Exploring Regioisomeric Indole-Furanone Tubulin Inhibitors

Abstract

Tubulin is involved in microtubule function and affects mitosis, cell shape, migration, and the movement of organelles. Consequently, tubulin inhibitors have emerged as promising targets for cancer treatment. We previously identified a novel antitubulin motif that combines a furanone, indole, and electron-rich dimethoxyphenyl ring. The lead indole-furanone compound (3) demonstrated submicromolar potency on cancer cells and inhibited tubulin polymerization. To advance these findings, we synthesized a small library of analogs of 3, analyzed their biological activities, and used molecular modeling to elucidate binding interactions in the tubulin colchicine binding site. To assess the impact on potency, we compared: (1) dimethoxy vs trimethoxy substitution of the phenyl A-ring, (2) N-indole substitution of the indole B-ring, and (3) regioisomers and anhydrides of the furanone C-ring. In the process of developing the synthesis of the furanone C-ring regioisomers, we identified that a modification of conditions (NaH/inert vs DBU/air) could be used to give either the corresponding furanones or maleic anhydrides. Of the 18 synthesized compounds, six are biologically active with two exhibiting submicromolar activity against HL-60 cells. Of the six active compounds, (1) three contained dimethoxyphenyl A-rings and three contained trimethoxyphenyl A-rings largely oriented toward the tubulin α-subunit, (2) the N-indole substitution appeared to be less impactful on activity although having the indole nitrogen pointing down into the colchicine binding site was favored, and (3) the furanone carbonyl group located cis to the di- or trimethoxyphenyl A-ring and pointing toward the α-subunit was favored.

1

(a) Colchicine-binding site (CBS) inhibitors; (b) previously studied compounds.

2

Rationally designed analogs of 3 investigated in this study.

1. Synthesis of Type-1 Furanones 10-12

2. Attempted Synthesis of Type-2 Furanones 20-21

3. Synthesis of Indolylmaleic Anhydrides 29-32

4. Synthesis of Indolylmaleic Anhydrides 33, 34

5. Synthesis of Type-2 Furanones 20, 21, 35, 36

3

Molecular modeling of 36. (A) The lowest predicted free energy of binding of 36 in the CBS. The α-subunit is in dark gray and the β-subunit is in light gray. (B) A stereoview of interactions between 36 and tubulin. 36 is shown in green (C = green, N = blue, O = red), GTP is in orange (C = orange, N = blue, O = red), and the side chains of the interacting residues are shown in gray (C = gray, N = blue, O = red). Hydrogen bonds in B are represented as dashed lines.

4

Cartoon representation of the main inhibitor-tubulin interactions of the most potent trimethoxy inhibitors (19, 35, and 36). Purple residues are from the tubulin α-subunit, and orange residues are from the tubulin β-subunit. Darker colors indicate interactions in all 3 inhibitors, while lighter colors represent interactions in 2 of 3 inhibitors. The dashed lines indicate hydrogen bonding, and the ellipses represent hydrophobic interactions.