Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues

FULL PAPER

Total Synthesis and Biological Evaluation of the Protein Phosphatase 2A

Inhibitor Cytostatin and Analogues

Laurent Bialy^{[a, b]}and Herbert Waldmann*^{[a, b]}

Abstract: The total synthesis of the

natural product cytostatin is described

which inhibits protein phosphatase 2A.

Cytostatin has anti-metastatic proper-

ties and induces apoptosis. On the

basis of this synthesis the relative and

absolute configuration of cytostatin

could be assigned. Key structural ele-

ments of cytostatin are an a,b-unsatu-

rated lactone group and a side chain

embodying a phosphate and a rather

unstable (Z,Z,E)-triene subunit. In ad-

dition, the natural product carries six

stereocenters. For the construction of

the stereocenters reagent-controlled

transformations were used in order to

ensure maximum stereochemical flexi-

bility. The Evans syn-aldol reaction was

chosen to establish the stereochemistry

at C-4, C-5, C-9 and C-10; C-6 was in-

troduced by means of the Evans asym-

metric alkylation. In all cases the same

chiral auxiliary was employed as ster-

eodirecting group. The stereocenter at

C-11 was established by an asymmetric

reduction using CBS-oxazaborolidine.

Temporary protection of the phosphate

group was achieved best by using the

base-labile 9-fluorenylmethyl group,

which could be cleanly cleaved by an

excess of triethylamine; this reaction

yielded analytically pure phosphates

after a simple aqueous work-up. The

(Z,Z,E)-triene embodied in cytostatin

was synthesized by means of a Stille

coupling as key transformation. The

synthesis sequence established in this

way readily gave access to a series of

analogues with simplified structure. Ini-

tial biological testing of these ana-

logues proved that the a,b-unsaturated

lactone, the C-11-hydroxy group and a

fully deprotected phosphate moiety at

C-9 are essential for the PP2A-inhibi-

tory activity of cytostatin. The rather

unstable triene moiety in the side chain

can be replaced by other lipophilic resi-

dues with only moderate decrease of

biological activity. Other phosphatases,

that is, PP1, VHR, PTP1B, CD45, were

not inhibited by cytostatin or any of

the analogues, demonstrating the high

selectivity of this compound. These

findings will be useful for the design

and synthesis of cytostatin-derived

chemical tools for the study of biologi-

cal processes influenced by PP2A.

Keywords: cytostatin

¥

natural

products ¥ phosphorylation ¥ protein

phosphatases ¥ total synthesis

Introduction

tein phosphorylation, whereas protein phosphatases (PPs)

are responsible for dephosphoshorylation. PKs are establish-

ed targets of drug discovery.^[2]However, the development of

small-molecule inhibitors of PPs is emerging only recently

as a very rapidly growing area of investigation in clinical bi-

ology and medicinal chemistry.^[3]Natural products, which

have widely been used to antagonize PP action in biological

experiments, can serve as invaluable starting points in struc-

tural space for the development of potent and selective PP

inhibitors.^[4]The serine-threonine phosphatase 2A (PP2A) is

one of the most important and abundant phosphatases and

is involved in the regulation of many crucial biological pro-

grams such as signal transduction cascades and the cell

cycle. Several toxic and biologically active natural products

have been identified as nanomolar PP2A inhibitors, in par-

ticular the diarrhetic shellfish poisoning toxin okadaic

acid,^[5]the microcystins^[6]and calyculin.^[7]However, the most

The reversible phosphorylation of proteins is employed by

living organisms for the regulation of innumerable cellular

processes, and aberrant protein phosphorylation contributes

to the development of many human diseases, including

cancer and diabetes.^[1]Protein kinases (PKs) catalyze pro-

[a] Dr. L. Bialy, Prof. Dr. H. Waldmann

Max-Planck-Institut f¸r molekulare Physiologie

Abteilung Chemische Biologie

Otto-Hahn-Strasse 11

44227 Dortmund (Germany)

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

[b] Dr. L. Bialy, Prof. Dr. H. Waldmann

Universit‰t Dortmund

Fachbereich 3

Organische Chemie

11]

selective PP2A inhibitors belong to the fostriecin family.^[8

They show an unprecedented discrimination between the

highly homologous serine-threonine phosphatases PP2A

44221 Dortmund (Germany)

Fax : (+49)231-133-2499

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FULL PAPER

(IC₅₀3.2nm 3.7mm) and PP1 (IC₅₀>100mm),^[8d,9d,10c]accom-

that an intramolecular hydrogen bond between O-9 and

OH-11 is formed,^[8e]one can deduce an anti-configuration

between H-10 and H-11 based on the Karplus relationship

(Figure 2). The configuration of stereocenter 6 was deter-

mined by synthesis of a short substructure of cytostatin.^[14]

The unknown configuration of cytostatin prompted us to

design the synthesis with a high degree of flexibility to allow

rapid and reliable variation of absolute and relative configu-

ration if desired. Therefore, we chose to employ the asym-

metric Evans aldol methodology (which gives access to syn-

and anti-aldol products in both enantiomeric forms^[15,16]), the

asymmetric Evans alkylation,^[17]and the enantioselective re-

duction of an acetylenic ketone (for which efficient reagent-

controlled processes that give rise to both possible stereo-

isomers are known^[18]) as synthesis methods for the genera-

tion of the stereocenters.

panied by interesting biological activities such as anti-can-

i,9b]

cer^[8f

and thrombopoietic activity.^[10d]

Cytostatin 1 was isolated from a new Streptomyces strain

by Ishizuka et al.^[9a]and belongs to the fostriecin family. It

inhibits PP2A with an IC₅₀value of 210nm,^[9d]blocks the ad-

hesion of B16 melanoma cells to components of the extra-

cellular matrix (laminin and collagen),^[9a]displays anti-meta-

static and cytotoxic activity,^[9]and induces apoptosis of B16

melanoma cells at submicromolecular concentrations.^[9d]Ish-

izuka et al. determined the constitution of the natural prod-

uct, but not its relative and absolute configuration.^[9e]In

order to develop chemical tools for the study of the biologi-

cal phenomena influenced by PP2A we embarked on a total

synthesis of cytostatin to prove the stereochemistry of the

natural product, get access to a variety of more stable ana-

logues and establish an initial structure activity relationship

(SAR).^[12]Herein we disclose a full account of this synthesis,

the assignment of the relative and absolute configuration of

cytostatin 1 and the synthesis and biological evaluation of

analogues of the natural product. These results demonstrate

which structural features of cytostatin are required for

PP2A inhibition.

Figure 2. Proposed configuration of cytostatin.

Results and Discussion

Initial retrosynthesic analysis of (2S,3S,4S,9S,10S,11S)-cy-

tostatin (1a) traced the molecule back to enediyne 4, from

which it should be accessible via partial hydrogenation

(Figure 3). The enediyne can be dissected into alkyne 5 and

alkynylhalide 6 (retro yne yne coupling). The a,b-unsaturat-

ed lactone 5 was traced back to b-hydroxy aldehyde 7, from

which it should be accessible via Still Gennari olefination^[19]

and subsequent lactonization. It was planned to generate

both syn-diols (corresponding to C-4/C-5 and C-9 and C-10

in the natural product) by means of an asymmetric aldol re-

action employing N-propionyloxazolidinone (8) and the

chiral aldehyde 9, which in turn is accessible by asymmetric

alkylation of 8.^[17]Conversion of the double bond into an al-

dehyde group and a second aldol reaction employing again

8 as chiral auxiliary would then be carried out. Finally, trans-

formation of the N-acyl group into an alkynyl ketone using

trimethylsilylacetylene (10) as nucleophile followed by

asymmetric reduction of the ketone would yield 7.

Retrosynthesic analysis: In planning the synthesis, we drew

from the structurally related fostriecin 2 and the phoslacto-

mycins 3, whose absolute configurations have been deter-

mined by NMR spectroscopy^[8e,10b]and total synthesis

(Figure 1).^[13]Based on the assumption that these natural

Synthesis of the (Z,Z,E)-triene unit–model studies: The

feasibility of the planned partial reduction of enediyne 4

was investigated employing a model analogue. Partial reduc-

tion of diynes has been carried out mainly by hydrogenation

with the Lindlar catalyst^[20]and zinc reduction.^[21]Pent-1-yn-

3-ol (11) was protected as p-methoxybenzyl ether 12

(Scheme 1). Trimethylsilylacetylene (10) and (E)-1-bromo-1-

propene (13) were coupled in a Sonogashira reaction to

yield trimethylsilylenyne 14,^[22]which was converted to bro-

moenyne 15^[23]by means of the Isobe protocol.^[24]The at-

tempted synthesis of the more reactive iodoalkyne led to

formation of substantial amounts of the undesired (Z)-

isomer. Enediyne 16a was then generated by palladium-cat-

alyzed coupling employing conditions previously described

Figure 1. Members of the fostriecin class and IC₅₀values for the inhibi-

tion of protein phosphatase 2A (PP2A).

products might have been synthesized via similar biosynthet-

ic pathways, the configuration of stereocenters 4, 5, 9, and

11 of cytostatin was chosen by analogy. The configuration of

stereocenter 10 was chosen based on the reported coupling

constant (³J=9.4 Hz) between H-10 and H-11.^[9e]Assuming

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2759 2780

with potassium cyanide^[28]were examined and led to overre-

duction and isomerization of 16a and 16b to yield complex

mixtures of compounds. In one case (with Zn/Cu/Ag), two

products 18 and 19 generated from addition of one molecule

1

H₂were identified as main products by H NMR and GC-

MS (Scheme 2). However, further treatment of the mixture

with fresh reagent and heating up led to a mixture of differ-

ent reduced products which could not be further separated.

Scheme 2. Attempted partial reduction of the endiynes 16a,b to the

(Z,Z,E)-trienes: a) Zn/Cu/Ag, MeOH, RT, 14 h; b) Zn/Cu/Ag, MeOH,

558C, 24 h.

Figure 3. Retrosynthesic analysis of (2S,3S,4S,9S,10S,11S)-cytostatin 1a.

As alternative starting material for the hydrogenation di-

enyne 23 was investigated (Scheme 3). (E)-Crotonic alde-

hyde (20) was transformed to dibromoalkene 21 following

Scheme 3. Synthesis and attempted partial reduction of the dienyne 23 to

the (Z,Z,E)-triene 17: a) 2 equiv CBr₄, 4 equiv PPh₃, CH₂Cl₂, 08C, 8 min,

87%; b) 0.04 equiv [Pd(PPh₃)₄], 1.07 equiv Bu₃SnH, THF, room tempera-

ture, 2 h; c) 0.2 equiv 12, 0.11 equiv CuI, DIPEA, THF, RT, 16 h, 36%

(based upon 12).

Scheme 1. Synthesis of the model diynes 16a/b: a) 1.1 equiv NaHMDS,

THF, DMF, 08C, 30 min; 1.2 equiv PMBCl, 0.05 equiv Bu₄N⁺I^ꢀ, room

temperature, 16 h, 67%; b) 0.05 equiv [Pd(PPh₃)₄], 2 equiv NEt₃,

0.1 equiv CuI, THF, RT, 18 h, 69%; c) 1.25 equiv NBS, 0.25 equiv

AgNO₃, DMF, 08C!RT, 4 h; ca. 50%; d) 0.03 equiv [Pd₂(dba)₃],

0.25 equiv CuI, 0.2 equiv LiI, 4.2 equiv 1,2,2,6,6-pentamethylpiperidine,

DMSO, room temperature, 0.66 equiv 11, 19 h, 50% (based upon 11);

e) 0.03 equiv [Pd₂(dba)₃], 0.25 equiv CuI, 0.2 equiv LiI, 4.2 equiv

1,2,2,6,6-pentamethylpiperidine, DMSO, room temperature, 0.66 equiv

12, 18 h, 56% (based upon 12).

the Corey Fuchs protocol.^[29]Selective reduction to (Z)-bro-

moalkene 22 and subsequent Sonogashira reaction with

alkyne 12 yielded the desired dienyne 23. This compound

could not be selectively hydrogenated to triene 17. Rather

overreduced compounds were formed under all reaction

conditions investigated. Similar difficulties were encoun-

tered in an early synthetic study towards fostriecin, although

no detailed description of the results was given.^[30]

by Vasella et al.^[25]The corresponding unprotected enediyne

16b was synthesized similarly from 11 and 14.

Unfortunately, all attempts to generate the corresponding

(Z,Z,E)-triene 17 from 16 failed. Different reaction condi-

tions using the Lindlar catalyst (palladium on charcoal,

lead-poisoned),^[26a]the Rosenmund catalyst (palladium on

barium sulfate),^[26b]freshly prepared Zn/Ag/Cu^[27]and Zn

At this stage we anticipated that a Stille coupling strat-

egy,^[31]which already had been successfully used to build up

similar trienes,^[32]would be a more promising methodology.

For this purpose the dibromoalkene 21 was converted into

the alkynylstannane 24 by the Corey Fuchs protocol

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ingly, the corresponding (Z)-bromoalkene did not react at

all with stannane 25 (not shown).

The p-methoxybenzyl group could serve as a potential

phosphate protecting group in the course of the synthesis.^[35]

Therefore, deprotection of triene 17 was investigated in

order to find conditions for its removal which are compati-

ble with the sensitivity of the triene moiety. Unfortunately

acidic and oxidative conditions examined led to extensive

decomposition of the material, probably by polymerization

of the triene. These results suggest that an acidic cleavage of

protecting groups should be avoided after installation of the

triene moiety.

Scheme 4. Synthesis and attempted deprotection of the triene 17:

a) 2 equiv nBuLi, THF, ꢀ788C, 1 h, RT, 70 min; b) 1.05 equiv Bu₃SnCl,

THF, ꢀ788C!RT, 15 h, 50% over 2 steps; c) 2.25 equiv Cp₂ZrHCl, THF,

RT, 30 min, silica gel, 99%; d) 0.20 equiv AgNO₃, 1.25 equiv NIS, ace-

tone, 4 h; 71%; e) 1.88 equiv potassium azodicarboxylate, 4.32 equiv pyri-

dine, 3.75 equiv HOAc in two portions, methanol, RT, 20 h, 92%;

f) 0.05 equiv [Pd(CH₃CN)₂Cl₂], DMF/THF 28:1, RT, 16 h, 74%;

Synthesis of the C1 C13 fragment of (2S,3S,4S,9S,10S,11S)-

cytostatin: After the general methodology for the synthesis

of the triene moiety had been established the construction

of the C1 C13 fragment was investigated (Scheme 5). Start-

ing from the known alcohol 28 which was obtained by asym-

g) 2.4 equiv DDQ, CH₂Cl₂/H₂O 20:1, RT, 30 min; h) 2 equiv cerium am-

ꢀ

monium nitrate, CH₃CN/H₂O 9:1, 08C, 45 min; i) 2 equiv Ph₃C⁺BF₄

,

DCM/H₂O 5:1, 08C, 5 min; j) CH₂Cl₂/TFA/thioanisol 43:1:1, ꢀ158C,

30 min.

(Scheme 4). Reduction of stan-

nane 24 to (Z)-alkenylstannane

25 was achieved by treatment

with zirconocene hydrochlo-

ride (Schwartz×s reagent).^[33]

Subsequent hydrolysis of the

zirconium species was best car-

ried out by filtration through

silica gel. Aqueous work-up

with saturated ammonium

chloride solution led to sub-

stantial isomerization to the

(E,E)-isomer.

Alkynyliodide

26 was obtained by silver cata-

lyzed iodination of alkyne 12.

Subsequent diimide reduc-

tion^[34]yielded (Z)-alkenyl

iodide 27. Overreduction to

the alkyl iodide could be large-

ly suppressed by adding the re-

agents (potassium azodicarbox-

ylate, acetic acid and pyridine)

in two portions, careful moni-

toring of the reaction by GC-

MS, and termination of the

conversion when the amounts

of alkinyl iodide and alkyl

iodide present were compara-

ble. The Stille coupling be-

tween 25 and 27 was effected

under ligand-free conditions as

described before^[31]and yielded

the desired (Z,Z,E)-triene 17

in high yield and stereoselec-

tively. For this reaction, the ad-

dition of a small amount of

THF as cosolvent proved to be

necessary since stannane 25 is

not soluble in DMF. Interest-

Scheme 5. Synthesis of the C3 C13 fragment with all stereocenters of (all-S)-cytostatin implemented:

a) 1.3 equiv TBSCl, 3 equiv imidazole, DMF, room temperature, 15 h, 100%; b) 1.2 equiv TBDPSCl, 2 equiv

imidazole, DMF, RT, 17 h, 97%; c) 1.1 equiv 9-BBN, THF, 08C, RT, 15 h, 3.5 equiv NaOH, H₂O₂, 24 h, RT,

83% (31a from 30a); d) 1.1 equiv 9-BBN, THF, RT, 19.5 h, 3.5 equiv NaOH, H₂O₂, RT, 24 h, 100% (31b from

30b); e) 2.6 equiv (COCl)₂, 6 equiv DMSO, CH₂Cl₂, ꢀ788C, 150 min, 7.5 equiv NEt₃, ꢀ78!08C (32a from

31a); f) 1.5 equiv DMP, 11.5 equiv NaHCO₃, CH₂Cl₂, 1 h (32b from 31b); g) 1.18 equiv Bu₂BOTf, 1.36 equiv

(iPr)₂NEt, CH₂Cl₂, 08C, 1 h, ꢀ788C, 8, 50 min, RT, 130 min, pH 7, H₂O, 0 8C, methanol, H₂O₂, H₂O, RT,

90 min, 89% over 2 steps (33 from 32a); h) 1.2 equiv Bu₂BOTf, 1.35 equiv (iPr)₂NEt, CH₂Cl₂, 08C, 45 min,

ꢀ788C, 8, RT, 90 min, H₂O₂, pH 7, 43% over 2 steps (34b from 31b); i) 2 equiv TBSCl, 4 equiv NEt₃,

0.1 equiv DMAP, CH₂Cl₂, 08C, 30 min, RT, 15 h, 97%; j) 4.6 equiv Me₃Al, 5 equiv Cl^ꢀ+H₂N(Me)OMe, THF,

ꢀ20!08C, 15 h, 84% (35a from 34a); k) 5 equiv Me₃Al, 5 equiv Cl^ꢀ+H₂N(Me)OMe, THF, ꢀ10!08C, 15 h

(35b from 34b); l) 10 equiv MOMCl, 13 equiv (iPr)₂NEt, CH₂Cl₂, 08C, 1 h, RT, 18 h, 94%; m) 10 equiv

MOMCl, 13 equiv (iPr)₂NEt, CH₂Cl₂, 08C, 1 h, RT, 19 h, 86% over 2 steps; n) 3 equiv BuLi, 6 equiv Me₃SiCꢁ

CH, THF, ꢀ78!ꢀ108C, 75 min (37a from 36a); o) 3 equiv BuLi, 6 equiv Me₃SiCꢁCH, THF, ꢀ78!ꢀ108C,

45 min, 84% (37b from 36b); p) 0.01 equiv Na₂B₄O₇/methanol 5:1, ꢀ108C!RT, 70 min, 95% over 2 steps;

q) 2 equiv 40, 5 equiv BH₃¥Me₂S, THF, ꢀ308C, 1 h, 84% (42a from 38a); r) 1 equiv 39, 1.2 equiv BH₃¥Me₂S,

THF, 08C, 40 min, 71% (41 from 38b); s) 1.1 equiv K₂CO₃, MeOH, 22 h, 84%; t) 1.35 equiv TBDPSCl,

2.5 equiv Imidazole, DMF, 15 h, RT (43a from 42a); u) 1.5 equiv TBDPSCl, 2.5 equiv imidazole, DMF, 21 h,

86% (43b from 42b); v) 1.1 equiv NaHMDS, DMF, 08C, 30 min, 1.2 equiv PMBCl, 0.05 equiv Bu₄N⁺I^ꢀ, RT,

24 h, 30%; w) 70% HF/py/THF 1:10, room temperature, 40 min, 88% over 2 steps.

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metric Evans alkylation followed by reduction,^[17]alcohol 29

was synthesized by oxidation to the corresponding aldehyde,

Evans aldol reaction with 8, MOM protection of the aldol

product and reduction of the acyloxazolidinone.^[14]For pro-

tection of the primary hydroxy function, initially the

TBDPS (tert-butyldiphenylsilyl) group was examined. How-

ever, further progression of the synthesis prompted us to

use TBS (tert-butyldimethylsilyl) group instead (see below).

Therefore, the synthesis of both the TBS and TBDPS pro-

tected compounds is described together to allow for a better

comparison of the results. Protection of alcohol 29 as silyl

ether to give 30a (TBS ether) and 30b (TBDPS ether)

turned out to be straightforward. After hydroboration with

9-BBN followed by oxidative work-up, alcohols 31a and

31b were oxidized by the Swern method^[36](for 32a) or with

the Dess Martin periodinane^[37](for 32b). In our hands this

hypervalent iodine compound gave a higher yield than the

tetrapropylammonium perruthenate catalyzed oxidation de-

veloped by Ley et al.^[38]Next, a syn-selective Evans aldol re-

action with freshly distilled dibutylboryl triflate and H¸nig×s

base was carried out. The commercially available solutions

of dibutylboryl triflate gave unreproducible results. Surpris-

ingly, the TBS group was cleaved off in the course of the re-

action (yielding diol 33), whereas this was not the case for

the TBDPS compound; 34b was obtained in high yield and

with complete stereoselectivity. Fortunately, selective repro-

tection of the primary hydroxy group was possible using

TBSCl, triethylamine and catalytic amounts of DMAP

(N,N-dimethylaminopyridine) in dichloromethane to give

the desired silyl ether 34a in high yield. Standard proce-

dures were used for the conversion of the oxazolidinones

34a,b to the Weinreb amides^[17,39]35a,b and the MOM

ethers 36a,b. The Weinreb amides were then converted to

alkynyl ketones which were suitable for an asymmetric re-

duction to the corresponding alkynols. Initial attempts with

commercial bromomagnesium acetylide were unsuccessful.

Upon treatment with lithium trimethylsilyl acetylide, pre-

pared in situ from n-butyllithium and trimethylsilylacetylene

(10), the TMS-protected alkynones 37a,b were obtained in

high yields. However, varying amounts of the terminal alky-

nones 38a and 38b were detected. These compounds are

probably generated through TMS cleavage by impurities in

the butyllithium solutions used. For example, lithium hy-

droxide could cleave the TMS group from 37a,b.

Next the asymmetric reduction of the ketone at C-11 was

investigated. In initial experiments with Midland×s Alpine

borane^[18a]no conversion of alkynone 37a was observed,

which is probably due to steric hindrance. Reduction with

(R)-phenylglycine-derived oxazaborolidine 39, which has

been reported to reduce TMS-alkynones with high stereose-

lectivity,^[18e]gave varying yields of alkynol 41 (71% in the

best case). However, problems were encountered in the pu-

rification of the product, especially since 41 could hardly be

separated from the chiral ligand by column chromatography.

Fortunately, Corey×s methyl CBS oxazaborolidine 40^[18c,d]

gave good yields of alkynol 42a with complete stereoselec-

1

tivity. The C-13 epimer could not be detected by H NMR

spectroscopy. The TBDPS-analogue 42b could be obtained

via deprotection of TMS-protected alkynol 41 with potassi-

um carbonate in methanol.

Next, the protection of the C-11-hydroxy group was inves-

tigated. The protecting group should be orthogonally stable

to the silyl protecting group of the primary alcohol and

resist the planned acidic deprotection of the MOM groups.

The first candidate which was examined was the PMB

group, which should be more stable under acidic conditions

than the MOM groups.^[41]However, deprotonation of the al-

cohol with a strong base such as sodium hexamethyldisila-

zide (NaHMDS) and subsequent treatment with p-methoxy-

benzyl chloride in the presence of catalytic amounts of tetra-

butylammonium iodide^[42]gave PMB ether 43c in only poor

yield (30%). Under those conditions, migration and cleav-

age of the TBDPS group by the intermediate alcoholate

probably occurred, leading to a mixture of compounds. Pro-

tection with PMB-trichloroacetimidate under various acidic

conditions (0.02 equiv triflic acid in diethyl ether, 0.1 equiv

camphorsulfonic acid in dichloromethane or 0.05 equiv

BF₃¥Et₂O complex in dichloromethane) was only sluggish.

Under forced conditions, the starting compound decom-

posed. An alternative protecting group would be the p-me-

thoxyphenyl group.^[43]However, no reaction was observed

when alcohol 42b was treated with 4-methoxyphenol under

Mitsunobu conditions (PPh₃, DEAD, THF, 908C). Next,

silyl protecting groups were examined. The TIPS (triisopro-

pylsilyl) group should be more stable than the TBDPS

group under basic deprotection conditions^[44]and than the

MOM protecting groups under acidic conditions. Unfortu-

nately, the attempted protection of the C-11 OH group in

42b by treatment with TIPSCl and imidazole in DMF was

not successful even at 908C. A further possibility was of-

fered by the TBDPS group, which should be more stable at

the C-11 position than the primary TBDPS-group at C-3

due to steric hindrance. Gratifyingly and–in the light of the

difficulties encountered with the other protecting groups ex-

amined–quite surprisingly, treatment of 42b with TBDPSCl

and imidazole in DMF gave the bis-silyl ether 43b in 86%

yield.

We therefore searched for a method to convert the mix-

tures of protected and deprotected alkynones to the depro-

tected alkynes. Treatment with potassium carbonate in

methanol led to rapid decomposition of alkynone 37a. Ex-

posure of alkynone 37a to catalytic amounts of borax

(sodium tetraborate) in aqueous methanol^[40]at room tem-

perature surprisingly led to substantial decomposition (up to

50%) via b-elimination of the MOM-protected hydroxy

group. Finally, it was discovered that slow addition of the

aqueous borax solution to a solution of alkynol 37a in meth-

anol precooled to ꢀ108C followed by warming to room tem-

perature, yielded deprotected alkynol 38a in very high yield

and purity. This modified procedure may be generally useful

for deprotection of TMS-alkynones prone to b-elimina-

tion.

The selective mono-deprotection of the bis(TBDPS)-ether

43b turned out to be problematic. Upon treatment with

TBAF, no selectivity was observed. A low selectivity for the

desired TBDPS ether 44 was observed with the HF/pyridine

complex. Surprisingly, a reversed selectivity was observed

with ammonium fluoride in methanol: the secondary

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TBDPS group was cleaved off faster than the primary silyl

ether. This result may be due to the propargylic character of

the C-11-OTBDPS group. To test this hypothesis a diol with

two primary TBDPS-protected hydroxy groups was synthe-

sized (Scheme 6). Hex-5-yn-1-ol (46) was protected as

Completion of the synthesis–Choice of the right phosphate

protecting group: For completion of the synthesis the trans-

formation of the alkyne moiety in 51 into a (Z)-alkenyl

iodide suitable for the planned Stille coupling, the deprotec-

tion and phosphorylation of the C-11-hydroxy group, and

the Stille coupling were considered the major problems to

be solved. It was planned to assemble the triene at a late

stage and to deprotect the phosphotriester at C-11 in the

final step to allow for facile purification of the intermedi-

ates. Since acid-mediated deprotection of model triene 17

led to extensive decomposition of the compound, an acid-

labile phosphate protecting group was ruled out. A Pd⁰

labile protecting group such as the allyl group^[48]would not

be compatible with the Stille coupling. A protecting group

sensitive to hydrogenation such as the benzyl group would

Scheme 6. Selective deprotection of a propargylic silyl ether: a) 1.2 equiv

TBDPSCl, 3 equiv imidazole, DMF, room temperature, 48 h, 95%;

b) 1.2 equiv BuLi, THF, ꢀ408C, 1 h, 3 equiv 1/x(HCHO)_x, ꢀ788C!RT,

5 h, 70%; c) 1.2 equiv TBDPSCl, 3 equiv imidazole, DMF, room temper-

ature, 15 h, 90%; d) 10 equiv NH₄F, MeOH, room temperature, 8 h,

82%.

TBDPS-ether 47, then hydroxy-

methylated to alcohol 48 which

was protected to give the

bis(TBDPS)-ether 49. Treat-

ment of 49 with ammonium

fluoride in methanol yielded

the expected alcohol 48 in a

non-optimized yield of 82%.

These results are in accordance

with previous observations

showing that allylic silyl-pro-

tected alcohols can be depro-

tected selectively in the pres-

ence of other silyl-protected al-

cohols.^[45]

Scheme 7. Synthesis of the lactone 51: a) 1.5 equiv DMP, 11.5 equiv NaHCO₃, CH₂Cl₂, RT, 85 min; b) 2 equiv

(CF₃CH₂O)₂P(O)CH₂C(O)OMe, 4 equiv [18]crown-6, 1.5 equiv KHMDS, THF, ꢀ788C, 35 min, aldehyde,

ꢀ788C, 260 min, 86% over 2 steps; c) 0.5 equiv CBr₄, 2-propanol, 828C, 14 h, 83%.

also not be a good choice due to the presence of the triene

Application of neutral alumina,^[46]which has been report-

ed to deprotect primary silyl ethers selectively, was tested

but failed to deprotect 43b. Finally, it was decided to change

the silyl protecting group at C-3 to the more labile TBS

group. Gratifyingly, the TBS group of compound 43a could

be removed in a highly selective manner by treatment with

HF/pyridine for 40 min with a yield of 88% (over two steps

from 42a).

unit. Therefore, base-labile protecting groups were investi-

gated which should be compatible to all structural features

of the molecule (for a detailed discussion see below). The

first choice was the well-established b-cyanoethyl group.^[49]

Iodination of alkyne 51 to alkynyl iodide 54 (Scheme 8)

could not be effected by treatment of 51 with N-iodosuccini-

mide and catalytic amounts of silver nitrate probably due to

steric hindrance by the adjacent TBDPS group. In contrast,

The synthesis of the lactone fragment of the natural prod-

uct is depicted in Scheme 7. Alcohol 44 was oxidized with

the Dess Martin periodinane. The resulting aldehyde was

submitted to a (Z)-selective Still Gennari olefination to

yield ester 50 in high yield and complete stereoselectivity.

No (E)-isomer was detected. It was planned to synthesize

lactone 51 by selective acid-mediated cleavage of the

MOM-protecting groups and concomitant ring closure.

While treating ester 50 with TMS-bromide for 1 h at ꢀ308C

in dichloromethane yielded deprotected ester 52 along with

starting material, treatment of this mixture with TMS-bro-

mide again at 08C for 40 min gave a 40:60 epimeric mixture

of the bromides 53, probably resulting from a Michael addi-

tion of bromide ion to the unsaturated lactone 51. In con-

trast, heating ester 50 with tetrabromomethane in 2-propa-

nol^[47]led to a clean reaction and a high yield of the desired

lactone 51. In this transformation probably HBr is generated

slowly thereby establishing conditions mild enough to de-

protect MOM groups in the presence of the TBDPS-ether.

Scheme 8. Synthesis of the phosphorylated alkynyl iodide 57: a) I₂,

5 equiv 15 equiv DMAP, toluene, 508C, 3 h, 76%; b) 6.1 equiv (b-CE)₂P-

NiPr₂, 4.45 equiv tetrazole, CH₃CN, 08C!RT, 105 min; 6.1 equiv I₂,

THF/pyridine/H₂O 7:2:1, RT, 5 min, quantitative (starting from 51);

c) 70% HF¥pyridine/THF 17:83, RT, 24 h, 84%; d) 1.5 equiv NIS,

0.15 equiv AgNO₃, DMF, RT, 90 min, 88%.

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2759 2780

treatment with iodine and morpholine in toluene at 508C

for 2.5 h (60% yield) or with iodine and DMAP (76%) was

successful.

sion. Finally, the use of the HF/pyridine complex (70% HF)

in THF led to a high yield of the desired alcohol 56. Iodina-

tion of alkyne 56 with N-iodosuccinimide in the presence of

catalytic amounts of silver nitrate gave a high yield of the

desired iodoalkyne 57.

At this stage of the synthesis it was decided to introduce

the phosphate in order to get access to diverse phosphory-

lated analogues for biological screening. For this purpose

phosphoramidite chemistry was employed.^[51]However,

upon treatment of alcohol 54 with (NCCH₂CH₂O)₂PN(iPr)₂

and tetrazole in acetonitrile and subsequent oxidation with

hydrogen peroxide, surprisingly the deiodinated, phosphory-

lated compound 55 was isolated. Changing the oxidant to

iodine in a pyridine/water solution, also resulted in a clean

conversion to 55 (71% yield). Thus, we speculate that the

phosphoramidite, a phosphorous(iii) reagent, had reduced

the iodoalkyne to the terminal alkyne in the presence of an

acid (in this case tetrazole). To test this hypothesis, a simple

alkynyl iodide was synthesized and exposed to an excess of

triphenylphosphine in the presence of tetrazole in acetoni-

trile. The corresponding terminal alkyne was obtained in

quantitative yield. To our knowledge, this is the first report

on the reduction of a iodoalkyne by phosphorous reagents

such as triphenylphosphine (Scheme 9).

With iodoalkyne 57 at hand, the diimide reduction to the

(Z)-iodoalkene was investigated. Treatment with diazodicar-

boxylate, acetic acid and pyridine in methanol led to a very

low yield along with extensive decomposition of the starting

compound (not shown). This was traced back to reduction

of the lactone double-bond, giving the saturated lactone 62

and probably to nucleophilic attack of hydrazine, which can

be formed by disproportionation of diimide,^[26c]to the a,b-

unsaturated lactone. This would lead to water-soluble com-

pounds. The last reaction was thought to be the main side

reaction, as considerable loss of material was observed

during diimide reduction of 57. Conditions for the diimide

reduction were optimized employing a mixture of model

compounds 59 and 63,^[53]and after variation of the solvent

(CH₃CN, DMSO, dioxane, 2-propanol), equivalents of

HOAc and azodicarboxylate added and reaction time condi-

tions were found under which the desired vinyl iodide 61

was obtained in 44% isolated yield (Scheme 10).

(Z)-Alkenyliodide 61 was then subjected to reaction con-

ditions developed for the synthesis of the model triene 17

(Scheme 11), and bis(b-cyanoethyl)protected cytostatin 66

was obtained in 40% yield by Stille coupling with stannane

25 in the presence of catalytic amounts of tris(dibenzylide-

neacetone)dipalladium.

For the final deprotection of bis-cyanoethyl cytostatin

problems were expected since phosphotriesters are readily

cleaved to phosphodiesters under basic conditions followed

by a much slower cleavage of the second phosphoester

Scheme 9. Reduction of

a

iodoalkyne with triphenylphosphine:

a) 1.2 equiv TBDPSCl, 2 equiv imidazole, DMF, RT, 77%; b) 1.25 equiv

NIS, 0.2 equiv AgNO₃, acetone, RT, 4.5 h; 96%; c) PPh₃, tetrazole,

CH₃CN, quantitative.

Consequently the reaction se-

quence was reversed and

alkyne 51 was treated with

phosphoramidite (b-CE)₂PNiPr₂

and subsequently oxidized to

phosphotriester 55. Because of

the base lability of the b-cya-

noethyl group, the conditions

established for the synthesis of

alkynyliodide 54 could not be

adopted. The C-13-OTBDPS

group was removed before iodi-

nation to reduce the steric hin-

drance. To avoid simultaneous

cleavage of the base-labile

phosphate protecting group,

acidic conditions had to be

used. Ammonium fluoride in

methanol led to the removal of

one b-cyanoethyl group, to give

phosphodiester 60 (Scheme 8).

Treatment of 55 with tetrabutyl-

ammonium fluoride (TBAF) in

the presence of an excess

Scheme 10. Reduction of alkynyl iodide 57 to vinyl iodide 61: a) 2 equiv K^+ꢀOOC N=N COO^ꢀ+K, 4 equiv

HOAc, 2-propanol, RT, 24 h, 44% (61) and 9% (62).

ꢀ

Scheme 11. Stille coupling and attempted deprotection of b-cyanoethyl protected (all-S) cytostatin:

a) 1.3 equiv 25, 0.05 equiv [Pd₂(dba)₃¥CHCl₃], DMF/THF 20:1, RT, 14 h, 40%; b) 8 equiv DBU, 4 equiv

TMSCl, CH₂Cl₂, room temperature, 8 h; c) (N,O)-bis-trimethylsilyl-acetamide, CH₂Cl₂, hn˜, 12 equiv DBU,

(400 equivalents)

of

acetic

acid^[52]resulted in no conver- CH₂Cl₂.

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H. Waldmann and L. Bialy

FULL PAPER

group.^[54]Indeed, conditions for the complete deprotection

of both cyanoethyl esters are harsh^[55]and competing attack

at the unsaturated lactone was feared. To find conditions

compatible with this structural element, model compound

68 was synthesized and exposed to a variety of different

conditions (Scheme 12). However, use of different amines

Scheme 12. Model reactions for the deprotection of bis(b-cyanoethyl)

phosphate triesters under non-nucleophilic conditions: a) 2 equiv tetra-

zole, 2 equiv (NCCH₂CH₂O)₂PNiPr₂, CH₃CN, 3 h, room temperature;

2 equiv I₂, pyridine/H₂O/THF, 5 min, room temperature, 74%.

Scheme 13. Investigation of the 9-fluorenylmethyl group on a C1 C9

fragment of cytostatin: a) 10 equiv MOMCl, 13 equiv (iPr)₂NEt, CH₂Cl₂,

08C, 1 h, room temperature, 13 h; b) 1.2 equiv TBAF, THF, room temper-

ature, 15 h, 90% over 2 steps; c) 1.5 equiv Dess Martin periodinane,

11.5 equiv NaHCO₃, CH₂Cl₂, room temperature, 90 min; d) 2 equiv

(CF₃CH₂O)₂P(O)CH₂C(O)Me, 4 equiv 18-crown-6, 1.5 equiv KHMDS,

THF, ꢀ788C, 35 min, aldehyde, ꢀ788C, 3 h, 80% over 2 steps; e) 1n

HCl, H₂O, THF, 15 h, 608C, 88%; f) 4 equiv (iPr₂)NP(OFm)₂(75),

3 equiv tetrazole, CH₂Cl₂, 270 min, 10 equiv m-CPBA, ꢀ788C, 08C,

90 min; 88%; g) NEt₃/CH₃CN 1:4.8 v/v, room temperature, 18 h, 75%.

(Me₂NEt, tBuNH₂) in different solvents (CH₃OH, 2-propa-

nol, DMF, neat, pyridine, acetonitrile) led to formation of

diester 69. Application of Me₂NEt/H₂O 1:1 for five days

gave the desired phophate 70 in quantitative yield but expo-

sure of 51 to these reaction conditions resulted in conjugate

addition of hydroxide to the enoate and ring opening. Final-

ly, the use of a base (NEt₃, DBU) together with TMSCl (to

temporarily mask the intermediary formed phosphodiester)

developed by Evans et al. in the synthesis of calyculin^[54]was

investigated. Indeed, 68 was converted to 70 when DBU/

TMSCl was applied but under these conditions bis-cya-

noethyl cytostatin 66 was only deprotected once. In the light

of these findings the use of the cyanoethyl phosphate pro-

tecting groups had to be abandoned.

The 9-fluorenylmethyl group was demonstrated by Wata-

nabe et al. to be an efficient base-labile phosphomonoester

protecting group, allowing for complete deprotection of

bis(9-fluorenylmethyl) protected phosphotriesters by means

of an excess of triethylamine under anhydrous conditions.^[56]

The suitability of this protecting group for the completion of

the cytostatin synthesis was investigated with a truncated,

C1 C9 fragment of cytostatin

gave the desired phosphotriester 76. Gratifyingly, treatment

of 76 with an excess of triethylamine in acetonitrile gave

completely deprotected compound 77 in high yield.

Encouraged by these results, the reaction sequence devel-

oped for the synthesis of cytostatin was carried out by analo-

gy (Scheme 14). Phosphorylation of alcohol 51 with phos-

phoramidite 75 had to be performed in a mixture of di-

chloromethane and acetonitrile. Phosphoramidite 75 is only

very poorly soluble in acetonitrile, but in CH₂Cl₂no conver-

sion was observed. TBDPS deprotection of phosphotriester

78 could be effected in high yield by using a higher concen-

tration of HF/pyridine in THF and longer reaction times in

comparison to the b-cyanoethyl protected phosphotriester

55. This lower reactivity is probably due to higher steric hin-

drance due to the presence of the larger neighboring 9-fluo-

(Scheme 13). To this end, alco-

hol 31b was protected as

MOM ether protected to give

71. After cleavage of the

TBDPS group, the alcohol 72

obtained was oxidized to the

corresponding aldehyde which

was immediately subjected to

a Still Gennari olefination to

yield the (Z)-configured unsat-

urated ester 73. Upon heating

in aqueous HCl and THF, lac-

Scheme 14. Final steps of the total synthesis: a) 3 equiv (iPr)₂NP(OFm)₂, 2.7 equiv tetrazole, CH₃CN/CH₂Cl₂

5:4 v/v, RT, 330 min, 3 equiv I₂, THF/pyridine/H₂O 7:2:1 (v/v), 5 min, 95%; b) HF/py/THF 1:4.75 v/v, RT, 24 h,

then 1:2.4, room temperature, 8 h, 82%; c) 1.5 equiv NIS, 0.15 equiv AgNO₃, DMF, 90 min, 100%;

d) 1.73 equiv K⁺(^ꢀOOCN=NCOO^ꢀ)⁺K, 3.47 equiv HOAc, 2-propanol/dioxane 11:1 v/v, 870 min, 63% (81),

(and 21% 80); e) 4.3 equiv (iPr₂)NP(OFm)₂(75), 0.24 equiv [PdCl₂(CH₃CN)₂], DMF/THF 17:1 v/v, RT, 20.5 h,

62%; f) NEt₃/CH₃CN 2:9 v/v, RT, 20 h, quantitative; g) Na⁺-Dowex, MeOH/H₂O 1:1 v/v, 85%.

tone 74 was formed. Phosphor-

ylation with phosphoramidite

(FmO)₂PNiPr₂(75) and subse-

quent oxidation with m-chloro-

perbenzoic acid (mCPBA)

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Cytostatin

2759 2780

renylmethyl groups. The synthesis of iodoalkyne 80 by reac-

tion of 79 with N-iodosuccinimide (NIS) and a catalytic

amount of silver nitrate in DMF was straightforward. The

diimide reduction could not be effected in 2-propanol as de-

veloped for the b-cyanoethyl protected compound 57 due to

the insolubility of iodoalkyne 80 in the solvent. However,

this problem was readily solved by addition of a small

amount of dioxane as cosolvent. Use of 2-propanol/dioxane

(11:1) gave a 63% yield of (Z)-iodoalkene 81 if the reaction

was stopped before the entire starting material had been

consumed (21% 80 recovered). The Stille coupling was per-

formed under ligand-free conditions with stannane 25 and a

catalytic amount of bis(acetonitrile)palladium(ii) chloride

and triene 82 was isolated in 62% yield after reversed-phase

HPLC. Gratifyingly, deprotection of the triester 82 proceed-

ed smoothly under the conditions developed for the truncat-

ed compound 76, that is, with an excess of triethylamine in

acetonitrile. The obtained monotriethylammonium salt was

converted to the sodium salt 1a by ion-exchange filtration

over Dowex resin.

To verify that the relative configuration of

(2S,3S,4S,9S,10S,11S)-cytostatin 1a was chosen correctly, we

made a direct NMR comparison of 1a with a sample of the

isolated natural product^[57]by mixing synthetic and natural

cytostatin in equimolar amounts. ¹H NMR, ³¹P NMR and

¹³C NMR spectroscopic (determined by HSQC) examination

in CD₃OD proved spectroscopic identity of the two com-

pounds. Unfortunately, the sample of the isolated material

provided by Ishizuka was seriously contaminated with im-

purities which could not be removed by reversed-phase

HPLC. The optical rotation of the isolated sample ([a]²_D⁰=

+20 (c=0.265, methanol)) was lower than the value record-

ed for synthetic 1a ([a]²_D⁰=+46 (c=0.265, methanol)). How-

ever, in the light of the NMR spectroscopic identity of the

compounds and the fact that the specific rotation is positive

for both samples we propose that the absolute configuration

of natural cytostatin is (4S,5S,6S,9S,10S,11S).

Scheme 15. Synthesis of cytostatin analogues: a) NEt₃/CH₃CN 1:4.6 v/v,

room temperature, 15 h, 98% (83), 49% (85), quantitative (86, 87), 92%

(89), 85% (90); b) Ac₂O, pyridine, cat. DMAP, 1 h, 60%.

the course of the diimide reduction of 80. It was deprotected

to give compound 89 in order to shed light on the impor-

tance of the electrophilic double bond in the lactone moiety

of the natural product. Finally, the bis(b-cyanoethyl) pro-

tected compound 56 was selectively monodeprotected to

diester 90 by treatment with an excess of triethylamine in

acetonitrile. Investigation of this compound would show if

the phosphate needs to be fully deprotected in order to

ensure PP2A inhibitory activity.

Biological evaluation of cytostatin analogues: The cytostatin

analogues were evaluated by means of in vitro inhibition

assays of the serine-threonine phosphatases of type 2A

(PP2A) and 1 (PP1). These are of particular interest be-

cause cytostatin has been described as a highly selective

PP2A-, but not PP1 inhibitor.^[9d]In addition, the inhibition

of protein tyrosine phosphatases PTP1B (which is consid-

ered as a potential target for diabetes therapy^[58]) and CD45

(a positive regulator of T-cell activation and therefore a po-

tential target for treatment of autoimmune diseases and sup-

pression of graft rejection^[59]), and the dual-specifity phos-

phatase VHR (which dephosphorylates ERK, a member of

the ras signal transduction pathway^[60]) was examined. The

enzymatic activity was determined by hydrolysis of p-nitro-

phenyl phosphate (p-NPP) in standard buffers for PP2A1,

PP1, VHR, PTP1B, and a commercially available peptide

for CD45 (see the Experimental Section).

Synthesis of cytostatin analogues: After successful comple-

tion of the total synthesis a set of analogues of the natural

product was prepared in order to unravel the decisive struc-

tural parameters of cytostatin. To this end, several synthesis

intermediates and model compounds were deprotected.

Since the phosphate moiety of the natural product is indis-

pensable for inhibition of PP2A,^[9d]it was decided to investi-

gate only phosphorylated analogues. The C1 C9 fragment

of cytostatin 77 was synthesized as shown in Scheme 13. The

synthesis of the other derivatives is depicted in Scheme 15.

All fluorenylmethyl-deprotection reactions were carried out

under the conditions described above, that is, with an excess

of triethylamine in acetonitrile and yielded analytically pure

compounds after simple aqueous work up. Alkyne 79, io-

doalkyne 80 and (Z)-iodoalkene 81 were converted to com-

pounds 83, 86 and 87 in very high yields. To evaluate the im-

portance of the C-11-hydroxy function, alcohol 79 was ace-

tylated with acetic anhydride in the presence of catalytic

amounts of DMAP in pyridine to give acetate 84. Deprotec-

tion yielded compound 85 acetylated at C-11. The saturated

lactone 88 was formed in small amounts as by-product in

Under the conditions described by Ishizuka et al.,^[9d]only

low enzymatic activity of PP2A in the hydrolysis of p-NPP

was observed. A much higher activity could be obtained by

using a different buffer at a higher pH value (8.1).^[61]The in-

hibition curves determined for the inhibition of PP2A₁are

shown in Figure 4, the determined IC₅₀values are given in

Table 1. Synthetic cytostatin 1a displays an IC₅₀value of

33 nm which is about seven times smaller than the value re-

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FULL PAPER

Figure 5. Structure activity relationship for cytostatin (PP2A₁inhibition).

ured double bond with a hydrophobic group attached (here

an iodine) seems to be sufficient for high PP2A-inhibitory

activity.

Clinical phase I trials of fostriecin have been stopped by

the NCI due to impurities in the natural product samples.^[62]

Most probably the triene moiety in fostriecin and cytostatin

is responsible for the instability of the natural products

upon storage. Thus, the finding

~

&

*

Figure 4. PP2A₁inhibition by cytostatin analogues 1a ( ), 83 ( ), 86 ( ),

!

^

89 ( ), and 90 ( ).

Table 1. IC₅₀values [mm] for inhibition of different phosphatases with cytostatin analogues.^[c]

that it may be replaced by sim-

pler structural elements with-

out loss of biological activity is

of particular importance for

the design of more stable but

still active analogues.

Notably protein phosphatase

1 (PP1) was not inhibited by

any of the cytostatin analogues

in a p-NPP based assay identi-

cal to the PP2A assay

(Table 1). This demonstrates

Entry

Compound

PP2A₁

PP1

VHR

PTP1B

CD45

1

2

3

4

5

6

7

8

1a

77

83

85

86

87

89

90

0.033ꢂ0.003

>20^[a]

>100^[a]

>20^[a]

>100^[a]

>20^[a]

>100^[a]

>20^[a]

>100^[a]

n.i.^[b]

>100^[a]

0.37ꢂ0.05

>100^[a]

0.079ꢂ0.009

0.039ꢂ0.004

ca. 100

n.i.^[b]

42ꢂ7

n.i.^[b]

[a] Highest concentration investigated. [b] Not investigated. [c] IC₅₀values were calculated on the basis of at

least three determinations.

ported for the isolated compound. Two reasons can be in-

voked to explain this deviation: 1) We used an enzyme from

a different source than Ishizuka et al. (see Experimental

Section), 2) we used different assay conditions (buffer, pH

value) than Ishizuka et al. The truncated cytostatin analogue

77 (C1 C9) is totally inactive (entry 2). The shortened frag-

ments of the natural product show a lower activity against

PP2A, albeit still in the submicromolecular range. It is inter-

esting to note that alkyne 83 is less active than the more hy-

drophobic iodoalkyne 86. This analogue in turn is less active

than the (Z)-iodoalkene 87 which is nearly as potent as the

natural product itself (entries 3, 5 and 6). The acetylated de-

rivative 85 is totally inactive (entry 4), while saturated lac-

tone 89 and phosphodiester 90 show very weak activities

with IC₅₀values in the high micromolar range.

From these results, the basic structure activity relation-

ship shown in Figure 5 can be derived. The unsaturated lac-

tone is necessary for the biological activity of the natural

product. It is prudent to speculate that covalent modifica-

tion of the enzyme may take place, for example by nucleo-

philic attack of a cysteine residue in a Michael-type reac-

tion. Furthermore, the phosphate group must be fully depro-

tected, suggesting that a very tight interaction of the phos-

phate with the enzyme is required for inhibition. The C-11-

hydroxy group seems to be essential for activity, as suggest-

ed by the lack of inhibition by the truncated compound 77

and the acetylated compound 85. The triene moiety is not

essential for PP2A inhibition. The presence of a (Z)-config-

that variation of the structure of the natural product can be

carried out without compromising the high selectivity descri-

bed for the natural products of the fostriecin class. Further-

more VHR, PTP1B and CD45 were also not inhibited by

any of the analogues. Thus, biological probes derived from

cytostatin promise to be highly selective tools for biological

investigations, for example in chemical proteomics studies.

Conclusion

We accomplished the first total synthesis of the PP2A-inhib-

itor cytostatin, thereby establishing its relative and absolute

stereochemistry. The natural product was obtained in a 24-

step sequence with an overall yield of 4.1% starting from

the known and easily accessible alcohol 28. The synthesis se-

quence was employed successfully to generate several ana-

logues of the natural product. Assaying the phosphatase-in-

hibiting activity of these analogues revealed the importance

of the unsaturated C-2,C-3 double bond, the phosphate, and

the C-11-hydroxy group for biological activity of cytostatin,

whereas the triene moiety could be replaced by other lipo-

philic groups. Our results demonstrate that highly selective

phosphatase inhibitors with enhanced properties are likely

to emerge from the synthesis of analogues of the natural

products of the fostriecin class. Such compounds could serve

as new molecular tools for the study of cellular phosphoryla-

tion processes.

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Chem. Eur. J. 2004, 10, 2759 2780

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2759 2780

and AgNO₃(10 mg, 58 mmol) were added and the mixture was stirred at

room temperature for 2 h. The reaction mixture was quenched by addi-

tion of ice-cold water (10 mL) and extracted with pentane (10 mL). The

combined organic layers were washed with brine (5 mL), then dried

(Na₂SO₄) and concentrated (500 mbar) to yield crude bromoalkyne

(26 mg) as a slightly yellow oil which was used immediately in the next

step without purification. R_f=0.9 (pentane); ¹H NMR (400 MHz,

CDCl₃): d = 6.21 (dq, J=16.1, 7.0 Hz, 1H), 5.46 (dq, J=16.1, 2.0 Hz,

1H), 1.77 (dd, J=7.0, 2.0 Hz, 3H); MS (EI, 70 meV): m/z (%): 146, 144

(100) [M]⁺, 65 (89) [MꢀBr]⁺.

1-[(E)-1-Ethyl-oct-6-ene-2,4-diynyloxymethyl]-4-methoxy-benzene (16a):

A solution of alkyne 12 (56 mg, 0.28 mmol) and bromoalkyne 15 (60 mg,

0.41 mmol) in degassed DMSO (3 mL, and 2î0.5 mL washings) and

1,2,2,6,6-pentamethylpiperidine (0.21 mL, 1.18 mmol) were added to a

mixture of CuI (14 mg, 0.07 mmol), LiI (8 mg, 0.06 mmol) and di-palladi-

um-tris-(dibenzylideneacetone)-chloroform complex (9 mg, 9 mmol). The

reaction mixture was stirred in the dark for 17 h at room temperature.

The reaction mixture was quenched with saturated aqueous NH₄Cl

(5 mL) and extracted with diethyl ether (2î10 mL). The combined or-

ganic layers were washed with water (2î5 mL), brine (2î5 mL), then

dried (Na₂SO₄), filtered through a short pad of silica gel and concentrat-

ed. The residue was purified by flash chromatography (silica gel, cyclo-

hexane/ethyl acetate 1:1) to give endiyne 16a (40 mg, 56%) as a yellow

oil. R_f=0.26 (cyclohexane/ethyl acetate 40:1); ¹H NMR (400 MHz,

CDCl₃): d = 7.28 (d, J=8.8 Hz, 2H), 6.88 (d, J=8.8 Hz, 2H), 6.35 (dq,

J=15.8, 7.0 Hz, 1H), 5.55 (dqd, J=15.8, 1.8, 0.8 Hz, 1H), 4.72 (d, J=

11.3 Hz, 1H), 4.43 (d, J=11.3 Hz, 1H), 4.08 (t, J=6.5 Hz, 1H), 3.80 (s,

3H), 1.83 (dd, J=6.8, 2.0 Hz, 3H), 1.82 1.70 (m, 2H), 1.00 (t, J=7.4 Hz,

3H); ¹³C NMR (100 MHz, CDCl₃): d = 159.4, 144.1, 130.0, 129.8, 113.9,

109.8, 81.1, 72.0, 71.2, 70.7, 70.5, 70.2, 55.4, 29.0, 19.1, 9.9; HRMS (FAB,

3-NBA): m/z: calcd for C₁₈H₂₀O₂: 268.1463, found 268.1473 [M]⁺.

Experimental Section

General: All reactions were carried out under an argon atmosphere. All

solvents were distilled using standard procedures before use. THF was

distilled under argon from molten potassium. Diethyl ether was distilled

under argon from molten Na/K alloy. CH₂Cl₂was distilled under argon

from CaH₂. Dibutylboryltriflate was synthesized as previously descri-

bed.^[64]All other chemicals were purchased from commercial sources and

used without further purification. Yields refer to isolated and pure com-

pounds unless otherwise stated. ¹H and ¹³C NMR data were recorded on

a Varian Mercury 400 spectrometer. The following abbreviations are

used to explain the multiplicities: s=singlet, d=doublet, t=triplet, q=

quartet, quint=quintet, sext=sextet, sept=septet, m=multiplet, brs=

broad signal. FAB and EI measurements were taken with a Jeol SX

102A using a 3-nitrobenzyl alcohol (3-NBA) matrix. GC-MS analysis was

performed on a Hewlett Packard HP 5890-Series II gaschromatograph

with a HP 5972-series mass selective detector. HPLC purifications were

performed using a Varian Pro Star machine with a Varian detector model

340. Optical rotations were measured with a Perkin Elmer Polarimeter

341. Flash chromatography was performed using silica gel from Merck

(silica gel 60). TLC was performed with aluminium-backed silica gel 60

F₂₅₄plates (Merck) using UV as a visualizing agent and a 0.5% aqueous

potassium permanganate solution or an ethanolic solution of phosphomo-

lybdic acid and heat as developing agents. Melting points were recorded

on a B¸chi B-540 apparatus and are uncorrected. Enzymatic assays were

measured on a dynatech MR 5000 photometer with Roth polysterene mi-

crotiter plates. The enzymes were purchased from Calbiochem (PP2A₁

(bovine kidney), PP1 (a-isoform, rabbit muscle, recombinant)), Biomol

(CD45 (human, recombinant) tyrosine phosphatase assay kit, PTP1B

(human, recombinant), VHR (human, recombinant)). Merck silica gel

(60, partical size 40 63 mm) and Merck aluminium oxide (type 506C)

were used for flash column chromatography.

(E)-Dec-8-ene-4,6-diyn-3-ol (16b): Endiyne 16b was synthesized by anal-

ogy to 16a starting from pent-1-yn-3-ol (11) (49 mL, 0.57 mmol) to yield a

brown oil (42 mg, 0.28 mmol). R_f=0.4 (pentane/diethyl ether 5:1);

¹H NMR (400 MHz, CDCl₃): d = 6.33 (dq, J=15.8, 7.0 Hz, 1H), 5.53

(dqd, J=15.8, 1.8, 0.8 Hz, 1H), 4.41 (t, J=6.3 Hz, 1H), 1.82 (dd, J=6.8,

1.8 Hz, 3H), 1.79 1.70 (m, 2H), 1.01 (t, J=7.4 Hz, 3H); ¹³C NMR

(100 MHz, CDCl₃): d = 144.3, 109.7, 82.2, 77.9, 71.7, 69.9, 64.4, 30.9,

19.0, 9.5; HRMS (EI, 70 meV): m/z: calcd for C₁₀H₁₂O: 148.0888; found:

148.0880 [M+H]⁺.

1-(1-Ethyl-prop-2-ynyloxymethyl)-4-methoxy-benzene (12):

A

1m

NaHMDS solution in THF (1.31 mL, 1.31 mmol) was added dropwise at

08C to a solution of pent-1-yn-3-ol (100 mg, 1.19 mmol) in DMF (2 mL)

and the mixture was stirred for 30 min at 08C. 4-Methoxybenzylchloride

(0.194 mL, 1.43 mmol) and tetrabutylammonium iodide (22 mg,

0.06 mmol) were added at 08C and the mixture was stirred for 16 h at

room temperature. The reaction mixture was quenched with 1m aqueous

KH₂PO₄(10 mL) and extracted with diethyl ether (3î20 mL). The com-

bined organic layers were washed with brine (10 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, pentane/diethyl ether 40:1 to 25:1) to give PMB-ether

12 (163 mg, 67%) as a colourless oil. R_f=0.86 (pentane/diethyl ether

5:1); ¹H NMR (400 MHz, CDCl₃): d = 7.30 (d, J=8.6 Hz, 2H), 6.89 (d,

J=8.6 Hz, 2H), 4.74 (d, J=11.4 Hz, 1H), 4.46 (d, J=11.4, 1H), 4.00 (dt,

J=2.0, 6.5 Hz, 1H), 3.81 (s, 3H), 2.47 (d, J=2.0 Hz, 1H), 1.81 1.72 (m,

2H), 1.02 (t, J=7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 159.4,

130.1, 129.8, 113.9, 83.0, 73.9, 70.2, 69.5, 55.4, 28.9, 9.7; HRMS (EI): calcd

for C₁₃H₁₆O₂: 204.1150; found: 204.1134 [M]⁺.

(E)-1,1-Dibromopenta-1,3-diene (21): The reaction was carried out in the

dark. CDCl₃was filtered through a layer of basic alumium oxide before

use. To a solution of (E)-crotonic aldehyde (20) (0.41 mL, 5 mmol) and

tetrabromomethane (3.32 g, 10 mmol) in CH₂Cl₂was added PPh₃(5.24 g,

20 mmol) in five portions at 08C over a period of 3 min and the bright

orange mixture was stirred for 8 min at 08C. The reaction mixture was di-

luted with pentane (100 mL), filtered through a layer of neutral alumini-

um oxide and carefully concentrated (100 mbar, room temperature). The

residue was purified by flash chromatography (neutral aluminium oxide,

pentane) to give dibromoalkene 21 (980 mg, 87%) as a yellow oil which

was immediately used for the next step due to its pronounced lability.

1

(E)-Trimethyl-pent-3-en-1-ynyl-silane

(14):

[Pd(PPh₄)]

(578 mg,

R_f=0.9 (pentane); H NMR (400 MHz, CDCl₃): d = 6.89 (d, J=10.0 Hz,

0.5 mmol) and copper iodide (190 mg, 1 mmol) were added at 08C to a

solution of (E)-2-bromopropene (0.86 mL, 10 mmol), triethylamine

(2.78 mL, 20 mmol), trimethylsilylacetylene (2.08 mL, 15 mmol), in de-

gassed THF (100 mL). The mixture was stirred for 15 h at 08C. The reac-

tion mixture was quenched with saturated aqueous NH₄Cl (5 mL) and ex-

tracted with diethyl ether (2î50 mL). The combined organic layers were

washed with brine (40 mL), then dried (Na₂SO₄) and concentrated. The

residue was purified by flash chromatography (silica gel, pentane) and

distilled (30 mbar, 808C) to give TMS-alkyne 14 (960 mg, 69%) as a col-

ourless oil. R_f=0.72 (pentane); ¹H NMR (400 MHz, CDCl₃): d = 6.22

(dq, J=16.1, 7.0 Hz, 1H), 5.51 (dq, J=16.1, 2.0 Hz, 1H), 1.78 (dd, J=7.0,

2.0 Hz, 3H), 0.17 (s, 9H); MS (EI, 70 meV): m/z (%): 138 (18) [M]⁺, 123

(100) [MꢀCH₃]⁺. The analytical data are in agreement with the litera-

ture.^[63]

1H), 6.14 6.07 (m, 1H), 5.92 (dq, J=15.1, 6.7 Hz, 1H), 1.77 (dd, J=1.5,

6.7 Hz, 3H). The analytical data are in agreement with the literature.^[22]

1-[(4Z,6E)-1-Ethylocta-4,6-dien-2-ynyloxymethyl]-4-methoxy-benzene

(23): Tri-n-butylstannane (2.0 mL, 7.7 mmol) was added dropwise to a

solution of dibromoalkene 22 (1.65 g, 7.3 mmol) and tetrakis-(triphenyl-

phosphine)-palladium (337 mg, 0.29 mmol) in degassed THF (70 mL) and

the reaction mixture was stirred for 1 h at room temperature. Another

portion of tri-n-butylstannane (0.1 mL, 0.385 mmol) was added dropwise

and the reaction solution was stirred for 1 h at room temperature. The re-

action progress was carefully monitored by GC-MS. To the reaction solu-

tion were added a solution of alkyne 12 (298 mg, 1.46 mmol) in degassed

THF (2 mL), degassed EtNiPr₂(7.5 mL) and CuI (150 mg, 0.79 mmol)

and the mixture was stirred for 16 h at room temperature. The mixture

was quenched with saturated NH₄Cl (250 mL) and extracted with diethyl

ether (300 mL). The combined organic layers were washed with brine

(2î50 mL), then dried (Na₂SO₄) and concentrated. The residue was puri-

fied by flash chromatography (silica gel, pentane to pentane/diethyl ether

10:1) to give dienyne 23 (142 mg, 36% over 2 steps) as a yellow oil. R_f=

(E)-1-Bromopent-3-en-1-yne (15): Recrystallized N-bromosuccinimide

(70 mg, 0.39 mmol) and AgNO₃(6 mg, 35 mmol) were added at 08C to a

solution of TMS-alkyne 14 (50 mg, 0.36 mmol) in DMF (2.5 mL) and the

mixture was stirred at 08C for 2 h and room temperature for 1 h. A

second portion of recrystallized N-bromosuccinimide (10 mg, 0.06 mmol)

0.30 (pentane); ¹H NMR (400 MHz, CDCl₃): d

=

7.32 (d, J=8.8 Hz,

2769

Chem. Eur. J. 2004, 10, 2759 2780

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Waldmann and L. Bialy

FULL PAPER

2H), 6.89 (d, J=8.8 Hz, 2H), 6.66 6.58 (m, 1H), 6.37 (t, J=10.6 Hz,

1H), 5.92 (dq, J=15.1, 7.0 Hz, 1H), 5.38 (d, J=10.8 Hz, 1H), 4.77 (d, J=

11.4 Hz, 1H), 4.49 (d, J=11.4 Hz, 1H), 4.20 (dt, J=1.8, 6.5 Hz, 1H), 3.81

(s, 3H), 1.86 1.78 (m, 5H), 1.04 (t, J=7.4 Hz, 3H); ¹³C NMR (100 MHz,

CDCl₃): d = 159.3, 140.5, 133.6, 130.4, 129.7, 129.2, 113.9, 106.3, 94.0,

83.2, 70.4, 70.2, 55.4, 29.2, 18.6, 10.0; HRMS (FAB, 3-NBA): m/z: calcd

for C₁₆H₁₇O₂: 241.1229, found 241.1268 [MꢀC₂H₅]⁺.

washed with saturated aqueous NaHCO₃(30 mL), brine (30 mL), dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 30:1) to give alkenyliodide 27

(576 mg, 92%) as a yellow oil. R_f=0.30 (cyclohexane/ethyl acetate 30:1);

¹H NMR (400 MHz, CDCl₃): d = 7.28 (d, J=8.7 Hz, 2H), 6.87 (d, J=

8.7 Hz, 2H), 6.47 (dd, J=7.8, 1.0 Hz, 1H), 6.19 (t, J=7.8 Hz, 1H), 4.51

(d, J=11.3 Hz, 1H), 4.34 (d, J=11.3 Hz, 1H), 4.05 (ddt, J=7.2, 1.0,

7.0 Hz, 1H), 3.81 (s, 3H), 1.75 1.53 (m, 2H), 0.95 (t, J=7.4 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃): d = 159.1, 142.1, 130.6, 129.5, 113.8, 84.0,

82.0, 70.5, 55.5, 27.8, 9.9; HRMS (FAB, 3-NBA): m/z: calcd for

C₁₃H₁₇IO₂: 332.0273, found 332.0285 [M]⁺.

(E)-Tributyl-pent-3-en-1-ynyl-stannane (24): CDCl₃was filtered through

a layer of basic alumium oxide before use. Silica gel was pretreated with

acetone/pyridine 1:1 and air-dried before use. A 2.5m BuLi solution in

hexanes (3.31 mL, 8.28 mmol) was added at ꢀ788C over a period of

45 min to a solution of dibromoalkene 21 (936 mg, 4.14 mmol) in THF

(20 mL). The mixture was stirred for 1 h at ꢀ788C and for 70 min at

room temperature. The mixture was recooled to ꢀ788C, tributylchloros-

tannane (1.18 mL, 4.35 mmol) was added dropwise at ꢀ788C and the

mixture was stirred for 15 h at room temperature. The reaction mixture

was quenched with saturated aqueous NH₄Cl and the mixture was ex-

tracted with pentane (50 mL). The combined organic layers were washed

with brine (50 mL), then dried (Na₂SO₄) and concentrated at room tem-

perature. The residue was purified by flash chromatography (silica gel,

cyclohexane) to alkinylstannane 24 (729 mg, 50%) as a colourless oil.

R_f=0.95 (pentane/diethyl ether 10:1); ¹H NMR (400 MHz, CDCl₃): d =

6.15 (dq, J=15.8, 6.8 Hz, 1H), 5.53 (dq, J=15.8, 2.0 Hz, 1H), 1.76 (dd,

J=6.8, 2.0 Hz, 1H), 1.60 1.52 (m, 6H), 1.33 (sext, J=7.8 Hz, 6H), 1.01

0.97 (m, 6H), 0.90 (t, J=7.4 Hz, 9H); ¹³C NMR (100 MHz, CDCl₃): d =

139.6, 111.6, 109.0, 91.3, 29.0, 27.1, 18.6, 13.8, 11.2; HRMS (FAB, 3-

NBA): m/z: calcd for C₁₄H₂₃Sn: 299.0822; found: 299.0808 [MꢀC₄H₉]⁺.

1-[(2Z,4Z,6E)-1-Ethylocta-2,4,6-trienyloxymethyl]-4-methoxy-benzene

(17): The reaction was carried out in the dark. CDCl₃was filtered

through a layer of basic alumium oxide before use. To a solution of alke-

nyliodide 27 (137 mg, 0.412 mmol) and alkenylstannane 25 (294 mg,

0.824 mmol) in degassed DMF (7 mL) and degassed THF (0.25 mL) was

added bis(acetonitrile)-palladium(ii)chloride (10 mg) and the mixture was

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

with 10% aqueous NH₃(5 mL) and water (25 mL) and extracted with di-

ethyl ether (3î30 mL). The combined organic layers were washed with

saturated aqueous 1m aqueous KH₂PO₄(20 mL), brine (20 mL), then

dried (Na₂SO₄) and concentrated. The residue was purified by flash chro-

matography (silica gel, pentane/diethyl ether 50:1 to 10:1) to give triene

17 (83 mg, 74%) as a yellow oil. R_f=0.28 (cyclohexane/ethyl acetate

1

30:1); H NMR (400 MHz, CDCl₃): d = 7.25 (d, J=8.6 Hz, 2H), 6.86 (d,

J=8.6 Hz, 2H), 6.67 (t, J=10.6 Hz, 1H), 6.60 6.52 (m, 1H), 6.08 5.97

(m, 2H), 5.80 (dq, J=14.7, 6.8 Hz, 1H), 5.38 (t, J=9.6 Hz, 1H), 4.51 (d,

J=11.5 Hz, 1H), 4.27 (d, J=11.5 Hz, 1H), 4.18 (q, J=6.8 Hz, 1H), 3.80

(s, 3H, OCH₃), 1.83 (dd, J=6.8 Hz, J=1.4 Hz, 1H), 1.78 1.43 (m, 2H),

0.89 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 159.2, 132.7,

132.0, 131.1, 131.1, 129.5, 126.8, 126.7, 121.8, 113.8, 75.1, 69.8, 55.4, 28.8,

18.6, 10.0; MS (EI, 70 meV): m/z (%): 272 (2) [M]⁺, 121 (100) [C₈H₉O]⁺.

(1Z,3E)-Tributyl-penta-1,3-dienyl-stannane (25): The reaction was carried

out in the dark. CDCl₃was filtered through a layer of basic alumium

oxide before use. A solution of alkinylstannane 24 (40 mg. 0.113 mmol)

in THF (0.5 mL, and 0.5 mL washing) was added to a mixture of zircono-

cene hydrochloride (65 mg, 0.254 mmol) in THF (0.75 mL). The orange

mixture was stirred for 30 min at room temperature, diluted with pentane

(5 mL), stirred for another 35 min at room temperature, filtered (silica

gel, 7 cm high pad, pentane/diethyl ether 30:1) and concentrated at room

temperature to yield crude alkenylstannane 25 (40 mg, 99%) as a slightly

yellow oil. Compound 25 was immediately used in the next step because

of its marked lability. R_f=0.95 (pentane/diethyl ether 10:1); ¹H NMR

(400 MHz, CDCl₃): d = 7.02 (dd, J=12.8, 10.4 Hz, 1H), 6.01 5.93 (m,

1H), 5.90 (dd, J=12.8, 0.6 Hz, 1H), 5.72 (dq, J=14.9, 6.6 Hz, 1H), 1.78

(dd, J=6.6, 1.6 Hz, 1H), 1.55 1.47 (m, 6H), 1.32 (sext, J=7.4, 6H), 0.96

0.92 (m, 6H), 0.89 (t, J=7.2 Hz, 9H); ¹³C NMR (100 MHz, CDCl₃): d =

146.7, 134.7, 131.2, 130.8, 29.3, 27.4, 18.4, 13.8, 10.6; MS (EI, 70 meV):

m/z (%): 357.0 (73) [MꢀH]⁺, 301 (100) [MꢀC(CH₃)₃]⁺, 245 (27)

[MꢀC₈H₁₇]⁺, 187 (26) [MꢀC₁₂H₂₇]⁺.

tert-Butyl-[(2S,3S,4S)-3-methoxymethoxy-2,4-dimethyl-hept-6-enyloxy]-

dimethylsilane (30a): tert-Butylchlorodimethylsilane (4.26 g, 28.3 mmol)

was added to a solution of alcohol 29 (4.09 g, 20.2 mmol) and imidazole

(4.26 g, 28.3 mmol) in DMF (25 mL) and the mixture was stirred for 15 h

at room temperature. The reaction mixture was quenched with brine

(150 mL) and extracted with diethyl ether (3î300 mL). The combined

organic layers were washed with brine (100 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, pentane/diethyl ether 50:1 to 10:1) to give TBS ether 30a

(6.46 g, quantitative) as a colourless oil. R_f=0.50 (pentane/diethyl ether

50:1); [a]²_D⁰=+7.65 (c=1.15, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d =

5.79 (dddd, J=17.2, 10.2, 8.0, 6.1 Hz, 1H), 5.05 4.98 (m, 2H), 4.68 (d,

J=6.5 Hz, 1H), 4.62 (d, J=6.5 Hz, 1H), 3.50 (dd, J=9.9, 6.3 Hz, 1H),

3.45 (dd, J=9.9, 7.8 Hz, 1H), 3.40 (dd, J=7.4, 2.7 Hz, 1H), 3.39 (s, 3H),

2.43 2.36 (m, 1H), 1.90 1.80 (m, 2H), 1.80 1.70 (m, 1H), 0.89 (s, 9H),

0.86 (d, J=6.8 Hz, 3H), 0.86 (d, J=6.6 Hz, 3H), 0.04 (s, 6H); ¹³C NMR

(100 MHz, CDCl₃): d = 137.9, 115.9, 98.6, 83.2, 66.0, 56.1, 37.8, 37.6,

36.0, 26.1, 18.4, 16.1, 10.9, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd

for C₁₇H₃₇O₃Si: 317.2512; found: 317.2521 [M+H]⁺.

1-(1-Ethyl-3-iodo-prop-2-ynyloxymethyl)-4-methoxy-benzene

(26):

AgNO₃(166 mg, 0.98 mmol) was added in the dark to a solution of

alkyne 13 (1.00 g, 4.90 mmol) and N-iodosuccinimide (1.38 g, 6.12 mmol)

in acetone (40 mL) and the mixture was stirred for 4 h at room tempera-

ture. The reaction mixture was quenched with ice-cold water (100 mL)

and extracted with ethyl acetate (3î150 mL). The combined organic

layers were washed with brine (100 mL), dried (Na₂SO₄) and concentrat-

ed. The residue was purified by flash chromatography (silica gel, cyclo-

hexane/ethyl acetate 100:1 to 30:1) to give iodoalkyne 26 (1.43 mg, 71%)

(2S,3S,4S)-7-(tert-Butyldimethylsilyloxy)-5-methoxymethoxy-4,6-dime-

thylheptan-1-ol (31a): A 0.5m solution of 9-BBN in THF (44.9 mL,

22.5 mmol) was added dropwise to a solution of alkene 30a (6.46 g,

20.4 mmol) in THF (67 mL) and the mixture was stirred for 15 h at room

temperature. The mixture was cooled to 08C, 3m aqueous NaOH (24 mL,

72 mmol) was added dropwise and the mixture was stirred for 24 h at

room temperature. The reaction mixture was diluted with water

(100 mL) and extracted with ethyl acetate (3î160 mL). The combined

organic layers were washed with brine (100 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, cyclohexane/ethyl acetate 5:2) to give alcohol 31a (5.67 g,

as

a

yellow oil. R_f=0.77 (cyclohexane/ethyl acetate 30:1); ¹H NMR

(400 MHz, CDCl₃): d = 7.28 (d, J=8.6 Hz, 2H), 6.88 (d, J=8.6 Hz, 2H),

4.72 (d, J=11.3 Hz, 1H), 4.43 (d, J=11.3 Hz, 1H), 4.12 (dt, J=6.5 Hz,

1H), 3.81 (s, 3H), 1.78 1.71 (m, 2H), 0.99 (t, J=7.4 Hz, 3H); ¹³C NMR

(100 MHz, CDCl₃): d = 159.2, 129.8, 129.6, 113.8, 94.1, 71.0, 70.3, 55.3,

28.9, 9.7, 1.4; HRMS (FAB, 3-NBA): m/z: calcd for C₁₃H₁₅INaO₂:

353.0014; found: 352.9987 [M+Na]⁺.

1-[(Z)-1-Ethyl-3-iodo-allyloxymethyl]-4-methoxy-benzene (27): Acetic

acid (0.27 mL, 4.73 mmol) over a period of 1 h was added to a mixture of

alkynyl iodide 26 (624 mg, 1.89 mmol), potassium azodicarboxylate

(459 mg, 2.36 mmol) and pyridine (0.45 mL, 5.44 mmol) in methanol

(7.9 mL) and the mixture was stirred for 5 h at room temperature. An-

other portion of potassium azodicarboxylate (230 mg, 1. 8 mmol) and

acetic acid (0.14 mL, 2.36 mmol) were added and the mixture was stirred

for further 15 h at room temperature. The pH of the reaction mixture

was adjusted to pH 3 with 1m aqueous HCl and the solution was extract-

ed with diethyl ether (3î30 mL). The combined organic layers were

83%) as a colourless oil. R_f=0.45 (cyclohexane/ethyl acetate 2:1); [a]_D²⁰

=

ꢀ4.9 (c=0.59, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 4.67 (d, J=

6.5 Hz, 1H), 4.62 (d, J=6.5 Hz, 1H), 3.66 3.60 (m, 2H), 3.50 3.39 (m,

3H), 3.38 (s, 3H), 1.88 1.79 (m, 1H), 1.73 1.57 (m, 4H), 1.55 1.45 (m,

1H), 1.23 1.14 (m, 1H), 0.88 (s, 9H), 0.88 (d, J=6.8 Hz, 3H), 0.84 (d,

J=6.8 Hz, 3H), 0.03 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d = 98.6,

83.3, 66.1, 63.4, 56.1, 37.6, 35.8, 30.5, 28.9, 26.0, 18.4, 16.4, 11.0, ꢀ5.2,

ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd for C₁₇H₃₈NaO₄Si: 357.2437;

found: 357.2435 [M+Na]⁺.

2770

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 2759 2780

Cytostatin

2759 2780

tert-Butyl-[(2S,3S,4S)-3-methoxymethoxy-2,4-diphenyl-hept-6-enyloxy]-

dimethyl-silane (30b): TBDPS ether 30b was synthesized by analogy to

30a starting from alcohol 28 (1.06 g, 5.54 mmol) to yield a colourless oil

(2.02 g, 97%). R_f=0.28 (cyclohexane/ethyl acetate 40:1); [a]²_D⁰=+10.0

(c=0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 7.71 7.66 (m, 4H),

7.46 7.36 (m, 6H), 5.79 (dddd, J=16.6, 10.0, 8.0, 6.0 Hz, 1H), 5.06 5.00

(m, 2H), 4.71 (d, J=6.5 Hz, 1H), 4.63 (d, J=6.5 Hz, 1H), 3.61 (dd, J=

10.0, 8.0 Hz, 1H), 3.54 3.49 (m, 2H), 3.34 (s, 3H), 2.44 2.37 (m, 1H),

1.96 1.82 (m, 2H), 1.82 1.71 (m, 1H), 1.08 (s, 9H), 0.88 (d, J=6.5 Hz,

3H), 0.86 (d, J=7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 137.7,

135.6, 133.9, 129.6, 127.6, 115.9, 98.5, 83.2, 66.8, 56.1, 37.7, 37.7, 36.0, 27.1,

19.6, 16.3, 11.0; HRMS (FAB, 3-NBA): m/z: calcd for C₂₇H₄₀NaO₃Si:

463.2644; found: 463.2623 [M+Na]⁺.

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 5:2 to 3:2) to give TBS ether

34a (7.46 g, 97%) as a pale yellow oil. R_f=0.20 (cyclohexane/ethyl ace-

tate 4:1); [a]²_D⁰=+ 8.7 (c=0.515, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d

= 7.44 7.35 (m, 3H), 7.31 7.29 (m, 2H), 5.68 (d, J=7.1 Hz, 1H), 4.79

(quint, J=7.1 Hz, 1H), 4.69 (d, J=6.5 Hz, 1H), 4.64 (d, J=6.5 Hz, 1H),

3.96 3.91 (m, 1H), 3.78 (dq, J=2.9, 7.0 Hz, 1H), 3.51 3.36 (m, 3H), 3.40

(s, 3H), 2.92 (d, J=3.3 Hz, 1H), 1.90 1.80 (m, 1H), 1.74 1.61 (m, 3H),

1.41 1.34 (m, 2H), 1.24 (d, J=7.0 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.89

(d, J=6.8 Hz, 3H), 0.89 (s, 9H), 0.85 (d, J=6.8 Hz, 3H), 0.04 (s, 6H);

¹³C NMR (100 MHz, CDCl₃): d = 177.3, 152.7, 133.3, 128.9, 125.8, 98.7,

83.3, 79.0, 72.0, 66.1, 56.1, 54.9, 42.7, 37.7, 35.8, 31.6, 29.2, 26.1, 18.4, 16.3,

14.5, 10.8, 10.5, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd for

C₃₀H₅₁NNaO₇Si: 588.3332, found 588.3325 [M+Na]⁺.

tert-Butyl-[(2S,3S,4S)-3-methoxymethoxy-2,4-diphenyl-hept-6-enyloxy]-

dimethyl-silane (31b): Alcohol 31b was synthesized by analogy to 31a

starting from alcohol 30b (1.87 g, 4.25 mmol) to yield a colourless oil

(1.67 g, 86%). R_f=0.33 (cyclohexane/ethyl acetate 2:1); [a]²_D⁰=ꢀ1.6 (c=

(4R,5S)-3-[(2R,3S,6S,7S,8S)-(tert-Butyldiphenylsilyloxy)-3-hydroxy-7-me-

thoxymethoxy-2,6,8-trimethyl-nonanoyl]-4-methyl-5-phenyl-oxazolidin-2-

one (34b): TBDPS ether 34b was synthesized by analogy to 34a starting

from alcohol 31b (753 mg, 1.64 mmol) to yield a colourless oil (486 mg,

43% over 2 steps). R_f=0.33 (cyclohexane/ethyl acetate 2:1); [a]²_D⁰=ꢀ1.6

(c=0.32, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 7.68 7.64 (m, 4H),

7.45 7.35 (m, 9H), 7.32 7.29 (m, 2H), 5.67 (d, J=7.2 Hz, 1H), 4.79

(quint, J=7.0 Hz, 1H), 4.69 (d, J=6.5 Hz, 1H), 4.62 (d, J=6.5 Hz, 1H),

3.95 3.90 (m, 1H), 3.78 (dq, J=2.9, 7.0 Hz, 1H), 3.59 (dd, J=10.0,

8.0 Hz, 1H), 3.53 3.48 (m, 2H), 3.33 (s, 3H), 1.94 1.88 (m, 1H), 1.73

1.61 (m, 3H), 1.39 1.34 (m, 2H), 1.24 (d, J=7.0 Hz, 3H), 1.06 (s, 9H),

0.89 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H);

1

0.32, CHCl₃); H NMR (400 MHz, CDCl₃): d = 7.69 7.64 (m, 4H), 7.45

7.36 (m, 6H), 4.69 (d, J=6.5 Hz, 1H), 4.62 (d, J=6.5 Hz, 1H), 3.65 3.47

(m, 5H), 3.33 (s, 3H), 1.90 (dsext, J=6.5, 2.5 Hz, 1H), 1.73 1.55 (m,

4H), 1.52 1.43 (m, 1H), 1.22 1.14 (m, 1H), 1.06 (s, 9H), 0.89 (d, J=

7.0 Hz, 3H), 0.84 (d, J=7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d =

135.7, 133.9, 129.7, 127.7, 98.5, 83.3, 66.7, 63.4, 56.0, 37.5, 35.7, 30.4, 28.8,

27.0, 19.4, 16.4, 11.0; HRMS (FAB, 3-NBA): m/z: calcd for

C₂₇H₄₂NaO₄Si: 481.2750; found: 481.2722 [M+Na]⁺.

(4R,5S)-3-[(2R,3S,6S,7S,8S)-9-(tert-Butyldimethylsilyloxy)-3-hydroxy-7-

methoxymethoxy-2,6,8-trimethyl-nonanoyl]-4-methyl-5-phenyl-oxazoli-

din-2-one (34a): A solution of DMSO (7.1 mL, 99.4 mmol) in CH₂Cl₂

(38 mL) was added dropwise at ꢀ788C to a solution of oxalylchloride

(3.64 mL, 42.4 mmol) in CH₂Cl₂(250 mL) and the mixture was stirred for

1 h at ꢀ788C. A solution of alcohol 31a (5.57 g, 16.6 mmol) in CH₂Cl₂

(49 mL) was added dropwise at ꢀ788C and the mixture was stirred for

2.5 h at ꢀ788C. Triethylamine (17.3 mL, 124 mmol) was added dropwise

at ꢀ788C and the mixture was warmed to 08C. The reaction mixture was

quenched with 1m aqueous KH₂PO₄(200 mL) and extracted with CH₂Cl₂

(3î200 mL). The combined organic layers were washed with brine

(100 mL), water (2î100 mL), then dried (Na₂SO₄) and concentrated to

give crude aldehyde 32a (6.35 g) as a pale yellow oil which was immedi-

ately used in the next step without purification. 32a: R_f=0.77 (cyclohex-

ane/ethyl acetate 2:1).

¹³C (100.6 MHz, CDCl₃): d

= 177.3, 152.8, 135.8, 134.0, 133.3, 129.7,

128.9, 127.8, 125.8, 98.7, 83.4, 79.0, 72.0, 66.8, 56.1, 54.9, 42.8, 37.6, 35.7,

31.5, 29.2, 27.0, 19.4, 16.4, 14.3, 10.9, 10.6.

(2R,3S,7S,8S,9S)-9-(tert-Butyldimethylsilyloxy)-3-hydroxy-N-methoxy-7-

methoxymethoxy-N-2,6,8-tetramethyl-nonanoic acid amide (35a): A 2m

solution of trimethylaluminium in toluene (30 mL, 60 mmol) was added

dropwise at 08C to a mixture of N,O-dimethylhydroxylamine hydrochlo-

ride (6.34 g, 65.0 mmol) in THF (50 mL) and the mixture was stirred for

30 min at room temperature. The solution was cooled to ꢀ208C and a

solution of oxazolidinone 34a (7.36 g, 13.0 mmol) in THF (25 mL) was

added dropwise at ꢀ208C. The mixture was warmed to 08C over a

period of 30 min and stirred for 15 h at 08C. The reaction mixture was

poured into a well-stirred mixture of 1m aqueous HCl (430 mL) and

chloroform (500 mL) at 08C. The pH of the aqueous layer should be 4 5.

The layers were separated and the aqueous layer was extracted with

CHCl₃(3î400 mL). The combined organic layers were washed with satu-

rated aqueous NaHCO₃(200 mL), brine (200 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, pentane/diethyl ether 1:3 to 1:8) to give Weinreb amide 35a

(4.89 g, 84%) as a pale yellow oil. R_f=0.50 (pentane/diethyl ether 1:4);

[a]²_D⁰=ꢀ6.4 (c=0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 4.67 (d,

J=6.5 Hz, 1H), 4.63 (d, J=6.5 Hz, 1H), 3.85 3.80 (m, 1H), 3.71 (brs,

1H), 3.69 (s, 3H), 3.51 3.38 (m, 3H), 3.38 (s, 3H), 3.19 (s, 3H), 2.85 2.80

(m, 1H), 1.89 1.80 (m, 1H), 1.70 1.60 (m, 3H), 1.32 1.27 (m, 2H), 1.17

(d, J=7.0 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H), 0.88 (s, 9H), 0.84 (d, J=

7.0 Hz, 3H), 0.03 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d = 178.5, 98.6,

83.3, 72.0, 66.1, 61.7, 56.1, 39.0, 37.6, 36.1, 32.1, 31.8, 29.3, 26.1, 18.4, 16.3,

10.9, 10.4, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd for

C₂₂H₄₈NO₆Si: 450.3251, found: 450.3241 [M+H]⁺.

EtNiPr₂(4.26 g, 18.3 mmol) and freshly distilled dibutylboryltriflate

(5.5 mL, 21.6 mmol) were added at 08C to a solution of N-propionyloxa-

zolidinone (8; 4.26 g, 18.3 mmol) in CH₂Cl₂(75 mL) and the solution was

stirred for 1 h at 08C. The solution was cooled to ꢀ788C and a solution

of aldehyde 33 in CH₂Cl₂(20 mL) was added dropwise. The mixture was

stirred for 50 min at ꢀ788C and for 130 min at room temperature. The

reaction mixture was cooled to 08C, quenched with 0.1m phosphate

buffer (pH 7, 200 mL) and extracted with CH₂Cl₂(300 mL). The organic

layer were concentrated, the residue was redissolved in methanol

(40 mL), 30% aqueous H₂O₂(47 mL) was added dropwise at 08C and

the mixture was stirred at room temperature for 90 min. The mixture was

diluted with brine (400 mL) and extracted with CH₂Cl₂(3î800 mL). The

combined organic layers were washed with brine (400 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 3:1 to 2:1) to give diol 33

(6.64 g, 89%) as a colourless oil, which was immediately used in the next

step without further characterization. R_f=0.32 (cyclohexane/ethyl acetate

3:1); ¹H NMR (400 MHz, CDCl₃): d = 7.44 7.35 (m, 3H), 7.31 7.28 (m,

2H), 5.68 (d, J=7.2 Hz, 1H), 4.79 (dq, J=7.2, 6.6 Hz, 1H), 4.72 (d, J=

6.8 Hz, 1H), 4.70 (d, J=6.8 Hz, 1H), 3.96 3.91 (m, 1H), 3.80 (dq, J=2.7,

6.8 Hz, 1H), 3.53 3.43 (m, 3H), 3.43 (s, 3H), 2.96 (t, J=6.6 Hz, 1H), 2.88

(d, J=3.1 Hz, 1H), 2.01 1.92 (m, 1H), 1.78 1.60 (m, 3H), 1.40 1.25 (m,

2H), 1.24 (d, J=7.2 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H), 0.87 (d, J=6.8 Hz,

3H), 0.80 (d, J=7.0 Hz, 3H).

(2R,3S,7S,8S,9S)-9-(tert-Butyldiphenylsilyloxy)-3-hydroxy-N-methoxy-

3,7-bis-methoxymethoxy-N-2,6,8-tetramethyl-nonanoic acid amide (36a):

Chloromethyl methyl ether (8.1 mL, 107 mmol) at 08C was added drop-

wise to a solution of alcohol 35a (4.79 g, 10. 7 mmol) in CH₂Cl₂(120 mL)

and EtNiPr₂(23.7 mL, 138.5 mmol). The solution was stirred for 1 h at

08C and for 18 h at room temperature. The orange solution was

quenched 1m KH₂PO₄(170 mL) and extracted with CH₂Cl₂(3î500 mL).

The combined organic layers were washed with water (100 mL), brine

(100 mL), then dried (Na₂SO₄) and concentrated. The residue was puri-

fied by flash chromatography (silica gel, pentane/diethyl ether 1:2) to

give MOM ether 36a (4.97 g, 94%) as a pale yellow oil. R_f=0.40 (pen-

tane/diethyl ether 1:2); [a]²_D⁰=ꢀ7.0 (c=0.63, CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d = 4.68 4.59 (m, 4H), 3.78 (q, J=5.6 Hz, 1H), 3.69

(s, 3H), 3.50 (dd, J=9.6, 7.6 Hz, 1H), 3.43 (dd, J=9.6, 6.3 Hz, 1H), 3.38

(s, 6H), 3.35 (dd, J=6.8, 2.0 Hz, 1H), 3.18 (s, 3H), 3.10 3.02 (m, 1H),

1.88 1.79 (m, 1H), 1.69 1.59 (m, 3H), 1.50 1.44 (m, 1H), 1.21 1.14 (m,

To a mixture of alcohol 33 (6.17 g, 13.7 mmol), N,N-dimethylaminopyri-

dine (167 mg, 1.37 mmol) and triethylamine (7.6 mL, 54.6 mmol) in

CH₂Cl₂(70 mL) was added dropwise a solution of tert-butylchlorodime-

thylsilane in CH₂Cl₂(30 mL) at 08C. The reaction mixture was stirred for

30 min at 08C, for 15 h at room temperature, quenched with 1m aqueous

KH₂PO₄(200 mL) and extracted with ethyl acetate (3î200 mL). The

combined organic layers were washed with brine (100 mL), then dried

2771

Chem. Eur. J. 2004, 10, 2759 2780

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Waldmann and L. Bialy

FULL PAPER

1H), 1.19 (d, J=7.0 Hz, 3H), 0.88 (s, 9H), 0.86 (d, J=6.8 Hz, 3H), 0.84

purified by flash chromatography (silica gel, cyclohexane/ethyl acetate

5:1) to give alkynone 38a (2.64 g, 95% over 2 steps) as a colourless oil.

R_f=0.68 (cyclohexane/ethyl acetate 2:1); [a]²_D⁰=ꢀ11.2 (c=1.09, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d = 4.68 (d, J=6.5 Hz, 1H), 4.66 (d, J=

6.8 Hz, 1H), 4.62 (d, J=6.5 Hz, 1H), 4.61 (d, J=6.8 Hz, 1H), 4.15 (dt,

J=3.7, 6.6 Hz, 1H), 3.49 (dd, J=9.8, 7.8 Hz, 1H), 3.44 (dd, J=9.8,

6.1 Hz, 1H), 3.41 (dd, J=7.2, 2.5 Hz, 1H), 3.39 (s, 3H), 3.33 (s, 3H), 3.25

(s, 1H), 2.74 (dq, J=3.7, 6.8 Hz, 1H), 1.89 1.75 (m, 2H), 1.71 1.64 (m,

1H), 1.55 1.63 (m, 1H), 1.50 1.39 (m, 1H), 1.26 1.15 (m, 1H), 1.19 (d,

J=7.0 Hz, 3H), 0.90 (d, J=7.0 Hz, 3H), 0.89 (s, 9H), 0.85 (d, J=6.8 Hz,

3H), 0.04 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d = 189.2, 98.5, 96.4,

83.1, 81.2, 79.3, 78.3, 66.0, 56.1, 56.0, 52.1, 37.7, 36.2, 30.5, 28.9, 26.0, 18.4,

16.4, 11.0, 9.3, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd for

C₂₄H₄₆NaO₆Si: 481.2961, found 481.2979 [M+Na]⁺.

(d, J=6.8 Hz, 3H), 0.03 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d

=

176.0, 98.4, 96.7, 83.5, 79.7, 66.2, 61.5, 56.1, 56.1, 39.6, 37.7, 36.3, 32.4,

30.9, 28.4, 26.1, 18.4, 16.2, 13.7, 11.1, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA):

m/z: calcd for C₂₄H₅₂NO₇Si: 494.3513, found 494.3521 [M+H]⁺.

(2R,3S,7S,8S,9S)-9-(tert-Butyldiphenylsilyloxy)-3-hydroxy-N-methoxy-

3,7-bis-methoxymethoxy-N-2,6,8-tetramethyl-nonanoic acid amide (36b):

Weinreb amide 35b was synthesized by analogy to 35a starting from al-

cohol 34b (221 mg, 0.320 mmol) to yield a colourless oil (241 mg). Com-

pound 35b was used without chromatographic purification in the next

step. 35b: R_f=0.40 (cyclohexane/ethyl acetate 2:1); ¹H NMR (400 MHz,

CDCl₃): d = 7.68 7.63 (m, 4H), 7.43 7.34 (m, 6H), 4.68 (d, J=6.4 Hz,

1H), 4.62 (d, J=6.4 Hz, 1H), 3.84 3.79 (m, 1H; CHOH), 3.66 (s, 3H;

CH₃ON), 3.59 (dd, J=10.0, 8.0 Hz, 1H), 3.52 3.47 (m, 2H), 3.31 (s, 3H),

3.19 (s, 3H), 2.93 2.81 (brs, 1H), 1.96 1.87 (m, 1H), 1.72 1.55 (m, 3H),

1.33 1.25 (m, 2H), 1.17 (d, J=7.2 Hz, 3H), 1.05 (s, 9H), 0.89 (d, J=

(3R,4S,5S,8S,9S,10S)-11-(tert-Butyldiphenylsilyloxy)-5,9-bis-methoxyme-

thoxy-4,8,10-trimethyl-1-trimethylsilyl-undec-1-yn-3-ol

(41):

THF

6.8 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H); ¹³C (100.6 MHz, CDCl₃): d

=

(0.2 mL) and a solution of TMS-alkynone 37b (73 mg, 0.111 mmol) in

THF (0.2 mL) were added dropwise at 08C to a 0.4m solution of oxaza-

borolidine 39^[18e]in toluene (0.28 mL, 0.133 mmol) and a 2m solution of

BH₃¥Me₂S in THF (67 mL, 0.134 mmol). The reaction mixture was stirred

for 40 min at 08C. The reaction was quenched by slow addition of metha-

nol (0.25 mL) at 08C. The mixture was stirred for 20 min at room temper-

ature and concentrated. The residue was purified by flash chromatogra-

phy (silica gel, cyclohexane/ethyl acetate 5:1 to 2:1) to give TMS-alkynol

41 (52 mg, 71%) as a colourless oil. R_f=0.24 (cyclohexane/ethyl acetate

6:1); ¹H NMR (400 MHz, CDCl₃): d = 7.67 7.63 (m, 4H), 7.44 7.36 (m,

6H), 4.69 4.65 (m, 3H), 4.60 (d, J=6.5 Hz, 1H), 4.30 (d, J=8.5 Hz, 1H),

3.88 (dt, J=2.5, 7.0 Hz, 1H), 3.57 (dd, J=10.0, 8.0 Hz, 1H), 3.52 3.46 (m,

2H), 3.40 (s, 3H), 3.32 (s, 3H), 1.92 1.84 (m, 2H), 1.78 1.62 (m, 2H),

1.57 1.49 (m, 1H), 1.42 1.31 (m, 1H), 1.18 1.09 (m, 1H), 1.05 (s, 9H),

0.99 (d, J=7.0 Hz, 3H), 0.88 (d, J=7.0 Hz, 3H), 0.83 (d, J=7.0 Hz, 3H),

0.17 (s, 9H); MS (MALDI-TOF): m/z: 679.6 [M+Na]⁺.

178.4, 135.7, 134.0, 129.7, 127.7, 98.5, 83.2, 72.0, 66.8, 61.6, 56.0, 39.0, 37.5,

36.1, 32.1, 31.8, 29.3, 27.0, 19.4, 16.3, 10.9, 10.4.

MOM ether 36b was synthesized by analogy to 36a starting from alcohol

35b to yield a colourless oil (171 mg, 86% over two steps). 36b: R_f=0.40

(cyclohexane/ethyl acetate 2:1); ¹H NMR (400 MHz, CDCl₃): d = 7.68

7.63 (m, 4H), 7.44 7.35 (m, 6H), 4.67 4.52 (m, 3H), 4.60 (d, J=6.4 Hz,

1H), 3.80 3.76 (m, 1H), 3.67 (CH₃ON), 3.59 (dd, J=10.1, 7.9 Hz, 1H),

3.50 3.45 (m, 2H), 3.37 (s, 3H), 3.30 (s, 3H), 3.18 (s, 3H), 3.12 3.02 (m,

1H), 1.90 (dsext, J=7.5, 2.6 Hz, 1H), 1.70 1.60 (m, 3H), 1.49 1.41 (m,

1H), 1.19 (d, J=7.1 Hz, 3H), 1.22 1.12 (m, 1H), 1.05 (s, 9H), 0.85 (d,

J=6.7 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H); ¹³C (100.6 MHz, CDCl₃): d =

176.2, 135.7, 134.0, 129.7, 127.7, 98.5, 96.7, 83.5, 79.6, 66.9, 61.5, 56.0, 56.0,

39.6, 37.7, 36.2, 32.4, 30.8, 28.3, 27.0, 19.4, 16.3, 13.7, 11.0; MS (ESI-MS):

m/z: 640.6 [M+Na]⁺.

(3R,4S,5S,8S,9S,10S)-11-(tert-Butyldiphenylsilyloxy)-5,9-bis-methoxyme-

thoxy-4,8,10-trimethyl-1-trimethylsilyl-undec-1-yn-3-one (37b): TMS-al-

kynone 37b was synthesized by analogy to 37a starting from Weinreb

amide 36a (18 mg, 0.029 mmol) to yield a colourless oil (16 mg, 84%).

R_f=0.46 (cyclohexane/ethyl acetate 10:1); ¹H NMR (400 MHz, CDCl₃):

d = 7.68 7.63 (m, 4H), 7.44 7.35 (m, 6H), 4.68 (d, J=6.5 Hz, 1H), 4.64

4.60 (m, 3H), 4.15 4.08 (m, 1H), 3.58 (dd, J=10.1, 7.9 Hz, 1H), 3.52

3.47 (m, 2H), 3.33 (s, 3H), 3.32 (s, 3H), 3.25 (dq, J=3.7, 6.7 Hz, 1H),

1.90 (dsext, J=2.7, 6.7 Hz, 1H), 1.82 1.74 (m, 1H), 1.70 1.62 (m, 1H),

1.61 1.53 (m, 1H), 1.44 1.37 (m, 1H), 1.23 1.15 (m, 1H), 1.17 (d, J=

6.7 Hz, 3H), 1.05 (s, 9H), 0.89 (d, J=6.7 Hz, 3H), 0.83 (d, J=7.1 Hz,

(3R,4S,5S,8S,9S,10S)-11-(tert-Butyldimethylsilyloxy)-5,9-bis-methoxyme-

thoxy-4,8,10-trimethyl-undec-1-yn-3-ol (42a): A 1m solution of (R)-2-

methyl-CBS-oxazolidinone 40 in toluene (11.2 mL, 11.2 mmol) was

evaporated to dryness. To the residue was added a solution of alkynone

38a (2.56 g, 5.58 mmol) in THF (70 mL) and the solution was cooled to

ꢀ308C. To this solution was added a 2m solution of BH₃¥Me₂S in THF

(14.0 mL, 28.0 mmol) and the mixture was stirred for 1 h at ꢀ308C. To

the reaction mixture was added dropwise methanol (22.5 mL) at ꢀ308C.

The mixture is diluted with saturated aqueous NH₄Cl (100 mL) and ex-

tracted with ethyl acetate (3î170 mL). The combined organic layers

were washed with saturated aqueous NaHCO₃(50 mL), brine (50 mL),

then dried (Na₂SO₄) and concentrated. The residue was purified by flash

chromatography (silica gel, cyclohexane/ethyl acetate 5:1) to give alkynol

42a (2.30 g, 89%) as a colourless oil. R_f=0.35 (cyclohexane/ethyl acetate

3:1); [a]²_D⁰=+ 29.1 (c=0.23, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d =

4.70 (d, J=6.5 Hz, 1H), 4.67 (d, J=6.5 Hz, 1H), 4.67 (d, J=6.5 Hz, 1H),

4.62 (d, J=6.5 Hz, 1H), 4.31 (ddd, J=8.0, 5.8, 2.2 Hz, 1H), 3.90 (dt, J=

2.7, 7.0 Hz, 1H), 3.70 (d, J=5.8 Hz, 1H), 3.50 3.40 (m, 3H), 3.41 (s, 3H),

3.38 (s, 3H), 2.45 (d, J=2.2 Hz, 1H), 1.94 1.86 (m, 1H), 1.86 1.63 (m,

3H), 1.59 1.50 (m, 1H), 1.43 1.34 (m, 1H), 1.20 1.09 (m, 1H), 1.02 (d,

J=7.0 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.89 (s, 9H), 0.84 (d, J=7.0 Hz,

3H), 0.04 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): d 98.5, 97.0, 84.8, 83.0,

79.4, 73.1, 66.0, 65.2, 56.1, 56.1, 42.5, 37.6, 36.2, 29.5, 29.1, 26.1, 18.4, 16.4,

10.9, 10.7, ꢀ5.2, ꢀ5.3; HRMS (FAB, 3-NBA): m/z: calcd for

C₂₄H₄₈NaO₆Si: 483.3118, found 483.3128 [M+Na]⁺.

3H), 0.25 (s, 9H); ¹³C (100.6 MHz, CDCl₃): d

= 189.7, 135.8, 134.0,

129.8, 127.8, 101.9, 99.0, 98.5, 96.4, 83.3, 78.4, 66.8, 56.1, 55.9, 51.8, 37.6,

36.2, 30.4, 28.7, 27.0, 19.4, 16.4, 11.0, 9.7, ꢀ0.6.

(3R,4S,5S,8S,9S,10S)-11-(tert-Butyldimethylsilyloxy)-5,9-bis-methoxyme-

thoxy-4,8,10-trimethyl-undec-1-yn-3-one (38a): A solution of n-butyllithi-

um in hexanes at ꢀ788C was added dropwise to a solution of trimethylsi-

lylacetylene (5.16 mL, 36.5 mmol) in THF (30 mL) and the reaction mix-

ture was stirred for 1 h at ꢀ788C and 15 min at room temperature. This

lithium trimethylsilyacetylide solution was added dropwise to a solution

of Weinreb amide 36a (3.00 g, 6.08 mmol) in THF (120 mL) and the mix-

ture was warmed to ꢀ108C over a period of 20 min. The mixture was stir-

red for 75 min at ꢀ108C, recooled to ꢀ788C, quenched with saturated

aqueous NH₄Cl (200 mL), diluted with diethyl ether (200 mL) and

warmed to room temperature. The mixture was diluted with water

(200 mL) and extracted with diethyl ether (3î300 mL). The combined

organic layers were washed with brine (100 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, cyclohexane/ethyl acetate 7:1 to 5:1) to give a mixture of

TMS-alkynone 37a and alkynone 38a (3.28 g) which was used in the next

step without separation. 37a: R_f=0.78 (cyclohexane/ethyl acetate 2:1).

(3R,4S,5S,8S,9S,10S)-11-(tert-Butyldiphenylsilyloxy)-5,9-bis-methoxyme-

thoxy-4,8,10-trimethylundec-1-yn-3-ol (42b): K₂CO₃(22 mg, 0.159 mmol)

was added to a solution of TMS-alkynone 41 (92 mg, 0.140 mmol) in

methanol (5 mL) and the mixture was stirred for 15 h at room tempera-

ture. The reaction mixture was quenched with saturated aqueous NH₄Cl

(20 mL), diluted with brine (100 mL) and extracted with diethyl ether

(3î200 mL). The combined organic layers were concentrated. The resi-

due was purified by flash chromatography (silica gel, cyclohexane/ethyl

acetate 5:1 to 3:1) to give alkynol 42b (67 mg, 82%) as a colourless oil.

A solution of disodium tetraborate (Borax, 12.6 mg) in water (120 mL) at

ꢀ108C was added dropwise to a solution of the mixture of 37a and 38a

in methanol (600 mL) and the mixture was stirred for 10 min at ꢀ108C,

then for 70 min at room temperature. The reaction progress is carefully

monitored by TLC. The reaction mixture was quenched with 1m aqueous

KH₂PO₄(100 mL), diluted with brine (400 mL) and extracted with dieth-

yl ether (3î500 mL). The combined organic layers were washed with

brine (300 mL), then dried (Na₂SO₄) and concentrated. The residue was

1

R_f=0.15 (cyclohexane/ethyl acetate 5:1); H NMR (400 MHz, CDCl₃): d

= 7.68 7.63 (m, 4H), 7.45 7.35 (m, 6H), 4.70 4.65 (m, 3H), 4.60 (d, J=

6.5 Hz, 1H), 4.31 (dd, J=8.2, 2.0 Hz, 1H), 3.90 (dt, J=2.7, 7.0 Hz, 1H),

3.58 (dd, J=10.0, 8.0 Hz, 1H), 3.52 3.46 (m, 2H), 3.41 (s, 3H), 3.32 (s,

3H), 2.45 (d, J=2.0 Hz, 1H), 1.93 1.85 (m, 2H), 1.80 1.63 (m, 2H),

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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Chem. Eur. J. 2004, 10, 2759 2780

Cytostatin

2759 2780

1.58 1.48 (m, 1H), 1.40 1.30 (m, 1H), 1.22 1.09 (m, 1H), 1.06 (s, 9H),

1.00 (d, J=7.0 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H);

MS (MALDI-TOF): m/z: 607.7 [M+Na]⁺.

The mixture was extracted with ethyl acetate (3î300 mL), the combined

organic layers were washed with brine (100 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, cyclohexane/ethyl acetate 2:1 to 1:1) to give alkynol 44 (2.43 g,

88% over two steps) as a colourless oil. R_f=0.32 (cyclohexane/ethyl ace-

tate 2:1); [a]²_D⁰=+77.1 (c=0.55, CHCl₃); ¹H NMR (400 MHz, CDCl₃):

d = 7.75 7.68 (m, 4H), 7.46 7.35 (m, 6H), 4.61 (d, J=6.6 Hz, 1H), 4.59

(d, J=6.6 Hz, 1H), 4.51 (d, J=6.8 Hz, 1H), 4.49 (d, J=6.8 Hz, 1H), 4.38

(dd, J=5.7, 2.2 Hz, 1H), 3.54 (q, J=5.9 Hz, 1H), 3.51 3.45 (m, 2H), 3.41

(s, 3H), 3.30 (s, 3H), 3.32 3.28 (m, 1H), 3.06 (t, J=6.5 Hz, 1H), 2.30 (d,

J=2.2 Hz, 1H), 1.95 1.82 (m, 2H), 1.58 1.44 (m, 3H), 1.37 1.25 (m,

1H), 1.14 (d, J=6.8 Hz, 3H), 1.09 1.07 (m, 1H), 1.08 (s, 9H), 0.75 (d,

J=7.0 Hz, 3H), 0.68 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d

= 136.2, 136.0, 133.7, 133.6, 130.0, 129.8, 127.8, 127.5, 99.2, 96.5, 83.9,

83.3, 79.3, 74.8, 65.2, 65.0, 56.3, 55.8, 43.5, 36.7, 36.5, 29.8, 27.9, 27.1, 19.5,

15.5, 10.6, 9.8; HRMS (FAB, 3-NBA): m/z: calcd for C₃₄H₅₂NaO₆Si:

607.3431, found 607.3405 [M+Na]⁺.

(3R,4R,5S,8S,9S,10S)-11-(tert-Butyldimethylsilyloxy)-3-(tert-butyldiphe-

nylsilyloxy)-5,9-bis-methoxymethoxy-4,8,10-trimethylundec-1-yne (43a):

tert-Butylchlorodiphenylsilane (1.70 mL, 6.53 mmol) was added dropwise

to a solution of alcohol 42a (2.23 g, 4.84 mmol) and imidazole (824 mg,

12.1 mmol) in DMF (10 mL) and the mixture was stirred for 15 h at

room temperature. The reaction mixture was diluted with brine (100 mL)

and extracted with diethyl ether (3î100 mL). The combined organic

layers were washed with brine (50 mL), then dried (Na₂SO₄) and concen-

trated. The residue was purified by flash chromatography (silica gel, cy-

clohexane/ethyl acetate 20:1 to 10:1) to give alkynol 43a (4.25 g) as a col-

ourless oil. The product was not totally pure and was used as such in the

next step. An analytical sample was obtained for characterization. R_f=

0.34 (cyclohexane/ethyl acetate 15:1); [a]²_D⁰=+17.2 (c=0.5, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d = 7.76 7.70 (m, 4H), 7.45 7.34 (m, 6H),

4.64 (d, J=6.5 Hz, 1H), 4.58 (d, J=6.5 Hz, 1H), 4.51 (d, J=6.5 Hz, 1H),

4.49 (d, J=6.5 Hz, 1H), 4.40 (dd, J=5.9, 2.2 Hz, 1H), 3.59 (q, J=5.5 Hz,

1H), 3.51 (dd, J=9.9, 7.8 Hz, 1H), 3.43 (dd, J=9.9, 6.4 Hz, 1H), 3.36 (s,

3H), 3.31 (dd, J=7.2, 2.3 Hz, 1H), 3.30 (s, 3H), 2.26 (d, J=2.2 Hz, 1H),

1.95 1.86 (m, 1H), 1.85 1.77 (m, 1H), 1.59 1.48 (m, 3H), 1.37 1.26 (m,

1H), 1.12 (d, J=6.8 Hz, 3H), 1.11 1.07 (m, 1H), 1.08 (s, 9H), 0.91 (s,

9H), 0.84 (d, J=6.8 Hz, 3H), 0.75 (d, J=6.8 Hz, 3H), 0.06 (s, 6H);

¹³C NMR (100 MHz, CDCl₃): d = 136.2, 136.0, 133.8, 133.6, 129.9, 129.7,

127.8, 127.4, 98.4, 96.6, 83.5, 83.3, 79.2, 74.8, 66.2, 65.2, 56.0, 55.8, 43.6,

37.6, 36.5, 30.2, 28.3, 27.1, 26.1, 19.5, 18.4, 16.1, 10.9, 10.5, ꢀ5.2, ꢀ5.2;

HRMS (FAB, 3-NBA): m/z: calcd for C₄₀H₆₆NaO₆Si₂: 721.4296, found

721.4294 [M+Na]⁺.

1,7-Bis(tert-butyldiphenylsilyloxy)hept-2-yne (49): tert-Butyldiphenylsilyl

chloride (86 mL, 0.326 mmol) tert-Butyldiphenylsilyl chloride (86 mL,

0.326 mmol) to a solution of alcohol 48^[64](100 mg, 0.272 mmol) and imi-

dazole (56 mg, 0.816 mmol) in DMF (0.4 mL) and the mixture was stirred

for 15 h at room temperature. The mixture was quenched with brine

(10 mL) and extracted with diethyl ether (3î20 mL). The combined or-

ganic layers were washed with brine (10 mL), then dried (Na₂SO₄) and

concentrated. The residue was purified by flash chromatography (silica

gel, cyclohexane/ethyl acetate 150:1) to give TBDPS ether 49 (149 mg,

91%) as

a colourless oil. R_f=0.83 (cyclohexane/ethyl acetate 2:1);

¹H NMR (400 MHz, CDCl₃): d = 7.72 7.64 (m, 8H), 7.43 7.34 (m, 12H),

4.30 (t, J=2.0 Hz, 2H), 3.65 (t, J=6.1 Hz, 2H), 2.15 (dt, J=6.6, 2.0 Hz,

2H), 1.65 1.52 (m, 4H), 1.67 1.60 (m, 4H), 1.05 (s, 9H), 1.04 (s, 9H);

¹³C NMR (100 MHz, CDCl₃): d = 135.8, 135.7, 134.1, 133.5, 129.8, 129.7,

127.8 (2îC), 85.8, 78.7, 63.5, 53.1, 31.8, 27.0, 26.9, 25.1, 19.4, 19.3, 18.7;

HRMS (FAB, 3-NBA): m/z: calcd for C₃₂H₄₃NaO₄Si: 627.3091, found

627.3110 [M+Na]⁺.

(3R,4R,5S,8S,9S,10S)-Bis-3,11-(tert-Butyldiphenylsilyloxy)-5,9-bis-me-

thoxymethoxy-4,8,10-trimethyl-undec-1-yne (43b): TBDPS ether 43b was

synthesized by analogy to 43a starting from alcohol 42a (39 mg,

0.067 mmol) to yield a colourless oil (47 mg, 85%). R_f=0.22 (cyclohex-

ane/ethyl acetate 20:1); ¹H NMR (400 MHz, CDCl₃): d = 7.75 7.64 (m,

8H), 7.44 7.33 (m, 12H), 4.62 (d, J=6.5 Hz, 1H), 4.55 (d, J=6.5 Hz,

1H), 4.49 (d, J=6.5 Hz, 1H), 4.46 (d, J=6.5 Hz, 1H), 4.40 (dd, J=5.7,

2.2 Hz, 1H), 3.61 3.55 (m, 2H), 3.47 (dd, J=10.0, 6.3 Hz, 1H), 3.38 (dd,

J=7.4, 2.5 Hz, 1H), 3.28 (s, 3H), 3.27 (s, 3H), 2.25 (d, J=2.2 Hz, 1H),

1.92 1.84 (m, 2H), 1.56 1.50 (m, 3H), 1.35 1.21 (m, 1H), 1.11 (d, J=

6.8 Hz, 3H), 1.08 1.05 (m, 1H), 1.07 (s, 9H), 1.06 (s, 9H), 0.82 (d, J=

6.8 Hz, 3H), 0.74 (d, J=6.7 Hz, 3H); MS (MALDI-TOF): 845.7

[M+Na]⁺.

7-(tert-Butyldiphenylsilyloxy)-hept-2-yn-1-ol

(48):

NH₄F

(30 mg,

0.83 mmol) was added to a solution of the TBDPS ether 48 (50 mg,

83 mmol) in methanol (5 mL) and the mixture was stirred for 8 h at room

temperature. The mixture was quenched with a 1m aqueous phosphate

buffer (pH 7) (10 mL), diluted with brine (10 mL) and extracted with

ethyl acetate (3î50 mL). The combined organic layers were washed with

brine (10 mL), then dried (Na₂SO₄) and concentrated. The residue was

purified by flash chromatography (silica gel, cyclohexane/ethyl acetate

10:1) to give the alcohol 48 (25 mg, 82%) as a colourless oil. R_f=0.28

(cyclohexane/ethyl acetate 4:1); ¹H NMR (400 MHz, CDCl₃): d = 7.68

7.65 (m, 4H), 7.45 7.36 (m, 6H), 4.23 (t, J=2.2 Hz, 2H), 3.68 (t, J=

5.9 Hz, 2H), 2.22 (tt, J=6.6, 2.2 Hz, 2H), 1.70 1.57 (m, 4H), 1.05 (s,

9H); ¹³C NMR (100 MHz, CDCl₃): d = 135.7, 134.1, 129.7, 127.8, 86.6,

78.6, 63.5, 51.6, 31.8, 27.0, 25.2, 19.4, 18.7.

(2S,3S,4S,7S,8S,9R)-tert-Butyl-[9-(4-methoxybenzyloxy)-3,7-bis-methoxy-

methoxy-2,4,8-trimethyl-undec-10-ynyloxy]-diphenyl-silane (43c): A 1m

solution of sodium hexamethyldisilazide (68 mL, 68 mmol) in THF at 08C

was added dropwise to a solution of alcohol 42a (36 mg, 62 mmol) in

DMF (0.85 mL) and the mixture was stirred for 30 min at 08C. 4-Methoxy-

benzylchloride (15 mL, 111 mmol) and tetrabutylammoniumiodide (1 mg,

3 mmol) were added at 08C and the mixture was stirred for 24 h at room

temperature. The reaction mixture was quenched with saturated aqueous

NH₄Cl (5 mL) and extracted with diethyl ether (3î10 mL). The com-

bined organic layers were washed with brine (5 mL), then dried

(Na₂SO₄), and concentrated. The residue was purified by flash chroma-

tography (silica gel, cyclohexane/ethyl acetate 50:1 to 1:1) to give PMB

ether 43c (13 mg, 30%) as a colourless oil and the starting compound

42a (4 mg, 11%). 43c: R_f=0.42 (cyclohexane/ethyl acetate 5:1);

¹H NMR (400 MHz, CDCl₃): d = 7.68 7.63 (m, 4H), 7.44 7.34 (m, 6H),

7.29 (d, J=8.8 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 4.74 (d, J=10.9 Hz,

1H), 4.65 (d, J=6.5 Hz, 1H), 4.59 (d, J=6.5 Hz, 1H), 4.56 (d, J=6.6 Hz,

1H), 4.54 (d, J=6.6 Hz, 1H), 4.41 (d, J=10.9 Hz, 1H), 4.07 (dd, J=8.4,

2.2 Hz, 1H), 3.79 (s, 3H), 3.81 3.75 (m, 1H), 3.58 (dd, J=10.0, 8.0 Hz,

1H), 3.50 3.44 (m, 2H), 3.32 (s, 3H), 3.29 (s, 3H), 2.48 (d, J=2.0 Hz,

1H), 1.98 1.84 (m, 2H), 1.75 1.45 (m, 3H), 1.40 1.30 (m, 1H), 1.15 1.03

(m, 4H), 1.05 (s, 9H), 0.85 (d, J=6.8 Hz, 3H), 0.83 (d, J=6.8 Hz, 3H).

The spectroscopical data are in accordance with the literature.^[63]

Methyl (4S,5S,6S,9S,10S,11R)-11-(tert-butyldiphenylsilyloxy)-5,9-bis-me-

thoxymethoxy-4,6,10-trimethyl-tridec-2-en-12-ynoate (50): A solution of

15% (by weight) Dess Martin periodinane in CH₂Cl₂(0.24 mL,

0.11 mmol) was added dropwise at room temperature to a mixture of the

alcohol 44 (43 mg, 74 mmol) and NaHCO₃(71 mg, 0.85 mmol) in CH₂Cl₂

(1.7 mL) and the mixture was stirred for 85 min at room temperature.

The mixture was added to a well-stirred mixture of saturated aqueous

NaS₂O₃(5 mL), saturated aqueous NaHCO₃(5 mL) and diethyl ether

(10 mL). The mixture was stirred for 1 h at room temperature, diluted

with saturated aqueous NaHCO₃(10 mL) and extracted with diethyl

ether (3î30 mL). The combined organic layers were washed with brine

(2î20 mL), then dried (Na₂SO₄) and concentrated at room temperature.

The residue was purified by flash chromatography (silica gel, pentane/di-

ethyl ether 1:1 to 0:1) to give the aldehyde (40 mg, 93%) as a colourless

oil which was directly used in the next step. R_f=0.50 (cyclohexane/ethyl

acetate 3:1).

(2S,3S,4S,7S,8R,9R)-9-(tert-Butyldiphenylsilyloxy)-3,7-bis-methoxyme-

thoxy-2,4,8-trimethyl-undec-10-yn-1-ol (44): The reaction was performed

in a teflon vessel. HF/py (11 mL) was added to a solution of TBS ether

43a in THF (110 mL) and the solution was stirred for 40 min at room

temperature. The reaction was carefully monitored by TLC. The mixture

was diluted with ethyl acetate (150 mL) and saturated aqueous NaHCO₃

was carefully added until no more CO₂formation was observed (pH>7).

A 0.5m solution of potassium hexamethyldisilazide in toluene (10.4 mL,

5.2 mmol) at ꢀ788C was added dropwise to a solution of [18]crown-6

(3.65 g, 13.8 mmol) and O,O’-bis(2,2,2-trifluoroethyl)-phosphono-acetic

acid methyl ester (1.46 mL, 6.90 mmol) in THF (65 mL) and the solution

was stirred at ꢀ788C for 35 min. A solution of the aldehyde synthesized

2773

Chem. Eur. J. 2004, 10, 2759 2780

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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Waldmann and L. Bialy

FULL PAPER

as described above (2.01 g, 3.45 mmol) in THF (12 mL) was added drop-

wise over a period of 45 min at ꢀ788C and the mixture was stirred for

260 min at ꢀ788C. The mixture was quenched with saturated aqueous

NH₄Cl (100 mL) and extracted with diethyl ether (3î500 mL). The com-

bined organic layers were washed with brine (100 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 5:1 to 2:1) to give ester 50

(2.05 g, 92% over 2 steps) as a colourless oil. R_f=0.71 (cyclohexane/ethyl

acetate 2:1); [a]²_D⁰=+53.2 (c=0.52, CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d = 7.75 7.69 (m, 4H), 7.44 7.34 (m, 6H), 6.18 (dd, J=11.5,

10.2 Hz, 1H), 5.73 (dd, J=11.5, 0.8 Hz, 1H), 4.60 (d, J=6.8 Hz, 1H),

4.57 (d, J=6.8 Hz, 1H), 4.49 (d, J=6.8 Hz, 1H), 4.46 (d, J=6.8 Hz, 1H),

4.38 (dd, J=5.9, 2.2 Hz, 1H), 3.85 3.75 (m, 1H), 3.69 (s, 3H), 3.56 (q,

J=5.3 Hz, 1H), 3.37 (s, 3H), 3.28 (s, 3H), 3.12 (dd, J=6.1, 5.1 Hz, 1H),

2.26 (d, J=2.2 Hz, 1H), 1.92 1.84 (m, 1H), 1.60 1.42 (m, 3H), 1.31 1.22

(m, 1H), 1.10 (d, J=6.8 Hz, 3H), 1.09 1.06 (m, 1H), 1.07 (s, 9H), 1.02

(d, J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (100 MHz,

CDCl₃): d = 166.6, 154.1, 136.2, 136.0, 133.8, 133.6, 129.9, 129.7, 127.8,

127.4, 118.0, 98.5, 96.5, 87.4, 83.5, 79.1, 74.8, 65.2, 56.2, 55.8, 51.2, 43.6,

36.8, 34.9, 30.3, 27.7, 27.1, 19.5, 16.3, 14.9, 10.4; HRMS (FAB, 3-NBA):

m/z: calcd for C₃₇H₅₄NaO₇Si: 661.3536, found 661.3549 [M+Na]⁺.

poured into

a

well-stirred mixture of saturated aqueous NaHCO₃

(5.3 mL), 0.1m Na₂S₂O₃(5.3 mL) and ethyl acetate (30 mL). The organic

phase was separated and the aqueous phase was extracted with ethyl ace-

tate (2î30 mL). The combined organic layers were washed with 1m

aqueous KH₂PO₄(25 mL), brine (20 mL), then dried (Na₂SO₄) and con-

centrated. The residue was purified by flash chromatography (silica gel,

CH₂Cl₂/ethanol 30:1) to give the phosphotriester 55 (49 mg, quantitative)

as a colourless oil. R_f=0.31 (CH₂Cl₂/ethanol 30:1); [a]²_D⁰=+76.6 (c=

1

0.27, CHCl₃); H NMR (400 MHz, CDCl₃): d = 7.75 7.68 (m, 4H), 7.45

7.35 (m, 6H), 6.99 (dd, J=9.6, 6.5 Hz, 1H), 5.95 (d, J=9.6 Hz, 1H),

4.50 4.43 (m, 1H), 4.34 4.15 (m, 5H), 3.85 (dd, J=10.4, 2.9 Hz, 1H),

2.78 (t, J=6.1 Hz, 2H), 2.70 (t, J=6.1 Hz, 2H), 2.41 (dquint, J=2.9,

7.0 Hz, 1H), 2.36 (d, J=2.2 Hz, 1H), 2.04 1.54 (m, 6H), 1.23 (d, J=

6.8 Hz, 3H), 1.08 (s, 9H), 0.98 (d, J=7.0 Hz, 3H), 0.66 (d, J=6.6 Hz,

3H); ¹³C NMR (100 MHz, CDCl₃): d = 164.8, 152.2, 136.2, 136.0, 133.3,

133.2, 130.0, 129.9, 127.9, 127.6, 120.0, 117.1, 116.7, 83.5, 82.3 (d,

J(¹³C,³¹P)=6.9 Hz), 81.9, 75.5, 64.1, 62.5 (d, J(¹³C,³¹P)=5.4 Hz), 62.3 (d,

J(¹³C,³¹P)=5.4 Hz), 43.4 (d, J(¹³C,³¹P)=6.2 Hz), 33.9, 30.4, 29.6 (d,

J(¹³C,³¹P)=2.3 Hz), 26.9, 26.2, 19.8 (d, J(¹³C,³¹P)=8.5 Hz), 19.7 (d,

J(¹³C,³¹P)=7.7 Hz), 19.5, 14.3, 10.9, 10.9; ³¹P NMR (162 MHz, CDCl₃): d

=

ꢀ2.07; HRMS (FAB, 3-NBA): m/z: calcd for C₃₈H₄₉N₂NaO₇PSi:

727.2944, found 727.2914 [M+Na]⁺.

(5S,6S)-6-[(1S,4S,5R,6R)-6-(tert-Butyldiphenylsilyloxy)-4-hydroxy-1,5-di-

methyl-oct-7-ynyl]-5-methyl-5,6-dihydro-pyran-2-one (51): A solution of

the ester 50 (1.97 g, 3.08 mmol) and CBr₄(510 mg, 1.54 mmol) in 2-prop-

anol (136 mL) was heated for 15 h to 828C, then concentrated. The resi-

due was purified by flash chromatography (silica gel, cyclohexane/ethyl

acetate 2:1 to 3:2) to give the lactone 51 (1.33 g, 83%) as a colourless oil.

R_f=0.6 (cyclohexane/ethyl acetate 1:1); [a]²_D⁰=+115 (c=0.56, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d = 7.76 7.69 (m, 4H), 7.47 7.36 (m, 6H),

6.98 (dd, J=9.6, 6.5 Hz, 1H), 5.97 (d, J=9.6 Hz, 1H), 4.35 (dd, J=4.1,

2.2 Hz, 1H), 4.18 4.13 (m, 1H), 3.98 (dd, J=10.4, 2.9 Hz, 1H), 2.62 (brs,

1H), 2.46 (dquint, J=3.1, 7.0 Hz, 1H), 2.33 (d, J=2.2 Hz, 1H), 1.98 1.90

(m, 1H), 1.80 1.56 (m, 3H), 1.26 1.20 (m, 2H), 1.09 (s, 9H), 1.03 (d, J=

6.5 Hz, 3H), 1.01 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H); ¹³C NMR

(100 MHz, CDCl₃): d = 165.0, 152.0, 136.4, 136.2, 132.9, 132.5, 130.2,

130.0, 127.9, 127.5, 120.2, 84.2, 83.6, 75.2, 71.8, 68.6, 44.1, 34.3, 32.2, 30.6,

29.2, 27.1, 19.5, 14.7, 10.9, 10.0; HRMS (FAB, 3-NBA): m/z: calcd for

C₃₂H₄₃O₄Si: 519.2930, found 519.2888 [M+H]⁺.

Bis(cyanoethyl)-{(1S,2S,3R)-3-hydroxy-2-methyl-1-[(3S,4S)-3-((3S)-3-

methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-ynyl} phosphate

(56): The reaction was carried out in

a teflon vessel. 70% HF/py

(0.54 mL) was added to a solution of TBDPS ether 55 (49 mg, 69 mmol)

in THF (2.6 mL) and the solution was stirred for 24 h at room tempera-

ture. The reaction mixture was added to a well-stirred mixture of saturat-

ed aqueous NaHCO₃(10 mL) and ethyl acetate (30 mL), the combined

organic layers were washed with 1m aqueous KH₂PO₄(6 mL), brine

(6 mL), then dried (Na₂SO₄), diluted with toluene (5 mL) and concentrat-

ed at room temperature. The residue was purified by flash chromatogra-

phy (silica gel, cyclohexane/ethyl acetate 1:8, then CH₂Cl₂/ethanol 10:1)

to give the alcohol 56 (27 mg, 84%) as a colourless oil. R_f=0.16 (cyclo-

hexane/ethyl acetate 1:8); [a]²_D⁰=+88.0 (c=0.57, CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d = 6.99 (dd, J=9.6, 6.5 Hz, 1H), 5.95 (d, J=9.8 Hz,

1H), 4.91 4.85 (m, 1H), 4.40 4.26 (m, 4H), 4.25 (dd, J=9.2, 2.2 Hz, 1H),

4.08 (dd, J=10.6, 3.1 Hz, 1H), 2.92 2.75 (m, 4H), 2.48 (dquint, J=2.9,

7.0 Hz, 1H), 2.48 (d, J=2.2 Hz, 1H), 1.95 1.85 (m, 4H), 1.56 1.41 (m,

2H), 1.07 (d, J=6.8 Hz, 3H), 1.03 (d, J=7.0 Hz, 3H), 0.91 (d, J=6.8 Hz,

3H); ¹³C NMR (100 MHz, CDCl₃): d = 164.9, 152.1, 120.0, 117.0, 116.7,

84.0, 83.4, 79.9 (d, J(¹³C,³¹P)=6.2 Hz), 63.6, 62.9 (d, J(¹³C,³¹P)=4.6 Hz),

62.8 (d, J(¹³C,³¹P)=6.2 Hz), 44.5 (d, J(¹³C,³¹P)=3.8 Hz), 33.4, 30.4, 30.1

(d, J(¹³C,³¹P)=3.8 Hz), 28.4, 19.9 (d, J(¹³C,³¹P)=8.5 Hz), 19.8 (d,

(5S,6S)-6-[(1S,4S,5R,6R)-6-(tert-Butyldiphenylsilyloxy)-4-hydroxy-8-

iodo-1,5-dimethyl-oct-7-ynyl]-5-methyl-5,6-dihydro-pyran-2-one (54):

A

solution of iodine (49 mg, 0.193 mmol) and N,N-dimethylaminopyridine

(71 mg, 58 mmol) in toluene (1.5 mL) was stirred in the dark for 1 h at

room temperature. To the dark brown mixture was added a solution of

the alkyne 51 (20 mg, 39 mmol) in toluene (3 mL) and the mixture was

heated for 3 h to 508C in the dark. The reaction mixture was cooled to

room temperature, diluted with 0.4m aqueous Na₂S₂O₃(8 mL), 1m aque-

ous KH₂PO₄(16 mL) and brine (30 mL). The mixture was extracted with

ethyl acetate (3î40 mL), the combined organic layers were washed with

brine (30 mL), then dried (Na₂SO₄) and concentrated. The residue was

purified by flash chromatography (silica gel, cyclohexane/ethyl acetate

5:1 to 3:1) to give the lactone iodoalkyne 54 (19 mg, 76%) as a colourless

oil. R_f=0.37 (cyclohexane/ethyl acetate 2:1); [a]²_D⁰=+131 (c=0.3,

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 7.73 7.68 (m, 4H), 7.46 7.38

(m, 6H), 6.98 (dd, J=9.6, 6.5 Hz, 1H), 5.97 (dd, J=9.6, 0.6 Hz, 1H), 4.45

(d, J=4.5 Hz, 1H), 4.17 4.12 (m, 1H), 3.99 (dd, J=10.4, 2.9 Hz, 1H),

2.46 (dquint, J=3.1, 7.2 Hz, 1H), 2.00 1.92 (m, 1H), 1.83 1.57 (m, 3H),

1.29 1.22 (m, 2H), 1.08 (s, 9H), 1.02 (d, J=7.0 Hz, 3H), 1.01 (d, J=

7.0 Hz, 3H), 0.90 (d, J=6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d =

165.0, 152.0, 136.3, 136.2, 132.8, 132.4, 130.3, 129.9, 127.9, 127.6, 120.2,

94.5, 84.2, 71.7, 70.2, 44.2, 34.3, 32.1, 30.6, 29.1, 27.1, 19.5, 14.7, 10.9, 10.0,

4.7; HRMS (FAB, 3-NBA): m/z: calcd for C₃₂H₄₂O₄SiI: 645.1897, found

645.1880 [M+H]⁺.

J(¹³C,³¹P)=7.7 Hz), 14.5, 10.9, 9.6; ³¹P NMR (162 MHz, CDCl₃): d

=

ꢀ0.29; HRMS (FAB, 3-NBA): m/z: calcd for C₂₂H₃₂N₂O₇P: 467.1947,

found 467.1943 [M+H]⁺.

Bis(cyanoethyl)-{(1S,2S,3R)-3-hydroxy-5-iodo-2-methyl-1-[(3S,4S)-3-

((3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-ynyl}

phosphate (57): Iodoalkyne 57 was synthesized by analogy to iodoalkyne

80 starting from alkyne 56 (11.0 mg, 23.6 mmol). The residue was purified

by flash chromatography (silica gel, cyclohexane/ethyl acetate 1:15 to

0:1) to give iodoalkyne 57 (15.6 mg, 84%) as a colourless oil. R_f=0.38

1

(cyclohexane/ethyl acetate 1:16); [a]²_D⁰=+65 (c=0.135, CHCl₃); H NMR

(400 MHz, CDCl₃): d = 6.98 (dd, J=9.6, 6.5 Hz, 1H), 5.94 (dd, J=9.6,

0.6 Hz, 1H), 4.86 4.80 (m, 1H), 4.40 4.27 (m, 5H), 4.07 (dd, J=10.6,

3.1 Hz, 1H), 2.91 2.75 (m, 4H), 2.45 (dquint, J=3.1, 7.0 Hz, 1H), 1.95

1.84 (m, 4H), 1.56 1.40 (m, 2H), 1.05 (d, J=6.8 Hz, 3H), 1.02 (d, J=

7.0 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d =

164.9, 152.1, 120.0, 117.0, 116.7, 94.7, 83.4, 79.9 (d, J(¹³C,³¹P)=6.2 Hz),

65.3, 62.9 (d, J(¹³C,³¹P)=5.4 Hz), 62.8 (d, J(¹³C,³¹P)=5.4 Hz), 44.7 (d,

J(¹³C,³¹P)=3.8 Hz), 33.4, 30.4, 30.0 (d, J(¹³C,³¹P)=4.6 Hz), 28.4, 19.9 (d,

J(¹³C,³¹P)=7.7 Hz), 19.8 (d, J(¹³C,³¹P)=7.7 Hz), 14.5, 10.9, 9.7, 2.0;

³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.35; HRMS (FAB, 3-NBA): m/z:

calcd for C₂₂H₃₁IN₂O₇P: 593.0914, found 593.0931 [M+H]⁺.

{(1S,2S,3R)-3-(tert-Butyldiphenylsilyloxy)-2-methyl-1-[(3S,4S)-3-((3S)-3-

methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-ynyl}-bis(2-cyano-

ethyl) phosphate (55):

(NCCH₂CH₂O)₂PNiPr₂

A

solution of the phosphoramidite

[65]

in acetonitrile (0.75 mL) at 08C was added

tert-Butyl-(1-ethyl-prop-2-ynyloxy)-diphenylsilane (58): tert-Butyldiphe-

nylsilyl chloride (1.86 mL, 7.12 mmol) was added dropwise to a solution

of pent-1-yn-3-ol (11, 500 mg, 5.94 mmol) and imidazole (809 mg,

11.9 mmol) in DMF (5 mL) and the mixture was stirred for 19.5 h at

room temperature. The reaction mixture was quenched with brine

(50 mL) and extracted with diethyl ether (3î50 mL). The combined or-

dropwise to a solution of the alcohol 51 (36 mg, 69 mmol) and tetrazol

(22 mg, 0.31 mmol) in acetonitrile (2.2 mL) and the mixture was stirred

for 105 min at room temperature and a 0.1m solution of iodine in pyri-

dine/THF/H₂O 2:7:1 (4.2 mL, 0.42 mmol) was added dropwise over

1 min. The mixture was stirred for 5 min at room temperature, then

2774

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 2759 2780

Cytostatin

2759 2780

ganic layers were washed with brine (50 mL), then dried (Na₂SO₄) and

concentrated. The residue was purified by flash chromatography (silica

gel, cyclohexane/ethyl acetate 100:1) to give the TBDPS ether 58 (1.48 g,

10.4, 2.9 Hz, 1H), 2.92 2.77 (m, 4H), 2.50 2.44 (m, 1H), 1.82 (dd, J=6.8,

1.4 Hz, 1H), 1.98 1.75 (m, 3H), 1.68 1.45 (m, 2H), 1.30 1.22 (m, 1H),

1.03 (d, J=7.2 Hz, 3H), 0.92 (d, J=6.8 Hz, 3H), 0.85 (d, J=7.0 Hz, 3H);

³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.33; MS (ESI): m/z (%): 557.1 (19)

[M+Na]⁺, 313.2 (100) [C₂₁H₂₉O₂]⁺.

77%) as

a colourless oil. R_f=0.7 (cyclohexane/ethyl acetate 50:1);

¹H NMR (400 MHz, CDCl₃): d = 7.77 7.68 (m, 4H), 7.46 7.35 (m, 4H),

4.31 (ddd, J=6.8, 5.5, 2.2 Hz, 1H), 2.31 (d, J=2.2 Hz, 1H), 1.74 1.62 (m,

2H), 1.09 (s, 9H), 0.96 (t, J=7.2 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃):

d = 136.0, 135.8, 133.7, 133.6, 129.7, 129.6, 127.6, 127.4, 85.0, 72.7, 65.0,

31.6, 27.2, 19.6, 9.4; HRMS (FAB, 3-NBA): m/z: calcd for C₂₁H₂₇OSi:

323.1831, found 323.1863 [M+H]⁺.

[7-(tert-Butyl-diphenylsilyloxy)-hept-2-ynyl]-bis(2-cyano-ethyl) phosphate

(68):

A solution of (NCCH₂CH₂O)₂PNiPr₂(64 mg, 0.175 mmol) in

CH₃CN (1 mL) at 08C was added to a solution of the alcohol 48 (64 mg,

0.175 mmol) and tetrazole (25 mg, 0.35 mmol) in CH₃CN (0.75 mL) and

the mixture was stirred for 3 h at room temperature. To the reaction mix-

ture was added dropwise a 0.1m solution of iodine in pyridine/THF/H₂O

2:7:1 (4.2 mL, 0.42 mmol) over 1 min and the mixture was stirred for

5 min at room temperature. The reaction mixture was poured onto a

well-stirred mixture of saturated aqueous NaHCO₃(4.4 mL), 0.1m aque-

ous Na₂S₂O₃(4.4 mL) and ethyl acetate (30 mL). The mixture was ex-

tracted with ethyl acetate (2î30 mL), the combined organic layers were

washed with 1m aqueous KH₂PO₄(30 mL), then dried (Na₂SO₄) and con-

centrated. The residue was purified by flash chromatography (silica gel,

cyclohexane/ethyl acetate 1:3 to 1:8) to give the phosphotriester 68

(72 mg, 74%) as a colourless oil. R_f=0.30 (cyclohexane/ethyl acetate

1:3); ¹H NMR (400 MHz, CDCl₃): d = 7.67 7.64 (m, 4H), 7.45 7.36 (m,

6H), 4.73 (dt, J=10.6, 2.2 Hz, 2H), 4.35 4.24 (m, 4H), 3.70 3.66 (m,

2H), 2.76 (t, J=6.3 Hz, 4H), 2.28 2.23 (m, 2H), 1.67 1.60 (m, 4H), 1.05

tert-Butyl-(1-ethyl-3-iodo-prop-2-ynyloxy)-diphenylsilane (59): Iodoal-

kyne 59 was synthesized by analogy to iodoalkyne 26 starting from

alkyne 58 (750 mg, 2.3 mmol) to give a colourless oil (990 mg, 96%).

R_f=0.36 (cyclohexane/ethyl acetate 100:1); ¹H NMR (400 MHz, CDCl₃):

d = 7.75 7.66 (m, 4H), 7.46 7.36 (m, 6H), 4.41 (dd, J=6.5, 5.7 Hz, 1H),

1.74 1.63 (m, 2H), 1.08 (s, 9H); 0.95 (t, J=7.4 Hz, 3H); ¹³C NMR

(100 MHz, CDCl₃): d = 136.0, 135.8, 133.5, 133.5, 129.7, 129.6, 127.6,

127.4, 95.9, 66.7, 31.8, 27.2, 19.6, 9.5, 1.4; HRMS (FAB, 3-NBA): m/z:

calcd for C₂₁H₂₅IOSi: 448.0719, found 448.0742 [M]⁺.

Bis(cyanoethyl)-{(1S,2S,3R,4Z)-3-hydroxy-5-iodo-2-methyl-1-[(3S,4S)-3-

((3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-enyl}

phosphate (61): A solution of acetic acid (2.8 mL, 48 mmol) in 2-propanol

(38 mL) over a period of 45 min was added to a mixture of iodoalkyne 57

(7.1 mg, 12.0 mmol) and potassium azodicarboxylate (4.7 mg, 24 mmol) in

2-propanol (88 mL) and the mixture was stirred for 24 h at room tempera-

ture. The reaction mixture was quenched with a 0.1m phosphate buffer

(1.5 mL) and extracted with ethyl acetate (3î5 mL). The combined or-

ganic layers were washed with brine (1 mL), then dried (Na₂SO₄) and

concentrated. The residue was purified by flash chromatography (silica

gel, cyclohexane/ethyl acetate 2:3 to 1:3) to give (Z)-iodoalkene 61

(3.9 mg, 44%) as a colourless oil which was contaminated with saturated

the lactone 62 (0.8 mg) and used in the next step without further purifica-

tion. R_f=0.31 (CH₂Cl₂/ethanol 20:1); ¹H NMR (400 MHz, CDCl₃): d =

6.99 (dd, J=9.6, 6.5 Hz, 1H), 6.47 (dd, J=7.4, 0.6 Hz, 1H), 6.23 (dd, J=

8.2, 7.6 Hz, 1H), 5.96 (dd, J=9.6, 0.6 Hz, 1H), 4.98 4.90 (m, 1H), 4.44

4.24 (m, 4H), 4.11 4.06 (m, 2H), 2.88 2.76 (m, 4H), 2.52 2.44 (m, 1H),

1.98 1.74 (m, 3H), 1.57 1.37 (m, 2H), 1.03 (d, J=7.2 Hz, 3H), 0.92 (d,

J=7.0 Hz, 6H); ³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.35.

(s, 9H); ¹³C NMR (100 MHz, CDCl₃): d

= 135.7, 134.0, 129.7, 127.8,

116.3, 89.9, 73.7 (d, J(¹³C,³¹P)=6.9 Hz), 64.0, 62.4 (d, J(¹³C,³¹P)=4.6 Hz),

57.1 (d, J(¹³C,³¹P)=5.4 Hz), 31.8, 27.0, 24.9, 19.8 (d, ³J(¹³C,³¹P)=6.9 Hz),

19.4, 18.7; ³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.33; HRMS (FAB, 3-

NBA): m/z: calcd for C₂₉H₃₇N₂NaO₅PSi: 575.2107, found 575.2091

[M+Na]⁺.

(2S,3S,4S)-7-Bis-methoxymethoxy-2,4-dimethyl-heptan-1-ol (72): MOM-

ether 71 was synthesized by analogy to MOM-ether 36a starting from al-

cohol 31b (208 mg, 0.453 mmol) to give a colourless oil after aqueous

work-up which was immediately used in the next step without purifica-

tion.

To a solution of TBDPS-ether 71 was added dropwise a 1m solution of

tetrabutylammonium fluoride in THF (0.54 mL, 0.54 mmol) at 08C and

the solution was stirred for 15 h at room temperature. The reaction mix-

ture was quenched with saturated aqueous NH₄Cl (10 mL) and H₂O

(10 mL), and extracted with ethyl acetate (3î25 mL). The combined or-

ganic layers were washed with brine (2î10 mL), then dried (Na₂SO₄)

and concentrated. The residue was purified by flash chromatography

(silica gel, cyclohexane/ethyl acetate 2:1) to give the alcohol 72 (108 mg,

tert-Butyl-(1-ethyl-3-iodo-allyloxy)-diphenylsilane (65): (Z)-Iodoalkene

65 was synthesized by analogy to (Z)-iodoalkene 27 starting from iodoal-

kyne 59 (848 mg, 1.88 mmol) to give a colourless oil (785 mg, 93%). R_f=

1

0.35 (cyclohexane/ethyl acetate 150:1); H NMR (400 MHz, CDCl₃): d =

7.70 7.64 (m, 4H), 7.45 7.33 (m, 6H), 6.26 (t, J=7.6 Hz, 1H), 6.09 (dd,

J=7.6, 1.0 Hz, 1H), 4.42 4.37 (m, 1H), 1.64 1.50 (m, 2H), 1.07 (s, 9H),

0.88 (t, J=7.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 143.6, 135.9,

135.9, 134.1, 134.1, 129.6, 129.5, 127.6, 127.5, 80.4, 77.2, 30.2, 27.3, 19.6,

9.3; HRMS (FAB, 3-NBA): m/z: calcd for C₁₇H₁₈IOSi: 393.0172, found

393.0187 [MꢀC(CH₃)₃]⁺.

Bis(2-cyanoethyl)-{(1S,2S,3R,4Z,6Z,8Z)-3-hydroxy-2-methyl-1-[(3S,4S)-3-

((3S)-3-methyl-3,6-dihydro-2H-hydropyran-2-yl)-butyl]-deca-4,6,8-trienyl}

phosphate (66): The reaction was carried out in the dark. To a solution of

the (Z)-alkenyl iodide 61 (3.3 mg, 5.55 mmol) was added a solution of the

freshly prepared (Z)-alkenylstannane 25 (2.6 mg, 7.2 mmol) in degassed

90%) as a colourless oil. R_f=0.28 (cyclohexane/ethyl acetate 2:1); [a]_D²⁰

=

+73.8 (c=0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 4.66 (s, 2H),

4.60 (s, 2H), 3.54 3.47 (m, 4H), 3.41 (s, 3H), 3.41 3.37 (m, 1H), 3.34 (s,

3H), 2.83 2.72 (brs, 1H), 1.99 1.90 (m, 1H), 1.78 1.60 (m, 3H), 1.55

1.45 (m, 1H), 1.17 1.08 (m, 1H), 0.85 (d, J=6.8 Hz, 3H), 0.78 (d, J=

6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d 99.1, 96.5, 84.0, 68.2, 65.2,

56.3, 55.2, 36.8, 36.0, 29.5, 27.5, 15.8, 10.0; HRMS (FAB, 3-NBA): m/z:

calcd for C₁₃H₂₉O₅: 265.2015, found 265.2026 [M+H]⁺.

Methyl (2Z,4S,5S,6S)-5,9-bis-methoxymethoxy-4,6-dimethyl-non-2-enoate

(73): A solution of 15% (by weight) Dess Martin periodinane in CH₂Cl₂

(1.9 mL, 0.891 mmol) was added dropwise at room temperature to a mix-

ture of alcohol 72 (157 mg, 0.594 mmol) and NaHCO₃(574 mg,

6.83 mmol) in CH₂Cl₂(13.5 mL) and the mixture was stirred for 90 min

at room temperature. The mixture was added to a well-stirred mixture of

saturated aqueous NaS₂O₃(15 mL), saturated aqueous NaHCO₃(15 mL)

and diethyl ether (30 mL). The mixture was stirred for 1 h at room tem-

perature and extracted with diethyl ether (2î50 mL). The combined or-

ganic layers were washed with brine (20 mL), then dried (Na₂SO₄) and

concentrated at room temperature. The crude aldehyde was immediately

used in the next step without purification.

DMF (0.10 mL) and degassed THF (6 mL), then

a solution of

[Pd₂dba₃¥CHCl₃] (0.3 mg) in degassed DMF (20 mL) at 08C. The mixture

was stirred for 14 h at room temperature, diluted simultaneously with

ethyl acetate (1 mL) and saturated aqueous NaHCO₃(1 mL), and ex-

tracted with ethyl acetate (3î5 mL). The combined organic layers were

washed with 0.1m pH 7 phosphate buffer (1 mL), brine (1 mL), then

dried (Na₂SO₄) and concentrated at room temperature. The residue was

purified by flash chromatography (silica gel, CH₂Cl₂/ethanol/pyridine

100:5:1 to 90:9:1), then preparative reversed-phase HPLC (VP 250/10

NUCLEOSIL 100-5 C18 HD, flow rate 2.5 mLmin^ꢀ1, t=0 min, 30%

CH₃CN, 70% H₂O!t=19 min, 80% CH₃CN, 20% H₂O!t=34 min,

80% CH₃CN, 20% H₂O!t=35 min, 99% CH₃CN, 1% H₂O, t_R=

21.5 min, UV detection 210 nm) to give triene 66 (1.2 mg, 40%) as a col-

ourless oil. R_f=0.31 (CH₂Cl₂/ethanol 20:1); ¹H NMR (400 MHz, CDCl₃):

d = 6.99 (dd, J=9.6, 6.1 Hz, 1H), 6.60 (t, J=11.3 Hz, 1H), 6.57 6.49 (m,

1H), 6.12 (t, J=11.3 Hz, 1H), 6.05 (t, J=10.8 Hz, 1H), 5.96 (d, J=

9.6 Hz, 1H), 5.80 (dq, J=14.9, 6.8 Hz, 1H), 5.42 (t, J=9.8 Hz, 1H), 5.00

4.92 (m, 1H), 4.47 (t, J=9.8 Hz, 1H), 4.41 4.31 (m, 4H), 4.08 (dd, J=

A 0.5m solution of potassium hexamethyldisilazide in toluene (1.78 mL,

0.89 mmol) over 15 min at ꢀ788C was added dropwise to a solution of

[18]crown-6 (630 mg, 2.38 mmol) and O,O’-bis(2,2,2-trifluoroethyl)-phos-

phono-acetic acid methyl ester (0.25 mL, 1.19 mmol) in THF (13 mL)

and the solution was stirred at ꢀ788C for 35 min. A solution of the alde-

hyde synthesized as described above in THF (1 mL, and 2î0.5 mL wash-

ings) was added dropwise over a period of 45 min at ꢀ788C and the mix-

ture was stirred for 3 h at ꢀ788C. The mixture was quenched with satu-

2775

Chem. Eur. J. 2004, 10, 2759 2780

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Waldmann and L. Bialy

FULL PAPER

rated aqueous NH₄Cl (15 mL), warmed to room temperature, diluted

with water (15 mL) and extracted with diethyl ether (3î100 mL). The

combined organic layers were washed with brine (40 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 3:1) to give the ester 73

(151 mg, 80% over 2 steps) as a colourless oil. R_f=0.37 (cyclohexane/

ethyl acetate 3:1); [a]_D²⁰=+ 64 (c=0.16, CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d = 6.16 (dd, J=11.7, 10.4 Hz, 1H), 5.73 (dd, J=11.5, 1.0 Hz,

1H), 4.65 4.61 (m, 2H), 4.60 (s, 2H), 3.86 3.78 (m, 1H), 3.70 (s, 3H),

3.51 3.47 (m, 2H), 3.39 (s, 3H), 3.35 (s, 3H), 3.21 (t, J=5.5 Hz, 1H),

1.75 1.57 (m, 4H), 1.52 1.42 (m, 1H), 1.21 1.10 (m, 1H), 1.04 (d, J=

6.8 Hz, 3H), 0.97 (d, J=7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d =

166.7, 153.8, 118.2, 98.5, 96.5, 87.4, 68.3, 56.2, 55.2, 51.2, 36.5, 35.1, 28.6,

27.8, 16.5, 15.3; HRMS (FAB, 3-NBA): m/z: calcd for C₁₆H₃₁O₆:

341.1940, found 341.1957 [M+Na]⁺.

1.65 (m, 3H), 1.60 1.49 (m, 1H), 1.21 1.09 (m, 1H), 1.00 (d, J=7.0 Hz,

3H), 0.83 (d, J=6.7 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 164.7,

151.9, 143.3, 143.2, 143.2, 141.5, 128.0, 128.0, 127.2, 127.2, 125.2, 120.1,

120.1, 120.1, 83.7, 69.2 (d, J(¹³C,³¹P)=6.2 Hz), 68.2 (d, J(¹³C,³¹P)=6.2 Hz),

48.1 (d, J(¹³C,³¹P)=8.0 Hz), 33.7, 30.5, 28.6, 27.4 (d, J(¹³C,³¹P)=6.9 Hz),

14.6, 10.9; ³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.50; HRMS (FAB, 3-

NBA): m/z: calcd for C₃₉H₃₉NaO₆P: 657.2382, found 657.2393 [M+Na]⁺.

(Triethylammonium)-monohydrogen-{(4S)-4-[(2S,3S)-3-methyl-6-oxo-3,6-

dihydro-2H-pyran-2-yl]-pentyl} phosphate (77): NEt₃(0.84 mL, 6.04 mL)

was added dropwise at 08C to a solution of the phosphotriester 76

(96 mg, 0.151 mmol) in CH₃CN (4 mL) and the solution was stirred for

18 h at room temperature. The solution was diluted with toluene (2 mL)

and concentrated at room temperature. The residue was diluted with

water (10 mL) and diethyl ether (5 mL). After separation from the or-

ganic layer, the aqueous layer was washed (no shaking!) with small por-

tions of diethyl ether (5 mL) until no UV-detectable material (TLC, R_f

ca. 1, ethyl acetate) was present in the aqueous layer. The aqueous layer

was lyophilized to yield the phosphate 77 as colourless oil (43 mg, 75%).

R_f=0 (ethyl acetate); [a]²_D⁰=+104 (c=0.45, MeOH); ¹H NMR

(400 MHz, CD₃OD): d = 7.16 (dd, J=9.6, 6.5 Hz, 1H), 5.93 (dd, J=9.6,

0.8 Hz, 1H), 4.08 (dd, J=10.4, 2.9 Hz, 1H), 3.90 (q, J=6.5 Hz, 2H), 3.18

(q, J=7.2 Hz, 6H), 2.59 (dquint, J=3.1, 6.8 Hz, 1H), 2.00 1.75 (m, 3H),

1.69 1.57 (m, 1H), 1.32 1.23 (m, 1H), 1.31 (t, J=7.4 Hz, 9H), 1.01 (d,

J=7.0 Hz, 3H), 0.95 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d

= 167.2, 155.0, 120.0, 85.5, 66.6 (d, J(¹³C,P)=5.4 Hz), 47.5, 34.9, 31.6,

29.8, 28.9 (d, ³J(¹³C,P)=7.7 Hz), 14.8, 10.9, 9.1; ¹³C NMR (100 MHz,

CD₃OD): d = 167.2, 155.0, 120.0, 85.5, 66.6 (d, ²J(¹³C,P)=5.4 Hz), 47.5,

34.9, 31.6, 29.8, 28.9 (d, ³J(¹³C,P)=7.7 Hz), 14.8, 10.9, 9.1; ³¹P NMR

(162 MHz, CD₃OD): d = 1.86; HRMS (FAB, 3-NBA): m/z: calcd for

C₁₁H₂₀O₆P: 279.0998, found 279.1009 [M+H]⁺.

(5S,6S)-6-((1S)-4-Hydroxy-1-methylbutyl)-5-methyl-5,6-dihydro-pyran-2-

one (74): 1m aqueous HCl (7.5 mL) was added to a solution of ester 73

(151 mg, 0.475 mmol) in THF (15 mL) and the mixture was stirred for

15 h at 608C. The mixture was cooled to room temperature, quenched

with saturated aqueous NaHCO₃, extracted with diethyl ether (3î

30 mL). The combined organic layers were washed with brine (20 mL),

then dried (Na₂SO₄) and concentrated. The residue was purified by flash

chromatography (silica gel, cyclohexane/ethyl acetate 2:5) to give lactone

74 (83 mg, 88%) as a colourless oil. R_f=0.25 (cyclohexane/ethyl acetate

2:1); ¹H NMR (400 MHz, CDCl₃): d = 6.98 (dd, J=9.6, 6.5 Hz, 1H),

5.94 (dd, J=9.6, 0.5 Hz, 1H), 3.99 (dd, J=10.4, 3.1 Hz, 1H), 3.69 3.60

(m, 2H), 2.46 (dquint, J=3.1, 6.6 Hz, 1H), 1.96 1.79 (m, 2H), 1.75 1.69

(m, 1H), 1.58 1.47 (m, 1H), 1.27 1.16 (m, 1H), 1.01 (d, J=7.0 Hz, 3H),

0.90 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 165.0, 152.1,

120.0, 84.0, 63.1, 33.8, 30.5, 29.7, 28.6, 14.7, 10.9; HRMS (FAB, 3-NBA):

m/z: calcd for C₁₁H₁₉O₃: 199.1334, found 199.1322 [M+H]⁺.

{(1S,2S,3R)-3-(tert-Butyldiphenylsilyloxy)-2-methyl-1-[(3S,4S)-3-((3S)-3-

methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-ynyl}-bis(9H-fluo-

Bis(9H-fluoren-9-ylmethyl)-diisopropylamidophosphite (75):^[56]CDCl₃

was filtered through a layer of basic alumium oxide before use. Diisopro-

pylaminophosphodichloridite (2.28 g, 11.3 mmol) at 08C Diisopropylami-

nophosphodichloridite (2.28 g, 11.3 mmol) at 08C to a solution of 9-fluo-

renylmethanol (4.44 g, 22.6 mmol) and EtNiPr₂(5.88 mL, 41.6 mmol) in

THF (25 mL). The mixture was stirred for 1 h at room temperature, then

filtered, and diluted with a 1m pH 7 buffer (pH of aqueous phase should

be 7 after shaking!) and ethyl acetate (200 mL). The mixture was extract-

ed with ethyl acetate (3î150 mL), then dried (Na₂SO₄) and concentrat-

ed. The residue (5.9 g, quantitative) was further purified by flash chroma-

tography (silica gel, cyclohexane/ethyl acetate/dimethylethylamine

20:1:0.2) to give the phosphoramidite 75 (551 mg starting from 1.0 g of

crude product, 53%) as a pale yellow oil. R_f=0.6 (cyclohexane/ethyl ace-

ren-9-ylmethyl) phosphate (78):

A solution of phosphoramidite 75

(905 mg, 1.73 mmol) in CH₂Cl₂(7.2 mL) at 08C was added to a solution

of alcohol 51 (300 mg, 0.58 mmol) and tetrazole (109 mg, 1.56 mmol) in

CH₃CN (9 mL) and the mixture was stirred for 5.5 h at room tempera-

ture. To the reaction mixture was added a 0.1m solution of iodine in pyri-

dine/THF/H₂O 2:7:1 (17.4 mL, 1.74 mmol) over 1 min and the mixture

was stirred for 5 min at room temperature. The reaction mixture was

poured to a well-stirred mixture of saturated aqueous NaHCO₃(30 mL),

0.1m aqueous Na₂S₂O₃(30 mL) and ethyl acetate (150 mL). The mixture

was extracted with ethyl acetate (2î150 mL), the combined organic

layers were washed with 1m aqueous KH₂PO₄(60 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 1:1 to 3:2) to give phospho-

triester 78 (542 mg, 95%) as a colourless oil. R_f=0.49 (cyclohexane/ethyl

acetate 1:1). ¹H NMR (400 MHz, CDCl₃): d = 7.79 7.76 (m, 8H), 7.56

7.16 (m, 18H), 6.95 (dd, J=9.6, 6.5 Hz, 1H), 5.97 (d, J=9.6 Hz, 1H),

4.38 4.03 (m, 8H), 3.73 (dd, J=10.4, 3.1 Hz, 1H), 2.35 (dquint, J=3.1,

6.8 Hz, 1H), 2.25 (d, J=2.2 Hz, 1H), 2.00 1.93 (m, 1H), 1.89 1.79 (m,

1H), 1.39 1.60 (m, 3H), 1.15 (d, J=6.6 Hz, 1H), 1.10 1.05 (m, 1H), 0.90

(d, J=7.0 Hz, 3H), 0.60 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz,

CDCl₃): d = 164.6, 151.7, 143.5, 143.4, 143.3, 143.3, 141.5, 141.5, 141.4,

136.2, 136.0, 133.5, 133.4, 129.9, 129.8, 127.9, 127.8, 127.8, 127.6, 127.2,

127.2, 127.2, 125.4, 125.4, 125.3, 120.3, 120.0, 120.0, 120.0, 83.7, 82.2, 81.2

(d, J(¹³C,³¹P)=6.2 Hz), 75.3, 69.2 (d, J(¹³C,³¹P)=6.2 Hz), 69.0 (d,

J(¹³C,³¹P)=5.8 Hz), 64.4, 48.1 (d, J(¹³C,³¹P)=8.5 Hz), 48.1 (d, J(¹³C,³¹P)=

8.1 Hz), 43.2 (d, J(¹³C,³¹P)=6.5 Hz), 34.2, 30.4, 29.8 (d, J(¹³C,³¹P)=

2.3 Hz), 27.0, 26.3, 19.5, 14.3, 10.9, 10.6; ³¹P NMR (162 MHz, CDCl₃): d

= ꢀ1.15; HRMS (FAB, 3-NBA): m/z: calcd for C₆₀H₆₄O₇PSi: 955.4159,

found 955.4144 [M+H]⁺.

1

tate/dimethylethylamine 20:1:0.2); H NMR (400 MHz, CDCl₃): d = 7.74

(t, J=6.8 Hz, 4H), 7.66 7.62 (m, 4H), 7.40 7.34 (m, 4H), 7.28 (dq, J=

7.6, 1.2 Hz, 4H), 4.17 (t, J=7.0 Hz, 2H), 4.00 (dt, J=10.0, 6.6 Hz, 2H),

3.80 (dt, J=10.0, 7.2 Hz, 2H), 3.70 3.60 (m, 2H), 1.15 (d, J=6.8 Hz,

12H); ¹³C NMR (100 MHz, CDCl₃): d = 144.8, 144.5, 141.3, 141.1, 127.3,

127.3, 126.8, 126.7, 125.4, 125.1, 119.8, 119.7, 66.0 (d, ²J(¹³C,³¹P)=

16.9 Hz), 49.3 (d, ³J(¹³C,³¹P)=7.7 Hz), 43.2 (d, J(¹³C,³¹P)=12.3 Hz, 24.8

(d, J(¹³C,³¹P)=6.9 Hz); ³¹P NMR (162 MHz, CDCl₃): d = 147.3.

Bis(9H-fluoren-9-ylmethyl)-{(4S)-4-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-

2H-pyran-2-yl]-pentyl} phosphate (76): A solution of the phosphorami-

dite 75 (209 mg, 0.400 mmol) in CH₂Cl₂(2 mL) at 08C was added to a

solution of the alcohol 74 (20 mg, 0.1 mmol) and tetrazole (21 mg,

0.3 mmol) in CH₂Cl₂(2 mL) and the mixture was stirred for 4.5 h at

room temperature. The reaction was cooled to ꢀ788C and 4-chloroper-

benzoic acid (247 mg, 70% purity by weight, 1 mmol) was added. The

mixture was stirred for 90 min at 08C, then quenched with 10% aqueous

sodium sulfite (15 mL), and extracted with CH₂Cl₂(3î50 mL). The com-

bined organic layers were washed with 1m aqueous KH₂PO₄(8 mL), sa-

turated aqueous NaHCO₃(20 mL), brine (2î10 mL), then dried

(Na₂SO₄) and concentrated. The residue was purified by flash chromatog-

raphy (silica gel, cyclohexane/ethyl acetate 2:1 to 2:3) to give the phos-

photriester 76 (56 mg, 88%) as a pale yellow oil. R_f=0.19 (cyclohexane/

ethyl acetate 2:3); [a]²_D⁰=+ 44.8 (c=0.25, CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d = 7.72 (t, J=7.6 Hz, 4H), 7.56 (d, J=7.6 Hz, 2H), 7.50 (d, J=

7.6 Hz, 4H), 7.41 7.23 (m, 8H), 6.97 (dd, J=9.4, 6.5 Hz, 1H), 5.96 (d,

J=9.4 Hz, 1H), 4.31 4.22 (m, 4H), 4.17 4.13 (m, 2H), 3.94 (dd, J=10.6,

3.2 Hz, 1H), 3.93 3.84 (m, 2H), 2.43 (dquint, J=2.9, 6.7 Hz, 1H), 1.90

Bis(9H-fluoren-9-ylmethyl)-{(1S,2S,3R)-3-Hydroxy-2-methyl-1-[(3S,4S)-3-

((3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-4-ynyl}

phosphate (79): The reaction was performed in a teflon vessel. 70% HF/

py (1.2 mL) and the solution was stirred for 24 h at room temperature

was added to a solution of the TBDPS-ether 78 (145 mg, 0.152 mmol) in

THF (5.7 mL). Another portion of 70% HF/py (1.2 mL) was added and

the solution was stirred for another 8 h at room temperature. The reac-

tion mixture was carefully (!) added to a well-stirred mixture of ethyl

acetate (100 mL) and saturated aqueous NaHCO₃(100 mL). The mixture

was extracted with ethyl acetate (3î100 mL). The combined organic

2776

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

Chem. Eur. J. 2004, 10, 2759 2780

Cytostatin

2759 2780

layers were washed with brine (75 mL), then dried (Na₂SO₄) and concen-

trated. The residue was purified by flash chromatography (silica gel, cy-

clohexane/ethyl acetate 2:1 to 1:1) to give alcohol 79 (89 mg, 82%) as a

white solid. R_f=0.16 (cyclohexane/ethyl acetate 1:1); [a]²_D⁰=+54.6 (c=

14.9, 10.9, 8.4; ³¹P NMR (162 MHz, CDCl₃): d = 1.29; HRMS (FAB, 3-

NBA): m/z: calcd for C₄₄H₄₇IO₇P: 845.2104, found 845.2109 [M+H]⁺.

Bis(9H-fluoren-9-ylmethyl)-{(1S,2S,3R,4Z,6Z,8Z)-3-hydroxy-2-methyl-1-

[(3S,4S)-3-((3S)-3-methyl-3,6-dihydro-2H-hydropyran-2-yl)-butyl]-deca-

4,6,8-trienyl} phosphate (82): All operations were done under exclusion

of slightly. To a solution of (Z)-alkenyliodide 81 (7.0 mg, 8.3 mmol) was

1

0.46, CHCl₃); m.p. 798C; H NMR (400 MHz, CDCl₃): d = 7.74 7.68 (m,

4H), 7.57 7.44 (m, 4H), 7.41 7.22 (m, 8H), 6.95 (dd, J=9.6, 6.5 Hz, 1H),

5.93 (dd, J=9.6, 0.4 Hz, 1H), 4.74 4.67 (m, 1H), 4.35 (dt, J=9.8, 6.3 Hz,

1H), 4.27 4.08 (m, 6H), 3.86 (dd, J=10.4, 2.9 Hz, 1H), 2.43 (d, J=

2.2 Hz, 1H), 2.40 (dquint, J=2.8, 6.9 Hz, 1H), 1.85 1.65 (m, 4H), 1.55

1.45 (m, 1H), 1.16 1.08 (m, 1H), 0.98 (d, J=7.0 Hz, 3H), 0.93 (d, J=

7.0 Hz, 3H), 0.79 (d, J=6.6 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d =

164.5, 151.7, 143.3, 143.0, 143.0, 141.5, 141.5, 141.4, 141.4, 128.0, 128.0,

128.0, 127.3, 127.2, 127.2, 125.2, 125.1, 120.2, 120.1, 120.1, 84.3, 83.7, 78.6

(d, J(¹³C,³¹P)=6.2 Hz), 73.1, 69.5 (d, J(¹³C,³¹P)=6.9 Hz), 69.4 (d,

J(¹³C,³¹P)=6.2 Hz), 63.6, 48.0 (d, J(¹³C,³¹P)=9.2 Hz), 47.9 (d, J(¹³C,³¹P)=

8.5 Hz), 43.8 (d, J(¹³C,³¹P)=3.8 Hz), 33.9, 30.7 (d, J(¹³C,³¹P)=4.6 Hz),

30.4, 28.6, 14.8, 10.8, 9.2; ³¹P NMR (162 MHz, CDCl₃): d = 0.96; HRMS

(FAB, 3-NBA): m/z: calcd for C₄₄H₄₆O₇P: 717.2981, found 717.2996

[M+H]⁺.

added

a solution of the freshly prepared (Z)-alkenylstannane 25

(12.7 mg, 35.6 mmol) in degassed DMF (0.10 mL) and degassed THF

(7 mL), then a 0.1m solution of Pd(CH₃CN)₂Cl₂in degassed DMF (20 mL,

2 mmol) was added at 08C. The mixture was stirred for 20.5 h at room

temperature, a total of 10 mL (8%) of the reaction solution was taken to

monitor reaction progress. The solution was diluted simultaneously with

ethyl acetate (5 mL) and saturated aqueous NaHCO₃(5 mL), and ex-

tracted with ethyl acetate (3î10 mL). The combined organic layers were

washed a 0.1m pH 7 phosphate buffer (3 mL), brine (3 mL), then dried

(Na₂SO₄) and concentrated at room temperature. The residue was puri-

fied by a SEPAK cartridge (C4-reversed phase material, H₂O to metha-

nol), then reversed-phase HPLC (VP 250/10 NUCLEOSIL 100-5 C18

HD, flow rate 2.5 mLmin^ꢀ1, t=0 min, 50% CH₃CN, 50% H₂O!t=

20 min, 90% CH₃CN, 10% H₂O!t=45 min, 90% CH₃CN, 10% H₂O!

t=50 min, 99% CH₃CN, 1% H₂O, t_R=34.7 min, UV detection 300 nm)

to give triene 82 (3.7 mg, 57%; 62% after 10% correction) as a colour-

less oil. R_f=0.30 (acetonitrile/water 3:1, RP-C8-TLC); [a]²_D⁰=+49 (c=

0.235 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 7.75 7.68 (m, 4H),

7.59 7.51 (m, 3H), 7.48 7.45 (m, 1H), 7.41 7.19 (m, 8H), 6.95 (dd, J=

9.6, 6.6 Hz, 1H), 6.54 (t, J=11.4 Hz, 1H), 6.54 6.46 (m, 1H), 5.94 (dd,

J=9.6, 0.6 Hz, 1H), 5.91 (t, J=11.3 Hz, 1H), 5.83 (t, J=11.0 Hz, 1H),

5.75 (dq, J=14.9, 6.8 Hz, 1H), 5.37 (t, J=10.6 Hz, 1H), 4.85 4.78 (m,

1H; CHOP), 4.35 4.21 (m, 5H), 4.17 4.11 (m, 2H), 3.88 (dd, J=10.4,

2.9 Hz, 1H), 2.80 2.67 (brs, 1H, OH), 2.41 (dquint, J=2.9, 7.0 Hz, 1H),

1.81 (dd, J=6.8, 1.0 Hz, 1H), 1.80 1.67 (m, 3H), 1.62 1.47 (m, 2H),

1.19 1.09 (m, 1H), 0.99 (d, J=7.0 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H), 0.72

(d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 164.6, 151.8, 143.2,

143.1, 143.1, 141.5, 141.5, 141.5, 141.5, 132.3, 132.0, 131.3, 128.0, 128.0,

128.0, 127.3, 127.3, 126.9, 125.7, 125.3, 125.2, 125.2, 121.9, 120.2, 120.1,

120.1, 120.1, 83.8, 79.5 (d, J(¹³C,³¹P)=6.2 Hz), 69.6 (d, J(¹³C,³¹P)=6.2 Hz),

69.5 (d, J(¹³C,³¹P)=6.2 Hz), 67.4 (CHOH), 48.1 (d, J(¹³C,³¹P)=5.4 Hz),

48.0 (d, J(¹³C,³¹P)=5.4 Hz), 43.1 (d, J(¹³C,³¹P)=3.8 Hz), 34.0, 30.8 (d,

J(¹³C,³¹P)=3.8 Hz), 30.5, 28.7, 18.6, 14.8, 10.9, 8.8; ³¹P NMR (162 MHz,

Bis(9H-fluoren-9-ylmethyl)-{(1S,2S,3R)-3-Hydroxy-5-iodo-2-methyl-1-

[(3S,4S)-3-((3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-

4-ynyl} phosphate (80): A solution of AgNO₃(7.5 mg, 44 mmol) in DMF

(0.23 mL) was added to a solution of the alkyne 79 (210 mg, 0.293 mmol)

and N-iodosuccinimide (99 mg, 0.440 mmol) in DMF (3.3 mL) and the

mixture was stirred for 90 min at room temperature. The reaction mix-

ture was quenched by addition of ice-cold water (50 mL) and extracted

with ethyl acetate (3î100 mL). The combined organic layers were

washed with brine (50 mL), then dried (Na₂SO₄) and concentrated. The

residue was purified by flash chromatography (silica gel, cyclohexane/

ethyl acetate 1:1 to 2:3) to give the iodoalkyne 80 (247 mg, quantitative)

as an off-white solid. R_f=0.30 (cyclohexane/ethyl acetate 2:3); [a]_D²⁰

=

+41.4 (c=0.5, CHCl₃); m.p. 1178C; ¹H NMR (400 MHz, CDCl₃): d =

7.74 7.68 (m, 4H), 7.56 7.43 (m, 4H), 7.41 7.21 (m, 8H), 6.95 (dd, J=

9.6, 6.5 Hz, 1H), 5.93 (dd, J=9.6, 0.6 Hz, 1H), 4.68 4.62 (m, 1H), 4.36

4.07 (m, 7H), 3.86 (dd, J=10.4, 3.1 Hz, 1H), 2.40 (dquint, J=3.1, 7.0 Hz,

1H), 1.85 1.67 (m, 4H), 1.52 1.43 (m, 1H), 1.15 1.06 (m, 1H), 0.98 (d,

J=7.0 Hz, 3H), 0.90 (d, J=6.8 Hz, 3H), 0.78 (d, J=6.8 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃): d = 164.5, 151.7, 143.3, 143.1, 143.0, 141.5,

141.5, 141.4, 128.1, 128.0, 128.0, 127.3, 127.3, 127.3, 125.2, 125.2, 125.1,

120.2, 120.2, 120.1, 120.1, 95.0, 83.7, 78.6 (d, J(¹³C,³¹P)=6.2 Hz), 69.5,

69.4, 65.3, 48.0 (d, J(¹³C,³¹P)=8.3 Hz), 48.0 (d, J(¹³C,³¹P)=8.2 Hz), 44.1

(d, J(¹³C,³¹P)=3.8 Hz), 34.0, 30.8 (d, J(¹³C,³¹P)=4.6 Hz), 30.5, 28.6, 14.9,

10.9, 9.3, 1.3; ³¹P NMR (162 MHz, CDCl₃): d=0.80; HRMS (FAB, 3-

NBA): m/z: calcd for C₄₄H₄₅IO₇P: 843.1948, found 843.1944 [M+H]⁺.

CDCl₃):

d 0.74; HRMS (FAB, 3-NBA): m/z: calcd for C₄₉H₅₄O₇P:

785.3607, found 785.3621 [M+H]⁺.

(4S,5S,6S,9S,10S,11S)-Cytostatin (1a): Only deionized water was used for

all operations. To a solution of phosphotriester 82 (11.7 mg, 15 mmol) in

CH₃CN (2.5 mL) was added dropwise NEt₃(0.55 mL) at 08C and the sol-

ution was stirred for 20 h at room temperature. The solution was diluted

with toluene (1 mL) and concentrated at room temperature. The residue

was diluted with water (10 mL) and diethyl ether (5 mL). After separa-

tion from the organic layer, the aqueous layer was washed (no shaking!)

with small portions of diethyl ether (5 mL) until no UV-detectable mate-

rial (TLC, R_fca. 1, ethyl acetate) was present in the aqueous layer. The

aqueous layer was lyophilized to yield the monotriethylammonium phos-

phate 1a as colourless oil (8 mg, quantitative). The sodium salt was pre-

pared by chromatography on ion exchange resin (DOWEX 50W2, 50

100 mesh, strongly acidic, 10 cm height, 0.8 cm diameter, methanol/water

1:1; the resin was sequentially washed with methanol/water 1:1, 1.0m

aqueous NaHCO₃(CO₂!), H₂O, methanol/water 1:1 before use) to yield

Bis(9H-fluoren-9-ylmethyl)-{(1S,2S,3R,4Z)-3-hydroxy-5-iodo-2-methyl-1-

[(3S,4S)-3-((3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-butyl]-pent-

4-enyl} phosphate (81): A solution of acetic acid (0.36 mL, 0.864 mmol)

in 2-propanol (4.6 mL) was added dropwise over a period of 1 h to a mix-

ture of iodoalkyne 80 (210 mg, 0.249 mmol) and potassium azodicarboxy-

late (84 mg, 0.432 mmol) in 2-propanol (1.56 mL) and 1,4-dioxane

(0.21 mL) and the mixture was stirred for 14.5 h at room temperature.

The reaction mixture was quenched with

a 0.1m phosphate buffer

(50 mL) and extracted with ethyl acetate (3î100 mL). The combined or-

ganic layers were washed with brine (30 mL), then dried (Na₂SO₄) and

concentrated. The residue was purified by flash chromatography (silica

gel, cyclohexane/ethyl acetate 2:3 to 1:3) to give (Z)-iodoalkene 81

(139 mg, 63%) as a white solid which was contaminated with saturated

lactone 88 (7 mg) and used in the next step without further purification.

Residual starting compound 80 (45 mg, 21%) could be reisolated after

chromatography. R_f=0.15 (cyclohexane/ethyl acetate 2:3); [a]²_D⁰=+49

(c=0.235, CHCl₃); m.p. 778C; ¹H NMR (400 MHz, CDCl₃): d = 7.75

7.67 (m, 4H), 7.61 7.50 (m, 4H), 7.41 7.20 (m, 8H), 6.95 (dd, J=9.6,

6.5 Hz, 1H), 6.39 (d, J=7.6 Hz, 1H), 6.16 (dd, J=8.4, 7.6 Hz, 1H), 5.94

(dd, J=9.6, 0.6 Hz, 1H), 4.82 4.76 (m, 1H), 4.45 4.23 (m, 4H), 4.21 4.13

(m, 3H), 3.87 (dd, J=10.4, 2.9 Hz, 1H), 2.41 (dquint, J=2.9, 6.8 Hz,

1H), 1.85 1.64 (m, 4H), 1.56 1.47 (m, 1H), 1.18 1.10 (m, 1H), 0.99 (d,

J=7.0 Hz, 3H), 0.81 (d, J=6.7 Hz, 3H), 0.78 (d, J=7.0 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃): d = 164.6, 151.7, 143.5, 143.2, 143.1, 142.0,

141.5, 141.5, 128.1, 128.0, 127.3, 125.5, 125.4, 125.3, 120.2, 120.2, 120.2,

120.1, 84.6, 83.8, 78.9, 74.2, 70.0, 69.8, 48.1, 42.7, 34.1, 30.7, 30.5, 28.7,

1a as an amorphous pale yellow powder. R_f=0 (ethyl acetate); [a]_D²⁰

=

+46 (c=0.285, methanol); ¹H NMR (400 MHz, CD₃OD): d = 7.15 (dd,

J=9.6, 6.6 Hz, 1H), 6.59 (t, J=11.5 Hz, 1H), 6.62 6.54 (m, 1H), 6.26 (t,

J=11.1 Hz, 1H), 5.99 (t, J=11.5 Hz, 1H), 5.93 (dd, J=9.6, 0.6 Hz, 1H),

5.76 (dq, J=14.9, 6.8 Hz, 1H), 5.41 (t, J=10.2 Hz, 1H), 4.59 (t, J=

10.0 Hz, 1H), 4.55 4.48 (m, 1H), 4.11 (dd, J=10.2, 2.9 Hz, 1H), 2.58

(dquint, J=3.1, 7.0 Hz, 1H), 2.05 1.96 (m, 1H), 1.86 1.75 (m, 2H), 1.80

(dd, J=6.8, 1.0 Hz, 3H), 1.58 1.46 (m, 2H), 1.30 1.24 (m, 1H), 1.01 (d,

J=7.0 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H);

¹³C NMR (100 MHz, CD₃OD, determined by HSQC and HMBC): d =

167.1, 154.9, 133.7, 132.0, 131.5, 127.9, 126.3, 123.3, 119.9, 85.4, 75.2, 68.7,

43.9, 35.2, 31.5, 31.4, 29.2, 18.4, 14.7, 10.9, 9.1; ³¹P NMR (162 MHz,

CD₃OD): d = 3.96; HRMS (FAB, 3-NBA): m/z: calcd for C₂₁H₃₃NaO₇P:

451.1862, found 451.1873 [M+Na]⁺.

2777

Chem. Eur. J. 2004, 10, 2759 2780

www.chemeurj.org

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Waldmann and L. Bialy

FULL PAPER

(Triethylammonium)-hydrogen-{(1S,2S,3R,4Z)-3-hydroxy-2-methyl-1-

{(3S)-3-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-butyl}-pent-4-

ynyl} phosphate (83): Phosphate 83 was synthesized by analogy to mono-

triethylammonium salt of 1a starting from phosphotriester 79 (5.0 mg,

7.0 mmol) to give an amorphous white powder (3.2 mg, 98%). R_f=0

(ethyl acetate); [a]²_D⁰=+75.6 (c=0.16, methanol); ¹H NMR (400 MHz,

CD₃OD): d = 7.16 (dd, J=9.6, 6.5 Hz, 1H), 5.93 (dd, J=9.6, 0.6 Hz,

1H), 4.44 (dddd, J=10.2, 8.0, 6.3, 2.3 Hz, 1H), 4.32 (dd, J=9.8, 2.2 Hz,

1H), 4.11 (dd, J=10.2, 2.9 Hz, 1H), 3.20 (q, J=7.4 Hz, 6H), 2.73 (d, J=

2.2 Hz, 1H), 2.59 (dquint, J=3.1, 7.0 Hz, 1H), 2.01 1.91 (m, 1H), 1.88

1.74 (m, 3H), 1.53 (ddt, J=16.8, 4.5, 8.0 Hz, 1H), 1.31 (t, J=7.2 Hz, 9H),

1.34 1.24 (m, 1H), 1.02 (d, J=6.8 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.97

45.2 (d, J(¹³C,³¹P)=4.6 Hz), 35.4, 31.6, 31.5, 29.4, 14.9, 11.0, 9.7, 9.2, 4.5;

³¹P NMR (162 MHz, CD₃OD): d = 2.87; HRMS (FAB, 3-NBA): m/z:

calcd for C₁₆H₂₄NaIO₇P: 509.0202, found 509.0211 [M+Na]⁺.

(Triethylammonium)-hydrogen-{(1S,2S,3R,4Z)-3-hydroxy-5-iodo-2-

methyl-1-{(3S)-3-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-

butyl}-pent-4-enyl} phosphate (87): Phosphate 87 was synthesized by

analogy to the monotriethylammonium salt of 1a starting from phospho-

triester 81 (5.0 mg, 5.9 mmol) to give an amorphous white powder

(3.5 mg, quantitative). R_f=0 (ethyl acetate); [a]²_D⁰=+76.0 (c=0.175,

MeOH); ¹H NMR (400 MHz, CD₃OD): d = 7.16 (dd, J=9.6, 6.5 Hz,

1H), 6.51 (dd, J=7.7, 0.8 Hz, 1H), 6.25 (dd, J=8.4, 7.7 Hz, 1H), 5.93

(dd, J=9.6, 0.8 Hz, 1H), 4.49 (dddd, J=10.4, 8.2, 5.9, 2.2 Hz, 1H), 4.37

(t, J=9.2 Hz, 1H), 4.10 (dd, J=10.2, 2.9 Hz, 1H), 3.20 (q, J=7.2 Hz,

6H), 2.62 2.54 (m, 1H), 2.05 1.94 (m, 1H), 1.87 1.67 (m, 3H), 1.53 (ddt,

J=17.2, 4.5, 8.2 Hz, 1H), 1.32 (t, J=7.2 Hz, 9H), 1.34 1.24 (m, 1H), 1.01

(d, J=7.0 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H);

¹³C NMR (100 MHz, CD₃OD): d = 167.4, 155.0, 143.9, 120.0, 85.5, 83.7,

75.8, 75.2 (d, J(¹³C,³¹P)=5.4 Hz), 47.8, 43.3 (d, J(¹³C,³¹P)=4.6 Hz), 35.4,

31.6, 31.6 (J(¹³C,³¹P)=3.1 Hz), 29.4, 14.9, 10.9, 9.2, 9.1; ³¹P NMR

(162 MHz, CD₃OD): d = 3.05; HRMS (FAB, 3-NBA): m/z: calcd for

C₁₆H₂₆INaO₇P: 511.0359, found 511.0351 [M+Na]⁺.

(d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CD₃OD): d

= 167.4, 155.1,

120.0, 85.9, 85.6, 74.9 (d, J(¹³C,³¹P)=5.4 Hz), 73.8, 64.8, 47.8, 44.9 (d,

J(¹³C,³¹P)=3.8 Hz), 35.4, 31.6, 31.6 (d, J(¹³C,³¹P)=3.1 Hz), 29.4, 14.9,

10.9, 9.6, 9.2; ³¹P NMR (162 MHz, CD₃OD): d = 2.96; HRMS (FAB, 3-

NBA): m/z: calcd for C₁₆H₂₅NaO₇P: 383.1236, found 383.1223 [M+Na]⁺.

{1-{(1S,2S,5S)-2-{Bis(9H-fluoren-9-ylmethoxy)-phosphoryloxy}-1-methyl-

5-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-hexyl}-prop-2-ynyl}

ethanoate (84): N,N-Dimethylaminopyridine (ca. 0.2 mg) and acetanhy-

dride (5 mL) were added to a solution of the alcohol 79 (10 mg, 14 mmol)

in pyridine (0.1 mL) and the mixture was stirred for 1 h at room tempera-

ture. The reaction mixture was quenched with 1m aqueous KH₂PO₄

(10 mL) and extracted with ethyl acetate (3î15 mL). The combined or-

ganic layers were washed with brine (15 mL), then dried (Na₂SO₄) and

concentrated. The residue was purified by flash chromatography (silica

gel, cyclohexane/ethyl acetate 1:1 to 1:2) to give the ester 84 (6.4 mg,

Bis(9H-fluoren-9-ylmethyl)-{(1S,2S,3R,4Z)-3-hydroxy-5-iod-2-methyl-1-

[(3S,4S)-3-((3S)-3-methyl-6-oxo-tetrahydropyran-2-yl)-butyl]-pent-4-enyl}

phosphate (88): Reduced lactone 88 was isolated as side-product during

the synthesis of 80 and was purified by reversed-phase HPLC (VP 250/10

NUCLEOSIL 100-5 C18 HD, flow rate 2.5 mLmin^ꢀ1, t=0 min, 50%

CH₃CN, 50% H₂O!t=20 min, 90% CH₃CN, 10% H₂O!t=45 min,

90% CH₃CN, 10% H₂O!t=50 min, 99% CH₃CN, 1% H₂O, t_R=

31.1 min, UV detection 300 nm) to give a colourless oil (2.2 mg). R_f=0.3

(cyclohexane/ethyl acetate 1:2); [a]²_D⁰=ꢀ10.2 (c=0.205, CHCl₃);

¹H NMR (400 MHz, CDCl₃): d = 7.77 7.67 (m, 4H), 7.62 7.50 (m, 4H),

7.42 7.15 (m, 8H), 6.39 (d, J=7.6 Hz, 1H), 6.16 (t, J=8.2 Hz, 1H), 4.83

4.76 (m, 1H), 4.47 4.40 (m, 1H), 4.37 4.29 (m, 2H), 4.27 4.14 (m, 4H),

4.77 4.73 (dd, J=10.0, 2.2 Hz, 1H), 2.48 2.41 (m, 2H), 2.14 2.08 (m,

1H), 2.04 1.95 (m, 1H), 1.88 1.72 (m, 2H), 1.71 1.58 (m, 3H), 1.54 1.45

(m, 1H), 1.18 1.10 (m, 1H), 0.90 (d, J=7.0 Hz, 3H), 0.80 (d, J=6.8 Hz,

3H), 0.78 (d, J=7.0 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d = 172.0,

143.4, 143.2, 143.1, 142.0, 141.5, 141.5, 128.1, 128.1, 127.3, 127.3, 125.5,

125.4, 120.3, 120.2, 120.1, 86.5, 84.6, 79.1 (d, J(¹³C,³¹P)=6.3 Hz), 74.3, 70.0

(d, J(¹³C,³¹P)=5.7 Hz), 69.9 (d, J(¹³C,³¹P)=6.3 Hz), 48.1 (J(¹³C,³¹P)=

8.6 Hz), 48.0 (J(¹³C,³¹P)=5.7 Hz), 42.6, 34.9, 30.8, 29.1, 27.0, 26.7, 26.3,

14.9, 11.5, 8.4; ³¹P NMR (162 MHz, CDCl₃): d 0.97; HRMS (FAB, 3-

NBA): m/z: calcd for C₄₄H₄₉O₇PI: 847.2261, found 847.2256 [M+H]⁺.

60%) as a colourless oil. R_f=0.65 (cyclohexane/ethyl acetate 1:3); [a]_D²⁰

=

+70.3 (c=0.32, CHCl₃); ¹H NMR (400 MHz, CDCl₃): d = 7.73 7.66 (m,

4H), 7.59 (d, J=7.5 Hz, 1H), 7.53 7.49 (m, 2H), 7.44 (d, J=7.5 Hz, 1H),

7.40 7.19 (m, 8H), 6.96 (dd, J=9.6, 6.6 Hz, 1H), 5.94 (d, J=9.6 Hz, 1H),

5.26 (dd, J=8.4, 2.2 Hz, 1H), 4.56 4.49 (m, 1H), 4.30 4.04 (m, 6H), 3.89

(dd, J=10.4, 2.9 Hz, 1H), 2.44 (d, J=2.2 Hz, 1H), 2.44 2.38 (m, 1H),

2.16 2.08 (m, 1H), 2.09 (s, 3H), 1.94 1.52 (m, 4H), 1.17 1.07 (m, 1H),

1.05 (d, J=6.8 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.79 (d, J=6.6 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃): d = 169.9, 164.6, 151.8, 143.4, 143.3, 143.2,

141.4, 128.1, 128.0, 128.0, 127.1, 127.0, 125.3, 125.1, 120.2, 120.0, 83.7,

80.1, 79.0, 74.8, 69.3, 69.2, 64.6, 48.0, 47.9, 40.3, 34.1, 30.3, 29.9, 27.8, 20.9,

14.5, 10.4, 9.6; ³¹P NMR (162 MHz, CDCl₃): d = ꢀ0.77; HRMS (FAB, 3-

NBA): m/z: calcd for C₄₆H₄₈O₈P: 759.3087, found 759.3119 [M+H]⁺.

Monotriethylammoniumsalt

of

{1-{(1S,2S,5S)-1-methyl-5-[(2S,3S)-3-

methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-2-phosphonooxy-hexyl}-prop-2-

ynyl}ethanoate (85): Phosphate 85 was synthesized by analogy to the

monotriethylammonium salt of 1a starting from phosphotriester 84

(6.4 mg, 8.4 mmol) to give an amorphous white powder (2.1 mg, 49%).

R_f=0 (ethyl acetate); [a]_D²⁰=+118 (c=0.10, methanol); ¹H NMR

(400 MHz, CD₃OD): d = 7.16 (dd, J=9.6, 6.5 Hz, 1H), 5.93 (d, J=9.6,

0.6 Hz, 1H), 5.28 (dd, J=8.2, 2.2 Hz, 1H), 4.31 (dddd, J=10.6, 7.0, 5.5,

3.7 Hz, 1H), 4.11 (dd, J=10.2, 2.9 Hz, 1H), 3.20 (q, J=7.2 Hz, 6H), 2.88

(d, J=2.2 Hz, 1H), 2.59 (dquint, J=7.0, 3.1 Hz, 1H), 2.17 2.10 (m, 1H),

2.07 (s, 3H), 2.03 1.95 (m, 1H), 1.93 1.78 (m, 2H), 1.63 (ddt, J=16.8,

4.3, 7.2 Hz, 1H), 1.31 (t, J=7.2 Hz, 9H), 1.33 1.24 (m, 1H), 1.13 (d, J=

7.0 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H); ¹³C NMR

(100 MHz, CD₃OD): d = 171.7, 167.3, 155.0, 120.0, 85.6, 81.8, 76.0 (d,

J(¹³C,³¹P)=6.2 Hz), 75.9, 66.6, 47.7, 41.4 (d, J(¹³C,³¹P)=6.2 Hz), 35.5,

31.6, 30.9, 28.9, 21.0, 14.8, 11.0, 10.3, 9.2; ³¹P NMR (162 MHz, CD₃OD):

d = 1.33; HRMS (FAB, 3-NBA): m/z: calcd for C₄₆H₄₈NaO₈P: 425.1341,

found 425.1328 [M+Na]⁺.

(Triethylammonium)-hydrogen-{(1S,2S,3R,4Z)-3-hydroxy-5-iodo-2-

methyl-1-{(3S)-3-[(2S,3S)-3-methyl-6-oxo-tetrahydro-pyran-2-yl]-butyl}-

pent-4-enyl} phosphate (89): Phosphate 89 was synthesized by analogy to

the monotriethylammonium salt of 1a starting from phosphotriester 81

(2.2 mg, 2.6 mmol) to give an amorphous white powder (1.4 mg, 92%).

R_f=0 (ethyl acetate); [a]_D²⁰=+32.9 (c=0.07, methanol); ¹H NMR

(400 MHz, CD₃OD): d = 6.51 (dd, J=7.6, 0.6 Hz, 1H), 6.24 (dd, J=8.4,

7.8 Hz, 1H), 4.48 (dddd, J=10.6, 8.2, 5.9, 2.3 Hz, 1H), 4.36 (t, J=8.8 Hz,

1H), 4.06 (dd, J=10.0, 2.2 Hz, 1H), 3.20 (q, J=7.2 Hz, 1H), 2.58 (ddd,

J=17.9, 8.6, 5.1 Hz, 1H), 2.47 (dt, J=17.9, 8.0 Hz, 1H), 2.26 2.18 (m,

1H), 2.18 2.08 (m, 1H), 2.03 1.94 (m, 1H), 1.80 1.60 (m, 4H), 1.57 1.45

(m, 1H), 1.32 (t, J=17.9 Hz, 9H), 1.32 1.20 (m, 1H), 0.95 (d, J=6.6 Hz,

3H), 0.93 (d, J=7.0 Hz, 3H), 0.89 (d, J=7.0 Hz, 3H); ¹³C NMR

(100 MHz, CD₃OD): d = 175.1, 143.8, 87.9, 83.6, 75.8 (d, J(¹³C,³¹P)=

10.0 Hz), 75.1, 47.8, 43.2, 36.2, 31.6, 29.8, 28.2, 27.5, 27.0, 15.1, 11.9, 9.3,

9.2; ³¹P NMR (162 MHz, CD₃OD): d = 3.07; = HRMS (FAB, 3-NBA):

m/z: calcd for C₁₆H₂₈INaO₇P: 513.0515, found 513.0502 [M+Na]⁺.

(Triethylammonium)-hydrogen-{(1S,2S,3R,4Z)-3-hydroxy-5-iodo-2-

methyl-1-{(3S)-3-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-

butyl}-pent-4-ynyl} phosphate (86): Phosphate 86 was synthesized by

analogy to the monotriethylammonium salt of 1a starting from phospho-

triester 80 (5.0 mg, 5.9 mmol) to give an amorphous white powder

(3.5 mg, quantitative). R_f=0 (ethyl acetate); [a]²_D⁰=+52.6 (c=0.175,

methanol); ¹H NMR (400 MHz, CD₃OD): d = 7.16 (dd, J=9.6, 6.6 Hz,

1H), 5.93 (dd, J=9.6, 0.6 Hz, 1H), 4.44 (d, J=9.6 Hz, 1H), 4.46 4.37 (m,

1H), 4.11 (dd, J=10.2, 2.9 Hz, 1H), 3.20 (q, J=7.4 Hz, 6H), 2.63 2.54

(m, 1H), 2.00 1.90 (m, 1H), 1.88 1.73 (m, 3H), 1.54 1.47 (m, 1H), 1.32

(t, J=7.2 Hz, 9H), 1.34 1.24 (m, 1H), 1.01 (d, J=6.8 Hz, 3H), 1.00 (d,

J=6.8 Hz, 3H), 0.96 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, CD₃OD):

d = 167.4, 155.1, 120.0, 95.9, 85.6, 74.9 (d, J(¹³C,³¹P)=6.2 Hz), 65.0, 47.8,

(Triethylammonium)-(2-cyanoethyl)-{(1S,2S,3R,4Z)-3-hydroxy-2-methyl-

1-{(3S)-3-[(2S,3S)-3-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl]-butyl}-pent-

4-ynyl} phosphate (90): Phosphate 90 was synthesized by analogy to the

monotriethylammonium salt of 1a starting fro phosphotriester 56

(11.3 mg, 24.2 mmol) to give a colourless oil (10.6 mg, 85%). R_f=0 (ethyl

acetate); [a]²_D⁰=+76.8 (c=0.525, MeOH).; ¹H NMR (400 MHz,

CD₃OD): d = 7.15 (dd, J=9.6, 6.5 Hz, 1H), 5.93 (d, J=9.6 Hz, 1H),

4.52 4.44 (m, 1H), 4.30 (dd, J=9.2, 2.0 Hz, 1H), 4.14 (dd, J=10.2,

2.9 Hz, 1H), 4.07 (q, J=6.6 Hz, 1H), 3.20 (q, J=7.2 Hz, 6H), 2.81 2.76

(m, 3H), 2.58 (dquint, J=2.9, 6.6 Hz, 1H), 1.90 1.76 (m, 4H), 1.61 1.51

(m, 1H), 1.39 1.30 (m, 1H), 1.32 (t, J=7.2 Hz, 9H), 1.04 (d, J=6.8 Hz,

2778

¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Chem. Eur. J. 2004, 10, 2759 2780

Cytostatin

2759 2780

3H), 1.01 (d, J=7.0 Hz, 3H), 0.97 (d, J=6.6 Hz, 3H); ¹³C NMR

(100 MHz, CD₃OD): d = 167.2, 154.9, 120.1, 119.4, 85.6, 85.4, 76.3 (d,

J(¹³C,³¹P)=6.2 Hz), 74.1, 64.6, 61.7 (d, J(¹³C,³¹P)=5.4 Hz), 47.8, 45.1 (d,

J(¹³C,³¹P)=4.6 Hz), 35.2, 31.6, 31.4 (d, J(¹³C,³¹P)=3.1 Hz), 29.4, 20.4 (d,

J(¹³C,³¹P)=8.5 Hz), 14.9, 11.0, 9.8, 9.2; ³¹P NMR (162 MHz, CD₃OD): d

= 1.55; HRMS (FAB, 3-NBA): m/z: calcd for C₁₉H₂₉NO₇P: 414.1682,

found 414.1720 [M+H]⁺.

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Enzymatic assays–PP2A₁inhibition: The enzyme (0.025 U) was pre-in-

cubated with the inhibitors in a buffer^[61](pH 8.1, 100 mL total assay

volume) containing Tris¥HCl (40 mm), KCl (20 mm), MgCl₂¥6H₂O

(30 mm), DTT (2 mm) and BSA (0.1%) for 10 min at room temperature.

Then p-NPP was added (end concentration 5 mm) and the read-out

(405 nm) was recorded on a microplate-reader after 10 15 min incubation

at room temperature.

Enzymatic assays–PP1 inhibition: The enzyme (0.025 U) was pre-incu-

bated with the inhibitors in

a

buffer^[61](pH 8.1, 100 mL total assay

volume) containing Tris¥HCl (40 mm), KCl (20 mm), MgCl₂¥6H₂O

(30 mm) and DTT (2 mm) and BSA (0.1%) for 10 min at room tempera-

ture. Then p-NPP was added (end concentration 5 mm) and the read-out

(405 nm) was recorded on a microplate-reader after 10 15 min incubation

at room temperature.

VHR inhibition: The enzyme (0.001 U) was pre-incubated with the inhib-

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(25 mm), EDTA (5 mm), NaCl (125 mm), DTT (2 mm) and BSA (0.1%)

for 10 min at room temperature. Then p-NPP was added (end concentra-

tion 5 mm) and the read-out (405 nm) was recorded on a microplate-

reader after 1 h incubation at 378C.

PTP1B inhibition: The enzyme (0.001 U) was pre-incubated with the in-

hibitors in a buffer^[66](pH 6.5, 100 mL total assay volume) containing

MOPS (25 mm), EDTA (5 mm), NaCl (125 mm), DTT (2 mm) and BSA

(0.1%) for 10 min at room temperature. Then p-NPP was added (end

concentration 5 mm) and the read-out (405 nm) was recorded on a micro-

plate-reader after 10 15 min incubation at room temperature.

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Received: September 16, 2003

Published online: April 16, 2004

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Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues

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