Solid-Phase Synthesis of Duocarmycin Analogues and the Effect of C-Terminal Substitution on Biological Activity

Abstract

The duocarmycins are potent antitumor agents with potential for use in the development of antibody-drug conjugates (ADCs) as well as being clinical candidates in their own right. In this article, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilization in solid-phase synthesis. The synthesis was performed on a large scale, and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers; its application to solid-phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to that in previous reports for both the monomeric and extended compounds. Substitution at the C-terminus of duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine, but this had no beneficial effects on biological activity.

A Peptide–Duocarmycin Conjugate Targeting the Thomsen-Friedenreich Antigen Has Potent and Selective Antitumor Activity

“…Previous work has shown that substitution on the C-terminus of DSA results in a loss of activity, and that a neutral C terminus is required. 20 Therefore, rink amide was chosen for the resin and Fmoc-DSA(OBn)-OH was loaded directly onto the resin, allowing for the production of a terminal amide. Studies of drug conjugate generation often debate "noncleavable" versus "cleavable" linkers.…”

“…The design of our peptide–drug conjugate aimed to fully exploit the ability to be prepared on the solid phase. Previous work has shown that substitution on the C-terminus of DSA results in a loss of activity, and that a neutral C terminus is required . Therefore, rink amide was chosen for the resin and Fmoc-DSA­(OBn)-OH was loaded directly onto the resin, allowing for the production of a terminal amide.…”

“…19 We previously reported the preparation of a molecule that enables the use of the alkylating unit of duocarmycin SA (termed DSA) in solid phase peptide synthesis (SPPS), referred to as Fmoc-DSA(OBn)-OH. 20,21 We demonstrated the use of this uncyclised seco-DSA unit in SPPS, allowing incorporation into the backbone of a peptide and, by extension, this approach allows the incorporation of the amino acid structure of the DSA alkylation subunit into any defined PDC structure. We report here a PDC targeting DSA to the TFα.…”

TCR-like antibody drug conjugates mediate killing of tumor cells with low peptide/HLA targets

“…16,22 All other TCR-like antibodies under study here also internalize following peptide/HLA binding based on subsequent cancer cell killing, presumably due to cathepsin B-specific release of pro-drug duocarmycin conjugates within lysosomes, which spontaneously rearrange to an active form and travel to the nucleus to mediate irreversible DNA damage. 34,49,50 Even in the case of YLSG-Ab3,5 ADCs, some degree of target cytotoxicity (»25%) was observed following co-culture with 4T1-CEA cells (Fig. 3).…”

Catalysis of Radical Cyclizations from Alkyl Iodides under H2: Evidence for Electron Transfer from [CpV(CO)3H]−

“…Polyhalogenated substrates react selectively at the iodide (for example, the aryl bromide in entry 5 remains unchanged). Entry 6 is the first 5-exo cyclization of an alkyl radical 21 onto a chlorinated double bond; Sharp and Zard have observed cyclizations promoted by Bu 3 SnH result in the dechlorination of such substrates. 22…”