Total synthesis of the bicyclic depsipeptide hdac inhibitors spiruchostatins a and b, 5″-epi-spiruchostatin b, fk228 (fr901228) and preliminary evaluation of their biological activity

Article abstract of DOI:10.1002/chem.200901552

The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors spiruchostatins A and B, 5″-ep(-spiruchostatin B and FK228 were efficiently synthesized in a convergent and unified manner. The synthetic method involved the following crucial steps : i) a JuliaKocienski olefination of a 1,3-propanediol-derived sulfone and a L- or Dmalic acid-derived aldehyde to access the most synthetically challenging unit, (35 or 3R,4E)-3-hydroxy-7- mercaptohept-4-enoic acid, present in a D-alanine- or D-valine-containing segment; ii) a condensation of a D-valine-D-cysteine- or D-allo-isoleucine-D- cysteinecontaining segment with a D-alanineor D-valine-containing segment to directly assemble the corresponding secoacids; and iii) a macrocyclization of a seco-acid using the Shiina method or the Mitsunobu method to construct the requisite 15- or 16-membered macrolactone. The present synthesis has established the C5″ stereochemistry of spiruchostatin B. In addition, HDAC inhibitory assay and the cell-growth inhibition analysis of the synthesized depsipeptides determined the order of their potency and revealed some novel aspects of structure-activity relationships. It was also found that unnatural 5″-epi-spiruchostatin B shows extremely high selectivity (ca. 1600-fold) for class I HDAC1 (IC₅₀ = 2.4 nM) over class II HDAC6 (IC ₅₀ = 3900nM) with potent cell-growth-inhibitory activity at nanomolar levels of IC₅₀ values.

Full text of DOI:10.1002/chem.200901552

DOI: 10.1002/chem.200901552
Total Synthesis of the Bicyclic Depsipeptide HDAC Inhibitors
Spiruchostatins A and B, 5’’-epi-Spiruchostatin B, FK228 (FR901228) and
Preliminary Evaluation of Their Biological Activity
Koichi Narita,[a] Takuya Kikuchi,[a] Kazuhiro Watanabe,[a] Toshiya Takizawa,[a]
Takamasa Oguchi,[a] Kyosuke Kudo,[a] Keisuke Matsuhara,[a] Hideki Abe,[a]
Takao Yamori,[b] Minoru Yoshida,[c] and Tadashi Katoh*[a]
Dedicated to Dr. Shiro Terashima on the occasion of his 70th birthday
Abstract: The bicyclic depsipeptide his-
tone deacetylase (HDAC) inhibitors
spiruchostatins A and B, 5’’-epi-spiru-
chostatin B and FK228 were efficiently
synthesized in a convergent and unified
manner. The synthetic method involved
the following crucial steps: i) a Julia–
Kocienski olefination of a 1,3-propane-
diol-derived sulfone and a l- or d-
malic acid-derived aldehyde to access
the most synthetically challenging unit,
(3S or 3R,4E)-3-hydroxy-7-mercapto-
hept-4-enoic acid, present in a d-ala-
nine- or d-valine-containing segment;
ii) a condensation of a d-valine-d-cys-
teine- or d-allo-isoleucine-d-cysteine-
containing segment with a d-alanine-
or d-valine-containing segment to di-
rectly assemble the corresponding seco-
acids; and iii) a macrocyclization of a
seco-acid using the Shiina method or
the Mitsunobu method to construct the
requisite 15- or 16-membered macro-
lactone. The present synthesis has es-
tablished the C5’’ stereochemistry of
spiruchostatin B. In addition, HDAC
inhibitory assay and the cell-growth in-
hibition analysis of the synthesized
depsipeptides determined the order of
their potency and revealed some novel
aspects of structure–activity relation-
ships. It was also found that unnatural
5’’-epi-spiruchostatin B shows extreme-
ly high selectivity (ca. 1600-fold) for
class I HDAC1 (IC50 =2.4 nm) over
class II HDAC6 (IC50 =3900 nm) with
potent cell-growth-inhibitory activity at
nanomolar levels of IC50 values.
Keywords: FK228 · histone deace-
tylase inhibitors · natural products ·
spiruchostatins · total synthesis
Introduction
[a] K. Narita, T. Kikuchi, Dr. K. Watanabe, Dr. T. Takizawa,
Dr. T. Oguchi, K. Kudo, K. Matsuhara, Dr. H. Abe,
Prof. Dr. T. Katoh
Recently, histone deacetylase (HDAC) inhibitors have re-
ceived considerable attention because of their biological and
pharmaceutical attributes.[1] HDAC enzymes are zinc metal-
loenzymes that catalyze the hydrolysis of acetylated lysine
residues on proteins, particularly on histones.[1] 18 HDACs
have been identified in humans; they have been grouped
into four classes according to their homology with the yeast
deacetylase.[2] Class I (HDACs 1–3 and 8), class II (HDACs
4–7, 9 and 10) and class IV (HDAC 11) enzymes are Zn+-
binding enzymes, while class III HDAC enzymes have an
NAD+-dependent mechanism of deacetylation.[1,2] It has
been reported that HDAC inhibitors can arrest the growth
of a wide range of transformed cells and can inhibit the
growth of human tumor xenografts.[1] Potent and selective
HDAC inhibitors, therefore, are anticipated to be promising
candidates for novel molecular-targeted anticancer agents.[1]
Laboratory of Synthetic Medicinal Chemistry
Department of Chemical Pharmaceutical Science
Tohoku Pharmaceutical University, 4-4-1 Komatsushima
Aoba-ku, Sendai, 981-8558 (Japan)
Fax : (+81)22-727-0135
E-mail: katoh@tohoku-pharm.ac.jp
[b] Prof. Dr. T. Yamori
Division of Molecular Pharmacology
Cancer Chemotherapy Center
Japanese Foundation for Cancer Research
3-10-6 Ariake, Koto-ku, Tokyo 135-8550 (Japan)
[c] Prof. Dr. M. Yoshida
Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa
Wako-shi, Saitama 351-0198 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901552: Additional synthetic
procedures and NMR spectra.
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Spiruchostatins A (1) and B (2) (Figure 1), isolated from
a culture broth of Pseudomonas sp. by Shin-ya et al. in
2001,[3] were found to exhibit potent HDAC inhibitory activ-
ity.[4] Structurally, these compounds are 15-membered bicy-
1996 followed by Williams et al.[12] and Ganesan et al.[13] in
2008. One total synthesis of FR901375 (4) has been reported
by Wentworth, Janda et al.[14] in 2003. In addition, three
total syntheses of spiruchostatin A (1) have been reported
by Ganesan et al.[15] in 2004, Doi, Takahashi et al.[16] in 2006
and Miller et al.[17] in 2009. However, the total synthesis of
spiruchostatin B (2) has not been mentioned in the litera-
ture, and the stereochemistry at C5’’ (spiruchostatin number-
ing) of 2 has not been clearly assigned. We have previously
reported our own preliminary results on the total synthesis
of spiruchostatins A and B that led to the establishment of
the stereochemistry at the C5’’ position of 2.[18] In this paper,
we describe in complete detail our total synthesis of 1–3 and
5’’-epi-spiruchostatin B (52) (cf. Scheme 9), using a highly
convergent and unified scheme. In addition, HDAC inhibi-
tion assay and the cell-growth inhibition analysis of the syn-
thesized compounds 1–3 and 52 are also described.
Results and Discussion
Synthesis of spiruchostatins A (1), B (2) and 5’’-epi-spiru-
chostatin B (52)
Synthetic plan: In our synthetic plan for spiruchostatins A
(1) and B (2), we envisioned that the target molecules 1 and
2 could be synthesized by macrolactonization of the corre-
sponding seco-acids 5 and 6, followed by a disulfide bond
formation according to the protocols described by previous
studies (Scheme 1).[15,16] The key feature of this Scheme was
Figure 1. Structures of spiruchostatins A (1), B (2), FK228 (FR901228)
(3) and FR901375 (4).
clic depsipeptides consisting of (3S,4R)-statine (in blue), d-
cysteine (in orange), d-alanine (in green), (3R,4E)-3-hy-
droxy-7-mercapto-4-heptenoic acid (in red) and a character-
istic disulfide bond linkage.[3] Structurally similar 16-mem-
bered bicyclic depsipeptides FK228 (3) (previously known
as FR901228) and FR901375 (4), isolated from a culture
broth of Chromobacterium violaceum (No. 968) and Psuedo-
monas chloroaphis (No. 2552), respectively, by Fujisawa
Pharmaceutical Co., Ltd. (now Astellas Pharma Inc.) in
1994,[5] also exhibit potent HDAC inhibitory activity.[6] Yosh-
ida et al. proposed a molecular mechanism by which FK228
inhibits HDAC.[7] In that mechanism, the depsipeptide
FK228, which itself serves as a stable prodrug, is activated
by reductive cleavage of the disulfide bond after incorpora-
tion into the cells, and the released sulfhydryl group in the
four-carbon side chain binds to the zinc cation located at the
active site of the HDACs, resulting in a potent inhibitory
effect. It has also been shown that FK228 exhibits promi-
nent in vivo antitumor activity against human tumor xeno-
graft models.[5b,6] This activity has led to its evaluation as an
anticancer agent in advanced clinical trials (phase II) in the
United States.[8] However, the phase II study was recently
terminated due to serious adverse cardiac events;[9] the un-
desirable side effects seem to arise from insufficient class se-
lectivity of this agent.[10]
The significant biological properties and unique structural
features have made 1–4 exceptionally intriguing and timely
targets for the total synthesis. To date, three total syntheses
of FK228 (3) have been reported: first by Simon et al.[11] in
Scheme 1. Synthetic plan for spiruchostatins A (1) and B (2). TBS=tert-
butyldimethylsilyl, Tr (trityl)=triphenylmethyl, Boc=tert-butoxycarbon-
yl, PMB=4-methoxybenzyl, PMP=4-methoxyphenyl.
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T. Katoh et al.
Table 1. Stereoselective reduction of ketone 19.
expected to be a highly convergent assembly of 5 and 6 by
direct condensation of segment 7 or 8 with segment 9. Seg-
ments 7 and 8 would be prepared via an aldol coupling of
N-Boc-d-valinal (10)[19] or d-allo-isoleucinal (11)[20] with
ethyl acetate (12), and subsequent condensation with d-cys-
teine dervative 13.[21] Segment 9, which contains the most
synthetically challenging unit, (3S,4E)-3-hydroxy-7-mercap-
tohept-4-enoic acid, would be produced via Julia–Kocienski
olefination[22] of sulfone 14 (accessible from 1,3-propanediol)
with aldehyde 15[23] (accessible from l-malic acid), followed
by condensation with d-alanine methyl ester (16).
Entry
Reducing agent
T [8C]
t [h]
Yield [%]
Ratio
18
17
18/17
1
2
3
4
LiBH4
NaBH4
KBH4
KBH4
0
0
0
ꢀ40
1
1
1
1
52
65
80
82
13
9
8
4:1
7:1
10:1
16:1
5
Synthesis of segment 7: We initially pursued the synthesis of
segment 7 (Scheme 2). Aldol coupling of the lithium enolate
of ethyl acetate (12) (generated in situ by reaction with
LDA) with the known N-Boc-d-valinal (10)[19] provided the
plained by the well-known Cramꢁs chelation model
(Scheme 3);[25] the stereoselectivity was consistent with the
chelation ability of alkali metal ions (K+ > Na+ > Li+) to
form a chelate in the proposed transition state 19A.
Scheme 3. Cramꢁs chelation model for the stereoselective reduction of
ketone 19A.
To continue the synthesis (cf. Scheme 2), ethyl ester 18
was then transformed to allyl ester 23 via a four-step se-
quence involving TBS protection of the hydroxy group in 18
(84%), saponification of the ester moiety in the resulting
TBS ether 20 (82%), formation of an allyl ester from the li-
berated carboxylic acid 21 (91%) and deprotection of the
N-Boc group in 22 (90%). Subsequent condensation of
amine 23 with N-Boc-S-trityl-d-cysteine (13)[21] proceeded
smoothly to produce amide 24 in 88% yield. Finally, depro-
tection of the N-Boc group in 24 furnished the desired seg-
ment 7 in quantitative yield.
Scheme 2. Synthesis of segment 7. a) LDA, CH3CO2Et (12), THF,
ꢀ788C; at ꢀ788C, add 10, 63% for 17, 30% for 18 (17/18 ca. 2:1);
b) Jones reagent, acetone, 08C to RT, 80%; c) KBH4, MeOH, ꢀ408C,
82% for 18, 5% for 17 (18/17 16:1) (see entry 4 in Table 1); d) TBSCl,
imidazole, DMF, RT, 84%; e) 1m NaOH, EtOH, RT, 82%; f) allyl bro-
mide, K2CO3, DMF, RT, 91%; g) TMSOTf, 2,6-lutidine, CH2Cl2, RT;
MeOH, RT; MeOH, RT, 90%; h) 13, PyBOP, iPr2NEt, MeCN, RT, 88%;
i) TMSOTf, 2,6-lutidine, CH2Cl2, RT, 99%. LDA=lithium diisopropyl-
ACHTUNGTRENNUNGamide, TMSOTf=trimethylsilyl trifluoromethanesulfonate, PyBOP=
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
Synthesis of segment 9: Next, we performed the synthesis of
segment 9, the condensation partner of 7, starting from the
desired coupling product 18 (30%) and the undesired ste-
reoisomer 17 (63%). Next, conversion of 17 to 18 by inver-
sion of the hydroxy group was investigated. The sequence
involved Jones oxidation[24] and subsequent stereoselective
reduction of the resulting ketone 19. In the reduction step,
several reducing agents, such as LiBH4, NaBH4 and KBH4,
were examined (Table 1). Among them, the use of KBH4
gave the best result (entry 3) and the desired alcohol 18 was
produced in 80% yield with high stereoselectivity (18/17
10:1). When the reaction was performed at a lower tempera-
ture (ꢀ408C), the stereoselectivity was apparently increased
(entry 4, 82% yield of 18 with 18/17 16:1). With the use of
LiBH4 or NaBH4 (entries 1 and 2), a lower stereoselectivity
of 18/17 was observed (4:1 and 7:1, respectively). The re-
markable stereoselectivity in the reduction of 19 can be ex-
known
3-(4-methoxybenzyloxy)propan-1-ol
(25)[26]
(Scheme 4). Sulfone 14, a substrate for the Julia–Kocienski
olefination,[22] was efficiently prepared from 25 via a four-
step operation involving the formation of tetrazole 26
(95%), molybdenum-mediated oxidation[27] of 26, deprotec-
tion of the PMB group in sulfone 27 (86% in two steps),
and substitution of the primary hydroxy group in 28 with a
S-trityl group (96%). The crucial Julia–Kocienski olefina-
tion[22] of 14 with the known aldehyde 15[23] (readily pre-
pared from l-malic acid) proceeded smoothly to form the
desired product 29 as an inseparable mixture of E/Z stereo-
1
isomers (E/Z ca. 5:1 by 400 MHz H NMR) in 66% yield.
Subsequent reductive opening of 29 with DIBAL[28] at 08C
provided the desired E-olefinic primary alcohol 30 as a
major product (60%) along with the undesired Z-olefinic
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Bicyclic Depsipeptide HDAC Inhibitors
FULL PAPER
target spiruchostatin A (1) by assembling the two segments
(Tables 2 and 3, Schemes 5 and 6). Initial attempts to
AHCTUNGTREGaNNNU chieve the crucial condensation of 7 and 9 using standard
condensing agents[31] (e.g., PyBOP, PyBOP/HOBt, EDCI/
HOBt or HATU in CH2Cl2 at ꢀ308C) resulted in partial ep-
imerization at the C2 stereogenic center (d-alanine part in
35) (entries 1–4, Table 2), while the condensation product 35
was obtained in high yield (82–91%).
Eventually, we overcame this epimerization problem by
using a combination of HATU and HOAt at a low tempera-
ture (entry 5). Thus, treating a mixture of 7 and 9 in CH2Cl2
with HATU (1.3 equiv) in the presence of iPr2NEt
(2.6 equiv) at ꢀ308C for 3 h resulted in the desired product
35 in 94% yield without appreciable epimerization at the
C2 position. In this condensation, the temperature was criti-
cal: when the reaction was performed at room temperature,
a small degree of epimerization (a-Me/b-Me 10:1) was ob-
served (entry 6). The condensation product 35 was then con-
verted to seco-acid 5, a substrate for macrolactonization, in
88% overall yield via alcohol 36 by successive deprotection
of the PMB and allyl groups (Scheme 5).
Having obtained seco-acid 5 in a convergent way, we next
examined the crucial macrolactonization (Table 3). In the
previous two total syntheses of spiruchostatin A (1), Gane-
san et al.[15] successfully achieved macrolactonization of the
O-triisopropylsilyl (TIPS) variant of 5 (R=TIPS) using the
Yamaguchi method (2,4,6-Cl3C6H2COCl, Et3N, MeCN/THF,
0 to 208C; DMAP, toluene, 508C, 53%); on the other hand,
Doi, Takahashi et al.[16] efficiently performed macrolactoni-
zation of O-non-protected variant of 5 (R=H) using the
Shiina method [2-methyl-6-nitrobenzoic acid (MNBA),
Scheme 4. Synthesis of segment 9. a) 1-phenyl-1H-tetrazole-5-thiol,
DEAD, PPh3, THF, RT, 95%; b) [Mo7O
EtOH, RT; c) DDQ, CH2Cl2/H2O, RT, 86% (2 steps); d) TrSH, DEAD,
PPh3, CH2Cl2, reflux, 96%; e) LiN
24ACHTUNGNERTN(UNG NH4)6]·4H2O, 30 % H2O2,
(SiMe3)2, DMF, ꢀ608C; at ꢀ608C, add.
15, ꢀ60 to 08C, 66% (E/Z ca. 5:1); f) DIBAL, toluene, 08C, 60% for 30,
12% for 31; g) Dess–Martin periodinane, NaHCO3, CH2Cl2, 08C to RT,
88%; h) NaClO2, NaH2PO4, DMSO/H2O, 0 8C to RT, 75%; i) d-alanine
methyl ester (16), PyBOP, iPr2NEt, CH2Cl2, 08C to RT, 90%; j) 1m
LiOH, MeOH, RT, 98%. DEAD=diethyl azodicarboxylate, DDQ=2,3-
dichloro-5,6-dicyano-1,4-benzoqui-
none, DIBAL=diisobutylaluminium
hydride.
Table 2. Condensation of segment 7 with segment 9.
isomer 31 (12%); at this stage,
the E/Z isomers could be sepa-
rated by silica gel column chro-
matography. Dess–Martin oxi-
dation[29] of 30 (86%) followed
by Pinnick oxidation[30] of the
resulting aldehyde 32 yielded
the corresponding carboxylic
acid 33. Finally, condensation
of 33 with d-alanine methyl
ester (16) followed by saponifi-
cation of the ethyl ester moiety
of the resulting amide 34, af-
forded the requisite segment 9
in 66% overall yield from 32.
Entry
Conditions
Yield [%][a]
35
Ratio[b]
a-Me/b-Me
1
2
3
4
5
6
PyBOP (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
82
85
91
89
94
91
3:1
ꢀ308C, 3 h
PyBOP (1.3 equiv), HOBt (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
5:1
ꢀ308C, 3 h
EDCI (1.3 equiv), HOBt (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
5:1
ꢀ308C, 3 h
HATU (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
6:1
ꢀ308C, 3 h
HATU (1.3 equiv), HOAt (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
1:0
ꢀ308C, 3 h
Completion of the total synthe-
HATU (1.3 equiv), HOAt (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2,
10:1
sis of spiruchostatin
A (1):
RT, 1 h
With the key segments 7 and 9
synthesized, we next investigat-
ed the synthesis of the first
[a] Isolated yield. [b] Determined by 400 MHz 1H NMR analysis. HOBt=1-hydroxybenzotriazole, EDC=1-
ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium hexafluorophosphate, HOAt=1-hydroxy-7-azabenzotriazole.
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T. Katoh et al.
Scheme 6. Synthesis of spiruchostatin A (1). a) I2, MeOH/CH2Cl2, RT,
80%; b) HF·pyridine, pyridine, RT, 92%.
Scheme 5. Synthesis of seco-acid 5. a) DDQ, CH2Cl2/H2O, RT, 89 %;
b) [Pd(PPh3)4], morpholine, THF, RT, 99%.
ACHTUNGTRENNUNG
1
DMAP, CH2Cl2, RT, 67%]. Given those previous results, we
investigated the macrolactonization of 5 under several con-
ditions (Table 3). The best result was obtained using the
Shiinaꢁs standard method (entry 1).[32] Thus, treatment of a
dilute solution of 5 in CH2Cl2 (0.001m) with MNBA
(1.3 equiv) and DMAP (3.0 equiv) at room temperature for
15 h produced the desired macrocyclic compound 37 in 90%
yield. The use of other Shiinaꢁs protocols[33] (entries 2 and 3)
resulted in almost the same yields (87% each). In contrast,
when macrolactonization was performed as per the Yamagu-
chi method[34] (entry 4) or the Mukaiyama–Corey–Nicolaou
method[35] (entry 5), the yield of 37 was lower (67% and
36%, respectively).
The final route that led to the completion of the total syn-
thesis of 1 is depicted in Scheme 6. Simultaneous S-Tr de-
protection and disulfide bond formation of 37 was per-
formed by brief exposure to iodine in dilute MeOH/CH2Cl2
solution (0.5 mm) at ambient temperature, as described in
previous studies,[11–16,36] producing the desired disulfide 38 in
80% yield. Finally, deprotection of the O-TBS group in 38
delivered 1, [a]D24 =ꢀ62.88 (c=0.14 in MeOH) {lit.[3] [a]D26 =
ꢀ63.68 (c=0.14 in MeOH)}, in 92% yield. The spectroscop-
ic properties (IR, H and 13C NMR, HRMS) of the synthetic
sample 1 were identical with those reported for natural 1.[3]
Synthesis of spiruchostatin B (2) and 5’’-epi-spiruchostatin B
(52): As mentioned previously, the stereochemistry at the
C5’’ position of spiruchostatin B (2) has not been clearly as-
signed; therefore, we decided to undertake the synthesis of
two possible C5’’ stereoisomers, namely, 2 (cf. Scheme 8)
and its C5’’-epimer 52 (cf. Scheme 9); the successful synthe-
sis revealed that natural 2 possesses the (5’’S)-configuration
as depicted in Scheme 8 (see below).
Synthesis of segment 8: As shown in Scheme 7, the synthesis
of segment 8 was conducted starting from N-Boc-d-allo-iso-
leucinal (11)[20] by employing a reaction sequence similar to
that described in the section on the synthesis of segment 7
(cf. Scheme 2). Thus, aldol coupling of 11 with the lithium
enolate of ethyl acetate (12) afforded the desired coupled
product 40 (31%) and the undesired stereoisomer 39
(62%). After inversion of the hydroxy group in 39 (85% in
two steps), the resulting alcohol 40 was further converted to
amine 44 in four steps via TBS ether 41, carboxylic acid 42
and allyl ester 43 (69% overall
yield). Subsequent condensa-
Table 3. Macrolactonization of seco-acid 5 leading to macrocyclic compound 37.[a]
tion of 44 with d-cysteine de-
rivative 13[21] provided the
requisite segment
8
(86%
overall yield) after N-Boc de-
protection of amide 45.
Completion of the total synthe-
sis of spiruchostatin
B (2):
After obtaining the key seg-
ment 8, we performed the syn-
thesis of the second target spir-
uchostatin B (2) (Scheme 8).
Condensation of 8 with the
common intermediate 9 pro-
ceeded smoothly and cleanly
under the optimized conditions
Entry Conditions
Yield 37
[%]
1
2
3
4
MNBA (1.3 equiv), DMAP (3.0 equiv), CH2Cl2, RT, 15 h
MNBA (1.3 equiv), 4-PPY (3.0 equiv), CH2Cl2, RT, 15 h
DMNBA (1.3 equiv), DMAP (3.0 equiv), CH2Cl2, RT, 15 h
2,4,6-trichlorobenzoyl chloride (5.0 equiv), Et3N (5.0 equiv), THF, 08C to RT; DMAP
(3.0 equiv), toluene, 608C, 16 h
90
87
87
67
described
previously
(cf.
5
2,2-dipyridyl disulfide (3.0 equiv), Ph3P (1.5 equiv), toluene, 808C, 10 h
36
entry 5, Table 2); the desired
product 46 was obtained in
[a] DMAP=4-dimethylaminopyridine, MNBA=2-methyl-6-nitrobenzoic anhydride, 4-PPY=4-piperidinopyri-
dine, DMNBA=2,6-dimethyl-4-nitrobenzoic anhydride.
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Bicyclic Depsipeptide HDAC Inhibitors
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Table 3) resulting in the desired cyclization product 48 in
89% yield. By employing a reaction sequence similar to that
described for the synthesis of 1 (cf. Scheme 6), compound 48
was transformed to 2, [a]2D0 =ꢀ59.88 (c=1.02 in MeOH)
{lit.[3] [a]2D6 =ꢀ58.68 (c=0.11 in MeOH)}, via O-TBS ether
1
49. The H and 13C NMR spectra of the synthetic sample 2
were essentially identical to those reported for natural spiru-
chostatin B.[3] To assure the C5’’ stereochemistry in 2, we
also pursued the synthesis of the other possible stereoiso-
mer, namely, 5’’-epi-spiruchostatin B (cf. 52, Scheme 9). We
describe this in the following section.
Completion of the total synthesis of 5’’-epi-spiruchostatin B
(52): As shown in Scheme 9, using (2R,3R)-N-Boc-d-isoleu-
cinal (50)[20] instead of (2R,3S)-N-Boc-d-allo-isoleucinal (11)
Scheme 7. Synthesis of segment 8. a) LDA, CH3CO2Et (12), THF,
ꢀ788C; at ꢀ788C, add 11, 62% for 39, 31% for 40 (39/40 2:1); b) Jones
reagent, acetone, 08C to RT, 95%; c) KBH4, MeOH, ꢀ408C, 90% for 40,
6% for 39 (40/39 15:1); d) TBSCl, imidazole, DMF, RT, 96%; e) 1m
NaOH, EtOH, RT, 80%; f) allyl bromide, K2CO3, DMF, RT, 96%;
g) TMSOTf, 2,6-lutidine, CH2Cl2, RT; MeOH, RT, 92%; h) 13, PyBOP,
iPr2NEt, MeCN, RT, 86%; i) TMSOTf, 2,6-lutidine, CH2Cl2, RT; MeOH,
RT, 99 %.
Scheme 9. Synthesis of 5’’-epi-spiruchostatin B (52).
as the starting material, 5’’-epi-spiruchostatin B (52) was
synthesized through condensation of segments 51 and 9 in
the same manner as described for the synthesis of spiruchos-
tatin B (2) (cf. Schemes 7 and 8). The 1H and 13C NMR
spectra of the synthesized 52 did not match those of natural
2. From these results, the C5’’ stereochemistry of 2 was un-
ambiguously determined to be S configuration as depicted
in Scheme 8.
Scheme 8. Synthesis of spiruchostatin B (2). a) HATU, HOAt, iPr2NEt,
CH2Cl2, ꢀ308C, 94%; b) DDQ, CH2Cl2/H2O, RT, 85%; c) [Pd
ACHTUNGTRNE(NUNG PPh3)4],
morpholine, THF, RT, 91%; d) MNBA, DMAP, CH2Cl2, RT, 89%; e) I2,
MeOH/CH2Cl2, RT, 94%; f) HF·pyridine, pyridine, RT, 93%.
Synthesis of FK228 (3): Having successfully synthesized spir-
uchostatins A (1) and B (2) as well as 5’’-epi-spiruchostatin
B (52) in a convergent and unified manner, we further in-
vestigated the synthesis of the final target FK228 (3) using
the same strategy as explored earlier.
94% yield. Subsequent removal of the PMB and allyl pro-
tecting groups from 46 furnished the seco-acid 6 in 77%
overall yield via allyl alcohol 47. The crucial macrolactoniza-
tion of 6 was also efficiently achieved under the same condi-
tions as explored for the preparation of 37 (cf. entry 1,
Primary synthetic plan: In our primary synthetic plan shown
in Scheme 10, we envisaged that the target molecule 3 could
Chem. Eur. J. 2009, 15, 11174 – 11186
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11179

T. Katoh et al.
Scheme 11. Synthesis of segment 54. a) FmocCl, 10% aq. Na2CO3, diox-
ane, RT; b) 57a, PyBOP, iPr2NEt, MeCN, RT, 96% (2 steps); c) Et2NH,
MeCN, RT; d) 13, PyBOP, iPr2NEt, MeCN, RT, 91% (2 steps); e) MsCl,
Et3N, CH2Cl2, 08C, quant.; f) DABCO, THF, 08C to RT, 90%;
g) BF3·Et2O, CH2Cl2, RT, 73%. FmocCl=9-fluorenylmethoxycarbonyl
chloride, MsCl=methanesulfonyl chloride, DABCO=1,4-diazabicyclo-
AHCTUNGTREG[NNUN 2.2.2]octane.
Scheme 10. Primary synthetic plan for FK228 (3).
be synthesized by macrolactonization of seco-acid 53, acces-
sible by condensation of segments 54 and 55, followed by a
disulfide bond formation. Segment 54 would be formed by
successive condensation of d-cysteine derivative 13, l-threo-
nine (56) and l-valine methyl ester (57). Segment 55 would,
in turn, be prepared through a condensation of the common
intermediate 33 and d-valine allyl ester (58).
Scheme 12. Synthesis of segment 55. a) 58a, PyBOP, iPr2NEt, MeCN, RT,
73%; b) [Pd(PPh3)4], morpholine, THF, RT, 94%.
AHCTUNGTRENNUNG
Synthesis of segment 54: First, the synthesis of segment 54
was performed starting from l-threonine (56) (Scheme 11).
After protection of the amino function in 56 with a 9-fluor-
Synthesis of seco-acid 53: Having obtained the requisite seg-
ments 54 and 55, we next pursued the synthesis of seco-acid
53 by assembling the two segments (Scheme 13). Thus, con-
densation of 54 with 55 proceeded efficiently under the
same conditions as mentioned previously (cf. entry 5,
Table 2), providing the desired product 66 in 81% yield.
This was further transformed to the requisite seco-acid 53
(77% yield in two steps) by removal of the two protecting
groups via alcohol 67.
ACHTUNGTRENNUNGenylmethoxycarbonyl (Fmoc) group, the resulting carbamate
59 was subjected to condensation with l-valine methyl ester
hydrochloride (57a), producing the desired amide 60 in
96% overall yield. Subsequent deprotection of the N-Fmoc
group in 60, followed by condensation of the liberated
amine 61 with d-cysteine derivative 13, provided tripeptide
62 in 91% overall yield. This was further converted to the
requisite segment 54 via a three-step sequence involving me-
sylation of the hydroxy group in 62 (quantitative yield),
elimination of the resulting mesylate 63 (96%) and depro-
tection of the N-Boc group in 64 (73%).
Initial attempts to achieve the macrolactonization of seco-
acid 53: With the key precursor in hand, we next attempted
the macrolactonization of seco-acid 53 (Scheme 14). We had
originally intended to achieve the macrolactonization by
using the powerful and reliable Shiina reagents that had
been very effective in the synthesis of spiruchostatins (cf.
5!37, entries 1–3, Table 3; 6!48, Scheme 8). However, to
our chagrin, all attempts to realize this macrolactonization
under the Shiina conditions [MNBA or DMNBA, DMAP or
4-PPY, CH2Cl2, RT] proved unsuccessful; none of the de-
Synthesis of segment 55: Segment 55, the condensation part-
ner of 54, was readily synthesized in 69% overall yield by
amide formation between the common intermediate 33 and
d-valine allyl ester p-toluenesulfonate (58a) followed by de-
protection of the allyl ester moiety in the resulting amide 65
(Scheme 12).
11180
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Chem. Eur. J. 2009, 15, 11174 – 11186

Bicyclic Depsipeptide HDAC Inhibitors
FULL PAPER
Scheme 13. Synthesis of seco-acid 53. a) HATU, HOAt, iPr2NEt, CH2Cl2,
ꢀ308C, 81%; b) DDQ, CH2Cl2/H2O, RT, 83%; c) LiOH, THF/H2O, 0 8C,
93%.
Scheme 15. Modified synthetic plan for FK228 (3).
employing the same reaction sequence (cf. 14 + ent-15 !
ent-33, ent-33 + 58 ! 70, 54 + 70 ! 69) as explored earli-
er.
Synthesis of segment 70: Segment 70, which corresponds to
the epimer of 55 (cf. Scheme 13) at the O-PMB group, was
efficiently synthesized via the same reaction sequence as de-
scribed for the preparation of 55 (Scheme 16). Thus, conden-
sation of sulfone 14 with aldehyde ent-15[22] (readily pre-
pared from d-malic acid) (66%) followed by reductive
acetal opening of the resulting olefin ent-29 provided alco-
hol ent-30 (57%). After twofold oxidation of ent-30, the re-
sulting carboxylic acid ent-33 was subjected to condensation
with d-valine derivative 58a to afford amide 71 (67% over-
all yield from ent-30). Finally, removal of the allyl protecting
group from 71 furnished the requisite segment 70 (92%).
Scheme 14. Attempts on the macrolactonization of seco-acid 53.
sired macrocyclization products 68 were obtained, and the
starting material 53 was always recovered with a small
amount of unidentified byproducts. We assumed that these
failures resulted from the very low reactivity of the activated
carboxylic acid, which we attributed to the presence of the
sterically hindered isopropyl substituent adjacent to the car-
bonyl reaction site. Incidentally, similar findings were also
reported recently by Ganesan et al.[13] in their total synthesis
of FK228. These unsuccessful results forced us to modify
our original synthetic plan. We describe this in the following
section.
Completion of the total synthesis of FK228 (3): With seg-
ment 70 synthesized, we next performed the synthesis of
FK228 (3) (Scheme 17). Thus, condensation of segments 54
and 70, under the same conditions as described previously,
proceeded smoothly to deliver the desired product 72
(76%), which was further converted to the requisite seco-
acid 74 (85% yield in two steps) via alcohol 73. Following
Simonꢁs procedure,[11] the macrocyclization of 74 under Mit-
sunobu conditions was next examined. Thus, treatment of
seco-acid 74 (~60 mg scale) with a large excess of Ph3P
(25 equiv) and diisopropyl azodicarboxylate (DIAD)
(20 equiv) in the presence of pTsOH (5 equiv) in dry THF
at 08C to room temperature for 4 h, and after purification
Modified synthetic plan: As mentioned in the preceding sec-
tion, our initial attempts to realize the macrolactonization of
seco-acid 53 resulted in failure; therefore, we settled on
modifying our original synthetic plan based on the Simonꢁs
pioneering work[11] (Scheme 15). The modified synthetic
plan involved Mitsunobu-based macrolactonization[14,37] of
seco-acid 69, which possesses the opposite stereochemistry
at the hydroxy group in the mercaptoheptenoic acid part.
The macrolactonization precursor 69 should be prepared
starting from aldehyde ent-15 (available from d-malic acid)
Chem. Eur. J. 2009, 15, 11174 – 11186
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11181

T. Katoh et al.
had encountered difficulties reproducing the 62% yield re-
ported by Simon and coworkers for this cyclization event in
their FK228 syntheses, whereas our results were in accord-
ance with the reported yield. Ultimately, disulfide bond for-
mation accompanied by oxidative S-Tr deprotection of 68
resulted in the production of 3, [a]2D0 =+37.68 (c=0.27 in
CHCl3) {lit.[5b] [a]D20 =+398 (c=1.0 in CHCl3)}, in 75% yield.
1
The spectroscopic properties (IR, H and 13C NMR, HRMS)
of the synthetic sample 3 were identical with those reported
for natural 3.[5b]
Biological evaluation: The synthesized spiruchostatins A (1)
and B (2), 5’’-epi-spiruchostatin B (52) and FK228 (3) were
evaluated for their HDAC inhibitory activity and their cell-
growth inhibitory activity to determine the order of the po-
tency and to reveal some novel aspects of structure–activity
relationships (SAR).
Scheme 16. Synthesis of segment 70. a) LiN
G
ꢀ608C, add ent-15, ꢀ60 to 08C, 66 % (E/Z ca. 5:1); b) DIBAL, toluene,
08C, 57%; c) Dess–Martin periodinane, NaHCO3, CH2Cl2, 08C to RT,
92%; d) NaClO2, NaH2PO4, DMSO/H2O, 0 8C to RT; e) 58a, PyBOP,
HDAC inhibition assay: It has been reported that specific
inhibition of class I HDACs is a useful mechanism for anti-
cancer agents,[38] and that inhibition of class II HDACs may
cause undesirable side effects such as the promotion of car-
diac hypertrophy.[10,39] Spiruchostatin A (1) and FK228 (3)
have been shown to display high selectivity for HDAC1
(class I) over HDAC6 (class II).[4a,7] Based on this informa-
tion, compounds 1–3 and 52 were tested for their HDAC in-
hibitory activity against HDAC1 and HDAC6 to determine
the degree of the potency and the isoform selectivity. Tri-
chostatin A (TSA) was used as a reference compound. As
summarized in Table 4, all compounds tested exhibited ex-
tremely potent inhibitory activity against HDAC1 in the low
nanomolar range (IC50 =2.2–3.6 nm). The order of the po-
tency was estimated to be 2 (IC50 =2.2 nm) ꢁ 52 (IC50 =
2.4 nm) ꢂ 1 (IC50 =3.3 nm) ꢁ 3 (IC50 =3.6 nm) @ TSA
(IC50 =20 nm). Interestingly, unnatural 52 showed almost the
same potency as compared with 2, highlighting that the C5’’
stereochemistry in 2 has no influence on HDAC1 inhibition
efficacy. As for HDAC6 inhibitory activity, all compounds
tested were essentially inactive (IC50 =390–3900 nm), as ex-
pected. Among the compounds tested, compound 52
showed the highest isoform selectivity, that is, 1625-fold dif-
ferential for HDAC1 over HDAC6. Isoform selectivity
iPr2NEt, MeCN, RT, 73% (2 steps); f) [PdACHTNUTRGEN(UNG PPh3)4], morpholine, THF,
RT, 92 %.
Table 4. HDAC Inhibitory activity of spiruchostatins A (1), B (2), 5’’-epi-
spiruchostatin B (52), and FK228 (3).
[a]
Compound
IC50 [nm]
EC1000[e] [nm]
cell HDAC
HDAC1[b]
HDAC6[b]
SI[d]
1
2
52
3.3
2.2
2.4
3.6
1600
1400
3900
390
485
636
1625
108
3
8.8
2.5
3.4
3.1
18
Scheme 17. Synthesis of FK228 (3). a) HATU, HOAt, iPr2NEt, CH2Cl2,
ꢀ208C, 76%; b) DDQ, CH2Cl2/H2O, RT, 91%; c) LiOH, THF/H2O, 0 8C,
93%; d) DIAD, PPh3, pTsOH, THF, 08C to RT, 62%; e) I2, MeOH/
CH2Cl2, RT, 75%. DIAD=diisopropyl azodicarboxylate.
3
TSA[c]
20
63
[a] The concentration that induces 50% inhibition against HDACs.
[b] The enzyme assay was performed in the presence of 100 mm dithio-
threitol (DTT). [c] Positive control as a representative HDAC inhibitor.
[d] Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity toward
class I HDAC1 over class II HDAC6. [e] The concentration that induces
the luciferase activity 10-fold higher than the basal level.
by silica gel column chromatography, the desired macrolac-
tone 68 was obtained in 62% yield. Recently, Williams
et al.[12] and Ganesan et al.[13] noted independently that they
11182
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Chem. Eur. J. 2009, 15, 11174 – 11186

Bicyclic Depsipeptide HDAC Inhibitors
FULL PAPER
Table 5. Growth inhibition of spiruchostatins A (1), B (2), 5’’-epi-spiru-
chostatin B (52) and FK228 (3) against a panel of 39 human cancer cell
lines.
(class I/II) is expressed, for convenience, as a selectivity
index (SI) value. The order of the selectivity was estimated
to be 52 (SI=1625) > 2 (SI=636) > 1 (SI=485) > 3 (SI=
108) @ TSA (SI=3). Notably, the isoform selectivity of 52
is 15-fold higher than that of FK228, which represents, to
the best of our knowledge, the highest level of the class I/II
selectivity among the natural depsipeptides and the synthe-
sized depsipeptide analogues known to date. In addition, a
good correlation between enzymatic (HDAC1) and cellular
HDAC inhibitory activity was found for all of compounds
tested. From these results, it was revealed that minor struc-
tural modification of the side chain on the statine part in
spiruchostatins is quite effective in improving the isoform
selectivity without loss of the potent HDAC inhibitory activ-
ity.
[a]
GI50 [nm]
Origin of cancer
breast
Cell line
1
2
52
3
HBC-4
BSY-1
HBC-5
8.5
19
20
5.6
8.3
12
6.9
15
17
6.9
8.5
13
MCF-7
MDA-MB-231
U-251
SF-268
SF-295
7.1
6.8
7.1
16
22
6.3
10
4.1
4.5
3.6
4.5
4.8
3.3
4.4
6.3
2.2
3.7
3.2
380[b]
2.6
4.8
9.3
1.3[c]
3.3
1.9
8.5
3.3
2.4
3.0
13
4.8
4.6
4.5
5.9
7.1
4.5
8.1
9.6
4.0
3.9
3.4
390[b]
3.2
4.9
17
4.2
5.5
3.9
4.9
4.0
3.6
7.2
9.6
3.1
3.4
3.3
450[b]
3.1
4.6
8.9
1.8[c]
3.0
2.6
5.8
3.6
2.5
4.6
20
central nervous
system (brain)
SF-539
SNB-75
SNB-78
HCC2998
KM-12
36
colon
lung
8.9
5.9
5.8
660[b]
5.1
13
HT-29
Cell-growth inhibition assay: The growth-inhibitory activity
of 1–3 and 52 was evaluated using a panel of 39 human
cancer cell lines in the Japanese Foundation for Cancer Re-
search.[41] The number of cell lines and their origin (organ)
are as follows: five breast, six central nervous system
(brain), one melanoma, five ovary, two kidney, six stomach
and two prostate cancers. Dose-response curves were mea-
sured at five different concentrations (10ꢀ10–10ꢀ6 m) for each
compound, and the concentration causing 50% cell-growth
inhibition (GI50) was compared with that of the control
(Table 5). It is evident that all compounds tested exhibited
extremely potent growth-inhibitory activity against almost
all 39 cell lines in the nanomolar range, which closely corre-
sponded to their ability to inhibit HDAC1. Although no dis-
tinct differences of the growth-inhibitory activity were ob-
served among the test compounds, the order of the potency
was estimated by the MG-MID value (mean value of GI50
over all cell lines tested) to be 2 (5.6 nm) ꢁ 3 (6.2 nm) ꢂ 52
(7.6 nm) > 1 (15 nm). Considering the HDAC inhibitory ac-
tivity and the antiproliferative activity together, compound
52, a novel congener of spiruchostatins, seems to be a prom-
ising candidate or a new possibility for the development of
novel anticancer agents targeting class I HDAC1.
HCT-15
HCT-116
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114
LOX-IMVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
RXF-631 L
ACHN
55
3.0[c]
8.5
4.6
38
1.5[c]
4.7
2.5
14
7.1
4.0
11
47
4.3
16
5.4
23
32
4.6
2.3
4.5
28
2.1
5.9
3.6
14
melanoma
ovary
2.5
4.6
2.2
8.7
17
2.8
5.5
3.3
6.6
20
kidney
23
stomach
St-4
48
26
46
22
MKN1
MKN7
6.8
25
69
45
10
21
29
15
2.6
10
22
3.6
11
58
3.2
4.9
17
MKN28
MKN45
MKN74
DU-145
PC-3
15
31
14
2.4
4.7
8.7
5.6
3.6
5.5
7.8
7.6
3.0
6.0
18
prostate
MG-MID[d]
6.2
[a] The concentration that induces 50% inhibition of cell growth com-
pared to control. [b] The least sensitive cell. [c] The most sensitive cell.
[d] Mean value of GI50 over all cell lines tested.
Conclusion
We have established unified synthetic routes to the bicyclic
depsipeptide HDAC inhibitors spiruchostatins A (1) and B
(2), 5’’-epi-spiruchostatin B (52) and FK228 (3). The method
explored features i) Julia-Kocienski olefination of sulfone 14
and aldehyde 15 or ent-15 to install the requisite E olefin
unit present in the critical segment 9 or 55 (14 + 15 ! 29,
Scheme 4; 14 + ent-15 ! ent-29, Scheme 16); ii) condensa-
tion of segment 7 or 8 with the common intermediate 9, and
segment 54 with segment 70 to directly assemble the crucial
seco-acid 5, 6 or 69 (7 + 9 ! 35, Table 2; 8 + 9 ! 46,
Scheme 8; 54 + 70 ! 72, Scheme 17); and iii) macrocycliza-
tion of 5, 6, or 69 using the Shiina method or the Mitsunobu
method to construct the desired macrolactone 37, 48 or 68
(5 ! 37, Table 3; 6 ! 48, Scheme 8; 74 ! 68, Scheme 17).
The C5’’ stereochemistry of 2 was determined by the present
synthesis. Preliminary biological evaluation of the synthe-
sized compounds 1–3 and 52 determined the order of their
efficacy and some novel aspects of the SAR study which
would be useful for designing and development of anticanc-
er agents with therapeutic potential that target isoform-se-
lective inhibition of HDACs. On the basis of the present
study further investigations concerning the synthesis of spir-
uchostatin analogs and the SAR study are currently under-
way and will be reported in due course.
Chem. Eur. J. 2009, 15, 11174 – 11186
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11183

T. Katoh et al.
FK228 (3): A solution of 68 (35.9 mg, 35 mmol) in MeOH (8 mL) was
Experimental Section
added dropwise to
a vigorously stirring solution of I2 (175 mg,
0.69 mmol) in CH2Cl2 (63 mL, 0.5 mm concentration) over 10 min at
room temperature. After 10 min, the reaction was quenched with 0.2m
ascorbic acid/citric acid buffer (7 mL, adjusted to pH 4.0) at room tem-
perature, and the resulting mixture was extracted with EtOAc (3ꢂ
70 mL). The combined extracts were washed with brine (2ꢂ30 mL) and
dried over Na2SO4. Concentration of the solvent in vacuo afforded a resi-
due, which was purified by column chromatography (EtOAc) to give 3
(14.2 mg, 75%) as a white solid. [a]2D0 =+35.68 (c=0.27 in CHCl3) {lit.[5b]
[a]2D0 =+398 (c=1.0 in CHCl3)}; 1H NMR (400 MHz, CDCl3/CD3OD
10:1): d=1.00 (d, J=6.8 Hz, 3H), 1.02 (d, J=6.8 Hz, 3H), 1.10 (d, J=
6.8 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H), 1.75 (d, J=6.8 Hz, 3H), 2.19–2.27
(m, 1H), 2.35–2.41 (m, 1H), 2.60–2.72 (m, 3H), 2.79–2.83 (m, 1H), 2.94–
3.01 (m, 1H), 3.11–3.21 (m, 3H), 4.00 (dd, J=4.2, 6.1 Hz, 1H), 4.55 (dd,
J=3.9, 7.8 Hz, 1H), 4.69–4.75 (m, 1H), 5.67–5.72 (m, 2H), 5.75–5.82 (m,
1H), 6.35 (q, J=6.8 Hz, 1H), 7.64 (d, J=7.8 Hz, 1H), 7.81 (d, J=6.1 Hz,
1H), 8.19 (d, J=3.4 Hz, 1H), 8.40 ppm (s, 1H); 13C NMR (100 MHz,
CDCl3/CD3OD 10:1): d=13.1, 18.1, 18.3, 19.0, 19.1, 28.9, 30.1, 31.7, 34.5,
37.7, 37.8, 56.6, 58.2, 62.4, 70.1, 129.2, 129.6, 129.7, 130.4, 165.7, 168.4,
169.4, 171.4, 172.3 ppm; IR (KBr): n˜ =3337, 2963, 2926, 1724, 1664, 1637,
1522, 1465, 1438, 1254, 1218, 1111, 1028, 979, 753 cmꢀ1; HRMS (FAB):
m/z: calcd for C24H37N4O6S2: 541.2155, found 541.2153 [M+H]+. The IR,
1H and 13C NMR, and HRMS spectrum are identical with those reported
for natural FK228.[5b]
General techniques: All reactions involving air- and moisture-sensitive
reagents were carried out using oven dried glassware and standard sy-
ringe-septum cap techniques. Routine monitoring of reaction were car-
ried out using glass-supported Merck silica gel 60 F254 TLC plates. Flash
column chromatography was performed on Kanto Chemical Silica Gel
60N (spherical, neutral 40–50 mm) with the solvents indicated.
All solvents and reagents were used as supplied with following excep-
tions. Tetrahydrofuran (THF), Et2O and dioxane were freshly distilled
from Na metal/benzophenone under argon. Toluene was distilled from
Na metal under argon. N,N-Dimethylformamide (DMF), dimethyl sulfox-
ide (DMSO), CH2Cl2, MeCN, pyridine, iPr2NH and iPr2NEt were dis-
tilled from calcium hydride under argon. Measurements of optical rota-
tions were performed with a JASCO DIP-370 automatic digital polarime-
ter. Melting points were taken on a Yanaco MP-3 micro melting point ap-
paratus and are uncorrected. 1H and 13C NMR spectra were measured
with a JEOL AL-400 (400 MHz) spectrometer. Chemical shifts were ex-
pressed in ppm using Me4Si (d=0) as an internal standard. The following
abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m) and broad (br). Infrared (IR) spectral measurements were
carried out with a JASCO FT/IR-4100 spectrometer. Low- and High-res-
olution mass (HRMS) spectra were measured on a JEOL JMS-DX 303/
JMA-DA 5000 SYSTEM high resolution mass spectrometer.
Spiruchostatin A (1): HF·pyridine (0.20 mL) was added to a stirred solu-
tion of 38 (19.5 mg, 33 mmol) in pyridine (1.5 mL) at room temperature.
After 14 h, the reaction mixture was diluted with EtOAc (60 mL), and
the organic layer was washed successively with 3% aqueous HCl (3ꢂ
15 mL), saturated aqueous NaHCO3 (2ꢂ15 mL) and brine (2ꢂ15 mL),
then dried over Na2SO4. Concentration of the solvent in vacuo afforded a
residue, which was purified by column chromatography (CHCl3/MeOH
10:1) to give 1 (spiruchostatin A) (14.4 mg, 92%) as a white amorphous
solid. [a]2D5 =ꢀ69.98 (c=0.14 in MeOH) {lit.[3] [a]2D6 =ꢀ63.68 (c=0.14 in
MeOH)}; 1H NMR (400 MHz, CDCl3): d=0.93 (d, J=6.8 Hz, 3H), 1.03
(d, J=6.8 Hz, 3H), 1.51 (d, J=7.3 Hz, 3H), 2.38–2.47 (m, 2H), 2.56 (d,
J=13.2 Hz, 1H), 2.70–2.77 (m, 5H), 3.09–3.28 (m, 4H), 4.25 (dq, J=3.9,
7.3 Hz, 1H), 4.54–4.59 (m, 1H), 4.89 (dt, J=3.9, 9.3 Hz, 1H), 5.50 (s,
1H), 5.65 (d, J=15.1 Hz, 1H), 5.92 (s, 1H), 6.28–6.31 (m, 4H), 6.71 (d,
J=9.3 Hz, 1H), 7.40 ppm (d, J=6.8 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d=16.7, 19.6, 20.6, 29.6, 33.3, 39.6, 40.7, 40.9, 52.3, 54.3, 63.8,
69.3, 70.5, 77.2, 128.6, 133.6, 169.0, 170.6, 170.9, 171.9 ppm; IR (neat): n˜ =
3375, 2933, 1633, 1542, 1160, 755 cmꢀ1; HRMS (FAB): m/z: calcd for
C20H32N3O6S2: 474.1732, found 474.1750 [M+H]+. The IR, 1H and
13C NMR, and HRMS spectrum are essentially identical with those re-
ported for natural spiruchostatin A.[3]
5”-epi-Spiruchostatin B (52): This compound was prepared through con-
densation of 51 and 9 in the same manner as described for the synthesis
of spiruchostatin B (2). [a]2D5 =ꢀ51.98 (c=0.10 in MeOH); 1H NMR
(400 MHz, CDCl3): d=0.82 (t, J=7.3 Hz, 3H), 0.97 (d, J=6.8 Hz, 3H),
1.02–1.11 (m, 1H), 1.46 (d, J=7.3 Hz, 3H), 1.50–1.56 (m, 1H), 2.07 (q,
J=7.3 Hz, 1H), 2.41–2.49 (m, 1H), 2.68 (d, J=12.6 Hz, 1H), 2.64–2.78
(m, 4H), 2.95 (q, J=7.8 Hz, 1H), 3.24 (dd, J=6.8, 13.2 Hz, 2H), 4.13–
4.19 (m, 1H), 4.45–4.47 (m, 1H), 4.72 (dd, J=8.3, 13.2 Hz, 1H), 5.50
(brs, 1H), 5.75 (d, J=15.6 Hz, 1H), 6.16–6.18 (m, 1H), 6.98 (d, J=
8.7 Hz, 1H), 7.07 (s, 1H), 7.42 ppm (d, J=7.3 Hz, 1H); 13C NMR
(100 MHz, CDCl3): d=10.8, 16.3, 16.4, 25.8, 32.6, 35.8, 39.5, 39.7, 39.9,
40.9, 52.2, 55.8, 61.7, 69.1, 71.0, 129.5, 132.8, 169.2, 171.1, 171.4,
171.9 ppm; IR (neat): n˜ =3375, 3320, 2964, 1731, 1660, 1652, 1539, 1040,
891, 753 cmꢀ1; HRMS (FAB): m/z: calcd for C21H34N3O6S2: 488.1889,
1
found 488.1886 [M+H]+. The H and 13C NMR spectrum are not identi-
cal with those reported for natural spiruchostatin B.[3]
HDACs preparation and enzyme inhibition assay:[40] In a 100 mm dish,
293T cells (1–2ꢂ106) were grown for 24 h and transiently transfected
with 10 mg each of the vector pcDNA3-HDAC1 for human HDAC1 or
pcDNA3-mHDA2/HDAC6 for mouse HDAC6, using the LipofectA-
MINE2000 reagent (Invitrogen). After successive cultivation in DMEM
for 24 h, the cells were washed with PBS and lysed by sonication in lysis
buffer containing 50 mm Tris-HCl (pH 7.5), 120 mm NaCl, 5 mm EDTA,
and 0.5% NP40. The soluble fraction collected by microcentrifugation
was precleared by incubation with protein A/G agarose beads (Roche).
After the cleared supernatant had been incubated for 1 h at 48C with
4 mg of an anti-FLAG M2 antibody (Sigma–Aldrich Inc.) for HDAC1
and HDAC6, the agarose beads were washed three times with lysis
buffer and once with histone deacetylase buffer consisting of 20 mm Tris-
HCl (pH 8.0), 150 mm NaCl, and 10% glycerol. The bound proteins were
released from the immune complex by incubation for 1 h at 48C with
40 mg of the FLAG peptide (Sigma–Aldrich Inc.) in histone deacetylase
buffer (200 mL). The supernatant was collected by centrifugation. For
the enzyme assay, 10 mL of the enzyme fraction was added to 1 mL of
fluorescent substrate (2 mm Ac-KGLGK(Ac)-MCA) and 9 mL of histone
deacetylase buffer, and the mixture was incubated at 378C for 30 min.
The reaction was stopped by the addition of 30 mL of tripsin
(20 mgmLꢀ1) and incubated at 378C for 15 min. The released amino
Spiruchostatin B (2): HF·pyridine (1.0 mL) was added to a stirring solu-
tion of 49 (68.3 mg, 0.11 mmol) in pyridine (2.0 mL) at room tempera-
ture. After 14 h, the reaction mixture was diluted with EtOAc (40 mL),
and the organic layer was washed successively with 3% aqueous HCl (3ꢂ
10 mL), saturated aqueous NaHCO3 (2ꢂ10 mL) and brine (2ꢂ10 mL),
then dried over Na2SO4. Concentration of the solvent in vacuo afforded a
residue, which was purified by column chromatography (CHCl3/MeOH
20:1) to give 2 (spiruchostatin B) (51.3 mg, 93%) as a white amorphous
solid. [a]2D5 =ꢀ59.88 (c=1.02 in MeOH) {lit.[3] [a]2D6 =ꢀ58.68 (c=0.11 in
MeOH)}; 1H NMR (400 MHz, CDCl3): d=0.89 (t, J=7.5 Hz, 3H), 0.90
(d, J=7.0 Hz, 3H), 1.18–1.29 (m, 2H), 1.50 (d, J=7.3 Hz, 3H), 1.54–1.59
(m, 1H), 2.04–2.11 (m, 1H), 2.42–2.51 (m, 1H), 2.61 (d, J=13.2 Hz, 1H),
2.69–2.78 (m, 4H), 2.92–2.95 (m, 1H), 2.94 (ddd, J=4.0, 7.0, 9.0 Hz, 1H),
3.11–3.24 (m, 2H), 3.33 (dd, J=7.3, 13.1 Hz, 2H), 4.22 (dq, J=3.9,
7.3 Hz, 1H), 4.60–4.65 (m, 1H), 4.87 (dt, J=3.4, 9.2 Hz, 1H), 5.50–5.51
(m, 1H), 5.68 (d, J=15.6 Hz, 1H), 6.27 (s, 1H), 6.37–6.42 (m, 1H), 6.78
(d, J=9.7 Hz, 1H), 7.29 ppm (d, J=9.3 Hz, 1H); 13C NMR (100 MHz,
CDCl3): d=11.5, 15.4, 16.6, 27.1, 33.3, 36.3, 39.5, 40.5, 40.7, 41.3, 52.2,
54.5, 61.7, 68.2, 70.6, 128.6, 133.4, 169.2, 170.6, 171.2, 171.8 ppm; IR
(neat): n˜ =3374, 3332, 1731, 1660, 1539, 1273, 990 cmꢀ1; HRMS (FAB):
m/z: calcd for C21H34N3O6S2: 488.1889, found 488.1900 [M+H]+. The IR,
1H and 13C NMR, and HRMS spectrum are essentially identical with
those reported for natural spiruchostatin B.[3]
methyl coumarin (AMC) was measured using
a fluorescence plate
reader. The 50% inhibitory concentrations (IC50) were determined as the
means with SD calculated from at least three independent dose-response
curves.
11184
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Bicyclic Depsipeptide HDAC Inhibitors
FULL PAPER
Cell HDAC inhibition assay (p21 promoter assay):[40] A luciferase report-
er plasmid (pGW-FL) was constructed by cloning the 2.4 kb genomic
fragment containing the transcription start site into HindIII and SmaI
sites of the pGL3-Basic plasmid (Promega Co., Madison, WI). Mv1Lu
(mink lung epithelial cell line) cells were transfected with the pGW-FL
and a phagemid expressing neomycin/kanamycin resistance gene (pBK-
CMV, Stratagene, La Jolla, CA) with the Lipofectamine reagent (Life
Technology, Rockville, MD, USA). After the transfected cells had been
selected by 400 mgmLꢀ1 Geneticin (G418, Life Technology), colonies
formed were isolated. One of the clones was selected and named MFLL-
9. MFLL-9 expressed a low level of luciferase, whose activity was en-
hanced by TSA in a dose-dependent manner. MFLL-9 cells (1ꢂ105) cul-
tured in a 96-well multi-well plate for 24 h were incubated for 20 h in the
medium containing various concentrations of drugs. The luciferase activi-
ty of each cell lysate was measured with a LucLite luciferase Reporter
Gene Assay Kit (Packard Instrument Co., Meriden, CT) and recorded
with a Luminescencer-JNR luminometer (ATTO, Tokyo, Japan). Data
were normalized to the protein concentration in cell lysates. Concentra-
tions at which a drug induces the luciferase activity 10-fold higher than
the basal level are presented as the 1000% effective concentration
1000% (EC1000). The human wild-type p21 promoter luciferase fusion
plasmid, WWP-Luc, was a kind gift from Dr. B. Vogelstein.
Cell-growth inhibition assay:[41] This experiment was carried out at the
Cancer Chemotherapy Center, Japanese Foundation for Cancer Re-
search. The screening panel consisted of the following 39 human cancer
cell lines (HCC panel): breast cancer HBC-4, BSY-1, HBC-5, MCF-7,
and MDA-MB-231; brain cancer U-251, SF-268, SF-295, SF-539, SNB-75,
and SNB-78; colon cancer HCC2998, KM-12, HT-29, HCT-15, and HCT-
116; lung cancer NCI-H23, NCI-H226, NCI-H522, NCI-H460, A549,
DMS273, and DMS114; melanoma LOX-IMVI; ovarian cancer
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SK-OV-3; renal
cancer RXF-631 L and ACHN; stomach cancer St-4, MKN1, MKN7,
MKN28, MKN45, and MKN74; prostate cancer DU-145 and PC-3. The
GI50 (50% cell-growth inhibition) value for these cell lines was deter-
mined by using the sulforhodamine B colorimetric method.
Med. Chem. 2005, 40, 1–13; m) O. Moradei, C. R. Maroun, I.
Paquin, A. Vaisburg, Curr. Med. Chem. Anti-Cancer Agents 2005, 5,
529–560; n) D. C. Drummond, G. K. Nobel, D. B. Kirpotin, Z. Guo,
G. K. Scott, C. C. Benz, Annu. Rev. Pharmacol. Toxicol. 2005, 45,
495–528; o) M. Biel, V. Wascholowski, A. Giannis, Angew. Chem.
2005, 117, 3248–3280; Angew. Chem. Int. Ed. 2005, 44, 3186–3216;
p) P. A. Marks, V. M. Richon, T. Miller, Adv. Cancer Res. 2004, 91,
137–168; q) T. A. Miller, D. J. Witter, S. Belvedere, J. Med. Chem.
2003, 46, 5097–5116.
[2] a) M. A. Holbert, R. Marmorstein, Curr. Opin. Struct. Biol. 2005, 15,
673–680; b) S. Voelt-Mahlknecht, A. D. Ho, U. Mahlnecht, Int. J.
Mol. Med. 2005, 8, 589–598; c) A. J. de Ruijter, A. H. van Gennip,
H. N. Caron, S. Kemp, A. B. Kuilenburg, J. Biochem. 2003, 370,
737–749; d) R. W. Johnstone, Nat. Rev. Drug Discovery 2002, 1,
287–299; e) C. M. Grozinger, C. A. Hassig, S. L. Schreiber, Proc.
Natl. Acad. Sci. USA 1999, 96, 4868–4873; f) J. Taunton, C. A.
Hassig, S. L. Schreiber, Science 1996, 272, 408–411.
[3] Y. Masuoka, A. Nagai, K. Shin-ya, K. Furihata, K. Nagai, K. Suzuki,
Y. Hayakawa, H. Seto, Tetrahedron Lett. 2001, 42, 41–44.
[4] a) S. J. Crabb, M. Howell, H. Rogers, M. Ishfaq, A. Yurek-George,
K. Carey, B. M. Pickering, P. East, R. Mitter, S. Maeda, P. W. W.
Johnson, P. Townsend, K. Shin-ya, M. Yoshida, A. Ganesan, G.
Packham, Biochem. Pharmacol. 2008, 76, 463–475; b) S. M. David-
son, P. A. Townsend, C. Carroll, A. Yurek-George, K. Balasubrama-
nyam, T. K. Kundu, A. Stephanou, G. Packham, A. Ganesan, D. S.
Latchman, ChemBioChem 2005, 6, 162–170.
[5] a) H. Ueda, H. Nakajima, Y. Hori, T. Fujita, M. Nishimura, T. Goto,
M. Okuhara, J. Antibiot. 1994, 47, 301–310; b) N. Shigematsu, H.
Ueda, S. Takase, H. Tanaka, J. Antibiot. 1994, 47, 301–314; c) H.
Ueda, T. Manda, S. Matsumoto, S. Mukumoto, F. Nishigaki, I. Ka-
wamura, K. Shimomura, J. Antibiot. 1994, 47, 315–323; d) M. Oku-
hara, T. Goto, T. Fujita, Y. Hori, H. Ueda (Fujisawa Pharmaceutical
Co. Ltd., Japan), JP 03141296A, 1991.
[6] H. Nakajima, Y. B. Kim, H. Terano, M. Yoshida, S. Horinouchi,
Exp. Cell Res. 1998, 241, 126–133.
[7] R. Furumai, A. Matsuyama, N. Kobashi, K.-H. Lee, M. Nishiyama,
H. Nakajima, A. Tanaka, Y. Komatsu, N. Nishino, M. Yoshida, S.
Horinouchi, Cancer Res. 2002, 62, 4916–4921.
[8] D. M. Vigushi, Curr. Opin. Invest. Drugs 2002, 1396–1402.
[9] M. H. Shah, P. Binkley, K. Chan, J. Xiao, D. Arbogast, M. Colla-
more, Y. Farra, D. Young, M. Grever, Clin. Cancer Res. 2006, 12,
3997–4003.
[10] a) A. Yurek-George, A. R. L. Ceil, A. H. K. Mo, S. Wen, H. Rogers,
F. Habens, S. Maeda, M. Yoshida, G. Packham, A. Ganesan, J. Med.
Chem. 2007, 50, 5720–5726; b) T. C. Karagiannis, A. El-Osta, Leu-
kemia 2007, 21, 61–65; c) M. Luedde, H. A. Katus, N. Frey, Recent
Pat. Cardiovasc. Drug Discovery 2006, 1, 1–20; d) T. A. McKinsey,
E. N. Olsen, J. Clin. Invest. 2005, 115, 538–546.
[11] K. W. Li, J. Wu, W. Xing, J. A. Simon, J. Am. Chem. Soc. 1996, 118,
7237–7238.
[12] T. J. Greshock, D. M. Johns, Y. Noguchi, R. M. Williams, Org. Lett.
2008, 10, 613–616.
[13] S. Wen, G. Packham, A. Ganesan, J. Org. Chem. 2008, 73, 9353–
9361.
Acknowledgements
We are especially grateful to Dr. Kazuo Shin-ya, National Institute of
Advanced Industrial Science and Technology, for providing us with
copies of the 1H and 13C NMR spectra of natural spiruchostatins A (1)
and B (2). We also thank Professor Takayuki Doi, Tohoku University
and Professor Isamu Shiina, Tokyo University of Science, for useful dis-
cussions and suggestions. In addition, Screening Committee of Anticanc-
er Drugs supported by Grant-in-Aid for Scientific Research on Priority
Area “Cancer” from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) for biological evaluation of compounds 1–3 and
52 is also acknowledged. This work was supported by a Grant-in-Aid for
Scientific Research on Priority Area “Creation of Biologically Functional
Molecules” (No. 17035073 and No. 18032065), a Grant-in-Aid for Scien-
tific Research (C) (No. 18590013 and No. 21590018) and a Grant-in-Aid
for High Technology Research Program from MEXT.
[14] Y. Chen, C. Gambs, Y. Abe, P. Wentworth, Jr., K. D. Janda, J. Org.
Chem. 2003, 68, 8902–8905.
[15] A. Yurek-George, F. Habens, M. Brimmell, G. Packham, A. Gane-
san, J. Am. Chem. Soc. 2004, 126, 1030–1031.
[16] a) Y. Iijima, A. Munakata, K. Shin-ya, A. Ganesan, T. Doi, T. Taka-
hashi, Tetrahedron Lett. 2009, 50, 2970–2972; b) T. Doi, Y. Iijima, K.
Shin-ya, A. Ganesan, T. Takahashi, Tetrahedron Lett. 2006, 47,
1177–1180.
[17] N. A. Calandra, Y. L. Cheng, K. A. Kocak, J. S. Miller, Org. Lett.
2009, 11, 1971–1974.
[18] a) T. Takizawa, K. Watanabe, K. Narita, T. Oguchi, H. Abe, T.
Katoh, Chem. Commun. 2008, 1677–1679; b) T. Takizawa, K. Wata-
nabe, K. Narita, K. Kudo, T. Oguchi, H. Abe, T. Katoh, Heterocycles
2008, 76, 275–290.
[1] a) L. Nolan, P. W. M. Jonson, A. Ganesan, G. Packham, S. J. Grabb,
Br. J. Cancer 2008, 99, 689–694; b) X.-J. Yang, E. Seto, Nat. Rev.
Mol. Cell Biol. 2008, 9, 206–218; c) M. Paris, M. Porcelloni, M. Bi-
naschi, D. Fattori, J. Med. Chem. 2008, 51, 1505–1529; d) M. Dok-
manovic, C. Clarke, P. A. Marks, Mol. Cancer Res. 2007, 5, 981–989;
e) P. Gallinai, S. Di Marco, P. Jones, M. Pallaoro, C. Steinkuhler,
Cell Res. 2007, 17, 195–211; f) D. Marchion, P. Mꢃnster, Expert Rev.
Anticancer Ther. 2007, 7, 583–598; g) G. Elaut, V. Rogiers, T. Van-
haecke, Curr. Pharm. Des. 2007, 13, 2584–2620; h) S. Minucci, P. G.
Pelicci, Nat. Rev. Cancer 2006, 6, 38–51; i) J. E. Bolden, M. J. Peart,
R. W. Johnstone, Nat. Rev. Drug Discovery 2006, 5, 769–784; j) T.
Suzuki, N. Miyata, Curr. Med. Chem. 2006, 13, 935–958; k) L. A.
Sorbera, Drugs Future 2006, 31, 335–344; l) C. Monneret, Eur. J.
[19] J.-A. Fehrentz, B. Castro, Synthesis 1983, 676–678.
Chem. Eur. J. 2009, 15, 11174 – 11186
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11185

T. Katoh et al.
[20] K. L. Rinehart, R. Sakai, V. Kishore, D. W. Sullins, K. M. Li, J. Org.
Chem. 1992, 57, 3007–3013.
[21] K. R. West, K. D. Bake, S. Otto, Org. Lett. 2005, 7, 2615–2618.
[22] a) P. R. Blakemore, W. J. Cole, P. J. Kocienski, A. Morley, Synlett
1998, 26–28; b) P. R. Blakemore, J. Chem. Soc. Perkin Trans. 1 2002,
2563–2585.
[23] R. W. Hoffmann, G. Mas, T. Brandl, Eur. J. Org. Chem. 2002, 3455–
3464.
[24] a) G. I. Poos, G. E. Arth, R. E. Beyler, L. H. Sarett, J. Am. Chem.
Soc. 1953, 75, 422–429; b) R. E. Jones, F. W. Kocher, J. Am. Chem.
Soc. 1954, 76, 3682–3683; c) J. A. Glinski, B. S. Joshi, Q. P. Jiang,
S. W. Pelletier, Heterocycles 1988, 27, 185–193.
R. Miyao, Heterocycles 2008, 76, 1313–1328; b) I. Shiina, M.
Kubota, H. Oshiumi, M. Hashizume, J. Org. Chem. 2004, 69, 1822–
1830.
[34] J. Inanaga, K. Hirata, H. Saeki, T. Katsuki, M. Yamaguchi, Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
[35] a) T. Sasaki, M. Inoue, M. Hirama, Tetrahedron Lett. 2001, 42,
5299–5303; b) E. J. Corey, K. C. Nicolaou, J. Am. Chem. Soc. 1974,
96, 5614–5616; c) T. Mukaiyama, R. Matsueda, M. Suzuki, Tetrahe-
dron Lett. 1970, 11, 1901–1904.
[36] a) S. Kato, Y. Hamada, T. Shioiri, Tetrahedron Lett. 1986, 27, 2653–
2656; b) B. Kamber, A. Hartmann, K. Eisler, B. Riniker, H. Rink, P.
Sieber, W. Rittel, Helv. Chim. Acta 1980, 63, 899–915.
[25] a) D. Castagnolo, S. Armaroli, F. Corelli, M. Botta, Tetrahedron
Asymmetry 2004, 15, 941–949; b) M. T. Reetz, Angew. Chem. 1984,
96, 542–555; Angew. Chem. Int. Ed. Engl. 1984, 23, 556–569;
c) D. J. Cram, D. R. Wilson, J. Am. Chem. Soc. 1963, 85, 1245–1249;
d) D. J. Cram, K. R. Kopecky, J. Am. Chem. Soc. 1959, 81, 2748–
2755.
[26] O. Barun, K. Kumar, S. Sommer, A. Langerak, T. U. Mayer, O.
Mꢃller, H. Waldmann, Eur. J. Org. Chem. 2005, 4773–4788.
[27] P. J. Kocienski, A. Bell, P. R. Blakemore, Synlett 2000, 365–366.
[28] a) S. Takano, M. Akiyama, S. Sato, K. Ogasawara, Chem. Lett. 1983,
1593–1596; b) Y. Ito, Y. Ohnishi, T. Ogawa, Y. Nakahara, Synlett
1998, 1102–1104.
[29] a) D. B. Dess, J. C. Martin, J. Am. Chem. Soc. 1991, 113, 7277–7287;
b) R. L. Amey, J. C. Martin, J. Am. Chem. Soc. 1979, 101, 5294–
5299; c) R. L. Amey, J. C. Martin, J. Am. Chem. Soc. 1978, 100, 300–
301.
[30] B. S. Bal, W. E. Childers, Jr., H. W. Pinnick, Tetrahedron 1981, 37,
2091–2096.
[37] a) R. Dembinski, Eur. J. Org. Chem. 2004, 2763–2772; b) O. Mitsu-
nobu, Synthesis 1981, 1–28.
[38] S. Senese, K. Zaragoza, S. Minardi, I. Muradore, S. Ronzoni, A. Pas-
safaro, L. Bernard, G. F. Draetta, M. Alcalay, C. Seiser, S. Chiocca,
Mol. Cell. Biol. 2007, 27, 4784–4795.
[39] a) S. Chang, T. A. McKinsey, C. L. Zhang, J. A. Richardson, J. A.
Hill, E. N. Olson, Mol. Cell. Biol. 2004, 24, 8467–8476; b) C. L.
Zhang, S. Chang, T. A. McKinsey, S. Chang, C. L. Antos, J. A. Hill,
E. N. Olson, Cell 2002, 110, 479–488.
[40] G. M. Shivashimpi, S. Amagai, T. Kato, N. Nishino, S. Maeda, T. G.
Nishino, M. Yoshida, Bioorg. Med. Chem. 2007, 15, 7830–7839.
[41] a) S. Yaguchi, Y. Fukui, I. Koshimizu, H. Yoshimi, T. Matsuno, H.
Gouda, S. Hirono, K. Yamazaki, T. Yamori, J. Natl. Cancer Inst.
2006, 98, 545–556; b) T. Yamori, Cancer Chemother. Pharmacol.
2003, 52 (Suppl. 1), 74–79; c) S. Dan, T. Tsunoda, O. Kitahara, R.
Yanagawa, H. Zembutsu, T. Katagiri, K. Yamazaki, Y. Nakamura, T.
Yamori, Cancer Res. 2002, 62, 1139–1147; d) T. Yamori, A. Matsu-
naga, S. Sato, K. Yamazaki, A. Komi, K. Ishizu, I. Mita, H. Edatsugi,
Y. Matsuba, K. Takezawa, O. Nakanishi, H. Kohno, Y. Nakajima, H.
Komatsu, T. Andoh, T. Tsuruo, Cancer Res. 1999, 59, 4042–4049.
[31] G. Benz in Comprehensive Organic Synthesis Vol. 6 (Eds.: B. M.
Trost, I. Fleming), Pergamon, Oxford, 1991, pp. 381–417.
[32] a) I. Shiina, Chem. Rev. 2007, 107, 239–273; b) I. Shiina, M. Kubota,
R. Ibuka, Tetrahedron Lett. 2002, 43, 7535–7539.
[33] 2,6-Dimethyl-4-nitrobenzoic anhydride (DMNBA) was a kind gift
from Professor I. Shiina, Tokyo University of Science; a) I. Shiina,
Received: June 8, 2009
Published online: September 16, 2009
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Chem. Eur. J. 2009, 15, 11174 – 11186