Fig. 4: Optimized PVP-057 Synthesis via a Scalable 3-Step Schema.

A (i) 3,5-dimethoxybenzoyl chloride, pyridine, 0–120 ᵒC, 10 min, pH 2; (ii) 10% NaOH, EtOH, RT, 1 h, pH 2; (iii) 2-amino-6-ethoxybenzothiazole, EDC, HOAt, 10:1 MeCN/DCM, RT 24 h. The structure and purity of the product were determined by UPLC-MS and ¹H-NMR (detailed synthesis and analytical information in supplementary). B PVP-057 was synthesized in increasing quantities and the final product tested for purity level variations as measured by UPLC-MS at four increasing amounts of scaled-up production. C Table summarizing the physicochemical properties of known and well-characterized TLR3 agonists. For some or all nucleotide agonists, the molecular weight is given as a referenced estimate due to the constitutional heterogeneity of these biologics. D–F Four batches of PVP-057 were synthesized separately, by four different chemists. THP-1 Dual cells (InvivoGen) were stimulated with 25 µM of each batch of PVP-057, and induction of the IRF (D) and NF-kB (E) pathways was quantified 22 h post-stimulation. N = 2 technical replicates, averaged per batch. Data are presented as mean ± SEM. F HEK-Blue hTLR3 cells and their parental HEK-Blue Null1 cells (InvivoGen) were stimulated with a dose-titration of each batch of PVP-057, and NF-kB was quantified 22 h post-stimulation. N = 2 technical replicates. Source data are provided as a Source Data file.