Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An "eye enhancer" was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.

A robust, green, and sustainable manufacturing process has been developed for the synthesis of gefapixant citrate, a P2X3 receptor antagonist that is under investigation for the treatment of refractory and unexplained chronic cough. The newly developed commercial process features low process mass intensity (PMI), short synthetic sequence, high overall yield, minimal environmental impact, and significantly reduced API costs. The key innovations are the implementation of a highly efficient two-step methoxyphenol synthesis, an innovative pyrimidine synthesis in flow, a simplified sulfonamide synthesis, and a novel salt metathesis approach to consistently deliver the correct active pharmaceutical ingredient (API) salt form in high purity.

The manufacturing route toward gefapixant citrate generates a trace amount of cyanide as a byproduct of a reaction employing the reagent chloroacetonitrile. In the development of a cyanide control strategy, conventional process and analytical approaches fell short because of challenges and incompatibilities with the matrices of the process and waste streams. To overcome these, we identified and adapted specific procedures for cyanide control. Our strategy ensured safety for patients, operators, and waste management.

The development of a safe, robust, and efficient manufacturing route for the synthesis of diaminopyrimidine 1, a key intermediate to gefapixant citrate (MK-7264), is described. A full mechanistic understanding of the cyclization step in the presence of guanidine was established by performing isotopic labeling experiments and identification of impurities. Guided by the mechanistic understanding, further attempts to modify the cyclization reaction by employing additives to reduce the triazine (9) formation and guanidine loading will also be presented. This newly developed method delivered compound 1 in 88−94% yield on a commercial scale and addressed the shortcomings of the early synthetic route including high PMI, low atom economy, long cycle-time, and multiple purifications to achieve the desired quality.

Two emerging concepts in cell biology are the back-and-forth trafficking of receptor proteins and nuclear transcription factors between the nucleus and the cell membrane, and the alternative splicing of messenger RNA to produce similar proteins targeted to different cell sites. Recent evidence suggests that the nucleolus is a dynamic structure whose components may be involved in both types of trafficking. In the nervous system of higher animals, the N-methyl-D-aspartate (NMDA)-specific glutamate receptor has various roles in development and cell communication. It is involved in learning, memory, axonal guidance and nerve regeneration. We have reported earlier that the NR1 subunit of the NMDA receptor is present in the cell periphery and the nucleus of stem cells, neurons and epitheliomuscular cells of the early-evolved cnidarian, Hydra vulgaris (Scappaticci et al., 2004. Cell Tissue Res 316:263-270); it is involved in coordinating hydra's neuroeffector systems (Kass-Simon and Scappaticci, 2003. Hydrobiologia 530/531:67-71; Pierobon et al., 2004. Eur J Neurosci 20:2598-2604; Scappaticci and Kass-Simon, 2008. Comp Biochem Physiol A 150:415-422; Kay and Kass-Simon, 2009. Bio Bull 216:113-129). Here we report immunocytochemical experiments, using a mouse monoclonal antibody raised against the mammalian NR1 receptor subunit, and an in silico genomic and gene expression analysis identifying the homologues in hydra of mammalian NR1 and fibrillarin (FBL) genes, and their expressed proteins. The experiments reveal that the NR1 antibody specifically labels the nucleoli of large and small interstitial cells (stem cells), nematoblasts, neuroblasts, and epitheliomusclar cells; antibody labeling of the nucleolar marker, FBL, confirms the nucleolar localization of NR1 antibody labeling. Genomic analysis reveals that NR1 and FBL genes are conserved in hydra, and suggests that there are at least two NR1 splice variants, one of which contains both nuclear and nucleolar targeting signals. The finding that an NR1 receptor subunit (or a portion of it) appears in nucleoli of hydra cells is unique, and has not been reported for any other organism. Its presence in nucleoli of hydra may signal the existence of a yet-undescribed shuttle mechanism between the cell surface and the nucleous, or the alternative deployment of NR1 splice variants to different cell sites.

The objective of the present study was to determine the in-vitro effect of Abietyl-Isothiocyanate (ABITC), a representative of a new class of anti-cancer drugs, on endometrial cancer (EC) cell lines. ABITC at concentrations ≥1 μM displayed dose-dependent and selective cytotoxicity to EC cell lines (ECC-1, AN3CA, RL95-2) in comparison to other cancer cell lines. After treatment with ABITC, ECC-1 unlike control cells displayed hallmark features of apoptosis including chromatin condensation and nuclear fragmentation. At concentrations below the IC50, ABITC exerted anti-proliferative effects by blocking cell-cycle progression through G0/G1 and S-phase. In addition, cells attempted to counteract drug treatment by pro-survival signaling such as deactivation of JNK/SAPK and p38 MAPK and activation of AKT and ErK1/2. ABITC also altered EGF-receptor phosphorylation. At a concentration of 5 μM ABITC generated an excess amount of reactive oxygen species (ROS) and displayed pro-apoptotic signaling such as activation of caspase-8, JNK-SAPK and deactivation of PARP-1. Co-treatment with an antioxidant blocked the drug effects by reducing ROS generation, cytotoxicity and pro-apoptotic signaling. In summary, novel isothiocyanate ABITC is an anti-proliferative and selectively cytotoxic drug to EC cells in-vitro. Key mechanisms during cell death are predominantly correlated to excess generation of ROS. We suggest the further development of ABITC as a potential therapeutic by studying the drug efficacy in EC in-vivo models.

A simple and efficient process to prepare Uprifosbuvir intermediate, 2′-deoxy-α-2′-chloro-β-2′-methyluridine (1), from bis-pivaloyl tertiary alcohol 5a is described. The key discoveries are a novel BSA-promoted anhydrouridine formation catalyzed by HCl as an additive and a milder safe Me 2 SiCl 2 -promoted chlorination of anhydrouridine. These discoveries collectively enabled the establishment of a robust process toward compound 1, which was demonstrated successfully at the plant scale.