Total Synthesis of the Depsipeptide FR901375 and Preliminary Evaluation of Its Biological Activity

Article abstract of DOI:10.1002/ejoc.201601023

The bicyclic depsipeptide FR901375 was efficiently synthesized in a highly convergent manner. The synthesis involved the condensation of a d-valine–d-valine–d-cysteine-containing segment with a (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid–l-threonine-containing segment to directly assemble the corresponding seco-amino acid, followed by cyclization to construct the desired 16-membered macrocyclic ring. The potency of the synthesized FR901375 was determined in assays for histone deacetylase (HDAC) inhibition and cell-growth inhibition, and the results were compared to those obtained for the clinically approved depsipeptide FK228 (romidepsin). It was found that FR901375 shows extremely high selectivity (957-fold) for a class I HDAC 1 (GI₅₀= 1.7 nm) over a class II HDAC 6 (GI₅₀= 1627 nm), and shows cell-growth inhibition GI₅₀values in the low nanomolar region. Furthermore, new aspects of the structure–activity relationship of bicyclic depsipeptide HDAC inhibitors were revealed.

Full text of DOI:10.1002/ejoc.201601023

A Journal of
Accepted Article
Title: Total Synthesis of the Depsipeptide FR901375 and Preliminary Evaluation of
its Biological Activity
Authors: Koichi Narita; Yuya Katoh; Ken-Ichi Ojima; Singo Dan; Takao Yamori; Aki-
hiro Ito; Minoru Yoshida; Tadashi Katoh
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601023
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601023
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European Journal of Organic Chemistry
10.1002/ejoc.201601023
FULL PAPER
Total Synthesis of the Depsipeptide FR901375 and Preliminary
Evaluation of its Biological Activity
Koichi Narita,[a] Yuya Katoh,[a] Ken-ichi Ojima,[a] Singo Dan,[b] Takao Yamori,[b,c] Akihiro Ito,[d] Minoru
Yoshida,[d] and Tadashi Katoh*[a]
Abstract: The bicyclic depsipeptide FR901375 was efficiently
synthesized in a highly convergent manner via the condensation of a
D-valine–D-valine–D-cysteine-containing segment with a (3S,4E)-3-
human cancer cells including A549, MCF-7 and SW480 and in
vivo antitumour activity against P388 leukemia cells.[1] The
structure of 1 is closely related to several reported bicyclic
depsipeptide histone deacetylase (HDAC) inhibitors, including
FK228 (romidepsin, 2)[2], spiruchostatins A–D (3–6)[3] and
burkholdacs A (7) and B (8)[4], with the main difference being the
position of the disulfide bridge. Thus, it is reasonable to assume
that the antitumor activity of 1 is due to its HDAC inhibitory
activity. However, no HDAC inhibitory data for FR901375 has
been reported in the literature.[1] The first total synthesis of 1 was
achieved in 2003 by Wentworth-Janda et al.,[5] and its biological
activity has not yet been assessed. Yoshida et al. demonstrated
a molecular mechanism by which FK228 inhibits HDAC.[6] In that
mechanism, the depsipeptide FK228, which itself serves as a
stable prodrug, is activated by reductive cleavage of the disulfide
bond after incorporation into the cells, and the released
sulfhydryl group in the butenyl side chain interacts with the zinc
cation located at the binding pocket of the HDACs, resulting in a
potent inhibitory effect.
hydroxy-7-mercapto-4-heptenoic
acid–L-threonine-containing
segment to directly assemble the corresponding seco-amino acid,
followed by cyclization to construct the desired 16-membered
macrocyclic ring. The potency of the synthesized FR901375 was
determined in histone deacetylase (HDAC) inhibition and cell-growth
inhibition assays, and the results were compared to those obtained
with the clinically approved depsipeptide FK228 (romidepsin). It was
found that FR901375 exhibits extremely high selectivity (957-fold) for
a class I HDAC1 (IC50 = 1.7 nM) over class II HDAC6 (IC50 = 1627
nM) and cell-growth inhibition IC50 values in the low nanomolar
region. Furthermore, the novel aspects of the structure–activity
relation of bicyclic depsipeptide HDAC inhibitors were revealed.
Keywords: natural products · total synthesis · depsipeptide ·
FR901375 · HDAC inhibitor
Introduction
In 1991, researchers from Fujisawa Pharmaceutical Co. Ltd.
(now Astellas Pharma Inc.) reported the isolation of FR901375
(1, Fig. 1) from the fermentation broth of Pseudomonas
chloroaphis No. 2522 and its structural elucidation.[1] FR901375
is a 16-membered bicyclic depsipeptide comprising L-threonine,
D-cysteine, two D-valine units, (3S,4E)-3-hydroxy-7-mercapto-4-
heptenoic acid (Hmh) and a characteristic disulfide bridge. This
natural product exhibits antiproliferative activity against several
[a]
Y. Katoh, Dr. K. Narita, K. Ojima, Dr. T. Katoh
Laboratory of Synthetic and Medicinal Chemistry, Faculty of
Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical
University
4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558 (Japan)
E-mail: katoh@tohoku-mpu.ac.jp
http: www.tohoku-mpu.ac.jp/laboratory/iyakugo/index.html
Dr. S. Dan, Dr. T. Yamori
[b]
Division of Molecular Pharmacology, Cancer Chemotherapy Center,
Japanese Foundation for Cancer Research
3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan)
Dr. T. Yamori
Pharmaceuticals and Medical Devices Agency (PMDA)
3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013 (Japan)
Dr. A. Ito, Dr. M. Yoshida
[c]
[d]
Fig. 1. Structures of FR901375 (1), FK228 (romidepsin, 2), spiruchostatins
A–D (3–6) and burkholdacs A (7) and B (8). iPr = isopropyl, sBu = sec-butyl,
iBu = isobutyl.
Chemical Genetics Laboratory, RIKEN
2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan)
Supporting information for this article is given via a link at the end of
the document.
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European Journal of Organic Chemistry
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During our research on the total synthesis and biological
evaluation of 2–8,[7] we became interested in synthesizing 1 and
evaluating its biological activity. Several total syntheses of 2–4,
7 and 8 have been published in the literature by other groups.[8]
In this study, we describe the total synthesis of 1 using a
macrolactamization strategy for the challenging construction of
the 16-membered macrocyclic ring. In addition, 1 was subjected
to an HDAC inhibition assay and antiproliferative analysis for
evaluating its biological potency as compared to the clinically
approved FK228.
Condensation of 14 with D-cysteine derivative 13 (83%) followed
by protection of the hydroxy group in the resulting dipeptide 15
(79%) affords the TBS ether 16. After deprotection of the N-
Fmoc group in 16, the liberated amine 17 is subjected to
condensation with the D-valine derivative 12, which results in the
tripeptide 18 in 88% yield over the two steps. Deprotection of the
N-Fmoc group in 18 affords the desired segment 10 in 78% yield.
Results and Discussion
Primary synthetic plan for FR901375 (1)
Our primary synthetic strategy is outlined in Scheme 1. With
reference to the synthetic approaches to 1 and 2 reported by
Wentworth-Janda et al.[5] and the present authors,[7c]
respectively, we envisaged that the target molecule 1 could be
synthesized by Mitsunobu-type macrolactonization[9] of the
corresponding seco-acid 9, followed by internal disulfide bond
formation. The key macrolactonization precursor 9 is accessible
by the direct condensation of the tripeptide segment 10 and the
D-valine-containing segment 11. Segment 10 is formed by the
successive condensation of D-valine derivative 12, D-cysteine
derivative 13 and L-threonine methyl ester hydrochloride (14).
The remaining segment 11 is obtained using the method
previously reported for our total synthesis of 2.[7c]
Scheme 2. Synthesis of segment 10. a) 13, PyBOP, iPr2NEt, MeCN, RT, 83%;
b) TBSCl, imidazole, DMF, RT, 79%; c) Et2NH, MeCN, RT; d) 12, PyBOP,
iPr2NEt, MeCN, RT, 88% (2 steps); e) Et2NH, MeCN, RT, 78%. PyBOP =
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, DMF =
N,N-dimethylformamide.
Synthesis and attempted macrolactonization of seco-acid 9
The synthesis of seco-acid 9 was performed by assembling the
two segments 10 and 11, as shown in Scheme 3. Treating a
mixture of 10 and 11 with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate (HATU) (1.3 equiv)
and 1-hydroxy-7-azabenzotriazole (HOAt) (1.3 equiv) in the
presence of iPr2NEt (2.6 equiv) in CH2Cl2 at −20 ºC for 6 h
affords the desired condensation product 19 in excellent yield
(95%) without appreciable epimerization at the C18 stereogenic
centre.[7c] The successive removal of the PMB and methyl
protecting groups from 19 affords the requisite seco-acid 9 with
an overall yield of 76%. This molecule corresponds to the key
intermediate in Wentworth-Janda’s total synthesis of 1.[10]
Scheme 1. Primary synthetic strategy for FR901375 (1). TBS
= tert-
butyldimethylsilyl, Tr = triphenylmethyl, PMB = 4-methoxybenzyl, Fmoc = 9-
fluorenylmethoxycarbonyl.
Synthesis of segment 10
The synthesis of segment 10 was performed from L-threonine
methyl ester hydrochloride (14), as shown in Scheme 2.
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European Journal of Organic Chemistry
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Scheme 3. Synthesis and attempted macrolactonization of seco-acid 9. a)
HATU, HOAt, iPr2NEt, CH2Cl2, −20 ºC, 95%; b) DDQ, CH2Cl2/H2O, RT, 88%;
c) LiOH·H2O, THF/H2O, 0 ºC, 87%; d) DIAD, Ph3P, pTsOH, THF, 0 °C to RT.
HATU
=
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
1-hydroxy-7-azabenzotriazole, DDQ 2,3-
hexafluorophosphate, HOAt
=
=
dichloro-5,6-dicyano-1,4-benzoquinone, DIAD = diisopropyl azodicarboxylate,
pTsOH = p-toluenesulfonic acid.
In the previous total synthesis of 1, Wentworth-Janda et al.
successfully achieved macrolactonization of
9 using the
Mitsunobu reaction, which provided the desired cyclization
product 21 in moderate yield (58%) with stereoinversion at the
C1 hydroxy group.[5] Therefore, we initially followed Wentworth-
Janda’s protocol to construct the key macrolactone ring. Thus,
treating seco-acid 9 (50 mg scale) with a large excess of PPh3
(25 equiv) and diisopropyl azodicarboxylate (DIAD) (20 equiv) in
the presence of pTsOH (5 equiv) in dry THF at 0 °C to room
temperature over 4 h afforded the cyclized product in 47% yield
after purification by silica gel column chromatography. However,
the spectral data of product 21 thus obtained did not match
those of the analogous compound 21 reported by Wentworth-
Janda et al.[5] All attempts to reproduce this macrolactonization
step using the Wentworth-Janda conditions were unsuccessful.
Although the reason for this irreproducibility is unclear, we
believe that the obtained product may be the C1 epimer of 21,
but its stereochemistry could not be assigned unambiguously.
This speculation is based on a study by De Brabander et al.,[11]
which reported that the Mitsunobu-type macrocyclization of an
allylic alcohol-containing seco-acid tends to produce the
corresponding macrolactone with retention of the alcohol
configuration. These unsuccessful results prompted us to modify
our original synthetic plan.
Scheme 4. Modified synthetic strategy for FR901375 (1). TIPS
triisopropylsilyl.
=
Modified synthetic plan
Synthesis of segment 23
As discussed above, our initial attempts to realize the
macrolactonization of seco-acid 9 under Mitsunobu conditions
resulted in failure; therefore, we modified our original synthetic
strategy, as shown in Scheme 4. The modified plan features a
macrolactamization strategy[12] (cf. 22 →1). We envisaged that 1
could be synthesized by internal amide bond formation of the
seco-amino acid 22 followed by disulfide bond formation. This
type of macrolactamization to construct a 16-membered-cyclic
ring was originally explored by Ganesan et al. in their total
synthesis of FK228.[8b] The key cyclization precursor 22 can be
The synthesis of segment 23 proceeds from the known D-
cysteine derivative 25,[13] as shown in Scheme 5. Condensation
of 25 with N-Fmoc-D-valine (12) affords dipeptide 28 in 82%
yield. After deprotection of the N-Fmoc group in 28, the liberated
amine 29 is subjected to condensation with 12 affording
tripeptide 30. Saponification of the methyl ester function in 30
results in the unintended deprotection of the N-Fmoc group;
therefore, its reprotection is required by treatment with N-(9-
fluorenylmethoxycarbonyloxy)succinimide (FmocOSu) and Et3N
after the reaction is quenched. The desired tripeptide 23 is
obtained in 58% overall yield over the three steps.
prepared in
a highly convergent manner by the direct
condensation of tripeptide segment 23 with ester segment 24.
Segment 23 is formed by the successive condensation of two D-
valine derivatives 12 and D-cysteine derivative 25. Segment 24
is obtained by the esterification of alcohol 26 with L-threonine
derivative 27.
Scheme 5. Synthesis of segment 23. a) 12, PyBOP, iPr2NEt, MeCN, RT, 82%;
b) Et2NH, MeCN, RT; c) 12, PyBOP, iPr2NEt, MeCN, RT; d) LiOH·H2O,
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European Journal of Organic Chemistry
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THF/H2O,
0 ºC; FmocOSu, Et3N 58% (3 steps). FmocOSu = N-(9-
which results in the production of FR901375 (1) in 89% yield
{[]20D = −27.3º (c = 0.90 in CHCl3); lit.[1] []20D = −25.3º (c = 1.0,
CHCl3); lit.[5] []25D = −24.4º (c = 0.7, CHCl3)}. The spectroscopic
properties (IR, 1H and 13C NMR, HRMS) of the product are
identical to those reported for natural 1[1] and synthetic 1[5].
fluorenylmethoxycarbonyloxy)succinimide.
Synthesis of segment 24
We next synthesized segment 24, the condensation partner of
23, starting from carboxylic acid 31,[7c] as shown in Scheme 6.
Protection of the carboxylic acid group in 31 with
a
triisopropylsilyl (TIPS)-oxymethyl group[14] (82%) followed by
removal of the PMB group in the resulting ester 32 (94%),
provides allyl alcohol 26. An ester bond-forming reaction
between 26 and L-threonine derivative 27 proceeds efficiently
using N,N’-dicyclohexylcarbodiimide (DCC) in the presence of 4-
dimethylaminopyridine (DMAP), affording ester 33 in excellent
yield (90%). Removal of the N-Fmoc group in 33 affords
segment 24 in 85% yield.
Scheme 6. Synthesis of segment 24. a) TIPSOCH2Cl, iPr2NEt, CH2Cl2, RT,
82%; b) DDQ, CH2Cl2/H2O, RT, 94%; c) 27, DCC, DMAP, CH2Cl2, RT, 90%; d)
Et2NH, CH2Cl2, RT, 85%. DCC = N,N’-dicyclohexylcarbodiimide, DMAP = 4-
dimethylaminopyridine.
Scheme 7. Completion of the total synthesis of FR901375 (1). a) HATU, HOAt,
iPr2NEt, CH2Cl2, −20 ºC, 80%; b) piperidine, CH2Cl2, RT, 72%; c) HF-pyridine,
pyridine, RT; d) HATU, iPr2NEt, THF, RT, 52% (2 steps); e) I2, CH2Cl2/MeOH,
RT, 89%.
Synthesis of FR901375 (1)
Biological evaluation
After obtaining the two key segments 23 and 24, we performed
the synthesis of FR901375 (1), as shown in Scheme 7. The
condensation of 23 with 24 proceeds well under the mild
conditions developed in our previous studies [i.e. HATU (1.3
equiv), HOAt (1.3 equiv), iPr2NEt (2.6 equiv), CH2Cl2, −20 °C, 2
h],[7c] with the desired product 34 being obtained in 80% yield.
After deprotection of the N-Fmoc group in 34 with piperidine
(72%), the TIPS-oxymethyl and TBS protecting groups in the
resulting amine 35 are removed simultaneously using HF-
pyridine, resulting in the hydroxy-containing seco-amino acid 22,
which represents the key macrolactamization precursor. Since
this product is a highly polar substance, it is used in the next
The synthesized FR901375 (1) was evaluated for its HDAC and
cell-growth inhibitory activity in order to determine its potency
and explore the structure-activity relationships (SAR) of bicyclic
depsipeptide HDAC inhibitors. Note that this evaluation
represents the first report on the HDAC inhibitory activity of 1.
HDAC inhibition assay
There are 18 human HDAC isoforms grouped into 4 major
classes. Class I (HDACs 1, 2, 3 and 8), class II (class IIa:
HDACs 4, 5, 7 and 9; class IIb: HDACs 6 and 10), and class IV
(HDAC 11) are Zn2+-dependent metallohydrolases, whereas
class III HDACs (SirTs 1–7) are NAD+-dependent sirtuins.[15]
Inhibiting class I HDACs is considered to be an effective
mechanism for anticancer agents, whereas inhibiting class II
HDACs may cause undesirable side effects such as serious
cardiac hypertrophy;[16] therefore, in cancer chemotherapy, the
potent and selective inhibition of class I HDACs is highly
desirable. The clinically approved FK228 (2) is known to be
reaction
without
further
purification.
The
crucial
macrolactamization is successfully achieved by treating a dilute
solution of 22 in dry THF (0.4 mM) with HATU (3 equiv) and
iPr2NEt (4.6 equiv) at room temperature for 18 h, which affords
the desired cyclized product 36 in 52% overall yield over the two
steps. Finally, simultaneous S-Tr deprotection and disulfide
bond formation of 36 is achieved by brief exposure to iodine in
dilute CH2Cl2/MeOH solution (0.5 mM) at room temperature,
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European Journal of Organic Chemistry
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FULL PAPER
associated with an unresolved cardiotoxicity issue that probably
arises from insufficient class selectivity.[17,18]
Table 2. Growth inhibition of FR901375 (1) against a panel of 39 human
cancer cell lines.
To elucidate its potency and selectivity, the synthesized 1
was tested for its HDAC inhibitory activity against HDAC1 (class
I) and HDAC6 (class IIb).[19] In this assay, FK228 was used as a
positive control. As summarized in Table 1, 1 exhibits inhibitory
activity against HDAC1 in the low-nanomolar range (IC50 = 1.7
nM). Its potency is ca. two-fold higher than that of 2 (IC50 = 3.6
nM). As we expected, 1 exhibits much lower HDAC6 inhibitory
activity (IC50 = 1627 nM). For convenience, isoform selectivity
(class I HDAC1/class IIb HDAC6) is expressed as a selective
index (SI) value (HDAC6 IC50/HDAC1 IC50). Note that 1 (SI =
957) exhibits ca. 8.8-fold higher isoform selectivity than 2 (SI =
108), which represents the highest level of class I/II selectivity
among the naturally occurring and synthetic bicyclic
depsipeptides known to date. Thus, changing of the bonding
position of the disulfide bridge to that in 1 is improves both the
HDAC inhibition and isoform selectivity.
GI50a [ nM ]
b
Origin of cancer
Breast
Cell line
FK228 (
2
)
FR901375 (1)
6.9
8.5
13
6.0
8.6
49
HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
U-251
SF-268
SF-295
SF-539
SNB-75
SNB-78
HCC2998
KM-12
4.2
5.5
3.9
4.9
4.0
3.6
7.2
9.6
3.1
3.4
3.3
450c
3.1
4.6
8.9
1.8d
3.0
2.6
5.8
3.6
2.5
4.6
20
4.9
3.8
7.4
8.0
7.0
2.9
5.2
8.2
6.3
6.1
4.1
1000c
4.8
4.6
7.0
1.3d
6.5
5.2
12
5.1
3.2
6.5
24
3.3
4.9
3.9
14
Central nervous
system (brain)
Colon
Lung
HT-29
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
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU-145
PC-3
Table 1. HDAC inhibitory activity of FR901375 (1).
[a]
IC50
(nM)
Compound
HDAC1 (class I)[b] HDAC6 (class IIb)[b]
SI[d]
[c]
3.6 ± 0.46
1.7 ± 0.19
108
957
390 ± 69.6
1627 ± 280
FK228 (
2)
FR901375 (
1
)
Melanoma
Ovary
[a] Concentration that induces 50% inhibition against HDACs.
2.8
5.5
3.3
6.6
20
[b] Enzyme assay was performed in the presence of 0.1 mM dithiothreitol
(DTT).
[c] Positive control used in this study was synthesized in our laboratory.[7c]
Kidney
29
37
6.3
22
34
[d] Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity toward class I
HDAC1 over class II HDAC6.
22
Stomach
3.2
4.9
17
Cell-growth inhibition assay
14
19
3.0
6.0
18
6.2
7.0
12
Prostate
Next, the growth-inhibitory activity of 1 was evaluated at the
Japanese Foundation for Cancer Research alongside FK228 (2)
as a reference compound using a panel of 39 human cancer cell
lines.[20] The number of cell lines and their organ of origin were 5
breast, 6 central nervous system (brain), 5 colon, 7 lung, 1
melanoma, 5 ovary, 2 kidney, 6 stomach and 2 prostate. Dose-
response curves were measured at five different concentrations
(from 10−10 to 10−6 M) for the compounds, and the
concentrations causing 50% cell growth inhibition (GI50) were
compared.
MG-MIDe
6.2
8.7
[a] Concentration that induces 50% inhibition of cell growth compared to the
control.
[b] Positive control used in this study was synthesized in our laboratory.[7c]
[c] The least sensitive cell.
[d] The most sensitive cell.
[e] Mean value of GI50 over all cell lines tested.
The GI50 values for 1 and FK228 are shown in Table 2.
Compound 1 exhibits nanomolar-range values for GI50 against
nearly all of the 39 cell lines. Although the MG-MID value, i.e.
the mean value for GI50 over all of the cell lines tested, for 1 (GI50
= 8.7 nM) is slightly lower than that for FK228 (GI50 = 6.2 nM),
the efficacy of 1 is superior to that of FK228 against several cell
lines, including HBC-4, MDA-MB-231, SF-539, SNB-75, SNB-78,
NCI-H226, NCI-H522, OVCAR-8 and PC-3. The pearson
correlation coefficient between fingerprints of 1 and FK228 was
extremely high (r = 0.914), suggesting that they share a common
mechanism of action in cell levels. Considering both its HDAC
inhibitory and antiproliferative activities, FR901375 seems to be
a promising candidate for a novel class I HDAC1-targeting
anticancer agent.
Conclusions
We have achieved the total synthesis of FR901375 (1) in a
reliable and efficient way starting from the known D-cysteine
derivative 25 having a 12.6% overall yield over a linear
sequence of nine steps. The key elements of the synthesis were
the following: (i) an amide condensation of the two segments 23
and 24 to directly assemble the seco-amino acid 22 (23 + 24 →
22, Scheme 7), and (ii) the macrocyclization by internal amide
bond formation of 22 (22 → 36, Scheme 7), which circumvented
the
difficulty
in
reproducing
the
Mitsunobu-based
macrolactonization (9 → 21, Scheme 3). The main advantage of
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European Journal of Organic Chemistry
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FULL PAPER
afforded a residue, which was purified by column chromatography
(hexane/EtOAc 4:1) to give 16 (0.90 g, 79%) as a colorless amorphous
solid. [α]D25 = +9.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ = 0.00
(3H, s), 0.03 (3H, s), 0.85 (9H, s), 1.17 (3H, d, J = 5.9 Hz), 2.69 (1H, dd,
J = 13.2, 7.8 Hz), 2.77 (1H, dd, J = 13.2, 5.9 Hz), 3.78 (3H, s), 3.96 (1H,
q, J = 6.7 Hz), 4.25 (1H, t, J = 6.8 Hz), 4.34 (1H, dd, J = 10.5, 7.1 Hz),
4.43–4.51 (3H, m), 5.11 (1H, br d, J = 6.8 Hz), 6.67 (1H, d, J = 8.8 Hz),
7.23–7.38 (10H, m), 7.40–7.49 (9H, m), 7.63 (2H, t, J = 7.3 Hz), 7.77 (1H,
d, J = 7.8 Hz), 7.80 ppm (1H, d, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3):
δ = −5.4, −4.5, 17.8, 20.8, 25.6 (3C), 33.8, 47.1, 52.2, 54.0, 57.8, 67.2,
67.3, 68.7, 119.94, 119.96, 125.10, 125.12, 126.9 (3C), 127.1 (2C),
127,7 (2C), 128.1 (6C), 129.5 (6C), 141.3 (2C), 143.6, 143.9, 144.3 (3C),
155.8, 170.3, 170.5 ppm. IR (neat): ν = 3315, 3060, 3018, 2952, 2929,
2896, 2856, 1730, 1677, 1514, 1493, 1446, 1317, 1253, 1093, 1034, 838,
741, 701 cm−1. HRMS (FAB): calcd for C48H55N2O6SSi [M+H]+ 815.3550;
found 815.3554.
the present synthesis compared with that previously reported by
Wentworth-Janda et al. (12.4% overall yield in 13 linear steps) is
the reduced number of reaction steps. Preliminary biological
evaluation of 1 determined its potency and novel aspects of the
SAR of bicyclic depsipeptides. These results may be useful for
designing and developing anticancer agents having therapeutic
potential that target the isoform-selective inhibition of HDACs.
Further investigation concerning the synthesis of unnatural
FR901375 analogues and study of their SAR is currently
underway in our group and will be reported in due course.
Experimental Section
General Techniques: All reactions involving air- and moisture-sensitive
reagents were carried out using oven dried glassware and standard
syringe-septum cap techniques. Routine monitorings of reaction were
carried 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 nm) with the solvents indicated.
Fmoc-D-Val–D-Cys(Tr)–L-Thr(TBS)-OMe (18): Et2NH (0.40 mL, 4.7
mmol) was added to a stirred solution of 16 (154 mg, 0.19 mmol) in
MeCN (4 mL) at room temperature. After 1 h, the reaction mixture was
concentrated in vacuo to afford NH2-D-Cys(Tr)-L-Thr(TBS)-OMe (17)
(112 mg) as a colorless oil, which was used for the next reaction without
further purification.
All solvents and reagents were used as supplied with following
exceptions. Tetrahydrofuran (THF) was freshly distilled from Na
metal/benzophenone under argon. N,N-Dimethylformamide (DMF),
CH2Cl2, MeCN, pyridine and iPr2NEt were distilled from calcium hydride
under argon.
iPr2NEt (0.15 mL, 0.88 mmol) was added dropwise to a stirred solution of
the crude product 17 (112 mg, 0.19 mmol) and Fmoc-D-Val-OH (12) (88
mg, 0.26 mmol) in MeCN (2 mL) containing PyBOP (171 mg, 0.33 mmol)
at room temperature under argon. After 1 h, concentration of the solvent
in vacuo afforded
a
residue, which was purified by column
Measurements of optical rotations were performed with a Anton Paar
MCP-100 automatic digital polarimeter. 1H and 13C NMR spectra were
measured with a JEOL AL-400 (400 MHz) and JEOL LA-600 (600 MHz)
spectrometers. Chemical shifts were expressed in ppm using Me4Si (δ =
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-resolution mass (HRMS)
spectra were measured on a JEOL JMS-700 high resolution mass
spectrometer.
chromatography (hexane/EtOAc 3:1) to give 18 (176 mg, 88%, 2 steps)
as a colorless amorphous solid. [α]D25 = +10.4 (c = 1.1, CHCl3); 1H NMR
(400 MHz, CDCl3): δ = −0.02 (3H, s), 0.03 (3H, s), 0.86 (9H, s), 0.88 (3H,
d, J = 6.8 Hz), 0.93 (3H, d, J = 6.3 Hz), 1.14 (3H, d, J = 5.9 Hz), 2.04–
2.06 (1H, m), 2.62 (1H, dd, J = 12.9, 6.1 Hz), 2.71 (1H, dd, J = 12.9, 6.6
Hz), 3.67 (3H, s), 4.00 (1H, dd, J = 8.3, 5.9 Hz), 4.18–4.23 (2H, m), 4.32–
4.36 (1H, m), 4.41–4.48 (3H, m), 5.36 (1H, d, J = 8.8 Hz), 6.14 (1H, d, J
= 6.8 Hz), 6.59 (1H, d, J = 9.3 Hz), 7.17–7.33 (11H, m), 7.38–7.44 (8H,
m), 7.60 (2H, t, J = 6.3 Hz), 7.77 ppm (2H, d, J = 7.3 Hz). 13C NMR (100
MHz, CDCl3): δ = −5.3, −4.5, 17.5, 17.8, 19.2, 20.8, 25.6 (3C), 31.4, 33.6,
47.2, 52.1, 52.2, 58.0, 59.8, 67.0, 67.2, 68.7, 119.92, 119.93, 125.1 (2C),
126.9 (3C), 127.1 (2C), 127,7 (2C), 128.0 (6C), 129.5 (6C), 141.3 (2C),
143.8, 143.9, 144.3 (3C), 156.2, 170.0, 170.4, 170.9 ppm. IR (neat): ν =
3309, 3059, 2956, 2855, 1648, 1523, 1447, 1388, 1249, 1096, 1034, 837,
741, 701 cm−1. HRMS (FAB): calcd for C53H64N3O7SSi [M+H]+ 914.4234;
found 914.4240.
Fmoc-D-Cys(Tr)–L-Thr-OMe (15): iPr2NEt (2.0 mL, 12 mmol) was added
dropwise to a stirred solution of 14 (503 mg, 3.0 mmol) and Fmoc-D-
Cys(Tr)-OH (13) (2.08 g, 3.6 mmol) in MeCN (30 mL) containing PyBOP
(2.32 g, 4.5 mmol) at room temperature under argon, and the mixture
was stiired for 4 h at the same temperature. Concentration of the solvent
in vacuo afforded
a
residue, which was purified by column
chromatography (hexane/EtOAc 2:1) to give 15 (1.71 g, 83%) as a
NH2-D-Val–D-Cys(Tr)–L-Thr(TBS)-OMe (10): Et2NH (0.90 mL, 0.86
mmol) was added to a stirred solution of 18 (786 mg, 0.86 mmol) in
MeCN (17 mL) at room temperature. After 1 h, the reaction mixture was
concentrated in vacuo afforded residue, which was purified by column
chromatography (hexane/EtOAc 1:1) to give 10 (466 mg, 78%) as a
colorless amorphous solid. [α]D25 = +18.1 (c = 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): δ = −0.03 (3H, s), 0.02 (3H, s), 0.81 (3H, d, J = 6.8 Hz),
0.83 (9H, s), 0.96 (3H, d, J = 6.8 Hz), 1.13 (3H, d, J = 6.3 Hz), 1.47 (2H,
br s), 2.12–2.29 (1H, m), 2.61 (2H, d, J = 6.8 Hz), 3.19 (1H, d, J = 3.9 Hz),
3.67 (3H, s), 4.27 (1H, q, J = 7.3 Hz), 4.38–4.45 (2H, m), 6.73 (1H, d, J =
8.8 Hz), 7.19–7.30 (9H, m), 7.41–7.43 (6H, m), 7.68 ppm (1H, d, J = 8.3
Hz). 13C NMR (100 MHz, CDCl3): δ = −5.4, −4.4, 16.1, 17.8, 19.7, 20.8,
25.6 (3C), 30.7, 33.5, 51.9, 52.2, 58.0, 60.1, 66.9, 68.6, 126.7 (3C),
128.0 (6C), 129.5 (6C), 144.5 (3C), 170.6, 170.7, 174.5 ppm. IR (neat): ν
= 3328, 2955, 2856, 1748, 1683, 1507, 1254, 836, 743, 700 cm−1. HRMS
(FAB): calcd for C38H54N3O5SSi [M+H]+ 692.3553; found 692.3562.
25
colorless amorphous solid. [α]D = 0.0 (c = 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): δ = 1.14 (3H, d, J = 5.9 Hz), 2.46 (1H, br s), 2.67 (2H, d, J
= 6.8 Hz), 3.68 (3H, s), 3.90 (1H, q, J = 6.7 Hz), 4.19 (1H, t, J = 6.8 Hz),
4.27 (1H, qd, J = 5.9, 2.9 Hz), 4.37 (2H, d, J = 6.8 Hz), 4,49 (1H, dd, J =
8.8, 2.4 Hz), 5.14 (1H, d, J = 6.8 Hz), 6.65 (1H, d, J = 8.8 Hz), 7.18–7.29
(10H, m), 7.34–7.43 (9H, m), 7.56 (2H, d, J = 6.8 Hz), 7.74 ppm (2H, t, J
= 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ = 19.9, 40.0, 47.0, 52.5, 53.9,
57.3, 67.1, 67.2, 67.9, 119.9 (2C), 125.0 (2C), 126.9 (2C), 127.0 (3C),
127.66, 127.68, 128.0 (6C), 129.5 (6C), 141.2 (2C), 143.5, 143.7, 144.2
(3C), 155.9, 170.5, 170.9 ppm. IR (neat): ν = 3327, 3060, 3017, 2952,
1716, 1669, 1594, 1521, 1508, 1446, 1319, 1249, 1217, 1150, 1082,
1034, 741, 701 cm−1. HRMS (FAB): calcd for C42H41N2O6S [M+H]+
701.2685; found 701.2685.
Fmoc-D-Cys(Tr)–L-Thr(TBS)-OMe (16): TBSCl (1.04 g, 6.9 mmol) was
added to a stirred solution of 15 (0.97 g, 1.4 mmol) in DMF (9 mL)
containing imidazole (0.66 g, 9.7 mmol) at 0 ºC under argon, and the
mixture was stirred for 24 h at room temperature. The reaction was
quenched with water (15 mL) and the resulting mixture was extracted
with Et2O (3 × 30 mL). The combined extracts were washed with brine (2
× 20 mL), then dried with Na2SO4. Concentration of the solvent in vacuo
(7R,11R,14R,17S,20S,E)-Methyl
20-[(R)-1-[(tert-
butyldimethylsiloxy)ethyl]-11,14-diisopropyl-7-(4-
methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-triphenyl-17-
tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (19):
iPr2NEt (0.19 mL, 1.1 mmol) was added dropwise to a stirred solution of
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601023
FULL PAPER
10 (315 mg, 0.46 mmol) and 11 (240 mg, 0.38 mmol) in CH2Cl2 (8.4 mL)
containing HATU (216 mg, 0.57 mmol) and HOAt (77 mg, 0.57 mmol) at
−20 °C under argon. After 6 h, the reaction mixture was diluted with
CHCl3 (80 mL). The organic layer was washed successively with 10%
aqueous HCl (2 × 20 mL), saturated aqueous NaHCO3 (2 × 20 mL) and
brine (2 × 20 mL), then dried over Na2SO4. Concentration of the solvent
2.39 (2H, m), 2.52 (1H, dd, J = 12.9, 7.8 Hz), 2.59 (1H, dd, J = 13.2, 6.8
Hz), 3.84–3.89 (1H, m), 4.01–4.05 (1H, m), 4.08–4.12 (1H, m), 4.21–4.25
(1H, m), 4.29–4.33 (1H, m), 4.37–4.42 (1H, m), 5.36 (1H, dd, J = 15.4,
5.9 Hz), 5.49 (1H, dt, J = 15.4, 6.3 Hz), 6.64 (1H, d, J = 9.3 Hz), 7.14–
7.25 (20H, m), 7.32–7.36 ppm (13H, m). 13C NMR (100 MHz,
CD3OD/CDCl3 9:1): δ = −5.6, −4.9, 17.68, 17.74, 17.9, 18.85, 18.92, 20.5,
25.4 (3C), 30.6, 30.7, 31.1, 31.3, 33.2, 42.6, 51.88, 51.92, 58.2, 58.4,
66.3, 66.8, 68.5, 68.7, 126.4 (3C), 126.6 (3C), 127.6 (6C), 127.8 (6C),
129.3 (12C), 129.4, 132.4, 144.1 (3C), 144.6 (3C), 170.4, 170.5, 171.0,
171.5, 171.8 ppm. IR (neat): ν = 3284, 3058, 3030, 2960, 2928, 2856,
1731, 1635, 1539, 1444, 1390, 1253, 1220, 1097, 1034, 965, 836, 743,
700 cm−1. HRMS (FAB): calcd for C68H85N4O8S2Si [M+H]+ 1177.5578;
found 1177.5565.
in vacuo afforded
a
residue, which was purified by column
chromatography (hexane/EtOAc 2:1) to give 19 (473 mg, 95%) as a
colorless amorphous solid. [α]D25 = +21.0 (c = 1.0, CHCl3); 1H NMR (400
MHz, CDCl3): δ = −0.03 (3H, s), 0.01 (3H, s), 0.74 (3H, s), 0.78 (3H, s),
0.79–0.82 (12H, m), 0.85 (3H, s), 1.09 (3H, d, J = 6.3 Hz), 1.93–2.13 (4H,
m), 2.19–2.22 (2H, m), 2.38 (1H, dd, J = 15.1, 3.4 Hz), 2.45 (1H, dd, J =
14.9, 8.5 Hz), 2.56 (1H, dd, J = 13.2, 6.3 Hz), 2.68 (1H, dd, J = 12.7, 6.3
Hz), 3.64 (3H, s), 3.77 (3H, s), 4.08–4.20 (4H, m), 4.23 (1H, d, J = 10.7
Hz), 4.37–4.48 (2H, m), 4.49 (1H, d, J = 11.2 Hz), 5.30 (1H, dd, J = 15.6,
8.3 Hz), 5.57 (1H, dt, J = 15.6, 6.8 Hz), 6.27 (1H, d, J = 7.8 Hz), 6.36 (1H,
d, J = 8.8 Hz), 6.56 (1H, d, J = 9.3 Hz), 6.75 (1H, d, J = 7.8 Hz), 6.80 (2H,
d, J = 8.3 Hz), 7.17–7.22 (8H, m), 7.24–7.28 (12H, m), 7.39–7.41 ppm
(12H, m). 13C NMR (100 MHz, CDCl3): δ = −5.3, −4.5, 17.7, 17.8, 18.1,
19.2, 19.3, 20.8, 25.6 (3C), 30.5, 30.9, 31.2, 31.5, 33.6, 43.2, 52.1, 52.2,
55.3, 57.9, 58.0, 58.9, 66.6, 67.2, 68.7, 70.0, 76.6, 113.7 (2C), 126.6
(3C), 126.9 (3C), 127.9 (6C), 128.0 (6C), 129.5 (6C), 129.6 (6C), 129.7
(2C), 129.9, 130.4, 133.1, 144.3 (3C), 144.9 (3C), 159.2, 170.1, 170.4,
170.6, 170.9, 171.0 ppm. IR (neat): ν = 3277, 3059, 2957, 1746, 1634,
1537, 1514, 1444, 1378, 1248, 1092, 1034, 836, 743, 700 cm−1. HRMS
(FAB): calcd for C77H95N4O9S2Si [M+H]+ 1311.6310; found 1311.6326.
Unsuccessful attempt at macrolactonization of seco-acid 9 to the
corresponding cyclization product 21: A solution of 1.9 M diisopropyl
azodicarboxylate (DIAD) in toluene (0.63 mL, 1.2 mmol) was added
slowly to a stirred solution of Ph3P (390 mg, 1.5 mmol) in THF (60 mL)
containing pTsOH·H2O (56.7 mg, 0.30 mmol) at 0 °C under argon, and
stirring was continued for 30 min at room temperature. A solution of 9
(70.1 mg, 60 mmol) in THF (6.0 mL) was added very slowly to the above
mixture at 0 °C over 2 h, and sttiring was continued for 2 h at 0 ºC.
Concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 2:3) to give product
(32.4 mg, 47%) as a colorless amorphous solid. The stereostructure of
the obtained product was tentatively asigned as the C1 epimer of 21.
[α]D25 = −14.4 (c = 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ = −0.03 (3H,
s), 0.03 (3H, s), 0.71 (3H, d, J = 6.8 Hz), 0.82–0.88 (15H, m), 0.91 (3H, d,
J = 6.8 Hz), 1.10 (3H, d, J = 6.3 Hz), 1.50–1.60 (1H, m), 1.69–1.76 (1H,
m), 2.10–2.23 (2H, m), 2.33–2.41 (1H, m), 2.45–2.52 (1H, m), 2.56 (1H,
dd, J = 12.7, 6.3 Hz), 2.65 (1H, dd, J = 12.7, 6.8 Hz), 2.87 (1H, dd, J =
15.1, 8.8 Hz), 2.94 (1H, dd, J = 14.9, 6.1 Hz), 3.47 (1H, dd, J = 8.5, 6.6
Hz), 3.96 (1H, q, J = 7.0 Hz), 4.18–4.22 (2H, m), 4.28 (1H, dd, J = 9.3,
2.4 Hz), 4.94 (1H, q, J = 6.7 Hz), 5.32 (1H, dd, J = 15.1, 6.8 Hz), 5.81–
5.89 (1H, m), 6.19 (1H, d, J = 8.8 Hz), 6.73 (1H, d, J = 8.8 Hz), 6.78 (1H,
d, J = 6.3 Hz), 7.18–7.42 ppm (31H, m). 13C NMR (100 MHz, CDCl3): δ =
−4.9, −4.3, 17.4, 17.9, 19.1, 19.52, 19.54, 20.8, 25.7 (3C), 27.4, 28.4,
29.7, 32.6, 33.2, 40.2, 52.1, 58.1, 59.6, 64.5, 66.9, 67.0, 68.5, 75.3,
126.7 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C), 129.6 (13C), 132.6,
144.4 (3C), 144.7 (3C), 170.1, 170.2, 170.7, 171.4, 172.2 ppm. IR (neat):
ν = 3285, 3058, 3031, 2960, 2928, 2855, 1748, 1654, 1522, 1444, 1254,
1185, 1105, 1034, 969, 837, 744, 700 cm−1. HRMS (FAB): m/z calcd for
C68H83N4O7S2Si [M+H]+ 1159.5472, found 1159.5466.
(7R,11R,14R,17S,20S,E)-Methyl
20-[(R)-1-(tert-
butyldimethylsiloxy)ethyl]-7-hydroxy-11,14-diisopropyl-9,12,15,18-
tetraoxo-1,1,1-triphenyl-17-tritylthiomethyl-2-thia-10,13,16,19-
tetraazahenicos-5-en-21-oate (20): DDQ (122 mg, 0.54 mmol) was
added in small portions to a stirred solution of 19 (350 mg, 0.27 mmol) in
CH2Cl2/H2O 9:1 (14 mL) at room temperature. After 3 h, the mixture was
diluted with CHCl3 (60 mL), and the organic layer was washed with
saturated aqueous NaHCO3 (2 × 20 mL) and brine (2 × 20 mL), then
dried over Na2SO4. Concentration of the solvent in vacuo afforded a
residue, which was purified by column chromatography (hexane/EtOAc
1:1) to give 20 (281 mg, 88%) as a colorless amorphous solid. [α]D
25
=
+19.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ = −0.03 (3H, s),
0.01 (3H, s), 0.80–0.85 (18H, m), 0.87 (3H, d, J = 6.8 Hz), 1.10 (3H, d, J
= 5.9 Hz), 1.99–2.08 (4H, m), 2.17–2.21 (2H, m), 2.33 (1H, dd, J = 15.1,
8.8 Hz), 2.43 (1H, dd, J = 15.1, 3.4 Hz), 2.57 (1H, dd, J = 13.2, 6.3 Hz),
2.68 (1H, dd, J = 13.2, 7.3 Hz), 3.53 (1H, d, J = 3.9 Hz), 3.66 (3H, s),
4.10 (1H, q, J = 7.2 Hz), 4.20–4.27 (2H, m), 4.36–4.44 (3H, m), 5.39 (1H,
dd, J = 15.6, 6.3 Hz), 5.54 (1H, dt, J = 15.6, 6.3 Hz), 6.40 (1H, br d, J =
5.9 Hz), 6.47 (1H, d, J = 8.8 Hz), 6.52 (1H, d, J = 8.8 Hz), 6.59 (1H, d, J =
8.3 Hz), 7.18–7.29 (17H, m), 7.36–7.42 ppm (13H, m). 13C NMR (100
MHz, CDCl3): δ = −5.3, −4.5, 17.8, 18.0, 18.3, 19.14, 19.16, 20.8, 25.6
(3C), 31.1, 31.3, 31.4, 31.5, 33.8, 42.7, 52.0, 52.2, 58.0 (2C), 58.7, 66.6,
67.2, 68.8, 69.1, 126.6 (3C), 126.9 (3C), 127.8 (6C), 128.1 (6C), 129.5
(6C), 129.6 (6C), 129.9, 132.3, 144.3 (3C), 144.9 (3C), 170.2, 170.3,
170.7, 170.9, 171.8 ppm. IR (neat): ν = 3274, 3059, 2959, 1745, 1632,
1539, 1444, 1390, 1254, 1092, 837, 743, 700 cm−1. HRMS (FAB): calcd
for C69H87N4O8S2Si [M+H]+ 1191.5735; found 1191.5740.
Fmoc-D-Val–D-Cys(Tr)-OMe (28): iPr2NEt (0.9 mL, 5.4 mmol) was
added dropwise to a stirred solution of 25 (375 mg, 0.91 mmol) and
Fmoc-D-Val-OH (12) (370 mg, 1.1 mmol) in MeCN (9 mL) containing
PyBOP (710 mg, 1.4 mmol) at room temperature under argon. After 1 h,
concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 2:1) to give 28 (516
25
mg, 82%) as a colorless amorphous solid. [α]D = −1.5 (c = 0.39 in
CHCl3); 1H NMR (400 MHz, CDCl3): δ = 0.91 (3H, d, J = 6.3 Hz), 0.96
(3H, d, J = 6.8 Hz), 2.05–2.10 (1H, m), 2.61 (H, dd, J = 12.7, 4.4 Hz),
2.74 (1H, dd, J = 12.2, 5.9 Hz), 3.71 (3H, s), 4.00 (1H, dd, J = 8.3, 5.4
Hz), 4.22 (1H, t, J = 6.8 Hz), 4.32–4.36 (1H, m), 4.43 (1H, dd, J = 10.7,
7.3 Hz), 4.51–4.55 (1H, m), 5.39 (1H, d, J = 8.8 Hz), 6.00 (1H, d, J = 7.8
Hz), 7.18–7.40 (19H, m), 7.55–7.60 (2H, m), 7.76 ppm (2H, d, J = 7.8 Hz).
13C NMR (100 MHz, CDCl3): δ = 17.5, 19.0, 31.6, 33.4, 47.2, 51.1, 52.6,
58.2, 59.7, 67.0, 119.95 (2C), 119.97 (2C), 125.1 (2C), 127.0 (3C), 127.1
(2C), 127.7 (2C), 128.1 (6C), 129.4 (6C), 141.3 (2C), 144.2 (3C), 156.2,
170.4, 170.7 ppm. IR (neat): ν = 3298, 3057, 3017, 2962, 1715, 1662,
1507, 1446, 1218, 1107, 1218, 1107, 1032, 758, 741, 701 cm−1. HRMS
(FAB): calcd for C43H43N2O5S [M+H]+ 699.2893; found 699.2904.
(7R,11R,14R,17S,20S,E)-20-[(R)-1-(tert-Butyldimethylsiloxy)ethyl]-7-
hydroxy-11,14-diisopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-17-
tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oic acid
(9): LiOH·H2O (54 mg, 1.3 mmol) was added dropwise to a stirred
solution of 20 (110 mg, 0.16 mmol) in THF/H2O 4:1 (32 mL) at 0 ºC. After
9 h, 10% aqueous HCl was added to the mixture at 0 °C until pH was 6.
The resulting mixture was extracted with EtOAc (3 × 30 mL), and the
combined extracts were washed with saturated aqueous NaHCO3 (2 × 15
mL) and brine (2 × 15 mL), then dried over Na2SO4. Concentration of the
solvent in vacuo afforded a residue, which was purified by column
chromatography (CHCl3/MeOH 15:1) to give 9 (330 mg, 87%) as a
colorless amorphous solid. [α]D25 = +34.3 (c = 1.0, CHCl3); 1H NMR (400
MHz, CD3OD/CDCl3 9:1): δ = −0.02 (3H, s), 0.00 (3H, s), 0.77–0.84 (21H,
m), 1.06 (3H, d, J = 5.9 Hz), 1.95–2.05 (4H, m), 2.13–2.17 (2H, m), 2.28–
Fmoc-D-Val–D-Val–D-Cys(Tr)-OH (23): Et2NH (0.8 mL, 7.4 mmol) was
added to a stirred solution of 28 (516 mg, 0.74 mmol) in MeCN (15 mL)
at room temperature. After 1 h, the reaction mixture was concentrated in
vacuo to afford NH2-D-Val–D-Cys-OMe (29) (353 mg) as a colorless oil,
which was used for the next reaction without further purification.
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European Journal of Organic Chemistry
10.1002/ejoc.201601023
FULL PAPER
iPr2NEt (0.51 mL, 3.0 mmol) was added dropwise to a stirred solution of
the crude product 29 (353 mg, 0.74 mmol) and Fmoc-D-Val-OH (12) (326
mg, 0.96 mmol) in MeCN (15 mL) containing PyBOP (576 mg, 1.1 mmol)
at room temperature under argon. After 2 h, concentration of the solvent
in vacuo afforded a residue, which was washed with EtOAc (2 × 100 mL)
to remove extra reaction reagents. The resulting residue containing
Fmoc-D-Val–D-Val–D-Cys-OMe (30) (591 mg) was used for the next
reaction without further purification.
MHz, CDCl3): δ = 11.8 (3C), 17.7 (6C), 31.3, 31.4, 41.5, 66.6, 68.4, 84.3,
126.6 (3C), 127.8 (6C), 129.6 (6C), 130.3, 131.8, 144.9 (3C), 171.5 ppm.
IR (neat): ν = 3460, 3057, 3030, 2944, 2866, 1736, 1595, 1489, 1464,
1444, 1158, 1115, 966, 883, 742, 700 cm−1. HRMS (FAB): calcd for
C36H47O4SSi [M–H]+ 603.2965; found 603.2961.
(S,E)-(Triisopropylsiloxy)methyl
3-(2S,3R)-2-[(9H-fluoren-9-
yl)methoxycarbonyl]amino-3- (tert-butyldimethylsiloxy)butanoyloxy-
7-(tritylthio)hept-4-enoate (33): DCC (9.1 mg, 44 μmol) and DMAP (0.4
mg, 3.4 μmol) was added to the stirred solution of 26 (23.5 mg, 39 μmol)
and Fmoc-L-Thr(OTBS)-OH (27) (31.8 mg, 0.70 mmol) in CH2Cl2 (1 mL)
at 0 ºC, and stirring was continued for 2 h at room temperature. The
LiOH (93 mg, 2.21 mmol) was added slowly to a stirred solution of the
crude product 30 (591 mg) in THF/H2O 4:1 (40 mL) at 0 °C. After 2 h, 1
M HCl was added to the mixture at 0°C until pH was 7. FmocOSu (324
mg, 0.96 mmol) was added to the resulting mixture, and pH was adjusted
to 8.0–9.0 using Et3N. After 1 h, the reaction was quenched with 1 M HCl
(10 mL) at 0 °C, and the resulting mixture was extracted with EtOAc (3 ×
30 mL). The combined extracts were washed with saturated aqueous
NaHCO3 (2 × 20 mL) and brine (2 × 20 mL), then dried with MgSO4.
Concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (CHCl3/MeOH 40:1) to give 23 (335
mg, 58%, 3 steps) as a colorless amorphous solid. [α]D25 = +4.7 (c = 0.34,
CHCl3); 1H NMR (400 MHz, CDCl3): δ = 0.81–0.86 (12H, m), 1.95–2.02
(2H, m), 2.70 (2H, d, J = 5.4 Hz), 4.07–4.11 (1H, m), 4.16 (1H, t, J = 6.8
Hz), 4.29–4.40 (4H, m), 5.78–5.80 (1H, m), 6.54–6.59 (1H, m), 7.15–7.37
(20H, m), 7.54 (2H, t, J = 7.6 Hz), 7.73 ppm (2H, d, J = 7.8 Hz). 13C NMR
(100 MHz, CDCl3): δ = 18.1, 18.2, 18.9, 19.1, 25.6, 29.7, 31.4, 33.1, 47.0,
51.5, 58.2, 60.4, 67.1, 119.9 (2C), 125.1 (2C), 126.9 (3C), 127.0 (2C),
127.7 (2C), 127.97 (2C), 128.03 (6C), 129.5 (6C), 141.2 (2C), 144.2 (3C),
156.5, 171.4, 171.8, 172.7 ppm IR (neat): ν = 3299, 3060, 2963, 2930,
2874, 1704, 1650, 1538, 1446, 1392, 1292, 1245, 1031, 933, 757, 741,
701 cm−1. HRMS (FAB): calcd for C47H50N3O6S [M+H]+ 784.3420; found
784.3451.
reaction mixture was filtered off through
a small pad of Celite.
Concentration of the filtrate in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 12:1) to give 33 (31.8
mg, 90%) as colorless amorphous solid. [α]D20 = −12.7 (c = 0.85, CHCl3);
1H NMR (400 MHz, CDCl3): δ = –0.09 (3H, s), 0.03, (3H, s), 0.82 (9H, s),
0.98–1.09 (21H, m), 1.14 (3H, d, J = 6.3 Hz), 1.96–2.03 (2H, m), 2.09–
2.16 (2H, m), 2.54 (1H, dd, J = 15.9, 7.1 Hz), 2.72 (1H, dd, J = 15.6, 6.3
Hz), 4.17 (1H, dd, J = 9.5, 1.7 Hz), 4.24 (1H, t, J = 7.3 Hz), 4.29–4.38
(3H, m), 5.34–5.44 (4H, m), 5.52 (1H, q, J = 7.0 Hz), 5.62 (1H, dt, J =
15.1, 6.8 Hz), 7.15–7.37 (19H, m) 7.58 (2H, t, J = 8.5 Hz), 7.73 ppm (2H,
d, J = 7.8 Hz). 13C NMR (100 MHz, CDCl3): δ = –4.9, –4.3, 11.9 (3C),
17.7 (6C), 17.9, 20.9, 25.7 (3C), 31.0, 31.5, 39.7, 47.2, 59.7, 66.6, 67.2,
68.6, 72.0, 84.3, 120.0 (2C), 125.16, 125.24, 126.6 (3C), 127.0, 127.1,
127.6, 127.7 (2C), 127.9 (6C), 129.6 (6C), 133.9, 141.3 (2C), 143.8,
144.0, 144.8 (3C), 156.5, 168.5, 169.7 ppm. IR (neat): ν = 2946, 2865,
1732, 1507, 1447, 1311, 1251, 1164, 1103, 970, 883, 837, 809, 777, 741,
700 cm−1. HRMS (FAB): calcd for C61H80NO8SSi2 [M+H]+ 1042.5143;
found 1042.5171.
(S,E)-(Triisopropylsiloxy)methyl
butyldimethylsiloxy)butanoyloxy]-7-(tritylthio)hept-4-enoate
3-[(2S,3R)-2-amino-3-(tert-
(24):
(S,E)-(Triisopropylsiloxy)methyl
3-(4-methoxybenzyloxy)-7-
tritylthiohept-4-enoate (32): TIPSOCH2Cl (84 μL, 0.36 mmol) was
added to a stirred solution of 31 (196 mg, 0.36 mmol) in CH2Cl2 (6 mL)
containing iPr2NEt (124 μL, 0.73 mmol) at room temperature under argon.
After 1 h, the reaction was quenched with 1 M HCl (3 mL), and the
resulting mixture was extracted with CHCl3 (3 × 5 mL). The combined
extracts were washed with saturated aqueous NaHCO3 (2 × 5 mL) and
brine (2 × 5 mL), then dried with MgSO4. Concentration of the solvent in
vacuo afforded a residue, which was purified by column chromatography
(Hexane/EtOAc 20:1) to give 32 (215 mg, 82%) as a colorless viscous
liquid. [α]D20 = −9.5 (c = 0.82, CHCl3); 1H NMR (400 MHz, CDCl3): δ =
1.04–1.13 (21H, m), 2.10–2.14 (2H, m), 2.20–2.23 (2H, m), 2.44 (1H, dd,
J = 15.6, 5.4 Hz), 2.64 (1H, dd, J = 15.4, 8.0 Hz), 3.78 (3H, s), 4.17 (1H,
td, J = 7.8, 5.4 Hz), 4.27 (1H, d, J = 1.2 Hz), 4.46 (1H, d, J = 11.2 Hz),
5.31 (1H, dd, J = 15.4, 8.0 Hz), 5.41 (1H, d, J = 3.9 Hz), 5.43 (1H, d, J =
4.4 Hz), 5.57 (1H, dt, J = 15.6, 6.8 Hz), 6.81 (2H, d, J = 8.8 Hz), 7.16–
7.29 (11H, m), 7.40–7.42 ppm (6H, m). 13C NMR (100 MHz, CDCl3): δ =
11.9 (3C), 17.7 (6C), 31.3, 31.5, 41.4, 55.2, 66.5, 69.8, 75.7, 84.2, 113.7
(2C), 126.6 (3C), 127.8 (6C), 129.3 (2C), 129.6 (6C), 130.4, 130.5, 132.8,
144.9 (3C), 159.1, 170.0 ppm. IR (neat): ν = 2944, 2866, 1747, 1613,
1514, 1489, 1464, 1444, 1300, 1248, 1171, 1112, 1036, 970, 882, 821,
743, 700 cm−1. HRMS (FAB) calcd for C44H57O5SSi [M+H]+ 725.3696;
found 725.3719.
Et2NH (47 μL, 0.47 mmol) was added to a stirred solution of 33 (31.8 mg,
31 μmol) in CH2Cl2 (1 mL) at room temperature. After 9 h, the reaction
mixture was concentrated in vacuo to afford a residue, which was purified
by column chromatography (hexane/EtOAc 4:1) to give 24 (20.9 mg,
85%) as a colorless amorphous solid. [α]D20 = −16.6 (c = 0.73, CHCl3); 1H
NMR (400 MHz, CDCl3): δ = −0.07 (3H, s), 0.01, (3H, s), 0.81 (9 H, s),
1.04–1.14 (21H, m), 1.19 (3H, d, J = 6.3 Hz), 1.63 (2H, br s), 1.99–2.09
(2H, m), 2.14–2.28 (2H, m), 2.57 (1H, dd, J = 15.6, 5.8 Hz), 2.70 (1H, dd,
J = 16.1, 7.3 Hz), 3.18 (1H, d, J = 2.0 Hz), 4.14–4.19 (1H, m), 5.37–5.43
(3H, m), 5.54 (1H, q, J = 6.7 Hz), 5.65 (1H, dt, J = 15.1, 6.8 Hz), 7.18–
7.40 ppm (15H, m). 13C NMR (100 MHz, CDCl3): δ = −4.9, −4.2, 11.9
(3C), 17.7 (6C), 17.9, 21.0, 25.7 (3C), 31.1, 31.4, 39.7, 60.7, 66.6, 69.5,
71.2, 84.3, 126.6 (3C), 127.8 (6C), 127.9, 129.6 (6C), 133.5, 144.8 (3C),
168.8, 173.4 ppm. IR (neat): ν = 2928, 2866, 1774, 1489, 1464, 1444,
1374, 1252, 1156, 1115, 1037, 954, 882, 837, 808, 775, 743, 700 cm−1
.
HRMS (FAB): calcd for C46H70NO6SSi2 [M+H]+ 820.4462, found 820.4496.
(5R,8R,11S,14S)-(S,E)-13,13-Diisopropyl-14-methyl-9-oxo-1,1,1-
triphenyl-10,12-dioxa-2-thia-13-silapentadec-5-en-7-yl 14-[(R)-1-(tert-
butyldimethylsiloxy)ethyl]-1-(9H-fluoren-9-yl)-5,8-diisopropyl-
3,6,9,12-tetraoxo-11-(tritylthio)methyl-2-oxa-4,7,10,13-
tetraazapentadecan-15-oate (34): iPr2NEt (58 μL, 0.34 mmol) was
added dropwise to a stirred solution of 23 (134 mg, 0.17 mmol) and 24
(70.1 mg, 85 μmol) in CH2Cl2 (1.7 mL) containing HATU (64.6 mg, 0.17
mmol) and HOAt (23.1 mg, 0.17 mmol) at −20 °C under argon. After 2 h,
the reaction mixture was diluted with CHCl3 (30 mL). The organic layer
was washed successively with 10% aqueous HCl (2 × 10 mL), saturated
aqueous NaHCO3 (2 × 10 mL) and brine (2 × 10 mL), then dried over
Na2SO4. Concentration of the solvent in vacuo afforded a residue, which
was purified by column chromatography (hexane/EtOAc 3:1) to give 12
(S,E)-(Triisopropylsiloxy)methyl
3-hydroxy-7-(tritylthio)hept-4-
enoate (26): DDQ (168 mg, 0.74 mmol) was added in small portions to a
stirred solution of 32 (214 mg, 0.30 mmol) in CH2Cl2/H2O 9:1 (10 mL) at
room temperature. After 1 h, the mixture was diluted with CHCl3 (60 mL),
and the organic layer was washed with saturated aqueous NaHCO3 (2 ×
20 mL) and brine (2 × 20 mL), then dried over Na2SO4. Concentration of
the solvent in vacuo afforded a residue, which was purified by column
chromatography (hexane/EtOAc 1:1) to give 26 (168 mg, 94%) as a
colorless viscous liquid. [α]D20 = –4.7 (c = 1.1, CHCl3). 1H NMR (400 MHz,
CDCl3): δ = 1.04–1.17 (21H, m), 2.04–2.10 (2H, m), 2.17–2.22 (2H, m),
2.50–2.52 (2H, m), 2.79 (1H, d, J = 3.4 Hz), 4.43–4.48 (1H, m), 5.41 (1H,
dd, J = 15.9, 6.3 Hz), 5.46 (1H, d, J = 4.3 Hz), 5.48 (1H, d, J = 4.3 Hz),
5.58 (1H, dt, J = 15.5, 6.8 Hz), 7.18–7.41 ppm (15H, m). 13C NMR (100
20
(108 mg, 80%) as a colorless amorphous solid. [α]D = +6.7 (c = 1.2,
CHCl3); 1H NMR (400 MHz, CDCl3): δ = −0.11 (3H, s), −0.05 (3H, s), 0.78
(9H, s), 0.85–0.88 (12H, m), 1.03–1.12 (24H, m), 1.95–2.07 (4H, m),
2.13–2.17 (2H, m), 2.49 (1H, dd, J = 15.9, 7.6 Hz), 2.56 (1H, dd, J = 12.9,
6.1 Hz), 2.63–2.70 (2H, m), 4.00 (1H, t, J = 7.8 Hz), 4.11–4.30 (4H, m),
4.33–4.44 (3H, m), 5.32–5.40 (3H, m), 5.45 (1H, q, J = 7.0 Hz), 5.51 (1H,
This article is protected by copyright. All rights reserved

European Journal of Organic Chemistry
10.1002/ejoc.201601023
FULL PAPER
d, J = 8.8 Hz), 5.61 (1H, dt, J = 15.6, 6.8 Hz), 6.23 (1H, d, J = 7.3 Hz),
6.40 (1H, d, J = 8.3 Hz), 6.49 (1H, d, J = 8.8 Hz), 7.18–7.41 (34H, m),
7.59 (2H, d, J = 7.3 Hz), 7.75 ppm (2H, d, J = 7.3 Hz). 13C NMR (100
MHz, CDCl3): δ = −5.0, −4.4, 11.9 (3C), 17.7 (6C), 17.82, 17.83, 18.0,
19.1, 19.3, 20.8, 25.7 (3C), 31.0, 31.3, 31.38, 31.44, 33.8, 39.6, 47.2,
52.0, 57.9, 58.0, 60.5, 66.6, 67.0, 67.2, 68.5, 71.9, 84.3, 119.92, 119.94,
125.07, 125.10, 126.6 (3C), 126.9 (3C), 127.1 (2C), 127.5, 127.7 (2C),
127.8 (6C), 128.0 (6C), 129.5 (12C), 133.9, 141.3 (2C), 143.8, 143.9,
144.3 (3C), 144.8 (3C), 156.3, 168.5, 168.6, 169.7, 170.5, 171.0 ppm. IR
(neat): ν = 3287, 3058, 2958, 2867, 1748, 1715, 1636, 1523, 1446, 1396,
1238, 1108, 1033, 956, 883, 836, 741, 700 cm−1. HRMS (FAB): calcd for
C93H117N4O11S2Si2 [M+H]+ 1585.7699; found 1585.7711.
(1H, d, J = 6.3 Hz). 13C NMR (100 MHz, CDCl3): δ = 18.5, 19.1, 19.3,
19.7, 20.3, 27.3, 29.7, 30.8, 30.9, 31.3, 33.5, 41.1, 52.9, 57.2, 59.0, 66.6,
67.1, 68.1, 72.3, 126.6 (3C), 126.9 (3C), 127.5, 127.9 (6C), 128.0 (6C),
129.6 (6C), 129.8 (6C), 133.2, 144.3 (3C), 144.8 (3C), 169.27, 169.32,
169.7, 173.5, 173.7 ppm. IR (neat): ν = 3298, 3057, 2964, 2928, 1734,
1653, 1525, 1490, 1444, 1389, 1274, 1185, 1082, 1034, 976, 743, 700,
621 cm−1. HRMS (FAB): calcd for C62H69N4O7S2 [M+H]+ 1045.4608
found; 1045.4600.
FR901375 (1): A solution of 36 (26.4 mg, 25 μmol) in CH2Cl2/MeOH 9:1
(4 mL) was added dropwise to a vigorously stirred solution of I2 (63.5 mg,
0.25 mmol) in CH2Cl2/MeOH 9:1 (157 mL, 0.5 mM concentration) over 10
min at room temperature. After 10 min, the reaction was quenched with
0.2 M ascorbic acid/citric acid buffer (3 mL, adjusted to pH 4.0) at room
temperature. The resulting mixture was diluted with CH2Cl2 (100 mL) and
water (50 mL). The organic layer was washed with brine (2 × 30 mL) and
dried over MgSO4. Concentration of the solvent in vacuo afforded a
residue, which was purified by column chromatography (CHCl3/MeOH,
60:1) to give 1 (12.5 mg, 89%) as a white solid. Recrystallization from
acetone afforded colorless prism, M.p. 221–227 ºC (decomp) {lit.[1] M.p.
(S,E)-(Triisopropylsiloxy)methyl
amino-3-methylbutanamido]}-3-methylbutanamido}-3-
3-{[(2S,3R)-2-{(S)-2-{(R)-2-[(R)-2-
(tritylthio)propanamido-3-(tert-butyldimethylsiloxy)butanoyloxy-7-
(tritylthio)hept-4-enoate (35): Piepridine (48 μL, 0.49 mmol) was added
to a stirred solution of 34 (19.3 mg, 12 μmol) in CH2Cl2 (1 mL) at room
temperature. After 1 h, the reaction was quenched with saturated
aqueous NH4Cl (2 mL), and the resulting mixture was extracted with
CHCl3 (3 × 4 mL). The combined extracts were washed with brine (2 × 4
mL), then dried with MgSO4. Concentration of the solvent in vacuo
afforded a residue, which was purified by column chromatography
(CHCl3/MeOH 40:1) to give 35 (11.9 mg, 72%) as a colorless amorphous
20
20
222–226 ºC (decomp)}. [α]D = −27.3 (c = 0.90, CHCl3), {lit.[1] [α]D
=
25
−25.3 (c = 1.0, CHCl3); lit.[5] [α]D = −24.4 (c = 0.7, CHCl3)}. 1H NMR
(600 MHz, CD3OD): δ = 0.98 (3H, d, J = 6.6 Hz), 0.99 (3H, d, J = 6.6 Hz),
1.01 (3H, d, J = 6.6 Hz), 1.04 (3H, d, J = 6.6 Hz), 1.17 (3H, d, J = 6.6 Hz),
2.08–2.14 (1H, m), 2.41–2.46 (1H, m), 2.58–2.62 (2H, m), 2.64 (1H, dd, J
= 13.9, 7.7 Hz), 2.85 (1H, dd, J = 13.7, 6.0 Hz), 2.89 (1H, ddd, J = 14.2,
6.2, 2.6 Hz), 3.01 (1H, ddd, J = 14.3, 9.9, 2.2 Hz), 3.38, (2H, d, J = 5.1
Hz), 3.59 (1H, d, J = 9.2 Hz), 4.04 (1H, d, J = 8.8 Hz), 4.32 (1H, dq, J =
12.9, 3.1 Hz), 4.44 (1H, d, J = 2.6 Hz), 4.69 (1H, t, J = 5.3 Hz), 5.59–5.61
(1H, m), 5.67 ( 1H, br d, J = 16.1 Hz), 5.82–5.87 ppm (1H, m). 13C NMR
(100 MHz, CD3OD): δ = 19.1, 19.87, 19.93, 20.2, 20.3, 29.5, 31.6, 34.7,
38.5, 40.6, 41.8, 55.1, 60.1, 62.1, 66.3, 68.5, 71.4, 127.3, 133.8, 170.4,
171.9, 172.4, 174.5, 175.3 ppm. IR (neat): ν = 3385, 2966, 2932, 1740,
20
solid. [α]D = +15.1 (c = 1.1, CHCl3). 1H NMR (400 MHz, CDCl3): δ =
−0.10 (3H, s), −0.03 (3H, s), 0.80–0.82 (12H, m), 0.88 (3H, d, J = 6.8 Hz),
0.92 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.8 Hz), 1.00–1.15 (24H, m),
1.44 (2H, br s), 1.96–2.06 (2H, m), 2.08–2.17 (3H, m), 2.23–2.31 (1H, m),
2.52 (1H, dd, J = 15.6, 7.3 Hz), 2.60–2.65 (2H, m), 2.71 (1H, dd J = 15.6,
5.9 Hz), 3.16 (1H, d, J = 3.9 Hz), 4.10 (1H, dd, J = 12.9, 7.1 Hz), 4.18
(1H, dd, J = 9.0, 6.6 Hz), 4.28 (1H, ddd, J = 12.6, 6.2, 1.8 Hz), 4.35 (1H,
dd, J = 9.0, 1.7 Hz), 5.33–5.49 (3H, m), 5.46 (1H, q, J = 7.0 Hz), 5.62 (1H,
dt, J = 15.6, 6.3 Hz), 6.34 (1H, d, J = 7.3 Hz), 6.59 (1H, d, J = 8.8 Hz),
7.18–7.41 (30H, m), 7.83 ppm (1H, d, J = 8.8 Hz). 13C NMR (100 MHz,
CDCl3): δ = −5.0, −4.4, 11.9 (3C), 16.1, 17.7 (6C), 17.8, 17.9, 19.69,
19.72, 20.8, 25.7 (3C), 30.0, 30.6, 31.0, 31.5, 33.8, 39.6, 52.1, 57.7, 57.9,
60.3, 66.6, 67.1, 68.4, 71.9, 84.3, 126.6 (3C), 126.9 (3C), 127.6, 127.8
(6C), 128.1 (6C), 129.5 (6C), 129.6 (6C), 133.8, 144.3 (3C), 144.8 (3C),
168.5, 168.7, 167.0, 171.4, 174.8 ppm. IR (neat): ν = 3275, 2957, 2311,
1748, 1684, 1636, 1541, 1508, 1472, 1387, 1121, 954, 837, 742, 699
cm–1. HRMS (FAB): m/z calcd for C78H107N4O9S2Si2 [M+H]+ 1363.7018
found, 1363.6985.
1671, 1523, 1419, 1264, 1112, 1072, 997, 971, 756, 663, 619 cm−1
.
HRMS (FAB): calcd for C24H39N4O7S2 [M+H]+ 559.2260 found, 559.2256.
The 1H and 13C NMR, IR, and MS spectra were identical to those
reported for natural[1] and a previously synthetic[5] FR901375.
Biological evaluation
HDACs preparation and enzyme inhibition assay:[19] In a 100-mm
dish, 293T cells (1–2 x 107) were grown for 24 h and transiently
transfected with 10 μg each of the vector pcDNA3-HDAC1 for human
HDAC1 or pcDNA3-mHDA2/HDAC6 for mouse HDAC6, using the
LipofectAMINE2000 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 4°C with 4 μg 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 4°C with 40 μg of the FLAG peptide (Sigma-Aldrich
Inc.) in histone deacetylase buffer (200 μL). The supernatant was
collected by centrifugation. For the enzyme assay, 10 μL of the enzyme
fraction was added to 1 μL of fluorescent substrate (2 mM Ac-
KGLGK(Ac)-MCA) and 9 μL of histone deacetylase buffer, and the
mixture was incubated at 37°C for 30 min. The reaction was stopped by
the addition of 30 μL of trypsin (20 mg/mL) and incubated at 37°C for 15
min. The released aminomethylcoumarin (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. The HDAC inhibitory activity of
FR901375 (1) and FK228 (2) was measured in the presence of 0.1 mM
DTT.
(3S,6S,9R,12R,16S)-3-[(R)-1-Hydroxyethyl]-9,12-diisopropyl-16-[(E)-
4-(tritylthio)but-1-en-1-yl]-6-(tritylthio)methyl-1-oxa-4,7,10,13-
tetraazacyclohexadecane-2,5,8,11,14-pentaone (36): HF·pyridine (0.5
mL) was added to a stirred solution of 35 (81.7 mg, 60 μmol) in pyridine
(1.0 mL) at room temperature. After 16 h, the reaction mixture was
azeotropic dried with benzene in vacuo. The resulting residue containing
seco-amino acid 22 (63.7 mg) was used for the next reaction without
further purification.
A solution of the crude product 22 (63.7 mg) in THF (18 mL) was added
very slowly to a stirred solution of HATU (68.4 mg, 0.18 mmol) in THF
(180 mL, 0.4 mM concentration) containing iPr2NEt (47 μL, 0.28 mmol) at
room temperature over 4 h. After 18 h, the mixture was diluted with
EtOAc (200 mL), and the organic layer was washed successively with
saturated aqueous NH4Cl (2 × 50 mL), water (2 × 50 mL) and brine (2 ×
50 mL), then dried over Na2SO4. Concentration of the solvent in vacuo
afforded a residue, which was purified by column chromatography
(CHCl3/MeOH, 60:1) to give 36 (40.0 mg, 52%, 2 steps) as a colorless
amorphous solid. [α]D20 = −37.3 (c = 0.86, CHCl3); 1H NMR (400 MHz,
CDCl3): δ = 0.88 (3H, d, J = 6.8 Hz), 0.95–0.97 (6H, m), 1.05 (3H, d, J =
6.8 Hz), 1.08 (3H, d, J = 6.8 Hz), 1.91–2.02 (3H, m), 2.09–2.18 (3H, m),
2.33 (1H, dd, J = 14.7, 2.7 Hz), 2.56 (1H, dd, J = 12.1, 4.3 Hz), 2.88–2.96
(2H, m), 3.28 (1H, dd, J = 10.4, 6.5 Hz), 4.12–4.19 (2H, m), 4.31–4.35
(1H, m), 4.44–4.49 (2H, m), 5.22 (1H, dd, J = 15.5, 6.8 Hz), 5.43–5.48
(1H, m), 5.58 (1H, dt, J = 15.5, 6.8 Hz), 5.92 (1H, d, J = 8.7 Hz), 6.39 (1H,
d, J = 9.7 Hz), 7.16–7.43 (30H, m), 7.80 (1H, d, J = 5.8 Hz), 8.55 ppm
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European Journal of Organic Chemistry
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Cell-growth inhibition assay:[20] This experiment was carried out at the
Cancer Chemotherapy Centre, Japanese Foundation for Cancer
Research. 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-631L 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 determined by using
the sulforhodamine B colorimetric method.
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Acknowledgements
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[10] The spectroscopic properties (IR, 1H and 13C NMR, HRMS) of our
synthesized seco-acid 9 are identical to those reported by Wentworth-
Janda et al.[5]
We are grateful to the Screening Committee of New Anticancer
Agents, which is supported by a Grant-in-Aid for Scientific
Research on Innovative Area ‘Scientific Support Programs for
Cancer Research (No. 221S001, 2010–2014)’ from the Ministry
of Education, Culture, Sports, Science and Technology, Japan
(MEXT), for assistance with the biological evaluation of
compounds 1 and 2. This study was also supported by a Grant-
in-Aid for the Strategic Research Foundation Program at Private
Universities (No. S1511001L, 2015–2019), a Grant-in-Aid for
Scientific Research (C) (No. 15K07865, 2015–2017), and a
Grant-in-Aid for Scientific Research (S) (No. 26221204, 2014–
2018) from MEXT.
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[12] In some cases, macrolactamization is preferable to macrolactonization
in the synthesis of macrocyclic depsipeptides. In this case, it can avoid
the difficulty in realizing the macrolactonization, due to the presence of
the sterically hindered O-TBS-protected 1-hydroxyethyl substituent
adjacent to the carbonyl reaction site in seco-acid 9. Recent examples
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Keywords: FR901375 • Histone deacetylase inhibitor • Natural
product • Bicyclic depsipeptide • Total synthesis
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Entry for the Table of Contents (Please choose one layout)
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Powerful and highly selective
HDAC inhibitor: FR901375, a bicyclic
depsipeptide natural product, was
efficiently synthesized in a highly
convergent manner. The synthesis
involved amide condensation of the
two segments (in blue and red)
followed by macrolactamization and
disulfide bond formation. Biological
evaluation revealed that FR901375
exhibits extremely high selectivity for a
class I HDAC1 (IC50 = 1.7 nM) over
class II HDAC6 (IC50 = 1627 nM).
Dr. Koichi Narita, Yuya Katoh, Ken-ichi
Ojima, Dr. Singo Dan, Dr. Takao Yamori.
Dr. Akihiro Ito, Dr. Minoru Yoshida and
Prof. Tadashi Katoh*
Page No. – Page No.
Total Synthesis of the Depsipeptide
FR901375 and Preliminary Evaluation
of its Biological Activity
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