n vitro Antiviral Activity of the Favipiravir and their 6- and 3-O-Substituted derivatives Against Coronovirus: Acetylation leads Improvement of Antiviral Activity

Synthesis and antiviral evaluation of a new series of functionalized favipiravir
The present manuscript described the optimization of the favipiravir as antiviral against in vitro bovine and human models of coronavirus, which was focused on two chemical functionalizations in pyrazine-structure: (i) functionalization at 6-position using halogens (F, Cl, Br and I) and hydrogen, and (ii) functionalization of 3-hydroxyl using different removal groups like acetyl, triflate, methanosulphonic and benzylic moieties. The first functionalization seeks to interpret the role of the tautomerism in the reactivity of the 3-hydroxypyrazine to form the active T07-RTP (T07-ribonucleoside 5′-triphosphate) metabolite, which was a question generated from previous reports in models of influenza (Huchting, J. et al. J. Med. Chem. 2018, 61, 6193-6210; De Almeida La Porta, F. et al. RSC Adv. 2021, 11, 35228 and other reports). Previously, we performed a full study of the tautomerism of this type of 3-hydroxy-2-pyrazinecarboxamides in solid state and in solution (J. Org. Chem. 2023, 88, 10735-10752) and we found that the keto-tautomerization in solution can be favored with diminution of halogen electronegativity as follows: 6-H >> 6-I > 6-Br >> 6-Cl > 6-F. That issue seeks to verify if the modulation of the keto-tautomerization by 6-halogen substitution can favor the antiviral response in coronavirus models, which is novel to the best of our knowledge.
Regarding the second functionalization, we seek to improve the cell penetration with the functionalization of the 3-hydroxyl moiety using removal lipophilicity moieties, whose final target was to enhance the antiviral response. The bioavailability of the favipiravir is one of its most significant disadvantages for infective in vitro models and more in particular, in vivo and clinical models (Nguyen TH, et al. PLoS Negl Trop Dis 2017;11(2):e0005389). With these chemical variations in favipiravir structure, we found that the modulation of the tautomer via 6-substitution did not provide an improvement in the antiviral response, whereas interestingly, from 3-O-functionalization, we found that the acetylation is a convenient removal moiety because it generated a compound 2-fold more active than favipiravir with a better selectivity against bovine and human model of coronavirus. Also, we demonstrated through NMR and fluorometric analysis that the diacetylated compound released in short time the favipiravir, being the acetylation a convenient chemical function to improve the penetration and accumulation into cell and its rapid release of favipiravir into cell favored the biological profile of the favipiravir as antiviral. This opens the door to the design of functionalized favipiravir and opens new perspectives on the importance of the lipophilicity to improve the antiviral profile of the favipiravir.

新型功能化法匹拉韦（favipiravir）的合成与抗病毒活性评价

本文针对法匹拉韦抗冠状病毒的体外牛源和人源模型的活性优化展开研究，重点围绕吡嗪（pyrazine）母核结构开展两处化学修饰：(i) 6位修饰：引入卤素（氟、氯、溴、碘）及氢原子；(ii) 3-羟基位点修饰：采用乙酰基、三氟甲磺酰基、甲磺酰基、苄基等不同离去基团进行衍生化。

第一处修饰旨在探究互变异构（tautomerism）对3-羟基吡嗪反应活性的调控作用，以生成活性代谢物T07-三磷酸核糖核苷（T07-ribonucleoside 5′-triphosphate, T07-RTP）——这一研究问题源于此前针对流感模型的相关报道（Huchting, J. et al. J. Med. Chem. 2018, 61, 6193-6210; De Almeida La Porta, F. et al. RSC Adv. 2021, 11, 35228及其他相关研究）。此前，我们已对该类3-羟基-2-吡嗪甲酰胺在固态及溶液中的互变异构现象开展了系统性研究（J. Org. Chem. 2023, 88, 10735-10752），并发现溶液中的酮式互变异构可随卤素电负性降低而增强，顺序为：6-H >> 6-I > 6-Br >> 6-Cl > 6-F。本研究旨在验证6位卤素取代对酮式互变异构的调控是否可增强冠状病毒模型中的抗病毒活性，据我们所知，这一思路尚未见报道。

关于第二处修饰，我们旨在通过3-羟基位点的亲脂性离去基团衍生化，提升细胞摄取能力，最终增强抗病毒活性。法匹拉韦的生物利用度是其在体外感染模型，尤其是体内及临床模型中应用的主要劣势之一（Nguyen TH, et al. PLoS Negl Trop Dis 2017;11(2):e0005389）。

通过上述法匹拉韦结构的化学改造，我们发现仅通过6位取代调控互变异构并未提升抗病毒活性；而有趣的是，经3-O-位功能化修饰后，乙酰基是一种较为理想的离去基团：其衍生化合物的抗病毒活性较法匹拉韦提升2倍，且对牛源和人源冠状病毒模型均表现出更优的选择性。此外，通过核磁共振（NMR）和荧光分析，我们证实该二乙酰化衍生物可在短时间内释放法匹拉韦——乙酰化作为一种便捷的化学修饰手段，可提升化合物的细胞摄取与胞内积累效率，而法匹拉韦的快速胞内释放则改善了其抗病毒生物学特性。本研究为功能化法匹拉韦的设计提供了新思路，也为通过调控亲脂性改善法匹拉韦的抗病毒活性开辟了新视角。