Synthesis and biological evaluation of structurally diverse indole-based analogues as inhibitors of tubulin polymerization and synthesis of drug-linker constructs cleavable by enzymes present in the tumor microenvironment.


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A sub-set of inhibitors of tubulin polymerization that bind to the colchicine site demonstrate dual functionality as both potent cytotoxins (antiproliferative agents) and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery of a potent inhibitor of tubulin polymerization and cancer cell growth, OXi8006, and its water-soluble phosphate prodrug salt, OXi8007, which functioned as a promising VDA in mouse models of cancer. To expand our understanding of the relationship between structure and activity, we generated analogues that incorporated indole modifications and separately incorporated a piperazine motif. A number of compounds from these focused libraries showed robust inhibition of tubulin polymerization and potent cytotoxic effects against human cancer cells. The most promising of these molecules were further assessed for their efficacy to function as tumor-selective vascular disrupting agents (VDAs). Antibody-drug conjugates (ADCs) have emerged as a promising therapeutic approach to the treatment of various cancer types. Several small-molecule inhibitors of tubulin polymerization that demonstrate dual-functionality as highly potent antiproliferative agents and VDAs offer promise as next generation payloads. Further selective targeting is provided by betabodies, which are engineered proteins that selectively target accessible phosphatidylserine (PS). Two small-molecule dual-mechanism payloads, KGP18 and KGP156, were synthetically tethered to drug-linker constructs featuring protease-cleavable short peptides. The most promising drug-linker constructs will be conjugated to PS-targeting betabodies in future studies. The potent payloads, KGP18 and KGP156, are capable of inducing tumor damage through two complementary and distinct mechanisms: inducing irreversible damage to tumor-associated vasculature to promote tumor necrosis and inhibiting proliferation in tumor cells. Although PS-targeting betabodies are highly specific for tumors and tumor-associated vasculature, they are not efficiently internalized within cells. As a result, the dual-mechanism payloads are designed to be released from BDCs through cleavage of linkers by extracellular proteases, such as urokinase-type plasminogen activator (uPA) or plasmin, that are upregulated or activated in the tumor microenvironment. We have incorporated plasmin-targeted (D-Ile-L-Phe-L-Lys) and uPA-targeted (Gly-Gly-L-Arg) short peptide sequences within the constructs bearing KGP18 and separately KGP156 payloads.