CHEMISTRY-DRIVEN OPTIMIZATION OF PURINE-BASED ANTIVIRALS FOR THE IDENTIFICATION OF NEW BROAD-SPECTRUM AGENTS

Background Emerging and re-emerging viruses represent a significant and unpredictable threat to global health, given their capacity for rapid transmission and selection of drug resistant and escape mutants. Current antiviral therapies are mostly virus-specific and limited in efficacy to some chronic infections, underscoring the urgent need for broad-spectrum antivirals (BSAs) capable of targeting a wide range of pathogens, including novel and re-emerging viruses1. Aims The risk of large outbreaks caused by emerging viruses such as Dengue, Zika, and West Nile Virus pose a global threat due to the absence of approved treatments or fully protective vaccines. Traditional target-based drug discovery is limited by resistance risks. To address this challenge, we employed a chemistry-driven phenotypic approach to optimize our previously identified flavivirus inhibitors based on the 2,6-diaminopurine scaffold. Methods Starting from our previous identification of 2,6-diaminopurine derivatives as potent flavivirus inhibitors we conducted a system-oriented optimization which involved the synthesis and screening of a library of analogs to discover compounds with broad-spectrum antiviral activity across multiple viral families2. Then, to enhance drug-like properties and the antiviral activity we applied a scaffold morphing strategy, converting the purine scaffold into novel chemotypes. We exploited the chemical versatility of the 2,4-diaminofuro[3,4-d]pyrimidin-7(5H)-one nucleus to generate multiple derivatives and explore the biologically relevant chemical space. Results In our previous work, we designed first-in-class multi-target 2,6-diaminopurine antivirals that inhibit both host cell factors c-Src/Fyn and the interaction between viral proteins NS5-NS33. Structure-activity relationship (2D-SAR) analysis guided the phenotypic hit-to-lead optimization, leading to MR-333 as a highly promising and cost-effective broad-spectrum antiviral (BSA). This compound demonstrated potent antiviral activity across multiple pathogens, including Dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Influenza A virus, and SARS-CoV-2, with IC₅₀ values ranging from 0.5 to 5.3 μM. Notably, MR-333 exhibited a high selectivity index, suggesting a favorable therapeutic window and limited cytotoxicity. To expand the chemical space around the original hits, we applied a phenotypic scaffold-morphing approach, transforming the flat purine derivatives into more complex chemotypes with increased saturation and improved drug-like properties4. Through this strategy, we selected 2,4-diaminofuro[3,4-d]pyrimidin-7(5H)-one (1) as a key scaffold, due to its capacity to access biologically relevant chemical space and the synthetic flexibility offered by its lactone ring. From this scaffold we designed a single synthetic strategy to obtain two different classes of derivatives: 2,4,5,6-tetrasubstituted pyridines (2) and 5H-pyrrolo[3,4-d]pyrimidin-7(6H)-one (3). All synthesized compounds were initially evaluated for their ability to inhibit DENV-2 (DENV serotype 2) replication in a virus yield reduction assay using ribavirin (RBV) as reference compound. While chemotype 1 derivatives were inactive, a few compounds endowed with a 5H-pyrrolo[3,4-d]pyrimidin-7(6H)-one chemotype (3) exhibited moderate inhibition of DENV replication. The most interesting results were obtained from compounds with a pyrimidine chemotype (2). Compounds belonging to chemotype 2 and 3 were also tested against EBOV, Sars-CoV-2 and H1N1 and some of them show EC50 in the low micromolar range. To get an overview on the quality of synthesized inhibitors compared to that of previous reported hits and reference drugs, key molecular descriptors of compounds were computed using Datawarrior. Fsp3 was calculated as the number of sp3 hybridized-carbon count and plotted against the total molecular weight for each molecule. Fsp3 has been shown to increase as compounds progress to clinical testing and is directly correlated with their molecular complexity and solubility. Conclusions We applied a phenotypic scaffold-morphing strategy to improve flat purine antivirals, expanding the original hits into new derivatives based on furo[3,4-d]pyrimidin-7(5H)-one with three distinct chemotypes. This strategy allows us to identify promising broad-spectrum antivirals with low shape index, and a good Fsp3. This work underscores the power of chemistry-driven drug discovery, demonstrating how a single chemotype can be evolved through rational modifications to get an optimized scaffold with improved properties. References 1Karim M, Lo CW, Einav S. Preparing for the next viral threat with broad-spectrum antivirals. J Clin Invest. 2023 Jun 1;133(11):e170236. 2Vicenti I, Martina MG, Boccuto A, et al. System-oriented optimization of multi-target 2,6-diaminopurine derivatives: Easily accessible broad-spectrum antivirals active against flaviviruses, influenza virus and SARS-CoV-2. Eur J Med Chem. 2021;224:113683. 3Kaptein SJF, Vincetti P, Crespan E, Rivera JIA, Costantino G, Maga G, Neyts J, Radi M. Identification of Broad-Spectrum Dengue/Zika Virus Replication Inhibitors by Functionalization of Quinoline and 2,6-Diaminopurine Scaffolds. ChemMedChem. 2018 Jul 18;13(14):1371-1376. 4Vincetti P, Kaptein SJF, Costantino G, Neyts J, Radi M. Scaffold Morphing Approach To Expand the Toolbox of Broad-Spectrum Antivirals Blocking Dengue/Zika Replication. ACS Med Chem Lett. 2019 Jan 23;10(4):558-563