"Reversine" and its 2-substituted adenine derivatives as potent and selective A3 adenosine receptor antagonists.
The dedifferentiation agent "reversine" [2-(4-morpholinoanilino)-N(6)-cyclohexyladenine 2] was found to be a moderately potent antagonist for the human A(3) adenosine receptor (AR) with a K(i) value of 0.66 microM. This result prompted an exploration of the structure-activity relationship of related derivatives, synthesized via sequential substitution of 6-chloro-2-fluoropurine with selected nucleophiles. Optimization of substituents at these two positions identified 2-(phenylamino)-N(6)-cyclohexyladenine (12), 2-(phenylamino)-N(6)-cycloheptyladenine (19), and 2-phenylamino-N(6)-endo-norbornyladenine (21) as potent A(3) AR ligands with K(i) values of 51, 42, and 37 nM, respectively, with 30-200-fold selectivity in comparison to A(1) and A(2A) ARs. The most selective A(3) AR antagonist (>200-fold) was 2-(phenyloxy)-N(6)-cyclohexyladenine (22). 9-Methylation of 12, but not 19, was well-tolerated in A(3) AR binding. Extension of the 2-phenylamino group to 2-benzyl- and 2-(2-phenylethylamino) reduced affinity. In the series of 2-(phenylamino), 2-(phenyloxy), and 2-(phenylthio) substitutions, the order of affinity at the A(3) AR was oxy > or = amino > thio. Selected derivatives, including reversine (K(B) value of 466 nM via Schild analysis), competitively antagonized the functional effects of a selective A(3) AR agonist, i.e., inhibition of forskolin-stimulated cAMP production in stably transfected Chinese hamster ovary (CHO) cells. These results are in agreement with other studies suggesting the presence of a lipophilic pocket in the AR binding site that is filled by moderately sized cycloalkyl rings at the N(6) position of both adenine and adenosine derivatives. Thus, the compound series reported herein comprise an important new series of selective A(3) AR antagonists. We were unable to reproduce the dedifferentiation effect of reversine, previously reported, or to demonstrate any connection between A(3) AR antagonist effects and dedifferentiation.
Perreira
Melissa
M
Chemical Biology Core Facility, and Molecular Recognition Section, Laboratory of Biological Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Jiang
Jian-Kang
JK
Klutz
Athena M
AM
Gao
Zhan-Guo
ZG
Shainberg
Asher
A
Lu
Changrui
C
Thomas
Craig J
CJ
Jacobson
Kenneth A
KA
D000225:Adenine / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000494:pharmacology; D058909:Adenosine A3 Receptor Agonists; D058918:Adenosine A3 Receptor Antagonists; D000818:Animals; D001667:Binding, Competitive; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D051379:Mice; D009025:Morpholines / Q000138:chemical synthesis* / Q000494:pharmacology; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects; D011687:Purines / Q000138:chemical synthesis* / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D013329:Structure-Activity Relationship; D014162:Transfection
D058909:Adenosine A3 Receptor Agonists; D058918:Adenosine A3 Receptor Antagonists; D009025:Morpholines; D011687:Purines; D000242:Cyclic AMP; D000225:Adenine; C484369:2-(4-morpholinoanilino)-6-cyclohexylaminopurine