The synthesis and antitumor activity of metal complexes of amine–carboxyborane adducts

The amine carboxyborane metal complexes, tetrakis - μ - (trimethylamine – boranecarbo - xylato)acetonitrile dicopper(II) and bis-μ- (morpholine–boranecarboxylato)zinc(II) dihydrate demonstrated cytotoxic activity against human Tmolt₃, HeLa-S³ and MB-9812 cell growth.Tetrakis-μ-(trimethylamine–boranecarboxylato)-acetonitrile dicopper(II) and bis - μ - (morpholine – boranecarboxylato)zinc (II) dihydrate inhibited L₁₂₁₀ DNA, RNA and protein syntheses, with greatest inhibitory effects on DNA synthesis. The reduction in DNA synthesis correlates well with inhibition of de novopurine synthesis and the key enzymes involved in this pathway, i.e. IMP dehydrogenase and PRPP amido transferase. These compounds were also observed to induce DNA strand scission but did not appear to intercalate between base pairs of DNA, alkylate bases or cause cross-linking of the strands of DNA. Tetrakis-μ-(trimethylamine – boranecarboxylato)acetonitrile dicopper(II) also demonstrated the ability to inhibit L₁₂₁₀ DNA topoisomerase II activity. © 1998 John Wiley & Sons, Ltd.     

The anti-inflammatory activity of metal complexes and salts of amine carboxyboranes

The metal complexes and salts of amine carboxyboranes were shown to be potent anti-inflammatory agents in rodents at 8 mg kg^?1. They were effective in blocking induced edema, pleurisy and endotoxic shock while blocking both local and central pain processes. The ability of the agents to function as anti-inflammatory agents is multi-fold. First, lysosomal enzymes of specific cells, e.g. macrophages, were inhibited with IC₅₀ values in the range of 10^?6 M . Collagenase I and II activities were inhibited with IC₅₀ values approximately equal to 10^?4 M . The anti-inflammatory activity of these agents at the molecular levels appears to be due to inhibition of the release of TNFα and Il-1 from macrophages which indirectly control chemotaxic migration of white blood cells as indicated by the suppression of PMN and macrophage invasion into sponges implanted subcutaneously (SC) in mice. Furthermore, in these invading cells, the agents' blockage of TNFα and Il-1 or Il-2 release down-regulates prostaglandin and leukotriene enzymatic synthetic rates and, consequently, their release, resulting in a reduction of the inflammation process.     

Diborane (4) derivatives via coupling of amine monobromocyanoboranes: Study of the bromination of amine cyanoborane and molecular structures of the amine dibromocyanoboranes

The first examples of diborane (4) compounds derived from amine cyanoboranes are described. A series of monobromo derivatives of amine cyanoboranes (A:BHBrCN), and dibromo derivatives (A:BBr₂CN), 1-7, were prepared. Lithiation of the monobromo derivative of trimethylamine cyanoborane, using n-BuLi, did not produce the C-lithiated intermediate Li⁺ [CH₂NMe₂BHBrCN]⁻, but instead the B-lithiated intermediate Li⁺ [Me₃NBHCN]⁻, was obtained. This intermediate, when allowed to react for 16 h, coupled with the un-lithiated trimethylamine monobromocyanoborane (Me₃NBHBrCN) and resulted in diborane (4) derivative formation as the 2LiBr complex. The same result was obtained when one equiv of the trimethylamine monobromocyanoborane was added to the reaction mixture 1 h after lithiation. Following the same procedure, novel diborane (4) derivatives of amine cyanoboranes were successfully obtained, 8-11, as their 2LiBr complexes from the monobromo derivatives of the corresponding amine cyanoboranes. Molecular structures of the trimethylamine dibromocyanoborane, 6, and the triethylamine dibromocyanoborane, 7, were determined using X-ray crystallography.     

Catalytic characteristics of enzyme-polyelectrolyte complexes based on hexahistidine-containing organophosphorus hydrolase

A number of covalent and non-covalent polyelectrolyte complexes based on enzyme such as hexahistidine-containing organophosphorus hydrolase was developed in this work. Polyanions and polycations were used in the form of block-copolymers with poly(ethylene glycol). To obtain covalent complexes of the enzyme, different coupling agents (glutaric aldehyde, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt) were tried. The best catalytic efficiency of the action of the covalently bound complex ((2.8 ± 0.3) × 10⁸ M^?1 s^?1) was obtained for samples with EDC. The modification of the protein surface was undertaken as an approach to its further biomedical application.     

Amino-phenol complexes of Fe(III) as promising T1 accelerators

Iron(III) complexes with N,O-ligands are compounds of high interest because they can be applied in catalysis and play an important role in living organisms, e.g., as models of catechol dioxygenase. Several N,O-ligands were studied: their synthesis, iron(III) complexation and the potential of the latter as T₁-MRI contrast agents. A route to the tetrapodal N₃O₂-naphthyl ligand was investigated. The resulting iron complex was obtained in 26% total yield and its relaxivity value was moderate (r₁ = 1.03 in water and 2.54 s^?1 mM^?1 in serum). Thus, phenyl isomeric salan complexes were obtained. These complexes differed in charge (positive and neutral) and in the presence of polar hydrogen-bonding substituents. The highest relaxivities (r₁ = 2.39 in water and 5.37 s^?1 mM^?1 in serum) were obtained for the Fe(III) cationic complex with MeO groups in the ligand. EPR studies confirmed a high spin configuration of rhombically distorted Fe(III) complexes.     

Sample preparation for the determination of radiocalcium by accelerator mass spectrometry

An accelerator mass spectrometric (AMS) technique has been developed for determining ⁴¹Ca/⁴⁰Ca ratios at terrestrial levels of 10^?16?10^?14. Calcium is extracted as calcium oxide from bone and rock samples. The calcium oxide is converted into calcium metal and subsequently into calcium hydride in a two-step reduction process. From these calcium hydride samples 5–10 μA of a negative-ion beam of CaH^?₃ is produced. A new gas-ionization detector and a high-resolution velocity detector allow discrimination against ⁴¹K and the stable isotopes of Ca, resulting in background levels of less than 6 × 10^?16. Hence it is possible (for the first time without pre-enrichment) to study the distribution of ⁴¹Ca in the terrestrial environment. The AMS method for ⁴¹Ca determination is applicable to samples of less than 1 g.     

Synthesis,crystal structures,DNA binding and cleavage properties and protein binding activities of three mononuclear cobalt(II) complexes

Three new mononuclear cobalt(II) complexes containing ligands with extended planar quinoxaline moieties, {dipyrido[3,2‐a:2′,3′‐c]phenazine (dppz) or dipyrido[3,2‐d:2′,3′‐f]quinoxaline (dpq)}, viz. [Co(dppz)(acac)₂] · CH₃OH ( 1 ), [Co(dpq)(tfnb)₂] ( 2 ) and [Co(dpq)(dbm)₂] ( 3 ), where acac = acetylacetonate, tfnb = benzoyltrifluoroacetone and dbm = dibenzoylmethane, have been synthesized and structurally characterized as octahedral complexes. The binding ability of the complexes with calf thymus (CT)‐DNA has been investigated by spectroscopic and viscosity measurements. Results indicate that all complexes bind to CT‐DNA via intercalative mode, and the DNA binding affinity of dppz complex 1 is apparently stronger than that of dpq complexes 2 and 3 . Furthermore, DNA photocleavage experiments indicate that these complexes are efficient DNA cleaving agents in UV‐A (365 nm) and hydroxyl radical (HO^·), singlet oxygen (¹O₂) and superoxide anion (¹O₂^?) serve as the major cleavage active species. In addition, interaction of the complexes with bovine serum albumin (BSA) was investigated using UV ? visible and fluorescence methods, which indicated that all complexes could quench the intrinsic fluorescence of BSA in a static quenching process. Copyright © 2014 John Wiley & Sons, Ltd.     

Design of active-site-directed fluorescent probes and their reactions with biopolymers

Fluorescent probes which are active-site-directed, reversible, competitive inhibitors of serum cholinesterase (ChE) enzymes have been designed and synthesized. Reversible inhibitors of enzyme active sites have a unique importance when they act as fluorescent probes, allowing fluorescence spectroscopic detection of conformation changes and activesite dynamics. 5-Dimethylamino-naphthalene-1-sulfonamido-N,N-dimethyl-n-propyl-amine and its aliphatic quaternary derivative are fluorescent probes for serum cholinesterase. The quaternary probe forms complexes with acetylcholinesterase (AChE). The dissociation constants K_d for the two probes with serum ChE are 6.0 × 10^?7 and 6.5 × 10^?7M. The inhibition constants K_i are 3.1 × 10^?6 and 6.3 × 10^?6M from the slopes of Lineweaver-Burk plots. The Michelis constant K_m for the enzyme was 8.8 × 10^?4M.     

Cytotoxicity,apoptosis, interaction with DNA,cellular uptake,and cell cycle arrest of ruthenium(II) polypyridyl complexes containing 4,4′-dimethyl-2,2′-bipyridine as ancillary ligand

Two new ruthenium(II) polypyridyl complexes, [Ru(dmb)₂(DNPIP)](ClO₄)₂ (1) (DNPIP?=?2-(2,4-dinitrophenyl)imidazo[4,5-f][1,10]phenanthroline, dmb?=?4,4′-dimethyl-2,2′-bipyridine) and [Ru(dmb)₂(DAPIP)](ClO₄)₂ (2) (DAPIP?=?2-(2,4-diaminophenyl)imidazo[4,5f][1,10]phenanthroline), were synthesized and characterized. The DNA-binding behaviors of these complexes have been studied by UV-Vis absorption titration, viscosity measurements, and photocleavage. The DNA-binding constants are 7.39 (±0.16)?×?10⁴ (s?=?2.68) and 2.73 (±0.16)?×?10^4?(mol?L^?1)^?1 (s?=?0.64) for 1 and 2, respectively. Their evaluation as cytotoxic agents on different cancer cell lines was investigated with IC₅₀ values of 59.5, 51.3, and 70.3?µmol?L^?1 for 1, >100, 87.9, and 77.9?µmol?L^?1 for 2 against BEL-7402, HepG-2, and MCF-7 cells, respectively. Complex 1 is more active than 2 against selected cancer cell lines. The apoptosis induced by these complexes was studied. Cellular uptake showed that these complexes could enter into the cytoplasm and accumulate in the nuclei. The cell cycle arrest and antioxidant activity against hydroxyl radicals were also investigated.     

Polarographic study of Eu(III)-succinate-ethylenediamine and Eu(III)-malonate-ethylenediamine mixed systems

The mixed complexes of Eu(III) with succinate (succ^2?) and malonate (mal^2?) and ethylenediamine (en) have been studied polarographically at 25°C and at constant ionic strength, μ = 0.1 (NaNO₃) and pH 6. The reduction of the complexes in each case is quasi-reversible and diffusion-controlled. In each system three mixed complexes are formed, viz. [Eu(succ)(en)]⁺, [Eu(succ)(en)2]⁺ and [Eu(succ)₂(en)]^? with stability constants log β₁₁ = 9.2, log β₁₂ = 17.5 and log β₂₁ = 11.7; and [Eu(mal)(en)]⁺, [Eu(mal)₂(en)₂]^? and [Eu(mal)₃(en)]^3? with stability constants log β₁₁ = 11.4, log β₂₂ = 19.08 and log β₃₁ = 13.5 respectively.     

Synthetic and antimicrobial studies on new gold(I) complexes of imidazolidin‐2‐ylidenes

Six new 1,3‐diorganylimidazolidin‐2‐ylidene (NHC) gold(I) complexes of the type [Au(NHC)₂]⁺ (1–6), were synthesized by reacting [AuCl(PPh)₃] with 1,3‐dimesitylimidazolidin‐2‐ylidene or bis(1,3‐dialkylimidazolidin‐2‐ylidene). The complexes 1–6 were fully characterized by elemental analyses and spectroscopic data. The placement of mesityl or para‐substituted benzyl groups on the nitrogen atoms of the ring of the complexes leads to the particularly active antibacterial agents evaluated in this work. It is worth noting that the p‐methoxybenzyl derivative (2) inhibited the growth of Pseudomona aeruginosa, Staphylococcus epidermidis, Staphylococcus aureus and Enterococcus faecalis with minimum inhibitory concentration (MIC) values of 3.12 µg ml^?1, 6.25 µg ml^?1, 3.12 µg ml^?1 and 3.12 µg ml^?1 respectively. In contrast, the analogous p‐dimethylaminobenzyl derivative (3) is effective only against Escherichia coli (MIC = 3.12 µg ml^?1). Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis and characterization of copper(II) complexes incorporating pyrazolyl-derived N,S-donor bidentate ligands

The novel bidentate N,S-donor anionic ligands [PhNCSIndz]^?, [PhNCSImz]^?, [PhNCSPz^Me3]^?, and [EtNCSPz]^?, where Indz?=?indazole, Imz?=?imidazole, Pz^Me3?=?3,4,5-trimethylpyrazole, and Pz?=?pyrazole, were synthesized and used to prepare Cu(II) complexes of general formula [Cu(N^S)₂]. The ligands were synthesized via direct addition of phenylisothiocyanate or ethylisothiocyanate into THF suspensions of the corresponding sodium-pyrazolate salts. All of the synthesized compounds, including the ligands and the complexes, were characterized by physico-chemical and spectroscopic methods, and crystal structures of [Cu(EtNCSPz)₂] and [Cu(PhNCSPz^Me2)₂] were determined by X-ray diffraction analysis, showing a trans-square planar geometry for [Cu(EtNCSPz)₂] and a distorted tetrahedral geometry for [Cu(PhNCSPz^Me2)₂].     

Ion-Molecule reactions in methylamine and dimethylamine and trimethylamine systems

Fourier transform ion cyclotron resonance mass spectrometry has been used to measure the reaction rates for ions derived from methylamine with dimethylamine or trimethylamine. The use of the selective ion ejection technique greatly simplifies the elucidation of the ion-molecule reaction channels. The rate constants for proton transfer from protonated metwlamine, CH₃NH ₃ ⁺ (m/z 32), to dimethylamine and trimethylamine are 16.1 ± 1.6 × 10^?10 and 9.3 ± 0.9 × 10^?10 cm³ molec^?1s^?1, respectively. The rate constants for charge transfer from methylamine molecular ion, CH₃NH ₂ ⁺ (m/z 31), to dimethylamine and trimethylamine are 9.3 ± 1.8 x 10^?10 and 15.0 ± 5 × 10^?10 cm³molec^?1s^?1, respectively.     

A thermal behaviour and structural study of bis(hydroxamato)oxovanadium(IV) complexes

The bis(hydroxamato)oxovanadium(IV) complexes of composition [VO(IAH)₂)] (I), [VO(IBH)₂)] (II) and [VO(ICH)₂)] (III) (where IAH = indole-3-acetohydroxamate; (C₉H₈NCONHO^?); IBH = indole-3-butyrohydroxamate; (C₁₁H₁₂NCONHO^?); ICH = indole-2-carbohydroxamate; (C₈H₆NCONHO^?)) synthesized form the reactions of VOSO₄·5H₂O with bi-molar amounts of potassium salts of the respective hydroxamic acids in methanol have been characterised by elemental analyses, magnetic moment measurements and IR spectral studies. The thermal behaviour of complexes has been studied by TG and DTA techniques. Thermograms indicated that all complexes decompose in two steps yielding [VO(IAH)], [VO(IBH)] and [VO(ICH)] as intermediate of respective complexes and VO₂ as the final product of decomposition in each case. From the initial decomposition temperatures (IDT), the order of thermal stability for the complexes has been inferred as II > I > III.     

Complexation Properties of N,N′,N″,N′′′‐[1,4,7,10‐Tetraazacyclododecane‐1,4,7,10‐tetrayltetrakis(1‐oxoethane‐2,1‐diyl)]tetrakis[glycine] (H4dotagl). Equilibrium,Kinetic, and Relaxation Behavior of the Lanthanide(III) Complexes

Eu³⁺, Dy³⁺, and Yb³⁺ complexes of the dota‐derived tetramide N,N′,N″,N′′′‐[1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrayltetrakis(1‐oxoethane‐2,1‐diyl)]tetrakis[glycine] (H₄dotagl) are potential CEST contrast agents in MRI. In the [Ln(dotagl)] complexes, the Ln³⁺ ion is in the cage formed by the four ring N‐atoms and the amide O‐atom donor atoms, and a H₂O molecule occupies the ninth coordination site. The stability constants of the [Ln(dotagl)] complexes are ca. 10 orders of magnitude lower than those of the [Ln(dota)] analogues (H₄dota=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). The free carboxylate groups in [Ln(dotagl)] are protonated in the pH range 1–5, resulting in mono‐, di‐, tri‐, and tetraprotonated species. Complexes with divalent metals (Mg²⁺, Ca²⁺, and Cu²⁺) are also of relatively low stability. At pH>8, Cu²⁺ forms a hydroxo complex; however, the amide H‐atom(s) does not dissociate due to the absence of anchor N‐atom(s), which is the result of the rigid structure of the ring. The relaxivities of [Gd(dotagl)] decrease from 10 to 25°, then increase between 30–50°. This unusual trend is interpreted with the low H₂O‐exchange rate. The [Ln(dotagl)] complexes form slowly, via the equilibrium formation of a monoprotonated intermediate, which deprotonates and rearranges to the product in a slow, OH^?‐catalyzed reaction. The formation rates are lower than those for the corresponding Ln(dota) complexes. The dissociation rate of [Eu(dotagl)] is directly proportional to [H⁺] (0.1–1.0M HClO₄); the proton‐assisted dissociation rate is lower for [Eu(H₄dotagl)] (k₁=8.1?10^?6 M ^?1 s^?1) than for [Eu(dota)] (k₁=1.4?10^?5 M ^?1 s^?1).     

Synthesis of Two Derivatives of 3,6,9‐tri(carboxymethyl)‐3,6,9‐triazaundecanedioic Acid and the Stabilities of Their Complexes with Ca2+, Cu2+, Zn2+, Dy3+ and Gd3+

Two N‐2‐hydroxy‐1‐phenylethyl and N‐2‐hydroxy‐2‐phenylethyl derivatives of DTPA (3,6,9‐tri(carboxymethyl)‐3,6,9‐triazaundecanedioic acid), DTPA‐H1P = 3,9‐di(carboxymethyl)‐6‐2‐hydroxy‐1‐phenylethyl‐3,6,9‐triazaundecanedioic acid, and DTPA‐H2P = 3,9‐di(carboxymethyl)‐6‐2‐hydroxy‐2‐phenylethyl‐3,6,9‐triazaundecanedioic acid were synthesized. Their protonation constants were determined by Potentiometric titration in 0.10 M Me₄NNO₃ and by NMR pH titration at 25.0 ± 0.1°C. The formations of lanthanide(III), copper(II), zinc(II) and calcium(II) complexes were investigated quantitatively by potentiometry. The stability constant for Gd(III) complex is larger than those for Ca(II), Zn(II) and Cu(II) complexes with these two ligands. The selectivity constants and modified selectivity constants of the DTPA‐H1P and DTPA‐H2P for Gd(III) over endogenously available metal ions were calculated. Comparing pM values at physiological pH 7.4 assesses effectiveness of these two ligands in binding divalent and trivalent metal ions in biological media. The observed water proton relaxivity values of [Gd(DTPA‐H1P)]^? and [Gd(DTPA‐H2P)]^? became constant with respect to pH changes over the range of 4‐10. ¹⁷O NMR shifts showed that the [Dy(DTPA‐H1P)]^? and [Dy(DTPA‐H2P)]^? complexes at pH 6.30 had 1.91 and 2.28 inner‐sphere water molecules, respectively. Water proton spin‐lattice relaxation rates of [Gd(DTPA‐H1P)]^? and [Gd(DTPA‐H2P)]^? complexes were also consistent with the inner‐sphere Gd(III) coordination.     

Interaction Between the Low Molecular Mass Components of Blood Serum and the Vanadium(III)–2,2′-Bipyridine System

The complex species formed between vanadium(III)?C2,2??-bipyridine (Bipy) and the small blood serum bioligands lactic (HLac), oxalic (H₂Ox), citric (H₃Cit) and phosphoric (H₃PO₄) acids were studied in aqueous solution by means of electromotive forces measurements emf(H) at 25?°C and 3.0?mol?dm^?3 KCl as the ionic medium. The data were analyzed using the least-squares computational program LETAGROP, taking into account the hydrolytic products of vanadium(III) and the binary complexes formed. Formation of the complexes [V(Bipy)(Lac)]²⁺, [V(Bipy)(Lac)₂]⁺, [V(OH)₂(Bipy)(Lac)] and [V₂O(Bipy)₂(Lac)₂]^? were observed in the vanadium(III)?CBipy?CHLac system. Also, the species [V(Bipy)(HOx)]²⁺, [V(Bipy)(Ox)]⁺, [V(OH)(Bipy)(Ox)], [V(OH)₂(Bipy)(Ox)]^? and [V(OH)₃(Bipy)(Ox)]^2? were found in the vanadium(III)?CBipy?CH₂Ox system, the complexes [V(Bipy)(HCit)]⁺, [V(Bipy)(Cit)], [V(OH)(Bipy)(Cit)]^? and [V(OH)₂(Bipy)(Cit)]^2? were found in the vanadium(III)?CBipy?CH₃Cit system, and the species [V(Bipy)(H₂PO₄)]²⁺ and [V(Bipy)(HPO₄)]⁺ were detected in the vanadium(III)?CBipy?CH₃PO₄ system. The stability constants of these complexes were determined.     

Tuning the Halogen/Hydrogen Bond Competition: A Spectroscopic and Conceptual DFT Study of Some Model Complexes Involving CHF2I

Insight into the key factors driving the competition of halogen and hydrogen bonds is obtained by studying the affinity of the Lewis bases trimethylamine (TMA), dimethyl ether (DME), and methyl fluoride (MF) towards difluoroiodomethane (CHF₂I). Analysis of the infrared and Raman spectra of solutions in liquid krypton containing mixtures of TMA and CHF₂I and of DME and CHF₂I reveals that for these Lewis bases hydrogen and halogen‐bonded complexes appear simultaneously. In contrast, only a hydrogen‐bonded complex is formed for the mixtures of CHF₂I and MF. The complexation enthalpies for the C?H ??? Y hydrogen‐bonded complexes with TMA, DME, and MF are determined to be ?14.7(2), ?10.5(5) and ?5.1(6) kJ mol^?1, respectively. The values for the C?I ??? Y halogen‐bonded isomers are ?19.0(3) kJ mol^?1 for TMA and ?9.9(8) kJ mol^?1 for DME. Generalization of the observed trends suggests that, at least for the bases studied here, softer Lewis bases such as TMA favor halogen bonding, whereas harder bases such as MF show a substantial preference for hydrogen bonding.     

Synthesis of the pentacarbonyl(chalcocarbonyl)chromium(0) complexes,Cr(CO)5(CX) (X = S,Se)

The arene complexes, (η⁶-C₆H₆)Cr(CO)₂(CX) (X = S, Se), react with excess CO gas under pressure in tetrahydrofuran at about 60° C to produce the Cr(CO)₅(CX) complexes in high yield. The IR and NMR (¹³C and ¹⁷O) spectra of these complexes are in complete accord with the expected C_4v molecular symmetry. Like the analogous W(CO)₅(CS) complex, both compounds react with cyclohexylamine to give Cr(CO)₅(CNC₆H₁₁). However, while W(CO)₅(CS) undergoes stereospecific CO substitution with halide ions (Y^? to form trans-[W(CO)₄(CS)Y]^?, the two chromium chalcocarbonyl complexes apparently undergo both CO and CX substitution to afford mixtures of [Cr(CO)₅Y]^? and trans-[Cr(CO)₄(CX)Y]^?.     

A Prospect for the Analysis of Species Acting as Enzyme Inhibitors as Illustrated by Heavy Metal Inhibition

Abstract

A flow system incorporating an amperometric glucose oxidase enzyme electrode has been used to study the inhibitory effects of 16 metal cations on glucose oxidase. Only copper(II), mercury(II) and silver(I) caused any significant inhibition. the enzyme electrode could be reactivated by EDTA, the reactivation being most effective for copper(II) and least so for silver(I). Other complexing agents were tried for reactivation but proved to be unsatisfactory.

The ability to reactivate the enzyme on the electrode following copper(II) inhibition, and the linear response of the system to the level of this inhibitor according to I/A = -9.49 × 10^?7 log([Cu]/M) + 4.84 × 10^?8; r = 0.994 between 2.5 × 10^?4M and 5 × 10^?3M [Cu]²⁺ indicates a prospect for the use of a flow system for determining enzyme inhibitors in samples.