C6-C8 bridged epothilones: Consequences of installing a conformational lock at the edge of the macrocycle

Article abstract of DOI:10.1002/chem.201102630

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.

Full text of DOI:10.1002/chem.201102630

DOI: 10.1002/chem.201102630
C6–C8 Bridged Epothilones: Consequences of Installing a Conformational
Lock at the Edge of the Macrocycle
Weiqiang Zhan,*[a] Yi Jiang,[a] Shubhada Sharma,[b] Peggy J. Brodie,[c] Susan Bane,[b]
David G. I. Kingston,[c] Dennis C. Liotta,[a] and James P. Snyder*[a]
Abstract: A series of conformationally
restrained epothilone analogues with a
short bridge between the methyl
groups at C6 and C8 was designed to
mimic the binding pose assigned to our
recently reported EpoA–microtubule
binding model. A versatile synthetic
route to these bridged epothilone ana-
logues has been successfully devised
and implemented. Biological evalua-
tion of the compounds against A2780
human ovarian cancer and PC3 pros-
tate cancer cell lines suggested that the
introduction of a bridge between C6–
C8 reduced potency by 25–1000 fold in
comparison with natural epothilone D.
Tubulin assembly measurements indi-
cate these bridged epothilone ana-
logues to be mildly active, but without
significant microtubule stabilization ca-
pacity. Molecular mechanics and DFT
energy evaluations suggest the mild ac-
tivity of the bridged epo-analogues
may be due to internal conformational
strain.
Keywords: antitumor agents · epo-
thilone · molecular modeling · natu-
ral products · total synthesis
Introduction
been proven to be very poor substrates for the phosphogly-
coprotein 170 (P-gp) efflux pump. Thus, they retain almost
full activity against P-gp overexpressing, Taxol-resistant cell
lines. Furthermore, epothilones are also active against cells
with tubulin mutations which induce paclitaxel resistance.[4a]
This suggests that epothilone-derived drugs might be useful
for treating certain drug resistant tumors. In addition, al-
though EpoA and EpoB were the major products isolated
from the myxobacterium, numerous other related structures
of the epothilone class have been identified as minor com-
ponents of the fermentation of myxobacteria, including, for
example, epothilone C (EpoC, 3) and D (EpoD, 4). These
compounds also exhibit potent anticancer properties
(Figure 1).[5] These exceptional biological advantages, com-
Epothilones are a family of cytotoxic polyketide natural
products originally isolated from the bacterium Sorangium
cellulosum.[1] Epothilone A (EpoA, 1) and epothilone B
(EpoB, 2) (Figure 1), two major representatives, were recog-
nized to be potent inhibitors against breast and colon cancer
cells shortly after their initial isolation.[1] The mechanism of
action of both EpoA and EpoB was established by the
Merck group to be induction of tubulin polymerization in
vitro resulting in the stabilization of microtubules under nor-
mally destabilizing conditions similar to the clinical anti-
cancer drugs Taxol and docetaxel.[2] While epothilones exert
their antiproliferative action in a similar way to Taxol, the
two classes of compounds are distinctly different in terms of
their potency and ability to inhibit the growth of multidrug-
resistant cancer cell lines.[2–4] In contrast to Taxol, the epo-
thilones are more efficacious promoters of cancer cell death
with EpoB being the most active. Epothilones have also
[a] Dr. W. Zhan, Dr. Y. Jiang, Prof. D. C. Liotta, Dr. J. P. Snyder
Department of Chemistry, Emory University
1515 Dickey Drive, Atlanta GA 30322 (USA)
Fax : (+1)404-712-8670
Figure 1. Structures of natural epothilones A–D.
E-mail: weiqiang.zhan@emory.edu
jsnyder@emory.edu
bined with the ease of synthesis by comparison with pacli-
taxel have evoked a vast effort within academic and phar-
maceutical research groups.[3] Numerous total and partial
epothilone syntheses have been published since the determi-
nation of absolute stereochemistry in 1996.[6] During the de-
velopment of these syntheses, a variety of methodologies
have enabled the development of diverse libraries of syn-
thetic analogues. In turn, these have contributed to mapping
[b] S. Sharma, Prof. S. Bane
Department of Chemistry, State University of New York
Binghamton, NY 13902-6016 (USA)
[c] P. J. Brodie, Prof. D. G. I. Kingston
Department of Chemistry
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102630.
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the extensive structure–activity relationship (SAR) profiles
of epothilones and to elucidating interactions between the
ligands and microtubules.[7–9] The tremendous efforts exerted
to generate epothilone SAR profiles have greatly aided our
understanding of the drug pharmacophore and the develop-
ment of natural/unnatural analogues with improved biologi-
cal activity and reduced toxicity. Significantly, these efforts
have delivered at least seven compounds in advanced clini-
cal trials, one of which recently won FDA approval for clini-
cal use as an anti-cancer drug (Ixabepilone).[10] Since the dis-
covery of the microtubule-stabilizing properties of epothi-
lones in 1995, the details of the binding poses for the struc-
turally diverse taxanes and epothilones have been pursued
in order to facilitate the rational design of improved and
perhaps structurally simplified analogues.[11–14] A variety of
epothilone conformations and tubulin-binding modes have
been proposed by pharmacophore mapping,[11,12] solution
NMR spectroscopy[15,16] and the superposition of epothilones
on taxanes in the electron crystallographic tubulin com-
plex.[13,14] By combining NMR spectroscopy, electron crystal-
lography and molecular modeling, our group proposed a
unique EpoA conformation and microtubule binding model
that offers an alternative to the common pharmacophore
model by describing the tubulin binding cavity as promiscu-
ous.[17] According to this model, epothilone and Taxol
occupy the same gross binding pocket, but the tubulin–
ligand binding is mediated through different sets of hydro-
gen bonds and hydrophobic interactions for the two com-
pounds. The electron crystallographic structure of epothi-
lone was superposed with that of Taxol bound to tubulin.
The overlap suggested that the thiazole moiety of epothi-
lone A and the benzoyloxy phenyl of Taxol do not reside in
the same region of the tubulin pocket. In addition, among
the five oxygen-containing polar groups in epothilone, only
the C7-OH falls near the similar C7-OH moiety in Taxol. In
this comparison, the latter is the only common center be-
tween the two molecules. An unusual feature of the EpoA
binding conformer derived by electron crystallography (EC)
is the presence of a syn-pentane interaction between the
methyl groups at C6 and C8. A conceivable test of the latter
structural feature introduces a short bridge between the ring
carbons to constrain the macrocycle to the EC model geom-
etry. The cyclohexane rings depicted in 5–8 (Figure 2) illus-
trate one solution to the problem.
pocket. To examine specific geometric details of the corre-
sponding structures, 5 and 6 in the proposed binding form
were optimized by molecular mechanics to show that both
reside in a stable local minimum. Subsequent docking of the
latter into the b-tubulin taxoid site suggested that the addi-
tional CH2 in the newly installed cyclohexane ring would
not experience undue steric congestion with the protein
(Figure 3). To our knowledge, modifications within the C6–
Figure 3. Docking poses of C6–C8 bridged epothilone analogues in the
electron crystallography-determined tubulin binding site: A) Docking
poses of 1 (yellow) and 5 (cyan); B) Docking poses of 1 (yellow) and 6
(blue). The shortest H—H contact between ligand and protein is 2.5 ꢁ,
an acceptable, though minimal, van der Waals separation.
C8 epothilone sector have received little previous attention
(Figure 4).[18–20] In a related study, however, Martin and co-
workers introduced a three carbon bridge between C4 and
C6 from the pro-R methyl at C4 in the EpoB framework (9,
Figure 4).[20] The compound had no effect when exposed to
the MCF-7 tumor cell line. Since the EC binding conformer
predicts the pro-S attachment to be the appropriate attach-
ment direction, stereochemical inversion may be responsible
for the lack of activity. Accordingly, bridged analogues 5–8
were selected as targets suitable for diagnostic tests of the
EC epothilone binding model. In addition, ZK-EPO (Sago-
pilone, 10) in which a vinyl moiety was introduced to the C6
methyl group and the C16–C21 side chain was replaced by a
benzothiazole moiety, is currently undergoing advanced clin-
ical development as the first fully synthetic epothilone can-
didate.[18] Previously, we briefly communicated the synthesis
of bridged 5, and reported the compound to be only weakly
active against the A2780 ovarian cancer cell line.[19] In the
present work, we describe the full synthetic details for 5–8.
All four analogues were further subjected to cytotoxicity as-
sessment against human ovarian (A2780) and prostate
cancer (PC3) cell lines, tubulin assembly and microtubule
cold stabilization.
A potential liability of this strategy is that the small ring,
expanding the volume of the epothilone, might introduce
steric congestion with the tubulin residues lining the binding
Figure 2. Structures of C6–C8 bridged epothilone analogues.
Figure 4. Selected epothilone analogues with modification around C6–C8.
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J. P. Snyder, W. Zhan et al.
Results and Discussion
presence of NaHCO3, homoallylic alcohol 17 was obtained
in 70% yield (d.r. >95% by 1H NMR).[24] The highly stereo-
selective epoxidation of 17 was first achieved by a homoal-
lylic alcohol-directed vanadium-catalyzed epoxidation strat-
egy to afford hydroxy epoxide 18 in 88% yield as a single
isomer. Further study indicated that mCPBA-based epoxida-
tion also delivered the desired epoxide 30 in 84% yield. The
relative configuration was confirmed by NOE. Considering
the difference between the optical rotation value of 17 and
that reported previously ([a]2D5 = ꢀ3.4, c = 1.0, CHCl3,
lit.:[23b] +6.83, l 0.5, neat), the p-nitrobenzoyl derivative 19
was prepared. X-ray crystallography of 19 unambiguously
confirmed the absolute configuration of alcohol 17 and hy-
droxy epoxide 18 (Scheme 2). The site of epoxide ring open-
ing by chloride anion further supports the original plan for
regioselective nucleophilic opening of hydroxy epoxide.
With successful stereoselective epoxidation, the next key
step in Scheme 2 is epoxide opening with an alkyl nucleo-
phile in a regioselective manner. Fortunately, this transfor-
mation was successfully performed by treatment of 18 with
freshly prepared 4-pentenylmaganesium bromide in the
presence of CuCN (10 mol%). The desired diol 20 was ob-
tained exclusively in 89% yield. We concur with Flippin and
co-workers[25] that the regioselectivity of this metal catalyzed
epoxide opening is not only controlled by the Fꢃrst-Plattner
rule favoring diaxial orientation, but also is most likely rein-
forced by a chelation process. Following the remarkable suc-
cess of the two key steps in the model study, we turned our
attention to probe the regioselective protection of the two
secondary hydroxy groups in 20 and the subsequent oxida-
tion of the sterically hindered secondary alcohol in the
model system. Selective silylation of the sterically less hin-
dered OH group in 20 was achieved by slow addition of tert-
butyldimethylsilyl triflate (TBSOTf) to a solution of 20 in
CH2Cl2 at ꢀ788C in the presence of 2,6-lutidine to provide
the mono-silyl ether 21 in 85% yield. Surprisingly, no silyla-
tion of the sterically hindered hydroxyl group was detected
even when 1.5 equiv of TBSOTf was added. At this stage, a
NOESY analysis for silyl ether 21 supported the previously
described regioselectivity of the oxirane opening and selec-
tive TBS protection (Scheme 2). Swern oxidation of the ster-
ically hindered alcohol afforded the desired olefinic ketone
22 in quantitative yield.
Synthesis of C4–C8 bridged epothilones: Retrosynthetic
analysis: The first-generation synthetic plan for the C6–C8
bridged epothilones, based on ring closure metathesis
(RCM) as a key step, is summarized in Scheme 1. Com-
pound 5 is used as an example. Although RCM is known to
give both cis and trans isomers during total syntheses of nat-
ural epothilones,[21] it was applied as a key step here consid-
ering that isolation of both isomers could contribute to the
structure–activity profile (SAR) for the compound series.
Applying a general disconnection strategy to epothilones,
the bridged target 5 could be traced back to alcohol 12 and
the advanced intermediate keto acid 13 after retrosynthetic
epoxidation, RCM and esterification. The preparation of
keto acid 13 was conceived as the key step along this route,
by which the cyclohexane core structure with three adjacent
chiral centers would be constructed. First, the stereochemis-
try at C7 and C8 in 13 was contemplated by means of se-
quential substrate directed epoxidation[22] and regiocontrol-
led epoxide opening from homoallylic alcohol 14. Moving
further along the retrosynthetic path, alcohol 14 was envi-
sioned to arise from aldehyde 15 by utilizing Brownꢂs asym-
metric cyclohexenylboration stratetgy.[23]
Scheme 1. Retrosynthetic analysis of C6–C8 bridged EpoA 5 by olefin
metathesis.
Construction of building blocks: Encouraged by the results
of the model studies, we proceeded to construct carboxylic
acid 13. In pursuit of this advanced intermediate, the previ-
ously reported silyl ether 23[19] was subjected to ozonolysis,
followed by acid-catalyzed acetal protection with ethylene
glycol and selective desilylation of the primary TBS silyl
ether to afford primary alcohol 24 in 56% yield over three
steps (Scheme 3). Exposure of 24 to Swern oxidation pro-
duced the desired aldehyde 15 in quantitative yield. At this
point, we were in a position to probe the feasibility of estab-
lishing the C5–C6 bond by Brownꢂs protocol.[23] Unfortu-
nately, when aldehyde 15 was subjected to the standard
Brown conditions, no workable amounts of product 14 could
Model study: To test the feasibility of the substrate-directed
epoxidation and subsequent regio-controlled epoxide open-
ing strategy, a simplified model system was studied as shown
in Scheme 2. The model study started from (ꢀ)-B-2-cyclo-
hexen-1-yldiisopinocampheylborane (16), prepared by treat-
ing cyclohexa-1,3-diene with diisopinocampheylborane de-
rived from (+)-a-pinene at ꢀ258C in tetrahydrofuran
(THF) as described by Brown et al.[23] The freshly prepared
solution of borane 16 in THF was cooled to ꢀ1008C and
treated with pivalaldehyde. After oxidation with H2O2 in the
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Epothilone Derivatives
FULL PAPER
desired homoallylic alcohol 26 was obtained in excellent
yield and selectivity (96%, d.r. >20:1 by 1H NMR) as
ꢀ
shown in Scheme 4. Surprisingly, both the C C bond forma-
ꢀ
tion and oxidative cleavage of the B O bond were unex-
pectedly sluggish, taking about three weeks. Stereochemistry
at C5 and C6 was assigned on the basis of the model study
(Scheme 2). With this chemistry in hand, the next phase in-
volved a crucial stereoselective epoxidation and subsequent
regioselective oxirane ring opening. Following the successful
strategy achieved in the model study, alcohol 26 underwent
chemoselective epoxidation by vanadium catalysis to pro-
vide hydroxy epoxide 27 in 93% yield (d.r. >20:1 by
1H NMR), followed by copper-catalyzed epoxide opening
with Grignard reagent to furnish diol 28 in 90% yield as the
sole diastereomer (Scheme 4). It is worth noting that an
excess of Grignard reagent (8–9 equiv) was required to min-
imize formation of the bromohydrin side product.[26] Selec-
tive silylation of the sterically less hindered OH group in 28
furnished silyl ether 29 (85% yield). The relative stereo-
chemistry of compound 29 was confirmed on the basis of
NOESY experiments. In practice, the conversion from 26 to
29 could be completed in 93% yield over three steps with-
out purification of the intermediates 27 and 28. To complete
Scheme 4, Swern oxidation converted the secondary alcohol
Scheme 2. Model study. a) Pivaldehyde, Et2O/THF, ꢀ1008C, 70%, d.r.
>95%. b) tBuOOH, VOACHTUNRGTNEUNG(acac)2 (cat.), CH2Cl2, 88%; or mCPBA,
CH2Cl2, 84%. c) CH2=CH
A
89%. d) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 85%. e) (COCl)2, Et3N,
DMSO, CH2Cl2, ꢀ788C ! RT, quant. f) 4-Nitrobenzoyl chloride, pyri-
dine, DAMP, THF, RT, 16%.
be separated (Scheme 3), while over 90% of aldehyde 15
was recovered before the oxidative quench. Attempts to fa-
cilitate the reaction by increasing temperature and reaction
Scheme 4. a) 1) HF/pyridine, THF, 08C ! RT, 88%; 2) (COCl)2, Et3N,
DMSO, CH2Cl2, ꢀ788C ! RT, 94 %. b) 16, THF, ꢀ788C, then H2O2,
Scheme 3. a) 1) O3, CH2Cl2, ꢀ788C, then PPh3, RT; 2) ethylene glycol,
PTSA (cat.), benzene, reflux; 3) HF/pyridine, THF, 08C ! RT, 53 % (3
steps). b) (COCl)2, Et3N, DMSO, CH2Cl2, ꢀ788C ! RT, quant.
NaHCO3, 408C, 92%, d.r. >20:1. c) tBuOOH, VOACHTUNRGTNE(GNU acac)2 (cat.), CH2Cl2,
93%, d.r. >20:1 d) CH2=CHACTHNUTRGNEUNG(CH2)3MgBr (8–9 equiv), CuCN (cat.), Et2O,
ꢀ55
! 08C, 90%. e) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 85%.
f) (COCl)2, Et3N, DMSO, CH2Cl2, ꢀ788C ! RT, 99 %.
time did not lead to satisfactory results. We presume the al-
lylboration is disfavored not only by steric hindrance from
the a-quaternary carbon of the aldehyde, but also by the co-
ordination between borane and the acetal oxygen atoms,
which in turn interrupts the interaction between borane and
aldehyde. To address this problem, we turned our attention
to an alternative aldehyde 25 (Scheme 4) in which a termi-
nal olefin replaces the acetal in 15 and, thereby, avoids the
potential coordination described above. Selective desilyla-
tion and subsequent Swern oxidation converted silyl ether
23 into the desired aldehyde 25. Upon treatment of the
modified aldehyde 25 with freshly prepared borane 16, the
to ketone 30 in quantitative yield. The stereochemical as-
signment for ketone 30 was confirmed subsequently by com-
parison with its analogue 50 (see Scheme 9), the stereo-
chemistry of which was determined by X-ray crystallography
of a derivative.[19] It is clear that aldehyde 25 has obvious ad-
vantages over aldehyde 15 in the context of cyclohexenylbo-
ration. However, application of 25 raised a second challeng-
ing problem, namely differentiation between the two termi-
nal olefins with high structural similarity in 30. As will be
shown, the terminal olefin homoallylic to OTBS could be se-
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J. P. Snyder, W. Zhan et al.
lectively converted to a carboxylic acid by taking advantage
of the masked homoallylic alcohol. Thus, we turned our at-
tention to the hydroxy-directed epoxidation to give a car-
boxylic acid precursor. To pursue this strategy, desilylation
of 30 with trifluoroacetic acid afforded diol 31 in 78% yield
(Scheme 5). Subsequently, vanadium catalyzed chemoselec-
tive epoxidation of 31, as expected, led to b-hydroxy epox-
ide 32 in 89% total yield as a mixture of two diastereomeric
elimination from the b-acetate of the carboxylic acid was re-
sponsible for the modest yield. However, b-elimination was
completely suppressed by a modified Yonemitsu–Yamaguchi
protocol,[30] giving keto ester 35 in 86% yield. Exposure of
35 to metathesis catalysts 39–42 under highly dilute condi-
tions resulted in clean formation of a single trans-macrocy-
clic olefin 36 (JH12–H13 =14.4 Hz). Grubbs catalysts 39 and 40
as well as the Hoveyda catalyst 41 gave the trans-product in
high yields, while the reaction was unresponsive to Hoveyda
catalyst 42 (entry 5, Scheme 6). It is widely known that the
E/Z selectivity of the ring closure metathesis depends on
many factors including substrate, solvent, temperature and
concentration.[31] In this specific case, attempts to modify the
geometric outcome of the reaction by choosing solvents and
temperatures as recorded in Table 1 were unsuccessful. The
1
epoxides (ca. 10:1 by H NMR). The stereochemistry of the
epoxide has been tentatively assigned in accord with the
proposed model by Mihelich and co-workers.[27] In view of
the subsequent cleavage of the epoxide, the diastereomeric
epoxides were subjected to the next step without separation.
At this stage, we initially attempted to reinstall the TBS silyl
ether onto 32 in order to pursue the original synthetic plan
(Scheme 1). However, all attempts with classical conditions
failed to give satisfactory results.[28] Fortunately, diacetyl ep-
oxide 33 was cleanly obtained in 93% yield by treatment of
alcohol 32 with acetic anhydride and 4-dimethylaminopyri-
dine (DMAP). At this point, it appeared timely to transfer
the primary epoxide to the carboxylic acid. The conversion
was accomplished with a three step sequence. The epoxide
first underwent tetrabutylammonium bisulfate catalyzed hy-
drolysis,[29] followed by NaIO4 cleavage of the resulting diol
to furnish an aldehyde intermediate. Purification of the al-
dehyde by silica gel flash column chromatography was un-
manageable due to the instability of this intermediate. Thus,
Pinnick oxidation of the crude aldehyde with NaClO2 in the
presence of 2-methyl-2-butene and NaH2PO4 in tBuOH/
H2O provided the desired carboxylic acid 34 in 45% yield
over the three-step procedure (Scheme 5).
Table 1. Ring closure metathesis of 35 gives trans-olefin 36 exclusively.
Entry
Cat. (10 mol%)
Conditions
Yield [%]
E/Z[a]
1
2
3
4
5
6
39
39
40
40
41
41
CH2Cl2, RT, 12 h
toluene, 808C, 12 h
CH2Cl2, RT, 12 h
toluene, 808C, 12 h
CH2Cl2, RT, 12 h
toluene, 808C, 12 h
1
84
76
quant
quant
<5
>20:1
>20:1
>20:1
>20:1
ND
95
>20:1
[a] The E/Z ratio was judged from crude H NMR.
disheartening geometric results from olefin metathesis tem-
porarily directed our attention to the 12,13-trans-6,8-bridged
epothilone C (37, Scheme 6). Considering previous SAR
studies suggesting that the non-natural epothilone analogue
12,13-trans-epothilone C is only slightly less active then the
natural epothilone C (3),[32] the C6–C8 bridged analogue 37
was regarded as potentially providing valuable structural in-
formation for the project. With this in mind, we turned to
the deacylation of 36 (Scheme 6). Surprisingly, none of the
reaction conditions applied was able to accomplish depro-
tection to produce the dihydroxyl lactone 37. In all cases,
either unreacted acetate was recovered or decomposition
took place. One of the major side reactions arising from at-
tempted deacylation was b-elimination leading to lactone
38, which could be alternatively prepared from 36 in 96%
yield by treatment with 8-diazabicycloACTHNUTRGNEUNG[5.4.0]undec-7-ene
Scheme 5. a) TFA, CH2Cl2, ꢀ20 ! 08C, 78%. b) tBuOOH, VO
ACHTUNGERTN(NUNG acac)2
(DBU). Such a process has been documented to suggest
that the approximate 1808 torsion angle around C2–C3
might be responsible for the elimination.[3,33] We presume
the unsuccessful deacylation could also arise from the com-
petition between hydrolysis and elimination of the b-acetate.
In this specific case, b-elimination might be much faster
than hydrolysis of the acetate. To facilitate deacylation, we
envisioned introducing a substituent to the acetyl moiety
which could increase the acetyl hydrolysis rate, while not
significantly altering its leaving group character. With this
scenario in mind, the chloroacetyl group was introduced,
recognizing that it might be 350–700 fold more quickly hy-
drolyzed than acetyl depending on the nature of the inter-
mediates.[34] As shown in Scheme 7, the required chloroace-
(cat.), CH2Cl2, 08C ! RT, 89%, d.r. 10:1 c) Ac2O, DMAP, CH2Cl2, 08C
! RT, 93 %. d) 1) nBu4NHSO4 (cat.), CH3CN/H2O, 50 8C; 2) NaIO4,
THF, RT; 3) NaClO4, NaH2PO4, 2-methyl-2-butene, tBuOH/H2O, 45% (3
steps).
Assembly of building blocks and synthesis of 12,13-trans-
6,8-bridged epothilone via olefin metathesis: With the key
building block 34 in hand, our attention was directed to the
feasibility of the olefin metathesis strategy. Therefore, the
coupling between alcohol 12 and carboxylic acid 34 was per-
formed under the influence of 1-ethyl-3-((dimethylamino)-
propyl)carbodiimide hydrochloride (EDCI) and DMAP to
furnish the proposed metathesis precursor 35 in 58% yield
as depicted in Scheme 6. In this coupling reaction, serious b-
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Epothilone Derivatives
FULL PAPER
keto acid 43 by alcohol 12 was achieved in modest yield
with the modified Yonemitsu–Yamaguchi protocol.[35] With-
out complication, the following olefin metathesis led cleanly
to trans-product 45 (JH12–H13 =14.8 Hz). For example, a 77%
yield of 45 was achieved with Hoveyda second-generation
metathesis catalyst 42. After screening various conditions
for the crucial deacylation step, we discovered that it could
be successfully performed by careful treatment of 45 with
ammonium hydroxide in methanol (1:10, v/v) followed by
treatment with ammonia in methanol to afford the 12,13-
trans-6,8-bridged EpoC analogue 37 in 57% yield
(Scheme 7). To bring the synthesis to a close, we initially at-
tempted to isomerize the C12–C13 trans double regioselec-
tively to the desired cis geometry. Unfortunately, following
attempts such as photoirradiated isomerization,[18] iodine-
catalyzed free radical isomerization[36] and Vedejs isomeriza-
tion,[37] no observable amounts of the isomerized product
could be separated (assuming isomerization occurred).
Either unreacted trans-olefin was recovered or decomposi-
tion took place.
Second-generation synthesis via Suzuki coupling: As dis-
cussed above, some surprising limitations surfaced in the
ring forming olefin metathesis reaction. Although there is
still much opportunity to optimize reaction conditions and
employ alternative methods such as the molybdenum-based
Schrock catalyst,[38] the epothilone literature teaches that
the stereochemical outcome of the RCM process is highly
substrate dependent.[21a,39] With such an ambiguous prece-
dent, it was imperative to select a reliable method for ac-
cessing the desired Z stereochemistry. An important alterna-
Scheme 6. a) 34, EDCI, DMAP, CH2Cl2, RT, 58%; or 2,4,6-trichloroben-
zoylchloride, DMAP, Et3N, toluene, ꢀ78 ! 08C, 86%. b) RCM, see text.
c) DBU, CH2Cl2, 08C ! RT, 96 %.
ꢀ
tive to introduce the Z-double bond at C12 C13 is Dani-
shefskyꢂs B-alkyl Suzuki coupling strategy.[40] Given the
widespread application of this strategy in epothilone synthe-
sis, we elected to explore its utility for our targets. The ret-
rosynthesis for bridged epothilones 5 and 6 via the second-
generation Suzuki coupling strategy is summarized in
Scheme 8.[41] The key disconnection for this route is at the
ꢀ
C11 C12 bond, leading to vinyl iodides 48/49 and olefin 50
as the Suzuki coupling partners. The keto diene 50 derives
from aldehyde 51 following a sequence similar to that for
the synthesis of dienyl ketone 30 in Scheme 4. In contrast to
keto diene 30, a gem-dimethyl moiety was introduced at the
right-side terminal olefin of 50 in order to easily differenti-
ate the two olefins.
Scheme 7. a) 1) (ClCH2CO)2O, DMAP, pyridine, CH2Cl2, 08C, quant.;
2) NaIO4/H5IO6, THF/H2O; 3) NaClO4, NaH2PO4, 2-methyl-2-butene,
tBuOH/H2O, 72% (2 steps). b) 2,4,6-trichlorobenzoylchloride, DMAP,
Et3N, toluene, ꢀ78 ! ꢀ358C, 49%. c) RCM, cat. 42, CH2Cl2, RT, 77%.
d) NH4OH/MeOH, 08C then NH3/MeOH, 08C, 57%.
Construction of building partners for Suzuki coupling: To
pursue this modified route, the preparation of Suzuki cou-
pling precursor 50 is illustrated in Scheme 9.[19] The modified
aldehyde 51 was first obtained by a four-step sequence from
23 in 58% yield, followed by allylboration of aldehyde 51
with freshly prepared (ꢀ)-B-2-cyclohexen-1-yldiisopinocam-
pheylborane (16) to give homoallylic alcohol 52 in 96%
tate was prepared from epoxy alcohol 32 in quantitative
yield. Subsequent exposure to NaIO4/H5IO6 mediated cleav-
age of the terminal epoxide to generate an aldehyde which
was subsequently subjected to Pinnick oxidation to furnish
the carboxylic acid 43 in 72% overall yield. Esterification of
1
yield (d.r. >20:1 by H NMR). Then, using the previously
validated vanadium-catalysis strategy, alcohol 52 was con-
verted to epoxy alcohol 53 in 93% yield (d.r. >20:1 by
1H NMR). Reaction of epoxide 53 with allylmagnesium bro-
Chem. Eur. J. 2011, 17, 14792 – 14804
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14797

J. P. Snyder, W. Zhan et al.
Scheme 8. Retrosynthesis of C6–C8 bridged EpoA/B by Suzuki coupling.
Scheme 9. a) See ref. [19]. b) 16, THF, ꢀ788C, then H2O2, NaHCO3,
408C, 96%, d.r >20:1. c) tBuOOH, VOACHTUNTRGNEUNG(acac)2 (cat.), CH2Cl2, 93%, d.r
mide in a copper-catalyzed fashion furnished epoxide-
opened product 54 (85% yield) along with a trace of the
C7-alkylated isomer and bromohydrin. The sterically less
hindered hydroxyl group from 54 was selectively converted
to TBS silyl ether 55 in 97% yield. At this point, a NOESY
analysis was executed to confirm the relative stereochemis-
try (Scheme 9). Finally, the sterically hindered secondary al-
cohol in diene 55 was transformed to dienyl ketone 50 by
Swern oxidation in 85% yield. The absolute configuration
of dienyl ketone 50 has been verified by X-ray crystal struc-
ture analysis of a derivative.[19] Preparation of the other
Suzuki coupling partners, vinyl iodides 48/49, was accom-
>20:1 d) AllylMgBr (8.0 equiv), CuCN (cat.), Et2O, ꢀ55 ! 08C, 85%.
e) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ788C, 97%. f) (COCl)2, Et3N, DMSO,
CH2Cl2, ꢀ788C ! RT, 85%. g) See ref. [39c, 42].
droxy lactones 60/61 by employing a procedure utilized by
Nicolaou et al. in the total synthesis of epothilones A and
B.[44] Selective desilylation with tetra-n-butylammonium
fluoride (TBAF), followed by Yamaguchi lactonization (62
in 51% yield, and 63 in 60% yield) and global desilylation
in the presence of a freshly prepared trifluoroacetic acid
(TFA) solution in CH2Cl2 (v/v, 1:4) gave C6–C8 bridged
epothilone C (7, 88% yield) and epothilone D (8, 91%
yield). Finally, we were pleased to obtain the C6–C8 bridged
epothilone A as a mixture of 5 and its cis-epoxide diastereo-
plished from alcohol 12 in
a
four-step sequence
(Scheme 9).[39c,42]
1
Completion of the synthesis of bridged epothilones A–D:
With the requisite coupling precursors in hand, the final
steps in the synthesis of bridged epothilones 5/6 were carried
out as depicted in Scheme 10. After regioselective hydrobo-
ration with 9-BBN, olefin 50 was coupled with vinyl iodides
48/49 in accordance with an approach reported by Danishef-
sky to furnish cis-olefins 46/47 (JH12–H13 =10.8 Hz, 46) in 92
and 57% yield, respectively.[40a] The following crucial regio-
selective dihydroxylation of trienes 46/47 was performed
under Sharpless asymmetric dihydroxylation conditions[43] to
selectively convert the gem-dimethyl olefin to diols 56/57 as
a mixture of diastereomers (56: 42% yield, 86% brsm, ca.
5:1 ratio by 1H NMR; 57: 42% yield, 87% brsm, ca. 4:1
ratio by 1H NMR). The stereochemistry of the hydroxyl
group was undefined. Without separation, the resulting mix-
ture of diols was subjected to NaIO4 mediated glycol cleav-
age and subsequent Pinnick oxidation to furnish the corre-
sponding carboxylic acids 58 and 59 in 78 and 58% yield, re-
spectively, after two steps (Scheme 10). As shown in
Scheme 10, final steps in the synthesis of the target com-
pounds 5/6 involved conversion of keto acids 58/59 to dihy-
mer 5a (84% total yield, ca. 2:1 ratio, H NMR) by treat-
ment with 3,3-dimethyldioxirane (DMDO) as described by
Danishefsky et al.[40a] Fortunately, these two diastereomers
were separable by preparative thin-layer chromatography.
In a similar fashion, the C6–C8 bridged epothilone D ana-
logue 8 was converted to the bridged epothilone B analogue
6 in 52% yield (d.r. >20:1 by H NMR). The stereochemis-
try of the epoxide of these bridged epothilones was deter-
mined by 1D and 2D NOE analysis (Scheme 10).
1
Bioactivity: Microtubule assembly and cytotoxicity: The
C6–C8 bridged epothilone analogues 5a, 5–8, 36–38 were
exposed to A2780 ovarian cancer and PC3 prostate cancer
cell lines to evaluate their antiproliferative properties
(Table 2). In general, these C6–C8 bridged compounds are
significantly less potent than the corresponding open chain
analogues. Against the A2780 cell line, compound 38 exhib-
ited the highest potency with an IC50 =1.1 mm, but it is still
27-fold less potent than EpoD. C6–C8 Bridged Epo B 6 ex-
hibited the highest potency against the PC3 prostate cancer
cell line with a 206-fold potency loss by comparison with the
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Epothilone Derivatives
FULL PAPER
Figure 5. Tubulin assembly and microtubule cold stabilizing activity of
^
^
bridged epothilones and PTX (!), 5a (~), 5 ( ), 6 ( ), 7 (*), 8 (~),
36 (!), 37 (&), 38 (&). Tubulin (10 mm) was incubated with 50 mm of a
bridged epothilone or 10 mm PTX in the presence of 1 mm GTP and 4%
DMSO in PME. Assembly was monitored in terms of apparent absorp-
tion at 350 nm. The arrow indicates temperature drop from 37 to 48C.
For reference, a tubulin sample in the absence of promoter (N) was also
included.
bridged epothilones showed significant ability to stabilize
the microtubules against cold induced disassembly.
Molecular mechanics and DFT energy evaluations: Obvious-
ly, these biological outcomes are not consistent with our
having installed a C6–C8 bridge that constrains 5–8 to an ef-
ficacious bioactive epothilone pose. There are several possi-
ble explanations for this: 1) the C6–C8 bridge has retained
the target conformation of the epothilone ring, but altered
the binding mode to prevent effective coordination with the
protein; 2) incorporation of the C6–C8 bridge has raised the
energy of the bound target conformations of 5–8 making
them inaccessible to microtubules; 3) the molecules have
been constrained as expected (Figure 3), but the target con-
formation is not the de facto bound form as proposed. Con-
cerning 1), there are precedents for loss of activity resulting
from internal bridge building in taxanes. In two cases, NMR
evidence supported the conclusion that the ligand conforma-
tions appear to be retained, but the additional tether inter-
feres sterically with the tubulin binding site, lifts the mole-
cule higher in the pocket and thereby reduces ligand bind-
ing.[45] In one of these reports involving cyclic constraints in
the C13 side chain, the differences in tubulin binding and
MCF7 cell cytotoxicity (5–10 fold and 37–120 fold, respec-
tively) arose from expansion of ring size from five members
to six.[50a] Thus, in spite of our initial assessment that a six-
membered ring in 5–8 would cause little steric congestion
(Figure 3), the degree of crowding may have been underesti-
mated. With respect to 2), we have examined the conforma-
tional landscapes of 1 and 5 to determine the energy differ-
ences between the respective bound conformations (Fig-
Scheme 10. a) 50, 9-BBN, Cs2CO3, AsPh3, [PdCl2ACHTNUGTRNEUNG(dppf)], DMF/H2O, RT,
46: 92%, 47: 57%. b) (DHQD)2PHAL, K2CO3, K3Fe(CN)6, CH3SO2NH2,
K2OsO4·2H2O, tBuOH/H2O, 0 8C! RT, 56: 42% (86%, BRSM), d.r. 5:1;
57: 42% (87%, brsm), d.r. 4:1. c) 1) NaIO4, THF/H2O, 0 8C; 2) NaClO4,
NaH2PO4, 2-methyl-2-butene, tBuOH/H2O, 58: 78% (2 steps), 59: 58%
(2 steps). d) TBAF, THF, 08C! RT, 60: 95%, 61: 90%. e) 2,4,6-Trichlor-
obenzoylchloride, Et3N, DMAP, toluene, RT, 62: 51%, 63: 60%. f) TFA,
CH2Cl2, ꢀ20 ! 08C, 7: 88%, 8: 91%. g) DMDO, CH2Cl2, ꢀ50
!
ꢀ308C, 5 (5a) 84%, d.r. 2:1, 6: 52%, d.r. >20:1.
activity of EpoD. The ability of C6–C8 bridged epothilones
to promote tubulin assembly and stabilize the microtubules
against cold induced disassembly was also studied using
50 mm of the compounds in 4% (v/v) DMSO. The results
were compared to that of 10 mm PTX under identical condi-
tions (Figure 5). Compounds 37
and 5a displayed the least
Table 2. Cytotoxicity of epothilone B, D and C6–C8 bridged epothilone analogues (IC50 [mm]).
effect on tubulin assembly.
Compounds 6 and 8 were the
most active, however, less so
than PTX. None of the C6–C8
Cpd.
A2780 0.04
PC3
EpoD
5a
5
6
7
8
36
37
38
24.3
8.5
3.6
19.0
5.1
5.6
9.6
1.1
0.016ꢁ0.001 10.3ꢁ1.6 11.6ꢁ0.3 3.3ꢁ0.2 16.3ꢁ1.8 6.7ꢁ0.2 7.6ꢁ0.2 10.7ꢁ0.6 9.8ꢁ0.8
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14799

J. P. Snyder, W. Zhan et al.
ure 3A) and their corresponding global minima (GM).
Thus, individual 25000 step conformational searches for the
two structures were performed with the MMFF/GBSA/H2O
protocol in Schrodingerꢂs Macromodel.[46] The GM were
then re-optimized with the OPLS-2005, MM3 and AMBER
molecular mechanics methods.[46] The EpoA EC structure
(1, Figure 3A) and that of 5 based on it were likewise opti-
mized with the same four methods to put the structures
(LM or local minimum forms) on the same energy scales. A
comparison of the EC-related structures and the optimized
variants demonstrated minimal average root mean square
deviations (RMSD) of 0.098 to 0.35 ꢁ for all heavy atoms.
Superposition of the EC-based structures and the optimized
OPLS-2005 conformers (average heavy-atom RMSD values
of 0.26 and 0.14 ꢁ, respectively) are provided in the Sup-
porting Information to demonstrate essentially a perfect
match between the EC bound and force-field optimized con-
formers. The energy differences between GM and LM for
lines. The cytotoxicity data implies that these epothilone an-
alogues are considerably less potent than the natural epothi-
lones. The tubulin assembly and microtubule cold stabiliza-
tion assay reveal that compounds 6 and 8 inhibit tubulin as-
sembly weakly, while none of the bridged epothilone ana-
logues show significant microtubule stabilization against
cold induced disassembly. Possible causes for the poor activ-
ity of the bridged epothilones include steric congestion be-
tween tubulin and the C6–C8 bridge, torsional strain in the
flexible portion of the epothilone ring raising the energy re-
quirement for binding and a mismatch with the empirical
binding pose. Insights into the first two explanations are
provided.
Experimental Section
General: Unless otherwise noted, commercial reagents and solvents were
used as received unless otherwise noted. Flash column chromatography
was performed by employing either Sorbent Technologies 200–400 mesh
or Waterman 230–400 mesh silica gel 60. Analytical thin-layer chroma-
tography (TLC) was performed on pre-coated silica gel 60 F254
(0.25 mm thick) from EM Science. TLC plates were visualized by expo-
sure to ultraviolet light (UV) and/or exposure to phosphomolybdic acid
or potassium permanganate TLC stains followed by brief heating on a
hot plate. Preparative TLC separation was performed on Analtech prepa-
rative plates pre-coated with silica gel 60 UV254 (0.5, 1.0 or 1.5 mm
thick). Melting points (m.p.), determined on a MEL-TEMP Melting
Point Apparatus from Laboratory Devices, are uncorrected. Optical rota-
tions were measured on a Perkin–Elmer Model 341 digital polarimeter
with a sodium lamp at room temperature. Infrared (IR) spectra were re-
corded on a Nicolet 370 with a diamond probe or an ASI ReactIR 1000
FI-IR Spectrophotometer with a silicone probe (wavenumbers (cmꢀ1)).
Where noted “neat”, the sample was loaded as a thin film. Proton nucle-
ar magnetic resonance (1H NMR) spectra and carbon nuclear magnetic
resonance (13C NMR) spectra were determined on an INOVA400
(1H NMR: 400 MHz, and 13C NMR: 100 MHz) or INOVA600 (1H NMR:
600 MHz, and 13C NMR: 150 MHz) instrument. Chemical shifts for
1H NMR are reported in parts per million (d=scale) with deuterated
chloroform (CDCl3) as the internal standard (7.26 ppm) and coupling
constants are in Hertz (Hz). The following abbreviations are used for
spin multiplicity: s=singlet, d=doublet, t=triplet, q=quartet, m=mul-
tiplet, brs=broad singlet. Chemical shifts for 13C NMR are reported in
parts per million (d=scale) relative to the central line of the triplet at
77.23 ppm for deuterated chloroform (CDCl3). High resolution mass
spectra (HRMS) were obtained on a JEOL JMS-SX102/SX102A/E or
Thermo Finnigan LTQ-FTMS instrument. Experimental details, charac-
terization data for all new compounds, NMR spectra of key intermedi-
ates, X-ray crystal structure data for 19 and results for energy calcula-
tions for 1 and 5 are available in the Supporting Information.
epoA and 5 for each method (DDE
N
ꢀ
LM)5) were computed to show that epoA falls lower by 3.0
(OPLS-2005), 2.4 (AMBER), 1.1 (MMFF) and 0.8 (MM3)
kcalmolꢀ1 (see the Supporting Information for details).
Thus, in the molecular mechanics regime, the bridged struc-
ture 5 is predicted to require an additional 1.8 kcalmolꢀ1 on
average relative to epoA to achieve the bound form on tu-
bulin in the context of the EC structure. In order to avoid
issues surrounding the variable parameterization of the
force field protocols, we calculated the energies of the
OPLS-2005 optimized GM and LM forms for EpoA (1) and
5 with the density functional protocol B3LYP/6-31G*. The
DDE=5.5 kcalmolꢀ1 reinforces the force field trend and
suggests that the weaker binding for compound 5 may be a
result of increased internal conformational strain energy rel-
ative to EpoA. The possibility remains that 3) is the under-
lying cause of the bio-data presented here. However, until a
definitive structure of the tubulin–epothilone complex is de-
termined, the current EC[17] and NMR[47] structures remain
the front-line contenders for the bound conformation of
epothilones to b-tubulin.
Conclusion
A series of conformationally restrained epothilone ana-
logues with a short bridge between methyl groups at C6 and
C8 was designed to mimic the binding pose derived for our
recently reported EpoA–microtubule binding model. A ver-
satile synthetic route to these bridged epothilone analogues
has been successfully devised and implemented. The key ste-
reochemistry within the bridged C6–C8 sector was con-
trolled by asymmetric allyboration followed by hydroxy-di-
rected epoxidation and regiocontrolled opening of the resul-
Molecular modeling and docking: The 3D structure of bridged epothi-
lones 5 and 6 were constructed based on the electron crystallographic
(EC) pose of EpoA bound to a b-tubulin.[17] The resulting structure of 5
and 6 was then fully optimized with the MMFF/GBSA/H2O force field to
provide the nearest local minimum. The latter was flexibly Glide-
docked[48] into the electron crystallographic structure of EpoA–tubulin.[17]
The best docking pose was chosen on the basis of the Glide scoring func-
tion together with visualization to ensure a reasonable binding mode and
match with the EC complex. Conformational analysis for 1 and 5 and
energy evaluations are described in the Supporting Information.
ꢀ
tant epoxide. The cis-C12 C13 double was constructed via
trans-12,13-Macrolactone 36
Suzuki coupling while the ring-closure metathesis exclusive-
ly gave trans selectivity. The C6–C8 bridged epothilones
were evaluated for their biological activity against the
A2780 human ovarian cancer and PC3 prostate cancer cell
Procedure A: To a solution of diene 35 (7.5 mg, 0.0125 mmol, 1.0 equiv)
in CH2Cl2 (12.5 mL, 0.001m) was added Grubbs catalyst
I (1.1 mg,
0.00125 mol, 10 mol%), and the reaction mixture was allowed to stir at
258C for 12 h. After the completion of the reaction as established by
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Epothilone Derivatives
FULL PAPER
TLC, the solvent was removed under reduced pressure and the crude
product was purified by preparative thin-layer chromatography (hexanes/
ethyl acetate 3:1) to afford the trans-lactone 36 (6.0 mg, 84%) as a white
foam.
08C. After being stirred for 48 h, 1H NMR suggested the reaction was
complete. The solvent was removed under reduced pressure and the resi-
due was purified by preparative thin-layer chromatography (hexanes/
ethyl acetate 15:4) to afford hydroxy lactone 37 (26 mg, 57%) as a white
foam. Rf =0.38 (CH2Cl2/MeOH 15:1); [a]2D2 = ꢀ11.6 (c = 0.85, CHCl3);
1H NMR (600 MHz, CDCl3): d=6.96 (s, 1H, SCH=C), 6.52 (s, 1H, CH=
CCH3), 5.48 (dd, J=9.8, 3.7 Hz, 1H, CHOC(O)), 5.45–5.40 (m, 1H, CH=
CH), 5.37–5.32 (m, 1H, CH=CH), 4.41 (dd, J=10.5, 1.8 Hz, 1H,
Procedure B: To a solution of diene 35 (15 mg, 0.0249 mmol, 1.0 equiv)
in toluene (25 mL) was added with Grubbs catalyst
I (2.1 mg,
0.00249 mol, 10 mol%), and the reaction mixture was allowed to stir at
808C for 12 h. After the reaction is complete, the mixture was worked up
according to the procedure described in procedure A to furnish 36
(10.9 mg, 76%).
CHOHC
ACHTUNGTRENNUNG
CHOHC
AHCTUNGTRENNUNG
10.4 Hz, 1H, CHC(O)), 2.70 (s, 3H, N=C(S)CH3), 2.50–2.42 (m, 3H),
2.24–2.19 (m, 1H), 2.17 (d, J=17.0 Hz, 1H, CH2CO2), 2.07 (s, 3H, CH=
CCH3), 1.93–1.83 (m, 3H), 1.73 (brs, 1H), 1.59–1.52 (m, 3H), 1.51–1.43
(m, 1H), 1.34–1.22 (m, 5H), 1.21–1.12 (m, 2H), 1.02 ppm (s, 3H, C-
Procedure C: Diene 35 (13 mg, 0.0216 mmol, 1.0 equiv) was converted to
36 (12.2 mg, 100%) in accordance with the procedure described in proce-
dure A except for the use of Grubbs catalyst II (1.8 mg, 0.0022 mol,
10 mol%).
AHCTUNGTRENNUNG
(CH3)2); 13C NMR (100 MHz, CDCl3): d=221.27, 173.30, 165.04, 152.42,
Procedure D: Diene 35 (13 mg, 0.0216 mmol, 1.0 equiv) was converted to
36 (10.1 mg, 100%) in accordance with the procedure described in proce-
dure B except for the use of Grubbs catalyst II (1.8 mg, 0.0022 mol,
10 mol%).
137.41, 134.02, 126.68, 120.60, 116.80, 79.52, 71.59, 71.53, 53.85, 43.83,
38.41, 37.97, 36.69, 31.70, 28.14, 27.45, 24.84, 24.27, 22.49, 20.64, 19.46,
16.05, 15.12 ppm; IR (Nujol): n˜ =3486 (br), 2930, 2860, 1729, 1679, 1505,
1444, 1405, 1374, 1336, 1297, 1247, 1177, 1123, 1085, 1046, 984, 915, 865,
726, 676 cmꢀ1; HRMS: m/z: calcd for C27H40NO5S: 490.26272 [M+H]+;
found 490.26064.
Procedure E: Diene 35 (12 mg, 0.02 mmol, 1.0 equiv) was converted to
36 (10.8 mg, 95%) in accordance with the procedure described in proce-
dure A except for the use of Hoveyda–Grubbs catalyst II (0.6 mg,
0.002 mol, 5 mol%). The crude reaction mixtures in procedures A, B, C,
D and E were determined to be >20:1 ratio of diastereomeric trans-
olefin by 1H NMR spectroscopy. Rf =0.37 (hexanes/ethyl acetate 2:1);
[a]2D2 = ꢀ47.8 (c = 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=6.97 (s,
1H, SCH=C), 6.50 (s, 1H, CH=CCH3), 5.87 (dd, J=7.2, 5.2 Hz, 1H,
CH2CHOAc), 5.57 (ddd, J=14.4, 7.2, 7.2 Hz, 1H, CH=CH), 5.47 (ddd,
J=14.4, 7.2, 7.2 Hz„ 1H, CH=CH), 5.31 (dd, J=9.4, 1.9 Hz, 1H, CHOC-
(O)CH2), 5.07 (s, 1H, CHCHOAc), 3.29–3.23 (m, 1H, CHC(O)), 2.69 (s,
3H, N=C(S)CH3), 2.71–2.68 (m, 1H, CH2CO2), 2.62–2.56 (m, 2H), 2.41
(dd, J=15.3, 4.9 Hz, 1H), 2.21–2.15 (m, 1H), 2.10 (s, 3H, ArCH=CCH3),
2.05 (s, 3H, CH3CO2), 2.03 (s, 3H, CH3CO2), 1.98–1.89 (m, 2H), 1.86–
1.76 (m, 1H), 1.72–1.67 (m, 2H), 1.57–1.493 (m, 3H), 1.40–1.35 (m, 2H),
Bridged epothilone C (7): A solution of Yamaguchi lactonization product
62 (89 mg, 0.124 mmol) in CH2Cl2 (0.1 mL) was treated with a freshly
prepared CF3CO2H/CH2Cl2 (0.73 mL, v/v, 1:4) at ꢀ208C. The reaction
mixture was allowed to reach 08C in 20 min and was stirred for addition-
al 1 h at that temperature at which time all silyl ether disappeared from
TLC plate. The mixture was diluted with CH2Cl2 (5 mL) and carefully
neutralized by saturated aqueous NaHCO3. After separation, the aque-
ous phase was further extracted with CH2Cl2 (2ꢄ5 mL). The combined
organics were dried over MgSO4, filtered and concentrated. The resulting
residue was purified by preparative thin-layer chromatography (CH2Cl2/
MeOH 20:1) to afford pure desired epothilone C analogue 7 (53.4 mg,
88%) as a colorless oil. Rf =0.36 (CH2Cl2/MeOH 15:1); [a]2D2 = ꢀ86.8 (c
= 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=6.97 (s, 1H, SCH=C),
6.62 (s, 1H, CH=CCH3), 5.50 (ddd, J=10.5, 10.5, 5.0 Hz, 1H, CH=CH),
5.40 (ddd, J=10.5, 10.5, 5.0 Hz, 1H, CH=CH), 5.22 (d, J=8.3 Hz, 1H,
1.33–1.21 (m, 2H), 1.14 (s, 3H, CACTHNUTRGENNUG(CH3)2), 1.07 ppm (s, 3H, CCAHTUNGTREN(NUNG CH3)2);
13C NMR (100 MHz, CDCl3): d=211.62, 170.71, 170.27, 169.21, 164.79,
152.88, 137.68, 132.67, 126.91, 119.64, 116.73, 79.62, 71.33, 70.48, 53.62,
41.94, 37.83, 37.71, 36.50, 31.30, 28.82, 26.94, 24.99, 24.09, 21.42, 21.24,
20.00, 19.89, 19.50, 18.87, 15.39 ppm; IR (Nujol): n˜ =2926, 2862, 1731,
1707, 1504, 1443, 1371, 1239, 1180, 1029, 972, 916, 731 cmꢀ1; HRMS: m/z:
calcd for C31H44NO7S: 574.28385 [MꢀH]+; found 574.28292.
CHOC(O)), 4.42 (dd, J=11.5, 1.8 Hz, 1H, CHOHCACHTNUGTRNEG(UN CH3)2), 4.20 (s, 1H,
CHCHOH), 3.89 (s, 1H, OH), 3.49 (s, 1H, OH), 2.98 (d, J=10.8 Hz, 1H,
CHC(O)), 2.76–2.63 (m, 4H), 2.50 (dd, J=14.7, 11.6 Hz, 1H, CH2CO2),
2.32 (dd, J=14.7, 2.3 Hz, 1H, CH2CO2), 2.30–2.24 (m, 1H), 2.18 (tt, J=
10.7, 7.6 Hz, 1H), 2.08 (s, 3H, CH=CCH3), 2.04–1.85 (m, 3H), 1.84–1.74
trans-2,3-Keto lactone 38:
A mixture of macrolactone 36 (21 mg,
(m, 1H), 1.66–1.43 (m, 4H), 1.36–1.29 (m, 2H), 1.28 (s, 3H, C
ACHTUNGTRENNUNG(CH3)2),
0.0366 mmol, 1.0 equiv) in anhydrous CH2Cl2 (2 mL) was treated with
DBU (55.7 mg, 0.366 mmol, 10.0 equiv) at room temperature. After
being stirred for 3 h, no more 36 was detected from TLC. The solvent
was removed under reduced pressure without further workup. The resul-
tant residue was purified by preparative thin-layer chromatography (hex-
anes/ethyl acetate 4:1) to furnish product 38 (18.4 mg, 96%) as a color-
less oil. Rf =0.51 (hexanes/ethyl acetate 2:1); [a]2D2 = +17.4 (c = 1.68,
CHCl3); 1H NMR (600 MHz, CDCl3): d=7.40 (d, J=16.0 Hz, 1H, CH=
CHC(O)), 6.96 (s, 1H, SCH=C), 6.61 (s, 1H, CH=CCH3), 6.07 (d, J=
16.0 Hz, 1H, CH=CHC(O)), 5.56 (dd, J=10.3, 2.2 Hz, 1H, CHOC-
(O)CH2), 5.53–5.37 (m, 2H, CH2CH=CH), 4.86 (s, 1H, CHCHOAc),
3.01–2.98 (m, 1H, CHC(O)), 2.71 (s, 3H, N=C(S)CH3), 2.52–2.49 (m,
1H), 2.44–2.39 (m, 1H), 2.19–2.14 (m, 1H), 2.11 (s, 3H, ArCH=CCH3),
2.00 (s, 3H, CH3CO2), 2.04–1.95 (m, 1H), 1.93–1.85 (m, 1H), 1.69–1.62
(m, 2H), 1.58–1.52 (m 1H), 1.50–1.37 (m, 3H), 1.26–1.12 ppm (m, 10H);
13C NMR (100 MHz, CDCl3): d=210.45, 170.59, 165.38, 164.88, 152.72,
152.39, 138.22, 132.55, 127.20, 121.98, 112.00, 116.39, 77.77, 71.34, 51.91,
43.51, 39.24, 36.58, 33.09, 28.54, 26.72, 23.64, 23.29, 23.00, 22.79, 21.38,
19.46, 15.61 ppm; IR (Nujol): n˜ =2929, 2861, 1713, 1645, 1503, 1444, 1379,
1362, 1294, 1242, 1177, 1048, 1017, 992, 970, 913, 879, 731 cmꢀ1; HRMS:
m/z: calcd for C29H40NO5S: 514.26272 [M+H]+; found 574.26186.
1.26–1.16 (m, 2H), 1.06 ppm (s, 3H, C
AHCTUNGTRENNUNG
CDCl3): d=220.74, 170.52, 165.35, 152.04, 139.43, 133.17, 125.23, 119.44,
115.82, 78.76, 73.05, 69.62, 54.22, 43.97, 39.79, 39.54, 31.91, 30.05, 28.83,
28.10, 25.17, 23.92, 23.58, 20.85, 19.25, 17.77, 16.26 ppm; IR (Nujol): n˜ =
3478 (br), 2928, 2860, 1736, 1678, 1507, 1443, 1409, 1291, 1248, 1187,
1084, 1046, 982, 913, 731 cmꢀ1; HRMS: m/z: calcd for C27H40NO5S:
490.26272 [M+H]+; found 490.26144.
Bridged epothilone A (5) and (5a): To a solution of bridged epothilone
C (7) (23 mg, 0.047 mmol, 1.0 equiv) in dry CH2Cl2 (2 mL) at ꢀ508C was
added a freshly prepared dry solution of 3,3-dimethyldioxirane (1.18 mL,
ca. 0.094 mmol, 0.08m in acetone, 2.0 equiv). The resulting solution was
allowed to warm to ꢀ308C for 2 h. A stream of argon was then bubbled
through the solution to remove excess dimethyldioxirane. The crude mix-
ture was determined to be a mixture of diastereomeric cis-epoxides (ca.
5:2 ratio by 1H NMR).Preparative thin-layer chromatography (CH2Cl2/
MeOH 20:1) to afford bridged epothilone A (5) (13.0 mg, 55%) as a
white foam and the cis-epoxide diastereomer 5a (7.0 mg, 29%) as a
white solid. 5: Rf =0.34 (CH2Cl2/MeOH 15:1); [a]2D2 = ꢀ26.9 (c = 0.87,
CHCl3); 1H NMR (600 MHz, CDCl3): d=6.98 (s, 1H, SCH=C), 6.61 (s,
1H, CH=CCH3), 5.35 (dd, J=9.9, 1.5 Hz, 1H, CHOC(O)), 4.38 (d, J=
10.3 Hz, 1H, CHOHC
OH), 3.83 (brs, 1H, CHCHOH), 3.03 (ddd, J=9.6, 3.0, 3.0 Hz, 1H,
CH2CH-O(epoxide)CH), 2.98 (d, J=10.6 Hz, 1H, CHC(O)), 2.96–2.93
(ddd, J=9.6, 3.0, 3.0 Hz, 1H, CH2CH-O(epoxide)CH), 2.68 (s, 3H, N=
ACHTUGNERTN(NUNG CH3)2), 4.31 (s, 1H, CHCHOH), 4.00 (s, 1H,
trans-12,13-Hydroxy lactone 37: A solution of chlorolactone 45 (60 mg,
0.093 mmol) in methanol (10 mL) at 08C was treated with ammonium
hydroxide (0.5 mL), and stirred at that temperature until the reaction
was complete (ca. 12 h). The solvent was removed under reduced pres-
sure to give white foam. Next, the white foam was dissolved in methanol
(10 mL), and treated with amino methanol (1 mL, 7n in methanol) at
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
C(S)CH3), 2.52 (dd, J=14.5, 11.3 Hz, 1H, CH2CO2), 2.30 (dd, J=14.5,
2.5 Hz, 1H, CH2CO2), 2.24–2.18 (m, 1H), 2.09 (s, 3H, CH=CCH3), 2.08–
2.02 (m, 1H), 1.95–1.84 (m, 3H), 1.80 (ddd, J=15.0, 9.9, 9.9 Hz, 1H),
Chem. Eur. J. 2011, 17, 14792 – 14804
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
14801

J. P. Snyder, W. Zhan et al.
1.68 (ddd, J=25.4, 12.1, 5.3 Hz, 1H), 1.63–1.56 (m, 2H), 1.56–1.46 (m,
53.94, 43.76, 40.21, 39.76, 33.06, 32.68, 30.92, 25.47, 25.33, 24.77, 23.50,
23.00, 21.14, 19.23, 17.70, 16.18 ppm; IR (Nujol): n˜ =3470 (br), 2934,
2864, 1737, 1679, 1505, 1467, 1444, 1413, 1382, 1324, 1293, 1251, 1177,
2H), 1.44–1.39 (m, 1H), 1.38–1.22 (m, 6H), 1.08 ppm (s, 3H, CACHTNUGTRNEG(UN CH3)2);
13C NMR (100 MHz, CDCl3): d=220.87, 170.45, 165.54, 151.68, 138.84,
120.16, 116.19, 76.96, 72.93, 68.08, 57.70, 55.75, 54.11, 43.80, 39.82, 39.65,
31.89, 30.90, 27.98, 25.34, 25.26, 24.74, 23.36, 21.04, 19.25, 17.96,
16.07 ppm; IR (Nujol): n˜ =3462 (br), 2930, 2864, 1731, 1679, 1509, 1444,
1154, 1073, 1042, 984, 941, 880, 760 cmꢀ1
; HRMS: m/z: calcd for
C28H42NO6S: 520.27328 [M+H]+; found 520.27228.
Tubulin purification: Tubulin was prepared by two cycles of temperature
dependent assembly-disassembly as described by Williams and Lee.[49]
Determination of protein concentration was carried out as previously de-
scribed.[50]
1413, 1390, 1293, 1258, 1181, 1154, 1085, 1046, 980, 919, 725 cmꢀ1
;
HRMS: m/z: calcd for C27H40NO6S: 506.25763 [M+H]+; found
506.25654. cis-Epoxide diastereomer 5a: Rf =0.34 (CH2Cl2/MeOH 15:1);
[a]2D2 = ꢀ58.9 (c = 1.0, CHCl3); 1H NMR (600 MHz, CDCl3): d=6.99 (s,
1H, SCH=C), 6.65 (s, 1H, CH=CCH3), 5.60 (t, J=3.9 Hz, 1H,
Tubulin polymerization assay: Tubulin assembly was monitored by appar-
ent absorption at 350 nm. Tubulin samples (10 mm) were incubated at
378C in PME buffer (100 mm PIPES, 1 mm MgSO4, and 2 mm EGTA)
containing 1 mm GTP in the spectrometer until a baseline was obtained.
The ligand to be tested in DMSO was added a final concentration of
50 mm (4% DMSO). After the assembly reached steady state, the temper-
ature was dropped to 48C. A tubulin sample (10 mm) with 4% DMSO
and no promoter was used as the reference.
CHOC(O)), 4.36 (dd, J=11.1, 1.9 Hz, 1H, CHOHC
CHCHOH), 3.99 (s, 1H, OH), 3.80 (brs, 1H, OH), 3.24 (ddd, J=7.4, 4.4,
4.4 Hz, 1H, CH2CH-O(epoxide)CH), 3.11 (dd, J=12.3, 1.7 Hz, 1H,
CHC(O)), 3.05–3.02 (m, 1H, CH2CH-O(epoxide)CH), 2.69 (s, 3H, N=
ACHTUGNTRENUN(NG CH3)2), 4.16 (s, 1H,
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
C(S)CH3), 2.56 (dd, J=14.7, 11.2 Hz, 1H, CH2CO2), 2.37 (dd, J=14.6,
2.6 Hz, 1H, CH2CO2), 2.10 (s, 3H, CH=CCH3), 2.09–1.96 (m, 3H), 1.93–
1.82 (m, 3H), 1.66–1.46 (m, 6H), 1.39–1.18 (m, 6H), 1.08 ppm (s, 3H, C-
Determination of cytotoxic activity: Cytotoxicity of bridged epothilones
in PC3 cells was assessed with the SRB assay.[51] The A2780 ovarian
cancer cell line assay was performed at Virginia Polytechnic Institute and
State University as previously reported.[52]
ACHTUNGTRENNUNG
(CH3)2); 13C NMR (100 MHz, CDCl3): d=221.01, 169.96, 165.32, 151.91,
136.90, 119.81, 116.12, 75.86, 72.88, 68.25, 57.23, 54.24, 53.79. 44.18, 39.78,
39.59, 30.83, 29.01, 28.28, 25.42, 25.06, 24.51, 22.20, 21.08, 19.27, 18.82,
16.31 ppm; IR (Nujol): n˜ =3466 (br), 2926, 2860, 1737, 1679, 1556, 1509,
1447, 1413, 1390, 1324, 1297, 1254, 1189, 1150, 1085, 1042, 1004, 984, 953,
919 cmꢀ1; HRMS: m/z: calcd for C27H40NO6S: 506.25763 [M+H]+; found
506.25659. The direction of the epoxide was further determined by 1D
and 2D NOE.
Bridged epothilone D (8): The desired C6–C8 bridged epothilone D (8)
was prepared from bis(silyl ether) macrolactone 63 (205 mg, 0.28 mmol)
by treatment with CF3CO2H according to the same procedure described
above for the preparation of 7, to obtain pure lactone 8 (137 mg, 91%)
Acknowledgements
The work was supported in part by the NIH Grant No. CA-69571. We
thank Kenneth Hardcastle (Emory University) for determination of the
X-ray crystal structure of compound 19.
as a colorless oil or white foam. Rf =0.46 (CH2Cl2/MeOH 15:1); [a]D22
=
ꢀ109.9 (c = 1.35, CHCl3); 1H NMR (600 MHz, CDCl3): d=6.95 (s, 1H,
SCH=C), 6.60 (s, 1H, ArCH=CCH3), 5.13 (m, 2H, CHOC(O), CH2CH=
CCH3), 4.62–4.39 (m, 1H, CHOHCACHTUNTRGENNUG(CH3)), 4.28 (s, 1H, CHCHOH), 3.87
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(s, 1H, OH), 3.69 (d, J=5.9 Hz, 1H, OH), 2.98 (dd, J=12.4, 2.2 Hz, 1H,
CHC(O)), 2.67 (s, 3H, N=C(S)CH3), 2.62 (ddd, J=15.2, 10.1, 10.1 Hz,
1H), 2.44 (dd, J=14.2, 11.4 Hz, 1H, CH2CO2), 2.35–2.30 (m, 1H), 2.26–
2.21 (m, 2H), 2.06 (d, J=1.1 Hz, 3H, ArCH=CCH3), 2.02–1.85 (m, 2H),
1.81–1.74 (m, 2H), 1.67 (s, 3H, CH=CCH3CH2), 1.62–1.44 (m, 5H), 1.38–
1.19 (m, 6H), 1.04 ppm (s, 3H, CACHTNUGRTNEUNG
(CH3)2); 13C NMR (100 MHz, CDCl3):
[3] For recent general reviews on epothilones, see: a) K. H. Altmann,
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see: a) A. Rivkin, Y. S. Cho, A. E. Gabarda, F. Yoshimura, S. J. Dan-
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d=221.06, 170.48, 165.33, 151.94, 140.02, 138.48, 121.17, 119.17, 115.61,
79.13, 73.02, 69.77, 54.28, 43.81, 40.03, 39.82, 32.89, 32.13, 29.93, 27.03,
25.26, 23.91, 23.88, 23.39, 20.89, 19.18, 17.02, 16.28 ppm; IR (Nujol): n˜
=3435 (br), 2934, 2864, 1733, 1679, 1509, 1447, 1413, 1378, 1336, 1293,
1251, 1185, 1143, 1081, 1046, 984, 938, 914, 849, 714, 683 cmꢀ1; HRMS:
m/z: calcd for C28H42NO5S: 504.27837 [M+H]+; found 504.27762.
C6–C8 bridged epothilone B (6): To a solution of bridged desoxyepothi-
lone B (8) (0.20 mg, 0.04 mmol, 1.0 equiv) in CH2Cl2 (0.4 mL) at ꢀ788C
was added freshly prepared 3,3-dimethyldioxirane (1.0 mL, ca.
0.08 mmol, ca. 0.08m in acetone, 2.0 equiv) dropwise. The resulting solu-
tion was warmed to ꢀ508C for 1 h, and another portion of dimethyldiox-
irane (0.4 mL, 0.032 mmol) was added. After stirring at ꢀ508C for addi-
tional 2.5 h, A stream of argon was then bubbled through the solution at
ꢀ508C to remove excess dimethyldioxirane and solvent. The crude reac-
tion mixture was determined to be >20:1 ratio of diastereomeric cis-ep-
oxides by 1H NMR spectroscopy. The resulting residue was purified by
preparative thin-layer chromatography (CH2Cl2/MeOH 30:1) to afford
bridged epothilone B (6) (10.8 mg, 52%) as a white foam. Rf =0.39
(CH2Cl2/MeOH 15:1); [a]D22
(600 MHz, CDCl3): d=6.98 (s, 1H, SCH=C), 6.61 (s, 1H, ArCH=CCH3),
5.33 (dd, J=8.9, 2.7 Hz, 1H, CHOC(O)), 4.43 (d, J=10.0 Hz, 1H,
=
ꢀ57.8 (c
=
1.0, CHCl3); 1H NMR
CHOHC
OH), 3.01 (dd, J=12.1, 1.8 Hz, 1H, CHC(O)), 2.79 (dd, J=9.1, 2.8 Hz,
1H, CH2CH-O(epoxide)C), 2.68 (s, 3H, N=C(S)CH3), 2.50 (dd, J=14.2,
ACHTUNGTRENNUNG(CH3)2), 4.37 (s, 1H, CHCHOH), 4.13 (s, 1H, OH), 4.00 (s, 1H,
ACHTUNGTRENNUNG
10.7 Hz, 1H, CH2CO2), 2.24 (dd, J=14.3, 2.6 Hz, 1H, CH2CO2), 2.16
(ddd, J=15.1, 2.8, 2.8 Hz, 1H), 2.08 (s, 3H, CH=CCH3), 2.07–2.00 (m,
1H), 1.97–1.72 (m, 5H), 1.72–1.47 (m, 5H), 1.41–1.20 (m, 9H), 1.07 ppm
(s, 3H,
165.59, 151.62, 138.88, 120.07, 116.10, 77.02, 72.99, 68.27, 62.51, 61.72,
CACHTUNGTRENNUNG
(CH3)2); 13C NMR (100 MHz, CDCl3): d=221.45, 170.43,
14802
www.chemeurj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2011, 17, 14792 – 14804

Epothilone Derivatives
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Received: August 24, 2011
Published online: November 30, 2011
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