Synthesis and Biological Evaluation of Benzocyclooctene-based and Indene-based Anticancer Agents that Function as Inhibitors of Tubulin Polymerization

Abstract

The natural products colchicine and combretastatin A-4 (CA4) have been inspirational for the design and synthesis of structurally related analogues and spin-off compounds as inhibitors of tubulin polymerization. The discovery that a water-soluble phosphate prodrug salt of CA4 (referred to as CA4P) is capable of imparting profound and selective damage to tumor-associated blood vessels paved the way for the development of a new therapeutic approach for cancer treatment utilizing small-molecule inhibitors of tubulin polymerization that also act as vascular disrupting agents (VDAs). Combination of salient structural features associated with colchicine and CA4 led to the design and synthesis of a variety of fused aryl-cycloalkyl and aryl-heterocyclic compounds that function as inhibitors of tubulin polymerization. Prominent among these compounds is a benzosuberene analogue (referred to as KGP18), which demonstrates sub-nM cytotoxicity against human cancer cell lines and functions (when administered as a water-soluble prodrug salt) as a VDA in mouse models. Structure activity relationship considerations led to the evaluation of benzocyclooctyl [6,8 fused] and indene [6,5 fused] ring systems. Four benzocyclooctene and four indene analogues were prepared and evaluated biologically. Three of the benzocyclooctene analogues were active as inhibitors of tubulin polymerization (IC₅₀ < 5 μM), and benzocyclooctene phenol 23 was comparable to KGP18 in terms of potency. The analogous indene-based compound 31 also functioned as an inhibitor of tubulin polymerization (IC₅₀ = 11 μM) with reduced potency. The most potent inhibitor of tubulin polymerization from this group was benzocyclooctene analogue 23, and it was converted to its water-soluble prodrug salt 24 to assess its potential as a VDA. Preliminary in vivo studies, which utilized the MCF7-luc-GFP-mCherry breast tumor in a SCID mouse model, demonstrated that treatment with 24 (120 mg/kg) resulted in significant vascular shutdown, as evidenced by bioluminescence imaging at 4 h post administration, and that the effect continued at both 24 and 48 h. Contemporaneous studies with CA4P, a clinically relevant VDA, were carried out as a positive control.

Keywords: benzocyclooctene analogues; bioluminescence imaging (BLI); indene analogues; inhibitors of tubulin polymerization; small-molecule synthesis; vascular disrupting agents (VDAs).

Figure 1

Representative small-molecule anti-proliferative agents including colchicine, paclitaxel,^, CA4, vinblastine,^, OXi6196,^, KGP18,^– KGP156,^,, OXi8006, and BNC105.^,

Figure 2

Structures of PR-104 and TH-302.

Figure 3

Several clinically relevant VDAs including CA4P,^– CA1P,^, BNC105,^,,,, AVE8062,^,– and ZD6126.^,,

Figure 4

Compilation of synthesized benzocyclooctene analogues 20, 21, 23, and 24 and indene analogues 28, 29, 31, and 32.

Figure 5

Dynamic bioluminescence with respect to vascular disruption. A) Graphs show evolution of light emission from individual MCF7-luc-GFP-mCherry breast tumors following administration of luciferin substrate subcutaneously in the fore-back region of each mouse at time 0. Respective curves are baseline (blue), 4 h (red), 24 h (green) and 48 h (purple) post administration of VDA. Left hand panel shows a representative mouse with 120 mg/kg analogue 24 and right hand panel shows CA4P (120 mg/kg). B) Photographs show selected images at the 10 min. time point. Left hand group for analogue 24 and right hand group for CA4P for the same mice used to generate the curves for part A. Respective images are each scaled to same values.

Figure 6

Histological assessment of tumor necrosis. H&E stained tumor cross sections showing necrotic (pink) and viable (purple) regions at 48 h post treatment for A: Analogue 24 (120 mg/kg) B: CA4P (120 mg/kg) C: Saline.

Scheme 1

Synthesis of benzocyclooctanone analogues 5 and 6.

Scheme 2

Synthesis of indanone analogues 11 and 12.

Scheme 3

Synthesis of TBS-protected benzocyclooctanone 14 and indanone 16.

Scheme 4

Synthesis of target benzocyclooctene analogues 20, 21, and 23.

Scheme 5

Conversion of analogue 23 to its corresponding phosphate prodrug salt 24.

Scheme 6

Synthesis of target indene compounds 28, 29, and 31.

Scheme 7

Synthesis of target indene water-soluble analogue 32.