Synthesis of a 35-member stereoisomer library of bistramide A: Evaluation of effects on actin state, cell cycle and tumor cell growth

Synthesis of a 35-Member Stereoisomer Library of Bistramide A:

Evaluation of Effects on actin State, Cell Cycle and Tumor Cell

Growth

Iwona E. Wrona,^†Jason T. Lowe,^†Thomas J. Turbyville,^‡,§Tanya R. Johnson,^‡

Julien Beignet,^†John A. Beutler,^‡and James S. Panek*^,†

Department of Chemistry and Center for Chemical Methodology and Library DeVelopment (CMLD-BU), Boston

UniVersity, 590 Commonwealth AVenue, Boston, Massachusetts 02215, and Molecular Targets DeVelopment

Program, National Cancer Institute at Frederick, Building 560-15, Frederick, Maryland 21702

panek@bu.edu

ReceiVed October 24, 2008

Synthesis and preliminary biological evaluation of a 35-member library of bistramide A stereoisomers

are reported. All eight stereoisomers of the C1-C13 tetrahydropyran fragment of the molecule were

prepared utilizing crotylsilane reagents 9 and 10 in our [4+2]-annulation methodology. In addition, the

four isomers of the C14-C18 γ-amino acid unit were accessed via a Lewis acid mediated crotylation

reaction with use of both enantiomers of organosilane 11. The spiroketal subunit of bistramide A was

modiﬁed at the C39-alcohol to give another point of stereochemical diversiﬁcation. The fragments were

coupled by using a standard peptide coupling protocol to provide 35 stereoisomers of the natural product.

These stereochemical analogues were screened for their effects on cellular actin and cytotoxicity against

cancer cell lines (UO-31 renal and SF-295 CNS). The results of these assays identiﬁed one analogue,

1.21, with enhanced potency relative to the natural product, bistramide A.

Introduction

While drugs that directly modulate actin have not progressed

to the stage of use as clinical cancer therapeutics, actin inhibitors

have demonstrated activity in many in vitro cancer models. In

addition, actin inhibitors have been extremely useful in elucidat-

ing the role of actin in cellular processes. Natural products, in

particular, have been a rich source of actin inhibitors, and many

have been cocrystallized with actin.²The cytochalasins and

phalloidins bind to F-actin,²while G-actin modulators include

latrunculins, mycalolide B, jasplakinolide, dolastatin 11, aply-

ronine A, swinholide A, and bistramide A.³Of the latter group,

bistramide A is unique in its binding mode to actin. Bistramide

A binds and sequesters G-actin and thus inhibits actin ﬁlament

formation, thereby promoting ﬁlament disruption.⁴A high-

resolution X-ray crystallographic structure of the bistramide

A-actin complex showed its binding site overlaps only slightly

Actin is a cytoskeletal protein that plays a crucial role in

maintenance of cell shape and other important cellular processes

including motility, cytokinesis, phagocytosis, and intracellular

transport. These functions are important for normal cell viability

as well as for the aberrant processes vital to cancer cell growth,

invasion, and metastasis. To function properly, actin is actively

switched in a dynamic equilibrium between the G-actin and

F-actin, both targets of natural products.¹The complex nature

of actin regulation is under active investigation, and cell

signaling pathways that include the small GTPases Rho, Rac,

and Cdc42 have been found to play coordinated, important roles

in the assembly and disassembly of actin-rich structures such

as stress ﬁbers, lamellipodia, ﬁlopodia, and focal adhesion

complexes.¹

(2) Cooper, J. A. J. Cell Biol. 1987, 105, 1473.

^†Boston University.

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2006, 63, 2119. (b) Saito, S.; Watabe, S.; Ozaki, H.; Kigoshi, H.; Yamada, K.;

Fusetani, N.; Karaki, H. J. Biochem. 1996, 120, 552. (c) Bubb, M. R.; Spector,

I.; Bershadsky, A. D.; Korn, E. D. J. Biol. Chem. 1995, 270, 3463.

^‡NCI at Frederick.

^§SAIC-Frederick.

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10.1021/jo802269q CCC: $40.75

Published on Web 02/03/2009

2009 American Chemical Society

J. Org. Chem. 2009, 74, 1897–1916 1897

Wrona et al.

SCHEME 1. Structures of Bistramides A-D and K

with that of other G-actin inhibitors.⁵Thus, bistramide A is a

novel actin inhibitor worthy of further investigation.

dropyran and a spiroketal subunit joined at the center by a

γ-amino acid linker. Subsequent spectroscopic and computa-

tional studies allowed for accurate prediction of relative and

absolute conﬁgurations of bistramides.¹¹Kozmin reported the

ﬁrst total synthesis of bistramide A in 2004, which conﬁrmed

the proposed stereostructure of the natural product.¹²To date,

there are several reports detailing fragment,^11b,13degradative,^11a,14

and total syntheses¹⁵of bistramides A-D and K.

Stereochemically complex natural products provide an op-

portunity to discover novel biologically relevant molecules.

Variation of chirality at selected stereocenters presents a method

for diversifying natural products as library templates or scaf-

folds.⁶Each member of a stereoisomer library should provide

a unique conformational proﬁle that could have a substantial

effect on the biological activity. It is well documented that a

conformation of a molecule often modulates speciﬁc interactions

with its biological target, either to enhance or attenuate

biological activity. The so-called stereostructure/activity rela-

tionships (SSAR) have only recently gained attention with

respect to complex natural products.^6bOur group has developed

and explored the utility of chiral organosilane reagents to prepare

stereochemically well-deﬁned and diversiﬁed intermediates for

natural product synthesis. Having access to stereochemical

iterations of these building blocks presents the opportunity to

construct multiple conformations of complex natural products.

In this article, we report preparation and use of these chiral

reagents to synthesize, in solution phase, a focused 35-member

library of the marine metabolite bistramide A 1.1, and the

associated biology of these new compounds.

In addition to the structural^9-11and synthetic challenges posed

by this family of natural products, numerous reports describing

their diverse bioactivity have recently emerged.¹⁶Bistramides

were shown to exhibit various biological activities, encompass-

ing neurotoxic,^7bantiproliferative,^16dand cytotoxic properties.^7b,8

Speciﬁcally, bistramide A displayed activity against a number

of tumor cell lines including KB, P388, P388/dox, B16, HT29,

and NSCLC-N6 cell lines with IC₅₀values in the 0.03-0.32

µg/mL range.^16aThe antiproliferative activity of bistramide A

(8) Biard, J.-F.; Roussakis, C.; Kornprobst, J.-M.; Gouiffe`s-Barbin, D.;

Verbist, J.-F.; Cotelle, P.; Foster, M. P.; Ireland, C. M.; Debitus, C. J. Nat. Prod.

1994, 57, 1336.

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Kornprobst, J. M.; Verbist, J. F.; Biard, J. F.; Ireland, C. M. J. Am. Chem. Soc.

1992, 114, 1110.

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Kitching, W. Tetrahedron Lett. 2002, 43, 531. (c) Wipf, P.; Uto, Y.; Yoshimura,

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In 1988, Verbist and co-workers isolated the highly potent

antiproliferative agent bistramide A, 1.1, from the marine

ascidian Lissoclinum bistratum.⁷Four additional congeners of

this class of natural products were reported by the same research

group in 1994.⁸The structure of bistramide A was originally

proposed to be a 19-membered macrolactam⁹but later revised

to a linear carbon framework (Scheme 1).¹⁰The skeletal

architecture of bistramide A consists of a substituted tetrahy-

(12) Statsuk, A. V.; Liu, D.; Kozmin, S. A. J. Am. Chem. Soc. 2004, 126,

9546.

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Wrona, I. E.; Panek, J. S. Org. Lett. 2007, 9, 327. (c) Crimmins, M. T.; DeBaillie,

A. C. J. Am. Chem. Soc. 2006, 128, 4936. (d) Wipf, P.; Hopkins, T. D. Chem.

Commun. 2005, 3421.

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C. M. Biochem. Biophys. Res. Commun. 1999, 260, 80. (b) Gautret, P.; Le Pape,

P.; Biard, J. F.; Menard, D.; Verbist, J. F.; Marjolet, M. Acta Parasitol. 1998,

43, 50. (c) Pusset, J.; Maillere, B.; Debitus, C. J. Nat. Toxins 1996, 5, 1. (d)

Riou, D.; Roussakis, C.; Robillard, N.; Biard, J. F.; Verbist, J. F. Biol. Cell

1993, 77, 261. (e) Sauviat, M. P.; Verbist, J. F. Gen. Physiol. Biophys. 1993,

12, 465. (f) Sauviat, M. P.; Chesnais, J. M.; Choukri, N.; Diacono, J.; Biard,

J. F.; Verbist, J. F. Cell Calcium 1993, 14, 301. (g) Sauviat, M. P.; Gouiffe`s-

Barbin, D.; Ecault, E.; Verbist, J. F. Biochim. Biophys. Acta, Biomembranes

1992, 1103, 109.

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P. A.; Kozmin, S. A. Nat. Chem. Biol. 2005, 1, 383.

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Soc. 2006, 128, 3882.

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2004, 126, 36. (b) Curran, D. P.; Zhang, Q.; Richard, C.; Lu, H.; Gudipati, V.;

Wilcox, C. S. J. Am. Chem. Soc. 2006, 128, 9561. (c) Dandapani, S.; Jeske, M.;

Curran, D. P. J. Org. Chem. 2005, 70, 9447.

(7) (a) Gouiffe`s, D.; Moreau, S.; Helbecque, N.; Bernier, J. L.; He´nichart,

J. P.; Barbin, Y.; Laurent, D.; Verbist, J. F. Tetrahedron 1988, 44, 451. (b)

Gouiffe`s, D.; Juge, M.; Grimaud, N.; Welin, L.; Sauviat, M. P.; Barbin, Y.;

Laurent, D.; Roussakis, C.; Henichart, J. P.; Verbist, J. F. Toxicon 1988, 26,

1129.

1898 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

FIGURE 1. A schematic drawing of the actin monomer showing the major subdomains (inset). Cross-section representation of 1 and 3 subdomains

of G-actin. The red portion of the molecules insert into the “cleft” of 1 and 3 subdomains of actin. The atoms of the natural products involved in

polar contacts with the protein are boxed.

was initially hypothesized to be due to selective activation of

protein kinase C-δ.¹⁷In 2006, however, Kozmin and co-workers

obtained a crystal structure of the natural product bound to a

single monomeric unit of G-actin.^4,5,18actin is a 43 kDa protein

consisting of four subdomains. ATP and Mg²⁺bind within a

deep cleft between subdomains 4 and 2 and polymerization

occurs asymmetrically at the pointed and barbed ends of the

monomer with 5-times greater polymerization occurring at the

pointed end. For natural products known to target actin directly,

the barbed end and the ATP-binding domain are the primary

sites of interaction. Bistramide A belongs to the class of natural

products that induce disassembly of microﬁlaments (or prevent

its formation) by interfering with the barbed end of the actin

monomer (inset, Figure 1). Within the bistramide A binding

pocket of actin, an intimate hydrogen bonding network was

observed between the protein and the central amino acid portion

of the molecule. In contrast to bistramide A, however, most

barbed end targeting molecules such as reidispongiolides,¹⁹

sphinxolides,¹⁹trisoxazoles,²⁰and others²¹have very few polar

contacts within the actin pocket. The cross-sectional representa-

tion of the 1 and 3 subdomains of G-actin shows the difference

in binding between bistramide A and other natural products

(Figure 1). The binding of most actin targeting natural products

relies mainly on hydrophobic interactions between their respec-

tive macrocycles and a shallow patch on the side of actin.^3a

Bistramide A, on the other hand, inserts deep into the pocket

and displays multiple polar contacts, thus allowing for a unique

binding mode among barbed end targeting natural products.²²

With a detailed model in hand, the present study examines

the biological and conformational effects of speciﬁc stereoiso-

mers of bistramide A.²³These epimers incorporate small, subtle

changes into a highly organized system, allowing us to determine

the importance of each polar contact as well as how the

stereochemical environment associated with these contacts

deﬁnes changes in biological activity and conformation within

the actin pocket. The notable differences in the way bistramide

A binds to actin through a highly ordered hydrogen bonding

network and few stereostructural activity relationship (SSAR)

studies for this speciﬁc class of molecules supports the relevance

of this study. By systematically altering the stereochemical

environment of this highly conserved portion of the molecule,

a framework is provided for designing potentially more potent

analogues of bistramide A.

(17) Grifﬁths, G.; Garrone, B.; Deacon, E.; Owen, P.; Pongracz, J.; Mead,

G.; Bradwell, A.; Watters, D.; Lord, J. Biochem. Biophys. Res. Commun. 1996,

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Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 4088.

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Acad. Sci. U.S.A. 2005, 102, 14527.

(20) Klenchin, V. A.; Allingham, J. S.; King, R.; Tanaka, J.; Marriott, G.;

Rayment, I. Nat. Struct. Biol. 2003, 10, 1058.

(21) (a) Hirata, K.; Muraoka, S.; Suenaga, K.; Kuroda, T.; Kato, K.; Tanaka,

H.; Yamamoto, M.; Takata, M.; Yamada, K.; Kigoshi, H. J. Mol. Biol. 2006,

356, 945. (b) Klenchin, V. A.; King, R.; Tanaka, J.; Marriott, G.; Rayment, I.

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(22) For a review see: (a) Yeung, K. S.; Paterson, I. Angew. Chem., Int. Ed.

2002, 41, 4632. (b) Spector, I.; Braet, F.; Shochet, N. R.; Bubb, M. R. Microsc.

Res. Tech. 1999, 47, 18. (c) Fenteany, G.; Zhu, S. Curr. Top. Med. Chem. 2003,

3, 593.

(23) For structural activity relationship studies on bistramide A, see ref

18.

J. Org. Chem. Vol. 74, No. 5, 2009 1899

Wrona et al.

SCHEME 2. Retrosynthetic Analysis of Bistramide Stereoisomers 1.1-1.36

Our initial interest was focused on reported points of hydrogen

bonding (C13, C13-N, C15, C18, C18-N, C39) of the

molecule and how subtle stereochemical changes would affect

the overall activity of bistramide A. Next, we considered the

stereochemical environment around the tetrahydropyran where

hydrophobic interactions are thought to play an important role

between bistramide A and the actin pocket. A recent account

has explored these potential binding sites through computational

measurements and highlighted the importance of tetrahydropyran

fragment in regards to binding and cytotoxicity.²⁴A greater

understanding of such molecular systems may allow for design

and synthesis of more efﬁcacious anticancer agents. Herein, we

report the synthesis and the associated biological activity of 35

stereoisomers of the bistramide A planar structure.

substituted tetrahydropyran system. The four isomers of the

central amino acid portion 7 were anticipated to arise from Lewis

acid-mediated additions using chiral (E)-silane reagents 11. The

spiroketal fragment, at the present time, was envisioned to

remain stereostructurally intact except for the C39 alcohol.

Enantioselective reduction of the precursor R,ꢀ-unsaturated

ketone with CBS reagent allowed for formation of the unnatural

C39-epimer.

Preparation of All Stereochemical Permutations of C1-

C13 Fragment 6. Recent accounts from our group detailed

formal [4+2]-annulations of enantioenriched allyl- and crotyl-

silanes with aldehydes to form highly functionalized dihydro-

pyran systems.²⁵This methodology was applied to the synthesis

of complex natural products, including (-)-apicularen A, (+)-

leucascandrolide A, callipeltoside A, GEX 1A, (+)-neopeltolide,

and most recently kendomycin.²⁶To synthesize bistramide A,

the then currently available silane reagents did not allow direct

access to the C9-C11-cis conﬁguration of the pyran moiety.

This can be explained by inspecting the proposed mechanism

of (E)-anti-crotylsilane ent-10b (Scheme 3). The absolute

stereochemical outcome of the annulation reaction has been

proposed to occur via an intramolecular anti-S_E′ mode of

addition.²⁷

Results/Discussion

Retrosynthetic Analysis. Recently, we reported a total

synthesis of bistramide A that utilizes 21 steps (longest linear

sequence) from readily available reagents.^15bThe strategy was

highlighted by the use of three different chiral crotylsilane

reagents to construct all principle fragments [(6R,9S,11R)-6,

(15S,16R)-7, and (39S)-8] and simultaneously install 8 of 11

stereogenic centers. Following our reported synthesis, we

decided to make several stereochemical iterations (35) of the

natural product in order to conduct SSAR studies. While

synthesis of this many stereoisomers of a complex natural

product might seem daunting, the highly convergent nature of

the synthesis together with ready access to a wide range of chiral

organosilane reagents greatly simpliﬁed the strategic plan.

Accordingly, disconnection at the amide bonds provided three

fragments of varying size and complexity possessing different

synthetic challenges (Scheme 2). The eight stereoisomers of

pyran 6 were envisioned to come from (E)- and (Z)-crotylsilane

reagents 9 and 10. This is the ﬁrst example of using one

methodology to access all possible stereoisomers of a 2,5,6-

The crotylsilane reagent ent-10b will react with an aldehyde

in the presence of a Lewis acid, forming a mixed acetal that

eventually leads to generation of an oxocarbenium ion I. This

reactive intermediate, if placed in a chairlike transition state

with the silyl group in a pseudoaxial orientation, allows for the

most effective orbital σ_C-Si-π overlap and stabilization of the

adjacent positive charge (ꢀ-effect). Electrophilic cyclization and

elimination of silicon group provides the 2,6-trans-5,6-trans-

dihydropyran 12 as the major diastereomer. To obtain the 5,6-

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1900 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

SCHEME 3. Proposed Mechanism for Formation of 2,6-trans-5,6-trans-Dihydropyran 12

SCHEME 4. Preparation of Enantiomers of Silyl glycidols

cis conﬁguration needed for bistramide A, however, the reaction

would have to proceed through a boat-like conformation II. This

alternate transition state suffers from a number of destabilizing

interactions that provide 2,6-cis-5,6-cis-dihydropyran 13 only

as the minor product.

To utilize organosilane-based [4+2]-annulation methodology

to construct the C1-C13 portion of bistramide A, a new

crotylsilane reagent was required. We reasoned, based on the

proposed transition state, that changing oleﬁn geometry from

(E) to (Z) could effectively allow access to the desired 5,6-cis-

dihydropyrans (minor isomer 13 in Scheme 3). The outlined

annulation approach would create a complementary reagent to

our well-established methodologies.

The (E)-syn and (E)-anti organosilane reagents 10 were

obtained through various Claisen rearrangement strategies.²⁸

Preparation of the proposed (Z)-crotylsilane reagent 9 with these

rearrangement reactions was anticipated to be problematic due

to potential oleﬁn isomerization. What was required was a route

that would allow access to all structural and stereochemical

variations of this new reagent. Inspired by a recent review,²⁹

we hoped epoxysilanes 15 would provide enantioenriched

crotylsilanes 9. Epoxysilanes are known for their ease of

preparation and serve as versatile building blocks and synthetic

intermediates.³⁰Methods to generate these substrates in their

enantioenriched form are also available.³¹The general interest

in epoxysilanes stems from their predictable behavior in

nucleophilic oxirane openings to provide a wide range of

functionalized ꢀ-hydroxysilanes.³²

Preparation of cis- and trans-epoxysilanes required for

synthesis of enantioenriched (Z)-crotylsilanes is depicted in

Scheme 4. As anticipated, the trans-silylglycidols 15a were

obtained from a modiﬁed Sharpless asymmetric epoxidation of

(E)-vinylsilane 14 with (+)- or (-)-diethyl tartrate, respec-

tively.³³The resulting epoxysilyl alcohols 15a and ent-15a were

produced in 87% yield (2 steps from propargyl alcohol) and

with >98% enantiomeric excess (eq 1 in Scheme 4).³⁴A similar

multigram synthesis of a derivative of enantioenriched epoxysi-

lane 15a was previously reported.³⁵

(30) (a) Chakraborty, T. K.; Laxman, P. Tetrahedron Lett. 2003, 44, 4989.

(b) Hudrlik, P. F.; Gebreselassie, P.; Tafesse, L.; Hudrlik, A. M. Tetrahedron

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1999, 10, 3601. (b) Kobayashi, Y.; Ito, T.; Yamakawa, I.; Urabe, H.; Sato, F.

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P. R.; Mungall, W. S.; Schroder, G.; Sharpless, K. B. J. Am. Chem. Soc. 1988,

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107, 5541.

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J. Org. Chem. Vol. 74, No. 5, 2009 1901

Wrona et al.

SCHEME 5. Synthesis of (Z)-Crotylsilanes 9

Generation of the cis-epoxysilanes 15b and ent-15b was

initially attempted with enantioselective epoxidation of the

complementary (Z)-vinylsilane 16. Subjection of this silane 16

to Sharpless asymmetric epoxidation resulted in production of

the desired product in less than 5% yield.³⁶Accordingly, we

explored the possibility of an enzymatic resolution of the

racemic epoxide (eq 2 in Scheme 4).³⁷Epoxidation of vinyl-

silanes 16 with m-CPBA gave a racemic mixture of epoxy

alcohols rac-15b in 85% yield. Several lipase enzymes were

surveyed to resolve the racemate.³⁸Best results were obtained

by using Amano PS-D lipase in the presence of vinyl acetate

to provide primary alcohol 15b and acetate 17 in high yields

and enantiomeric excess. Base-catalyzed methanolysis of the

acetate group gave the desired cis-epoxysilane ent-15b in 97%

yield.³⁹To the best of our knowledge this is the ﬁrst example

of enzymatically resolved silylglycidols.

A straightforward, 3-step sequence of oxidation, oxirane

opening, and Lindlar reduction was used for completion of the

(Z)-crotylsilane reagent 9 (Scheme 5). Primary alcohol 15a was

subjected to NaIO₄and catalytic RuCl₃⁴⁰to give an acid, which

was immediately esteriﬁed⁴¹with DCC to provide trans-silyl

glycidate 18a (72%, 2 steps). Applying the oxidation/esteriﬁ-

cation sequence to the cis-epoxide 15b proved troublesome.

Upon scale-up, a signiﬁcant amount of aldehyde was observed

(ca. 20%) resulting in a two-step 55% isolated yield for ester

18b. Fortunately, two sequential catalytic ruthenium oxidations⁴²

followed by esteriﬁcation with DCC/DMAP afforded the silyl

glycidate 18b in 66% yield over 2 steps. Both the cis- and trans-

silylglycidates 18 have been prepared in >25 g quantities.

Regioselective epoxide ring opening with diethylpro-

pynylaluminum^30cgave 2,3-syn- and 2,3-anti-hexyne methyl

esters 19a and 19b, respectively, in moderate yields and good

selectivities. Minor reaction byproducts included alkyne addition

to the methyl ester and chloride opening of the silyl glycidate.

The latter is a common byproduct obtained from metal additions

to epoxysilanes (e.g., Grignard).²⁹Fortunately, addition of the

alkyne to the ꢀ-carbon of the silicon functionality was not

detected in the described conditions. Several explanations have

been put forth on the selective opening of epoxysilanes.^29,43-45

One interesting hypothesis includes the formation of a penta-

coordinated silicon followed by 1,2-migration,⁴⁵though more

recent studies with allyl cuprates do not support this pathway.⁴³

Alternatively, prior coordination of nucleophile with both the

R-carbon and silicon functionality has also been postulated.⁴⁴

Lastly, X-ray crystal structure⁴⁶and gas phase and theoretical

studies⁴⁷showed the R C-O bond (adjacent to the Si) to be

longer and weaker than the ꢀ C-O bond. Our particular system

has the added advantage of a Lewis basic site on the adjacent

carbonyl to aid in opening via coordination of resident metal

centers. Lindlar reduction of alkynes 19 followed by protection

of the secondary alcohols as their TMS ethers gave the syn-

and anti-(Z)-crotylsilanes 9a and 9b and their enantiomers. Each

stereoisomer was prepared in >10 g quantities.

The preparation of the required 5,6-cis-dihydropyran of the

C1-C13 fragment of bistramide A was explored next. Recently,

we described a [4+2]-annulation of syn-(Z)-crotylsilane reagent

9a with ꢀ-benzyloxy aldehyde 20 to provide the desired 5,6-

cis-pyran 21a in 66% yield (Table 1, entry 1). Extension of

this methodology to the other isomer silanes would afford all

possible stereochemical iterations of 2,5,6-dihydropyrans. Sub-

jection of all syn- and anti-(Z)- and (E)-silane reagents 9 and

10 to aldehyde 20⁴⁸under TMSOTf-catalyzed conditions

afforded the eight possible stereochemical analogues of the

(32) (a) Adam, J.-M.; de Fays, L.; Laguerre, M.; Ghosez, L. Tetrahedron

2004, 60, 7325. (b) Landais, Y.; Mahieux, C.; Schenk, K.; Surange, S. S. J.

Org. Chem. 2003, 68, 2779. (c) Ohi, H.; Inoue, S.; Iwabuchi, Y.; Irie, H.;

Hatakeyama, S. Synlett 1999, 11, 1757. (d) Fleming, I.; Barbero, A.; Walter, D.

Chem. ReV. 1997, 97, 2063. (e) Chauret, D. C.; Chong, J. M. Tetrahedron Lett.

1993, 34, 3695. (f) Russell, A. T.; Procter, G. Tetrahedron Lett. 1987, 28, 2041.

(g) Tamao, K.; Nakajo, E.; Ito, Y. J. Org. Chem. 1987, 52, 4412. (h) Hudrlik,

P. F.; Peterson, D.; Rona, R. J. J. Org. Chem. 1975, 40, 2263.

(33) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.;

Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765.

(34) Lowe, J. T.; Youngsaye, W.; Panek, J. S. J. Org. Chem. 2006, 71, 3639.

The enantiomeric excess was determined to be 98% for both 15a and ent-15a

as observed with Chiralcel OD 99/1 (hexanes/IPA) at 1 mL/min (t_R35:92 for

15a and 45:17 for ent-15a).

(42) During the course of oxidation, the reaction solution became a dark

green color with a black suspension, suggesting an inactive catalytic system. To

drive the reaction to completion, catalyst loading was increased and extended

reaction times were explored with little success. Only a reset of the catalytic

system provided full conversion.

(35) For preparation of (2R,3R)-3-(triphenylsilyl)-2,3-epoxypropane-l-ol see:

Raubo, P.; Wicha, J. Tetrahedron: Asymmetry 1995, 6, 577.

(36) Elevated temperatures (-20, 0, and 25 °C) under the described conditions

for the (E)-vinylsilane did not provide the desired epoxide.

(37) A Shi epoxidation of 16 was also considered; however, literature

precedent showed attenuated ee values for similar systems, see ref 32d for more

information.

(38) Lipase AK “Amano” 20, Lipase PS “Amano”, and Lipase PS-D

“Amano” were initially screened. Lipase PS-D was found to be superior.

(39) The enantiomeric excess was determined to vary between 94% and 96%

for both 15b and ent-15b as observed with Chiralcel OD 85/15 (hexanes/IPA)

at 1 mL/min (t_R5:03 for 15b and 7.08 for ent-15b).

(40) Carlsen, P. H. J.; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J. Org.

Chem. 1981, 46, 3936.

(41) Decomposition of cis and trans epoxide acids was noticed upon

prolonged storage or column chromatography.

(43) Hudrlik, P. F.; Ma, D.; Bhamidipati, R. S.; Hurdlik, A. M. J. Org. Chem.

1996, 61, 8655.

(44) (a) Eisch, J. J.; Galle, J. E. J. Org. Chem. 1976, 41, 2615. (b) Eisch,

J. J.; Trainor, J. T. J. Org. Chem. 1963, 28, 2870.

(45) (a) Davis, A. P.; Hughes, G. J.; Lowndes, P. R.; Robbins, C. M.; Thomas,

E. J.; Whitham, G. H. J. Chem. Soc., Perkin Trans. 1981, 1, 1934. (b) Eisch,

J. J.; Chiu, C. S. J. Organomet. Chem. 1988, 358, C1.

(46) Siriwardane, U.; Chu, S. S. C.; Buynak, J. D. Acta Crystallogr., Sect.

C: Cryst. Struct. Commun. 1989, C45, 531.

(47) Fristad, W. E.; Bailey, T. R.; Paquette, L. A.; Gleiter, R.; Boehm, M. C.

J. Am. Chem. Soc. 1979, 101, 4420.

(48) Cloarec, J.-M.; Charette, A. B. Org. Lett. 2004, 6, 4731.

1902 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

TABLE 1. [4+2]-Annulation of both (Z)- and (E)-Crotylsilanes 9

and 10 with Aldehyde 20

SCHEME 6. Possible Mechanism for (Z)-Crotylsilane ent-9b

annulation of isopropyl ester anti-(E)-crotysilanes 22 and ent-

22 afforded a 2-fold increase in selectivity for the desired 2,6-

trans-5,6-trans-dihydropyrans 23 and ent-23 (entries 8 and 9).

The stereochemical outcome of the annulation reaction with

(Z)-crotylsilanes was not entirely anticipated. In sharp contrast

to the proposed mechanism of (E)-crotylsilanes (Scheme 3), the

silyl group of (Z)-crotyl reagents presumably adopts a pseu-

doequatorial orientation. The oxocarbenium ion of ent-9b prefers

the chairlike intermediate IV to give dihydropyran 13 as the

major diastereomer (Scheme 6).⁵⁰The potential A^1,3destabiliz-

ing interactions arising from the (Z)-oleﬁn and axial silicon in

III⁵¹prevent formation of the 2,6-trans-5,6-cis product 24.⁵²

Though there is a turnover in selectivity about the 2,6-position

between the (E)- and (Z)-crotylsilanes, the reagents remain

completely complementary in their stereochemical outcome.

With ample quantities of all eight stereochemical analogues of

2,5,6-dihydropyrans in hand, we were positioned to complete

the synthesis of each of the desired isomers of the C1-C13

portion of bistramide A.

^aReactions were performed with 1.0 equiv of silane, 1.0 equiv of

aldehyde 20, and 1.0 equiv of TMSOTf in CH₂Cl₂at -50 °C. ^bIsolated

yield after SiO₂chromatography. ^cRatio determined by ¹H NMR

analysis of crude reaction mixtures.

Each dihydropyran stereoisomer was taken through an eight-

step sequence to access the diastereomeric C1-C13 fragments

6 (Scheme 7). Catalytic hydrogenation with Adam’s catalyst

concomitantly reduced the double bonds and deprotected the

benzyl ethers. The resulting alcohols were protected as TBDPS

ethers, and the methyl esters were reduced with LiBH₄. The

newly generated alcohols were treated with (PhO)₃P⁺CH₃I^-to

afford primary iodides 25. The primary iodides were displaced

with the lithium anion of 2-(1-propenyl)-1,3-dithiane to give

26 as masked R,ꢀ-unsaturated systems.⁵³The dithiane inter-

mediates 26 were unveiled to give ketones 27 by using a mild

method developed by Panek employing Dess-Martin periodi-

nane for the oxidative removal of thioacetals.⁵⁴Removal of the

TBDPS ether was accomplished with wet HF in acetonitrile

and oxidation of the resulting alcohols to carboxylic acids 6

occurred with catalytic chromium in wet acetonitrile. Comple-

tion of the eight stereoisomers of the C1-C13 tetrahydropyran

2,5,6-dihydropyran system (Table 1). The 2,3-syn-(Z)-crotylsi-

lanes 9a and ent-9a provided 2,6-trans-5,6-cis-dihydropyrans

21a and ent-21a in useful yields and selectivities (entries 1 and

2). In a complementary fashion, the 2,3-anti-(Z)-crotylsilanes

9b and ent-9b gave rise to dihydropyrans 21b and ent-21b

bearing all cis-2,5,6-stereochemical relationships in equally good

yields and selectivities (entries 3 and 4). Similarly, the 2,3-syn-

(E)-crotylsilanes 10a and ent-10a yielded 2,6-cis-5,6-trans-

dihydropyrans 21c and ent-21c with excellent yields and

selectivities (entries 5 and 6). The 2,3-anti-(E)-crotylsilane 10b

underwent annulation to provide dihydropyran 21d in moderate

yield, but with a 1:1 mixture of diastereomers (entry 7). This

result, however, was not entirely surprising. A similar problem

was encountered during a second generation approach to a late-

stage intermediate of leucascandrolide A.^26cAnnulations with

2,3-anti-(E)-crotylsilane 10b and protected 3-hydroxypropi-

onaldehyde derivatives provided dihydropyran products in poor

yields and selectivities. The problem was addressed through

modiﬁcation of the organosilane reagents to affect the resident

A values of the postulated intermediate oxocarbenium ion. This

analysis was loosely based on the A values of ethyl (1.1 kcal/

mol) versus that of a methyl ester (1.3 kcal/mol) of a mono-

substituted cyclohexane ring.⁴⁹The 0.2 kcal/mol difference

between methyl and ethyl esters should translate into even higher

activation energy for the isopropyl ester. Experimentally,

(50) It has been suggested that silicon prefers an axial orientation for both

electronic and steric reasons but may still eliminate even without optimal orbital

overlap. For a detailed discussion and relevant examples see: Lambert, J. B.

Tetrahedron 1990, 46, 2677.

(51) Intramolecular silyl-modiﬁed Sakurai condensations of vinylsilanes

suggest allylic strain plays a role in stereochemical outcome: Marko´, I. E.; Dobbs,

A. P.; Scheirmann, V.; Chelle´, F.; Bayston, D. J. Tetrahedron Lett. 1997, 38,

2899.

(52) It is conceivable that an oxonia-Cope rearrangement could play a role

in the stereochemical outcome of these reactions; however, no byproducts from

this pathway were observed. For an example of oxonia-Cope rearrangements in

allyl systems, see: Roush, W. R.; Dilley, G. J. Synlett 2001, 955.

(53) (a) Corey, E. J.; Seebach, D. Angew. Chem., Int. Ed. 1965, 4, 1075. (b)

Gro¨bel, B. T.; Seebach, D. Synthesis 1977, 357.

(49) Smith, M. B.; March, J. AdVanced Organic Chemistry; John Wiley and

Sons: New York, 2001, 5th ed.; p 174.

(54) Langille, N. F.; Dakin, L. A.; Panek, J. S. Org. Lett. 2003, 5, 575.

J. Org. Chem. Vol. 74, No. 5, 2009 1903

Wrona et al.

SCHEME 7. Synthesis of Eight Stereoisomers of the C1-C13 Tetrahydropyran Fragment 6

SCHEME 8. Initial Synthetic Approach to the C14-C18 Amino Acid Fragment 7

fragment was accomplished in a nine-step synthetic sequence

to provide the desired tetrahydropyran isomers in 20-30%

overall yield.

both monodentate and bidentate Lewis acids were utilized.⁵⁶

Since the anti-C15-C16 stereochemistry was needed as well,

an alternate protecting group was required that would be capable

of providing a sufﬁciently active site for chelation. To that end,

reaction of the Cbz-protected aldehyde 28c with silane (R)-11

afforded the desired alcohol 29c, albeit in low yield and

selectivity (30% yield, dr 2:1). Modiﬁcation of reaction condi-

tions (solvents, temperatures, Lewis acids) failed to improve

the selectivity and yield of this reaction. Thus, we turned our

efforts toward preparation of all stereoisomers of the C14-C18

amino fragment utilizing an R-alkoxy aldehyde. In this case, a

benzyl ether protecting group was employed to provide a second

chelation site with a bidentate Lewis acid.⁵⁷

Synthesis of C14-C18 Subunits 7. Our initial strategy for

the preparation of C14-C18 fragments of bistramide analogues

exploited a crotylation reaction between protected glycine

aldehyde 28 and organosilane reagent (R)-11 (Scheme 8). The

newly formed homoallylic alcohols 29 were thought to be

attainable with syn and anti relationships about the newly formed

bond. This “turnover” in diastereoselectivity depends on the

nature of Lewis acid (monodentate vs bidentate) and the

presence of a heteroatom on the aldehyde capable of providing

a second contact point with a Lewis acid. The latter was

showcased in the synthesis of mycalolide A where the C8-C9

anti-conﬁguration was obtained through chelation controlled

addition of silane (S)-11 with an R-nitrogen bearing aldehyde.⁵⁵

Functionalization of the resulting alcohols 29 via a four-step

sequence would afford the desired protected amino acids 30 in

only ﬁve steps.

The crotylation reaction was ﬁrst explored by using tert-butyl

carbamate (Boc) protected aldehyde 28a. Exposure of this

volatile and unstable aldehyde to our standard crotylation

conditions (BF₃· OEt₂, CH₂Cl₂) resulted in decomposition of

both starting materials, including protodesilylation of crotylsilane

reagent (R)-11. We next turned to condensation of phthalimide

protected amino aldehyde 28b with the organosilane reagent

(R)-11 in the presence of TiCl₄. The desired homoallylic alcohol

29b was obtained in 70% yield and high diastereoselectivity

(dr 20:1). This alcohol was then protected as a TBS ether and

the double bond was oxidized to afford the amino acid fragment

30b in just three steps (68% yield). Unfortunately, only the syn-

homoallylic alcohols were observed with aldehyde 28b when

The desired syn and anti homoallylic alcohols 33 were

accessed via a two-step sequence: [3+2]-annulation followed

by furan opening reactions (Scheme 9).^26aUse of the protected

acetaldehyde 31 allowed for generation of all possible isomers

of the amino acid fragment, where the primary alcohol would

later be exchanged for the desired amine functionality. Con-

densation of (R)-11 with R-benzyloxyacetaldehyde 31 in the

presence of SnCl₄provided 2,5-anti-tetrahydrofuran 32a in 73%

yield and excellent diastereoselectivity (>20:1). Use of

BF₃·OEt₂as the Lewis acid promoter, on the other hand,

effected formation of 2,5-cis-tetrahydrofuran ent-32b in 76%

yield and 20:1 selectivity. These furans were then subjected to

E₂-type elimination mediated by SbCl₅. The newly emerged

homoallylic alcohols, 33a and ent-33b, respectively, were

obtained in moderate to good yields, 62-85%, and greater than

10 g quantities. The remaining two isomers, alcohols ent-33a

(56) The homoallylic substrates were converted to 1,1-dimethyl acetonides

in a three-step sequence.

(57) (a) Panek, J. S.; Yang, M. J. Am. Chem. Soc. 1991, 113, 9868. (b) Panek,

J. S.; Beresis, R. J. Org. Chem. 1993, 58, 809.

(55) Liu, P.; Panek, J. S. J. Am. Chem. Soc. 2000, 122, 1235.

1904 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

SCHEME 9. Preparation of C15-Homoallylic Alcohols 33

SCHEME 10. Synthesis of Four Isomers of γ-Amino Acid Subunit 7

and 33b, were also obtained in this fashion following the same

reaction pathway utilizing enantiomeric crotylsilane (S)-11.

Completion of the synthesis of all isomers of the γ-amino

acid fragment 7 is depicted in Scheme 10. Protection of the

C15-homoallylic alcohols 33 as silyl ethers was followed by

an ozonolysis/NaBH₄reduction sequence to yield primary

alcohols in good yields (87-92%, 2 steps). Subsequent protec-

tion of the primary alcohols as TBS-ethers afforded the desired

intermediates 34 in high yields. Cleavage of benzyl ethers (Pd/

C, H₂) gave primary alcohols which were subjected to Mit-

sunobu conditions by using (PhO)₂P(O)N₃to install primary

azides in high yields (86-93% yields). Selective removal of

the primary TBS ether was effected by using CSA to provide

alcohol products 35 in 87-92% yields. The required TIPS esters

of the C14-C18 subunit were prepared in moderate yields via

a TEMPO-mediated oxidation and protection sequence (65-89%).

Reduction of the azide functionality (Pd/C, H₂) gave the fully

functionalized isomers of the amino acid fragment 7 in 15-41%

yield over 10 steps.

Preparation of the left-hand side of bistramide A analogues

proceeded as anticipated (Table 2). Union of eight isomers of

the C1-C13 pyran fragment 6 with the four diastereomers of

the γ-amino acid segments 7 was achieved in good yield by

using the coupling reagent PyBOP in the presence of triethy-

lamine. The TIPS-ester fragments 36 were obtained in yields

ranging between 53% and 81%. It is important to note that there

appears to be no stereochemical dependence on the extent of

conversion and the overall yield of the coupling reactions for

either the pyran or the amino acid fragments. Each isomeric

fragment 36 was prepared in greater than 40 mg quantities and

showed little decomposition after prolonged storage at low

temperature.

Preparation of the Spiroketal Moiety 8. Construction of

the C29-C40 segment of the spiroketal subunit utilized starting

materials obtained from a chiral pool source (Scheme 11).

Subjection of the commercially available (S)-1,2,4-butanetriol

derivative 37⁵⁸to a Wittig oleﬁnation reaction with phospho-

nium salt 38⁵⁹provided oleﬁn 39 as a mixture of geometric

isomers in 70% yield (Z:E ) 10:1). The exocyclic oleﬁn was

reduced and concomitant deprotection of the benzyl ether with

Raney nickel yielded a primary alcohol in 78% yield. Swern

oxidation of this material provided an aldehyde, which was

directly converted to the R,ꢀ-unsaturated ketone 40 by using

the Horner-Emmons reagent, diethyl 1-methyl-2-oxopropyl

phosphonate.⁶⁰The afforded ketone was enantioselectively

reduced with Corey’s oxazaborolidine reagent [(R)-CBS]⁶¹to

afford the (S)-alcohol, which was protected as a TBS ether 41

in 77% yield (2 steps). Reductive opening of the PMP acetal

41 with Dibal-H generated a secondary PMB ether in 98% yield.

Conversion of this material to the triphenyl phosphine salt 42

(58) Blakemore, P. R.; Kim, S. K.; Schulze, V. K.; White, J. D.; Yokochi,

A. F. T. J. Chem. Soc., Perkin Trans. 2001, 1, 1831.

(59) Gaeta, F. C. A.; Lehman de Gaeta, L. S.; Kogan, T. P.; Or, Y. S.; Foster,

C.; Czarniecki, M. J. Med. Chem. 1990, 33, 964.

(60) Altenbach, H. J.; Korff, R. Tetrahedron Lett. 1981, 22, 5175.

(61) Corey, E. J.; Helal, C. J. Angew. Chem., Int. Ed. 1998, 37, 1986, and

references found therein. .

J. Org. Chem. Vol. 74, No. 5, 2009 1905

Wrona et al.

TABLE 2. Structures of C1-C18 Isomeric Fragments 36

^aIsolated yields after SiO₂column chromatography.

1906 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

SCHEME 11. Reaction Sequence for the Synthesis of Phosphine Salt 42

SCHEME 12. Coupling of Tetrahydropyran 44 with the Phosphine Salt 42

SCHEME 13. Synthesis of C19-C40 Spiroketal Fragment 49

was carried out through a standard two-step sequence: bromi-

nation of the primary alcohol was followed by displacement

with PPh₃to afford the advanced phosphine salt partner 42.

Synthesis of the spiroketal portion of C19-C40 fragment 45

was initiated with a [4+2]-annulation employing syn-(E)-

crotylsilane reagent 10a and 4-(benzyloxy)butanal (Scheme

12).⁶²The desired 2,6-cis-dihydropyran 43 was obtained in 97%

isolated yield and high diastereoselectivity (dr 20:1). The pyran

double bond was isomerized into conjugation with tetra-n-butyl

ammonium hydroxide and subsequent treatment with methanolic

CSA afforded the methyl glycoside (78% over 2 steps). This

material was then reduced with Dibal-H to provide the

intermediate aldehyde 44, which was subjected to a Wittig

oleﬁnation with phosphonium salt 42. Gratifyingly, phosphorus-

based oleﬁnation provided the PMB-protected (Z)-alkene 45 in

86% yield and as a single geometric isomer. Incorporation of a

fully functionalized C40-C32 side chain prior to spirocycliza-

tion underscores the highly convergent nature of our synthesis.

Our initial approach for spirocycle formation exploited a two-

step sequence: deprotection of the C31-PMB group of the

C28-C29 unsaturated analogue 45 followed by spirocyclization

(Scheme 13).⁶³Unmasking the alcohol, however, proved to be

very difﬁcult as deprotection under standard DDQ conditions

(CH₂Cl₂/H₂O) did not provide the free C31-secondary alcohol

as hoped, but instead gave a complex mixture of compounds.

Interestingly, the major product identiﬁed from this reaction was

spirocycle 46, isolated in 17% yield after puriﬁcation (Scheme

13).⁶⁴Attempts to optimize the spirocyclization led to 45% yield

of the desired spiroketal intermediate 46.⁶⁵Selective hydrogena-

tion of 46 with Wilkinson’s catalyst gave the required spirocycle

(63) Fe´ne´zou, J. P.; Gauchet-Prunet, J.; Julia, M.; Pancrazi, A. Tetrahedron

Lett. 1988, 29, 3667.

(64) For a previous example of spirocyclization in the presence of DDQ see:

Paterson, I.; Chen, D. Y.-K.; Franklin, A. S. Org. Lett. 2002, 4, 391.

(62) Cloarec, J.-M.; Charette, A. B. Org. Lett. 2004, 6, 4731.

J. Org. Chem. Vol. 74, No. 5, 2009 1907

Wrona et al.

TABLE 3. Spiroketal Formation with the Saturated Framework

48

Having the desired spiroketal 47 in hand, a three-step

sequence was required for completion of the C19-C40 fragment

(Scheme 13). Birch reduction of the primary benzyl ether was

followed by a Mitsunobu displacement of the resulting alcohol

with (C₆H₅O)₂P(O)N₃. The desired primary azide was formed

in 86% yield and subjected to Me₃P to yield a free amine 49

via in situ hydrolysis of the phosphine imine intermediate.⁷⁵

With the fully functionalized spiroketal 49 in hand, we were

poised to begin construction of the complete carbon frameworks

of stereochemical analogues. Accordingly, union of deprotected

C1-C18 fragments 36i-l with the spirocycle 49 was ac-

complished by using the PyBOP peptide coupling reagent

(Scheme 14). The resulting silyl protected analogues were

obtained in 41-84% yield. Applying the previously reported

TBS-deprotection conditions (PPTS, MeOH) to these stereo-

chemical derivatives proved difﬁcult. Signiﬁcant decomposition

of a subset of analogues 50.16-50.24 was observed. Closer

examination of the crude reaction proﬁle suggested that the

C1-C4-R,ꢀ-unsaturated system of the isomers was affected.⁷⁶

Removal of the C15- and C39-silyl ethers with HF, HF ·pyridine,

CSA, or TBAF was unsuccessful. Interestingly, use of TBAF

in THF selectively removed the C15-TBS ether without

decomposition of the product. With this key observation, slight

modiﬁcation of the spirocycle 49 was performed prior to

coupling allowing access to stereochemical analogues without

incident. With that accomplished, azide 51 was treated with CSA

in methanol to expose alcohol (S)-8 in 85% yield (Scheme 15).

Reduction of azide (S)-8 to the primary amine with trimeth-

ylphosphine afforded the natural spiroketal fragment (S)-52.

Enantioselective inversion of the allylic alcohol intermediate

(S)-8 provided an additional point of stereochemical induction.

The C39-hydroxyl group has been shown to have a bridged

contact point with a water molecule of the Tyr143 residue of

actin; this residue is also hydrogen bonded to the C18 amide of

the natural product. Inversion of the C39-stereocenter was

achieved in a two-step process. Oxidation of the alcohol (S)-8

with Dess-Martin periodinane afforded an R,ꢀ-unsaturated

ketone (92% yield), which was reduced with (S)-CBS reagent

to give the C39-epimeric alcohol (R)-8 in good yield and

diastereoselectivity (81%, dr 20:1). Staudinger reaction and

hydrolysis of the resulting iminophosphorane provided the

unnatural spiroketal fragment (R)-52, epimeric at C39.

entry

conditions^a

yield^b

1

2

3

4

5

3 equiv of DDQ, CH₂Cl₂/H₂O (10/1)

3 equiv of DDQ, CH₂Cl₂/pH 7.0 buffer

3 equiv of DDQ, CH₂Cl₂

2 equiv of DDQ, CH₂Cl₂, 6 equiv pyridine

21

23

<5

76

44^c

^aReactions were carried out at 0 °C. ^bIsolated yields after SiO₂

column chromatography. ^cReaction was warmed to room temperature.

47 in 95% yield. However, the low yield obtained in spirocy-

clization prompted us to investigate alternative conditions for

this transformation.

To optimize the yield of the spirocyclization by using DDQ,

the C28-C29 oleﬁn was reduced as it may promote premature

oxonium ion formation. Selective hydrogenation of the C28-C29

oleﬁn with Wilkinson’s catalyst provided the monounsaturated

system 48 in 75% yield. Exposure of the C28-C29 dihydro

framework 48 to DDQ under aqueous or anhydrous conditions

provided low yields of the desired spiroketal 47 (entries 1-3,

Table 3). However, exposure of the intermediate 48 to DDQ

in the presence of pyridine under anhydrous conditions provided

the desired spirocycle 47 in 76% yield (entry 4).⁶⁶Warming

the reaction mixture to room temperature caused signiﬁcant

erosion in chemical yield (entry 5). Other conditions screened

for PMB deprotection/spiroketal formation include the follow-

ing: TMSI,⁶⁷I₂in MeOH,⁶⁸BCl₃·Me₂S complex,⁶⁹and

MgBr₂·Et₂O.⁷⁰

The role of pyridine in the reaction was not explored in detail

but it may behave as a simple buffer for any adventitious acid

that might cause premature formation of oxonium ion.⁷¹

Mechanistically, DDQ behaves as an electron acceptor to the

local PMB group to form a charge transfer complex. Upon

dissociation of this complex, the hydroquinone species 2,3-

dichloro-5,6-dicyano-1,4-dihydroxybenzene (DDQH₂) is gener-

ated. The DDQH₂molecule is somewhat acidic (pK_a) ∼5)

and was considered to be the potential cause of premature

oxonium ion generation under traditional conditions (water/

dichloromethane).⁷²An additional mechanistic role of pyridine

may involve production of an intermediate charge-transfer

complex between pyridine and DDQ.⁷³Charge-transfer com-

plexes have been documented with electron-rich nitrogen

heterocycles including pyrimidines and imidazoles.⁷⁴

Completion of the skeletal backbones of stereochemical

analogues was achieved in two steps (Scheme 16). Coupling

of C1-C18 acids 36a-p and ent-36a-p with the natural

spiroketal (S)-52 was mediated with PyBOP after initial

treatment of TIPS-acids with TBAF. The resulting C15-protected

alcohol products 53.1-53.32 were obtained in 60-80% yield.

Subjection of these advanced intermediates to TBAF afforded

the bistramide A as well as the desired 31 unnatural products

(73) (a) Bhattacharya, A.; DiMichele, L. M.; Dolling, U. H.; Grabowski,

E. J. J.; Grenda, V. J. J. Org. Chem. 1989, 54, 6118. (b) Bhattacharya, A.;

DiMichele, L. M.; Dolling, U. H.; Douglas, A. W.; Grabowski, E. J. J. J. Am.

Chem. Soc. 1988, 110, 3318. (c) Wallace, D. J.; Gibb, A. D.; Cottrell, I. F.;

Kennedy, D. J.; Brands, K. M. J.; Dolling, U. H. Synthesis 2001, 12, 1784.

(74) Charge-transfer complexes with other nitrogen heterocycles including

imidazoles and pyrimidines have been reported, see: (a) Salman, H. M. A.; Rabie,

U. M.; Abd-Alla, E. M. Can. J. Anal. Sci. Spectrosc. 2004, 49, 1. (b) Boraei,

A. A. A. Spectrochim. Acta, Part A 2002, 58, 1895. (c) Khashaba, P. Y.; El-

Shabouri, S. R.; Emara, K. M.; Mohamed, A. M. J. Pharm. Biomed. Anal. 2000,

22, 363.

(75) For a review of the Staudinger reaction see: Gololobov, Y. G.; Kasukhin,

L. F. Tetrahedron 1992, 48, 1353.

(76) The unknown product was obtained as a mixture of isomers with a

loss of C2-C3 oleﬁn. We were unable to separate the mixture for

characterization.

(65) Anhydrous conditions resulted in no reactions while using phosphate

buffer (pH 7) provided 19% yield of the desired spirocycle.

(66) Conditions found in Table 3 were also applied to 44 with little success.

(67) Gordon, D. M.; Danishefsky, S. J. J. Am. Chem. Soc. 1992, 114, 659.

(68) Vaino, A. R.; Szarek, W. A. Synlett 1995, 1157.

(69) Congreve, M. S.; Davison, E. C.; Fuhry, M. A. M.; Holmes, A. B.;

Payne, A. N.; Robinson, R. A.; Ward, S. E. Synlett 1993, 663.

(70) Onoda, T.; Shirai, R.; Iwasaki, S. Tetrahedron Lett. 1997, 38, 1443.

(71) The use of collidine as a buffer (against DDQH₂) in the DDQ oxidation

of silyl enol ethers has also been noted, see: (a) Fleming, I.; Paterson, I. Synthesis

1979, 736. (b) Siewert, J.; Textor, A.; Grond, S.; von Zezschwitz, P. Chem.

Eur. J. 2007, 13, 7424. (c) Banwell, M. G.; Hockless, D. C. R.; McLeod, M. D.

New J. Chem. 2003, 27, 50. (d) Fevig, T. L.; Elliott, R. L.; Curran, D. P. J. Am.

Chem. Soc. 1988, 110, 5064.

(72) Akutagawa, T.; Saito, G. Bull. Chem. Soc. Jpn. 1995, 68, 1753.

1908 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

SCHEME 14. Coupling of Acids 36 and Spiroketal 49 and Silyl Deprotection

SCHEME 15. Preparation of the C39-Epimer of the Spiroketal Subunit 52

SCHEME 16. Completion of the Synthesis of Stereoisomers 1.1-1.36

1.2-1.32 in good yields and greater than 3 mg quantities. On

the other hand, union of (R)-spiroketal 52 with the isomers of

the natural pyran 36a-d provided four additional epimers

1.33-1.36 of the natural product.

of the natural product in the barbed end binding pocket. A recent

publication describes computational docking studies using the

X-ray structure concerning the structural determinants of mac-

rolide-actin binding.¹⁴In this study, ﬂexible docking of the

bistramide A pyran fragment in the hydrophobic cleft of actin

revealed at least three hydrophobic interactions between the

protein and tetrahydropyran/enone portion of the molecule. The

highest scoring ﬂexibly docked structure was thus shown to have

rotation around the C12-C13 bond of the natural product. Thus,

the results of the computational docking experiments may help

to explain the enhanced activity of analogue 1.21 in the UO-31

cell line.

Six other epimers demonstrated submicromolar potency

against the UO-31 cell line (Table 4). Similar to the 1.21 variant,

the derivatives 1.5, 1.9, 1.17, 1.25, and 1.29 differ only in

stereochemistry at the C6, C9, and C11 positions of the pyran

moiety (Figure 2). Interestingly, compound 1.33, the C39-epimer

of the natural product, also showed good micromolar potency.

Biological Evaluation of Bistramide A Stereochemical

Analogues. The 35-membered stereochemical library of bis-

tramide A was screened for cytotoxicity and disruption of

cellular actin. The stereostructure activity relationships (SSAR)

of bistramide A 1.1 and its derivatives (1.2-1.36) were explored

by using cell growth assays: renal carcinoma line UO-31 and

the CNS tumor cell line SF-295 (Table 4). By using a 2-day

assay for cell growth inhibition of UO-31 cells, analogue 1.21

was shown to be approximately twice as potent (44 nM) as the

natural product 1.1 (77 nM). Interestingly, compound 1.21

differs stereochemically from bistramide A 1.1 at the C6 and

C9 positions of the pyran moiety (Figure 2). The C1-C13

portion of the bistramide A is not involved in polar contacts

with the protein but was suggested to only attenuate lipophilicity

J. Org. Chem. Vol. 74, No. 5, 2009 1909

Wrona et al.

TABLE 4. Cell-Growth Inhibition of Bistramide Stereochemical

Analogues 1.1-1.36 Against UO-31 and SF-295 Cancer Cell Lines

FIGURE 2. Representative structures of potent stereochemical ana-

logues of Bistramide A.

TABLE 5. Pearson Correlation Coefﬁcients between Compounds

1.1 and 1.21 and Other Known actin-Binding Natural Products^a

name

NSC

GI₅₀to 1.1

GI₅₀to 1.21

aplyronine A

687160

174119

107658

209835

175151

305222

675858

606195

613009

613011

V4827

V4828

685570

-0.11

0.55

0.71

0.69

0.37

0.34

0.58

0.28

0.30

0.49

-0.03

0.42

0.49

0.63

0.66

0.56

0.49

0.45

0.41

0.44

0.50

0.61

cytochalasin A

cytochalasin B

cytochalasin D

cytochalasin E

cytochalasin H

cytochalasin Q

dolastatin 11

jasplakinolide

latrunculin A

mycalolide C, D mixture

mycalolide E

^aCompounds were not tested. ^bEstimated IC50 value.

The remainder of the stereoisomers exhibited lower activity for

the UO-31 cell line, with the least potent analogue 1.7 having

an IC₅₀value of 45.3 µM (Table 4).

swinholide H

^aCorrelations were obtained for the GI₅₀level of response.

A subset of the compounds was also tested against the CNS

tumor cell line SF-295 under the same protocol (Table 4). In

general, the compounds inhibited cell growth to a lesser degree,

but the relative rank potency was similar to the UO-31 cell line.

Stereoisomer 1.33 showed a slightly better inhibition (0.34 µM)

in comparison to bistramide A (0.47 µM) for the SF-295 cell

line.

The effects on cell cycle were assessed for two of the most

potent cell growth inhibiting compounds (1.1 and 1.33), as well

as for the least active one (1.7). A dose responsive reduction of

the proportion of cells in S phase and increase in G1 phase by

analogues 1.1 and 1.33 was observed. These effects were

demonstrated in both the UO-31 and SF-295 cell lines indicating

blockade of the cell cycle by the two isomers. Stereoisomer

1.7, which was essentially inactive as an inhibitor of cell growth,

also had no effect on the cell cycle. As such, cell cycle data are

presented for a relatively potent library member, the natural

product (bistramide A), as well as a very weak binding member

of the collection. The essential point is that potent members of

the library appear to block the cell cycle in a manner consistent

with actin inhibition, while weak members of the collection do

not.

The inhibition of cell cycle by bistramide A 1.1 and analogue

1.33 was conﬁrmed with confocal microscopy examination of

FITC-phalloidin stained cells. At concentrations that inhibited

cell growth and cell cycle progression, the actin cytoskeleton

was disrupted as indicated by the loss of ﬂuorescence and

alteration of the cytoskeletal structure. These effects were

consistent with both cell lines.

Finally, the natural product 1.1 and C6,C9-stereochemical

analogue 1.21 were subjected to the NCI 60-cell assay. The

obtained results provide further insight into the action of

bistramide A and its epimers. Stereoisomer 1.21 was ap-

proximately twice as potent as the natural product 1.1, with

mean GI₅₀values of 9 and 18 nM, respectively. The patterns

of cell growth inhibition were highly correlated (Pearson

coefﬁcient of 0.81 at the GI₅₀level and 0.81 at the TGI level

of response). The most sensitive panels were the renal cancer

cell lines and the CNS cancer cell lines. The difference

between the least and most sensitive cell lines was between

2 and 2.5 log units at the GI₅₀level of response, and

approximately 3.5 log units at the TGI level. These data were

compared to that of other compounds known to bind to and

1910 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

FIGURE 3. Cell-growth inhibition of stereochemical analogues 1.1-1.10 against renal carcinoma cell line UO-31.

FIGURE 4. Cell cycle analysis of the UO-31 cell line for the most potent cell growth inhibiting isomers 1.1 and 1.33 and least active isomer 1.7.

interfere with actin (Table 5). With the notable exception of

aplyronine A, the data for all other natural products showed

signiﬁcant, albeit modest, correlations in 60-cell patterns to

the data for compounds 1.1 and 1.21.

Biological Activity: Experimental. Cytotoxicity Assays. A

tetrazolium dye [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-

2H-tetrazolium-5-carboxanilide; XTT]-based colorimetric as-

say was used in 96-well plates to measure inhibition of the

proliferation/survival of tumor cell lines in vitro (Figure 3).⁷⁷

All test compounds were formulated in DMSO and applied

to cells such that the ﬁnal DMSO concentration was e0.1%.

Cell Cycle Analysis. SF-295 and UO-31 cells were harvested

from ﬂasks at 75-85% conﬂuency, counted, and plated into

(77) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney,

S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988,

48, 4827.

J. Org. Chem. Vol. 74, No. 5, 2009 1911

Wrona et al.

FIGURE 5. Cell cycle analysis of the SF-295 cell line for the most potent cell growth inhibiting analogues 1.1 and 1.33 and least active stereochemical

compound 1.7.

FIGURE 6. Confocal microscopy using phalloidin staining images of UO-31 cells. Bistramide 1.1 causes disruption of the F-actin in a dose

dependent manner, while the least active epimer 1.7 does not.

35-mm dishes at a density of approximately 1 × 10⁶cells/dish,

as a 5 mM stock solution in DMSO and applied to cells at ﬁnal

concentrations of 1, 0.1, and 0.001 µM. A control dish was

then allowed to reattach overnight. Compounds were prepared

1912 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

FIGURE 7. Confocal microscopy using phalloidin staining images of SF-295 cells. Bistramide 1.1 and its stereochemical analogue 1.33 cause

disruption of the F-actin in dose dependent manner, while the least active epimer 1.7 does not.

treated with vehicle (DMSO) alone. Cells were incubated for

24 h in the continuous presence of the various treatments, at

which times they were rinsed with PBS, harvested by trypsiniza-

tion, resuspended in complete medium, pelleted by centrifuga-

tion, rinsed once with PBS, and resuspended in 1 mL of

Krishan’s buffer (0.1% sodium citrate, 0.02 mg/mL RNase, 0.3%

NP-40 and 50 mg/mL of propidium iodide (PI) at pH 7.4). Cells

were analyzed for relative DNA content by ﬂow cytometry by

using a Becton Dickinson FACScan (Becton Dickinson Immu-

nocytometry Systems, San Jose, CA) equipped with a DDM

using CELLQuest software. The analysis was done with

MODFIT LT 2.0 (Verity Software House, Inc., Topsham, ME).

Confocal Images. UO-31 and SF-295 cell monolayers were

established on glass coverslips and incubated overnight with

the compounds (Figures 4-7). After treatment, the cells were

ﬁxed in 3.7% formaldehyde, permeabalized with 0.1% Triton

X-100, and stained with ﬂuorescein phalloidin (Molecular

Probes, Invitrogen Corporation, Carlsbad, CA) at a ﬁnal

concentration of 165 nM. After staining, slides were rinsed with

PBS, and mounted in a DAPI containing mounting medium

(Molecular Probes). Confocal images were acquired using a 63×

oil objective (Zeiss UV510, Oberkochen, Germany).

Conclusions

We have shown that by employing a uniﬁed strategy, the

creation of a library of selected stereoisomers of the natural

product bistramide A is viable, providing the reaction methodol-

ogy is sufﬁciently robust. We have learned that the generation

of unique biological information on stereostructure-activity

relationships (SSAR) is a consequence of such an approach. In

the present case, we have sampled a small number of stereoi-

somers (35 of the 2048 possible compounds), selected where

crystallographic studies have indicated that the chirality might

be critical for binding to actin. Our synthetic strategy was

highlighted by preparation of the stereochemical variants of the

principle fragments with three structurally and stereochemically

different enantioenriched organosilane reagents. The compounds

were screened for growth inhibition against UO-31 and SF-

295 cell lines as well as for binding to actin. Six steroisomers

(1.5, 1.9, 1.17, 1.29, and 1.33) inhibited growth in the micro-

molar range, while compound 1.21 was shown to be the most

potent in the UO-31 cell line assay. Stereochemical analogue

1.21 was shown to be approximately twice as potent as the

natural bistramide A 1.1 with mean GI₅₀values of 9 and 18

nM, respectively, in the NCI 60 cell assay. Given that

stereoisomer libraries are generally difﬁcult to achieve by

solution phase synthesis is a testament to the versatility of

chiral silane-based bond construction methodology. Enabling

enhanced facility in asymmetric synthesis and more efﬁcient

NCI 60-Cell Assay. The NCI 60-cell assay was conducted

as previously described.⁷⁸Compounds were tested ﬁrst at a

single 10^-5M dose, then at two separate times in full dose

response format in ﬁve 10-fold dilutions. Comparison analyses

were performed as previously described.⁷⁹

(78) Monks, A.; Scudiero, D. A.; Johnson, G. S.; Paull, K. D.; Sausville,

E. A. Anti-Cancer Drug Des. 1997, 12, 533.

(79) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.;

Rubinstein, L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989, 81, 1088.

J. Org. Chem. Vol. 74, No. 5, 2009 1913

Wrona et al.

¹H NMR (CDCl₃, 400 MHz) δ 7.64 (m, 4H), 7.36 (m, 5H), 3.91

(dt, J ) 4.8, 9.6 Hz, 1H), 3.84 (dd, J ) 2.2, 11.8, 1H), 3.76 (m, 1H),

3.72 (s, 3H), 3.17 (dt, J ) 2.2, 9.4 Hz, 1H), 2.00-1.81 (m, 3H),

1.67-1.53 (m, 2H), 1.40 (m, 1H), 1.23 (m, 1H), 1.02 (s, 9H), 0.81

(d, J ) 6.4 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 172.1, 135.56,

135.55, 134.1, 134.0, 129.47, 129.44, 127.55, 127.53, 80.2, 60.0, 51.9,

35.7, 34.6, 32.7, 29.4, 26.9, 19.2, 17.7; IR (neat) ν_max2955, 2930,

2856, 1428, 1112, 702 cm^-1; HRMS (CI, NH₃) m/z calcd for

C₂₆H₃₆O₄Si [M]⁺440.2383, found 440.2334.

modular tactics in convergent assembly make it possible to

elucidate the role of each chiral center in bioactivity. With

bistramide A, the correlation to crystallographic data vali-

dated the approach, but it should be possible to empirically

probe stereochemical factors in the absence of a target as

well. For compounds with large numbers of chiral centers,

it may not be desirable to synthesize all possible stereoiso-

mers, but an iterative approach which samples several centers

in each stage to guide successive syntheses should facilitate

a complete analysis of SSAR, when coupled with high

throughput biological testing methods.

(2S,5R,6S)-6-(2-(tert-Butyldiphenylsilyloxy)ethyl)-5-methyltet-

rahydro-2H-pyran-2-yl)methanol. To a solution of ester (1.24 g,

28.1 mmol) in diethyl ether (25.0 mL) at 0 °C was added lithium

tetrahydroborate (1.23 g, 5.60 mmol) in one portion. The reaction

mixture was allowed to warm to room temperature and stirred for

an additional 2 h. The reaction was then quenched by addition of

water and the solution was stirred until bubbling ceased. Layers

were separated and the aqueous layer was extracted with Et₂O (3×).

The combined organic layers were dried with magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by

ﬂash chromatography (silica, 10% EtOAc/hexanes) provided 88%

Experimental Section

The following experimental information is representative and

describe the complete details of the convergent synthesis of

bistramide A stereoisomer 1.21.

(2S,5R,6S)-Methyl 6-(2-(Benzyloxy)ethyl)-5-methyl-5,6-dihy-

dro-2H-pyran-2-carboxylate (ent-21c). To a solution of 3-(ben-

zyloxy)propanal 20 (1.87 g, 11.4 mmol) and (E)-syn crotylsilane

ent-10a (2.00 g, 5.70 mmol) in methylene chloride (120 mL) at

-50 °C was added trimethylsilyl triﬂuoromethanesulfonate (2.08

mL, 11.4 mmol) dropwise. The reaction was stirred for 24 h at

-50 °C before it was quenched with saturated NaHCO₃and warmed

to room temperature. The layers were separated and the aqueous

layer was extracted with methylene chloride (3×). The combined

organic layers were dried with magnesium sulfate, ﬁltered, and

concentrated under reduced pressure. Puriﬁcation by column

chromatography (silica, 10% EtOAc/hexanes) afforded dihydro-

pyran ent-21c in 72% yield. [R]²⁵_D-116.7 (c 2.0, CHCl₃); ¹H NMR

(CDCl₃, 400 MHz) δ 7.33-7.25 (m, 5H), 5.73 (tq, J ) 2.0, 10.2,

22.6 Hz, 2H), 4.67 (m, 1H), 4.50 (q, J ) 12.0, 24.0 Hz, 1H), 3.75

(s, 3H), 3.72- 3.61 (m, 2H), 3.32 (dt, J ) 2.4, 9.6 Hz, 1H), 2.21

(m, 1H), 2.08-2.00 (m, 1H), 1.84-1.75 (m, 1H), 0.93 (d, J ) 7.2

Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 170.6, 138.6, 134.0, 128.3,

127.6, 127.4, 133.3, 77.3, 74.7, 72.9, 66.7, 52.3, 34.0, 33.2, 17.1;

IR (neat) ν_max2958, 2870, 1736, 1263, 1097, 700 cm^-1; HRMS

(CI, NH₃) m/z calcd for C₁₇H₂₂O₄[M + 23]⁺313.1416, found

313.1354.

of the desired product as a clear oil. [R]²⁵-26.0 (c 1.1, CHCl₃);

D

1H NMR (CDCl₃, 400 MHz) δ 7.66 (m, 4H), 7.38 (m, 6H), 3.78

(m, 2H), 3.50 (dt, J ) 3.0, 8.8 Hz, 1H), 3.43-3.30 (m, 2H), 3.12

(dt, J ) 2.0, 9.6 Hz, 1H), 1.96 (m, 1H), 1.78 (m, 2H), 1.54 (m, 1H),

1.45 (m, 1H), 1.34-1.11 (m, 2H), 1.03 (s, 9H), 0.80 (d, J ) 6.8 Hz,

3H); ¹³C NMR (CDCl₃, 75 MHz) δ 135.5, 134.0, 129.5, 127.6,

79.8, 77.5, 66.3, 60.3, 36.1, 35.1, 32.4, 27.6, 26.9, 19.2, 17.7; IR

(neat) ν_max3448, 3069, 2931, 2858, 1428, 1102, 703 cm^-1; HRMS

(CI, NH₃) m/z calcd for C₂₅H₃₈O₃Si [M + 23]⁺414.2593, found

414.2560.

tert-Butyl(2-((2S,3R,6S)-6-(iodomethyl)-3-methyltetrahydro-

2H-pyran-2-yl)ethoxy)diphenylsilane (ent-25c). To a solution of

alcohol (3.60 g, 8.72 mmol) and 2,6-lutidine (2.38 mL, 20.6 mmol)

in N,N-dimethylformamide (70.0 mL) at 0 °C was added methyl-

triphenoxyphosphonium iodide (8.48 g, 18.8 mmol) in one portion.

The reaction was stirred for 1 h at 0 °C before diethyl ether and

water were added. Layers were separated and the aqueous layer

was extracted with diethyl ether (3×). The combined organic layers

were dried with magnesium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by ﬂash chromatography (silica, 3%

EtOAc/hexanes) provided ent-25c in 79% as a clear oil. [R]²⁵_D-3.9

(c 1.5, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.66 (m, 4H), 7.35

(m, 5H), 3.85 (m, 2H), 3.23 (m, 1H), 3.12 (dt, J ) 2.0, 9.6 Hz,

1H), 3.05 (d, J ) 5.6 Hz, 2H), 1.95 (m, 1H), 1.83-1.74 (m, 2H),

1.53 (m, 2H), 1.30-1.16 (m, 2H), 1.04 (s, 9H), 0.80 (d, J ) 6.8

Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 135.8, 134.4, 129.7, 127.8,

80.6, 77.7, 60.6, 36.3, 35.0, 32.9, 31.9, 27.2, 19.5, 17.7, 10.0; IR

(neat) ν_max3068, 2931, 2857, 1465, 1097, 702 cm^-1; HRMS (CI,

NH₃) m/z calcd for C₂₅H₃₅IO₂Si [M + 23]⁺545.1349, found

545.1363.

(2S,5R,6S)-Methyl 6-(2-Hydroxyethyl)-5-methyltetrahydro-

2H-pyran-2-carboxylate. To a solution of pyran ent-21c (0.880

g, 3.01 mmol) in methanol (19 mL) was added PtO₂(0.171 g, 25

mol %). The reaction ﬂask was then placed under an atmosphere

of hydrogen and stirred for 12 h. The heterogeneous mixture was

ﬁltered over Celite, washed with methanol, and concentrated in

vacuo. Puriﬁcation by ﬂash chromatography (silica, 50% EtOAc/

hexanes) provided 95% of the desired product as a clear oil. [R]²⁵

D

1

-60.1 (c 1.0, CHCl₃); H NMR (CDCl₃, 400 MHz) δ 3.94 (dd, J

) 2.6, 11.8 Hz, 1H), 3.76 (m, 2H), 3.69 (s, 3H), 3.21 (dt, J ) 2.8,

9.8 Hz, 1H), 1.97-1.80 (m, 3H), 1.73 (m, 1H), 1.55 (dq, J ) 3.8,

13.0, 25.0 Hz, 1H), 1.43 (m, 1H), 1.22 (dt, J ) 3.8, 13.0, 25.2 Hz,

1H), 0.79 (d, J ) 6.4 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ

171.5, 84.7, 76.1, 61.3, 52.0, 34.7, 34.6, 32.0, 28.8, 17.3; IR (neat)

tert-Butyl(2-((2S,3R,6S)-3-methyl-6-((2-((E)-prop-1-enyl)-1,3-

dithian-2-yl)methyl)tetrahydro-2H-pyran-2-yl)ethoxy)diphenyl-

silane (ent-26c). To a solution of 2-((E)-1-propenyl)-[1,3]-dithiane

(2.02 g, 12.6 mmol) and hexamethylphosphoramide (3.30 mL, 18.9

mmol) in tetrahydrofuran (50.0 mL) at -78 °C was added tert-

butyllithium in pentane (1.70 M, 7.43 mL) dropwise. The reaction

mixture was stirred for 1 h at -78 °C before a solution of iodide

ent-25c (3.30 g, 6.32 mmol) in tetrahydrofuran (50.0 mL) was added

dropwise. The reaction mixture was then allowed to stir for 1 h at

-78 °C, warmed to 0 °C, and stirred for an additional hour.

Reaction was quenched by addition of water and extracted with

diethyl ether (3×). The combined organic layers were dried with

magnesium sulfate, ﬁltered, and concentrated under reduced pres-

sure. Puriﬁcation by ﬂash chromatography (silica, 1% EtOAc/

ν

_max3460, 2954, 2929, 2876, 1753, 1439, 1212, 1088 cm^-1; HRMS

(CI, NH₃) m/z calcd for C₁₀H₁₉O₄[M + 1]⁺203.1283, found

203.1286.

(2S,5R,6S)-Methyl 6-(2-(tert-Butyldiphenylsilyloxy)ethyl)-5-

methyltetrahydro-2H-pyran-2-carboxylate. To a solution of

alcohol (2.90 g, 14.3 mmol) in N,N-dimethylformamide (22.0 mL)

at 0 °C was added 1H-imidazole (1.20 g, 17.9 mmol) followed by

tert-butylchlorodiphenylsilane (4.10 mL, 15.8 mmol) dropwise. The

reaction was allowed to warm to room temperature on its own and

was stirred for 16 h at room temperature. The reaction mixture

was then diluted with water and extracted with Et₂O (3×). The

combined organic layers were dried with magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by

ﬂash chromatography (silica, 3% EtOAc/hexanes) provided 99%

hexanes) provided ent-26c in 90% as a clear oil. [R]²⁵-7.9

D

(c 2.5, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.65 (m, 4H), 7.37

(m, 6H), 5.80 (m, 1H), 5.43 (d, J ) 15.2 Hz, 1H), 3.83 (m, 2H),

3.38 (m, 1H), 2.99 (dt, J ) 2.0, 9.6 Hz, 1H), 2.76 (m, 1H),

2.67-2.56 (m, 3H), 2.09 (dd, J ) 6.8, 14.8 Hz, 1H), 1.91-1.75

of the desired product as a clear oil. [R]²⁵-34.1 (c 2.0, CHCl₃);

D

1914 J. Org. Chem. Vol. 74, No. 5, 2009

A 35-Member Library of Bistramide A Stereoisomers

(m, 4H), 1.70 (m, 1H), 1.64 (d, J ) 6.4 Hz, 3H), 1.55 (m, 2H),

1.35-1.13 (m, 3H), 1.02 (s, 9H), 0.76 (d, J ) 6.4 Hz, 3H); ¹³C

NMR (CDCl₃, 75 MHz) δ 135.6, 134.3, 133.7, 129.4, 128.4, 127.5,

80.4, 74.3, 61.4, 53.1, 48.1, 36.3, 34.8, 33.2, 33.1, 27.1, 27.0, 26.9,

25.2, 19.2, 17.8, 17.5; IR (neat) ν_max3068, 2928, 2856, 1428, 1087,

703 cm^-1; HRMS (CI, NH₃) m/z calcd for C₃₂H₄₆O₂S₂Si [M]⁺

554.2708, found 554.2720.

J ) 5.0, 15.6 Hz, 1H), 2.38 (B of ABX, J ) 9.6, 15.6 Hz, 1H),

1.87 (dd, J ) 1.6, 6.8 Hz, 3H), 1.79 (m, 1H), 1.68 (m, 1H),

1.41-1.20 (m, 3H), 0.83 (d, J ) 6.8 Hz, 3H); ¹³C NMR (CDCl₃,

75 MHz) δ 198.2, 174.5, 143.8, 132.2, 79.9, 74.4, 45.9, 38.7, 34.8,

32.1, 31.6, 18.3, 17.4; IR (neat) ν_max2929, 1713, 1631, 1438, 1194,

1082, 971 cm^-1; HRMS (CI, NH₃) m/z calcd for C₁₃H₂₀O₄[M +

23]⁺263.1259, found 263.1249.

(2S,3R)-Triisopropylsilyl 3-(tert-Butyldimethylsilyloxy)-2-

methyl-4-(2-((2S,3R,6S)-3-methyl-6-((E)-2-oxopent-3-enyl)tet-

rahydro-2H-pyran-2-yl)ethanamido)butanoate (ent-36j). A so-

lution of azide (1.00 mg, 0.233 mmol) in tetrahydrofuran (10.0 mL)

was treated with 10 wt % palladium on activated carbon (5.0 mg).

The reaction ﬂask was ﬂushed with argon and hydrogen atmo-

spheres sequentially. The reaction was then placed under an

atmosphere of hydrogen and stirred for 12 h. The heterogeneous

mixture was ﬁltered over Celite, rinsed with tetrahydrofuran, and

concentrated in vacuo. The resulting thick oil of 7a was used in

the next step without further puriﬁcation.

To a solution of the crude amine 7a in methylene chloride (10.0

mL) was added crude acid ent-6c (50.0 mg, 0.208 mmol), PyBOP

(119 mg, 0.229 mmol), and triethylamine (34.8 µL, 0.250 mmol).

The reaction mixture was allowed to stir for 24 h at room

temperature. Upon completion of the reaction, diethyl ether was

added. The organic layer was washed sequentially with water,

saturated sodium bicarbonate, and brine solutions. The combined

organic layers were dried with magnesium sulfate, ﬁltered, and

concentrated under reduced pressure. Puriﬁcation by ﬂash chro-

(E)-1-((2S,5R,6S)-6-(2-(tert-Butyldiphenylsilyloxy)ethyl)-5-me-

thyltetrahydro-2H-pyran-2-yl)pent-3-en-2-one (ent-27c). To a

solution of dithiane ent-26c (100 mg, 0.180 mmol) in a mixture of

acetonitrile (4.00 mL), methylene chloride (0.500 mL), and water

(0.500 mL) was added Dess-Martin periodinane in methylene

chloride (0.30 M, 1.20 mL) dropwise. The reaction was allowed to

stir at room temperature for 5 h before ethyl acetate and water were

added. Layers were separated and water was extracted with ethyl

acetate (3×). The combined organic layers were dried with

magnesium sulfate, ﬁltered, and concentrated under reduced pres-

sure. Puriﬁcation by ﬂash chromatography (silica, 2-5% EtOAc/

hexanes) provided ent-27c in 60% as a clear oil. [R]²⁵-22.7

D

(c 1.1, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 7.64 (m, 4H), 7.34

(m, 6H), 6.76 (m, 1H), 6.07 (d, J ) 15.6 Hz, 1H), 3.79-3.63 (m,

3H), 3.09 (dt, J ) 1.6, 9.2 Hz, 1H), 2.71 (A of ABX, J ) 6.0, 14.8

Hz, 1H), 2.46 (B of ABX, J ) 6.0, 15.2 Hz, 1H), 1.91 (m, 1H),

1.79 (d, J ) 6.8 Hz, 3H), 1.74-1.63 (m, 2H), 1.50 (m, 1H), 1.22

(m, 3H), 1.02 (s, 9H), 0.78 (d, J ) 6.0 Hz, 3H); ¹³C NMR (CDCl₃,

75 MHz) δ 198.7, 143.0, 135.5, 134.1, 132.5, 129.4, 127.5, 80.2,

74.3, 60.4, 46.6, 36.2, 35.0, 32.8, 32.2, 29.7, 26.9, 19.2, 18.2; IR

(neat) ν_max3068, 2954, 2857, 1672, 1632, 1431, 1108, 703 cm^-1

;

matography (silica, 20% EtOAc/hexanes) provided product ent-

HRMS (CI, NH₃) m/z calcd for C₂₉H₄₀O₃Si [M + 23]⁺487.2644,

found 487.2681.

1

36j in 71% as a clear oil. [R]²⁵-9.5 (c 1.0, CHCl₃); H NMR

D

(CDCl₃, 400 MHz) δ 6.81 (m, 2H), 6.06 (dd, J ) 1.6, 16.0 Hz,

1H), 4.07 (q, J ) 6.4, 10.4 Hz, 1H), 3.79 (m, 1H), 3.48 (quint, J

) 6.8, 13.2 Hz, 1H), 3.29 (dt, J ) 1.8, 9.2 Hz, 1H), 3.17 (dt, J )

5.4, 13.6 Hz, 1H), 2.77 (A of ABX, J ) 7.0, 16.8 Hz, 1H), 2.63

(m, 1H), 2.56 (B of ABX, J ) 5.8, 16.8 Hz, 1H), 2.49 (A of ABX,

J ) 2.0, 15.6 Hz, 1H), 2.20 (B of ABX, J ) 9.2, 15.6 Hz, 1H),

1.84 (dd, J ) 1.6, 6.8 Hz, 3H), 1.73 (m, 1H), 1.62 (m, 1H), 1.24

(m, 6H), 1.13 (d, J ) 7.6 Hz, 3H), 1.03 (d, J ) 7.6 Hz, 18 H),

0.82 (s, 9H), 0.79 (d, J ) 6.4 Hz, 3H), 0.04 (s, 3H), 0.03 (s, 3H);

¹³C NMR (CDCl₃, 75 MHz) δ 197.6, 173.7, 171.3, 142.9, 132.1,

80.6, 73.7, 71.9, 46.1, 45.1, 42.6, 40.5, 34.6, 32.3, 31.5, 25.8, 18.2,

18.0, 17.8, 17.7, 12.8, 11.8, -4.5, -4.9; IR (neat) ν_max3361, 2932,

2867, 1674, 1464, 1194, 1085, 837 cm^-1; HRMS (CI, NH₃) m/z

calcd for C₃₃H₆₄O₆NSi₂[M + 1]⁺626.4272, found 626.4299.

(2S,5S,E)-7-((2S,6S,8R,9S)-8-(3-Azidopropyl)-9-methyl-1,7-

dioxaspiro[5.5]undecan-2-yl)-3,5-dimethylhept-3-en-2-ol ((S)-

8). To a solution of 51 (50.0 mg, 0.0986 mmol) in a mixture of

methylene chloride (1.0 mL) and methanol (1.0 mL) at 0 °C was

added 10-camphorsulfonic acid (7.0 mg, 0.03 mmol). The reaction

was allowed to stir at 0 °C for 2 h before it was quenched with a

saturated solution of NaHCO₃. The aqueous layer was extracted

with dichloromethane (3×). The combined organic layers were dried

with magnesium sulfate, ﬁltered, and concentrated under reduced

pressure. Puriﬁcation by column chromatography (silica, 15%

(E)-1-((2S,5R,6S)-6-(2-Hydroxyethyl)-5-methyltetrahydro-2H-

pyran-2-yl)pent-3-en-2-one. To a solution of ent-27c (100 mg,

0.215 mmol) in a nalgene vial in acetonitrile (10.0 mL) at room

temperature was added hydroﬂuoric acid (1.00 mL) dropwise. The

reaction was allowed to stir for 3 h at room temperature before it

was quenched by slow addition of saturated NaHCO₃. The aqueous

layer was extracted with ethyl acetate (3×). The combined organic

layers were dried with magnesium sulfate, ﬁltered, and concentrated

under reduced pressure. Puriﬁcation by ﬂash chromatography (silica,

50% EtOAc/hexanes) provided product in 95% as a clear oil. [R]²⁵

D

-25.3° (c 2.9, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 6.85 (dq, J

) 6.8, 13.6 Hz, 1H), 6.11 (ddd, J ) 1.6, 3.2, 15.8 Hz, 2H), 3.81

(m, 1H), 3.71 (dt, J ) 1.6, 4.4 Hz, 2H), 3.20 (dt, J ) 2.8, 9.6 Hz,

1H), 2.79 (A of ABX, J ) 8.0, 15.6 Hz, 1H), 2.51 (B of ABX, J

) 4.8, 15.6 Hz, 1H), 1.88 (dd, J ) 1.8, 7.0 Hz, 3H), 1.84 (m, 1H),

1.76 (dq, J ) 2.8, 6.8 Hz, 1H), 1.66-1.52 (m, 2H), 1.40-1.17 (m,

3H), 0.79 (d, J ) 6.8 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ

198.3, 143.6, 132.5, 84.6, 74.3, 61.5, 46.0, 35.0, 34.7, 32.5, 31.8,

18.3, 17.6; IR (neat) ν_max3447, 2924, 2873, 1670, 1631, 1441, 1085,

973 cm^-1; HRMS (CI, NH₃) m/z calcd for C₁₃H₂₃O₃[M + 1]⁺

227.1647, found 227.1670.

2-((2S,3R,6S)-3-Methyl-6-((E)-2-oxopent-3-enyl)tetrahydro-

2H-pyran-2-yl)ethanoic Acid (ent-6c). To a solution of alcohol

(45.0 mg, 0.199 mmol) in acetonitrile (1 mL), and water (15 µL)

at 0 °C was added CrO₃/H₅IO₆stock solution (500 µL) dropwise.

The reaction was stirred at 0 °C for 0.5 h before another equivalent

of CrO₃/H₅IO₆stock solution (500 µL) was added dropwise and

then the reaction was stirred for 0.5 h at 0 °C. The reaction was

then quenched by addition of Na₂HPO₄(60 mg/1 mL of H₂O),

diluted with ethyl ether, and stirred for 30 min at room temperature.

Layers were separated and the aqueous layer was extracted with

ethyl ether (3×). Combined organic layers were dried with

magnesium sulfate, ﬁltered, and concentrated under reduced pres-

sure. Puriﬁcation by ﬂash chromatography (silica, 50% EtOAc/

EtOAc/hexanes) affords alcohol (S)-8 in 85% as a clear oil. [R]²⁵

D

-36.9 (c 0.3, CHCl₃); ¹H NMR (CDCl₃, 400 MHz) δ 5.16 (d, J )

9.2 Hz, 1H), 4.17 (q, J ) 6.0, 12.4 Hz, 1H), 3.41 (br t, 1H), 3.30

(m, 2H), 3.14 (t, J ) 9.6 Hz, 1H), 2.33 (m, 1H), 1.91 (m, 1H),

1.75 (m, 2H), 1.60 (s, 3H), 1.58-1.28 (m, 15H), 1.23 (d, J ) 6.4

Hz, 3H), 0.91 (d, J ) 6.8 Hz, 3H), 0.82 (d, J ) 6.4 Hz, 3H); ¹³C

NMR (CDCl₃, 100 MHz) δ 137.2, 131.4, 95.5, 74.1, 73.4, 69.2,

51.9, 36.0, 35.4, 35.0, 34.1, 33.6, 31.9, 31.3, 30.3, 27.9, 25.5, 21.6,

21.0, 19.2, 18.0, 11.8; IR (neat) ν_max3407, 2932, 2868, 2095, 1457,

1225, 1096, 985 cm^-1; HRMS (CI, NH₃) m/z calcd for C₂₂H₃₉O₃N₃

[M + 23]⁺416.2889, found 416.2913.

hexanes) provided ent-6c in 80% as a clear oil. [R]²⁵-35.1 (c

(S,E)-7-((2S,6S,8R,9S)-8-(3-Azidopropyl)-9-methyl-1,7-

dioxaspiro[5.5]undecan-2-yl)-3,5-dimethylhept-3-en-2-one. To a

solution of (S)-8 (38.0 mg, 0.0966 mmol) in methylene chloride

(9.70 mL) at 0 °C was added Dess-Martin periodinane (0.552 mL,

0.35 M in methylene chloride) dropwise. The reaction was allowed

D

1

1.0, CHCl₃); H NMR (CDCl₃, 400 MHz) δ 6.84 (dq, J ) 7.0,

13.8, 15.6 Hz, 1H), 6.10 (dq, J ) 1.6, 3.2, 15.6 Hz, 1H), 3.86 (m,

1H), 3.42 (dt, J ) 3.0, 9.4 Hz, 1H), 2.80 (A of ABX, J ) 7.6, 15.6

Hz, 1H), 2.67 (A of ABX, J ) 2.8, 15.6 Hz, 1H), 2.57 (B of ABX,

J. Org. Chem. Vol. 74, No. 5, 2009 1915

Wrona et al.

to warm to room temperature over 1 h and was diluted with ethyl

acetate. The organic layer was washed with a saturated solution of

sodium bicarbonate followed by sodium chloride solution. The

combined organic layers were dried with magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. Puriﬁcation by

column chromatography (silica, 10% EtOAc/hexanes) gives product

as a colorless glass (35.0 mg, 0.0894 mmol, 92.5%). [R]²⁵_D+37.8

with magnesium sulfate, ﬁltered, and concentrated under reduced

pressure. The crude acid was used in the coupling step without

further puriﬁcation.

To a solution of C1-C18 acid and C19-C40 amine (S)-52 (6.00

mg, 0.0163 mmol) in methylene chloride (1.60 mL) at room

temperature was added PyBOP (9.34 mg, 0.0180 mmol) and

triethylamine (3.18 µL, 0.0228 mmol). The reaction mixture was

allowed to stir for 2 h at room temperature before it was

concentrated in vacuo. Puriﬁcation by column chromatography

(silica, 80% EtOAc/hexanes) affords coupled product 53.1 as a

colorless oil (10.0 mg, 0.0122 mmol, 74.8%, 2 steps).

1

(c 0.8, CHCl₃); H NMR (CDCl₃, 400 MHz) δ 6.37 (d, J ) 9.6

Hz, 1H), 3.43 (m, 1H), 3.31 (d quint, 2H), 3.13 (dt, J ) 2.4, 9.6

Hz, 1H), 2.55 (m, 1H), 2.30 (s, 3H), 1.92 (m, 1H), 1.80 (m, 1H),

1.76 (s, 3H), 1.71 (m, 1H), 1.62-1.44 (m, 8H), 1.36 (m, 7H), 1.13

(dq, J ) 3.6, 12.8, 25.2 Hz, 1H), 1.03 (d, J ) 6.8 Hz, 3H), 0.82 (d,

J ) 6.8 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 200.2, 149.2,

136.3, 95.5, 74.2, 69.0, 51.9, 36.0, 35.4, 34.9, 34.2, 33.7, 32.9, 31.3,

30.3, 27.8, 25.5, 25.4, 20.0, 19.1, 18.0, 11.4; IR (neat) ν_max2932,

2869, 2095, 1670, 1457, 1096, 986 cm^-1; HRMS (CI, NH₃) m/z

calcd for C₂₂H₃₇O₃N₃[M + 23]⁺414.2733, found 414.2733.

(2R,5S,E)-7-((2S,6S,8R,9S)-8-(3-Azidopropyl)-9-methyl-1,7-

dioxaspiro[5.5]undecan-2-yl)-3,5-dimethylhept-3-en-2-ol ((R)-

8). To a solution of ketone (13.0 mg, 0.0332 mmol) in toluene

(2.00 mL) at -78 °C was added (S)-CBS (39.8 µL, 0.0398 mmol,

1.0 M in toluene) dropwise. After 5 min, catecholborane (66.4 uL,

0.0664 mmol, 1.0 M in toluene) was added dropwise and the

reaction mixture was allowed to stir at -78 °C for 24 h. Reaction

was quenched by addition of methanol (0.070 mL) and was allowed

to warm to room temperature and then stirred for 1 h. The mixture

was diluted with ethyl acetate and a saturated solution of sodium

bicarbonate. The aqueous layer was extracted with ethyl acetate

(3×). The combined organic layers were dried with magnesium

sulfate, ﬁltered, and concentrated under reduced pressure. Puriﬁca-

tion by column chromatography (silica, 15% EtOAc/hexanes)

To a solution of 53.1 (10.0 mg, 0.0122 mmol) in tetrahydrofuran

(1.20 mL) at 0 °C was added TBAF (14.6 µL, 1.00 M in THF)

dropwise. The reaction was stirred for 2 h at 0 °C before it was

diluted with ethyl ether. The organic layer was washed with 0.1 M

HCl and brine solutions sequentially. The combined organic layers

were dried with magnesium sulfate, ﬁltered, and concentrated under

reduced pressure. Puriﬁcation by ﬂash chromatography (silica, 100%

EtOAc) provided product 1.21 in 63% (3 steps) as a clear oil. [R]²⁵

D

1

+18.5 (c 0.6, CHCl₃); H NMR (C₆D₆, 400 MHz) δ 7.66 (t, J )

5.6 Hz, 1H), 7.24 (obs t, 1H), 6.59 (dq, J ) 6.8, 13.6, 16.0 Hz,

1H), 5.88 (dd, J ) 1.6, 16.0 Hz, 1H), 5.37 (dd, J ) 0.8, 8.0 Hz,

1H), 5.27 (d, J ) 6.0 Hz, 1H), 4.11 (q, J ) 6.4, 12.8 Hz, 1H), 3.89

(m, 1H), 3.81 (t, J ) 4.8 Hz, 1H), 3.72 (m, 1H), 3.58 (m, 2H),

3.42 (t, J ) 9.4 Hz, 1H), 3.27 (m, 1H), 3.03 (t, J ) 9.8 Hz, 1H), 2.47

(m, 3H), 2.23 (B of ABX, J ) 10.0, 16.4 Hz, 1H), 2.00 (m, 3H),

1.85-0.85 (m, 25H), 1.65 (s, 3H), 1.43 (dd, J ) 1.6, 6.8 Hz, 3H),

1.33 (d, J ) 6.8 Hz, 3H), 1.26 (d, J ) 6.4 Hz, 3H), 1.07 (d, J )

6.8 Hz, 3H), 0.82 (d, J ) 6.4 Hz, 3H), 0.53 (d, J ) 6.4 Hz, 3H);

¹³C NMR (C₆D₆, 75.0 MHz) δ 197.5, 175.4, 172.7, 143.4, 138.4,

132.0, 130.5, 95.5, 80.4, 74.9, 74.2, 73.3, 72.8, 69.4, 45.9, 45.3,

43.2, 40.2, 39.5, 36.6, 36.0, 35.4, 34.7, 34.6, 34.1, 32.5, 32.3, 31.7,

31.5, 30.6, 28.5, 26.3, 22.3, 21.4, 19.8, 18.2, 17.9, 17.5, 16.0, 12.6;

IR (neat) ν_max3328, 2928, 2868, 1645, 1549 cm^-1; HRMS (CI,

NH₃) m/z calcd for C₄₀H₆₈N₂NaO₈[M + Na]⁺m/z calcd for

727.4873, found 727.4865.

affords alcohol (R)-8 as a colorless glass (13.0 mg, 0.0330 mmol,

1

99%). [R]²⁵+39.6 (c 0.3, CHCl₃); H NMR (CDCl₃, 400 MHz)

D

δ 5.16 (d, J ) 9.2 Hz, 1H), 4.17 (q, J ) 6.0, 12.4 Hz, 1H), 3.41

(br. t, 1H), 3.30 (m, 2H), 3.14 (t, J ) 9.6 Hz, 1H), 2.33 (m, 1H),

1.91 (m, 1H), 1.75 (m, 2H), 1.60 (s, 3H), 1.58-1.28 (m, 15H),

1.23 (d, J ) 6.4 Hz, 3H), 0.91 (d, J ) 6.8 Hz, 3H), 0.82 (d, J )

6.4 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 137.2, 131.4, 95.5,

74.1, 73.4, 69.2, 51.9, 36.0, 35.4, 35.0, 34.1, 33.6, 31.9, 31.3, 30.3,

27.9, 25.5, 21.6, 21.0, 19.2, 18.0, 11.8; IR (neat) ν_max3407, 2932,

2868, 2095, 1457, 1225, 1096, 985 cm^-1; HRMS (CI, NH₃) m/z

calcd for C₂₂H₃₉O₃N₃[M + 23]⁺416.2889, found 416.2913.

(2S,3R)-3-Hydroxy-N-(3-((2R,3S,6S,8S)-8-((3S,6S,E)-6-hydroxy-

3,5-dimethylhept-4-enyl)-3-methyl-1,7-dioxaspiro[5.5]undecan-

2-yl)propyl)-2-methyl-4-(2-((2S,3R,6S)-3-methyl-6-((E)-2-oxopent-

3-enyl)tetrahydro-2H-pyran-2-yl)ethanamido)butanamide (1.21).

To a solution of azide (S)-8 (5.00 mg, 0.0127 mmol) in a mixture

of tetrahydrofuran (0.900 mL) and water (0.300 mL) at room

temperature was added trimethyl phosphine (63.5 µL, 1.0 M in

tetrahydrofuran) dropwise. The reaction was allowed to stir for 1 h

and then diluted with a saturated solution of sodium chloride. The

aqueous layer was extracted with ethyl ether (3×). The combined

organic layers were dried with magnesium sulfate, ﬁltered, and

concentrated under reduced pressure. The crude, clear oil was used

directly in the coupling step without further puriﬁcation.

Acknowledgment. Financial support for this research was

obtained from NIH CA56304. J.S.P. is grateful to Amgen,

Johnson & Johnson, Merck Co., Novartis, Pﬁzer, and GSK for

ﬁnancial support. I.E.W. acknowledges a Novartis Graduate

Fellowship. J.T.L. acknowledges an ACS Graduate Fellowship

(sponsored by Bristol-Myers Squibb) and Merck Co. Graduate

Fellowship, Supported by the Intramural Research Program of

the National Cancer Institute, NIH, Center for Cancer Research.

Flow cytometry and cell cycle analysis were done by Kathleen

Noer, Roberta Matthai, and Samantha Bauchiero of the CCR-

Frederick Flow Cytometry Core, SAIC-Frederick, Inc. We thank

the Developmental Therapeutics Program, NCI, for 60-cell

testing. This project has been funded in part with federal funds

from the National Cancer Institute, National Institutes of Health,

under contract N01-CO-12400.

Supporting Information Available: Complete spectroscopic

and analytical data including copies of ¹H and ¹³C NMR spectra.

This material is available free of charge via the Internet at

http://pubs.acs.org.

To a solution of TIPS acid ent-36j (8.00 mg, 0.0128 mmol) in

THF (1.30 mL) at 0 °C was added TBAF (14.7 µL, 1.00 M in

THF) dropwise. The reaction was stirred for 0.5 h before it was

diluted with ethyl acetate. The organic layer was washed with 0.01

M HCl and brine solutions. The combined organic layers were dried

JO802269Q

1916 J. Org. Chem. Vol. 74, No. 5, 2009

Article Doi

Synthesis of a 35-member stereoisomer library of bistramide A: Evaluation of effects on actin state, cell cycle and tumor cell growth

DOI: 10.1021/jo802269q

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