A general route to cyclopeptide alkaloids: total syntheses and biological evaluation of paliurines e and F, ziziphines N and Q, abyssenine A, mucronine E, and analogues

FULL PAPER

DOI: 10.1002/ejoc.200900122

A General Route to Cyclopeptide Alkaloids: Total Syntheses and Biological

Evaluation of Paliurines E and F, Ziziphines N and Q, Abyssenine A,

Mucronine E, and Analogues

Mathieu Toumi,^[a]Vincent Rincheval,^[b]Ashley Young,^[c]Danielle Gergeres,^[c]

Edward Turos,^[c]François Couty,^[a]Bernard Mignotte,^[b]and Gwilherm Evano*^[a]

Dedicated to Dr. Jean-Paul Mazaleyrat on the occasion of his retirement

Keywords: Alkaloids / Biological activity / Copper catalysis / Cyclopeptide alkaloids / Macrocycles / Total synthesis

A full account of the total syntheses of the cyclopeptide alka-

loids paliurine E and F, ziziphine N and Q, abyssenine A, and

mucronine E is provided. A key feature of the syntheses in-

volves an intramolecular amidation of a vinyl iodide, which

allows us simultaneously to address two synthetic challenges

associated with cyclopeptide alkaloids: the formation of the

enamide and macrocyclization. We also document the use of

other strategies for the macrocyclization step, as well as the

evaluation of the antibacterial and cytotoxic properties of the

natural products and analogues obtained.

Germany, 2009)

Introduction

Cyclopeptide Alkaloids

Cyclopeptide alkaloids form a group of closely related

cyclic polyamide bases of plant origin. Although they were

mentioned in the literature as early as 1884,^[1]the isolation

and structural elucidation of pandamine in 1966^[2]marks

the beginning of a growing interest in these natural prod-

ucts, which nowadays encompass over 200 compounds. Sev-

eral characteristic features are common to the majority of

these alkaloids. They generally possess a 13- (1), 14- (2),

or 15-membered (3) cycle containing an aromatic ring. The

remainder of the macrocycle consists of a peptide unit that

is connected to the aromatic ring in either a 1,4- or a 1,3-

orientation through enamide and alkyl aryl ether (or meth-

ylene) linkages (Figure 1).^[3]

Figure 1. Representative cyclopeptide alkaloids.

[a] Institut Lavoisier de Versailles, UMR CNRS 8180, Université

de Versailles Saint Quentin en Yvelines – PRES UniverSud

Paris,

Although cyclopeptide alkaloids are found in the roots,

leaves, and bark of a number of plants, extraction tech-

niques usually afford complex mixtures, making their isola-

tion an extremely tedious adventure. Yields from dried

plants vary from 0.0002–1% depending on the plant source,

location, method of isolation, and plant maturity. Their

common occurrence has, however, led to a rich history of

service in folk medicine, in which sources of cyclopeptide

alkaloids have been used as remedies for diarrhea, dysen-

tery, or insomnia.^[4]Recently, they have been shown to dis-

45 avenue des Etats-Unis, 78035 Versailles Cedex, France

Fax: +33-1-39254452

E-mail: evano@chimie.uvsq.fr

[b] Laboratoire de Génétique et Biologie Cellulaire, UMR CNRS

8159, Université de Versailles Saint Quentin en Yvelines –

PRES UniverSud Paris,

45 avenue des Etats-Unis, 78035 Versailles Cedex, France

[c] Center for Molecular Diversity in Drug Design, Discovery,

and Delivery, Department of Chemistry, University of South

Florida,

4202 East Fowler Avenue, Tampa, Florida 33620, USA

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.200900122.

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A General Route to Cyclopeptide Alkaloids

play numerous forms of biological activity, including seda- paper we report in detail on the development of a general

tive/stimulant,^[5]antibacterial,^[6]antifungal,^[6a–6c,7]and anti- route to cyclopeptide alkaloids, as well as on its application

plasmodial.^[8]However, the limited supplies of cyclopeptide to the preparation of two additional natural products: zizi-

alkaloids have not been sufficient for extensive pharmaco- phines N and Q. We also document the use of other strate-

logical investigations, and the lack of efficient synthetic gies for the macrocyclization step, together with evaluation

strategies has limited structure–activity relationship studies. of the antibacterial and cytotoxic properties of the natural

Their biological properties, together with their intriguing products and analogues obtained.

structures, have resulted in a steady stream of studies di-

rected towards the synthesis of this class of compounds

during the past decades.^[9]These studies have revealed that

the synthetic challenges include elaboration of the aryl alkyl

Strategic Considerations

ether, formation of the strained macrocycle, and introduc-

The design of a new synthetic route to cyclopeptide alka-

tion of the enamide unit. The initial report by Schmidt uti- loids has to meet the following criteria: it must be as

lized the macrolactamization of a pentafluorophenyl es- straightforward as possible, and it should allow the prepa-

ter,^[10]which was also later used in the total syntheses by ration of useful quantities of the target molecules and be

Joullié^[11]and by Han,^[12]whereas Zhu’s synthesis incorpo- flexible enough to permit the efficient construction of ana-

rated an intramolecular S_NAr reaction as macrocyclization logues. It is also important to consider the generality of the

protocol.^[13]All these syntheses chose elaboration of the en- process and its reliability.

amide moiety by different stepwise elimination methods af-

Our proposed route, which meets these criteria, is sum-

ter macrocyclization, which rather decreased the overall marized in Scheme 1 and Scheme 2 in a retrosynthetic fash-

synthetic efficiency. We have developed an alternative cycli- ion starting from the sedative cyclopeptide alkaloid paliur-

zation strategy based on an intramolecular amidation of a ine F (1),^[5c,18]which was chosen as our primary target for

vinyl iodide and have successfully implemented it in ef- this study. From a retrosynthetic perspective, we envisioned

ficient asymmetric syntheses of paliurine F,^[14]abyssen- the installation of the peptidic side chain late in the synthe-

ine A,^[15]and mucronine E.^[16]Besides being convergent, the sis, thereby permitting the construction of paliurine F from

salient feature of our approach is that two synthetic chal- the advanced precursor 4, as depicted in Scheme 1. In the

lenges associated with cyclopeptide alkaloids – the forma- planning of this synthesis, the choice of macrocyclization

tion of the enamide and macrocyclization – have been re- site and the reaction to be employed to carry out this trans-

duced to a single operation with high efficiency.^[17]In this formation appeared to be of the utmost importance. In or-

Scheme 1. Key macrocyclization step: the possibilities.

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der to minimize the overall number of steps and to avoid a with phenanthroline and cesium carbonate as a base, in tol-

painful stepwise installation of the enamide in 4 after elabo- uene at 110 °C (Table 1, Entry 1). Although all the starting

ration of the macrocycle, we decided to investigate strategies material had been consumed after 12 h, it rapidly turned

to allow for their concomitant formation.

out that the desired phenyl pyrrolidinyl ether had indeed

Table 1. Optimization of the hydroxypyrrolidine reaction partner

for the copper-mediated arylation.

Scheme 2. Retrosynthetic analysis of acyclic precursors.

We therefore decided that the macrocycle would be

closed at the enamide bond variously through a cyclodehy-

dration reaction starting from α-amido ω-aldehyde 5,

through an intramolecular oxidative amidation starting

from acyclic precursor 6,^[19]through a ring-closing metathe-

sis starting from ene-enamide 7,^[20]or through an intramo-

lecular copper-mediated amidation starting from

8

(Scheme 1).^[21,22]

All precursors 5–8, bearing different functionalities,

would be derived from the corresponding carboxylic acids

of general formula 9 (Scheme 2) by a simple peptide cou-

pling with isoleucine amide or derivatives. Finally, further

analysis suggested a disconnection at the aryl ether bond to

give the hydroxypyrrolidines 10 and aromatic fragments 11,

which would be coupled in copper-^[23]or palladium-medi-

ated^[24]arylation reactions.

Results and Discussion

Installation of the Aryl Alkyl Ether

Our synthesis started with the optimization of the reac-

tion partners and conditions for the formation of the highly

substituted aryl alkyl ether moiety. Initial studies focused

on the determination of the structural requirements for the

hydroxypyrrolidine fragment 10 with use of iodobenzene

(12) as a model arylating agent (Table 1). To this end, a set

of seven hydroxypyrrolidines 10 were easily prepared from

commercially available trans-3-hydroxyproline (10d)^[25]by

classical transformations. They were then subjected to the

conditions reported by Buchwald for the copper-mediated

arylation of alcohols^[23]or amino alcohols (Table 1).^[26]In

an initial experiment, the Boc-protected 3-hydroxyproline

methyl ester 10a was treated with iodobenzene (12) in the

[a] Yields based on pure materials isolated by column chromatog-

presence of catalytic amounts of copper(I) iodide, together raphy.

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A General Route to Cyclopeptide Alkaloids

been formed in the reaction mixture but that it had immedi-

ately undergone β-elimination to yield the endocyclic en-

amide 13 and phenol. The use of lower temperatures or of

other bases such as potassium phosphate or carbonate did

not block the undesired elimination. We therefore decided

to examine the reactivities of the other substrates 10b–e,

variously possessing a free amine (Table 1, Entries 2 and 3),

a free carboxylic acid (Table 1, Entry 7), both (Table 1, En-

tries 5 and 6), or a trityl protecting group on the nitrogen

(Table 1, Entry 4) in order to diminish or suppress the acid-

ity of the labile hydrogen involved in the β-elimination reac-

tion. Whereas the trityl group in 10c did not survive the

reaction conditions and extensive degradation was ob-

served, we faced a total lack of reactivity with other sub-

strates 10b, 10d, and 10e, which were fully recovered at the

end of the reactions. At this point of screening, we decided

to replace the carboxylic acid or ester groups in 10a–e with

a protected hydroxymethyl precursor, hoping to obtain

products that would be stable under the reaction conditions.

Although the absence of a protecting group on the nitrogen

again inhibited the reaction, we were delighted to note that

substrate 10g could be smoothly coupled with iodobenzene

(12), giving the long-awaited phenyl pyrrolidinyl ether 14 in

70% yield (Table 1, Entry 10). This yield could further be

improved simply by raising the reaction temperature from

110 to 125 °C (Table 1, Entry 11). A notable feature of this

arylation reaction is that, unlike in the originally published

procedure, which required an excess of the alcohol, the stoi-

chiometry could be reversed without significantly lowering

the yield, provided that the excess of iodobenzene

(1.5 equiv.) was added in two portions.

Scheme 4. Synthesis of aromatic fragments.

Table 2. Optimization of the aromatic reaction partner for the cop-

per-mediated arylation.

Before we moved on to the optimization of the second

reaction partner, we needed a suitable synthetic route to

provide useful quantities of the hydroxypyrrolidine frag-

ment 10g easily. Indeed, the preparation of this fragment

from commercially available trans-3-hydroxyproline (10d),

as used for the model studies, was not practical on large

scale because of the prohibitive price of this starting mate-

rial. Our goal was therefore to design a gram-scale, sub-

strate-controlled asymmetric route to 10g with a limited

[a] Yields based on pure materials isolated by column chromatog-

raphy. [b] Reaction performed at 110 °C to avoid polymerization of

11c and 19c (the yield drops to 27% when the reaction is run at

125 °C).

Scheme 3. Gram-scale synthesis of the hydroxypyrrolidine fragment

10g.

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number of steps and protecting groups. After exploring RCM or an aza-Wacker reaction starting from 19c, or

many synthetic routes based on RCM, intramolecular through intramolecular amidation starting from 19a or 19b.

Michael addition, or intramolecular epoxide-opening, we Unfortunately, the reaction with bis-iodide 11e, which

eventually came across the synthesis depicted in Scheme 3, would have avoided the installation of the vinyl iodide re-

which we later on applied to the efficient preparation of quired for a macrocyclization through intramolecular amid-

other hydroxypyrrolidines.^[27]From the starting protected ation after the installation of the aryl alkyl ether, turned

N-Boc--serine 15, the desired fragment could easily be ob- out to be non-selective because of competing vinylation of

tained on a multigram scale, in 54% overall yield and by a the alcohol (Table 2, Entry 5).

rather simple sequence. A Tegner reaction allowed for the

transformation of the carboxylic acid in 15 into the corre-

sponding unconjugated ketone 16, which was reduced to 17

with high levels of diastereoselectivity. Two-step formation

of the five-membered ring heterocycle finally provided the

desired fragment 10g.^[14]

Before moving on to the next steps of the synthesis, we

briefly evaluated other protocols based on palladium-medi-

ated coupling or nucleophilic aromatic substitution reac-

tions for the arylation of 10g, to compare their efficiencies

with that of the copper-mediated arylation procedure.

Therefore, 10g was treated variously with aromatic iodide

With gram quantities of hydroxypyrrolidine 10g to hand, 11b or with bromide 20 in the presence of palladium ace-

screening of different aromatic iodides 11 compatible with tate, cesium carbonate, and phosphane ligands L^[24b]or

our retrosynthetic analysis was initiated. Except for com- with aromatic fluoride 21 in the presence of KHMDS or

pound 11a, which was prepared from 5-iodosalicylic TBAF (Scheme 5). However, the palladium-mediated reac-

acid,^[28]the other aromatic iodides 11b–f were all easily syn- tion mostly led to reduction of the aryl halide and left

thesized from salicylaldehyde in excellent yields by iodin- alcohol 10g unaffected, which can certainly be attributed to

ation/methylation/olefination sequences (Scheme 4).

steric crowding at the reacting center.

These compounds were then treated under the optimized

The nucleophilic aromatic substitution did not afford the

conditions developed for the coupling of 10g with iodo- desired coupled product either, which is easily understand-

benzene: copper(I) iodide (10 mol-%) and 1,10-phenan- able due to the low reactivity of 21. Since the copper-medi-

throline (20 mol-%) in the presence of cesium carbonate as ated arylation procedure seemed to be the reaction of

base in toluene at 125 °C. Results from this screening re- choice for the formation of 19b, which could be synthesized

vealed that all iodides were suitable reaction partners in useful quantities, we decided to explore the next impor-

(Table 2), therefore enabling the macrocyclization to be ef- tant step of the synthesis: the formation of the macrocycle

fected, after functionalization, variously through cyclodehy- through an intramolecular copper-mediated amidation re-

dration starting from 19d or 19f, through an ene-enamide action.

Scheme 5. Attempted fragment coupling by a palladium-mediated arylation or by a S_NAr reaction.

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A General Route to Cyclopeptide Alkaloids

Scheme 6. Elaboration of the acyclic skeleton and copper-mediated macroenamidation.

Macrocyclization by Copper-Mediated Macroenamidation

inamide (24), yielding acyclic substrate 25. In a similar way,

styrenoxyproline 26 was coupled with isoleucine derivatives

24 and 27^[31]to give the substrates required for the aza-

Wacker reaction and for the ene-enamide RCM: 6 and 28,

respectively.

Several chemical manipulations were necessary before

macrocyclization. To that end, the Z vinyl iodide required

for the final macroamidation step was stereoselectively in-

stalled by use of Stork–Zhao olefination reagent (97%, de

Ͼ 95%),^[29]the TBS ether was deprotected with TBAF in

THF, and the resulting primary alcohol was converted into

the acid in a two-step sequence (Swern/buffered NaClO₂

oxidations) in good overall yield (Scheme 6). Finally, the

second constitutive amino acid of the macrocycle, together

with the amide group necessary for the intramolecular am-

idation, were introduced through an EDC/HOBt-mediated

coupling with isoleucinamide.

The efficient preparation of the acyclic precursor 8 set

the stage for the crucial macrocyclization step. The iodo

amide 8 was treated with catalytic copper(I) iodide and

N,NЈ-dimethylethylenediamine^[21c]in THF under high-di-

lution conditions at 60 °C and smoothly provided the 13-

membered ring 4 in 70% yield (89% based on recovered

starting material). The most striking feature of this ap-

proach is that this mild intramolecular amidation protocol

proceeds without any epimerization at the two amino acid

stereocenters^[30]or isomerization of the Z vinyl iodide and

without dimerization or formation of higher oligomers

(Scheme 6).^[14]

Macrocyclization by Cyclodehydration, Ene-Enamide RCM,

or Intramolecular Oxidative Amidation

During the course of our study of the synthesis of paliur-

ine F, we decided also to investigate an alternate route for

the preparation of macrocycle 4 by means variously of a

cyclodehydration, an ene-enamide RCM, or an intramolec-

ular aza-Wacker reaction. This would have been a good op-

portunity to test the efficiency of our copper-based macro-

cyclization procedure and, in the best-case scenario, would

have provided more efficient access to the target macrocycle

4. To this end, the required precursors 25, 6, and 28 were

prepared from pyrrolidines 19d and 19c by the sequence

previously established for the preparation of acyclic frag-

ment 8. Because the enol ether in 19d did not survive the

two consecutive oxidations (Swern/NaClO₂), we decided to

protect it as a cyclic acetal that could be cleaved in situ

during the cyclodehydration (Scheme 7). Subsequent depro-

Scheme 7. Elaboration of the acyclic substrates for macrocycliza-

tion by cyclodehydration, intramolecular aza-Wacker reaction, or

ene-enamide RCM.

The preparation of acyclic fragments finally set the stage

tection of the primary alcohol and oxidation gave acid 23, for their cyclization (Scheme 8). We began this study with

which was then engaged in a peptidic coupling with isoleuc- the cyclization of the masked aldehyde 25. After consider-

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Scheme 8. Macrocyclization by cyclodehydration, ene-enamide RCM, or intramolecular oxidative amidation.

able experimentation with various promoters (TsOH, HCl, cycle together with the installation of the enamide. We

oxalic acid, iodine) and conditions for deprotection and/or therefore decided to use this option to complete the synthe-

activation of the acetal and for the cyclization/dehydration sis of the target molecule, paliurine F, as well as the related

sequence, we eventually found that this cyclodehydration alkaloid paliurine E, possessing a different side-chain.

was best effected with use of 60 mol-% p-toluenesulfonic

acid in mixture of toluene and water (8:2) at 100 °C over

7 d.

Synthesis of Paliurines E and F

Under these conditions, the cyclized product 4 could be

isolated in 34% yield, a low yield that could be attributed

To install the side chains of paliurines E and F, the Boc

to extensive degradation of both starting material and group was first carefully removed in 80% yield by use of

macrocyclic enamide under the reaction conditions. We TMSOTf and 2,6-lutidine (Scheme 9). Next, a HATU/

next focused on the intramolecular aza-Wacker reaction, HOAt-mediated coupling of the resulting free amine 29

using a procedure that had been shown to provide β-amino- with N,N-dimethyl--phenylalanine gave the desired paliur-

styrenes regioselectively by intermolecular amidation.^[19]ine E (30) in 97% yield. The synthetic (–)-paliurine E exhib-

Upon treatment with a mixture of PdCl₂(CH₃CN)₂and ited physical, spectroscopic, and spectrometric characteris-

CuCl₂under O₂in DME at 60 °C, amidostyrene 6 indeed tics (¹H NMR, ¹³C NMR, IR, [α]_D, UV, and MS) identical

furnished the desired macrocycle 4, but in a disappointing to those reported for the natural product.^[18]

21% yield. The ene-enamide ring-closing metathesis proved

Elaboration of paliurine F (1) proved to be more chal-

to be a bit more efficient but turned out to be especially lenging than originally anticipated, and an extensive review

substrate-dependent and required extensive optimization of of conditions to promote the coupling of 29 with dipeptide

both reaction conditions and substitution pattern of the en- 31 was undertaken. Among the conditions surveyed were

amide.^[31]In the best case, the methyl-substituted enamide the use of HATU/K₂CO₃, HATU/iPr₂NEt, DCC/HOBt/

28 could be cyclized in 49% yield in the presence of the iPr₂NEt, or EDC/HOAt/iPr₂NEt. All of these proved inef-

second-generation Grubbs catalyst in 1,2-dichloroethane at fective, giving back starting material and an impressive

reflux. Attempts to optimize this yield by use of additives number of side-products among which paliurine F could

that should prevent either degradation of the catalyst or not even be detected. This might be attributed to the pres-

chelation with the substrate or by use of other metathesis ence of the dimethylamino group in 31, which probably in-

promoters failed to give better results. Again it was clear terferes with the activated carboxylic acid under the cou-

that the copper-mediated intramolecular amidation reac- pling conditions to give highly nucleophilic and epimeriz-

tion was the most viable option for formation of the macro- able cyclic species such as 32. A more predictable method

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A General Route to Cyclopeptide Alkaloids

Synthesis of Ziziphines N and Q and Analogues

The success met with for the synthesis of the paliurines

prompted us to apply our synthesis to the preparation of

other cyclopeptide alkaloids. We therefore targeted zizi-

phines N (38) and Q (39), members of the ziziphine family

isolated from Ziziphus oenoplia that display significant anti-

plasmodial activity.^[8,32]The synthesis started from the com-

mon intermediate 33 (Scheme 10). Treatment with -prolin-

amide (34) in the presence of EDC and HOBt gave the acy-

clic precursor 35, which was smoothly cyclized, by use of

catalytic copper iodide and N,NЈ-dimethylethylenediamine,

in 82% yield. Removal of the Boc group finally gave the

fully elaborated macrocycle 37, which served as a platform

for the synthesis of ziziphines N (38) and Q (39), obtained

in 52 and 64% yields, respectively, after installation of the

peptidic side chains.^[33]

Following an observation by Han and co-workers, who

demonstrated that epimerization of the side chains could

have a deep impact on the biological activities of related

compounds, we next turned our attention to stereochemical

variations at the side-chain residues. Macrocyclic com-

pound 37 was therefore engaged in peptidic coupling with

all combinations of N-Fmoc--leucine or N-Fmoc--leu-

cine (“AA₁” in Scheme 10) and N,N-dimethyl--isoleucine

or N,N-dimethyl--allo-isoleucine (“AA₂” in Scheme 10),

giving the ziziphine N diastereoisomers 40, 41, and 42. In

order to allow assessment of the role of the enamide, zizi-

phine Q was reduced by treatment with hydrogen in the

presence of palladium on carbon in methanol to furnish

dihydroziziphine Q (43) in quantitative yield. Biological

evaluation of all these compounds is discussed at the end

of this article.

Scheme 9. Synthesis of paliurines E and F.

was then used to attach the side-chain by a stepwise pro-

cedure: coupling of 29 with N-Fmoc--isoleucine, followed

by removal of the Fmoc group by treatment with dieth-

ylamine in acetonitrile and subsequent coupling with N,N-

dimethyl--leucine, gave the desired paliurine F (1) in 57%

yield over the three final steps.^[14]

Synthesis of the 15-Membered Ring Cyclopeptide Alkaloids

Abyssenine A and Mucronine E

When we started this project, only slight attention had

been paid to the synthesis of 15-membered ring alkaloids.

Scheme 10. Synthesis of ziziphines N, Q, and analogues.

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Their structures are slightly different from those of their it would be interesting to distinguish between necrotic and

13-membered ring homologues because they incorporate a non-necrotic cell death within global cell death, because ne-

methylene group in place of the ether bond and possess crosis is considered to be basically harmful to the organism,

three amino acids within the macrocycle. Whereas much unlike non-necrotic cell death. Results from flow cytometry

work has been devoted to the synthesis of the 13- or 14- analysis are given in Figure 2 and show that paliurine F (1),

membered ring cyclopeptide alkaloids,^[9–15]a single synthe- abyssenine A (3), and mucronine E (48) are cytotoxic,

sis of a 15-membered ring compound, mucronine B,^[10b,10c]whereas the other compounds are no different from con-

featuring a macrolactamization reaction and a stepwise in- trols.^[37]Of the active compounds, mucronine E (48) is the

stallation of the enamide, had been reported.^[34]We there- most efficient in triggering cell death and clearly is the most

fore initiated studies directed towards the extension of our interesting because it mostly induces non-necrotic cell de-

synthetic route to the preparation of this subclass of cyclo- ath. Cell morphology is shown in Figure 2: floating dead

peptide alkaloids and chose abyssenine A^[6c,35](3) and muc- cells can be observed for treatments with 1, 3, and 48

ronine E^[6c](48) as target molecules (Scheme 11). Acyclic whereas living cells reach confluence in controls and other

precursors 44^[15]and 45^[16]were therefore prepared and sub- treatments, except in the case of paliurine E (30). For this

jected to the copper-mediated “macroenamidation” pro- compound it can be noted that the cells do not reach con-

cedure. Cyclization proceeded smoothly in both cases, yield- fluence although no significant cell death was indicated,

ing the 15-membered ring macrocycles 46 and 47 in excel- which suggests that 30 specifically inhibits cellular prolifera-

lent yields, and therefore expending the scope of this proto- tion.

col for the preparation of macrocyclic enamides. Careful

removal of the Boc groups in 46 and 47 by treatment with

TMSOTf and 2,6-lutidine provided synthetic abyssenine A

(3) and mucronine E (48), respectively and completed our

synthetic studies on cyclopeptide alkaloids. Biological

evaluation of all these compounds, with a focus on their

antibacterial and cytotoxic properties, is discussed below.

Scheme 11. Synthesis of the 15-membered ring cyclopeptide alka-

loids abyssenine A and mucronine E.

Figure 2. Cellular viability (top) and cell morphology (bottom) af-

ter 48 h treatment with synthetic cyclopeptides at 1 m.

Cytotoxicity Assays

The human HT1080 tumoral cell line was used to test

the cytotoxicities of the ten compounds. These cells are able

We next determined the IC₅₀values after incubation of

to undergo physiological cell death, a process termed cells with various concentrations of the three cytotoxic

apoptosis,^[36]and are sensitive to a wide variety of cytotoxic compounds (Table 3). These values confirmed that mucron-

drugs such as etoposide, cisplatin, staurosporine, or TNF- ine E (48) was the most cytotoxic and globally showed that

α. The ten synthetic cyclopeptides 1, 3, 30, 38–43, and 48 compounds 1, 3, and 48 were indeed cytotoxic, although

were first tested at high concentration (1 m) over 48 h on at relatively high concentrations. These results suggest that

HT1080 cells, and the percentages of dead cells were deter- whereas the size of the macrocycle does not seem to have a

mined by flow cytometry analysis. In addition, we thought crucial influence, the constitutive amino acids do, because

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A General Route to Cyclopeptide Alkaloids

the paliurines and ziziphines show dramatically different above compounds against B. anthracis and MRSA. This

cytotoxicities. Comparison of the effects of paliurines F (1) was done by mixing different amounts of the test com-

and E (30) demonstrates that the nature of the peptidic side pound into agar to afford final compound concentrations

chain also seems to be of great importance and provides ranging from 512 µg mL^–1down to 4 µg mL^–1. The agar

the first insights into SARs of cyclopeptide alkaloids.

was then inoculated with the microbe, and incubation was

carried out for 24 h at 37 °C to determine whether the com-

pounds could inhibit bacterial replication on the surface of

the agar. The MIC value was determined as being the low-

est concentration of test compound capable of completely

preventing opaqueness or growth of bacteria colonies on

the surface of the agar. None of the compounds, including

paliurine F, had a MIC value below 512 µg mL^–1, demon-

strating the lack of significant antibacterial activity.

Table 3. Concentrations inducing 50% cellular viability (IC₅₀) in

HT1080 cells after 48 h treatment.

Entry

Synthetic cyclopeptide

IC₅₀[m]

1

2

3

paliurine F (1)

abyssenine A (3)

mucronine E (48)

0.82

1.03

0.68

Antimicrobial Assays

Conclusions

A selection of test compounds including paliurines E (30)

and F (1), ziziphines N (38) and Q (39), α-epi-ziziphine N

(40), β-epi-ziziphine N (41), α,β-diepi-ziziphine N (42), and

the deprotected macrocycle 29 were also evaluated for in

vitro antimicrobial activity, to assess whether the human

cytotoxicity would also be observed in prokaryotic cells.

These compounds were individually subjected to in vitro

susceptibility assays on agar against three representative

test microbes: a methicillin-resistant strain of Staphylococ-

cus aureus (MRSA), Bacillus anthracis, and Escherichia coli.

First we ran Kirby–Bauer well diffusion experiments on

agar plates to look for visible signs of growth inhibition

appearing around the wells. We evaluated each of the com-

pounds at three different amounts (20, 50, and 100 µg) in

each well. None of the compounds produced completely

cleared inhibition zones at these quantities, whereas the

control antibiotic, penicillin G, produced boldly visible

growth inhibition zones even against the MRSA. Neverthe-

less, close inspection of the plates indicated that B. anthracis

was somewhat more susceptible to the inhibitory effects of

the compounds than either MRSA or E. coli. The strongest

bioactivity of all the compounds screened appeared to be

for paliurine F, whereas the rest of the analogues were all

about equivalent in activity against B. anthracis, and were

all completely inactive against MRSA and E. coli. Despite

having weak bioactivity against B. anthracis, all of the com-

pounds gave only very pale zones that were largely filled in

with bacterial colonies, indicating partial growth inhibition,

but the clearing became more pronounced as the drug

amounts were increased from 20 µg to 50 µg and 100 µg.

For paliurine F, the zone size also increased from 12 mm to

18 mm to 22 mm, but the zones were again still largely

In conclusion, we have achieved the total syntheses of

the cyclopeptide alkaloids paliurines E and F, ziziphines N

and Q, abyssenine A, and mucronine E in average overall

yields of about 10%. Our approach featured a key intramo-

lecular cycloenamidation reaction for the construction of

the paracyclophane ring. This original macrocyclization

procedure allowed us simultaneously to address two syn-

thetic challenges associated with cyclopeptide alkaloids: the

formation of the enamide and of the macrocycle, which

were reduced to a single operation. We have also documen-

ted the use of other strategies for the macrocyclization step,

which highlighted the high efficiency of the intramolecular

copper-mediated vinylation reaction that allowed us to de-

velop a unified strategy for these natural products. Prelimi-

nary results of the biological screening showed that none of

the synthetic cyclopeptides displayed significant antibacte-

rial activity, whereas paliurine F and mucronine E turned

out to be moderately cytotoxic. Further studies on the use

of copper-mediated cyclization reactions^[38]for the prepara-

tion of other alkaloids are underway and will be reported

in due time.

Experimental Section

General: All reactions were carried out in oven- or flame-dried

glassware under argon with use of standard techniques for handling

air-sensitive materials.

All solvents were reagent grade. Tetrahydrofuran (THF) and tolu-

ene were freshly distilled from sodium/benzophenone under argon

immediately prior to use. Dichloromethane, 1,2-dimethoxyethane,

DMSO, and DMF were freshly distilled from calcium hydride.

opaque, indicating that bacterial growth was only partially Methanol was distilled from magnesium turnings and iodine.

inhibited within these zones even at 50 µg well loading. In

Diethylamine, diisopropylethylamine, N-methylmorpholine, and

summary, the Kirby–Bauer data indicate that the com-

pounds have only very weak antibacterial properties, with

B. anthracis being the most vulnerable of the three microbes

examined and paliurine F having the strongest potency of

all the ring analogues.

triethylamine were distilled from calcium hydride. Copper(I) iodide

(99.999% purity) was purchased from Aldrich and used as sup-

plied. Finely powdered potassium carbonate (325 mesh) and ce-

sium carbonate were used for copper-mediated coupling reactions.

All other reagents were used as supplied.

To investigate this further, we conducted additional in

vitro bacterial susceptibility experiments by measuring the

Unless otherwise noted, reaction mixtures were magnetically stirred

and reactions were monitored by thin layer chromatography

minimum inhibitory concentration (MIC) values of the (Merck Kieselgel 60F₂₅₄plates). Chromatography was performed

Eur. J. Org. Chem. 2009, 3368–3386

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3377

G. Evano et al.

FULL PAPER

with silica gel 60 (particle size 35–70 µm) supplied by SDS. Yields

refer to chromatographically and spectroscopically pure com-

pounds, unless otherwise noted.

ESIHRMS: m/z calcd. for C₁₁H₁₉NO₅Na [M + Na]⁺268.1161;

found 268.1150.

(2R,3S)-1-(Triphenylmethyl)-3-hydroxyproline Methyl Ester (10c):

Triethylamine (845 µL, 6.0 mmol) and trityl chloride (613 mg,

2.2 mmol) were added to a solution of 10b (363 mg, 2.0 mmol) in

dichloromethane (10 mL). The resulting mixture was stirred at

room temp. for 5 h, quenched with water, and extracted with

dichloromethane. The combined organic layers were washed with

brine, dried with MgSO₄, filtered, and concentrated. The crude res-

idue was purified by flash chromatography over silica gel (EtOAc/

PE 1:1) to yield the desired protected pyrrolidine 10c as a white

solid (512 mg, 1.32 mmol, 66%); m.p. 107 °C. [α]²_D⁰= –54 (c = 1.0,

Proton NMR spectra were recorded with use of an internal deute-

rium lock at ambient temperature with a Bruker 300 MHz spec-

trometer. Internal references of δ_H= 7.26 ppm and δ_H= 2.50 ppm

were used for residual CHCl₃in CDCl₃and residual [D₅]DMSO

in [D₆]DMSO, respectively. Data are presented as follows: chemical

shift (in ppm on the δ scale relative to δ_TMS= 0 ppm), multiplicity

(s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintu-

plet, m = multiplet, br = broad, app = apparent), coupling constant

(J/Hz) and integration. Resonances that are either partially or fully

obscured are denoted obscured (obs.). Carbon-13 NMR spectra

were recorded at 75 MHz. Internal references of δ_C= 77.16 ppm

and δ_C= 39.52 ppm were used for CDCl₃and [D₆]DMSO, respec-

tively.

1

CHCl₃). H NMR (300 MHz, CDCl₃): δ = 7.63 (d, J = 7.7 Hz, 6

H), 7.33 (t, J = 7.7 Hz, 6 H), 7.23 (t, J = 7.3 Hz, 3 H), 4.05 (br. s,

1 H), 3.80 (s, 1 H), 3.70 (s, 3 H), 3.50 (td, J = 10.7, 3.3 Hz, 1 H),

2.79–2.88 (m, 1 H), 2.05–2.17 (m, 1 H), 1.21–1.33 (m, 1 H) ppm.

¹³C NMR (75 MHz, CDCl₃): δ = 175.7, 143.8, 129.6, 127.9, 126.7,

Infrared spectra were recorded with a Nicolet OPUS IR (impact

400D) spectrophotometer. Optical rotations were recorded with a

Perkin–Elmer 341 polarimeter at 589 nm and are reported as fol-

lows: [α]²_D⁰, concentration (c in g/100 mL), and solvent. Melting

points were recorded with a Büchi B-545 instrument. UV spectra

were recorded in methanol with a Shimadzu UV-160A instrument.

Mass spectra were obtained with a GCMS Hewlett–Packard 5989B

spectrometer (EIMS: 70eV, CIMS: 230 eV, ESIMS: analytica

Brandford ionization interface, capex 180 V, skimmer 30 V).

70.2, 51.9, 47.0, 33.3 ppm. IR (KBr): ν

= 3488, 2971, 2863,

˜

max

1721, 1440, 1258, 1163, 968, 877, 733, 657 cm^–1. ESIMS (positive

mode): m/z = 410.3, 243.2, 166.1. ESIHRMS: m/z calcd. for

C₂₅H₂₅NO₃Na [M + Na]⁺410.1732; found 410.1738.

(2S,3S)-1-(tert-Butoxycarbonyl)-3-hydroxyproline (10e): A solution

of commercially available (2S,3S)-3-hydroxyproline (10d, 400 mg,

3.0 mmol) in a mixture of THF (7 mL) and aqueous NaOH (1 ,

7 mL) was treated with di-tert-butyl dicarbonate (800 mg,

3.7 mmol). The resulting mixture was vigorously stirred at room

temp. for 2 h, acidified to pH 1 with HCl (1 ), and extracted with

diethyl ether. The combined organic layers were dried with MgSO₄,

filtered, and concentrated to yield the desired carbamate as a white

solid (708 mg, quantitative crude yield), which was used in the next

step without further purification; m.p. 157 °C. [α]²_D⁰= –9 (c = 1.4,

Because of the presence of complex rotameric mixtures, it was nec-

1

essary to record H and ¹³C NMR spectra of some compounds in

[D₆]DMSO at 345–355 K, provided that they were stable enough

at these temperatures. Even at those temperatures, some ¹³C peaks

were poorly resolved and are denoted “broad” (br).

(2S,3S)-3-Hydroxyproline Methyl Ester Hydrochloride (10b): Thio-

nyl chloride (780 µL, 10.7 mmol) was slowly added at 0 °C to a

suspension of commercially available (2S,3S)-3-hydroxyproline

(10d, 1.0 g, 7.6 mmol) in methanol (10 mL). The resulting mixture

was allowed to warm to room temp., heated at reflux for 2 h, and

concentrated under vacuum to yield the desired methyl ester as a

white solid (1.4 g, quantitative crude yield), which was used in the

next step without further purification. [α]²_D⁰= +11 (c = 1.5, MeOH).

¹H NMR (300 MHz, D₂O): δ = 4.66–4.69 (obs. m, 1 H), 4.29 (d, J

= 2.7 Hz, 1 H), 3.76 (s, 3 H), 3.46–3.51 (m, 2 H), 1.96–2.17 (m, 2

H) ppm. ¹³C NMR (75 MHz, D₂O): δ = 168.4, 72.9, 66.4, 54.0,

1

MeOH). H NMR (300 MHz, MeOD): δ = 4.88 (br. s, 2 H), 4.37

(app. t, J = 1.9 Hz, 1 H), 4.14 and 4.07 (rotamers, s, s, 1 H), 3.44–

3.57 (m, 2 H), 1.96–2.08 (m, 1 H), 1.83–1.90 (m, 1 H), 1.45 and

1.40 (rotamers, s, s, 1 H) ppm. ¹³C NMR (75 MHz, MeOD): δ =

175.7 and 175.4 (rotamers), 157.8 and 157.5 (rotamers), 82.9 and

82.7 (rotamers), 77.2 and 76.4 (rotamers), 70.8 and 70.5 (rotamers),

47.2 and 46.8 (rotamers), 34.8 and 34.2 (rotamers), 30.1 and 30.0

(rotamers) ppm. IR (KBr):

ν

= 3207, 1755, 1661, 1434,

˜

max

1252 cm^–1. CIMS (NH₃gas): m/z = 249, 232, 193, 176, 132, 86.

ESIHRMS: m/z calcd. for C₁₀H₁₇NO₅Na [M + Na]⁺254.1004;

found 254.1009.

44.4, 31.4 ppm. IR (KBr): ν

= 3288, 1741, 1588, 1444,

˜

max

1235 cm^–1. CIMS (NH₃gas): 146, 86. HRMS (CI, NH₃gas): m/z

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)meth-

yl]-3-hydroxypyrrolidine (10g): Lithium borohydride (44 mg,

2.0 mmol) was added at 0 °C to a solution of 10a (246 mg,

1.0 mmol) in THF (5 mL). The resulting mixture was stirred over-

night at room temp., quenched by slow addition of a saturated

solution of NH₄Cl, basified with NaOH (1 ) and extracted with

diethyl ether. The combined organic layers were washed with HCl

solution (1 ) and brine, dried with MgSO₄, filtered, and concen-

trated to yield the intermediate diol as a white solid (220 mg, quan-

titative crude yield). This solid was dissolved in THF (1 mL) and

treated with imidazole (82 mg, 1.20 mmol) and tert-butyldimethyl-

silyl chloride (146 mg, 0.97 mmol). The resulting white slurry was

vigorously stirred at 0 °C for 2 h and quenched by addition of

water. The organic layer was separated and the aqueous layer was

extracted with diethyl ether. The combined organic layers were

washed with brine, dried with MgSO₄, filtered, and concentrated

under vacuum. The crude residue was purified by flash chromatog-

raphy over silica gel (petroleum ether/EtOAc 8:2) to yield the pro-

tected product 10g as a white solid (303 mg, 0.92 mmol, 92%); m.p.

110 °C. [α]²_D⁰= –38 (c = 2.3, CHCl₃). ¹H NMR (300 MHz, [D₆]-

calcd. for C₆H₁₂NO₃[M + H]⁺146.0817; found 146.0812.

(2S,3S)-1-(tert-Butoxycarbonyl)-3-hydroxyproline (10a): Triethyl-

amine (3.0 mL, 21.5 mmol) and di-tert-butyl dicarbonate (1.9 g,

8.6 mmol) were successively added to a solution of 10b (1.3 g,

7.2 mmol) in dichloromethane (30 mL). The resulting mixture was

stirred at room temp. overnight, quenched with water, and ex-

tracted with dichloromethane. The combined organic layers were

washed with HCl solution (1 ) and brine, dried with MgSO₄, fil-

tered, and concentrated to yield the desired carbamate as a color-

less oil (1.8 g, quantitative crude yield), which was used in the next

1

step without further purification. [α]²_D⁰= –21 (c = 1.4, CHCl₃). H

NMR (300 MHz, [D₆]DMSO): δ = 4.42 (br. s, 1 H), 4.28 and 4.16

(rotamers, 2 br. s, 1 H), 3.72 (s, 3 H), 3.54–3.66 (m, 2 H), 2.80 (dd,

J = 13.0, 3.3 Hz, 1 H), 2.03–2.16 (m, 1 H), 1.85–1.93 (m, 1 H), 1.45

and 1.39 (rotamers, s, s, 9 H) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO): δ = 171.9 and 171.5 (rotamers), 154.4 and 154.1 (rota-

mers), 80.4, 75.3, 68.1 and 67.9 (rotamers), 52.5 and 52.3 (rota-

mers), 44.7 and 44.3 (rotamers), 32.7 and 32.4 (rotamers), 28.5 and

28.4 (rotamers); CIMS (NH₃gas): 246, 207, 190, 186, 146.

3378

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Eur. J. Org. Chem. 2009, 3368–3386

A General Route to Cyclopeptide Alkaloids

DMSO, 343 K): δ = 4.66 (s, 1 H), 4.18 (br. s, 1 H), 3.67 (app. q, J

(NH₃gas): m/z = 408, 352, 308, 294. ESIHRMS: m/z calcd. for

= 6.8 Hz, 1 H), 3.53 (br. s, 2 H), 3.39 (td, J = 9.8, 7.3 Hz, 1 H), C₂₂H₃₇NO₄SiNa [M + Na]⁺430.2390; found 430.2402.

3.23 (td, J = 9.8, 2.6 Hz, 1 H), 1.92–2.04 (m, 1 H), 1.62–1.73 (m,

4-Iodo-1-methoxy-2-vinylbenzene (11c): n-BuLi (1.6  in hexanes,

32.0 mL, 51.2 mmol) was added at –78 °C to a suspension of meth-

yltriphenylphosphonium bromide (20.5 g, 57.3 mmol) in THF

(200 mL). The resulting orange solution was stirred at –78 °C for

1 H), 1.42 (s, 9 H), 0.88 (s, 9 H), 0.05, 0.03 (s, s, 3 H, 3 H) ppm.

¹³C NMR (75 MHz, [D₆]DMSO, 343 K): δ = 153.4, 77.7, 71.7 (br),

66.6, 61.8 (br), 44.4, 31.4 (br), 27.8, 25.3, 17.4, –5.9, –6.0 ppm. IR

(KBr): ν

= 3375, 2925, 2756, 2592, 1675, 1424, 1096, 835 cm^–1.

˜

max

1 h and a solution of 5-iodo-2-methoxy-benzaldehyde (11b,^[14]

5.0 g, 19.1 mmol) in THF (50 mL) was added by cannula. The reac-

tion mixture was warmed to 0 °C over 2 h and quenched with satu-

rated aqueous NH₄Cl solution (100 mL). The organic layer was

separated and the aqueous layer was extracted with EtOAc. The

combined organic layers were washed with brine, dried with

MgSO₄, filtered, and concentrated. The crude residue was purified

ESIMS (positive mode): m/z = 354.3, 298.3, 254.2. HRMS (CI,

NH₃): m/z calcd. for C₁₆H₃₄NO₄Si [M + H]⁺332.2257; found

332.2252.

(2R,3S)-2-[(tert-Butyldimethylsilyloxy)methyl]-3-hydroxypyrrolidine

(10f): Anhydrous zinc bromide (1.0 g, 4.5 mmol) was added to a

solution of 10g (331 mg, 1.0 mmol) in dichloromethane (30 mL).

The resulting slurry was stirred overnight at room temp., concen- by flash chromatography over silica gel (Et₂O/petroleum ether 2:8)

trated, and purified by flash chromatography over silica gel

(EtOAc) to yield the desired deprotected pyrrolidine 10f as a white

solid (152 mg, 0.66 mmol, 66%); m.p. 110 °C. [α]²_D⁰= –38 (c = 2.3,

CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ = 4.07–4.16 (m, 1 H), 3.69

to give the desired substituted styrene 11c (4.1 g, 15.8 mmol, 83%)

as a pale yellow liquid. H NMR (300 MHz, CDCl₃): δ = 7.65 (d,

1

J = 2.2 Hz, 1 H), 7.42 (dd, J = 8.6, 2.2 Hz, 1 H), 6.84 (dd, J =

17.7, 11.1 Hz, 1 H), 6.54 (d, J = 8.6 Hz, 1 H), 5.63 (d, J = 17.7 Hz,

(A of ABX syst., J = 10.0, 5.1 Hz, 1 H), 3.56 (B of ABX syst., J 1 H), 5.21 (d, J = 11.1 Hz, 1 H), 3.74 (s, 3 H) ppm. ¹³C NMR

= 10.0, 6.2 Hz, 1 H), 3.0–3.12 (m, 3 H), 2.33 (s, 2 H), 1.95–2.06

(75 MHz, CDCl₃): δ = 156.6, 137.4, 135.2, 130.4, 129.4, 115.8,

= 2991, 1480, 1240, 1122,

(m, 1 H), 1.70–1.80 (m, 1 H), 0.89 (s, 9 H), 0.1 (s, 6 H) ppm. ¹³C 113.2, 83.2, 55.7 ppm. IR (neat): ν

˜

max

NMR (75 MHz, CDCl₃): δ = 74.9, 67.8, 64.4, 44.9, 35.1, 26.1, 18.4, 1025 cm^–1. ESIMS (positive mode): m/z = 283.1 ppm. ESIHRMS:

–5.3 ppm. ESIHRMS: m/z calcd. for C₁₁H₂₅NO₂SiNa [M + Na]⁺

m/z calcd. for C₉H₉IONa [M + Na]⁺282.9596; found 282.9603.

254.1552; found 254.1544.

4-Iodo-1-methoxy-2-(2-methoxyvinyl)benzene (11d): Potassium bis-

(trimethylsilyl)amide (0.5  solution in toluene, 115 mL, 58 mmol)

was added over 30 min to a cooled solution (–78 °C) of methoxy-

methyltriphenylphosphonium chloride (24.5 g, 71.5 mmol) in dry

THF (200 mL). The resulting red solution was stirred at –78 °C for

30 min and at 0 °C for 20 min, and was cooled back to –78 °C. 5-

Iodo-2-methoxybenzaldehyde (11b,^[14]4 g, 15.3 mmol) in dry THF

(60 mL) was then added dropwise at –78 °C by cannula and the

resulting orange-red solution was allowed to warm to room temp.

and to stir overnight. The mixture was quenched at 0 °C with water

and diluted with diethyl ether. The aqueous layer was extracted

Methyl 1-(tert-Butoxycarbonyl)-4,5-dihydropyrrole-2-carboxylate

(13): A 15 mL pressure tube was charged with 10a (245 mg,

1.0 mmol), iodobenzene (135 µL, 1.2 mmol), cesium carbonate

(650 mg, 2.0 mmol), 1,10-phenanthroline (38 mg, 0.2 mmol), and

copper(I) iodide (19 mg, 0.1 mmol). Toluene (0.5 mL) was added,

the pressure tube was closed, and the brownish suspension was

heated to 110 °C for 24 h. The crude reaction mixture was filtered

through a plug of silica gel (washed with EtOAc), concentrated,

and purified by flash chromatography over silica gel (EtOAc/petro-

leum ether 2:8) to yield elimination product 13 as a pale yellow oil

(227 mg, 0.73 mmol, 73%). H NMR (300 MHz, CDCl₃): δ = 5.78 with diethyl ether, and the combined organic layers were washed

1

(t, J = 3.0 Hz, 1 H), 3.93 (t, J = 8.8 Hz, 2 H), 3.80 (s, 3 H), 2.62

with brine, dried with MgSO₄, filtered, and concentrated. The

crude residue was finally purified by flash chromatography over

(td, J = 8.8, 3.0 Hz, 2 H), 1.44 (s, 9 H) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 162.8, 152.9, 136.7, 119.4, 81.1, 52.1, 48.4, 28.6, silica gel (EtOAc/EP 5:95) to give the desired 2-methoxyvinyl prod-

28.2 ppm. ESIHRMS: m/z calcd. for C₁₁H₁₇NO₄Na [M + Na]⁺

250.1055; found 250.1039.

uct as a pale yellow, oily solid (4.2 g, 14.5 mmol, 95%) and as a

1:1 mixture of Z and E isomers; m.p. 41 °C. H NMR (300 MHz,

1

CDCl₃): δ = 8.30 and 7.53 (stereoisomers, d, J = 2.3 Hz, d, J =

2.2 Hz, 1 H), 7.40 and 7.37 (stereoisomers, dd, J = 8.6, 2.3 Hz, dd,

J = 8.6, 2.2 Hz, 1 H), 7.12 and 6.19 (stereoisomers, d, J = 13.0 Hz,

d, J = 7.2 Hz, 1 H), 6.58 and 6.57 (stereoisomers, d, J = 8.6 Hz, d,

J = 8.6 Hz, 1 H), 5.91 and 5.54 (stereoisomers, d, J = 13.0 Hz, d,

J = 7.2 Hz, 1 H), 3.80 and 3.69 (stereoisomers, s, s, 3 H), 3.78 and

3.77 (stereoisomers, s, s, 3 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ

= 155.7 and 155.4 (stereoisomers), 150.4 and 148.8 (stereoisomers),

137.9 and 135.3 (stereoisomers), 135.0 and 134.3 (stereoisomers),

127.2 and 128.0 (stereoisomers), 112.9 and 112.4. (stereoisomers),

99.4 and 97.4 (stereoisomers), 83.3, 60.9, 56.5 and 55.6 (stereoiso-

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-phenoxypyrrolidine (14): A 15 mL pressure tube was

charged with 10g (330 mg, 1.0 mmol), iodobenzene (110 µL,

1.0 mmol), cesium carbonate (650 mg, 2.0 mmol), 1,10-phenan-

throline (38 mg, 0.2 mmol), and copper(I) iodide (19 mg,

0.1 mmol). Toluene (0.5 mL) was added, the pressure tube was

closed, and the brownish suspension was heated to 125 °C for 24 h.

Another portion of iodobenzene (110 µL, 1.0 mmol) was then

added and the reaction mixture was heated for an additional 24 h

and allowed to cool to room temp. The crude reaction mixture was

finally filtered through a plug of silica gel (washed with EtOAc),

concentrated, and purified by flash chromatography over silica gel

(EtOAc/petroleum ether 3:7) to yield ether 14 a pale yellow sticky

oil (306 mg, 0.75 mmol, 75%). [α]²_D⁰= –13 (c = 1.0, CHCl₃). ¹H

NMR (300 MHz, CDCl₃): δ = 7.13–7.20 (m, 2 H), 6.80–6.92 (m, 3

H), 4.79 (dd, J = 14.1, 3.5 Hz, 1 H), 3.95–3.98 and 3.76–3.83 (rota-

mers, m, m, 0.3, 1.7 H), 3.33–3.58 (m, 3 H), 1.97–2.21 (m, 2 H),

1.36 (s, 9 H), 0.84 (s, 9 H), 0.00 (s, 3 H) ppm. ¹³C NMR (75 MHz,

CDCl₃): δ = 157.5, 154.6, 129.5, 120.9, 115.6, 79.7 and 79.3 (rota-

mers) ppm. IR (neat): ν

= 3068, 2997, 2930, 2843, 1639, 1573,

˜

max

1481, 1245, 1219, 1097, 933, 790 cm^–1. CIMS (NH₃gas): m/z =

290, 289, 232, 148, 133, 90, 77. ESIHRMS: m/z calcd. for

C₁₀H₁₁IO₂Na [M + Na]⁺312.9701; found 312.9708.

(Z)-4-Iodo-2-(2-iodovinyl)-1-methoxybenzene (11e): A solution of

NaHMDS (2.0  solution in THF, 7.6 mL, 15.2 mmol) was added

dropwise at room temp. to a suspension of iodomethyltriphenyl-

phosphonium iodide (8.6 g, 16.2 mmol) in THF (90 mL). The re-

mers), 79.0 and 78.5 (rotamers), 64.1, 63.1, and 62.5 (rotamers), sulting red-orange solution was stirred at room temp. for 20 min

45.4 and 45.1 (rotamers), 30.1 and 29.0 (rotamers), 28.6, 26.0, 18.3,

and cooled to –78 °C before addition of HMPA (10 mL) and a

–5.4 ppm. IR (neat): ν

= 2955, 1695, 1600, 1393 cm^–1. CIMS

solution of 11b^[14](2.5 g, 9.5 mmol) in THF (60 mL). The reaction

˜

max

Eur. J. Org. Chem. 2009, 3368–3386

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3379

G. Evano et al.

FULL PAPER

mixture was stirred at –85 °C for three hours and quenched at

–78 °C by addition of saturated aqueous NaHCO₃. The mixture

was allowed to warm to room temp., diluted with Et₂O, and filtered

through a plug of Celite^®, which was thoroughly washed with di-

ethyl ether. The biphasic filtrate was separated and the organic

layer was dried with MgSO₄, filtered, and concentrated. The crude

residue was purified by flash chromatography over silica gel (Et₂O/

petroleum ether 3:97) to give the desired vinyl iodide 11e as a pale

yellow solid (3.1 g, 8.0 mmol, 76%). The diastereoisomeric excess

(rotamers), 29.7 and 30.6 (rotamers), 28.6, 25.8, 18.2, –5.5 ppm. IR

(neat): ν

= 2958, 1731, 1693, 1588, 1485, 1397, 1118 cm^–1. ES-

˜

max

IMS (positive mode): m/z = 630.1, 614.2, 518.3, 492.1. HRMS (CI,

NH₃) m/z calcd. for C₂₅H₄₁NO₇Si [M]⁺495.2652; found 495.2639.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-(3-formyl-4-methoxyphenoxy)pyrrolidine (19b): Solvent

system for purification by flash chromatography over silica gel, elu-

ent gradient from CH₂Cl₂to CH₂Cl₂/EtOH 95:5; pale yellow sticky

oil (scale: 1.9 g of compound obtained; yield 75%). [α]²_D⁰= –5 (c =

1.2, CHCl₃). ¹H NMR (300 MHz, [D₆]DMSO, 345 K): δ = 10.33

(s, 1 H), 7.27 (d, J = 3.1 Hz, 1 H), 7.22–7.25 (m, 1 H), 7.13 (d, J

= 8.8 Hz, 1 H), 4.82 (d, J = 4.1 Hz, 1 H), 3.78–3.93 (m, 2 H), 3.88

(s, 3 H), 3.58–3.63 (m, 1 H), 3.34–3.51 (m, 2 H), 2.23 (app. qt, J =

9.8, 4.7 Hz, 1 H), 2.10 (app. dd, J = 13.4, 6.2 Hz, 1 H), 1.42 (s, 9

H), 0.88 (s, 9 H), 0.06 (s, 6 H) ppm. ¹³C NMR (75 MHz, [D₆]-

DMSO, 345 K): δ = 186.9, 155.2, 152.2, 149.4, 123.8, 123.0, 113.0,

112.5, 78.8 (br), 77.2, 62.6, 60.7 (br), 55.0, 43.3, 27.0 (br), 26.7,

1

was determined by analysis of H NMR spectra of the crude reac-

tion mixture and was found to be higher than 95%; m.p. 67 °C. ¹H

NMR (300 MHz, CDCl₃): δ = 7.91 (d, J = 2.2 Hz, 1 H), 7.50 (dd,

J = 8.7, 2.0 Hz, 1 H), 7.19 (d, J = 8.5 Hz, 1 H), 6.56 (d, J = 8.6 Hz,

2 H), 3.73 (s, 3 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 156.7,

138.2, 137.3, 133.7, 128.4, 122.9, 82.5, 82.0, 55.7 ppm. ESIHRMS:

m/z calcd. for C₉H₈I₂ONa [M + Na]⁺408.8562; found 408.8570.

2-(5-Iodo-2-methoxybenzyl)-1,3-dioxolane (11f): Ethylene glycol

(3 mL) and concentrated HCl (35 wt.-% in H₂O, 4 drops) were

added dropwise to a solution of enol ether 11d (200 mg, 0.69 mmol)

in Et₂O (3 mL). The resulting mixture was stirred at room temp.

for 3 d, carefully quenched with aqueous Na₂CO₃(7 wt.-% in

H₂O), and diluted with diethyl ether. The aqueous layer was ex-

tracted with Et₂O and the combined organic layers were washed

with brine, dried with MgSO₄, filtered, and concentrated. The

crude product was purified by flash chromatography over silica gel

(EtOAc/petroleum ether/Et₃N 10:89.5:0.5) to give the desired diox-

olane 11f (211 mg, 0.66 mmol, 96%) as a white solid; m.p. 64 °C.

¹H NMR (300 MHz, CDCl₃): δ = 7.44 (d, J = 2.0 Hz, 1 H), 7.40

(dd, J = 8.5, 2.1 Hz, 1 H), 6.52 (d, J = 8.6 Hz, 1 H), 5.01 (t, J =

5.0 Hz, 1 H), 3.86–3.91 (m, 2 H), 3.74–3.77 (m, 2 H), 3.70 (s, 3 H),

2.83 (d, J = 5.0 Hz, 2 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

157.5, 139.7, 136.7, 127.5, 112.7, 103.2, 82.8, 64.9, 55.6, 34.7 ppm.

24.2, 16.4, –7.1 ppm. IR (neat): ν

= 2883, 2755, 1694, 1490,

˜

max

1398 cm^–1. ESIMS (positive mode): m/z = 970.0, 953.7, 599.5,

583.5, 488.4. HRMS (CI, NH₃) m/z calcd. for C₂₄H₃₉NO₆Si [M]⁺

465.2547; found 465.2552.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-(4-methoxy-3-vinylphenoxy)pyrrolidine (19c): Solvent sys-

tem for purification by flash chromatography over silica gel, eluent

EtOAc/petroleum ether 15:85; colorless oil (scale: 1.1 g of com-

pound obtained; yield 79%). [α]²_D⁰= –2 (c = 1.5, CHCl₃). ¹H NMR

(300 MHz, CDCl₃): δ = 6.96–7.06 (m, 2 H), 6.85–6.91 (m, 1 H),

6.78 and 6.77 (rotamers, d, J = 8.8 Hz, d, J = 8.8 Hz, 1 H), 5.72

and 5.71 (rotamers, d, J = 17.7 Hz, d, J = 17.7 Hz, 1 H), 5.28 and

5.27 (rotamers, d, J = 11.1 Hz, d, J = 11.1 Hz, 1 H), 4.84 and 4.80

(rotamers, d, J = 3.0 Hz, d, J = 4.1 Hz, 1 H), 3.80–4.07 (m, 2 H),

3.81 (s, 3 H), 3.46–3.73 (m, 3 H), 2.05–2.29 (m, 2 H), 1.49 (s, 9 H),

0.92 (s, 9 H), 0.08 (s, 6 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ =

154.7, 151.6, and 151.3 (rotamers), 151.4, 131.5, and 131.4 (rota-

mers), 128.0, 115.5, 115.1 and 114.6 (rotamers), 115.0, 112.2, and

112.1 (rotamers), 80.0 and 79.8 (rotamers), 79.6 and 79.4 (rota-

mers), 64.2, 63.1, and 62.5 (rotamers), 56.3 and 56.2 (rotamers),

45.4 and 45.1 (rotamers), 30.1 and 28.9 (rotamers), 28.7, 26.0, 18.4,

IR (KBr): ν

= 2935, 2765, 1490, 1244, 1122, 1072, 805 cm^–1.

˜

max

EIMS: m/z = 320, 247, 73. ESIHRMS: m/z calcd. for C₁₁H₁₃IO₃Na

[M + Na]⁺342.9807; found 342.9801.

Typical Procedure for Copper-Mediated Arylation of Hydroxypyr-

rolidine (10g): A 15 mL pressure tube was charged with 10g

(330 mg, 1.0 mmol), aryl iodide 11 (1.0 mmol), cesium carbonate

(652 mg, 2.0 mmol), 1,10-phenanthroline (36 mg, 0.2 mmol), and

copper(I) iodide (19 mg, 0.1 mmol). Toluene (0.5 mL) was added,

the pressure tube was closed, and the brownish suspension was

heated to 125 °C (110 °C in the case of styrene derivative 11c) for

24 h. Another portion of aryl iodide 11 (0.5 mmol) was then added

and the reaction mixture was heated for an additional 24 h and

allowed to cool to room temp. The crude reaction mixture was

finally filtered through a plug of silica gel (washed with EtOAc),

concentrated, and purified by flash chromatography over silica gel

to give the desired ethers 19.

–5.3, –5.4 ppm. IR (neat): ν

= 1694, 1682, 1494, 1393,

˜

max

1180 cm^–1. CIMS (NH₃gas): m/z (%) = 464 (37), 408 (31), 364 (26),

350 (100), 306 (12). ESIHRMS: m/z calcd. for C₂₅H₄₁NO₅SiNa [M

+ Na]⁺486.2652, found 486.2638.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-[3-(2-methoxyvinyl)-4-methoxyphenoxy]pyrrolidine (19d):

Solvent system for purification by flash chromatography over silica

gel, eluent gradient from EtOAc/petroleum ether 1:9 to 2:8; color-

less oil (scale: 321 mg of compound obtained; yield 65%). This

compound was obtained as a 7:3 mixture of isomers and each iso-

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)- mer exists as a mixture of rotamers. [α]²_D⁰= –2 (c = 1.1, CHCl₃).

methyl]-3-(4-methoxy-3-methoxycarbonylphenoxy)pyrrolidine (19a):

Solvent system for purification by flash chromatography over silica

gel, eluent EtOAc/petroleum ether 3:7; pale sticky oil (scale: 332 mg

of compound obtained; yield 67%). [α]²_D⁰= +4 (c = 1.4, CHCl₃).

¹H NMR (300 MHz, CDCl₃): δ = 7.92 (d, J = 3.6 Hz, 1 H), 7.65

(dd, J = 7.6, 1.0 Hz, 1 H), 6.67 (dd, J = 8.8, 3.3 Hz, 1 H), 5.38 (t,

¹H NMR (300 MHz, CDCl₃): δ = 7.60 (app. s, 0.3 H), 7.12 (dd, J

= 13.0 and 3.1 Hz, 0.7 H), 6.82 (br. s, 0.7 H), 6.70–6.78 (m, 2 H),

6.09 (dd, J = 7.1 and 4.1 Hz, 0.4 H), 5.89 (d, J = 11.9 Hz, 0.6 H),

5.52 (d, J = 7.2 Hz, 0.3 H), 4.71 (dd, J = 14.2 and 2.7 Hz, 1 H),

4.03 (br. s, 0.5 H), 3.75–3.91 (m, 5.5 H), 3.67 (s, 3 H), 3.44–3.60

(m, 2 H), 1.97–2.18 (m, 2 H), 1.38 (s, 9 H), 0.84 (s, 9 H), 0.00 (s, 6

J = 4.2 Hz, 1 H), 3.66–3.97 (m, 2 H), 3.81 (s, 3 H), 3.78 (s, 3 H), H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 154.6, 151.4, 151.3,

3.64 (dd, J = 10.8, 4.9 Hz, 1 H), 3.39–3.54 (m, 2 H), 2.24–2.33 (m,

1 H), 1.97–2.0 (m, 1 H), 1.40 (s, 9 H), 0.84 (s, 9 H), 0.0 (s, 6 H) ppm.

¹³C NMR (75 MHz, CDCl₃): δ = 171.0, 164.2, and 164.4 (rota-

mers), 159.0, 154.4, 142.0, and 142.2 (rotamers), 139.8 and 140.0

(rotamers), 122.1 and 122.4 (rotamers), 114.4, 81.7, 79.4, and 79.7

(rotamers), 65.0, 62.0, and 62.7 (rotamers), 56.1, 45.2, and 45.5

151.1, 150.9, 150.8, 150.5, 150.4, 150.1, 150.0, 148.3, 148.2, 126.6,

125.8, 125.7, 118.1, 117.4, 114.5, 114.3, 113.6, 113.3, 112.9, 100.3,

98.7, 98.6, 80.0, 79.9, 79.7, 79.6, 79.4, 79.3, 79.2, 64.3, 64.2, 63.0,

62.4, 62.3, 60.7, 56.4, 56.3, 56.2, 56.1, 56.0, 45.5, 45.4, 45.2, 45.0,

30.3, 30.1, 29.8, 29.0, 28.8, 28.6, 26.0, 18.3, –5.3 ppm. IR (neat):

ν

= 3000, 1705, 1694, 1681, 1650, 1494, 1392, 1224, 1096,

˜

max

3380

www.eurjoc.org

Eur. J. Org. Chem. 2009, 3368–3386

A General Route to Cyclopeptide Alkaloids

777 cm^–1. CIMS (NH₃gas): m/z = 493, 438, 380. ESIHRMS: m/z

stirred at –78 °C for 30 min and a solution of (2R,3S)-1-(tert-but-

oxycarbonyl)-2-(hydroxymethyl)-3-[3-(1,3-dioxolan-2-ylmethyl)-4-

methoxyphenoxy]pyrrolidine (500 mg, 1.22 mmol) in dichloro-

methane (6.5 mL) was added dropwise by cannula. The reaction

mixture was stirred for 40 min at –78 °C before dropwise addition

of triethylamine (740 µL, 5.2 mmol), and the mixture was warmed

to –10 °C over 2 h. The reaction was next quenched at –10 °C with

water and diluted with CH₂Cl₂. The aqueous layer was extracted

with CH₂Cl₂and the combined organic layers were washed with

brine, dried with MgSO₄, filtered, and concentrated to give the de-

sired aldehyde as pale yellow oil (497 mg, 1.22 mmol, quant.),

which was used without purification in the next step. An analytical

sample was purified by flash chromatography over silica gel

(EtOAc/petroleum ether 1:1) to give a pale yellow oil. [α]²_D⁰= –25

(c = 0.8, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ = 9.69 and

9.63 (rotamers, s, s, 1 H), 6.83 (br. s, 1 H), 6.76 (br. s, 2 H), 5.11 (dd,

J = 10.1, 5.1 Hz, 1 H), 4.84 (br. s, 1 H), 4.50 and 4.32 (rotamers, s,

s, 1 H), 3.82–4.01 (m, 4 H), 3.78 (s, 3 H), 3.60–3.76 (m, 2 H), 2.95

(d, J = 5.0 Hz, 2 H), 2.20 (dd, J = 13.5, 5.8 Hz, 1 H), 1.88–2.00

(m, 1 H), 1.43 and 1.49 (rotamers, s, s, 9 H) ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 200.1 and 200.0 (rotamers), 155.0, 150.3,

and 150.2 (rotamers), 150.6, 126.5, 119.8, and 119.7 (rotamers),

114.0 and 113.9 (rotamers), 111.5 and 111.4 (rotamers), 103.6,

80.6–80.0 (rotamers), 78.8, 70.9, and 70.6 (rotamers), 65.0, 56.0,

45.3, and 45.0 (rotamers), 35.3 and 35.2, 31.1 and 30.5 (rotamers),

calcd. for C₂₆H₄₃NO₆SiNa [M + Na]⁺516.2757, found 516.2773.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-[3-(1,3-dioxolan-2-ylmethyl)-4-methoxyphenoxy]pyrroli-

dine (19f): Solvent system for purification by flash chromatography

over silica gel, eluent EtOAc/ petroleum ether/Et₃N 14.5:85:0.5;

orange oil (scale: 126 mg of compound obtained; yield 47 %).

[α]²_D⁰= –0.3 (c = 1.4, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ =

6.78–6.86 (m, 2 H), 6.75 and 6.72 (rotamers, s, s, 1 H), 5.11 (dd, J

= 10.4, 5.1 Hz, 1 H), 4.78 (dd, J = 13.2, 2.9 Hz, 1 H), 3.98–4.03

(m, 1 H), 3.96–3.98 (m, 2 H), 3.86–3.91 (m, 1 H), 3.83–3.85 (m, 2

H), 3.78–3.81 (m, 1 H), 3.77 (s, 3 H), 3.44–3.57 (m, 2 H), 2.94–2.97

(m, 2 H), 2.01–2.26 (m, 2 H), 1.46 (s, 9 H), 0.91 (s, 9 H), 0.01 (s, 6

H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 154.7, 152.2, 151.2 and

151.1 (rotamers), 126.3 and 126.2 (rotamers), 120.0 and 119.7 (rot-

amers), 113.9 and 113.7 (rotamers), 111.2 and 111.4 (rotamers),

103.7 (C₃), 79.7 and 79.3 (rotamers), 65.0, 64.2, 63.1, 56.1 and 56.0

(rotamers), 45.5 and 45.1 (rotamers), 35.3 and 35.2 (rotamers), 30.2

and 29.8 (rotamers), 28.9 and 28.6 (rotamers), 26.0, 18.4, –6.3 ppm.

IR (neat): ν

= 2966, 1731, 1690, 1501, 1383, 1117 cm^–1. CIMS

˜

max

(NH₃gas): m/z = 524, 468, 424, 410.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-[(tert-butyldimethylsilyloxy)-

methyl]-3-[3-(1,3-dioxolan-2-ylmethyl)-4-methoxyphenoxy)pyrroli-

dine (19f): Preparation from enol ether 19d. p-Toluenesulfonic acid

monohydrate (50 mg, 0.25 mmol) and ethylene glycol (840 µL,

15.1 mmol) were added to a solution of 19d (150 mg, 0.30 mmol)

in toluene (7 mL). The solution was heated at reflux under a Dean–

Stark apparatus, monitored by TLC until all starting material was

consumed, concentrated, and diluted with water. The aqueous solu-

tion was extracted with CH₂Cl₂and the combined organic layers

were washed with brine, dried with MgSO₄, filtered, and concen-

trated. The crude residue was finally purified by flash chromatog-

raphy over silica gel (EtOAc/petroleum ether/Et₃N 14.5:85:0.5) to

give acetal 19f (110 mg, 0.21 mmol, 70%) as an orange oil.

28.5 and 28.4 ppm. IR (neat): ν_max= 2991, 1731, 1685, 1650, 1511,

˜

1496, 1424 cm^–1. CIMS (CH₄gas): m/z = 407, 352, 308, 290, 246,

211, 142, 114. ESIHRMS: m/z calcd. for C₂₁H₂₉NNaO₇[M +

Na]⁺430.1842, found 430,1823.

(2S,3S)-1-(tert-Butoxycarbonyl)-3-[3-(1,3-dioxolan-2-ylmethyl)-4-

methoxyphenoxy]proline (23): 2-Methylbut-2-ene (90 %, 1.3 mL,

11.0 mmol), followed by a solution of sodium chlorite (80 %,

335 mg, 2.96 mmol) and sodium dihydrogen phosphate dihydrate

(400 mg, 2.6 mmol) in water (8 mL), was added to a solution of

(2S,3S)-1-(tert-butoxycarbonyl)-2-formyl-3-[3-(1,3-dioxolan-2-yl-

methyl)-4-methoxyphenoxy]pyrrolidine (497 mg, 1.22 mmol) in a

mixture of THF (4 mL) and tert-butyl alcohol (12 mL). The yellow

reaction mixture was then stirred for 1 h, carefully quenched with

HCl solution (1 ), and diluted with diethyl ether. The aqueous

layer was extracted with diethyl ether, and the combined organic

layers were dried with MgSO₄, filtered, and concentrated. The

crude residue was purified by flash chromatography over silica gel

(EtOH/DCM 5:95) to give the free carboxylic acid as a yellow, oily

solid (423 mg, 1.0 mmol, 82% over two steps). [α]²_D⁰= –32 (c = 1.0,

CHCl₃). ¹H NMR (300 MHz, [D₆]DMSO, 350 K): δ = 6.84–6.93

(m, 3 H), 5.03 (t, J = 3.9 Hz, 1 H), 4.86 (app. d, J = 3.2 Hz, 1 H),

4.24 (s, 1 H), 3.88–3.93 (m, 2 H), 3.74–3.79 (m, 2 H), 3.75 (s, 3 H),

3.44–3.60 (m, 2 H), 2.84 (d, J = 5.1 Hz, 2 H), 2.00–2.20 (m, 2 H),

1.39 (br. s, 9 H) ppm. ¹³C NMR (75 MHz, [D₆]DMSO, 350 K): δ

= 170.8, 153.1 (br. s), 152.1, 149.7, 125.9, 119.1, 113.9, 111.8, 102.6,

80.2 (br), 78.7, 64.6 (br), 63.7, 55.8, 44.0, 34.0, 29.0 (br), 27.6 ppm.

(2R,3S)-1-(tert-Butoxycarbonyl)-2-(hydroxymethyl)-3-[3-(1,3-dioxol-

an-2-ylmethyl)-4-methoxyphenoxy]pyrrolidine: A solution of 19f

(440 mg, 0.84 mmol) in THF (9 mL) was treated at –10 °C with a

solution of TBAF (1  solution in THF, 1.25 mL, 1.25 mmol). The

resulting light orange mixture was allowed to warm to room temp.

over 60 min and quenched with water. The aqueous layer was ex-

tracted with Et₂O and the combined organic layers were washed

with brine, dried with MgSO₄, filtered, and concentrated. The

crude residue was finally purified by flash chromatography over

silica gel (gradient from EtOAc/petroleum ether/Et₃N 34.5:65:0.5

to EtOAc/petroleum ether/Et₃N 44.5:55:0.5) to give the unprotec-

ted alcohol (344 mg, 0.84 mmol, quant) as an orange oil. [α]²_D⁰

=

1

–16 (c = 1.0, CHCl₃). H NMR (300 MHz, CDCl₃): δ = 6.85 (br.

s, 1 H), 6.74 (br. s, 2 H), 5.07 (t, J = 4.9 Hz, 1 H), 4.80 and 4.60

(rotamers, 2 br. s, 1 H), 3.87–4.13 (m, 2 H), 3.81–3.95 (m, 4 H),

3.81–3.83 (m, 1 H), 3.74 (s, 3 H), 3.43–3.58 (m, 2 H), 2.97 (A of

ABX syst., J = 13.7, 4.8 Hz, 1 H), 2.89 (B of ABX syst., J = 13.8,

5.1 Hz, 1 H), 2.08 (br. s, 2 H), 1.44 (s, 9 H) ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 156.2, 152.3, 150.6, 126.0, 119.3, 114.3,

111.4, 103.6, 80.2 and 80.0 (rotamers), 79.6, 65.0, 64.9, 64.3, 56.0,

IR (neat): ν_max= 3441, 2930, 1757, 1629, 1424 cm^–1. ESIMS (posi-

˜

tive mode): m/z = 462.2, 446.2, 390.2, 362.2, 346.2. ESIHRMS: m/z

calcd. for C₂₁H₂₉NNaO₈[M + Na]⁺446.1791, found 446.1805.

(2S,3S)-{1-(tert-Butoxycarbonyl)-3-[3-(1,3-dioxolan-2-ylmethyl)-4-

methoxyphenoxy]prolyl}isoleucinamide (25): 1-Hydroxybenzotri-

azole (HOBt, 103 mg, 0.76 mmol) was added to a solution of 23

(305 mg, 0.72 mmol) and isoleucinamide acetic acid salt^[39]

(138 mg, 0.72 mmol) in DMF (6.5 mL). 1-[3-(Dimethylamino)pro-

pyl]-3-ethylcarbodiimide hydrochloride (EDC, 152 mg, 0.79 mmol)

45.4, 35.0, 29.9, 28.5 ppm. IR (neat): ν_max= 3455, 3037, 1711, 1667,

˜

1514, 1503, 1454, 1416, 1123, 944, 875 cm^–1. ESIMS (positive

mode): m/z = 841.6, 432.3, 376.3. ESIHRMS: m/z calcd. for

C₂₁H₃₁NNaO₇[M + Na]⁺432.1998, found 432.2014.

(2S,3S)-1-(tert-Butoxycarbonyl)-2-formyl-3-[3-(1,3-dioxolan-2-yl-

methyl)-4-methoxyphenoxy]pyrrolidine: DMSO (220 µL, 3.1 mmol) and N-methylmorpholine (200 µL, 1.80 mmol) were next added at

was added at –78 °C to a solution of oxalyl chloride (230 µL,

0 °C and the solution was stirred for 16 h while allowed to warm

2.6 mmol) in dichloromethane (7 mL). The resulting solution was

progressively to room temp. The yellow reaction mixture was

Eur. J. Org. Chem. 2009, 3368–3386

www.eurjoc.org

3381

G. Evano et al.

FULL PAPER

quenched with water and diluted with diethyl ether. The aqueous

layer was extracted with diethyl ether and the combined organic

layers were successively washed with aqueous HCl (1 ), saturated

aqueous NaHCO₃, and brine, dried with MgSO₄, filtered, and con-

centrated. The crude residue was purified by flash chromatography

over silica gel (EtOAc) to give the desired peptide (300 mg,

0.56 mmol, 78%) as a pale yellow solid; m.p. 72 °C. [α]²_D⁰= –43 (c

EtOH 99:1) to give the desired cyclized product 4^[14](3 mg,

6.3 µmol, 34%) as a white solid.

Paliurine Core 4 by Oxidative Amidation from 6: PdCl₂(CH₃CN)₂

(2.6 mg, 0.01 mmol) and copper(II) chloride (1.3 mg, 0.01 mmol)

were successively added to a solution of 6 (47 mg, 0.10 mmol) in

1,2-dimethoxyethane (10 mL). The resulting mixture was stirred at

60 °C for 3 d under O₂, filtered through a plug of silica gel, and

concentrated. The crude residue was purified by flash chromatog-

raphy over silica gel (Et₂O/EtOH 99:1) to give the desired cyclized

product 4^[14](10 mg, 0.021 mol, 21%) as a white solid.

1

= 0.8, CHCl₃). H NMR (300 MHz, [D₆]DMSO, 350 K): δ = 7.18

(d, J = 8.5 Hz, 1 H), 7.15 (br. s, 1 H), 6.86–6.91 (m, 4 H), 5.02 (t,

J = 5.0 Hz, 1 H), 4.77 (br. s, 1 H), 4.37 (s, 1 H), 4.20 (t, J = 8.1 Hz,

1 H), 3.84–3.92 (m, 2 H), 3.82–3.74 (m, 5 H), 3.41–3.55 (m, 2 H),

2.84 (d, J = 4.9 Hz, 2 H), 2.06 (app. d, J = 5.8 Hz, 2 H), 1.72–1.85

(m, 1 H), 1.41 (s, 9 H), 1.03–1.19 (m, 2 H), 0.83–0.90 (m, 6 H) ppm.

¹³C NMR (75 MHz, [D₆]DMSO, 360 K): δ = 172.1, 168.7, 153.6

(br), 152.0, 149.8, 125.8, 119.2, 114.1, 111.9, 102.6, 80.2 (br), 79.1,

65.8 (br), 63.7, 56.6, 55.8, 44.4, 36.4, 34.0, 28.9 (br), 27.6, 24.0,

N,N-Dimethyl-L-leucinyl-L-isoleucine Benzyl Ester: O-(7-Azabenzo-

triazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate

(HATU, 913 mg, 2.4 mmol) was added at 0 °C to a cooled solution

of N,N-dimethyl--leucine (500 mg, 2.6 mmol) in DMF (20 mL).

The resulting beige solution was stirred at 0 °C for 20 min and a

mixture of -isoleucine benzyl ester p-toluenesulfonate (787 mg,

2 mmol) and diisopropylethylamine (1.45 mL, 8.2 mmol) in DMF

(15 mL) was added dropwise by cannula at 0 °C. The resulting yel-

low solution was allowed to warm to room temp. and stirred over-

night. The mixture was quenched at 0 °C with saturated aqueous

NH₄Cl and diluted with diethyl ether. The aqueous layer was ex-

tracted with diethyl ether and the combined organic layers were

washed with brine, dried with MgSO₄, filtered, and concentrated.

The crude residue was purified by flash chromatography over silica

gel (EtOAc/EtOH 96:4) to give the desired dipeptide (580 mg,

1.6 mmol, 80%) as an orange oil. [α]²_D⁰= +0.5 (c = 1.0, CHCl₃).

¹H NMR (300 MHz, CDCl₃): δ = 7.31 (d, J = 9.1 Hz, 1 H), 7.20–

7.26 (m, 5 H), 5.12 (A of AB syst., J = 12.2 Hz, 1 H), 5.01 (B of

AB syst., J = 12.2 Hz, 1 H), 4.52 (dd, J = 9.1, 4.8 Hz, 1 H), 2.79

(dd, J = 8.8, 5.0 Hz, 1 H), 2.18 (s, 6 H), 1.82–1.90 (m, 1 H), 1.56–

1.67 (m, 1 H), 1.42–1.51 (m, 1 H), 1.23–1.37 (m, 2 H), 0.92–1.12

(m, 1 H), 0.76–0.85 (m, 12 H) ppm. ¹³C NMR (75 MHz, CDCl₃):

δ = 173.7, 172.0, 135.5, 128.6, 128.4, 67.8, 66.9, 56.1, 42.5, 37.8,

15.1, 10.6 ppm. IR (KBr): ν

= 3406, 3196, 2960, 1675, 1501,

˜

max

1404, 1219, 1173, 1122, 1035 cm^–1. CIMS (NH₃gas): m/z = 535,

436, 278, 211, 86. ESIHRMS: m/z calcd. for C₂₇H₄₁N₃O₈Na [M +

Na]⁺558.2791, found 558.2805.

(2S,3S)-[1-tert-Butoxycarbonyl-3-(4-methoxy-3-vinylphenoxy)prolyl]-

isoleucinamide (6): 1-Hydroxybenzotriazole (HOBt, 59 mg,

0.43 mmol) was added to a solution of 26^[31](150 mg, 0.41 mmol)

and isoleucinamide acetate^[39](79 mg, 0.41 mmol) in DMF (4 mL).

1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide

hydrochloride

(EDC, 86 mg, 0.45 mmol) and N-methylmorpholine (110 µL,

1.02 mmol) were next added at 0 °C, and the solution was stirred

for 16 h while being allowed to warm progressively to room temp.

The yellow reaction mixture was quenched with water and diluted

with diethyl ether. The aqueous layer was extracted with diethyl

ether and the combined organic layers were successively washed

with aqueous HCl (1 ), saturated aqueous NaHCO₃, and brine,

dried with MgSO₄, filtered, and concentrated. The crude residue

was purified by flash chromatography over silica gel (EtOAc/petro-

37.5, 25.9, 25.1, 23.6, 22.1, 15.8, 11.6 ppm. IR (neat): ν

= 3350,

˜

max

leum ether 8:2) to give the desired peptide (138 mg, 0.29 mmol, 2955, 1742, 1675, 1496, 1189, 738, 691 cm^–1. ESIMS (positive

71%) as a pale yellow solid; m.p. 85 °C. [α]²_D⁰= –25 (c = 0.7,

mode): m/z = 385.4, 363.4, 114.1. ESIHRMS: m/z calcd. for

1

CHCl₃). H NMR (300 MHz, [D₆]DMSO, 345 K): δ = 7.61 (d, J

C₂₁H₃₅N₂O₃[M + H]⁺363.2648; found 363.2662.

= 8.7 Hz, 1 H), 7.16 (d, J = 2.7 Hz, 1 H), 6.88–7.12 (m, 4 H), 5.82

and 5.26 (rotamers, dd, J = 17.7, 1.5 Hz, dd, J = 11.2, 1.5 Hz, 1

H), 4.84 (br. s, 1 H), 4.39 (app. s, 1 H), 4.21 (dd, J = 8.5, 6.6 Hz,

1 H), 3.78 (s, 3 H), 3.57 (app. tt, J = 10.6, 2.9 Hz, 1 H), 3.42–3.51

(m, 1 H), 2.07–2.10 (br. m, 2 H), 1.76–1.85 (m, 1 H), 1.40–1.54 (m,

1 H), 1.41 (s, 9 H), 1.04–1.18 (m, 1 H), 0.89 (d, J = 6.8 Hz, 3 H),

0.86 (d, J = 7.4 Hz, 3 H) ppm. ¹³C NMR (75 MHz, [D₆]DMSO,

345 K): δ = 172.1, 168.7, 153.5, 151.2, 150.3, 130.8, 127.5, 116.1,

114.8, 113.7, 112.9, 80.4 (br), 79.0, 65.9, 56.6, 55.9, 44.4, 36.4, 29.0

N,N-Dimethyl-

(10 wt.-% on activated carbon, 55 mg) was added to a solution of

N,N-dimethyl--leucinyl--isoleucine benzyl ester (510 mg,

L-leucinyl-L-isoleucine (31): Palladium on carbon

1.41 mmol) in methanol (80 mL). The system was purged three

times under vacuum and stirred under hydrogen for 90 min. The

mixture was filtered through a plug of Celite^®and concentrated

under vacuum to give the deprotected dipeptide (391 mg,

1.41 mmol, quant.) as a yellow solid, which was used without puri-

fication for the next step; m.p. 74 °C. [α]²_D⁰= +10 (c = 1.6, CHCl₃).

¹H NMR (300 MHz, CDCl₃): δ = 8.10 (br. s, 1 H), 4.50 (dd, J =

7.7, 3.8 Hz, 1 H), 3.82 (br. s, 1 H), 2.64 (s, 6 H), 1.81–1.96 (m, 2

H), 1.42–1.62 (m, 3 H), 1.18–1.32 (m, 1 H), 0.87–0.98 (m, 12

H) ppm; ¹³C NMR (75 MHz, CDCl₃): δ = 175.6 (br), 168.0 (br),

66.9 (br), 58.2, 41.0 (br), 38.4, 37.4, 25.9, 25.4, 23.1, 21.6, 15.4,

(br), 27.7, 24.0, 15.1, 10.6 ppm. IR (KBr): ν_max= 3319, 2966, 1690,

˜

1486, 1388, 1219, 1173 cm^–1. ESIMS (positive mode): m/z = 499.3,

498.3. ESIHRMS: m/z calcd. for C₂₅H₃₇N₃O₆Na [M + Na]⁺

498.2580; found 498.2589.

Paliurine Core 4 by Cyclodehydration from 25: p-Toluenesulfonic

acid monohydrate (1.2 mg, 5.6 µmol) was added to a solution of

25 (10 mg, 18.6 µmol) in mixture of toluene (2.5 mL) and water

(250 µL). The mixture was progressively heated from room temp.

to 80 °C during five days. After five days, water (250 µL) and p-

toluenesulfonic acid monohydrate (1.2 mg, 5.6 µmol) were added

and the solution was heated to 100 °C for 2 d. The light orange

mixture was then hydrolyzed with saturated aqueous NaHCO₃and

diluted with diethyl ether. The aqueous layer was extracted with

diethyl ether and the combined organic layers were washed with

brine, dried with MgSO₄, filtered, and concentrated. The crude res-

idue was purified by flash chromatography over silica gel (Et₂O/

12.1 ppm. IR (neat): ν

= 3432, 2966, 2935, 1665, 1593, 1470,

˜

max

1388 cm^–1. CIMS (NH₃gas): m/z = 273, 114. ESIHRMS: m/z

calcd. for C₁₄H₂₉N₂O₃[M + Na]⁺273.2178; found 273.2185.

(2S,3S,Z)-{1-(tert-Butoxycarbonyl)-3-[3-(2-iodovinyl)-4-methoxyphen-

oxy]prolyl}prolinamide (35): 1-Hydroxybenzotriazole (HOBt,

265 mg, 1.96 mmol) was added to a solution of 33^[14](600 mg,

1.23 mmol) and prolinamide (183 mg, 1.60 mmol) in DMF

(12 mL). 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydro-

chloride (EDC, 330 mg, 1.72 mmol) and N-methylmorpholine

(405 µL, 3.68 mmol) were next added at 0 °C and the solution was

stirred for 16 h while being allowed to warm progressively to room

3382

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Eur. J. Org. Chem. 2009, 3368–3386

A General Route to Cyclopeptide Alkaloids

temp. The yellow reaction mixture was quenched with water and

diluted with diethyl ether. The aqueous layer was extracted with

diethyl ether and the combined organic layers were successively

washed with aqueous HCl (1 ), saturated aqueous NaHCO₃, and

brine, dried with MgSO₄, filtered, and concentrated. The crude res-

idue was purified by flash chromatography over silica gel (EtOAc/

3.44–3.5 (m, 1 H), 3.03–3.21 (m, 2 H), 2.70 (br. s, 1 H), 2.19–2.34

(m, 2 H), 2.07–2.15 (m, 1 H), 1.81–2.03 (m, 3 H) ppm. ¹³C NMR

(75 MHz, CDCl₃): δ = 171.6, 167.4, 154.1, 151.4, 151.1, 124.0,

121.3, 117.6, 113.8, 111.2, 107.1, 80.6, 67.5, 61.2, 56.0, 47.8, 35.9,

29.3, 25.0 ppm. IR (KBr): ν

= 3395, 3309, 3150, 3114, 3068,

˜

max

2940, 2873, 1696, 1644, 1624, 1511, 1419, 1306, 1255, 1219, 1184,

EtOH 88:12) to give the desired peptide 35 (530 mg, 0.91 mmol, 1086, 1025, 815, 799, 784, 730, 661 cm^–1. ESIMS (positive mode):

74%) as a pale yellow solid; m.p. 93 °C. [α]²_D⁰= –11 (c = 0.53,

m/z

=

737.5, 380.4, 358.4. ESIHRMS: m/z calcd. for

CHCl₃). ¹H NMR (300 MHz, DMSO, 355 K): δ = 7.39 (d, J = C₁₉H₂₄N₃O₆N₄[M + H]⁺358.1767; found 358.1760.

8.5 Hz, 1 H), 7.33 (d, J = 2.6 Hz, 1 H), 7.06 (dd, J = 8.5, 2.5 Hz,

Typical Procedure for the Synthesis of Ziziphines N (38) and Q (39)

1 H), 7.01 (br. s, 1 H), 6.72–6.96 (br. s, 2 H), 6.78 (d, J = 8.5 Hz,

and Stereoisomers 40–42: O-(7-Azabenzotriazol-1-yl)-N,N,NЈ,NЈ-

1 H), 4.86 (br. d, J = 3.5 Hz, 1 H), 4.57 (br. s, 1 H), 4.33 (br. d, J

tetramethyluronium hexafluorophosphate (HATU, 134 mg,

= 6.2 Hz, 1 H), 3.78 (s, 3 H), 3.40–3.60 (m, 4 H), 1.85–2.28 (m, 6

0.35 mmol) and diisopropylethylamine (185 µL, 1.05 mmol) were

H), 1.39 (s, 9 H) ppm. ¹³C NMR (75 MHz, DMSO, 355 K): δ =

added at 0 °C to a cooled solution of N-Fmoc-protected amino

172.6, 167.6, 151.6, 149.2, 133.9, 126.4, 118.0, 117.2, 112.5, 82.5,

acid (“N-Fmoc-AA₁” in Scheme 10, 0.38 mmol) and 1-hydroxyaza-

81.2, 78.5, 63.6, 59.2, 55.9, 46.2, 44.3, 28.7, 27.7, 24.1 ppm. IR

benzotriazole (HOAt, 62 mg, 0.45 mmol) in DMF (3 mL). The re-

(KBr): ν_max= 3426, 3109, 2981, 1690, 1650, 1496, 1404, 1280, 1255,

˜

sulting yellow solution was stirred at 0 °C for 20 min and added

dropwise by cannula at 0 °C to a cooled solution of 37 (90 mg,

0.25 mmol) in DMF (4 mL). The flask containing the activated

acid was rinsed with an additional portion of DMF (2.5 mL),

which was cannulated into the solution of the amine. The resulting

yellow solution was allowed to warm to room temp. and stirred

overnight. The mixture was quenched at 0 °C with saturated aque-

ous NH₄Cl and diluted with diethyl ether. The aqueous layer was

extracted with diethyl ether and the combined organic layers were

washed with brine, dried with MgSO₄, filtered, and concentrated

to give an orange oil that was used in the next step without further

purification.

1219, 1163 cm^–1. ESIMS (positive mode): m/z = 608.3. ESIHRMS:

m/z calcd. for C₂₄H₃₂IN₃O₆Na [M + Na]⁺608.1234; found

608.1245.

Ziziphine Core 36: A 100 mL flask was charged with iodo-amide

35 (400 mg, 0.68 mmol), copper(I) iodide (39 mg, 0.21 mmol), and

cesium carbonate (334 mg, 1.03 mmol). The flask was evacuated

under high vacuum, backfilled with argon, and closed with a rub-

ber septum. Dry and degassed THF (92 mL) and N,NЈ-dimethyl-

ethylene-1,2-diamine (45 µL, 0.41 mmol) were next added, the rub-

ber septum was replaced by a glass stopper, and the light green

suspension was heated to 60 °C for 64 h. The reaction mixture was

allowed to cool to room temp., filtered through a plug of silica gel

(washed with EtOAc), and concentrated. The crude residue was

purified by flash chromatography over silica gel (Et₂O/EtOH 99:1)

to give the desired cyclized product 35 (255 mg, 0.56 mmol, 82%)

as a white solid; m.p. 94 °C. [α]²_D⁰= –363 (c = 0.6, CHCl₃). ¹H

NMR (300 MHz, CDCl₃): δ = 8.40 (d, J = 11.6 Hz, 1 H), 6.80–

6.97 (m, 4 H), 5.94 (d, J = 8.3 Hz, 1 H), 5.25–5.32 (m, 1 H), 4.64

(dd, J = 9.1, 4.2 Hz, 1 H), 4.29 (d, J = 4.8 Hz, 1 H), 4.28–4.36 (m,

1 H), 3.79 (s, 3 H), 3.73–3.86 (m, 1 H), 3.39–3.48 (m, 1 H), 3.24–

3.33 (m, 1 H), 2.33–2.41 (m, 1 H), 2.15–2.26 (m, 2 H), 1.93–2.04

(m, 2 H), 1.71–1.86 (m, 1 H), 1.43 (s, 9 H) ppm; ¹³C NMR

(75 MHz, CDCl₃): δ = 172.7, 168.0, 154.1, 151.4, 151.2, 124.3,

121.8, 117.2, 114.0, 110.9, 106.7, 80.4, 79.9, 62.5, 62.0, 56.1, 48.0,

This oil was dissolved in CH₃CN (4.5 mL) and diethylamine was

added (950 µL, 9.2 mmol) at room temp. After 50 min, the crude

yellow mixture was concentrated under vacuum and residue was

immediately engaged in the next step without further purification.

O-(7-Azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexa-

fluorophosphate (HATU, 134 mg, 0.35 mmol) and diisopropylethy-

lamine (185 µL, 0.72 mmol) were added at 0 °C to a cooled solution

of N,N-dimethylamino acid (“N,N-Me₂-AA₂” in Scheme 10,

0.38 mmol) and 1-hydroxyazabenzotriazole (HOAt, 62 mg,

0.45 mmol) in DMF (3 mL). The resulting yellow solution was

stirred at 0 °C for 20 min and added dropwise by cannula at 0 °C

to a solution of the previously deprotected amine in DMF (4 mL).

The flask containing the activated acid was rinsed with an ad-

ditional portion of DMF (2.5 mL), which was cannulated into the

solution of the amine. The yellow solution was allowed to warm to

room temp. and stirred overnight. The mixture was hydrolyzed at

0 °C with saturated aqueous NH₄Cl and diluted with diethyl ether.

The aqueous layer was extracted with diethyl ether and the com-

bined organic layers were washed with brine, dried with MgSO₄,

filtered, and concentrated. The crude residue was purified by flash

chromatography over silica gel to give natural cyclopeptides and

stereoisomers 38–42.

45.4, 32.6, 29.2, 28.5, 25.0 ppm. IR (KBr): ν_max= 3411, 3155, 2981,

˜

1690, 1634, 1506, 1475, 1399, 1316, 1260, 1219, 1168, 1132, 1061,

902, 769 cm^–1. ESIMS (positive mode): m/z = 937.7, 480.4.

ESIHRMS: m/z calcd. for C₂₄H₃₁N₃O₆Na [M + Na]⁺480.2111;

found 480.2931.

Deprotected Ziziphine Core 37: 2,6-Lutidine (66 µL, 0.56 mmol)

and a solution of trimethylsilyl trifluoromethanesulfonate (1.4 

solution in dichloromethane, 1.6 mL, 2.24 mmol) were added at

–10 °C to a solution of 36 (255 mg, 0.56 mmol) in dichloromethane

(12.5 mL). The resulting light pink solution was stirred for 1 h

while progressively warming to 0 °C. The mixture was next hy-

drolyzed at 0 °C by addition of saturated aqueous NaHCO₃and

diluted with dichloromethane. The aqueous layer was extracted

with dichloromethane and the combined organic layers were

washed with brine, dried with MgSO₄, filtered, and concentrated.

The crude residue was purified by flash chromatography over silica

gel (EtOAc/EtOH/NH₃85:10:5) to give the desired N-deprotected

macrocycle 37 (182 mg, 0.51 mmol, 91%) as a white solid; m.p.

196 °C. [α]²_D⁰= –351 (c = 0.54, CHCl₃). ¹H NMR (300 MHz,

CDCl₃): δ = 8.24 (d, J = 11.4 Hz, 1 H), 6.68–6.91 (m, 4 H), 5.91

Ziziphine N (38): Solvent system for purification by flash

chromatography over silica gel, eluent EtOAc/EtOH 96:4; white so-

lid (scale: 79 mg of compound obtained; yield 52% over three

steps). R_f= 0.28 (Merck Kieselgel 60F₂₅₄TLC plates, EtOAc/EtOH

95:5); m.p. 82 °C {ref. 117–119 °C}.^[8][α]²_D⁰= –127 (c = 0.68,

CHCl₃) {ref. natural ziziphine N: [α]²_D⁰

= –327 (c = 0.18,

CHCl₃)};^{[8,33] 1}H NMR (300 MHz, CDCl₃):^[40]δ = 8.35 (d, J =

11.5 Hz, 1 H), 6.94 (d, J = 9.0 Hz, 1 H), 6.81–6.92 (m, 3 H), 6.79

(br. s, 1 H), 5.94 (d, J = 8.7 Hz, 1 H), 5.21–5.27 (m, 1 H), 4.78

(app. q, J = 7.5 Hz, 1 H), 4.54 (dd, J = 9.1, 4.0 Hz, 1 H), 4.38 (d,

(d, J = 9.0 Hz, 1 H), 5.20–5.25 (m, 1 H), 4.55 (dd, J = 8.4, 5.1 Hz, J = 5.6 Hz, 1 H), 4.21–4.29 (m, 2 H), 3.79 (s, 3 H), 3.57–3.66 (m,

1 H), 3.76 (s, 3 H), 3.72–3.84 (m, 1 H), 3.61 (d, J = 3.7 Hz, 1 H), 1 H), 3.26–3.33 (m, 1 H), 2.57 (br. s, 1 H), 2.43–2.49 (m, 1 H),

Eur. J. Org. Chem. 2009, 3368–3386

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3383

G. Evano et al.

FULL PAPER

2.35–2.39 (m, 1 H), 2.25 (s, 6 H), 2.20–2.30 (m, 1 H), 1.91–2.04 (m,

2 H), 1.76–1.86 (m, 2 H), 1.42–1.67 (m, 4 H), 1.11–1.21 (m, 1 H),

0.92 (d, J = 6.3 Hz, 6 H), 0.92 (t, J = 6.3 Hz, 3 H), 0.87 (d, J =

(d, J = 8.5 Hz, 1 H), 5.91 (d, J = 8.8 Hz, 1 H), 5.22 (app. ddd, J =

12.2, 10.2, 6.9 Hz, 1 H), 4.78 (app. dt, J = 9.3, 5.1 Hz, 1 H), 4.50

(dd, J = 9.1, 4.2 Hz, 1 H), 4.34 (d, J = 5.8 Hz, 1 H), 4.19–4.27 (m,

6.6 Hz, 3 H) ppm. ¹³C NMR (75 MHz, CDCl₃):^[40]δ = 171.7, 2 H), 3.76 (s, 3 H), 3.58 (ddd, J = 10.4, 5.7, 2.1 Hz, 1 H, H_21b),

171.4, 167.9, 151.4, 151.1, 124.2, 121.8, 117.1, 114.0, 110.9, 106.8,

78.8, 74.5, 62.9, 62.2, 56.1, 48.0 (2C), 45.5, 43.3, 41.0, 34.5, 32.9,

3.24–3.31 (m, 1 H), 2.50 (d, J = 6.9 Hz, 1 H), 2.37–2.47 (m, 1 H),

2.29–2.37 (m, 1 H), 2.23 (s, 6 H), 2.15–2.28 (m, 1 H), 1.89–2.0 (m,

2 H), 1.72–1.84 (m, 3 H), 1.49–1.62 (m, 2 H), 1.41–1.48 (m, 2 H),

0.99 (d, J = 6.3 Hz, 6 H), 0.93 (t, J = 6.4 Hz, 3 H), 0.80 (d, J =

29.2, 27.1, 25.1, 24.8, 23.3, 21.9, 14.8, 12.1 ppm. IR (KBr): ν

=

˜

max

3400, 2955, 2925, 2868, 2781, 1690, 1649, 1506, 1224, 1050,

767 cm^–1. UV: λ_max(log ε) = 273 (4.42), 324 (4.31) nm. CIMS (NH₃6.7 Hz, 3 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.6, 171.4,

gas): m/z = 612. ESIHRMS: m/z calcd. for C₃₃H₅₀N₅O₆[M + H]⁺

171.2, 167.8, 151.4, 151.0, 124.1, 121.7, 117.0, 113.9, 110.8, 106.7,

78.7, 75.3, 62.9, 62.1, 56.0, 48.0, 47.8, 45.1, 42.7, 41.2, 34.6, 32.8,

29.1, 25.0, 24.9, 24.8, 23.1, 22.0, 16.2, 11.9 ppm. ESIMS (positive

mode): m/z = 634.5, 612.5. ESIHRMS: m/z calcd. for C₃₃H₅₀N₅O₆

[M + H]⁺612.3761, found 612.3741.

612.3761, found 612.3735.

Ziziphine Q (39): Solvent system for purification by flash

chromatography over silica gel, eluent EtOAc/EtOH 96:4; white so-

lid (scale: 96 mg of compound obtained; yield 64% over three

steps). R_f= 0.25 (Merck Kieselgel 60F₂₅₄TLC plates, EtOAc/EtOH

95:5); m.p. 96 °C {ref. 140–142 °C}.^[8][α]²_D⁰= –234 (c = 0.54,

α,β-Diepiziziphine N (42): Solvent system for purification by flash

chromatography over silica gel, eluent CH₂Cl₂/EtOH/30 wt.-%

aqueous ammonia 95:4.5:0.5; white solid (scale: 35 mg of com-

CHCl₃) {lit. natural ziziphine Q: [α]²_D⁰

CHCl₃)};^{[8,33] 1}H NMR (300 MHz, CDCl₃):^[41]δ = 8.33 (d, J =

= –325 (c = 0.16,

pound obtained; yield 65% over three steps); m.p. 99 °C. [α]²_D⁰

=

11.5 Hz, 1 H), 6.94 (dd, J = 8.8, 11.7 Hz, 1 H), 6.89–6.95 (obs. m, –209 (c = 0.53, CHCl₃). ¹H NMR (300 MHz, CDCl₃): δ = 8.42 (d,

1 H), 6.79–6.88 (m, 3 H), 5.94 (d, J = 8.8 Hz, 1 H), 5.24 (dt, J = J = 11.6 Hz, 1 H), 6.89 (dd, J = 11.5, 8.9 Hz, 1 H), 6.87 (app. d, J

9.6, 6.6 Hz, 1 H), 4.47–4.54 (m, 2 H), 4.38 (d, J = 6.0 Hz, 1 H), = 9.1 Hz, 1 H), 6.78 (br. s, 1 H), 6.65 (d, J = 8.5 Hz, 1 H), 5.92 (d,

4.20–4.31 (m, 2 H), 3.78 (s, 3 H), 3.59–3.68 (m, 1 H), 3.27–3.34 (m, J = 8.8 Hz, 1 H), 5.25 (app. ddd, J = 12.7, 9.6, 6.9 Hz, 1 H), 4.89

1 H), 2.58 (d, J = 5.1 Hz, 1 H), 2.41–2.49 (m, 1 H), 2.30–2.38 (m, (app. dt, J = 9.4, 4.9 Hz, 1 H), 4.49 (dd, J = 8.8, 3.7 Hz, 1 H), 4.30

1 H), 2.24 (s, 6 H), 2.21–2.26 (m, 1 H), 1.90–2.03 (m, 3 H), 1.77– (d, J = 5.5 Hz, 1 H), 4.20–4.26 (m, 1 H), 3.80–3.92 (m, 2 H), 3.83

1.86 (m, 2 H), 1.48–1.62 (m, 1 H), 1.15–1.22 (m, 1 H), 0.94 (d, J (s, 3 H), 3.24–3.31 (m, 1 H), 2.48–2.55 (m, 1 H), 2.46 (d, J = 6.0 Hz,

= 7.3 Hz, 3 H), 0.92 (t, J = 6.7 Hz, 3 H), 0.92 (d, J = 6.4 Hz, 3 H),

0.90 (d, J = 6.5 Hz, 3 H) ppm. ¹³C NMR (75 MHz, CDCl₃):^[41]

= 172.1, 171.5, 170.8, 167.7, 151.2, 150.9, 124.0, 121.6, 116.9, 113.5,

1 H), 2.23–2.35 (m, 1 H), 2.22 (s, 6 H), 2.09–2.18 (m, 1 H), 1.88–

2.01 (m, 1 H), 1.70–1.83 (m, 3 H), 1.50–1.68 (m, 2 H), 1.29–1.48

(m, 2 H), 1.06–1.21 (m, 1 H), 0.98 (d, J = 6.4 Hz, 6 H), 0.94 (t, J

δ

110.7, 106.6, 78.5, 74.5, 62.6, 62.1, 55.9, 54.9, 47.8, 45.5, 43.0, 34.3, = 6.7 Hz, 3 H), 0.84 (d, J = 6.3 Hz, 3 H) ppm. ¹³C NMR (75 MHz,

32.6, 30.8, 29.0, 26.9, 24.9, 19.0, 18.5, 14.7, 11.9 ppm. IR (KBr):

= 3401, 2966, 2925, 2878, 2776, 1639, 1506, 1412, 1225,

CDCl₃): δ = 171.43, 171.38, 171.3, 167.8, 151.5, 151.0, 124.3, 121.7,

ν

117.1, 114.0, 110.8, 106.6, 78.7, 75.8, 63.1, 62.0, 56.1, 48.0, 47.4,

˜

max

1051 cm^–1. UV: λ_max(log ε) = 270 (3.60), 318 (3.48) nm. CIMS 45.8, 43.1, 42.2, 34.8, 32.8, 29.1, 25.0, 24.9, 24.8, 23.1, 22.0, 16.5,

(NH₃gas): m/z = 598, 358. ESIHRMS: m/z calcd. for C₃₂H₄₈N₅O₆

12.3. ESIMS (positive mode): m/z = 634.4, 612.3. ESIHRMS: m/z

[M + H]⁺598.3605, found 598.3594.

calcd. for C₃₃H₅₀N₅O₆[M + H]⁺612.3761, found 612.3744.

α-Epiziziphine N (40): Solvent system for purification by flash

chromatography over silica gel, eluent EtOAc/EtOH 96:4; white so-

lid (scale: 46 mg of compound obtained; yield 42% over three

steps); m.p. 59 °C. [α]²_D⁰= –418 (c = 0.53, CHCl₃). ¹H NMR

(300 MHz, CDCl₃): δ = 8.45 (d, J = 11.5 Hz, 1 H), 6.92 (d, J =

Dihydroziziphine Q (43): Pd/C (10% wt, 10 mg) was added to a

solution of ziziphine Q (39, 9.9 mg, 0.0165 mmol) in dry methanol

(7 mL). The solution was purged three times, placed under hydro-

gen, stirred for 1 h, filtered through a pad of Celite^®, and concen-

trated in vacuo to give dihydroziziphine Q (10.0 mg, 0.0165 mmol,

1

9.0 Hz, 1 H), 6.91 (dd, J = 11.5, 9.0 Hz, 1 H), 6.87 (br. s, 1 H), quant) as a white solid. H NMR (300 MHz, CDCl₃): δ = 6.89 (d,

6.81 (app. d, J = 9.5 Hz, 2 H), 5.93 (d, J = 8.8 Hz, 1 H), 5.29 (app.

ddd, J = 11.8, 9.5, 6.8 Hz, 1 H), 4.89 (dt, J = 9.3, 5.1 Hz, 1 H),

4.52 (dd, J = 8.5, 3.5 Hz, 1 H), 4.33 (d, J = 5.2 Hz, 1 H), 4.27–4.32

J = 9.0 Hz, 1 H), 6.76 (br. s, 1 H), 6.68–6.74 (m, 2 H), 6.43 (app.

d, J = 4.9 Hz, 1 H), 5.37 (app. dt, J = 11.1, 7.1 Hz, 1 H), 4.59 (d,

J = 7.3 Hz, 1 H), 4.45 (t, J = 8.5 Hz, 1 H), 4.30 (t, J = 9.3 Hz, 1

(m, 1 H), 3.83–3.90 (m, 2 H), 3.79 (s, 3 H), 3.23–3.31 (m, 1 H), H), 4.18 (d, J = 7.6 Hz, 1 H), 3.84–3.93 (m, 2 H), 3.74–3.83 (m, 1

2.64 (br. s, 1 H), 2.49–2.57 (m, 1 H), 2.27–2.37 (m, 1 H), 2.25 (s, 6 H), 3.77 (s, 3 H), 3.64–3.72 (m, 1 H), 3.13–3.20 (m, 1 H), 3.07 (app.

H), 2.08–2.18 (m, 1 H), 1.90–2.05 (m, 2 H), 1.73–1.85 (m, 2 H), t, J = 7.8 Hz, 2 H), 2.38–2.48 (m, 1 H), 2.30–2.36 (m, 1 H), 2.22

1.53–1.64 (m, 2 H), 1.39–1.51 (m, 2 H), 1.06–1.21 (m, 1 H), 0.99 (s, 6 H), 2.17–2.27 (m, 1 H), 1.92–2.02 (m, 1 H), 1.67–1.87 (m, 5

(d, J = 6.3 Hz, 6 H), 0.93 (t, J = 6.4 Hz, 3 H), 0.80 (d, J = 6.7 Hz, H), 1.47–1.62 (m, 1 H), 1.09–1.33 (m, 1 H), 0.98 (d, J = 6.7 Hz, 6

3 H) ppm. ¹³C NMR (75 MHz, CDCl₃): δ = 171.4, 171.3, 170.8,

167.9, 151.5, 151.0, 124.3, 121.7, 117.1, 114.0, 110.8, 106.6, 78.7,

74.1, 63.1, 62.0, 56.1, 47.9, 47.4, 45.9, 43.0, 42.0, 34.8, 32.8, 29.1,

H), 0.95 (d, J = 6.6 Hz, 3 H), 0.90 (t, J = 7.9 Hz, 3 H) ppm. ¹³C

NMR (75 MHz, CDCl₃): δ = 171.5, 171.3, 171.1, 170.4, 152.6,

150.0, 127.5, 117.3, 116.3, 112.3, 78.5, 74.7, 62.6, 60.0, 56.1, 47.8,

45.5, 43.3, 36.7, 34.5, 31.6, 31.2, 30.8, 27.2, 26.6, 25.1, 19.2, 19.0,

14.9, 12.1 ppm. ESIMS (positive mode): m/z = 600.4, 408.3, 361.0,

359.0, 183.0. ESIHRMS: m/z calcd. for C₃₂H₅₀N₅O₆[M + H]⁺

600.3761; found 600.3785.

26.7, 25.1, 25.0, 23.5, 22.0, 14.6, 12.1 ppm. IR (KBr): ν_max= 3503,

˜

2955, 2920, 2873, 1701, 1644, 1516, 1455, 1419, 1260, 1219, 1178,

1045 cm^–1. ESIMS (positive mode): m/z = 634.2, 612.3. ESIHRMS:

m/z calcd. for C₃₃H₅₀N₅O₆[M + H]⁺612.3761, found 612.3739.

β-Epiziziphine N (41): Solvent system for purification by flash

chromatography over silica gel, eluent CH₂Cl₂/EtOH/30 wt.-%

aqueous ammonia 95:4.5:0.5; white solid (scale: 98 mg of com-

Cellular Viability: The human HT1080 fibrosarcoma adherent cell

line was cultured at 37 °C under a humidified atmosphere contain-

ing CO₂(5%) in Dulbecco’s modified Eagle’s medium (DMEM/

F12) supplemented with fetal bovine serum (10%) together with

penicillin (100 µg mL^–1), streptomycin (100 U mL^–1), and glutamax

(1% v/v) from Invitrogen. Stock solutions of the nine compounds

in DMSO (100 m) were prepared and stored at –20 °C. Cells were

pound obtained; yield 70% over three steps); m.p. 103 °C. [α]²_D⁰

=

–478 (c = 0.57, CHCl₃). ¹H NMR (300 MHz, CDCl₃): 8.32 (d, J =

11.6 Hz, 1 H), 6.89 (dd, J = 11.5, 8.8 Hz, 1 H), 6.85 (app. d, J =

4.0 Hz, 1 H), 6.80 (app. d, J = 3.0 Hz, 1 H), 6.77 (br. s, 1 H), 6.65

3384

www.eurjoc.org

Eur. J. Org. Chem. 2009, 3368–3386

A General Route to Cyclopeptide Alkaloids

seeded in 12-well plates (5ϫ10⁴cells per well). After 24 h, the me-

dium was replaced in each well by complete medium (1 mL) with

the appropriate concentrations of the tested drugs and cells were

incubated for 48 h. Corresponding controls with analogous concen-

trations of DMSO were carried out in parallel. After drug treat-

ment, the media from each well were kept in centrifuge tubes. The

adherent cells were detached (trypsin), pooled with the correspond-

ing media, centrifuged, and resuspended in complete medium. Cells

were then loaded with fluorescein diacetate (FDA, 1 µg mL^–1)

30 min and propidium iodide (PI 5 µg mL^–1) 5 min prior to analy-

sis. FDA (Polysciences) is a nonfluorescent compound that be-

comes fluorescent (free fluorescein) when cleaved by esterases in

living cells, and PI (Sigma) specifically penetrates in necrotic cells

after loss of their plasma membrane integrity.^[42]Percentages of cell

deaths were determined with a XL3C flow cytometer (Beckman–

Coulter, France). Pictures of cells were taken in culture wells before

trypsinization under a Nikon TMS microscope equipped fitted with

a Nikon F601 camera.

Supporting Information (see also the footnote on the first page of

1

this article): Copies of H and ¹³C NMR spectra for all new com-

pounds and synthetic ziziphines. Ziziphines N and Q spectroscopic

data and comparison with reported data.

Acknowledgments

We thank the Centre National de la Recherche Scientifique

(CNRS) for financial support. M. T. acknowledges the Ministère

de la Recherche for a graduate fellowship. We are indebted to Dr.

Sunit Suksamrarn (Srinakharinwirot University) for kindly provid-

ing NMR spectra of ziziphine Q and for a gift of natural zizi-

phine N. We also thank Dr. Karen Wright, Dr. Bruno Drouillat,

and Dr. Jean-Paul Mazaleyrat for helpful and stimulating dis-

cussions.

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Antimicrobial Susceptibility Testing: MRSA ATCC 43300, B. an-

thracis Sterne strain, and E. coli ATCC23590 were purchased from

ATCC sources. A culture of each microorganism from a freezer

stock in tryptic soy broth (Difco Laboratories, Detroit, MI) and

glycerol (20%) was grown on tryptic soy agar (TSA) plates

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Received: February 5, 2009

Published Online: May 19, 2009

3386

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Eur. J. Org. Chem. 2009, 3368–3386

Article Doi

A general route to cyclopeptide alkaloids: total syntheses and biological evaluation of paliurines e and F, ziziphines N and Q, abyssenine A, mucronine E, and analogues

DOI: 10.1002/ejoc.200900122

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