No abstract

The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug’s monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing.

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.

The design and synthesis of novel, orally active, potent, and selective inhibitors of influenza neuraminidase differing structurally from existing neuraminidase inhibitors are described. X-ray crystal structures of complexes of neuraminidase with known five- and six-membered ring inhibitors revealed that potent inhibition of the enzyme is determined by the relative positions of the interacting inhibitor substituents (carboxylate, glycerol, acetamido, hydroxyl) rather than by the absolute position of the central ring. This led us to design potential neuraminidase inhibitors in which the cyclopentane ring served as a scaffold for substituents (carboxylate, guanidino, acetamido, alkyl) that would interact with the four binding pockets of the neuraminidase active site at least as effectively as those of the established six-membered ring inhibitors such as DANA (2), zanamivir (3), and oseltamivir (4). A mixture of the isomers was prepared initially. Protein crystallography of inhibitor-enzyme complexes was used to screen mixtures of isomers in order to identify the most active stereoisomer. A synthetic route to the identified candidate 50 was developed, which featured (3 + 2) cycloaddition of 2-ethylbutyronitrile oxide to methyl (1S,4R)-4[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (43). Structures of the synthetic compounds were verified by NMR spectroscopy using nuclear Overhauser effect methodology. Two new neuraminidase inhibitors discovered in this work, 50 and 54, have IC(50) values vs neuraminidase from influenza A and B of <1 and <10 nM, respectively. These IC(50) values are comparable or superior to those for zanamivir and oseltamivir, agents recently approved by the FDA for treatment of influenza. The synthetic route used to prepare 50 and 54 was refined so that synthesis of pure active isomer 54, which has five chiral centers, required only seven steps from readily available intermediates. Further manipulation was required to prepare deoxy derivative 50. Because the activities of the two compounds are comparable and 54 [RWJ-270201 (BCX-1812)] is the easier to synthesize, it was selected for further clinical evaluation.