Design, synthesis and biological evaluation of bridged epothilone D analogues

Article abstract of DOI:10.1039/b814823f

Six epothilone D analogues with a bridge between the C4-methyl and the C12-methyl carbons were prepared in an attempt to constrain epothilone D to its proposed tubulin-binding conformation. Ring-closing metathesis (RCM) was employed as the key step to bui

Full text of DOI:10.1039/b814823f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Design, synthesis and biological evaluation of bridged
epothilone D analogues†
Qiao-Hong Chen,‡a Thota Ganesh,‡a,b Peggy Brodie,a Carla Slebodnick,a Yi Jiang,c Abhijit Banerjee,b
Susan Bane,c James P. Snyderb and David G. I. Kingston*a
Received 26th August 2008, Accepted 26th September 2008
First published as an Advance Article on the web 6th November 2008
DOI: 10.1039/b814823f
Six epothilone D analogues with a bridge between the C4-methyl and the C12-methyl carbons were
prepared in an attempt to constrain epothilone D to its proposed tubulin-binding conformation.
Ring-closing metathesis (RCM) was employed as the key step to build the C4–C26 bridge. In
antiproliferative assays in the human ovarian cancer (A2780) and prostate cancer (PC3) cell lines, and
also in tubulin assembly assay, all these compounds proved to be less active than epothilone D.
chemistry, which has been reviewed on several occasions.3 At this
Introduction
time one epothilone derivative, the semisynthetic epoB analogue
ixabepilone (3), has been approved for clinical use,4 and at least six
The epothilones are a class of macrolide natural products isolated
from the soil myxobacterium Sorangium cellulosum.1 Epothilones
A (1) and B (2) (epoA and B) (Fig. 1) were discovered in 1986 based
on their antifungal activity,1 but they attracted only moderate
scientific interest until the report by Bollag et al. in 1995 that
they had the same mechanism of action as paclitaxel, stabilizing
the tubulin polymer and causing apoptotic cell death.2 This
report triggered a large amount of research on their biology and
other epothilone analogues are in advanced clinical trials.5 EpoB
is in phase III trials and EpoD (4) is currently in Phase II clinical
trials. Additional new and exciting analogues, such as fludelone,6
have emerged in recent years.
The nature of the binding of the epothilones to tubulin has been
extensively investigated. The early observation that epothilones
can displace tubulin-bound paclitaxel suggested that both are
bound by the same or overlapping binding sites.7 A combined
paclitaxel-epothilone hybrid construct has been prepared and
shown to promote tubulin assembly 3-fold less effectively than
paclitaxel, lending support to the hypothesis that paclitaxel and
the epothilones share a common binding site and mechanism of
action.8 The X-ray structure of epoB has been published,9 and
two distinct solution conformations have been detected for epoA
and epoB,10 but neither result addressed the conformation of
epothilone on the tubulin polymer. A conformation of tubulin-
bound epothilone has been proposed based on solution NMR
spectroscopy.11 The NMR experiments were however performed
on unassembled tubulin, and it is uncertain if the conformation
of epoA bound to tubulin is the same as its conformation bound
to microtubules. Finally, minireceptor modeling approaches led to
3D QSAR models of epothilone.12
Recently one of us proposed an entirely different epothilone
A conformation based on crystallographic electron density maps
Fig. 1 Structures of epothilones A (1) and B (2), ixabepilone (3), and
epothilone D (4).
˚
of Zn-stabilized tubulin sheets that diffract to 2.9 A, molecular
modeling, and NMR-NAMFIS treatment.13,14 The proposed
binding conformation from this study explains most of the SAR
studies generated for the epothilones, and also accounts for the
drug resistance of mutated cell lines. Analysis of this Nettles
et al. model for epoA reveals juxtaposition of a C4 methyl carbon
aDepartment of Chemistry, M/C 0212, Virginia Polytechnic Institute and
State University, Blacksburg, Virginia 24061, USA. E-mail: qchen06@
vt.edu, dkingston@vt.edu; Fax: 1 540 231 3255; Tel: 1 540-231-6570
bDepartment of Chemistry, Emory University, Atlanta, GA 30322, USA.
E-mail: jsnyder@emory.edu, tganesh@emory.edu
˚
and C12-H at a distance of 4.5 A (Fig. 2). The corresponding
cDepartment of Chemistry, State University of New York of Binghamton,
Binghamton, NY 13902, USA. E-mail: sbane@binghamton.edu
˚
separation between C4-Me and C12-Me (epoB) is 5.5 A. To test
the proposed binding modes, we designed epoD analogues with
built-in conformational restrictions between these positions by
introducing a bridge between C4 and C26.
Herein, we report the synthesis of six new bridged
epothilone analogues and their antiproliferative activities towards
† Electronic supplementary information (ESI) available: Experimental
procedures for the synthesis of compounds 7, 9, 14a, 14b, 15, 16, 18, 19a–e,
1
20a, 21–23, 31, 37–40, 41a–d, 42, 43, 54, and 55, and selected H NMR
spectra. CCDC reference numbers 699878. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/b814823f
‡ These authors contributed equally to the synthetic work reported.
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alternative diene precursor 10 could be synthesized from fragments
9 and 11.
Synthesis
The synthesis of fragment 8 was carried out as previously
described.16,17 Ketone 9 was prepared as shown in Scheme 2.
Treatment of methyl acetoacetate with benzyl chloromethyl
ether provided diketone 12,18 which was subjected to catalytic
asymmetric reduction to provide hydroxyester 13.19 The benzyl
group was deprotected and the resulting diol 14a was selectively
protected with tert-butyldimethylsilyl chloride to give ester 14b
in 95% overall yield. Compound 14b was treated with LDA
and allyl iodide to provide an allyl derivative in excellent yield
and diastereoselectivity, which was subjected to LDA and methyl
iodide to provide compound 15 as the major diastereomer in 53%
yield (dr. 3:1).
Fig. 2 (a) The epoA model proposed by Nettles et al., based on EC
density with juxtaposition of the C4-methyl and C12-H (methyl for epoB);
˚
r(C–C) = 4.5 A. (b) MMFF-energy minimized structures of the EC
template and the designed bridged epoD analogue 5. (Overlaid with
3 point selection in PyMol).
human-ovarian (A2780) and prostate cancer (PC3) cell lines, as
well as their tubulin-assembly activities.
Results
Synthetic strategy
The introduction of a bridge between C4-Me and C26 in
epothilone D was planned with a ring closing metathesis reaction
as the key step, by analogy with our successful synthesis of bridged
paclitaxel derivatives.15 Thus, the synthesis of bridged analogue 5
was designed to proceed from diene precursors 7 or 10 (Scheme 1).
The diene precursor 7 could be derived from fragments 8 and 9 in
a convergent manner. Fragment 8 has previously been synthesized
by us employing Nicolaou’s method16 as an intermediate in the
synthesis of fluorescently labeled epothilone analogues.17 The
Scheme 2 Synthesis of ketone 9.
Scheme 1 Retrosynthetic analysis for bridged epothilone 5.
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The configuration of allyl derivative 15 was initially assigned
based on literature precedent;19 this assignment was subsequently
established by converting it to the ketal 17. Selective irradiation of
the C4-methyl group (Fig. 3) resulted in signal enhancement of the
protons Ha-Hc on the adjacent carbons. Irradiation of proton Ha
of the (S) chiral center gave signal enhancement of the C4-methyl
group and the Hb and Hc protons, but not of the CH2 protons of
the allyl group.
with either first- or second- or third-generation Grubbs catalyst
and also the second-generation Hoveyda-Grubbs catalyst. The
reaction was very sluggish, and when the reaction time was
prolonged the starting material decomposed and no product 6
was obtained. Similar results were observed for diene 22. Diene
23, however, with the longest olefin chain, underwent ring closing
metathesis with the second-generation Grubbs catalyst, albeit in
low yield, to furnish the desired product 25 (Scheme 4).
The configuration of the double bond in 25 was established
as trans based on 1D NOE experiments. Selective irradiation of
either of its olefinic bridge protons resulted in signal enhancement
of the CH2 protons adjacent to the other side of the double
bond, thus confirming the trans orientation of double bond. The
bridged iodide 25, on coupling with the thiazolyl stannane 26 in the
presence of Pd(MeCN)2Cl2, provided the final bridged epothilone
D analogue 27 in 70% yield.
At this point we reconsidered option 1, introduction of the
thiazole unit on the triol 21 followed by acylation at the C26
position and then ring closing metathesis, with the notion that
replacing the iodide with the thiazole unit might bring the
ring closing olefinic units together. Treatment of triol 21 with
thiazolyl stannane 26 provided the intermediate 28, which then was
acylated with 3-butenoyl chloride to give 29. Gratifyingly, when 29
was subjected to ring closing metathesis with second-generation
Grubbs catalyst, the ring closed epothilone D analogue 30 was
obtained, albeit in low yield (Scheme 5). In an effort to make the
shorter bridged analogue 5 by this route, diene 31 was likewise
prepared from 28. However, this diene did not undergo normal
ring closing metathesis with second-generation Grubbs catalyst to
give the expected product 5.
Since conjugation of double bonds with carbonyl groups
reduces their reactivity towards ring-closing metathesis, we next
investigated cyclization of the diene precursor 33 (Scheme 6) with
a C26 allyloxy group instead of a C26 acryloyl group. The (4R)-4-
allyl-4-demethyl-epothilone analogue 32 was prepared by selective
deprotection of the trityl group of lactone 20 followed by coupling
with thiazolyl stannane 26. Alcohol 32 was then converted to its
sodium alkoxide with sodium hydride in THF and allylated with
allyl iodide to give diene 33 in 23% yield, together with 55% starting
material 32. The yield could not be improved by prolonging the
reaction time, which only led to the formation of elimination
byproduct. Diol 34 was prepared by standard desilylation of 33
with HF·Py complex for comparison of its bioactivity with that
of the final cyclized product. Happily, ring closing metathesis
of 33 with second-generation Grubbs catalyst gave the expected
Fig. 3 Key NOE correlations of ketal 17.
These results confirm the (R) configuration of the newly intro-
duced center at the C4 carbon. The remaining transformations
from 15 to 9 were straightforward and proceeded via alcohol 16 in
52% overall yield for the five steps.
Aldol coupling of aldehyde 8 and ketone 9 using LDA as the
base furnished 18 in 85% yield and high diastereoselectivity (11:1).
The stereochemistries at the C6 and C7 positions of the major
and minor diastereomers were assigned by analogy to literature
precedents.20 Presumably this aldol coupling step proceeds via the
favoured Cram/Felkin-Ahn transition state leading to a 6R,7S
major diastereomer. Alcohol 18 was converted to hydroxyacid
19e and then to lactones 20a and then 21 in seven synthetic
steps and 51% overall yield by methods similar to those described
previously16 (Scheme 3).
Having made the advanced C4-allyl-C4-demethyl-triol inter-
mediate 21, we acylated it separately with acryloyl chloride, 3-
butenoyl chloride, and 4-pentenoyl chloride by a known protocol18
to yield the diene precursors 7, 22 and 23 in good yields
(Scheme 4).
At this point, two options were available for the last two steps
of the synthesis. The first was to attach the thiazole unit and then
do the ring closing metathesis, while the second reverses the order
of these steps. The second option was attractive, since it would
generate the macrocyclic iodide building blocks 6, 24, or 25 that
could be exploited by attaching various side chain units such as
pyridine and benzothiazoles, in addition to the parent thiazole
unit. Initially, diene 7 was subjected to ring closing metathesis21
Scheme 3 Synthesis of lactone 21. i. LDA, THF, -78 ◦C to -40 ◦C, 80%; ii. TBSOTf, 2,6-lutidine, 0 ◦C, 94%; iii. HF·Py in Py, 0 ◦C–RT, 86; iv. SO3.Py,
87%; v. NaClO2, NaH2PO4, 98%; vi. TBAF, THF, 0 ◦C–RT, 97%; vii. Et3N, 2,4,6-trichlorobenzoyl chloride, DMAP, 78%.
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Scheme 4 Synthesis of bridged epothilone D analogue 27.
Scheme 5 Synthesis of bridged epothilone D analog 30.
Scheme 6 Synthesis of bridged epothilone D analog 36.
bridged epothilone analogue 35 in 79% yield. Desilylation of 35
with HF·Py complex in THF provided the final epothilone D
analogue 36 in 84% yield, with a 5-atom bridge between C4 and
and this conclusion was supported by the fact that some pairs
of signals collapsed to single signals when the temperature was
increased from 22 ◦C to 50 ◦C in CDCl3.
We also investigated the effect of the stereochemistry at C4 on
biological activity by preparing bridged epothilone analogues with
the 4S stereochemistry. Compound 14b was treated with LDA and
methyl iodide to provide a methyl derivative, which was reacted
with LDA and allyl iodide to furnish compound 37 as the major
diastereomer in 62% yield. The remaining transformations from
1
C26. Interestingly, the H NMR spectrum of the TBS ether 35
of the bridged analogue showed only one signal for each type of
proton. However, the bridged analogue 36 with two free hydroxyl
groups showed pairs of signals for each group of protons in its
1H NMR spectrum in both CDCl3 and CD3CN. We attribute
this pairing of signals to the existence of two atropisomers of 36,
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Scheme 7 Synthesis of fragment C1-C6 with 4S stereochemistry.
Scheme 8 Synthesis of allyl epothilone derivative 43.
37 to 39 were straightforward and proceeded in 20% overall yield
for the five steps (Scheme 7).
To our surprise, aldol coupling of aldehyde 11 and ketone
39 employing LDA as the base furnished the unexpected 6S,7R
diastereoisomer 40 as the major product (46%), as well as two
other diastereomers (28%) (Scheme 8).
The indicated stereochemistry of 40 was confirmed by X-ray
crystallographic analysis of its derivative 56 described below.
Alcohol 40 was converted to hydroxyacid 41d and thence to
lactones 42 and 43 in seven steps and 19% overall yield by methods
similar to the preparation of compound 21 (Scheme 8). By the
same procedures as used for the preparation of 4R,6R,7S bridged
analogue 36, and in similar yields, the 4S,6S,7R bridged analogue
47 was prepared from compound 43 via intermediates 44 and 46
(Scheme 9). The configuration of the bridging double bond in 47
was established as cis based in 1D NOE experiments. Selective
irradiation of one of the olefinic bridge protons resulted in signal
enhancement of the other olefinic bridge proton. The 4S,6S,7R
bridged analogue 47 also exists as a pair of atropisomers, showing
paired signals for each group of protons in its proton NMR
spectrum in CD3CN as solvent.
In an additional effort to investigate the effect of the 4S,6S,7R
stereochemistry on metathesis of a C26 acryloxyl group, the C26
acryloyl derivative 48 was prepared from 43. Diene 48 did not
undergo ring closing metathesis with second-generation Grubbs
catalyst. However, dienes 49 and 50, with one or two free hydroxyl
groups, underwent ring closing metathesis with second-generation
Grubbs catalyst to furnish the bridged products 51 (62%) and
53 (93%). The latter compound was contaminated by a minor
epothilone impurity which could not be removed by normal
chromatography; the major compound was estimated to be 86.5%
pure by 1H NMR spectroscopy (Scheme 9).
Scheme 9 Synthesis of bridged epothilone D analog 47, 52, and 53.
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Scheme 10 Synthesis of 4S,6S,7R epothilone D derivative 56.
Table 1 Bioactivity data of epothilone D and its analogues
Desilylation of 51 with HF·Py complex in THF provided the
bridged analogue 52 with the 4S,6S,7R stereochemistry. The
configurations of the double bonds in 51 and 52 were established
as cis based on the coupling constant (J = 11.5 Hz) of the olefinic
proton adjacent to the carbonyl group of the newly made bridge.
The coupling constant (J = 17.0 Hz) of the corresponding olefinic
proton of 53 indicated that the configuration of its bridged double
bond was trans.
EC50, tubulin
assembly (mM)
Cmpd
IC50, A2780 (mM)
IC50, PC3 (mM)
EpoD
7
0.04
0.016 0.001
ND
ND
0.44 0.02
ND
ND
19.3 2.5
25.6 7.0
22.0 1.1
15.6 3.0
2.1 1.1
4.7 0.3
3.2 0.1
0.24 0.02
0.10 0.04
3.9 0.5
6.2 0.8
22.9 0.9
5.2 0.2
4.9 0.3
31.2 1.2
23
25
27
28
29
30
31
34
36
45
47
50
52
53
ND
ND
2.1 0.15
1.2 0.4
1.2 0.1
0.93 0.06
ND
0.077 0.016
1.2 0.3
ND
ND
ND
ND
ND
6.9 0.9
1.3 0.1
0.88 0.06
2.4 0.1
0.58 0.06
0.54 0.07
1.62 0.13
ND
ND
ND
ND
ND
Assignment of the stereochemistry of the C6 and C7 chiral
centers of the 4S series of compounds could not readily be achieved
by conventional NMR methods, and so the benzoyloxy derivative
56 was prepared from 42 via intermediate 54 and 55 (Scheme 10).
Global deprotection of 42 with HF·Py complex yield triol 54
in 51% yield. Then using the Sharpless conditions [L-(+)-DET,
Ti(iPrO)4, tBuOOH],22 allylic alcohol 54 was converted to 12b,13b-
1
epoxide 55 (d.e. > 95% as judged by H NMR spectroscopy).
The primary hydroxyl group at C26 of triol 55 was selectively
acylated with benzoyl chloride to yield the benzoyloxy derivative
56 as colorless needles from hexanes-acetone. Single crystal X-ray
crystallography of 56 confirmed its absolute stereochemistry as
C3(S), C4(S), C6(S), C7(R), C8(S), C12(S), C13(S) and C15(S)
(Fig. 4).23
some of the analogues on tubulin assembly was also investigated.
The trend in the ability of the analogue to induce microtubule
assembly correlated with the trends in antiproliferative activities
(Table 1).
Molecular modeling and discussion
The bridged epothilone D analogues 27, 30 and 36 were docked
into the tubulin binding site of epothilone using the Glide docking
program.24 Neither 27 nor 30 adopts a docking pose similar to
that of epothilone A in the electron crystallographic (EC) binding
model.13 This is most likely due to the longer and less compatible
length of the cross-ring bridges in 27 and 30. As expected, the
shorter bridge in 36 permits the analogue to readily adopt the
epothilone A EC binding model in the binding site (Fig. 5). In the
cell-free tubulin assembly assay, the reduction in EC50 compared to
EpoD correlates with bridge length, 36 < 30 < 27, while the most
active analog is only 3–4 fold less active than EpoD (Table 1). One
possible explanation for the lack of potency improvement in the
bridged series is that the bridge alters the conformational profile of
the ligands to introduce a greater degree of global conformational
strain relative to the monocyclic standard, thereby decreasing Ka
Fig. 4 Displacement ellipsoid drawing (50%) of compound 56.
Bioactivity
The in vitro antiproliferative activities of the open and constrained
epothilone analogues were determined by employing the A2780
ovarian cancer and PC-3 prostate cancer cell lines (Table 1).
The activities of the constrained analogues 27, 30 and 52 were
comparable to those of their respective open chain analogues, but
they were significantly less than that of epothilone D. The effect of
and increasing DGbind
.
The cell-based data shows another activity pattern. That is,
the EC50 values for all three compounds in both A2780 and PC3
cytotoxicty assays are 75–390 fold greater than that for EpoD,
while the corresponding tubulin polymerization range is 4–15 fold
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1H NMR (400 MHz, CDCl3) d 6.44 (s, 1H), 6.00 (m, 1H), 5.82
(m, 1H), 5.43 (s, 1H), 5.42 (t, J = 6.0 Hz, 1H), 5.00 (d, J = 14.0
Hz, 1H), 4.40 (m, 1H), 4.07 (d, J = 14.0 Hz, 1H), 3.49 (d, J =
2.4 Hz, 1H), 3.18 (dq, J = 5.8, 2.4 Hz, 1H), 2.88 (d, J = 2.8 Hz,
1H), 2.70–2.58 (m, 3H), 2.50–2.38 (m, 6H), 2.30 (m, 2H), 2.10
(m, 2H), 1.88 (s, 3H), 1.84 (m, 1H), 1.70 (m, 1H), 1.40 (m, 1H),
1.27 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.95 (s, 3H);
13C NMR (100 MHz, CDCl3) d 220.0, 172.6, 169.1, 143.7, 137.3,
134.4, 128.2, 118.0, 78.2, 77.4, 76.0, 75.6, 67.9, 63.8, 57.0, 42.7,
39.9, 39.1, 38.6, 32.0, 29.9, 29.8, 28.8, 27.7, 26.9, 24.0, 22.8, 16.9,
14.4, 14.3; HRFABMS: calcd for C28H42O7I (M + H) 617.1975,
found 617.1982.
Bridged epothilone D 27. A solution of iodide 25 (3 mg,
0.0048 mmol), and Pd(MeCN)2(Cl)2 (2 mg) were added to a
degassed solution of stannane 26 (8 mg, 0.02 mmol, 4 eq) in DMF
(0.8 mL) at 25 ◦C and the resulting solution was stirred over 24 h.
The reaction mixture was filtered off through a short plug of silica
gel eluting with ethyl acetate. The filtrate was concentrated and
the residue was subjected to preparative thin layer chromatography
over silica gel, eluting with 25% ethyl acetate in hexanes, to furnish
27 (2 mg, 70%). [a]D -68.2 (c 0.08, CHCl3); 1H NMR (400 MHz,
CDCl3) d 7.00 (s, 1H), 6.59 (s, 1H), 6.06 (m, 1H), 6.00 (m, 1H),
5.67 (t, J = 10.8 Hz, 1H), 5.39 (br.s, 1H), 4.88 (d, J = 13.6 Hz, 1H),
4.55 (d, J = 10.8 Hz, 1H), 4.20 (d, J = 13.6 Hz, 1H), 3.54 (br.s,
1H), 3.36 (m, 1H), 3.22 (q, J = 6.8 Hz, 1H), 2.99 (br.s, 1H), 2.82
(m, 1H), 2.44 (s, 3H), 2.56–2.42 (m, 6H), 2.28 (m, 2H), 2.16 (m,
2H), 2.06 (s, 3H), 1.88 (m, 2H), 1.38 (m, 2H), 1.27 (d, J = 6.8 Hz,
3H), 1.25 (s, 3H), 0.99 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 221.0, 173.0, 169.6, 164.8, 152.1, 136.5, 133.8, 128.3,
120.1, 119.5, 115.2, 77.4, 75.8, 75.7, 67.2, 64.9, 57.4, 42.5, 40.0,
39.2, 38.8, 32.0, 30.1, 29.9, 28.7, 28.1, 26.5, 24.2, 19.1, 17.1, 16.9,
14.4, 14.1; HRFABMS: calcd for C32H46NO7S (M + H) 588.2995,
found 588.2960.
Fig. 5 Docking poses of 1 (yellow) and 36 (cyan) in the EC determined
tubulin binding site.
(Table 1). We attribute the differences provisionally to physical
property variations associated with membrane permeability.
Two additional observations can be made from the data. The
diastereomeric compounds 47, 52, and 53 were all significantly
less active in the A2780 antiproliferative assay than compounds
with the normal epothilone stereochemistry. This finding, while
unsurprising, confirms the importance of stereochemistry in the
bioactivity of the epothilones. A second observation is that the
open chain 4-allyl analogue 34 is the most active of all the
analogues prepared, and approaches the activity of epothilone
D in the tubulin assembly assay. It is likewise only 2–5 fold
less cytotoxic than EpoD (Table 1). Epothilones with additional
substitution at C4 are suggested as a class of analogs that is worth
investigating further.
Experimental
Genaral synthetic procedures
(4R)-4-Allyl-4-demethyl-26-hydroxyepothilone 28. Iodide 21
was converted to 28 (33 mg, 89%) by a similar procedure to that
described previously for the conversion of 25 to 27. [a]D -79 (c
0.6, CHCl3); 1H NMR (400 MHz, CDCl3) d 6.94 (s, 1H), 6.62 (s,
1H), 5.63 (m, 1H), 5.44 (dd, J = 9.8, 4.8 Hz, 1H), 5.30 (d, J =
8.4 Hz, 1H), 5.13 (dd, J = 17.2, 1.6 Hz, 1H), 5.10 (dd, J = 10.4,
1.6 Hz, 1H), 4.45 (d, J = 11.2 Hz, 1H), 4.06 (d, J = 13.2 Hz,
1H), 4.00 (d, J = 13.2 Hz, 1H), 3.90 (br.s, 1H), 3.66 (d, J = 6.0
Hz, 1H), 3.22 (qt, J = 6.8, 1.2 Hz, 1H), 3.13 (br.s, 1H), 2.67 (s,
3H), 2.60 (m, 3H), 2.50 (m, 1H), 2.38–2.30 (m, 3H), 2.05 (s, 3H),
1.96 (m, 3H), 1.80 (m, 1H), 1.62 (m, 2H), 1.45 (m, 2H), 1.30 (m,
2H), 1.17 (d, J = 6.8 Hz, 3H), 1.02 (s, 3H), 1.00 (d, J = 6.8 Hz,
3H); 13C NMR (100 MHz, CDCl3) d 220.0, 170.5, 165.3, 152.0,
142.1, 139.2, 133.5, 122.2, 119.2, 119.1, 115.8, 78.5, 73.3, 71.6,
66.5, 58.0, 41.5, 40.2, 39.8, 37.4, 31.6, 28.2, 27.0, 24.7, 17.7, 15.7,
14.5, 13.8; HRFABMS: calcd for C29H44NO6S (M + H) 534.2889,
found 534.2925.
Optical rotations were recorded on a Perkin-Elmer 241 Polarime-
ter. Infrared spectra were measured using a SENSIR ATR on
MIDAC M2004 Series spectrometer. NMR spectra were obtained
on a JEOL Eclipse 500, a Varian Unity 400, or a Varian Inova 400
spectrometer in CDCl3 or CD3CN. The chemical shifts are given
in d (ppm), and coupling constants are reported in Hz. High-
resolution FAB mass spectra were obtained on a JEOL HX110
Double Focusing Mass Spectrometer. THF was distilled from
sodium-benzophenone and dichloromethane was distilled from
calcium hydride. Other reagents and solvents were purchased from
commercial sources and were used without further purification.
Silica gel column chromatography was performed using flash silica
gel (32–63 m). Preparative thin-layer chromatography (PTLC)
separations were carried out on 500 m or 1000 m Uniplate thin
layer chromatography plates. All reactions were carried out under
a nitrogen atmosphere unless otherwise noted.
Bridged macrolactone iodide 25. To a solution of diene 23
(13 mg, 0.02 mmol) in dichloromethane (3 mL) was added
second-generation Grubbs catalyst (6 mg, 0.007 mmol) at room
temperature for 3 h and the resulting reaction mixture was
stirred for 3 d. The dichloromethane was evaporated to give
crude product, which was subjected to preparative thin layer
chromatography over silica gel, eluting with 20% ethyl acetate in
hexanes, to furnish 25 (3.7 mg, 30%). [a]D -68.6 (c 0.035, CHCl3);
(4R)-4-Allyl-4-demethyl-26-(3-butenoyloxy)epothilone
D
29.
Epothilone derivative 28 was acylated to provide 29 (10 mg, 50%)
by a similar procedure to that described for the conversion of 21
1
to 22 (ESI). H NMR (400 MHz, CDCl3) d 6.95 (s, 1H), 6.58 (s,
1H), 5.90 (m, 1H), 5.66 (m, 1H), 5.46 (dd, J = 10.2, 5.2 Hz, 1H),
5.30 (d, J = 9.6 Hz, 1H), 5.17 (dd, J = 17.2, 1.2 Hz, 1H), 5.15 (dd,
J = 10.4, 1.2 Hz, 1H), 5.14 (dd, J = 17.2, 10.4 Hz, 1H), 5.12 (dd,
4548 | Org. Biomol. Chem., 2008, 6, 4542–4552
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J = 10.4, 1.2 Hz, 1H), 4.55 (dd, J = 12.4 Hz, 1H), 4.46 (d, J =
12.4 Hz, 1H), 4.44 (m, 1H), 3.67 (d, J = 5.6 Hz, 1H), 3.62 (d,
J = 5.2 Hz, 1H), 3.20 (q, J = 5.6 Hz, 1H), 3.12 (d, J = 7.2 Hz,
1H), 3.09 (dt, J = 7.2, 1.2 Hz, 1H), 2.69 (s, 3H), 2.62–2.42 (m,
3H), 2.40–2.22 (m, 3H), 2.06 (s, 3H), 2.02 (m, 1H), 1.80 (m, 1H),
1.64 (m, 1H), 1.42 (m, 2H), 1.30 (m, 2H), 1.14 (d, J = 6.8 Hz,
3H), 1.03 (s, 3H), 1.00 (d, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 219.8, 170.4, 165.4, 152.0, 139.0, 137.1, 133.5, 130.3,
125.3, 119.4, 119.1, 118.9, 115.9, 78.2, 73.4, 71.7, 67.9, 57.9, 41.4,
40.2, 39.8, 39.3, 37.7, 32.5, 31.6, 28.3, 24.7, 19.2, 16.1, 15.7, 14.6,
12.2; HRFABMS: calcd for C33H48NO7S (M + H) 602.3152, found
602.3141.
171.0, 164.8, 152.5, 143.9, 138.5, 133.0, 120.7, 119.6, 118.9, 116.2,
79.5, 74.6, 66.7, 60.7, 57.0, 48.2, 41.0, 39.2, 37.8, 32.6, 31.5, 28.3,
27.4, 26.6, 26.4, 19.6, 19.4, 18.9, 15.5, 14.4, -3.0, -3.25, -3.4, -5.3;
HRFABMS: calcd for C41H72NO6SSi2 (M + H) 762.4619, found
762.4654.
(4R)-4-Allyl-4-demethyl-26-(allyloxy)epothilone D bis-TBS ether
33. To a solution of 32 (20 mg, 0.026 mmol) in THF (1.5 mL)
was added sodium hydride (6.3 mg, 60%, 0.158 mmol) at 0 ◦C,
and the resultant mixture was allowed to stir at 25 ◦C for 30 min.
Allyl iodide (48 mL, 0.525 mmol) was added to the above reaction
mixture, and the reaction was allowed to proceed at 25 ◦C for
additional 1.5 h. The reaction mixture was filtered off via a pad
of siliga gel eluting with ether to provide crude product, which
was subjected to preparative thin layer chromatography over silica
gel, eluting with 30% ether in hexanes, to furnish allyl ether 33
(4.84 mg, 23%) and starting material (11 mg, 55%). Compound
Bridged epothilone D 30. A silimar procedure was employed
to prepare bridged epothilone D (3 mg, 25%) as that described
1
previously for the conversion of 23 to 25. H NMR (400 MHz,
CDCl3) d 7.07 (s, 1H), 6.89 (s, 1H), 5.90 (m, 1H), 5.68 (t, J = 10.8
Hz, 1H), 5.32 (d, J = 4.8 Hz, 1H), 4.78 (d, J = 11.2 Hz, 1H), 4.48
(d, J = 10.8 Hz, 1H), 4.20 (d, J = 11.2 Hz, 1H), 3.46 (br.s, 1H),
3.36 (m, 1H), 3.00 (m, 2H), 2.90 (br.s, 1H), 2.80 (m, 1H), 2.62–2.45
(m, 3H), 2.30 (d, J = 15.6 Hz, 1H), 2.17–2.00 (s, 3H), 2.10 (s, 3H),
1.96 (s, 3H), 1.81 (m, 2H), 1.65 (m, 1H), 1.22 (m, 3H), 1.08 (d, J =
6.8 Hz, 3H), 0.99 (s, 3H), 0.86 (d, J = 7.2 Hz, 3H); 13C NMR (100
MHz, CDCl3) d 221.1, 171.0, 170.4, 169.5, 145.2, 144.3, 137.0,
129.1, 126.6, 125.4, 114.7, 112.8, 74.5, 74.1, 71.3, 66.1, 57.7, 42.8,
39.0, 38.9, 38.4, 38.3, 31.8, 30.4, 29.7, 28.9, 25.9, 17.2, 16.4, 15.2,
13.6; HRFABMS: calcd for C31H44NO7S (M + H) 574.2838, found
574.2848.
1
33 was obtained as a colorless oil; H NMR (500 MHz, CDCl3)
d 6.96 (s, 1H), 6.54 (s, 1H), 5.94–5.85 (m, 1H), 5.66–5.58 (m, 1H),
5.46 (dd, J = 6.8, 5.5 Hz, 1H), 5.26 (d, J = 13.6 Hz, 1H), 5.17
(d, J = 8.3 Hz, 1H), 5.04–5.01 (m, 3H), 4.16–4.08 (m, 1H), 4.04
(d, J = 9.4 Hz, 1H), 3.99–3.85 (m, 3H), 3.72 (d, J = 9.4 Hz, 1H),
3.10–3.04 (m, 1H), 2.85–2.64 (m, 3H), 2.70 (s, 3H), 2.45–2.31 (m,
3H), 2.19–1.98 (m, 3H), 2.11 (s, 3H), 1.83–1.76 (m, 1H), 1.32–1.02
(m, 2H), 1.11 (d, J = 6.8 Hz, 3H), 1.10 (s, 3H), 1.02–0.80 (m, 1H),
0.96 (d, J = 6.8 Hz, 3H), 0.95 (s, 9H), 0.82 (s, 9H), 0.10 (s, 3H),
0.07 (s, 6H), -0.15 (s, 3H); 13C NMR (100 MHz, CDCl3) d 214.8,
171.3, 165.1, 152.9, 138.9, 135.3, 133.4, 124.2, 119.8, 119.2, 117.5,
116.5, 79.8, 77.7, 74.9, 73.9, 71.3, 57.2, 48.7, 41.3, 40.2, 39.8, 37.8,
32.9, 31.8, 30.2, 28.7, 27.6, 26.8, 26.6, 19.7, 19.1, 19.0, 15.8, 0.49,
-2.7, -2.96, -3.18, -5.00; HRFABMS: calcd for C44H76NO6SSi2
(M + H) 802.4932, found 802.4968.
(4R)-4-Allyl-4-demethyl-26-hydroxyepothilone D bis-TBS ether
(32). To a solution of macrolactone 20a (28 mg, 0.027 mmol) in
ether (0.56 mL) at -5 ◦C was added formic acid (0.56 mL) and
the reaction was allowed to proceed with stirring for 5 h at -5 ◦C.
Water (10 mL) and then solid NaHCO3 were sequently added until
cessation of effervescence. The mixture was extracted with ether
(10 mL ¥ 3), the combined organic extracts were dried (Na2SO4),
and the solvents were removed in vacuo. Purification of the crude
product via preparative thin layer chromatography eluting with
50% ether in hexanes to yield lactone iodide 20b (15 mg, 71%).
[a]D -21.6 (c 0.7, CHCl3); 1H NMR (400 MHz, CDCl3) d 7.26 (s,
1H), 6.39 (s, 1H), 5.69–5.59 (m, 1H), 5.43 (dd, J = 9.6, 6.0 Hz,
1H), 5.17–5.05 (overlap, 1H), 5.10 (d, J = 10.4 Hz, 1H), 5.05 (d,
J = 17.2 Hz, 1H), 4.17 (d, J = 8.8 Hz, 1H), 4.09 (br.d, J = 12.4 Hz,
1H), 3.96 (d, J = 13.2 Hz, 1H), 3.89 (d, J = 8.8 Hz, 1H), 3.09–3.05
(m, 1H), 2.75–2.56 (m, 3H), 2.48–2.28 (m, 3H), 2.15–1.92 (m, 2H),
1.88 (s, 3H), 1.76–1.62 (m, 1H), 1.56–1.43 (m, 1H), 1.32–1.15 (m,
7H), 1.12 (d, J = 6.8 Hz, 3H), 1.11 (s, 3H), 0.96 (d, J = 6.8 Hz,
3H), 0.93 (s, 9H), 0.83 (s, 9H), 0.09 (two singlet, 9H), 0.07 (s, 3H);
13C NMR (100 MHz, CDCl3) d 214.0, 170.4, 146.0, 132.9, 118.7,
79.8, 77.7, 74.1, 66.3, 56.7, 40.9, 31.9, 31.1, 29.6, 28.1, 27.1, 26.3,
26.1, 20.5, 18.6, 18.5, -3.3, -3.5, -3.6, -5.3. HRFABMS: calcd for
C37H68O6Si2I (M + H) 791.3599, found 791.3676. Iodide 20b was
converted to 32 by a similar procedure to that described previously
for the conversion of 25 to 27. 1H NMR (400 MHz, CDCl3) d 6.93
(s, 1H), 6.51 (s, 1H), 5.58 (m, 1H), 5.43 (m, 1H), 5.00–4.97 (m,
3H), 3.94 (dd, J = 13.2, 4.4 Hz, 1H), 4.10–4.05 (m, 2H), 3.86 (dd,
J = 8.4, 4.8 Hz, 1H), 3.04 (m, 1H), 2.76–2.53 (m, 3H), 2.67 (s, 3H),
2.46–2.20 (m, 3H), 2.18–1.92 (m, 3H), 2.08 (s, 3H), 1.75–1.55 (m,
4H), 1.06 (s, 3H), 0.89 (s, 9 H), 1.20–0.74 (m, 7 H), 0.77 (s, 9H),
0.03 (s, 9H), -0.19 (s, 3H); 13C NMR (100 MHz, CDCl3) d 214.4,
(4R)-4-Allyl-4-demethyl-26-(allyloxy)epothilone D 34. A simi-
lar procedure was employed to prepare 34 (2.39 mg, 83%, colorless
oil) as that described previously for the conversion of 20a to 21
1
(ESI). H NMR (500 MHz, CDCl3) d 6.96 (s, 1H), 6.58 (s, 1H),
5.93–5.85 (m, 1H), 5.68–5.59 (m, 1H), 5.43 (dd, J = 10.4, 4.4 Hz,
1H), 5.34 (d, J = 9.9 Hz, 1H), 5.25 (dt, J = 17.0, 1.6 Hz, 1H),
5.16 (br.d, J = 10.7 Hz, 1H), 5.13 (br.d, J = 17.9 Hz, 1H), 5.10
(br.d, J = 10.4 Hz, 1H), 4.44 (d, J = 10.4 Hz, 1H), 3.95–3.86 (m,
3H), 3.78 (d, J = 11.8 Hz, 1H), 3.66 (m, 2H), 3.21 (q, J = 6.8 Hz,
1H), 3.14 (br.s, 1H), 2.73–2.47 (m, 4H), 2.68 (s, 3H), 2.34–2.23 (m,
3H), 2.09–2.03 (m, 1H), 2.06 (s, 3H), 1.83–1.76 (m, 1H), 1.70–1.62
(m, 1H), 1.48–1.37 (m, 2H), 1.32–1.24 (m, 2H), 1.14 (d, J = 6.6
Hz, 3H), 1.02 (s, 3H), 1.01 (d, J = 6.8 Hz, 3H); 13C NMR (100
MHz, CDCl3) d 218.0, 170.5, 165.4, 152.0, 139.4, 134.9, 133.5,
124.2, 119.3, 119.1, 117.2, 115.8, 78.6, 73.7, 73.4, 71.8, 71.2, 57.9,
41.4, 40.2, 39.8, 37.8, 32.6, 31.8, 28.3, 24.7, 19.2, 16.1, 15.7, 14.7,
12.2; HRFABMS: calcd for C32H48NO6S (M + H) 574.3202, found
574.3188.
Bridged epothilone D 35. To a solution of diene 33 (1.1 mg,
0.00137 mmol) in dichloromethane (3 mL) was added second-
generation Grubbs catalyst (0.41 mg, 0.00048 mmol, 0.35 eq) in
dichloromethane (2 mL) for a period of 1.5 h via syringe pump
under N2. The resulting reaction mixture was allowed to stir
for 0.5 h at 25 ◦C, and then the dichloromethane was removed
under reduced pressure. The residue obtained was subjected to
preparative thin layer chromatography over silica gel, eluting with
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30% ether in hexanes, to furnish 35 (0.84 mg, 79%) as a pale yellow
oil. 1H NMR (500 MHz, CDCl3) d 6.88 (s, 1H), 6.46 (s, 1H), 5.62
(br.t, J = 10.4 Hz, 1H), 5.56–5.48 (m, 1H), 5.44 (br.s, 1H), 5.29
(d, J = 4.4 Hz, 1H), 4.61 (br.s, 2H), 4.49 (br.s, 2H), 4.23 (dd, J =
10.4, 2.5 Hz, 1H), 3.73 (d, J = 6.3 Hz, 1H), 3.32–3.26 (m, 1H),
3.10–3.04 (m, 1H), 2.71 (s, 3H), 2.70–2.65 (m, 1H), 2.54–2.45 (m,
2H), 2.16–2.03 (m, 4H), 2.15 (s, 3H), 1.70–1.62 (m, 1H), 1.46 (s,
3H), 1.42–1.26 (m, 2H), 1.26 (s, 3H), 1.14–1.02 (m, 1H), 1.07 (d,
J = 6.8 Hz, 3H), 0.91, 0.87 (each s, 18H), 0.86 (overlap, 3H), 0.174
(s, 3H), 0.170 (s, 3H), 0.05 (s, 3H), 0.02 (s, 3H); HRFABMS: calcd
for C42H72NO6SSi2 (M + H) 774.4619, found 774.4644.
(m, 4H), 2.09–2.00 (m, 2H), 2.08 (s, 3H), 1.49–1.38 (m, 2H), 1.09
(d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.95 (s, 3H),
0.95–0.82 (m, 2H); 13C NMR (125 MHz, CDCl3) d 222.0, 170.9,
165.1, 152.1, 138.7, 134.8, 132.5, 123.9, 120.3, 119.3, 117.0, 116.6,
78.4, 74.7, 73.1, 72.0, 71.1, 56.1, 42.4, 39.3, 38.6, 35.6, 33.3, 32.5,
29.7, 28.3, 24.2, 19.5, 16.4, 16.0, 15.6, 11.7; HRFABMS: calcd for
C32H48NO6S (M + H) 574.3202, found 574.3223.
4S, 6S, 7R-Bridged epothilone D bis-TBS ether 46. Bridged
analogue 46 was prepared as a colorless oil (2.4 mg, 71%) via
ring closing metathesis reaction with second-generation Grubbs
catalyst from terminal diene 44, according to the procedure
described previously for the synthesis of bridged compound 35.
1H NMR (400 MHz, CDCl3) d 6.92 (s, 1H), 6.50 (s, 1H), 5.43
(br.s, 1H), 5.50–5.32 (m, 2H), 5.29 (d, J = 5.2 Hz, 1H), 4.75 (dd,
J = 11.2, 4.4 Hz, 1H), 4.61 (br.s, 2H), 4.48 (br.s, 2H), 3.93 (d,
J = 9.6 Hz, 1H), 3.13–2.98 (m, 2H), 2.72 (s, 3H), 2.66 (dd, J =
14.0, 4.0 Hz, 1H), 2.45, 2.42 (ABq, J = 11.2 Hz, 2H), 2.20 (dd,
J = 16.0, 12.0 Hz, 1H), 2.13 (s, 3H), 2.16–1.99 (m, 2H), 1.79–
1.72 (m, 1H), 1.44 (s, 3H), 1.46–1.10 (m, 2H), 1.25 (s, 3H), 0.99
(d, J = 7.2 Hz, 3H), 0.90, 0.83 (each s, 18H), 0.80 (d, J = 6.8
Bridged epothilone 36. The epothilone D bridged analogue
36 was obtained from bridged epothilone D bis-TBS ether
(35), according to the procedure described previously for the
preparation of 21 (ESI), as a colorless oil in 84.5% yield.1H NMR
(500 MHz, CDCl3) d 7.01, 7.00 (s, 1H), 6.67, 6.57 (s, 1H), 5.77–
5.70 (m, 1H), 5.62–5.36 (m, 3H), 4.64 (br.s, 2H), 4.53 (br.s, 2H),
4.54–4.0, 4.15–4.11 (m, 1H), 3.65, 3.20 (br.s, 1H), 3.55, 3.39 (d, J =
9.5 Hz, 1H), 3.40–3.31 (m, 2H), 3.10–3.04 (m, ~0.5 H), 2.93 (br.d,
J = 13.5 Hz, ~0.5H), 2.78, 2.71 (br.s, 1H), 2.74 (s, 3H), 2.75–2.46
(m, 3H), 2.25–2.06 (m, 3H), 2.15 (s, 3H), 1.85–1.80 (m, 1H), 1.51–
1.40 (m, 4H), 1.26, 1.16 (s, 3H), 1.20, 1.11 (d, J = 6.5 Hz, 3H),
0.89, 0.86 (d, J = 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) d
218.0, 170.2, 165.4, 152.3, 140.7, 132.0, 125.8, 119.2, 118.9, 116.6,
116.3, 77.4, 77.2, 76.2, 73.2, 72.6, 58.4, 44.3, 41.2, 35.6, 33.0, 32.8,
27.7, 25.0, 19.4, 16.9, 15.9, 15.79, 15.70; HRFABMS: calcd for
C30H44NO6S (M + H) 546.2889, found 546.2914.
Hz, 3H), 0.18 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H), 0.01 (s, 3H); 13
C
NMR (100 MHz, CDCl3) d 213.4, 169.9, 165.0, 152.9, 140.5, 135.7,
129.5, 124.2, 119.2, 119.1, 116.5, 77.4, 76.2, 74.7, 70.8, 57.0, 45.2,
40.8, 38.0, 35.3, 32.5, 29.9, 27.3, 26.4, 26.0, 19.5, 18.8, 18.2, 17.4,
16.7, 15.6, 13.0, -3.10, -3.31, -4.00, -5.28; HRFABMS: calcd for
C42H72NO6SSi2 (M + H) 774.4619, found 774.4568.
Bridged epothilone 47. A similar procedure was used to
prepare 47 (1.16 mg, 71.6%, colorless oil) from bis-TBS ether
46 based on the previously described method for the conversion
of 20a to 21 (ESI). 1H NMR (500 MHz, CD3CN) d 7.17, 7.15 (s,
1H), 6.61, 6.55 (s, 1H), 5.65–5.27 (m, 4H), 4.62 (dd, J = 10.5, 4.0
Hz, 1H), 4.51 (br.s, 2H), 4.42 (br.s, 2H), 3.60–3.54, 3.40–3.35 (m,
2H), 3.32–3.28, 3.21–3.13 (m, 1H), 2.98–2.92 (m, 1H), 2.76 (dd,
J = 10.5, 4.0 Hz, 1H), 2.66 (s, 3H), 2.56, 2.52; 2.41, 2.38 (ABq, J =
11.5 Hz, 2H), 2.27–2.05 (m, 4H), 2.15 (s, 3H), 1.58–1.36 (m, 6H),
1.27, 1.22 (s, 3H), 1.05, 1.04 (d, J = 7.0 Hz, 3H), 0.92, 0.86 (d, J =
6.5 Hz, 3H); 13C NMR (125 MHz, CD3CN) d 218.6, 169.7, 165.0,
152.6, 140.7, 135.3, 129.3, 124.3, 119.0, 118.6, 116.7, 76.4, 75.4,
74.6, 74.2, 68.8, 56.8, 42.8, 39.2, 35.4, 33.3, 33.1, 26.9, 26.4, 24.7,
18.3, 16.3, 15.9, 13.5, 12.5; HRFABMS: calcd for C30H44NO6S
(M + H) 546.2889, found 546.2908.
(4S)-4-Allyl-4-demethyl-26-(allyloxy)-6S,7R-epothilone D bis-
TBS ether (44). Allyl ether 44 was synthesized as a colorless oil
(4.0 mg, 19%), as well as 55% starting material was recovered, from
primary alcohol 43 following the procedure described previously
for the synthesis of allyl ether 33. 1H NMR (400 MHz, CDCl3) d
6.96 (s, 1H), 6.33 (s, 1H), 5.91–5.82 (m, 1H), 5.75–5.62 (m, 1H),
5.35 (t, J = 8.8 Hz, 1H), 5.24–5.06 (m, 4H), 4.17 (d, J = 8.8 Hz,
1H), 3.99 (d, J = 2.8 Hz, 1H), 3.93–3.83 (m, 2H), 3.79 (d, J =
11.6 Hz, 1H), 3.47–3.35 (m, 1H), 2.70 (s, 3H), 2.65 (t, J = 6.8
Hz, 1H), 2.59–2.41 (m, 3H), 2.55–2.19 (m, 1H), 2.12 (s, 3H), 1.98–
1.88 (m, 1H), 1.55–1.42 (m, 4H), 1.28–1.24 (m, 2H), 1.07 (d, J =
7.2 Hz, 3H), 0.96 (s, 3H), 0.92 (s, 9H), 0.90 (s, 9H), 0.88 (d, J =
6.4 Hz, 3H), 0.20 (s, 3H), 0.16 (s, 3H), 0.12 (s, 3H), 0.04 (s, 3H);
13C NMR (100 MHz, CDCl3) d 214.8, 170.5, 164.7, 152.7, 140.2,
136.7, 135.1, 134.1, 134.2, 122.2, 119.2, 118.1, 117.0, 116.3, 77.4,
75.0, 74.8, 73.8, 70.9, 56.6, 48.7, 42.4, 40.2, 38.9, 34.9, 31.1, 29.9,
28.2, 28.1, 26.5, 26.4, 19.4, 18.9, 18.8, 16.1, 14.3, -3.3, -3.8, -4.2,
-4.6; HRFABMS: calcd for C44H76NO6SSi2 (M + H) 802.4932,
found 802.4903.
(4S)-4-Allyl-4-demethyl-26-(acryloyloxy)-6S,7R-epothilone
D
bis-TBS ether 48. Acryloyloxy derivative 48 was synthesized
(6 mg, 56%) as a colorless oil from primary alcohol 43, according
to the procedure described previously for the preparation of 7
1
(ESI). H NMR (500 MHz, CDCl3) d 6.96 (s, 1H), 6.36 (d, J =
(4S)-4-Allyl-4-demethyl-26-(allyloxy)-6S,7R-epothilone D 45.
A similar procedure was employed to prepare 45 (0.65 mg, 29%,
colorless oil), together with 64% mono-TBS ether, from 44 as that
17.3 Hz, 1H), 6.34 (s, 1H), 6.10 (dd, J = 17.3, 10.4 Hz, 1H), 5.77
(d, J = 10.4 Hz, 1H), 5.74–5.65 (m, 1H), 5.42 (t, J = 8.2 Hz, 1H),
5.16 (t, J = 4.1 Hz, 1H), 5.08 (d, J = 18.4 Hz, 1H), 5.07 (d, J =
9.6 Hz, 1H), 4.56, 4.51 (ABq, J = 12.6 Hz, 2H), 4.18 (br.d, J =
8.5 Hz, 1H), 3.98 (d, J = 3.3 Hz, 1H), 3.45–3.38 (m, 1H), 2.69 (s,
3H), 2.65 (dd, J = 14.5, 6.5 Hz, 1H), 2.56–2.43 (m, 4H), 2.25–2.20
(m, 2H), 2.11 (s, 3H), 1.95–1.87 (m, 1H), 1.60–1.44 (m, 5H), 1.07
(d, J = 7.1 Hz, 3H), 0.96 (s, 3H), 0.90 (s, 9H), 0.88 (s, 9H), 0.86
(d, J = 6.3 Hz, 3H), 0.19 (s, 3H), 0.15 (s, 3H), 0.12 (s, 3H), 0.02
(s, 3H); 13C NMR (125 MHz, CDCl3) d 220.1, 170.4, 166.0, 164.6,
152.5, 137.9, 136.4, 134.1, 130.7, 128.5, 123.6, 119.4, 118.0, 116.3,
1
described for the conversion of 20a to 21 (ESI). H NMR (500
MHz, CDCl3) d 6.97 (s, 1H), 6.51 (s, 1H), 5.95–5.86 (m, 1H),
5.64–5.57 (m, 2H), 5.47 (dd, J = 9.9, 3.5 Hz, 1H), 5.28 (dd, J =
17.0, 1.6 Hz, 1H), 5.19 (dd, J = 10.4, 1.6 Hz, 1H), 5.12 (d, J =
11.5 Hz, 1H), 5.11 (d, J = 15.4 Hz, 1H), 4.16 (br.d, J = 11.0 Hz,
1H), 3.96–3.94 (m, 2H), 3.91, 3.82 (ABq, J = 11.8 Hz, 2H), 3.58–
3.50 (m, 2H), 3.31 (br.s, 1H), 2.92 (d, J = 3.6 Hz, 1H), 2.74–2.63
(m, 2H), 2.70 (s, 3H), 2.57 (dd, J = 16.2, 1.9 Hz, 1H), 2.48–2.20
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77.6, 74.8, 73.8, 68.5, 56.3, 48.6, 42.0, 40.2, 38.6, 34.8, 31.1, 28.3,
27.8, 26.3, 21.1, 19.3, 18.8, 18.6, 16.1, 15.8, 14.3, -3.5, -4.0, -4.3,
-4.7; HRFABMS: calcd for C44H74NO7SSi2 (M + H) 816.4725,
found 816.4722.
Cis bridged epothilone 52. A similar procedure was used to
prepare 52 (1.36 mg, 47%, colorless oil) from mono-TBS ether 51
based on the previously described method for the conversion of
20a to 21 (ESI). 1H NMR (500 MHz, CDCl3) d 6.99 (s, 1H), 6.56
(s, 1H), 6.25–6.15 (m, 1H), 5.90 (dd, J = 11.8, 1.9 Hz, 1H), 5.72
(d, J = 9.3 Hz, 1H), 5.65 (dd, J = 11.5, 1.7 Hz, 1H), 4.64, 4.45
(ABq, J = 11.4 Hz, 2H), 4.52–4.30 (m, 1H), 3.63–3.54 (m, 1H),
3.44–3.34 (m, 4H), 2.81–2.67 (m, 2H), 2.70 (s, 3H), 2.44–2.41 (m,
3H), 2.09 (s, 3H), 1.91–1.82 (m, 1H), 1.71–1.58 (m, 2H), 1.45–1.38
(m, 2H), 1.31–1.19 (m, 2H), 1.25 (s, 3H), 1.07 (d, J = 6.6 Hz,
3H), 0.93 (d, J = 7.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) d
221.5, 169.9, 166.2, 165.1, 152.0, 141.8, 137.0, 136.8, 130.8, 123.1,
120.7, 116.9, 78.8, 76.8, 70.9, 70.7, 58.2, 41.1, 35.8, 33.2, 32.7, 31.6,
30.9, 29.7, 26.5, 22.7, 19.3, 15.3, 14.2, 10.8; HRFABMS: calcd for
C30H42NO7S (M + H) 561.2760, found 560.2720.
(4S)-4-Allyl-4-demethyl-26-(acryloyloxy)-6S,7R-epothilone ana-
logues 49 and 50. A similar procedure was employed to prepare
mono-TBS ether 49 (3.0 mg, 50%, colorless oil) and diol 50
(2.0 mg, 40%) from bis-TBS ether 48 as previously described
1
for the conversion of 20a to 21 (ESI). Compound 49: H NMR
(500 MHz, CDCl3) d 6.94 (s, 1H), 6.38 (d, J = 17.6 Hz, 1H), 6.33
(s, 1H), 6.10 (dd, J = 17.6, 10.0 Hz, 1H), 5.80 (d, J = 10.4 Hz,
1H), 5.55–5.45 (m, 2H), 5.32 (br.s, 1H), 5.10 (d, J = 9.2 Hz, 1H),
5.07 (d, J = 16.0 Hz, 1H), 4.63, 4.58 (ABq, J = 12.8 Hz, 2H), 4.17
(br.d, J = 8.8 Hz, 1H), 3.78–3.72 (m, 1H), 3.62 (d, J = 8.4 Hz,
1H), 3.54 (br.s, 1H), 2.78 (dd, J = 14.4, 6.4 Hz, 1H), 2.70 (s, 3H),
2.57–2.41 (m, 3H), 2.32–2.02 (m, 3H), 2.07 (s, 3H), 1.66–1.45 (m,
3H), 1.28–1.20 (m, 3H), 1.13 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8
Hz, 3H), 0.94 (s, 9H), 0.20 (s, 3H), 0.10 (s, 3H); 13C NMR (125
MHz, CDCl3) d 222.4, 170.4, 166.0, 164.8, 152.3, 137.0, 136.3,
132.8, 130.9, 128.4, 124.4, 119.1, 119.0, 116.2, 75.0, 73.8, 68.4,
67.6, 57.3, 44.2, 40.2, 39.5, 35.2, 33.5, 31.1, 29.7, 28.2, 26.4, 23.5,
19.5, 19.3, 18.6, 16.3, 15.7, 11.5, -3.5, -4.5; HRFABMS: calcd for
C38H60NO7SSi (M + H) 702.3860, found 702.3855. Compound 50:
1H NMR (500 MHz, CDCl3) d 6.96 (s, 1H), 6.51 (s, 1H), 6.43 (d,
J = 17.3 Hz, 1H), 6.15 (dd, J = 17.5, 10.4 Hz, 1H), 5.87 (d, J =
10.4 Hz, 1H), 5.62–5.54 (m, 2H), 5.42 (t, J = 6.8 Hz, 1H), 5.13
(d, J = 18.1 Hz, 1H), 5.12 (d, J = 9.3 Hz, 1H), 4.67, 4.54 (ABq,
J = 13.2 Hz, 2H), 4.20 (d, J = 11.3 Hz, 1H), 3.58 (q, J = 6.8 Hz,
1H), 3.53 (d, J = 9.3 Hz, 1H), 3.31 (br.s, 1H), 2.93 (d, J = 3.0
Hz, 1H), 2.73–2.66 (m, 1H), 2.70 (s, 3H), 2.55 (d, J = 15.9 Hz,
1H), 2.48 (dd, J = 14.2, 6.3 Hz, 1H), 2.42–2.30 (m, 4H), 2.09–2.00
(m, 1H), 2.07 (s, 3H), 1.66–1.40 (m, 3H), 1.26–1.22 (m, 1H), 1.09
(d, J = 6.6 Hz, 3H), 1.02 (d, J = 6.3 Hz, 3H), 0.95 (s, 3H), 0.97–
0.85 (m, 1H); 13C NMR (125 MHz, CDCl3) d 222.1, 170.7, 166.2,
165.2, 152.1, 136.8, 136.3, 132.5, 131.2, 128.3, 124.6, 120.3, 119.2,
116.7, 78.3, 74.7, 71.9, 70.3, 67.1, 56.2, 42.2, 39.0, 38.5, 35.6, 33.3,
32.6, 28.5, 24.3, 19.3, 16.0, 15.9, 15.5, 11.8; HRFABMS: calcd for
C32H46NO7S (M + H) 588.2995, found 588.3037.
Trans bridged epothilone 53. Ring closing metathesis reaction
of terminal diene 50 with second-generation Grubbs catalyst,
according to the procedure described previously for the synthesis
of bridged compound 35, produced a mixture (0.86 mg, ~93%
total yield) as a colorless oil. This mixture consists of 86.5% of 53
based on the integration of its 1H NMR spectrum. Compound 53:
1H NMR (500 MHz, CDCl3) d 6.95 (s, 1H), 6.55 (s, 1H), 5.94 (d,
J = 17.0 Hz, 1H), 5.70 (dd, J = 17.0, 9.9 Hz, 1H), 5.46 (dd, J =
12.1, 3.0 Hz, 1H), 4.71 (t, J = 7.4 Hz, 1H), 4.59, 4.52 (ABq, J =
11.3 Hz, 2H), 3.43 (d, J = 10.4 Hz, 1H), 3.25 (q, J = 6.6 Hz, 1H),
2.82–2.71 (m, 1H), 2.70 (s, 3H), 2.55–2.31 (m, 6H), 2.09 (s, 3H),
2.08–2.02 (m, 1H), 1.75–1.65 (m, 1H), 1.50–1.20 (m, 3H), 1.23 (s,
3H), 1.06 (s, 3H), 1.03 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.9 Hz,
3H), 0.97–0.85 (m, 1H); HRFABMS: calcd for C30H42NO7S (M +
H) 560.2682, found 560.2593.
(4R)-4-Allyl-4-demethyl-26-(benzoyloxy)-6S,7R-epothilone B 56.
26-benzoyloxy-b-epoxide 56 was synthesized from b-epoxide triol
55 employing the same procedure as that described for compound
22 (ESI). Compound 56 was obtained as a colorless needles from
hexanes-acetone in 94% yield. 1H NMR (500 MHz, CDCl3) d 7.95
(d, J = 7.2 Hz, 2H), 7.53 (t, J = 7.4 Hz, 1H), 7.35 (t, J = 7.4 Hz,
2H), 6.93 (s, 1H), 6.65 (s, 1H), 5.65 (br.s, 1H), 5.56–5.48 (m,
1H), 5.15–5.08 (overlap, 1H), 5.10 (d, J = 11.2 Hz, 1H), 5.09
(d, J = 16.0 Hz, 1H), 4.48–4.41 (m, 1H), 4.20 (br.s, 1H), 4.18
(d, J = 12.0 Hz, 1H), 3.79 (d, J = 9.0 Hz, 1H), 3.47 (br.s, 1H),
3.31 (q, J = 7.0 Hz, 1H), 3.25 (dd, J = 9.4, 3.3 Hz, 1H), 2.70
(s, 3H), 2.70–2.68 (m, 1H), 2.56 (d, J = 12.3 Hz, 1H), 2.52 (d,
J = 9.1 Hz, 1H), 2.41 (dd, J = 14.3, 5.5 Hz, 1H), 2.25 (dt, J =
15.4, 3.8 Hz, 1H), 2.16 (s, 3H), 1.99–1.86 (m, 1H), 1.73–1.38 (m,
8H), 1.10 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.4 Hz, 3H), 1.01 (s,
3H). Colorless needles of compound 56 (0.03 ¥ 0.03 ¥ 0.80 mm3)
were crystallized from hexanes/acetone at room temperature. The
chosen crystal was centered on the goniometer of an Oxford
Diffraction Gemini Ultra diffractometer equipped with a Sapphire
3 CCD detector and operating with CuKa radiation. The data
collection routine, unit cell refinement, and data processing were
carried out with the program CrysAlis.25 The Laue symmetry
and systematic absences were consistent with the orthorhombic
space group P212121. Structure solution was performed with the
graphical user interface WinGX.26 The structure was solved by
direct methods using SIR9227 and refined using SHELXTL-NT.28
The asymmetric unit of the structure comprises one crystallo-
graphically independent molecule. The final refinement model
Cis bridged epothilone 51. Compound 51 was prepared as
a colorless oil with a cis double bond on the newly made
bridge (2.8 mg, 62%) via ring closing metathesis with second-
generation Grubbs catalyst from terminal diene 49, according to
the procedure described previously for the synthesis of bridged
1
compound 35. H NMR (500 MHz, CDCl3) d 6.97 (s, 1H), 6.58
(s, 1H), 6.09 (dt, J = 12.1, 3.0 Hz, 1H), 5.82 (dd, J = 11.5, 2.2
Hz, 1H), 5.78 (t, J = 6.8 Hz, 1H), 5.76 (d, J = 11.5 Hz, 1H), 4.72
(d, J = 8.5 Hz, 1H), 4.68, 4.33 (ABq, J = 11.8 Hz, 2H), 3.93 (t,
J = 13.5 Hz, 1H), 3.42 (q, J = 6.6 Hz, 1H), 3.29 (d, J = 10.4 Hz,
1H), 2.88–2.73 (m, 2H), 2.71 (s, 3H), 2.59 (d, J = 11.0 Hz, 1H),
2.46–2.41 (m, 2H), 2.33 (t, J = 12.6 Hz, 1H), 2.15 (s, 3H), 1.99 (d,
J = 14.3 Hz, 1H), 1.79–1.72 (m, 1H), 1.52–1.37 (m, 3H), 1.30–1.17
(m, 2H), 1.21 (s, 3H), 1.08 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6
Hz, 3H), 0.83 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR (125
MHz, CDCl3) d 221.5, 169.9, 166.1, 165.1, 152.2, 141.6, 136.9,
136.1, 131.4, 123.1, 121.2, 117.1, 79.7, 74.9, 72.2, 68.1, 59.7, 40.0,
36.3, 32.7, 32.6, 31.1, 29.7, 26.7, 25.8, 19.4, 19.3, 18.2, 15.0, 10.4,
1.11, 0.09, -2.6, -3.8; HRFABMS: calcd for C36H56NO7SSi (M +
H) 674.3547, found 674.3593.
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involved anisotropic displacement parameters for non-hydrogen
atoms and a riding model for all hydrogen atoms. The absolute
configuration was established from anomalous dispersion effects
(Flack x = 0.01(6)). The configuration of chiral centers were
assigned as C3(S), C4(S), C6(S), C7(R), C8(S), C12(S), C13(S)
and C15(S).
6 T.-C. Chou, H. Dong, A. Rivkin, F. Yoshimura, A. E. Gabarda, Y. S.
Cho, W. P. Tong and S. J. Danishefsky, Angew. Chem. Int. Ed. Engl.,
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Kuduk and S. J. Danishefsky, Proc. Natl. Acad. Sci. USA, 1999, 96,
4256.
9 G. Ho¨fle, N. Bedorf, H. Steinmetz, D. Schomburg, K. Gerth and H.
Reichenbach, Angew. Chem. Int. Engl., 1996, 35, 1567.
10 R. E. Taylor and J. Zajicek, J. Org. Chem., 1999, 64, 7224; R. E. Taylor,
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F. Petersen, D. Schinzer, K.-H. Altmann and C. Griesinger, Angew.
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2515; (c) M. Reese, V. M. Snchez-Pedregal, K. Kubicek, J. Meiler, M.
J. J. Blommers, C. Griesinger and T. Carlomagno, Angew. Chem. Int.
Ed., 2007, 46, 1864.
12 F. Manetti, S. Forli, L. Maccari, F. Corelli and M. Botta, Il Farmaco,
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Downing, Science, 2004, 305, 866.
Biological data. Tubulin was prepared by two cycles of temper-
ature dependent assembly–disassembly as described by Williams
and Lee.29 ED50 values for tubulin assembly were determined as
described in Chatterjee et al.30
The A2780 ovarian cancer cell line assay was performed at
Virginia Polytechnic Institute and State University as previously
reported.31 The A2780 cell line is a drug-sensitive ovarian cancer
cell line.32 The MTT assay33 was used at SUNY for PC3 cells.
Molecular modeling and design. Structures of bridged
epothilone analogues 27, 30 and 36 were constructed in Maestro34
by using the electron crystallographic structure of epo-A as
template. The structures were subsequently optimized with the
MMFF/GBSA/H2O force field. The optimized structures of 27,
30 and 36 were separately and flexibly docked into tubulin binding
site by using the Glide docking protocol.24 The best docking poses
were chosen on the basis of the Emodel scoring function together
with visualization to ensure reasonable binding modes.
14 D. W. Heinz, W.-D. Schubert and G. Ho¨fle, Angew. Chem. Int. Ed.,
2005, 44, 1298.
15 T. Ganesh, R. C. Guza, S. Bane, R. Ravindra, N. Shanker, A. S.
Lakdawala, J. P. Snyder and D. G. I. Kingston, Proc. Natl. Acad. Sci.
USA, 2004, 101, 10006.
16 K. C. Nicolaou, N. P. King, M. R. V. Finlay, Y. He, F. Roschavgar,
D. Vourloumis, H. Vallberg, F. Sarabia, S. Nincovic and D. Hepworth,
Bioorg. Med Chem., 1999, 7, 665; K. C. Nicolaou, S. Nincovic, F.
Sarabia, D. Vourloumis, Y. He, H. Vallberg, M. R. V. Finlay and Z.
Yang, J. Am. Chem. Soc., 1997, 119, 7974.
17 T. Ganesh, R. J. K. Schilling, R. K. Palakodety, R. Ravindra, N.
Shanker, S. Bane and D. G. I. Kingston, Tetrahedron, 2003, 59, 9979.
18 E. C. Taylor and J. L. LaMattina, J. Org. Chem., 1978, 43, 1200.
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E. A. Reiff, T. C. Boge and G. I. Georg, J. Org. Chem., 2000, 65, 7792.
20 K. C. Nicolaou, D. Hepworth, N. P. King, M. R. V. Finlay, R. Scarpelli,
M. M. A. Pereira, B. Bollbuck, A. Bigot, B. Werschkun and N.
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Acknowledgements
This work was supported by a grant awarded by the National
Cancer Institute (CA 69571), and this support is gratefully
acknowledged. We also thank Mr. William Bebout and Mr.
Tom Glass, Virginia Polytechnic Institute and State University
(VPI&SU), for assistance in acquiring some MS and NMR
data, Mr. Chao Yang (VPI&SU) for carrying out a synthetic
reaction, and Dr. Shugeng Cao (VPI&SU) for assistance with
the interpretation of NMR spectra.
21 For leading references, see: T. M. Trnka and R. H. Grubbs, Acc. Chem.
Res., 2001, 34, 18, and references sited therein; A. Fu¨rstner, Angew.
Chem. Int. Ed. Engl., 2000, 39, 3012; R. H. Grubbs and S. Chang,
Tetrahedron, 1998, 54, 4413, and references cited therein.
22 T. Katsuki and K. Sharpless, J. Am. Chem. Soc., 1980, 102, 5976.
23 Crystal data for 56: C36H47NO8S, Mr = 653.8, orthorhombic, space
Notes and references
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˚
˚
˚
group P212121, a = 6.0080(11) A, b = 17.848(2) A, c = 30.989(4) A,
3
˚
˚
V = 3323.0(8) A , Z = 4, CuKa radiation (1.54178 A), T = 100(2) K,
10746 reflections collected; 3558 unique (Rint = 0.1684); R1 = 0.0602
(1655 I > 2s(I)), wR2 = 0.1492 (all data), Flack = 0.01(6).
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4552 | Org. Biomol. Chem., 2008, 6, 4542–4552
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©