Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion.

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA )-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms,electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glycoprofiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

Discovery and SAR of small molecule PAR1 antagonists.

High-throughput screening resulted in the identification of a small molecule inhibitor of PAR1. Optimisation of the initial hit led to the discovery of compounds 34 and 49, which displayed antithrombotic activity in an arteriovenous shunt model in the rat after iv administration.

Kinetic studies on the stability and reactivity of beta-amino alkylzinc iodides derived from amino acids.

Beta-amino alkylzinc iodides are intrinsically unstable toward beta-elimination and protonation. The aim of this study was to determine the rates of these processes and also to understand how the reactivity of a range of beta-amino alkylzinc iodides in Negishi cross-coupling reactions is influenced by the presence of functional groups within the zinc reagent. Decomposition of beta-benzamido alkylzinc iodides occurs by protonation, and the first-order rate constant for the self-protonation of the carbon-zinc bond in reagent 4b was determined to be 5.2 x 10(-6) s(-1) (at 291 K). In contrast, the carbamate derivative 2 decomposes by a first-order elimination process. The homologous reagent 3, derived from glutamic acid, decomposes more quickly by beta-elimination, with a first-order rate constant of 24 x 10(-6) s(-1) (at 291 K). Reagents 23 and 25, in which the Boc group has been replaced with a trifluoroacetyl group, are more stable toward beta-elimination than the corresponding reagents 2 and 3, a striking outcome given that the trifluoroacetamido group is a better leaving group. Moreover, this replacement also changes the mechanism of the elimination to a second order process. Pseudo-second-order rate constants for the Negishi cross-coupling of reagents 2, 3, 23, and 25 with iodobenzene have been determined, revealing the higher reactivity of the glutamic acid-derived reagents 3 and 25. The main factor influencing reactivity, therefore, is determined to be the proximity of the ester group, rather than the nature of the nitrogen protecting group. Finally, beta-amino alkylzinc iodides 46-48 containing Weinreb amides have been prepared, rate constants for their decomposition through elimination determined, and their synthetic potential for the preparation of beta-amino ketones established.

Direct synthesis of unprotected phenols using palladium-catalysed cross coupling reactions of functionalised organozinc reagents.

Palladium-catalysed reaction of unprotected 2-, 3-, and 4-iodophenols with a range of amino acid derived organozinc reagents (not used in excess) gives the expected products in good to excellent yield, demonstrating that carbon-zinc bonds are not protonated by acidic phenols under the conditions of palladium-catalysed coupling reactions.

The rate of elimination of a beta-amino zinc reagent is reduced by using a better leaving group.

Replacing the N-Boc-protecting group on a beta-amino organo-zinc reagent with a trifluoroacetyl group, which would be expected to make the nitrogen a better leaving group, results in a reagent that is more stable towards elimination.