Dibromotyrosine derivatives from the ethanol extract of the marine sponge Aplysina sp.: Structures, transformations, and origin

Article abstract of DOI:10.1007/s11172-011-0088-9

Twenty nine 3,5-dibromotyrosine derivatives were isolated from the ethanol extract of the marine sponge Aplysina sp. (South China Sea) including the earlier unknown compounds, in particular, p-hydroxycyclohexadienone and p-hydroxycyclohexenone ketals. The isolated enones, dienones, and ketals can be transformation products of aeroplysinin-1 in the course of its reactions with water and alcohols.

Full text of DOI:10.1007/s11172-011-0088-9

Russian Chemical Bulletin, International Edition, Vol. 60, No. 3, pp. 570—580, March, 2011
570
Dibromotyrosine derivatives from the ethanol extract
of the marine sponge Aplysina sp.: structures, transformations, and origin
E. A. Santalova, V. A. Denisenko, V. P. Glazunov, A. I. Kalinovskii, S. D. Anastyuk, and V. A. Stonik
Pacific Institute of Bioorganic Chemistry, FarꢀEastern Branch of the Russian Academy of Sciences,
159 prosp. 100 let Vladivostoku, 690022 Vladivostok, Russian Federation.
Fax: +7 (423) 231 4050. Eꢀmail: santalova@piboc.dvo.ru
Twenty nine 3,5ꢀdibromotyrosine derivatives were isolated from the ethanol extract of the
marine sponge Aplysina sp. (South China Sea) including the earlier unknown compounds, in
particular, pꢀhydroxycyclohexadienone and pꢀhydroxycyclohexenone ketals. The isolated
enones, dienones, and ketals can be transformation products of aeroplysininꢀ1 in the course of
its reactions with water and alcohols.
Key words: marine sponges, dibromotyrosines, alkaloids, aeroplysininꢀ1, ketals, cyclohexaꢀ
dienones.
Representatives of sponges of the genus Aplysina beꢀ
from the ethanol extract of the sponge Aplysina sp. by
column chromatography on Sephadex LHꢀ20 and silica
gel with subsequent purification by the reversedꢀphase
HPLC. The earlier described compounds were identified
by comparison of their spectral data and physical conꢀ
stants with those given in the literature.4,6—13 The strucꢀ
long to a small group of marine invertebrates possessing
mechanism of "activated chemical defense".1 Thus, when
the tissues of Aplysina sponge are injured, its deterrent
isoxazoline alkaloids are enzymatically cleaved to the more
lowꢀmolecularꢀweight antibacterial derivatives with the
skeleton of 3,5ꢀdibromotyrosine, that can protect the
sponge organism from penetration of pathogenic bacteꢀ
ria.2 Aeroplysininꢀ1 (1) is the most familiar among diꢀ
bromotyrosines formed and attracts attention as a potenꢀ
tial medicine.3
The concept of existence of the mechanism of activatꢀ
ed chemical defense in sponges Aplysina sp. appeared comꢀ
paratively recently. Chemistry of the formation of many
dibromotyrosine derivatives (enones, dienones, ketals),
which together with compound 1 are found in the extracts
of Aplysina, remains poorly studied. In particular, some
authors still suggest the presence of hypothetical arenꢀ
oxide or imino ether precursors in sponges,4,5 from which
all the spectrum of these simple dibromotyrosines is
formed. The insufficient knowledge of chemical processes
leading from compoundsꢀprecursors to numerous prodꢀ
ucts of their transformation prompted us to carry out deꢀ
tailed structural studies of dibromotyrosine derivatives of
the sponge Aplysina sp. (the order Verongida, the family
Aplysinidae; South China Sea) and demonstrate a key role
of aeroplysininꢀ1 (1) in the sequence of their transformaꢀ
tions suggested on the basis of mechanism of activated
chemical defense.
1
tures of compounds 16—29 were established by H and
13C NMR spectroscopy (including the COSY, HMBC,
and NOESY experiments), IR spectroscopy, ESI and EI
mass spectrometry, as well as quantum chemical calculaꢀ
tions. Based on the common origin with (+)ꢀaeroplysiꢀ
ninꢀ1 (1),6 the same absolute configuration of the asymꢀ
metric centers C(1) and C(6) was assigned to the rest of
the found by us chiral compounds.
pꢀHydroxycyclohexadienone ketals. Analysis of the
1H NMR spectra (Table 1), IR spectra, and EI mass specꢀ
tra of configurational isomers 13 and 14 shows that both
compounds are the methyl ethyl ketals of pꢀhydroxycycloꢀ
hexadienone 8. The NOESY spectrum of 2ꢀ(Eꢀ3,5ꢀdibroꢀ
moꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycyclohexaꢀ2,5ꢀdienꢀ
1ꢀyl)acetamide (13) exhibits a crossꢀpeak between the
H2C(7) at δH 2.51 (s) and —OCH2CH3 at δH 3.27 (q),
that indicates the cisꢀorientation of these group with reꢀ
spect to the plane of the double bond of the ring. Conꢀ
versely, the NOESY spectrum of 2ꢀ(Zꢀ3,5ꢀdibromoꢀ4ꢀ
ethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)ꢀ
acetamide (14) exhibits crossꢀpeaks between the H2C(7)
at δH 2.51 (s) and —OCH3 at δH 3.16 (s), as well as beꢀ
tween the —OH at δH 5.17 (br.s) and —OCH2CH3 at δH
1
3.36 (q). Comparison of the H NMR spectra of isomers
Results and Discussion
13 and 14 shows that the main difference between them
consists in position of the signals for the protons of the
ether groups at the C(4) atom. Thus, the Eꢀisomer 13 has
the difference in the chemical shift (CS) values ΔδH of the
The already described dibromotyrosine derivatives 1—15
and new compounds of this group 16—29 were isolated
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 557—567, March, 2011.
1066ꢀ5285/11/6003ꢀ570 © 2011 Springer Science+Business Media, Inc.

Sponge dibromotyrosines
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
571
Comꢀ
pound
8
9
10
R
Comꢀ
pound
12
13
14
15
16
17
R1
R2
Comꢀ
pound
18
19
20
21
22
23
R1
R2
Comꢀ
pound
24
25a
25b
25c
26
R1
R2
NH2
OH
OMe
OEt
Me
Me
Et
Bu
Me
Et
Me
Et
Me
Me
Bu
Et
Me
Et
Me
Et
Et
Me
Et
Me
Et
Me
Et
Et
H
Ac
Ac
Me
Me
Me
H
H
Ac
Me
Et
11
Et
27
signals for the protons —OCH2— and —OCH3 equal to
0.06 ppm, whereas the corresponding value for Zꢀisomer
14 is equal to 0.20 ppm. Judging from the ΔδH value, the
latter isomer has been described earlier9 without assignꢀ
ment of configuration, but with indication on the presꢀ
ence of admixture of a diastereoisomer in it.
13C NMR spectra (Tables 1, 2) exhibit signals for the two
ethoxy groups at the C(4) atom, whereas the rest of the
NMR spectra of homologous ketals 13 and 17 are identical.
According to the 13C NMR spectroscopic and (+)ꢀESI
mass spectrometric data, the mixed ketals 18 and 19
containing an acetonitrile side moiety instead of the acetꢀ
amide one have molecular formulas C11H13Br2NO3.
1H and 13C NMR spectra (see Tables 1 and 2) of these
compounds are close to the corresponding spectra of diꢀ
methyl ketal 7,4 however, the signals for the methoxy and
ethoxy groups are present instead of signals for the two
methoxy ones at the C(4) atom. The NOESY spectrum of
(Eꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycycloꢀ
hexaꢀ2,5ꢀdienꢀ1ꢀyl)acetonitrile (18) exhibits a crossꢀpeak
between H2C(7) (δH 2.73, s) and —OCH2CH3 (δH 3.33, q).
The NOESY spectrum of its Zꢀisomer (19) shows corꢀ
relations between the H2C(7) (δH 2.73, s) and —OCH3
(δH 3.21, s), as well as between the —OH (δH 2.37, br.s)
and —OCH2CH3 (δH 3.35, q).
Aplysinketal A (15)10 and its earlier unknown isomer
2ꢀ(Eꢀ3,5ꢀdibromoꢀ4ꢀbutoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycycloꢀ
1
hexaꢀ2,5ꢀdienꢀ1ꢀyl)acetamide (16) have close H NMR
spectra (see Table 1), IR spectra, and EI mass spectra.
However, the NOESY spectrum of compound 15 conꢀ
tains a crossꢀpeak between the H2C(7) (δH 2.51, s) and
—OCH3 (δH 3.16, s), whereas the NOESY spectrum of
compound 16 has a crossꢀpeak between the H2C(7)
(δH 2.51, s) and —OCH2CH2CH2CH3 (δH 3.18, t).
Molecular formula C12H17Br2NO4 for 2ꢀ(3,5ꢀdibroꢀ
moꢀ4,4ꢀdiethoxyꢀ1ꢀhydroxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)ꢀ
acetamide (17) was found based on the 13C NMR spectroꢀ
scopic and (+)ꢀESI mass spectrometric data. The 1H and

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Santalova et al.
Table 1. 1H NMR spectra of compounds 13—29*
Comꢀ
pound
1H NMR, δ (J/Hz)
13
14
15
1.28 (t, 3 Н, С(4)OCH2СН3, J = 6.7); 2.51 (s, 2 Н, С(7)Н2); 3.21 (s, 3 Н, С(4)ОМе); 3.27 (q, 2 Н, С(4)OCH2СН3,
J = 6.7); 5.10 (br.s, 1 Н, C(1)OH); 5.59, 5.76 (both br.s, 1 Н each, О=C(8)NН2); 6.73 (s, 2 Н, С(2)Н, С(6)Н)
1.275 (t, 3 Н, С(4)OCH2СН3, J = 6.8); 2.51 (s, 2 Н, С(7)Н2); 3.16 (s, 3 Н, С(4)ОМе); 3.36 (q, 2 Н, С(4)OCH2СН3,
J = 6.8); 5.17 (br.s, 1 Н, C(1)OH); 5.67, 5.825 (both br.s, 1 Н each, О=C(8)NН2); 6.73 (s, 2 Н, С(2)Н, С(6)Н)
0.91 (t, 3 Н, С(4)OСН2СН2CH2СН3, J = 7.3); 1.45 (m, 2 Н, С(4)OСН2СН2CH2СН3); 1.62 (m, 2 Н,
С(4)OСН2СН2CH2СН3); 2.51 (s, 2 Н, С(7)Н2); 3.16 (s, 3 Н, С(4)ОМе); 3.28 (t, 2 Н, С(4)OСН2СН2CH2СН3,
J = 6.2); 5.17 (br.s, 1 Н, C(1)OH); 5.56, 5.73 (both br.s, 1 Н each, О=C(8)NН2); 6.73 (s, 2 Н, С(2)Н, С(6)Н)
16
17
0.92 (t, 3 Н, С(4)O(СН2)3СН3, J = 7.2); 1.46 (m, 2 Н, С(4)O(СН2)2CH2СН3); 1.62 (m, 2 Н,
С(4)OСН2СН2CH2СН3); 2.51 (s, 2 Н, С(7)Н2); 3.18 (t, 2 Н, С(4)OСН2(СН2)2СН3, J = 6.1); 3.21 (s, 3 Н,
С(4)ОМе); 5.13 (br.s, 1 Н, C(1)OH); 5.56, 5.74 (both br.s, 1 Н each, О=C(8)NН2); 6.73 (s, 2 Н, С(2)Н, С(6)Н)
1.28, 1.29 (both t, 3 Н each, both С(4)OCH2СН3, J = 7.0); 2.50 (s, 2 Н, С(7)Н2); 3.24, 3.34 (both q, 2 Н each,
both С(4)OCH2СН3, J = 7.0); 5.10 (br.s, 1 Н, C(1)OH); 5.62, 5.77 (both br.s, 1 Н each, О=C(8)NН2); 6.70 (s, 2 Н,
С(2)Н, С(6)Н)
18
19
20
1.29 (t, 3 Н, С(4)OCH2СН3, J = 7.0); 2.37 (br.s, 1 Н, C(1)OH); 2.73 (s, 2 Н, С(7)Н2); 3.22 (s, 3 Н, С(4)ОМе);
3.33 (q, 2 Н, С(4)OCH2СН3, J = 7.0); 6.72 (s, 2 Н, С(2)Н, С(6)Н)
1.30 (t, 3 Н, С(4)OCH2СН3, J = 7.0); 2.37 (br.s, 1 Н, C(1)OH); 2.73 (s, 2 Н, С(7)Н2); 3.21 (s, 3 Н, С(4)ОМе);
3.35 (q, 2 Н, С(4)OCH2СН3, J = 7.0); 6.72 (s, 2 Н, С(2)Н, С(6)Н)
1.28 (t, 3 Н, С(4)OCH2СН3, J = 7.0); 1.32 (t, 3 Н, О=C(8)ОСН2СН3, J = 7.2); 2.62 (s, 2 Н, С(7)Н2); 3.21 (s, 3 Н,
С(4)ОМе); 3.24 (q, 2 Н, С(4)OCH2СН3, J = 7.0); 4.21 (br.s, 1 Н, C(1)OH); 4.24 (q, 2 Н, О=С(8)ОСН2СН3,
J = 7.2); 6.72 (s, 2 Н, С(2)Н, С(6)Н)
21
22
1.28 (t, 3 Н, С(4)OCH2СН3, J = 7.0); 1.32 (t, 3 Н, О=C(8)ОСН2СН3, J = 7.2); 2.625 (s, 2 Н, С(7)Н2); 3.14 (s, 3 Н,
С(4)ОМе); 3.35 (q, 2 Н, С(4)OCH2СН3, J = 7.0); 4.21 (br.s, 1 Н, C(1)OH); 4.24 (q, 2 Н, О=С(8)ОСН2СН3,
J = 7.2); 6.72 (s, 2 Н, С(2)Н, С(6)Н)
1.28, 1.285 (both t, 3 Н each, both С(4)OCH2СН3, J = 6.9); 1.32 (t, 3 Н, О=C(8)ОСН2СН3, J = 7.1); 2.61 (s, 2 Н,
С(7)Н2); 3.23, 3.34 (both q, 2 Н each, both С(4)OCH2СН3, J = 6.9); 4.17 (br.s, 1 Н, C(1)OH); 4.24 (q, 2 Н,
О=С(8)ОСН2СН3, J = 7.1); 6.68 (s, 2 Н, С(2)Н, С(6)Н)
23**
24**
25a
1.28 (t, 3 Н, С(4)OCH2СН3, J = 6.9); 2.70 (s, 2 Н, С(7)Н2); 3.21 (s, 3 Н, С(4)ОМе); 3.26 (q, 2 Н, С(4)OCH2СН3,
J = 6.9); 6.755 (s, 2 Н, С(2)Н, С(6)Н)
1.28 (t, 3 Н, С(4)OCH2СН3, J = 6.9); 2.70 (s, 2 Н, С(7)Н2); 3.15 (s, 3 Н, С(4)ОМе); 3.355 (q, 2 Н, С(4)OCH2СН3,
J = 6.9); 6.755 (s, 2 Н, С(2)Н, С(6)Н)
2.74, 2.82 (both d, 1 Н each, С(7)Н2, J = 16.4); 2.88 (s, 1 Н, С(1)OН); 3.46, 3.51 (both s, 3 Н each, both С(4)ОМе);
4.01 (d, 1 Н, C(6)OH, J = 9.3); 4.32 (dd, 1 Н, С(6)Н, J = 4.6, J = 9.3); 4.47 (d, 1 Н, C(5)Н, J = 4.6);
6.50 (d, 1 Н, С(2)Н, J = 1.2)
26
27
2.465 (br.s, 1 Н, C(3а)OH); 2.72, 2.91 (both d, 1 Н each, С(3)Н2, J = 18.6); 3.33, 3.43 (both s, 3 Н each, both С(4)ОМе);
4.41 (d, 1 Н, С(7)H, J = 3.2); 5.02 (d, 1 Н, C(7а)Н, J = 3.2); 6.57 (s, 2 Н, С(4)Н)
1.18 (t, 3 Н, С(6)ОСН2СН3, J = 7.0); 2.45 (br.s, 1 Н, С(3а)ОН); 2.71, 2.92 (both d, 1 Н each, С(3)Н2, J = 18.5);
3.43 (s, 3 Н, С(6)ОМе); 3.49, 3.62 (both m, 1 Н each, C(6)OСH2СН3); 4.43 (d, 1 Н, С(7)H, J = 3.3); 5.03 (d, 1 Н,
C(7а)Н, J = 3.3); 6.55 (s, 2 Н, С(4)Н)
28
2.65, 2.99 (both d, 1 Н each, С(3)Н2, J = 18.2); 3.37, 3.43 (both s, 3 Н each, both С(4)ОМе); 4.74 (d, 1 Н, С(7)H,
J = 4.6); 4.95 (d, 1 Н, C(7а)Н, J = 4.6); 6.46 (s, 2 Н, С(4)Н)
29**
2.28, 2.35 (both d, 1 Н each, С(7)Н2, J = 15.1); 3.60 (s, 3 Н, С(4)ОМе); 3.95 (s, 1 Н, С(6)Н); 6.28 (s, 1 Н, С(2)Н)
* 1H NMR spectra were recorded at 700 MHz for compounds 13, 14, 26, 27 (in CDCl3), at 500 MHz for compounds 28 (in CDCl3)
and 29 (in DMSOꢀd6), and at 300 MHz for compounds 15—19 and 25a (in CDCl3); the signals were assigned using the HMBC twoꢀ
dimensional experiment.
** No signals for the hydroxyl protons were observed.
The peaks of pseudomolecular ions in the (+)ꢀESI
mass spectra of derivatives 20 and 21 having a CH2COOEt
side moiety correspond to the molecular formula
C13H18Br2O5. The EI mass spectra of these compounds
contain a strong peak for the fragment ion at m/z 327
[M – CH2COOCH2CH3]+, whereas the HMBC spectra
exhibit correlations between the C(8) atom and protons of
methylene (H2C(7)) and ester (—OCH2CH3) units. The

Sponge dibromotyrosines
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
573
Table 2. 13C NMR spectra of compounds 13 and 16—19 (CDCl3)*
Atom
δ
or group
13
16
17
18
19
С(1)
70.9
139.5
124.4
96.2
43.8
172.4
51.2
70.8
138.9
124.2
96.0
43.5
171.8
51.2
—
70.9
138.9
125.2
95.6
43.8
172.3
—
59.1, 15.0
59.5, 15.1
—
70.0
137.6
127.4
95.7
30.1
114.9
51.2
70.0
137.7
127.3
95.7
30.1
115.0
51.4
С(2)Н, С(6)Н
С(3), С(5)
С(4)
С(7)Н2
С(8)
С(4)ОMe
C(4)OEt
15.1
60.0, 15.0
59.9, 15.0
C(4)OBu
—
63.5, 31.2,
19.4, 13.7,
—
*
13C NMR spectra were recorded at 75 MHz for compounds 13 and 18, at 125 MHz for compounds 17 and 19;
13C NMR spectrum of compound 16 was obtained through the CHꢀcorrelation in the HMBC spectrum; the
signals in the spectra of compounds 13, 16, 18, and 19 were assigned using the HMBC twoꢀdimensional
experiment.
NOESY spectrum of ethyl (Eꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀ
hydroxyꢀ4ꢀmethoxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)acetate (20)
has a crossꢀpeak between the H2C(7) (δH 2.62, s) and
—OCH2CH3 at C(4) (δH 3.24, q). Conversely, the crossꢀ
peaks between the H2C(7) (δH 2.63, s) and —OCH3
(δH 3.14, s), as well as between the —OH (δH 4.22, br.s)
and —OCH2CH3 (δH 3.35, q) are found in the NOESY
spectrum its Zꢀisomer (21).
In the HMBC spectrum of homologous diethyl ketal 22,
the protons of both ethoxy groups —OCH2CH3 correlate
to C(4). The data of its 1H and 13C NMR spectra (Tables 1
and 3) and (+)ꢀESI mass spectrum (molecular formula
C14H20Br2O5) correspond to the structure of ethyl (3,5ꢀdiꢀ
bromoꢀ4,4ꢀdiethoxyꢀ1ꢀhydroxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)ꢀ
acetate.
The 1H and 13C NMR spectra of isomeric acids 23 and
24 (see Tables 1 and 3) are close to the corresponding
spectra of their derivatives 20 and 21, however, they have
no signals for the ester ethoxyl. These data together with
the results of the (+)ꢀ and (–)ꢀESI mass spectrometric
studies allows us to draw a conclusion that molecular forꢀ
mula of compounds 23 and 24 is C11H14Br2O5. The IR
spectra of compounds 23 and 24 exhibit a broad absorpꢀ
tion band at 3300—2400 cm–1 characteristic of the stretchꢀ
ing vibrations of hydroxy group of carboxylic acid dimers.
The NOESY spectrum of (Eꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀ
hydroxyꢀ4ꢀmethoxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)acetic acid
(23) contains a crossꢀpeak between the H2C(7) (δH 2.70,
s) and —OCH2CH3 (δH 3.26, q), whereas the correspondꢀ
ing spectrum of its Zꢀisomer (24) — between the H2C(7)
(δH 2.70, s) and —OCH3 (δH 3.15, s).
Table 3. 13C NMR spectra of compounds 20—22 and 24
(CDCl3)*
Atom
δ
or group
20
21
22
24
Note that earlier attempts to separate mixtures of steꢀ
reoisomeric dibromotyrosine mixed ketals were unsuccessꢀ
ful. Because of this, aplysinketal A (15) isolated by recrysꢀ
tallization still is the only dibromotyrosine ketal with the
known stereochemistry found by Xꢀray diffraction analysis.10
The isolated pairs of stereoisomeric mixed ketals are
examples of a rare type of geometrical isomers. Their molꢀ
ecules are achiral, since they have a plane of symmetry, in
which substituents at the two saturated carbon atoms of
the cycle are placed. This plane of symmetry goes through
the atoms C(1) and C(4) perpendicular to the plane of the
double bonds of the ring. The isomers obtained can be
С(1)
70.2
139.3
124.4
96.2
43.9
170.6
50.9
59.2
15.1
61.7
13.5
70.2
139.3
124.7
96.1
43.0
170.9
50.8
59.4 59.3, 59.3**
14.4 14.7, 14.7**
61.6
14.1
70.3
138.9
125.3
95.6
43.6
171.3
—
70.0
138.5
124.8
95.8
42.8
172.0
50.8
59.5
14.9
—
С(2)Н, С(6)Н
С(3), С(5)
С(4)
С(7)Н2
С(8)
С(4)ОСН3
C(4)СН2СН3
C(4)СН2СН3
C(8)ООСН2СН3
C(8)ООСН2СН3
61.7
14.0
—
*
13C NMR spectra were obtained through the CHꢀcorrelations
in the HMBC spectra.
1
distinguished based on the specificities of their H NMR
spectra. Thus, the signals for the protons of the ester groups
** In the HMBC spectrum of compound 22, the corresponding
signals for the carbon atoms of two ethoxyls at C(4) were undisꢀ
tinguished.
1
at C(4) atom in the H NMR spectrum of Eꢀisomer are
closer as compared to the corresponding signals in the

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Santalova et al.
Table 4. The chemical shift values of the signals for the
αꢀprotons of the alkoxy groups at the C(4) atom in the
1H NMR spectra (CDCl3) of configurational isomers
13—16, 18—21, 23, and 24*
rial) protons H(5) and H(6) of the ring equal to 4.6 Hz,
whereas in the H NMR spectrum of diacetate 25c it inꢀ
1
creases to 11.3 Hz. Obviously, acetylation of compound
25a causes changes of the halfꢀchair conformation of the
cyclohexene ring (see Ref. 14 and references cited thereꢀ
in), which results in the fact that diequatorial H(5) and
H(6) atoms become diaxial due to the introduction of
bulky Oꢀacetyl groups at the neighboring positions. In this
case, acetylation of the hydroxyl at HC(6) is a decisive
factor for the change of the ring conformation, which is
Comꢀ
pound
δ (J/Hz)
Н3СОС(4) (с)
—Н2СОС(4)
Eꢀ13
Zꢀ14
Zꢀ15
Eꢀ16
Eꢀ18
Zꢀ19
Eꢀ20
Zꢀ21
Eꢀ23
Zꢀ24
3.21
3.16
3.16
3.21
3.22
3.21
3.21
3.14
3.21
3.15
3.27 (q, J = 6.7)
3.36 (q, J = 6.7)
3.28 (t, J = 6.1)
3.18 (t, J = 6.1)
3.33 (q, J = 7.0)
3.35 (q, J = 7.0)
3.24 (q, J = 7.0)
3.35 (q, J = 7.0)
3.26 (q, J = 6.9)
3.355 (q, J = 6.9)
1
indicated by the H NMR spectrum of monoacetate 25b
with J5,6 = 10.5 Hz.
According to the results of our quantum chemical calꢀ
culations (performed based on the literature data15,16), the
cyclohexene rings of compounds 25a and 25c have conꢀ
formations close to the halfꢀchair 6HC5 and halfꢀboat HB6,
respectively. Therefore, it appears that no complete inverꢀ
sion of the ring 6HC5 → 5HC6 take place upon acetylation
(Scheme 1). The calculated values of the dihedral angle
between the vicinal H(5) and H(6) atoms for compounds
25a (64.3°) and 25c (175.3°) in the corresponding conforꢀ
mations agree with the values of spinꢀspin coupling conꢀ
stants of these protons observed in the 1H NMR spectra of
the ketal and its diacetate.
* 1H NMR spectra were recorded at 300 MHz for comꢀ
pounds 15, 16, 18, 19 and at 700 MHz for the rest of
compounds.
spectrum of Zꢀisomer (Table 4). This difference in CS, as
well as the difference in the signals for the protons of
1
similar substituents at C(4) atom in the H NMR spectra
of dimethoxy ketals 7 and 12 and diethoxy ketals 17 and
22, are explained by the ability of different substituents at
C(1) atom to differently shield the cisꢀalkoxy groups at
C(4) atom.
Scheme 1
Ketals of pꢀhydroxycyclohexenone derivatives. Ketals
25—28 belong to the earlier unknown type of monoene
ketals from extracts of verongid sponges.
The (+)ꢀESI mass spectrum of compound 25a conꢀ
tains a peak at m/z 391.9074 [M + Na]+ corresponding to
the molecular formula C10H13Br2NO4. The 1H—1H COSY
spectrum of compound 25a shows correlation between the
vicinal protons H(6) and H(5), as well as a crossꢀpeak
between the olefin proton H(2) and the equatorial proton
H(6) with characteristic Wꢀarrangement; the HMBC
spectrum exhibits correlational peaks between the ketal
C(4) and the protons of two —OCH3 groups, H(2) and
H(5). Despite that the signal for the —C≡N group is not
observed in the 13C NMR spectra because of the small
amount of isolated compound, the presence of the nitrile
is indicated by the value of CS and splitting of the signals
for the neighboring H2C(7) group in the 1H (see Table 1)
and 13C NMR spectra, which are characteristic of the
corresponding spectra of aeroplysininꢀ1 (1).
Domination of different conformations of the cycloꢀ
hexene ring in solutions of diol ketal 25a and its diacetate
25c was confirmed by the change in intensity of responses
of H2C(7) protons to the H(6) and H(5) in the NOESY
experiments. Thus, in the NOESY spectrum of diol 25a
intensity of the correlation peak between the H2C(7) and
H(6) is higher than between the H2C(7) and H(5), whereꢀ
as in the corresponding spectrum of diacetate 25c the picꢀ
ture is the opposite. To sum up, the diequatorial positions
of the H(5) and H(6) atoms in diol 25a indicates the Sꢀconꢀ
figuration of C(5). Therefore, the structure of [(1S,5S,6R)ꢀ
Though analysis of the NOESY spectrum of compound
25a and the absence of the data on the closely related
structures do not allow us to unambiguously establish conꢀ
figuration of the C(5) atom, results of acetylation of this
1
ketal are very informative. Thus, the H NMR spectrum
of compound 25a exhibits the spinꢀspin coupling constant
for two vicinal (presumably, diequatorial or axialꢀequatoꢀ

Sponge dibromotyrosines
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
575
3,5ꢀdibromoꢀ1,6ꢀdihydroxyꢀ4,4ꢀdimethoxycyclohexꢀ2ꢀ
enꢀ1ꢀyl]acetonitrile (25a) corresponds to the compound
obtained.
Ketals 26—28 are unstable and easily convert to keto
acid 9, which is characteristic of aeroplysininꢀ2 (2)
as well.11
Signals for the substituted hydroxylated ring of γꢀlacꢀ
tone in the 1H (see Table 1) and 13C NMR spectra and IR
spectra of dimethyl ketal 26 and aeroplysininꢀ2 (2)11 looks
alike. The HMBC spectrum of compound 26 exhibits corꢀ
relation peaks between the two —OCH3 groups and the
ketal C(6), and in this case the olefin (H(4)) and the vicinal
(H(7) and HC(7a)) protons also respond to the C(6). The
NOESY spectrum of the compound under consideration
contains a crossꢀpeak between the cisꢀoriented H(3β) at
δH 2.72 (d, J = 18.6 Hz) and H(7) at δH 4.41 (d, J = 3.2 Hz)
protons, that together with the EI mass spectrometric data
correspond to the structure of (3aS,7S,7aR)ꢀ5,7ꢀdibromoꢀ
3aꢀhydroxyꢀ6,6ꢀdimethoxyꢀ3a,6,7,7aꢀtetrahydroꢀ1ꢀbenzoꢀ
furanꢀ2(3H)ꢀone (26).
The NMR spectra, IR and EI mass spectra of comꢀ
pound 27 indicate that it is a homolog of ketal 26. The key
NOESY correlations for (3aS,6R,7S,7aR)ꢀ5,7ꢀdibromoꢀ
6ꢀethoxyꢀ3aꢀhydroxyꢀ6ꢀmethoxyꢀ3a,6,7,7aꢀtetrahydroꢀ1ꢀ
benzofuranꢀ2(3H)ꢀone (27) include the crossꢀpeaks beꢀ
tween the H(3β) at δH 2.71 (d, J = 18.5 Hz) and H(7) at
δH 4.43 (d, J = 3.3 Hz), —OCH2CH3— at δH 3.49 (m),
—OCH2CH3 at δH 1.18 (t, J = 7.0 Hz).
Cyclohexadienol derivative. The 13C NMR spectra of
compound 29 and its known analog with the acetamide
side chain4 are close. However, the presence of the abꢀ
sorption band at 1581 cm–1 in the IR spectrum of comꢀ
pound 29 indicates that it contains a carboxylate group. In
addition, the peak of pseudomolecular ion in the (–)ꢀESI
mass spectrum of this compound at m/z 354.8834 [M]–
corresponds to the molecular formula C9H9O5Br2. The
presence of a carboxylate at position 8, secondary hydrꢀ
oxyl at CH(6), and tertiary hydroxyl at C(1) was demonꢀ
strated by the HMBC correlation and confirmed by the
results of acetylation of compound 29. Thus, treatment of
this compound with acetic anhydride in pyridine at –17 °C
gives a mixture of aeroplysininꢀ2 (2) and its acetate11 (2 : 1)
identified using 1H NMR and HMBC spectra, IR and EI
mass spectra. Moreover, the HMBC spectrum of [(1S,6R)ꢀ
3,5ꢀdibromoꢀ1,6ꢀdihydroxyꢀ4ꢀmethoxycyclohexaꢀ2,4ꢀ
dienꢀ1ꢀyl]acetate (29) shows the presence of admixture of
aeroplysininꢀ2 (2) in it as a result of ready transformation
of compound 29 to γꢀlactone 2.
On origin of isolated compounds. Isoxazoline alkaloids
including aerophobinꢀ2 (32) capable to be cleaved to comꢀ
pounds 1 and 8 when sponges tissues are injured2 (Scheme 2)
have been found by us earlier17 in the extract of Aplysina sp.
If the product of "activated chemical defense" aeroplysiꢀ
ninꢀ1 (1) is a precursor of other simple dibromotyrosines
found in the extract, then numerous enones and ketals
mentioned above should be formed after addition of water
or ethanol, respectively, to the double bond of the fragꢀ
ment of enol ether 1.
Ketals 26 and 28 have the same molecular weight and
fragmentation in the EI mass spectra. The main difference
in the NMR spectra of compounds 26 and 28 as epimers
at the C(7) atom consists in the position of signals for the
HC(7) proton and carbon. Thus, in the 13C NMR specꢀ
trum of compound 28 the signal for C(7) is shifted by
5.2 ppm to the low field as compared to the corresponding
signal for C(7) in the spectrum of epimer 26. In fact,
according to our quantum chemical calculation in the
13C NMR spectrum of compound 28 (conformation of
In fact, hydrolysis of compound 1 occurs under mild
1
conditions. Thus, when the H NMR spectrum of aeroꢀ
7
the cyclohexene ring is close to the halfꢀboat HB) the
plysininꢀ1 (1) is recorded in CDCl3 at room temperature, the
disappearance of usual residual signal for water protons
and appearance of an intense signal for the protons of free
methanol and signals for the protons of compounds 4 and
signal for the C(7) atom should be downfield shifted by
9.4 ppm as compared to the corresponding signal in analꢀ
ogous spectrum of epimer 26 (the halfꢀchair conformation
7aHC7). In addition, in the 1H NMR spectrum of
compound 26 (see Table 1) the difference in CS of
the signals for the H2C(3) geminal protons in the lactone
ring is 0.19 ppm, whereas for compound 28 it is 0.34 ppm.
Such an increase in the nonequivalence of methylene
protons in epimer 28 can be explained by the effect of their
approximation to the pseudoaxial bulky bromine. In
the IR spectra, the absorption band of the lactone carbonꢀ
yl group (1797 cm–1) of compound 28 is shifted toward
somewhat higher frequencies than for compounds 26
and 27 (1792 and 1791 cm–1, respectively). This sugꢀ
gests that in (3aS,7R,7aR)ꢀ5,7ꢀdibromoꢀ3aꢀhydroxyꢀ6,6ꢀ
dimethoxyꢀ3a,6,7,7aꢀtetrahydroꢀ1ꢀbenzofuranꢀ2(3H)ꢀ
one (28), the electronegative bromine is closer to the
carbonyl group (the known "field effect" or dipolar inꢀ
teraction).
1
5 are observed with time. The H NMR spectrum of the
hydrolysis products of 1, accelerated by heating to 50—55 °C
in CDCl3, contains signals not only for compounds 4 and
5, but also for pꢀhydroxycyclohexadienones 6 and 8. It is
obvious that dehydration of compounds 4 and 5 to diꢀ
enone 6, then giving 8, is facilitated by the presence of the
labile H(5) atom at αꢀposition to the hydroxy group at the
C(6) atom. Rechromatographic conditions for aeroplysiꢀ
ninꢀ1 (1) upon the reversedꢀphase HPLC in aqueous methꢀ
anol also lead to the formation of compounds 4—6 and 8.
To sum up, addition of water to the enol ether fragment of
aeroplysininꢀ1 (1) does not require participation of Oꢀdeꢀ
alkylating enzymes, as it has been suggested earlier.18 In
addition, it is obvious that hydration of the nitrile group of
compound 1 occurs rather readily and does not require
interfering postulated by nitrile hydratases.18

576
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
Santalova et al.
Scheme 2
The fact that rechromatography of aeroplysininꢀ1 (1)
Apparently, ethoxy analog of aeroplysininꢀ2 (2) in the
presence of ethanol is transformed with the lactone ring
opening to diethyl ketal 22, whereas aeroplysininꢀ2 (2)
itself — to methyl ethyl ketals 20 and 21 (while in the
presence of water — to keto acid 9).11
in aqueous methanol in addition gives ketals 7 and 12,
whereas in aqueous ethanol ketals 13 and 14, indicates
that addition of alcohols to compound 1 can also proceed
under rather mild conditions. However, storage of aeroꢀ
plysininꢀ1 (1) in CD3OD over two months shows no signs
of its significant transformation to the corresponding ketal.
Only acidification of a solution of aeroplysininꢀ1 (1) in
CD3OD with HCl after heating at 55 °C gives rise to ketals
with the nitrile, amide, and carboxy groups in the side
chain and two —OCD3 residues at the C(4) atom as folꢀ
Compound 29 can be considered as the final hydrolysis
product of aeroplysininꢀ1 (1) at the nitrile group, howevꢀ
er, it is doubtful that such a deep hydrolysis of compound
1 can occur under mild enough conditions of isolation,
while the labile enol fragment remained intact. The more
real seems formation of acid derivative 29 in the step of
isoxazoline ring opening in the precursor of aerophobinꢀ2
(32) type after addition of two water molecules and exꢀ
1
lows from the analysis of the H, HMBC NMR spectra
and the EI mass spectra. It is obviously that transketalizaꢀ
tion takes place in acidic medium19 with the exchange of
the methoxyl with —OCD3. Thus, aeroplysininꢀ1 (1) can
be a precursor of ketals, rather than hypothetical comꢀ
pounds 30 and 31. The presence of a number of new comꢀ
pounds also indicates formation of ketals from enol ethers
Aplysina sp. For instance, ketal 25a can be a product of
addition of methanol to compound 1, whereas ketals 26—28
can be products of addition of the corresponding alcohol
to aeroplysininꢀ2 (2).
Diethyl ketals 17 and 22 are apparently products of
ethanolysis of either the starting enol ether, or the correꢀ
sponding methyl ethyl ketals. At least, we found the correꢀ
sponding signs of transformation of aeroplysininꢀ2 (2)
upon rechromatography of its ethanol solution in aqueous
ethanol, which showed that one of the fractions obtained
contained, together with diethyl ketal 22, a homolog of
aeroplysininꢀ2 (2) with the ethoxyl instead of methoxyl at
the corresponding position. The presence of ethoxy anaꢀ
log of compound 2 was indicated by the 1H, HMBC NMR
and IR data, but, unfortunately, we were unable to register
the mass spectrum of this derivative due to its lability.
+
change of counter ion NH4 with sodium. Since aeroꢀ
plysininꢀ2 (2) can simultaneously be formed in this proꢀ
cess through lactonization, then no postulation of hypoꢀ
thetical imino ether 31 is required to explain the origin of 2.
Scheme 2 illustrates a suggested sequence of transformaꢀ
tions leading from isolated alkaloids to their derivatives.
In conclusion, isoxazoline alkaloids and product of
their bioconversion, aeroplysininꢀ1 (1),2 can play a key
role in the formation of simple dibromotyrosine derivaꢀ
tives (enones, dienones, ketals, and lactones) isolated from
the extracts of sponges of the family Aplysina. In this case,
existence of the mechanism of activated chemical defense
in Aplysina assumes that all the simple dibromotyrosines
of its extracts are not constitutive. However, can they be
considered as artifacts? Conditions used for the isolation
allowed us to obtain a set of compounds typical of the
extracts of Aplysina. All the ketals, esters 10 and 11, which
showed no antimicrobal activity,10,20,21 are obvious artiꢀ
facts, since they contain residues of alcohols used in the
isolation process. This question cannot be answered unꢀ
ambiguously for other simple dibromotyrosines, since they

Sponge dibromotyrosines
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
577
20—38 m near Ladd Reef in the South China Sea (8°39,9 N,
111°41,6 E) during a cruise of the R/V "Academik Oparin" in
May, 2007. The sponge was identified by V. B. Krasokhin
(PIBOC FEB RAS, Vladivostok, Russian Federation). A vouchꢀ
er specimen (034—164) is deposited with the Pacific Institute of
Bioorganic Chemistry collection, Vladivostok, Russia. The
ethanol extract of Aplysina sp. exhibited antibacterial and antiꢀ
fungal activity.
Isolation of compounds 1—29. The collected sponge material
(~4 kg of crude weight) was frozen, stored at –15 °C and then
cut and extracted with ethanol at room temperature. The extract
was concentrated in vacuo, the residue was dissolved in water
and extracted with nꢀhexane and then with nꢀbutanol. The seꢀ
quence of isolation of compounds 1—29 from the butanol fracꢀ
tion of the sponge is given in Scheme 3.
Acetylation of compound 25a. Pyridine (0.2 mL) and acetic
anhydride (0.2 mL) were added to compound 25a (0.2 mg), the
solution was cooled to –17 °C and kept for 3 days. A mixture of
monoacetate 25b and diacetate 25c in the ratio ~7 : 10 was obꢀ
tained after concentration at 55 °C. For exhaustive acetylation,
the same amounts of Py and Ac2O were added to this mixture,
which was kept for ~14 h at +3 °C.
Acetylation of compound 29. Pyridine (0.2 mL) and acetic
anhydride (0.2 mL) were added to compound 29 (0.5 mg), the
solution was cooled to –17 °C and kept for 44 h, then concenꢀ
trated to dryness in vacuo at 55 °C to obtain a mixture of aeroꢀ
plysininꢀ2 (2) and aeroplysininꢀ2 acetate (~2 : 1).21
can be formed on injuring the sponge tissues under natural
conditions and, as a rule, possess antibacterial properꢀ
ties.6,7,13,21,22 The formation of numerous related antiꢀ
bacterial compounds from the labile precursors after addiꢀ
tion/elimination of water can be "programmed" by reꢀ
quirements of the sponge organisms: a wide spectrum of
structures of these antibiotics decreases the risk of developꢀ
ment of resistance to them in microbs.
Experimental
1
H and 13C NMR spectra were recorded on Bruker DРXꢀ300,
Bruker DRXꢀ500, and Bruker Avance III 700 spectrometers
(300.13, 500.13, and 700.13 MHz for the 1H NMR spectra, 75.48
and 125.75 MHz for the 13C NMR spectra), Me4Si was used
as an internal standard. Optical rotation was measured on
a Perkin—Elmer 343 polarimeter; specific rotation is given in
deg mL g–1 dm–1, the concentration of solution is given in
g (100 mL)–1. IR spectra were recorded on Bruker Vector 22 and
Bruker Equinox 55 Fourierꢀspectrophotometers with the resoluꢀ
tion 2 cm–1 in CDCl3 in CaF2 cuvettes (the layer width was
0.61—1.00 mm) or in KBr pellets. Measurements of frequencies
and values of expansion of the contour of the stretching vibraꢀ
tions absorption bands ν(OH) to the components were performed
using the OPUS/IR 02 program package the 3.0.2 version; reꢀ
production of frequency measurements was at least 0.5 cm–1
.
Alcoholysis of aeroplysininꢀ1 (1). Methanolꢀd4 (0.6 mL) and
concentrated HCl (2 drops) were added to compound 1 (0.5 mg),
the solution in the NMR tube was heated for 5.5 h at 55 °C to
obtain a mixture of ketones, ketals, and the starting compound
in the ratio 1 : 1.5 : 2.
2ꢀ(Eꢀ3,5ꢀDibromoꢀ4ꢀbutoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycycloꢀ
hexaꢀ2,5ꢀdienꢀ1ꢀyl)acetamide (16), amorphous compound. IR
(CDCl3), ν/cm–1: 3523, 3406 (NH2), 3362 (OH), 1682 (C=O),
1099, 1079 (OMe, OBu). 1H and 13C NMR spectra (CDCl3)
of compounds 16—19 are given in Tables 1 and 2, respecꢀ
UV spectra were recorded on a Shimadzu UVꢀ1601PC spectroꢀ
photometer in methanol, EI mass spectra were recorded on
a AMDꢀ604S instrument. Mass spectra ESI were recorded on
a quadrupleꢀtimeꢀofꢀflight mass spectrometer with the double
source of electrospray ionization 6510 (Agilent, USA), the neeꢀ
dle potential was 4 kV in the negative and 3.5 kV in the positive
modes of recording of ions at the temperature of the drying gas
325 °C and potential of fragmentator 243 V. Quantum chemical
calculations were accomplished using the functional density theory
with the B3LYP exchange correlation functional (see Ref. 15) in
the 6ꢀ31G(d) and 6ꢀ311G(d) basis of atomic orbitals using the
GAUSSIAN 03 program.16 Electron energies (E) and constants
of magnetic shielding of hydrogen and carbon atoms were found
by the complete optimization of the geometry in the 6ꢀ311G(d)
basis of atomic orbitals in the ground electron state of comꢀ
pounds. Corrections for the energy of zero vibrations (ZPE),
temperature corrections GT and HT were calculated in the same
6ꢀ311G(d) basis. The Gibbs energy G and enthalpy H were calꢀ
culated with allowance for all the electronic, translational, rotaꢀ
tional, and vibrational degrees of freedom at 298.15 К. Column
chromatography was carried out on Sephadex LHꢀ20 (25—100 μm,
Pharmacia, Sweden) and silica gel L (40/100 μm, Chemapol,
Czechoslovakia), TLC was performed on Sorbfil plates (Sorbꢀ
polimer, Krasnodar, Russia) with the foilꢀbound STKhꢀ1A
(5—17 μm) silica gel layer. HPLC was performed on a Du Pont
Series 8800 Instrument chromatograph with the RIDKꢀ102
refractometer as a detector on the ZORBAX Carbohydrate
(4.6×250 mm) columns for compound 29, Supelco Discovery®C8
(4.6×250 mm) for compounds 4, 5, 8, and 12, and Agilent
ZORBAX Eclipse XDBꢀC8 (4×150 mm) for the rest of comꢀ
pounds in the mixtures of MeOH, EtOH, or MeCN with water.
Biological material: The sponge Aplysina sp. (order Verongiꢀ
da, family Aplysinidae) was collected by scuba at the depth
tively. HMBC (CDCl3, 500 MHz), H/C (δH, δ HC(2)
C
and HC(6), C(3) and C(4) overlap): 2 (6)/3 (5), 4, 6 (2), 7; 7/1,
2, 6, 8; OCH3/4; OCH2(CH2)2CH3/4, OCH2CH2CH2CH3;
OCH2CH2CH2CH3/OCH2CH2CH2CH3; O(CH2)2CH2CH3/
OCH2(CH2)2CH3; O(CH2)3CH3/OCH2CH2CH2CH3. COSY
(CDCl3, 700 MHz), H/H: O(CH2)3CH3/O(CH2)2CH2CH3;
O(CH2)2CH2CH3/OCH2CH2CH2CH3; OCH2CH2CH2CH3/
OCH2CH2CH2CH3; OCH2(CH2)2CH3/OCH2CH2CH2CH3.
MS (EI, 70 eV), m/z (Irel (%)): 380/382/384 [M – MeO]+ (4/10/4),
338/340/342 [M – BuO]+ (15/28/12), 306/308/310 [M – MeO –
– BuO – H]+ (13/25/15), 293/295/297 (8/18/13), 274/276/278
(22/33/22), 242/244 (33/47), 215/217 (33/48), 185/187 (19/18),
59 (100), 32 (70). MS (+)ꢀESI, m/z: 433.9562 [M + Na]+.
C13H1979Br2NO4Na. Calculated: m/z 433.9579.
2ꢀ(3,5ꢀDibromoꢀ4,4ꢀdiethoxyꢀ1ꢀhydroxycyclohexaꢀ2,5ꢀdienꢀ
1ꢀyl)acetamide (17), amorphous compound. IR (CDCl3), ν/cm–1
:
3523, 3406 (NH2), 3362 (OH), 1680, 1591 (NH2), 1113, 1089,
1062, 1033 (OEt). MS (EI, 70 eV), m/z (Irel (%)): m/z 397/399/
401 [M]+ (0.5/1/0.5), 352/354/356 [M – EtO]+ or [M – CONH2 –
– H]+ (45/88/45), 334/336/338 [M – EtO – H2O]+ (10/19/11),
318/320 [M – Br]+ (26/25), 307/309/311 [M – 2 EtO]+ (22/40/26),
293/295/297 (10/17/9), 279/281/283 (14/27/17), 272/274
[M – EtO – HBr]+ (50/61), 256/258 (72/71), 244/246 (44/44),
229/231 (100/99), 201/203 (58/63), 185/187 (35/34), 59 (49).

578
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
Santalova et al.
Scheme 3*
a. HPLC; b. HPLC of the fraction after storage; c. HPLC, then recrystallization (CHCl3—hexane).
* The weight (mg) is given in parentheses, BE is a butanol extract.
MS (+)ꢀESI, m/z: 419.9429 [M + Na]+. C13H1979Br2NO4Na.
Calculated: m/z 419.9422.
298 [M – CH2CN – MeO]+ (21/37/19), 286/288 [M – Br]+
(73/73), 280/282/284 (59/97/50) [M – CH2CN – EtO]+, 265/267/
269 (41/82/49), 255/257 [M – MeO – Br]+ (97/97), 246/248
[M – CH2CN – Br]+ (92/94), 241/243 [M – EtO – Br]+ (100/100),
227/229 (69/64), 185/187 (27/30). MS (+)ꢀESI, m/z: 387.9168
[M + Na]+. C11H1379Br2NO3Na. Calculated: m/z 387.9160.
(Zꢀ3,5ꢀDibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycyclohexaꢀ
2,5ꢀdienꢀ1ꢀyl)acetonitrile (19), amorphous compound. IR (CDCl3),
ν/cm–1: 3586 (OH), 2834 (OMe), 1658, 1629 (cisꢀC=C), 1108,
1090, 1065, 1027, 1018 (OMe, OEt). MS (EI, 70 eV), m/z (Irel (%)):
m/z 365/367/369 [M]+ (1/2/1), 334/336/338 [M – MeO]+
(5/13/9), 320/322/324 [M – EtO]+ (17/34/17), 304/306/308
(6/10/5), 294/296/298 [M – CH2CN – MeO]+ (7/12/7), 286/288
(Eꢀ3,5ꢀDibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycyclohexaꢀ
2,5ꢀdienꢀ1ꢀyl)acetonitrile (18), amorphous compound. IR (CDCl3),
ν/cm–1: 3586 (OH), 2836 (OMe), 1659, 1629 (cisꢀC=C), 1110,
1093, 1063, 1026, 1017 (OMe, OEt). (The absorption band of
the nitrile group overlaps with the absorption of CDCl3, that is
observed in the IR spectra of all the dibromotyrosine nitriles
studied.) HMBC (CDCl3, 300 MHz), H/C: 2 (6)/3 (5), 4, 6 (2),
7; 7/1, 2, 6, 8; OCH3/4; OCH2CH3/4, OCH2CH3; OCH2CH3/
OCH2CH3. MS (EI, 70 eV), m/z (Irel (%)): m/z 365/367/369
[M]+ (2/4/2), 334/336/338 [M – MeO]+ (29/63/38), 320/322/
324 [M – EtO]+ (28/53/26), 304/306/308 (15/29/17), 294/296/

Sponge dibromotyrosines
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 3, March, 2011
579
[M – Br]+ (35/35), 280/282/284 (31/51/25) [M – CH2CN –
– EtO]+, 265/267/269 (23/46/27), 255/257 [M – MeO – Br]+
(38/38), 246/248 [M – CH2CN – Br]+ (53/52), 241/243
[M – EtO – Br]+ (100/100), 227/229 (37/32), 185/187 (23/25).
MS (+)ꢀESI, m/z: 387.9153 [M + Na]+. C11H1379Br2NO3Na.
Calculated: m/z 387.9160.
Ethyl (Eꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycycloꢀ
hexaꢀ2,5ꢀdienꢀ1ꢀyl)acetate (20), amorphous compound. IR
(CDCl3), ν/cm–1: 3495 (OH), 1715 (C=O), 1665, 1633 (cisꢀC=C),
1204 (O=C—OEt), 1163, 1102, 1065, 1018 (OMe, OEt). 1H and
13C NMR spectra (CDCl3) of compounds 20—22 and 24 are
given in Tables 1 and 3, respectively. HMBC (CDCl3, 500 MHz),
H/C: 2 (6)/3 (5), 4, 6 (2), 7; 7/1, 2, 6, 8; OCH3/4; OCH2CH3/4,
OCH2CH3; OCH2CH3/OCH2CH3; OH/1, 2, 6, 7;
COOCH2CH3/8, COOCH2CH3; COOCH2CH3/COOCH2CH3.
MS (EI, 70 eV), m/z (Irel (%)): 380/382/384 [M – MeOH]+
(8/20/12), 366/368/370 [M – EtOH]+ (3/6/3), 325/327/329
[M – CH2COOEt]+ (34/64/32), 307/309/311 [M – CH2COOEt –
– H2O]+ (16/20/10), 246/248 [M – CH2COOEt – Br]+ (99/100),
185/187 (16/16). MS (+)ꢀESI, m/z: 434.9418 [M + Na]+.
C13H1879Br2O5Na. Calculated: m/z 434.9419.
Ethyl (Zꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycycloꢀ
hexaꢀ2,5ꢀdienꢀ1ꢀyl)acetate (21), amorphous compound. IR
(CDCl3), ν/cm–1: 3489 (OH), 2833 (OMe), 1714 (C=O), 1665,
1632 (cisꢀC=C), 1205 (O=C—OEt), 1163, 1103, 1094, 1064,
1019 (OMe, OEt). HMBC (CDCl3, 500 MHz), H/C: 2 (6)/3
(5), 4, 6 (2), 7; 7/1, 2, 6, 8; OCH3/4; OCH2CH3/4, OCH2CH3;
OCH2CH3/OCH2CH3; OH/1, 2, 6, 7; COOCH2CH3/8,
COOCH2CH3; COOCH2CH3/COOCH2CH3. MS (EI, 70 eV),
m/z (Irel (%)): 380/382/384 [M – MeOH]+ (4/7/3), 366/368/
370 [M – EtOH]+ (9/20/9), 325/327/329 [M – CH2COOEt]+
(25/44/21), 307/309/311 [M – CH2COOEt – H2O]+ (10/17/10),
246/248 [M – CH2COOEt – Br]+ (98/100), 185/187 (18/19).
MS (+)ꢀESI, m/z: 434.9421 [M + Na]+. C13H1879Br2O5Na. Calꢀ
culated: m/z 434.9419.
(Irel (%)): 352/354/356 [M – MeOH]+ (9/16/12), 338/340/342
[M – EtOH]+ (14/21/10), 32 (100). MS (+)ꢀESI, m/z: 406.9107
[M + Na]+. C11H1479Br2O5Na. Calculated: m/z 406.9106. MS
(–)ꢀESI, m/z: 382.9135 [M – H]–. C11H1379Br2O5. Calculated:
m/z 382.9130.
[(1S,5S,6R)ꢀ3,5ꢀDibromoꢀ1,6ꢀdihydroxyꢀ4,4ꢀdimethoxyꢀ
cyclohexꢀ2ꢀenꢀ1ꢀyl]acetonitrile (25a), amorphous compound. IR
(CDCl3), ν/cm–1: 3562 (OH), 3467 (OH), 1121, 1046 (OMe).
1H NMR spectrum (CDCl3) is given in Table 1. 13C NMR
(CDCl3), δ: 26.3 (C(7)); 48.4 (C(5)); 49.6, 52.3 (2 —OCH3);
73.4 (C(1)); 76.3 (C(6)); 96.9 (C(4)); 124.2 (C(3)); 134.5 (C(2)).
HMBC (CDCl3, 500 MHz), H/C: 2/3, 4, 6, 7; 5/3, 4, 6, 1; 7/1,
2; OCH3/4. MS (+)ꢀESI, m/z: 391.9102 [M
C10H1379Br2NO4Na. Calculated: m/z 391.9109.
+
Na]+.
[(1S,5S,6R)ꢀ1,6ꢀDiacetoxyꢀ3,5ꢀdibromoꢀ4,4ꢀdimethoxyꢀ
cyclohexꢀ2ꢀenꢀ1ꢀyl]acetonitrile (25c), amorphous compound.
1H NMR (CDCl3, 700 MHz), δ: 2.06 (s, 3 H, C(1)OAc); 2.22
(s, 3 H, C(6)OAc); 2.875, 2.92 (both d, 1 H each, C(7)H2, J =
= 16.8 Hz); 3.42, 3.62 (both s, 3 H each, both C(4)OMe); 4.355
(d, 1 H, C(5)H, J = 11.3 Hz); 6.07 (d, 1 H, C(6)H, J = 11.3 Hz);
6.67 (s, 1 H, C(2)H). 13C NMR (CDCl3), δ: 20.4 (C(6)OCOCH3);
21.5 (C(1)OCOCH3); 24.2 (C(7)); 51.6 (C(5)); 70.9 (C(6)); 79.2
(C(1)); 97.1 (C(4)); 125.8 (C(3)); 133.6 (C(2)); 168.8
(C(6)OCOCH3); 169.1 (C(1)OCOCH3). HMBC (CDCl3,
700 MHz), H/C: 2/3, 4, 6, 7; 5/4, 6, 1; 6/1, 4, 5, 7, C(6)—OC=O;
7/1, 2, 6; OCH3/4; C(1)—COOCH3/1, C(1)—OC=O;
C(6)—COOCH3/6, C(6)—OC=O. MS (+)ꢀESI, m/z: 475.9316
[M + Na]+. C14H1779Br2NO6Na. Calculated: m/z 475.9320.
(3aS,7S,7aR)ꢀ5,7ꢀDibromoꢀ3aꢀhydroxyꢀ6,6ꢀdimethoxyꢀ
3a,6,7,7aꢀtetrahydroꢀ1ꢀbenzofuranꢀ2(3H)ꢀone (26), amorphous
compound. IR (CDCl3), ν/cm–1: 1792 (C=O), 1196 (O=C—O),
1
1120, 1093, 1062, 1035 (OMe). H NMR spectrum (CDCl3) is
given in Table 1. 13C NMR (CDCl3), δ: 43.0 (C(3)); 46.5 (C(7));
49.2, 51.5 (2 —OCH3); 74.6 (C(3a)); 87.6 (C(7a)); 96.4 (C(6));
124.1 (C(5)); 135.6 (C(4)); 171.4 (C(2)). HMBC (CDCl3,
500 MHz), H/C: 3/2, 3a, 4, 7a; 4/5, 6, 7a; 7/3a, 5, 6, 7a; 7a/2,
3a, 4, 6, 7; OCH3/6. MS (EI, 70 eV), m/z (Irel (%)): 370/372/374
[M]+ (13/21/11), 339/341/343 [M – MeO]+ (59/100/50),
290/292 [M – Br]+ (45/46), 270/272/274 (43/75/40), 259/261
[M – MeO – Br]+ (22/20), 191/193 (89/68), 44 [CO2]+ (53).
(3aS,6R,7S,7aR)ꢀ5,7ꢀDibromoꢀ6ꢀethoxyꢀ3aꢀhydroxyꢀ6ꢀmethꢀ
oxyꢀ3a,6,7,7aꢀtetrahydroꢀ1ꢀbenzofuranꢀ2(3H)ꢀone (27), amorꢀ
phous compound. IR (CDCl3), ν/cm–1: 3578 (OH), 1791 (C=O),
1199 (O=C—O), 1123, 1096, 1056, 1037 (OMe). 1H NMR specꢀ
trum (CDCl3) is given in Table 1. 13C NMR (CDCl3), δ: 14.8
(—OCH2CH3); 43.0 (C(3)); 46.7 (C(7)); 49.0 (—OCH3); 61.1
(—OCH2CH3); 74.5 (C(3a)); 87.5 (C(7a)); 96.0 (C(6)); 124.4
(C(5)); 135.3 (C(4)); 171.6 (C(2)). HMBC (CDCl3, 500 MHz),
H/C: 3/2, 3a, 4, 7a; 4/3, 3a, 5, 6, 7a; 7/3a, 5, 6, 7a; 7a/2, 3a, 4, 6,
7; OCH3/6; OCH2CH3/6, OCH2CH3; OCH2CH3/OCH2CH3.
MS (EI, 70 eV), m/z (Irel (%)): 384/386/388 [M]+ (8/15/9),
353/355/357 [M – MeO]+ (8/14/8), 339/341/343 [M – EtO]+
(48/100/51), 304/306 [M – Br]+ (23/30), 284/286/288 (22/46/18),
191/193 (75/66), 44 [CO2]+ (43), 32 [MeOH]+ (75). MS (+)ꢀESI,
m/z: 406.9095 [M + Na]+. C11H1479Br2O5Na. Calculated:
m/z 406.9106.
Ethyl (3,5ꢀdibromoꢀ4,4ꢀdiethoxyꢀ1ꢀhydroxycyclohexaꢀ2,5ꢀ
dienꢀ1ꢀyl)acetate (22), amorphous compound. HMBC (CDCl3,
500 MHz), H/C: 2 (6)/3 (5), 4, 6 (2), 7; 7/1, 2, 6, 8; OCH2CH3/4,
OCH2CH3;
OCH2CH3/OCH2CH3;
COOCH2CH3/8,
COOCH2CH3; COOCH2CH3/COOCH2CH3. MS (+)ꢀESI, m/z:
448.9566 [M + Na]+. C14H2079Br2O5Na. Calculated: m/z 434.9575.
(Eꢀ3,5ꢀDibromoꢀ4ꢀethoxyꢀ1ꢀhydroxyꢀ4ꢀmethoxycyclohexaꢀ
2,5ꢀdienꢀ1ꢀyl)acetic acid (23) and (Zꢀ3,5ꢀdibromoꢀ4ꢀethoxyꢀ1ꢀ
hydroxyꢀ4ꢀmethoxycyclohexaꢀ2,5ꢀdienꢀ1ꢀyl)acetic acid (24),
amorphous mixture (~1 : 2). IR (CDCl3), ν/cm–1: 3495 (OH),
3300—2400 (COOH dimers of acids), 2834 (OMe), 1734 (C=O
monomer), 1714 (C=O dimer), 1664, 1632 (cisꢀC=C), 1141,
1104, 1096, 1063 (OMe, OEt). 1H NMR spectrum (CDCl3) is
given in Table 1. HMBC (CDCl3, 500 MHz), H/C: 2 (6)/3 (5),
4, 6 (2), 7; 7/1, 2, 6, 8; OCH3/4; OCH2CH3/4, OCH2CH3;
OCH2CH3/OCH2CH3. MS (EI, 70 eV), m/z (Irel (%)): 352/
354/356 [M – MeOH]+ (9/16/12), 338/340/342 [M – EtOH]+
(14/21/10), 32 (100). MS (+)ꢀESI, m/z: 406.9108 [M + Na]+.
C11H1479Br2O5Na. Calculated: m/z 406.9106. MS (–)ꢀESI, m/z:
382.9138 [M – H]–. C11H1379Br2O5. Calculated: m/z 382.9130.
Acid 24, amorphous compound. IR (CDCl3), ν/cm–1: 3497
(OH), 3300—2400 (COOH dimers of acids), 2834 (OMe), 1733
(C=O of monomer), 1713 (C=O of dimer), 1664, 1631 (cisꢀC=C),
1142, 1104, 1063 (OMe, OEt). HMBC (CDCl3, 500 MHz),
H/C: 2 (6)/3 (5), 4, 6 (2), 7; 7/1, 2, 6, 8; OCH3/4; OCH2CH3/4,
OCH2CH3; OCH2CH3/OCH2CH3. MS (EI, 70 eV), m/z
(3aS,7R,7aR)ꢀ5,7ꢀDibromoꢀ3aꢀhydroxyꢀ6,6ꢀdimethoxyꢀ
3a,6,7,7aꢀtetrahydroꢀ1ꢀbenzofuranꢀ2(3H)ꢀone (28), amorphous
compound. IR (CDCl3), ν/cm–1: 1797 (C=O), 1122, 1098, 1062
(OMe). 1H NMR spectrum (CDCl3) is given in Table 1. 13C NMR
(CDCl3), δ: 42.1 (C(3)); 51.7 (C(7)); 47.9, 51.7 (2 —OCH3);

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Santalova et al.
75.5 (C(3a)); 83.2 (C(7a)); 97.2 (C(6)); 126.0 (C(5)); 132.0
(C(4)); 173.6 (C(2)). HMBC (CDCl3, 500 MHz), H/C: 3/2, 3a,
4, 7a; 4/3, 5, 6, 7a; 7/3a, 5, 6, 7a; 7a/2, 3a, 4, 6, 7; OCH3/6. MS
(EI, 70 eV), m/z (Irel (%)): 370/372/374 [M]+ (4/5/3), 339/341/343
[M – MeO]+ (43/90/47), 290/292 [M – Br]+ (15/19), 270/272/274
(16/25/17), 259/261 [M – MeO – Br]+ (21/20), 191/193 (66/54),
44 [CO2]+ (100).
7. G. M. Sharma, P. R. Burkholder, Tetrahedron Lett.,
1967, 4147.
8. G. M. Sharma, B. Vig, P. R. Burkholder, J. Org. Chem.,
1970, 35, 2823.
9. R. J. Andersen, D. J. Faulkner, Tetrahedron Lett., 1973, 1175.
10. F. Cruz, L. Quijano, F. GуmezꢀGaribay, T. Rios, J. Nat.
Prod., 1990, 53, 543.
Sodium [(1S,6R)ꢀ3,5ꢀdibromoꢀ1,6ꢀdihydroxyꢀ4ꢀmethoxyꢀ
cyclohexaꢀ2,4ꢀdienꢀ1ꢀyl]acetate (29), amorphous compound,
[α]D +20 (c 0.075, MeOH). UV (MeOH), λmax/nm: 281.
11. L. Minale, G. Sodano, W. R. Chan, A. M. Chen, J. Chem.
Soc., Chem. Commun., 1972, 674.
12. C. W. J. Chang, A. J. Weinheimer, Tetrahedron Lett.,
1977, 4005.
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14. A. N. Vereshchagin, V. E. Kataev, A. A. Bredikhin, A. P.
Timosheva, G. I. Kovylyaeva, E. Kh. Kazakova, Konforꢀ
matsionnyi analiz uglevodorodov i ikh proizvodnykh [Conforꢀ
mational Analysis of Hydrocarbons and their Derivatives],
Nauka, Moscow, 1990, 296 pp. (in Russian).
25
IR (KBr), ν/cm–1: 2853 (=C—OCH3), 1581 (COO–), 1025
(=C—OCH3). 1H NMR spectrum (DMSOꢀd6) is given in Table 1.
13C NMR (DMSOꢀd6, 125 MHz), δ: 42.4 (C(7)); 59.2
(C(4)—OCH3); 74.0 (C(1)); 78.0 (C(6)); 113.8 (C(5)); 116.5
(C(3)); 137.0 (C(2)); 146.4 (C(4)); 176.1 (C(8)). HMBC
(DMSOꢀd6, 500 MHz), H/C: 2/1, 3, 4, 6, 7; 6/1, 2, 4, 5, 7; 7/1,
2, 6, 8; OCH3/4. MS (EI, 70 eV), m/z (Irel (%)): 338/340/342
[M – H2O]+ (21/39/17), 320/322/324 [M – 2 H2O]+ (38/80/39),
306/308/310 (7/16/8), 292/294/296 [M – 2 H2O – CO]+
(40/79/37), 278/280/282 (10/28/13), 264/266/268 (12/25/12),
221/223/225 (9/17/9), 213/215 [M – 2 H2O – CO – HBr]+
(16/14), 44 (61), 32 (100). MS (–)ꢀESI, m/z: 354.8834 [M]–.
C9H9O5Br2. Calculated: m/z 354.8822.
15. P. J. Stephens, F. J. Devlin, C. F. Chabalowski, M. J. Frisch,
J. Phys. Chem., 1994, 98, 11623.
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M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr. T.
Vreven, K. N. Kudin, J. C. Burant, J. M. Millam, S. S.
Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene,
X. Li, J. E. Knox, H. P. Hratchian, J. B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterꢀ
ski, P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador, J. J.
Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels,
M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck,
K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G.
Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J.
Fox, T. Keith, M. A. AlꢀLaham, C. Y. Peng, A. Nanayakꢀ
kara, M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen,
M. W. Wong, C. Gonzalez, J. A. Pople, GAUSSIAN 03,
Revision D.01, Gaussian Inc., Wallingford, 2004.
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Commun., 2010, 5, 377.
The authors are grateful to the administration of the
Institute of Oceanography of the Vietnam Academy of
Sciences and Technology (Nha Trang, Vietnam) for asꢀ
sistance in collection of the sponge and V. B. Krasokꢀ
hin (PIBOC, FEB of RAS, Vladivostok) for its identiꢀ
fication.
This work was financially supported by the Russian
Academy of Sciences (Program of the Presidium of RAS
"Molecular and Cell Biology") and by the Council on
Grants at the President of the Russian Federation (Proꢀ
gram of State Support for Leading Scientific Schools of
the Russian Federation, Grant NShꢀ3531.2010.4).
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Received July 20, 2010;
in revised form January 14, 2011