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Molecules 2011, 16,4681-4694; doi:10.3390/molecules16064681

moleculesISSN 1420-3049

www.mdpi.com/journal/moleculesReview

Synthetic Routes and Biological Evaluation of Largazole and

Its Analogues as Potent Histone Deacetylase Inhibitors

Shang Li1,2,3

, Hequan Yao1, Jinyi Xu

1,* and Sheng Jiang

2,3,*

1 School of Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China;

Tel.: +86 25 83302827; Fax: +86 25 832714452 Shanghai Institute of Technology, Shanghai 210032, China

3Laboratory of Peptide Chemistry, Guangzhou Institute of Biomedicine and Health, CAS,

Guangzhou, Guangdong 510530, China; Tel.: +86 18688888237; Fax: +86 20 32015299

* Authors to whom correspondence should be addressed; E-Mails: jinyixu@china.com (J.X.);

jiang_sheng@gibh.ac.cn (S.J.).

Received: 25 April 2011; in revised form: 13 May 2011 / Accepted: 18 May 2011 /

Published: 7 June 2011

Abstract: Natural products with interesting biological properties and structural diversity

have often served as valuable lead drug candidates for the treatment of various human

diseases. Largazole, isolated from the marine cyanobacterium Symploca sp. has exhibited

potent inhibitory activity against many cancer cell lines. Besides, it shows remarkable

selectivity between transformed and nontransformed cells, which is the main disadvantage

of other antitumor natural products such as paclitaxel and actinomycin D. Due to its

potential as a potent and selective anticancer drug candidate, a great deal of attention has

been focused on largazole and its analogues. It is the aim of this review to highlight

synthetic aspects of largazole and its analogues as well as their preliminary structure

activity relationship studies.

Keywords: largazole; histone deacetylase inhibitor; natural products; total synthesis;

biological evaluation

Abbreviations

BOP = (1H-benzotriazol-1-yloxy) tris(dimethylamino)-phosphonium hexafluorophosphate

DCC = dicyclohexylcarbodiimide

OPEN ACCESS

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DIAD = diisopropyl azodicarboxylate

DMAP = 4-(dimethylamino)pyridine

DMF = dimethylformamide

DMSO = dimethylsulfoxide

EDC = 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide

HATU = -[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)-N-methyl-

methanaminium hexafluorophosphateN-oxide

HBTU =N-[(1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium

hexafluorophosphateN-oxide

HOAt = 1-hydroxy-7-azabenzotriazole

HOBt = 1-hydroxybenzotriazole

TBAF = tetrabutylammonium fluoride

TEMPO = 2,2,6,6-tetramethylpiperidine 1-oxyl

TFA = trifluoroacetate

THF = tetrahydrofuran

1. Introduction

Chromatin template activities, including DNA transcription, replication, and repair, are regulated by

a variety of posttranslational modifications, among which histone acetylation plays a prominent role.

Histone deacetylase inhibitors (HDACIs) have long been used in psychiatry and neurology as mood

stabilizers and antiepileptics. More recently, HDACIs are being studied as targeted therapies for the

treatment of neurodegenerative diseases [1]. Largazole (1, Figure 1) is a natural macrocyclic

depsipeptide isolated from a Floridian marine cyanobacterium Symploca sp. by Luesch and co-workers

[2]. The growth-inhibitory activity of largazole is shown considerably higher for cancer cell lines

(GI50 = 7.7 nm) than for the corresponding nontransformed cells (GI50 = 122 nm). It has shown

promising selective biological activity for differential growth inhibition in a number of transformed

and nontransformed human and murine derived cell lines. The remarkable selectivity of largazole

against cancer cells has prompted research on its mode of action and its importance as a potential

cancer chemotherapeutic agent, and several research groups have completed its total synthesis. This

review focuses on recently developed novel synthetic routes and their applications in the development

of conformational constrained analogues of largazole, as well as biological evaluation and the

preliminary structureactivity relationships of largazole and its analogues are also discussed.

2. Synthetic Routes and Biological Evaluation of Largazole and Its Analogues

Largazole is a densely functionalized macrocyclic depsipeptide consisting of an -methylcysteine-

derived thiazoline coupled to a thiazole embedded within a 16-member macrocycle and a caprylic

acid-derived thioester, which is rarely found in natural products. The retrosynthetic analysis of

largazole shows that two cyclization sites A and B exist in its structure (Figure 1). To synthesize

largazole, most research groups have used the following three key building blocks: valine (2),

-hydroxy ester3a, and thiazolylthiazoline 4.

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Figure 1. Synthesis strategies and key building fragments.

OO

NH

O

NH

SN

S

N

S

O

O

H2NOH

S

N

S

N

O

1

2

3a

4

OHO

NH2

OH O

OR1

macrolactamization A

Hong, Doi

macrolactamization B

Cramer, Williams, Phillips, Ye,Ghosh, Doi, Forsyth, Jiang

cross-metathesis

Cramer, Hong, Phillips

S

O

3b

TrtS

3c

Julia-Kocienski olefination

Jiang

Luesch and co-workers were not only the first to isolate the natural largazole, but also the first to

complete the total synthesis of largazole in collaboration with Hong and co-workers [3]. The

condensation of compound 5 with (R)-2-methylcysteine methyl ester provided the key building block

4a. Removal of the Boc group, followed by coupling of amine 4b with 6, which was prepared by a

Nagao aldol reaction [4,5], Yamaguchi esterification or DCC-coupling reaction, with N-Boc-L-valine

(2a) provided 8. Subsequent hydrolysis and deprotection provided a precursor to the 16-member cyclic

depsipeptide core. The macrocyclization of the precursor using HATUHOAt (HATU, HOAt,

i-Pr2NEt) proceeded smoothly to give 9 in 64% yield (in three steps). Thioester 10 was prepared by

coupling the thioacid with 4-bromo-1-butene. The olefin cross-metathesis reaction of thioester10 with

the macrocycle 9 in the presence of 50 mol% of Grubbs second-generation catalyst in refluxing

toluene provided largazole in 41% yield, a better result compared with that obtained in the presence of

Hoveyda-generation catalyst (Scheme 1).

By using this strategy, Luesch and his collaborators also synthesized several key analogues [6]. The

acetyl analogue 12 was prepared by the olefin cross-metathesis reaction of the macrocycle 9 with

thioacetic acid S-but-3-enyl ester (50 mol % of Grubbs second-generation catalyst, toluene, reflux,

50%). The same procedure was used to synthesize 13, except 1-triisopropylsiyloxyl-3-butene was used

instead of thioacetic acid S-but-3-enyl ester, followed by deprotection. In addition, the aminolysis of1

provided the thiol analogue 14 in 70%80% yield (Scheme 2).

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Scheme 1. Synthetic route of largazole developed by Luesch and co-workers in collaboration

with Hong and co-workers.

BocHN

O

SN

SN

OHOOC NHBoc

NS

OS OH

BocHN

SN

NC

H2N

O

SN

SN

O

TFA

OH

NH

OSN

SN

MeOOC

5 4a 4b

6

7

2a

51%

TFA

DMAP,CH2Cl2

OSHNH2

O

CH2Cl2

SC7H15

O

OO

NH

O

NH

SN

SN

O

OO

NH

O

NHBocSN

SN

MeOOC

10

98

3) HATU, HOAt, DIPA,DCM, 64% (3 steps)

toluene, reflux50% Grubbs' II catalyst

1) LiOH, THF, H2O;

2) TFA, DCM;

Largazole

Scheme 2. Synthesis of largazole analogues 1214.

S

O

toluene, reflux50% Grubbs' IIcatalyst

NH3, MeCN

9OO

NH

O

NH

SN

S

N

O

12

S

O OO

NH

O

NH

SN

S

N

O

13

HO

TIPSO

Grubbs' II catalyst

TBAF, THF

1)

2)

OO

NH

O

NH

SN

S

N

O

14

HS

Largazole

The olefin cross-metathesis reaction of the macrocycle 9 with the products obtained from coupling

the thioester with 3-bromo-1-propene, 5-bromo-1-pentene, and 6-bromo-1-hexene in the presence of

Grubbs second-generation catalyst provided 15, 16, and 17, respectively (Scheme 3).

Scheme 3. Synthesis of largazole analogues 1517.

OO

NH

O

NH

SN

S

N

S

O

O

n

C7H15 SH

OBrn

NaH, THF C7H15 S

O

n toluene, reflux

15, n=1 16, n=3 17, n=410 11

9, Grubbs' II catalyst

Alanine analogue 20 and C17-epimer25 were prepared by replacing the valine and (3S)-hydroxy-

carboxylic acid with the alanine and (3R)-hydroxycarboxylic acid, respectively (Scheme 4).

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Scheme 4. Synthesis of largazole analogues 20 and 25.

SC7H15

O

7toluene, reflux50% Grubbs'II catalyst

1). L-Boc-Ala-OH,Yamaguchi esterification

2). LiOH, THF, H2O

3). TFA, CH2Cl2

OO

NH

O

NH2

SN

S

N

O

18

HO

HATU, HOAt

DIPA, DMF OO

NH

O

NH

SN

S

N

O

19

OO

NH

O

NH

SN

S

N

O

20

SC7H15

O

SC7H15

O

toluene, reflux50% Grubbs'II catalyst

1). L-Boc-Val-OH,Yamaguchi esterification

2). LiOH, THF, H2O

3). TFA, CH2Cl2

HATU, HOAt

DIPA, DMF

OO

NH

O

NH

SN

S

N

O

24

OO

N

H

O

NH

SN

S

N

O

25

SC7H15

O

NS

OS OH

21

4a, TFA

NH

OSN

SN

MeOOC

22

OHOO

NH

O

NH2SN

S

N

O

23

HO

Luesch and co-workers showed that histone deacetylase (HDAC) is the cellular target for

largazoles antiproliferative activity. Largazole is a prodrug, and 14 is the reactive metabolite.

Largazole, its thiol analogue 14, and its acetyl analogue 12 all exhibited similar cellular and

antiproliferative activities against HDACs. However, the hydroxyl analogue 13 and the macrocycle 9

did not show any inhibitory activity. This result suggests that the thiol group is indispensable for both

activities. Alanine analogue 20 showed approximately a 2- to 3-fold decrease in both activities

compared with 1. The five- and six-atom linkers in 16 and 17, respectively, reduced the cell growth

and HDAC inhibitory activity by several orders of magnitude, whereas 15 with the shorter chainshowed no activity. The C17-epimer25 lacked significant HDAC inhibitory activity compared with 1,

suggesting that the four-atom linker between the macrocycle and the octanoyl group in the side chain

and the (S)-configuration at the C17-position are all critical to the potent HDAC inhibitory activity of1.

The valine residue in the macrocycle can be replaced with the alanine without compromising its

activity to a large extent.

Cramer and co-workers [7] have developed a short route for the synthesis of largazole (1), in which

largazole was obtained in 19% overall yield. The -hydroxy ester fragment 3c, which was obtained by

an enzymatic resolution of racemic alcohol, was subjected to the esterification with Fmoc- L-valine

followed by the deprotection of the Fmoc group to provide 26. Nitrile 5 was allowed to react with

(R)--methylcysteine hydrochloride under mild aqueous conditions to provide the thiazoline acid 4a in

an excellent yield. The intermediate 27 was prepared by coupling 4a with the amine 26 in the presence

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of HATU. The deprotection of 27 provided the lactam 9 in 77%89% yield in a dilute solution of

HATU and Hings base. In the optimization of the cross-metathesis reaction, Cramer and co-worker

found a yield increased from 44% to 75% when the p-nitro-substituted catalyst was used instead of the

Hoveyda-Grubbs second-generation catalyst with the catalyst loading of 15% in CH2Cl2 at 80100 C.

Thus, Largazole and its analogues 14, 16, 17, 28, 29, and 30 were prepared in comparable yields

(37%92%) under the optimized conditions (Scheme 5).

Scheme 5. Synthetic route of largazole and its analogues developed by Cramer and co-workers.

1). Fmoc-L-valineDIPEA, DMAPCH2Cl2

2). piperidine DMF

O

OtBu

OO

NH24a, HATU,DIPEA, DMF,

26

OH

OtBu

O

3c

OH

OtBu

O

3

1) Amano lipase PS,vinyl acetate

2) K2CO3, MeOH,82%

OO

NH

O

NH

SN

SN

O

R

OO

NH

O

NH

SN

S

N

R

O

1 R=CH2SC(O)(CH2)6CH328 R=(CH2)9CH3; 29R=CH2Br

30 R=CH2OC(O)(CH2)6CH3

16 R=(CH2)2SC(O)(CH2)6CH317 R=(CH2)3SC(O)(CH2)6CH314 R=CH2SH

9

OO

OtBu

O

NH

SN

SN

O

NHBoc

1). TFA, Et3SiHCH2Cl2

2). HATUDIPEATHF

27

The findings of the antiproliferative activity of 1 and its analogues showed that the octanoic

thioester of 1 acts as a protecting group and that the free thiol 14 has slightly lower potency but

significantly higher specificity compared with the thioester of1. The intermediate 9 and the analogue

28 showed no growth inhibitory activity. The replacement of the thioester with an ester group in the

compound 30 led to a complete loss of activity. Thioester derivatives 16 and 17 showed no activity at

all. It suggests the importance of positioning thio functionality at the right distance from the cyclic core

and the necessity of the thiobutenyl group for its antiproliferative activity.

Ye and co-workers [8] have accomplished the total synthesis of 1 in 5.8% overall yield from

3-[(tert-butyldimethylsilyl) oxy] propane 33. The allylic alcohol fragment 37 was prepared by a series

of reactions: oxidation of alcohol 33, Wittig reaction, reduction of ester 34, oxidation of34 to give

the enal 35, and then reaction with Nagaos chiral N-acetylthiazolidine-2-thione 36 [4,5]. The

displacement of the auxiliary of the alcohol 37, followed by the esterification of 39, provided the

intermediate 40, which was converted to its corresponding disulfide 41. The fragment 4a was prepared

according to Pattendens procedure (Et3N, MeOH, 50 C) [9]. Hydrolysis of the methyl ester 4a

provided the acid, and it was coupled with the product obtained from the removal of the Fmoc group of

the disulfide 41 to give the linear depsipeptide 42. The deprotection of both Boc and TMSE ester

groups of the depsipeptide 42 followed by treatment with HATU provided the cyclodepsipeptide 43.

Largazole was prepared by the reductive cleavage of the disulfide bond in the cyclodepsipeptide 43

followed by the reaction with octanoyl chloride (Scheme 6).

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Scheme 6. Synthetic route of largazole developed by Ye and co-workers.

H2N

S

NHBoc

31

S

N

S

NO

BocHN

O

SN

BocHN

NC

SN

BocHN

EtOOC

BrCH2COCO2EtTFAA, 2,6-lutidine

1.NH3-MeOHMeOH

2. POCl3, pyridineCH2Cl2Et3N, MeOH

4a32 5

KHCO3, 91%

(R)-2-methylcysteinemethyl ester

N

OH O

S

SN-Fmoc-L-valine

DCC, CH2Cl2, 91%

O

O

OTBS

SN

SO

TiCl4, DIPEA, CH2Cl2,83% (dr = 14:1)

OTBS

OTMSE

OH O

OTBS OTMSE

O O

TBSO

O

NHFmoc

OH

OTBS

H

O

OTBS

1.trichloroisocyanuric acid,TEMPO, CH2Cl2, 91%

2.Ph3PCHCOOMe,CH2Cl2, 92%

1. DABAL-H, THF, 83%

2.DMP,CH2Cl2, 81%

TMSEOH

CH2

Cl2

, 94%

33 3435

37 38 39

36

OTMSE

O O

AcS

O

NHFmoc1. HF-py,py,THF

2. TsCl, Et3N, DMAPCH2Cl2

3. KSAc, DMF,64% (3 steps)

1. K2CO3,MeOH,66%

OTMSE

O O

But-S-S

O

NHFmoc

40 41

2. (a) DTNP, CH2Cl2(b) t-BuSH, Et3N,

MeOH, 75% (2 steps)

OO

NH

SN

S

N

S

O

OTMSEO

1.Et3N,MeCN

2, 4a, LiOH, H2O,THF

3.Mukaiyama reagentDIPEA, CH2Cl2,91% (from 42)

1. TFA, CH2Cl2

2. HATU, HOAt

DIPEA, DMF,61% (2 steps)

O

O

NH

O

NH

SN

S

N

S-StBu

O

1. PBu3, THF,H2O

2. n-C7H15COCl,

DIPEA,DMAP,CH2Cl2,78% (2 steps)

4243

S

tBu

BocHN

Largazole

Ghosh and Kulkarni have also completed an enantioselective total synthesis of largazole [10]. The

condensation of the thiazole acid 48 and protected (R)-2-methylcysteine (49) gave 50, and then

treatment of50 according to Kellys procedure [11]followed by reduction and protection provided the

thiazole ester 4a. The requisite thioester was obtained by reaction of 3-butene-1-thiol (44) with

octanoyl chloride. A cross-metathesis of thioester and alcohol 3c was performed followed the same

way used by Cramer and his co-workers in the presence of 3 mol % of Grubbs second-generationcatalyst. The Yamaguchi esterification ofN-Boc-valine, the deprotection of the Boc group, and the

saponification of the thiazole ester 4a were performed. The coupling of the amine 46 to the acid 4a

was carried out by using HATU and HOAt to prepare 51. By removing the protecting Boc and

tert-butyl groups of51, compound 1 was synthesized under dilute conditions with HATU (2 equiv.)

and HOAt (2 equiv.) in the presence of diisopropylethylamine (Scheme 7).

Doi and co-workers [12] synthesized unit 4d using Kellys method, as did Ghosh and co-workers.

They also carried out one-pot bisthiazoline formation using Ishiharas method [13]. Unit 57 was

prepared by the asymmetric aldol reaction of aldehyde 55 and a modified Nagao reagent, N-acetyl-

thiazolidinethione 56 [14]. The subsequent amidation of57 with the amine prepared from 4d by the

removal of the Fmoc group provided a thiazolinethiazole alcohol 58. To synthesize 59, the thiazoline

thiazole alcohol 58 was subjected to the Yamaguchi esterification of the hydroxyl group with

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Fmoc-Val-OH, hydrolysis of methyl ester, and then removal of the Fmoc group. The desired cyclic

depsipeptide 67 was obtained by using HATU and DIPEA under high-dilution conditions. The

deprotection of the trityl group of60 provided thiol 14, and then it was allowed to react with octanoyl

chloride to give 1. The analogous 12 and 61 were prepared by treating the thiol 14 with acetic

anhydride and 22-dipyridyl disulfide, respectively (Scheme 8).

Scheme 7. Synthetic route of largazole developed by Ghosh and Kulkarni.

OO

OtBu

O

NH2

SC7H15

O

SN

SN

O

BocHN

O

OH

OtBu

O

SH C7H15COCl

Grubbs' II catalystCH2Cl2, 67%

OH

OtBu

OS

1. 2,4,6-Cl3-C6H2-COClBoc-L-valine, DIPEA,DMAP, 91%

2. 30% TFA, CH2Cl2

N3NH2

O 1. Lawesson'sreagent, THF

2. BrCH2COCO2EtEtOH, reflux, 55%

S

N

N3

COOEt 1. 1M LiOH

2. EDC, HOBt,DIPEA, 96%

COOMeH2N

TrtS

S

N

N3

O HNSTrt

COOMe

1.Ph3PO, Tf2O,CH2Cl2, 89%

2. PPh3, MeOHBoc2O, CH2Cl2,95%

44 453c 46

47

48 50 4a

1.

2.

C7H15

O

49

1. 1M LiOH

2. HATU, HOAt, 46DIPEA, CH2Cl2, 66%

OO

OtBu

O

NH

SN

S

N

O

NHBocS

O

1. TFA, CH2Cl2

2. HATU, HOAt, DIPEA,CH2Cl2, 40%

51

Largazole

Scheme 8. Synthetic route of largazole and its analogous by Doi and co-workers.

N

S

NHFmoc

O

OH EDCI, HOAtDIPEA, CH2Cl2 HN COOMe

TrtS

1. TFA, Et3SiH,CH2Cl2

2.(NH4)2MnO4,toluene 120'C

Ph3PO, Tf2O,CH2Cl2 0'C

52

534d

54

COOMeH2N

TrtS

49

S

N

NHFmoc

OHN

STrt

COOMe

SN

S

N

O

FmocHN

O

O

HN

STrt

O

FmocHN

OH

N

O

TrtSS

S

N

O

S

STiCl4, DIPEA

CH2Cl2 -78'C 1. Et3N, MeCN

2.DIPEA, CH2Cl2

OH

NH

OSN

S

N

STrt

MeOOC

O

NH

OSN

S

N

STrt

HOOC

NH2

OO

NH

O

NH

SN

S

N

O

STrt

1.Yamaguchiesterification

1. TFA, Et3SiH,CH2Cl2

2. Me3SnOHDCE

3. Et3N, MeCN

HATU, DIPEACH2Cl2

OO

NH

O

NH

SN

S

N

O

SH OO

NH

O

NH

SN

S

N

O

RS

1 R=Me(CH2)6CO

12 R=Ac

61 R=N

S

1.octanoyl chloride, DIPEA, CH2Cl2

12. Ac2O, DIPEA, DMAP, CH2Cl2 61. (2-Py)-S-S-(2-Py),CH2Cl2, MeOH

5556

57

58 59 60

14

4dO

TrtS H

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The HDAC inhibitory activity of Largazole and its analogous 12, 14, and 61 was similar, whereas

the thiol 14 was slightly more potent than the S-substituted compounds, suggesting that thiol 14 is

probably a real active form that is released within the cells.

Phillips [15] and co-workers synthesized the macrocycle 9 started with the Boc-protected glycine

thioamide 31 and ethyl -bromopyruvate converted to thiazolyl amide 62 by Hantzsch thiazole

synthesis [16]. Dehydration of amide 62 produced the nitrile 5, and it was allowed to react with

-methylcysteine to give thiazolylthiazoline 4c under mild aqueous conditions, the same as Cramer

and his co-workers. Coupling of Fmoc-Val-OH with the -hydroxy ester3c, which was obtained by

enzymatic resolution of its corresponding racemic aldol adduct, and the subsequent removal of the

Fmoc group provided amine 26. Condensation of amide 26 and 4c was accomplished with DCC and

pentafluorophenol. After removal of Boc and tert-butyl groups, 27 was subjected to macrolactamization

with PyAOP and DMAP in acetonitrile to provide macrocycle 9 in 50% yield. 1 was obtained in 34%

yield in the presence of 20 mol% of Grubbs second-generation catalyst (Scheme 9).

Scheme 9. Synthetic route of largazole and some analogues developed by Phillips and co-workers.

BocHNNH2

S

BocHN

SN

H2NOC

BocHN

SN

NC

EtOH, NH4OH

then NH4OH

(CF3CO)2O

Et3N, CH2Cl2

COOH

NH2HS

NaHCO3,phosphate bufferMeOH, reflux 77%

BocHN

OH

SN

S

N

O

31 62 5 63 4c

OtBu

OOO

NH2

OtBu

OOH1.Fmoc-Val-OH

EDCI, DMAP

2.Et2NH, CH2Cl2

4c DCC

PFP, THFOO

OtBu

O

NH

SN

S

N

O

NHBoc

TFA, CH2Cl2

then PyAOPDMAP, MeCN

OO

NH

O

NH

SN

S

N

O

X

O

20% Grubbs' IIPhMe 34%

OO

NH

O

NH

SN

S

N

X

O

O

OH

NH

O

NH

SN

S

N

S

O

O

1 X= S 30 X= O 64 X=CH2

COOMe

3c

26

27

9 65

The prepared analogues such as ester30, ketone 64, seco-ester65, macrocycle 9, and largazole (1)were tested for differential inhibitory activity. The results showed that only largazolepreferentially

acted on tumor cells. It is shown that the cellular target of largazole is HDAC and that the

conformation of the depsipeptide is important for targeting HDAC, as 9, 30, 64, and 65 did not show

any activity. Phillips and co-workers also confirmed the preliminary conformation of largazole.

Forsyth and Wang [17] developed a novel synthesis strategy. The Julia olefination using ,-epoxy

aldehyde 75 and thioester 74 containing a tetrazolyl sulfone moiety, which was prepared by the

Mitsunobu reaction, gave alkenes. Removal of the TBS protecting group and the oxidation of the

primary alcohol of76 provided the unstable ,-epoxy aldehyde 77. The requisite 79 was prepared

from ,-epoxy aldehyde 77 by N-heterocyclic carbenemediated esterification with fluoren-9-

ylmethanol. The azido thioester71 was prepared from 66 by a series of reactions: amide formation,

thiol substitution, and thioesterification. Treatment of the azido thioester 71 with PPh3 in acetonitrile

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under microwave irradiation gave the polypeptide. The acid fragment 72 was obtained by the

deprotection of Boc and tert-butyl groups of the polypeptide and by an efficient Fmoc derivatization.

Largazole was obtained by the Yamaguchi esterification of79 and acid 72, followed by deprotection

and macrolactamization (Scheme 10).

Scheme 10. Synthetic route of largazole developed by Forsyth and Wang.

OH OH

O

N3H2N

O Ot-Bu

NH

O Ot-BuO

HO

N3BOP, i-Pr2NEt,CH2Cl2

1. Ph3P, DIADAcSH, THF

2. LiOH, MeOHH2O

NH

O Ot-BuO

HS

N3

66 67 68 69

S

N

BocHN

O

OH

EDCI.HCl, CH2Cl2O

NHS

N

SO

OtBu

BocHN

O

N3

1.Ph3P, MeCNmicrowave, 65'C

O

NH

SN

S

N

O

OH

FmocHN

2. TFA, CH2Cl23.FmocCl, dioxane

NaHCO3

70

7172

OH

S

NN

N

NO

O

Ph

n-C7H15COSH

Ph3P, DIAD,THF, O'C

S

S NN

NN

OO Ph

O

C7H15

1.KHMDS, THF-78'C

OH

O

S O

C7H15OTBS

OH

O

2. TBAF, THF

H

O

SO

C7H15

O

DMSO,SO3.Pyi-Pr2NEt,CH2Cl2

0'C

FmOH,i-Pr2NEt,CH2Cl2

S

N Bn

Cl73

74

75

76 7778

OFm

OH

SO

C7H15

O OO

OFm

O

NH

SN

S

N

SC7H15

O

O

2,4,6-Cl3PhCOCl

i-Pr2NEt, THFthen DMAP

1. Et2NH, CH2Cl2

2.HATU, HOAti-Pr2NEt, MeCN

NHFmoc79

80

72

Largazole

Williams and co-workers [18] synthesized the thiazolinethiazole subunit 4c by condensation of

nitrile 5 and (R)-2-methylcysteine methyl ester hydrochloride under basic conditions (triethylamine in

methanol, Scheme 3). Thiazolidinethione 81 was treated with 2-(trimethylsilyl)ethanol to prepare

TSE-protected acid, and then it was allowed to couple with N-Fmoc-L-Val to prepare 82. The

deprotection of the Fmoc group of 82, followed by PyBOP-mediated coupling with the thiazoline

thiazole subunit 4c provided 83. Removal of protecting Boc and TMSE groups of 83 under high

dilution in the presence of HATU (2 equiv) and HOBt (2 equiv) gave the macrolactamization product

60. Removal of the protecting S-trityl group from 60 provided the thiol 14. The acylation of14 with

octanoyl chloride under standard conditions produced largazole (Scheme 11).

During the synthesis of largazole, a wide range of analogues were obtained by Williams and

co-workers [19]. According to the assay data of these analogues (Figure 2), the potency of the

enantiomer of largazole decreased by almost three orders of magnitude. The C-2 epimer, the valine-to-

proline substitution, and the thiazolethiazole derivative showed intermediate potency. It shows the

importance of the obligate stereochemical and conformation activity relationships between the naturalproduct and its protein targets. The oxazolineoxazole analogue showed a similar activity compared

with largazole. It suggests that the role of the natural 3-hydroxy-7-mercaptohept4-enoic acid moiety in

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Largazole is significant for its activity, which can also be known in FK228 and spiruchostatin. The

thiazolepyridine substitution possessed subnanomolar activity, which was three to four times more

potent than largazole[20]. It seems that the methyl substituent of the thiazoline ring is not essential for

the potency of the natural product.

Scheme 11. Synthesis of largazole developed by Williams and co-workers.

OO

NH

O

NH

SN

SN

O

TrtS

OO

O

O

NH

SN

S

N

O

TrtS NHBoc

SiMe3

OO

OTMSE

O

TrtS

NHFmoc

STrt

N

OH

O

S

Bn

STSEOH

CH2Cl2

N-Fmoc-L-Valine

EDCI, DMAPCH2Cl2

1 Et3N, CH3CN

2.PyBOP,iPr2NEt,CH2Cl2

1.TFA, CH2Cl2

2.HATU,HOBtiPr2NEt,CH2Cl2

iPr3SiH,TFA, CH2Cl2

OO

NH

O

NH

SN

S

N

O

HS

C7H15COCl

Et3N,CH2Cl2

4c

8182

6014

largazole

83

Figure 2. Thestructural modification pathways of largazole.

OO

NH

O

NH

SN

S

N

S

O

O

i-Pr, i-Bu, Bn, p-OH-Bn

C-17 enantiomer

NH

N

O

NS

N

nn=0,1,2

STrt, HN

O

R

N O

Proline

Recently, our efforts have been focused on the total synthesis of largazole [21], we performed the

solid-phase synthesis of key fragment byusing the 2-chlorotrityl chloride resin 84. The thiazoline

thiazole fragment 4a was successfully prepared by tandem deprotectioncyclodehydration and

subsequent oxidation with activated manganese dioxide (Scheme 12).

The primary alcohol 92 was prepared from commercially available ()malic acid (88). Swern

oxidation of the alcohol and the JuliaKocienski olefination followed by coupling with sulfone gave

the key intermediate 94 in a favorable E/Zratio (8/1). The selective deprotection of the primary TBS

and the Mitsunobu reaction provided 95. The allylic alcohol was coupled with enantiomerically pure

amino acids to prepare the intermediate 97. Removal of the Fmoc group followed by coupling with

thiazolinethiazole acid 4a gave cyclization precursor 98. The 16-member cycloamide was prepared

by removal of protected group and subsequent macrolactamization (Scheme 13).

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Scheme 12. Synthesis of thiazolinethiazole fragment 4a.

Cl O

O

STrt

NHFmoc

1) 20% piperidine in DMF, 10min

2) Fmoc-Cys(Trt)-OH, HBTUHOBt, DIPEA, DMF, 1H;

3) 20% piperidine in DMF, 10min

4) Boc-Gly-OH, HBTU, HOBt,DIPEA, DMF, 1h

O

O

STrt

HN

O

N

H

STrt

HO

NHBoc

1% TFA in DCM,2 min, 20 times

HO

O

STrt

HN

O

NH

STrt

HO

NHBoc1)TiCl4,DCM, 5h

2) activated MnO2DCM, 45% overall yield

N

S

N

S

OOH

NHBoc

84 85 86

87 4a

Scheme 13. Synthesis of largazole developed by our group.

HOOH

O

O

OH 1)SOCl2, MeOH

2) BH3Me2S,NaBH4, 91%

OCH3

O

OH

OH

OCH3

O

OTBS

TBSOTBSCl, imidazole

DMF, 92%

1)KOH, THF/H2O, 90%

2) DCC, TMSEOH, 89%88 89 90

O

O

OTBS

TBSO

Si CSA

80%O

O

OH

TBSO

Si1)(COCl)2, DMSO, Et3N, 86%

2)TBSO S

N N

NN

O

O

Ph

NaHMDS, THF, 80%

O

OOTBS

SiTBSO

1) CSA, CH2Cl2/ MeOH(9:1)

2) octanethioic acid, DEAD,Ph3P THF, 90%

O

OOTBS

SiS

O

5

91 92 93

94 95

CSA, CH2Cl2/MeOH (2:1)

80% O

OOH

Si

S

O Fmoc-L-Valine, EDCI, HOAt

DIPEA, CH2Cl2, rt , 79%5 96

O

OO

SiS

O

5

O

NHFmocR

R

OO

NH

O

NH

SN

S

N

S

O

O

5Si

NHBoc

1) piperidine, DMF20min, rt, 96%

2)17 EDC, HOAtDIPEA, CH2Cl2 79%

97

98

1)TFA, TES, CH2Cl2, 2h

2) HATU, HOAt, DIPEACH2Cl2, 2days, rt

30%-50% (2steps)

R

OO

NH

O

NH

SN

S

N

S

O

O1:R= i-Pr, 18E 99:R= i-Pr, 18Z

100:R= i-Bu, 18E 101:R= i-Bu, 18Z

102:R= Bn, 18E

103:R= Bn, 18Z

104:R= p-OH-Bn, 18E 105:R= p-OH-Bn, 18Z

Using this strategy, a small library of largazole analogs was developed. The analogues with cis-

alkenes showed no activity against either human tumor cell lines or normal cell lines. Compound 104

showed slightly lower potency, but much improved selectivity for cancer cell lines. Structure-activity

relationships studies suggested that the geometry of the alkene in the side chain is critical. While the

largazole analogues with trans-alkene are potent for the antiproliferative effect, those with cis-alkene

moieties are completely inactive. Most importantly, replacement of valine with tyrosine in largazole

increased selectivity toward human cancer cells over human normal cells more than 100-fold.

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3. Conclusions

The recent progresses in synthesis of largazole and its analogues are reviewed, in which several

competitive synthetic routes, and its preliminary structureactivity relationships are discussed. Studies

on the biological evaluation of largazole and its analogues have revealed that largazole is a pro-drug,which inhibits the growth of cells by releasing active free thiol group within the target site. A large

body of investigations has provided important insight into the structural, functional, stereochemical, and

conformational aspects of the largazole molecular scaffold which will constitute the basis for the further

design and synthesis of extraordinarily potent HDAC inhibitors with potential therapeutic significance.

Acknowledgments

We thank the National Major Scientific and Technological Program for Drug Discovery (Grants

2009ZX09103-101), NSFC (Grants 20802078, 20972160), Shanghai University DistinguishedProfessor (Eastern scholars) Program (DF2009-02), Pujiang Talent Plan Project (09PJ1409200) and

MOST (Grants 2009CB940900) for financial support.

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Sample Availability:Not available.

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