The plakotenins: Biomimetic diels-alder reactions, total synthesis, structural investigations, and chemical biology

DOI: 10.1002/chem.201201585

The Plakotenins: Biomimetic Diels–Alder Reactions, Total Synthesis,

Structural Investigations, and Chemical Biology

Emmanuel Bourcet,^[a]Larissa Kaufmann,^[b]Stephanie Arzt,^[a]Angela Bihlmeier,^[c]

Wim Klopper,^[c]Ute Schepers,^[b]and Stefan Brꢀse*^{[a, b]}

Dedicated to Prof. Dr. Dr. hc. Lutz F. Tietze on the occasion of his 70th birthday

Abstract: The total synthesis of plakotenin, a cytotoxic marine natural product,

using a biomimetic Diels–Alder reaction is described in detail. Two approaches

were used, whereby the Diels–Alder reaction occurs at different stages of the syn-

thesis. Homo- and nor-plakotenin, related natural products, were also prepared, as

well as iso-plakotenin, a diastereoisomer of plakotenin. The syntheses prove the

relative and absolute stereochemistry of the latter. The chemical biology of the

plakotenins was investigated on selected compounds.

Keywords: biomimetic synthesis ·

chemical biology · cycloaddition ·

natural products · polyketides

Introduction

Plakotenin (1, Figure 1) is a secondary metabolite of polyke-

tide origin, which was isolated in 1992 from an Okinawan

marine sponge of the genus Plakortis.^[1]It showed in vitro

cytotoxicity against murine lymphoma L1210 and human ep-

idermoid carcinoma KB cells. The marine natural product 1

possesses a [4.3.0] bicyclic core and six stereogenic centers,

one of which is quaternary. The relative stereochemistry of

1 was deduced by extensive 2D NMR analysis. Later, Faulk-

ner et al. isolated and characterized 1 again along with its

closely related compounds nor-plakotenin (2) and homo-pla-

kotenin (3, Figure 1) from another marine sponge, Plakortis

lita from Palau.^[2,3]Compounds 1, 2, and 3 were found to sig-

Figure 1. Plakotenin (1), nor- (2), homo- (3), and iso-plakotenin (4).

nificantly reduce proliferation of rheumatoid synovial fibro-

blasts. It is proposed that 1 might be produced biosyntheti-

cally through an intramolecular Diels–Alder (IMDA) reac-

tion of a linear precursor.^[1]Herein, we describe the total

synthesis^[4]of 1 and its congeners 2, and 3 as well as its dia-

stereoisomer iso-plakotenin (4) (Figure 1).^[5–7]In addition to

our already published total synthesis of 1,^[8]in this manu-

script we want to show a second expedient method and

reveal studies to understand the chemical biology of the pla-

kotenins. The results of computational studies, which were

made to allow prediction and support of the synthetic work,

were recently published^[9]and showed that the stereochemis-

try had to be revised. The correct stereochemistry is shown

in Figure 1.

[a] Dr. E. Bourcet, Dr. S. Arzt, Prof. Dr. S. Brꢀse

Institute of Organic Chemistry and

Center for Functional Nanostructures (CFN)

Karlsruhe Institute of Technology (KIT), Campus South

Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany)

Fax : (+49)721-608-48581

E-mail: braese@kit.edu

[b] L. Kaufmann, Dr. U. Schepers, Prof. Dr. S. Brꢀse

Institute of Toxicology and Genetics

Karlsruhe Institute of Technology (KIT), Campus North

Hermann-von-Helmholtz-Platz 1

76344 Eggenstein-Leopoldshafen (Germany)

[c] Dr. A. Bihlmeier, Prof. Dr. W. Klopper

Institute of Physical Chemistry and

Results and Discussion

Center for Functional Nanostructures (CFN)

Karlsruhe Institute of Technology (KIT), Campus South

Fritz-Haber-Weg 2, 76131 Karlsruhe (Germany)

Retrosynthetic analysis: The synthesis of 1 was envisaged by

construction of the bicyclic core using a biomimetic intramo-

lecular Diels–Alder reaction.^[10–12]Retrosynthetic analysis re-

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem.201201585.

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Scheme 2. a) i) LDA, LiCl, THF, À788C; ii) 10, 08C, 91%; b) LiNH₂·BH₃,

THF, 08C, 91%; c) IBX, DMSO, quant; d) C, toluene, 1008C, 88%;

e) LiAlH₄, THF, 08C, 90%; f) IBX, DMSO, quant; g) B, LiN

ACHTUNGTRENNUNG(TMS)₂,

THF, À788C, 80%.

Synthesis of the required central chiral fragment 8 was

achieved by employing an asymmetric alkylation approach

reported by Myers et al. (Scheme 2).^[14]An anti-selective

aldol reaction between commercially available pseudoephe-

drine auxiliary E and iodide (S)-10^[15], prepared in 3 steps

from (S)-Roche ester 9^[16], produced amide 11 as a single di-

astereoisomer (in >98% diastereoselectivity (ds)). Reduc-

tive removal of the chiral auxiliary was effected with lithium

amidotrihydroborate to afford alcohol 12 in 91% yield.^[17]

Primary alcohol 12 was oxidized to aldehyde 8 in quantita-

tive yield using iodoxybenzoic acid (IBX). Wittig olefination

of aldehyde 8 with ylide C gave the E-unsaturated ester 13

Scheme 1. Retrosynthetic analysis of plakotenin (1).

veals that the linear precursor of 1 could be the tetraene 5

that is, unlike most polyketides^[13], not completely E config-

ured but includes one Z double bond (Scheme 1). Further

in-depth retrosynthetic analysis however shows that triene 7

would already be suitable for cyclization. Therefore, two

synthetic approaches are possible: Diels–Alder reaction of

tetraene 5 (“late-stage Diels–Alder” strategy) or alternative-

ly Diels–Alder reaction of triene 7 followed by appropriate

functionalization of the ring system (“early-stage Diels–

Alder” strategy).

1

stereoselectively (E/Z>14:1 based on H NMR analysis of

For both methods, a suitable first target is the C₂-symmet-

rical dimethyl building block 8, which becomes apparent by

taking a closer look at triene 7 (Scheme 1). This central

chiral fragment 8 should be accessible from commercially

available (S)-Roche ester (9). Subsequent bidirectional ex-

the crude reaction mixture) in 88% yield. The reduction of

the ester 13 with lithium aluminium hydride (90% yield)

and oxidation of the resulting alcohol 14 with IBX (quant)

gave a,b-unsaturated aldehyde 15. Extension of 15 and

hence introduction of the styryl group was accomplished by

a Julia–Kocienski olefination reaction with tetrazole B using

lithium bis(trimethylsilyl)amide as base in 80% yield (EE/

ZE 10:1).^[18]

The alcohol function in diene 16 was then deprotected

with camphorsulfonic acid in good yield (Scheme 3), which

was followed by oxidation of the resulting alcohol 17 to al-

dehyde 18 with Dess–Martin periodinane (DMP) in quanti-

tative yield. Introduction of the third double bond, the only

Z-configured bond in linear precursor 5, was now possible

through a mild (to avoid in situ cyclization) Z-selective var-

iation of the Horner–Wadsworth–Emmons reaction using

tension of

8

by Wittig, Horner–Wadsworth–Emmons

(HWE), or Julia–Kocienski olefination reactions should de-

liver triene 7 or/and tetraene 5. In this manuscript we want

to describe the synthesis of 1 by a late-stage Diels–Alder, as

well as an early-stage Diels–Alder strategy.

Synthesis of

1 by a

late-stage Diels–Alder strategy:^[8]

Aiming for 1 by this approach we first carried out the com-

plete synthesis of linear precursor 5 and only then utilized

À

the intramolecular Diels–Alder reaction as the last C C

bond forming reaction of the synthetic route (Scheme 1).

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we report a way of synthesizing 1, in which cyclization takes

place directly after introduction of the third double bond.

This method offers the following advantages. Not only the

careful handling of sensitive compounds prone to cyclization

becomes unnecessary, but also derivatization of the core

structure could be achieved more easily. This would give

access to a vast variety of compounds derived from plakote-

nin that can be interesting for biological studies.

Thus, with triene 7 in hand (obtained in 13 steps from (S)-

Roche ester (9) with an overall yield of 27.2%), the IMDA

was realized by heating in toluene overnight (Scheme 4).

Cycloadduct 6 could be obtained in very good yield and its

relative stereochemistry was identical to plakotenin (de-

duced by 2D NMR analysis on compound 22).

Scheme 3. a) CSA, CH₂Cl₂/MeOH (2:1), 08C, 88%; b) DMP, CH₂Cl₂,

quant; c) D or F, NaH, THF, 08C, 76%; d) LiAlH₄, THF, 08C, 61%;

e) DMP, CH₂Cl₂, quant; f) A, NaH, THF, 08C, 88%; g) toluene, 1108C,

91%; h) NaOH (2m), THF/MeOH, 408C, 86%.

phosphonate D (ethyl 2-((bis(o-tolyloxy))phosphoryl)buta-

noate).^[19]Ester 7 was obtained in 64% yield with a Z/E se-

lectivity of 6:1. Classical HWE reaction conditions using

phosphonate F proceeded also smoothly and surprisingly de-

livered again the Z isomer 7 as the major product with a 4:1

ratio in 76% yield. The Z stereochemistry of this newly

formed double bond was proven by NOESY experiments re-

alized on alcohol 19. Again a reduction/oxidation sequence

of the ester part in 7 furnished aldehyde 20, which was sub-

sequently elongated to the desired tetraene 5 using a second

HWE olefination with phosphonate A.

On heating in toluene, tetraene 5 cyclized smoothly in ex-

cellent yield to plakotenin ethyl ester 21 detected as single

diastereoisomer. Finally, saponification yielded synthetic

plakotenin (1). Extensive spectroscopic studies were con-

ducted on 1 to confirm its structure and relative stereochem-

istry. The spectroscopic data, as well as the optical rotation

of synthetic plakotenin were identical with the reported

data of the natural product. Hence, plakotenin (1) was suc-

cessfully synthesized in 15 steps (from (S)-iodide 10) in an

overall yield of 14.7%.

Scheme 4. a) Toluene, 1108C, 85%; b) LiAlH₄, THF, 08C, 95%; c) DMP,

CH₂Cl₂, quant; d) isopropenylmagnesium bromide, THF, 08C then 508C,

87% (d.r.=10:1); e) SOCl₂, pentane/ether (1:1) 08C to RT; f) NMO,

DMSO, RT, 61% (two steps); g) NaClO₂, H₂O₂, NaH₂PO₄, MeCN/H₂O

(6:1), RT, 63%.

After the reduction/oxidation sequence on cycloadduct 6,

which gave aldehyde 23 various olefination attempts

(Wittig, HWE; results not shown) were made but did not

deliver the desired elongated bicycle. This reaction failure

can be attributed to the steric hindrance of cyclic aldehyde

23, which prevents the attack of bulky olefination reagents.

To overcome this problem, aldehyde 23 was treated with iso-

propenylmagnesium bromide. Addition of the Grignard re-

agent on the aldehyde moiety was found to be slow, and

heating the reaction mixture up to 508C was required to ob-

serve complete conversion. Allylic alcohol 24 could be even-

tually isolated in good yield, as a 10:1 mixture of insepara-

ble diastereoisomers. To form the tri-substituted double

bond, we first attempted a palladium-catalyzed rearrange-

ment of the corresponding allylic acetate^[20](results not

shown). However, this rearrangement did not proceed, and

only starting material was recovered after workup. We then

Synthesis of 1 by an early-stage Diels–Alder strategy: In

contrast to the method described in the previous paragraph,

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Total Synthesis of the Plakotenins

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turned our attention to the use of thionyl chloride, which is

known to convert secondary allylic alcohols into their iso-

meric primary allylic chlorides.^[21]To our delight, the rear-

rangement took place with good selectivity, and the primary

chloride was directly used without purification for the next

oxidation into the corresponding aldehyde. This reaction

was performed using a modification of the Ganem oxida-

tion.^[22]Indeed, when the primary chloride was treated with

N-methylmorpholine-N-oxide (NMO) in DMSO, aldehyde

25 could be isolated with 61% yield over the two steps. Fi-

nally, Pinnick oxidation furnished the second synthetic pla-

kotenin (1), which showed identical spectroscopic data to

that previously obtained following the late-stage strategy.

This early-stage strategy allowed us to obtain 1 in 17 steps

(from (S)-iodide 10) in an overall yield of 9.4%.

chemistry again deduced using 2D NMR analysis. Compar-

ing spectroscopic data of synthetic 1 and 4 with the spectra

of the natural plakotenin allowed unambiguous approval of

the proposed structure of isolated plakotenin.

Synthesis of 2: For the synthesis of 2, a congener of 1, again

a late-stage Diels–Alder strategy was used. With alcohol 17

already in hand, triene 31 was readily prepared using a

HWE reaction between the resulting aldehyde 18 and phos-

phonate A, which allowed introduction of a methyl instead

of an ethyl group (Scheme 6). A 3:1 selectivity for the de-

Synthesis of 4: During the course of the synthesis of 1, we

also planned to prepare an isomer of the proposed structure

of isolated plakotenin, in which all double bonds of the

linear precursor are E configured. Because substances of

polyketide origin only rarely include Z configured double

bonds, the synthesis of 4 should help to account for the cor-

rect relative stereochemistry of isolated plakotenin.

For this purpose, triene 26 was separated, from the major

Z isomer obtained by the HWE reaction to introduce the

third double bond, by purification of the crude mixture on

column chromatography and isolated with 18% yield. Next

the synthesis of iso-plakotenin (Scheme 5) was completed in

Scheme 6. a) A, NaH, THF, 08C, 64%; b) LiAlH₄, THF, 08C, 78%;

c) DMP, CH₂Cl₂, quant; d) A, NaH, THF, 08C, 89%; e) toluene, 1108C,

92%; f) NaOH (2m), THF/MeOH, 408C, 88%.

sired Z olefin was observed for this new HWE reaction and

triene 31 could be isolated with 64% yield. Tetraene 34, the

linear precursor of nor-plakotenin, was then synthesized by

performing the reduction/oxidation/HWE sequence descri-

bed above on triene 31. On heating, the intramolecular

Diels–Alder reaction proceeded smoothly and further sapo-

nification of the ethyl ester gave 2 in very good yield (over-

all yield of 16.6% (from (S)-iodide 10)). The optical rota-

tion was measured in chloroform and methanol, and was in

accordance with the literature and with synthetic plakotenin

itself.^[2]

Scheme 5. a) F, NaH, THF, 08C, 18%; b) LiAlH₄, THF, 08C, 68%;

c) DMP, CH₂Cl₂, quant; d) A, NaH, THF, 08C, 84%; e) toluene, 1108C,

82%; f) NaOH (2m), THF/MeOH, 408C, 75%.

Synthesis of 3: For the synthesis of 3 a change in the syn-

thetic route of 1 is necessary at a much earlier stage com-

pared with 2 because the ethyl substituent must be intro-

duced in the first Wittig reaction. When the reaction be-

tween aldehyde 8 and ylide G was performed, we observed

a very slow conversion, and the mixture had to be refluxed

for 4 days to go to completion (Scheme 7). As a conse-

quence of the long period of heating, epimerization at the a

position of the newly created double bond occurred. To

avoid this, phosphonate F was used to install the first double

bond, and surprisingly a 5:1 mixture in favor of the Z

isomer was obtained. We decided to continue the sequence

with this mixture, hoping that isomerization of this double

bond could be possible at a later stage of the synthesis. As

previously described, compound 37 was reduced into the

a method analogous to that described for the synthesis of 1

by

a late-stage Diels–Alder strategy. Interestingly, the

IMDA of the all E-configured linear precursor 29 proceeded

with less selectivity, since a 6.6:1 ratio of inseparable diaster-

1

eoisomers could be determined by analysis of the H NMR

spectrum of ester 30. Unfortunately, the relative stereo-

chemistry of the minor diastereoisomer could not be deter-

mined, since the corresponding carboxylic acid could not be

isolated. iso-Plakotenin (4) was successfully synthesized in

an overall yield of 2.9% (from (S)-iodide 10) and its stereo-

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Scheme 7. a) G, toluene, 1008C, 18%; b) A, NaH, THF, 08C, 73%;

c) LiAlH₄, THF, 08C, 93%; d) DMP, CH₂Cl₂, quant; e) B, LiN

THF, À788C, 78%; f) I₂, CHCl₃, RT.

ACHTUNGERTN(NUNG TMS)₂,

Scheme 8. a) CSA, CH₂Cl₂/MeOH (2:1), 08C, 45% (2 steps); b) DMP,

CH₂Cl₂, quant; c) F, NaH, THF, 08C, 73%; d) LiAlH₄, THF, 08C, 77%;

e) DMP, CH₂Cl₂, quant; f) A, NaH, THF, 08C, 68%; g) toluene, 1108C,

90%; h) NaOH (2m), THF/MeOH, 408C, 88%.

primary alcohol 38 and further elongated to the diene 40 by

a Julia–Kocienski olefination reaction with tetrazole B. The

newly created double bond in 40 was assigned as having E

configuration (based on the coupling constant J=16.4 Hz)

and obtained with a ratio of 7.7:1. At this stage, the geomet-

rical isomers could not be separated and the mixture was

treated with iodine in chloroform,^[23]to attempt isomeriza-

tion into the required (E,E) diene. Isomerization occurred

and after 1 hour led to a 2:1 mixture (with respect to the

second double bond) of the desired (E,E) diene 41. Pro-

longed reaction time did not improve this ratio, and only

degradation of the product was observed.

Computational studies: The described total syntheses of 1,

its congeners 2 and 3, as well as its diastereomer 4 proceed

by an intramolecular Diels–Alder reaction following either

the early-stage or the late-stage strategy. The reaction may

lead to various products because there are four possible

transition states for the precursor molecule (Figure 2).

Since we were able to remove the minor (Z,E) isomer

after trityl deprotection in the course of the synthesis of 1,

we directly treated compound 41 with camphorsulfonic acid

(Scheme 8). Eventually, primary alcohol 42 could be isolated

as a single diastereoisomer with 45% yield over the two

steps. Then, construction of the linear tetraene 47 was ach-

ieved using the same reaction sequence as described above.

Similar yields were obtained, and compound 47 was cyclized

under thermal conditions to yield homo-plakotenin ethyl

ester 48 as a single diastereoisomer. The ester moiety was

eventually hydrolyzed using the same method as described

earlier to give 3. As a consequence of the unexpected selec-

tivity observed in the first HWE olefination and of the parti-

al isomerization of diene 40, the overall yield of this last nat-

ural product is much lower (overall yield of 6.0% (from (S)-

iodide 10)) than for its two congeners (1 and 2).

Figure 2. Possible transition states (shown as triene conformers) of the in-

tramolecular Diels–Alder reaction that is applied to the synthesis of pla-

kotenin and iso-plakotenin.

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To understand the stereoselectivity of the intramolecular

Diels–Alder reaction we have computed the relative ener-

gies of the transition states TS1 to TS4 for the synthesis of

plakotenin and iso-plakotenin. The results for the late-stage

Diels–Alder strategy (R¹=CH=CCH₃COOEt, R²=Et and

vice versa) have been published in previous work, together

with the spectroscopic properties of the final products, and

were the basis for the structure revision of plakotenin and

its congeners.^[9]In the present work, we additionally present

our results for the early-stage Diels–Alder strategy

(Table 1). We find that transition state TS1 is lowest in

energy for both the (E,E,E) and (E,E,Z) precursors. This is

in agreement with our experimental findings, since the struc-

tures of plakotenin and iso-plakotenin derive from TS1.

Moreover, the values given in Table 1 correspond nicely

with the data obtained for the late-stage Diels–Alder strat-

egy, implying that both methods are equally well suited to

deliver the desired product.

Table 1. Relative energies of the various transition states (in kJmol^À1

)

that may occur during the synthesis of plakotenin and iso-plakotenin by

the early-stage Diels–Alder strategy.

A

ACHTUNGTRNE(NUNG E,E,Z)

R¹=Et, R²=COOMe

R¹=COOMe, R²=Et

TS1

TS2

TS3

TS4

0

42

30

24

0

43

35

25

Biological tests: As previously reported, plakotenin displays

a decent cytotoxicity on tumor cells such as murine lympho-

cytic leukemia cells (L1210) with IC₅₀Tox values of 15.4–

21.1 mm.^[1]

Figure 3. Plakotenin derivatives tested for biological activity.

Eventually, a small selection of new cyclic and acyclic pla-

kotenin derivatives 49–55 was prepared using standard

methods (esterification, conversion to an azide etc.). They

are derived from the natural isomers along the lines descri-

bed above; this is the reason we looked first at isomers di-

rectly emerging from the natural product syntheses.

To test, whether the newly synthesized plakotenin deriva-

tives have stronger cytotoxic effects on tumor cells or on

their cell cycle than plakotenin itself, a variety of tumor cell

lines were incubated with different concentrations of plako-

tenin and its derivatives (Figures 3 and 4) as well as some

linear synthesis intermediates (see the Supporting Informa-

tion). Cytotoxicity was analyzed using the MTT assay, by

which mitochondrial integrity is measured as a mean of via-

bility. The viability of different tumor cell lines (human

cervix carcinoma (HeLa), human glioma (U251)) was then

compared after treatment with 20 mm of the compounds,

which is around the IC₅₀Tox value of plakotenin itself

(Figure 5 and Figures S1 and S2 in the Supporting Informa-

tion). Interestingly, changing the stereochemistry at the qua-

ternary position to the opposite configuration decreases the

cytotoxic activity of all iso-plakotenin derivatives 4, 30, 51,

54. Likewise, the E-configured linear precursors (E)-26, (E)-

Figure 4. Acyclic plakotenin precursors tested for biological activity.

27, (4E)-29, which lead to the synthesis of iso-plakotenin de-

rivatives, also display a lower cytotoxicity than their Z iso-

mers 5, (Z)-26, (Z)-27, 31, 32, 34. Further, changing the

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potent derivatives, which show

an enhanced cell cycle arrest in

the G2M phase and a decrease

in the G1 phase indicating an

antiproliferating activity of the

plakotenin intermediates (Fig-

ure S3 in the Supporting Infor-

mation). It is interesting to

point of out that the acyclic de-

rivative 5 is the precursor of 21

containing an ester moiety at

the stereogenic center.

Conclusion

We presented two different ap-

proaches towards the total syn-

thesis of members of the plako-

tenin family. Besides the natu-

rally occurring plakotenin, for

the first time we were able to

synthesize its natural isomers

Figure 5. Comparison of the toxicology of plakotenin (1) and plakotenin derivatives in HeLa or U251 tumor

cell lines. In a 96 well plate 5ꢂ10⁵cells per well were incubated with 20 mm of plakotenin derivatives for 72 h.

Eventually, the viability of the cells was determined by using an MTT assay (Promega). Absorption of the for-

mazan dye as a means to test for viability was measured at 595 nm. Full diagrams with standard error (SE)

and also the 10 mm concentrations are shown in Figures S1 and S2 (in the Supporting Information).

chain length at the quaternary stereogenic center in plakote-

nin derivatives as in 1, 6 and 53 or in 22 and 50 does not

seem to interfere with the cytotoxicity to a large extent,

however introducing a polar residue such as an alcohol se-

verely increases the cytotoxicity of the plakotenin deriva-

tives 22 and 50 as well as their precursors (Z)-27 and 32

with an IC₅₀Tox =5.24 mm for the alcohol 22. However, the

hydroxyl group should be at the terminus of the aliphatic

residue for an enhancement of the cytotoxicity as the activi-

ty in 24 is not better than in plakotenin itself. By introducing

an aldehyde moiety at the side chain, the cytotoxicity was

also fourfold increased with an IC₅₀Tox =5.56 mm as meas-

ured for aldehyde 25.

Furthermore, fluorescence assisted cell sorting (FACS)

Figure 6. Determination of the apoptotic rates by FACS measurements in

L1210 cells using 2, 3, and 5 mm plakotenin (1), iso-plakotenin (4), nor-

plakotenin (2), and the aldehyde 25 (P values pꢀ0.5 “*significant” pꢀ

0.01 “**significant” pꢀ0.001).

and a quantification of caspase 8 cleavage was performed to

measure the apoptotic potential of the compounds and their

influence on the cell cycle of tumor cells especially by meas-

uring the G2M arrest. Even if the cytotoxicity of a com-

pound is low, it can lead to a deceleration of tumor growth

by decreasing the rate of cell division. For the FACS meas-

urements we used concentrations of 1, 2, 3, and 5 mm, which

were below the reported IC₅₀Tox values (15.4–21.1 mm) of

plakotenin in murine lymphocytic leukemia cells (L1210).^[1]

Figure 6 shows the comparison between plakotenin and its

natural isomers, as well as with the aldehyde 25, which has

been shown to exhibit a higher toxicity in the MTT assay.

As expected, plakotenin (1) itself shows a minor increase of

apoptosis, whereas the aldehyde 25 enhances the apoptosis

rate three-fold. iso-Plakotenin (4) and also nor-plakotenin

(2) do not show any increase in apoptosis at these concen-

trations.

homo-plakotenin, iso-plakotenin, and nor-plakotenin. The

biological studies of the natural isomers revealed that plako-

tenin itself displays a cytotoxicity to tumor cells with an

IC₅₀Tox value of about 15–21 mm depending on the tumor

cell type. Interestingly, the natural isomers of plakotenin are

mostly inactive. Synthesis of a variety of plakotenin deriva-

tives and their isomers as well as their linear precursors led

to more potent cytotoxic derivatives than plakotenin itself.

By replacing the carboxy-group-containing moiety at the

quaternary stereogenic center with different polar and less-

polar residues we found that moieties with a terminal hy-

droxyl group are the most efficacious derivatives. They show

a decent increase in tumor cell cytotoxicity with IC₅₀Tox

values up to 5.21 mm. Introducing more lipophilic moieties at

Additionally, the influence on cell cycle was measured by

FACS. Besides 1, the intermediates 5 and 21 were the most

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Total Synthesis of the Plakotenins

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mined using the polarimeter Perkin–Elmer 241. Melting points were reg-

istered on a Mel-Temp II melting point microscope from Laboratory De-

vices Inc. and are not corrected.

this position led to a reduction of activity. Analogous to the

natural isomers of plakotenin, iso-plakotenin, and nor-pla-

kotenin, all derivatives with the same stereochemistry at the

benzylic position are far less active as plakotenin and its de-

rivatives. Furthermore, the linear precursors act in the same

way proposing a binding and folding in an active center of a

receptor on tumor cells. The biological data suggest the exis-

tence of a plakotenin binding receptor, which requires the

correct stereochemistry at the quaternary position but is to

a certain extent flexible in the length of the moiety. Polar

residues at the terminus of this moiety might interact with

polar amino acids in their proximity by enhancing either the

binding, the turnover of the ligand–receptor complex or the

conformational change of the ligand–receptor complex. In-

troducing an aldehyde moiety also increases the cytotoxic

activity probably by crosslinking the ligand with the recep-

tor. However, these speculations have to be proven by

future experiments after the characterization of the putative

receptor. It is interesting to note that, so far, there are no

similar compounds known being structurally comparable

(arylhydroindanes) with the plakotenin substructure.

Amide 11: A solution of n-butyllithium in hexanes (2.5m, 10.8 mL,

27.0 mmol) was slowly added to a suspension of lithium chloride (3.64 g,

85.9 mmol) and diisopropylamine (4.10 mL, 3.00 g, 29.0 mmol) in THF

(15 mL) at À788C. The resulting suspension was warmed to 08C briefly

and was then cooled to À788C again. An ice-cooled solution of N-

((1S,2S)-1-hydroxy-1-phenylpropan-2-yl)-N-methylpropionamide

(E)

(3.14 g, 14.2 mmol) in THF (35 mL) was added. The mixture was stirred

at À788C for 2 h, at 08C for 15 min and at RT for 5 min. The mixture

was cooled to 08C and iodide 10 (2.99 g, 6.76 mmol) in THF (35 mL) was

added. After being stirred for 2 d at 458C the reaction mixture was treat-

ed with half saturated aqueous NH₄Cl (70 mL) and the resulting mixture

was extracted with EtOAc (3ꢂ40 mL). The combined organic extracts

were dried (MgSO₄) and evaporated under reduced pressure. The crude

product was purified by column chromatography on silica using cyclohex-

ane/ethyl acetate 2:1 to give amide 11 (3.31 g, 91%) as white foam. R_f=

0.22 (cyclohexane/EtOAc 2:1); m.p. 508C; [a]²_D⁰= +33.5 (c=0.99 in

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.92 (d, J=2.7 Hz, 3H), 0.94

(d, J=2.7 Hz, 3H), 1.00 (d, J=6.9 Hz, 3H), 1.31–1.44 (m, 2H), 1.50–1.61

(m, 1H), 2.35–2.43 (m, 1H), 2.50 (s, 3H), 2.70 (dd, J=8.8 Hz, J=6.0 Hz,

1H), 2.88 (dd, J=8.8 Hz, J=4.7 Hz, 1H), 4.24 (brs, 1H), 4.49 (t, J=

7.2 Hz, 1H), 7.11–7.24 (m, 14H), 7.33–7.39 ppm (m, 6H); ¹³C NMR

(100 MHz, CDCl₃): d=14.3 (p), 17.5 (p), 18.2 (p), 31.7 (t), 34.1 (t), 37.8

(s), 67.4 (s), 76.4 (t), 86.0 (q), 126.1 (t), 126.8 (t), 127.5 (t), 127.6 (t), 128.2

(t), 128.7 (t), 142.5 (q), 144.3 (q), 179.0 ppm (q) (the ¹H- and ¹³C NMR

spectra are complex due to amide geometrical isomerism); IR (neat): n˜ =

3382, 3059, 3030, 2969, 2930, 2871, 1620, 1490, 1449, 1410, 1373, 1318,

1221, 1155, 1070, 988, 926, 899, 839, 764, 747, 705, 648, 633, 506 cm^À1; MS

(FAB, Matrix: 3-NBA): m/z (%): 558 [M+Na]⁺, 536 [M+H]⁺, 243 (100);

HRMS (FAB, Matrix: 3-NBA) m/z: calcd for C₃₆H₄₂NO₃: 536.3165;

found: 536.3167.

In future studies, we will use the plakotenin substructure

as a lead structure for further optimization.

Experimental Section

General methods: ¹H and ¹³C NMR spectra were recorded on a Bruker

AM 400 (400 MHz/100 MHz), Bruker DRX 500 (500 MHz/125 MHz) or

Bruker Avance 600 (600 MHz/150 MHz) instrument using CDCl₃as sol-

vent. Chemical shifts are expressed in parts per million (ppm, d) down-

field from tetramethylsilane (TMS) and are referenced to CHCl₃(d=

7.26 ppm) as an internal standard. All coupling constants are absolute

values and J values are expressed in Hertz (Hz). For assigning signal sep-

aration of ¹H NMR spectra the following abbreviations were used: s=

singlet, brs=broad singlet, d=doublet, t=triplet, q=quartet, sext=

sextet, m=multiplet, dd=doublet of doublets, dq=doublet of quartets,

ddq=doublet of dq and Ar-H=aromatic proton. For assigning signals of

¹³C NMR spectra the following abbreviations were used: p=primary

(RCH₃), s=secondary (R₂CH₂), t=tertiary (R₃CH), q=quaternary

(R₄C). The assignment was supported by analysis of DEPT90 and

DEPT135 spectra. MS (EI) (electron impact mass spectrometry), MS

(FAB) (fast atom bombardment mass spectrometry) and HRMS: Finni-

gan MAT 90. The molecular fragments are quoted as the relation be-

tween mass and charge (m/z), the intensities as a percentage value rela-

tive to the intensity of the base signal (100%). The molecular ion has the

abbreviation [M⁺]. IR (infrared spectroscopy): FTIR Bruker IFS 88. IR

spectra of oils were recorded as thin films on KBr, in the case of solids

the neat substance was used. The deposit of the absorption band is given

in wavenumbers in cm^À1. Solvents and chemicals used for reactions were

purchased from commercial suppliers. Solvents were dried under stand-

ard conditions; chemicals were used without further purification. All the

reactions were performed in standard glassware. All reactions were car-

ried out under Argon in flame-dried glassware. Evaporation of solvents

and concentration of reaction mixtures were performed in vacuo on a

Bꢃchi rotary evaporator. Column chromatography was performed using

silica gel 60 (purchased from Merck) under flash conditions. For thin

layer chromatography, aluminum foils layered with silica gel with fluores-

cence indicator (silica gel 60 F₂₅₄) produced by Merck were employed.

The detection was carried out with a UV lamp from Heraeus, model

Fluotest. The Seebach reagent (molybdophosphoric acid (2.5 w%), ceriu-

m(IV) sulfate tetrahydrate (1.0 w%), H₂SO₄concd (6.0 w%), water

(90.5 w%)) was used as dipping reagent. Specific rotations were deter-

Alcohol 12: A solution of n-butyllithium in hexanes (2.5m, 9.30 mL,

23.0 mmol) was added to a solution of diisopropylamine (3.50 mL, 2.50 g,

25.0 mmol) in THF (30 mL) at À788C. The resulting solution was stirred

at À788C for 10 min, was then warmed to 08C and held at that tempera-

ture for 20 min. Lithium amidotrihydroborate (819 mg, 23.9 mmol) was

added in one portion. The suspension was stirred at 08C for 15 min and

then was warmed to RT. After 15 min, the suspension was cooled to 08C.

A solution of amide 11 (3.20 g, 5.97 mmol) in THF (30 mL) was slowly

added. The reaction mixture was warmed to RT, held at that temperature

for 2 h and was then cooled to 08C where excess hydride was quenched

by careful addition of 3m aqueous HCl (70 mL). The mixture was stirred

for 10 min at 08C and was then extracted with Et₂O (3ꢂ50 mL). The

combined extracts were washed sequentially with 3m aqueous HCl

(10 mL), 2m aqueous NaOH (10 mL) and brine (50 mL). The organic

layer was dried (MgSO₄) and evaporated under reduced pressure. The

crude product was purified by column chromatography on silica using cy-

clohexane/ethyl acetate 9:1 to give alcohol 12 (2.03 g, 91%) as a white

solid. R_f=0.18 (cyclohexane/EtOAc 6:1); m.p. 728C; [a]²_D⁰= +13.0 (c=

0.81 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.87 (d, J=6.6 Hz, 3H;

CH₃), 0.93 (d, J=6.7 Hz, 3H; CH₃), 1.10–1.23 (m, 2H; CHCH₂CH), 1.31

(t, J=5.5 Hz, 1H; OH), 1.61–1.69 (m, 1H; HOCH₂CH), 1.79–1.87 (m,

1H; TrtOCH₂CH), 2.89–2.96 (m, 2H; TrtOCH₂), 3.34–3.46 (m, 2H;

HOCH₂), 7.20–7.31 (m, 9H; Ar-H), 7.43–7.46 ppm (m, 6H; Ar-H);

¹³C NMR (100 MHz, CDCl₃): d=16.3 (p), 17.1 (p), 31.2 (t), 33.0 (t), 37.2

(s), 68.9 (s), 69.0 (s), 86.2 (q), 126.8 (t), 127.6 (t), 128.7 (t), 144.4 ppm (q);

IR (neat): n˜ =3299, 3056, 3031, 2958, 2925, 2871, 1595, 1491, 1449, 1387,

1323, 1220, 1177, 1151, 1062, 1033, 985, 949, 931, 897, 821, 765, 752, 709,

697, 648, 632, 540, 484, 422 cm^À1; MS (EI, 70 eV): m/z (%): 374 (1) [M⁺],

259 (12), 243 (100), 183 (17), 165 (35), 105 (11), 77 (4); HRMS m/z: calcd

for C₂₆H₃₀O₂: 374.2246; found: 374.2249.

Ester 13: To a solution of alcohol 12 (2.03 g, 5.42 mmol) in DMSO

(20 mL) was added 2-iodobenzoic acid (3.79 g, 13.6 mmol). The reaction

mixture was stirred at RT for 2.5 h, after which it was diluted with H₂O

(100 mL) and the resulting precipitate filtered off. The aqueous layer was

extracted with Et₂O (3ꢂ40 mL). The combined organic extracts were

Chem. Eur. J. 2012, 18, 15004 – 15020

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

15011

S. Brꢀse et al.

dried (MgSO₄) and evaporated under reduced pressure to yield the crude

aldehyde 8 (2.02 g, 5.42 mmol, assumed to be quantitative) as colorless

oil, which was used without further purification for the following olefina-

tion reaction. To a solution of the crude aldehyde 8 (2.02 g, 5.42 mmol)

in toluene (20 mL) was added methyl 2-(triphenylphosphoranylidene)-

propanoate (C) (2.83 g, 8.13 mmol) at RT. The reaction mixture was

heated under reflux for 17 h. The crude product was purified by column

chromatography on silica using cyclohexane/ethyl acetate 50:1 to give

ester 13 (2.11 g, 88%) as colorless oil. A 14:1 ratio of E/Z products was

obtained which was measured from the ¹H NMR spectrum of the crude

reaction mixture. R_f=0.31 (cyclohexane/EtOAc 18:1); [a]²_D⁰=À41.2 (c=

0.26 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.91 (d, J=6.6 Hz, 3H;

TrtOCH₂CHCH₃), 1.02 (d, J=6.7 Hz, 3H; C=CHCHCH₃), 1.11–1.18 (m,

1H; CHCH_AH_BCH), 1.46–1.53 (m, 1H; CHCH_AH_BCH), 1.60 (d, J=

1.4 Hz, 3H; MeCO₂CCH₃), 1.64–1.75 (m, 1H; TrtOCH₂CH), 2.29–2.40

(m, 1H; C=CHCH), 2.81 (dd, J=8.7 Hz, J=6.4 Hz, 1H; TrtOCH_AH_B),

3.00 (dd, J=8.7 Hz, J=4.7 Hz, 1H; TrtOCH_AH_B), 3.71 (s, 3H; CO₂CH₃),

6.50 (dd, J=10.1 Hz, J=1.4 Hz, 1H; C=CH), 7.19–7.30 (m, 9H; Ar-H),

7.42–7.45 ppm (m, 6H; Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=12.3 (p),

18.3 (p), 20.0 (p), 30.6 (t), 31.8 (t), 40.8 (s), 51.6 (p), 67.3 (s), 86.0 (q),

125.8 (q), 126.8 (t), 127.6 (t), 128.7 (t), 144.4 (q), 148.3 (t), 168.8 ppm (q);

IR (film): n˜ =3058, 3023, 2956, 2926, 2869, 1715, 1649, 1597, 1490, 1448,

1386, 1315, 1271, 1212, 1154, 1092, 1070, 1032, 989, 898, 813, 764, 748,

707, 648, 632 cm^À1; MS (EI, 70 eV): m/z (%): 442 (0.02) [M⁺], 243 (100),

183 (9), 165 (15), 105 (15), 77 (4); HRMS m/z: calcd for C₃₀H₃₄O₃:

442.2508; found: 442.2505.

evaporated under reduced pressure. The crude product was purified by

column chromatography on silica using cyclohexane/ethyl acetate 50:1 to

give compound 16 (190 mg, 80%) as colorless oil. A 10:1 ratio of E/Z

products was obtained. R_f=0.30 (cyclohexane/EtOAc 50:1); [a]²_D⁰=À75.0

(c=0.71 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.91 (d, J=6.6 Hz,

3H; TrtOCH₂CHCH₃), 1.03 (d, J=6.7 Hz, 3H; C=CHCHCH₃), 1.09–1.16

(m, 1H; CHCH_AH_BCH), 1.45–1.49 (m, 1H; CHCH_AH_BCH), 1.63 (s, 3H;

PhCH=CHCCH₃), 1.70–1.80 (m, 1H; TrtOCH₂CH), 2.36–2.47 (m, 1H;

C=CHCH), 2.81 (dd, J=8.6 Hz, J=6.7 Hz, 1H; TrtOCH_AH_B), 3.02 (dd,

J=8.7 Hz, J=4.7 Hz, 1H; TrtOCH_AH_B), 5.36 (d, J=9.6 Hz, 1H; PhCH=

CHC=CH), 6.38 (d, J=16.1 Hz, 1H; PhCH), 6.74 (d, J=15.6 Hz, 1H;

PhCH=CH), 7.16–7.24 (m, 5H; Ar-H), 7.26–7.32 (m, 9H; Ar-H), 7.40–

7.46 ppm (m, 6H; Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=12.4 (p), 18.5

(p), 21.0 (p), 30.2 (t), 31.8 (t), 41.7 (s), 67.6 (s), 86.0 (q), 125.6 (t), 126.1

(t), 126.7 (t), 126.8 (t), 127.7 (t), 128.5 (t), 128.8 (t), 132.1 (q), 134.2 (t),

138.0 (q), 140.9 (t), 144.5 ppm (q); IR (film): n˜ =3084, 3058, 3025, 2957,

2924, 2866, 2851, 1597, 1560, 1542, 1491, 1448, 1386, 1314, 1220, 1182,

1155, 1070, 1031, 959, 926, 899, 826, 774, 763, 746, 706, 647, 632,

530 cm^À1; MS (EI, 70 eV): m/z (%): 486 (50) [M⁺], 374 (73), 414 (100),

485 (95); HRMS m/z: calcd for C₃₆H₃₈O: 486.2922; found: 486.2917.

Alcohol 17: To a solution of 16 (3.01 g, 6.19 mmol) in CH₂Cl₂/MeOH

(200/100 mL) at 08C camphorsulfonic acid (2.44 g, 10.5 mmol) was added

in one portion. The mixture was stirred at RT for 90 min and was then

neutralized by the addition of saturated aqueous NaHCO₃(60 mL). The

layers were separated and the aqueous layer extracted with CH₂Cl₂(3ꢂ

50 mL). The combined organic layers were dried (MgSO₄) and then con-

centrated. The crude product was then purified by flash chromatography

using cyclohexane/ethyl acetate 6:1 to yield alcohol 17 (1.33 g, 88%) as

Alcohol 14: To a solution of ester 13 (1.92 g, 4.34 mmol) in THF (80 mL)

at 08C was slowly added lithium aluminium hydride (168 mg, 4.43 mmol).

The reaction mixture was stirred at RT for 19 h, after which it was cooled

to 08C and quenched with H₂O (50 mL) followed by Rochelleꢄs salt solu-

tion (50 mL) and the reaction mixture stirred at RT for 15 h. The aque-

ous layer was extracted with EtOAc (3ꢂ50 mL). The combined organic

extracts were dried (MgSO₄) and evaporated under reduced pressure.

The crude product was purified by column chromatography on silica

using cyclohexane/ethyl acetate 9:1 to give alcohol 14 (1.62 g, 90%) as

colorless oil. R_f=0.06 (cyclohexane/EtOAc 12:1); [a]²_D⁰=À25.1 (c=1.08

in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.86 (d, J=6.6 Hz, 3H;

TrtOCH₂CHCH₃), 1.01 (d, J=6.7 Hz, 3H; C=CHCHCH₃), 1.03–1.09 (m,

1H; CHCH_AH_BCH), 1.28 (t, J=6.1 Hz, 1H; OH), 1.36–1.41 (m, 1H;

CHCH_AH_BCH), 1.44 (d, J=1.3 Hz, 3H; HOCH₂CCH₃), 1.69–1.77 (m,

1H; TrtOCH₂CH), 2.20–2.31 (m, 1H; C=CHCH), 2.81 (dd, J=8.6 Hz,

J=6.7 Hz, 1H; TrtOCH_AH_B), 2.99 (dd, J=8.6 Hz, J=4.8 Hz, 1H; TrtO-

CH_AH_B), 3.92 (d, J=5.6 Hz, 2H; HOCH₂), 5.12 (dd, J=9.5 Hz, J=

1.2 Hz, 1H; C=CH), 7.19–7.30 (m, 9H; Ar-H), 7.43–7.46 ppm (m, 6H;

Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=13.6 (p), 18.3 (p), 20.9 (p), 29.4

(t), 31.6 (t), 41.5 (s), 67.7 (s), 69.0 (s), 86.0 (q), 126.8 (t), 127.6 (t), 128.8

(t), 132.9 (t), 133.0 (q), 144.5 ppm (q); IR (film): n˜ =3331, 3086, 3058,

3032, 2955, 2920, 2867, 1597, 1490, 1448, 1385, 1317, 1265, 1220, 1182,

1154, 1068, 1032, 1003, 926, 899, 849, 801, 774, 764, 746, 707, 648, 633,

618, 482 cm^À1; MS (EI, 70 eV): m/z (%): 414 (0.03) [M⁺], 243 (100), 183

(3), 165 (12), 105 (3); HRMS m/z: calcd for C₂₉H₃₄O₂: 414.2559; found:

414.2561.

colorless solid. R_f=0.16 (cyclohexane/EtOAc 6:1); m.p. 438C; [a]_D²⁰

=

À44.5 (c=0.92 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.95 (d, J=

6.7 Hz, 3H; HOCH₂CHCH₃), 0.99 (d, J=6.6 Hz, 3H; C=CHCHCH₃),

1.11–1.19 (m, 1H; CHCH_AH_BCH), 1.27 (brs, 1H; OH), 1.39–1.45 (m,

1H; CHCH_AH_BCH), 1.63–1.71 (m, 1H; HOCH₂CH), 1.88 (s, 3H;

PhCH=CHCCH₃), 2.61–2.72 (m, 1H; C=CHCH), 3.42 (dd, J=10.5 Hz,

J=6.5 Hz, 1H; HOCH_AH_B), 3.53 (dd, J=10.5 Hz, J=5.2 Hz, 1H; HO-

CH_ACH_B), 5.43 (d, J=9.5 Hz, 1H; PhCH=CHC=CH), 6.45 (d, J=

16.1 Hz, 1H; PhCH), 6.79 (d, J=16.1 Hz, 1H; PhCH=CH), 7.17–7.21 (m,

1H; Ar-H), 7.29–7.32 (m, 2H; Ar-H), 7.39–7.41 ppm (m, 2H; Ar-H);

¹³C NMR (100 MHz, CDCl₃): d=12.6 (p), 17.2 (p), 20.8 (p), 30.3 (t), 33.5

(t), 41.0 (s), 68.1 (s), 125.8 (t), 126.1 (t), 126.9 (t), 128.5 (t), 132.1 (q),

134.1 (t), 137.9 (q), 140.8 ppm (t); IR (neat): n˜ =3307, 3078, 3055, 3028,

2959, 2921, 2870, 1658, 1630, 1596, 1575, 1492, 1447, 1370, 1269, 1232,

1153, 1074, 1037, 990, 961, 924, 909, 883, 830, 747, 690, 647, 532, 456, 438,

427, 409 cm^À1; MS (EI, 70 eV): m/z (%): 244 (47) [M⁺], 171 (100), 143

(29), 129 (31), 99 (37), 91 (31); HRMS m/z: calcd for C₁₇H₂₄O: 244.1827;

found: 244.1829.

Ester 7: Dess–Martin periodinane (3.56 mL, 1.65 mmol) was added at

08C to a solution of alcohol 17 (310 mg, 1.27 mmol) in CH₂Cl₂(15 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(5 mL) and saturated aqueous NaHCO₃

(5 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ10 mL). The organic layer was dried (MgSO₄) and concen-

trated to yield the crude aldehyde 18 (310 mg, 1.27 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. To a solution of sodium hydride

(60% in mineral oil, 152 mg, 3.87 mmol) in THF (15 mL) at 08C was

added dropwise ethyl 2-(diethoxyphosphoryl)butanoate (F) (960 mg,

3.87 mmol) (or ethyl 2-((bis(o-tolyloxy))phosphoryl)acetate (D) for

method according to Ando et al.^[19a–d]). The solution was stirred at RT for

1 h before cooling to 08C and addition of the crude aldehyde 18 (310 mg,

1.27 mmol) in THF (5 mL). The solution was stirred for 1 h at 08C,

warmed slowly to RT and stirred for an additional 3 h. The reaction was

quenched by pouring on to saturated aqueous NH₄Cl (80 mL). The prod-

uct was extracted with Et₂O and the combined extracts were dried

(MgSO₄) and concentrated. The crude product was then purified by

column chromatography on silica using n-pentane/Et₂O 30:1 to yield

ester 7 (328 mg, 76%) as a colorless oil. A 4:1 ratio of Z/E products was

obtained. R_f=0.50 (cyclohexane/EtOAc=18:1); [a]²_D⁰=À10.0 (c=1.36 in

Diene 16: To a solution of alcohol 14 (201 mg, 485 mmol) in DMSO

(10 mL) was added 2-iodobenzoic acid (340 mg, 1.21 mmol). The reaction

mixture was stirred at RT for 1 h, after which it was diluted with H₂O

(50 mL) and the resulting precipitate was filtered off. The aqueous layer

was extracted with Et₂O (3ꢂ20 mL). The combined organic extracts

were dried (MgSO₄) and evaporated under reduced pressure to yield the

crude aldehyde 15 (200 mg, 485 mmol, assumed to be quantitative) as col-

orless oil, which was used without further purification for the following

olefination reaction. To a solution of 5-(benzylsulfonyl)-1-phenyl-1H-tet-

razole (B) (218 mg, 728 mmol) in THF (5 mL) was slowly added lithium

bis(trimethylsilyl)amide (1m in THF, 600 mL, 600 mmol) at 08C. After stir-

ring for 20 min at RT, it was cooled to À788C and a solution of crude al-

dehyde 15 (200 mg, 0.485 mmol) in THF (5 mL) was added. The reaction

mixture was stirred overnight while slowly warming to RT. It was diluted

with Et₂O (5 mL) and the reaction quenched by addition of saturated

aqueous NaHCO₃(5 mL). The aqueous layer was extracted with EtOAc

(3ꢂ15 mL). The combined organic extracts were dried (MgSO₄) and

1

CHCl₃); H NMR (400 MHz, CDCl₃): d=0.94 (d, J=6.4 Hz, 3H; PhCH=

15012

www.chemeurj.org

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2012, 18, 15004 – 15020

Total Synthesis of the Plakotenins

FULL PAPER

CHC=CHCHCH₃), 1.00 (d, J=7.2 Hz, 3H; EtCO₂C=CHCHCH₃), 1.04

(t, J=7.2 Hz, 3H; EtCO₂CCH₂CH₃), 1.21 (t, J=6.8 Hz, 3H;

CO₂CH₂CH₃), 1.29–1.36 (m, 2H; CHCH₂CH), 1.79 (s, 3H; PhCH=

CHCCH₃), 2.22–2.33 (m, 2H; EtCO₂CCH₂CH₃), 2.43–2.59 (m, 1H;

PHCH=CHC=CHCH), 2.98–3.01 (m, 1H; EtCO₂C=CHCH), 4.01–4.15

(m, 2H; CO₂CH₂CH₃), 5.34 (d, J=9.6 Hz, 1H; PhCH=CHC=CH), 5.58

(d, J=9.6 Hz, 1H; EtCO₂C=CH), 6.41 (d, J=16.0 Hz, 1H; PhCH), 6.77

(d, J=16.0 Hz, 1H; PhCH=CH), 7.17–7.21 (m, 1H; Ar-H), 7.28–7.32 (m,

2H; Ar-H), 7.38–7.40 ppm (m, 2H; Ar-H); ¹³C NMR (100 MHz, CDCl₃):

d=12.6 (p), 13.9 (p), 14.3 (p), 21.1 (p), 21.5 (p), 27.8 (s), 31.3 (t), 32.0 (t),

45.8 (s), 60.2 (s), 125.7 (t), 126.2 (t), 126.9 (t), 128.6 (t), 132.6 (q), 132.9

(q), 134.3 (t), 138.2 (q), 140.5 (t), 145.9 (t), 168.3 ppm (q); IR (film): n˜ =

3027, 2961, 2868, 1727, 1599, 1493, 1450, 1381, 1211, 1180, 1030, 1029,

704 cm^À1; MS (EI, 70 eV): m/z (%): 340 (6) [M⁺], 267 (14), 171 (8), 91

(6), 43 (100); HRMS m/z: calcd for C₂₃H₃₂O₂: 340.2402; found: 340.2400.

Ar-H), 7.39 ppm (d, J=7.2 Hz, 2H; Ar-H); ¹³C NMR (100 MHz, CDCl₃):

d=12.7 (p), 13.4 (p), 14.3 (p), 14.4 (p), 21.1 (p), 21.5 (p), 30.0 (s), 30.9

(t), 31.9 (t), 45.9 (s), 60.7 (s), 125.6 (t), 126.2 (t), 126.9 (t), 128.5 (q), 128.6

(t), 132.6 (q), 134.3 (t), 134.9 (t), 136.3 (q), 138.2 (q), 139.1 (t), 141.0 (t),

168.4 ppm (q); IR (film): n˜ =3027, 2962, 2926, 2869, 1711, 1631, 1598,

1493, 1450, 1368, 1254, 1115, 1034, 959, 747, 692 cm^À1; MS (EI, 70 eV): m/

z (%): 380 (4) [M⁺], 225 (4), 171 (4), 91 (3), 43 (100); HRMS m/z: calcd

C₂₆H₃₆O₂: 380.2715; found: 380.2717.

Plakotenin ethyl ester 21:

A solution of linear ester 5 (85.0 mg,

223 mmol) in toluene (20 mL) was heated to reflux and stirred in a sealed

vial for 20 h. The mixture was concentrated. The crude product was puri-

fied by column chromatography on silica using n-pentane/Et₂O 40:1 to

yield plakotenin ethyl ester 21 (77.0 mg, 91%) as colorless oil. R_f=0.42

(cyclohexane/EtOAc=18:1); [a]²_D⁰= +203.8 (c=1.46 in CHCl₃); (NMR

assignment according to numbering system used in Ref. [1]) ¹H NMR

(400 MHz, CDCl₃): d=0.77 (t, J=7.2 Hz, 3H; 18-H), 0.98 (d, J=6.0 Hz,

3H; 14-H), 1.00–1.10 (m, 1H; 17-H), 1.15 (d, J=6.0 Hz, 3H; 15-H), 1.32

(t, J=7.5 Hz, 3H; 20-H), 1.54 (t, J=8.2 Hz, 2H; 7-H), 1.72–1.78 (m, 3H;

5-H, 9-H and 17-H), 1.82 (s, 3H; 16-H), 1.85–1.92 (m, 2H; 8-H and 6-H),

2.05 (s, 3H; 13-H), 3.69 (d, J=4.0 Hz, 1H; 12-H), 4.22 (q, J=7.2 Hz, 2H;

19-H), 5.23 (d, J=4.0 Hz, 1H; 11-H), 6.86 (s, 1H; 3-H), 7.20–7.24 (m,

1H; Ar-H), 7.28–7.31 ppm (m, 4H; Ar-H); ¹³C NMR (100 MHz, CDCl₃):

d=9.4 (p), 14.3 (p), 14.4 (p), 21.9 (p), 22.5 (p), 23.3 (p), 27.2 (s), 31.7 (t),

34.7 (t), 45.1 (s), 47.9 (q), 52.3 (t), 52.4 (t), 55.5 (t), 60.8 (s), 125.4 (t),

126.6 (t), 127.8 (t), 128.1 (q), 131.0 (t), 137.1 (q), 142.3 (q), 146.6 (t),

169.5 ppm (q); MS (EI, 70 eV): m/z (%): 380 (22) [M⁺], 225 (16), 171

(13), 91 (12), 43 (100); HRMS m/z: calcd C₂₆H₃₆O₂: 380.2715; found:

380.2714.

Alcohol 19: To a solution of ester 7 (325 mg, 954 mmol) in THF (10 mL)

at 08C was slowly added lithium aluminium hydride (37.0 mg, 974 mmol).

After stirring at 08C for 2 h the reaction mixture was quenched carefully

at 08C with H₂O followed by Rochelleꢄs salt solution. The aqueous layer

was extracted with Et₂O (4ꢂ10 mL). The combined organic extracts

were dried (MgSO₄) and concentrated. The crude product was then puri-

fied by column chromatography on silica using n-pentane/Et₂O 9:1 to

yield alcohol 19 (175 mg, 61%). R_f=0.52 (cyclohexane/EtOAc=6:1);

[a]²_D⁰=À102.6 (c=0.61 in CHCl₃); ¹H NMR (500 MHz, CDCl₃): d=0.94

(d, J=6.5 Hz, 3H; HOCH₂C=CHCHCH₃), 0.97 (d, J=6.5 Hz, 3H;

PhCH=CHC=CHCHCH₃), 1.05 (t, J=7.5 Hz, 3H; HOCH₂CCH₂CH₃),

1.14 (brs, 1H; OH), 1.27–1.38 (m, 2H; CHCH₂CH), 1.80 (s, 3H; PhCH=

CHCCH₃), 2.14 (q, J=7.5 Hz, 2H; HOCH₂CCH₂CH₃), 2.41–2.56 (m,

2H; CHCH₂CH), 4.02 (s, 2H; HOCH₂), 5.05 (d, J=10.0 Hz, 1H;

HOCH₂C=CH), 5.41 (d, J=9.5 Hz, 1H; PhCH=CHC=CH), 6.44 (d, J=

16.0 Hz, 1H; PhCH), 6.79 (d, J=16.0 Hz, 1H; PhCH=CH), 7.19 (t, J=

7.5 Hz, 1H; Ar-H), 7.29–7.32 (m, 2H; Ar-H), 7.40 ppm (d, J=7.5 Hz,

2H; Ar-H); ¹³C NMR (125 MHz, CDCl₃): d=12.7 (p), 13.2 (p), 21.7 (p),

22.7 (p), 28.0 (s), 30.5 (t), 31.2 (t), 46.0 (s), 60.7 (s), 126.2 (t), 126.3 (t),

127.1 (t), 128.7 (t), 133.0 (q), 133.7 (t), 133.9 (t), 138.0 (q), 139.3 (q),

140.8 ppm (t); IR (film): n˜ =3343, 3027, 2959, 2923, 1598, 1493, 1450,

1385, 1016, 958, 747, 692 cm^À1; MS (EI, 70 eV): m/z (%): 298 (67) [M⁺],

171 (100), 153 (69), 107 (60); HRMS m/z: calcd for C₂₁H₃₀O: 298.2297;

found: 298.2296.

Plakotenin 1: To

a solution of Plakotenin ethyl ester 21 (25.0 mg,

66.0 mmol) in THF/MeOH (1.6/0.8 mL) was added NaOH (2m) (160 mL,

328 mmol) and the resulting mixture was heated to 408C and stirred for

20 h. After cooling to RT, the mixture was acidified with aqueous HCl

(1m) and then extracted with EtOAc. The combined organic extracts

were backwashed with brine, dried (MgSO₄), and concentrated. The

crude product was purified by column chromatography on silica using n-

pentane/Et₂O 2:1 to yield plakotenin 1 (20.0 mg, 86%) as colorless oil.

R_f=0.40 (cyclohexane/EtOAc=2:1); [a]²_D⁰= +193 (c=0.95 in CHCl₃);

(NMR assignment according to numbering system used in Ref. [8])

¹H NMR (500 MHz, CDCl₃): d=0.78 (t, J=7.0 Hz, 3H; 18-H), 0.98 (d,

J=6.5 Hz, 3H; 14-H), 1.03–1.12 (m, 1H; 17-H), 1.16 (d, J=6.5 Hz, 3H;

15-H), 1.56 (t, J=8.5 Hz, 2H; 7-H), 1.73–1.80 (m, 3H; 5-H, 9-H and 17-

H), 1.83 (s, 3H; 16-H), 1.86–1.89 (m, 1H; 8-H), 1.89–1.92 (m, 1H; 6-H),

2.07 (s, 3H; 13-H), 3.71 (d, J=4.0 Hz, 1H; 12-H), 5.23 (s, 1H; 11-H),

7.03 (s, 1H; 3-H), 7.21–7.25 (m, 1H; Ar-H), 7.29–7.30 ppm (m, 4H; Ar-

H); ¹³C NMR (125 MHz, CDCl₃): d=9.4 (p), 14.0 (p), 21.9 (p), 22.5 (p),

23.3 (p), 27.1 (s), 31.7 (t), 34.7 (t), 45.1 (s), 48.2 (q), 52.2 (t), 52.5 (t), 55.4

(t), 125.3 (t), 126.6 (t), 127.3 (q), 127.9 (t), 131.0 (t), 137.2 (q), 142.1 (q),

149.7 (t), 174.8 ppm (q); IR (film): n˜ =2929, 2868, 1683, 1629, 1492, 1451,

1419, 1377, 1281, 877, 762, 745, 703 cm^À1; MS (EI, 70 eV): m/z (%): 352

(100) [M⁺], 261 (48), 225 (84), 171 (70); HRMS m/z: calcd for C₂₄H₃₂O₂:

352.2402; found: 352.2401.

Ester 5: Dess–Martin periodinane (1.41 mL, 653 mmol) was added at 08C

to a solution of alcohol 19 (150 mg, 503 mmol) in CH₂Cl₂(10 mL). After

stirring for 2 h at RT, the reaction mixture was quenched by adding satu-

rated aqueous Na₂S₂O₃(5 mL) and saturated aqueous NaHCO₃(5 mL).

The layers were separated and the aqueous layer was extracted with

CH₂Cl₂(4ꢂ10 mL). The organic layer was dried (MgSO₄) and concen-

trated to yield the crude aldehyde 20 (150 mg, 503 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. To a solution of sodium hydride

(60% in mineral oil, 66.0 mg, 1.66 mmol) in THF (10 mL) at 08C was

added dropwise ethyl 2-(diethoxyphosphoryl)propanoate (A) (350 mL,

1.61 mmol). The solution was stirred at RT for 1 h before cooling to 08C

and addition of crude aldehyde 20 (150 mg, 503 mmol) in THF (5 mL).

The solution was warmed slowly to RT and after stirring overnight the re-

action was quenched by pouring on to saturated aqueous NH₄Cl

(10 mL). The product was extracted with Et₂O and the combined extracts

were dried (MgSO₄) and concentrated. The crude product was then puri-

fied by column chromatography on silica using n-pentane/Et₂O 40:1 to

yield ester 5 (169 mg, 88%) as colorless oil. R_f=0.44 (cyclohexane/

EtOAc=18:1); [a]²_D⁰= +17.3 (c=0.84 in CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d=0.92 (d, J=6.4 Hz, 3H; PhCH=CHC=CHCHCH₃), 0.93 (d,

J=6.8 Hz, 3H; EtCO₂C=CHC=CHCHCH₃), 0.99 (t, J=7.6 Hz, 3H;

EtCO₂C=CHCCH₂CH₃), 1.13 (t, J=7.2 Hz, 3H; CO₂CH₂CH₃), 1.30–1.34

(m, 2H; CHCH₂CH), 1.80 (s, 3H; EtCO₂CCH₃), 1.83 (s, 3H; PhCH=

CHCCH₃), 2.13 (q, J=7.6 Hz, 2H; EtCO₂C=CHCCH₂CH₃), 2.17–2.23

(m, 1H; EtCO₂C=CHC=CHCH), 2.44–2.55 (m, 1H; PhCH=CHC=

CHCH), 3.94–4.08 (m, 2H; CO₂CH₂CH₃), 5.12 (d, J=10.0 Hz, 1H;

EtCO₂C=CHC=CH), 5.35 (d, J=9.6 Hz, 1H; PhCH=CHC=CH), 6.41 (d,

J=16.0 Hz, 1H; PhCH), 6.76 (d, J=16.0 Hz, 1H; PhCH=CH), 7.05 (s,

1H; EtCO₂C=CH), 7.15 (t, J=7.2 Hz, 1H; Ar-H) 7.28 (d, J=8.0 Hz, 2H;

Bicyclic ester 6: A solution of linear ester 7 (100 mg, 294 mmol) in tol-

uene (30 mL) was heated to reflux and stirred in a sealed vial for 20 h.

The mixture was concentrated. The crude product was purified by

column chromatography on silica using n-pentane/Et₂O 40:1 to yield

cyclic ester 6 (85.0 mg, 85%) as colorless oil. R_f=0.40 (cyclohexane/

EtOAc=18:1); [a]²_D⁰= +173.5 (c=0.34 in CHCl₃); (NMR assignment ac-

cording to numbering system used in Ref. [1]) ¹H NMR (400 MHz,

CDCl₃): d=0.78 (t, J=7.2 Hz, 3H; 15-H), 0.94 (d, J=6.4 Hz, 3H; 11-H),

1.00–1.06 (m, 1H; 14-H), 1.14 (d, J=6.0 Hz, 3H; 12-H), 1.19–1.24 (m,

1H; 11-H), 1.31 (t, J=7.5 Hz, 3H; 17-H), 1.52–1.62 (m, 3H; 3-H and 5-

H), 1.80 (brs, 3H; 13-H), 1.83–1.92 (m, 2H; 6-H and 7-H), 2.43–2.51 (m,

1H; 4-H), 4.12 (brs, 1H; 10-H), 4.22 (ddq, J=24, 9.6, 7.2 Hz, 2H; 16-H),

5.28 (s, 1H; 9-H), 7.22–7.25 (m, 1H; Ar-H), 7.26–7.30 ppm (m, 4H; Ar-

H); ¹³C NMR (100 MHz, CDCl₃): d=9.4 (p), 14.6 (p), 21.6 (p), 22.3 (p),

24.1 (p), 26.8 (s), 31.2 (t), 35.4 (t), 45.0 (s), 48.0 (t), 53.1 (t), 53.6 (t), 54.6

(q), 60.2 (s), 126.2 (t), 126.6 (t), 127.9 (t), 130.8 (t), 136.2 (q), 141.9 (q),

175.7 ppm (q); IR (film): n˜ =3083, 3059, 3027, 2959, 2867, 1727, 1600,

Chem. Eur. J. 2012, 18, 15004 – 15020

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

15013

S. Brꢀse et al.

1494, 1450, 1379, 1212, 1180, 1130, 1110, 1029, 768, 704 cm^À1; MS (EI,

70 eV): m/z (%) 340 (43) [M⁺], 267 (100), 249 (42), 171 (40), 136 (69),

121 (44), 91 (52); HRMS m/z: calcd for C₂₃H₃₂O₂: 340.2302; found:

340.2404.

0.797 mmol) was added to a solution of the primary chloride (47.5 mg,

0.133 mmol) in dry DMSO (0.15 mL) and the resulting mixture was stir-

red overnight. The reaction was quenched with brine (1 mL) and diluted

with Et₂O (3 mL). The layers were separated and the aqueous layer ex-

tracted with Et₂O (4ꢂ5 mL). The combined organic layers were back-

washed with brine (5 mL) and then dried (MgSO₄) and concentrated.

The crude product was purified by column chromatography on silica

using n-pentane/Et₂O 40:1 to yield aldehyde 25 (30.4 mg, 0.090 mmol,

68% yield for the two steps) as colorless oil. R_f=0.38 (cyclohexane/

EtOAc=18:1); [a]²_D⁰= +197.5 (c=0.42 in CHCl₃); (NMR assignment ac-

cording to numbering system used in Ref. [8]); ¹H NMR (600 MHz,

CDCl₃): d=0.82 (t, J=7.2 Hz, 3H, 18-H), 0.96 (d, J=6.0 Hz, 3H, 14-H),

1.00–1.06 (m, 1H, 17-H), 1.11 (d, J=6.6 Hz, 3H, 15-H), 1.50–1.53 (m,

2H, 7-H), 1.63–1.68 (m, 1H, 9-H), 1.80 (s, 3H, 16-H), 1.82–1.86 (m, 4H,

17-H, 5-H, 8-H, 6-H), 1.93 (s, 3H, 13-H), 3.71 (s, 1H, 12-H), 5.23 (s, 1H,

11-H), 6.42 (s, 1H, 3-H), 7.18–7.23 (m, 2H, Ar-H), 7.25–7.27 (m, 3H, Ar-

H_.) 9.38 (s, 1H, 1-H, CHO);.¹³C NMR (100 MHz, CDCl₃): d=9.3 (p),

11.1 (p), 21.8 (p), 22.5 (p), 23.3 (p), 27.0 (s), 31.7 (t), 34.7 (t), 45.0 (s),

49.1 (q), 51.8 (t), 52.6 (t), 55.0 (t), 125.3 (t), 126.7 (t), 127.9 (2 t), 130.9 (2

t), 137.3 (q), 139.6 (q), 141.6 (q), 161.0 (t), 197.2 (q); IR (film): n˜ =3082,

3059, 3026, 2932, 2868, 1688, 1630, 1599, 1493, 1451, 1378, 1216, 1031,

912, 882, 773, 704 cm^À1; MS (EI, 70 eV): m/z (%) 336 (1) [M⁺], 226 (4),

171 (4), 111 (12), 88 (10), 84 (100), 43 (34); HRMS m/z: calcd for

C₂₄H₃₂O: 336.2453; found 336.2455.

Bicyclic alcohol 22: To a solution of ester 6 (85.0 mg, 250 mmol) in THF

(3 mL) at 08C was slowly added lithium aluminium hydride (9.70 mg,

255 mmol). After stirring at 08C to RT overnight the mixture was

quenched carefully at 08C with H₂O followed by Rochelleꢄs salt solution.

The aqueous layer was extracted with EtOAc (4ꢂ5 mL), the combined

organic extracts were dried (MgSO₄) and concentrated. The crude prod-

uct was purified by column chromatography on silica using n-pentane/

Et₂O 9:1 to yield alcohol 22 (71.0 mg, 95%) as colorless oil. R_f=0.34 (cy-

clohexane/EtOAc=6:1); [a]_D²⁰= +184.6 (c=0.28 in CHCl₃); (NMR as-

signment according to numbering system used in Ref. [8]) ¹H NMR

(600 MHz, CDCl₃): d=0.82 (t, J=7.2 Hz, 3H; 15-H), 0.93 (d, J=6.6 Hz,

3H; 11-H), 1.04 (sext, J=7.2 Hz, 1H; 14-H), 1.20 (d, J=5.4 Hz, 3H; 12-

H), 1.28 (brs, 1H; OH), 1.35 (sext, J=7.2 Hz, 1H; 14-H), 1.50–1.54 (m,

1H; 5-H), 1.57–1.62 (m, 1H; 5-H), 1.71 (t, J=10.8 Hz, 1H; 3-H), 1.83 (s,

3H; 13-H), 1.86–1.90 (m, 2H; 6-H and 7-H), 2.17–2.23 (m, 1H; 4-H),

4.12 (d, J=4.2 Hz, 1H; 10-H), 3.60 (d, J=11.4 Hz, 1H; 1-H), 3.84 (d, J=

10.8 Hz, 1H; 1-H), 5.19 (d, J=3.6 Hz, 1H; 9-H), 7.20–7.23 (m, 1H; Ar-

H), 7.25–7.29 ppm (m, 4H; Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=8.8

(p), 21.6 (p), 22.5 (p), 24.0 (p), 25.7 (s), 31.4 (t), 35.2 (t), 43.4 (q), 45.3 (s),

49.8 (t), 51.7 (t), 54.3 (t), 65.2 (s), 126.1 (t), 126.3 (t), 127.7 (t), 130.7 (t),

136.5 (q), 141.3 ppm (q); IR (film): n˜ =3394, 3081, 3059, 3025, 2928, 1657,

1599, 1491, 1451, 1376, 1032, 909, 872, 771, 747, 703 cm^À1. - MS (EI,

70 eV): m/z (%) 298 (26) [M⁺], 267 (100), 171 (37), 162 (32), 133 (26), 91

(47), 43 (56); HRMS m/z: calcd for C₂₁H₃₀O: 298.2297; found: 298.2299.

Plakotenin 1: NaH₂PO₄·2H₂O (9.6 mg, 0.062 mmol, 0.6 equiv), H₂O₂

(0.044 mL, 0.384 mmol, 3.75 equiv), and NaClO₂(55.6 mg, 0.615 mmol,

6.0 equiv) were successively added at 08C to a solution of aldehyde 25

(34.5 mg, 0.018 mmol) in MeCN/H₂O (3.0/0.5 mL) and the mixture was

stirred at RT for 20 h. The reaction mixture was then quenched by addi-

tion of water (0.5 mL). The layers were separated and the aqueous layer

extracted with EtOAc (4ꢂ3 mL). The combined organic layers were

backwashed with brine (3 mL) and then dried (MgSO₄) and concentrat-

ed. The crude product was purified by column chromatography on silica

using n-pentane/Et₂O 2:1 to yield 1 (22.3 mg, 0.063 mmol, 61% yield) as

colorless oil. R_f=0.40 (cyclohexane/EtOAc=2:1); [a]²_D⁰= +193 (c=0.95

in CHCl₃); (NMR assignment according to numbering system used in

Ref. [8]) ¹H NMR (500 MHz, CDCl₃): d=0.78 (t, J=7.0 Hz, 3H; 18-H),

0.98 (d, J=6.5 Hz, 3H; 14-H), 1.03–1.12 (m, 1H; 17-H), 1.16 (d, J=

6.5 Hz, 3H; 15-H), 1.56 (t, J=8.5 Hz, 2H; 7-H), 1.73–1.80 (m, 3H; 5-H,

9-H and 17-H), 1.83 (s, 3H; 16-H), 1.86–1.89 (m, 1H; 8-H), 1.89–1.92 (m,

1H; 6-H), 2.07 (s, 3H; 13-H), 3.71 (d, J=4.0 Hz, 1H; 12-H), 5.23 (s, 1H;

11-H), 7.03 (s, 1H; 3-H), 7.21–7.25 (m, 1H; Ar-H), 7.29–7.30 ppm (m,

4H; Ar-H);¹³C NMR (125 MHz, CDCl₃): d=9.4 (p), 14.0 (p), 21.9 (p),

22.5 (p), 23.3 (p), 27.1 (s), 31.7 (t), 34.7 (t), 45.1 (s), 48.2 (q), 52.2 (t), 52.5

(t), 55.4 (t), 125.3 (t), 126.6 (t), 127.3 (q), 127.9 (t), 131.0 (t), 137.2 (q),

142.1 (q), 149.7 (t), 174.8 ppm (q); IR (film): n˜ =2929, 2868, 1683, 1629,

1492, 1451, 1419, 1377, 1281, 877, 762, 745, 703 cm^À1; MS (EI, 70 eV): m/z

(%): 352 (100) [M⁺], 261 (48), 225 (84), 171 (70); HRMS m/z: calcd for

C₂₄H₃₂O₂: 352.2402; found: 352.2401.

Allylic alcohol 24: Dess–Martin periodinane (0.83 mL, 0.395 mmol) was

added at 08C to a solution of alcohol 22 (90.0 g, 0.304 mmol) in CH₂Cl₂

(3 mL). After stirring for 2 h at RT, the reaction mixture was quenched

by adding saturated aqueous Na₂S₂O₃(1.5 mL) and saturated aqueous

NaHCO₃(1.5 mL). The layers were separated and the aqueous layer ex-

tracted with CH₂Cl₂(4ꢂ3 mL). The organic layer was dried (MgSO₄) and

concentrated to yield the crude aldehyde 23 (89.5 mg, 0.304 mmol, as-

sumed to be quantitative) as colorless oil, which was used without further

purification for the following Grignard reaction. Under argon, to a solu-

tion of aldehyde 23 (88.0 mg, 0.297 mmol) in THF (5 mL) was added iso-

propenylmagnesium bromide (0.5m in THF, 0.89 mL, 0.445 mmol) at

08C. The reaction was then warmed to 508C and stirred for 3 h and then

quenched with 1m HCl (5 mL) and diluted with Et₂O. The layers were

separated and the aqueous layer extracted with Et₂O (3ꢂ5 mL). The

combined organic layers were washed with brine (5 mL) dried (MgSO₄)

and then concentrated. The crude product was purified by column chro-

matography on silica using n-pentane/Et₂O 18:1 to yield allylic alcohol 24

(89.5 mg, 0.264 mmol, 87% yield) as a colorless oil. R_f=0.40 (cyclohex-

ane/EtOAc=9:1); 10:1 mixture of diastereoisomers. Description of the

major diastereoisomer: (NMR assignment according to numbering

system used in Ref. [8]); ¹H NMR (400 MHz, CDCl₃): d=0.39 (t, J=

7.2 Hz, 3H, 18-H), 0.95 (d, J=6.4 Hz, 3H, 14-H), 1.16–1.21 (m, 1H, 17-

H), 1.22 (d, J=6.4 Hz, 3H, 15-H), 1.53 (t, J=8.4 Hz, 2H, 7-H), 1.64–1.71

(m, 2H, 9-H and 17-H), 1.85 (s, 3H, 16-H), 1.89–1.91 (m, 1H, 5-H), 1.94

(s, 3H, 12-H), 2.11 (t, J=11.0 Hz, 1H, 9-H), 2.36–2.47 (m, 1H, 6-H), 3.36

(brs, 1H, 12-H), 4.52 (s, 1H, 3-H), 5.02 (s, 1H, 1-H), 5.08 (s, 1H, 1-H),

5.13 (brs, 1H, 11-H), 7.17–7.25 (m, 5H, Ar-H);¹³C NMR (150 MHz,

CDCl₃): d=9.7 (p), 21.3 (p), 22.4 (p), 22.6 (p), 22.8 (p), 25.7 (s), 33.7 (t),

34.3 (t), 45.1 (s), 46.8 (q), 51.2 (t), 52.2 (t), 52.8 (t), 80.0 (t), 115.7 (s),

125.7 (t), 126.4 (t), 127.8 (2t), 131.1 (2t), 137.1 (q), 143.3 (q), 147.3 (q);

IR (film): n˜ =3471, 3061, 3024, 2927, 2867, 1633, 1599, 1492, 1452, 1374,

1039, 983, 903, 879, 762, 702 cm^À1; MS (EI, 70 eV): m/z (%) 338 (18) [M⁺

] 320 (31), 267 (100), 195 (27), 131 (38), 105 (28), 91 (56), 43 (42); HRMS

m/z: calcd for C₂₄H₃₄O: 338.2610; found: 338.2613.

Ester 26: The compound was isolated during the purification of ester 7

and purified by column chromatography to give a colorless oil (77.8 mg,

0.028 mmol 18%). R_f=0.46 (cyclohexane/EtOAc=18:1); [a]²_D⁰=À33.1

(c=0.61 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): d=0.94 (t, J=7.8 Hz,

3H, EtCO₂C=CH₂CH₃), 0.97 (d, J=7.2 Hz, 3H, PhCH=CHC=

CHCHCH₃), 1.01 (d, J=6.6 Hz, 3H, EtCO₂C=CHCHCH₃), 1.31 (t, J=

7.2 Hz, 3H, CO₂CH₂CH₃), 1.40–1.43 (m, 2H, CHCH₂CH), 1.77 (s, 3H,

PhCH=CHCCH₃), 2.17–2.26 (m, 2H, EtCO₂CCH₂CH₃), 2.47–2.55 (m,

2H, PhCH=CHC=CHCH and EtCO₂C=CHCH), 4.20 (q, J=7.2 Hz, 2H,

CO₂CH₂CH₃), 5.37 (d, J=9.0 Hz, 1H, PhCH=CHC=CH), 6.43 (d, J=

16.2 Hz, 1H, PhCH), 6.49 (d, J=10.2 Hz, 1H, EtCO₂C=CH), 6.79 (d, J=

16.2 Hz, 1H, PhCH=CH), 7.18–7.21 (m, 1H, Ar-H), 7.31 (t, J=7.8 Hz,

2H, Ar-H), 7.40 ppm (d, J=7.2 Hz, 2H, Ar-H); ¹³C NMR (125 MHz,

CDCl₃): d=12.8 (p), 14.4 (2 p), 20.2 (s), 21.3 (p), 21.7 (p), 31.3 (t), 31.5

(t), 45.6 (s), 60.4 (s), 125.9 (t), 126.3 (2t), 127.0 (t), 128.7 (2t), 133.1 (q),

133.2 (q), 134.1 (t), 138.1 (q), 140.3 (t), 147.6 (t), 168.3 ppm (q); IR

(film): n˜ =3026, 2960, 2925, 2869, 1711, 1645, 1493, 1452, 1377, 1223,

1183, 1032, 959, 693 cm^À1; MS (EI, 70 eV): m/z (%) 340 ([M⁺], 93), 267

(100), 171 (61), 91 (40), 43 (26); HRMS m/z: calcd for C₂₃H₃₂O₂:

340.2402; found: 340.2404.

Aldehyde 25: Under argon, to

a solution of aldehyde 25 (45 mg,

0.133 mmol) in dry Et₂O/n-pentane (1.5 mL) was added at 08C freshly

distilled SOCl₂(0.048 mL, 0.665 mmol), and the mixture was slowly

warmed up to RT and stirred for 6 h. Solvents were then removed under

vacuum, and the crude residue directly used for the next step without fur-

ther purification. Under argon, one portion of NMO (93.4 mg,

15014

www.chemeurj.org

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2012, 18, 15004 – 15020

Total Synthesis of the Plakotenins

FULL PAPER

Alcohol 27: Lithium aluminium hydride (8.6 mg, 0.226 mmol) was slowly

added to a solution of ester 26 (70 mg, 0.206 mmol) in THF (4 mL) at

08C. After stirring at 08C for 2 h the reaction mixture was quenched

carefully at 08C with H₂O followed by Rochelleꢄs salt solution. The aque-

ous layer was extracted with Et₂O (4ꢂ5 mL), dried (MgSO₄), and con-

centrated. The crude product was then purified by column chromatogra-

phy on silica using n-pentane/Et₂O 9:1 to yield alcohol 27 (41.7 mg,

0.140 mmol, 68% yield). R_f=0.44 (cyclohexane/EtOAc=6:1). R_f=0.52

(cyclohexane/EtOAc=6:1); [a]²_D⁰=À107.6 (c=0.25 in CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d=0.94–0.97 (m, 9H, HOCH₂C=CHCHCH₃,

HOCH₂C=CH₂CH₃, and PhCH=CHC=CHCHCH₃), 1.29–1.34 (m, 2H,

CHCH₂CH), 1.79 (s, 3H, PhCH=CHCCH₃), 1.91–1.98 (m, 1H,

HOCH₂CCH₂CH₃), 2.05–2.14 (m, 1H, HOCH₂CCH₂CH₃), 2.35–2.47 (m,

1H, HOCH₂C=CHCHCH₃), 2.49–2.56 (m, 1H, PhCH=CHC=

CHCHCH₃), 4.05 (s, 2H, HOCH₂), 5.14 (d, J=9.6 Hz, 1H, HOCH₂C=

CH), 5.39 (d, J=9.6 Hz, 1H, PhCH=CHC=CH), 6.44 (d, J=16.0 Hz, 1H,

PhCH), 6.80 (d, J=16.0 Hz, 1H, PhCH=CH), 7.19 (t, J=7.2 Hz, 1H, Ar-

H), 7.31 (t, J=7.6 Hz, 2H, Ar-H), 7.40 ppm (d, J=7.6 Hz, 2H, Ar-H);

¹³C NMR (100 MHz, CDCl₃): d=12.8 (p), 13.7 (p), 21.3 (s), 21.6 (p), 22.2

(p), 30.2 (t), 31.2 (t), 46.1 (s), 67.0 (s), 125.7 (t), 126.3 (2t), 127.0 (t),

128.7 (2 t), 132.6 (t), 132.7 (q), 134.3 (t), 138.1 (q), 139.7 (q), 141.1 ppm

(t); IR (film): n˜ =3315, 3030, 2960, 1597, 1493, 1451, 1372, 1313, 1075,

1036, 1013, 961, 747, 690 cm^À1; MS (EI, 70 eV): m/z (%) 298 ([M⁺], 23),

171 (100), 143 (47), 129 (66), 91 (91), 43 (46); HRMS m/z: calcd for

C₂₁H₃₀O: 298.2297; found: 298.2295.

40:1 to yield cyclic ester 30 (24.6 mg, 0.065 mmol, 82% yield) as colorless

oil. R_f=0.42 (cyclohexane/EtOAc=18:1). [a]²_D⁰= +179.1 (c=0.23 in

CHCl₃); (NMR assignment according to numbering system used in

Ref. [8]) ¹H NMR (500 MHz, CDCl₃): d=0.80 (d, J=6.0 Hz, 3H, 14-H),

1.02 (t, J=7.0 Hz, 3H, 18-H), 1.06 (t, J=7.0 Hz, 3H, 20-H), 1.22 (d, J=

6.0 Hz, 3H, 15-H), 1,51 (dt, J=2.5, 8.5 Hz, 2H, 7-H), 1.68 (t, J=10.5 Hz,

1H, 5-H), 1.86 (s, 3H, 16-H), 1.82–1.94 (m, 4H, 17-H, 9-H, and 8-H),

1.98 (s, 3H, 13-H), 1.98–2.04 (m, 1H, 6-H), 3.36 (brs, 1H, 12-H), 3.92–

4.02 (m, 2H, 19-H), 5.22 (brs, 1H, 11-H), 6.11 (s, 1H, 3-H), 7.06 (d, J=

7.0 Hz, 2H, Ar-H), 7.16 (t, J=7.5 Hz, 1H, Ar-H), 7.20 ppm (d, J=

7.5 Hz, 2H, Ar-H); ¹³C NMR (125 MHz, CDCl₃): d=10.9 (p), 14.2 (p),

14.3 (p), 21.2 (p), 22.4 (p), 22.7 (p), 26.1 (s), 33.5 (t), 33.9 (t), 44.5 (s),

47.1 (q), 50.9 (t), 54.1 (t), 54.4 (t), 60.3 (s), 124.1 (t), 125.7 (q), 126.4 (t),

127.6 (2 t), 130.5 (2 t), 136.6 (q), 141.8 (q), 148.2 (t), 169.2 ppm (q); IR

(film): n˜ =3025, 2930, 2867, 1705, 1639, 1600, 1492, 1450, 1378, 1262,

1105, 1033, 743, 702 cm^À1; MS (EI, 70 eV): m/z (%) 380 ([M⁺], 44), 225

(34), 171 (28), 91 (21), 58 (30), 43 (100); HRMS m/z: calcd for C₂₆H₃₆O₂:

380.2715; found: 380.2712.

iso-Plakotenin 4: NaOH (2m) (0.13 mL, 0.263 mmol) was added to a sol-

ution of cyclic ester 30 (20.0 mg, 0.053 mmol) in THF/MeOH (1.6/

0.8 mL) and the resulting mixture was heated to 408C and stirred for

20 h. After cooling to RT, the mixture was acidified with aqueous HCl

(1m), and then extracted with EtOAc (5ꢂ3 mL). The combined organic

extracts were washed with brine, dried (MgSO₄), and concentrated. The

crude product was purified by column chromatography on silica using n-

pentane/Et₂O 2:1 to yield carboxylic acid 4 (13.9 mg, 0.039 mmol, 75%

yield) as colorless oil. R_f=0.38 (cyclohexane/EtOAc=2:1). [a]²_D⁰= +

184.6 (c=0.52 in CHCl₃); (NMR assignment according to numbering

system used in Ref. [8]); ¹H NMR (600 MHz, CDCl₃): d=0.82 (d, J=

6.0 Hz, 3H, 14-H), 1.01 (t, J=7.5 Hz, 3H, 18-H), 1.21 (d, J=6.0 Hz, 3H,

15-H), 1.47–1.55 (m, 2H, 7-H), 1.71 (t, J=10.8 Hz, 1H, 5-H), 1.86 (s, 3H,

16-H), 1.82–1.93 (m, 3H, 17-H, 9-H, and 8-H), 1.95 (s, 3H, 13-H), 1.98–

2.05 (m, 2H, 6-H and 17-H), 3.36 (d, J=3.6 Hz, 1H, 12-H), 5.22 (brs,

1H, 11-H), 6.27 (s, 1H, 3-H), 7.05 (d, J=7.8 Hz, 2H, Ar-H), 7.16 (t, J=

7.2 Hz, 1H, Ar-H), 7.21 ppm (t, J=7.2 Hz, 2H, Ar-H); ¹³C NMR

(125 MHz, CDCl₃): d=11.1 (p), 14.1 (p), 21.1 (p), 22.3 (p), 22.9 (p), 26.5

(s), 33.7 (t), 33.8 (t), 44.3 (s), 47.4 (q), 51.0 (t), 52.3 (2t), 124.1 (t), 125.1

(q), 126.6 (t), 127.7 (2t), 130.4 (2 t), 136.5 (q), 141.5 (q), 151.1 (t),

173.98 ppm (q); IR (film): n˜ =3394, 3027, 2957, 2867, 1678, 1452, 1377,

1271, 1031, 746, 701 cm^À1; MS (EI, 70 eV): m/z (%) 352 ([M⁺], 4), 225

(2), 171 (6), 107 (10), 77 (15), 58 (15), 43 (100); HRMS m/z: calcd for

C₂₄H₃₂O₂: 352.2402; found: 352.2404.

Ester 29: Dess–Martin periodinane (0.39 mL, 0.157 mmol) was added at

08C to a solution of alcohol 27 (36.0 mg, 0.121 mmol) in CH₂Cl₂(3 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(2 mL) and saturated aqueous NaHCO₃

(2 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ5 mL). The organic layer was dried (MgSO₄) and concentrat-

ed to yield the crude aldehyde 28 (35.0 mg, 0.121 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. Ethyl 2-(diethoxyphosphoryl)pro-

panoate (0.08 mL, 0.386 mmol) was added dropwise to a solution of

sodium hydride (60% in mineral oil, 15.4 mg, 0.386 mmol) in THF

(4 mL) at 08C. The solution was stirred at RT for 1 h before cooling to

08C and addition of crude aldehyde 28 (35.0 mg, 0.121 mmol) (from pre-

vious reaction) in THF (2 mL). The solution was warmed slowly to RT

and stirred for 4 h. The reaction was then quenched by pouring on to sa-

turated aqueous NH₄Cl (3 mL). The product was extracted with Et₂O

(4ꢂ5 mL) and the combined extracts were dried (MgSO₄) and concen-

trated. The crude product was then purified by column chromatography

on silica using n-pentane/Et₂O 40:1 to yield ester 29 (38.6 mg,

0.101 mmolmg, 84% yield) as colorless oil. R_f=0.42 (cyclohexane/

EtOAc=18:1). [a]²_D⁰À215.46 (c=0.28 in CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d=0.90 (t, J=7.6 Hz, 3H, EtCO₂C=CHCCH₂CH₃), 0.98 (d, J=

7.2 Hz, 3H, EtCO₂C=CHC=CHCHCH₃), 0.99 (d, J=6.8 Hz, 3H, PhCH=

CHC=CHCHCH₃), 1.32 (t, J=7.2 Hz, 3H, CO₂CH₂CH₃), 1.28–1.36 (m,

2H, CHCH₂CH), 1.80 (s, 3H, PhCH=CHCCH₃), 1.99 (d, J=1.2 Hz, 3H,

EtCO₂CCH₃), 2.00–2.06 (m, 1H, EtCO₂C=CHCCH₂CH₃), 2.15–2.24 (m,

1H, EtCO₂C=CHCCH₂CH₃), 2.46–2.56 (m, 2H, PhCH=CHC=

CHCHCH₃and EtCO₂C=CHC=CHCHCH₃), 4.22 (q, J=7.2 Hz, 2H,

CO₂CH₂CH₃), 5.23 (d, J=9.6 Hz, 1H, EtCO₂C=CHC=CH), 5.40 (d, J=

9.6 Hz, 1H, PhCH=CHC=CH), 6.43 (d, J=16.0 Hz, 1H, PhCH), 6.81 (d,

J=16.0 Hz, 1H, PhCH=CH), 7.09 (s, 1H, EtCO₂C=CH), 7.19 (t, J=

7.2 Hz, 1H, Ar-H) 7.31 (d, J=8.0 Hz, 2H, Ar-H), 7.41 ppm (d, J=7.2 Hz,

2H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=12.8 (p), 13.8 (p), 14.1 (p),

14.5 (p), 21.7 (p), 22.1 (p), 23.6 (s), 31.1 (t), 31.4 (t), 46.1 (s), 60.7 (s),

125.8 (t), 126.3 (2t), 126.7 (q), 127.0 (t), 128.7 (2t), 133.0 (q), 134.2 (t),

137.0 (q), 138.1 (q), 140.0 (t), 140.7 (t), 142.1 (t), 169.2 ppm (q); IR

(film): n˜ =3025, 2961, 2924, 2867, 1706, 1627, 1598, 1493, 1449, 1366,

1252, 1112, 1033, 960, 748, 692 cm^À1; MS (EI, 70 eV): m/z (%) 380 ([M⁺],

15), 225 (13), 171 (18), 91 (13), 58 (39), 43 (100); HRMS m/z: calcd for

C₂₆H₃₆O₂: 380.2715; found: 380.2713.

Ester 31: Dess–Martin periodinane (1.71 mL, 0.798 mmol) was added at

08C to a solution of alcohol 17 (150.0 mg, 0.614 mmol) in CH₂Cl₂(6 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(3 mL) and saturated aqueous NaHCO₃

(3 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ10 mL). The organic layer was dried (MgSO₄) and concen-

trated to yield the crude aldehyde 18 (150.0 mg, 0.614 mmol, assumed to

be quantitative) as colorless oil, which was used without further purifica-

tion for the following olefination reaction. Ethyl 2-(diethoxyphosphoryl)-

propanoate (0.39 mL, 1.842 mmol) was added dropwise to a solution of

sodium hydride (60% in mineral oil, 75 mg, 1.842 mmol) in THF (15 mL)

at 08C. The solution was stirred at RT for 1 h before cooling to 08C and

addition of the crude aldehyde 18 (150.0 mg, 0.614 mmol) (from previous

reaction) in THF (5 mL). The solution was stirred for 1 h at 08C, warmed

slowly to RT and stirred for an additional 3 h. The reaction was quenched

by pouring on to saturated aqueous NH₄Cl (10 mL). The product was ex-

tracted with Et₂O (3ꢂ10 mL) and the combined extracts were dried

(MgSO₄) and concentrated. The crude product was then purified by

column chromatography on silica using n-pentane/Et₂O 30:1 to yield

ester 31 (128.3 mg, 0.393 mmol, 64%) as a colorless oil. A 3:1 ratio of Z/

E products was obtained. R_f=0.52 (cyclohexane/EtOAc=18:1). [a]_D²⁰

=

À20.1 (c=0.57 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.98 (d, J=

6.4 Hz, 3H, PhCH=CHC=CHCHCH₃), 0.99 (d, J=6.4 Hz, 3H, EtCO₂C=

CHCHCH₃), 1.21 (t, J=7.2 Hz, 3H, CO₂CH₂CH₃), 1.30–1.35 (m, 2H,

CHCH₂CH), 1.77 (d, J=1.2 Hz, 3H, PhCH=CHCCH₃), 1.90 (d, J=

1.2 Hz, 3H, EtCO₂CCH₃), 2.45–2.56 (m, 1H, PhCH=CHC=CHCH),

3.07–3.18 (m, 1H, EtCO₂C=CHCH), 4.03–4.11 (m, 2H, CO₂CH₂CH₃),

iso-Plakotenin ethyl ester 30: A solution of linear ester 29 (30.0 mg,

0.079 mmol) in toluene (8 mL) was heated to reflux and stirred in a

sealed vial for 20 h. The mixture was concentrated. The crude product

was purified by column chromatography on silica using n-pentane/Et₂O

Chem. Eur. J. 2012, 18, 15004 – 15020

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

15015

S. Brꢀse et al.

5.34 (d, J=9.6 Hz, 1H, PhCH=CHC=CH), 5.66 (dd, J=10.0, 1.2 Hz, 1H,

EtCO₂C=CH), 6.41 (d, J=16.0 Hz, 1H, PhCH), 6.77 (d, J=16.0 Hz, 1H,

PhCH=CH), 7.17–7.21 (m, 1H, Ar-H), 7.28–7.32 (m, 2H, Ar-H), 7.38–

7.40 ppm (m, 2H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=12.6 (p), 14.3

(p), 20.9 (p), 21.0 (p), 21.5 (p), 31.3 (t), 32.0 (t), 45.7 (s), 60.2 (s), 125.7

(t), 126.2 (2t), 126.3 (q), 126.9 (t), 128.6 (2t), 132.6 (q), 134.3 (t), 138.2

(q), 140.4 (t), 148.4 (t), 168.2 ppm (q); IR (film): n˜ =3059, 3062, 2958,

2927, 2868, 1720, 1451, 1376, 1222, 1174, 1097, 1024, 770, 704 cm^À1; MS

(EI, 70 eV): m/z (%) 326 ([M⁺], 42), 253 (100), 252 (47), 171 (39), 136

(41), 121 (36), 91 (48); HRMS m/z: calcd C₂₂H₃₀O₂: 326.2246; found:

326.2242.

(q); IR (film): n˜ =3079, 3058, 3025, 2958, 1709, 1631, 1598, 1492, 1449,

1382, 1258, 1114, 1036, 958, 825, 747, 692 cm^À1; MS (EI, 70 eV): m/z (%)

366 ([M⁺], 100), 225 (77), 171 (88), 128 (21), 107 (41), 91 (95); HRMS m/

z: calcd for C₂₅H₃₄O₂: 366.2559; found: 366.2560.

nor-Plakotenin ethyl ester 35: A solution of linear ester 34 (65.0 mg,

0.177 mmol) in toluene (18 mL) was heated to reflux and stirred in a

sealed vial for 20 h. The mixture was concentrated. The crude product

was purified by column chromatography on silica using n-pentane/Et₂O

40:1 to yield cyclic ester 35 (59.8 mg, 0.163 mmol, 92% yield) as colorless

oil. R_f=0.40 (cyclohexane/EtOAc=18:1). [a]²_D⁰= +189.4 (c=0.24 in

CHCl₃); (NMR assignment according to numbering system used in

Ref. [8]) ¹H NMR (400 MHz, CDCl₃): d=0.90 (s, 3H, 17-H), 0.93 (d, J=

6.8 Hz, 3H, 14-H), 1.21 (d, J=6.0 Hz, 3H, 15-H), 1.34 (t, J=7.2 Hz, 3H,

19-H), 1.50–1.63 (m, 3H, 7-H and 5-H), 1.87 (s, 3H, 16-H) 1.88–1.93 (m,

3H, 9-H, 8-H and 6-H), 2.14 (d, J=1.2 Hz, 3H, 13-H), 3.63 (d, J=4.0 Hz,

1H, 12-H), 4.23 (q, J=7.2 Hz, 2H, 18-H), 5.23 (brs, 1H, 11-H), 7.11 (d,

J=1.2 Hz, 1H, 3-H), 7.24–7.34 ppm (m, 5H, Ar-H); ¹³C NMR (100 MHz,

CDCl₃): d=14.4 (p), 14.8 (p), 22.0 (p), 22.4 (p), 22.9 (p), 24.9 (p), 32.1

(t), 34.6 (t), 41.7 (q), 44.8 (s), 51.6 (t), 53.2 (t), 55.6 (t), 60.8 (s), 124.9 (t),

126.5 (t), 127.8 (2t), 128.0 (q), 130.7 (2t), 137.4 (q), 142.2 (q), 148.3 (t),

169.5 (q); IR (film): n˜ =3025, 2929, 2868, 1708, 1491, 1451, 1379, 1240,

1098, 744, 703 cm^À1; MS (EI, 70 eV): m/z (%) 366 ([M⁺], 18), 225 (11),

171 (11), 107 (14), 91 (14), 43 (100); HRMS m/z: calcd for C₂₅H₃₄O₂:

366.2559; found: 366.2555.

Alcohol 32: Lithium aluminium hydride (15.3 mg, 0.404 mmol) was

slowly added to a solution of ester 31 (120 mg, 0.368 mmol) in THF

(5 mL) at 08C. After stirring at 08C for 2 h the reaction mixture was

quenched carefully at 08C with H₂O followed by Rochelleꢄs salt solution.

The aqueous layer was extracted with Et₂O (4ꢂ5 mL), dried (MgSO₄)

and concentrate. The crude product was then purified by column chroma-

tography on silica using n-pentane/Et₂O 9:1 to yield alcohol 32 (81.5 mg,

0.287 mmol, 78% yield). R_f=0.48 (cyclohexane/EtOAc=6:1). [a]_D²⁰

=

À126.4 (c=0.58 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.92 (d, J=

6.4 Hz, 3H, HOCH₂C=CHCHCH₃), 0.96 (d, J=6.8 Hz, 3H, PhCH=

CHC=CHCHCH₃), 1.25–1.37 (m, 2H, CHCH₂CH), 1.79 (d, J=1.2 Hz,

3H, HOCH₂CCH₂CH₃), 1.80 (d, J=1.2 Hz, 2H, PhCH=CHCCH₃), 2.36–

2.46 (m, 1H, HOCH₂C=CHCH), 2.47–2.56 (m, 1H, PhCH=CHC=

CHCH), 3.99 (d, J=1.6 Hz, 2H, HOCH₂), 5.03 (d, J=9.6 Hz, 1H,

HOCH₂C=CH), 5.40 (d, J=9.6 Hz, 1H, PhCH=CHC=CH), 6.44 (d, J=

16.0 Hz, 1H, PhCH), 6.80 (d, J=16.0 Hz, 1H, PhCH=CH), 7.19 (t, J=

7.2 Hz, 1H, Ar-H), 7.30 (t, J=8.0 Hz, 2H, Ar-H), 7.40 ppm (d, J=

7.2 Hz, 2H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=12.7 (p), 21.5 (p),

21.7 (p), 22.6 (p), 30.6 (t), 31.2 (t), 45.9 (s), 62.0 (s), 126.2 (t), 126.3 (2t),

127.1 (t), 128.7 (2t), 132.9 (q), 133.5 (q), 133.9 (t), 134.8 (t), 138.0 (q),

140.8 ppm (t); IR (film): n˜ =3427, 2960, 1721, 1600, 1494, 1452, 1377,

1265, 1176, 1004, 750, 699 cm^À1; MS (EI, 70 eV): m/z (%) 284 ([M⁺], 90),

253 (53), 171 (100), 169 (41), 143 (42), 129 (44); HRMS m/z: calcd for

C₂₀H₂₈O: 284.2140; found: 284.2137.

nor-Plakotenin 2: NaOH (2m) (0.34 mL, 0.682 mmol) was added to a sol-

ution of cyclic ester 35 (50.0 mg, 0.136 mmol) in THF/MeOH (3.2/

1.6 mL) and the resulting mixture was heated to 408C and stirred for

20 h. After cooling to RT, the mixture was acidified with aqueous HCl

(1m), and then extracted with EtOAc (5ꢂ5 mL). The combined organic

extracts were backwashed with brine, dried (MgSO₄), and concentrated.

The crude product was purified by column chromatography on silica

using n-pentane/Et₂O 2:1 to yield carboxylic acid 2 (40.6 mg, 0.120 mmol,

88% yield) as colorless oil. R_f=0.38 (cyclohexane/EtOAc=2:1); [a]_D²⁰

=

+1018 (0.19 g/100 mL, CHCl₃); [a]²_D⁰= +1288 (0.22 g/100 mL, MeOH);

(NMR assignment according to numbering system used in Ref. [8])

¹H NMR (600 MHz, CDCl₃): d=0.90 (s, 3H, 17-H), 0.91 (d, J=6.6 Hz,

3H, 14-H), 1.20 (d, J=5.4 Hz, 3H, 15-H), 1.50–1.63 (m, 3H, 7-H and 5-

H), 1.85 (s, 3H, 16-H) 1.86–1.94 (m, 3H, 9-H, 8-H and 6-H), 2.13 (s, 3H,

13-H), 3.62 (s, 1H, 12-H), 5.21 (s, 1H, 11-H), 7.20 (s, 1H, 3-H), 7.22–7.24

(m, 3H, Ar-H), 7.28–7.31 (m, 2H, Ar-H), 10.18 ppm (brs, 1H, -OH);

¹³C NMR (100 MHz, CDCl₃): d=14.7 (p), 22.0 (p), 22.5 (p), 22.9 (p), 24.8

(p), 32.2 (t), 34.7 (t), 41.9 (q), 44.8 (s), 51.7 (t), 53.3 (t), 55.6 (t), 124.9 (t),

126.6 (t), 127.8 (2t and q), 130.7 (2t), 137.5 (q), 142.2 (q), 150.1 (t),

174.9 ppm (q); IR (film): n˜ =3025, 2928, 2869, 1683, 1628, 1492, 14512,

1378, 1277, 998, 703 cm^À1; MS (EI, 70 eV): m/z (%) 338 ([M⁺], 1), 86 (4),

84 (6), 58 (36), 43 (100); HRMS m/z: calcd for C₂₃H₃₀O₂: 338.2246;

found: 338.2243.

Ester 34: Dess–Martin periodinane (0.57 mL, 0.274 mmol) was added at

08C to a solution of alcohol 32 (60.0 mg, 0.211 mmol) in CH₂Cl₂(5 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(2 mL) and saturated aqueous NaHCO₃

(2 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ5 mL). The organic layer was dried (MgSO₄) and concentrat-

ed to yield the crude aldehyde 33 (60.0 mg, 0.211 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. Ethyl 2-(diethoxyphosphoryl)pro-

panoate (0.14 mL, 0.633 mmol) was added dropwise to a solution of

sodium hydride (60% in mineral oil, 25.0 mg, 0.633 mmol) in THF

(5 mL) at 08C. The solution was stirred at RT for 1 h before cooling to

08C and addition of crude aldehyde 33 (60.0 mg, 0.211 mmol) (from pre-

vious reaction) in THF (3 mL). The solution was warmed slowly to RT

and stirred for an additional 3 h. The reaction was then quenched by

pouring on to saturated aqueous NH₄Cl (5 mL). The product was extract-

ed with Et₂O (4ꢂ5 mL) and the combined extracts were dried (MgSO₄)

and concentrated. The crude product was then purified by column chro-

matography on silica using n-pentane/Et₂O 40:1 to yield ester 34

(68.8 mg, 0.188 mmol, 89% yield) as colorless oil. R_f=0.40 (cyclohexane/

EtOAc=18:1); [a]²_D⁰= +14.9 (c=0.40 in CHCl₃); ¹H NMR (400 MHz,

CDCl₃): d=0.93 (d, J=6.8 Hz, 3H, EtCO₂C=CHC=CHCHCH₃), 0.96 (d,

J=6.8 Hz, 3H, PhCH=CHC=CHCHCH₃), 1.12 (t, J=6.8 Hz, 3H,

CO₂CH₂CH₃), 1.26–1.34 (m, 2H, CHCH₂CH), 1.80 (d, J=1.2 Hz, 3H,

PhCH=CHCCH₃), 1.87–1.88 (m, 6H, EtCO₂CCH₃and EtCO₂C=

CHCCH₃), 2.26–2.37 (m, 1H, EtCO₂C=CHC=CHCH), 2.45–2.56 (m, 1H,

PhCH=CHC=CHCH), 3.93–4.05 (m, 2H, CO₂CH₂CH₃), 5.18 (d, J=

10.4 Hz, 1H, EtCO₂C=CHC=CH), 5.36 (d, J=9.6 Hz, 1H, PhCH=CHC=

CH), 6.41 (d, J=16.0 Hz, 1H, PhCH), 6.78 (d, J=16.0 Hz, 1H, PhCH=

CH), 7.17–7.20 (m, 2H, EtCO₂C=CH and H_arom), 7.29 (d, J=7.6 Hz, 2H,

Ar-H), 7.39 ppm (d, J=7.2 Hz, 2H, Ar-H); ¹³C NMR (100 MHz, CDCl₃):

d=12.6 (p), 14.3 (2 p), 21.3(p), 21.5 (p), 23.2 (p), 31.0 (t), 31.9 (t), 45.8

(s), 60.6 (s), 125.6 (t), 126.2 (2 t), 126.9 (t), 127.7 (q), 128.6 (2 t), 130.2

(q), 132.8 (q), 134.2 (t), 138.1 (q), 138.6 (t), 138.9 (t), 140.7 (t), 168.7 ppm

Ester 37: Dess–Martin periodinane (2.47 mL, 1.145 mmol) was added at

08C to

a solution of alcohol 12 (330.0 mg, 0.881 mmol) in CH₂Cl₂

(10 mL). After stirring for 2 h at RT, the reaction mixture was quenched

by adding saturated aqueous Na₂S₂O₃(5 mL) and saturated aqueous

NaHCO₃(5 mL). The layers were separated and the aqueous layer ex-

tracted with CH₂Cl₂(4ꢂ10 mL). The organic layer was dried (MgSO₄)

and concentrated to yield the crude aldehyde 8 (330.0 mg, 0.881 mmol,

assumed to be quantitative) as colorless oil, which was used without fur-

ther purification for the following olefination reaction. To a solution of

sodium hydride (60% in mineral oil, 106 mg, 2.143 mmol) in THF

(15 mL) at 08C was added ethyl 2-(diethoxyphosphoryl)butanoate

(668 mg, 2.143 mmol) dropwise. The solution was stirred at RT for 1 h

before cooling to 08C and addition of the crude aldehyde 8 (330.0 mg,

0.881 mmol) (from previous reaction) in THF (5 mL). The solution was

stirred for 1 h at 08C, warmed slowly to RT and stirred for an additional

3 h. The reaction was quenched by pouring on to saturated aqueous

NH₄Cl (10 mL). The product was extracted with Et₂O (3ꢂ10 mL) and

the combined extracts were dried (MgSO₄) and concentrated. The crude

product was then purified by column chromatography on silica using cy-

clohexane/ethyl acetate 30:1 to yield ester 37 (300.7 mg, 0.639 mmol,

73%) as a colorless oil. A 5:1 ratio of Z/E products was obtained. R_f=

15016

www.chemeurj.org

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2012, 18, 15004 – 15020

Total Synthesis of the Plakotenins

FULL PAPER

0.50 (cyclohexane/EtOAc=18:1). Description of major diastereomer (Z):

¹H NMR (400 MHz, CDCl₃): ¹H NMR (400 MHz, CDCl₃): d=0.94 (d,

J=6.8 Hz, 3H, EtO₂CC=CHCHCH₃), 0.95 (d, J=6.4 Hz, 3H,

TrtOCH₂CHCH₃), 1.01 (t, J=7.2 Hz, 3H, EtO₂CCH₂CH₃), 1.07–1.12 (m,

1H, CHCH_AH_BCH), 1.24 (t, J=7.2 Hz, 3H, CO₂CH₂CH₃), 1.36–1.44 (m,

1H, CHCH_AH_BCH), 1.74–1.82 (m, 1H, TrtOCH₂CH), 2.23 (q, J=7.2 Hz,

2H, EtO₂CCH₂CH₃), 2.85 (dd, J=6.4, 8.4 Hz, 1H, TrtOCH_AH_B), 2.94

(dd, J=5.6, 8.4 Hz, 1H, TrtOCH_AH_B), 2.99–3.07 (m, 1H, C=CHCH),

4.12 (q, J=7.2 Hz, 2H, CO₂CH₂CH₃), 5.57 (d, J=10.0 Hz, 1H, C=CH),

7.20–7.31 (m, 9H, Ar-H), 7.43–7.45 ppm (m, 6H, Ar-H); ¹³C NMR

(100 MHz, CDCl₃): d=13.7 (p), 14.3 (p), 17.6 (p), 20.3 (p), 27.6 (s), 31.0

(t), 31.8 (t), 41.2 (s), 59.9 (s), 68.5 (s), 86.1 (q), 126.8 (t), 126.7 (t), 128.8

(t), 131.9 (q), 144.6 (t), 146.0 (q), 168.3 ppm (q); IR (film): n˜ =3086,

3059, 3023, 2964, 2928, 2871, 1712, 1643, 1597, 1491, 1449, 1373, 1303,

1215, 1158, 1070, 1032, 988, 899, 764, 746, 706, 648, 633 cm^À1; MS (EI,

70 eV): m/z (%): 470 (0.01) [M⁺], 440 (0.04), 243 (100), 165 (16), 105 (6),

77 (2); HRMS m/z: calcd for C₃₂H₃₈O₃: 470.2821; found: 470.2825.

(m, 1H, TrtOCH_AH_B), 2.83–2.87 (m, 1H, TrtOCH_AH_B), 5.17 (d, J=

9.6 Hz, 1H, PhCH=CHC=CH), 6.48 (d, J=16.2 Hz, 1H, PhCH), 6.90 (d,

J=16.2 Hz, 1H, PhCH=CH), 7.12–7.27 (m, 14H, Ar-H), 7.36–7.43 ppm

(m, 6H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=13.9 (p), 18.4 (p), 21.6

(p), 26.7 (s), 29.4 (t), 32.0 (t), 42.0 (s), 67.9 (s), 86.1 (q), 125.4 (t), 126.4

(t), 126.9 (t), 127.2 (t), 127.6 (t), 127.7 (t), 128.7 (t), 128.9 (t), 136.4 (q),

137.3 (t), 138.2 (q), 144.7 ppm (q); IR (film): n˜ =3058, 3025, 2961, 2924,

2871, 1627, 1597, 1491, 1448, 1373, 1315, 1219, 1182, 1155, 1069, 1032,

961, 899, 763, 747, 706, 633 cm^À1

.

Diene 41: Under argon, I₂(10.1 mg, 0.040 mmol) was added to a solution

of compound 39 (200.0 mg, 0.399 mmol) in CHCl₃(4 mL) at RT. After

stirring at RT for 1 h, the reaction mixture was quenched with a saturated

solution of sodium metabisulfite (5 mL). The aqueous layer was extracted

with CH₂Cl₂(3ꢂ5 mL). The combined organic extracts were washed with

brine, dried (MgSO₄), and concentrated to give crude compound 41

(200.0 mg, 0.399 mmol), which was used for the next step without further

purification. R_f=0.56 (cyclohexane/EtOAc 18:1); mixture of 3 diaster-

Alcohol 38: lithium aluminium hydride (24.8 mg, 0.654 mmol) was slowly

added to a solution of ester 37 (280.0 mg, 0.595 mmol) in THF (6 mL) at

08C. The reaction mixture was stirred at RT overnight, after which it was

cooled to 08C and quenched with H₂O (5 mL) followed by Rochelleꢄs

salt solution (5 mL) and the reaction mixture stirred at RT for 15 h. The

aqueous layer was extracted with EtOAc (3ꢂ10 mL). The combined or-

ganic extracts were dried (MgSO₄) and evaporated under reduced pres-

sure. The crude product was purified by column chromatography on

silica using cyclohexane/ethyl acetate 9:1 to give alcohol 38 (237.1 mg,

0.553 mmol, 93%) as colorless oil. A 5:1 ratio of Z/E products was ob-

tained. R_f=0.24 (cyclohexane/EtOAc 6:1). Description of major diaster-

eomer (Z): ¹H NMR (400 MHz, CDCl₃): d=0.85 (d, J=6.4 Hz, 3H,

EtO₂CC=CHCHCH₃), 0.98 (t, J=7.2 Hz, 3H, EtO₂CCH₂CH₃), 1.01 (d,

J=6.4 Hz, 3H, TrtOCH₂CHCH₃), 1.02–1.06 (m, 1H, CHCH_AH_BCH),

1.34–1.43 (m, 1H, CHCH_AH_BCH), 1.72 (sext, J=6.4 Hz, 1H,

TrtOCH₂CH), 2.06 (q, J=7.2 Hz, 2H, EtO₂CCH₂CH₃), 2.23–2.36 (m, 1H,

C=CHCH), 2.79 (dd, J=7.2, 8.0 Hz, 1H, TrtOCH_AH_B), 2.98 (dd, J=4.8,

8.8 Hz, 1H, TrtOCH_AH_B), 3.86 (dd, J=12,0, 19.2 Hz, 2H, CCH₂OH),

4.98 (d, J=9.6 Hz, 1H, C=CH), 7.19–7.30 (m, 9H, Ar-H), 7.43–7.45 ppm

(m, 6H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=13.0 (p), 18.6 (p), 22.0

(p), 27.7 (s), 29.4 (t), 31.8 (t), 42.1 (s), 60.5 (s), 67.8 (s), 86.2 (q), 126.9 (t),

127.8 (t), 128.9 (t), 134.0 (t), 138.4 (q), 144.6 ppm (q); IR (film): n˜ =3342,

3086, 3058, 3032, 2961, 2924, 2869, 1597, 1491, 1449, 1384, 1219, 1182,

1155, 1068, 1032, 899, 764, 746, 706, 633 cm^À1; HRMS m/z: calcd for

C₃₀H₃₆O₂: 428.2715; found: 428.2712.

eoisomers: (E,E)/

astereomers (E,E)/AHCTUNGTRENNUNG

G

ACHTUNGTREN(NUNG Z,Z)=6.6/3.3/1; description of the two major di-

(400 MHz, CDCl₃): d=0.91 (d, J=6.4 Hz, 3H, C=CHCHCH₃, (E,E)),

0.95–1.03 (m, 9H, C=CHCHCH₃, (E,Z); TrtOCH₂CHCH₃, (E,E) and

(E,Z); CCH₂CH₃, (E,E)), 1.06–1.12 (m, 3H, CHCH_AH_BCH, (E,E);

CHCH_AH_BCH, (E,Z); and CCH₂CH₃, (E,Z)), 1.45–1.52 (m, 1.5H,

CHCH_AH_BCH, (E,E); and CHCH_AH_BCH, (E,Z)), 1.71–1.79 (m, 1.5H,

TrtOCH₂CH, (E,E); and TrtOCH₂CH, (E,Z)), 2.12 (dq, J=3.2, 7.6 Hz,

2H, CCH₂CH₃, (E,E)), 2.27 (q, J=7.2 Hz, 1H, CCH₂CH₃, (E,Z)), 2.33–

2.43 (m, 1H, C=CHCH, (E,E)), 2.66–2.73 (m, 0.5H, C=CHCH, (E,Z)),

2.83 (dd, J=6.4, 8.8 Hz, 1H, TrtOCH_AH_B, (E,E)), 2.87 (dd, J=6.0,

8.4 Hz, 0.5H, TrtOCH_AH_B, (E,Z)), 2.94 (dd, J=5.2, 8.8 Hz, 1H, TrtO-

CH_AH_B, (E,Z)), 3.01 (dd, J=4.8, 8.8 Hz, 1H, TrtOCH_AH_B, (E,E)), 5.19

(d, J=9.6 Hz, 0.5H, PhCH=CHC=CH, (E,Z)), 5.29 (d, J=10.0 Hz, 1H,

PhCH=CHC=CH, (E,E)), 6.41 (d, J=16.2 Hz, 1H, PhCH, (E,E)), 6.50

(d, J=16.0 Hz, 0.5H, PhCH, (E,Z)), 6.62 (d, J=16.2 Hz, 1H, PhCH=CH,

(E,E)), 6.92 (d, J=16.0 Hz, 0.5H, PhCH=CH, (E,Z)), 7.15–7.30 (m, 14H,

Ar-H), 7.36–7.45 ppm (m, 6H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=

13.9 (p), 14.2 (p), 18.4 (p), 18.5 (p), 20.1 (s), 21.5 (p), 21.6 (p), 26.7 (s),

29.5 (t), 30.3 (t), 32.0 (t), 41.9 (s), 42.0 (s), 67.8 (s), 67.9 (s), 86.1 (q),

125.3 (t), 125.4 (t), 126.2 (t), 126.4 (t), 126.9 (t), 127.2 (t), 127.7 (t), 127.8

(t), 128.7 (t), 128.9 (t), 132.9 (t), 136.4 (q), 137.3 (t), 138.2 (q), 138.3 (q),

138.4 (q), 140.6 (t), 144.6 (q), 144.7 ppm (q).

Alcohol 42: Camphorsulfonic acid (134 mg, 0.577 mmol) was added in

one portion to a solution of compound 41 (170 mg, 0.340 mmol) in

CH₂Cl₂/MeOH (16/8 mL) at 08C. The mixture was stirred at RT for 1 h

30 min and was then neutralized by the addition of saturated aqueous

NaHCO₃(10 mL). The layers were separated and the aqueous layer ex-

tracted with CH₂Cl₂(3ꢂ10 mL). The combined organic layers were

washed with brine, dried (MgSO₄) and then concentrated. The crude

product was then purified by flash chromatography using cyclohexane/

ethyl acetate 6:1 to yield alcohol 42 (39.5 mg, 0.153 mmol, 45%) as color-

less solid. R_f=0.16 (cyclohexane/EtOAc 6:1 [a]²_D⁰=À31.4 (c=0.14 in

CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.96 (d, J=6.8 Hz, 3H,

HOCH₂CHCH₃), 1.00 (d, J=6.8 Hz, 3H, C=CHCHCH₃), 1.11 (t, J=

7.6 Hz, 3H, PhCH=CHCCH₂CH₃), 1.13–1.17 (m, 1H, CHCH_AH_BCH),

1.38 (brs, 1H, OH), 1.39–1.46 (m, 1H, CHCH_AH_BCH), 1.65–1.72 (m,

1H, HOCH₂CH), 2.38 (q, J=7.6 Hz, 2H, PhCH=CHCCH₂CH₃), 2.59–

2.70 (m, 1H, C=CHCH), 3.42 (dd, J=6.4 Hz, J=10.4 Hz, 1H, HO-

CH_AH_B), 3.53 (dd, J=5.2 Hz, J=10.4 Hz, 1H, HOCH_ACH_B), 5.38 (d, J=

10.0 Hz, 1H, PhCH=CHC=CH), 6.48 (d, J=16.2 Hz, 1H, PhCH), 6.68

(d, J=16.2 Hz, 1H, PhCH=CH), 7.20 (t, J=7.4 Hz, 1H, Ar-H), 7.31 (t,

J=8.0 Hz, 2H, Ar-H), 7.41 ppm (d, J=7.6 Hz, 2H, Ar-H); ¹³C NMR

(100 MHz, CDCl₃): d=14.3 (p), 17.3 (p), 20.2 (s), 21.2 (p), 30.3 (t), 33.7

(t), 41.3 (s), 68.3 (s), 125.6 (t), 126.3 (2 t), 127.0 (t), 128.7 (2 t), 132.7 (t),

138.1 (q), 138.4 (q), 140.3 ppm (t); IR (neat): n˜ =3345, 3026, 2961, 2925,

2872, 1627, 1598, 1493, 1450, 1375, 1030, 961, 749, 693 cm^À1; MS (EI,

70 eV): m/z (%) 258 ([M⁺], 33), 185 (72), 169 (100), 145 (39), 129 (48),

99 (60), 91 (61); HRMS m/z: calcd for C₁₈H₂₆O: 258.1984; found:

258.1982.

Diene 40: Dess–Martin periodinane (1.51 mL, 0.698 mmol) was added at

08C to a solution of alcohol 39 (230.0 mg, 0.537 mmol) in CH₂Cl₂(5 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(3 mL) and saturated aqueous NaHCO₃

(3 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ5 mL). The organic layer was dried (MgSO₄) and concentrat-

ed to yield the crude aldehyde 39 (230.0 mg, 0.537 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. Lithium bis(trimethylsilyl)amide

(1m in THF, 0.91 mL, 0.91 mmol) was slowly added, at 08C, to a solution

of 5-(benzylsulfonyl)-1-phenyl-1H-tetrazole (B) (282 mg, 0.939 mmol) in

THF (8 mL). After stirring for 20 min at RT, it was cooled to À788C and

a solution of crude aldehyde 39 (230.0 mg, 0.537 mmol) in THF (2 mL)

was added. The reaction mixture was stirred overnight while slowly

warming up to RT. It was diluted with Et₂O (5 mL) and the reaction

quenched by addition of saturated aqueous NaHCO₃(5 mL). The aque-

ous layer was extracted with EtOAc (3ꢂ15 mL). The combined organic

extracts were dried (MgSO₄) and evaporated under reduced pressure.

The crude product was purified by column chromatography on silica

using cyclohexane/ethyl acetate 50:1 to give compound 40 (209.2 mg,

78%) as colorless oil. R_f=0.36 (cyclohexane/EtOAc 50:1); mixture of 4

diastereoisomers: (E,Z)/ACHTNUGTREN(NNUG E,E)/ACHTUNRTGENN(GUN Z,Z)/ACHTGUNTREN(NUGN E,Z)=25/5.3/3.3/1. Description of

major diastereomer (E,Z): ¹H NMR (400 MHz, CDCl₃): d=0.93 (d, J=

6.8 Hz, 3H, C=CHCHCH₃), 0.98 (d, J=6.8 Hz, 3H, TrtOCH₂CHCH₃),

1.06 (t, J=7.2 Hz, 3H, CCH₂CH₃), 1.07–1.12 (m, 1H, CHCH_AH_BCH),

1.45–1.52 (m, 1H, CHCH_AH_BCH), 1.71–1.77 (m, 1H, TrtOCH₂CH), 2.24

(q, J=7.2 Hz, 2H, CCH₂CH₃), 2.64–2.71 (m, 1H, C=CHCH), 2.83–2.87

Chem. Eur. J. 2012, 18, 15004 – 15020

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

15017

S. Brꢀse et al.

Ester 44: Dess–Martin periodinane (0.38 mL, 0.176 mmol) was added at

08C to a solution of alcohol 42 (35.0 mg, 0.135 mmol) in CH₂Cl₂(3 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(2 mL) and saturated aqueous NaHCO₃

(2 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ5 mL). The organic layer was dried (MgSO₄) and concentrat-

ed to yield the crude aldehyde 43 (35.0 mg, 0.135 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. Ethyl 2-(diethoxyphosphoryl)buta-

noate (103 mg, 0.407 mmol) was added dropwise to a solution of sodium

hydride (60% in mineral oil, 16.3 mg, 0.407 mmol) in THF (3 mL) at

08C. The solution was stirred at RT for 1 h before cooling to 08C and ad-

dition of the crude aldehyde 46 (35.0 mg, 0.135 mmol) (from previous re-

action) in THF (1 mL). The solution was stirred for 1 h at 08C, warmed

slowly to RT and stirred for an additional 3 h. The reaction was quenched

by pouring on to saturated aqueous NH₄Cl (3 mL). The product was ex-

tracted with Et₂O (3ꢂ3 mL) and the combined extracts were washed

with brine, dried (MgSO₄), and concentrated. The crude product was

then purified by column chromatography on silica using n-pentane/Et₂O

30:1 to yield ester 44 (35.1 mg, 0.099 mmol, 73%) as a colorless oil. A

4:1 ratio of Z/E products was obtained. R_f=0.50 (cyclohexane/EtOAc=

18:1); [a]²_D⁰=À6.3 (c=0.16 in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=

0.97–1.06 (m, 12H, PhCH=CHC=CHCHCH₃, EtCO₂C=CHCHCH₃,

PhCH=CHCCH₂CH₃, and EtCO₂CCH₂CH₃), 1.21 (t, J=6.8 Hz, 3H,

CO₂CH₂CH₃), 1.27–1.32 (m, 2H, CHCH₂CH), 2.22–2.32 (m, 4H, PhCH=

CHCCH₂CH₃, and EtCO₂CCH₂CH₃), 2.43–2.55 (m, 1H, PhCH=CHC=

CHCH), 2.99–3.11 (m, 1H, EtCO₂C=CHCH), 4.05–4.17 (m, 2H,

CO₂CH₂CH₃), 5.30 (d, J=10.0 Hz, 1H, PhCH=CHC=CH), 5.59 (d, J=

10.0 Hz, 1H, EtCO₂C=CH), 6.44 (d, J=16.2 Hz, 1H, PhCH), 6.65 (d, J=

16.2 Hz, 1H, PhCH=CH), 7.18 (t, J=7.6 Hz, 1H, Ar-H), 7.30 (t, J=

7.2 Hz, 2H, Ar-H), 7.39 ppm (d, J=7.6 Hz, 2H, Ar-H); ¹³C NMR

(100 MHz, CDCl₃): d=13.9 (p), 14.3 (p), 14.4 (p), 21.2 (s), 21.1 (p), 21.8

(p), 27.8 (s), 31.1 (t), 31.9 (t), 45.9 (s), 60.1 (s), 125.3 (t), 126.2 (2 t), 126.9

(t), 128.6 (2 t), 132.7 (q), 132.8 (t), 138.2 (q), 138.8 (q), 139.9 (t), 146.1

(t), 168.3 ppm (q); IR (film): n˜ =3027, 2965, 2871, 1715, 1598, 1494, 1450,

1378, 1220, 1184, 1130, 1030, 961, 749, 693 cm^À1; MS (EI, 70 eV): m/z

(%) 354 ([M⁺], 35), 281 (84), 227 (49), 185 (74), 136 (41), 129 (66), 121

(61), 105 (47), 91 (100); HRMS m/z: calcd for C₂₄H₃₄O₂: 354.2559;

found: 354.2557.

(1 mL). The layers were separated and the aqueous layer extracted with

CH₂Cl₂(4ꢂ3 mL). The organic layer was dried (MgSO₄) and concentrat-

ed to yield the crude aldehyde 46 (24.0 mg, 0.077 mmol, assumed to be

quantitative) as colorless oil, which was used without further purification

for the following olefination reaction. ethyl 2-(diethoxyphosphoryl)pro-

panoate (0.049 mL, 0.230 mmol) was added dropwise to a solution of

sodium hydride (60% in mineral oil, 9.2 mg, 0.230 mmol) in THF (3 mL)

at 08C. The solution was stirred at RT for 1 h before cooling to 08C and

addition of crude aldehyde 46 (24.0 mg, 0.077 mmol) (from previous reac-

tion) in THF (1.5 mL). The solution was warmed slowly to RT and stirred

for an additional 3 h. The reaction was quenched then by pouring on to

saturated aqueous NH₄Cl (2 mL). The product was extracted with Et₂O

(4ꢂ3 mL) and the combined extracts were washed with brine, dried

(MgSO₄), and concentrated. The crude product was then purified by

column chromatography on silica using n-pentane/Et₂O 40:1 to yield

ester 47 (20.6 mg, 0.052 mmol, 68% yield) as colorless oil. R_f=0.44 (cy-

clohexane/EtOAc=18:1 [a]²_D⁰= +87.5 (c=0.08 in CHCl₃); ¹H NMR

(400 MHz, CDCl₃): d=0.92 (d, J=6.4 Hz, 3H, EtCO₂C=CHC=

CHCHCH₃), 0.96 (d, J=6.8 Hz, 3H, PhCH=CHC=CHCHCH₃), 0.99 (t,

J=7.6 Hz, 3H, EtCO₂C=CHCCH₂CH₃), 1.07 (t, J=7.6 Hz, 3H, PhCH=

CHCCH₂CH₃), 1.16 (t, J=7.0 Hz, 3H, CO₂CH₂CH₃), 1.25–1.29 (m, 2H,

CHCH₂CH), 1.80 (t, J=1.2 Hz, 3H, EtCO₂CCH₃), 2.12 (q, J=7.6 Hz,

2H, EtCO₂C=CHCCH₂CH₃), 2.16–2.24 (m, 1H, EtCO₂C=CHC=CHCH),

2.27–2.38 (m, 2H, PhCH=CHCCH₂CH₃), 2.44–2.55 (m, 1H, PhCH=

CHC=CHCH), 4.04 (ddq, J=12.4, 3.6, 7.6 Hz, 2H, CO₂CH₂CH₃), 5.14

(dd, J=1.2, 10.0 Hz, 1H, EtCO₂C=CHC=CH), 5.31 (d, J=10.0 Hz, 1H,

PhCH=CHC=CH), 6.44 (d, J=16.0 Hz, 1H, PhCH), 6.65 (d, J=16.0 Hz,

1H, PhCH=CH), 7.08 (s, 1H, EtCO₂C=CH), 7.17 (t, J=7.2 Hz, 1H,

H_arom), 7.29 (t, J=8.0 Hz, 2H, Ar-H), 7.39 ppm (d, J=7.2 Hz, 2H, Ar-H);

¹³C NMR (100 MHz, CDCl₃): d=13.3 (p), 14.3 (2 p), 14.4 (p), 20.3 (s),

21.0 (p), 21.8 (p), 30.0 (s), 30.7 (t), 31.8 (t), 46.0 (s), 60.7 (s), 125.3 (t),

126.2 (2 t), 126.9 (t), 128.6 (2 t), 128.7 (q), 132.8 (t), 135.0 (t), 136.2 (q),

138.2 (q), 138.7 (q), 139.2 (t), 140.3 (t), 168.4 ppm (q); MS (EI, 70 eV):

m/z (%) 394 ([M⁺], 74), 239 (79), 185 (88), 129 (49), 121 (62), 91 (100),

43(52); HRMS m/z: calcd for C₂₇H₃₈O₂: 394.2872; found: 394.2870.

homo-Plakotenin ethyl ester 48: A solution of linear ester 47 (16.0 mg,

0.041 mmol) in toluene (4 mL) was heated to reflux and stirred in a

sealed vial for 20 h. The mixture was concentrated. The crude product

was purified by column chromatography on silica using n-pentane/Et₂O

40:1 to yield cyclic ester 48 (14.4 mg, 0.036 mmol, 90% yield) as colorless

oil. R_f=0.42 (cyclohexane/EtOAc=18:1). [a]²_D⁰= +168.9 (c=0.09 in

CHCl₃); (NMR assignment according to numbering system used in

Ref. [8]) ¹H NMR (400 MHz, CDCl₃): d=0.79 (t, J=7.2 Hz, 3H, 19-H),

0.97 (d, J=6.4 Hz, 3H, 14-H), 1.01 (t, J=7.2 Hz, 3H, 17-H), 1.03–1.10

(m, 1H, 18-H), 1.17 (d, J=5.6 Hz, 3H, 15-H), 1.31 (t, J=7.2 Hz, 3H, 21-

H), 1.55 (t, J=8.0 Hz, 2H, 7-H), 1.74–1.90 (m, 5H, 18-H, 5-H, 9-H, 8-H,

and 6-H), 2.05 (s, 3H, 13-H), 2.10–2.23 (m, 2H, 16-H), 3.71 (d, J=4.0 Hz,

1H, 12-H), 4.17–4.25 (m, 2H, 20-H), 5.25 (d, J=4.4 Hz, 1H, 11-H), 6.87

(s, 1H, 3-H), 7.21–7.24 (m, 1H, Ar-H), 7.28–7.30 ppm (m, 5H, Ar-H);

¹³C NMR (100 MHz, CDCl₃): d=9.4 (p), 13.2 (p), 14.3 (p), 14.4 (p), 22.0

(p), 23.3 (p), 27.0 (s), 27.9 (s), 31.6 (t), 34.8 (t), 45.4 (s), 48.0 (q), 51.3 (t),

52.2 (t), 55.6 (t), 60.8 (s), 123.3 (t), 126.5 (t), 127.8 (2 t), 127.9 (q), 131.0

(2 t), 142.3 (q), 142.9 (q), 146.6 (t), 169.5 ppm (q); IR (film): n˜ =2927,

2865, 1698, 1493, 1451, 1375, 1260, 1246, 1139, 1103, 874, 763, 741,

704 cm^À1; MS (EI, 70 eV): m/z (%) 394 ([M⁺], 100), 239 (60), 185 (56),

129 (39), 91 (84); HRMS m/z: calcd for C₂₇H₃₈O₂: 394.2872; found:

394.2870.

Alcohol 45: Lithium aluminium hydride (4.1 mg, 0.109 mmol) was slowly

added to a solution of ester 44 (35.0 mg, 0.099 mmol) in THF (3 mL) at

08C. After stirring at 08C for 2 h the reaction mixture was quenched

carefully at 08C with H₂O (1 mL) followed by Rochelleꢄs salt solution

(1 mL). The aqueous layer was extracted with Et₂O (4ꢂ3 mL). The com-

bined organics were washed with brine, dried (MgSO₄), and concentrat-

ed. The crude product was then purified by column chromatography on

silica using n-pentane/Et₂O 9:1 to yield alcohol 45 (24.0 mg, 0.077 mmol,

77% yield). R_f=0.48 (cyclohexane/EtOAc=6:1); [a]²_D⁰=À90.0 (c=0.11

in CHCl₃); ¹H NMR (400 MHz, CDCl₃): d=0.94 (d, J=6.4 Hz, 3H,

HOCH₂C=CHCHCH₃), 0.99 (d, J=6.8 Hz, 3H, PhCH=CHCCH₂CH₃),

1.05 (t, J=7.4 Hz, 3H, HOCH₂CCH₂CH₃), 1.06 (t, J=7.6 Hz, 3H,

PhCH=CHC=CHCHCH₃), 1.32 (ddd, J=2.4, 5.6, 8.4 Hz, CHCH₂CH),

2.13 (dq, J=1.2, 7.4 Hz, 3H, HOCH₂CCH₂CH₃), 2.20–2.37 (m, 2H,

PhCH=CHCCH₂CH₃), 2.41–2.55 (m, 2H, HOCH₂C=CHCH and PhCH=

CHC=CHCH), 4.02 (brs, 2H, HOCH₂), 5.05 (d, J=10.0 Hz, 1H,

HOCH₂C=CH), 5.35 (d, J=9.6 Hz, 1H, PhCH=CHC=CH), 6.46 (d, J=

16.4 Hz, 1H, PhCH), 6.68 (d, J=16.4 Hz, 1H, PhCH=CH), 7.19 (t, J=

7.2 Hz, 1H, Ar-H), 7.30 (t, J=7.6 Hz, 2H, Ar-H), 7.40 ppm (d, J=

7.6 Hz, 2H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=13.1 (p), 14.4 (p),

20.3 (s), 22.1 (p), 22.7 (p), 28.0 (s), 30.4 (t), 31.0 (t), 46.2 (s), 60.8 (s),

125.7 (t), 126.3 (2 t), 127.0 (t), 128.7 (2 t), 132.5 (t), 133.8 (t), 138.0 (q),

139.1 (q), 139.2 (q), 140.2 ppm (t); IR (film): n˜ =3423, 3027, 2960, 2924,

1598, 1494, 1452, 1383, 1029, 960, 748, 692 cm^À1.; MS (EI, 70 eV): m/z

(%) 312 ([M⁺], 38), 281 (91), 185 (61), 149 (44), 133 (42), 105 (34), 91

(48), 43 (100); HRMS m/z: calcd for C₂₂H₃₂O: 312.2453; found: 312.2452.

homo-Plakotenin 3: NaOH (2m) (0.076 mL, 0.152 mmol) was added to a

solution of cyclic ester 48 (12.0 mg, 0.030 mmol) in THF/MeOH (1.2/

0.6 mL), and the resulting mixture was heated to 408C and stirred for

20 h. After cooling to RT, the mixture was acidified with aqueous HCl

(1m), and then extracted with EtOAc (5 times). The combined organic

extracts were backwashed with brine, dried (MgSO₄), and concentrated.

The crude product was purified by column chromatography on silica

using n-pentane/Et₂O 2:1 to yield carboxylic acid 1a (9.8 mg, 0.027 mmol,

Ester 47: Dess–Martin periodinane (0.21 mL, 0.100 mmol) was added at

08C to a solution of alcohol 45 (24.0 mg, 0.077 mmol) in CH₂Cl₂(2 mL).

After stirring for 2 h at RT, the reaction mixture was quenched by adding

saturated aqueous Na₂S₂O₃(1 mL) and saturated aqueous NaHCO₃

88% yield) as colorless oil. R_f=0.40 (cyclohexane/EtOAc=2:1); [a]_D²⁰

=

+1838 (0.09 g/100 mL, CHCl₃); [a]²_D⁰= +1858 (0.14 g/100 mL, MeOH);

(NMR assignment according to numbering system used in Ref. [8])

¹H NMR (400 MHz, CDCl₃): d=0.80 (t, J=7.4 Hz, 3H, 19-H), 0.97 (d,

15018

www.chemeurj.org

ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2012, 18, 15004 – 15020

Total Synthesis of the Plakotenins

FULL PAPER

J=6.0 Hz, 3H, 14-H), 1.02 (t, J=7.2 Hz, 3H, 17-H), 1.06–1.11 (m, 1H,

18-H), 1.17 (d, J=5.6 Hz, 3H, 15-H), 1.56 (dt, J=2.4, 8.0 Hz, 2H, 7-H),

1.75–1.91 (m, 5H, 17-H, 5-H, 9-H, 8-H and 5-H), 2.06 (d, J=1.2 Hz, 3H,

13-H), 2.17 (q, J=7.2 Hz, 2H, 16-H), 3.75 (d, J=4.4 Hz, 1H, 12-H), 5.24

(d, J=4.0 Hz, 1H, 11-H), 7.03 (s, 1H, 3-H), 7.21–7.25 (m, 1H, Ar-H),

7.29–7.30 (m, 4H, Ar-H); ¹³C NMR (100 MHz, CDCl₃): d=9.4 (p), 13.0

(p), 14.0 (p), 22.0 (p), 23.3 (p), 27.2 (s), 27.8 (s), 31.5 (t), 34.8 (t), 45.4 (s),

48.3 (q), 51.6 (t), 51.8 (t), 55.5 (t), 123.1 (t), 126.6 (t), 127.2 (q), 127.9

(2t), 131.1 (2t), 142.2 (q), 142.9 (q), 149.6 (t), 174.8 (q); IR (film): n˜ =

2927, 2868, 1679, 1628, 1451, 1418, 1376, 1276, 1030, 908, 883, 762, 734,

702, 556 cm^À1; MS (EI, 70 eV): m/z (%) 366 ([M⁺], 100), 239 (29), 185

(31), 145 (21), 91 (33); HRMS m/z: calcd for C₂₅H₃₄O₂: 366.2559; found:

366.2557.

Rꢅgion Rhꢆne-Alpes (fellowship to E.B.). Financial support by the Deut-

sche Forschungsgemeinschaft through the Center for Functional Nano-

structures (CFN, Project C3.3 and E1.1) is gratefully acknowledged. We

thank Dr. A. Qureshi for copies of NMR spectra and Prof. Sir J. E. Bald-

win, as well as Dr. D. Keck for valuable advice in the initial phase of this

project.

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FACS measurements: For cell cycle analyses cells were treated with 1, 2,

3, and 5 mm of Plakotenin, Plakotenin derivatives or intermediates for

72 h, harvested, mixed with ice-cold 70% ethanol and fixed overnight at

48C. Cells were pelleted at 530 g for 5 min, washed once with PBS and

stained with Draq5 (Biostatus td) at a final concentration of 10 mm for

15 min in the dark. The DNA content of cells was determined using a

flow cytometer (FACScan; Becton Dickinson).

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Western blot analysis: For testing cell lysate against antibodies^[24]cells

were washed twice in ice-cold PBS, scraped into PBS, and centrifuged at

1000 g for 5 min. Cells were lysed by incubation in NP-40 lysis buffer

(150 mm NaCl, 50 mm Tris [pH 8], 5 mm EDTA, 1% NP-40, 1 mm phenyl-

methylsulfonyl fluoride) on ice for 30 min. The protein extract was

cleared by centrifugation at 13000 g at 48C for 20 min, and the protein

concentration of the supernatant (protein extract) was determined by the

method of Bradford (Biorad). SDS-polyacrylamide gel electrophoresis

(PAGE) and Western blotting were performed with 12% SDS PAGE

Gel, proteins were disconnected with 100 V in running buffer (0.18m gly-

cine, 24 mm Tris base) and blotted on PVDF membrane at 30 V overnight

in transfer buffer (0.18m glycine, 24 mm Tris base, 10% MEOH) and

blocked 30 min with 5% low-fat milk solution.

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We used antibodies against cleaved caspase-8 (Cell Signaling) in a 1:500

dilution (in 5% low fat milk solution) and Tubulin (abcam) in an 1:1000

dilution as loading control. Secondary antibody was used in a 1:1000 solu-

tion anti rabbit (for Caspase-8) and anti-mouse for Tubulin. Secondary

antibody was bound to horseradish peroxidase and incubated with lumi-

nol peroxide solution (PIERCE,Thermo-scientific) bonds were made visi-

ble on a film (Kodak). As a positive control cells were incubated with

50 mm LiCl for 2 days.^[25]

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Statistical analysis of FACS data: Values were tested for significance by a

single-sided t-test of paired samples (n=6). Values of plakotenin were

compared to values of intermediates.

MTT-tests: 5ꢂ10⁵HeLa wild type (wt) cells were seeded in a 96 well-

plate and incubated with 1, 2, 3, and 5 mm of plakotenin derivatives for

two days. Later cells were treated with MTT (Promega) for 4 h and lysed

with lysis buffer (Promega). Absorption of formazan was measured at

595 nm. For blank measurements (positive control) cells were treated

with 5% TritonX100 15 min before MTT was added. Negative control

(untreated cells) is set to 100% cell vitality.

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Computational details: The density functional calculations in this work

were carried out with the Turbomole program package.^[26]Transition-

state structures were optimized using the B3LYP hybrid functional^[27]in

combination with a TZVP basis set^[28]and employing tight convergence

À1

criteria (SCF energy:10^À8E_h, energy gradient: 10^À4E_ha₀and inclusion

of the derivatives of quadrature weights). Force constants and vibrational

frequencies were computed to ensure that all structures are first-order

saddle points with the desired imaginary mode. The reported relative en-

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This work has been supported by the Karlsruhe Institute of Technology

(KIT), the Landesgraduiertenfçrderung (fellowship to S.A.) and the

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Received: May 7, 2012

Published online: October 4, 2012

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Article Doi

The plakotenins: Biomimetic diels-alder reactions, total synthesis, structural investigations, and chemical biology

DOI: 10.1002/chem.201201585

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