Synthesis of N-Hydroxycytidine and Dimethyl Dioxol Impurities, Method Development and Validation for Their Simultaneous Analysis in Molnupiravir

Abstract

WHO declared the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus as a global pandemic (coronavirus disease 2019 (COVID-19)) in March 2020. Molnupiravir (MPV) is an oral antiviral drug that received use authorization for mild to moderate COVID-19 treatment in adults. However, the global and national drug testing and research of MPV is still difficult due to lack of standards and validated procedures. Therefore, this work aims to synthesize the standard substance of two main molnupiravir impurities, N-hydroxycytidine (NHC) and dimethyl dioxol (DMDO), followed by development of a HPLC-photo diode array (PDA) for their simultaneous analysis. The procedure was validated in compliance with the international pharmaceutical analysis guideline (ICH), and employed to test these compounds in molnupiravir on the market. As a result, NHC and DMDO were successfully synthesized by a hydrolysis in an alkaline environment, and acetalization in an acid environment with very high yields of 83.76 and 73.51%, respectively, along with the purities of over 99%. NHC was detected below the allowable threshold whereas DMDO could not be detected in our samples. The findings show the high applicability of our synthesis and determination procedures in the large-scale production and quality control of impurities of commercial molnupiravir medicines.

Keywords: N-hydroxycytidine; dimethyl dioxol; impurity; method validation; molnupiravir; synthesis.