key: cord-0896013-xopefz0o authors: Wang, Mo; Zhang, Lu; Huo, Xiaohong; Zhang, Zhenfeng; Zhang, Wanbin title: Chemical Synthesis of the Anti‐COVID‐19 Drug Remdesivir date: 2021-12-08 journal: Curr Protoc DOI: 10.1002/cpz1.303 sha: ead4ee0b4a0efb295e4e29972d253fade5c1c380 doc_id: 896013 cord_uid: xopefz0o Remdesivir has become an important compound for the treatment of COVID‐19. Here, we describe the catalytic asymmetric synthesis of this anti‐COVID‐19 drug. First, the P‐racemic phosphoryl chloride is synthesized in a facile procedure. Then, it is possible to obtain the protected remdesivir via the organocatalytic asymmetric phosphorylation of protected nucleoside GS441524 with P‐racemic phosphoryl chloride catalyzed by chiral bicyclic imidazole. Finally, remdesivir is easily prepared by deprotection. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2‐ethylbutyl (chloro(phenoxy)phosphoryl)‐l‐alaninate rac‐4 Basic Protocol 2: Synthesis of chiral bicyclic imidazole Ad‐DPI Basic Protocol 3: Synthesis of remdesivir Recently, remdesivir, developed by Gilead Sciences using ProTide technology, has become one of the most effective anti-COVID-19 drugs. Thus far, two synthetic methods for remdesivir have been established (Siegel et al., 2017; Warren et al., 2016) , but both of these methods require chiral resolution and additional synthetic steps, leading to resource waste and low synthetic efficiency. In 2012, our group developed the first catalytic asymmetric synthesis of P-stereogenic phosphoric acid derivatives using chiral bicyclic imidazole organocatalysts (Liu et al., 2012) . Inspired by this methodology for the construction of P-stereocenters, we developed the first catalytic asymmetric synthesis of remdesivir via chiral bicyclic imidazole-catalyzed phosphorylation . The core strategy in this protocol is to generate the S P -isomer of protected remdesivir via catalytic asymmetric phosphorylation. Basic Protocol 1 describes the synthesis of 2-ethylbutyl (chloro(phenoxy)phosphoryl)-L-alaninate rac-4. Basic Protocol 2 describes the synthesis of the chiral bicyclic imidazole Ad-DPI. Basic Protocol 3 describes the synthesis of remdesivir. Figures and some textual material are taken from our recently published work and rewritten as step-by-step protocols to support reproduction of the original work. Current Protocols Cryogenic cooling circulation pump Vacuum filtration system 1 H, 13 C, and 31 P NMR equipment or facilities 1. Add 75 ml (609.3 mmol) 2-ethylbutan-1-ol 1 io a dry 200-ml two-necked roundbottomed flask. 2. Add 10.9 ml (150.0 mmol) SOCl 2 dropwise over 10 min to the flask containing 2-ethylbutan-1-ol 1 at 0°C and stir the mixture for 1 hr at 0°C. 3. Weigh out 8.9 g (100.0 mmol) of L-alanine into the flask containing the mixture at 0°C. 4. Raise the temperature of the reaction mixture to 90°C, stir the mixture for 16 hr at 90°C, and then allow it to cool to 25°C. 5. Transfer the reaction mixture into a dry 200-ml round-bottomed flask. 6. Evaporate the reaction mixture using a rotary evaporator. 7. Wash the reaction mixture with 20 ml petroleum ether twice. 8. Dry the resulting white solid for 1 hr in vacuo to obtain 2-ethylbutyl L-alaninate hydrochloride salt 2. 9. Weigh out 3.15 g (15.0 mmol) of 2 into a dry 150-ml two-necked round-bottomed flask. 10. Add 50 ml anhydrous CH 2 Cl 2 to the flask using a syringe needle. 11. Cool the temperature of reaction mixture to -80°C under dry nitrogen. 12. Add 2.24 ml (15.0 mmol) phenyl dichlorophosphate to the flask using a syringe needle. 13. Add 4.17 ml (30.0 mmol) Et 3 N to the flask using a syringe needle over 10 min, and stir the mixture for 30 min. 14. Raise temperature of reaction mixture to 25°C and stir it for 2 hr. 15. Transfer the reaction mixture into a dry 150-ml round-bottomed flask. 16. Evaporate the reaction mixture using a rotary evaporator to obtain a white solid. 17. Add 50 ml anhydrous Et 2 O to the flask and stir the mixture for 10 min. 18. Vacuum filter the mixture using a vacuum filtration system with the 150-ml roundbottomed flask to remove the solid. 19. Evaporate the filtrate using a rotary evaporator. 20. Dry the resulting colorless oil for 1 hr in vacuo to provide 2-ethylbutyl (chloro(phenoxy)phosphoryl)-L-alaninate rac-4. 21. Characterize the product by 1 H NMR, 13 C NMR, and 31 P NMR. Product rac-4 is a colorless oil (4.9 g, 94% yield 172.9, 172.8, 172.7, 149.9, 149.9, 149.9, 149.8, 149.8, 130.0, 129.9, 129.9, 129.9, 126.0, 126.0, 126.0, 120.6, 120.6, 68.0, 67.9, 50.9, 50.6, 50.6, 40.3, 23.3, 23.3, 23.3, 20.6, 20.6, 20.6, 20.6, 11.0, and 11.0. 31 This protocol describes the synthesis of chiral bicyclic imidazole Ad-DPI Wang, Zhang, Ling, Zhang, & Zhang, 2017; Zhang, Xie, Jia, & Zhang, 2010) , the catalyst for asymmetric phosphorylation prepared from imidazole (Fig. 2) . The synthesis involves three steps: (1) synthesis of (rac)-HO-DPI from acrolein and imidazole, (2) synthesis of (S)-HO-DPI via kinetic resolution catalyzed by Novozyme 435, and (3) synthesis of Ad-DPI from (S)-HO-DPI. Imidazole ( Syringe needle 1 H and 13 C NMR, HPLC, and high-resolution mass spectrometry (HRMS) equipment or facilities Additional reagents and equipment for silica gel flash chromatography (see Current Protocols article: Meyers, 2001) Synthesis of (rac)-HO-DPI 1. Weigh out 1.6 g (23.9 mmol) imidazole into a 100-ml two-necked round-bottomed flask. 2. Add 25 ml 1,4-dioxane to this flask. 3. Add 0.1 ml (1.7 mmol) AcOH to the flask. 4. Add 2.5 ml (36.7 mmol) acrolein to the flask and stir the mixture. 5. Reflux the reaction mixture for 36 hr. 6. Transfer the reaction mixture into a 100-ml round-bottomed flask. 7. Evaporate the reaction mixture using a rotary evaporator. 8. Purify the crude product by silica gel chromatography (100-200 Mesh; Meyers, 2001) using 3/1 (v/v) EtOAc/MeOH to obtain (rac)-HO-DPI. 9. Characterize the product by 1 H NMR, 13 C NMR, HRMS, and HPLC. 14. Raise temperature of reaction mixture to 35°C and stir the mixture gently for 12 hr. 15. Vacuum filter the mixture using a vacuum filtration system with the 250-ml roundbottomed flask to remove the solid. This protocol describes the synthesis of remdesivir , the ProTide prodrug prepared from rac-4 and nucleoside 5 (Fig. 3) . The synthesis involves two steps: (1) catalytic asymmetric phosphorylation of nucleoside GS441524 5 with 2-ethylbutyl (chloro(phenoxy)phosphoryl)-L-alaninate (rac-4) catalyzed by Ad-DPI to prepare protected remdesivir S P -6. (2) deprotection of S P -6 with 37% HCl in THF to provide remdesivir (Warren et al., 2016) . GS441524 (5, 98.0%, Aikonchem, AK01FWCB) (S)-6,7-Dihydro-5H-pyrrolo[1,2-a]imidazol-7-yl adamantan-1-ylcarbamate (Ad-DPI, Basic Protocol 2) 4 Å molecular sieves Dry nitrogen Dichloromethane (CH 2 Cl 2 , >99.5%, General-Reagent, G81014C) 2,6-Lutidine (99.0%, Energy Chemical, W330004) 2-Ethylbutyl (chloro(phenoxy)phosphoryl)-L-alaninate (rac-4; Basic Protocol 1) Silica gel (100- 1. Add 10.0 g (30.2 mmol) 5 to a dry 1000-ml two-necked round-bottomed flask equipped with a magnetic stir bar. 2. Add 909.6 mg (3.0 mmol) Ad-DPI to the flask. 3. Add 11.0 g 4 Å molecular sieve to the flask. 4. Evacuate and backfill the flask with dry nitrogen three times. 5. Add 300 ml CH 2 Cl 2 to the flask with the aid of a syringe needle. 6. Add 7.0 ml (60.4 mmol) 2,6-lutidine to the flask with the aid of a syringe needle. 7. Cool the temperature of reaction mixture to -40°C under dry nitrogen and stir the mixture for 10 min. 8. Add 15.7 g (45.3 mmol) rac-4 to the flask with the aid of a syringe needle and stir the reaction mixture for 48 hr. 9. Add 30 ml water to the flask to quench the reaction. 10. Transfer the reaction mixture into a 1000-ml round-bottomed flask. 11. Evaporate the reaction mixture using a rotary evaporator and concentrate the residue in vacuo using a high-vacuum oil pump. 12. Analyze the crude product by 31 P NMR showing a d.r. of 21.2:1. 13. Purify the crude product by silica gel chromatography (100-200 Mesh) using 1/5 v/v petroleum ether/ethyl acetate and then recrystallize the product from dichloromethane/isopropyl ether at -30°C to provide protected remdesivir S P -6 as a white solid (16.5 g, 85% yield, >99:1 d.r.). 14. Purify the residue from mother liquor by preparative HPLC (Daicel CHIRALCEL IE, 1 cm × 25 cm, 10 ml/min, isopropanol/hexane = 30/70, 220 nm, collected from 49.2 to 63.9 min) to yield S p -6 as a white solid (0.8 g, 4% yield, >99:1 d.r.). 15. Combine two parts of product providing 17.3 g S p -6 (89% yield, >99:1 d.r.). 16. Characterize the product by 1 H NMR, 13 C NMR, 31 P NMR, HPLC, and HRMS. 9, 174.8, 156.6, 152.0, 151.9, 147.6, 130.6, 126.0, 124.9, 121.3, 121.2, 118.0, 117.7, 116.9, 112.5, 103.0, 85.7, 84.8, 84.8, 83.0, 82.5, 68.1, 67.0, 66.9, 51.4, 41.6, 26.5, 25.6, 24.2, 24.1, 20.5, 20.4, 11.3, and 11.3. 31 22. Transfer the reaction mixture into a 500-ml round-bottomed flask and dilute the reaction mixture with 60 ml water. 23. Add saturated aqueous sodium bicarbonate solution to adjust the pH of reaction mixture to 8. 24. Extract the resulting mixture with 60 ml ethyl acetate three times. 25. Dry the combined organic phase over Na 2 SO 4 and remove the solid by filtration. Wang et al. Current Protocols 26. Evaporate the reaction mixture using a rotary evaporator. 27. Purify the crude product by silica gel chromatography (100-200 Mesh) using 1/3 v/v) petroleum ether/ethyl acetate to provide remdesivir. 28. Characterize the product by 1 H NMR, 13 C NMR, 31 P NMR, and high-resolution mass spectrometry (HRMS). 0, 174.9, 157.1, 152.1, 152.1, 148.2, 130.7, 126.0, 125.5, 121.3, 121.3, 117.9, 117.6, 112.3, 102.7, 84.3, 84.2, 81.2, 75.6, 71.6, 68.1, 67.2, 67.1, 51.5, 41.6, 24.2, 24.2, 20.6, 20.5, 11.3, 11.3. 31 Remdesivir is one of the most effective treatments for COVID-19 (Holshue et al., 2020) , which attracts researchers to develop an effective synthetic approach for this compound. Until now, two synthetic methods for remdesivir have been established (Siegel et al., 2017; Warren et al., 2016) . Both of these methods require not only chiral resolution but also additional synthetic steps, which lead to the waste of resources and low synthetic efficiency. In 2010, a novel chiral bicyclic imidazole organocatalyst was developed by our group (Zhang et al., 2010) . Over the past decade, this kind of organocatalyst was successfully applied in a number of asymmetric reactions (Liu et al., 2012; Wang et al., 2017; Wang, Zhang, Liu, Xie, & Zhang, 2014; Wang, Zhou, Zhang, Zhang, & Zhang, 2020; Zhang et al., 2019; Zhang, Wang, Xie, Sun, & Zhang, 2014; Zhou et al., 2019; Zhou et al., 2021) . In 2012, the first catalytic asymmetric synthesis of P-stereogenic phosphoric acid derivatives was developed by our group using the chiral bicyclic imidazole catalyst (Liu et al., 2012) . Later, scientists at Merck & Co. utilized bicyclic imidazole catalyst in the asymmetric synthesis of nucleoside-based phosphoramidate prodrugs (DiRocco et al., 2017) . Inspired by our previous work, we successfully developed the first catalytic asymmetric synthesis of remdesivir using the chiral bicyclic imidazole Ad-DPI . In Basic Protocol 1, both steps are sensitive to water. In Basic Protocol 2, the step for syn-thesis of Ad-DPI is sensitive to water. In Basic Protocol 3, the catalytic asymmetric synthesis of S P -6 step is sensitive to water. Anhydrous reaction conditions are required for the aforementioned steps. Glassware needs to be ovendried, and anhydrous solvents should be prepared or purchased in Sure/Seal bottles. In the step for synthesis of S P -6 in Basic Protocol 3, 4 Å molecular sieves are necessary for the reaction. After the synthesis, the purified compounds are identified by 1 H-NMR, 13 C-NMR, and HRMS as shown in each synthesis. The d.r. value of crude S p -6 is analyzed by 31 P-NMR. The optically pure S p -6 is verified by 31 P-NMR and HPLC. Synthesis of 2-ethylbutyl (chloro(phenoxy)phosphoryl)-L-alaninate (rac-4) (Basic Protocol 1) can be completed within 28 hr. Synthesis of chiral bicyclic imidazole Ad-DPI (Basic Protocol 2) can be completed within 4 days. Synthesis of remdesivir (Basic Protocol 3) can be completed within 3 days. 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Advanced Synthesis & Catalysis Chiral bicycle imidazole nucleophilic catalysts: Rational design, facile synthesis, and successful application in asymmetric Steglich rearrangement Chiral bicyclic imidazolecatalyzed acylative dynamic kinetic resolution for the synthesis of chiral phthalidyl esters Development of a new bicyclic imidazole nucleophilic organocatalyst for direct enantioselective C-acylation The authors declare no conflict of interest. Data openly available in a public repository that issues datasets with DOIs.