A two-step synthesis of molnupiravir (1) is presented. This work focuses on the development of practical reaction and purification conditions toward a manufacturing route. The sequence commences from highly available cytidine (2), and molnupiravir is formed through direct hydroxamination of the cytosine ring and esterification of the sugar’s primary alcohol without use of protecting or activating groups. A highly crystalline hydrate of N-hydroxycytidine (3) resulted in an easily purified intermediate, and a practical, off-the-shelf enzyme was selected for the acylation. The yield was increased through a chemically promoted, selective ester cleavage, which converted a byproduct, molnupiravir isobutyryl oxime ester (4), into the final API. Both reactions proceed in >90% assay yield, and crystallization procedures are used to afford intermediates and active pharmaceutical ingredients in purities above 99% with an overall yield of 60%. Excellent throughput and sustainability are achieved by limiting the total concentration to 7 volumes of solvent in the course of the two reactions with an overall PMI of 26 including work-up and isolation. Environmentally friendly solvents, water and 2-methyl tetrahydrofuran, enhance sustainability of the operation.

Chiral separations depend upon column efficiency and chiral selectivity. Supercritical fluid chromatography (SFC) has been shown to have pronounced advantages in chiral separations due to its enhanced column efficiency at normal flow rates. Examination of factors affecting selectivity in SFC is crucial to systematic chiral method development. Selectivity is a compromise between differences in enantiomeric binding enthalpy and disruptive entropic effects. Increased temperature will decrease the effect of differences in enantiomer binding enthalpy, eventually decreasing selectivity to a point where the enantiomers coelute. Extension of this thermodynamic theory predicts that further increases in temperature lead to selectivity values of less than 1.0, and elution order of the enantiomers reverses. In this region separations are said to be "entropically driven", and selectivity increases (decreases from 1.0) with temperature. Performing separations in this region is attractive because column efficiency is also expected to increase with temperature. Such entropically driven separations have been observed only in gas chromatography. Most data used to support the application of this theory in SFC have been generated at subcritical temperatures, while theory purports to predict behavior above the critical temperature (T(c)). This approach ignores the effects of traversing the critical temperature in SFC, which is known to have a variety of unpredictable consequences. Work presented here shows the effect of temperatures above T(c) on chromatographic behavior. Contrary to theory, capacity factors increase near T(c) and column efficiency declines. Use of pressures well above the critical pressure lessens these effects. In accordance with theory, selectivity does decrease with temperature through T(c) and isoelution temperatures and two instances of elution order reversal are observed here for the first time in SFC.

A number of common mobile phase modifiers were characterized by chromatographing monofunctional probe solutes on a cyano-bonded stationary phase under subcritical and supercritical conditions. Solvatochromic analysis techniques were used to characterize the relative selectivity, efficiency, and eluotropic strengths of the various modified mobile phases. Analysis of the combined results suggests that modifier choice may be made in a rational manner to either enhance or mask a particular type of molecular interaction. This control is necessary for challenging applications such as chiral separations. The relative effects of each modifier property change depending on whether the chromatographic system is subcritical or supercritical, thus making knowledge of the phase equilibria associated with the mobile phase essential.

The effect of mobile phase additives is investigated for a variety of compounds under subcritical and supercritical conditions using packed columns. Retention of hydrogen bond donor/acceptor analytes was found to be more dependent on the presence of mobile phase additives than weak hydrogen bond acceptor analytes. The temperature and pressure of the mobile phase are major factors in the extent of this dependence. Consequently, selectivity between homologous compounds is dependent on both the additive used and the state of the mobile phase. Efficiency is nearly always improved by the presence of mobile phase additives, more so under supercritical conditions than under subcritical conditions. These observations suggest that surface molar excesses of mobile phase additives play a large part in the resulting character of the supercritical chromatographic system.