Complex formation of ML324, the histone demethylase inhibitor, with essential metal ions: Relationship between solution chemistry and anticancer activity.

N-(3-(dimethylamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide (ML324, HL) is a potent inhibitor of the iron-containing histone demethylase KDM4, a recognized potential target of cancer therapeutics. Herein, we report the proton dissociation and complex formation processes of ML324 with essential metal ions such as Fe(II), Fe(III), Cu(II) and Zn(II) using UV-visible, fluorescence, electron paramagnetic resonance and 1H NMR spectroscopic methods. The electrochemical behaviour of the copper and iron complexes was characterized by cyclic voltammetry and spectroelectrochemistry. The solid phase structure of ML324 analysed by X-ray crystallography is also provided. Based on the solution equilibrium data, ML324 is present in solution in H2L+ form with a protonated dimethylammonium moiety at pH 7.4, and this (N,O) donor bearing ligand forms mono and bis complexes with all the studied metal ions and the tris-ligand species is also observed with Fe(III). At pH 7.4 the metal binding ability of ML324 follows the order: Fe(II) < Zn(II) < Cu(II) < Fe(III). Complexation with iron resulted in a negative redox potential (E'1/2 = -145 mV vs. NHE), further suggesting that the ligand has a preference for Fe(III) over Fe(II). ML324 was tested for its anticancer activity in chemosensitive and resistant human cancer cells overexpressing the efflux pump P-glycoprotein. ML324 exerted similar activity in all tested cells (IC50 = 1.9-3.6 µM). Co-incubation and complexation of the compound with Cu(II) and Zn(II) had no impact on the cytotoxicity of ML324, whereas Fe(III) decreased the toxicity in a concentration-dependent manner, and this effect was more pronounced in the multidrug resistant cells.

A Comparative Study on the Complexation of the Anticancer Iron Chelator VLX600 with Essential Metal Ions.

As cancer cells exhibit an increased uptake of iron, targeting the interaction with iron has become a straightforward strategy in the fight against cancer. This work comprehensively characterizes the chemical properties of 6-methyl-3-{(2E)-2-[1-(2-pyridinyl)ethylidene]hydrazino}-5H-[1,2,4]triazino[5,6-b]indole (VLX600), a clinically investigated iron chelator, in solution. Its protonation processes, lipophilicity, and membrane permeability as well as its complexation with essential metal ions were investigated using UV-visible, electron paramagnetic resonance, and NMR spectroscopic and computational methods. Formation constants revealed the following order of metal binding affinity at pH 7.4: Cu(II) > Fe(II) > Zn(II). The structures of VLX600 (denoted as HL) and the coordination modes in its metal complexes [Cu(II)(LH)Cl2], [Cu(II)(L)(CH3OH)Cl], [Zn(II)(LH)Cl2], and [Fe(II)(LH)2](NO3)2 were elucidated by single-crystal X-ray diffraction. Redox properties of the iron complexes characterized by cyclic voltammetry showed strong preference of VLX600 toward Fe(II) over Fe(III). In vitro cytotoxicity of VLX600 was determined in six different human cancer cell lines, with IC50 values ranging from 0.039 to 0.51 µM. Premixing VLX600 with Fe(III), Zn(II), and Cu(II) salts in stoichiometric ratios had a rather little effect overall, thus neither potentiating nor abolishing cytotoxicity. Together, although clinically investigated as an iron chelator, this is the first comprehensive solution study of VLX600 and its interaction with physiologically essential metal ions.

Structural and solution speciation studies on selected [Cu(NN)(OO)] complexes and an investigation of their biomimetic activity, ROS generation and their cytotoxicity in normoxic, hypoxic and anoxic environments in MCF-7 breast cancer-derived cells.

Reactive oxygen species(ROS) generation with subsequent DNA damage is one of the principle mechanisms of action assigned to copper-based anticancer complexes. The efficacy of this type of chemotherapeutic may be reduced in the low oxygen environment of tumours. In this study the cytotoxicity of three complexes, [Cu(dips)(phen)] (1), [Cu(ph)(phen)]·2H2O (2) and [Cu(ph)(bpy)]·H2O (3) (disp: 3,5-diisopropylsalicylate, phen: 1,10- phenanthroline, ph: phthalate, bpy: 2,2'-bipyridyl) were assessed for anticancer activity in the breast-cancer derived MCF-7 line under normoxic, hypoxic and anoxic conditions. In an immortalised keratinocyte HaCaT cell line, the cytotoxicity of complexes 2 and 3 was significantly reduced under both normoxic and hypoxic conditions, whilst the cytotoxicity of complex 1 was increased under hypoxic conditions. The ability of the complexes to generate ROS in the MCF-7 cell line was evaluated as was their ability to act as superoxide dismutase(SOD) and catalase mimics using a yeast cell assay. ROS generation was significant for complexes 2 and 3, less so for complex 1 though all three complexes had SOD mimetic ability. Given the ternary nature of the complexes, solution speciation studies were undertaken but were only successful for complex 3, due to solubility issues with the other two complexes. The concentration distribution of various species, formed in aqueous solution, was evaluated as a function of pH and confirmed that complex 3 is the dominant species at physiological pH in the mM concentration range. However, as its concentration diminishes, it experiences a progressive dissociation, leading to the formation of binary complexes of bpy alongside unbound phthalate.

Cu(II) and Zn(II) Complexes of New 8-Hydroxyquinoline Schiff Bases: Investigating Their Structure, Solution Speciation, and Anticancer Potential.

We report the synthesis and characterization of three novel Schiff bases (L1-L3) derived from the condensation of 2-carbaldehyde-8-hydroxyquinoline with amines containing morpholine or piperidine moieties. These were reacted with CuCl2 and ZnCl2 yielding six new coordination compounds, with the general formula ML2, where M = Cu(II) or Zn(II) and L = L1-L3, which were all characterized by analytical, spectroscopic (Fourier transform infrared (FTIR), UV-visible absorption, nuclear magnetic resonance (NMR), or electron paramagnetic resonance (EPR)), and mass spectrometric techniques, as well as by single-crystal X-ray diffraction. In the solid state, two Cu(II) complexes, with L1 and L2, are obtained as dinuclear compounds, with relatively short Cu-Cu distances (3.146 and 3.171 Å for Cu2(L1)4 and Cu2(L2)4, respectively). The free ligands show moderate lipophilicity, while their complexes are more lipophilic. The pKa values of L1-L3 and formation constants of the complex (for ML and ML2) species were determined by spectrophotometric titrations, with the Cu(II) complexes showing higher stability than the Zn(II) complexes. EPR indicated the presence of several species in solution as pH varied and binding modes were proposed. The binding of the complexes to bovine serum albumin (BSA) was evaluated by fluorescence and circular dichroism (CD) spectroscopies. All complexes bind BSA, and as demonstrated by CD, the process takes several hours to reach equilibrium. The antiproliferative activity was evaluated in malignant melanoma cells (A375) and in noncancerous keratinocytes (HaCaT). All complexes display significant cytotoxicity (IC50 < 10 µM) but modest selectivity. The complexes show higher activity than the free ligands, the Cu(II) complexes being more active than the Zn(II) complexes, and approximately twice more cytotoxic than cisplatin. A Guava ViaCount assay corroborated the antiproliferative activity.

Peptide-based chemical models for lytic polysaccharide monooxygenases.

Copper(II) complexes of HPH-NH2 (L1) and HPHPY-NH2 (L2) peptides have been studied as small molecular models of lytic polysaccharide monooxygenases by pH-potentiometry and UV-vis, CD and EPR spectroscopy. The coordination properties of these ligands are fundamentally different from those of other non-protected N-terminal HXH-sequences concerning the metal binding ability of amide nitrogens. The proline units prevent the formation of fused chelates with the participation of amide nitrogens; therefore, instead of ATCUN-type {NH2,2N-,Nim} coordination, dimer complexes (Cu2HxL2, where x = -1, -2, and -3 for L1 and 1, 0, and -1 for L2) are formed in equimolar systems above pH 5. Using H2O2 as the oxidant and PNPG as the activated substrate, these dimer complexes were proved to be relevant functional models of LPMOs, even at neutral pH. Although the tyrosine residue in L2 participates in the coordination at pH 7-9.6, it does not seem to play a role in the oxidation process. In the presence of H2O2, the dimer complexes partially dissociate to form mononuclear hydroperoxo complexes, which are stable for 1-2 hours in equimolar concentrations of H2O2. On the other hand, with excess H2O2 both their formation and their decomposition are faster. The decay of (hydro)peroxo complexes, after longer reaction times, results in the evolution of dioxygen bubbles and the formation of Cu(I) (probably through catalytic disproportionation). However, in the presence of PNPG, the formation of dioxygen bubbles was not observed. Therefore, we assumed that the formed Cu(I) complexes bind H2O2 and enter into a similar catalytic cycle as suggested recently for native LPMOs.

Thiosemicarbazone Derivatives Developed to Overcome COTI-2 Resistance.

COTI-2 is currently being evaluated in a phase I clinical trial for the treatment of gynecological and other solid cancers. As a thiosemicarbazone, this compound contains an N,N,S-chelating moiety and is, therefore, expected to bind endogenous metal ions. However, besides zinc, the metal interaction properties of COTI-2 have not been investigated in detail so far. This is unexpected, as we have recently shown that COTI-2 forms stable ternary complexes with copper and glutathione, which renders this drug a substrate for the resistance efflux transporter ABCC1. Herein, the complex formation of COTI-2, two novel terminal N-disubstituted derivatives (COTI-NMe2 and COTI-NMeCy), and the non-substituted analogue (COTI-NH2) with iron, copper, and zinc ions was characterized in detail. Furthermore, their activities against drug-resistant cancer cells was investigated in comparison to COTI-2 and Triapine. These data revealed that, besides zinc, also iron and copper ions need to be considered to play a role in the mode of action and resistance development of these thiosemicarbazones. Moreover, we identified COTI-NMe2 as an interesting new drug candidate with improved anticancer activity and resistance profile.

Solution Equilibrium Studies on Salicylidene Aminoguanidine Schiff Base Metal Complexes: Impact of the Hybridization with L-Proline on Stability, Redox Activity and Cytotoxicity.

The proton dissociation processes of two tridentate salicylidene aminoguanidine Schiff bases (SISC, Pro-SISC-Me), the solution stability and electrochemical properties of their Cu(II), Fe(II) and Fe(III) complexes were characterized using pH-potentiometry, cyclic voltammetry and UV-visible, 1H NMR and electron paramagnetic resonance spectroscopic methods. The structure of the proline derivative (Pro-SISC-Me) was determined by X-ray crystallography. The conjugation of L-proline to the simplest salicylidene aminoguanidine Schiff base (SISC) increased the water solubility due to its zwitterionic structure in a wide pH range. The formation of mono complexes with both ligands was found in the case of Cu(II) and Fe(II), while bis complexes were also formed with Fe(III). In the complexes these tridentate ligands coordinate via the phenolato O, azomethine N and the amidine N, except the complex [Fe(III)L2]+ of Pro-SISC-Me in which the (O,N) donor atoms of the proline moiety are coordinated beside the phenolato O, confirmed by single crystal X-ray crystallographic analysis. This binding mode yielded a stronger Fe(III) preference for Pro-SISC-Me over Fe(II) in comparison to SISC. This finding is also reflected in the lower redox potential value of the iron-Pro-SISC-Me complexes. The ligands alone were not cytotoxic against human colon cancer cell lines, while complexation of SISC with Cu(II) resulted in moderate activity, unlike the case of its more hydrophilic counterpart.

Copper(II) Complexes of Pyridine-2,6-dicarboxamide Ligands with High SOD Activity.

Copper(II) complexes of pyridine-based ligands functionalized with alanine (PydiAla) and tyrosine (PydiTyr) moieties have been synthesized as novel superoxide dismutase mimics. The complexes were characterized by pH-potentiometric, spectroscopic (UV-vis, circular dichroism, mass spectrometry, electron paramagnetic resonance spectroscopy), computational (DFT), and X-ray diffraction methods. Both ligands form high stability copper(II) complexes via the (Npy,N-,N-) donor set supported by the binding of the carboxylate pendant arms. Although the coordination mode is the same for the two systems, the tyrosine containing counterpart exhibits increased copper(II) binding affinity, which is most likely due to the presence of the aromatic moiety of the side chains. Both copper(II) complexes are capable of binding N-methylimidazole, and the formation of the corresponding ternary species was observed at physiological pH. The binary and ternary copper(II) complexes exhibit high SOD activity. The PydiTyr complex exhibits about 1 order of magnitude higher activity than the PydiAla complex. This is probably due to the presence of the phenolic OH group in the former species, which promotes the binding of the superoxide anion radical to the metal center. The results serve as a basis for designing highly efficient copper(II) mimics for medical and practical applications.

Metal Complexes of a 5-Nitro-8-Hydroxyquinoline-Proline Hybrid with Enhanced Water Solubility Targeting Multidrug Resistant Cancer Cells.

Multidrug resistance (MDR) in cancer is one of the major obstacles of chemotherapy. We have recently identified a series of 8-hydroxyquinoline Mannich base derivatives with MDR-selective toxicity, however with limited solubility. In this work, a novel 5-nitro-8-hydroxyquinoline-proline hybrid and its Rh(η5-C5Me5) and Ru(η6-p-cymene) complexes with excellent aqueous solubility were developed, characterized, and tested against sensitive and MDR cells. Complex formation of the ligand with essential metal ions was also investigated using UV-visible, circular dichroism, 1H NMR (Zn(II)), and electron paramagnetic resonance (Cu(II)) spectroscopic methods. Formation of mono and bis complexes was found in all cases with versatile coordination modes, while tris complexes were also formed with Fe(II) and Fe(III) ions, revealing the metal binding affinity of the ligand at pH 7.4: Cu(II) > Zn(II) > Fe(II) > Fe(III). The ligand and its Rh(III) complex displayed enhanced cytotoxicity against the resistant MES-SA/Dx5 and Colo320 human cancer cell lines compared to their chemosensitive counterparts. Both organometallic complexes possess high stability in solution, however the Ru(II) complex has lower chloride ion affinity and slower ligand exchange processes, along with the readiness to lose the arene ring that is likely connected to its inactivity.

Estradiol-Based Salicylaldehyde (Thio)Semicarbazones and Their Copper Complexes with Anticancer, Antibacterial and Antioxidant Activities.

A series of novel estradiol-based salicylaldehyde (thio)semicarbazones ((T)SCs) bearing (O,N,S) and (O,N,O) donor sets and their Cu(II) complexes were developed and characterized in detail by 1H and ¹³C nuclear magnetic resonance spectroscopy, UV-visible and electron paramagnetic resonance spectroscopy, electrospray ionization mass spectrometry and elemental analysis. The structure of the Cu(II)-estradiol-semicarbazone complex was revealed by X-ray crystallography. Proton dissociation constants of the ligands and stability constants of the metal complexes were determined in 30% (v/v) DMSO/H2O. Estradiol-(T)SCs form mono-ligand complexes with Cu(II) ions and exhibit high stability with the exception of estradiol-SC. The Cu(II) complexes of estradiol-TSC and its N,N-dimethyl derivative displayed the highest cytotoxicity among the tested compounds in MCF-7, MCF-7 KCR, DU-145, and A549 cancer cells. The complexes do not damage DNA according to both in vitro cell-free and cellular assays. All the Cu(II)-TSC complexes revealed significant activity against the Gram-positive Staphylococcus aureus bacteria strain. Estradiol-TSCs showed efficient antioxidant activity, which was decreased by complexation with Cu(II) ions. The exchange of estrone moiety to estradiol did not result in significant changes to physico-chemical and biological properties.

8-Hydroxyquinoline-Amino Acid Hybrids and Their Half-Sandwich Rh and Ru Complexes: Synthesis, Anticancer Activities, Solution Chemistry and Interaction with Biomolecules.

Solution chemical properties of two novel 8-hydroxyquinoline-D-proline and homo-proline hybrids were investigated along with their complex formation with [Rh(η5-C5Me5)(H2O)3]2+ and [Ru(η6-p-cymene)(H2O)3]2+ ions by pH-potentiometry, UV-visible spectrophotometry and 1H NMR spectroscopy. Due to the zwitterionic structure of the ligands, they possess excellent water solubility as well as their complexes. The complexes exhibit high solution stability in a wide pH range; no significant dissociation occurs at physiological pH. The hybrids and their Rh(η5-C5Me5) complexes displayed enhanced cytotoxicity in human colon adenocarcinoma cell lines and exhibited multidrug resistance selectivity. In addition, the Rh(η5-C5Me5) complexes showed increased selectivity to the chemosensitive cancer cells over the normal cells; meanwhile, the Ru(η6-p-cymene) complexes were inactive, most likely due to arene loss. Interaction of the complexes with human serum albumin (HSA) and calf-thymus DNA (ct-DNA) was investigated by capillary electrophoresis, fluorometry and circular dichroism. The complexes are able to bind strongly to HSA and ct-DNA, but DNA cleavage was not observed. Changing the five-membered proline ring to the six-membered homoproline resulted in increased lipophilicity and cytotoxicity of the Rh(η5-C5Me5) complexes while changing the configuration (L vs. D) rather has an impact on HSA or ct-DNA binding.

Estrone-salicylaldehyde N-methylated thiosemicarbazone hybrids and their copper complexes: solution structure, stability and anticancer activity in tumour spheroids.

The terminal N-mono- and dimethylated derivatives of an estrone-salicylaldehyde thiosemicarbazone hybrid and their highly cytotoxic Cu(II) complexes were synthesized and characterized in addition to their structurally related simpler bicyclic analogues. Solution stability and structure of the complexes were determined by UV-visible spectrophotometry and electron paramagnetic resonance spectroscopy. The monomethylation has a minor influence on the pKa values, while the dimethylation results in somewhat more acidic derivatives compared to the non-methylated derivatives, although all the compounds are neutral at physiological pH. Based on the speciation studies performed in a 30% (v/v) dimethyl sulfoxide/water mixture, the four novel ligands form fairly high-stability complexes with Cu(II) ions, in which they coordinate in mono-anionic (O‒,N,S) or di-anionic (O‒,N,S‒) binding modes. [CuLH‒1] species with (O‒,N,S‒)(H2O) coordination mode are present in solution at neutral pH, and these complexes were isolated and further studied. The Cu(II) complexes formed with the estrone hybrids were more stable in comparison with the bicyclic analogues. The terminal N-dimethylation results in the most stable complexes in a given ligand series. In vitro cytotoxicity of all the Cu(II) complexes was measured in 3D spheroids of HCT-116, A-549 and CH-1 human cancer cells which showed fairly low IC50 values (3.9‒17.1 µM). The Cu(II) complexes caused reduced tumour growth, and they activated the caspase-3 and caspase-7 endoproteases leading to apoptosis except the case of the complex formed with the monomethylated bicyclic derivative, where other type of mechanisms of action seems to induce the cell death. Anticancer Cu(II) complexes of mono- and dimethylated salicylaldehyde thiosemicarbazone-estrone hybrids possessing high solution stability and strong cytotoxic effect against 3D spheroids of a series of human cancer cells. 398x273 mm (150 x 150 DPI).

Copper(II) Complexes of Sulfonated Salan Ligands: Thermodynamic and Spectroscopic Features and Applications for Catalysis of the Henry Reaction.

Copper(II) complexes formed with sulfonated salan ligands (HSS) have been synthesized, and their coordination chemistry has been characterized using pH-potentiometry and spectroscopic methods [UV-vis, electron paramagnetic resonance (EPR), and electron-electron double resonance (ELDOR)-detected NMR (EDNMR)] in aqueous solution. Several bridging moieties between the two salicylamine functions were introduced, e.g., ethyl (HSS), propyl (PrHSS), butyl (BuHSS), cyclohexyl (cis-CyHSS, trans-CyHSS), and diphenyl (dPhHSS). All of the investigated ligands feature excellent copper(II) binding ability via the formation of a (O-,N,N,O-) chelate system. The results indicated that the cyclohexyl moiety significantly enhances the stability of the copper(II) complexes. EPR studies revealed that the arrangement of the coordinated donor atoms is more symmetrical around the copper(II) center and similar for HSS, BuHSS, CyHSS, and dPhHSS, respectively, and a higher rhombicity of the g tensor was detected for PrHSS. The copper(II) complexes of the sulfosalan ligands were isolated in solid form also and showed moderate catalytic activity in the Henry (nitroaldol) reaction of aldehydes and nitromethane. The best yield for nitroaldol production was obtained for copper(II) complexes of PrHSS and BuHSS, although their metal binding ability is moderate compared to that of the cyclohexyl counterparts. However, these complexes possess larger spin density on the nitrogen nuclei than that for the other cases, which alters their catalytic activity.

Copper Coordination Chemistry of Sulfur Pendant Cyclen Derivatives: An Attempt to Hinder the Reductive-Induced Demetalation in 64/67Cu Radiopharmaceuticals.

The Cu2+ complexes formed by a series of cyclen derivatives bearing sulfur pendant arms, 1,4,7,10-tetrakis[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO4S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (DO3S), 1,4,7-tris[2-(methylsulfanyl)ethyl]-10-acetamido-1,4,7,10-tetraazacyclododecane (DO3SAm), and 1,7-bis[2-(methylsulfanyl)ethyl]-4,10-diacetic acid-1,4,7,10-tetraazacyclododecane (DO2A2S), were studied in aqueous solution at 25 °C from thermodynamic and structural points of view to evaluate their potential as chelators for copper radioisotopes. UV-vis spectrophotometric out-of-cell titrations under strongly acidic conditions, direct in-cell UV-vis titrations, potentiometric measurements at pH >4, and spectrophotometric Ag+-Cu2+ competition experiments were performed to evaluate the stoichiometry and stability constants of the Cu2+ complexes. A highly stable 1:1 metal-to-ligand complex (CuL) was found in solution at all pH values for all chelators, and for DO2A2S, protonated species were also detected under acidic conditions. The structures of the Cu2+ complexes in aqueous solution were investigated by UV-vis and electron paramagnetic resonance (EPR), and the results were supported by relativistic density functional theory (DFT) calculations. Isomers were detected that differed from their coordination modes. Crystals of [Cu(DO4S)(NO3)]·NO3 and [Cu(DO2A2S)] suitable for X-ray diffraction were obtained. Cyclic voltammetry (CV) experiments highlighted the remarkable stability of the copper complexes with reference to dissociation upon reduction from Cu2+ to Cu+ on the CV time scale. The Cu+ complexes were generated in situ by electrolysis and examined by NMR spectroscopy. DFT calculations gave further structural insights. These results demonstrate that the investigated sulfur-containing chelators are promising candidates for application in copper-based radiopharmaceuticals. In this connection, the high stability of both Cu2+ and Cu+ complexes can represent a key parameter for avoiding in vivo demetalation after bioinduced reduction to Cu+, often observed for other well-known chelators that can stabilize only Cu2+.

Binding Models of Copper(II) Thiosemicarbazone Complexes with Human Serum Albumin: A Speciation Study.

Copper(II) complexes of thiosemicarbazones (TSCs) often exhibit anticancer properties, and their pharmacokinetic behavior can be affected by their interaction with blood transport proteins. Interaction of copper(II) complexes of an {N,N,S} donor α-N-pyridyl TSC (Triapine) and an {O,N,S} donor 2-hydroxybenzaldehyde TSC (STSC) with human serum albumin (HSA) was investigated by UV-visible and electron paramagnetic resonance spectroscopy at physiological pH. Asp-Ala-His-Lys and the monodentate N-methylimidazole were also applied as binding models. Conditional formation constants were determined for the ternary copper(II)-TSC complexes formed with HSA, DAHK, and N-methylimidazole based on the spectral changes of both charge transfer and d-d bands. The neutral N-methylimidazole displays a similar binding affinity to both TSC complexes. The partially negatively charged tetrapeptide binds stronger to the positively charged Triapine complex in comparison to the neutral STSC complex, while the opposite trend was observed for HSA, which demonstrates the limitations of the use of simple ligands to model the protein binding. The studied TSC complexes are able to bind to HSA in a fast process, and the conditional constants suggest that their binding strength is only weak-to-moderate.

Complex formation of an estrone-salicylaldehyde semicarbazone hybrid with copper(II) and gallium(III): Solution equilibria and biological activity.

The solution chemical properties such as proton dissociation, complex formation with copper(II) and gallium(III) ions in addition to antibacterial and antitumor activity of a novel tridentate salicyaldehyde semicarbazone-estrone hybrid (estrone-SC) and a related bicyclic compound (thn-SC) were investigated. The crystal structure of complex [Cu(thn-SCH-1)Cl] was studied by single crystal X-ray diffraction method. Estrone-SC and thn-SC form mono-ligand complexes with Cu(II) characterized by relatively high stability, however, they are much less stable than their thiosemicarbazone analogues. The neutral Cu(II) complexes with (O-,N,O-)(H2O) coordination mode predominate at physiological pH. Estrone-SC and thn-SC are more efficient Ga(III) binders in comparison with thiosemicarbazones, although the complexes also suffer dissociation at pH 7.4. The Cu(II) complex of estrone-SC displayed significant cytotoxicity in A549, SW480 and CH1/PA cancer cells, and moderate apoptosis induction and ROS formation. The semicarbazone compounds did not exhibit antibacterial effect; unlike the related Cu(II)-thiosemicarbazone complexes represented by the fairly low MIC values (3-50 µM) obtained on the Gram-positive Staphylococcus aureus and Enterococcus faecalis bacteria.

Half-sandwich organometallic Ru and Rh complexes of (N,N) donor compounds: effect of ligand methylation on solution speciation and anticancer activity.

A series of half-sandwich polypyridyl complexes was synthesized and compared focusing on structural, cytotoxic and aqueous solution behaviour. The formula of the synthesized complexes is [M(arene)(N,N)Cl]Cl, where M: Ru or Rh, arene: p-cymene, toluene or C5Me5-, (N,N): 2,2'-bipyridine (bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 1,10-phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (neo). The structures of five half-sandwich complexes were determined by X-ray crystallography. It was found that introducing methyl groups next to the coordinating nitrogen atoms of the bidentate ligand causes steric congestion around the metal centre which changes the angle between ligand planes. The ligands and the Rh complexes showed significant cytotoxicity in A2780 and MES-SA cancer cell lines (IC50 = 0.1-56 µM) and in the cisplatin-resistant A2780cis cells. Paradoxically, phen and dmb as well as their half-sandwich Rh complexes showed increased toxicity against multidrug resistant MES-SA/Dx5 cells. In contrast, coordination to Ru caused loss of toxicity. Solution equilibrium constants showed that the studied metal complexes have high stability, and no dissociation was found for Ru and Rh complexes even at micromolar concentrations in a wide pH range. However, in the case of Ru complexes a slow and irreversible decomposition, namely arene loss, was also observed, which was more pronounced in light exposure in aqueous solution. In the case of neo, the methyl groups next to the nitrogen atoms significantly decrease the stability of complexes. For Rh complexes, the order of the stability constants corrected with ligand basicity (log K*): 9.78 (phen) > 9.01 (dmb) > 8.89 (bpy) > 3.93 (neo). The coordinated neo resulted in an enormous decrease in the chloride ion affinity of Ru compounds. Based on the results, a universal model was introduced for the prediction of chloride ion capability of half-sandwich Rh and Ru complexes. It combines the effects of the bidentate ligand and the M(arene) part using only two terms, performing multilinear regression procedure.

Synthesis of dihydrotestosterone derivatives modified in the A-ring with (hetero)arylidene, pyrazolo[1,5-a]pyrimidine and triazolo[1,5-a]pyrimidine moieties and their targeting of the androgen receptor in prostate cancer.

One of the main directions of steroid research is the preparation of modified derivatives in which, in addition to changes in physicochemical properties, receptor binding is significantly altered, thus a bioactivity different from that of the parent compound predominates. In the frame of this work, 2-arylidene derivatives were first synthesized by regioselective modification of the A-ring of natural sex hormone, 5α-dihydrotestosterone (DHT). After Claisen-Schmidt condensations of DHT with (hetero)aromatic aldehydes in alkaline EtOH, heterocyclizations of the α,ß-enones were performed with 3-amino-1,2,4-triazole, 3-aminopyrazole and 3-amino-5-methylpyrazole in the presence of t-BuOK in DMF to afford 7'-epimeric mixtures of A-ring-fused azolo-dihydropyrimidines, respectively. Depending on the electronic demand of the substituents of the arylidene moiety, spontaneous or 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ)-induced oxidation of the heteroring led to triazolo[1,5-a]pyrimidines and pyrazolo[1,5-a]pyrimidines in good yields, while, using the Jones reagent as a strong oxidant, 17-oxidation also occurred. The crystal structures of an arylidene and a triazolopyrimidine product have been determined by single crystal X-ray diffraction and both were found to crystallize in the monoclinic crystal system at P21 space group. Most derivatives were found to diminish the transcriptional activity of androgen receptor (AR) in reporter cell line. The candidate compound (17ß-hydroxy-2-(4-chloro)benzylidene-5α-androstan-3-one, 2f) showed to suppress androgen-mediated AR transactivation in a dose-dependent manner. We confirmed the cellular interaction of 2f with AR, described the binding in AR-binding cavity by the flexible docking and showed the ability of the compound to suppress the expression of AR-regulated genes in two prostate cancer cell lines.

Catalytic antioxidant nanocomposites based on sequential adsorption of redox active metal complexes and polyelectrolytes on nanoclay particles.

An antioxidant nanocomposite was prepared by successive adsorption of redox active metal complexes (copper(ii)-bipyridyl and iron(iii)-citrate) and polyelectrolytes (poly(styrene sulfonate) and poly(diallyldimethyl ammonium)) on layered double hydroxide nanoclay. The experimental conditions were optimized in each preparation step and thus, the final composite formed highly stable colloids, i.e., excellent resistance against salt-induced aggregation was achieved. Due to the synergistic effect of the metal complexes, the developed composite showed remarkable activity in the dismutation of superoxide radicals, close to the one determined for the native superoxide dismutase enzyme. The obtained composite is highly selective for superoxide radical dismutation, while its activity in other antioxidant tests was close to negligible. Structural characterization of the composite revealed that the excellent superoxide radical scavenging ability originated from the advantageous coordination geometry around the copper(ii) center formed upon immobilization. The structure formed around the metal centers led to optimal redox features and consequently, to an improved superoxide dismutase-like activity. The catalytic antioxidant composite is a promising candidate to reduce oxidative stress in industrial manufacturing processes, where natural enzymes quickly lose their activity due to the harsh environmental conditions.

Complex formation and cytotoxicity of Triapine derivatives: a comparative solution study on the effect of the chalcogen atom and NH-methylation.

α-N-Heterocyclic thiosemicarbazones are an important class of investigational anticancer drugs. The most prominent representative is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine), which has shown promising results in clinical trials and is currently evaluated in phase III. In this study, we investigated the influence of a chalcogen atom exchange from S (Triapine) to O (O-Triapine) and Se (Se-Triapine) and the methylation of the hydrazonic NH moiety (Me-Triapine) on their complexation with Fe(ii), Fe(iii) and Cu(ii) ions and their cytotoxicity. The main aim of this study was to characterize and compare the most feasible chemical forms in solution, their stability and redox properties, as well as to reveal the relationships of the solution speciation and kinetic data with cytotoxic activity. The complex equilibria and redox properties of the complexes were characterized by the combined use of pH-potentiometry, UV-visible spectrophotometry, electron paramagnetic resonance spectroscopy, and cyclic voltammetry. These revealed that Se-Triapine forms Cu(ii) complexes with higher, and O-Triapine with lower stability as compared with Triapine. Me-Triapine, which is not able to coordinate via the typical (N,N,S-) donor set, nevertheless coordinates to Cu(ii) with unexpected high stability. The Cu(ii) complexes of Se-Triapine and Me-Triapine can be relatively slowly reduced by glutathione at pH 7.4 (but not by ascorbate), similarly to Cu(ii)-Triapine. In contrast, the Cu(ii)-O-Triapine complex can be reduced by both reducing agents in rapid redox reactions. Se-Triapine and Triapine form high stability complexes with both Fe(ii) and Fe(iii) ions, while O-Triapine has a much stronger preference towards Fe(iii) and Me-Triapine towards Fe(ii). This difference in the iron preference of the ligands seems to have a strong impact on their cytotoxic effects, which was measured in a human uterine sarcoma cell line (MES-SA) and its multidrug-resistant subline (MES-SA/Dx5). The Cu(ii) complexes of these calcogensemicarbazones are moderately toxic, and the highest level of ROS generation was found for the Cu(ii) complex of O-Triapine, which is the most reducible.