Concise synthesis and structure-activity relationship of furospinosulin-1, a hypoxia-selective growth inhibitor from marine sponge

Article abstract of DOI:10.1016/j.tet.2011.05.009

Structure-activity relationship of furospinosulin-1 (1), a hypoxia-selective growth inhibitor isolated from marine sponge, was investigated. Concise synthetic method of 1 was developed, and some structurally modified analogues were prepared. Biological evaluation of them revealed that the whole chemical structure was important for the hypoxia-selective growth inhibitory activity of 1. Among prepared, the desmethyl analogue 30 showed excellent hypoxia-selective inhibitory activity similar to that of 1 and also exhibited in vivo anti-tumor activity with oral administration.

Full text of DOI:10.1016/j.tet.2011.05.009

Tetrahedron 67 (2011) 6673e6678
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Concise synthesis and structureeactivity relationship of furospinosulin-1,
a hypoxia-selective growth inhibitor from marine sponge
*
Naoyuki Kotoku , Shinichi Fujioka, Chiaki Nakata, Masaki Yamada, Yuji Sumii, Takashi Kawachi,
*
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:
Received 9 April 2011
Received in revised form 2 May 2011
Accepted 3 May 2011
Available online 10 May 2011
Structureeactivity relationship of furospinosulin-1 (1), a hypoxia-selective growth inhibitor isolated
from marine sponge, was investigated. Concise synthetic method of 1 was developed, and some struc-
turally modified analogues were prepared. Biological evaluation of them revealed that the whole
chemical structure was important for the hypoxia-selective growth inhibitory activity of 1. Among
prepared, the desmethyl analogue 30 showed excellent hypoxia-selective inhibitory activity similar to
that of 1 and also exhibited in vivo anti-tumor activity with oral administration.
Keywords:
Furospinosulin-1
Ó 2011 Elsevier Ltd. All rights reserved.
Hypoxia-selective growth inhibitor
Marine sponge
Analogue compound
1. Introduction
biological study and to develop a more promising drug lead for the
treatment of cancer. Here we report the practical synthesis of
Hypoxic environment in tumor is now recognized to be an im-
portant factor for tumor growth, angiogenesis, metastasis, and re-
sponse to chemotherapy and irradiation.1 Then, the compounds,
which exhibit growth inhibitory activity against tumor cells under
hypoxic environment selectively, are expected to be novel and
promising lead for anti-cancer drugs and to be important bioprobe
to find new responsible molecules for the adaptation of hypoxia in
tumor cells.2
furospinosulin-1 (1) and its analogues together with the biological
evaluation of them (Fig. 1).
S
HN
O
In the course of our search for hypoxia-selective growth
inhibitors, we have isolated a furanosesterterpene, furospinosulin-
1 (1),3 from the Indonesian marine sponge of Dactylospongia ele-
gans. It revealed that the compound 1 showed selective growth
inhibitory activity against human prostate cancer cells DU145 un-
der 1% of low oxygen atmosphere dose-dependently, while 1 did
O
furospinosulin-1 (1)
n
R
not inhibit production of Hypoxia Inducible Factor-1
a (HIF-1a),
R = H, Et
n = 0~2
which is considered to be a major transcription factor to adapt
hypoxic environment in tumor. Moreover, orally administered
furospinosulin-1 (1) suppressed tumor growth in the mice s.c.-in-
oculated mouse sarcoma S180 cells.4 These intriguing bioactivities
of furospinosulin-1 (1) prompted us to engage in synthetic study, in
order to supply sufficient amount of the compound for further
Fig. 1. Furospinosulin-1 (1).
2. Results and discussions
2.1. Synthesis of furospinosulin-1 and analogues
Furospinosulin-1 (1) has an aromatic furan ring and a long side
chain consisting of four iterative isoprene units. As there has been
noinformationaboutstructure-requirementforthehypoxia-selective
* Corresponding authors. Tel.: þ81 6 6879 8215; fax: þ81 6 6879 8219; e-mail
addresses: kotoku@phs.osaka-u.ac.jp (N. Kotoku), kobayasi@phs.osaka-u.ac.jp
(M. Kobayashi).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.009

6674
N. Kotoku et al. / Tetrahedron 67 (2011) 6673e6678
growth inhibitory activity of 1, we decided to elucidate structur-
eeactivity relationship of 1 through synthesis and biological evalua-
tion of some structurally modified analogues.
a,b
c,d
CO2Et
R
2
O
O
12: R = OH
13: R = Br
11
Firstly, geometrically non-selective synthesis of 1 was exam-
ined, to analyze participation of the olefin geometry in the side
chain of 1. Wittig reaction between the phosphonium salt 3,5 which
was prepared from 3-(3-furyl)propan-1-ol (2),6 and commercially
available farnesylacetone (9, a mixture of C-5 isomers) provided
a mixture of four geometric isomers of 1 (Scheme 1). The natural
type (E,E,E)-isomer and the other isomers (10) were separated by
using reversed-phase HPLC. Analogues 4 and 5 having a side chain
of different lengths were also prepared as a mixture of isomers,
through the coupling reaction between 3 and geranylacetone (8) or
teplenone (10, a mixture of isomers), respectively. Furthermore
coupling reaction between 9 and a known phosphonium salt 67
gave a phenyl-analogue 7.
e,f
O
R
14: R = SO2Ph
1: R = H
PhO2S
15
Scheme 2. Reagents and conditions: (a) PCC, CH2Cl2, 81%; (b) Ph3P]C(Me)CO2Et,
toluene, 60 ꢀC, 80%; (c) DIBAL, CH2Cl2, 0 ꢀC, quant.; (d) PPh3, CBr4, CH2Cl2, quant.; (e)
15, t-BuOK, THF, ꢁ30 ꢀC, 96%; (f) LiBHEt3, Pd(dppp)Cl2, THF, 0 ꢀC, 96%.
d
a-c
O
OH
PPh3+Br-
d
1
O
O
O
2
3
O
d
1' (mixture of unnatural three isomers)
O
O
5 (mixture of four isomers)
4 (mixture of two isomers)
8
PPh3+Br-
d
O
n
6
9: n = 1
10: n = 2
7 (mixture of four isomers)
Scheme 1. Reagents and conditions: (a) TsCl, pyridine, 89%; (b) LiBr, acetone, 94%; (c) PPh3, toluene, 120 ꢀC, 57%; (d) 8 or 9 or 10, n-BuLi, THF, 1: 23%; 4: 26%; 5: 31%.
Secondly, geometry-selective total synthesis of furospinosulin-
1 (1) was examined using sulfone-mediated coupling method.8
In order to apply to the syntheses of various analogues,
furospinosulin-1 (1) was divided into the two fragments of the
allylic bromide 13 and the known farnesyl phenylsulfone 15.9 PCC
oxidation of the alcohol 2 and subsequent Wittig olefination
provided an enoate 11 (E/Z¼20:1). The undesired Z-isomer could
be removed by SiO2 column chromatography easily. Then, DIBAL
reduction of the ester moiety in 11 and following bromination of
the hydroxyl group of 12 with CBr4/PPh3 gave a desired allylic
bromide 13 in quantitative yield. Coupling reaction between
compounds 13 and 15 proceeded smoothly using t-BuOK as
a base10 to yield a compound 14, and final reductive desulfony-
lation11 of 14 with LiBHEt3 in the presence of Pd(dppp)Cl2 affor-
ded furospinosulin-1 (1), by 60% overall yield with six linear
reaction steps from the known compound 2. No olefin isomeri-
zation was observed throughout the synthesis.
We next executed the syntheses of some structurally modified
analogues of 1 using the same synthetic method shown in Scheme 2.
Various analogues could be prepared systematically by chang-
ing structure of the allylic bromide, to analyze the detailed struc-
tureeactivity relationship around the aromatic ring. Scheme 3
shows the syntheses of the analogues having a different heterocycle.
The thiophene, pyrrole, and tetrahydrofuran-containing allylic
bromides (18, 21, and 24) were prepared in seven steps from the
commercially available materials (16, 19, and 22), respectively.12,13
Then, coupling reaction with the sulfone 15 and subsequent desul-
fonylation provided analogues 25e27 in good yield.
Two analogues (30 and 31) having a different substituent were
also prepared, as depicted in Scheme 4. The use of the different
Wittig or HornereEmmons reagents as an elongation unit provided
desired fragments 28 and 29, and the following steps similar to
those of the other analogues afforded compounds 30 and 31 having
a proton or ethyl group in place of the methyl group nearest to the
furan ring, respectively.
2.2. Biological evaluation of synthetic analogues
The growth inhibitory effects of the synthetic analogues against
DU145 cells under normoxic or hypoxic conditions were evaluated,
which were briefly summarized in Fig. 2. A mixture (10) of three
geometrical isomers of 1 showed very weak and non-selective
growth inhibition. Analogue 4 having a shorter side chain showed
non-selective toxicity at higher dose, and analogue 5 having a lon-
ger chain showed moderate hypoxia-selective activity similar to 1.
These results show that the both geometry and length of the side
chain in 1 were important for exhibiting hypoxia-selective growth
inhibitory activity. Phenyl analogue 7 showed only weak toxicity in
normoxic condition. Thiophene analogue 25 showed weak but
hypoxia-selective activity, and pyrrole analogue 26 and tetrahy-
drofuran analogue 27 were toxic even in lower dose. These evi-
dences clearly indicate that the furan moiety might be the best
structure in aromatic part. Ethyl analogue 31 showed no hypoxia-
selective activity, maybe due to the steric repulsion, while des-
methyl analogue 30 showed excellent hypoxia-selective activity
both in lower and higher dose. These results revealed that the

N. Kotoku et al. / Tetrahedron 67 (2011) 6673e6678
6675
H
H
n,o
n-p
n,o
d-g
a-c
OH
Br
Br
O
O
S
S
N
S
S
25
26
27
17
18
16
d-g
h-j
OH
N
HN
19
HN
O
TIPS
TIPS
20
21
k-m
d-g
OH
Br
O
O
24
O
23
22
Scheme 3. Reagents and conditions: (a)Ph3P]CHCO2Et, CH2Cl2, 95%; (b) DIBAL, CH2Cl2, 0 ꢀC, 98%; (c) H2/PdeC, EtOH, quant.; (d) PCC, CH2Cl2; (e) Ph3P]C(Me)CO2Et, toluene, 60 ꢀC;
(f) DIBAL, CH2Cl2, 0 ꢀC; (g) PPh3, CBr4, CH2Cl2, 18: 49%; 21: 66%; 24: 41% (each for four steps); (h) TIPSCl, n-BuLi, THF, ꢁ78 ꢀC, 98%; (i) NBS, THF, ꢁ78 ꢀC, 93%; (j) n-BuLi, THF, ꢁ78 ꢀC;
trimethylene oxide, BF3$Et2O, 83%; (k) (EtO)2P(O)CH2CO2Et, NaOH, CH2Cl2/H2O, 51%; (l) H2/PdeC, AcOEt, quant.; (m) DIBAL, CH2Cl2, 0 ꢀC, 75%; (n) 15, t-BuOK, THF, ꢁ30 ꢀC; (o)
LiBHEt3, Pd(dppp)Cl2, THF, 0 ꢀC, 25: 56%; 27: 50% (each for two steps); (p) TBAF, THF, 26: 72% (three steps).
inhibited growth of tumor, and 72% reduction of the tumor weight
was observed at the dose of 50 mg/kg compared with that of the
control (Fig. 4). Moreover, analogue 30 exhibited no significant acute
toxicity, such as body weight loss or diarrhea, up to 100 mg/kg
administration.
a, b or c
CO2Et
2
O
R
28: R = H
29: R = Et
3. Conclusion
d-g
We investigated structureeactivity relationship of furospinosulin-
1 (1) through concise syntheses of some structurally modified ana-
logues. It revealed that the whole chemical structure was important
for the hypoxia-selective growth inhibition of 1. We found that des-
methyl analogue 30 showed excellent hypoxia-selective growth
inhibitory activity similar to that of 1, and also exhibited in vivo anti-
tumor effect with oral administration. Development of more prom-
ising hypoxia-targeting anti-tumor drug candidate is now under
investigation.
O
R
30: R = H
31: R = Et
Scheme 4. Reagents and conditions: (a) PCC, CH2Cl2, 81%; (b) Ph3P]CHCO2Et, toluene,
55%; (c) (EtO)2P(O)CH(Et)CO2Et, NaH, THF, 50%; (d) DIBAL, CH2Cl2, 0 ꢀC; (e) PPh3, CBr4,
CH2Cl2; (f) 15, t-BuOK, THF, ꢁ30 ꢀC; (g) LiBHEt3, Pd(dppp)Cl2, THF, 0 ꢀC, 30: 75%; 31:
69% (each for four steps).
100
80
60
40
20
0
hypoxia
normoxia
10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100
1’ 25 26 27
4
5
7
30
31
1
concentration (µM)
Fig. 2. Growth inhibition of synthetic analogues against DU145 cells.
whole chemical structure of 1 should be needed for its hypoxia-
selective growth inhibitory activity. The binding molecule (pro-
tein) of 1 might recognize the whole structure of 1 strictly.
The detailed growth inhibition of desmethyl analogue 30
against DU145 cells was shown in Fig. 3. Comparing with that of 1,
analogue 30 exhibited greater growth inhibition at lower dose
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 in-
ternal standard. Mass spectra were obtained with a Waters Q-Tof
Ultima API using MeOH as a solvent. HPLC was performed using
a Hitachi L-6000 pump equipped with Hitachi L-4000H UV de-
(1e10
(300
candidate.
Then, in vivo anti-tumor testing of analogue 30 was also exam-
mM) and lower toxicity in normoxic condition at higher dose
mM). These properties of 30 might be better than 1 for drug
ined. Murine sarcoma S180 cells were implanted subcutaneously,
and the testing compound was administrated orally. The effective-
ness of the compound was determined by weighing the surged tu-
mor. It was found that the 10e50 mg/kg dose of analogue 30
tector. Silica gel (Kanto, 40e100 mm) 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 phosphomolybdic

6676
N. Kotoku et al. / Tetrahedron 67 (2011) 6673e6678
100
80
60
40
20
0
purified by SiO2 column chromatography (n-hexane/AcOEt¼3:1) to
give 2 (156 mg, 61%) as a colorless oil. IR (KBr): 3378, 2984, 1684,
1520, 1419, 1143 cmꢁ1 1H NMR (500 MHz, CDCl3)
: 7.35 (1H,
O
.
d
30
t, J¼1.8 Hz), 7.23 (1H, s), 6.28 (1H, s), 3.68 (2H, t, J¼6.4 Hz), 2.52 (2H,
t, J¼7.3 Hz), 1.83 (2H, dt, J¼14.9, 6.7 Hz), 1.42 (1H, br s). 13C NMR
hypoxia
(125 MHz, CDCl3) d: 142.8, 138.9, 124.4, 110.9, 62.2, 32.8, 21.0.
normoxia
4.2.2. [3-(Furan-3-yl)propyl]triphenylphosphonium bromide (3).
p-Toluenesulfonyl chloride (1.95 g, 10.2 mmol) was added to a so-
lution of 2 (644 mg, 5.10 mmol) in pyridine (12 mL) at ꢁ20 ꢀC, and
the whole mixture was stirred for 6 h. H2O was added to the
mixture at 0 ꢀC, and the whole mixture was extracted with Et2O.
Removal of the solvent from the Et2O extract under reduced pres-
sure gave a crude product, which was purified by SiO2 column
chromatography (n-hexane/AcOEt¼5:1) to give 3-(furan-3-yl)pro-
pyl p-toluenesulfonate (1.27 g, 89%) as a colorless oil. IR (KBr): 2955,
1
3
10
30
100
300
concentration (µM)
100
80
60
40
20
0
1360, 1177 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d
: 7.79 (2H, d, J¼8.5 Hz),
7.35 (2H, d, J¼7.9 Hz), 7.32 (1H, s), 7.11 (1H, s), 6.18 (1H, s), 4.04 (2H,
O
t, J¼6.4 Hz), 2.48 (2H, d, J¼7.3 Hz), 2.46 (3H, s), 1.92e1.86 (2H, m).
furospinosulin-1(1)
13C NMR (125 MHz, CDCl3)
d: 144.7, 143.0, 139.2, 133.1, 129.8 (2C),
hypoxia
127.9 (2C), 123.1, 110.6, 69.5, 29.2, 21.6, 20.5.
Lithium bromide (228 mg, 2.6 mmol) was added to a solution of
the above product (147 mg, 0.53 mmol) in acetone (5.0 mL), and the
whole mixture was stirred for 4 h at 60 ꢀC. H2O was added to the
mixture at 0 ꢀC, and the whole mixture was extracted with Et2O.
Removal of the solvent from the Et2O extract under reduced pres-
sure gave a crude product, which was purified by SiO2 column
chromatography (n-hexane/AcOEt¼10:1) to give 3-(3-bromo-
propyl)furan (93.4 mg, 94%) as a colorless oil. IR (KBr): 2934, 1503,
normoxia
1
3
concentration (µM)
10
30
100
300
1437, 1260 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d
: 7.37 (1H, t, J¼1.5 Hz),
7.26 (1H, s), 6.28 (1H, s), 3.41 (2H, t, J¼6.7 Hz), 2.60 (2H, t, J¼7.3 Hz),
Fig. 3. Detailed growth inhibition of desmethyl analogue (30) and furospinosulin-1 (1).
2.12e2.07 (2H, m). 13C NMR (125 MHz, CDCl3)
d: 143.0, 139.7, 123.1,
110.8, 33.0, 32.7, 23.0.
Triphenylphosphine (957 mg, 3.65 mmol) was added to a solu-
tion of the above product (276 mg, 1.46 mmol) in toluene (5.0 mL),
and the whole mixture was stirred for 12 h at 120 ꢀC. Removal of
the solvent from the reaction mixture gave a crude product, which
was purified by SiO2 column (CH2Cl2/MeOH¼10:1) to give 3
(307 mg, 57%) as a colorless amorphous solid. IR (KBr): 2899,
1.2
0.8
0.4
1439 cmꢁ1 1H NMR (500 MHz, CDCl3)
. d: 7.85e7.75 (9H, m),
7.70e7.66 (6H, m), 7.31 (1H, d, J¼1.2 Hz), 7.26 (1H, s), 6.18 (1H, d,
J¼1.2 Hz), 3.97e3.91 (2H, m), 2.87 (2H, t, J¼7.0 Hz), 1.88 (2H, td,
J¼15.6, 7.5 Hz).
0
10
25
50 (mg/kg)
30
4.2.3. Furospinosulin-1 (1) E,Z mixture. n-BuLi (1.65 M in n-hexane,
0.17 mL, 0.39 mmol) was added dropwise to a solution of 3 (119 mg,
0.26 mmol) in THF (0.6 mL) at ꢁ78 ꢀC, and the whole mixture was
stirred for 30 min. A solution of farnesylacetone (9) (49.3 mg,
0.19 mmol) in THF (0.3 mL) was added to the solution via cannula,
and the whole mixture was stirred for 3 h at 0 ꢀC. Satd NH4Cl 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 a crude product, which was purified by SiO2
column (n-hexane/AcOEt¼30:1) to give 1 (15.4 mg, 23%) as a col-
orless oil (1:1:1:1 mixture of four geometrical isomers). 1H NMR
Fig. 4. anti-Tumor effect of desmethyl analogue (30).
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 purchased
reagents and solvents without further purification. After workup,
the organic phases were dried over Na2SO4.
4.2. Geometry non-selective synthesis of furospinosulin-1
and analogues
(500 MHz, CDCl3) d: 7.34 (1H, s), 7.21 (1H, s), 6.27 (1H, s), 5.17 (1H, t,
J¼7.3 Hz), 5.13e5.10 (3H, m), 2.45 (2H, q, J¼7.1 Hz), 2.25 (2H, q,
J¼7.5 Hz), 2.06e2.00 (12H, m), 1.69e1.61 (15H, s). ESI-MS m/z: 377
[MþNa]þ. HR-ESI-MS: 377.2820, calcd for C25H38ONa. Found:
377.2836. Compound 1 ((E,E)-isomer) and other three-isomeric
mixture were separated by reversed-phase HPLC (COSMOSIL 5C18
MS-II, MeOH/H2O¼9:1).
4.2.1. 3-(Furan-3-yl)propan-1-ol (2). n-BuLi (1.65 M in n-hexane,
1.3 mL, 2.12 mmol) was added to a solution of 3-bromofuran
(297 mg, 2.02 mmol) in THF (4.5 mL) at ꢁ78 ꢀC, and the whole
mixture was stirred for 30 min. Trimethylene oxide (0.15 mL,
2.32 mmol) and BF3$Et2O (0.31 mL, 2.42 mmol) were added to the
mixture, and the whole mixture was further stirred for 3 h
at ꢁ78 ꢀC. H2O was added to the mixture, 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
Analogues 4 and 5 were obtained from geranylacetone (8) or
teplenone (10), respectively, using the same procedure as that for 1.
Analogue 7 was also obtained from known phosphonium salt 67
and 9.

N. Kotoku et al. / Tetrahedron 67 (2011) 6673e6678
6677
4.3. Geometry-selective synthesis of furospinosulin-1 and
analogues
gave a crude product, which was purified by SiO2 column (n-hexane/
AcOEt¼10:1) to give 15 (464 mg, 70%) as a colorless oil. IR (KBr):
2920, 1447, 1306, 1146 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 7.88e7.86
4.3.1. (E)-5-(Furan-3-yl)-2-methylpent-2-enoic acid ethyl ester
(11). A solution of 2 (460 mg, 3.64 mmol) in CH2Cl2 (6 mL) was
added to the suspension of Celite (864 mg) and PCC (864 mg,
4.00 mmol) in CH2Cl2 (30 mL) via cannula, and the whole mixture
was stirred for 3 h at rt. Then the mixture was diluted with Et2O
(50 mL), and the whole mixture was filtered through short SiO2
pad. Removal of the solvent from the filtrate under reduced pres-
sure to give a crude product, which was purified by SiO2 column
(n-hexane/Et2O¼1:1) to give 3-(furan-3-yl)propionaldehyde
(365 mg, 81%) as a colorless oil. IR (KBr): 2926, 1726, 1502,
(2H, m), 7.64e7.63 (1H, m), 7.55e7.52 (2H, m), 5.19 (1H, td, J¼8.1,
1.4 Hz), 5.08e5.05 (2H, m), 3.81 (2H, d, J¼7.9 Hz), 2.05e1.99 (8H, m),
1.68 (3H, s), 1.60 (3H, s), 1.58 (3H, s), 1.31 (3H, s). 13C NMR (125 MHz,
CDCl3) d: 146.4, 138.7, 135.7, 133.5, 131.4, 128.9 (2C), 128.6 (2C), 124.2,
123.3, 110.3, 56.1, 39.7, 26.7, 26.2, 25.7, 17.7, 16.2, 16.0.
4.3.5. ((3E,7E,11E)-4,8,12,16-Tetramethyl-6-(phenylsulfonyl)hepta-
deca-3,7,11,15-tetraen-1-yl)furan (14). t-BuOK (1.0
M in THF,
0.16 mL, 0.16 mmol) was added to a solution of 13 (30.1 mg,
0.13 mmol) and 15 (41.2 mg, 0.12 mmol) in THF (0.6 mL) at ꢁ30 ꢀC,
and the whole mixture was stirred for 1 h. Satd NH4Cl aq was
added to the mixture at 0 ꢀC, 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/AcOEt¼20:1) to give 14 (62.4 mg, 96%) as
a colorless oil.
1389 cmꢁ1 1H NMR (500 MHz, CDCl3)
. d: 9.81 (1H, s), 7.35 (1H, t,
J¼1.8 Hz), 7.23 (1H, s), 6.28e6.26 (1H, m), 2.78e2.77 (2H, m),
2.72e2.69 (2H, m). 13C NMR (125 MHz, CDCl3)
d: 201.5, 143.1, 139.0,
123.3, 110.7, 44.0, 17.5.
Ph3P]C(Me)CO2Et (665 mg, 1.91 mmol) was added to the so-
lution of the above product (198 mg, 1.59 mmol) in toluene (16 mL),
and the whole mixture was stirred for 8 h at 60 ꢀC. Removal of the
solvent from the reaction mixture under reduced pressure gave
a crude product, which was purified by SiO2 column (n-hexane/
AcOEt¼5:1) to give 11 (247 mg, 80%) as a colorless oil. IR (KBr):
IR (KBr): 2920, 1447, 1306, 1146 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d
: 7.84 (2H, d, J¼6.7 Hz), 7.62 (1H, t, J¼7.6 Hz), 7.51 (2H, t, J¼7.6 Hz),
7.31 (1H, t, J¼1.5 Hz), 7.15 (1H, s), 6.22 (1H, s), 5.19 (1H, t, J¼7.0 Hz),
5.08e5.04 (2H, m), 4.91 (1H, d, J¼10.4 Hz), 3.89e3.87 (1H, m), 2.89
(1H, d, J¼12.2 Hz), 2.39 (2H, t, J¼7.6 Hz), 2.29 (1H, dd, J¼13.7,
6.9 Hz), 2.20 (2H, q, J¼7.3 Hz), 2.04e1.97 (8H, m), 1.68 (3H, s), 1.60
(3H, s), 1.58 (3H, s), 1.52 (3H, s), 1.15 (3H, s). 13C NMR (125 MHz,
2982, 1711, 1447, 1368, 1275 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 7.35
(1H, dd, J¼2.4, 1.2 Hz), 7.23 (1H, d, J¼1.8 Hz), 6.79e6.75 (1H, m),
6.28 (1H, s), 4.19 (2H, q, J¼7.1 Hz), 2.57 (2H, t, J¼7.3 Hz), 2.43 (2H, q,
J¼7.5 Hz), 1.81 (3H, s), 1.29 (3H, t, J¼7.3 Hz). 13C NMR (125 MHz,
CDCl3) d: 145.1, 142.5, 138.7, 137.9, 135.5, 133.3, 131.3, 130.6, 129.2
CDCl3)
d
: 168.1, 142.9, 140.9, 138.9, 128.5, 124.1, 110.8, 60.5, 29.2,
(2C), 128.6 (2C), 127.6, 124.5, 124.1, 123.4, 117.2, 110.8, 63.4, 39.63,
39.61, 37.3, 28.3, 26.6, 26.2, 25.6, 24.7, 17.6, 16.3, 15.92, 15.88. ESI-MS
m/z: 517 [MþNa]þ. HR-ESI-MS: 517.2752, calcd for C31H42O3NaS.
Found: 517.2727.
23.8, 14.3, 12.4. ESI-MS m/z: 231 [MþNa]þ. HR-ESI-MS: 231.0997,
calcd for C12H16O3Na. Found: 231.1005.
4.3.2. (E)-5-(Furan-3-yl)-2-methylpent-2-en-1-ol (12). DIBAL (1.0 M
in n-hexane, 1.62 mL, 1.62 mmol) was added to a solution of 11
(96.0 mg, 0.46 mmol) in CH2Cl2 (1.0 mL) at 0 ꢀC, and the whole
mixture was stirred for 20 min. 5% HCl was added to the mixture,
and the whole mixture was extracted with CH2Cl2. Removal of the
solvent from the CH2Cl2 extract under reduced pressure gave a crude
product, which was purified by SiO2 column (n-hexane/AcOEt¼5:1)
to give 12 (77.4 mg, quant.) as a colorless oil. IR (KBr): 3333, 2929,
4.3.6. Furospinosulin-1 (1). LiBHEt3 (1.0
M in THF, 0.34 mL,
0.34 mmol) was added dropwise to a solution of 14 (49.3 mg,
0.097 mmol) and Pd(dppp)Cl2 (11.4 mg, 0.019 mmol) in THF
(0.48 mL) at 0 ꢀC, and the whole mixture was stirred for 30 min.
Satd NH4Cl aq was added to the mixture at 0 ꢀC, 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/AcOEt¼10:1) to give 1
(33.0 mg, 96%) as a colorless oil. IR (KBr): 2920, 1447, 1306,
1503,1449,1383 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 7.35 (1H, s), 7.22
(1H, s), 6.28 (1H, s), 5.45 (1H, td, J¼7.0, 1.4 Hz), 4.00 (2H, s), 2.49 (2H,
t, J¼7.6 Hz), 2.30 (2H, q, J¼7.5 Hz), 1.66 (3H, s). 13C NMR (125 MHz,
1146 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 7.33 (1H, s), 7.21 (1H, s),
CDCl3)
d
: 142.7, 138.8, 135.5, 125.3, 124.7, 111.0, 68.8, 28.0, 24.7, 13.7.
6.27 (1H, s), 5.17 (1H, t, J¼6.5 Hz), 5.11 (3H, s), 2.45 (2H, t, J¼7.3 Hz),
ESI-MS m/z: 189 [MþNa]þ. HR-ESI-MS: 189.0891, calcd for
2.25 (2H, q, J¼7.3 Hz), 2.07 (6H, m), 2.00 (6H, m), 1.68 (3H, s), 1.60
C10H14O2Na. Found: 189.0917.
(12H, s). 13C NMR (125 MHz, CDCl3)
d: 142.5, 138.8, 135.8, 135.0,
134.9, 131.3, 125.0, 124.4, 124.3, 124.2, 123.7, 110.1, 39.7, 28.4, 26.8,
26.6, 26.5, 25.7, 25.0, 17.7, 16.04, 16.00. ESI-MS m/z: 377 [MþNa]þ.
HR-ESI-MS: 377.2820, calcd for C25H38ONa. Found: 377.2801.
Analogues 25e27, 30, and 31 were prepared using the same
procedure as that for 1, using compounds 17, 20, 23, 28, and 29 as
substrates.
4.3.3. (E)-3-(5-Bromo-4-methylpent-3-enyl)furan (13). PPh3 (147 mg,
0.55 mmol) and CBr4 (186 mg, 0.55 mmol) were successively added
to the solution of 12 (78.0 mg, 0.46 mmol) in CH2Cl2 (2.3 mL) at 0 ꢀC,
and the whole mixture was stirred for 30 min. H2O was added to the
mixture, and the whole mixture was extracted with CH2Cl2. Removal
of the solvent from the reaction mixture under reduced pressure
gave a crude product, which was purified by SiO2 column (n-hexane/
AcOEt¼30:1) to give 13 (97.3 mg, quant.) as a colorless oil. IR (KBr):
4.4. Biological evaluation of furospinosulin-1 analogues
2920,1501,1437,1206 cmꢁ1. 1H NMR (500 MHz, CDCl3)
d: 7.35 (1H, s),
4.4.1. Cell cultures. Human prostate cancer cell line DU145 and
mouse sarcoma cell S180 were cultured in RPMI 1640 supplemented
with heat-inactivated 10% fetal bovine serum (FBS) and kanamycin
7.21 (1H, s), 6.27 (1H, s), 5.63 (1H, td, J¼7.0, 1.0 Hz), 3.97 (2H, s), 2.49
(2H, t, J¼7.6 Hz), 2.29 (2H, q, J¼7.3 Hz), 1.74 (3H, s). 13C NMR
(125 MHz, CDCl3)
24.2, 14.7.
d: 142.7, 138.9, 132.7, 130.4, 124.3, 110.9, 41.5, 28.7,
(50 m
g/mL) in a humidified atmosphere of 5% CO2 at 37 ꢀC.
4.3.4. (2E, 6E)-(3,7,11-Trimethyldodeca-2,6,10-triene-1-sulfonyl)ben-
zene (15). PhSO2Na (411 mg, 2.51 mmol) was added to the solution
of farnesyl bromide (549 mg, 1.93 mmol) in DMF (10 mL) at 0 ꢀC, and
the whole mixture was stirred for 1 h. Satd NH4Cl aq 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
4.4.2. Assay for anti-proliferative activity under hypoxic con-
dition. The cells in the culture medium was plated into each well
of 96-well plates (1ꢂ104 cells/well/200
mL) for 4 h in a humidified
atmosphere of 5% CO2 at 37 ꢀC (normoxic condition). Then, the
plates were incubated for 12 h in the 94% nitrogen, 5% CO2, and
1% O2 (hypoxic condition) inducing hypoxia related genes, such

6678
N. Kotoku et al. / Tetrahedron 67 (2011) 6673e6678
as HIF-1
a
. After 12 h incubation, testing compounds were added,
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan.
and then the plates were incubated for an additional 24 h in the
hypoxic condition. The cell proliferation was detected according
to established MTT method as previously described.4 The growth
inhibition rate was calculated as percentage of parallel negative
controls. The anti-proliferative activity of testing compounds
under normoxic condition was also evaluated by colorimetric
reagent of MTT.
Supplementary data
The experimental procedures and physical data for compounds
4e31, together with the 1H and 13C NMR spectra of compounds
1e31, are available. Supplementary data associated with this ar-
ticle can be found, in the online version, at doi:10.1016/
j.tet.2011.05.009.
4.4.3. In vivo anti-tumor effect of analogue 30. All animal pro-
cedures were approved by the Committee on Animal Experimen-
tation of Osaka University. Mouse sarcoma S180 cells (1ꢂ106 cells/
body) were implanted subcutaneously into the right ventral flank
of female ddY mice (6 weeks old). After one week from implanta-
tion, analogue 30 was administered on every other day for 2 week
(total seven times) by oral administration as a suspension in 1%
sodium carboxymethyl cellulose (CMCeNa). Then, tumor was iso-
lated and weighed to calculate the inhibition ratio. The control
group and the group treated with analogue 30 consisted of six mice
each in this study.
References and notes
1. Dewhirst, M. W.; Cao, Y.; Moeller, B. Nat. Rev. Cancer 2008, 8, 425.
2. Wouters, B. G.; Koritzinsky, M. Nat. Rev. Cancer 2008, 8, 851.
3. Cimino, G.; De Stefano, S.; Minale, L. Tetrahedron 1972, 28, 1315.
4. Arai, M.; Kawachi, T.; Setiawan, A.; Kobayashi, M. ChemMedChem 2010, 5,
1919.
5. Magatti, C. V.; Kaminski, J. J.; Rothberg, I. J. Org. Chem. 1991, 56, 3102.
6. Sperry, J. B.; Wright, D. L. Tetrahedron 2006, 62, 6551.
7. Kaiser, S.; Smidt, S. P.; Pfaltz, A. Angew. Chem., Int. Ed. 2006, 45, 5194.
8. Grieco, P. A.; Masaki, Y. J. Org. Chem. 1974, 39, 2135.
9. Robustell, B.; Abe, I.; Prestwich, G. D. Tetrahedron Lett. 1998, 39, 957.
10. Zheng, Y.; Lin, J.; Li, C.; Li, J. J. Chem. Res. 2007, 686.
Acknowledgements
11. Mohri, M.; Kinoshita, H.; Inomata, K.; Kotake, H. Chem. Lett. 1985, 451.
12. Martins, A.; Lautens, M. J. Org. Chem. 2008, 73, 8705.
13. Morrison, M. D.; Hanthorn, J. J.; Pratt, D. A. Org. Lett. 2009, 11, 1051.
This study was financially supported by the Uehara Memorial
Foundation, and by the Grant-in-Aid for Scientific Research from