(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (K obs /[I]) of 223,000 M؊1 s ؊1 }. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC 50 ) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (C max ) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC 50 required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the C max at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that C max rather than the C min might be the key variable predicting clinical efficacy.The Picornaviridae comprise one of the largest families of known human and animal pathogens. Included in the picornavirus family are the human rhinoviruses (HRV) and human enteroviruses (HEV). Consisting of over 100 different serotypes, the HRV are the single most important etiological agents of the common cold (7,23,26). In most individuals, HRV infections are mild and self-limiting; however, in patients with underlying respiratory disorders, infections may result in exacerbations of asthma, cystic fibrosis, bronchitis, and chronic obstructive pulmonary disease (1,2,6,12,18,25,28). To date, there are no marketed antiviral therapies available for the prevention or treatment of HRV-related illnesses.We have focused our drug discovery efforts on the HRV 3C protease, an enzyme that is absolutely required for the majority of the proteolytic cleavage events that occur during the viral life cycle (7,17,19,23,26). DNA sequence comparisons among numerous HRV serotypes and several related picornaviruses have demonstrated a significant degree of homology among amino acid residues involved in key 3C protease inhibitor-binding interactions, providing an additional rationale for targeting research activities (3,20,22). Early drug discovery efforts led to the identification of ...
