Table 4.

Summary of results of modifications to the ribofuranosyl moiety

Furan conformation There was an absolute requirement for the sugar moiety to be in a furanosyl-like conformation. Neither of the acyclo-adenosine analogs tested (50–51), nor any of the twelve pyranoses assayed (52–63) were substrates for Ado kinase. It is unlikely that many of these compounds were recognized by the active site despite the presence of the adenine base as evidenced by the fact that only 9- (2-deoxy-β-D-erythro-pentopyranosyl)-adenine (56) poorly inhibited the phosphorylation of 0.1 μM Ado at 100 μM. None of the other pyranoses or acyclo-Ado analogs were either substrates for, or inhibitors of, M. tuberculosis Ado kinase. Only modifications that maintained a β-D-ribofuranosyl-like conformation were substrates for M. tuberculosis Ado kinase.

2′ A trans-2′-hydroxyl group was preferred, although a cis-2′-hydroxyl group (araA, 15) and 2′-deoxy-Ado (9) were also poor substrates. Compounds with 2′-modifications were poor inhibitors unless they also had a second substitution at the 2-position.

3′ A trans-3′-hydroxyl group was nearly a requirement for substrate and inhibitor recognition, although 9-[β-D-xylofuranosyl]-adenine (23), which has a cis-3′-hydroxyl group, was a poor substrate with 1 nmol/mg-min of activity. Modifications at this position may be limited by steric hindrance.

4′-O The 4′-oxygen was the most flexible of the substitutions to the ribose moiety. Carbocyclic-Ado (3) was a good substrate for both human and M. tuberculosis Ado kinases. The selectivity for carbocyclic-Ado analogs can be improved with the addition of an exocyclic substitution at the 2-position of the adenine base.

5′ Substitutions to the 5-position were promising in terms of substrate activity and antitubercular activity. The best substrates in this category were 9-[α-L- lyxofuranosyl]-adenine (34) and 9-[α-L-lyxofuranosyl]-2-fluoroadenine (35), both maintained about 3% of the activity of Ado. Compounds with an additional 5′-methyl group in the allo and talo conformations (36–39) revealed that the allo- conformations resulted in better substrates and inhibitors of Ado kinase. Although it has yet to be confirmed, 5′-amino-5′-deoxy-Ado (32) appeared to be a substrate for this enzyme. This compound was also the best inhibitor of all of the substitutions to the ribose moiety.

Glycosidic bond position The preferred position for the N-glycosidic bond was at the 9-position of Ado. However, activity was measured with 3-[β-D-ribofuranosyl]-adenine (47) as well. Ado kinase did not recognize compounds as substrates when the bond was moved to the 7 or 8-positions on Ado (45, 46, and 48).