SummaryCellular senescence contributes to organismal development, aging, and diverse pathologies, yet available assays to detect senescent cells remain unsatisfactory. Here, we designed and synthesized a lipophilic, biotin‐linked Sudan Black B (SBB) analogue suitable for sensitive and specific, antibody‐enhanced detection of lipofuscin‐containing senescent cells in any biological material. This new hybrid histo‐/immunochemical method is easy to perform, reliable, and universally applicable to assess senescence in biomedicine, from cancer research to gerontology.

Drugs targeting adenosine receptors (AR) can provide treatment for diseases. We report the identification of 7-(phenylamino)-pyrazolo­[3,4-c]​pyridines L2–L10, A15, and A17 as low-micromolar to low-nanomolar A1R/A3R dual antagonists, with 3-phenyl-5-cyano-7-(trimethoxyphenylamino)-pyrazolo­[3,4-c]​pyridine (A17) displaying the highest affinity at both receptors with a long residence time of binding, as determined using a NanoBRET-based assay. Two binding orientations of A17 produce stable complexes inside the orthosteric binding area of A1R in molecular dynamics (MD) simulations, and we selected the most plausible orientation based on the agreement with alanine mutagenesis supported by affinity experiments. Interestingly, for drug design purposes, the mutation of L2506.51 to alanine increased the binding affinity of A17 at A1R. We explored the structure–activity relationships against A1R using alchemical binding free energy calculations with the thermodynamic integration coupled with the MD simulation (TI/MD) method, applied on the whole G-protein-coupled receptor–membrane system, which showed a good agreement (r = 0.73) between calculated and experimental relative binding free energies.

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H2S slow-releasing moiety, coupled via a stable ether bond. The H2S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H2S donor, were selected for further in vivo evaluation. Their ability to release H2S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.

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775The discovery of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir, Fig. 1) 1) as the first potent and selective inhibitor of a human herpes virus 2) has stimulated continuing research into the preparation and evaluation of novel acyclic analogues of nucleosides as potential substrates of virus-specific thymidine kinase (TK) and/or inhibition of DNA polymerase after activation by the viral TK and subsequent phosphorylation by cellular enzymes to the 5Ј-triphosphates.3,4) Structureactivity relationship studies have been explored extensively and revealed that both the nature of the heterocyclic base and the chemical structure of the aliphatic substituent have a pronounced effect on the antiviral activity of these molecules. Purine nucleosides structurally related to acyclovir that are currently used in the clinic are ganciclovir, 5,6) which is useful against cytomegalovirus (HCMV) infections and the penciclovir prodrug famciclovir, 7) which is marketed for shingles. The antiviral spectrum of these agents is limited to the herpes virus genus. On the other hand, 3-deazaadenosine as well as a number of acyclic and carbocyclic adenosine analogues, such as the S enantiomer of 9-(2,3-dihydroxypropyl)-adenine (S-DHPA), aristeromycin, neplanocin A and 3-deazaneplanocin A (Fig. 1) were found to act as reversible inhibitors of S-adenosyl-L-homocysteine (SAH) hydrolase. 8) These SAH hydrolase inhibitors exhibit potent antiviral activity against a number of DNA and RNA viruses, which heavily depend on methylation reactions for viral replication. More interestingly, acyclic nucleoside phosphonates have been developed, which retain marked activity against thymidine kinase deficient viral strains and some of them are useful for the treatment of HCMV, retrovirus (HIV) and human hepatitis B virus (HBV) infections. 9)In the course of our studies, concerning the preparation of a number of C-nucleosides structurally related to inosine and adenosine 10,11) and in conjunction with the stimulating results reported for acyclic nucleosides, we were interested in determining whether antiviral activity is retained when the (2-hydroxyethoxy)methyl group of acyclovir is employed in combination with a base structurally related to 3-deazaadenine. Within this context we report here the synthesis and the in vitro antiviral evaluation of some new acyclic C-nucleoside analogues, bearing the 8-aza-1,9-dideazapurine or the 8-aza-3,9-dideazapurine skeleton. Results and DiscussionChemistry For the preparation of the pyrazolopyridinone 13 (Chart 1) we used commercial 2-picoline N-oxide (1) which was converted to the picoline 4 through known procedures.12) Subsequent nitration of 4 provided a mixture of the isomeric nitroderivatives 5 and 6 and from this mixture only the desired compound 6 could be isolated in pure form by column chromatography, whereas 5 was contaminated with traces of 6. The isomer 6 was reduced to the aminoderivative 7, which has been previously reported as a sideproduct of the ammonolysis of 3-methoxy-2-acylfuran. The preparation of...

Substituted pyrazolopyridines are potent inhibitors of phosphodiesterases and cyclin-dependent kinases. In this study, NMR was used to investigate the potential N1-H and N2-H tautomerism of 5-substituted pyrazolo[3,4-c]pyridine derivatives. Six compounds were fully characterized by using (1)H, (13)C, and (15)N chemical shifts and indirect (1)H--(13)C and (1)H--(15)N coupling constants. The (1)H NMR spectra were measured over a broad range of temperatures. All of the compounds were shown to exist predominantly in the N1-H tautomeric form. Complementary quantum-chemical calculations of the chemical shieldings and indirect spin-spin couplings support the structural conclusions drawn.

Non-selective and A1 but not A2A and A3 ARs agonists are essential for triggering cardioprotection. The molecular mechanism involves both RISK and the JAK/STAT pathways.