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.

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.

Promising anticancer agents based on 8-hydroxyquinoline hydrazone copper(II) complexes.

We report the synthesis and characterization of a group of benzoylhydrazones (Ln) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing distinct para substituents (R = H, Cl, F, CH3, OCH3, OH and NH2, for L1-7, respectively; in L8 isonicotinohydrazide was used instead of benzylhydrazide). Cu(II) complexes were prepared by reaction of each benzoylhydrazone with Cu(II) acetate. All compounds were characterized by elemental analysis and mass spectrometry as well as by FTIR, UV-visible absorption, NMR or electron paramagnetic resonance spectroscopies. Complexes isolated in the solid state (1-8) are either formulated as [Cu(HL)acetate] (with L1 and L4) or as [Cu(Ln)]3 (n = 2, 3, 5, 6, 7 and 8). Single crystal X-ray diffraction studies were done for L5 and [Cu(L5)]3, confirming the trinuclear formulation of several complexes. Proton dissociation constants, lipophilicity and solubility were determined for all free ligands by UV-Vis spectrophotometry in 30% (v/v) DMSO/H2O. Formation constants were determined for [Cu(LH)], [Cu(L)] and [Cu(LH-1)] for L = L1, L5 and L6, and also [Cu(LH-2)] for L = L6, and binding modes are proposed, [Cu(L)] predominating at physiological pH. The redox properties of complexes formed with L1, L5 and L6 are investigated by cyclic voltammetry; the formal redox potentials fall in the range of +377 to +395 mV vs. NHE. The binding of the Cu(II)-complexes to bovine serum albumin was evaluated by fluorescence spectroscopy, showing moderate-to-strong interaction and suggesting formation of a ground state complex. The interaction of L1, L3, L5 and L7, and of the corresponding complexes with calf thymus DNA was evaluated by thermal denaturation. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. The complexes show higher activity than the corresponding free ligand, and most complexes are more active than cisplatin. Compounds 1, 3, 5, and 8 were selected for additional studies: while these complexes induce reactive oxygen species and double-strand breaks in both cancer cells, their ability to induce cell-death by apoptosis varies. Within the set of compounds tested, 8 emerges as the most promising one, presenting low IC50 values, and high induction of oxidative stress and DNA damage, which eventually lead to high rates of apoptosis.

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 chemical properties and anticancer potential of 8-hydroxyquinoline hydrazones and their oxidovanadium(IV) complexes.

We report the synthesis and characterization of a family of benzohydrazones (Ln, n = 1-6) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing different substituents in the para position. Their oxidovanadium(IV) complexes were prepared and compounds with 1:1 and 1:2 metal-to-ligand stoichiometry were obtained. All compounds were characterized by elemental analyses and mass spectrometry as well as FTIR, UV-visible absorption, NMR (ligand precursors) and EPR (complexes) spectroscopies, and by DFT computational methods. Proton dissociation constants, lipophilicity and solubility in aqueous media were determined for all ligand precursors. Complex formation with V(IV)O was evaluated by spectrophotometry for L4 (Me-substituted) and L6 (OH-substituted) and formation constants for mono [VO(HL)]+, [VO(L)] and bis [VO(HL)2], [VO(HL)(L)]-, [VO(L)2]2- complexes were determined. EPR spectroscopy indicates the formation of [VO(HL)]+ and [VO(HL)2], with this latter being the major species at the physiological pH. Noteworthy, the EPR data suggest a different behaviour for L4 and L6, which confirm the results obtained in the solid state. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. All complexes show much higher activity on A-375 (IC50 < 6.3 µM) than in A-549 cells (IC50 > 20 µM). Complex 3 (F-substituted) shows the lowest IC50 on both cell lines and lower than cisplatin (in A-375). Studies identified this compound as the one showing the highest increase in Annexin-V staining, caspase activity and induction of double stranded breaks, corroborating the cytotoxicity results. The mechanism of action of the complexes involves reactive oxygen species (ROS) induced DNA damage, and cell death by apoptosis.

The coordination modes of (thio)semicarbazone copper(II) complexes strongly modulate the solution chemical properties and mechanism of anticancer activity.

Thiosemicarbazones are promising candidates for anticancer therapy and their mechanism of action is often linked to their metal chelating ability. In this study, five (thio)semicarbazones with different donor sets (NNS, NNO, ONS, ONO) were selected and their behaviour in aqueous solution, the stability of their copper(II) complexes in addition to their cytotoxicity, DNA-binding, DNA cleavage ability and inhibition of topoisomerase IIα were investigated and compared. We aimed to reveal relationships between the structural variations, the significantly different physico-chemical properties, solution speciation and biological activity. The cytotoxicity of the ligands did not show correlation with the solubility, lipophilicity and permeability; and the decreased activity of the oxygen donor containing compounds was explained by their stronger preference towards chelation of iron(III) over iron(II). Meanwhile, among the copper complexes the most lipophilic species with the highest stability and membrane permeability exhibited the highest cytotoxicity. The studied copper(II) complexes interact with DNA, and reaction with glutathione led to heavy DNA cleavage in the case of the highly stable complexes which could be reduced in a reversible reaction with moderate rate. All the tested copper complexes inhibited topoisomerase IIα, however, this property of the complexes with low stability is most probably linked to the liberated free copper(II).

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.

Cancer Cell Resistance Against the Clinically Investigated Thiosemicarbazone COTI-2 Is Based on Formation of Intracellular Copper Complex Glutathione Adducts and ABCC1-Mediated Efflux.

COTI-2 is a novel anticancer thiosemicarbazone in phase I clinical trial. However, the effects of metal complexation (a main characteristic of thiosemicarbazones) and acquired resistance mechanisms are widely unknown. Therefore, in this study, the copper and iron complexes of COTI-2 were synthesized and evaluated for their anticancer activity and impact on drug resistance in comparison to metal-free thiosemicarbazones. Investigations using Triapine-resistant SW480/Tria and newly established COTI-2-resistant SW480/Coti cells revealed distinct structure-activity relationships. SW480/Coti cells were found to overexpress ABCC1, and COTI-2 being a substrate for this efflux pump. This was unexpected, as ABCC1 has strong selectivity for glutathione adducts. The recognition by ABCC1 could be explained by the reduction kinetics of a ternary Cu-COTI-2 complex with glutathione. Thus, only thiosemicarbazones forming stable, nonreducible copper(II)-glutathione adducts are recognized and, in turn, effluxed by ABCC1. This reveals a crucial connection between copper complex chemistry, glutathione interaction, and the resistance profile of clinically relevant thiosemicarbazones.

Triapine Derivatives Act as Copper Delivery Vehicles to Induce Deadly Metal Overload in Cancer Cells.

Thiosemicarbazones continue to attract the interest of researchers as potential anticancer drugs. For example, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, or triapine, is the most well-known representative of this class of compounds that has entered multiple phase I and II clinical trials. Two new triapine derivatives HL1 and HL2 were prepared by condensation reactions of 2-pyridinamidrazone and S-methylisothiosemicarbazidium chloride with 3-N-(tert-butyloxycarbonyl) amino-pyridine-2-carboxaldehyde, followed by a Boc-deprotection procedure. Subsequent reaction of HL1 and HL2 with CuCl2·2H2O in 1:1 molar ratio in methanol produced the complexes [CuII(HL1)Cl2]·H2O (1·H2O) and [CuII(HL2)Cl2] (2). The reaction of HL2 with Fe(NO3)3∙9H2O in 2:1 molar ratio in the presence of triethylamine afforded the complex [FeIII(L2)2]NO3∙0.75H2O (3∙0.75H2O), in which the isothiosemicarbazone acts as a tridentate monoanionic ligand. The crystal structures of HL1, HL2 and metal complexes 1 and 2 were determined by single crystal X-ray diffraction. The UV-Vis and EPR spectroelectrochemical measurements revealed that complexes 1 and 2 underwent irreversible reduction of Cu(II) with subsequent ligand release, while 3 showed an almost reversible electrochemical reduction in dimethyl sulfoxide (DMSO). Aqueous solution behaviour of HL1 and 1, as well as of HL2 and its complex 2, was monitored as well. Complexes 1-3 were tested against ovarian carcinoma cells, as well as noncancerous embryonic kidney cells, in comparison to respective free ligands, triapine and cisplatin. While the free ligands HL1 and HL2 were devoid of antiproliferative activity, their respective metal complexes showed remarkable antiproliferative activity in a micromolar concentration range. The activity was not related to the inhibition of ribonucleotide reductase (RNR) R2 protein, but rather to cancer cell homeostasis disturbance-leading to the disruption of cancer cell signalling.

High Copper Complex Stability and Slow Reduction Kinetics as Key Parameters for Improved Activity, Paraptosis Induction, and Impact on Drug-Resistant Cells of Anticancer Thiosemicarbazones.

Aims: Due to their significant biological activity, thiosemicarbazones (TSCs) are promising candidates for anticancer therapy. In part, the efficacy of TSCs is linked to their ability to chelate essential metal ions such as copper and iron. Triapine, the best-studied anticancer TSC, has been tested clinically with promising results in hematological diseases. During the past few years, a novel subclass of TSCs with improved anticancer activity was found to induce paraptosis, a recently characterized form of cell death. The aim of this study was to identify structural and chemical properties associated with anticancer activity and paraptosis induction of TSCs. Results: When testing a panel of structurally related TSCs, compounds with nanomolar anticancer activity and paraptosis-inducing properties showed higher copper(II) complex solution stability and a slower reduction rate, which resulted in reduced redox activity. In contrast, TSCs with lower anticancer activity induced higher levels of superoxide that rapidly stimulated superoxide dismutase expression in treated cells, effectively protecting the cells from drug-induced redox stress. Innovation: Consequently, we hypothesize that in the case of close Triapine derivatives, intracellular reduction leads to rapid dissociation of intracellularly formed copper complexes. In contrast, TSCs characterized by highly stable, slowly reducible copper(II) complexes are able to reach new intracellular targets such as the endoplasmic reticulum-resident protein disulfide isomerase. Conclusion: The additional modes of actions observed with highly active TSC derivatives are based on intracellular formation of stable copper complexes, offering a new approach to combat (drug-resistant) cancer cells.

Drug likeness prediction of 5-hydroxy-substituted coumarins with high affinity to 5-HT1A and 5-HT2A receptors.

One of the latest trends is search for the new anti-psychotic drugs among coumarin derivatives with piperazine moiety. Their therapeutic potential can be hampered by poor physico-chemical parameters as low brain penetration or limited transport in the body fluid. Herein, we predicted the drug likeness of six coumarins with high affinity towards 5-HT1A and 5-HT2A receptors. Subsequent experimental determination of their binding constants to human serum albumin (HSA) revealed the binding with a moderate strength (logK=4.8-5.8) at the Sudlow's site 1, which represents a possibility of temporary storage of tested coumarins on HSA. Computational mapping of the binding of coumarins - HSA complexes showed that the coumarin rings of all tested compounds were similarly located within the hydrophobic binding pocket of HSA, while the rest of molecules (composed with alkyl chains, piperazine and benzene rings) decided about the difference in binding modes by the hydrogen bonding interactions. The proton dissociation constants (pKa) of the compounds were also determined by UV-vis spectrophotometric titrations to obtain the distribution of the species in the different protonation states at physiological pH of 7.4. A good agreement of the computationally-determined free enthalpy values of the ligand - HSA complexes with the values determined by experimental fluorescence quenching data could be a promising prospect for proposed theoretical strategy.