Cecilia Bottecchia

Herein we disclose a concise synthesis of inhaled soluble guanylate cyclase (isGC) stimulator Frespaciguat (MK-5475) that relies exclusively on complexity-increasing transformations from commodity chemicals. Key transformations include a solvent-free photochemical reaction that converts two gaseous reactants into a liquid product in high yield; a direct ester-to-amidine conversion using HMDS, water, and triflic acid; and a rare example of enantioselective methylation via copper-catalyzed asymmetric conjugate addition (ACA) to install a quaternary center using trimethylboroxine as a nucleophile. Given the general challenge of asymmetric methyl installation, we further demonstrate the utility of this methodology beyond the present setting and suggest its strategic value in synthetic planning where nonracemic, quaternary methyl-containing stereocenters are required.

An enantioselective seven-step synthesis of MK-7845, an investigational protease inhibitor for the treatment of COVID-19 is described. Key features of the synthesis include the preparation of a chiral isonitrile intermediate utilizing an asymmetric ruthenium-catalyzed reductive amination reaction to set the key amine stereocenter; a highly enantioselective, biocatalytic oxidation reaction with a monoamine oxidase enzyme to furnish a bicyclic imine; and, finally, the union of these fragments via a diastereoselective three-component Joullié-Ugi coupling reaction en route to MK-7845.

The chemical properties of disulfides are leveraged in a wide array of applications, ranging from protein-drug conjugates for cancer treatment to self-healing materials. However, disulfide reduction strategies remain severely underdeveloped despite being the key to efficiently accessing the desired targets. Specifically, no homogeneous catalyst has been reported for this reaction, and conditions that allow the use of mild and green reductants (e.g., via electrochemical reduction) are not known. Herein, we unveil a vitamin B₁₂-catalyzed, electrochemically driven protocol for efficiently reducing disulfide bonds in various aqueous buffers over a broad pH range. This robust and simple method is suitable for disulfide reductions of substrates ranging from small molecules to large proteins. Finally, one-pot reduction and conjugation of disulfide bonds in a monoclonal antibody were demonstrated to produce antibody conjugates.

In most FDA-approved antibody-drug conjugates, cysteines generated through reduction of the native interchain di-sulfide bonds in monoclonal antibodies (mAbs) are conjugated with maleimide-based cytotoxic payloads. Despite being key to efficiently producing well-defined conjugates, selective disulfide reduction strategies are severely underdevel-oped. Herein, we report a vitamin B12-catalyzed, electrochemically driven protocol that efficiently reduces disulfide bonds in various aqueous buffers and at a broad pH range. This robust and simple method is suitable for disulfide reduc-tions of substrates ranging from biologically relevant small molecules to large proteins. Finally, one-pot reduc-tion/conjugation of disulfide bonds in mAbs was achieved to access antibody conjugates.

This manuscript reports the identification of hydrophobic interaction chromatography (HIC)-shifting, non-toxic linker-payload surrogates as tool molecules for the optimization of maleimide/cysteine conjugations relevant to antibody-drug conjugates (ADCs). These tool molecules are demonstrated to allow conjugation measurement via HIC with a mAb (mono-clonal antibody) bearing engineered cysteines, and for conjugation to mAb interchain cysteines. The linker/payload (LP) mimics were employed to optimize conjugations via high-throughput experimentation and employed to facilitate the development of continuous flow conjugations in a microfluidic reactor and on larger scale. Putative identification of the novel ADC mimics by HIC was confirmed by mass spectrometry. Overall, our studies provide confidence that commercially available, non-toxic LP mimics can be employed successfully to optimize ADC-type conjugations in batch and flow, while minimizing materials needs and experimental work in specialized facilities required for potent compound handling.