Total synthesis, structural revision, and biological evaluation of calafianin, a marine spiroisoxazoline from the sponge, Aplysina gerardogreeni

Article abstract of DOI:10.1246/bcsj.79.134

The reported structure of the natural calafianin (1) and its isomer in the epoxide region 13 were successfully synthesized through the cis- and trans-spiroisoxazoline compounds, which were produced by electrochemical oxidation of the hydroxyimino-phenol d

Full text of DOI:10.1246/bcsj.79.134

134 Bull. Chem. Soc. Jpn. Vol. 79, No. 1, 134–139 (2006)
ꢀ 2006 The Chemical Society of Japan
Total Synthesis, Structural Revision, and Biological
Evaluation of Calafianin, a Marine Spiroisoxazoline
from the Sponge, Aplysina gerardogreeni
ꢀ1
Takahisa Ogamino,1 Rika Obata,1 Hiroshi Tomoda,2 and Shigeru Nishiyama
1Department of Chemistry, Faculty of Science and Technology, Keio University,
3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522
2School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641
Received August 4, 2005; E-mail: nisiyama@chem.keio.ac.jp
The reported structure of the natural calafianin (1) and its isomer in the epoxide region 13 were successfully syn-
thesized through the cis- and trans-spiroisoxazoline compounds, which were produced by electrochemical oxidation of
the hydroxyimino-phenol derivative 7, followed by Zn[BH4]2 reduction. Comparison of the spectroscopic data of the
synthetic (ꢁ)-1 and (ꢁ)-13 resulted in structural revision of the natural calafianin to 13, possessing the trans-relationship
between an epoxide and an oxygen of the isoxazoline. In addition, a significant difference of antimicrobial activity
between 1 and 13 is discussed.
A series of the spiroisoxazoline natural products, such as
aerothionin (2),1 homoaerothionin (3), and aerophobin-1 (4),
have been isolated from marine sponges since the early
1970’s (Fig. 1).2
(TTN)3 or anodic oxidation4 of the corresponding hydroxy-
imino-phenol 7, followed by reduction of the spirodienone 8
with Zn[BH4]2 (Scheme 1). The relative stereochemistries of
5 and 6 were determined by conversion into natural products,
such as aerothionin (2),3 the stereochemistry of which was
unambiguously determined by X-ray crystallographic analy-
sis.1b In addition, exposure of the unnatural 6 to bases caused
ꢀ-elimination to provide 7.
From among the spiroisoxazoline natural products, calafia-
nin (1), isolated from the sponge Aplysina gerardogreeni n. sp
(aplysinidae), possesses a dimeric structure of the typical spi-
roisoxazoline fused with the cyclohexenone carrying epoxide
rings.6 From a stereochemical viewpoint, it was reported that
the cis-relationship of two oxygen atoms between the epoxide
and the isoxazoline was deduced by the NOE correlation
These spiroisoxazolines share a spiro skeleton, which con-
sists of an isoxazoline ring and a cyclohexadiene moiety A.
Biosynthetic condensation of A with alkyl amines or diamines
produces a variety of structural diversities of these class natu-
ral products, which display a wide range of biological activi-
ties, such as antimicrobial, cytotoxic, and antiinflammatory
activities. Accordingly, spiroisoxazoline natural products have
been recognized as attractive synthetic targets. Indeed, many
research groups have endeavored in their total synthesis.3–5
In this context, we constructed the natural trans-form 5 and un-
natural cis-form 6 of spiroisoxazolines by thallium(III) nitrate
O
O
O
O
NH(CH2)4NH
NH(CH2)nNH
N
O
N
O
N
O
N
O
4'
HO
Br
1
OH
Br
O
O
Br
Br
Br
Br
O
OMe
O
OMe
1
2
3
n = 4
n = 5
O
O
O
O
NH
NH(CH2)4NH
N
R
N
N
N
O
O
O
O
HO
Br
NH
HO
Br
HO
Br
OH
Br
N
Br
Br
Br
Br
OMe
OMe
OMe
OMe
A
4
2'
Fig. 1. Proposed structure of calafianin (1) and related natural products.
Published on the web January 13, 2006; DOI 10.1246/bcsj.79.134

T. Ogamino et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 1 (2006)
135
NOH
COOMe
COOMe
Zn[BH4]2
COOMe
COOMe
N
O
N
O
N
O
CCE
n-Bu4NClO4
O
HO
Br
HO
H
HO
Br
+
MeCN
Br
Br
Br
Br
Br
Br
OMe
OMe
OMe
OMe
7
8
5
6
Scheme 1. Synthesis of the spiroisoxazoline structure.
COOMe
COOR
Br
COOMe
N
O
N
O
N
O
HO
Br
HO
Br
AcOH
DBU
O
CH2Cl2
80 °C
Br
Br
32% in 2 steps
O
O
OMe
( )-6
9
10 R = Me
12 R = H
1 M KOH
quant.
O
COOMe
3''
1''
N
O
2''
N
H
3'
4''
N
O
2
PivCl
4'
3
+
7
1,4-diaminobutane
1
2
5
O
Br
O
Br
4
py/CH2Cl2
32%
6
Br
O
41%
11
1
8%
Scheme 2. Synthesis of proposed structure of calafianin (1).
between H-1 and H-40 protons,6 as well as that no significant
biological activity against the multidrug-resistant clinical iso-
late M. tuberculosis H37Rv, occurred in contrast to that of
2.7 In spite of closely related structures, the clear biological
difference between 1 and 2 prompted us to synthesize 1 by uti-
lizing the spiroisoxazoline derivatives mentioned above and to
examine its biological activity. We describe herein synthesis of
calafianin in (ꢁ)-form and its antimicrobial activity.8
od was available than this approach using 6 as an intermediate.
Alkaline hydrolysis of 10 in aq 1,4-dioxane afforded the car-
boxylic acid 12 in high yield, while reaction in MeOH was un-
successful owing to undesired decarboxylation.4b Accordingly,
compound 12 should be submitted to the next step without
purification for its instability. In the final step, upon using such
reagents as carbonyl diimidazole (CDI), 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodimide (EDCI), and 1H-benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP), coupling of 12 with 1,4-diaminobutane gave calafianin
(1) at the most in 12% yield. After extensive evaluation to
improve the yield, the final condensation could be achieved
in 41% yield by using pivaloyl chloride (PivCl) as a coupling
reagent.
Results and Discussion
At the outset, synthesis of (ꢁ)-1 was attempted by chemical
modification of (ꢁ)-cis-aerothionin (20);3,9 however, an appro-
priate method could not be found under the several reaction
conditions examined, owing to the labile character of the same
cis-spiroisoxazoline structure as that of the ꢀ-elimination-
prone 6. Accordingly, as an alternative method, (ꢁ)-6 prepared
from selective reduction of 8 with Na[BH4]10 was converted
into the epoxy ketone 10 carrying the partial-structure of 1
(Scheme 2). Thus, cleavage of a methyl enol ether of 6 under
acidic conditions gave 9 as a diastereomeric mixture, which
upon treatment with DBU afforded the epoxy derivative 10
in 2 steps. Upon using refluxing AcOH, the epoxy compound
10 was obtained in 32% yield, along with the dienone 119 (8%)
and phenol 7 (32%), whereas no desired reaction proceeded
in MsOH or TFA. The main reason of the low yield might be
the labile property of the cis-spiroisoxazoline 6 under basic or
even acidic conditions, which gave the spirodienone 11 by ꢀ-
elimination, along with 7 by the ring-opening reaction of iso-
xazoline. Despite the accompanying problems, no better meth-
1
As shown in Table 1, comparison of the H and 13C NMR
data of the synthetic sample with the reported data6 exhibited
clear differences in the regions of H-3, H-6, and H-40 and C-1,
C-4, C-6, and C-100. This observation indicated that the report-
ed configuration of the epoxide residue should be revised.
As mentioned above, the reported calafianin has the cis
vicinal relationship of two oxygen atoms. However, other spi-
roisoxazoline natural products, such as aerothionin (2), homo-
aerothionin (3), and aerophobin-1 (4), have the trans vicinal
relationship (Fig. 1). In hypothesis of their biosynthesis, the
fundamental framework A is constructed by oxidation of the
p-methoxyphenyl derivative B, followed by an intramolecular
nucleophilic attack on the epoxide part of the arene oxide C
(Scheme 3).11
Based on the assumption that natural calafianin was assem-

136 Bull. Chem. Soc. Jpn. Vol. 79, No. 1 (2006)
Synthesis of Calafianin
Table 1. Comparison of the 1H and 13C NMR data (ꢁ, DMSO-d6) of the Synthetic 1 and 13 with the Reported
Data (Ref. 6)
Synthetic 1
1H
(mult, J in Hz)
Synthetic 13
Natural data (Ref. 6)
1H
(mult, J in Hz)
1H
(mult, J in Hz)
No.
13C
13C
13C
1
2
4.12 (dd, 2.8, 4.0)
55.0
85.4
4.12 (dd, 2.4, 4.0)
56.8
83.9
4.12 (dd, 2.6, 3.7)
56.9
84.0
3
4
5
6
30
40
7.29 (d, 2.8)
3.81 (d, 4.0)
144.2
119.7
185.8
51.8
154.6
43.3
7.48 (d, 2.4)
143.7
122.7
185.9
52.9
154.8
43.3
7.49 (d, 2.6)
3.94 (d, 3.5)
143.7
122.8
186.0
53.0
154.9
43.6
3.92 (d, 3.6)
3.56 (d, 18.4)
3.41 (d, 18.4)
3.67 (d, 18.0)
3.60 (d, 18.0)
3.68 (d, 17.8)
3.61 (d, 17.9)
100
200
300
400
159.7
158.2
158.3
8.61 (t, 6.0)
3.19 (br)
1.52 (br)
8.64 (t, 6.0)
3.20 (br)
1.48 (br)
8.63 (t, 5.7)
3.19 (br)
1.50 (br)
38.5
26.3
38.5
26.3
38.6
26.4
O
R
O
R
O
N
O
R
NOH
NOH
O
intramolecular
cyclization
HO
Br
[O]
Br
Br
Br
Br
Br
OMe
OMe
OMe
B
C
A
Scheme 3. Biosynthetic hypothesis of the natural spiroisoxazolines.
bled by a similar biosynthetic pathway to those of other spiro-
isoxazoline derivatives, we expected the natural product should
possess the relative stereochemistry of 13, which has the trans
relationship. Accordingly, synthesis of 13 was carried out start-
ing from the trans-alcohol 5 by employing essentially the same
approach as that of 1 (Scheme 4).
In conclusion, (ꢁ)-1 carrying the proposed structure of cal-
afianin and the isomeric (ꢁ)-13 were successfully synthesized
by using the spiroisoxazolines 5 and 6 as an efficient demon-
stration of chemical modification of a spiroisoxazoline struc-
ture. This investigation enabled the structural revision of 1 to
13, which has the trans-relationship between the epoxy ring
and the oxygen atom in the spiroisoxazoline residue. In addi-
tion, biological assessment of the synthetic samples showed a
significant difference in antimicrobial activity between 1 and
13 involving MRSA.
The epoxy derivative 15,12 possessing the trans-relationship
between two oxygen atoms, was synthesized from (ꢁ)-5 by
acidic hydrolysis followed by epoxidation of the ketone 14
without production of by-products such as 11. Finally, 13 was
produced in 39% yield by condensation of the carboxylic acid
form (16) of 15 with 1,4-diaminobutane. As expected, the spec-
tral data of the synthetic (ꢁ)-13 was superimposable to those
Experimental
General. Melting points were measured on a Yanaco MP-S3
and are uncorrected. IR spectra were recorded on a JASCO Model
A-202 spectrophotometer. 1H NMR and 13C NMR spectra were
obtained on JEOL JNM EX-270 and JEOL JNM GX-400 spec-
trometers in deuteriochloroform using tetramethylsilane as an
internal standard, unless otherwise stated. High-resolution mass
spectra were obtained on JEOL JMS-700 (FAB) and Hitachi M-
80 B GC-MS spectrometers. Preparative and analytical TLC were
carried out on silica-gel plates (Kieselgel 60 F254, E. Merck AG.,
Germany) using UV light and/or 5% molybdophosphoric acid in
ethanol for detection. Kanto Chemical silica 60N (spherical, neu-
tral, 63–210 mm) was used for column chromatography.
reported (Table 1).6,13 Against the report by Encarnacion et al.,
´
the clear NOE correlation between H-1 and H-40 (2%) was ob-
served even in the trans-relationship. Accordingly, the relative
stereochemisty of calafianin should be revised to 13, as shown
in Scheme 4. Also, 13 was obtained from aerothionin (ꢁ)-(2),1
which was prepared from 53 in 2 steps as an alternative access
(Scheme 5).
Bioactivity. Despite of the weak activity of calafianin
reported,6 the synthetic samples were submitted for biological
assessment. Consequently, 13 showed antimicrobial activity
against Gram-positive bacteria including MRSA, while 1
showed no activity (Table 2). Although 1 and 13 possess
closely similar structures, a large difference in biological activ-
ity against several Gram-positive bacteria between them was
observed. Further biological investigation is in progress.
Synthesis of Methyl (1R,2S,6S)-4-Bromo-5-oxo-7-oxaspiro-
[bicyclo[4.1.0]hept-3-ene-2,50(40H)isoxazole]-30-carboxylate (10).
A solution of 6 (680 mg, 1.7 mmol) in AcOH (20 mL) was stirred
at refluxing temperature for 6 h. After evaporation, the mixture

T. Ogamino et al.
Bull. Chem. Soc. Jpn. Vol. 79, No. 1 (2006)
137
COOMe
COOR
N
COOMe
N
N
O
O
O
HO
Br
HO
Br
AcOH
DBU
O
CH2Cl2
80 °C
Br
Br
Br
66% in 2 steps
O
O
OMe
( )-5
14
15 R = Me
16 R = H
1 M KOH
quant.
O
N
N
O
2
H
PivCl
4'
1
1,4-diaminobutane
O
py/CH2Cl2
Br
O
39%
13
Scheme 4. Synthesis of 13, revised structure of calafianin (1).
O
COOMe
N
N
O
N
O
H
2
1) MsOH, 0 °C
HO
Br
1,4-diaminobutane
CH2Cl2
HO
Br
13
2) DBU
Br
Br
OMe
OMe
aerothionin (2)
11%
40%
5
Scheme 5. Synthesis of 13, prepared through aerothionin (2).
was submitted to the next step without further purification.
A mixture of the crude and DBU (0.4 mL) in CH2Cl2 (20 mL)
was stirred for 1 h. The mixture was diluted with H2O, and
extracted with CHCl3. The organic layer was washed with brine,
dried (Na2SO4), and evaporated. Purification by silica-gel column
chromatography (hexane/Et2O = 1/1) gave 10 (166 mg, 32%), 7
(224 mg, 32%), and 11 (52 mg, 8%).
10: mp 159–160 ꢂC (needles, EtOAc–hexane); IR (KBr) 1705,
1603 cmꢃ1; 1H NMR ꢁ 3.41 (1H, d, J ¼ 17:5 Hz), 3.45 (1H, d, J ¼
17:5 Hz), 3.74 (1H, d, J ¼ 3:6 Hz), 3.83 (1H, dd, J ¼ 2:6, 3.6 Hz),
3.93 (3H, s), 6.99 (1H, d, J ¼ 2:6 Hz); 13C NMR ꢁ 43.0, 51.5, 53.2,
54.9, 86.6, 121.0, 141.9, 150.5, 159.5, 185.2; HRMS found m=z
300.9580, calcd for C10H879BrNO5: M, 300.9585. Found: C,
39.47; H, 2.71; N, 4.50%. Calcd for C10H8BrNO5: C, 39.76; H,
2.67; N, 4.64%.
Synthesis of Calafianin (Proposed Structure) (1). A solution
of 10 (78 mg, 0.26 mmol) in 1,4-dioxane (4 mL), H2O (1.6 mL),
and 1 M (1 M = 1 mol dmꢃ3) KOH (0.5 mL) was stirred at 0 ꢂC for
1 h. After treatment with Amberlite IR-120B (Hþ), the mixture was
filtered and the filtrate was evaporated to give 12 (77 mg, quant.) as
a colorless solid: 1H NMR (CD3OD) ꢁ 3.38 (1H, d, J ¼ 18:0 Hz),
3.49 (1H, d, J ¼ 18:0 Hz), 3.68 (1H, d, J ¼ 4:0 Hz), 3.93 (1H, dd,
J ¼ 2:4, 4.0 Hz), 7.16 (1H, d, J ¼ 2:4 Hz); 13C NMR (CD3OD) ꢁ
45.3, 53.1, 56.6, 86.9, 121.1, 145.4, 157.3, 164.8, 187.2.
A mixture of 12 (77 mg, 0.26 mmol) and PivCl (32 mL, 0.26
mmol) in CH2Cl2 (4 mL) and pyridine (1.6 mL) was stirred at ꢃ10
ꢂC for 10 min, and then 1,4-diaminobutane (13 mL, 0.13 mmol) was
added. After further stirring for 1 h, the mixture was diluted with
H2O and extracted with EtOAc. The organic layer was washed
with brine, dried (Na2SO4), and evaporated. The residue was puri-
fied by preparative TLC (hexane/acetone = 1/1) to give 1 (33
mg, 41%) as a colorless solid: IR (KBr) 3375, 1703, 1666 cmꢃ1
;
1H NMR (DMSO-d6) ꢁ 1.52 (4H, br), 3.19 (4H, br), 3.41 (2H, d,
J ¼ 18:4 Hz), 3.56 (2H, d, J ¼ 18:4 Hz), 3.81 (2H, d, J ¼ 4:0 Hz),
4.12 (2H, dd, J ¼ 2:8, 4.0 Hz), 7.29 (2H, d, J ¼ 2:8 Hz), 8.61 (2H,
t, J ¼ 6:0 Hz); 13C NMR (DMSO-d6) ꢁ 26.3, 38.5, 43.3, 51.8, 55.0,
85.4, 119.7, 144.2, 154.6, 159.7, 185.8; HRMS m=z 629.9620,
calcd for C22H2081Br2N4O8: M, 629.9614.
Synthesis of Methyl (1S,2S,6R)-4-Bromo-5-oxo-7-oxaspiro-
[bicyclo[4.1.0]hept-3-ene-2,50(40H)isoxazole]-30-carboxylate (15).
A solution of 5 (331 mg, 0.83 mmol) in AcOH (10 mL) was stirred
at refluxing temperature for 4 h. After concentration of the mix-
ture, the residue was diluted with CH2Cl2 (10 mL) and then
DBU (0.4 mL) was added at room temperature. After being stirred
for 1 h, the mixture was diluted with H2O and extracted with
CHCl3. The organic layer was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by silica-gel column
chromatography (hexane/EtOAc = 4/1) to give 15 (165 mg, 66%):
mp 156–158 ꢂC (needles, EtOAc–hexane); IR (KBr) 1708, 1608
cmꢃ1; 1H NMR ꢁ 3.43 (1H, d, J ¼ 17:6 Hz), 3.74 (1H, d, J ¼ 17:6
Hz), 3.78 (1H, d, J ¼ 3:4 Hz), 3.80 (1H, dd, J ¼ 2:4, 3.4 Hz),
3.95 (3H, s), 6.93 (1H, d, J ¼ 2:4 Hz); 13C NMR ꢁ 43.7, 53.28,
53.29, 57.0, 85.2, 125.1, 140.8, 151.2, 159.6, 184.3; HRMS: m=z
300.9575, calcd for C10H879BrNO5: M, 300.9585. Anal. Found:
C, 39.57; H, 2.90; N, 4.42%. Calcd for C10H8BrNO5: C, 39.76;
H, 2.67; N, 4.64%.

138 Bull. Chem. Soc. Jpn. Vol. 79, No. 1 (2006)
Table 2. Antimicrobial Activities of Calafianin Derivatives
Synthesis of Calafianin
Diameter of inhibition zoneaÞ/mm
Microorganism
1
13
Bacillus subtilis
Staphylococcus aureus
Micrococcus luteus
—
—
—
—
—
14.6
—
—
—
—
—
—
—
—
7.9
20.9
11.8
8.1
7.9
—
18.0
—
7.4
—
—
—
—
—
—
17.5
Esherichia coli
Pseudomonas aeruginosa
Xanthomonas campestris pv. oryzae
Bacteroides fragilis
Acholeplasma laidlawii
Pyricularia oryzae
Aspergillus niger
Mucor racemosus
Candida albicans
Saccharomyces cerevisiae
Mycobacterium smegmatis
methicillin-resistant Staphyloccocus aureus (MRSA)
a) —: No inhibition zone.
Synthesis of Calafianin (Revised Structure) (13). Method A:
Hydrolysis of 15 (98 mg, 0.33 mmol) in 1,4-dioxane (5 mL), H2O
(2 mL), and 1 M KOH (0.6 mL) was undertaken by essentially
the same procedure as in the case of 10 to give 16 (97 mg, quant.)
(Nissui Seiyaku Co.) for B. fragilis; Bacto PPLO agar (Difco) sup-
plemented with 15% horse serum, 0.1% glucose, 0.25% phenol red
(5 mg mLꢃ1), and 1.5% agar for A. laidlawii; Mueller-Hinton broth
(Difico) and 1.5% agar (Shimizu Shokuhin Co.) for MRSA; Taiyo
agar (Shimizu Syokuhin Kaisya Ltd.) for the other bacteria; a
medium composed of 1.0% yeast extract and 0.8% agar for fungi
and yeasts. A paper disk containing 10 mg of a sample was placed
on an agar plate. Bacteria, except for X. oryzae, were incubated at
37 ꢂC for 24 h. Yeasts and X. oryzae were incubated at 27 ꢂC for
24 h. Fungi were incubated at 27 ꢂC for 48 h. Antimicrobial activity
was expressed as diameter (mm) of the inhibitory zone.
1
as a colorless solid: H NMR (CD3OD) ꢁ 3.48 (1H, d, J ¼ 18:0
Hz), 3.66 (1H, d, J ¼ 18:0 Hz), 3.74 (1H, d, J ¼ 3:6 Hz), 3.93 (1H,
dd, J ¼ 2:4, 3.6 Hz), 7.22 (1H, d, J ¼ 2:4 Hz); 13C NMR (CD3OD)
ꢁ 46.0, 54.5, 57.4, 85.3, 124.4, 144.6, 157.1, 165.0, 187.1.
Condensation of 16 (97 mg, 0.33 mmol) with 1,4-diaminobutane
(16 mL, 0.17 mmol) as in the case of 1 afforded 13 (40 mg, 39%) as
a colorless solid: IR (KBr) 3410, 1701, 1670 cmꢃ1 1H NMR
;
(DMSO-d6) ꢁ 1.48 (4H, br), 3.20 (4H, br), 3.60 (2H, d, J ¼ 18:0
Hz), 3.67 (2H, d, J ¼ 18:0 Hz), 3.92 (2H, d, J ¼ 3:6 Hz), 4.12 (2H,
dd, J ¼ 2:4, 4.0 Hz), 7.48 (2H, d, J ¼ 2:4 Hz), 8.64 (2H, t, J ¼ 6:0
Hz); 13C NMR (DMSO-d6) ꢁ 26.3, 38.5, 43.3, 52.9, 56.8, 83.9,
122.7, 143.7, 154.8, 158.2, 185.9; HRMS m=z 650.9520, calcd
for C22H2079Br80BrN4O8Na: M þ Na, 650.9527.
This work was supported by Grant-in-Aid for the 21st Cen-
tury COE program ‘‘Keio Life Conjugate Chemistry,’’ as well
as Scientific Research C from the Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan. The authors are
grateful to the COE program and JSPS Research Fellowships
for Young Scientests for financial support to TO.
Method B: To a solution of aerothionin (2) (55 mg, 0.068
mmol) in CH2Cl2 (1 mL) was added MsOH (0.5 mL) at 0 ꢂC. After
being stirred for 1 h, the reaction mixture was diluted with H2O
and extracted with CHCl3. The organic layer was washed with
brine, dried (Na2SO4), and evaporated. The residue was diluted
with CH2Cl2 (1 mL) and then DBU (0.1 mL) was added at room
temperature. After being stirred for 1 h, the mixture was diluted
with H2O and extracted with CHCl3. The organic layer was wash-
ed with brine, dried (Na2SO4), and evaporated. The residue was
purified by silica-gel column chromatography (toluene/acetone =
2/1) to give 13 (4 mg, 11%).
References
1
a) E. Fattorusso, L. Minale, G. Sodano, K. Moody, R. H.
Thomson, J. Chem. Soc., Chem. Commun. 1970, 752. b) J. A.
McMillan, I. C. Paul, Y. M. Goo, K. L. Rinehart, Jr., W. C.
Krueger, L. M. Pschigoda, Tetrahedron Lett. 1981, 22, 39. c) G.
Cimino, S. De Rosa, S. De Stefano, R. Self, G. Sodano, Tetra-
hedron Lett. 1983, 24, 3029.
2 D. J. Faulkner, Nat. Prod. Rep. 2002, 19, 1, many refer-
ences are cited therein.
Bioassay.14 Antimicrobial activity against 15 species of micro-
organisms was measured by the agar diffusion method using paper
disks (i.d. 6 mm, ADVANTEC). The microorganisms were as fol-
lows; Bacillus subtilis PCI 219, Staphylococcus aureus FDA 209P,
methicillin-resistant S. aureus K-24 (a clinica isolate, MRSA),
Micrococcus luteus PCI 1001, Mycobacterium smegmatis ATCC
607, Escherichia coli NIHJ, Pseudomonas aeruginosa P-3, Xan-
thomonas campestris pv. oryzae KB 88, Bacteroides fragilis
ATCC 23745, Acholeplasma laidlawii PG 8, Pyricularia oryzae
KF 180, Aspergillus niger ATCC 6275, Mucor racemosus IFO
4581, Candida albicans ATCC 64548, and Saccharomyces cerevi-
siae. Media for the microorganisms were as follows: GAM agar
3
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