Engineered Ribosyl-1-Kinase Enables Concise Synthesis of Molnupiravir, an Antiviral for COVID-19.

Molnupiravir (MK-4482) is an investigational antiviral agent that is under development for the treatment of COVID-19. Given the potential high demand and urgency for this compound, it was critical to develop a short and sustainable synthesis from simple raw materials that would minimize the time needed to manufacture and supply molnupiravir. The route reported here is enabled through the invention of a novel biocatalytic cascade featuring an engineered ribosyl-1-kinase and uridine phosphorylase. These engineered enzymes were deployed with a pyruvate-oxidase-enabled phosphate recycling strategy. Compared to the initial route, this synthesis of molnupiravir is 70% shorter and approximately 7-fold higher yielding. Looking forward, the biocatalytic approach to molnupiravir outlined here is anticipated to have broad applications for streamlining the synthesis of nucleosides in general.

Unlocking the Potential of High-Throughput Experimentation for Electrochemistry with a Standardized Microscale Reactor.

Organic electrochemistry has emerged as an enabling and sustainable technology in modern organic synthesis. Despite the recent renaissance of electrosynthesis, the broad adoption of electrochemistry in the synthetic community, and especially in industrial settings, has been hindered by the lack of general, standardized platforms for high-throughput experimentation (HTE). Herein, we disclose the design of the HTe - Chem, a high-throughput microscale electrochemical reactor that is compatible with existing HTE infrastructure and enables the rapid evaluation of a broad array of electrochemical reaction parameters. Utilizing the HTe - Chem to accelerate reaction optimization, reaction discovery, and chemical library synthesis is illustrated using a suite of oxidative and reductive transformations under constant current, constant voltage, and electrophotochemical conditions.

Efficient synthesis of antiviral agent uprifosbuvir enabled by new synthetic methods.

An efficient route to the HCV antiviral agent uprifosbuvir was developed in 5 steps from readily available uridine in 50% overall yield. This concise synthesis was achieved by development of several synthetic methods: (1) complexation-driven selective acyl migration/oxidation; (2) BSA-mediated cyclization to anhydrouridine; (3) hydrochlorination using FeCl3/TMDSO; (4) dynamic stereoselective phosphoramidation using a chiral nucleophilic catalyst. The new route improves the yield of uprifosbuvir 50-fold over the previous manufacturing process and expands the tool set available for synthesis of antiviral nucleotides.

Biocatalytic oxidation of alcohols using galactose oxidase and a manganese(III) activator for the synthesis of islatravir.

Galactose oxidase (GOase) is a Cu-dependent metalloenzyme that catalyzes the oxidation of alcohols to aldehydes. An evolved GOase variant was recently shown to catalyze a desymmetrizing oxidation as the first enzymatic step in the biocatalytic synthesis of islatravir. Horseradish peroxidase (HRP) is required to activate the GOase, introducing cost and protein burden to the process. Herein we describe that complexes of earth-abundant Mn(iii) (e.g. Mn(OAc)3) can be used at low loadings (2 mol%) as small molecule alternatives to HRP, providing similar yields and purity profiles. While an induction period is observed when using Mn(OAc)3 as the activator, employment of alternative Mn(iii) sources, such as Mn(acac)3 and K3[Mn(C2O4)3], eliminates the induction period and provides higher conversions to product. We demonstrate that use of the Mn(OAc)3 additive is also compatible with subsequent biocatalytic steps in the islatravir-forming cascade. Finally, to exhibit the wider utility of Mn(OAc)3, we show that Mn(OAc)3 functions as a suitable activator for several commercially available variants of GOase with a series of alcohol substrates.

Metal-Free Electrochemical Synthesis of Sulfonamides Directly from (Hetero)arenes, SO2 , and Amines.

Sulfonamides are among the most important chemical motifs in pharmaceuticals and agrochemicals. However, there is no methodology to directly introduce the sulfonamide group to a non-prefunctionalized aromatic compound. Herein, we present the first dehydrogenative electrochemical sulfonamide synthesis protocol by exploiting the inherent reactivity of (hetero)arenes in a highly convergent reaction with SO2 and amines via amidosulfinate intermediate. The amidosulfinate serves a dual role as reactant and supporting electrolyte. Direct anodic oxidation of the aromatic compound triggers the reaction, followed by nucleophilic attack of the amidosulfinate. Boron-doped diamond (BDD) electrodes and a HFIP-MeCN solvent mixture enable selective formation of the sulfonamides. In total, 36 examples are demonstrated with yields up to 85 %.

New Mechanism for Cinchona Alkaloid-Catalysis Allows for an Efficient Thiophosphorylation Reaction.

An efficient synthesis of nucleoside 5'-monothiophosphates under mild reaction conditions using commercially available thiophosphoryl chloride was achieved with a cinchona alkaloid catalyst. A detailed mechanistic study of the reaction was undertaken, employing a combination of reaction kinetics, NMR spectroscopy, and computational modeling, to better understand the observed reactivity. Taken collectively, the results support an unprecedented mechanism for this class of organocatalyst.

Multi-column ultra-high performance liquid chromatography screening with chaotropic agents and computer-assisted separation modeling enables process development of new drug substances.

Modern process research and development can often be hampered by the tedious method development required to chromatographically resolve mixtures of chemical species with very similar physical properties. Herein, we describe a simple approach for the development and implementation of an efficient ultra-high performance liquid chromatography (UHPLC) assay that is extensively applied to the separation and analysis of multicomponent reaction mixtures of closely related pharmaceutical intermediates and impurities. Methods are optimized using multi-column and multi-solvent UHPLC screening in conjunction with chromatography simulation software (ACD Labs/LC Simulator). This approach is implemented to enable the separation, identification, mapping and control of impurities formed within the process chemistry optimization of the dimeric catalyst used in the synthesis of new drug substances. The final method utilized a sub-2 µm C18 stationary phase (2.1 mm I.D. × 50 mm length, 1.7 µm particle size ACQUITY UPLC BEH C18) with a non-conventional chaotropic mobile phase buffer (35 mM potassium hexafluorophosphate in 0.1% phosphoric acid/acetonitrile) in order to achieve baseline separation of all reaction components. The chromatographic simulation and modeling strategy served to generate 3D resolution maps with robust separation conditions that match the outcome of subsequent experimental data (overall ΔtR < 0.35%). Our multi-column UHPLC screening with computer-assisted chromatographic modeling is a great addition to the toolbox of synthetic chemists and can be a powerful tool for streamlining process chemistry optimization in organic chemistry laboratories across both academic and industrial sectors.

Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances.

Impurity fate and purge studies are critical in order to establish an effective impurity control strategy for approval of the commercial filing application of new medicines. Reversed phase liquid chromatography-diode array-mass spectrometry (RPLC-DAD-MS) has traditionally been the preferred tool for impurity fate mapping. However, separation of some reaction mixtures by LC can be very problematic requiring combination LC-UV for area % analysis and a different LC-MS method for peak identification. In addition, some synthetic intermediates might be chemically susceptible to the aqueous conditions used in RPLC separations. In this study, the use of supercritical fluid chromatography-photodiode array-electrospray ionization mass spectrometry (SFC-PDA-ESIMS) for fate and purge of two specified impurities in the 1-uridine starting material from the synthesis of a bis-piv 2'keto-uridine, an intermediate in the synthesis of uprifosbuvir, a treatment under investigation for chronic hepatitis C infection. Readily available SFC instrumentation with a Chiralpak IC column (4.6 × 150 mm, 3 µm) and ethanol: carbon dioxide based mobile phase eluent enabled the separation of closely related components from complex reaction mixtures where RLPC failed to deliver optimal chromatographic performance. These results illustrate how SFC combined with PDA and ESI-MS detection can become a powerful tool for direct impurity fate mapping across multiple reaction steps.

A multifunctional catalyst that stereoselectively assembles prodrugs.

The catalytic stereoselective synthesis of compounds with chiral phosphorus centers remains an unsolved problem. State-of-the-art methods rely on resolution or stoichiometric chiral auxiliaries. Phosphoramidate prodrugs are a critical component of pronucleotide (ProTide) therapies used in the treatment of viral disease and cancer. Here we describe the development of a catalytic stereoselective method for the installation of phosphorus-stereogenic phosphoramidates to nucleosides through a dynamic stereoselective process. Detailed mechanistic studies and computational modeling led to the rational design of a multifunctional catalyst that enables stereoselectivity as high as 99:1.

Mechanism-Based Solution to the ProTide Synthesis Problem: Selective Access to Sofosbuvir, Acelarin, and INX-08189.

A general and efficient method for the synthesis of pronucleotide (ProTide) 5'-phosphoramidate monoesters is reported. This method consists of a highly stereoselective 5'-phosphorylation mediated by dimethylaluminum chloride to afford the desired target ProTides in excellent yields without employing 3'-protection strategies. The application of this methodology to the synthesis of a number of pharmaceutically relevant compounds currently marketed or under investigation in clinical research is demonstrated.

Synthesis of the GPR40 Partial Agonist MK-8666 through a Kinetically Controlled Dynamic Enzymatic Ketone Reduction.

A scalable and efficient synthesis of the GPR40 agonist MK-8666 was developed from a simple pyridine building block. The key step to set the stereochemistry at two centers relied on an enzymatic dynamic kinetic reduction of an unactivated ketone. Directed evolution was leveraged to generate an optimized ketoreductase that provided the desired trans alcohol in >30:1 dr and >99% ee. Further, it was demonstrated that all four diastereomers of this hydroxy-ester could be prepared in high yield and selectivity. Subsequently, a challenging intramolecular displacement was carried out to form the cyclopropane ring system with perfect control of endo/exo selectivity. The endgame coupling strategy relied on a Pd-catalyzed C-O coupling to join the headpiece chloropyridine with the benzylic alcohol tailpiece.

Asymmetric Synthesis of N-Boc-(R)-Silaproline via Rh-Catalyzed Intramolecular Hydrosilylation of Dehydroalanine and Continuous Flow N-Alkylation.

An asymmetric synthesis of a silicon-containing proline surrogate, N-Boc-(R)-silaproline (1), is described. Starting from N-Boc-dehydroalanine ester, deprotonation, followed by N-alkylation with chloromethyldimethylsilane under flow conditions, afforded the N-alkylated product 8 in 91% yield. An unprecedented enantioselective (NBD)2RhBF4/Josiphos 404-1 catalyzed 5-endo-trig hydrosilylation afforded the silaproline ester in 85-90% yield and >95% ee. Subsequent saponification and salt formation upgraded 1 to >99% ee.

A highly efficient asymmetric synthesis of vernakalant.

A novel synthesis of vernakalant is described. Using inexpensive and readily available reagents, the key transformations involve (1) an efficient zinc-amine-promoted etherification, (2) a highly stereoselective enzyme-catalyzed dynamic asymmetric transamination to set up the two contiguous chiral centers in the cyclohexane ring, and (3) a pyrrolidine ring formation via alkyl-B(OH)2-catalyzed amidation and subsequent imide reduction.

Enantioselective synthesis of an HCV NS5a antagonist.

A concise, enantioselective synthesis of the HCV NS5a inhibitor MK-8742 (1) is reported. The features of the synthesis include a highly enantioselective transfer hydrogenation of an NH imine and a dynamic diastereoselective transformation. The synthesis of this complex target requires simple starting materials and nine linear steps for completion.

Enantioselective, palladium-catalyzed α-arylation of N-Boc pyrrolidine: in situ react IR spectroscopic monitoring, scope, and synthetic applications.

A comprehensive study of the enantioselective Pd-catalyzed α-arylation of N-Boc pyrrolidine has been carried out. The protocol involves deprotonation of N-Boc pyrrolidine using s-BuLi/(-)-sparteine in TBME or Et(2)O at -78 °C, transmetalation with ZnCl(2) and Negishi coupling using Pd(OAc)(2), t-Bu(3)P-HBF(4) and the aryl bromide. This paper reports several new features including in situ React IR spectroscopic monitoring of the process; use of (-)-sparteine and the (+)-sparteine surrogate to access products with opposite configuration; development of a catalytic asymmetric lithiation-Negishi coupling reaction; extension to a wide range of heteroaromatic bromides; total synthesis of (R)-crispine A, (S)-nicotine and (S)-SIB-1508Y via short synthetic routes; and examples of α-vinylation of N-Boc pyrrolidine using vinyl bromides exemplified by the total synthesis of naturally occurring (+)-maackiamine (thus establishing its configuration as (R)). In this way, the full scope and limitations of the methodology are delineated.

Enantioselective, biocatalytic reduction of 3-substituted cyclopentenones: application to the asymmetric synthesis of an hNK-1 receptor antagonist.

A convergent and enantioselective route to the hNK-1 receptor antagonist (1) is described, which sets all six stereogenic centers with high diastereoselectivity and delivers 1 in only 11 steps and 23% overall yield. The process was enabled by the development of the enantioselective enzymatic reduction of 3-functionalized cyclopentenones and stereospecific Pd-catalyzed etherification coupling of fragments 6 and 7.

Identification and structural elucidation of process impurities generated in the end-game synthesis of taranabant (MK-0364) via cyanuric chloride.

Taranabant (MK-0364) is a highly potent and selective cannabinoid-1 receptor (CB-1R) inverse agonist. It is being developed at Merck & Company to treat obesity. The chemical synthesis of MK-0364 drug substance involved the direct coupling of chiral amine and pyridine acid side chains mediated by cyanuric chloride. Four major process impurities were observed and characterized using high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detectors. The exact mass data was used for structural elucidation which suggests that the impurities are derivatives of cyanuric chloride formed in the coupling step. Owing to the reactive nature of these impurities, an interesting degradation phenomenon was observed during stability testing of MK-0364 drug substance when stored at 40 degrees C/75% RH and 25 degrees C/60% RH conditions. Degradation pathways were proposed to explain the changes observed in the HPLC impurity profile. Forced degradation experiments were also conducted to confirm the degradation pathways and assess the stability of the impurities. Finally, the complete stability data of the bulk drug are reported to support the hypothesis.

Enantioselective Pd-catalyzed alpha-arylation of N-Boc-pyrrolidine: the key to an efficient and practical synthesis of a glucokinase activator.

A short and practical synthesis of glucokinase activator 1 was achieved utilizing a convergent strategy involving S(N)Ar coupling of activated aryl fluoride 11 with hydroxypyridine 9. The key to the success of the synthesis was the development of a novel method for enantioselective formation of alpha-arylpyrrolidines during the course of the project. In this method, (-)-sparteine-mediated enantioselective lithiation of N-Boc-pyrrolidine was followed by in situ transmetalation to zinc and Pd-catalyzed coupling with aryl bromide 3, proceeding in 92% ee. This transformation allowed the preparation of compound 1 in a 31% overall yield over six steps.

N-silyl protecting groups for labile aziridines: application toward the synthesis of N-H aziridinomitosenes.

Hindered N-silylamines were examined for their utility to serve as protecting groups for the labile aziridine nitrogen found within the highly sensitive aziridinomitosene framework. tert-Butyldiphenylsilyl and modified tert-butyldiphenylsilyl groups were the most resistant to nitrogen-silicon bond cleavage under various reaction conditions and were thus employed in transformations relevant to aziridinomitosene synthesis. The N-silylaziridines 7a, 21a, and 21b underwent azomethine ylide cycloaddition and afforded, upon deprotection, the N-H aziridine 24 in 18-32% overall yield for the three steps.

Enantioselective, palladium-catalyzed alpha-arylation of N-Boc-pyrrolidine.

This communication discloses the first instance of the enantioselective Pd-catalyzed alpha-arylation of N-Boc-pyrrolidine. The methodology relies on Beak's sparteine-mediated, enantioselective deprotonation of N-Boc-pyrrolidine to form the 2-pyrrolidinolithium specices in high enantiomeric ratio (er). Transmetalation of this intermediate with zinc chloride generates the stereochemically rigid, 2-pyrrolidinozinc reagent, which was readily coupled to a variety of functionalized aryl halides at room temperature using a catalyst generated from Pd(OAc)2 and PtBu3-HBF4. A diverse array of 2-aryl-N-Boc-pyrrolidines was synthesized using this methodology, providing adducts consistently in a 96:4 er. A survey of the stoichiometry revealed that as little as 0.3 equiv of zinc could be used in the coupling reaction, and the 2-pyrrolidinozinc reagent was found to exhibit stereochemical stability up to 60 degrees C. The method allows for the most convergent and reliable preparation of a broad range of functionalized 2-aryl-N-Boc-pyrrolidines in high enantioselectivity, which is highlighted in this report by the enantioselective synthesis of (R)-nicotine.