Shreesha Bhat, 10 April 2020
Synthesis of Remdesivir
Mackman
D. Siegel, H. C. Hui, E. Doerffler, M. O. Clarke, K. Chun, L. Zhang, S. Neville, E. Carra, W. Lew, B. Ross, Q. Wang, L. Wolfe, R. Jordan, V. Soloveva, J. Knox, J. Perry, M. Perron, K. M. Stray, O. Barauskas, J. Y. Feng, Y. Xu, G. Lee, A. L. Rheingold, A. S. Ray, R. Bannister, R. Strickley, S. Swaminathan, W. A. Lee, S. Bavari, T. Cihlar, M. K. Lo, T. K. Warren, R. L. Mackman, J. Med. Chem. 2017, 60, 1684-1661.
DOI: 10.1021/acs.jmedchem.6b01594
B. K. Chun, et al., US 2016/0122374 A1, United States Patent and Trademark Office, May 5, 2016.
History and Development of Remdesivir
In the quest towards finding a potent drug against the Ebola virus (EBOV), scientists from Gilead Sciences and the United States Army Medical Research Institute of Infectious Diseases screened more than 1000 nucleoside analogues focused towards cyclic ribose or ribose like core that could target RNA viruses in 2014.
This led to the identification of the parent nucleoside 6 and its potent phosphoramidate prodrug diastereoisomer 9a (GS-5734). Structure activity relationship studies pointed towards the criticality of 1'-CN functional group as it provided lower toxicity and selectivity towards viral polymerase as compared to the unmodified nucleoside. The prodrug diastereoisomer 9a had potent in vitro activity against HCV (Hepatitis C virus), human and zoonotic coronaviruses like RCV (Rat Coronavirus), SARS-CoV, MERS- CoV (Middle east respiratory syndrome) in addition to anti-EBOV activity found in 6. This broad-spectrum antiviral activity of 9a (GS-5734) has led it to being tested against the 2020 pandemic Covid-19 caused by SARS-Cov-2.
GS-5734 (9a) - also called Remdesivir - is an RNA polymerase inhibitor with promising efficacy data in non-human primate models against EBOV, RCV, MERS, etc. Remdesivir converts into its triphosphate metabolites 9tp in human cells which is taken up in place of adenosine triphosphate by the viral RNA polymerase, thereby crippling the virus replication.
First Generation Synthesis of Remdesivir
The scientists at Gilead started with the synthesis of their best lead and the single Sp phosphoramidate prodrug with a commercially available tribenzyl protected lactol 1 followed by oxidation to its corresponding lactone 2. The next key step was the C-C bond forming glycosylation reaction of the ribolactone 2 with a bromo pyrrolotriazine nucleus 3. This was facilitated by the N-silyl protection in 3, followed by a lithium-halogen exchange using excess BuLi at -78°C. The lithiated pyrrolotriazine was coupled with ribolactone 2 to provide a mixture of 1' isomers of nucleoside 4 followed by 1'- cyanation to give the β-anomer 5 after chromatographic purification. Tribenzyl deprotection gave the 1-cyano modified adenine nucleoside 6.
The diastereomeric mixture of the phosphoramidoyl chloridate prodrug moiety 8 was prepared from the L-alanine analogue 7.
Finally, coupling of nucleoside 6 and chloridate 8 provided the phosphoramidate prodrug mixture 9 in ~ 1:1 diastereomeric ratio. The two diastereomers were resolved using chiral HPLC to afford the Sp isomer 9a and Rp isomer 9b, respectively.
Second Generation Synthesis of Remdesivir
The use of cryogenic temperatures, dependency on rate of addition of n-BuLi, unpredictable yields and need for chiral chromatography deemed the first-generation synthetic route unscalable. Efforts were directed towards using milder reagents and temperature and obtaining enhanced selectivity.
The foremost changes in the method proceeded with replacement of the inconsistent n-BuLi method for the glycosylation reaction towards a coupling accelerated by the Turbo Grignard reagent i-PrMgCl·LiCl. The use of PhMgCl and TMSCl led to better control in the amino protection, and the iodo base 11 enabled a more facile metal-halogen exchange than its bromo equivalent. This method of the nucleoside synthesis allowed for consistent yields at milder temperatures, hence making it scale-up friendly.
The 1'-cyanation of C-nucleoside 4 gave the product 5 in >95:5 anomeric ratio favoring the desired β-anomer. The inclusion of TfOH was found to be responsible for the high yield and high selectivity, thereby bypassing the need for chiral separation. Henceforth, a crucial change in the protection-deprotection strategy was undertaken whereby after the initial debenzylation, 2',3'-acetonide protection of the hydroxyl moieties was carried out to give 12. It was found that the coupling of nucleoside 12 with the prodrug counterpart 11 provided far better yields as compared to the unprotected glycoside 6.
Opting for a p-nitrophenolate prodrug precursor 10 instead of chloridate 8 afforded a single Sp isomer 11 after resolution through solvent crystallization, which proved to be the key step towards the stereoselective synthesis of the final product.
The final reaction of the p-nitrophenolate 2-ethylbutyl-L-alaninate prodrug coupling partner 11 with the acetonide protected nucleoside 12 proceeded in the presence of MgCl2 to give a diastereoselective product (exclusive Sp isomer) through SN2 type inversion of the phosphorus stereocenter. In both cases, the Sp isomer was established through single X-ray crystallography. Final deprotection of the acetonide yielded Remdesivir (9a) in 69% yield.
The second-generation synthesis of Remdesivir thus was a far better improvement in terms of scalability, yields and stereoselectivity bypassing the bottleneck of inconsistent yields and chiral separation.
