Synthesis of 8-Substituted Analogues of Cyclic ADP-4-Thioribose and Their Unexpected Identification as Ca2+-Mobilizing Full Agonists.

A series of 8-substituted analogues of cyclic ADP-4-thioribose (cADPtR, 3), which is a stable equivalent of Ca2+-mobilizing second messenger cyclic ADP-ribose (cADPR, 1), were designed as potential pharmacological tools for studies on cADPR-modulated Ca2+ signaling pathways. These 8-amino analogue (8-NH2-cADPtR, 4), 8-azido analogue (8-N3-cADPtR, 5), and 8-chloro analogue (8-Cl-cADPtR, 6) were efficiently synthesized, where the stereoselective N1-ß-thioribosyladenine ring closure reaction via an α/ß-equilibrium of the 1-aminothioribose derivative and construction of the characteristic 18-membered pyrophosphate ring by Ag+-promoted activation of a phenyl phosphorothioate type substrate were the two key steps. Although 8-NH2-cADPR (2) is a well-known potent antagonist against cADPR-inducing Ca2+-release, the 4-thioribose congener 8-NH2-cADPtR turned out unexpectedly to be a full agonist in sea urchin egg homogenate evaluation system. This important finding suggested that the ring-oxygen in the N1-ribose of cADPR analogues is essential for the antagonistic activity in the Ca2+-signaling pathway, which can contribute to clarify the structure-agonist/antagonist activity relationship.

Design, Synthesis, and Identification of 4″α-Azidoethyl-cyclic ADP-Carbocyclic-ribose as a Highly Potent Analogue of Cyclic ADP-Ribose, a Ca(2+)-Mobilizing Second Messenger.

Cyclic adenosine diphosphate-carbocyclic-ribose (cADPcR, 2) is a stable equivalent of cyclic adenosine diphosphate-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. On the basis of the structure-activity relationship of cADPR-related compounds and three-dimensional structural modeling of cADPcR, we designed and synthesized cyclic-ADP-4″α-azidoethyl carbocyclic-ribose (N3-cADPcR, 3) to demonstrate that it has a highly potent Ca(2+)-mobilizing activity (EC50 = 24 nM). N3-cADPcR will be a useful precursor for the preparation of biological tools effective to investigate cADPR-mediated signaling pathways.

Synthesis of 7-Deaza-cyclic Adenosine-5'-diphosphate-carbocyclic-ribose and Its 7-Bromo Derivative as Intracellular Ca(2+)-Mobilizing Agents.

Cyclic ADP-carbocyclic-ribose (cADPcR, 3) is a biologically and chemically stable equivalent of cyclic ADP-ribose (cADPR, 1), a Ca(2+)-mobilizing second messenger. We became interested in the biological activity of the 7-deaza analogues of cADPcR, i.e., 7-deaza-cADPcR (7) and its 7-bromo derivative, i.e., 7-deaza-7-Br-cADPcR (8), because 7-deazaadenosine is an efficient bioisostere of adenosine. The synthesis of 7 and 8 required us to construct the key N1-carbocyclic-ribosyl-7-deazaadenosine structure. Therefore, we developed a general method for preparing N1-substituted 7-deazaadenosines by condensing a 2,3-disubstituted pyrrole nucleoside with amines. Using this method, we prepared the N1-carbocyclic ribosyl 7-deazaadenosine derivative 10a, from which we then synthesized the target 7-deaza-cADPcR (7) via an Ag(+)-promoted intramolecular condensation to construct the 18-membered pyrophosphate ring structure. The corresponding 7-bromo derivative 8, which was the first analogue of cADPR with a substitution at the 7-position, was similarly synthesized. Biological evaluation for Ca(2+)-mobilizing activity in the sea urchin egg homogenate system indicated that 7-deaza-cADPcR (7) and 7-deaza-7-Br-cADPcR (8) acted as a full agonist and a partial agonist, respectively.

Design, Synthesis, and Chemical and Biological Properties of Cyclic ADP-4-Thioribose as a Stable Equivalent of Cyclic ADP-Ribose.

Here we describe the successful synthesis of cyclic ADP-4-thioribose (cADPtR, 3), designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, in which the key N1-ß-thioribosyladenosine structure was stereoselectively constructed by condensation between the imidazole nucleoside derivative 8 and the 4-thioribosylamine 7 via equilibrium in 7 between the α-anomer (7α) and the ß-anomer (7ß) during the reaction course. cADPtR is, unlike cADPR, chemically and biologically stable, while it effectively mobilizes intracellular Ca2+ like cADPR in various biological systems, such as sea urchin homogenate, NG108-15 neuronal cells, and Jurkat T-lymphocytes. Thus, cADPtR is a stable equivalent of cADPR, which can be useful as a biological tool for investigating cADPR-mediated Ca2+-mobilizing pathways.

Synthesis of 5''-branched derivatives of cyclic ADP-carbocyclic-ribose, a potent Ca2+-mobilizing agent: The first antagonists modified at the N1-ribose moiety.

The 5''-branched cyclic ADP-carbocyclic-ribose derivatives were designed and synthesized. These target compounds were identified as the first antagonists of cADPR without a substituent at the adenine 8-position, and were shown to be stable due to the N1-carbocyclic-ribosyl structure.