Molecular Pharmacology, Vol 3, 386-395, Copyright © 1967 by the American Society for Pharmacology and Experimental Therapeutics

Inhibition of the Steroid-Induced Synthesis of ⁵-3-Ketosteroid Isomerase in Pseudomonas testosteroni by a New Purine Deoxyribonucleoside Analog: 6-Chloro-8-aza-9-cyclopentylpurine

MORRIS S. ZEDECK ¹, ALAN C. SARTORELLI ¹, PAULINE K. CHANG ¹, KAREL RAKA JR. ¹, ROLAND K. ROBINS ¹, and ARNOLD D. WELCH ¹

¹ Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

Analogs of 2'-deoxyadenosine (2'-AdR) were used to determine the structural requirements for inhibition of the steroid-induced synthesis of ⁵-3-ketosteroid isomerase in Pseudomonas testosteroni. Compounds more active than 2'-AdR included 4-aminopyrazolo[3,4-d]pyrimidine-2'-deoxyribonucleoside and 6-chloro-9-cyclopentylpurine. The data obtained with many compounds indicate that: (a) substitution of the amino group of adenine with chlorine enhances activity, (b) replacement of C with N in position 8 of the purine ring increases activity, (c) replacement of deoxyribose in position 9 of 2'-AdR with substituents containing a 2'-hydroxyl (as in adenosine, 3'-AdR, and 9-(2'-hydroxy-cyclopentyl)adenine) causes a loss of activity, while nonhydroxyl-containing substituents (as in 2',3',5'-trideoxyadenosine and 9-cyclopentyladenine) retain activity (thus, direct phosphorylation of the analogs is not prerequisite to inhibitory activity), and (d) unnatural derivatives of 2'-AdR containing either L-deoxyribose in -configuration or D-deoxyribose in -configuration are inactive. These findings were used to guide the design and synthesis of 6-chloro-8-aza-9-cyclopentylpurine (689). This compound, which cannot be phosphorylated directly, markedly inhibited the synthesis of induced enzymes in P. testosteroni at a concentration (0.3 mM) that was significantly less inhibitory to the synthesis of total protein and to the incorporation into protein of L-leucine-1-¹⁴C. The inhibition of ⁵-3-ketosteroid isomerase activity was not attributable either to prevention of uptake of the inducer or to direct inhibition of enzyme activity. These data suggest that 689 inhibits relatively selectively a process critically involved in the inductive synthesis in P.testosteroni of ⁵-3-ketosteroid isomerase.

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ACKNOWLEDGMENTS This investigation was supported by Research Grant CA-02817 from the U.S. Public Health Service. We are grateful to Dr. J. A. Montgomery (Southern Research Institute, Birmingham, Alabama) and to Dr. H. J. Schaeffer (State University of New York, Buffalo), who supplied many valuable compounds. We wish to thank Miss Ingrid Grove for technical assistance and Dr. L. J. Sciarini, who was very helpful in the preparation of compounds; during the early phase of the work, Mrs. Heidi Landau also was very helpful.