Drug-Linker Constructs Incorporating Unique Dual-Mechanism Payloads: Indole-based Analogues of OXi8006 Bearing Aryl Ring Bridge Modifications
Introduction
Antibody-drug conjugates and related entities bearing engineered proteins remain a promising therapeutic approach for the treatment of certain types of cancer. The development of new drug-linker constructs featuring payloads with diverse mechanisms of action offers promise for next generation therapeutics. A variety of small-molecule payloads (e.g. OXi8006) designed and synthesized by the Pinney Research Group, were structurally inspired by the tubulin-binding natural products colchicine and combretastatin A-4 (CA4). These molecules function as inhibitors of tubulin polymerization (colchicine site) and feature a unique dual-mechanism of action as both potent antiproliferative agents (cytotoxins) and promising tumor-selective vascular disrupting agents (VDAs). The indole-based analogue, OXi8006, demonstrates potent inhibition of tubulin polymerization (IC50 = 0.84 µM) and strong cytotoxicity against various human cancer cell lines [e.g. MCF-7 (breast): GI50 = 48 nM]. OXi8006 analogues were designed and synthesized to expand structure-activity relationship (SAR) knowledge regarding both tubulin binding and VDA efficacy. Indole analogues incorporating modifications to the carbonyl group situated between the 2-arylindole moiety and the 3-aroyl (trimethoxybenzoyl) group of OXi8006 were synthesized, and several aniline congeners were also prepared. These OXi8006 analogues offer promise as payloads in drug-linker constructs, which for these studies included a conjugation site (maleimidocaproyl, Mc), a dipeptide linker (valine-citrulline, Val-Cit, or related), a spacer (para-aminobenzyloxycarbonyl, PABC), and a payload.
Methods
The OXi8006 analogues were prepared by chemical synthesis featuring a variety of approaches including Fisher indole synthesis and Buchwald-Hartwig amination. Following purification by silica gel chromatography, structural confirmation was obtained through a combination of NMR spectroscopy and mass spectrometry. The OXi8006 analogues were evaluated biologically for their ability to inhibit tubulin polymerization with the most active molecules undergoing determination of their colchicine binding. Antiproliferative activity (cytotoxicity) was determined against the MCF-7 human breast carcinoma cell line. The release of payloads from drug-linker constructs was assessed by LC-MS (cathepsins B and L).
Results
KGP555, bearing a sulfide bridge, demonstrated significant inhibition of tubulin assembly (IC50 = 0.66 µM) and potent cytotoxicity against the MCF-7 human breast cancer cell line (GI50 = 5.0 nM), comparable to CA4 (IC50 = 0.75 µM; GI50 = 9.0 nM respectively). The potent biological activity of KGP555 inspired the design and synthesis of OXi8006 analogues featuring structural modifications to replace the carbonyl group, which included one-atom bridges (C, N, O, Se) and two-atom bridges [trans(E)-ethylene, amide, imine]. Among these OXi8006 analogues, KGP608, bearing a methylene bridge, demonstrated potent inhibition of tubulin polymerization (IC50 = 0.16 µM) and strong cytotoxicity against the MCF-7 human breast cancer cell line (GI50 = 4.0 nM). Drug-linker constructs (Mc-Val-Cit-PABC-DMED-Payload) were synthesized that incorporated either KGP555 or KGP608 as payload. Enzyme cleavage (cathepsin B) studies demonstrated minimal payload release (26% for KGP555 and 15% for KGP608). This was not entirely unanticipated as phenolic-based payloads often demonstrate low release in our hands under these enzyme-mediated cleavage conditions.
Conclusion
KGP555 and KGP608, bearing a sulfide and a methylene bridge, respectively, demonstrated potent inhibition of tubulin polymerization (IC50 = 0.75 µM and 0.16 µM) and were strongly cytotoxic against the MCF-7 breast cancer cell line (GI50 = 6.0 nM and 4.0 nM, respectively). Corresponding drug-linker constructs (Mc-Val-Cit-PABC-DMED-Payload) were synthesized and evaluated for enzyme-mediated payload release.