Eudistomidin G, a new β-carboline alkaloid from the Okinawan marine tunicate Eudistoma glaucus and structure revision of eudistomidin B

Article abstract of DOI:10.1016/j.bmcl.2010.05.071

A new β-carboline alkaloid, eudistomidin G (1), has been isolated from the Okinawan marine tunicate Eudistoma glaucus, and the structure was elucidated from spectroscopic data. Furthermore, the structure of eudistomidin B (2), which has been isolated from the same tunicate, was revised from 2a to 2b by detailed analyses of spectroscopic data. Asymmetric synthesis of the revised structure (2b) of eudistomidin B (2) and its (1S,10S)-diastereomer (2c) has been accomplished with the Noyori catalytic asymmetric hydrogen-transfer reaction. The absolute configuration of eudistomidin B (2) was confirmed to be 2b possessing (1R,10S)-configuration, from comparison of the ¹H NMR data, CD spectra, [α]_D values, and HPLC analysis of 2b, 2c, and natural eudistomidin B.

Full text of DOI:10.1016/j.bmcl.2010.05.071

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4100–4103
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Eudistomidin G, a new b-carboline alkaloid from the Okinawan marine tunicate
Eudistoma glaucus and structure revision of eudistomidin B
*
Yohei Takahashi, Haruaki Ishiyama, Takaaki Kubota, Jun’ichi Kobayashi
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new b-carboline alkaloid, eudistomidin G (1), has been isolated from the Okinawan marine tunicate
Eudistoma glaucus, and the structure was elucidated from spectroscopic data. Furthermore, the structure
of eudistomidin B (2), which has been isolated from the same tunicate, was revised from 2a to 2b by
detailed analyses of spectroscopic data. Asymmetric synthesis of the revised structure (2b) of eudistom-
idin B (2) and its (1S,10S)-diastereomer (2c) has been accomplished with the Noyori catalytic asymmetric
hydrogen-transfer reaction. The absolute configuration of eudistomidin B (2) was confirmed to be 2b pos-
Received 19 April 2010
Revised 14 May 2010
Accepted 18 May 2010
Available online 10 June 2010
Keywords:
b-Carboline alkaloids
Tunicate
Eudistoma glaucus
Eudistomidins B and G
sessing (1R,10S)-configuration, from comparison of the 1H NMR data, CD spectra, [
analysis of 2b, 2c, and natural eudistomidin B.
a] values, and HPLC
D
Ó 2010 Elsevier Ltd. All rights reserved.
Marine tunicates are well known to be a rich source of second-
ary metabolites with interesting structures and various biological
activities.1 Among them, many b-carboline alkaloids have been iso-
lated from tunicates of the genus Eudistoma so far.2 In our search
for new metabolites from marine tunicates, a new b-carboline
alkaloid, eudistomidin G (1), has been isolated from the Okinawan
tunicate Eudistoma glaucus together with a known related b-carbo-
line alkaloid, eudistomidin B (2). In this Letter, we describe the iso-
Eudistomidin G (1)4 was obtained as an optically active amor-
phous solid. The ESIMS spectrum of eudistomidin G (1) showed
the pseudomolecular ion peaks at m/z 398 and 400 (1:1), indicating
the presence of a bromine atom, and the molecular formula,
C
21H24N379Br, was established by HRESIMS data (m/z 398.1235
[M+H]+,
D +0.9 mmu). UV absorptions (kmax 296, 286, and 231 nm)
of 1 implied the presence of tetrahydro-b-carboline ring, while the
existence of NH functionality was suggested by the IR absorptions
(3211 and 3049 cmꢀ1). The 1H NMR (Table 1) spectrum of 1 included
signals due to one NH (dY 11.12), eight aromatic protons (dY 7.73–
6.59), two sp3 methines (dY 5.14 and 4.13), six sp3 methylenes (dY
3.28–2.42), and two N-methyls (dY 2.92 and 2.87), while the 13C
NMR (Table 1) spectrum of 1 showed signals due to six sp2 quater-
nary carbons, eight sp2 methines, two sp3 methines, three sp3 meth-
ylenes, and two sp3 methyls.
Connectivities of C-1 to C-10, C-3 to C-4, C-5 to C-6, C-10 to
C-11, and C-13 to C-17 were deduced from the 1H–1H COSY, TOCSY,
and HMQC spectra of 1 (Fig. 1). HMBC correlations for H-5 to C-7
and C-8a, H-6 to C-4b, H-8 to C-7, NH-9 to C-4b, and H-1 to
C-9a, and NOESY correlations for H-1/NH-9, H-4/H-5, and H-8/
NH-9 revealed the presence of a 1,2,7-trisubstituted tetrahydro-
b-carboline ring. 13C chemical shifts of C-6–C-8 (dC 123.88,
117.67, and 115.40, respectively) suggested that a bromine atom
was attached to C-7, while the connection of N-2 and C-19 was dis-
closed by 1H and 13C chemical shifts for CH3-19 (dH 2.92; dC 39.79)
and HMBC correlations for H-19 to C-1 and C-3. The HMBC cross-
peak of H-18 to C-10 and 1H and 13C chemical shifts for CH-10
(dH 4.13; dC 63.80) and CH3-18 (dH 2.87; dC 32.45) indicated that
CH3-18 was attached to C-10 via 10-NH. H-11 showed HMBC cor-
relations for C-12 and C-13 (C-17), suggesting the connectivity of
lation and structure elucidation of eudistomidin
G (1) and
structure revision of eudistomidin B (2) from 2a to 2b as well as to-
tal synthesis of 2b.
Br
Br
6
6
5
7
4
4b
Br
4a
9a
8
8a
9
N
H
NMe
19
H
NMe
NMe
H
R
R
S
1
N
H
N
H
1
1
S H
S
S
10
10
10
MeHN
MeHN
H2N
18
12
15
15
15
Me
eudistomidin G (1)
proposed structure (2a) of
eudistomidin B (2)
revised structure (2b) of
eudistomidin B (2)
According to the previous purification procedure,3 fractions
containing eudistomidins were purified by repeated reversed-
phase HPLC to afford eudistomidins G (1) and B (2).
* Corresponding author. Tel.: +81 11 706 3239; fax: +81 11 706 4989.
E-mail address: jkobay@pharm.hokudai.ac.jp (J. Kobayashi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.071

Y. Takahashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4100–4103
4101
Table 1
Table 2
1H and 13C NMR data for eudistomidin G (1) in CDCl3
1H and 13C NMR data for the revised structure (2b) of eudistomidin B (2) in CDCl3
Position
dC
dH
Position
dC
dH
1
3
63.83c
45.65
CH
CH2
5.14
3.28
2.95e
2.87e
2.42
(d, 3.2)
(dd, 13.0, 6.0)
(m)
(m)
(dd, 16.3, 5.2)
1
3
63.63
45.85
CH
CH2
5.07
3.30
2.94c
2.86c
2.43
(d, 2.9)
(m)
(m)
(m)
(br d, 10.5)
4
15.01
CH2
4
14.92
CH2
4a
4b
5
6
7
8
8a
9
105.98
123.82d
119.53
123.88d
117.67
115.40
137.53
C
C
4a
4b
5
6
7
8
8a
9
105.59
126.61
121.06
113.71
127.06
113.82
135.53
C
C
CH
CH
C
CH
C
7.23
7.27
(d, 8.5)
(dd, 8.5, 1.6)
CH
C
CH
CH
C
7.54
(s)
7.41
7.43
(d, 8.5)
(d, 8.5)
7.73
(d, 1.6)
(s)
11.12
11.19
(s)
9a
10
11
121.62
63.80c
33.06
C
CH
CH2
9a
10
11
122.09
63.86
33.37
C
CH
CH2
4.13
3.24
3.09
(m)
(dd, 15.0, 4.8)
(dd, 15.0, 9.7)
4.01
3.22
3.07
(m)
(m)
(dd, 15.0, 9.4)
12
134.12
128.46
128.86
127.77
32.45
C
12
133.82
128.46
128.93
127.93
32.54
C
13, 17
14, 16
15
18
19
CH
CH
CH
CH3
CH3
6.59a
7.04a
7.12
(d, 7.5)
(dd, 7.5, 7.5)
(dd, 7.5, 7.5)
(s)
13, 17
14, 16
15
18
19
CH
CH
CH
CH3
CH3
6.61a
7.07a
7.15
(d, 7.5)
(dd, 7.5, 7.5)
(dd, 7.5, 7.5)
(s)
2.87b
2.92b
2.85b
2.90b
39.79
(s)
39.81
(s)
a
a
b
c
2H.
3H.
2H.
3H.
b
c,d
Interchangeable.
Interchangeable.
e
J-values were not determined since overlapping with other signals.
C-13 to C-17. HMBC and NOESY correlations indicated the presence
of a 6-brominated 1,2,6-trisubstituted tetrahydro-b-carboline ring.
The connectivity of C-11 and C-12 was suggested by HMBC corre-
lations for H-11 to C-12 and C-13 (C-17). HMBC correlations for
H3-19 to C-1 and C-3 disclosed that one methyl group (C-19) was
connected to N-2, while the HMBC correlation for H3-18 to C-10 re-
vealed that another methyl group (C-18) was not attached to C-15
but attached to N-10. (Fig. 2). These data suggested that the gross
structure of eudistomidin B (2) was 2b but not 2a.9
7
4
Br
3
NMe19
1
N
H
10
MeHN
18
1H-1H COSY and TOCSY
HMBC
NOESY
13
The relative stereochemistry of revised structure (2b) of eudis-
3
tomidin B (2) was not able to be assigned from the JH-1/H-10 value
1
15
(2.9 Hz), and NOESY correlations for H-10/H-19, H-1/H-10, and
NH-9/H-11b. The absolute configuration at C-1 in eudistomidin B
(2) was deduced to be R from a positive Cotton effect at 231 nm
Figure 1. Selected 2D NMR correlations for eudistomidin G (1).
(De
+2.4) in the CD spectrum of 2.5,6
C-11 and C-12. Thus, the gross structure of eudistomidin G was
elucidated to be 1.
To elucidate the relative and absolute configurations of eudis-
The relative stereochemistry of 1 was not able to be elucidated
from the 3JH-1/H-10 value (3.2 Hz) and NOESY correlations of H-1/H-
10, H-10/H-19, and NH-9/H-11b. The absolute configuration at C-1
tomidin B, the revised structure (2b) of eudistomidin B (2) and
its (1S,10S)-diastereomer (2c) were prepared in optical active form
employing the Noyori catalytic asymmetric hydrogen-transfer
reaction10 (Scheme 1). Treatment of 5-bromotryptamine 37 with
carboxylic acid 411 in the presence of EDC and HOBt provided
amide 5. The Bischler–Napieralski reaction12 in benzene afforded
dihydro-b-carboline (6). Noyori asymmetric hydrogen-transfer
reaction of 6 in DMF afforded tetrahydro-b-carboline, which was
in 1 was deduced as R from a positive Cotton effect at 228 nm (
De
+5.6) in the CD spectrum of 1.5
The structure of eudistomidin B (2) was reinvestigated, since
the total synthesis of the proposed structure (2a) of eudistomidin
B (2) revealed that the NMR data of the synthetic compound were
inconsistent with those of natural eudistomidin B (2).7 The molec-
ular fomula of eudistomidin B (2)8 was shown to be C21H24N379Br
Br
6
(m/z 398.1235 [M+H]+,
D +0.9 mmu), and UV and IR absorptions of
2 were the same as those reported previously.3 The 1H NMR (Ta-
ble 2) spectrum of 2 measured in CDCl3 showed signals due to
one NH (dY 11.19), eight aromatic protons (dY 7.54–6.61), two sp3
methines (dY 5.07 and 4.01), six sp3 methylenes (dY 3.30–2.43),
and two methyls (dY 2.90 and 2.85). The 13C NMR (Table 2) spec-
trum of 2 included 19 signals derived from 21 carbons due to six
sp2 quaternary carbons, eight sp2 methines, two sp3 methines,
three sp3 methylenes, and two sp3 methyls. Among them, two
methyls (dC 39.81, dY 2.90, and dC 32.54, dY 2.85) were ascribed
to those bearing a nitrogen.
4
3
NMe19
N
H
10
MeHN
18
1H-1H COSY and TOCSY
13
HMBC
NOESY
2b
15
The 1H–1H COSY, TOCSY, and HMQC spectra of 2 revealed con-
nectivities of C-1 to C-10, C-3 to C-4, C-7 to C-8, C-10 to C-11, and
Figure 2. Selected 2D NMR correlations for the revised structure (2b) of eudis-
tomidin B (2).

4102
Y. Takahashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4100–4103
Br
Br
CO2H
Br
Fmoc
a
b
N
S
Me
N
+
N
H
Fmoc
N
H
O
N
NH
NH2
N
H
N
S
Me
Fmoc
S
4
3
Me
6
5
Br
Br
Br
NMe
H
1
N
H
MeHN
NMe
H
NMe
H
1
1
S
N
H
Fmoc
N
H
MeHN
10
S
R
R
e
f, g, h
c, d
10
S
10
S
6
N
Me
2c
2b
7
Scheme 1. Synthesis of the revised structure (2b) of eudistomidin B (2) and its (1S,10S)-diastereomer (2c). Reagents and conditions: (a) HOBt, EDC, CH2Cl2, 94%; (b) POCl3,
benzene, 12%; (c) (S,S)-TsDPEN–Ru(II) complex, HCO2H/Et3N (5:2), DMF; (d) HCHO aq, NaBH3CN, MeCN, 89% for two steps; (e) DBU, CH2Cl2, 85%; (f) (R,R)-TsDPEN–Ru(II)
complex, HCO2H/Et3N (5:2), DMF; (g) HCHO aq, NaBH3CN, MeCN, 92% for two steps; (h) DBU, CH2Cl2, 78%.
methylated with HCHO and NaBH3CN to yield 7 in 89% (dr >10:1
from 1H NMR analysis). Removal of Fmoc group in 7 with DBU fur-
nished the revised structure (2b) of eudistomidin B (2), having
(1R,10S)-configuration. Similarly, by starting from dihydro-b-carb-
oline (6), (1S,10S)-diastereomer (2c) was synthesized.
at 280 nm], and it was found that the retention time of natural
eudistomidin B (2) (tR 13.0 min) was identical with that of 2b (tR
13.0 min) but not that of 2c (tR 10.8 min). These results also sup-
port that natural eudistomidin B (2) has (1R,10S)-configuration.
The relative stereochemistry of eudistomidin G (1) was eluci-
dated from comparison of 1H NMR data of 1 with those of synthetic
diastereomers (2b and 2c) of eudistomidin B (2). 1H NMR data of 1
were quite similar to those of 2b but not those of 2c, indicating that
the relative stereochemistry of eudistomidin G (1) was the same as
that of the revised structure (2b) of eudistomidin B (2). Since the
absolute configuration at C-1 in eudistomidin G (1) was deduced
to be R from the CD spectrum, the absolute configuration at C-10
in eudistomidin G (1) was assigned as S.
The spectral data (1H and 13C NMR, CD, and optical rotation)13
of the synthetic revised structure (2b) were coincident with those
of natural eudistomidin B (2), whereas the spectral data14 for its
synthetic (1S,10S)-diastereomer (2c) were inconsistent with those
of natural eudistomidin B (2) (Table 3). Thus, the structure of
eudistomidin B (2) was revised from 2a to 2b. Furthermore, syn-
thetic compounds 2b and 2c, and natural eudistomidin B (2) were
subjected to C18 HPLC [Luna Phenyl-Hexyl, 4.6 ꢁ 250 mm; flow
rate 0.5 mL/min: eluent; MeOH/H2O/TFA (80:20:0.1); UV detection
Eudistomidin G (1) corresponds to the 7-bromo analog of eudis-
tomidin B (2). Biosynthetically both 1 and 2 might be derived from
tryptophan and
L-phenylalanine. Eudistomidins G (1) and B (2)
Table 3
showed cytotoxicity against L1210 murine leukemia cells (IC50
,
1H NMR data for synthetic revised structure (2b) of eudistomidin B (2) and its
synthetic (1S,10S)-diastereomer (2c) in CDCl3
4.8 and 4.7 lg/mL, respectively) in vitro. Since eudistomidin alka-
loids show various biological activity,3,15 further biological studies
of eudistomidins B and G are in progress.
Position
2b
(d, 2.0)
2c
(d, 2.3)
1
3
5.10
3.32
2.94
2.86
2.42
5.27
3.52
2.98
2.88
2.30
(m)
(m)
(m)
(m)
Acknowledgments
4
(m)
(m)
(br d, 10.6)
(dd, 16.9, 4.9)
Authors thank Ms. A. Tokumitsu, Center for Equipment Man-
agement Center, Hokkaido University, for measurements of ESIMS.
This work was partly supported by a research fellowship for young
scientists from the Japan Society for the Promotion of Science (to
Y.T.) and Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
4a
4b
5
6
7
8
8a
9
7.53
(s)
7.49
(d, 2.0)
7.43
7.45
(d, 8.6)
(d, 8.6)
7.48
7.42
(d, 9.0)
(dd, 9.0, 2.0)
11.04
(s)
11.27
(s)
9a
10
11
3.99
3.22
3.07
(m)
(m)
(dd, 14.9, 9.3)
4.10
3.27
3.26
(m)
(m)
(m)
References and notes
1. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat.
Prod. Rep. 2010, 27, 165.
2. Wang, W.; Nam, S.-J.; Lee, B.-C.; Kang, H. J. Nat. Prod. 2008, 71, 163. and
references cited therein.
12
13, 17
14, 16
15
18
19
6.56a
7.07a
7.15
(d, 7.3)
(dd, 7.3, 7.3)
(dd, 7.3, 7.3)
(s)
6.50a
7.02a
7.12
(d, 7.5)
(d, 7.5)
(d, 7.5)
(s)
3. Kobayashi, J.; Cheng, J.-F.; Ohta, T.; Nozoe, S.; Ohizumi, Y.; Sasaki, T. J. Org.
Chem. 1990, 55, 3666.
2.87b
2.91b
2.92b
2.94b
4. Eudistomidin G (1): pale yellow amorphous solid; ½a D21
ꢂ
+6.4 (c 0.2, CHCl3); IR
(s)
(br s)
(film) mmax 3211, 3049, 2922, 2851, and 1458 cmꢀ1; UV (MeOH) kmax 366 (
e
a
2H.
3H.
200), 296 (sh 1870), 286 (2190), 231 (10,200), and 203 nm (10,400); CD
b
(MeOH) kext 293 (
D
e
+0.11), 228 nm (+5.6), 213 nm (ꢀ2.5); 1H and 13C NMR

Y. Takahashi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4100–4103
4103
(CDCl3) see Table 1; ESIMS (pos.) m/z 398 and 400 ([M+H]+, 1:1); HRESIMS
(pos.) m/z 398.1235 ([M+H]+, calcd for C21H25N379Br, 398.1226).
11. Biron, E.; Chatterjee, J.; Ovadia, O.; Langenegger, D.; Brueggen, J.; Hoyer, D.;
Schmid, H. A.; Jelinek, R.; Gilon, C.; Hoffman, A.; Kessler, H. Angew. Chem., Int.
Ed. 2008, 47, 2595.
5. Stöckigt, J.; Zenk, M. H. J. Chem. Soc., Chem. Commun. 1977, 646.
6. Though we have previously reported3 that the absolute configuration at C-1 in
eudistomidin B (2) was assigned to be S from a negative Cotton effect at
12. Bischler, A.; Napieralski, B. Chem. Ber. 1893, 26, 1891.
13. Synthetic 2b: pale yellow amorphous solid; ½a D20
ꢂ
+8.2 (c 0.2, CHCl3); UV (MeOH)
kmax 361 (e 280), 301 (sh 1770), 293 (2320), 284 (sh 2700), 227 (12,700), and
216 nm
(
D
e
ꢀ14.4) in the CD spectrum5 of 2, the 1R configuration of
eudistomidin B (2) was deduced from a positive Cotton effect at 231 nm (
+2.4), considering red shift due to bromine substitution in the indole ring.
7. Ito, T.; Kitajima, M.; Takayama, H. Tetrahedron Lett. 2009, 50, 4506.
D
e
205 nm (12,800); CD (MeOH) kext 276 (De ꢀ0.6), 231 (+4.7), and 213 nm
(ꢀ4.0); 1H NMR (CDCl3) see Table 3; ESIMS (pos.) m/z 398 and 400 ([M+H]+,
1:1); HRESIMS (pos.) m/z 398.1230 ([M+H]+, calcd for C21H25N379Br, 398.1226);
13C NMR (CDCl3) d:135.43 (C-8a), 133.93 (C-12), 128.95 (C-14,16), 128.41 (C-
13,17), 127.94 (C-15), 127.15 (C-7), 126.52 (C-4b), 121.90 (C-9a), 121.00 (C-5),
114.02 (C-8), 113.76 (C-6), 105.37 (C-4a), 64.22 (C-10), 64.03 (C-1), 45.61 (C-3),
39.93 (C-19), 33.76 (C-11), 32.54 (C-18), 15.03 (C-4).
8. Eudistomidin B (2): pale yellow amorphous solid; ½a D21
ꢂ
+7.5 (c 0.5, CHCl3); IR
(film) mmax 3211, 3029, 2916, 2855, and 1454 cmꢀ1; UV (MeOH) kmax 364 (
e
170), 301 (sh 1950), 291 (2490), 284 (sh 2410), 230 (12,100), and 204 nm
(11,500); CD (MeOH) kext 281 (
D
e
+0.39), 231 nm (+2.4), and 213 nm (ꢀ1.7); 1
H
and 13C NMR (CDCl3) see Table 2; ESIMS (pos.) m/z 398 and 400 ([M+H]+, 1:1);
HRESIMS (pos.) m/z 398.1235 ([M+H]+, calcd for C21H25N379Br, 398.1226).
9. In this study, eudistomidin B reported previously3 has proved to be a (2:1)
mixture of eudistomidins B (2) and G (1).
14. Synthetic 2c: pale yellow amorphous solid; ½a D20
ꢀ11.5 (c 0.5, CHCl3); UV
ꢂ
(MeOH) kmax 300 (e 1710), 290 (2180), 279 (2190), 227 (11,700), and 205 nm
(11,100); CD (MeOH) kext 278 (
D
e
+0.7), 231 (ꢀ3.3), and 215 nm (+1.3); 1H
NMR (CDCl3) see Table 3; ESIMS (pos.) m/z 398 and 400 ([M+H]+, 1:1);
HRESIMS (pos.) m/z 398.1227 ([M+H]+, calcd for C21H25N379Br, 398.1226).
15. Kobayashi,J.;Nakamura,H.;Ohizumi,Y.;Hirata,Y.TetrahedronLett. 1986,27,1191.
10. Uematsu, N.; Fujii, A.; Hashiguchi, S.; Ikariya, T.; Noyori, R. J. Am. Chem. Soc.
1996, 118, 4916.