Cytokinin activity of ureidopurine derivatives related to a modified nucleoside found in transfer RNA.

By use of a radioactive labeling technique N-(nebularin-6-ylcarbamoyl)threonine has been detected in plant transfer RNA. Derivatives of this nucleoside promote cell division of a cytokinin-requiring soya bean tissue.

Identification of cis-dichloro-bis-isopropylamine platinum(II) as a major metabolite of iproplatin in humans.

Iproplatin is a quadrivalent second-generation platinum complex undergoing clinical evaluation. In plasma and urine of patients receiving this drug, iproplatin- and platinum-containing metabolites of iproplatin were separated by reverse-phase gradient high performance liquid chromatography. One of the metabolites was identified by cochromatography and electron impact mass spectrometry as cis-dichloro-bisisopropylamineplatinum(II), a metabolite formed by reduction of iproplatin. Incubation of iproplatin with ascorbic acid and cysteine, in vitro, indicates that iproplatin can be easily reduced to cis-dichloro-bis-isopropylamineplatinum(II) by reducing agents. It is hypothesized that the reduction of the quadrivalent complex iproplatin to cis-dichloro-bisisopropylamineplatinum(II) occurs intracellularly.

Isolation and characterization of a novel nucleoside, 7-beta-D-ribofuranosylhypoxanthine, from the urine of a chronic myelogenous leukemia patient.

A novel nucleoside, in the amount of 400 micrograms, was isolated from a 24-h collection of urine of a chronic myelogenous leukemia patient. On the basis of ultraviolet, nuclear magnetic resonance, and mass spectrometry and chromatography, its structure was established to be 7-beta-D-ribofuranosylhypoxanthine. The ultraviolet and mass spectral data and the thin layer chromatographic mobilities of the natural material were identical to those of a synthetic sample. High performance liquid chromatographic retention times and the coinjection high performance liquid chromatography of the natural material with the synthetic samples of the alpha and beta-anomers of 7-ribofuranosylhypoxanthines further confirmed the identity of the isolated material as 7-beta-D-ribofuranosylhypoxanthine.

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.

Quantitative aspects of metal ion binding to certain transfer RNA anticodon loop modified nucleosides.

Magnesium and manganese ions bind strongly to the unusual transfer RNA anticodon loop nucleotides, N-[(9-beta-D-ribofuranosyl-9H-purin-6-yl)carbamoyl]-L-threonine 5'-monophosphate (pt6A) and uridine-5-oxyacetic acid 5'-monophosphate (pV). Potentiometric measurements have shown that the delta G for metal ion-pt6A complex formation is 2-3-times more exothermic than for AMP. Electron-nuclear longitudinal dipolar relaxation data yielded manganese-ligand atom distances which permit a three-dimensional construct of the complex in which metal is coordinated to the phosphate, carboxylate of the threonine side-chain (with the nucleotide in the anti glycosidic conformation) and N7 of the adenine ring. Similarly, manganese binds strongly to pV, involving phosphate and carboxylate functions. It is possible that a facet of the functional role of these unusual residues is to chelate magnesium ions and in so doing permit optimum anticodon loop conformational stability and stability of tRNA-mRNA-ribosome complexes.

Purinylhydantoins. Facile conversion of the naturally occurring N-(purin-6-ylcarbamoyl)-L-amino acids into 3-pruin-6-ylhydantoins and 3-cyclohexyl-1-(purin-6-ylcarbamoyl)hydantoins.

The naturally occurring N-(purin-6-ylcarbamoyl)-L-threonine (PCT, 1b), N-(purin-6-ylcarbamoyl)glycine (PCG, 1a), and some of their analogs were converted into novel purine derivatives, the purinylhydantoins. The PCT and PCG underwent intramolecular cyclization in the presence of N,N-dicyclohexylcarbodiimide (CDD) to give the 3-purin-6-ylhydantoins (2a-c). The same hydantoins were also obtained when the PCT and PCG were allowed to react through the mixed anhydride formed from cyclohexyl isocyanate or ethyl chloroformate. 1,3-Dicyclohexyl-1-(N-(purin-6-ylcarbamoyl)aminoacyl)ureas 3a and 3c, by-products obtained from the DCC reaction, were rapidly converted in aqueous NaOH to another type of purinylhydantoins, the 3-cyclohexyl-1-(purin-6-ylcarbamoyl)hydantoins 4a and 4b. Compound 4a when heated in base underwent hydrolysis of the hydantoin ring giving biuret N-(cyclohexylcarbamoyl)-N-(purin-6-ylcarbamoyl)glycine (5a) and N-(purin-6-ylcarbamoyl)glycine cyclohexylamide (6a). The characterization of these hydantoins was carried out by uv, nmr, and mass spectrometry. The 3-purin-6-ylhydantoins and 3-cyclohexyl-1-(purin-6-ylcarbamoyl)hydantoins showed growth inhibitory activity in the cultured leukemic cells, while the parent amino acid compounds were inactive.

Synthesis and biological activities of some N6-(nitro- and -aminobenzyl)adenosines.

Synthesis and biological activities of 12 analogs of N6-benzyladenosine are described. The compounds were prepared by two methods: (1) direct alkylation of adenosine with an appropriately substituted benzyl bromide to give the N1-substituted derivative which was then rearranged in base to give the N6-substituted compound, and (2) by nucleophilic displacement of chlorine in 6-chloropurine ribonucleoside, 6-chloro-2-aminopurine ribonucleoside, and 6-chloro-2-aminopurine with an amine. These analogs were examined for their growth inhibitory effect in cultured leukemic cells and also for their effect on adenosine aminohydrolase activity. N6-p-Nitrobenzyladenosine and its 2'-deoxy analog were competitive inhibitors (K1 65, 22 MUM). The 2-amino-N6-p-nitrobenzyladenine and its ribonucleoside were found to be noncompetitive inhibitors of adenosine aminohydrolase. In cultured L1210 leukemia, 2-amino-6-p-nitrobenzylaminopurine and the corresponding ribonucleoside were better growth inhibitors than N6-benzyladenosine, while N6-p-nitrobenzyladenosine, its 2'-deoxy analog, and N6-p-fluorobenzyladenosine were as active as N6-benzyladenosine.

Synthesis and biological activities of some N4-substituted 4-aminopyrazolo(3,4-d)pyrimidines.

Syntheses and biological activities of 26 N4-substituted 4-aminopyrazolo[3,4-d]pyrimidines as analogs of naturally occurring modified nucleic acid bases, N-(purin-6-ylcarbamoyl)-L-threonine and N6-(delta2-isopentenyl)adenine, are described. 4-Aminopyrazolo[3,4-d]pyrimidine was converted into the desired intermediate, ethyl pyrazolo[3,4-d]pyrimidine-4-carbamate (2). 4-Ureidopyrazolo[3,4-d]pyrimidines (6-26) were prepared by displacement of the ethoxy group of the carbamate 2 by amino acids and a variety of amines and by a reaction of 4-aminopyrazolo[3,4-d]pyrimidine (1) with isocyanates. N4-Alkylaminopyrazolo[3,4-d]pyrimidines were generally prepared by displacement of the chlorine from 4-chloropyrazolo[3,4-d]pyrimidine with various amines. Several analogs exhibited moderate to very good growth inhibitory activities against cultured L1210 leukemia and 6410 human leukemic myeloblasts.

Synthesis and antitumor activity of 5'-phosphates and cyclic 3',5'-phosphates derived from biologically active nucleosides.

Syntheses and biological activities of 12 N6-substituted adenosine 5'-phosphates and 15 cyclic 3',5'-phosphates are described. Included among these are the cyclic phosphates of the naturally occurring anticodon adjacent modified nucleosides, N6-(delta2-isopentenyl)adenosine and N-(purin-6-ylcarbamoyl)-L-threonine ribonucleoside. Also reported in this paper are the 5'-phosphates and cyclic phosphates of the cytokinins, N6-benzyladenosine, kinetin ribonucleoside, 3-(chloro-trans-2-buten-2-yl)adenosine,6-o-chlorophenylureidopurine ribonucleoside, and 6-allylureidopurine ribonucleoside. The 5'-nucleotides were prepared by direct phosphorylation of the corresponding ribonucleosides with POCl3 and triethyl phosphate. These compounds were converted to the cyclic 3',5'-phosphates by cyclization of the corresponding 5'-nucleotides with dicyclohexylcarbodiimide. Comparison of the cytotoxicity of the ribonucleosides with their 5'-nucleotides and cyclic 3',5'-nucleotides showed that some of the 5'-phosphates and cyclic phosphates were almost as active as the parent nucleosides. The 5'-nucleotides and the cyclic phosphates were more soluble than the parent nucleosides. The cyclic 3',5'-nucleotides were examined as alternate activators of cAMP-dependent protein kinase from beef heart. While all of the analogs studied showed some activity toward this enzyme, several compounds were more effective than cAMP itself. The analogs were also tested as substrates for cyclic 3',5'-nucleotide phosphodiesterase from beef heart. The N6-alkyl-cAMP analogs were poor substrates for the enzyme, while N6-carbamoyl-cAMP derivatives were inert toward this enzyme. These compounds did not inhibit the phosphodiesterase. Some of the cyclic phosphates exhibited marginal effect in the inhibition of glycogen synthesis in skin slices.

Radioimmunoassay for a novel urinary nucleoside, N6-succinyladenosine.

A sensitive radioimmunoassay has been developed for an unusual urinary nucleoside, N6-succinyladenosine (N6-SAR). Antibodies were raised in rabbits and the antibody specificity was established by binding inhibition radioimmunoassay of compounds with structural similarity to N6-SAR. The assay is sensitive enough to detect up to a nanomole of N6-SAR in 1 ml of urine. The mean N6-SAR urinary excretion value for normal adults was 4.09 mg/gm of creatinine with a standard deviation of 1.22. The measurement of N6-SAR in the urines of a number of subjects with metastatic malignant disease suggests that these patients excreted in their urines elevated amounts of N6-SAR in comparison to that in the urines of normal subjects.

Synthesis and biological activity of N6-(n-alkylureido)purine ribonucleosides and their 5'-phosphates.

Syntheses and biological activities of 12 N6-(n-alkylureido)purine ribonucleosides (alkyl chain length of 1--10, 16, and 18 carbons) and three N6-(n-alkylureido)purine ribonucleoside 5'-phosphates (chain length of 4, 9, and 10 carbons) are described. The N6-(n-alkylureido)purine ribonucleosides were prepared by a reaction of (2',3',5'-tri-O-acetyl-beta-D-ribofuranosyl)-9H-purine-6-carbamate and n-alkylamine in refluxing pyridine. The 5'-nucleotides were prepared by direct phosphorylation of the corresponding ribonucleoside with phosphorus oxychloride and triethyl phosphate. Some N6-(n-alkylureido)purine ribonucleosides (n-octyl, n-nonyl, and n-decyl) and their nucleotides showed a marked antiproliferative activity against L-1210 cells in culture.

Metabolic fate of N6-benzyladenosine and N6-benzyladenosine-5'-phosphate in rats.

The radiolabeled antitumor nucleoside (14C-8)-N6-benzyladenosine and its (14C-8)-5'-phosphate were administered to rats intravenously, and their metabolic fate was studied. Twenty-nine percent of the radioactivity was recovered in the 48-hr urine collection after (14C-8)-N6-benzyladenosine administration. The following metabolites were isolated: unchanged N6-benzyladenosine (20%), adenine (12%), uric acid (5%), and N6-benzyladenine (0.3%). In the case of (14C-8)-N6-benzyladenosine-5'-phosphate, a total of 28% of the radioactivity was recovered in the 48-hr urine collection and the following metabolites were isolated: N6-benzyladenosine (40%), uric acid (12%), adenine (trace), and unidentified urea derivatives (30%). Metabolism of N6-benzyladenosine appears to involve N-debenzylation to some extent, followed by conversion to adenine and uric acid. N6-Benzyladenosine and its 5'-phosphate differ from other adenosine analogs in being retained in significant amounts by the animals.