1
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,
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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:
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.
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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.
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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.
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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
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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
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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
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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
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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.
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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.
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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.
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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
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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
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eH
3po
sitiv
ece
lls
OPMB
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+
4 top fragmentC18–C26
a
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6
O
TBSO
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3 middle fragmentC10–C17
TBS
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TBSO
O
R
O
OTBSI
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ClO
7a,b bottom fragmentsC1–C9, both epimers at C6
f
8a,b, R = CH2OTBS9a,b, R = CHO
O
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O
TBSO
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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
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(–)-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
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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)
Vollmer et al. Figure 2
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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
Vollmer et al. Figure 3
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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
Vollmer et al. Figure 4
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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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