A general and expedient method has been developed for the solid-phase synthesis of 1,4-benzodiazepine-2,5-diones 1 from three commercially available components; anthranilic acids, α-amino esters, and alkylating agents. Reaction conditions have been developed to prepare either racemic compounds for lead identification efforts or optically pure compounds for lead optimization efforts. The incorporation of diverse functionality into the benzodiazepine products has also been demonstrated. On the basis of the scope and generality of the synthesis sequence, a library of 1,4-benzodiazepine-2,5-diones has been prepared from 11 alkylating agents, 12 anthranilic acids, and 19 α-amino esters (nine sets of enantiomeric pairs and glycine methyl ester hydrochloride). The library was prepared in a spatially separate format using a microtiter-based apparatus that is inexpensive and straightforward to construct from ordinary items found in an organic or bioorganic laboratory. The high quality of the 1,4-benzodiazepine-2,5-dione library has been demonstrated by evaluating representative compounds by HPLC analysis and 1H NMR.

GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, while potency, metabolic, and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50 = 0.43 nM was found toward a panel of 75 RSV A and B clinical isolates and dose-dependent antiviral efficacy in the cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46 to 100%, with evidence of efficient penetration into lung tissue. In healthy human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a mean 4.2 log10 reduction in peak viral load and a significant reduction in disease severity compared to placebo. In conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat RSV infection in infants and adults is reported.

GS-9148 [(5-(

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Hydrogenation of the C(4') exocyclic olefin of the pacidamycins has been shown to produce a series of semisynthetic compounds, the dihydropacidamycins, with antimicrobial activity similar to that of the natural products. Elucidation of stereochemistry in the pacidamycins has been completed through a campaign of natural product degradation experiments in combination with the total synthesis of the lowest-molecular weight dihydropacidamycin, dihydropacidamycin D. The stereochemical identities of the tryptophan and two alanine residues contained in pacidamycin D have been shown to be of the natural (S) configuration, and the unique 3-methylamino-2-aminobutyric acid contained in this series of antibiotics has been shown to be of the (2S,3S) configuration. Finally, the stereochemistry obtained by hydrogenation of the C(4')-C(5') exocyclic olefin has been shown to be (R) at the C(4') nucleoside site.

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GS-9131 is a phosphonoamidate prodrug of the novel ribose-modified phosphonate nucleotide analog GS-9148 that demonstrates potent anti-human immunodeficiency virus type 1 (HIV-1) activity and an excellent resistance profile in vitro. Prodrug moieties were optimized for the efficient delivery of GS-9148 and its active diphosphate (DP) metabolite to lymphoid cells following oral administration. To understand the intracellular pharmacology of GS-9131, incubations were performed with various types of lymphoid cells in vitro. The intracellular accumulation and antiviral activity levels of GS-9148 were limited by its lack of cellular permeation, and GS-9131 increased the delivery of GS-9148-DP by 76-to 290-fold relative to that of GS-9148. GS-9131 activation was saturable at high extracellular concentrations, potentially due to a high-affinity promoiety cleavage step. Once inside the cells, GS-9148 was efficiently phosphorylated, forming similar amounts of anabolites in primary lymphoid cells. The levels of GS-9148-DP formed in peripheral blood mononuclear cells infected with HIV-1 were similar to that in uninfected PBMCs, and approximately equivalent intracellular concentrations of GS-9148-DP and tenofovir (TVF)-DP were required to inhibit viral replication by 90%. Once it was formed, GS-9148-DP was efficiently retained in activated CD4؉ cells, with a half-life of 19 h. In addition, GS-9131 showed a low potential for drug interactions with other adenine nucleoside/nucleotide reverse transcriptase inhibitors, based on the lack of competition for anabolism between suprapharmacologic concentrations of GS-9148 and TVF and the lack of activity of GS-9131 metabolites against purine nucleoside phosphorylase, an enzyme involved in the clearance of 2,3-dideoxyinosine. Together, these observations elucidate the cellular pharmacology of GS-9131 and illustrate its efficient loading of lymphoid cells, resulting in a prolonged intracellular exposure to the active metabolite GS-9148-DP.The 2Ј-fluorine ribose-modified nucleotide phosphonate analog [5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid (GS-9148 or Fd4AP) (Fig. 1) is a selective inhibitor of wild-type and drug-resistant forms of human immunodeficiency virus type 1 (HIV-1) in vitro (5). Strains carrying mutations that cause resistance to nucleoside/ nucleotide reverse transcriptase inhibitor (NRTI) therapy showed either a lack of resistance to GS-9148 (K65R, L74V, and M184V) or a less marked effect on the susceptibility to GS-9148 relative to that of FDA-approved NRTIs in vitro (multiple thymidine analog mutations) (5). Fluorine at the 2Ј position was rationally designed into the molecule in order to increase the selectivity for HIV-1 reverse transcriptase (RT) over that of mitochondrial DNA polymerase ␥ (23), and enzymatic studies have illustrated that the diphosphate metabolite of GS-9148 (GS-9148-DP) is a potent inhibitor of wild-type and resistant RT while showing minimal inhibition of host polymerases (5).Despite the promising in vitro p...