Optimization of Novel Indole-2-carboxamide Inhibitors of Neurotropic Alphavirus Replication

Abstract

Neurotropic alphaviruses, which include western equine encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. We previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted Sindbis virus infection in mice. We describe here further development of this series resulting in 10-fold improvement in potency in a WEEV replicon assay and up to 40-fold increases in half-lives in mouse liver microsomes. Using a rhodamine123 uptake assay in MDR1-MDCKII cells we were able to identify structural modifications that markedly reduce recognition by P-glycoprotein, the key efflux transporter at the blood brain barrier. In a preliminary mouse PK study we were able to demonstrate that two new analogs could achieve higher and/or longer plasma drug exposures than our previous lead, and that one compound achieved measurable drug levels in the brain.

Novel Indole-2-Carboxamide Compounds Are Potent Broad-Spectrum Antivirals Active against Western Equine Encephalitis Virus In Vivo

“…Synthesis of compounds CCG205432, CCG206381, and CCG209023 has been previously described (13,20). Ribavirin, mycophenolic acid (MPA), antimycin A, phosphonoacetic acid (PAA), and cycloheximide were all purchased from Sigma-Aldrich (St. Louis, MO).…”

“…1C) to reduce molecular weight. Compared to the lead second-generation compound, CCG203926, the thirdgeneration compound, CCG205432, had 10-fold-increased potency, 2-fold-increased aqueous solubility, and 6-fold-increased metabolic stability measured by mouse liver microsome assay (20). For CCG206381, potency and aqueous solubility increased by 10-fold and 15-fold, respectively, and susceptibility to efflux by P glycoprotein was reduced, whereas there was little change in metabolic stability compared to that for CCG203926 (20).…”

“…We directed subsequent SAR development toward continued improvement in potency, but we also sought to enhance crucial chemical parameters predictive of in vivo activity, such as solubility, metabolic stability, and reduced efflux by P glycoprotein, in efforts to advance preclinical development. We produced a series of third-generation compounds with submicromolar IC 50 values against WEEV replicons (20), and we initiated detailed antiviral studies on two potent derivatives, CCG205432 and CCG206381. Structures for these compounds, plus IC 50 values, half-maximal cytotoxicity (CC 50 ) values, and selectivity indices (CC 50 /IC 50 ) for the replicon assay in BSR-T7 cells are shown in Fig.…”

“…Structures for these compounds, plus IC 50 values, half-maximal cytotoxicity (CC 50 ) values, and selectivity indices (CC 50 /IC 50 ) for the replicon assay in BSR-T7 cells are shown in Fig. 1B and C. Both compounds contain a terminal 4-pyridylethyl carboxamide, which proved to be a key functional determinant for potency (13,20). The only molecular difference between CCG205432 and CCG206381 is the core ring structure, which was maintained as a bicyclic indole in CCG205432 (Fig.…”

“…Through extensive structure-activity relationship (SAR) analyses and targeted medicinal-chemistry efforts, we produced second-generation indole-2-carboxamide derivatives that display increased potency and striking enantiospecific activity against WEEV and related alphaviruses in cultured cells and that also improve survival in mice infected with a neuroadapted strain of Sindbis virus (SINV) (13). We have now produced third-generation analogues through continued medicinal-chemistry-directed efforts that have increased antiviral potency, where we have achieved submicromolar half-maximal inhibitory concentrations (IC 50 ) against WEEV replicons in cultured cells (20). In this report, we detail the antiviral activity of three closely related third-generation compounds, CCG205432, CCG206381, and CCG209023.…”