Reactions of 3-ethoxy-2-oxobutyraldehyde bis(N4-dimethylthiosemicarbazonato) cadmium with tumor cells.

The cytotoxic properties of a bis(thiosemicarbazonato) cadmium complex are studied. Preincubation of Ehrlich cells with the complex prevents growth of the ascites tumor in mice. Although the complex inhibits tumor growth without undue initial toxocity, longer-term side effects limit the use of the compound. The complex inhibits incorporation of 3H-thymidine into DNA and the respiration of tumor cells. It is shown in the principal correlation that the complex is more inhibitory of the above biochemical processes than cadmium ion at equal cellular concentrations of the metal. In addition the cellular reactions of the cadmium, zinc, and copper bis(thiosemicarbazonato) complexes are compared. It is shown that subtle chemical differences in their chelate structures appear to be responsible for their marked differences in cellular reactivity.     

Properties of the binding of copper by bleomycin

The glycopeptide, bleomycin, binds metal ions including Cu²⁺. It is the copper complex of this material that is isolated from Streptomyces verticillus. Both free ligand and copper complex are excellent antitumor agents in animals. The biochemical and pharmacological relationship between these compounds has not been established. The present study begins an analysis of the chemistry and biochemistry of copper-bleomycin with structural and equilibrium properties of the complex. Potentiometric and fluorometric titrations of bleomycin confirm three acidic groups with pK_a values of 7.50, 4.93, and 2.72. The conjugate nitrogen bases of these groups, comprise three of the binding sites for Cu²⁺ according to similar titrations of copper-bleomycin. The fourth is a conjugate base of an acid with a very large pK_a that cannot be measured by these techniques. The participation of a fourth such group is inferred from both proton release studies of the binding of metal and ligand above pH 8 and from several studies of the thermodynamic stability of copper bleomycin. At low pH binding of copper to bleomycin occurs in two steps, as observed by several independent techniques which monitor either the metal or the ligand. Log stability constants for the reactions Cu²⁺ + H_kBlm ? CuH_k-nBlm + nH⁺ and CuH_k-nBlm ? CuH_k-n-rBlm + rH⁺ are 1.32 and ?4.31, respectively, with n of 2.21 in the first equation and r of 2.07 in the second equation. The derived logarithm of the pH independent stability constant for copper bleomycin multiplied by the protonation constant for the unknown fourth ligand in the binding site is 12.16. This agrees closely with values obtained from measurements of conditional formation constants. One of the groups which binds in the second reaction is the substituted pyrimidine.     

Electronic substituent effects upon properties of 5-substituted-2-formylpyridine thiosemicarbazones and their metal complexes

The protonation constants of several 5-substituted-2-formylpyridine thiosemicarbazones, formation constants for their copper complexes, adduct formation constants of these complexes with ethylenediamine, protonation constants of the copper complexes, and half-wave reduction potentials of the copper and corresponding iron complexes have been determined. The electronic effect of substituents has been examined through the calculation of linear free energy correlations utilizing Hammet substituent constants as the independent parameter in the relationships. The effect of substituents upon the pharmacological properties of thiosemicarbazones is reconsidered here. The current results are used to suggest new experiments involving the reaction of 5-substituted-2-formylpyridine thiosemicarbazonato copper(II) complexes with Ehrlich cells.     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of bleomycin.     

Early reactions of cadmium with Ehrlich cells.

Ehrlich ascites tumor cells accumulate cadmium against a concentration gradient in a bisphasic uptake process. There is little efflux of the metal from preloaded cells into a cadmium-free medium. Incorporation of 3H-thymidine into DNA is markedly inhibited by cadmium ion at 5-100 ng atoms of Cd/mg of cell protein, but uptake of the nucleoside label into cells is not depressed in this concentration range. Cell respiration is much less affected by cadmium ion despite the sensitivity of isolated mitochondria to the metal. Model experiments using several cadmium complexes with known conditional formation constants show that bovine heart mitochondria have strong affinity for cadmium ion. The contrast between this result and the resistance of cells to respiratory inhibition with cadmium ion is discussed to illustrate the difficulty in relating in vitro studies to the cell. The behavior of cadmium ion with the Ehrlich cell is compared with data for zinc ion to reveal similarities in inhibition of nucleoside metabolism and respiration but a sharp difference in transport properties.