Terresterol, a polyoxygenated lanostanoid, isolated from the oomycete Saprolegnia terrestris, and its innate immune-promoting activity

Article abstract of DOI:10.1016/j.tet.2013.02.088

Oomycetes are widely spread eukaryotic microorganisms. However, there have only been a few reports on the secondary metabolites produced by them. In the course of screening for innate immune regulators from natural resources, we isolated a new polyoxygenated lanostanoid, terresterol (1), from the oomycetes Saprolegnia terrestris. Compounds bearing a 21,22,24,25-tetraoxygenated side chain similar to 1 have only been rarely described, and the isolation of such compounds shows that oomycetes are promising resources for natural product chemistry. In addition, terresterol (1) has potential to be used as a lead compound for novel immunostimulating agents against bacterial infections and as probe for the innate immune system.

Full text of DOI:10.1016/j.tet.2013.02.088

Tetrahedron 69 (2013) 3536e3542
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Terresterol, a polyoxygenated lanostanoid, isolated from the
oomycete Saprolegnia terrestris, and its innate immune-promoting
activity
*
Haruhisa Kikuchi , Yuichi Sato, Shoichiro Kurata, Yasuhiro Katou, Yoshiteru Oshima
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oomycetes are widely spread eukaryotic microorganisms. However, there have only been a few reports
on the secondary metabolites produced by them. In the course of screening for innate immune regulators
from natural resources, we isolated a new polyoxygenated lanostanoid, terresterol (1), from the oomy-
cetes Saprolegnia terrestris. Compounds bearing a 21,22,24,25-tetraoxygenated side chain similar to 1
have only been rarely described, and the isolation of such compounds shows that oomycetes are
promising resources for natural product chemistry. In addition, terresterol (1) has potential to be used as
a lead compound for novel immunostimulating agents against bacterial infections and as probe for the
innate immune system.
Received 31 January 2013
Received in revised form 19 February 2013
Accepted 23 February 2013
Available online 1 March 2013
Keywords:
Oomycetes
Innate immunity
Immunostimulating agents
Lanostanoid
Ó 2013 Elsevier Ltd. All rights reserved.
Structural elucidation
1. Introduction
innate immunity, and our culture system has proven to be useful for
identifying immune regulators that act on human innate immunity.5
Oomycetes are eukaryotic microorganisms that are widely
found in water and soil.1 Since they were observed to form fungus-
like mycelium, they were classified as fungi in the past. However,
the cell walls of them are composed of cellulose rather than chitin,
as in the fungi.2 In the present, ultrastructures, biochemistry, and
genomic sequences indicate that oomycetes belong to the Kingdom
Chromista, not the Kingdom Fungi, and are related to organisms
such as brown algae and diatoms. Because many species of oomy-
cetes have plant pathogenicity and cause plant diseases, they are
agriculturally and economically important.3 However, they have
not been exploited in natural product chemistry. Furthermore,
there have only been reports on a few compounds produced by
these organisms, including antheridiol, oogoniol and their de-
rivatives, a sex hormone in oomycetes Achlya.4
We have used this system to search for natural substances that reg-
ulate innate immunity.8 Here we describe the isolation of terresterol
(1) (Fig. 1), a polyoxygenated lanostanoid from the oomycetes, Sap-
rolegnis terrestris, which also functions as an innate immune
promoter.
Recently, we have focused oninnateimmunity, and we established
an ex vivo culture system based on the Drosophila IMD signaling
pathway to screen pharmaceuticals that target innate immunity.5
Because of the striking conservation between the mechanisms that
regulate insect immunity and mammalian innate immunity,6,7 Dro-
sophila is a model organism for genetic and molecular studies of
Fig. 1. Structure of terresterol (1).
2. Results and discussion
2.1. Isolation
S. terrestris NRBC102124 was cultivated in yeast extractemalt
extract broth. The mycelial cake of culture broth (9.0 L) was
* Corresponding author. Tel.: þ81 22 795 6824; fax: þ81 22 795 6821; e-mail
address: hal@mail.pharm.tohoku.ac.jp (H. Kikuchi).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.02.088

H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
3537
lyophilized and extracted with methanol at room temperature to
yield an extract (9.86 g). The ethyl acetate solubles (1.47 g) in the
methanol extract were separated by repeated column chromatog-
raphy over silica gel and ODS column to yield terresterol (1) (25 mg)
along with fucosterol and 24-methylenecholesterol.
2.2. Structural elucidation
HRFABMS (m/z 577.4078 [MþH]þ and m/z 599.3901 [MþNa]þ)
indicated the molecular formula of 1 as C34H56O7. The 13C NMR
spectrum of 1 showed the presence of two ester carbonyl, two
quaternary olefinic, one oxygenated quaternary, three oxymethine,
one oxymethylene, four quaternary, three methine, nine methy-
lene, and nine methyl carbons (Table 1). The cross-peaks in 1He1H
COSY implied that C-1eC-2eC-3, C-5eC-6eC-7, C-11eC-12, C-
15eC-16eC-17eC-20eC-21, and C-22eC-23eC-24 were connected
(Fig. 2A). HMBC correlations for H-22 to C-21; H2-21 to C-22; H3-26
to C-24 and C-25; and H3-27 to C-24 indicated a side chain moiety
consisting of C-20eC-27. HMBC correlations were observed for H2-
21 and H3-32 to C-31; H-24 and H3-34 to C-33, suggesting that an
acetoxy group was attached to C-21 and C-24, respectively. The
existence of a cyclopenta[a]phenanthrene skeleton, except at the
bond between C-8 and C-9, was inferred from the HMBC correla-
tions for H2-6 to C-8; H2-12 to C-9; H3-18 to C-12, C-13, C-14 and C-
17; H3-19 to C-1, C-5, C-9 and C-10; H3-28 to C-3, C-4 and C-5; H3-
Fig. 2. Structural elucidation of terresterol (1). A. Planar structure of 1. B. Relative
structure of the tetracyclic moiety of 1.
29 to C-3, C-4 and C-5; H3-30 to C-8, C-13, C-14 and C-15. Finally,
two remaining olefinic carbons, C-8 and C-9, were supposed to be
connected, and the planar structure of 1 was elucidated as
21,24-diacetoxylanostan-3,22,25-triol.
Table 1
1H and 13C NMR spectra of terresterol (1)
Terresterol (1)a
The cross-peaks of H3-18eH3-1913 and H3-19eH3-29 in the
NOESY spectrum of 1 indicated that the configurations of three
13C
35.5
1H
1
2
1.61e1.67 (1H, m)
1.11e1.16 (1H, m)
1.56e1.63 (1H, m)
1.47e1.52 (1H, m)
methyl groups at C-18, C-19, and C-29 were b-axial (Fig. 2B). The
cross-peaks of H-3eH-5 and H-17eH3-30 revealed that the orien-
tations of protons at C-3, C-5, C-17, and the 30-methyl group were
27.6
a-axial. Thus, the relative configuration of the tetracyclic moiety of
3
4
5
6
78.8
38.8
50.3
18.1
3.15 (1H, dd, J¼11.7, 4.5 Hz)
1 was determined as 3S , 5R , 10S , 13R , 14R , and 17R . The relative
*
*
*
*
*
*
0.97 (1H, dd, J¼12.7, 1.8 Hz)
1.57e1.64 (1H, m)
1.41e1.46 (1H, m)
configuration of the side chain moiety consisting of C-21eC-27
could not be directly established because of the flexibility of bond
rotation.
To determine its relative configuration of the side chain, we have
fixed its conformation. When terresterol (1) was treated with
2,2-dimethoxypropane under acidic conditions, the acetoxy group
at C-24 was hydrolyzed and a 22,24-O-isopropylidene derivative 2
was afforded (Scheme 1). The NOESY spectrum of 2 showed the
cross-peaks of H-22eH3-36 and H-24eH3-37 (Fig. 3A). In the 1H
7
8
9
10
11
12
26.4
133.9
134.6
37.0
20.9
29.9
1.90e2.02 (2H, m)
1.82e1.91 (2H, m)
1.55e1.64 (1H, m)
1.33e1.42 (1H, m)
13
14
15
44.5
49.4
30.7
NMR spectrum of 2, the signal of H-24 (
that the dihedral angle between H-24 and H-23
d
3.99, d, J¼4.4 Hz) implied
1.53e1.59 (1H, m)
1.10e1.14 (1H, m)
1.72e1.77 (1H, m)
1.32e1.38 (1H, m)
1.73e1.79 (1H, m)
0.67 (3H, s)
b
was almost 90ꢀ
(Fig. 3B). These facts indicated that the conformation of the
1,3-dioxane moiety was the twisted-boat form due to an alleviation
of 1,3-diaxial interactions,9 and the relative configurations of C-22
16
26.7
17
18
19
20
21
42.5
15.9
19.1
45.6
63.0
and C-24 were R
*
and R , respectively.
*
0.90 (3H, s)
After deacetylation of 1 using sodium methoxide, the treatment
with 2,2-dimethoxypropane under acidic conditions afforded
a 21,22:24,25-di-O-isopropylidene derivative 3 (Scheme 1). The
1.86e1.91 (1H, m)
4.26 (1H, dd, J¼11.7, 2.9 Hz)
4.07 (1H, dd, J¼11.7, 5.4 Hz)
3.55 (1H, br d, J¼12.2 Hz)
1.55e1.69 (2H, m)
4.86 (1H, dd, J¼9.5, 3.1 Hz)
22
23
24
25
26
27
28
29
30
31
32
33
34
68.4
32.1
77.9
71.7
25.8
25.7
27.9
15.3
24.4
171.1
21.0
171.9
21.0
NOESY spectrum of 3 showed the cross-peak between H-21
H-22 (Fig. 4). In the 1H NMR spectrum, the signals of H-21
H-21 were observed at
b
a
and
and
b
d
4.00 (dd, J¼11.6, 3.9 Hz) and 3.93 (dd,
J¼11.6, 3.7 Hz), respectively. These facts indicate that H-20 was an
1.14 (3H, s)
1.14 (3H, s)
0.93 (3H, s)
0.74 (3H, s)
0.83 (3H, s)
equatorial proton in the chair-formed 1,3-dioxane moiety, and the
relative configuration of C-20 was R .
*
Since the relative structures of the tetracyclic moiety and the
side chain moiety were determined separately, the absolute
structures of these moieties were also separately determined. We
1.99 (3H, s)
2.06 (3H, s)
employed the esterification of 1 with (R)-a-methoxyphenylacetic
acid in the presence of MNBA10 to yield 3O,22O-(R)-MPA diester 4a
(Scheme 1). In a similar manner, (S)-MPA diester 4b was produced.
a
600 MHz for 1H and 150 MHz for 13C in CDCl3.

3538
H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
Scheme 1. Conversion of terresterol (1) into 2, 3, 4a, and 4b.
C-3 and C-22 had S- and R-configurations, respectively, and the
absolute configuration of 1 was determined to be 3S, 5R, 10S, 13R,
14R, 17R, 20R, 22R, 24R.
2.3. Promoting activity on innate immune response
We evaluated the effect of terresterol (1) on the innate immune
response using the ex vivo Drosophila culture system as previously
described.5 Although compound 1 showed cytotoxicity against
Fig. 3. Relative structure of the 22,24-O-isopropylidene derivative 2. A. Conformation
of the 1,3-dioxane moiety. B. Newman projection for the C-24eC-23 bond.
Drosophila S2 cells (CC50 40 mg/mL), 10 mg/mL of 1 increased the
Drosophila innate immune response (Fig. 5A) more than two-fold.
Furthermore, in order to evaluate the innate immune activity of 1
at the cellular level, Drosophila S2 cells, which were stably trans-
fected with luciferase reporter gene driven by attacin promoter,
were used.12 Attacin is one of the antimicrobial peptides regulated
by the Drosophila IMD pathway. Using attacin reporter, compound 1
showed concentration-dependent promoting activity at up to
0.1 mg/mL dosage (Fig. 5B).
Fig. 4. Conformation of the 1,3-dioxane moiety of the 21,22:24,25-di-O-isopropylidene
derivative 3.
2.4. Derivatization of terresterol (1)
To reveal the structural requirements of terresterol (1) for the
innate immune-promoting activity, we synthesized several de-
rivatives from 1. By the treatment of 1 with sodium methoxide,
both of the 22- and 24-acetyl groups were removed to produce
a pentaol 5 (Scheme 2). A 3,21,22,24-tetraacetate 6 was afforded by
The DdRS value of each proton was calculated from the difference in
the chemical shifts of 4a and 4b in 1H NMR spectra, and the
structure of 1 was fit into the proposed model of
a-methox-
yphenylacetate11 in accordance with the sign of DdRS. As a result,

H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
3539
the acetylation of 1. Dihydroxylation of lanosterol (7), using os-
mium tetroxide, yielded a 1:1 mixture of inoterpene A (8) and B (9),
which were reported as the constituents of the fungus Inonotus
obliquus.13 The activities of these compounds on the Drosophila
innate immune response were evaluated by the use of attacin re-
porter (Fig. 6). For compound 6, the innate immune response was
completely inhibited at 10 mg/mL. However, the viability of Dro-
sophila S2 cells was reduced to 15% of control at the same con-
centration, indicating that compound 6 showed only cytotoxicity,
and not inhibitory activity on innate immune response. The activity
of the mixture of inoterpene A (8) and B (9) was almost the same as
that of 6. On the other hand, the deacetylated derivative 5 inhibited
the activity on Att-Luc system in a concentration-dependent man-
Fig. 5. Innate immune-promoting activity of terresterol (1). A. Effects of terresterol (1)
on DAP-type peptidoglycans-mediated activation of Drosophila Dpt-lacZ. DAP-type
peptidoglycans-mediated activation of Dpt-lacZ (open squares) and Drosophila S2 cell
viability (black circles) are represented as the percent relative to the control (DMSO). B.
Effects of terresterol (1) on attacin promoter activity in Drosophila S2 cells. S2 cells
stably transfected with a luciferase reporter gene driven by an attacin promoter were
treated with terresterol (1) at indicated concentrations, and stimulated by DAP-type
peptidoglycan. The luciferase activity (open triangles) and Drosophila S2 cell viability
(black circles) are represented as the percent relative to the control (DMSO). The bars
indicate the standard errors of three independent measurements.
ner (IC50 1.7 mg/mL). Since the CC50 value (23 mg/mL) for S2 cells was
10 times higher than the IC50 value for the Att-Luc system, com-
pound 5 had selective immunosuppressive effects. These results
implied that the hydroxy groups and acetoxy groups in the side
chain of 1 were necessary for regulating innate immune responses,
and the number of acetoxy groups may switch over from a pro-
moting activity to an inhibitory activity.
Scheme 2. Synthesis of 5, 6, 8, and 9.
Fig. 6. Effects of compounds 5 (A), 6 (B), and the mixture of 8 and 9 (C) on attacin promoter activity in Drosophila S2 cells. The luciferase activity (open triangles) and Drosophila S2
cell viability (black circles) are represented as the percent relative to the control (DMSO). The bars indicate the standard errors of three independent measurements.

3540
H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
Although many polyoxygenated lanostanoids and tetracyclic tri-
terpenoids have been reported, compounds bearing a 21,22,24,25-
tetraoxygenated side chain similar to terresterol (1) have been
watereacetonitrile solvent system to give terresterol (1) (25.3 mg)
as water/acetonitrile (3:7) elutant.
Data for 1: colorless amorphous solid; mp 252e255 ꢀC (dec);
rarely described, as is the case with 1a,7a-diacetoxyapotirucall-14-
[
a
]
þ27.1 (c 0.461, CHCl3); IR (film) nmax (cmꢁ1) 3416, 2935, 1737,
D
ene-3
a
,21,22,24,25-pentaol (10) (Fig. 7), which was isolated from
1243; 1H NMR and 13C NMR data are shown in Table 1; HRFABMS
m/z 599.3901 [MþNa]þ (599.3924 calcd for C34H56O7Na) and
577.4078 [MþH]þ (577.4101 calcd for C34H57O7).
Azadirachta indica.14 Therefore, the isolation of such compounds
shows that oomycetes are promising resources for natural product
chemistry. Additionally, the isolation of fucosterol and 24-
methylenecholesterol supports the phylogenetic relationship be-
tween oomycetes and brown algae.15
3.4. Conversion of 1 into 22,24-O-isopropylidene derivative 2
To a solution of 1 (1.2 mg, 2.1 mmol) in 2,2-dimethoxypropane
(2 mL) was added p-toluenesulfonic acid (2.1 mg). After being
stirred for 14 h at room temperature, the reaction mixture was
poured into saturated sodium bicarbonate solution, and extracted
with ethyl acetate three times. The combined organic layer was
washed with water and brine, dried over sodium sulfate, and
evaporated. The residue was chromatographed over silica gel
eluted by hexane/ethyl acetate (1:1) to give 2 (0.6 mg, 1.1
57%). Data for 2: colorless amorphous solid; 1H NMR (600 MHz,
CDCl3)
mmol,
d
4.29 (1H, dt, J¼11.8, 3.8 Hz), 4.21 (2H, d, J¼2.1 Hz), 3.99 (1H,
d, J¼3.2 Hz), 3.20 (1H, dd, J¼11.8, 4.3 Hz), 1.88e2.09 (8H, m),
1.45e1.76 (14H, m), 1.38 (3H, s), 1.27 (3H, s), 1.18e1.36 (7H, m), 1.02
(1H, dd, J¼12.7, 2.1 Hz), 0.98 (3H, s), 0.95 (3H, s), 0.88 (3H, s), 0.79
Fig. 7. Structure of 1a,7a-diacetoxyapotirucall-14-ene-3a,21,22,24,25-pentaol (10).
(3H, s), 0.72 (3H, s); 13C NMR (150 MHz, CDCl3)
d 171.0, 134.8, 134.0,
118.9, 80.7, 79.3, 79.0, 69.1, 63.0, 50.4, 49.5, 44.6, 43.1, 41.6, 38.9, 37.1,
35.6, 30.9, 29.7, 28.0 (2C), 27.9, 27.4, 27.0, 26.5, 25.7, 24.5, 23.5, 21.2,
21.1, 20.4, 19.2, 18.2, 16.0, 15.4; HRFABMS m/z 575.4332 [MþH]þ
(575.4312 calcd for C35H59O6).
Terresterol (1) showed innate immune-promoting activity.
Thus, terresterol (1) has potential to be used as a lead compound for
novel immunostimulating agents against bacterial infections and as
probes for the innate immune system.
3.5. Conversion of 1 into 21,22:24,25-di-O-isopropylidene de-
rivative 3
3. Experimental section
3.1. General methods
To a solution of 1 (5.0 mg, 8.7 mmol) in methanol (1 mL) was
added sodium methoxide (5.0 mg). After being stirred for 3 h at
room temperature, the mixture was acidified with Dowex 50w (Hþ
form) until the pH reached 2, and filtered. Then, the filtrate was
evaporated, and the residue was dissolved in 2,2-dimethoxypropane
(2 mL). p-Toluenesulfonic acid (4.0 mg) was added to the solution.
After being stirred for 3 h at room temperature, the reaction mixture
was poured into saturated sodium bicarbonate solution, and
extracted with ethyl acetate three times. The combined organic layer
was washed with water and brine, dried over sodium sulfate, and
evaporated. The residue was chromatographed over silica gel eluted
Analytical TLC was performed on silica gel 60 F254 (Merck).
Column chromatography was carried out on silica gel 60 (70e230
mesh, Merck). NMR spectra were recorded on JEOL ECA-600 and
AL-400. Chemical shifts for 1H and 13C NMR are given in parts per
million (d) relative to tetramethylsilane (dH 0.00) and residual sol-
vent signals (dC 77.0 and 49.0 for CDCl3 and CD3OD, respectively) as
internal standards. Mass spectra were measured on JEOL JMS-700
and JMS-DX303. IR spectra were measured on JASCO FT/IR-4200.
by hexane/ethyl acetate (9:1) to give 3 (3.1 mg, 5.4
steps)). Data for 3: colorless amorphous solid; 1H NMR (600 MHz,
CDCl3)
mmol, 62% (two
3.2. Organism and culture conditions
d
4.19 (1H, d, J¼11.0 Hz), 4.00 (1H, dd, J¼11.6, 3.9 Hz), 3.93 (1H,
The oomycete, S. terrestris NRBC102124 was supplied by Bi-
ological Resource Center (NBRC), National Institute of Technology
and Evaluation, Chiba, Japan. This strain was cultured in YM me-
dium (glucose 1%, Bacto Peptone 0.5%, Bacto Malt extract 0.3%, yeast
extract 0.3%) at 28 ꢀC for 2 weeks on a rotary shaker at 150 rpm.
dd, J¼11.6, 3.7 Hz), 3.87 (1H, d, J¼10.1 Hz), 3.15 (1H, dd, J¼11.7,
4.4 Hz), 2.25 (1H, q, J¼8.6 Hz),1.94e2.08 (5H, m),1.86 (1H, dt, J¼13.4,
8.7 Hz), 1.72 (1H, dt, J¼12.8, 2.9 Hz), 1.19e1.67 (12H, m), 1.40 (6H, s),
1.37 (3H, s), 1.31 (3H, s), 1.24 (3H, s), 1.07 (3H, s), 1.04 (1H, d,
J¼12.6 Hz), 0.99 (3H, s), 0.97 (3H, s), 0.93 (3H, s), 0.80 (3H, s), 0.72
(3H, s); 13C NMR (150 MHz, CDCl3)
d 134.9, 134.0, 106.4, 99.0, 79.9,
3.3. Isolation of terresterol (1)
79.6, 79.0, 71.4, 63.9, 50.4, 49.4, 45.3, 42.9, 40.6, 38.9, 37.0, 35.6, 34.0,
31.6, 30.6, 29.7, 29.1, 28.6, 28.0, 27.9, 26.8, 26.6, 25.7, 24.3, 23.1, 21.2,
21.0, 19.2, 18.2, 15.9, 15.4; HRFABMS m/z 572.4431 [M]þ (572.4438
calcd for C36H60O5).
The mycelia of S. terrestris, which was obtained from the cul-
tured broth (9.0 L), were extracted three times with methanol (2 L)
at room temperature to give the extract (9.86 g). This extract was
partitioned with ethyl acetate and water to yield ethyl acetate
solubles (1.47 g). The ethyl acetate solubles were chromatographed
over SiO2, and the column eluted with n-hexane/ethyl acetate
mixtures with increasing polarity to afford hexane/ethyl acetate
(2:1) eluent (fraction A, 750 mg) and hexane/ethyl acetate (1:9)
eluent (fraction B, 54 mg). Fraction A was further separated by ODS
column using water/acetonitrile solvent system to give the mixture
of fucosterol and 24-methylenecholesterol (1:1) (25.5 mg). Fraction
3.6. Conversion of 1 into di-(R)-a-methoxyphenylacetate 4a
To a solution of 1 (1.1 mg, 1.9
were added triethylamine (20
nitrobenzoic anhydride (8.2 mg), and (R)-
m
m
mol) in dichloromethane (0.5 mL)
L), DMAP (2.9 mg), 2-methyl-6-
-methoxyphenylacetic
a
acid (7.1 mg) at room temperature. After being stirred for 6 h, the
mixture was poured into saturated ammonium chloride solution,
and extracted with ethyl acetate three times. The combined organic
layer was washed with water and brine, dried over sodium sulfate,
B
was also further separated by ODS column using

H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
3541
and evaporated. The residue was chromatographed over silica gel
eluted by hexane/ethyl acetate (1:1) to give 4a (1.2 mg, 1.4 mol,
72%). Data for 4a: colorless oil; 1H NMR (600 MHz, CDCl3)
7.41e7.43 (2H, m), 7.36e7.39 (2H, m), 7.29e7.35 (6H, m),
Data for the mixture of 8 and 9: colorless amorphous solid; 1H NMR
and 13C NMR data were identical to those of 8 and 9 reported in the
literature;13 HRFABMS m/z 460.3895 [M]þ (460.3914 calcd for
C30H52O3).
m
d
4.94e4.97 (1H, m), 4.74e4.76 (1H, m), 4.73 (1H, s), 4.72 (1H, s), 4.52
(1H, dd, J¼11.3, 4.7 Hz), 3.68 (1H, dd, J¼11.9, 4.2 Hz), 3.62 (1H, dd,
J¼11.9, 2.8 Hz), 3.44 (3H, s), 3.41 (3H, s), 2.09 (3H, s), 1.98e2.02 (2H,
m), 1.91 (3H, s), 1.17e1.86 (18H, m), 1.15 (3H, s), 1.14 (3H, s), 1.08 (1H,
dd, J¼12.7, 2.1 Hz), 0.90 (3H, s), 0.83 (3H, s), 0.82 (3H, s), 0.80 (3H, s),
0.55 (3H, s); HRFABMS: m/z 872.5081 [M]þ (872.5071 calcd for
C52H72O11).
3.11. Ex vivo Drosophila culture assay
The detailed procedure was described previously.5b Briefly, the
abdominal cavity of third-instar larva was opened using fine pinc-
ettes. Individual whole larval tissues were cultured in Schneider’s
Drosophila medium (Gibco-BRL, Invitrogen, Carlsbad, CA) containing
20% fetal bovine serum (Valley Biomedical, Winchester, VA) and 1%
antibiotics/antimycotics (Gibco-BRL) in each well of a 96-well plate
at 25 ꢀC. For each condition, six females were cultured to produce six
replicates. The test compounds were dissolved in DMSO and added
to the culture medium. To determine the effects of the test com-
pounds on the innate immune response, Dpt-lacZ larvae were cul-
tured in the presence of 100 ng/mL peptidoglycans from Escherichia
coli (InvivoGen, San Diego, CA) and the compound at 25 ꢀC for 12 h.
3.7. Conversion of 1 into di-(S)-a-methoxyphenylacetate 4b
In the same manner as the synthesis of 4a, compound 4b
(1.2 mg, 1.4 mol, 88%) was synthesized from 1. Data for 4b: col-
orless oil; 1H NMR (600 MHz, CDCl3)
7.43e7.45 (4H, m), 7.29e7.38
m
d
(6H, m), 4.89e4.91 (1H, m), 4.72 (1H, s), 4.69 (1H, s), 4.49 (1H, dd,
J¼11.6, 4.3 Hz), 4.11 (1H, dd, J¼12.1, 2.3 Hz), 4.02e4.07 (2H, m), 3.40
(3H, s), 3.38 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 1.94e1.99 (2H, m),
1.14e1.81 (18H, m), 1.03 (1H, dd, J¼12.7, 2.0 Hz), 0.94 (3H, s), 0.92
(3H, s), 0.91 (3H, s), 0.84 (3H, s), 0.68 (3H, s), 0.65 (3H, s), 0.42 (3H,
s); HRFABMS: m/z 872.5032 [M]þ (872.5071 calcd for C52H72O11).
The cultured individual larvae were sonicated with 200 mL reaction
buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM
MgCl2) using an Ultrasonic Processor (Misonix, New York, NY). After
centrifugation (10,000ꢂg) at 4 ꢀC for 10 min, supernatant was har-
vested, and b-galactosidase activity and total protein amount of su-
pernatant were determined as previously described.5a
Galactosidase activity was normalized to total protein amount.
b-
3.8. Conversion of 1 into pentaol 5
To a solution of 1 (2.2 mg, 3.8 mmol) in methanol (1 mL) was
added sodium methoxide (5.0 mg). After being stirred for 3 h at
room temperature, the mixture was acidified with Dowex 50w (Hþ
form) until the pH reached 2, and filtered. Then, the filtrate was
evaporated, and the residue was chromatographed over silica gel
3.12. Measurement of cytotoxicity and luciferase activity
Drosophila S2 cells and S2att-luc cells, harboring luciferase re-
porter gene driven by attacin promoter,12 were cultured in
Schneider’s Drosophila medium (Gibco-BRL) supplemented with
10% FBS and 1% antibiotics/antimycotics at 25 ꢀC. Cytotoxicity was
measured using the colorimetric thiazoyl blue conversion assay
using WST-8 solution (nacalai tesque) as described previously.5b To
determine the effect of compounds on attacin expression via IMD
pathway, S2att-luc cells were pre-treated with compounds for 1.5 h
and stimulated by 100 ng/mL peptidoglycans. At 8 h after stimu-
lation, cells were lysed with Glo-lysis buffer (Promega), and lucif-
erase activities were measured by One-Glo (Promega).
eluted by ethyl acetate/methanol (9:1) to give 5 (1.8 mg, 3.7
97%). Data for 5: colorless amorphous solid; 1H NMR (400 MHz,
CD3OD) 3.96e4.02 (2H, m), 3.57e3.65 (2H, m), 3.14e3.18 (1H, m),
mmol,
d
1.98e2.19 (5H, m), 1.42e1.79 (14H, m), 1.20e1.26 (1H, m), 1.18 (3H,
s), 1.17 (3H, s), 1.05 (1H, dd, J¼12.3, 1.7 Hz), 1.02 (3H, s), 0.98 (3H, s),
0.90 (3H, s), 0.82 (3H, s), 0.80 (3H, s); HRFABMS: m/z 493.3901
[MþH]þ (493.3890 calcd for C30H53O5).
3.9. Conversion of 1 into tetraacetate 6
To a solution of 1 (1.2 mg, 2.1
mmol) in pyridine (1 mL) was
Acknowledgements
added acetic anhydride (300 L). After being stirred for 5 h at room
m
temperature, the reaction mixture was evaporated. The residue was
chromatographed over silica gel eluted by hexane/ethyl acetate
We thank Dr. J.L. Imler for Att-luc reporter construct. This work
was supported in part by Grant-in-Aid for Scientific Research (No.
23710247) from the Ministry of Education, Science, Sports and
Culture of Japan, and the SUNBOR GRANT from the Suntory In-
stitute for Bioorganic Research.
(3:2) to give 6 (0.6 mg, 0.9
m
mol, 37%). Data for 6: colorless amor-
phous solid; 1H NMR (400 MHz, CDCl3)
d
4.91 (1H, dt, J¼10.9,
3.0 Hz), 4.87 (1H, dd, J¼10.6, 3.0 Hz), 4.49 (1H, dd, J¼11.8, 4.5 Hz),
4.21 (1H, dd, J¼11.8, 2.6 Hz), 4.11 (1H, dd, J¼11.8, 5.1 Hz), 2.09 (3H,
s), 2.06 (3H, s), 2.05 (3H, s), 2.03 (3H, s), 1.23e1.95 (20H, m), 1.20
(6H, s), 1.14 (1H, dd, J¼12.5, 2.1 Hz), 1.00 (3H, s), 0.91 (3H, s), 0.88
(6H, s), 0.72 (3H, s); HRFABMS: m/z 660.4223 [MþH]þ (660.4233
calcd for C38H60O9).
Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.02.088.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
3.10. Synthesis of the mixture of inoterpene A (8) and B (9)
To a solution of lanosterol (7) (31.6 mg, 74.2 mmol) in acetone/
acetonitrile/water (1:1:1) (4 mL) was added trimethylamine N-
References and notes
oxide (15.9 mg) and 4% osmium tetroxide solution in water
(100 mL). After being stirred for 24 h at room temperature, the re-
1. Sleigh, M. A. Protozoa and Other Protists; Cambridge University Press: London,
1989.
2. Van der Auwera, G.; De Baere, R.; Van de Peer, Y.; De Rijk, P.; Van den Broeck, I.;
De Wachter, R. Mol. Biol. Evol. 1995, 12, 671e678.
3. (a) William, F. Mol. Plant Pathol. 2008, 9, 385e402; (b) McDonald, B. A.; Linde, C.
Annu. Rev. Phytopathol. 2002, 40, 349e379.
4. (a) McMorris, T. C.; Barksdale, A. W. Nature 1967, 215, 320e321; (b) Arsenault,
G. P.; Biemann, K.; Barksdale, A. W.; McMorris, T. C. J. Am. Chem. Soc. 1968, 90,
5635e5636; (c) Green, D. M.; Edwards, J. A.; Barksdale, A. W.; McMorris, T. C.
Tetrahedron Lett. 1971, 27, 1199e1203.
action mixture was poured into 10% sodium thiosulfate solution,
and extracted with ethyl acetate three times. The combined organic
layer was washed with saturated sodium bicarbonate solution and
brine, dried over sodium sulfate, and evaporated. The residue was
chromatographed over silica gel eluted by hexane/ethyl acetate
(1:1) to give the 1:1 mixture of 8 and 9 (8.5 mg, 18.5 mmol, 25%).

3542
H. Kikuchi et al. / Tetrahedron 69 (2013) 3536e3542
5. (a) Yajima, M.; Takada, M.; Takahashi, N.; Kikuchi, H.; Natori, S.; Oshima, Y.;
Kurata, S. Biochem. J. 2003, 371, 205e210; (b) Sekiya, M.; Ueda, K.; Fujita, T.;
Kitayama, M.; Kikuchi, H.; Oshima, Y.; Kurata, S. Life Sci. 2006, 80, 113e119.
6. Hoffmann, J. A.; Reichhart, J. M. Nat. Immunol. 2002, 3, 121e126.
Isobe, M.; Kurata, S.; Katou, Y.; Oshima, Y. Tetrahedron 2012, 68,
6218e6223.
9. Rychnovsky, S. D.; Yang, G.; Powers, J. P. J. Org. Chem. 1993, 58, 5251e5255.
10. Shiina, I.; Kobota, M.; Oshiumi, H.; Hashizume, M. J. Org. Chem. 2004, 69,
1822e1830.
7. Hultmark, D. Curr. Opin. Immunol. 2004, 15, 12e19.
8. (a) Sekiya, M.; Ueda, K.; Okazaki, K.; Kikuchi, H.; Kurata, S.; Oshima, Y.
Biochem. Pharmacol. 2008, 75, 2165e2174; (b) Kikuchi, H.; Sekiya, M.;
Katou, Y.; Ueda, K.; Kabeya, T.; Kurata, S.; Oshima, Y. Org. Lett. 2009, 11,
1693e1695; (c) Kikuchi, H.; Okazaki, K.; Sekiya, M.; Uryu, Y.; Ueda, K.;
Katou, Y.; Kurata, S.; Oshima, Y. Eur. J. Med. Chem. 2011, 46, 1263e1273;
(d) Sekiya, M.; Ueda, K.; Okazaki, K.; Terashima, J.; Katou, Y.; Kikuchi, H.;
Kurata, S.; Oshima, Y. Int. Immunopharmacol. 2011, 11, 1497e1503; (e)
Kikuchi, H.; Isobe, M.; Sekiya, M.; Abe, Y.; Hoshikawa, T.; Ueda, K.; Kurata,
S.; Katou, Y.; Oshima, Y. Org. Lett. 2011, 13, 4624e4627; (f) Kikuchi, H.;
11. Trost, B. M.; Balletire, J. L.; Godleski, S.; McDougal, P. G.; Balkovec, J. M. J. Org.
Chem. 1986, 51, 2370e2374.
12. Goto, A.; Matsushita, K.; Gesellchen, V.; Chamy, L. E.; Kuttenkeuler, D.; Take-
uchi, O.; Hoffmann, J. A.; Akira, S.; Boutros, M.; Reichhart, J.-M. Nat. Immunol.
2008, 9, 97e104.
13. Nakamura, S.; Iwami, J.; Matsuda, H.; Mizuno, S.; Yoshikawa, M. Tetrahedron
2009, 65, 2443e2450.
14. Luo, X.-D.; Wu, S.-H.; Ma, Y.-B.; Wu, D.-G. Fitoterapia 2000, 71, 668e672.
15. Gaulin, E.; Bottin, A.; Dumas, B. Plant Signal. Behav. 2010, 5, 258e260.