Although both 4,5- and 4, 6-linked aminoglycosides target the same ribosomal site, they appear to bind and effect antibiotic activity in different manners. The aminoglycosides might recognize different RNA conformations or the interaction might involve different RNA tertiary structures that are not equally sampled in our ribosome-free model. These results imply that models of ribosomal RNA must be carefully designed if the data are expected to accurately reflect biological activity.

The structure and activity of the pseudodisaccharide core found in aminoglycoside antibiotics was probed with a series of synthetic analogues in which the position of amino groups was varied around the glucopyranose ring. The naturally occurring structure neamine was the best in the series according to assays for in vitro RNA binding and antibiotic activity. With this result in hand, neamine was used as a common core structure for the synthesis of new antibiotics, which were evaluated for binding to models of the Escherichia coli 16S A-site ribosomal RNA, in vitro protein synthesis inhibition, and antibiotic activity. Analysis of RNA binding revealed some correlation between the relative affinity and specificity of RNA binding and antibacterial efficacy. However, the correlation was not linear. This result led us to develop the in vitro translation assay in an effort to better understand aminoglycoside−RNA interactions. A linear correlation between in vitro translation inhibition and antibiotic activity was observed. In addition, IC50s in the protein synthesis assay were typically lower than the K ds obtained for RNA binding, suggesting that binding of these compounds to intact ribosomes is tighter in these cases than binding to the model RNA oligonucleotides. This reflects possible differences in RNA conformation between intact ribosomes and the free RNA of the model system, or possible high-affinity ribosomal binding sites in addition to the A-site RNA.

Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care.

The specificity of neomycin B and related aminoglycoside antibiotics in their interaction with the Rev responsive element (RRE) of HIV-1 mRNA has been studied by directly observing the aminoglycoside-RNA complexes using surface plasmon resonance. Several different RNA sequences, each with a biotin tag, have been prepared using T7 RNA polymerase-catalyzed transcription of synthetic DNA templates and have been immobilized on a streptavidin-coated surface for the binding study. The results indicate that neomycin B is not specific for the G-rich bubble region in RRE. Rather, it appears to interact with three different sites, each with a submicromolar dissociation constant, within the 67-nucleotide domain II of RRE. Further analysis of neomycin B binding with three short synthetic RNA hairpins showed binding with submicromolar affinity and 1:1 stoichiometry in each case. This suggests that neomycin B may generally bind with this affinity to regular A-form RNA or hairpin loops. The approach described here is generally useful for understanding the fundamental interactions involved in the specific recognition of nucleic acids by small molecules which is the basis of rational drug design.

Et THFlNMP Ph-CZC-Et t -35 'C, 3 h 13 : 88 %; Z : € 9 9 : 1 2) 12 0c IPd(dba)n] (4 rnol%) Ph,P (16 rnol%) THF. 55 'C, 10 h. then HCI n HN+Me2 C I -14 : (Z)-tamoxiten 75%;Z: € > 9 9 : 1Scheme 4. Synthesis of (2)-tamoxifen (14).In summary, we have reported a new intramolecular syn carbonickelation leading to alkylated exo-alkylidenecyclopentane derivatives, as well as an intermolecular carbozincation of substituted phenylacetylenes that allows stereoselective (> 98 % syn addition) synthesis of tri-and tetrasubstituted phenylalkenes. Experimental Procedures(12b): [Ni(acac),] (320 mg, 1.25 mmol, 25 mol%) and 8d (0.89 g, 5 mmol, 1 equiv) were dissolved in T H F (3 75mL) and NMP (1.25mL) at -4O'C under argon. Diethylzinc ( I .O mL. 10 mmol, 2 equiv) was carefully added via syringe at -78 "C. The reaction mixture was allowed to warm to -35'C and stirred for 2.5 h. Meanwhile a mixture of CuCN (1 79 g, 20 mmol, 4 equiv) and LiCl (1 69 g, 40 mmol, 8 equiv) was dried in VaCUO at 130 "C for 2 h and then dissolved in T H F (10 mL). The solution was cooled to -60°C and added by syringe to the reaction mixture at -78 C. The resulting dark solution was warmed to 0°C for a few minutes and then again cooled to -78'C. Ethyl (a-brom~methyl)acrylate'~' (4.82 g, 25 mmol, 5 equiv) was added, and the reaction mixture warmed to 25°C and worked up. The crude product was purified by flash-chromatography (hexanesiether 20/1), affording the ester 12b (1.13 g, 3.53 mmol, 71 % yield; Z:E>99:1) as a white powder. (5c): [Ni(acac)J (96 mg. 0.37 mmol, 7 mol%) was dissolved in T H F (3.75 mL) and NMP (1 2 5 mL) at -40 'C under argon, and 1 -iodo-4-phenyl-5-hexyne (4b) (I .41 g, 5 mmol, 1 equiv) was added At z 7 8 " C , Pent& (2.0 mL, 10 mmol, 2 equiv) was carefully added by syringe. The reaction mixture was stirred for 30 h at -40°C. After the usual workup. the solvents were distilled off, and the crude residue was purified by chromatography (hexanes) to give the cyclized product 5c (0.74g 3.24mmol. 65% yield: E:Z>99:1) as a colorless oil.