Antitumor activities of newly synthesized 5-carbamoyl-1 H-imidazol-4yl 1-adamantanecarboxylate and 5-carbamoyl-1H-imidazol-4yl piperonylate.
In synthetic studies on the chemical modification of the nucleoside antibiotic bredinin, two new derivatives, 5-carbamoyl-1H-imidazol-4-yl 1-adamantanecarboxylate and 5-carbamoyl-1H-imidazol-4-yl piperonylate, were found to possess a potent antitumor activity in several experimental tumor systems, even though bredinin itself shows only in vitro cytotoxicity and thus lacks therapeutic effectiveness. These two derivatives of bredinin exhibited antitumor activity against a wide variety of tumors, including leukemias L1210 and P388, Lewis lung carcinoma, B16 melanoma, Colon 26 and 38 adenocarcinomas. Ehrlich carcinoma, and Sarcoma 180. It is noteworthy that these agents showed good therapeutic effects not only against ascitic types of tumors but also against a number of slow-growing solid tumor lines, particularly the ascitic and solid forms of Ehrlich carcinoma. At their optimal doses, both compounds effected a complete cure of all or most of the mice treated. Although the mechanisms of action of these compounds remain unknown, they are able to suppress in vivo tumor growth, presumably by being slowly anabolized in vivo to an active form and inhibiting purine de novo synthesis as bredinin does.
Mode of action of bredinin with guanylic acid on L5178Y mouse leukemia cells.
Moderate concentrations of bredinin (1.2 X 10(-5) M) strongly inhibited growth of L5178Y cells, with the effect being reversed by guanylic acid (GMP). However, at higher concentrations of breeding the inhibition was not reversed completely by GMP added in excess. Bredinin was cytocidal at concentrations above 2 X 10(-5) M, but 5 X 10(-5) M bredinin in the presence of excess GMP, bredinin was cytostatic. Bredinin inhibited nucleic acid synthesis of L5178Y cells, but bredinin itself was not incorporated into the nucleic acid. Inhibition of nucleic acid synthesis was clearly reversed by GMP. Similarly chromosomal aberrations in L5178Y cells caused by bredinin were reversed by GMP. In contrast, the effect of ahigh concentration of bredinin on cell multiplication was not reversed by GMP. The modal volume of L5178Y cells increased during incubation in the presence of bredinin and GMP for 24 hours, 5 X 10(-5) M bredinin with GMP causing a 70% increase in cell volume. This increase in cell volume was mainly due to an increase in the protein content of the cells. The cytostatic effect of bredinin with GMP was reversed completely by adenosine-3',5'-cyclic monophosphate (cyclic AMP). Other cyclic nucleotides and nucleotides were ineffective. The reversing effect of cyclic AMP on cell survival depended upon the concentration of GMP, and was not seen in the absence of GMP. It was concluded that cyclic AMP influences the secondary cytostatic effect of bredinin, and not the primary cytotoxic effect reversed by GMP.
New method for analysis of enzymatic reactions: Thermolysin-catalyzed synthesis of aspartame precursor
Aspartame is an artificial sweetener which is 200 times sweeter than sucrose. A precursor of aspartame, N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-APM) was synthesized and hydrolyzed with thermolysin in an aqueous monophasic system. Graphical plotting methods and/or nonlinear regression have been used to analyze enzymatic reactions because of the nonlinearity of enzymatic rate equations. There are, however, problems: the former require accurate measurement of the initial reaction rate and the latter gives no analytical information. The authors proposed a method to solve the nonlinear enzymatic rate equations with respect to the concentration of a product with the Taylor expansion, especially for the reversible Theorell-Chance mechanism (Murakami, Y., Hirata, M., and Hirata, A., J. Ferment. Bioeng., 82, 246–252, 1996). This solution, however, is complex for practical use. In this paper, a simpler solution to explain the thermolysin-catalyzed synthesis of Z-APM is obtained. New methods to determine the kinetic parameters, especially for the reversible Theorell-Chance mechanism, based on the approximated solution are proposed. The results calculated using the obtained kinetic parameters can satisfactorily explain the experimental results, which demonstrates the effectiveness of the proposed analysis. We must know the relation between the concentrations of substrates and a product, and accurately evaluate of the result obtained using Taylor expansion when we determine the kinetic parameters using our newly developed methods. The methods to determine the kinetic parameters of the reversible Theorell-Chance mechanism described in this paper is, however, expected to be applied to general enzymatic reactions.