Lead Optimization of Spiropyrazolopyridones: A New and Potent Class of Dengue Virus Inhibitors

Abstract

Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model. KEYWORDS: Dengue virus (DENV), spiropyrazolopyridones, structure−activity relationship, lead optimization D engue fever is a febrile disease caused by dengue virus (DENV), which is transmitted by Aedes aegypti, a mosquito that feeds on humans.1 DENV threatens up to 2.5 billion people in more than 100 endemic countries. According to a World Health Organization (WHO) report, 2 there are 50− 100 million infections annually (and yet this number could be far underestimated), 3 with approximately 500,000 cases of dengue hemorrhagic fever and 22,000 deaths globally. 4,5 Despite the clear unmet medical need, currently there is no clinically approved vaccine or antiviral therapy available for treatment of dengue.6−10 Therefore, it is urgent to develop safe and effective therapeutics.High-throughput phenotypic screening is a powerful tool to identify compounds that are active against DENV by inhibiting either host pathways and/or viral proteins, which are essential for viral replication.11−16 As a result of DENV-2 screening on Novartis compound library, a new chemical class spiropyrazolopyridone 1 was identified to be an interesting starting point for further optimization (Figure 1). As compound 1 was a racemate, two enantiomers were subsequently separated by chiral high-performance purification chromatography (HPLC). The structure of R enantiomer 1a was unambiguously determined by X-ray crystallography (Figure 2). Interestingly, when tested in the DENV-2 in vitro assay, these two individual enantiomers displayed remarkably different potency where R enantiomer 1a was greater than 200-fold more potent than the S enantiomer 1b (Figure 1). Very recently, genetic analysis demonstrated that mutations in dengue viral NS4B conferred the resistance of compound 1a, suggesting that this class of compounds inhibited DENV replication by targeting NS4B protein. 11,17−19 In this report, lead optimization efforts to explore the structure−activity relationships (SARs) and improve the physicochemical properties are described.Following the identification of compound 1, which was subsequently found to be poorly soluble in aqueous media, we initiated a lead optimization effort in an attempt to improve the solubility while maintaining the potency. The general synthetic route to the spiropyrazolopyridone derivatives is outlined in Scheme 1. The synthesis, featuring a three component...

Therapeutic Potential of Spirooxindoles as Antiviral Agents

“…Utilizing a three-component condensation reaction of aminopyrazoles, commercially available isatin derivatives, and Meldrum’s acid, Zou et al quickly provided access to a series of spiropyrazolopyridone analogues for SAR investigation mainly focused on A-and B-ring modifications. 73 It has been further confirmed by chiral column HPLC separation of racemates that R enantiomers tend to be significantly more potent than S enantiomers. Meanwhile, it has remained that both of the amide carbonyl groups appear to be important for the activity, whereas neither of the NH groups is critical for the potency.…”

Discovery of Dengue Virus NS4B Inhibitors

“…Compound 14a showed a better pharmacological profile than did compound 1a. The detailed PK parameters were recently reported (19). The improved pharmacological parameters of compound 14a are most likely due to the improved compound solubility (Table 1).…”

“…All animal studies were approved by the IACUC. The pharmacokinetic (PK) profiles of compounds 1a and 14a (Table 1) (19) were determined in male Wistar rats (weighing 200 to 400 g) following intravenous (i.v.) and oral (p.o.)…”

“…However, compound 14a showed improved aqueous solubility, from 11 M (compound 1a) to 504 M, and an improved logP value, from 4.9 (compound 1a) to 2.8 ( Table 1). The detailed structure-activity relationship of medicinal chemistry was recently reported (19).…”

A Novel Benzodiazepine Compound Inhibits Yellow Fever Virus Infection by Specifically Targeting NS4B Protein

“…These efforts have resulted in identification of a variety of small molecules that inhibit viral replication through targeting distinct viral and host cellular functions (17)(18)(19). Small molecules that specifically target viral capsid assembly (20), NS3/2A proteases (21)(22)(23), NS3 RNA helicase (24), NS4B protein (25,26), NS5 methyltransferase (27), and RNA-dependent RNA polymerase (28,29) have been discovered, and the in vivo antiviral efficacies for four of these direct-acting antiviral (DAA) agents have been demonstrated in animal models (24,(30)(31)(32); however, none of them has reached human clinical trials. Balapiravir, a tri-isobutyrate ester prodrug of 4=-azidocytidine (R1479) that was originally developed for treatment of chronic hepatitis C virus (HCV) infection by Hoffmann-La Roche, is active against DENV in cultured cells.…”