Cysteine Proteases and Their Inhibitors.

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor alpha and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-kappaB. However, TLR8 activation by R-848 and TLR2 activation by [S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)] were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

https://www.pnas.org/content/pnas/100/11/6646.full.pdf

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7

Palladium-assisted routes to nucleosides.

The importance of nucleoside analogues in chemotherapy and in other potential therapeutic approaches as immunomodulation or regulation of gene expression, is reviewed.

Nucleoside Analogues as Chemotherapeutic Agents: A Review

https://aasldpubs.onlinelibrary.wiley.com/doi/pdf/10.1002/hep.20839

Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection

Novel analogues of the naturally occurring purine nucleosides were synthesized in the thiazolo[4,5-d]pyrimidine ring system to determine the immunomodulatory effects of insertion of a sulfur atom in place of nitrogen at position 7 of the purine ring. In particular, 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (7, guanosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,5,7(3H,4H,6H) trione (8, xanthosine analogue), 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H)-dione (10, inosine analogue), and 7-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidin-2(3H)-one (32, adenosine analogue) were prepared, as well as the 8-mercaptoguanosine (14) and 6-mercaptoguanosine (17) analogues. Single-crystal X-ray studies confirmed the structural assignment of 17 and 32 as having the beta-configuration with the site of glycosylation at N3. The nucleosides were evaluated for their ability to potentiate various murine immune functions in direct comparison to the known active agents 8-bromoguanosine (1), 8-mercaptoguanosine (2), and 7-methyl-8-oxoguanosine (3). Two of the guanosine analogues, 7 and 14, were found to exhibit significant immunoactivity relative to the positive control compounds (1-3), while the adenosine, inosine, xanthosine, and 6-mercaptoguanosine analogues were devoid of activity. Compound 7 exhibited greater immunoactivity than any of the other guanosine analogues and derivatives in all test systems. Specifically, 7 was shown to be about twice as potent as 3 in the murine spleen cell mitogenicity assay. In addition, treatment with 7 produced about a 4-fold increase in natural killer cell cytotoxicity, while treatment with 3 afforded a 3-fold increase over controls. Finally, 7 provided excellent protection (92% survivors compared to 0% for placebo controls) against Semliki Forest virus in mice. Induction of interferon may account for the major mode of action of these guanosine analogues.

Thiazolo[4,5-d]pyrimidine nucleosides. The synthesis of certain 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidines as potential immunotherapeutic agents.

Synthesis of certain pyrazolo[3,4-d]pyrimidin-3-one nucleosides

Several sugar-modified nucleoside derivatives of the purine analogue 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7-dione (1) were synthesized. Phosphorylation of 1 using POCl3 resulted in 5'-monophosphate 2, which was subsequently converted to 3',5'-cyclic phosphate 3, by reported methods. 5'-Sulfamoyl derivative 4 was synthesized by treatment of the 2,3-O-isopropylidene derivative of 1 with chlorosulfonamide followed by acid deprotection. Compounds 5-7, the 5'-deoxy, the tri-O-acetyl, and the 2'-deoxy derivatives of 1, respectively, were synthesized by glycosylation of 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, the aglycon of 1, with the appropriate sugar moieties, utilizing the Vorbruggen procedure. Oxidative cleavage of the C2'-C3' bond in 1 followed by reduction with sodium borohydride led to "seco" analogue 8. Nucleosides 2-8 were evaluated for antiviral activity in vivo against the Semliki Forest virus. The activity of compounds 2, 5, and 7 were similar to that of 1. Cyclic phosphate 3 was toxic at the high dose and weakly active at the lower dose. Compounds 4, 6, and 8 were inactive in this system.

Synthesis and antiviral activity of certain guanosine analogues in the thiazolo[4,5-d]pyrimidine ring system.

Several guanosine analogues were synthesized in the pyrazolo[3,4-d]pyrimidine ring system with various substituents at the 3-position. The new analogues prepared here include the CH3 (2-amino-3-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13a), the phenyl (2-amino-3-phenyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13b), and the NH2 (3,6-diamino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)- one, 17) substituted derivatives. These new agents, as well as several other 3-substituted derivatives including H, Br, OCH3, COOH, and oxo, were evaluated for their ability to potentiate certain murine immune functions relative to the known active agent 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (4, 7-thia-8-oxoguanosine). The biological evaluation included the (1) ex vivo determination of increased natural killer cell function and (2) in vivo antiviral protection against a lethal challenge of Semliki Forest virus. The 3-unsubstituted (5a) and the 3-bromo (5c) derivatives were found to be the most active immunopotentiators in this series.

Guanosine analogues. Synthesis of nucleosides of certain 3-substituted 6-aminopyrazolo[3,4-d]pyrimidin-4(5H)-ones as potential immunotherapeutic agents.

Tubercidin (7-deazaadenosine, 1a) and several 6-chlorotuber-cidin derivatives were synthesized including 4-amino-6-chloro-7-β-D-ribofuranosylpyrrolo[2,3-d]pyrimidine-3′,5′-cycyclic phosphate 9. Isolation of a side product found in the glycosylation step of the reaction sequence proved to be the N-1 ribosyl-attached isomer as shown by X-ray diffraction analysis. All derivatives were tested for in vitro antiviral and antitumor activity.

Jack D. Anderson

Papers

Thiazolo[4,5-d]pyrimidine nucleosides. The synthesis of certain 3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidines as potential immunotherapeutic agents.

Synthesis of certain pyrazolo[3,4-d]pyrimidin-3-one nucleosides

Synthesis and antiviral activity of certain guanosine analogues in the thiazolo[4,5-d]pyrimidine ring system.

Guanosine analogues. Synthesis of nucleosides of certain 3-substituted 6-aminopyrazolo[3,4-d]pyrimidin-4(5H)-ones as potential immunotherapeutic agents.

Synthesis of Tubercidin, 6-Chlorotubercidin and Related Nucleosides