Total synthesis of bistramide A and its 36(Z) isomers: Differential effect on cell division, differentiation, and apoptosis

Article abstract of DOI:10.1002/chem.201102462

The total synthesis of bistramide A and its 36(Z),39(S) and 36(Z),39(R) isomers shows that these compounds have different effects on cell division and apoptosis. The synthesis relies on a novel enol ether-forming reaction for the spiroketal fragment, a kinetic oxa-Michael cyclization reaction for the tetrahydropyran fragment, and an asymmetric crotonylation reaction for the amino acid fragment. Preliminary biological studies show a distinct pattern of influence of each of the three compounds on cell division, differentiation, and apoptosis in HL-60 cells, thus suggesting that these effects are independent activities of the natural product. Copyright

Full text of DOI:10.1002/chem.201102462

FULL PAPER
DOI: 10.1002/chem.201102462
Total Synthesis of Bistramide A and Its 36(Z) Isomers: Differential Effect on
Cell Division, Differentiation, and Apoptosis
Loꢀc Tomas,[a] Gustav Boije af Gennꢁs,[b] Marie Aude Hiebel,[c] Peter Hampson,[d]
David Gueyrard,[a] Bꢂatrice Pelotier,[c] Jari Yli-Kauhaluoma,[b] Olivier Piva,[c]
Janet M. Lord,*[d] and Peter G. Goekjian*[a]
Dedicated to Professor Edwin Vedejs on the occasion of his 70th birthday
Abstract: The total synthesis of
bistramide A and its 36(Z),39(S) and
tion for the tetrahydropyran fragment,
and an asymmetric crotonylation reac-
tion for the amino acid fragment. Pre-
liminary biological studies show a dis-
tinct pattern of influence of each of the
three compounds on cell division, dif-
ferentiation, and apoptosis in HL-60
cells, thus suggesting that these effects
are independent activities of the natu-
ral product.
ACHTUNGTRENNUNG
36(Z),39(R) isomers shows that these
compounds have different effects on
cell division and apoptosis. The synthe-
sis relies on a novel enol ether-forming
reaction for the spiroketal fragment,
a kinetic oxa-Michael cyclization reac-
Keywords: alkenes · bistramide A ·
protein kinase C · total synthesis
Introduction
cells, thus resulting in inhibition of cytokinesis and growth
arrest.[5] However, Kozmin and co-workers more recently
identified covalent modification of actin and disruption of
filamentous actin as a primary mode of action for bistram-
Bistramide A (bistratene A; Scheme 1) is a marine metabo-
lite that was isolated from Lissoclinum bistratum in 1988 by
Verbist and co-workers;[1] furthermore, four additional
members of the family (bistramides B, C, D, and K) were
isolated in 1994.[2] This class of compounds shows significant
cytotoxicity and neurotoxicity and plays a complex role in
cell-cycle regulation, differentiation, and apoptosis.[2–4] It
was initially shown that bistramide A activates the d isoform
of protein kinase C (PKC) in human promyelocytic leuke-
mia (HL-60) and human malignant melanoma (MM96E)
AHCTUNGTRENNUNG
ide A in various cancer-cell lines.[6] There is currently little
consensus on the activation of PKC-d. It was determined
early on that bistramide A does not activate PKC-d in vitro,
nor does it compete with phorbol esters in binding to the di-
acylglycerol activation site.[6,7] However, although bistram-
AHCTUNGTREGiNNUN de A failed to translocate green fluorescent protein (GFP)/
PKC-d in rat basophilic leukemia (RBL) cells,[6] immunos-
taining and fractionation studies showed migration of PKC-
d to the perinuclear region rather than to the nuclear or cy-
toplasmic membrane.[5a] Furthermore, only certain substrates
of PKC-d were phosphorylated in whole cells, notably talin
but not vimentin, vinculin, or tubulin,[5b] thus suggesting an
atypical activation mode for this important family of
second-messenger-activated protein kinases. Therefore, the
synthesis of bistramide A analogues with modulated biologi-
cal activities may serve to shed light on the biochemical
basis of these different biological properties.
[a] Dr. L. Tomas, Dr. D. Gueyrard, Prof. P. G. Goekjian
Universitꢀ de Lyon, ICBMS - UMR CNRS 5246
LCO2 - Glycochimie, Universitꢀ Claude Bernard Lyon 1
Bat. 308 CPE Lyon, 43, bd du 11 Novembre 1918
69622 Villeurbanne Cedex (France)
Fax : (+33)472 43 2752
E-mail: goekjian@univ-lyon1.fr
[b] Dr. G. Boije af Gennꢁs, Prof. J. Yli-Kauhaluoma
Division of Pharmaceutical Chemistry
Faculty of Pharmacy, University of Helsinki
PO Box 56 (Viikinkaari 5 E), 00014 Helsinki (Finland)
The first total synthesis of bistramide A was reported by
Kozmin and co-workers 2004.[8] Later, Crimmins,[9] Panek,[10]
and Yadav[11] and their respective co-workers also per-
formed total syntheses of the natural compound, whereas
the synthesis of bistramide C was realized by Wipf et al.[12]
We report herein the synthesis of bistramide A and its
36(Z),39(R) and 36(Z),39(S) isomers. Inspection of the X-
ray crystal structure of the bistramide A/actin complex
[c] Dr. M. A. Hiebel, Dr. B. Pelotier, Prof. O. Piva
Universitꢀ de Lyon, ICBMS - UMR CNRS 5246
Surcoof, Universitꢀ Claude Bernard Lyon 1
Bat. Raulin, 43, bd du 11 Novembre 1918
69622 Villeurbanne Cedex (France)
[d] Dr. P. Hampson, Prof. J. M. Lord
School of Immunity and Infection
The Medical School, University of Birmingham
Birmingham B152TT (UK)
ꢀ
shows a water-relayed hydrogen bond between the C39 OH
unit and tyrosine143 (Tyr143) residue[13] (Figure 1). Inver-
sion of the stereochemistry of the double bond might place
the same hydroxy group in proximity to the Tyr143 carbonyl
E-mail: j.m.lord@bham.ac.uk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102462.
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Synthesis of the spiroketal frag-
ment: The spiroketal core of
the C19–C40 fragment of the
bistramides can be obtained
through an exocyclic enol ether
by extending our recent meth-
odology for the synthesis of
functionalized exo-glycals from
sugar-derived lactones[16] to the
coupling of a non-carbohydrate
lactone 8 with the benzothiazol-
yl sulfone 9 (Scheme 2), which
can
be
prepared
from
a common precursor, namely,
1,2:5,6-dicyclohexylidene-d-
mannitol.[14] Such an approach
might eventually allow for the
synthesis of analogues substitut-
ed at the C28 position or kinet-
ic spiroketal stereoisomers.
Scheme 1. Retrosynthetic analysis for the C19–C36 fragment. Boc=tert-butyloxycarbonyl, Fmoc=9-fluorenyl-
methyloxycarbonyl, TBDPS=tert-butyldiphenylsilyl.
Figure 1. Left: X-ray structure of the bistramide A/actin complex that
highlights the water-relayed hydrogen bond to the Tyr143 residue. Right:
a model of the 36(Z),39(S) isomer obtained by substitution and MM2
energy minimization in the X-ray structure of the bistramide A/actin
complex, thus showing the expected proximity of the hydroxy group to
Scheme 2. Synthetic precursors of bistramide A.
the Tyr143 carbonyl unit (Inte:ligand LigandScout).
The synthesis of lactone 8 is shown in Schemes 3 and 4.
Initial studies focused on the stereoselective hydrogenation
of the methyl-substituted unsaturated esters 10a,b. In the
case of the E isomer, the stereoselectivity is in favor of the
undesired syn stereoisomer as reported, presumably due to
1,2-allylic strain.[17] Although the Z isomer might have been
expected to favor the desired anti isomer if 1,3-allylic strain
were to dominate, we obtained a disappointing 1:1 mixture
with palladium on carbon as the catalyst. Although better
group, thus allowing the formation of a direct hydrogen
bond. Therefore, we sought to prepare these isomers to test
this hypothesis.
Results and Discussion
Bistramide A and its isomers were prepared by coupling spi-
roketal-containing amines (e.g., 1) with the appropriately
protected homothreonine central fragment 2 and the tetra-
hydropyran-containing carboxylic acid 3. The spiroketal
C19–C40 fragment 1 was obtained by using a novel synthe-
sis of exocyclic enol ethers from lactones.[14] The amino acid
fragment 2 was accessed by asymmetric crotyltitanation in
a variation of the procedure developed by Kozmin and co-
workers,[8] whereas the tetrahydropyran fragment 3 was pre-
pared by a kinetically controlled oxa-Michael cyclization re-
action (Scheme 1).[15]
Scheme 3. Stereoselective hydrogenation.
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
was obtained by cuprate addition rather than hydrogenation
in the first step, followed by the same reaction sequence.
The key step of the construction of the spiroketal frag-
ment is the coupling of lactone 8 and the Julia–Kocienski re-
agent 9 followed by ipso substitution and spontaneous re-
ductive elimination in the presence of 1,8-diazabicyclo-
AHCTUNGTREG[NNNU 5.4.0]undec-7-ene (DBU) to afford the intermediate enol
ether 19. The pseudosymmetry of the spiroketal fragment
also allows an alternative combination (e.g., 20), in which
the methyl group is attached to the sulfone 17 rather than
the lactone. The extension of the enol ether synthesis to
enol ethers from non-carbohydrate lactones required the de-
velopment of newly optimized conditions (Table 1) because
significant differences were observed in the reactivity of the
lactone, the stability of the resulting enol ether, and the spi-
rocyclization step. Indeed, under the conditions reported for
sugar lactones,[16] the reaction gave disappointing yields in
the region of 20% (Table 1, entries 1 and 2). The use of an
alternative combination (e.g., sulfone 17 and lactone 18),[21]
did not improve the reaction (Table 1, entry 3), so subse-
quent studies focused on lactone 8 and sulfone 9. As in the
case of carbohydrates, the use of KHMDS (Table 1, entry 4)
gave little or no exo-glycal and neither did the use of other
lithium salts such as LiClO4 (Table 1, entries 5 and 6). The
Scheme 4. Synthesis of lactone 8. DHP=3,4-dihydro-2H-pyran, DMAP=
4-dimethylaminopyridine, HMDS=hexamethyldisilazide, PCC=pyridini-
um chlorochromate, PPTS=pyridinium para-toluenesulfonate, THP=
tetrahydropyran-2-yl, TMS=trimethylsilyl.
use of
a diethyl ether/THF solvent mixture (Table 1,
results might be obtained with other catalysts,[18] we did not
pursue this approach further.
entry 6) provided no improvement over THF alone. As the
aliphatic lactone appeared to be significantly less reactive
than the carbohydrate lactones, the addition of boron tri-
fluoride etherate improved the degree of conversion consid-
erably; although the resulting enol ether species was isolated
almost exclusively in this case in its hydrated form 21
(Table 1, entry 7). Indeed, in addition to the lower reactivity
of the lactone, the enol ether itself was considerably more
sensitive than its carbohydrate counterparts. Therefore, we
tried to isolate the enol ether moiety by chromatography on
basic alumina (Table 1, entry 8) or to cyclize the crude enol
ether moiety directly under thermodynamic conditions
(Table 1, entry 9). This last option provided the differentially
Therefore, the methyl group was introduced by using the
approach of Hanessian and Sumi.[19] Ester 12, obtained from
dicyclohexylidene d-mannitol, was deprotected and then dif-
ferentially protected with a silyl and tetrahydropyranyl
group to yield 13 in 77% overall yield. The addition of lithi-
um dimethylcuprate to unsaturated ester 13 in the presence
of trimethylsilyl chloride provided the desired isomer 14.
Consistent with the report of Hanessian and Sumi,[19] a full
six equivalents of the cuprate are required because a smaller
excess proved ineffective, even under equal reaction concen-
trations. Homologation of the ester by reduction with diiso-
butylaluminum
hydride
(DIBAL-H) and the Wittig re-
action furnished enol ether 15.
Finally, acidic hydrolysis and
oxidation of the lactol inter-
mediate provided the lactone 8.
The synthesis of the benzo-
thiazolyl sulfone reagents are
shown in Scheme 5.[16] Ester 12
was hydrogenated, reduced,
and converted into the tosylate
derivative 16, which provided
the sulfone species upon substi-
tution with 2-mercaptobenzo-
thiazole and oxidation of the
sulfur atom with ammonium
molybdate and hydrogen perox-
ide.[20]
The
corresponding
methyl-substituted sulfone 17 Scheme 5. Synthesis of the benzothiazolyl sulfones. Ts=tosyl.
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P. G. Goekjian, J. M. Lord et al.
Table 1. Optimization of the synthesis of the enol ether 19 and spiroketal 7.
Entry
Reaction conditions[a]
Isolation
Product
Yield
[%]
1
2
3
4
5
6
7
Barbier, LiHMDS (2.4 equiv), THF, ꢀ788C
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
chromatography on silica gel (5% Et3N)
19
19
20
19
19
19
19
21
19
7
20
23
23
<5
<5
14
5
70
14
43
40
40
69
Barbier, LiHMDS (1.2 equiv), LiCl, THF, ꢀ788C
Barbier, LiHMDS (1.2 equiv), LiCl, THF, ꢀ788C
Barbier, KHMDS (2.4 equiv), THF, ꢀ788C
premetallation, LiHMDS (1.5 equiv), LiClO4 (2 equiv), THF, ꢀ788C
Barbier, LiHMDS (2.0 equiv), LiCl (2 equiv), THF/Et2O, ꢀ788C
premetallation, LiHMDS (2.0 equiv), BF3 etherate (2.0 equiv), THF, ꢀ788C
8
9
10
11
12
premetallation, LiHMDS (2.0 equiv), BF3 etherate (1.0 equiv), THF, ꢀ788C
Barbier, LiHMDS (2.0 equiv), BF3 etherate (1.0 equiv), THF, ꢀ788C
Barbier, LiHMDS (2.0 equiv), BF3 etherate (1.0 equiv), THF, ꢀ1008C
Barbier, LiHMDS (2.0 equiv), BF3 etherate (1.0 equiv), THF, ꢀ508C
Barbier, LiHMDS (2.0 equiv), BF3 etherate (1.0 equiv), THF, ꢀ788C
chromatography on basic alumina
p-TSA, MeOH
p-TSA, MeOH
p-TSA, MeOH
p-TSA, CH2Cl2
7
7
7
[a] Conditions for the first step. The reaction was quenched at low temperature with acetic acid and, after aqueous workup, the crude product was treat-
ed with DBU (2 equiv) in THF at room temperature. p-TSA=para-toluenesulfonic acid.
protected spiroketal 7 in somewhat better yield (43%).
Under the same cyclization conditions, changing the temper-
ature of the enol ether-forming reaction did not improve the
results (Table 1, entries 10 and 11). However, we noted that
the spirocyclization step, which was systematically quantita-
tive in the carbohydrate series under these conditions, was
hampered in this case by sluggish methanolysis of the cyclo-
hexylidene group, thus leading to the methyl ketal of 21.
Forcing conditions led to the loss of the silyl protective
group and thus to a spiroketal product that cannot be de-
symmetrized efficiently. Finally, cyclization with p-TSA in
dichloromethane instead of methanol prevented hydrolysis
of the silyl group and provided the differentially protected
spiroketal 7 as a single stereoisomer in 69% yield over the
two steps on a gram scale (Scheme 6).
sults were obtained by performing the coupling step at
ꢀ788C and then heating directly to 608C, instead of warm-
ing progressively. The resulting olefin 24 was thus obtained
in 85% yield.
Compound 24 was deprotected to give the corresponding
spiroketal alcohol, which was oxidized to undergo
a
Horner–Emmons olefination with triACTHUNGTERNNUeG thylphosphono-
acetate, thus providing the a,b-unsaturated ester 25 in good
yield. The treatment of 25 with palladium on charcoal under
a hydrogen atmosphere provided alcohol 26, which was sub-
sequently protected as the silyl ether by using TBDPSCl
and submitted to reduction of the ester function to obtain 6,
which was previously described by Crimmins and
DeBaillie.[9] The structure was confirmed by comparison of
the NMR spectroscopic data.
The synthesis of the natural
C19–C40 spiroketal fragment
was completed from intermedi-
ate 6[9] by generally following
the route developed by Crim-
min and DeBaillie (Scheme 8).
A phthalimide group was intro-
duced in two steps via the cor-
responding iodo derivative 27.
Deprotection of the tert-butyl-
diphenylsilyl protective group
by using the 3HF·Et3N complex
followed by oxidation of the al-
cohol under Swern conditions
provided aldehyde 29. At this
stage, we decided to prepare
Scheme 6. Spiroketal synthesis. DBU=1,8-diazabicycloACTHNUGRTENUNG[5.4.0]undec-7-ene.
the natural bistramide A frag-
ment 32, but also the C36(Z)
Further elaboration of spiroketal 7 is shown in Scheme 7.
Swern oxidation provided aldehyde 22, which was subjected
to a modified Julia reaction[22] with sulfone 23[9] to provide
alkene 24. This traditional version of the reaction gave sur-
prisingly poor results under the reported experimental con-
ditions, and the reaction therefore also required optimiza-
tion (see the Supporting Information). Finally, the best re-
analogues with both configurations at C39 via the Z enone
31.
The E enone of the bistram
ACHTUNGERTNiNUNG de spiroketal system 30
(Scheme 8) was prepared from 29, as described previously
using (EtO)2POCHACTHNUGTRNE(GNU CH3)COCH3 and Ba(OH)2 in THF/
water,[9] although the mixture of BaO and Ba(OH)2 was
more robust and gave a slightly better selectivity in our
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
led to the E isomer 30 with
good selectivity, but surprisingly
the reaction was accompanied
by racemization of the adjacent
C35 methyl stereocenter, even
at low conversion.
The Z isomer 31 was ob-
tained
a Still–Gennari reaction with
(CF3CH2O)2APOCH(CH3)-
selectively
through
C
ACHTUNGTRENNUNG
COCH3.[23] Interestingly, the
use of BaO/Ba(OH)2 in THF
gave significantly higher Z se-
lectivities (24:1) and better
yields in the authentic system
than with the use of KHMDS,
[18]-crown-6, and THF or
tBuOK alone, which gave Z se-
lectivities of 7:1 and 4:1, respec-
tively (Scheme 9). Surprisingly,
we did not observe the same
trend in the stereoselectivity of
the formation of the enone
Scheme 7. Functionalization of the spiroketal 7. DMSO=dimethyl sulfoxide, TBAF=tetra-n-butylammonium
fluoride, Bn=benzyl.
when
using
3-benzyloxy-2-
methylpropanal as a model (see
the Supporting Information). A
significant b-alkoxy effect is
thus observed with barium, but
not potassium bases, thereby re-
flecting differences either in the
relative diastereoselectivity or
in the degree of reversibility of
the initial step.[24]
The E and Z unsaturated ke-
tones 30 and 31 were reduced
under
Corey-Bakshi-Shibata
conditions to give the C19–C40
spiroketal fragment with the
natural 36(E),39(S) configura-
tion in 32 and the unnatural
configurations 36(Z),39(R) and
36(Z),39(S) in isomers 33a,b,
respectively. The 36(E),39(R)
isomer has been reported previ-
ously by Panek and co-workers
as part of an impressive library
of stereoisomers and therefore
was not prepared in this
study.[10a]
Amino acid fragment: The
amino acid fragment 2 was pre-
pared in five steps from the N-
Boc-protected aminoacetalde-
hyde by using a variation of the
Scheme 8. Synthesis of the C19–C40 fragments. CBS=Corey–Bakshi–Shibata catalyst.
hands (see the Supporting Information). The Wittig–Horner
phosphorane (Ph3P=CACTHNUGTRNE(UGN CH3)COCH3, toluene, reflux) also
asymmetric crotonylation route developed by Kozmin
et al.[8] by using TADDOL instead as a chiral ligand
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P. G. Goekjian, J. M. Lord et al.
over two steps. A diastereose-
lective allylation of 40 was first
attempted under the conditions
developed by Brown and
Jadhav with (ꢀ)-B-allyldiisopi-
nocamphenylborane.[28] A high
diastereoselectivity was ob-
served, but homoallylic alcohol
41 could be isolated in only
46% yield. Therefore, we per-
formed the allylation reaction
with the Roush chiral diiso-
propyl tartrate allylboronate.[29]
In this case, alcohol 41 was ob-
tained in 91% yield as a 4:1
diastereomeric mixture, from
which the major isomer was iso-
lated in 73% yield.
Scheme 9. b-Alkoxy effect in the barium-catalyzed Still–Gennari reaction.
The target Michael acceptor
38 was prepared from 41 as fol-
lows: The secondary alcohol
was protected as a tert-butyldi-
methylsilyl (TBS) ether, and
the primary alcohol was depro-
tected with DDQ and oxidized
to the corresponding aldehyde,
which was engaged in a Wittig
reaction to provide the geomet-
ric E isomer 42 in 70% yield
over four steps (Scheme 13).
Removal of the TBS protecting
Scheme 10. Synthesis of the C14–C18 fragment. DMP=2,2-dimethoxypropane, Fmoc-OSu=N-(9-fluorenyl-
A
ACHTUNGTNERmNUNG eth-
ACHTUNGTRENNUNG
(Scheme 10). The first step consists of the formation of
chiral amino alcohol 36 by asymmetric crotyltitanation.[25,26]
The amino alcohol was protected as a 1,3-oxazolidine and
cleavage of the double bond with rutheniumACTHUNRTGNEUNG(III) chloride/
sodium periodate provided amino acid 37, which was fully
deprotected under acidic conditions. Finally, the introduc-
tion of an Fmoc group gave the enantiomerically pure N-
protected amino acid 2.
Synthesis of the tetrahydropyran fragment: The C1–C13
fragment of bistramides can be obtained from the core
trans-2,6-disubstituted tetrahydropyran 4, which can be pre-
pared by a kinetically controlled intramolecular oxa-Michael
cyclization of hydroxyester 38 (Scheme 11).[15] The oxa-Mi-
chael precursor was synthesized in a few steps from 5-hexe-
noic acid by using classical asymmetric methodologies.
By starting from 5-hexenoic acid, an asymmetric alkyla-
tion reaction that employed the Davies Superquat auxili-
ary[27] led to the methylated product 39, which could be iso-
lated as a single diastereoisomer in 86% yield after recrys-
tallization (Scheme 12). After reductive removal of the
chiral auxiliary, the resulting alcohol was protected as
a para-methoxybenzyl ether, and aldehyde 40 was obtained
by oxidative cleavage of the terminal alkene in 94% yield
Scheme 11. Retrosynthetic analysis of the C1–C13 fragment.
group under acidic conditions furnished 38 in quantitative
yield.
The key step of the C1–C13 fragment synthesis is the in-
tramolecular oxa-Michael cyclization reaction of 38. Previ-
ous studies on nor-methyl derivatives have shown that the
conjugate addition could lead to the major formation of the
thermodynamically less-favoured 2,6-trans-disubstituted tet-
rahydropyran, when the reaction was performed under ki-
netic conditions.[30] In our case, the presence of a methyl
substituent could be an issue, and indeed when the reaction
was first attempted at ꢀ708C for 20 minutes with one equiv-
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
the oxidation/isomerization in
one pot (Scheme 14). Unfortu-
nately, deprotection of ester 44
was unsuccessful under a variety
of conditions due to the insta-
bility of the a-(tetrahydropyra-
nyl)enone system. The activat-
ed tetrahydropyran acid 3 was
therefore prepared from the in-
termediate
4 in four steps.
Saponification of the ester gave
the corresponding carboxylic
acid, which was oxidized under
Swern conditions. At this stage,
Scheme 12. Synthesis of chiral homoallylic alcohol intermediate 41. CSA=camphorsulfonic acid, LDA=lithi- the a,b-unsaturated ketone
um diisopropylamide, PMB=para-methoxybenzyl, NMO=N-methylmorpholine-N-oxide.
moiety was successfully put in
place, yet a Pummerer rear-
rangement of the Swern inter-
mediate also led to the formation of the (methylthio)methyl
(MTM) ester of the acid functional group to provide 45 in
good yield.[31] The MTM ester facilitated the purification of
the product and was subsequently deprotected with magne-
sium bromide, followed by EDCI-mediated coupling of N-
hydroxysuccinimide to provide the activated ester 3 in 40%
yield over four steps.
Synthesis of bistramide A and its Z isomers: To complete
the synthesis of bistramide A (Scheme 15), cleavage of the
phthalimide protecting group of 32 followed by benzotria-
zol-1-yl-oxytripyrrolidinophosphonium (PyBOP)-mediated
condensation with acid 2 led us to the desired amide 46 in
78% yield. Removal of the Fmoc group and subsequent re-
action of the amine with activated ester 3 provided bistram-
AHCTUNGTRENNUNG
ide A in excellent yield (87%).[8,9] The 36(Z),39(R) and
Scheme 13. Synthesis of key intermediate 4. DDQ=2,3-dichloro-5,6-di-
cyano-1,4-benzoquinone, TES=triethylsilyl.
36(Z),39(S) analogues 48 and 49 (Scheme 14) were obtained
by the same route from 47a,b, respectively. The total syn-
thesis of bistramide A and its 36(Z),39(R) and 36(Z),39(S)
isomers was thus completed.
alent of tBuOK, only the 2,6-cis-diACTHNUGTRNEUNGsubstituted tetrahydropyr-
an epi-43 (Scheme 13) was obtained. When the reaction was
repeated at temperatures between ꢀ100 and ꢀ858C, the
cyclization did not occur even after prolonged reaction
times. Finally, the desired kinetic Michael adduct 43 could
be obtained as the major isomer when the reaction was per-
formed at ꢀ80/ꢀ788C with 1.1 equivalents of tBuOK in
THF and was isolated in 72%
Preliminary biological studies of bistramide A and its
Z isomers: We performed a preliminary biological evalua-
tion of 36(E),39(S) bistramide A and the isomers
36(Z),39(R) 48,and 36(Z),39(S) 49. The overall antiprolifer-
ative effect was evaluated with the leukaemic cell line HL-
yield. The C1–C13 fragment
precursor 4 was prepared in
three steps by oxidative cleav-
age of the terminal alkene and
allylation of the resulting alde-
hyde under Barbier conditions
in 92% overall yield.
The ethyl ester of the natural
tetrahydropyran fragment was
next prepared under Swern
conditions with an excess of
triethylamine base to effectuate Scheme 14. Completion of the C1–C13 fragment. EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
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P. G. Goekjian, J. M. Lord et al.
differentiation. A more detailed
analysis by flow cytometry was
therefore undertaken to gain
further insight into the relative
contributions of these effects on
the observed growth inhibition
(Figure 3). The results suggest
that although the overall anti-
proliferative potencies are simi-
lar, there are significant differ-
ences as to the origin of this ac-
tivity for the three compounds.
The results for bistramide A
show accumulation in G2M
that starts at a concentration of
1 mm, which is similar to that re-
ported by Panek and co-work-
ers[10] for bistramide A-treated
renal carcinoma UO-31 cells.
Previous reports for HL-60
cells treated with naturally de-
rived bistramide A have also re-
ported accumulation of cells in
G2M, although at much lower
concentrations
(50 nm).[5a]
Scheme 15. Completion of the synthesis of bistramide A and the analogues 36(Z),39(R) 48 and 36(Z),39(S) 49.
These data are at odds with
those reported by Verbist and
60, as used in previous studies,[5a] by employing a 3-(4,5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium) (MTS) assay to evaluate the
number of viable cells (Figure 2). The natural bistramide A
co-workers, who saw irreversible accumulation in the G1
stage.[3b] This disparity most likely reflects a difference be-
tween the cell lines used, in that the nonsmall cell lung
cancer (NSCLC) line used in the study of Verbist and co-
workers is an adherent cell line, whereas HL-60 is not and
bistramide A has actions upon the cytoskeleton that could
influence the biological outcome. It may also reflect differ-
ent sensitivities to differentiation and inhibition of cytokine-
sis between the two cell lines. Strong accumulation of 4n
polyploid cells is seen at concentrations of 10 mm, which has
been confirmed by differential staining and light microscopy
(data not shown), and a corresponding decrease in G0/G1
cells, which is consistent with the inhibition of cytokinesis as
reported by Kozmin and co-workers.[6] However, at higher
concentrations (i.e., 100 mm), an accumulation of cells is ob-
served in the sub-G0 region, which is consistent with the in-
duction of apoptosis. The results for the isomer 36(Z),39(S)
49 show a similar strong accumulation in the 4n polyploid
cells, with no corresponding accumulation in the sub-G0
region, and thus little apparent corresponding contribution
of the proapoptotic activity. Interestingly, the isomer
36(Z),39(R) 48, which has similar overall antiproliferative
activity to 49, shows a marked proapoptotic effect as evi-
denced by a strong accumulation in the sub-G0 region at
100 mm and thus has a biological profile more similar to bis-
tramide A.
Figure 2. Evaluation of the antiproliferative effect in the leukaemic cell
line HL-60 by using the MTS assay. ^: bistramide A; &: 36(Z),39(R)
isomer 48; ~: 36(Z),39(S) isomer 49
isomer showed IG50 =1.6 mm, which is generally in accord
with that reported for this particular cell line with natural[33]
and synthetic[6a] bistramide A. The Z isomers showed slightly
lower potency, with IG50 values of 7.8 and 3.7 mm for the iso-
mers 39(R) 48 and 39(S) 49, respectively.
Additional studies were performed to evaluate the pro
ACHTUNGTRENNUNGap-
The observed growth inhibition could be due to a combi-
nation of factors, including inhibition of cell cycling (anti-
ACHTUNGTRENNUNGmitotic effect), a proapoptotic effect, and/or the induction of
G
E
ACHTUNGTRENNUNG
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
Figure 3. Cell-cycle analysis for HL-60 cells treated with synthetic bistramide A and its Z isomers 48 and 49. The HL-60 cells were incubated with these
compounds at the concentrations shown, stained with propidium iodide, and analyzed by flow cytometry to assess the proportion of cells at each stage of
the cell cycle (G1, S, G2M) or in the sub-G0 (apoptotic) phase.
Figure 4. Annexin V/sytox analysis of apoptosis on synthetic bistramide A and its Z isomers 48 and 49. The HL-60 cells were treated with these com-
pounds at the concentrations shown and stained with annexin V/phycoerythrin (PE) and sytox green and analyzed by flow cytometry. The cells that were
positive for annexin V and negative for sytox green were taken as early apoptotic cells, and double positive cells were in late apoptosis. The percentage
of viable cells is shown in the top right-hand corner of each flow plot.
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P. G. Goekjian, J. M. Lord et al.
Figure 5. CD11b expression in cells treated with synthetic bistramide A and isomers 48 and 49. The HL-60 cells were treated with these compounds at
the concentrations shown and stained with an antibody to CD11b. The immunostaining was analyzed by flow cytometry. The percentage of CD11b posi-
tive cells is shown in each flow plot.
tramide A (77% viable cells at 100 mm), whereas little or no
apoptosis was induced by the isomer 36(Z),39(S) 49 at the
highest concentration tested (85% viable cells at 100 mm).
On the other hand, a definite proapoptotic effect was seen
for the isomer 36(Z),39(R) 48 (57% viable cells at 100 mm),
with cells seen in both early and late apoptosis.
methodologies, for example, the synthesis of enol ethers
from lactones, provides the impetus for new insight and can
provide access to alternative analogues of the spiroketal
moiety. In addition, we have shown that the three synthetic
stereoisomers have similar overall antiproliferative activities,
yet each shows a distinct distribution of antimitotic, pro
ACHTUNGTRENNUNGapACHTUNGTRENNUNGo-
Finally, CD11b immunostaining studies were performed
to evaluate CD11b expression as an early marker of HL-60
cell differentiation (Figure 5). Bistramide A showed a mild
induction of CD11b expression at lower concentrations
(36% of cells were positive for CD11b at 10 mm), which is
consistent with a mild prodifferentiation activity reported
previously for the natural product.[5a] Interestingly, the
isomer 36(Z),39(S) 49, which showed little or no proapop-
totic effect, induced a marked expression of CD11b (66%
CD11b were positive cells at 100 mm). Conversely, the
isomer 36(Z),39(R) 48 showed only a weak induction of
CD11b expression (24% CD11b were positive cells at
100 mm). CD11b expression should not be overinterpreted,
and additional experiments are necessary to correlate these
results to the potential contribution of cell differentiation to
the antiproliferative effects of the compounds.
ACHTUNGTRENpNUNG totic, and prodifferentiation effects. These results confirm
the existence of all three reported effects and confirm that
accumulation of 4n polyploid cells represents the primary
mode of action of the natural product. Our results also sug-
gest that the antimitotic and proapoptotic activities are inde-
pendent effects of the natural product, as opposed to
common effects of actin binding, because the activity pro-
files would be parallel in the latter case. Additional ana-
logues will be needed to determine whether induction of dif-
ferentiation is dependent or independent of actin binding.
As the effects of bistramide A and isomers 48 and 49 were
seen at high concentrations in HL-60 cells, it is not possible
to state categorically whether they could be achieved in
vivo, and, if so, whether different profiles (polyploidy, ap
ACHTUNGTRENNUNGo-
A
ACHTUNGTRENNUNG
compounds in vivo. A more detailed report of the biological
studies on these and related compounds will be reported in
due course.
Conclusion
We have reported the total synthesis of bistramide A and its
36(Z),39(R) and 36(Z),39(S) isomers. Although our synthe-
sis is slightly more linear than previous ones (29 steps, 2%
overall yield), it is convenient and robust. The use of new
Experimental Section
Experimental procedures are provided only for selected key steps. The
general experimental procedures; detailed descriptions of biological
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
assays; synthesis of compounds 2, 6, 8, 9, 10a, 10b, 11a, 11b, 13–15, 22,
25–30, 32, 37, 39, 40, 46, and other synthetic intermediates; and the de-
tails of the optimization of various olefination reactions are provided in
the Supporting Information.
69.6, 65.4, 36.6, 36.0, 35.2, 31.2, 31.0, 28.0, 26.9, 19.5, 19.1, 17.7, 17.0 ppm;
IR (film): n˜ =2930, 2857, 1455, 1428, 1380, 1360, 1272, 1224, 1111, 983,
822, 738 cmꢀ1; MS (ESI): m/z: 613 [M+H]+, 635.4 [M+Na]+; HRMS
(ESI): m/z calcd for C39H52O4SiNa: 635.3531 [M+Na]+; found: 635.3531.
{(2S,6S,8S,9S)-8-[(tert-Butyldiphenylsilyloxy)methyl]-9-methyl-1,7-dioxa-
({(2S,3S,6S,8S)-8-[(S,Z)-4-(Benzyloxy)-3-methylbut-1-enyl]-3-methyl-1,7-
spiro
G
dioxaspiroACTHNGUTERN[UNG 5.5]undecan-2-yl}methoxy)(tert-butyl)diphenylsilane ((Z)-24):
3.16 mmol, 1 equiv) and benzothiazolyl sulfone
9
[a]2D5 = +43.3 (c=1.0 in CHCl3); 1H NMR (400 MHz, CDCl3, 258C,
TMS): d=0.86 (d, J=5.9 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 1.05 (s, 9H),
1.22–1.69 (m, 10H), 1.99–2.10 (m, 1H), 2.79–2.90 (m, 1H), 3.22 (d,
6.5 Hz, 2H), 3.40 (ddd, J=2.1, 5.0, 8.8 Hz, 1H), 3.74 (dd, J=5.5, 10.9 Hz,
1H), 3.83 (dd, J=2.2, 10.9 Hz, 1H), 4.41 (s, 2H), 4.48 (ddd, J=1.8, 8.1,
10.6 Hz, 1H), 5.33 (pdd, J=10.3, 11.1 Hz, 1H), 5.42 (dd, J=8.2, 11.1 Hz,
1H), 7.23–7.28 (m 5H), 7.25–7.40 (m, 6H), 7.69–7.79 ppm (m, 4H).
1.2 equiv) were added to a solution of 8 (1.21 g, 3.2 mmol, 1 equiv; see
the Supporting Information) in THF (5.3 mL). The mixture was cooled
to ꢀ788C and lithium hexamethyldisilazide (1m in THF, 6.32 mL,
6.3 mmol, 2 equiv) was added dropwise. The reaction mixture was stirred
at ꢀ788C for 30 min, hydrolyzed at ꢀ788C with acetic acid (542 mL,
9.48 mmol, 3 equiv), stirred at room temperature for 15 min, and extract-
ed with ethyl acetate. The organic layers were combined, washed with
brine, and dried over anhydrous sodium sulfate. After filtration and evap-
oration in vacuo, the residue was dissolved in THF (30 mL), and 1,8-
2-(3-{(2R,3S,6S,8S)-8-[(S,Z)-3,5-Dimethyl-6-oxohept-4-en-1-yl]-3-methyl-
1,7-dioxaspiroACTHNUTRGNEUGN[5.5]undecan-2-yl}propyl)isoindoline-1,3-dione (31): DMSO
(10 mL, 0.11 mmol, 4 equiv) in dichloromethane (0.1 mL) was added to
a solution of oxalyl chloride (30 mL, 0.57 mmol, 2 equiv) in dichlorome-
thane (0.2 mL) at ꢀ788C over 15 min and then stirred for an additional
diazabicycloACHTUNGTRENNUNG[5.4.0]undec-7-ene (943 mL, 3.32 mmol, 2 equiv) was added to
the reaction mixture over 10 min. The reaction mixture was stirred at
room temperature for 30 min, quenched by the addition of aqueous
NH4Cl solution, and diluted and extracted with ethyl acetate. The organic
layers were combined, washed with brine, and dried over anhydrous
sodium sulfate. Filtration and evaporation in vacuo provided a mixture of
diastereomeric enol ethers 19 as a pale yellow oil, which was used with-
out further purification. p-TSA (300 mg, 1.6 mmol) was added to the mix-
ture of enol ethers 19 in dichloromethane (45 mL). The reaction mixture
was stirred at room temperature for 25 min, hydrolyzed by the addition
of aqueous NaHCO3 solution, and extracted with ethyl acetate. The or-
ganic layers were combined, washed with brine, dried over anhydrous
magnesium sulfate, and filtered. The solvent was evaporated in vacuo
and the crude product was purified by flash chromatography on silica gel
(petroleum ether/ethyl acetate 85:15) to afford spiroketal 7 as a colorless
15 min.
2-(3-{(2R,3S,6S,8S)-8-[(S)-4-Hydroxy-3-methylbutyl]-3-methyl-
1,7-dioxaspiro
G
(11 mg,
0.03 mmol, 1 equiv; see the Supporting Information for the preparation
from 26) was dissolved in dichloromethane (0.6 mL) was added dropwise
to the reaction mixture, which was stirred for 30 min. Dry triethylamine
(25 mL, 0.17 mmol, 6 equiv) was added dropwise to the reaction mixture,
which was allowed to warm to room temperature for 30 min, brine was
added, and the aqueous phase was extracted with diethyl ether. The com-
bined organic layers were washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The resulting oil was
used without further purification. Ba(OH)2·8H2O (21 mg, 0.068 mmol,
2 equiv) and BaO (21 mg, 0.136 mmol, 4 equiv) were added to a solution
of MeCOCH(Me)P(O)ACHTUNGTRNEG(UN OCH2CF3)2 (16 mg, 0.041 mmol, 1.4 equiv) in
oil (1.03 g, 2.21 mmol, 69% over 2 steps). [a]D25
= +26.2 (c=1.0 in
THF at 08C under argon and the reaction mixture was stirred at 08C for
45 min. A solution of aldehyde 29 (0.034 mmol, 1 equiv) in THF was
added slowly to the reaction mixture, which was left for 1 h at 08C and
aged at room temperature for 1 h. The reaction was quenched with satu-
rated sodium bicarbonate solution and diluted with ethyl acetate. The
aqueous phase was extracted with ethyl acetate and the combined organ-
ic layers were washed with brine, dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The crude compound was purified by
column chromatography on silica gel (toluene/diethyl ether 9:1) to afford
the corresponding enone (Z)-31 as a colorless oil (9.5 mg, 0.019 mmol,
60%). [a]D25 = +12.4 (c=0.2 in CHCl3); 1H NMR (400 MHz, CDCl3,
258C, TMS): d=0.82 (d, J=6.3 Hz, 3H), 0.98 (d, J=6.2 Hz, 3H), 1.10–
1.75 (m, 17H), 1.78–1.87 (m, 1H), 1.90 (br d, 3H), 1.97–2.03 (m, 1H),
2.22 (s, 3H), 2.74–2.78 (m, 1H), 3.15 (ddd, J=1.4, 9.7, 9.7 Hz, 1H), 3.45–
3.50 (m, 1H), 3.66–3.79 (m, 2H), 5.42 (dd, J=1.0, 9.9 Hz, 1H), 7.71 (dd,
J=3.0, 5.4 Hz, 2H), 7.85 ppm (dd, J=3.0, 5.4 Hz, 2H); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d=204.3, 168.6, 144.3, 135.1, 134.0,
132.4, 123.3, 95.6, 74.2, 69.3, 38.6, 36.2, 35.6, 35.2, 34.4, 33.8, 33.7, 31.5,
30.8, 30.3, 28.0, 25.3, 21.2, 21.1, 19.3, 18.2 ppm; IR (film): n˜ =2925, 2855,
1773, 1714, 1456, 1396, 885, 720 cmꢀ1; MS (ESI): m/z: 518 [M+Na]+;
HRMS (ESI): m/z calcd for C30H41NO5Na: 518.2877 [M+Na]+; found:
518.2873.
CHCl3); 1H NMR (300 MHz, CDCl3, 258C, tetramethylsilane (TMS)):
d=0.84 (d, J=6.2 Hz, 3H), 1.05 (s, 9H), 1.22–2.05 (m, 12H), 3.33 (ddd,
J=3.1, 4.4, 9.4 Hz, 1H), 3.48 (dd, J=6.9, 11.2 Hz, 1H), 3.58 (dd, J=3.4,
11.3 Hz, 1H), 3.73–3.84 (m, 3H), 7.35–7.42 (m, 6H), 7.73–7.78 ppm (m,
4H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=135.8, 135.7, 134.1,
134.0, 129.6, 127.7, 127.6, 96.0, 76.1, 69.5, 66.3, 65.2, 35.7, 35.4, 30.7, 28.0,
26.8, 26.6, 19.4, 18.5, 17.7 ppm; IR (film): n˜ =2931, 2858, 1456, 1428,
1382, 1225, 1105, 984, 738 cmꢀ1; MS (ESI): m/z: 491 [M+Na]+; HRMS
(ESI): m/z calcd for C28H40O4SiNa: 491.2594 [M+Na]+; found: 491.2593.
({(2S,3S,6S,8S)-8-[(S,E or Z)-4-(Benzyloxy)-3-methylbut-1-enyl]-3-meth-
ACHTUNGTRENNUNGyl-1,7-dioxaspiroACHTUNGTRENNUNG[5.5]undecan-2-yl}methoxy)(tert-butyl)diphenylsilane
(24): Lithium hexamethyldisilazide (1m in THF, 1.84 mL, 1.84 mmol,
1.2 equiv) was added dropwise to a solution of sulfone 23 (665 mg,
1.84 mmol, 1.2 equiv) in THF (4 mL) at ꢀ788C for 30 min. A solution of
22 (1.53 mmol, 1 equiv; see the Supporting Information) in THF (4 mL)
was slowly added to the reaction mixture, which was heated directly to
608C for 1.5 h. The reaction was quenched by pouring the mixture into
an aqueous solution of ammonium chloride, which was then extracted
with ethyl acetate. The organic layers were combined, washed with brine,
and dried over anhydrous sodium sulfate. After filtration and evapora-
tion in vacuo, the residue was purified by column chromatography on
silica gel (petroleum ether/ethyl acetate 97:3) to afford a diastereomeric
mixture (E/Z=7:3) of 24 as a colorless oil (584 mg, 0.95 mmol, 62% over
2 steps). Analytical samples of the two isomers were obtained by repeat-
ed chromatography on silica gel.
2-(3-{(2R,3S,6S,8S)-8-[(3S,6R,Z)-6-Hydroxy-3,5-dimethylhept-4-en-1-yl)-
3-methyl-1,7-dioxaspiroACTHNUTRGENUG[N 5.5]undecan-2-yl}propyl)isoindoline-1,3-dione
(33a): (S)-CBS (1.0m in toluene, 60 mL, 0.060 mmol, 2 equiv) was added
to a solution of ketone 31 (11 mg, 0.022 mmol, 1 equiv) in distilled tolu-
ene (0.4 mL). The reaction mixture was cooled under argon at ꢀ788C
and stirred for 30 min. A solution of catecholborane (1.0m in THF,
40 mL, 0.040 mmol, 2 equiv) was added to the reaction mixture, which
was stirred for 12 h at ꢀ788C, quenched with methanol (0.1 mL), and al-
lowed to warm to room temperature. The reaction mixture was diluted
with diethyl ether and washed with 1m NaOH saturated with NaHCO3
until the aqueous washings were colorless. The aqueous phase was ex-
tracted three times with diethyl ether and the combined organic layers
were washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated in vacuo. The crude compound was purified by column
chromatography on silica gel (petroleum ether/diethyl ether 6:4) to
({(2S,3S,6S,8S)-8-[(S,E)-4-(Benzyloxy)-3-methylbut-1-enyl]-3-methyl-1,7-
dioxaspiroACHTUNGTRENNUNG[5.5]undecan-2-yl}methoxy)(tert-butyl)diphenylsilane ((E)-24):
1
[a]2D5 = +9.3 (c=1.0 in CHCl3); H NMR (400 MHz, CDCl3, 258C, TMS):
d=0.81 (d, J=6.2 Hz, 3H), 1.03–1.05 (m, 12H), 1.20–1.68 (m, 10H),
1.98–2.08 (m, 1H), 2.45–2.55 (m, 1H), 3.26 (dd, J=7.5, 9.1 Hz, 1H),
3.32–3.36 (m, 1H), 3.39 (dd, J=6.0, 9.1 Hz, 1H), 3.74 (dd, J=5.5,
10.9 Hz, 1H), 3.79 (dd, J=2.5, 10.9 Hz, 1H), 4.17 (ddd, J=2.1, 4.9,
11.4 Hz, 1H), 4.50 (d, J=2.9 Hz, 2H), 5.50 (dd, J=5.2, 15.8 Hz, 1H),
5.56 (dd, J=5.7, 15.8 Hz, 1H), 7.32–7.43 (m, 11H), 7.74–7.76 ppm (m,
4H); 13C NMR (100 MHz, CDCl3, 258C, TMS): d=135.9, 135.8, 134.2,
134.1, 133.4, 131.4, 129.6, 128.5, 127.7, 127.7, 127.6, 96.2, 76.1, 75.4, 73.1,
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P. G. Goekjian, J. M. Lord et al.
afford the corresponding reduced ketone (Z)-33a as a colorless oil
(9.7 mg, 0.019 mmol, 85%). [a]2D5 = +23.3 (c=0.18 in CHCl3); 1H NMR
(400 MHz, CDCl3, 258C, TMS): d=0.81 (d, J=6.5 Hz, 3H), 0.94 (d, J=
6.6 Hz, 3H), 1.09–1.14 (m, 1H), 1.22 (d, J=6.4 Hz, 3H), 1.31–1.66 (m,
16H), 1.71 (d, J=1.1 Hz, 3H), 1.78–1.89 (m, 1H), 1.97–2.06 (m, 1H),
2.42–2.50 (m, 1H), 3.14 (ddd, J=1.8, 9.8, 9.8 Hz, 1H), 3.45–3.48 (m, 1H),
3.67–3.80 (m, 2H), 4.76 (q, J=6.5 Hz, 1H), 4.95 (br d, J=9.6 Hz, 1H),
5.37 (br s, 1H), 7.71 (dd, J=3.0, 5.4 Hz, 2H), 7.85 ppm (dd, J=3.0,
5.4 Hz, 2H); 13C NMR (100 MHz, CDCl3, 258C, TMS): d=168.7, 136.7,
134.0, 133.7, 132.3, 123.4, 95.6, 74.3, 68.6, 65.9, 38.7, 36.2, 35.6, 35.3, 34.2,
33.5, 31.6, 31.4, 30.9, 28.0, 25.5, 22.0, 21.52, 19.3, 18.2, 17.2 ppm; IR
product was purified by column chromatography on silica gel (hexane/
EtOAc/dichloromethane 16:4:1) to give 36 as
a clear oil (0.42 g,
1.97 mmol, 57%). [a]2D5 ꢀ2.93 (c=5.8 in CHCl3); 1H NMR (300 MHz,
CDCl3, 258C, TMS) d=1.05 (d, J=6.8 Hz, 3H), 1.44 (s, 9H), 2.23 (sext,
J=7.0 Hz, 2H), 3.06 (app ddd, J=13.8, 8.2, 4.9 Hz, 1H), 3.42–3.28 (m,
1H), 3.48 (ddd, J=7.9, 6.7, 2.9 Hz, 1H), 4.94 (br s, 1H), 5.16–5.09 (m,
2H), 5.81–5.69 ppm (m, 1H); 13C NMR (75 MHz, CDCl3, 258C, TMS)
d=156.8, 140.0, 116.6, 79.6, 74.3, 44.4, 42.5, 28.5, 16.3 ppm.
AHCTUNGTERG(NNUN 4R,7S)-8-(4-Methoxybenzyloxy)-7-methyloct-1-en-4-ol (41): A solution
of Roush allylboronate[28] (0.12m in toluene, 15 mL, 1.8 mmol) was added
to activated powdered 4 ꢄ molecular sieves under argon. The mixture
was stirred for 30 min at room temperature and cooled to ꢀ788C. A solu-
tion of aldehyde 40 (344 mg, 1.45 mmol) in dry toluene (2 mL) was
added dropwise to the reaction mixture, which was stirred for 2.5 h at
ꢀ788C. After hydrolysis with an aqueous solution of NaOH (2.5m,
25 mL), the mixture was stirred for 2 h at room temperature. Diethyl
ether (15 mL) was added to the reaction mixture, the phases were sepa-
rated, and the aqueous phase was extracted with diethyl ether (3ꢃ
10 mL). The combined organic layers were dried over MgSO4, filtered,
and evaporated in vacuo. The residue was purified by flash column chro-
matography on silica gel (hexanes/EtOAc 80:20) to allow isolation of al-
cohol 41 (295 mg, 1.06 mmol, 73%) and its diastereoisomer (73 mg
0.26 mmol, 18%) in 91% total yield. Rf =0.6 (hexanes/EtOAc 70:30).
1H NMR (300 MHz, CDCl3, 258C, TMS): d=0.92 (d, J=6.6 Hz, 3H),
1.15–1.30 (m, 1H), 1.40–1.70 (m, 3H), 1.71–1.79 (m, 1H), 2.10–2.18 (m,
1H), 2.28–2.34 (m, 1H), 3.23 (dd, JAB=9.1 Hz, J=6.4 Hz, 1H), 3.30 (dd,
JAB=9.1 Hz, J=6.2 Hz, 1H), 3.58–3.65 (m, 1H), 3.80 (s, 3H), 4.44 (s, 2H),
5.11–5.16 (m, 2H), 5.82 (dddd, J=17.5, 9.4, 7.9, 6.6 Hz, 1H), 6.88 (d, J=
8.7 Hz, 2H), 7.25 ppm (d, J=8.6 Hz, 2H); 13C NMR (75 MHz, CDCl3,
258C, TMS): d=17.3, 29.7, 33.6, 34.2, 41.9, 55.4, 71.1, 72.8, 76.7, 113.9,
118.2, 129.3, 129.4, 135.0, 159.2 ppm; IR (film): n=3700–3090, 3077, 2933,
2893, 1641, 1614, 1587, 1514, 1463, 1361, 1302, 1249, 1172, 1090, 1036,
914 cmꢀ1; HRMS (ESI): m/z calcd for C17H26O3Na+: 301.1780 [M+Na]+;
found: 301.1780.
(film): n˜ =3464, 2926, 2855, 1772, 1713, 1438, 1267, 1072, 984, 720 cmꢀ1
;
MS (ESI): m/z: 520 [M+Na]+; HRMS (ESI): m/z calcd for
C30H43NO5Na: 520.3033 [M+Na]+; found: 520.3019.
2-(3-{(2R,3S,6S,8S)-8-[(3S,6S,Z)-6-Hydroxy-3,5-dimethylhept-4-en-1-yl]-
3-methyl-1,7-dioxaspiroACHTUNGTRENNUNG[5.5]undecan-2-yl}propyl)isoindoline-1,3-dione
(33b): (R)-CBS (1.0m in toluene, 60 mL, 0.051 mmol, 2.2 equiv) was
added to a solution of ketone 31 (11.6 mg, 0.023 mmol, 1 equiv) in dis-
tilled toluene (0.80 mL). The reaction mixture was cooled under argon at
ꢀ788C and stirred for 30 min. A solution of catecholborane (1.0m in
THF, 46 mL, 0.046 mmol, 2 equiv) was added. The reaction mixture was
stirred for 12 h at ꢀ788C, quenched with methanol (100 mL), and allowed
to warm to room temperature. The reaction mixture was diluted with di-
ethyl ether and washed with 1m NaOH solution saturated with NaHCO3
until the aqueous washings were colorless. The aqueous phase was ex-
tracted three times with diethyl ether and the combined organic layers
were washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated in vacuo. The crude compound was purified by column
chromatography on silica gel (petroleum ether/diethyl ether 6:4) to
afford the corresponding reduced ketone (Z)-33b as a colorless oil
(9.8 mg, 0.020 mmol, 85%). [a]2D5 +18.0 (c=0.1 in CHCl3); 1H NMR
(400 MHz, CDCl3, 258C, TMS): d=0.82 (d, J=6.5 Hz, 3H), 0.92 (d, J=
6.5 Hz, 3H), 1.09–1.15 (m, 1H), 1.23 (d, J=6.5 Hz, 3H; H20), 1.24–1.62
(m, 17H), 1.71 (d, J=1.3 Hz, 3H; H18), 1.71–1.85 (m, 1H), 1.94–2.04 (m,
1H), 2.41–2.47 (m, 1H), 3.17 (ddd, J=2.0, 9.9, 9.9 Hz, 1H), 3.46–3.50 (m,
1H), 3.66–3.81 (m, 2H), 4.78 (q, J=6.4 Hz, 1H), 4.96 (br d, J=9.6 Hz,
1H), 7.71 (dd, J=3.0, 5.5 Hz, 2H), 7.85 ppm (dd, J=3.0, 5.5 Hz, 2H);
13C NMR (100 MHz, CDCl3, 258C, TMS): d=168.7, 136.5, 134.0, 133.5,
132.3, 123.4, 95.6, 74.3, 69.2, 66.0, 38.6, 36.2, 35.6, 35.3, 34.3, 33.8, 31.7,
31.5, 30.8, 28.0, 25.4, 21.74, 21.70, 19.3, 18.2, 17.2 ppm; IR (film): n˜ =
3464, 2926, 2855, 1772, 1713, 1438, 1267, 1072, 984, 720 cmꢀ1; MS (ESI):
m/z: 520 [M+Na]+; HRMS (ESI): m/z calcd for C30H43NO5Na: 520.3038
[M+Na]+; found: 520.3038.
AHCTUNGTERG(NNUN 4S,7R)-Ethyl 7-(tert-butyldimethylsilyloxy)-4-methyldeca-2,9-dienoate
(42): (Carbethoxymethylene)triphenylphosphorane (711 mg, 2.04 mmol)
was added to a solution of (2S,5R)-5-(tert-butyldimethylsilyloxy)-2-meth-
yloct-7-enal (39 mg, 0.144 mmol) in dichloromethane (15 mL) cooled to
08C. The reaction mixture was allowed to warm to room temperature
and stirred for 16 h. Water (15 mL) was added to the reaction mixture,
and the aqueous phase was extracted with dichloromethane (4ꢃ8 mL).
The organic phase was dried over MgSO4, filtered, and concentrated in
vacuo. Ester 42 (46 mg, 0.135 mmol) was isolated after flash column chro-
matography on silica gel (hexanes/EtOAc 97:3) in 94% yield as a color-
less oil. Rf =0.5 (hexanes/EtOAc 95:5); [a]2D5 = +20.8 (c=0.92, CHCl3);
1H NMR (300 MHz, CDCl3, 258C, TMS): d=0.03 (s, 6H), 0.87 (s, 9H),
1.03 (d, J=6.6 Hz, 3H), 1.28 (t, J=7.5 Hz, 3H), 1.32–1.55 (m, 4H), 2.16–
2.26 (m, 3H), 3.65–3.69 (m, 1H), 4.17 (q, J=7.2 Hz, 2H), 4.99–5.04 (m,
2H), 5.70–5.84 (m, 2H), 6.84 ppm (dd, J=15.6, 7.7 Hz, 1H); 13C NMR
(75 MHz, CDCl3, 258C, TMS): d=ꢀ4.4, ꢀ4.2, 14.4, 18.2, 19.6, 26.0, 31.5,
34.2, 36.7, 41.9, 60.3, 71.9, 116.9, 119.9, 135.3, 154.5, 167.0 ppm; IR (film):
n=3078, 2954, 2931, 2858, 1723, 1652, 1463, 1652, 1464, 1368, 1257, 1177,
tert-Butyl [(2R,3R)-2-hydroxy-3-methylpent-4-enyl]carbamate (36):
solution of (4S,5S)-2,2-dimethyl-a,a,a’,a’-tetraphenyldioxolane-4,5-di-
methanol (TADDOL; 2.0 g, 4.29 mmol, 1 equiv) in diethyl ether (10 mL)
was added to mixture of cyclopentadienyltitanium(IV) trichloride
A
A
ACHTUNGTRENNUNG
a
(0.94 g, 4.29 mmol, 1 equiv) and diethyl ether (20 mL). An additional por-
tion of diethyl ether (4 mL) was used to rinse all the diol into the reac-
tion mixture. The resulting mixture was stirred for 2 min at room temper-
ature and a solution of Et3N (1.5 mL) in diethyl ether (5 mL) was added
dropwise to the reaction mixture over 55 min. An additional portion of
diethyl ether (5 mL) was used to rinse all the Et3N into the reaction mix-
ture. The resulting mixture was stirred for 23 h protected from light at
room temperature and was filtered under argon. The solid was washed
with diethyl ether (4ꢃ5 mL), the yellow filtrate was cooled to 08C, and
1145, 1046, 987, 914, 836, 775 cmꢀ1
C19H36O3SiNa+: 363.2331 [M+Na+]; found: 363.2331.
(4S,7R)-Ethyl 7-hydroxy-4-methyldeca-2,9-dienoate (38): A catalytic
; HRMS (ESI): m/z calcd for
G
amount of CSA (6 mg, 0.026 mmol) was added to a solution of 42
(44 mg, 0.135 mmol) in methanol/dichloromethane (3:1, 4 mL). The reac-
tion mixture were stirred for 3 h at room temperature, the solvents were
evaporated, and the residue purified by flash column chromatography on
silica gel (hexanes/EtOAc 80:20). Alcohol 38 was isolated in quantitative
yield as a colorless oil (30 mg, 0.135 mmol). Rf =0.6 (hexanes/EtOAc
80:20); [a]2D5 = +27.0 (c=0.6 in CHCl3); 1H NMR (300 MHz, CDCl3,
258C, TMS): d=1.05 (d, J=6.6 Hz, 3H), 1.28 (t, J=7.2 Hz, 3H), 1.40–
1.55 (m, 4H), 2.07–2.17 (m, 1H), 2.26–2.31 (m, 2H), 3.55–3.65 (m, 1H),
4.17 (q, J=7.2 Hz, 2H), 5.10–5.15 (m, 2H), 5.74–5.85 (m, 1H), 5.77 (br d,
J=15.2 Hz, 1H), 6.84 ppm (dd, J=15.6, 7.9 Hz, 1H); 13C NMR (75 MHz,
CDCl3, 258C, TMS): d=14.4, 19.6, 32.0, 34.4, 36.6, 42.1, 60.3, 70.6, 118.5,
120.1, 134.7, 154.2, 167.0 ppm; IR (film): n=3700–3090, 3077, 2972, 2957,
a
solution of 2-butenylmagnesium chloride in THF (0.5m, 7.72 mL,
3.89 mmol, 0.9 equiv) was added dropwise to the filtrate over 20 min. The
resulting red mixture was stirred for 60 min at 08C and cooled to ꢀ788C.
N-Boc-2-aminoacetaldehyde (0.55 g, 3.43 mmol, 0.8 equiv) was added to
the reaction mixture, which was stirred at ꢀ788C for 20 h. A 40% aque-
ous solution of NH4F (8.71 mL, 94.1 mmol, 22 equiv) was added dropwise
to the resulting orange reaction mixture, which was left to warm to room
temperature and stirred for 24 h. The white reaction mixture was filtered
through celite 545, the pad was washed with THF and dichloromethane,
and the filtrate was evaporated in vacuo. Water (40 mL) was added to
the solid residue, the aqueous phase was extracted with EtOAc (3ꢃ
40 mL), and the combined organic layers were washed with brine (2ꢃ
40 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The crude
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Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
2933, 2881, 1716, 1652, 1456, 1369, 1269, 1198, 1037, 987, 914 cmꢀ1
;
was dried over MgSO4, filtered, and concentrated in vacuo. The crude
residue was purified by flash column chromatography on silica gel (hex-
anes/EtOAc 85:15) to yield enone 44 (16 mg, 0.060 mmol, 77%) as a col-
orless oil. Rf =0.5 (hexanes/EtOAc 80:20); [a]2D5 =ꢀ50.0 (c=0.4 in
CHCl3; [a]2D5 =ꢀ54.0 (c=0.1 in CHCl3) for the acid derivative);[10a]
1H NMR (300 MHz, CDCl3, 258C, TMS): d=0.81 (d, J=7.0 Hz, 3H),
1.25 (t, J=7.1 Hz, 3H), 1.27–1.43 (m, 2H), 1.59–1.78 (m, 2H), 1.87–1.95
(m, 1H), 1.89 (dd, J=7.0, 1.7 Hz, 3H), 2.34 (dd, JAB=14.5 Hz, J=4.7 Hz,
1H), 2.51 (dd, JAB=15.2 Hz, J=6.2 Hz, 1H), 2.71 (dd, JAB=14.5 Hz, J=
10.0 Hz, 1H), 2.78 (dd, JAB=15.2 Hz, J=6.4 Hz, 1H), 4.05–4.32 (m, 3H),
4.28 (bdt, J=9.8, 4.9 Hz, 1H), 6.11 (dq, J=15.7, 1.7 Hz, 1H), 6.83 ppm
(dq, J=15.7, 6.8 Hz, 1H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=
14.3, 16.7, 18.4, 26.6, 30.6, 32.8, 33.1, 45.9, 60.6, 66.7, 74.3, 132.6, 143.3,
172.0, 198.4 ppm; HRMS (ESI) calcd for C15H24O4Na+: 291.1572 [M+
Na+]; found: 291.1572.
HRMS (CI): m/z calcd for C13H22O3H+: 227.1647 [M+H+]; found:
227.1647.
(2’S,3’S,6’R)-Ethyl 2-(6’-allyl-3’-methyltetrahydro-2H-pyran-2’-yl)acetate
(43): tBuOK (33 mg, 0.29 mmol) was added to a solution of alcohol 38
(60 mg, 0.27 mmol) in THF (2 mL) at ꢀ788C, and the reaction mixture
was stirred at ꢀ788C over 25 min. A saturated solution of NH4Cl (3 mL)
was added to the reaction mixture, which was warmed to room tempera-
ture and extracted with diethyl ether (3ꢃ3 mL). The organic phase was
dried over MgSO4, filtered, and concentrated in vacuo. The purification
of the residue by flash column chromatography on silica gel (hexanes/
EtOAc 98:2) furnished the Michael adduct 43 (43 mg, 0.19 mmol, 72%)
as a colorless oil. Rf =0.7 (hexanes/EtOAc 90:10); [a]2D5 =ꢀ60.4 (c=0.98
in CHCl3); 1H NMR (300 MHz, CDCl3, 258C, TMS): d=0.81 (d, J=
7.0 Hz, 3H), 1.26 (t, J=7.1 Hz, 3H), 1.27–1.40 (m, 2H), 1.61–1.67 (m,
2H), 1.86–1.98 (m, 1H), 2.07–2.26 (m, 2H), 2.31 (dd, JAB=14.0 Hz, J=
4.5 Hz, 1H), 2.71 (dd, JAB=14.0 Hz, J=10.6 Hz, 1H), 3.60–3.68 (m, 1H),
4.14 (qt, J=7.1, 1.1 Hz, 2H), 4.30 (dt, J=10.7, 4.7 Hz, 1H), 4.98 (br d,
J=10,4 Hz, 1H), 5.03 (br d, J=17.5 Hz, 1H), 5.78 ppm (ddt, J=17.1,
10.2, 7.0 Hz, 1H); 13C NMR (75 MHz, CDCl3, 258C, TMS): d=14.3, 16.7,
26.7, 30.3, 32.9, 33.2, 40.2, 60.5, 69.2, 74.2, 116.4, 135.3, 172.1 ppm; IR
(film): n=3071, 2976, 2952, 2932, 1737, 1438, 1370, 1285, 1191, 1711,
1037, 911 cmꢀ1; HRMS (ESI) calcd for C13H22O3Na+: 249.1467 [M+
(Methylthio)methyl-2-{(2S,3S,6R)-3-methyl-6-[(E)-2-oxopent-3-enyl]-tet-
rahydropyran-2-yl}acetic acid (45): A solution of NaOH (5 wt%, 0.2 mL)
was added to a solution of pyran 4 (8 mg, 0.0296 mmol, 1 equiv) in meth-
anol (0.3 mL), and the reaction mixture was stirred and monitored by
TLC. The reaction mixture was diluted with water and washed with di-
ethyl ether. The aqueous layer was then acidified until pH 3 with 3m HCl
and extracted with diethyl ether. The combined organic layers were
washed with brine, dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The crude compound was used without further
purification. DMSO (9 mL, 0.12 mmol, 4 equiv) in dichloromethane
(0.1 mL) was added to a solution of oxalyl chloride (30 mL, 0.06 mmol,
2 equiv) in dichloromethane (0.2 mL) at ꢀ788C over 15 min and stirred
for 15 min. The crude alcohol (0.03 mmol, 1 equiv) dissolved in dichloro-
methane (0.5 mL) was added dropwise to this mixture, which was stirred
for 30 min before dry triethylamine (200 mL, 1.5 mmol, 50 equiv) was
added dropwise. The reaction mixture was allowed to warm to room tem-
perature overnight and diluted with brine. The aqueous phase was ex-
tracted with diethyl ether. The combined organic layers were washed
with brine, dried over anhydrous sodium sulfate, filtered, and concentrat-
ed in vacuo. The crude compound was purified by column chromatogra-
phy on silica gel (petroleum ether/ethyl acetate 95:5!7:3) to afford 45 as
a colorless oil (5.9 mg, 0.0197 mmol, 66%). [a]2D5 =ꢀ64.3 (c=0.14 in
CHCl3); 1H NMR (400 MHz, CDCl3, 258C, TMS): d=0.81 (d, 9.3 Hz,
3H), 1.19–1.42 (m, 2H), 1.62–1.67 (m, 1H), 1.72–1.77 (m, 1H), 1.89 (dd,
J=1.6, 6.8 Hz, 3H), 1.88–2.00 (m, 1H), 2.23 (bs, 3H), 2.40 (dd, J=4.7,
14.7 Hz, 1H), 2.52 (dd, J=6.3, 15.3 Hz, 1H), 2.78 (dd, J=10.4, 14.7 Hz,
1H), 2.80 (dd, J=6.5, 15.3 Hz, 1H), 4.06–4.12 (m, 1H), 4.30 (ddd, J=4.8,
4.8, 9.9 Hz, 1H), 5.15 (dd, J=11.8, 24.9 Hz, 2H), 6.11 (ddd, J=1.6, 3.2,
15.7 Hz, 1H), 6.83 ppm (dddd, J=6.8, 6.8, 6.8, 15.7 Hz, 1H); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d=198.3, 171.7, 143.3, 132.6, 74.3, 68.4,
66.9, 45.9, 33.1, 32.8, 30.6, 26.6, 18.5, 16.8, 15.6 ppm; IR (film): n˜ =2924,
Na+];
ACHTUNGTRENNUNG found: 249.1468.
(2’S,3’S,6’R)-Ethyl 2-[6’-(2-hydroxypent-4-enyl)-3’-methyltetrahydro-2H-
pyran-2-yl]acetate (4): A solution of NMO (12 mg, 0.092 mmol) in water
(0.5 mL) followed by OsO4 (4% in water, 0.0008 mmol, 1 drop) was
added to cycloadduct 43 (19 mg, 0.084 mmol) dissolved in acetone
(2 mL). The reaction mixture was stirred for 24 h at room temperature,
water (5 mL) was added, and the aqueous phase was extracted with ethyl
acetate (5ꢃ5 mL). The organic layer was washed with brine (5 mL),
dried over MgSO4, filtered, and concentrated in vacuo. NaIO4 (19 mg,
0.088 mmol) was added to the crude residue (23 mg) dissolved in acetoni-
trile/water (5:1, 3 mL). After sthe had been stirred at room temperature
for 2 h, the solvents were evaporated and the residue dissolved in ethyl
acetate/water (1:1, 10 mL). The organic layer was separated and the
aqueous phase was further extracted with ethyl acetate (5ꢃ5 mL). The
combined organic phases were washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. Allyl bromide (0.18 mmol, 15 mL) and
zinc powder (0.18 mmol, 11 mg) were added to the crude residue (20 mg)
in THF/saturated NH4Cl solution (1.5:1, 2.5 mL). The reaction mixture
was stirred for 21 h at room temperature, diethyl ether (5 mL) was
added, the phases were separated, and the aqueous phase was further ex-
tracted with diethyl ether (5ꢃ3 mL). The combined organic phases were
washed with brine (5 mL), dried over MgSO4, filtered, and concentrated
in vacuo. The crude residue was purified by flash column chromatogra-
phy on silica gel (hexanes/EtOAc 80:20) to give alcohol 4 as a colorless
oil (21 mg, 0.078 mmol, 92% over 2 steps). Rf =0.7 (hexanes/EtOAc
70:30); 1H NMR (300 MHz, CDCl3, 258C, TMS): (mixture of isomers)
d=0.81 (d, J=7.0 Hz, 3H), 1.24–1.60 (m, 7H), 1.90–2.04 (m, 2H), 2.14–
2.37 (m, 4H), 2.74–2.84 (m, 1H), 3.42–3.91 (m, 2H), 4.09–4.35 (m, 3H),
5.01–5.10 (m, 2H), 5.75–5.92 ppm (m, 1H); 13C NMR (75 MHz, CDCl3,
258C, TMS): (mixture of isomers) d=14.3 (2C), 17.4, 17.5, 26.6, 27.1,
29.9, 30.5, 31.9, 32.1, 32.5, 32.9, 33.3, 41.7, 41.9, 42.9, 61.0, 61.1, 65.7, 66.7,
70.9, 71.4, 74.5, 74.9, 116.8, 117.1, 135.3, 135.7, 172.1, 173.3 ppm; HRMS
(ESI) calcd for C15H27O4+: 271.1909 [M+H+]; found: 271.1909.
2875, 1739, 1693, 1670, 1629, 1437, 1396, 1146, 1073, 972, 949, 916 cmꢀ1
;
MS (ESI): m/z: 323 [M+Na]+; HRMS (ESI): m/z calcd for
C15H24O4SNa: 323.1293 [M+Na]+; found: 323.1292.
2,5-Dioxopyrrolidin-1-yl
2-{(2S,3S,6R)-3-methyl-6-[(E)-2-oxopent-3-
enyl]tetrahydropyran-2-yl}-acetate (3): MgBr2·Et2O (30 mg, 0.12 mmol,
6 equiv) was added to a solution of the unsaturated enone 45 (5.9 mg,
0.0197 mmol, 1 equiv) in dry diethyl ether at room temperature under
argon. The reaction mixture was stirred for 3 h, diluted with water and
diethyl ether, and stirred for a further 15 min. The aqueous layer was ex-
tracted with diethyl ether and the organic layers were washed with brine,
dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo.
The crude compound was used without further purification. N-Hydroxy-
succinimide (4 mg, 0.03 mmol, 1.5 equiv) and 1-ethyl 3-(3-dimethylamino-
propyl) carbodiimide hydrochloride (6 mg, 0.03 mmol, 1 equiv) were
added to a solution of the crude acid (0.0197 mmol, 1 equiv) in freshly
distilled dichloromethane (0.6 mL). The reaction mixture was stirred at
room temperature overnight under argon and quenched with a solution
of saturated NaHCO3. The mixture was extracted with dichloromethane,
and the combined organic layers were washed with brine, dried over an-
hydrous sodium sulfate, filtered, and concentrated in vacuo. The crude
compound was purified by column chromatography on silica gel (petro-
(2’S,3’S,6’R,3’’E)-Ethyl 2-[3’-methyl-6’-(2-oxopent-3-enyl)tetrahydro-2H-
pyran-2’-yl]acetate (44): Dry DMSO (13 mL, 0.19 mmol) was added to
a solution of freshly distilled oxalyl chloride (8 mL, 0.09 mmol) in dry di-
chloromethane (1.2 mL) cooled at ꢀ788C, and the reaction mixture was
stirred for 15 min. A solution of alcohol 4 (21 mg, 0.078 mmol) in dry di-
chloromethane (0.8 mL) was added to the reaction mixture at ꢀ788C,
which was stirred for a further 20 min at that temperature. A solution of
freshly distilled Et3N (550 mL, 4 mmol) in dichloromethane (2 mL) was
added dropwise to the reaction mixture, which was allowed to warm to
room temperature and stirred for 24 h. The mixture was diluted with di-
chloromethane (10 mL) and washed with saturated aqueous solutions of
NaHCO3 (10 mL), NH4Cl (10 mL), and NaCl (5 mL). The organic layer
AHCTUNGERTGlNNUN eum ether/ethyl acetate 6:4) to afford N-hydroxysuccinimide ester 3 as
a
colorless oil (3.6 mg, 0.011 mmol, 55%). [a]2D5 =ꢀ26.3 (c=0.9 in
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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P. G. Goekjian, J. M. Lord et al.
CHCl3); 1H NMR (400 MHz, CDCl3, 258C, TMS): d=0.87 (d, 7.0 Hz,
3H), 1.28–1.42 (m, 2H), 1.64–1.72 (m, 1H), 1.79–1.86 (m, 1H), 1.89 (dd,
J=1.6, 6.8 Hz, 3H), 1.98–2.03 (m, 1H), 2.63 (dd, J=7.9, 15.8 Hz, 1H),
2.69 (dd, J=5.2, 15.0 Hz, 1H), 2.82 (s, 4H), 2.98 (dd, J=4.9, 15.8 Hz,
1H), 3.03 (dd, J=9.5, 15.0 Hz, 1H), 4.06–4.15 (m, 1H), 4.30 (ddd, J=5.0,
5.0, 9.7 Hz, 1H), 6.11 (ddd, J=1.6, 3.2, 15.8 Hz, 1H), 6.86 ppm (dddd, J=
6.8, 6.8, 6.8, 15.8 Hz, 1H); 13C NMR (100 MHz, CDCl3, 258C, TMS): d=
198.5, 169.0, 167.2, 143.5, 132.6, 73.9, 67.2, 45.5, 32.7, 30.5, 29.9, 26.5, 25.8,
18.5, 16.6 ppm; IR (film): n˜ =2923, 2852, 1812, 1785, 1741, 1691, 1633,
1463, 1377, 1208, 1067, 803 cmꢀ1; MS (ESI): m/z: 360 [M+Na]+; HRMS
(ESI): m/z calcd for C17H23NO6Na: 360.1423 [M+Na]+; found: 360.1423.
CD3CN, 258C, TMS): d=176.3, 157.7, 145.2, 142.1, 138.3, 133.2, 128.7,
128.1, 126.1, 121.0, 96.0, 75.2, 73.8, 68.8, 67.0, 65.7, 48.1, 45.9, 43.7, 40.0,
36.8, 35.9, 34.8, 33.9, 32.3, 31.7, 31.4, 30.4, 28.7, 26.9, 22.16, 22.10, 20.0,
18.3, 17.3, 15.6 ppm; IR (film): n˜ =3316, 2926, 2856, 1705, 1645, 1547,
1451, 1377, 1255, 1225, 1099, 1073, 1033, 985, 740 cmꢀ1; MS (ESI): m/z:
727 [M+Na]+; HRMS (ESI): m/z calcd for C42H60N2O7Na: 727.4293
[M+Na]+; found: 727.4296.
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
methyl-4-(-{(2S,3S,6R)-3-methyl-6-[(E)-2-oxopent-3-enyl]tetrahydro-2H-
pyran-2-yl}-ethanamido)butanamide (48): Diethylamine (90 mL) was
added to a solution of 47a (5.1 mg, 0.007 mmol, 1 equiv) in dry DMF
(0.4 mL), and the reaction mixture was stirred at room temperature until
total consumption of the starting material. The reaction mixture was con-
centrated in vacuo and DMF was removed by coevaporation with tolu-
ene. A solution of the crude amine and the N-hydroxysuccinimide ester
fragment 3 (3.5 mg, 0.010 mmol, 1.4 equiv) in dry DMF (0.1 mL) was
stirred 24 h at room temperature. The reaction mixture was concentrated
in vacuo, and the crude compound was purified by column chromatogra-
phy on silica gel (chloroform/methanol 88:12) to afford 48 (2.5 mg,
ACHTUNGTRENNUNG
A
E
N
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
2H-pyran-2-yl}-ethanamido)butanamide (bistramide A): Diethylamine
(68 mL) was added to a solution of 46 (3.2 mg, 0.005 mmol, 1 equiv) in
dry DMF (0.25 mL), and the reaction mixture was stirred at room tem-
perature until total consumption of the starting material. The reaction
mixture was concentrated in vacuo, and DMF was removed by coevapo-
ration with toluene. A solution of the crude amine and the N-hydroxy-
succinimide ester fragment 3 (3.0 mg, 0.010 mmol, 1.4equiv) in dry DMF
(0.25 mL) was stirred for 24 h at room temperature, and the reaction mix-
ture was concentrated in vacuo. The crude compound was purified by
column chromatography on silica gel (chloroform/methanol 88:2) to
1
0.003 mmol, 43%). [a]2D5 = +9.2 (c=0.13 in CH2Cl2); H NMR (400 MHz,
CDCl3, 258C, TMS): d=0.82 (d, J=6.5 Hz, 3H), 0.86 (d, J=6.9 Hz, 3H),
0.96 (d, J=6.9 Hz, 3H), 1.11–1.72 (m, 32H), 1.92 (dd, J=1.1, 6.8 Hz,
3H), 1.99–2.05 (m, 1H), 2.14 (dd, J=1.3, 15.2 Hz, 1H), 2.35–2.38 (m,
1H), 2.48–2.57 (m, 2H), 2.70–2.77 (m, 2H), 2.92 (dd, J=8.9, 17.1 Hz,
1H), 3.10–3.31 (m, 3H), 3.39–3.53 (m, 3H), 3.70–3.75 (m, 1H), 4.06–4.10
(m, 1H), 4.18–4.21 (m, 1H), 4.58–4.66 (bs, 1H), 4.76 (q, J=6.3 Hz, 1H),
4.91 (d, J=9.6 Hz, 1H), 6.12 (dd, J=1.6, 15.8 Hz, 1H), 6.90 (dq, J=6.8,
15.6 Hz, 1H), 7.16 (br t, J=5.5 Hz, 1H), 7.26 ppm (br t, 1H); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d=199.3, 175.4, 173.6, 144.9, 136.7,
133.5, 132.3, 95.6, 74.89, 74.85, 73.9, 67.8, 65.7, 65.0, 45.3, 44.6, 43.4, 39.8,
36.2, 35.7, 35.2, 33.8, 33.4, 33.0, 32.6, 31.8, 31.0, 30.81, 30.75, 28.1, 26.7,
25.7, 22.1, 21.6, 19.4, 18.6, 18.2, 17.21, 17.19, 15.7 ppm; IR (film): n˜ =
3335, 2925, 2855, 1697, 1649, 1554, 1452, 1378, 1260, 1097, 1026, 798,
711 cmꢀ1; MS (ESI): m/z: 727 [M+Na]+; HRMS (ESI): m/z calcd for
C40H68N2O8Na: 727.4868 [M+Na]+; found: 727.4859.
afford bistramide A as a colorless oil (2.8 mg, 0.004 mmol, 87%). [a]D25
=
+5.3 (c=0.32 in CH2Cl2); 1H NMR (400 MHz, CDCl3, 258C, TMS): d=
0.81 (d, J=6.5 Hz, 3H), 0.86 (d, J=7.0 Hz, 3H), 0.95 (d, J=6.7 Hz, 3H),
1.07–1.75 (m, 31H), 1.78–1.83 (m, 2H), 1.92 (dd, J=1.6, 6.8 Hz, 3H),
1.98–2.06 (m, 1H), 2.14 (dd, J=1.5, 15.2 Hz, 1H), 2.31–2.40 (m, 2H),
2.52 (dd, J=2.9, 17.0 Hz, 1H), 2.75 (dd, J=11.7, 15.2 Hz, 1H), 2.90 (dd,
J=9.0, 17.0 Hz, 1H), 3.15 (dt, J=2.2, 9.6 Hz, 1H), 3.23 (dt, J=5.7,
13.8 Hz, 1H), 3.30 (dt, J=6.6, 12.8 Hz, 2H), 3.41–3.47 (m, 1H), 3.50
(ddd, J=5.3, 6.5, 13.9 Hz, 1H), 3.71 (dt, J=5.1, 10.4 Hz, 1H), 4.06 (dd,
J=4.6, 11.0 Hz, 1H), 4.17–4.22 (m, 2H), 4.61 (br s, 1H), 5.18 (d, J=
9.4 Hz, 1H), 6.12 (dd, J=1.6, 15.8 Hz, 1H), 6.90 (dq, J=6.8, 15.5 Hz,
1H), 6.94 (br t, J=5.5 Hz, 1H), 7.30–7.31 ppm (br t, 1H); 13C NMR
(100 MHz, CDCl3, 258C, TMS): d=199.1, 175.3, 173.7, 144.7, 137.3,
132.3, 131.6, 95.6, 75.0, 74.4, 74.1, 73.5, 69.2, 64.9, 45.4, 44.9, 43.5, 39.6,
36.3, 35.6, 35.0, 34.3, 33.6, 33.5, 32.5, 32.0, 31.5, 30.9, 30.6, 28.1, 26.7, 26.0,
21.9, 21.1, 19.4, 18.6, 18.2, 17.3, 15.7, 12.0 ppm; IR (film): n˜ =3361, 2921,
2852, 1740,1658, 1464, 1378, 1261, 1097, 887, 720 cmꢀ1; MS (ESI): m/z:
727 [M+Na]+; HRMS (ESI): m/z calcd for C40H68N2O8Na: 727.4868
[M+Na]+; found: 727.4834.
(9H-Fluoren-9-yl)methyl
[(3S,6S,Z)-6-hydroxy-3,5-dimethylhept-4-en-1-yl]-3-methyl-1,7-dioxa-
spiro[5.5]undecan-2-yl}propyl)amino]-3-methyl-4-oxobutyl}carbamate
{(2R,3S)-2-hydroxy-4-[(3-{(2R,3S,6S,8S)-8-
A
ACHTUNGTRENNUNG
(47b): A solution methylamine in water (40% wt, 70 mL) was added to
a solution of 33b (9.8 mg, 0.020 mmol, 1 equiv) in ethanol (0.6 mL). The
reaction mixture was stirred at 508C for 3 h and then concentrated in
vacuo. A solution of the amino acid fragment 2 (10.0 mg, 0.03 mmol,
1.5 equiv) in DMF (0.4 mL), followed by PyBOP (11.4 mg, 0.022 mmol,
1.5 equiv) and diisopropylethylamine (7 mL), was added to a solution of
the crude amine in dry DMF (0.4 mL) at room temperature. The reaction
mixture was stirred overnight at room temperature and diluted with di-
ethyl ether. The organic layer was washed with a saturated NaHCO3 so-
lution. The aqueous phase was extracted with diethyl ether (3ꢃ), and the
combined organic layers were washed with brine, dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo. The crude compound
was purified by column chromatography on silica gel (petroleum ether/
ethyl acetate 8:2) to afford 47b as a colorless oil (8.5 mg, 0.012 mmol,
60%). [a]2D5 = +14.2 (c=0.1 in CHCl3); 1H NMR (400 MHz, CD3CN,
258C, TMS): d=0.79 (d, J=6.5 Hz, 3H), 0.86 (d, J=6.8 Hz, 3H), 1.12–
1.14 (m, 6H), 1.17–1.56 (m, 15H), 1.63 (d, J=1.3 Hz, 3H), 1.66–1.83 (m,
4H), 2.22–2.30 (m, 1H), 2.39–2.48 (m, 1H), 2.86 (d, J=3.4 Hz, 1H),
3.03–3.08 (m, 1H), 3.11–3.22 (m, 4H), 3.45–3.50 (m, 1H), 3.52–3.56 (m,
1H), 4.01 (d, J=6.9 Hz, 1H), 4.22 (t, J=6.9 Hz, 1H), 4.33 (dd, J=2.6,
6.8 Hz, 2H), 4.61–4.67 (m, 1H), 4.85 (d, J=9.7 Hz, 1H), 5.74–5.78 (br t,
1H), 6.66–6.68 (br t, 1H), 7.33 (t, J=7.5 Hz, 2H), 7.41 (t, J=7.4 Hz,
2H), 7.65 (d, J=7.5 Hz, 2H), 7.83 ppm (d, J=7.5 Hz, 2H); 13C NMR
(100 MHz, CD3CN, 258C, TMS): d=176.2, 157.7, 145.2, 142.1, 138.5,
132.8, 128.6, 128.1, 126.1, 120.9, 96.0, 75.0, 73.7, 69.3, 67.0, 66.0, 48.1, 46.0,
43.6, 40.0, 36.8, 35.9, 34.8, 34.3, 32.2, 31.9, 31.2, 30.3, 28.8, 26.6, 22.4, 22.0,
19.9, 18.2, 17.4, 15.6 ppm; IR (film): n˜ =2922, 2853, 1708, 1647, 1544,
1462, 1260, 1099, 984, 740 cmꢀ1; MS (ESI): m/z: 727 [M+Na]+; HRMS
(ESI): m/z calcd for C42H60N2O7Na: 727.4293 [M+Na]+; found:
727.4285.
(9H-Fluoren-9-yl)methyl
[(3S,6R,Z)-6-hydroxy-3,5-dimethylhept-4-en-1-yl]-3-methyl-1,7-dioxa-
spiro[5.5]undecan-2-yl}propyl)amino]-3-methyl-4-oxobutyl}carbamate
{(2R,3S)-2-hydroxy-4-[(3-{(2R,3S,6S,8S)-8-
A
ACHTUNGTRENNUNG
(47a): A solution of methylamine in water (40% wt, 6 mL) was added to
a solution of 33a (9.7 mg, 0.019 mmol, 1 equiv) in ethanol (0.6 mL). The
reaction mixture was stirred at 508C for 3 h and then concentrated in
vacuo. A solution of PyBOP (9.9 mg, 0.019 mmol, 1 equiv) and diisopro-
pylethylamine (7 mL) were added to a solution of the crude amine and
the amino acid fragment 2 (7.5 mg, 0.020 mmol, 1 equiv) in dry DMF
(0.4 mL) at room temperature. The reaction mixture was stirred over-
night at room temperature and diluted with diethyl ether. The organic
layer was washed with a saturated NaHCO3 solution. The aqueous phase
was extracted with diethyl ether (3ꢃ), and the combined organic layers
were washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated in vacuo. The crude compound was purified by column
chromatography on silica gel (petroleum ether/ethyl acetate 8:2) to
afford 47a as a colorless oil (5.1 mg, 0.007 mmol, 40%). [a]2D5 = +21.8
(c=0.11 in CHCl3); 1H NMR (400 MHz, CD3CN, 258C, TMS): d=0.80
(d, J=6.5 Hz, 3H), 0.94 (d, J=6.6 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H), 1.14
(d, J=6.4 Hz, 3H), 1.20–1.53 (m, 15H), 1.64 (d, J=1.3 Hz, 3H), 1.70–
1.85 (m, 4H), 2.23–2.33 (m, 1H), 2.43–2.52 (m, 1H), 2.94 (d, J=3.1 Hz,
1H), 3.02–3.17 (m, 3H), 3.20–3.30 (m, 2H), 3.44–3.50 (m, 1H), 3.52–3.59
(m, 1H), 4.00 (d, J=6.4 Hz, 1H), 4.23 (t, J=6.8 Hz, 1H), 4.34 (dd, J=
2.6, 6.8 Hz, 2H), 4.62–4.69 (m, 1H), 4.84 (d, J=9.2 Hz, 1H), 5.75 (br t,
1H), 6.72 (br t, 1H), 7.34 (t, J=7.1 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.66
(d, J=7.6 Hz, 2H), 7.84 ppm (d, J=7.5 Hz, 2H); 13C NMR (100 MHz,
&
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www.chemeurj.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!

Total Synthesis of Bistramide A and Synthetic Isomers
FULL PAPER
A
[7] C. Stanwell, A. Gescher, D. Watters, Biochem. Pharmacol. 1993, 45,
1753–1761.
R
E
N
ACHTUNGTRENNUNG
A
[8] A. V. Statsuk, D. Liu, S. A. Kozmin, J. Am. Chem. Soc. 2004, 126,
9546–9547.
2H-pyran-2-yl}ethanamido)butanamide (49): Diethylamine (110 mL) was
added to a solution of 47b (5.1 mg, 0.007 mmol, 1 equiv) in dry DMF
(0.35 mL), and the reaction mixture was stirred at room temperature
until total consumption of the starting material. The reaction mixture
was concentrated in vacuo and DMF was removed by coevaporation with
toluene. A solution of the crude amine and the N-hydroxysuccinimide
ester fragment 3 (4.7 mg, 0.014 mmol, 2equiv) in dry DMF (0.2 mL) was
stirred 24 h at room temperature. The reaction mixture was concentrated
in vacuo, and the crude compound was purified by column chromatogra-
phy on silica gel (chloroform/methanol 88:12) to afford 49 (4.6 mg,
0.0065 mmol, 90%). [a]D25 =ꢀ3.0 (c=0.1 in CH2Cl2); 1H NMR
(400 MHz, CDCl3, 258C, TMS): d=0.82 (d, J=6.5 Hz, 3H), 0.86 (d, J=
6.9 Hz, 3H), 0.92 (d, J=6.9 Hz, 3H), 1.08–1.69 (m, 28H), 1.71 (d, J=
1.2 Hz, 3H), 1.75–1.87 (m, 2H), 1.92 (dd, J=1.5, 6.8 Hz, 3H), 2.12 (dd,
J=1.4, 15.3 Hz, 1H), 2.34–2.41 (m, 1H), 2.48–2.56 (m, 2H), 2.70–2.80 (m,
2H), 2.89 (dd, J=8.9, 17.0 Hz, 1H), 3.12–3.38 (m, 3H), 3.45–3.55 (m,
3H), 3.68–3.75 (m, 1H), 4.01–4.08 (m, 1H), 4.15–4.23 (m, 1H), 4.64–4.71
(b s, 1H), 4.78 (q, J=6.3 Hz, 1H), 4.96 (d, J=9.7 Hz, 1H), 6.12 (dd, J=
1.6, 15.8 Hz, 1H), 6.90 (dq, J=6.8, 15.6 Hz, 1H), 7.08 (br t, J=5.5 Hz,
1H), 7.26 ppm (br t, 1H); 13C NMR (100 MHz CDCl3), d=199.3, 175.4,
173.6, 144.7, 136.7, 133.8, 133.2, 95.6, 75.0, 74.4, 73.9, 68.6, 65.7, 64.9, 45.5,
44.8, 43.4, 40.1, 36.2, 35.7, 35.2, 34.0, 33.5, 33.2, 32.0, 31.6, 31.0, 30.45,
30.34, 28.1, 26.7, 26.3, 22.3, 21.8, 19.3, 18.6, 18.1, 17.35, 17.27, 15.8 ppm;
IR (film): n˜ =3333, 2924, 2854, 1724, 1654, 1550, 1455, 1378, 1261, 1225,
1098, 985, 740 cmꢀ1; MS (ESI): m/z: 727 [M+Na]+; HRMS (ESI): m/z
calcd for C40H68N2O8Na: 727.4868 [M+Na]+; found: 727.4885.
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Acknowledgements
Financial support from the European Commission (contract No. LSHB-
CT-2004–503467, research fellowship for M.A.H. and G.B.aG) and from
the Ministꢅre de la Recherche et de la Technologie (research fellowship
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twicklungs- und Consulting GmbH (Maria Enzersdorf, Austria) for pro-
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Received: August 8, 2011
Revised: December 7, 2011
Published online: && &&, 2012
Chem. Eur. J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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ÞÞ
These are not the final page numbers!

P. G. Goekjian, J. M. Lord et al.
Natural Products
L. Tomas, G. Boije af Gennꢀs,
M. A. Hiebel, P. Hampson,
D. Gueyrard, B. Pelotier,
J. Yli-Kauhaluoma, O. Piva,
J. M. Lord,*
P. G. Goekjian* ............... &&&& — &&&&
Total Synthesis of Bistramide A and
Its 36(Z) Isomers: Differential Effect
on Cell Division, Differentiation, and
Apoptosis
Actin versus apoptosis–not so EZ: The
total synthesis of bistramide A and its
36(Z),39(S) and 36(Z),39(R) isomers
(see figure) relies on a novel synthesis
of exocyclic enol ethers for the spiro-
ketal fragment, a kinetic oxa-Michael
cyclization for the tetrahydropyran
fragment, and an asymmetric crotony-
lation for the amino acid fragment.
These compounds show distinctly dif-
ferent effects on cell division, differen-
tiation, and apoptosis, thus suggesting
that there are multiple independent
biological activities of the natural
product.
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