Flexible synthesis and evaluation of diverse anti-apicomplexa cyclic peptides

Article

pubs.acs.org/joc

Flexible Synthesis and Evaluation of Diverse Anti-Apicomplexa Cyclic

Peptides

Mariam Traore,^†Flore Mietton,^‡Daniele Maubon,^‡,§Marine Peuchmaur,^†Flaviane Francisco Hilario,^†,∥,⊥

́

̀

́

Rossimiriam Pereira de Freitas,^∥Alexandre Bougdour,^‡Aurelie Curt,^‡,§Marjorie Maynadier,^#Henri Vial,^#

́

Herve Pelloux,^‡,§Mohamed-Ali Hakimi,^‡and Yung-Sing Wong*^,†

́

^†Dep

bat

^‡Laboratoire Adaptation et Pathogen

F-38042 Grenoble Cedex 9, France

^§Laboratoire de Parasitologie-Mycologie, Dep

Grenoble cedex 9, France

́

artement de Pharmacochimie Molec

iment Andre Rassat, 470 rue de la Chimie, F-38041 Grenoble Cedex 9, France

ie des Micro-organismes, Universite Joseph Fourier-Grenoble 1, CNRS UMR 5163, BP 170,

́ ́

ulaire, Universite Joseph Fourier-Grenoble 1, CNRS UMR 5063, CNRS ICMG FR 2607,

̂

́

artement des Agents Infectieux, Centre Hospitalier Universitaire, BP 217, 38043

∥

́

Departamento de Quımica, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil

^⊥CAPES Foundation, Ministry of Education of Brazil, Brasilia DF 70040-020, Brazil

^#Dynamique des Interactions Membranaires Normales et Pathologiques, Universite

Place E. Bataillon, F-34095 Montpellier Cedex 5, France

́

de Montpellier 2, CNRS UMR 5235, CP 107,

S

* Supporting Information

ABSTRACT: A modular approach to synthesize anti-

Apicomplexa parasite inhibitors was developed that takes

advantage of a pluripotent cyclic tetrapeptide scaﬀold capable

of adjusting appendage and skeletal diversities in only a few

steps (one to three steps). The diversiﬁcation processes make

use of selective radical coupling reactions and involve a new

example of a reductive carbon−nitrogen cleavage reaction with

SmI₂. The resulting bioactive cyclic peptides have revealed new

insights into structural factors that govern selectivity between Apicomplexa parasites such as Toxoplasma and Plasmodium and

human cells.

Most recently, the close natural product analogue AS1387392 (R

= H)⁷was reported to display a better oral absorption with a

plasma concentration higher than that of FR235222.

INTRODUCTION

■

The Apicomplexa phylum of protozoa includes important

human pathogens such as Plasmodium falciparum and

Toxoplasma gondii, responsible for malaria and toxoplasmosis,

respectively. Targeting epigenetic mechanisms is an emerging

and attractive area for therapeutic applications, particularly those

involved in histone modiﬁcations.¹Histone deacetylase

(HDAC) holds a prominent position as a drug target due to its

role in the regulation of the dynamic post-translational level of

acetylation in lysine residues located at the histone tail. The

inhibition of this key event in chromatin remodeling results in

alteration of gene expression regulation.²This approach has been

validated by the discovery of selective histone deacetylase

inhibitors (HDACis) against Apicomplexa parasites such as P.

falciparum.^3,4For many years, naturally occurring cyclic

tetrapeptides or depsipeptides have served as rich sources for

the discovery of new HDACis.⁵Apicidin (Figure 1) was the ﬁrst

described HDACi that exhibits an inhibition eﬀect on

Apicomplexa parasite proliferation, albeit with low selectivity.^4a

FR235222,⁶another cyclic tetrapeptide isolated in 2003 from the

fermentation broth of the fungi Acremonium, was reported to

exhibit immunosuppressive properties and HDAC inhibitions.

Recently, Hakimi and co-workers showed that FR235222 was

highly eﬀective against diﬀerent Apicomplexa.⁸With P.

falciparum and berghei parasites, the growth and diﬀerentiation

were blocked in red blood cells. FR235222 also inhibits T. gondii

intracellular growth at a very low nanomolar level (ED₅₀= 9.7

nM) and is much more potent than pyrimethamine, a current

toxoplasmosis drug, evaluated under the same assay conditions.

At sublethal concentrations, this molecule induces T. gondii

diﬀerentiation from replicative (tachyzoite) to cystogenic

(bradyzoite) form. Importantly, T. gondii HDAC3 (TgHDAC3),

a class I HDAC zinc-dependent enzyme, was identiﬁed as the

target of FR235222. Moreover, the infectious capacity of

Toxoplasma cysts was abolished when they were treated with

FR235222, broadening the therapeutic applications from acute

to chronic toxoplasmosis, particularly for immunocompromised

patients.⁹

Received: January 25, 2013

Published: March 27, 2013

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Figure 1. Natural cyclic tetrapeptides and depsipeptides HDACi.

a

Scheme 1. Synthetic Analogues and Their Biological Activities on Cells

a

Selectivity index (SI): EC₅₀human foreskin ﬁbroblasts (HFF)/EC₅₀T. gondii RH strain (T.g. RH). MCF-7 (breast cancer), HeLa (cervical cancer),

HL-60 (acute promyelocytic leukemia).

With regard to their key pharmacophoric features, these

natural products can be divided into two regions (Figure 1), the

cyclic peptide part (cap region) involved in the recognition or

cell permeability¹⁰and the zinc binding group (ZBG), both parts

being joined by a hydrophobic linker of about 7.8 Å length.

Carbonyl groups are found to be a privileged ZBG in many

HDACi. Recently, we reported modiﬁcations of FR235222 at its

ZBG by converting its chiral hydroxyl group located at the

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Scheme 2. Connecting Bio-Relevant Analogues by Short Reaction Pathways

position α to the ketone group to either acetate (1) or thioacetate

(2) derivatives (Scheme 1).⁹These products appeared to be

signiﬁcantly less toxic to human foreskin ﬁbroblasts (HFF) while

keeping eﬃciency at a low nanomolar concentration against T.

gondii. Conversely, removal of these chiral hydroxyl groups to

give 3, having the apicidin-like ZGB (ethyl ketone), resulted in

reduced potency against T. gondii. The possibility of altering such

selectivity warrants the development of further new carbonyl

ZBGs. Thioester, which is found in the HDACi natural cyclic

depsipeptide largazole¹¹(Figure 1) or synthetic HDACi (4),¹²is

a novel and emergent masked ZBG. Like the disulﬁde group (e.g.

5¹³), thioester is expected to act as a prodrug group by the

delivery in cell medium of a free thiol capable of chelating the zinc

cation in the catalytic pocket.¹⁴The exploration of the cyclic

peptide part by iterative variations of amino acid residues has

been reported along the conventional α₄ring system (12-

membered ring)¹⁵or with enlarged β-amino acid containing

scaﬀolds such as α₃/β and α₂/β₂(13- or 14-membered ring).¹⁶

The results showed that larger cyclic α₃/β pseudotetrapeptides

can give rise to selective HDACis.¹⁷Moreover, it was recently

demonstrated that the cyclic α₃/β-pseudotetrapeptide 6 without

ZBG can retain HDACi activity by itself by interacting with the

active-site opening.¹⁸

FR235222. Moreover, short adjustable/customizable syntheses

capable of constructing structural extensions through fragments

and/or skeletal variations according to the diversity-oriented

synthesis (DOS) principles^19,20are highly attractive in drug

design, making an ever-growing diverse and relevant bioactive

library easily accessible.

Our strategy rests on the design of cyclic tetrapeptide building

blocks 7 (Scheme 2), presenting two major features for

diversiﬁcation: a terminal oleﬁn group as a generic acceptor for

the grafting of a collection of an assortment of carbonyl xanthates

8 or thioacids 9 ZBG (fragment diversity) using known radical

reactions and a cyclic tetrapeptide scaﬀold capable of increasing

readily its ring size in only one step (skeletal diversity). In the

course of our work, we found indeed that our cyclic tetrapeptide

scaﬀold was potent for delivering a new enlarged ring system,

promoted by a novel reductive samarium diiodide (SmI₂) bond

cleavage at the proline unit of 10 and 11. Previous DOS

approaches had already employed bond cleavage to achieve the

increment of diversity through the formation of new skeletons by

oxidative²¹or retro-Diels−Alder reactions.²²Taken together,

these selective reactions bring modularity and ﬂexibility and

connect each analogue to each parent structure in not more than

one or two steps. This short distance between derivatives should

allow a fast navigation and selection between analogues, avoiding

the synthesis of tremendous numbers of intermediates. These

reactions provide a new, convenient, and direct means to

combine fragments and skeletal diversities onto bioactive cyclic

Our current interest in exploring and improving cyclic

tetrapeptide HDACis as potent antitoxoplasmosis agents

prompted us to conceive a concise and modular pathway to

synthesize existing or new cyclic tetrapeptides closely related to

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Scheme 3. Syntheses of cyclic Tetrapeptide (−)-17 and the Collection of Carbonyl Xanthate Fragments 8

Table 1. Synthesis of Carbonyl ZBG Cyclic Tetrapeptides 20

entry

R⁶

19 (yield, %)

20 (yield, %)

1

2

3

4

5

6

CH₂Cl

19a (65)

19b (90)

19c (−)

19d (−)

19e (96)

19f (95)

20a (92)

OBn

20b (69%)

a

CH₂CH₃

CH₂CO₂Et

20c (50)

a

20d (52)

(R)-CHCOTBDMSCH₃

(S)-CHCOTBDMSCH₃

20e (60)

20f (59)

a

Overall yield in a “one vessel” reaction.

peptides. As a result, this collection of novel analogues by their

evaluation on cell assays has shed light on new structural features

that will help in the design of more potent and selective

antiparasitic agents.

The cyclic tetrapeptide (−)-17 was taken as a model and was

prepared from the N-(Boc)-homoallylglycine (+)-14²⁷and

tripeptide 15²⁸in an overall yield of 56%. When (−)-17 was

reacted with diﬀerent xanthates 8 (Table 1) in reﬂuxing 1,2-

dichloroethane and in the presence of dilauroyl peroxide (DLP),

the radical transfer reaction allowed xanthates 19 to be obtained

in good yields (Table 1, entries 1 and 2 and entries 5 and 6). The

reductive removal of the resulting xanthate group was

accomplished by reﬂuxing 19 with DLP in isopropyl alcohol to

yield the ﬁnal cyclic tetrapeptides 20. Alternatively, this

conversion in two steps can be performed in the same vessel

by removing the solvent 1,2-dichloroethane once 19 was formed

and by replacing it by isopropyl alcohol (entries 3 and 4).

The α-hydroxyl ketones cyclic tetrapeptides 21²⁹and 23^29d

were obtained from 20e,f by silyl deprotection, and 21 was

further converted into the corresponding acetylated product

22^29b(Scheme 4). Hemisynthesis from FR235222 has focused

on adding an aliphatic acyl group to the α-hydroxyl ketone ZBG

to give 24, which mimics the thioester group in largazole.

RESULTS AND DISCUSSION

■

To gain a better understanding of the positive contribution to the

antiparasitic activity from each part of the molecule, we ﬁrst

planned to identify the best ZBG. Previous grafting strategies of

carbonyl ZBG onto cyclic peptide or depsipeptide scaﬀolds have

mainly involved cross-metathesis reactions. However, this

assembling process usually proceeded with moderate yields

due to competitive homocoupling reactions.^14,23In addition, the

xanthate ZBG intermediates 8a−f²⁴(Scheme 3) were easily

obtained by reacting potassium ethyl xanthate with commercially

available α-halogen carbonyls 18a−d or with chiral precursors

18e,f accessible within a few steps.²⁵Radical transfer of either

xanthates (Zard’s reaction)²⁶or thioacid derivatives with 7 would

allow the preparation of 10 and 11 more selectively.

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Scheme 4. Further Carbonyl ZBG Analogues Derived from 20e,f and FR235222

Scheme 5. Thioacetylation by Photoreaction of (−)-17 To Give Thioester Analogues

Scheme 6. Cyclic Tetrapeptide Scaﬀolds Tested under the Reductive Carbon−Nitrogen Cleavage Conditions

Alternatively, the terminal oleﬁn in the cyclic tetrapeptide

(−)-17 can selectively react with thioacids 26 and 27³⁰under

photoreaction conditions using AIBN as radical initiator to give

the corresponding thioesters 28 and 29, respectively, in good

yields (Scheme 5).

In our attempts to directly remove the hydroxyl group from

FR235222 to obtain 3 (see Scheme 1) by reductive

deoxygenation using SmI₂,³¹an unexpected reductive carbon−

nitrogen cleavage reaction simultaneously took place in the 3-

methylproline core. Instead of the desired product 3, the

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Table 2. In Vitro Proliferation Assays

a

b

RH = type 1 T. gondii RH strain. HFF = human foreskin ﬁbroblast.

Table 3. In Vitro Proliferation Assays

a

b

c

RH = type 1 T. gondii RH strain. HFF = human foreskin ﬁbroblast. No inhibition eﬀect at 4 μM.

enlarged cyclic structure 30 was obtained in good yield (Scheme

6) in 30 min at 0 °C. In comparison with the FR235222 structure,

30 displays a ¹³C NMR spectrum (DEPT 135) that clearly shows

a change in the ratio of CH and CH₂with the lack of a CH and

the appearance of a new CH₂at 34.1 ppm. High-resolution mass

spectrometry (ESI+) conﬁrms the reduction of FR235222 to

give 30 by the presence of major peaks for [M + Na]⁺(100%)

and [M + H]⁺(10%). Works by Honda showed that SmI₂

promotes reductive deamination of α-amino carbonyl groups in

the presence of HMPA and a proton source such as methanol.³²

To the best of our knowledge, clean reductive deamination of

proline located in a peptide backbone has never been reported. In

addition, the use of HMPA in our case was unnecessary. To

deﬁne the scope of this reaction, we applied the same conditions

for the analogue 20c, which lacks a methyl group on the proline

unit. The enlarged ring system 31 was obtained in a fair yield of

66%. With CJ-15,208,³³which lacks a quaternary center in the

cyclopeptide backbone, conversion was much slower, as shown

by TLC monitoring. However, increasing the reaction time up to

3 h resulted in product decomposition. It seems that this reaction

has to proceed quickly to avoid any side reactions. In contrast, for

apicidin with its piperidine motif fused within the 12-membered

ring, no reaction occurred and only starting material was

recovered. The eﬀectiveness of this pyrrolidine reductive ring

cleavage under SmI₂conditions seems to proceed eﬃciently

through a breaking strain induced by the constrained 12−5-

membered ring system having a quaternary center.

Having in hand this ﬁrst generation of analogues with diverse

ZBGs and two kinds of ring sizes (12- and 15-membered rings),

their potency to inhibit cell proliferation in vitro with T. gondii

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a

Scheme 7. Syntheses of Homochiral α-Amino Methyl Ester

a

Reagents and conditions: (a) LHMDS, THF, −78 °C; (b) HCl; (c) SOCl₂, MeOH; (d) NaOH, CH₃CN, 4 h; (e) Boc₂O, Et₃N, CH₃CN.

RH strain was evaluated (Tables 2 and 3). A screening at 90 nM

was conducted to distinguish molecules having high or low

eﬃcacy. The new ester analogue 24 derived from FR235222

bearing the same aliphatic acyl group found in largazole (Table 2,

entry 3), although being highly active (EC₅₀14.3 nM), showed

no improvement in term of selectivity index. The α₄cyclic

tetrapeptide of (−)-17 alone (without ZBG) showed no activity

(Table 3, entry 1), whereas α₃/β cyclic scaﬀolds lacking a ZBG

can be potent.¹⁸Analogues derived from (−)-17 and having

either the new α-chloro ketone 20a (entry 2), the benzyl ester

20b (entry 3), the new β-keto ester 20d (entry 5), the silyl

protected α-hydroxyl ketones 20e,f (entries 6 and 7) and the

thioesters 28 and 29 (entries 11 and 12) turned out to be also

inactive or weakly active at this concentration. To check further

putative activity on human cells, the compound 28 was tested on

human foreskin ﬁbroblast strain (HFF) and no inhibition was

detected even at 4 μM (entry 11). The results obtained with

these thioesters conﬁrmed previous works by Nishino and co-

workers, who demonstrated that the bioactive masked thiol ZBG

with disulﬁde derivatives such as 5 (see Scheme 1) requires a

longer aliphatic linker.³⁴For the other analogues that induced

cell inhibition higher than 40% at 90 nM with T. gondii RH strain,

their EC₅₀values on T. gondii RH and HFF strains were

investigated to determine their eﬃciency and their selectivity

indexes. Again, the acetylated (R)-hydroxyl ketone ZBG from

analogue 22 was identiﬁed as being the most eﬃcient ZBG

against T. gondii (EC₅₀= 17 nM, entry 9). Analogue 21 (entry 8),

having an (R)-hydroxyl ketone conﬁguration, showed eﬃciency

slightly higher than that of its (S)-hydroxyl ketone diastereomer

23 (entry 10), demonstrating a preferential conﬁguration for a

better antiparasitic proﬁle. Finally, the potencies of each

cyclopeptide scaﬀold to act as an antiparasitic agent can be

compared, given that apicidin, 20c, 3, and 30 share the same ZBG

(ethyl ketone). The best cyclopeptide framework is apicidin

(EC₅₀= 15 nM; Table 2, entry 1), followed by FR235222 (3;

EC₅₀= 95.3 nM, see Scheme 1) and the synthetic model (20c;

EC₅₀= 172 nM, Table 3, entry 4), whereas the enlarged 15-

membered ring 30 (Table 2, entry 4) exhibited only a weak

inhibitory eﬀect.

We next turned our attention to the modiﬁcation of residues

located at the cyclic tetrapeptide part. New homochiral α-amino

acids were synthesized. Quaternary α-amino methyl esters

(+)-34 and (−)-34 were readily obtained according to Seebach

and Mutter procedures³⁵by derivation of their corresponding

chiral synthons 32.³⁶For the tertiary α-amino acids (+)-37 and

(−)-14³⁷(Scheme 7), we chose the Belokon’ procedure,³⁸which

oﬀers a more general and practical access to each kind of

conﬁguration.

Aromatic groups were found in many synthetic or natural

HDACis, suggesting a critical and speciﬁc hydrophobic

interaction involved in their contact with the enzyme external

surface that provides a stabilizing interaction for such

complexes.¹³To evaluate the contribution of hydrophobic

phenyl residues, we synthesized the three novel cyclic building

blocks 47−49 by replacing the existing phenyl group with a

cyclohexyl group or by adding an additional phenyl group at the

Aib position with a ^Dor L conﬁguration (Scheme 8). We chose a

convergent and more modular pathway by coupling dipeptides

39−41 with dipeptides 42 and 43 to give the linear tetrapeptides

44−46. Cyclization of 44 gave 47 in a fair yield of 48%, whereas

48 and 49 were obtained in lower yields of 22% and 36%,

respectively.³⁹The cyclic tetrapeptide building block (+)-17,

having a reverse conﬁguration, was obtained from dipeptides

(+)-39 and (−)-42.

With regard to our previous results, the (R)-hydroxyl ketone

head was chosen as the standard ZBG. The xanthate 8e was

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Scheme 8. Convergent Synthesis of New Cyclic Building Blocks 47−49 and (+)-17 and Their Derivations To Give the Carbonyl

a

ZBG Cyclic Derivatives 50−53

a

Reagents and conditions: (a) LiOH, THF/H₂O; (b) HOBt, DCC, Et₃N, DMF, 18 h; (c) H₂, Pd/C, acetic acid; (d) LiOH, THF/MeOH/H₂O; (e)

HOBt, EDC, N-methylmorpholine, DMF, 18 h; (f) TFA, 0 °C, 3 h; (g) HATU, DIEA, DMF, 18 h; (h) DLP, 1,2-dichloroethane, reﬂux; (i) DLP,

isopropyl alcohol, reﬂux; (j) TBAF, THF.

selectively grafted on 47−49 and (+)-17. Subsequent reductive

xanthate removal and silyl deprotection aﬀorded the new

analogues 50−52 and 53. The cyclic peptide 56,⁴⁰diﬀering

from (−)-17 by only one extra methylene group in the aliphatic

chain, was also obtained according to our strategy (Scheme 9).

This building block gave access to new thioester analogues 57

and 58 with the desired length in the aliphatic linker.^12,13The

corresponding thiol analogue 59 was obtained from 58 by

removal of the acetyl group followed by dithiothreitol (DTT)

reduction of the disulﬁde bond formed by the resulting dimer.³⁴

The enlarged cyclic depsipeptide thioester analogue 60 was

readily obtained by treating 57 with SmI₂.

These new analogues were evaluated on T. gondii RH and HFF

strains (Table 4). The cyclic tetrapeptides 50−52 showed no

inhibition at 90 nM, indicating that the lack (50, entry 1) or the

presence of too many (51 and 52, entries 2 and 3) hydrophobic

aromatic groups disfavors activity against the parasite. A

decreased activity on the HFF strain was also observed. Only a

preferential conﬁguration found in the product 51 retained

signiﬁcant activity on human cells (EC₅₀= 438 nM). The

analogue 53 (entry 4), which has on one side the cyclic

tetrapeptide with a conﬁguration reverse to that of natural

HDCAi and on the other side the most eﬃcient (R)-hydroxyl

ketone ZBG, oﬀers the opportunity to interrogate cell

penetrating mechanisms. An active membrane permeability

process that involves the chiral recognition of FR235222 may

prevent penetration of 53 into cells. This compound was indeed

active at 90 nM on the RH strain, with a good EC₅₀value of 98

nM, and showed a weaker impact on human cells (EC₅₀= 735

nM on the HFF strain). A passive membrane permeability

property by virtue of the cyclic tetrapeptide unit could explain

this cell permeability,¹⁰although a nonchiral selective and active

process cannot be ruled out. From a pharmacodynamic point of

view, this result also pointed out the conﬁguration tolerance for

ligands (substrates) interacting with the enzyme.

With the analogues 57−60, we wanted to check whether new

largazole-like analogues with a longer aliphatic chain could confer

inhibiting properties against T. gondii. Surprisingly, the thioesters

57 and 58 showed no activity on T. gondii at 90 nM while keeping

a good inhibition on human cell proliferation (Table 4, entries 5

and 6). Under these test conditions, the thioester ZBG with the

optimized aliphatic chain length exhibits a reversal of selectivity

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a

Scheme 9.

a

Reagents and conditions: (a) LiOH, THF/H₂O; (b) HOBt, DCC, Et₃N, DMF, 18 h; (c) LiOH, THF/MeOH/H₂O; (d) TFA, 0 °C, 3 h; (e)

HATU, DIEA, DMF, 18 h; (f) AIBN cat., THF, reﬂux, light; (g) NH₃in MeOH, DMF. 67 h; (h) DTT, DMF, 37°C, 24 h; (i) SmI₂, THF/MeOH, 0

°C, 30 min.

Table 4. In Vitro Proliferation Assays with New Analogues

a

b

c

RH = Type 1 T. gondii RH strain. HFF = human foreskin ﬁbroblast. No inhibition eﬀect at 4 μM.

for human cell versus T. gondii parasite. The putative in vivo

active species, the free thiol analogue 59, has no eﬀect on T.

gondii and a weak activity on HFF, very likely due to its instability

in cell medium.

Again, the ring size contribution of the cyclic peptide part can

be evaluated by using 60 as a comparative element. We observed

that 60 has no eﬀect on the RH strain at 90 nM and has lost

activity on HFF (entry 8), demonstrating the critical role of a

more constrained and smaller cyclic peptide structure for

eﬃcient parasitic inhibition. To conﬁrm the reversal of selectivity

for 57 and 59, these products and the most representative

antiparasitic ZBG ketone analogues 1, 21 and 22 were also

evaluated against P. falciparum 3D7 strain, Jurkat cell line, and

erythroblast cells (K-562) (Table 5).

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antiparasitic eﬀect. Future directions to improve the selectivity

between species will mainly explore new modulations on new

positions of the cyclic tetrapeptide scaﬀold to increase the

structural diversity of our analogues. Further analogue

optimizations to better target Apicomplexa and novel synthetic

strategies to increase diversity are now underway.

Table 5

IC₅₀P. falciparum 3D7

CC₅₀h.e. K-

IC₅₀Jurkat cell

line (nM)

entry product

strain (nM)

562 (nM)

1

2

3

4

5

1

7.1

17.4

17

18

48

21

22

57

59

22.1

61

127

40

9450

6200

67

EXPERIMENTAL SECTION

723

763

■

General Considerations. All reagents were used as purchased from

commercial suppliers. Solvents were puriﬁed or dried by conventional

methods prior to use. For reactions performed under anhydrous

conditions, glassware was oven-dried and reactions were performed

under an argon atmosphere. Reactions were monitored by thin-layer

chromatography on precoated aluminum sheets (silica gel 60, F₂₅₄).

Flash column chromatography was performed on silica gel 0.04−0.063

mm (230−400 mesh). ¹H and ¹³C NMR spectra were recorded at room

temperature in deuterated solvents. Chemical shifts δ are given relative

to TMS as internal standard or relative to the solvent ¹³C δ(CDCl₃)

77.23 ppm. Accurate mass measurements (HRMS) were carried out on

a TOF spectrometer.

Once again, for the thioester (57) and thiol (59) analogues

(Table 5, entries 4 and 5), we observed a dramatic decrease in

eﬃciency against Apicomplexa parasite such as Plasmodium

falciparum, whereas the activity remains high (7.1−22.1 nM) for

the analogues 1, 21, and 22 (entries 1−3). Against the Jurkat cell

line and erythroblast cells, the high activity observed with 1, 21,

22, and 57, and to a lesser extent 59, correlates well with the

previous results obtained with HFF (Table 4).

CONCLUSION

■

(−)-(R)-8-[(3S,6R,9S,13S,14aR)-9-Benzyl-6-ethyl-6,13-dimeth-

yl-1,4,7,10-tetraoxotetradecahydropyrrolo[1,2-a][1,4,7,10]-

tetraazacyclododecin-3-yl]-3-oxooctan-2-yl Acetate (1). To

FR235222 (20 mg, 0.036 mmol) in CH₂Cl₂(2 mL) were added acetic

anhydride (5 mg, 0.049 mmol), Et₃N (7.5 mg, 0.072 mmol), and DMAP

(9 mg, 0.072 mmol) at 0 °C. The solution was stirred for 1 h at 0 °C and

16 h at room temperature. EtOAc was added, and the mixture was

washed with a saturated solution of NH₄Cl. The organic layer was dried

(MgSO₄) and was removed in vacuo. After ﬂash chromatography (silica

gel, 2% MeOH/CH₂Cl₂), the product 1 (20 mg, 93%) was isolated as a

colorless ﬁlm: R_f= 0.18 (2% MeOH/CH₂Cl₂); [α]_D²⁰= −87.5° (c = 2.4,

CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3538, 3293, 2929, 2864, 1728,

1680, 1621, 1520, 1433, 1237, 1053 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 0.84 (t, J = 7.4 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 1.25−1.35 (m,

7H), 1.36 (m, 1H), 1.39 (d, J = 7.1 Hz, 3H), 1.53−1.66 (m, 3H), 1.81

(m, 1H), 2.13 (s, 3H), 2.16 (m, 1H), 2.28−2.56 (m, 4H), 2.63 (m, 1H),

2.73 (dd, J = 9.6, 8.1 Hz, 1H), 2.96 (dd, J = 13.5, 6.0 Hz, 1H), 3.24 (dd, J

= 13.5, 9.9 Hz, 1H), 4.06 (dd, J = 9.6, 7.6 Hz, 1H), 4.21 (ddd, J = 10.2,

7.6, 7.6 Hz, 1H), 4.67 (d, J = 6.5 Hz, 1H), 5.08 (q, J = 7.1 Hz, 1H), 5.16

(ddd, J = 10.1, 9.9, 6.0 Hz, 1H), 5.87 (s, 1H, NH), 7.16 (d, J = 10.2 Hz,

1H, NH), 7.19−7.29 (m, 5H), 7.57 (d, J = 10.1 Hz, 1H, NH); ¹³C NMR

(100 MHz, CDCl₃) δ (ppm) 8.6 (CH₃), 16.3 (CH₃), 18.3 (CH₃), 21.0

(CH₃), 22.5 (CH₃), 23.1 (CH₂), 25.5 (CH₂), 28.3 (CH₂), 28.8 (CH₂),

28.9 (CH₂), 33.0 (CH), 33.2 (CH₂), 35.9 (CH₂), 38.2 (CH₂), 53.5

(CH), 54.0 (CH₂), 54.6 (CH), 58.2 (CH), 63.2 (C), 74.8 (CH), 126.9

(CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C), 170.6 (C), 172.0

(C), 173.2 (C), 174.4 (C), 175.8 (C), 207.7 (C); LRMS (ESI+) m/z

(%) 621 (100) [M + Na]⁺, 599 (15); HRMS (ESI+) m/z calcd for

C₃₂H₄₆N₄O₇Na 621.3264, found 621.3257.

The design and discovery of short postmodiﬁcations on a

pluripotent cyclopeptide scaﬀold have given an easy access to

useful and relevant functional and skeletal diversiﬁcations. Such

an approach addresses the issue of extracting maximum

biological information with a limited number of molecules.

The number of synthetic steps is minimized by maintaining

connectivity between each synthetic and bioactive intermediates.

By targeting Apicomplexa inhibition with our assortment of

cyclic peptides, we have gained a better insight into important

molecular features aﬀecting selectivity between species. This

work has conﬁrmed that the (R)-hydroxyl ketone ZBG and its

acetylated analogue connected to a cyclic tetrapeptide backbone

are the most potent and selective ZBGs against T. gondii. In

contrast, the substitution with thioester ZBG located at the

proper distance from the peptide results in high aﬃnity against

human cells, while showing no or little eﬀect on T. gondii RH and

P. falciparum strains. This inversion in selectivity with thioester

ZBG is signiﬁcant and deserves further investigations to better

understand the mechanisms underlying this new phenotype.

During this work, we also focused our attention on the

remarkable properties arising from this natural cyclic tetrapep-

tide scaﬀold. In particular, the strain in this ring system has

revealed new reactivity with SmI₂. We believe that this

constrained preorganized structure is essential for bioactivity,

as the new analogues (30 and 60) arising from this ring

expansion reaction showed a decrease or loss of cell bioactivity in

comparison to their analogues with a 12-membered ring (3 and

57, respectively). This gives rise to questions as to whether the

cell permeability or ligand−protein interaction is aﬀected by this

structural change. Indeed, ligand−protein interactions can also

beneﬁt from the reduced entropic cost of a more constrained

system. Another noteworthy feature of this cyclic peptide scaﬀold

seems to be its good cell permeation, regardless of their

conﬁguration (53 compared to 21), which may also contribute to

the nanomolar eﬃcacy in cell assays. Other structural features

deserve attention as well. From the cyclic tetrapeptide part,

involved in cell permeability and also in ligand−protein

recognition, the benzyl residue on the phenylalanine is beneﬁcial,

as its change to a greatly hindered aliphatic ring (cyclohexyl

group) led to loss of bioactivity (21 versus 50). In contrast, the

presence of too many benzyl groups on the cyclopeptide seems

to be detrimental. We have also conﬁrmed that the methyl group

of the proline found in FR235222 is not necessary for the

(−)-O-(R)-8-[(3S,6R,9S,13S,14aR)-9-Benzyl-6-ethyl-6,13-di-

methyl-1,4,7,10-tetraoxotetradecahydropyrrolo[1,2-a]-

[1,4,7,10]tetraazacyclododecin-3-yl]-3-oxooctan-2-yl O-Phenyl

Carbonothioate (2). Phenyl chlorothiono carbonate (34 mg, 0.2

mmol) was added to a solution of FR235222 (83 mg, 0.15 mmol) in

CH₂Cl₂(2 mL) at 0 °C, followed by DMAP (38 mg, 0.32 mmol). The

mixture was stirred for 1 h at 0 °C and at room temperature for 16 h.

After solvent evaporation, the residue was directly puriﬁed by ﬂash

chromatography (silica gel, 25% EtOAc/cyclohexane) to give 2 (76 mg,

20

74%) as a colorless ﬁlm: R_f= 0.15 (30% EtOAc/cyclohexane); [α]_D

=

−71.9° (c = 1.1, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3303, 2924, 1735,

1683, 1662, 1627, 1525, 1274, 1222, 1207 cm⁻¹; ¹H NMR (400 MHz,

CDCl₃) δ (ppm) 0.83 (t, J = 7.4 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 1.25−

1.50 (m, 8H), 1.57 (d, J = 7.2 Hz, 3H), 1.59−1.71 (m, 3H), 1.80 (m,

1H), 2.15 (m, 1H), 2.31 (m, 1H), 2.37 (m, 1H), 2.45−2.77 (m, 3H),

2.73 (dd, J = 9.6, 8.0 Hz, 1H), 2.96 (dd, J = 13.5, 6.0 Hz, 1H), 3.24 (dd, J

= 13.5, 9.9 Hz, 1H), 4.06 (dd, J = 9.6, 7.6 Hz, 1H), 4.19 (ddd, J = 10.0,

7.6, 7.6 Hz, 1H), 4.66 (dd, J = 7.8, 1.6 Hz, 1H), 5.16 (ddd, J = 10.1, 9.9,

6.0 Hz, 1H), 5.56 (q, J = 7.2 Hz, 1H), 5.78 (s, 1H, NH), 7.13−7.34 (m,

9H, NH), 7.43 (dd, J = 7.8, 7.8 Hz, 2H), 7.54 (d, J = 10.1 Hz, 1H, NH);

3664

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Article

¹³C NMR (100 MHz, CDCl₃) δ ppm 8.6 (CH₃), 16.6 (CH₃), 18.3

(−)-(R)-S-3-(tert-Butyldimethylsilyloxy)-2-oxobutyl O-ethyl car-

bonodithioate (8e): Product 8e was puriﬁed by ﬂash chromatography

(5% ether/cyclohexane): 96% yield (pale yellow oil); R_f= 0.25 (4%

ether/cyclohexane); [α]_D²⁰= −7.3° (c = 1.15, CHCl₃); IR ν_max(ﬁlm)

(CH₃), 22.6 (CH₃), 23.0 (CH₂), 25.6 (CH₂), 28.0 (CH₂), 28.9 (CH₂),

29.0 (CH₂), 33.1 (CH), 33.2 (CH₂), 35.9 (CH₂), 38.4 (CH₂), 53.6

(CH), 54.1 (CH₂), 54.6 (CH), 58.2 (CH), 63.2 (C), 83.4 (CH), 122.0

(2 × CH), 126.9 (CH), 127.0 (CH), 128.8 (2 × CH), 129.3 (2 × CH),

129.8 (2 × CH), 137.3 (C), 153.6 (C), 172.1 (C), 173.3 (C), 174.4 (C),

175.9 (C), 194.8 (C), 206.7 (C); LRMS (ESI+) m/z (%) 715 (100) [M

+ Na]⁺, 693 (25) [M + H]⁺; HRMS (ESI+) m/z calcd for

C₃₇H₄₈N₄O₇NaS 715.3141, found 715.3154.

(−)-(3S,6R,9S,13S,14aR)-9-Benzyl-6-ethyl-6,13-dimethyl-3-

(6-oxooctyl)decahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (3). To the product 2 (4.4 mg, 6.4

μmol) in a degassed solution of toluene (1 mL) were added nBu₃SnH

(5.5 mg, 19 μmol) and AIBN (catalytic). The mixture was warmed at

100 °C under argon for 3 h. The solvent was removed in vacuo, and the

residue was directly puriﬁed by ﬂash chromatography (10% KF w/w in

1

2955, 2931, 1735, 1728, 1363, 1224, 1112, 1051, 836 cm⁻¹; H NMR

(400 MHz, CDCl₃) δ (ppm) 0.13 (s, 3H), 0.14 (s, 3H), 0.95 (s, 9H),

1.38 (d, J = 6.8 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H), 4.29 (d, J = 17.9 Hz,

1H), 4.34 (q, J = 6.8 Hz, 1H), 4.35 (d, J = 17.9 Hz, 1H), 4.62 (q, J = 7.1

Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) −4.9 (CH₃), −4.4

(CH₃), 13.9 (CH₃), 18.1 (C), 21.0 (CH₃), 25.9 (2 × CH₃), 42.8 (CH₂),

70.6 (CH₂), 75.0 (CH), 205.7 (C), 213.6 (C); LRMS (ESI+) m/z (%)

345 (100) [M + Na]⁺, 323 (5); HRMS (ESI+) m/z calcd for

C₁₃H₂₆O₃NaSiS₂345.0990, found 345.0987.

(+)-(S)-S-3-(tert-Butyldimethylsilyloxy)-2-oxobutyl O-ethyl car-

bonodithioate (8f): Product 8f was puriﬁed by ﬂash chromatography

(5% ether/cyclohexane), 94% yield (pale yellow oil): R_f= 0.25 (4%

20

ether/cyclohexane); [α]_D= +7.4° (c = 1.2, CHCl₃); IR ν_max(ﬁlm)

silica gel, 30% EtOAc/cyclohexane) to give the product 3 (2.3 mg, 67%)

as a colorless ﬁlm: R_f= 0.12 (30% EtOAc/cyclohexane); [α]_D

=

2960, 2930, 1735, 1725, 1363, 1232, 1110, 1048, 835 cm⁻¹; H NMR

20

1

−117.6° (c = 1.3, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3307, 2925, 1714,

(400 MHz, CDCl₃) δ (ppm) 0.13 (s, 3H), 0.14 (s, 3H), 0.95 (s, 9H),

1.38 (d, J = 6.8 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H), 4.29 (d, J = 17.9 Hz,

1H), 4.34 (q, J = 6.8 Hz, 1H), 4.35 (d, J = 17.9 Hz, 1H), 4.62 (q, J = 7.1

Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) −4.9 (CH₃), −4.4

(CH₃), 13.9 (CH₃), 18.1 (C), 21.0 (CH₃), 25.9 (2 × CH₃), 42.8 (CH₂),

70.6 (CH₂), 75.0 (CH), 205.7 (C), 213.6 (C); LRMS (ESI+) m/z (%)

345 (100) [M + Na]⁺, 323 (8); HRMS (ESI+) m/z calcd for

C₁₃H₂₆O₃NaSiS₂345.0990, found 345.0989.

1

1682, 1662, 1628, 1524, 1456, 1378, 1248, 914 cm⁻¹; H NMR (400

MHz, CDCl₃) δ (ppm) 0.84 (t, J = 7.4 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H),

1.05 (t, J = 7.3 Hz, 3H), 1.23−1.42 (m, 8H), 1.52−1.65 (m, 3H), 1.80

(m, 1H), 2.15 (m, 1H), 2.27−2.46 (m, 6H), 2.62 (m, 1H), 2.73 (dd, J =

9.7, 8.0 Hz, 1H), 2.96 (dd, J = 13.5, 6.0 Hz, 1H), 3.24 (dd, J = 13.5, 9.9

Hz, 1H), 4.06 (dd, J = 9.7, 7.6 Hz, 1H), 4.19 (ddd, J = 10.2, 7.6, 7.6 Hz,

1H), 4.67 (dd, J = 8.0, 2.0 Hz, 1H), 5.16 (ddd, J = 10.1, 9.9, 6.0 Hz, 1H),

5.77 (s, 1H, NH), 7.14 (d, J = 10.2 Hz, 1H, NH), 7.18−7.32 (m, 5H),

7.54 (d, J = 10.1 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

8.1 (CH₃), 8.6 (CH₃), 18.3 (CH₃), 22.6 (CH₃), 23.8 (CH₂), 25.6

(CH₂), 28.1 (CH₂), 29.0 (CH₂), 29.1 (CH₂), 33.1 (CH), 33.3 (CH₂),

35.9 (CH₂), 36.1 (CH₂), 42.4 (CH₂), 53.6 (CH), 54.1 (CH₂), 54.6

(CH), 58.2 (CH), 63.3 (C), 126.9 (CH), 128.8 (2 × CH), 129.3 (2 ×

CH), 137.3 (C), 172.1 (C), 173.2 (C), 174.4 (C), 175.9 (C), 211.9 (C);

LRMS (ESI+) m/z (%) 563 (100) [M + Na]⁺; HRMS (ESI+) m/z calcd

for C₃₀H₄₄N₄O₅Na 563.3209, found 563.3209.

General Procedure for 8: Example with Benzyl 2-(Ethoxy-

carbonothioylthio)acetate (8b). To a solution of benzyl 2-

chloroacetate 18b (2.76 g, 15 mmol) in acetone (50 mL) was added

at 0 °C a solution of potassium ethyl xanthate (2.60 g, 16 mmol) in

acetone (50 mL). The mixture was stirred for 4 h at room temperature.

The organic solvent was removed in vacuo and CH₂Cl₂was added. The

organic layer was washed with brine, dried (MgSO₄) and then

evaporated. After Kugelrohr distillation (13 mbar, 180−200 °C), the

product 8b (2.74 g, 68%) was isolated as a pale yellow oil (for 8e and 8f,

puriﬁcation was performed by ﬂash chromatography): R_f= 0.21 (5%

EtOAc/cyclohexane); IR ν_max(ﬁlm) 2982, 1740, 1615, 1498, 1455,

1376, 1232, 1150 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.35 (t, J

= 7.1 Hz, 3H), 3.93 (s, 2H), 4.58 (q, J = 7.1 Hz, 2H), 5.18 (s, 2H), 7.32−

7.36 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 13.7 (CH₃), 37.9

(CH₂), 67.6 (CH₂), 70.8 (CH₂), 128.5 (CH), 128.6 (2 × CH), 128.7 (2

× CH), 135.4 (C), 167.9 (C), 212.4 (C); LRMS (ESI+) m/z (%) 293

(100) [M + Na]⁺; HRMS (ESI+) m/z calcd for C₁₂H₁₄O₃NaS₂

293.0282, found 293.0281.

O-Ethyl S-2-Oxobutyl carbonodithioate (8c): Product 8c was

puriﬁed by distillation (Kugelrohr, 2.2 mbar, 100 °C): 63% yield (pale

yellow oil); IR ν_max(ﬁlm) 2980, 1729, 1717, 1457, 1376, 1224, 1113,

1052 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.11 (t, J = 7.1 Hz,

3H), 1.42 (t, J = 7.1 Hz, 3H), 2.65 (q, J = 7.1 Hz, 2H), 4.00 (s, 2H), 4.63

(q, J = 7.1 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 7.9 (CH₃),

13.9 (CH₃), 35.4 (CH₂), 45.3 (CH₂), 71.0 (CH₂), 204.0 (C), 213.5 (C);

LRMS (ESI+) m/z (%) 215 (100) [M + Na]⁺.

Ethyl 4-(ethoxycarbonothioylthio)-3-oxobutanoate (8d): Product

8d was puriﬁed by distillation (Kugelrohr, 2.2 mbar, 150 °C): 67% yield

(pale yellow oil); IR ν_max(ﬁlm) 2983, 1745, 1616, 1376, 1225, 1113,

1048 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.30 (t, J = 7.1 Hz,

3H), 1.42 (t, J = 7.1 Hz, 3H), 3.65 (s, 2H), 4.12 (s, 2H), 4.20 (q, J = 7.1

Hz, 2H), 4.64 (q, J = 7.1 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ

(ppm) 13.9 (CH₃), 14.3 (CH₃), 45.6 (CH₂), 48.3 (CH₂), 61.8 (CH₂),

71.2 (CH₂), 166.9 (C), 196.3 (C), 213.0 (C); LRMS (ESI+) m/z (%)

273 (100) [M + Na]⁺.

(+)-(S)-Methyl 2-(tert-butoxycarbonylamino)hex-5-enoate

((+)-14). Into zinc powder (2.48 g, 37.9 mmol) in dry DMF (28 mL)

was injected 1,2-dibromoethane (0.17 mL) under argon. The mixture

was stirred for 20 min at room temperature. TMSCl (50 μL) was

injected, and the solution was stirred at 60 °C for 30 min. (R)-Methyl 2-

(tert-butoxycarbonylamino)-3-iodopropanoate⁴¹(2.0 g, 6.08 mmol) in

DMF (8 mL) was added dropwise. The solution was stirred for 20 min at

60 °C. LiCl (587 mg, 13.8 mmol) and CuCN (619 mg, 6.9 mmol) in

DMF (6.5 mL) were injected at −55 °C, and the mixture was warmed

with stirring for 10 min at 0 °C. The solution was cooled again to −55

°C, and allyl bromide (1.05 mL, 12 mmol) was injected. After 5 min, the

mixture was warmed to 0 °C and then stirred for 2 h at this temperature.

The unreacted zinc was removed by Celite ﬁltration, and the ﬁltrate was

quenched with a saturated aqueous NH₄Cl solution. The product was

extracted with EtOAc. The combined organic layer was washed with

brine, dried (MgSO₄), and evaporated. After ﬂash chromatography

(silica gel, 8% EtOAC/cyclohexane), the product (+)-14 (1.38 g, 93%)

was isolated as a colorless oil: R_f= 0.25 (10% EtOAc/cyclohexane);

[α]_D²⁰= +20.5° (c = 1.0, CHCl₃); IR ν_max(ﬁlm, CH₂Cl₂) 3358, 2978,

1

1745, 1716, 1518, 1453, 1366, 1249, 1163, 914 cm⁻¹; H NMR (400

MHz, CDCl₃) δ (ppm) 1.45 (s, 9H), 1.74 (m, 1H), 1.90 (m, 1H), 2.12

(m, 2H), 3.74 (s, 3H), 4.32 (m, 1H), 5.00 (bd, J = 10.3 Hz, 1H), 5.05

(bd, J = 17.0 Hz, 1H), 5.18 (bd, J = 7.2 Hz, 1H, NH), 5.79 (m, 1H); ¹³C

NMR (100 MHz, CDCl₃) δ (ppm) 28.4 (3 × CH₃), 29.6 (CH₂), 32.0

(CH₂), 52.3 (CH₃), 53.0 (CH), 79.8 (C), 115.7 (CH₂), 137.0 (CH),

155.4 (C), 173.4 (C); LRMS (ESI+) m/z (%) 266 (100) [M + Na]⁺,

210 (43), 166 (38); HRMS (ESI+) m/z calcd for C₁₂H₂₁NO₄Na

266.1368, found 266.1374.

(−)-(3S,9S,14aR)-9-Benzyl-3-(but-3-enyl)-6,6-dimethyldeca-

hydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,10-

tetraone ((−)-17). To a cooled solution of tripeptide 15 (H-Aib-^L-Phe-

D-Pro-O-t-Bu; 1.38 g, 3.42 mmol) and methyl (S)-2-(benzyloxycarbo-

nylamino)-5-hexenoate ((+)-14; 784 mg, 3.42 mmol) in dry DMF (7

mL) were added HOBt·H₂O (525 mg, 3.89 mmol), DCC (850 mg, 3.8

mmol), and triethylamine (0.5 mL), and the mixture was stirred at room

temperature overnight (18 h). DMF was evaporated, and the residue

was diluted with EtOAc and washed with 10% citric acid, 4% sodium

carbonate, and brine. The organic phase was dried (MgSO₄) and

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

1.5% MeOH/CH₂Cl₂) to give the corresponding tetrapeptide 16 (1.58

g, 75%) as a colorless ﬁlm: R_f= 0.09 (2% MeOH/CH₂Cl₂); LRMS (ESI

+) m/z (%) 637 (100) [M + Na]⁺, 615 (20) [M + H]⁺; HRMS (ESI+)

m/z calcd for C₃₃H₅₀N₄O₇Na 637.3577, found 637.3567. This

tetrapeptide (1.57 g, 2.55 mmol) was dissolved in TFA (7 mL) at 0

3665

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The Journal of Organic Chemistry

Article

°C and stirred for 3 h at this temperature. After evaporation, the product

was precipitated in dry ether to give after ﬁltration the deprotected

product (1.08 g, 74%) as a TFA salt. To a solution of this tetrapeptide

triﬂuoroacetic acid (900 mg, 1.57 mmol) in DMF (140 mL) were added

HATU (666 mg, 1.75 mmol) and DIEA (0.81 mL) in ﬁve aliquots with a

time interval of 30 min under vigorous stirring. Then the reaction

mixture was stirred for 1 h at room temperature. The DMF was

evaporated, and EtOAc was added. After it was washed with a solution of

10% citric acid followed by 4% NaHCO₃, the organic phase was dried

(MgSO₄) and evaporated. After ﬂash chromatography (silica gel, 1%

(CH₃), −4.5 (CH₃), 14.0 (CH₃), 18.2 (C), 21.1 (CH₃), 23.7 (CH₃),

24.9 (CH₂), 25.2 (CH₂), 25.9 (3 × CH₃), 26.4 (CH₂), 26.7 (CH₂), 27.6

(CH₂), 31.4 (CH₂), 34.5 (CH₂), 36.0 (CH₂), 47.2 (CH₂), 51.1 (CH),

53.6 (CH), 54.3 (CH), 58.0 (CH), 59.0 (C), 70.2 (CH₂), 75.0 (CH),

126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C), 172.1 (C),

173.0 (C), 174.1 (C), 175.8 (C), 213.5 (C), 214.4 (C); LRMS (ESI+)

m/z (%) 785 (90) [M + Na]⁺, 763 (70), 413 (100); HRMS (ESI+) m/z

calcd for C₃₇H₅₈N₄O₇NaSiS₂785.3414, found 785.3407.

General Method To Obtain 20 from 19. Method B. Example

with (−)-(3S,9S,14aR)-9-Benzyl-3-(7-chloro-6-oxoheptyl)-6,6-

dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-

1,4,7,10-tetraone (20a). A solution of product 19a (12 mg, 0.018

mmol) in isopropyl alcohol (1 mL) was reﬂuxed for 5 h with DLP (11

mg, 0.027 mmol). After evaporation and direct ﬂash chromatography

(silica gel, 30% EtOAc/cyclohexane), the product 20a (9 mg, 92%) was

MeOH/CH₂Cl₂), the cyclotetrapeptide (−)-17 (512 mg, 74%) was

20

isolated as a white foam: R_f= 0.17 (40% EtOAc/cyclohexane); [α]_D

=

−110.0° (c = 0.84, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3303, 2930,

1

1678, 1663, 1630, 1663, 1529, 1427, 1251, 1181, 913 cm⁻¹; H NMR

(400 MHz, CDCl₃) δ (ppm) 1.31 (s, 3H), 1.67−1.82 (m, 6H), 1.92 (m,

1H), 2.09 (m, 2H), 2.16 (m, 1H), 2.30 (m, 1H), 2.94 (dd, J = 13.5, 5.8

Hz, 1H), 3.19−3.28 (m, 2H), 3.86 (m, 1H), 4.30 (ddd, J = 10.1, 7.5, 7.5

Hz, 1H), 4.66 (m, 1H), 4.97 (m, 1H), 5.00 (m, 1H), 5.17 (ddd, J = 10.1,

10.0, 5.8 Hz, 1H), 5.78 (ddt, J = 16.8, 10.2, 6.6 Hz, 1H), 6.18 (s, 1H,

NH), 7.16−7.28 (m, 6H, NH), 7.61 (d, J = 10.1 Hz, 1H, NH); ¹³C NMR

(100 MHz, CDCl₃) δ (ppm) 23.4 (CH₃), 24.6 (CH₂), 24.8 (CH₂), 26.3

(CH₃), 28.2 (CH₂), 29.6 (CH₂), 35.7 (CH₂), 46.7 (CH₂), 53.2 (CH),

53.7 (CH), 57.6 (CH), 58.5 (C), 115.5 (CH₂), 126.5 (CH), 128.4 (2 ×

CH), 128.9 (2 × CH), 136.9 (C), 136.9 (CH), 171.5 (C), 172.6 (C),

174.2 (C), 175.4 (C); LRMS (ESI+) m/z (%) 463 (100) [M + Na]⁺;

HRMS (ESI+) m/z calcd for C₂₄H₃₂N₄O₄Na 463.2321, found

463.2316.

20

obtained as a colorless ﬁlm: R_f= 0.07 (40% EtOAc/cyclohexane); [α]_D

= −80° (c = 1.0, CHCl₃); IR ν_max(ﬁlm) 3304, 2928, 1733, 1678, 1663,

1

1628, 1525, 1434, 1390, 1179 cm⁻¹; H NMR (400 MHz, CDCl₃) δ

(ppm) 1.29−1.34 (m, 7H), 1.63 (m, 2H), 1.69−1.87 (m, 7H), 2.17 (m,

1H), 2.32 (m, 1H), 2.56 (t, J = 7.3 Hz, 2H), 2.95 (dd, J = 13.5, 5.7 Hz,

1H), 3.21 (dd, J = 10.0, 7.0 Hz, 1H), 3.26 (dd, J = 13.5, 10.0 Hz, 1H),

3.86 (m, 1H), 4.07 (s, 2H), 4.19 (ddd, J = 10.2, 7.6, 7.6 Hz, 1H), 4.67 (m,

1H), 5.16 (ddd, J = 10.1, 10.0, 5.7 Hz, 1H), 5.95 (s, 1H, NH), 7.11 (bd, J

= 10.1 Hz, 1H, NH), 7.19−7.29 (m, 5H), 7.51 (bd, J = 10.1 Hz, 1H,

NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.4 (CH₂), 23.7 (CH₃),

24.9 (CH₂), 25.2 (CH₂), 25.4 (CH₂), 26.7 (CH₃), 28.8 (CH₂), 28.9

(CH₂), 36.0 (CH₂), 39.6 (CH₂), 47.1 (CH₂), 48.3 (CH₂), 53.6 (CH),

54.4 (CH), 57.9 (CH), 59.0 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2

× CH), 137.2 (C), 172.0 (C), 173.0 (C), 175.0 (C), 175.8 (C), 202.7

(C); LRMS (ESI+) m/z (%) 555 (100) [M + Na]⁺, 533 (10); HRMS

(ESI+) m/z calcd for C₂₇H₃₇N₄O₅ClNa 555.2350, found 555.2343.

Benzyl 6-[(3S,9S,14aR)-9-benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecin-3-

yl]-4-(ethoxycarbonothioylthio)hexanoate (19b). According to meth-

od A (8 h reﬂux) and starting from (−)-17 (100 mg, 0.23 mmol) and

xanthate 8b (92 mg, 0.34 mmol), the product 19b (145 mg, 90%) was

obtained as a colorless ﬁlm (ﬂash chromatography: 1% MeOH/

CH₂Cl₂): R_f= 0.06 (1% MeOH/CH₂Cl₂); IR ν_max(ﬁlm) 3308, 2940,

1734, 1681, 1663, 1629, 1525, 1454, 1436, 1215, 1111, 1049, 910 cm⁻¹;

¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.34 (s, 3H), 1.41 (t, J = 7.1 Hz,

General Method To Obtain 19. Method A. Example with (S)-{1-

[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetraoxotetradeca-

hydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecin-3-yl]-7-chloro-6-

oxoheptan-3-yl} O-Ethyl Carbonodithioate (19a). A solution of

cyclotetrapeptide (−)-17 (75 mg, 0.17 mmol) and carbonyl xanthate 2a

(72 mg, 0.34 mmol) in 1,2-dichloroethane (0.3 mL) was reﬂuxed for 30

min under argon. Dilauroyl peroxide (7 mg) was added every 2 h (ﬁve to

seven times). After completion (8−18 h at reﬂux), the solvent was

removed in vacuo and the residue was directly puriﬁed by ﬂash

chromatography (silica gel, 40% EtOAc/cyclohexane for 19a) to give

19a (72 mg, 65%) as a colorless ﬁlm: R_f= 0.16 (40% EtOAc/

cyclohexane); IR ν_max(ﬁlm) 3302, 2925, 1734, 1678, 1663, 1628, 1525,

1

1435, 1390, 1217, 1111, 1048 cm⁻¹; H NMR (400 MHz, CDCl₃) δ

3H), 1.65−1.81 (m, 8H), 1.86−2.00 (m, 2H), 2.05−2.26 (m, 2H), 2.32

(m, 1H), 2.52 (m, 2H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.23 (m, 1H),

3.26 (dd, J = 13.5, 10.1 Hz, 1H), 3.79 (m, 1H), 3.86 (m, 1H), 4.21 (m,

1H), 4.62 (q, J = 7.1 Hz, 2H), 4.66 (m, 1H), 5.12 (s, 2H), 5.17 (m, 1H),

5.92 (s, 1H, NH), 7.13 (bd, J = 10.2 Hz, 1H, NH), 7.19−7.39 (m, 10H),

7.47 (bd, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

14.0 (CH₃), 23.7 (CH₃), 24.9 (CH₂), 25.2 (CH₂), 26.4 (CH₂), 26.7

(CH₃), 29.5 (CH₂), 30.8 (CH₂), 31.7 (CH₂), 36.0 (CH₂), 47.2 (CH₂),

50.6 (CH), 53.6 (CH), 54.3 (CH), 58.0 (CH), 59.0 (C), 66.7 (CH₂),

70.3 (CH₂), 126.9 (CH), 128.5 (2 × CH), 128.8 (2 × CH), 128.9 (2 ×

CH), 129.2 (3 × CH), 136.0 (C), 137.2 (C), 172.1 (C), 172.9 (C),

173.0 (C), 174.1 (C), 175.8 (C), 214.1 (C); LRMS (ESI+) m/z (%) 733

(100) [M + Na]⁺, 711 (46), 463 (25); HRMS (ESI+) m/z calcd for

C₃₆H₄₆N₄O₇NaS₂733.2706, found 733.2709.

(ppm) 1.35 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H), 1.62−1.96 (m, 10H), 2.07−

2.23 (m, 2H), 2.32 (m, 1H), 2.76 (m, 2H), 2.95 (dd, J = 13.5, 5.7 Hz,

1H), 3.23 (m, 1H), 3.26 (dd, J = 13.5, 10.2 Hz, 1H), 3.76 (m, 1H), 3.86

(m, 1H), 4.08 (s, 2H), 4.22 (m, 1H), 4.65 (q, J = 7.1 Hz, 2H), 4.66 (m,

1H), 5.17 (m, 1H), 5.92 (s, 1H, NH), 7.15 (bd, J = 10.1 Hz, 1H, NH),

7.19−7.32 (m, 5H), 7.47 (bd, J = 10.1 Hz, 1H, NH); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 14.0 (CH₃), 23.7 (CH₃), 24.9 (CH₂), 25.2

(CH₂), 26.7 (CH₃), 28.0 (CH₂), 29.9 (CH₂), 31.1 (CH₂), 36.0 (CH₂),

37.0 (CH₂), 47.2 (CH₂), 48.4 (CH₂), 50.7 (CH), 53.6 (CH), 54.3

(CH), 58.0 (CH), 59.0 (C), 70.5 (CH₂), 126.9 (CH), 128.8 (2 × CH),

129.2 (2 × CH), 137.2 (C), 172.1 (C), 173.1 (C), 174.1 (C),175.7 (C),

202.0 (C), 214.3 (C); LRMS (ESI+) m/z (%) 675 (100) [M + Na]⁺,

653 (32); HRMS (ESI+) m/z calcd for C₃₀H₄₁N₄O₆ClNaS₂675.2054,

found 675.2053.

S-(R)-1-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetraoxo-

tetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecin-3-yl]-

7-(tert-butyldimethylsilyloxy)-6-oxo-octan-3-yl O-ethyl carbonodi-

thioate (19e). According to method A and starting from (−)-17 (60 mg,

0.14 mmol) and xanthate 8e (110 mg, 0.34 mmol), the product 19e

(100 mg, 96%), was obtained as a colorless ﬁlm (ﬂash chromatography:

0.8% MeOH/CH₂Cl₂): R_f= 0.10 (1% MeOH/CH₂Cl₂); IR ν_max(thin

ﬁlm, CH₂Cl₂) 3474, 3295, 2920, 2855, 1712, 1666, 1628, 1525, 1438,

1251, 1111 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.07 (s, 6H),

0.91 (s, 9H), 1.28 (m, 3H), 1.34 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.62−

1.88 (m, 9H), 1.92−2.11 (m, 2H), 2.18 (m, 1H), 2.33 (m, 1H), 2.76 (m,

2H), 2.96 (dd, J = 13.5, 5.7 Hz, 1H), 3.23 (m, 1H), 3.27 (dd, J = 13.5,

10.1 Hz, 1H), 3.77 (m, 1H), 3.86 (m, 1H), 4.14 (q, J = 6.7 Hz, 1H), 4.22

(m, 1H), 4.63 (q, J = 7.1 Hz, 2H), 4.66 (m, 1H), 5.17 (m, 1H), 5.96 (s,

1H, NH), 7.15 (d, J = 10.1 Hz, 1H, NH), 7.18−7.30 (m, 5H), 7.51 (bd, J

= 10.1 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) −4.8

S-(S)-1-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetraoxo-

tetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecin-3-yl]-

7-(tert-butyldimethylsilyloxy)-6-oxo-octan-3-yl O-ethyl carbono-

dithioate (19f). According to method A and starting from (−)-17 (80

mg, 0.18 mmol) and xanthate 8f (145 mg, 0.45 mmol), the product 19f

(126 mg, 95%) was obtained as a colorless ﬁlm (ﬂash chromatography:

0.8% MeOH/CH₂Cl₂): R_f= 0.1 (1% MeOH/CH₂Cl₂); IR ν_max(thin

ﬁlm, CH₂Cl₂) 3463, 3305, 2955, 2932, 1715, 1671, 1625, 1522, 1444,

1

1257, 1216, 1111, 1047, 837 cm⁻¹; H NMR (400 MHz, CDCl₃) δ

(ppm) 0.07 (s, 6H), 0.91 (s, 9H), 1.26 (m, 3H), 1.35 (s, 3H), 1.42 (t, J =

7.1 Hz, 3H), 1.60−1.86 (m, 9H), 1.90−2.08 (m, 2H), 2.17 (m, 1H),

2.33 (m, 1H), 2.76 (m, 2H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.23 (m,

1H), 3.26 (dd, J = 13.5, 10.1 Hz, 1H), 3.77 (m, 1H), 3.86 (m, 1H), 4.13

(q, J = 6.7 Hz, 1H), 4.24 (m, 1H), 4.63 (q, J = 7.1 Hz, 2H), 4.67 (m, 1H),

5.17 (m, 1H), 6.00 (bs, 1H, NH), 7.15 (d, J = 10.1 Hz, 1H, NH), 7.18−

7.30 (m, 5H), 7.50 (bd, J = 10.1 Hz, 1H, NH); ¹³C NMR (100 MHz,

CDCl₃) δ (ppm) −4.9 (CH₃), −4.5 (CH₃), 14.0 (CH₃), 18.2 (C), 21.1

3666

dx.doi.org/10.1021/jo4001492 | J. Org. Chem. 2013, 78, 3655−3675

The Journal of Organic Chemistry

Article

(CH₃), 23.7 (CH₃), 24.9 (CH₂), 25.2 (CH₂), 25.9 (3 × CH₃), 26.4

(CH₂), 26.6 (CH₃), 27.6 (CH₂), 31.2 (CH₂), 34.4 (CH₂), 36.0 (CH₂),

47.1 (CH₂), 51.0 (CH), 53.6 (CH), 54.5 (CH), 58.0 (CH), 59.0 (C),

70.2 (CH₂), 75.0 (CH), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH),

137.2 (C), 172.0 (C), 173.0 (C), 174.1 (C), 175.8 (C), 213.6 (C), 214.3

(C); LRMS (ESI+) m/z (%) 785 (100) [M + Na]⁺, 763 (30); HRMS

(ESI+) m/z calcd for C₃₇H₅₈N₄O₇NaSiS₂785.3414, found 785.3431.

(−)-Benzyl 6-[(3S,9S,14aR)-9-benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecin-3-

yl]hexanoate (20b). According to method B and starting from 19b (124

mg, 0.17 mmol), the product 20b (69 mg, 69%) was obtained as a

colorless ﬁlm (ﬂash chromatography: silica gel, 1% MeOH/CH₂Cl₂): R_f

= 0.17 (40% EtOAc/cyclohexane); [α]_D²⁰= −75.0° (c = 1.4, CHCl₃); IR

ν_max(ﬁlm) 3309, 2934, 1735, 1678, 1663, 1629, 1525, 1424, 1257, 1173

cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.25−1.40 (m, 7H), 1.65

(m, 2H), 1.72 (m, 1H), 1.77−1.87 (m, 6H), 2.17 (m, 1H), 2.29 (m, 1H),

2.35 (t, J = 7.5 Hz, 2H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.19−3.29 (m,

2H), 3.86 (ddd, J = 10.3, 8.6, 4.6 Hz, 1H), 4.18 (ddd, J = 10.2, 7.6, 7.6 Hz,

1H), 4.65 (m, 1H), 5.11 (s, 2H), 5.16 (ddd, J = 10.2, 10.1, 5.7, 1H), 5.90

(s, 1H, NH), 7.09 (bd, J = 10.2 Hz, 1H, NH), 7.17−7.40 (m, 10H), 7.51

(bd, J = 10.3 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.7

(CH₃), 24.86 (CH₂), 24.93 (CH₂), 25.2 (CH₂), 25.4 (CH₂), 26.7

(CH₃), 28.9 (2 × CH₂), 34.3 (CH₂), 36.0 (CH₂), 47.2 (CH₂), 53.6

(CH), 54.5 (CH), 58.0 (CH), 59.0 (C), 66.3 (CH₂), 126.9 (CH), 128.4

(3 × CH), 128.7 (2 × CH), 128.8 (2 × CH), 129.2 (2 × CH), 136.2 (C),

137.2 (C), 172.0 (C), 173.0 (C), 173.6 (C), 174.5 (C), 175.8 (C);

LRMS (ESI+) m/z (%) 613 (100) [M + Na]⁺, 591 (18); HRMS (ESI+)

m/z calcd for C₃₃H₄₂N₄O₆Na 613.3002, found 613.2991.

58.9 (C), 75.1 (CH), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH),

137.2 (C), 172.0 (C), 173.0 (C), 174.5 (C), 175.8 (C), 214.3 (C);

LRMS (ESI+) m/z (%) 665 (100) [M + Na]⁺, 643 (30); HRMS (ESI+)

m/z calcd for C₃₄H₅₄N₄O₆NaS_i665.3710, found 665.3705.

General Method To Obtain 20 from (−)-17. Method C. Example

with 20e. A solution of cyclotetrapeptide (−)-17 (31 mg, 0.07 mmol)

and carbonyl xanthate 8e (45 mg, 0.14 mmol) in 1,2-dichloroethane (0.2

mL) was reﬂuxed for 30 min under argon. Dilauroyl peroxide (3 mg)

was added every 2 h (ﬁve to seven times). After completion (18 h

reﬂux), the solvent was removed in vacuo and isopropyl alcohol (1.5

mL) was added. After the mixture was reﬂuxed for 30 min under argon,

DLP (28 mg, 0.07 mmol) was added and the solution was reﬂuxed for 1

h. Additional DLP (14 mg, 0.035 mmol) was then added again, and the

solution was reﬂuxed for 3 h. After evaporation, the residue was directly

puriﬁed by ﬂash chromatography (silica gel, 25% EtOAc/cyclohexane)

to give the product 20e (22 mg, 48%).

(−)-(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-3-(6-oxooctyl)deca-

hydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,10-tet-

raone (20c). According to method C and starting from (−)-17, product

20c was obtained after ﬂash chromatography (silica gel, 30% EtOAc/

cyclohexane) as a colorless ﬁlm: 50% yield; R_f= 0.1 (40% EtOAc/

cyclohexane); [α]_D²⁰= −65.0° (c = 0.47, CHCl₃); IR ν_max(ﬁlm) 2923,

1716, 1683, 1540, 1522, 1457, 1177 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 1.05 (t, J = 7.3 Hz, 3H), 1.25−1.34 (m, 4H), 1.34 (s, 3H),

1.51−1.70 (m, 4H), 1.77 (m, 2H), 1.77 (s, 3H), 2.18 (m, 1H), 2.32 (m,

1H), 2.39 (t, J = 7.3 Hz, 2H), 2.41 (q, J = 7.3 Hz, 2H), 2.95 (dd, J = 13.5,

5.7 Hz, 1H), 3.22 (m, 1H), 3.26 (dd, J = 13.5, 9.9 Hz, 1H), 3.86 (m, 1H),

4.18 (m, 1H), 4.66 (bd, J = 8.0 Hz, 1H), 5.16 (ddd, J = 10.1, 10.1, 5.8 Hz,

1H), 5.89 (s, 1H, NH), 7.08 (d, J = 10.2 Hz, 1H, NH), 7.18−7.31 (m,

5H), 7.51 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ

(ppm) 8.1 (CH₃), 23.8 (CH₃), 25.0 (CH₂), 25.2 (CH₂), 25.6 (CH₂),

26.7 (CH₃), 29.0 (CH₂), 29.1 (CH₂), 29.9 (CH₂), 36.0 (CH₂), 36.1

(CH₂), 42.4 (CH₂), 47.2 (CH₂), 53.6 (CH), 54.5 (CH), 58.0 (CH),

59.0 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C),

172.0 (C), 173.0 (C), 174.6 (C), 175.9 (C), 211.8 (C); LRMS (ESI+)

m/z (%) 535 (100) [M + Na]⁺, 437 (33); HRMS (ESI+) m/z calcd for

C₂₈H₄₀N₄O₅Na 535.2896, found 535.2896.

(−)-Ethyl 8-[(3S,9S,14aR)-9-benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetra-azacyclododecin-3-

yl]-3-oxooctanoate (20d). According to method C and starting from

(−)-17, product 20d was obtained after ﬂash chromatography (silica gel,

35% EtOAc/cyclohexane) as a colorless ﬁlm: 52% yield; R_f= 0.05 (40%

EtOAc/cyclohexane); [α]_D²⁰= −71.0° (c = 0.68, CHCl₃); IR ν_max(ﬁlm)

3305, 2928, 1741, 1714, 1681, 1666, 1629, 1529, 1434, 1315, 1232,

1178, 1030 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.28 (t, J = 7.1

Hz, 3H), 1.25−1.34 (m, 4H), 1.34 (s, 3H), 1.54−1.67 (m, 4H), 1.77 (m,

2H), 1.77 (s, 3H), 2.17 (m, 1H), 2.32 (m, 1H), 2.53 (t, J = 7.3 Hz, 2H),

2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.23 (m, 1H), 3.26 (dd, J = 13.4, 10.0 Hz,

1H), 3.42 (s, 2H), 3.86 (m, 1H), 4.17 (m, 1H), 4.20 (q, J = 7.1 Hz, 2H),

4.66 (bd, J = 7.8 Hz, 1H), 5.16 (ddd, J = 10.1, 10.1, 5.8 Hz, 1H), 5.92 (s,

1H, NH), 7.09 (d, J = 10.2 Hz, 1H, NH), 7.19−7.32 (m, 5H), 7.51 (d, J =

10.1 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 14.3 (CH₃),

23.3 (CH₂), 23.8 (CH₃), 25.0 (CH₂), 25.2 (CH₂), 25.5 (CH₂), 26.7

(CH₃), 28.8 (CH₂), 28.9 (CH₂), 36.0 (CH₂), 43.0 (CH₂), 47.2 (CH₂),

49.5 (CH₂), 53.6 (CH), 54.5 (CH), 58.0 (CH), 59.0 (C), 61.6 (CH₂),

126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C), 167.4 (C),

172.0 (C), 173.0 (C), 174.5 (C), 175.8 (C), 202.9 (C); LRMS (ESI+)

m/z (%) 593 (100) [M + Na]⁺, 521 (20); HRMS (ESI+) m/z calcd for

C₃₀H₄₂N₄O₇Na 593.2951, found 593.2942.

(−)-(3S,9S,14aR)-9-Benzyl-3-[(R)-7-(tert-butyldimethylsilyloxy)-6-

oxooctyl]-6,6-dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraaza-

cyclododecine-1,4,7,10-tetraone (20e). According to method B and

starting from 19e (71 mg, 0.093 mmol), the product 20e (36 mg, 60%)

was obtained as a colorless ﬁlm (ﬂash chromatography: silica gel, 0.8%

MeOH/CH₂Cl₂): R_f= 0.32 (40% EtOAc/cyclohexane); [α]_D²⁰= −47°

(c = 2.3, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3520, 3314, 2925, 2853,

1

1716, 1678, 1630, 1525, 1427, 1246, 1165, 1117, 836 cm⁻¹; H NMR

(400 MHz, CDCl₃) δ (ppm) 0.07 (s, 6H), 0.91 (s, 9H), 1.26−1.34 (m,

7H), 1.34 (s, 3H), 1.55−1.73 (m, 4H), 1.75 (m, 2H), 1.77 (s, 3H), 2.17

(m, 1H), 2.32 (m, 1H), 2.47−2.64 (m, 2H), 2.95 (dd, J = 13.5, 5.7 Hz,

1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5, 10.1 Hz, 1H), 3.86 (m, 1H), 4.13

(q, J = 6.8 Hz, 1H), 4.19 (ddd, J = 10.1, 7.6, 7.6 Hz, 1H), 4.66 (bd, J = 7.8

Hz, 1H), 5.16 (m, 1H), 6.03 (s, 1H, NH), 7.12 (d, J = 10.1 Hz, 1H, NH),

7.18−7.29 (m, 5H), 7.55 (bd, J = 10.1 Hz, 1H, NH); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) −4.8 (CH₃), −4.5 (CH₃), 18.2 (C), 21.1 (CH₃),

22.9 (CH₂), 23.2 (CH₂), 23.8 (CH₃), 25.0 (CH₂), 25.2 (CH₂), 26.0 (3 ×

CH₃), 26.7 (CH₂), 29.8 (CH₂), 29.9 (CH₂), 36.0 (CH₂), 36.9 (CH₂),

47.2 (CH₂), 53.7 (CH), 54.6 (CH), 58.0 (CH), 59.0 (C), 75.1 (CH),

126.9 (CH), 128.8 (2 × CH), 129.3 (2 × CH), 137.3 (C), 172.0 (C),

173.0 (C), 174.6 (C), 175.9 (C), 214.4 (C); LRMS (ESI+) m/z (%) 665

(17) [M + Na]⁺, 551 (100); HRMS (ESI+) m/z calcd for

C₃₄H₅₄N₄O₆NaS_i665.3710, found 665.3717.

(−)-(3S,9S,14aR)-9-Benzyl-3-[(S)-7-(tert-butyldimethylsilyloxy)-6-

oxooctyl]-6,6-dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraaza-

cyclododecine-1,4,7,10-tetraone (20f). According to method B and

starting from 19f (114 mg, 0.15 mmol), the product 20f (57 mg, 59%)

was obtained as a colorless ﬁlm (ﬂash chromatography: silica gel, 0.8%

20

MeOH/CH₂Cl₂): R_f= 0.32 (40% EtOAc/cyclohexane); [α]_D

=

−77.8° (c = 1.1, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3515, 3311, 2920,

1

2850, 1718, 1677, 1631, 1526, 1427, 1251, 1170, 1117, 835 cm⁻¹; H

NMR (400 MHz, CDCl₃) δ (ppm) 0.07 (s, 6H), 0.91 (s, 9H), 1.25−

1.34 (m, 10H), 1.55 (m, 2H), 1.63 (m, 2H), 1.76 (m, 2H), 1.77 (s, 3H),

2.17 (m, 1H), 2.32 (m, 1H), 2.47−2.64 (m, 2H), 2.95 (dd, J = 13.5, 5.7

Hz, 1H), 3.21 (m, 1H), 3.26 (dd, J = 13.5, 10.1 Hz, 1H), 3.86 (m, 1H),

4.13 (q, J = 6.8 Hz, 1H), 4.20 (ddd, J = 10.1, 7.6, 7.6 Hz, 1H), 4.67 (bd, J

= 7.6 Hz, 1H), 5.17 (m, 1H), 6.11 (bs, 1H, NH), 7.15 (d, J = 10.1 Hz,

1H, NH), 7.18−7.29 (m, 5H), 7.57 (bd, J = 10.1 Hz, 1H, NH); ¹³C

NMR (100 MHz, CDCl₃) δ (ppm) −4.9 (CH₃), −4.5 (CH₃), 18.2 (C),

21.1 (CH₃), 22.9 (CH₂), 23.1 (CH₂), 23.8 (CH₃), 24.9 (CH₂), 25.2

(CH₂), 25.9 (3 × CH₃), 26.6 (CH₃), 29.8 (CH₂), 29.9 (CH₂), 36.0

(CH₂), 36.9 (CH₂), 47.1 (CH₂), 53.6 (CH), 54.6 (CH), 58.0 (CH),

(−)-(3S,9S,14aR)-9-Benzyl-3-[(R)-7-hydroxy-6-oxooctyl]-6,6-

dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (21). Three drops of TBAF (1 M in

THF, 20 μL) were added to a solution of 20e (12.6 mg, 0.0196 mmol) in

THF (1 mL). The mixture was stirred for 30 min at room temperature.

After evaporation, the residue was directly puriﬁed by ﬂash

chromatography (silica gel, 2% MeOH/CH₂Cl₂) to give 21 (8.1 mg,

20

78%): R_f= 0.16 (2% MeOH/CH₂Cl₂); [α]_D= −98° (c = 0.58,

CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3288, 2920, 2850, 1710, 1666,

1

1625, 1523, 1426, 1368, 1187 cm⁻¹; H NMR (400 MHz, CDCl₃) δ

(ppm) 1.26−1.35 (m, 4H), 1.34 (s, 3H), 1.38 (d, J = 7.1 Hz, 3H), 1.60−

3667

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The Journal of Organic Chemistry

Article

1.69 (m, 4H), 1.77 (m, 2H), 1.77 (s, 3H), 2.18 (m, 1H), 2.33 (m, 1H),

2.40−2.57 (m, 2H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.21 (m, 1H), 3.27

(dd, J = 13.5, 10.1 Hz, 1H), 3.86 (m, 1H), 4.19 (m, 1H), 4.24 (q, J = 7.1

Hz, 1H), 4.66 (bd, J = 7.7 Hz, 1H), 5.17 (ddd, J = 10.1, 10.1, 5.8 Hz, 1H),

6.00 (s, 1H, NH), 7.13 (d, J = 10.2 Hz, 1H, NH), 7.18−7.30 (m, 5H),

7.51 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

20.1 (CH₃), 23.5 (CH₂), 23.8 (CH₃), 25.0 (CH₂), 25.2 (CH₂), 25.4

(CH₂), 26.7 (CH₃), 28.9 (CH₂), 29.0 (CH₂), 36.0 (CH₂), 37.5 (CH₂),

47.2 (CH₂), 53.7 (CH), 54.5 (CH), 58.0 (CH), 59.0 (C), 72.8 (CH),

126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C), 172.1 (C),

173.0 (C), 174.5 (C), 175.8 (C), 212.6 (C); LRMS (ESI+) m/z (%) 551

(100) [M + Na]⁺, 529 (10); HRMS (ESI+): m/z calcd for

C₂₈H₄₀N₄O₆Na 551.2846, found 551.2847.

(−)-(R)-8-(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl)-3-oxooctan-2-yl Acetate (22). To a stirred solution

of 21 (9.7 mg, 0.018 mmol) in CH₂Cl₂(1 mL) were added DMAP

(catalytic amount) and Ac₂O (5.5 mg, 0.054 mmol) at 0 °C. The mixture

was stirred overnight at room temperature. After solvent evaporation,

the residue was directly puriﬁed by ﬂash chromatography (40% EtOAc/

cyclohexane) to give 22 (9.2 mg, 88%) as a colorless ﬁlm: R_f= 0.09 (40%

EtOAc/cyclohexane); [α]_D²⁰= −73.1° (c = 0.9, CHCl₃); IR ν_max(thin

ﬁlm, CH₂Cl₂) 3518, 3293, 2929, 1723, 1679, 1625, 1528, 1425, 1368,

1233, 1082 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.25−1.34 (m,

4H), 1.34 (s, 3H), 1.39 (d, J = 7.1 Hz, 3H), 1.54−1.67 (m, 4H), 1.77 (m,

2H), 1.77 (s, 3H), 2.14 (s, 3H, Ac), 2.18 (m, 1H), 2.32 (m, 1H), 2.42 (m,

1H), 2.51 (m, 1H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.23 (m, 1H), 3.26

(dd, J = 13.5, 10.1 Hz, 1H), 3.86 (m, 1H), 4.19 (m, 1H), 4.66 (bd, J = 7.7

Hz, 1H), 5.08 (q, J = 7.1 Hz, 1H), 5.16 (ddd, J = 10.1, 10.1, 5.8 Hz, 1H),

6.02 (s, 1H, NH), 7.12 (d, J = 10.2 Hz, 1H, NH), 7.20−7.30 (m, 5H),

7.53 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

16.3 (CH₃), 21.0 (CH₃), 23.1 (CH₂), 23.8 (CH₃), 25.0 (CH₂), 25.2

(CH₂), 25.5 (CH₂), 26.7 (CH₃), 28.9 (CH₂), 29.0 (CH₂), 36.0 (CH₂),

38.2 (CH₂), 47.2 (CH₂), 53.6 (CH), 54.5 (CH), 58.0 (CH), 59.0 (C),

74.8 (CH), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C),

170.6 (C), 172.0 (C), 173.0 (C), 174.6 (C), 175.9 (C), 207.8 (C);

HRMS (ESI+) m/z calcd for C₃₀H₄₂N₄O₇Na 593.2946, found

593.2946.

(−)-(3S,9S,14aR)-9-Benzyl-3-[(S)-7-hydroxy-6-oxooctyl]-6,6-

dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (23). TBAF (1 M in THF, 34 μL) was

added to a solution of 20f (21.5 mg, 0.0334 mmol) in THF (1.5 mL).

The mixture was stirred for 30 min at room temperature. After

evaporation, the residue was directly puriﬁed by ﬂash chromatography

(2% MeOH/CH₂Cl₂) to give 23 (13.7 mg, 78%): R_f= 0.09 (60%

EtOAc/cyclohexane); [α]_D²⁰= −65.3° (c = 0.98, CHCl₃); IR ν_max(thin

ﬁlm, CH₂Cl₂) 3290, 2925, 2840, 1716, 1666, 1625, 1525, 1424, 1188

cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.25−1.35 (m, 4H), 1.34

(s, 3H), 1.38 (d, J = 7.1 Hz, 3H), 1.62−1.69 (m, 4H), 1.77 (m, 2H), 1.77

(s, 3H), 2.17 (m, 1H), 2.32 (m, 1H), 2.38−2.58 (m, 2H), 2.95 (dd, J =

13.5, 5.7 Hz, 1H), 3.22 (m, 1H), 3.26 (dd, J = 13.5, 10.1 Hz, 1H), 3.86

(m, 1H), 4.19 (m, 1H), 4.22 (q, J = 7.1 Hz, 1H), 4.67 (bd, J = 7.7 Hz,

1H), 5.16 (ddd, J = 10.1, 10.1, 5.8 Hz, 1H), 6.12 (bs, 1H, NH), 7.17 (d, J

= 10.2 Hz, 1H, NH), 7.20−7.30 (m, 5H), 7.54 (d, J = 10.2 Hz, 1H, NH);

¹³C NMR (100 MHz, CDCl₃) δ (ppm) 20.1 (CH₃), 23.5 (CH₂), 23.8

(CH₃), 25.0 (CH₂), 25.2 (CH₂), 25.5 (CH₂), 26.7 (CH₃), 28.9 (CH₂),

29.0 (CH₂), 36.0 (CH₂), 37.5 (CH₂), 47.2 (CH₂), 53.7 (CH), 54.5

(CH), 58.0 (CH), 59.0 (C), 72.8 (CH), 126.9 (CH), 128.8 (2 × CH),

129.2 (2 × CH), 137.2 (C), 172.1 (C), 173.0 (C), 174.5 (C), 175.8 (C),

212.6 (C); LRMS (ESI+) m/z (%) 551 (45) [M + Na]⁺, 242 (100);

HRMS (ESI+): m/z calcd for C₂₈H₄₀N₄O₆Na 551.2846, found

551.2847.

in vacuo. After ﬂash chromatography (silica gel, 25% EtOAc/

cyclohexane), the product 24 (47.5 mg, 91%) was isolated as a colorless

20

ﬁlm: R_f= 0.30 (30% EtOAc/cyclohexane); [α]_D= −88.6° (c = 1.0,

CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3444, 2933, 2855, 1720, 1646,

1

1630, 1523, 1421, 1384, 1164 cm⁻¹; H NMR (400 MHz, CDCl₃) δ

(ppm) 0.83 (t, J = 7.4 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H), 0.89 (t, J = 6.8

Hz, 3H), 1.24−1.35 (m, 15H), 1.36 (m, 1H), 1.38 (d, J = 7.1 Hz, 3H),

1.51−1.71 (m, 5H), 1.81 (m, 1H), 2.16 (m, 1H), 2.27−2.45 (m, 5H),

2.51 (dt, J = 17.4, 7.2 Hz, 1H), 2.62 (m, 1H), 2.73 (dd, J = 9.7, 8.1 Hz,

1H), 2.96 (dd, J = 13.5, 6.5 Hz, 1H), 3.24 (dd, J = 13.5, 9.8 Hz, 1H), 4.06

(dd, J = 9.6, 7.6 Hz, 1H), 4.20 (ddd, J = 10.2, 7.6, 7.6 Hz, 1H), 4.67 (d, J =

6.5 Hz, 1H), 5.08 (q, J = 7.1 Hz, 1H), 5.16 (ddd, J = 10.2, 9.9, 6.0 Hz,

1H), 5.86 (s, 1H, NH), 7.15 (d, J = 10.2 Hz, 1H, NH), 7.18−7.31 (m,

5H), 7.56 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ

(ppm) 8.6 (CH₃), 14.2 (CH₃), 16.3 (CH₃), 18.3 (CH₃), 22.5 (CH₃),

22.8 (CH₂), 23.1 (CH₂), 25.0 (CH₂), 25.5 (CH₂), 28.0 (CH₂), 28.9

(CH₂), 29.0 (CH₂), 29.1 (CH₂), 29.2 (CH₂), 31.8 (CH₂), 33.0 (CH),

33.2 (CH₂), 34.2 (CH₂), 35.9 (CH₂), 38.2 (CH₂), 53.5 (CH), 54.0

(CH₂), 54.6 (CH), 58.1 (CH), 63.2 (C), 74.6 (CH), 126.8 (CH), 128.7

(2 × CH), 129.2 (2 × CH), 137.2 (C), 172.0 (C), 173.2 (C), 173.4 (C),

174.3 (C), 175.8 (C), 207.9 (C); HRMS (ESI+) m/z calcd for

C₃₈H₅₈N₄O₇Na 705.4203, found 705.4200.

(−)-(S)-4-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl]butyl Ethanethioate (28). To a solution of (−)-17 (32

mg, 0.073 mmol) in dry THF (10 mL) was added thioacetic acid 26 (22

mg, 0.29 mmol). The mixture was reﬂuxed for 30 min under argon. A

catalytic amount of AIBN was added, and the mixture was stirred at

reﬂux for 16 h while the reaction mixture was held up to the light. After

evaporation, the residue was directly puriﬁed by ﬂash chromatography

(1% MeOH/CH₂Cl₂) to give 28 (34 mg, 91%) as a colorless ﬁlm: R_f=

0.12 (1% MeOH/CH₂Cl₂); [α]_D²⁰= −85.1° (c = 1.1, CHCl₃); IR ν_max

(ﬁlm) 3307, 2934, 1684, 1630, 1528, 1428, 1274, 1187, 915 cm⁻¹; ¹H

NMR (400 MHz, CDCl₃) δ (ppm) 1.34 (s, 3H), 1.39 (m, 2H), 1.53−

1.71 (m, 4H), 1.72 (m, 5H), 2.11−2.22 (m, 2H), 2.32 (s, 3H), 2.85 (t, J

= 7.2 Hz, 2H), 2.95 (dd, J = 13.4, 5.6 Hz, 1H), 3.23 (m, 1H), 3.26 (m,

1H), 3.86 (m, 1H), 4.21 (m, 1H), 4.68 (m, 1H), 5.17 (s, 1H), 6.02 (s,

1H, NH), 7.13 (d, J = 10.1 Hz, 1H, NH), 7.19−7.29 (m, 5H), 7.53 (d, J =

10.1 Hz, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.7 (CH₃),

24.8 (CH₂), 24.9 (CH₂), 25.1 (CH₂), 26.6 (CH₂), 28.6 (CH₂), 28.9

(CH₂), 29.33 (CH₂), 30.8 (CH₃), 35.9 (CH₂), 47.1 (CH₂), 53.6 (CH),

54.4 (CH), 57.9 (CH), 58.9 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2

× CH), 137.2 (C), 172.0 (C), 173.0 (C), 174.4 (C),175.8 (C), 196.0

(C); LRMS (ESI+) m/z (%) 539 (100) [M + Na]⁺, 517 (10); HRMS

(ESI+) m/z calcd for C₂₆H₃₆N₄O₅NaS 539.2304, found 539.2302.

(−)-(S)-4-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl]butyl Octanethioate (29). To a solution of (−)-17 (42

mg, 0.095 mmol) in dry THF (12 mL) was added thioacid 27 (61 mg,

0.38 mmol). The mixture was reﬂuxed for 30 min under argon. A

catalytic amount of AIBN was added, and the mixture was stirred at

reﬂux for 16 h while the reaction mixture was held up to the light. After

evaporation, the residue was directly puriﬁed by ﬂash chromatography

(1% MeOH/CH₂Cl₂) to give 29 (38.7 mg, 68%) as a colorless ﬁlm: R_f=

0.19 (8% EtOAc/CH₂Cl₂); [α]_D²⁰= −56.5° (c = 1.2, CHCl₃); IR ν_max

(thin ﬁlm, CH₂Cl₂) 3297, 2945, 2921, 2855, 1687, 1658, 1620, 1524,

1420, 1225, 1176 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.88 (t, J

= 6.9 Hz, 3H), 1.23−1.33 (m, 10H), 1.34 (s, 3H), 1.58 (m, 2H), 1.60−

1.70 (m, 5H), 1.77 (s, 3H), 1.78 (m, 2H), 2.17 (m, 1H), 2.53 (t, J = 7.5

Hz, 2H), 2.85 (t, J = 7.3 Hz, 2H), 2.95 (dd, J = 13.5, 5.7 Hz, 1H), 3.18−

3.31 (m, 2H), 3.86 (m, 1H), 4.19 (dt, J = 10.2, 7.6 Hz, 1H), 4.66 (m,

1H), 5.16 (dt, J = 10.2, 5.7 Hz, 1H), 6.00 (s, 1H, NH), 7.12 (d, J = 10.2

Hz, 1H, NH), 7.17−7.35 (m, 5H), 7.51 (d, J = 10.2 Hz, 1H, NH); ¹³C

NMR (100 MHz, CDCl₃) δ (ppm) 14.3 (CH₃), 22.8 (CH₂), 23.8

(CH₃), 24.9 (CH₂), 25.0 (CH₂), 25.2 (CH₂), 25.9 (CH₂), 26.6 (CH₃),

28.6 (CH₂), 28.7 (CH₂), 29.1 (2 × CH₂), 29.5 (CH₂), 31.8 (CH₂), 36.0

(CH₂), 44.4 (CH₂), 47.2 (CH₂), 53.6 (CH), 54.4 (CH), 58.0 (CH),

59.0 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C),

172.1 (C), 173.0 (C), 174.4 (C), 175.8 (C), 199.9 (C); HRMS (ESI+)

m/z calcd for C₃₂H₄₈N₄O₅NaS 623.3243, found 623.3244.

(−)-(R)-8-[(3S,6R,9S,13S,14aR)-9-Benzyl-6-ethyl-6,13-dimeth-

yl-1,4,7,10-tetraoxotetradecahydropyrrolo[1,2-a][1,4,7,10]-

tetraazacyclododecin-3-yl]-3-oxooctan-2-yl Octanoate (24). To

a solution of FR235222 (42.6 mg, 0.077 mmol) in dry CH₂Cl₂(2 mL)

were added octanoyl chloride (22 mg, 0.13 mmol) and DMAP (36 mg,

0.29 mmol). The mixture was stirred for 18 h and was quenched by a

saturated solution of NaHCO₃. The product was extracted with EtOAc.

The organic layer was dried (MgSO₄), and the solvent was concentrated

3668

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Article

(−)-(3S,6R,9S,14S)-3-Benzyl-6-ethyl-6,14-dimethyl-9-(6-ox-

ooctyl)-1,4,7,10-tetraazacyclopentadecane-2,5,8,11-tetraone

(30). FR235222 (15 mg, 0.027 mmol) in a degassed solution of THF/

methanol (1:1; 1 mL) under argon was cooled to 0 °C. SmI₂(0.1 M in

THF)⁴²was injected until the persistence of a deep blue coloration

(about 3 mL). The mixture was stirred for 30 min at 0 °C. A THF/H₂O

solution (1:1) was added, and the product was extracted with EtOAc.

The combined organic layers were dried (MgSO₄). After evaporation,

the residue was puriﬁed by ﬂash chromatography (silica gel, 3% MeOH/

CH₂Cl₂) to give 30 (13 mg, 89%) as a colorless ﬁlm: R_f= 0.3 (4%

MeOH/CH₂Cl₂); [α]_D²⁰= −56° (c = 1.3, CHCl₃); IR ν_max(thin ﬁlm,

CH₂Cl₂) 3309, 2933, 2864, 1707, 1638, 1520, 1454, 1260, 1180 cm⁻¹;

¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.46 (t, J = 7.4 Hz, 3H), 0.94 (d, J

= 6.6 Hz, 3H), 1.06 (t, J = 7.3 Hz, 3H), 1.25−1.42 (m, 8H), 1.42−1.79

(m, 6H), 1.80−2.03 (m, 2H), 2.08 (m, 1H), 2.25 (m, 1H), 2.40−2.47

(m, 4H), 2.68 (m, 1H), 2.88 (dd, J = 14.2, 11.0 Hz, 1H), 3.49 (m, 1H),

3.54 (m, 1H), 3.80 (m, 1H), 4.74 (m, 1H), 6.18 (s, 1H, NH), 6.44 (bd, J

= 6.8 Hz, 1H, NH), 6.55 (bd, J = 9.5 Hz, 1H, NH), 7.19 (m, 1H), 7.26−

7.27 (m, 4H), 7.63 (bd, J = 7.6 Hz, 1H, NH); ¹³C NMR (100 MHz,

CDCl₃) δ (ppm) 7.9 (CH₃), 8.1 (CH₃), 18.3 (CH₃), 21.6 (CH₃), 23.7

(CH₂), 26.3 (CH₂), 28.8 (CH₂), 29.2 (CH₂), 29.7 (CH₂), 31.6 (CH₂),

33.2 (CH), 34.1 (CH₂), 36.2 (CH₂), 37.7 (CH₂), 42.2 (CH₂), 44.3

(CH₂), 54.7 (CH), 55.2 (CH), 61.7 (C), 126.7 (CH), 128.6 (2 × CH),

129.3 (2 × CH), 138.2 (C), 171.7 (C), 172.6 (C), 173.2 (C), 175.0 (C),

211.9 (C); LRMS (ESI+) m/z (%) 565 (100) [M + Na]⁺, 543 (10) [M +

H]⁺, 291 (8); HRMS (ESI+) m/z calcd for C₃₀H₄₆N₄O₅Na 565.3366,

found 565.3364.

(−)-(3S,9S)-3-Benzyl-6,6-dimethyl-9-(6-oxooctyl)-1,4,7,10-

tetraazacyclopentadecane-2,5,8,11-tetraone (31). According to

the procedure used for 30 and starting from 20c (5 mg, 0.098 mmol), 31

(3.3 mg, 66%) was obtained after ﬂash chromatography (silica gel, 4%

MeOH/CH₂Cl₂) as a colorless ﬁlm: R_f= 0.10 (4% MeOH/CH₂Cl₂);

[α]_D²⁰= −56.4° (c = 0.33, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3311,

2927, 2855, 1646, 1547, 1452, 1361, 1266 cm⁻¹; ¹H NMR (400 MHz,

CDCl₃) δ (ppm) 1.05 (t, J = 7.3 Hz, 3H), 1.25−1.45 (m, 9H), 1.45−1.86

(m, 9H), 2.05−2.21 (m, 2H), 2.39−2.46 (m, 4H), 3.12 (m, 1H), 3.17

(dd, J = 14.0, 6.0 Hz, 1H), 3.42 (m, 1H), 3.46 (dd, J = 14.0, 4.9 Hz, 1H),

3.91 (m, 1H), 4.49 (m, 1H), 6.19 (d, J = 7.3 Hz, 1H, NH), 6.44 (s, 1H,

NH), 6.50 (m, 1H, NH), 7.16−7.30 (m, 5H), 7.37 (bd, J = 8.4 Hz, 1H,

NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 8.05 (CH₃), 22.1 (CH₂),

23.4 (CH₂), 24.8 (CH₃), 25.7 (CH₃), 25.9 (CH₂), 27.6 (CH₂), 28.7

(CH₂), 29.8 (CH₂), 35.9 (CH₂), 36.2 (CH₂), 36.6 (CH₂), 38.0 (CH₂),

42.1 (CH₂), 54.9 (CH), 55.8 (CH), 58.0 (C), 126.7 (CH), 128.6 (2 ×

CH), 129.5 (2 × CH), 138.4 (C), 171.6 (C), 173.3 (C), 173.9 (C),

175.1 (C), 212.0 (C); HRMS (ESI+) m/z calcd for C₂₈H₄₂N₄O₅Na

537.3047, found 537.3046.

CH), 128.6 (2 × CH), 129.1 (2 × CH), 129.8 (CH), 129.9 (CH), 130.2

(2 × CH), 136.1 (C), 136.2 (C), 136.4 (C), 169.3 (C), 175.0 (C);

LRMS (ESI+) m/z (%) 394 (10) [M + Na]⁺, 372 (100) [M + H]⁺, 238

(15); HRMS (ESI+) m/z calcd for C₂₄H₂₂NO₃372.1594, found

372.1598.

(−)-(2S,4R)-3-Benzoyl-4-benzyl-4-methyl-2-phenyl-1,3-oxa-

zolidin-5-one ((−)-33). The product (−)-33 was obtained as a white

solid following the experimental procedure described above, starting

from (−)-(2S,4R)-3-benzoyl-4-methyl-2-phenyl-1,3-oxazolidin-5-one

((−)-32):³⁶[α]_D= −251.1° (c = 1.2, CHCl₃); LRMS (ESI+) m/z

20

(%) 394 (8) [M + Na]⁺, 372 (100) [M + H]⁺, 238 (15); HRMS (ESI+)

m/z calcd for C₂₄H₂₂NO₃372.1594, found 372.1598.

(+)-(2S)-Methyl-2-amino-2-methyl-3-phenylpropanoate Hy-

drochloride ((+)-34/HCl). A suspension of oxazolidinone (+)-33

(1.50 g, 4.04 mmol) in concentrated HCl (18 mL) was reﬂuxed under

nitrogen overnight. After the mixture was cooled, the precipitate was

removed by ﬁltration and the ﬁltrate was evaporated in vacuo. The

residue was then dissolved in methanol (12 mL), and thionyl chloride

(0.88 mL, 12.1 mmol) was slowly added at 0 °C. The reaction mixture

was stirred overnight at room temperature and was then concentrated in

vacuo. The oily residue was precipitated in Et₂O to give the salt of the

amino ester (+)-34 (789 mg, 85%) as a white solid: [α]_D²⁰= +8.5° (c =

2.5, MeOH); IR ν_max(thin ﬁlm) 3408, 2075, 1654 cm⁻¹; ¹H NMR (400

MHz, CD₃OD) δ (ppm) 1.64 (s, 3H), 3.17 (d, J = 12.0 Hz, 1H), 3.29 (d,

J = 12.0 Hz, 1H), 3.83 (s, 3H), 7.20−7.23 (m, 2H), 7.32−7.51 (m, 3H);

¹³C NMR (100 MHz, CD₃OD) δ (ppm) 22.6 (CH₃), 44.2 (CH₂), 54.0

(CH₃), 62.1 (C), 129.3 (CH), 130.2 (2 × CH), 131.4 (2 × CH), 134.5

(C), 172.3 (C); LRMS (ESI+) m/z (%) 194 (100) [M + H]⁺; HRMS

(ESI+) m/z calcd for C₁₁H₁₆NO₂194.1176, found 194.1177.

(−)-(2R)-Methyl-2-amino-2-methyl-3-phenylpropanoate Hy-

drochloride ((−)-34/HCl). The product (−)-34/HCl was obtained as

a white solid (725 mg, 78%) following the experimental procedure

20

described above, starting from (−)-33 (1.50 g, 4.04 mmol): [α]_D

=

−8.1° (c = 1.1, MeOH); LRMS (ESI+) m/z (%) 194 (100) [M + H]⁺;

HRMS (ESI+) m/z calcd for C₁₁H₁₆NO₂194.1176, found 194.1178.

(R)-Glycine Nickel (R)-2-[N-(N′-Benzylpropyl)amino]-

benzophenone pent-1-ene Complex (36). To a suspension of

(+)-35 (9.9 g, 19.8 mmol) and NaOH (2.0 g, 50 mmol) in acetonitrile

(100 mL) was added 5-bromopent-1-ene (4.4 g, 29.72 mmol). The

mixture was stirred at room temperature for 4 h. An aqueous solution of

0.1 N HCl (155 mL) was added, and the product was extracted with

CH₂Cl₂(3 × 100 mL), dried over MgSO₄, ﬁltered, and concentrated in

vacuo. The product 36 (9.5 g, 85%) was obtained after ﬂash

20

chromatography (silica gel, 30% acetone/CH₂Cl₂): [α]_D= +6.1° (c

= 1.0, CHCl₃); R_f= 0.4 (30% acetone/CH₂Cl₂); IR ν_max(ﬁlm) 2957,

1

1671, 1637, 1589, 1439, 1330, 1256, 1165, 1063, 913, 752 cm⁻¹; H

NMR (400 MHz, CDCl₃) δ (ppm) 1.77−1.58 (m, 1H), 2.11−1.87 (m,

4H), 2.13−2.19 (m, 2H), 2.31−2.17 (m, 1H), 2.60−2.45 (m, 1H),

2.81−2.71 (m, 1H), 3.49 (m, 2H), 3.57 (d, J = 12.7 Hz, 1H), 3.91 (dd, J

= 8.1, 3.5 Hz, 1H), 4.42 (d, J = 12.7 Hz, 1H), 4.95 (m, 2H), 5.72 (tdd, J =

16.9, 10.2, 6.7 Hz, 1H), 6.64 (m, 2H), 6.91 (d, J = 7.43 Hz, 1H), 7.12 (m,

1H), 7.18 (m, 1H), 7.26 (m, 1H), 7.34 (m, 2H), 7.42−7.47 (m, 2H),

7.51 (m, 2H), 8.05 (m, 2H), 8.13 (d, J = 8.3 Hz, 1H); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 23.7 (CH₂), 24.6 (CH₂), 30.8 (2 × CH₂), 33.3

(CH₂), 34.8 (CH₂), 57.1 (CH₂), 63.2 (CH), 70.3 (CH), 115.3 (CH),

120.7 (CH), 123.7 (CH), 126.5 (CH), 127.2 (CH), 127.6 (2 × CH),

128.9 (4 × CH), 129.7 (2 × CH), 131.6 (C), 132.1 (CH), 133.3 (CH),

133.8 (CH), 137.7 (2 × C), 142.3 (C), 170.3 (C), 179.3 (C), 180.3 (C);

HRMS (ESI+) m/z calcd for C₃₂H₃₃N₃NaNiO₃588.1512 found

588.1512.

(+)-(2R,4S)-3-Benzoyl-4-benzyl-4-methyl-2-phenyl-1,3-oxa-

zolidin-5-one ((+)-33). To a solution of hexamethyldisilazane (5.7 mL,

26.7 mmol) in THF (25 mL) was added n-butyllithium (2.5 M in

hexane, 7.8 mL, 19.6 mmol) at −78 °C. After 5 min at this temperature,

the reaction mixture was warmed to 0 °C for 30 min and then cooled to

−78 °C again. A solution of (+)-(2R,4S)-3-benzoyl-4-methyl-2-phenyl-

1,3-oxazolidin-5-one ((+)-32;³⁶5.0 g, 17.8 mmol) in THF (60 mL) was

slowly added under nitrogen to this reaction mixture. After the mixture

was stirred for 3 h at this temperature, a solution of benzyl bromide (2.76

mL, 23.1 mmol) in THF (50 mL) was slowly added. The reaction

mixture was warmed to room temperature over 4 h and was stirred

overnight. The solvent was evaporated in vacuo, and the residue was

dissolved in 10% NH₄Cl and extracted with dichloromethane. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated under

vacuum. After ﬂash chromatography (silica gel, 10% EtOAc/cyclo-

hexane), the product (+)-33 (4.42 g, 67%) was isolated as a white solid:

(+)-(2S)-tert-Butyl 1-(Methoxycarbonyl)hex-4-enylcarba-

mate ((+)-37). According to the procedure reported for (−)-14 (vide

infra) and starting from 36 (9.48 g, 16.75 mmol), the product (+)-37

20

R_f= 0.48 (20% EtOAc/cyclohexane); [α]_D= +246.9° (c = 2.5,

CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3554, 1795, 1650, 1447, 1393,

1358, 1226, 1170, 1032 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm)

2.18 (s, 3H), 3.38 (d, J = 13.6 Hz, 1H), 3.94 (d, J = 13.6 Hz, 1H), 5.64 (s,

1H), 6.65 (d, J = 7.0 Hz, 2H), 6.75 (d, J = 7.4 Hz, 2H), 7.02−7.11 (m,

4H), 7.15−7.22 (m, 2H), 7.33−7.39 (m, 2H), 7.40−7.45 (m, 3H); ¹³C

NMR (100 MHz, CDCl₃) δ (ppm) 24.6 (CH₃), 41.3 (CH₂), 65.6 (C),

90.6 (CH), 126.0 (2 × CH), 127.0 (2 × CH), 128.0 (CH), 128.5 (2 ×

(3.19, 12.4 mmol, 74%) was obtained after ﬂash chromatography (5%

20

EtOAc/cyclohexane): R_f= 0.15 (5% EtOAc/cyclohexane); [α]_D

=

+11.45° (c = 0.2, CHCl₃); IR ν_max(ﬁlm, CH₂Cl₂) 3359, 2977, 1743,

1718, 1520, 1450, 1366, 1249, 1163, 915 cm⁻¹; NMR (400 MHz,

CDCl₃) δ (ppm) 1.34 (s, 11H), 1.53 (m, 1H), 1.71 (m, 1H), 1.96 (m,

2H), 3.63 (s, 3H), 4.18 (dd, J = 12.9, 7.5 Hz, 1H), 4.86 (m, 2H), 5.16 (d,

J = 8.12 Hz, 1H, NH), 5.66 (m, 1H); ¹³C NMR (100 MHz; CDCl₃) δ

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(ppm) 24.5 (CH₂), 27.2 (3 × CH₃), 31.9 (CH₂), 33.0 (CH₂), 51.9

(CH₃), 53.2 (CH), 79.4 (C), 114.9 (CH₂), 137.8 (CH), 155.3 (C),

173.1 (C); LRMS (ESI+) m/z (%) 258 (100) [M + H]⁺; HRMS (ESI+)

m/z calcd for C₁₃H₂₃NO₄Na 280.1519, found 280.1520.

NMR (100 MHz, CDCl₃) δ (ppm) 13.5 (CH₃); 18.9 (CH₂), 24.5

(CH₃), 25.0 (CH₃), 28.2 (3 × CH₃), 29.6 (CH₂), 30.4 (CH₂), 31.7

(CH₂), 53.7 (CH), 56.0 (C), 64.9 (CH₂), 79.4 (C), 115.1 (CH₂), 137.4

(CH), 155.7 (C), 171.5 (C), 174.0 (C); HRMS (ESI+) m/z calcd for

C₁₉H₃₄N₂O₅Na 393.2365, found 393.2365.

(+)-(R)-Butyl 2-{2-[(tert-Butoxycarbonyl)amino]hex-5-enami-

do}-2-methylpropanoate ((+)-39). According to the procedure

reported for (−)-39, and starting from (−)-14 (1.0, 4.11 mmol), the

product (+)-39 was obtained (1.23 g, 81% yield): [α]_D²⁰= +16.5° (c =

1.0, CHCl₃); HRMS (ESI+) m/z calcd for C₁₉H₃₄N₂O₅Na 393.2365,

found 393.2360.

(S)-Glycine Nickel (S)-2-[N-(N′-Benzylpropyl)amino]benzo-

phenone But-1-ene Complex (38). According to the procedure

reported for 36 and starting from (−)-35 (6.36 g, 12.8 mmol) and 4-

bromobut-1-ene (2.44 g, 19.52 mmol), the product 38 (5.33 g, 9.67

mmol, 76%) was obtained after ﬂash chromatography (silica gel, 30%

20

acetone/CH₂Cl₂): [α]_D= −9.9° (c = 1.0, CHCl₃), R_f= 0.4 (30%

acetone/CH₂Cl₂); IR ν_max(ﬁlm) 2957, 1672, 1637, 1589, 1438, 1330,

1257, 1165, 1063, 913, 752 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm)

1.72 (m, 1H), 1.82 (dd, J = 6.5, 1.4 Hz, 1H), 2.08 (m, 1H), 2.17 (m, 1H),

2.29 (m, 1H), 2.49 (m, 1H), 2.74 (m, 2H), 3.48 (m, 2H), 3.57 (d, J =

12.7 Hz, 1H), 3.90 (dd, J = 8.6, 3.5 Hz, 1H), 4.43 (dd, J = 12.7, 5.6 Hz,

1H), 4.87 (dd, J = 10.2, 1.7 Hz, 1H), 4.96 (qd, J = 17.1, 1.5 Hz, 1H), 5.49

(m, 1H), 6.64 (m, 2H), 6.92 (d, J = 7.4 Hz, 1H), 7.16 (m, 2H), 7.26 (m,

1H), 7.35 (m, 2H), 7.42−7.48 (m, 2H), 7.51 (m, 2H), 8.05 (m, 2H),

8.12 (d, J = 8.3 Hz, 1H) ; ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.9

(CH₂), 29.5 (CH₂), 30.9 (2 × CH₂), 35.1 (CH₂), 57.2 (CH₂), 63.2

(CH), 70.4 (CH), 115.9 (CH), 120.9 (CH), 123.8 (CH), 126.6 (CH),

127.4 (CH), 127.6 (2 × CH), 128.9 (2 × CH), 129.0 (2 × CH), 129.8 (2

× CH), 131.6 (C), 132.2 (CH), 133.3 (CH), 13 3.8 (CH), 136.6 (2 ×

C), 142.3 (C), 170.5 (C), 179.3 (C), 180.5 (C); HRMS (ESI+) m/z

calcd for C₃₁H₃₁N₃NaNiO₃574.1611, found 574.1611.

(+)-(2R,5S)-Methyl 2-Benzyl-5-[(tert-butoxycarbonyl)amino]-

2-methyl-4-oxonon-8-enoate ((+)-40). To a solution of methyl

ester (+)-14 (655 mg, 2.69 mmol) in THF (10 mL) was added a

solution of LiOH (161 mg, 6.73 mmol) in water (10 mL). After it was

stirred for 2 h at room temperature, the reaction mixture was quenched

by addition of a solution of 5% H₃PO₄until pH 3. After extraction with

EtOAc, the organic layer was dried over MgSO₄and evaporated to give

the corresponding carboxylic acid. The hydrochloride salt of the amino

ester (+)-32 (789 mg, 3.43 mmol) was neutralized using a saturated

aqueous solution of NaHCO₃, and the free amine was extracted with

EtOAc. The organic layer was dried over MgSO₄and evaporated. To a

solution of carboxylic acid and free amine in dichloromethane (10 mL)

was added DCC (555 mg, 2.69 mmol) at 0 °C. The reaction mixture was

stirred overnight at room temperature, the precipitate was ﬁltered oﬀ,

and the ﬁltrate was concentrated in vacuo. After ﬂash chromatography

(silica gel, 10% EtOAc/cyclohexane), the product (+)-40 (1.0 g, 92%)

was isolated as a colorless oil: R_f= 0.48 (30% EtOAc/cyclohexane);

[α]_D²⁰= +5.8° (c = 1.8, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3313, 2974,

1740, 1666, 1523, 1249, 1168, 1123 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 1.47 (s, 9H), 1.65−1.72 (m, 4H), 1.92−2.02 (m, 1H), 2.16 (q, J

= 7.2 Hz, 2H), 3.22 (d, J = 13.5 Hz, 1H), 3.55 (d, J = 13.5 Hz, 1H), 3.80

(s, 3H), 4.02−4.12 (m, 1H), 4.95−5.11 (m, 3H, NH), 5.82 (ddt, J =

17.0, 10.2, 6.6 Hz, 1H), 6.71 (bs, 1H, NH), 7.06 (dd, J = 7.8, 2.0 Hz, 2H),

7.21−7.29 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.3

(CH₃), 28.5 (3 × CH₃), 29.9 (CH₂), 31.8 (CH₂), 41.7 (CH₂), 52.8

(CH₃), 54.5 (CH), 61.3 (C), 80.2 (C), 115.9 (CH₂), 127.2 (CH), 128.5

(2 × CH), 130.0 (2 × CH), 136.2 (C), 137.4 (CH), 155.7 (C), 171.5

(C), 174.2 (C); LRMS (ESI+) m/z (%) 405 (58) [M+H]⁺, 349 (100)

[M − t-Bu + 2H]⁺, 305 (69) [M − Boc + 2H]⁺; HRMS (ESI+) m/z

calcd for C₂₂H₃₃N₂O₅405.2384, found 405.2386.

(+)-(2R)-tert-Butyl 1-(methoxycarbonyl)pent-4-enylcarba-

mate ((+)-14). To a suspension of compound 38 (5.3 g, 9.67 mmol)

in MeOH (80 mL) was added 2 M aqueous HCl (100 mL), and the

mixture was stirred under reﬂux for 2 h. The green solution was cooled

to room temperature and basiﬁed to pH 9−10 with 25% ammonia

solution. After extraction with CH₂Cl₂the aqueous layer was

concentrated in vacuo to aﬀord a green solid. Thionyl chloride (1.2

mL, 14.81 mmol) was added dropwise to a suspension of the following

green solid (1.25 g, 9.67 mmol) in dry methanol (100 mL) under

nitrogen at 0 °C, and the reaction mixture was stirred under reﬂux for 3

h. The solvent was removed under reduced pressure, and the solid

residue was dissolved in acetonitrile (75 mL) and was placed in an ice/

water bath. Triethylamine (2.10 mL, 14.81 mmol) and di-tert-butyl

dicarbonate (3.23 g, 14.81 mmol) were added, and the suspension was

stirred for 16 h. The solvent was removed under reduced pressure, and

the residue was dissolved in water (50 mL) and EtOAc (75 mL). The

organic layer was separated, and the aqueous layer was extracted with

EtOAc (2 × 75 mL). The combined organic extracts were dried over

MgSO₄, ﬁltered, and concentrated under reduced pressure to give an oil

(−)-(2S,5S)-Methyl 2-Benzyl-5-[(tert-butoxycarbonyl)amino]-

2-methyl-4-oxonon-8-enoate ((−)-41). According to the procedure

reported for (+)-40 and starting from (+)-14 (420 mg, 1.73 mmol) and

(−)-34/HCl (594 mg, 2.59 mmol), the product (−)-41 (532 mg, 76%)

which was puriﬁed by silica gel column chromatography (5% EtOAc/

20

cyclohexane) to aﬀord (+)-14 (2.30 g, 9.47 mmol, 98%): [α]_D

=

was obtained after ﬂash chromatography as a colorless oil (silica gel, 10%

20

−19.2° (c = 1, CHCl₃); HRMS (ESI+) m/z calcd for C₁₂H₂₁NO₄Na

266.1368, found 266.1368.

EtOAc/cyclohexane): R_f= 0.55 (30% EtOAc/cyclohexane); [α]_D

=

−14.4° (c = 1.6, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3415, 1650, 1527,

1258, 1168, 1119 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.46 (s,

9H), 1.63 (s, 3H), 1.64−1.73 (m, 1H), 1.81−1.93 (m, 1H), 2.10 (q, J =

7.0 Hz, 2H), 3.28 (d, J = 13.6 Hz, 1H), 3.46 (d, J = 13.6 Hz, 1H), 3.79 (s,

3H), 4.00−4.12 (m, 1H), 4.99−5.07 (m, 2H), 5.08−5.16 (m, 1H), 5.80

(ddt, J = 17.0, 10.3, 6.6 Hz, 1H), 6.64 (bs, 1H, NH), 7.08 (dd, J = 8.0, 1.4

Hz, 2H), 7.24−7.32 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

23.3 (CH₃), 28.4 (3 × CH₃), 29.8 (CH₂), 31.8 (CH₂), 41.6 (CH₂), 52.8

(CH₃), 54.3 (CH), 60.9 (C), 80.0 (C), 115.8 (CH₂), 127.2 (CH), 128.5

(2 × CH), 130.1 (2 × CH), 136.1 (C), 137.4 (CH), 155.7 (C), 171.5

(C), 174.2 (C); LRMS (ESI+) m/z (%) 405 (63) [M + H]⁺, 349 (100)

[M − t-Bu + 2H]⁺, 305 (73) [M − Boc + 2H]⁺; HRMS (ESI+) m/z

calcd for C₂₂H₃₃N₂O₅405.2385, found 405.2384.

(+)-(S)-tert-Butyl 1-[(R)-2-Amino-3-phenylpropanoyl]-

pyrrolidine-2-carboxylate ((+)-42). To a solution of (Z)-Phe-OH

(2.88 g, 9.6 mmol) and H-^L-Pro-O-t-Bu (2.0 g, 9.6 mmol) in DMF (20

mL) were added at 0 °C HOBt·H₂O (1.5 g, 11.1 mmol), DCC (2.38 g,

11.5 mmol), and Et₃N (1,43 g, 9.6 mmol). The mixture was stirred at 0

°C for 1 h and then at room temperature overnight. DMF was

evaporated, and the residue was diluted with EtOAc and washed with

10% citric acid, 4% sodium carbonate, and brine. The organic phase was

dried (MgSO₄) and evaporated. After ﬂash chromatography (silica gel,

2% MeOH/CH₂Cl₂), the corresponding dipeptide was obtained (4.0 g,

(−)-(S)-Butyl 2-{2-[(tert-Butoxycarbonyl)amino]hex-5-enami-

do}-2-methylpropanoate ((−)-39). To a solution of (+)-14 (959 mg,

3.95 mmol) in THF (15 mL) was added LiOH (434 mg, 9.88 mmol) in

water (15 mL). The mixture was stirred at room temperature for 2−3 h

until completion. A 5% aqueous solution of H₃PO₄was added until pH

3, and the organic phase was extracted with EtOAc, dried (MgSO₄), and

evaporated to give the corresponding carboxylic acid (915 mg, 4 mmol).

To a cooled solution of the following residue and H-Aib-OnBu (636 mg,

4 mmol) in dry DMF (20 mL) were added HOBt·H₂O (540 mg, 4

mmol), DCC (929 mg, 4.80 mmol), and Et₃N (0.6 mL), and the mixture

was stirred at room temperature overnight (18 h). DMF was evaporated,

and the residue was diluted with EtOAc and washed with 10% citric acid,

4% sodium carbonate, and brine. The organic phase was dried (MgSO₄)

and evaporated. The residue was puriﬁed by ﬂash chromatography

(silica gel, 20% EtOAc/cyclohexane) to give the corresponding

dipeptide (−)-39 (1.17 g, 3.16 mmol, 80%): [α]_D²⁰= −17.0° (c = 1.0,

CHCl₃); R_f= 0.23 (20% EtOAc/cyclohexane); IR ν_max(ﬁlm, CH₂Cl₂)

3358, 2978, 2890, 1745, 1750, 1518, 1453, 1366, 1220, 1070, 914 cm⁻¹;

¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.82 (t, J = 7.4 Hz, 3H), 1.26 (m,

2H), 1.32 (ds, 9H), 1.41 (ds, 6H), 1.50 (m, 2H), 1.61 (m, 1H), 1.77 (m,

1H), 2.04 (dd, J = 13.7, 6.7 Hz, 2H), 4.00 (m, 2H), 4.1 (td, J = 12.0, 5.9

Hz, 1H), 4.90 (m, 2H), 5.57−5.79 (m, 2H, NH), 7.28 (s, 1H, NH); ¹³C

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20

8.84 mmol, 92%). To a solution of this protected dipeptide in acetic acid

(18 mL) was added Pd/C 10% (520 mg, 0.48 mmol), and the mixture

was stirred under H₂at room temperature overnight. The mixture was

ﬁltered on Celite, washed with EtOAc, and concentrated in vacuo. After

ﬂash chromatography (silica gel, 3% MeOH/CH₂Cl₂), the product

(+)-42 (2.39 g, 85%) was isolated as a colorless ﬁlm: [α]_D²⁰= +43.0° (c

= 1.0, CHCl₃); R_f= 0.23 (5% MeOH/CH₂Cl₂); IR ν_max(ﬁlm, CH₂Cl₂)

3358, 2978, 2929, 1724, 1646, 1454, 1364, 1254, 1152, 1090, 910, 849,

756, 694 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.39 (s, 9H), 1.49

(m, 1H), 1.71−1.90 (m, 3H), 2.62 (m, 1H), 2.74 (m, 2H), 3.44 (m, 1H),

3.72 (dd, J = 7.9, 6.8 Hz, 1H), 4.18 (dd, J = 8.3, 3.9 Hz, 1H), 7.05−7.30

(m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 24.3 (CH₂), 27.8 (3 ×

CH₃), 28.7 (CH₂), 42.0 (CH₂), 46.4 (CH₂), 54.7 (CH), 59.3 (CH),

81.0 (C), 126.5 (CH), 128.2 (2 × CH), 129.2 (2 × CH), 137.5 (C),

171.1 (C), 173.1 (C).

(−)-(R)-tert-Butyl 1-[(S)-2-Amino-3-phenylpropanoyl]-

pyrrolidine-2-carboxylate ((−)-42). According to the procedure

reported for (+)-42 and starting from (Z)-^D-Phe-OH (3.0 g, 10.0 mmol)

and H-Pro-O-t-Bu (2.1 g, 10.0 mmol), the protected and unprotected

dipeptides (−)-42 were subsequently obtained in yields of 92% (4.1 g)

and 79% (2.17 g), respectively: [α]_D²⁰= −42.6° (c = 1.0, CHCl₃); IR

ν_max(ﬁlm, CH₂Cl₂) 3358, 2978, 2929, 1724, 1646, 1454, 1364, 1254,

1152, 1090, 910, 849, 756, 694 cm⁻¹; HRMS (ESI+) m/z calcd for

C₁₈H₂₆N₂O₃Na 341.1841, found 341.1838.

tetrapeptide 47 (35 mg, 48%) was isolated as a white foam: [α]_D

=

−80.0° (c = 0.5, CHCl₃); R_f= 0.23 (20% EtOAc/cyclohexane); IR ν_max

(ﬁlm, CH₂Cl₂) 330, 2921, 2852, 1650, 1630, 1585, 1458, 1380, 719 cm⁻¹

; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.87−1.01 (m, 2H), 1.07−1.28

(m, 4H), 1.34 (s, 3H), 1.54−1.63 (m, 2H), 1.64−1.75 (m, 5H), 1.77 (s,

3H), 1.79−1.97 (m, 4H), 2.09 (m, 2H), 2.26 (m, 1H), 2.39 (m, 1H),

3.51 (ddd, J = 10.2, 7.4, 7.4 Hz, 1H), 3.97 (ddd, J = 10.2, 8.4, 8.4 Hz, 1H),

4.23 (ddd, J = 10.3, 7.5, 7.5 Hz, 1H), 4.74 (dd, J = 7.9, 2.0 Hz, 1H), 4.88−

5.09 (m, 3H), 5.77 (ddd, J = 16.9, 10.2, 6.6 Hz, 1H), 5.92 (s, 1H, NH),

7.13 (d, J = 10.3 Hz, 1H), 7.31 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 23.8 (CH₂), 25.0 (CH₂), 25.4 (CH₂), 26.3

(CH₃), 26.6 (CH₃), 26.8 (CH₂), 28.3 (CH₂), 29.8 (CH₂), 33.4 (CH),

33.5 (CH₂), 34.1 (CH₂), 34.6 (CH), 37.1 (CH₂), 47.3 (CH₂), 50.3

(CH), 54.0 (CH), 58.0 (CH), 59.0 (C), 115.9 (CH₂), 137.2 (CH),

172.1 (C), 173.7 (C), 174.4 (C), 175.8 (C); HRMS (ESI+) m/z calcd

for C₂₄H₃₈N₄O₄Na 469.2785, found 469.2781.

(−)-(3S,6S,9S,14aR)-6,9-Dibenzyl-3-(but-3-enyl)-6-methyl-

decahydropyrrolo[1,2-α][1,4,7,10]tetraazacyclododecine-

1,4,7,10-tetraone (48). To a solution of methyl ester (+)-40 (610 mg,

1.51 mmol) in THF (10 mL) was added a solution of LiOH (90 mg, 3.77

mmol) in water (10 mL). The reaction mixture was stirred overnight at

room temperature, and the reaction was quenched by addition of a

solution of 5% H₃PO₄until pH 3. After extraction with EtOAc, the

organic layer was dried over MgSO₄and evaporated to give the

corresponding carboxylic acid. To a solution of this carboxylic acid and

amine (+)-42 (480 mg, 1.51 mmol) in DMF (4.5 mL) were added

HOBt·H₂O (245 mg, 1.81 mmol), EDC (347 mg, 1.81 mmol), and N-

methylmorpholine (0.33 mL, 3.02 mmol). The reaction mixture was

stirred overnight at room temperature, and the solvent was evaporated.

The residue was dissolved in EtOAc, and the organic layer was washed

with 10% citric acid, 5% NaHCO₃, and brine. The organic layer was

dried over Na₂SO₄and concentrated in vacuo. After ﬂash chromatog-

raphy (silica gel, 20−30% EtOAc/cyclohexane), the product 45 (832

mg, 80%) was isolated as a colorless ﬁlm: R_f= 0.38 (40% EtOAc/

cyclohexane); [α]_D²⁰= +24.1° (c = 1.1, CHCl₃); LRMS (ESI+) m/z (%)

713 (8) [M + Na]⁺, 691 (100) [M + H]⁺; HRMS (ESI+): m/z calcd for

C₃₉H₅₅N₄O₇691.4065, found 691.4065. The tetrapeptide 45 (150 mg,

0.22 mmol) was dissolved in TFA (2 mL) at 0 °C and stirred for 3 h at

this temperature. After evaporation, the product was precipitated in dry

ether. To a solution of this tetrapeptide triﬂuoroacetic acid in DMF (13

mL) were added HATU (91 mg, 0.24 mmol) and DIEA (0.11 mL, 0.63

mmol) in ﬁve aliquots with a time interval of 30 min under vigorous

stirring. The reaction mixture was stirred overnight. The solvent was

then removed, and EtOAc was added. After it was washed with a

solution of 10% citric acid followed by 5% NaHCO₃, the organic layer

was dried over MgSO₄and evaporated. After ﬂash chromatography

(silica gel, 30−40% EtOAc/cyclohexane), the cyclotetrapetide 48 (25

mg, 22%) was isolated as a white foam: R_f= 0.34 (40% EtOAc/

(R)-tert-Butyl 1-[(S)-2-Amino-3-cyclohexylpropanoyl]pyrrol-

idine-2-carboxylate ((+)-43). According to the procedure reported

for (+)-42, and starting with (Z)-Cha-OH (1.0 g, 3.28 mmol) and H-^D-

Pro-O-t-Bu (679 mg, 3.28 mmol), the protected and unprotected

dipeptides (+)-43 were subsequently obtained in yields of 93% (1.4 g)

and 97% (964 mg), respectively: [α]_D²⁰= +67.2° (c = 1.0, CHCl₃); R_f

=0.21 (20% EtOAc/cyclohexane); IR ν_max(ﬁlm, CH₂Cl₂) 2974, 2925,

1

2851, 1723, 1638, 1442, 1364, 1217, 1148, 841 cm⁻¹; H NMR (400

MHz, CDCl₃) δ (ppm) 0.70−0.91 (m, 2H), 1.03 (m, 1H), 1.18 (m, 2H),

1.26 (m, 1H), 1.36 (s, 9H), 1.57 (m, 4H), 1.75 (m, 1H), 1.79−1.92 (m,

3H), 2.04 (m, 2H), 3.37 (m, 1H), 3.51 (m, 1H), 3.60 (m, 1H), 4.01 (dd,

J = 14.3, 7.1 Hz, 1H), 4.23 (dd, J = 8.3, 3.4 Hz, 1H); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 24.6 (CH₂), 25.9 (CH₂), 26.2 (CH₂), 26.4

(CH₂), 27.9 (3 × CH₃), 28.9 (CH), 32.5 (CH₂), 33.9 (CH₂), 34.2

(CH₂), 42.4 (CH₂), 46.8 (CH₂), 50.4 (CH), 59.6 (CH), 81.0 (C), 171.3

(C), 174.9 (C); LRMS (ESI+) m/z (%) 325 (100) [M + H]⁺, 269 (75),

649 (20); HRMS (ESI+) m/z calcd for C₁₈H₃₂N₂NaO₃347.2305, found

347.2301.

(−)-(3S,9S,14aR)-9-Cyclohexan-3-(but-3-enyl)-6,6-dimethyl-

decahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-

1,4,7,10-tetraone (47). To a solution of the dipeptide (−)-39 (120

mg, 0.32 mmol) in THF/H₂O/MeOH (1/1/0.5, 2.5 mL) was added

LiOH (36 mg, 0.82 mmol), and the mixture was stirred at room

temperature for 2 h. A 5% aqueous solution of H₃PO₄was added until

pH 3, and the organic phase was extracted with EtOAc, dried (MgSO₄),

and evaporated to give the corresponding unprotected dipeptide. To a

cooled solution of this dipeptide and (Z)-Cha-^D-Pro-O-t-Bu ((+)-43;

131 mg, 0.32 mmol) in dry DMF (5 mL) were added HOBt·H₂O (44

mg, 0.32 mmol), DCC (80 mg, 0.39 mmol), and Et₃N (45 μL), and the

mixture was stirred at room temperature overnight (18 h). DMF was

evaporated, and the residue was diluted with EtOAc and washed with

10% citric acid, 4% sodium carbonate, and brine. The organic phase was

dried (MgSO₄) and evaporated. The residue was puriﬁed by ﬂash

chromatography (silica gel, 20% EtOAc/cyclohexane) to give the

corresponding tetrapeptide 44 (101 mg, 50%): HRMS (ESI+) m/z

calcd for C₃₃H₅₀N₄O₇Na 637.3577, found 637.3577. This tetrapeptide

was dissolved in TFA (1 mL) at 0 °C and stirred for 3 h at this

temperature. After evaporation, the product was precipitated in dry

ether to give after ﬁltration the unprotected product. The tetrapeptide

triﬂuoroacetic acid, HATU (75 mg, 0.19 mmol), and DIEA (0.1 mL)

were added in ﬁve aliquots with a time interval of 30 min under vigorous

stirring in DMF (100 mL). Then the reaction mixture was stirred for 18

h at room temperature. The DMF was evaporated, and EtOAc was

added. After washing with a solution of 10% citric acid followed by 4%

NaHCO₃, the organic phase was dried (MgSO₄) and evaporated. After

ﬂash chromatography (silica gel, 30% EtOAc/cyclohexane), the cyclic

20

cyclohexane); [α]_D= −134.7° (c = 0.7, CHCl₃); IR ν_max(thin ﬁlm,

CH₂Cl₂) 3375, 1626, 1654, 1532, 1430, 1246, 993, 911 cm⁻¹; ¹H NMR

(400 MHz, CDCl₃) δ (ppm) 1.22 (s, 3H), 1.75−1.92 (m, 4H), 2.01−

2.27 (m, 3H), 2.34−2.41 (m, 1H), 3.14 (dd, J = 13.8, 7.0 Hz, 1H), 3.29−

3.42 (m, 2H), 3.42 (d, J = 13.9 Hz, 1H), 3.70 (d, J = 13.9 Hz, 1H), 3.87−

3.95 (m, 1H), 4.25−4.32 (m, 1H), 4.73−4.77 (m, 1H), 5.02−5.09 (m,

2H), 5.27−5.36 (m, 1H), 5.77−5.87 (m, 1H), 5.89 (bs, NH), 7.08−7.10

(m, 2H), 7.20 (d, J = 10.3 Hz, NH), 7.22−7.47 (m, 8H), 7.91 (d, J = 10.3

Hz, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.4 (CH₃), 25.3

(CH₂), 27.1 (CH₂), 28.5 (CH₂), 29.8 (CH₂), 36.0 (CH₂), 40.7 (CH₂),

47.2 (CH₂), 53.6 (CH), 54.1 (CH), 58.1 (CH), 62.9 (C), 116.1 (CH₂),

127.0 (CH), 127.1 (CH), 128.4 (2 × CH), 128.9 (2 × CH), 129.3 (2 ×

CH), 130.4 (2 × CH), 135.6 (C), 137.2 (CH), 137.3 (C), 171.9 (C),

173.0 (C), 174.8 (C), 175.4 (C); LRMS (ESI+) m/z (%) 517 (100) [M

+ H]⁺; HRMS (ESI+) m/z calcd for C₃₀H₃₇N₄O₄517.2809, found

517.2808.

(−)-(3S,6R,9S,14aR)-6,9-Dibenzyl-3-(but-3-enyl)-6-methyl-

decahydropyrrolo[1,2-α][1,4,7,10]tetraazacyclododecine-

1,4,7,10-tetraone (49). According to the procedure reported for 48

and starting from dipeptide (−)-41 (504 mg, 1.25 mmol) and dipeptide

(+)-42 (397 mg, 1.25 mmol), the tetrapeptide 46 (635 mg, 74%) was

obtained after ﬂash chromatography (silica gel, 20−30% EtOAc/

3671

dx.doi.org/10.1021/jo4001492 | J. Org. Chem. 2013, 78, 3655−3675

The Journal of Organic Chemistry

Article

cyclohexane) as a colorless oil: R_f= 0.14 (30% EtOAc/cyclohexane);

[α]_D²⁰= +33.8° (c = 1.1, CHCl₃); LRMS (ESI+) m/z (%) 691 (100) [M

+ H]⁺; HRMS (ESI+): m/z calcd for C₃₉H₅₅N₄O₇691.4065, found

691.4060. Starting from the tetrapeptide 46 (480 mg, 0.70 mmol), the

cyclotetrapeptide 49 (80 mg, 36%) was obtained after ﬂash

chromatography (silica gel, 20−30% EtOAc/cyclohexane) as a white

foam: R_f= 0.36 (40% EtOAc/cyclohexane); [α]_D²⁰= −41.5° (c = 1.4,

CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3309, 2921, 2851, 1679, 1625,

1527, 1450, 1262, 739, 702 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm)

1.65−1.86 (m, 6H), 1.88−1.98 (m, 1H), 2.02−2.21 (m, 3H), 2.28−2.35

(m, 1H), 2.99 (d, J = 14.1 Hz, 1H), 3.01 (dd, J = 13.4, 5.7 Hz, 1H), 3.19

(d, J = 14.1 Hz, 1H), 3.22−3.34 (m, 2H), 3.80−3.87 (m, 1H), 4.22 (dt, J

= 15.1, 7.6 Hz, 1H), 4.66−4.71 (m, 1H), 4.94−4.98 (m, 1H), 4.99−5.01

(m, 1H), 5.20 (dt, J = 10.1, 5.8 Hz, 1H), 5.77 (ddt, J = 16.5, 10.8, 6.8 Hz,

1H), 6.10 (bs, NH), 7.08 (d, J = 10.3 Hz, NH), 7.23−7.42 (m, 10H),

7.48 (d, J = 10.2 Hz, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 22.1

(CH₃), 25.0 (CH₂), 27.1 (CH₂), 28.1 (CH₂), 29.9 (CH₂), 36.1 (CH₂),

44.0 (CH₂), 47.0 (CH₂), 53.8 (CH), 53.9 (CH), 58.0 (CH), 61.8 (C),

116.0 (CH₂), 126.9 (CH), 127.4 (CH), 128.6 (2 × CH), 128.8 (2 ×

CH), 129.3 (2 × CH), 131.2 (2 × CH), 135.5 (C), 137.1 (CH), 137.2

(C), 171.8 (C), 172.9 (C), 174.5 (C), 175.8 (C); LRMS (ESI+) m/z

(%) 517 (100) [M + H]⁺; HRMS (ESI+) m/z calcd for C₃₀H₃₇N₄O₄

517.2809, found 517.2806.

from this xanthate intermediate (20 mg, 0.026 mmol), the next silylated

intermediate (10.5 mg, 62%) was obtained after ﬂash chromatography

(silica gel, 0.8% MeOH/CH₂Cl₂) as a colorless ﬁlm: R_f= 0.30 (30%

EtOAc/cyclohexane); [α]_D²⁰= −64.7° (c = 0.8, CHCl₃); HRMS (ESI+)

m/z calcd for C₃₄H₆₀N₄NaO₆Si 671.416615, found 671.417433.

Starting from this silylated intermediate (10.5 mg, 0.016 mmol), the

product 50 (10.3 mg, 100%) was obtained after ﬂash chromatography

(silica gel, 1% MeOH/CH₂Cl₂) as a colorless oil: R_f= 0.30 (30%

EtOAc/cyclohexane); [α]_D²⁰= −78.0° (c = 1.0, CHCl₃); IR ν_max(ﬁlm,

CH₂Cl₂) 3465, 3281, 2983, 2925, 2860, 1724, 1643, 1458, 1368, 1225,

1

1152, 1368, 907, 841, 747, 703 cm⁻¹, H NMR (400 MHz, CDCl₃) δ

(ppm) 1.02−0.84 (m, 2H), 1.10−1.24 (m, 2H), 1.26 (m, 2H), 1.26−

1.32 (m, 3H), 1.34 (s, 3H), 1.38 (d, J = 7.1 Hz, 3H), 1.63−1.57 (m, 7H),

1.73−1.64 (m, 4H), 1.77 (s, 3H), 1.78−1.97 (m, 3H), 2.33−2.14 (m,

1H), 2.40−2.55 (m, 2H), 3.51 (td, J = 10.2, 7.4 Hz, 2H), 3.98 (ddd, J =

10.2, 8.5, 4.8 Hz, 1H), 4.27−4.14 (m, 2H), 4.73 (dd, J = 7.9, 2.0 Hz, 1H),

4.97 (ddd, J = 10.0, 8.3, 7.2 Hz, 1H), 5.93 (s, 1H, NH), 7.12 (d, J = 10.2

Hz, 1H, NH), 7.29 (s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm)

20.1 (CH₃), 23.5 (CH₂), 23.8 (CH₃), 25.0 (CH₂), 25.4 (CH₂), 25.5

(CH₂), 26.3 (CH₃), 26.6 (CH₂), 26.8 (CH₃), 28.9 (CH₂), 29.0 (CH₂),

29.9 (CH), 33.4 (CH₂),, 36.6 (CH₂), 34.7 (CH₂), 37.2 (CH₂), 37.5

(CH₂), 47.3 (CH₂), 50.3 (CH), 54.5 (CH), 58.0 (C), 59.0 (CH), 72.8

(CH), 172.2 (C), 173.7 (C), 174.4 (C), 175.8 (C) 212.6 (C); LRMS

(ESI+) m/z (%) 535 (100) [M + H]⁺, 225 (70); HRMS (ESI+) m/z

calcd for C₂₈H₄₆N₄O₆Na 557.3305, found 557.3310.

(−)-(3S,6S,9S,14aR)-6,9-Dibenzyl-3-[(R)-7-hydroxy-6-oxooc-

tyl]-6-methyldecahydropyrrolo[1,2-α][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (51). A solution of cyclotetrapeptide 48

(87 mg, 0.17 mmol) and carbonyl xanthate 8e (109 mg, 0.34 mmol) in

1,2-dichloroethane (0.3 mL) was reﬂuxed for 30 min under argon.

Dilauroyl peroxide (DLP, 8 mg) was added every 1 h (ﬁve to seven

times). After the mixture was reﬂuxed overnight, the solvent was

removed in vacuo and the residue was directly puriﬁed by ﬂash

chromatography (silica gel, 20−30% EtOAc/cyclohexane) to give the

xanthate intermediate (82 mg, 58%) as a colorless ﬁlm: R_f= 0.35 (40%

EtOAc/cyclohexane); LRMS (ESI+) m/z (%) 839 (100) [M + H]⁺;

HRMS (ESI+): m/z calcd for C₄₃H₆₃N₄O₇S₂Si 839.3902, found

839.3892. A solution of this intermediate (75 mg, 0.09 mmol) in

isopropyl alcohol (4 mL) was reﬂuxed for 5 h with DLP (50 mg). After

evaporation and direct ﬂash chromatography (silica gel, 30−40%

EtOAc/cyclohexane), the silylated intermediate (40 mg, 62%) was

(−)-(3S,6R,9S,14aR)-6,9-Dibenzyl-3-[(R)-7-hydroxy-6-oxooc-

tyl]-6-methyldecahydropyrrolo[1,2-α][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (52). According to the procedure

reported for 51 and starting from cyclic tetrapeptide 49 (70 mg, 0.14

mmol), the xanthate intermediate (80 mg, 70%) was obtained after ﬂash

chromatography (silica gel, 20−30% EtOAc/cyclohexane) as a colorless

ﬁlm: R_f= 0.36 (40% EtOAc/cyclohexane); LRMS (ESI+) m/z (%) 856

(100) [M + H₂O + H]⁺, 839 (46) [M + H]⁺, 391 (35); HRMS (ESI+):

m/z calcd for C₄₃H₆₃N₄O₇S₂Si 839.3902, found 839.3897. Starting from

this xanthate intermediate (75 mg, 0.09 mmol), the next silylated

intermediate (49 mg, 76%) was obtained after ﬂash chromatography

(silica gel, 30% EtOAc/cyclohexane) as a colorless ﬁlm: R_f= 0.42 (40%

EtOAc/cyclohexane); [α]_D²⁰= −13.6° (c = 1.9, CHCl₃); LRMS (ESI+)

m/z (%) 736 (100) [M + H₂O + H]⁺, 719 (69) [M + H]⁺, 391 (86);

HRMS (ESI+) m/z calcd for C₄₀H₅₉N₄O₆Si 719.4198, found 719.4190.

Starting from this silylated intermediate (30 mg, 0.04 mmol), the

product 52 (21 mg, 85%) was obtained after ﬂash chromatography

(silica gel, 1% MeOH/CH₂Cl₂) as a colorless oil: R_f= 0.24 (4% MeOH/

CH₂Cl₂); [α]_D²⁰= −32.1° (c = 0.7, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂)

20

obtained as a colorless ﬁlm: R_f= 0.40 (40% EtOAc/cyclohexane); [α]_D

= −90.1° (c = 0.8, CHCl₃); LRMS (ESI+) m/z (%) 719 (100) [M+H]⁺;

HRMS (ESI+): m/z calcd for C₄₀H₅₉N₄O₆Si 719.4198, found 719.4190.

TBAF (1 M in THF, 42 μL) was added to a solution of the silylated

intermediate (30 mg, 0.042 mmol) in THF (1.5 mL). The mixture was

stirred for 30 min at room temperature. After evaporation, the residue

was directly puriﬁed by ﬂash chromatography (1% MeOH/CH₂Cl₂) to

give 51 (23 mg, 91%) as a colorless oil: R_f= 0.24 (4% MeOH/CH₂Cl₂);

[α]_D²⁰= −117.0° (c = 0.9, CHCl₃); IR ν_max(thin ﬁlm, CH₂Cl₂) 3297,

2921, 2847, 1674, 1654, 1523, 698 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ

(ppm) 1.21 (s, 3H), 1.25−1.41 (m, 4H), 1.41 (d, J = 7.1 Hz, 3H), 1.61−

1.98 (m, 7H), 2.17−2.26 (m, 1H), 2.33−2.62 (m, 3H), 3.13 (dd, J =

13.8, 7.0 Hz, 1H), 3.29−3.40 (m, 2H), 3.43 (d, J = 13.7 Hz, 1H), 3.69 (d,

J = 13.7 Hz, 1H), 3.86−3.94 (m, 1H), 4.29 (q, J = 7.1 Hz,1H), 4.76 (dd, J

= 7.9, 2.4 Hz, 1H), 5.27−5.36 (m, 1H), 6.01 (s, 1H, NH), 7.04−7.10 (m,

2H), 7.20−7.36 (m, 9H), 7.91 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 20.1 (CH₃), 23.3 (CH₂), 23.5 (CH₃), 24.9

(CH₂), 25.2 (CH₂), 25.5 (CH₂), 29.0 (CH₂), 29.8 (CH₂), 36.0 (CH₂),

37.5 (CH₂), 40.7 (CH₂), 47.2 (CH₂), 53.6 (CH), 54.7 (CH), 58.0

(CH), 62.9 (C), 72.8 (CH), 126.9 (CH), 127.1 (CH), 128.4 (2 × CH),

128.9 (2 × CH), 129.3 (2 × CH), 130.4 (2 × CH), 135.6 (C), 137.2

(C), 172.0 (C), 173.0 (C), 174.9 (C), 175.4 (C), 212.6 (C); LRMS (ESI

+) m/z (%) 627 (6) [M + Na]⁺, 605 (100) [M + H]⁺, 517 (8); HRMS

(ESI+) m/z calcd for C₃₄H₄₅N₄O₆605.3334, found 605.3326.

1

3305, 2925, 2851, 1674, 1527, 1450, 1258, 698 cm⁻¹; H NMR (400

MHz, CDCl₃) δ (ppm) 1.25−1.36 (m, 4H), 1.42 (d, J = 7.1 Hz, 3H),

1.60−1.71 (m, 4H), 1.71−1.86 (m, 5H), 2.08−2.17 (m, 1H), 2.28−2.36

(m, 1H), 2.38−2.57 (m, 2H), 3.00 (m, 2H), 3.17−3.24 (m, 2H), 3.29

(dd, J = 13.4, 10.1 Hz, 1H), 3.78−3.86 (m, 1H), 4.15−4.25 (m, 1H),

4.28 (q, J = 7.0 Hz,1H), 4.69 (dd, J = 7.6, 1.8 Hz, 1H), 5.19 (ddd, J =

10.1, 10.1, 5.8 Hz,1H), 6.18 (s, 1H, NH), 7.09 (d, J = 10.2 Hz, 1H, NH),

7.23−7.40 (m, 10H), 7.45 (d, J = 10.1 Hz, 1H, NH); ¹³C NMR (100

MHz, CDCl₃) δ (ppm) 20.1 (CH₃), 22.9 (CH₂), 23.5 (CH₃), 24.9

(CH₂), 25.0 (CH₂), 25.4 (CH₂), 28.8 (CH₂), 29.0 (CH₂), 36.2 (CH₂),

37.5 (CH₂), 43.9 (CH₂), 47.0 (CH₂), 53.9 (CH), 54.4 (CH), 58.1

(CH), 61.9 (C), 72.8 (CH), 127.0 (CH), 127.4 (CH), 128.6 (2 × CH),

128.8 (2 × CH), 129.3 (2 × CH), 131.3 (2 × CH), 135.6 (C), 137.2

(C), 172.0 (C), 173.0 (C), 174.6 (C), 175.8 (C), 212.6 (C); LRMS (ESI

+) m/z (%) 605 (100) [M + H]⁺; HRMS (ESI+) m/z calcd for

C₃₄H₄₅N₄O₆605.3334, found 605.3330.

(+)-(3R,9R,14aS)-9-Benzyl-3-(but-3-enyl)-6,6-dimethyldeca-

hydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,10-

tetraone ((+)-17). According to the procedure reported for 47 and

starting from dipeptide (+)-39 (1.11 g, 3.06 mmol) and dipeptide

(−)-42 (1.08 g, 2.68 mmol), the corresponding tetrapeptide (1.12 g,

68%) was obtained after ﬂash chromatography (silica gel, 20% EtOAc/

cyclohexane) as a white foam: HRMS (ESI+) m/z calcd for

C₃₃H₅₀N₄O₇Na 637.3577, found 637.3577. This tetrapeptide was

converted into cyclic tetrapeptide (+)-17 (350 mg, 51%, ﬂash

chromatography: silica gel, 20% EtOAc/cyclohexane) as a white foam:

(−)-(3S,9S,14aR)-9-(Cyclohexylmethyl)-3-[(R)-7-hydroxy-6-

oxooctyl]-6,6-dimethyldecahydropyrrolo[1,2-a][1,4,7,10]-

tetraazacyclododecine-1,4,7,10-tetraone (50). According to the

procedure reported for 51 and starting from cyclic tetrapeptide 47 (36

mg, 0.081 mmol) and carbonyl xanthate 8e (65 mg, 0.2 mmol), the

xanthate intermediate (40.5 mg, 65%) was obtained after ﬂash

chromatography (silica gel, 20−30% EtOAc/cyclohexane). Starting

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20

[α]_D= +109° (c = 1, CHCl₃); HRMS (ESI+) m/z calcd for

(2 × CH), 137.3 (C), 138.2 (CH), 172.0 (C), 173.0 (C), 174.5 (C),

175.8 (C); HRMS (ESI+) m/z calcd for C₂₅H₃₄N₄O₄Na 477.2472,

found 477.2469.

C₂₄H₃₂N₄O₄Na 463.2321, found 463.2321.

(+)-(3R,9R,14aS)-9-Benzyl-3-[(R)-7-hydroxy-6-oxooctyl]-6,6-

dimethyldecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecine-1,4,7,10-tetraone (53). According to the procedure

reported for 51 and starting from cyclotetrapeptide (+)-17 (63.6 mg,

0.14 mmol) and carbonyl xanthate 8e (112 mg, 0.35 mmol), the

xanthate intermediate (60 mg, 57%) was obtained after ﬂash

chromatography (silica gel, 20−30% EtOAc/cyclohexane) as a colorless

ﬁlm. Starting from this xanthate intermediate (48 mg, 0.063 mmol), the

next silylated intermediate (30 mg, 75%) was obtained after ﬂash

chromatography (silica gel, 20% EtOAc/cyclohexane) as a colorless

(−)-(S)-4-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl]pentyl Octanethioate (57). To a solution of 56 (92.2

mg, 0.20 mmol) in dry THF (10 mL) was added thioacetic acid 27

(129.5 mg, 0.89 mmol). The mixture was reﬂuxed for 30 min under

argon. A catalytic amount of AIBN was added, and the mixture was

stirred at reﬂux for 16 h while the reaction mixture was held up to the

light. After evaporation, the residue was directly puriﬁed by ﬂash

chromatography (silica gel, 1% MeOH/CH₂Cl₂) to give 57 (80 mg,

20

ﬁlm: R_f= 0.30 (30% EtOAc/cyclohexane); [α]_D= +78.5° (c = 0.8,

65%) as a colorless ﬁlm: R_f= 0.12 (1% MeOH/CH₂Cl₂); [α]_D

=

CHCl₃); HRMS (ESI+) m/z calcd for C₃₄H₅₄N₄O₆NaS_i665.3710,

found 665.3705. Starting from this silylated intermediate (11.8 mg,

0.018 mmol), the product 53 (10.3 mg, 100%) was obtained after ﬂash

chromatography (silica gel, 1% MeOH/CH₂Cl₂) as a colorless oil:

[α]_D²⁰= +64.7° (c = 0.43, CHCl₃); R_f= 0.16 (2% MeOH/CH₂Cl₂); ¹H

NMR (400 MHz, CDCl₃) δ (ppm) 1.22−1.30 (m, 4H), 1.34 (s, 3H),

1.38 (dd, J = 7.1 Hz, 3H), 1.57−1.69 (m, 4H), 1.77 (m, 2H), 1.77 (m,

3H), 2.18 (m, 1H), 2.33 (m, 1H), 2.38−2.58 (m, 2H), 2.95 (dd, J = 13.5,

5.7 Hz, 1H), 3.11−3.39 (m, 2H), 3.8 (m, 1H), 4.21 (m, 2H), 4.66 (d, J =

7.3 Hz, 1H), 5.16 (ddd, J = 10.1, 10.1, 5.8 Hz, 1H), 6.0 (s, 1H, NH), 7.13

(d, J = 10.2 Hz, 1H, NH), 7.17−7.31 (m, 5H), 7.50 (d, J = 10.2 Hz, 1H,

NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 20.1 (CH₃), 23.5 (CH₂),

23.8 (CH₃), 25.0 (CH₂), 25.2 (CH₂), 25.4 (CH₂), 26.7 (CH₃), 28.9

(CH₂), 29.0 (CH₂), 36.0 (CH₂), 37.5 (CH₂), 47.2 (CH₂), 53.7 (CH),

54.5 (CH), 58.0 (CH), 59.0 (C, C9), 72.8 (CH, C24), 126.9 (CH, C17),

128.8 (2 × CH, 2 × C15), 129.2 (2 × CH, 2 × C16), 137.2 (C), 172.1

(C), 173.0 (C), 174.5 (C), 175.8 (C), 212.6 (C); LRMS (ESI+) m/z

(%) 551 (100) [M + Na]⁺, 529 (10); HRMS (ESI+) m/z calcd for

C₂₈H₄₀N₄O₆Na 551.2846, found 551.2847.

−60.7° (c = 0.7, CHCl₃); IR ν_max(ﬁlm) 3296, 2945, 2920, 2855, 1689,

1658, 1620, 1524, 1420, 1225, 1176 cm⁻¹; ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 0.88 (t, J = 5.9, Hz, 3H), 1.25−1.33 (m, 10H), 1.34 (s, 3H),

1.41 (m, 2H), 1.56 (m, 2H), 1.59−1.69 (m, 2H), 1.71−1.86 (m, 7H),

2.18 (m, 1H), 2.32 (m, 1H), 2.50 (t, J = 7.5 Hz, 2H), 2.85 (t, J = 7.3 Hz,

2H), 2.95 (dd, J = 13.5, 1H), 3.16−3.30 (m, 2H), 3.86 (dt, J = 10.1, 4.6

Hz, 1H), 4.18 (td, J = 10.2, 7.6 Hz, 1H), 4.66 (d, J = 5.7 Hz, 1H), 5.16

(dt, J = 10.1, 5.8 Hz, 1H), 5.93 (s, 1H, NH), 7.09 (d, J = 10.2 Hz, 1H,

NH), 7.13−7.35 (m, 5H), 7.51 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR

(100 MHz; CDCl₃) δ (ppm) 14.3 (CH₃), 22.8 (CH₂), 23.8 (CH₃), 25.0

(CH₂), 25.2 (CH₂), 25.9 (CH₂), 26.7 (CH₃), 28.6 (CH₂), 28.8 (CH₂),

29.0 (CH₂), 29.1 (2 × CH₂), 29.6 (CH₂), 31.8 (CH₂), 34.0 (CH₂), 36.0

(CH₂), 44.4 (CH₂), 47.2 (CH₂), 53.7 (CH), 54.5 (CH), 58.0 (CH),

59.0 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2 (C),

172.0 (C), 173.0 (C), 174.5 (C), 175.8 (C), 199.9 (C); HRMS (ESI+)

m/z calcd for C₃₃H₅₀N₄NaO₅S 637.3394, found 637.3387.

(−)-(S)-4-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl]pentyl Ethanethioate (58). To a solution of 56 (10

mg, 0.022 mmol) in dry THF (10 mL) was added thioacetic acid 26 (7

mg, 0.092 mmol). The mixture was reﬂuxed for 30 min under argon. A

catalytic amount of AIBN was added, and the mixture was stirred at

reﬂux for 16 h while the reaction mixture was held up to the light. After

evaporation, the residue was directly puriﬁed by ﬂash chromatography

(silica gel, 1% MeOH/CH₂Cl₂) to give 58 (7 mg, 59%) as a colorless

ﬁlm: R_f= 0.12 (1% MeOH/CH₂Cl₂); [α]_D²⁰= −54.3° (c = 0.7, CHCl₃);

IR ν_max(ﬁlm) 3307, 2934, 1684, 1630, 1528, 1428, 1274, 1187, 915

cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.24 (m, 2H), 1.34 (s, 3H),

1.40 (m, 2H), 1.57 (m, 2H), 1.61 (s, 3H), 1.77−1.87 (m, 5H), 2.17 (m,

1H), 2.32 (s, 3H), 2.85 (t, J = 7.2 Hz, 2H), 2.95 (dd, J = 13.5, 5.7 Hz,

1H), 3.25 (m, 1H), 3.75 (m, 1H), 3.85 (m, 1H), 4.18 (td, J = 10.3, 7.6,

7.6 Hz, 1H), 4.66 (dd, J = 7.7, 2.3 Hz, 1H), 5.16 (dt, J = 10.1, 10.1, 5.8

Hz, 1H), 5.95 (s, 1H, NH), 7.09 (d, J = 10.3 Hz, 1H, NH), 7.18−7.34

(m, 5H), 7.50 (d, J = 10.2 Hz, 1H, NH); ¹³C NMR (100 MHz; CDCl₃) δ

(ppm) 23.8 (CH₃), 25.0 (CH₂), 25.3 (2 × CH₂), 26.7 (CH₃), 28.6

(CH₂), 29.0 (CH₂), 29.1 (CH₂), 29.5 (CH₂), 30.9 (CH₃), 36.1 (CH₂),

47.2 (CH₂), 53.7 (CH), 54.6 (CH), 58.0 (CH), 59.1 (C), 126.9 (CH),

128.8 (2 × CH), 129.3 (2 × CH), 137.3 (C), 172.1 (C), 173.0 (C),

174.5 (C), 175.9 (C), 196.2 (C); HRMS (ESI+) m/z calcd for

C₂₇H₃₈N₄NaO₅S 553.2450, found 553.2455.

(+)-(S)-Butyl 2-{2-[(tert-Butoxycarbonyl)amino]hept-6-enam-

ido}-2-methylpropanoate (54). According to the procedure

reported for (−)-39, and starting from (+)-37 (135 mg, 0.53 mmol)

and H-Aib-O-n-Bu (84.3 mg, 0.53 mmol), the product 54 (157 mg,

77%) was obtained as a colorless ﬁlm: [α]_D²⁰= −16.9° (c = 1.0, CHCl₃);

R_f= 0.23 (20% EtOAc/cyclohexane); IR ν_max(ﬁlm, CH₂Cl₂) 3358,

1

2978, 2890, 1745, 1750, 1518, 1453, 1366, 1220, 1070, 914 cm⁻¹; H

NMR (400 MHz, CDCl₃) δ (ppm) 0.91 (t, J = 7.4 Hz, 3H), 1.35 (m,

2H), 1.41 (m, 2H), 1.43 (s, 9H), 1.51 (s, 6H), 1.60 (m, 3H), 1.78 (m,

1H), 2.05 (m, 2H), 4.09 (m, 2H), 4.95 (m, 2H), 5.27 (d, J = 7.8 Hz, 1H),

5.75 (tdd, J = 16.9, 10.2, 6.7 Hz, 1H), 7.0 (s, 1H, NH); ¹³C NMR (100

MHz; CDCl₃) δ (ppm) 13.8 (CH₃), 19.1 (CH₂), 24.7 (CH₂), 24.8

(CH₂), 26.9 (CH₃), 28.4 (3 × CH₃), 30.6 (CH₃), 32.1 (CH₂), 33.4

(CH₂), 54.4 (CH), 56.4 (C), 65.3 (CH₂), 79.8 (C), 114.9 (CH₂), 138.2

(CH), 155.9 (C), 171.4 (C), 174.4 (C); HRMS (ESI+) m/z calcd for

C₂₀H₃₆N₂NaO₅407.2516, found 407.2515.

(−)-(3S,9S,14aR)-9-Benzyl-3-(pent-3-enyl)-6,6-dimethyldeca-

hydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,10-

tetraone (56). According to the procedure reported for 47 and starting

from dipeptide 54 (157 mg, 0.4 mmol) and (+)-42 (161 mg, 0.4 mmol),

the tetrapeptide (220 mg, 90%) was obtained after ﬂash chromatog-

raphy (silica gel, 30% EtOAc/cyclohexane) as a white foam: HRMS (ESI

+) m/z calcd for C₃₄H₅₂N₄O₇Na 651.3728, found 651.3721. This

tetrapeptide (220 mg, 0.38 mmol) was converted into cyclic

tetrapeptide 56 (100 mg, 58%, ﬂash chromatography: silica gel, 30%

EtOAc/cyclohexane) as a white foam: [α]_D²⁰= −86.6° (c = 1, CHCl₃);

R_f= 0.22 (30% EtOAc/cyclohexane); IR ν_max(ﬁlm, CH₂Cl₂) 3304,

(−)-(S)-4-[(3S,9S,14aR)-9-Benzyl-6,6-dimethyl-1,4,7,10-tetra-

oxotetradecahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclo-

dodecin-3-yl]pentyl Ethanethioate (59). The cyclic tetrapeptide 58

(63 mg, 0.12 mmol) was dissolved in DMF (2 mL), NH₃/MeOH (1.2

mL of 7 N solution) was added, and the mixture was kept at room

temperature under an argon atmosphere for 67 h. DMF was evaporated

in vacuo, and the crude product was puriﬁed using ﬂash chromatography

(silica gel, 50% EtOAc/cyclohexane) to give a mixture of monomer and

dimer (45 mg) as a colorless ﬁlm. From this mixture, 31 mg was

dissolved in DMF (1 mL), dithiotreitol (98 mg, 0.63 mmol) was added,

and the mixture was kept at 37 °C under an argon atmosphere for 24 h.

DMF was evaporated in vacuo, and the product was extracted with

EtOAc and washed with water. After evaporation, the crude product was

puriﬁed using column chromatography (alumina, 99.75/0.25 CH₂Cl₂/

MeOH) to give 59 (18 mg, 0.037 mmol, 59%) as a colorless ﬁlm: R_f=

1

2930, 1678, 1663, 1630, 1663, 1529, 1428, 1251, 1181, 914 cm⁻¹; H

NMR (400 MHz, CDCl₃) δ (ppm) 1.33 (s, 3H), 1.55 (m, 2H), 1.61−

1.79 (m, 5H), 1.84 (m, 1H), 1.99 (m, 2H), 2.09 (m, 1H), 2.23 (m, 1H),

2.25 (m, 1H), 2.85 (dd, J = 13.5, 5.7 Hz, 1H), 3.10−3.20 (m, 2H), 3.76

(m, 1H), 4.10 (td, J = 10.3, 7.6, 7.6 Hz, 1H), 4.56 (dd, J = 7.7, 2.3 Hz,

1H), 4.85−4.94 (m, 2H), 5.68 (td, J = 10.1, 10.0, 5.8 Hz, 1H), 5.80 (dt, J

= 16.8, 10.2, 6.6 Hz, 1H), 6.99 (s, 1H, NH), 7.07−7.22 (m, 6H, NH),

7.41 (d, J = 10.26 Hz, 1H, NH); ¹³C NMR (100 MHz; CDCl₃) δ (ppm)

23.8 (CH₃), 24.9 (CH₂), 25.0 (CH₂), 25.2 (CH₂), 26.7 (CH₃), 28.6

(CH₂), 33.5 (CH₂), 36.0 (CH₂), 47.2 (CH₂), 53.7 (CH), 54.7 (CH),

58.0 (CH), 59.0 (C), 115.3 (CH₂), 126.9 (CH), 128.8 (2 × CH), 129.2

20

0.24 (silica gel, 30% EtOAc/cyclohexane); [α]_D= −92.5° (c = 1.1,

CHCl₃); IR ν_max(ﬁlm, CH₂Cl₂) 3309, 3284, 2929, 2358, 2337, 1659,

3673

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1626, 1524, 1422 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 1.25−

1.34 (m, 7H), 1.38−1.46 (m, 2H), 1.58−1.65 (m, 1H), 1.72−1.87 (m,

6H), 2.16−2.21 (m, 1H), 2.29−2.34 (m, 1H), 2.52 (q, J = 7.3 Hz, 2H),

2.95 (dd, J = 13.4, 5.8 Hz, 1H), 3.19−3.29 (m, 2H), 3.48 (s, 1H, SH),

3.83−3.89 (m, 1H), 4.19 (td, J = 10.2, 7.6, 7.6 Hz, 1H), 4.66−4.68 (m,

1H), 5.16 (td, J = 10.1, 5.7, 5.7 Hz, 1H), 6.02 (s, 1H, NH), 7.13 (d, J =

10.4 Hz, 1H, NH), 7.19−7.30 (m, 5H), 7.52 (d, J = 10.4 Hz, 1H, NH);

¹³C NMR (100 MHz, CDCl₃) δ (ppm) 23.8 (CH₃), 24.6 (CH₂), 24.9

(CH₂), 25.2 (CH₂), 26.7 (CH₃), 28.1 (CH₂), 29.0 (CH₂), 29.9 (CH₂),

33.9 (CH₂), 36.0 (CH₂), 47.2 (CH₂), 53.6 (CH), 54.5 (CH), 58.0

(CH), 59.0 (C), 126.9 (CH), 128.8 (2 × CH), 129.2 (2 × CH), 137.2

(C), 172.0 (C), 173.0 (C), 174.5 (C), 175.8 (C); LRMS (ESI+) m/z

(%) 489 (100) [M + H]⁺; HRMS (ESI+) m/z calcd for C₂₅H₃₇N₄O₄S

489.2530, found 489.2530.

(−)-(S)-{5-[(2S,8S)-8-Benzyl-5,5-dimethyl-3,6,9,15-tetraoxo-

1,4,7,10-tetraazacyclopentadecan-2-yl]pentyl} Octanethioate

(60). According to the procedure used for 30 and starting from 57

(9.7 mg, 0.0157 mmol), 60 (6.0 mg, 62%) was obtained after ﬂash

chromatography (3% MeOH/CH₂Cl₂) as a colorless ﬁlm: R_f= 0.10 (3%

MeOH/CH₂Cl₂); [α]_D²⁰= −42.8° (c = 0.6, CHCl₃); IR ν_max(thin ﬁlm,

CH₂Cl₂) 3313, 2925, 2859, 1646, 1548, 1450, 1360, 1266, 1249 cm⁻¹;

¹H NMR (400 MHz, CDCl₃) δ (ppm) 0.88 (t, J = 6.8 Hz, 3H), 1.25−

1.30 (m, 12H), 1.31 (s, 3H), 1.32 (s, 3H), 1.34−1.85 (m, 10H), 2.06−

2.24 (m, 2H), 2.54 (t, J = 7.4 Hz, 2H), 2.85 (t, J = 7.3 Hz, 2H), 3.06−3.19

(m, 2H), 3.39−3.49 (m, 2H), 3.91 (m, 1H), 4.50 (m, 1H), 6.28 (m, 1H,

NH), 6.42 (s, 1H, NH), 6.50 (m, 1H, NH), 7.15−7.29 (m, 5H), 7.37 (m,

1H, NH); ¹³C NMR (400 MHz, CDCl₃) δ (ppm) 14.3 (CH₃), 22.1

(CH₂), 22.8 (2 × CH₂), 24.8 (CH₃), 25.5 (CH₂), 25.6 (CH₃), 25.9

(CH₂), 27.6 (CH₂), 28.2 (CH₂), 28.5 (CH₂), 29.1 (CH₂), 29.5 (CH₂),

30.0 (CH₂), 31.8 (CH₂), 35.8 (CH₂), 36.7 (CH₂), 38.0 (CH), 44.4

(CH₂), 55.0 (CH), 55.6 (CH), 58.0 (C), 126.7 (CH), 128.6 (2 × CH),

129.5 (2 × CH), 138.4 (C), 171.6 (C), 173.2 (C), 173.9 (C), 175.2 (C),

200.4 (C); LRMS (ESI+) m/z (%) 617 (100) [M + H]⁺; HRMS (ESI+)

m/z calcd for C₃₃H₅₃N₄O_5S617.3731, found 617.3729.

(2) (a) Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug

Discovery 2006, 5, 769−784. (b) Marks, P. A.; Xu, W. S. J. Cell. Biochem.

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Mech. 2010, 1799, 717−725. (d) Andrews, K. T.; Gupta, A. P.; Tran, T.

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Fairlie, D. P. Curr. Top. Med. Chem. 2009, 9, 292−308. (b) Andrews, K.

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M.; Crumley, T. M.; Allocco, J. J.; Cannova, C.; Meinke, P. T.; Colletti,

S. L.; Bednarek, M. A.; Singh, S. B.; Goetz, M. A.; Bombrowski, A. W.;

Polishook, J. D.; Schmatz, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,

13143−13147. (b) Dow, G. S.; Chen, Y.; Andrews, K. T.; Cardiha, D.;

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ASSOCIATED CONTENT

■

S

* Supporting Information

Text and ﬁgures giving ¹H and ¹³C NMR spectra and biological

evaluation conditions. This material is available free of charge via

the Internet at http://pubs.acs.org.

(10) Rezai, T.; Bock, J. E.; Zhou, M. V.; Kalyanaraman, C.; Lokey, R. S.;

Jacobson, M. P. J. Am. Chem. Soc. 2006, 128, 14073−14080.

(11) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,

1806−1807.

AUTHOR INFORMATION

Corresponding Author

*E-mail for Y.-S.W.: yung-sing.wong@ujf-grenoble.fr.

■

(12) (a) Suzuki, T.; Kouketsu, A.; Itoh, Y.; Hisakawa, S.; Maeda, S.;

Yoshida, M.; Nakagawa, H.; Miyata, N. J. Med. Chem. 2006, 49, 4809−

4812. (b) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.; Maeda, S.;

Yoshida, M.; Nakagawa, H.; Miyata, N. J. Med. Chem. 2007, 50, 5425−

5438.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Financial support by the “Agence Nationale de la Recherche”

under Grant ANR-MIME-2006 and Grant ANR-EMMA-2010

and “programme Labex” (ARCANE project no. ANR-11-LABX-

(13) Huang, D.; Li, X.; Sun, L.; Xiu, Z.; Nishino, N. J. Pept. Sci. 2012,

18, 242−251.

(14) Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc.

2008, 130, 8455−8459.

003) and by Region Rho

acknowledged. F.F.H. was supported by a fellowship (PDSE

9712-11-9) from the CAPES Foundation (Coordenacao de

́

Aperfeico̧ amento de Pessoal de Nivel Superior), Ministry of

Education of Brazil. We thank Astella Parma Inc. for kindly

providing FR235222 and Dr. Sandrine Py and Dr. Olga N.

Burchak for the gift of SmI₂reagent. The P. falciparum 3D7 strain

was obtained through the Malaria Research and Reference

Reagent Resource Center (MR4; http://www.mr4.org/).

̂

ne-Alpes (Cluster 5) is gratefully

(15) Shivashimpi, G. M.; Amagai, S.; Kato, T.; Nishino, N.; Maeda, S.;

Nishino, T. G.; Yoshida, M. Bioorg. Med. Chem. 2007, 15, 7830−7839.

(16) Montero, A.; Beierle, J. M.; Olsen, C. A.; Ghadiri, M. R. J. Am.

Chem. Soc. 2009, 131, 3033−3041.

̧

̃

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(18) Vickers, C. J.; Olsen, C. A.; Leman, L. J.; Ghadiri, M. R. ACS Med.

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Flexible synthesis and evaluation of diverse anti-apicomplexa cyclic peptides

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