(A–C) Synthesis of ciliobrevin derivatives. (A) Pyrroloquinazolinone derivative. Reagents, conditions, and yield (%): (i) malononitrile, ammonium acetate, toluene, 100°C, 13 hr, 72%; (ii) bromine, carbon tetrachloride, 70°C, 9 hr, 38%; (iii) methyl anthranilate, isopropanol, 100°C, 20 hr, 15%. Selected atoms are numbered for reference. (B–C) Pyrazoloquinazolinone derivatives. Reagents, conditions, and yield (%): (iv) acetic acid, 150°C (microwave), 30 min, 26%. (v) malononitrile, sodium hydride, tetrahydrofuran, 0°C, 1 hr, 96%; (vi) dimethyl sulfate, N,N-diisopropylethylamine, dioxane, 60°C, 23 hr, 27%; (vii) hydrazine hydrate, ethanol, 80°C, 6 hr, 82%; (viii) methyl 2-fluorobenzoate, potassium carbonate, dimethylformamide, 140°C, 30 min, 11%. (D) Gel filtration trace (Superose 6) for GFP-dynein 2, with volume at elution peak indicated. Vo, void volume. (E) SDS-PAGE analysis (Coomassie blue stain) of GFP-dynein 2, ~0.5 µg protein loaded. (F) Schematic of microtubule motility assay. Anti-GFP antibody (Ab), GFP-dynein (dyn), and microtubule (MT) are indicated. (G) Montages of fluorescent microtubules moving on GFP-dynein-2-coated glass slides in the solvent control (2% DMSO) or in the presence of ciliobrevin D (20 µM). (H) Montages of fluorescent microtubules moving on GFP-dynein-2-coated glass coverslips in the presence of compounds 2–4 (20 µM). (I) Mean velocity of dynein-2-driven microtubule gliding in the presence of control solvent (2% DMSO), ciliobrevin D, or compounds 2–4 (mean. + S.D., n ≥ 3). Number of microtubules quantified: DMSO-327, Cil. D-85, 2–98, 3–90, 4–77. All motility assays were run at 1 mM MgATP, 0.05 mg/mL casein, and 2% DMSO. For all montages, the interval between successive images is 2 s and total time elapsed is 20s. Horizontal scale bar, 5 µm.