Discovery of Cytotoxic Dolastatin 10 Analogues with N-Terminal Modifications

Abstract

Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.

Design, Synthesis, and Cytotoxic Evaluation of Novel Tubulysin Analogues as ADC Payloads

“…This is quite intriguing and clearly demonstrates a divergence of SAR between auristatins and tubulysins, as such substituents have yielded very potent auristatins. 14 Overall, tertiary amine 8g, containing an α-methyl tertiary amine designed based on our auristatin SAR, 14 provided the best balance of potency against a variety of cell lines, including the MDR1 expressing KB 8.5 line. It should be noted that 8g was predicted to overlay better with compound 2, especially the position of the terminal N atom, than compound 8f (Figure 2C vs D), thus giving support for our design approach.…”

“…We selected ester 4 rather than the corresponding natural acid-containing Tup C-terminus for exploring N-termini SAR, primarily to minimize possible diminished activity in a cellbased assay, known to be reliant on permeability. 14 The Ntermini modified library (analogues 8a−g) was generated by coupling of the amine 4, prepared from known intermediate 7, 17 with selected Boc-or Fmoc-protected amino acids and removal of the protecting group (Scheme 1). N87 cells (liver metastasis from gastric carcinoma) and MDA-MB-361-DYT2 cells (breast carcinoma) were employed for cytotoxic evaluation of tubulysin analogues.…”

“…There were two consistent observations during the library design using shape-based molecular overlay. First, the amide bond between Ile and Tuv residues in tubulysins appeared to be trans as compared to the cis configuration 14 between corresponding Val and Dil residues in the tubulin-bound auristatin ( Figure 2B). Second, the N-terminal backbone stereochemical differences between the compound classes ( Figure 2A,B) seem to have a direct impact on the position of the terminal N atom, especially in compounds with cyclic monomers 17f ( Figure 2C) and 17g ( Figure 2D).…”

Novel Anti-TM4SF1 Antibody–Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature

“…S1A; ref. 27). However, a key difference of MMAF is the presence of a N-methylated a,a-dimethyl amino acid (Aib) that replaces the N-methyl valine on the N-terminus of MMAF.…”

“…To test this hypothesis, we humanized L6, generating antibody v1.10; L6 and v1.10, unlike 8G4, cross-react with cynomolgus monkey TM4SF1 (10). We then armed v1.10 with LP2 (chemical name mc-3377), a synthetic analog of dolastatin-10 (26); dolastatin-10 and synthetic analogs, termed auristatins, inhibit tubulin polymerization, and ultimately induce G 2 -M cell-cycle arrest and cell death at low picomolar intracellular concentrations (27). Tubulin inhibitors have been extensively investigated as vascular targeting agents and seem to be preferentially toxic against tumor and tumor vascular endothelial cells with high proliferation rates, while sparing the nondividing endothelial cells of normal tissues (28)(29)(30).…”