes of gold, 
platinum and palladium, Curr. Opin. Chem. Biol. 43 (2018) 30–36. 
[17] W.M. Motswainyana, et al., Imino-phosphine palladium(II) and platinum(II) 
complexes: synthesis, molecular structures and evaluation as antitumor agents, 
J. Inorg. Biochem. 129 (2013) 112–118. 
[18] A.R. Katritzky, D.L. Ostercamp, T.I. Yousaf, The mechanisms of heterocyclic ring 
closures, Tetrahedron 43 (22) (1987) 5171–5186. 
[19] T.R. De Moura, et al., Palladium (ii) complexes bearing 1-iminothiolate-3, 5- 
dimethylpyrazoles: synthesis, cytotoxicity, DNA binding and enzymatic inhibition 
studies, New J. Chem. 44 (45) (2020) 19891–19901. 
[20] S.J. Berners-Price, P.J. Sadler, Phosphines and metal phosphine complexes: 
relationship of chemistry to anticancer and other biological activity. Bioinorganic 
Chemistry, Springer, 2005, pp. 27–102. 
[21] C. Icsel, et al., Trans-Pd/Pt (II) saccharinate complexes with a phosphine ligand: 
Synthesis, cytotoxicity and structure-activity relationship, Bioorg. Med. Chem. 
Lett. 30 (9) (2020), 127077. 
[22] S.L. Queiroz, A. Batista, Complexos fosfínicos e suas aplicações na medicina, 
Química Nova 19 (6) (1996) 651–659. 
[23] F. You, C. Gao, Topoisomerase Inhibitors and Targeted Delivery in Cancer Therapy, 
Curr. Top Med. Chem. 19 (9) (2019) 713–729. 
[24] A.K. Jadhav, S.M. Karuppayil, Molecular docking studies on thirteen 
fluoroquinolines with human topoisomerase II a and b, In Silico Pharmacol. 5 (1) 
(2016) 4. 
[25] S.M. Vos, et al., All tangled up: how cells direct, manage and exploit topoisomerase 
function, Nat. Rev. Mol. Cell Biol. 12 (12) (2011) 827–841. 
[26] V. Vutey, et al., Human topoisomerase IB is a target of a thiosemicarbazone copper 
(II) complex, Arch. Biochem. Biophys. 606 (2016) 34–40. 
[27] C.G. Oliveira, et al., Palladium(ii) complexes with thiosemicarbazones derived 
from pyrene as topoisomerase IB inhibitors, Dalton Trans. 48 (44) (2019) 
16509–16517. 
[28] F.V. Rocha, et al., N-Methyl-2-(1-methyl-3-phenylprop-2-en-1-ylidene) 
hydrazinecarbothioamide, Acta Crystallogr. Sect. E Struct. Rep. Online 70 (7) 
(2014) o800. 
[29] A.M. Bego, et al., Immunomodulatory effects of palladium (II) complexes of 1, 2, 4- 
triazole on murine peritoneal macrophages, J. Braz. Chem. Soc. 20 (2009) 
437–444. 
[30] H.A. Mohamad, et al., Novel palladium (II) complex derived from mixed ligands of 
dithizone and triphenylphosphine synthesis, characterization, crystal structure, 
and DFT study, Bull. Chem. Soc. Ethiop. 36 (3) (2022) 617–626. 
[31] F.V. Rocha, et al., Computational studies, design and synthesis of Pd(II)-based 
complexes: allosteric inhibitors of the human topoisomerase-IIα, J. Inorg. Biochem. 
199 (2019), 110725. 
[32] P.I.d.S. Maia, et al., Palladium (II) complexes with thiosemicarbazones: syntheses, 
characterization and cytotoxicity against breast cancer cells and anti- 
mycobacterium tuberculosis activity, J. Braz. Chem. Soc. 21 (2010) 1177–1186. 
[33] W. Hernández, et al., Synthesis and characterization of new palladium(II) 
thiosemicarbazone complexes and their cytotoxic activity against various human 
tumor cell lines, Bioinorg. Chem. Appl. 2013 (2013), 524701. 
[34] J.L. Burmeister, F. Basolo, Inorganic linkage isomerism of the thiocyanate ion, 
Inorg. Chem. 3 (11) (1964) 1587–1593. 
[35] R. Prabhakaran, et al., Topoisomerase II inhibition activity of new square planar Ni 
(II) complexes containing N-substituted thiosemicarbazones: Synthesis, 
spectroscopy, X-ray crystallography and electrochemical characterization, Inorg. 
Chim. Acta 374 (1) (2011) 647–653. 
A.O. Akinyemi et al. 
https://doi.org/10.1016/j.molstruc.2023.136460
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0001
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0001
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0002
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0002
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0003
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0003
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0004
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0004
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0005
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0005
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0005
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0006
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0006
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0007
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0007
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0007
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0008
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0008
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0008
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0009
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0009
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0010
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0010
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0010
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0011
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0011
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0011
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0012
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0012
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0013
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0013
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0013
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0014
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0014
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0014
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0015
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0015
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0016
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0016
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0017
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0017
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0017
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0018
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0018
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0019
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0019
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0019
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0020
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0020
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0020
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0021
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0021
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0021
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0022
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0022
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0023
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0023
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0024
http://refhub.elsevier.com/S0022-2860(23)01550-8/sbref0024