Copper--ligand interactions and the physiological free radical processes. Part 3. Influence of histidine, salicylic acid and anthranilic acid on copper-driven Fenton chemistry in vitro

With a view to the possible use of copper(II)-^·OH inactivating ligand (OIL) complexes as regulators of inflammation, the reactivity of the copper(II)-ascorbate system with hydrogen peroxide has been investigated in the presence of three key substances: histidine (the main copper(II) low molecular mass ligand in extracellular fluid), salicylic acid (the well-known non-steroidal antiinflammatory drug, previously shown to be potentiated by copper(II) in animal models of inflammation), and anthranilic acid (an inactive substance by itself, known to be activated by copper(II) in the same models) at physiological pH (7.4) and inflammatory pH (5.5).

Such substances may affect the amount of TBARS detected in solution following copper-mediated Fenton-like reactions through three distinct mechanisms: (i) by decreasing the Cu(II)/Cu(I) redox potential, i.e. at the expense of ^·OH radical production, (ii) by scavenging ^·OH radicals in the body of the solution, and/or (iii) by acting as a true OIL, i.e. at the expense of ^·OH detection. Redox potential measurements of initial solutions have been performed in parallel to TBARS determinations to help discriminate between different ligand influences. Computer-aided speciation has been used to understand the role of copper(II) distribution on the ligand effects characterised.

Contrary to previous interpretations, histidine has been found to mainly affect ^·OH production by lowering the redox potential of the Cu(II)/Cu(I) couple. Salicylate, which has no effect on ^·OH production, has been confirmed to mainly scavenge ^·OH radicals in the body of the solution. Anthranilate, which both increases ^·OH production and decreases ^·OH detection, behaves as a potential OIL.

These results tend to confirm our previous hypothesis that copper potentiation of antiinflammatory substances is indirect, i.e. independent of any interaction between metal and drug, whereas copper activation of substances that are inactive by themselves results from specific metal-substance interactions taking place at inflammatory sites.     

Antibacterial,antifungal and in vitro antileukaemia activity of metal complexes with thiosemicarbazones

1‐phenyl‐3‐methyl‐4‐benzoyl‐5‐pyrazolone 4‐ethyl‐thiosemicarbazone (HL) and its copper(II), vanadium(V) and nickel(II) complexes: [Cu(L)(Cl)]·C₂H₅OH·( 1 ), [Cu(L)₂]·H₂O ( 2 ), [Cu(L)(Br)]·H₂O·CH₃OH ( 3 ), [Cu(L)(NO₃)]·2C₂H₅OH ( 4 ), [VO₂(L)]·2H₂O ( 5 ), [Ni(L)₂]·H₂O ( 6 ), were synthesized and characterized. The ligand has been characterized by elemental analyses, IR, ¹H NMR and ¹³C NMR spectroscopy. The tridentate nature of the ligand is evident from the IR spectra. The copper(II), vanadium(V) and nickel(II) complexes have been characterized by different physico‐chemical techniques such as molar conductivity, magnetic susceptibility measurements and electronic, infrared and electron paramagnetic resonance spectral studies. The structures of the ligand and its copper(II) ( 2 , 4 ), and vanadium(V) ( 5 ) complexes have been determined by single‐crystal X‐ray diffraction. The composition of the coordination polyhedron of the central atom in 2 , 4 and 5 is different. The tetrahedral coordination geometry of Cu was found in complex 2 while in complex 4 , it is square planar, in complex 5 the coordination polyhedron of the central ion is distorted square pyramid. The in vitro antibacterial activity of the complexes against Escherichia coli, Salmonella abony, Staphylococcus aureus, Bacillus cereus and the antifungal activity against Candida albicans strains was higher for the metal complexes than for free ligand. The effect of the free ligand and its metal complexes on the proliferation of HL‐60 cells was tested.     

Potentiometric and spectroscopic studies on the solution molecular structures of copper(II)-[S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine complexes, potential non-steroidal anti-inflammatory agents (NSAIDs)

The purpose of this study was to investigate the complexes formed by copper(II) with potential non-steroidal anti-inflammatory agents (NSAIDs) under physiological conditions. A former study suggested that 2-benzylaminomethylpyrrolidine ligands could be good candidates as potential OIL (OH-inactivating ligand) when complexed to copper(II). In order to assess the chemical behavior as OIL, [S]-2-[N-(2′-hydroxybenzyl)aminomethyl]pyrrolidine (OHbamp) was synthesized and bound to copper(II). Physico-chemical properties were determined at 37 °C in 0.15 M NaCl using glass electrode potentiometry, UV-Vis and circular dichroism spectroscopies, before and after copper(II) complexation. [Cu(OHbamp)(H₂O)₃]⁺ was the main complex found at both physiological and inflammatory pH values, showing appreciable stability at pathological pH compared to copper(II) complexes of histidine, the predominant low-molar-mass ligand of copper(II) in blood plasma. However, neutral species such as [Cu(OHbamp)₂(H₂O)₂] and [Cu(OHbamp)(OH)(H₂O)₃] are predominant only above pH 8, preventing a significant amount of drug from diffusing through membranes at inflammatory pH. In conclusion, copper(II)-OHbamp system does not meet all the requirements to be an OIL. Nevertheless, these results allow us to better identify the chemical features needed for a good OIL candidate.     

Chronotoxicity of a Copper (II) Complex with a Linear Tetradentate Ligand in Mice

In vitro copper (II) complex presents antimitotic effects. In this work, we have studied the in vivo seasonal toxic effects of copper (II), ligand (H₂L) and the complex [Cu(H₂L)(H₂O)₂]Cl₂·4H₂O in male Swiss mice. During spring, an i.p. injection of CuCl₂ in aqueous NaCl (9 g·l^-1) up to 0.05 µmol·kg^-1 b.w. (body weight) killed 60% of the rodents after 6 days. LD100 was up to 0.3 µmol·kg^-1; H₂L was well tolerated, while the complex was 30% lethal with 50 µmol·kg^-1. In autumn, mice were less sensitive to CuCl₂, and both ligand and complex were equally tolerated and this leads to the conclusion that, in vivo, chronotoxicities of copper (II) and complex in NaCl aqueous solutions are quite different in spring and autumn seasons.     

Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Effect of feeding supplemental copper on performance,fatty acid profile and on cholesterol contents and oxidative stability of meat of rabbits

One hundred and four rabbits, five weeks old at the beginning of the experiment, were divided into four groups according to a feed additive treatment. Rabbits of the 1st, 2nd, 3rd and 4th group were fed a basal granulated feed (control), basal feed supplemented with CuSO₄ · 5H₂O at 50mg Cu · kg^‐1, basal feed supplemented with 150mg Cu · kg^‐1, and the latter feed supplemented with 100mg · kg^‐1 vitamin E, respectively. The duration of the experiment was 42 days. Addition of Cu at 150mg · kg.^‐1 increased weight gain non‐significantly by 9.1%. This effect was the most pronounced in the first two weeks of fattening. The lowest mortality was observed in rabbits fed the highest amount of additives (7.7% vs. 19.2% in the control). Rabbits were slaughtered at the age of 11 weeks. Neither treatment influenced proportions of saturated, monounsaturated and polyunsaturated fatty acids in lipids extracted from the loin and hindleg muscles. In rabbits fed the highest amount of copper and vitamin E, the cholesterol concentration was significantly decreased by 13.6% and 17.9% in the loin and hindleg meat, respectively. Effects of Cu added at 50mg · kg^‐1 were marginal. Copper had no effect on the oxidative stability of meat, measured as thiobarbituric acid‐reactive substances in meat stored at 4°C for 0, 3 and 8 days. Vitamin E added in excess of nutritional requirement improved the oxidative stability of meat. In copper‐fed rabbits, Cu accumulated in the liver, but not in muscles. Feeding of the basal feed for 7 days to rabbits previously fed copper sulphate decreased the hepatic Cu concentration by 14.0 to 24.4%     

In Vitro Cytotoxic Activities,DNA‐, and BSA‐Binding Studies of a New Dinuclear Copper(II) Complex with N‐[3‐(Dimethylamino)propyl]‐N′‐(2‐carboxylatophenyl)‐ Oxamide as Ligand

A new dinuclear copper(II) complex bridged by N‐[3‐(dimethylamino)propyl]‐N′‐ (2‐carbo‐xylatophenyl)oxamide (H₃dmapob), and endcapped with 2,2′‐diamino‐4,4′‐bithiazole (dabt), namely [Cu₂(dmapob)(dabt)(CH₃OH)(pic)]·(DMF)_0.75·(CH₃OH)_0.25 has been synthesized and characterized by elemental analysis, molar conductivity measurement, infrared and electronic spectra studies, and single‐crystal X‐ray diffraction. In the crystal structure, both copper(II) ions have square–pyramidal coordination geometries. The Cu···Cu separation through the oxamido bridge is 5.176(9) Å. A two‐dimensional supramolecular framework is formed through hydrogen bonds and π–π stacking interactions. The reactivities toward herring sperm DNA and bovine serum albumin (BSA) show that the complex can interact with the DNA via intercalation mode and bind to the BSA responsible for quenching of tryptophan fluorescence by the static quenching mechanism. The in vitro anticancer activities suggest that the copper(II) complex is active against the selected tumor cell lines. The influence of different bridging ligands in dinuclear complexes on the DNA‐ and BSA‐binding properties as well as anticancer activities is preliminarily discussed.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Structure–activity relationship study of copper(II) complexes with 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4′-methylbenzoyl) hydrazone: synthesis, structures, DNA and protein interaction studies, antioxidative and cytotoxic activity

Novel 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (4′-methylbenzoyl) hydrazone (H₂L) (1) and its two copper(II) complexes have been synthesized. Single-crystal X-ray diffraction studies revealed that the structure of the new copper(II) chloride complex, [Cu(H₂L)Cl₂]·2H₂O (2), is square pyramidal and that of the copper(II) nitrate complex, [Cu(HL)NO₃]·DMF (3), is square planar. In 2, the copper atom is coordinated by the ligand with ONO donor atoms, one chloride ion in the apical position, and the other chloride in the basal plane. In 3, the ligand coordinates as a uninegative tridentate ONO⁻ species and with one nitrate ion in the basal plane. DNA binding experiments indicated that the ligand and copper(II) complexes can interact with DNA through intercalation. Bovine serum albumin binding studies revealed that the compounds strongly quench the intrinsic fluorescence of bovine serum albumin through a static quenching process. Antioxidative activity tests showed that 1 and its copper(II) complexes have significant radical scavenging activity against free radicals. Cytotoxic activities of the ligand and copper(II) complexes showed that the two copper(II) complexes exhibited more effective cytotoxic activity against HeLa and HEp-2 cells than the corresponding ligand. The entire biological activity results showed that the activity order was 1 < 2 < 3.     

Anthracycline-copper(II) complexes: Structure-dependent coordination pattern as evidenced by ESR

The copper(II) coordination properties of different anthracycline antitumor agents were studied by ESR spectroscopy. Three classes of drugs have been identified: (i) adria-like, which contains adriamycin, daunomycin, 4′-deoxydaunomycin, 4′-deoxy- 4′-iododaunomycin and 4-demethoxydaunomycin; (ii) 4′-epiadria-like, which contains 4′-epiadriamycin, 4′-epidaunomycin, 4-demethoxy-6-deoxydaunomycin and 4-demethoxy-11-deoxydaunomycin; and (iii) border-line type, which contains carminomycin, 6-O- methylcarminomycin, 11-deoxycarminomycin and 6- deoxycarminomycin. They have been characterized by their different Cu(II) chelation properties.The adria-like class members give rise to multi- nuclear complexes, where both the CO and COH functionalities of both sides of the hydroxyanthra- quinone system are involved in the coordination. By contrast, the 4′-epiadria-like drugs form monomeric Cu(II) complexes because of the unavailability of the C(6)OH group. The border-line type compounds yield more than one monomeric Cu(II)adduct because of the presence of OH at the C(4) position.All Cu(II) derivatives are characterized by a very strong ligand field, the unpaired electron lying in the d_x²−_y² orbital. The onset of comptexation is oxygen dependent and this is related to the σ and π bond properties of the anthracycline ligand.     

Copper(II) interactions with non-steroidal anti-inflammatory agents. III--3-Methoxyanthranilic acid as a potential *OH-inactivating ligand: a quantitative investigation of its copper handling role in vivo

Pharmacological activities of copper(II) complexes are a direct function of the nature of their ligands associated with the metal ion in vivo. Some of these, defined as *OH-inactivating ligands (G. Berthon, Agents Actions 39 (1993) 210-217), may act as specific "lures" for hydroxyl radicals at inflammatory sites and behave as pseudo-catalase-like agents. This property has been advanced for anthranilic acid (H. Miche, V. Brumas, G. Berthon, J. Inorg. Biochem. 68 (1997) 27-38). With a view to improve the chemical features required to render such inactive substances effective anti-inflammatory drugs through their association with copper(II), an in vitro investigation into copper(II) interactions with the anionic form of an anthranilic acid derivative, namely 3-methoxyanthranilate (Man), has been performed under experimental conditions pertaining in vivo. Copper(II)-Man complex equilibria have been determined using glass electrode potentiometry, then checked by UV-vis and mass spectrometries. Given the prime role of histidine as a copper(II) ligand in blood plasma, copper(II)-histidine-Man ternary equilibria have also been studied. Subsequent computer simulations of the distribution of copper(II) in the extracellular fluid revealed that Man can specifically mobilize Cu(II) ions under inflammatory conditions without affecting their distribution under normal physiological conditions. Thiobarbituric acid reactive substances (TBARS) tests conducted with respect to standardized copper-mediated Fenton-type reactions (P. Maestre, L. Lambs, J.P. Thouvenot, G. Berthon, Free Rad. Res. 20 (1994) 205-218) have shown that, like anthranilic acid, Man can effectively both increase the Fenton-like reactivity of copper and decrease the amount of TBARS detected in solution, i.e., act as a potential *OH-inactivating ligand.     

Free radical scavenging activity of the Japanese herbal medicine Toki-Shakuyaku-San (TJ-23) and its effect on superoxide dismutase activity,lipid peroxides,glutamate, and monoamine metabolites in aged rat brain

The free radical scavenging activity of the Japanese herbal medicine, Toki-Shakuyaku-San (TJ-23; TSUMURA & Co., Tokyo, Japan), was examined using electron spin resonance (ESR) spectrometry. TJ-23 scavenged 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), superoxide (O₂ ⁻), and hydroxyl radicals (·OH) dose-dependently. It also diminished carbon centered radicals (·C) generated by oxidative stress and inhibited thiobarbituric acid-reactive substances (TBARS) formation in mouse cortex homogenate. In addition, the effect of TJ-23 on the concentration of neurotransmitters and TBARS formation, and superoxide dismutase (SOD) activity in the cortex, hippocampus and striatum of the aged rat brain was studied. The concentrations of the metabolites of monoamines, glutamate and glutamine were decreased by 4 weeks of oral administration of TJ-23. The SOD activity of mitochondrial fraction was increased and TBARS formation was significantly suppressed. These results suggest that TJ-23 has an antioxidant action and would have a prophylactic effect against free radical-mediated neurological diseases associated with aging.     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

The peptides Asp-Ala-His-Lys (DAHK) and Gly-His-Lys (GHK) are naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid. Here, the sensitive thermodynamic technique isothermal titration calorimetry was used to study the energetics of Cu(II) binding to DAHK and GHK peptides in the presence of the weaker ligand glycine as a competitor. DAHK and GHK bind Cu(II) predominantly in a 1:1 stoichiometry with conditional dissociation constants [i.e., at pH 7.4, in the absence of the competing chelators glycine and 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid buffer] of 2.6 ± 0.4 × 10⁻¹⁴ M and 7.0 ± 1.0 × 10⁻¹⁴ M, respectively. Furthermore, the apparent ΔH values were measured and the number of protons released upon Cu(II) binding was determined by performing experiments in different buffers. This allowed us to determine the conditional ΔG, ΔH, and ΔS, i.e., corrected for the contributions of the weaker ligand glycine and the buffer at pH 7.4. We found that the entropic and enthalpic contributions to the Cu(II) binding to GHK and DAHK are distinct, with a enthalpic contribution for GHK. The thermodynamic parameters obtained correspond well to those in the literature obtained by other techniques, suggesting that the use of the weaker ligand glycine as a competitor in isothermal titration calorimetry provides accurate data for Cu(II) binding to high-affinity peptides, which cannot be accurately determined without the use of a competitor ligand.     

Spectroscopic,structural and antibacterial properties of copper(II) complexes with bio-relevant 5-methyl-3-formylpyrazole N(4)-benzyl-N(4)-methylthiosemicarbazone

The coordination behaviour of the title ligand, 5-methyl-3-formylpyrazole N(4)-benzyl-N(4)-methylthiosemicarbazone(HMPz4BM), is reported with solid state isolation of copper(II) complexes, [Cu(HMPz4BM)X₂] (X = Cl, Br, NO₃, ClO₄ and BF₄) which have been spectroscopically and structurally characterised. I.r. data for the free ligand and its Cu(II) complexes indicate that HMPz4BM exhibits a neutral NNS tridentate function via the pyrazolyl nitrogen(tertiary), azomethine nitrogen and thione sulphur. Electronic spectral data are suggestive of a square pyramidal environment for the seemingly pentacoordinated Cu(II) species. E.s.r parameters (RT and LNT) of the reported copper(II) complexes are indicative of a dx_x2–y2 ground state for the reported species. Cyclic voltammograms of Cu(II) complexes show a quasireversible Cu^II/Cu^III couple and also an irreversible Cu^II/Cu^I couple. X-ray crystallography of a representative species, [Cu(HMPz4BM)(NO₃)₂] (C2/c, monoclinic ), has unambiguously documented the conjectural findings from i.r. data that coordinating sites of the title ligand are pyrazolyl (tertiary)nitrogen, azomethine nitrogen and the thione sulphur (NNS); and the oxygen of one of the nitrate ions has occupied the basal plane; the fifth coordination position has been occupied by the oxygen of another nitrate ion in a square pyramidal geometry. The antibacterial properties of the ligand and its copper(II) complexes studied on microorganism, Staphylococcus aureus have pointed out that most of the complexes have higher activities than that of the free ligand.     

Copper(II) interaction with 3,5-diisopropylsalicylic acid (Dips): new insights on its role as a potential *OH inactivating ligand

The purpose of this study was to identify the low molecular mass complexes formed between copper(II) and 3,5-diisopropylsalicylic acid (Dips) in physiological conditions. Copper(II)-Dips complex equilibria were determined using glass electrode potentiometry and their solution structures checked by UV-visible (UV-vis) spectrophotometry. Because of the low solubility of Dips in water, the equilibria were investigated in different water/ethanol mixtures. Formation constants were extrapolated to 100% water and then compared with the values obtained for the other anti-inflammatory drugs previously studied. Given the prime role of histidine as the copper(II) ligand in blood plasma, copper(II)-histidine-Dips ternary equilibria were studied under similar experimental conditions. Computer simulations of copper(II) distribution relative to different biofluids, gastrointestinal (g.i.) fluid and blood plasma, show that like salicylic and anthranilic acids, Dips favors g.i. copper absorption, but cannot exert any significant influence on plasma copper distribution. Moreover, Dips can mobilize increasing fractions of copper(II) as the pH decreases. In conclusion, Dips seems to correspond to the notion of *OH-inactivating ligand (OIL) as determined for anthranilic acid.     

Copper complexes with bioactive ligands

Biological properties of new copper(II) complexes of 2-methylthionicotinate (2-MeSNic) of composition Cu(2-MeSNic)₂(MeNia)₂·4H₂O (where MeNia isN-methylnicotinamide), Cu(2-MeSNic)₂(Nia)₂·2H₂O (where Nia is nicotinamide) and Cu(2-MeSNic)₂(₂ (where L is isonicotinamide (iNia) or ethyl nicotinate (EtNic)) are reported. Gram⁻-bacteria (Escherichia coli) are more resistant against Cu(II) complexes than Gram⁺-bacteria (Staphylococcus aureus)—significant antistaphylococcal activity was found with Cu(2-MeSNic)₂(MeNia)₂·4H₂O (IC₅₀ 1.3 mmol/L).Caddida parapsilosis was most inhibited by Cu(2-MeSNic)₂·H₂O and Cu(2-MeSNic)₂(MeNia)₂·4H₂O (IC₅₀ 1.4 mmol/L and 1.5 mmol/L, respectively). Biosynthesis of nucleic acids influenced by Cu(2-MeSNic)₂-(Nia)₂·2H₂O indicated by incorporation of¹⁴C-adenine (IC_50(Ade) 0.31 mmol/L) is more sensitive than biosynthesis of proteins indicated by incorporation of¹⁴C-leucine (IC_50(Leu) 9.94 mmol/L). Cu(II) complexes with expressed antimicrobial activity showed no mutagenic activity.     

Intramolecular hydrogen bonding: synthesis and crystal structure of nickel(II) and copper(II) complexes of a tetradentate amine oxime

In the reaction of the tetradentate ligand 3,3′-(1,4- butanediyldiamino) bis (3-methyl-2-butanone)-dioxime (BnAO) with nickel(II) and copper(II), the monomeric [Ni(BnAO-H)]I·H₂O and a mixed monomer/dimer salt [Cu(BnAO-H)H₂O]₂[(Cu(BnAO-H))₂](ClO₄)₄, respectively, are formed, and all complexes have an intramolecular hydrogen bond between cis oxime groups. The OHO bonds give the characteristic infrared absorptions as well as the downfield proton-NMR signal (Ni complex). [Ni(BnAO-H)]I·H₂O crystallizes in space group P2₁/a with a=13.511(2), b=10.599(2), c=14.096(2) Å, β=97.52°, Z=4 and D_c=1.623 g/cm³. The structure was solved by Patterson and Fourier methods and refined by full-matrix least-squares techniques to a final R of 0.021 for 2124 reflections with I 2σ(I). The nickel(II) atom in the complex has slightly distorted square planar geometry with an intramolecular O···O contact of 2.417(7) Å. The copper(II) complex crystallizes in space group P2₁/c with a =13.425(2), b=21.446(3), c=14.349(4) Å, β= 104.4(5)°, Z=8 (monomers) and D_c=1.485 g/cm³. The final R value for this complex was 0.053 for 3033 reflections with I 2σ(I). This structure contains a monomeric [Cu(BnAO-H)H₂O]⁺ ion and a dimeric [(Cu(BnAO-H))₂]²⁺ ion, having intramolecular O···O hydrogen bonds of 2.421(5) and 2.531(5) Å, respectively. The copper(II) ions have square-pyramidal coordination with the axial positions occupied by an oxygen of the water of hydration in the monomer and by an oxime oxygen atom in the dimer. A center of symmetry relates the two halves of the dimer. The copper atom in each case is out of the plane of the four nitrogen atoms toward the axial site. The copper(II) complex is unusual in that the crystal contains both a monomer and a dimer.