Diversely functionalized, fused aryl-alkyl ring systems hold a prominent position as well-established molecular frameworks for a variety of anti-cancer agents. The benzosuberene (6,7 fused, also referred to as dihydro-5H-benzo[7]annulene and benzocycloheptene) ring system has emerged as a valuable molecular core component for the development of inhibitors of tubulin assembly, which function as antiproliferative anti-cancer agents and, in certain cases, as vascular disrupting agents (VDAs). Both a phenolic-based analogue (known as KGP18, compound 39) and its corresponding amine-based congener (referred to as KGP156, compound 45), which demonstrate strong inhibition of tubulin assembly (low micromolar range) and potent cytotoxicity (picomolar range for KGP18 and nanomolar range for KGP156) are noteworthy examples of such benzosuberene-based compounds. In order to extend the structure-activity relationship (SAR) knowledge base related to benzosuberene anti-cancer agents, a series of eleven analogues (including KGP18) were prepared in which the methoxylation pattern on the pendant aryl ring as well as functional group incorporation on the fused aryl ring were varied. The synthetic approach to these compounds featured a sequential Wittig olefination, reduction, Eaton's reagent-mediated cyclization strategy to achieve the core benzosuberone intermediate, and represented a higher-yielding synthesis of KGP18 (which we prepared previously through a ring-expansion strategy). Incorporation of a fluorine or chlorine atom at the 1-position of the fused aryl ring or replacement of one of the methoxy groups with hydrogen (on the pendant aryl ring of KGP18) led to benzosuberene analogues that were both strongly inhibitory against tubulin assembly (IC50 approximately 1.0 M) and strongly cytotoxic against selected human cancer cell lines (for example, GI50 = 5.47 nM against NCI-H460 cells with fluorobenzosuberene analogue 37). A water-soluble phosphate prodrug salt of KGP18 (referred to as KGP265, compound 44) and a water-soluble serinamide salt (compound 48) of KGP156 were also synthesized and evaluated in this study.

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The discovery of 3-methoxy-9-(3′,4′,5′-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-ol (a benzosuberene-based analogue referred to as KGP18) was originally inspired by the natural products colchicine and combretastatin A-4 (CA4). The relative structural simplicity and ease of synthesis of KGP18, coupled with its potent biological activity as an inhibitor of tubulin polymerization and its cytotoxicity (in vitro) against human cancer cell lines, has resulted in studies focused on new analogue design and synthesis. Our goal was to probe the relationship of structure to function in this class of anticancer agents. A series of twenty-two new benzosuberene-based analogues of KGP18 was designed and synthesized. These compounds vary in their methoxylation pattern and separately incorporate trifluoromethyl groups around the pendant aryl ring for the evaluation of the effect of functional group modifications on the fused six-membered aromatic ring. In addition, the 8,9-saturated congener of KGP18 has been synthesized to assess the necessity of unsaturation at the carbon atom bearing the pendant aryl ring. Six of the molecules from this benzosuberene-series of compounds were active (IC50 < 5 μM) as inhibitors of tubulin polymerization while four analogues were comparable (IC50 approximately 1 μM) in their tubulin inhibitory activity to CA4 and KGP18. The potency of a bis-trifluoromethyl analogue 74 and the unsaturated KGP18 derivative 73 as inhibitors of tubulin assembly along with their moderate cytotoxicity suggested the potential utility of these compounds as vascular disrupting agents (VDAs) to selectively target microvessels feeding tumors. Accordingly, water-soluble and DMSO-soluble phosphate prodrug salts of each were synthesized for preliminary in vivo studies to assess their potential efficacy as VDAs.

The discovery of a benzosuberene‐based, small‐molecule anti‐cancer agent (KGP18) with remarkable cytotoxicity against selected human cancer cell lines (BMC 2008 16, 8161–8171) provided the inspiration for a collaborative research program to investigate the mechanism of action of this compound and related benzosuberene analogues. The structure of KGP18 and its ability to inhibit tubulin polymerization into microtubules in vitro suggests that it may act as a vascular disrupting agent (VDA). An adequate blood supply is required for tumor growth and metastasis. VDAs represent a promising new class of anti‐cancer agents that function by selectively disrupting the tumor vasculature, thus starving the tumor of nutrients and oxygen via an adequate blood supply. Tubulin‐binding VDAs inhibit microtubule assembly in the activated endothelial cells, lining the tumor vasculature, leading to cytoskeletal reorganization, activation of RhoA and RhoA kinase, increased vascular permeability, cell rounding and detachment, and vessel occlusion. The lead benzosuberene analogue (KGP18) binds to tubulin at the colchicine binding site as determined by a competitive radiometric assay. Flow cytometry indicates that MDA‐MB‐231 cells treated with KGP18 are arrested at the G2/M phase of the cell cycle. (Supported by Oxigene Inc., the NIH, and CPRIT).

KGP18 is a tubulin‐binding, benzosuberene‐based small molecule with remarkable cytotoxicity against selected human cancer cell lines (BMC 2008 16, 8161–8171). Its structure together with its ability to inhibit tubulin polymerization into microtubules in vitro suggest that it may act as a vascular disrupting agent (VDA). An adequate blood supply is required for tumor growth and metastasis. VDAs represent a promising new class of anti‐cancer agents that function by selectively disrupting the tumor vasculature, thus starving the tumor of nutrients and oxygen. Tubulin‐binding VDAs inhibit microtubule assembly in the activated endothelial cells, lining the tumor vasculature, leading to cytoskeletal reorganization, increased vascular permeability, cell rounding and detachment, and vessel disruption. The lead benzosuberene analogue (KGP18) and several analogues were assessed for their ability to disrupt microtubules, and cause a rearrangement of F‐actin in human umbilical vein endothelial cells (HUVECs) in cell culture. The disruption of pre‐established tubules formed by HUVECs on Matrigel™ by these benzosuberene analogues was also evaluated. (Supported by Oxigene Inc., the NIH, and CPRIT).