A simplified synthesis of novel dictyostatin analogs with in ......2011/04/13 · A simplified...

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A simplified synthesis of novel dictyostatin analogs with in vitro activity against epothilone B

resistant cells and antiangiogenic activity in zebrafish embryos

Laura L. Vollmer1,$, Maria Jiménez2,$ , Daniel P. Camarco1, Wei Zhu2, Hikmat N. Daghestani3,

Raghavan Balachandran4, Celeste E. Reese1, John S. Lazo1,, Neil A. Hukriede5, Dennis P. Curran2,

Billy W. Day1,2,4, and Andreas Vogt1,6*

University of Pittsburgh Drug Discovery Institute1 and Departments of Chemistry2, Molecular

Biophysics & Structural Biology3, Pharmaceutical Sciences4, Developmental Biology5,

and Computational and Systems Biology6, Pittsburgh, Pennsylvania 15260

$ These authors contributed equally to the work

Running title: Potent dictyostatin analogs by convergent synthesis

Key words: dictyostatin, high-content screening, multidrug resistance, zebrafish, angiogenesis.

Abbreviations: Cy3, indocarbocyanine dye with three-methine linker; DDQ, 2,3-dichloro-5,6-

dicyano-1,4-benzoquinone; ddGTP, 2’,3’-dideoxyguanosine-5’-triphosphate; DMSO,

dimethylsulfoxide; DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; Et2BOMe,

diethylmethoxyborane; FITC, fluorescein isothiocyanate; HBSS, Hank’s Balanced Salt Solution;

HCS, high-content screening; hpf, hours post fertilization; HWE, Horner-Wadsworth-Emmons;

ISV, intersegmental vessels; MDEC, minimal detectable effective concentration; Mes,

on June 7, 2021. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 13, 2011; DOI: 10.1158/1535-7163.MCT-10-1048

http://mct.aacrjournals.org/

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morpholineethanesulfonate; MSG, monosodium glutamate; MTs, microtubules; MTS, 3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium; NaHMDS ,

sodium bis(trimethylsilyl)amide; NHK, Nozaki-Hiyama-Kishi; NiCl2(dppf), dichloro[1,1'-

bis(diphenylphosphino)ferrocene]nickel(II); PCV, posterior cardinal vein; PMB, para-

methoxybenzyl; SAR, structure activity relationship; TBS, tert-butyldimethylsilyl; TBSOTf, tert-

butyldimethylsilyl trifluoromethanesulfonate

Notes

This work was supported by the National Institutes of Health [Grants CA78039 to A.V and J.S.L.,

HD053287 to N.A.H.], and the Fiske Drug Discovery Fund.

*Corresponding author: Andreas Vogt, Department of Computational and Systems Biology,

University of Pittsburgh Drug Discovery Institute, 10047 Biomedical Science Tower 3, University

of Pittsburgh, Pittsburgh, PA 15260; Telephone: 412-383-5856; Fax: 412-648-9009; Email:

[email protected].

Present address for J.S. Lazo : University of Virginia School of Medicine, Charlottesville, VA 22908




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Abstract

The natural product (–)-dictyostatin is a microtubule stabilizing agent that potently

inhibits the growth of human cancer cells including paclitaxel-resistant clones. Extensive

structure-activity relationship studies have revealed several regions of the molecule that could

be altered without loss of activity. The most potent synthetic dictyostatin analog described to

date, 6-epi-dictyostatin, has in vivo antitumor activity against human breast cancer xenografts

superior to paclitaxel. Despite their encouraging preclinical activities, the complex chemical

structure of the dictyostatins presents a major obstacle in their development into novel

antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26

dihydrodictyostatins and found several agents that compared with 6-epi-dictyostatin retained

nanomolar activity in cellular microtubule bundling assays but showed cross-resistance to

paclitaxel in cells with mutations in beta-tubulin. Extending these studies, we applied the new,

highly convergent synthesis to generate 25,26-dihydrodictyostatin and 6-epi-25,26-

dihydrodictyostatin. Both compounds were potent microtubule perturbing agents that induced

mitotic arrest and microtubule assembly in vitro and in intact cells. In vitro radioligand binding

studies showed that 25,26-dihydrodictyostatin and its C-6 epimer were able to displace

[3H]paclitaxel and [14C]epothilone B from microtubules with potencies comparable to (–)-

dictyostatin and discodermolide. Both compounds inhibited the growth of paclitaxel- and

epothilone B-resistant cell lines at low nanomolar concentrations, synergized with paclitaxel in

MDA-MB-231 human breast cancer cells, and had antiangiogenic activity in transgenic zebrafish

larvae. The data identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as

candidates for scale-up synthesis and further preclinical development.




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Introduction

Microtubules (MTs) are an important component in cell division and mitosis.

Interference with MT dynamics causes a block in cell cycle progression and eventually

programmed cell death (apoptosis), desirable results for treating rapidly dividing cancer cells.

MT perturbing agents such as taxanes, epothilones, or vinca alkaloids, which stabilize or

destabilize MTs, are successfully used in the treatment of solid or hematologic malignancies (1).

The clinical successes of these anticancer agents have made MTs one of the most validated

molecular cancer targets. Current, FDA-approved MT stabilizing agents are the taxanes

paclitaxel (TaxolTM), docetaxel (TaxotereTM), cabazitaxel (JevtanaTM), an albumin-bound form of

paclitaxel (AbraxaneTM), and a semi-synthetic analog of epothilone B, ixabepilone (IxempraTM).

Despite their success, the development of drug resistance reduces the effectiveness of these

agents (2), resulting in a continued effort to develop novel MT perturbing agents.

Several MT stabilizing agents are currently under investigation as potential anticancer

therapies (3). A particularly promising agent, (+)-discodermolide, a potent microtubule

stabilizer with activity superior to paclitaxel, entered into Phase I clinical trials, but

disappointingly failed due to pulmonary toxicity (4). Previously overshadowed by (+)-

discodermolide, (–)-dictyostatin, a closely related compound, has recently gained attention as a

potential anticancer agent. A decade after isolation, the complex structure was finally resolved

(5), and two total syntheses (6, 7) provided enough sample for a detailed characterization (7, 8).

Extensive structure activity relationship (SAR) studies have provided important information for




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the development of several (–)-dictyostatin analogs (9-11). These studies culminated in the

discovery of 6-epi-dictyostatin, which was shown to have antitumor activity superior to

paclitaxel in mice bearing human breast cancer MDA-MB-231 xenografts (12). In spite of these

promising preclinical results, the complex structure and difficult synthesis of (–)-dictyostatin

and analogs present major obstacles in their further preclinical development.

We recently reported a streamlined synthesis that generated new 16-desmethyl-25,26-

dihydrodictyostatins that were considerably easier to make and in preliminary biological studies

retained much of the potency of (–)-dictyostatin (13). Based on the biological activity of the

series, which suggested reduction of the C25, C26 double bond is well tolerated but removal of

the C16 methyl group results in loss of activity against paclitaxel-resistant cells (13), we applied

the new streamlined synthesis to generate 25,26-dihydrodictyostatin (1a) and 6-epi-25,26-

dihydrodictyostatin (1b). High-content cellular analysis revealed that 25,26-dihydrodictyostatin

and 6-epi-25,26-dihydrodictyostatin induced mitotic arrest and stabilized cellular MTs with

potencies similar to that of the natural product. In vitro, both agents caused tubulin assembly

with potency similar to paclitaxel and displaced [3H]paclitaxel and [14C]epothilone B from

preformed MTs. The new analogs inhibited the growth of human cancer cells at low nanomolar

concentrations, retained antiproliferative activity in epothilone B- and paclitaxel-resistant

cancer cell lines, were able to synergize with paclitaxel, and had antiangiogenic activity in a

zebrafish model. The data validate 25,26-dihydrodictyostatin and 6-epi-25,26-

dihydrodictyostatin as bona fide MT stabilizing agents and identify them as candidates for




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continued preclinical development.




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Materials and Methods

Compounds. The dictyostatin analogs 1a and 1b were prepared by full syntheses. The

Supporting Information contains complete characterization details and copies of NMR spectra.

Full experimental details of the synthesis will be published elsewhere. [3H]Paclitaxel was

obtained from the Drug Synthesis and Chemistry Branch, NCI. [14C]Epothilone B was a gift from

Novartis Pharma.

Cells and culture. HeLa human cervical carcinoma cells (ATCC, Manassas, VA), A549 human

lung cancer cells, and their epothilone B-resistant counterparts EpoB40/A549 (a gift from Susan

Horwitz, Albert Einstein College of Medicine) were maintained in Dulbecco’s modified Eagle

medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS, Cellgro), 2 mM L-

glutamine (Invitrogen), and 1% penicillin-streptomycin (Invitrogen). Maintenance medium for

EpoB40/A549 cells contained 40 nM epothilone B, which was removed prior to experimental

setup. The HeLa/DZR cell line was generated as previously described (14) using ethyl methane

sulfonate mutagenesis followed by stepwise increased concentrations of the antimitotic, tubulin

assembly inhibiting, macrocyclic polyketide disorazole C1 (0.1-10.8 nM), leading to ~30-fold

resistance to disorazole C1. These cells were valuable in our studies because they are resistant

to natural products at least in part due to the overexpression of the ATP-binding cassette

ABCB1 transporter (14). Thus, HeLa/DZR cells are cross resistant to the natural products

vinblastine, doxorubicin and paclitaxel but not to cisplatin (14). Cells were cultured as

previously described (14).




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MDA-MB-231 human breast cancer cells (ATCC), 1A9 human ovarian carcinoma cells and their

paclitaxel-resistant clones 1A9/PTX10 and 1A9/PTX22 (a gift from Drs. Tito Fojo and Paraskevi

Giannakakou) were maintained in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine

serum. Maintenance medium for 1A9/PTX10 and 1A9/PTX22 cells was further supplemented

with 17 nM paclitaxel and 10 μM verapamil. Forty-eight hours prior to test agent analyses,

paclitaxel and verapamil were removed and the cells placed into phenol red-free RPMI 1640

medium supplemented with 10% FBS and antibiotics. All cells were maintained in a humidified

atmosphere of 95% air-5% CO2 at 37oC. The identities of the HeLa and MDA-MB-231 cell lines

were confirmed by The Research Animal Diagnostic Laboratory (RADIL) at the University of

Missouri, Columbia, MO (http://www.radil.missouri.edu), using a PCR based method that

detects 9 short tandem repeat (STR) loci, followed by comparison of results to the ATCC STR

database.

High-content analysis of mitotic arrest and microtubule stabilization. We used our previously

reported cell-based immunofluorescence assay (11, 15) for high-content analysis of mitotic

arrest and microtubule stabilization. In brief, 7,500 HeLa cells per well were seeded into the

wells of two 384-well collagen-coated microplates (Becton Dickinson), allowed to adhere for 5

h, and treated for an additional 21 h with either vehicle control (DMSO) or test agents. Cells

were fixed with 4% formaldehyde containing 20 μg/mL Hoechst 33342, permeabilized with

0.2% Triton X-100 and immunostained with the following antibody combinations: anti- -tubulin

(Sigma Aldrich, T9026, mouse monoclonal, 1:3000 dilution)/ fluorescein isothiocyanate (FITC)-




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labeled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-095-150, 1:500 dilution) and

anti-phosphohistone H3 (Millipore, 06-570, rabbit polyclonal, 1:500 dilution)/Cy3-labeled

donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-165-152, 1:500 dilution) for mitotic

arrest, or anti-acetylated tubulin (Sigma Aldrich, T7451, mouse monoclonal, 1:1000

dilution)/Cy-3-labeled donkey anti-mouse IgG (Jackson ImmunoResearch, 715-165-150, 1:500

dilution) for quantitation of stabilized cellular MTs. Cells were imaged on the ArrayScan II HCS

reader (Thermo Fisher Cellomics, Pittsburgh, PA) using a 20X objective and an Omega XF93

filter set at excitation/emission wavelengths of 350/461 nm (Hoechst), 494/519 nm (FITC), and

556/573 nm (Cy3). For each condition images of 1,000 cells were acquired and analyzed using a

Target Activation Bioapplication algorithm (Thermo Fisher Cellomics, Pittsburgh, PA) essentially

as described (16). An image mask was generated from the Hoechst-stained nuclei. MT density

and acetylation were defined as the average pixel intensity in an area defined by the nuclear

mask. For determination of mitotic index and nuclear condensation, thresholds for Hoechst

33342 and phosphohistone-H3 intensities were defined as one S.D. above the average Hoechst

33342 or Cy3 intensity obtained from 28 vehicle-treated wells located in the center of the

microplate. Cells were classified as positive if their average Hoechst 33342 or Cy3 intensity

exceeded this threshold. Minimal detectable effective concentrations (MDEC) were estimated

from concentration-response curves as described (17).

Antiproliferative activities.

Epothilone B-resistant cells. Growth inhibition of A549 and EpoB40/A549 cells was assessed

over three days using a modified version of our previously described high-content cytotoxicity




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assay (18). Cells were plated in 384-well collagen-coated plates at 1,000 cells per well, allowed

to adhere overnight, and treated in quadruplicate with 10-point 2-fold serial dilutions of

individual test agents or vehicle control (DMSO) for an additional 72 h. After the 72 h

treatment period, cells were fixed and nuclei stained with 10 μg/mL Hoechst 33342. Four

imaging fields were acquired on the ArrayScan II at excitation/emission wavelengths of 350/461

nm using a 10x objective, and nuclei enumerated as described (18). Cell densities were

calculated as objects per imaging field and normalized to vehicle control density at the end of

the study.

Paclitaxel-resistant cells. Growth inhibition of 1A9 human ovarian cancer cells and the

paclitaxel-resistant clones 1A9/PTX10 and 1A9/PTX22 was assessed over three days using a

previously described colorimetric assay (8). Cells were seeded at a low density into 96-well

plates. Following a 48 h attachment and growth period, the cells were treated with a

concentration range of individual test agents in quadruplicate or vehicle control (DMSO, n=8)

for an additional 72 h. Cell proliferation was assessed spectrophotometrically after exposure to

3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium and

N-methyloxyphenazine methylsulfate (MTS) followed by an absorbance reading at 490 nm

minus the absorbance reading at 630 nm. One full microplate was developed at the end of the

attachment period to determine cell numbers at the time of treatment. The 50% growth

inhibitory concentrations (GI50) of test agents were calculated from the spectrophotometrically

determined expansion of the control cells over the 72 h period.




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siRNA knockdown of ABCB1. HeLa/DZR cells were transfected with 20 nM ABCB1 siRNA or

scrambled siRNA (Stealth siRNA Negative Control Hi GC, both from Invitrogen) as described

previously (14). Treatment with this ABCB1 siRNA caused >75% decrease in ABCB1 protein

levels at 24- and 72-h after transfection as measured by Western blotting (14). Briefly,

HeLa/DZR cells were plated at a density of 7.5 x 104 cells/well into a six-well tissue culture plate

and transfected 24 h thereafter with 20 nM ABCB1 siRNA or scrambled siRNA using 5 μL/well

Dharmafect 1 reagent (Dharmacon, Lafayette, CO) and 480 μL/well Optimem transfection

medium (Invitrogen) in a total volume of 2 mL/well. After 5 h, the transfection medium was

replaced with fresh medium. Twenty-four h later cells were detached with 0.05% trypsin,

seeded into 96 well plates at a density of 1,000 cells/well, and allowed to attach overnight. Cells

were then treated with test agents or vehicle control for 72 h. Growth inhibition was

determined by measuring Hoechst 33342-stained nuclei as described above.

Combination cytotoxicity studies. Combination cytotoxicity studies were performed

essentially as described (19). MDA-MB-231 cells were treated in quadruplicate for 96 h with

10-point 2-fold serial dilutions of paclitaxel, test agents, or a fixed ratio of test agent and

paclitaxel based on the GI50 values of the individual agents. Images were acquired on the

ArrayScan II and nuclei enumerated as described above. Affected fractions (Fa) were calculated

as Fa = cell density of drug treated cells / cell density of vehicle treated cells. The data were

analyzed using the median-effect analysis of Chou and Talalay (20), assuming mutually exclusive

drug effects. The degree of synergism, additivity, and antagonism was measured by calculating

combination indices (CI) over a range of affected fractions exactly as described previously (19).




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Radioligand displacement studies. Experiments were performed as previously described (11)

using tubulin purified in our laboratory from bovine brains by the method of Hamel (21). MTs

were preformed by incubating 2 μM bovine tubulin with 40 μM ddGTP in 0.75 M MSG, pH 6.6,

at 37 °C for 30 min. In separate tubes, a 50 μL solution of 8 μM test agent and 4 μM

radiolabeled [3H]paclitaxel or [14C]epothilone B in 0.75 M MSG, pH 6.6, with a final DMSO

content of 1%, was incubated for 10 min at 37 °C. An aliquot (50 μL) of the preformed MTs was

added to the radioligand/test agent mixture and incubated at 37 °C for an additional 30 min.

Final concentrations of tubulin, radioligand and test agents were 1 μM, 2 μM, and 4 μM,

respectively. Reaction mixtures were then centrifuged at 17,000 x g for 30 min at room

temperature and the amount of unbound radioligand determined by analyzing 50 μL of the

supernatant by scintillation spectrometry (Beckman-Coulter LS6500). To account for non-

specific radioligand binding, the amount of bound radioligand was calculated by subtracting the

amount of radioligand in the supernatant in the presence of test agent from the amount of

radioligand in the supernatant in the presence of a large molar excess of the agent with the

highest binding affinity (20 μM (–)-dictyostatin) (8, 11). The extent of displacement was then

calculated as percent inhibition = (1-(radioligand bound with test agent/radioligand bound with

DMSO))*100.

Tubulin assembly assay. Tubulin assembly was monitored turbidimetrically at 350 nm in

temperature-controlled, multichannel Beckman-Coulter 7400 or Gilford 250

spectrophotometers as described previously (8, 22). Reaction mixtures without test




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compounds consisted of bovine brain tubulin (1 mg/mL) in 0.1 M (4-morpholino)ethane

sulfonate (Mes) and were cooled to 2.5oC to establish baselines. Compounds predissolved in

DMSO were added to give the indicated final concentrations and each reaction mixture (0.25

mL final volume) was subjected to a temperature gradient. From the precooled state, the

temperature was rapidly raised to 30°C (in approximately 1 min) and maintained for 20 min.

The temperature was then rapidly lowered back to 0.25–2.5°C. Absorbance at 350 nm was

monitored every 15 s.

Antiangiogenesis assay. The Tg(Fli1:EGFP)y1 transgenic zebrafish line (obtained from Dr. Brant

Weinstein) was maintained as described (23). Embryos were collected at 24 h post fertilization

(hpf) and staged according to (24). For each condition, five Tg(Fli1:EGFP)y1 transgenic zebrafish

embryos were placed in 500 μL E3 medium (5 mM NaCl, 0.33 mM CaCl2, 0.17 mM KCl, 0.33 mM

MgSO4) and treated with vehicle (DMSO, 0.5%) or various concentrations of test agents (1 μM

to 25 μM) for an additional 24 h. After manual removal of the chorions, single embryos were

transferred to wells of a 96-well half area plate (Greiner, Monroe, NC) containing 40 μg/ml

MS222 (tricaine methanesulfonate, Sigma) in E3 for imaging.

Photomicrographs of fluorescent ISV were acquired with the ImageXpress Ultra high-content

reader (Molecular Devices, Sunnyvale, CA) using a 4X objective and the 488 nm argon laser.

Images were uploaded into the Definiens Developer software suite (Definiens AG, Germany)

and analyzed with a custom designed Cognition Network Technology (CNT) ruleset as described

(25). Thresholding modifications were made to the CNT ruleset to accommodate the higher




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resolution and pixel depth of the ImageXpress system compared with the previously used

ArrayScan (25). Total embryo size and intensity measurements were used to identify dead

embryos, plate-loading artifacts, and autofluorescent compounds. Wells that contained no

embryos, or embryos in which no dorsal region could be detected were eliminated. For the

remaining wells, the ruleset provided numerical measurements of ISV development (area,

length, and shape). The parameter that most robustly measured ISV development was the total

ISV area (in pixels). Data were normalized to vehicle controls. Experiments were repeated at

least three times.




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Results

Synthesis of novel dictyostatins analogs. We recently reported a streamlined synthesis of

dictyostatin and used it to prepare two 16-desmethyl-25,26-dihydrodictyostatins epimeric at C6

(13). Based on the biological activity of the series, we concluded that the reduction of the C25–

C26 double bond is well tolerated but that removal of the C16 methyl group causes loss of

activity against paclitaxel resistant cells (13). Accordingly, we selected 25,26-

dihydrodictyostatin 1a and 6-epi-25,26-dihydrodictyostatin 1b as target compounds.

The streamlined route, which features high convergence, modularity, a relative ease with which

structurally complex new analogs of DCT can be prepared without ambiguity in the C2-C3

configuration, and reliability of the fragment couplings, was used to make the new analogs 1a

and 1b. Fragment couplings and completion of the syntheses are summarized in Figure 1.

Briefly, a Horner-Wadsworth-Emmons (HWE) reaction (26) was used to couple the known top

fragment 4 (13) with new middle fragment 3 to give 5. 1,4-Reduction of the enone, removal of

the para-methoxybenzyl (PMB) group, stereoselective ketone reduction and mono-silylation

then provided 6. Intermolecular esterification with epimeric acid chlorides 7a,b incorporated

the bottom fragment (27) to give 8a,b. Selective removal of the primary tert-butyldimethylsilyl

(TBS) group and oxidation provided aldehydes 9a,b that were substrates for an intramolecular

Nozaki-Hiyama-Kishi (NHK) reaction (13) to give macrolactone 10a,b. Selectivity in the

formation of the new stereocenter at C9 depended on the configuration at C6 with the b

isomer being more selective (10b, 10/1; 10b, 3/1). Desilylation and careful purification to

remove the C9 epimers provided the target products 1a and 1b. The strategy enabled the total




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synthesis of both analogs in a total of 39 steps, with a longest linear sequence of 11 steps from

commercially available starting material.

High-content analysis of mitotic arrest. We first characterized the novel agents for mitotic

arrest and microtubule perturbation using our multiparameter high-content analysis assay (11,

15) as described in the Materials and Methods Section. Immunofluorescence images of HeLa

cells treated with test agents for 21 h show that the new analogs, like 6-epi-dictyostatin, caused

MT bundling (shown in green), chromatin condensation (blue), and elevated levels of phospho-

histone H3 (red) at nanomolar concentrations (Figure 1B). All agents showed concentration-

dependent changes (Figure 1C). From the range of concentrations tested, a minimum

detectable effective concentration (MDEC) value was determined (28). The data indicate that

the new agents were equipotent to 6-epi-dictyostatin and paclitaxel. A detailed summary of

the mitotic arrest assay results can be found in Table S1 in the Data Supplements Section.

Stabilization of cellular MTs and tubulin assembly in vitro. We next asked if the new agents

stabilized MTs in cells and caused MT assembly of isolated tubulin in vitro. It was previously

shown that acetylated tubulin is a marker for stabilized cellular MTs (29). Cells were stained

with antibodies against alpha tubulin or acetylated tubulin, respectively, to visualize cellular

MTs and MT acetylation. Figure 2A shows distinct differences in the concentration-response

curves of tubulin and acetylated tubulin staining obtained with (–)-dictyostatin, a known MT

stabilizer, or vincristine, a known MT destabilizer. In cells treated with (–)-dictyostatin, we

observed a steady increase in cellular MT density as well as acetylated MTs that plateaued at




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high concentrations. In contrast, vincristine caused an initial increase in cellular MT density and

MT acetylation at low concentrations that was lower in magnitude and that reversed at higher

concentrations. This bimodal response is characteristic for MT destabilizing agents: the initial

increase results from morphological changes (i.e., cell rounding); the subsequent decrease is

due to extraction of monomeric tubulin into the permeabilization buffer during cell processing

and staining (15). Both the shape and the magnitude of MT and acetylated MT density curves

caused by the dictyostatin analogs (Figure 2A) were identical to that elicited by (–)-dictyostatin,

suggesting 25,26-dihydrodictyostatin (1a) and 6-epi-25,26-dihydrodictyostatin (1b) caused MT

stabilization. Immunofluorescence micrographs of acetylated MTs confirmed the results of the

automated analysis (Figure 2B).

In vitro tubulin assembly. To further confirm the MT stabilizing activity of the new analogs, we

performed in vitro tubulin assembly studies using a turbidity assay (22) and paclitaxel as a

positive control. Isolated tubulin from bovine brain was incubated with vehicle (DMSO) or

various concentrations of test agents and subjected to a temperature gradient as shown in

Figure 2C. The new agents induced rapid and vigorous tubulin assembly with potency similar to

paclitaxel and (–)-dictyostatin (Figure 2C). Assembly was concentration-dependent and the

resulting polymer was cold-stable, similar to paclitaxel and consistent what we had previously

observed with 6-epi-dictyostatin (11).

In vitro radioligand displacement. We previously showed that (–)-dictyostatin competes with

[3H]paclitaxel and [14C]epothilone B for binding to tubulin polymer formed in the presence of




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ddGTP (8). We therefore tested whether the new analogs retained this ability. Discodermolide,

(–)-dictyostatin, and the new analogs were incubated with preformed MTs labeled with

[3H]paclitaxel and [14C]epothilone, and the amount of unbound tracer measured by scintillation

spectrometry. Table 1 shows that the new analogs displaced [3H]paclitaxel and [14C]epothilone

B with similar potency to discodermolide or (–)-dictyostatin. These experiments provided

conclusive evidence that the new dictyostatin analogs bind the taxoid site on tubulin polymer

with affinities similar to that of (–)-dictyostatin.

Antiproliferative activity in paclitaxel-, epothilone B-, and disorazole C1-resistant cell lines.

(–)-Dictyostatin has antiproliferative activity in paclitaxel-resistant cells (11). To assess if the

analogs remained active in drug resistant cancer cell lines, we tested 25,26-dihydrodictyostatin

and 6-epi-25,26-dihydrodictyostatin in paclitaxel-resistant 1A9 human ovarian cancer cells with

beta-tubulin mutations (Phe270 –> Val) and (Ala364 –>Thr) (30) induced by long-term culture

with paclitaxel, and in epothilone B-resistant A549 human lung cancer cells that harbor a point

mutation in beta-tubulin (292Gln–>Glu) as a result of long-term exposure to epothilone (31).

Table 2 shows that cross-resistance to paclitaxel in the 1A9/PTX10 cells was reduced from 49-

fold, to 15-fold with (–)-dictyostatin and further reduced with the new analogs (7- and 8-fold for

1a and 1b, respectively). Similarly, cross resistance to epothilone B was reduced with (–)-

dictyostatin (from 94-fold for epothilone B and 18-fold for paclitaxel to 10-fold with (–)-

dictyostatin), and further diminished with the new analogs (5-fold and 3-fold, respectively, for

1a and 1b). Diminished cross-resistance was also observed in a recently described disorazole

C1-resistant human cervical carcinoma cell line that overexpresses the ABCB1 P-glycoprotein




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pump (14). Consistent with previously published data (14), these cells were 1395- and 502-fold

resistant to paclitaxel and vinblastine, respectively (Table 2). In contrast, the new dictyostatin

analogs showed greatly reduced cross-resistance to disorazole C1 compared with paclitaxel and

vinblastine, with a residual 12- and 18-fold resistance respectively, for 1a and 1b. To

investigate further if the new analogs were affected by multidrug transport proteins, we

performed siRNA knockdown of ABCB1, which reversed the residual cross-resistance in the

disorazole C1 resistant cells (Table 2).

Combination cytotoxicity studies of dictyostatins and paclitaxel. Discodermolide and

paclitaxel represent a synergistic drug combination in human cancer cells (32). We therefore

examined the novel dictyostatin analogs in combination with paclitaxel to determine if they

also resulted in synergy. We used our previously described growth inhibition assay (18)

together with median effect analysis (20) to quantify synergism, additivity, and antagonism.

MDA-MB-231 cells were treated with comprehensive concentration gradients of paclitaxel,

discodermolide, 6-epi-dictyostatin, 25,26-dihydrodictyostatin 1a, 6-epi-25,26-

dihydrodictyostatin 1b, or equipotent, fixed mixtures thereof with paclitaxel for four days, and

cell densities quantified by counting Hoechst 33342-stained nuclei. Median effect (Dm), slopes

(m), and correlation coefficients (r) for the individual agents and the combinations can be found

in Table S2 in the Supporting Information Section. Combination indices were then calculated for

various effect levels by the method of Chou and Talalay (20, 33) as described previously (18).




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As shown in Figure 3, we reproduced the results of Martello et al. (32), who found the

combination of paclitaxel and discodermolide to be synergistic at lower effect levels and

antagonistic at high effect levels. The dictyostatins had combination index profiles similar to

that of discodermolide, although the degree of synergism was lower. The least potent

combination was with 6-epi-25,26-dihydrodictyostatin 1b (Figure 3D), which was additive over

much of the effect range. The data consistently repeated over the course of multiple

independent experiments. The data suggest that (–)-dictyostatin and the new analogs share

the ability of discodermolide to synergize with paclitaxel, a feature that is potentially favorable

for clinical use.

Inhibition of angiogenesis in zebrafish embryos. Some MT perturbing agents have

antiangiogenic activity that contributes to in vivo anticancer activity (34). Solid tumors require

an adequate supply of blood vessels to survive, grow, and metastasize (reviewed in (35)), and

agents targeting tumor angiogenesis are now FDA-approved anti-cancer medicines (e.g.,

bevacizumab, Avastin ®). We therefore asked if the dictyostatin analogs had antiangiogenic

activity. We used the Tg(fli1:EGFP)y1 zebrafish line that expresses EGFP under the control of

the Fli1 promoter, thereby labeling all blood vessels and providing a live visual marker for

vascular development (36). Zebrafish have a stereotypical vertebrate vasculature that develops

in response to the same signals that guide mammalian blood vessel development (37, 38).

Zebrafish vasculature recruitment also occurs in response to human glioma xenografts (39, 40),

mimicking conditions found in mammals.




21

Tg(fli1:EGFP)y1 zebrafish embryos at 24 hpf were treated for 24 h with vehicle or various

concentrations of test agents and imaged. Figure 4A shows that, as expected, vehicle-treated

embryos had well-established intersegmental vessels (ISV) that extended from the dorsal aorta

(DA) and connected to the dorsal longitudinal anastomotic vessel (DLAV) (Figure 4A, (Isogai et

al., 2001)). Visually, all of the dictyostatin analogs stunted ISV outgrowth and prevented the

establishment of the DLAV (Figure 4A, upper panels). Our previously described image analysis

algorithm (25) quantified the antiangiogenic phenotype (Figure 4A, lower panels). All agents

concentration-dependently inhibited angiogenesis (Figure 4B), with concentrations required to

reduce ISV area by 50% compared with control (IC50) of 8.8, 6.1, and 6.7 μM for 6-epi-

dictyostatin, 25,26-dihydrodictyostatin 1a, and 6-epi-25,26-dihydrodictyostatin 1b, respectively.

Importantly, at concentrations that were antiangiogenic, we observed no obvious signs of

toxicity such as the appearance of necrotic opaque cells. At the highest concentration tested

(25 μM, data not shown), the test agents caused a bent-tail phenotype, suggesting that the

compounds at this concentration would likely cause developmental defects in the embryo.




22

Discussion

An improved synthetic route to dictyostatin analogs. The complex chemical structure and

difficult synthesis of the dictyostatins is a major impediment to their development into novel

antineoplastic agents. This work validates that our recently described synthetic route (13) can

be used to rapidly make new analogs. The streamlined route features a bimolecular

esterification to make the C1–O21 bond in place of the usual macrolactonization. This bypasses

a major problem of Z/E isomerization of the C2–C3 alkene that has plagued the

macrolactonization. In turn, the large ring is closed by a mild Nozaki-Hiyama-Kishi reaction to

make the C9–C10 bond. It should be possible to access many more analogs thanks to the

modularity of this route and the reliability of the fragment couplings and end game steps.

Predictions based on existing SAR are validated. Consistent with prior findings, removal of the

C16 methyl moiety did not dramatically affect antiproliferative activity in human tumor cells

expressing wild-type tubulin but diminished the ability of the compounds to inhibit the growth

of paclitaxel-resistant clones harboring mutations within beta-tubulin (10). We therefore

reasoned that retaining the C16 methyl group would preserve the lack of cross-resistance to

paclitaxel and selected 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as target

compounds. Consistent with existing SAR, both new agents showed low nanomolar

antiproliferative activity in HeLa, A-549, and MDA-MB-231 cells, and reduced cross-resistance

to paclitaxel and epothilone B in cells with mutant tubulin.




23

Dictyostatin analogs occupy the taxane binding site on tubulin. To confirm that the new

analogs directly interact with their proposed target, we performed radioligand binding studies.

These experiments show the new analogs have affinities for the taxane site similar to paclitaxel,

epothilone B, or discodermolide. The precise location of the dictyostatin binding site has not

been established, because the interaction of the dictyostatins or discodermolide with tubulin

has not been solved by cryoelectron microscopy as it has for paclitaxel and epothilone A (41,

42). Furthermore, two binding sites have been described for taxanes: an internal luminal

binding site and an external transient binding site of unknown structure. The radioligand

competition studies are unable to distinguish the two sites. However, growth inhibition studies

of the natural product (8) and on the 16-desmethyl analogs using 1A9/PTX10 ovarian cancer

cells with the Phe270 —>Val mutation that we performed previously (13) are consistent with

dictyostatin and analogs binding to the internal site.

Similarities and dissimilarities to (+)-discodermolide. The new analogs retained some but not

all of the ability of discodermolide to synergize with paclitaxel in human breast cancer cells.

Modeling studies based on NMR structures have suggested that the bound conformer of

dictyostatin resembles that of discodermolide and provides similar contacts with tubulin (43).

Because it is unusual for two drugs that bind to identical sites on the same target to show

synergy, the combination cytotoxicity data do support the previously proposed model of

overlapping binding sites for paclitaxel and the dictyostatins (43). The extent of synergy varied

with the analogs; the least potent agent was 1b, although all of them showed a trend towards

higher synergy at lower effect levels. Therefore, our results confirmed a synergistic




24

relationship specifically at the lower concentrations of the two drugs as reported by Horwitz’s

group (32). The reasons for the differential activity of the analogs in this assay are unknown.

The fact that the dictyostatins were essentially equivalent in all of our assays, including the in

vitro radioligand binding studies, makes it seem unlikely that differences in binding affinity or

cellular distribution would account for the observed differences. To formulate a valid

hypothesis based on structural terms, however, physical evidence such as a high resolution

cryoelectron microscopy structure of the dictyostatins and discodermolide is needed.

Alternatively, the different degree of synergy of the dictyostatins compared with

discodermolide may be a result of off-target effects. As pointed out by Martello et al. (32),

discodermolide induces apoptosis by mechanisms unrelated to MT binding, and it is currently

not known whether the dictyostatins share these activities. The data do suggest, however, that

the combination of paclitaxel with either 6-epi-dictyostatin or 1a merits exploration in in vivo

antitumor studies.

Dictyostatins lack cross-resistance to paclitaxel, epothilone B, and disorazole C1. Drug

resistance is a major problem with MT perturbing agents in clinical use. One clinically

important resistance mechanism is overexpression of p-glycoprotein efflux pumps (44). In

cultured cells, additional resistance mechanisms have been observed that involve tubulin

mutations induced by long-term culture of cell lines in the presence of MT perturbing agents

(31, 45), although such drug-induced mutations have not been found in clinical samples. In

three such cellular models with mutant tubulin, the new analogs retained activity against both

paclitaxel- and epothilone B-resistant cells, and appeared less cross-resistant than the natural

product. The 1A9/PTX10 cell line harbors a Phe270 —> Val mutation that is located within the




25

taxane binding site (42) and confers 49-fold resistance to paclitaxel. Consistent with our

previous studies with (–)-dictyostatin and 6-epi-dictyostatin (13), cross-resistance was reduced

to Thr mutation that is adjacent to the taxane binding

pocket. In epothilone B resistant A-549 cells with a 292Gln —> Glu mutation, which is located

at the periphery of the taxane pocket and makes contact with epothilone but not paclitaxel

(42), the analogs showed only a 12-18-fold cross resistance compared with epothilone B (94-

fold resistance). The data indicate that reduction of the terminal double bond does not alter

the mode of tubulin binding. They are consistent with a mode of binding to tubulin as

proposed by Canales et al. (43) that involves the taxane binding pocket but not residues outside

the pocket that make contact with the taxane side chain.

The analogs showed a unique behavior toward cells with acquired resistance against the natural

product disorazole C1 (14), which owe their resistance phenotype at least in part to

overexpression of the ABCB1 p-glycoprotein pump. All agents were subnanomolar inhibitors of

wild-type HeLa cells. Paclitaxel and vinblastine were 1395- and 502-fold less active,

respectively, in the resistant cells (HeLa/DZR, Table 2). Knockdown of the P-glycoprotein pump,

ABCB1, restored most, of their activity (HeLa/DZR/ABCB1 siRNA, Table 2). In contrast, the

HeLa/DZR cells showed only minor cross-resistance to the dictyostatin analogs (12- to 18-fold,

HeLa/DZR, Table 2) that was fully reversed by ABCB1 knockdown. The data suggest that the

dictyostatins may be only weak substrates for ABCB1. Moreover, because the HeLa/DZR cells

were generated by a single exposure to the mutagen ethyl methane sulfonate followed by a




26

stepwise increased disorazole C1 exposure, it is likely that resistance mechanisms other than

elevated ABCB1 exist, but these do not appear to influence cellular sensitivity to the

dictyostatin analogs.

Antiangiogenic activity in vivo. We had previously shown that microtubule-perturbing agents

inhibit angiogenesis in Tg(fli1:EGFP)y1 transgenic fluorescent zebrafish embryos (15). Here we

demonstrate that the new analogs also have this property, which is thought to be beneficial for

clinical activity (34, 46). In the Tg(fli1:EGFP)y1 model, the agents appeared to have

antiangiogenic rather than antivascular activity. During development, intersegmental vessels

(ISV)s sprout from the dorsal aorta (DA) at 24 hpf, and at 48 hpf are fully established and

connected to the dorsal longitudinal anastomotic vessel (DLAV). To assess the effect of test

agents on new vessel outgrowth (angiogenesis), embryos were treated at 24 hpf (when ISV are

just beginning to sprout and are barely visible (15)), and analyzed for ISV formation 24 h

thereafter. While the analogs caused a concentration-dependent inhibition of new vessel

growth, they did not affect existing blood vessels as the head and large trunk vessels were

intact. Furthermore, heart beat, circulation, and twitch response (assessed visually) were all

normal (data not shown). We also did not observe tissue necrosis, which would show as

opaque cells in the fluorescence micrographs (see Figure 4). Test agent-treated embryos also

showed little difference in gross morphology when compared with control embryos (Figure 4),

although we did observe a bent tail phenotype at the highest concentration tested (25 μM).

While the model is currently not well enough characterized to suggest therapeutic safety in the




27

context of angiogenesis inhibition, the data indicate the new dictyostatins have antiangiogenic

activity in a zebrafish model of angiogenesis at nontoxic concentrations.

In summary, we have used our previously reported, highly convergent, streamlined

synthesis (13) to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin, two

new analogs of the highly complex natural product, (–)-dictyostatin. Consistent with existing

SAR studies and a mode of action involving high affinity binding to the taxane site on tubulin,

the new analogs retained essentially all of the biological activities of (–)-dictyostatin and 6-epi-

dictyostatin, the only analog whose activity in adult mammals has been described to date (12).

While the new analogs do not represent a significant simplification from a structural

standpoint, reduction of the exposed double bond eliminates chemical reactivity and a

potential metabolic “soft spot”, as has been shown for discodermolide (47). Future

experiments should focus on this issue. The results identify 25,26-dihydrodictyostatin and 6-

epi-25,26-dihydrodictyostatin as candidates for scale-up using the improved synthesis

procedure and for further preclinical development.

Acknowledgments

We thank Dr. Brant Weinstein for the transgenic Tg(fli1:EGFP)y1 line, Dr. Susan B. Horwitz for

the epothilone B-resistant cells, Drs. Tito Fojo and Paraskevi Giannakakou for the paclitaxel-

resistant clones, the National Cancer Institute for [3H]paclitaxel, and Novartis Pharma for

[14C]epothilone B.




28

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Table 1. In vitro radioligand displacement.

Percent inhibition of[3H]Paclitaxel binding

Percent inhibition of [14C]Epothilone B binding

4 μM test agent 20 μM test agent 4 μM test agent 20 μM test agent25,26-dihydrodictyostatin 1a 61 ± 4 (2) 68 ± 5 (2) 66 ± 13 (3) 101 ± 9 (3) 6-epi-25,26-dihydrodictyostatin 1b 83 ± 2 (2) 98 ± 11 (2) 41 ± 3 (3) 70 ± 9 (3) (–)-dictyostatin 75 ± 6 (2) 100 (2) 53 ± 6 (3) 100 (3) (+)-discodermolide 86 ± 13 (2) ND 65 ± 3 (3) 112 ± 17 (3)

Preformed MTs were labeled with 2 μM [3H]paclitaxel or 2 μM [14C]epothilone in the presence or absence of test agents, and the

amount of unbound radioligand measured by scintillation spectrometry as described in the Materials and Methods Section. Data

were normalized to free radiolabel measured in the presence of vehicle (DMSO) and in the presence of 20 μM (–)-dictyostatin. Data

represent the average ± S.D. of (n) independent experiments. ND, not determined.

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Table 2. Antiproliferative activity in paclitaxel-, epothilone B-, and disorazole C1-resistant cell lines.

A549 aEpoB40/ A549 a

1A9 b 1A9/PTX10 b 1A9/PTX22b HeLa c HeLa/DZR cHeLa/DZR

ABCB1siRNA c

25,26-dihydrodictyostatin1a

2.3 ± 0.5 12.4 ± 1.5 (5)

6.8 ± 0.7 44.9 ± 12.9 (7)

12.2 ± 4.8 (2)

0.6 ± 0.3 7.0 ± 3.2 (12)

0.4 ± 0.1 (1)

6-epi-25,26-dihydrodictyostatin 1b

4.5 ± 1.0 15.1 ± 1.8 (3)

5.0 ± 3.5 42.1 ± 20.1 (8)

9.6 ± 4.8 (2) 1.0 ± 0.7 17.5 ± 9.1 (18)

0.9 ± 0.5 (1)

6-epi-dictyostatin0.7 ± 0.3 4.5 ± 1.0

(6)0.8 ± 0.1 14.2 ± 5.6

(18)1.9 ± 0.7 (2) 0.4 ± 0.0 5.9 ± 2.6

(15)0.7 ± 0.2

(2)

(–)-dictyostatin 0.5 ± 0.0 5.1 ± 0.5

(10)1.3 ± 1.0 18.8 ± 2.2

(15)5.1 ± 0.6 (4) ND d ND ND

(+)-discodermolide 2.2 ± 0.1 22.0 ± 2.2

(10)ND ND ND ND ND ND

Paclitaxel0.4 ± 0.1 7.0 ± 1.0

(18)1.7 ± 0.3 83.5 ± 6.4

(49)58.7 ± 13.7

(35)0.2 ± 0.0 279 ± 13

(1395) 4.5 ± 2.4

(23)

Epothilone B 0.2 ± 0.1 18.7 ± 3.8

(94)ND ND ND ND ND ND

VinblastineND ND ND ND ND 0.4 ± 0.0 201 ± 1

(502)8.3 ± 0.5

(21)

Cells were exposed to vehicle or test agents for 72-120 h and cell growth determined as described in the Materials and Methods

Section. Data represent average 50% growth inhibitory concentrations (GI50) ± S.D. (nM) from at least three independent

experiments. Values in parentheses denote fold resistance compared with wild-type cells. a 3-day HCS assay; b 3-day MTS assay; c 5-

day HCS assay; ND, not determined

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Figure Legends

Figure 1. Mitotic arrest and alterations in MT morphology by 25,26-dihydrodictyostatin (1a)

and 6-epi-25,26-dihydrodictyostatin (1b) prepared by a highly convergent synthesis. A) A

highly convergent chemical synthesis of 25,26-dihydrodictyostatin (1a) and its C6-epimer (1b).

Reaction conditions: (a) Ba(OH)2, 70%; (b) [Ph3PCuH]6; (c) 2,3-dichloro-5,6-dicyano-1,4-

benzoquinone (DDQ), 76% over 2 steps; (d) NaBH4, Diethylmethoxyborane (Et2BOMe), 90%; (e)

tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), 86%. (f) sodium

bis(trimethylsilyl)amide (NaHMDS), b isomer 57%; (g) HF•pyridine, a isomer 70% (over 2 steps),

b isomer 84%; (h) Dess-Martin, NaHCO3, a isomer 95%%, b isomer 94%; (i) CrCl2, dichloro[1,1'-

bis(diphenylphosphino)ferrocene]nickel(II) (NiCl2(dppf)), 4,4'-di-tert-butyl-2,2'-dipyridyl, a

isomer 22%, b isomer 42%; (f) HF•pyridine, a isomer 54%, b isomer 82%. (B-C),

Multiparametric analysis of mitotic arrest. HeLa cells were treated for 21 h with concentration

gradients of 6-epi-dictyostatin, 25,26-dihydrodictyostatin 1a, or 6-epi-25,26-dihydrodictyostatin

1b, and stained with anti-tubulin and anti-phosphohistone H3 antibodies. Nuclei were

counterstained with Hoechst 33342. B) Images documenting microtubule perturbation and

mitotic arrest. Fluorescence micrographs of tubulin (green), nuclei (blue), and phospho-histone

H3 (red) were acquired on the ArrayScan II. Images are from one representative field of view.

All agents are shown at 15.6 nM. C) Quantification of cellular response. MT density, nuclear

condensation, and histone H3 phosphorylation were measured in 1,000 individual cells after

treatment with 6-epi-dictyostatin ( ), 25,26-dihydrodictyostatin 1a ( ), or 6-epi-25,26-

dihydrodictyostatin 1b ( ). Data points represent the average of quadruplicate wells ± S.E.

from one representative experiment that was repeated at least three times.




35

Figure 2. Stabilization of cellular MTs and tubulin assembly in vitro. A) HeLa cells were

treated for 21 h with vincristine, (–)-dictyostatin, 25,26-dihydrodictyostatin, or 6-epi-25,26-

dihydrodictyostatin. MT density ( ) or acetylated MT density ( ) was quantified on the

ArrayScan II as described in Materials and Methods. Data are the averages ± S.E. of

quadruplicate wells from a single experiment that has been repeated three times. B)

Immunofluorescence images of acetylated MTs in cells treated with dictyostatin analogs (31

nM) or vincristine (25 nM). C) In vitro tubulin assembly. Electrophoretically homogenous

bovine brain tubulin was incubated with vehicle (DMSO) or the indicated concentrations of test

agents and subjected to a temperature gradient as indicated. Tubulin assembly was measured

by turbidimetry at 350 nm.

Figure 3. Combination cytotoxicity of dictyostatin analogs and paclitaxel. MDA-MB-231 cells

were treated with concentration gradients of paclitaxel, discodermolide, 6-epi-dictyostatin,

25,26-dihydrodictyostatin, 6-epi-25,26-dihydrodictyostatin, or equipotent combinations of

paclitaxel and dictyostatin analog for four days. Cell densities were quantified on the ArrayScan

II as described in Materials and Methods. Data were analyzed by the median-effect analysis of

Chou and Talalay, assuming mutually exclusive drug effects. Combination Indices (CI) were

calculated for each effect level. CI values of < 1, 1 (solid line), and >1 are synergistic, additive,

and antagonistic, respectively. Data points represent average CI ± S.E. of at least three

independent experiments.




36

Figure 4. Antiangiogenic activity in zebrafish embryos. Tg(fli1:EGFP)y1 larvae at 24 hpf were

treated with vehicle (DMSO) or various concentrations of test compounds for an additional 24 h

and imaged on a Molecular Devices ImageXpress high-content confocal imager. (A-D)

representative fluorescence micrographs of embryos treated with A) vehicle (DMSO) B) 6-epi-

dictyostatin (12.5 μM), C) 25,26-dihydrodictyostatin 1a (5 μM) or D) 6-epi-25,26-

dihydrodictyostatin 1b (5 μM). Top panel, raw fluorescence micrographs (shown inverted for

visual clarity); Bottom panel, images with Cognition Network Technology (CNT) analysis applied.

DMSO concentration was 0.5% in all cases. ISVs are shown in red. Green, purple, pink, yellow,

and white areas represent the zebrafish body, large trunk vessels, head, yolk, and dorsal

regions, respectively. E) Quantification of ISVs. Images were analyzed with a CNT ruleset as

described (25). Total ISV area was calculated for each condition, and normalized to pooled

vehicle controls. Each data point represents the average ± S.E. from at least three independent

experiments, performed in quintuplicate




B

Microtubule Density Chromatin Condensation Mitotic IndexC

DMSO (–)-dictyostatin 1a 1b

6-epi-dictyostatin1a1b

0 1 2 30

200

400

log nM

mea

n pi

xel i

nten

sity

, nu

clea

r reg

ion

0 1 2 30

20

40

60

log nM

%co

nden

sed

nucl

ei

0 1 2 30

20

40

60

log nM

%ph

osph

o-hi

ston

eH

3po

sitiv

ece

lls

OPMB

O(MeO)2OP

OTBS

TBSO

O

I

OH

TBSO

O

I

+

4 top fragmentC18–C26

a

5

b–e

6

O

TBSO

I

3 middle fragmentC10–C17

TBS

O

TBSO

O

R

O

OTBSI

OTBSTBSO

ClO

7a,b bottom fragmentsC1–C9, both epimers at C6

f

8a,b, R = CH2OTBS9a,b, R = CHO

O

HO

OH

O

OH OH

O

TBSO

O

O

OH OTBS

10a,b

TBS TBS

g-i j

1a, C6-(R), Me (down)1b, C6-(S), Me (up)C25-C26 saturated

(–)-dictyostatin, C6-(R), Me (down)C25-C26 unsaturated

9

1625

26

6 6 6

6

A

Vollmer et al. Figure 1




(–)-dictyostatin

DMSO

PTX (10 μM)5 μM10 μM20 μM

1a(–)-dictyostatin 1b

C

(–)-dictyostatin

0 1 2 30

200

400

600

800

mea

n pi

xel i

nten

sity

, nu

clea

r reg

ion

vincristine

0 1 2 30

200

400

600

800

log nM

1a

0 1 2 30

200

400

600

800

1b

0 1 2 30

200

400

600

800

B

1a 1b

A

log nM log nM log nM

vincristine

0 10 20 30

0.0

0.4

0.8

2.5 oC 2.5 oC30 oC

Abs

350

nm

time (min)

0 10 20 30

0.0

0.4

0.8

2.5 oC 2.5 oC30 oC

0 10 20 30

0.0

0.4

0.8

2.5 oC 2.5 oC30 oC

time (min) time (min)


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(+)-discodermolide

0.0 0.2 0.4 0.6 0.8 1.00.0

0.5

1.0

1.5

2.0

affected fraction

Com

bina

tion

Inde

x

6-epi-dictyostatin

0.0 0.2 0.4 0.6 0.8 1.00.0

0.5

1.0

1.5

2.0

affected fraction

Com

bina

tion

Inde

x

25,26-dihydro-dictyostatin

0.0 0.2 0.4 0.6 0.8 1.00.0

0.5

1.0

1.5

2.0

affected fraction

Com

bina

tion

Inde

x

6-epi-25,26-dihydro-dictyostatin

0.0 0.2 0.4 0.6 0.8 1.00.0

0.5

1.0

1.5

2.0

affected fraction

Com

bina

tion

Inde

x





A. B. C. D.

E.

ISV

DA

DLAV

0

50

100

totalISV

area

(%ofvehiclecontrol)

6-epi- 1a 1bdictyostatin

1 μM2.5 μM5 μM12.5 μM25 μM


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Published OnlineFirst April 13, 2011.Mol Cancer Ther Laura L Vollmer, Maria Jimenez, Daniel P Camarco, et al. activity in zebrafish embryosactivity against epothilone B resistant cells and antiangiogenic A simplified synthesis of novel dictyostatin analogs with in vitro

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