Ianthesines A-D, four novel dibromotyrosine-derived metabolites from a marine sponge, Ianthella sp.

Article abstract of DOI:10.1016/S0040-4020(00)00544-5

Novel dibromotyrosine-derived metabolites, ianthesines A, B, C, and D, were isolated from an Australian marine sponge of the genus Ianthella sp. Their structures were elucidated using chemical and spectroscopic techniques. Ianthesines A, B, and D were derived from two dibromotyrosines. Ianthesine C is a tetramer possessing eight bromine atoms and its molecular weight is 1606. Ianthesines B-D showed Na,K-ATPase inhibitory activity in the range of 50-440 μM, whereas ianthesine A is inactive. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00544-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5813±5818
Ianthesines A±D, Four Novel Dibromotyrosine-Derived
Metabolites from a Marine Sponge, Ianthella sp.
²
Yoshihiro Okamoto, Makoto Ojika, Shigemasa Kato and Youji Sakagami
*
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received 17 May 2000; accepted 14 June 2000
AbstractÐNovel dibromotyrosine-derived metabolites, ianthesines A, B, C, and D, were isolated from an Australian marine sponge of the
genus Ianthella sp. Their structures were elucidated using chemical and spectroscopic techniques. Ianthesines A, B, and D were derived from
two dibromotyrosines. Ianthesine C is a tetramer possessing eight bromine atoms and its molecular weight is 1606. Ianthesines B±D showed
Na,K-ATPase inhibitory activity in the range of 50±440 mM, whereas ianthesine A is inactive. q 2000 Elsevier Science Ltd. All rights
reserved.
Introduction
titioned between EtOAc and water. The EtOAc portion,
which inhibited the dog kidney Na,K-ATPase with an IC50
of 10 mg/ml, was repeatedly subjected to bioassay-guided
fractionation by silica gel chromatography (CHCl3/MeOH/
H2O system) and reversed-phase HPLC to give ianthesines
A (1) (0.069% of wet sponge), B (2) (0.019%), C (3)
(0.019%), and D (4) (0.011%).
Marine sponges of the Verongida order, which include the
genera Aplysina, Ianthella, Verongia, Psammaplysilla, etc.
have proved to be a rich source of secondary metabolites
derived from dibromotyrosine,1 such as aeroplysinin-1,2
aerothionin,3 ®stularins,4 bastadins,5,6 araplysillins,7 pure-
alin,8 aerophobin-1,9 lipopurealins,10 purealidins,11,12 and
others.13,14 Some of them show biological activities such
as antibacterial, cytotoxic, and ATPase modulating
activities. During the course of our search for biologically
active compounds in marine invertebrates, our attention
became focused on the Na,K-ATPase inhibitory con-
stituents of a sponge of the genus Ianthella, because the
organic extract of the sponge (class Demospongiae, order
Verongida, family Ianthellidae) collected in Australia
showed a potent Na,K-ATPase activity. We have recently
reported a novel dimeric polybrominated benzofuran,
iantheran A, as an active principal component from this
sponge.15 This paper describes the isolation of four novel
dibromotyrosine-derived metabolites, named ianthesines A
(1), B (2), C (3), and D (4), from the same sponge and their
structural determination by spectral analysis and chemical
derivatizations (Scheme 1).
Ianthesine A (1), mp 154±1568C, showed pseudomolecular
ions at m/z 786, 788, 790, 792, and 794 (M1H)1 in the ratio
of ca. 1:4:6:4:1 in the positive FABMS spectrum, indicating
the presence of four bromine atoms in the molecule. The
molecular formula of
1
was determined to be
C24H27Br4N3O7 based on the high-resolution positive
FABMS and elemental analysis, implying 11 of unsaturation
index. The IR bands at 1660 and 1540 cm21 showed the
presence of a secondary amide group. The proton±carbon
connectivities were determined as shown in Tables 1 and 2
by the HMQC experiment. The 1H±1H COSY spectrum of 1
revealed the presence of a 1,3-disubstituted propane moiety
(cross-peaks of H-10/H-11 and H-11/H-12) and a 1,1,2-
trisubstituted ethane moiety (cross-peaks of H-19a/H-19b,
H-19a/H-20, and H-19b/H-20). The proton spin±spin
coupling of the AB quartet at d 3.09 and 3.79 could be
assigned to an isolated methylene group that is adjacent to
a p-electron system because of the large geminal J value of
18.2 Hz.16 Since there were numerous quaternary carbons,
extensive HMBC experiments were required for deter-
mining the carbon±carbon connectivities in 1. The H±C
correlations obtained by the HMBC spectrum are sum-
marized in Fig. 1, revealing the presence of a 1,2,3,5-tetra-
Results and Discussion
The MeOH extract of the sponge (1.8 kg wet weight),
collected at the Great Barrier Reef in Australia, was par-
Keywords: amino acids and derivatives; enzyme inhibitors; marine meta-
bolites; natural products.
substituted benzene and a 1,2,3,5,5,6-hexasubstituted
1,3-cyclohexadiene moiety. The position of the oxygen
substituents was determined to be C-1, C-3, C-6, C-12,
C-13, and 3-OMe based on the relatively low-®eld shifts
of these carbons. The relatively high-®eld shifts at the sp2
* Corresponding author. Tel.: 181-52-789-4117; fax: 181-52-789-4118;
e-mail: ojika@agr.nagoya-u.ac.jp
Present address: Agroscience Research Laboratories, Sankyo Co. Ltd.,
²
Yasu-cho, Yasu-gun, Shiga 520-2342, Japan.
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00544-5

5814
Y. Okamoto et al. / Tetrahedron 56 (2000) 5813±5818
Scheme 1. Structures 1±4.
carbons of C-2, C-4, C-14, and C-18 indicated the location
of four bromine atoms at these carbons. The HMBC correla-
tions of OMe/C-3 and H-12/C-13 indicated the connectivity
between C-3 and OMe and between C-12 and C-13 via ether
bonds. The HMBC correlations of H-10/C-9 and H-20/
NMe2 revealed the connectivities between C-9 and C-10
and between C-20 and NMe2 via a nitrogen atom. The
presence of a spiroisoxazoline structure was deduced by
comparing the 1H and 13C NMR of 1 with those previously
reported for the Verongida sponge constituents.2±13 The
trans stereochemistry of the vicinal oxygen atoms at C-1
and C-6 was established by the chemical shifts of H-1 and
Table 1. 1H NMR data for 1±4. Spectra were recorded at 400 MHz in methanole-d4 for 1 and 2 or at 600 MHz in DMSO-d6 for 3 (at 558C) and 4 (at 408C)
Position
1
2
3a
4
1
5
7a
7b
10
11
12
15,17
19a
19b
4.10 s
6.40 s
3.09 d (18.2)
3.79 d (18.2)
3.57 t (7.0)
2.09 m
4.02 t (6.0)
7.57 s
3.09 dd (8.8, 13.9)
3.17 dd (5.2, 13.9)
3.74 dd (5.2, 8.8)
4.07 s
6.41 s
3.09 d (18.2)
3.77 d (18.2)
3.58 t (7.0)
2.11 m
4.07 t (6.0)
7.55 s
2.98 dd (8.1, 14.7)
3.20 dd (4.7, 14.7)
3.75 dd (4.7, 8.1)
3.96 brs
6.54 s
3.94 brs
6.56 s
3.64 d (17.5)
3.17 d (17.5)
3.40 q (6.6)
2.00 quint (6.6)
3.96 m
7.52 s, 7.44 s
2.94 m, 2.79 m
2.79 m, 3.01 dd (4.7, 13.6)
3.87 m, 3.89 m
6.24 brs
3.63 d (18.2)
3.20 d (18.2)
3.40 dt (5.8, 6.5)
1.99 quint (6.5)
3.95 m
7.53 s
2.98 dd (4.0, 13.0)
2.86 dd (4.0, 13.0)
3.82 brs
6.32 brs
3.65 s
20
1-OH
3-OMe
9-NH
20-NH
20-Nme2
(right half of 3)
150,170
190a
3.71 s
2.89 s
3.72 s
3.66 s
8.35 m
4.91 brs, 4.52 brs
8.46 t (5.8)
- (not detected)
7.38 s, 7.47 s
2.95 m, 2.64 dd (7.5, 14.0)
2.86 dd (6.2, 13.4), 2.81 m
4.26 m, 4.24 m
190b
200
200-NH
7.90 brd (6.4), 8.06 brs
a 3 is a 3:2 diastereomeric mixture (the former is the major isomer).

Y. Okamoto et al. / Tetrahedron 56 (2000) 5813±5818
5815
Table 2. 13C NMR data for 1±4. Spectra were recorded at 100 MHz in
methanol-d4 for 1 and 2 or at 150 MHz in DMSO-d6 for 3 (at 558C) and 4 (at
408C)
which agreed with those of the known compound aero-
thionin,3 whose absolute con®guration was established by
X-ray analysis and CD spectroscopy.18 The con®guration of
the chiral center at C-20 has been determined as follows.
Ianthesine A (1) was hydrolyzed with hydrobromic acid
to give 3,5-dibromo-N,N-dimethyltyrosine, which was
subjected to catalytic hydrogenation to afford N,N-
dimethyltyrosine.19 Chiral HPLC analysis using synthetic
samples revealed that the obtained tyrosine had a D con-
®guration. The structure of ianthesine A was thus elucidated
as shown by formula 1.
Position
1
2
3a
4
1
2
3
4
5
6
7
75.4 d
75.5 d
73.7 d
73.7 d
113.1 s
147.2 s
120.7 s
131.4 d
90.3 s
39.4 t
114.2 s 114.2 s 113.0 s
149.2 s 149.3 s 147.1 s
122.7 s 122.8 s 120.6 s
132.3 d 132.3 d 131.3 d
92.4 s
40.2 t
92.4 s
40.2 t
90.3 s
39.3 t
8
9
10
11
12
13
14,18
155.3 s 155.3 s 154.3 t
161.4 s 161.6 s 158.9 s
154.5 t
159.0 s
36.3 t
29.5 t
71.2 t
Ianthesine B (2), mp 154±1578C, showed pseudomolecular
ions at m/z 758, 760, 762, 764, and 766 (M1H)1 in the ratio
of ca. 1:4:6:4:1 in the positive FABMS spectrum, indicating
the presence of four bromine atoms like ianthesine A (1).
The molecular formula of 2 was determined to be
C22H23Br4N3O7 based on the high-resolution positive
38.0 t
30.6 t
72.2 t
38.0 t
30.6 t
72.2 t
36.2 t
29.4 t
71.2 t
153.3 s 153.7 s 150.6, 150.7, 150.8 sb 150.4 s
119.1 s 119.4 s 116.7, 116.8, 116.9 sb 116.5 s
15,17
16
19
20
21
OMe
20-NMe2
134.9 d 135.0 d 133.5 d
136.9 s 136.6 s 138.0, 138.2 sb
134.0 d
138.8 s
36.3 t
57.2 d
174.3 s
59.6 q
1
FABMS and elemental analysis. The H and 13C NMR
33.7 t
72.6 d
171.4 s 173.0 s 171.8, 172.5 s
36.8 t
57.1 d
36.7, 37.0 t
57.9, 57.3 d
data (Tables 1 and 2) for 2 were similar to those for 1 except
for the absence of the N,N-dimethyl resonance and for the
observed higher ®eld shift of C-20 (dC 57.1) than that (dC
60.4 q
42.3 q
60.4 q
59.5 q
1
72.6) of 1. The HMBC and H±1H COSY spectra of 2
(right half of 3)
190
200
210
36.7, 36.2 t
54.5, 54.5 d
172.0, 172.3 s
suggest that 2 was the N,N-didesmethyl derivative of 1.
The absolute stereochemistry of the spirocyclohexadienyl-
isoxazoline moiety of 2 was deduced to be the same as that
of 1 based on a minus optical rotation and the negative
Cotton effects in the CD spectrum of 2. The con®guration
at C-20 has been determined as follows. Ianthesine B (2)
was subjected to the same reaction sequence as that for 1 to
afford tyrosine, which was found to be a mixture of the l-
and d-forms in the ratio of 7:3. These ®ndings indicate that 2
is a 7:3 mixture of two diastereomers (1S,6R,20S and
1S,6R,20R). The chiral HPLC analysis of 2 itself also
showed two peaks in the ratio of 7:3 and no epimerization
during the hydrolysis.
a 3 is a 3:2 diastereomeric mixture (the former is the major isomer).
b Assignments for major/minor isomer and right/left half are unclear.
Ianthesine C (5), mp 2008C (dec.), showed a symmetrical
isotope peak pattern around m/z 1583 (M2Na)2 and 1503
(M2SO3Na)2 in the negative FABMS spectrum, indicating
the presence of several bromine atoms and a sulfate group in
the molecule. The presence of SO23 was supported by the
intense bands at 1257 and 1047 cm21 in the IR spectrum. It
was dif®cult to determine the molecular formula of 3 by
high-resolution MS due to the low intensity of the lowest-
weight isotope peak of the pseudomolecular ions (M2Na)2
at m/z 1575 containing eight 79Br atoms. This compound 3
appears to be an unsymmetrical dimer of ianthesine B (2)
Figure 1. HMBC correlations of 1.
H-817 and no NOE observation between H-1 and H-7. The
absolute stereochemistry of the spirocyclohexadienylisoxa-
zoline moiety was deduced to be 1S,6R based on a large
minus optical rotation, [a]2D2ˆ2118, and the negative
Cotton effects at 248 and 285 nm in the CD spectrum,
Figure 2. The observed (left) and simulated (right) FABMS patterns for the (M±Na)2 ion of 3.

5816
Y. Okamoto et al. / Tetrahedron 56 (2000) 5813±5818
spirocyclohexadienylisoxazoline moiety was deduced to be
the same as those of 1±3 from the comparison of the NMR
data and CD spectra.
A considerable number of dibromotyrosine-derived metabo-
lites with a spirocyclohexadienylisoxazoline ring have been
isolated from the marine Verongida sponges. Most of these
known compounds are the terminal amine-type, whereas, to
the best of our knowledge, ianthesines A±D (1±4) are the
®rst examples of the amino acid-type among this class of
metabolites. Some of these types of metabolites are known
to show Na,K-ATPase inhibitory activity.7,8,10 The dose
response curves for 1±4 against the dog kidney Na,K-
ATPase is shown in Fig. 3. The IC50 values of 2, 3 and 4
are 440, 50 and 280 mM, respectively, while 1 was inactive
(.2.5 mM), whereas a cardiac steroid glycoside, ouabain,
showed an IC50 of 0.2 mM under the same conditions. The
amino group of the terminal a amino acid (in 1, 2 and 4) or
the dipeptide moiety (in 3) seems to be important for the
inhibitory activity, because the activity decrease in the
order, ±NHSO23 (3), ±NHSO32 (4), ±NH2 (2), ±NMe2 (1).
Figure 3. Na,K-ATPase inhibition by 1±4. IC50 values for 1±4 and ouabain
are .2,500, 440, 50, 280, and 0.2 mM, respectively.
based on the NMR data (Tables 1 and 2), and the ion peak at
m/z 1583 (M2Na)2 of 3 coincides with `2£761 (molecular
weight of 2)218 (H2O)180 (SO3)2H'. These data suggest
that 3 is a dipeptide comprised of two molecules of 2 and
one sulfate group, and consequently, the molecular formula
of 3 is C44H43Br8NaN6O16S. This was proved from the
elemental analysis and the good agreement between the
observed FABMS and a simulated isotope pattern (Fig. 2).
The observation of one NH proton at d 4.91/4.52 (3:2 ratio)
indicates that the sulfate group is located on the amino
group at C-20. The NMR chemical shifts of the left half
of the molecule (acid part as a dipeptide) were quite similar
to or partially the same as the corresponding chemical shifts
of the right half (amino part). However, we could assign
most of the signals as shown in Tables 1 and 2 based on
the observation of an NOE between the inter-residual aH
(H20) and 200-NH, which is well-known in the NMR of
peptides. Ianthesine C (3) seems to be a diastereomeric
mixture in the ratio of ca. 3:2, because several NMR signals
around the central part of the molecule (positions 13±21)
make a pair in the ratio of 3:2. The con®gurations of C-20
and C-200 of these diastereomers are unclear. The stereo-
chemistry of the two spirocyclohexadienylisoxazoline
moieties of 3 were deduced to be the same as those of 1
and 2 because of the approximately 2-fold intensity of the
Cotton effects as those of 1.
Experimental
General methods
Melting points were uncorrected. HPLC was performed on a
Shimadzu LC-8A preparative liquid chromatograph. IR
spectra were recorded on a JASCO FT/IR-7000S. UV spec-
tra were recorded on a JASCO Ubest-50 UV/VIS spectro-
photometer. Optical rotations were measured on a JASCO
DIP-370 digital polarimeter using a 10 cm cell. CD spectra
were recorded on a JASCO J-720WI spectrometer. NMR
spectra were recorded on a Brucker-ARX400 (400 MHz)
or Brucker-AMX600 (600 MHz) spectrometer. Chemical
shifts were referenced to the solvent peak at dH 3.30
(residual CHD2OD), 2.49 (residual CD3SOCD3), dC 49.0
(CD3OD), or 39.7 (CD3SOCD3). FAB Mass spectra were
recorded on a JMS DX-705 or a MStation JMS-700 (high-
resolution) mass spectrometer using glycerol or thioglycerol
as the matrix.
Na,K-ATPase inhibitory assay
To 70 ml of 30 mM Tris±HCl buffer (pH 7.4) supplemented
with 5 mM MgCl2, 100 mM NaCl, 10 mM KCl, and 3 mM
EDTA was added a sample solution (10 ml) in DMSO (or
10 ml of DMSO as a control). This mixture was kept at 378C
for 10 min. Na,K-ATPase (0.002 units, from dog kidney,
Sigma) in 10 ml of Tris±mannitol buffer (250 mM mannitol,
20 mM Tris±HCl, 1 mM EDTA, pH 7.4) was added to the
mixture, and after pre-incubation for 8 min at 378C, 10 mM
ATP (Oriental Yeast) in water (10 ml) was added. The
resulting mixture was incubated for 1.5 h at 378C. The reac-
tion mixture was quenched with 20% trichloroacetic acid
(2 ml) and cooled on ice, then analyzed by reversed-phase
HPLC (Develosil ODS-UG-5 (4.6 i.d.£250 mm, Nomura
Chemical) with 50 mM sodium phosphate buffer (pH 7.0)
at a ¯ow rate of 1.0 ml/min, detected at 260 nm) to
quantify the content of ADP (tRˆ5.9 min) and ATP
(tRˆ5.0 min). The inhibition of the ADP production from
Ianthesine D (4), mp 1908C (dec.), showed NMR spectra
quite similar to those of ianthesine B (2) (Tables 1 and 2).
The ion peaks at m/z 836, 838, 840, 842, and 844 (M2Na)2
in the ratio of ca. 1:4:6:4:1 in the negative FABMS spectrum
indicated the presence of four bromine atoms like 2. The
molecular formula of 4, C22H22Br4NaN3O10S, which was
determined by the high-resolution FABMS and elemental
analysis, is more than that of 2 by `SO3Na±H'. The presence
of a sulfate group was supported by the fragment peaks of
(M2SO3Na)2 at m/z 756, 758, 760, 762, and 76 and the
intense bands at 1217 and 1046 cm21 in the IR spectrum.
Although the proton signal due to 20-NH could not be
observed, the position of the sulfate group was assumed to
be on the amino group at C-20 based on the presence of the
1
signals due to both 1-OH and 9-NH in the H NMR. The
con®guration at C-20 is unclear. The stereochemistry of the

Y. Okamoto et al. / Tetrahedron 56 (2000) 5813±5818
5817
ATP was given by the following equation: (12[ADP]sample
[ADP]control)£100%, [ADP] indicates the peak area of ADP.
/
Anal. Calcd for C22H23Br4N3O7: C, 34.72; H, 3.05; N,
5.52%. Found: C, 34.73; H, 2.80; N, 5.23%.
Ianthesine C (3). Yellow powder; mp 2008C (dec.) (pre-
cipitated from H2O±DMSO); [a]2D6 293 (c 0.86, DMSO);
IR (KBr) nmax 3700±2500 (br), 1660, 1584, 1541, 1257,
1218, 1183, 1047, and 990 cm21; UV (MeOH±0.5%
DMSO) lmax 231 (e 26900) and 283 (11900) nm; CD
Collection, extraction, and isolation
The sponge Ianthella sp. (1.8 kg wet weight), collected at a
depth of 12 m in the Great Barrier Reef, Australia, was
homogenized in MeOH (4 l), and the resulting mixture
was then ®ltered. The residue was washed three times
with MeOH (2 l). The ®ltrates were combined and concen-
trated to 1 l, and the resulting aqueous residue was extracted
three times with EtOAc (1.5 l). The combined EtOAc layers
were evaporated to give a pale brown powder (14.4 g),
which inhibited Na,K-ATPase with an IC50 of 10 mg/ml.
A portion (4.0 g) of the EtOAc extract was chromato-
graphed on silica gel (200 g) with CHCl3±MeOH±H2O
(7:3:1 lower phase, 65:35:10 lower phase, and then 6:4:1)
to give seven fractions. The third fraction was pure 1
[182 mg, Rfˆ0.43 on silica gel TLC developed with
CHCl3±MeOH±H2O (65:35:10 lower phase)]. The second
fraction (687 mg) was chromatographed on silica gel
(137 g) with CHCl3±MeOH±H2O (10:3:1 lower phase).
The fractions containing 1, 2 (Rfˆ0.33 under the same
conditions), and 3 (Rfˆ0.28 under the same conditions)
were combined and then chromatographed on silica gel
(63 g) to give 1 (163 mg), 2 (63 mg), and 3 (94 mg) as
powders. An other portion (3.0 g) of the above EtOAc
extract was chromatographed on silica gel in the same
manner to give seven fractions. The third fraction
(503 mg) contained 1, 2, and 3. The fourth fraction
(990 mg) contained iantheran A15 (Rfˆ0.37 under the
same conditions) as the major component. The ®fth fraction
(322 mg) containing 4 (Rfˆ0.13 under the same conditions)
was chromatographed on silica gel (97 g) with CHCl3±
MeOH±H2O (7:3:1 lower phase and then 65:35:10 lower
phase) to give four fractions. A portion (41.4 mg) of the
third fraction (total 86.4 mg) was puri®ed by reversed-
phase HPLC [Develosil ODS-HG-5 (10 i.d.£250 mm,
Nomura Chemical) with MeOH±H2O (3:2) at a ¯ow rate
of 2.5 ml/min, detected at 254 nm] to give 4 (20 mg,
tRˆ6.8 min) as a powder.
1
(MeOH) lext 252 nm (De 216.0), 289 (216.9); H and
13C NMR data, see Tables 1 and 2; FABMS (matrix:
glycerol), see Fig. 2. Anal. Calcd for C44H43Br8N6O16SNa:
C, 32.90; H, 2.70; N, 5.23%. Found: C, 32.48; H, 2.81; N,
4.84%.
Ianthesine D (4). Colorless powder; mp 1908C (dec.);
[a]2D5ˆ269 (c 0.19, MeOH); IR (KBr) nmax 3700±2500
(br), 1662, 1596, 1541, 1258, 1217, and 1046 cm21; UV
(MeOH) lmax 206 (e 53200), 220 (sh, 25000), and 282 nm
(6760); CD (MeOH) lext 254 nm (De 9.14), 289 (28.74);
1H and 13C NMR data, see Tables 1 and 2; FABMS (matrix:
glycerol) m/z 836, 838, 840, 842, and 844 (rel. int. 1:4:6:4:1)
(M2Na)2. Anal. Calcd for C22H22Br4N3O7SNa: C, 30.61;
H, 2.57; N, 4.87%. Found: C, 30.60; H, 2.53; N, 4.87%.
Degradation of ianthesine A (1)
A solution of 1 (10.7 mg) in 48% hydrobromic acid (1 ml)
was heated under re¯ux for 24 h. The resulting solution was
evaporated, and the residue was puri®ed by reversed-phase
HPLC [Develosil ODS-HG-5 (10 i.d.£250 mm), 33% aq.
MeOH, ¯ow rate 2.5 ml/min, detected at 210 nm] to afford
3,5-dibromo-N,N-dimethyltyrosine (3.9 mg, tRˆ10 min) as
1
a white powder: H NMR (400 MHz, CD3OD) d 2.66 (s,
6H), 2.93 (m, 1H), 3.05 (m, 1H), 3.49 (m, 1H), and 7.41 (s,
2H). A solution of 3,5-dibromo-N,N-dimethyltyrosine
(3.9 mg) in MeOH±H2O (1:2) (1.0 ml) was stirred with
10% Pd±C (1.7 mg) under a hydrogen atmosphere at
room temperature for 1.5 h. After ®ltration, the ®ltrate
was evaporated to give a crude product, which was puri®ed
by reversed-phase HPLC [Develosil ODS-HG-5 (10
i.d.£250 mm), 33% aq. MeOH, ¯ow rate 2.5 ml/min,
detected at 210 nm] to afford N,N-dimethyltyrosine
(0.9 mg, tRˆ5.7 min) as a white powder: [a]2D3ˆ273 (c
0.075, water); IR (KBr) nmax 3700±2500 (br), 1615, 1595,
1515, and 1250 cm21; 1H NMR (400 MHz, CD3OD) d 2.79
(s, 6H), 3.09 (dd, Jˆ6.9, 14.7 Hz, 1H), 3.22 (dd, Jˆ6.7,
14.7 Hz, 1H), 3.74 (dd, Jˆ6.7, 6.9 Hz, 1H), 6.73 (d,
Jˆ8.5 Hz, 2H), and 7.16 (d, Jˆ8.5 Hz, 2H); HRMS
(FAB) calcd for C11H16NO3 (M1H) m/z 210.1130, found
210.1146.
Ianthesine A (1). Colorless ®ne crystals; mp 154±1568C
(MeOH±H2O); [a]2D2ˆ2118 (c 1.02, MeOH); IR (KBr)
nmax 3700±2300 (br), 1660, 1630, 1540, 1260, and
1045 cm21; UV (MeOH) lmax 210 (e 29500) and 283 nm
(7390); CD (MeOH) lext 248 nm (De 210.2), 285 (29.94);
1H and 13C NMR data, see Tables 1 and 2; FABMS (matrix:
thioglycerol) m/z 786, 788, 790, 792, and 794 (rel. int.
1:4:6:4:1) (M1H)1; HRMS (FAB) calcd for
C24H2879Br4N3O7 (M1H) m/z 785.8661, found 785.8649.
Anal. Calcd for C24H27Br4N3O7: C, 36.53; H, 3.45; N,
5.33%. Found: C, 36.53; H, 3.18; N, 5.23%.
The absolute stereochemistry was determined to be D by
chiral HPLC analysis [CHIRALPAK WH (4.6
i.d.£250 mm, Daicel Chemical), column temp. 508C,
0.25 mM CuSO4, ¯ow rate 1.0 ml/min, detected at
230 nm], in which the retention times of the authentic
samples of N,N-dimethyl-d and l-tyrosines19 were 13.3
and 25.4 min, respectively.
Ianthesine B (2). Colorless ®ne crystals; mp 154±1578C
(MeOH±H2O); [a]2D2ˆ297 (c 0.58, MeOH); IR (KBr)
nmax 3700±2300 (br), 1660, 1635, 1540, 1260, and
1045 cm21; UV (MeOH) lmax 207 (e 45400) and 283 nm
(7300); CD (MeOH) lext 248 nm (De 211.6), 284 (211.2);
1H and 13C NMR data, see Tables 1 and 2; FABMS (matrix:
thioglycerol) m/z 758, 760, 762, 764, and 766 (rel. int.
1:4:6:4:1) (M1H)1; HRMS (FAB) calcd for
C22H2479Br4N3O7 (M1H) m/z 757.8348, found 757.8359.
Degradation of ianthesine B (2)
A solution of 2 (10.3 mg) in 48% hydrobromic acid (1 ml)
was heated under re¯ux for 24 h. The resulting solution was

5818
Y. Okamoto et al. / Tetrahedron 56 (2000) 5813±5818
evaporated, and the residue was puri®ed by reversed-
phase HPLC under the same conditions as those for 1
to afford 3,5-dibromotyrosine (2.8 mg, tRˆ12.0 min)
together with 3-bromotyrosine (1.1 mg, tRˆ8.0 min) as
white powders. 3,5-Dibromotyrosine: 1H NMR
(400 MHz, CD3OD) d 2.90 (dd, Jˆ8.3, 14.6 Hz, 1H),
3.16 (dd, Jˆ4.4, 14.6 Hz, 1H), 3.70 (dd, Jˆ4.4, 8.3 Hz,
1H), and 7.43 (s, 2H); 3-bromotyrosine: 1H NMR
(400 MHz, CD3OD) d 2.89 (dd, Jˆ8.4, 14.7 Hz, 1H),
3.17 (dd, Jˆ4.4, 14.7 Hz, 1H), 3.69 (dd, Jˆ4.4, 8.4 Hz,
1H), 6.86 (d, Jˆ8.3 Hz, 1H), 7.10 (dd, Jˆ2.1, 8.3 Hz,
1H), and 7.42 (d, Jˆ2.1 Hz, 1H).
References
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Acknowledgements
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This work was ®nancially supported in part by a grant,
Research for the Future Program from the Japan Society
for the Promotion of Science (JSPS).
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