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.
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Synthesis of 3-(trifluoromethylsulfonyloxi)-6-fluoropyrazin-2-carboxamide 2a and 3-(trifluoromethylsulfonyloxi)-6-fluoropyrazin-2-carbonitrile 2b. 3-Hydroxy-2-pyrazinecarboxamide (0.10 g; 0.64 mmol) was dissolved in dry dichloromethane (3 mL). The mixture was cooled at 0 ºC, triethylamine (0.12 g; 1.27 mmol) and subsequently triflic anhydride (0.36 g; 1.27 mmol) was added under nitrogen atmosphere. Then, the mixture was stirred for 1 h. The reaction was monitored by TLC using ethyl acetate eluent. The solvent was removed under reduced pressure to yield a yellowish oil, which was purified through a chromatographic column. From chromatography, two products 2a and 2b were obtained through the solvent gradient technique. Compound 2b (Rf= 0.92 in ethyl acetate) was firstly obtained from n-hexane eluent, whereas compound 2a (Rf= 0.75 in ethyl acetate) was purified using n-hexane/ethyl acetate (8:2). Synthesis of 3-(methylsulfonyloxi)-6-fluoropyrazin-2-carboxamide 2c. 3-Hydroxy-2-pyrazinecarboxamide (0.10 g; 0.64 mmol) was dissolved in dry dichloromethane (3 mL). The mixture was cooled at 0 ºC, triethylamine (0.20 g; 1.9 mmol) and subsequently methane-sulfonic chloride (0.10 g; 0.83 mmol) was added under nitrogen atmosphere. Then, the mixture was stirred for 1 h. The reaction was monitored by TLC using ethyl acetate eluent. The solvent was removed under reduced pressure to yield a white solid, which was recrystallized from acetonitrile. Synthesis of 3-(acetylcarbamoyl)-5-fluoropyrazin-2-yl acetate 2d. 3-Hydroxy-2-pyrazinecarboxamide (0.10 g; 0.64 mmol) was dissolved in dry acetic anhydride (3 mL). To the mixture, phosphoric acid 85 % (0.5 mL) was added and then, heated at 60 ºC for 30 minutes. The reaction was monitored by TLC using ethyl acetate eluent. The mixture was cooled at 5 ºC and cold water was added (5 mL) yielding a yellowish solid, which was filtered under vacuum. The solid was recrystallized from acetonitrile. Synthesis of 3-(benzyloxy)-6-fluoropyrazin-2-carboxamide 2e. 3-Hydroxy-2-pyrazinecarboxamide (0.10 g; 0.64 mmol) was dissolved in dry dichloromethane (1 mL). To the mixture, triethylamine (0.12 g; 1.27 mmol) and benzyl chloride (0.60 g; 4.7 mmol) were added. Then, the mixture was stirred for 5 h at 100 ºC. The reaction was monitored by TLC using ethyl acetate eluent. The solvent was removed under reduced pressure to yield an orange oil, which was washed with n-hexane to remove the excess of benzyl chloride. The resulting mixture was purified through chromatographic column using n-hexane and n-hexane/AcOEt mixture to separate efficiently all mixture components. n-Hexane (100%) was used to remove benzyl chloride, n-hexane/AcOEt (95:5- 90:10) to remove the rest of the triethylamin chloride salts (Et3NH+) and, finally, n-hexane/AcOEt (8:2) to obtain the compound 2e.
Universidad de la República Uruguay