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Vol. 1, 1305-1313, December 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

Synthesis and High Content Cell-based Profiling of Simplified Analogues of the Microtubule Stabilizer (+)-Discodermolide 1

Jose M. Minguez, Kenneth A. Giuliano, Raghavan Balachandran, Charitha Madiraju, Dennis P. Curran and Billy W. Day2

Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 [J. M. M., D. P. C., B. W. D.] Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 [R. B., C. M., B. W. D.] and Cellomics, Inc., Pittsburgh, Pennsylvania 15219 [K. A. G.]


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Biology
 Results
 Discussion
 References
 
(+)-Discodermolide, a C24:4, trihydroxylated, octamethyl, carbamate-bearing fatty acid lactone originally isolated from a Caribbean sponge, has proven to be the most potent of the microtubule-stabilizing agents. Recent studies suggest that it or its analogues may have advantages over other classes of microtubule-stabilizing agents. (+)-Discodermolide’s complex molecular architecture has made structure-activity relationship analysis in this class of compounds a formidable task. The goal of this study was to prepare simplified analogues of (+)-discodermolide and to analyze their biological activities to expand structure-activity relationships. A small library of analogues was prepared wherein the (+)-discodermolide methyl groups at C-14 and C-16 and the C-7 hydroxyl were removed, and the lactone was replaced by simple esters. The library components were analyzed for microtubule-stabilizing actions in vitro, antiproliferative activity against a small panel of human carcinoma cells, and cell signaling, microtubule architecture and mitotic spindle alterations by a multiparameter fluorescence cell-based screening technique. The results show that even drastic structural simplification can lead to analogues with actions related to microtubule targeting and signal transduction, but that these subtle effects were illuminated only through the high information content cell-based screen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Biology
 Results
 Discussion
 References
 
The dynamic assembly and disassembly of tubulin {alpha}-ß heterodimers and their polymer, microtubules, are an integral component of cell structure, intracellular communication, and cell division. Perturbation of microtubule dynamics by tubulin/microtubule-interactive agents leads to cell cycle arrest at mitosis and, depending on the given agent and cell type, the induction of apoptosis. Tubulin/microtubules comprise the microtubule cytoskeleton and are therefore an attractive target for anticancer drug development. Two major effects are elicited by tubulin/microtubule-interactive agents: inhibition of tubulin assembly and microtubule-stabilization. Colchicine, 2-methoxyestradiol, and the Vinca alkaloids are representative assembly inhibitors. The taxanes (e.g., paclitaxel) are the prototype microtubule-stabilizers. A few other natural products and analogues have been shown to stabilize assembled tubulin. Among these, (+)-discodermolide is one of the most potent discovered to date. Beyond its potent antiproliferative, microtubule-stabilizing, and apoptosis-inducing actions, (+)-discodermolide may have advantages over other classes of microtubule-stabilizing agents. Importantly, (+)-discodermolide is active against cancer cells expressing P-glycoprotein efflux pumps (the transporter encoded by ABC1) and those expressing altered ß-tubulins that make the cells resistant to taxanes (1–5).

Several total syntheses of (+)-discodermolide and its enantiomer have been developed (6–12). These syntheses have not proven simple, all requiring >=30 transformations from commercially available starting materials to arrive at the final product or its analogues. Consequently, few congeners have been reported, and structure-activity relationship data for (+)-discodermolide are sparse (12–16). Our goal in this study was to prepare simpler analogues of (+)-discodermolide in fewer synthetic steps than necessary for the natural product and the full-length congeners reported to date. Herein, we describe a first library of simplified (+)-discodermolide analogues. All of the analogues retain the C8-C14 core of (+)-discodermolide; the three stereocenters and two (Z)-alkenes of this core give the molecule a characteristic shape (17, 18). The right side diene display matched that of the natural product, as did the geometries of the three nonterminal double bonds (at C-8, C-13, and C-22) and the relative positions of all four alkene links. These analogues differed from the natural product in that methyl groups at C-14 and C-16, as well as the C-7 hydroxyl, were omitted, and the left side display of a lactone moiety was replaced by simple esters (Fig. 1).



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  		Fig. 1. Structure of (+)-discodermolide and regions simplified in this library of analogues.

 
The library members, (+)-discodermolide and paclitaxel, were tested for biological activity by rapid and high information content biochemical and cell-based screening methods. The results show that even drastic structural simplification can lead to analogues with microtubule targeting actions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Biology
 Results
 Discussion
 References
 
Chemistry
General.   Unless otherwise noted, all reactions were performed under an argon atmosphere. All reagents used in chemical syntheses were purchased from Aldrich Chemical Co. and used without additional purification. NMR3 spectra were obtained on Bruker DPX-300 or DPX-500 spectrometers at ambient temperature in the solvent specified. Chemical shifts are reported in parts/million ({delta}) downfield from tetramethylsilane and proton-proton coupling constants (J) in Hz. IR spectra were recorded on an ATI Mattson Genesis Series Fourier transform spectrometer. Low resolution EI mass spectra were obtained on a Hewlett Packard-9000 GC-MS, and high resolution spectra were obtained on a VG 70-G or Micromass Autospec double-focusing instrument under EI or FAB modes. Flash chromatography purifications were done on ICN silica gel 60, 230–400 mesh, with the designated solvents. Reactions were monitored by thin layer chromatography on Kieselgel 60 F254 silica gel plates. Optical rotations were recorded on a Perkin-Elmer 241 digital polarimeter with a sodium lamp at ambient temperature and are reported as [{alpha}]o{lambda} (c = g/100 ml).

General Synthetic Procedures for Compounds 4–8.   Pyridine (50 µmol) and the corresponding acyl chloride 2 (30 µmol) were added to a solution of the alcohol 1 (6.77 mg, 10 µmol) in 1 ml of CH2Cl2. The mixture was stirred at room temperature for 12–18 h, concentrated by rotary evaporation, and purified by flash column chromatography (CH2Cl2 to CH2Cl2/EtOAc 9:1 as eluent) to yield the corresponding esters 3. Each of the individual esters 3 (10 µmol) in 1 ml of CH2Cl2 was treated with NaHCO3 (40 mg) and DDQ (30 µmol). The mixture was stirred at room temperature for 1 h, concentrated under vacuum, and the crude was purified by column chromatography (CH2Cl2 to CH2Cl2/EtOAc 1:1) to provide the final products.

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraenyl benzoate 4.   1H NMR (CDCl3), {delta} 8.06 (d, 2H, J = 7.4 Hz), 7.61–7.55 (m, 1H), 7.48–7.43 (m, 2H), 6.61 (ddd, 1H, J = 16.7, 11.0, 10.1 Hz), 6.05 (dd, 1H, J = 11.0, 11.0 Hz), 5.57–5.49 (m, 1H), 5.42–5.31 (m, 4H), 5.23 (d, 1H, J = 16.7 Hz), 5.13 (d, 1H, J = 10.1 Hz), 4.76 (dd, 1H, J = 6.5, 4.6 Hz), 4.60 (br s, 2H), 4.37–4.29 (m, 2H), 3.67–3.62 (m, 1H), 3.23 (dd, 1H, J = 5.7, 5.7 Hz), 3.00 (ddq, 1H, J = 10.3, 6.9, 3.3 Hz), 2.68–2.60 (m, 2H), 2.32–2.05 (m, 4H), 1.92–1.71 (m, 3H), 1.53–1.46 (m, 2H), 1.01 (d, 3H, J = 6.9 Hz), 0.98 (d, 3H, J = 6.9 Hz), 0.97 (d, 3H, J = 6.9 Hz), 0.93 (d, 3H, J = 6.9 Hz); MS (FAB, glycerol matrix) m/z 542 (M + H)+, 524, 463, 393, 225, 185; [{alpha}]20D +64.4 (c 0.09, CHCl3).

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraen-1-yl acetate 5.   1H NMR (CDCl3), {delta} 6.63 (ddd, 1H, J = 16.8, 11.0, 10.1 Hz), 6.06 (dd, 1H, J = 11.0, 11.0 Hz), 5.52–5.33 (m, 5H), 5.23 (d, 1H, J = 16.8 Hz), 5.14 (d, 1H, J = 10.1 Hz), 4.76 (dd, 1H, J = 6.7, 4.5 Hz), 4.58 (br s, 2H), 4.10–4.05 (m, 2H), 3.69–3.62 (m, 1H), 3.24 (dd, 1H, J = 5.8, 5.8 Hz), 3.06–2.97 (m, 1H), 2.70–2.58 (m, 2H), 2.20–2.03 (m, 4H), 2.06 (s, 3H), 1.78–1.64 (m, 3H), 1.57–1.48 (m, 2H), 1.01 (d, 3H, J = 6.9 Hz), 1.00 (d, 3H, J = 6.8 Hz), 0.99 (d, 3H, J = 6.9 Hz), 0.94 (d, 3H, J = 6.9 Hz); MS (FAB, glycerol/NaCl matrix) m/z 502 (M + Na)+, 469, 301, 207; [{alpha}]20D +73.7 (c 0.08, CHCl3).

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraen-1-yl 2,2-dimethylpropanoate 6.   IR (thin film, NaCl) 3437, 2916, 2846, 1713, 1653, 1457, 1162, 1045 cm-1; 1H NMR (CDCl3), {delta} 6.62 (ddd, 1H, J = 16.8, 11.0, 10.1 Hz), 6.07 (dd, 1H, J = 11.0, 11.0 Hz), 5.58–5.46 (m, 1H), 5.42–5.32 (m, 4H), 5.25 (d, 1H, J = 16.8 Hz), 5.16 (d, 1H, J = 10.1 Hz), 4.78 (dd, 1H, J = 6.7, 4.6 Hz), 4.60 (br s, 2H), 4.15–4.05 (m, 2H), 3.69–3.64 (m, 1H), 3.25 (dd, 1H, J = 5.7, 5.7 Hz), 3.02 (ddq, 1H, J = 10.0, 6.8, 3.3 Hz), 2.71–2.58 (m, 2H), 2.23–2.04 (m, 4H), 1.81–1.66 (m, 3H), 1.59–1.51 (m, 2H), 1.23 (s, 9H), 1.03 (d, 3H, J = 6.8 Hz), 1.01 (d, 3H, J = 6.8 Hz), 1.00 (d, 3H, J = 6.9 Hz), 0.96 (d, 3H, J = 6.9 Hz); 13C NMR (CDCl3) {delta} 178.3, 157.3, 133.5, 133.4, 132.4, 132.1, 130.2, 130.0, 128.7, 118.0, 79.7, 79.0, 72.8, 63.8, 39.9, 38.7, 35.3, 34.9, 34.7, 34.5, 28.7, 27.2, 24.2, 24.1, 18.0, 17.6, 15.3, 7.9; MS (FAB, glycerol/NaCl matrix) m/z 544 (M + Na)+, 443, 301, 245, 191; [{alpha}]20D +67.0 (c 0.27, CHCl3).

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraen-1-yl thiophene-2-carboxylate 7.   IR (thin film, NaCl) 3418, 2962, 2925, 1712, 1695, 1600, 1418, 1265, 1101 cm-1; 1H NMR (CDCl3), {delta} 7.81 (dd, 1H, J = 3.7, 1.2 Hz), 7.56 (dd, 1H, J = 5.0, 1.2 Hz), 7.11 (dd, 1H, J = 5.0, 3.7 Hz), 6.62 (ddd, 1H, J = 16.9, 11.0, 10.1 Hz), 6.05 (dd, 1H, J = 11.0, 11.0 Hz), 5.55–5.47 (m, 1H), 5.42–5.31 (m, 4H), 5.22 (d, 1H, J = 16.9 Hz), 5.13 (d, 1H, J = 10.1 Hz), 4.76 (dd, 1H, J = 6.7, 4.6 Hz), 4.58 (br s, 2H), 4.37–4.25 (m, 2H), 3.67–3.62 (m, 1H), 3.23 (dd, 1H, J = 5.7, 5.7 Hz), 3.05–2.95 (m, 1H), 2.69–2.61 (m, 2H), 2.30–2.05 (m, 3H), 1.88–1.72 (m, 4H), 1.53–1.45 (m, 2H), 1.00 (d, 3H, J = 7.0 Hz), 0.98 (d, [3H], J = 7.0 Hz), 0.97 (d, 3H, J = 6.8 Hz), 0.93 (d, 3H, J = 7.0 Hz); MS (FAB, 3-nitrobenzoic acid matrix) m/z 548 (M + H)+, 487, 391, 257, 154, 136; [{alpha}]20D +60.5 (c 0.185, CHCl3).

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraen-1-yl furan-2-carboxylate 8.   IR (thin film, NaCl) 3405, 2964, 2919, 1713, 1580, 1122, 1046 cm-1; 1H NMR (CDCl3), {delta} 7.58 (dd, 1H, J = 1.7, 0.8 Hz), 7.18 (dd, 1H, J = 3.5, 0.8 Hz), 6.62 (ddd, 1H, J = 16.8, 10.9, 10.1 Hz), 6.52 (dd, 1H, J = 3.5, 1.7 Hz), 6.05 (dd, 1H, J = 10.9, 10.9 Hz), 5.55–5.32 (m, 5H), 5.23 (dd, 1H, J = 16.8, 1.8 Hz), 5.13 (d, 1H, J = 10.1 Hz), 4.76 (dd, 1H, J = 6.6, 4.6 Hz), 4.59 (br s, 2H), 4.37–4.25 (m, 2H), 3.68–3.62 (m, 1H), 3.23 (dd, 1H, J = 5.7, 5.7 Hz), 3.04–2.94 (m, 1H), 2.69–2.60 (m, 2H), 2.30–2.09 (m, 3H), 1.92–1.71 (m, 4H), 1.56–1.46 (m, 2H), 1.01 (d, 3H, J = 7.0 Hz), 0.98 (d, 6H, J = 6.8 Hz), 0.93 (d, 3H, J = 7.0 Hz); MS (FAB, 3-nitrobenzoic acid matrix) m/z 532 (M + H)+, 507, 391, 257, 154; [{alpha}]20D +71.6 (c 0.19, CHCl3).

Synthetic Procedure for 9.   A mixture of alcohol 1 (6.8 mg, 10 µmol), (4-methoxybenzyloxy)acetic acid (20 µmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (20 µmol), and 4-dimethylaminopyridine (0.5 µmol) in 1 ml of CH2Cl2 was stirred at room temperature for 40 h. The mixture was concentrated under vacuum and purified by flash chromatography using 9:1 CH2Cl2/EtOAc as eluent to afford ester 3. The ester (3.8 mg, 4.4 µmol) was dissolved in 0.5 ml of CH2Cl2 and treated with NaHCO3 (30 mg) and DDQ (5 mg, 22 µmol). The mixture was stirred at room temperature for 6 h, concentrated and the residue was purified by flash chromatography (EtOAc) to provide the final product 9 (19).

(Z,Z,Z)-(6S,7S,8S,13R,14S,15S,16S)-15-Carbamoyloxy-7,13-dihydroxy-6,8,14,16-tetramethyleicosa-4,9,17,19-tetraen1–1-yl hydroxyacetate 9.   1H NMR (CDCl3), {delta} 6.62 (ddd, 1H, J = 17.0, 11.0, 10.2 Hz), 6.04 (dd, 1H, J = 11.0, 11.0 Hz), 5.50–5.30 (m, 5H), 5.23 (dd, 1H, J = 17.0, 1.3 Hz), 5.14 (d, 1H, J = 10.2 Hz), 4.77–4.72 (m, 1H), 4.61 (br s, 2H), 4.21 (t, 2H, J = 6.4 Hz), 4.17 (s, 2H), 3.67–3.62 (m, 1H), 3.24 (dd, 1H, J = 5.7, 5.7 Hz), 3.03–2.95 (m, 1H), 2.68–2.54 (m, 2H), 2.18–2.05 (m, 2H), 1.78–1.47 (m, 7H), 1.01 (d, 3H, J = 6.8 Hz), 1.00 (d, 3H, J = 6.8 Hz), 0.99 (d, 3H, J = 6.8 Hz), 0.94 (d, 3H, J = 7.0 Hz); [{alpha}]20D +76.0 (c 0.025, CHCl3).


   Biology
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 Abstract
 Introduction
 Materials and Methods
 Biology
Results
 Discussion
 References
 
Materials.
Tubulin without microtubule-associated proteins was prepared from fresh bovine brains (20). Paclitaxel was provided by the Drug Synthesis and Chemistry Branch, National Cancer Institute or was purchased from Sigma Chemical Company (St. Louis, MO). (+)-Discodermolide was a gift from Dr. Kenneth Bair (Novartis Corp.). Ca2+- and Mg2+-free HBSS was from either Life Technologies, Inc., or BioWhittaker. Fetal bovine serum was from Hyclone. RPMI 1640 culture medium was obtained from Life Technologies, Inc. Formaldehyde was from Polysciences. Antiphospho-histone H3 was obtained as part of a HitKit Reagent Kit (Cellomics, Inc., Pittsburgh, PA) and antiphospho-RSK1 (RSK90[S363]) was obtained from Upstate Biotechnology (Lake Placid, NY). The secondary antibodies were from Jackson ImmunoResearch. The ArrayScan HCS System was from Cellomics, Inc. All other reagents, materials, and antibodies were from Sigma Chemical Co. The paclitaxel resistant and their parental ovarian carcinoma cell lines were obtained from Drs. Tito Fojo and Paraskevi Giannakakou at the National Cancer Institute. Other cell lines were obtained from American Type Culture Collection (Manassas, VA). Cell lines were used within five passages of their receipt from the respective source.

Tubulin Polymerization.
Tubulin assembly was followed in a Beckman-Coulter 7400 spectrophotometer, equipped with an electronic Peltier temperature controller, reading absorbance (turbidity) at 350 nm (21). Reaction mixtures (0.25 ml of final volume) contained tubulin (final concentration 10 µm; 1 mg/ml), monosodium glutamate (0.8 m from a stock solution adjusted to pH 6.6 with HCl), DMSO (final volume 4% v/v), and test agent (10 µM) where indicated. Reaction mixtures without test agent were cooled to 0°C and added to cuvettes held at 0.25–0.5°C in the spectrophotometer. Test agent in DMSO was then rapidly mixed in the reaction mixture. Each run contained one positive control (paclitaxel, 10 µM final concentration) and one negative control (DMSO only). Baselines were established at 0.25–2.5°C, and the temperature was rapidly raised to 30°C (in ~1 min) and held there for 20 min. The temperature was then rapidly lowered back to 0.25–2.5°C. The change in absorbance 20 min after samples reached 30°C was used to calculate extent of polymerization. The change in absorbance at this time point for the addition of vehicle plus paclitaxel was considered 100% assembly (positive control), whereas the change in absorbance for addition of vehicle alone (negative control) was taken as 0% assembly. Each series of determinations included one positive and one negative control.

Cell Growth Inhibition.
Cells were plated (500–2000 cells/well depending on the cell line) in 96-well microplates, allowed to attach and grow for 48 h, then treated with vehicle (4% DMSO, a concentration that allowed doubling times of <=24 h) or test agent [50, 10, 2, 0.4, and 0.08 µM for the new agents; 0.10, 0.05, 0.010, 0.002, and 0.0004 µM for paclitaxel and (+)-discodermolide] for the given times (22). One plate consisted of cells from each line used for a time zero cell number determination. The other plates in a given determination contained eight wells of control cells, eight wells of medium, and each agent concentration tested in quadruplicate. Cell numbers were obtained spectrophotometrically (absorbance at 490 nm minus that at 630 nm) in a Dynamax plate reader after treatment with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium using phenazine methanesulfonate as the electron acceptor. After initial screening with the above 5-fold dilutions, GI50S were determined for each agent by repeating the screen using 2-fold dilutions (five concentrations) centered on the initial estimated GI50 concentration, again in quadruplicate.

Paclitaxel Binding Site Inhibition Assay.
A stock solution of [3H]paclitaxel (26.8 µM, 16.2 Ci/mmol), obtained from the National Cancer Institute, was prepared in 37% (v/v) DMSO (3,21). The test agents were prepared in 25% (v/v) DMSO-0.75 M monosodium glutamate (prepared from a 2 M stock solution adjusted to pH 6.6 with HCl). The radiolabeled paclitaxel and test agents, as indicated in terms of final concentrations, were mixed in equal volumes and warmed to 37°C. A reaction mixture (50 µl) containing 0.75 M monosodium glutamate, 4.0 µM tubulin, and 40 µM ddGTP (a nonhydrolyzable analogue of GTP) was prepared and incubated at 37°C for 30 min to preform microtubules. An equivalent volume of drug mixture with [3H]paclitaxel was added to preformed polymer and incubated for 30 min at 37°C. Bound [3H]paclitaxel was separated from free drug by centrifugation of the reaction mixtures at 14,000 rpm for 20 min at room temperature. Radioactive counts from the supernatant (50 µl) were determined by scintillation spectrometry. Bound [3H]paclitaxel was calculated from the following: total paclitaxel added to each reaction mixture minus the paclitaxel present in the supernatant (free drug). The percentage of bound values were normalized to the control values with no inhibitor added.

High Content Profiling.
PC-3 or HeLa cells were plated at 6000–8000 cells/well in 384-well microplates and incubated at 37°C in a 5% CO2 humidified incubator 3–8 h to allow attachment and spreading (22, 23). Wells were treated with vehicle (DMSO) or concentration gradients of test agents and incubated for an additional 14.5 h at 37°C. Cells were fixed in 4% (v/v) aqueous formaldehyde, and nuclei/chromatin were simultaneously labeled with 10 µg/ml Hoechst 33342 in HBSS. Wells were rinsed with HBSS, then cells were permeabilized with 0.5% (w/w) aqueous Triton X-100 for 5 min at room temperature. After rinsing with HBSS, the cells were incubated with primary antibody solution for 1 h at room temperature. Although several mixtures were surveyed (as noted in the "Results") for the photomicrographs and numerical data in this paper, the antibody mixture consisted of a HBSS solution containing rabbit polyclonal antiphospho-histone H3 (1:1000), polyclonal sheep polyclonal antiphosphoRSK1 [RSK90(S363), 1:1000], and mouse monoclonal mouse anti-{alpha}-tubulin (1:2500). After rinsing with HBSS, all wells in the microplate were incubated for 1 h in an HBSS solution containing a mixture of the following fluorescently labeled secondary antibodies: FITC-labeled donkey antimouse IgG (1:250); Cy3-labeled donkey antirabbit IgG (1:250); and Cy5-labeled donkey antisheep IgG (1:250). After the incubation, cells were rinsed once with HBSS and stored at 4°C in HBSS before analysis. This protocol yielded cells labeled with four distinct fluorophores in 384-well microplates ready for HCS. HCS microplates were analyzed with the ArrayScan HCS System using the general screening application. Within the screening application, multiple fields in each well were imaged at four different wavelengths each. Proprietary algorithms provided both morphological information (e.g., nuclear condensation) or, because primary antibodies directed against the phosphorylated (activated) form of several signaling kinase substrates were used, a population distribution of cells containing a range of activated kinases.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Biology
 Results
Discussion
 References
 
Synthesis of (+)-Discodermolide Analogues.
Fig. 2 shows the general synthetic route to the preparation of compounds 4–9. The alcohol intermediate 1 (synthesis will be reported elsewhere), whose fully deprotected form was found to be biologically inactive, was coupled with several acyl chlorides or carboxylic acids 2 to provide the corresponding esters 3. These esters were treated with DDQ to remove the 4-methoxybenzyl alcohol-protecting groups (24) affording the final products 4–9.



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  		Fig. 2. Synthesis of (+)-discodermolide analogues 4–9.

 
Antiproliferative Activities, Induction of Tubulin Assembly, and Paclitaxel Displacement.
The full length (+)-discodermolide analogues 4–9 were tested for antiproliferative effects against human breast (MDA-MB231), prostate (PC-3), and ovarian (2008) carcinoma cell lines. The results showed that although dramatically simplified structurally, compounds 4–7 and 9 had antiproliferative activity, albeit considerably weaker than that of paclitaxel and (+)-discodermolide (Table 1; Refs. 2–5). In an in vitro assay to measure intrinsic microtubule-inducing actions wherein added compound is tested for its ability to cause nucleation of bovine brain tubulin in the absence of the normal initiator of tubulin assembly, GTP, compounds 4–8 were weakly active. The conditions of this assay have been previously shown to detect microtubule-stabilizers less potent than paclitaxel (21). Compounds were also tested for their ability to compete with [3H]paclitaxel for binding to microtubules. Compounds 4–6, the benzoyl, acetate, and pivaloyl esters, were the most potent of the new agents in the cell proliferation and, in general, the paclitaxel displacement assays, followed by compounds 7 > 8 > 9. Compounds 7 and 8 have heteroaromatic ester functions (thiophenoyl and furanoyl, respectively), whereas the least active analogue, compound 9, had a 2-hydroxyacetate ester moiety. It is therefore clear that to retain antiproliferative and paclitaxel displacement activities, replacement of the lactone left-side display of (+)-discodermolide requires a relatively hydrophobic ester with no hydrogen-bonding substituents.


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  		Table 1 Antiproliferative, paclitaxel-displacing, microtubule assembly, and antimitotic effects of (+)-discodermolide analogs

 
Although compounds 4–8 were weak nucleators of isolated tubulin, the sum of these results suggested that their antiproliferative actions could be related to stabilization of the microtubule cytoskeleton and resulting changes in signaling pathways. This hypothesis was then tested in living cells using a high content profiling approach.

High Content Profiling: Activation of Multiple Targets by Microtubule-perturbing Agents.
Several cell-based, multiparameter fluorescence screens were performed using HeLa (cervical) and PC-3 (prostate) human carcinoma cells. In these cells, microtubule-perturbing agents caused phosphorylation of several stress- and G2-M-related markers in cells with condensed nuclei and altered microtubule cytoskeletons. These markers included mitogen-activated protein kinase (antiphospho-T183/Y185 antibody), P38 (antiphospho-T182/Y184), c-Jun NH2-terminal kinase (antiphospho-T183/Y185), retinoblastoma (antiphospho-S780, antiphospho-S795, and antiphospho-S807/S811), c-Jun (antiphospho-S73), and histone H3 (antiphospho-S10). Targets that were not phosphorylated included nuclear factor {kappa}B, Stat-1, and Oct-1. Almost without exception, this same pattern of marker phosphorylation occurred in control mitotic cells, but subcellular distributions of the markers differed from agent-treated cells. Histone H3 phosphorylation, a clear marker of the G2-M interface, was chosen as a metric for agent-induced actions.

An example of the organellar, cytoskeletal, and molecular signaling effects of one of the (+)-discodermolide analogues is shown in Fig. 3. HeLa cells, which remained relatively well attached to the microtiter plates during mitosis, were treated for 14.5 h with 25 µM 6. Multiparameter fluorescent images were obtained from the same microscopic field and included Hoechst 33342-labeled nuclei and immunolocalized tubulin, phosphorylated histone H3, and phosphorylated RSK90. This concentration of 6 produced a heterogeneous cellular response that included abnormally condensed nuclear chromatin, tubulin frozen into a morphology reminiscent of newly formed mitotic spindles, multi-/nonbipolar spindles, and activation of at least one cell stress kinase pathway. In addition, 6 induced the appearance of a subpopulation of cells with microtubule bundles that, in several cases, condensed around the nuclei of affected cells (Fig. 3B).



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  		Fig. 3. Examples of the organellar, cytoskeletal, and molecular signaling effects of (+)-discodermolide analogue 6 in HeLa cells. Cells were treated for 14.5 h with 25 µM 6. Images were obtained from the same microscopic field: Hoechst 33342-labeled nuclei (A); immunolocalized tubulin (B); phosphorylated histone H3 (C); and phosphorylated RSK90 (D). This concentration of 6 produced a heterogeneous cellular response that included abnormally condensed nuclear chromatin (A and C), tubulin frozen into morphologies representative of both normal bipolar and abnormal multipolar mitotic spindles (B), cells blocked at early mitosis (C), and activation of a cell stress pathway (D). Scale bar = 20 µM.

 
The heterogeneous microtubule reorganization induced by compound 6 had characteristics consistent with the condensation of microtubules during spindle formation as well as the bundling of microtubules induced by the drug paclitaxel. The cellular images in Fig. 4 illustrate examples of these phenomena. In mitotic cells undergoing normal metaphase, microtubules were highly condensed during spindle formation (Fig. 4C; arrows). This was in contrast to the mostly asymmetric and less condensed spindle-like microtubule morphology induced in a subpopulation of cells treated with 6 (Fig. 3B). Furthermore, a subpopulation of cells treated with 6, which remained relatively well spread, exhibited bundles of microtubules, especially around the nucleus Fig. 3B), that were similar to but not as extensively bundled as microtubules in cells treated with paclitaxel (Fig. 4D).



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  		Fig. 4. Nuclear condensation and tubulin reorganization in normally cycling cells, and cells treated with paclitaxel. HeLa cells were treated either with vehicle (A and C) or with 1 µM paclitaxel (B and D) for 14.5 h. Images obtained from the same microscopic fields included Hoechst 33342-labeled nuclei (A and B) and immunolocalized tubulin (C and D). Normal cycling cells contained a subpopulation in various stages of mitosis that displayed both chromatin and tubulin condensation (A and C; arrows), whereas paclitaxel treatment caused subpopulations of cells with bundled microtubules, including those surrounding the nucleus but without necessarily exhibiting condensed nuclei (B and D; arrows). Scale bar = 20 µM.

 
The (+)-discodermolide analogues exhibited mitosis-blocking activity as well as toxic effects. Fig. 5 shows in graphical format that the phosphorylation status of histone H3 in HeLa cells could be used to measure the subpopulation of cells either undergoing mitosis or blocked at some stage of the process as a result of agent action (22). Paclitaxel, which traps cells at the G2-M border, gave a sigmoidal concentration-response curve with this metric. The (+)-discodermolide analogues exhibited activities with respect to histone H3 phosphorylation that mirrored their antiproliferative potencies. The minimum detectable concentration at which each agent caused histone H3 phosphorylation (in excess over that seen in vehicle-treated, control cultures) is given in Table 1. For example, compound 6 was ~8-fold less active than paclitaxel in enhancing microtubule assembly in vitro, yet it had substantial growth inhibitory activity in several cell lines. Although less potent than (+)-discodermolide or paclitaxel, 6 also caused mitotic block in cells.



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  		Fig. 5. (+)-Discodermolide analogues exhibited mitosis-blocking activity as well as toxic effects. The phosphorylation state of histone H3 was used to measure the subpopulation of cells either undergoing mitosis or blocked at some stage of the process as a result of agent action (22). The top panel shows the titration of HeLa cells with paclitaxel, which traps cells at the G2-M border. The center and bottom panels show the activity of two (+)-discodermolide analogues 6 and 8, which exhibited different potencies. As described earlier (22, 23), the preferential loss of agent-sensitive cells from the substrate attributable to the toxic effects of the two analogues was demonstrated by the dramatic decrease in the histone H3 phosphorylation ratio at relatively high analogue concentrations. Error bars show SDs at each point.

 
Differentiation of Microtubule Stabilizing Agent Target Specificities Using Cells with Mutant ß-Tubulin.
The appearance of a relatively large number of near-normal looking mitotic spindles in samples of HeLa cells treated with 6 (Fig. 3B) suggested that this compound might have properties that distinct from those of (+)-discodermolide and paclitaxel. Thus, two cell lines, PTX10 and PTX22, which express mutant ß-tubulin with alterations in the paclitaxel binding site and are therefore resistant to paclitaxel treatment, and their parental human ovarian carcinoma line (1A9) were used to test this hypothesis. Fig. 6 shows the high content profiling of tubulin mass in these cell lines using the three agents to induce microtubule cytoskeleton stabilization. The stabilization of the microtubule cytoskeleton in these cell lines (e.g., increased cellular tubulin mass values) induced by paclitaxel reflected the altered paclitaxel binding properties of the mutant. That is, the microtubule cytoskeleton in the parental cell line was stabilized to a greater extent by paclitaxel than was the microtubule cytoskeleton of the two mutant lines. (+)-Discodermolide also stabilized the microtubule cytoskeleton of the parental line, but relatively high concentrations of this agent stabilized the microtubule cytoskeleton of one of the mutant cell lines (PTX22) to a greater extent than in the parental line (Fig. 6B). Analogue 6 showed microtubule cytoskeleton stabilizing activity, but with virtually the same efficacy in all three cell lines (Fig. 6C), suggesting that 6 was unaffected by the mutations in ß-tubulin that cause paclitaxel resistance.



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  		Fig. 6. ß-Tubulin mutant cell lines revealed mechanistic differences between the microtubule stabilizing agents paclitaxel, (+)-discodermolide, and its simplified analogues. Two cell lines expressing mutated ß-tubulin, PTX10 (circles), and PTX22 (triangles), and their parental line 1A9 (squares) were titrated with microtubule-stabilizing agents and their tubulin mass changes determined with high content profiling. A, paclitaxel induced stabilized microtubules in 1A9 cells but produced only a muted response in the two mutant lines. B, (+)-discodermolide had a lower potency stabilizing effect than paclitaxel in 1A9 cells, but at relatively high concentrations, the agent stabilized the microtubule cytoskeleton to a greater extent in PTX22 cells than in 1A9 cells and to a lesser extent in PTX10 cells than in 1A9 cells. C, compound 6 induced a moderate microtubule cytoskeleton stabilization compared with (+)-discodermolide but with no significant selectivity for mutant tubulin-expressing cells. Error bars show SDs at each point.

 
Microtubule-perturbing Agent Specificity in the Coupling of Cellular Signaling Pathways.
Figs. 7 and 8 show additional high content profiling features from the experiment that yielded the data shown in Fig. 6. Data in Figs. 7 A and 8A indicate that paclitaxel-resistant cells did not exhibit the same signaling response that the parental cells did. Paclitaxel-induced activation of mitotic and cell stress pathways was inhibited in both mutant lines compared with the parental line, consistent with a mechanism of action requiring coupled regulation of the microtubule cytoskeleton and mitotic and stress kinase pathways. In the case of (+)-discodermolide (Figs. 7B and 8B), the mitotic and stress kinase pathway activation was greater in the mutant lines relative to the parental line when compared with paclitaxel treatment of the same cells. Again, this is consistent with subtle differences in the molecular activities of paclitaxel and (+)-discodermolide in living cells. Finally, analogue 6 caused activation of histone H3 and RSK90 phosphorylation to a significantly greater extent in the parental line than in the two mutant cell lines (Figs. 7C and 8C), although it was able to equally stabilize the microtubule cytoskeleton in all three cell lines (Fig. 6C).



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  		Fig. 7. A key phosphorylation event in mitosis was coupled to mutations in proteins that comprise the microtubule cytoskeleton. Two cell lines expressing mutated ß-tubulin molecules, PTX10 (circles) and PTX22 (triangles) and their parental line 1A9 (squares), were titrated with microtubule-stabilizing agents, and the status of an early mitotic marker, histone H3 phosphorylation, was measured with HCS. A, paclitaxel induced a mitotic block in 1A9 cells but produced only a muted response in the two mutant lines. B, (+)-discodermolide induced a lower potency mitotic blocking effect than paclitaxel in 1A9 cells, but an overall larger magnitude in these cells. The mutant cell lines were not as responsive as the parental cell line. (+)-Discodermolide effected a larger magnitude response in these cells than did paclitaxel at the same concentrations. C, compound 6 produced a maximal effect that was nearly half that of (+)-discodermolide in 1A9 cells, but it showed more selectivity for the inhibition of mitosis in the mutant lines than it did for microtubule stabilization in the same cells (Fig. 6C). Error bars show SDs at each point.

 


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  		Fig. 8. Phosphorylation of a stress pathway kinase downstream target (RSK90) was coupled to mutations in microtubule proteins. Two cell lines expressing mutant ß-tubulin, PTX10 (circles) and PTX22 (triangles), and their parental line 1A9 (squares), were titrated with microtubule-stabilizing agents, and the phosphorylation level of RSK90 was measured with HCS. A, paclitaxel increased RSK90 phosphorylation in parental 1A9 cells and exhibited an ~4-fold lower magnitude response in the two mutant lines, similar to the effect paclitaxel had on histone H3 phosphorylation in the same cells. B, (+)-discodermolide increased RSK90 phosphorylation in 1A9 cells as well as a muted increase in the two mutant cell lines. C, compound 6 produced a maximal effect that was once again nearly half that of (+)-discodermolide, but the agent showed more selectivity for the activation of RSK90 in the mutant cell lines than it did for microtubule stabilization. Error bars show SDs at each point.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Biology
 Results
 Discussion
References
 
Six new derivatives of the microtubule-stabilizing natural product (+)-discodermolide were prepared from a common precursor, compound 1. These analogues contained the intact right side diene display of the lead compound but differed from it by substituent simplification: methyl groups at C-14 and C-16 were removed as was the hydroxyl at C-7, and the left-side lactone moiety was replaced by simple esters.

A high throughput screen for antiproliferative activity showed that analogues 4–8 were growth inhibitory. Another rapid screen for microtubule assembly induction with isolated bovine brain tubulin showed that the new agents were at best weak hypernucleators of tubulin in vitro. On the other hand, the analogues showed some ability to displace [3H]paclitaxel from microtubules. Because of this and the antiproliferative activities shown by the analogues, we suspected that their molecular activity might be masked by the artificial conditions in the tubulin assembly assay and therefore sought to determine whether they had the desired activity, microtubule stabilization and mitotic block, within cells.

A high content, multiparameter fluorescent cell profile was therefore developed to provide a cellular context in which the activities of the new analogues, their parent compound, and paclitaxel could be dissected within a living milieu. The high content profile was designed as a cell-based assay to measure the assembly state of the microtubule cytoskeleton and two other signaling events associated with the activity several microtubule cytoskeleton-perturbing agents. The profile revealed that the new (+)-discodermolide analogues did indeed modulate all three cellular targets to some degree. This targeting was translated into G2-M block as evidenced by sustained histone H3 phosphorylation, stabilization of the microtubule cytoskeleton, including perturbed mitotic figures and increased tubulin mass after detergent extraction, and initiation of a mitogen-activated stress kinase pathway with the readout being phosphorylated RSK90, a downstream target of the extracellular signal-regulated kinase-MAP/ERK kinase survival signal transduction cascade (25). For example, all of the analogues, although nearly inactive in the in vitro tubulin assembly assays, were able to block a significant population of cells in early mitosis (Table 1). This cell-based screening method also showed that analogue 9, as well as the other more potent (+)-discodermolide analogues, showed significant toxicity (e.g., cell loss from the substrate) at concentrations >10 µM. The results of the high content profiling were consistent with the new analogues having cytotoxic activity while also inducing specific cellular events that would be difficult to observe in a highly defined assay system with purified cellular components.

Paclitaxel, (+)-discodermolide, and its new analogues showed subtle differences in mechanism of action. That paclitaxel, (+)-discodermolide, and its simplified analogue 6 exhibited differences in their efficacy and potency in modulating the microtubule cytoskeleton of paclitaxel-resistant and parental cell lines is consistent with at least two scenarios. The first is that each agent interacts differently with its putative intracellular target, tubulin/microtubules. Support for this assertion comes from comparing the in vitro data with the high content profiling data. Agents that did not have significant paclitaxel displacement or microtubule assembly activity were active to some degree in stabilizing the microtubule cytoskeleton of cells. Furthermore, the paclitaxel-resistant cell lines used here have previously been shown to have differential sensitivity to other microtubule-perturbing agents (3, 26). The second scenario is that each agent exhibits a unique spectrum of intracellular activity that depends on cellular target specificity. Paclitaxel, (+)-discodermolide, and analogue 6 individually produced a unique high content profile in the paclitaxel-resistant and parental cell lines, indicating that multiple targets for these agents could exist, or the coupling between the activities of the target and its effect on down stream molecular processes may be differentially modulated by each agent. It is likely, however, that the cellular responses we measured are attributable to a combination of these two circumstances because, in all cases described here, the activation of mitosis and cell stress specific kinase pathways did not occur in the absence of agent-induced microtubule cytoskeleton stabilization.

(+)-Discodermolide has shown exciting preclinical activity. The rarity of (+)-discodermolide in nature combined with its complex structure makes it an attractive lead agent for structural simplification. Our results show that even dramatically simplified analogues of (+)-discodermolide can retain microtubule-targeting actions in living cells. It must be noted, however, that classical antiproliferative and isolated tubulin assays would not have determined these actions. High-content profiling was necessary to dissect the desired activity from the toxic actions of the new compounds. This screening approach therefore will help us make better decisions during the drug design and discovery process by better identifying potential lead compounds that would have been missed if the standard in vitro assays were the only screen for activity. For example, high content profiling revealed an unusual morphology (frozen spindles) induced by some of the new analogues that could not be detected in cells treated with (+)-discodermolide. Although some aspects of the actions of the (+)-discodermolide analogues, the parent compound and paclitaxel are similar, the unusual spindle effect induced by the analogues coupled with a multiparameter cell physiological profile will provide a metric for evaluation of future analogues.


   Acknowledgments
 
J. M. M. thanks the Ministerio de Educacion y Cultura, Spain for a Postdoctoral Fellowship. We thank Dr. Kenneth Bair and Novartis Corp. for the generous gift of (+)-discodermolide.


   Footnotes
 
1 Supported by National Cancer Institute Grant CA78039. Back

2 To whom requests for reprints should be addressed, at 726 Salk Hall, 3501 Terrace Street, Pittsburgh, PA 15261. Phone: (412) 648-9706; Fax: (412) 624-1850; E-mail: bday{at}pitt.edu. Back

3 The abbreviations used are: NMR, nuclear magnetic resonance; CH2Cl2, dichloromethane; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; EI, electron ionization; FAB, fast atom bombardment; HCS, high content screening; IR, infrared spectroscopy; MS, mass spectrometry; PMB, 4-methoxybenzyl; RSK90, 90-kDa ribosomal s6 kinase (a.k.a. mitogen-activated protein kinase-activated protein kinase-1). Back

Received 2/11/02; revised 9/16/02; accepted 10/23/02.


   References
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 Biology
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 Discussion
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