Natural product-guided synthesis of a spiroacetal collection reveals modulators of tubulin cytoskeleton integrity

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Natural Product-Guided Synthesis of a Spiroacetal Collection Reveals

Modulators of Tubulin Cytoskeleton Integrity

Okram Barun,^[a]Kamal Kumar,^[a]Stefan Sommer,^[a,b]Anette Langerak,^[c]

Thomas U. Mayer,^[d]Oliver Müller,^[c]and Herbert Waldmann*^[a,b]

Keywords: Asymmetric synthesis / Chemical biology / Natural products / Solid-phase synthesis / Spiroketals

The spiro[5.5]ketal moiety forms the underlying structural

skeleton of numerous biologically active natural products.

Since simplified but characteristic spiroketals derived from

the parent natural products retain biological activity, the spi-

ro[5.5]ketal unit can be regarded as a biologically prevali-

dated framework for the development of natural product-de-

rived compound collections. We report an enantioselective

synthesis of spiro[5.5]ketals on solid support. The reaction

sequence employs asymmetric boron enolate aldol reactions

with the enolate bound to the polymer or in solution as the

key enantiodifferentiating step. It proceeds in up to 12 steps

on solid support, makes the desired spiroketals available in

high overall yields and with high stereoselectivities and is

amenable to structural variation of the products. The small

spiroketal collection synthesized contains phosphatase inhib-

itors and compounds that modulate the formation of the tub-

ulin cytoskeleton in human cancer cells without directly tar-

geting microtubules.

Germany, 2005)

leads in medicinal chemistry programs. This requires the

availability of efficient and practical solid-phase synthesis

methods and multi-step synthesis sequences (typically Ͼ10

linear steps) that will provide access to frameworks of biolo-

gically promising natural products and will proceed with

degrees of efficiency and selectivity comparable to those of

competing solution-phase techniques. To date this goal has

been achieved in only a few cases.^[1,3,4]

Introduction

The solid-phase synthesis of collections of compound

with predetermined profiles of properties has evolved to be-

come one of the crucial enabling techniques in post-geno-

mic chemical biology and medicinal chemistry research.

Paramount to success in this area is that the underlying

chemical structures of the compound libraries are meaning-

ful to nature. Ideally they should be biologically prevali-

dated^[1]and/or embody so-called privileged structures:

structures that enable the library members to bind to se-

veral different proteins.^[2]

The precondition of biological validation is fulfilled by

biologically active natural products that can be regarded as

evolutionarily selected ligands for structurally conserved yet

genetically mobile protein domains.^[1]This insight points to

the synthesis of natural product-derived compound libraries

as promising starting points for initiation of research in

chemical biology as well as in the development of hits and

Spiroketals are found in abundance, generally across the

insect kingdom, and are known for their pheromonal activi-

ties.^[5]In particular, the importance of the spiro[5.5]ketal

has been increasing due to its rigid molecular framework

and its occurrence as a fragment in various complex natural

products displaying a wide range of biological activities. Ex-

amples include the extraordinarily potent tubulin polymeri-

zation-inhibiting spongistatins,^[6]the protein phosphatase

inhibitors okadaic acid,^[7]tautomycin,^[8]and the HIV-1 pro-

tease inhibitors integramycin.^[9]Many groups have already

started investigations into the roles of spiroketal fragments

in their parent molecules. Interestingly, structurally simpli-

fied but characteristic spiroketals derived from the parent

natural products retain biological activity (Figure 1).^[10–12]

The spiroketal motifs present in okadaic acid and tautomy-

cin are enantiomers, and the stereochemistry of the spiro-

ketal moieties could be the major determining factor for

the affinity characteristics of okadaic acid and tautomycin

towards the PP1 and PP2 phosphatases.^[13]The above find-

ings validate the choice of the spiro[5.5]ketal as an underly-

ing structural framework for the development of promising

natural product-derived compound collections. The useful-

[a] Department of Chemical Biology and Department of Structural

Biology, Max-Planck Institute of Molecular Physiology,

Otto-Hahn-Str. 11, 44227 Dortmund, Germany

E-mail: herbert.waldmann@mpi-dortmund.mpg.de

[b] Department of Chemistry, University of Dortmund,

44227 Dortmund, Otto-Hahn-Str. 6, Germany

[c] Department of Structural Biology, Max-Planck Institute of

Molecular Physiology,

44227 Dortmund, Germany

[d] Department of Chemical Biology, Independent Research

Group, Max-Planck Institute of Biochemistry,

Am Klopferspitz 18, 82152 Martinsried, Germany

Supporting information for this article is available on the

WWW under http://ww w .eurjoc.org or from the author.

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DOI: 10.1002/ejoc.200500605

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Figure 1. Biologically active natural products with spiroketal substructure and structurally simplified yet biologically active analogues

embodying the spiroketal substructure.

ness of spiroketals in combinatorial chemistry^[10]and a

method for their synthesis on solid support have already adduct 2. One of the hydroxy groups required for acetal

been reported by Ley and co-workers.^[14]

formation was chosen for attachment to the solid support,

Spiroketal 1 was retrosynthetically traced back to aldol

Here we describe in detail the development of an asym- while the other alcohol would have to be protected by an

metric synthesis of spiro[5.5]ketals on solid support through appropriate blocking function orthogonally stable to the

the use of stereoselective boron-mediated aldol reactions as other protecting groups employed. We envisioned that, if

key stereodefining transformations.^[15]

this protecting group were chosen such that it were of the

same chemical type as the anchor to the solid support, final

release from the polymeric carrier should be accompanied

by cleavage of the blocking function. Also, under appropri-

ate conditions spontaneous ketalization might be induced,

giving rise to the desired spiroketals 1. We expected that

these preconditions should be fulfilled by the p-meth-

oxybenzyl (PMB) group and its analogue incorporated into

the Wang linker for syntheses on solid supports (Scheme 1).

We reasoned that the immobilized aldol intermediate 2

might be formed through an asymmetric aldol reaction be-

Results and Discussion

In planning the synthesis we chose as targets compounds

of the general structure 1 (Scheme 1), since they incorporate

up to eight sites of possible diversification, thereby opening

up opportunities to synthesize a fairly diverse compound

library.

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tion proceeds analogously to the stereochemical course ob-

served for related cases.^[20]The alcohol was again masked

as a TBS ether, with an overall yield of 89% for two steps.

Simultaneous oxidative cleavage of both PMB protecting

groups proceeded smoothly on treatment with DDQ for 3 h

and was followed – as hoped – by spontaneous formation

of spiroketal 13, which was obtained as a single isomer in

88% yield.

Furthermore, to ascertain the structure of compound 13

it was deprotected by treatment with TBAF to yield spi-

roketal 14 in 81% yield. This compound’s analytical data

were compared with those recorded for closely related com-

pounds,^[21]and additional NOE-spectroscopic investi-

gations also confirmed the stereochemical course of the

overall reaction sequence. Clear NOE signal enhancements

were detected between the two methyl groups and between

the two protons at C-5 and C-11, respectively indicating

close proximity (Scheme 2). In addition, an NOE enhance-

ment was detected between the methyl group at C-11 and

the proton at H-9, but not between the methyl group at C-

5 and the proton at H-2, so the methyl group at C-5 is in

the equatorial position whereas the methyl group at C-11 is

in the axial orientation. This conclusion is further sup-

ported by the observation of a fairly high melting point and

by the fact that the signal of the hydroxy protons in the ¹H

NMR spectrum appears at δ = 1.75–1.52 (D₂O exchange),

which has been shown to be diagnostic of equatorial hy-

droxy protons in related systems.^[21]

After development of this reaction sequence we investi-

gated whether it could be successfully transferred to the so-

lid support. To this end, Merrifield resin modified with the

Wang linker 15 (loading 1.2 mmolg^–1) was activated as

Scheme 1. Retrosynthetic analysis of the spiro[5.5]ketal structure.

tween an enolate generated from polymer-bound ketone 3 trichloroacetimidate 16 (Scheme 3),^[22]and this was then

and aldehyde 4. Similarly, the immobilized ketone 3 was subjected to nucleophilic displacement by mono-TBS-pro-

traced back to the polymer-linked aldehyde 5 and ketone tected propanediol 17. This two-step sequence was conve-

6, which would again be subjected to an asymmetric aldol niently monitored by FT-IR spectroscopy. After cleavage

reaction. This strategy requires the application of asymmet- of the TBS group the primary alcohol was oxidized to the

ric aldol reactions employing both polymer-bound^[16]and corresponding aldehyde 19 by use of IBX as oxidant. At

soluble chiral enolates.^[17]A few examples of stereoselective this stage, resin loading was determined to be 0.75 mmolg^–1

aldol reactions with soluble and polymer-bound chiral eno- by application of a method developed earlier by us, based

lates have been described, but the use of polymer-bound on formation of the corresponding dinitrophenylhydra-

chiral enolates in aldol reactions has not yet been explored. zone.^[4]At this stage we wanted to attempt the anti-aldol

The feasibility of the strategy was first investigated in reactions with the enolate generated in solution phase, in

solution (Scheme 2). To this end, p-methoxybenzyl-pro- order subsequently to construct the spiroketal. Preformed

tected aldehyde 8 was synthesized in two steps from com- (E)-enolate 11 derived from 10 in solution phase as de-

mercially available propane-1,3-diol (7) and treated with scribed above was allowed to react with aldehyde-bound

(Z)-diisopinocampheyl borinate 9,^[18]generated from (–)- Wang resin at 0 °C in diethyl ether. Oxidative workup with

Ipc₂BOTf and pentan-3-one. Further oxidative workup H₂O₂in a pH 7 buffer/MeOH/DMF mixture at 0 °C yielded

with 30% aq. H₂O₂in MeOH/pH 7 buffer solution afforded anti-aldol 20 (detected by IR; strong absorption at

the syn-aldol adduct with very high stereoselectivity (de Ͼ 3504 cm^–1and 1714 cm^–1). Notably, raising the temperature

98%, ee Ͼ 91%). The aldol OH group was then protected during oxidative workup resulted in partial cleavage from

as a tert-butyldimethyl silyl ether to give 10 in 69% yield in the resin. After oxidative workup to cleave the B–O bond,

two steps. The (E) enolate 11 was then generated by treat- 20 was treated with DDQ in a CH₂Cl₂/buffer (pH 7) mix-

ment of 10 with dicyclohexylboron chloride by established ture to afford spiroketal 21 along with unidentified mix-

methods.^[19]It was subjected to a further aldol reaction with tures. Since the separation of 21 from anisaldehyde was dif-

aldehyde 8. Oxidative workup yielded anti-aldol adduct 12 ficult the resin-bound intermediate 20 was protected as a

(de Ͼ97%). The absolute configuration of aldol adduct 12 TBS ether to yield the corresponding immobilized aldol

was assigned on the basis of the assumption that the reac- product (monitored by FT-IR spectroscopy). Treatment of

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Scheme 2. Solution-phase synthesis of spiroketals 13 and 14. a) NaH (1.5 equiv.), CH₃OC₆H₄CH₂Cl (1.1 equiv.), DMF, 0 °C to room

temp., 24 h, 63%. b) COCl₂(1.5 equiv.), DMSO (2.5 equiv.), Et₃N (4 equiv.), CH₂Cl₂, –78° to 0 °C, 2 h, 93%. c) Pentan-3-one (1 equiv.),

(–)-Ipc₂BOTf (1.2 equiv.), iPr₂NEt (1.5 equiv.), CH₂Cl₂, –78° to –30 °C, 20 h. d) 30% aq. H₂O₂/MeOH/pH 7 buffer (1.5:5:1), 0 °C to room

temp., 2 h, 69%. e) TBSCl (1.3 equiv.), imidazole (2.1 equiv.), DMF, room temp., 24 h, 91%. f) Dicyclohexylboron chloride (1.3 equiv.),

Et₃N (1.5 equiv.), Et₂O, 0 °C, 4 h. g) Compound 8 (1.4 equiv.), Et₂O, –78° to –30 °C, 24 h. h) 30% aq. H₂O₂, MeOH, pH 7 buffer (1.5:5:1),

0 °C to room temp., 2 h, 89%. i) TBSCl (1.3 equiv.), imidazole (2.5 equiv.), DMF, room temp., 24 h 92%. j) DDQ (2.8 equiv.), CH₂Cl₂,

pH 7 buffer, 0 °C to room temp., 3 h, 88%. k) TBAF (3 equiv.), THF, room temp., 48 h 81%.

this intermediate with DDQ in a CH₂Cl₂/buffer (pH 7) mix- was filtered and the whole process was repeated once. After

ture resulted in simultaneous cleavage of the PMB ether, oxidative workup to cleave the B–O bond, the secondary

release from the Wang resin and spiroketalization. After pu- alcohol was protected as TBS ether to yield immobilized

rification by filtration through a short silica gel column, aldol product 22 (monitored by FT-IR spectroscopy).

spiroketal 13 was obtained (42% overall yields in 7 steps)

For the crucial second aldol reaction on the solid sup-

1

as a single stereoisomer as determined by HPLC and H/ port, employing a polymer-bound chiral boron enolate,

¹³C NMR spectroscopy. Comparison of the spectroscopic ketone resin 22 was swollen in diethyl ether and a solution

data and the specific rotation, together with NOE spectro- of dicyclohexylboron chloride and triethylamine in diethyl

scopic investigation, showed it to be identical to the spiro- ether was then added to the resin at 0 °C. After 6 h the resin

ketal synthesized in solution as described above.

was washed and the procedure was repeated once again. It

We next attempted to perform all transformations on so- was expected that, analogously with enolate formation in

lid support by the same strategy as in solution-phase syn- solution (see above and Ref.^[15]), (E)-dicyclohexylboron

thesis (Scheme 4). The polymer-bound aldehyde 19 enolate 23 would be formed on the solid support.

(0.75 mmolg^–1) was treated at –78 °C with the preformed

Boron enolate resin 23 was then treated at –78 °C with

(Z)-enolate 9 in dichloromethane for 1.5 h. After the reac- aldehyde 8 and, after oxidative workup as described above,

tion mixture had been stored at –27 °C for 16 h, the resin the secondary alcohol 24 (strong absorptions at 3504 cm^–1

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Scheme 3. anti-Aldol reaction on solid phase with boron enolate in solution phase. a) Cl₃CCN (8 equiv.), DBU (3 mol-%), CH₂Cl₂, 0 °C,

40 min. b) TBSCl (1.2 equiv.), imidazole (2.0 equiv.), DMF, room temp., 24 h. c) Compound 16, BF₃·Et₂O (3 mol-%), cyclohexane,

CH₂Cl₂, room temp., 15 min. d) TBAF (8 equiv.), THF, room temp., 14 h. e) IBX (8 equiv.), DMSO, room temp., 36 h. f) 11 (6 equiv.),

Et₂O, –78° to –27 °C 20 h (two cycles). g) 30% aq. H₂O₂/MeOH/DMF/pH 7 buffer (1.5:4:4:1, 0 °C, 3 h. h) DDQ (10 equiv.), CH₂Cl₂/

pH 7 buffer (20:1), 0 °C to room temp. 6 h. i) TBSCl (10 equiv.), DMAP (1 mol-%), imidazole (10 equiv.), DMF/CH₂Cl₂(1:1), room

temp., 24 h (two cycles).

and 1714 cm^–1in the IR spectrum) was liberated bound resin 24 was protected with an acetyl group by treat-

(Scheme 4). The intermediate 24 was then protected with a ment with acetic anhydride in THF in the presence of pyri-

TBS group, and subsequent treatment with DDQ in a dine and DMAP. Oxidative cleavage with DDQ gave spiro-

CH₂Cl₂/buffer (pH 7) mixture resulted in simultaneous ketal 25 in 14% overall yield.

cleavage of the PMB ether, release from the Wang resin and

In order to demonstrate that the reaction sequence

spiroketalization to give 13. The compound was found to shown in Scheme 4 is amenable to the synthesis of com-

be identical to that synthesized in solution phase as de- pound collections, seven further building blocks were syn-

scribed above and in Scheme 3.

thesized and subjected to solid-phase aldol chemistry. All

The spiroketal was obtained in a 12-step solid-phase syn- protected β-hydroxyaldehydes were available in a few simple

thesis in an overall yield of 16%, which corresponds to an transformations from chiral pool compounds or by apply-

average yield of 86% yield for each step. This compares very ing enantioselective allylations to aldehydes with subse-

favourably with the overall yield of 27% recorded for the quent alcohol protection and oxidative cleavage of the

10-step solution synthesis described above. The fact that the double bond (Scheme 5).^[23]The spiroketals 38 to 53 were

configurations of the spiroketals obtained both from the obtained by the established route in overall yields ranging

solid-phase and from the solution-phase syntheses are iden- from 5 to 13% (Scheme 6).

tical confirmed that both aldol reactions on the polymeric

To gain further insight into the stereoselectivity of the

support proceed completely analogously to the correspond- asymmetric boron-mediated aldol reactions on solid sup-

ing asymmetric transformations in solution. In addition, port, we investigated whether the principle of double dias-

the finding that the desired product was obtained with very tereodifferentiation was also operative in this case. There-

high stereoselectivity convincingly demonstrates that the de- fore, both enantiomers of chiral aldehyde 27 were used on

gree of stereodifferentiation is also very comparable in both the solid support. After treatment with DDQ, spiroketali-

cases. To extend the scope of the synthesis the alcohol- zation gave compounds 38, 39 and 40 as single isomers,

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Scheme 4. Synthesis of spiroketals through syn and anti aldol reactions on solid support. a) Pentan-3-one (6 equiv.), (–)-Ipc₂BOTf

(6.1 equiv.), iPr₂NEt (7 equiv.), CH₂Cl₂, –78° to 0 °C, 20 h; filter and wash (two cycles). b) 30% aq. H₂O₂/MeOH/DMF/pH 7 buffer

(1.5:4:4:1, 0 °C room temp., 4 h. c) TBSCl (10 equiv.), DMAP (1 mol-%), imidazole (10 equiv.), DMF/CH₂Cl₂(1:1), room temp., 24 h

(two cycles). d) Dicyclohexylboron chloride (8 equiv.), Et₃N (9 equiv.), Et₂O, 0 °C, 24 h, filter and wash (two cycles). e) Compound 8

(10 equiv.), Et₂O, –78° to 20 °C, 26 h (2 cycles). f) 30% aq. H₂O₂/MeOH/DMF/pH 7 buffer (1.5:4:4:1), 0 °C, 4 h. g) TBSCl (10 equiv.),

DMAP (1 mol-%), imidazole (10 equiv.), DMF/CH₂Cl₂(1:1), room temp., 24 h (two cycles). h) DDQ (10 equiv.), CH₂Cl₂/pH 7 buffer

(20:1), 0 °C to room temp., 6 h. i) TBAF (3 equiv.), THF, room temp., 48 h, 81%. j) Ac₂O (30 equiv.), pyridine (30 equiv.), DMAP (cat.),

THF, room temp., 20 h.

whereas 41 and 42 were isolated along with two minor in- subsequent reduction to the alcohol by DIBAH. The

separable isomers (detected by GC-MS) (Scheme 6). Thus, alcohol was oxidized with IBX to give the aldehyde 56 with

in aldol reactions of the chiral enolate 23 with the two enan- a loading of 0.7 mmolg^–1. A syn-aldol reaction of this alde-

tiomeric aldehydes 27a and 27b, the anti-aldol adduct is hyde with pentan-3-one, followed by TBS protection of the

formed as the major product. Consistently with related fin- alcohol, yielded intermediate 57.

dings,^[24]the combination of 23 and 27b represents the

The resin-bound aldol intermediates 55 and 57 were em-

matched case and the combination of 23 and 27a the mis- ployed to synthesize further spiroketal molecules. When

matched case. Spiroketals 43 to 53 were obtained as single resin-bound intermediate 55 was used in the corresponding

isomers.

sequence (i.e., an anti-aldol reaction with protected β-hy-

Furthermore, decoration of the spiroketal skeleton with droxy aldehyde 27b, acylation of the alcohol, simultaneous

stereogenic centres was achieved through the use of the cor- deprotection of the PMB group and cleavage from the

responding substituted resin-bound aldol intermediates. To resin), a mixture of products was obtained. Along with

this end, two additional aldol intermediates were prepared other impurities, spiroketal isomers were formed in low

on solid support (Scheme 7) through syn-aldol reactions be- yield. This is notable, since the aldehyde 27b had yielded

tween resin-bound β-hydroxy aldehydes and pentan-3-one. spiroketals in good yields in the reaction sequence described

The secondary alcohol obtained after the stereoselective above (Scheme 6). In the reaction sequence involving 55,

ring-opening of O-benzyl-(S)-glycidol by vinyl Grignard however, at least four spiroketal isomers were formed (in a

reagent was attached to Wang resin via the resin trichlo- ratio of 55:35:8:2, determined by GC). The major isomer

roacetimidate under acid catalysis conditions, followed by 58 could be purified after TBS group deprotection, and the

ozonolysis to give aldehyde 54 with

a loading of minor isomer 59, which was found to be the 10-epimer of

0.45 mmolg^–1(see Supporting Information). This aldehyde the major product, could be isolated by preparative HPLC.

was used for a syn-aldol reaction with pentan-3-one with The structure of the minor product 59 was determined from

subsequent TBS protection of the formed alcohol to yield NMR spectroscopic data and NOE experiments. In the spiro-

intermediate 55. Commercially available ethyl (S)-3-hy- ketal 59, the proton at C-10 appears relatively upfield (at

droxybutyrate (36) was directly attached to Wang resin with 4.64 ppm) relative to where it usually appears in the case

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reaction, as many as four spiroketals were again observed

after the reaction sequence. This result suggests that both

the aldol reactions in this sequence might have been mis-

matches, resulting in isomer formation. The next sequence

was therefore tested with resin-bound aldol intermediate 57,

starting with an anti-aldol reaction with aldehyde 27b. After

the reaction sequence we were, to our delight, able to ob-

serve only two isomers of spiroketals (in a ratio of 5:1),

which could be purified by HPLC. As the aldol reactions

in this sequence appear to be the matching cases, further

spiroketals were synthesized by this sequence through varia-

tion of the β-hydroxy aldehydes for the anti-aldol reaction

(Scheme 8). In most cases the major product was ac-

companied by varying amounts of its 10-epimer. The major

product could be easily separated by HPLC, and 16 highly

substituted spiroketals were added to the collection by this

reaction sequence (Scheme 8).

In order to determine whether the synthesized spiroketals

displayed biological activity associated with some of the ac-

tivities characteristic of naturally occurring spiroacetals (see

above) they were tested in different assays. In particular,

we investigated whether the spiroacetal collection contained

phosphatase inhibitors and modulators of microtubule for-

mation.

The protein tyrosine phosphatase PTP1b, the dual speci-

ficity phosphatase VHR and Cdc25a and the serine-threo-

nine phosphatase PP1 were chosen as representative en-

zymes. PTP1B is a key negative regulator of insulin receptor

activity and PTP1B-inhibitors are expected to enhance in-

sulin sensitivity and act as effective therapeutic treatments

for Type II diabetes, insulin resistance and obesity. The vac-

cinia virus VH1-related phosphatase VHR is a physiologi-

cal regulator of extracellular regulated kinases of the MAP

(mitogen-activated protein) kinase family and influences

signalling through the MAP kinase pathway. Cdc25a is in-

volved in cell cycle regulation and PP1 influences various

biological processes. Great attention has been paid to the

development of inhibitors of these phosphatases for bio-

logical studies and drug development.^[25]

Scheme 5. Synthesis of building blocks 27a, 27b, 30, 32, 33, 35 and

37. a) CH₃OC₆H₄CH₂OC(=NH)CCl₃(1.5 equiv.), BF₃·Et₂O (cat.),

pentane/CH₂Cl₂(5:1), 0 °C to room temp., 16 h, 75%. b) OsO₄

(0.01 equiv.), N-methylmorpholine oxide (1.1 equiv.), tBuOH/H₂O

(2:1), 0 °C to room temp., 6 h, 94%. c) NaIO₄(2.5 equiv.), CH₂Cl₂/

H₂O (1:1), 0 °C to room temp., 6 h, 93%.

The results of the assays are shown in Table 1. Spiroace-

tal 39 inhibits VHR with an IC₅₀value in the low micromo-

lar range and weakly inhibits PTP1b. Compound 42 turned

out to be a moderate inhibitor of VHR, and compounds 50

and 53 proved to be moderate inhibitors of PTP1b and

VHR. None of the synthesized spiroacetals inhibited

of the major isomers (Ϸ 5.02–5.20 ppm), suggesting that it Cdc25a or PP1.

occupies an equatorial position. Furthermore, the coupling

Comparison between spiroacetals 39 and 42 shows that

constant value of 8.4 Hz for this proton clearly indicates the the configurations of the stereocenters embedded in the spi-

absence of a diaxial arrangement. The latter observation roacetal rings markedly influence phosphatase inhibition.

was further strengthened by the absence of any NOE signal The observation that analogues 38, 40 and 41 (Scheme 6)

between protons at C10, C2 and C8. Moreover, the NOE are not active modulators indicates that the variation of the

signal enhancement between methyl protons of the acetyl substituents attached to the basic skeleton of the molecules

group and the proton at C-8 confirms this conformation appears to be of similar importance.

(Scheme 8).

In order to determine whether some of the spiroacetals

The low stereoselectivity recorded above may reflect a described above would affect microtubule formation, their

mismatch in at least one of the aldol reactions. Similarly, influence on the tubulin and the actin cytoskeletons of the

when β-hydroxyaldehyde 8, which does not possess an ad- human breast carcinoma cell line MDA-MB-231 was inves-

ditional stereogenic centre, was used in the second aldol tigated.

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Scheme 6. Solid-phase synthesis of spiroketals 38–53. a) Aldehyde (10 equiv.), Et₂O, –78° to 20 °C, 26 h (2 cycles). b) 30% aq. H₂O₂/

MeOH/DMF/pH 7 buffer (1.5:4:4:1), 0 °C 4 h. c) TBSCl (10 equiv.), DMAP (1 mol-%), imidazole (10 equiv.), DMF/CH₂Cl₂(1:1), room

temp., 24 h (two cycles) or Ac₂O (30 equiv.), pyridine (30 equiv.), DMAP (cat.), THF, room temp., 20 h. d) DDQ (10 equiv.), CH₂Cl₂/

pH 7 buffer (20:1), 0 °C to room temp., 6 h. e) TBAF (5 equiv.), THF, room temp., 24 h; overall yields are given in parentheses.

Scheme 7. Synthesis of aldol intermediates 55 and 57 on solid support. a) NaH (1.2 equiv.), DMF, benzyl bromide (1.25 equiv.), 0 °C to

room temp., overnight. b) Vinylmagnesium bromide (1.3 equiv.), CuI (10 mol-%), –30 °C. c) Compound 16, BF₃·Et₂O (3 mol-%), cyclohex-

ane, CH₂Cl₂, room temp., 15 min. d) O₃, CH₂Cl₂, –78 °C, PPh₃, –78 °C to room temp., overnight. e) Pentan-3-one (6 equiv.), (–)-Ipc₂BOTf

(6.1 equiv.), iPr₂NEt (7 equiv.), CH₂Cl₂, –78° to 0 °C, 20 h; filter and wash (two cycles). f) 30% aq. H₂O₂/MeOH/DMF/pH 7 buffer

(1.5:4:4:1, 0 °C room temp., 4 h. g) TBSCl (10 equiv.), DMAP (1 mol-%), imidazole (10 equiv.), DMF/CH₂Cl₂(1:1), room temp., 24 h

(two cycles). h) DIBAH (5.0 equiv.), THF, 5 h. i) IBX (8 equiv.), DMSO, room temp., 36 h (see Supporting Information).

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Scheme 8. Solid-phase synthesis of spiroketals 58–73: a) Aldehyde (10 equiv.), Et₂O, –78° to 20 °C, 26 h (2 cycles). b) 30% aq. H₂O₂/

MeOH/DMF/pH 7 buffer (1.5:4:4:1), 0 °C 4 h. c) Ac₂O (30 equiv.), pyridine (30 equiv.), DMAP (cat.), THF, room temp., 20 h. d) DDQ

(10 equiv.), CH₂Cl₂/pH 7 buffer (20:1), 0 °C to room temp., 6 h. e) TBAF (3 equiv.), THF, room temp., 24 h; overall isolated yields are

given in parentheses.

Figure 2 shows that untreated cells displayed intact tubu- ized fluorescently labelled microtubules in vitro in the pres-

lin and actin networks. Upon addition of spiroacetals 39 ence of compounds 39, 42 or DMSO as a solvent control.

and 42, the microscopically visible tubulin structure was af- Microscopic analyses revealed that neither compound sig-

fected. Cells treated with 5 μm 39 displayed irregular and nificantly affected microtubule polymerization in vitro at

folded tubulin cytoskeletons, whereas the tubulin fibres in concentrations as high as 200 μm (Figure 3). To confirm

cells treated with 5 μm 42 were rather incomplete. The com- that our in vitro microtubule polymerization assay is sensi-

pounds had no effect on the actin structure or on the over- tive to microtubule-depolymerizing molecules we included

all cell morphology. Lower concentrations of 39 or 42 and nocodazole as a positive control. As shown in Figure 3, no-

all other tested compounds had no effect.

codazole inhibited microtubule polymerization in vitro al-

Uckun et al.^[11]recently reported that a model spiroacetal most completely at concentrations as low as 100 nm. Taken

derived from spongistatin 1 prevents microtubule formation together, these data suggest that the compounds 39 and 42

in cells. This conclusion was subsequently questioned by affect the organization of the microtubule cytoskeleton in

Smith III. et al.^[26]

In order to determine whether the microscopically visible bules.

cells by a mechanism other than direct targeting of microtu-

effect of compounds 39 and 42 (Figure 2) was due to direct

It has not escaped our attention that the influence on

targeting of microtubules by these spiroacetals we polymer- microtubule integrity is paralleled by phosphatase-in-

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H. Waldmann et al.

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Table 1. IC₅₀values of phosphatase inhibitors.

[a] All IC₅₀values were calculated from at least three independent determinations. IC₅₀: concentration required for 50% inhibition.

Figure 3. Compound 39 and 42 do not significantly affect microtu-

bule polymerization in vitro. Microscope images of fluorescently

labelled microtubules polymerized in vitro in the presence of the

indicated concentrations of compounds or DMSO as a solvent con-

trol (scale bar: 10 mm).

Figure 2. Effects of spiroketals on tubulin skeleton in MDA-MB-

231 breast tumour cells; DNA immmunostained (blue). Upper row:

tubulin immunostained (orange). Lower row: actin immunostained

(yellow). Left column: control cells show intact tubulin and actin

networks. Middle column: cells treated with compound 39 at 5 μm

final concentration show an irregular and folded tubulin network

and an intact actin network. Right column: cells treated with com-

pound 42 at 5 μm final concentration show incomplete tubulin

fibres and an intact actin network.

hibiting activity in the cases of both 39 and 42. While this

observation alone does not prove a link between the two

phenomena it suggests avenues for subsequent research

aimed at identifying the cellular targets of these compounds

responsible for the influence on the tubulin cytoskeleton.

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Spiroacetal Collection Revealing Modulators of Tubulin Cytoskeleton Integrity

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stirred for 4 h at the same temperature. After the system had been

cooled to –78 °C, the enolate solution was transferred by cannula

to the Wang resin-bound aldehyde (1.66 g, 1.25 mmol), swollen in

CH₂Cl₂(10 mL) at –78 °C. It was stirred for another 1.5 h at

–78 °C, and the reaction mixture was then stored for 18 h at –27 °C.

The solution was filtered and the resin was washed with CH₂Cl₂

and dried under vacuum for 5 h. A second aldol reaction cycle

under the same reaction conditions was carried out. After the solu-

tion from the second cycle had been filtered, the resin was washed

with CH₂Cl₂, pH 7 buffer (phosphate), DMF and MeOH. It was

then swollen in a mixture of MeOH (12 mL), DMF (12 mL) and

pH 7 buffer (phosphate, 3 mL). After the system had been cooled

to 0 °C, aq. H₂O₂(30%, 4.5 mL) was added, and the mixture was

kept shaking for another 8 h at the same temperature. The solution

was filtered and the resin was washed with H₂O, pH 7 buffer, THF/

H₂O, THF, CH₂Cl₂and MeOH, and was then dried under vacuum

Conclusions

In conclusion, we have developed an efficient solid-phase

synthesis of spiro[5.5]ketals. It proceeds on a polymeric car-

rier in up to 12 steps, makes the desired spiroketals available

in high overall yields and with high stereoselectivities, and

is amenable to structural variation of the products. We have

demonstrated that demanding transformations in solution-

phase synthesis can be adapted to solid-support conditions

for library synthesis, generating a small collection of

spiro[5.5]ketals. This sequence should allow the develop-

ment of structurally diverse spiroketal libraries and other

related compound libraries as new tools and reagents for

chemical biology and medicinal chemistry research.

Research along this line is particularly strongly moti-

vated and justified by the finding that the small collection

of spiroketals described here is enriched in bioactivity.

Analogously with the activities described for some of the

natural product guides, it contains phosphatase inhibitors

and modulators affecting the tubulin cytoskeletons of

breast cancer cells.

for 5 h to afford yellowish resin 20. IR (SiC): ν

= 3506, 3059,

˜

max

2930, 1720, 1604, 1450 cm^–1.

Generation of Enolate 9 in Solution and Reaction with Wang Resin-

Bound Aldehyde 19 (19 to 22 in Scheme 4): Diisopropylethylamine

(1.73 mL, 10.5 mmol) was added at –78 °C to a solution of (–)-

Ipc₂BOTf (3.96 g, 9.15 mmol) in CH₂Cl₂(20 mL). After the reac-

tion mixture had been stirred for 30 min, a solution of pentan-3-

one (952 μL, 9.0 mmol) in CH₂Cl₂was added at the same tempera-

ture. After 4 h, the solution was carefully transferred by cannula to

the aldehyde bound to Wang resin 19 (2 g, 1.5 mmol), swollen in

CH₂Cl₂(10 mL) at –78 °C. The reaction mixture was stirred for

another 1.5 h at –78 °C and was then stored for 18 h at –27 °C. The

solution was filtered and the resin was washed with CH₂Cl₂and

dried under vacuum for 5 h. A second aldol reaction cycle was

carried out under the same reaction conditions . After the solution

from the second cycle had been filtered, the resin was washed with

CH₂Cl₂, pH 7 buffer (phosphate), DMF and MeOH and was then

swollen in a mixture of MeOH (4 mL), DMF (4 mL) and pH 7

buffer (phosphate, 1 mL). After the system had been cooled to

0 °C, aq. H₂O₂(30%, 1.5 mL) was added, and the mixture was kept

shaking for another 8 h at the same temperature. The solution was

filtered and the resin 22 was washed with H₂O, pH 7 buffer, THF/

H₂O, THF, CH₂Cl₂and MeOH, and was then dried under vacuum

Experimental Section

General: ¹H NMR and ¹³C spectra were recorded on a Varian Mer-

cury 600 or a Bruker DRX 500 spectrometer. GC-MS (EI) were

measured on a Hewlett–Packard 6890 series gas chromatograph

connected to a Hewlett–Packard 5973 series mass spectrometer;

column:

H&W

19091σ-102

HP-5MS

capillary:

25.0 m×201 μm×0.33 μm nominal. LC-MS was performed on a

Hewlett–Packard 1100 series connected to a Finnigan LCQ ESI-

Spectrometer; column: VP 50/10 Nucleosil C18PPN-column (Ma-

cherey–Nagel); gradient: 90:10 (v/v) H₂O/acetonitrile (0.1% formic

acid) to 10:90 (v/v) in 30 min, flow 1.00 mLmin^–1. Preparative

HPLC was conducted with an Agilent 1100 series; column: VP 125/

21 NUCLEODUR C18 Gravity, 5 μ (Macherey–Nagel); gradient:

60:40 (v/v) H₂O/acetonitrile (no acid!) to 100% acetonitrile in

29 min, flow 10 mLmin^–1. High-resolution mass spectra (HR-MS)

were measured on a Finnigan MAT 8200 spectrometer. IR spectra

were measured on a Bruker Vector 22 spectrometer with an A527

diffuse reflectance head from Spectra Tech. UV spectra were mea-

sured on a Perkin–Elmer Cary 50 spectrometer. HPLC was mea-

sured on Hewlett–Packard 1100 HPLC. The optical rotation was

determined with a Perkin–Elmer 241 polarimeter. Chiral GC was

measured on an Agilent Technologies 6890N; column: Lipodex-E

(25 m, 0.025 mm); 85 °C isotherm. Wang resin (1.1 mmolg^–1, 1%

DVB, 100–200 mesh) was purchased from Novabiochem. All reac-

tions were performed under argon with freshly distilled and dried

solvents. All solvents were distilled by standard procedures. Unless

otherwise noted, reagents were obtained from Aldrich, Acros,

Fluka, Lancaster and Strem and were used without further purifi-

cation.

for 5 h. IR (SiC): ν

= 3504, 3062, 2928, 1714, 1604, 1585,

˜

max

1453 cm^–1.

Generation of Enolate 23 on Solid Support and Reaction with Alde-

hyde 8: The intermediate resin 22 (1.5 g, 1.15 mmol) was swollen in

diethyl ether (8 mL) and cooled to 0 °C. Triethylamine (1.44 mL,

10.35 mmol) was added at 0 °C to a flask containing dicyclohex-

ylboron chloride (9 mL, 9.0 mmol) in diethyl ether (3 mL). After

the mixture had been stirred for 1 h it was transferred to the resin

by cannula at 0 °C. The shaking was continued for 10 h and the

solution was removed (by syringe) while the resin was still kept at

0 °C. The resin was washed with cold diethyl ether (3×15 mL) by

syringe. Fresh diethyl ether (10 mL) was added and the above pro-

cess was repeated again but with a shorter reaction time (4 h). The

solution was removed carefully by syringe and was washed with

cold diethyl ether (4×15 mL). Throughout the process the resin

was kept at 0 °C. Fresh cold diethyl ether (10 mL) was again added

and the mixture was then cooled to –78 °C. A solution of aldehyde

8 (2.13 g) in diethyl ether (5 mL) was added by cannula and the

Procedure for Boron-Mediated Aldol Reactions on Wang Resin

Generation of Enolate 11 in Solution and anti-Aldol Reaction with

Resin-Bound Aldehyde 19: Triethylamine (1.56 mL, 11.25 mmol) mixture was shaken for 2 h at –78 °C before storage at –27 °C for

was added at 0 °C to a solution of dicyclohexylboron chloride

(9.60 mL of 1 m in hexane, 9.60 mmol) in diethyl ether (10 mL).

The reaction mixture was stirred for 1 h at the same temperature.

A solution of TBS-protected aldol 10 (3 g, 7.50 mmol) in diethyl

ether (5 mL) was then added by cannula and the mixture was

24 h. After the mixture had been washed with diethyl ether and

dried under vacuum, a second cycle of the process, under the same

reaction conditions but with half the equivalent of the reagents, was

carried out on this resin sample swollen in diethyl ether. It was then

swollen in a mixture of MeOH (5 mL), DMF (5 mL) and pH 7

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4783

H. Waldmann et al.

FULL PAPER

buffer (phosphate, 1.7 mL). After the system had been cooled to

0 °C, aq. H₂O₂(30%, 2 mL) was added, and it was kept shaking

for another 6 h. The solution was filtered and the resin was washed

with H₂O, pH 7 buffer, THF/H₂O, THF, CH₂Cl₂and MeOH, and

29.53, 25.93, 21.23, 18.13, 13.72, 13.34, 10.67, –4.08, –4.54 ppm.

IR (KBr): ν

= 2838, 1732, 1143, 1127 cm^–1. GC-MS (m/z,%)

˜

max

372 [M]⁺(5), 352 [M – 15]⁺(10).

Spiroketal (+)-38: Yield: 14.2 mg (from 200 mg resin), 13.0%. R_f=

was then dried under vacuum for 5 h. IR (SiC): ν_max= 3510, 3059,

˜

1

0.26 (silica gel, cyclohexane). [α]²_D⁰= +109.5 (CHCl₃, c = 0.49). H

2926, 1713, 1575, 1442 cm^–1.

3

NMR (400 MHz, CDCl₃): δ = 4.30 (dt, J = 11.3, J = 4.7 Hz, 1

H), 3.68–3.59 (m, 4 H), 3.51–3.49 (m, 1 H), 2.20–2.15 (m, 1 H),

A similar reaction procedure was followed for all other anti-aldol

reactions of the ketones 22, 55 and 57 (Scheme 6 and 8) with β-

hydroxy aldehydes.

3

1.79–1.50 (m, 4 H), 1.20 (d, J = 6.1, 3 H), 1.13 (d, J = 6.7 Hz, 3

3

H), 1.03 (d, J = 6.8 Hz, 3 H), 0.87 (s, 9 H), 0.89 (s, 9 H), 0.53 (s,

3 H), 0.04 (s, 9 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 102.59,

72.61, 66.90, 59.35, 58.00, 44.40, 43.47, 35.53, 29.58, 26.17, 26.12,

18.34, 18.24, 18.07, 14.07, 9.61, –3.80, –4.33, –4.40, –4.42 ppm. IR

General Procedure for the Protection of Secondary Alcohols with a

tert-Butyldimethylsilyloxy Group on Solid Support: The resin-bound

secondary alcohol (1 g) was swollen at room temperature in a mix-

ture of CH₂Cl₂(10 mL) and DMF (10 mL). After addition of

TBSCl (1.13 g, 7.5 mmol), DMAP (5 mg, 0.075 mmol) and imida-

zole (0.51 g, 7.5 mmol), the resin was shaken for 24 h. The resin

was filtered and a second cycle was performed with half the equiva-

lents of the reagents in order to complete the reaction (monitored

by FT-IR until the bands at 3504 cm^–1and 3062 cm^–1had com-

pletely disappeared). The resin was washed with CH₂Cl₂, DMF,

THF/H₂O, THF and CH₂Cl₂and dried under vacuum for 5 h.

(KBr): ν

= 2880, 1156, 1132 cm^–1. GC-MS (m/z,%) 458 [M]⁺

˜

max

(3), 401 [M– 57]⁺(16). HR-MS (EI) (70 eV): calcd. for C₂₄H₅₀O₄Si₂

458.3248; found 458.3243.

Spiroketal (+)-39: Yield: 9.1 mg (from 200 mg resin), 10.0%. R_f=

0.26 (silica gel, ethyl acetate/cyclohexane 0.1:10). [α]²_D⁰= +104.5

1

3

(CHCl₃, c = 0.69). H NMR (400 MHz, CDCl₃): δ = 5.08 (dd, J

= 11.12, ³J = 4.8 Hz, 1 H), 3.74–3.67 (m, 1 H), 3.61–3.46 (m, 3 H),

3.34–3.29 (m, 1 H), 2.45–2.38 (m, 1 H), 2.02 (s, 3 H), 1.95–1.89 (m,

1 H), 1.77–1.71 (m, 1 H), 1.64–1.57 (m, 1 H), 1.51–1.42 (m, 1 H),

General Procedure for the Protection of a Secondary Alcohol with

an Acetyl Group on Solid Support: The resin-bound secondary

alcohol (1 g) was swollen at room temperature in THF (10 mL).

After the addition of pyridine (3.3 mL, 22.5 mmol), DMAP (5 mg,

0.075 mmol) and acetic anhydride (1.53 g, 22.5 mmol), the resin

was shaken for 24 h. The resin was filtered (monitored by FT-IR

until the bands at 3504 cm^–1and 3062 cm^–1had completely disap-

peared, with the appearance of sharp peak at 1725 to 1730 cm^–1).

The resin was washed in turn with THF, THF/H₂O, THF and

CH₂Cl₂and was dried under vacuum for 5 h.

3

1.06 (d, J = 6.6 Hz, 3 H), 0.99 (d, J = 7.0 Hz, 3 H), 0.88 (s, 9 H),

0.77 (d, ³J = 6.6 Hz, 3 H), 0.05 (s, 3 H), 0.04 (s, 3 H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 170.55, 102.54, 75.57, 72.54, 65.09,

58.17, 43.82, 40.10, 33.23, 29.52, 25.96, 21.28, 18.13, 13.73, 13.38,

10.69, –4.09, –4.59 ppm. IR (KBr): ν

= 2878, 1729, 1143,

˜

max

1127 cm^–1. GC-MS (m/z,%) 386 [M]⁺(9). HR-MS (EI) (70 eV):

calcd. for C₂₀H₃₈O₅Si 386.2489; found 386.2471.

Spiroketal (+)-40: Yield: 5.0 mg, 8.0%. R_f= 0.4 (silica gel, cyclo-

hexane/EtOAc 10:0.3). [α]²_D⁰= +105 (CHCl₃, c = 0.21). H NMR

1

(400 MHz, CDCl₃): δ = 5.11 (dd, ³J = 11.1, ³J = 5.08 Hz, 1 H),

3.73–3.70 (m, 1 H), 3.60–3.50 (m, 3 H), 3.32–3.26 (m, 1 H), 2.30–

2.26 (m, 1 H), 2.24 (s, 3 H), 1.99–1.84 (m, 2 H), 1.59 (br., 3 H),

General Procedure for Spiroketalization and Cleavage from the Solid

Support: The intermediate resin (200 mg) was swollen in a mixture

of CH₂Cl₂(10 mL) and pH 7 buffer (phosphate, 0.5 mL), and

recrystallized DDQ (425 mg, 7.5 mmol) was added at 0 °C. Shaking

was continued for 1 h at 0 °C followed by 6 h at room temperature.

The solution was filtered and the resin was washed repeatedly with

CH₂Cl₂and aqueous NaHCO₃(aq. sat.). The combined organic

solution was washed with brine (8 mL). It was then dried with

MgSO₄and the solvents were evaporated in vacuo to yield a mix-

ture of spiroketal with 4-methoxybenzaldehyde, detected by GC-

MS [HP-MS (0.33 μm, 25 m×0.2 mm ID), t_R= 13.45 min]. The

pure single spiroketal was obtained by silica gel column chromatog-

raphy.

3

1.16 (d, ³J = 6.4 Hz, 3 H), 1.01 (d, J = 7.2 Hz, 3 H), 0.78 (d, J =

6.4 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 170.59,

102.32, 75.53, 71.88, 65.15, 57.93, 45.15, 41.66, 33.47, 29.45, 21.30,

13.70, 13.32, 10.61 ppm. IR (KBr): ν

= 3430, 1731, 1153,

˜

max

1132 cm^–1. GC-MS (m/z,%) 272 [M]⁺(7), 254 [M – 18]⁺(15). HR-

MS (EI) (70 eV): calcd. for C₁₄H₂₄O₅272.1624; found 272.1649.

Spiroketal (+)-41: Yield: 10.9 mg (from 200 mg resin), 10.0%. R_f=

0.26 (silica gel, cyclohexane). [α]²_D⁰= +86.5 (CHCl₃, c = 0.42). H

1

NMR (400 MHz, CDCl₃): δ = 4.20 (t, ³J = 4.4 Hz, 1 H), 3.67–3.59

(m, 2 H), 3.55–3.46 (m, 2 H), 3.27–3.21 (m, 1 H), 2.23–2.19 (m, 1

H), 1.79–1.62 (m, 4 H), 1.05 (d, ³J = 6.6 Hz, 3 H), 1.01 (d, ³J =

7.2 Hz, 3 H), 0.89 (s, 9 H), 0.88 (s, 9 H), 0.77 (d, ³J = 6.4 Hz, 3

H), 0.05 (s, 3 H), 0.04 (s, 9 H) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ = 102.93, 72.98, 72.62, 65.49, 57.93, 43.97, 43.17, 33.22, 32.06,

26.04, 26.03, 18.26, 18.16, 14.03, 13.87, 10.11, –4.10, –4.13, –4.52,

General Procedure for the Removal of a TBS Group on a Spiroketal:

TBAF (0.017 mL, 0.017 mmol, 1 m solution in THF, 5 equiv.) was

added at 0 °C to a stirred solution of the TBS-protected spiroketal

(0.0033 mmol) in THF (2 mL). The reaction mixture was stirred at

room temperature for 24 h. Saturated NH₄Cl solution (3 ml) was

added, and it was extracted with ethyl acetate, washed with water

and dried with MgSO₄and concentrated to afford crude hydroxy

spiroketal. This was purified by silica gel column chromatography

with ethyl acetate/cyclohexane (0.5:10) as eluent.

–4.65 ppm. IR (KBr): ν

= 2891, 1166, 1152 cm^–1. GC-MS

˜

max

(m/z,%) 458 [M]⁺(3), 401 [M – 57]⁺(16). HR-MS (EI) (70 eV):

calcd. for C₂₄H₅₀O₄Si₂458.3248; found 458.3263.

Spiroketal (+)-42: Yield: 10.0 mg (from 200 mg resin), 11.0%. R_f=

0.26 (silica gel, ethyl acetate/cyclohexane 0.1:10). [α]²_D⁰= +98.5

1

3

Spiroketal (–)-25: Yield: 12.5 mg (from 200 mg resin), 14.0%. R_f=

0.21 (silica gel, ethyl acetate/cyclohexane 0.1:10). [α]²_D⁰= +114.5

(CHCl₃, c = 0.25). H NMR (400 MHz, CDCl₃): δ = 5.29 (t, J =

3

5.2 Hz, 1 H), 3.89 (dd, J = 11.3, J = 5.1 Hz, 1 H), 3.65–3.54 (m,

3 H), 3.35 (dd, J = 11.4, J = 2.8 Hz, 1 H), 2.19–2.16 (m, 1 H),

= 11.2, J = 4.6 Hz, 1 H), 3.64–3.60 (m, 1 H), 3.51–3.26 (m, 3 H), 2.08 (s, 3 H), 1.72–1.71 (m, 1 H), 1.61–1.58 (m, 3 H), 1.10 (d, J =

1

3

(CHCl₃, c = 0.39). H NMR (400 MHz, CDCl₃): δ = 5.00 (dd, J

3

3.44–3.30 (m, 1 H), 2.35–2.28 (m, 1 H), 2.00 (s, 3 H), 1.91–1.84 (m,

2 H), 1.69–1.51 (m, 1 H), 1.62–1.53 (m, 1 H), 1.49–1.45 (m, 1 H),

1.15 (d, J = 6.6 Hz, 3 H), 0.99 (d, J = 7.0 Hz, 3 H), 0.91 (s, 9 H),

0.25 (s, 3 H), 0.04 (s, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ

= 170.64, 102.44, 75.47, 72.53, 65.08, 58.16, 43.83, 40.12, 33.21,

6.6 Hz, 3 H), 1.07 (d, ³J = 7.6 Hz, 3 H), 1.02 (d, ³J = 7.2 Hz, 3 H),

0.89 (s, 9 H), 0.07 (s, 3 H), 0.05 (s, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.83, 102.63, 72.40, 71.64, 63.42, 58.62, 44.33, 42.67,

34.75, 32.02, 26.00, 21.28, 18.17, 14.82, 13.62, 13.34, –3.19,

3

–4.54 ppm. IR (KBr): ν_max= 2880, 1726, 1155, 1122 cm^–1. GC-MS

˜

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Spiroacetal Collection Revealing Modulators of Tubulin Cytoskeleton Integrity

FULL PAPER

(m/z,%) 386 [M]⁺(3). HR-MS (EI) (70 eV): calcd. for C₂₀H₃₈O₅Si

1215 cm^–1. GC-MS (m/z,%) 516 [M]⁺(7), 459 [M – 57]⁺(10). HR-

386.2489; found 386.2463.

MS (EI) (70 eV): calcd. for C₂₆H₅₂O₆Si₂516.3302; found 516.3339.

Spiroketal (+)-43: Yield: 7.8 mg (from 200 mg resin), 6.0%. R_f=

0.32 (silica gel, ethyl acetate/cyclohexane 0.2:10). [α]²_D⁰= +114.5

Spiroketal (+)-48: Yield: 4.7 mg (from 200 mg resin), 4.0%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 0.3:10). [α]²_D⁰= +94

1

(CHCl₃, c = 0.23). ¹H NMR (400 MHz, CDCl₃): δ = 7.26–7.40 (m, (CHCl₃, c = 0.3). H NMR (400 MHz, CDCl₃): δ = 7.36–7.26 (m,

5 H), 4.29 (dt, ³J = 11.4, ³J = 4.8 Hz, 1 H), 3.67–3.52 (m, 4 H),

5 H), 4.99 (dt, ³J = 11.7, ³J = 4.9 Hz, 1 H), 4.48 (s, 2 H), 4.39–

4.33 (m, 1 H), 3.81–3.79 (m, 1 H), 3.62–3.48 (m, 3 H), 2.31–2.2.20

(m, 2 H), 2.15–1.12 (m, 1 H), 2.03 (s, 3 H), 1.94–1.82 (m, 2 H),

2.67–2.52 (m, 2 H), 2.14–2.02 (m, 1 H), 1.81–1.73 (m, 1 H), 1.71–

3

1.63 (m, 2 H), 1.52–1.30 (m, 4 H), 1.04 (d, J = 6.7 Hz, 3 H), 0.98

(d, ³J = 6.8 Hz, 3 H), 0.92 (s, 9 H), 0.89 (s, 9 H), 0.07 (s, 3 H), 0.05

1.81–1.72 (m, 1 H), 1.69–1.51 (m, 1 H), 1.36 (dd, J = 13.7, J =

2

3

(s, 9 H). IR (KBr): ν

= 2933, 1260, 1245 cm^–1. HR-MS (EI) 2.1 Hz, 1 H), 1.04 (d, ³J = 7.2 Hz, 3 H), 0.90 (d, ³J = 6.8 Hz, 3 H),

˜

max

(70 eV): calcd. for C₃₁H₅₆O₄Si₂548.3371; found 548.3725.

0.87 (s, 9 H), 0.04 (s, 6 H) ppm. ¹³C NMR (CDCl₃, 100 MHz,

CDCl₃): δ = 170.67, 138.68, 128.46, 127.78, 127.61, 100.48, 73.13,

71.90, 69.88, 67.55, 67.11, 56.74, 37.36, 36.31, 34.54, 30.55, 28.70,

Spiroketal (+)-44: Yield: 10.2 mg (from 200 mg resin), 9.0%. R_f=

0.2 (silica gel, ethyl acetate/cyclohexane 0.1:10). [α]²_D⁰= +121.5

(CHCl₃, c = 0.35). ¹H NMR (400 MHz, CDCl₃): δ = 7.27–7.25 (m,

2 H), 7.19–7.17 (m, 3 H), 5.04 (dt, ³J = 11.9, ³J = 4.7 Hz, 1 H),

4.44–4.38 (m, 1 H), 3.84–3.77 (m, 2 H), 3.53–3.50 (m, 1 H), 2.74–

2.60 (m, 1 H), 2.33–2.25 (m, 1 H), 2.03 (s, 3 H), 1.83–1.73 (m, 2

26.30, 21.44, 18.66, 15.47, 6.40, –4.55, –4.59 ppm. IR (KBr): ν

˜

max

= 2961, 1727, 1238, 1210 cm^–1. GC-MS (m/z,%) 492 [M]⁺(4), 407

[M – 85]⁺(10). HR-MS (EI) (70 eV): calcd. for C₂₆H₅₂O₆Si₂

492.2907; found 492.2928.

H), 1.62–1.53 (m, 1 H), 1.38 (dd, ²J = 12.8, ³J = 2.4 Hz, 1 H), 1.07 Spiroketal (+)-49: Yield: 8.0 mg (from 200 mg resin), 7.0%. R_f=

3

(d, J = 7.4 Hz, 3 H), 1.02–0.99 (m, 1 H), 0.93 (d, J = 6.6 Hz, 3

H), 0.91 (s, 9 H), 0.89 (d, ³J = 5.6 Hz, 3 H), 0.64 (s, 3 H), 0.04 (s, 3

H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 170.20, 142.43, 128.45,

128.33, 125.80, 100.49, 72.01, 69.92, 69.20, 60.47, 56.71, 38.37,

37.40, 34.60, 31.55, 30.49, 28.74, 21.40, 21.15, 18.70, 15.51, 14.31,

0.35 (silica gel, ethyl acetate/cyclohexane 1.5:10). [α]²_D⁰= +52

1

3

(CHCl₃, c = 0.15). H NMR (400 MHz, CDCl₃): δ = 4.28 (dt, J

3

= 11.3, J = 5.0 Hz, 1 H), 4.00–3.90 (m, 1 H), 3.79–3.77 (m, 2 H),

3.73–3.68 (m, 1 H), 3.62–3.51 (m, 2 H), 2.68 (br., 1 H), 2.21–2.17

(m, 1 H), 1.85–1.67 (m, 4 H), 1.53–1.38 (m, 3 H), 1.04 (d, ³J =

6.41, –4.48, –4.67 ppm. GC-MS (m/z,%) 476 [M]⁺(6), 419 [M – 57]⁺6.6 Hz, 3 H), 0.97 (d, ³J = 7.0 Hz, 3 H), 0.88 (s, 9 H), 0.87 (s, 9

(10). HR-MS (EI) (70 eV): calcd. for C₂₇H₄₄O₅Si 476.2958; found

476.2972.

H), 0.05 (s, 3 H), 0.04 (s, 9 H) ppm. ¹³C NMR (CDCl₃, 100 MHz,

CDCl₃): δ = 102.91, 72.75, 69.36, 66.59, 61.77, 58.22, 51.60, 43.44,

42.27, 37.60, 35.14, 32.79, 32.56, 25.99, 25.97, 18.19, 18.13, 14.03,

Spiroketal (+)-45: Yield: 4.2 mg, 5.0%. R_f= 0.41 (silica gel, ethyl

9.34, –4.19, –4.45, –4.60, 4.71 ppm. IR (KBr): ν

= 2965, 1251,

˜

1

acetate/cyclohexane 1.5:10). [α]²_D⁰= +118.5 (CHCl₃, c = 0.22). H

max

1233 cm^–1. GC-MS (m/z,%) 488 [M]⁺(6), 492 [M – 129]⁺(35). HR-

NMR (400 MHz, CDCl₃): δ = 7.30–7.27 (m, 2 H), 7.20–7.15 (m, 3

MS (EI) (70 eV): calcd. for C₂₅H₅₂O₅Si₂488.3353; found 488.3327.

H), 5.01 (dt, ³J = 11.7, ³J = 4.5 Hz, 1 H), 4.30–4.23 (m, 1 H), 3.99–

3.92 (m, 1 H), 3.70 (dd, ³J = 11.7, ³J = 5.6 Hz, 1 H), 2.85–2.77 (m, Spiroketal (+)-50: Yield: 17.0 mg (from 200 mg resin), 14.0%. R_f=

1 H), 2.70–2.62 (m, 1 H), 2.47 (dt, ²J = 14.1, ³J = 6.8 Hz, 1 H),

0.30 (silica gel, ethyl acetate/cyclohexane 0.2:10). [α]²_D⁰= +83

1

2.32–2.58 (m, 1 H), 2.06 (s, 3 H), 2.05–1.93 (m, 3 H), 1.89–1.80 (m,

(CHCl₃, c = 0.3). H NMR (400 MHz, CDCl₃): δ = 7.32–7.24 (m,

2

3

1 H), 1.77–1.61 (m, 3 H), 1.49 (d, J = 14.0 Hz, 1 H), 1.06 (d, J = 5 H), 5.02 (dt, ³J = 11.7, ³J = 4.5 Hz, 1 H), 4.59–4.40 (m, 2 H),

7.0 Hz, 3 H), 0.92 (d, ³J = 6.9 Hz, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.20, 141.20, 128.29, 128.03, 125.83, 102.30, 70.72, (dd, J = 9.3, J = 3.5 Hz, 1 H), 3.50–3.46 (m, 2 H), 2.29–2.20 (m,

4.37–4.33 (m, 1 H), 3.90–3.81 (m, 1 H), 3.80–3.79 (m, 1 H), 3.62

3

2

70.24, 68.82, 55.99, 38.04, 36.70, 33.10, 31.39, 30.17, 26.93, 21.04,

14.19, 5.85 ppm. GC-MS (m/z,%) 362 [M]⁺(10), 303 [M – 59]⁺(8).

HR-MS (EI) (70 eV): calcd. for C₂₁H₃₀O₅362.2093; found

362.2078.

2 H), 2.03 (s, 3 H), 1.81–1.70 (m, 4 H), 1.31 (br., J = 13.68 Hz, 1

3

H), 1.03 (d, J = 7.2 Hz, 3 H), 0.90 (d, J = 6.64 Hz, 3 H), 0.85 (s,

9 H), 0.012 (s, 3 H), 0.0005 (s, 3 H) ppm. ¹³C NMR (CDCl₃,

100 MHz, CDCl₃): δ = 170.66, 138.57, 128.44, 127.85, 127.64,

100.67, 73.76, 73.15, 71.72, 69.82, 68.77, 56.77, 37.42, 34.19, 28.45,

28.20, 26.14, 21.43, 18.45, 15.24, 6.28, –4.68, –4.77 ppm. IR (KBr):

Spiroketal (+)-46: Yield: 9.8 mg (from 200 mg resin), 7.0%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 0.1/20). [α]²_D⁰= +119

ν

_max= 2961, 1727, 1238, 1210 cm^–1. GC-MS (m/z,%) 506 [M]⁺(6),

˜

1

3

(CHCl₃, c = 0.54). H NMR (400 MHz, CDCl₃): δ = 4.27 (dt, J

492 [M – 99]⁺(10). HR-MS (EI) (70 eV): calcd. for C₂₈H₄₆O₆Si

3

= 11.3, J = 4.9 Hz, 1 H), 3.61–3.58 (m, 4 H), 3.51–3.48 (m, 2 H),

506.3064; found 506.3046.

2.03–2.02 (m, 1 H), 1.81–1.73 (m, 1 H), 1.52–1.26 (m, 4 H), 1.03

(d, ³J = 6.6 Hz, 3 H), 0.92 (d, ³J = 6.7 Hz, 3 H), 0.90 (s, 9 H), 0.89

Spiroketal (+)-51: Yield: 5.5 mg (from 10 mg of 50), 10.0%. R_f=

(s, 9 H), 0.87 (s, 9 H), 0.07 (s, 6 H), 0.05 (s, 3 H), 0.04 (s, 9 H) ppm. 0.23 (silica gel, ethyl acetate/cyclohexane 1.0:1.0). [α]²_D⁰= +103.8

¹³C NMR (100 MHz, CDCl₃): δ = 102.72, 72.58, 69.59, 67.19,

66.63, 57.87, 44.77, 43.35, 36.04, 33.92, 31.39, 26.15, 26.02, 18.44,

18.22, 18.15, 13.81, 9.48, –4.03, –4.40, –4.56, –4.61, –5.07,

(CHCl₃, c = 0.3). H NMR (400 MHz, CDCl₃): δ = 7.33–7.24 (m,

1

3

5 H), 5.02 (dt, J = 11.6, J = 4.8 Hz, 1 H), 4.57 (s, 2 H), 4.29 (dt,

³J = 12.0, J = 2.8 Hz, 1 H), 4.17–4.09 (m, 2 H), 3.75 (br., 1 H),

3

–5.16 ppm. IR (KBr): ν

= 2930, 1260, 1245 cm^–1. GC-MS

3.66 (dd, ³J = 11.6, ²J = 5.6 Hz, 1 H), 3.53 (dd, ²J = 9.8, ³J =

˜

max

(m/z,%) 588 [M]⁺(0.5), 531 [M – 57]⁺(14). HR-MS (EI) (70 eV):

8.0 Hz, 1 H), 3.45 (dd, J = 9.8, J = 2.8 Hz, 1 H), 2.53–2.47 (m,

1 H), 2.31–2.27 (m, 1 H), 2.07–1.98 [m, with a singlet at 2.03 (3

H), 4 H], 1.59–1.46 (m, 5 H), 1.04 (d, ³J = 7.2 Hz, 3 H), 0.89 (d, ³J

= 6.8 Hz, 3 H) ppm. ¹³C NMR (CDCl₃, 100 MHz, CDCl₃): 170.43,

138.08, 128.56, 127.80, 127.78, 127.62, 102.27, 73.48, 73.06, 71.07,

69.97, 69.22, 56.59, 36.99, 33.31, 27.37, 26.75, 24.28, 21.33, 16.64,

14.57, 6.18 ppm. GC-MS (m/z,%) 392 [M]⁺(5), 375 (5), 146 (58),

91 (100). HR-MS (EI) (70 eV): calcd. for C₂₂H₃₂O₆392.2198;

found 392.2173.

2

3

calcd. for C₃₀H₆₄O₅Si₃588.4062; found 588.4069.

Spiroketal (+)-47: Yield: 6.0 mg (from 200 mg resin), 5.0%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 0.1:10). [α]²_D⁰= +114

1

3

(CHCl₃, c = 0.3). H NMR (400 MHz, CDCl₃): δ = 4.94 (dt, J =

11.2, ³J = 4.5 Hz, 1 H), 4.31–4.38 (m, 1 H), 3.82–3.75 (m, 1 H),

3.62–3.45 (m, 4 H), 2.31–2.2.20 (m, 2 H), 2.14–1.10 (m, 1 H), 2.03

(s, 2 H), 1.94–1.82 (m, 1 H), 1.81–1.72 (m, 1 H), 1.69–1.51 (m, 1

2

3

H), 1.36 (dd, J = 13.7, J = 2.1 Hz, 1 H), 1.04 (d, J = 7.2 Hz, 3

H), 0.90 (d, J = 6.8 Hz, 3 H), 0.89 (s, 9 H), 0.87 (s, 9 H), 0.06 (s,

3

Spiroketal (+)-52: Yield: 6.5 mg (from 200 mg resin), 6.0%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 0.2:10). [α]²_D⁰= +64

6 H), 0.04 (s, 6 H) ppm. IR (KBr): ν

= 2941, 1730, 1223,

˜

max

Eur. J. Org. Chem. 2005, 4773–4788

www.eurjoc.org

4785

H. Waldmann et al.

FULL PAPER

1

3

(CHCl₃, c = 0.15). H NMR (400 MHz, CDCl₃): δ = 4.25 (dt, J (FAB, 70 eV) calcd. for C₂₁H₄₀O₅Si 400.2645; found 401.2699 [M

= 12.12, ³J = 4.8 Hz, 1 H), 3.82–3.63 (m, 3 H), 3.54–3.49 (m, 1 H),

+ H]⁺.

2.20–2.17 (m, 1 H), 1.83–1.79 (m, 1 H), 1.72–1.65 (m, 1 H), 1.46–

Spiroketal (–)-61: Yield: 7.0 mg (from 12 mg of 60), 3.7%. R_f=

0.35 (silica gel, ethyl acetate/cyclohexane 3:10). ¹H NMR

(400 MHz, CDCl₃): δ = 5.10 (dd, ³J = 11.2 and 4.8 Hz, 1 H), 3.61–

3.44 (m, 3 H), 3.13 (t, ³J = 11.2 Hz, 1 H), 2.28 (dd, ³J = 7.6

and 7.2 Hz, 1 H), 2.12 (dd, ³J = 7.2 and 5.2 Hz, 1 H), 1.99 (s, 3 H),

1.91–1.80 (m, 3 H), 1.13 (d, ³J = 6.4 Hz, 3 H), 1.11 (d, ³J = 6.4 Hz,

3 H), 0.95 (d, J = 7.6 Hz, 3 H), 0.71 (d, J = 6.4 Hz, 3 H) ppm.

GC-MS (m/z,%) 268 [M – 18]⁺(0.1), 209 (4), 127 (100). HR-MS

(FAB, 70 eV) calcd. for C₁₅H₂₆O₅286.178; found 227.1663 [M –

OAc]⁺.

3

1.25 (m, 3 H), 1.13 (d, ³J = 6.24 Hz, 3 H), 1.03 (d, J = 6.64, 3 H),

3

0.95 (d, J = 7.04, 3 H), 0.89 (s, 9 H), 0.87 (s, 9 H), 0.06 (s, 3 H),

0.04 (s, 9 H) ppm. GC-MS (m/z,%) 458 [M]⁺(6), 401 [M – 57]⁺

(19). HR-MS (EI) (70 eV): calcd. for C₂₄H₅₀O₄Si₂458.3248; found

458.3225.

3

Spiroketal (+)-53: Yield: 6.5 mg (from 200 mg resin), 7.0%. R_f=

0.39 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= +79 (CHCl₃,

1

3

c = 0.20). H NMR (400 MHz, CDCl₃): δ = 4.96 (dt, J = 12.12,

³J = 4.8 Hz, 1 H), 4.40–4.34 (m, 1 H), 3.58–3.45 (m, 3 H), 2.30–

2.25 (m, 1 H), 2.22–2.18 (m, 1 H), 2.12–1.97 (m, 1 H), 2.00 (s, 3

H), 1.95–1.85 (m, 1 H), 1.62–1.58 (m, 1 H), 1.51–1.42 (m, 1 H),

Spiroketal (+)-62: Yield: 2.8 mg (from 800 mg resin), 0.6%. R_f=

0.48 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= +20 (CHCl₃,

c = 0.10). ¹H NMR (400 MHz, CDCl₃): δ = 4.65 (dd, ³J = 7.6

1.20 (d, ³J = 6.0 Hz, 3 H), 1.00 (d, ³J = 7.4 Hz, 3 H), 0.85 (d, J =

3

5.9 Hz, 3 H), 0.84 (s, 9 H), 0.02 (s, 3 H), 0.01 (s, 3 H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 170.69, 100.47, 71.73, 69.92, 65.52,

56.70, 37.17, 34.23, 32.74, 28.48, 26.10, 22.07, 21.43, 18.43, 15.33,

6.30, –4.68, –4.76 ppm. GC-MS (m/z,%) 386 [M]⁺(12), 301 [M –

85]⁺(15). HR-MS (EI) (70 eV): calcd. for C₂₀H₃₈O₅Si 386.2489;

found 386.2465.

2

3

and 4.0 Hz, 1 H), 4.01 (dd, J = 12.0, J = 5.2 Hz, 1 H), 3.90–3.77

3

(m, 1 H), 3.63 (dt, J = 10.4 and 6.4 Hz, 1 H), 3.28 (dd, J = 12.0

and 6.4 Hz, 1 H), 2.24 (dq, ³J = 7.2 and 4.0 Hz, 1 H), 2.03 (s, 3 H),

3

1.93–1.86 (m, 2 H), 1.75 (dq, J = 6.0 and 6.4 Hz, 1 H), 1.61 (dd,

³J = 12.0 and 11.6 Hz, 1 H), 1.14 (d, J = 6.0 Hz, 3 H), 1.04 (d, J

3

= 7.2 Hz, 3 H), 0.97 (d, J = 7.2 Hz, 3 H), 0.90 (d, J = 6.8 Hz, 3

H), 0.83 (s, 9 H), 0.02 (s, 3 H), 0.00 (s, 3 H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 170.30, 102.61, 75.63, 72.97, 64.77, 63.05,

45.01, 42.24, 29.58, 26.85, 25.80, 21.32, 21.17, 17.99, 13.92, 13.33,

10.70, –3.56, –4.31 ppm. GC-MS (m/z,%) 400 [M]⁺(0.1), 382 (0.5),

283 (14), 127 (100). HR-MS (FAB, 70 eV) calcd. for C₂₁H₄₀O₅Si

400.2645; found 401.2709 [M + H]⁺.

Spiroketal (–)-58: Yield: 8.2 mg (from 800 mg resin), 2.2%. R_f=

0.18 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= –7.2 (CHCl₃,

c = 0.25). ¹H NMR (400 MHz, CDCl₃): δ = 7.35–7.28 (m, 5 H),

5.23 (dd, ³J = 11.2 and 5.2 Hz, 1 H), 4.61 (br.s, 2 H), 3.77–3.70

3

2

(m, 1 H), 3.60–3.48 (m, 4 H), 3.33 (appt, J = 11.2, J = 11.6 Hz,

3

1 H), 2.21 (dd, ³J = 7.2, J = 5.2 Hz, 1 H), 2.06 (s, 3 H), 1.96–1.89

3

(m, 2 H), 1.51–1.31 (m, 3 H), 1.17 (d, J = 6.4 Hz, 3 H), 1.02 (d,

³J = 7.2 Hz, 3 H), 0.72 (d, ³J = 6.4 Hz, 3 H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 174.24, 138.72, 128.56, 127.65, 127.65,

102.56, 75.62, 73.73, 73.39, 72.11, 67.19, 65.18, 45.46, 42.35, 36.08,

29.56, 21.35, 13.80, 13.35, 10.58 ppm. GC-MS (m/z,%) 392 [M]⁺

(0.1), 251 (17), 139 (21), 91 (100). HRMS-FAB (70 eV) calcd. for

C₂₂H₃₂O₆392.2199; found 415.2087 [M + Na]⁺.

Spiroketal (+)-63: Yield: 10.0 mg (from 610 mg resin), 3.0%. R_f=

0.37 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= +10 (CHCl₃,

c = 0.25). ¹H NMR (400 MHz, CDCl₃): δ = 7.35–7.32 (m, 5 H),

5.49 (dd, ³J = 12.0 and 4.8 Hz, 1 H), 4.64 (s, 2 H), 3.86–3.43 (m, 6

H), 2.18–2.11 (m, 1 H), 1.92 (s, 3 H), 1.83–1.71 (m, 2 H), 1.49–1.42

3

(m, 1 H), 1.37–1.28 (m, 3 H), 1.10 (d, J = 6.4 Hz, 3 H), 0.95 (d,

³J = 6.8 Hz, 3 H), 0.86 (s, 9 H), 0.04 (s, 3 H), 0.03 (s, 3 H) ppm.

GC-MS (m/z,%) 492 [M]⁺(0.8), 375 [M – 117]⁺(13), 91 (100). HR-

MS (EI) (70 eV): calcd. for C₂₇H₄₄O₆Si 492.2907; found 493.2986

[M + H]⁺.

Spiroketal (+)-59: Yield: 9.7 mg (from 800 mg resin), 2.0%. R_f=

0.63 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= +19 (CHCl₃,

c = 0.43). ¹H NMR (400 MHz, CDCl₃): δ = 7.30–7.22 (m, 5 H),

3

4.64 (dd, J = 8.4 and 4.0 Hz, 1 H), 4.58 (brs, 2 H), 3.95–3.88 (m,

3

2

3

Spiroketal (–)-64: Yield: 5.9 mg (from 10 mg of 63), 2.3%. R_f=

0.31 (silica gel, ethyl acetate/cyclohexane 3:10). ¹H NMR

(400 MHz, CDCl₃): δ = 7.34–7.24 (m, 5 H), 5.48 (dd, ³J = 11.6

and 4.8 Hz, 1 H), 4.60 (s, 2 H), 3.73–3.64 (m, 2 H), 3.58–3.47 (m,

4 H), 2.22–2.13 (m, 1 H), 1.99 (s, 3 H), 1.97–1.91 (m, 1 H), 1.75

2 H), 3.67 (dt, J = 9.2 and 5.6 Hz, 1 H), 3.57 (dd, J = 9.6, J =

2

3

2

5.6 Hz, 1 H), 3.44 (dd, J = 10.0, J = 5.6 Hz, 1 H), 3.29 (dd, J =

12.0, ³J = 7.8 Hz, 1 H), 2.27–2.22 (m, 1 H), 2.05–2.03 (m, 1 H),

3

2.00 (s, 3 H), 1.94–1.86 (m, 2 H), 1.74–1.66 (m, 1 H), 1.07 (d, J =

7.2 Hz, 3 H), 0.98 (d, ³J = 6.8 Hz, 3 H), 0.87 (d, ³J = 6.8 Hz, 3 H),

0.85 (s, 9 H), 0.03 (s, 3 H), 0.01 (s, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 171.03, 138.71, 128.48, 127.82, 127.67, 102.78, 76.30,

74.77, 73.54, 72.06, 70.70, 64.80, 42.74, 35.99, 35.01, 30.36, 25.96,

21.26, 18.10, 15.06, 14.52, 9.83, –4.13, –4.55 ppm. GC-MS (m/z,%)

506 [M]⁺(0.1), 389 [M – 117]⁺(14), 115 (21), 91 (100). HR-MS

(FAB, 70 eV) calcd. for C₂₈H₄₆O₆Si 506.3064; found 529.2986 [M

+ Na]⁺.

2

3

(dd, J = 12.0, J = 5.6 Hz, 1 H), 1.67–1.61 (m, 2 H), 1.41 (br., 1

H), 1.39–1.31 (m, 1 H), 1.17 (d, ³J = 6.8 Hz, 3 H), 1.05 (d, ³J =

7.2 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 171.39,

137.29, 128.40, 127.51, 127.47, 102.21, 73.49, 73.01, 71.87, 70.11,

66.83, 58.84, 45.46, 42.09, 35.84, 31.11, 21.35, 13.62, 11.13 ppm.

GC-MS (m/z,%) 378 [M]⁺(1.0), 319 (5), 173 (19), 91 (100).

Spiroketal (–)-65 (as a mixture of 65/63 in a ratio of 86:14): Yield:

6.4 mg (from 610 mg resin), 1.8%. R_f= 0.54 (silica gel, ethyl ace-

tate/cyclohexane 1:10). ¹H NMR (400 MHz, CDCl₃): δ = 7.25–7.21

Spiroketal (–)-60: Yield: 22.0 mg (from 800 mg resin), 4.6%. R_f=

0.55 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= –9.0 (CHCl₃,

c = 1.0). ¹H NMR (400 MHz, CDCl₃): δ = 5.14 (dd, ³J = 11.2 (m, 5 H), 4.99 (dd, ³J = 8.8 and 5.2 Hz, 1 H), 4.57 (dd, ²J =

and 5.2 Hz, 1 H), 3.58–3.46 (m, 3 H), 3.15 (appt, ³J = 11.6

12.0 Hz, 2 H), 4.12–4.06 (m, 1 H), 4.02–3.96 (m, 1 H), 3.74 (ddd,

3

and 11.2 Hz, 1 H), 2.13 (dd, J = 7.2 and 5.2 Hz, 1 H), 2.02 (s, 3

³J = 10.0, 7.6 and 5.6 Hz, 1 H), 3.60 [m, 2 H (with a ddd, ²J = 9.6,

2

3

H), 1.95–1.86 (m, 1 H), 1.75–1.69 (m, 1 H), 1.45–1.36 (m, 2 H),

³J = 5.6 Hz, 1 H)], 3.44 (dd, J = 9.6, J = 5.6 Hz, 1 H), 2.20–2.13

3

1.14 (d, ³J = 6.8 Hz, 3 H), 1.08 (d, J = 6.4 Hz, 3 H), 0.99 (d, J =

6.4 Hz, 3 H), 0.85 (s, 9 H), 0.75 (d, ³J = 6.4 Hz, 3 H), 0.03 (s, 3

H), 0.02 (s, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 170.30,

102.61, 75.63, 72.97, 64.77, 63.05, 45.01, 42.24, 29.58, 26.85, 25.80,

21.32, 21.17, 17.99, 13.92, 13.33, 10.70, –3.56, –4.31 ppm. GC-MS

(m/z,%) 400 [M]⁺(0.1), 382 (0.5), 283 (14), 127 (100). HR-MS

(m, 1 H), 1.98 (s, 3 H), 1.97–1.91 (m, 3 H), 1.87 (appt, ³J = 7.6

and 7.2 Hz, 1 H), 1.77–1.73 (m, 2 H), 1.06 (d, J = 7.2 Hz, 3 H),

3

1.03 (d, ³J = 7.2 Hz, 3 H), 0.87 (s, 9 H), 0.05 (s, 3 H), 0.04 (s, 3

H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 171.01, 138.49, 128.34,

127.66, 127.55, 102.19, 77.20, 74.46, 73.39, 71.41, 71.08, 70.82,

45.63, 37.73, 36.22, 27.95, 25.81, 21.16, 17.97, 13.85, 11.88, –4.35,

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Eur. J. Org. Chem. 2005, 4773–4788

Spiroacetal Collection Revealing Modulators of Tubulin Cytoskeleton Integrity

–3.84 ppm. GC-MS (m/z,%) 492 [M]⁺(0.1), 375 [M – 117]⁺(9), (m, 3 H), 1.15 (d, ³J = 6.4 Hz, 3 H), 1.09 (d, ³J = 6.2 Hz, 3 H),

FULL PAPER

287 (8), 115 (18), 91 (100).

0.99 (d, ³J = 7.2 Hz, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ =

170.22, 138.36, 128.34, 127.67, 127.56, 101.88, 73.11, 72.23, 70.49,

66.47, 64.44, 63.10, 45.31, 41.59, 41.43, 35.76, 31.22, 29.69, 21.28,

13.45, 10.17 ppm. GC-MS (m/z,%) 389 [M + 1 – H₂O]⁺(3), 307

(4), 207 (4), 145 (9), 91 (100).

Spiroketal (–)-66: Yield: 3.6 mg, 1.3%. R_f= 0.31 (silica gel, ethyl

1

acetate/cyclohexane 3:10). [α]²_D⁰= –12 (CHCl₃, c = 0.6). H NMR

(400 MHz, CDCl₃): δ = 7.33–7.26 (m, 5 H), 4.98 (dd, ³J = 6.8

3

and 3.6 Hz, 1 H), 4.57 (s, 2 H), 3.84–3.77 (m, 1 H), 3.69 (dt, J =

2

3

10.8 and 4.8 Hz, 1 H), 3.55 (dd, J = 10.4, J = 5.6 Hz, 1 H), 3.44

Spiroketal (–)-71: Yield: 25 mg (from 800 mg resin), 5.1%. R_f=

0.40 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= –33 (CHCl₃,

c = 1.0). ¹H NMR (400 MHz, CDCl₃): δ = 7.34–7.31 (m, 5 H),

2

3

2

3

(dd, J = 11.2, J = 4.4 Hz, 1 H), 3.40 (dd, J = 11.2, J = 5.2 Hz,

1 H), 1.98–1.91 (m, 1 H), 1.87 (s, 3 H), 1.82–1.75 (m, 1 H), 1.65–

1.61 (m, 1 H), 1.52–1.48 (m, 3 H), 1.29–1.18 (m, 2 H), 0.98 (d, ³J =

6.8 Hz, 3 H), 0.87 (d, ³J = 7.2 Hz, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 171.18, 138.68, 128.37, 127.55, 127.46, 101.21, 73.54,

70.09, 69.50, 67.50, 54.82, 53.40, 42.87, 37.41, 36.09, 30.20, 21.20,

11.98, 11.42 ppm. GC-MS (m/z,%) 378 [M]⁺(1.0), 319 (5), 173

(19), 91 (100).

3

2

5.46 (dt, J = 11.2 and 5.2 Hz, 1 H), 4.61 (d, J = 12.4 Hz, 1 H),

4.57 (d, ²J = 12.4 Hz, 1 H), 3.82–3.76 (m, 1 H), 3.59–3.45 (m, 4

H), 2.09 (dq, ³J = 7.2 and 5.6 Hz, 1 H), 2.00 (s, 3 H), 1–75–1.70

2

(m, 2 H), 1.48–1.40 (m, 2 H), 1.24 (q, J = 11.2 Hz, 1 H), 1.13 (d,

³J = 6.0 Hz, 3 H), 1.11 (d, J = 6.4 Hz, 3 H), 1.01 (d, J = 7.2 Hz,

3 H), 0.86 (s, 9 H), 0.04 (s, 3 H), 0.03 (s, 3 H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 170.23, 138.66, 128.35, 127.46, 127.29,

102.53, 73.36, 73.05, 72.96, 70.37, 67.92, 63.15, 45.21, 42.30, 41.77,

27.65, 25.86, 21.34 (2 Cs), 18.00, 13.92, 10.32, –3.83, –4.64 ppm.

MS-FAB (m/z,%) 507 [M + 1]⁺(3), 506 [M]⁺(1), 447 (50), 145

(15), 91 (100). HR-MS (FAB, 70 eV) calcd. for C₂₈H₄₆O₆Si

506.3064; found 507.3147 [M + H]⁺.

3

Spiroketal (–)-67: Yield: 20.0 mg (from 1.5 g resin), 2.8%. R_f= 0.60

(silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= –34.1 (CHCl₃, c

= 1.0). ¹H NMR (400 MHz, CDCl₃): δ = 5.42 (dd, ³J = 11.8

and 4.8 Hz, 1 H), 3.70–3.63 (m, 1 H), 3.55–3.45 (m, 2 H), 2.04 (dd,

³J = 7.0 and 5.8 Hz, 1 H), 2.00 (s, 3 H), 1.76–1.67 (m, 2 H), 1.42–

3

1.19 (m, 3 H), 1.14 (d, J = 6.0 Hz, 3 H), 1.12 (d, J = 6.0 Hz, 3

3

H), 1.09 (d, J = 6.4 Hz, 3 H), 1.00 (d, J = 6.44 Hz, 3 H), 0.86 (s,

9 H), –0.05 (s, 3 H), –0.04 (s, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.32, 102.40, 72.97, 70.69, 63.86, 62.84, 45.25, 42.38,

41.51, 32.86, 25.86, 21.39, 21.33 (2 Cs), 18.00, 13.86, 10.34, –3.83,

–4.62 ppm. GC-MS (m/z,%) 400 [M]⁺(0.1), 382 (0.5), 341 (4), 269

(14), 127 (100), HR-MS (FAB, 70 eV) C₂₁H₄₀O₅Si 400.2645; found

401.2703 [M + H]⁺.

Spiroketal (–)-72: Yield: 9.0 mg (from 15 mg of 71), 4.0%. R_f=

0.35 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= –43 (CHCl₃,

c = 0.9). ¹H NMR (400 MHz, CDCl₃): δ = 7.34–7.31 (m, 5 H),

3

2

5.46 (dt, J = 11.8 and 5.0 Hz, 1 H), 4.59 (q, J = 12.3 Hz, 2 H),

2

3

3.83–3.76 (m, 1 H), 3.65–3.57 (m, 3 H), 3.53 (dd, J = 10.5, J =

2

3

5.8 Hz, 1 H), 3.46 (dd, J = 10.5, J = 4.1 Hz, 1 H), 2.12 (dq, J =

7.0 and 5.2 Hz, 1 H), 2.01 (s, 3 H), 1.89 (dq, J = 7.2 and 2.1 Hz,

1 H) 1.77–1.70 (m, 1 H), 1.29–1.21 (m, 2 H), 1.20 (d, J = 6.2 Hz,

3

Spiroketal (–)-68: Yield: 5.7 mg (from 10 mg of 67), 2.2%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= –31 (CHCl₃,

c = 0.5). ¹H NMR (400 MHz, CDCl₃): δ = 5.42 (dd, ³J = 11.8

and 4.9 Hz, 1 H), 3.72–3.64 (m, 1 H), 3.59–3.51 (m, 2 H), 2.09 (dq,

³J = 7.2 and 5.2 Hz, 1 H), 2.01 (s, 3 H), 1.88 (dq, ³J = 7.2

and 4.9 Hz, 1 H), 1.72–1.67 (m, 1 H), 1.43–1.32 (m, 3 H), 1.24 (br.,

3

3 H), 1.17 (d, J = 6.2 Hz, 3 H), 1.02 (d, J = 7.2 Hz, 3 H) ppm.

¹³C NMR (100 MHz, CDCl₃): δ = 170.22, 138.56, 128.35, 127.49,

127.33, 102.24, 73.42, 73.02, 72.13, 70.25, 68.03, 63.26, 53.40,

45.17, 41.68, 41.43, 27.58, 21.34, 13.54, 10.15 ppm. GC-MS

(m/z,%) 372 [M – H₂O]⁺(0.1), 281 (32), 207 (100), 115 (27), 91

(42). HR-MS (FAB, 70 eV) calcd. for C₂₂H₃₂O₆392.2199; found

393.2275 [M + H]⁺.

3

1 H), 1.17 (d, J = 7.2 Hz, 3 H), 1.15 (d, J = 7.2 Hz, 3 H), 1.14

(d, J = 6.6 Hz, 3 H), 1.01 (d, J = 7.2 Hz, 3 H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 170.31, 102.12, 72.12, 70.56, 64.05, 63.00,

45.24, 41.47, 41.40, 32.75, 21.37, 21.34, 21.28, 13.45, 10.18 ppm.

GC-MS (m/z,%) 268 [M – H₂O]⁺(0.1), 209 (4), 127 (100). HR-MS

(FAB, 70 eV) calcd. for C₁₅H₂₆O₅286,178; found 227.1022 [M –

OAc]⁺.

3

Spiroketal (–)-73: Yield: 8.2 mg (from 800 mg resin), 1.7%. R_f=

0.60 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= –22.4

1

(CHCl₃, c = 0.5). H NMR (400 MHz, CDCl₃): δ = 7.34–7.31 (m,

3

5 H), 5.06 (dd, J = 6.8 and 3.2 Hz, 1 H), 4.59 (s, 2 H), 4.00–3.96

(m, 1 H), 3.79–3.71 (m, 2 H), 3.50–3.40 (m, 2 H), 2.01 (s, 3 H),

3

1.93 (dq, J = 6.8 and 3.6 Hz, 1 H), 1.81–1.70 (m, 2 H), 1.57–1.53

Spiroketal (–)-69: Yield: 22.4 mg (from 750 mg resin), 4.2%. R_f=

0.41 (silica gel, ethyl acetate/cyclohexane 1:10). [α]²_D⁰= –48 (CHCl₃,

c = 0.6). ¹H NMR (400 MHz, CDCl₃): δ = 7.33–7.27 (m, 5 H),

3

(m, 1 H), 1.24–1.17 (m, 2 H), 1.12 (d, J = 6.0 Hz, 3 H), 0.94 (d,

3

³J = 6.8 Hz, 3 H), 0.90 (d, J = 6.8 Hz, 3 H), 0.87 (s, 9 H), 0.04 (s,

3 H), 0.03 (s, 3 H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 171.20,

138.66, 128.35, 127.46, 127.28, 102.55, 73.41, 73.17, 7058, 70.45,

63.73, 63.18, 43.25, 42.65, 36.14, 32.57, 25.88, 21.55, 21.15, 18.06,

11.72, 11.62, –4.01, –4.63 ppm. GC-MS (m/z,%) 506 [M]⁺(4.0),

407 (15), 207 (100), 91 (86). HR-MS (FAB, 70 eV) calcd. for

C₂₈H₄₆O₆Si 506.3064; found 507.3149 [M + H]⁺.

3

2

5.46 (dt, J = 12.0 and 4.8 Hz, 1 H), 4.45 (q, J = 12.0 Hz, 2 H),

3.79–3.70 (m, 1 H), 3.62–3.43 (m, 4 H), 2.10–2.00 (m, 1 H), 1.99

(s, 3 H), 1.80–1.72 (m, 2 H), 1.69–1.63 (m, 2 H), 1.43–1.37 (m, 2

3

H), 1.20 (appq, J = 12.4 and 11.2 Hz, 1 H), 1.07 (d, J = 6.4 Hz,

3

3 H), 1.06 (d, J = 6.0 Hz, 3 H), 0.98 (d, J = 7.2 Hz, 3 H), 0.87

(s, 9 H), 0.04 (s, 3 H), 0.03 (s, 3 H) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.28, 138.41, 130.13, 128.35, 127.62, 102.18, 73.11,

73.06, 70.61, 66.61, 64.31, 62.99, 53.40, 45.33, 42.33, 41.66, 35.77,

31.26, 25.87, 21.27, 18.02, 13.82, 10.34, –3.82, –4.63. MS-FAB

(m/z,%) 521 [M + 1]⁺(1.1), 520 [M]⁺(0.7), 461 (21), 389 (14), 145

(21), 91 (100). HR-MS (FAB, 70 eV) calcd. for C₂₉H₄₈O₆Si

520.3220; found 461.3073 [M – OAc]⁺.

Supporting Information (for details see footnote on the first page of

this article): Spectroscopic and analytical data, selected experimen-

tal procedures and procedure for different enzymatic assays are

available.

Spiroketal (–)-70: Yield: 9.2 mg (from 15 mg of 69), 3.4%. R_f=

0.30 (silica gel, ethyl acetate/cyclohexane 3:10). [α]²_D⁰= –15 (CHCl₃,

c = 0.8). ¹H NMR (400 MHz, CDCl₃): δ = 7.33–7.28 (m, 5 H),

5.43 (dt, ³J = 11.8 and 4.8 Hz, 1 H), 4.45 (appq, ²J = 11.7 and

Acknowledgments

We thank Dr. H. Prinz, Dr. R. Gail and H. Rimpel for performing

11.4 Hz, 2 H), 3.81–3.73 (m, 1 H), 3.64–3.42 (m, 4 H), 2.10 (dq, ³J the phosphatase assays and Priv. Doz. Dr. B. Costisella for support

= 7.0 and 5.6 Hz, 1 H), 2.00 (s, 3 H), 1.85–1.62 (m, 5 H), 1.49–1.36

in NOE experiments. This research was supported by the Max

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FULL PAPER

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Planck Gesellschaft and the Fonds der Chemischen Industrie. O. B.

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tion, S. S. by a Kekulé Fellowship from the Fonds der Chemischen

Industrie and T. U. M. by the Emmy Noether Fellowship of the

DFG.

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Received: August 9, 2005

Published Online: October 13, 2005

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Article Doi

Natural product-guided synthesis of a spiroacetal collection reveals modulators of tubulin cytoskeleton integrity

DOI: 10.1002/ejoc.200500605

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Article abstract of DOI:10.1002/ejoc.200500605

Full text of DOI:10.1002/ejoc.200500605

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