Total synthesis of burkholdacs A and B and 5,6,20-tri-epi-burkholdac A: HDAC inhibition and antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2014.02.044

The bicyclic depsipeptide histone deacetylase (HDAC) inhibitors burkholdacs A and B were efficiently synthesized in a highly convergent and unified manner. The synthesis features the amide coupling of a d-valine-d-cysteine- or d-allo-isoleucine-d-cysteine-containing segment with a d-methionine-containing segment to directly assemble the corresponding seco-acids, key precursors for macrolactonization. Using the same methodology, 5,6,20-tri-epi-burkholdac A was also synthesized. HDAC inhibitory assays and cell-growth inhibition analyses of the synthesized depsipeptides demonstrated the potency order of this class of bicyclic depsipeptides as compared to the clinically approved depsipeptide FK228 (romidepsin). Novel structure-activity relationships within this class of compounds were also revealed.

Full text of DOI:10.1016/j.ejmech.2014.02.044

European Journal of Medicinal Chemistry 76 (2014) 301e313
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Total synthesis of burkholdacs A and B and 5,6,20-tri-epi-burkholdac
A: HDAC inhibition and antiproliferative activity
,
Yurie Fukui a, Koichi Narita a, Singo Dan b, Takao Yamori b c, Akihiro Ito d, Minoru Yoshida d,
,
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 Pharmaceuticals and Medical Devices Agency (PMDA), 3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo 100-0013, Japan
d 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 burkholdacs A and B were efficiently
Received 17 January 2014
Received in revised form
12 February 2014
Accepted 16 February 2014
Available online 18 February 2014
synthesized in a highly convergent and unified manner. The synthesis features the amide coupling of a
D-
valine- -cysteine- or -allo-isoleucine- -cysteine-containing segment with a -methionine-containing
D
D
D
D
segment to directly assemble the corresponding seco-acids, key precursors for macrolactonization. Using
the same methodology, 5,6,20-tri-epi-burkholdac A was also synthesized. HDAC inhibitory assays and
cell-growth inhibition analyses of the synthesized depsipeptides demonstrated the potency order of this
class of bicyclic depsipeptides as compared to the clinically approved depsipeptide FK228 (romidepsin).
Novel structureeactivity relationships within this class of compounds were also revealed.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Burkholdacs
Histone deacetylase inhibitors
Natural products
Bicyclic depsipeptide
Total synthesis
1. Introduction
including FK228 (romidepsin) (5) [4] and the spiruchostatins A (6)
[4,5], B (7) [4,6], C (8) [7] and D (9) [7], we became interested in the
In 2011, Brady et al. reported the isolation and structural eluci-
dation of the two new bicyclic depsipeptide histone deacetylase
(HDAC) inhibitors, burkholdacs A (1) and B (2) (Fig. 1) [1]. These
compounds were identified through the systematic overexpression
of transcription factors associated with natural product gene clus-
ters encoded within Burkholderia thilandensis E264 [1]. The ste-
reochemistry at the C5, C6 and C20 positions could not be
unambiguously assigned at that time. The stereostructures of 1 and
2 were proposed as compounds 3 and 4 in Fig. 1, respectively, based
on the biosynthesis gene cluster that contains only one epimerase
domain [1]. Through two independent total syntheses by Ganesan
et al. in 2011 [2] and by Ye et al. in 2012 [3], the stereochemistry of
the two new HDAC inhibitors, which includes their absolute con-
figurations, was determined, as shown in structures 1 and 2.
In the course of our continuing research on the synthesis and
biological evaluation of bicyclic depsipeptide HDAC inhibitors
synthesis of 1 and 2 and their analogues with the aim of identifying
novel mechanism-based anticancer agents [8]. Several total syn-
theses of 5e7 by other groups have been published in the literature
[9]. In this study, we describe our total synthesis of 1 and 2 and the
earlier proposed stereostructure 3 by applying a synthetic strategy
developed previously in our laboratory [4e7]. The synthesized
compounds 1e3 were subjected to HDAC inhibition assays and
antiproliferative analyses to establish the potency order for this
class of bicyclic depsipeptide HDAC inhibitors (1e3 and 5e9).
2. Results and discussion
2.1. Chemistry
2.1.1. Synthetic plan for burkholdacs A (1) and B (2)
Our synthetic plan for 1 and 2 is outlined in Scheme 1. We
envisioned that target molecules 1 and 2 could be synthesized via
the macrolactonization of the corresponding seco-acids 10 and 11,
followed by disulfide bond formation. The key macrolactonization
* Corresponding author.
E-mail address: katoh@tohoku-pharm.ac.jp (T. Katoh).
http://dx.doi.org/10.1016/j.ejmech.2014.02.044
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
oxidative deprotection step (cf. DDQ, CH2Cl2/H2O, rt, 3 h). The
critical macrolactonization of 10 was successfully achieved by
employing the Shiina method [10] [MNBA (1.3 equiv), DMAP
(3.0 equiv), CH2Cl2 (10 mM), rt, 12 h], which afforded the expected
cyclization product 20 in 79% yield. Subsequent disulfide bond
formation accompanied by oxidative S-Tr deprotection of 20 was
efficiently achieved by brief exposure to I2 in a dilute CH2Cl2/MeOH
solution (0.5 mM) at ambient temperature, which resulted in the
production of the desired disulfide 21 in 81% yield. Finally, depro-
tection of the TBS group in 21 (89%) led to the completion of the
total synthesis of 1. The spectroscopic properties (IR, 1H and 13C
NMR, and MS) of the synthesized sample 1 were identical to those
reported for natural 1 [1].
2.1.3. Synthesis of burkholdac B (2)
As shown in Scheme 4, using segments 13 [4,6] and 14 as the
starting materials, 2 was synthesized in a manner similar to that
described for the synthesis of 1 (cf. Scheme 3). The spectroscopic
properties (IR, 1H and 13C NMR, and MS) of the synthesized sample
2 were identical with those reported for natural 2 [1].
2.1.4. Synthesis of 5,6,20-tri-epi-burkholdac A (3)
Moreover, we synthesized the Brady’s proposed stereoisomer,
5,6,20-tri-epi-burkholdac A (3), to assess its biological activity.
Using N-Boc- -valinal (26) [11] as the starting material, the key
L
segment 35 was synthesized according to a method developed
previously in our laboratory [4,5] (Scheme 5). The other key
segment 37 was readily prepared via the condensation of
commercially available -methionine methyl ester (ent-15) with
L
Fig. 1. Revised (1, 2) and Brady’s original (3, 4) structures of burkholdacs A and B,
FK228 (romidepsin) (5) and spiruchostatins AeD (6e9). i-Pr ¼ isopropyl, s-Bu ¼ sec-
butyl, i-Bu ¼ isobutyl.
precursors 10 and 11 could be prepared through the direct coupling
of the
isoleucine-
the -methionine-containing segment 14. The segment 14 was to
be formed by condensation of commercially available -methionine
methyl ester (15) with the known carboxylic acid 16 (previously
prepared from -malic acid in our laboratory [4e6]). The two seg-
D
-valine-
D-cysteine-containing segment 12 and
D-allo-
D
-cysteine-containing segment 13, respectively, with
D
D
L
ments 12 and 13 were obtained as previously described in our total
synthesis of 6 and 7 [4e6].
2.1.2. Synthesis of burkholdac A (1)
We initially pursued the synthesis of 1. For the synthesis, the key
segment 14 was efficiently prepared, as shown in Scheme 2. The
condensation of 15 with 16 [4e6] afforded the desired coupling
product 17 in excellent yield. Subsequent saponification of the
methyl ester moiety in 17 furnished the requisite segment 14 in
quantitative yield.
Subsequently, the synthesis of 1 was accomplished by assem-
bling the two segments 12 [4,5] and 14, as shown in Scheme 3.
Thus, treatment of 12 and 14 with HATU (1.3 equiv) and HOAt
(1.3 equiv) in the presence of i-Pr2NEt (2.6 equiv) in CH2Cl2
at ꢀ30 ꢁC for 3 h afforded the desired condensation product 18 in
83% yield without appreciable epimerization at the C20 stereogenic
centre (burkholdacs numbering). The condensation product 18 was
then converted to the requisite seco-acid 10 with an overall yield of
74% using alcohol 19 via the successive removal of both the PMB-
and allyl-protecting groups. Notably, the sensitive methyl sulfide
function present in the C20 side chain remained intact during the
Scheme 1. Synthetic plan for burkholdacs A (1) and B (2). TBS ¼ tert-butyldime-
thylsilyl, Tr (trityl) ¼ triphenylmethyl, PMB ¼ 4-methoxybenzyl.

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
303
determine the order of their potency and reveal some novel aspects
of structureeactivity relationships (SARs) within this class of
compounds.
2.2.1. HDAC inhibition assay
HDAC enzymes are zinc metalloenzymes that catalyze the hy-
drolysis of acetylated lysine residues on proteins, particularly his-
tones [12]. There are 18 human HDAC isoforms, which are grouped
into the following four 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 Zn2þ-dependent metallohydrolases, while
class III HDACs (7 members) are NADþ-dependent sirtuins [13]. The
inhibition of class I HDACs is considered to be a useful mechanism
for anticancer agents, whereas the inhibition of class IIb HDACs may
cause undesirable side effects such as serious cardiac hypertrophy
[14]. In 2009, FK228 has been approved by the Food and Drug
Administration in USA for treatment of cutaneous T-cell lymphoma
[15]. However, this anticancer agent is associated with an unre-
solved cardiotoxicity that probably arises from insufficient class
selectivity [15,16]. Therefore, the potent and selective inhibition of
class I enzymes is highly desirable in next-generation cancer
chemotherapy [17].
Scheme 2. Synthesis of segment 14. (a) PyBOP, i-Pr2NEt, MeCN, rt, 99%; (b) LiOH,
MeOH, rt, 100%. PyBOP
¼
(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate.
Compounds 1e3 were tested for their HDAC inhibitory activity
against HDAC1 (class I) and HDAC6 (class IIb) enzymes to determine
their degree of potency and isoform selectivity. In this assay, FK228
was used as a positive control. As summarized in Table 1, 1 and 2
exhibited extremely potent inhibitory activity against HDAC1 in the
low nanomolar range (IC50 ¼ 1.0e1.2 nM). Their potencies were
observed to be approximately three-fold higher than that of FK228
(IC50 ¼ 3.6 nM). In contrast, the unnatural stereoisomer 3 exhibited
much lower inhibitory activity (IC50 ¼ 336.6 nM) than the natural
products (1: IC50 ¼ 1.0 nM, 2: IC50 ¼ 1.2 nM), suggesting that the
stereochemistry at the C5, C6 and C20 positions plays a key role in
determining the pronounced HDAC1 inhibition efficacy. Combined
with our previous data for spiruchostatins A (6), B (7), C (8) and D
(9) [4,7], the potency order was estimated to be
9
(IC50 ¼ 0.75 nM) ꢂ 8 (IC50 ¼ 0.93 nM) z 1 (IC50 ¼ 1.0 nM) ꢂ 2
(IC50 ¼ 1.2 nM) > 7 (IC50 ¼ 2.2 nM) > 6 (IC50 ¼ 3.3 nM) ꢂ FK228 (5)
(IC50 ¼ 3.6 nM). As expected for HDAC6 inhibitory activity, both 1
and
2
were essentially inactive (1: IC50
¼
1975 nM, 2:
IC50 ¼ 992 nM). Isoform selectivity (class I/IIb) is expressed, for
convenience, as a selective index (SI) value (HDAC6 IC50/HDAC1
IC50). Along with our previous data [4,7], the order of the selectivity
was then determined to be 1 (SI ¼ 1645) > 2 (SI ¼ 992) > 7
(SI ¼ 636) > 6 (SI ¼ 485) > 8 (SI ¼ 346) ꢂ 9 (SI ¼ 320) > FK228 (5)
(SI ¼ 108). Notably, the isoform selectivity of 1 and 2 is nine-to-
fifteen-fold higher than that of FK228, which represents, to the
best of our knowledge, the highest level of class I/II selectivity
among the naturally occurring bicyclic depsipeptides known to
date. In addition, a good correlation between enzymatic (HDAC1)
activity and cellular HDAC inhibition efficacy [expressed as the
EC10000 (effective concentration for 100 times increased induction)
value measured via the p21 promoter assay] was determined for all
of the compounds. From these results, it was revealed that struc-
tural alteration of the side chain at the C20 position is quite effec-
tive for improving the isoform selectivity without loss of the potent
HDAC inhibitory activity.
Scheme 3. Synthesis of burkholdac A (1). (a) HATU, HOAt, i-Pr2NEt, CH2Cl2, ꢀ30 ꢁC,
83%; (b) DDQ, CH2Cl2/H2O, rt, 87%; c) Pd(PPh3)4, morpholine, THF, rt, 85%; (d) MNBA,
DMAP, CH2Cl2, rt, 79%; (e) I2, CH2Cl2/MeOH, rt, 81%; (f) HF$pyridine, pyridine, rt, 89%.
HATU
¼
O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
hexa-
fluorophosphate, HOAt
¼
1-hydroxy-7-azabenzotriazole, DDQ 2,3-dichloro-5,6-
¼
dicyano-1,4-benzoquinone, MNBA ¼ 2-methyl-6-nitrobenzoic anhydride, DMAP ¼ 4-
dimethylaminopyridine.
carboxylic acid 16 (Scheme 6). The final target molecule 3 was
synthesized, as shown in Scheme 7, through condensation of the
two segments 35 and 37 in a manner similar to that described for
the synthesis of 1 (cf. Schemes 2 and 3).
2.2.2. Cell-growth inhibition assay
Next, the growtheinhibitory activity of compounds 1e3 was
evaluated at the Japanese Foundation for Cancer Research simul-
taneously with FK228 as a reference compound using a panel of 39
human cancer cell lines [18]. 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.
2.2. Biological evaluation
The synthesized compounds 1e3 were evaluated for their HDAC
inhibitory activity and their cell-growth inhibitory activity to

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Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
Scheme 4. Synthesis of burkholdac B (2). (a) HATU, HOAt, i-Pr2NEt, CH2Cl2, ꢀ30 ꢁC, 92%; (b) DDQ, CH2Cl2/H2O, rt, 82%; (c) Pd(PPh3)4, morpholine, THF, rt, 94%; (d) MNBA, DMAP,
CH2Cl2, rt, 81%; (e) I2, CH2Cl2/MeOH, rt, 79%; (f) HF$pyridine, pyridine, rt, 97%.
Dose-response curves were measured at five different concentra-
tions (from 10ꢀ10 to 10ꢀ6 M or from 10ꢀ8 to 10ꢀ4 M) for each
compound, and the concentration causing 50% cell growth inhibi-
tion (GI50) was compared with that of the control.
The GI50 values of the tested compounds 1e3 along with those
for FK228 are shown in Table 2. Compounds 1 and 2 exhibited
extremely potent growtheinhibitory activity against nearly all of
the 39 cell lines in the sub-nanomolar to nanomolar range. Notably,
the efficacy of 1 and 2 was superior to that of FK228 on brain cancer
(SF-539, 1: GI50 ¼ 0.72 nM, 2: GI50 ¼ 0.70 nM), colon cancer
(HCC2998, 1: GI50 ¼ 0.67 nM; KM-12, 1: GI50 ¼ 0.76 nM; HT-29, 1:
GI50 ¼ 0.65 nM, 2: GI50 ¼ 0.74 nM; HCT-116, 1: GI50 ¼ 0.51 nM, 2:
GI50 ¼ 0.51 nM), lung cancer (NCI-H23, 1: GI50 ¼ 0.64 nM; NCI-
H226, 1: GI50 ¼ 0.47 nM; NCI-H522, 1: GI50 ¼ 0.20 nM, 2:
GI50 ¼ 0.42 nM; NCI-H460, 1: GI50 ¼ 0.91 nM, 2: GI50 ¼ 0.53 nM;
A549, 1: GI50
¼
0.57 nM, 2: GI50
¼
0.85 nM; DMS114, 1:
GI50 ¼ 0.97 nM), melanoma cancer (LOX-IMVI, 1: GI50 ¼ 0.50 nM),
ovary cancer (OVCAR-8, 1: GI50 ¼ 0.68 nM, 2: GI50 ¼ 0.72 nM; SK-
OV-3, 1: GI50
¼
0.78 nM), stomach cancer (MKN1, 1:
GI50 ¼ 0.67 nM) and prostate cancer (DU-145, 1: GI50 ¼ 0.82 nM)
cells. In contrast, 3 exhibited fairly decreased activity against all of
the 39 cell lines (GI50 ¼ 230e15000 nM). The potency order of the
bicyclic depsipeptides, including 5e9 (based on our previous data
[4,7]), was estimated using the MG-MID value (mean value of GI50
over all of the cell lines tested) to be 1 (2.1 nM) ꢂ 2 (3.2 nM) ꢂ 9
(3.8 nM) > 7 (5.6 nM) z FK228 (5) (6.2 nM) > 6 (15 nM) > 8
(28 nM). These results indicate that the antiproliferative activity of
1 and 2 is two-to-three-fold higher than that of FK228. Considering
both the HDAC inhibitory and antiproliferative activities, 1 and 2
seem to be promising candidates for the development of novel
anticancer agents with higher efficacy and lower toxicity.
Scheme 5. Synthesis of segment 35. (a) LDA, CH3CO2Et, THF, ꢀ78 ꢁC; at ꢀ78 ꢁC, add.
26, 57% for 27, 30% for 28 (27/28 2:1); (b) Jones reagent, acetone, 0 ꢁC to rt, 86%; (c)
KBH4, MeOH, ꢀ40 ꢁC, 79% for 28, 5% for 27 (28/27 16:1); (d) TBSCl, imidazole, DMF, rt,
99%; (e) 1 M NaOH, EtOH, rt; (f) allyl bromide, K2CO3, DMF, rt, 81% (2 steps); (g)
TMSOTf, 2,6-lutidine, CH2Cl2, rt; MeOH, rt; (h) 33, PyBOP, i-Pr2NEt, MeCN, rt, 68% (2
steps); (i) TMSOTf, 2,6-lutidine, CH2Cl2, rt; MeOH, rt; 98%. LDA ¼ lithium diisopropy-
lamide, TMSOTf ¼ trimethylsilyl trifluoromethanesulfonate.
3. Conclusion
The total synthesis of burkholdacs A (1) and B (2) and 5,6,20-tri-
epi-burkholdac A (3) was achieved in a highly convergent and

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
305
Table 1
HDAC inhibitory activity of burkholdacs A (4) and B (5) and 5,6,20-tri-epi-bur-
kholdac A (3).
a
e
Compound
IC50 (nM)
EC10000
(nM)
HDAC1
HDAC6
SId
Cell
HDAC
(class I)b
(class IIb)b
1
2
3
1.2
1.0
336.6
3.6
1975
992
>10,000
390
1645
992
e
1.4
0.76
>200
3.8
Scheme 6. Synthesis of segment 37. (a) PyBOP, i-Pr2NEt, CH2Cl2, 0 ꢁC to rt, 81%; (b) 1 M
LiOH, MeOH, rt, 98%.
FK228c
108
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 [4].
Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity towards class I
b
c
d
HDAC1 over class IIb HDAC6.
e
The concentration that induces luciferase activity 100-fold higher than basal
level.
Table 2
Growth inhibition of burkholdacs A (1) and B (2) and 5,6,20-tri-epi-burukholdac A
(3) against a panel of 39 human cancer cell lines.
FK228b
a
Origin of cancer
Cell line
GI50 (nM)
1
2
3
Breast
HBC-4
BSY-1
HBC-5
MCF-7
3.0
4.0
7.8
2.0
1.8
3.8
5.0
2.3
2.8
2.6
1300
1600
1900
1800
1800
6.9
8.5
13
4.2
5.5
MDA-MB-231
Central nervous system (Brain)
U-251
2.2
1.8
3.0
0.72
3.6
4.4
4.1
2.3
0.70
1.4
11
2.6
3.6
410
960
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
SF-268
SF-295
SF-539
SNB-75
SNB-78
HCC2998
KM-12
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
1600
1800
1000
1700
1700
1600
1400
460c
0.67
0.76
0.65
300
0.51
0.64
0.47
0.20d
0.91
0.57
3.0
Colon
Lung
0.74
Scheme 7. Synthesis of 5,6,20-tri-epi-burkholdac
A (3) (Brady’s proposed stereo-
100c
15,000c
540
1600
1700
360
structure for 1). (a) HATU, HOAt, i-Pr2NEt, CH2Cl2, ꢀ30 ꢁC, 81%; (b) DDQ, CH2Cl2/H2O, rt,
83%; (c) Pd(PPh3)4, morpholine, THF, rt, 74%; (d) MNBA, DMAP, CH2Cl2, rt, 84%; (e) I2,
CH2Cl2/MeOH, rt, 85%; (f) HF$pyridine, pyridine, rt, 89%.
0.51
3.3
2.7
0.42d
0.53
0.85
2.7
1800
1400
2300
1300
230d
1800
1800
1300
1900
1700
4000
4400
3400
1400
2100
1900
3700
1500
1500
1600
1600
unified manner. Using the results of a preliminary biological eval-
uation of the synthesized compounds 1e3 and our previously ob-
tained data for FK228 (5) and the spiruchostatins AeD (6e9), the
order of the efficacy of the naturally occurring bicyclic depsipeptide
HDAC inhibitors was established. Notably, the burkholdacs (1 and
2) were determined to be superior to 5 with respect to their HDAC1
inhibitory activity, isoform selectivity towards HDAC1 over HDAC6
and antiproliferative activity. These results should be useful for the
design and development of anticancer agents with therapeutic
potential that target the isoform-selective inhibition of HDACs. We
are currently synthesizing additional analogues of burkholdacs
with the aim of exploring their SARs. In addition, further investi-
gation of the activity of the synthetic samples using animal models
is in progress.
0.97
0.50
2.2
5.8
1.0
0.68
0.78
1.3
3.4
8.1
0.67
4.1
8.5
7.6
1.8
0.82
4.4
2.1
3.2
2.5
2.8
5.0
1.6
0.72
1.9
2.3
6.6
1.1
2.5
10
16
14
Melanoma ovary
2.8
5.5
3.3
6.6
20
Kidney
Stomach
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU-145
PC-3
22
3.2
4.9
17
14
2.9
3.0
6.0
18
Prostate
3.1
8.3
3.2
4. Experimental
MG-MIDe
6.2
a
Concentration that induces 50% inhibition of cell growth compared to the
4.1. Chemistry
control.
b
c
Positive control used in this study was synthesized in our laboratory [4].
The least sensitive cell.
The most sensitive cell.
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
d
e
Mean value of GI50 over all cell lines tested.

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Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
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 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.
129.7 (2C), 130.0, 133.1, 144.8 (3C), 159.2, 171.7, 175.2; IR (neat):
3358, 3085, 2916, 1729, 1644, 1613, 1514, 1443, 1248, 1175, 1077,
1034, 970, 748, 701 cmꢀ1
; HRMS (FAB): m/z calcd for
C
39H43NO5S2Na (Mþ þ Na) 692.2480, found 692.2454.
4.1.3. (3S,4R)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)hept-4-enoylamino]-4-
(methylthio)butanoylamino]-3-tritylthio(propionylamino)}-5-
methylhexanoate (18)
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-
i-Pr2NEt (71 mL, 0.42 mmol) was added dropwise to a stirred
solution of 12 (106 mg, 0.16 mmol) and 14 (107 mg, 0.16 mmol) in
CH2Cl2 (3.2 mL) containing O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-
tetramethyluronium hexafluorophosphate (HATU) (79.1 mg,
0.21 mmol) and 1-hydroxy-7-azabenzotriazole (HOAt) (28.3 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 18
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 SYS-
TEM high resolution mass spectrometer.
4.1.1. (R)-Methyl 2-[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)
hept-4-enamide]-4- (methylthio)butanoate (17)
(175 mg, 83%) as a colorless viscous liquid. [
a
]25
D
þ10.8 (c 1.02,
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.01 (3H, s), 0.05 (3H, s), 0.79e
i-Pr2NEt (0.32 mL, 1.9 mmol) was added dropwise to a stirred
solution of 16 (146 mg, 0.27 mmol) and D-methionine methyl ester
0.84 (6H, m), 0.82 (9H, s), 1.66e1.73 (1H, m), 1.88e1.98 (2H, m), 1.97
(3H, s), 2.05e2.23 (4H, m), 2.30e2.45 (5H, m), 2.47e2.55 (2H, m),
2.73 (1H, dd, J ¼ 8.3, 13.2 Hz), 3.74e3.80 (1H, m), 3.79 (3H, s), 3.87
(1H, dd, J ¼ 5.4, 7.8 Hz), 4.01 (1H, dt, J ¼ 3.4, 8.1 Hz), 4.09e4.17 (1H,
m), 4.15 (1H, d, J ¼ 10.2 Hz), 4.35e4.42 (1H, m), 4.37 (1H, d,
J ¼ 10.7 Hz), 4.45e4.55 (2H, m), 5.18e5.31 (3H, m), 5.45 (1H, dt,
J ¼ 6.8, 15.4 Hz), 5.82e5.92 (1H, m), 5.99 (1H, d, J ¼ 10.2 Hz), 6.63
(1H, d, J ¼ 7.3 Hz), 6.83 (2H, d, J ¼ 8.8 Hz), 7.05 (1H, d, J ¼ 7.3 Hz),
(15) (108 mg, 0.54 mmol) in MeCN (15 mL) containing (benzo-
triazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) (281 mg, 0.54 mmol) at 0 ꢁC under argon, and stirring was
continued for 2 h at room temperature. The reaction mixture was
diluted with EtOAc (120 mL), and the organic layer was washed
successively with 10% aqueous HCl (2 ꢃ 40 mL), saturated aqueous
NaHCO3 (2 ꢃ 40 mL) and brine (2 ꢃ 40 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc 2:1) to give 17
7.13e7.44 (32H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.7, e4.6, 15.1,
16.8, 17.9, 20.4, 25.8 (3C), 27.9, 30.1, 30.6, 31.3, 31.4, 33.0, 39.6, 42.5,
52.1, 52.6, 55.3, 58.3, 65.3, 66.6, 67.1, 69.5, 70.1, 76.5,114.0 (3C),118.4
(3C), 126.6 (6C), 126.8 (2C), 127.9 (6C), 128.1 (6C), 128.7, 129.5 (6C),
129.7, 129.8 (2C), 130.0, 132.1, 133.1, 144.3 (3C), 144.8 (3C), 159.4,
169.7, 171.21, 171.22, 171.6; IR (neat): 3435, 2929, 2855, 1683, 1638,
1556, 1541, 1515, 1248, 1171, 1082, 1035, 828, 744, 701 cmꢀ1; HRMS
(FAB): m/z calcd for C77H94N3O8S3Si (Mþ þ H) 1312.5972, found
1312.5957.
(183 mg, 99%) as a colorless oil. [
NMR (400 MHz, CDCl3):
a
]25
D
ꢀ15.2 (c 1.00, CHCl3); 1H
d
1.82e1.91 (1H, m),1.99e2.17 (3H, m), 2.02
(3H, s), 2.21e2.25 (2H, m), 2.36e2.43 (3H, m), 2.51 (1H, dd, J ¼ 8.8,
15.1 Hz), 3.73 (3H, s), 3.79 (3H, s), 4.10 (1H, dt, J ¼ 2.9, 8.3 Hz), 4.31
(1H, d, J ¼ 11.2 Hz), 4.51 (1H, d, J ¼ 10.7 Hz), 4.65e4.70 (1H, m), 5.32
(1H, dd, J ¼ 7.8, 15.6 Hz), 5.59 (1H, dt, J ¼ 6.8, 15.1 Hz), 6.83 (2H, d,
J ¼ 8.3 Hz), 7.01 (1H, d, J ¼ 8.3 Hz), 7.19e7.45 (17H, m); 13C NMR
(100 MHz, CDCl3):
d
15.4, 29.9, 31.2, 31.4, 31.8, 42.7, 51.3, 52.3, 55.3,
4.1.4. (3S,4R)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]-4-(methylthio)
butanoylamino]-3-tritylthio(propionylamino)}-5-methylhexanoate
(19)
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (64 mg,
0.23 mmol) was added in small portions to a stirred solution of 18
(146 mg, 0.11 mmol) in CH2Cl2/H2O 9:1 (11 mL) at room tempera-
ture. After 3 h, the 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. Con-
centration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (hexane/EtOAc, 1:1) to give 19
66.6, 70.0, 76.7, 113.8 (3C), 126.6 (2C), 127.9 (6C), 129.6 (6C), 129.7
(2C), 129.9, 130.3, 133.0, 144.9 (3C), 159.3, 170.6, 172.4; IR (neat):
3438, 1734, 1647, 1514, 1443, 1363, 1300, 1248, 1174, 1077, 1032,
972 cmꢀ1; HRMS (EI): m/z calcd for C40H45NO5S2 (Mþ) 683.2739,
found 683.2741.
4.1.2. (R)-2-[(S,E)-3-(4-Methoxybenzyloxy)-7-(tritylthio)hept-4-
enamide]-4-(methylthio)butanoic acid (14)
1 M LiOH (0.64 mL, 0.64 mmol) was added dropwise to a stirred
solution of 17 (110 mg, 0.16 mmol) in MeOH (3.2 mL) at room
temperature. After 3 h, 10% aqueous HCl was added to the mixture
at 0 ꢁC until the pH was 6. The resulting mixture was extracted with
EtOAc (3 ꢃ 20 mL), and the combined extracts were washed with
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 (CHCl3/
MeOH 10:1) to give 14 (107 mg, 100%) as a white amorphous solid.
(114 mg, 87%) as a colorless viscous liquid. [
a
]25
D
þ6.4 (c 1.00,
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.00 (3H, s), 0.05 (3H, s), 0.79e
0.84 (6H, m), 0.81 (9H, s),1.86e1.97 (2H, m), 2.02e2.10 (3H, m), 2.06
(3H, s), 2.17e2.23 (3H, m), 2.30e2.33 (1H, m), 2.40 (1H, dd, J ¼ 7.3,
15.6 Hz), 2.51e2.55 (3H, m), 2.63 (1H, dd, J ¼ 8.3, 12.9 Hz), 3.30 (1H,
br s), 3.89e3.97 (1H, m), 3.92e3.97 (1H, m), 4.14e4.18 (1H, m),
4.33e4.37 (1H, m), 4.42e4.54 (3H, m), 5.19e5.31 (2H, m), 5.36 (1H,
dd, J ¼ 6.3,15.4 Hz), 5.49 (1H, dt, J ¼ 6.8,15.1 Hz), 5.81e5.91 (2H, m),
6.49 (1H, d, J ¼ 7.8 Hz), 7.14e7.45 (32H, m); 13C NMR (100 MHz,
[
a
]25
D
ꢀ3.2 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d 1.84e1.91
(1H, m), 1.99 (3H, s), 2.10e2.24 (5H, m), 2.40e2.52 (4H, m), 3.76
(3H, s), 4.07e4.12 (1H, m), 4.26 (1H, d, J ¼ 10.7 Hz), 4.41 (1H, d,
J ¼ 10.3 Hz), 4.57e4.61 (1H, m), 5.29 (1H, dd, J ¼ 7.3, 15.4 Hz), 5.57
CDCl3):
d
ꢀ4.8, ꢀ4.7, 15.4, 16.9, 17.9, 20.2, 25.7 (3C), 28.0, 30.3, 30.4,
(1H, dt, J ¼ 6.8, 15.1 Hz), 6.80e6.84 (3H, m), 7.15e7.4 (17H, m); 13
C
31.25, 31.33, 33.2, 39.4, 44.1, 52.6, 53.1, 58.5, 65.3, 66.6, 67.0, 69.3,
69.8, 118.4, 126.6 (3C), 126.8 (3C), 127.8 (6C), 128.0 (6C), 129.48 (6C),
129.53 (6C), 129.9, 132.0, 132.5, 144.3 (3C), 144.8 (3C), 170.0, 170.9,
NMR (100 MHz, CDCl3): d 15.2, 29.8, 31.2, 31.3, 42.3, 51.8, 55.2, 66.5,
66.9, 70.0, 76.4, 113.8 (3C), 126.5 (2C), 127.8 (6C), 128.5, 129.6 (6C),

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
307
171.5, 171.6; IR (neat): 3438, 2958, 2930, 2854, 1736, 1635, 1550,
4.1.7. (1S,5S,6R,9S,20R,E)-5-(Tert-butyldimethylsilyloxy)-6-
isopropyl-20-[2-(methylthio)ethyl]-2-oxa-11,12-dithia-7,19,22-
triazabicyclo[7.7.6]docos-15-ene-3,8,18,21-tetraone (21)
1489, 1443, 1252, 1181, 1036, 828, 741, 701 cmꢀ1; HRMS (FAB): m/z
calcd for C69H86N3O7S3Si (Mþ þ H) 1192.5397, found 1192.5396.
A solution of 20 (89 mg, 78 mmol) in CH2Cl2/MeOH 9:1 (20 mL)
was added dropwise to a vigorously stirred solution of I2 (198 mg,
0.78 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 (10 mL,
adjusted to pH 4.0) at room temperature, and the resulting mixture
was extracted with CHCl3 (3 ꢃ 50 mL). The combined extracts were
washed with brine (2 ꢃ 50 mL), then dried over Na2SO4. Concen-
tration of the solvent in vacuo afforded a residue, which was pu-
rified by column chromatography (CHCl3/MeOH 30:1) to give 21
4.1.5. (3S,4R)-3-(Tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-[(S,E)-
3-hydroxy-7-(tritylthio)hept-4-enoylamino]-4-(methylthio)
butanoylamino]-3-tritylthio(propionylamino)}-5-methylhexanoic
acid (10)
Morpholine (18.8 L, 0.22 mmol) was added dropwise to a stirred
solution of 19 (117 mg, 97
mmol) in THF (10 mL) containing
Pd(PPh3)4 (13.0 mg, 11 mol) at room temperature under argon.
m
After 30 min, the reaction mixture was diluted with EtOAc (30 mL).
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 10 (98.9 mg, 85%) as a
(40.8 mg, 81%) as a white amorphous solid. [
a
]25
D
þ27.4 (c 0.81,
CHCl3); 1H NMR (400 MHz, CD3OD):
d
ꢀ0.07 (3H, s), 0.00 (3H, s),
0.72 (3H, d, J ¼ 6.8 Hz), 0.74 (9H, s), 0.79 (3H, d, J ¼ 6.8 Hz), 1.87e
1.95 (1H, m), 1.92 (3H, s), 2.00e2.06 (1H, m), 2.19e2.25 (1H, m),
2.37e2.61 (6H, m), 2.67e2.75 (2H, m), 2.79e2.83 (2H, m), 3.01e
3.24 (3H, m), 4.06e4.09 (1H, m), 4.59e4.62 (2H, m), 5.46e5.47 (1H,
m), 5.73e5.85 (2H, m), 6.84 (1H, d, J ¼ 8.8 Hz), 7.47 (1H, d,
white amorphous solid. [
a
]25
D
þ2.2 (c 1.00, CHCl3); 1H NMR
(400 MHz, CDCl3):
d
0.05 (6H, s), 0.80e0.87 (15H, m), 1.91e2.12 (5H,
m), 2.04 (3H, s), 2.19e2.25 (3H, m), 2.33 (1H, dd, J ¼ 2.9, 13.4 Hz),
2.40 (1H, dd, J ¼ 4.4, 15.9 Hz), 2.49e2.53 (4H, m), 2.72 (1H, dd,
J ¼ 8.3, 12.4 Hz), 3.75e3.84 (1H, m), 4.07e4.13 (2H, m), 4.33e4.38
(1H, m), 4.49e4.54 (1H, m), 5.34 (1H, dd, J ¼ 5.9, 15.6 Hz), 5.47 (1H,
dt, J ¼ 6.8, 15.1 Hz), 6.32 (1H, d, J ¼ 10.3 Hz), 6.81 (1H, d, J ¼ 7.3 Hz),
J ¼ 7.3 Hz), 8.38 (1H, br s); 13C NMR (100 MHz, CD3OD):
ꢀ4.7, ꢀ4.0,
d
15.1, 18.1, 18.9, 21.6, 26.3 (3C), 30.5, 31.0, 31.3, 33.4, 41.4, 41.6, 42.1,
56.7, 57.3, 63.9, 69.1, 71.4, 79.5,130.1,132.4,170.5, 170.9, 173.9,174.3;
IR (neat): 3342, 2957, 2928, 2856, 1744,1662, 1542,1432, 1257,1158,
1137, 1080, 833, 776, 755 cmꢀ1; HRMS (FAB): m/z calcd for
7.15e7.44 (31H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.5, 15.4,
C
28H50N3O6S3Si (Mþ þ H) 648.2631, found 648.2629.
16.4, 17.9, 20.3, 25.7 (3C), 27.7, 29.7, 30.2, 31.28, 31.32, 33.2, 39.7,
44.2, 53.30, 53.34, 59.3, 66.2, 67.0, 69.0, 69.8, 126.6 (3C), 126.9 (3C),
127.9 (6C), 128.1 (6C), 129.5 (6C), 129.6 (6C), 130.1, 132.3, 144.3 (3C),
144.8 (3C), 170.4, 171.5, 172.0, 173.9; IR (neat): 3435, 2955, 1646,
1557, 1541, 1507, 1437, 1396, 1255, 1184, 1088, 1011, 953, 742,
702 cmꢀ1; HRMS (FAB): m/z calcd for C66H82N3O7S3Si (Mþ þ H)
1152.5084, found 1152.5062.
4.1.8. Burkholdac A (1)
HF$pyridine (2.5 mL) was added to a stirred solution of 21
(32.0 mg, 49 mmol) in pyridine (5 mL) at room temperature. After
14 h, the reaction mixture was diluted with EtOAc (60 mL), and the
organic layer was washed successively with 3% aqueous HCl
(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 1 (23.4 mg, 89%) as a white
4.1.6. (2S,6R,9S,12R,13S)-13-(Tert-butyldimethylsilyloxy)-12-
isopropyl-6-[2-(methylthio)ethyl]-2-[(E)-4-(tritylthio)but-1-enyl]-
9-tritylthiomethyl-1-oxa-5,8,11-triazacyclopentadecane-4,7,10,15-
tetraone (20)
amorphous solid. [
a
]25
D
ꢀ57.3 (c 0.16, MeOH), {lit [3]. [
a
]20
D
ꢀ54 (c
0.23 in MeOH)}. 1H NMR (400 MHz, CD3CN):
d
0.78 (3H, d,
J ¼ 6.3 Hz), 0.88 (3H, d, J ¼ 6.8 Hz), 1.92e1.99 (1H, m), 2.02 (3H, s),
2.13e2.21 (1H, m), 2.36e2.43 (1H, m), 2.48e2.75 (9H, m), 2.88 (1H,
dd, J ¼ 7.3, 13.2 Hz), 3.02e3.05 (1H, m), 3.14e3.24 (3H, m), 4.04e
4.09 (1H, m), 4.33e4.39 (1H, m), 4.62 (1H, dt, J ¼ 3.9, 9.3 Hz), 5.43e
5.45 (1H, m), 5.80 (1H, d, J ¼ 12.2 Hz), 6.01e6.07 (1H, m), 6.78 (1H,
d, J ¼ 8.8 Hz), 7.17 (1H, br s), 7.29 (1H, d, J ¼ 6.8 Hz); 13C NMR
A solution of 10 (119 mg, 0.10 mmol) in CH2Cl2 (10 mL) was
added very slowly to a stirred solution of 2-methyl-6-nitrobenzoic
anhydride (MNBA) (46.3 mg, 0.14 mmol) in CH2Cl2 (100 mL, 1.0 mM
concentration) containing 4-dimethylaminopyridine (DMAP)
(38 mg, 0.31 mmol) at room temperature over 14 h. After 1 h, the
mixture was diluted with CH2Cl2 (100 mL), and the organic layer
was washed successively with saturated aqueous NaHCO3
(2 ꢃ 40 mL), water (2 ꢃ 40 mL) and brine (2 ꢃ 40 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 (89.0 mg, 79%) as a white amorphous solid.
(100 MHz, CD3CN): d 15.1, 19.7, 21.1, 30.4, 30.6, 31.2, 33.3, 40.8, 40.9
(2C), 41.7, 56.3, 56.8, 63.2, 69.1, 71.6, 131.4, 132.4, 169.9, 171.7, 171.8,
172.8; IR (neat): 3361, 3335, 2960, 2921, 2849, 1733, 1717, 1683,
1654, 1541, 1523, 1508, 1263, 1162, 1020, 983, 754 cmꢀ1; HRMS
(FAB): m/z calcd for C22H36N3O6S3 (Mþ þ H) 534.1766, found
534.1769. The 1H and 13C NMR, and MS spectrum are identical with
those reported for natural burkholdac A [1].
[
a]25
D
ꢀ7.7 (c 1.01, CHCl3); 1H NMR (400 MHz, CDCl3):
d 0.00 (3H,
s), 0.08 (3H, s), 0.81e0.84 (6H, m), 0.87 (9H, s), 2.02e2.26 (7H, m),
2.06 (3H, s), 2.35e2.52 (4H, m), 2.58e2.70 (2H, m), 2.75 (1H, dd,
J ¼ 4.4, 12.7 Hz), 3.26e3.31 (1H, m), 3.40e3.46 (1H, m), 3.66e3.70
(1H, m), 4.08e4.12 (1H, m), 4.34e4.37 (1H, m), 5.40 (1H, dd, J ¼ 6.8,
15.1 Hz), 5.56e5.60 (1H, m), 5.70 (1H, dt, J ¼ 6.8, 15.1 Hz), 6.45 (1H,
br s), 6.88 (1H, d, J ¼ 5.9 Hz), 7.11 (1H, d, J ¼ 10.3 Hz), 7.18e7.41 (30H,
4.1.9. (3S,4R,5R)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-(4- methoxybenzyloxy)-7-(tritylthio)hept-4-enoylamino]-
4-(methylthio)butanoylamino]-3- tritylthio(propionylamino)}-5-
methylheptanoate (22)
i-Pr2NEt (0.17 L, 1.0 mmol) was added dropwise to a stirred so-
lution of 13 (260 mg, 0.39 mmol) and 14 (258 mg, 0.39 mmol) in
CH2Cl2 (7.7 mL) containing HATU (190 mg, 0.50 mmol) and HOAt
(68.1 mg, 0.50 mmol) at ꢀ30 ꢁC under argon. After 3 h, the reaction
mixture was diluted with CHCl3 (40 mL). The organic layer was
washed successively with 10% aqueous HCl (2 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 chromatography (hexane/EtOAc 1:1)
m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.1, 15.1, 15.5, 17.9, 20.5,
25.7 (3C), 27.3, 29.3, 30.3, 31.0, 31.3, 31.7, 42.0, 42.1, 52.9, 58.0, 58.9,
66.6, 66.9, 68.8, 71.5, 126.6 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C),
128.6, 129.49 (6C), 129.52 (6C), 133.3, 144.4 (3C), 144.8 (3C), 169.8,
170.16, 170.18, 171.8; IR (neat): 3430, 2955, 2928, 2857, 1733, 1715,
1648, 1575, 1540, 1507, 1490, 1445, 1260, 1082, 832, 771, 747,
699 cmꢀ1; HRMS (FAB): m/z: calcd for C66H80N3O6S3Si (Mþ þ H)
1134.4979, found 1134.4996.

308
Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
to give 22 (471 mg, 92%) as a colorless viscous liquid. [
a
]25
D
þ12.8
1.75e1.81 (1H, m), 1.89e1.96 (1H, m), 2.01e2.10 (3H, m), 2.01 (3H,
s), 2.18e2.34 (4H, m), 2.39 (1H, dd, J ¼ 3.9, 16.1 Hz), 2.46e2.56 (4H,
m), 2.70 (1H, dd, J ¼ 8.3, 11.9 Hz), 3.89e3.94 (1H, m), 4.07e4.11 (2H,
m), 4.32e4.37 (1H, m), 4.52 (1H, dd, J ¼ 7.8, 12.7 Hz), 5.34 (1H, dd,
J ¼ 5.9, 15.6 Hz), 5.47 (1H, dt, J ¼ 6.8, 15.1 Hz), 6.41 (1H, d,
J ¼ 10.2 Hz), 6.88 (1H, d, J ¼ 7.3 Hz), 7.14e7.42 (31H, m); 13C NMR
(c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.00 (3H, s), 0.06 (3H,
s), 0.78e0.85 (6H, m), 0.83 (9H, s), 1.01e1.12 (1H, m), 1.15e1.24 (1H,
m), 1.65e1.79 (2H, m), 1.88e1.97 (1H, m), 1.97 (3H, s), 2.04e2.15
(2H, m), 2.17e2.23 (2H, m), 2.30e2.47 (5H, m), 2.50e2.55 (2H, m),
2.73 (1H, dd, J ¼ 8.3, 13.2 Hz), 3.78 (3H, s), 3.84e3.94 (2H, m), 4.00
(1H, dt, J ¼ 3.4, 8.3 Hz), 4.10e4.17 (1H, m), 4.14 (1H, d, J ¼ 10.7 Hz),
4.35e4.42 (1H, m), 4.37 (1H, d, J ¼ 11.2 Hz), 4.43e4.54 (2H, m),
5.17e5.30 (3H, m), 5.44 (1H, dt, J ¼ 6.8, 15.1 Hz), 5.81e5.91 (1H, m),
5.99 (1H, d, J ¼ 10.2 Hz), 6.61 (1H, d, J ¼ 7.8 Hz), 6.83 (2H, d,
J ¼ 8.3 Hz), 7.04 (1H, d, J ¼ 7.3 Hz), 7.13e7.44 (32H, m); 13C NMR
(100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.4, 11.8, 13.5, 15.3, 17.8, 25.7 (3C), 27.1,
30.2, 30.4, 31.2, 31.3, 33.0, 34.1, 40.1, 44.0, 53.1, 53.4, 56.9, 66.5, 66.9,
68.8, 69.7, 126.6 (3C), 126.8 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C),
129.5 (6C), 129.9, 132.3, 144.2 (3C), 144.8 (3C), 170.3, 171.6, 171.9,
174.3; IR (neat): 3285, 3059, 3018, 2957, 2927, 2856, 1714, 1642,
1539,1489,1471,1443,1252,1085, 836, 751, 700 cmꢀ1; HRMS (FAB):
m/z calcd for C67H83N3O7S3SiNa (Mþ þ Na) 1188.5060, found
1188.5057.
(100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.5, 11.7, 13.6, 15.0, 17.9, 20.4, 25.7 (3C),
27.2, 30.0, 30.6, 31.2, 31.3, 32.9, 34.0, 40.0, 42.5, 51.9, 52.5, 55.2, 55.9,
65.2, 67.0, 69.4, 70.0, 76.4, 113.9 (2C), 118.3, 126.6 (3C), 126.7 (3C),
127.8 (6C), 128.0 (6C), 129.47 (6C), 129.49 (6C), 129.6, 129.8 (2C),
130.0, 132.0, 133.0, 144.2 (3C), 144.8 (3C), 159.3, 169.6, 171.1, 171.2,
171.5; IR (neat): 3287, 3059, 2957, 2928, 2856, 1733, 1639, 1539,
1514, 1443, 1248, 1172, 1084, 831, 744, 700 cmꢀ1; HRMS (FAB): m/z
calcd for C78H96N3O8S3Si (Mþ þ H) 1326.6129, found 1326.6110.
4.1.12. (2S,6R,9S,12R,13S)-12-[(S)-Isobutyl]-13-(tert-
butyldimethylsilyloxy)-6-[2-(methylthio)ethyl]-2-[(E)-4-(tritylthio)
but-1-en-1-yl]-9-tritylthiomethyl-1-oxa-5,8,11-
triazacyclopentadecane-4,7,10,15-tetraone (24)
A solution of 11 (320 mg, 0.27 mmol) in CH2Cl2 (27 mL) was
added very slowly to a stirred solution of MNBA (123 mg,
0.36 mmol) in CH2Cl2 (270 mL, 1.0 mM concentration) containing
DMAP (101 mg, 0.82 mmol) at room temperature over 14 h. After
1 h, the mixture was diluted with CH2Cl2 (200 mL), and the organic
layer was washed successively with saturated aqueous NaHCO3
(2 ꢃ 80 mL), water (2 ꢃ 80 mL) and brine (2 ꢃ 80 mL), then dried
over Na2SO4. Concentration of the solvent in vacuo afforded a
residue, which was purified by column chromatography (hexane/
4.1.10. (3S,4R,5R)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-
2-[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]-4-
(methylthio)butanoylamino]-3-tritylthio(propionylamino)}-5-
methylheptanoate (23)
DDQ (161 mg, 0.71 mmol) was added in small portions to a
stirred solution of 22 (471 mg, 0.36 mmol) in CH2Cl2/H2O 9:1
(18 mL) at room temperature. After 3 h, the mixture was diluted
with CHCl3 (60 mL), and the organic layer was washed with satu-
rated 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 24 (256 mg, 81%) as a white amorphous solid. [a]
25D
ꢀ6.0 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d 0.00 (3H, s),
0.08 (3H, s), 0.83 (3H, d, J ¼ 6.8 Hz), 0.89e0.93 (3H, m), 0.89 (9H, s),
1.04e1.12 (1H, m), 1.18e1.27 (1H, m), 1.87e1.92 (1H, m), 2.00e2.20
(4H, m), 2.05 (3H, s), 2.22e2.30 (2H, m), 2.36e2.66 (4H, m), 2.59e
2.66 (2H, m), 2.72 (1H, dd, J ¼ 10.2, 18.3 Hz), 3.34e3.41 (2H, m), 3.82
(1H, dt, J ¼ 2.0, 9.8 Hz), 4.16 (1H, dt, J ¼ 4.4, 9.8 Hz), 4.34 (1H, dd,
J ¼ 7.3, 14.6 Hz), 5.41 (1H, dd, J ¼ 6.8, 15.6 Hz), 5.59e5.64 (1H, m),
5.71 (1H, dt, J ¼ 6.8, 15.1 Hz), 6.48 (1H, d, J ¼ 7.3 Hz), 7.00 (1H, d,
J ¼ 4.9 Hz), 7.16 (1H, d, J ¼ 10.3 Hz), 7.25e7.49 (30H, m); 13C NMR
EtOAc, 1:1) to give 23 (353 mg, 82%) as a colorless viscous liquid. [a]
25D
þ12.3 (c 1.01, CHCl3); 1H NMR (400 MHz, CDCl3):
d 0.00 (3H, s),
0.06 (3H, s), 0.78e0.86 (6H, m), 0.83 (9H, s),1.05e1.12 (1H, m),1.17e
1.24 (1H, m), 1.72e1.78 (1H, m), 1.86e1.95 (1H, m), 2.01e2.09 (3H,
m), 2.04 (3H, s), 2.17e2.23 (3H, m), 2.32 (1H, dd, J ¼ 2.4, 13.7 Hz),
2.41 (1H, dd, J ¼ 7.3, 16.1 Hz), 2.49e2.63 (5H, m), 3.40 (1H, d,
J ¼ 2.9 Hz), 3.87e3.97 (2H, m), 4.14 (1H, dt, J ¼ 3.4, 7.2 Hz), 4.33e
4.37 (1H, m), 4.39e4.52 (3H, m), 5.18e5.30 (2H, m), 5.39 (1H, dd,
J ¼ 6.3, 15.4 Hz), 5.49 (1H, dt, J ¼ 6.8, 15.6 Hz), 5.79e5.88 (1H, m),
5.91 (1H, d, J ¼ 10.3 Hz), 6.52 (1H, d, J ¼ 7.8 Hz), 7.15e7.43 (31H, m);
(100 MHz, CDCl3):
d
ꢀ4.9, ꢀ4.0, 12.0, 12.9, 15.1, 17.8, 25.6 (3C), 27.3,
29.5, 30.2, 30.9, 31.3, 31.8, 34.0, 41.8, 42.2, 52.9, 57.0, 57.8, 66.6, 66.8,
68.6, 71.5, 126.6 (3C), 126.8 (3C), 127.8 (6C), 128.0 (6C), 128.1, 129.45
(6C), 129.48 (6C), 133.1, 144.4 (3C), 144.7 (3C), 169.8, 170.17, 170.24,
171.9; IR (neat): 3286, 3057, 2956, 2928, 2855, 1732, 1683, 1669,
1652,1541,1444,1255,1184,1084, 834, 742, 699 cmꢀ1; HRMS (FAB):
m/z calcd for C67H81N3O6S3SiNa (Mþ þ Na) 1170.4954, found
1170.4971.
13C NMR (100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.5, 11.6, 13.7, 15.3, 17.9, 25.7
(3C), 27.0, 30.3, 30.4, 31, 2, 31.3, 33.1, 34.1, 39.9, 44.0, 52.6, 53.0, 56.1,
65.2, 66.6, 66.9, 69.3, 69.8, 118.3, 126.6 (3C), 126.8 (3C), 127.8 (6C),
128.0 (6C), 129.45 (6C), 129.52 (6C), 129.9, 132.0, 132.5, 144.2 (3C),
144.8 (3C), 170.0, 170.9, 171.5, 171.6; IR (neat): 3277, 3059, 2956,
2927, 2855, 1736, 1636, 1543, 1490, 1443, 1387, 1251, 1084, 836, 743,
700 cmꢀ1; HRMS (FAB): m/z calcd for C70H88N3O7S3Si (Mþ þ H)
1206.5554, found 1206.5562.
4.1.13. (1S,5S,6R,9S,20R,E)-6-[(S)-Isobutyl]-5-(tert-
butyldimethylsilyloxy)-20-[2-(methylthio)ethyl]-2-oxa-11,12-
dithia-7,19,22-triazabicyclo[7.7.6]docos-15-ene-3,8,18,21-tetraone
(25)
4.1.11. (3S,4R,5R)-3-(Tert-butyldimethylsiloxy)-4-{(S)-2-[(R)-2-
[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]-4-(methylthio)
butanoylamino]-3-tritylthio(propionylamino)}-5-methylheptanoic
acid (11)
A solution of 24 (246 mg, 0.21 mmol) in CH2Cl2/MeOH 9:1
(50 mL) was added dropwise to a vigorously stirred solution of I2
(544 mg, 2.1 mmol) in CH2Cl2/MeOH 9:1 (420 mL, 0.5 mM con-
centration) over 10 min at room temperature. After 10 min, the
reaction was quenched with 0.2 M ascorbic acid/citric acid buffer
(20 mL, adjusted to pH 4.0) at room temperature, and the resulting
mixture was extracted with CHCl3 (3 ꢃ 50 mL). The combined ex-
tracts were washed with brine (2 ꢃ 50 mL), then dried over Na2SO4.
Concentration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (CHCl3/MeOH 30:1) to give 25
Morpholine (51
solution of 23 (353 mg, 0.29 mmol) in THF (15 mL) containing
Pd(PPh3)4 (33.8 mg, 29 mol) at room temperature under argon.
mL, 0.59 mmol) was added dropwise to a stirred
m
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. Con-
centration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (CHCl3/MeOH 20:1) to give 11
(112 mg, 79%) as a white amorphous solid. [
a
]25
D
þ13.1 (c 1.01,
(320 mg, 94%) as a white amorphous solid. [
CHCl3); 1H NMR (400 MHz, CDCl3):
0.04 (3H, s), 0.05 (3H, s), 0.77e
0.85 (6H, m), 0.85 (9H, s), 1.04e1.11 (1H, m), 1.16e1.23 (1H, m),
a
]25D
þ4.8 (c 1.13,
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.09 (3H, s), 0.16 (3H, s), 0.86e
d
0.96 (6H, m), 0.93 (9H, s), 1.20e1.27 (1H, m), 1.36e1.43 (1H, m), 1.88
(1H, ddd, J ¼ 2.4, 6.8, 14.0 Hz), 2.02e2.10 (1H, m), 2.19 (3H, s), 2.28e

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
309
2.36 (1H, m), 2.49e2.83 (8H, m), 2.96e3.14 (4H, m), 3.54e3.65 (1H,
Compound 28: colorless oil; [
NMR (400 MHz, CDCl3):
a
]25
D
þ9.0 (c 1.02, CHCl3); 1H
m), 4.41 (1H, dt, J ¼ 4.4, 8.8 Hz), 4.86 (1H, dt, J ¼ 3.4, 10.3 Hz), 4.98e
5.02 (1H, m), 5.67e5.71 (2H, m), 6.33e6.41 (1H, m), 6.76 (1H, d,
J ¼ 6.8 Hz), 7.13 (1H, d, J ¼ 6.8 Hz), 7.52 (1H, d, J ¼ 3.9 Hz); 13C NMR
d
0.88 (3H, d, J ¼ 7.3 Hz), 0.95 (3H, d,
J ¼ 6.8 Hz), 1.28 (3H, t, J ¼ 7.3 Hz), 1.44 (9H, s), 2.09e2.17 (1H, m),
2.46 (1H, dd, J ¼ 9.8, 17.1 Hz), 2.59 (1H, dd, J ¼ 2.0, 17.1 Hz), 3.30 (1H,
d, J ¼ 4.9 Hz), 3.50e3.56 (1H, m), 3.90e3.96 (1H, m), 4.18 (2H, dq,
J ¼ 6.8, 2.0 Hz), 4.43 (1H, br d, J ¼ 10.2 Hz); 13C NMR (100 MHz,
(100 MHz, CDCl3):
d
ꢀ5.0, ꢀ4.3, 11.9, 14.5, 15.5, 17.9, 25.7 (3C), 25.8,
27.7, 28.0, 31.3, 34.5, 36.2, 40.0, 40.4, 41.3, 53.7, 57.3, 61.0, 67.0, 68.9,
129.3, 132.4, 168.9, 169.0, 170.1, 171.0; IR (neat): 3341, 2957, 2929,
2857, 1745, 1662, 1543, 1432, 1257, 1158, 1138, 1080, 949, 837,
756 cmꢀ1; HRMS (FAB): m/z calcd for C29H52N3O6S3Si (Mþ þ H)
662.2788, found 662.2783.
CDCl3):
d 14.1, 16.2, 20.1, 27.5, 28.3 (3C), 38.4, 58.7, 60.8, 69.2, 79.5,
156.4, 173.2; IR (neat): 3450, 3369, 2965, 1716, 1698, 1524, 1391,
1367, 1308, 1251, 1173, 1069, 984, 869, 775 cmꢀ1; HRMS (EI): m/z
calcd for C14H27NO5 (Mþ) 289.1889, found 289.1891.
4.1.16. Conversion of compound 27 to compound 28
4.1.14. Burkholdac B (2)
2.6 M Jones reagent (5.2 mL, 14 mmol) was added dropwise to a
stirred solution of 27 (2.68 g, 9.2 mmol) in acetone (80 mL) at 0 ꢁC,
and stirring was continued for 1 h at room temperature. The
mixture was diluted with Et2O (200 mL). The organic layer was
washed with saturated aqueous NaHCO3 (2 ꢃ 50 mL) and brine
(2 ꢃ 50 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded a residue, which was purified by column chro-
matography (hexane/EtOAc 8:1 / 4:1) to give (S)-ethyl 4-(tert-
butoxycarbonylamino)-5-methyl-3-oxohexanoate (2.26 g, 86%) as a
colorless oil.
HF$pyridine (1.2 mL) was added to a stirred solution of 25
(77.3 mg, 0.12 mmol) in pyridine (2.3 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 2 (62.0 mg, 97%) as a white
amorphous solid. [
a]25
D
ꢀ57.8 (c 1.00, MeOH), {lit. [2]. [
a]32D
ꢀ62.6
(c 0.34, MeOH)}. 1H NMR (400 MHz, CD2Cl2):
d
0.88 (3H, d,
KBH4 (2.13 g, 40 mmol) was added in small portions to a stirred
solution of the above product (2.26 g, 7.9 mmol) in MeOH (80 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 (30 mL) was added, and the
resulting mixture was extracted with CH2Cl2 (4 ꢃ 30 mL). The
combined extracts were washed with 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 28 (1.80 g, 79%) along with 27 (112 mg,
5%). The IR, 1H and 13C NMR, mass spectra of these samples were
identical with those recorded for 27 and 28, respectively.
J ¼ 6.8 Hz), 0.91 (3H, t, J ¼ 7.3 Hz), 1.16e1.24 (1H, m), 1.48e1.54 (1H,
m), 1.99e2.09 (2H, m), 2.17 (3H, s), 2.20e2.29 (1H, m), 2.42e2.51
(1H, m), 2.61e2.68 (5H, m), 2.73e2.79 (2H, m), 2.88e2.93 (2H, m),
3.11 (1H, d, J ¼ 13.7 Hz), 3.20e3.28 (2H, m), 3.32e3.36 (1H, m), 4.29
(1H, dt, J ¼ 8.3, 4.4 Hz), 4.55e4.57 (1H, m), 4.83 (1H, dt, J ¼ 8.8,
3.4 Hz), 5.49e5.51 (1H, m), 5.72 (1H, d, J ¼ 15.6 Hz), 6.31 (1H, t,
J ¼ 11.7 Hz), 6.77 (1H, d, J ¼ 9.3 Hz), 7.25 (1H, d, J ¼ 7.3 Hz), 7.46 (1H,
d, J ¼ 2.9 Hz); 13C NMR (100 MHz, CD2Cl2):
d 11.7, 15.56, 15.58, 27.5,
28.6, 31.5, 33.7, 36.6, 39.9, 41.1, 41.4, 41.8, 55.0, 57.6, 61.8, 68.5, 71.1,
129.5, 133.4, 169.4, 170.5, 171.4, 172.1; IR (neat): 3376, 2957, 2924,
2855, 1736, 1685, 1656, 1543, 1524, 1439, 1273, 1162, 980, 753 cmꢀ1
;
HRMS (FAB): m/z calcd for C23H38N3O6S3 (Mþ þ H) 548.1923, found
548.1924. The 1H and 13C NMR, and MS spectrum are identical with
those reported for natural burkholdac B [1].
4.1.17. (3R,4S)-Ethyl 4-(tert-butoxycarbonylamino)-3-(tert-
butyldimethylsiloxy)-5-methylhexanoate (29)
tert-Butyldimethylsilyl chloride (TBSCl) (1.26 g, 8.4 mmol) was
added to stirred solution of 28 (807 mg, 2.8 mmol) in DMF (20 mL)
containing imidazole (1.14 g, 17 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-
matography (hexane/EtOAc 10:1 / 5:1) to give 29 (1.11 g, 99%) as a
4.1.15. (3S,4S)-Ethyl 4-(tert-butoxycarbonylamino)-3-hydroxy-5-
methylhexanoate (27) and its (3R,4S)-isomer (28)
A solution of EtOAc (6.0 mL, 62 mmol) in THF (8 mL) was added
slowly to a stirred solution of lithium diisopropylamide (LDA)
(62 mmol) [prepared from n-BuLi in hexane (1.6 M solution,
38.7 mL, 62 mmol) and i-Pr2NH (9.3 mL, 65 mmol)] in THF (30 mL)
at ꢀ78 ꢁC. After 30 min, (2S)-2-(tert-butoxycarbonylamino)-3-
methylbutylaldehyde (N-Boc-L-valinal) (26) [11] (3.66 g,
18 mmol) in THF (30 mL) was added to the above mixture at ꢀ78 ꢁC.
After 40 min, the reaction was quenched with 2 M HCl (20 mL)
at ꢀ78 ꢁC, and the resulting mixture was extracted with EtOAc
(2 ꢃ 100 mL). The combined extracts were washed with saturated
aqueous NaHCO3 (2 ꢃ 50 mL) and brine (2 ꢃ 50 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 27 (2.98 g, 57%, less polar) and 28 (1.56 g,
30%, more polar).
colorless oil. [
a]25
D
ꢀ5.0 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.04 (3H, s), 0.09 (3H, s), 0.87e0.88 (12H, m), 0.92 (3H, d,
J ¼ 6.8 Hz), 1.27 (3H, t, J ¼ 7.3 Hz), 1.43 (9H, s), 1.93e1.99 (1H, m),
2.43 (1H, dd, J ¼ 5.9, 15.1 Hz), 2.53 (1H, dd, J ¼ 6.2, 15.6 Hz), 3.47e
3.53 (1H, m), 4.07e4.22 (3H, m), 4.46 (1H, br d, J ¼ 10.7 Hz);
13CNMR (100 MHz, CDCl3):
d
ꢀ4.9, ꢀ4.7, 14.1, 16.8, 18.0, 20.6, 25.7
(3C), 27.9, 28.4 (3C), 40.0, 59.5, 60.5, 70.1, 78.9, 155.9, 171.9; IR
(neat): 3455, 3377, 2960, 2931, 1719, 1703, 1390, 1366, 1304, 1254,
1174, 1082, 837, 777 cmꢀ1; HRMS (EI): m/z calcd for C20H41NO5Si
(Mþ) 403.2754, found 403.2850.
Compound 27: colorless oil; [
NMR (400 MHz, CDCl3):
a
]25
D
ꢀ39.8 (c 1.03, CHCl3); 1H
d
0.96 (3H, d, J ¼ 6.8 Hz), 1.00 (3H, d,
J ¼ 6.8 Hz), 1.28 (3H, t, J ¼ 7.1 Hz), 1.44 (9H, s), 1.83e1.91 (1H, m),
2.45 (1H, dd, J ¼ 2.7,16.7 Hz), 2.55 (1H, dd, J ¼ 9.8,16.7 Hz), 3.15 (1H,
t, J ¼ 9.5 Hz), 3.29 (1H, d, J ¼ 2.9 Hz), 4.17 (2H, q, J ¼ 6.8 Hz), 4.24e
4.28 (1H, m), 4.85 (1H, d, J ¼ 9.3 Hz); 13C NMR (100 MHz, CDCl3):
4.1.18. (3R,4S)-Allyl 4-(tert-butoxycarbonylamino)-3-(tert-
butyldimethylsiloxy)-5-methylhexanoate (31)
1 M NaOH (14 mL, 14 mmol) was added dropwise to a stirred
solution of 29 (1.11 g, 2.8 mmol) in EtOH (30 mL) at room tem-
perature. After 6 h, the mixture was diluted with 10% aqueous
HCl (30 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
d
14.1, 19.5, 19.8, 28.4 (3C), 30.4, 39.1, 59.6, 60.8, 67.1, 79.1, 156.4,
173.7; IR (neat): 3393, 2977, 1716, 1507, 1392, 1367, 1309, 1279, 1247,
1174, 1042, 1023, 869, 777 cmꢀ1; HRMS (EI): m/z calcd for
C
14H27NO5 (Mþ) 289.1889, found 289.1882.

310
Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
solvent in vacuo afforded (3R,4S)-4-(tert-butoxycarbonylamino)-
3-(tert-butyldimethylsiloxy)-5-methylhexanoic acid (30) (1.03 g),
which was used for the next reaction without further
purification.
4.1.20. (3R,4S)-Allyl 4-[(S)-2-amino-3-(tritylthio)propanamido]-3-
(tert-butyldimethylsiloxy)-5- methylhexanoate (35)
TMSOTf (0.29 mL, 1.6 mmol) was added dropwise to a stirred
solution of 34 (241 mg, 0.32 mmol) in CH2Cl2 (4 mL) containing 2,6-
lutidine (0.37 mL, 3.2 mmol) at room temperature. After 1 h, MeOH
(0.7 mL) was added to the reaction mixture at 0 ꢁC, and stirring was
continued for 1 h at room temperature. The mixture was concen-
trated in vacuo to afford a residue, which was purified by column
chromatography (hexane/EtOAc 5:1) to give 35 (206 mg, 98%) as a
Allyl bromide (0.48 mL, 5.5 mmol) was added to a stirred so-
lution of the crude carboxylic acid 30 (1.03 g, 2.8 mmol) in DMF
(30 mL) containing K2CO3 (1.16 g, 8.3 mmol) at room temperature.
After 6 h, the reaction was diluted with water (10 mL), and the
resulting mixture was extracted with Et2O (4 ꢃ 30 mL). The
combined 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 31 (927 mg, 81%, 2 steps)
white amorphous solid. [
(400 MHz, CDCl3):
a
]25
D
ꢀ4.2 (c 1.00, CHCl3); 1H NMR
d
0.00 (3H, s), 0.03 (3H, s), 0.82e0.85 (15H, m),
1.30 (2H, br s), 1.93e2.01 (1H, m), 2.45 (1H, dd, J ¼ 6.8, 15.6 Hz),
2.47e2.56 (2H, m), 2.75 (1H, dd, J ¼ 3.9, 12.7 Hz), 3.04 (1H, dd,
J ¼ 3.9, 8.8 Hz), 3.74e3.79 (1H, m), 4.14 (1H, dd, J ¼ 6.3,11.2 Hz), 4.46
(1H, dd, J ¼ 5.4, 13.2 Hz), 4.53 (1H, dd, J ¼ 5.9, 13.2 Hz), 5.20 (1H, d,
J ¼ 10.7 Hz), 5.28 (1H, dd, J ¼ 1.5, 17.1 Hz), 5.81e5.90 (1H, m), 7.08
(1H, d, J ¼ 9.3 Hz), 7.18e7.44 (15H, m); 13C NMR (100 MHz, CDCl3):
as a pale yellow oil. [
a
]25
D
ꢀ6.0 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3):
d
0.04 (3H, s), 0.10 (3H, s), 0.87e0.88 (12H, m), 0.93 (3H, d,
J ¼ 6.8 Hz), 1.43 (9H, s), 1.91e2.00 (1H, m), 2.47 (1H, dd, J ¼ 7.3,
15.6 Hz), 2.57 (1H, dd, J ¼ 5.8, 15.6 Hz), 3.48e3.53 (1H, m), 4.21
(1H, dd, J ¼ 6.8, 12.7 Hz), 4.43 (1H, br d, J ¼ 10.7 Hz), 4.53e4.63
(2H, m), 5.23e5.35 (2H, m), 5.88e5.97 (1H, m); 13C NMR
d
ꢀ4.7, ꢀ4.6, 16.9, 17.9, 20.6, 25.7 (3C), 27.8, 37.4, 40.0, 54.1, 57.7,
65.2, 66.9, 69.9, 118.4, 126.7 (3C), 127.9 (6C), 129.6 (6C), 132.0, 144.6
(3C), 171.5, 172.8; IR (neat): 3369, 3312, 2958, 2929, 2856, 1736,
(100 MHz, CDCl3):
d
ꢀ4.9, ꢀ4.7, 16.7, 17.9, 20.5, 25.7 (3C), 27.9, 28.4
1673, 1508, 1255, 1174, 1092, 936, 836, 777, 743, 701, 676 cmꢀ1
;
(3C), 40.0, 59.5, 65.3, 70.1, 79.0, 118.5, 132.0, 155.9, 171.5; IR (neat):
3453, 3374, 2960, 2931, 2858, 1714, 1704, 1504, 1390, 1366, 1254,
1172, 1083, 990, 837, 777, 666 cmꢀ1; HRMS (EI): m/z calcd for
HRMS (FAB): m/z calcd for C38H53N2O4SSi (Mþ þ H) 661.3466, found
661.3476.
C
21H41NO5Si (Mþ) 415.2754, found 415. 2752.
4.1.21. (S)-Methyl 2-[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)
hept-4-enamide]-4-(methylthio)butanoate (36)
i-Pr2NEt (0.15 L, 0.91 mmol) was added dropwise to a stirred
solution of 16 (70.9 mg, 0.13 mmol) and L-methionine methyl ester
(ent-15) (51.9 mg, 0.26 mmol) in MeCN (6.5 mL) containing PyBOP
(135 mg, 0.26 mmol) at 0 ꢁC under argon, and stirring was
continued for 2 h at room temperature. The reaction mixture was
diluted with EtOAc (30 mL), and 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 2:1) to give 36
4.1.19. (3R,4S)-Allyl 4-[(S)-2-(tert-butoxycarbonylamino)-3-
(tritylthio)propanamido]-3-(tert-butyldimethylsiloxy)-5-
methylhexanoate (34)
Trimethylsilyl trifluoromethanesulfonate (TMSOTf) (0.69 mL,
3.8 mmol) was added to a stirred solution of 31 (200 mg,
0.48 mmol) in CH2Cl2 (10 mL) in the presence of 2,6-lutidine
(0.56 mL, 4.8 mmol) at room temperature. After 30 min, MeOH
(1.6 mL) was added to the reaction mixture at 0 ꢁC, and stirring was
continued for 3 h at room temperature. The reaction mixture was
concentrated in vacuo to afford (3R,4S)-allyl 4-amino-3-(tert-
butyldimethylsiloxy)-5-methylhexanoate (32) (151 mg) as a color-
less oil, which was immediately used for the next reaction due to its
(87.0 mg, 98%) as a colorless oil. [
(400 MHz, CDCl3): 1.84e1.91 (1H, m), 2.04 (3H, s), 2.05e2.16 (3H,
a]25D
1.8 (c 1.00, CHCl3); 1H NMR
d
m), 2.21e2.23 (2H, m), 2.36e2.49 (4H, m), 3.70 (3H, s), 3.78 (3H, s),
4.08e4.13 (1H, m), 4.25 (1H, d, J ¼ 12.2 Hz), 4.52 (1H, d, J ¼ 11.2 Hz),
4.68 (1H, dd, J ¼ 7.3,12.7 Hz), 5.35 (1H, dd, J ¼ 8.3,15.1 Hz), 5.59 (1H,
dt, J ¼ 6.8, 15.6 Hz), 6.83 (2H, d, J ¼ 8.8 Hz), 6.87 (1H, d, J ¼ 8.3 Hz),
instability (prone to form a g-lactam ring).
i-Pr2NEt (0.25 mL, 1.5 mmol) was added dropwise to a stirred
solution of the crude amine 32 (151 mg, 0.48 mmol) and N-Boc-S-
7.19e7.45 (17H, m); 13C NMR (100 MHz, CDCl3):
d 15.3, 29.8, 31.2,
31.4, 31.8, 43.0, 51.3, 52.3, 55.2, 66.5, 69.8, 76.3, 113.8 (3C), 126.6
(2C), 127.8 (6C), 129.5 (6C), 129.6 (2C), 129.9, 130.2, 133.1, 144.8 (3C),
159.2, 170.3, 172.2; IR (neat): 3327, 3057, 2952, 2917, 1743, 1652,
1443, 1248, 1174, 1080, 1034, 822, 744, 701 cmꢀ1; HRMS (EI): m/z
calcd for C40H45NO5S2 (Mþ) 683.2739, found 683.2737.
trityl- -cysteine (33) (333 mg, 0.72 mmol) in MeCN (16 mL) con-
D
taining PyBOP (300 mg, 0.58 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 to afford a residue, which was purified by column chro-
matography (hexane/EtOAc 1:1) to give 34 (247 mg, 68%, 2 steps) as
4.1.22. (S)-2-[(S,E)-3-(4-methoxybenzyloxy)-7-(tritylthio)hept-4-
enamide]-4-(methylthio)butanoic acid (37)
1 M LiOH (1.2 mL, 1.2 mmol) was added dropwise to a stirred
solution of 36 (213 mg, 0.31 mmol) in MeOH (6.2 mL) at room
temperature. After 3 h, 10% aqueous HCl was added to the mixture
at 0 ꢁC until the pH was 6. The resulting mixture was extracted with
EtOAc (3 ꢃ 20 mL), and the combined extracts were washed with
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 (CHCl3/
MeOH 10:1) to give 37 (203 mg, 98%) as a white amorphous solid.
a colorless oil. [
CDCl3):
a
]25
D
ꢀ3.2 (c 1.00, CHCl3); 1H NMR (400 MHz,
d
0.00 (3H, s), 0.05 (3H, s), 0.81 (3H, d, J ¼ 6.3 Hz), 0.83 (9H,
s), 0.86 (3H, d, J ¼ 6.8 Hz), 1.42 (9H, s), 1.91e1.99 (1H, m), 2.44 (1H,
dd, J ¼ 6.3, 17.1 Hz), 2.50e2.58 (2H, m), 2.70e2.78 (1H, m), 3.80e
3.86 (1H, m), 3.92 (1H, dt, J ¼ 6.3, 5.4 Hz), 4.14 (1H, dt, J ¼ 6.8,
4.4 Hz), 4.44e4.57 (2H, m), 4.70e4.76 (1H, m), 5.18e5.30 (2H, m),
5.80e5.90 (1H, m), 6.08 (1H, d, J ¼ 10.7 Hz), 7.19e7.43 (15H, m); 13
C
NMR (100 MHz, CDCl3):
d
ꢀ4.8, ꢀ4.6, 16.6, 17.9, 20.4, 25.7 (3C), 27.9,
28.2 (3C), 33.7, 39.7, 53.8, 57.9, 65.2, 67.2, 69.8, 80.2, 118.4, 126.8
(3C), 128.0 (6C), 129.5 (6C), 132.0, 144.4 (3C), 155.2, 170.3, 171.5; IR
(neat): 3327, 2959, 2930, 2857, 1717, 1683, 1491, 1367, 1254, 1173,
[
a
]25
D
ꢀ4.6 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d 1.84e1.93
(1H, m), 2.00 (3H, s), 2.07e2.18 (3H, m), 2.21e2.25 (2H, m), 2.39e
2.51 (4H, m), 3.77 (3H, s), 4.11 (1H, dt, J ¼ 8.3, 3.4 Hz), 4.24 (1H, d,
J ¼ 10.7 Hz), 4.51 (1H, d, J ¼ 11.7 Hz), 4.63e4.67 (1H, m), 5.32 (1H,
dd, J ¼ 8.3, 15.6 Hz), 5.59 (1H, dt, J ¼ 6.8, 15.1 Hz), 6.82 (2H, d,
1093, 937, 836, 751, 701 cmꢀ1
;
HRMS (EI): m/z calcd for
C
43H60N2O6SSi (Mþ) 760.3941, found 760.3934.

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
311
J ¼ 8.3 Hz), 7.07 (1H, d, J ¼ 8.3 Hz), 7.05e7.45 (17H, m); 13C NMR
131.9, 132.3, 144.3 (3C), 144.8 (3C), 169.5, 170.8, 171.8, 172.0; IR
(neat): 3340, 3318, 2958, 2928, 1732, 1634, 1539, 1444, 1255, 1179,
(100 MHz, CDCl3): d 15.2, 29.8, 31.2, 31.3, 31.4, 42.7, 51.6, 55.2, 66.5,
69.8, 76.2, 113.8 (3C), 126.6 (2C), 127.8 (6C), 129.5 (6C), 129.6, 129.7
(2C), 130.0, 133.4, 144.8 (3C), 159.2, 171.7, 175.1; IR (neat): 3336,
3015, 2918, 1733, 1614, 1541, 1514, 1489, 1444, 1248, 1177, 1034, 822,
747, 701 cmꢀ1; HRMS (FAB): m/z calcd for C39H44NO5S2 (Mþ þ H)
670.2661, found 670.2684.
1094, 836, 743, 700 cmꢀ1
; HRMS (FAB): m/z: calcd for
C
69H86N3O7S3Si (Mþ þ H) 1192.5397, found 1192.5406.
4.1.25. (3R,4S)-3-(Tert-butyldimethylsiloxy)-4-{(S)-2-[(S)-2-[(S,E)-3-
hydroxy-7-(tritylthio)hept-4-enoylamino]-4-(methylthio)butanoyl-
amino]-3-tritylthio(propionylamino)}-5-methylhexanoic acid (40)
4.1.23. (3R,4S)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(S)-2-
[(S,E)-3-(4- methoxybenzyloxy)-7-(tritylthio)hept-4-enoylamino]-
4-(methylthio)butanoylamino]-3-tritylthio(propionylamino)}-5-
methylhexanoate (38)
Morpholine (15.6
red solution of 39 (100 mg, 84
Pd(PPh3)4 (11 mg, 9.5 mol) at room temperature under argon.
mL, 0.18 mmol) was added dropwise to a stir-
mmol) in THF (10 mL) containing
m
i-Pr2NEt (66 mL, 0.39 mmol) was added dropwise to a stirred
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. Con-
centration of the solvent in vacuo afforded a residue, which was
purified by column chromatography (CHCl3/MeOH 20:1) to give 40
solution of 35 (99.1 mg, 0.15 mmol) and 37 (100 mg, 0.15 mmol) in
CH2Cl2 (3.0 mL) containing HATU (74.1 mg, 0.20 mmol) and HOAt
(26.5 mg, 0.20 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)
(71.6 mg, 74%) as a white amorphous solid. [
a
]25
D
ꢀ13.8 (c 1.00,
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 9:1):
d
0.03 (6H, s), 0.74
(3H, d, J ¼ 7.3 Hz), 0.81 (3H, d, J ¼ 7.3 Hz), 0.84 (9H, s),1.83e1.92 (1H,
m), 1.95e2.01 (1H, m), 2.01 (3H, s), 2.03e2.09 (3H, m), 2.17e2.20
(2H, m), 2.29 (1H, dd, J ¼ 9.3,14.1 Hz), 2.32e2.39 (3H, m), 2.48e2.52
(2H, m), 2.54e2.57 (2H, m), 3.71e3.76 (1H, m), 4.02e4.06 (2H, m),
4.33e4.38 (1H, m), 4.44 (1H, t, J ¼ 7.1 Hz), 5.39 (1H, dd, J ¼ 5.9,
15.1 Hz), 5.51 (1H, dt, J ¼ 6.3, 15.6 Hz), 6.59 (1H, d, J ¼ 9.8 Hz), 7.15e
to give 38 (159 mg, 81%) as a colorless viscous liquid. [
a
]25
D
ꢀ1.2 (c
1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.00 (3H, s), 0.04 (3H, s),
0.72 (3H, d, J ¼ 6.3 Hz), 0.80 (9H, s), 0.85 (3H, d, J ¼ 6.3 Hz), 1.72e
1.81 (2H, m), 1.94e2.00 (1H, m), 1.94 (3H, s), 2.11e2.15 (2H, m),
2.19e2.44 (7H, m), 2.54 (1H, dd, J ¼ 3.9, 15.6 Hz), 2.65 (2H, d,
J ¼ 6.8 Hz), 3.76e3.78 (1H, m), 3.78 (3H, s), 4.05 (1H, dt, J ¼ 8.3,
3.4 Hz), 4.17e4.22 (4H, m), 4.44e4.56 (3H, m), 5.17e5.33 (3H, m),
5.56 (1H, dt, J ¼ 6.3, 15.6 Hz), 5.80e5.90 (1H, m), 6.25e6.31 (2H, m),
6.80e6.90 (3H, m), 7.17e7.42 (32H, m); 13C NMR (100 MHz, CDCl3):
7.44 (32H, m); 13C NMR (100 MHz, CDCl3/CD3OD 9:1):
d
ꢀ5.1, ꢀ4.7,
15.1, 16.1, 17.7, 20.3, 25.6 (3C), 27.6, 29.5, 29.9, 30.8, 31.2, 31.3, 33.4,
43.0, 52.5, 52.7, 59.3, 66.4, 67.0, 68.6, 69.5, 126.5 (3C), 126.7 (3C),
127.7 (6C), 127.9 (6C), 129.3 (6C), 129.4 (6C), 131.2, 132.4, 144.2 (3C),
144.7 (3C), 170.2, 171.9, 172.2, 172.5; IR (neat): 3420, 2956, 2927,
2855, 1646, 1577, 1489, 1444, 1253, 1184, 1083, 1034, 969, 836, 743,
700, 622 cmꢀ1; HRMS (FAB): m/z calcd for C66H81N3O7S3SiK
(Mþ þ K) 1190.4643, found 1190.4653.
d
ꢀ4.8, ꢀ4.7, 15.2, 17.5, 17.9, 20.3, 25.7 (3C), 28.4, 30.0, 30.6, 31.2,
31.4, 33.7, 39.4, 42.9, 52.6, 53.1, 55.3, 59.0, 65.3, 66.6, 67.2, 69.3, 69.9,
76.4, 113.9 (2C), 118.3, 126.6 (3C), 126.8 (3C), 127.9 (6C), 128.0 (6C),
129.52 (6C), 129.54 (6C), 129.6 (2C), 129.9, 130.2, 132.1, 133.2, 144.4
(3C), 144.8 (3C), 159.3, 169.3, 171.05, 171.09, 172.0 ppm; IR (neat):
3319, 2957, 2929, 2856, 1733, 1645, 1514, 1444, 1250, 1176, 1083,
1035, 836, 743, 701 cmꢀ1
C
;
HRMS (FAB): m/z calcd for
4.1.26. (2S,6S,9S,12S,13R)-13-(Tert-butyldimethylsilyloxy)-12-
isopropyl-6-[2-(methylthio)ethyl]-2-[(E)-4-(tritylthio)but-1-enyl]-
9-tritylthiomethyl-1-oxa-5,8,11-triazacyclopentadecane-4,7,10,15-
tetraone (41)
77H94N3O8S3Si (Mþ þ H) 1312.5972, found 1312.5948.
4.1.24. (3R,4S)-Allyl 3-(tert-butyldimethylsiloxy)-4-{(S)-2-[(S)-2-
[(S,E)-3-hydroxy-7-(tritylthio)hept-4-enoylamino]-4-(methylthio)
butanoylamino]-3-tritylthio(propionylamino)}-5-
A solution of 40 (81.2 mg, 70
mmol) in CH2Cl2 (7 mL) was added
very slowly to a stirred solution of MNBA (31.3 mg, 91
mmol) in
methylhexanoate (39)
CH2Cl2 (70 mL, 1.0 mM concentration) containing DMAP (25.7 mg,
0.21 mmol) at room temperature over 14 h. After 1 h, the mixture
was diluted with CH2Cl2 (100 mL), and the organic layer was
washed successively with saturated aqueous NaHCO3 (2 ꢃ 40 mL),
water (2 ꢃ 40 mL) and brine (2 ꢃ 40 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 41
DDQ (93.1 mg, 0.34 mmol) was added in small portions to a
stirred solution of 38 (212 mg, 0.16 mmol) in CH2Cl2/H2O 9:1
(16 mL) at room temperature. After 3 h, the mixture was diluted
with CHCl3 (60 mL), and the organic layer was washed with satu-
rated 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/
(66.3 mg, 84%) as a white amorphous solid. [
a
]25
D
ꢀ7.6 (c 1.00,
EtOAc, 1:1) to give 39 (158 mg, 83%) as a colorless viscous liquid. [
a
]
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 9:1):
d
ꢀ0.03 (3H, s), 0.14
25D
ꢀ7.7 (c 1.01, CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.00 (3H, s),
(3H, s), 0.90e0.98 (15H, m), 1.85e1.94 (1H, m), 1.97e2.04 (1H, m),
2.10e2.14 (1H, m), 2.10 (3H, s), 2.25e2.37 (6H, m), 2.46e2.55 (3H,
m), 2.63e2.75 (3H, m), 3.65 (1H, dd, J ¼ 7.8, 14.6 Hz), 3.78 (1H, dt,
J ¼ 10.7, 2.4 Hz), 4.07 (1H, dt, J ¼ 10.2, 3.4 Hz), 4.45 (1H, dd, J ¼ 7.3,
16.1 Hz), 5.45e5.54 (2H, m), 5.67 (1H, dt, J ¼ 5.9, 14.6 Hz), 6.52 (1H,
d, J ¼ 10.2 Hz), 7.02 (1H, d, J ¼ 8.3 Hz), 7.28e7.51 (30H, m), 7.93 (1H,
0.04 (3H, s), 0.78 (3H, d, J ¼ 6.3 Hz), 0.80 (9H, s), 0.87 (3H, d,
J ¼ 6.8 Hz), 1.81e1.88 (1H, m), 1.90e1.99 (1H, m), 2.02e2.14 (3H, m),
2.03 (3H, s), 2.18e2.22 (2H, m), 2.27 (1H, dd, J ¼ 9.3, 15.1 Hz), 2.55e
2.69 (5H, m), 2.59 (1H, dd, J ¼ 5.4, 12.7 Hz), 2.66 (1H, dd, J ¼ 7.8,
13.2 Hz), 3.25 (1H, d, J ¼ 3.4 Hz), 3.80 (1H, dt, J ¼ 9.8, 6.3 Hz), 4.14e
4.18 (2H, m), 4.29 (1H, dd, J ¼ 6.8, 13.2 Hz), 4.35e4.41 (1H, m), 4.40
(1H, dd, J ¼ 4.9, 13.7 Hz), 4.54 (1H, dd, J ¼ 5.9, 13.2 Hz), 5.18e5.30
(2H, m), 5.38 (1H, dd, J ¼ 6.3, 15.6 Hz), 5.53 (1H, dt, J ¼ 6.8, 15.1 Hz),
5.80e5.90 (1H, m), 6.17 (1H, d, J ¼ 10.2 Hz), 6.51e6.56 (2H, m),
d, J ¼ 8.3 Hz); 13C NMR (100 MHz, CDCl3/CD3OD 9:1):
d
ꢀ5.4, ꢀ4.6,
14.4, 15.2, 17.8, 20.2, 25.6 (3C), 26.4, 30.0, 31.0, 31.2, 31.3, 31.9, 40.5,
41.5, 51.7, 53.0, 57.9, 66.5, 67.1, 67.7, 70.8,126.5 (3C),126.7 (3C),127.7
(6C), 127.9 (6C), 128.1, 129.4 (12C), 132.5, 144.3 (3C), 144.7 (3C),
169.1, 170.1, 171.4, 171.8; IR (neat): 3443, 2959, 2930, 1733, 1645,
1540, 1444, 1387, 1256, 1219, 1180, 1126, 1084, 1035, 834, 743,
700 cmꢀ1; HRMS (FAB): m/z calcd for C66H80N3O6S3Si (Mþ þ H)
1134.4979, found 1134.4974.
7.18e7.52 (30 H, m); 13C NMR (100 MHz, CDCl3):
d
ꢀ4.8 (2C), 15.2,
17.3, 17.9, 20.3, 25.7 (3C), 28.3, 30.15, 30.21, 31.3, 31.4, 33.6, 39.5,
42.9, 52.7, 52.9, 58.8, 65.4, 66.6, 67.1, 68.9, 69.2, 118.5, 126.6 (3C),
126.8 (3C), 127.8 (6C), 128.0 (6C), 129.47 (6C), 129.52 (6C), 130.0,

312
Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
4.1.27. (1S,5R,6S,9S,20S,E)-5-(Tert-butyldimethylsilyloxy)-6-
isopropyl-20-[2-(methylthio)ethyl]-2-oxa-11,12-dithia-7,19,22-
triazabicyclo[7.7.6]docos-15-ene-3,8,18,21-tetraone (42)
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 in-
A solution of 41 (56 mg, 49 mmol) in CH2Cl2/MeOH 9:1 (13 mL)
was added dropwise to a vigorously stirred solution of I2 (127 mg,
0.49 mmol) in CH2Cl2/MeOH 9:1 (98 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 (10 mL,
adjusted to pH 4.0) at room temperature, and the resulting mixture
was extracted with CHCl3 (3 ꢃ 50 mL). The combined extracts were
washed with brine (2 ꢃ 50 mL), then dried over Na2SO4. Concen-
tration of the solvent in vacuo afforded a residue, which was pu-
rified by column chromatography (CHCl3/MeOH 30:1) to give 42
cubation for 1 h at 4 ꢁC with 40
Aldrich Inc.) in histone deacetylase buffer (200
was collected by centrifugation. For the enzyme assay, 10
enzyme fraction 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 incu-
m
g of the FLAG peptide (Sigmae
L). The supernatant
L of the
m
m
m
m
m
bated 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 doseeresponse
curves.
(27.4 mg, 85%) as a white amorphous solid. [
a
]25
D
ꢀ105.5 (c 1.01,
CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD 9:1):
d
0.05 (3H, s), 0.15
(3H, s), 0.77 (3H, d, J ¼ 5.4 Hz), 0.80e0.82 (12H, m), 2.02 (3H, s),
2.09e2.27 (3H, m), 2.31e2.47 (5H, m), 2.54e2.60 (3H, m), 2.80e
2.87 (2H, m), 3.32e3.46 (2H, m), 3.75e3.83 (2H, m), 4.02e4.07 (1H,
m), 4.78e4.83 (1H, m), 5.56 (1H, d, J ¼ 4.4 Hz), 5.65e5.73 (2H, m),
7.04 (1H, d, J ¼ 7.3 Hz), 7.13 (1H, d, J ¼ 10.2 Hz), 7.96 (1H, d,
4.2.2. Cell HDAC inhibition assay (p21 promoter assay) [19]
A luciferase reporter plasmid (pGW-FL) was constructed by
cloning the 2.4 kb genomic fragment containing the transcription
start site into HindIII and SmaI sites of the pGL3-Basic plasmid
(Promega Co., Madison, WI). Mv1Lu (mink lung epithelial cell line)
cells were transfected with the pGW-FL and a phagemid expressing
neomycin/kanamycin resistance gene (pBK-CMV, Stratagene, La
Jolla, CA) with the Lipofectamine reagent (Life Technology, Rock-
ville, MD, USA). After the transfected cells had been selected by
J ¼ 5.9 Hz); 13C NMR (100 MHz, CDCl3/CD3OD 9:1):
d
ꢀ5.5, ꢀ4.4,
14.6, 14.7, 17.8, 20.5, 25.7 (3C), 26.3, 27.9, 29.6, 30.8, 31.4, 40.0, 40.3,
42.9, 53.9, 56.7, 60.0, 69.0, 69.6, 130.5 (2C), 170.0, 170.1, 170.3, 171.3;
IR (neat): 3389, 3308, 2957, 2928, 2856, 1741, 1660, 1541, 1430, 1301,
1249, 1167, 1092, 947, 838, 778, 756 cmꢀ1; HRMS (FAB): m/z: calcd
for C28H50N3O6S3Si (Mþ þ H) 648.2631, found 648.2639.
400 m
g mLꢀ1 Geneticin (G418, Life Technology), colonies formed
4.1.28. 5,6,20-Tri-epi-burkholdac A (3)
HF$pyridine (2.0 mL) was added to a stirred solution of 42
(26 mg, 40 mmol) in pyridine (4 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 3 (18.9 mg, 89%) as a white
were isolated. One of the clones was selected and named MFLL-9.
MFLL-9 expressed a low level of luciferase, whose activity was
enhanced by TSA in a dose-dependent manner. MFLL-9 cells
(1 ꢃ 105) cultured in a 96-well multi-well plate for 24 h were
incubated for 20 h in the medium containing various concentra-
tions of drugs. The luciferase activity of each cell lysate was
measured with a LucLite luciferase Reporter Gene Assay Kit
(Packard Instrument Co., Meriden, CT) and recorded with a
Luminescencer-JNR luminometer (ATTO, Tokyo, Japan). Data were
normalized to the protein concentration in cell lysates. Concen-
trations at which a drug induces the luciferase activity 100-fold
higher than the basal level are presented as the 10,000% effective
concentration 10,000% (EC10000). The human wild-type p21 pro-
moter luciferase fusion plasmid, WWP-Luc, was a kind gift from Dr.
B. Vogelstein.
amorphous solid. [
CD3CN):
a
]25
D
ꢀ114.3 (c 0.38, CHCl3); 1H NMR (400 MHz,
d
0.74 (3H, d, J ¼ 6.8), 0.75 (3H, d, J ¼ 6.8 Hz), 2.00 (3H, s),
2.00e2.09 (4H, m), 2.20 (1H, dd, J ¼ 3.4,13.7 Hz), 2.24e2.33 (2H, m),
2.36e2.44 (2H, m), 2.47e2.56 (3H, m), 2.61 (1H, dd, J ¼ 6.8,12.9 Hz),
3.13e3.35 (2H, m), 3.41 (1H, d, J ¼ 7.3 Hz), 3.60 (1H, dt, J ¼ 2.9,
10.2 Hz), 3.73e3.78 (1H, m), 3.84e3.90 (1H, m), 4.67 (1H, br s),
5.49e5.51 (1H, m), 5.69 (1H, d, J ¼ 15.6 Hz), 5.84 (1H, br s), 6.88e
6.90 (1H, m), 6.96 (1H, d, J ¼ 9.8 Hz), 7.37 ppm (1H, d, J ¼ 4.9 Hz);
4.2.3. Cell-growth inhibition assay [18]
13C NMR (100 MHz, CD3CN):
d
15.1, 15.6, 20.9, 28.3, 29.1, 30.3, 31.5,
This experiment was carried out at the Cancer Chemotherapy
Center, 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 deter-
mined by using the sulforhodamine B colorimetric method.
41.07, 41.13, 41.30, 41.33, 43.8, 54.9, 60.0, 69.8, 69.9, 131.0, 131.4,
170.6, 171.0, 171.3, 172.1; IR (neat): 3383, 3301, 2961, 2923, 1733,
1717, 1653, 1541, 1507, 1429, 1286, 1245, 1166, 1021, 974, 754 cmꢀ1
;
HRMS (FAB): m/z: calcd for C22H36N3O6S3 (Mþ þ H) 534.1766, found
534.1764.
4.2. Biological evaluation
4.2.1. HDACs preparation and enzyme inhibition assay [19]
In a 100-mm dish, 293T 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
TriseHCl (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
Acknowledgments
We are grateful to the Screening Committee of New Anticancer
Agents, which is supported by a Grant-in-Aid for Scientific Research
on Priority Area ‘Cancers’ from the Ministry of Education, Culture,
Sports, Science and Technology, Japan (MEXT), for assistance with
the biological evaluation of compounds 1e3. This study was also
cleared supernatant had been incubated for 1 h at 4 ꢁC with 4
an anti-FLAG M2 antibody (SigmaeAldrich Inc.) for HDAC1 and
mg of

Y. Fukui et al. / European Journal of Medicinal Chemistry 76 (2014) 301e313
313
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