Synthesis and biological evaluation of fluorescently labeled epothilone analogs for tubulin binding studies

Article abstract of DOI:10.1016/j.tet.2003.10.024

The two fluorescently labeled epothilones 14 and 15 have been synthesized using a modification of Nicolaou's macrolactonization and Stille coupling strategy. The cytotoxicities of the compounds were 6.1 and 2.7 μg/mL, respectively, against the A2870 ovari

Full text of DOI:10.1016/j.tet.2003.10.024

Tetrahedron 59 (2003) 9979–9984
Synthesis and biological evaluation of fluorescently labeled
epothilone analogs for tubulin binding studies
Thota Ganesh,a Jennifer K. Schilling,a Radha K. Palakodety,a Rudravajhala Ravindra,b
Natasha Shanker,b Susan Baneb and David G. I. Kingstona
,
*
aDepartment of Chemistry, Virginia Polytechnic Institute and State University, 3111 Hahn Hall, MC 0212, Blacksburg, VA 24061, USA
bDepartment of Chemistry, State University of New York, Binghamton, NY 13902, USA
Received 12 August 2003; revised 9 October 2003; accepted 9 October 2003
Abstract—The two fluorescently labeled epothilones 14 and 15 have been synthesized using a modification of Nicolaou’s
macrolactonization and Stille coupling strategy. The cytotoxicities of the compounds were 6.1 and 2.7 mg/mL, respectively, against the
A2870 ovarian cancer cell line, and 0.5 and 1.0 mg/mL, respectively, against the PC-3 prostate cancer cell line. The critical concentration of
tubulin was 0.5 and 1.0 mM in the presence of 14 and 15, respectively, compared with 0.3 mM for paclitaxel. The fluorescent properties of the
two molecules in solution and bound to microtubules are described.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
An exciting new discovery in the cancer chemotherapy area
has been the emergence of the epothilones as potential new
chemotherapeutic agents. Though the gross structure was
revealed in a German patent by Hofle et al. 1993,1 scientific
interest in them was greatly stimulated by the discovery of
their tubulin polymerization activity in 1995.2 Studies of the
bioactivity of epothilones A and B have shown that they
stabilize microtubules in the same way as paclitaxel, but that
they were superior to paclitaxel in treating vinblastin-resistant
CCRF-CEM tumors in mice.3 Also epothilone D was curative
against paclitaxel-resistant CCRF-CEM tumors.4 The chem-
istry, biology and structure activity relationships of the
epothilones have been extensively reviewed.5,6
Not only do epothilones act as tubulin-polymerization
agents in the same way that paclitaxel does, but they also
compete for the same binding site on the polymer, since they
act as competitive inhibitors for the binding of [3H]
paclitaxel to tubulin polymer.7 Recently, a bridged pacli-
taxel was prepared as a proposed paclitaxel–epothilone
hybrid construct. It was shown to promote tubulin assembly
nearly as well as paclitaxel, although it was significantly less
cytotoxic that either compound alone.8 This finding was
taken as lending support to the hypothesis that paclitaxel
and epothilone share a common pharmacophore, but the
activity differences noted suggest that critical aspects of the
pharmacophore have yet to account for ligand–protein
binding. For this reason a study of the interaction of the
epothilones with tubulin polymer will not only provide
information on the conformation of the epothilones on the
tubulin polymer, but may also well provide further
Keywords: epothilone; tubulin; fluorescence; bioactivity.
*
Corresponding author. Tel.: þ1-5402316570; fax: þ1-5402317702;
e-mail: dkingston@vt.edu
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.10.024

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T. Ganesh et al. / Tetrahedron 59 (2003) 9979–9984
information on the nature of the paclitaxel tubulin
interaction.
envisioned in two steps, namely coupling of 7 or 8 with
m-nitrobenzoic acid, followed by reduction of the nitro
group. The EDCI (ethyl-(3-dimethylaminopropyl)carbodi-
imide hydrochloride) coupling of epothilone analog 7 with
3-nitrobenzoic acid at room temperature provided the
desired derivative 10 in nearly 60% yield, but reduction of
the nitro group to the amino group under various
hydrogenation conditions only gave complex mixtures of
products. It was clear from the proton NMR spectra of the
crude product mixture that the double bonds and/or the
lactone ring were being affected, and we were unable to find
hydrogenation conditions that were selective for the nitro
group. Since the supply of compound 10 was exhausted by
these preliminary experiments, we elected to pursue a
protecting group strategy, starting with N-protected benzoic
acids. Most of the N-protecting groups require strongly
acidic or strongly basic reagents, for their deprotection.19 Of
the available amino protecting groups, the trimethylsilyl-
ethoxycarbonyl (Teoc) and fluorenemethyloxycarbonyl
(Fmoc) groups require comparably mild conditions
for their deprotection.19 The coupling of 3-(N-
trimethylsilylethoxycarbonyl) amino benzoic acid, prepared
from 3-aminobenzoic acid with succinimidyl Teoc in
aqueous dioxane,20 with 7 furnished 11 in nearly 70%
yield. Regrettably, however, deprotection posed unprece-
dented problems. Deprotection of 11 with HF·Py, in
pyridine, at 0–608C gave only TBS deprotected product
(12), with the N-Teoc group intact. With TBAF at 08C, the
result was the same, and higher temperatures gave a
complex mixture. Evidently the lactone ring of epothilone
11 was not compatible with TBAF at high temperatures
(Scheme 1).
Knowledge of the molecular conformation helps in the
development of drugs with the necessary three-dimensional
features to bind to the protein target, but with minimal
complexity in their structures. Although an X-ray structure
of epothilone B has been published,9 it does not define the
structure of the molecule in the bound state on tubulin.
Recently, the Taylor group investigated the conformational
properties of epothilones in solution, based on compu-
tational and 2D NMR methods. They concluded that
epothilone A and B prefer two distinct conformations,10
but this work did not address the conformation of epothilone
on the tubulin polymer. We thus elected to approach the
question of the interaction of epothilone and tubulin through
fluorescence spectroscopic studies using the FRET tech-
nique and a REDOR NMR study, similar to the previous
studies we have done for paclitaxel.11
Fluorescence spectroscopy is widely used to investigate
ligand–receptor interactions in biological systems.12–14
The studies are performed using fluorescently labeled
molecules. In this paper we describe the synthesis and
cytotoxicity of epothilone analogs and two of their
fluorescent congeners. Based on previous structure–activity
studies of epothilones,5,6 structural changes at C21 and C26 are
less detrimental to their activity than changes at other
locations, and so we envisioned attaching the fluorescent
labels at these sites. Our synthesis was based primarily on
Nicolaou’s macrolactonization and Stille coupling strategy.15
In view of the difficulties encountered in the synthesis of the
free amino derivative, we elected to prepare the N,N-
dimethylamino benzoate derivatives as our first target
compounds for fluorescence studies.
2. Results and discussion
The synthesis of advanced precursor 4 was accomplished
from the fragments 1, 2, and 3, which were in turn prepared
from commercially available starting materials using
literature procedures.15–17
The analogs 7 and 8 were subjected to EDCI coupling with
3-(N,N-dimethylamino)benzoic acid to furnish TBS ether
13 and compound 14. Deprotection of 13 with HF·Py in
pyridine at 08C provided 15 in 92% yield.
The vinyl iodide (4) on coupling with stannanes, 515 and 6
(4.5 equiv.) in the presence of Pd(AcCN)2Cl2 provided the
epothilone analogs 7 and 8,18 respectively.
The cytotoxicities of the epothilone analogs 8, 9, 14, and 15
were determined towards the A2780 ovarian cancer and
PC-3 prostate cancer cell lines (Table 1). Compound 8 was
the most cytotoxic, with an activity comparable to that of
paclitaxel in the A2780 cell line. The 21-hydroxyl
derivative 9 was less active, as were the fluorescent
derivatives 14 and 15. Although these analogs are less
cytotoxic than most of the naturally occurring epothilones,
their diminished cytotoxicity should not prove a barrier to
carrying out the proposed binding studies.
Having synthesized the epothilone analogs with a C26
hydroxyl group, incorporation of the fluorescent tags
remained. We decided to use the 3-aminobenzoyl or 3-
(N,N-dimethylamino)benzoyl group as fluorescent tags,
which are the smallest organic fluorophores routinely used
in biological systems. Such environmentally sensitive
groups on paclitaxel have been successfully used to monitor
ligand binding to microtubules, to probe the polarity of
the binding site environment, and as donor fluorophores
in measurements of FRET.11 The manipulation was
Table 1. Cytotoxicities of epothilones 8, 9, 14, and 15
Compound
IC50, A2780 (mM)
IC50, PC-3 (mM)
Paclitaxel
8
9
0.23
0.59
4.0
0.05
0.25
1.1
0.50
1.0
14
15
6.1
2.7

T. Ganesh et al. / Tetrahedron 59 (2003) 9979–9984
9981
Scheme 1. (a) Pd(AcCN)2Cl2, DMF, rt (69% for 7, 80% for 8). (b) 7, 3-(N,N-dimethylamino)benzoic acid, EDCl, DMAP, CH2Cl2, (13, 79%) 8, 3 (N,N-
dimethylamino)benzoic acid, EDCl, DMAP, CH2Cl2, (14, 54%). (c) 13, HF–Py in Py, THF, 08C, 92–95%.
All compounds induced tubulin to assemble into normal
microtubules, as confirmed by electron microscopy. Pacli-
taxel binding to polymerized tubulin affects the confor-
mation of the protein in a way that favors tubulin assembly,
i.e. by increasing the equilibrium constant for polymer
growth (Kp). The reciprocal of the critical concentration is a
very close approximation of Kp.21 The critical concentration
of tubulin in the presence of 14 and 15 was 0.5 and 1.0 mM,
respectively, compared to 0.3 mM for paclitaxel. These data
indicate that microtubule binding by these ligands and
paclitaxel affect the conformation of tubulin in the same
manner.
The fluorescent properties of 14 and 15 were examined as a
function of solvent and of microtubule binding. The Stokes
shift was a linear function of solvent polarity (ET30). The
optical properties of the fluorophore can therefore, be
described by general solvent effects.22 The emission
spectrum of 15 in the presence of excess polymerized
tubulin is shown in Figure 1. The emission maximum of the
microtubule-bound species was similar to that of the
fluorophore in DMSO, indicating that the fluorophore’s
environment on the microtubule is of intermediate polarity.
Microtubule binding also greatly increased the quantum
yield of the fluorophore; this property will be useful for
quantitative analysis of the microtubule binding parameters
of the fluorescent epothilone and for FRET studies.
3. Conclusions
We have synthesized two pairs of epothilone analogs with
their fluorescent conjugates (8, 14 and 9, 15) for
comparative fluorescent study; the environment of the
fluorophore on the microtubule is in a region of intermediate
polarity.
Figure 1. Emission spectrum of 15 bound to microtubules. Tubulin
(15 mM) in buffer (0.1 M PIPES, 1 mM MgSO4, 2 mM EGTA, 0.1 mM
GTP, pH 6.90) was assembled by incubation at 378C for 30 min. Compound
15 in DMSO was added to a final concentration of 0.3 mM. The emission
spectrum is shown (solid curve). The emission spectrum of 30 mM 15 in
ethanol is shown for comparison (dashed curve). The excitation wavelength
was 320 nm, and the background has been subtracted in each spectrum.
Comparable results were obtained with 14.
4. Experimental
4.1. General experimental procedures
1H and 13C NMR spectra were obtained on Varian Unity
400 or JEOL Eclipse 500 spectrometers in CDCl3 at 400 and

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T. Ganesh et al. / Tetrahedron 59 (2003) 9979–9984
100 MHz or at 500 and 125 MHz, respectively. HRFAB and
LRFAB mass spectra were obtained on a JEOL HX-110
instrument. NMR and other characterization data for known
compounds were identical with reported values.
16.0, 13.8, 25.2. HRFABMS m/z 638.35620; calcd for
C33H56NO7SSi, 638.3547.
4.1.5. Synthesis of 8. Treatment of 4 with thiazole 6 by the
same procedure as described above for reaction of 4 and 5,
furnished 818 with some minor tin impurities (7.5 mg, 80%).
[a]D¼225.08 (c 0.185, CHCl3). 1H NMR (400 MHz) d 6.96
(s, 1H), 6.60 (s, 1H), 5.44 (dd, J¼7, 5 Hz, 1H), 5.24 (dd,
J¼7, 2 Hz, 1H), 4.30 (m, 1H), 4.15 (dd, J¼13, 2 Hz, 1H),
4.06 (dd J¼13, 2 Hz, 1H), 3.69 (bs, 2H), 3.19 (qd, J¼6.8,
3 Hz, 1H), 3.01 (bs, 1H), 2.68 (s, 3H), 2.52 (ddd, J¼16, 7,
7 Hz, 1H), 2.45 (dd, J¼15, 10 Hz, 1H), 2.36–2.30 (m, 3H),
2.10 (m, 1H), 2.06 (s, 3H), 1.80 (bs, 1H), 1.75 (s, 3H), 1.35
(s, 3H), 1.18 (d, J¼6.8 Hz, 3H), 1.06 (s, 3H), 1.01 (d,
J¼7.2 Hz, 3H). 13C NMR (100 MHz) d 220.9, 170.5, 165.3,
152.0, 142.1, 139.0, 122.0, 119.4, 115.8, 78.5, 74.0, 72.2,
66.4, 53.9, 41.7, 39.8, 38.1, 32.2, 31.8, 28.0, 25.4, 23.0,
19.2, 17.7, 16.2, 16.0, 13.3. HRFABMS m/z 508.2730; calcd
for C27H42NO6S, 508.2733.
4.1.1. Compound 4. [a]D¼2328 (c 0.2, CHCl3), literature
value [a]D¼232.1 (c 0.2, CHCl3).15
4.1.2. Thiazole 5. Compound 5 was prepared by the
literature procedure.15
4.1.3. Synthesis of thiazole 6. (a) To a solution of 2,4-
dibromo-thiazole (1 g, 4.13 mmol) in ether (20 mL, 0.2 M)
was added dropwise n-BuLi (1.6 M, 2.84 mL, 4.54 mmol,
1.1 equiv.) at 2788C for 5 min. After complete addition, the
reaction mixture was stirred for 45 min. MeSO3CF3
(0.934 mL, 8.26 mmol, 2 equiv.) was added and stirred for
15 min. Sat. NaHCO3 was added to quench the reaction,
followed by water, and the mixture was extracted with ether
(3£20 mL). The combined organic extracts were dried and
evaporated. The resultant crude mixture was purified by
chromatography over silica gel, eluting with 5% ether in
hexane, to furnish 2-methyl-4-bromothiazole (300 mg,
4.1.6. Synthesis of 9. To a solution of 7 (7.1 mg,
0.011 mmol), in THF (1.4 mL) was added a stock solution
of HF·Py in pyridine, prepared by addition of HF·Py
(0.1 mL) to pyridine (0.28 mL) in THF (0.5 mL, at 08C, and
the resulting solution was stirred for 2.5 h at 08C. Sat.
NaHCO3 solution was added to quench the reaction
followed by extraction with EtOAc (15 mL£3). The
combined organics were dried and evaporated. The resulting
residue was purified by preparative TLC on silica gel,
eluting with EtOAc, to give 9 (5.5 mg, 95%). [a]D¼227.88
1
40%). H NMR (400 MHz): d 7.00 (s, 1H), 2.68 (s, 3H).
13C NMR (100 MHz, CDCl3): d 167.4, 124.2, 116.3, 19.5.
(b) To a solution of the above compound (280 mg,
1.55 mmol) in ether (10 mL) was added dropwise n-BuLi
(1.6 M, 1.73 mL, 1.1 equiv.) at 2788C and the resulting
solution was stirred for 15 min at 2788C. Tributyltin
chloride (0.53 mL, 1.89 mmol, 1.2 equiv.) was added and
the solution stirred for 2 h. Hexane was added and the
mixture filtered through a short plug of silica gel, eluting
with 30% EtOAc in hexane. Evaporation of the solvent and
purification of the resulting crude product on preparative
TLC over silica gel with 4% EtOAc in hexane gave 6
1
(c 0.165, CHCl3). H NMR (400 MHz, CDCl3) d 7.21 (s,
1H), 6.61 (s, 1H), 5.46 (dd, J¼9, 5 Hz, 1H), 5.28 (d,
J¼7.2 Hz, 1H), 4.9 (s, 2H), 4.27 (d, J¼8.4 Hz, 1H), 4.08 (d,
J¼13.2 Hz, 1H), 4.00 (d, J¼13.2 Hz, 1H), 3.68 (dd, J¼4,
2.6 Hz, 1H), 3.55 (bs, 1H), 3.15 (qd, J¼7, 2.5 Hz, 1H), 3.00
(bs, 1H), 2.65 (m, 1H), 2.48 (d, J¼11 Hz, 1H), 2.45 (d,
J¼11 Hz, 1H), 2.35 (dd, J¼14, 3 Hz, 1H), 2.29 (d,
J¼2.4 Hz, 1H), 2.25 (m, 1H), 2.12 (m, 1H), 2.05 (s, 3H),
1.75 (m, 2H), 1.34 (m, 1H), 1.32 (s, 3H), 1.15 (d, J¼6.8 Hz,
3H), 1.05 (s, 3H), 1.00 (d, J¼7.2 Hz, 3H). 13C NMR
(100 MHz) d 220.7, 170.4, 142.1, 139.4, 133.1, 127.3,
121.6, 118.9, 116.6, 78.5, 74.1, 72.2, 66.4, 53.8, 41.9, 39.8,
38.1, 31.9, 31.8, 28.3, 25.6, 23.1, 17.9, 16.2, 16.0, 13.5.
HRFABMS m/z 544.4951; calcd for C27H41NO7SNa,
546.2501.
1
(250 mg, 40% yield). H NMR (400 MHz): d 7.18 (s, 1H),
2.78 (s, 3H), 1.58 (m, 6H), 1.36 (m, 6H), 1.12 (m, 6H), 0.88
(t, J¼7 Hz, 9H). 13C NMR (100 MHz, CDCl3): d 166.0,
159.5, 125.2, 29.2, 27.4, 18.9, 13.8, 10.3. HRFABMS m/z
390.1299; calcd for C16H32NSSn, 390.1279.
4.1.4. Synthesis of 7.15 To a solution of compound 4
(24 mg, 0.044 mmol) and Pd(AcCN)2Cl2 (3 mg) was added
a degassed solution of stannane 5 (105 mg, 0.20 mmol,
4.5 equiv.) in DMF (1 mL) at room temperature, and the
resulting solution was stirred for 24 h. The reaction mixture
was then filtered through a short plug of silica gel, eluting
with EtOAc. The EtOAc was concentrated and the resulting
residue was purified by preparative TLC on silica gel,
eluting with 58% EtOAc in hexane, to provide 7 (19.3 mg,
69%). [a]D¼244.48 (c 0.5, CHCl3). 1H NMR (500 MHz) d
7.06 (s, 1H), 6.58 (s, 1H), 5.44 (dd, J¼9, 5 Hz, 1H), 5.26
(dd, J¼9.5, 2 Hz, 1H), 4.90 (s, 2H), 4.25 (m, 1H), 4.06 (d,
J¼13 Hz, 1H), 4.02 (d, J¼13 Hz, 1H), 3.69 (bs, 1H), 3.42
(d, J¼5.7 Hz, 1H), 3.15 (qd, J¼6.8, 3 Hz, 1H), 2.97 (bs,
1H), 2.68 (ddd, J¼15, 9.7, 9.7 Hz, 1H), 2.46 (dd, J¼15,
10 Hz, 1H), 2.29 (m, 2H), 2.06 (m, 1H) 2.05 (s, 3H), 1.63 (s,
3H), 1.67 (m, 1H), 1.65 (s, 3H), 1.32 (s, 3H), 1.28 (m, 3H),
1.18 (d, J¼6.8 Hz, 3H), 1.06 (s, 3H), 0.99 (d, J¼7.2 Hz,
3H), 0.95 (s, 9H), 0.12 (s, 6H). 13C NMR (125 MHz) d 220,
172.7, 170.5, 152.3, 142.1, 138.7, 122.0, 119.7, 116.2,
78.8, 74.0, 72.4, 66.5, 63.2, 53.7, 41.9, 39.8, 38.1, 32.2,
31.9, 28.1, 28.0, 27.0, 25.9, 25.6, 22.9, 18.3, 17.7, 16.1,
4.1.7. Synthesis of 13. To a solution of 3-dimethyl-
aminobenzoic acid (6.7 mg, 0.042 mmol) and DMAP
(2–3 mg) in CH2Cl2 (1 mL) was added 7 (10 mg,
0.01564 mmol) in CH2Cl2 (1 mL), followed by EDCI
(10 mg, 0.052 mmol, 1.2 equiv. to acid) and the resulting
solution was stirred for 6 h. EtOAc was then added to
quench the reaction and the solution concentrated. The
resulting residue was purified by preparative TLC on silica
gel, eluting with 30% EtOAc in hexane gave 13 (9.8 mg,
1
79%). H NMR (400 MHz, CDCl3) d 7.38 (d, J¼2.6 Hz,
1H), 7.37 (d, J¼7.6 Hz, 1H), 7.25 (t, J¼8.8 Hz, 1H), 6.98 (s,
1H), 6.88 (dd, J¼8, 2 Hz, 1H), 6.54 (s, 1H), 5.56 (dd, J¼9,
6 Hz, 1H), 5.27 (dd, J¼9, 5 Hz, 1H), 4.92 (s, 3H), 4.82 (d,
J¼12.7 Hz, 1H), 4.64 (d, J¼12.7 Hz, 1H), 4.26 (d,
J¼10 Hz, 1H), 3.69 (bs, 1H), 3.16 (qd, J¼6.8, 2.4 Hz,
2H), 2.98 (s, 6H), 2.89 (bs, 1H), 2.49 (m, 1h), 2.45 (m, 2H),
2.27 (m, 2H), 2.19 (m, 1H) 2.08 (s, 3H), 1.74 (bs, 3H), 1.38

T. Ganesh et al. / Tetrahedron 59 (2003) 9979–9984
9983
(m, 1H), 1.37 (s, 3H), 1.25 (s, 1H), 1.17 (d, J¼6.8 Hz, 3H),
1.06 (s, 3H), 1.00 (d, J¼7 Hz, 3H), 0.96 (s, 9H), 0.13 (s,
6H). 13C NMR (100 MHz) d 220.7, 172.6, 170.4, 167.2,
152.4, 150.69, 138.4, 137.3, 131.0, 129.2, 124.5, 119.8,
117.6, 117.0, 116.3, 113.4, 78.4, 77.4, 74.2, 72.4, 68.0, 63.3,
53.6, 42.0, 40.7, 39.7, 38.2, 32.1, 31.8, 29.9, 28.6, 25.9,
25.6, 22.9, 18.4, 18.3, 16.07, 16.03, 13.6, 25.1. HRFABMS
m/z 785.4243; calcd for C42H65N2O8SSi 785.4231.
28 mM 14 or 15. The concentrations of ligand were chosen
to ensure that the receptor site was saturated with ligand.23
The extent of assembly was measured at different tubulin
concentrations (0.5–6 mM). Critical concentrations were
calculated from the x-intercepts of plots of apparent A350 nm
vs tubulin concentration.
Absorption and emission spectra of the fluorophores in
solvent and bound to microtubules were measured and the
Stokes shifts were calculated in standard ways, described in
detail in Han et al.24 for a fluorescent derivative of
paclitaxel.
4.1.8. Synthesis of 14. Treatment of 8 by the same as
described above yielded 14 (5.2 mg, 54%). 1H NMR
(400 MHz, CDCl3) d 7.39 (d, J¼2.6 Hz, 1H), 7.36 (d,
J¼7.8 Hz, 1H), 7.26 (t, J¼7.8 Hz, 1H), 6.90 (s, 1H), 6.89 (d,
J¼3 Hz, 1H), 6.58 (s, 1H), 5.58 (dd, J¼9, 5.7 Hz, 1H), 5.26
(dd, J¼8.7, 1.9 Hz, 1H), 4.80 (d, J¼12.7 Hz, 1H), 4.66 (d,
J¼12.7 Hz, 1H), 4.31 (bd, J¼11.3 Hz, 1H), 3.68 (m, 2H),
3.16 (qd, J¼6.8, 2.2 Hz, 2H), 2.98 (s, 6H), 2.88 (s, 1H), 2.69
(s, 3H), 2.66 (m, 1H), 2.42 (m, 2H), 2.28 (m, 3H), 2.06 (s,
3H), 1.78 (bs, 1H), 1.38 (m, 2H), 1.35 (s, 3H), 1.26 (s, 1H),
1.16 (d, J¼6.8 Hz, 3H), 1.05 (s, 3H), 1.00 (d, J¼7 Hz, 3H).
13C NMR (100 MHz) d 220.9, 170.4, 167.1, 151.1, 137.3,
131.0, 129.3, 124.9, 117.1, 115.7, 113.5, 78.1, 77.4, 74.2,
71.9, 68.1, 54.0, 41.7, 40.8, 39.8, 38.3, 32.1, 31.8, 30.0,
28.6, 25.5, 23.1, 19.0, 17.6, 16.5, 15.9, 13.5. HRFABMS m/z
655.3412; calcd for C36H51N2O7S, 655.3417.
References
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4.1.9. Synthesis of 15. To a solution of 13 (6.4 mg,
0.0081 mmol), in THF (1.5 mL) was added a stock solution
of HF·Py in pyridine, prepared by addition of HF·Py
(0.1 mL) to pyridine (0.28 mL) in THF (0.5 mL, at 08C, and
the resulting solution was stirred for 6 h at 08C. Aqueous
NaHCO3 was added to quench the reaction, and the
resulting solution was extracted with EtOAc (3£15 mL).
The combined organic extracts were dried and evaporated.
The resulting residue was purified by preparative TLC on
silica gel, eluting with 65% EtOAc in hexane, to give 15
(5 mg, 92%). [a]D¼221.88 (c 0.11, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.38 (d, J¼2 Hz, 1H), 7.36 (d,
J¼7.6 Hz, 1H), 7.25 (t, J¼6.8 Hz, 1H), 7.0 (s, 1H), 6.90
(dd, J¼7.5, 2.6 Hz, 1H), 6.55 (s, 1H), 5.60 (dd, J¼9, 6.4 Hz,
1H), 5.30 (dd, J¼8.3, 2.4 Hz, 1H), 4.89 (d, J¼3.5 Hz), 4.83
(d, J¼13 Hz, 1H), 4.65 (d, J¼13 Hz, 1H), 4.28 (m, 1H),
3.68 (bd, J¼3.7 Hz, 1H), 3.45 (d, J¼6 Hz, 1H), 3.26 (bs,
1H), 3.16 (qd, J¼2.68, 1H), 2.97 (s, 6H), 2.88 (d, J¼2 Hz,
1H), 2.66 (m, 1H), 2.46 (m, 2H), 2.26 (m, 3H), 2.0 (m, 3H),
1.70 (m, 2H), 1.68 (s, 3H), 1.37 (m, 1H), 1.36 (s, 3H), 1.25
(s, 1H), 1.17 (d, J¼6.8 Hz, 3H), 1.06 (s, 3H), 1.00 (d,
J¼7 Hz, 3H). 13C NMR (100 MHz) d 220.5, 170.3, 170.0,
167.2, 152.4, 150.7, 138.8, 137.2, 129.2, 127.7, 124.2,
119.2, 117.7, 117.2, 116.7, 113.4, 78.2, 77.4, 74.4, 72.2,
68.0, 62.1, 53.7, 42.0, 40.7, 39.7, 38.2, 31.9, 31.7, 28.6,
25.6, 23.8, 18.0, 16.2, 15.9, 13.7. HRFABMS m/z 671.3350;
calcd for C36H51N2O8S, 671.3336.
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