Greater control is desirable in the stochastic conjugation technology used to synthesize antibody-drug conjugates (ADC). We have shown recently that a fluorescent dye can be stably conjugated to a mAb using a bifunctional platinum(II) linker. Here, we describe the general applicability of this novel linker technology for the preparation of stable and efficacious ADCs. The ethylenediamine platinum(II) moiety, herein called Lx, was coordinated to Desferal (DFO) or auristatin F (AF) to provide storable "semifinal" products, which were directly conjugated to unmodified mAbs. Conjugation resulted in ADCs with unimpaired mAb-binding characteristics, DAR in the range of 2.5 to 2.7 and approximately 85% payload bound to the Fc region, presumably to histidine residues. To evaluate the in vivo stability of Lx and its effect on pharmacokinetics and tumor targeting of an ADC, Lx-DFO was conjugated to the HER2 mAb trastuzumab, followed by radiolabeling with Zr. Trastuzumab-Lx-DFO-Zr was stable in vivo and exhibited pharmacokinetic and tumor-targeting properties similar to parental trastuzumab. In a xenograft mouse model of gastric cancer (NCI-N87) or an ado-trastuzumab emtansine-resistant breast cancer (JIMT-1), a single dose of trastuzumab-Lx-AF outperformed its maleimide benchmark trastuzumab-Mal-AF and FDA-approved ado-trastuzumab emtansine. Overall, our findings show the potential of the Lx technology as a robust conjugation platform for the preparation of anticancer ADCs. Cancer Res; 77(2); 257-67. ©2016 AACR.

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore and trastuzumab (Herceptin™) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L-NBD)Cl](NO3 ) (en=ethylenediamine), with L=N-heteroaromatic, N-alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO4 or Na2 SO4 in tricine or HEPES buffer. Reaction of N-coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5-2.9 that were stable in phosphate-buffered saline at room temperature for at least 48 h. In contrast, thioether-coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.

The synthesis of fluoro‐ and trifluoromethyl‐containing N‐sulfonylated nitrogen heterocycles is described. A first crucial step is a Mitsunobu functionalization of intermediate sulfonamides using commercially available unsaturated alcohols, which gives efficient access to fluorinated ring‐closing metathesis (RCM) precursors. Key step is an RCM reaction of these precursors leading to the corresponding fluoro‐ and trifluoromethyl‐containing heterocyclic building blocks. Furthermore, aminopalladation of the same sulfonamide intermediates provides access to trifuoromethyl‐containing pyrrole derivatives.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Bacteria under pressure! An efficient synthesis of the enantiopure tricyclic core of a recently discovered antibiotic, platencin, has been developed. This synthesis highlights a novel high‐pressure Diels–Alder reaction of the flavoring agent (S)‐(−)‐perillaldehyde and an efficient SmI2‐mediated pinacol cyclization for the construction of the bicyclo[2.2.2]octane framework (see scheme).

A comprehensive study on the synthesis and ring-closing metathesis (RCM) of a,b-dehydroamino acids is described. This sequence has led to the formation of a range of biologically relevant functionalized nitrogen heterocycles. The incorporation of chiral building blocks in the RCM precursors eventually resulted in the formation of optically active 4-substituted cyclic dehydroamino acids. In ad-dition, olefin isomerization under metathesis conditions was observed for a number of compounds, which could be successfully inhibited either by the introduction of allylic substituents or by the addition of a ruthenium hydride scavenger. Scheme 1. Ring-closing metathesis of dehydroamino acids.Scheme 4. Synthesis of allylically substituted RCM precursors.Scheme 5. RCM of allylically substituted dehydroamino acid-based precursors.