Design, synthesis and biological evaluation of a simplified fluorescently labeled discodermolide as a molecular probe to study the binding of discodermolide to tubulin

Article abstract of DOI:10.1016/j.bmc.2011.06.082

The design, synthesis, and biological evaluation of a simplified fluorescently labeled discodermolide analogue possessing a dimethylaminobenzoyl fluorophore has been achieved. Stereoselective Suzuki coupling and Horner-Wadsworth-Emmons reaction comprised

Full text of DOI:10.1016/j.bmc.2011.06.082

Bioorganic & Medicinal Chemistry 19 (2011) 5247–5254
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of a simplified fluorescently labeled
discodermolide as a molecular probe to study the binding of discodermolide
to tubulin
Jun Qi a, Adam R. Blanden b, Susan Bane b, David G. I. Kingston a,
⇑
a Department of Chemistry, M/C 0212, Virginia Tech, Blacksburg, VA 24061, USA
b Department of Chemistry, State University of New York at Binghamton, Binghamton, NY 13902, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis, and biological evaluation of a simplified fluorescently labeled discodermolide ana-
logue possessing a dimethylaminobenzoyl fluorophore has been achieved. Stereoselective Suzuki cou-
pling and Horner–Wadsworth–Emmons reaction comprised the key tactics for its construction. The
analogue exhibited qualitatively similar activity to paclitaxel in a tubulin assembly assay, and it can thus
be used as a fluorescent molecular probe to explore the local environment of the discodermolide binding
site on tubulin. The results of fluorescence measurements on the tubulin-bound analogue are reported.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 14 April 2011
Revised 24 June 2011
Accepted 28 June 2011
Available online 2 July 2011
Keywords:
Discodermolide
Tubulin
Synthesis
Fluorescence
1. Introduction
If further development of discodermolide is to become a reality,
it would be most useful to have a detailed understanding of the
The polyketide natural product discodermolide (1, Fig. 1) was
isolated from the marine sponge Discodermia dissoluta.1 It had po-
tent cytotoxicity to human and murine cell lines,1 and its mecha-
nism of action was found to be very similar to that of the
important anticancer drug paclitaxel (2, Fig. 1). It promotes the
assembly and stabilization of microtubules, causing cell cycle ar-
rest at the G2/M phase boundary and subsequent cell death by
apoptosis.2
These facts made discodermolide a prime candidate for preclin-
ical and eventually clinical development. The low yield of disco-
dermolide from its producing organism, and the difficulty of
aquaculture approaches, made synthesis the only viable route to
the quantities needed for this work. Several effective synthetic
routes have been developed, including practical large-scale synthe-
ses by the Smith3 and Paterson groups4; these studies have been
reviewed by Smith and Freeze.5 Extensive preclinical studies were
carried out by Novartis, using material prepared by modifications
of these routes,6 and initial Phase I clinical results were encourag-
ing.7 Sadly further development was discontinued because of lack
of efficacy and toxicity issues,8 but it is possible that modified ana-
logues might still prove useful anticancer drugs.
24
21
OH
O
OH
HO
O
NH2
HO
O
5
O
discodermolide (1)
O
AcO
OH
O
Ph
NH
O
O
OAc
OCOPh
H
Ph
O
HO
OH
paclitaxel (2)
Figure 1. Discodermolide (1) and paclitaxel (2).
⇑
Corresponding author.
E-mail address: dkingston@vt.edu (D.G.I. Kingston).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.082

5248
J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
evaluation of two simplified fluorescently labeled discodermolide
analogues as potential fluorescent molecular probes to explore
the local environment of the discodermolide-tubulin binding site
by fluorescence spectroscopy.
OH
O
O
OH
O
O
OH
OH
In previous studies in our group, the small environmentally sen-
sitive dimethylaminobenzoyl fluorophore was successfully used to
explore the local environment of the paclitaxel binding site on
microtubules.15 In the case of paclitaxel, this group could replace
the side chain N-benzoyl group or the C-2 O-benzoyl group with
minimal impact on the overall structure and activity, but the incor-
poration of such a fluorophore into discodermolide must inevitably
cause major structural changes, which could lead to the loss of
activity. We thus explored incorporating the fluorophore as a
replacement for the d-lactone ring of discodermolide, and were
encouraged by the fact that several analogues of this nature,
including the simple analogue 3, had submicromolar cytotoxic
activity against various cell lines (Fig. 2).5 Analogue 4 was thus
designed to accomplish this (Fig. 2).
NH2
NH2
HO
O
5
O
Me2N
O
3
4
Figure 2. Simplified discodermolides 3 and 4.
binding of discodermolide to the tubulin polymer, and in particular
to know the binding conformation of discodermolide. Unlike pac-
litaxel and other tubulin-binding agents with polycyclic or macro-
cyclic skeletons, discodermolide consists of a flexible chain, and
thus can adopt a seemingly infinite number of conformations both
in solution and when binding to tubulin. Since the discodermolide-
tubulin complex has not been crystallized, the nature of the bind-
ing conformation must be determined by indirect methods. Exten-
sive NMR studies on the conformation of free discodermolide or
tubulin-bound discodermolide have been conducted, and have
provided valuable but not fully conclusive information on the
question.9 A recent study by Horwitz and co-workers probed
hydrogen–deuterium exchange between tubulin and discodermo-
lide by mass spectrometry. This study indicated that unlike paclit-
axel, which interacts primarily with the M-loop of tubulin,
discodermolide orients itself towards the N-terminal H1-S2 loop.10
Finally, the combination of force-field conformational searches
with NMR deconvolution in different solvents led to the conclusion
that the hairpin conformation of discodermolide predominates.
Docking studies then identified two distinct docking poses, and
one of them was identified as better accommodating the available
SAR data.11
2. Results and discussion
2.1. Synthesis of fluorescent discodermolide analogue 4
In the design of analogue 4 the terminal diene at the C21-24 po-
sition was replaced by a saturated butyl group to simplify the syn-
thesis, since this conjugated diene system does not contribute
significantly to the activity of discodermolide.14,16 The retrosyn-
thetic analysis of analogue 4 is shown in Scheme 1.
The synthesis of analogue 4 involved the final installation of the
fluorophore 7 preceded by the Horner–Wadsworth–Emmons cou-
pling of the phosphonate 6 and aldehyde 5, which was prepared
from iodide 8 and vinyl iodide 9by Suzuki coupling.
The precursors 8 and 9 were synthesized by the convergent ap-
proach developed by Smith and his group.16b,17 Their connection
was achieved via Suzuki coupling to afford TBS ether 10 in 77%
yield. Subsequent TBS deprotection and Swern oxidation provided
aldehyde 5 in 65% yield (Scheme 2).
The synthesis of subunit 6 began with propane-1,3-diol, which
was mono protected as its TBS ether, and then oxidized by Dess–
Martin oxidation and further oxidation by NaClO2 and NaH2PO4
to give acid 11. This was then converted to acid chloride 12 by
treatment with 1-chloro-N,N,2-trimethyl-1-propenylamine,18 and
12 was transformed to phosphonate 6 in 40% yield over two steps
(Scheme 3).
In addition to these NMR and mass spectrometric methods,
indirect evidence of the orientation of a ligand in the tubulin bind-
ing pocket can be obtained from the fluorescence energy transfer
spectroscopy of the ligand-protein complex.12 Fluorescence spec-
troscopy is widely used to study ligand–receptor interactions and
the local environment of a binding site.13 At this time such studies
have not been reported on discodermolide, although the synthesis
of a fluorescent discodermolide derivative has been reported by
Smith.14 In this paper, we describe the synthesis and biological
As shown in Scheme 4, HWE coupling between subunits 5 and 6
afforded TBS ether 13 in 85% yield, which was then subjected to
I
OH
O
O
OH
PMBO
OTBS
OMOM
O
PMBO
OTBS
I
8
NH2
5
HO
O
TBSO
P(OCH2CF3)2
O
O
TBSO
OMOM
6
9
Me2N
O
NMe2
4
COOH
7
Scheme 1. Retrosynthesis of analogue 4.

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
5249
I
8
1. , t-BuLi,
1. 1% HCl in EtOH
PMBO
OTBS
I
PMBO
OTBS
PMBO
OTBS
MeO-9-BBN,
OMOM
OTBS
OMOM
O
o
8
THF, —78 C
2.Swern oxidation,
65% over 2 steps
2. 9, Cs2CO3 in
H2O, Pd(dppf)2Cl2,
Et2O, DMF, 77%
10
5
TBSO
OMOM
9
Scheme 2. Synthesis of aldehyde 5.
1. CH3P(O)(OCH2CF3)2,
LiHMDS, THF, -98oC
TBSO
O
TBSO
Me2C=C(NMe2)Cl, DCM
TBSO
O
P(OCH2CF3)2
2. 12 in THF, -98oC,
40% over 2 steps
O
O
OH
Cl
11
12
6
Scheme 3. Synthesis of phosphonate 6.
PMBO
OTBS
PMBO
OTBS
OMOM
OMOM
OMOM
OR OTBS
1. K2CO3,18-crown-6,
1. DDQ, DCM,
toluene, HMPA, rt
5
pH 7 buffer, 75%
O
O
O
2. Cl3CCONCO,
Al2O3, DCM, 82%
2. 5 and 6 in toluene
TBSO
and HMPA, -10 to -5 oC,
85%
P(OCH2CF3)2
O
O
14
R = H
OTBS
OTBS
6
15 R = H2NCO
13
Scheme 4. Synthesis of fragment 15.
oxidation with DDQ to remove the PMB protecting group to give
alcohol 14. Subsequent installation of the carbamate moiety on
alcohol 14 gave carbamate15 (Scheme 4).
activity in the cells is reflected in the tubulin assembly activity,
since compound 4 is an order of magnitude less potent than paclit-
axel in promoting microtubule assembly.
Reduction of the keto group of carbamate 15 by K-selectride™
gave alcohol 16, whose configuration was determined by Mosher
ester analysis.19 Alcohol 16 was coupled separately with R-2-
methoxyphenylacetic acid (R-MPA) and S-MPA to form the S and
R Mosher esters via EDC coupling (Scheme 5). Their 1H NMR spec-
tra were obtained and the dR-S values were calculated for adjacent
protons to the chiral center. The results are summarized in Figure 3,
where the dR-S values of the top part of the structure were positive,
while the value for the bottom part was negative. According to the
Mosher ester model (Fig. 3), the configuration was confirmed to be
the desired S.
Compound 16 was then treated with HCl (4 N) in small portions
over 4 h to remove all the protecting groups, and the resulting
alcohol 17 was coupled with 3-dimethylaminobenzoic acid via
EDC coupling to give the final product 4 in 16% yield over two steps
(Scheme 6).
The emission energy and intensity of the m-dimethylamino
benzoate fluorophore is sensitive to solvent polarity.21 This fluoro-
phore has been used previously to probe the paclitaxel site on
tubulin by Sengupta et al.,22 who also noted that the emission
maximum of this probe in various solvents was affected by both
the polarity of the solvent and its hydrogen bonding capabilities.
We evaluated the emission maximum and intensity of 4 in various
solvents (Table 2). In general, decreasing the polarity of the solvent
increased the emission intensity and shifted the emission maxi-
mum to shorter wavelength. We were unable to observe fluores-
cence of the probe in buffer. This type of behavior has been
noted in m-aminobenzonitriles, whose fluorescence properties
are very similar to those of m-amino benzoates.21,23 We observed
very weak fluorescence of the molecules when 2% DMSO was in-
cluded in the buffer. Increasing the concentration of DMSO in the
buffer increased the emission intensity to detectable levels.
As hoped, tubulin binding induced a large increase in the emis-
sion intensity of the environmentally sensitive dimethylamino ben-
zoate fluorophore for 4 (Fig. 4). The increase in emission intensity
and the shorter wavelength of the emission maximum point to a
hydrophobic environment for the microtubule-bound fluorophore.
The fluorescent signal was used to determine the affinity of the
compound for GMPcPP-stabilized microtubules. Plots of the in-
crease in ligand fluorescence as a function of concentration dis-
played saturation behavior and were fit a single rectangular
hyperbola. The dissociation constant for 4 binding to microtubules
2.2. Biological evaluation of fluorescent discodermolide
analogue
The simplified, fluorescently-labeled discodermolide analogue 4
was evaluated for antiproliferative, tubulin assembly and tubulin
binding activities. The assay results are summarized in Table 1.
Analogue 4 is significantly less active than paclitaxel in the
A2780 and PC3 assays, respectively. Compound 4 is almost 400-
fold less toxic than paclitaxel toward A2780 cells. This relative

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J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
OMOM
O
O
OTBS
O
NH2
MeO
OH
O
Ph
S-MPA
S-MPA
OMOM
O
O
OTBS
OMOM
O
O
OTBS
EDCI,
S-MPA ester
OTBS
NH2
NH2
DMAP, DCM
K-selectride,
O
toluene
HO
O
-78 oC, 90%
MeO
OH
15
OTBS
OMOM
O
O
OTBS
OTBS
16
Ph
R-MPA
NH2
O
R-MPA
R-MPA ester
OTBS
Scheme 5. Reduction of fragment 15 and Mosher ester formation from alcohol 16.
Figure 3. Determination of the absolute configuration of 16.
COOH
OH
OMOM
OH
O
O
OH
O
O
OTBS
O
O
OH
4N HCl,
NMe2
MeOH, rt
NH2
NH2
NH2
DMAP,
88%
EDCI, DC
HO
HO
HO
S
18%
O
O
OTBS
OH
16
17
Me2N
4
Scheme 6. Finale of the synthesis of analogue 4.
Table 1
Cytotoxicity and tubulin binding activity of analogue 4
Compound
A2780 assay IC50 (nM)
14
5400 220
Tubulin assembly assay EC50
(lM)
Binding assay Kd (lM)
0.015 0.004a
Paclitaxel 2
4
2
0.49 0.22
5.2 1.2
24
5
a
From Li et al.20
is 1600-fold greater than that of paclitaxel (Table 1). Since the lac-
tone moiety of discodermolide is contained within the protein
binding site, it is not surprising that a significant structural modi-
fication at this position decreases the affinity of a ligand for this

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
5251
Table 2
also support the idea that, like the parent molecule discodermo-
lide, the microtubule binding site for the fluorescent discodermo-
lide overlaps with the paclitaxel site on microtubules.
Emission maxima of 4 in different environments
a
Solventa
kmax (nm)
RFIb
We note that 4 is the second type of fluorescent probe based on
discodermolide.14 The earlier probes possess a dansyl group ap-
pended to the C-24 carbon by a spacer that contains 5 or 6 meth-
ylene groups. The activities of these molecules are either identical
or very similar to discodermolide with respect to promotion of
microtubule assembly and cytotoxicity. The high activity is to be
expected since the bulky dansyl group is tethered to the parent
molecule rather than included as an integral part of the structure.
No fluorescence data have been reported for these probes. We
speculate that the environment experienced by the sensitive dan-
syl group is primarily on the exterior of the receptor site.
Dioxane
DMSO
MeCN
EtOH
MeOH
431
439
443
453
460
438
15
12
12
3
1
c
4 with microtubules
—
a
b
c
Excitation at 345 nm.
Relative fluorescence intensity (RFI) at emission maximum.
RFI not meaningful in this context.
In conclusion, a simplified fluorescently-labeled discodermolide
analogue possessing a dimethylaminobenzoyl fluorophore was de-
signed, synthesized, and biologically evaluated for antiproliferative
activity, tubulin assembly activity, and microtubule binding. The
molecule maintained some of the activity of the parent discodermo-
lide, but to a diminished extent. The large increase in fluorescence
intensity that occurs with microtubule binding is indicative of a
hydrophobic local environment for the fluorophore in the disco-
dermolide binding site on tubulin. This probe is suitable for direct
observation of the discodermolide analogue binding to stabilized
microtubules. It should be useful as donor molecules for fluores-
cence resonance energy transfer measurements and for assessing
potential discodermolide-site microtubule stabilizers in vitro.
1.0
PME
Tubulin
0.8
0.6
0.4
0.2
0.0
3. Experimental
400
450
500
550
3.1. General experimental methods
Wavelength (nm)
All chemicals obtained from commercial sources were used
without further purification. The anhydrous reactions were per-
formed under nitrogen or argon. All solvents were of reagent grade
or HPLC grade. Tetrahydrofuran (THF) was distilled over sodium/
benzophenone, and dichloromethane (DCM) was distilled over cal-
cium hydride. The reactions were monitored by the analytical thin
layer chromatography (TLC) plates (Silica Gel 60 GF) and analyzed
with 254 nm UV light, KMnO4 stain, and/or vanillin/sulfuric acid
spray. Preparative thin layer chromatography (PTLC) plates were
purchased from Analtech. All NMR spectral data were obtained
on JEOL Eclipse spectrometer at 500 MHz, Varian Unity or Varian
Inova spectrometer at 400 MHz. Chemical shifts reported as d-val-
ues relative to known solvent residue peaks. High-resolution mass
spectra (HRMS) were obtained in the analytical service in the
Department of Chemistry at Virginia Tech. Known compounds
were prepared through the reported procedures, and the NMR data
of these compounds matched the literature values. All UV–vis
spectroscopy was conducted in HP 8453 UV–vis spectrophotome-
ter equipped with a multi-cell thermostated cuvette holder, and
all fluorescence spectroscopy in a Jobin Yvon Fluorolog 3 spectro-
fluorometer in a thermostatted cell. All compounds were more
than 95% pure as judged by TLC and 1H NMR.
Figure 4. Representative fluorescence spectra of analogue 4 in the presence and
absence of tubulin. The spectra shown are of 2 M 4 in PME with 4% v/v DMSO
alone (solid line) and with 10 M tubulin (dashed line). The excitation wavelength
l
l
was 345 nm. Background fluorescence due to buffer and protein were subtracted.
6
5
4
3
2
1
0
380 400 420 440 460 480 500 520 540
Wavelength, nm
Figure 5. Fluorescence of 4 bound to microtubules in the presence and absence of
paclitaxel. Tubulin (10
90 lM 4 at 37 °C. Paclitaxel was added to a total concentration of 10
l
M) in PME containing 1 mM GTP was incubated with
3.2. Synthesis
lM. The final
concentration of DMSO was 4% v/v. The excitation wavelength was 345 nm. Dotted
line: tubulin only; solid line: tubulin plus 4; dashed line: tubulin plus 4 after
addition of paclitaxel.
3.2.1. (2R,3R,4S,5Z,8S,9R,10R,11S,12S,Z)-9-(tert-
Butyldimethylsilyloxy)-11-(4-methoxybenzyloxy)-3-(O-
methoxymethyl)-2,4,6,8,10,12-hexamethylhexadec-5-enal (5)
To a solution of iodide 816b (87 mg, 0.15 mmol) in ether
(1.74 mL) was added t-BuLi (0.18 mL, 1.7 M in pentane, 0.3 mmol),
followed by MeO-9-BBN (0.3 mL, 1 M in hexane, 0.3 mmol) and
THF (1.74 mL) at ꢀ78 °C. After stirring at rt for 1 h, Cs2CO3 solution
(0.13 mL, 3 M in water, 0.38 mmol), a solution of vinyl iodide 917d
receptor site. Although compound 4 is not a high affinity ligand for
this site, the micromolar dissociation constant should still be suffi-
cient for competition binding assays in vitro. This possibility is
illustrated with in Figure 5. Addition of paclitaxel to microtubules
bound to probe 4 greatly decreased ligand fluorescence. These data

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J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
(67 mg, 0.15 mmol) in DMF (1.4 mL) and Pd(dppf)Cl2 (6.2 mg) were
added successively. The resulting solution was covered with alumi-
num foil and stirred at rt for 20 h. After dilution with water and
ether, the mixture was extracted with ether, washed by brine,
dried over Na2SO4, concentrated and purified by flash chromatog-
raphy (5% EtOAc in hexanes) to afford TBS ether 10 (86 mg, 77%) as
afford acid 11 (431 mg, 68% over 2 steps) as a colorless oil. 1H
NMR (500 MHz, CDCl3) d 3.91 (2H, t, J = 6.1 Hz), 2.58 (2H, t,
J = 6.1 Hz), 0.89 (9H, s), 0.09 (6H, s).
To a solution of acid 11 (166 mg, 0.81 mmol) in DCM (9 mL)
was added 1-chloro-N,N,2-trimethyl-1-propenylamine (0.24 mL,
1.8 mmol). After stirring for 1 h, the solution was concentrated
and the crude acid chloride 12 was dried in vacuo for 30 min.
Meanwhile, LiHMDS (2.7 mL, 1.0 M in THF, 2.7 mmol) was added
dropwise to a solution of methylphosphonic acid bis(2,2,2-trifluo-
roethyl) ester (700 mg, 2.67 mmol) in THF (2 mL) at ꢀ98 °C. After
stirring for 15 min, a solution of the crude acid chloride 12 in
THF (5.8 mL) was added at ꢀ98 °C. The resulting solution was stir-
red for 1 h at ꢀ98 °C before adding NH4Cl solution to quench the
reaction. The solution was then warmed to rt, and more NH4Cl
solution was added. The mixture was extracted with DCM, dried
over Na2SO4, filtered, concentrated and purified by flash chroma-
a colorless oil. ½a D23
ꢁ
+4.0 (c 10.0, CHCl3); 1H NMR (400 MHz, CDCl3)
d 7.28 (2H, d, J = 7.2 Hz), 6.87 (2H, d, J = 8.3 Hz), 5.12 (1H, d,
J = 10.2 Hz), 4.64 (2H, q, J = 6.5 Hz), 4.49 (2H, dd, J = 33.5,
10.7 Hz), 3.80 (3H, s), 3.64 (1H, dd, J = 10.0, 3.8 Hz), 3.52–3.47
(2H, m), 3.41 (3H, s), 3.21 (1H, t, J = 5.8 Hz), 3.14 (1H, t,
J = 5.3 Hz), 2.64–2.49 (1H, m), 2.22 (1H, t, J = 13.3 Hz), 1.95–1.80
(3H, m), 1.80–1.73 (2H, m), 1.63 (3H, s), 1.45–1.23 (4H, m), 1.20–
1.12 (2H, m), 0.99 (3H, d, J = 6.6 Hz), 0.96 (3H, d, J = 6.8 Hz), 0.95–
0.87 (9H, m), 0.94 (9H, s), 0.89 (9H, s), 0.75 (3H, d, J = 6.7 Hz), 0.07
(3H, s), 0.06 (3H, s), 0.03 (6H, s).
TBS ether 10 (86 mg, 0.11 mmol) was dissolved in HCl/EtOH
(1:99, 3.8 mL) at rt and stirred for 20 min. The reaction was
quenched with NaHCO3 and extracted with DCM, dried over
Na2SO4, filtered, and concentrated to give the crude alcohol with-
out purification. To the crude alcohol (50 mg, 0.077 mmol) in
DCM (0.4 mL) and DMSO (0.4 mL) was added Et3N (0.11 mL,
0.77 mmol) and pyridine sulfur trioxide complex (122 mg,
0.77 mmol) at 0 °C. After stirring at 0 °C for 1 h, NH4Cl solution
and water was added. The mixture was extracted with DCM,
washed by water and brine, dried over Na2SO4, filtered, concen-
trated and purified by flash chromatography (15% EtOAc in hex-
anes) to afford aldehyde 5 (33 mg, 65% over 2 steps) as a
colorless oil. 1H NMR (500 MHz, CDCl3) d 9.62 (1H, d, J = 1.2 Hz),
7.27 (2H, d, J = 8.0 Hz), 6.88 (2H, d, J = 8.6 Hz), 4.85 (1H, d,
J = 10.5 Hz), 4.67 (2H, dd, J = 8.2, 7.1 Hz), 4.53 (1H, d, J = 10.8 Hz),
4.43 (1H, d, J = 10.8 Hz), 3.80 (3H, s), 3.62 (1H, dd, J = 8.1, 4.0 Hz),
3.46 (1H, dd, J = 5.2, 3.5 Hz), 3.37 (3H, s), 3.13 (1H, t, J = 5.2 Hz),
2.69–2.59 (2H, m), 2.18 (1H, t, J = 12.4 Hz), 1.95–1.81 (2H, m),
1.77–1.65 (2H, m), 1.58–1.56 (3H, m), 1.52 (1H, ddd, J = 10.3, 7.6,
3.1 Hz), 1.45–1.23 (3H, m), 1.19–1.13 (2H, m), 1.09 (3H, d,
J = 7.0 Hz), 1.01 (3H, d, J = 6.6 Hz), 0.98 (3H, d, J = 6.9 Hz), 0.96
(3H, d, J = 6.8 Hz), 0.93 (9H, s), 0.89 (3H, t, J = 7.2 Hz), 0.72 (3H, d,
J = 6.7 Hz), 0.07 (3H, s), 0.06 (3H, s); 13C NMR (500 MHz, CDCl3) d
202.7, 159.0, 135.7, 131.3, 129.1, 128.6, 113.7, 97.4, 85.3, 84.1,
77.5, 74.5, 56.0, 55.3, 49.5, 39.0, 37.3, 35.9, 35.2, 35.1, 31.1, 29.9,
26.3, 23.1, 18.5, 17.4, 17.2, 14.2, 13.5, 11.4, 9.9, ꢀ3.4, ꢀ3.4; HRMS
(APCI+) calcd for C38H69O6Si [M+H]+ 649.4863, found 649.4865.
tography (30% EtOAc in hexanes) to afford phosphonate 6
(135 mg, 40% over 2 steps) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d 4.50–4.39 (4H, m), 3.90 (2H, t, J = 6.0 Hz), 3.38 (1H, s),
3.34 (1H, s), 2.74 (2H, td, J = 6.0, 0.8 Hz), 0.88 (9H, s), 0.06 (6H,
s); 13C NMR (500 MHz, CDCl3) d 200.7, 122.6 (dd, J = 551.5,
16.6 Hz), 62.5 (dq, J = 75.8, 11.1 Hz), 58.6, 47.2 (d, J = 10.4 Hz),
42.8, 41.7, 25.9, 18.3, ꢀ5.5; HRMS (ESI+) calcd for C14H26F6O5PSi
[M+H]+ 447.1191, found 447.1186.
3.2.3. (8Z,10S,11S,12S,13Z,16S,17R)-17-((2R,3S,4S)-3-
(4-Methoxybenzyloxy)-4-methyloctan-2-yl)-11-(O-
methoxymethyl)-2,2,3,3,10,12,14,16,19,19,20,20-dodecamethyl-
4,18-dioxa-3,19-disilahenicosa-8,13-dien-7-one (13)
K2CO3 (60 mg, 0.43 mmol) and 18-crown-6 (228 mg,
0.86 mmol) were dissolved in toluene (0.6 mL) and HMPA
(0.06 mL) at rt and the mixture was stirred for 3 h before adding
a solution of aldehyde 5 (38 mg, 0.07 mmol) and phosphonate 6
(103 mg, 0.23 mmol) in toluene (0.6 mL) and HMPA (0.06 mL) at
ꢀ10 °C. After stirring at ꢀ5 °C for 18 h, NH4Cl solution was added
to quench the reaction and the mixture was extracted with EtOAc,
washed with brine, dried over Na2SO4, filtered, concentrated and
purified by flash chromatography (20% EtOAc in hexanes) to afford
the new TBS ether 13 (51 mg, 85%) as a colorless oil. 1H NMR
(500 MHz, CDCl3) d 7.27 (2H, d, J = 8.5 Hz), 6.87 (2H, d, J = 8.7 Hz),
6.15–6.08 (2H, m), 4.90 (1H, d, J = 10.3 Hz), 4.73 (2H, dd, J = 36.7,
6.7 Hz), 4.48 (2H, dd, J = 36.0, 10.8 Hz), 3.95–3.84 (2H, m), 3.80
(3H, s), 3.75–3.64 (1H, m), 3.49–3.46 (1H, m), 3.41 (3H, s), 3.25
(1H, d, J = 7.3, 3.8 Hz), 3.13 (1H, t, J = 5.4 Hz), 2.73–2.55 (2H, m),
2.54–2.42 (1H, m), 2.16 (1H, t, J = 12.5 Hz), 1.95–1.80 (2H, m),
1.74–1.65 (2H, m), 1.57 (3H, s), 1.51 (1H, ddd, J = 10.7, 8.0,
2.7 Hz), 1.45–1.25 (3H, m), 1.20–1.13 (2H, m), 1.02 (3H, d,
J = 6.9 Hz), 0.98 (3H, d, J = 6.9 Hz), 0.96 (3H, d, J = 6.8 Hz), 0.96
(3H, d, J = 6.6 Hz), 0.93 (9H, s), 0.89 (3H, t, J = 7.3 Hz), 0.87 (9H,
s), 0.72 (2H, d, J = 6.7 Hz), 0.07 (3H, s), 0.06 (3H, s), 0.04 (3H, s),
0.04 (3H, s); 13C NMR (500 MHz, CDCl3) d 199.6, 159.0, 151.1,
133.0, 131.4, 130.0, 129.0, 125.7, 113.7, 97.6, 86.9, 85.6, 77.5,
74.6, 59.1, 56.2, 55.3, 47.2, 38.8, 36.7, 36.2, 35.9, 35.6, 35.4, 30.9,
29.9, 29.7, 26.3, 25.9, 23.1, 18.5, 18.3, 17.5, 17.3, 16.9, 14.2, 14.0,
11.4, ꢀ3.4, ꢀ3.5, ꢀ5.4, ꢀ5.4; HRMS (ESI+) calcd for C48H88O7Si2Na
[M+Na]+ 855.5966, found 855.5944.
3.2.2. 1-(Bis(2,2,2-trifluoroethyl)phosphoryl)-4-(tert-
butyldimethylsilyloxy)butan-2-one (6)
To a solution of 1,3-propanediol (3 mL, 40 mmol) in THF
(60 mL) was added NaH (1.2 g, 60%, 20 mmol) at rt. After stirring
for 2.5 h, TBSCl (630 mg, 4.3 mmol) was added and the mixture
was stirred overnight before the addition of NaHCO3 solution in
MeOH. The solution was then extracted with DCM, washed with
water and brine, dried over Na2SO4, filtered, concentrated and
purified by flash chromatography (30% EtOAc in hexanes) to afford
2-((tert-butyldimethylsilyl)oxy)ethanol ether (800 mg, 90% based
on TBSCl) as a colorless oil. To a solution of this TBS ether
(590 mg, 3.11 mmol) in DCM (29 mL) was added NaHCO3 and
Dess–Martin periodinane at rt. After stirring for 30 min, the
solution was concentrated, diluted with hexane to participate
byproduct, filtered and concentrated carefully to give crude
2-((tert-butyldimethylsilyl)oxy)acetaldehyde. A solution of this
crude aldehyde in t-BuOH (13 mL) and 2-methyl-2-butene
(1.5 mL) was treated with a solution of NaClO2 (500 mg, 5.5 mmol)
and NaH2PO4 (1.85 g, 15.4 mmol) in water (12 mL) dropwise. After
stirring for 1 h, the reaction was partitioned between brine and
DCM. The organic layer was dried over Na2SO4, filtered, concentrated
and purified by flash chromatography (30% EtOAc in hexanes) to
3.2.4. (8Z,10S,11S,12S,13Z,16S,17R)-17-((2R,3S,4S)-3-
Hydroxy-4-methyloctan-2-yl)-11-(methoxymethoxy)-
2,2,3,3,10,12,14,16,19,19,20,20-dodecamethyl-4,18-dioxa-3,19-
disilahenicosa-8,13-dien-7-one (14)
To a vigorously stirred solution of PMB ether 13 (45 mg,
0.06 mmol) in DCM (2.7 mL) and pH 7 buffer (0.3 mL) was added
DDQ (25 mg, 0.11 mmol) at 0 °C. After stirring for 1.5 h, NaHCO3
solution was added and the mixture was extracted with DCM,
washed by Na2S2O3 solution, water and brine, dried over Na2SO4,

J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
5253
filtered, concentrated and purified by flash chromatography (20%
EtOAc in hexanes) to give alcohol 14 (28 mg, 75%) as a white solid.
1H NMR (500 MHz, CDCl3) d 6.21–6.08 (2H, m), 4.92 (1H, d,
J = 10.2 Hz), 4.73 (2H, dd, J = 24.2, 6.7 Hz), 3.90 (2H, qt, J = 10.3,
6.6 Hz), 3.71 (1H, ddd, J = 9.0, 6.9, 3.7 Hz), 3.65 (1H, t, J = 3.9 Hz),
3.41 (3H, s), 3.26 (2H, dd, J = 7.2, 3.8 Hz), 2.70 (1H, dt, J = 15.9,
6.7 Hz), 2.60 (1H, dt, J = 15.9, 6.4 Hz), 2.54–2.45 (1H, m), 2.14
(1H, t, J = 12.1 Hz), 1.90–1.79 (3H, m), 1.70 (1H, br s), 1.66–1.61
(1H, m), 1.59 (3H, d, J = 0.9 Hz), 1.55–1.50 (1H, m), 1.43–1.26
(3H, m), 1.24–1.15 (1H, m), 1.13–1.05 (1H, m), 1.03 (3H, d,
J = 6.9 Hz), 0.97 (3H, d, J = 6.6 Hz), 0.92 (9H, s), 0.90 (3H, t,
J = 7.3 Hz), 0.89 (3H, d, J = 7.0 Hz), 0.87 (9H, s), 0.84 (3H, d,
J = 6.7 Hz), 0.74 (3H, d, J = 6.9 Hz), 0.10 (3H, s), 0.10 (3H, s), 0.05
(3H, s), 0.04 (3H, s); 13C NMR (500 MHz, CDCl3) d 199.6, 151.0,
133.3, 130.0, 125.8, 97.8, 87.1, 79.7, 79.1, 59.0, 56.2, 47.2, 36.9,
36.4, 36.2, 36.0, 35.8, 35.3, 31.9, 29.2, 26.2, 25.9, 23.2, 23.2, 18.4,
18.3, 17.5, 17.1, 16.0, 14.5, 14.2, 8.8, ꢀ3.2, ꢀ4.1, ꢀ5.4, ꢀ5.4; HRMS
(ESI+) calcd for C40H80O6Si2Na [M+Na]+ 735.5391, found 735.5401.
ꢀ5.5; HRMS (ESI+) calcd for C41H83NO7Si2Na [M+Na]+ 780.5606,
found 780.5615.
3.2.7. (3S,4Z,6S,7S,8S,9Z,12S,13R,14S,15S,16S)-15-
(Carbamoyloxy)-3,7,13-trihydroxy-6,8,10,12,14,16-
hexamethylicosa-4,9-dienyl 3-(dimethylamino)benzoate (4)
TBS ether 16 (18 mg, 0.024 mmol) was dissolved in MeOH
(3.6 mL). After stirring for 15 min, HCl solution (3.6 mL, 4 M in
water) was added to the solution in small portions (0.15 mL per
10 min in 4 h) at rt before NaHCO3 was added carefully to quench
reaction. The solution was extracted with EtOAc, washed with
brine, dried over Na2SO4, filtered and concentrated. The residue
was purified by flash chromatography (80% EtOAc in hexanes) to
give alcohol 17 (10 mg, 88%) as a colorless oil.
To
mol) and DMAP (1.5 mg, 12.3
added solution of alcohol 17 (4.2 mg, 8.7
(0.6 mL), followed by EDCI (3.9 mg, 20 mol). After stirring at rt
a
solution of 3-(dimethylamino)benzoic acid (2.8 mg,
mol) in DCM (0.6 mL) was
mol) in DCM
17
l
l
a
l
l
for 12 h, water was added and the mixture was extracted with
EtOAc. The organic layer was then washed by brine, dried over
Na2SO4, filtered, concentrated and purified by flash chromatogra-
phy (50% EtOAc in hexanes) and to give 4 (1 mg, 18%) as a colorless
3.2.5. (5S,6S,7R,8R,9S,11Z,13S,14S,15S,16Z)-8,20-Bis(tert-
butyldimethylsilyloxy)-14-(methoxymethoxy)-5,7,9,11,13,15-
hexamethyl-18-oxoicosa-11,16-dien-6-yl carbamate (15)
To a solution of alcohol 14 (28 mg, 0.04 mmol) in DCM (3.7 mL)
oil. ½a 2D5
ꢁ
ꢀ12.5 (c 0.2, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.40 (1H,
was added Cl3CCONCO (24
lL, 0.2 mmol) at rt. After stirring for
dd, J = 2.6, 1.4 Hz), 7.35 (1H, dd, J = 7.6, 1.2 Hz), 7.29 (1H, t,
J = 7.9 Hz), 7.00 (1H, ddd, J = 8.2, 2.7, 0.9 Hz), 5.60 (1H, d,
J = 10.3 Hz), 5.42 (1H, dd, J = 10.7, 9.2 Hz), 5.03 (1H, d,
J = 10.0 Hz), 4.65 (1H, t, J = 5.9 Hz), 4.53 (1H, td, J = 8.6, 3.6 Hz),
4.42 (2H, dd, J = 7.3, 5.6 Hz), 3.20 (1H, t, J = 5.7 Hz), 3.14 (1H, dd,
J = 7.7, 3.8 Hz), 2.99 (6H, s), 2.72–2.67 (1H, m), 2.52–2.37 (2H, m),
2.18–2.08 (2H, m), 1.97–1.89 (3H, m), 1.88–1.78 (2H, m), 1.74
(1H, dd, J = 11.6, 2.6 Hz), 1.59 (3H, s), 1.46–1.41 (2H, m), 1.22–
1.11 (4H, m), 1.07 (3H, d, J = 7.0 Hz), 0.95 (3H, d, J = 6.6 Hz), 0.93
(3H, t, J = 7.2 Hz), 0.91–0.89 (6H, m), 0.78 (3H, d, J = 6.6 Hz); 13C
NMR (500 MHz, CDCl3) d 167.6, 159.1, 150.8, 133.0, 132.9, 132.1,
130.7, 130.2, 128.8, 117.4, 117.0, 112.9, 79.2, 76.4, 64.1, 61.6,
48.3, 39.5, 36.8, 36.5, 36.3, 36.1, 35.6, 34.4, 32.8, 30.4, 29.1, 22.7,
22.2, 18.2, 16.4, 15.3, 13.2, 13.0, 8.3; HRMS (ESI+) calcd for
1.5 h, the solution was loaded onto a neutral Al2O3 plug and left
for 2 h. The Al2O3 plug was then flushed with EtOAc (100 mL)
and the solution was concentrated and purified by flash chroma-
tography (30% EtOAc in hexanes) to afford carbamate 15 (24 mg,
82%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d 6.17–6.06
(2H, m), 4.86 (1H, d, J = 10.3 Hz), 4.73 (2H, dd, J = 33.8, 6.7 Hz),
4.69 (2H, br s), 4.61 (1H, dd, J = 7.2, 4.6 Hz), 3.91 (2H, t,
J = 6.8 Hz), 3.68 (1H, dqd, J = 10.3, 6.9, 3.6 Hz), 3.40 (3H, s), 3.40–
3.38 (1H, m), 3.22 (1H, dd, J = 7.8, 3.5 Hz), 2.67 (2H, td, J = 6.8,
2.7 Hz), 2.50–2.41 (1H, m), 2.15 (1H, t, J = 12.4 Hz), 1.96–1.87
(2H, m), 1.73–1.62 (2H, m), 1.59 (3H, s), 1.45–1.23 (4H, m), 1.17–
1.03 (2H, m), 1.01 (3H, d, J = 7.0 Hz), 0.94 (3H, d, J = 6.6 Hz), 0.91
(9H, s), 0.90–0.85 (9H, m), 0.86 (9H, s), 0.69 (3H, d, J = 6.8 Hz),
0.07 (3H, s), 0.06 (3H, s), 0.04 (3H, s), 0.04 (3H, s); 13C NMR
(500 MHz, CDCl3) d 199.4, 157.1, 151.1, 133.4, 129.6, 125.7, 97.8,
87.2, 79.9, 77.1, 59.1, 56.2, 47.1, 37.4, 36.6, 36.3, 36.0, 34.9, 34.9,
31.1, 29.4, 26.2, 25.9, 23.0, 22.9, 18.5, 18.3, 17.7, 17.0, 16.1, 14.1,
13.0, 10.5, ꢀ3.4, ꢀ3.6, ꢀ5.4; HRMS (ESI+) calcd for C41H81NO7Si2Na
[M+Na]+ 778.5449, found 778.5462.
C
36H60N2O7Na [M+Na]+ 655.4298, found 655.4306.
3.3. Biological experiments
3.3.1. Handling and storage
Stock solutions of analogue 4 were made in DMSO and stored at
ꢀ20 °C until use. Concentrations were determined spectrophoto-
3.2.6. (5S,6S,7R,8R,9S,11Z,13S,14S,15S,16Z,18S)-8,20-Bis(tert-
butyldimethylsilyloxy)-18-hydroxy-14-(O-methoxymethyl)-
5,7,9,11,13,15-hexamethylicosa-11,16-dien-6-yl carbamate (16)
A solution of ketone 15 (15 mg, 0.02 mmol) in toluene (3.7 mL)
metrically using a molar absorptivity of
as determined in the SUNY laboratory.
e
345 nm = 1800 Mꢀ1 cmꢀ1
3.3.2. Antiproliferative activities
was treated with K-selectride™ (40
lL, 1 M in THF, 0.04 mmol) at
Antiproliferative activity against the A2780 cell line24 was
determined as described previously.
ꢀ78 °C. After stirring for 2 h, one drop of AcOH was added to
quench the reaction. The solution was diluted with EtOAc, washed
with water and brine, dried over Na2SO4, filtered and concentrated.
The residue was purified by flash chromatography (15% EtOAc in
hexanes) to give alcohol 16 (14 mg, 90%) as a colorless oil. 1H
3.3.3. Tubulin isolation
Tubulin was isolated from bovine brains by two cycles of tem-
perature-dependent polymerization and depolymerization fol-
lowed by phosphocellulose ion-exchange chromatography.25 The
protein containing fractions were then pooled, drop frozen, and
stored on liquid nitrogen until use. Prior to use, frozen aliquots
were gently thawed and desalted into PME buffer (0.1 M Pipes,
1 mM MgSO4, 2 mM EDTA, pH 6.90) The concentration was deter-
mined spectrophotometrically using an extinction coefficient of
1.23 (mg/mL)ꢀ1 cmꢀ1 at 278 nm.26
NMR (500 MHz, CDCl3)
d 5.49–5.38 (2H, m), 5.03 (1H, d,
J = 10.1 Hz), 4.63 (2H, s), 4.62–4.54 (3H, m), 3.91 (1H, dt, J = 10.3,
5.3 Hz), 3.88–3.81 (1H, m), 3.43 (1H, dd, J = 5.9, 3.0 Hz), 3.39 (3H,
s), 3.14–3.07 (1H, m), 2.81 (1H, dd, J = 13.8, 6.8 Hz), 2.58–2.50
(1H, m), 2.19 (1H, t, J = 12.3 Hz), 1.98–1.90 (2H, m), 1.79–1.66
(4H, m), 1.64 (3H, s), 1.44–1.27 (4H, m), 1.19–1.04 (2H, m), 1.02
(3H, d, J = 6.9 Hz), 0.95 (3H, d, J = 6.7 Hz), 0.92 (9H, s), 0.90 (9H,
s), 0.90–0.85 (9H, m), 0.73 (3H, d, J = 6.8 Hz), 0.08 (3H, s), 0.07
(3H, s), 0.07 (3H, s), 0.06 (3H, s); 13C NMR (500 MHz, CDCl3) d
157.1, 134.3, 132.9, 131.8, 130.3, 98.2, 87.3, 80.0, 77.2, 68.2, 62.4,
56.1, 38.8, 37.3, 36.4, 35.7, 35.1, 35.0, 31.1, 29.7, 29.4, 26.2, 25.9,
23.1, 23.0, 18.5, 18.3, 18.2, 16.6, 16.1, 14.1, 13.1, 10.5, ꢀ3.4, ꢀ3.7,
3.3.4. EC50 measurement
The EC50 is defined as the concentration of the molecule re-
quired to assemble the protein to 50% of the activity at saturation.
This was quantified for the discodermolide analogues in reference

5254
J. Qi et al. / Bioorg. Med. Chem. 19 (2011) 5247–5254
4. Paterson, I.; Delgado, O.; Florence, G. J.; Lyothier, I.; O’Brien, M.; Scott, J. P.;
Sereinig, N. J. Org. Chem. 2005, 70, 150.
5. Smith, A. B., III; Freeze, B. S. Tetrahedron 2008, 64, 261.
to the standard paclitaxel. Analogue 4 was evaluated by light scat-
tering.27 Samples containing 5
M soluble tubulin in PME with
l
0.1 mM GTP were equilibrated to 37 °C and polymerization in-
duced by addition of varying concentrations of drug dissolved in
DMSO to a final concentration of 10% v/v. Polymerization activity
was monitored as apparent absorption increase at 350 nm, taking
the plateau value with the baseline subtracted to be polymeriza-
tion extent. The plateau value was graphed on a linear axis as a
function of drug concentration and the EC50 value extracted from
a square hyperbolic fit in SigmaPlot 10.0 (Systat Software Inc.,
San Jose, CA).
6. (a) Mickel, S. J.; Niederer, D.; Daeffler, R.; Osmani, A.; Kuesters, E.; Schmid, E.;
Schaer, K.; Gamboni, R.; Chen, W.; Loeser, E.; Kinder, F. R., Jr.; Konigsberger, K.;
Prasad, K.; Ramsey, T. M.; Repic, O.; Wang, R.-M.; Florence, G.; Lyothier, I.;
Paterson, I. Org. Process Res. Dev. 2004, 8, 122; (b) Mickel, S. J.; Sedelmeier, G. H.;
Niederer, D.; Daeffler, R.; Osmani, A.; Schreiner, K.; Seeger-Weibel, M.; Berod,
B.; Schaer, K.; Gamboni, R.; Chen, S.; Chen, W.; Jagoe, C. T.; Kinder, F. R., Jr.; Loo,
M.; Prasad, K.; Repic, O.; Shieh, W.-C.; Wang, R.-M.; Waykole, L.; Xu, D. D.; Xue,
S. Org. Process Res. Dev. 2004, 8, 92; (c) Mickel, S. J.; Sedelmeier, G. H.; Niederer,
D.; Schuerch, F.; Grimler, D.; Koch, G.; Daeffler, R.; Osmani, A.; Hirni, A.; Schaer,
K.; Gamboni, R.; Bach, A.; Chaudhary, A.; Chen, S.; Chen, W.; Hu, B.; Jagoe, C. T.;
Kim, H.-Y.; Kinder, F. R., Jr.; Liu, Y.; Lu, Y.; McKenna, J.; Prashad, M.; Ramsey, T.
M.; Repic, O.; Rogers, L.; Shieh, W.-C.; Wang, R.-M.; Waykole, L. Org. Process Res.
Dev. 2004, 8, 101; (d) Mickel, S. J.; Sedelmeier, G. H.; Niederer, D.; Schuerch, F.;
Koch, G.; Kuesters, E.; Daeffler, R.; Osmani, A.; Seeger-Weibel, M.; Schmid, E.;
Hirni, A.; Schaer, K.; Gamboni, R.; Bach, A.; Chen, S.; Chen, W.; Geng, P.; Jagoe, C.
T., ; Kinder, F. R., Jr.; Lee, G. T.; McKenna, J.; Ramsey, T. M.; Repic, O.; Rogers, L.;
Shieh, W.-C.; Wang, R.-M.; Waykole, L. Org. Process Res. Dev. 2004, 8, 107; (e)
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K.; Daeffler, R.; Osmani, A.; Bixel, D.; Loiseleur, O.; Cercus, J.; Stettler, H.; Schaer,
K.; Gamboni, R.; Bach, A.; Chen, G.-P.; Chen, W.; Geng, P.; Lee, G. T.; Loeser, E.;
McKenna, J.; Kinder, F. R., Jr.; Konigsberger, K.; Prasad, K.; Ramsey, T. M.; Reel,
N.; Repic, O.; Rogers, L.; Shieh, W.-C.; Wang, R.-M.; Waykole, L.; Xue, S.;
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3.3.5. Microtubule binding properties
Because the analogue showed a fluorescence increase upon
binding to tubulin, the increase in fluorescence at 440 nm was used
to measure binding. Dissociation constants were assessed by titra-
tion of ligand fluorescence at 25 °C using a fixed concentration of
ligand and varying concentrations of the receptor, GMPcPP micro-
tubules (prepared as described by Vulevic and Correia)28 in PME
buffer containing 0.1 mM GTP and 10% DMSO. The data were fit
to the equation
f ¼ Fmax ꢂ ½MTꢁ=ðKd þ ½MTꢁÞ
where f is the fluorescence intensity at the emission maximum of
the ligand at each concentration, Fmax is the maximum fluorescence,
[MT] is the total concentration of tubulin in GMPcPP-microtubules,
and Kd is the dissociation constant. Saturation was not achieved for
compound 4; therefore the value for Fmax was obtained from double
reciprocal plots. At the end of the titration, competition by paclit-
axel was assessed in the following manner: paclitaxel in DMSO
was added to a total concentration equal to the concentration of
tubulin and a fluorescence spectrum was taken.
Competition with paclitaxel was also assessed with GTP-tubu-
lin. Tubulin (10 lM) in PME buffer containing 1 mM GTP was
equilibrated to 37 °C in the spectrofluorometer and an emission
spectrum was collected. Excess probe in DMSO was added to a
12. Stryer, L. Ann. Rev. Biochem. 1978, 47, 819.
13. Souto, A. A.; Acuna, A. U.; Andreu, J. M.; Barasoain, I.; Abal, M.; Amat-Guerri, F.
Angew. Chem., Int. Ed. 1995, 34, 2710.
14. Smith, A. B., III; Rucker, P. V.; Brouard, I.; Freeze, B. S.; Xia, S.; Horwitz, S. B. Org.
Lett. 2005, 7, 5199.
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Kingston, D. G. I.; Bane, S. Biochemistry 1996, 35, 14173; (b) Han, Y.; Malak, A.
G.; Chaudhary, A. G.; Chordia, M. D.; Kingston, D. G. I.; Bane, S. Biochemistry
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total concentration of 90
an additional 30 min. After collection of the emission spectrum,
paclitaxel in DMSO was added to final concentration of
10 M and 4% DMSO. The emission spectrum was collected
lM and the sample was incubated for
a
l
5 min after the addition of paclitaxel. The excitation wavelength
was 345 nm.
Acknowledgments
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We are grateful for support of this work by NIH Grant CA-69571
and by NSF Grant CHE-0619382 for purchase of the Bruker Avance
600 spectrometer. We thank William Bebout (VT) for mass spectro-
scopic determinations, and Peggy Brodie (VT) for A2780 cytotoxic-
ity assays.
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