Total synthesis of bicyclic depsipeptides spiruchostatins C and D and investigation of their histone deacetylase inhibitory and antiproliferative activities

Article abstract of DOI:10.1016/j.ejmech.2012.12.023

The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors spiruchostatins C and D were synthesized for the first time in a highly convergent and unified manner. The method features the amide coupling of a d-leucine-d-cysteine- or d-valine-d-cysteine-containing segment with a d-alanine- or d-valine-containing segment to directly assemble the corresponding seco-acids, key precursors of macrolactonization. The HDAC inhibitory assay and cell-growth inhibition analysis of the synthesized depsipeptides determined the order of potency of spiruchostatins A-D in comparison with the clinically approved depsipeptide FK228 (romidepsin). Novel aspects of structure-activity relationships (SAR) were revealed.

Full text of DOI:10.1016/j.ejmech.2012.12.023

European Journal of Medicinal Chemistry 60 (2013) 295e304
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Total synthesis of bicyclic depsipeptides spiruchostatins C and D and
investigation of their histone deacetylase inhibitory and
antiproliferative activities
,
Koichi Narita a, Yurie Fukui a, Yui Sano a, Takao Yamori b, Akihiro Ito c, Minoru Yoshida c, Tadashi Katoh a
*
a Laboratory of Synthetic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
b Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan
c Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors spiruchostatins C and D were synthe-
Received 27 October 2012
Received in revised form
8 December 2012
Accepted 11 December 2012
Available online 21 December 2012
sized for the first time in a highly convergent and unified manner. The method features the amide
coupling of a D-leucineeD-cysteine- or D-valineeD-cysteine-containing segment with a D-alanine- or D-
valine-containing segment to directly assemble the corresponding seco-acids, key precursors of mac-
rolactonization. The HDAC inhibitory assay and cell-growth inhibition analysis of the synthesized dep-
sipeptides determined the order of potency of spiruchostatins AeD in comparison with the clinically
approved depsipeptide FK228 (romidepsin). Novel aspects of structureeactivity relationships (SAR) were
revealed.
Keywords:
Spiruchostatins
Histone deacetylase inhibitors
Natural products
Bicyclic depsipeptide
Total synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
potent and selective inhibition of class I enzymes is highly desir-
able in cancer chemotherapy.
Histone deacetylase (HDAC) inhibitors have attracted signifi-
cant attention because of their potentials as novel mechanism-
based anticancer agents [1]. It has been reported that HDAC
inhibitors exhibit prominent antitumour activity in several human
tumour xenograft models by inducing transcriptional events
involved in growth arrest, differentiation and apoptotic cell death
[1,2]. HDAC enzymes remove an acetyl group from the lysine
residues of the core histones, leading to a changed chromatin
structure that impacts transcriptional activity [3]. There are 18
human HDAC isoforms, which are grouped into four major classes:
class I (HDACs 1, 2, 3 and 8), class II (class IIa: HDACs 4, 5, 7 and 10;
In 2009, the Food and Drug Administration (FDA) in the USA
approved the bicyclic depsipeptide HDAC inhibitor FK228 (romi-
depsin, 1, Fig. 1) for the treatment of cutaneous T cell lymphoma
(CTCL) [6]. However, this anticancer agent is associated with an
unresolved cardiotoxicity issue [6b,7], which highlights the need
for further identification and development of new HDAC inhibitors
with high efficacy and low toxicity [1n,4a]. FK228 was originally
isolated from a culture broth of Chromobacterium violaceum (No.
968) by Fujisawa Pharmaceutical Co. Ltd. (now Astellas Pharma
Inc.) in 1994 [8]. The structural features of this natural product
include a 16-membered bicyclic depsipeptide consisting of (Z)-2-
class IIb: HDACs 6 and 10), and class IV (HDAC 11) are Zn2þ
-
amino-2-butenoic acid, L-valine, D-cysteine, D-valine, (3R,4E)-3-
dependent metallohydrolases, while class III HDACs (7 members)
are NADþ-dependent sirtuins [4]. The inhibition of class I HDACs is
considered to be a useful mechanism for anticancer agents,
whereas the inhibition of class II HDACs may cause undesirable
side effects such as serious cardiac hypertrophy [5]. Therefore, the
hydroxy-7-mercapto-4-heptenoic acid and a characteristic disul-
fide bond linkage [8b]. Subsequent to the discovery of FK228, the
structurally similar 15-membered bicyclic depsipeptide HDAC
inhibitors d spiruchostatins A (2) [9], B (3) [9], C (4) [10,11] and D
(5) [12] d were successively isolated from a culture broth of the
Pseudomonas sp. by Shin-ya and Yamanouchi Pharmaceutical Co.
Ltd. (now Astellas Pharma Inc.).
The attractive biological properties and unique structural
features of 1e5 have made them intriguing and timely targets for
* Corresponding author. Tel./fax: þ81 22 727 0135.
E-mail address: katoh@tohoku-pharm.ac.jp (T. Katoh).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.023

296
K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
Fig. 1. Structures of FK228 (romidepsin) (1) and spiruchostatins A (1), B (2), C (3) and D
(4). i-Pr ¼ isopropyl, s-Bu ¼ sec-butyl, i-Bu ¼ isobutyl.
total synthesis. Several total syntheses of FK228 (1) [13], spir-
uchostatins A (2) [13a,14] and B (3) [13a,15] have been published.
However, to the best of our knowledge, the total synthesis of spir-
uchostatins C (4) and D (5) has not been reported, and their bio-
logical properties have not been described in the patent literatures
[10,12]. We have previously presented the total synthesis of 1e3
using a highly convergent and unified scheme [13a,14c,15b], and
demonstrated that 1e3 exhibit a potent class I HDAC inhibitory
activity (IC50 ¼ 2.2e3.6 nM) [13a]. During our efforts on the
synthesis and biological evaluation of the bicyclic depsipeptide
HDAC inhibitors, we became interested in the biological potencies
of 4 and 5 as compared with those of 1e3. In this paper, we describe
the total synthesis of 4 and 5 for the first time applying the
synthetic strategy developed in our laboratory [13a]. HDAC inhi-
bition assay and antiproliferative analysis of the synthesized 4 and
5 were also performed to establish the potency order of the bicyclic
depsipeptides family (1e5).
Scheme 1. Synthetic plan for spiruchostatins C (4) and D (5). TBS ¼ tert-butyldime-
thylsilyl, Tr (trityl)
methoxybenzyl.
¼
triphenylmethyl, Boc
¼
tert-butoxycarbonyl, PMB
¼
4-
2. Results and discussion
2.1. Chemistry
2.1.1. Synthetic plan for spiruchostatins C (4) and D (5)
Our synthetic plan for spiruchostatins C (4) and D (5) is outlined
in Scheme 1. We envisioned that the targeted molecules 4 and 5
could be synthesized by the macrolactonization of the corre-
sponding seco-acids 6 and 7, followed by disulfide bond formation.
The key element of this scheme is the highly convergent assembly
of 6 and 7 by the direct coupling of a
containing segment 8 and a -alanine-containing segment 10 and
-valinee -cysteine-containing segment 9 and a -valine-con-
taining segment 11, respectively. The segment 8 was to be prepared
by the aldol coupling of N-Boc- -leucinal (12) with ethyl acetate
(13) and subsequent condensation with the -cysteine derivative
D-leucineeD-cysteine-
D
a
D
D
D
D
D
14. The remaining three segments 9, 10 and 11 were obtained by
our previously described total synthesis of 1e3 [13a].
2.1.2. Synthesis of segment 8
The synthesis of 8, starting from the known N-Boc-D-leucinal
(12) [16], is shown in Scheme 2; the route is based on our previous
study [13a]. Thus, the aldol coupling of 12 with the lithium enolate
of ethyl acetate (13) gave the desired coupling product 16 (33%)
and the undesired stereoisomer 15 (54%). The conversion of 15 to
16 was achieved by Jones oxidation (80%) and the stereoselective
reduction of the resulting ketone 17 (90%). The ethyl ester 16 was
then transformed to the allyl ester 21 via a four-step operation
involving the tert-butyldimethylsilyl (TBS) protection of the
hydroxy group in 16 (99%), the saponification of the ester moiety
in the resulting TBS ether 18, the formation of an allyl ester from
Scheme 2. Synthesis of segment 8. (a) LDA, CH3CO2Et (13), THF, ꢀ78 ꢁC; at ꢀ78 ꢁC,
add. 12, 54% for 15, 33% for 16 (15/16 ca. 3:2); (b) Jones reagent, acetone, 0 ꢁC to rt, 80%;
(c) KBH4, MeOH, ꢀ40 ꢁC, 90%; (d) TBSCl, imidazole, DMF, rt, 99%; (e) 1 M NaOH, EtOH,
rt; (f) allyl bromide, K2CO3, DMF, rt. 77% (2 steps); (g) TMSOTf, 2,6-lutidine, CH2Cl2, rt;
MeOH, rt; (h) 14, PyBOP, i-Pr2NEt, MeCN, rt, 69% (2 steps); (i) TMSOTf, 2,6-lutidine,
CH2Cl2, rt, 88%. LDA
¼
lithium diisopropylamide, TMSOTf
¼
trimethylsilyl tri-
fluoromethanesulfonate, PyBOP ¼ (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate.

K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
297
the liberated carboxylic acid 19 (77% in two steps) and the
deprotection of the N-Boc group in 20. The condensation of the
(IR, 1H and 13C NMR, and MS) of the synthetic sample 4 were
identical with those reported for natural 4 [10].
amine 21 with N-Boc-S-trityl- -cysteine (14) furnished the desired
L
coupling product 22 with a 69% yield from 20. Finally, the
deprotection of the N-Boc group in 22 afforded the requisite
segment 8 in 88% yield.
2.1.4. Synthesis of spiruchostatin D (5)
As shown in Scheme 4, using segments 9 [13a] and 11 [13a] as the
starting materials, the second target spiruchostatin D (5) was
synthesized in a manner similar to that described for 4 (cf. Scheme
3). The spectroscopic properties (IR, 1H and 13C NMR, and MS) of the
2.1.3. Synthesis of spiruchostatin C (4)
After obtaining the key segment 8, we performed the synthesis
of the first target spiruchostatin C (4), as shown in Scheme 3. The
condensation of 8 with segment 10 proceeded well under the mild
conditions [HATU (1.3 equiv), HOAt (1.3 equiv), i-Pr2NEt (2.6
equiv), ꢀ30 ꢁC, 6 h] explored in our previous studies [13a]. The
desired coupling product 23 was obtained in 84% yield without
synthesized sample 5 were identical with those reported for natural
25
5 [12]. The optical rotation of synthesized 5 {[
in MeOH)} corresponded to that of natural 5 {[
(c ¼ 0.20 in MeOH)} [12].
a
]
¼ ꢀ59.2ꢁ (c ¼ 0.29
D
25
a]
¼ ꢀ65.3ꢁ
D
2.2. Biological evaluation
appreciable epimerization at the C2 stereogenic centre (D-alanine
part in 23). The coupling product 23 was converted to the requisite
seco-acid 6 with an overall yield of 98% using the alcohol 24 by the
successive removal of both the PMB and allyl protecting groups. The
subsequent macrolactonization of 6 was achieved by employing the
Shiina method [17] [MNBA (1.3 equiv), DMAP (3.0 equiv), CH2Cl2
(10 mM), rt, 12 h], which resulted in the desired cyclization product
25 in 75% yield. Finally, simultaneous S-Tr deprotection and disul-
fide bond formation of 25 (95%) followed by the deprotection of the
2.2.1. HDAC inhibition assay
The synthesized spiruchostatins C (4) and D (5) were tested for
their HDAC inhibitory activity against HDAC1 (class I) and HDAC6
(class IIb) to determine the degree of potency and isoform selec-
tivity. In this assay, FK228 was used as a positive control. As
summarized in Table 1, the two compounds 4 and 5 that were
tested exhibited extremely potent inhibitory activity against
HDAC1 in the sub-nanomolar range (IC50 ¼ 0.75e0.93 nM). The
potencies of 4 and 5 were observed to be 3-to-4-fold higher than
that of FK228. Combined with our previous data for spiruchostatins
A (2) and B (3) [13a], the order of potency was estimated to be 5
(IC50 ¼ 0.75 nM) ꢂ 4 (IC50 ¼ 0.93 nM) > 3 (IC50 ¼ 2.2 nM) ꢂ 2
(IC50 ¼ 3.3 nM) zFK228 (1) (IC50 ¼ 3.6 nM). Spiruchostatin D (5)
TBS group of the resulting disulfide 26 (91%) furnished spir-
25
uchostatin C (4), [
a]
¼ ꢀ59.4ꢁ (c ¼ 0.20 in CHCl3) {lit. [10]
D
25
[
a]
¼ ꢀ60.0ꢁ (c ¼ 0.10 in CHCl3)}. The spectroscopic properties
D
Scheme 3. SynthesisofspiruchostatinC(4). (a)HATU, HOAt,i-Pr2NEt,CH2Cl2,ꢀ30ꢁC,84%;
(b) DDQ, CH2Cl2/H2O, rt, 99%; (c) Pd(PPh3)4, morpholine, THF, rt, 99%; (d) MNBA, DMAP,
CH2Cl2, rt, 75%; (e) I2, MeOH/CH2Cl2, rt, 95%;(f) HF$pyridine, pyridine, rt, 91%. HATU¼ O-(7-
azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate, HOAt
¼
1-
2,3-dichloro-5,6-dicyano-1,4-benzoquinone,
MNBA ¼ 2-methyl-6-nitrobenzoic anhydride, DMAP ¼ 4-dimethylaminopyridine.
Scheme 4. Synthesis of spiruchostatin D (5). (a) HATU, HOAt, i-Pr2NEt, CH2Cl2, ꢀ30 ꢁC,
94%; (b) DDQ, CH2Cl2/H2O, rt, 79%; (c) Pd(PPh3)4, morpholine, THF, rt, 90%; (d) MNBA,
DMAP, CH2Cl2, rt, 85%; (e) I2, MeOH/CH2Cl2, rt, 88%; (f) HF$pyridine, pyridine, rt, 93%.
hydroxy-7-azabenzotriazole, DDQ
¼

298
K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
Table 1
Table 2
HDAC inhibitory activity of spiruchostatins C (4) and D (5).
Growth inhibition of spiruchostatins C (4) and D (5) against a panel of 39 human
cancer cell lines.
Compound
IC50 (nM)
SId
a
Origin of cancer
Breast
Cell line
GI50a (nM)
HDAC1 (class I)b
HDAC6 (class IIb)b
4
5
FK228b
4
5
0.93
0.75
3.6
320
240
390
346
320
108
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
20
25
150
47
14
52
4.1
5.2
18
6.9
8.5
13
FK228c
a
Concentration that induces 50% inhibition against HDACs.
Enzyme assay was performed in the presence of 100 mM dithiothreitol (DTT).
Positive control used in this study was synthesized in our laboratory.
Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity toward class I
2.9
1.7
2.7
3.5
5.1
0.58
1.9
41
4.2
5.5
3.9
4.9
4.0
3.6
7.2
9.6
3.1
3.4
3.3
450d
3.1
4.6
8.9
1.8c
3.0
2.6
5.8
3.6
2.5
4.6
20
b
c
Central nervous system (brain)
d
25
HDAC1 over class IIb HDAC6.
32
10c
10c
78
exhibited the most potent HDAC1 inhibitory activity among the
spiruchostatins family (2e5), suggesting that sterically bulky
terminal-branched alkyl groups are preferable for the R1 and R2
side chains in the spiruchostatins (cf. Fig. 1). As expected for HDAC6
inhibitory activity, both 4 and 5 were essentially inactive (4:
IC50 ¼ 320 nM, 5: IC50 ¼ 240 nM), which is similar to the behavior
exhibited by FK228 (IC50 ¼ 390 nM). Isoform selectivity (class I/IIb)
is expressed, for convenience, as a selective index (SI) value (HDAC6
IC50/HDAC1 IC50). The isoform selectivity of 4 (SI ¼ 346) and 5
(SI ¼ 320) is approximately 3-fold higher than that of FK228
(SI ¼ 108). These results suggested that the minor structural
alteration of the R1 and R2 side chains in spiruchostatins improves
HDAC inhibitory activity and isoform selectivity. This is in agree-
ment with a previous SAR study on synthetic FK228 analogues
reported by Ganesan et al. [18].
Colon
Lung
11
1.5
2.6
2.2
210d
1.3
2.5
4.0
0.39c
2.8
0.97
5.1
3.6
1.8
2.2
4.7
1.2
2.7
2.3
5.9
21
10c
10c
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-631L
ACHN
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU-145
PC-3
3800d
10c
10c
53
10c
16
10c
32
10c
10c
16
Melanoma
Ovary
58
10c
10c
15
2.8
5.5
3.3
6.6
20
Kidney
35
2.2.2. Cell-growth inhibition assay
130
190
16
The growth-inhibitory activity of compounds 4 and 5 was
evaluated simultaneously with FK228, a reference compound,
using a panel of 39 human cancer cell lines at the Japanese Foun-
dation for Cancer Research [19]. The number of cell lines and their
origin (organ) are as follows: 5 breast, 6 central nervous system
(brain), 1 melanoma, 5 ovary, 2 kidney, 6 stomach and 2 prostate.
Doseeresponse curves were measured at five different concentra-
tions (10ꢀ10e10ꢀ6 or 10ꢀ8e10ꢀ4 M) for each compound, and the
Stomach
16
22
2.0
5.8
9.5
9.6
2.9
4.0
8.7
3.8
3.2
4.9
17
150
130
100
15
14
3.0
6.0
18
Prostate
10c
23
28
MG-MIDe
6.2
a
concentration causing 50% cell-growth inhibition (GI50
) was
Concentration that induces 50% inhibition of cell growth compared to the
control.
compared with that of the control. The GI50 values of the tested
compounds 4 and 5 along with FK228 are shown in Table 2.
Compound 5 exhibited extremely potent growth-inhibitory activity
against almost all 39 cell lines in the sub-nanomolar to nanomolar
range and its efficacy was superior to that of FK228. In contrast,
compound 4 exhibited decreased activity compared to FK228. The
order of potency, combined with our previous data for 2 and 3
[13a], was estimated by the MG-MID value (mean value of GI50 over
all cell lines tested) to be 5 (3.8 nM) ꢂ 3 (5.6 nM) zFK228 (1)
(6.2 nM) > 2 (15 nM) > 4 (28 nM).
Considering the HDAC inhibitory and antiproliferative activities,
spiruchostatin D (5) seems to be a promising candidate for the
development of novel anticancer agents with high efficacy and low
toxicity. Interestingly, the order of potency in antiproliferative
activity is not necessarily in agreement with that in HDAC inhibi-
tory activity. This is probably influenced by bioavailability and/or
cell permeability. Another possible explanation for this discrepancy
is that this class of HDAC inhibitors has an additional mechanism of
action, albeit the molecular target is unknown at present. This
hypothetical issue is now under investigation in our laboratories.
b
c
Positive control used in this study was synthesized in our laboratory.
The most sensitive cell.
The least sensitive cell.
d
e
Mean value of GI50 over all cell lines tested.
present synthesis demonstrated that our explored synthetic
strategy has the potential for producing spiruchostatin analogues
that possess various alkyl side chains. The preliminary biological
evaluation of the synthesized 4 and 5 together with our previous
data for 2 and 3 determined the order of the efficacy of the spir-
uchostatins family (2e5). These results would be useful for the
design and development of anticancer agents with therapeutic
potential that target the isoform-selective inhibition of HDACs.
Further investigations concerning the synthesis of unnatural spir-
uchostatin analogues and SAR study are currently underway and
will be reported in due course.
4. Experimental
4.1. Chemistry
3. Conclusion
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
We have achieved the first total synthesis of spiruchostatins C
(4) and D (5) in a highly convergent and unified manner. The

K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
299
plates. Flash column chromatography was performed on Kanto
Chemical Silica Gel 60N (spherical, neutral 40e50 nm) with the
solvents indicated.
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, i-Pr2NH, and i-Pr2NEt were distilled from
calcium hydride under argon.
in vacuo afforded a residue, which was purified by column chro-
matography (hexane/EtOAc 8:1 / 4:1) to give 17 (0.98 g, 86%) as
25
a colorless oil. [
a]
D
þ18.4 (c 1.03, CHCl3); IR (neat): 3358, 2961,
2935, 2872, 1748, 1714, 1513, 1391, 1367, 1317, 1252, 1167, 1044,
1030 cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d
0.95 (3H, d, J ¼ 3.1 Hz),
0.96 (3H, d, J ¼ 3.4 Hz), 1.28 (3H, t, J ¼ 7.3 Hz), 1.36e1.42 (1H, m),
1.45 (9H, s), 1.56e1.77 (1H, m), 3.55 (2H, dd, J ¼ 15.6, 15.6 Hz), 4.20
(2H, dd, J ¼ 7.3, 7.3 Hz), 4.34e4.39 (1H, m), 4.93 (1H, d, J ¼ 7.3 Hz);
Measurements of optical rotations were performed with
a JASCO DIP-370 automatic digital polarimeter. 1H and 13C NMR
spectra were measured with a JEOL AL-400 (400 MHz) spectrom-
13C NMR (100 MHz, CDCl3):
d 14.1, 21.6, 23.2, 24.8, 28.3 (3C), 39.9,
46.3, 58.2, 61.4, 80.1, 155.5, 167.0, 172.7; HRMS (EI): m/z calcd for
C15H27NO5 (Mþ) 301.1889, found 301.1883.
eter. Chemical shifts were expressed in ppm using Me4Si (
d
¼ 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-DX 303/JMA-DA 5000
SYSTEM high-resolution mass spectrometer.
4.1.3. Conversion of compound 17 to compound 16 by
stereoselective reduction
KBH4 (1.30 g, 4.3 mmol) was added in small portions to a stirred
solution of 17 (1.30 g, 4.3 mmol) in MeOH (40 mL) at ꢀ40 ꢁC. After
5 h, the reaction was quenched with 10% aqueous citric acid at 0 ꢁC
(adjusted to pH 3). After concentration of the reaction mixture in
vacuo, water (15 mL) was added, and the resulting mixture was
extracted with CH2Cl2 (4 ꢃ 15 mL). The combined extracts were
washed with brine (2 ꢃ 15 mL), then dried over Na2SO4. Concen-
tration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 5:1 / 4:1) to
give 16 (1.04 g, 90%). The IR, 1H and 13C NMR, mass spectra of this
sample were identical with those recorded for compound 16.
4.1.1. (3R,4R)-ethyl 4-(tert-butoxycarbonylamino)-3-hydroxy-6-
methylheptanoate (15) and its (3S,4R)-isomer (16)
A solution of EtOAc (3.05 mL, 31 mmol) in THF (3 mL) was added
slowly to a stirred solution of lithium diisopropylamide (LDA)
(31 mmol) [prepared from n-BuLi in hexane (1.6 M solution,
20.6 mL, 33 mmol) and i-Pr2NH (4.4 mL, 31 mmol)] in THF (15 mL)
at ꢀ78 ꢁC. After 30 min, (2R)-2-(tert-butoxycarbonylamino)-4-
methylpentylaldehyde (N-Boc-D-leucinal) (12) [16] (1.98 g,
4.1.4. (3S,4R)-ethyl 4-(tert-butoxycarbonylamino)-3-(tert-
9.2 mmol) in THF (9 mL) was added to the above mixture at ꢀ78 ꢁC.
After 40 min, the reaction was quenched with 2 M HCl (10 mL)
at ꢀ78 ꢁC, and the resulting mixture was extracted with EtOAc
(2 ꢃ 50 mL). The combined extracts were washed with saturated
aqueous NaHCO3 (2 ꢃ 30 mL) and brine (2 ꢃ 30 mL), then dried
over Na2SO4. Concentration of the solvent in vacuo afforded
a residue, which was purified by column chromatography (hexane/
EtOAc 5:1 / 4:1) to give 15 (1.51 g, 54%, less polar) and 16 (0.93 g,
butyldimethylsiloxy)-6-methylheptanoate (18)
tert-butyldimethylsilyl chloride (TBSCl) (1.15 g, 7.7 mmol) was
added to a stirred solution of 16 (0.78 g, 2.3 mmol) in DMF (30 mL)
containing imidazole (1.04 g, 15 mmol) at room temperature. After
24 h, the reaction mixture was diluted with Et2O (120 mL), and the
organic layer was washed successively with 3% aqueous HCl
(2 ꢃ 30 mL), saturated aqueous NaHCO3 (2 ꢃ 30 mL) and brine
(2 ꢃ 30 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded a residue, which was purified by column chro-
33%, more polar).
25
Compound 15: colorless oil; [
a
]
þ33.8 (c 1.00, CHCl3); IR
matography (hexane/EtOAc 10:1 / 5:1) to give 18 (1.08 g, 99%) as
D
25
(neat): 3441, 3381, 2957, 2933, 2870, 1714, 1695, 1513, 1506, 1470,
1455, 1392, 1367, 1327, 1251, 1173, 1114, 1098, 1046, 1027 cmꢀ1 1H
NMR (400 MHz, CDCl3):
a colorless oil. [
a]
þ15.5 (c 1.01, CHCl3); IR (neat): 3371, 3273,
D
;
2957, 2931, 2901, 2857, 1703, 1504, 1472, 1390, 1366, 1284, 1253,
1174, 1094, 1051, 1028, 1007, 836, 777 cmꢀ1 1H NMR (400 MHz,
CDCl3): 0.01 (3H, s), 0,07 (3H, s), 0.84 (9H, br s), 0.84e0.90 (6H, m),
d
0.93 (6H, d, J ¼ 6.8 Hz), 1.25e1.28 (1H, m),
;
1.27 (3H, t, J ¼ 7.3 Hz),1.44e1.56 (1H, m), 1.44 (9H, s), 1.63e1.68 (1H,
m), 2.47e2.58 (2H, m), 3.41 (1H, d, J ¼ 2.9 Hz), 3.62 (1H, d,
J ¼ 5.4 Hz), 4.02 (1H, s), 4.17 (2H, dd, J ¼ 6.8, 6.8 Hz), 4.79 (1H, d,
d
1.21e1.25 (5H, m), 1.40 (9H, s), 1.57e1.65 (1H, m), 2.40 (1H, d,
J ¼ 6.4 Hz), 3.61 (1H, s), 4.06e4.16 (3H, m), 4.39 (1H, d, J ¼ 8.3 Hz);
J ¼ 9.8 Hz); 13C NMR (100 MHz, CDCl3):
d
14.0, 22.2, 22.9, 24.7, 28.3
13C NMR (100 MHz, CDCl3):
25.7 (3C), 28.3 (3C), 38.5, 39.8, 53.0, 60.4, 71.7, 78.8, 155.3, 171.2;
HRMS (EI): m/z calcd for C21H43NO5Si (Mþ) 417.2911, found
417.2893.
d
ꢀ4.9, ꢀ4.8, 14.1, 18.0, 21.5, 23.7, 24.6,
(3C), 38.8, 41.6, 51.9, 60.7, 69.7, 79.0, 156.0, 173.2; HRMS (EI): m/z
calcd for C15H29NO5 (Mþ) 303.2046, found 303.2039.
25
Compound 16: colorless oil; [
a]
þ20.2 (c 1.00, CHCl3); IR
D
(neat): 3444, 3371, 2977, 2957, 2935, 2871, 1714, 1695, 1522, 1391,
1367,1252,1174,1043,1026 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
0.91
4.1.5. (3S,4R)-allyl 4-(tert-butoxycarbonylamino)-3-(tert-
(3H, d, J ¼ 6.3 Hz), 0.94 (3H, d, J ¼ 6.8 Hz), 1.27 (3H, t, J ¼ 7.3 Hz),
1.32e1.35 (2H, m) 1.44 (9H, s), 1.64e1.71 (1H, m), 2.46e2.48 (2H,
m), 3.54 (1H, d, J ¼ 2.9 Hz), 3.67 (1H, br s), 4.01 (1H, br s), 4.17 (2H,
dd, J ¼ 6.8, 7.3 Hz), 4.68 (1H, d, J ¼ 8.8 Hz); 13C NMR (100 MHz,
butyldimethylsiloxy)-6-methylheptanoate (20)
1 M NaOH (16 mL, 16 mmol) was added dropwise to a stirred
solution of 18 (1.30 g, 3.1 mmol) in EtOH (30 mL) at room
temperature. After 6 h, the mixture was diluted with 10% aqueous
HCl (20 mL) at 0 ꢁC, and the resulting mixture was extracted with
EtOAc (3 ꢃ 30 mL). The combined extracts were washed with brine
(2 ꢃ 20 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded (3S,4R)-4-(tert-butoxycarbonylamino)-3-(tert-
CDCl3):
d 14.1, 21.5, 23.6, 24.7, 28.3 (3C), 38.1, 38.8, 52.7, 60.7, 71.3,
79.4, 156.1, 172.7; HRMS (EI): m/z calcd for C15H29NO5 (Mþ)
303.2046, found 303.2039.
4.1.2. (R)-ethyl 4-(tert-butoxycarbonylamino)-6-methyl-3-
oxoheptanoate (17)
butyldimethylsiloxy)-6-methylheptanoic acid (19) as a white
amorphous solid, which was used for the next reaction without
further purification.
Allyl bromide (0.54 mL, 6.2 mmol) was added to a stirred
solution of 19 (1.21 g, 3.1 mmol) in DMF (30 mL) containing K2CO3
(1.31 g, 9.4 mmol) at room temperature. After 6 h, the reaction was
diluted with water (15 mL) at room temperature, and the resulting
mixture was extracted with Et2O (4 ꢃ 30 mL). The combined
2.6 M Jones reagent (2.21 mL, 5.8 mmol) was added dropwise to
a stirred solution of 15 (1.16 g, 3.8 mmol) in acetone (40 mL) at 0 ꢁC,
and stirring was continued for 1 h at room temperature. The
mixture was diluted with Et2O (150 mL). The organic layer was
washed with saturated aqueous NaHCO3 (2 ꢃ 40 mL) and brine
(2 ꢃ 40 mL), then dried over Na2SO4. Concentration of the solvent

300
K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
extracts were washed successively with 3% aqueous HCl
(2 ꢃ 20 mL), saturated aqueous NaHCO3 (2 ꢃ 20 mL) and brine
(2 ꢃ 20 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded a residue, which was purified by column chro-
matography (hexane/EtOAc 5:1) to give 20 [1.03 g, 77% (2 steps
d 0.82e0.89 (6H, m), 0.86 (9H, s), 1.25e1.37 (2H, m), 1.49e1.51 (1H,
m), 2.42e2.52 (2H, m), 2.58 (1H, dd, J ¼ 8.3, 8.3 Hz), 2.69 (1H, dd,
J ¼ 3.4, 3.9 Hz), 3.10 (1H, dd, J ¼ 3.4, 3.9 Hz), 3.89e3.94 (1H, m),
4.11e4.15 (1H, m) 4.55 (2H, d, J ¼ 5.9 Hz), 5.22e5.33 (2H, m), 5.85e
5.94 (1H, m), 7.14 (1H, d, J ¼ 9.3 Hz), 7.21 (2H, d, J ¼ 7.3 Hz), 7.26e
from 18)] as a pale yellow oil. [
a
]
D
25 þ16.1 (c 1.03, CHCl3); IR (neat):
7.44 (15H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.7, ꢀ4.6, 17.9, 21.6,
2956, 2930, 2898, 2857, 1739, 1717, 1703, 1389, 1366, 1253, 1171,
23.8, 24.6, 25.8 (3C), 37.3, 38.3, 40.1, 51.5, 53.7, 65.3, 66.8, 71.8,
118.5, 126.7 (3C), 127.9 (6C), 129.6 (6C), 132.0, 144.6, 171.0, 172.2;
HRMS (FAB): m/z calcd for C39H55N2O4SSi (Mþ þ H) 674.3574,
found 675.3662.
1095, 836, 777 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
ꢀ0.02 (3H,
s), ꢀ0.05 (3H, s), 0.82 (9H, s), 0.82e0.88 (6H, m), 1.19e1.24 (2H, m),
1.38 (9H, s), 1.55e1.63 (1H, m), 2.42 (2H, d, J ¼ 6.4 Hz), 3.59 (1H, br
s), 4.16 (1H, br s), 4.35 (1H, d, J ¼ 8.3 Hz), 4.48e4.57 (2H, m), 5.17e
5.29 (2H, m), 5.82e5.92 (1H, m); 13C NMR (100 MHz, CDCl3):
4.1.8. (3S,4R)-allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)hept-4-enoylamino]
propionylamino]-3-tritylthio(propionylamino)}-6-
methylheptanoate (23)
d
ꢀ4.8, ꢀ4.7, 18.0, 21.6, 23.7, 24.6, 25.8 (3C), 28.3 (3C), 38.5, 39.7,
53.1, 65.3, 70.5, 71.7, 118.4, 132.0, 155.3, 171.0; HRMS (EI): m/z calcd
for C22H43NO5Si (Mþ) 429.2911, found 429. 2927.
i-Pr2NEt (73 mL, 0.43 mmol) was added dropwise to a stirred
4.1.6. (3S,4R)-allyl 4-[(S)-2-(tert-butoxycarbonylamino)-3-
(tritylthio)propanamido]-3-(tert-butyldimethylsiloxy)-6-
methylheptanoate (22)
solution of 8 (108 mg, 0.16 mmol) and (R)-2-[(S,E)-3-(4-met
hoxybenzyloxy)-7-(tritylthio)hept-4-enamido]propanoic acid (10)
[13a] (100 mg, 0.16 mmol) in CH2Cl2 (3.2 mL) containing O-(7-
azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluoroph
osphate (HATU) (81.2 mg, 0.21 mmol) and 1-hydroxy-7-
azabenzotriazole (HOAt) (28.6 mg, 0.21 mmol) at ꢀ30 ꢁC under
argon. After 3 h, the reaction mixture was diluted with CHCl3
(30 mL). The organic layer was washed successively with 10%
aqueous HCl (2 ꢃ 10 mL), saturated aqueous NaHCO3 (2 ꢃ 10 mL)
and brine (2 ꢃ 10 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 23 (169 mg, 84%) as
Trimethylsilyl trifluoromethanesulfonate (TMSOTf) (2.13 mL,
12 mmol) was added to a stirred solution of 20 (632 mg, 1.5 mmol)
in CH2Cl2 (30 mL) in the presence of 2,6-lutidine (1.70 mL,15 mmol)
at room temperature. After 1 h, MeOH (5.0 mL) was added to the
reaction mixture at 0 ꢁC, and stirring was continued for 1 h at room
temperature. The reaction mixture was concentrated in vacuo to
afford (3S,4R)-allyl 4-amino-3-(tert-butyldimethylsiloxy)-6-meth
ylheptanoate (21) as a colorless oil, which was immediately used
for the next reaction due to its instability (prone to form a
ring).
i-Pr2NEt (0.37 mL, 2.21 mmol) was added dropwise to a stirred
solution of the crude amine 21 (480 mg, 15 mmol) and N-Boc-S-
g-lactam
a colorless viscous liquid. [
a]
25 þ12.0 (c 1.00, CHCl3); IR (neat): 282,
D
2956, 2930, 1735, 1626, 1541, 1512, 1444, 1247, 1172, 1083, 835, 743,
699 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
ꢀ0.01 (3H, s), 0.05 (3H, s),
trityl-
D-cysteine (14) [13a] (1.02 g, 2.21 mmol) in MeCN (50 mL)
0.81e0.86 (15H, m), 1.16 (3H, d, J ¼ 7.3), 1.32 (2H, m), 1.45e1.48 (1H,
m), 2.08e2.12 (2H, m), 2.19e2.23 (2H, m), 2.31 (1H, dd, J ¼ 3.4,
2.9 Hz), 2.40e2.52 (4H, m), 2.83 (1H, dd, J ¼ 7.3, 7.8 Hz), 3.79 (3H, s),
3.89e4.02 (2H, m), 4.06e4.14 (3H, m), 4.19e4.27 (1H, m), 4.36 (1H,
d, J ¼ 10.7 Hz), 4.53e4.55 (2H, m), 5.19e5.45 (4H, m), 5.85e5.94
(1H, m), 6.19 (1H, d, J ¼ 9.3 Hz), 6.53 (1H, d, J ¼ 8.3 Hz), 6.82e
6.84 (2H, m), 6.96 (1H, d, J ¼ 6.3 Hz), 7.14e7.29 (20H, d), 7.36e
containing (benzotriazol-1-yloxy)tripyrrolidinophosphonium hex-
afluorophosphate (PyBOP) (918 mg, 1.8 mmol) at room temperature
under argon. After 3 h, the mixture was diluted with Et2O (80 mL),
and the organic layer was washed successively with 3% aqueous
HCl (2 ꢃ 30 mL), saturated aqueous NaHCO3 (2 ꢃ 30 mL) and brine
(2 ꢃ 30 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded a residue, which was purified by column chro-
matography (hexane/EtOAc 1:1) to give 22 (781 mg, 69%, 2 steps
7.43 (12H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.7, ꢀ4.6, 17.0, 18.0,
21.5, 23.8, 24.7, 25.8 (3C), 29.6, 31.2, 33.1, 39.1, 39.6, 42.4, 49.0, 52.1,
52.2, 55.3, 65.2, 66.6, 66.9, 70.0, 71.7, 76.4, 113.9 (2C), 118.3, 126.6
(3C), 126.8 (3C), 127.8 (6C), 128.0 (6C), 129.48 (6C), 129.52 (8C),
129.6, 129.8 (2C), 132.1, 144.3 (3C), 144.8 (3C), 159.4, 169.3, 171.2,
171.23, 172.0; HRMS (FAB): m/z calcd for C76H92N3O8S2Si (Mþ þ H)
1266.7665, found 1266.6104.
from 20) as a colorless oil. [
a
]
25 þ5.0 (c 1.00, CHCl3); IR (neat): 2956,
D
2930,1736, 1692, 1512, 1492, 1366, 1252, 1169, 833, 777, 700 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
ꢀ0.01 (3H, s), 0.05 (3H, s), 0.83e0.88
(6H, m), 0.85 (9H, s), 1.25e1.31 (2H, m), 1.40 (9H, s), 1.52e1.58 (1H,
m), 2.41e2.51 (3H, m), 2.76 (1H, dd, J ¼ 6.8, 6.8 Hz), 3.88e3.98 (2H,
m), 4.10e4.14 (1H, m), 4.53e4.56 (2H, m), 4.72 (1H, d, J ¼ 6.8 Hz),
5.21e5.33 (2H, m), 5.85e5.94 (1H, m), 6.08 (1H, d, J ¼ 8.3 Hz), 7.19e
4.1.9. (3S,4R)-allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]propionylamino]-
3-tritylthio(propionylamino)}-6-methylheptanate (24)
7.42 (15H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.7, ꢀ4.6, 18.0, 21.5,
23.9, 24.5, 25.8 (3C), 28.2 (3C), 33.4, 38.7, 39.8, 51.8, 53.5, 65.3, 67.1,
71.7, 80.3, 118.5, 126.8 (3C), 128.0 (6C), 129.6 (6C), 132.0, 144.5 (3C),
149.8, 170.0, 170.9 ppm; HRMS (FAB): m/z calcd for C44H62N2O6SSi
(Mþ) 774.4098, found 774.4091.
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (73.5 mg,
0.27 mmol) was added in small portions to a stirred solution of 23
(160 mg, 0.13 mmol) in CH2Cl2/H2O 9:1 (13 mL) at room temperature.
After 3 h, the reaction mixture was diluted with CHCl3 (60 mL), and
the organic layer was washed with saturated aqueous NaHCO3
(2 ꢃ 20 mL) and brine (2 ꢃ 20 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 3:2) to give 24
4.1.7. (3S,4R)-allyl 4-[(S)-2-amino-3-(tritylthio)propanamido]-3-
(tert-butyldimethylsiloxy)-6-methylheptanoate (8)
TMSOTf (0.90 mL, 5.0 mmol) was added dropwise to a stirred
solution of 22 (781 mg, 1.0 mmol) in CH2Cl2 (11 mL) containing
2,6-lutidine (0.91 mL, 5.0 mmol) at room temperature. After 1 h,
MeOH (2.2 mL) was added to the reaction mixture at 0 ꢁC, and
stirring was continued for 1 h at room temperature. The mixture
was concentrated in vacuo to afford a residue, which was purified
(147 mg, 99%) as a colorless viscous liquid. [
a
]
25 þ10.4 (c 1.00, CHCl3);
D
IR (neat): 3283, 2955, 2929, 1734, 1626, 1525, 1489, 1444, 1253, 1169,
1090, 833, 742, 699, 675 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d 0.01 (3H,
s), 0.06 (3H, s), 0.83e0.88 (15H, m),1.26e1.34 (5H, m),1.47e1.54 (1H,
m), 2.04e2.09 (2H, m), 2.17e2.26 (2H, m), 2.31 (1H, dd, J ¼ 2.4,
2.4 Hz), 2.41e2.54 (4H, m), 2.71 (1H, dd, J ¼ 7.3, 7.3 Hz), 3.08 (1H, d,
J ¼ 1.9 Hz), 3.91e3.96 (1H, m), 4.09e4.22 (2H, m), 4.30e4.35 (2H, m),
4.48e4.57 (2H,m), 5.20e5.48 (4H, m), 5.84e5.94 (1H, m), 6.06 (1H, d,
by column chromatography (hexane/EtOAc 2:1) to give 8 (593 mg,
25
88%) as a white amorphous solid. [
a
]
þ3.8 (c 1.00, CHCl3); IR
D
(neat): 2954, 2929, 2895, 2856, 1736, 1671, 1508, 1471, 1445, 1254,
1172, 1084, 835, 777, 743, 700 cmꢀ1 1H NMR (400 MHz, CDCl3):
;

K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
301
J ¼ 7.3 Hz), 6.14 (1H, d, J ¼ 9.3 Hz), 6.89 (1H, d, J ¼ 8.3 Hz), 7.17e7.31
(18H, m), 7.37e7.43 (12H, m); 13C NMR (100 MHz, CDCl3):
4.1.12. (1S,5S,6R,9S,20R,E)-5-(tert-butyldimethylsilyloxy)-6-
isobutyl-20-methyl-2-oxa-11,12-dithia-7,19,22-triazabicyclo[7.7.6]
docos-15-ene-3,8,18,21-tetraone (26)
d
ꢀ4.7, ꢀ4.6, 17.5, 18.0, 21.5, 23.8, 24.7, 25.8 (3C), 31.28, 31.34, 33.3,
39.3, 39.6, 43.9, 49.6, 52.3 (2C), 65.3, 66.6, 66.9, 69.7, 71.7, 118.3,126.6
(3C), 126.9 (3C), 127.9 (6C), 128.1 (6C), 129.5 (6C), 129.6 (6C), 130.0,
132.1, 132.5, 144.3 (3C), 144.9 (3C), 169.5, 171.3, 171.6, 171.8; HRMS
(FAB): m/z calcd for C68H84N3O7S2Si (Mþ þ H) 1146.6180, found
1146.5529.
A solution of 25 (33.5 mg, 31 mmol) in CH2Cl2/MeOH 9:1 (7.5 mL)
was added dropwise to a vigorously stirred solution of I2 (78.4 mg,
0.31 mmol) in CH2Cl2/MeOH 9:1 (62 mL, 0.5 mM concentration)
over 10 min at room temperature. After 10 min, the reaction was
quenched with 10% aqueous Na2S2O3 (30 mL) at room temperature.
The resulting mixture was diluted with CH2Cl2 (90 mL), and the
organic layer was washed with saturated aqueous NaHCO3
(2 ꢃ 30 mL) and brine (2 ꢃ 30 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
4.1.10. (7S,11R,14S,17R,18S,E)-18-(tert-butyldimethylsilyloxy)-7-
hydroxy-17-isobutyl-11-methyl-9,12,15-trioxo-1,1,1,-triphenyl-14-
tritylthiomethyl-2-thia-10,13,16-triazaicos-5-en-20-oic acid (6)
Morpholine (7.3
solution of 24 (47.8 mg, 42
Pd(PPh3)4 (4.8 mg, 4.2 mol) at room temperature under argon.
After 30 min, the reaction mixture was diluted with EtOAc (30 mL),
and the organic layer was washed with 10% aqueous HCl (2 ꢃ 10 mL)
and brine (2 ꢃ 10 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 6 (45.7 mg, 99%) as
m
L, 84
m
mol) was added dropwise to a stirred
purified by column chromatography (CHCl3/MeOH 30:1) to give 26
25
mmol) in THF (4.2 mL) containing
(17.6 mg, 95%) as a white amorphous solid. [
a
]
ꢀ3.4 (c 1.00,
D
m
CHCl3); IR (neat): 3426, 2953, 2857, 1747, 1653, 1541, 1259, 1146,
1127, 833 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
0.09 (3H, s), 0.13 (3H,
s), 0.89e0.93 (15H, m), 1.50 (3H, d, J ¼ 7.3 Hz), 1.54e1.66 (2H, m),
1.96e2.05 (1H, m), 2.44e2.57 (3H, m), 2.64e2.70 (3H, m), 3.03e
3.11 (2H, m), 3.21 (2H, dd, J ¼ 7.3, 7.3 Hz), 3.41 (1H, br s), 4.24e
4.31 (1H, m), 4.50e4.54 (1H, m), 4.91 (1H, br s), 5.64e5.68 (2H,
m), 5.88 (1H, s), 6.08 (1H, br s) 6.72 (1H, d, J ¼ 9.8 Hz), 7.47 (1H, d,
a white amorphous solid. [
a
]
25 ꢀ11.2 (c 1.00, CHCl3); IR (neat): 3295,
D
3081, 3058, 3018, 2955, 2928, 2856, 1714, 1659, 1651, 1644, 1635,
J ¼ 6.8 Hz); 13C NMR (100 MHz, CDCl3):
ꢀ4.8, ꢀ4.3, 16.7, 17.9, 21.1,
d
1538, 1488, 1444, 1256, 1094, 835, 752, 700 cmꢀ1
;
1H NMR
0.07 (6H, d, J ¼ 4.9 Hz), 0.79e0.91 (15H, m), 1.35
23.7, 25.3, 25.7 (3C), 33.3, 38.8, 38.9, 39.9, 40.6, 40.9, 52.2, 54.5, 55.9,
69.3, 70.8, 129.6, 132.5, 168.5, 168.8, 171.2, 171.3; HRMS (FAB): m/z
calcd for C27H48N3O6S2Si (Mþ þ H) 601.8941, found 602.2757.
(400 MHz, CDCl3):
d
(3H, d, J ¼ 7.3 Hz), 1.38e1.49 (2H, m), 1.52e1.57 (1H, m), 2.02e2.07
(2H, m), 2.17e2.26 (3H, m), 2.31e2.37 (2H, m), 2.44 (1H, dd, J ¼ 3.4,
3.9 Hz), 2.53 (1H, dd, J ¼ 6.3, 6.3 Hz), 2.82 (1H, dd, J ¼ 6.8, 6.8 Hz),
3.86e3.96 (2H, m), 4.25e4.35 (3H, m), 5.28e5.44 (2H, m), 6.38 (1H,
d, J ¼ 5.9 Hz), 6.54 (1H, d, J ¼ 8.3 Hz), 7.06 (1H, d, J ¼ 8.3 Hz), 7.15e
7.30 (18H, m), 7.34e7.43 (12H, m); 13C NMR (100 MHz, CDCl3):
4.1.13. Spiruchostatin C (4)
HF pyridine (0.55 mL) was added to a stirred solution of 26
(35.4 mg, 58 mmol) in pyridine (1.1 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
(3 ꢃ 15 mL), saturated aqueous NaHCO3 (2 ꢃ 15 mL) and brine
(2 ꢃ 15 mL), then dried over Na2SO4. Concentration of the solvent in
vacuo afforded a residue, which was purified by column chroma-
tography (CHCl3/MeOH 10:1) to give 4 (spiruchostatin C) (26.0 mg,
d
ꢀ4.7, ꢀ4.5, 17.2, 17.9, 21.4, 24.0, 24.8, 25.8 (3C), 29.7, 31.3, 33.1, 39.9,
39.96, 44.03, 50.3, 52.6, 53.1, 66.7, 66.8, 69.7, 71.7, 126.7 (3C), 127.0
(3C), 127.9 (6C), 128.1 (6C), 129.4 (6C), 129.6 (6C), 132.0, 132.1, 144.1
(3C), 144.8, 170.4, 172.25, 172.30, 172.4; HRMS (FAB): m/z calcd for
C65H80N3O7S2Si (Mþ þ H) 1106.5542, found 1106.5216.
93%) as a white amorphous solid. [
a
]
25 ꢀ59.4 (c 0.20, CHCl3) {lit. [10]
D
4.1.11. (2S,6R,9S,12R,13S)-13-(tert-butyldimethylsiloxy)-12-
isobutyl-6-methyl-2-[(E)-4-(tritylthio)but-1-enyl]-9-
tritylthiomethyl-1-oxa-5,8,11-triazacyclopentadecane-4,7,10,15-
tetraone (25)
[a]
25 ꢀ60.0 (c 0.10, CHCl3)}; IR (neat): 3455, 3369, 3311, 2998, 2953,
D
2868, 1728, 1659, 1546, 1530, 1275, 1161 cmꢀ1 1H NMR (400 MHz,
;
CDCl3):
d
0.92 (6H, d, J ¼ 6.3), 1.50e1.56 (4H, m), 1.59e1.68 (1H, m),
2.05e2.13 (1H, m), 2.42 (1H, dd, J ¼ 11.7, 11.7 Hz), 2.57 (1H, d,
J ¼ 13.2 Hz), 2.64e2.76 (5H, m), 2.99e3.06 (1H, m), 3.17e3.20 (2H,
m), 3.30 (1H, br s), 3.41 (1H, dd, J ¼ 7.3, 7.3 Hz), 4.23e4.29 (1H, m),
4.34e4.41 (1H, m), 4.84e4.89 (1H, m) 5.46 (1H, br s), 5.67 (1H, d,
J ¼ 15.1 Hz), 5.93 (1H, d, J ¼ 3.4 Hz), 6.36 (1H, t, J ¼ 12.2,13.7 Hz), 6.72
(1H, d, J ¼ 9.3 Hz), 7.49 (1H, d, J ¼ 7.3 Hz); 13C NMR (100 MHz,
A solution of 6 (45.7 mg, 41
very slowly to a stirred solution of 2-methyl-6-nitrobenzoic
anhydride (MNBA) (18.5 mg, 54 mol) in CH2Cl2 (41 mL, 1.0 mM
concentration) containing 4-dimethylaminopyridine (DMAP)
(18.5 mg, 54 mol) at room temperature over 14 h. After 1 h, the
mmol) in CH2Cl2 (4.1 mL) was added
m
m
mixture was diluted with CH2Cl2 (50 mL), and the organic layer was
washed successively with saturated aqueous NaHCO3 (2 ꢃ 20 mL),
water (2 ꢃ 20 mL) and brine (2 ꢃ 20 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which
CDCl3): d 16.6, 21.2, 23.5, 25.2, 29.7, 32.9, 38.7, 38.8, 40.7, 41.1, 52.3,
54.8, 56.3, 70.6, 70.9, 128.7, 133.5, 168.8, 170.8, 171.0, 171.6; HRMS
(FAB): m/z calcd for C21H34N3O6S2 (Mþ þ H) 487.6332, found
488.1890. The IR, 1H and 13C NMR, and HRMS spectrum are essen-
tially identical with those reported for natural spiruchostatin C [10].
was purified by column chromatography (hexane/EtOAc 1:1) to
25
give 25 (33.5 mg, 75%) as a white amorphous solid. [
a
]
ꢀ9.3 (c
D
1.01, CHCl3); IR (neat): 3468, 3323, 2954, 2927, 1735, 1651, 1537,
1489, 1444, 1259, 1094, 832, 743, 700, 668 cmꢀ1 1H NMR
(400 MHz, CDCl3): 0.01 (3H, s), 0.05 (3H, s), 0.78e0.93 (15H, m),
4.1.14. (3S,4R)-allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)hept-4-enoylamino]
isovaleroylamino]-3-tritylthio(propionylamino)}-5-
;
d
1.22e1.40 (5H, m), 1.72e1.83 (1H, m), 1.94e2.08 (2H, m), 2.16e2.20
(2H, m), 2.33e2.41 (2H, m), 2.47e2.53 (2H, m), 2.71 (1H, d,
J ¼ 13.2 Hz), 3.28e3.35 (2H, m), 3.70 (1H, br s), 4.18 (1H, t, J ¼ 6.3,
6.8 Hz), 4.30e4.33 (1H, m), 5.29e5.35 (1H, m), 6.23 (1H, br s), 6.60
(1H, br s), 6.94 (1H, d, J ¼ 7.3 Hz), 7.18e7.40 (30H, m); 13C NMR
methylhexanoate (27)
i-Pr2NEt (0.17 mL, 1.0 mmol) was added dropwise to a stirred
solution of 4-[(S)-2-amino-3-(tritylthio)propanamido]-3-(tert-
butyldimethylsiloxy)-5-methylhexanoate (9) [13a] (258 mg,
0.39 mmol) and (R)-2-[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)
hept-4-enamido]-3-methylbutanoic acid (11) [13a] (247 mg,
0.39 mmol) in CH2Cl2 (8.0 mL) containing HATU (193 mg,
0.51 mmol) and HOAt (69.2 mg, 0.51 mmol) at ꢀ30 ꢁC under argon.
After 3 h, the reaction mixture was diluted with CHCl3 (50 mL). The
organic layer was washed successively with 10% aqueous HCl
(2 ꢃ 20 mL), saturated aqueous NaHCO3 (2 ꢃ 20 mL) and brine
(100 MHz, CDCl3):
d
ꢀ4.7, ꢀ4.3, 17.20, 17.9, 21.3, 23.8, 25.0, 25.8
(3C), 31.1, 31.3, 32.5, 38.8, 39.4, 41.5, 50.4, 53.1, 54.4, 66.7, 69.8, 70.2,
77.2, 126.7 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C), 128.5, 129.50 (6C),
129.54 (6C), 131.9, 144.5 (3C), 144.8 (3C), 169.4, 169.9, 170.5, 171.5;
HRMS (FAB): m/z calcd for C65H78N3O6S2Si (Mþ þ H) 1088.5389,
found 1088.5118.

302
K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
(2 ꢃ 20 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded a residue, which was purified by column chro-
matography (hexane/EtOAc 2:1) to give 27 (469 mg, 94%) as
(3H, d, J ¼ 6.8 Hz), 1.94e2.02 (3H, m), 2.12e2.36 (6H, m), 2.41e2.50
(2H, m), 2.63e2.68 (1H, m), 3.69e3.75 (1H, m), 4.02e4.07 (2H, m),
4.18e4.21 (1H, m), 4.27e4.32 (1H, m), 5.32 (1H, dd, J ¼ 5.9, 15.4 Hz),
5.41 (1H, dt, J ¼ 6.4, 15.1 Hz), 6.22 (1H, d, J ¼ 9.8 Hz), 6.33 (1H, br s),
7.02 (1H, br s), 7.10e7.42 (30H, m); 13C NMR (100 MHz, CDCl3):
a colorless viscous liquid. [
a]
25 þ5.6 (c 1.00, CHCl3); IR (neat): 3290,
D
3058, 2958, 2930, 2857, 1734, 1637, 1540, 1514, 1444, 1389, 1248,
1173, 1092, 1036, 830, 743, 700 cmꢀ1
;
1H NMR (400 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.5, 16.7, 17.4, 17.9, 19.5, 20.3, 25.7 (3C), 27.9, 29.6, 31.3, 33.2,
d
0.02 (3H, s), 0.06 (3H, s), 0.68 (3H, d, J ¼ 6.8 Hz), 0.76e0.86 (9H,
39.7, 43.9, 53.2, 58.8, 59.0, 66.5, 66.9, 69.1, 69.6, 126.6 (3C), 126.7
(3C), 128.5 (6C), 128.6 (6C), 129.45 (6C), 129.51 (6C), 131.5, 132.5,
144.3 (3C), 144.8 (3C), 170.4, 171.5, 172.2, 174.0; IR (neat): 282, 3058,
2958, 1717, 1637, 1544, 1489, 1438, 1388, 1255, 1181, 1120, 1093, 1034,
m), 0.82 (9H, s), 1.86e2.13 (4H, m), 2.17e2.23 (2H, m), 2.34e2.57
(6H, m), 2.76 (1H, dd, J ¼ 7.8, 12.9 Hz), 3.75e3.82 (1H, m), 3.78
(3H, s), 3.93e4.02 (2H, m), 4.09 (1H, dd, J ¼ 5.9, 7.3 Hz), 4.15e4.22
(2H, m), 4.39 (1H, d, J ¼ 10.7 Hz), 4.46 (1H, dd, J ¼ 5.9, 13.2 Hz), 4.53
(1H, dd, J ¼ 5.9, 12.7 Hz), 5.18e5.31 (3H, m), 5.49 (1H, dt, J ¼ 6.8,
15.5 Hz), 5.81e5.91 (1H, m), 6.01 (1H, d, J ¼ 10.2 Hz), 6.30 (1H, d,
J ¼ 7.8 Hz), 6.80 (2H, d, J ¼ 8.3 Hz), 7.03 (1H, d, J ¼ 7.8 Hz), 7.16e7.45
836, 744, 723, 699 cmꢀ1; HRMS (FAB): m/z calcd for C66H81N3O7S2
-
SiK (Mþ þ K) 1158.4922, found 1158.4901.
4.1.17. (2S,6R,9S,12R,13S)-13-(tert-butyldimethylsiloxy)-6,12-
diisopropyl-2-[(E)-4-(tritylthio)but-1-enyl]-9-tritylthiomethyl-1-
oxa-5,8,11-triazacyclopentadecane-4,7,10,15-tetraone (29)
(32H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.7, ꢀ4.6, 16.9, 17.6, 17.9,
19.5, 20.3, 25.7 (3C), 28.0, 29.9, 31.2, 31.3, 33.2, 39.4, 42.5, 52.4, 55.3,
58.4, 58.5, 65.2, 66.5, 67.1, 69.4, 70.0, 76.5, 113.8 (2C), 118.3, 126.6
(3C), 126.8 (3C), 127.9 (6C), 128.1 (6C), 129.5 (12C), 129.7, 129.9 (2C),
129.9, 132.1, 133.2, 144.3 (3C), 144.8 (3C), 159.3, 169.8, 171.1, 171.2,
171.6; HRMS (FAB): m/z calcd for C77H93N3O8S2SiNa (Mþ þ Na)
1318.5810, found 1318.5787.
A solution of 7 (34.8 mg, 31
added very slowly to a stirred solution of MNBA (13.9 mg,
31 mol) in CH2Cl2 (30 mL, 1.0 mM concentration) containing
DMAP (11.4 mg, 93 mol) at room temperature over 14 h. After
mmol) in CH2Cl2 (3.0 mL) was
m
m
1 h, the mixture was diluted with CH2Cl2 (50 mL), and the organic
layer was washed successively with saturated aqueous NaHCO3
(2 ꢃ 20 mL), water (2 ꢃ 20 mL) and brine (2 ꢃ 20 mL), then dried
over Na2SO4. Concentration of the solvent in vacuo afforded
4.1.15. (3S,4R)-allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]isovaleroylamino]-
3-tritylthio(propionylamino)}-5-methylhexanate (28)
a
residue, which was purified by column chromatography
DDQ (166 mg, 0.73 mmol) was added in small portions to
a stirred solution of 27 (469 mg, 0.37 mmol) in CH2Cl2/H2O 9:1
(18 mL) at room temperature. After 3 h, the reaction mixture was
diluted with CHCl3 (60 mL), and the organic layer was washed with
saturated aqueous NaHCO3 (2 ꢃ 20 mL) and brine (2 ꢃ 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 28 (336 mg, 79%) as a colorless viscous liquid.
(hexane/EtOAc 1:1) to give 29 (28.9 mg, 85%) as a white amor-
phous solid. [
a
]
25 ꢀ6.3 (c 0.64, CHCl3); 1H NMR (400 MHz, CDCl3):
D
d
ꢀ0.10 (3H, s), 0.00 (3H, s), 0.76e0.80 (6H, m), 0.80 (9H, s), 0.95
(3H, d, J ¼ 6.8 Hz), 1.00 (3H, d, J ¼ 6.8 Hz), 1.93e2.09 (4H, m),
2.16e2.20 (2H, m), 2.31 (1H, dd, J ¼ 6.3, 16.1 Hz), 2.38 (1H, dd,
J ¼ 2.9, 16.6 Hz), 2.45e2.52 (2H, m), 2.61 (1H, d, J ¼ 8.3 Hz), 3.24e
3.29 (2H, m), 3.54e3.60 (1H, m), 3.94e3.99 (2H, m), 5.37 (1H, dd,
J ¼ 6.8, 15.4 Hz), 5.44e5.49 (1H, m), 5.67 (1H, dt, J ¼ 6.3, 15.6 Hz),
6.11 (1H, d, J ¼ 7.8 Hz), 6.60 (1H, d, J ¼ 4.9 Hz), 7.20e7.43 (31H, m);
[
a]
D
25 þ11.2 (c 1.00, CHCl3); IR (neat): 3270, 3058, 2958, 2928, 2856,
1736, 1673, 1633, 1549, 1490, 1444, 1390, 1254, 1174, 1093, 837, 743,
13C NMR (100 MHz, CDCl3):
d
ꢀ4.9, ꢀ3.9, 15.6, 17.9, 18.6, 19.4, 20.5,
700, 674 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d
0.00 (3H, s), 0.05 (3H,
25.8 (3C), 27.2, 29.7, 30.4, 30.9, 31.4, 32.0, 41.2, 42.1, 58.3, 59.6,
66.6, 66.9, 68.9, 72.2, 126.6 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C),
128.2, 129.47 (6C), 129.52 (6C), 133.4, 144.4 (3C), 144.8 (3C), 169.6,
170.1, 170.6, 173.1 ppm; IR (neat): 3468, 3323, 2954, 2927, 1735,
1651, 1537, 1489, 1444, 1259, 1094, 832, 743, 700, 668 cmꢀ1; HRMS
(FAB): m/z calcd for C66H79N3O6S2SiNa (Mþ þ Na) 1124.5077,
found 1124.5040.
s), 0.79e0.85 (9H, m), 0.81 (9H, s), 0.92 (3H, d, J ¼ 6.8 Hz), 1.87e1.92
(1H, m), 2.05 (1H, d, J ¼ 7.3 Hz), 2.17e2.26 (4H, m), 2.33 (1H, dd,
J ¼ 2.7, 13.7 Hz), 2.41 (1H, dd, J ¼ 7.3, 15.6 Hz), 2.49e2.61 (4H, m),
3.43 (1H, br s), 3.73e3.78 (1H, m), 3.99 (1H, dd, J ¼ 8.3,14.9 Hz), 4.17
(1H, dt, J ¼ 3.4, 7.2 Hz), 4.23 (1H, dd, J ¼ 4.9, 8.3 Hz), 4.33e4.37 (1H,
m), 4.49 (1H, dd, J ¼ 5.9, 13.3 Hz), 4.52 (1H, dd, J ¼ 5.9, 13.2 Hz),
5.19e5.31 (2H, m), 5.36 (1H, dd, J ¼ 5.9,15.4 Hz), 5.51 (1H, dt, J ¼ 6.3,
15.5 Hz), 5.81e5.93 (2H, m), 6.04 (1H, d, J ¼ 8.3 Hz), 6.93 (1H, d,
J ¼ 8.3 Hz), 7.17e7.43 (30H, m); 13C NMR (100 MHz, CDCl3):
4.1.18. (1S,5S,6R,9S,20R,E)-5-(tert-butyldimethylsilyloxy)-6,20-
diisopropyl-2-oxa-11,12-dithia-7,19,22-triazabicyclo[7.7.6]docos-
15-ene-3,8,18,21-tetraone (30)
d
ꢀ4.7, ꢀ4.7, 16.9, 17.4, 17.9, 19.5, 20.2, 25.7 (3C), 28.1, 29.7, 31.30,
31.34, 33.3, 39.34, 44.0, 52.6, 58.5, 58.6, 65.3, 66.6, 67.0, 69.3, 69.8,
118.4, 126.6 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C), 129.48 (6C),
129.54 (6C), 129.9, 132.0, 132.5, 144.3 (3C), 144.8 (3C), 170.2, 170.9,
171.6, 171.7 ppm; HRMS (FAB): m/z calcd for C69H86N3O7S2Si
(Mþ þ H) 1159.5598, found 1160.5654.
A solution of 29 (45.0 mg, 42 mmol) in CH2Cl2/MeOH 9:1 (17 mL)
was added dropwise to a vigorously stirred solution of I2 (170 mg,
0.67 mmol) in CH2Cl2/MeOH 9:1 (84 mL, 0.5 mM concentration)
over 10 min at room temperature. After 10 min, the reaction was
quenched with 10% aqueous Na2S2O3 (30 mL) at room temperature.
The resulting mixture was diluted with CH2Cl2 (90 mL), and the
organic layer was washed with saturated aqueous NaHCO3
(2 ꢃ 30 mL) and brine (2 ꢃ 30 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
4.1.16. (7S,11R,14S,17R,18S,E)-18-(tert-butyldimethylsilyloxy)-7-
hydroxy-11,17-diisopropyl-9,12,15-trioxo-1,1,1-triphenyl-14-
tritylthiomethyl-2-thia-10,13,16-triazaicos-5-en-20-oic acid (7)
Morpholine (46
solution of 28 (308 mg, 0.27 mmol) in THF (13 mL) containing
Pd(PPh3)4 (30.7 mg, 27 mol) at room temperature under argon.
After 30 min, the reaction mixture was diluted with EtOAc (30 mL),
and the organic layer was washed with 10% aqueous HCl (2 ꢃ 10 mL)
and brine (2 ꢃ 10 mL), then dried over Na2SO4. Concentration of the
solvent in vacuo afforded a residue, which was purified by column
m
L, 0.53 mmol) was added dropwise to a stirred
purified by column chromatography (CHCl3/MeOH 30:1) to give 30
25
(36.1 mg, 88%) as a white amorphous solid. [
a
]
þ13.6 (c 0.28,
D
m
CHCl3); IR (neat): 3335, 2956, 1747, 1670, 1541, 1258, 1158, 948, 831,
781 cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d
0.10 (3H, s), 0.16 (3H, s),
0.79 (3H, d, J ¼ 6.8 Hz), 0.83 (9H, s), 0.87 (3H, d, J ¼ 6.8 Hz), 0.95 (3H,
d, J ¼ 6.8 Hz), 0.99 (3H, d, J ¼ 6.8 Hz), 2.06e2.14 (1H, m), 2.18e2.26
(1H, m), 2.47e2.53 (3H, m), 2.58e2.91 (6H, m), 3.04 (1H, dd, J ¼ 7.3,
13.2 Hz), 3.70e3.72 (1H, m), 4.07 (1H, t, J ¼ 5.2 Hz), 4.75 (1H, dt,
J ¼ 3.4, 9.8 Hz), 4.95e4.99 (1H, m), 5.54 (1H, d, J ¼ 15.1 Hz), 5.61e
5.62 (1H, m), 5.71 (1H, d, J ¼ 4.4 Hz), 6.50 (1H, t, J ¼ 13.4 Hz), 6.62
chromatography (CHCl3/MeOH 20:1) to give 7 (270 mg, 90%) as
25
a white amorphous solid. [
a
]
ꢀ0.8 (c 1.00, CHCl3); 1H NMR
D
(400 MHz, CDCl3):
d
0.00 (6H, s), 0.74e0.79 (9H, m), 0.79 (9H, s), 0.87

K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
303
(1H, d, J ¼ 9.8 Hz), 7.00 (1H, J ¼ 6.8 Hz); 13C NMR (100 MHz, CDCl3):
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 deter-
mined by using the sulforhodamine B colorimetric method.
d
ꢀ4.8, ꢀ4.2, 17.0, 18.0, 19.1, 19.8, 21.0, 25.7 (3C), 29.0, 29.2, 35.2,
39.2, 40.6, 41.3, 44.2, 53.6, 61.8, 62.8, 67.4, 68.7, 128.3, 133.4, 168.8,
169.1, 170.0, 171.2 ppm; HRMS (FAB): m/z calcd for C28H49N3O6S2Si
(Mþ þ H) 615.2832, found 615.2914.
4.1.19. Spiruchostatin D (5)
HF$pyridine (0.55 mL) was added to a stirred solution of 30
(35.4 mg, 58 mmol) in pyridine (1.1 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
(3 ꢃ 15 mL), saturated aqueous NaHCO3 (2 ꢃ 15 mL) and brine
(2 ꢃ 15 mL), then dried over Na2SO4. Concentration of the solvent in
vacuo afforded a residue, which was purified by column chroma-
Acknowledgements
tography (CHCl3/MeOH 10:1) to give 5 (spiruchostatin D) (26.8 mg,
We are grateful to the Screening Committee of New Anticancer
Agents supported by a Grant-in-Aid for Scientific Research on
Priority Area ‘Cancer’ from the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT) for the biological
evaluation of compounds 4 and 5. This study was supported by
a Grant-in-Aid for the Strategic Research Foundation Program at
Private Universities (2010e2014) from MEXT and a Grant-in-Aid for
Scientific Research (C) (Nos. 21590018 and 24590017) from MEXT.
25
93%) as a white amorphous solid. [
a]
ꢀ59.2 (c 0.29, MeOH) {lit.
D
[12] [
a
]
D
25 ꢀ65.3 (c 0.20, MeOH)}; 1H NMR (400 MHz, CDCl3):
d 0.93
(3H, d, J ¼ 6.8 Hz), 1.04 (3H, d, J ¼ 6.8 Hz), 1.07 (3H, d, J ¼ 6.8 Hz),
1.09 (3H, d, J ¼ 7.3 Hz), 2.25e2.34 (1H, m), 2.36e2.44 (1H, m), 2.46e
2.54 (1H, m), 2.62 (1H, d, J ¼ 13.2 Hz), 2.66e2.79 (5H, m), 2.98e3.15
(3H, m), 3.38 (1H, d, J ¼ 6.8, 13.2 Hz), 3.51e3.57 (1H, m), 4.10 (1H, t,
J ¼ 5.1 Hz), 4.56e4.58 (1H, m), 4.93e4.98 (1H, m), 5.50e5.52 (1H,
m), 5.68 (1H, d, J ¼ 15.1 Hz), 5.92e5.95 (1H, m), 6.54 (1H, t,
J ¼ 12.7 Hz), 6.74 (1H, d, J ¼ 9.3 Hz), 7.39 (1H, d, J ¼ 7.3 Hz); 13C NMR
(100 MHz, CDCl3):
d
19.3, 19.6, 19.7, 20.6, 29.1, 29.6, 34.0, 39.7, 40.2,
Appendix A. Supplementary data
40.8, 42.6, 54.3, 62.1, 63.4, 69.4, 70.4, 128.1, 133.9, 168.9, 170.0, 171.0,
171.8 ppm; IR (neat): 3375, 3342, 2964, 2932, 2875, 1731, 1660,
1534, 1519, 1266, 1162, 979, 911, 755, 733 cmꢀ1; HRMS (FAB): m/z
calcd for C20H32N3O6S2 (Mþ þ H) 501.196, found 501.2057. The IR,
1H and 13C NMR, and HRMS spectrum are essentially identical with
those reported for natural spiruchostatin D [12].
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.12.023.
References
[1] (a) S. Kanno, N. Maeda, A. Tomizawa, S. Yomogida, T. Katoh, M. Ishikawa, Int. J.
Oncol. 41 (2012) 862e868;
4.2. Biological evaluation
(b) S. Kanno, N. Maeda, A. Tomizawa, S. Yomogida, T. Katoh, M. Ishikawa, Int. J.
Oncol. 40 (2012) 1391e1396;
(c) T. Ai, H. Cui, L. Chen, Curr. Med. Chem. 19 (2012) 475e487;
(d) O. Khan, N.B. La Thangue, Immunol. Cell. Biol. 90 (2012) 85e94;
(e) P. Perego, V. Zuco, L. Gatti, F. Zunino, Biochem. Pharmacol. 83 (2012)
987e994;
4.2.1. HDACs preparation and enzyme inhibition assay [20]
In a 100-mm dish, 293 T cells (1e2 ꢃ 107) 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 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 Trise
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
(f) B.N. Singh, H. Zhou, J. Li, T. Tipton, B. Wang, G. Shao, E.N. Gilbert, Q. Li, S.-
W. Jiang, Future Oncol. 7 (2011) 1415e1428;
(g) R. Harrod, Leuk. Res. 35 (2011) 1436e1437;
(h) B. Venugopal, T.R.J. Evans, Curr. Med. Chem. 18 (2011) 1658e1671;
(i) E.M. Bertino, G.A. Otterson, Expert Opin. Inv. Drugs 20 (2011) 1151e1158;
(j) S. Consalvi, V. Saccone, L. Giordani, G. Minetti, C. Mozzetta, P.L. Puri, Mol.
Med. 17 (2011) 457e465;
(k) V. Carafa, A. Nebbioso, L. Altucci, Recent Pat. Anti-Cancer Drug Discov. 6
(2011) 131e145;
supernatant had been incubated for 1 h at 4 ꢁC with 4
mg of an anti-
(l) A. Quintás-Cardama, F.P.S. Santos, G. Garcia-Manero, Leukemia 25 (2011)
226e235;
FLAG M2 antibody (SigmaeAldrich 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 TriseHCl (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
(m) P.A. Marks, Expert Opin. Inv. Drugs 19 (2010) 1049e1066;
(n) P. Bertrand, Eur. J. Med. Chem. 45 (2010) 2095e2116;
(o) A.A. Lane, B.A. Chabner, J. Clin. Oncol. 27 (2009) 5459e5468;
(p) X. Ma, H.H. Ezzeldin, R.B. Diasio, Drugs 69 (2009) 1911e1934.
[2] (a) G.R. Leggatt, B. Gabrielli, Immunol. Cell. Biol. 90 (2012) 33e38;
(b) A.K. Oyelere, P.C. Chen, W. Guerrant, S.C. Mwakwari, R. Hood, Y. Zhang,
Y. Fan, J. Med. Chem. 52 (2009) 456e468;
with 40
deacetylase buffer (200
centrifugation. For the enzyme assay, 10
was added to 1 L of fluorescent substrate (2 mM Ac-KGLGK(Ac)-
MCA) and 9 L of histone deacetylase buffer, and the mixture was
incubated at 37 ꢁC for 30 min. The reaction was stopped by the
addition of 30
L of trypsin (20 mg/mL) and incubated at 37 ꢁC for
m
g of the FLAG peptide (SigmaeAldrich Inc.) in histone
L). The supernatant was collected by
L of the enzyme fraction
m
(c) L.M. Butler, D.B. Agus, H.I. Scher, B. Higgins, A. Rose, C. Cordon-Cardo,
H.T. Thaler, R.A. Rifkind, P.A. Marks, V.M. Richon, Cancer Res. 60 (2000)
5165e5170;
(d) A. Saito, T. Yamashita, Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando,
T. Suzuki, T. Tsuruo, O. Nakanishi, Proc. Natl. Acad. Sci. U S A 96 (1999)
4592e4597;
m
m
m
(e) R.D. Glick, S.L. Swendeman, D.C. Coffey, R.A. Rifkind, P.A. Marks,
V.M. Richon, M.P. La Quaglia, Cancer Res. 59 (1999) 4392e4399.
[3] (a) X.J. Yang, E. Seto, Nat. Rev. Mol. Cell. Biol. 9 (2008) 206e218;
(b) T. Kouzarides, Cell 128 (2007) 693e705;
(c) B.E. Bernstein, A. Meissner, E.S. Lander, Cell 128 (2007) 669e681;
(d) P. Trojer, D. Reinberg, Cell 125 (2006) 213e217.
[4] (a) O. Witt, H.E. Deubzer, T. Milde, I. Oehme, Cancer Lett. 277 (2009) 8e21;
(b) M. Haberland, R.L. Montgomery, E.N. Olson, Nat. Rev. Genet. 10 (2009)
32e42;
m
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 doseeresponse curves.
4.2.2. Cell-growth inhibition assay [19]
This experiment was carried out at the Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research. The screening
(c) M.A. Holbert, R. Marmorstein, Curr. Opin. Struct. Biol. 15 (2005) 673e680;
(d) S. Voelt-Mahlknecht, A.D. Ho, U. Mahlnecht, Int. J. Mol. Med. 16 (2005)
589e598;

304
K. Narita et al. / European Journal of Medicinal Chemistry 60 (2013) 295e304
(e) A.J.M. De Ruijter, A.H. Van Gennip, H.N. Caron, S. Kemp, A.B.P. Van Kui-
lenburg, Biochem. J. 370 (2003) 737e749.
[13] (a) K. Narita, T. Kikuchi, K. Watanabe, T. Takizawa, T. Oguchi, K. Kudo,
K. Matsuhara, H. Abe, T. Yamori, M. Yoshida, T. Katoh, Chem. Eur. J. 15 (2009)
11174e11186;
[5] (a) T.A. McKinsey, J. Mol. Cell. Cardiol. 51 (2011) 491e496;
(b) A.P. Kozikowski, K.V. Butler, Curr. Pharm. Des. 14 (2008) 505e528;
(c) A.V. Bieliauskas, M.K.H. Pflum, Chem. Soc. Rev. 37 (2008) 1402e1413;
(d) S. Senese, K. Zaragoza, S. Minardi, I. Muradore, S. Ronzoni, A. Passafaro,
L. Bernard, G.F. Draetta, M. Alcalay, C. Seiser, S. Chiocca, Mol. Cell. Biol. 27
(2007) 4784e4795;
(b) S. Wen, G. Packham, A. Ganesan, J. Org. Chem. 73 (2008) 9353e9361;
(c) T.J. Greshock, D.M. Johns, Y. Noguchi, R.M. Williams, Org. Lett. 10 (2008)
613e616;
(d) K.W. Li, J. Wu, W. Xing, J.A. Simon, J. Am. Chem. Soc. 118 (1996) 7237e7238.
[14] (a) N.A. Calandra, Y.L. Cheng, K.A. Kocak, J.S. Miller, Org. Lett. 11 (2009)
1971e1974;
(e) P. Kahnberg, A.J. Lucke, M.P. Glenn, G.M. Boyle, J.D.A. Tyndall, P.G. Parsons,
D.P. Fairlie, J. Med. Chem. 49 (2006) 7611e7622;
(b) Y. Iijima, A. Munakata, K. Shin-ya, A. Ganesan, T. Doi, T. Takahashi,
(f) S. Chang, T.A. McKinsey, C.L. Zhang, J.A. Richardson, J.A. Hill, E.N. Olson, Mol.
Cell. Biol. 24 (2004) 8467e8476.
[6] (a) K.M. Van der Molen, W. McCulloch, C.J. Pearce, N.H. Oberlies, J. Antibiot. 64
(2011) 525e531;
(b) C. Grant, F. Rahman, R. Piekarz, C. Peer, R. Frye, R.W. Robey, E.R. Gardner,
W.D. Figg, S.E. Bates, Expert Rev. Anticancer Ther. 10 (2010) 997e1008.
[7] M.H. Shah, P. Binkley, K. Chan, J. Xiao, D. Arbogast, M. Collamore, Y. Farra,
D. Young, M. Grever, Clin. Cancer Res. 12 (2006) 3997e4003.
[8] (a) H. Ueda, H. Nakajima, Y. Hori, T. Fujita, M. Nishimura, T. Goto, M. Okuhara,
J. Antibiot. 47 (1994) 301e310;
Tetrahedron Lett. 50 (2009) 2970e2972;
(c) T. Takizawa, K. Watanabe, K. Narita, K. Kudo, T. Oguchi, H. Abe, T. Katoh,
Heterocycles 76 (2008) 275e290;
(d) T. Doi, Y. Iijima, K. Shin-ya, A. Ganesan, T. Takahashi, Tetrahedron Lett.
47 (2006) 1177e1180;
(e) A. Yurek-George, F. Habens, M. Brimmell, G. Packham, A. Ganesan, J. Am.
Chem. Soc. 126 (2004) 1030e1031.
[15] (a) S. Fuse, K. Okada, Y. Iijima, A. Munakata, K. Machida, T. Takahashi,
M. Takagi, K. Shin-ya, T. Doi, Org. Biomol. Chem. 9 (2011) 3825e3833;
(b) T. Takizawa, K. Watanabe, K. Narita, T. Oguchi, H. Abe, T. Katoh, Chem.
Commun. (2008) 1677e1679.
[16] E.P. Johnson, M.P. Hubieki, A.P. Combs, C.A. Teleha, Synthesis (2011) 4023e4026.
[17] (a) I. Shiina, T. Katoh, S. Nagai, M. Hashizume, Chem. Rec. 9 (2009) 305e320;
(b) I. Shiina, A. Sasaki, T. Kikuchi, H. Fukui, Chem. Asian J. 3 (2008) 462e472;
(c) I. Shiina, Chem. Rev. 107 (2007) 239e273;
(b) N. Shigematsu, H. Ueda, S. Takase, H. Tanaka, J. Antibiot. 47 (1994)
311e314;
(c) H. Ueda, T. Manda, S. Matsumoto, S. Mukumoto, F. Nishigaki,
I. Kawamura, K. Shimomura, J. Antibiot. 47 (1994) 315e323;
(d) M. Okuhara, T. Goto, T. Fujita, Y. Hori, H. Ueda (Fujisawa Pharmaceu-
tical Co. Ltd., Japan), JP 3141296A (1991).
(d) I. Shiina, M. Hashizume, Tetrahedron 62 (2006) 7934e7939;
(e) I. Shiina, M. Kubota, H. Oshiumi, M. Hashizume, J. Org. Chem. 69 (2004)
1822e1830.
[9] Y. Masuoka, A. Nagai, K. Shin-ya, K. Furihata, K. Nagai, K. Suzuki, Y. Hayakawa,
H. Seto, Tetrahedron Lett. 42 (2001) 41e44.
[10] N. Shindo, A. Terada, M. Mori, N. Amino, K. Hayata, K. Nagai, Y. Hayakawa, K.
Shin-ya, Y. Masuoka (Yamanouchi Pharmaceutical Co. Ltd., Japan), JP 348340A
(2001).
[11] Recently, Klausmeyer et al. reported the isolation and structural elucidation of
a novel spiruchostatin congener from Burkholderia thailandensis and assigned
it an improper name ‘spiruchostatin C’. To avoid nomenclatural confusions,
maybe it should be named as one of a series of burkholdacs such as ‘bur-
kholdac C’, see: P. Klausmeyer, S.M. Shipley, K.M. Zuck, T.G. McCloud J. Nat.
Prod. 74 (2011) 2039e2044.
[18] 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. 50 (2007) 5720e5726.
[19] (a) S. Yaguchi, Y. Fukui, I. Koshimizu, H. Yoshimi, T. Matsuno, H. Gouda,
S. Hirono, K. Yamazaki, T. Yamori, J. Natl. Cancer Inst. 98 (2006) 545e556;
(b) T. Yamori, Cancer Chemother. Pharmacol. 52 (Suppl. 1) (2003) 74e79;
(c) S. Dan, T. Tsunoda, O. Kitahara, R. Yanagawa, H. Zembutsu, T. Katagiri,
K. Yamazaki, Y. Nakamura, T. Yamori, Cancer Res. 62 (2002) 1139e1147;
(d) T. Yamori, A. Matsunaga, 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. 59 (1999) 4042e4049.
[20] G.M. Shivashimpi, S. Amagai, T. Kato, N. Nishino, S. Maeda, T.G. Nishino,
M. Yoshida, Bioorg. Med. Chem. 15 (2007) 7830e7839.
[12] K. Nagai, M. Taniguchi, N. Shindo, Y. Terada, M. Mori, N. Amino, K. Suzumura, I.
Takahashi, M. Amase (Yamanouchi Pharmaceutical Co. Ltd., Japan), PCT WO
20460 A1 (2004).