22. molecules (2011), 16 4681-4694
TRANSCRIPT
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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: [email protected] (J.X.);
[email protected] (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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Molecules 2011, 16 4692
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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