Synthesis and evaluation of effective photoaffinity probe molecule of furospinosulin-1, a hypoxia-selective growth inhibitor

Article abstract of DOI:10.1016/j.bmc.2014.02.026

The synthesis and evaluation of a photoaffinity probe molecule for furospinosulin-1, a hypoxia-selective growth inhibitor that we identified from marine sponge, was studied. An analogue carrying an alkyne tail showed potent hypoxia-selective inhibitory activity exceeding that of the parent molecule, and exhibited in vivo anti-tumor activity following oral administration. The alkyne moiety in the analogue was also found to be a good anchoring group for the preparation of probe molecules; a photoaffinity probe molecule having an optimized spacer length was selected through the systematic synthesis of several probes and the evaluation of their hypoxia-selective growth inhibitory activity and electrophoretic mobility shift properties.

Full text of DOI:10.1016/j.bmc.2014.02.026

Bioorganic & Medicinal Chemistry 22 (2014) 2102–2112
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of effective photoaffinity probe molecule
of furospinosulin-1, a hypoxia-selective growth inhibitor
⇑
Naoyuki Kotoku , Chiaki Nakata, Takashi Kawachi, Takanori Sato, Xiu-Han Guo, Aoi Ito, Yuji Sumii,
⇑
Masayoshi Arai, Motomasa Kobayashi
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of a photoaffinity probe molecule for furospinosulin-1, a hypoxia-selective
growth inhibitor that we identified from marine sponge, was studied. An analogue carrying an alkyne tail
showed potent hypoxia-selective inhibitory activity exceeding that of the parent molecule, and exhibited
in vivo anti-tumor activity following oral administration. The alkyne moiety in the analogue was also
found to be a good anchoring group for the preparation of probe molecules; a photoaffinity probe mol-
ecule having an optimized spacer length was selected through the systematic synthesis of several probes
and the evaluation of their hypoxia-selective growth inhibitory activity and electrophoretic mobility shift
properties.
Received 25 January 2014
Revised 15 February 2014
Accepted 18 February 2014
Available online 4 March 2014
Keywords:
Furospinosulin-1
Hypoxia-selective growth inhibitor
Marine sponge
Ó 2014 Elsevier Ltd. All rights reserved.
Analogue compound
Photoaffinity probe
1. Introduction
important chemical tools to identify new molecule(s) responsible
for the adaptation of tumor cells to hypoxia.
The identification of target molecules of bioactive compounds is
one of the most important and attractive topics in the fields of
chemical biology and drug discovery. Affinity purification using
synthetic probe molecules that mimic the structure of the bioac-
tive compounds is a straightforward and powerful method for
identifying target molecules.1,2 In particular, target analyses of
the active compounds identified through phenotypic screening
have high potential in the discovery of new biological systems
and drug targets.3,4
The hypoxic environment in tumors is now considered an
important factor for tumor growth, angiogenesis, metastasis, and
response to chemotherapy and irradiation.5 The system by which
tumor cells adapt to hypoxia has been extensively investigated
with particular regard to hypoxia inducible factor-1 (HIF-1), which
is now considered a key mediator in the adaptation to hypoxic
environment. Anti-cancer drug discovery studies targeting HIF-1
are ongoing.6 However, the complete adaptation system remains
unclear. Therefore, compounds that selectively exhibit growth
inhibitory activity against tumor cells under hypoxic environments
are expected to be novel and promising anti-cancer drug leads and
Marine natural products have proved to be a rich and promising
source of drug candidates, especially in the field of anti-cancer
drug discovery.7 While searching for new bioactive substances
from marine organisms, we established a new screening system
to identify hypoxia-selective growth inhibitors as potential anti-
cancer drug leads that exhibit novel mechanisms of action, and iso-
lated a furanosesterterpene, furospinosulin-1 (1), from the Indone-
sian marine sponge Dactylospongia elegans.8 Furospinosulin-1 (1)
exhibits dose-dependent selective growth inhibitory activity
against human prostate cancer cells DU145 in a 1% oxygen atmo-
sphere (Fig. 1). Several marine natural products targeting hypoxia
inhibit HIF-1 and/or its signalling pathway.9 Interestingly, com-
pound 1 did not inhibit the accumulation of HIF-1
a but did sup-
press the expression of insulin-like growth factor-2 (IGF-2)
through the inhibition of complex formation of nuclear proteins
with the Sp1 consensus sequence in the IGF-2 promoter region.
Moreover, orally administered 1 suppressed tumor growth in sub-
cutaneously inoculated mouse sarcoma S180 cells.8 The intriguing
bioactivity of 1 prompted us to identify its target molecules, which
could lead to the identification of new molecules responsible for
adaptation to hypoxia, and to the development of a promising
anti-cancer drug lead through structure-based design. Here, we re-
port the systematic synthesis of a photoaffinity probe molecule of
1 by synthesizing its tail-modified analogues and evaluating their
biological activity.
⇑
Corresponding authors. Tel.: +81 6 6879 8215; fax: +81 6 6879 8219.
E-mail addresses: kotoku@phs.osaka-u.ac.jp (N. Kotoku), kobayasi@phs.osaka-u.
ac.jp (M. Kobayashi).
http://dx.doi.org/10.1016/j.bmc.2014.02.026
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
2103
a,b
PhO2S
2
100
80
60
40
20
0
Br
O
5
PhO2S
OR
hypoxia
3
c-f
3: R = H
normoxia
4: R = TIPS
R2
R1
O
6: R1 = SO2Ph, R2 = OTIPS
7: R1 = H, R2 = OTIPS
8: R1 = H, R2 = OH
1
3
10
30
100
300
i-k
g or h
concentration (µM)
9: R1 = H, R2 = Br
R
Figure 1. Structure of furospinosulin-1 (1) and its growth inhibition activity against
DU145 cells.
n
R
O
12a: n = 0, R = SiMe3
13a: n = 0, R = H
2. Results and discussions
10: R = COCH3
11: R = 2-propynyl
12b: n = 1, R = SiMe3
13b: n = 1, R = H
2.1. Synthesis and evaluation of tail-modified analogues
14: n = 1, R = CH3
Furospinosulin-1 (1) is a simple molecule consisting of an aro-
matic furan ring covalently linked to a long side chain consisting
of four iterative isoprene units, and lacks functional groups such
as alcohol or amine moieties that can be used for its derivatization
to affinity probe molecules. Therefore, de novo synthesis of an ana-
logue compound equipped with an appropriate anchoring group
for binding to functional units without affecting the bioactivity of
the parent compound is needed. We have previously analyzed
the structure–activity relationship of 1 through the concise synthe-
sis and biological evaluation of 1 and some analogue compounds.10
Most of the structural modifications attempted, such as change of
the aromatic ring, elongation of the methyl group near the furan
ring, and truncation of the side chain, resulted in significant loss
of hypoxia-selective growth inhibitory activity, clearly indicating
that the whole chemical structure was important for the binding
to the target molecule. On the other hand, an analogue having a
longer side chain was found to partially retain hypoxia-selective
growth inhibitory activity. We hypothesized that the methyl group
on the side chain tail could be modified to prepare an effective
affinity probe molecule without loss of activity, and decided to
synthesize and evaluate various tail-modified analogues of 1.
The synthesis of the tail-modified analogues was examined as
depicted in Scheme 1. The allylic oxidation of (E,E)-farnesyl phen-
ylsulfone (2)11 using SeO2 and t-BuOOH12,13 proceeded predomi-
nantly at the terminal methyl group to give compound 3, in
which the hydroxyl group was protected as triisopropylsilyl (TIPS)
ether to provide compound 4. Based on our previous report,10 the
coupling reaction between 4 and allylic bromide 5 using t-BuOK14
and the successive reductive desulfonylation of 6 with LiBHEt3/
Scheme 1. Synthesis of tail-modified analogues. Reagents and conditions: (a) SeO2
(cat.), t-BuOOH, salicylic acid, CH2Cl2; NaBH4, MeOH, 38%; (b) TIPSCl, imidazole,
DMF, 98%; (c) t-BuOK, THF, ꢀ40 °C, 90%; (d) LiBHEt3, Pd(dppp)Cl2, THF, 0 °C, 85%; (e)
TBAF, THF, 94%; (f) MsCl, Et3N, CH2Cl2, ꢀ40 °C; LiBr, THF, quantitative; (g) Ac2O,
pyridine, quantitative; (h) 3-bromo-1-propyne, NaH, THF, 56%; (i) 1-trimethylsi-
lylacetylene or 1-trimethylsilylpropyne, n-BuLi, THF, ꢀ40 °C, 41% for 12a, 68% for
12b; (j) TBAF, THF, 92% for 13a, quantitative for 13b; (k) n-BuLi, MeI, THF, ꢀ78 °C,
41%.
The results of the biological evaluation of these analogues are
displayed in Figure 2. Most of the analogues showed non-selective
growth inhibitory activity (hypoxia/normoxia) or hypoxia-selec-
tive growth inhibitory activity in a narrow concentration range,
which clearly indicates that the modification of the terminal
methyl group is highly restricted. On the other hand, the C-propar-
gylated analogue 13b showed dose-dependent and hypoxia-selec-
tive growth inhibitory activity. A detailed evaluation revealed that
the analogue 13b exhibited hypoxia-selective growth inhibitory
activity in the wide concentration range from 1 to 300 lM and,
surprisingly, its potency is much higher than that of the natural
product (1) in the low concentration range (Fig. 3a). Furthermore,
the analogue 13b showed potent in vivo antitumor effects after
oral administration; doses of 5, 10, and 25 mg/kg of the analogue
13b suppressed tumor growth to 45%, 26%, and 11% of the control,
respectively (Fig. 3b). In addition, side effects such as weight loss or
diarrhea were not observed during the experimental period.
2.2. Systematic preparation of the photoaffinity probes
15
Pd(dppp)Cl2 proceeded smoothly to give compound 7, which
was further treated with tetra-n-butylammonium fluoride (TBAF)
to produce a tail-hydroxylated furospinosulin-1 (8). Then, we pre-
pared some analogue compounds through modification of the hy-
droxyl group of 8, to identify an appropriate anchoring group. Thus,
O-esterification and O-alkylation provided acetate ester 10 and 2-
propynyl ether 11, respectively, while the conversion to the corre-
sponding bromide 9 and the following alkylation with silyl-pro-
tected lithium acetylide or 2-propynyllithium16 gave the two
terminal C-alkylated products 13a or 13b, respectively. The termi-
nal alkyne of 13b was further methylated to give 14.
Once the tail-modified analogue of 1 that maintained hypoxia-
selective growth inhibitory activity was synthesized, we examined
the synthesis of an affinity probe molecule derived from the ana-
logue 13b, to find the target molecule of 1. Because the only avail-
able information about the binding affinity of 1 to its target
molecule was that the two molecules might interact exclusively
through hydrophobic interactions, we used a photo-crosslinking
strategy to capture the target molecule reliably by forming a cova-
lent bond. To maximize the cross-linking efficiency of the photore-
active group, the distance between the bioactive ligand and

2104
N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
100
80
60
40
20
0
hypoxia
normoxia
*
*
*
*
*
*
10 100 10 100 10 100 10 100 10 100 10 100 10 100
10 11 13a 13b 14
1
8
concentration (µM)
Figure 2. Growth inhibition activity of the synthetic analogues of furospinosulin-1 (1) against DU145 cells. ⁄P < 0.05.
(A)
100
80
60
40
20
0
hypoxia
*
normoxia
*
*
*
*
*
Figure 4. Working hypothesis for the structural optimization of the photoaffinity
probe.
group could be as close as possible to the surface of the target pro-
tein (Scheme 2). Thus, the common functional unit composed of
biotin tag for detection/purification20 and the benzophenone moi-
ety for photo-crosslink,21 and the alkyne-tailed furospinosulin-1
analogue 13b were assembled through a spacer unit. Starting from
1
3
10
30
100
300
concentration (µM)
(B)
2.0
1.6
Boc-protected L-cystine (15), the commercially available azide-
tailed polyethylene glycol (PEG) linker was introduced to give
compound 16. Removal of the Boc group and condensation with
4-benzoylbenzoic acid provided compound 18, and the subsequent
copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction22
with the biotin derivative 1923 afforded compound 20, a precursor
of the common functional unit. Treatment of 20 with tris(2-car-
boxyethyl)phosphine (TCEP)24 in aqueous dimethylformamide
(DMF) cleaved the disulfide bond of the cystine to generate a reac-
tive form of the common functional unit, and the liberated thiol
was trapped in situ by the iodoacetamide moiety of the spacer unit
21a to give 22a. Finally, the alkyne-tailed furospinosulin-1 13b was
connected by a second CuAAC reaction to the azide moiety of 22a
precast on the end of the spacer unit to furnish the desired photo-
affinity probe molecule of furospinosulin-1 (23a). By changing the
spacer unit, three more photoaffinity probes (23b–d) were system-
atically obtained through the easy two-step assembling method
mentioned above.
1.2
0.8
*
*
0.4
0
*
5
10
25 (mg/kg)
13b
Figure 3. (A) Growth inhibition against DU145 cells and (B) in vivo anti-tumour
effect of C-propargyl analogue (13b). ⁄P < 0.05.
photoreactive group should be optimized (Fig. 4a). If the distance is
too short, the bulky photoreactive group might hinder the binding
of the ligand to its target molecule (Fig. 4b), whereas if it is too
long, the desired cross-linking with the target molecule might have
a low yield because of non-specific linking with the solvent (water)
or some other molecule (Fig. 4c).17–19
2.3. Evaluation of the synthetic probe molecules
Next, we evaluated the hypoxia-selective growth inhibitory
activity of the synthesized affinity probes against DU145 cells. As
summarized in Figure 5, the photoaffinity probes 23a and 23b,
Therefore, we decided to prepare various affinity probes by
using spacer units with different lengths so that the photoreactive

N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
2105
O
O
NHR
HN
NHBoc
CO2H
d
a-c
O
S
NH
O
S
N3
N
2
O
N
N
2
NH
O
S
O
HN
3
H
16: R = Boc
17: R = H
2
15
HN
HN
O
H
3
20
S
18: R = 4-benzoylbenzoyl
O
O
O
NH
O
HN
H
HN
e
H
O
NH
N3
X
NH
N
S
O
N
19
NH
S
O
HN
O
N
H
HN
HN
O
O
H
22a: X = –(CH2OCH2)3–
22b: X = –(CH2OCH2)2–
22c: X = –(CH2)3–
3
I
S
N3
X
N
H
f
21a: X = –(CH2OCH2)3–
21b: X = –(CH2OCH2)2–
21c: X = –(CH2)3–
22d: X = –CH2–
O
O
21d: X = –CH2–
NH
O
NH
X
N
N
O
N
N
S
NH
H
O
HN
N
N
O
HN
HN
O
H
O
4
3
23a: X = –(CH2OCH2)3–
23b: X = –(CH2OCH2)2–
23c: X = –(CH2)3–
S
23d: X = –CH2–
Scheme 2. Systematic synthesis of the photoaffinity probe molecules 23a–d. Reagents and conditions: (a) 11-azido-3,6,9-trioxaundecane-1-amine, EDCIꢁHCl, HOBt, THF,
quantitative; (b) TFA, CH2Cl2, 88%; (c) 4-benzoylbenzoic acid, EDCIꢁHCl, HOBt, THF, 97%; (d) 19, CuSO4, sodium ascorbate, TBTA, t-BuOH/H2O, 90%; (e) TCEPꢁHCl, NaHCO3,
DMF/H2O; 21a–d, 74% for 22a, 70% for 22b, 64% for 22c, 72% for 22d; (f) 13b, CuSO4, sodium ascorbate, t-BuOH/H2O, 48% for 23a, 40% for 23b, 65% for 23c, 60% for 23d.
100
probes 23a and 23b could be used as effective probe molecules
in pulldown studies.
hypoxia
80
We further evaluated the probe molecules by electrophoretic
mobility shift assay (EMSA) analysis. We have previously reported
that 1 abrogates the formation of the complex among the nuclear
proteins of DU145 cells cultured under hypoxic condition and the
oligonucleotide probe containing the Sp1 consensus sequence
responsible for the transcription of IGF–2.8 This result strongly sug-
gests that the target protein of 1 might be a part of the transcrip-
tion factor complex, and that the probe molecule is expected to
bind to nuclear proteins, resulting in the inhibition of complex
formation.
normoxia
*
60
40
20
0
*
*
*
3
30
3
30
3
30
23c
3
30
23d
23a
23b
concentration (µM)
The results of the EMSA analysis using probes 23a–d are shown
in Figure 6. As expected, the supershifted band derived from the
complex of the nuclear proteins with the Sp1 oligonucleotide
Figure 5. Growth inhibition activity of the synthetic probe molecules 23a–d
against DU145 cells. ⁄P < 0.05.
probe (lane 2) significantly diminished in the presence (100 lM)
of the probes 23a and 23b (lanes 4 and 6), and these bands com-
pletely disappeared upon treatment at a higher concentration
which have the longest spacer units, showed a hypoxia-selective
growth inhibitory activity resembling that of the parent compound
13b and natural furospinosulin-1 (1), whereas the probes 23c and
23d, which have the shortest spacer units, showed only weak or
non-selective activity. This result implies that the photoaffinity
(300 lM) (lanes 5 and 7). In contrast, the shortest probes 23c
and 23d did not affect complex formation at all (lanes 8 and 9).
These results agree with those of the cell growth inhibitory assay
(Fig. 5). Therefore, the probes 23a and 23b are expected to bind
the target protein contained in the transcription factor complex.

2106
N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
(68.4 mg, 0.50 mmol) in CH2Cl2 (3.8 mL) at 0 °C. After stirring for
10 min, (E,E)-farnesyl phenylsulfone (2, 1.72 g, 4.95 mmol) was
added to the mixture and the whole mixture was stirred for 20 h
at 4 °C. Toluene was added to the mixture, and the whole mixture
was extracted with AcOEt. The combined AcOEt extract was suc-
cessively washed with satd NaHCO3 aq and satd Na2S2O3 aq Re-
moval of the solvent from the AcOEt extract under reduced
pressure gave a crude product, which was dissolved to MeOH
(5.0 mL). NaBH4 (37.5 mg, 0.990 mmol) was added to the solution
at 0 °C and the whole mixture was stirred for 90 min at 0 °C. Satd
NH4Cl aq was added to the mixture and the whole mixture was ex-
tracted with Et2O. Removal of the solvent from the Et2O extract un-
der reduced pressure gave a crude product, which was purified by
SiO2 column (n-hexane/AcOEt = 2:1) to give 3 (658 mg, 38%) as a
lane :
1
2
3
4
5
6
7
8
9
colorless oil. IR (KBr): 3547, 2922, 1447, 1306, 1238 cmꢀ1 1H
.
NMR (500 MHz, CDCl3) d: 7.86 (2H, d, J = 7.8 Hz), 7.63 (1H, t,
J = 7.8 Hz), 7.53 (2H, t, J = 7.8 Hz), 5.38 (1H, t, J = 6.7 Hz), 5.19
(1H, t, J = 7.9 Hz), 5.06 (1H, t, J = 5.5 Hz), 3.98 (2H, br s), 3.80 (2H,
d, J = 7.9 Hz), 2.12 (2H, q, J = 7.3 Hz), 2.03–2.00 (6H, m), 1.65 (3H,
s), 1.58 (3H, s), 1.30 (3H, s). 13C NMR (125 MHz, CDCl3) d: 146.3,
138.7, 135.3, 134.8, 133.5, 128.9 (2C), 128.5 (2C), 125.7, 123.6,
110.3, 68.8, 56.1, 39.6, 39.3, 26.1 (2C), 16.1, 15.9, 13.6. HR-ESI
MS: m/z 385.1794, calcd for C21H30O3SNa. Found: 385.1813.
nuclear protein :
– +
– –
+
1
+
+
+
+
+
+
compound
(100 µM)
:
23a 23a* 23b 23b* 23c 23d
Figure 6. Evaluation of the synthetic probe molecules 23a–d by electrophoretic
mobility shift assay analysis. ⁄The concentration of these probes was 300
lM.
3. Conclusions
4.2.2. Triisopropyl(((2E,6E,10E)-2,6,10-trimethyl-12-
(phenylsulfonyl)dodeca-2,6,10-trien-1-yl)oxy)silane (4)
We synthesized and evaluated photoaffinity probe molecules of
furospinosulin-1 (1) through the systematic assembly of a common
functional unit and tail-modified analogues carrying spacer units
of different lengths. Our results revealed that the anchoring moiety
and the length of the spacer unit affect the hypoxia-selective
growth inhibitory activity and the binding affinity to the target
protein. Finally, we synthesized the probes 23a and 23b having
optimized spacer length that bind to the target protein of 1. Fur-
thermore, the systematic preparation method for probe molecules
described here could be applied to other bioactive ligands to ana-
lyze their target molecules. The pulldown experiment using those
probes and the detailed analysis of the target protein(s) of furospi-
nosulin-1 (1), which might be a novel molecule responsible for the
adaptation of tumor cells against hypoxia, are now underway.
TIPSCl (0.7 mL, 3.43 mmol) and imidazole (467 mg, 6.86 mmol)
were added to a solution of 3 (622 mg, 1.72 mmol) in DMF (4.3 mL)
and the whole mixture was stirred for 16 h at 0 °C. H2O was added
to the mixture and the whole mixture was extracted with Et2O. Re-
moval of the solvent from the Et2O extract under reduced pressure
gave a crude product, which was purified by SiO2 column (n-hex-
ane/AcOEt = 10:1) to give 4 (841 mg, 98%) as a colorless oil. IR
(KBr): 2946, 2865, 1447, 1385, 1307, 1238 cmꢀ1
.
1H NMR
(500 MHz, CDCl3) d: 7.87 (2H, d, J = 7.7 Hz), 7.63 (1H, t,
J = 7.7 Hz), 7.53 (2H, t, J = 7.7 Hz), 5.41 (1H, t, J = 6.7 Hz), 5.19
(1H, t, J = 7.9 Hz), 5.06 (1H, t, J = 5.8 Hz), 4.07 (2H, s), 3.81 (2H, d,
J = 7.9 Hz), 2.12 (2H, q, J = 7.3 Hz), 2.04–2.00 (6H, m), 1.60 (3H, s),
1.59 (3H, s), 1.32 (3H, s), 1.12–1.02 (21H, m). 13C NMR (125 MHz,
CDCl3) d: 146.9, 139.2, 136.1, 134.9, 134.0, 129.4 (2C), 129.1 (2C),
123.9 (2C), 110.8, 68.9, 56.6, 40.2, 39.9, 26.7, 26.6, 18.5 (6C),
16.7, 16.5, 13.9, 12.5 (3C). HR-ESI MS: m/z 541.3135, calcd for C30-
H30O3SiSNa. Found: 541.3148.
4. Experimental
4.1. General experimental
A JEOL JNM LA-500 (1H: 500 MHz, 13C: 125 MHz) and a Varian
NMR system (1H: 600 MHz, 13C: 150 MHz) spectrometer were used
to obtain 1H and 13C NMR data using tetramethylsilane as an internal
standard. IR spectra were obtained with a JASCO FT/IR-5300 infrared
spectrometer. Mass spectra were obtained with a Waters Q-Tof Ulti-
4.2.3. (((2E,6E,10E,14E)-17-(Furan-3-yl)-2,6,10,14-tetramethyl-
12-(phenylsulfonyl)heptadeca-2,6,10,14-tetraen-1-
yl)oxy)triisopropylsilane (6)
t-BuOK (1.0 M in THF, 1.02 mL, 1.02 mmol) was added dropwise
to a solution of 4 (510 mg, 1.02 mmol) and 5 (256 mg, 1.12 mmol)
in THF (5.0 mL) at ꢀ40 °C and the whole mixture was stirred for
30 min at that temperature. Satd NH4Cl aq was added to the mix-
ture and the whole mixture was extracted with AcOEt. Removal of
the solvent from the AcOEt extract under reduced pressure gave a
crude product, which was purified by SiO2 column (n-hexane/
AcOEt = 10:1) to give 6 (612 mg, 90%) as a colorless oil. IR (KBr):
ma API using MeOH as a solvent. Silica gel (Kanto, 40–100
lm) and
pre-coated thin layer chromatography (TLC) plates (Merck, 60F254
)
were used for column chromatography and TLC. Spots on TLC plates
were detected by spraying phosphomolybdic acid solution (5 g phos-
phomolybdic acid in 100 mL of EtOH) and acidic p-anisaldehyde
solution (p-anisaldehyde: 25 mL, c-H2SO4: 25 mL, AcOH: 5 mL,
EtOH: 425 mL) with subsequent heating. Unless otherwise noted,
all the reactions were performed under N2 atmosphere using pur-
chased reagents and solvents without further purification. After
workup, the organic phases were dried over Na2SO4.
2944, 2865, 1447, 1385, 1306 cmꢀ1 1H NMR (500 MHz, CDCl3) d:
.
7.85 (2H, d, J = 7.5 Hz), 7.61 (1H, t, J = 7.5 Hz), 7.51 (2H, t,
J = 7.5 Hz), 7.31 (1H, s), 7.15 (1H, s), 6.22 (1H, s), 5.41 (1H, t,
J = 6.7 Hz), 5.19 (1H, t, J = 7.0 Hz), 5.05 (1H, t-like, J = 5.5 Hz), 4.90
(1H, d, J = 10.6 Hz), 4.07 (2H, s), 3.88 (1H, td, J = 10.6, 2.8 Hz),
2.90 (1H, d, J = 13.0 Hz), 2.39 (2H, t, J = 7.6 Hz), 2.29 (1H, t,
J = 13.0 Hz), 2.19 (2H, q, J = 7.3 Hz), 2.12 (2H, q, J = 7.5 Hz), 2.00
(2H, t, J = 7.6 Hz), 1.93 (4H, d-like, J = 5.5 Hz), 1.59 (3H, s), 1.58
(6H, s), 1.52 (3H, s), 1.15 (3H, s), 1.10–1.05 (21H, m). 13C NMR
(125 MHz, CDCl3) d: 145.1, 142.6, 138.8, 135.5, 134.4, 133.3,
130.7, 129.2 (2C), 128.6 (2C), 127.6, 124.6, 123.6, 123.4, 117.2,
4.2. Synthesis of tail-modified analogues
4.2.1. (2E,6E,10E)-2,6,10-Trimethyl-12-(phenylsulfonyl)dodeca-
2,6,10-trien-1-ol (3)
t-BuOOH (70% (w/v) solution in water, 2.55 mL, 17.8 mmol) was
added to a solution of SeO2 (55.0 mg, 0.50 mmol) and salicylic acid

N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
2107
110.9, 68.4, 63.5, 39.7, 39.4, 37.4, 28.4, 26.3, 26.1, 24.7, 18.0 (6C),
4.2.7. 3-((3E,7E,11E,15E)-4,8,12,16-Tetramethyl-17-(prop-2-yn-
16.3, 16.0, 15.9, 13.4, 12.1 (3C). HR-ESI MS: m/z 689.4042, calcd
1-yloxy)heptadeca-3,7,11,15-tetraen-1-yl)furan (11)
for C40H62O4SiSNa. Found: 689.4036.
NaH (60% (w/w) in oil, 2.5 mg, 0.06 mmol) was added to a solu-
tion of 8 (15.6 mg, 0.04 mmol) in THF (0.6 mL) at 0 °C and the
whole mixture was stirred for 1 h. Propargyl bromide (80% (w/v)
solution in toluene, 0.006 mL, 0.05 mmol) was added to the mix-
ture and the whole mixture was further stirred for 7 h. Satd NH4Cl
aq was added to the mixture and the whole mixture was extracted
with Et2O. Removal of the solvent from the Et2O extract under re-
duced pressure gave a crude product, which was purified by SiO2
column (n-hexane/AcOEt = 8:1) to give 11 (9.7 mg, 56%) as a color-
4.2.4. (((2E,6E,10E,14E)-17-(Furan-3-yl)-2,6,10,14-tetrameth-
ylheptadeca-2,6,10,14-tetraen-1-yl)oxy)triisopropylsilane (7)
LiBHEt3 (1.0 M in THF, 2.1 mL, 2.10 mmol) was added drop-
wise to a solution of 6 (561 mg, 0.841 mmol) and Pd(dppp)Cl2
(95.0 mg, 0.16 mmol) in THF (4.2 mL) at 0 °C and the whole mix-
ture was stirred for 15 min. Satd NaHCO3 aq was added to the
mixture and the whole mixture was extracted with AcOEt. Re-
moval of the solvent from the AcOEt extract under reduced pres-
sure gave a crude product, which was purified by SiO2 column
(n-hexane/AcOEt = 50:1) to give 7 (380 mg, 85%) as a colorless
less oil. IR (KBr): 2922, 2855, 2363, 1507, 1446, 1383 cmꢀ1 1H
.
NMR (500 MHz, CDCl3) d: 7.33 (1H, d, J = 1.8 Hz), 7.21 (1H, s),
6.27 (1H, s), 5.43 (1H, t, J = 7.0 Hz), 5.17 (1H, t, J = 6.4 Hz), 5.12
(1H, t, J = 7.0 Hz), 5.10 (1H, t, J = 6.8 Hz), 4.07 (2H, d, J = 2.4 Hz),
3.93 (2H, s), 2.45 (2H, t, J = 7.6 Hz), 2.40 (1H, t, J = 2.4 Hz), 2.24
(2H, q, J = 7.5 Hz), 2.14 (2H, q, J = 7.5 Hz), 2.08–1.96 (10H, m),
1.65 (3H, s), 1.60 (6H, s), 1.59 (3H, s). 13C NMR (125 MHz, CDCl3)
d: 142.5, 138.8, 135.8, 135.0, 134.5, 131.1, 129.4, 125.0, 124.6,
124.2, 123.7, 111.1, 80.1, 75.9, 74.0, 56.2, 39.7 (2C), 39.2, 28.5,
26.7, 26.6, 26.4, 25.0, 16.0 (3C), 14.0. HR-ESI MS: m/z 431.2918,
calcd for C28H40O2Na. Found: 431.2926.
oil. IR (KBr): 2928, 2865, 1504, 1458, 1383 cmꢀ1 1H NMR
.
(500 MHz, CDCl3) d: 7.34 (1H, t, J = 1.5 Hz), 7.21 (1H, s), 6.28
(1H, s), 5.43 (1H, td, J = 7.0, 1.4 Hz), 5.18 (1H, t, J = 6.4 Hz), 5.13
(1H, t, J = 7.0 Hz), 5.11 (1H, t, J = 6.8 Hz), 4.08 (2H, s), 2.45 (2H,
t, J = 7.3 Hz), 2.25 (2H, q, J = 7.3 Hz), 2.24–2.05 (6H, m), 2.03–
1.97 (6H, m), 1.61 (1H, s), 1.60 (3H, s), 1.11–1.05 (21H, m). 13C
NMR (125 MHz, CDCl3) d: 142.5, 135.0, 138.8, 135.8, 134.8,
134.4, 125.0, 124.4, 124.1, 123.7 (2C), 111.1, 68.5, 39.7 (2C),
39.5, 28.5, 26.7, 26.6, 26.2, 25.0, 18.0 (6C), 16.0 (3C), 13.4, 12.1
(3C). HR-ESI MS: m/z 549.4077, calcd for C34H58O2SiNa. Found:
549.4104.
4.2.8. ((4E,8E,12E,16E)-19-(Furan-3-yl)-4,8,12,16-tetramethylno-
nadeca-4,8,12,16-tetraen-1-yn-1-yl)trimethylsilane (12a)
Et3N (0.31 mL, 2.26 mmol) and methanesulfonyl chloride
(0.09 mL, 1.13 mmol) were successively added to a solution of 8
(349 mg, 0.941 mmol) in CH2Cl2 (5.0 mL) at ꢀ40 °C and the whole
mixture was stirred for 3 h. The resultant mixture was diluted with
THF (15 mL), and LiBr (409 mg, 4.71 mmol) was added to the mix-
ture. After stirring for 1 h, satd NH4Cl aq was added to the mixture
and the whole mixture was extracted with Et2O. Removal of the
solvent from the Et2O extract under reduced pressure gave a crude
bromide 9 (408 mg, quant), which was used for the next reaction
without further purification. 1H NMR (500 MHz, CDCl3) d: 7.34
(1H, s), 7.21 (1H, s), 6.28 (1H, s), 5.58 (1H, t, J = 7.0 Hz), 5.17 (1H,
t, J = 7.0 Hz), 5.13–5.08 (2H, m), 3.97 (2H, s), 2.45 (2H, t,
J = 7.6 Hz), 2.25 (2H, q, J = 7.5 Hz), 2.14–1.97 (12H, m), 1.75 (3H,
s), 1.60 (9H, s).
4.2.5. (2E,6E,10E,14E)-17-(Furan-3-yl)-2,6,10,14-tetramethylhep-
tadeca-2,6,10,14-tetraen-1-ol (8)
TBAF (1.0 M in THF, 1.1 mL, 1.11 mmol) was added to a solu-
tion of 7 (450 mg, 0.85 mmol) in THF (8.5 mL) and the whole
mixture was stirred for 2 h. H2O was added to the mixture and
the whole mixture was extracted with AcOEt. Removal of the sol-
vent from the AcOEt extract under reduced pressure gave a crude
product, which was purified by SiO2 column (n-hexane/
AcOEt = 5:1) to give 8 (349 mg, 94%) as a colorless oil. IR (KBr):
3295, 2920, 2855, 1505, 1447, 1383 cmꢀ1 1H NMR (500 MHz,
.
CDCl3) d: 7.34 (1H, s), 7.21 (1H, s), 6.28 (1H, s), 5.39 (1H, t,
J = 7.0 Hz), 5.17 (1H, t, J = 6.7 Hz), 5.12 (1H, t, J = 7.0 Hz), 5.11
(1H, t, J = 7.0 Hz), 3.99 (2H, s), 2.45 (2H, t, J = 7.6 Hz), 2.24 (2H,
q, J = 7.3 Hz), 2.15–2.06 (6H, m), 2.03–1.97 (6H, m), 1.67 (3H, s),
1.60 (6H, s), 1.59 (3H, s). 13C NMR (125 MHz, CDCl3) d: 142.5,
138.8, 135.7, 134.9, 134.7, 134.5, 126.2, 125.0, 124.5, 124.2,
123.7, 111.1, 69.0, 39.7 (2C), 39.3, 28.4, 26.6 (2C), 26.2, 25.0,
16.0 (3C), 13.7. HR-ESI MS: m/z 393.2758, calcd for C25H38O2Na.
Found: 393.2770.
1-Trimethylsilylacetylene (0.03 mL, 0.22 mmol) was added to a
solution of n-BuLi (1.67 M in n-hexane, 0.13 mL, 0.22 mmol) in THF
(0.7 mL) at ꢀ40 °C and the whole mixture was stirred for 15 min at
ꢀ40 °C and for 20 min at ꢀ20 °C. After cooling the mixture again at
ꢀ40 °C, the above bromide 9 (31.6 mg, 0.74 mmol) was added to
the mixture and the whole mixture was gradually warmed to rt
with stirring for 14 h. Satd NH4Cl aq was added to the mixture
and the whole mixture was extracted with Et2O. Removal of the
solvent from the Et2O extract under reduced pressure gave a crude
product, which was purified by SiO2 column (n-hexane/tolu-
ene = 10:1) to give 12a (13.6 mg, 41%) as a colorless oil. IR (KBr):
4.2.6. (2E,6E,10E,14E)-17-(Furan-3-yl)-2,6,10,14-
tetramethylheptadeca-2,6,10,14-tetraen-1-yl acetate (10)
Acetic anhydride (0.1 mL) was added to a solution of 8 (11.5 mg,
0.03 mmol) in pyridine (0.2 mL) and the whole mixture was stirred
for 2 h. H2O was added to the mixture and the whole mixture was
extracted with AcOEt, and the combined AcOEt extract was washed
with 5% HCl. Removal of the solvent from the AcOEt extract under
reduced pressure gave a crude product, which was purified by SiO2
column (n-hexane/AcOEt = 8: 1) to give 10 (12.8 mg, quant) as a
2922, 2176, 1507, 1447, 1385, 1255 cmꢀ1 1H NMR (500 MHz,
.
CDCl3) d: 7.34 (1H, t, J = 1.5 Hz), 7.21 (1H, s), 6.28 (1H, s), 5.39
(1H, t, J = 7.0 Hz), 5.17 (1H, t, J = 7.0 Hz), 5.13 (1H, t, J = 7.8 Hz),
5.11 (1H, t, J = 7.5 Hz), 2.91 (2H, s), 2.45 (2H, t, J = 7.6 Hz), 2.25
(2H, q, J = 7.5 Hz), 2.12–2.06 (6H, m), 2.02–1.97 (6H, m), 1.67
(3H, s), 1.60 (6H, s), 1.59 (3H, s), 0.17 (9H, s). 13C NMR (125 MHz,
CDCl3) d: 142.5, 138.8, 135.8, 135.0, 134.7, 129.5, 125.9, 125.0,
124.5, 124.2, 123.7, 111.1, 104.7, 86.6, 39.7 (2C), 39.4, 30.0, 28.5,
26.7 (2C), 26.6, 25.0, 16.1, 16.0 (3C), 0.1 (3C). HR-ESI MS: m/z
473.3237, calcd for C30H46OSiNa. Found: 473.3216.
colorless oil. IR (KBr): 2922, 1742, 1507, 1447, 1381, 1235 cmꢀ1
.
1H NMR (500 MHz, CDCl3) d: 7.33 (1H, s), 7.21 (1H, s), 6.27 (1H,
s), 5.45 (1H, t, J = 6.7 Hz), 5.17 (1H, t, J = 6.7 Hz), 5.12 (1H, t,
J = 6.5 Hz), 5.11 (1H, t, J = 6.5 Hz), 4.45 (2H, s), 2.45 (2H, t,
J = 7.6 Hz), 2.24 (2H, q, J = 7.3 Hz), 2.14 (2H, q, J = 7.3 Hz), 2.08–
2.05 (4H, m), 2.07 (3H, s), 2.03–1.97 (6H, m), 1.65 (3H, s), 1.60
(6H, s), 1.59 (3H, s). 13C NMR (125 MHz, CDCl3) d: 171.0, 142.5,
138.8, 135.7, 135.0, 134.3, 130.0, 129.6, 125.0, 124.7, 124.2,
123.7, 111.1, 70.3, 39.7 (2C), 39.0, 28.4, 26.7, 26.6, 26.4, 25.0,
21.0, 16.0 (3C), 14.0. HR-ESI MS: m/z 435.2883, calcd for C27H40O3-
Na. Found: 435.2875.
4.2.9. 3-((3E,7E,11E,15E)-4,8,12,16-Tetramethylnonadeca-3,7,11,
15-tetraen-18-yn-1-yl)furan (13a)
TBAF (1.0 M in THF, 0.04 mL, 0.04 mmol) was added dropwise
to a solution of 12a (13.4 mg, 0.03 mmol) in THF (0.3 mL) and
the whole mixture was stirred for 3 h. Satd NH4Cl aq was added

2108
N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
to the mixture and the whole mixture was extracted with AcOEt.
Removal of the solvent from the AcOEt extract under reduced pres-
sure gave a crude product, which was purified by SiO2 column (n-
hexane/toluene = 10:1) to give 13a (10.3 mg, 92%) as a colorless oil.
IR (KBr): 3297, 2922, 2855, 1505, 1447, 1385, 1165, 1026 cmꢀ1. 1H
NMR (500 MHz, CDCl3) d: 7.34 (1H, t, J = 1.5 Hz), 7.21 (1H, s), 6.28
(1H, s), 5.41 (1H, td, J = 7.0, 1.2 Hz), 5.17 (1H, t, J = 6.4 Hz), 5.12 (1H,
t, J = 6.5 Hz), 5.11 (1H, t, J = 6.8 Hz), 2.87 (2H, s), 2.45 (2H, t,
J = 7.6 Hz), 2.24 (2H, q, J = 7.3 Hz), 2.12–2.05 (7H, m), 2.02–1.97
(6H, m), 1.68 (3H, s), 1.60 (6H, s), 1.59 (3H, s). 13C NMR
(125 MHz, CDCl3) d: 142.5, 138.8, 135.8, 135.0, 134.6, 129.3,
126.2, 125.0, 124.5, 124.2, 123.7, 111.1, 82.2, 70.0, 39.7, 39.4,
29.7, 28.6, 28.5, 26.7 (2C), 26.6, 25.0, 16.1, 16.0 (2C), 15.9. HR-ESI
MS: m/z 401.2827, calcd for C27H38ONa. Found: 401.2820.
tracted with Et2O. Removal of the solvent from the Et2O extract un-
der reduced pressure gave a crude product, which was purified by
SiO2 column (n-hexane/toluene = 10:1) to give 14 (9.3 mg, 41%) as
a colorless oil. IR (KBr): 2924, 2855, 1507, 1447, 1383, 1026 cmꢀ1
.
1H NMR (500 MHz, CDCl3) d: 7.34 (1H, t, J = 1.5 Hz), 7.21 (1H, s),
6.28 (1H, s), 5.17 (1H, t, J = 7.8 Hz), 5.15 (1H, t, J = 7.5 Hz), 5.11
(2H, t, J = 7.0 Hz), 2.45 (2H, t, J = 7.6 Hz), 2.27–2.13 (6H, m), 2.10–
2.05 (6H, m), 2.02–1.97 (6H, m), 1.77 (3H, t, J = 2.4 Hz), 1.60
(12H, s). 13C NMR (125 MHz, CDCl3) d: 142.5, 138.8, 135.8, 135.0,
134.8, 133.7, 125.2, 125.0, 124.3, 124.2, 123.7, 111.1, 79.1, 75.6,
39.7, 39.6 (2C), 39.1, 28.5, 26.7, 26.6 (2C), 25.0, 18.0, 16.0 (3C),
15.8, 3.5. HR-ESI MS: m/z 429.3136, calcd for C29H42ONa. Found:
429.3133.
4.3. Synthesis of photoaffinity probes
4.2.10. ((5E,9E,13E,17E)-20-(Furan-3-yl)-5,9,13,17-
tetramethylicosa-5,9,13,17-tetraen-1-yn-1-yl)trimethylsilane
(12b)
4.3.1. Di-tert-butyl ((14R,19R)-1,32-diazido-13,20-dioxo-3,6,9,
24,27,30-hexaoxa-16,17-dithia-12,21-diazadotriacontane-14,
19-diyl)dicarbamate (16)
1-Trimethylsilylpropyne (0.28 mL, 1.88 mmol) was added drop-
wise to
a
solution of n-BuLi (1.67 M in n-hexane, 1.13 mL,
11-Azido-3,6,9-trioxaundecane-1-amine (45.8 mg, 0.21 mmol),
HOBt (36.7 mg, 0.24 mmol), and EDCIꢁHCl (46 mg, 0.24 mmol)
1.88 mmol) in THF (5.2 mL) at ꢀ40 °C, and the whole mixture
was stirred for 15 min at ꢀ40 °C and for 20 min at ꢀ20 °C. 9
(408 mg, 0.94 mmol) was added to the mixture at ꢀ40 °C and the
whole mixture was gradually warmed to rt with stirring for 16 h.
Satd NH4Cl aq was added to the mixture and the whole mixture
was extracted with Et2O. Removal of the solvent from the Et2O ex-
tract under reduced pressure gave a crude product, which was
purified by SiO2 column (n-hexane/toluene = 10:1) to give 12b
(296 mg, 68%) as a colorless oil. IR (KBr): 2922, 2176, 1507, 1447,
were added to a solution of Boc-L-cystine (15, 44 mg, 0.1 mmol)
in THF (1 mL) and the whole mixture was stirred for 6 h at room
temperature. After evaporation under reduced pressure to dryness,
the crude product was purified by SiO2 column (CHCl3/MeOH/
H2O = 60:3:1, lower phase) to give 16 (87.5 mg, quant). IR (KBr):
3327, 2872, 2106, 1712, 1672, 1350, 1105 cmꢀ1
.
1H NMR
(125 MHz, CDCl3) d: 7.67 (2H, br s), 5.58 (2H, d, J = 9.2 Hz), 4.77
(2H, br s), 3.67–3.53 (24H, m), 3.47–3.41 (4H, m), 3.37 (4H, t,
J = 5.0 Hz), 2.95 (4H, m), 1.44 (18H, s). 13C NMR (500 MHz, CDCl3)
d: 170.3, 155.6, 79.9, 70.4, 70.3, 70.1, 69.8, 69.4, 54.2, 50.5, 46.0,
39.2, 28.2. ESI MS m/z: 863 [M+Na]+. HR-ESI MS m/z: 863.3731,
calcd for C32H60N10O12S2Na. Found: 863.3740.
1385, 1250 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 7.34 (1H, s), 7.21
.
(1H, s), 6.28 (1H, s), 5.17 (1H, t, J = 6.0 Hz), 5.16 (1H, t, J = 6.2 Hz),
5.11 (2H, t, J = 6.4 Hz), 2.45 (2H, t, J = 7.6 Hz), 2.31–2.22 (4H, m),
2.18 (2H, t, J = 7.6 Hz), 2.10–2.04 (6H, m), 1.99 (6H, q-like), 1.60
(12H, s), 0.14 (9H, s). 13C NMR (125 MHz, CDCl3) d: 142.8, 139.1,
136.0, 135.2, 135.0, 133.6, 125.8, 125.2, 124.6, 124.4, 124.0,
111.3, 107.7, 84.8, 40.0 (2C), 39.9, 38.9, 28.7, 26.9 (2C), 26.8,
25.3, 19.5, 16.3 (3C), 16.1, 0.4 (3C). HR-ESI MS: m/z 487.3375, calcd
for C31H48OSiNa. Found: 487.3372.
4.3.2. (2R,20R)-3,30-Disulfanediylbis(2-amino-N-(2-(2-(2-(2-azido-
ethoxy)ethoxy)ethoxy)ethyl)propanamide) (17)
TFA (0.8 mL) was added to a solution of 16 (87.5 mg, 0.1 mmol)
in CH2Cl2 (2 mL) at 0 °C and the whole mixture was stirred for 12 h
at rt. After evaporation under reduced pressure to dryness, satd
NaHCO3 aq was added to the mixture and the whole mixture
was extracted with CH2Cl2. The CH2Cl2 layer was dried over anhy-
drous Na2SO4, and the solvent was removed under reduced pres-
sure to give 17 (58.5 mg, 88%). IR (KBr): 3368, 2870, 2106, 1664,
4.2.11. 3-((3E,7E,11E,15E)-4,8,12,16-Tetramethylicosa-3,7,11,15-
tetraen-19-yn-1-yl)furan (13b)
TBAF (1.0 M in THF, 0.75 mL, 0.75 mmol) was added dropwise
to a solution of 12b (290 mg, 0.62 mmol) in THF (6.2 mL) and the
whole mixture was stirred for 3 h. Satd NH4Cl aq was added to
the mixture and the whole mixture was extracted with AcOEt. Re-
moval of the solvent from the AcOEt extract under reduced pres-
sure gave a crude product, which was purified by SiO2 column
(n-hexane/toluene = 10:1) to give 13b (244 mg, quant) as a color-
less oil. IR (KBr): 3306, 2922, 2855, 1505, 1447, 1383, 1165,
1525, 1350, 1105 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 7.74 (2H, t-
.
like), 3.65–3.55 (22H, m), 3.52 (4H, t, J = 4.8 Hz), 3.44–3.36 (4H,
m), 3.33 (4H, t, J = 4.8 Hz), 3.21 (2H, dd, J = 13.8, 4.0 Hz), 2.71
(2H, dd, J = 13.7, 8.6 Hz), 1.93 (4H, br). 13C NMR (125 MHz, CDCl3)
d: 173.1, 70.5, 70.4, 70.1, 69.8, 69.5, 53.7, 50.5, 43.5, 38.9. ESI MS
m/z: 641 [M+H]+. HR-ESI MS m/z: 641.2863, calcd for C22H45N10O8-
S2. Found: 641.2895.
1026 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 7.34 (1H, s), 7.21 (1H,
.
s), 6.28 (1H, s), 5.17 (2H, t, J = 7.0 Hz), 5.11 (2H, t, J = 7.0 Hz), 2.45
(2H, t, J = 7.6 Hz), 2.29–2.19 (6H, m), 2.11–2.04 (6H, m), 2.00–
1.96 (6H, m), 1.94 (1H, t, J = 2.4 Hz), 1.61 (3H, s), 1.60 (9H, s). 13C
NMR (125 MHz, CDCl3) d: 142.5, 138.8, 135.7, 135.0, 134.7, 133.1,
125.6, 125.0, 124.4, 124.2, 123.7, 111.1, 84.4, 68.2, 39.7 (2C),
39.6, 38.4, 28.5, 26.7, 26.6 (2C), 25.0, 17.6, 16.0 (3C), 15.8. HR-ESI
MS: m/z 415.2969, calcd for C28H40ONa. Found: 415.2977.
4.3.3. N,N0-((14R,19R)-1,32-Diazido-13,20-dioxo-3,6,9,24,27,
30-hexaoxa-16,17-dithia-12,21-diazadotriacontane-14,19-diyl)
bis(4-benzoylbenzamide) (18)
4-Benzoylbenzoic acid (85.9 mg, 0.38 mmol), HOBt (56.8 mg,
0.41 mmol), and EDCIꢁHCl (79.5 mg, 0.41 mmol) were added to a
solution of 17 (110.7 mg, 0.17 mmol) in THF (0.86 mL) and the
reaction mixture was stirred for 5 h. Removal of the solvent under
reduced pressure gave a crude product, which was purified by SiO2
column (CHCl3/MeOH/H2O = 40:3:1, lower phase) to give 18
(177.4 mg, 97%) as a colorless oil. IR (KBr): 3300, 2870, 2106,
4.2.12. 3-((3E,7E,11E,15E)-4,8,12,16-Tetramethylhenicosa-
3,7,11,15-tetraen-19-yn-1-yl)furan (14)
n-BuLi (2.69 M in n-hexane, 0.02 mL, 0.06 mmol) was added
dropwise to a solution of 13b (21.7 mg, 0.06 mmol) in THF
(0.3 mL) at ꢀ78 °C and the whole mixture was gradually warmed
1637, 1280, 1138 cmꢀ1 1H NMR (500 MHz, CDCl3) d: 8.39 (2H,
.
m), 7.98 (2H, d, J = 9.0 Hz), 7.91 (4H, d, J = 8.5 Hz), 7.75–7.70 (8H,
m), 7.54 (2H, t, J = 7.5 Hz), 7.42 (4H, t, J = 7.5 Hz), 5.67 (2H, m),
3.57–3.41 (28H, m), 3.24 (4H, t, J = 5.0 Hz), 3.07 (4H, m). 13C NMR
(125 MHz, CDCl3) d: 195.6, 170.3, 166.6, 140.2, 136.7, 136.4,
to rt with stirring. Iodomethane (4 lL, 0.06 mmol) was added to
the mixture at ꢀ78 °C and the whole mixture was stirred for
17 h. H2O was added to the mixture and the whole mixture was ex-

N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
2109
132.8 (4C), 129.9 (4C), 129.8 (4C), 128.3 (4C), 127.2, 70.4, 70.3,
70.1, 69.8, 69.4, 53.8, 50.4, 46.1, 39.4. ESI MS m/z: 1079 [M+Na]+.
HR-ESI MS m/z: 1079.3731, calcd for C50H60N10O12S2Na. Found:
1079.3759.
4.3.8. N-(3-Azidopropyl)-2-iodoacetamide (21d)
Using the same procedure as that for 21a, 3-azidopropan-1-
amine27 (20.5 mg, 0.205 mmol) was converted to 21d (46.9 mg,
85%). 1H NMR (600 MHz, CDCl3) d: 6.56 (1H, br), 3.69 (2H, s), 3.40–
3.33 (4H, m), 1.80 (2H, quint, J = 6.6 Hz). 13C NMR (150 MHz, CDCl3)
d: 167.4, 49.2, 38.0, 28.4, ꢀ0.6. ESI MS m/z: 291 [M+Na]+. HR-ESI MS
m/z: 290.9719, calcd for C5H9N4OINa. Found: 290.9707.
4.3.4. N,N0-((14R,19R)-13,20-Dioxo-1,32-bis(4-((5-((3aS,4S,6aR)-
2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)
methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,24,27,30-hexaoxa-16,17-dithia-
12,21-diazadotriacontane-14,19-diyl)bis(4-benzoylbenzamide)
(20)
4.3.9. N-((R)-1-Azido-13,18-dioxo-30-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-
CuSO4 (0.04 M in water, 8.1
1,2,3-triazol-4-yl)methyl]amine (TBTA, 1.7 mg, 3.22
(18.5 mg, 66.0 mol) were added to a solution of 18 (34.0 mg,
32.2 mol) in t-BuOH/H2O (1:1, 0.4 mL). The reaction mixture
was degassed using the freeze-pump-thaw cycle method and so-
dium ascorbate (10 mM in degassed water, 0.161 mL, 1.61 mol)
l
L, 0.322
l
mol), tris[(1-benzyl-1H-
yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,22,25,28-
hexaoxa-15-thia-12,19-diazatriacontan-17-yl)-4-
benzoylbenzamide (22a)
l
mol), and 19
l
l
TCEPꢁHCl (3.5 mg, 0.012 mmol) and NaHCO3 (4.1 mg,
0.05 mmol) were added to a solution of 20 (19.7 mg, 0.012 mmol)
in DMF/H2O (4:1, 0.25 mL) and the whole mixture was stirred for
1 h. 21a (11.3 mg, 0.029 mmol) was added to the mixture and
the whole mixture was further stirred for 1 h. Removal of the sol-
vent under reduced pressure gave a crude product, which was
purified by SiO2 column (CHCl3/MeOH/H2O = 15:3:1, lower phase)
to give 22a (19.3 mg, 74%) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d: 8.54 (1H, d, J = 7.9 Hz), 8.10 (1H, t, J = 5.2 Hz), 8.02 (2H,
d, J = 8.0 Hz), 7.85 (2H, d, J = 8.0 Hz), 7.85 (1H, s), 7.83 (2H, d,
J = 7.5 Hz), 7.61 (1H, t, J = 7.5 Hz), 7.56 (1H, t, J = 5.2 Hz), 7.50
(2H, t, J = 7.5 Hz), 7.30 (1H, t, J = 5.2 Hz), 6.80 (1H, br s), 5.49 (1H,
br s), 4.89 (1H, q, J = 7.1 Hz), 4.57–4.45 (5H, m), 4.31 (1H, dd-like),
3.92–3.87 (2H, m), 3.66–3.45 (24H, m), 3.46 (2H, t, J = 5.5 Hz), 3.37
(2H, t, J = 4.9 Hz), 3.34 (2H, d, J = 2.4 Hz), 3.13–3.02 (3H, m), 2.88
(1H, dd, J = 12.8, 4.9 Hz), 2.71 (1H, d, J = 12.8 Hz), 2.18–2.07 (2H,
m), 1.64–1.25 (6H, m). 13C NMR (150 MHz, CDCl3) d: 196.0,
173.4, 170.8, 169.8, 166.8, 164.2, 144.6, 140.3, 137.0, 136.7, 132.9
(2C), 130.1 (2C), 130.0 (2C), 128.4 (2C), 127.5, 123.7, 70.6, 70.5,
70.42, 70.40, 70.3, 70.2, 70.1, 69.9, 69.53, 69.45, 69.2, 61.8, 60.2,
55.6, 53.4, 50.6, 50.3, 40.5, 39.7, 39.5, 35.9, 35.3, 35.1, 34.8, 31.9,
29.7, 29.6, 27.9, 25.2. ESI MS m/z: 1091 [M+Na]+. HR-ESI MS m/z:
1091.4460, calcd for C48H68N12O12S2Na. Found: 1091.4419.
l
was added. The reaction mixture was stirred for 24 h at room tem-
perature. Removal of the solvent under reduced pressure gave a
crude product, which was purified by SiO2 column (CHCl3/MeOH/
H2O = 15:3:1, lower phase) to give 20 (47.0 mg, 90%) as a colorless
oil. IR (KBr): 3279, 2924, 2858, 1657, 1539, 1450, 1277, 1118 cmꢀ1
.
1H NMR (500 MHz, CDCl3) d: 8.39 (2H, d, J = 8.0 Hz), 8.27 (2H, m),
7.95 (4H, d, J = 8.0 Hz), 7.74–7.69 (10H, m), 7.55 (2H, d,
J = 5.5 Hz), 7.52 (2H, d, J = 8.0 Hz), 7.42 (4H, t, J = 8.0 Hz), 7.01
(2H, s), 6.20 (2H, s), 5.28 (2H, m), 4.42–4.38 (10H, m), 4.19 (2H,
m), 3.75 (4H, t, J = 5.0 Hz), 3.52–3.41 (22H, m), 3.36 (2H, m), 3.12
(4H, d, J = 7.0 Hz), 3.00 (2H, m), 2.77 (2H, dd, J = 12.5, 4.5 Hz),
2.62 (2H, d, J = 12.5 Hz), 2.09–2.07 (4H, m), 1.60–1.45 (8H, m),
1.38–1.18 (4H, m). 13C NMR (125 MHz, CDCl3) d: 195.9, 173.2,
170.8, 167.0, 164.5, 144.8, 140.2, 136.9, 136.8, 132.9 (4C), 130.1
(4C), 130.0 (4C), 128.4 (4C), 127.6, 123.5, 70.5, 70.3, 70.2, 69.5,
69.2, 61.8, 60.2, 55.7, 53.7, 50.2, 43.3, 40.5, 39.5, 35.5, 34.6, 28.1,
28.0, 25.3. ESI MS m/z: 832 [M+2Na]2+. HR-ESI MS m/z: 832.3012,
calcd for C76H98N16O16S4Na2. Found: 832.3023.
4.3.5. N-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl)-2-iodo-
acetamide (21a)
Iodoacetic anhydride (100 mg, 0.28 mmol) and Et3N (100
l
L,
4.3.10. N-((R)-1-Azido-9,14-dioxo-26-(4-((5-((3aS,4S,6aR)-2-oxo-
hexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)methyl)-
1H-1,2,3-triazol-1-yl)-2,5,18,21,24-pentaoxa-11-thia-8,
0.71 mmol) were added to a solution of 11-azido-3,6,9-trioxaunde-
cane-1-amine (52.5 mg, 0.24 mmol) in CH2Cl2 (1 mL) at 0 °C and
the whole mixture was stirred for 30 min. Satd NaHCO3 aq was
added to the mixture and the whole mixture was extracted with
AcOEt. Removal of the solvent from the AcOEt extract under re-
duced pressure gave 21a (76.8 mg, 83%). 1H NMR (600 MHz, CDCl3)
d: 6.85 (1H, br), 3.64 (2H, s), 3.62–3.56 (10H, m), 3.50 (2H, t,
J = 5.2 Hz), 3.38 (2H, q, J = 5.2 Hz), 3.33 (2H, t, J = 5.2 Hz). 13C
NMR (150 MHz, CDCl3) d: 167.4, 70.62, 70.57, 70.5, 70.3, 70.0,
69.3, 50.6, 40.1, ꢀ0.5. ESI MS m/z: 409 [M+Na]+. HR-ESI MS m/z:
409.0349, calcd for C10H19N4O4INa. Found: 409.0334.
15-diazahexacosan-13-yl)-4-benzoylbenzamide (22b)
Using the same procedure as that for 22a, compound 22b
(16.2 mg, 70%) was obtained from 21b (8.6 mg, 0.025 mmol) and
20 (18.4 mg, 0.011 mmol) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d: 8.48 (1H, d, J = 7.3 Hz), 8.09 (1H, t, J = 4.9 Hz), 8.02 (2H,
d, J = 8.2 Hz), 7.84 (1H, s), 7.83 (2H, d, J = 8.2 Hz), 7.80 (2H, d,
J = 7.6 Hz), 7.61 (1H, t, J = 7.6 Hz), 7.50 (2H, t, J = 7.6 Hz), 7.42
(2H, t-like), 6.87 (1H, s), 5.60 (1H, br s), 4.89 (1H, q, J = 7.3 Hz),
4.58–4.46 (5H, m), 4.31 (1H, dd-like), 3.92–3.85 (2H, m), 3.66–
3.41 (22H, m), 3.37 (2H, t, J = 4.9 Hz), 3.34 (2H, s), 3.12–3.02 (3H,
m), 2.88 (1H, dd, J = 12.8, 4.9 Hz), 2.71 (1H, d, J = 12.8 Hz), 2.19–
2.08 (2H, m), 1.65–1.25 (6H, m). 13C NMR (150 MHz, CDCl3) d:
196.0, 173.4, 170.7, 169.8, 166.8, 164.3, 144.7, 140.2, 137.0,
136.7, 132.9 (2C), 131.0, 130.1 (2C), 130.0 (2C), 128.4 (2C), 127.5,
123.7, 70.42, 70.41, 70.3, 70.2, 70.1, 69.6, 69.5, 69.4, 69.3, 61.8,
60.2, 55.6, 53.3, 50.6, 50.2, 40.5, 39.6, 39.5, 36.0, 35.4, 35.2, 34.7,
29.7, 29.3, 28.0, 27.9, 25.3. ESI MS m/z: 1047 [M+Na]+. HR-ESI MS
m/z: 1047.4175, calcd for C46H64N12O11S2Na. Found: 1047.4157.
4.3.6. N-(2-(2-(2-Azidoethoxy)ethoxy)ethyl)-2-iodoacetamide
(21b)
Using the same procedure as that for 21a, 2-(2-(2-azidoeth-
oxy)ethoxy)ethanamine25 (35 mg, 0.2 mmol) was converted to
21b (55 mg, 80%). All physical data are identical to those in the
literature.25
4.3.7. N-(5-Azidopentyl)-2-iodoacetamide (21c)
Using the same procedure as that for 21a, 5-azidopentan-1-
amine26 (25.6 mg, 0.2 mmol) was converted to 21c (49.2 mg,
83%). 1H NMR (600 MHz, CDCl3) d: 6.24 (1H, br), 3.68 (2H, s),
3.29–3.24 (4H, m), 1.64–1.52 (4H, m), 1.43–1.38 (2H, m). 13C
NMR (150 MHz, CDCl3) d: 166.9, 51.2, 40.2, 28.8, 28.4, 23.9, –0.3.
ESI MS m/z: 319 [M+Na]+. HR-ESI MS m/z: 319.0032, calcd for C7-
H13N4OINa. Found: 319.0021.
4.3.11. N-((R)-24-Azido-13,18-dioxo-1-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)
methyl)-1H-1,2,3-triazol-1-yl)-3,6,9-trioxa-16-thia-12,
19-diazatetracosan-14-yl)-4-benzoylbenzamide (22c)
Using the same procedure as that for 22a, compound 22c
(17.6 mg, 64%) was obtained from 21c (10 mg, 0.034 mmol) and
20 (22.9 mg, 0.014 mmol) as a colorless oil. 1H NMR (500 MHz,

2110
N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
CDCl3) d: 8.35 (1H, d, J = 7.3 Hz), 8.09 (1H, br s), 7.99 (2H, d,
J = 8.0 Hz), 7.82 (2H, d, J = 8.0 Hz), 7.81 (1H, s), 7.78 (2H, d,
J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.57 (1H, br s), 7.49 (2H, t,
J = 7.6 Hz), 7.29 (1H, t, J = 5.8 Hz), 6.91 (1H, br s), 5.85 (1H, br s),
4.90 (1H, q, J = 6.9 Hz), 4.56–4.47 (5H, m), 4.29 (1H, dd, J = 6.5,
5.5 Hz), 3.90–3.83 (2H, m), 3.60–3.36 (12H, m), 3.33 (2H, d,
J = 6.1 Hz), 3.29–3.20 (4H, m), 3.10–3.00 (3H, m), 2.87 (2H, dd,
J = 12.8, 4.9 Hz), 2.70 (1H, d, J = 12.8 Hz), 2.21–2.09 (2H, m), 1.65–
1.31 (12H, m). 13C NMR (150 MHz, CDCl3) d: 196.0, 173.4, 170.7,
169.5, 166.8, 164.3, 144.6, 140.3, 136.9, 136.6, 132.9 (2C), 130.1
(2C), 130.0 (2C), 128.5 (2C), 127.5, 123.7, 70.44, 70.40, 70.3, 70.2,
69.4, 69.2, 61.8, 60.4, 60.2, 55.6, 53.3, 51.2, 50.3, 40.5, 39.7, 39.6,
36.0, 35.3, 34.7, 29.7, 29.3, 28.9, 28.4, 27.9, 25.3, 24.0. ESI MS m/
z: 1001 [M+Na]+. HR-ESI MS m/z: 1001.4133, calcd for C45H62N12-
O9S2Na. Found: 1001.4102.
m). 13C NMR (150 MHz, CDCl3) d: 196.0, 173.3, 170.8, 169.7,
166.8, 164.2, 147.9, 144.7, 142.5, 140.3, 138.8, 137.0, 136.7,
135.7, 135.0, 134.8, 133.9, 132.9 (2C), 130.1 (2C), 130.0 (2C),
128.4 (2C), 127.5, 125.1, 125.0, 124.3, 124.1, 123.7, 123.5, 121.7,
111.1, 70.5, 70.43, 70.41, 70.39, 70.3, 70.2, 70.1, 69.6, 69.5, 69.4,
69.3, 61.8, 60.2, 55.6, 53.5, 50.2, 50.0, 40.5, 39.7, 39.67, 39.6, 39.5,
39.3, 36.0, 35.2, 35.1, 34.8, 29.7, 28.4, 27.9, 27.85, 26.73, 26.69,
26.6, 25.2, 25.0, 24.5, 16.05, 16.03, 16.0. ESI MS m/z: 1484
[M+Na]+. HR-ESI MS m/z: 1483.7583, calcd for C76H108N12O13S2Na.
Found: 1483.7498.
4.3.14. 4-Benzoyl-N-((R)-1-(4-((3E,7E,11E,15E)-18-(furan-3-yl)-
3,7,11,15-tetramethyloctadeca-3,7,11,15-tetraen-1-yl)-1H-1,2,3-
triazol-1-yl)-10,15-dioxo-27-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,19,22,25-
pentaoxa-12-thia-9,16-diazaheptacosan-14-yl)benzamide (23b)
Using the same procedure as that for 23a, compound 23b
(8.9 mg, 40%) was obtained from 22b (16.2 mg, 0.016 mmol) and
13b (6.0 mg, 0.016 mmol) as a colorless oil. 1H NMR (500 MHz,
CDCl3) d: 8.49 (1H, d, J = 7.3 Hz), 8.09 (1H, t, J = 4.9 Hz), 8.01 (2H,
d, J = 8.5 Hz), 7.83 (1H, s), 7.81 (2H, d, J = 8.5 Hz), 7.79 (2H, d,
J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.49 (3H, t, J = 7.6 Hz), 7.42
(1H, br s), 7.39 (1H, br s), 7.32 (1H, s), 7.20 (1H, s), 6.78 (1H, br
s), 6.27 (1H, s), 5.62 (1H, br s), 5.15 (2H, q, J = 6.9 Hz), 5.10 (2H, t,
J = 6.4 Hz), 4.89 (1H, q, J = 7.7 Hz), 4.57–4.47 (7H, m), 4.29 (1H,
dd-like), 3.89–3.85 (4H, m), 3.64–3.46 (16H, m), 3.42 (4H, q,
J = 5.5 Hz), 3.36 (2H, s), 3.12–3.04 (3H, m), 2.88 (1H, dd, J = 12.8,
4.3 Hz), 2.79 (2H, t, J = 7.9 Hz), 2.70 (1H, d, J = 12.8 Hz), 2.44 (2H,
t, J = 7.6 Hz), 2.30 (2H, t, J = 7.9 Hz), 2.23 (2H, q, J = 7.3 Hz), 2.18–
2.11 (2H, m), 2.07–1.93 (12H, m), 1.63 (3H, s), 1.57 (9H, s), 1.58–
1.25 (6H, m). 13C NMR (150 MHz, CDCl3) d: 196.0, 173.3, 170.8,
169.7, 166.8, 164.2, 142.5, 140.3, 138.8, 136.9, 136.7, 135.7,
135.0, 134.8, 133.8, 132.9 (2C), 130.1 (2C), 130.0 (2C), 128.4 (2C),
127.5, 125.1, 125.0, 124.2, 124.1, 123.7, 111.1, 70.40, 70.36, 70.3,
70.24, 70.18, 69.52, 69.45, 69.2, 61.7, 60.8, 55.9, 53.4, 50.3, 50.2,
40.9, 39.69, 39.67, 39.6, 39.5, 39.2, 36.1, 35.4, 35.2, 34.6, 29.7,
28.4. 27.8. 26.8. 26.7. 25.2, 25.0, 24.5, 16.05, 15.96. ESI MS m/z:
1440 [M+Na]+. HR-ESI MS m/z: 1439.7236, calcd for C74H104N12O12-
S2Na. Found: 1439.7236.
4.3.12. N-((R)-22-Azido-13,18-dioxo-1-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)
methyl)-1H-1,2,3-triazol-1-yl)-3,6,9-trioxa-16-thia-12,
19-diazadocosan-14-yl)-4-benzoylbenzamide (22d)
Using the same procedure as that for 22a, compound 22d
(22.5 mg, 72%) was obtained from 21d (10.0 mg, 0.037 mmol)
and 20 (26.6 mg, 0.016 mmol) as a colorless oil.
1H NMR (500 MHz, CDCl3) d: 8.41 (1H, d, J = 7.3 Hz), 8.22 (1H, t,
J = 4.9 Hz), 8.01 (2H, d, J = 8.5 Hz), 7.84 (2H, d, J = 8.5 Hz), 7.83 (1H,
s), 7.80 (2H, d, J = 7.5 Hz), 7.61 (1H, t, J = 7.5 Hz), 7.57 (1H, t,
J = 5.8 Hz), 7.50 (2H, t, J = 7.5 Hz), 7.39 (1H, t-like), 6.86 (1H, br s),
5.48 (1H, br s), 4.92 (1H, q, J = 7.3 Hz), 4.57–4.44 (5H, m), 4.31
(1H, dd, J = 7.5, 5.0 Hz), 3.93–3.83 (2H, m), 3.62–3.38 (12H, m),
3.36–3.31 (6H, m), 3.11–3.01 (3H, m), 2.88 (1H, dd, J = 12.8,
4.9 Hz), 2.70 (1H, d, J = 12.8 Hz), 2.21–2.08 (2H, m), 1.80 (2H, quint,
J = 6.7 Hz), 1.67–1.25 (6H, m). 13C NMR (150 MHz, CDCl3) d: 196.0,
173.4, 170.7, 169.8, 166.9, 164.3, 144.6, 140.3, 136.9, 136.6, 132.9
(2C), 130.1 (2C), 130.0 (2C), 128.4 (2C), 127.5, 123.7, 70.42,
70.39, 70.3, 70.2, 69.4, 69.2, 61.8, 60.2, 55.6, 53.2, 50.3, 49.1, 40.5,
39.5, 37.3, 36.0, 35.5, 35.3, 34.7, 29.7, 29.3, 28.6, 28.4, 27.9, 25.2.
ESI MS m/z: 973 [M+Na]+. HR-ESI MS m/z: 973.3793, calcd for C43-
H58N12O9S2Na. Found: 973.3789.
4.3.13. 4-Benzoyl-N-((R)-1-(4-((3E,7E,11E,15E)-18-(furan-3-yl)-
3,7,11,15-tetramethyloctadeca-3,7,11,15-tetraen-1-yl)-1H-1,2,3-
triazol-1-yl)-13,18-dioxo-30-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,22,25,28-
hexaoxa-15-thia-12,19-diazatriacontan-17-yl)benzamide (23a)
4.3.15. 4-Benzoyl-N-((R)-24-(4-((3E,7E,11E,15E)-18-(furan-3-yl)-
3,7,11,15-tetramethyloctadeca-3,7,11,15-tetraen-1-yl)-1H-1,2,3-
triazol-1-yl)-13,18-dioxo-1-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-
CuSO4 (0.04 M in water, 22
l
L, 0.88
l
mol) was added to a solu-
yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9-trioxa-16-
thia-12,19-diazatetracosan-14-yl)benzamide (23c)
tion of 13b (6.4 mg, 0.016 mmol) and 22a (15.9 mg, 0.015 mmol) in
t-BuOH/H2O (1:1, 0.6 mL). The reaction mixture was degassed
using the freeze-pump-thaw method (three cycles) and sodium
Using the same procedure as that for 23a, compound 23c
(13.9 mg, 65%) was obtained from 22c (15.3 mg, 0.016 mmol)
ascorbate (1.7 mg, 8.86
lmol) was added. The reaction mixture
and 13b (9.0 mg, 0.023 mmol) as a
colorless oil. 1H NMR
was stirred for 24 h at room temperature. Removal of the solvent
under reduced pressure gave a crude product, which was purified
by SiO2 column (CHCl3/MeOH/H2O = 15:3:1, lower phase) to give
23a (10.3 mg, 48%) as a colorless oil. 1H NMR (500 MHz, CDCl3)
d: 8.51 (1H, d, J = 7.3 Hz), 8.04 (1H, t, J = 4.3 Hz), 8.02 (2H, d,
J = 8.0 Hz), 7.83 (2H, d, J = 8.0 Hz), 7.83 (1H, s), 7.79 (2H, d,
J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.57 (1H, t, J = 5.2 Hz), 7.49
(2H, t, J = 7.6 Hz), 7.41 (1H, s), 7.35 (1H, br s), 7.33 (1H, s), 7.20
(1H, s), 6.77 (1H, br s), 6.27 (1H, s), 5.60 (1H, br s), 5.15 (2H, q,
J = 6.1 Hz), 5.10 (2H, t, J = 6.7 Hz), 4.89 (1H, q, J = 7.1 Hz), 4.55–
4.45 (7H, m), 4.30 (1H, dd-like), 3.90–3.83 (4H, m), 3.61–3.37
(22H, m), 3.47–3.42 (2H, m), 3.34 (2H, d, J = 1.8 Hz), 3.13–3.02
(3H, m), 2.87 (1H, dd, J = 12.8, 4.9 Hz), 2.79 (2H, t, J = 8.2 Hz),
2.71 (1H, d, J = 12.8 Hz), 2.44 (2H, t, J = 7.6 Hz), 2.31 (2H, t,
J = 7.9 Hz), 2.23 (2H, q, J = 7.3 Hz), 2.17–2.11 (2H, m), 2.09–1.94
(12H, m), 1.63 (3H, s), 1.59 (6H, s), 1.58 (3H, s), 1.53–1.25 (6H,
(500 MHz, CDCl3) d: 8.55 (1H, d, J = 7.3 Hz), 8.12 (1H, t-like), 8.02
(2H, d, J = 8.5 Hz), 7.83 (2H, d, J = 8.5 Hz), 7.80 (1H, s), 7.79 (2H,
d, J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.49 (2H, t, J = 7.6 Hz), 7.45
(1H, t-like), 7.33 (1H, s), 7.25 (1H, s), 7.20 (1H, s), 6.87 (1H, br s),
5.73–5.66 (1H, m), 5.72 (1H, s), 5.15 (2H, q, J = 7.9 Hz), 5.10 (2H,
t, J = 6.7 Hz), 4.92 (1H, q, J = 7.1 Hz), 4.54–4.43 (5H, m), 4.32–4.27
(3H, m), 3.90–3.86 (2H, m), 3.61–3.35 (12H, m), 3.32 (2H, s), 3.23
(2H, q, J = 6.9 Hz), 3.11–2.99 (3H, m), 2.88 (1H, dd, J = 12.2,
4.9 Hz), 2.80–2.73 (3H, m), 2.44 (2H, t, J = 7.6 Hz), 2.30 (2H, t,
J = 7.9 Hz), 2.23 (2H, q, J = 7.5 Hz), 2.19–2.10 (2H, m), 2.07–1.95
(12H, m), 1.89–1.83 (2H, m), 1.63 (3H, s), 1.59 (6H, s), 1.58 (3H,
s), 1.67–1.29 (10H, m). 13C NMR (150 MHz, CDCl3) d: 196.0,
173.4, 170.8, 169.5, 166.9, 164.2, 148.0, 144.6, 142.5, 140.3,
138.8, 136.9, 136.6, 135.7, 135.0, 134.8, 133.8, 132.9 (2C), 130.1
(2C), 130.0 (2C), 128.4 (2C), 127.5, 125.2, 125.0, 124.3, 124.1,
123.7, 123.6, 120.7, 111.1, 70.42, 70.39, 70.3, 70.2, 69.4, 69.2,

N. Kotoku et al. / Bioorg. Med. Chem. 22 (2014) 2102–2112
2111
61.8, 60.2, 55.6, 53.4, 50.2, 49.9, 40.5, 39.69, 39.67, 39.6, 39.52,
39.47, 39.2, 36.0, 35.2, 34.8, 29.8, 29.7, 28.6, 28.4, 27.94, 27.90,
26.73, 26.69, 26.6, 25.2, 25.0, 24.4, 23.7, 16.05, 16.04, 15.9. ESI
MS m/z: 1394 [M+Na]+. HR-ESI MS m/z: 1393.7245, calcd for C73-
H102N12O10S2Na. Found: 1393.7181.
one week from implantation, analogue 13b was administered on
every other day for 2 week (total 7 times) by oral administration
as a suspension in 1% sodium carboxymethyl cellulose (CMC-Na).
Then, tumor was isolated and weighed to calculate the inhibition
ratio. The control group and the group treated with analogue 13b
consisted of four mice each in this study.
4.3.16. 4-Benzoyl-N-((R)-22-(4-((3E,7E,11E,15E)-18-(furan-3-yl)-
3,7,11,15-tetramethyloctadeca-3,7,11,15-tetraen-1-yl)-1H-1,2,3-
triazol-1-yl)-13,18-dioxo-1-(4-((5-((3aS,4S,6aR)-2-
oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9-trioxa-16-
thia-12,19-diazadocosan-14-yl)benzamide (23d)
Using the same procedure as that for 23a, compound 23d
(14.4 mg, 60%) was obtained from 22d (16.6 mg, 0.02 mmol) and
13b (6.7 mg, 0.02 mmol) as a colorless oil.
4.4.4. Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from DU145 cells were prepared under norm-
oxic or hypoxic conditions according to the method described in
our previous report.8
The 50-biotinylated double-stranded oligonucleotides, which
contained the binding sites of Sp1 (Sp1_F: 50-CAG CCC GCA CC
CCCCGCCCCG CTC TTG G-biotin-30; Sp1_R: 50-C CAA GAG
CGGGGCGGGG GG TGC GGG CTG-30), were synthesized by Hokka-
ido System Science Co. Ltd (Hokkaido, Japan) and used as probes.
The underlined sequence is the consensus sequence of Sp1.
EMSA was performed using a LightShift chemiluminescent
EMSA kit (Pierce Biotechnology, Rockford, IL, USA), according to
the manufacturer’s instructions. Briefly, the nuclear proteins
1H NMR (500 MHz, CDCl3) d: 8.58 (1H, d, J = 7.9 Hz), 8.13 (1H, t,
J = 5.5 Hz), 8.02 (2H, d, J = 8.5 Hz), 7.90 (1H, t, J = 4.9 Hz), 7.83 (2H,
d, J = 8.5 Hz), 7.82 (1H, s), 7.80 (2H, d, J = 7.6 Hz), 7.60 (1H, t,
J = 7.6 Hz), 7.49 (2H, t, J = 7.6 Hz), 7.38 (1H, br s), 7.36 (1H, s),
7.33 (1H, s), 7.20 (1H, s), 6.93 (1H, s), 6.27 (1H, s), 5.83 (1H, s),
5.15 (2H, q, J = 8.3 Hz), 5.10 (2H, t, J = 6.1 Hz), 4.95 (1H, q,
J = 7.0 Hz), 4.53 (4H, br s), 4.44 (1H, dd, J = 15.3, 4.3 Hz), 4.38 (2H,
t, J = 6.1 Hz), 4.31 (1H, t-like), 3.91–3.87 (2H, m), 3.60 (2H, d,
J = 4.3 Hz), 3.56–3.48 (12H, m), 3.34 (2H, br s), 3.34–3.22 (2H, m),
3.12–3.07 (2H, m), 3.02 (1H, dd, J = 14.0, 5.5 Hz), 2.89 (1H, dd,
J = 12.8 Hz, 4.3 Hz), 2.79–2.74 (3H, m), 2.44 (2H, t, J = 7.6 Hz),
2.29 (2H, t, J = 7.9 Hz), 2.23 (2H, q, J = 7.3 Hz), 2.15–1.93 (14H,
m), 1.62 (3H, s), 1.58 (9H, s), 1.62–1.25 (6H, m). 13C NMR
(150 MHz, DMSO-d6) d: 195.9, 174.4, 172.6, 170.6, 169.6, 166.1,
143.3, 143.0, 139.8, 139.3, 137.6, 137.1, 135.3, 134.74, 134.70,
133.5 (2C), 130.1 (2C), 129.8 (2C), 129.1 (2C), 128.2, 124.9,
124.32, 124.27, 124.1, 111.6, 70.1, 70.04, 70.03, 70.0, 69.3, 69.0,
61.3, 59.4, 56.4, 53.4, 50.1, 49.0, 47.9, 41.1, 39.2, 39.0, 36.5, 35.6,
35.2, 34.7, 34.6, 30.1, 29.4, 28.5, 28.4, 28.2, 26.6, 26.5, 26.4, 25.6,
24.9, 24.4, 16.27, 16.26, 16.23, 16.20. ESI MS m/z: 1366 [M+Na]+.
HR-ESI MS m/z: 1365.6858, calcd for C71H98N12O10S2Na. Found:
1365.6868.
(2
TrisꢁHCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol,
1% (v/v) glycerol, 100 g/mL BSA, and 50 g/mL poly(deoxyino-
lg) were pre-incubated on ice with binding buffer [10 mM
l
l
sine-deoxycytosine)] in the presence or absence of testing com-
pound (furospinosulin-1 (1) or affinity probes 23a–d, 100 or
300
lM) for 20 min, then incubated with the labeled oligonucleo-
tide probe (4 fmol per 20
lL reaction mixture) for 15 min at room
temperature. The reaction mixtures were separated on a 5% non-
denaturing polyacrylamide gel in 0.5ꢂ Tris-borate-EDTA buffer at
100 V for 60 min, then transferred to a nylon membrane (Hy-
bond-N+, GE Healthcare). The membrane was incubated with
LightShift-stabilized streptavidin—horseradish peroxidase conju-
gate, and the probe–protein complexes were visualized using an
ECL kit (Pierce Biotechnology).
Acknowledgments
This study was financially supported by a Grant-in-Aid for Sci-
entific research from the Ministry of Education, Culture, Sports, Sci-
ence, and Technology of Japan. The authors are also grateful to the
Uehara Memorial Foundation for financial support.
4.4. Biological evaluation of furospinosulin-1 analogues
4.4.1. Cell cultures
Human prostate cancer cell line DU145 and mouse sarcoma cell
S180 were cultured in RPMI 1640 supplemented with heat-inacti-
Supplementary data
vated 10% fetal bovine serum (FBS) and kanamycin (50 lg/ml) in a
humidified atmosphere of 5% CO2 at 37 °C.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.026.
4.4.2. Assay for anti-proliferative activity under hypoxic
condition
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