Cytokinins: Development of a Potent Antagonist

Abstract

A systematic search has resulted in the synthesis of a class of cytokinin antimetabolites. The development and biological properties of the anticytokinins are discussed in terms of one member of the class, 3-methyl -7 -(3 -methylbutylamino)pyrazolo [4,3 -dl-pyrimidine. way through which the cytokinin itself exerts its effect. We wish to report the discovery of a series of such compounds, the activity of which is exemplified here by one member of that series, 3-methyl-7-(3-methylbutylamino)pyrazolo [4,3-dipyrimidine (6).The distribution of cytokinins (factors promoting cell division and growth) in natural systems is widespread (1, 2) and includes compounds at the purine, ribonucleoside, ribonucleotide, and tRNA levels (3, 4). Plants presumably require cytokinins for growth, because growth of excised plant tissues is often dependent on an exogenous cytokinin supply. One cytokinin, 6-(3-methyl-2-butenylamino)purine (1) has been shown to be the active factor in the plant pathogen Corynelbacterium fascian8 (5). In addition, phytohemagglutininstimulated human lymphocyte cells are both stimulated and inhibited by 6-(3-methyl-2-butenylamino)-9-1-D-ribofuranosylpurine (2) with respect to DNA synthesis, transformation, -and mitosis (6). The-effect depended upon the stage of the cell cycle and the concentration of 2 employed. Compound 2 has antitumor activity in experimental animals (7, 8) and in preliminary clinical trials (9). In spite of this broad range of physiological activity and the occurrence of several different cytokinins in nature (1), including four as components of tRNA (10), as well as hundreds of synthetic cytokinins (1, 2), only one example of cytokinin antagonism has been reported in a compound that bears an obvious structural relationship to the known cytokinins (5). Although the structural relationship between this compound (6-methylaminopurine) and the -cytokinins suggests its potential utility in studies of the mechanism of cytokinin action, it would probably not be suitable, since it has a very slight activity as an antagonist. To date, no systematic search for compounds that would oppose the action of the cytokinins has been reported.The synthesis of a potent cytokinin antagonist is of considerable interest, since it could extend the study of cytokinins to biological systems that do not require any exogenously added cytokinins, presumably because they produce their own. The antagonist that blocks the action of the endogenous cytokinins in these systems would thus make the tissue cytokinin-dependent. The antagonist would be of greatest utility, of course, if it acted in a reversible manner on the same path- (12). In order to detect anticytokinin activity, the growth of tobacco callus was observed on a standard medium to which either 6-(3-methyl-2-butenylamino)purine or 6-benzylaminopurine had been added (over a range of concentrations) and to which the other compounds (3-6) were also added in different concentrations. RESULTS AND DISCUSSIONThe combined effects of cytokinin and antagonist ...

Competitive Inhibition of Beef Heart Cyclic AMP Phosphodiesterase by Cytokinins and Related Compounds

“…In spite of the widespread natural occurrence of cytokinins and the diverse metabolic effects which they are now known to promote in both plants and animals (1-13), relatively little has been learned about their mechanism of action at the molecular level. This prompted the design and synthesis of a class of potent anticytokinins, structurally related to the cytokinins, in the hope that the antimetabolites might extend the study of cytokinins to new biological systems and provide useful information pertinent to the mechanism of cytokinin action (14)(15)(16)(17).…”

“…13 t Many of these structural analogs of the cytokinins (e.g., 9 and 10), which bind competitively to the cyclic AMIP phosphodiesterase and mimic the effects of the cytokinins in human lymphocytes and mouse fibroblasts (S. M. Hecht and R. B. Frye, in preparation), are also anticytokinins in the tobacco bioassay. Although their mode of action in relation to the cytokinins cannot be exactly the same in the plant and mammalian assays, it is gratifying to note that the anticytokinins, which were postulated to function in the tobacco bioassay by binding to the same cellular "receptors" as the cytokinins (15,16), actually can act as structural analogs of the cytokinins in certain mammalian systems.…”

“…This prompted the design and synthesis of a class of potent anticytokinins, structurally related to the cytokinins, in the hope that the antimetabolites might extend the study of cytokinins to new biological systems and provide useful information pertinent to the mechanism of cytokinin action (14)(15)(16)(17).…”

“…In spite of the widespread natural occurrence of cytokinins and the diverse metabolic effects which they are now known to promote in both plants and animals (1-13), relatively little has been learned about their mechanism of action at the molecular level. This prompted the design and synthesis of a class of potent anticytokinins, structurally related to the cytokinins, in the hope that the antimetabolites might extend the study of cytokinins to new biological systems and provide useful information pertinent to the mechanism of cytokinin action (14)(15)(16)(17).On the basis of the similar effects obtained with N6-(A2-isopentenyl)adenosine and dibutyryl cyclic AMP, the cytokinins have been postulated to mediate their effects in phytohemagglutinin-treated human lymphocytes by involvement in cyclic AMP metabolism (13). This postulate was consistent with the finding that the cytokinin trans-zeatin ribonucleoside was inhibitory to the cyclic AMP phosphodiesterases from beef brain and crown-gall tumor cells, the latter of which was Abbreviations: Dibutyryl cyclic AM1P, N6,02'-(dibutyryl)adenosine cyclic 3': 5'-monophosphate; cyclic AMP, adenosine cyclic 3': 5'-monophosphate; trans-zeatin ribonucleoside, N6-(4-hydroxy-3-methyl-trans-2-butenyl)adenosine; 8-bromo …”

Biological Effects of Cytokinin Antagonists 7-(Pentylamino) and 7-(Benzylamino)-3-Methylpyrazolo(4,3-d)Pyrimidines on Suspension-cultured Tobacco Cells

“…On the basis that the growth inhibition observed at a given combination of analog and cytokinin concentrations was abolished or reduced in presence of higher concentrations of cytokinins, these analogs were described as cytokinin antagonists (1). The first to be described was 3-methyl-7-(3-methylbutylamino)pyrazolo (4,3-d)pyrimidine (1) and the most potent in a series of 7-substituted 3-methylpyrazolo(4,3-d)pyrimidines described later by Skoog et al (7), was 3-methyl-7-(pentylamino)pyrazolo(4,3-d)pyrimidine (I). Indeed, in the presence of 0.003 LM N6-(A2-isopentenyl)adenine, it was lethal at a concentration of 0.2 jAM and antagonist activity was detected at the minimal concentration of 0.03 lsm in the tobacco callus bioassay system (7).…”